Protein Malnutrition in Children: Advances in Knowledge in the Last Ten Years

PROTEIN MALNUTRITION IN CHILDREN: ADVANCES IN KNOWLEDGE IN THE LAST TEN YEARS By J

. C. WATERLOW*

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and G A . 0 ALLEYNE

Medical Research Council of Great Britain. Tropical Metabolism Research Unit. University of the West Indies. Jamaica

I. Introduction

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I1. Clinical Aspects . . . . . . . . . . . . . A . Classification and Nomenclature . . . . . . . . B . Geographical Differences in Clinical Picture . . . . . C . Advances in Treatment and Prognosis . . . . . . D . The Long-Term Effects of Infantile Malnutrition . . . . E. The Assessment of Marginal Protein Malnutrition . . . I11. Protein Requirements and the Prevention of Protein Malnutrition

A . Requirements in Terms of Reference Protein B. Measurement of Protein Value . . . . C . General Conclusion . . . . . . . IV . Body Composition, Body Fluids, and Electrolytes . A . Total Body Protein and Lean Body Mass . B . Body Fluids and Electrolytes . . . . . V . Functional Changes . . . . . . . . A . Cardiac Function . . . . . . . . B. Renal Function . . . . . . . . C. Intestinal Function . . . . . . . D . Changes in Endocrine Activity . . . . VI . Metabolic Changes . . . . . . . . A . Oxygen Consumption . . . . . . . B . Carbohydrate Metabolism . . . . . C . Fat Metabolism . . . . . . . . D . Protein Metabolism . . . . . . . VII . Conclusion . . . . . . . . . . References . . . . . . . . . .

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I. INTRODUCTION Over ten years ago a review was published in Advances in Protein Chemistry summarizing the characteristics of protein malnutrition in

children. with special emphasis on the biochemical and metabolic changes (Waterlow et al., 1960). The aim of the present article is to describe the advances t h a t have been made in our knowledge of the subject in ten years . I n doing this we must inevitably build on the foundations of the earlier review. and wherever necessary will simply refer to it. rather than

* Present address: Department of Human Kutrition. London School of Hygiene and Tropical Medicine. London. England . 117

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repeat facts and arguments which have already been set out. Other comprehensive articles published since 1960 are those by Viteri et al. (1964) and by Metcoff (1967). A complete account of all the contributions in this very wide field would be far too long, so our approach is necessarily selective and subjective. One important aspect that we have not attempted to discuss is the interaction between nutrition and infection, because this is well covered in a recent comprehensive review by Scrimshaw and his co-workers (1968). A noteworthy characteristic of the last decade is the increasing use of animal “models” for the study of human protein malnutrition (Kirsch et al., 196813). Nererthcless, in this review the main emphasis has been given to studies on human beings, since it is not feasible to summarize all the relevant experimental work. Although it is impossible t o give any worthwhile figures for the ineidence of protein malnutrition, there is no doubt that i t is still a very serious problem in large areas of the world-in Africa, Asia, and Central and South America. This does not mean that there has been no practical progress, but increases in food production and improvements in food distribution tend all the time t o be overtaken by rises in population, and by adverse social factors, such as migration from country to town. In this review we only touch on these aspects of the problem. We are primarily concerned, as before, in examining the metabolic and biochemical changes produced by protein malnutrition. At the outset the question has to be faced, because many people ask it, whether there is any point in these detailed investigations, and whether the clinical research worker and the biochemist have any longer a useful contribution to make. It is widely held that we possess enough knowledge to solve the problem, and that what is lacking is the application of this knowledge, not by physicians and laboratory scientists, but by administrators, economists, agriculturists, sociologists, etc. It is not for us to try to counter this point of view. It is enough t o point out that there is no sharp dividing line between malnutrition and good nutrition; in this review we are concerned with nutritional processes, the study of which has been stimulated by the disease protein malnutrition, but which in fact have a wider relevance in medicine and biology. One cannot suppose that there is only one “normal” nutritional state of the organism. Clearly there is a wide range of adaptations to different nutritional conditions, all of which are compatible with health. Protein malnutrition, when it reaches the stage of clinical illness, represents a breakdown of adaptive mechanisms (Waterlow, 1968 ; Monckeberg, 1968a). It seems to us axiomatic that progress in detecting and prcventing protein malnutrition depends ultimately upon a better under-

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standing of adaptive changes a t the level both of the whole organism and of the cell. The concept of adaptation is crucial to the understanding of nutritional problems. This is the theme for the next decade, and this is the justification for the continued scientific study of what is certainly a preventable disease.

11. CLINICAL ASPECTS A . Classification and Nomenclature 'The question of the names used for the different clinical forms of infantile malnutrition was discussed in some detail in the previous review. Nevertheless, i t still causes difficulty and confusion. Most workers continue to recognize two syndromes-kwashiorkor and marasmus-and believe that the former is caused by a relative deficiency of protein in relation to calories,' the latter by a total deficiency of both protein and calories. It would obviously be better if we could make a diagnosis in terms of processes rather than clinical signs (Waterlow, 1955) and refer to protein deficiency and calorie deficiency rather than to kwashiorkor and marasmus (McCance and Widdowson, 1966). Unfortunately, there is still no real proof that these simple causal relationships which have been postulated for twenty years are in fact true. The most convincing evidence comes from animal experiments, such as those of Platt et al. (1964), who produced edema and fatty liver, the typical signs of kwashiorkor, in pigs on a low-protein high-calorie diet, whereas pigs whose total food intake was restricted did not show these signs. Many authors, as far back as Kohman in 1920, have produced edema with low plasma albumin levels in rats on a low-protein high-carbohydrate diet, provided that the animals eat enough of the food, or are force-fed (Kirsch et al., 1968a; Edozien, 1968). In man the experimental production of the two syndromes by different diets is clearly not justifiable, and retrospective studies of the dietary histories of individual patients are of little value. Gopalan (1968) has concluded from a survey in India that there is no detectable difference between diets that lead to The Committee on Nomenclature of the International Union of Nutritional Sciences, meeting in Belgrade and Prague in August 1969, unanimously recommended that the joule should gradually replace the calorie in nutritional nomenclature, the conversion factor being 4.19 joules per calorie. The old terminology has been retained in this review, because it is used in all the papers quoted. The word calorie is used to refer to units of energy in general; kilocalorie (kcal) is used when actual values are given.

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marasmus and those t ha t lead to kwashiorkor. He regards the one as an adaptation to dietary insufficiency, the other as a breakdown of adaptation. We still, therefore, have to fall back on arbitrary definitions of the names t h a t are used, based on the presence or absence of certain clinical signs. For example, Garrow (1966) analyzed a series of 343 cases of primary malnutrition in Jamaica. His definitions were as follows: a child was said t o have marasmus if his weight was less than half that of a normal child of the same age, with no edema and no depigmentation of skin and hair. A child was said to have kwashiorkor only if he had gross edema, with depigmentation of skin or hair and enlargement of the liver. According to these rather strict criteria, 21% of the cases were classified as marasmus and 11% as kwashiorkor. The remaindertwo-thirds of the total-were mixed cases which did not fall neatly into either group. I n many countries there is a preponderance of mixed cases, as in Jamaica. For this reason some workers prefer to abandon distinct names, and t o group all patients under the comprehensive umbrella of “protein-calorie malnutrition” (PCM) . This name draws attention to the fact t h a t in man, although perhaps not in experimental animals, protein deficiency is almost always accompanied by some degree of calorie deficiency. Others feel that to use a single name is a retrograde step, because it ignores differences t ha t are important, and discourages the search for specific causal processes and specific metabolic effects. I n any attempt t o classify or subdivide protein-calorie malnutrition, we have to distinguish between classifications based on quality and on severity. The distinction between kwashiorkor and marasmus is a qualitative one; both conditions may also vary in severity. All workers seem to agree that edema is a sine qua n o n for the diagnosis of kwashiorkor. The other signs that may be found-hepatomegaly, skin and hair changes, mental changes-are given different emphasis in different regions. They were classified among the “primary” signs in the 1960 report, but are difficult to assess objectively. As an example of a qualitative classification, McLaren et al. (1967) have proposed a scoring system in which the main emphasis is given to edema and hypoalbuminemia, with less weight attached t o the other signs mentioned above. This method of scoring is intended to be applied to severe cases and has the advantage of objectivity. It makes no attempt to differentiate according to severity. The earliest and best known classification according to severity is that of Gomez et al. (1956b), who divided protein-calorie malnutrition into lst, 2nd, and 3rd degrees, corresponding to body weights of 90-75, 75-

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60, and less than 60% of the Boston 50th percentile.

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The presence or absence of clinical characteristics such as edema was not taken into account. D. B. Jelliffe (1966133 proposed a modification of this classification into 4 groups, a t intervals of 10% body weight deficit. McLaren and co-workers (1970) have introduced an “index of thriving” which combines in a single score deficits in weight, height, head circumference, and midarm circumference. Classifications of this type are particularly valuable for overall assessment of the nutritional state of children in field surveys, etc., because they include milder cases which may show no specific clinical signs. I n some communities classifications of this type cannot be used because the children’s ages are not known, and therefore deficits in comparison with a standard cannot be calculated. T o get round this difficulty, the suggestion has been made that the arm circumference may be used as an index of nutritional status instead of the body weight (E. F. P. Jelliffe and Jelliffe, 1969) because in normal children i t varies only slightly between 1 and 4 years of age. McLaren et al. (1970) as a refinement of this use the ratio of arm circumference to head circumference, since the latter will not be affected by nutritional state. The classification of choice depends on the purpose for which it is needed-whether for hospital studies or field surveys. A recent working party (Lancet, 1970) proposed a very simple classification which combines some indication of both severity and type. The “normal” or “expected” weight for age is taken as the 50th percentile of the Boston standards (Nelson, 1959). A child is considered to be at risk from malnutrition if his weight is 80% or less of the normal for age (this corresponds approximately to the Boston 3rd percentile) ; he is classified as marasmic if his weight is less than 60% of the expected weight for age. This leads to a classification in 4 groups, as shown in Table IA. This simple approach may be useful for comparing types of malnutrition in different countries, provided that birth weights are known. It must, however, be emphasized that from a strict scientific point of view there is no real justification for putting the emphasis on edema, which is common to all clinical classifications, since children with marasmus, as well as those with kwashiorkor, have increased amounts of body water (Smith, 1960; McLaren and Pellett, 1970). A serious criticism of any classification based on clinical findings a t one given moment is that it ignores the time factor. For example, South African and Mexican workers have shown that “marasmic7’ children have on average serum albumin levels somewhat higher than those of patients with L L k ~ a ~ h i o r k obut r , 7 7lower than those of normal children. They believe, and we agree, that a child diagnosed today as kwashiorkor

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TABLE I

SiniplifLed Classi$cations of Znfantzle Malnutrition

Part A

Edema absent Edema present

Part B Kwashiorkor Marasmus “Nutritional dwarfism” a

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Body weight, as percent of standard“ for age 80-60

Less than 60

“Underweight child” Kwashiorkor

Marasmus Marasmic kwashiorkor

Weight for ageb

Height for ageb

Weight for heightb

11 111 111

1 11

11

111

1

-

50th percentile of Boston standard (Nelson, 1959). Symbols: 1 indicates severity of deficit; = indicates little or no deficit

may present tomorrow as marasmus, if he happens to have lost fluid, and the opposite may also be true. I n fact, it was suggested more than 20 years ago that one type of clinical picture could easily be transformed into the other (Waterlow, 1948). The sequence of events leading to the state of malnutrition is obviously very important, although it is usually difficult to determine accurately. For example, if a child a t one year is grossly underweight, it must undoubtedly make a great difference whether he grew normally for a time, and then lost weight, perhaps as a result of infection and consequent anorexia, or whether he simply grew slowly from birth, without ever losing weight. It has been recognized in recent years that the way in which weight loss or weight deficit is expressed can throw some light on the natural history of malnutrition. A child may lose weight, but he cannot reduce his stature or height. Therefore a child who grows normally for a time and then loses weight mill be underweight for his age, and also underweight compared with a normal child of the same height. A baby whose growth has been retarded from birth will also be underweight for his age, but may have a normal weight in relation to his height. It is likely that these two children, although they may have the same weight deficit in relation to their age, will be very different both in body composition and in metabolic state. As a broad generalization, therefore, one might say that the degree of weight deficit in comparison with a normal child of the same height is a

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measure of the severity of malnutrition; the degree of stunting in height in comparison with a normal child of the same age is a measure of chronicity. When this method is used, differences become apparent between patients diagnosed clinically as kwashiorkor and those diagnosed as marasmus. This is shown schematically in Table IB, based on our experience (Garrow, 1966) and that of Frenk (1969) in Mexico. The same trend is shown in the patients described by Graham (1968) in Peru. He expresses the weight, height, and head circumference each as a ‘(developmental age” which is the age of a normal child of the same weight, height, etc. The developmental age divided by the chronological age gives a ‘(developmental quotient.” With these quotients a realistic comparison can be made of the degree of retardation in the various aspects of growth, in a way which is not possible if deficits are calculated simply in relation to the normal value for a child of the same age. Since height is one-dimensional, head circumference and arm circumference are twodimensional, and weight is three-dimensional, deficits in these parameters are not numerically comparable, whereas the developmental quotients are comparable. It is also worth pointing out that the sensitivity of these measurements to nutritional change will obviously vary according to their dimension. In general, children with kwashiorkor are less under height for their age than those with marasmus, suggesting that the latter is a more chronic process, but the weight for height is not very different in the two groups. Table I B shows a third type of malnutrition, which has been well described by Monckeberg (1968a) in Chile. These are patients who, presumably through general underfeeding, have never grown normally from birth, and at 12 months may hardly have exceeded their birth weight, yet they are normally proportioned babies, with a normal weight for height. They are probably best referred to as cases of “nutritional dwarfism” to be distinguished from cases of marasmus, who are severely underweight for their height. It is probable that some of the conflicting results in the recent literature result from failure to make this distinction. In conclusion, in our view controversy about nomenclature is a waste of time. What matters is the existence or nonexistence of associations between the different features of malnutrition. Weight deficit accompanied by height deficit is not the same as weight deficit without height deficit. It is of little importance what name is given to the patients who present these features; what is important is the mechanism of the association. It is only by a study of associations and their underlying mechanisms that we shall get some insight into the operation of different

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causal processes. I n the remainder of this review, we shall make little use of the names “kwa~hiorkor’~ and ‘ L m a r a ~ m ~except ~ , 7 7 when quoting the results of other authors who have used them.

B. Geographical Differences in Clinical Picture I n the 1960 review (Waterlow et al., 1960), we stated that the essential clinical features of protein malnutrition occur independently of the geographical area or ethnic group. This statement now needs modification and change of emphasis. I n the 1940’s and 1950’s it was important to establish that we were dealing with a condition which was broadly the same throughout the world; that the differences between patients in Europe, Africa, the Far East, and Latin America were superficial rather than real, and did not represent a whole series of different diseases to which it was appropriate to attach separate names. I n general this remains true. However, it has been repeatedly stated that protein-calorie malnutrition represents a spectrum in which the relative importance of either deficiency-of protein or of calories-may vary (see, for example, Viteri et al., 1964). This results not only in differences between individual patients, but also in differences in the characteristic clinical picture from one region to another. The age of onset is another factor which seems to have an important influence on the prevailing form of infantile malnutrition. This probably depends very much upon the duration of breast-feeding, and this in turn depends upon social conditions which vary from region to region. I n one sense these differences complicate the problem, but in another way they are an advantage because they offer a n opportunity for comparative studies, which may help to identify the importance of different causal factors. Such comparisons have been impeded by the lack of agreed criteria for the description and classification of patients. There are not many publications which make possible a clear comparison between conditions in different countries, and much of the descriptive account which follows is based on unpublished exchanges of information. I n regions where most of the population is rural, living mainly off their own resources, breast-feeding is often continued into the second year of life; malnutrition tends to develop in the second or third year, and the predominant form is kwashiorkor. Uganda and Guatemala provide examples of this situation. I n Uganda in a recent series of patients admitted to hospital with kwashiorkor, the peak age was 19-24 months (Whitehead, 1969a) ; in a similar series in Guatemala the average age was 41 months (Viteri, 1969). As people move into towns they are exposed to the pressures of a more sophisticated way of life even though they cannot afford it. More women work away from home, so that

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breast-feeding is reduced in frequency and ends earlier. Other forms of milk are too expensive to provide a n adequate substitute. As a result malnutrition begins to develop a t an earlier age, and the clinical picture is more often that of marasmus or marasmic kwashiorkor (Table I). This kind of situation seems to be occurring in regions such as Mexico, South Africa, India, the Middle East, and the Caribbean. I n Mexico the transition from the rural picture with a predominance of kwashiorkor to an urban or semiurban picture with a high frequency of marasmus and mixed forms has been very noticeable in the last ten years (Frenk, 1969). I n the Middle East the commonest clinical picture is marasmus (McLaren, 1966), and this is now true also in Jamaica. On t5e whole, marasmus develops at an earlier age than kwashiorkor, although there are exceptions to this generalization. I n India Gopalan (1968) gives the age of maximal incidence of marasmus as 6-18 months, of kwashiorkor 1 2 4 8 months. I n Jordan the average age for marasmus was 7.7 months, that of marasmic kwashiorkor 21 months (McLaren, 1966). On the other hand, in the series described by Garrow (1966) in Jamaica, the average age of the group classified as marasmus was 11.7 months, that of kwashiorkor 12.5 months-an insignificant difference. The overall age range was 3-28 months. I n South Africa also no difference has been observed in age incidence between the various clinical groups (Hansen, 1969). The age range again is very wide, from 6 to 48 months. The extreme case of early malnutrition is the infant who from birth or very soon afterward receives too little food-too few calories as well as too little protein. This leads to growth failure and so-called “nutritional dwarfing,” as described by Monckeberg (1968a) in Chile. This syndrome seems to differ in a number of ways from marasmus developing at a later age. We must, therefore, accept that there are well-marked regional differences in the characteristics of infantile malnutrition. These differences depend upon factors such as the age of onset; the duration, nature, and severity of dietary deficiency ; and the incidence of aggravating infections. It is inevitable that the importance of these several factors should vary from place to place. It depends upon one’s objective and point of view whether more attention should be given to the differences or to the similarities. Taking the world picture as a whole, it seems that, in the words of McLaren (1966), “present trends all indicate increasing dominance of marasmus in the future.” They also indicate a shift toward an earlier age of onset. This has consequences of great importance, because of the accumulating evidence that the earlier the onset the greater the danger of

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permanent aftereffects, particularly impairment of mental development (see Section 11,D). I n the past decade the emphasis in preventive programs has been on meeting the protein needs of the preschool child (National Research Council, 1966). If the analysis of trends made by McLaren and others is correct, an alteration in policy is needed to meet the changing situation.

C. Advances in Treatment and Prognosis Treatment falls into two stages, the initial, in which the main consideration is the correction of fluid and electrolyte disturbances and the control of diarrhea and infection; and the recovery stage, in which the objective is to provide enough calories and protein to produce optimum growth rates. 1. Treatment in the Initial Stage

The fluid and electrolyte disturbances which are characteristic of severe cases were reviewed in the 1980 report (Waterlow et al., 1960). Measurements of total body potassium in a whole-body counter (Garrow, 1965) have reinforced the evidence available a t that time that K deficiency is a common and serious complication of infantile malnutrition (see Garrow et al., 1968). Progress has been made in defining the requirements for adequate treatment. Initially, daily K supplements of 6-8 meq/kg are advisable for about a week, or longer if there is diarrhea. The Guatemalan workers (Nichols et al., 1969) have claimed that K repletion cannot occur, even in the initial stage, unless adequate amounts of protein are given a t the same time, but this has not been our experience in Jamaica (Alleyne, 1970). After the K deficit has been corrected, daily supplements of 1-2 meq/kg, in addition to the K provided by the diet, are enough to cover the requirement for growth. During this stage retention of K parallels that of X. Further reports (Back et al., 1962; Caddell, 1967; Caddell and Goddard, 1967) have confirmed the original finding of Montgomery (1960) that protein-malnourished children may be depleted of magnesium. Some patients present clinical signs, but i t is likely that in many others there is an asymptomatic deficiency. This is difficult to diagnose with certainty, because a reduction in serum magnesium level is not invariable, and urine collections, as recommended by Montgomery, may not be practicable. For this reason some workers routinely give supplementary Mg, 1-2 meq/kg per day by mouth. Intramuscular Mg is effective when clinical signs are present. Evidence is beginning to appear that in some parts of the world these patients may be deficient in certain trace elements (see Section IV,B).

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As yet there are no indications for specific treatment, except in the case of copper for refractory anemia (Cordano et al., 1964). I n some countries severe hypoglycemia has been reported (Sloane et al., 1961; Whitehead and Harland, 1966), and intravenous glucose may be life-saving. The possible mechanisms of hypoglycemia and the general subject of carbohydrate metabolism in these patients are discussed below (Section V1,B). Hypoglycemia does not seem to be related in any way to fatty infiltration of the liver, which is one of the most striking factors of the kwashiorkor syndrome. The cause of the fatty liver has still not been established (see Section V1,C) and therefore there is no specific treatment for it. The importance of diarrhea in aggravating the effects of protein malnutrition and precipitating the onset of illness is now well recognized. It plays a major part in causing potassium and fluid loss, and many patients present the clinical signs of dehydration. One of the main dangers in treatment is overhydration and overloading with sodium, which may cause death from pulmonary edema. Wharton et al. (1967) found that high sodium diets could precipitate cardiac failure, and Alleyne (1966a) showed that these infants cannot excrete sodium efficiently. Therefore high sodium intakes should be avoided. Most workers agree that usually no specific pathogenic agent can be found to account for the diarrhea, which has to be treated symptomatically. Bowie et al. (1963) pointed out that a carbohydrate-free diet caused a n instant decrease in stool weight, and were the first to suggest the possibility of an intestinal disaccharidase deficiency (see Section V,C) . The general problem of the interaction between nutrition and infection has been discussed in detail by Scrimshaw et al. (1968). In malnutrition the normal bodily responses to infection may be reduced or disguised. I n children who die it is not uncommon to find a t postmortem evidence of infection, such as otitis or bronchopneumonia, which had not provoked fever or leukocytosis. For this reason it is now common practice, although not universally accepted, to give preventive antibiotic therapy as a routine.

2. Prognosis in the Initial Stage Here we consider the short-term prognosis of malnourished children admitted to hospital. The long-term effects are discussed in Section I1,D. The mortality varies widely in different places, and perhaps reflects the incidence of complicating factors such as gastroenteritis and vitamin deficiencies, particularly that of vitamin A (McLaren et al., 1968, 1969).

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The Mexican workers report a mortality of 30% (Ramos-Galvan and Calderon, 1965) ; in a small series from Teheran the mortality was about 35% (Sadre and Donoso, 1969). I n 146 cases reported by McLaren et al. (1969) from Jordan, the mortality was 28%. It was higher in those with kwashiorkor than in those classified as marasmus, but in Mexico the presence or absence of edema made no difference to the mortality rate. I n Jamaica the mortality in our ward was 15% in the first years of the decade (Garrow and Pike, 1967), falling more recently t o about 67% (Alleyne, 1971). The majority of deaths occur during the first week, and are usually attributed to infections, electrolyte disturbances or liver failure. RamosGalvan and Caideron (1965) analyzed the records of 2400 children admitted to their hospital and showed that 40-50% of the deaths occurred during the first 48 hours. They concluded t hat children with electrolyte imbalance or infection were the most likely to die. From an analysis of 343 children with severe malnutrition in Jamaica, Garrow and Pike (1967) showed that the most important factors contributing to a bad prognosis were a low serum sodium concentration and a raised serum bilirubin. The former is evidence of severe disturbance of electrolyte and body fluid regulation (see Section IV,B), the latter of liver failure. It seems to be a finding peculiar to Jamaica that some children die apparently of hepatic failure. Our earlier data (see Waterlow et al., 1960) showed t h a t the mortality rate is rather high in patients with severe fatty livers (fat more than 40% of wet weight). McLean (1962, 1966) found evidence of functional damage-raised levels of serum bilirubin and of glutamic-pyruvate transaminase. Garrow and Pike (1967) in their review emphasized tha t even without other signs of hepatic failure, a serum bilirubin level of more than 1.0 mg/100 ml indicates a very poor prognosis. The cause of the liver failure is not clear. Many years ago (Waterlow, 1948) i t was shown that bromosulfalein excretion is impaired. This is a measure of liver blood flow as well as of liver cell function. From the histological picture it looks as though the fat takes up so much space that blood flow must be impeded, leading to secondary cell damage. Some children still die in spite of adequate fluid and electrolyte replacement and antibiotic therapy, and without evidence of liver failure. Metcoff and collaborators (1966) have tried, by analyzing muscle biopsy samples, to define the underlying metabolic changes that may be responsible. They showed that in children recovering from malnutrition there was a rise in the levels of potassium, pyruvate, isocitrate, a-oxoglutarate, and oxaloacetate in muscle. I n children who died the opposite changes were found. Unfortunately studies of this kind cannot determine whether the changes found are the cause or the result of cell death.

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3. Indices of Response t o Treatment

There is no single objective index by which the response to treatment can be assessed. Edema is usually lost or greatly reduced within a week, and the serum albumin level rises rapidly once the child begins to take food. These two changes were aptly called by Brock et al. (1955) “initiation of cure.” The serum transferrin concentration also increases at an early stage (Antia et al., 1968). At the same time the pattern of free amino acids in the plasma is rapidly restored toward normal. I n the charts published by Arroyave and Bowering (1968) the fall in ratio of nonessential to essential amino acids is almost the mirror image of the rise in plasma albumin. Both indices may have reached almost normal levels within 3 weeks. The body weight usually behaves rather differently. After the loss of edema it reaches a minimum level, and then may increase very little in the next 2-3 weeks, in spite of an adequate intake of protein and calories. This stationary phase is probably due to the concurrent loss of extracellular fluid as new tissue is laid down (Smith, 1960). That growth is actually occurring is shown by nitrogen retention (Waterlow and Wills, 1960), and by increases in the excretion of creatinine and of hydroxyproline peptides (Picou et al., 1965) (see Section 11,E). Some workers believe that the most sensitive sign that a child has turned the corner and begun to recover is an improvement in his psychological state. It is to be hoped that in the next decade there will be many more objective studies of this aspect of childhood malnutrition.

4. Protein and Calorie Requirements during Recovery Some years ago it was the custom in some centers to treat children recovering from protein malnutrition with very large quantities of protein, up to 6 gm/kg per day. More recently it has been realized that such large amounts cannot be utilized, and that i t is just as important to provide a generous supply of calories as of protein. From the practical point of view it is the calorie supply which is the critical factor, since the amount of food which a child can take is limited by his appetite, gastric capacity, etc. I n order to reach a satisfactory level of calorie intake it is necessary to provide a high proportion of the energy intake in the form of fat, which the infants appear to tolerate very well. We have found that on an energy intake of 160-180 kcal/kg per day and a protein intake of 3-4 gm/kg per day children recovering from malnutrition gain weight a t 1&15 times the rate of normal children of the same height (Ashworth et al., 1968). Similar results have been obtained by Graham and co-workers (1963) with even lower protein intakes.

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Measuremeiits of N balance and of total body potassium showed that the children were laying down tissue of balanced composition and not simply becoming obese. A multiple regression analysis relating weight gain to intakes of protein and calories showed that, within the range of intakes studied, although the level of protein intake had some effect upon the rate of weight gain, that of calories was far more important (Ashworth et al., 1968), so that the calorie intake seems to be the limiting factor determining the rate of growth. At present i t is difficult to understand why such large amounts of energy should be needed. I n our series, during the phase of rapid weight gain, the averagc calorie cost of growth was 12.7 kcal per gram weight gain. If a correction is made for the basal metabolic rate the cost falls to about 10 kcal per gram weight gain. This fits in with the results of nutritional experiments in animals. According to McCracken (1968), the energy cost of fat formation is about 16 kcal/gm. D. S. Miller and Payne (1963), in a theoretical analysis of the rate of protein utilization under different conditions, used a figure of 24 kcal/gm for the energy cost of laying down protein. This value is based on experiments in which N retention is related to calorie intake. On this basis, if 1 gm tissue contains 0.2 gm protein and 0.2 gm fat, 4.8 kcal would be needed for the formation of protein and 3.2 kcal for that of fat, making a total of 8 ltcal per gram of tissue. This agrees quite well with the value found in our infants, which is not surprising since the measurements on both infants and animals were made in a similar way, and based on the same principles. The difficulty arises from the fact that there is no biochemical or thermodynamic basis for such a high energy cost of protein synthesis. The energy requirement for protein synthesis is essentially that for amino acid activation and peptide bond formation. From the known amounts of ATP and G T P needed it can be calculated that the energy cost is less than 1 kcal per gram of protein (Grisolia and Kennedy, 1966; Krebs, 1969). This estimate differs by an order of magnitude from that based on nutritional measurements, and a t present there is no explanation for this difference. When the patients in our series were classified on clinical grounds as kwashiorkor or marasmus, it appeared that the efficiency of growth (weight gain per kilocalorie ingested) was significantly greater in those with marasmus. This suggests that calorie starvation in these infants may have produced some kind of adaptive change in metabolic pathways, but its nature remains to be defined. As the children approach full recovery they voluntarily reduce their food intake, their physical activity increases, and their rate of weight

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gain falls off (Ashworth, 1969a). Sometimes this occurs quite abruptly a t the point when the child has regained his expected weight for height. It remains a mysterious question how the organism knows that it is the “right” size. Tanner’s (1963) concept of growth as “target-seeking” is one way of phrasing the question. Clinical observation suggests that the immediate controlling factor is the child’s appetite. Harper (1969) has put forward the interesting idea that the reduction in food intake which characteristically occurs in rats receiving a diet imbalanced in amino acids is provoked or controlled by changes in the plasma amino acid pattern. We do not know whether repletion of the protein stores, to use Allison’s terminology, brings about a change in the pattern; this is a matter for future investigation.

D . The Long-Term Effects of Infantile Malnutrition Clear-cut information about the long-term effects of malnutrition in infancy is still scanty. The problem would be more tractable if we could have confidence in applying the results of animal experiments to man. In the rat a deficient diet imposed after weaning seems to have no permanent effects, and “catch-up” growth may be complete once an adequate intake is restored. On the other hand, if the intake is deficient during the suckling period, or even during pregnancy, growth may be permanently impaired (Widdowson and McCance, 1960; Chow and Lee, 1964; Venkatachalam and Ramanathan, 1966). I n human populations where there is much malnutrition in infancy, the heights and weights of children usually fall well below the average levels in well-nourished countries (e.g., Jackson, 1966; Ashcroft et al., 1966), and in many of these communities adults also tend to be small in stature. It is well established that there is a relationship between height and social class (Illsley, 1955; Schreider, 1964), and that in some countries there has been a secular increase in height. A brilliant analysis of the causes and consequences of poor growth has been published by Birch and Gussow (1970). They conclude: “There is a growing body of evidence tha,t among groups who are endemically short, increase in stature follows a n improvement in economic status, and that the shortness of such groups under their original environmental conditions arises not from ‘short genes’ but from social and environmental inadequacies.” Although in populations as a whole, economic improvement is accompanied by an increase in stature, we do not know whether in the individual the stunting produced by malnutrition in infancy is permanent, or whether complete “catch-up” growth can occur. Garrow and Pike (1967) made a follow-up study in Jamaica of children who had been admitted to hospital with severe malnutrition 3-5 years previously. Their

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heights and weights were no different from those of siblings who, as far as was known, had never been malnourished. However, in a more recent investigation, also in Jamaica (Birch and Tizard, 1971), children who had been malnourished 5-8 years before were found to be significantly shorter than their siblings, and these in turn were shorter than control children from the same school. These results in one country illustrate the justice of Graham’s conclusion (1968) : “The few existing reports of long-term observations on the later growth of malnourished infants and children are not in agreement and seldom comparable.” I n Peru severely malnourished children who were weaned during the first 3 months of life and then kept on a starvation regime until the age of 1 year or more, after 3 years of intensive dietary management still had severe deficits in height and head circumference which seemed likely to be permanent (Graham, 1968). It is possible that in man, as in the rat, the long-term effects are more severe the earlier the time a t which malnutrition develops. Perhaps differences in the age of onset account for some of the discrepancies which have been observed. Since a small person needs less food, smallness in size may be regarded as an adaptation to shortage of food. I n itself it presumably has little importance unless it is accompanied by functional changes which diminish health and productivity. A few physiological studies have been done on subjects in countries where infantile malnutrition is common. In Ethiopia, Areskog et al. (1969) studied cardiovascular function in school children and adult men, and found little difference in functional efficiency between different social groups, or in comparison with Swedish subjects. I n Guatemala, Viteri (1969) measured the efficiency of physical work in peasants from backward rural communities where malnutrition is common. The working efficiency of these people, smalI as they are, was no less than that of subjects in well-nourished countries. The same, unfortunately, does not seem to apply t o the very important function of childbearing. Thomson (1959), working in Aberdeen, concluded that “The fetus of a short woman has lower vitality and is less likely to be well-grown and to survive than that of a tall woman.” This effect is independent of social class, and clearly leads immediately to a vicious circle. The question of whether malnutrition in infancy affects the incidence and pattern of disease later in life is a key problem for clinicians, pathologists, and epidemiologists, but i t is not one which can be dealt with adequately in this review. If the important experimental work of Ross (1969) could be applied to man, it would give some grounds for optimism, since i t suggests that adaptation to marginal undernutrition may well

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have beneficial effects. Ross showed that in the rat certain liver enzymes change with age according to a definite pattern. Rats whose diet was restricted, so that throughout life they were smaller than controls fed ad libitum, retained an enzyme pattern characteristic of youth and had a longer life-span. Unfortunately, for human beings the position cannot be so simple, since shortage of food is always accompanied by other factors which are adverse. Some years ago a great deal of attention was devoted to the possibility that malnutrition in infancy may cause permanent and progressive damage to the liver-cirrhosis and even primary carcinoma. This seemed possible from animal experiments (see review by Waterlow and Bras, 1957). However, the geographical distribution of kwashiorkor is very much wider than that of hepatic cirrhosis or carcinoma (Brock, 1954), so that other factors must enter in, such as malaria, viral infections, or toxins, e.g., aflatoxin. Moreover, biopsy studies in children who had previously had fatty livers have shown that structure and histology return to normal (Suckling and Campbell, 1957). Cook and Hutt (1967), working in Uganda, concluded that “there is no evidence that progressive liver disease and cirrhosis are long-term sequelae of treated kwashiorkor.’’ The difficulty, of course, is to rule out the possible sequelae of untreated and unrecognized malnutrition. Recent studies of carbohydrate metabolism in malnourished infants (see Section V1,B) have shown that even after 3 months treatment, when clinical recovery is complete, intravenous glucose tolerance tests are still abnormal, and the insulin output in response to a glucose load is less than in comparable children without a history of malnutrition (James and Coore, 1970). We do not yet know whether these changes are permanent. It is well established (see Waterlow et al., 1960) that exocrine pancreatic function is reduced in malnourished infants. Barbezat (1967) has obtained evidence that it may be permanently impaired with chronic malnutrition, but not after an acute episode in infancy. Undoubtedly the most serious aspect of the problem is the possible effect of malnutrition in infancy on brain development and mental capacity. This subject has been extensively covered in several recent reviews (Scrimshaw and Gordon, 1968; Winick, 1969; Platt and Stewart, 1970) ; nevertheless, because of its importance 1971; Birch and GUSSOW, and its difficulty it merits some attention here. Animal experiments show that when malnutrition occurs after weaning the brain is spared compared with the rest of the body, and there is little reduction in the content of protein (Waterlow and Stephen, 1966) , DNA, or cholesterol (Dickerson and Walmsley, 1967). However, if the dietary deficiency is imposed early enough in life it can affect both the chemical

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composition of the brain and the pattern of behavior, and it is entirely possible that these effects are irreversible. Different parts of the brain develop a t different rates and a t different times, the pattern varying from one species to another. The experimental work has established the general principle that the brain is most vulnerable a t the time when it is developing most rapidly (Dobbing, 1964). This has been shown for two processes, the formation of myelin and of DNA. Davison and Dobbing (1966) conclude that “the effect of undernutrition during the myelination period is certainly to retard the process, and may be to produce a permanent deficit.” Winick and Noble (1966) showed that in the rat food restriction in the first weeks of life leads to a reduction, which is probably permanent, in the amount of DNA and number of cells in cerebrum and cerebellum. Feeding a low-protein diet during pregnancy decreased the total number of cells in the whole body and liver as well as in the brain (Zerman and Stanbrough, 1969). Other authors have confirmed that there is a critical period during which low protein diets cause a reduction in brain DNA, and that later feeding of a good diet will not restore the DNA content to normal (Culley and Lineberger, 1968; Guthrie and Brown, 1968; Chase et al., 1969). Platt and Stewart (1968, 1971) have reviewed various types of behavioral changes produced in pigs, dogs, and rats by low-protein diets. The same principle seems to hold, that the earlier the period of malnutrition, the worse the effects. The most severe changes were found in pups who were the offspring of chronically protein-depleted mothers. A curious observation is that in rats females seem to be more resistant than males to the effects of undernutrition on learning capacity (Barnes et at., 1966). The experimental work points to the possibility that in man also protein-calorie malnutrition may cause adverse effects on the brain which are permanent and irreversible, but the evidence is even less clear-cut than in animals. The brain seems to be smaller than normal in malnouriskied children; this has been shown by direct measurements made a t autopsy (Brown, 1966) and indirectly from measurements of head circumference (Stock and Smythe, 1967; Winick and ROSSO, 1969). Information about the composition of the human brain in malnutrition is scanty. At autopsy the potassium content and the ratio of K to N may be low (Garrow et al., 1965), but this is by no means invariable (Alleyne et al., 196913). Since the significance of electrolyte measurements made after death is uncertain, of greater importance is the demonstration by Garrow (1967), by whole-body counting, of a large loss of K from the brains of malnourished infants in vivo (see Sectian IV,B, Table IX). This could well account for some of the mental changes, particularly apathy, which are characteristic of kwashiorkor, and perhaps the enceph-

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alopathy described by Balmer et a,?, (1968). The potassium loss is reversible, but we do not know whether, like temporary anoxia or hypoglycemia, i t can lead to permanent damage. I n a small series of infants in Chile dying of malnutrition, predominantly of a marasmic type, the total DNA content of cerebrum and cerebellum a t any given age was smaller than in children dying of nonnutritional causes (Winick, 1969) , suggesting a reduction in cell number. Similar results were found in infants in Jamaica (Winick et al., 1970). The cholesterol concentration in these brains was normal, but the amount and concentration of gangliosides were reduced (Dickerson, 1971). Since these compounds are preferentially located in the synaptic membrane, a loss of them might have serious consequences for cerebral function. Fishman et al. (1969) found a reduction of about 25% in the cerebroside concentration in the brains of 3 malnourished infants studied in Puerto Rico. The really important questions are whether it is possible to extrapolate from results obtained in children who die to those who are less severely malnourished and recover; and whether changes in size and chemical composition of the brain, if they occur, have any functional significance. Chase et al. (1969) remark: “We have recently examined two children who suffered from severe malnutrition in the first 8 months of life and who showed evidence of cerebellar damage a t age 4 and 5.” This must represent an exceptionally severe effect. In principle, the problem needs to be attacked by psychological as well as neurological methods-that is, by tests of performance. The pioneer work of Cravioto and co-workers was summarized in a review published in 1966. They took stunting in height as evidence of previous malnutrition. Stunted children had significantly poorer neurosensory coordination than childen whose height was more nearly normal for their age. Similar investigations have been made, or are in progress, in a number of different countries, e.g., India (Champakam et al., 19681, South Africa (Stock and Smythe, 1967, 1968) , Chile (Monckeberg, 1968b), and Colombia (Lerna et al., 1968). Various tests of mental capacity have been applied to children of different socioeconomic groups, and without exception it has been shown that those from the poorer and worst-nourished communities have an inferior performance. As all who work on this subject know well, results of this kind are difficult to interpret, and there is a danger of attributing to malnutrition effects which are either genetic or result from social deprivation. People genetically a t the lower end of the scale of mental ability will tend to form a pool of families who are underprivileged and more liable to malnutrition. Again, malnutrition is a concomitant of poverty and ignorance; children brought up in such conditions may not only be short of food but

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also deprived of the stimulus and opportunities for learning, and for the full development of their mental capacities. It is clearly very difficult to control these factors in such a way that a causal relation between malnutrition and impairment of mental capacity can be established unequivocally, if it exists. I n several centers particular emphasis is being laid on longitudinal studies in which children are being followed from birth, with adequate controls of the same genetic stock and social background, e.g., the Human Growth and Development Project in Guatemala (Canosa, 1968). Such investigations are extremely complex, but i t may not be too optimistic to hope that definitive answers may be available within ten years. I n the meantime the information obtained so far in man, taken together with the experimental work, already has practical implications. Since the human brain develops most rapidly in the last 3 months of fetal life and the first 6 months after birth, i t may well be that early malnutrition leading to “nutritional dwarfing” may be more important in its permanent effects than the more dramatic forms of kwashiorkor and marasmus which are typically seen a t about 1 year of age. Since, in the words of D. B. Jelliffe (1966a), “An increasing flood of truly infantile malnutrition is going to be a feature of the next decade in developing regions of the world,” i t becomes of great importance to define as closely as possible the critical period for man, during which dietary deficiency can cause permanent impairment of cerebral function. At the same time added importance must attach to the question of how far maternal undernutrition affects the growth and development of the fetus. The old idea is losing ground, that the fetus is a parasite on the mother, who contributes from her own stores even when her diet is inadequate. The babies of undernourished mothers tend to have a low birth weight (Thomson, 1968), and evidence is beginning to appear that LLsmallfor dates” babies may be handicapped in later life (see Platt and Stewart, 1971 ; PAHO, 1969; Birch and GUSSOW, 1970).

E . The Assessment of Marginal Protein Malnutrition ‘The recognition and assessment of marginal protein malnutrition presents conceptual as well as practical difficulties. If the term “marginal” means anything, i t is a state of malnutrition which does not yet present recognizable clinical signs. It might be better to call it “potential” malnutrition. It is reasonable to suppose that there may be biochemical changes which precede the onset of clinical signs, and i t is just these changes which we are looking for. A measurement which is sensitive must, by definition, give a positive result a t a time when other tests are negative. How then is it possible to show that the positive result has

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any significance? Moreover, to use the words “positive result” begs the question: i t suggests t ha t we can start by defining a normal level or a range of variation, when i t is precisely this which is not known. There seem to us to be three ways of tackling the problem. The first is by accumulating experience of the natural history of the disease. For example, clinicians are justified in regarding a certain level of blood sugar as prediabetic from long experience of the evolution of diabetes and its complications. The second way is by experiment, by imposing a dietary deficiency and following the sequence of the changes which occur. If i t could be shown that a certain biochemical alteration was always followed by accepted signs of malnutrition, then it would be reasonable to regard that alteration as a sensitive index of impending malnutrition. This is the method which has been adopted by Whitehead and co-workers in experiments on rats and pigs, whose particular aim was to establish the significance of changes in the plasma amino acid pattern (Widdowson and Whitehead, 1966; Grimble and Whitehead, 1969, 1970a,b). Arroyave e t al. (1969) made a study which is very relevant for our purpose, although its aim was different. They gave infants diets in which the protein intake was progressively decreased, each level being maintained for 2 weeks. During each period a variety of biochemical measurements were made. The purpose was t o determine the protein requirement by identifying the point a t which the tests became “abnormal.” One could, however, look a t the experiment from the opposite point of view: assume a certain level for the protein requirement, based on the considerations outlined below in Section 111, and try to determine which tests respond most sensitively to intakes below the requirement. A similar study has been published recently by Grimble and Whitehead (1970b). The third approach is by a better understanding of the physiological meaning of the parameters which are measured. There is a wide range of protein intakes compatible with health, and therefore we must suppose that in the physiological sense there is a range of adaptation, and only when this is exceeded does breakdown occur. Understanding of adaptation will not by itself distinguish between “normal” and “abnormal,” but it will help to direct attention t o those changes which are likely to be significant. By an extension of Claude Bernard’s concept, adaptation may be regarded as a process by which not only cell composition but also metabolic function is maintained constant. If that is so, we can distinguish between those parameters which are closely controlied and those which can be allowed t o vary. A familiar example is that of acid-base regulation in the blood; the pH is maintained very constant, but bicarbonate concentration may vary widely according to circumstances. Another example of a quantity whose constancy seems to be important,

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because there are regulatory mechanisms which maintain it, is the intravascular albumin mass (see Section V1,D). A change in one of these “fixed” quantities must be regarded as physiologically significant. The measurements and tests which have been used or proposed for the assessment of marginal protein malnutrition have been described in a recent review (Waterlow, 1969a), in which a survey is made of the results obtained by different workers. Here, therefore, the tests will be summarized briefly, mainly from thc point of view of what they measure. Special attention, however, is given to the plasma amino acid pattern, because this is a promising approach, and one which has attracted some controversy in the last few years. 1, Serum Proteins a. Albumin. The classical biochemical finding in malnourished infants (but not adults) is a reduction in serum albumin concentration. There is some conflict of opinion about whether this is a late event, or whether it develops gradually as protein deficiency progress. Certainly during dietary treatment the albumin concentration rises very rapidly, long before body weight or muscle mass are restored to normal (Brock et al., 1955). Whitehead and Dean (1964) regarded serum albumin as a rather insensitive index of early protein malnutrition, because in children attending a rural clinic the albumin levels were normal, but the plasma amino acid ratios were raised. The children were therefore considered to be suffering from subclinical kwashiorkor. I n Cape Town, Hansen and his colleagues have established quite clearly that there are small but statistically significant differences in average serum albumin concentration between groups of apparently healthy children from different socioeconomic classes (Wittmann et al., 1967), and in children receiving different levels of dietary protein intake (Schendel e t al., 1962). According to the data of the latter authors, the range in well-fed subjects is quite narrow, the coefficient of variation being of the order of 10%. Therefore in an individual a reduction of 20% below the normal mean would be suspicious. This is quite sensitive in comparison with other clinical biochemical measurements. From what is known about the metabolism of albumin one would expect a fall in concentration to be a rather late event, because of the protective mechanisms which tend to maintain the circulating albumin mass (see Section V1,D). Schendel et al. (1962) found that in children on a poor quality diet, in which the protein was derived from maize, the serum albumin did not begin to fall for several weeks. It seems likely, therefore, that a moderate decrease in albumin concentration could be interpreted as a sign of long-continued, though not necessarily seyere, protein deficiency.

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b. Transferrin. Antia et aZ. (1968) showed that in kwashiorkor the serum transferrin (siderophilin) concentration may fall to one-fifth of the level found in controls-a depression much greater than that of total protein or albumin. These workers claimed that the transferrin level gave a more accurate assessment of the severity and response to treatment of patients with protein-calorie malnutrition than any of the other biochemical tests which are currently used (H. McFarlane et aZ., 1969). 2. E n z y m e s

Not very much work has been done in the last ten years on enzyme measurements as a tool for the detection and assessment of early protein malnutrition, and that which has been done contributes little to the solution of the problem. There are three quite distinct lines of investigation. The first is the attempt to identify in tissues from malnourished subjects enzymatic changes that will indicate the nature of the underlying biochemical lesion or adaptation. Examples of this approach are the work of Waterlow (1961) on oxidative phosphorylation in the liver, of Metcoff and his colleagues (1966) on intermediates and enzymes of carbohydrate metabolism in muscle, and of Stephen and Waterlow (1968) on adaptive enzyme changes in the liver. These studies are not very relevant to the problem of early diagnosis and assessment. The second line of approach is one which has been exploited with great success in general medicine in recent years-the measurement of enzymes which appear in excessive amounts in serum as a result of damage to tissues and leakage from cells. McLean (1966) found that in severely malnourished children serum levels of glutamic-pyruvic transaminase and isocitric dehydrogenase were increased. The greatest rises occurred in patients who died. In the liver, in contrast to the serum, glutamic-pyruvic transaminase activity was reduced. This appeared to be the result of two processes: in the human infant, as in the rat, low protein feeding lowers the activity of this enzyme in the liver. At the same time the high serum levels presumably indicate leakage from damaged cells. Obviously in this situation we could not expect a clear correlation between enzyme level in the serum and the severity of protein malnutrition. Ittyerah and co-workers in India have explored the possibility of lysosoma1 damage in kwashiorkor. They found a raised serum level and an increased urinary excretion of the lysosomal enzyme arylsulfatase, which returned to normal with recovery (Ittyerah e t al., 1967; Begum and Ittyerah, 1970). Other lysosomal enzymes which were studied-acid phosphatase and P-glucuronidase-did not show these changes. Moreover, the excretion of arylsulfatase was greatly increased by infection,

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so that i t would be difficult to establish a close relation between enzyme changes and severity of malnutrition. It seems likely that these effects, which depend upon leakage from damaged cells or intracellular organelles, are late events in the progress of a deficiency state. The third line of approach is based on the hypothesis that since enzymes are proteins their level in the bloodstream may give some indication of the rate of protein synthesis in the organ from which they are derived. For pseudocholinesterase, this is the liver; for amylase, the exocrine pancreas ; for alkaline phosphatase, in all probability, bone. The basis of the hypothesis is clearly insecure, since the level in the blood must depend on the rate of disappearance as well as on the rate of entry of the enzyme. Moreover, it is not necessarily true that protein deficiency will reduce the rate of production of an enzyme. It is now clear that within a single tissue the synthesis rates of different proteins may be altered in different directions by the same dietary stimulus (Rechcigl, 1968; see also Section V1,D). Empirically, fairly well-defined changes have been found in certain enzymes, of which pseudocholinesterase is one of the best examples as well as one of the first to be investigated, The literature has been summarized recently (Waterlow, 1969a ; Waterlow and Stephen, 1969). Most of the studies relate to patients with kwashiorkor or marasmus, very few to subjects who are not clinically ill but may be considered a t risk to malnutrition. Schendel et al. (1962) showed that there was a slight reduction in pseudocholinesterase, paralleling that of serum albumin, in children receiving a low protein intake, but Behar e t al. (1960) found normal levels of pseudocholinesterase and alkaline phosphatase in preschool children of a low income group. Evidently, therefore, these enzyme changes in the serum are rather insensitive, and it is a natural step t o search for enzymes which are more sensitive, using animal experiments as a guideline. In the liver some of the enzymes of amino acid catabolism, for example, threonine dehydrase, are extremely sensitive to the level of protein intake (Pitot and Peraino, 1964) and variations of 100-fold in activity may be produced by appropriate dietary conditions. Unfortunately this enzyme is not present in the serum, as shown by Flores (1970) in our laboratory. The latest addition to the enzymes which have been studied from this general point of view is creatine kinase, which occurs mainly in muscle and to a lesser extent in brain. Reindorp (1970) found that in rats on different diets serum creatine kinase activity was correlated with creatinine output and with muscle mass. In children with kwashiorkor, serum creatine kinase is extremely low and rises 10-fold or more on treatment (Balmer and Rutishauser, 1968; Reindorp and Whitehead, 1971).

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This enzyme therefore does seem to be a sensitive indicator of the nutritional state; unfortunately its activity in serum is also very much affected by nonnutritional factors, such as infections. Further work needs to be done on the value of this enzyme measurement as an indicator of early malnutrition. We conclude that so far measurements of enzyme activity have not achieved the objective of providing a method of assessing protein deficiency which is both specific and sensitive. The practical requirement that measurements should be made on blood or urine is a serious limitation; it is possible that more useful results could be obtained from white blood cells (Pineda, 1968). 3. Urinary Nitrogen and Urea

The urinary output of nitrogen or urea is a measure of the preceding N intake. If 24-hour specimens are not available, early morning samples may be taken and total N or urea N related to creatinine. Another index which has been used is the relation of urea N to total N. The smaller the total N excretion, the smaller is the contribution of urea and the larger that of the “endogenous” N-containing compounds. I n subjects on a normal protein intake urea N forms about 80% of total urinary N, on a low intake about 50%. These urinary indices do show differences between groups living on different levels of protein (Luyken and Luyken-Koning, 1960), and in several surveys a good correlation has been reported between the N: creatinine ratio in urine and dietary protein intake (e.g., Powell et al., 1960; Simmons and Bohdal, 1970), although this was not our experience in a survey in Jamaica. It seems not to be known how far the N content of a morning sample of urine reflects the immediately previous intake, i.e., the meal of the evening before, or whether it gives an indication of the general level of intake over several days. This must depend upon the speed with which the adaptive mechanisms come into operation, adjusting output to match intake. In children the adjustment is more rapid than in adults. Martin and Robison (1922) took about 6 days to reach minimum urinary N output on changing from a normal to a protein-free diet, whereas the infants studied by Chan (1968) adapted in 2 days. Such experiments, however, do not tell us very much about the effect of day-to-day fluctuations. Some results which we obtained in a study of acclimatization to high altitude may be relevant to this question (Waterlow and Bunje, 1966). Urine was collected in 12-hour periods, i.e., day and night, for 25 days, during which time the protein intakes fluctuated widely as a result of mountain sickness, intense physical activity, etc. The N in the morning urine samples did tend to reflect the

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previous day’s intake, but with smaller variations, so that the fluctuations of intake were smoothed out. Clearly the urinary nitrogen indices are most likely to be useful as a measure of protein nutrition in people whose dietary pattern is regular and constant.

4. Urinary Creatinine Output The validity of urinary creatinine excretion as a measure of muscle mass is discussed in Section IV,A. The experience gained in children recovering from malnutrition indicates t ha t the deficit in muscle mass is relatively greater than t ha t of body weight (Standard et al., 1959; Picou et al., 1965), and therefore it should be a fairly sensitive index of protein depletion. The difficulty is the practical one, that the excretion has to be measured over a timed interval. Arroyave and Wilson (1961) showed that excretion over a period of 3 hours was proportionately the same as in 24 hours. Using this shortened collection period, they showed that creatinine output was below the normal level in children from low income groups in Guatemala. Other workers do not seem to have followed this lead.

5. Excretion of Hydroxyproline Peptides Hydroxyproline, mainly in peptide form, is excreted in the urine as a by-product of collagen metabolism, and the rate of excretion seems to be closely related to the rate of growth (Jasin et al., 1962; Smiley and Ziff, 1964). The excretion of hydroxyproline is greatly reduced in malnourished children (Picou et al., 1965; Whitehead, 1965), and this has been widely used, particularly by Whitehead, as the basis of a field test for the evaluation of nutritional status. Since in the field i t is seldom practicable to make timed urine collections, Whitehead (1965) related the hydroxyproline concentration in the urine to that of creatinine to give the hydroxyproline ratio. This, when corrected for body weight, he called the hydroxyproline index. McLaren et al. (1970) recommend an additional correction for age. I n principle, the hydroxyproline output gives a measure of the rate of growth a t one moment of time. This would be a great advantage, because single measurements of weight and height in children tell what has happened in the past, but not what is happening now, and repeated measurements may not be practicable. As an illustration of this, Rutishauser and Whitehead (1969) report a survey on children of three tribes in Uganda. The group with the lowest hydroxyproline excretion was by a considerable margin taller than the other 2 groups, but their body fat, measured by skinfold thickness, was less (see Table 11). The interpreta-

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tion of these findings was that this tribe normally lived on a diet providing adequate protein, and the children were therefore well grown, but because of seasonal variations, they were very short of food a t the actual time of the survey. Thus the hydroxyproline index supplemented the information provided by weight and height. On the other hand, McLaren and co-workers (1970), in a study over a period of 4 months of children recovering from marasmus, found no correlation between hydroxyproline output and rate of gain in height. They emphasized the pitfalls in the use of this test and concluded with a pessimistic appreciation of its value. If hydroxyproline excretion is related to collagen metabolism (Weiss and Klein, 1969), it may seem strange that the excretion should be reduced in malnutrition, in spite of the fact that in malnourished animals and children the collagen content of the body and of tissues such as muscle is increased, and the absolute amount is close to that expected in a normal child of the same height (Mendes and Waterlow, 1958; Dickerson and McCance, 1964; Picou et al., 1965). Collagen is certainly not a totally inert tissue, particularly in young subjects. The most likely explanation of the facts is that the catabolic rate of collagen is decreased in malnutrition, but this does not necessarily imply a reduction in the rate of net synthesis or growth. 6. Plasma Amino Acid Ratio

Changes in the pattern of free amino acids in blood in relation to protein nutrition have been studied by many workers. This is a new development in the last ten years, and one which seems to offer much promise for the diagnosis of marginal protein malnutrition. The first complete study was that of Arroyave et al. (1962) in Guatemala. A year later Holt and co-workers (1963) described the plasma “aminogram” of patients with kwashiorkor from nine different countries. The pattern was fairly constant, regardless of the dietary background. The salient changes compared with the normal pattern were a fall in the concentrations of most of the essential amino acids, particularly the branched-chain amino acids, with lysine and phenylalanine less affected ; the inessentials were well maintained or even increased in concentration. Holt concluded that this pattern was characteristic of a situation in which the limiting factor in the diet was not any single essential amino acid, but total nitrogen. Shortly afterward Whitehead and Dean (1964) suggested that the degree of distortion of pattern might be an index of the severity of protein depletion. Whitehead (1964b) developed a simplified paper chromatographic method by which he estimated the ratio of the concentrations of two groups of amino acids (N:E ratio).

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J. C. WATERLOW AND G . A, 0. ALLEYNE

The inessential group (N) consisted of glycine, serine, glutamine, and taurine, and the essential group (E) of leucine, isoleucine, valine, and methionine. The ratio was greatly increased in kwashiorkor, and high values compared with controls were found in ambulant children attending a rural clinic, who for this reason were considered t o have mild protein malnutrition. Much less striking changes were found in marasmic patients (Whitehead, 1965), and therefore Whitehead and Dean claimed that the test was diagnostic of protein deficiency. Not all workers have found raised N : E ratios in malnourished children (e.g., McLaren e t al., 1965) ; Saunders e t al. (1967), in contrast t o Whitehead, found no difference in the amino acid pattern between children with kwashiorkor and those with marasmus. The explanation put forward for these discrepancies was that in calorie deficiency the levels of essential amino acids in the plasma are kept up because of the breakdown of protein to supply energy, and the same may occur in infection. Many patients diagnosed clinically as kwashiorkor may in fact be suffering from a mixed deficiency of protein and of calories, or from complicating infections. Grimble and Whitehead (1969, 1970a) produced evidence to confirm this hypothesis in experiments on pigs fed decreasing levels of protein. At a stage when growth had almost ceased because of the low protein content of the diet, but the calorie intake was normal, the ratio of nonessentials increased, mainly because of a rise in alanine and a fall in the branched-chain amino acids. I n the terminal phase loss of appetite led to reduced calorie intake, and the ratio then tended to fall, largely because of a decrease in the nonessential component. These two papers bring out another important point. Although estimation of what may be called the “Whitehead ratio” by paper chromatography has the advantage of technical simplicity and avoids errors inherent in the measurement of absolute concentrations, it can also give confusing results because a rise may be caused by an increase in the numerator or a decrease in the denominator or both, and the two may vary independently. Moreover, in the simplified test the nonessential group does not include alanine, the alterations in which are very important. Perhaps the most clear-cut and specific test for protein deficiency is a decrease in the concentration of branched-chain amino acids, particularly valine, in the plasma. The significance of the changes in individual amino acids will be considered in more detail in Section V1,D. The question to be examined here is: How useful are measurements of plasma amino acids for the practical assessment of protein malnutrition? Observations in the field suggest t h a t this type of test does give specific information not provided

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PROTEIN MALNUTRITION I N CHILDREN

TABLE I1 Comparison of Anthropomctric and Biochemical Indices of Nutrition in Children of S Communities in Uganda0 Community Index

A

B

C

Standard

Weight (kg) Height (cm) Triceps skin-fold (mm) Plasma albumin (gm/100 ml) Amino acid ratio Hydroxyproline index

11.3 82.5 8.3 5.7 3.2 2.1

11.6 91.3 6.9 6.3 2.3 1.6

11.9 84.9 9.6 3.3 2.6 1.9

13.5 91.8 9.8 -

-

a Data from Rutishauser and Whitehead (1969) for children aged 25-36 months, by courtesy of the Editor of British Journal of Medicine.

by other measurements. Some of the findings in the survey of three tribes already referred to (Rutishauser and Whitehead, 1969) are summarized in Table 11. The children of group B normally live on a diet of cereals and milk, but were suffering from an overall shortage of food. They were tall but thin, with the highest serum albumin levels and the lowest N : E ratio. In group C, whose staple diet is millet and plantain, the children were short but fat, and had the highest incidence of minor clinical signs of malnutrition. They had the lowest serum albumin level and an intermediate N : E ratio. I n this region malaria and hookworm infestation are very common. I n group A the staple is again plaintain, but the incidence of infections is less. These children were also short, and had the highest N : E ratio, but no depression of serum albumin. The conclusion drawn was t ha t in both groups A and C there was a specific deficiency of protein, and that “the biochemical measurements indicated the reason for the nutritional difficulties, which could otherwise only have been surmised from the food habits of the area.” This study has been discussed in detail, because it is an excellent example of the application of these measurements to nutritional problems in the field. The surveys made by the Institute of Nutrition for Central America and Panama (INCAP, 1971) have shown that in groups who are a t risk from protein deficiency-that is, with intakes a t the border-line of the estimated requirement-the N : E ratio tends to be higher than in those who are certainly well-fed. There is a clear correlation between the average N : E ratio and social class, but the elevations found are not great, and only become significant with numbers. For example, children in an orphanage receiving a good diet were compared with those from a poor rural community (Arroyave and Bowering, 1968). The mean N: E ratio was 2.07 in the former and 2.89 in the latter. This difference was

146

J. C. TVATERLOIV AND G. A. 0.ALLEPKE

highly significant statistically, but it was not reflected in any great difference in weight or heights. If the hypothesis of Holt and of Whitehead is accepted, that these changes in amino acid ratio or pattern are diagnostic of the process of protein deficiency, the more difficult question then has to be considered, whether they reflect the state of protein nutrition, and are a sign of an existing or impending pathological process, or whether they represent an adaptive change reflecting the prevailing level of protein intake. It is certain that, even in the absence of calorie deficiency, the ratio of nonessentials to essentials may be normal in a subject who is severely protein depleted, in the sense discussed in Section I V below. This is shon-n by the response of patients with kwashiorkor to treatment ; Saunders et al. (1967) observed t h a t after only 1 day of treatment the fasting plasma aminogram of patients with kwashiorkor returned to normal. I n the patients clescribed by Arroyave and Bowering (1968), the N:E ratio, initially high, fell within 2 or 3 weeks to normal levels long before body weight or lean body mass had been restored. Conversely, when protein deficiency is imposed experimentally in human subjects, an alteration in amino acid pattern occurs quite rapidly. Su-endseid e t al. (1966) found that when adult men were given a diet providing 20 gm of protein daily, within 10-15 days the ratio’ of nonessential to essential amino acids increased from an average initial level of 2.1 to about 3. This occurred a t a time when the cumulative N loss from the body was only about 25 gm, or little more than 1% of total body N. Scrimshaw et al. (1966) gave young men a diet containing 0.4 gm of protein per kilogram per day, which produced a small negative N balance. Initially the average value of the ratio of nonessentials t o essentials was 1.43, rising to 1.83 after 15 days. I n a later study Young and Scrimshaw, 1968) young men were fed a protein-free diet for 16 days. The ratio of nonessentials to essentials increased from 1.34 to 2.22, in spite of the fact that over this period the lean body mass, measured by whole body K counting, decreased only by 0.5 kg-an insignificant degree of depletion. Holt e t a l . (1968) reported that premature infants fed on low intakes of milk protein (2 gm/kg) promptly developed amino acid patterns simulating kwashiorkor, although they were gaining weight normally. Holt therefore asked the key question: “Must we enlarge our concept of ‘In these experimental studies the concentrations of all the amino acids in the plasma were measured, and expressed as micromoles per liter. The values quoted here represent the sum of the nonessentials divided by the sum of the essentials. They are therefore not strictly comparable nith the N : E ratios derived by Whitehead’s simplified method.

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what is a normal aminogram, or must we assume that a deviation from the accepted normal is in some respects an indication of a menace which does not immediately manifest itself clinically?” Animal experiments have so far not provided much illumination on this point. In the rat experiments of Widdowson and Whitehead (1966), the protein deficiency was so severe that the rats immediately stopped growing, and one cannot tell whether the rise in N:E ratio has any prognostic significance. I n experiments in which pigs were given progressively lower protein intakes the ratio did not become abnormal until growth had slowed and serum albumin concentration had begun to fall (Grimble and Whitehead, 1969). In puppies fed a low-protein diet, Heard et al. (1969) found that the N : E ratio rose only slowly, and was a less sensitive index of deficiency than the albumin concentration. One reason why animal models may not provide an accurate parallel to the conditions in man may be the much more rapid growth rate of the rat, pig, and puppy. A falling off in growth rate might therefore occur at a marginal level of biochemical abnormality not detectable by present methods. Two recent studies on children go a long way toward answering Holt’s question. The first is the experiment of Arroyave et al. (1969) referred to above. These workers found that the N : E ratio began to rise when the protein intake of the children fell below 1 gm/kg. This level of intake is a little below that proposed by WHO/FAO (1965) as the requirement of children aged 1 year (see Section 111). If one accepts this estimate of requirement, the lower intake does represent a menace, and the rise in N : E ratio is a warning of it. A similar study was made by Grimble and Whitehead (1970b) ; they gave children who had recovered from kwashiorkor progressively lower levels of protein for periods of 1 week at a time. A clearly abnormal amino acid pattern was established when the protein intake was reduced to 1.4 gm/kg per day. At this point there was a significant fall in serum albumin level, but the children continued to gain weight, although not as fast as on the higher protein intakes. On 0.9 gm/kg per day the amino acid pattern became even more distorted and the children lost a small amount of weight. This level of protein intake is just above that considered adequate for maintenance, but is not enough for growth. To summarize, it seems fair to conclude that in man an increased ratio of nonessential to essential amino acids, and in particular a fall in the concentration of branched-chain amino acids in the plasma, is a fairly sensitive indication of a low protein intake. An abnormal pattern does not, however, necessarily mean that the subject is a t that particular moment suffering from any significant degree of protein depletion.

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J . C. WATERLOW AND G . A. 0. ALLEYNE

Rather i t seems to mean t ha t the amino acid supply is inadequate to match the requirement, and therefore i t indicates impending malnutrition. It has also been suggested t ha t the altered amino acid pattern may in itself have harmful effects (Arroyave et al., 1962), but on the available evidence this seems unlikely. The metabolic significance of these changes in pattern will be discussed in more detail in Section V1,D.

7. Hair Changes Changes in the hair-dyspigmentation, sparseness, and fragility-have long been regarded as an important feature of kwashiorkor (see Waterlow e t al., 1960). Chemical analysis has not been revealing: earlier studies on the cystine content of the hair in kwashiorkor gave conflicting results (Nutrition Reviews, 1968; Bradfield, 1968). The zinc content has also been measured, because mineral deficiencies in animals cause hypochromotrichia, but no difference was found between malnourished children and controls (Bradfield et al., 1969a). On the other hand, very interesting work has been done in the last few years on the morphological characteristics of hair in malnourished subjects. Although in this section we are primarily concerned with the biochemical assessment of protein-calorie malnutrition, hair changes should be considered because they have an obvious metabolic basis. Bradfield (1968) points out t ha t the germinative cells of hair follicles proliferate a t a rate greater than any other tissue except possibly bone marrow. Waterlow and Stephen (1966) obtained evidence that in the rat the cellular protein of skin, to which the hair follicles must make a large contribution, turns over rapidly, so that 50% was lost after 3 days on a protein-free diet. Sims (1968) and Bradfield (1968) showed that in malnutrition the diameter of the hair shaft is reduced; as a result, the tensile strength of the hair is low (Latham and Velez, 1966). Bradfield and co-workers went on to study the morphological characteristics of the hair root. I n malnourished children the diameter of the hair root or bulb was greatly decreased, the proportion of hairs in the anagen or growing phase was reduced, and the hair sheaths absent or abnormal (Bradfield et al., 1968, 1969b). The changes were more severe in marasmus than in marasmic kwashiorkor; this was considered to be an effect of the duration of malnutrition. Both in children and in protein-depleted pigs a correlation was found between hair root diameter and serum albumin level (Bradfield, 1968). I n human adults on a protein-free diet the hair roots become abnormal before there was any change in serum albumin (Bradfield et al., 1967). I n less severely malnourished children there was a relationship between root diameter and deficit in weight for age, a significant decrease in diam-

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149

eter being found with a weight deficit of only 10-2096 (Bradfield and E. F. P . Jelliffe, 1970). Sims (1968) has stressed the value of studies on hair for the information they give about previous events. By measuring the distance from the root a t which the shaft became abnormal, together with the rate of hair growth, he was able to estimate the time of onset of the deficiency state. Because hair is so easily obtainable, studies of this kind should have much practical value and further work on the underlying biochemical changes is obviously indicated. 8. Conclusion

It will be evident that the biochemical tests outlined above measure different things-level of protein intake, muscle mass, growth rate, albumin depletion, and possibly, in the case of serum enzymes, the functional state of certain organs. It is therefore difficult to compare the relative sensitivity of these tests for the assessment of protein malnutrition. Some workers regard a fall in the plasma albumin concentration as a more sensitive and reliable index than the amino acid ratio, others the reverse. Perhaps things are being compared which are not truly comparable. 111. PROTEIN REQUIREMENTS AND THE PREVENTION OF PROTEIN MALNUTRITION The objective of preventive measures is the fulfilment of human protein needs, and all actions in the fields of public health, agriculture, and food technology must be based on our estimates of what these needs are. A great deal of attention has been given to this question in the last ten years, but many uncertainties and contradictions remain, and it has therefore seemed worthwhile in this review to discuss the state of knowledge on this subject in some detail. I n 1965 WHO/FAO published revised recommendations on human protein requirements, and several other national bodies have since made their own recommendations. The WHO/FAO estimates were reached by a two-stagc process: first, computation of the obligatory3 losses of N which have to be replaced for maintenance, together with the amounts laid down during the processes of growth, pregnancy, and lactation. This computation-the so-called factorial method-gives the requirement The word “obligatory” is more appropriate than the older term “endogenous” urinary and fecal N loss. Folin’s concept (1905) of a fixed component and a variable component in urinary r\’ excretion is still valid, but the implication of the words endogenous and exogenous-that the one is derircd from the tissues and the other direct from the food-is no longer acceptable.

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J . C. WATERLOW AND G . A. 0. ALLEYNE

in terms of N utilized in the body; it is converted to protein by multiplying by the conventional factor 6.25, and expressed as reference protein, defined as protein which is 100% utilized. The second stage is to evaluate the efficiency with which, under any given conditions, food protein is in fact utilized to cover obligatory losses and synthesis of body protein. Both steps depend upon estimates which may be challenged, and assumptions which are open to question.

A . Requirements in Terms of Reference Protein 1. Maintenance Requirement

Except in very young infants, and perhaps in lactating women, the amount of N needed for maintenance is much greater than the amount laid down in the formation of new protein. The latter can be calculated from the known rates of growth of infants and children, from the weights of fetal and adnexal tissues formed during pregnancy and from the amounts of milk protein produced daily in lactation. The main problem, therefore, is to obtain an accurate and meaningful estimate of the maintenance requirement. This is sometimes called the “minimum” requirement, and is defined as the minimal intake needed to maintain N balance, and so to cover the obligatory or irreducible N losses from the body. These losses occur by three main routes-kidney, intestinal tract, and skin-and estimates of their magnitude vary widely. Of the three, the first is the most important. a. Urine. Direct measurements of urinary N output in subjects on a protein-free diet or very low protein intake are summarized in Table 111. TABLE I11 Direct Measurement of “Endogenous” Urinary and Fecal N Loss in Subjects on a Protein-Free or Low-Protein Intake ~

Subjects Infants, 4-6 months Children 3-4 years Adults Adults

Previous protein intake

Daily urinary N loss AWkg M d k c a l

Daily fecal N loss (mg/kg)

~~~~

Reference

Low

37

0.6

20

Fomon et a!. (1965)

Low

46

0.9

23

Fomon et al. (1965)

Low High

29 37

1.2 1.6

11 9

Hawley et al. (1948) Young and Scrimshaw (1968)

PROTEIN MALNUTRITION IN CHILDREN

151

Such studies are not numerous, and not all age groups are covered. Therefore it has been customary to estimate the obligatory urinary N loss indirectly from the basal metabolic rate (BMR), relying upon the observations of Terroine and his co-workers some forty years ago, which indicated that the “endogenous” urinary fecal N excretion bore a constant relationship to BMR in all types of homoeothermic animals examined. Smuts in 1935 proposed the numerical ratio of 2 mg N per basal kilocalorie to cover the urinary N loss, but excluding the fecal loss. The details of Smuts’ experiments, which were made on 5 animal species but not on man, have been examined and criticized by Holmes (1965). It is clear that although Terroine’s “law” may represent a useful biological generalization, the data are inadequate as a basis for estimating requirements with any approach to precision. Anyone reading Holmes’ critique must find it remarkable that national and international bodies should erect a superstructure of recommendations upon foundations which appear so flimsy. The data summarized in Table I11 show that in adults the obligatory urinary loss is about 1.5 mg N per kilocalorie, falling to about half that level in infants. The same value, therefore, does not apply a t all ages, and use of the ratio 2 mg N per kilocalorie will overestimate the loss, particularly in younger children. It seems that in man the obligatory N is fairly constant over a wide age range at a level of about 30-40 mg/kg per day. b. Feces. Table 111 also shows values for the obligatory fecal N loss. I n adults i t amounts to about 30% of the urinary loss, in children to somewhat more. In addition to the N recovered in the feces, Steggerda and Dimmick (1966) found that subjects on a normal diet excreted an average of 0.25 gm N per day as gaseous nitrogen in flatus. On a diet of beans the average amount of N in flatus rose to 0.87 gm per day. Losses of this order could be significant; it is not known whether they occur in people who habitually consume a diet high in legumes, or whether there are adaptive changes in the intestinal bacterial flora. c. Skin. Nitrogen is lost from the skin in 2 ways-in sweat and by exfoliation of epidermis, hair, nails, etc. Many direct measurements have been made by collecting sweat from the whole body, and sometimes epithelial debris as well. The procedures are difficult and the results vary widely. Some representative values are shown in Table IV. An indirect estimate of losses from the skin and by routes other than urine and feces can be derived from N balances. Mitchell (1949) measured N balances for 6 months in healthy young men and found a mean positive balance of 1.38 gm per day with no change in body weight. This apparent N retention he attributed to dermal and desquamative losses.

+

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J. C. WATERLOW AND G . A. 0. ALLEYNE

TABLE IV Estimates of Nitrogen Losses f r o m the Skin in Adults Sample

Conditions

N loss (gm/day)

Normal Normal Heat, exercise

0.36 0.25 0.4

Normal Normal

0.14 1.38

Mitchell et al. (1949) Darke (1960) Ashworth and Harrower (1967) Sirbu et al. (1967) Mitchell (1949)

Normal

0.9

Isaksson and Sjogren (1967)

Author ~~

Sweat Sweat Sweat

+

Sweat debris Total, by N balance Total, by N balance

Isaksson and Sjogren (1967) estimated N loss from the skin as the difference between the apparent N retention and the expected retention calculated from measurements of total body K, on the assumption that the ratio of N t o K is fixed at 3 meq K per gram of N. By this method they obtained a mean value for skin loss of 0.9 gm of N per day in subjects under normal conditions. Since many of the countries where protein malnutrition occurs are tropical, it is a question of some practical importance whether losses of N from the skin are increased at high rates of sweating. Consolazio et al. (1963) observed very high N losses in men exercising in hot conditions, so much so that the N balance was negative even on an excellent intake. However, the subjects were probably unacclimatized and the conditions artificial. Ashworth and Harrower (1967) in Jamaica found that young men working hard in a hot climate and sweating profusely had a mean daily loss in sweat of only 0.5 gm of N. The N concentration in the sweat was very low, presumably because the subjects were West Indians and fully acclimatized. The greater part of the N in sweat is urea N, and one would expect on physiological grounds that an increased rate of loss from the skin would be compensated by a decreased renal excretion. Ashworth and Harrower obtained some evidence of this, and i t is supported by the finding of Sirbu et al. (1967) of a close correlation between dermal N loss and blood urea concentration. Clearly, however, the real extent of the loss of N from the skin under different conditions is still one of the main unknown factors, nor is there any information about skin losses in children. d. Other Routes of Loss. The possibility has been raised that N may be lost from the body as atmospheric nitrogen (Costa et al., 1968).

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PROTEIN MALNUTRITION I N CHILDREN

However, Hoffmann and Schiemann (1964) in experiments on rats given food enriched with ammonium-lzN salts found no evidence of any exchange with atmospheric N,. e . Effects of Stress. I n the present context we are concerned with the protein requirements of healthy people. It is well known that severe illness or injury can cause very large losses of N (Cuthbertson, 1964), but mild infections and even psychological strain may also be accompanied by an increase in urinary N output (Scrimshaw, 1963). For this reason WHO/FAO (1965) added an extra 10% to the obligatory losses to cover the stresses of ordinary life. f. Individual Variation. Data such as thosc in Table 111 represent averages of individuals in a group. The coefficient of variation has been estimated as 10-15%; therefore an addition of 2&30% ( 2 SD) should cover the requirements of practically all individuals in the population. It will be seen that there is a rather wide range of variation in estimates of all the components which make up the basal losses. This has led to substantial differences in the conclusions on the minimal requirements of adults reached in various recent reports (Table V ) . TABLEV Estimates of Minimal Nitrogen Requirement for Maintenance in Adult Males ~

Weight (kg)

BMR (kcal/day) N loss in urine (gm/day) N loss in feces (gm/day) N loss by skin (gm/day) Allowance for stress Total (gm Nlday) Percentage addition for individual variation or margin of safety Total (gmN/day) As reference protein (gm/day) As reference protein (gm/kg/day)

~

~

_

_

_

_

U.K:

_

Present review

WHO/FAOa

U.S.A.0

65 1500 3.OOb 1.30 1.30 0.56 6.16 20

1750 3. 50b 0.70 1.40 0 5.60 30

65 1600 3.20b 0.91 0.13 0 4.24 20

70 2.31" 0.70" 0 .50d 0 3.51 67

7.40 46.5

7.30 45.5

5.05 31.5

5.85 36.5

0.49

0.52

0.71

70

,065

Sources: WHO/FAO (1965); U.S.A. National Research Council (1968); U.K. Department of Health and Social Security (1969). * Calculated as 2 mg N per basal kcal. From direct measurements (Table 111). Compromise between estimates in Table IV.

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J. C . WATERLOW AND G . A.

0. ALLEYNE

2. Protein Requirement of Infants

It is fortunate that in infants, the group who are most vulnerable to protein deficiency, requirements are more firmly established and there is less disagreement. The reason is that in infancy the rate of growth provides an easily measured and reasonably reliable and sensitive index of whether requirements are being met. Table VI, taken from WHO/ F A 0 (1965), shows the protein requirements of infants a t two ages, derived by the factorial method. Several types of direct measurement have been made by a-hich these estimates can be checked. (i) The observed protein intakes of infants given breast milk ad libitum and growing normally were 2.3 gm/kg a t 0-3 months and 1.25 gm/kg a t 9-12 months TABLE VI Calcztlation of the Protein Requirements of Infants b y the Factorial il1ethod.J

Basal urinary and fecal loss Sweat loss Requirement for growth and maturation Addition of 10% for stress

As grams reference protein per kg Addition of 20% for individual variation Requirement based on actual intakes

Aged 0-3 months

Aged 9-12 months

70 5 140 __ 215 22 __ 237 1.46 1.76 2.3

70 5 38 __ 113 11 124 0.77 0.93 1.25

Data from WHO/FAO (1965).

* Values are expressed as milligrams of nitrogen per kilogram per day. (WHO/FAO, 1965). These values are somewhat higher than the theoretical estimates, even after 20% has been added for individual variation. (ii) Balance and growth measurements in infants a t about 1 year suggested that the maintenance requirement mas approximately 0.7 gm/kg per day, and t h a t 1.25 gm/kg per day was enough to support growth a t even more than the normal rate (Chan and Waterlow, 1966). (iii) The amino acid pattern in the plasma has been regarded as a fairly sensitive index of the adequacy of the protein intake (Holt and Snyderman, 1965). Arroyave and co-workers (1969) gave graded amounts of protein to infants, and found that the plasma amino acid pattern began to change toward that characteristic of deficiency when the intake fell below 1.25 gm/kg per day. This fits well with the findings of Chan and Waterlow.

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PROTEIN MALNUTRITION I N CHILDREN

3. Critique of the Principles

Apart from any uncertainties in the data, the method of estimating requirements by balancing the obligatory N losses raises two difficulties of principle. First, the obligatory or endogenous N output is not a fixed quantity with a single value. When a subject is put on a protein-free diet, the urinary N output falls rapidly for a few days and then more slowly (Martin and Robison, 1922), but it never reaches a truly constant level. For example, Deuel in a classical experiment, lived for 30 days on a protein-free diet (Deuel et al., 1928). Between the 9th and 30th day the urinary N output fell from 3.12 to 2.10 gm per day (Fig. 1 ) . The cumulative N loss from the body over this interval was 101 gm. This continued loss of N must represent depletion of body protein, which is a pathological process. On the other hand the WHO/FAO committee regarded the initial rapid loss as a physiological adaptation. I n that case the obligatory N loss would be the output a t the point of inflexion of the curve, a t the dividing line between physiological and pathological. The choice of this point can only be arbitrary. I n fact, the value found for the “endogenous” N depends not only on the time of the measurement, but also on the previous protein intake (Fomon e t d.,1965). I n practice, therefore, the obligatory N loss, like the basal metabolic rate can be defined only in operational terms, and it is not surprising that estimates should vary when conditions have not been rigidly standardized.

I

0

FIG. 1. Daily of from Dew1 et al. of World Review

0-0, grams

5

I

I

I

I

10 15 20 25 Days on low N dlet (0.24 - 0.51 grn/day)

I

30

nitrogen excretion by subject on low protein diet for 30 days. N per day; milligrams of N per basal kilocalorie. Data (1928) ; reproduced from Holmes (1965), by courtesy of the Editor of Nutrition and Dietetics.

so,

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J . C. WATERLOW AND G . A. 0. ALLEYNE

‘The second difficulty is that the change from a high to a low protein intake leads to a net loss of N from the body during the first few days. This is regarded by many authors as a loss of “labile protein reserves” (see Munro, 1964). Therefore i t may be held that subjects who are brought into N balance a t the minimum level of intake are a t a disadvantage compared with those receiving more protein, because they have lost reserves. I n the rat this labile protein, which is mainly derived from liver and intestine, is said to amount to 3-5% of total body protein (Munro, 1964). However, recent measurements on adult humans (Young et al., 1968) and on infants (Chan, 1968) showed that the net loss was only about 1% of total body N. Moreover, i t was no greater in well-nourished than in malnourished infants-a finding difficult to fit in with the concept of “reserves” as a characteristic of good nutrition. I n animal experiments several authors have failed to show any adverse effect of low protein feeding on subsequent response to dietary stress (Holt et al., 1962). It would, however, be hazardous to apply these results directly t o man. An alternative explanation of the N loss when the protein intake is reduced is that it results from a lag during the change from one metabolic equilibrium to another (Waterlow, 1968). On this view i t would represent an adaptation rather than a loss of reserves, which implies a harmful effect. It seems that the only way to resolve this question is by a better understanding of the mechanism of the N loss.

4. Conclusion Sixty years ago Chittenden in the United States was advocating a daily protein intake of some 30 gm, and Voit in Germany one of 120 gm. Cathcart (1912) quoted Chittenden as believing that “this amount is much too abundant, and that any person who lives up t o this standard and who encourages others to do so is encouraging individual and racial suicide.” Nowadays the divergence of opinion is not so great, but the range of variation in the official estimates of minimal requirement (Table V) is still large enough to produce serious practical problems. Moreover, all these estimates are based on the ratio 2 mg of N per basal kilocalorie as the measure of the obligatory urinary loss. If the values obtained by direct observation (Table 111) were used instead, the estimates of minimal requirement would be lower. The importance of accuracy in these estimates is very great. If the requirement is set too high, targets for food production may be unrealistic, leading to a burden on the economies of developing countries. If requirements are set too low the prevalence of protein deficiency will be seriously

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underestimated. A very large number of people are living on marginal intakes which come within what has been called the “area of ignorance” (Ministry of Health, 1964). Autret and co-workers (1968) have examined the national food supplies of 84 countries representing 60% of the world’s population. According to their data, in 39 countries the average amount of protein available per head falls short of the requirement, as recommended by WHO/FAO in 1965. If the estimate of the requirement was reduced by 20%, the average intake4 would become apparently satisfactory in all but seven countries. Therefore, the need to reduce the area of ignorance is rather urgent. Until more knowledge is obtained an empirical approach might be based on the relatively secure data which exist for infants. I n theory, at 9-12 months the requirement for niaintenance and growth should be about 0.7 gm of protein per kilogram per day (made up of 75 mg of N per kilogram to cover obligatory losses and 38 mg of N per kilogram for growth (WHO/FAO, 1965). Observation shows that this is not in fact enough, and that infants will not gain weight on such an intake (Snyderman et al., 1962; Chan and Waterlow, 1966). The observed requirement for normal weight gain, which, from all pediatric experience, is a very sensitive criterion of health, is about 1.2 gm/kg per day, or approximately 1% times the theoretical requirement. If the same safety factor were applied to the adult’s theoretical maintenance requirement, this would give the value shown in the last column of Table V, based on observed rather than derived figures for the obligatory loss. One may wonder, however, whether such exercises can ever be regarded as a legitimate basis for worldwide recommendations. Perhaps the time has come when the old approach can take us no further, and in the future we should concentrate on finding more sensitive tests of protein deficiency in man.

B. Measurement of Protein Value The second step in matching protein intakes t o needs is evaluation of the efficiency with which food protein is utilized for maintenance and growth. The most important factors governing it are the quality of the protein, its concentration in the food, and the adequacy or otherwise of the caloric intake. While quality is a fixed characteristic of a protein, the efficiency of utilization may vary according to the circumstances. ‘Even when the average intake is equal to the requirement, the position is not satisfactory, because a t least half the population will receive less than the amount recommended, and there is no way of knowing whether these are individuals who happen to have lower than average needs.

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1. Protein Quality

It is now accepted that the quality of a protein or of a mixture of pro-

teins depends upon its amino acid composition. The first F A 0 report on protein requirements (FAO, 1957a) summarized what was known about the amounts of each essential amino acid required by man, for maintenance in adults and for maintenance plus growth in children. On the basis of these figures an ideal pattern was drawn up which showed the relative proportions in which the essential amino acids are needed. This is usually referred to as the “ F A 0 pattern.” The quality of a protein is assessed by comparing its amino acid composition with the ideal pattern. I n this comparison one amino acid is identified as limiting, and the “protein score” is then calculated from the extent to which the limiting amino acid is lacking in the test protein compared with the ideal pattern. Alternatively, protein quality can be measured biologically, by its effectiveness in promoting growth or N retention. The many test procedures which have been devised are fully described by Allison (1964). Probably the most accurate method is measurement by carcass analysis of the amount of N retained in the body by rats given the test diet for a standard period, compared with rats fed a protein-free diet for the same period. Since food N cannot be utilized with maximal efficiency if it is given in excess, the protein must be fed a t a low level if a true measure of quality is t o be obtained. D. S. Miller and Payne (1961a) have designated as NPU (standardized) the net protein utilization under standard test conditions, when protein is fed a t a maintenance level. I n the second report on protein requirements (WHO/FAO, 1965) various adjustments, based on newer knowledge, were made to the ideal pattern of amino acid requirements. These brought the ideal pattern into close correspondence with t h a t of egg protein, and it was proposed to adopt as the reference pattern the essential amino acid composition of whole egg. The choice of reference pattern is important, because it determines which amino acid is considered to be limiting. Egg protein is rich in sulfur-amino acids, and therefore when this pattern is used for calculating chemical scores the limiting amino acid in most foods is methionine. On the other hand, if the pattern of human or cow’s milk is used, lysine is the amino acid most often found to be limiting. Cresta et al. (1969) made a mathematical analysis in a series of 274 diets of the correlation between chemical score, calculated from several different reference patterns, and protein quality measured biologically. Three different kinds of tests were evaluated: two, net protein utilization (NPU) and protein efficiency ratio (PER), measure the efficiency for growth;

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and one, biological value (BV) , measures the protein efficiency for maintenance. Scores based on egg protein gave the best correlation with the growth tests, and scores based on milk protein the best correlation with the maintenance test. These findings suggest that the amino acid requirements for maintenance and for growth are not the same: for maintenance, lysine is likely to be limiting; for growth, methionine cystine. This conclusion is perhaps a t variance with the earlier work of Allison (1963), who was unable to show any clear differences in the amino acid requirements for maintenance, growth, or repletion. However, i t seems likely that the statistical approach based on a larger sample may uncover differences that cannot easily be determined experimentally. Kofranyi and Jekat (1966) have even proposed that the concept of a single limiting amino acid should be abandoned. A second modification made in the 1965 report was to separate the N requirement into two components, that for essential amino acids and that for total N. I n the original F A 0 pattern, the sum of all the essentials amounted to only 32% of the total amino acids. I n many natural foods, particularly those of animal origin, the essential amino acids form a larger proportion of the total N ; e.g., in egg protein, 51% of the total (Scrimshaw e t aZ., 1966). Therefore, if the F A 0 pattern correctly represents human requirements, i t should be possible to “dilute” proteins such as those of egg with nonessential N, without any loss of protein quality. Snyderman e t al. (1962) showed that when infants were fed decreasing amounts of milk protein, so that growth fell off, weight gain and N retention could be restored to normal by the administration of unessential N in the form of glycine or even of urea. Scrimshaw et aZ. (1966, 1969) reported that in young men fed just enough egg protein to maintain balance, 30% of the N from egg could be replaced with nonspecific N-ammonium citrate glycine-without producing a negative balance. However, other workers have obtained contrary results. Romo and Linkswiler (1969) concluded that “increasing the proportion of essential aminoacids, even up to 100% of the total intake, significantly improves N retention, particularly a t levels of intake which are not much greater than those needed for maintenance.” It is doubtful whether the possibility of utilizing nonspecific N has much practical application. Autret et al. (1968), in their study of the diets of 84 countries, remarked: “Considering the difference in diets, the share of essential aminoacids in relation to total proteins is remarkably constant. It represents about 40% of total proteins both in countries with a small protein allowance of vegetable origin and in countries with a high protein allowance rich in animal proteins.’’ However, if it were feasible to fortify certain foods, such as skim milk powder, with nonessen-

+

+

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J . C. WATERLOW AND G . A. 0. ALLEYNE

tial N without decreasing their protein value, this would be a useful advance, since in infants the margin between adequate and inadequate intake is so narrow that even a small supplement may make a difference. 2. The Effect of Protein Concentration and of Caloric Intake on Protein Utilization

If a diet which fulfills the caloric requirement contains more than enough protein to cover the needs for maintenance and growth, this extra protein will be wasted, and the overall efficiency of protein utilization be reduced. I n order to correct for this, D. S. Miller and Payne (1961b) introduced the term NPU (operative) to designate the efficiency of utilization under any set of conditions, not just under the standardized conditions used for measuring the protein quality. The protein value is also affected by the caloric intake. If this is below the requirement, some protein will be metabolized to provide energy, and the efficiency of N utilization will fall. D. S. Miller and Payne (1961~ ) determined the magnitude of this effect by measuring the N P U of various diets fed to rats a t different levels of caloric intake. A typical result was that with a, 10% casein diet a reduction in the daily caloric intake from 150 to 100 kcal/kg0’3 caused a fall of about 30% in the protein value. Similar results were obtained by Morrison and Narayana Rao (1967). This effect of caloric deficiency would be reflected in a lower N P U (operative) . Miller and Payne believe that these findings in the rat can be applied to man, provided that caloric intakes are related to the three-fourths power of the body weight, since this method of expression leads to broadly similar values for the basal metabolic rate over a wide range of animal species (Brody, 1945; see also Munro, 1964). 3. Expression of Protein Value

The protein value of a diet is given by the protein concentration multiplied by the NPU (operative). Platt and Miller (1959) proposed that i t would be logical to express i t in terms of protein calories as a percentage of the total calorie supply, rather than as grams per 100 gm of food. This method of expression can be applied also to requirements. For example, the infant at 3 months needs 7.5% of his calories in the form of utilizable protein. This figure is based on the international recommendations for calories and protein (FAO, 1957b; WHO/FAO, 1965). One’s confidence in these estimates is fortified by the fact that the proteincalorie content of human milk, the natural food for young infants, exactly fits this figure. (Protein, calculated as N 6.25 = 1.25 gm or 5 kcal per 100 ml; total calories = 67 kcal per 100 ml; protein calories = 7.5%; NP U = approximately 100.)

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161

Platt and Miller’s approach is useful for matching intakes and requirements, but it raises some theoretical questions. The calorie requirements of individuals vary widely, even when their levels of physical activity are comparable; some people can satisfy their calorie needs with smaller intakes than others. Do these people also need less protein? To fix the ratio of protein to calorie requirements implies that the two always vary together. It may be so: i t seems that in a general may not only the endogenous urinary N output but also the total N turnover may be related to the basal metabolic rate (see below). However, in the present context the question of practical importance is whether this is simply a broad generalization, or whether it is close enough to hold in individual cases. It is noteworthy that, in the small series of subjects described by Young and Scrimshaw (1968), when the endogenous urinary N was related to the basal metabolic rate the coefficient of variation was much larger than when it was related to the body cell mass.

4. Practical Applications A good example of how these estimates of protein value are used in practice is the study by Autret et al. (1968) of the diets in 84 countries. The countries were divided into seven groups according to the main source of protein: animal products, wheat, millet and sorghum, maize, mixed cereals, rice, or roots and tubers. I n the first five groups the protein value of the diet was in most cases high enough to satisfy the requirements of all except special groups (infants and lactating women) provided that the quantity eaten was adequate. Only the diets based on rice and tubers were so low in utilizable protein that they probably could not fulfill requirements without the addition of supplementary protein. However, in a great many countries, although the protein vaIue of the diet was adequate, the actual intakes were so low that on the average protein requirements were not met. At the same time the deficiency of protein would be made more severe by the inadequate calorie intake, which reduces the efficiency of protein utilization. Sukhatme (1970) has emphasized the importance of this concept in India, where calorie intakes as well as protein intakes are commonly very low. The practical implication of this is that in many populations or groups there would be no need of a special protein supplement or of amino acid fortification to fill a protein gap or to supply a limiting amino acid; what is needed is simply a larger intake of the type of food which the people already eat.

C . General Conclusion The subject of protein requirements has been dealt with a t some length because, in spite of its practical importance, the state of knowledge is far

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from satisfactory. Rational measures for preventing protein malnutrition must depend upon an accurate knowledge of requirements, but in fact there are a great many uncertainties and unknowns, some of which are listed below:

i. The arbitrary conditions under which the basal urinary loss is measured ii. Uncertainty about the size of losses by routes other than urine and feces, especially skin iii. Uncertainty about the significance of thc loss of so-called “labile

N”

iv. The virtual impossibility of estiiiiating the extent in real life of losses caused by stress v. Errors and variability in the detcrinination of protein quality vi. Uncertainty about the relationship bctween calorie needs and protein needs vii. Uncertainty about the requirement for essential amino acids in relation to total N Finally, as all workcrs on this subject are well aware, recommendations based on small-scale studies under controlled conditions must be applied with reserve to world populations as a whole.

IV. BODYCOMPOSITION, BODYFLUIDS, AXD ELECTROLYTES McCance has said that the study of the chemical composition of the body and the study of malnutrition are complementary. I n dealing with protein-calorie malnutrition we would go further than this: a knowledge of the composition of the body is, in principle, thc essential basis for undcrstanding the disease, assessing its severity, and comparing one form with another. Questions of the type: Is this patient more malnourished than that one? or Does such and such a measurement correlate with the severity of malnutrition? are really questions about body composition. The simplest measure of severity is the deficit in weight for height, but body weight is affected by variations in water and fat content, and weight deficit or failure to grow is entirely nonspecific. A . Total B o d y Protein and L e a n B o d y M a s s 1 . General Considerations

Since me are concerned with protein nutrition, severity should logically be graded by the extent to which the body is depleted of protein. It

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163

would be interesting, for example, to know whether children with kwashiorkor are more or less protein depleted than those with marasmus. The concept of protein depletion was discussed in detail in the 1960 review (Waterlow et al., 1960), and again more recently (Waterlow, 1969a). As a baseline for measurements in vivo, Garrow and co-workers (1965; Picou et al., 1966; Halliday, 1968a) analyzed the cadavers of 10 children aged 9-18 months who died from malnutrition and other causes. These appear to be the only direct measurements of body composition which have ever been made in children of this age group, and they are an important source of information on the amounts of water, fat, protein, and minerals in the whole body and in various tissues. [Earlier analyses by German workers (see Garrow et al., 1968) were mainly on neonates.] I n interpreting the findings, the problem immediately arose of the appropriate standard of reference. This general question, particularly in relation to measurements of body composition, has been examined by Garrow et al. (1968) and by Garrow (1970). I n our work we consider that a child has recovered from malnutrition when he has regained the expected weight for his height, even though he may still be small for his age. Therefore a suitable standard of reference would be the total protein content of a normal child of the same height. I n the living subject a test of the existence of protein depletion is the ability to retain more nitrogen than is needed for normal growth and maintenance, but, in order to get a quantitative measure of the extent of depletion, i t would be necessary to determine the cumulative nitrogen retention up to the time of recovery. This is clearly not practicable. The analogous measurement of potassium depletion by the cumulative K balance is easier, because the deficit is restored more quickly (Hansen, 1956). Total body protein (TP) is not equivalent to lean body mass (LBM) because the LBM includes water as well as protein and minerals. Methods of measuring LBM in man and animals were reviewed in detail by a conference of the New York Academy of Sciences (1963) and more recently by Cheek (1968). I n malnutrition the proportion of water to protein in the body may vary widely, and therefore, as pointed out in the 1960 report, to determine protein content in vivo i t is necessary to have independent measurements of body fat and body water. These, subtracted from body weight, give the nonfat solids (protein and minerals). Mineral, including bone salts, accounts for about 3% of body weight (Garrow and Fletcher, 1964) or about 16% of nonfat solids. A great deal of attention has been devoted in the last ten years to the problem of measuring TP, without conspicuous success.

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2. Measurement of Total B o d y Fat The fat content can be calculated from the specific gravity of the body if the water content is also known. The classical method of measuring specific gravity by underwater weighing cannot be applied to severely ill children. Attempts have been made to measure the body volume, and hence the specific gravity, by the method first developed by Siri (1955), in which the subject is placed in a closed chamber of known capacity, and the volume of residual air measured by helium dilution. The application of this method to infants has not been successful, because the helium does not mix with gas in the stomach and gut, the volume of which, in relation to the residual air space, is large enough to introduce serious errors (Halliday, 1968b, summarized by Waterlow, 1969a). Attempts to measure fat content by the uptake of fat-soluble gases such as cyclopropane (Lesser et al., 1960) or krypton (Hytten et al., 1966) were unsuccessful, because equilibrium was usually not attained, particularly in recovered children (Halliday, 1968b). Moreover, the results with cyclopropane did not differentiate between malnourished infants with very little subcutaneous fat and recovered ones with plenty of fat. T h e probable reason for this is that the greater part of the cyclopropane uptake was by lipid in brain and liver; uptake by the fat depots is slower, and they do not become saturated within a reasonable time because the blood supply to them is relatively poor. I n malnourished children the brain forms a larger proportion of the body than normal (Montgomery, 1962a). The liver also may be very fatty; Garrow et al. (1965) showed that as much as one-third of the total body fat might be in the liver. Thus the increased uptake by these two organs masks the decreased uptake by the shrunken fat depots. 3. Total B o d y Protein from Measurement of Total B o d y Water

Calculation of total body protein from total body water (TW) alone assumes a constant water content of the lean tissue (Pace and Rathbun, 1945). I n malnutrition this assumption does not hold good. McCance and Widdowson (1951) attempted to correct for the error by separate measurement of the extracellular fluid volume, which accounts for most of the excess fluid (see Section IV,B), but the assumption still has to be made of a constant ratio of solids to intracellular water, and this also may not be true. If in fact there is intracellular over-hydration, TP calculated on this basis will be overestimated.

4. Total B o d y Protein from Measurement of Total B o d y Potassium Calculation of TP from total body potassium (TK) depends upon the assumption of a fixed value for the K:N ratio in lean tissue. Since

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malnourished infants are often depleted of K, so that the K concentration of the body is reduced, this assumption will again introduce an error, and calculations based on it will underestimate TP. However, since specific K depletion is usually corrected within about a week (Section IV,B) , this source of error is not too serious in practice. A further complication is that the factor used for calculating TP from T K assumes a “normal” distribution of K within the body. This is certainly not true in malnutrition; the brain, for instance, which contains 15-200/, of total body K in a normal 1-year-old child, may contain more than 30% in one who is severely malnourished (Garrow et al., 1968; Alleyne, 1971). Table VII shows a comparison of estimates of total body protein made by two methods: A, measurement of total body K ; B, measurement of total water and extracellular fluid volume. Initially, as would be expected, method A gives lower values than B (initial A/B: mean = 0.72) ; TABLE VII Total Body Protein in Malnourished and Recovered Infants. Calculated ( A ) from Total Body K ( B )from Total Body Water and Extracellular Fluid. Vo2umea.b

Patient MS

DT HM

CB ML AG

LK BP

Days in hospital 2 69 2 53 13 47 8 29 1 49 2 68 3 40 6 62

Total body K Weight (kg) (meq/kg) 4.90 7.66 5.82 8.01 4.14 5.30 4.20 5.00 7.12 8.48 5.62

38.5 50.0 35.8 44.8 41.9 53.6 44.5 48.0 35.0 46.7 38.7

9.01 8.73 5.06 7.74

21.4 42.0 38.7 50.3

8.34

43.3

Total protein (kg)

A

B

0.58 1.18 0.64 1.11 0.53 0.87 0.575 0.74 0.77 1.22 0.67 1.11 0.69 1.13 0.605 1.19

0.75 1.28 0.98 1.38 0.94 0.96 0.715 0.77 0.88 1.40 0.99 1.25 0.865 1.40 0.75 1.545

Original data from Alleyne (1968). Calculation of total body protein: protein assumed to be 84’34 of nonfat solids (Garrow and Fletcher, 1964). (A) Calculated from total body K on the assumption that K = 60 meq/kg lean body mass, and that nonfat solids = 22 gm/100 gm lean body mass. (B) Calculated from total body water (T,) and extracellular fluid volume (corrected bromide space = E,) according to the formula: nonfat solids = 0.612 T,” - 0.54 E, (McCance and Widdowson, 1951; Wedgwood, 1963). b

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as the children recover the difference between the two values becomes less (final A/B: mean = 0.87). A consistent difference between the two estimates remains, which is presumably due to errors in the factors used. From our experience we believe that once K depletion has been overcome, T K gives the best estimate of TP, and it has the great advantage that serial measurements can be made throughout the period of recovery. Parallel measurements of N balance show that during the rapid growth phase approximately 3 meq of K are retained per gram of N (Ashworth et al., 1968) ; this represents the average K : N ratio in normal tissue. However, even if total body protein can be measured with reasonable accuracy in this way, that is not the end of the storyl because what is important is not the total protein but the amount of “mobile” protein (see Waterlow et al., 1960). Picou et al. (1966) showed by direct analysis t h a t in infants dying of malnutrition collagen accounted for nearly 50% of total body protein compared with 27% in 2 well-nourished children. This dilution by collagen masks the reduction in mobile protein. 5 . Muscle Mass Muscle is the largest single protein reservoir of the body, and muscle wasting is one of the prominent clinical signs in protein malnutrition. Kerpel-Fronius and Frank (1949) showed by direct dissection that in infants dying of marasmus the muscle mass was only 30% of the mass normal for the age, and Montgomery (196213) found by measurement of photomicrographs that in a malnourished child the cross-section area of the sartorius muscle bundles was reduced to about one-tenth of that found in a control child. There is now much evidence that muscle protein is more mobile than used to be thought (see Section VI,D,3), and that muscle, in addition to its contractile function, has a kind of homeostatic role, acting as a buffer for other tissues when protein is in short supply. Therefore the measurement of muscle mass may be an excellent index of the true extent of protein depletion. Since Folin (1905) first suggested t h a t the urinary creatinine output could be used as an index of active cell mass, and more specifically of muscle mass, a large literature has accumulated on this subject, reviewed by Cheek (1968). Not all authors accept the relationship (e.g., Parot, 1965; H. Fishcr, 1965), but empirically it seems to be useful (see Waterlow et al., 1960, a i d Section 11,E). Viteri et al. (1966; Viteri and Alvarado, 1970) introduced the creatinine-height index as a method for comparing muscle mass in different individuals and groups. This index is defined as (24-hour creatinine excretion of patient) / (24-hr creatinine excrction of a normal subject of the same height). It is interesting that the index shows a good correlation with various physiological parameters,

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such as red cell mass (Viteri et al., 1968) and glucose absorption (Viteri, 1969). By analogy, a potassium-height index can be calculated as (total body K of patient)/(total body K of normal) child of same height (Alleyne et al., 1970b). This index, of course is valid only after any K deficit has been corrected. Height is used in these indices rather than weight because i t is less affected by variables such as edema and changes in body fat. There is a good correlation between the creatinine index and the potassium index (Fig. 2 ) , suggesting that during recovery from malnutrition the deficit in muscle mass bears a close relationship to the deficit in total cell mass. The data compiled by Brody (1945) from a range of animal species led him to conclude that the daily creatinine excretion varies almost directly as the body weight (as Wt0.9),whereas most other functions, such as the basal metabolic rate, are related to the 3/4 power of the weight (see also Munro, 1969). The rate of creatinine excretion in adult man is about 20 mg, and in a normal 1-year-old child 13.5 mg, per kilogram per day (Stearns et al., 1958). If in the adult, muscle represents 40% of the body weight, this means that 1 mg of creatinine corresponds to 20 gm

1.4

1

1.2

-

.B 1.0

-

x D w

._ +

1

L

117 observations

..

.

0

C ._ c ._ '0 0.8

F

~0.713

-

0

0.6

I

1

0.6

0.8

1.0

1.2

1.4

Potassium - height index

FIG.2. Correlation between creatinine-height index and potassium-height index in malnourished children at different stages of recovery. Data of Alleyne et al. (1970h). Reproduced by courtesy of the Editor of American Journal of Clinical Nutrition.

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J . C. WATERLOW AND G . A. 0. ALLEYNE

muscle. Chinn (1966) cites very similar estimates from the older literature, going back to Schaffer in 1908. If the same factor applies a t all ages, i t means that in the normal child a t 1 year the muscle mass should be about 27% of body weight. In the malnourished child, with a creatinine excretion of about 5 mg/kg per day (Standard et al., 1959), the muscle mass on this calculation would be only 10% of body weight. It would probably be more accurate to regard creatinine excret>ionas a measure of collagen-free or “true” muscle mass. Halliday (1968a) dissected the muscle from a malnourished child who died, and found that it amounted to 25% of the body weight, but 47% of the muscle protein was collagen, so that the true muscle mass would be only about 13% of body weight. This gives a dramatic indication of the severity of the depletion, since the body weight itself was very low for the child’s age and height. 6. Creatine Turnover

The hypothesis t ha t the same factor relating creatinine output to true muscle mass applies in different species, a t different ages and in different nutritional states, raises some interesting problems. Since creatinine is derived from phosphocreatine by an irreversible reaction, it means that the turnover rate of creatine5 per unit weight of muscle must be always the same. This in turn implies either that the concentration and fractional turnover rate of creatine do not vary in different species and different dietary states, or t ha t they always vary in opposite directions. Either of these alternatives would represent an unusual biological situation. The evidence from the literature is rather conflicting. Chinn (1966) lists some results obtained by various workers for creatinine output and total body creatine in sheep, dog, rabbit, guinea pig, and rat. The range of values found by different authors for creatinine excretion in one species, the rat, was 24-39 mg per kilogram per day ; there was an equally large range in the estimates of the creatine content of the body per kilogram weight. These discrepancies probably result froin differences in analytical techniques. Chinn calculated from his data that the rate constant of creatine turnover in the rat was 0.0076 day-l, corresponding t o a half-life of 91 days. There was no difference in the turnover rate in males and females, nor between normally fed rats and those given restricted amounts of food for 2 weeks. A similar calculation of creatine turnover in the rat, based on creatinine output and total body creatine, can be made from the data of Chanutin and Kinard (1932) and of Bloch et al. (1941). These two sets ’Turnover has to be defined in relation to both the precursor and the product. By creatine turnover we mean the overall rate of conversion of creatiiie via phosphocreatine to creatinine.

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TABLEVIII Isotopic Measurenzents of Turnover Rate of Muscle Creatine in Different Species Species

Isotope ~~

Mouse Rat Dog Man

14

c

I5N 2H '4C 1 4 c

'5N 15N

'4C '4C

Half-life (days) 20 29 36 30 38 42 48 38 37

Author Fitch el al. (1961) Bloch et al. (1941) Cohn et al. (1946) Waterlow et al. (1971) Quoted by Fitch and Sinton (1964) Hoberman at a!. (1948a) Hoberman et al. (194813) Fitch and Sinton (1964) Fitch et al. (1968)

of results are in close agreement, giving much faster turnover rates of about 0.02 day-l, and a half-life of 35 days. Chinn attributes this difference to technical shortcomings in the earlier work, by which the creatinine output was overestimated and the body creatine content underestimated. However, it is noteworthy that in the experiments of Bloch e t al. l5N-labeled creatine was given, and so it was possible to estimate the turnover rate independently, from the decay curve of the isotope in urinary creatinine. This gave a half-life of 29 days, in reasonable agreement with that derived from nonisotopic data. Preliminary measurements in our laboratory with 14C-labeled creatine show that in rats the turnover rate of creatine was the same in young and in old animals and was not significantly affected by low-protein diets or starvation. It was, however, greatly increased in some rats which had an infection. The average half-life in uninfected rats was 30 days, in good agreement with the results of Bloch e t al. (1941). Results obtained by isotopic methods for the half-life of creatine in various species are summarized in Table VIII. They are remarkably constant, considering the very wide range of body weights. This must strengthen confidence in the value of the creatinine output as a measure of muscle mass. However, we know nothing about the turnover rate of creatine in human infants, and very little about how it is affected by dietary deficiency. 7. Muscle Composition

In the 1960 review (Waterlow et al., 1960), attention was drawn to changes in the distribution of protein at the cellular level, and particularly to the reduction in the amount of protein per cell, in muscle as well as in liver, shown by a reduced ratio of N to DNA. Cheek (1968)

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has explored this subject extensiyely, and has obtained evidence that in rats calorie deficiency causes a reduction in the total number of cells in muscular tissue, with an increase in their size and the ratio of protein to DNA, whereas with protein deficiency the number of cells is normal, but the size and N:DNA ratio are reduced (Mendes and Watcrlow, 1958). This seems a promising approach to the study of human malnutrition, and to the differentiation of marasmus and kwashiorkor. Cheek (1968) stated: “In recent studies on the muscles of marasmic infants it was found that the ratio of protein:DNA was grossly reduced, but in early rehabilitation the incrcrnents in cell size were more remarkable than increments in cell number.”

B. Body Fluids and Electrolytes 1. Fluid Spaces

Recent studies hare confirmed the previous work, which showed that malnutrition leads to overhydration of the body (Kerpel-Fronius and Kovach, 1948; Schneiden et al., 1958; Smith, 1960). Simultaneous measurements of total body water and extracellular fluid were made in Capetomn and Jamaica, with similar results. Tritiated water and thiosulfate were used by Brinkinan et al. (1965) to measure total body water and extracellular space, whereas Alleyne (1968) used tritiated water and bromide. With the quantities used and the rapid turnover of water (Smith, 1960), the radiation dosage is well below safety limits. The results of both studies confirmed the increase in total body water and showed that most of it was the result of expansion of the extracellular space. I n several children without edema there was an increase in extracellular space. It is probably this loss of excess extracellular fluid which causes the initial weight loss observed in almost all children a t the beginning of treatment, irrespective of the presence or absence of edema. Plasma volume is increased relative to body weight (Gollan, 1948; S. Cohen and Hansen, 1962; Picou, 1962; Alleyne, 1 9 6 6 ~ ) . Workers from Mexico (Gomez e t al., 1950) and India (Pate1 e t al., 1960) have reported that in severe kwashiorkor the blood volume is increased, but in Jamaica it was found that the increase in plasma volume was associated with a decrease in venous hematocrit; thus there was no change in blood volume with recovery (Alleyne, 1 9 6 6 ~ ) . 2. Sodium

The presence of edema and the expansion of the extracellular fluid space suggest that there is an increase in total body sodium. Evidence of this was obtained by Hansen (1956) by balance studies. As yet no

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measurements of total exchangeable sodium have been made in malnourished infants. Serum sodium has been reported as being normal (Hansen, 1956; Politzer and Wayburne, 1957) or low (Chirinos and Ramos-Galvan, 1964; Ward, 1964). It is agreed, however, that a low serum sodium is a bad prognostic sign (Kahn, 1959; Garrow and Pike, 1967). Muscle analysis has consistently shown an increase in calculated intracellular sodium (Frenk et al., 1957; Metcoff et al., 1966), which is not a function of intracellular dehydration, since cell sodium was still increased when referred to fat free solids. The cause of this intracellular accumulation of sodium is still unknown. Estimations of the intracellular concentration of any substance must be viewed with caution, since there is no good evidence that the usual extracellular markers, such as chloride or bromide, maintain their normal distribution when cell function is disturbed. 3. Potassium

Much more information has been accumulated on the potassium status of malnourished infants. The early findings of a low serum K (Hansen and Brock, 1954), a large retention of K early in the treatment phase (Hansen, 1956), low body K as measured with 42K (Smith and Waterlow, 1960), and low muscle K concentration (Waterlow and Mendes, 1957; Frenk et al., 1957; Smith and Waterlow, 1960; Metcoff et al., 1966) all indicated some degree of K deficiency. However, some workers have doubted the existence of K deficiency; Vis et al. (1965), who studied older children in the Belgian Congo, could not substantiate Hansen’s evidence of K depletion, as judged by retention of K in excess of N. Vis et al. (1965) also reported that K concentration in muscle was not reduced. Since a whole-body counter for infants has been installed in Jamaica, it has been possible to reconcile these differences and to present a clearer picture of potassium metabolism in malnutrition. I n general, malnourished children have a low total body K (TBK) which rises to normal levels after about 6 weeks of therapy (Fig. 3) (Garrow, 1965; Alleyne, 1970). Garrow showed that TBK was initially lower in those children who had edema, and subsequent experience has shown this to be true, but there is often overlap between those children with edema and those without edema. The concentration of K in the intracellular water of muscle is low (Metcoff et al., 1966), and the whole body intracellular K concentration (total body K/total intracellular water) is also low (Alleyne, 1968). Nichols et al. (1969a) measured muscle K and showed it to be related to TBK, but the relationship is not a simple linear one. T o explain these

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f

a

=76 53 44 47

0

2

34

33

29

21

19

4 6 8 Weeks after admission

19

12 10

12

8

12

8

14

FIG.3. Rate of repletion of total body potassium in infants during recovery from malnutrition. Data of Alleyne (1970). Reproduced by courtesy of the Editor of British Journal of Nutrition.

and subsequent findings of the same kind, it has been proposed that K status be considered in terms of two components-the body’s potassium capacity, and the extent t o which the body content of potassium saturates that capacity (Alleyne et aE., 1970a; Alleyne, 1970). I n children with TBK over 35 meq/kg there is a low body capacity for K because of tissue wasting, but that capacity is saturated; thus there is little true K depletion and muscle K per unit muscle weight is very little reduced. When the body potassium is below 35 meq/kg there is usually severe K depletion and muscle K is greatly reduced. This state of depletion rarely lasts for more than 1 week if adequate therapy is given, and thereafter a low TBK indicates t ha t the body’s K capacity is still low. On this concept the discrepancy between the findings of Vis and those of other workers is apparent rather than real. The malnourished children from the Congo may ha re had a low TBK but need not have been K depleted, and therefore would not show increased retention of K compared with N. I n the recovery phase of malnutrition approximately 3 meq of K are retained pri- gram of N (Hansen, 1956; Alleyne, 1970). Unfortunately, nieasi’reqent of total body K cannot give information about the distribution of K loss within the body. The major part of the total loss must be frorn muscle, as the largest K reservoir in the body, but, as the muscle ma-s wastes, its K represents a progressively smaller proportion of total body K, and brain K represents a larger proportion. Garrow et al. (1965, 1968) and Alleyne et al. (1969b) have shown this in analyses made post mortem, and it remains true even though the K concentration in the brain is reduced. Garrow (1967) devised an ingenious method for measurement in vivo of the K content of the head,

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Tanm IX Loss of Potassium jrom lhe Brain in Malnourished Injantsa Potassium depleted Number Age (months) Height (cm) Initial weight (kg) Total body K (meq/kg) Before treatment After treatment Head K (meq) Before treatment After treatment a

5

Not potassium depleted

5 11.2

9.4 68 5.9

67

31.3 43.5

41.9 42.2

44 77

84 78

6.0

Data from Garrow (1967). Reproduced by courtesy of the Editor of The Lancet.

wliicli is virtually cquivalent to the K content of the brain. His results are summarizcd in Tablc IS. Tlic loss of more than 30 meq of K from tlic brain is cnougli to linvc a significant cffcct on the total body K. The iniportancc of this loss is probably cven greater from the physiological point of view; pcrliaps it accounts for thc cxtremc mental apathy which is such a cliaractcristic fcaturc of kwashiorkor.

4. Magnesium Scrum magncsium is iisually normal (hlontgomery, 1960) but may fall in the presence of severe gastroenteritis (Ward, 1964). Tlie evidence for magnesium dcplction in malnutrition comes from balance studies and muscle biopsics. Montgomery (1961) observed increased retention of magnesium in short balances, and Lindcr et al. (1963) in a morc detailed study sliowcd that tlic rctcntion of magncsium was greater than would be prcdictcd from nitrogen balances. Muscle magnesium is low (Montgomery, 1960; Mctcoff et al., 1960; .4llcyne et al., 1970a), though tlie reduction is not as grcat as that of potassium, pcrliaps because of the large store of magncsium i n bonc. Workers in Nigeria (Caddell, 1967; Caddell and Goddard, 1967) claimed that specific elcctrocardiograpliic changes in mnlnourishcd cliildrcn may be attributable to magnesium deficiency, and in a controllcrl trial Caddcll (1967) obtained cvidencc that tlie outcoriic of trcatmcnt is worsc if mngncsium is omitted. Clinical features attributablc to swcre magncsium dcplction occur in Jamaica (Back e t al., 1962), but not as frequently as Caddell appears to have fniinrl in A f r i o n

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6. Trace Elements-Copper

and Zinc Copper has been measured in the serum of malnourished infants from Nigeria (Edozien and Udeozo, 1961), Guatemala (Lahey et al., 1958), India (Gopalan et al., 1965),Egypt (Sandstead et al., 1965), and South Africa (Hansen and Lehmann, 1969). The reduction appears to be greater in children with kwashiorkor than in those with marasmus (Gopalan et al., 1965). Hansen and Lehmann (1969) found the following levels in serum: in kwashiorkor 61,in marasmus 93,and in controls 140 &lo0 ml. The copper content of the liver is also reported to be low in kwashiorkor (Macdonald and Warren, 1961) but not in marasmus (Godette and Warren, 1967); the results in liver thus parallel those in serum. However, in other tissues no reduction in copper content was found (Warren et al., 1969),and the South African workers consider that a true copper deficiency has not yet been demonstrated. A possible relationship between zinc deficiency and the growth failure of malnutrition has been proposed by Sandstead et al. (1965) and by Prasad (1967). These workers found that plasma zinc concentrations were low in Egyptian children with kwashiorkor, and, although the values rose, they did not reach normal levels even after complete recovery. Hanscn and Lehmann (1969)also found very low concentrations of zinc in the serum in both kwashiorkor and marasmus. There was a correlation with serum albumin concentration, but no relationship to the degree of weight deficit or of stunting. The urinary output of zinc was not reduced, compared with the values found after treatment. Hansen and Lehmann therefore concluded: “In our group of children protein and calorie deficiency per se is sufficient to explain growth retardation. It would thus be difficult to attribute it to zinc deficiency, even if we found unequivocal evidence of this.” 6. The Cawre of the Disturbances in Fluid and Electrolytes

It is clear that there can be no single cause for all the electrolyte disturbances that occur in malnutrition. The Indian workers have insisted over many years that the main cause of the increased body water and edema is the inability of the damaged liver to inactivate ferritin, which stimulates release of antidiuretic hormone from the neurohypophysis (Srikantia, 1958, 1959; Srikantia and Gopalan, 1959; Belavsdy, 1965). In a recent paper they showed that the levels of antidiuretic hormone activity in plasma and urine in children with kwashiorkor were significantly higher than those in normal or marasmic children (Srikantia and Mohanram, 1970). Two objections may be raised to this theory of the cause of edema and overhydration in kwashiorkor. First, increased anti-

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diuretic hormone activity would lead to elaboration of a concentrated urine, and the urine of malnourished children is typically more dilute than that of well nourished controls (Gordillo et al., 1957; Alleyne, 1966a). I n addition, there is no evidence that increase of antidiuretic activity leads to edema in any clinical or experimental situation. An alternative theory is that the body fluid changes are basically renal in origin. There is an inability to excrete water (Alleyne, 1966a) and, in subjects whose diet consists entirely of dilute liquids, there is abnormal accumulation of fluid, leading to hypotonicity. Edema is simply a clinical sign which indicates an increased total body sodium. This arises either from an increased dietary intake or from failure of renal elimination. I n view of the other abnormalities of renal function which have been described (Kerpel-Fronius et al., 1954; Gordillo et al., 1957; Ward, 1964; Alleyne, 1967), the second possibility is more likely. The presence of edema in the more severely potassium-deficient children and a diuresis in response to potassium therapy have been described (Garrow, 1965; Hansen, 1956), but the mechanism by which potassium deficiency causes extracellular fluid expansion and edema is still obscure. The relationship between hypoproteinemia and edema in infantile malnutrition has been examined frequently. It is clear that total serum proteins, and specifically serum albumin, are low in almost all children with edema. Montgomery (1963), in an analysis of 173 cases, showed that the serum total protein and albumin levels varied inversely with the degree of edema, whether this was assessed clinically or by the extent of weight loss at the beginning of recovery. I n 50 children with the most severe edema the serum albumin was almost invariably less than 2.5 gm per 100 ml. However, the converse did not hold: of 60 cases whose albumin level was below 2 gm, only half had severe edema. I n the series examined by Garrow (1966), the mean serum protein concentration was significantly lower in patients classified as kwashiorkor, all of whom had edema, than in those diagnosed as marasmus, in whom there was no clinical edema, but the range of values was the same in the two groups. This overlap makes i t unlikely that there is a strict causal relationship between hypoalbuminemia and edema. The opinion expressed in the 1960 review (Waterlow et al., 1960) still seems to be valid, that “on the balance of the evidence i t seems probable that hypoproteinemia is a modifying factor and not the basic cause of the water and salt retention.” Aldosterone has also been implicated in the production of the edema, but its role is obscure. Lurie and Jackson (1962a) showed that a significant increase in urinary aldosterone levels only occurred when the edema was being lost. The unbound and presumably physiologically active fraction of aldosterone increases in kwashiorkor as a result of the hypo-

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A.

0. ALLEYNE

albuminemia (Leonard and MacWilliam, 1965), but this is still no proof that hyperaldosteronisni exists. This question will be answered only when aldosterone secretory rates are measured. The electrolyte depletion, when it does occur, is almost certainly the result of gastrointestinal loss, and a correlation has been found between the severity of diarrhea and the level of the total body potassium (Alleyne, 1970). Another possibility is that there is a renal Yeak” of potassium. Smith (1961) found that 6 out of 13 children, when put on a potassium-restricted diet, had a urinary potassium excretion of more than 2 meq per 24 hours. Since the period of dietary potassium restriction was necessarily short, no definite conclusion can be drawn that renal potassium loss may be a factor in the production of potassi_um depletion. Routine collections of urine from patients with low TBK-rarely if ever show abnormally high potassium concentrations. Finally, there is the “metabolic” theory of the production of the electrolyte disturbances, based on the elegant work of Metcoff and his colleagues (1966) in Mexico. They attempted to correlate levels of intracellular ions and metabolites and measured sodium, potassium, magnesium, phosphate, phosphoenolpyruvate, pyruvate, a-oxoglutarate, and oxaloacetate in biopsy samples of muscle from malnourished children. They found that pyruvatc and oxaloacetate were reduced. There was an increase in cell water, which was correlated with reduced levels of potassium, organic phosphate, and phosphoenolpyruvate. They suggested that there is inefficient synthesis or utilization of key glycolytic intermediates, leading t o impaired energy production, as a result of which intracellular sodium increases and potassium decreases.

V. FUNCTIONAL CHANGES A . Cardiac Function

Typically, children with severe malnutrition have cold, pale extremities, which Smythe et al. (1962) attributed to circulatory insufficiency. Kerpel-Fronius and colleagues (Kerpel-Fronius and Varga, 1949 ; Kei-pelFronius et al., 1954) studied the circulation in malnutrition and noted a prolongation of the circulation time, hypotension, and diminished cardiac output as measured by the Fick principle. Smythe et al. (1962) showed by radiography that the heart was small in kwashiorkor and increased in size with clinical improvement. Electrocardiographically there was sinus tachycardia with changes in the S-T segment and T wave, and occasionally in the U waves. Wharton e t al. (1969) also did electrocardiograms in patients with kwashiorkor on admission and during recovery, and noted T-wave changes suggestive of

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PROTEIN MALiVJTRITION I N CHILDREN

nonspecific myocardial damage. Toward the end of the first week of therapy, a t a time when patients were most prone to develop heart failure, there was a tendency to left ventricular predominance. Frank congestive heart failure has been proposed as a possible cause of death (Garrow et al., 1962; Viteri et al., 1964). Wharton et al. (1967), and Smythe et al. (1962) have suggested t ha t some of the sudden deaths seen in kwashiorkor may result from acute heart failure. It is probable that some of these cases of congestive failure were iatrogenic and related to excess sodium intakes. Wharton et al., (1967) noted a reduction in the occurrence of heart failure when the dietary sodium intake was reduced. The cardiac status of malnourished children has been investigated in some detail in .Jamaica (,4llcyne, 196613). A dye dilution technique was used to measure cardiac output, circulation time, and dye appearance time. Some of the data are shown in Table X. These results indicate some degree of circulatory failure in malnutrition. The reduction in cardiac output was related to the weight deficit. The child with the lowest cardiac output (619 ml/min or 2.06 liter/min x m3ht) in fact died shortly afterward. The reduced cardiac output and prolonged circulation times are even more striking when it is realized that these children were on the whole anemic, with a mean hemoglobin of 8.1 gm per 100 ml. There are several studies which show that there is normally a marked increase in cardiac output in the presence of anemia (Sharpey-Schafer, 1944; Roy et al., 1963). Histological studies on hearts from patients dying of kwashiorkor have not usually revealed any typical lesion (Smythe et al., 1962; Wharton et al., 1969). The fact that children who have recovered from malnutrition have normal cardiac status shows that there is no severe structural change initially. 'There is no real evidence that potassium deficiency is the cause of the cardiac impairment, because the ECG changes found in malnutrition are TABLE X Circulatory Changes in Malnourished Infantsa Malnourished Cardiac output (L/min X m3ht) Pulse rate (beats/min) Systemic recirculation time (seconds) Dye appearance time (seconds) a

4.77

110

13.7 4.7

Recovered 6.90

131 10.5 3.7

From Alleyne (1966b), by courtesy of the Editor of Clinical Science.

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J . C. WATERLOW AND C. A. 0.ALLEYNE

not typical of those produced by hypokalemia. Caddell (1967) attributed several of the EGG changes to inagesiuin deficiency, and found that they regressed after treatment with magnesium.

B. Renal Function All studies on the kidney in malnutrition have been confined to morphology and excretory function, and so far there has been no attempt to investigate the effects of malnutrition on renal metabolism. I n the discussion which follows we will consider together all the results froin children who were edematous and thosc u-ithout edema, since it has not been possible to find any difference in renal function between the two groups (Alleyne, 1965). 1. Gloinerular Filtration Rate and Renal Plasma Flow Recent work (M7ard, 1964; Allcync, 19671 has confirmed the results of earlier invcstigators (Kerpel-Fronius et al., 1954; Gordillo et al., 1957) that there is a reduction of gloinerular filtration rate and renal plasma flow in malnutrition. With gastroenteritis there is an even greater depression of these two functions. Arroyave et al. (1961) also found a depression of crcatinine clcarancc in malnourished infants, with values as low as 7.0 compared with the normal of 30-40 ml per minute per square meter of surface area. Thcy believed that their values were too low to be accounted for only by a depression of filtration rate, and suggested that there might be some tubular reabsorption of creatinine. Alleyne (1967) showed that these low Yalues for gloinerular filtration rate and renal plasma flow returned to normal with recovery. 2. Tubular Function

a. Concentrating Ability. Malnourished children may pass a hypotonic urine even when they are dehydrated (Gordillo et al., 1957; Metcoff et al., 1957). This may indicate an impairment of renal concentrating ability. hlleyne (1967) tested the effect of intramuscular aqueous Pitressin and found that nialnourished children responded by an increase in urine osmolality which was still significantly below that achieved after recovery. I n a more detailed study, McCance et al. (1969) used a period of water dcprivatioii to assess thc power of malnourished children to concentrate the urine. They found that although the patients could elaborate urine more conccntratcd than plasma, their concentrating ability was not as good as when they had recovered. This concentration defect was not the result of structural change because it could be reversed by feeding urea. The concentrating ability of the parents of these children could also be improved by feeding urea. McCance and his colleagues pos-

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179

tulated t h a t the concentration defect of malnourished children is a result of a low protein intake; they supported this by showing that previously normal individuals, when put on the local diet, could not produce as concentrated a urine as on their usual diet. Metcoff (1967), on the other hand, attributed the concentration defect to K deficiency. He pointed out that impairment of the concentrating mechanism is the most profound and consistent physiological effect of K depletion on renal function. b. Urinary Acidity. The urine is usually acid in malnourished children. Metcoff (1966) has suggested that the excretion of relatively large amounts of H' in the presence of K depletion may be the reason why these children are not as severely acidotic as well-nourished children with the same degree of diarrhea. Although the urine is acid, the ability to excrete a maximally acid urine after a standard ammonium chloride load is reduced, and the defect seems to be related to the degree of potassium depletion (Smith, 1959; Smith and Waterlow, 1960). Alleyne (1967) measured titratable acid and ammonium excretion after ammonium chloride and showed that not only was the maximum total hydrion excretion reduced in malnutrition, but the fraction contributed by ammonium was higher. It is doubtful if this defect has any significant effect on acid-base balance, since metabolic acidosis is not found except in the presence of severe diarrhea. c. Phosphaturia and Aminoaciduria. Renal phosphaturia occurs infrequently (Careddu, 1955; Alleyne, 1967), but aminoaciduria has been described by several authors (Cheung et al., 1955; Sarrouy et al., 1957; Kean and Picou, 1962; Schendel et al., 1959; Edozien e t al., 1960). It has been attributed to a specific proximal renal tubular defect, since plasma amino acid levels are in general normal or low (Edozien et al., 1960). Aminoaciduria is rather variable from one patient to another, and sometimes i t may appear during the recovery phase (Cheung et al., 1955; Schendel and Hansen, 1959, 1960, 1962; Alleyne, 1967). Schendel and Hansen claimed tha t initially there is a close correlation between nitrogen intake and urinary amino acid excretion. They also showed that when their patients were given a test meal of protein, plasma amino acids rose to a peak slowly and then fell slowly, and aminoaciduria, when i t occurred, was a reflection of these sustained high plasma levels. Awwaad et al. (1962) also showed that after intravenous administration of methionine, plasma levels remained elevated for an abnormally long time in Egyptian children with kwashiorkor. These results suggest t ha t there is some diminution in peripheral utilization of amino acids which would tend to aggravate aminoaciduria in the presence of impaired tubular function. d. T h e Causes of Impaired Renal Function. Protein deprivation in

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J . C. WATERLOW AND G . A. 0. ALLEYNE

previously healthy adults may cause reductions in glomerular filtration rate and renal plasma flow (Pullman et al., 1954), and it is possible that some of the abnormalities of renal function described could be attributable directly t o loss of protein from the kidneys. The fact that all the lesions are reversible makes it likely tha t there are no marked structural changes. Stirling (1962) has demonstrated increased capsular swelling, but it is not known to what extent this was attributable to protein depletion, or the result of attendant fluid and electrolyte disturbances which were present in many of the cases. Potassium deficiency may cause a fall in glomerular filtration rate (W. B. Schwartz and Relman, 1953) although other workers dispute this (Fourman and Hervey, 1955). The disturbances of hydrogen ion excretion, renal phosphaturia, aminoaciduria and failure to concentrate the urine may also all be attributable to potassium depletion (Milne et al., 1957 ; Mahler and Stanbury, 1956; Metcoff, 1967). The inability to concentrate urine may be related as well to the low urinary urea which results from the low protein intake (Edozien and Phillips, 1961; Waterlow, 1963). It has been suggested that the low levels of blood and urine urea characteristic of protein malnutrition indicate decreased amino acid deamination (Viteri et al., 1964) (see also Section V1,D).

C. Intestinal Function

I. Structural Changes It has been known for many years that malnutrition may produce profound changes in the structure and function of the intestine. Observations on adults in India (Passmore, 1947), and more recent investigations with jejunal biopsies in Africa (Banwell et al., 1964; Burman, 1965; Stanfield et al., 1965; Barbeaat et al., 1967), South and Central America (Mayorel et al., 1967; Garcia, 1968), and the Caribbean (Sparlte and James, 1968) have shown that the jejunal mucosa is often abnormal and may be completely flat and devoid of villi. Usually in a group of malnourished children there is a continuous spectrum of abnormality from the milder jejunal changes, often seen in the general population of the area, to the most severe mucosal atrophy. Brunser et al. (1966, 1968) in Chile, by studying only cases with typical kwashiorkor and typical marasmus, have suggested t ha t severe mucosal atrophy is a specific effect of protein deficiency. Ten of eleven children with kwashiorkor had severe mucosal atrophy, whereas only 1 of 18 cases of marasmus showed this appearance. In the kwashiorkor group the crypts appeared elongated, and the mitotic index of the crypt cells was normal. In contrast, the mucosa of the marasmic children had a low mitotic index compatible

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181

with a reduced rate of cell generation in the crypts. These observations suggested that in protein deficiency the generation of crypt cells can occur a t a normal rate, but that the cells formed are unable to maintain the villous structure, whereas with dietary calorie restriction the mucosa adapts by reducing the production and loss of cells, with preservation of the villous pattern. These observations apply to the two extremes of the clinical spectrum of protein-calorie malnutrition, but in practice the majority of patients may have signs of both kwashiorkor and marasmus, so that the variability in mucosal abnormalities reported in other studies is not incompatible with the observations of Brunser et al. I n malnutrition, recovery of the jejunal mucosa may take a long time. Often there is a failure of the mucosa to return to normal, but these cases are usually malnourished children studied months or years after admission to hospital, with no control of their diet during the intervening period (Stanfield et al., 1965; Cook and Lee, 1966). I n Colombia malnourished adults were fed in hospital for 6-9 months and reversal of the jejunal abnormalities did occur (Mayorel et al., 1967). Improvement in mucosal structure, especially in children with severe mucosal atrophy, has also been seen after 3 months in hospital on an optimal diet (Sparke and James, 1968). This emphasizes that when intestinal damage occurs in malnutrition its reversal may be very slow and improvement may not occur if patients return to their previous environment with an inadequate diet after only a short period of treatment. 2. Diarrhea

Diarrhea has always been described as one of the salient features of protein-calorie malnutrition. The problem of investigating the nature of the diarrhea has been complicated by the close interrelationship between gastroenteritis and malnutrition (Behar et al., 1958; Gordon et d., 1964a). Diarrhea without any obvious fecal pathogen is not only common in malnutrition, but also during an acute apparently infective episode of gastroenteritis (Gordon et al., 196413). Children in developing countries often have recurrent episodes of acute diarrhea as well as a more chronic looseness of stool. The acute episodes of diarrhea in malnourished children may well prove to be infective in origin. Dammin (1964, 1965) has found in postmortem studies on malnourished children that large numbers of bacteria colonized the upper intestine in children with diarrhea, but this overgrowth =as not found in those without diarrhea. Further work on this is clearly needed and may help to distinguish between an acute infective process and a more chronic overgrowth of “nonpathogenic” bacteria within the upper intestine, such as that re-

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S. C. WATERLOW AND G . A. 0. ALLEYNE

ported in tropical sprue (Gorbach et al., 1969). The exacerbation of diarrhea on feeding malnourished children probably represents the combined effects of previous infections as well as of malnutrition itself on the function of the intestine. 3. Carbohydrate Absorption and Disaccharide Intolerance

Dean in 1952 noted that malnourished children had a fermentative diarrhea and proposed that lactose should be eliminated from the diet. Bowie et al. (1967), in an investigation of carbohydrate absorption in cases with typical kwashiorkor, found markedly reduced levels of lactase, sucrase, and maltase in the jejunal mucosa of 7 of 10 children biopsied. Children with low disaccharidase activities and poor absorption had more diarrhea and a greater stool weight on a milk diet than on a disaccharidefree diet. The stool on a milk diet contained not only increased amounts of lactic acid, but also several unabsorbed sugars. Bowie et al. (1965) concluded t h a t lactose intolerance played a role in 60% of the malnourished children with diarrhea. Studies on children with malnutrition in East Africa confirm that lactase deficiency occurs and that the level of lactase in the mucosa is related to the extent of the pathological damage (Stanfield et al., 1965). Studies in Jamaica suggest that children with kwashiorkor or marasmic-kwashiorkor tend to have lower activities of lactase than marasmic children (James, 1969). This would be consistent with the findings of Brunser et al. of greater pathological changes in patients with kwashiorkor. Perfusion studies show not only a decreased capacity to hydrolyze disaccharide in the malnourished state, but also a reduction in monosaccharide absorption (James, 1968a). The unabsorbed sugar within the bowel acts as an osmotic load retaining water within the intestinal lumen and producing rapid intestinal transit. With treatment in hospital the activities of jejunal disaccharidases may increase and absorption of monosaccharidases improve (James, 1968a), but the jejunal lactase activities are the slowest to be restored and may remain subnormal (James, 196813). It has been suggested that in some parts of the world there is a genetic basis for lactase deficiency which will predispose children to marasmus in early life (Cook, 1967b). This awaits confirmation.

4, Absorption of Fat F at absorption is frequently impaired in malnutrition (Dean, 1955; Gomez et al., 1956a), but the exact nature of the defect remains obscure. Many malnourished children, when first admitted to hospital, do not have obvious steatorrhea because their fat intake is usually very low both before admission and in the first few days of treatment. A marked

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183

depression of pancreatic exocrine secretion, including lipase secretion, is known to occur (Barbezat, 1967), but the role of abnormal bile salt metabolism and the presence of an abnormal bacterial flora as contributory factors are still unexplored. Similarly, mucosal malabsorption or defective P-lipoprotein production for transport of fat into the lymph may all contribute to the steatorrhea; those factors are more likely to affect children with mucosal damage caused by protein deficiency. Sugar intolerance, by speeding the flow of food through the small intestine, may contribute to steatorrhea so that fat absorption and xylose absorption may improve when disaccharides are removed from the diet (Bowie et al., 1963). After the first few days of treatment, fat is usually well tolerated and exocrine pancreatic function returns to normal (Barbezat, 1967). 5 . Absorption of Nitrogen

Despite the fall in pancreatic digestive enzymes in malnutrition, nitrogen absorption is usually reported as good (e.g., De Mayer and Vanderborght, 1958; Waterlow and Wills, 1960) unless either severe diarrhea is present (Gomez et al., 1856a), or disaccharides are fed to sugar-intolerant subjects (Bowie et al., 1963). It may also be poor when vegetable protein is the main source of protein in the diet (Cravioto, 1958). The generally good retention of nitrogen probably reflects not only the greater resistance of pancreatic trypsin secretion to dietary restriction (Barbezat, 1967), but also the large reserve for protein absorption in the intestine. There is also little evidence that malnourished children become more protein depleted by exudation of protein into the intestine. Studies with '"1-labeled polyvinylpyrrolidone injected intravenously show only occasional cases with increased fecal excretion of label (H. Cohen et al., 1962; Purves and Hansen, 1962). This is confirmed by the low values found for the catabolic rate of circulating albumin in malnutrition (see Section VI,D). 6. Conclusion

We still do not know whether the diarrhea which is such a prominent feature of protein malnutrition is mainly caused by a hidden infection or by structural and functional changes in the gut which are themselves the result of malnutrition. I n effect, these causal factors interact, as Scrimshaw and his colleagues have emphasized for many years (Scrimshaw et al., 1968). One aspect which perhaps needs further attention is the effect on intestinal function of infections outside the gut. For example, in studies on iron absorption we have found that iron is usually well absorbed by malnourished children, but even a minor infective process, as shown by

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a slight pyrexia, will temporarily abolish absorption completely (Beresford et al., 1971). Results of the same kind were obtained in rats given endotoxin by Cortell and Conrad (1967).

D . Changes in Endocrine Activity 1. Growth Hormone

Histology of the pituitary gland from malnourished children has shown a variable increase of some of the secretory cells (Tejada and Russfield, 1957). The recent development of an immunoassay has led to the measurement of growth hormone levels in plasma. Pimstone et al. (1966, 1967, 1968) in South Africa have established conclusively that plasma growth hormone levels are high in malnourished infants and the fall that occurs with recovery is associated with a rise in plasma albumin. After intravenous glucose there was a greater fall in growth hormone levels in recovered than in malnourished children. Milner in Jamaica (1971) has shown similar increases in plasma growth hormone levels, which rise even further after an intravenous injection of glucagon. The metabolic significance of the elevated plasma growth hormone is not clear, but it is definitely not related to growth, since, at the time when the children are growing most rapidly, growth hormone levels are decreasing. I n contrast, the Chilean workers (Monckeberg et al., 1963; Monckeberg, 196th) suggested that marasmic children were deficient in growth hormone, since after giving the hormone they found increased retention of nitrogen. Hadden and Rutishauser (1967), however, could not demonstrate any significant metabolic effect of exogenous growth hormone in children with kwashiorkor. There appeared, therefore, to be a conflict between the findings in Africa and South America, but a recent paper by the Chilean group (Beas et al., 1971) resolves it. They measured plasma growth hormone levels before and after stimulation with arginine in a group of children with classical kwashiorkor and in another group of infants, from 6 to 13 months old, who had hardly grown a t all since birth. I n the former plasma growth hormone levels were high, as in African kwashiorkor; in the latter they were low, and showed no rise after arginine. Now t h a t this clear-cut difference has been found between the two clinical groups, the concepts of the Capetown and Chilean workers, which seemed to be diametrically opposite, can be reconciled. I n kwashiorkor an elevation of plasma growth hormone may be regarded as part of an adaptive or homeostatic response to protein deficiency of relatively recent onset; this response promotes the conservation of nitrogen

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185

when supplies are short. I n contrast, in Monckeberg’s patients with marasmus, or perhaps more aptly, nutritional dwarfing, there is a longstanding deficiency of calories as well as of protein. I n addition to the low growth hormone levels, the Chilean workers obtained evidence, summarized by Beas et al. (1971), of reduced adrenal and thyroid activity, suggesting a general hypofunction of the pituitary. They concluded that this is an adaptive mechanism which promotes survival by reducing the rate of growth, the rate of protein synthesis, and the metabolic rate. It will be evident that some of the confusion on this subject arose from misunderstandings about nomenclature : marasmus in South America is not necessarily the same condition as marasmus in Africa (see Section 11,A). Some discrepancies may also result from differences in the age a t which malnutrition begins. The experiments of Chow and Lee (1964) are of interest in this connection. ‘They studied rats whose growth was retarded by restriction of the food intake of the mothers during pregnancy and lactation. Administration of growth hormone to the rats from “restricted” mothers for 3 weeks after birth caused them to catch up in body weight and thereafter maintain a normal rate of growth long after the hormone treatment had ended. Cheek and Graystone (1969) have proposed that increase in cell number is a process which is growth hormone dependent. Since this process is most rapid just before and after birth, children who are malnourished very early in life, as seems to be the case in Chile, may be more sensitive to growth hormone. 2. Cortisol

Trowel1 et al. (1954) reported atrophy of the adrenal glands, but Gillman and Gillman (1951) suggested t ha t the final picture of atrophy may not reflect the situation in vivo. They presented some evidence that initially there was hyperfunction, and only terminally exhaustion and atrophy. Urinary steroid measurements have given conflicting results. Castellanos and Arroyave (1961) measured 17-ketosteroids and 17-hydroxysteroids in the urine and showed that marasmic infants had higher levels of glucocorticoid hormones than those with kwashiorkor. Lurie and Jackson (196213) also measured urinary stcroids and concluded that malnourished infants as compared to normal children had low steroid excretion, but they found no difference between those children who were acutely malnourished and those who had completely recovered from malnutrition. They concluded that there was rarely any evidence of diminished adrenal function in malnutrition. The difficulties in equating urinary steroid excretion with adrenal function, especially in the presence of impaired kidney function, are now clear (Cope and Pearson, 1963).

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J. C. WATERLOW AND G . A. 0 . ALLEYKE

T a n m XI Plasma Corlrsol Levels and Turnover Rates an Malnourishcd and Recovered Chrldrena

Plasma cortisol a t 9 A M (pg/100 ml) Plasma cortisol a t midnight (pg/100 ml) Plasma cortisol 1 hour after injection of Synacthen (pg/100 ml) Half-life of exogenous cortisol (min) Cortisol production rate (mg/kg X day) ~~~

~

RIalnourished

Recovered

28.2 20 8

11.5 4.9

58.4

44.6

180 0.2s

so

0.36

~

Data from Alleyne arid Young (1967).

Alleync and Young (1967) have investigated adrenal function in malnourished children, and some of their data are shown in Table XI. Plasma cortisol was consistently high in malnourished children and fell with recovery. An altered diurnal rhythm was also found, and there was impaired clearance of exogenous cortisol. There was good functional reberye, as shomi hy the prompt response to Synacthcn ( / 3 - 2 4 - ~ ~ r t i ~ ~ tropin) ~vliich directly stimulates the adrenal cortex. The prolonged half-life of exogenous cortisol is probably a reflection of impaired hepatic conjugation. In spite of the high levels of circulating hormone, cortisol production rates in the malnourished children were the same as in those who had recovered. The high plasma levels of cortisol could be partially suppressed ivith dexamethasone, and there was a good linear relationship between plasma cortisol and fasting blood glucose. Similar findings have also been reported from India (Rao et al., 1968). Cortisol levels were significantly higher in marasmic children than in those with kwashiorkor; in both groups the lcrels fell after treatment. The response to P-corticotropin was smaller in the children with kwashiorkor. Rao et al. (1968) interpreted this as further evidence that kwashiorkor represents a state in which the power of adaptive response is reduced. Plasma 17-OH corticosteroids have been measured in malnourished Egyptian children and also found to be elevated (Abassy e t al., 1967). There is decreased binding of cortisol in the plasma of malnourished infants (Leonard and ;\lacWilliam, 1964) ; therefore in effect they must have functional hypercorticism, and some of the features of infantile malnutrition, such as increase in liver fat and abnormal glucose tolerance, may be caused or potentiated by these high levels of free cortisol in the plasma.

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3. Other Hormones

Insulin production and plasma insulin levels are best discussed in relation to the changes in carbohydrate metabolism (see Section V1,B). The scanty information available about aldosterone activity in malnourished infants has been considered in the section on body fluids and electrolytes (Section IV,B). A few studies of thyroid function have been made in malnourished infants, particularly in relation to measurements of oxygen consumption. Montgomery (1962b) used the radioiodine excretion test, and found normal levels in malnourished infants soon after admission to hospital. The excretion was sometimes reduced during recovery, but Montgomery concluded that increased thyroid activity did not play any part in the dramatic rise in oxygen consumption which he found in recovering children (Section V1,A). Beas et al. (1966) found that in children with severe marasmus there was a diminished iodine uptake by the thyroid and decreased serum levels of protein-bound iodine and butanol-extractable iodine-all indications of decreased thyroid function. The low values were corrected by administration of thyroid-stimulating hormone. It was concluded that decreased thyroid activity was part of the general pituitary hypofunction discussed in Section V,D,l. VI. METABOLIC CHANGES

A . Oxygen Consumption A number of papers on the oxygen consumption or basal metabolism of malnourished infants were published in the 1920’s (listed by Montgomery, 1962a), but until the last few years interest in this subject seems to have lapsed. These early studies were made on marasmic infants, and they showed that the basal metabolic rate, when related to the body weight, tended to be raised above the normal range, although there was rather a wide variation. Montgomery (1962a) made serial measurements of oxygen uptake in infants with kwashiorkor and marasmus, and reported that the initial oxygen consumption per unit body weight was approximately normal, being slightly lower in kwashiorkor than in marasmus. Monckeberg et al. (1964) also found that on the average the oxygen uptake per kilogram was normal in a group of severely marasmic infants with stationary body weight. These results contrast with the universal experience in adults, that starvation reduces the basal metabolic rate.

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J . C. WATERLOW AND G . A, 0. ALLEYNE

I n the measurements on infants the question of the standard of reference presents great difficulty (see Section IV,A) . Montgomery related his results not only to weight but also to surface area, classically used in adults, but this may not have much meaning in severely malnourished infants. Monckeberg et al. (1964) showed that when O2 uptake was related to height it was greatly depressed in the malnourished child. However, what we are interested in is not the deficit in O2 uptake compared with that of a normal child of the same height, but the question whether the respiratory metabolism of the active cell mass is normal or not. Montgomery measured total body water in some of his cases; the 0, uptake related to total body solids was normal or high, presumably because of the reduced amount of body fat in these infants. He made the important point that the brain forms a larger proportion of the body in malnourished than in well-nourished children ; on the assumption that brain metabolism continues a t the normal rate regardless of the degree of malnutrition, Montgomery calculated that the 0, uptake of the remainder of the lean body mass must be reduced. However, he did not allow for dilution of the lean body mass by excess collagen (Picou et al., 1966), so that the active cell mass would be less than he had supposed. Nichols et al. (1968~)found that the basal 0, consumption was reduced in infants with a low total body K. The 0, uptake of the whole body showed a correlation with that of muscle homogenates measured in the Cartesian diver. However, although in muscle the Qo, per milligram wet weight was reduced, when related to noncollagen protein it was normal. This suggests once again that the apparently low oxygen uptakes may be the result of dilution with inert materials-water and collagenboth in the whole body and in muscle. Recently Alvarado (unpublished) has shown that in malnourished infants studied very soon after admission to hospital the whole body 0, consumption was reduced when related t o creatinine output as a measure of true muscle mass (see Section IV,A). With treatment the metabolic rate rose rather rapidly. We may conclude that there is some evidence, admittedly not very secure, of a reduction in overall metabolic activity in severely malnourished children. This fits in with the clinical observation of hypothermia (Brenton et al., 1967) ; i t is also in keeping with observations made at the tissue level-impairment of energy-producing mechanisms in muscle (Metcoff et al., 1966), and of oxidative phosphorylation in liver (Waterlow, 1961). The metabolic rate per kilogram of body weight increases rapidly in the early stages of recovery. Montgomery (1962a) claimed that i t rose to a level significantly higher than normal, in some cases to 90 kcal/kg per day, compared with 60 in control infants. The 0, uptake fell within a

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few days if the infants were given a low-protein diet. Later work suggests that perhaps some of these very high levels resulted from infants being studied too soon after a meal. Ashworth (1969) defined a “standard” metabolic rate in infants as that measured 4 hours after a feed. I n recovering infants the standard metabolic rate was 60-70 kcal/kg per day, and it was not affected by the previous level of protein or calorie intake, or by the rate of growth a t the time of the test. However, Ashworth found that, in children who were growing rapidly, within 1 hour after a feed the oxygen uptake rose by 30%, falling gradually toward the basal level after 3 hours (Fig. 4). It was concluded that this increase was not an example of classical specific dynamic action because the extent of it was the same with different amounts of protein in the test meal, and no such increase occurred in fully recovered children, tested in the same way, who were gaining weight much less rapidly. We interpret these results to mean that in children showing very rapid weight gain, the process of growth, i.e., the formation of new tissue, is not

40

t

0 L

a3

t

.A-

0

20 L

t

c

E

a

-10 0

2

I

3

Time (hours)

FIG.4. Response of oxygen consumption to a meal in infants during and after recovery from malnutrition. 0-0, During catch-up growth: test meal varied according to body weight, mean= 194 kcal and 4.5 gm protein; e----., during catch-up growth: test meal constant = 146 kcal and 3.3 gm protein; 0-0, after recovery: test meal varied according to body weight, mean = 283 kcal and 6.5 gm after recovery: test meal = 146 kcal and 3.3 gm protein. From protein; O----O Ashworth (1969), by courtesy of the Editor of Nature.

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continuous but occurs in bursts after each meal. These manifest themselves by an increased oxygen uptake because the process of protein synthesis seems to require a relatively large amount of energy (see Section I1,C).

B. Carbohydrate Metabolism I n the ten years since the 1960 review (Waterlow et al., 1960), there has been a good deal of activity in various parts of the world in the study of carbohydrate metabolism in malnourished infants. I n some cases the findings of different groups are contradictory, and we do not know whether these reflect real differences in the nature of the malnutrition, the proteincalorie balance, etc. On general grounds one might expect that there would be rather marked differences between kwashiorkor and marasmus in some aspects of carbohydrate mctabolism, depending on the degree of caloric deficiency, but so far a clear-cut picture has not emerged. 1. Blood Glucose Levels and Glucose Tolerance

It is a fairly constant finding that blood glucose is lower than normal in malnourished children. Sloane et al. (1961) and Baig and Edozien (1965) reported hypoglycemia in kn-ashiorkor, and Hadden (1967) showed that blood glucose was low in both kwashiorkor and marasmus. The actual levels of blood sugar found seem to vary from one center to another. Kerpel-Fronius and Kaiser (1967) in Hungary studied marasmic children, some of whom died, with blood sugar levels between 0 and 25 mg/100 ml, and Whitehead and Harland (1966) in Uganda described several cases of kwashiorkor with blood glucose levels lower than 40 mg/100 ml, but in Jamaica such profound hypoglycemia is rare. James and Coore (1970) found that in a series of 26 malnourished children the mean fasting blood glucose was 55 mg/100 ml initially, compared with 70 mg/100 ml after recovery. In a smaller series AIleyne and Scullard (1969) found similar values. There is variation also in the results of glucose tolerance tests. Sloane e t al. (1961 1 , Baig and Edozien (1965), and Hadden (1967), all working in Africa, showed clearly that there is impairment of glucose tolerance in patients with kwashiorkor, but the last authors, as well as Bowie (1964), found it to be normal in marasmus. This is in direct contrast to the results in Chile, where Oxman et al. (1968) reported marked glucose intolerance in marasmic infants. Baig and Edoeien (1965) were able to follow their patients after treatment and found a progressive improvement; Hadden (1967) showed that there was a return to normal by the 14th day of treatment. On the other hand, James and Coore (1970) in Jamaica found that tolerance was still

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PROTEIN MALNUTRITION I N CHILDREN

TABLEXI1 Rate of Glucose Utilization by Malnourished and Recovered Children Group

No. of children

Malnourished Recovered Normal*

26 28 5

Ka (mean

_+

SEM)

1.95 0.11 2.53 k 0.16 3.56 i- 0.36

P <0.01 <0.05

a K = rate of glucose disappearance (percent per minute) after an intravenous glucose load. Children who were never malnourished.

impaired even when the children were in all appearance fully recovered, after 2-3 months of treatment. Their results are summarized in Table XII. I n the initial malnourished stage no differences were observed between patients with the clinical features of kwashiorkor or marasmus, and therefore they have been treated as a single group. Cook (1967a) examined children who had been treated for kwashiorkor 6-12 years previously and found that there was still a significant impairment of glucose tolerance. Recently Hopkins et al. (1968) have attributed glucose intolerance in malnutrition to chromium deficiency. I n a study in Jordan and Nigeria they showed that in areas where there is a low concentration of chromium in the drinking water, glucose intolerance is more pronounced. They claimed that oral administration of chromium led to marked improvement, sometimes after only 1 day of treatment, but Carter et al. (1968), working in Egypt, were unable to confirm this. 2. Insulin Activity

The development of immunoassay techniques has made it possible to study plasma insulin levels as well as the insulin response to various stimuli. Baig and Edozien (1965) first showed that fasting plasma insulin levels were low in kwashiorkor and rose with recovery, but in their study there was a normal insulin response to intravenous glucose. Hadden in Uganda (1967) on the contrary reported that plasma insulin levels were low in marasmus, but normal in kwashiorkor. I n Jamaica James and Coore (1970) and Milner (1971) have shown that plasma insulin is low initially and that the rise in response to intravenous glucose is small or absent. After clinical recovery the response, although significantly improved, is still much lower than in normal children (Fig. 5 ) . Milner (1971) could not demonstrate an increase in plasma insulin after intravenous injection of glucagon. The experimental work of Heard and Henry (1969) poses the question

192

J . C. WATERLOW AND G . A. 0. ALLEYNE

*l

0

I"

' 9,

I I f

I

I

\

\

\

\

T

iA

I

OJ

,

0

\

\

\

1

10

20

30

Minutes Time after intravenous glucose injection

FIG.5. Serum insulin levels after an intravenous glucose load in malnourished

( X - - - X ) and recovered (0-0) children, and in children who had never been

malnourished (O---O). Data of James and Coore (1970). Reproduced by courtesy of the Editor of the American Journal of Clinical Nutrition.

whether the impairment of glucose tolerance does in fact result from insulin deficiency. In dogs fed a low protein diet they found poor glucose tolerance in the presence of a high insulin response to a glucose load; they therefore concluded that the deficiency state reduced the sensitivity to insulin. This might be regarded as a reversion to the normal state of the newborn animal. 3. Metabolism of Glycogen

a. Liver. Low levels of blood sugar may perhaps result from impaired glycogenolysis, so that studies on the metabolism of liver and muscle glycogen are of importance. Earlier histological and chemical assessments of liver glycogen in biopsy specimens gave rather inconsistent results (Waterlow and Weisz, 1956; Salazar de Souza, 1959; K. L. Stuart et al., 1958). I n our more recent studies (Alleyne and Scullard, 1969), we found that the amount of liver glycogen was significantly reduced in malnourished children (Table XIII) . Whitehead and Harland (1966) suggested that there was some im-

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PROTEIN MALNUTRITION I N CHILDREN

TABLEXI11 Glycogen Concentration and Gluwse--6-phosphatase Activity in the Liver in Malnutrition Subject

Gly cogene (mg/100 mg wet wt)

Glucose-6-phosphatasea (units/100 mg protein)

Malnourished Recovering Recovered

2.02 f 0.28 (18) 4.58 f 0.88 (12) 5.85 0.50 (13)

4.85 f 0.64 (14) 2.47 f 0.23 (11) 2.98 k 0.27 (10)

a Mean i -SEM. Number of patients in parentheses. units = milligrams of inorganic P released per 15 minutes.

Glucose-6-phosphatase

pairment of hepatic glycogenolysis, since the rise in blood glucose in response to subcutaneous epinephrine (adrenaline) or intravenous glucagon was less in malnourished than in recovering children. Fletcher (1966) also claimed that glycogenolysis might be impaired, since he found subnormal levels of hepatic glucose-6-phosphatase. Other authors have supported Fletcher’s finding (Mukerjee and Nath, 1957; Salazar de Souza, 1959), but our results show the opposite effect-an increase in the activity of this enzyme in the livers of malnourished infants (Table XIII) . With recovery there was a n increase in glycogen and a decrease in glucose-6-phosphatase activity. We have attempted to assess the integrity of hepatic glycogenolysis and the functional capacity of glucose-6-phosphatase by measuring blood glucose, lactate, and pyruvate after intravenous injection of glucagon (Alleyne and Scullard, 1969). After glucagon there was a prompt rise in blood glucose in all children, but the peak value was higher in those who had recovered. Milner (1971) obtained similar results. This proves that there can be no impairment of hepatic glycogenolysis. There was no rise in blood lactate or pyruvate after glucagon injection, which shows that glucose-6-phosphatase activity is probably not impaired. This was further confirmed by showing that glucose concentration reached a normal peak after injection of galactose. Liver phosphorylase activity showed no change in the malnourished children (Alleyne and Scullard, 1969). b. Muscle. Muscle carbohydrate metabolism has also been investigated recently. The glycogen content of muscle is low in malnutrition and is correlated with the concentration of potassium (Alleyne e t al., 1969a). With recovery there is a marked overshoot to supranormal levels, but these return to normal in the fully recovered child (Alleyne and Scullard, 1969; Nichols et al., 196913). It is possible that there is

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J . C. WATERLOW AND G . A. 0. ALLEYNE

an impairment of glucose metabolism in muscle, which contributes to the glucose intolerance described in the previous section.

4. Glycolysis and Gluconeogeneszs Oxman et al. (1968) showed t h a t blood pyruvate levels in marasmic infants were higher than normal, but did not rise after intravenous glucose injection. This suggested t h a t there might be a block in the pathways in both directions at some point between glucose and pyruvate. Whitehead and Harland (1966) also report elwated blood pyruvate levels in kwashiorkor, but in .Jamaica malnourished children have been found to have normal blood pyruvatc (Allcyne and Scullard, 1969; Flores et al., 1970). nlletcoff et al. (1966) measured metabolic intermediates as well as the enzyme pyruvate kinase in muscle biopsy samples from nialnourished children. They found decreased levels of phosphoenol pyruvate and oxaloacetate but normal levels of pyruvate and reduced activity of pyruvate kinase. They also showed altered kinetic properties of this enzyme when it was assayed zn vitro with certain substrates and cofactors a t concentrations similar to those which exist in the muscle of malnourished children. I t is rather difficult to explain why in malnutrition the level of phosphoenol pyruvate should bc low and that of pyruvate unchanged. I n the presence of a block at the pyruvate kiiiase step one might expect the opposite changes. A decrease in activity of pyruvate kinase and a reduction in ATP, pyruvate and oxaloacetate have also been described in leukocytes of severely malnourished children (Yoshida e t al., 1967, 1968). Their contention is that a block of terminal glycolysis may lead to a decrease in ATP-formation and impaired energy production This method of studying energy-producing processes and relating them to more conventional static measurements is a welcome approach t o the study of metabolism in malnutrition. Another approach which has been used in the investigation of carbohydrate metabolism is the infusion of labeled substrates, followed by measurement of incorporation of label into other substances. Gillman e t al. (1961) infused gluc~sc-'~C,p y r ~ v a t c - ~ ~and C , acetate-l-14C into patients with kwashiorkor and showed that the conversion of glucose t o pyruvate and thence to acetyl-CoA was markedly depressed. There was, however, a more ready synthesis of glucose from pyruvate in malnourished infants, which may be evidence of enhanced gluconeogenesis. There is therefore some conflict with the observations of Oxman e t al. (1968) referred t o above. It will be important to repeat these studies

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195

with stable isotopes, since 14C compounds cannot be used with safety in children. 5. General Conclusions

Glucose intolerance is an almost universal finding in infantile malnutrition, but the cause is not yet clear. A deficiency of insulin and a poor P-cell response to glucose probably play some part. I n addition, there may well be a block in peripheral glucose utilization caused by reduction in activity of one of the more important glycolytic enzymes. The increased activity of hepatic glucose-6-phosphatase as well as the elevated plasma cortisol levels already described make it likely that there is increased gluconeogenesis. The finding that some functional impairment remains even after clinical recovery is apparently complete underlines the need for long-term follow-up studies of children who have been malnourished in infancy.

C. Fat Metabolism 1. Liver Lipids

Fatty liver was one of the features of kwashiorkor as it was originally described by Williams (1933), and it is one of the criteria which is often used to differentiate kwashiorkor from marasmus. It seems t o be more common and more severe in some places than in others. However, only exceptionally has the actual fat content been measured chemically, other than in specimens obtained after death, so that quantitative comparison between different series is difficult. I n Jamaica we have found fat contents as high as 50% of the wet weight of the liver (Waterlow et al., 1960). I n the analysis of cadavers made by Garrow et al. (1965) , there were three children in whom more than 30% of the total body fat was in the liver, and in one child analyzed by Halliday (1968) the liver contained 44% of the total body fat. Chatterjee and Mukherjee (1968) in India found comparable levels of fatty infiltration; in cases classified as kwashiorkor the average liver fat, measured in biopsy samples, was 39% of the wet weight. The measurements made by Macdonald (1960) on the livers of Bantu children a t autopsy show equally severe fatty change in some patients. It seems that fatty liver is a fairly widespread feature of protein malnutrition throughout the world. All authors agree that the excess fat is mainly triglyceride. Macdonald (1960) found that there was a reduction in the concentration of phospholipid, but not in the total amount. Chatterjee and Mukherjee (1968) measured phospholipid fractions in liver biopsies from malnourished children. Again, there was a decrease in the concentration of phospho-

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A.

0. ALLEYNE

lipid in the fatty livers, but a rough calculation from their figures suggests that there was no great reduction in the ratio of phospholipid to fat-free solids. These workers found tha t in kwashiorkor the proportion of phosphatidylethanolamine, expressed as percentage of total phospholipids, was significantly lower than in marasmic patients or in children who had recovered. These observations are important because they indicate the possibility of changes in the structural lipids of cell membranes. Macdonald (1960) was the first to study the fatty acid composition of the liver lipids. I n a series of samples obtained post mortem from children in Nigeria and Jamaica he found an increase in the proportion of palmitoleic acid and a decrease in t ha t of linoleic acid. Similar changes were found in the depot fat. These findings were confirmed in a later series of measurements made on biopsy samples (Macdonald et al., 1963). The changes resembled closely those found in rabbits fed a high-carbohydrate diet. Macdonald concluded t ha t the excess fat in the liver was not derived from the depots, but was synthesized from carbohydrate. Lewis et al. (1964) also measured the fatty acid pattern of the liver lipids in kwashiorkor; their results agreed reasonably well with those of Macdonald, but they came to precisely the opposite conclusion-that the excess fat reaches the liver from the depots. The difference between these two views is more apparent than real: the fat must have been originally synthesized from carbohydrate or protein, since the dietary fat intake of these children is certainly low. The point in dispute, therefore, is the site of synthesis.

2. Serum Lipids There is almost universal agreement on the pattern of plasma or serum lipids in malnutrition. In both marasmus and kwashiorkor, free fatty acids in plasma are elevated and there is a rapid fall early in the recovery phase (Lewis et al., 1964; Fletcher, 1966; Hadden, 1967; Milner, 1971). Lewis et al. (1966) also measured the flux of fatty acids through the plasma and showed that it was increased 10 times in kwashiorkor and some 4 times in marasmus. I n order to explain the elevated fatty acid levels in kwashiorkor, which is supposed to be produced by a high calorie low protein intake, they postulated that a t the stage of admission to hospital these children were in a phase of acute starvation because of anorexia and diarrhea. The severe calorie deprivation a t this time would lead to increased peripheral lipolysis and increased plasma free fatty acid flux. Rao and Prasad (1966) studied the fatty acid response to epinephrine. In both malnourished and recovered children there was a prompt increase in serum fatty acids after subcutaneous injection of epinephrine, although

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197

the percentage increase was higher in those who had recovered. But after 60 minutes, when the fatty acid levels had returned almost to normal in the recovered children, they were still rising in the malnourished patients. Serum triglycerides are initially low in kwashiorkor (R. Schwartz and Dean, 1957; Lewis et al., 1964; Monckeberg, 1968a), and with treatment rise rapidly to levels which may be above normal. I n marasmus there is less change in triglyceride levels. Serum cholesterol is also low in kwashiorkor and rises with treatment. 3. The Mechanism of Fatty Liver

There is very little disagreement about the facts, but there are several different interpretations of those facts. Three possible mechanisms have been proposed to explain the fatty liver. The first is increased production of fat in the liver from carbohydrate. This is the hypothesis put forward by Macdonald (1960, 1963). Some support was provided by the work of Fletcher (1966), who obtained evidence of impaired glucose utilization in the liver, and suggested that unused Carbohydrate may be converted to fat. By measurements made in vitro on biopsy samples he was able to show that liver tissue from malnourished children is capable of converting acetate to fatty acids. It cannot be denied that the liver fat is formed from carbohydrate, but there is no direct evidence that the rate of formation is increased, to such an extent that the transport mechanisms are overloaded. The second hypothesis is that there is increased transport of fatty acids to the liver from the depots. The evidence for this is the finding by Lewis e t al. (1966) of a greatly enhanced flux of free fatty acids in kwashiorkor. If, as these authors suggested, this results from calorie starvation in the later stages of the illness, it would mean that fatty infiltration of the liver must be a rapid and late event, but on the whole clinical observation does not bear this out (Waterlow, 1948). That there should be an increased fatty acid flux in marasmus is to be expected, but why there is an even greater increase in kwashiorkor is not yet explained. The observations of Rao and Prasad (1966) suggest that possibly the peripheral utilization of fatty acids may be impaired. The third explanation of the fatty liver, and the one which a t the moment seems most likely, is that there is a failure of fat transport out of the liver. Long ago R. Schwartz and Dean (1957) suggested that the increase of serum triglycerides and cholesterol which occurs with treatment represents a clearing of fat from the liver. Recently Flores et al. (1967) in Chile have measured liver and plasma lipids simultaneously, and separated two lipoprotein fractions with the ultracentrifuge. This

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work gives perhaps the clearest picture of the correlation between the changes in plasma and liver lipids. The serum triglycerides were initially low in kwashiorkor but not in marasmus ; the reduction was mainly in the low density lipoprotein fraction ( d < 1.063), which is responsible for the transport of triglycerides in the plasma. Phospholipid and cholesterol were reduced only in these low density lipoproteins. After 8-15 days of treatment there was a reduction of up to 50% in the liver fat; a t the same time there was an increase in serum lipids, accounted for mainly by a rise in the low density lipoprotein fraction. This was accompanied by a rise in the serum P-globulins (Monckeberg, 1968a). The work in Chile agrees well with the recent demonstration by Truswell et al. (1969) by electrophoresis of a pre-P-lipoprotein band in the plasma of children recovering from kwashiorkor. Roheim et al. (1965) showed that rat plasma contains a lipid-free protein which acts as a precursor for lipoproteins of density less than 1.019, and which therefore may be regarded as an apoprotein. Lees (1967) obtained evidence that this npoprotein is included in the @-globulin fraction of serum proteins, so that the well-known finding of low P-globulin levels in malnourished children gains added significance. Flores et al. (1971) have shown that the fraction of plasma protein containing this apoprotein, when injected into protein-depleted rats with fatty livers, causes a rise in the serum triglyceride level and increased incorporation of labeled fatty acids into the triglycerides of low density lipoprotein. Some years earlier Robinson and Seakins (1962) had shown the opposite effect: that injection of puromycin in rats caused a rise in liver lipid and a fall in plasma lipids, resulting from depression of lipoprotein synthesis. The hypothesis, therefore, is that the fatty liver of kwashiorkor is caused by failure of fat transport out of the liver. This in turn is caused by decreased synthesis of the apoprotein part of the lipid-transporting mechanism. It appears that, as in the case of albumin, the synthesis of this protein must be rather sensitive to dietary protein supply.

D. Protein Metabolism 1 , Amino Acid Metabolism

a. Abnormalities of Amino Acid Metabolism. Whitehead and coworkers in Uganda (summarized by Whitehead, 1969b) and Edozien and Obasi (1965) in Nigeria have obtained evidence of abnormalities in the metabolism of histidine, phenylalanine, tyrosine, and tryptophan in severe kwashiorkor. After a test load of these amino acids a number of metabolites appear in the urine which would normally be oxidized. Whitehead (1964a) also descrihed an unidentified substance in the

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serum which was thought to be a metabolite of lysine. Edozien and Obasi were inclined to attribute the blocks in the metabolism of histidine and phenylalanine to deficiency of cofactors, notably folic acid and pyridoxine, whereas Whitehead and Holt e t al. (1963) thought that there might be an acquired deficiency of some of the enzymes of amino acid catabolism, analogous to the genetic deficiency in phenylketonuria. This excretion of abnormal amino acid metabolites appears to be a breakdown phenomenon which is transient, and disappears rapidly once the children begin to respond to treatment. Whitehead (1969b) reported that loading tests with histidine and phenylalanine were valueless for the diagnosis of early protein malnutrition. It was this interest in the wider problem which led him to concentrate instead on the distortion of the plasma amino acid pattern as a more sensitive diagnostic tool. b. Mechanism of the Changes in Plasma Amino Acid Pattern. I n Section II,E a general account was given of the alterations in plasma amino acid pattern found in protein malnutrition, and the discussion centered on their use as a test or measure of protein deficiency. Here we shall consider in more detail the changes in individual amino acids, the possible ways in which these changes are brought about, and their significance in the overall metabolism of the body. These questions were discussed very thoroughly by Holt et al. (1963) in the light of the knowledge then available, and more recently by Grimble and Whitehead (1970a). It immediately appears t ha t there is no single explanation for all the alterations in amino acid pattern; different influences affect the individual amino acids in different ways according to their metabolic role. Table XIV shows the main changes found in plasma amino acid pattern in infants with protein malnutrition, summarized from four representative sets of data (Arroyave et al., 1962; Holt et al., 1963; Saunders et al., 1967; Grimble and Whitehead, 1970b). This table is not intended to be complete; the extensive studies of Vis (1963), for example, have not been included because he was dealing with rather older children. I n the second part of the table are comparable data from experimental studies in adults who were starved or given protein-free diets (Adibi, 1968; Young and Scrimshaw, 1968; Felig et al., 1969a), and in children who were given different levels of protein intake (Grimble and Whitehead, 19TOb). For the sake of simplicity, only those amino acids are shown whose concentrations change consistently and significantly. Valine is taken as an example of the branched-chain amino acids. It is not easy to decide on the most illuminating way of expressing the figures. Arroyave and Holt, in their original papers, in addition to giving the concentrations of

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TABLE XIV Plasma-Free Amino Acid Concentrations: ( A )In Children with Kwashiorkor or Marasmus, or on Low Protein Diet. ( B ) I n Adults Starved or on Protein-Free Diet

Subject

Total NH2-N

A . Infants (mg/100 ml) 15.5 Normal (1)" 21 .o Normal (2) 21.2 Recovered (3) 16.1 Recovered (8)b 10.15 Kwashiorkor (1) 7.35 Kwashiorkor (2) 9.5 Kwashiorkor (3) 11.6 Kwashiorkor 8.5 Marasmus (1) 14.1 Low protein (8)* B . Adulk (:pmoles/l) 2.31 Normal (4A) 2.20 Normal (4B) 2.14 Normal (5) 2.45 Normal (6) 2.41 Normal (7) 2.24 Starved 2 wk (4A) 1.90 Starved 6 wk (5) 2.38 Starved 6 wk (7) 2.67 Protein-free 2 wk (4B) 2.45 Protein-free 2 wk (6)

Percent of total NH2-N Alanine

Glycine

Valine

Phenylalanine : tyrosine

11.3 9.0 10.0 20.6 10.6 13.2 8.65 19.6 10.8 37.3

8.4 5.1 7.6 12.6 17.7 13.9 16.6 25.1 14.6 16.7

11.2 13.7 7.9 11.8 6.1 4.1 2.9 5.0 6.6 7.35

0.83 0.61 0.79 0.84 1.96 2.46 3.56 2.46 1.92 1.14

17.7 20.1 13.6 12.9 14.9 12.5 5.85 8.2 27.0 26.1

8.8 9.7 12.3 11.5 7.2 13.2 19.0 16.3 12.2 13.4

11.6 8.5 9.6 8.8 11.2 11.0 6.8 7.5 6.2 5.9

0.90 1.00 1.00 1.03 0.81 0.95 0.68 0.80 1.16 1.10

a Numbers in parentheses refer to references. Key: (1) Saunders el al. (1967); (2) Holt et al. (1963); (3) Arroyave et al. (1962); (4) Adibi (1968): A: starved; B: on protein-free diet; ( 5 ) Felig et al. (1969a); (6) Young and Scrimshaw (1968); (7) Swendseid et al. (1969); (8) Grimble and Whitehead (1970b). Only 14 amino acids measured.

each amino acid, have calculated the values in malnourished infants as percentages of those in controls. Since throughout we have been discussing the amino acid pattern i t seemed more revealing to express the value for each amino acid as a percentage of the total free amino acid concentration in the plasma. (i) Total amino acids. I n malnourished children the total is greatly reduced, t o about half the normal level, whereas this is not found in adults or infants subjected experimentally to starvation or protein deficiency. One likely reason is that the depletion period was relatively much shorter. A second reason is that the child's free amino acid pool is turning over more rapidly, so t ha t it is more sensitive to changes in

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input and output. We have no information about the pool sizes in infants, but data of Nyhan quoted by Young (1970) show that in oIder children, although the plasma amino acid levels are about the same as in adults, the concentrations in muscle are much lower. At the same time the turnover rate of protein is greater, which implies a faster turnover of free amino acids. Young (1970) has compared the size of the free amino acid pool with the amino acid requirement. He calculates that in the adult the pool is large enough to cover the essential amino acid requirements for about a day, whereas in the child i t would cover the requirements for only a few hours. It is natural, therefore, that the pool size should be more labile in the child. (ii) Alanine. The changes in the relative concentration of alanine in protein malnutrition are not very striking (Table XIV). This is interesting, and possibly revealing, because in the adult experiments the picture is quite different: starvation causes a substantial fall in plasma alanine, and a protein-free diet an equally substantial rise. Cahill and his group (Felig et al., 1969, 1970) have emphasized the importance of alanine for gluconeogenesis, and have postulated an “alanine cycle” in which this amino acid acts as an NH, carrier from the periphery to the liver. In starvation there is an increased uptake of alanine by the liver for gluconeogenesis. Later there is also a fall in the output of amino acids, and particularly of alanine, from muscle as gluconeogenesis is reduced to conserve body protein. Both these effects tend to cause a fall in plasma alanine concentration. On a low-protein, high-carbohydrate diet there is a different situation. If alanine acts as a carrier for NH, groups released from other amino acids in peripheral tissues, one may suppose that the route by which they are converted to urea in the liver proceeds via glutamic-pyruvate transaminase and glutamic dehydrogenase (Krebs, 1964). It is well established that on low-protein diets the activity of both these enzymes in the liver is reduced (Rosen et al., 1959; Muramatsu and Ashida, 1962; Harper, 1965). At the same time, since such diets tend to be high in carbohydrate, there is no need for gluconeogenesis from amino acids. Thus one may speculate that if both liberation of the amino group for urea formation and utilization of the carbon skeleton for glucose formation are partially blocked, the result is a fall in the utilization of alanine and a rise in its plasma concentration. Perhaps the fact that in most cases of protein malnutrition, as it occurs naturally, there are no striking changes in alanine concentration indicates that the two opposing processes of protein deficiency and calorie deficiency are operating together. Only in the Ugandan series (Grimble and Whitehead, 1970b) was there a marked increase in the relative alanine

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concentration, and the general dietary evidence suggests that these patients represent a fairly purc form of protein deficiency. (iii) GZycme. Table S I V shoxvs that the glycine concentration is very greatly increased in protein malnutrition. Experimentally, unlike alaninc, it is increased in both starvation and protein deficiency. Glycine may be catabolized by several different pathways (Krebs, 1964), and it is also a precursor of many biologically important substances such as porphyrins, nuclcotides, and creatine. However, unlike alanine i t docs not enter the major pathways of energy metabolism; muscle, for example, is apparently unable to oxidize glycine (hilanchester, 1965). Therefore it seeins likely that quantitatively the main routes of glycine utilization are uptake into protein or degradation to urea. Collagen is particularly rich in glycine, and one might therefore expect that changes in the rate of collagen turnover could have an important influence on the glycine concentration in body fluids. To fit the facts it would be necessary to postulate that collagcn synthesis is inhibited, so that free glycine accumulates, and that later the breakdown rate of collagen is reduced to produce a new steady state. The same inferences about collagen metabolism were drawn from the data on hydroxyproline output-that in malnutrition the turnover rate of collagen is reduced without any significant change in its amount (see Section 11,E). It is noteworthy also that both the increase in plasina glycine and the decrease in urinary hydroxyproline occur equally in protein deficiency and in caloric deficiency. (iv) PhenyZaZanirze:tyrosine ratio. It is apparent from Table XIV that one of the most striking features of the amino acid pattern in kwashiorkor is a w r y great increase in the ratio of phenylalanine to tyrosine, caused mainly by a fall in the tyrosine concentration, that of phenylalanine being well maintained. Holt et nl. (1963) concluded that there may be interference with the hydroxylation of phenylalanine, a possibility originally suggested by Cheung e t al. (1955) on the basis of observations on the urine, and one which fits in well with the findings of Whitehead referred to above. This change in the phenylalanine: tyrosinc ratio was not found in either the starved or the protein-deficient subjects, which tends to confirm Whitehead’s view (1969b) that it is a very late event. Several authors have suggested that depigmentation of the hair, which is a frequent, although not invariable, feature of kwashiorkor, may be a specific effect of tyrosine deficiency. (v) Branched-chain amino acids. All authors are agreed that in protein malnourished infants the branched chain amino acids are the ones most decreased, particularly valine. This is sho\vil in Table XIV. Leucine and isoleucine follow the same pattern as valine, but the changes in them are not quite so great. I n adults, both in starvation and in experi-

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mental protein depletion, the concentrations of valine, leucine, and isoleucine fall, but not as much as in children with kwashiorkor. Swendseid et al. (1966) pointed out that in severe obesity, in some cases of diabetes, and in healthy subjects on a ketogenic diet, the ratio of nonessential to essential amino acids in plasma changes in the opposite direction to that found in protein malnutrition. The ratio falls, mainly because of an increase in the concentrations of the branched-chain amino acids. All these conditions are associated with a high rate of gluconeogenesis, and Swendseid suggested that a low ratio might be indicative of increased gluconeogenesis. She added “apparently not only the amount of calories but also the type of calories may influence plasma aminoacid patterns.” The work of Heard et al. (1969) supported this concept. They found that in pups on a low-protein diet the ratio of nonessentials to essentials rose significantly only if the diet was high in carbohydrate, but not if i t was high in fat. The ratio was also low in marasmus, and Heard et al. suggested that in both cases a low ratio reflected a deficiency of carbohydrate, since carbohydrate can provide the carbon skeletons of nonessential amino acids, but fat cannot. These ideas do not explain the greater decrease in the branched-chain amino acids than in the other essentials. The three branched-chain amino acids do not have the same metabolic fate: valine is glucogenic, leucine is ketogenic, and isoleucine may give rise to both glucose and fat (Krebs, 1964; Meister, 1965). Nevertheless, the important point is brought out that the characteristic amino acid pattern found in protein deficiency may have no direct relation to changes in protein metabolism, but may be a side-effect of the balance of the other main dietary constituents. It also seems possible that the changes in the branched-chain amino acids may in the main be reflections of metabolic events in muscle. Experiments by liver perfusion (L. L. Miller, 1962; M . M. Fisher and Kerly, 1964; Rothschild et al., 196913) and by hepatic vein catheterization (Elwyn, 1968) have indicated that the liver metabolizes branched-chain amino acids rather slowly, whereas they are extensively oxidized by extrahepatic tissues (L. L. Miller, 1962). The first step in the catabolic pathway of these amino acids is transamination, and i t is therefore of great interest that the branched-chain amino acid transaminases occur predominantly in muscle (Mimura et at., 1968). According to the calculations of Young (1970), the total quantity of these transaminases in muscle is some 100 times greater than that in liver. Mimura et al. (1968) found that in rats on a low protein diet the activity of these muscle transaminases is approximately doubled ; this could provide an explanation for the fall in the concentrations of the branched-chain amino acids

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in plasma. On the other hand, I. G. McFarlane and von Holt (1969) have shown that in protein-deficient rats CO, production from leucine by the whole animal is reduced. Presumably in these circumstances, if more leucine is deaminated but less is oxidized, there must be a greater utilization of the carbon residues for fat synthesis. This in turn fits in with the observations of Heard and of Swendseid referred to above. c. Conclusions. Some tentative generalizations may be made from the work described. 1. The amino acid pattern in protein malnutrition does not depend upon the nature of the limiting amino acid in the diet, since there is a remarkable similarity of pattern in patients from different countries with different diets (Holt et al., 1963). 2. The pattern is affected by the absolute and relative intakes of carbohydrate and fat, as well as by the protein intake. 3. The alterations in pattern may predominantly reflect alterations in muscle metabolism. Beyond this it is hard to go. Arroyave e t al. (1962) suggested that the changes in the plasma aminoacids must per se have an adverse effect on cellular function. They supposed that the intracellular amino acid concentration and pattern must also be altered, and referred to the earlier work of Christensen, which showed that “enriched aminoacid environments are associated with accelerated growth.’’ However, the situation does not seem to be as simple as this, because sometimes the opposite conditions apply. I n children intracellular amino acid concentrations in muscle are apparently less than in adults, yet growth is faster. An interesting example is from the work of Betheil et al. (1965), who showed that cortisone increases the rate of protein synthesis in liver, but decreases the amino acid concentration, whereas the opposite changes occur in muscle. Moreover, although the literature on the point is rather conflicting, i t seems that a reduced amino acid supply from the food does not necessarily lead to a fall in cellular amino acid concentrations. For example, in the experiments of Jost et al. (1968), the concentrations of all amino acids were higher in the livers of rats on a protein-free diet than in those fed protein. On the other hand, Wannemacher and co-workers (1968) showed that in rats kept on a low-protein diet for a long period ( 7 weeks) there was a moderate fall in the concentration of most of the essential amino acids in the liver.6 ‘This statement is based on a recalculation of the data of Wannemacher et aE. (1968) to give the concentrations of amino acids per gram of liver. Their method of expressing amounts of amino acid per cell or per unit DNA is not relevant in the present context.

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Amino acid concentrations can themselves give little information about the function which is really important-the rate of turnover or of uptake into protein. I n theory it would be possible to have a plasma concentration almost equal to zero with a normal turnover, and the reverse may also happen, On the other hand, there can be no doubt that amino acid supply does influence protein synthesis rates. A clear-cut example is the close dependence of albumin synthesis on the dietary protein intake (see Section VI,D,2). The relationship of amino acid supply to protein synthesis a t the level of the ribosome has been discussed by Munro (1968) , but consideration of this problem is beyond the scope of the present review. The point which we wish to emphasize is that the amino acid concentration bears no simple relationship either to the rate of supply or to the rate of utilization. It would be a great simplification if there was such a relationship, and if, for example, we could use the demonstration by Wurtman e t al. (1968) of diurnal variations in plasma amino acid levels to support the idea put forward above (Section V1,A) that protein synthesis may be an intermittent and not a continuous process. Finally, the work of Harper (1969) suggests that di'stortions of the plasma amino acid pattern may influence overall metabolism through quite a different mechanism-an effect on the appetite. The imbalances produced by Harper in rats do not lead to quite the same changes in plasma amino acid pattern as those found in human protein malnutrition; nevertheless, his theory raises an important question for future investigation, because of the well-known fact that anorexia is one of the milestones in the downhill path of children whose diet is deficient in protein. 2. Plasma Protein Turnover

a. Albumin. In the ten years since the last review a large number of papers have appeared on albumin metabolism in man under various conditions, but we shall consider only those in which it has been studied in relation to nutritional state. The first such study was that of Gitlin et al. (1958) in Mexico. They calculated the catabolic rate from the decay curve of radioactivity in the plasma after the injection of 1311-labeled albumin, and found the same half-life-about 10 days-in malnourished and recovered children. It was suggested in the 1960 review (Waterlow et al., 1960) that the initial measurement was probably in error, because during the period of the test the children, who were in the early stages of recovery, would be increasing their circulating albumin mass and diluting the pool of labeled albumin. The same criticism applies to our earlier studies (Picou and Waterlow, 1960). The catabolic rate cannot be determined from the plasma slope unless

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there is a steady state, but the error due t o nonsteady states can be reduced, if not eliminated completely, by calculating it from the urinary excretion of labeled iodide (S. Cohen et al., 1961). A further advantage of this method, particularly for work on children, is that a valid result can be obtained in a shorter time, 7-10 days, than by the plasma slope method. A disadvantage is that unless whole-body counting is possible, continuous urine collections are necessary in order that the amount of labeled albumin remaining in the body may be accurately k n o v n By this method S. Cohen and Hansen (1962) in Cape Town and Picou and Waterlow (1962) in Jamaica showed that in malnourished children the catabolic rate of albumin was greatly reduced, t o about half the rate after recovery. Although the results of these two studies agreed closely, there were also some important differences. The Cape Town workers kept the children on a low-protein diet throughout the first test; they were therefore able t o assume a steady state, and this meant that they could calculate the distribution of albumin between the intravascular (IV) and extravascular (EV) pools. I n the malnourished children the total albumin mass was about 50% of that found after recovery, the EV pool bcing more depleted than the I V pool. I n the Jamaican study a high-protein diet was fed, so that a steady state could not be assumed, and a valid calculation of the albumin pool sizes could not be made. However, judging by the body weight deficits, the Jamaican infants were more malnourished than those in Capetown, and the point remaining unresolved was whether the reduction in catabolic rate resulted from a low protein intake or from a state of protein depletion. The effect of low protein intakes on albumin metabolism was explored in more detail by Hoffenberg et al. (1962, 1966) in studies on adults who were given a diet containing 10 gm of protein per day for 3-6 weeks. This period of mild depletion produced a small fall in the plasma albumin concentration and in the IV albumin pool, and a larger fall in the EV pool. Some evidence was obtained of increased transfer of albumin from the E V pool, and it was inferred that the synthesis rate of albumin was reduced, but this could not be measured directly under the conditions of the experiments. By far the most dramatic effect was a fall in the catabolic rate, from an average of 149 mg/kg per day initially to 92 after depletion (Hoffenberg et al., 1962). This reduction of some 40% in the catabolic rate occurred a t a time when the mean cumulative loss of N from the body, measured by N balance, was only 58 gm, or about 3% of total body N. James and H a y (1968) separated the two factors of protein intake and protein depletion by altering the level of protein intake for successive periods of 7-10 days after a single injection of a l b ~ m i n - ' ~ l I .These

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studies were made on both malnourished and recovered children. The experimental design meant that a nonsteady state was deliberately produced. The catabolic rates were calculated from the urinary output of 1311,and the synthetic rates from the curves for total EV and I V activity. I n a steady state these two curves are parallel; if there is net synthesis of albumin they tend to diverge, and if there is net loss to converge. A digital computer was used to generate simulated curves, and by adjustment of the appropriate rate coefficients the slopes of the generated curves could be modified until a close fit to the experimental curves was obtained. The results of this study are summarized in Table XV. They show that in both malnourished and recovered children the synthesis rate of albumin responds rapidly to an alteration in the level of protein intake, while the catabolic rate changes more slowly, after a lag period of several days. The changes are more extreme in the malnourished children. The data also indicate, in agreement with the findings of Hoffenberg et al. (1966), that on the low protein intake there was a net transfer of albumin from the EV to the I V pool. I n the work described so far the synthesis rate of albumin has either been taken as equal to the catabolic rate, a steady state being assumed, or, as in the studies of James and Hay (1968),it has been calculated under TABLEXV The Influence of the Level of Protein Intake on the Absolute Rates of Albumin Catabolism and Sunthesis in Malnourished and Recovered Infantsapb Days of study Protein intake: Recovered children

C

S No. of cases Malnourished children

C S

No. of cases

0-10 High

10-17 Low

17-24'

Low

17-24' High

219 222

178 148

140 138

156 236

166 233 9

171 101

131 87

178 288 5

9

9

9

4

4

5

Reproduced by courtesy of the Editor of The Lancet. Data from James and Hay (1968). Synthetic rates were derived by a computer technique suitable for nonsteady-state conditions. All values are expressed as milligrams of albumin per kilogram per day. C = catabolism; S = synthesis. In the final dietary period (days 17-24) t:ach group of 9 children was divided into two subgroups: in one subgroup the low-protein diet was continued; in the other it was replaced by a high protein intake.

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nonsteady state conditions in a way which can give only an approximate answer. It was therefore an important advance when A. S McFarlane in 1963 introduced an independent method of measuring the synthesis rate of albumin. The principle of this method is that if carbonate-’*C is given, some of i t will be incorporated into urea, and some into arginine by the Krebs-Henseleit cycle, and thence into albumin and other liverproduced proteins. From the rule that the products of the same precursor must have the same specific activity, the amount of albumin synthesized in a given period can be calculated from the specific activities of albumin and urea, and the amount of urea formed over the same time-interval. I n adult human subjects (Tavill et al., 1968) and in rats (Kirsch et al., 1968c), excellent agreement has been obtained between the synthesis rate measured in this way and the catabolic rate measured simultaneously by the conventional method. I n children, however, the use of 14C is not considered justifiable, and so the McFarlane method has not been applied. Kirsch e t al. (1968~)showed that in rats on a normal protein intake the synthetic and catabolic rates of albumin were equal, and amounted to 90 mg per 100 gm body weight per day. After 60 days on a low protein diet the rate of synthesis had fallen to one-fourth of the normal level, but the rats had adapted by an equal reduction in catabolic rate. When serial measurements were made during protein depletion and repletion the synthesis rate altered rapidly in response to changes in protein intake, while changes in the catabolic rate lagged behind by several days. These results are therefore in excellent agreement with those obtained in infants by James and Hay. From this body of work a fairly clear picture emerges of the relationship of albumin metabolism to protein supply. The immediate reaction to a reduced intake is a fall in the synthesis rate. This leads to a small reduction in the plasma albumin concentration and the IV albumin mass, but two compensatory or adaptive mechanisms come into play-a shift of albumin from the E V to the IV pool, and a fall in catabolic rate. This concept of the compensatory mechanisms was first proposed by Hoffenberg and his co-workers (1962, 1966), and all that has been done since supports it. It seems that, following Claude Bernard, the plasma albumin concentration may be regarded as an “e’le’ment constant,” while the turnover rate is an “e‘le’ment variable.” Hoffenberg e t al. (1962) published a very interesting figure in which they showed a linear relationship between serum albumin concentration and absolute catabolic rate, expressed in milligrams per kilogram per day. The nature of this relationship was such that a large change in catabolic rate corresponded to a small change in serum albumin; e.g., when albumin fell from 4 to 3.5 gm/lOo ml, a reduction of one-eighth, the turnover rate fell from 150 to

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100 mg/kg per day, a reduction of one-third. Thus the dynamic measurement of turnover rate could be regarded as a much more sensitive index of protein depletion than the static measurement of albumin concentration. Unfortunately, it is not a test that can be applied on a routine scale. The sequence of events described above makes it almost certain that there are separate mechanisms which regulate the rates of albumin catabolism and synthesis. Freeman and Gordon (1964) proposed that the catabolic rate may be controlled by the plasma albumin concentration, but i t is clear from the work of Matthews (1965) on rabbits and that of James and H a y on infants that, the two may vary independently. It also seems that the catabolic rate does not respond directly to a change in synthesis rate, since it is possible to produce situations in which they alter in opposite directions (Matthews, 1965; James and Hay, 1968). James and Hay cite evidence t ha t growth hormone causes a fall in the rate of albumin catabolism, and therefore the finding of Pimstone et al. (1966) of increased levels of circulating growth hormone in kwashiorkor is very relevant. I n a later report Pimstone and co-workers (1969) have shown that in the untreated patient there is an inverse relationship between the concentrations of growth hormone and albumin in the plasma, but they do not believe that this is cause and effect, because after 24 hours of treatment the growth hormone level falls rapidly, long before that of albumin rises. The rate of albumin synthesis responds very rapidly to the level of protein intake, and therefore it seems that it must be controlled directly by the rate of amino acid supply. Rothschild e t al. (1969a), using McFarlane’s method, showed that in rabbits fasted for 18-36 hours the rate of albumin synthesis was decreased by 3370. I n experiments with perfused livers they showed that the synthesis rate was markedly reduced when the donor animal was starved or fed a low-protein diet, although the blood used for the perfusion came from well-fed animals. I n other experiments (Rothschild et al., 196913) they perfused the liver with amino acid solutions; they found, as before, that if the liver donors had been fasted the rate of albumin synthesis was reduced by 5070, but it was restored to normal by the addition of tryptophan to the perfusion fluid. Isoleucine had a smaller effect, and the other essential amino acids had none. Kirsch et al. (196913) performed the opposite experiment; they perfused the livers of well-fed rats with blood from rats deprived of protein for 48 hours. The rate of albumin synthesis, measured by the carbonate-14C method, was reduced, but there was a significant improvement when branched-chain amino acids were added to the perfusing blood.

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Although there are some discrepancies hetween these results, they do show a close dependence of albumin synthesis on amino acid supply. I n the opinion of Rothschild and co-workers this is not simply because one or other amino acid is rate-limiting. They conclude that “the effects of even a short-term fast are directed or transmitted to the microsomes, which appear to remain inhibited for hours in vitro after the fast has ended.” Their experiments suggest that the mechanism of inhibition and its reversal by tryptophan is that described by Munro (1968), and depends upon the state of aggregation of the polysomes. However, it seems that Rothschild and his group do not regard amino acids as the only factor controlling albumin synthesis. They have shown in rabbits that increasing the plasma oncotic pressure by infusions of dextran reduced the rate of albumin productioii (Oratz et al., 1970). At the subcellular level the dextran infusions, like fasting, produced a decrease in polysome aggregation. The effect of hormones may also be important. John and Miller (1969) showed that albumin production by the perfused rat liver was doubled when insulin and cortisol together with an amino acid mixture were added to the perfusates. Neither the amino acids nor the hormones were effective by themselves. They suggest that “there are diurnal variations in the rate of hepatic synthesis of plasma proteins, with increases during active assimilation of an adequate diet.” This fits in with evidence cited earlier for the concept of protein synthesis as a function which waxes and wanes with protein supply. b. Conclusion. The response of albumin metabolism to changes in protein intake represents a most important and interesting example of a homoeostatic mechanism, which plays a part in the body’s adaptation to different nutritional conditions. It is for this reason that we have discussed it in some detail. A t the same time, one must bear in mind that albumin metabolism is not necessarily representative of the metabolism of all or even most proteins in the body. This will become apparent in the next sections. Because the proteins of the plasma are the only ones whose turnover rates can a t all easily be studied in the human subject, there is a tendency for protein metabolism to be equated with plasma protein or even albumin metabolism. For example, in a recent symposium with the title “Radioisotope Techniques in the Study of Protein Metabolism” (International Atomic Energy Agency, 19651, 20 out of 24 papers were concerned with plasma proteins. This emphasizes how little is still known about the metabolism of tissue proteins in man. c. 7-Globulin. S. Cohen and Hansen (1962) showed that in malnourished children the metabolic behavior of y-globulin was quite different from that of albumin. The synthesis rate was unaffected by the state of

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nutrition, but in children whose illness was complicated by infection i t was greatly increased. I n adults also, low protein diets imposed experimentally caused no change in y-globulin metabolism (Hoffenberg et al., 1966). S. Cohen and Hansen (1962) point out t h a t the production of large amounts of 7-globulin by protein-depleted subjects in response to an infection shows that the cells which form y-globulin must have preference in the utilization of available amino acids. They go on to suggest t h a t “as a result, the synthesis of other biologically important proteins may be seriously limited, and this may be one reason why the clinical manifestations of kwashiorkor are often precipitated by infection.” However, from what we have learned since then it does not seem likely that this hypothesis can hold in any simple form. The rate of y-globulin synthesis in infected children was about 200 mg/kg per day. The data given in the next section show that this represents only about 3% of the total protein turnover in the body, and therefore could hardly act as a significant drain on the overall amino acid supply. 3. Turnover and Synthesis of Tissue Proteins

The last ten years have seen enormous advances in our understanding of the mechanism of protein synthesis a t the subcellular level. I n this review we propose to treat the subject strictly from the physiological point of view, confining ourselves to an examination of what is known about the effects of protein nutrition on protein turnover, synthesis and catabolism in the whole animal or in individual tissues. a. Total Protein Turnover. Total protein turnover is the sum of the turnovers of all the individual proteins of the body; from the physiological standpoint it is analogous to the total oxygen consumption or basal metabolic rate which is the sum of the oxygen uptakes of all cells, and we may suppose that it is of equal physiological interest. I n the 1940’s Rittenberg and his co-workers began their studies of the metabolism of N compounds in man using 15N as a label, and developed methods for the measurement of total N turnover (Sprinson and Rittenberg, 194913; San Pietro and Rittenberg, 1953). I n the years t h a t followed a number of studies were made of N turnover in human subjects under different conditions of diet, endocrine activity, etc. (for references, see Waterlow, 1967). Latterly this approach seems to have fallen from favor, probably for two reasons: the results did not seem to be particularly informative, and there are theoretical objections to the method of San Pietro and Rittenberg which was used by most workers. It seemed to us of importance to find out more about total protein turnover and overall rates of synthesis and catabolism in children with

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J . C. WATERLOW AND G .

A. 0. ALLEYNE

protein malnutrition. The practical and theoretical problems of such studies were reviewed by Neuberger and Richards (1964), and again more recently (Waterlow, 1969a). The interpretation of results obtained after a single dose of a labeled amino acid is very difficult, and therefore a more promising approach seemed to be t o produce an isotopic steady state by continuous infusion of the labeled material, as has been done in studies of the metabolism of fatty acids and steroid hormones. The method adopted for the measurements on infants involved infusion of I5N-labeled glycine for 30 hours, and measurement of the steady-state rate of isotope excretion in the urine (Picou and Taylor-Roberts, 1969). For practical reasons it seemed advisable to give the infusion by the intragastric rather than the intravenous route, and the first problem to be faced was whether the body handles amino acids entering from the gut in the same way as it handles those entering the general circulation from catabolism of body protein. It seems a reasonable possibility that ingested amino acids, which pass immediately to the liver, should be more readily degraded to urea than those which enter the peripheral circulation from the tissues. Picou and Taylor-Roberts (1969) tested this point by comparing the turnover rates obtained by intragastric and intravenous infusions made consecutively in the same infant under the same conditions. Two experiments were done and reasonably close agreement was found, which means that for practical purposes we can assume that amino acids entering by the two routes mix in a homogeneous pool. A second question was whether glycine is a representative tracer for the total amino-N pool. Wu and co-workers measured total N turnover in different subjects with single doses of glycine-15N, aspartic acid-15N or phenylalanine-l5N, and found no consistent differences with these amino acids (Wu and Snyderman, 1950; Wu and Bishop, 1959; Wu and Sendroy, 1959; Wu et al., 1959). Nevertheless, the point had to be tested. T o do this the results were compared of consecutive intragastric infusions in the same subject of g1ycine-l5N plus milk and of egg protein uniformly labeled with laN. Again there was satisfactory agreement, which shows that the behavior of glycine is reasonably representative of that of the total amino acid mixture. The constant infusion method is designed to measure the total amino acid flux, i.e., the amount entering or leaving the pool in unit time. Since the pool size can be presumed to be constant, one can write Q = I C= S E , where Q is the flux, I and E are the rates of nitrogen intake and excretion and S and C are the rates of synthesis and catabolism. I and E are easily measured, and if Q is determined the total rates of N synthesis and catabolism are then known. The results obtained by Picou and Taylor-Roberts (1969) are sum-

+

+

213

PROTEIN MALNUTRITION I N CHILDREN

TABLE XVI Effect of Protein Intake and of Nutritional State on Rates of Total Protein Synthesis and Catabolism in InjantsaJ‘ Children

I. Recovered

11. Malnourished Recovered

Intake

Synthesis

Catabolism

1.2 5.2 3.4 3.6

6.2 6.5 11.2 6.1

4.2 9.7 4.7

5.5

Data of Picou and Taylor-Roberts (1969). Reproduced by courtesy of the Editor of The Lancet. * Values are expressed in grams of protein per kilogram per day. Measurements by constant intragastric infusion of glycine-lSN.

marized in Table XVI. The first point is that the rates of synthesis and catabolism were the same regardless of the level of protein intake. The low intake provided 1.2 gm of protein per kilogram per day, and the high intake 4 times as much. The diets were fed for long enough to allow N balance to be achieved at the new level of intake before the infusion was given. This result suggests that albumin, the synthesis of which is so sensitive to the amino acid supply, may be a special case, and not representative of the body as a whole. The second finding was that in the malnourished children both synthetic and catabolic rates per kilogram of body weight were greatly increased. If account were taken of the “dilution” of active tissue protein discussed in Section IV,A, the difference between malnourished and recovered children would be even greater. This result is surprising, and clearly more experience must be obtained before i t is accepted as conclusive. As far as we know, there is no systematic source of error in the method which would apply in malnourished but not in recovered children. Comparison with other results in the literature is not particularly helpful, because only one other study has been made in the same age group. Wu and Snyderman (1950) measured total N turnover in 3 infants with a single dose of aspartic acid-15N, and obtained values of 3.3, 3.4, and 7.8 gm of protein per kilogram per day. The general range found in adults by the single-dose method is about 2-4 gm/kg per day, although both higher and lower values occur (summarized by Waterlow, 1967). The results of Picou and Taylor-Roberts (1969) are therefore not out of line with those of earlier workers, and they are probably more consistent. Labels other than 15N have on occasion been used for measurement of protein or amino acid turnover in man. We estimated the total protein turnover in 3 adults by intravenous infusion of lysine-14C and other 14Clabeled amino acids have been useful in studies of phenylketonuria

214

J. C. WATERLOW AND G . A. 0. ALLEYNE

(Grumer e t al., 1961, 1962) and hyperglycineniin (Sylian and Childs, 19641, hut they have not heen given to infants because of the radiation hazard. Picou niatlc some studies of methionine turnover in malnourished and recovered infants, but the results m r e difficult to interpret, probahly I)ecauFe of tlic >l)c'cialnietal)olic patlin :iys of tlic iulfur-containing amino acids iPicoii and Waterlow, 1967). The work of Aivw.ad e t cxl. (1967) indicated that the iiicthioiiinc aiialog ~cleiiomctliionineis utilized for protein syntlie&, and siiicc " S e is a gnimnn emitter it seemed p o s i h l e that ~elenomctliioniiic-'S c might he a useful tool for the measurement of total protein turnover hy whole-hody counting (WatcrloTv e t nl., 1969). This proved not to be the case. A large proportion of the trace1 wa5 excreted in tlie few>.with a higher bpccific activity than that 111 the urine. This invalidates tlic model used for calculating the turnover rate from the curve of whole-body nctivity. Second,it appcarecl that sclcnomethioninc is reutilized to a much greater degree than other amino acids; an experiinent i n which it ivas given together with glycine-1'X sho~vedthat the tn-o amino acids I)eliaved in quite different ways. Siiicc we have not l m n able to develop an independent method of measuring total protein turnover in infants, the only other ivay of cvaluating the results is by comparison with animal experiments. I n the rat total protein turnover was measured by constant intrarenous infusion of lysine-14C (Waterlow and Stephen, 1967, 196%). The turnover per kilogram of body weight was higher in young animals than in old ones, and higher in males than in females. When our estimates of the total protein turnover in normal adult nian, infant, and rat are compared with tlie basal metabolic rate, there is a rough parallelism which makes sense in biological terms (Table XVII). In the rat the turnover was not reduced by short periods of starvation or protein deprivation, but therc was R fall of about 302, after 5-13 nccks on a lorn-protein diet. We have not been able to reproduce 111 the rat the increased total turnover rates found in malnourished children. One possible explanation of the fall in urinary hi exerction which follows a reduction in protein intake would be that it reflects an equally TABLE XVII Protein Turnover and Basal Metabolic Ra.tc=

a

Subject

Body weight (kg)

Protein turnover (gm/kg/day)

Basal metabolic rate (kcal/kg/day)

Rat Infant Adult

0.1 10 70

25 6 2-3

130 45 20

Data from Waterlow (196913). Reproduced by courtesy of the Editor of T h e Lancet.

PROTEIN MALNUTRITION IN CHILDREN

215

great fall in overall N turnover. The results obtained in both man and rat rule out this explanation. A low protein intake does not cause a general slowing down of protein turnover, and we have to look elsewhere for the mechanism of the adaptive change in urinary N output. b. Utilization of Ammonia Nitrogen. Sprinson and Rittenberg (1949a) showed that when I5NH,+ was given to rats and to human subjects] some 50% of the dose was retained and presumably utilized for protein synthesis. Since then it has been very well documented] both in rats and man, that ammonium-N can supply a large proportion of the nonessential N requirement (see Section II1,B). Sprinson and Rittenberg observed that more of the tracer was retained by rats or humans on a low protein intake. One might also expect from the studies of Allison (1951) on dogs that a t any given level of intake the retention would be greater in subjects who were protein depleted. This, therefore, might form the basis of a method for assessing the extent of protein depletion. Read et al. (1969) gave 15N-diammonium citrate to children with kwashiorkor and marasmus a t intervals during recovery, and measured the cumulative excretion of isotope in the total urinary N, urea, and NH, over a period of 48 hours. They found that as the children recovered the proportion of isotope retained progressively fell. However, the patients were also receiving an increasing protein intake, so that the reduced retention of 15N reflects both a diminishing avidity of the tissues for N and a greater dilution by N from the food. I n our Unit we have given 15NH4Cland analyzed the isotope excretion curve according to a simple 2-pool model. It is possible to derive from the curve an approximate estimate of the fraction (F) of the total N entering the metabolic pool which is excreted. (l-F) then represents the fraction utilized for protein synthesis. The same information can be obtained more accurately by the constant infusion technique; the idea behind the studies with amrnonia-l5N was that the simplicity of the method might compensate for its lack of precision. A possible source of error, which is difficult to assess, is that NH,' may to some extent be preferentially converted to urea, and is therefore not an ideal tracer for amino-N. Nevertheless, the results may be useful for comparative purposes. It appears that the fraction of N excreted (F) varies more or less directly with the total N intake or total N excretion. This is not just another way of stating what is well known-that the N excretion falls as the intake falls. What it means is that as the intake falls a larger proportion of the N entering the free amino acid pool is taken up into protein and a smaller proportion is degraded to urea. This is also shown by the data of Picou and Taylor-Roberts (1969). Under normal conditions it is probable that two-thirds to three-fourths

216

J. C. WATERLOW AND G. A. 0. ALLEYNE

TABLE XVIII Utilization of Nitrogen Entering the Body Pool at Different Levels of Protein Intakea

N derived from food N derived from catabolism Total N entering pool N excreted in urine Fraction of N entering pool excreted in urine

A. High protein intakeb

B. Low protein intakeb

0.5

0.2 1.0 1.2 0.1 0.083

1.0

1.5 0.4 0.27

B/A

70

40 100 80 25 31

a A decrease of 20% in the total N entering the pool causes a reduction of 70p% in the proportion excreted in the urine. Values are expressed as milligrams of nitrogen per kilogram per day.

of the total N entering the pool is derived from the catabolism of body protein, and one-third to one-fourth from the food. It was shown above t h a t a low protein intake does not cause any reduction in the rate of catabolism. Therefore a large change in the dietary component represents only a small change in the total rate a t which N enters the pool. Yet this small change is enough to alter profoundly the relative rates of nitrogen flow through the two pathways-uptake into protein and degradation to urea. This point is illustrated numerically in Table XVIII. c. Utilization of Urea Nitrogen. Snyderman et al. (1962) showed that when normal infants were given an inadequate intake of protein (0.7 gm/kg per day) so t h a t they stopped growing, weight gain could be restored by the addition of large amounts of urea to the diet. They proved that this represented a real utilization of urea N because when 15N-urea was given there was some incorporation of isotope into plasma and red cell proteins. Although no precise calculations can be made, the data suggest that the utilization of exogenous urea must be very inefficient. One could not, on this evidence, propose that in man, as in ruminants, urea might be a useful supplementary source of nitrogen. The utilization of urea N is of more interest from the point of view of urea kinetics and their bearing on turnover studies. Walser and Bodenlos (1959) and Jones et al. (1968) have investigated the extent of recycling of endogenous urea in human subjects. From their results it seems that normally about 30% of the urea formed in the body is reutilized. It is probable that the urea is broken down by bacteria in the intestine, and the N reabsorbed as ammonia or amino-N, since sterilization of the gut with antibiotics reduced the extent of recycling. Recent work of Read et al. (1969) shows that reutilization of endogenous urea occurs in infants also. Forty-eight hours after a dose of urea-

PROTEIN MALNUTRITION I N CHILDREN

217

15N 3 marasmic infants had retained 15, 25, and 31% of the dose, whereas two controls only retained 1% and 11%. The marasmic children also excreted a larger proportion of the 16Nin compounds other than urea. These authors showed very ingeniously that the retention of urea did not result simply from failure of excretion by the kidneys. They found that when W I Wurea was given, throughout the next 24 hours an increasing proportion of the isotope was excreted as 15N14Nurea. This is proof that the urea molecule must have been broken down and resynthesized. The effect was more marked in the marasmic children. It is not possible from the data of Read et al. to calculate the absolute quantities of urea reutilized. If the marasmic child reutilizes 30% of its urea production, this would be a significant amount, but the question remains whether it represents a saying of N which would otherwisc be lost from the body. Presumably this could only happen if essential amino acids are present in adequate amounts and the limiting factor is the supply of nonspecific N (see Section 111,B). d. Meast~reinentof the Turnover Rates of Tissue Proteins. For a fuller understanding of the physiology of protein metabolism in protein depletion determination of total protein turnover needs to be supplemented by measurements of turnover in different organs and tissues, and ultimately of the turnover of individual tissue proteins. C p to the present time studies of this kind have only been done on animals. Accurate determination of the synthesis rate from the incorporation of labeled amino acid into protein requires that the specific activity of the precursor as well as of the product should be known throughout the period of the measurement. After a single injection of a labeled amino acid the specific activity of the free amino acid changes so rapidly that both in theory and in practice the problem of calculating the synthesis rate becomes very difficult. To avoid this the method of constant intravenous infusion was developed, which produces an approximation to an isotopic steady state (Gan and Jeffay, 1967; Waterlow and Stephen, 1968a;Aub and Waterlow, 1970).

On the other hand, if the turnover rate is estimated from the rate of decay of radioactivity in tissue proteins, the results can be seriously invalidated by reutilization of amino acids liberated by catabolism of labeled protein. The difficulty can be overcome only by the use of labels that are not reutilized. For studies on proteins produced in the liver arginine labeled in the guanidine carbon atom fulfills this criterion, because as fast as labeled arginine is liberated by protein catabolism the guanidine C is removed in the urea cycle (Swick, 1958; A. S. McFarlane, 1963). This method cannot be applied to muscle, which has no urea cycle, but

218

J . C . WATERLOW AND G . A . 0. ALLEYNE

Millm-arcl (1970a) 11:~s shon-n that for nieasurmient of n i u ~ c l cprotein turnover c a r l ) ~ n a t e - ~ ~can C / lie used as a 1aI)cl which is yirtnally not rcutilized. I4C is taken up into the carboxyl groups of aspartic and glutamic acids b y the reactions of the Krcbs cycle. Wlien tliese aniino acids are lilwratcd in the free forin hy catabolism of protein, the lahelcd C atoms exchange with the carbonate pool of the body, which, because of its large size and rapid turnover, is virtually un1al)cletl. By this mctliod Millward showed that’ in gron-ing rats weighing 80-100 gm the fractional synthesis rate of mixed iiiusrlc protein T T ~ Sahout 11% per day, corresponding t o a half-life of 6 days, and the fractioiial catabolic rate was about GC/O per day, corresponding to a half-life of 11 days. The turnover rates of tlic myofibrillar proteins wcrc 2-3 times slower than those of the sarcoplasniic proteins. Tlicsc result:: agree very well with those obtained by coiistant intravenous infusion of lysinc (Waterlow and Stephen, 1968a), or of glycine (Garlick, 1969). Thc constant infusion method is essentially a measure of syntliesis rate ; n i t h botli amino acids it gave idcnticnl half-lives for mixed muscle protein, of 5-7 days. It as satisfactory to find this agreement 1)ctw-ccn int1el)cndeiit methods, bccansc the concept of muscle as a mohile protein rcserw makes it all the more important that cstiniates of its turnover ratc should he correct. e. Changes in the Distribution of Protein Synthesis. *4ltIiough the total rate of protein synthesis inay be unchanged in protein depletion, this docs not incan simply the preservation of the status quo. I n tlie 1960 revien- cxperiinciits were dcscrihed which suggested that in protein deplctioii “there is a concentration of protcin synt.hesis in the iiiteriial organs at tlic expense of muscle ant1 skin.” Since then a number of studies have lieen publislicd xyliicli in a gciicrnl way support this idea ( c g , llurxmatsu ef nl., 1963; Gactxni e t nl., 1961), hut thc methods used were subject to the errors clescribctl in tlie previous scction. M’c bclieyc that the coilstant infusion iiicthotl giyw a better cstiniatc of tlie true rates of protein synthesis, although it also depends upon assumptioiis which may he in error, notably that tlie intracellular amino acid pool is liomogcneous iWaterlow and Stephen, 1968a) . Measurements made on rats h y constant intravenous infusion of l y s i ~ i e - ~showed ~C that low protein feeding for 3-10 days caused no reduction, or e w n a small increaac, in the fractional ratc of synthesis of mixed liver protein (Table X I S ) . There is an npliarent discrepancy liere n-it11 tlie wellknown fact, discussed in detail by l l u n r o (1964), that in the rat an immediate effect of protein deprivation is a substantial loss of protcin from the liver, u-liicli must result from a net difference hetween synthctic and catabolic rates. This is not brought out by the constant infusion method,

219

PROTEIN MALNUTRITION I N CHILDREN

TABLEXIX Synthesis Rates of Liver and Muscle Proteins in Male Rats under Different Dietary Conditions Measured by Continuous Intravenous Infusion of Lysine-1%' ~~

Synthesis rate (% of control) Conditions

Liver

Muscle

Starved 2 days Protein-free 3 days Low protein 3 days Low protein 10 days Low protein 10 days Low protein 5 weeks

105 113 110 125 165 80

69 65 50 48 56

+ insulin

a Data of Waterlow and Stephen (1968a). Reproduced by courtesy of the Editor of The Lancet.

because it gives an estimate of the synthesis rate only. Moreover, the experiments were done after 3 days or more of low protein feeding, and by this time adaptation of liver protein metabolism would probably be virtually complete. The point to be emphasized is that in the adapted state the synthesis rate of mixed liver protein was not decreased. By contrast, in muscle after only 3 days on the low-protein diet the synthesis rate of mixed protein had fallen by 50%. Preliminary results of Millward (1970b) obtained by the carbonate-14C method show that a short period of starvation or a protein-free diet caused a fall in the rate of protein synthesis in muscle with little change in the rate of catabolism. This applied both to the sarcoplasmic and to the myofibrillar fractions. It is of interest that, as in the case of plasma albumin (Section VI,B) rates of synthesis and catabolism varied independently. These findings support the concept of muscle as a kind of buffer which adjusts its protein metabolism to meet the needs of the body as a whole. I n the rat the experimental evidence suggests that the skin, which contains about 25% of the total body protein, also acts as a protein reserve (Waterlow and Stephen, 1966). Whether this applies in human beings we do not know, because no measurements of the turnover rate of tissue proteins have yet been made in man. Munro (1969) has advanced arguments to show that in a large animal such as man muscle metabolism makes a greater contribution to overall metabolism than in a small animal like the rat. f. Amino Acid Economy. I n Section VI,D,3,d amino acid recycling was mentioned as a source of error in the measurement of turnover rates. Its importance is wider than this, because it probably plays a very real part in the process of adaptation to low protein intakes.

220

J. C. WATERLOW AND G. A, 0. ALLEYNE

TABLE XX Half-Lives of Serum and Liver Proteins in the Rat, Measured with Arginine-IaC ~~

Half-life Sample Mixed serum-proteins Control Protein-depleted Liver proteins Control Protein-depleted a

in days

(T1iz)

True

Apparent

2.0 3.3

2.7 5.5

1.9 2.3

5.5 9.2

Data of Stephen and Waterlow (1966). Reproduced by courtesy of the Editor of

Nature.

I n the 1960 review (Waterlow et al., 1960) a distinction was made between “apparent” and “true” half-lives of proteins, the difference between the two being a measure of the extent of amino acid reutilization. By the use of uniformly labeled arginine-l*C it was possible to measure the 2 half-lives simultaneously for proteins produced in the liver (Stephen and Waterlow, 1966). The results are summarized in Table XX. They show that on a low protein diet the true half-life of mixed serum proteins was somewhat increased, while that of liver protein was not affected. I n both tissues the apparent half-life was lengthened, a finding indicative that there was more recycling of labeled amino acid. It is also possible to estimate the extent of amino acid reutilization by the constant infusion technique, which has the advantage that it can be applied to any tissue, not only to the liver. I n the steady state the proportion of amino acid reutilized is given by the ratio of the specific activities of the free amino acid in tissue and plasma after they have reached a plateau (Gan and Jeffay, 1967; Waterlow and Stephen, 1968a; Aub and Waterlow, 1970). From this type of measurement i t appeared that normally in rat liver about 50% of the amino acids used for protein synthesis are derived from protein catabolism. In muscle the extent of recycling is less: 10-30%. This method provides an estimate of the internal recycling within a particular tissue; it does not take account of the utilization of amino acids t h a t have been liberated by catabolism elsewhere in the body. The calculation depends also on the assumption that, within the cell, amino acids from the two sources-those which enter from the blood-stream and those derived from the breakdown of the cell protein-mix in a homogeneous pool, and this may not be true (Kipnis e t al., 1961). We found that the half-lives of liver and serum proteins measured by constant

PROTEIN MALNUTRITION IN CHILDREN

221

amino acid infusion were shorter than the “true” half-lives measured by the arginine method (Waterlow and Stephen, 1968a). This could result from “compartmentation” of intracellular amino acid. It is clear, however, that reutilization of amino acids is a normal process, particularly in liver. It is enhanced when protein supplies are short, and this may make a significant contribution to the conservation of amino acids, especially the essentials. Penn e t al. in 1957 measured the apparent half-lives of serum proteins with different labeled amino acids; their results indicate that the essential amino acids lysine and leucine were reutilized to a greater degree than the inessential alanine and glycine. This effect may contribute to the reduced concentration of essential amino acids in the plasma in protein deficiency. g . Adaptive Enzyme Changes. I n preceding sections three elements were identified in the response of the organism to a low protein intake: (i) Diversion of the pathways of amino acid utilization, so that a smaller proportion is degraded to urea and a large proportion incorporated into protein. Since urea formation occurs only in the liver, the primary site of this change must be the liver. (ii) A more economical use of amino acids, particularly by the liver. This effect is presumably a reflection of the previous one. (iii) A decreased rate of amino acid uptake by muscle and perhaps by other peripheral tissues. From the experimental work of the last few years we can suppose that at least the effects in the liver result from adaptive enzyme changes. The pioneer experiments of Schimke (1962) showed that in rats on low protein intakes there is a decrease in all the enzymes of the urea cycle. There is also a reduction in the activity of those enzymes which make NH2groups available for entry into the urea cycle-glutamic-pyruvic transaminase and glutamic dehydrogenase (Rosen et al., 1959; Muramatsu and Ashida, 1962; Harper, 1965). The former must play a key role if the theory of Felig et al. (196913) is correct, that alanine acts as a carrier of NH, groups from the periphery. At the same time the work of Spadoni and her group in Rome showed that in protein-depleted rats the activity of amino acid synthetases in the liver is more than doubled (Mariani et al., 1963; Gaetani et al., 1964). These two sets of enzyme changes would have just the effects which have been observed in the whole animal. There are a few observations in malnourished children which confirm that the results obtained in rats apply also in man. Glutamic dehydrogenase, unlike malic and lactic dehydrogenases, was found to be moderately reduced in the liver of malnourished infants (Waterlow and Patrick, 1954). Stephen measured the activity of amino acid synthetases and of the urea-cycle enzyme argininosuccinate lyase in biopsy samples

222

J. C. WATERLOW AND G . A. 0. ALLEYNE

TABLE XXI Activity of A m i n o Acid Synthetases and of Argininosuccinate-Lyase in Liver Biopsies of Malnourished and Treated Children"

Subjects

Synthetase activity: 18 children (pmoles P exchanged per mg protein per hr)

I Malnourished I1 Recovering I11 Recovered Significance of difference I and I1 I and I11 a

Argininosuccinate-ly ase activity: 11 children (pmoles urea per mg protein per hr)

1.44 1.04 0.91 0.05 0.01

> P > 0.02 > P > 0.002

1.06 1.31 1.46

P

NS =

0.02

Data from Waterlow and Stephen (1968b). By courtesy of the Editor of The Lancet

of liver from children before, during and after recovery from malnutrition (Stephen and Waterlow, 1968). The results are shown in Table XXI. The changes are in the same direction as those found in rats, but are not so great. Much less is known about the effect of protein depletion on enzymes of amino acid metabolism in muscle. The increase in branched-chain amino acid transaminases (blimura e t al., 1968) has already been referred to. Young (1970) points out tha t the total activity of glutamic-pyruvic and glutamic-oxaloacetic transaminases in muscle is several times greater than in liver. This again emphasizes the important role of muscle in overall amino acid metabolism. Gaetani e t al. (1964) measured the synthetase activity in muscle as well as in liver; they found that in protein-depleted rats the activity per unit weight was unchanged, but the activity per unit DNA was decreased. Thus synthetases were lost in parallel with the other soluble proteins of the muscle cell. Stephen (1968) confirmed tha t in protein-depleted rats there was no change in the synthetase activity per unit weight of muscle, but when the rats were refed the synthetase activity rose, reaching a peak after 6 days. During the same period the activity of the synthetases in liver was falling. I n all the experiments referred to the enzyme changes were measured after depletion periods of several days. However, when the protein intake is reduced the urea excretion falls very rapidly. We know of no observations of its time-course in the rat, but in the human infant adaptation to a new level of intake is complete after about 2 days (Chan, 1968), and in the adult after about 6 days. The question is whether the adaptive enzyme changes occur quickly enough for a cause-and-effect relation-

PROTEIN MALNUTRITION I N CHnDREN

223

ship to be accepted. This seems not to have been investigated directly, but there is no doubt that under appropriate experimental conditions large changes in enzyme activity can be produced in a few hours. For example, the data of Jost et al. (1968) show that 10 hours after a single feed of amino acids the activity of serine dehydratase in rat liver was increased 30-fold. It is apparent from the work reviewed by Schimke et al. (1968) and by Rechcigl (1968) that such increases in enzyme activity represent a new synthesis of enzyme protein. When the stimulus is removed, the activity decays very quickly because many enzyme proteins have halflives of only a few hours. The observations from Potter’s laboratory of diurnal fluctuations in the activity of liver enzymes in relation to feeding are of great interest in this context (Watanabe et al., 1968; Baril and Potter, 1968). They lead to the concept that changes in enzyme activity in the liver represent a normal and continuous process of adjustment to changes in the rate of amino acid supply. Wurtman et al. (1968) have suggested that diurnal rhythms of cortisol and insulin production may be responsible for the regular fluctuations which they have observed in plasma amino acid levels. Whether the alterations in enzyme activity are related to food intake or to these hormonal rhythms, one may suppose that they result in a periodicity of protein synthesis and urea formation which is obscured in the conventional type of nutritional study. It will be recalled that Ashworth’s observations (1969b) on the oxygen uptake of infants suggested that protein synthesis and growth may occur in bursts after a meal (Section V,A). The older work, showing that a complete amino acid mixture must be presented simultaneously if nitrogen is to be retained, fits in with this concept. It seems, therefore, that we should regard the enzymatic adaptations to low protein intakes as extensions of a normal response, in which the controls are set at a different level. h. The Control of Adaptation. Any attempt to explain the regulation of the adaptive response to low protein intakes must take account of the fact that different tissues, of which liver and muscle have been used as examples, react in different and complementary ways. We think it is premature to consider in any detail the possible role of the endocrines in this regulation, because the picture is too complicated. In infants with protein malnutrition, plasma growth hormone and cortisol levels are raised, and the rate of insulin secretion is reduced (Section V1,B). The known effects of these endocrine changes fit some parts of the metabolic picture but not others. A decreased rate of amino acid uptake by muscle is quite consistent with the effects of insulin deficiency (e.g., Castles et al., 1965). On the other hand, an increased level of circulating

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cortisol would be expected to promote amino acid catabolism in the liver and the production of amino acid catabolizing enzymes (Schimke, 1963; Schimke e t al., 1965; John and Miller, 1969). Presumably the initial stimulus to the process of adaptation, and the continuing stimulus by which the state of adaptation is maintained, is a reduced supply of amino acids from the gut. I n Section VI,D,l we discussed briefly the relation, or absence of re!ation, between changes in amino acid concentration and amino acid supply. Peraino and Harper (1963) have shown that after a protein meal the changes in amino acid concentration are much greater in the portal than in the peripheral circulation. Allison e t al. (1963) observed that in rats on a protein-free diet the changes in amino acid concentration were much smaller in muscle than in liver. Therefore the main site a t which amino acid concentration might be expected to exert a n effect is the liver. Are the adaptive changes in muscle brought about by the same stimulus? If so, they must be sensitive to much smaller changes in amino acid concentration. The studies of Young and Alexis (1968) on protein synthesis by muscle ribosomes in vitro show that they are affected by the previous diet of the animal in much the same way as liver ribosomes, but we have no information about the responsiveness of muscle protein synthesis in vivo. On the whole it seems to us most likely that it is controlled by some other signal, not by the amino acid A further difficulty in trying to make any unitary hypothesis about the regulatory mechanisms is t h a t within a tissue different proteins respond differently to the same stimulus. The use of naive phrases such as “liver protein turnover” can lead to serious error. One example has already been given: the sensitiveness of albumin synthesis to the level of protein intake, and the insensitiveness of y-globulin synthesis. The differential effect of the same stimulus is particularly clearly shown in relation to enzyme synthesis in the liver. For example, protein restriction depresses the activity of the catabolic enzyme serine dehydrase, and increases the formation of the synthetic enzyme 3-glycerophosphate dehydrogenase (Fallon et al., 1966). The experiments of Jost e t al. (1968) provide another very striking illustration: 10 hours after an amino acid meal there was a 30-fold increase in the uptake of labeled amino acid into the protein of serine dehydrase, but no increase a t all in the uptake into mixed liver protein. Observations such as this make it clear that a t the cellular level the mechanism of regulation must be very complex. ‘Since this review was written, recent experiments by Garlick (1971), in which the rate of muscle protein synthesis in rats was measured by constant infusion of t~rosine-’~C, have shown that the synthesis rate is affected by the preceding pattern of feeding, and falls to a minimum 18 hours after a meal. This does not, of course, prove that the controlling factor is the amino acid supply.

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Finally, one aspect of the protein of regulation which is still entirely mysterious is the mechanism by which catabolic rates are controlled. The question has been discussed briefly by Schimke et al. (1968). It is clear that protein catabolism is regulated independently of anabolism, and it is generally accepted that catabolic rates behave as first-order reactions, but this is almost the whole extent of our knowledge. The problem, however, is one of great importance for a better understanding of protein malnutrition. i. Conclusion. It is difficult to define adaptation. We hare used the term in a teleological sense, to mean a metabolic adjustment to altered circumstances compatible with normal function. This is not very satisfactory, because the word “normal” begs the question. Nevertheless, everyone would admit that a person who has changed from a high to a low protein intake, and who has come into nitrogen equilibrium a t the new level, has to Qhat extent adapted successfully. The controversial point is whether N balance is a sufficient criterion of normality; whether the subject is a t any disadvantage because he has suffered a loss of protein from the body during the process of becoming adapted. The origins and significance of the so-called “labile protein” (defined as protein which is easily lost and easily restored) were discussed in detail by Munro (1964). The question is of great practical importance, because if the loss of labile protein is a disadvantage, the whole basis on which human protein requirements have been estimated is destroyed (see Section 111). The only way of getting out of the difficulty, we believe, is by a better understanding of the mechanisms of adaptation. We draw the conclusion from the evidence available so far that the loss of labile protein results from a lag in the process of adjustment from one metabolic Ievel to another. There is no reason a priom to suppose that one level is “better” or more “normal” than the other; they are simply different. On this interpretation, it is misleading to think of labile protein as a kind of reserve which ought to be maintained a t a high level.

VII. CONCLUSION Some aspects of protein malnutrition have not been covered in this review, and others have been dealt with in a very cursory way. The scope has expanded very greatly, and we are well aware that much important and relevant work has been neglected. It is clear that in the last ten years a great deal of new knowledge has been gained about the effects of protein deficiency and protein-calorie imbalance in human beings. It will be obvious from the papers cited that a large part of this new knowledge has come from developing countries, where the existence of malnutrition has acted as a powerful stimulus to basic research. At the same time there has been an increased awareness of the problem in developed

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countries, and a greater output of experimental work which is consciously related to it. I n this respect the aim expressed a t the end of the 1960 reviex (Waterlow et al., 1960) is being achieved, of informing “those who are primarily experimentalists of the nature and severity of the problem, so that their researches may contribute more directly to its solution.” As a result of this cxpanding interest, the study of protein malnutrition is making an increasing contribution to human biology and medical science in general. This is well seen in relation to growth, endocrine interactions, and disturbances of electrolytes and body fluids. It seems that just as the disease diabetes has been a powerful stimulus t o the better understanding of carbohydrate and fat metabolism, so protein malnutrition is beginning to stimulate work in the relatively neglected field of protein and amino acid metabolism in man. However, we have to face the question raised in the introduction: in what way are these advances in knowledge contributing to the practical problem, and what is the scientific strategy for the future? There haye undoubtedly been improvements in the treatment of severe malnutrition and a reduction in the mortality; but, this is not, a matter for very great satisfaction when we are dealing with an essentially preventable disease. It is now more than ever apparent t h a t the major problem is not treatment but prevention. Scientific interest is concentrated less on the acute and severe deficiency state, and more on the mild and marginal forms of protein malnutrition. We cannot. define or recognize these early stages without more sensitive and specific biochemical measurements. Whitehead (1969b) in his Drummond Lecture, referring to biochemical tests of subclinical protein deficiency, said with truth: “Any abnormality must first be shown t o have significance or potential significance in terms of essential bodily function. I n other words, subclinical malnutrition can only he diagnosed in terms of malfunction.” This brings in the epidemiologist, to trace the connection between nutritional patterns and disease ; the physiologist ; and the psychologist. The next ten years will surely see a great extension of functional and long-term studies of this kind. These, however, are not. diagnostic tools. There are two factors in Whitchead’s equation : biochemical abnormality and functional change. The interpretation of the biochemical findings depends upon an understanding of the metabolic adjustments to different levels of protein intake. Only in this may is it possible to distinguish between changes that are trivial and changes that are significant from the metabolic point of view. Perhaps, therefore, the most important contribution of the intensive work on protein malnutrition, and one which will have very great prac-

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tical consequences in the long run, is that it is forcing a reappraisal of the concept of ‘‘normal” in nutrition. “In protein malnutrition there is no sharp line between health and disease” (Waterlow et al., 1960). Ten years ago the word “adaptation” was hardly mentioned in this context; now it recurs again and again. Even though i t is not defined with any precision, its general meaning is clear enough: if man can adapt successfully to different patterns and levels of food intake, i t is then entirely arbitrary to select as “normal” the characteristics of one particular group, or to suppose that the standards of one particular society are in any way universal. We believe, therefore, that in this field the scientific strategy for the future is the study of the nature and mechanism of adaptive processes. This kind of understanding will provide the basis for rational standards and rational targets in nutrition.

ACKNOWLEDGMENTS We wish to record our thanks to the members of the Wellcome Trust Working Party held in Jamaica in May, 1969, who allowed us to make use of their data and to draw on their experience. We are also grateful to members of the staff of the Tropical Metabolism Research Unit for help with many parts of this review, particularly H. Flores, W. P. T. James, and D. Picou.

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