Development of Intestinal Enzyme Systems, and Its Relation to Diarrhoea

Development of Intestinal Enzyme Systems,and Its Relation to Diarrhoea A. HOLZEL, M.D.

In the ancient civilisations of the East medical men were already preoccupied with infant feeding, and the Papyrus of Ebers, written about 3500 years ago, contained advice on measures helpful in increasing the supply of milk in the breast of the nursing mother. But right through the millenia wet nurses were employed when lactation in the mother failed. Until three centuries ago physicians were often more concerned with the character of the wet nurse than her physical suitability or the composition of the milk. It seems that in Europe only in the course of the sixteenth and seventeenth centuries was cow's milk widely used as an alternative to human milk. English physicians who wrote on handrearing of infants in the eighteenth century recommended the introduction of cereals and sugar as additives to the cow's milk feed of the newborn. For the past 200 years the amount, type and number of carbohydrates have varied with the medical fashion and knowledge of the day. Many of the proprietary milk preparations available today for infant feeding have milk sugar added to them to bring their level close to that of human milk. Lactose thus provides in the breast-fed and in the artificially fed baby over 40 per cent of the total caloric supply. A number of formulae contain sucrose or dextrose-maltose as additional sugars. As the child grows older the amount of carbohydrate in his diet increases, and the percentage of total calories from carbohydrate rises to approximately 50 per cent. Dietary carbohydrates are largely ingested as disaccharides and polysaccharides. The polysaccharides starch and glycogen are hydrolysed by salivary and pancreatic amylase to the disaccharides maltose and small quantities of isomaltose. The disaccharides with which the human intestinal mucosa has to deal are thus lactose, sucrose, maltose and isomaltose. It is therefore surprising that until a few years ago little interest was taken in the physiology as well as the pathology of disaccharide absorption.

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INTRAMURAL LOCATION OF CARBOHYDRASES

It has been known for a long time that the disaccharides have to be hydrolysed into their component monosaccharides before being metabolised. It was believed that the disaccharidases, lactase, sucrase (invertase) and maltase, were secreted by unspecified cells of the intestinal mucosa into the lumen of the gut where hydrolysis occurred. But during recent studies of digestion and absorption in the human intestine it was found that the succus entericus had practically no disaccharidase activity (Borgstrom et al., 8 Dahlquist and Borgstrom12). After a test meal, for instance, the rapid disappearance of the sugars from the gut and rapid rise of the blood glucose levels could not be accounted for by the hydrolytic activity of the intestinal content. Fridhandler and QuasteJ22 demonstrated that perfusion of a sucrose solution through the lumen of an isolated surviving guinea pig intestine leads to the appearance on the serosal side of glucose, fructose and small amounts of sucrose. After comparison with glucose and fructose perfusion they arrived at the conclusion that the hydrolysis of sucrose takes place within the intestinal tissue. Since disaccharides in the circulation are excreted by the kidneys largely unhydrolysed, and since disacchariduria is uncommon, it could also be inferred that the sugars pass from the lumen into the cell of the intestinal mucosa where hydrolysis occurs.

INTRACELLULAR LOCALISATION OF DISACCHARIDASES

Miller and Crane,38 after having separated the brush border from the rest of the intestinal mucosa of the hamster, were able to show that it contained all the enzyme activity as compared with a homogenate of the whole mucosa. They therefore claimed the brush border as the structure containing the hydrolytic enzymes. Doell and Kretchmer,17 by fractionating centrifugally homogenates of rat and human intestinal mucosa, obtained four principal components: a nuclear, a mitochondrial, a microsomal and a soluble fraction. The specific beta-galactosidase (lactase) action was always associated with the particulate fraction, and it was suggested that they were linked with the microsomes. Using histochemical staining techniques for bringing into view glycosidase activities, Rutenburg et a1. 43 . 44 thought to have proved their presence in the cytoplasm of the epithelial cells, whilst Dahlquist and Brun,13 using different histochemical methods, found the stain accumulating in the granules of the cytoplasm. No irrefutable information is yet available to link glycosidase activity definitely with any particular intracellular structure. Just as there is some uncertainty as to the exact localisation of the disaccharidases within the cell, there is also some question as to their

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distribution along the consecutive parts of the small intestine. Recent animal studies suggest that there are some species differences also in this aspect. In the rat the lactase activity is stronger in the middle part of the small intestine than in its proximal or distal regions. Maltase activity is uniformly distributed, whilst sucrase (invertase) and isomaltase activities are strongest in the proximal areas (Dahlquist15 ). According to investigations by Auricchio and his colleagues9 on human intestinal mucosa obtained at operation or by intestinal biopsy in the adult, it seems that sucrase, isomaltase and lactase show a lower degree of activity in the first part of the duodenum than in the remainder of the duodenum. In the first part of the jejunum and the last part of the ileum disaccharidase activities appear to be of the same order of magnitude. In fact there is every likelihood that the glycosidases are active through the entire length of the small intestine with the exception of the first part of the duodenum.

FOETAL AND NEONATAL INTESTINAL DISACCHARIDASE ACTIVITY

The prenatal and postnatal development of disaccharidase activities is not only of theoretical interest, but has also its practical implications in the management of very small premature infants. Langstein and Meyer,32 in their book on the physiological metabolism of the infant, stated that lactase activity is already present in the newborn, but often absent in premature babies. This they saw as the reason for the frequently encountered lactosuria in the immature infant. They also mentioned that maltase and invertase activities can be ascertained early in foetal life, whilst lactase activity becomes manifest rather later. Auricchio, Rubino and Murset,6 studying intestinal glycosidase activity in the human embryo, foetus and newborn, found these activities present by the third month of intrauterine life, when the intestinal mucosa is already differentiated. All alpha-disaccharidase activities (namely, those of the maltases and sucrase) reach a maximum during the sixth or seventh month of intrauterine life, whilst the beta-glycosidases (namely, lactase and cellobiase) develop less rapidly during antenatal existence and reach their peak at the end of normal gestation in the perinatal period. Premature infants have thus a low level of betagalactosidase activity which rises rapidly during the early period of extrauterine life independent of milk intake. In some mammals such as rat, pig, cow, lactase activity is highest in the newborn and decreases gradually to its lowest level in the adult specimen, whilst in man activity remains high, provided it has not been reduced by external factors. In rats and pigs invertase activity is not

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evident at birth, but rather later in their development. Doell and Kretchmer18 reported that injection of young rats with hydrocortisone caused precocious appearance of invertase activity in the small intestine; it is assumed that hydrocortisone does not simply hasten tissue maturation, but must act by another, still unknown mechanism.

SPECIFICITY OF THE INTESTINAL CARBOHYDRASES

Pig and rat intestinal disaccharidases have been studied by heat inactivation and anion exchange, and at least six different enzymes have been isolated. Five separate maltase activities have been recognised by Dahlquist14 and Semenza and Auricchi0 45 in their recent investigations into this problem. From their studies it becomes clear that two maltases (I and II) exert their hydrolytic action on a common substrate, namely, maltose, whilst maltases III and IV have sucrase activity and maltase V ( isomaltase) hydrolyses isomaltose. Doell and Kretchmer were able to show that the intestinal mucosa of the rabbit and the rat contained two beta-galactosidase activities, one attached to the particulate fraction which hydrolyses two substrates, lactose and ortho-nitrophenyl-,B galactoside, whilst the soluble enzyme splits only the latter. Semenza and Auricchi0 45 suggested, on the basis of chromatographic separation, also the presence of two lactases in the human intestinal mucosa. Both lactases are equally active in splitting cellobiose, a disaccharide resulting from the breakdown of cellulose and consisting of two molecules of glucose with a 1-4 ,B linkage as it exists between the galactose and glucose molecules of lactose.

SUGAR TRANSPORT

Assuming that the disaccharide-splitting enzymes are intracellular, the question as to how these sugars enter the mucosal cells remains to be answered. Maybe by diffusion, i.e. from a point of higher to a lower concentration across the cell membrane. The rapid hydrolysis of the disaccharide within the cell might continuously keep the concentration difference between it and the lumen. But there might also exist an active carrier system as it operates in the absorption of glucose. The latter is transported against the concentration gradient, i.e. from a lower to a higher concentration. A definite structure has been postulated for actively transported sugars. They have got to have a pyranose ring, an oxygen bridge between the first and fifth carbons, an aldehyde group at the first carbon, and a free hydroxyl group in the second position. Another essential requirement is the presence of sodium ions on the membrane of the mucosal cells facing the lumen. The driving force is

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regarded as a form of biological pump. Adenosine triphosphate (ATP) acts as the instantaneously available energy source of the cell which provides the power for the pumping action. The cell membrane contains a specific enzyme which releases a large amount of energy by splitting a molecule of inorganic phosphate off ATP. This adenosine triphosphatase is dependent upon· sodium for its function and is specifically inhibited by digitalis, as is the active transport of sugar. The pump part of the active transport is then an ATP-adenosine triphosphatase sodium complex with a specific carrier for sugar. Semenza et al. 46 have demonstrated the sodium activation of human intestinal sucrase, from which one might deduce a similar mechanism to the sodium ion glucose pump being responsible for the transport of sucrose. By analogy one might extend it also to apply to the other nutritionally important disaccharides until further evidence becomes available.

SUCROSE-MALTOSE MALABSORPTION

Since the publication by Weijers et al. 48 of three cases of sucrose and maltose malabsorption, well over 30 similar cases have been recorded in the paediatric literature. Some of the studies (Prader et al.,40 Gorouben et aJ.25 and Burgess et a1. 9 ) related to groups of patients, thus indicating that this disorder is by no means uncommon. The clinical and laboratory data available are adequate to provide a well defined nosological entity. The onset of the disturbance depends on the time of introduction of the offending sugar into the infant's diet. In the breast-fed baby this will coincide with the start of weaning, and in the bottle-fed child with the addition of sucrose and starch to the formula used or the regular administration of sugar water between feeds. Diarrhoea is the most characteristic manifestation, which, though not always very severe, is nevertheless intractable and ceases only when the relevant disaccharide is removed from the formula. The chronic diarrhoea leads to malnutrition of varying degrees. In the very young infant the water loss may be heavy enough to necessitate intravenous fluid replacement. The stools are loose and watery, of low pH due to the accumulation of relatively large quantities of lactic and low-molecular weight organic acids. The normal faecal pH is between 7 and 8, in the fermentative diarrhoeas between 5 and 6. A useful screening test therefore is the determination of faecal pH. Occasionally refusal of feeds and abdominal distension have been noticeable features. There is as a rule no steatorrhoea, no abnormal mellituria, a normal xylose absorption and excretion and the microscopical appearance of the intestinal mucosa within bounds of the normal.

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Auricchio et aU were the first to point out that sucrose malabsorption in their patients was always associated with isomaltose malabsorption, which led to poor tolerance of starch and dextrin mixtures, often used as a carbohydrate addition to infant feeding formulae. Intestinal symptoms due to these carbohydrates were slight, since isomaltose forms only a small part of starch hydrolysis. These observations have subsequently been confirmed by a number of investigators (Anderson et al.,! Nordio and Lamedica,39 Burgess et al., 9 Bertrand, 7 Launiala et aJ.33). The course of the disorder in the untreated patient varies appreciably. In some, spontaneous improvement seems to occur after many years; in others the manifestations may persist into adult life. The diagnosis is based on the result of absorption tests with the suspected sugar and the component monosaccharides. Blood glucose levels after a sucrose load are compared with those following a tolerance test with a glucose-fructose mixture. It is important to remember that these tests should not be carried out during an acute phase of diarrhoea, that adequate quantities of glucose should be given for several days before the test, and that the result should be reproducible. Since it is difficult to obtain sufficiently large amounts of isomaltose for tolerance tests, Prader et al. 41 introduced as an alternative palatinose, an artificial disaccharide which consists of one molecule of glucose and one of fructose, but with the same 1-6 a linkage as isomaltose, and is therefore split by isomaltase (palatinase). After the sucrose loading test the faeces contain relatively large quantities of the sugar, and their pH will fall below 5. This is due to the accumulation of lactic acid and other low-molecular weight organic acids in the stools and can be used as a diagnostic criterion. According to Weijers and van de Kamer,50 the excretion of more than 50 mg. of lactic acid per 100 gm. of faeces indicates the presence of pathological fermentation. This determination can be undertaken on a random portion of faeces according to the method of Long.37 The diagnosis may be further substantiated, where feasible, by qualitative or quantitative estimation of enzyme activity in intestinal biopsy specimens (Auricchio et al., 5 Anderson et aI., l Burgess et al. 9). Dahlquist,14 on the basis of his investigations, assumes that the human isomaltase and invertase activities are exerted by two different enzymes. It would therefore seem that two enzymes are absent in the same disorder, and this poses an interesting genetic problem, since it contradicts the so far accepted theory of "one gene, one enzyme." Three hypotheses (Prader41 ) have been advanced to explain such a finding: ( 1) The presence of a genetically determined inhibitor blocking both enzymes; (2) that a polypeptide chain genetically defective (Wyngaarden51 ) and common to both enzymes abolishes their activity; ( 3) that a regulator gene common to both enzymes underwent mutation. None of these hypotheses is particularly attractive.

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Launiala et al. 33 put forward as an alternative explanation the concept of one major a-glucosidase, which can hydrolyse more than one substrate, and that a genetically determined abnormality of its structure may affect its various enzymatic activities in different degrees. The mode of inheritance of the sucrose-isomaltose malabsorption is, in spite of the rapidly increasing number of recorded cases, still problematic. Prader et aJ.4° and Auricchio et aJ.3 suggested that the condition might be inherited as an autosomal dominant because in one family the mother of three patients also suffered from typical sucrose malabsorption; in other families the father or mother of investigated children gave a history of persistent diarrhoea which eventually improved and later responded normally to sucrose loading tests. In one of the parents examination of the intestinal biopsy (Prader et al. 41 ) yielded normal disaccharidase activities. In Anderson'sl report the father of the patient had suggestive symptoms and a flat sucrose tolerance curve. Gorouben et al.,25 Rey et al.,42 and Burgess et al. 9 could find no evidence for dominance of the trait and believe it to be transmitted as an autosomal recessive. Several authors had noticed the familial occurrence of sucrose malabsorption, and Weijers et al.,48 Auricchio et al.,3 Gorouben etaP5 and Burgess et aJ.9 found two siblings affected. Consanguinity of the parents was noted only in Lelong and Alagille's45 case cited by Rey et aJ.42 There seems to be no difference regarding the sex incidence.

HEREDITARY LACTOSE MALABSORPTION (HEREDITARY ALACTASIA)

In 1959 Holzel, Schwarz and Sutcliffe27 published the first observation of two siblings with lactose malabsorption. Since then we have studied three further infants with deficient lactase activity, and nine additional observations in the literature provide a characteristic clinical picture. The disorder is likely to be more severe in the purely breast-fed and in the artificially fed infant, to whose cow's milk formula lactose is added. Most of the proprietary dried milks contain additional lactose. The clinical manifestations become apparent within a few days after birth, as soon as feeding is well established. Affected babies cry persistently, partly because of hunger, since they lose half their total caloric supply, and partly because of painful intestinal colic. Diarrhoea is one of the earliest signs to appear. The stools are frequent, watery, often green and voided with force-almost explosively. They are frothy and sour-smelling, and the pH is between 4.5 and 6. The high content of lactic and other organic acids in the stools-a result of bacterial lactose fermentation-leads to severe excoriation of the buttocks which resists any form

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of treatment until the underlying disorder is dealt with. In all the cases examined there was no lactose in the urine and only traces of it in the stools, except after loading tests. Random blood sugar estimations give low readings for as long as the patient receives his carbohydrates in the form of lactose. There is no steatorrhoea, and xylose absorption tests give normal values. The degree of malnutrition varied considerably in our five cases, and this depended to some extent on the maternal attitude. Babies who were fed whenever they appeared hungry, even every two hours, were in much better shape than those of mothers who rigidly adhered to a predetermined feeding schedule. The last of our patients was 2 pounds below birth weight at the age of five weeks. Their appetite is generally excellent, they are prepared to take large feeds, and often will ingest up to 100 calories per pound per day-twice the normal amount-without gaining weight. Vomiting, though rare, is occasionally present from the start in the more severe cases (Holzel,28 Durand21 ). The explanation of this symptom is by no means easy. It is therefore not surprising that these infants are often diagnosed as suffering from an infective form of gastroenteritis and treated accordingly. During the period that they receive a glucose electrolyte mixture they improve rapidly, but the diarrhoea returns with the commencement of milk feeding. Diagnosis. Once the abnormality has been borne in mind, the diagnosis is not difficult. Lactose loading tests do not lead to any increase in blood glucose level, whilst glucose or glucose plus galactose absorption tests produce a corresponding rise of the blood sugar curve. The young age of our patients made intestinal biopsies impracticable. Figure 24 shows the blood glucose levels in four patients following lactose loading tests.

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Course and Prognosis. With weaning, the milk intake decreases and diarrhoea seems to abate in the untreated patients. But the enzyme defect seems to be permanent, and large quantities of milk or lactose are likely to provoke diarrhoea even in the older child (Holzel et aJ.27 and unpublished data; Jean, as quoted by Durand;21 Cozzetto10 ). Genetics. The occurrence of the disorders was noted in siblings by Holzel et al.,27 Weijers and van de Kamer4 9 and Durand. 21 No instance of consanguinity of the parents has so far been published. Among the 14 cases recorded there were 11 males and three females (our own series consists of one girl and four boys), a circumstance which suggests some preponderance of the male sex. Yet the small number of cases recorded does not permit any valid conclusions to be drawn from this observation. In view of the recessive character of most inborn errors of metabolism, one might expect a similar mode of inheritance to operate also in this particular example of deficient enzyme activity. Lactose tolerance tests in the parents of three of our patients did not reveal any abnormality in the hyperglycaemic response. Treatment. The state of malnutrition in this disorder is the result of the caloric deficit that arises when the only sugar in the formula is lactose. Weight gain can be easily achieved by introducing other carbohydrates. The vomiting, the diarrhoea and the skin manifestations may necessitate a reduction of milk sugar content by replacing the usual proprietary brand by a special low lactose milk. D.Th. was the fourth child of healthy parents; the older children were well and developing satisfactorily. Birth weight was 8 pounds 4 ounces. At the age of six weeks he was admitted to hospital because of an acute respiratory infection which had been present for three days and gradual loss of weight over several weeks in spite of insatiable hunger and feeding practically all day. There was no vomiting, and the stools were loose, but not frequent. Weight on entry was 7 pounds 8 ounces. On examination he was noted to be a crying, hungry and pathetically thin baby, with signs of bronchitis and bilateral catarrhal otitis media. There were no other abnormal findings. Laboratory investigations gave the following data: white blood cell count 12,000 per cubic millimeter; differential count, neutrophils 23, lymphocytes 68, monocytes 8, eosinophils 1. The erythrocyte sedimentation rate was 3, the hemoglobin level H.8 gm. per 100 mI. There was slight anisocytosis and poikilocytosis of the red cells. Tryptic activity in the faeces was normal in dilutions of 1:5 to 1:80. The faeces had a pH value of 5, with no Giardia lamblia, fat globules or pathogens. The urine had a pH value of 6.1 and was negative for protein and reducing substances; microscopically, it contained 2 plus calcium phosphate crystals. On culture there was a light growth of faecal contaminants. Two-dimensional amino acid chromatography showed an amino acid pattern accepted as normal for the age. On treatment with oxytetracycline the respiratory infection cleared rapidly, but the infant did not gain weight in spite of a daily intake of 28 to 32 ounces of a half-cream dried milk formula with a lactose content of 7.1 per cent. Calorically, this was far in excess of his requirements. Lactose absorption studies showed practically no rise of blood glucose levels on repeated testing (Fig. 25). The results were similar on administration of 3 gm. of lactose per

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kilogram of body weight as well as on 2 gm. per kilogram of body weight in 10 per cent solution. A glucose loading test produced a normal response. Introduction of low lactose milk preparation led to rapid gain in weight, but after an intercurrent gastroenteritis it was badly tolerated (Fig. 26). Replacement of the low lactose milk by the sugar, egg, cereal, margarine mixture used in

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the treatment of galactosaemia, combined with a milk-free weaning diet, was followed by a satisfactory increase in weight as well as a striking improvement in the infant's general condition.

LACTASE DEFICIENCY IN THE ADULT A syndrome, not unlike that in young infants with deficient lactase activity, has been described in nine adults by Hammerli et al. 26 These are patients whose milk intolerance developed later in life and who within two hours after milk intake are subject to abdominal discomfort, flatulence, colicky pains and watery diarrhoea. On exhaustive investigation, including intestinal biopsies, it could be proved that in these patients lactase activity was almost completely absent. Removal of lactose from their diet would bring them rapid relief. In view of the history that they could tolerate milk well in childhood, one has to accept that the patients have subsequently lost their ability to digest and absorb lactose. Cuatrecasas et al.11 found that 55 per cent of 60 male and female adults were deficient in lactase activity and intolerant of milk and lactose. There is little evidence at present to attribute this loss of function to a reduced intake of milk, just as there is hardly any support for the opposite belief that lactase activity can be increased by a process of enzyme adaptation after prolonged administration of large quantities of lactose. Only one study (Girardet et al. 23 • 24) in adult animals (rat) demonstrates that a fourfold increase in specific hydrolytic activity of lactase has followed the prolonged feeding of lactose.

HEREDITARY INFANTILE LACTOSE INTOLERANCE (WITH LACTOSURIA)

In 1958 Durand19 published the case history of a 15-month-old girl who had died of chronic enteritis, and had had signs of renal acidosis, proteinuria and lactosuria. The findings at autopsy were atrophic enteritis, atrophy of liver and adrenals and degeneration of the convoluted tubules. Since then approximately a dozen cases have found their way into the paediatric literature under a variety of titles such as idiopathic lactosuria and amino-aciduria, idiopathic lactosuria, congenital lactose intolerance, and others. This is an extremely serious illness which leads to renal and hepatic involvement and haemorrhagic manifestations and ends fatally unless there is timely and complete withdrawal of lactose from the diet. The lactose intolerance, however, is not permanent, and after intervals varying from several months to two years on a lactose-free diet these children can digest normal quantities of milk without any ill effect. A summary of the clinical data gives the following picture. The disease begins in the neonatal period, often in the first or second week of life, irrespective of breast or bottle feeding, with diarrhoea, vomiting, refusal of feeds and gross malnutrition. The abdominal distension, the loss of subcutaneous fat and the muscular hypotonia produce often a coeliac-like syndrome. The renal disturbances observed were acidosis, hypercalcaemia,

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BLOOD (LACTOSE TOL. TEST)

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Lactose lactose

Galactose Galactose

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Figure 27. Plasma sugar chromatogram whilst the patient was on a milk diet shows lactosaemia following a lactose tolerance test.

proteinuria, amino-aciduria and increases in blood urea. The lactosuria, though sometimes accompanied by sucrosuria or glycosuria, is the most constant feature of this syndrome and probably its guiding sign towards pathogenesis and aetiology. It was the lactosuria and a limited rise of the blood sugar curve in response to a lactose tolerance test that caused Durand to propose as pathogenesis for the syndrome deficient lactase activity-a suggestion which has been widely, but rather uncritically, accepted. The systemic manifestations have been attributed to the lactosaemia, which is of a relatively minor degree. Lactosuria and lactosaemia can occur in perfectly healthy newborns, in premature babies and in lactating women without any signs of ill effect. There is no evidence at present that the lactosaemia and the resulting lactosuria can cause the organ damage reported in patients with congenital lactose intolerance; even more difficult is it to explain its relation to the pyloric stenosis observed in the cases of Darling et al,16 and Inall and Burkinshaw. 31 Flat blood sugar curves after a lactose absorption test were found in

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only two out of eight cases in which it was carried out; in the other six, increases in blood sugar levels from 26 to 108 mg. per 100 ml. could be registered. In our experience an interval as short as three weeks on a low lactose diet altered the tolerance to such an extent that a single loading dose of lactose was absorbed in a normal manner with a satisfactory rise in blood glucose (Fig. 27) ~ Comparison of a plasma sugar chromatogram during the first lactose tolerance test when the patient was still on a milk diet with the second carried out after three weeks on the low lactose food clearly demonstrates the disappearance of the lactosaemia (Fig. 28). Continued administration of lactose in very small quantities, however, leads to rapid deterioration in the patient's condition. The rapid recovery of absorptive capacity (three weeks) is in our opinion difficult to reconcile with a hereditary defect of enzyme activity. Furthermore, the clinical pattern in this condition differs strikingly from the other forms of disaccharide malabsorption, in which disacchariduria is not a feature. The self-limited nature of the disease is another reason against the lactase deficiency hypothesis. The sucrosuria and glucosuria occasionally associated with the lactose excretion, as well as the rarer steatorrhoea, support the view that lactose may well exert a toxic effect SPECIMEN MARKERS

BLOOD (LACTOSE TOL. TEST)

Lactose Lactose

Galactose Galactose

Glucose

Figure 28. Disappearance of the lactosaemia during a repetition of the test after 23 days on a low lactose formula.

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on the intestinal mucosa. The pathogenesis, however, seems to us unknown, particularly regarding the damage to the various parenchymatous organs, and its clarification will depend on further studies. Consanguinity of the parents in Durand's19 case and of the great grandparents in Darling's patients, as well as the occurrence of the disorder in siblings, suggests the implication of genetic factors, probably of a recessive nature. Treatment of lactose intolerance requires as strict an adherence to a low lactose diet as applies in galactosaemia. Apart from possibly immediate requirements of rehydration, dietary treatment is satisfactory and will in time lead to complete recovery. The prognosis depends on the early recognition of the disease and institution of treatment. Delay in diagnosis may have disastrous consequences. Five out of the 12 known patients succumbed to their illness.

MONOSACCHARIDE MALABSORPTION

A newly recognised inborn defect of intestinal monosaccharide absorption adds to the difficulties of the differential diagnosis in disaccharidase deficiency. Lindquist and Meeuwisse36 described in 1962 a form of chronic diarrhoea in a female child, born of consanguineous parents, which started on the fourth day of life. The infant was breast-fed and in spite of taking normal amounts of milk was losing weight, became severely dehydrated and required parenteral rehydration. For as long as the infant's food contained lactose, dextrin-maltose or sucrose the diarrhoea persisted; when, however, these sugars were replaced by a formula with fructose as the main carbohydrate, she started to gain weight, and her condition improved rapidly. The frequent, loose stools were of low pH and contained large quantities of sugar which, however, were not the dietary disaccharides, but their component monosaccharides. These findings, as well as sugar balance studies, suggested to the authors that the hexoses formed by intracellular hydrolysis of the disaccharides may have diffused back into the lumen of the gut where they caused a partly fermentative and partly osmotic diarrhoea. Laplane et a1. 34 reported independently in the same year similar observations in two newborn infants in whom diarrhoea developed shortly after birth when they were offered sucrose water or a lactose-containing formula. The babies took their feeds well, seemed thirsty, but rapidly lost weight. Within a few days they became dehydrated and appeared to be suffering from painful colic; abdominal distension developed. Surprising was the large volume of the liquid stools, which contained fine floccules of mucus, a fact also noticed by Lindquist and Meeuwisse. Lactic acid was present in abnormally high concentrations. The pH ranged between

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4 and 5. Analysis of the faecal sugars showed only traces of lactose, but glucose and galactose could be detected in appreciable amounts. Anderson et aJ.2 studied another example of the same disorder. The patient was a seven-day-old baby girl who was admitted to hospital because of severe diarrhoea and moderate dehydration. The parents of the child had already lost one infant of a diarrhoeal disease which had started on the third day of life and persisted until the fatal end at the age of three months. The pattern of the disease was practically identical with that encountered by the French and Swedish authors, and once more lactose malabsorption seemed the most likely diagnosis. But glucose and galactose were found to be poorly absorbed and largely excreted in the stools. Among disaccharides, sucrose was by far the best tolerated, probably because fructose, one of the component hexoses, can be satisfactorily absorbed. Urine samples, examined whilst the patient was still receiving cow's milk, showed the presence of small quantities of glucose as in the patient of Lindquist et al. Extensive balance studies with a number of actively absorbed sugars, such as glucose, galactose and 3-0 methyl glucose, indicated that only they were affected by the disordered metabolism, whilst sugars passing through the cell membrane by diffusion, such as fructose, were utilised normally. Estimations of carbohydrase activities in intestinal biopsy specimens (Anderson et aJ.2) gave normal values. Although the pathogenesis of the disorder remains unknown, it is fair to assume that there may be a genetically determined defect in the sugar transport mechanism. The familial incidence, already manifest in the available data, lends further support to this view. Without a distinct clinical pattern the differentiation of this condition from disaccharide malabsorption will have to be based on sugar tolerance test, analysis of urine and faeces for sugars, pH and lactic acid measurements, and finally on the assessment of trial diets.

INTESTINAL MALABSORPTION SYNDROMES

In addition to the congenital and probably hereditary deficient disaccharidase activity, malabsorption of sugar may follow a variety of insults to the intestinal mucosa. During the past three years we exaInined our patients with gluten-induced enteropathy. It was, of course, not surprising to find in a condition with such profound structural alteration of the intestinal mucosa a gross reduction of carbohydrase activity. In the florid phase of the disease all the enzyme activities were greatly diminished, but lactase more so than sucrase and the maltases. During the recovery stage, however, sugar absorption rapidly improves with the

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exception of lactose, which rather lags behind. In the patient with inadequate dietary control, although he may appear clinically well, poor response to disaccharide tolerance tests indicates an incomplete recovery of intestinal absorptive capacity. Temporary disaccharide intolerance associated with infantile diarrhoea has been observed in six infants whose ages ranged from five weeks to 12 months (Sunshine and Kretchmer 46a ). Two infants could not hydrolyse lactose, three others sucrose, and one was unable to absorb either lactose or sucrose; the symptomatology was similar to that of the hereditary disorders. Two of the patients had pathogenic bacteria in their stools, and one was suffering from cystic fibrosis of the pancreas. The diarrhoea ceased after the exclusion of the specific sugars from the diet. Re-evaluation of the intestinal absorptive capacity in these children after some time showed normal tolerance. The occurrence of such transitory disturbances of disaccharide digestion makes a correct diagnosis more difficult and elucidation of their aetiology an essential project for future study. A further striking example was the severe disaccharide malabsorption in the course of an extremely serious, presumably viral, infection of the lungs in a two-month-old male infant. Malabsorption of sucrose, maltose and lactose was accompanied by a moderate degree of steatorrhoea. Improvement and eventual recovery followed the introduction of extraneous sugar-splitting enzymes (HolzePO). The nutritional sequelae of acquired forms of disaccharide malabsorption are likely to be more severe than in the hereditary types. D i sacchari de

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MilK Allergy

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0' Figure 29.

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Flat blood glucose curve following lactose tolerance test in case of

milk allergy whilst sucrose absorption is unimpaired.

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DEVELOPMENT OF INTESTINAL ENZYME SYSTEMS

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It has been stated that giardiasis producing a coeliac-like syndrome may selectively inhibit lactase (Durand and Lamedica20 ). In our experience of this protozoal infestation all disaccharidase activities were grossly reduced (HolzePO).

MILK ALLERGY

An acquired form of lactose malabsorption was encountered in two infants towards the end of the neonatal period, in the fourth week of life. Both children had failed to thrive, and were considerably below birth weight. A moderate degree of wasting, abdominal distension, vomiting and diarrhoea with the passage of sour-smelling stools of low pH completed the picture of an infant suffering from some form of malabsorption. Investigation revealed that these babies indeed suffered from lactose malabsorption, but associated with milk allergy. Improvement followed promptly on a non-allergenic low lactose milk. When after several weeks of dietary treatment lactose was added to the formula, increasing it up to 7 per cent, it seemed to be well tolerated, whilst reintroduction of milk precipitated a return of the initial symptoms within a few days (Fig. 29). A lactose tolerance test gave a completely Hat curve, whilst a sucrose load produced a normal rise in blood sugar levels. Weare as yet unable to offer an explanation as to the operative mechanism. If one assumes that oedema of the intestinal mucosa may develop as a reaction to gastrointestinal milk allergy, one might have expected the absorptive capacity of the mucosa to have suffered in all its aspects and not just to find lactose absorption selectively impaired. Whatever the pathogenesis, this acquired form of lactose malabsorption, occurring so early in life, augments the difficulties encountered in the diagnosis of deficient enzyme activities in early infancy.

SUMMARY

The discovery of deficient carbohydrase activities as a cause of serious ill health in early infancy as well as in later life has given new momentum to the study of the physiology and pathology of these enzymes. As a result the intracellular localisation of the disaccharidases is now generally accepted, and the brush border of the intestinal epithelial cells the more or less undisputed site of their activities. With the exception of the first part of the duodenum, the enzymes seem fairly evenly distributed over the whole length of the small intestine. The access mechanism of the disaccharides into the cell is still uncertain, but there is some evidence in favour of an active transport from the lumen of the gut similar to that operating for glucose and galactose. Investigation

A.

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of the foetal development of the disaccharidases has shown the a-glycosidases to reach the maximum of activity at seven months of gestation, whilst ,B-glycosidases, in particular lactase, attain their peak in the perinatal period. Disaccharidase denciencies can be hereditary or acquired. The failure of disaccharidase activity leads to malabsorption of the respective sugar and, in its wake, to fermentative diarrhoea and malnutrition. Sucrose and isomaltose malabsorption seem to be closely linked; the genetic implications contradict the "one gene, one enzyme" theory. Dencient lactase activity leading to lactose malabsorption and failure to thrive has been more frequently seen in the male than in the female infant. Lactose intolerance with lactosuria and associated involvement of parenchymatous organs is a separate entity of still obscure aetiology which can be fatal unless treated with complete lactose withdrawal. The newly recognised syndrome of monosaccharide malabsorption adds to the differential diagnostic difficulties.

REFERENCES 1. Anderson, C. M., Messer, M., Townley, R R W., and Freeman, M.: Intestinal Sucrase and Isomaltase Deficiency in Two Siblings. Pediatrics, 31: 1003, 1963. 2. Anderson, C. M., Kerry, K. R, and Townley, R R W.: An Inborn Defect of Intestinal Absorption of Certain Monosaccharides. Arch. Dis. Childhood, 40:i, 1965. 3. Auricchio, S., Prader, A., Murset, T., and Witt, C.: Saccharose Intoleranz. Durchfall infolge hereditaren Mangels au intestinaler Saccharase Aktivitat. Helv. Paed. Acta, 16:483, 1961. 4. Auricchio, S., Dahlquist, A., Murset, C., and Prader, A.: Intestinal Isomaltase Deficiency in Patients with Hereditary Sucrose and Starch Intolerance. Lancet, 1: 1303, 1962. 5. Auricchio, S., and others: Disaccharidase Activities in Human Intestinal Mucosa. Enzym. Biol. Clin., 3: 193, 1963. 6. Auricchio, S., Rubino, A., and Murset, G.: Intestinal Glycosidase in the Human Embryo, Foetus and Newborn. Pediatrics, to be published. 7. Bertrand, M.: L'intolerance congenitale au saccharose. These Lyon, Imprinnerie Bose Freres, 1963. 8. Borgstrom, B., Dahlquist, A., Lundh, G., and Sjoval, J.: Studies on Intestinal Digestion and Absorption in the Human. J. CUn. Invest., 36: 1521, 1957. 9. Burgess, E. A., Levin, B., Mahalanabis, D., and Tongue, R E.: Hereditary Sucrose Intolerance: Levels of Sucrase Activity in Jejunal Mucosa. Arch. Dis. Childhood, 39:431, 1964. 10. Cozetto, F. J.: Co-existing Cystic Fibrosis and Intestinal Lactase Deficiency Confirmed by Enzyme Assay. Pediatrics, 32:228, 1963. 11. Cuatrecasas, P., Lockwood, D. H., and Caldwell, J. R: Lactase Deficiency in the Adult. Lancet, 1:14, 1965. 12. Dahlquist, A., and Borgstrom, B.: Digestion and Absorption of Disaccharides in Man. Biochem. J., 81 :411, 1961. 13. Dahlquist, A., and Brun, A.: A Method for the Histochemical Demonstration of Disaccharidase Activities. Application to Invertase and Trehalase in Some Animal Tissues. J. Histochem. Cytochem., 10:294, 1962. 14. Dahlquist, A.: Specificity of the Human Intestinal Disaccharidases and Implications for Hereditary Disaccharide Intolerance. J. Clin. Invest., 41:463, 1962.

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15. Idem: Intestinal Disaccharidases in Disorders Due to Intestinal Defective Carbohydrate Digestion and, Absorption. Editeq by P. Durand. Rome, II Pensiero Scientmco, 1964. 16. Darling, S., Mortensen, 0., and Sondergaard, G.: Lactosuria and Amino-aciduria in Infancy. A New Inborn Error of Metabolism. Acta Paediat., 49:281, 1960. 17. Doell, R. G., and Kretchmer, N.: Studies of Small Intestine during Development. I. Distribution and Activity of B Galactosidase. Biochim. Biophys. Acta, 353, 1962. 18. Idem: Intestinal Invertase: Precocious Development of Activity after Injection of Hydrocortisone. Science, 143:42, 1964. 19. Durand, P.: Lattosuria idiopatica in una paziente condiarrea cromica ed acidosi. Minerva Pediat., 10:706, 1958. 20. Durand, P., and Lamedica, G. M.: Disaccharide Intolerance. Helv. Paediat. Acta, 17:395, 1962. 21. Durand, P.: Lactose Intolerance in Disorders Due to Intestinal Defective Carbohydrate Digestion and Absorption. Edited by P. Durand. Rome, II Pensiero Scientifico, 1964. 22. Fridhandler, L., and Quastel, J. H.: Absorption of Sugars from Isolated Surviving Intestine. Arch. Biochim. Biophys., 56:412, 1955. 23. Girardet, P., Richterich, R., and Anterier, 1.: Adaptation de la lactase intestinale a l'administration de lactose chez Ie rat adulte. Helv. Phys. Acta, 22:7,1964. 24. Girardet, P.: Absorption et malabsorption du lactose. Berne et Stuttgart, Hans Huber, 1965. 25. Gorouben, J. C., and others: L'intolerance au saccharose. Etude clinique et biologique de 5 cas. Arch. frant;s. pediat., 20:53, 1963. 26. Haemmerli, U. P., and others: Acquired Milk Intolerance in the Adult Caused by Lactose Malabsorption Due to a Selective Deficiency of Intestinal Lactase Activity. Amer. J. Med., 38:7, 1965. 27. Holzel, A., Schwarz, V., and Sutcliffe, K. W. Defective Lactose Absorption Causing Malnutrition in Infancy. Lancet, 1:1126, 1959. 28. Holzel, A.: Alactasia; in P. Linneweh (Ed.): Erbliche StofJwechsel Krankheiten. Miinchen, Urban und Schwarzenberg, 1962. 29. Holzel, A., Mereu, T., and Thomson, M. L.: Severe Lactose Intolerance in Infancy. Lancet, 2:1346, 1962. 30. Holzel, A.: Nutritional Consequences of Altered Carbohydrate Absorption in Infancy and Childhood. Proc. Nutr. Soc., 23:123, 1964. 31. Inall, J. A., and Burkinshaw, J. H.: Lactosuria and Sucrosuria with Failure to Thrive. Proc. Roy. Soc. Med., 53:318, 1960. 32. Langstein, L., and Meyer, S. F.: Sauglingsernahrung und Sauglings Stoffwechsel. Wiesbaden, J. F. Bergmann, 1914. 33. Laliniala, K., Perheentupa, J., Visakorpi, J., and Hallman, N.: Disaccharidases of Intestinal Mucosa in a Patient with Sucrose Intolerance. Pediatrics, 34:615, 1964. 34. Laplane, R., and others: L'intolerance aUK sucres a transfert intestinal active. Les rapports avec !'intolerance au lactose et Ie syndrome coeliaque. Arch. frant;. pediat., 19:895, 1962. 35. Lelong, M., and Alagille, D.: quoted by Rey et al. 42 36. Lindquist, B., and Meeuwisse, C. W.: Chronic Diarrhoea Caused by Monosaccharide Malabsorption. Acta paediat., 51:674, 1962. 37. Long, C.: The Stabilization and Estimation of Lactic Acid in Blood Samples. Biochem. J., 40:27, 1946. 38. Miller, D., and Crane, R. K.: The Digestive Function of the Epithelium of the Small Intestine. II. Localization of Disaccharide Hydrolysis in the Isolated Brush Border Portion of Intestinal Epithelial Cells. Biochim. et Biophys. Acta, 52:293, 1961. 39. Nordio, S., and Lamedica, G. M.: Intolerance to Sucrose, Maltose, Isomaltose and Starch in Disorders Due to Intestinal Defective Carbohydrate Digestion and Absorption. Edited by P. Durand, Rome, II Pensiero Scientifica, 1964. 40. Prader, A., Auricchio, S., and Murset, G.: Durchfall infolge hereditaren Mangels am intestinaler Saccharase Aktivitat (Saccharose Intoleranz). Schweiz. med. Wchnschr., 91:465, 1961.

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41. Prader, A., Auricchio, S., and Semenza, G.: Die hereditiire Saccharose und Isomaltose-Malabsorption. Mschr. f. kindlk., 112:177, 1964. 42. Rey, J., Frezel, J., Jos, J., Bauche, P., and Lanry, M.: Diarrhee par trouble de l'hydrolyse intestinale du saccharose, du maltose et de !'isomaltose. Arch. fran~. pediat., 20:381, 1963. 43. Rutenburg, A. M., Goldberg, J. A., Rutenburg, S. H., and Lang, R. T.: The Histochemical Demonstration of a-D-Glucosidase in Mammalian Tissues. J. Histochem. Cytochem., 8:268, 1960. . 44. Rutenburg, A. M., Rutenburg, S. H., Morris, B., Teague, R., and Seligman, A. M.: Histochemical Demonstration of i3-D-Galactosidase in the Rat. J. Histochem. Cytochem., 6: 122, 1958. 45. Semenza, G., and Auricchio, S.: Chromatographic Separation of Human Intestinal Disaccharidases. Biochim. Biophys. Acta, 65:173, 1962. 46. Semenza, G., Tosi, R., Valloton-Delachaux, M. C., and Muhlhaupt, E.: Sodium Activation of Human Intestinal Sucrase and Its Possible significance in the Enzymic Organization of Brush Borders. Biochim. Biophys. Acta, 89:109, 1964. 46a. Sunshine, P., and Kretchmer, N.: Studies of Small Intestine During Development. III. Infantile Diarrhea AssoCiated with Intolerance to Disaccharides. Pediatrics, 34:38, 1964. 47. Weijers, H. A., van de Kamer, J. H., Mossel, D. A. A., and Dicke, W. K.: Diarrhoea Caused by DefiCiency of Sugar Splitting Enzymes. (Preliminary Communication.) Lancet, 2:296, 1960. 48. Weijers, H. A., van de Kamer, J. H., Dicke, W. K., and Ijsseling, J.: Diarrhoea Caused by DefiCiency of Sugar Splitting Enzymes. Acta paediat., 50:55, 1961. 49. Weijers, H. A., and van de Kamer, J. H.: Diarrhoea Caused by Deficiency of Sugar Splitting Enzymes. Acta paediat., 51:371, 1962. 50. Idem: Fermentative Diarrhoeas in Disorders Due to Intestinal Defective Carbohydrate Digestion and Absorption. Edited by P. Durand. Rome, II Pensiero SCientifica, 1964. 51. Wyngaarden, J. B.: Xanthinuria; in J. B. Stanbury, J. B. Wyngaarden and D. S. Fredrickson: The Metabolic Basis of Inherited Disease. New York, McGraw-Hill Book Company, Inc., 1960. St. Mary's Hospital Whitworth Park Manchester 13, Lanes. England