Laboratory Tests and Nutritional Assessment: Protein-Energy Status

Selected Topics in Pediatric Pathology

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Laboratory Tests and Nutritional Ass~ssment Protein-Energy Status

Denis R. Benjamin, BSc, MB, BCh*

The requirements for improved nutritional assessment in infants and children have become more complex and perhaps more important than they were in the past. This is due to the aggressive enteral and parenteral nutritional therapies we now have available; the successful long-term management of diseases that predispose to nutrient depletion; the increased risk of certain treatments such as radiation or extensive surgery in an even marginally malnourished child; the successful management of some hereditary enzyme deficiencies that require dietary manipulation such as phenylketonuria; and the appreciation that moderate depletion of some nutrients may have significant functional and other clinical consequences before the development of the late, clinically evident, easily diagnosed deficiency states. Although poorly defined at present, there is gathering evidence from a variety of sources that marginal levels of nutrition may contribute to both increased morbidity and mortality in hospitalized patients by predisposing to infection and other complications such as poor wound healing. 39, 62 There are convincing data to demonstrate that hospitalization is prolonged in these situations. 2, 3 We cannot be concerned merely with the obvious, final stages of malnutrition, such as delayed growth and impaired development. Although this is of immense concern in some countries, it occurs in relatively few of our patients. It is evident that suboptimal nutrition can affect cognitive ability, influence behavior, and lead to poor performance in a number of areas. It is important to remember that health is not simply defined as an absence of disease but as a positive sense of well-being. All these factors have shifted the emphasis toward developing specific, sensitive, reproducible tests that objectively quantitate developing nutrient *Associate Professor, Departments of Pathology and Laboratory Medicine, and Adjunct Associate Professor, Department of Pediatrics, University of Washington; Director of Laboratories, Children's Hospital and Medical Center, Seattle, Washington

Pedi(ltric Clinics of North America-Vol. 36, No.1, February 1989

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deficiency and can monitor its repletion if therapy is instituted. All attempts over the past couple of decades have failed to meet this goal. Today, the role of the laboratory in the evaluation and management of the state of nutrition in general and protein-calorie nutrition specifically remains problematic at best. By understanding the limitations of currently available tests, by clearly defining the clinical questions that need laboratory answers, and with an increased knowledge of nutrition and metabolism, it is possible to develop a more satisfactory and systematic approach to using the laboratory for nutritional assessment. Clinicians should be wary and skeptical of any claim that a single test, an index, or a group of tests is the way to monitor nutritional status.

STAGES IN THE DEVELOPMENT OF MALNUTRITION As the nutrient supply decreases and/or the demand increases, the organism undergoes a series of adaptive changes, often designed to protect more vital functions. In the usual situation, tissue stores or other available pools are first utilized and depleted before there are any significant alterations in other systems. During the next phase, there are a variety of hormonal adaptations. The extent and spectrum of hormonal changes is quite variable and dependent on the duration, severity, and type of nutrient deficiency. For example, chronic protein-calorie malnutrition will be associated with a spectrum of hormonal changes quite different from that accompanying acute starvation or fasting. When these adaptive phenomena begin failing, functional clinical consequences become evident. We are only now beginning to appreciate the extent and importance of these. Finally, the terminal stage is reached when growth and development are impaired and vital functions may become compromised. By the time the patient reaches this late stage, the diagnosis is clinically evident and the laboratory has little role in diagnosis, but may be very useful in assessing prognosis and providing monitoring information. Figure 1 illustrates the stages during which the laboratory can play its major roles.

KEY QUESTIONS IN NUTRITIONAL ASSESSMENT

A frequent problem leading to the misuse of laboratory data is the failure of the clinician to ask specific questions and his or her tendency to rely on a standardized group of tests because that is what their laboratory has available. In dealing with individual patients, the approach must be unique and customized. It is also important to remember that the clinical laboratory alone cannot provide the answer to many of the questions. Clinicians may have expectations of the laboratory out of proportion to the real value of the results.

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Dietary Assessment

Inadequate intake Malabsorrption Incr. excretion Incr. requirements Incr. destruction

~

Laboratory Assessment

Depletion of reserves

~

Physiologic/metabolic alterations Anthropometric _ _ _ _• Assessment

1 1

ADAPTATION

~

Wasting or decreased growth

~

Clinical Assessment

CAUSES

CLINICAL CONSEQUENCES

Specific anatomic changes Figure 1. Role of the laboratory in nutritional assessment.

In many situations, one would like answers to one or more of the following questions: 1. What are the child's nutritional requirements-that is, is the child hypo-, hyper-, or eumetabolic? 2. Are sufficient protein/calories and other nutrients being provided-that is, what is the child's intake? 3. Is absorption adequate? 4. Is there excess loss of nutrients? 5. How are nutrients being utilized (metabolized)-that is, is the child in an anabolic or catabolic state? 6. What are the stores or reserves? 7. What are the clinical consequences of the nutritional state-that is, is any "harm" being done? Are growth, development, cognitive ability, and so forth being compromised? Is the child at increased risk for complications such as wound healing or infection? 8. Does the child require additional nutritional support? If so, how much of what? 9. Is nutritional support effective and how should one monitor it? Does it need to be monitored?

In this article, only a few of these questions will be addressed and only those specifically relating to protein-energy nutrition. Tests that are used to investigate gastrointestinal function and absorptive capacity are not reviewed, nor are the clinical methods used to assess nutrient requirements. Moreover, it will become clear that the test selected to answer one question may be inappropriate for another.

COMPARISON OF TESTS The types of tests available can be loosely grouped into a few categories each having its own set of applications and limitations: static tests, functional

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INTAKE SYNTHESIS

TISSUE STORES

"

1 /

• I BLOO_DJ

1

\

CATABOLISM

EXCRETION Figure 2. Factors influencing blood concentrations of analytes.

tests, turnover studies, adaptive responses, and anthropometric measurements. , Static tests are usually based on measurements of a nutrient or related metabolite in the blood. These are some of the most widely available tests and often the least satisfactory. The major problem is that the "level" in the blood is carefully controlled and maintained by many homeostatic systems. Seldom does the value in the serum bear much relationship to tissue stores. As illustrated in Figure 2, many factors may impact on a single measured value. For example, serum calcium remains unchanged until the terminal phase of rickets or osteomalacia, at which time almost all the body stores of calcium have been depleted. Albumin decreases only late in the courSe of protein deficiency. Moreover, there are usually many other factors, such as concomitant illness or therapy, that can affect the measured value. These will be discussed with each individual analyte. Functional indices of nutritional status have been widely investigated and actively promoted over the past decade. These are based on the idea that the final outcome of a nutrient deficiency and its biologic importance are not merely a measured level in a tissue or blood, but the failure of one or more physiologic processes that rely on that nutrient for optimal performance. The best examples of these indices and the most routinely employed relate to the irrimune system. Deficits of a number of nutrients have been associated with a panoply of abnormalities in immune function, all leading to some level of immunocompromise and increased susceptibility of infection. But there are many other examples, including nerve conduction, dark adaptation, and sleep pattern, which have been discussed in recent reviews. 90 • 91 These functional indices, although very attractive from a total biologic standpoint, are not without their problems. They are often dependent on far more than a simple nutrient deficiency, making them

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very nonspecific. Most are still largely experimental. There are not good normative data at various ages and developmental stages. This is an area of potential, however, and one in which future research is likely to be fruitful. Turnover, metabolic, or dynamic studies are perhaps the most "sophisticated" investigative tools available, because they can incorporate information about intake, excretion, synthesis, and catabolism. They are extraordinarily difficult to perform on a routine basis and have not found widespread clinical application. They do, however, have a major role in research. These may become more useful with the introduction of nutrient molecules labeled with stable isotopes. Measurement of the adaptive response of an individual to a developing nutrient deficiency is an intriguing approach and one that has potential in specific instances. For example, somatomedin C falls rather precipitously when calories are restricted. Such early changes are useful in acute situations, in hospitalized patients, and in monitoring the success of therapy. Weare currently learning about the limitations and value of this group of tests. The anthropometric measurements are the final arbiters of proteinenergy nutriture. The problems with these are numerous. They change very slowly and only late in the development of malnutrition. They can be regarded in the same sense as other functional tests, because growth is the physiologic response to the genetic, nutritional, hormonal, and health endowments of an individual. On the other hand, because of the slow changes, the measurements are often used in a static manner. In addition, the reliability of measurement is often in question.

CURRENTLY AVAILABLE TESTS FOR EVALUATION OF PROTEIN-ENERGY STATUS There has been a tendency to divide the protein stores of the body, which account for approximately 15 per cent of the body mass, into two "compartments"-the "somatic" compartment, which consists of the protein in skeletal muscle, and the "visceral" compartment, which contains everything else. This distinction is arbitrary and artificial. Although the somatic compartment is relatively homogeneous, the visceral is composed of hundreds of different proteins serving many different structural and functional (for example, enzymatic) roles. The relationship between these two "compartments" is ill-defined in terms of synthetic rates, catabolism, and response to both nutritional deficiency and other stresses or diseases. In fact, it is questionable whether this distinction has any great merit.

SERUM PROTEIN MEASUREMENTS

The use of serum protein measurements is widespread. All suffer to some degree from similar problems, in that their concentration in the blood is dependent not only on protein deficiency but on a large number of physiologic and pathologic variables. For example, hepatic disease and

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Table 1. Comparison of Serum Protein Measurements in the Evaluation of Protein-Energy Status CONSTITUENT

HALF· LIFE (MW)

CLINICAL USE

LIMITATIONS

Albumin

18-20 days (66,000)

For prognosis; severity of malnutrition

Hydration; liver and renal disease; inflammation

Transferrin

8-9 days (77,000)

None

Inflammation; disorders of iron metabolism

Prealbumin

2 days (54,400)

Acute depletion; monitoring; prognosis?

Inflammation; responds more to calories than protein

Retinol-binding protein

12 hr (21,000)

Similar to prealbumin

Inflammation; vitamin A and zinc deficiency; liver disease

Fibronectin

4-24 hr (440,000)

Acute depletion; prognosis?

Inflammation; reference ranges not well studied

Monitoring parenteral nutrition?

Many

Amino-acid profiles

renal excretion will affect them. The hydrational state of the patient is an important physiologic variable, as is circadian rhythm and posture. Perhaps the most significant influence on all the protein measurements is the presence of the acute phase reaction in response to trauma, infection, neoplasia, or any other tissue damage. 51 The "ideal" protein for measurement, as stated by Haider, should be one "with a short biologic half-life, should respond to a protein-deficient diet and should reflect its deficiency by decreased concentration in the blood, should have small stores or reserves, a rapid rate of synthesis, a fairly constant metabolic rate, and be responsive only to protein and energy restriction. "38 Unfortunately, we are far from finding this ideal. Table 1 compares the major characteristics of the more commonly measured proteins. Albumin

Serum albumin was one of the first biochemical indicators of malnutrition to find almost universal use. Because of this, a large body of data has accumulated and most nutritional studies still include serum albumin as one parameter. The serum concentration is the net result of hepatic synthesis and degradation. The body pool of albumin is large. As much as 60 per cent of total body albumin is extravascular. The biologic half-life is long (18 to 20 days). The control of hepatic synthesis is incompletely known. Infection and inflammation may inhibit albumin synthesis. On the other hand, recent studies have demonstrated that synthesis can be increased following injury and the low serum values are due to peripheral redistribution or increased metabolism. 19 Similar studies in children with tumors have confirmed these observations. 59 Because of these factors, the change in serum albumin following protein depletion is slow to develop. The albumin is first mobilized

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from the extravascular pool and will maintain serum concentration for some time. The long half-life also contributes to this delay. The result is that a decrease in serum albumin only develops late in the course of malnutrition and then only in the more serious cases. The relationship of hypoalbuminemia to the edema associated with such malnourished states as kwashiokor is not clear cut. Edema is not, as many of us were taught, a direct result of the low albumin and decreased colloid osmotic pressure but has far more complex mechanisms. 29 , 31 Serum albumin is highly dependent on the hydrational state of the patient, in addition to showing mild circadian fluctuations and changes dependent on posture. Albumin synthesis and catabolism may be altered as part of the acute phase response to sepsis or trauma, so they are difficult to interpret in patients with any cause of inflammation. Hypoalbuminemia is common in children being treated for malignant tumors, is unrelated to dietary intake, but dependent on inflammation. 59 Hepatic disease is often associated with low albumin values. Both renal and gastrointestinal protein loss can result in profound depression in serum values. Albumin should not be used to diagnose either recent or mild-tomoderate degrees of protein-energy malnutrition. Its major role is in the assessment of the severity of chronic malnutrition and in estimating prognosis. But even here its clinical use is limited, because most patients with low serum albumin (below 3.5 g per dl) have obvious clinical signs. Marasmus, in which the caloric insufficiency is far more severe than the protein insufficiency is often associated with normal serum albumin concentrations. 26, 52 In population studies or in groups of hospitalized patients, it is evident that low albumin concentrations are associated with increased morbidity and mortality. 2, 3, 16, 71, 84, 99 However, translating these group findings to an individual patient is always difficult and, like all laboratory tests, must be interpreted in clinical context. Serum albumin is neither a sensitive nor a specific indicator of acute protein-energy malnutrition. Transferrin Transferrin, the major transport protein for iron, is the second best investigated protein "marker" in malnutrition. 33, 47, 71 Transferrin is synthesized in the liver, has a much smaller pool than albumin, most of it being intravascular, and has a considerably shorter halflife (8 to 9 days). Its main function is to bind and transport iron. In the usual situation, about one third of the available transferrin is bound to the serum iron. In many laboratories, estimation of the total iron binding capacity of serum approximates the transferrin value and has been used as an alternative. 99 However, there are excellent methods for direct measurement such as radial immunodiffusion and nephelometry. This method is no longer recommended. Although it initially showed some promise, especially compared with albumin, the problems (vide infra) and better alternatives have rendered it obsolete for this purpose. Transferrin is affected by many factors other than protein-calorie deficiency. Iron deficiency, a common accompaniment to protein-calorie malnutrition, results in increased hepatic synthesis and very high levels. On the other hand, decreases are seen in many inflammatory states, liver

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disease, exogenous protein loss such as the nephrotic syndrome, and in some anemias, especially those associated with hemolysis. Pre albumin Prealbumin (thyroxin-binding prealbumin, transthyretin) is an important transport protein with a much shorter half-life (2 days) than albumin. It too is synthesized in the liver and has a very small pool size. Its only well-known functions include carrying a portion of thyroxine (for which it transports about one third, the rest being largely transported on thyroxinebinding globulin) and, together with retinol-binding protein, transporting vitamin A. This latter relationship, the complexing of prealbumin with retinol-binding protein appears to be important in stabilizing the binding of vitamin A (retinol) and protecting it during transport. 56 Prealbumin decreases rapidly when calories and/or protein intake is below normal. Even if protein intake is adequate, severe calorie restriction will result in a decrease in prealbumin in as few as 3 to 4 days. 23, 96 It is clearly sensitive to the early phases of decreased nutrition. Moreover, its concentration will rapidly return to expected levels once adequate nutritional therapy has been instituted. 14, 47, 53 In some respects, it may be a better indicator of recent dietary intake than an accurate assessment of nutritional status. 22 There is some evidence that the failure of prealbumin to return to normal despite adequate intake portends a poor prognosis. 13 In young adults and older patients, its use appears reasonably satisfactory. 80, 86 A variety of studies in premature infants have produced contradictory results, some claiming that prealbumin is useful in nutritional management56 and others concluding that its measurement adds little information, being either too insensitive or too variable. 64, 89 It is fair to suggest that the jury is still out on the value of prealbumin measurements in the individual patient in pediatrics. A number of studies, including one at our institution (the premature infant· nursery of the University of Washington), may help to clarify this particular issue in the next year or two. Like albumin, prealbumin is very sensitive to the inflammatory response, and serum concentrations will decrease dramatically because of the inhibition of protein synthesis. 72 This limits the use of the test, because many seriously ill hospitalized patients have significant sepsis, tissue trauma, or some other stimulus of the inflammatory reaction. Patients with a wide spectrum of liver disorders have low values, such that one of its suggested uses is as a liver function test. 37, 45 Abnormal values may also be related to iron metabolism 20 and increased levels have been observed in renal disease. Because values will rapidly return to normal in many patients when sufficient calories are provided, it cannot be used as an endpoint for stopping nutritional support. Retinol-binding Protein This small protein (MW 21,000 daltons) is produced by the liver, has an extraordinarily short half-life (12 hours), a smaller pool size than even prealbumin, and very low serum concentrations. It is responsible, when complexed with prealbumin, for the transport of vitamin A.

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Most clinical studies have demonstrated a direct and close correlation between retinol-binding protein and prealbumin in response to proteincalorie deprivation and nutritional therapy.48, 86 Although some have suggested a theoretical advantage over prealbumin because of its shorter biologic half-life and small pool size, its very low concentrations in normal blood and the greater technical difficulties in its precise measurement have not encouraged its widespread use. There has yet to be a convincing study illustrating a distinct advantage over prealbumin. Like prealbumin, retinol-binding protein in blood will increase when the diet is sufficient in calories but still deficient in protein. 30 It too may be a better indicator of recent dietary intake than protein status. Low values are also seen in vitamin-A deficiency, hyperthyroidism, zinc deficiency, liver disease and, as usual, as part of the acute phase response.

Fibronectin Fibronectin (cold-insoluble globulin, nonspecific or nonimmune serum opsonin, serum opsonic factors, and so on) is the most recent addition to the spectrum of available protein measurements. As can be deduced from the synonyms, it has been known for a number of years for its role in opsonizing bacteria or other particulate matter. Its relationship to nutritional deficiency is in part serendipitous. Plasma fibronectin is a large glycoprotein (MW 440,000 daltons) synthesized by many cell types, especially endothelial cells, fibroblasts, and hepatocytes. Unlike the other serum proteins, however, nonhepatic sources are more important. It has a short half-life of somewhere between 4 to 24 hours. 78 It apparently serves many functions related to its extraordinary "stickiness," playing important roles in cell-matrix interactions, cell adhesion, wound healing, hemostasis, and macrophage function. Many of its biologic functions are currently under active investigation. A number of recent clinical studies have demonstrated low fibronectin levels in states of acute nutritional deprivation and its return to normal levels when nutritional therapy was instituted. 55, 83, 100 The mechanism underlying the decrease of fibronectin in plasma in this situation is unknown, Further clinical studies are required to validate the use of this measurement, especially in relation to chronic nutritional defiCiency and prognosis. One study has suggested that the composition of the parenteral diet affects the rapidity with which fibronectin returns to normal. 43 It is intriguing to speCUlate that some of the complications of malnutrition, such as an increased susceptibility to infection and poor wound healing, may in part be mediated by fibronectin deficiency. Clearly, many other factors are also crucial to these complex phenomena, Considering the diversity of cells capable of producing this protein and its multifunctional nature, it is not surprising that variations in plasma concentration may be due to a host of disorders. These include trauma, 79 burns,35 shock, 77 sepsis,36 disseminated intravascular coagulation,61 and many disorders associated with the acute phase response. 12 Interpreting values in such situations requires careful clinical and laboratory exclusion of any of these possibilities. As discussed further, it may be advisable to always include a measure of an acute phase reactant such as C-reactive protein.

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In addition to these biological variables, there are also methodologic considerations. Specimen handling is crucial because of the tendency of this molecule to adhere to almost any surface. Controls and calibrators for the various assays are not well standardized, and samples should not be stored for any length of time unless frozen at - 20° C. Finally, there is a dearth of data relating to reference values at various ages and stages of development. Despite all those caveats, plasma fibronectin may yet find a clinically useful role. Amino Acid Profiles Despite some claims for their usefulness,81 there is no convincing evidence that the measurement of plasma amino concentrations or their profiles in blood has any specific role in the diagnosis or management of protein-energy malnutrition. Very little is known about how serum concentrations are regulated, and there seems to be little relationship to protein intake and turnover. Although some interesting alterations in amino acid profiles have been recorded, these are usually late in the clinical sequence of malnutrition and we do not know what they mean. 4 , 63 Monitoring plasma amino acids has found some application in patients being managed with total parenteral nutrition.

TESTS OF THE HORMONAL RESPONSE AND ADAPTATION TO DIMINISHED NUTRITION

As soon as the fuel supply falls below the requirements for a growing child, a wide variety of metabolic changes develop, usually mediated through alterations in hormone synthesis, secretion, clearance transport, and binding. These appear to be adaptive responses to protect vital physiologic functions. The final consequence, if the inadequate nutrition persists or worsens, is that growth is compromised, mediated in part by even more profound disturbances in hormonal regulation. Many abnormalities in serum hormone values have been reported, and the data are conflicting and variable. This is largely owing to studies of patients with different levels of malnutrition, at different stages of the process, and with different underlying conditions. But there is increasing evidence from both animal experimental studies and even uncontrolled human studies of a definite pattern of hormonal response. In this section, only those hormone measurements that may have a useful role in clinical management are examined. Many other hormones are affected in this intricate and complex arrangement. For details, consult two excellent reviews. 6, 76 Growth Hormone The finding that growth hormone levels are increased in children with protein-calorie malnutrition came as a surprise, because it had been assumed for many years that growth failure was mediated by a decrease in growth hormone. 7, 50, 66 Although there is variability in the reported studies, most demonstrate high basal levels of growth hormone secretion and in the

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majority of cases, a relatively normal response to the usual stimuli, such as hypoglycemia. Even acute starvation will increase growth hormone secretion. 58 Although this may be the general trend, there is enough conflicting data to make the growth hormone assay difficult to interpret in many clinical situations. For example, some patients with cystic fibrosis do not increase their growth hormone secretion appropriately, 34 and basal levels may be low in anorexia nervosa. 44 In longstanding protein-calorie deprivation, growth hormone secretion may be reduced. There is certainly little value in any of the provocative tests, at least in respect to assessing malnutrition. The mechanism for this seeming paradox of elevated growth hormone levels in many patients with poor nutrition is not fully understood. It does appear to be due to increased synthesis and secretion by the pituitary, because its biologic half-life and clearance are normal. It may be related to another important change in the group of growth regulatory hormones, the somatomedins. Growth hormone measurement is only useful if measured in concert with somatomedin C. In these circumstances, a basal level is often adequate. Somatomedins Regardless of how they are measured, somatomedins, the growthpromoting pep tides produced by the liver in response to growth hormone stimulation, are uniformly decreased in patients with protein-calorie malnutrition. l7 , 32, 42, 73, 88 Moreover, the experimental administration of growth hormone will not increase the levels. 85 They will only be restored to normal with adequate nutrition, especially the provision of sufficient protein. 49 This is distinctly different from the rapid return to normal of many of the serum proteins such as prealbumin and fibronectin, which are more dependent on energy intake than protein. There has been much speculation concerning the mechanism for this striking decrease in somatomedins in states of inadequate nutrition. Suggestions have been made that it is due to inhibitors or that biologically inactive growth hormone may be secreted. Currently, the two most attractive hypotheses are that the receptors for growth hormone on hepatocytes are altered in malnutrition and/or that it is merely another manifestation of the general inhibition or decrease in protein synthesis that accompanies protein-calorie deprivation. Whatever the mechanism, it is likely that the low levels of somatomedin are partly responsible for the increase in growth hormone secretion as part of the usual negative-feedback mechanism. There are a variety of causes for depressed somatomedin values, but there are relatively few clinical circumstances in which somatomedins are low and growth hormone is elevated (Table 2). Most of these circumstances, such as renal failure and cirrhosis, can be readily diagnosed on a clinical basis, or with a few simple laboratory tests. In patients with renal failure, special care with the measurement is required in order to remove interfering compounds. 68 The combination of low levels of plasma somatomedin C as measured by specific immunoassay and normal or raised levels of basal growth

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Table 2. Causes of Decreased Somatomedin C Concentrations GROWfH HORMONE CONCENTRATION

Growth hormone deficiency Psychosocial growth failure Malnutrition: Acute Chronic Hypothyroidism Renal failure Cirrhosis Drugs (eg, bromocriptine, prednisolone) Peripheral growth hormone resistance

Low Low Normal or increased Increased Increased Increased Increased

? Increased

hormone is very suggestive of protein-calorie malnutrition. It can help to distinguish growth failure due to growth hormone deficiency and establish a diagnosis of malnutrition in those patients at high risk (hospitalized patients,97 for example, or patients on parenteral nutrition). It can also be applied to distinguish malnutrition from other causes of growth retardation. Psychosocial growth failure, at least early on, is usually associated with both decreased growth hormone and decreased somatomedin C. Other Hormonal Changes Alterations have been described in a variety of clinical situations in all hormonal systems, the hypothalamo-pituitary, thyroid, gonadal and adrenal axes, the endocrine pancreas, and adrenal medulla. These adaptive responses become clinically evident and pathologic in the late stages of malnutrition when the physiologic functions they normally support are finally affected. For example, delayed puberty, amenorrhea, or testicular dysfunction is the consequence of the disturbance in gonadal function. Although these alterations are crucial to our understanding of the development of malnutrition and our appreciation of the impact they have on a developing child, currently we cannot apply this knowledge to the routine laboratory and clinical diagnosis or management.

NITROGEN BALANCE AND OTHER DYNAMIC MEASUREMENTS

A number of methods have been devised to estimate protein status from more dynamic measurements. Although many of these have helped considerably in our understanding of malnutrition, few have found much clinical application outside the meticulous care that can be provided by a metabolic research ward. The problems lie in the inherent difficulty in collecting the necessary specimens and the many factors that may influence both the values and the measurements. Table 3 illustrates the use of the major techniques. Creatinine Excretion and Creatinine Height Index Creatinine, the breakdown product from creatine, is almost exclusively (but not entirely) derived from muscle. The rate of conversion of creatine

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Table 3. Comparison of Dynamic Measurements TEST

24-hour creatinine excretion Creatinine/height index Nitrogen balance 3-Methylhistidine excretion Amino acid infusions (stable or radioactive)

MAJOR CLINICAL USE

Lean body mass Protein reserves Short-term total body protein metabolism Short-term skeletal muscle turnover Protein synthesis; protein turnover

to creatinine is relatively constant, and although there is considerable diurnal variation in its excretion by the kidney, the total excreted over 24 hours is also relatively stable. This measurement has been regarded as a reasonable assessment of lean body mass. 24 If well performed in a carefully controlled situation in a patient without other significant problems, it also correlates with oxygen consumption92 and anthropometric estimates of muscle mass. Unfortunately, there are multiple factors that can influence the result (Table 4). Moreover, there are logistical difficulties in collecting urine over 24 hours, especially in young infants, making measurements notoriously unreliable. It is also not a test that can be adapted to an outpatient setting. This measurement of creatinine excretion has been combined with the simple measurement of height to obtain an index that is an estimate of body protein reserve. The creatinine/height index (CHI) is calculated as follows: CHI -

24-hour urine creatinine excretion (mg) x 100 Normal value for 24-hour urine creatinine excretion for height (mg)

The normal values used in this index have been published. 10, 98 These are based on relatively small numbers of patients. A CHI under 40 per cent is regarded as evidence of severe protein depletion, 40 to 60 per cent moderate, and 60 to 80 per cent mild. Clearly, this index is absolutely dependent on the accuracy of the 24-hour urine collection and creatinine measurment in the patient and the reliability of reference tables. 3-Methylhistidine 3-Methylhistidine (N-methylhistidine) has been advocated as another measure of skeletal protein stores and turnover. 50, 101 This amino acid is largely derived from the breakdown of skeletal muscle proteins, especially Table 4. Factors Influencing Creatinine Excretion The interpretation of the test assumes the following: Normal renal function Normal hydration Normal urine output (no diuretics) No prolonged bedrest No recent high-protein meal (eg, meat) No extremes of age (eg, less than 2 months old) Patient not catabolic (no surgery, stress, infection)

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actin, and is excreted unaltered in the urine without being further metabolized. Unfortunately, there is also a significant pool of 3-methylhistidine outside of skeletal muscle. 75 The problems with its measurement include all those relating to any 24-hour urine collection and to creatinine in particular. There are no established reference ranges for children; diet and renal function will significantly influence the measurement. Although it would be very useful to have an estimate of skeletal muscle catabolism, especially in hospitalized patients, 3-methylhistidine cannot be recommended for pediatric patients. Nitrogen Balance Studies In tP-e normal adult, the intake of protein and its loss from the body is so precisely controlled that neither predominates, and the rate of protein synthesis (anabolism) is the same rate as its destruction (catabolism). In infants and actively growing children, there should be a positive nitrogen balance during periods of protein accretion. The same can be true during recovery from an illness or trauma. Ideally, nitrogen-balance studies should involve a 24-hour measurement of protein intake and a measurement of its total loss from the body. In practice, a number of assumptions are made to correct for or estimate the many variables that are not directly measured. In addition, there is the tendency to measure only urine urea nitrogen, which only accounts for approximately 90 per cent of the nitrogen in urine. More precise measurements are available. . al NItrogen B ance =

Protein intake (gm/24 hr) Urine urea ( / h) 5 -'t gm 24 r 6.2 m rogen

+4

The nitrogen intake is estimated by how much protein there is in the diet and dividing by 6.25, which is the average amount of nitrogen in most dietary protein. The constant 4 is used to account for other losses of nitrogen in stool, through the skin, or elsewhere. During states of catabolism, more nitrogen is lost than is present in the diet, and the balance will be negative. As seen in Table 5, a negative balance may be due to at least three major mechanisms: an insufficient dietary intake of protein, an active catabolic state, and unmeasured protein loss. The goal of nutritional support is to achieve a positive balance. The problems of performing accurate intake and output measurement and urine collections are considerable, and the number of assumptions used in the calculation make this a crude test at best. Liver disease, renal disease, disturbances in hydration, and many drugs will render the results even more questionable. Nitrogen balance gives no information about protein stores or nutritional state. It merely reflects very short-term protein metabolism and dietary intake. Measurement of Protein Metabolism Using Radioactive or Stable Isotopes The difficulties in interpreting these indirect and rather crude estimates of protein metabolism have led a number of investigators to measure the

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Table 5. Nitrogen Balance Positive balance +2 and> +2

Anabolic state (e.g., recovery) Drugs Growth

Neutral balance +1 to -1

Anabolism = catabolism Intake = output

Negative balance -2 and < -2

Deficient intake Catabolic state Inflammation Trauma Neoplasia Excess losses Burns Protein-losing enteropathy Renal disease

incorporation of an amino acid directly into protein. 57 For example, muscle protein synthesis has been measured with the stable isotope L_[1_13C] leucine, but some of these rely on muscle biopsy and tissue analysis. 74 Using radio labeled amino acids such as L_[V4C] leucine, it is possible to estimate whole body protein turnover. 9 None of these techniques are trivial and at this time are only research tools. Certainly the development of a stable isotopic technique would be more routinely employed and might .find wider application in pediatrics.

"FUNCTIONAL" TESTS IN NUTRITIONAL ASSESSMENT

Investigation into the many physiologic consequences of inadequate nutrition has revealed a myriad of cellular, tissue, organ, and system dysfunctions. These have formed the basis of the so-called "functional" tests. Although they are very interesting from a biological standpoint, it is obvious that none are dependent solely on the state of nutrition. Some of them may be rather sensitive indicators of poor nutrition, but they are extraordinarily nonspecific. One should always remember that the result of increasing the sensitivity of any test is very often accompanied by a sacrifice of its specificity. There are a number of excellent reviews of functional tests. 90, 91 In many respects these assays can be regarded as investigating the physiologic abnormalities that integrate many processes, whereas the ones already discussed examine the biochemical and hormonal alterations. It.would be a mistake, however, to somehow regard these tests as being better than the others. They suffer many of the same limitations and drawbacks. Some of the more useful functional tests are those relating to single nutrient deficiencies in which there is a more direct relationship between the nutrient and the physiologic process. Excellent examples of these include vitamin-K intake, absorption, and utilization with the prothrombin time; vitamin A and dark adaptation; and vitamin E and peroxide-induced red cell hemolysis. Although other factors playa role in each of these, the limiting factor in the usual situation is the vitamin status.

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Table 6. Frequent Immune System Abnormalities Accompanying Protein-C alone M alnutntion Cell-mediated system (T cells) T lymphoctes decreased Change in T4rrS ratio Defects in mitogenesis (eg, to PHA) Diminished cutaneous delayed hypersensitivity Decreased lymphoid tissue (eg, tonsils, nodes, thymus) Elevated terminal deoxynucleotidyl transferase (Tdt)

Humoral system (B cells) Decreased secretory IgA (in secretions) Phagocytes (neutrophil function) Diminished microbial killing Impaired chemotaxis Decreased 6bronectin Complement components Decreased C3

As far as protein-calorie nutrition is concerned, there is no clear-cut functional test. The disturbances in growth develop late and are assessed by anthropometry. However, monitoring change in growth velocity by frequent sequential measurement remains a very important clinical tool. It has been widely known in developing countries that malnutrition is associated with an extraordinary incidence of infection. This enhanced susceptibility and decrease in host defenses have been carefully dissected over the past decade and dozens of separate defects have been described, all of which limit the organism's ability to defend itself. The relationship between nutrition and the immune system is both close and complex. Poor nutrition leads to defects in immune function and infection. Infection or inflammation can in turn have a profound effect on general body metabolism and nutrition. The immune system, however, is also dependent on many other factors, some of which are clearly as important, such as infection, stress, genetics, and vitamin and mineral metabolism. A complete catalog of immune system abnormalities is not supplied here because they are discussed elsewhere,15, 18, 28, 93, 94 but the common abnormalities are listed in Table 6. From the available data, it is evident that the defects in cell-mediated immunity are more frequent, develop earlier, and are more clinically significant than the other major components of host defense. However, the reliability of anyone or a group of these to detect malnutrition is still in considerable doubt. Certainly there is no evidence that the total lymphocyte count is of any clinical value. 25 The elevation of the terminal deoxynucleotidyl transferase (Tdt) , an indirect estimate of the impairment in T-cell differentiation,21 is also nonspecific and variable. Apart from those relating to growth and immune function, no other functional tests have found much application in protein-calorie malnutrition in children. There is still considerable interest in muscle, nerve, and brain function. It is possible that useful tests may yet be devised to evaluate these areas. For example, nerve conduction and other electroencephalographic changes have been documented. 90 Many more studies are required, however. OTHER TESTS

A number of other procedures have been designed to assist in analyzing body composition. Computed tomography, for example, has been used to

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measure various organ volumes. 4o • 41 The limitations of this on a routine basis are obvious. Total body potassium or exchangeable potassium are closely related to body composition, but require either whole body gamma counters or radioactive isotope-dilution methodsY' 87 Neutron activation analysis, stable isotopes, and nuclear magnetic resonance techniques (both in vivo and in vitro) may find useful application in the future. Even total body electrical conductivity has been studied as a possible test in nutritional assessment. 70

RECOMMENDATIONS From the descriptions of the various tests, it should be clear there are considerable difficulties in using the laboratory for the clinical evaluation and management of patients with poor nutrition. In circumstances in which help is most needed, namely hospitalized patients, many tests are least useful, because they are profoundly affected by the underlying disease, therapy, or inflammation. In the outpatient setting, office, or clinic, they are often needed less, because clinical evaluation is generally adequate. It is here that they are more reliable. For many of the questions posed by the clinician, there is no simple laboratory test. Answering the questions, "What are this child's requirements? Is he hyper- or hypometabolic?" requires either direct or indirect calorimetry. Direct calorimetry is available at only a few medical centers, because it requires a special thermally insulated chamber that can measure heat exchange directly. Indirect calorimetry, on the other hand, is based on oxygen consumption. It is merely an estimate and difficult to perform on small children. Such questions as these are often answered with a rough clinical assessment, an even rougher knowledge of metabolism, and reference to a set of normal tables, the validity of which is often questionable. This raises a very fundamental issue. If we usually base our therapy and management on a set of assumptions with wide limits, how much precision and accuracy is required of a laboratory test? To quantitate a specific parameter, when there is great imprecision in our management, places an unwarranted value on that number. It sometimes leads one to rely too heavily on the laboratory and to ignore important clinical features. Another problem is that many of these tests may be more appropriate for population surveys and group studies. 69 There is a great gap between applying a series of observations made by an investigator knowledgeable about nutrition who is studying mechanisms in a carefully defined set of patients in a controlled situation to the individual patient in one's office. We need many more studies that directly investigate the use and application of specific tests in the type of clinical setting in which one practices, 1 not the mere extrapolation of research data, most of which are analyzed retrospectively. There are, however, situations in which the laboratory can be most helpful. In each of these, the tests must be used to complement clinical evaluation, and not all tests may be needed in each case. With time and experience, these will almost surely be refined and modified.

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When investigating a child with possible early protein-calorie malnutrition or when attempting to distinguish growth failure related to nutrition from other causes, the most useful tests are standard anthropometric measurements, albumin, prealbumin, somatomedin C (and a basal growth hormone if necessary), and C-reactive protein. The same test panels can be used to assess if a child is at increased risk for postsurgical complications. In this situation, a measurement of fibronectin might be found useful, although it is unlikely to provide significant additional information. Monitoring the success or progress of nutritional therapy can be accomplished with this same small group of tests. The frequency of testing depends on the specific test. Prealbumin, fibronectin and somatomedin C change quite rapidly and usually normalize within 3 to 5 days. Weekly testing is usually adequate. Albumin need not be repeated more often than once every 2 weeks or even less frequently. None of these tests should be used as a guide to end supplemental nutritional therapy. The C-reactive protein has been included as a measure of the inflammatory reaction because the presence of the acute phase response profoundly influences the interpretation of all the tests. In the last few years, it has become evident that the cachexia and negative nitrogen balance associated with both infections and neoplasia are mediated by a polypeptide synthesized by macrophages, variously known as cachectin or tumor necrosis factor.8 The mechanism of its effect on energy metabolism is under investigation at present. There is suppression of fat synthesis and interference with enzymes such as lipoprotein lipase. 95 There is still a great deal to learn about how this group of molecules influences energy, fat, and protein metabolism. A number of indices have been devised that rely on many different tests. 39 One index also includes an assessment of inflammation. 46 The main valu~ of these indices is not their precision or even their predictive capabilities; rather, they are important because they highlight nutrition as an important variable in outcome. No indices have been applied in a prospective fashion in infants and children, so it is difficult to assess their real value in the uncontrolled clinical situation. There are a number of studies comparing the use of laboratory data to clinical evaluation in assessing nutrition. These illustrate rather nicely the limitations of the laboratory and the importance of clinical examination. 5. 65 Such studies add further weight to the preceding recommendations and the conclusions that follow.

SUMMARY 1. There is no one definitive laboratory test, group of tests, or indices that are satisfactory for the assessment of protein-calorie status. 2. Clinical evaluation remains the simplest, most widely available, most reproducible, and wisest method. It is satisfactory for the majority of clinical situations.

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3. The laboratory should be used selectively and to complement clinical evaluation. Routine testing must be relegated to research protocols. 4. Always include some assessment of inflammation (for example, Creactive protein, erythrosedimentation rate [ESR]), because its presence affects the interpretation of all the other tests. 5. The pathophysiologic effects of the underlying disease, especially in hospitalized patients, will affect the interpretation of every one of the laboratory tests. 6. Nutritional status often impacts more on the interpretation of commonly performed laboratory tests than laboratory tests impact on the assessment of nutrition. ACKNOWLEDGMENTS I wish to thank Ms. Fran Denham, for her secretarial assistance, and Dr. Kent Opheim, Director of Clinical Chemistry, for his ideas and suggestions.

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17. Clemmons DR, Klibanski A, Underwood LE, et al: Reduction of plasma immunoreactive somatomedin-C during fasting in humans. J Clin Endocrinol Metab 53:1247, 1981 18. Cunningham-Runells S: Effect of nutritional status on immunological function. Am J Clin Nutr 35:1202, 1982 19. Dahn MS, Jacobs L, Smith S, et al: The significance of hypoalbuminemia following injury and infection. Am Surg 51:340, 1985 20. Delpeuch F, Cornu A, Chevalier P: The effect of iron defiCiency anemia on two indices of nutritional status, pre-albumin, and transferrin. Br J Nutr 43:375, 1980 21. Dionigi R: Immunological factors in nutritional assessment. Proc Nutr Soc 41:355, 1982 22. Farthing MJG: Serum thyroxine binding pre-albumin may reflect energy and nitrogen intake rather than overall nutritional status in chronic intestinal disease [letter J. N utr Res 3:618, 1983 23. Fischer JE: Plasma proteins as indicators of nutritional status. In Lewenson SM (ed): Nutritional Assessment, Present Status, Future Directions and Prospects. Report on the Second Ross Conference on Medical Research, Ross Laboratories, Columbus, Ohio, 1982, p 25. 24. Forbes GB, Bruining GJ: Urinary creatinine excretion and lean body mass. Am J Clin Nutr 29:1359, 1976 25. Forse RA, Rompre C, Crosilla P, et al: Reliability of the total lymphocyte count as a parameter of nutrition. Can J Surg 28:216, 1985 26. Forse RA, Shizgal HM: Serum albumin and nutritional status. J Pediatr Endocrinol Nutr 4:450,1980 27. Garrow JS: New approaches to body composition. Am J Clin Nutr 35:1152, 1982 28. Gershwin ME, Beach RS, Hurley LS: Nutrition and Immunity. Orlando, Florida, Academic Press Inc., 1985 29. Golden MHN: Protein deficiency, energy deficiency and the oedema of malnutrition. Lancet 1:1261, 1982 30. Golden MHN: Transport proteins as indices of protein status. Am J Clin Nutr 35:1159, 1982 31. Golden MHN, Golden BE, Jackson AA: Albumin and nutritional oedema. Lancet 1:114, 1980 32. Grant DB, Hambley J, Becker D, et al: Reduced sulphation faction in undernourished children. Arch Dis Child 48:596, 1973 33. Grant JP, Custer PB, Thurlow J: Current techniques of nutritional assessment. Surg Clin North Am 61:437, 1981 • 34. Green OL, Fefferman R, Nau S: Plasma growth hormone levels in cystic fibrosis and short stature. Unresponsiveness to hypoglycemia. J Clin Endocrinol Metab 27:1059, 1967 35. Grossman JE, Demling RH, Duy ND, et al: Response of plasma fibronectin to major bum. J Trauma 20:967, 1980 36. Grossman J, Pohlman T, Koerner F, et al: Plasma fibronectin concentration in animal models of sepsis and endotoxemia. J Surg Res 34:134, 1983 37. Haellen J, Laurell CB: Plasma protein pattern in cirrhosis of the liver. Scand J Clin Lab Invest 29 (suppl 124):97, 1972 38. Haider M, Haider SQ: Assessment of protein-calorie malnutrition. Clin Chern 30:1286, 1984 39. Harvey KB, Moldawer LL, Bistrian BR, et al: Biological measures for the formulation of a hospital prognostic index. Am J Clin Nutr 34:2013, 1981 40. Heymsfeld SB, Fulenwidker T, Nordlinger B: Accurate measurement of liver, kidney, and spleen volume and mass by computed axial tomography. Ann Intern Med 90:185, 1979 41. Heymsfeld SB, Olafson RP, Kutner MH, et al: A radiographic method of quantifying protein-calorie malnutrition. Am J Clin Nutr 32:639, 1972 42. Hintz RL, Suskind R, Amatayalrul K, et al: Plasma somatomedin and growth hormone values in children with protein-calorie malnutrition. J Pediatr 92:153, 1978 43. Horwitz GD, Groeger JS, Legaspi A, et al: The response of fibronectin to differing parenteral calorie sources in normal man. J Pediatr Endocrinol Nutr 9: 435, 1985 44. Huseman C, Johanson A: Growth hormone deficiency in anorexia nervosa. J Pediatr 87:946, 1975

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Director of Laboratories Children's Hospital and Medical Center P.O. Box C-5371 Seattle, W A 98105