Laboratory Assessment of Nutritional Status

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Laboratory Assessment of Nutritional Status Evaluating nutritional status by laboratory methods is a more objective and precise approach than the community assessment, dietary methodology, or clinical assessment methods. It utilizes biochemical tests, performed in a hospital, commercial or other laboratory, to measure levels of nutrients in biological fluids (blood or urine) or to evaluate certain biochemical functions which are dependent on an adequate supply of essential nutrients. However, the interpretation of laboratory data is often difficult and does not necessarily always correlate with either clinical or dietary findings. Not all nutrients can or should be assessed by laboratory methods. In general, laboratory methods are used to determine deficiencies in: 1. Serum protein, particularly albumin level; 2. The blood-forming nutrients: iron, folacin, vitamin B6, and vitamin B12; 3. Water-soluble vitamins: thiamine, riboflavin, niacin, and vitamin C; 4. The fat-soluble vitamins: A, D, E, and K; 5. Minerals: iron, iodine and other trace elements; 6. Levels of blood lipids such as cholesterol and triglycerides, glucose and various enzymes which are implicated in heart disease, diabetes, and other chronic diseases. Various surveys have shown significant deficiencies in many of these nutrients. For example, in the 1965 USDA Food Intake Study of approximately 15,000 individuals, insufficient dietary intakes of viamins A and C, pyridoxine, thiamine, riboflavin, iron, and calcium were reported for relatively large numbers of persons. Laboratory tests conducted during the Ten State Nutrition Survey also revealed considerable dietary, clinical, and laboratory evidence of clinical and sub-clinical nutritional deficiency. Composite surveys reported at the White House Conference on Food, Nutrition and Health revealed similar findings.

Objectives of Laboratory Assessment The use of laboratory tests has two primary functions: * To detect marginal nutritional deficiencies in individuals, particularly when 28 AJPH SUPPLEMENT, Vol. 63, NOVEMBER, 1973

dietary histories are questionable or unavailable; their use is especially important before overt clinical signs of disease appear, thus permitting the initiation of appropriate remedial steps. * To supplement or enhance other studies, such as dietary or community assessment among specific population groups, in order to pinpoint nutritional problems that these modalities may have suggested or failed to reveal. Laboratory investigation is of little use if it merely confirms a known clinical diagnosis. Often, laboratory values will be obtained suggesting marginal or acute deficiencies when the patient appears clinically normal since clinical signs usually -occur only after prolonged inadequate intake of nutrients. The probability, then, is that the subject may be in various stages of depletion and, if this state continues, will become ill. Most importantly, a deficiency in one nutrient can be considered an almost certain indicator of other nutritional inadequacies; these too should be rigorously investigated.

Methodology Generally two types of tests are employed in laboratory surveys-measurement of circulating levels of nutrients in blood or urine, and/or functional tests. The first may identify the presence of a nutritional problem; the latter will be a superior indicator of its severity. In other words, the functional tests measure the effect, or lack thereof, on the enzymes by which the body makes use of its nutrient intake. For example, a thiamine deficiency can be detected in urine, but measurement of the enzyme transketolase in red blood cells will be a more accurate indicator of its severity. At times, certain other types of highly sophisticated laboratory tests can be undertaken, for example, microbiological assays. These would likely be undertaken under special circumstances -with the advice and under the supervision of highly qualified personnel. However, the accompanying text and charts in the appendix do indicate where they can be used.

Planning a Laboratory Assessment While laboratory assessment of nutritional status may seem formidable, it can be undertaken

if appropriate advice is sought and proper preliminary planning takes place. The best single source for advice is the Nutritional Biochemistry Section, Center for Disease Control (CDC) in Atlanta, Georgia, although other laboratories can be consulted as well. Some of the primary considerations in planning laboratory studies are: 1. Laboratory assessment requires a method of coordinating the collection of !samples of blood ana urine from subjects to be surveyed. Further, an appropriate laboratory must be selected for analysis, and arrangements made to provide samples and accumulate data. Such a medical analytical laboratory should have facilities for colorimetry, spectrophotometry, fluorometry, chromatography, flame and atomic absorption spectrophotometry, and microbiological assay. 2. Medical or paramedical personnel obtain and process the blood and urine samples. Specific instructions are described in Appendix A to this section. 3. All subjects should be informed about the purpose of the study and their permission obtained. Parental permission is mandatory in the case of minors. 4. The methods utilized for nutritional assessment vary in cost, degree of technical expertise required, and reliability. They are also constantly being revised and improved. Some methods are essentially research tools, others are in common use. Unless the laboratory is supervised by a qualified person, advice should be obtained before determining which tests should be undertaken and how the data are to be interpreted.

Standards for Interpretation of Laboratory Data The interpretation of laboratory data will always be a matter for some disagreement, since the prime objective is to detect "risk of deficiency" before clinical evidence of disease develops. The standards may also vary somewhat with the specific methods used, since the methods vary in specificity and reproducibility. All methods used in a survey should be standardized and an appropriate design developed so that the results do not vary beyond acceptable limits during the course of the survey. This is best accomplished by repeated evaluation of standards previously checked by a recognized standards laboratory. The criteria used in the Ten State Nutrition Survey are included in Appendix B for current information and for reference purposes. These, in

turn, are based, in large part, on data compiled as part of the classic studies conducted by the Interdepartmental Committee on Nutrition for National Defense which are not expressed by age as in the Ten State Nutrition Survey. These standards may well be modified in the future as better methods and more data upon the physiological significance of different levels of intake or function are obtained.

Precautions in Laboratory Evaluation There are some essential precautions that should be kept in mind when undertaking and evaluating laboratory nutritional assessments. Various biochemical tests differ considerably in their reproducibility. Urinary excretion levels of nutrients, for example, vary more than plasma levels and are therefore less definitive. In any case, nutrient levels may vary from time to time and reflect immediate rather than usual intake. Biological fluid levels and functional tests may vary even in individuals seemingly on similar diets or suffering from equally apparent degrees of nutritional depletion. Further, inter-current disease may affect nutrient levels. Indeed, nutritional tests may provide "signposts" of disease such as neoplasms, wasting neurological diseases, etc. Finally, the "cut-off points" selected as representing some degree of risk of deficiency (see Appendix B) are, and will presumably always be, a matter of some argument and arbitrary decision. The controversies will be settled when, and if, there are: * More simple and reliable tests; * Extension of the range and specificity of laboratory evaluation of nutrients; * Better data on the physiological significance of the test used.

Laboratory Assessment for Individual Nutrients The following is a description of methods employed to assess those nutrients which can be measured by laboratory evaluations, as well as an indication of when and why they should be employed. The specific tests that might be used are not named, since they vary from laboratory to laboratory. This comprises a brief summary of laboratory methods; specific references for each nutrient are listed in Appendix C to this section and should be consulted before tests are undertaken. Circulating levels of most of these elements can be measured in blood or plasma, and deficiencies can be estimated. Protein-Protein deficiency in the United States appears to be uncommon. Reduced levels have been reported in pregnant women, but it is uncertain whether the criteria for "normal" levels LABORATORY ASSESSMENT 29

in non-pregnant women also apply in pregnancy. Protein deficiency produces a fall in serum protein levels, especially serum albumin, but this is not a particularly sensitive index and is not specific for protein deficiency. Serum protein levels may be maintained for a considerable period of time, despite limited protein intake. Total serum protein and serum albumin determinations are standard clinical chemical procedures. Recently, nitrogen/creatinine ratios in the urine and ratios of specific amino acids in plasma have been recommended as measures of proteincalorie malnutrition, but have not been extensively studied in the United States. Water-Soluble Vitamins 1. Vitamin C (Ascorbic Acid)-Clinical scurvy, the disease associated with severe vitamin C deficiency, is uncommon in the United States, although infants, alcoholics, the elderly, and neglected persons may be scorbutic. Nevertheless, reduced plasma levels have been reported in a significant portion of people in many nutrition surveys. Serum levels of vitamin C vary substantially and depend, to a considerable degree, on the intake immediately preceding the test. This must be borne in mind in interpreting the results of plasma vitamin C levels. 2. Thiamine-Although clinical evidence of thiamine deficiency is very uncommon in most United States populations, it is probably an important cause of morbidity in alcoholics. The usual test is the estimation of thiamine excretion in the urine, ordinarily made on "spot samples" collected in the field or in the clinic, rather than 24hour collection. However, thiamine content of spot samples in the same individual vary substantially, and this is a relatively insensitive test of the nutritional status. A functional enzyme test is preferable. Transketolase is an enzyme which requires thiamine (as thiamine pyrophosphate, TPP) for its function. Transketolase in red blood cells and the "TPP effect" (the increase in activity due to the addition of TPP) may be measured, and is probably the method of choice since activity does change with moderate depletion of thiamine. It has not yet been applied to broadly-based surveys. Microbiologic assays are also available to estimate thiamine in blood. 3. Riboflavin-A variety of lesions associated with riboflavin deficiency are not uncommon in many parts of the world. The specificity of these lesions as indicators of riboflavin depletion is, however, in doubt. The usual method of estimating riboflavin adequacy is by examination of urinary excretion. Such tests are not completely satisfactory due to the variability in riboflavin excretion. Recently, an enzyme functional test has been proposed. This is the estimation of gluta30 AJPH SUPPLEMENT, Vol. 63, NOVEMBER, 1973

thione reductase in red blood cells and the "FAD effect" (the increase in activity due to the addition of flavin adenine dinucleotide, FAD). This may become the method of choice, although experience with it is limited. Microbiological and chemical methods are also available to measure riboflavin in blood. 4. Niacin-Pellagra, the deficiency disease caused by niacin deficiency, is now rare in the United States, although it may occasiopially be seen in alcoholics or other persons with severely restricted diets. Niacin is derived from the amino acid, tryptophan, and thus pellagra is ordinarily associated with populations consuming little tryptophan (corn and sorghum-based diets). Estimation of N'-methylnicotinamide in the urine has been the traditional method of determining adequacy of niacin intake. As with thiamine, urinary excretion is not a generally satisfactory method for surveys. Microbiologic methods are available for the estimation of circulating niacin. 5. Folacin-Folate deficiency results in anemia. Low circulating levels have been reported to be common in pregnant women and in women taking birth control pills and other estrogenic medications. The circulating level of folate in plasma or red blood cells is utilized in estimating adequacy of intake, although the standards for interpreting data are controversial. Folate deficiency increases excretion of FIGLU (formimino-glutamic acid) in urine, but this also may occur in vitamin B12 deficiency. 6. Vitamin BB (Pyridoxine)-Vitamin B6 has been too little studied in relation to its effects on nutritional status. This is unfortunate because dietary surveys have indicated that vitamin B6 intake may be a rginal in some population groups in the United ctates. Evidence is also accumulating that Vitami B6 requirements may be markedly increased durin g pregnancy and in women taking birth contr Il pills. Tests suggested for evaluating vitamin B6 status include: measurement of various B6 metabol ites in the urine, estimation of tryptophan metabo ites in the urine after a tryptophan dose, estimatioi of transaminase in blood cells or plasma and its response to the addition of pyridoxal phospha te, and estimation of vitamin Be in the blood. 7. Vitamin B1l-Vitamin B12 deficiency causes anemia, and inability to utilize the vitamin B12 in food is the cause of pernicious anemia. Vitamin B12 deficiency due to inadequate intake has been reported in some vegetarians. Reduced blood levels of vitamin B12 have been reported in pregnant women and those taking birth control pills. Analytical estimation of vitamin B12 is by microbiologic techniques or by radioisotopic methods. 8. Other Water-Soluble Vitamins-Panto-

thenic acid, biotin, and choline are the other watersoluble vitamins. There is little doubt that they are essential, but they do not seem to present practical nutritional problems in most populations. However, this may be a false assumption since methods for estimating nutritional status with regard to these nutrients have not been developed and little is known of the probable requirement. Microbiologic or chemical methods for their estimation are available. Fat-Soluble Vitamins The fat-soluble vitamins A, D, E, and K are best absorbed in the presence of some fat in the diet. Thus, diseases which interfere with fat absorption may also impair absorption of fat-soluble vitamins. Patients with sprue, gluten enteropathy, and other absorption problems may manifest deficiencies even though the dietary intake appears adequate. It should also be noted that vitamins A and D are well known to be toxic when consumed in excess, and represent a potential problem in our vitamin-conscious society. 1. Vitamin A and Beta-Carotene-Vitamin A does not occcur directly in plant foods, but the body converts plant pigment, beta-carotene, into vitamin A. Both are ordinarily measured in serum as the criteria of vitamin A adequacy. A low carotene level, of course, only indicates limited consumption of green leafy and yellow vegetables, not necessarily vitamin A deficiency. Surprisingly, substantial numbers of people examined in the Ten State Nutrition Survey had low serum vitamin A levels, indicating some degree of vitamin A deficiency. There are also reports that a significant number of children in Canada and the United States (at autopsy) had no vitamin reserves in their livers, the primary site of vitamin A storage. Xerophthalmia, caused by severe vitamin A deficiency, is rare in the United States. The significance of the low levels of vitamin A are thus open to some debate. Night blindness, an. early sign of vitamin A deficiency, which may be estimated by dark adaptation tests, has not yet been adequately investigated. 2. Vitamin D-Rickets, the childhood deficiency disease resulting from an inadequate vitamin D intake, is relatively rare in the United States, but does occur occasionally. Osteomalacia in the elderly, possibly due to a lack of vitamin D, is reported to be relatively common in many countries. Elevated serum alkaline phosphatase levels were once thought to be indicative of vitamin D deficiency, but this is not an infallible test. No suitable methods are available to survey populations. Although there is relatively little evidence to indicate that vitamin D deficiency is a general problem, the situation is somewhat uncertain. Since vitamin D may be supplied by synthesis invoked

by exposure to sunlight, dietary evaluation is also unsatisfactory. 3. Vitamin E-Clinical evidence of vitamin E deficiency has only been reported in infants. Deliberate restriction of vitamin E in adults may result in increased red blood cell fragility under certain conditions. Since there is abundant evidence in animals that increased consumption of unsaturated fats increases the need for vitamin E, this vitamin deserves more study than it has received in the past. Vitamin E may be measured in the serum directly. Methods also exist for estimating the fragility of red cells. 4. Vitamin K-Since vitamin K is synthesized by the flora in the intestinal tract, deficiency is thought to occur only in very young infants before the 'lora are established, and in diseases in which fat absorption or the utilization of vitamin K is abnormal. There is no evidence that tests for vitamin K function need to be included in general nutrition surveys. It might be noted that while most nutrients -carbohydrate, fat, protein, vitamins, and minerals-are supplied by diet, some portion of man's vitamin needs is supplied from synthesis by gastrointestinal microorganisms. Such is the case for vitamins K, B1, B12, folacin, biotin, and other micronutrients. Thus, any intestinal tract pathology will reduce availability of these vitamins, as will antibiotic therapy which modifies intestinal flora. Minerals 1. Iron-A deficit of iron eventually results in anemia. The most common method of detecting anemia is by the measurement of the hemoglobin level in the blood or the hematocrit. Anemia may be caused by a variety of nutritional or nonnutritional factors other than iron deficiency. Modest degrees of anemia caused by inadequate iron intake are common in the United States (10% of the population according to one estimate), especially in women and children since iron requirements are increased by growth and menstrual blood loss. The extent of iron deficiency in adult men is a matter of considerable interest and debate. Several different standards have been recommended for the evaluation of hemoglobin and hematocrit, and the extent of anemia observed in any population depends, of course, upon the standards used. a. Hematocrit-The hematocrit, the percentage of packed red cells in whole blood, is a standard clinical procedure available in all community hospitals and other laboratories. b. Hemoglobin-This determination is a simple colorimetric procedure that is standard in all clinical laboratories. Ordinarily, both hemoglobin and hematocrit are determined and are prefLABORATORY ASSESSMENT 31

erable to blood cell counts since they involve less laboratory error. c. Serum Iron and Transferrin-Iron is carried in the plasma by a specific protein called transferrin. A reduced saturation of transferrin and a reduced serum iron level provide more specific evidence of iron deficiency and will detect reduced iron stores before anemia develops. If iron levels and transferrin saturation are normal in the face of anemia, the anemia does not represent iron deficiency. 2. Calcium-Although substantial numbers of people in the United States consume less calcium than ordinarily recommended, there is little or no evidence of calcium deficiency. Blood levels of calcium are essentially constant over a wide range of intakes, and measurement of blood calcium does not provide adequate evaluation of dietary calcium. No suitable laboratory or clinical methods for surveys are available for monitoring the adequacy of calcium intakes.. 3. Iodine-The adequacy of iodine intake is usually estimated by the clinical evaluation of thyroid enlargement or goiters. However, not all goiters are the result of iodine insufficiency. An approximation of iodine intake may be determined by relating urinary iodine to urinary creatinine. Other clinical determinations, for example, plasmabound iodine (PBI), reflect the functional utilization of iodine and are standard clinical laboratory tests. 4. Other Minerals-A number of essential minerals are not discussed in detail here. These include magnesium, manganese, zinc, fluoride, chromium, selenium, copper, sodium, potassium, phosphorus, chloride, and others. Currently, there is widespread interest and research into the nutritional and metabolic aspects of certain of these trace elements. Clinical evidence of magnesium and zinc deficiencies has been found in hospital populations, and relatively low levels of intake of both minerals are not uncommon in the United States. However, methodology to evaluate nutritional status of these and

32 AJPH SUPPLEMENT, Vol. 63, NOVEMBER, 1973

other trace elements has not been standardized, and criteria for adequacy have not been developed. Lipids and other Serum DeterminationsCirculating levels of Qholesterol, triglycerides, glucose, various enzymes, and hormones such as insulin, glucagon, etc., also have important implications with regard to nutritional status and to certain disease states, especially coronary heart disease and diabetes. These and other measurements have been included in some surveys and should be considered as integral parts of nutrition surveys in the future. The National Heart and Lung Institute, Bethesda, Md., is sponsoring Lipid Research Centers and Centers for Multiple Risk Factor Intervention Trials for the prevention of coronary heart disease. This illustrates the present and future public health significance of the use of serum cholesterol and triglyceride determinations in the screening of persons with elevated levels of lipids who may be at high risk of developing coronary heart disease. Moreover, physicians in evaluation of intervention programs are using serum cholesterol and triglyceride levels to assess response to diets designed to lower these lipid factors. The current great interest in serum lipids has been generated by: a. The identification by the Framingham Study and other studies of serum cholesterol level as a risk factor in coronary heart disease (along with hypertension, smoking, obesity, and other determinants); b. The indication by prospective studies (such as the Anti-Coronary Club of the City of New York Department of Health), that the lowering of serum cholesterol by nutritional means has been attended by reduced coronary heart disease morbidity and mortality; c. The relatively recent emergence of the hyperlipidemias (a condition in which both genetic and environmental factors collaborate to raise serum lipids) as having possible public health significance.

Appendix A Sample Collection and Preservation

While specific instructions for sample collection and preparation vary according to the specific assays to be done, certain general rules apply to all procedures. Blood and urine samples will deteriorate rapidly unless they are properly managed and preserved for transmission to the laboratory for assay. Optimal attention must be given to specimen collection, preservation, and transportation. No level of excellence in clinical technique can correct for changes in perishable nutrients resulting from faulty or careless collection, preservation, or shipping of specimens. No laboratory, regardless of the degree of sophistication, can improve the quality of inadequate specimens. The following general procedures should be followed: Blood drawn into vacuum tubes can be placed directly on ordinary ice, and urine can be acidified (with acetic or hydrochloric acid) and chilled similarly. Where necessary, due to the lability of the nutrient, such as ascorbic acid, it will be necessary to make an acid filtrate of blood immediately, and store by freezing the filtrate (in this case, the acid would be trichloracetic or metaphosphoric). Similarly, separated serum should be properly preserved and frozen immediately for folic acid evaluation. Certain enzymes are unstable to freezing, necessitating immediate assay, while others are stable in the freezer for longer periods of time. To avoid spurious results, therefore, it is mandatory to carefully review each analytical technique to be used with a view toward its specific needs for sample collection and preservation. As much of the sample preparation as possible, including chilling and/or freezing, should be done at the collection site. When properly prepared, most samples can be frozen, at least for a short period of time to await assay. It is most important that the samples remain frozen until assayed, particularly if they are to be shipped some distance to the analytical laboratory. This can be done effectively only by shipping with dry ice in a properly insulated polystyrofoam container. The dry ice does not usually last more than 60-72 hours, under the best conditions. Some enzymes, i.e. transaminases, are destroyed by refreezing, so that these precautions are imperative. The time of day that samples are to be ob-

tained from the subject may influence the findings, particularly if this is done shortly after the individual has eaten or taken a vitamin supplement. Optimally, blood samples would be taken in the morning before breakfast or any food or drink is consumed. For urinalysis, the optimal sample is a total 24-hour collection. If this is not possible, the best sample is the first upon arising. When that is not feasible, compromises must be made, and considered, in interpreting the data. The best compromise would be to obtain samples at least 2-3 hours after the last meal. Ideally, all samples are to be collected under the same circumstances. As indicated above, some indication of nutritional status may be obtained on the basis of a urine sample alone for a variety of nutrients. Accordingly, much can be accomplished toward evaluating nutritional status even if the evaluating team does not include personnel to draw blood samples. Moreover, it is also noteworthy that many nutritional evaluation procedures may be available in hospital clinic laboratories, i.e. hematocrit, hemoglobin, iron, transferrin saturation, folic acid, B12, and often others. A crucial need in laboratory nutritional assessment is the coordinated standardization of the various tests. This is essential in order to assure that data obtained from different laboratories and/or localities can be compared with each other in order to draw valid conclusions. Indeed, this is essential if one is to use uniform criteria for judging nutrient adequacy. For the routine clinical chemical assay work, there are standards, calibrators, and plasma or serum controls available which the laboratory can use to assure accuracy and precision of its own analytical procedures. This assures the laboratory that its data relate to similar work done in other clinical laboratories. No similar synthetic standards are available as yet for the nutritional evaluation techniques. Rather, it is necessary to coordinate one's work with that of established laboratories. This may be accomplished by comparing the analytical values obtained by the laboratory involved in the local evaluation program, with values obtained from submitting replicate samples under code to a standard reference laboratory. LABORATORY ASSESSMENT 33

Appendix B Table of Current Guidelines for Criteria of Nutritional Status for Laboratory Evaluation Nutrient and Units

*Hemoglobin (gm/i OOml)

Age of Subject (years)

Deficient

Criteria of Status Marginal

Acceptable

6-23 mos. 2-5 6-12 13-16M 13-16F 16+M 16+F Pregnant (after 6+ mos.)

Upto Upto Upto Upto Upto Upto Up to

9.0 10.0 10.0 12.0 10.0 12.0 10.0

9.0- 9.9 10.0-10.9 10.0-11.4 12.0-12.9 10.0-11.4 12.0-13.9 10.0-11.9

10.0+ 11.0+ 11.5+ 13.0+ 11.5+ 14.0+ 12.0+

Upto

9.5

9.5-10.9

11.0+

*Hematocrit (Packed cell volume in percent)

Up to 2 2-5 6-12 13-16M 13-16F 16+M 16+F Pregnant

Up to Upto Upto Upto Upto Up to Upto Up to

*Serum Albumin (gm/i OOml)

Up to 1 1-5 6-16 16+ Pregnant

*Serum Protein (gm/1OOml)

Upto Upto

28 30 30 37 31 37 31 30

2.8 3.0

28-30 30-33 30-35 37-39 31-35 37-43 31-37 30-32

31+ 34+ 36+ 40+ 36+ 44+ 33+ 33+

Up to 2.5 Up to 3.0 Up to 3.5 2.8-3.4 3.0-3.4

2.5+ 3.0+ 3.5+ 3.5+ 3.5+

Up to 5.0 Up to 5.5 Up to 6.0 6.0-6.4 5.5-5.9

5.0+ 5.5+ 6.0+ 6.5+ 6.0+

0.1-0.19

0.2+

Up to 1 1-5 6-16 16+ Pregnant

Upto Upto

6.0 5.5

*Serum Ascorbic Acid (mg/ 1 OOmI) *Plasma vitamin A (mcg/100 ml) *Plasma Carotene (mcg/100 ml)

All ages

Up to

0.1

All ages

Upto 10

10-19

20+

All ages Pregnant

Up to 20

20-39 40-79

40+ 80+

*Serum Iron (mcg/100 ml)

Up to 2 2-5 6-12 12+M 12+F

Up to Up to Up to Up to Up to

30 40 50 60 40

Upto Upto Up to Upto Up to

15.0 20.0 20.0 15.0

*Transferrin Saturation Up to 2 2-12 (percent) 12+M 12+F All ages **Serum Folacin (ng/ml) All ages **Serum vitamin B12

(pg/mI)

* Adapted from the Ten State Nutrition Survey ** Criteria may vary with different methodology.

34 AJPH SUPPLEMENT, Vol. 63, NOVEMBER, 1973

2.0

Up to 100

30+ 40+ 50+ 60+ 40+ 15.0+ 20.0+ 20.0+ 15.0+ 2.1-5.9

6.0+

100+

Appendix B Table of Current Guidelines for Criteria of Nutritional Status for Laboratory Evaluation (continued) Nutrient and Units

Age of Subject (years)

Deficient

*Thiamine in Urine (mcg/g creatinine)

1-3 4-5 6-9 10-15 16+ Pregnant

*Riboflavin in Urine (mcg/g creatinine)

1-3 4-5 6-9

10-16

**RBC TransketolaseTPP-effect (ratio)

16+ Pregnant All ages

**RBC Glutathione All ages Reductase-FAD-effect (ratio) **Tryptophan Load Adults (mg Xanthurenic (Dose: 100mg/kg acid excreted) body weight) **Urinary Pyridoxine 1-3 (mcg/g creatinine) 4-6

Critera of Status Marginal

Acceptable

Up to 120 Upto 85 Up to 70 Up to 55 Up to 27 Upto 21

120-175 85-120 70-180 55-150 27- 65 21- 49

175+ 120+ 180+ 150+ 65+ 50+

Up to 150 Up to 100 Upto 85 Up to 70 Up to 27 Up to 30 25+

150-499 100-299 85-269 70-199 27- 79 30- 89 15- 25

500+ 300+ 270+ 200+ 80+ 90+ Up to 15

1.2+

Up to 1.2

25+(6 hrs.) 75+(24 hrs.)

Up to 25 Up to 75

10-12 13-15 16+

All ages Pregnant

Up to 0.8

**Urinary Pantothenic Acid (mcg)

All

Up to 200

**Plasma vitamin E (mg/ 1OOml)

All ages

7-9

*Urinary N'methyl nicotinamide

(mg/g creatinine)

*

ages

Up to

Up to

90+ 80+ 60+ 40+ 30+ 20+

90 80 60 40 30 20

Upto Up to Up to Up to Upto Upto

0.2

0.2

0.2-5.59 0.8-2.49

0.6+ 2.5+

200+ 0.2-0.6

0.6+

*Transaminase Index (ratio) tEGOT

Adult

2.0 +

Up to 2.0

tEGPT

Adult

1.25+

Up to 1.25

* Adapted from the Ten State Nutrition Survey Criteria may vary with different methodology t Erythrocyte Glutamic Oxalacetic Transaminase

**

Erythrocyte Glutamic Pyruvic Transaminase

LABORATORY ASSESSMENT 35

Appendix C Special Selected References for Nutritional Laboratory Assessment A. General References for Clinical Chemistry and Nutrients Fundamentals of Clinical Chemistry, edited by Norbert W. Tietz, W. B. Saunders Co., 1970, Philadelphia, London, Toronto (new edition coming Spring, 1974). The Vitamins, Sebrell/Harris, Vols. 1 & 4 (in preparation). P. Gyorgy and W. M. Pearson, Academic Press, 7 vols. 1967-1973. Natelson, S. Techniques of Clinical Chemistry (3rd ed.) C. C. Thomas Co. 1971. B. References for Individual Nutrients 1. Protein Electrophoretic separation of serum proteins, Manual for Nutrition Surveys, ICNND, 2nd edition, p. 147, U.S. Government Printing Office, Washington, D.C. 1963.* Oberman, et al. Electrophoretic analysis of serum proteins in infants and children. N. Eng. J. Med., 225:743, 1956. Total serum protein, albumin and globulin by a modified Biuret Tec4nique: Manual tor Nutrition Surveys, ICNND, 2nd edition, p. 133, U.S. Govemment Printing Office, Washington, D.C. 1963.* 2. Hematocrit Macro: Manual for Nutrition Surveys, ICNND, 2nd edition, p. 116, U.S. Government Printing Office, Washington, D.C. 1963.* Micro: Clinical Diagnosis by Laboratory Methods, 14th edition, Todd and Sanford, eds., p. 146, W. B. Saunders Co., Philadelphia, 1969. 3. Hemoglobin Manual for Nutrition Surveys, ICNND, 2nd edition, p. 115, U.S. Govemment Printing Office, Washington, D.C. 1963* 4. Iron Brit. J. Hematol., 20:451, 1971. Ramsey, W. N. M. The determination of the total iron binding capacity of serum. 2:221, 1957. Clin. Chim. Acta. 2:221, 1954. Scarlata, R. W. and Moore, E. W. A micromethod for the determination of serum iron and serum ironbinding capacity. Clin. Chem. 8:360, 1962. Woodruff, C. W. A Micromethod tor serum iron determination, J. Lab. Clin. Med. 53:955, 1959. 5. Ascorbic Acid Cheraskin, E., et al. A lingual vitamin C test. Int. J. Vit. Res. 38:114, 1968. Serum vitamin C (ascorbic acid)-Dinitrophenylhyrrazine Method. Manual for Nutrition Surveys, ICNND, 2nd edition, p. 117, U.S. Government Printing Office, 1963.* Serum vitamin C-Micro procedure, Ibid. p. 119.* * Out of print.

36 AJPH SUPPLEMENT, Vol. 63, NOVEMBER, 1973

6. Pyridoxine Baker, H. and Frank, 0. Vitamin B6 in "Clinical Vitaminology: Methods and Interpretation." lnterscience Publications, New York, N.Y. pp. 66-81, 1968. Brin, M. A simplified Toepfer-Lehmann Assay for the three Vitamin Be Vitamers. Method in Enzymology XVIII, 519-523, 1970. Hamfelt, A. Age variation of vitamin B6 metabolism in man. Clin. Chim. Acta. 10:48, 1964. Luhby, A. Leonard, Brin, M., Gordon, M., Davis, P., Murphy, M. and Spiegel, H. Vitamin Be metabolism in users of oral contraceptive agents 1. Abnormal urinary xanthurenic acid excretion and its correction by pyridoxine. Amer. J. Clin. Nutr. 24: pp. 684-93, June 1971. Price, S. A. et al. Effects of dietary vitamin B6 deficiency and oral contraceptives on the spontaneous urinary excretion of 3-hydroxy anthranilic acid. Am. J. Clin. Nutri. 25:494, 1972. Sauberlich, H. E. et al. Biochemical Assessment of the Nutritional Status of Vitamin Be in the human. Am. J. Clin. Nutr. 25:629, 1972. Tryptophan load test-xanthurenic acid in serum. Manual for Nutrition Surveys, ICNND, 1st edition, p. 88, U.S. Government Printing Office, Washington, D.C. 1963.* Note: The test is now modified to giving a load of 2 gm L-tryptophan. Approximately 67% of the xanthurenic acid is excreted in the first 8 hours. 7. Folacin Jukes, T. H. Assay of compounds with tolic acid activity. Meth. Bioch. Anal. 2:121, 1955. Luhby, A. L. and J. M. Cooperman. Folic acid deflciency and its Inter-relationship with vitamin B,2 metabolism. Adv. Metab. Discord. 1:263-334, 1964. Water, et al. J. Clin. Path. 14:335, 1961. 8. Vitamin B13, Baker, H. and Frank, 0. Vitamin B,2 In "Clinical Vitaminology: Methods and Interpretation," Interscience Pubs., New York, N.Y., pp. 116-141, 1968. Lau, K. S., Gottlieb, C., Wasserman, L. R. and Herbert, V. Measurement of serum Vitamin B2 Level using radioisotope dilution and coated charcoal. Blood 26:202, 1965. Skeggs, H. R., Microbiological Assay for Vitamin Ba,. Methd. Bioch. Anal. 14:53, 1966. 9. Thiamine Baker, H. and Frank, 0. Thiamine In "Clinical Vitaminology: Methods and Interpretation," Interscience Pubs., Inc., New York, N.Y., p. 7-19, 1968. Brin, M. "Transketolase and the TPP-Effect in Assessing Thiamine Adequacy, In "Vitamins and Coen-

Appendix C (continued) zymes: Methods in Enzymology" Academic Press, N.Y., Vol. XVIII, pp. 125-133, 1970. Dreyfus, P., Clinical Application of Blood Transketolase Determinations, N. Eng. J. Med. 267:596, 1962. (microassay) Erythrocyte Transketolase Activity. M. Brin In Methods of Enzymatic Analysis, H. U. Bergmeyer, ed., In press 1974. Thiamine in Urine, Manual for Nutrition Surveys, ICNND, 2nd edition, p. 136, U.S. Gov't Print. Off., 1963.* Creatinine in Urine, Picrate Method, ibid, p. 135.* 10. Riboflavin Baker, H. and Frank, 0. Riboflavin In "Clinical Vitaminology: Methods and Interpretation," Interscience Pubs., New York, N.Y., pp. 43-52, 1968. Bamji, M. S., Glutathione Reductase Activity in Red Blood Cells and Riboflavin Nutritional Status in Humans, Clin. Chim. Acta. 26:263, 1969. Glatzle, D., et al, Method for the Detection of a Biochemical Riboflavin Deficiency. Investigations of the Vitamin B2 Status in Healthy People and Geriatric Patients, Int'l J. Vit. Res., 40:166, 1970. Urinary Riboflavin, Manual for Nutrition Surveys, ICNND, 2nd edition, p. 140, U.S. Gov't Print. Off. 1963.* Creatinine in Urine-Picrate Method, ibid, p. 135.*

11. Niacin Baker, H. and Frank, 0. Nicotinic Acid In "Clinical Vitaminology: Methods and Interpretation," Interscience Pubs., New York, N.Y., pp. 87-108, 1968. N'Methyl Nicotinamide in Urine, Manual for Nutrition Surveys, ICNND, 2nd edition, p. 142, U.S. Gov't Print. Off., 1963.* 12. Pantothenic Acid Baker, H., and Frank, 0. Pantothenic Acid in "Clinical Vitaminology: Methods and Interpretation," Interscience Pubs., New York, N.Y., pp. 54-63, 1968. Hatano, M., Microbiological Assay of Pantothenic Acid in Blood and Urine, J. Vitaminol. 8:134, 1962. 13. Vitamin A Gary, P. J., et al, Vitamin A. Fluorometry and Uses of Silicic Acid Technique. Clin. Chem. 16:766, 1970. Neeld, J. B., and Pearson, W. N. Macro- and MicroMethods for the Determinations of Serum Vitamin A using Trifluoracetic Acid, J. Nutr. 79:454, 1963. 14. VItamin E Baker, H. and Frank, 0. Vitamin E In "Clinical Vitaminology: Methods and Interpretation," Interscience Pubs., New York, N.Y., pp. 169-75, 1968. Hashim, S. A. and Schruttinger, G. R., Rapid Determination of Tocopherol in macro- and micro-quantities of Plasma. Am. J. Clin. Nutr. 19:137, 1966. *

Out of print.

LABORATORY ASSESSMENT 37