Laboratory Assessment

of
Nutritional Status

Evaluating nutritional status by laboratory dietary histories are questionable or un-

methods is a more objective and precise approach available; their use is especially impor-
than the community assessment, dietary method- tant before overt clinical signs of disease
ology, or clinical assessment methods. It utilizes appear, thus permitting the initiation of
biochemical tests, performed in a hospital, com- appropriate remedial steps.
mercial or other laboratory, to measure levels of * To supplement or enhance other studies,
nutrients in biological fluids (blood or urine) or such as dietary or community assess-
to evaluate certain biochemical functions which ment among specific population groups,
are dependent on an adequate supply of essential in order to pinpoint nutritional problems
nutrients. However, the interpretation of labora- that these modalities may have sug-
tory data is often difficult and does not neces- gested or failed to reveal.
sarily always correlate with either clinical or dietary Laboratory investigation is of little use if it
findings. merely confirms a known clinical diagnosis. Often,
Not all nutrients can or should be assessed laboratory values will be obtained suggesting mar-
by laboratory methods. In general, laboratory ginal or acute deficiencies when the patient ap-
methods are used to determine deficiencies in: pears clinically normal since clinical signs usually
1. Serum protein, particularly albumin -occur only after prolonged inadequate intake of
level; nutrients. The probability, then, is that the sub-
2. The blood-forming nutrients: iron, fola- ject may be in various stages of depletion and, if
cin, vitamin B6, and vitamin B12; this state continues, will become ill. Most impor-
3. Water-soluble vitamins: thiamine, ribo- tantly, a deficiency in one nutrient can be con-
flavin, niacin, and vitamin C; sidered an almost certain indicator of other nutri-
4. The fat-soluble vitamins: A, D, E, and K; tional inadequacies; these too should be rigor-
5. Minerals: iron, iodine and other trace ously investigated.
elements;
6. Levels of blood lipids such as choles- Methodology
terol and triglycerides, glucose and Generally two types of tests are employed
various enzymes which are implicated in laboratory surveys-measurement of circulating
in heart disease, diabetes, and other levels of nutrients in blood or urine, and/or func-
chronic diseases. tional tests. The first may identify the presence of
Various surveys have shown significant deficien- a nutritional problem; the latter will be a superior
cies in many of these nutrients. indicator of its severity. In other words, the func-
For example, in the 1965 USDA Food Intake tional tests measure the effect, or lack thereof, on
Study of approximately 15,000 individuals, insuffi- the enzymes by which the body makes use of its
cient dietary intakes of viamins A and C, pyridox- nutrient intake. For example, a thiamine defi-
ine, thiamine, riboflavin, iron, and calcium were ciency can be detected in urine, but measurement
reported for relatively large numbers of persons. of the enzyme transketolase in red blood cells
Laboratory tests conducted during the Ten State will be a more accurate indicator of its severity.
Nutrition Survey also revealed considerable die- At times, certain other types of highly
tary, clinical, and laboratory evidence of clinical sophisticated laboratory tests can be undertaken,
and sub-clinical nutritional deficiency. Composite for example, microbiological assays. These would
surveys reported at the White House Conference likely be undertaken under special circumstances
on Food, Nutrition and Health revealed similar -with the advice and under the supervision of
findings. highly qualified personnel. However, the accom-
panying text and charts in the appendix do indi-
Objectives of Laboratory Assessment cate where they can be used.
The use of laboratory tests has two primary
functions: Planning a Laboratory Assessment
* To detect marginal nutritional defi- While laboratory assessment of nutritional
ciencies in individuals, particularly when status may seem formidable, it can be undertaken
28 AJPH SUPPLEMENT, Vol. 63, NOVEMBER, 1973
if appropriate advice is sought and proper pre- turn, are based, in large part, on data compiled as
liminary planning takes place. The best single part of the classic studies conducted by the Inter-
source for advice is the Nutritional Biochemistry departmental Committee on Nutrition for National
Section, Center for Disease Control (CDC) in Defense which are not expressed by age as in the
Atlanta, Georgia, although other laboratories can Ten State Nutrition Survey. These standards may
be consulted as well. well be modified in the future as better methods
Some of the primary considerations in and more data upon the physiological signifi-
planning laboratory studies are: cance of different levels of intake or function are
1. Laboratory assessment requires a obtained.
method of coordinating the collection of
!samples of blood ana urine from sub- Precautions in Laboratory Evaluation
jects to be surveyed. Further, an appro- There are some essential precautions that
priate laboratory must be selected for should be kept in mind when undertaking and
analysis, and arrangements made to evaluating laboratory nutritional assessments. Var-
provide samples and accumulate data. ious biochemical tests differ considerably in their
Such a medical analytical laboratory reproducibility. Urinary excretion levels of nutri-
should have facilities for colorimetry, ents, for example, vary more than plasma levels
spectrophotometry, fluorometry, chro- and are therefore less definitive. In any case, nu-
matography, flame and atomic absorp- trient levels may vary from time to time and reflect
tion spectrophotometry, and microbio- immediate rather than usual intake. Biological
logical assay. fluid levels and functional tests may vary even in
2. Medical or paramedical personnel ob- individuals seemingly on similar diets or suffering
tain and process the blood and urine from equally apparent degrees of nutritional de-
samples. Specific instructions are de- pletion. Further, inter-current disease may affect
scribed in Appendix A to this section. nutrient levels. Indeed, nutritional tests may pro-
3. All subjects should be informed about vide "signposts" of disease such as neoplasms,
the purpose of the study and their per- wasting neurological diseases, etc.
mission obtained. Parental permission Finally, the "cut-off points" selected as rep-
is mandatory in the case of minors. resenting some degree of risk of deficiency (see
4. The methods utilized for nutritional as- Appendix B) are, and will presumably always be,
sessment vary in cost, degree of tech- a matter of some argument and arbitrary decision.
nical expertise required, and reliability. The controversies will be settled when, and if,
They are also constantly being revised there are:
and improved. Some methods are es- * More simple and reliable tests;
sentially research tools, others are in * Extension of the range and specificity of
common use. Unless the laboratory is
supervised by a qualified person, advice laboratory evaluation of nutrients;
should be obtained before determining * Better data on the physiological signifi-
which tests should be undertaken and cance of the test used.
how the data are to be interpreted.
Laboratory Assessment for Individual
Standards for Interpretation of Nutrients
Laboratory Data The following is a description of methods
The interpretation of laboratory data will employed to assess those nutrients which can be
always be a matter for some disagreement, since measured by laboratory evaluations, as well as an
the prime objective is to detect "risk of defi- indication of when and why they should be em-
ciency" before clinical evidence of disease de- ployed. The specific tests that might be used are
velops. The standards may also vary somewhat not named, since they vary from laboratory to lab-
with the specific methods used, since the methods oratory. This comprises a brief summary of labo-
vary in specificity and reproducibility. All methods ratory methods; specific references for each nutri-
used in a survey should be standardized and an ent are listed in Appendix C to this section and
appropriate design developed so that the results should be consulted before tests are undertaken.
do not vary beyond acceptable limits during the Circulating levels of most of these elements can
course of the survey. This is best accomplished be measured in blood or plasma, and deficiencies
by repeated evaluation of standards previously can be estimated.
checked by a recognized standards laboratory. Protein-Protein deficiency in the United
The criteria used in the Ten State Nutri- States appears to be uncommon. Reduced levels
tion Survey are included in Appendix B for current have been reported in pregnant women, but it is
information and for reference purposes. These, in uncertain whether the criteria for "normal" levels
LABORATORY ASSESSMENT 29
in non-pregnant women also apply in pregnancy. thione reductase in red blood cells and the "FAD
Protein deficiency produces a fall in serum effect" (the increase in activity due to the addition
protein levels, especially serum albumin, but this of flavin adenine dinucleotide, FAD). This may be-
is not a particularly sensitive index and is not come the method of choice, although experience
specific for protein deficiency. Serum protein with it is limited. Microbiological and chemical
levels may be maintained for a considerable methods are also available to measure riboflavin
period of time, despite limited protein intake. in blood.
Total serum protein and serum albumin determi- 4. Niacin-Pellagra, the deficiency disease
nations are standard clinical chemical procedures. caused by niacin deficiency, is now rare in the
Recently, nitrogen/creatinine ratios in the urine United States, although it may occasiopially be
and ratios of specific amino acids in plasma have seen in alcoholics or other persons with severely
been recommended as measures of protein- restricted diets. Niacin is derived from the amino
calorie malnutrition, but have not been extensively acid, tryptophan, and thus pellagra is ordinarily
studied in the United States. associated with populations consuming little
Water-Soluble Vitamins tryptophan (corn and sorghum-based diets).
1. Vitamin C (Ascorbic Acid)-Clinical Estimation of N'-methylnicotinamide in the urine
scurvy, the disease associated with severe vita- has been the traditional method of determining
min C deficiency, is uncommon in the United adequacy of niacin intake. As with thiamine,
States, although infants, alcoholics, the elderly, urinary excretion is not a generally satisfactory
and neglected persons may be scorbutic. Never- method for surveys. Microbiologic methods are
theless, reduced plasma levels have been re- available for the estimation of circulating niacin.
ported in a significant portion of people in many 5. Folacin-Folate deficiency results in ane-
nutrition surveys. Serum levels of vitamin C vary mia. Low circulating levels have been reported to
substantially and depend, to a considerable de- be common in pregnant women and in women
gree, on the intake immediately preceding the test. taking birth control pills and other estrogenic
This must be borne in mind in interpreting the re- medications. The circulating level of folate in
sults of plasma vitamin C levels. plasma or red blood cells is utilized in estimating
2. Thiamine-Although clinical evidence of adequacy of intake, although the standards for in-
thiamine deficiency is very uncommon in most terpreting data are controversial. Folate deficiency
United States populations, it is probably an impor- increases excretion of FIGLU (formimino-glutamic
tant cause of morbidity in alcoholics. The usual acid) in urine, but this also may occur in vitamin
test is the estimation of thiamine excretion in the B12 deficiency.
urine, ordinarily made on "spot samples" col- 6. Vitamin BB (Pyridoxine)-Vitamin B6 has
lected in the field or in the clinic, rather than 24- been too little studied in relation to its effects on
hour collection. However, thiamine content of nutritional status. This is unfortunate because di-
spot samples in the same individual vary substan- etary surveys have indicated that vitamin B6 in-
tially, and this is a relatively insensitive test of the take may be a rginal in some population groups
nutritional status. in the United ctates. Evidence is also accumulat-
A functional enzyme test is preferable. ing that Vitami B6 requirements may be markedly
Transketolase is an enzyme which requires thia- increased durin g pregnancy and in women tak-
mine (as thiamine pyrophosphate, TPP) for its ing birth contr Il pills. Tests suggested for evaluat-
function. Transketolase in red blood cells and the ing vitamin B6 status include: measurement of vari-
"TPP effect" (the increase in activity due to the ous B6 metabol ites in the urine, estimation of tryp-
addition of TPP) may be measured, and is probably tophan metabo ites in the urine after a tryptophan
the method of choice since activity does change dose, estimatioi of transaminase in blood cells or
with moderate depletion of thiamine. It has not plasma and its response to the addition of pyri-
yet been applied to broadly-based surveys. Micro- doxal phospha te, and estimation of vitamin Be in
biologic assays are also available to estimate thia- the blood.
mine in blood. 7. Vitamin B1l-Vitamin B12 deficiency
3. Riboflavin-A variety of lesions associ- causes anemia, and inability to utilize the vitamin
ated with riboflavin deficiency are not uncommon B12 in food is the cause of pernicious anemia.
in many parts of the world. The specificity of Vitamin B12 deficiency due to inadequate intake
these lesions as indicators of riboflavin depletion has been reported in some vegetarians. Reduced
is, however, in doubt. The usual method of esti- blood levels of vitamin B12 have been reported in
mating riboflavin adequacy is by examination of pregnant women and those taking birth control
urinary excretion. Such tests are not completely pills. Analytical estimation of vitamin B12 is by
satisfactory due to the variability in riboflavin ex- microbiologic techniques or by radioisotopic
cretion. Recently, an enzyme functional test has methods.
been proposed. This is the estimation of gluta- 8. Other Water-Soluble Vitamins-Panto-
30 AJPH SUPPLEMENT, Vol. 63, NOVEMBER, 1973
thenic acid, biotin, and choline are the other water- by exposure to sunlight, dietary evaluation is also
soluble vitamins. There is little doubt that they are unsatisfactory.
essential, but they do not seem to present prac- 3. Vitamin E-Clinical evidence of vitamin
tical nutritional problems in most populations. E deficiency has only been reported in infants.
However, this may be a false assumption since Deliberate restriction of vitamin E in adults may
methods for estimating nutritional status with re- result in increased red blood cell fragility under
gard to these nutrients have not been developed certain conditions. Since there is abundant evi-
and little is known of the probable requirement. dence in animals that increased consumption of
Microbiologic or chemical methods for their esti- unsaturated fats increases the need for vitamin E,
mation are available. this vitamin deserves more study than it has re-
Fat-Soluble Vitamins ceived in the past.
The fat-soluble vitamins A, D, E, and K are Vitamin E may be measured in the serum
best absorbed in the presence of some fat in the directly. Methods also exist for estimating the fra-
diet. Thus, diseases which interfere with fat ab- gility of red cells.
sorption may also impair absorption of fat-soluble 4. Vitamin K-Since vitamin K is synthe-
vitamins. Patients with sprue, gluten enteropathy, sized by the flora in the intestinal tract, deficiency
and other absorption problems may manifest de- is thought to occur only in very young infants be-
ficiencies even though the dietary intake appears fore the 'lora are established, and in diseases in
adequate. It should also be noted that vitamins A which fat absorption or the utilization of vitamin
and D are well known to be toxic when consumed K is abnormal. There is no evidence that tests for
in excess, and represent a potential problem in vitamin K function need to be included in general
our vitamin-conscious society. nutrition surveys.
1. Vitamin A and Beta-Carotene-Vitamin A It might be noted that while most nutrients
does not occcur directly in plant foods, but the -carbohydrate, fat, protein, vitamins, and min-
body converts plant pigment, beta-carotene, into erals-are supplied by diet, some portion of man's
vitamin A. Both are ordinarily measured in serum vitamin needs is supplied from synthesis by gas-
as the criteria of vitamin A adequacy. A low caro- trointestinal microorganisms. Such is the case for
tene level, of course, only indicates limited con- vitamins K, B1, B12, folacin, biotin, and other micro-
sumption of green leafy and yellow vegetables, nutrients. Thus, any intestinal tract pathology will
not necessarily vitamin A deficiency. reduce availability of these vitamins, as will anti-
Surprisingly, substantial numbers of people biotic therapy which modifies intestinal flora.
examined in the Ten State Nutrition Survey had Minerals
low serum vitamin A levels, indicating some de- 1. Iron-A deficit of iron eventually results
gree of vitamin A deficiency. There are also re- in anemia. The most common method of detect-
ports that a significant number of children in ing anemia is by the measurement of the hemo-
Canada and the United States (at autopsy) had no globin level in the blood or the hematocrit. Anemia
vitamin reserves in their livers, the primary site may be caused by a variety of nutritional or non-
of vitamin A storage. Xerophthalmia, caused by nutritional factors other than iron deficiency.
severe vitamin A deficiency, is rare in the United
States. The significance of the low levels of vita- Modest degrees of anemia caused by in-
min A are thus open to some debate. Night blind- adequate iron intake are common in the United
ness, an. early sign of vitamin A deficiency, which States (10% of the population according to one
may be estimated by dark adaptation tests, has estimate), especially in women and children since
not yet been adequately investigated. iron requirements are increased by growth and
2. Vitamin D-Rickets, the childhood defi- menstrual blood loss. The extent of iron deficiency
ciency disease resulting from an inadequate vita- in adult men is a matter of considerable interest
min D intake, is relatively rare in the United and debate. Several different standards have been
States, but does occur occasionally. Osteomalacia recommended for the evaluation of hemoglobin
in the elderly, possibly due to a lack of vitamin D, and hematocrit, and the extent of anemia observed
is reported to be relatively common in many in any population depends, of course, upon the
countries. standards used.
Elevated serum alkaline phosphatase levels a. Hematocrit-The hematocrit, the per-
were once thought to be indicative of vitamin D centage of packed red cells in whole blood, is a
deficiency, but this is not an infallible test. No suit- standard clinical procedure available in all com-
able methods are available to survey populations. munity hospitals and other laboratories.
Although there is relatively little evidence to indi- b. Hemoglobin-This determination is a
cate that vitamin D deficiency is a general prob- simple colorimetric procedure that is standard in
lem, the situation is somewhat uncertain. Since all clinical laboratories. Ordinarily, both hemo-
vitamin D may be supplied by synthesis invoked globin and hematocrit are determined and are pref-
LABORATORY ASSESSMENT 31
erable to blood cell counts since they involve less other trace elements has not been standard-
laboratory error. ized, and criteria for adequacy have not been
c. Serum Iron and Transferrin-Iron is car- developed.
ried in the plasma by a specific protein called Lipids and other Serum Determinations-
transferrin. A reduced saturation of transferrin and Circulating levels of Qholesterol, triglycerides, glu-
a reduced serum iron level provide more specific cose, various enzymes, and hormones such as
evidence of iron deficiency and will detect reduced insulin, glucagon, etc., also have important im-
iron stores before anemia develops. If iron levels plications with regard to nutritional status and to
and transferrin saturation are normal in the face certain disease states, especially coronary heart
of anemia, the anemia does not represent iron disease and diabetes. These and other measure-
deficiency. ments have been included in some surveys and
2. Calcium-Although substantial numbers should be considered as integral parts of nutrition
of people in the United States consume less cal- surveys in the future.
cium than ordinarily recommended, there is little The National Heart and Lung Institute,
or no evidence of calcium deficiency. Blood levels Bethesda, Md., is sponsoring Lipid Research Cen-
of calcium are essentially constant over a wide ters and Centers for Multiple Risk Factor Interven-
range of intakes, and measurement of blood cal- tion Trials for the prevention of coronary heart
cium does not provide adequate evaluation of disease. This illustrates the present and future
dietary calcium. No suitable laboratory or clinical public health significance of the use of serum
methods for surveys are available for monitoring cholesterol and triglyceride determinations in the
the adequacy of calcium intakes.. screening of persons with elevated levels of lipids
3. Iodine-The adequacy of iodine intake is who may be at high risk of developing coronary
usually estimated by the clinical evaluation of heart disease. Moreover, physicians in evaluation
thyroid enlargement or goiters. However, not all of intervention programs are using serum choles-
goiters are the result of iodine insufficiency. An ap- terol and triglyceride levels to assess response to
proximation of iodine intake may be determined diets designed to lower these lipid factors. The
by relating urinary iodine to urinary creatinine. current great interest in serum lipids has been
Other clinical determinations, for example, plasma- generated by:
bound iodine (PBI), reflect the functional utiliza- a. The identification by the Framingham
tion of iodine and are standard clinical laboratory Study and other studies of serum cholesterol level
tests. as a risk factor in coronary heart disease (along
4. Other Minerals-A number of essential with hypertension, smoking, obesity, and other
minerals are not discussed in detail here. These determinants);
include magnesium, manganese, zinc, fluoride, b. The indication by prospective studies
chromium, selenium, copper, sodium, potassium, (such as the Anti-Coronary Club of the City of
phosphorus, chloride, and others. New York Department of Health), that the lowering
Currently, there is widespread interest and of serum cholesterol by nutritional means has been
research into the nutritional and metabolic aspects attended by reduced coronary heart disease mor-
of certain of these trace elements. Clinical evi- bidity and mortality;
dence of magnesium and zinc deficiencies has c. The relatively recent emergence of the
been found in hospital populations, and relatively hyperlipidemias (a condition in which both genetic
low levels of intake of both minerals are not un- and environmental factors collaborate to raise
common in the United States. However, method- serum lipids) as having possible public health
ology to evaluate nutritional status of these and significance.

32 AJPH SUPPLEMENT, Vol. 63, NOVEMBER, 1973

 Appendix A
Sample Collection and Preservation

While specific instructions for sample col- tained from the subject may influence the findings,
lection and preparation vary according to the spe- particularly if this is done shortly after the indi-
cific assays to be done, certain general rules ap- vidual has eaten or taken a vitamin supplement.
ply to all procedures. Blood and urine samples Optimally, blood samples would be taken in the
will deteriorate rapidly unless they are properly morning before breakfast or any food or drink is
managed and preserved for transmission to the consumed. For urinalysis, the optimal sample is a
laboratory for assay. Optimal attention must be total 24-hour collection. If this is not possible, the
given to specimen collection, preservation, and best sample is the first upon arising. When that is
transportation. No level of excellence in clinical not feasible, compromises must be made, and con-
technique can correct for changes in perishable sidered, in interpreting the data. The best com-
nutrients resulting from faulty or careless collec- promise would be to obtain samples at least 2-3
tion, preservation, or shipping of specimens. No hours after the last meal. Ideally, all samples are
laboratory, regardless of the degree of sophistica- to be collected under the same circumstances.
tion, can improve the quality of inadequate speci- As indicated above, some indication of nu-
mens. The following general procedures should tritional status may be obtained on the basis of a
be followed: urine sample alone for a variety of nutrients. Ac-
Blood drawn into vacuum tubes can be cordingly, much can be accomplished toward eval-
placed directly on ordinary ice, and urine can be uating nutritional status even if the evaluating team
acidified (with acetic or hydrochloric acid) and does not include personnel to draw blood samples.
chilled similarly. Where necessary, due to the Moreover, it is also noteworthy that many nutri-
lability of the nutrient, such as ascorbic acid, it tional evaluation procedures may be available in
will be necessary to make an acid filtrate of blood hospital clinic laboratories, i.e. hematocrit, hemo-
immediately, and store by freezing the filtrate (in globin, iron, transferrin saturation, folic acid, B12,
this case, the acid would be trichloracetic or meta- and often others.
phosphoric). Similarly, separated serum should be
properly preserved and frozen immediately for A crucial need in laboratory nutritional as-
folic acid evaluation. Certain enzymes are unstable sessment is the coordinated standardization of the
to freezing, necessitating immediate assay, while various tests. This is essential in order to assure
others are stable in the freezer for longer periods that data obtained from different laboratories
of time. To avoid spurious results, therefore, it is and/or localities can be compared with each other
mandatory to carefully review each analytical tech- in order to draw valid conclusions. Indeed, this is
nique to be used with a view toward its specific essential if one is to use uniform criteria for judg-
needs for sample collection and preservation. ing nutrient adequacy. For the routine clinical
As much of the sample preparation as pos- chemical assay work, there are standards, cali-
sible, including chilling and/or freezing, should brators, and plasma or serum controls available
be done at the collection site. When properly pre- which the laboratory can use to assure accuracy
pared, most samples can be frozen, at least for a and precision of its own analytical procedures.
short period of time to await assay. It is most im- This assures the laboratory that its data relate to
portant that the samples remain frozen until as- similar work done in other clinical laboratories.
sayed, particularly if they are to be shipped some No similar synthetic standards are available as
distance to the analytical laboratory. This can be yet for the nutritional evaluation techniques. Rath-
done effectively only by shipping with dry ice in er, it is necessary to coordinate one's work with
a properly insulated polystyrofoam container. The that of established laboratories. This may be ac-
dry ice does not usually last more than 60-72 complished by comparing the analytical values
hours, under the best conditions. Some enzymes, obtained by the laboratory involved in the local
i.e. transaminases, are destroyed by refreezing, so evaluation program, with values obtained from
that these precautions are imperative. submitting replicate samples under code to a
The time of day that samples are to be ob- standard reference laboratory.
LABORATORY ASSESSMENT 33
 Appendix B
Table of Current Guidelines for Criteria of Nutritional Status
for Laboratory Evaluation
Nutrient Age of Subject Criteria of Status
and Units (years) Deficient Marginal Acceptable

*Hemoglobin 6-23 mos. Upto 9.0 9.0- 9.9 10.0+

(gm/i OOml) 2-5 Upto 10.0 10.0-10.9 11.0+
6-12 Upto 10.0 10.0-11.4 11.5+
13-16M Upto 12.0 12.0-12.9 13.0+
13-16F Upto 10.0 10.0-11.4 11.5+
16+M Upto 12.0 12.0-13.9 14.0+
16+F Up to 10.0 10.0-11.9 12.0+
Pregnant
(after 6+ mos.) Upto 9.5 9.5-10.9 11.0+
*Hematocrit Up to 2 Up to 28 28-30 31+
(Packed cell volume 2-5 Upto 30 30-33 34+
in percent) 6-12 Upto 30 30-35 36+
13-16M Upto 37 37-39 40+
13-16F Upto 31 31-35 36+
16+M Up to 37 37-43 44+
16+F Upto 31 31-37 33+
Pregnant Up to 30 30-32 33+
*Serum Albumin Up to 1 Up to 2.5 2.5+
(gm/i OOml) 1-5 Up to 3.0 3.0+
6-16 Up to 3.5 3.5+
16+ Upto 2.8 2.8-3.4 3.5+
Pregnant Upto 3.0 3.0-3.4 3.5+
*Serum Protein Up to 1 Up to 5.0 5.0+
(gm/1OOml) 1-5 Up to 5.5 5.5+
6-16 Up to 6.0 6.0+
16+ Upto 6.0 6.0-6.4 6.5+
Pregnant Upto 5.5 5.5-5.9 6.0+
*Serum Ascorbic Acid All ages Up to 0.1 0.1-0.19 0.2+
(mg/ 1 OOmI)
*Plasma vitamin A All ages Upto 10 10-19 20+
(mcg/100 ml)
*Plasma Carotene All ages Up to 20 20-39 40+
(mcg/100 ml) Pregnant 40-79 80+
*Serum Iron Up to 2 Up to 30 30+
(mcg/100 ml) 2-5 Up to 40 40+
6-12 Up to 50 50+
12+M Up to 60 60+
12+F Up to 40 40+
*Transferrin Saturation Up to 2 Upto 15.0 15.0+
(percent) 2-12 Upto 20.0 20.0+
12+M Up to 20.0 20.0+
12+F Upto 15.0 15.0+
**Serum Folacin All ages Up to 2.0 2.1-5.9 6.0+
(ng/ml)
**Serum vitamin B12 All ages Up to 100 100+
(pg/mI)
* Adapted from the Ten State Nutrition Survey
** Criteria may vary with different methodology.

34 AJPH SUPPLEMENT, Vol. 63, NOVEMBER, 1973

 Appendix B
Table of Current Guidelines for Criteria of Nutritional Status
for Laboratory Evaluation (continued)
Nutrient Age of Subject Critera of Status
and Units (years) Deficient Marginal Acceptable
*Thiamine in Urine 1-3 Up to 120 120-175 175+
(mcg/g creatinine) 4-5 Upto 85 85-120 120+
6-9 Up to 70 70-180 180+
10-15 Up to 55 55-150 150+
16+ Up to 27 27- 65 65+
Pregnant Upto 21 21- 49 50+
*Riboflavin in Urine 1-3 Up to 150 150-499 500+
(mcg/g creatinine) 4-5 Up to 100 100-299 300+
6-9 Upto 85 85-269 270+
10-16 Up to 70 70-199 200+
16+ Up to 27 27- 79 80+
Pregnant Up to 30 30- 89 90+
**RBC Transketolase- All ages 25+ 15- 25 Up to 15
TPP-effect (ratio)
**RBC Glutathione All ages 1.2+ Up to 1.2
Reductase-FAD-effect
(ratio)
**Tryptophan Load Adults 25+(6 hrs.) Up to 25
(mg Xanthurenic (Dose: 100mg/kg 75+(24 hrs.) Up to 75
acid excreted) body weight)
**Urinary Pyridoxine 1-3 Upto 90 90+
(mcg/g creatinine) 4-6 Up to 80 80+
7-9 Up to 60 60+
10-12 Up to 40 40+
13-15 Upto 30 30+
16+ Upto 20 20+
*Urinary N'methyl All ages Up to 0.2 0.2-5.59 0.6+
nicotinamide Pregnant Up to 0.8 0.8-2.49 2.5+
(mg/g creatinine)
**Urinary Pantothenic All ages Up to 200 200+
Acid (mcg)
**Plasma vitamin E All ages Up to 0.2 0.2-0.6 0.6+
(mg/ 1OOml)
*
*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 6. Pyridoxine
Fundamentals of Clinical Chemistry, edited by Norbert Baker, H. and Frank, 0. Vitamin B6 in "Clinical Vita-
W. Tietz, W. B. Saunders Co., 1970, Philadelphia, minology: Methods and Interpretation." lnterscience
London, Toronto (new edition coming Spring, 1974). Publications, New York, N.Y. pp. 66-81, 1968.
The Vitamins, Sebrell/Harris, Vols. 1 & 4 (in prepara- Brin, M. A simplified Toepfer-Lehmann Assay for the
tion). P. Gyorgy and W. M. Pearson, Academic Press, three Vitamin Be Vitamers. Method in Enzymology
7 vols. 1967-1973. XVIII, 519-523, 1970.
Natelson, S. Techniques of Clinical Chemistry (3rd ed.) Hamfelt, A. Age variation of vitamin B6 metabolism in
C. C. Thomas Co. 1971. man. Clin. Chim. Acta. 10:48, 1964.
B. References for Individual Nutrients Luhby, A. Leonard, Brin, M., Gordon, M., Davis, P.,
1. Protein Murphy, M. and Spiegel, H. Vitamin Be metabolism in
Electrophoretic separation of serum proteins, Manual users of oral contraceptive agents 1. Abnormal
for Nutrition Surveys, ICNND, 2nd edition, p. 147, urinary xanthurenic acid excretion and its correction
U.S. Government Printing Office, Washington, D.C. by pyridoxine. Amer. J. Clin. Nutr. 24: pp. 684-93,
1963.* June 1971.
Oberman, et al. Electrophoretic analysis of serum pro- Price, S. A. et al. Effects of dietary vitamin B6 defi-
teins in infants and children. N. Eng. J. Med., ciency and oral contraceptives on the spontaneous
225:743, 1956. urinary excretion of 3-hydroxy anthranilic acid. Am.
Total serum protein, albumin and globulin by a modi- J. Clin. Nutri. 25:494, 1972.
fied Biuret Tec4nique: Manual tor Nutrition Surveys, Sauberlich, H. E. et al. Biochemical Assessment of
ICNND, 2nd edition, p. 133, U.S. Govemment Printing the Nutritional Status of Vitamin Be in the human.
Office, Washington, D.C. 1963.* Am. J. Clin. Nutr. 25:629, 1972.
2. Hematocrit Tryptophan load test-xanthurenic acid in serum. Man-
Macro: Manual for Nutrition Surveys, ICNND, 2nd edi- ual for Nutrition Surveys, ICNND, 1st edition, p. 88,
tion, p. 116, U.S. Government Printing Office, Wash- U.S. Government Printing Office, Washington, D.C.
ington, D.C. 1963.* 1963.*
Micro: Clinical Diagnosis by Laboratory Methods, 14th Note: The test is now modified to giving a load of
edition, Todd and Sanford, eds., p. 146, W. B. Saun- 2 gm L-tryptophan. Approximately 67% of the
ders Co., Philadelphia, 1969. xanthurenic acid is excreted in the first 8 hours.
3. Hemoglobin 7. Folacin
Manual for Nutrition Surveys, ICNND, 2nd edition, p. Jukes, T. H. Assay of compounds with tolic acid ac-
115, U.S. Govemment Printing Office, Washington, tivity. Meth. Bioch. Anal. 2:121, 1955.
D.C. 1963* Luhby, A. L. and J. M. Cooperman. Folic acid defl-
4. Iron ciency and its Inter-relationship with vitamin B,2
Brit. J. Hematol., 20:451, 1971. metabolism. Adv. Metab. Discord. 1:263-334, 1964.
Ramsey, W. N. M. The determination of the total iron Water, et al. J. Clin. Path. 14:335, 1961.
binding capacity of serum. 2:221, 1957. Clin. Chim. 8. Vitamin B13,
Acta. 2:221, 1954. Baker, H. and Frank, 0. Vitamin B,2 In "Clinical Vita-
Scarlata, R. W. and Moore, E. W. A micromethod for minology: Methods and Interpretation," Interscience
the determination of serum iron and serum iron- Pubs., New York, N.Y., pp. 116-141, 1968.
binding capacity. Clin. Chem. 8:360, 1962. Lau, K. S., Gottlieb, C., Wasserman, L. R. and Herbert,
Woodruff, C. W. A Micromethod tor serum iron deter- V. Measurement of serum Vitamin B2 Level using
mination, J. Lab. Clin. Med. 53:955, 1959. radioisotope dilution and coated charcoal. Blood
5. Ascorbic Acid 26:202, 1965.
Cheraskin, E., et al. A lingual vitamin C test. Int. J. Skeggs, H. R., Microbiological Assay for Vitamin Ba,.
Vit. Res. 38:114, 1968. Methd. Bioch. Anal. 14:53, 1966.
Serum vitamin C (ascorbic acid)-Dinitrophenylhyrra- 9. Thiamine
zine Method. Manual for Nutrition Surveys, ICNND, Baker, H. and Frank, 0. Thiamine In "Clinical Vita-
2nd edition, p. 117, U.S. Government Printing Office, minology: Methods and Interpretation," Interscience
1963.* Pubs., Inc., New York, N.Y., p. 7-19, 1968.
Serum vitamin C-Micro procedure, Ibid. p. 119.* Brin, M. "Transketolase and the TPP-Effect in Assess-
* Out of print. ing Thiamine Adequacy, In "Vitamins and Coen-

36 AJPH SUPPLEMENT, Vol. 63, NOVEMBER, 1973

 Appendix C (continued)
zymes: Methods in Enzymology" Academic Press, 11. Niacin
N.Y., Vol. XVIII, pp. 125-133, 1970. Baker, H. and Frank, 0. Nicotinic Acid In "Clinical
Dreyfus, P., Clinical Application of Blood Transketolase Vitaminology: Methods and Interpretation," Inter-
Determinations, N. Eng. J. Med. 267:596, 1962. science Pubs., New York, N.Y., pp. 87-108, 1968.
(microassay) N'Methyl Nicotinamide in Urine, Manual for Nutrition
Erythrocyte Transketolase Activity. M. Brin In Methods Surveys, ICNND, 2nd edition, p. 142, U.S. Gov't Print.
of Enzymatic Analysis, H. U. Bergmeyer, ed., In Off., 1963.*
press 1974. 12. Pantothenic Acid
Thiamine in Urine, Manual for Nutrition Surveys, Baker, H., and Frank, 0. Pantothenic Acid in "Clinical
ICNND, 2nd edition, p. 136, U.S. Gov't Print. Off., Vitaminology: Methods and Interpretation," Inter-
1963.* science Pubs., New York, N.Y., pp. 54-63, 1968.
Creatinine in Urine, Picrate Method, ibid, p. 135.* Hatano, M., Microbiological Assay of Pantothenic Acid
10. Riboflavin in Blood and Urine, J. Vitaminol. 8:134, 1962.
Baker, H. and Frank, 0. Riboflavin In "Clinical Vita- 13. Vitamin A
minology: Methods and Interpretation," Interscience Gary, P. J., et al, Vitamin A. Fluorometry and Uses of
Pubs., New York, N.Y., pp. 43-52, 1968. Silicic Acid Technique. Clin. Chem. 16:766, 1970.
Bamji, M. S., Glutathione Reductase Activity in Red Neeld, J. B., and Pearson, W. N. Macro- and Micro-
Blood Cells and Riboflavin Nutritional Status in Methods for the Determinations of Serum Vitamin A
Humans, Clin. Chim. Acta. 26:263, 1969. using Trifluoracetic Acid, J. Nutr. 79:454, 1963.
Glatzle, D., et al, Method for the Detection of a Bio- 14. VItamin E
chemical Riboflavin Deficiency. Investigations of the Baker, H. and Frank, 0. Vitamin E In "Clinical Vitamin-
Vitamin B2 Status in Healthy People and Geriatric ology: Methods and Interpretation," Interscience
Patients, Int'l J. Vit. Res., 40:166, 1970. Pubs., New York, N.Y., pp. 169-75, 1968.
Urinary Riboflavin, Manual for Nutrition Surveys, Hashim, S. A. and Schruttinger, G. R., Rapid Determi-
ICNND, 2nd edition, p. 140, U.S. Gov't Print. Off. nation of Tocopherol in macro- and micro-quantities
1963.* of Plasma. Am. J. Clin. Nutr. 19:137, 1966.
Creatinine in Urine-Picrate Method, ibid, p. 135.* * Out of print.

LABORATORY ASSESSMENT 37