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