Caballero B. (ed.) Encyclopaedia of Food Science, Food Technology and Nutrition. Ten-Volume Set

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progress over time. In addition, these techniques are

useful in population-based field research of nutrition-

related disorders.

See also: Body Composition; The Problem of

Malnutrition; Nutritional Assessment: Importance of

Measuring Nutritional Status; Obesity: Etiology and

Diagnosis

Further Reading

Bjorntorp P (1987) Classification of obese patients and

complications related to the distribution of surplus fat.

American Journal of Clinical Nutrition 45: 1120–1125.

Durnin JVGA and Womersley J (1974) Body fat assessed

from the total body density and its estimated skinfold

thickness: measurements on 481 men and women aged

from 16 to 72 years. British Journal of Nutrition 32:

77–97.

Forbes GB (1990) The abdomen:hip ratio. Normative data

and observations on selected patients. International

Journal of Obesity 14: 149–157.

Gibson RS (1990) Principles of Nutritional Assessment.

London: Oxford University Press.

Heymsfield S, McManus CB, Smith J, Stevens V and Dixon

DW (1982) Anthropometric measurement of muscle-

mass: revised equations for calculating bone-free arm

muscle area. American Journal of Clinical Nutrition

36: 680–690.

James WPT, Ferro-Luzzi A and Waterlow JC (1988) Defin-

ition of chronic energy deficiency in adults. European

Journal of Clinical Nutrition 42: 909–981.

Lapidus L, Bengtsson Q, Larsson B et al. (1984) Distribu-

tion of adipose tissue and risk of cardiovascular death: a

12 year follow-up of participants in the population of

women in Gothenburg, Sweden. British Medical Journal

289: 1257–1261.

Larsson L, Svardsudd K, Welin L et al. (1984) Abdominal

adipose tissue distribution and risk of cardiovascular

disease and death: a 13 year follow-up of participants

in the study of men born in 1913. British Medical

Journal 288: 1401–1404.

Lohman TG (1981) Skinfolds and body density and their

relation to body fatness: a review. Human Biology 53:

181–225.

Lohman TG (1992) Advances in body composition assess-

ment. Current Issues in Exercise Science. Monograph

No. 3. Champaign, IL: Human Kinetics.

Siri WE (1961) Body composition from fluid spaces and

density: Analysis of methods. Techniques for Measuring

Body Composition, pp. 223–244. Washington, DC:

National Academy of Sciences, National Research

Council.

Smalley KJ, Knerr AN, Kendrick ZV, Colliver JA, and

Owen OE (1990) Reassessment of body mass indices.

American Journal of Clinical Nutrition 52: 405–408.

Physical Status. The Use and Interpretation of Anthropom-

etry. (1995) WHO Technical Report 854. Geneva:

WHO.

Biochemical Tests for Vitamins

and Minerals

A V Lakshmi and M S Bamji

, National Institute of

Nutrition, Hyderabad, AP, India

Introduction

0001Increasing attention is being given to the definition

of marginal malnutrition since the elimination of

clinical signs of deficiency per se is not sufficient for

optimal health. Biochemical measurements on easily

available body fluids represent the most objective

assessment of nutritional status and often provide

subclinical information. It also provides information

on micronutrient toxicity. During the development of

any deficiency disease, biochemical changes precede

clinical symptoms and hence biochemical tests help to

identify the disease at the subclinical stage. They also

help to confirm the clinical diagnosis.

0002An ideal biochemical test should be specific, sensi-

tive, and indicative of tissue depletion at an early

stage but not immediate dietary intake. However,

we do not have an ideal biochemical test for most

nutrients and the method selected depends on the

situation in which it is applied. Often, more than

one test is required. Classification of an individual

as severely deficient, marginally deficient, normal,

or intoxicated may require different tests. In this

chapter biochemical tests used for the nutritional

status assessment of fat- and water-soluble vitamins

and of a few minerals – sodium, potassium, calcium,

iron, iodine, zinc, copper, and selenium are discussed.

Types of Laboratory Tests

0003Laboratory tests for the assessment of vitamin or

mineral nutritional status can be placed in the

following categories:

0004Measurement of excretion of nutrient or its

metabolite in urine, without or after a bolus load

(load return test)

0005Measurement of nutrient level and/or its active

metabolites in blood

0006Assay of activity of a vitamin-dependent enzyme

and its in vitro stimulation with corresponding

coenzyme in the blood. (See Coenzymes.)

0007Measurement of rise in the concentration of a

metabolite in the blood or urine (resulting from

inadequate intake of the vitamin) after adminis-

tering a load of the precursor

Current address: Dangoria Charitable Trust, 1–7–1074, Hydera-

bad 500020, India.

4184 NUTRITIONAL ASSESSMENT/Biochemical Tests for Vitamins and Minerals

0008 Measurement of excretion of vitamin or its metab-

olite in urine can be carried out on a 24-h sample,

random sample, or in the first voided morning sample

of urine. Although a 24-h specimen provides the most

accurate estimate, it is difficult to collect it in the free-

living population. Measurements in random samples

or a first voided urine sample, and expressing the

value per gram of creatinine, may be useful for popu-

lation studies but are not very reliable for individual

assessment.

0009 The limitation regarding the interpretation of urin-

ary data is that it is influenced by immediate dietary

intake and may not reflect tissue stores. Load return

tests may yield better information regarding the state

of tissues but they are seldom used now. Certain

physiological and pathological conditions, as well as

ingestion of drugs, influence urinary excretion of

certain vitamins and minerals.

0010 Either whole blood or different components such

as plasma, erythrocytes, leukocytes, and platelets has

been used for the assessment of vitamin and mineral

status. The choice of the blood components is decided

by the concentration of the nutrient and its sensitivity

to vitamin or mineral depletion. High-performance

liquid chromatography (HPLC) technique has revived

interest in these measurements due to improved

sensitivity. (See Chromatography: High-performance

Liquid Chromatography.)

0011 Details regarding enzymatic tests and metabolite

tests will be discussed while dealing with the individ-

ual vitamins.

Guidelines for Interpretation of Values

0012 Availability of standard reference material (serum

and erythrocyte hemolysates), use of standardized

procedures, and strict quality control measures are

necessary for arriving at interpretative guidelines.

Since reference quality control samples are not

available for many vitamins, and different laboratory

procedures are adopted by various workers, only ten-

tative guidelines based on the values obtained with

apparently healthy, well-nourished controls are avail-

able at present for interpretation of data. (See Vita-

mins: Determination.)

Fat-soluble Vitamins

Vitamin A

0013 Serum vitamin A level is not a sensitive indicator of

vitamin A stores. The levels are however indicative of

vitamin A status when the stores are either fully replete

or totally deplete and in the absence of infection. Serum

levels below 0.35 mmol l

1

are usually associated with

clinical signs of deficiency. Serum vitamin A levels

above 0.70 mmol l

1

are considered satisfactory and

reflect adequate liver reserves (Table 1).

0014Marginal vitamin A status can be assessed by

the relative dose–response (RDR) test or the modified

relative dose–response (MRDR) test. The RDR test

consists of determination of the plasma levels of vita-

min A at baseline and 5 h after the administration of a

small oral or intravenous dose of retinylpalmitate or

retinyl acetate. An increase of more than 20% is

indicative of inadequate hepatic reserves.

0015In the MRDR test, 3,4-didehydroretinol (vitamin

) is administered instead of retinol. This test re-

quires only one blood sample for analysis. The ratio

of dehydroretinol to retinol is used to assess liver

vitamin A concentrations (Table 1).

0016Use of deuterated vitamin A for measuring the total

body pool by means of isotope dilution is currently

being refined.

Toxicity

0017High plasma levels of retinyl esters in the presence of

high plasma levels of vitamin A is a sign of vitamin A

toxicity. In conditions of normal vitamin A status, the

plasma levels of retinyl esters is about 5%.

0018Spectrophotometric, HPLC fluorometric, and cal-

orimetric techniques are used for measuring plasma

vitamin A level. (See Retinol: Properties and Deter-

mination.)

Vitamin D

0019Measurement of serum 25-hydroxycholecalciferol,

the major circulating metabolite of vitamin D, is the

best indicator of vitamin D status. In healthy adults

its concentration in serum varies from 20 to 130 nmol

1

and less than 12.5 nmol l

1

is generally considered

inadequate. It can be measured by HPLC or by com-

petitive protein-binding assay. (See Cholecalciferol:

Properties and Determination.)

Vitamin E

0020Vitamin E status is normally assessed by measuring

levels of the vitamin in plasma or serum and other

tbl0001Table 1 Tentative guidelines for the interpretation of

biochemical parameters of vitamin A status

Biochemicalmeasurement Acceptable Mediumrisk High risk

Serum (mmol l

1

) > 70 35–70 < 35

Liver (mmol g

1

) > 0.70 0.17–0.70 < 0.17

Relative dose–response (%) < 20 > 20

Modified relative

dose–response (DR/R)

< 0.03 > 0.03

DR, 3,4-didehydroretinol; R, retionol.

NUTRITIONAL ASSESSMENT/Biochemical Tests for Vitamins and Minerals 4185

tissues (red cells, platelets, adipocytes) by spectro-

photometric, fluorometric, or HPLC methods. Since

the levels of vitamin E and lipids in the serum are

directly correlated, their ratio is a better measure of

status than serum vitamin E alone (Table 2). The ratio

of plasma vitamin E to cholesterol þ triglyceride or

vitamin E to cholesterol has also been reported to be

as good as that of vitamin E to total lipids. (See

Tocopherols: Properties and Determination.)

0021 In vitro hemolysis of red blood cells in the presence

of hydrogen peroxide, quantitating the formation of

malondialdehyde from hydroperoxides generated by

the in vitro peroxidation of polyunsaturated fatty

acids or erythrocytes exposed to hydrogen peroxide

is considered a functional test for assessing vitamin E

status. The latter tests, based on quantitation of mal-

ondialdehyde formed, are considered to be more

sensitive than the hemolysis test.

Vitamin K

0022 In the past, vitamin K status has been assessed by

measuring prothrombin time and other clotting

times. These measures are however not sensitive to

detect subclinical vitamin K deficiency since clotting

time does not change until the concentration of pro-

thrombin decreases by about 50%.

0023 Vitamin K is needed for the g-carboxylation of

certain glutamic acid residues in proteins. During

vitamin K deficiency, the vitamin K-dependent pro-

teins occur in an undercarboxylated form. Milder

forms of vitamin K deficiency can be assessed by

measuring urinary g-carboxyglutamic acid excretion

or circulating levels of undercarboxylated prothrom-

bin and osteocalcin. The last parameter is considered

to be a very sensitive marker of vitamin K status.

Plasma level of phyloquinone has also been used as

a measure of vitamin K status. Plasma phyloquinone

and urinary g-carboxyglutamic acid can be deter-

mined by HPLC separation followed by fluorometric

detection. Undercarboxylated prothrombin and

osteocalcin can be determined by radioimmunoassay.

(See Immunoassays: Radioimmunoassay and Enzyme

Immunossay.)

Thiamin

0024The biochemical tests used for the assessment of

thiamin nutritional status include measurement of:

(1) erythrocyte transketolase activation coefficient

(ETK-AC); (2) urinary excretion of thiamin; and (3)

measurement of whole blood or erythrocyte thiamin

pyrophosphate (TPP) concentration.

0025TPP is the coenzyme for transketolase and deter-

mination of ETK-AC is the most widely accepted

procedure for assessing thiamin status. Activity of

this enzyme is measured in erythrocyte hemolysate

without (basal) and with a saturating amount of

TPP added in vitro (stimulated). The ratio of stimu-

lated to basal activity is called the activity coefficient

(ETK-AC). Basal activity falls and ETK-AC increases

in thiamin deficiency (Table 2). Activity can be meas-

ured by determining the rate of disappearance of

pentose or appearance of hexose. However interpret-

ation of the results is complicated under certain dis-

ease conditions (some cancers, uremia, neuropathy,

and diabetes) and drug treatment (diuretics, antacids)

since ETK activity is affected under these conditions.

(See Enzymes: Uses in Analysis.)

0026Normal adults receiving adequate dietary thiamin

excrete more than 0.3 mmol per 24 h. Urinary thiamin

can be measured by fluorometric method. (See Thia-

min: Properties and Determination.)

0027Measurement of blood or erythrocyte TPP by

HPLC appears to be a more sensitive and promising

indicator of thiamin status and needs further verifica-

tion.

Riboflavin

0028Measurement of urinary excretion of riboflavin, red

blood cell riboflavin, and erythrocyte glutathione

reductase activation coefficient (EGR-AC) are used

for the biochemical assessment of riboflavin nutri-

tional status.

0029The urinary excretion method is useful for popula-

tion surveys. On an adequate dietary intake of

riboflavin, normal adults excrete more than

0.21 mmol g

1

of creatinine. Riboflavin concentration

tbl0002 Table 2 Tentative guidelines for interpretation of blood vitamin levels and enzyme tests

Parametermeasured Acceptable Mediumrisk Highrisk

Serum vitamin E (mmol l

1

) > 16.2 11.6–16.2 < 11.6

Serum vitamin E total lipid ratio > 0.8 < 0.8

ETK-AC < 1.15 1.15–1.25 > 1.25

EGR-AC < 1.2 1.2–1.4 > 1.4

Serum folate (nmol l

1

) > 11.0 6.7–11.0 < 6.7

Erythrocyte folate (nmol l

1

) > 360 315–359 < 315

Plasma ascorbate (mmol l

1

) 17–85 11–17 < 11

Leukocyte ascorbate (mmol 10

6

cells) 113–300

ETK-AC, erythrocyte transketolase activation coefficient; EGR-AC, erythrocyte glutathione reductase activation coefficient.

4186 NUTRITIONAL ASSESSMENT/Biochemical Tests for Vitamins and Minerals

in urine can be measured by fluorometric or micro-

biological assay or HPLC. (See Riboflavin: Properties

and Determination.)

0030 Red cell riboflavin level may be a better index of

tissue status than urinary riboflavin. Fluorometric or

HPLC technique can be used to estimate red cell

riboflavin and the value can be expressed per gram

hemoglobin or packed cell volume. However this test

is not commonly used because tissue saturation is

difficult to define and the value varies between indi-

viduals. Measurement of red cell flavin adenine

dinucleotide (FAD) level using HPLC may improve

the index.

0031 At present, the EGR-AC test is the most accepted

biochemical method for the assessment of riboflavin

status. FAD, the coenzyme form of riboflavin, is

necessary for the activity of glutathione reductase.

The activity of this enzyme is measured in erythrocyte

hemolysate or blood hemolysate without (basal) and

with added FAD (stimulated). The ratio of stimulated

to basal activity is called the activation coefficient

(EGR-AC). This ratio increases in riboflavin defi-

ciency (Table 2). EGR activity is assayed by rate

reaction techniques. However the EGR-AC test fails

to measure riboflavin status when glucose 6-phos-

phate dehydrogenase deficiency is also present.

0032 During respiratory infections, urinary and blood

levels of riboflavin are raised due to mobilization of

the vitamin from the liver. There is a transient reduc-

tion in EGR-AC values also. Thus assessment of ribo-

flavin status during such infection is difficult.

Niacin

0033 Measurements of the urinary excretion of N

-methyl-

nicotinamide (NMN) and its oxidation product,

-myethyl-2-pyridone-5-carboxamide (2-pyridone)

have been used to determine niacin status. Normal

adults excrete more than 17 mmol NMN per day,

whereas during deficiency its excretion is less than

5.8 mmol per day.

0034 A more recent study suggested that erythrocyte

nicotinamide adenine dinucleotide (NAD) concentra-

tion may serve as a sensitive indicator of niacin status

and that a ratio of erythrocyte NAD to NAD phos-

phate (NADP) < 1.0 may suggest a risk of developing

niacin deficiency. (See Niacin: Properties and Deter-

mination.)

Vitamin B

0035 Several direct and indirect measures are used for the

assessment of vitamin B

status.

0036 Measurement of the plasma pyridoxal phosphate

(PLP) level, the coenzyme form of the vitamin, is the

most commonly used method and a concentration of

30 nmol l

1

is considered to be adequate. However,

proper interpretation of plasma PLP data must take

into account the factors which affect this value. High

protein intake, pregnancy, age, smoking, and an in-

crease in alkaline phosphatase activity are known to

reduce PLP concentration. The measurement of both

PLP and pyridoxal is suggested to be a better index of

vitamin B

status during pregnancy. Pyridoxal phos-

phate levels in plasma can be measured by enzymatic

or HPLC techniques.

0037The second direct measure is urinary excretion of

4-pyridoxic acid. It correlates well with dietary vita-

min B

intake and this parameter is useful for popu-

lation surveys. An excretion of > 3.0 mmol per day is

considered to be associated with adequate vitamin

status and it can be measured by fluorometric

method. (See Vitamin B

: Properties and Deter-

mination.)

0038Functional tests for assessing vitamin B

status in-

clude measurement of PLP-dependent transaminases,

such as erythrocyte aspartate aminotransferase

(EAST) and alanine aminotransferase (EALT) and

their activation coefficient (AC), and tryptophan or

methionine load test. They reflect tissue levels of PLP.

0039EAST or EALT activity is measured without and

with added PLP to give an activation coefficient

(EAST-AC or EALT-AC) – an enzymatic test analo-

gous to that used for thiamin and riboflavin. Al-

though widely used for assessing vitamin B

status,

the interpretation of EAST-AC value is not standard-

ized. Values ranging from 1.7 to 2.0 have been used to

differentiate between adequacy and deficiency. The

EALT-AC value suggested for adequacy of vitamin

status is less than 1.25. These two enzymes can

be assayed by colorimetric or rate reaction technique.

0040The tryptophan load test has been used in popula-

tion surveys as a functional index of B

status. An

inadequate status results in an increased excretion of

tryptophan metabolites such as kynurenine and

xanthurenic acid, since vitamin B

-dependent enzyme

kynureninase is required for their metabolism. This

test is usually carried out after administering an oral

load of 2–5 g tryptophan and measuring the urinary

excretion of xanthurenic acid in a 6–9-h period.

However excretion of tryptophan metabolites is in-

fluenced by factors like protein intake and pregnancy.

Folic Acid and Vitamin B

0041Serum as well as red blood cell folate reflect folate

status. While serum folate reflects the dietary intake

and readily available tissue reserves, red blood cell

folate reflects long-term folate status. Microbio-

logical or competitive protein-binding radioassay is

used to measure folate levels in serum and red blood

NUTRITIONAL ASSESSMENT/Biochemical Tests for Vitamins and Minerals 4187

cells. The guidelines for interpreting these values are

given in Table 2.(See Folic Acid: Properties and

Determination.)

0042 The metabolism of histidine to glutamic acid is

impaired in folate deficiency, leading to an increase

in urinary excretion of formiminoglutamic acid

(FIGLU). This occurs particularly after a loading

dose of histidine. Normal adults excrete less than

35 mg FIGLU in 24 h after 5 g histidine load.

Deoxyuridine Suppression Test

0043 In folic acid deficiency the activity of the enzyme

thymidilate synthalase is impaired. This enzyme cata-

lyzes the conversion of deoxyuridine to thymidine.

In this test bone marrow cells or peripheral blood

lymphocytes are preincubated with nonradioactive

deoxyuridine and then with

H thymidine. The

amount of radioactivity incorporated into the DNA

of the cells is increased in folate as well as in vitamin

deficiency. These two deficiencies can be differen-

tiated by in vitro incubation with respective vitamins.

0044 Hypersegmentation of circulating neutrophils is a

morphological change that occurs in folate deficiency

due to slowed DNA synthesis. There are studies to

suggest that this change occurs when plasma folate

levels drop lower than 10 nmol l

1

0045 Measurement of plasma homocysteine level may

also indicate the adequacy of folate and, to a lesser

extent, vitamin B

status. Vitamin B

deficiency also

raises plasma homocysteine concentration.

0046 Vitamin B

status is generally assessed by measur-

ing its concentration in serum by microbiological

or competitive protein-binding radioassay. Radioas-

say using pure intrinsic factor measures true vitamin

, whereas microbiological assay measures a

few analogs of vitamin B

as well. The lowest ac-

ceptable serum level using microbiological assay is

150 pmol l

1

, whereas for the radioassay specific for

cobalamine, serum level < 75 pmol l

1

is considered

deficient. Measurement of plasma vitamin B

bound

to transcobalamine II is considered to be more sensi-

tive than total vitamin B

for the assessment of mar-

ginal vitamin B

deficiency.

0047 The urinary or plasma methylmalonic acid level,

preferably after an oral load of valine (its precursor),

is a metabolic test for detecting vitamin B

defi-

ciency. Normal adults excrete less than 100 mmol

in 24 h.

Pantothenic Acid

0048 Urinary excretion of pantothenic acid or blood levels

of the vitamin can be used to assess pantothenic acid

status. Adults taking an adequate amount of dietary

pantothenate excrete between 9 and 24 mmol of the

vitamin per gram creatinine and the blood levels

range between 1.2 and 1.8 m mol l

1

. Pantothenic

acid levels in blood and urine can be measured by

microbiological or radioimmunoassay. (See Panto-

thenic Acid: Properties and Determination.)

Biotin

0049Biotin nutritional status can be assessed by using the

following sensitive biochemical parameters: (1) urin-

ary excretion of organic acids such as 3-hydroxyiso-

valeric, 3-hydroxypropionic, and methylcitric acids,

or of biotin and its metabolite bisnorbiotin; (2) assay

of the activity of biotin-dependent enzyme propionyl

coenzyme A carboxylase with and without in vitro

addition of biotin in lymphocytes; and (3) measure-

ment of plasma biotin level. Plasma biotin is a less

sensitive indicator compared to the other two

methods. The normal reported range for plasma

biotin is 140–356 pmol l

1

. Biotin levels in blood

and urine can be determined by microbiological or

isotopic dilution assay. (See Biotin: Properties and

Determination.)

Ascorbic Acid

0050Plasma and leukocyte ascorbate levels indicate ascor-

bic acid status. The simplicity and reliability of

plasma ascorbic acid determination make it prefer-

able for identifying individuals at risk of deficiency

due to chronic low intake of the vitamin. However,

plasma ascorbic acid levels may be less useful for

defining the ascorbic acid status of individuals with

adequate or high intake. Leukocyte ascorbic acid

concentration reflects more accurately tissue stores

of vitamin than plasma level.

0051Plasma and leukocyte ascorbic acid levels are de-

termined by HPLC or colorimetric techniques.

Thresholds for interpretation of ascorbic acid status

are given in Table 2.(See Ascorbic Acid: Properties

and Determination.)

Mineral Nutrition

0052In this section, biochemical methods used for the

nutritional assessment of a few macro- and micro-

mineral elements are given. However, the list is not

comprehensive. Only those elements for which a

laboratory test can give meaningful data from the

nutritional point of view are included.

Sodium and Potassium

0053Dietary deficiency of sodium and potassium cations

might occur after increased losses under certain con-

ditions such as vomiting and diarrhea. Their status is

generally assessed by measuring serum or plasma

levels by atomic emission flame photometry or

4188 NUTRITIONAL ASSESSMENT/Biochemical Tests for Vitamins and Minerals

atomic absorption spectrophotometry (Table 3). (See

Sodium: Properties and Determination.)

Calcium

0054 In blood, calcium is present almost entirely in the

plasma. As ionized calcium in the plasma is under

strict homeostatic control (1.2 mmol l

1

), it cannot

serve as a sensitive parameter to assess nutritional

deficiency of calcium. (See Calcium: Properties and

Determination.)

0055 Bone density is an indirect measure of calcium

status, since 99% of it is present in the bones. It can

be measured by radiographic, single- or dual-photon

absorptiometry techniques.

Iron

0056 Iron status can be assessed by three types of biochem-

ical tests, reflecting different stages of deficiency.

First, bone marrow iron and serum ferritin concen-

trations indicate iron stores. Serum ferritin levels less

than 12 mgl

1

suggest depletion. As an acute-phase

reactant, serum ferritin is elevated by acute and

chronic infections, inflammatory diseases, malignan-

cies, and liver disorders.

0057 In the second stage of iron deficiency, referred to as

iron-deficiency erythropoiesis, the supply of iron to

the erythroid marrow is reduced. This can be assessed

by the transferrin saturation index. An increase in red

cell protoporphyrin level also indicates restricted iron

supply to the developing red cell (Table 4).

0058 Recent studies suggest that serum transferrin recep-

tor is a sensitive and quantitative index of tissue

iron status (Table 4). The levels are elevated in

iron-deficiency anemia. Inflammation or liver disease

does not affect serum transferrin receptor levels.

0059The third stage of iron deficiency is anemia, which

can be assessed by hemoglobin level or hematocrit.

Hemoglobin levels less than 13 g dl

1

of blood for

adult males and 12 g dl

1

blood for adult females

are considered deficient, whereas during pregnancy

hemoglobin concentration less than 11 g dl

1

is con-

sidered to be anemia. (See Anemia (Anaemia): Iron-

deficiency Anemia.)

Iodine

0060Urinary excretion of iodine reflects the dietary intake.

An excretion of more than 100 mg per day suggests an

adequate dietary intake of iodine. It can be measured

by the kinetic method, based on iodide-catalyzed re-

duction of ceric to cerus by arsenic. (See Iodine: Prop-

erties and Determination.)

0061The only known requirement of iodine in humans

is in the synthesis of thyroid hormones. Measure-

ments of serum levels of thyrotropic hormone or

triiodothyronine and thyroxine are sensitive func-

tional tests for iodine status.

Zinc

0062Zinc levels in plasma, urine, hair and cellular com-

partments of blood, activity of zinc-dependent

enzymes such as carbonic anhydrase in red cells and

plasma levels of zinc-binding protein metallothionin

have been used as indices of zinc nutrition status.

0063Plasma zinc appears to be the most widely used

parameter. It decreases in cases of severe and moder-

ate deficiency of zinc. Plasma zinc concentrations are

susceptible to a number of pathophysiological influ-

ences. Since erythrocytes and hair have a longer half-

life, their zinc content does not indicate acute zinc

deficiency. Zinc levels in cells with rapid turnover

(neutrophils and monocytes) can be used to detect

mild zinc deficiency. Serum thymulin level without

and with in vitro addition of zinc has been suggested

as a sensitive method to assess mild zinc deficiency.

Atomic absorption spectrometry is used for the analy-

sis of zinc in urine and blood. Table 5 gives the

normal range of tissue zinc. (See Zinc: Properties

and Determination.)

tbl0003 Table 3 Normal ranges of sodium, potassium, copper, and

selenium in blood

Parameter Range

Plasma sodium (mmol l

1

) 136–145

Plasma potassium (mmol l

1

) 3.5–5.0

Plasma copper (mg l

1

) 0.8–1.75

Serum ceruloplasmin (mmol l

1

) 1.7–2.9

Plasma selenium (mgl

1

) 60–120

Erythrocyte selenium (mgl

1

) 90–190

tbl0004 Table 4 Assessment of iron status (commonly used cut-off

values)

Parameter Value

Serum ferritin (mgl

1

)12

Transferrin saturation (%) 16

Red cell protoporphyrin (mmol l

1

) 1.24

Serum transferrin receptor (mg l

1

: normal range) 2.5–8.5

Hemoglobin (g l

1

)

Men 130

Women 120

tbl0005Table 5 Normal range of tissue zinc

Parameter Range

Plasma (mg l

1

) 0.7–1.4

Erythrocyte (mg l

1

) 10–14

Urine (mg per day) 400–600

Leukocytes (mg per 10

cells) 80–130

Hair (mgg

1

) 124–320

NUTRITIONAL ASSESSMENT/Biochemical Tests for Vitamins and Minerals 4189

Copper

0064 Copper status is commonly assessed by estimating

serum copper concentration or by assaying the activ-

ity of the copper-dependent enzyme ceruloplasmin in

serum (Table 3). Serum copper levels tend to be

higher in women. The values are influenced by certain

physiological and pathological conditions. About

95% of copper in serum is in ceruloplasmin, which

is an acute-phase reactant protein. Erythrocyte

copper-zinc superoxide dismutase has been suggested

to be a better index of copper status than serum

copper or ceruloplasmin.

0065 Serum copper can be estimated by atomic absorp-

tion spectrometry. (See Copper: Properties and Deter-

mination.)

Selenium

0066 Selenium status can be assessed by measuring its

levels in urine, plasma, erythrocytes, and blood or

by assaying the selenium-dependent enzyme gluta-

thione peroxidase in erythrocytes or platelets.

0067 Plasma selenium is a sensitive index of short-term

changes until the plasma level reaches a plateau.

Erythrocyte selenium and glutathione peroxidase

activity indicates long-term changes because of the

long life span of these cells.

0068 The commonly used techniques for measuring sel-

enium in biological fluids include fluorimetry, atomic

absorption spectrometry, and mass spectrometry.

Table 3 gives the frequently reported normal ranges

in plasma and erythrocytes. (See Selenium: Properties

and Determination.)

0069 Selenium is an integral part of glutathione peroxid-

ase and measurement of the activity of this enzyme is

a functional test for the assessment of selenium status.

The minimal erythrocyte selenium level required to

reach a plateau of glutathione peroxidase activity has

been estimated to be about 141 mgl

1

Conclusions

0070 The value of biochemical tests can be improved with

a better understanding of their limitation and their

relationship to functional consequences. What level

of biochemical insult can an individual accommodate

without any ill effects? Can generalized interpretative

guidelines be applied to all population groups or are

there substantial variations? Despite these dilemmas,

biochemical tests have an important place in nutri-

tional diagnosis.

See also: Ascorbic Acid: Properties and Determination;

Biotin: Properties and Determination; Calcium:

Properties and Determination; Copper: Properties and

Determination; Folic Acid: Properties and Determination;

Iodine: Properties and Determination; Niacin: Properties

and Determination; Riboflavin: Properties and

Determination; Selenium: Properties and Determination;

Sodium: Properties and Determination; Thiamin:

Properties and Determination; Tocopherols: Properties

and Determination; Vitamin B

: Properties and

Determination; Vitamins: Determination; Zinc:

Properties and Determination

Further Reading

Bamji MS (1981) Vitamins in Human Biology and Medi-

cine, pp. 1–27, Boca Raton, Florida: CRC Press.

Flair EC Programme (1993) The measurement of micronu-

trient absorption and status. Flair concerted action no.

10 status papers. International Journal of Vitamins and

Nutrition Research 63: 247–316.

Sauberlich HE (1984) Implications of nutritional status on

human biochemistry, physiology and nutrition. Clinical

Biochemistry 17: 132–142.

Shils ME and Young VR (eds) (1988) Modern Nutrition in

Health and Disease, VII edn, pp. 142–446. Philadelphia:

Lea & Febiger.

Sokoll LJ, Booth SL, O’Brien ME, Davidson KW, Tsaioin KI

and Sadowski JA (1997) Changes in serum osteocalcin,

plasma phylloquinone and urinary r-carboxyglutamic

acid in response to altered intakes of dietary phylloquin-

one in human subjects. American Journal of Clinical

Nutrition 65: 779–784.

Ziegler EE and Filer LJ Jr (eds) (1996) Present Knowledge

in Nutrition, VII edn, pp. 120–236. Washington, DC:

ILSI Press.

Functional Tests

N W Solomons, CeSSIAM, Guatemala City, Guatemala

Background

0001Reasons abound for assessing human nutritional

status, ranging from the population level (epidemi-

ology, public health) to assess risk of nutrient defi-

ciency and excess, and to monitor changes and

impacts of interventions to the clinical level, where a

practitioner is interested in the patient’s nutriture for

routine maintenance, as a clue to disease, and in

titrating nutritional and disease-specific therapies.

(See Epidemiology.)

0002One of the options for assessing nutritional status is

the approach of functional assessment. The purpose

of this chapter is to discuss the history, origins, con-

cepts, applications, advantages and disadvantages,

4190 NUTRITIONAL ASSESSMENT/Functional Tests

and modern advances in the arena of functional tests

of human nutriture.

Historical Perspective

0003 Clinical assessment of nutritional status by examin-

ing signs and symptoms is the most venerable ap-

proach; in fact, the syndromes of scurvy, beriberi,

pellagra, rickets, and anemia had been recognized

clinically long before the chemical nature and etio-

logical roles of ascorbic acid, thiamin, nicotinic acid,

cholecalciferol, and the hematinic nutrients (iron,

folic acid, vitamin B

) were discovered. (See Anemia

(Anaemia): Iron-deficiency Anemia; Scurvy; refer to

individual nutrients.)

0004 As sensitivity (the ability of a test to find all

affected individuals) and specificity (the ability of a

test to reject all unaffected individuals) of a diagnostic

approach must always be considered, clinical exam-

ination has its limitations. In terms of sensitivity,

clinical examination is a poor index of nutritional

deficiency, as clinical manifestations emerge late in

the process and only in those most deficient. They

represent the tip of the iceberg. The specificity of

clinical diagnosis is variable. Whereas scurvy and

rickets are unmistakable, anemia has multiple

nutritional bases and a host of causes unrelated to

nutrition.

0005 The twentieth century saw the chemical identifica-

tion of the essential nutrients, producing reliable

assays to measure nutrients or their metabolites in

biological fluids and tissues. The first-line approach

for the detection of deficiencies and excess of nutri-

ents shifted from clinical examination to laboratory

tests, with the realization that changes in body

reserves and tissue content of nutrients decrease

before the clinical manifestations of deficiency are

expressed. Similarly, in terms of excessive accumula-

tion, an increased burden of a nutrient occurs before

overt toxic signs and symptoms develop. Most, but

not all, biochemical procedures generate static indices

of nutritional status. Assessment of individual status

is pursued by constructing a normative reference dis-

tribution for a healthy, well-nourished population as

the standard, and assigning cut-off criteria on the

high and low ends that represent excess and defi-

ciency, respectively. For a population assessment, the

deviation of its biochemical distribution from that of

the reference population is gauged, or the prevalence

of individuals with values outside the criteria levels is

tabulated.

0006 The concept of functional assessment was added to

the lexicon of nutritional status evaluation in the

1970s, although its origins go much further back to

the Hess tourniquet test for capillary fragility for

incipient scurvy of the early twentieth century, dark-

adaptation tests for vitamin A status, and prothrom-

bin- and coagulation-time tests for vitamin K

adequacy of the middle of the century. However, in

1978 Study Team IX of the Committee on Inter-

national Nutrition Programs of the National Acad-

emy of Sciences of the USA, chaired by the late

Professor Doris Howes Calloway, issued a report

arguing that the intactness of the physiological and

behavioral functions that depended on nutrients was

of more interest to policy-makers in governments and

development assistant agencies than were the levels of

nutrients in the body. In 1983, Noel Solomons and

Lindsay Allen produced a systematic classification of

tests of nutritional status based on measures of

physiological performance or behavioral responses.

Definitions

0007This article compares and contrasts two types of in-

dices that seek to determine the nutriture of

individuals: static and functional indices.

Static Indices

0008Static indices are tests directed at assessing the quan-

titative content of a nutrient in the organism, either

as whole-body reserves or as tissue concentrations.

They represent chemically measurements of the

nutrient itself, some active or inactive metabolite, or

a complex, such as hemoglobin, that contains the

nutrient. In the instance of iron status, for example,

hemoglobin is a surrogate for iron in target tissues,

whereas ferritin reflects its presence in body iron

stores. Serum iron, transform saturation, and iron

in hair or nails are all examples of static indices of

iron status. (See Anemia (Anaemia): Iron-deficiency

Anemia.)

Functional Indices

0009Functional indices of nutritional status are those

behavioral, physiological, or biochemical functions

of the organism dependent on the adequate availabil-

ity of a nutrient or resulting from the homeostatic

regulatory processes that maintain body stores and

harmonic internal distribution of some nutrients.

0010As mentioned, perhaps the first example of a func-

tional test of nutritional status was the capillary fra-

gility test of Hess, followed in the 1930s by dark

adaptation tests based on Wald’s elucidation of the

dependence of retinal cone function on vitamin A.

With the advent of isolated radioisotopes and

whole-body counting, tests of the absorption, reten-

tion, and distribution of several nutrients have been

developed. With the molecular biology, genomic,

and proteonomic revolutions, new and unexplored

NUTRITIONAL ASSESSMENT/Functional Tests 4191

opportunities for functional tests have emerged. (See

Ascorbic Acid: Physiology; Retinol: Physiology.)

Classification of Functional Indices

Classification by System

0011 Functional tests can be conveniently classified

according to the body system. The categories are as

follows: (1) structure and structural integrity; (2) im-

munity and host defense; (3) transport; (4) hemo-

stasis; (5) reproductive biology; (6) nervous system

function; (7) hemodynamics and physical capacity.

Some functional tests cannot be classified into any

of these, and reside in a ‘miscellaneous’ catchment

category.

0012 Structure and structural integrity Under ‘structure

and structural integrity’ are classified all of those tests

that relate to growth and development of children

and adolescents. At the level of specific tissues, tests

of the integrity of somatic membranes, red cell mem-

branes, capillary walls, and collagen and osseous

structures also pertain to this category. The destruc-

tion (lipid peroxidation) of membranes is presumably

related to the adequacy of vitamin E and of other

putative antioxidant nutrients such as selenium,

zinc, and copper. (See Growth and Development;

refer to individual nutrients.)

0013 Immunity and host defense In the category of im-

munity and host defense are included the multitude of

tests of phagocytic, humoral, and cellular immuni-

ty,as well as the newer tests of messenger (hormonal)

function of the cytokine mediators of the inflamma-

tory response and of autoimmunity and autoimmune

surveillance. Almost all macronutrients, and most of

the micronutrients that relate to cellular proliferation

or metabolism, influence the function at some level of

host immunity. (See Immunology of Food.)

0014 Transport Tests of transport function begin at the

intestinal level, specifically with its homeostatic regu-

lation of the body reserves. Additionally, at the level

of transmembrane movement of nutrients, we have

different uptake potentials based on the nutrient

status of the organism. In both cases, deficiency

should enhance a net inward flux, and sufficiency

should balance inward and outward movement.

Among the nutrients with regulated uptake at the

gut are iron and zinc. Cobalt ion can be used as a

surrogate for iron in an isotopic absorption test. The

conversion of carotene to vitamin A by the gut is also

regulated by the level of body vitamin A stores, and

theoretically could be measured by a clinical test

that measures the outcome of its uptake and intestinal

metabolism. Isotopic turnover studies also fall into the

category of tests of nutrient transport, as do the spe-

cific tests of mobilization from stores to the periphery,

such as the relative dose–response and the modified

relative dose–response for vitamin A status assess-

ment. (See Cobalt.)

0015Hemostasis Nutrition with respect to vitamin C,

vitamin K, and all micronutrients that influence plate-

let aggregation can be reflected in tests of hemostasis

related to capillary structure, bleeding time, or clot-

ting capacity. It should be noted that capillary integ-

rity can be classified under ‘structural integrity’ or

under ‘hemostasis.’

0016Reproductive biology Spermatogenesis and gon-

adal–pituitary hormone axis function, as well as

lactation and reproduction performance in the indi-

vidual women and, at the population level, fertility

and fecundity of the community, birthweights, and

peripartum hemorrhage rates constitute functional

tests of reproductive biology. (See Menstrual Cycle:

Nutritional Aspects; Premenstrual Syndrome: Nutri-

tional Aspects; Lactation: Physiology; Lactic Acid

Bacteria.)

0017Nervous system function From cognitive perform-

ance, social competence, scholastic performance,

through sleep pattern to peripheral nerve transmis-

sion and, finally, to the specific sensory reception in

eyes, ears, nose, and tongue, functional tests in the

nervous system cover a ganut of nervous functions.

Both macro- and micronutrient deficiencies have been

associated with impairment of higher cerebral func-

tions. Micronutrients, such as thiamin with respect to

the sixth cranial nerve function, vitamin A, zinc, and

vitamin E for retinal function, and zinc and vitamin A

for olfactory acuity, are some of the nutrients that can

be evaluated in tests of the special senses.

0018Hemodynamics and physical capacity Functional

tests of hemodynamics and physical capacity are

based on techniques of noninvasive and work perfor-

mance physiology. Doubly labeled water (

can be used as a long-term marker of energy expend-

iture. Protein, energy, and iron are the nutrients most

regularly thought to be determinant of the perform-

ance of the individual in efforts of physical exertion.

(See Exercise: Metabolic Requirements.)

Classification by Type of Test

0019The alternative form of classification of functional

test of nutritional status is by type of test, i.e., by the

manner in which the function is approached. These

include four levels: in vitro tests of in vivo functions;

4192 NUTRITIONAL ASSESSMENT/Functional Tests

induced responses and load tests in vivo; spontaneous

in vivo activities and responses of organs and tissues;

and responses at the level of the individual or popula-

tion. A selected array of functional tests are listed

under the respective classifications described in the

following four sections.

0020 In vitro tests of in vivo functions

0020 erythrocyte transketolase activity coefficient;

0021 erythrocyte glutathione reductase activity coeffi-

cient;

0022 erythrocyte transaminase activity coefficient;

0023 circulating homocysteine concentration;

0024 granulocyte chemotactic response;

0025 lymphocyte chemotactic response;

0026 bactericidal capacity of granulocytes;

0027 leucocyte glycolysis;

0028 granulocyte reduction of nitroblue tetrazolium;

0029 serum opsonin activity;

0030 T-cell blastogenesis;

0031 monocyte interferon production;

0032 monocyte monokine (interleukin-1; tumor necrosis

factor) production;

0033 erythrocyte fragility;

0034

Se uptake by erythrocytes;

0035

Zn uptake by erythrocytes;

0036 thymulin activity reconstitution by zinc in vitro;

0037 prothrombin time;

0038 descarboxy prothrombin levels;

0039 platelet aggregation;

0040 d()-uridine suppression test;

0041

C-formate conversion in lymphocytes;

0042 tensile strength of skin strips.

This group of functional tests requires some form of

biological sample, either blood to separate the vari-

ous cellular elements or a skin biopsy. Where specific

cellular elements such as platelets, lymphocytes, and

granulocytes are concerned, the volumes of blood

to be extracted can be considerable. The samples

required for erythrocytes are usually small.

0043 In vivo induced responses and load tests

0043 capillary fragility (Hess) test;

0044 experimental wound healing;

0045 collagen accumulation in subcutaneous sponge

implant;

0046 mobilization of inflammatory cells (Rebuck skin

window);

0047 delayed cutaneous hypersensitivity;

0048 de novo antibody formation;

0049 vasopressor response;

0050 total-body energy expenditure with

metab-

olism;

0051isotopic turnover studies with whole-body

counting;

0052radioiron absorption;

0053radiocobalt absorption;

0054zinc absorption tests;

0055intravenous magnesium infusion tolerance (urinary

excretion) test;

0056retinol relative dose–response;

0057dihydroxyretinol modified relative dose–response;

0058b-carotene bioconversion to retinyl ester challenge

0059thyroid radioiodine uptake;

0060glucose tolerance test;

0061postglucose plasma chromium response;

0062postglucose urine chromium response;

0063glucose load with exercise for lactate and pyruvate;

0064urinary malonaldehyde excretion;

0065mixed-function oxidase (

) breath test;

0066

C-histidine (

) breath test;

0067

C-serine (

) breath test;

0068histidine load for urinary formimino glutamic acid;

0069histidine load test for urinary hydantoin propionic

acid;

0070purine load test for urinary xanthine;

0071sulfur amino acid load test for abnormal sulfur

metabolites;

0072sodium bisulfite load test for abnormal sulfur

metabolites;

0073tryptophan load test for urinary xanthurenic acid;

0074leucine load test for urinary 3-hydroxyisovaleric

acid.

In this class of test, some form of substrate, either

natural or artificial, and unlabeled, labeled with a

stable isotope, or labeled with a radioisotope, is

administered, and its metabolism is monitored.

The metabolism is either governed by a nutrient-

dependent function of the nutrient in question or

related to the body’s homeostatic regulation of

nutrient reserves.

0075Spontaneous in vivo activities and responses of

organs and tissues

0075capillary bleeding tendency;

0076dark adaptation;

0077visual recovery time;

0078central scotoma size;

0079color discrimination;

0080taste acuity;

0081olfactory acuity;

0082auditory acuity;

0083abducens (sixth cranial nerve) function;

0084nerve conduction time;

0085skin conductivity;

0086electroencephalography;

0087sleep pattern;

NUTRITIONAL ASSESSMENT/Functional Tests 4193