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close this bookThe Management of Nutrition in Major Emergencies (WHO - OMS, 2000, 250 p.)
View the document(introduction...)
View the documentPreface
View the documentAcknowledgements
Open this folder and view contentsChapter 1. Meeting nutritional requirements
Open this folder and view contentsChapter 2. Major nutritional deficiency diseases in emergencies
Open this folder and view contentsChapter 3. Assessment and surveillance of nutritional status
Open this folder and view contentsChapter 4. Nutritional relief: general feeding programmes
Open this folder and view contentsChapter 5. Nutritional relief: selective feeding programmes
Open this folder and view contentsChapter 6. Prevention, treatment, and control of communicable diseases
Open this folder and view contentsChapter 7. The context: emergency preparedness and response programmes
View the documentAnnex 1. Nutritional requirements
View the documentAnnex 2. Basic facts about food and nutrition
View the documentAnnex 3. Nutritional anthropometry in emergencies
View the documentAnnex 4. Statistical procedures for nutritional surveys
View the documentAnnex 5. Use of particular foods in emergencies
View the documentAnnex 6. Guiding principles for feeding infants and young children in emergencies
View the documentAnnex 7. Programme indicators
View the documentAnnex 8. Biochemical assessment of micronutrients
View the documentAnnex 9. Human resource development for the management of nutrition in major emergencies: outline of an educational programme
View the documentBack Cover

Annex 8. Biochemical assessment of micronutrients

Assessment of thiamine deficiency

The clinical signs of beriberi are described in Chapter 2, but there are several biochemical and other methods that provide a more sensitive assessment of thiamine status. The most common determinations are the following:

· urinary thiamine excretion, per 6- or 24-hour period or per gram of creatinine (see Table A 8.1);

· erythrocyte transketolase levels, and the increase that occurs on addition of thiamine pyrophosphate (TPP), expressed as a percentage of the basal level; this increase is termed the thiamine pyrophosphate effect (TPPE) or erythrocyte ketolase activity coefficient (ETK-AC) (see Table A 8.2);

· breast-milk thiamine levels, used as a measure of both maternal and infant thiamine status (see Table A 8.3);

· thiamine levels in whole blood and in erythrocytes;

· blood pyruvate levels;

· dietary thiamine intake;

· increases in monthly mortality rates among infants aged 2-5 months.

Criteria for assessing the public health significance of thiamine deficiency are summarized in Table A 8.4.

Assessment of niacin deficiency

The clinical signs of pellagra are described in Chapter 2, but there are biochemical and other methods that provide a more sensitive assessment of niacin status. The most common determinations are the following:

· urinary excretion of N1 - methylnicotinamide, per 24-hour period or per gram of creatinine (see Table A 8.5);

· ratio of niacin metabolites in urine - (6-pyridone): (N1-methylnicotinamide);

· loading tests with nicotinic acid, nicotinamide, or tryptophan;

· dietary intake of niacin equivalents.

Interpretation of these biochemical parameters is somewhat equivocal, and dietary surveys are difficult to organize. Provisional criteria for assessing the public health significance of niacin deficiency in the absence of other available tests are summarized in Table A 8.6.

Table A8.1 Guidelines for the interpretation of urinary excretion of thiaminea

Parameter

Deficient (high risk)

Low (medium risk)

Acceptable (low risk)

Urinary thiamine (µg/g creatinine)




Children:





1-3 years

<120

120-175

176


4-6 years

<85

85-120

121


7-9 years

<70

70-180

181


10-12 years

<60

60-180

181


13-15 years

<50

50-150

151

Adults

<27

27-65

66

Pregnancy





2nd trimester

<23

23-54

55


3rd trimester

<21

21-49

50

Urinary thiamine (µg)




Adults





per 6-hour period

<10

10-24

25


per 24-hour period

<40

40-99

100

Load test (% return of 5-mg thiamine dose in 4-hour period)




Adults

<20

20-79

80

a Adapted, with permission, from: Sauberlich HE, Skala JH, Dowdy RP. Laboratory tests for the assessment of nutritional status. Cleveland, OH, CRC Press, 1974.

Table A8.2 Classification and interpretation of TPPE (thiamine pyrophosphate effect) levels in individualsa

Thiamine status

TPPE (%)

Normal

0-14

Marginally deficient

15-24

Severely deficient (with clinical signs)

³25

a Reproduced, with permission, from: Brin M et al. Some preliminary findings on the nutritional status of the aged in Onondaga county, New York. American journal of clinical nutrition, 1965, 17:240-258.

© American Society for Clinical Nutrition.

Assessment of ascorbic acid deficiency

The clinical signs of scurvy are described in Chapter 2, but there are several biochemical and other methods that provide a more sensitive assessment of ascorbic acid (vitamin C) status. The most common determinations are the following:

· serum or whole plasma levels of ascorbic acid (see Table A 8.7);

· whole blood levels of ascorbic acid (see Table A 8.7);

· leukocyte ascorbic acid levels (see Table A 8.7);

· urinary excretion of ascorbic acid or ascorbate-2-sulfate, per 24-hour period or per gram of creatinine;

· ascorbic acid saturation or loading tests.

Table A8.3 Breast-milk thiamine levels: provisional criteria for assessment in individuals

Thiamine status

Breast-milk thiamine (µg/litre)

Normal

100-200

Marginally deficient

50-99

Severely deficient

<50

Table A8.4 Provisional criteria for severity of public health problem of thiamine deficiency

Indicator

Severity of public health problem


Mild

Moderate

Severe

Clinical signs

³1 clinical case:
<1% of population in age group concerned

1-4% of population in age group concerned

³5% of population in age group concerned

TPPE test ³ 25%

5-19%

20-49%

³50%

Urinary thiamine (µg/g creatinine)

Adults:

% below 27

5-19%

20-49%

³50%

Children:






1-3 years

% below 120





4-6 years

% below 85





7-9 years

% below 70





10-12 years

% below 60





13-15 years

% below 50




Pregnancy:






2nd trimester

% below 23





3rd trimester

% below 21




Breast-milk thiamine <50 µg/litre

5-19%

20-49%

³50%

Dietary intake <0.33 mg/1000 kcalth

5-19%

20-49%

³50%

Mortality among infants aged 2-5 months

No decline in rates

Slight peak in rates

Marked peak in rates

Table A 8.5 Guidelines for the interpretation of urinary excretion of niacin metabolitesa

Parameter

Deficient

Low

Acceptable

High

Urinary N1 -methylnicotinamide






(mg per g creatinine)





Adults (men; women, non-pregnant

<0.5

0.5-1.59

1.6-4.29

³4.3


or 1st trimester pregnancy)





Pregnant women:






2nd trimester

<0.6

0.6-1.99

2.0-4.99

³5.0


3rd trimester

<0.8

0.8-2.49

2.5-6.49

³6.5

Ratio 2-pyridone: N1 -methylnicotinamide

<0.5

<1.0

1.0-4.0


Dietary intake of niacin equivalents (mg/day)

<5

5-9

³10


a Sources:

Sauberlich HE, Skala JH, Dowdy RP. Laboratory tests for the assessment of nutritional status. Cleveland, OH, CRC Press, 1974.

Dillon JC et al. Les metabolites urinaires de la niacine au cours de la pellagre. [Urinary metabolites of niacin in pellagra.] Annals of nutrition and metabolism, 1992, 36:181-185.

Malfait P et al. An outbreak of pellagra related to changes in dietary niacin among Mozambican refugees in Malawi. International journal of epidemiology, 1993, 22:504-511.

Table A8.6 Provisional criteria for severity of public health problem of niacin deficiency

Indicator

Severity of public health problem


Mild

Moderate

Severe

Clinical signs

³11 clinical case;
<1% of population in age group concerned

1-4% of population in age group concerned

³5% of population in age group concerned

Urinary N1-methylnicotinamide
<0.50 mg per gram creatinine

5-19%

20-49%

³50%

Ratio 2-pyridone:
N1-methylnicotinamide <1.0

5-19%

20-49%

³50%

Dietary intake of niacin equivalents <5 mg/day

5-19%

20-49%

³50%

Table A8.7 Guidelines for the interpretation of ascorbic acid levels in individuals of all agea

Indicator

Deficient (high risk)

Low (medium risk)

Acceptable (low risk)

Serum ascorbic acid (mg/100 ml)

<0.20

0.20-0.29

>0.30

Leukocyte ascorbic acid (nmol/108 cells)

<57

57-114

>114

Whole blood ascorbic acidb (mg/100 ml)

<0.30

0.30-0.49

>0.50

a Reproduced, with permission, from: Sauberlich HE, Skala JH, Dowdy RP. Laboratory tests for the assessment of nutritional status. Cleveland, OH, CRC Press, 1974. Data kindly updated by Dr H.E. Sauberlich.

b This classification may not be valid for individuals with marked anaemia.

Table A8.8 Provisional criteria for severity of public health problem of ascorbic acid deficiencya

Indicator

Severity of public health problem


Mild

Moderate

Severe

Clinical signs

³1 clinical case;
<1 % of population in age group concerned

1-4% of population in age group concerned

³5% of population in age group concerned

Serum ascorbic acid (mg/100 ml):





<0.20

10-29%

30-49%

³50%


<0.30

30-49%

50-69%

³70%

a Sources:

Sauberlich HE, Skala JH, Dowdy RP. Laboratory tests for the assessment of nutritional status. Cleveland, OH, CRC Press, 1974.

Desenclos JC et al. Epidemiological patterns of scurvy among Ethiopian refugees. Bulletin of the World Health Organization, 1989, 67:309-316.

Only the first of these tests is widely used, reliable, and practicable in field surveys. Care must be taken both in conserving serum/plasma samples and to avoid haemolysis (which can lead to false results). Criteria for assessing the public health significance of ascorbic acid deficiency are summarized in Table A 8.8.

Summary of laboratory methods and samples required

General principles

Micronutrients are essentially labile or are found in labile compounds, and analysis is technically difficult, both because of this lability and because of the presence in blood, urine, and breast milk of substances that interfere with analysis. Thus, although it is not difficult to specify the analytical method or methods commonly used, great care is required to obtain reliable and accurate results. It would be wise for any laboratory starting up new work in this area to contact a well established facility with experience in the assessment of the nutrient concerned and to carry out a calibration procedure; this should ensure that equivalent and internationally acceptable results are obtained. On request, WHO can provide a list of leading laboratories with experience in the assessment of the various nutrients.1

1 Available on request from Programme of Nutrition, World Health Organization, 1211 Geneva 27, Switzerland.

The equipment and reagents required, references to the basic biochemical methods for the assessment of iron, iodine, and vitamin A status, and general requirements for a micronutrient laboratory are detailed in the following publication:

· May WA et al. Micronutrient laboratory: equipment manual. Atlanta, GA, Programme against Micronutrient Malnutrition, 1996.

For specific nutrients, a summary of the main methods and references is given in the publications or documents cited in the following paragraphs.

Anaemia and iron status

Assessing anaemia: haemoglobin and haematocrit (erythrocyte volume fraction)

These are two basic and widely used tests for assessing anaemia, which are simple to perform and can provide reasonably good information about iron status. However, the need for proper standardization, supervision, and quality control of these procedures, plus training in their use, should not be overlooked. Various methods are discussed in the following publication:

· Anemia detection in health services: guidelines for program managers. Seattle, WA, Program for Appropriate Technology in Health, 1996.

Haemoglobin

The cyanmethaemoglobin method2 is considered to be the most accurate and reliable method of determining haemoglobin. Details are contained in the following publication:

· Procedure for the quantitative determination of haemoglobin in blood. Villanova, PA, National Committee for Clinical Laboratory Standards, 1984.

2 The method requires a well trained laboratory technician, routine instrument calibration, careful execution, and attention to proper method standardization and quality control practices.

In the field, dried blood spot samples can be used for performing the test. These are prepared by applying a drop of whole blood to a special filter paper, which is then allowed to dry. Once dried, the samples remain fairly stable, even without freezing. Small battery-operated colorimeters are now available that allow determinations to be made outside a laboratory setting.

A simple, transportable, robust haemoglobin photometer is now available that can be used for accurate haemoglobin testing in the field. It uses disposible sample cuvettes into which blood is drawn, and which are then placed in the instrument for automatic haemoglobin measurement. Results are available in 1-2 minutes and are comparable to laboratory methods in terms of accuracy and precision. The instrument can be used by non-laboratory personnel after minimal training. The high cost of the test cuvettes limits routine use, but the instrument can play a valuable role in rapid nutrition surveys or sentinel site surveillance. The instrument is reviewed in the following publication:

· Johns WL, Lewis SM. Primary health screening by haemoglobinometry in a tropical community. Bulletin of the World Health Organization, 1989, 67(6):627-633.

In the filter-paper method, a spot of capillary blood is collected directly on filter paper and compared with a printed set of colour standards. This method is inexpensive, simple, portable, and rapid, and therefore useful for screening in rural settings. However, it is highly subjective and inappropriate as a stand-alone test. Recently, WHO has developed a reliable haemoglobin colour scale for use with standard-quality filter paper; its suitability and reliability for use in various field conditions are currently being assessed. Details are given in the following publication:

· Stott GM, Lewis SM. A simple and reliable method for estimating haemoglobin. Bulletin of the World Health Organization 1995, 73(3):369-373.

Haematocrit (erythrocyte volume fraction)1

1 The method requires a well trained laboratory technician, routine instrument calibration, careful execution, and attention to proper method standardization and quality control practices.

Determination of haematocrit can be used as an alternative to haemoglobin testing. The method involves spinning a small volume of blood collected into a heparinized glass capillary tube, using a special microhaematocrit centrifuge. Its advantages are technical simplicity, low operational costs, and the fact that it can be done outside the laboratory. Details are given in the following publication:

· Procedure for determining packed cell volume by the microhematocrit method, 2nd ed. Villanova, PA, National Committee for Clinical Laboratory Standards, 1993.

Assessing iron status

Serum ferritin, erythrocyte protoporphyrin, transferrin saturation, transferrin receptors, and red cell indices, especially mean corpuscular volume, provide much more insight into iron status. The tests are more sensitive than those for determining haemoglobin and haematocrit and confirm the presence of underlying iron deficiency. However, because of the cost and the sophistication of these methods, large-scale use is not common; usage is generally limited to special surveys or to settings where resources are relatively abundant.

A general reference on assessment of iron status is:

· Measurements of iron status. Washington, DC, International Nutritional Anaemia Consultative Group, 1985.

Serum ferritin

Serum ferritin is the most specific and sensitive biochemical test for iron deficiency; when used in combination with haemoglobin, it may offer the greatest potential for monitoring iron deficiency. However, serum ferritin is often elevated in response to infectious or inflammatory conditions. Methods are detailed in the following publications:

· Miles LEM et al. The measurement of serum ferritin by a 2-site immunoradiometric assay. Annals of biochemistry, 1974, 61:209-224. (This is the conventional immunoradiometric assay method.1)

· Pintar J et al. A screening test for assessing iron status. Blood, 1982, 59:110-113. (A semi-quantitative ferritin measurement based on a modification of a two-site enzyme-linked immunoassay. It requires only 2 drops of blood, i.e. 35 µl serum, and a total incubation time of 90 minutes. It can be performed without laboratory facilities and is therefore suitable for use in fieldwork.)

· May WA. Micronutrient laboratory equipment manual. Atlanta, GA, Program Against Micronutrient Malnutrition, 1996. (Details are provided for newer non-radioactive enzyme-linked immunoassay.)

1 The method requires a well trained laboratory technician, routine instrument calibration, careful execution, and attention to proper method standardization and quality control practices.

Erythrocyte protoporphyrin

Protoporphyrin is a precursor of haem and accumulates in red cells when there is insufficient iron to make haem. Elevated levels occur in the second stage of iron deficiency and after iron stores have been depleted. Erythrocyte protoporphyrin correlates well with low serum ferritin and can serve as a screening test for moderate iron deficiency. However, elevated levels can also be difficult to interpret in areas where infection rates are high. The method is described in the following publication:

· Blumberg WE, Doleiden FH, Lamol AA. Haemoglobin determined in 15 µl of whole blood by "front-face" fluorometry. Clinical chemistry, 1980, 26:409-413.

Transferrin saturation

Almost all of the iron in the serum is bound to the iron-binding protein, transferrin. Transferrin saturation is calculated by measuring both serum iron and total iron-binding capacity using spectrophotometric techniques, dividing the iron concentration by the iron-binding capacity, and multiplying the result by 100 to yield a percentage. The method is described in the following publication:

· Summary of a report on assessment of the iron nutritional status of the United States population. Expert Scientific Working Group. American journal of clinical nutrition, 1985, 42:1318-1330.

Serum transferrin receptors

This newer test for serum transferrin receptors has the advantage of being unaffected by the presence of infection; however, it is affected by any increase in red-cell turnover, e.g. haemolysis. The method is described in the following publication:

· Flowers CH et al. The clinical measurement of serum transferrin receptors. Journal of laboratory and clinical medicine, 1988, 114:368-377.

Red cell indices

When measured by electronic blood counters, mean corpuscular volume and red cell distribution width are reliable indices of iron deficiency; the indices are not very sensitive, however, and change only when anaemia is also apparent.

Iodine

Indicators of iodine deficiency are discussed in the following publication:

· Indicators for assessing iodine deficiency disorders and their control through salt iodization. Geneva, World Health Organization, 1994 (unpublished document WHO/NUT/94.6).1

1 Available on request from Programme of Nutrition, World Health Organization, 1211 Geneva 27, Switzerland.

The main test of thyroid function is the thyroid-stimulating hormone level. Methods are outlined in the following publication:

· May WA. Micronutrient laboratory: equipment manual. Atlanta, GA, Program Against Micronutrient Malnutrition, 1996.

Vitamin A

Indicators for assessing vitamin A deficiency are discussed in the following publication:

· Indicators for assessing vitamin A deficiency and their application in monitoring and evaluating intervention programmes. Geneva, World Health Organization, 1996 (unpublished document WHO/NUT/96.10).1

1 Available on request from Programme of Nutrition, World Health Organization, 1211 Geneva 27, Switzerland.

Thiamine

The most commonly used procedure for assessing thiamine status has been the measurement of erythrocyte transketolase activity and its stimulation in vitro by the addition of thiamine pyrophosphate (TPP effect).

The erythrocyte transketolase activity assay involves the incubation of haemolysed whole blood samples in a buffered medium with an excess of ribose-5-phosphate, in both the presence and absence of excess thiamine pyrophosphate.

Details of the erythrocyte transketolase activity assay are given in the following publications:

· Duffy P, Morris P, Neilson G. Thiamin status of a Melanesian population. American journal of clinical nutrition, 1981, 343:1584-1592. (Using a bichromatic analyser to provide semiautomated measurement of the stimulation of erythrocyte transketolase by thiamine pyrophosphate.)

· Smeets E, Muller H, de Wael J et al. A NADH-dependent transketolase assay in erythrocyte hemolysates. Clinica chimica acta, 1971, 33:379 - 86. (Manual method for TPP effect.)

· Waring P et al. A continuous-flow (AutoAnalyzer II) procedure for measuring erythrocyte transketolase activity. Clinical chemistry, 1982, 28:2206-2213. (Simple, rapid, semiautomated assay for transketolase activity.)

· Bayoumi RA, Rosalki SB. Evaluation of methods of coenzyme activation of erythrocyte enzymes for detection of deficiency of vitamins B1, B2, and B6. Clinical chemistry, 1976, 22:327-35. (Ultraviolet spectrophotometric procedure.)

· Basu TK, Patel DR, Williams DC et al. A simplified microassay of transketolase in human blood. International journal for vitamin and nutrition research, 1974, 44:319-326. (Calorimetric method.)

· Kimura M, Itokawa Y. Determination of blood transketolase by high-performance liquid chromatography. Journal of chromatography, 1982, 239:707-710. (Measures the amount of transketolase present rather than the enzyme activity. A micromethod that requires further development and validation.)

· Vuilleumier JP, Keller HE, Keck E. Clinical chemical methods for the routine assessment of the vitamin status in human populations. Part III: The apoenzyme stimulation tests for vitamin B1, B2 and B6 adapted to the Cobas-Bio analyzer. International journal for vitamin and nutrition research, 1989, 60:126-135. (Assay using centrifugal analyser.)

The following publications provide details of the determination of thiamine in blood:

· Fidanza F. Nutritional assessment: a manual for population studies. London, Chapman & Hall, 1991.

· Tietz NW. Clinical guide to laboratory tests, 3rd ed. Philadelphia, W.B. Saunders, 1995.

· Finglas PM. Thiamin. International journal for vitamin and nutrition research, 1993, 63:270-274.

The following publications provide details of the determination of thiamine in urine:

· Roser RL et al. Determination of urinary thiamin by high-pressure liquid chromatography utilizing the thiochrome fluorescent method. Journal of chromatography, 1978, 146:43-53. (Method comparable to using the microbiological assay.)

· Dong MH, Green MD, Sauberlich HE. Determination of urinary thiamin by the thiochrome method. Clinical biochemistry, 1981, 14:16-18.

· Pearson W. Biochemical appraisal of nutritional status in man. American journal of clinical nutrition, 1962, 11:462-476.

· O'Neal RM, Johnson OC, Schaefer AE. Guidelines for classification and interpretation of group blood and urine data collected as part of the National Nutrition Survey. Pediatric research, 1970, 4:103-106.

· Hennessy DJ, Cerecedo LR. Determination of free phosphorylated thiamin by a modified thiochrome assay. Journal of the American Chemical Society, 1939, 61:179-183.

· Nail PA, Thomas MR, Eakin R. The effect of thiamin and riboflavin supplementation on the level of those vitamins in human breast milk and urine. American journal of clinical nutrition, 1980, 33:198-204.

Methods for the determination of thiamine in breast milk are detailed in the following publications:

· Kendall N. Thiamin content of various milks. Journal of pediatrics, 1942, 20:65-73. (The thiochrome method.)

· Valyasevi A et al. Chemical compositions of breast milk in different locations of Thailand. Journal of the Medical Association of Thailand, 1968, 51:348-353.

· Hennessy DJ et al. Determination of free phosphorylated thiamin by a modified thiochrome assay. Journal of the American Chemical Society, 1939, 61:179-183. (Standard method of the Infant Formula Council.)

· Nail PA, Thomas MR, Eakin R. The effect of thiamin and riboflavin supplementation on the level of those vitamins in human breast milk and urine. American journal of clinical nutrition, 1980, 33:198-204.

Niacin

Only a few laboratory procedures are available for assessing niacin status. The general procedure is to measure one or more urinary excretion products of niacin metabolism, most commonly N1-methylnicotinamide (N1-MN) and 2-pyridone. The excretion ratio of 2-pyridone to N1-MN is the most reliable indicator of niacin nutritional status. More recently, high-pressure liquid chromatography techniques have been applied to plasma and urine samples to measure nicotinic acid, nicotinamide, and their metabolites. There have been reports on the use of modified microbiological assay procedures for determining the biologically active forms of niacin in plasma and blood. For laboratories lacking appropriate chromatography equipment, an extremely sensitive fluorometric procedure has been reported for the measurement of N1-MN and nicotinamide in serum.

In practice, laboratory assessment of niacin nutritional status is still limited to the measurement of niacin metabolites in urine. The availability of high-pressure liquid chromatography simplifies the process and enhances the speed, accuracy, and sensitivity of determinations of 2-pyridone and N1-MN in urine. General reference to determination of niacin may be found in the following publication:

· Sauberlich HE. Newer laboratory methods for assessing nutriture of selected B-complex vitamins. Annual reviews of nutrition, 1984, 4:377-407.

Methods for assessing niacin nutritional status using urine samples is detailed in the following publications:

· Goldsmith GA et al. Studies of niacin requirement in man. I. Experimental pellagra in subjects on corn diets low in niacin and tryptophan. Journal of clinical investigation, 1952, 31:533-542. (Microbiological methods for the determination of niacin and tryptophan and fluorometric methods for the determination of N1-MN and 6-pyridone.)

· Dillon JC et al. Les metabolites urinaires de la niacine au cours de la pellagre. [Urinary metabolites of niacin in pellagra.] Annals of nutrition and metabolism, 1992, 36:181-185. (High-pressure liquid chromatography method for measuring excretion ratio of 2-pyridone to N1-MN.)

Methods for assessing niacin nutritional status using plasma and blood samples are described in the following publications:

· Gravesen J. pH metric method for the determination of nicotinic acid in plasma. Journal of clinical microbiology, 1977, 5:390-392. (Microbiological assay procedure for determination of the biologically active forms of niacin in plasma and blood.)

· Kertcher JA et al. A radiometric microbiologic assay for the biologically active forms of niacin. Journal of nuclear medicine, 1979, 20:419-423. (Radiometric microbiological assay requiring use of tracer amounts of a radioisotope.)

Ascorbic acid

The measurement of serum levels of ascorbic acid is the most commonly used and practical procedure for evaluating vitamin C nutritional status in population groups or individuals. White blood cell levels of ascorbic acid provide information concerning the body stores of the vitamin, but the measurement is technically difficult to perform and its use is confined to clinical situations as an aid in the diagnosis of scurvy. Information on urinary levels of ascorbic acid and the use of vitamin C loading tests can also be helpful in the clinical diagnosis of scurvy.

Assessment of vitamin C status using serum, blood, or urine samples is described in the following publications:

· Schaffert RR, Kingsley GR. A rapid, simple method for the determination of reduced, dehydro-, and total ascorbic acid in biological material. Journal of biological chemistry, 1955, 212:59. (Using either dinitrophenylhydrazine or 2,6-dichloroindophenol reagent.)

· Deutsch MJ, Weeks CE. Microfluorimetric assay for vitamin C. Journal - Association of Official Analytical Chemists, 1965, 48:1248-1256. (Method shows a high degree of specificity.)

The following publication describes the assessment of vitamin C status using urine samples:

· March SC. A quantitative procedure for the assay of ascorbate-3-sulfate in biological samples. Federation proceedings, 1972, 31: 705.

Assessment of vitamin C status using serum/plasma samples is detailed in the following publications:

· Goad WC et al. A semiautomated technique for the determination of vitamin C in serum or plasma samples. US Army Medical Research and Nutrition Laboratory Report, Denver, CO, June 1972.

· Brubacher G, Vuilleumier JP, Vitamin C. In: Curtis HC, Roth M, eds. Clinical biochemistry - principles and methods. Berlin, Walter de Gruyter, 1974.

· Bates CJ. Plasma vitamin C assays: a European experience. International journal for vitamin and nutrition research, 1994, 64:283-287.