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close this bookMethods for the Evaluation of the Impact of Food and Nutrition Programmes (UNU, 1984, 287 p.)
close this folder4. Measuring impact using laboratory methodologies
View the document(introduction...)
View the documentIntroduction
View the documentDevelopment of a primary nutritional deficiency
View the documentChoice of tissue for laboratory assessment
View the documentSelection of laboratory methodologies for nutritional impact evaluation
View the documentLaboratory methods for assessment of nutritional impact
View the documentAnnex A. Laboratory evaluation of protein nutriture
View the documentAnnex B. Laboratory evaluation of vitamin A nutriture
View the documentAnnex C. Suggested methods for hematology
View the documentReferences

Laboratory methods for assessment of nutritional impact

The major nutrient deficiencies of public health significance which food or nutrient-specific distribution programmes have most often been designed to alleviate include inadequate food energy intake, protein-energy malnutrition, iron deficiency anaemia. vitamin A deficiency, and iodine deficiency. Evaluation by laboratory measurement of the nutritional impact of food programmes to combat these five nutrient-specific problems is possible using several methods that vary in degree of sophistication, reliability, and accuracy. Individual laboratories should evaluate their own resources and select the method best suited to their situation. We have chosen to include in this chapter those methods that are most practical and least costly, that required a moderate level of training of personnel, yet provide the degree of precision and accuracy required for programme evaluation. Other methods might be more appropriate for research purposes, or where laboratories are well-equipped and have a highly trained staff.

 

Protein-Energy Malnutrition (PEM)

Energy balance is best assessed by non-laboratory measurements (see chapter 3), while protein status can be reflected in biochemical measurements. The speed at which changes in protein status occur will vary according to the turnover rate of the protein in question. In contrast to iron and vitamin A, for which there are tissues that accumulate reserve stores (step 2, fig. 4.1.), there is no true storage tissue for protein, only variations in the amount of total active protein mass. Knowledge of turnover rates of various protein species found in blood is needed, therefore, to select the parameter appropriate for use in evaluating nutritional impact of nutritional interventions, i.e., long-term effects rather than short-term responses to the relative availability of a balanced amino acid supply for protein synthesis. In this respect, blood levels of rapidly turning over transport proteins (retinol-binding protein, or RBP, transferring and prealbumin) are known to be sensitive to short-term changes in the available protein and energy supply (step 3. fig. 1), but do not necessarily reflect depletion in protein mass (step 2, fig. 1 ) or decreased functional level (step 4, fig. 1).

The slower turning over albumin level in blood best reflects longer term protein status (3). The transport proteins, for example, are most useful for evaluating the immediate responsiveness to an intervention that provides an energy or protein supplement, or that decreases the burden of infections that stress protein-energy requirements; albumin levels, on the other hand, better reflect true nutritional impact on protein status. Because some transport proteins, such as transferring and retinol-binding protein (RBP), are at least partially dependent upon the availability of the nutrient they carry, their use for assessing protein nutriture may be confounded by concurrent deficits of the dependent nutrient. Prealbumin, though a carrier of the RBP-retinol complex as well as the iodine-containing thyroxine, is not dependent on either of these nutrients for its hepatic synthesis or secretion, yet still has a short half-life (2-days) that makes it sensitive to the immediate availability of a balanced amino acid supply (3). Blood levels of prealbumin, like albumin and other transport proteins, are dependent upon adequate liver function, and therefore are depressed by liver disease independent of dietary adequacy.

To evaluate programme impact on protein status, a combined assessment of prealbumin and albumin provides information on both short and long-term dietary effects, respectively (4). This methodology is most appropriately applied for evaluation of intervention programmes targeted to vulnerable groups such as preschool children, pregnant and lactating mothers. Unless evidence exists for substantially inadequate protein or energy intake among other recipient populations, such as school children and adult male workers, prior to the intervention, laboratory assessment of protein nutriture is unlikely to reflect responsiveness to a dietary change achieved through food aid programmes.

Suggested methods

Both prealbumin and albumin can be determined by relatively inexpensive, reliable laboratory technique requiring a routinely trained laboratory technician (see appendix). Radial immuno-diffusion (RID) is used to determine blood levels of prealbumin, and kits are available commercially for this purpose (5). The kits contain complete instructions and all the materials necessary for the assay, including the protein standards, with the exception of a microliter syringe and measuring ruler or caliper. The plates should be stored before use at refrigerator temperatures. The assay can be completed in as little as 18 hours.

There are several methods for determination of serum albumin, including standard electrophoresis, dyebinding, and salt fractionation. Specificity is highest for electrophoresis, followed by dye-binding and salt fractionation, and the relative cost for the analysis and sophistication of required equipment follow the same order. For evaluation purposes, the specificity and precision of the dye-binding procedure is adequate.

 

Vitamin A Nutriture

Interpretation of the biochemical measurement of vitamin A in blood short of deficient (< 10 µg/dl) or excess (> 70-80 µg/dl) levels is confounded by homeostatic controls, partially independent of diet, that modulate the release of liver reserve supplies (1). Hence, blood levels (step 3, fig. 1 ) do not necessarily reflect the level in the liver reserve (step 2, fig. 1 ) and, therefore, the relative level of vitamin A nutriture. Blood values that lie between about 15-30 µg/dl. particularly in young children and for some specific individuals, may reflect physiological conditions unrelated to vitamin A reserve stores. Under such circumstances, improved dietary intake of vitamin A through an intervention will not necessarily change the level in the blood. On the other hand, if the blood level for individuals is in a range that is difficult to interpret (15-30 µg/dl), for reasons of chronic inadequate intake and low liver reserves (step 2, fig. 1), blood levels will increase in response to an increased intake of vitamin A. Still, it cannot be assumed that adequate reserve stores have been established as a result of increased circulating levels, since blood levels must exceed a threshold before stores are replenished.

Therefore, to evaluate the impact of a food programme that seeks to improve vitamin A nutriture, it is important to look at changes in the lower end of the population distribution curve of blood levels rather than means or absolute values (6). When the lower end of the distribution curve shifts to the right following an intervention programme, this can be interpreted as programme impact even though there may be no significant change in mean or median values (7). Figures 4.2 (see



FIG. 4.2. Effect of Sugar Fortification with Vitamin A on the Distribution of Serum Retinol Levels of Rural Preschool Children. A: 1975 1976. B: 1975,1977.) and 4.3 (see


FIG. 4.3. Pre-supplementation and Post-supplementation Distribution of Serum Retinol Concentration (A) and Total Vitamin A Concentration (B) in a Group of Normal Elderly Men.) illustrate this point for, respectively, children in a national programme of vitamin A fortification of sugar and a shorter duration of vitamin A supplementation programme for elderly men. To assess biochemically the programme impact on vitamin A status, change in the distribution of blood levels under 30 µg/dl in the latter case, and especially those under 15 µg/dl in the former case, were found to be most useful.

Retinol-binding protein has been suggested as a vitamin A adequacy indicator that can be simply assayed in the field by RID techniques. However, as noted above, RBP synthesis is influenced by acute protein deficiency (4) and liver function, as well as by the availability of vitamin A from the diet or reserve tissue stores (8). Furthermore, the RID assay determines total RBP, including that not bound to retinol. The unbound or apo-RBP is physiologically unimportant with respect to vitamin A status. It is important to note that total RBP may remain in the low normal range while available vitamin A (holo-RBP) has declined dangerously (1).

Suggested Methods

The analytic method of choice for determination of vitamin A status uses high-pressure liquid chromatography (HPLC), which is fast, determines retinol directly, and minimizes opportunity for oxidative losses, but is expensive.

Several other analytic methods are available, such as spectrophotometry, fluorometry, and colorimetry, and with care they can be used interchangeably, depending upon available laboratory resources. All methods require a carefully trained and standardized technician. These methods are described in detail elsewhere (1). When a spectrophotometer equivalent to a DU is available, the procedure of Bessey and Lowry, based on UV inactivation, is likely to be least expensive and most reliable. In the absence of a DU spectrophotometer, which involves a relatively high initial investment, the colorimetric assay using trifluoracetic acid, trichloracetic acid, or antimony bichloride can be satisfactory, provided a reliable vitamin A standard is available and proper precautions are exercised (1). Fluorometric procedures are more sensitive than the colorimetric ones. However. spurious high results are notable because of fluorescent contaminants difficult to avoir under most laboratory conditions in developing countries.

Serum levels of vitamin A obtained cross-sectionally and displayed in distribution curves can provide information on the percentage of individuals with low levels of vitamin A who may be subclinicaily malnourished. When evaluated against an appropriately matched comparison group, this information is useful in assessing differences in the magnitude of the "at risk" group among recipients of an intervention programme. The best way to determine the nutritional impact of a programme on vitamin A status is to have before and after treatment laboratory measurements. An alternative is to measure the response of a subsample of recipients with plasma values that are in the lower portion of the distribution to an additional short-term supplement (8).

Laboratory assessments of vitamin A status are appropriate for evaluation of nutrition intervention programmes in which the daily intake of vitamin A is increased (vitamin A-containing food fortification programmes), in single, massive-dose intervention programmes, and in programmes to correct severe forms of protein-energy malnutrition. Usefulness in the latter types of programme stems from the intimate interrelationship between protein and vitamin A status. Intervention programmes to correct serious protein deficiency should always provide sources of vitamin A concurrently, Since there is no practical way of knowing whether serum levels of vitamin A are lowered by depletion of tissue reserves or are secondary to protein deficiency and impairment of mobilization. Stimulation of growth by correcting protein deficiency elevates the need for vitamin A, and if the latter is not supplied, irreversible eye damage can be precipitated in a very short time.

 

Iodine Nutriture

Clinical examination and palpation of the thyroid gland are generally sufficient to evaluate the success of programmes to correct iodine deficiency and control endemic goiter. However, iodine nutriture can be assessed in the laboratory by measuring blood levels of protein-bound iodine (PBI), urinary excretion of iodine, and radioiodine uptake. Since all three of these parameters may be influenced by various physiological states and drugs, interpretation of iodine nutriture by biochemical methods must be done with caution. As for vitamin A, it is necessary to evaluate the iodine impact of food programmes by looking for shifts in the lower range of PBI and urinary excretion values rather than for absolute values for means or medians.

 

Iron Deficiency Anaemia

Nutritional anaemia is one of the most common and significant nutritional problems in the world today. It is likely, therefore, that anaemia will be prevalent in areas where nutrition intervention programmes take place. Iron deficiency, folate deficiency, protein-calorie malnutrition, acute infection, and chronic disease can all contribute to the occurrence of anaemia. Studies in several parts of the world have demonstrated, however, that iron deficiency is, in most situations, the main etiologic factor.

Definition of Anaemia

Anaemia is usually defined using criteria established by population studies. These studies have determined, for individuals of different sex, age, and physiological condition, levels of haemoglobin concentration under which anaemia is likely to be present. It must be borne in mind, however, that there is an overlap of haemoglobin concentration figures between normal and anaemic individuals. Therefore the use of fixed limits of normality will misdiagnose as anaemic a certain proportion of individuals with adequate haemoglobin concentration and include among the normal group some anaemic subjects (see



FIG. 4.4. Fitted Double Population of Haemoglobin Concentrations in the Third Trimester of Pregnancy in Latin American Women: The vertical line depicts the haemoglobin value used to define anaemia according to WHO criteria. The higher curve represents the distribution of the normal population of pregnant women, and the lower curve, the anaemic group. The crosshatched area above 11 g/dl represents subjects found anaemic by distribution analysis but considered normal by WHO criteria. The shaded area below 11 g/dl represents that portion of the population found normal by distribution analysis but considered anaemic by WHO criteria. (Redrawn from Cook et al. 19] by Bothwell et al. 110].)). A more precise definition of the prevalence of anaemia in a population can be obtained by determining the proportion of individuals that show a significant haemoglobin response to supplementation with hematinics.

Stages in the Development of Iron Deficiency

A measureable decrease in haemoglobin concentration is a late effect of iron deficiency. The iron-replete individual not only has sufficient iron to synthesize haemoglobin and other essential iron containing compounds, but also has some iron reserves. The amount of iron stores in normal adult males have been estimated at 500 to 1,000 mg. Stores are lower in women of reproductive age and in infancy and childhood.

The first consequence of a negative iron balance is a decrease in the amount of storage iron, a condition known as iron depletion. Once stores are depleted there may not be a sufficient supply of iron for erythropoiesis, and haemoglobin synthesis is impaired. This state is known as iron-deficient erythropoiesis. After some time, this is reflected in a decrease in haemoglobin concentration and iron deficiency anaemia.

Laboratory Tests

The most useful tests for the evaluation of iron nutritional status are the plasma ferritin, the per cent saturation of transferrin, the concentration of free erythrocyte protoporphyrin, and the haemoglobin concentration. There is a general correlation between the stages of iron deficiency and the changes in these laboratory tests.

Valuable information on the iron status of individuals can be obtained by the measurement of plasma or serum ferritin. It has been shown that the concentration of this compound (step 3, fig. 4.1.) reflects the amount of storage iron (step 2, fig. 4.1.), and that 1 µg/l of serum ferritin is roughly equivalent to 10 mg. of iron stores. With progressive depletion of iron stores there is a parallel drop in serum ferritin, with serum values below 12 µg/l representing absence of storage iron.

After iron stores are depleted there is a drop in the amount of iron being transported in the plasma (plasma iron). The amount of the transport protein transferrin that is saturated with iron (or total binding capacity) concomitantly increases, so that per cent saturation of transferrin with iron falls from values above 30 per cent to less than 15 per cent. At the same time, since not all the protoporphyrin synthesized by erythrocyte precursors in the bone marrow is formed into heme because of the insufficient iron supply, there is a rise in the amount of free erythrocyte protoporphyrin in red cells from normal values of about 30 µg/dl to above 100 µ/dl. Finally, there is a measurable drop in haemoglobin concentration.

The relationship of these measurements to iron stores and the values found in the different stages of iron deficiency are depicted in figure 4.5.(see



FIG. 4.5. Measurements of Iron Status in Relationship to Body Iron Stores (mg). Negative iron stores indicate the amount of iron that must be replaced in circulating red cells before iron reserves can re-accumulate (Data from Cook and Finch [11]).). Extremely useful information can be obtained by the measurement of all four parameters. The most important, however, are probably haemoglobin and plasma ferritin, which permit evaluation of both extremes of the spectrum of iron status.

Monitoring Results of Intervention Programmes

As already mentioned, the two most useful laboratory tests for measuring the effect of interventions on nutritional anaemias are the haemoglobin concentration and the serum or plasma ferritin. In situations where there is a high prevalence of anaemia and supplementation strategies are used, effects will be most readily measured by changes in haemoglobin. With food fortification, on the other hand, especially in populations where there is little anaemia, one can expect relatively modest increases in the amount of daily absorbed iron, and results may be better monitored by measuring changes in iron stores as reflected in serum ferritin.

The effects of intervention programmes on iron deficiency anaemia can be better evaluated in vulnerable groups such as infants, preschool children, and pregnant women. Ideally, studies should be conducted in representative samples of the target populations and should include appropriate control groups.