The plasma amino acid ratio as an indicator of the protein nutrition status: a review of recent work.

480 NOTES The Plasma Amino Acid Ratio as an Indicator of the Protein Nutrition Status: A Review of Recent Work by W. K. SIMMONS 1 In many investigations, biochemical measure- ments for the determination of protein nutrition have been suggested and tested. Some measurements have been shown to be useful while others have not. The following discussion presents a review of the latest work on a biochemical test that the author believes to be a useful tool in the evaluation of protein nutrition. Cravioto (1958) found in Mexico that the total free amino acid concentration was low in the plasma of 13 malnourished infants. However, he made no correlation between the total level of amino acids and the clinical type of malnutrition, its duration or degree. In the same year, Westall et al. (1958), also in Mexico, reported on a study of 4 children with protein-calorie malnutrition. They observed that the essential and non-essential amino acids were no longer in balance. Children with severe protein malnutrition (Arroyave et al., 1962) or on a nitrogen- free diet (Arroyave, 1962) showed that the total amount of a-amino acids present in the plasma was half that in healthy children. As a generalization, they stated that the amino acids most affected were the essential ones with the exception of lysine and phenylalanine. Of the non-essential amino acids, tyrosine, cystine and arginine were the lowest in concentration. A similar pattern of the imbalance of essential and non-essential amino acids has also been found in other parts of the world (Edozien, Phillips & Collis, 1960; Vis et al., 1958). The most striking feature is the great reduction in the amounts of the essential amino acids leucine, isoleucine and valine. Of the non-essential amino acids, tyrosine, arginine, citralline and a-amino-isobutyric acid are far below normal, whereas the other non-essential amino acids are at, or even above, normal levels (Snyderman et al., 1963). A study made in 1963 has been reported (Holt et al., 1963) in which 64 patients from 9 countries (Mexico, Haiti, Jamaica, Senegal, 1 Formerly Biochemist, WHO Nutrition Survey and Campaign against Malnutrition, Nutritional Biochemistry Laboratory, Nairobi, Kenya. Present address: Institute of Nutrition, University of Recife, Recife, Brazil. Ghana, Nigeria, Egypt, Jordan and India) with clinical signs of protein-calorie malnutrition were examined to obtain direct and definite evidence of the limiting amino acids in the disease. It was found that a uniform pattern of the plasma amino acids occurred on each occasion, regardless of the charac- teristic diet of the country. The plasma concentra- tions of the essential amino acids and of certain non-essential ones, notably tyrosine and arginine, were strikingly reduced, whereas the concentrations of most of the non-essential amino acids remained at their usual levels, and at times even exhibited abnormally high values. It was also noted that even the mildest cases already showed a slight fall in concentration of the essential amino acids and in tyrosine levels and an increase above normal of the non-essential amino acid levels. In the most severe cases there is a marked fall in concentration of all amino acids. Of the essential amino acids, the branched-chain amino acids-leucine, isoleucine and valine-are subject to the most marked changes, while lycine and phenylalanine are the least affected. The same imbalance of plasma amino acids in children with protein malnutrition has been reported from Uganda (Whitehead & Dean, 1964a). Therefore, it is possible that a study of the con- centrations of free amino acids of the plasma may be helpful in the early detection of protein malnutrition. There are large falls in the concentration of most of the essential amino acids with the result that the ratio of the non-essential to essential amino acids is increased before any other alterations of bio- chemistry so far known can be detected. White- head (Dean & Whitehead, 1963; Whitehead, 1964; Whitehead & Dean, 1964b) introduced a test making use of this principle. Approximately 100 pl-200 /tl of blood are taken by means of a finger-prick. The amino acids are estimated by 1-way paper-chromato- graphy, in which the group of branched-chain essential amino acids (leucine, isoleucine, valine and methionine) is separated from another group that contains the non-essential amino acids (glycine, serine, taurine and glutamine). The spots containing the amino acids are cut out and eluted and the ratio 2488G THE PLASMA AMINO ACID RATIO AND PROTEIN NUTRITION STATUS is obtained from the extinction coefficients of the colours given by the 2 groups. This method was applied to several hundred children and was found to be helpful in the recogni- tion of cases of subclinical kwashiorkor, in that the height of the ratio could be related to the incidence of the clinical signs (Whitehead & Dean, 1964b). Probably more important was the observation that the ratio was well correlated with the percentage weight deficit. Whitehead (1967) again tested the plasma amino acid ratio and found that it was raised in children with kwashiorkor. However, the ratio was only slightly raised in marasmic children. Some exceptions to this conclusion have been found; in a field study of 260 children in Lebanon (McLaren, Kamel & Ayyoub, 1965), no correlation was found between the plasma amino acid ratio and the percentage weight deficit and it was not found to be helpful in identifying subclinical cases. The reason may be that throughout the Middle East the " summer diarrhoea" season dominates and determines malnutrition in infants and young children. It causes repeated attacks of gastroenteritis and prolonged semistarvation and therefore pro- duces the predominant entity of nutritional maras- mus and, to a much lesser extent, typical kwashior- kor. In contrast, the diets seen in Buganda, Uganda, are high in carbohydrate and low in protein. The effect of season is also not as dramatic. Similar experiences have been noted in Turkey and in South Africa. In Turkey, the plasma amino acid ratio was tested in children with severe kwashiorkor of the marasmic type and the value was found to be 1.41 L0.57, which would be expected in normal children (Gurson & Heyzi, 1966). It was concluded that the amino acid ratio was not an effective test in children with kwashiorkor of the marasmic type. In South Africa the reason why the plasma amino acid ratio was ineffective for detecting subclinical cases of kwashiorkor was the supervention of acute starvation in the course of chronic protein mal- nutrition (Truswell et al., 1966). Prolonged fasting in adults leads to an increase of plasma branched- chain amino acids accompanied by reduced non- essential amino acids (Swenseid, Friedrich & Tuttle, 1961; Tuttle et al., 1962). The opposite pattern is seen in typical kwashiorkor. It was shown that in rats (Widdowson & White- head, 1966) the plasma amino acid ratio was raised when animals on a low-protein diet were compared with the control rats. In the rats fed a diet low in calories, the amino acid ratio was raised slightly but not to the same extent as in the rats on a low- protein diet. Therefore, in rats under experimental conditions, an increased amino acid ratio is an indicator of protein deficiency rather than calorie deficiency. This more or less supports the views of other investigators concerning children. Even though inconsistencies have been noted, it has been found (Bohdal, Gibbs & Simmons, 1968; Bohdal & Simmons, 1969; Simmons & Bohdal, unpublished data) that the plasma amino acid ratio is a useful test under both hospital and field con- ditions. The plasma amino acid ratio test was first used, along with several other biochemical tests, to aid the evaluation of a low-cost, high-protein food for use in Kenya (Bohdal, Gibbs & Simmons, 1968). A total of 12 children with severe kwashiorkor was divided into 2 control and test groups with 6 children in each group. The control group was given enzyma- tically digested casein with dried skimmed milk. In the test group, the casein was replaced by the low- cost, high-protein food in amounts corresponding to the nitrogen of the digested casein. The diets were given to the children for 4 weeks. As can be seen from Fig. 1, the amino acid ratio averaged 3.0-4.0 in the kwashiorkoric children. In 2 weeks of treatment, the amino acid ratios in both groups were in the normal range (below 2.0) thus assisting the evaluation of the study. The plasma amino acid ratio was subsequently used (Bohdal & Simmons, 1969) as an indicator of the protein nutrition status of children in an elite kindergarten in Nairobi. As was expected (Table 1), TABLE 1 PLASMA AMINO ACID RATIOS IN THE BLOOD OF NORMAL EUROPEAN, AFRICAN AND ASIAN CHILDRENa Euro- |peanb African b Asian b Averageb Number examined 28 45 14 87 Mean ratio 1.6 1.6 1.7 1.6 Standard deviation 0.33 0.36 0.40 0.37 Standard error 0.06 0.05 0.07 0.04 a Taken from Bohdal & Simmons (1969). b Non-significant differences. 10 481 NOTES FIG. I CHANGES IN THE PLASMA AMINO ACID RATIO IN KWASHIORKORIC CHILDREN DURING 4 WEEKS OF TREATMENT a 40 I-t 30 0 <10 0 1 2 3 . 4 WEEKS OF TREATMENT te 9 o o Test ^ 6 Control ...... Normal a Taken from Bohdal, Gibbs & Simmons (1968). TABLE 2 PLASMA AMINO ACID RATIOS DISTRIBUTION BY LOCATION IN CHILDREN AGED 1-15 YEARS a Control West Koguta J Masumbi Uthiuni Nyaani Number examined 87 84 57 80 78 Mean ratio 1.6 2.42 2.16 2.83 2.18 Standard deviation 0.37 0.49 0.57 0.87 0.49 Standard error 0.04 0.05 0.08 0.10 0.06 Control v. West Koguta P<0.001; highly significant Control v. Masumbi P<0.001; highly significant Control v. Uthiuni P<0.001; highly significant Control v. Nyaani P<0.001; highly signlficant West Koguta v. Masumbi P<0.01; signiflcant West Koguta v. Uthiuni P<0.001; highly significant West Koguta v. Nyaani P<0.001; highly significant Masumbi v. Uthiuni P<0.001; highly significant Masumbi v. Nyaani Not significant Uthiuni v. Nyaani P<0.001; highly significant a From Simmons, W. K. & Bohdal, M. (unpublished data). 482 THE PLASMA AMINO ACID RATIO AND PROTEIN NUTRITION STATUS 483 FIG. 2 POSSIBLE EFFECTIVENESS OF THE PLASMA AMINO ACID RATIO IN THE DETECTION OF PROTEIN-CALORIE MALNUTRITION Kwashiorkor Maofasmus (protein deficiency) Marasmic kwvashiorkor (calorie deficiency) \wmO 917,9 Possible range of an abnormal amino acid ratio the ratio for each group was 1.6, which is the normal value. When an attempt was made to evaluate the protein nutrition status of communities it was found that the plasma amino acid ratio was extremely useful. The values ranged from 2.1 to 2.8 and there was a highly significant difference (P<0.001) in each case when the various locations were compared with the kindergarten (control) group (Table 2). The plasma amino acid ratios were found to agree with the results of the dietary surveys (Simmons & Bohdal, unpublished data). It is interesting to note that even though some of the dietary data suggested a deficiency of calories as well as of proteins, the amino acid ratio was still useful in determining differences among the areas surveyed. Therefore, the plasma amino acid ratio could be a useful test to help in the evaluation of the protein nutritional status of communities. Other workers (Rutishauser & Whitehead, 1969; Arroyave & Bowering, 1968; Bjornesjo, 1968) have also tested the plasma amino acid ratio under field conditions and found it useful in evaluating the protein nutritional status of communities. It seems therefore that the plasma amino acid ratio is a useful test with which to detect alterations in the free-amino-acid pool consequent to an in- sufficient supply of dietary protein. However, if a deficiency of calories, as well as a shortage of protein, or a general deprivation of food, is suspected, the test may be of little value. In Fig. 2 a scheme is presented to show the possible effectiveness of the plasma amino acid ratio in the detection of cases of protein-calorie mal- nutrition. If it is accepted that " kwashiorkor " is caused mainly by a deficiency of protein in the diet, that " marasmus" results principally from calorie deficiency and that " marasmic kwashiorkor " is the intermediate form, the amino acid ratio may be elevated in the range shown. However, it must be noted that all biochemical tests are subjected to intercurrent factors. In the detection of protein- calorie malnutrition, such factors as infections or vitamin or mineral deficiencies could greatly influence the effectiveness of the biochemical test. More re- search is needed under controlled conditions to evaluate the plasma amino acid ratios under different dietary regimes. REFERENCES Arroyave, G. (1962) Amer. J. clin. Nutr., 11, 493 Arroyave, G. & Bowering, J. (1968) Arch. lat.-amer. Nutr., 18, 341 Arroyave, G., Wilson, D., Funes, C. de & Behar, M. (1962) Amer. J. clin. Nutr., 11 517 Bjomesjo, K. B. (1968) Calorie deficiencies and protein deficiencies, London, Churchill, p. 117 Bohdal, M., Gibbs, N. E. & Simmons, W. K. (1968) Nutrition survey and campaign against malnutrition in Kenya, 1964-1968, Nairobi, Ministry of Health, Kenya, p. 185 Bohdal, M. & Simmons, W. K. (1969) Bull. Wld HIth Org., 40, 166 Cravioto, J. (1958) Amer. J. clin. Nutr., 6, 495 Dean, R. F. A. & Whitehead, R. G. (1963) Free amino acids in the blood of malnourished children: a simplified method of analysis. In: Proceedings of the Sixth International Congress of Nutrition, Edinburgh, Living- stone, p. 597 Edozien, J. C., Phillips, E. J. & Collis, W. R. F. (1960) Lancet, 1, 615 Gurson, C. T. & Neyzi, 0. (1961) Plasma amino acid ratio in infants with severe chronic malnutrition. In: Abstracts of Proceedings of the Seventh International Congress of Nutrition, Hamburg, p. 48 484 NOTES Holt, L. E., Snyderman, S. E., Norton, P. M., Roitman, E. & Finch, J. (1963) Lancet, 2, 1343 McLaren, D. S., Kamel, W. W. & Ayyoub, N. (1965) Amer. J. clin. Nutr., 17, 152 Rutishauser, I. H. E. & Whitehead, R. G. (1969) Brit. J. Nutr., 23, 1 Snyderman, S. E., Holt, L. E., Norton, P. M., Roitman, E. & Finch, J. (1963) Amer. J. clin. Nutr., 12, 333 Swenseid, M. E., Friedrich, B. W. & Tuttle, S. G. (1961) Fed. Proc., 20, No. 1, Pt 1, p. 8 Truswell, A. S., Wannenburg, P., Wittman, W. & Hansen, J. D. L. (1966) Lancet, 1, 1162 Tuttle, S. G., Swenseid, M. E., Friedrich, B. W. & Griffith, W. H. (1962) Fed. Proc., 21, No. 2, p. 395 Vis, H., Dubois, R., Leob. H., Vincent, M. & Bigwood, E. J. (1958) Ann. Soc. Belge Med. trop., 38, 991 Westall, R. G., Roitman, E., de la Pena, C., Rasmussen, H., Cravioto, J., Gomez, F. & Holt, L. E., Jr (1958) Arch. Dis. Childh., 33, 499 Widdowson, E. M. & Whitehead, R. G. (1966) Nature (Lond.), 212, 683 Whitehead, R. G. (1964) Lancet, 1, 250 Whitehead, R. G. (1967) Arch. Dis. Childh., 42, 479 Whitehead, R. G. & Dean, R. F. A. (1964a) Amer. J. clin. Nutr., 14, 313 Whitehead, R. G. & Dean, R. F. A. (1964b) Amer. J. clin. Nutr., 14, 320 A Study on the Minimal Amount of Serum Necessary for the Determination of the Plasma Amino Acid Ratio by W. K. SIMMONS 1 Many workers have indicated that the free amino acids of the serum of children acutely ill with kwashiorkor are affected in such a way that the concentration of essential amino acids is reduced while the concentration of non-essential amino acids remains normal or is elevated (Arroyave & Bowering, 1968; Cravioto et al., 1960; Edozien, Phillips & Collis, 1960; Norton, 1960; Saunders et al., 1967; Westall et al., 1958; Whitehead & Dean, 1964a). Using this principle, Whitehead (1964) and White- head & Dean (1964b) developed a simple paper- chromatographic technique in which some essential amino acids, principally valine, leucine and iso- leucine, were compared with a group of non-essential amino acids. The method was used to help evaluate the protein! nutritional status (Whitehead & Dean, 1964b). It Lwas suggested (Whitehead, 1964) that 100 pl-200 I-l of blood were necessary for the deter- mination. In this study, the smallest amount of serum that would be required for an accurate determina- tion was investigated. Methods A group of 20 children from Kenyatta Hospital, Nairobi, was selected for the study; 5 ml of blood 1 Formerly Biochemist, WHO Nutrition Survey and Campaign against Malnutrition, Nutritional Biochemistry Laboratory, Nairobi, Kenya. Present address: Institute of Nutrition, University of Recife, Recife, Brazil. were taken from each child. The amino acid ratio was determined according to the method of White- head (Whitehead, 1964; Whitehead & Dean, 1964b). From the 20 samples of blood, 6 samples which were representative of both normal and abnormal values were chosen. Serum samples were not taken from kwashiorkoric children because such cases are not usually found in the field. From each of the 6 serum samples, 120 ,l were taken and an amino acid ratio determination was made in triplicate from each sample. Each sample was then estimated in triplicate, the volume being reduced by 10 ,lI on each occasion until a volume of only 60 ,ul was used. Results In Table 1, the amino acid ratios together with their ranges are shown. The ratios range from 1.2 to 2.9; these are values found in healthy individuals and persons living on a rural African diet (Bohdal & Simmons, 1969; Simmons & Bohdal, unpublished data). In Table 2, the mean value of the amino acid ratios in percentages of the mean at 100 pl are given. It can be seen that the mean values of the amino acid ratio are constant down to a sample size of 90 IlI when the amino acid ratio is in the range 1.9-2.9. In samples D and F where the amino acid ratio is only 1.2, the values are constant only down to a sample size of 100 ul. 2488H