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Principles and Methods of Estimating Protein
Requirements during Growth

Dr. G. Gopalan and B.S. Narasinga Rao

1. Methods of estimating protein requirements

1.1. Growth and nitrogen balance:

The logical approach for estimating protein requirements for growth would be to determine the level of protein intake which could help achieve normal growth and development. In early infancy, when growth-rate is rapid, growth could in fact be a useful and practical criterion for assessing protein adequacy. However, in children beyond one year of age when growth is not rapid, the use of growth as a criterion of protein adequacy would imply studies of long duration in a large number of subjects. Such studies are difficult to organize and it may be unethical to maintain children for long duration on diets providing marginal or inadequate levels of protein. These considerations necessitate resort to the conventional approach of measurements of nitrogen balance on diets providing different levels of protein and amino acids. The cumulative errors involved in the usual nitrogen balance studies and the other limitations of this approach are now well-recognized. Despite these limitations, controlled feeding experiments employing measurements of growth and/or nitrogen retention provide, at present, the most feasible approach for the estimation of protein requirements for growth. In fact, most of the data available today with regard to protein requirements for growth are based on such studies.

There is, however, a need for identifying other biochemical criteria of protein adequacy during growth. Arroyave (1) has tried to use the ratio of non-essential/ essential amino acids in plasma for this purpose. Hydroxyproline excretion may also merit investigation. The validity of these approaches still remains to be established.

1.2. The factorial approach:

Protein requirements during growth may also be computed through the so-called "factorial" approach. The overall protein requirement in infancy and childhood may be considered to represent the sum total of the following: (a) "endogenous" or "obligatory nitrogen losses in urine and feces"; (b) cutaneous and sweat losses in nitrogen; (c) protein need for chemical maturation or changing body composition; and (d) protein needs for actual growth.

The computation of protein requirement through this approach involves some uncertainties and assumptions, but may be employed as a check on the values determined by other procedures.

Direct determinations of endogenous nitrogen loss in infants and children by maintaining them on protein-free regimes are generally not feasible. Endogenous urinary and faecal nitrogen losses may, however, be computed from prediction equations based on the observed linear relationship between nitrogen balance and nitrogen retention at different ages in several nitrogen metabolism studies.

Direct estimates of integumental and sweat losses are also difficult but there are indirect indications from reported studies of nitrogen balance and growth that these losses may not be significant.

Body nitrogen content which is about 2% at birth increases to the adult values of about 3% by the age of 4, but 90% of this increase is believed to occur during early infancy. In the interpretation of data from nitrogen balance studies during early infancy, this factor of chemical maturation has to be taken into account. The observations (Holt et al (2)) that (a) in infants nitrogen retention tends to increase with increasing protein intake, and (b) that such retentions are not necessarily reflected in growth, have been interpreted as indicating that it is possible to accelerate chemical maturation through higher levels of protein. It is, however, doubtful whether such acceleration is in fact desirable.

1.3. Total body protein turnover:

Protein requirement during growth may be considered as the requirement for replacing body proteins and for laying down new tissues. On this basis, an estimate of total body protein turnover or total body protein synthesis rate in normally growing infants and children should theoretically provide information on protein requirmenets during growth. Several attempts have been made to study total protein turnover in man using different labelled amino acids. The values for the rate of protein synthesis in these studies have varied over a wide range depending on the method employed. This would imply that a valid and universally acceptable method for measuring protein synthesis and turnover rate has yet to be established. Moreover, even if total protein synthesis rate can be estimated accurately by an appropriate isotopic method, caution is necessary in interpreting the results. Both tissue proteins and the diet contribute to the amino acid pool. The level of dietary amino acid needed to maintain a particular level of synthetic rate may be expected to depend on the efficienty of re-utilization of tissue amino acids.

Picou and Taylor-Roberts (3) found that the synthesis rate in children recovering from malnutrition, using glycine labelled with N15 was the same with mean intakes of 0.84 g N/kg/day and 0.19 g N/kg/day. Presumably the lowest dietary protein intake which could maintain normal synthetic rate could provide the indication of the actual protein requirement.

1.4. Estimation of total body potassium:

The two criteria often employed in estimating protein requirement during growth viz., body weight increase and nitrogen retention have their limitations. Changes in body composition may vitiate the significance of body weight changes; nitrogen balance techniques are not always feasible in young children and also often tend to over-estimate nitrogen retention. Measurements of lean body mass may, under these circumstances, provide more reliable estimate of tissue growth. This measurement can be achieved through determination of total body potassium by whole body counting of natural radiative potassium40. Though this technique involves expensive equipment and careful standardisation, it has other practical advantages in studies in infants and young children.

The possible approaches to the estimation of protein requirements during growth have been summarized in Table 1.

2. Review of available data  

2.1. Protein requirement in infancy:

2.1.1. Data from growth and nitrogen balance studies:

Human milk has been traditionally considered as the ideal food for infants at least up to the age of six months. Macy and co-workers (4,5) had indicated that in infants growing normally protein intake from breast milk during early weeks of infancy was 2.3 g/kg and declined to 1.5 g/kg by six months.

Fomon and May (6) fed bottled pasteurized human milk to infants between the ages of 8 and 180 days ad lib and determined growth and nitrogen retention. From the amount of milk consumed and its composition, protein and calorie intakes were calculated. The protein intake was found to decrease from 2.4 g/kg during the first month to 1.5 g/kg between 4½ and 6 months. The calorie intake ranged from 143 Kcal/kg during the first month to 90 Kcal/kg during the sixth month. On these intakes, the infants were growing satisfactorily. Nitrogen balance carried out at different periods indicated that the mean N retention at one month was 180 mg/kg and it gradually decreased to 45 mg/kg. by six months of age. Employing cow's milk or soya-based formula these authors (7, 9) observed that daily protein intake by infants of 4 ½ - 6 months old, for satisfactory growth, was 1.46 g. and 1.73 g. per kg. body weight respectively.

In another study Fomon and Filer (10) fed soya-based formula in which protein contributed 6.1 % of calories to 20 normal full-size infants from a few days after birth until approximately 112 days of age. Increases in length and weight of 18 of the 20 infants were similar to those of breast fed infants. Protein intake ranged from 1.25 to 2.17 g/kg per day. However, serum albumin concentrations at 112 days of age in these children were significantly lower than those of normal breast fed infants. Fomon and Filer (11) repeated this study with another group of 22 infants with a milk-based formula providing 6 % of calories from protein. All these infants showed normal growth rates and all but 2 had normal serum albumin concentration. The average protein intakes on this formula, between 8 and 112 days of age by 12 male infants ranged from 1.26 to 1.83 g/kg and in 10 female subjects from 1.36 to 1.60 g/kg/day.

Gopalan (12) studied growth of 14 breast fed infants of Indian women belonging to the low socio-economic group from one week to 22 weeks. From the amount of breast milk ingested which was determined by test-feeding technique and the protein concentration in breast-milk, protein intakes by these infants were determined. The daily protein intake ranged from 2.0 g/kg/day in the first fortnight to 1.1 g/kg at the sixth month. Though these children doubled their birth-weight in 4 months, the overall growth rate was slightly lower than that reported for infants fed breast milk ad lib. It was calculated that the intake of breast-milk in these infants was also probably insufficient to meet their calorie needs, providing as they did only 106 Kcal/kg in the first week and 58 Kcal/kg in the 22nd week. Considering the satisfactory growth rate during the first three months, an intake of 2.0 g/kg may be considered adequate at that period while the observed intake of 1.1 g/kg at 6 months may have been below requirement.

The results of the above studies are summarised in Table 2. They amply demonstrate that protein intake ranging from 1.5 to 2.3 g/kg is adequate for satisfactory growth up to six months of life. It makes no difference whether the protein is derived from breastmilk or cow's milk. It is also evident from these studies that a milk preparation containing protein providing 6 % of calories is adequate in early infancy.

2.1.2. Data from the "factorial approach":

The computed endogenous urinary and faecal nitrogen losses, on the basis of prediction equations for different age periods in the several N metabolic studies carried out by Fomon and his group (9) (Table 3) varied between 44 mg/kg to 67 mg/kg. The average endogenous loss during the first 6 months would work out to 60 mg/kg/day.

Fomon et al (13) had also determined endogenous urine N and metabolic faecal N in infants up to 6 months of age by feeding a low-protein diet in 6 day metabolic studies (Table 3). These studies involved two groups of infants, those whose earstwhile protein intake was low or intermediate (2.5 g/kg) and those whose earstwhile intake was high (4.0 g/kg or more). They reported endogenous urinary nitrogen excretion of 0.6 mg per basal calorie or 37 mg/kg for faecal excretion of 20 mg N/kg in the first group. In the second group, the corresponding values were 0.8 mg/basal calories (or 42 mg/kg) for urinary loss and 33 mg/kg for faecal loss. Since the protein intake of the second group was rather high, a three-day adjustment period may not have been sufficient for the urinary excretion to reach a steady state thus leading to higher levels. Therefore, the values obtained in the first group with normal protein intake may be considered a more reasonable estimate.

An interesting observation in the above study was that endogenous urinary N excretion was only about 0.6 mg per basal calorie, which is much lower than in adults. However, urinary N loss expressed per kg body weight was comparable to the adult values (37 mg/kg).

From the above studies, it may be concluded that obligatory loss of N through urine and faeces in infants up to 6 months is around 70 mg/kg/day.

Cutaneous loss: There have been no attempts to directly measure cutaneous loss of N in infants. Cook et al (14) had reported a sweat loss of 5 mg N/kg/day in infants. It may, however, be surmised that cutaneous losses of N are negligible since, in spite of over-estimation arising from inherent cumulative errors of estimation in N balance studies, observed N retentions in Fomon's subjects were actually lower than that predicted for the growth.

Growth needs: An estimate of the expected nitrogen retention for growth and maturation is given in Table 4. Fomon and May (6) have reported from their N balance studies in breast milk fed babies, a nitrogen retention of 180 mg/kg/day during the first month which decreased to 47 mg/kg by the sixth month. The mean gain in weight per g. N retained was found to be 42.1 g &+-; 11.5, indicating that the protein content of body weight gain is 15 % during this period.

Based on 70 mg of endogenous loss through urine and faeces and an average growth requirement of 130 mg N/kg/day, average protein requirement during the first 6 months would work out to 1.25 g reference protein per kg/day. An allowance of 30 % for individual variation and stress would bring the figure to 1.6 g/kg. If the utilization is about 80 %, the figure in terms of nett protein would be 2.0 g/kg/day. This is in good agreement with the estimations for protein requirement in infancy arrived at by N balance and growth studies.

It may be concluded on the basis of available evidence that protein requirement in infancy during first six months will range from 2.3 g/kg/day, in the early weeks, to 1.5 g/ kg/day at about six months.

2.2. Protein requirements of children:

2.2.1. Data from growth and nitrogen balance studies:

The previous FAO/WHO Committee (15) employed the factorial method to estimate protein requirements of children. Since then, there have been some studies to estimate protein requirement of children employing growth and N balance as criteria.

Chan and Waterlow (16) studied N balance and growth in children at one year (age range 6-18 months) on different levels of protein intake. These children had recovered from malnutrition and they were fed a formula in which protein was derived from skimmilk at a constant intake of 120 Cal/kg. They carried out both maintenance and growth studies. In the former, the children were given 200 mg N/kg/day and N intake progressively reduced till N balance was just maintained. In the growth study, N intake was varied between 190-275 mg N/kg/day.

It was observed that an intake of 200 mg N/kg/day was sufficient to promote satisfactory growth and N retention. At this intake, growth and N retention were maximal and beyond this intake N retention was constant. The growth rate was 3 g/kg/day, which was more than twice the expected growth rate for this age group. Nitrogen retention per 100 g. body weight gain was 2.91 g. which was close to the expected figure. At an intake of 1.25 g/protein/kg, NPU was 88.5 %. It should, however, be pointed out that at this intake, the growth rate was twice the normal figure. It may, therefore, be safely assumed that a level of 1.25 g/kg of a protein of NPU 90 or reference protein of 1.1 g/kg/day would be adequate for normal growth. It is even possible that the actual requirement for normal growth is somewhat lower than this.

Arroyave (1) has recently studied protein requirement in children of 2-3 years of age employing N balance and plasma ratios of non-essential to essential amino acids as the criteria. The children were fed varying levels of egg protein both in the descending and the ascending order. A positive N balance of 30 mg/kg or more and a normal plasma amino acid ratio, were observed at intakes of 1.0 - 1.20 g. of egg protein per kg of body weight. This level has been considered adequate to meet the protein requirement of this age group, thus confirming the correctness of earlier estimates.

Nitrogen balance study carried out recently in Hyderabad, (17) in moderately undernourished children aged 3-5 years on a diet exclusively based on wheat indicated that an intake of 280 mg N/kg/day from wheat was sufficient to promote satisfactory N retention in these children. These children were fed "chappathis" made with either unfortified or lysine fortified wheat, for a period of one month and N balances were determined on three occasions at ten day intervals. The calorie intake was 100 Kcal/kg/day. It was observed that these children on an average intake of 280 mg N/kg (or 1.75 g protein/kg) retained 40 mg or more of nitrogen per kg body weight per day. On the basis of nitrogen retained, this intake of 1.75 g. of wheat protein/kg/day can be considered adequate to meet the protein requirement of these children. Assuming an NPU of 50 for wheat protein, the intake in terms of reference protein would be 0.88 g/kg/day.

Begum et al (18) studied growth and nitrogen balance in apparently healthy Indian children of 2-5 years of age on wheat and rice based diets, the proteins in these diets being exclusively of plant origin. These children (13 on each diet) were fed 2.0 g. protein and 100 Cal/kg/day for a period of six months. Growth of these children on these two diets was quite satisfactory and was comparable to the North American standard. A short term nitrogen balance study in 10 of the children on each of the diets indicated a positive balance of about 100 mg/kg/day. This study would thus indicate that 2.0 g. protein per kg/day derived mainly from cereals is more than adequate to meet the protein requirement of pre-school children. This intake in terms of reference protein would be roughly 1.0 g/kg/day assuming an average NPU of 50 % for these diets. This level may not necessarily be the minimal level since lower protein levels were not employed in this study.

Aberanthy et al (19) carried out extensive nitrogen balance studies in girls of 7-9 years on natural diets at different levels of protein intakes. Their data indicate that an intake of 210 mg N/kg/day (or 1.3 g protein) is adequate to promote nitrogen retention of the order of 30 mg/kg/day which may be considered as adequate. An intake of 1.3 g. of dietary protein or 0.8 g. of reference protein per kg/day would thus appear to be adequate for this age group.

Several N balance studies have been carried out at Mysore (20 - 26) in Indian children of 8 - 12 years of age on poor diets predominantly based on cereals or millets with NPU ranging from 45 - 60. Nitrogen retention of 30 mg/kg/day or more was observed on intakes of about 250 mg N/kg/day (1.6 g protein/kg) from plant proteins or 0.8 g. reference protein per kg. per day. These figures are in accordance with the estimated requirement employing the factorial method.

Results of the above studies are summarized in Table 5. From these data it can be concluded that protein requirement of children in terms of reference protein would vary from 1.1 g/kg/day at about 1 year to 0.8 g/kg/day during 10 - 12 years.

2.2.2 Data from factorial approach:

Available information on endogenous urinary and faecal loss in children is summarized in Table 6. Waterlow and Wills (27) carried out nitrogen balance studies in malnourished infants with an average age of 12 months (3 months - 24 months), on different levels of protein intakes derived from milk protein and found a linear relationship between N intake and N absorbed which on extrapolation yielded a value of 33 mg/kg for the metabolic fecal N loss. They also found a linear relationship between absorbed N and N retention from which the endogenous urinary nitrogen excretion was estimated to be 37 mg/kg/day.

In another study by Chan and Waterlow (16) on children between 6 and 18 months of age, who had recovered from malnutrition, obligatory N loss through urine and faeces was estimated from the regression equation relating intake and retention to be 97 mg/kg/day.

Fomon et al (13) determined endogenous urinary and fecal nitrogen in children of 3 - 4 years of age by estimating urinary and fecal N during 4th - 6th days on a protein-free diet. In a group of 12 children with prior protein intake of 0.5 - 1.3 g/kg/day endogenous urinary and faecal nitrogen excretion was found to be 46 mg/kg (0.9 mg/basal calories) and 23 mg/kg respectively. In another group with prior protein intake of 1.4 - 1.7 g/kg/day, the corresponding values were 53 mg/kg (1.1 mg/basal cal.) and 29 mg/kg. In eight children with prior high protein intakes of 4.5 - 6.3 g/kg/day, still higher values of 68 mg/kg (1.4 mg/basal cal.) for urinary N and 31 mg/kg for faecal N were obtained. In the last group excretion may not have reached the steady state after three days of protein-free diet. It would appear that in this group the preceding level of protein intake had definitely influenced the urinary N excretion. Considering this limitation, this study would suggest that obligatiory loss of urinary N is about 50 mg/kg or 1.0 mg/basal cal. and faecal N loss is 30 mg/kg in children of 3 - 4 years of age.

DeMaeyer and Vanderborght (28) have reported nitrogen balance studies in rural African children of 3 - 8 years of age. Nitrogen balance was done on a protein-free diet and with different proteins at various levels. From the regression equation relating intake and N balance with milk and soya protein diets, the estimated endogenous nitrogen was found to range from 64 - 75 mg/kg with an average of 70 mg/kg. They also reported that endogenous urinary N loss on a protein-free diet was 1.0 mg/basal calorie.

Aberanthy et al (19) estimated from their N balance studies in girls of 7 - 9 years, metabolic faecal nitrogen loss in the children studied to be 19.3 mg/kg/day. This was estimated from the regression equation relating intake and apparent absorption. Workers at Mysore (24, 25) determined endogenous urinary N and metabolic faecal N in girls and boys aged between 8 and 12 years, after feeding protein-free diets. The reported average values for endogenous urinary N were 61 mg. and 70 mg. per kg body weight in boys and girls respectively. When expressed on the basis of basal calories (computed from reported BMR for this age group) these values were 1.38 mg. and 1.47 mg. per basal calorie respectively. Metabolic faecal N loss in these children was found to be 32 mg. and 40 mg. per kg body weight for boys and girls respectively.

Cutaneous loss of Nitrogen: There is no information on cutaneous loss of nitrogen in children. Available information in adults has to be projected to arrive at the figures for children. Whether this is a valid procedure is not definite. Some of the earlier estimations of cataneous losses of N in adults are now suspected to be too high.

Nitrogen required for growth: Nitrogen required for growth forms only a small proportion of total nitrogen requirement in children. Based on normal growth rate (body weight increment) and expected body N content, the estimated N required for growth between the ages of 1 and 12 years would range from 20 to 10 mg per kg. body weight per day (Table 4).

Macy and co-workers (29) who carried out 593 N balance studies in 29 children aged between 4 and 12 years on high level of protein (60 - 80 g. protein) observed mean retention of 27 mg N/kg/day, a figure which considerably exceeds that which may be expected to be actually necessary for growth. The difference between the actual N retention and the expected N retention is not likely to be solely attributable to cutaneous losses of N in view of the well known inherent limitations of N balance studies resulting in overestimation of N retention. It was pointed out earlier that in their studies on children between 6 and 18 months of age, Chan and Waterlow (16) found that the nitrogen retention per 100 g. body weight gain was 2.9 g. which was close to the expected figure.

It may, therefore, be predicted that the assumption of a figure 20 mg N/kg for cutaneous loss in the earlier FAO/WHO report probably represents an over-estimate.

It would appear that in children endogenous losses of N through urine and faeces together may be generally around 80 mg/kg and in any case may not exceed 100 mg/kg. The requirement for actual growth may not exceed from 10 mg/kg to 20 mg/kg between 1 and 12 years of age and cutaneous losses of nitrogen may be around 10 mg N/kg. On this basis the protein requirement would range from 0.75 g. to 0.8 g/kg. With an allowance of 30% for individual variation and stress the overall protein requirement would work out to 0.9 - 1.1 g/kg, which is in agreement with the conclusion arrived at from the growth and nitrogen balance studies.

3. Essential amino acid requirement for growth

Human protein needs can be defined in terms of essential amino acid requirements and total nitrogen requirements. Determination of essential amino acid requirements of humans is indeed a difficult task. Some information on essential amino acid requirements of adults, infants and school children is available at present. However, there is no information on the amino acid requirement of children between infancy and 10 years.

3.1. Essential amino acid requirements of infants:

Holt and co-workers (30) had determined the amino acid requirements of infants. These figures are shown in Table 7. These currently used figures need critical evalution.

In these studies the infants were fed nitrogen equivalent to 3.0 g/protein/kg whereas it is known that even 2.0 g/kg protein derived from breast milk is adequate for satisfactory growth. Several animal experiments suggest that amino acid requirement for growth is related to protein level in the diet. It is not known whether this applies to human infants also. If it were so, actual essential amino acid requirement will be lower than that estimated by Holt et al (30). Even according to Holt et al (30), the values determined by them do not represent close approximation to absolute minimum, but are in excess of them. These values were not determined under conditions of maximum sparing. Subsequent evidence has shown that true minima for some of the amino acids are of the order of one half of the earlier estimate. Snyderman et al (31) have reported that amount of milk protein needed for normal growth of infants of 3 weeks to 20 weeks of age, can be reduced to 1.1 g/kg provided additional non-essential nitrogen is supplied to make up the total nitrogen equivalent to 3.0 g. protein/kg. Even 0.82 g/kg of milk protein was shown to be sufficient to satisfy requirements of all amino acids except methionine. The essential amino acids contained in 1.1 g. cow's milk protein are given in Table 7. Compared with the original estimates, these values are significantly lower in respect of all amino acids. It must be pointed out that the above study was done in only 4 infants. It has been pointed out by Holt (32) that the original estimate of isoleucine requirement was unduly high, having been carried out with supplements of isoleucine subsequently found to be highly contaminated with alloisoleucine. In a subsequent study with infants with maple syrup disease, isoleucine requirement was found to be only 45 - 50 mg/kg, while the requirements of other branched chain amino acids were comparable to that of normals.

Having in view some of the criticisms of the earlier work like:

  1. the use of amino acid mixtures which may not be equivalent to feeding proteins;
  2. feeding relatively high level of protein; and
  3. observation being made on a small number of infants and for short durations of time.

Fomon and co-workers (11) have attempted to determine amino acid requirement for growth employing a different approach. They fed 22 infants a milk formula providing 1.01 g protein/100 ml from the 8th day to 112th day. The infants grew satisfactorily and the protein intake varied from 1.2 - 1.6 g/kg/day. On the basis of this intake essential amino acid requirements were computed. These values are also shown in Table 7. These values do not by any means represent minimum requirement figures, but indicate that they are above the minimal requirement. But one is not certain how much above the minimal levels they are. Even so, these intake figures are lower than the figures provided by Holt et al (30), particularly with regard to isoleucine, phenylalanine, methionine, tryptophan and valine, suggesting thereby that the estimates of Holt et al are on the higher side.

In view of these observations, currently used figures of Holt et al (30) for essential amino acid requirement for growth have to be given up in favour of lower figures. Perhaps, the amino acids provided by 1.1 g cow's milk protein per kg. may be a more realistic estimate of essential amino acid requirement for growth.

3.2. Amino acid requirements of children:

Nakagawa et al (33, 35) reported amino acid requirements of children aged between 10 - 12 years. The children were fed a basal diet containing 11 g. of total nitrogen of which essential amino acid contributed 0.8 g. Each amino acid was fed at a few levels, the minimal level which promoted positive N balance to the extent of 5 - 8 % of intake was considered to be the requirement. These values are given in Table 7. These values are intermediate between those reported for infants and adults. It should be pointed out, however, that the levels of total N used in these studies are rather high, much higher than the actual requirement for this age group. The correctness of these estimates has yet to be clearly established.

3.2.1. Amino acid requirement of pre-school children:

Pre-school children contribute the most vulnerable section from the point of view of protein nutrition. It is among these that widespread protein deficiency is observed. Therefore a precise knowledge about protein and essential amino acid requirement of this group is apre-requisite in combatting protein malnutrition among them. Unfortunately essential amino acid requirements of preschool children have not so far been determined. However, one can obtain an idea of their amino acid requirement from the figures available for infants, assuming that the amino acid requirements run parallel to protein requirements. Such an estimate is given in Table 8. These are derived from the three sets of figures for infants (Holt's, Fomon's and amino acid content of 1.1 g/kg milk protein) assuming that the average protein requirements of infants and pre-school children are 1.7 and 1.0 g. reference protein per kg body weight per day respectively.

The correctness of these estimates can be judged by comparing these with the amino acid contained in proteins which are known to satisfy the minimal requirements. Use of vegetable proteins which contain a lower proportion of essential amino acids than animal proteins for such a comparison may be more realistic.

In a study (17) at the National Institute of Nutrition, it was observed that an intake of 1.75 g. wheat protein per kg body weight would satisfy the protein requirement of preschool children as judged by the N balance criterion. Lysine supplementation of this wheat diet did not improve N balance indicating that lysine present in 1.75 g. wheat was sufficient to meet the lysine requirement of these children. Essential amino acid intakes corresponding to 1.75 g. wheat protein/kg are given in Table 8. In the study of Begum et al (18) a protein intake of 2.0 g/kg from wheat-based or rice based diet was found to satisfy the protein requirement of pre-school children as judged by growth and N balance. Essential amino acid intakes corresponding to 2.0 g. protein/kg, calculated from the amino acid analysis of the diets are also given in Table 8. A comparison of these values with the derived value from Holt's figures indicate that the derived values are higher in case of certain amino acids viz. isoleucine, lysine and phenyl a lanine. The lowest values for the amino acids from the three diets are comparable with the values derived from Fomon's figures or from amino acid content of 1.1 g. of milk protein. Based on these comparisons a more realistic estimate of essential amino acid requirement of pre-school children has been attempted in Table 8.

4. Practical considerations

4.1. 'Normal' growth and development:

Protein requirement during growth may be defined as the level of protein intake which would ensure "normal" growth and development. This would imply the availability of universally acceptable yardsticks of "normal" growth and development. There is, at present, considerable information on the pattern of growth and development of infants and children belonging to affluent communities in Europe and North America, living under good environmental conditions and free from nutritional constraints imposed by economic factors. The nutrient intakes of children of such prosperous communities may be expected to exceed "physiological" requirements and it may also be argued that their growth, for this reason, may not necessarily reflect 'normal' or 'optimal' performance. Moreover, in view of possible genetic variations in growth potential, the applicability of data on growth and development obtained in Europe and North America for the rest of the world has to be verified.

Extensive studies on growth and development in India in recent years have covered not only children of the "general population" the majority of whom belong to the poor socio-economic groups, but also a significant number (7000) of children belonging to the upper socio-economic groups (Vijayaraghavan et al) (36). These studies show that the 50th percentile figures for heights and weights of well-to-do Indian children correspond closely to the 50th percentile figures for similar measurements in American children at all ages up to 12 years in boys and 14 years in girls. It may be concluded on the basis of these that similar studies among other population groups (Ashcroft and Lovell) (37), that genetic variations in growth potential may not be significant and may be much less important than environmental factors as determinants of growth performance. For all practical purposes, therefore, the parameters of normal growth and development currently being employed may be considered as generally acceptable.

4.2. Protein requirement in undernourished population:

The question whether protein requirement of chronically undernourished population is different from that of the well-nourished population is often raised. This question is important for developing countries where a considerable proportion of children at any time are in fact in various stages of undernutrition and malnutrition.

So far as the minimum protein requirement for nitrogen equilibrium of adults is concerned, there appears to be no difference between well-nourished and undernourished subjects. Studies by Pasricha et al (38) have shown that minimum protein required for N equilibrium in well-nourished and poor women is essentially the same. Similarly Gopalan and Narasinga Rao (39) have shown that obligatory urinary N loss in undernourished male adults is not different from that reported for well-nourished Western adults.

However, when one considers the protein requirement of infants and children, this question may assume greater importance. Undernourished children have subnormal body weights compared to well-to-do children of the corresponding age. It may be argued that by providing higher than normal amounts of proteins, growth rate of these children may be accelerated to catch up with those of well-to-do children. This possibility was investigated at the National Institute of Nutrition, Hyderabad, both in breast fed infants and young children. Breast fed infants of women belonging to the low socio-economic group were given extra milk so that their breast milk and supplement together provided nearly 2 to 2.5 g. protein per kg. of expected body weight for a period of nine months (Venkatachalam et al) (40). In another study lasting for nine months a group of children aged between 5 and 10 years, from a local orphanage were given protein supplement of 18 g. so that their total daily protein intake corresponded to the recommended allowance on the basis of expected body weights (Darshan Singh and Swaminathan) (41). In both the studies it was observed that such a supplementation failed to bring about any improvement in growth over that observed in unsupplemented group.

There is another side to this question. Undernourished subjects tend to utilize dietary protein better than to well-nourished subjects. Several N balance studies, both in adults and children, have demonstrated that undernourished subjects do retain lot more nitrogen on a given level of intake than the well-nourished subjects. Hence a compensatory mechanism may help undernourished children to utilize better and grow faster on levels of protein intakes corresponding to normal requirement. This is well illustrated by the study of Chan and Waterlow (16). They fed children with an average age of 1 year, 1.2 g. milk protein per kg and 120 cals/kg, which corresponded to normal requirement level. These children had recovered from malnutrition but with body weights still below normal. On such intakes these children retained more nitrogen and grew at twice the normal rate.

It can be concluded from the above observations that there is no theoretical justification for increased protein allowance per kg. body weight to undernourished children. The above studies also demonstrated that there is no particular advantage in recommending total protein intake on the basis of ideal body weight to such children.

4.3. Influence of infection on protein requirement:

While recommending protein allowances, the question is often raised whether population groups in which infection is quite common need more protein than normal children. This is based on the finding that protein requirement during infection may be raised.

Acute infection and trauma, no doubt, bring about negative nitrogen balance, leading to body protein depletion. It has been observed, however, that the degree of negative nitrogen balance brought about by infection or trauma is much less in malnourished individuals than in well-nourished subjects.

Although there is body protein loss during trauma or the acute phase of infection, a compensatory response comes into play during convalescence. During this phase, body attempts to retain higher proportion of ingested nitrogen than during the normal state. The extent of this retention can be augmented to some extent by increasing the protein intake during this period. It has been suggested for this reason that children belonging to communities in which infections are frequent may be provided extra allowance of protein as a blanket coverage. Such a proposal would appear to be unsound and undesirable for the following reasons:

(a) It is not possible to quantify precisely the increased protein requirements necessitated by infection since this is likely to be determined by the type, severity and duration of the infection and the immunological response and the nutritional status of the host.

(b) Even in a poor community not all sections of the population are uniformly affected by infection and a blanket recommendation covering the entire community is not desirable. Even in the section exposed to infection, it may be expected that the incidence of several infections are seasonal and erratic. During the actual infectious episodes in a population, while a proportion of the subjects may be actually suffering from infection a proportion would be in the convalescent or the incubation stages. The protein requirement in these different stages would be very different.

(c) Infection not only modifies protein requirements but also the requirements of calories and a number of other nutrients including vitamin A. Unilateral recommendation with regard to one nutrient under these circumstances may not be desirable and could result in undesirable distortions of the dietary pattern.

(d) It is wrong public health strategy to attempt to combat infection and to provide for it by recommending increased nutrients especially for communities which already cannot afford even the basic minimum requirements. The accepted public health approach is to control and if possible eradicate the infections by improving environmental sanitation (when the pot is leaky the answer is to plug the leak and not to pour more water).

The rational approach would be to deal with the question of protein requirements in infection and post-infection on an individual and clinical basis and not through blanket recommendations covering the entire community.

4.4. Strategy for ensuring protein requirement:

In translating the recommendations regarding protein requirements into actual practice, among populations in whom protein-calorie malnutrition is widespread, two different strategies may have to be followed.

In some parts of the world where protein-calorie malnutrition exists, the diets are based on such staples as Cassava and banana. In such situations it will not be possible to meet the protein requirement for growth and development through the existing diets, even if thes diets are fed at levels sufficient to meet the calorie needs. In these situations there would seem to be a legitimate case for special protein-rich supplements.

On the other hand in India and other parts of Asia the diets of poor communities among whom protein-calorie malnutrition is common are cereal-based. Available evidence indicates that it would be possible to meet protein requirement during growth and development with these diets if they are fed at levels sufficient to meet the calorie needs. Protein-calorie malnutrition in these situations arises largely from insufficient intake of food. Thus, a "food-gap" rather than a "protein-gap" is at the root of the problem in these areas. This problem cannot be solved by providing protein concentrates or through amino acid supplementation but by bridging the "food-gap". The important question, however, is whether, in fact, young children will be able to consume bulky cereal based diets in amounts sufficient to meet their calorie needs. Studies carried out on children between 2 and 3 years of age at Hyderabad indicate that this is possible provided the mothers are educated to divide the daily diet into several meals.

The failure to clearly distinguish these two different situations and to appreciate the need for different strategies to meet them has led to many unfortunate false starts and expensive exercises in futility in many developing countries.



Method Principle Critique
Growth Minimum protein intake for "normal" growth Useful during infancy when growth is rapid, difficult to employ in older children
Nitrogen balance Minimum protein intake for N retention required for growth Suffers from cumulative errors and over-estimates retention.
Factorial Obligatory nitrogen loss through urine, faeces and skin + nitrogen required for growth and chemical maturation Based on several assumptions, validity of which may be questioned
Total body protein turnover Dietary protein required to maintain body protein synthesis during normal growth Acceptable method for determing body protein synthesis, has yet to be established
(a) Plasma amino acid ratio of non-essential to essential amino acids These can be used as biochemical parameters of growth Usefulness of these has yet to be established beyond doubt
(b) Hydroxyproline excretion in urine
(c) Total body potassium

Table - 2


Protein source Age of infants Protein intake gm/kg/day Criteria for nutritional adequacy Reference
Breastmilk   2.3-1.5 Growth Macy & coworkers 4, 5 (1934, 41)
" 1 - 22 weeks 2.0-1.1


Gopalan (1956)12
" 8 - 180 days 2.4-1.5 Growth and N balance Fomon & May (1958)6
Cow's milk 4½-6 months 1.46


Fomon & May (1960)8 (1961)9
" " Male 8 - 112 days 1.83-1.26 Growth, N balance & serum proteins Fomon & Filer (1967)11
Female 1.60-1.36

" "

Soy milk 4½-6 months 1.73 " " Fomon (1959)7

" "

8 - 112 days 2.17-1.25 " " Fomon & Filer (1967)10



Age Obligatory N loss mg/kg/day Method of measurement Reference
Urine Feces Total
31-60 days     66.8 Extrapolation from N balance data Fomon (1961)9
61-90 days     43.9 " " "
91-120 days     61.3 " " "
121-150 days     58.3 " " "
151-182 days     62.3 " " "
Mean     58.5 " " "
0-6 m:       (a) 37 (0.6) 20 57 Protein free diet (c) Fomon et al13 (1961)


42 (0.8) 33 75 " " "

(a) Prior protein intake 2.5 g/kg/day

(b) Prior protein intake 4.0 g/kg/ or more

(c) the infants were on protein free (low protein) diet for 6 days, urine and feces collected from 4th to 6th day. Figures in parenthesis indicate mg N excreted per basal calories.

Table - 4


Age Mean body weight kg. (a) N content of body g/100 g. (b) Nitrogen for growth mg N/kg/day (c)
0-3 m. 4.55 2.00 154
3-6 m. 6.55 2.30 104
6-9 m. 8.15 2.60 77.4
9-12 m. 9.40 2.80 35.5
1 yr. 11.15 2.85 19.9
2" 13.45 2.92 13.8
3" 15.50 2.96 11.8
4-6 yrs. 18.40 3.00 12.2
7-9 " 27.26 3.0 12.3
10-12 " 35.20 3.0 9.9

(a) From Nelson's Text-book of Pediatrics 8th Ed. (1964)

(b) Composition at the beginning of each period from Holt et al.2

(c) Body N-content at the end of the period - Body N content of the beginning of the period

Interval in days × Mean body weight




Protein source

Intake: g/kg/day

Criteria for adequacy


Dietary protein

Reference protein

About 1 yr. (6-18 m.) Milk 1.25 1.1 Growth & N balance Chan & Waterlow16 (1966)
2-3 yrs. Egg 1.0-1.25 1.0 Plasma amino acid ratio & N balance Arroyave (1970)1
3-5 " Wheat 1.75(a) 0.88 N balance 40 mg/kg/day N.I.N. (1970)17 Hyderabad
2-5 " Wheat or rice based diets 2.0(a) 1.0 Growth & N balance Begum et al. (1970)18
7-9 " Mixed diet 1.3(b) 0.8 N balance of 30 mg/kg/day Aberanthy (1966)19
8-12 " Cereal based diets 1.56(c) 0.74

" "

C.F.T.R.I. (1962-65)20, 26 Mysore

(a) NPU about 50

(b) NPU 62

(c) NPU 50




Daily N loss

Method of assessment






mg/basal Cal



About 1 yr. (3-24 months)





Extrapolation from N balance data

Waterlow and Wills27 (1960)

About 1 yr. (6-18 months)      


" "

Chan and Waterlow16 (1966)

3-4 yrs.





Low protein diet

Fomon et al. (1965)13













3-8 yrs.  




Low protein diet and extrapolation from N balance data

De Maeyer et al. (1961)28

7-9 yrs.    



Extrapolation from N balance data

Aberanthy et al. (1966)19

9-10 yrs. (boys) 61.1


32.1 93.2 Protein free diet

Tasker et al. (1962)25

8-9 " (girls)






Joseph et al. (1963)21








Children + 10-12 yrs.
Holt's Data Fomon's Data Based on 1.1 g. milk protein
Arginine - 34-50 - -
Histidine 34 19-28 25 -
Isoleucine 119 48-70 67 28
Leucine 150 111-161 118 49
Lysine 103 111-161 86 59
Phenylalanine 90a 42-61 61 27c
Methionine 45b 23-29 32 27d
Cystine - 9-13 - -
Threonine 87 80-116 48 34
Tryptophan 22 11-17 17 3.7
Valine 105 64-93 73 33

(a) In presence of tyrosine

(b) in presence of cystine

(c) in absence of tyrosine

(d) in absence of cystine

+ Nakagawa et al.33-35







Holt's data (1) Fomon's (c) data (2) 1.1 g/kg proteins Wheat diet (3) Wheat based diet (4) Rice-based diet (4) Suggested levels
Histidine 20 16.5 14.7 40 - - 20
Isoleucine 73 41 40 57 37 45 40
Leucine 90 93 70 117 77 89 70
Lysine 61 93 51 50 44 72 50
Phenylalanine 54a 36 36 79 50 32 36
Methionine 27b 17 19 26 7 20 20
Cystine - 7.7 - 45 40 23 20
Threonine 52 68 28 51 28 55 30
Tryptophan 13 10 10 19 17 27 10
Valine 62 55 43 77 55 67 55

Ref: (1) Holt and Snyderman30

(2) Fomon and Filer11

(3) N.I.N., Hyderabad17

(4) Begum et al.18

(a) in presence of tyrosine

(b) in presence of cystine

(c) upper value used in calculation


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