International Recommendations on Protein Intakes in Infancy: Some Points for Discussion

Brian A. Wharton

Old Rectory, Belbroughton, Worcestershire, DY9 9TF, United Kingdom

BACKGROUND

Populations

The definitive international report on protein requirements is the one published by the WHO in 1985 (1). It forms the basis of many other recommendations, including the American ones published in 1988 (2) and the recent British Dietary Reference Values (3). These reports also include energy requirements, which are obviously closely related to protein requirements.

Foods

In addition, there are other recommendations and in some cases legislation concerning the composition of infant formula, follow-on milk, and weaning foods. Since these foods are consumed by infants to meet their nutritional requirements, the reports are in effect recommendations concerning the infants' nutritional intake, or at least part of it. A few reports are "international" (e.g., Codex Alimentarius of WHO/ FAO, reports of the European Society of Pediatric Gastroenterology and Nutrition, and reports of the Committee on Food of the European Community). There is a plethora of national reports concerning infant foods. References will be given to individual reports at relevant points in the paper. There is little point in doing a line-by-line comparison of all these reports, whether for populations or foods. Instead, 1 shall discuss five areas where there is room for debate.

NUMERATOR

For numerical recommendations a numerator and a denominator are involved (e.g., protein per person per day). In this section the numerator is considered. 67 68 RECOMMENDATIONS ON PROTEIN INTAKES

Multiples of Nitrogen

The major problem with the numerator is its expression as weight or as a weight of nitrogen contained within the food. The use of nitrogen arises from the chemical analysis of foods, but it extends into other nutritional considerations when assessing balance data (e.g., amount of nitrogen in feces). In parenteral and enteral nutrition it has become the custom to express protein, peptides, and amino acids as nitrogen. The conversion of weight of nitrogen to weight of protein uses a conventional multiple of 6.25. For milk the more appropriate factor 6.38 is often used. For weaning foods containing cereal protein only, the appropriate figure is 5.7-6.0, depending on the cereal. In practice, the content of protein stated on a label of a weaning food is likely to be calculated from food composition tables rather than from direct chemical analyses, and these tables will have used multiples of analyzed nitrogen appropriate for the source of protein.

Non-protein Nitrogen

The multiple of nitrogen method does, of course, count non-protein nitrogen as protein and where this is amino nitrogen it is likely to be available for synthesis of protein by the baby. The problem of how to account for the non-protein nitrogen of breast milk is well known. In the British national "pooled sample" of breast milk (4), total nitrogen was 210 mg per 100 ml, of which non-protein non-amino-acid nitrogen was 46 mg. If total nitrogen found in the milk is multiplied by 6.38, a figure of 1.3 g of protein per 100 ml is obtained, but when non-protein non-amino-acid nitrogen is excluded, the figure is 1.05 g. It is not clear, however, that non-protein nitrogen compounds, particularly urea, should be excluded. Urea reaching the colon either directly from the food or via diffusion from the blood is hydrolyzed by the gut flora releasing ammonia, which travels via the portal vein to the liver for production of non-essential amino acids. The concept of urea cycling in protein economy, particularly during rapid catch-up growth, has been developed by Jackson (5). We should beware of assuming that non- protein nitrogen is of no nutritional significance. To regard the protein and amino acid nitrogen content of breast milk as true protein and to use this as a basis for determining protein requirements may lead to an underestimate because it ignores urea. Heine has explored the bioavailability of urea nitrogen in breast milk (6). Further caution is necessary if we are tempted to deduct from this "true protein" the immunological proteins such as lactoferrin and secretory IgA (on the basis that they are not absorbed and are not nutritionally available), to give a final nutritionally available protein content in breast milk that is perhaps as low as 0.8 g per 100 ml. Two studies have found that relatively small amounts of the secretory IgA and lactoferrin ingested in breast milk appear in the feces of infants a few weeks old (7,8). This suggests that large amounts of these protective proteins are broken down and so become nutritionally available higher in the gut. Our own observations were that RECOMMENDATIONS ON PROTEIN INTAKES 69 only 1% of estimated lactoferrin intake appeared in the feces of breast-fed babies aged 2 weeks; the figure for bovine lactoferrin was around 0.5% (9). In summary, neither non-protein nitrogen nor the "immunological proteins" should be discarded when assessing the amount of protein available to and required by an infant.

DENOMINATOR

Individuals and Their Weight

Recommendations on protein intake in children, which are part of national or international recommendations for all ages, use either an individual or his weight as the denominator; that is, the "safe level," "advisable intake," or "reference nutrient level" is expressed as grams per person at a particular age or as grams per kilogram of body weight. The WHO figures from which the others follow are based on the factorial method, or at least a modification of it, since the determination of obligatory nitrogen loss on very low protein diets is not relevant for a rapidly growing infant. A typical calculation for 0-4 months quoted in the WHO report (1) is an increase in tissue protein of 3.5 g per day plus the equivalent of 0.5 g each day lost from desquamating skin and sweat. At this age average retention is 45% of intake. Therefore, an intake of 8.9 g is necessary to retain 4 g; that is, for an average weight at this age of 5.25 kg, an intake of 1.7 g/kg is needed. Some factor may then be applied to allow for safety to give an advisable or safe intake of around 1.9 g/kg.

Volume, Weight, and Energy of Food

In recommendations for food, values expressed as grams per infant or grams per kilogram of infant clearly will not do. For infant formulas the choices of denominator are weight of powder, volume of feed, or energy content of the feed. Few people will think in terms of the weight of powder in a formula (about 13 g per 100 ml in most), and the volume fed is the obvious measurement. Furthermore, if nutrients are expressed per unit volume, this stresses the role of water as an essential nutrient, which is vital when considering the renal solute load of a diet and its effect on the concentration of urine and on water balance. This type of reasoning led to British recommendations on the composition of infant formulas being expressed per unit volume (10). However, volume is not a suitable denominator for a weaning food; the consistency of a cereal and sugar gruel, whether homemade or provided by a manufacturer, could vary considerably. In addition, as weaning progresses renal function, including concentrating ability, increases, so the renal solute load of a diet becomes less important unless there are very abnormal losses of water from lungs, skin, or gut. Of the possible denominators, therefore, only energy can be applied to food for both suckling 70 RECOMMENDATIONS ON PROTEIN INTAKES and weanling infants. Apart from this pragmatic reason there are also physiological reasons for the choice of energy. It recognizes the interrelationships between the requirements of protein and energy and the relatively large proportion of both energy and protein used for growth in the rapidly growing infant. Many international reports therefore express protein per unit energy, both for infant formulas [ESPGAN (11), Codex Alimentarius (12), European Community (13)] and for weaning foods (14).

Protein/Energy Ratio in Physiological States

Since there are physiological as well as pragmatic reasons for expressing protein content of foods in terms of energy, should this concept be extended to the recommendations for individuals? In other words, should the reference, safe, or advisable intakes of protein by groups of people be expressed not per person or per kilogram of body weight, but per 100 kcal of food ingested? Fomon (15) has argued in favor of this method of calculation and we have also used it as a way of expressing advisable intakes of many nutrients for children of various ages (see Table 1). Where recommendations for protein and energy are expressed separately, then calculation of the ratio from these figures is, in theory at least, invalid since the protein figure is chosen to meet the requirements of 95% of the population (that is, more than most people need) while the energy figure is the observed average intake of the population (that is, less than what 50% of the population consumes). The mathematics are complex since the ratio involves two factors, each with its own variance. The concept and statistical manipulations are discussed in some detail by Beaton & Swiss (17). Table 2 shows the protein/energy ratios calculated in this way for infants. The ratios are all above the ratio found in breast milk. It is probably unwise to adopt the very low protein/energy ratio in breast milk as a minimum safe standard, since the nitrogen present is utilized with unusual efficiency [an argument developed by Mill- ward (19)]. For older children the "safe" protein/energy ratios calculated this way are well below those seen in the normal diet of Western children. In my opinion there should be some caution in accepting very low ratios of 1.7 and 1.5 g per 100 kcal as "safe" in a community until there have been extensive observations of children living on such diets. Therefore, in our recommendations shown in Table 1 (16), we adopted higher protein/energy ratios (as consumed by normal children) than the international recommendations imply.

Protein/Energy Ratios in Weaning Diets

Consideration of the protein/energy ratio can also help in unscrambling the vexing question of the composition of milk or formula feeds given during weaning. Figure 1 shows the mathematical relationships of protein/energy ratios in milks, formulas, and solid foods used during weaning to achieve the advisable intake of not less than 1.7 g per 100 kcal. We have described the mathematical derivation of these RECOMMENDATIONS ON PROTEIN INTAKES 71

TABLE 1. Advisable intake of nutrients per unit energy

Age of child 1 year-1 year Nutrient/100 kcal 0-11 months 11 months 2-10 years

Protein (g) 2.25-3.0 2.25-3.0 2.25-3.5 N2(g) 0.36-0.48 0.36-0.48 0.4-0.56 Non-protein Kcal N2 ratio 160:1-238:1 193:1-228:1 194:1-175:1 Fat(g) 3.3-6.5 3.3-5.5 3.3-4.2 C8:0 % total fat Max. 20% C10:0% total fat C12:0% total fat Max. 15% C14:0% total fat Max. 15% Linoleic acid (g) 0.3-1.2 0.3-1.2 0.3-1.2 Total carbohydrate (CHO) (g) 7-14 10-15 12-15 Lactose Min. 3.5 Sucrose <20% total CHO Fructose Not usually added Precooked starch and/or <30% total CHO gelatinized starch Minerals Iron (mg) 0.5-1.5 0.6-1.2 0.6-1.2 Calcium (mg) Min. 45 Min. 45 Min. 35 Phosphorus (mg) 25-90 50-100 50-100 Sodium (mmol) 1.0-2.6 1.0-3.0 1.0-3.0 Potassium (mmol) 1.6-3.8 1.6-5.7 1.6-5.7 Chloride (mmol) 1.4-3.5 1.4-3.5 1.4-4.0 Magnesium (mg) 5-15 10-20 10-20 Copper (p.g) 60-120 Min. 100 Min. 100 Zinc (mg) 0.3-1.0 0.5-1.0 0.5-1.0 Manganese (p.g) 2-8 Min. 100 Min. 100 Iodide (p.g) Min. 5 Min. 5 Min. 5 Chromium (p.g) 3-10 3-10 3-10 Molybdenum (|xg) 5-15 5-15 5-15 Selenium (|j.g) 3-10 3-10 3-10 Fluoride (mg) 0.02-0.1 0.04-0.15 0.04-0.15 Vitamins Vitamin A (p.g RE) 60-180 25-45 20-45 Vitamin 0 (\ig cholecalciferol) 1-2 0.8-1.0 0.6-1.0 Vitamin E (mg) Min. 0.5 Min. 0.4 Min. 0.4 (mg/g PUFA) Min. 0.5 Min 0.4 Min. 0.4 Vitamin K (p.g) 2.5-15.0 1.5-15.0 1.5-15.0 Vitamin C (mg) Min. 5 Min. 1.5 Min. 1.5 Thiamine (mg) 0.04-0.09 0.04-0.09 0.04-0.09 Riboflavin (mg) 0.06-0.4 0.1-0.4 0.1-0.4 Nicotinic acid (mg equivalent) 0.250-1.25 0.6-1.25 0.6-1.25 Folic acid (M-9) 4-10 8-20 8-20 B-12 (ng) 0.1-0.5 0.15-0.5 0.15-0.5 Pyridoxine (mg) 0.035-0.16 0.08-0.16 0.08-0.16 Biotin (p.g) 1.5-5.00 Min. 5 Min. 5 Panthothenic acid (mg) 0.30-0.75 0.20-0.75 0.20-0.75

Energy 110 - (3 x age) x kg body weight

From Wharton BA, & Clark B Med Int 1990; 3375-81. 72 RECOMMENDATIONS ON PROTEIN INTAKES

TABLE 2. Protein, energy, and protein/energy (Pr.En) ratio in recommendations and observed intakes

Age Energy Protein Source (months) (kcal) (g/100 kcal) Pr:En ratio

WHO (1) (calculated) kg"1 1-2 116 2.25* 1.9 4 kg" 464 9 kg 3.6 99 1.86 1.9 7 kg 693 13 kg 6-9 97 1.65 1.7 8 kg 776 13 kg 9-12 100 1.48 1.5 9 kg 900 13 (recommended) Fomon (15) 0-4 1.9 4-12 1.7 Wharton & Clark (16) 0-12 2.25-3.0

Intakes from milks/formula (% energy intake) Breast milk at 150 ml/kg 3 105 1.95a 1.9= (100%) Infant formula at 150 ml/ 3 100 2.25-3.0 2.25-3.0" kg (100%) Follow-on milk providing 8 300 8-16 2.25-4.5" 300 kcal (40%)

Observed intakes in British (calculated) weanlings (18) Total intake 6-9 815 27.4 3.4 Intakes from milks/ 384 13.1 3.4 formula % total 47 48 Intake from other foods 431 14.3 3.3 % total 53 52 Total intake 9-12 928 34.4 3.7 Intake from milks/ 335 14.7 4.4 formula % total 36 43 Intake from other foods 593 19.7 3.3 % total 64 57

• Reference protein for WHO figures; other figures refer to actual protein. * Typical weight for age. c Protein at nitrogen x 6.38 (4). " Pr:En ratio advised by European Community (13). RECOMMENDATIONS ON PROTEIN INTAKES 73

British weanling ) Advisable protein intake: Rice, Oil, Egg

Wheat

a) 4 Rice, Oil

I— 0% 20% 40% 60% 80% 100% Proportion of total energy supplied by milk/formula

FIG. 1. Protein/energy (PR: En) ratios in milks, infant formulas, and solid foods to achieve advisable protein intake of 1.7 g per 100 kcal in older infants. For example, if an older infant received 40% of his energy intake from a follow-on milk (protein 2.7 g per 100 kcal) and the remainder of his energy intake from rice (protein 1.1 g per 100 kcal), then since the intersecting point is just above the line for a follow-on milk, the advisable protein intake of 1.7 g per 100 kcal would be achieved. Intersecting points below and to the left of the line would not achieve the advisable intake. All Pr:En ratios in the milks, formula, and solid foods have been converted for net protein utilization in children: breast milk 1.0, cow's milk 0.9, wheat 0.49, rice 0.63, maize 0.36. [Modified from Wharton BA, et at. Ada Paediatr 1994 (in press).] relationships previously (20). A brief description is given in the legend to Fig. 1. As an example, a child getting 40% of his energy from an infant formula (2.25 g per 100 ml) would need to consume weaning foods with a protein/energy ratio of 1.5 g per 100 kcal to meet the desirable ratio in the overall diet of 1.7 g per 100 kcal. The protein/energy ratio in British weanlings' supplementary food (3.3 g per 100 kcal or 2.5 g after corrections for NPU; see Table 2) is well above the 1.7 g "advisable" figure even if these infants consumed no milk of any kind. If, however, the supplementary food were entirely rice or entirely maize, as, for example, in Asia or Africa, the safety limits are met very much earlier; for example, rice alone is satisfactory only if breast milk is still providing about 70% of the energy intake; if the child receives an infant formula (providing 2.25 g protein per 100 kcal), rice is satisfactory as long as the formula meets 55% of the energy requirement, and so on. The protein/energy ratio of rice plus oil is much lower, so that if the infant were receiving less than 85% of his energy intake from breast milk, the safe ratio would not be met; a weaning food that provided only 15% of energy intake would be of limited value. From this unsatisfactory position (point a in Fig. 1), the protein/energy ratio of the diet could be improved in two ways: 74 RECOMMENDATIONS ON PROTEIN INTAKES

1. By the use of a milk or formula with higher protein/energy ratio. This child's position moves horizontally from point (a) to, say, point (b). A safe protein/energy ratio is achieved if a follow-on milk accounts for only 55% of the energy intake. 2. By the use of a weaning food with enhanced protein as well as enhanced energy. The child's position moves vertically upward from point (a) to (c). This is achieved by using a "double mix" weaning food based on rice and oil plus another protein source, such as egg. Which of these strategies is better will depend on local factors such as availability and cost of the additional foods. In both instances care is necessary to ensure microbiological safety of the milk and rice gruel used.

Protein/Energy Ratio in Pathological States

The ratio thus deals effectively with safe or advisable intakes in infants and with guidelines or directives for foods designed for infants. A further advantage is that the concept of the protein/energy ratio can be extended to pathological states of nutrition.

Hypercatabolic States

Protein supplementation when there is already an adequate energy intake is often ineffective because the liver and muscles cannot synthesize any more protein from an increased amino acid intake even though abnormal losses of nitrogen are occurring. Even in hypercatabolic syndromes it is probable that the protein/energy ratio should not exceed the upper suggested limit of 3 g per 100 kcal (3.5 g per 100 kcal for 2- to 10-year-olds). One rule of thumb for dealing with the increased requirements following burns is to increase energy intake over "normal" requirements by 20 kcal for every percent of surface area burned (for example, 50% burns would require an extra 1000 kcal), and protein intake by 1 g of protein for every percent of burned area. The same effect is achieved by increasing total energy intake as indicated and main- taining the protein/energy ratio of the intake at 3.0-3.5 g per 100 kcal. In practice, if a suitable enteral feed is chosen (3.0-3.5 g per 100 kcal), the volume of feed can be increased according to the proportion of surface area burned, without individual consideration of protein and energy.

Diets for Catch-up Growth

A further use of the protein/energy ratio is as a check on diets that have been manipulated in some way. It has become a common practice in British hospitals when trying to induce catch-up growth in a malnourished child to give an infant formula to which extra fat and carbohydrate have been added. A brief consideration of protein/energy ratios shows that this maneuver has potential difficulties. RECOMMENDATIONS ON PROTEIN INTAKES 75

Using Fomon's figures (15,21), deposition of new tissue from birth to 4 months of age on average requires 5.6 kcal/g and the tissue contains 11.4% protein. The following protein/energy ratios are necessary after allowing for varying levels of the efficiency of protein utilization: 100% efficiency = 2.04 g per 100 kcal (0.114 g per 5.8 kcal) 90% efficiency = 2.26 g per 100 kcal 80% efficiency = 2.54 g per 100 kcal 75% efficiency = 2.71 g per 100 kcal Clearly, the deposition of new tissue will require all essential nutrients as well as energy, and the figures above indicate that when supplementing a standard formula to support faster growth in early infancy, the supplement should contain at least 2.26 g of protein per 100 kcal and probably more. Use of an energy-only supplement is of limited value.

AMINO ACIDS

The quantity of protein in any recommendation must necessarily be accompanied by some statement on quality. This generally involves the use of a defined reference protein with a stated amino acid composition. For the suckling infant there is no doubt that the reference protein should be breast milk protein. For the weanling infant the choice is less clear, but the FAO reference protein is widely used for amino acid scoring. An additional measure, the protein efficiency ratio, may be used. This is weight gain (g) divided by protein consumed (g) in rats under standardized condi- tions. The reference protein used is usually casein, and a protein/efficiency ratio of 70% is regarded as suitable (14). Casein may also be used as a reference protein for amino acid scoring. Questions of protein quality and amino acid content become more important as protein quantity is reduced. This has been most apparent as the protein content of infant formulas has fallen. Cow's milk protein contains less cysteine (about 50%) and less tryptophan (about 65%) than breast milk protein.

Cysteine

The story of cysteine is well known, so I shall deal with it only briefly. The lower intake of cysteine with cow's milk formulas is not accompanied by lower plasma cysteine concentrations in either low birthweight or normal-sized babies (20,22,23), but plasma concentrations may not be a good guide to requirements. An increase in the cow's milk whey fraction in infant formulas increases the cysteine content of the formula, so that when the total protein intake is 2.25 g per 100 kcal, the cysteine intake matches that of breast-fed babies. The FAO protein standard combines methio- nine and cysteine and should not be used for reference in the first 6 months of life. 76 RECOMMENDATIONS ON PROTEIN INTAKES

Tryptophan

While cysteine has received considerable attention, tryptophan has only recently been examined in any detail, probably because of problems in estimation. In 1990 we published reference values for plasma amino acids in early life using reversed- phase HPLC (2i). This method allows satisfactory determination of plasma tryptophan. Normal bottle-fed babies receiving a standard formula (2.25 g protein per 100 kcal) had lower plasma tryptophan concentrations during the first 3 weeks of life than those of breast-fed babies (Fig. 2). These biochemical differences were not associated with clinical or biochemical disadvantages. Growth velocity and other biochemical measures of protein nutrition (plasma albumin, transfer™, urea, amino acid ratios), although differing in detail, were broadly similar to those in breast-fed babies. Nevertheless, tryptophan has many biological functions, such as a role in neurotransmitter metabolism, so we should be wary of casting aside differences in plasma concentrations in the newborn. It has recently been shown that if low protein formulas (1.3 g per 100 ml, 1.95 g

PLASMA TRYPTOPHAN

JJLMOULITER

DAY DAY WEEK WEEK WEEK 11 7 11

FIG. 2. Plasma tryptophan in normal breast-fed babies (A) and in bottle-fed babies receiving either a casein-predominant (O) or whey-predominant (•) formula ad libitum (protein: 2.25 g per 100 kcal). "Results significantly different from those in breast-fed babies. Original data from Scott et al. (23) [Modified from Wharton BA, et al. Ada Paediatr Scand 1994 (in press).] RECOMMENDATIONS ON PROTEIN INTAKES 77 per 100 kcal) are supplemented with tryptophan, plasma tryptophan concentrations are similar to those in breast-fed infants (24,25).

Recommendation

The metabolic importance of cysteine and tryptophan illustrate the importance of amino acid composition in determining protein quality. Recommendations for amino acid requirements at present use little or none of the data available from kinetic studies and estimates of turnover. Denne & Kalhan (26) have used this approach in the newborn and Young & Cortiella (27) in older children. The nutritional properties of whole proteins and peptides are not just the sum of their constituent amino acids. This makes me wary of the recommendation that a low total protein intake is allow- able as long as the intake of the constituent amino acids is not less than (say) that of a breast-fed baby.

ARE INTESTINAL EVENTS IMPORTANT?

Consideration of intestinal events in classical determinations of protein quality and requirements is limited. Net absorption of nitrogen is considered only in so far as it contributes to net protein utilization (NPU). In simple terms, where / is intake, F is fecal nitrogen, and U is urinary nitrogen, j - f - U NPU = j = retention as a proportion of intake

Net absorption (/ - F) is therefore included. It is, however, completely excluded in such indices as the chemical score, which depends solely on a comparison of the amino acid content of the protein with that of a reference protein. Different proteins do, however, have different effects on the gastrointestinal tract, and four examples that have attracted my interest are discussed next.

Net Nutrient Absorption

Table 3 shows the results of two nitrogen balance investigations that compared the quality of proteins when quantity was held constant. In low birthweight babies a whey-predominant protein was better absorbed than a casein-predominant protein. In children recovering from kwashiorkor, soya protein was less well absorbed than whole cow's milk protein with added casein (28,29). Apart from direct effects on nitrogen absorption, however, the type (quality) of the protein affects the absorption of other nutrients. The intestinal effects of soya protein are complex and even in soya protein isolates, the phytate present alters the absorption of phosphorus, iron, and zinc. Fat absorption from a whey-predominant formula is greater than from a 78 RECOMMENDATIONS ON PROTEIN INTAKES

TABLE 3. Nitrogen and fat absorption in children undergoing rapid catch-up growth*

Children studied: Low birthweight babies aged 3 weeks" Toddlers recovering Quality of Drotein* Casein- Whey- from kwashiorkor* predominant predominant Cows' milk formula formula plus casein Soya flour

Fecal weight (g) 11.8 7.8 Nitrogen Intake (mmol) 35.3 35.5 49.2 51.2 Net absorption" (mmol) 31.9 33.1 38.8 31.3 % intake 90 93 79 61 Fat intake (mmol) 27.8s 29* Net absorption* (mmol) 20.4 24.7 % intake 73 85 Fecal bile acids (^mol) 12.1 8.2

• All values per kg/24 h. " Berger ef al. (29). c Rutishauser & Wharton (28). d Net absorption = intake minus fecal excretiori • Brown ef al. (30). casein-predominant formula (see Table 3), so the metabolizable energy available to the baby is greater (30).

Other Effects in the Lumen

Casein delays gastric emptying, induces lactobezoar formation, reduces the secre- tion of pancreatic carboxypeptidases, reduces binding of bile salts, and reduces the bioavailability of divalent cations such as calcium and iron. Fragments produced during digestion can act as gut hormones, inducing intestinal movement, insulin secre- tion, and so on [see Miller et al. for a review (31)].

Fecal Flora

A modern infant formula has metabolic effects similar to those of breast milk. Babies grow at about the same rate whichever food they receive, and plasma and urinary concentrations of various metabolites are similar in the two groups. This similarity is not found in microbiological observations, however (see Fig. 3). The fecal flora of breast-fed babies are very different from those of formula-fed babies, despite all the modifications aimed at making the composition of cow's milk formula similar to that of breast milk. Bifidobacteria and lactobacilli are the most common organisms in the feces of breast-fed babies, while coliforms, enterococci, and bacteroides predominate in the feces of bottle-fed babies (see Whey, Fig. 3). Casein- predominant formulas cause a change in the fecal flora even further from the breast- fed baby. The addition of bovine lactoferrin also has the same effect (see Whey + RECOMMENDA TIONS ON PROTEIN I NT A KES 79

Breast milk

EcSt Bac

Bac Bac

FIG. 3. Fecal flora (mean proportion of total bacterial counts) at 14 days of age in breast-fed babies and in bottle-fed babies receiving either a whey-predominant or a whey plus bovine lactoferrin formula, protein 2.25 g per 100 kcal. B/L, bifidobacteria and lactobacilli; Ec, Escherichia coli; St, staphylococci; Bac, bacteroides; Sf, enterococci, including Streptococcus faecalis; Cl, clostridia. Original data from Balmer & Wharton (32) and Balmer et al. (33). [Modified from Wharton BA, & Balmer SE New perspectives in infant nutrition. Stuttgart: Georg Thiem Verlag, 1992; 89-94.]

L, Fig. 3). It seems that different proteins have different effects on the fecal flora (32-34). The nutritional consequences of these microbiological differences are not known. There is evidence of effects on the digestion and absorption of fat (35). The hydrolysis of urea and the production of short-chain fatty acids in the colon may also be affected, resulting in changes in both protein and energy metabolism.

Metabolic Factors Affecting Gut Function

Once the whole protein and products of digestion have left the intestinal lumen, the amount and type of peptides and amino acids absorbed may affect gut function. It is well known that breast milk contains more taurine (as a free amino acid, not as part of the protein) than is contained in cow's milk. In 1978 we showed that breast-fed babies preferentially conjugate their bile acids with taurine, whereas those receiving a formula, which in those days contained no added taurine, conjugated predominantly with glycine (36). There have subsequently been several investigations relating diet to changes in bile composition and its effect on intestinal function, for example, that by Jarvenpaa (37). 80 RECOMMENDATIONS ON PROTEIN INTAKES

To summarize, if some present regulations and recommendations are based on the aim of mimicking the effect of breast milk on intermediary metabolism, in the future should we not also aim to mimic its effect on gastrointestinal function and on the microbial flora?

THE REAL WORLD

Recommendations, guidelines, regulations, and directives are all very well, but can the food industry meet them? What are the tolerance limits around a stated concentration on a label? If in the regulation the denominator is volume (or weight of powder), then the numerator (i.e., nitrogen x 6.38) is the only variable. If the denominator is energy, the variability of the other energy-providing proximates in the food (i.e., fat and carbohydrate) will cause a substantial increase in the variation of the actual content around the value claimed on the label. If the "regulation" is, say, 1.8-3.0 g per 100 kcal and for a particular product the label claim is, say, 2.2 g, what are the limits of variability around this in individual batches of the product? A variation of, say, +15% would give a range of 1.9-2.5, values still within the regulatory range but somewhat different from those the pediatri- cian thinks that he has prescribed. In practice, a manufacturer usually aims at a concentration above that claimed on the label. This approach probably has its roots in the fight against diluted and adulterated foods which began in the nineteenth cen- tury. There are two other factors that will cause manufacturers to provide excess nutrients in their products. Many nutrients are only given a lower limit in composition recommendations, so label claims will always tend to err on the generous side; some nutrients, such as vitamins, deteriorate on storage, so the original amount in the product has to be higher, to meet the label claim when the product is used many months later. Whatever the reason, however, the actual amount of protein in a product, as determined by nitrogen x 6.38, may well be somewhat different from the label claim. Since the amount is usually higher, the nutritional implications are related more to the effects of higher protein intakes than to protein deficiency. The higher than ex- pected intake of casein, for example, may increase gastric emptying time and the buffering capacity within the intestine. The effects on intermediary metabolism will be different; for example, casein-predominant formulas give rise to higher plasma concentrations of aromatic amino acids, while whey-predominant formulas lead to increased threonine. The renal solute load, and hence the obligatory water losses, will be higher with a higher protein intake. Pediatricians are fond of discussing and studying quite small changes in the protein intake of infants. This is relevant in nutritional terms, but whether it is relevant in the real world, where variations around stated label concentrations could be greater than the fine tuning suggested by the physiological experiments, remains to be shown. Manufacturers need to improve the understanding of pediatricians and nutritional scientists on these matters. Cook (38) has started this process and it needs to be RECOMMENDATIONS ON PROTEIN INTAKES 81 continued. In the meantime, if a label states, say, that the protein content is 1.5 g per 100 ml, what are the 95% confidence limits after allowing for all variations due to formulation, nutrient stability, analytical methods, and so on?

CONCLUSIONS AND SUMMARY

1. International recommendations concerning protein for normal infants are based primarily on the WHO report of 1985 (1). Five points deserving further attention are discussed. 2. Neither non-protein nitrogen nor the immunological proteins in breast milk should be ignored when assessing the amount of protein available to and required by an infant. 3. There are many advantages, both pragmatic and physiological, in considering protein requirements in relation to energy intake (i.e., as grams per 100 kcal) both in an individual and in a food. This method can be applied to situations as diverse as weaning diets, catch-up growth, and hypercatabolic states. 4. The nutritional properties of whole proteins and peptides are not just the sum of the constituent amino acids. We should be wary of making recommendations that a low total protein intake is acceptable as long as the intake of each of the constituent amino acids is not less than, say, that of a breast-fed baby. 5. Since some recommendations and regulations are based on the effect of breast milk on intermediary metabolism, should we in future also aim to mimic the effects of breast milk on gastrointestinal function and microbial flora? 6. Pediatricians are fond of discussing and studying quite small changes in protein intake of infants. This is relevant in nutritional terms but it is less certain that it is relevant in the real world, where variations around label concentrations could be greater than the fine tuning suggested by physiological experiments.

REFERENCES

1. World Health Organization. Energy and protein requirements. WHO Technical Report Series No. 742. Geneva: WHO, 1985; 98-105. 2. National Academy of Sciences. Recommended dietary allowances, 10th ed. NAS, Washington. DC, 1989. 3. Department of Health. Dietary reference values for food energy and nutrients for the United Kingdom. London: HMSO, 1991; 78-84. 4. Christie AA, Darke SJ, Paul AA, Wharton BA, Widdowson EM. The composition of mature human milk. Department of Health & Social Security. Report on Health and Social Subjects No 12. London: HMSO, 1977; 19-20. 5. Jackson AA, Doherty J, de Benoist M-H, Hibbert J, Persaud C. The effect of the level of dietary protein, carbohydrate and fat on urea kinetics in young children during rapid catch up weight gain. Br J Nutr 1990; 64: 371-85. 6. Heine W, Tiess M. Wutzke KD. 15N tracer investigations of the physiological availability of urea protein in mothers' milk. Ada Paediatr Scand 1986; 75: 439-43. 7. Prentice A, Ewing G, Roberts SB. The nutritional role of breast milk IgA and lactoferrin. Acta Paediatr Scand 1987; 76: 592-8. 82 RECOMMENDATIONS ON PROTEIN INTAKES

8. Davidson LA, Lonnerdal B. Persistence of human milk proteins in breast fed infants. Ada Paediatr Scand 1987; 76: 733-40. 9. Balmer SE, Scott PH, Wharton BA. Diet and faecal flora in the newborn: lactoferrin. Arch Dis Child 1989; 64: 1685-90. 10. Oppe TE, Barltrop D, Belton NR, et al. Artificial feeds for the young infant. DHSS Report on Health and Social Subjects No 18. London: HMSO, 1980: 1-10. 11. ESPGAN. Recommendations for the composition of an adapted formula. Ada Paediatr Scand 1977; 262: 1-20. 12. Codex Alimentarius Commission. Recommended international standards for foods for infants and children. FAO/WHO publication CAC/RS 72/74. Rome: FAO, 1976; 3-11. 13. Rey J, Astier-Dumas M, Fernandes J, et al. First report of the Scientific Committee for Food on the essential requirements of infant formulae and follow up milks based on cow's milk proteins. 14th Series, EUR 8752 EN. Luxembourg: Commission of the European Communities, 1983; 198. 14. Wharton BA, Oppe TE, Astier-Dumas M, et al. First report of the Scientific Committee for Food on the essential requirements for weaning foods. 24th Series, EUR 131140 EN. Luxembourg: Com- mission of the European Communities, 1990; 9-38. 15. Fomon SJ. Infant nutrition. Philadelphia: WB Saunders, 1974; 139-41. 16. Wharton BA, Clark B. Childhood nutrition. Med Int 1990; 3375-81. 17. Beaton GH, Swiss LD. Evaluation of the nutritional quality of food supplies: prediction of desirable or safe protein:calorie ratio. Am J Clin Nutr 1974; 27: 485-504. 18. Mills A, Tyler H. Food and nutrient intakes of British infants aged 6-12 months. London: HMSO, 1992; 12-44. 19. Millward DJ. Protein requirements of infants. Am J Clin Nutr 1989; 50: 406-7. 20. Wharton BA, Balmer SE, Berger HM, Scott PH. Faecal flora, protein nutrition and iron metabolism: the role of milk based formulas. Ada Paediatr Scand 1994 (in press). 21. Fomon SJ, Thomas LN, Filer LJ, Ziegler EE, Leonard MT. Food consumption and growth of infants fed milk based formulas. Ada Paediatr Scand 1971; 223: 1-36. 22. Scott PH, Berger HM, Wharton BA. Growth velocity and plasma amino acids in the newborn. Paediatr Res 1985; 5: 446-50. 23. Scott PH, Sandham S, Balmer SE, Wharton BA. Diet related reference values for plasma amino acids in newborn measured by reversed-phase HPLC. Clin Chem 1990; 36: 1992-7. 24. Hanning RM, Paes B, Atkinson SA. Protein metabolism and growth of term infants in response to a reduced protein, 40:60 whey casein formula with added tryptophan. Am J Clin Nutr 1992; 56: 1004-11. 25. Fazzolari-Nesci A, Domianella D, Sotera V, Raiha NCR. Tryptophan fortification of adapted formula increases trytophan concentrations to levels not different from those found in breast fed infants. J Pediatr Gastroenterol Nutr 1992; 14: 456-9. 26. Denne SC, Kalhan SC. Leucine metabolism in human newborns. Am J Physiol 1987; 253: E608-15. 27. Young VR, Cortiella J. Protein and amino acid requirements of healthy 6-12 month old infants. In: Heird WC, ed. Nutritional needs of the six to twelve month old infant. New York: Raven Press, 1991: 149-74. 28. Rutishauser IHE, Wharton BA. Toasted full-fat soya flour in treatment of kwashiorkor. Arch Dis Child 1968; 43: 463-7. 29. Berger HM, Scott PH, Kenward C, Scott P, Wharton BA. Curd and whey proteins in the nutrition of low birth weight babies. Arch Dis Child 1979; 54: 98-104. 30. Brown GA, Berger HM, Brueton MJ, Scott PH, Wharton BA. Nonlipid formula components and fat absorption in the low birthweight newborn. Am J Clin Nutr 1989; 49: 55-61. 31. Miller MJS, Witherlys A, Clark DA. Casein: a milk protein with diverse biological consequences. Proc Soc Exp Biol Med 1990; 195: 143-59. 32. Balmer SE, Wharton BA. Diet and faecal flora in the newborn: breast milk and infant formula. Arch Dis Child 1989; 64: 1672-7. 33. Balmer SE, Scott PH, Wharton BA. Diet and faecal flora in the newborn: casein and whey proteins. Arch Dis Child 1989; 64: 1678-84. 34. Wharton BA, Balmer SE. Diet and faecal flora in the newborn. In: Renner B, Sawatzki Q, eds. New perspectives in infant nutrition. Stuttgart: Georg Thiem Verlag, 1992; 89—94. 35. Combe E, Demarne Y, Guegen L, lvorec Szylit O, Meslin JC, Sacquet E. Some aspects of the relationships between gastrointestinal flora and host nutrition. World Rev Nutr Diet 1976; 24: 1-57. 36. Brueton KJ, Berger HM, Brown GA. Ablitt C. lynkaran N, Wharton BA. Duodenalbile acid conjuga- tion patterns and dietary sulphur amino acids in the newborn. Gut 1978; 19: 95-8. RECOMMENDA T1ONS ON PROTEIN INTAKES 83

37. Jarvenpaa A-L. Feeding the low birth weight infant. IV. Fat absorption as a function of diet and duodenal bile acids. Pediatrics 1983; 72: 684-9. 38. Cook DA. Nutrient levels in infant formulas: technical considerations. J Nutr 1989; 119: 1773-8.

DISCUSSION FOLLOWING THE PRESENTATION OF DR. WHARTON

Dr. Guesry: Concerning formula feeds, Dr. Wharton has asked me to comment on the discrepancy that is often found between what is on the label and what is in the container. Of greatest importance for us is the daily requirement. What is in the can should be more than or equal to what is on the label. This is why, to be on the safe side, we tend to add a little more. The second point is that we work with an unstable raw material. Cow's milk is not constant. In spring there is more fat; during the winter, when the animals receive other types of food, there is less fat. Because of this, the protein/energy ratio is quite variable. We try to measure what is in the raw material and to adapt it, but this is not very easy to do on a day-to-day basis. This explains one type of variability. The law is also different from country to country and we have to take this into account. Dr. Wharton also suggested that the quality of the formula should be adapted to the quality of the rest of the weaning food, the beikost. That is what we are doing. We have different types of formulation for different situations. For developing countries we tend to have higher levels of protein than for the industrialized countries. In spite of this the protein intake may be very high in industrialized countries, due to the fact that the beikost is rich in protein, and that mixed feeding is often started early, for example in the USA. So the trend now in industrialized countries is for a further decrease in the protein quantity. Years ago ESPGAN recommended that follow-on formulas in developing countries should cover the daily protein needs of the baby (i.e., around 15 g of protein) in a volume of half a liter. This means that we need to provide about 4.5 g of protein per 100 kcal. Such a high protein concentration may still be necessary in certain developing countries. Dr. Pettifor: I am interested in your comments about the need to maintain an adequate protein intake in your milk formula whatever formula you use, as you approach the weaning age, particularly in developing countries, where the protein is often relatively poorly available or of low biological value. Related to that is the issue of repeated infections. Does infection change the ratio of protein to energy requirements? Dr. Wharton: With an infection you will certainly get nitrogen loss. We heard this morning that both synthesis and degradation will be increased but there will be a brisk nitrogen loss even in the mildest infection or even following immunization procedures. So the protein/ energy ratio should presumably be increased following such an episode. Dr. Bremen Much more is now known about specific functions of individual amino acids. Is there any indication that we should now be deriving new figures for amino acid requirements based on the functions of individual amino acids? Dr. Wharton: I am not aware of any new work on this other than that of Vemon Young. He has started to argue that we should be using kinetic data as opposed to net balance data in determining requirements of specific amino acids (1). Dr. Garlick: One has to recognize that requirements derived from kinetic methods are strikingly different from those derived by traditional methods. This discrepancy has not yet been resolved, and until it is I don't think we can really rely on kinetic data. Dr. Wharton: Could you give us some examples? Dr. Garlick: From what has been published, the estimates for leucine requirements in adults by the kinetic method appear to be considerably higher than by traditional nitrogen 84 RECOMMENDATIONS ON PROTEIN INTAKES balance methods. The kinetic method measures leucine oxidation on different leucine intakes and attempts to derive a value for the amount of leucine required to maintain leucine balance. Until the reasons for these differences have been investigated thoroughly it is hard to comment on them further. Dr. Uauy: Kinetic data provide values for specific amino acid requirements. For several amino acids these may be twice the values obtained from nitrogen balance data. I agree that these data are not yet generally accepted, but the point that remains to be resolved is the functional implication of a given level of protein oxidation or amino acid oxidation. I think until this is settled we shall not be able to abandon the nitrogen balance data. Up to 6 months of age the model was human milk. The real problem starts after 6 months, and up to 1 year the only values available were the 1-year data, partly from Fomon and partly from Wang. So the values from 6 months to 1 year are just a direct interpolation of the 6-month value on the 1-year value, and they are mainly derived from short-term nitrogen balances. We now know much more about the limitations of such short-term studies. Probably not more than 10 or 15 infants have been studied on long-term nitrogen balances, and this is the work of Tontiserin in Thailand. With the information we have today we are going to be more demanding on what we are going to call an acceptable level of protein intake. We are going to be demanding data on immune function and on growth, and not only growth in weight gain but in body composition. In particular, as Waterlow points out, we should be considering the protein requirements for optimal linear growth. I don't think we are prepared to answer this question right now, but it is the question that comes up whenever and wherever stunting occurs. There is quite a strong correlation between linear growth and protein intake, specifically animal protein intake. I am not saying that we have the answer but I think we should probably keep an open mind when considering both the right kind of metabolic studies and the right kind of epidemiologic studies. Nitrogen balances have already given an answer but probably not the final answer. Dr. Axelsson: I was surprised by the protein content in the supplementary food you showed.-Is it homemade or canned baby food? The protein value is much lower than we have in Sweden and lower than the ESPGAN recommendations. I agree with Dr. Guesry that the recommendations for formulas for weaning must be based on the composition of available food supplements. Dr. Wharton: The weaning foods in Fig. 1 are both homemade (e.g., rice, rice with oil, rice with oil and egg) and commercial products (included in the British weanling diet). In many countries almost all of the weaning food would be rice. In South Africa I think there are many children whose only weaning food will be maize, perhaps with minor changes. This provides a lowish, but not bad, protein/energy ratio, but then you have to make your adjustment for net protein utilization to derive the figures I presented. Foods such as cassava or plantain, which is used in Uganda, have crude protein/energy ratios well below 1 g per 100 kcal and when you apply the adjustment for net protein utilization, the value becomes very low indeed. In Jamaica, historically, the weaning food was sucrose. Most manufactured infant foods available in Europe based on rice have other foods added. Sometimes high-protein foods are added to the rice, such as a milk powder or soya. Dr. Yamashiro: I understand that protein requirements in infants are higher in developing countries than in highly developed countries. I wonder if babies in their first 3 months still require more protein than those in developed countries. Dr. Wharton: As far as I know the answer to that is no. The recommendations relate to increased requirements resulting mainly from infections, which are much more prevalent after 3 months than before. RECOMMENDATIONS ON PROTEIN INTAKES 85

Dr. Uauy: There is only one recommended value for healthy individuals the world over. Of course, there is a long list of environmental stress factors, infection being number one, chronic gastrointestinal problems being number two, and energy deficit being number three, which all affect protein requirements. So as soon as the infant stops being breast fed, or even before because of inadequate weaning foods, he is likely to end up requiring more protein in developing countries, but this is not because he is biologically or genetically different. Dr. Rassin: I have a couple of technical comments. First, with respect to Olson Schneide- manen's work, I think we sometimes put too much trust in some of the earlier amino acid studies. Some of these studies involved only one infant. You need to be really careful about how you interpret such data; such studies might not even be published today. The second point is about tryptophan. I am concerned about the methodology related to tryptophan measurements. In our experience it is extremely difficult to get accurate tryptophan measurements in blood unless you use very specific HPLC or fluorometric methodologies. You see very different results depending on the methods used. Dr. Wharton: Our results quoted (2) were determined by reversed-phase HPLC, using a quaternary solvent system, and fluorimetry. Dr. Marini: I would like to present our experience with growth of babies fed with a hypoallergenic diet. We had a program for very high risk atopic babies, giving breast milk until 1 year, during which time the mother's diet was supervised, or hypoallergenic formula containing 1.5 g of protein per 100 ml. We started solid foods at about 6 months of age, beginning with rice alone because it is hypoallergenic, and graduating to meat products such as turkey, rabbit, or lamb, which are also not very allergenic. The only dairy product during the first year was Parmesan cheese because it consists of partially hydrolyzed protein and is rich in calcium. We calculated the intakes of these elements in comparison with the WHO recommendations and they are very similar. These babies have grown very well. Some of them are now 5 years old and have no problems at all. I believe it is quite possible to achieve good growth in developed countries following the WHO recommendations. Of course, it is not easy to obtain full cooperation from the family, but if there are good medical reasons this can usually be achieved. Dr. Wharton: Do you think it is possible to get as good growth with protein hydrolysates? Dr. Marini: Yes, in terms of both stature and body composition. Dr. Wharton: The question of protein quality and protein hydrolysates is an intriguing and difficult area. I think the standards used should not just rely on amino acid scores, and so on, which is the common approach in legislation relating to infant formulas; it should also include some type of biological assessment, such as the protein efficiency ratio. Dr. Heine: I should like to add something to the information Dr. Wharton has given us relating to gut microflora. Alan Jackson showed that there is recycling of ammonia from the splitting of urea in the large bowl, and it is certainly possible that intestinal bacteria may produce essential amino acids from the ammonia that is split by bacterial urease. We don't know whether this has any importance and if it has, to what degree. But it is well known that you can harvest bacteria on culture media which contain ammonium salts exclusively and that they are capable of producing essential amino acids in large amounts, whereas if the ammonia is absorbed only non-essential amino acids can be produced. Further work is needed to decide whether this is of importance for nutrition, especially in situations of undernutrition. Dr. Uauy: I should like to make a point about adaptation to low protein intakes. In the case of children "adaptation" usually means growing less well and being more likely to become infected. In adults it has been very well demonstrated that you can reduce the protein 86 RECOMMENDATIONS ON PROTEIN INTAKES intake to half and if you wait long enough nitrogen balance will be regained. But this is a very artificial situation, in which the subjects need to be protected from environmental stress. So I don't think that we should consider adaptation as being something potentially good. It is a survival state and should not be the basis of any sort of assessment of a reasonable protein requirement. Dr. Marini: We have been considering protein, but there are of course many other food constituents that are also important for growth. I did a study on growing preterm babies of about 2 kg birthweight who grew rather well on about 2 g of protein per kilogram per day, achieving weight gains of about 16 g/kg/d with good retention. However, when these babies had an inadequate intake of chlorine or potassium, they gained weight at only 13 g/kg/d instead of 16 g/kg/d. So when we consider protein we should not only be considering the amount of protein but the other constituents of the diet as well. Dr. Wharton: That is absolutely right. We tend to think mainly of protein and energy in relation to limiting nutrients for growth, but there are certainly situations where other factors may be naturally limiting: for example, zinc. I think that very occasionally vitamin D could be a limiting nutrient for growth. We should not be obsessed by the macro elements of the diet.

REFERENCES

1. Young VR, Yu YM, Fukagawa NK. Protein and energy interactions throughout life. Ada Paediatr Scand 1991; 373: 5-24. 2. Scott PH, Sandham S, Balmer SE. Wharton BA. Diet related reference values for plasma amino acids in newborn measured by reversed-phase HPLC. Clin Chem 1990; 36: 1992-7.