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Articles

Dietary Protein, Growth and Urea Kinetics in Severely Malnourished Children and During Recovery¹

Asha Badaloo^*, Michael Boyne^*, Marvin Reid^*, Chandarika Persaud, Terrence Forrester^*, D. Joe Millward and Alan A. Jackson^,2

^* Tropical Metabolism Research Unit, University of the West Indies, Mona, Kingston 7, Jamaica, Institute of Human Nutrition, University of Southampton, Southampton SO16 7PX, United Kingdom, and Centre for Nutrition and Food Safety, School of Biological Sciences, University of Surrey, GU2 5XH, United Kingdom

²To whom correspondence should be addressed at Institute of Human Nutrition, University of Southampton, Bassett Crescent East, Southampton SO16 7PX, United Kingdom.

	ABSTRACT

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The case mortality for severe malnutrition in childhood remains high, but established best approaches to treatment are not used in practice. The energy and protein content of the diet at different stages of treatment appears important, but remains controversial. The effect on growth, urea kinetics and the urinary excretion of 5-L-oxoproline was compared between a standard infant formula (HP group) provided in different quantities at each stage of treatment and a recommended dietary regimen, which differentiates the requirements of protein and energy during the acute phase of resuscitation (maintenance intake of energy and protein, relatively low protein to energy ratio, LP group) from those during the restoration of a weight deficit (energy and nutrient dense). The energy required to maintain weight was less in the HP than the LP group, but the HP group was not able to achieve as high an energy intake during repletion of wasting because of the high volume which would have had to be consumed. Compared to the LP group, in the HP group during catch-up growth there was significantly greater deposition of lean tissue and higher rates of urea production, hydrolysis and salvage of urea-nitrogen. These, together with higher rates of 5-L-oxoprolinuria, suggest a greater constraint of the formation of adequate amounts of nonessential amino acids, especially glycine, in the face of enhanced demands. Although more effective rehabilitation might be achieved using a standard formula, there is the need to determine the extent to which it might impose metabolic stress compared with the modified formulation.

KEY WORDS: • energy • marasmus • kwashiorkor • protein • 5-L-oxoproline • children

	INTRODUCTION

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Acute, severe malnutrition during childhood remains a common health problem in many parts of the world and makes a large contribution, both directly and indirectly, to childhood mortality (Pelletier 1995 ). In specialized centers, mortality was reduced to low levels, but for nonspecialized centers the case mortality has changed little over the last 50 y (Jackson and Golden 1987 , Schofield and Ashworth 1996 ), because of a failure to put established understanding into practice (Bredow and Jackson 1994 , Khanum et al. 1994 ). Successful regimens of treatment recognize that nutrient requirements are not constant for different phases of recovery and differ importantly between the needs of the acute phase of resuscitation compared with the phase of dietary rehabilitation, or the restoration of the weight deficit (Ashworth 1980 , Bredow and Jackson 1994 , Jackson and Golden 1987 , Khanum et al. 1994 ). The objectives of the resuscitation phase is to provide an intake of energy and protein adequate to satisfy the requirements for maintenance without promoting tissue deposition, while infections are treated, fluid and electrolyte balance reestablished and specific nutrient deficiencies corrected. During the restoration of weight deficit, generous amounts of energy and protein are provided with other nutrients to promote net tissue deposition (Ashworth 1969 , Ashworth 1974 , Waterlow 1961 ).

For the rapid restoration of weight loss and shortening the time to recovery, the emphasis of care is on the provision of a generous intake of energy, with the presumption that the availability of protein would not limit rapid rates of weight gain and optimal tissue restoration (Ashworth 1975 ). However, the relationship between the needs for energy and for protein is not linear across a range of intakes (Ashworth 1980 , Jackson and Wootton 1990 ). At lower rates of weight gain, balanced tissue can be readily deposited. At high rates the weight gained tends to contain an excess of adipose tissue with incomplete repair of the deficit in lean tissue and muscle mass (Castilla-Serna et al. 1996 , Fjeld et al. 1989 , Jackson and Wootton 1990 , MacLean and Graham 1980 , Reeds et al. 1978 ) and catch-up in height has been very difficult to achieve in the short term (Ashworth 1975 , Walker and Golden 1988 ).

With the adequate provision of energy, a constraint in the full recovery of lean tissue implies a limitation in the availability of a constituent of lean tissue (Rudman et al. 1975 ). Thus, when additional zinc is provided to infants consuming a low-zinc formula, there is enhanced partitioning of nutrients to lean tissue formation (Golden and Golden 1981 ). The protein content of the diet might be limiting (MacLean and Graham 1979 , Jackson and Wootton 1990 ), but earlier adverse experience with diets containing relatively large amounts of protein generate some caution especially as it seems unlikely that the diet is short of a specific essential amino acid (Jackson and Grimble 1990 ). More recent understanding of the metabolic importance of nonessential amino acids, such as glutamine and arginine, raises the possibility that nonessential amino acids or nonessential nitrogen might be limiting in availability at times when the metabolic demands are high (Jackson 1993 , Jackson 1995 ). At all ages there is a substantial need for nonessential nitrogen, and when the protein content of the diet is reduced to levels below which nitrogen balance can be maintained a supplement of nitrogen can effectively restore balance, in part through the enhanced utilization of urea-nitrogen, salvaged through the metabolic activity of the colonic microflora (Jackson 1995 , Kies 1972 , Meakins and Jackson 1996 ). In infants consuming diets which contain too little protein to support normal growth and nitrogen balance, the addition of a supplement as either glycine or urea will restore normal rates of weight gain (Snyderman et al. 1962 ). An increase in the urinary excretion of 5-L-oxoproline, marking poor glycine status, is seen in children with severe malnutrition and is brought toward normal levels with a dietary supplement of glycine (Persaud et al. 1997 ). Further, malnourished children salvage urea-nitrogen at increased rates at all stages of recovery (Doherty and Jackson 1992 , Jackson et al. 1990 , Picou and Phillips 1972 ). Possibly during rapid weight gain, there is a constraint on the availability, or the ability to utilize nitrogen effectively for the formation of nonessential amino acids.

As children develop severe malnutrition in situations where the facilities for medical care are poor, there is the need to identify the most simple and straightforward approach to care. It has been proposed that case management would be more straightforward if a single formulation, based on a readily available infant formula, were used in treatment. In the present study we compared the effect of the approach to nutritional management used in a specialized center, with an alternative in which different amounts of a commercial infant formula are offered at the different stages of recovery. We measured urea kinetics and 5-L-oxoproline excretion to compare metabolic function between the two dietary regimens.

	METHODS

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Subjects

The studies were carried out in 20 male subjects who had been admitted for the treatment of severe undernutrition to the Tropical Metabolism Research Unit. They were between 6 and 24 mo of age, with the diagnosis of marasmus, marasmic-kwashiorkor, kwashiorkor, or undernutrition on admission (Wellcome Trust Working Party 1970 ). Patients with secondary malnutrition were excluded. The study protocol was approved by the Ethical Committee of the University Hospital of the West Indies, and for each subject written, informed consent was given by the parent or guardian. All clinical decisions regarding care and treatment were taken by the attending physicians. On admission a full clinical history was taken, a clinical examination completed and specimens taken for hematology, biochemical tests and investigations for infection. Experienced staff measured recumbent body length using a calibrated length board and weight on an electronic balance accurate to 1 g. The weight of each subject was measured at the same time each day and length was measured weekly. Each subject was studied on three separate occasions.

Study design

Once entered into the study, the subjects were randomly allocated to two groups based on the diet offered for consumption during Stages 1, 2 and 3 (Table 1 ).All the diets were milk-based, and one group was offered a formula with a higher protein to energy density (HP, n = 7)³ and the other a formula with a lower protein to energy density (LP, n = 7). At the completion of the study, the protein content of the formula was lower than had been expected. As it was possible that this small difference around maintenance levels of protein intake could have made a critical difference to the results, a further group of subjects was recruited to the protocol, receiving slightly more protein (n = 6), [stage of rehabilitation (MAL), 0.52 and 0.62 g protein · kg^-1 · d^-1; stage of rapid weight gain (RWG), 3.06 and 3.05 g protein · kg^-1 · d^-1; stage after recovery (REC), 0.53 and 0.69 g protein · kg^-1 · d^-1]. As there were no differences for any of the results between the two groups, they were combined to form the LP group (Table 2 ).For each study period, at each stage, the study formula was offered to the subject in controlled amounts for a period of 5 d. Over the last 36 h of the 5-d period, urea kinetics was determined, and the urinary excretion of 5-L-oxoproline was measured. For each subject the rate of weight gain was calculated for the 5-d period based upon the linear regression of daily weight on time. The energy cost of growth over the period of rapid catch-up growth was calculated.

Diets

The LP formula was based upon the standard formula and the approach used for the nutritional rehabilitation of severely malnourished subjects admitted to Tropical Metabolism Research Unit (Jackson and Golden 1987 ). The LP formula offered during Stage 1 is designed to provide a metabolizable energy intake adequate to maintain body weight, 418 kJ · kg^-1 · d^-1 (100 kcal · kg^-1 · d^-1) (Kerr et al. 1973 ) and to satisfy the maintenance requirement for protein, 0.6 g protein · kg^-1 · d^-1 (Chan and Waterlow 1966 ), with generous supplements of minerals and vitamins. Once acute problems were successfully managed, appetite returned. During Stage 2 patients are offered a formula which is energy- and nutrient-dense and is designed to promote rapid catch-up growth. The HP group was offered a single formula during the entire period of rehabilitation, using an unmodified commercial milk powder (Pelargon; Nestlé, Vevey, Switzerland) reconstituted to the instructions of the manufacturer. At each stage of treatment the two groups were offered diets that contained similar amounts of energy, but different amounts of protein (Table 1) . Thus, during MAL (Stage 1), both groups of subjects were offered 418 kJ · kg^-1 · d^-1 (100 kcal · kg^-1 · d^-1), but the formula for the HP group provided 3 g of protein/kg/d compared with 0.6 g of protein/kg/d in the LP group. During RWG (Stage 2), both groups were offered an amount of formula that would provide 711 kJ · kg^-1 · d^-1 (170 kcal · kg^-1 · d^-1), and in the HP group this provided 4.6 g protein · kg^-1 · d^-1, compared with 3.1 g protein · kg^-1 · d^-1 in the LP group. For the duration of Stage 2, except the 5 day study period, all subjects consumed the formulation ad libitum, until their weight deficit had been corrected. At this time the REC (Stage 3) study was carried out, and each subject was offered the same milk preparations they had taken during MAL, with one difference, the energy intake estimated to satisfy the maintenance requirements at recovery is 459 kJ · kg^-1 · d^-1 (110 kcal · kg^-1 · d^-1) (Ashworth 1974 ). The diets were supplemented with vitamins and minerals: 2 mL/d of Tropivite (retinyl palmitate 1.8 mg retinol equivalents, calciferol 40 µg, thiamine HCl 12 mg, riboflavine 5' phosphate Na 3.2 mg, ascorbic acid 120 mg, pyridoxine HCl 4 mg, and nicotinamide 28 mg); folic acid, 5 mg/d (Federated Pharmaceutical, Kingston, Jamaica); potassium, 2 mEq · kg^-1 · d^-1, and magnesium, 1 mEq · kg^-1 · d^-1, (74.6 g KCl + 8 MgCl₂ · 6H₂O/L H₂O; BDH Chemicals, Lutterworth, UK). The subjects were offered the formula every 3 h, eight times during each 24-h period during MAL, or every 4 h, six times during the 24 h during REC. The amount consumed at each feed was determined gravimetrically by weighing the cups before and after feeding on an electronic balance.

Urea kinetics

Urea kinetics were measured over a period of 36 h using prime and intermittent oral doses of [¹⁵N¹⁵N]urea (99.8 atom percentage; Isotope Laboratories, Cambridge, MA). A known quantity of isotope was accurately weighed on an electronic microbalance and made up to a known concentration. At 0600 h a priming dose of isotope (40 mg), equivalent to 12 h continuous infusion, was given to shorten the time taken to achieve plateau in the isotopic enrichment of urinary urea. From 1200 h, three hourly doses of 10 mg isotope were given for 30 h. Each dose of isotope was weighed in a disposable syringe and was administered to the back of the subject's throat to ensure that all the dose was swallowed.

In all studies, a specimen of urine was collected before any isotope was given for the measurement of baseline enrichment. From 0600 h, urine was collected by continuous aspiration from a perineal urine bag into chilled containers for periods of approximately 6 h for 36 h. The weight of each urine collection was determined, and an aliquot was acidified to pH 2 with 6 mol/L HCl and stored at -20°C for later analysis.

Biochemical analyses and mass spectrometry

The concentration of urea nitrogen in urine was measured by the Berthelot method (Kaplan 1965 ), and urea was isolated from urine for mass spectrometry using short column ion-exchange chromatography (Jackson et al. 1980 ). Nitrogen gas was liberated from urea in vacuo by reaction with alkaline hypobromite. Nitrogen is released from urea in a monomolecular reaction, and it is thus possible to determine the proportions of different isotopic species of urea molecules by measuring the relative ion beams of nitrogen liberated with mass 28, 29 and 30 (Walser et al. 1954 ) using a triple collector isotope ratio mass spectrometer (SIRA 10; VG Isogas, Cheshire, United Kingdom).

Urinary 5-L-oxoproline was measured in a three-step method, ensuring that 5-L-oxoproline is measured without any contamination from 5-D-oxoproline. 5-Oxoproline was isolated by short-column ion-exchange chromatography, free from glutamate and other amino acids. The 5-L-oxoproline in the eluate was hydrolyzed in hot acid to glutamic acid and the resulting L-glutamic acid measured enzymatically with glutamate dehydrogenase (EC 1.4.1.2.) (Jackson et al. 1996 ).

Theoretical considerations and calculations

    Energy cost of growth. The energy cost of growth (ECG_t2-t1), kcal/g, the energy required for net tissue deposited between time t1 and t2, was estimated from energy intake (total El_t2-t1) and weight gain during the period of catch-up growth .

The maintenance energy expenditure (maintenance EE) is the energy required to maintain constant body weight under usual conditions of diet and activity. It is not possible to know what this is with certainty during the period of RWG. Based upon the studies by Spady et al. (1976) and our balance data (Jackson et al. 1983 , Kennedy et al. 1990 ), we took this to be a metabolizable energy intake of 376 kJ · kg^-1 · d^-1 (90 kcal · kg^-1 · d^-1). The energy cost of growth comprises two components: the energy required for net tissue formation plus the energy contained in the net tissue deposited. The energy available for weight gain was taken to be the difference between the total energy intake and the energy required to maintain body weight. An index of the composition of tissue laid down during recovery was obtained from an established linear relationship between the energy available for weight gain and the proportion of weight gain attributed to an increase in muscle mass in children recovering from severe malnutrition (Jackson et al. 1977 ). It has been assumed that the energy cost of net tissue deposition is 8.8 kJ/g (2.1 kcal/g) for protein and 4.2 kJ/g (1.01 kcal/g) for fat and that the energy content of protein is 23.8 kJ/g (5.7 kcal/g) and the energy content of fat is 38.9 kJ/g (9.3 kcal/g) (Coyer et al. 1987 ). Thus, to deposit 1 g of lean tissue containing 20% protein will require 10 kJ (2.4 kcal), and to deposit 1 g of adipose tissue containing 90% lipid and 2% protein will require 36.4 kJ (8.7 kcal). On this basis the fraction of tissue deposited as lean tissue is given by the expression :

The relative height [height for age H/A)], weight [weight for age (W/A)], and weight for height (W/H) for each subject were calculated in relation to the reference standard (Hamill et al. 1979 ).

    Urea kinetics. The kinetics of urea metabolism were derived using a stochastic model that assumes an isotopic and biological steady state in which the dilution of an intermittent dose of [¹⁵N¹⁵N]urea gives a measure of the rate of urea production in the body (Jackson et al. 1984 ), according to the relationship :

where dose [¹⁵N¹⁵N]urea is expressed as mmol N · kg^-1 · d^-1 and the ratio of tracer to tracee as moles excess. A proportion of this urea is excreted in urine, and a negligible amount is excreted in stool (Jackson et al. 1984 ). Therefore the difference between urea production and urea excreted in urine is presumed to have passed into the colon and to have been hydrolyzed by the resident flora with the nitrogen being returned to the general metabolic pool. A part of this nitrogen is recycled into urea synthesis, and the remainder is available for amino acid synthesis. The urea entering the colon is doubly labeled and on hydrolysis yields [¹⁵N]ammonia. The preponderance of naturally occurring [¹⁴N]ammonia, permits a very small chance that two labeled ammonia molecules would be incorporated into one molecule of urea. Therefore, nitrogen from urea hydrolysis which is recycled to urea synthesis is determined from measurements of the molar ratio of singly labeled urea to unlabeled urea, [¹⁵N¹⁴N]urea to [¹⁴N¹⁴N]urea, in the urine (Jackson et al. 1984 ).

Statistics

Statistical analyses were carried out using the Systat statistical package (Evanston, IL 1988). Variables with unequal variances were log-transformed. Comparisons between dietary groups (HP, LP) were carried out by one way analysis of variance. To assess differences between Stages within the groups, repeated measures analysis of variance were performed with post-hoc Scheffé's test to determine differences between particular groups. Differences were considered to be statistically significant for P < 0.05.

	RESULTS

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Twenty subjects ages between 6 and 15 mo were entered into the protocol, and a total of 60 studies was completed. The clinical diagnosis of the subjects at admission and the anthropometric characteristics at admission and discharge are given in Table 2 . On admission, nine subjects were diagnosed as marasmus, five as kwashiorkor, five as marasmic-kwashiorkor and two as underweight. The HP and LP groups were similar in terms of diagnosis, age, height and weight. When the results for subjects with different diagnoses at admission were analyzed separately, there were no differences. The duration of stay in a hospital was similar for both the LP (1.9 ± 0.5 mo) and the HP (2.0 ± 1.3 mo) groups. As a group, the subjects were stunted at admission with a height for age of about 87% of the reference (Hamill et al. 1979 ). Both groups gained significantly in height during the admission at a rate appropriate for their age, about 2.3 cm, so that the degree of stunting was not changed. There were no differences between the LP and HP groups in height at admission or discharge. Although not significant, it may be noteworthy that compared with a loss in height for age of 0.5% in the LP group, the HP group gained 2.7% (P = 0.176). There was a significant increase in weight, expressed absolutely (LP 2.03 ± 0.84 kg; HP 2.01 ± 0.62 kg) or in relation either to age or to height, with treatment in both dietary groups. At recovery the weight for height of the subjects was not different between the two groups.

Dietary intake

There was no difference in weight of the subjects in each group at the commencement of each stage of study, and for each group, weight was significantly increased for each successive stage (Table 3 ).The energy consumed at each stage approximated that planned, except during RWG for the HP group, where the consumption was only 648 kJ · kg^-1 · d^-1 (155 kcal · kg^-1 · d^-1), compared with the intended 711 kJ · kg^-1 · d^-1 (170 kcal · kg^-1 · d^-1). The LP formula was energy-dense (Table 1) , but the volume of the HP formula which the subject had to consume to obtain 170 kJ · kg^-1 · d^-1 was significantly greater than for the LP group, and bulk may have contributed to some refusal (Table 1) . As planned, protein consumption was significantly different between groups at each stage. The proportion of total energy derived from protein in the HP formula was about 12%. For the LP formula this ratio was 2.8% during MAL and REC, and 7.4% during RWG.

Crude nitrogen balance was taken as the difference between nitrogen consumed and the urinary loss of nitrogen as urea, which would give an overestimate of nitrogen retention. At all stages, regardless of the diet consumed, crude nitrogen balance was positive, but at each stage crude balance was significantly greater whilst consuming the HP diet than the LP diet. While consuming the LP diet, crude balance was five times that during RWG, than either MAL or REC, which did not differ from each other (P < 0.01). There was a significantly positive crude nitrogen balance at all stages while consuming the HP diet (P < 0.01), although this was significantly greater during RWG than MAL (P < 0.05), and during MAL than REC (P < 0.01).

Weight gain

The subjects did not gain any weight while consuming the LP diet at maintenance levels of protein and energy intake, MAL and REC, but they did have a significantly greater rate of weight gain when this diet was enriched with energy and protein and consumed in excess of maintenance requirements, RWG (P < 0.01). For the subjects who consumed the HP diet during MAL, there was a significant positive rate of weight gain compared with the subjects consuming the LP diet during MAL (P < 0.01) and the subjects consuming the HP diet during REC (P < 0.05). Similar to the LP group, in the HP group the highest rate of weight gain was during RWG. There was no difference in the rate of weight gain between the subjects consuming the two diets during RWG. Over the 5 d of study, the tissue deposited had an energy density of about 24 kJ/g (5.7 kcal/g) in both groups, indicating that the composition was ~50% lean and 50% adipose. However, when the same calculation was made for the whole period of Stage 2, the energy density for the LP group was 26 kJ/g ± 7 (6.2 kcal/g) and for the HP group 15 ± 7 kJ/g (3.6 kcal/g). Suggesting that overall, the proportion of weight gained as lean tissue in the HP group, 80%, was significantly greater than in the LP group, 40% (P < 0.01).

Urinary 5-L-oxoproline

For the LP and HP groups combined, the excretion of 5-L-oxoproline in urine was significantly greater during MAL (4.33 ± 0.36 µmol/h) than during RWG (3.22 ± 0.27 µmol/h, P < 0.01) or during REC (2.72 ± 0.14 µmol/h, P < 0.001), and excretion during RWG was significantly greater than during REC (P < 0.01). Excretion was greater in the HP group than the LP group during RWG (P < 0.05) (Table 3) . In the LP group, excretion in MAL was significantly greater than either RWG, 30%, or REC, 40% (P < 0.01).

Urea kinetics

The achievement of a plateau in the ratio of tracer to tracee was determined by visual inspection for [¹⁵N¹⁵N]urea to [¹⁴N¹⁴N]urea and for [¹⁵N¹⁴N]urea to [¹⁴N¹⁴N]urea in the samples of urine with time. The coefficient of variation for [¹⁵N¹⁵N]urea to [¹⁴N¹⁴N]urea was 9% on average (range 2 to 23%). The results for urea kinetics are shown in Tables 4 and 5,and for every aspect of urea kinetics the values were greatest during RWG than either the MAL or REC periods.

    Urea hydrolysis. At all stages there were significant differences in urea hydrolysis which was greater in the HP group than the LP group (Table 4) . In the HP group, hydrolysis was significantly greater during RWG and REC than during MAL (P < 0.05). In the LP group, urea hydrolysis during RWG was three times that during MAL and five times that during REC (P < 0.001). When compared with the rate of urea production, on the HP diet significantly more of production was hydrolyzed in the HP group during MAL, 59%, than during RWG or REC, 47% (P < 0.05) (Table 5) . In the LP group, hydrolysis as a percentage of production was not different in MAL compared RWG.

    Fate of urea-N derived from hydrolysis. On all diets and at every stage of rehabilitation, only a small proportion of the nitrogen derived from urea hydrolysis was returned to urea formation, with the majority, over 85%, being utilized for synthetic pathways. More was available for synthetic pathways in the HP group than the LP group. Thus, for example during RWG, in the HP group, 734 mg N · kg^-1 · d^-1 was consumed with 189 mg N · kg^-1 · d^-1, available from urea salvage, 20% of the total. Similarly, in the LP group during RWG, consumption was 490 mg N · kg^-1 · d^-1 with 123 mg N · kg^-1 · d^-1, from urea salvage, 20% of the total. The group in which the utilization of urea-nitrogen was lowest was the LP group at REC. Here consumption of 90 mg of N · kg^-1 · d^-1 was complemented by salvage of 24 mg N · kg^-1 · d^-1, 21% of the total. The greatest relative contribution of nitrogen derived from urea salvage was during MAL in the LP group, where utilization of urea-nitrogen was 53% of the nitrogen consumed, significantly greater than the relative proportion in the LP group during RWG, 25% (P < 0.01).

The protein available for consumption was similar for three study periods, the LP group during RWG, and the HP group during MAL and REC. In Figure 1, a comparison is drawn for each of these periods for the weight gain, nitrogen balance, urea kinetics and 5-L-oxoproline excretion. Although the protein consumed was similar during each study period, there were marked differences in the energy consumed and the metabolic state of the subjects. Crude nitrogen balance for the HP group during MAL was not different to the LP group during RWG. The rate of weight gain was greatest during RWG in the LP group and 5-L-oxoproline excretion greatest during MAL in the HP group. Urea production and urea excretion were progressively greater at each stage of recovery.

View larger version (28K): [in this window] [in a new window]   Figure 1. A comparison of crude nitrogen balance, rate of weight gain, urinary 5-L-oxoproline (top panel) and aspects of urea kinetics, rate of urea production, excretion and hydrolysis (bottom panel) in groups of infants and young children consuming similar amounts of protein, but different amounts of energy at different stages of recovery from severe malnutrition. The level of protein consumption for each group was about 3 g of protein · kg-1 · d-1, but the energy consumption differed with stage of recovery: stage of rehabilitation of a malnourished child (MAL) 414 kJ · kg-1 · d-1 (99 kcal · kg-1 · d-1); stage of rapid weight gain of a malnourished child (RWG) 694 kJ · kg-1 · d-1 (166 kcal · kg-1 · d-1); stage after recovery of a malnourished child (REC) 426 kJ · kg-1 · d-1 (102 kcal · kg-1 · d-1). Values are means ± SD. The subjects in the MAL and REC groups (n = 7) are the same, and different from those in the RWG group (n = 13).

	DISCUSSION

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Urea kinetics was measured in malnourished and recovered children while consuming diets either high or low in protein and during catch-up growth in subjects consuming high energy diets, enriched with carbohydrate or lipid (Doherty and Jackson 1992 , Jackson et al. 1990 , Picou and Phillips 1972 ). At any stage of recovery the consumption of energy determines the potential for lean tissue formation and determines the demand for nitrogen, but the intake of protein determines the extent to which that demand might be met (Jackson and Wootton 1990 ). At higher absolute levels of energy consumption, the dietary protein may be insufficient to satisfy the demand, and under these circumstances the ability to increase the rate at which urea-nitrogen is salvaged appears to play an important part in helping to satisfy the body's nitrogen requirements. Thus, a diet with a protein-to-energy ratio which satisfies lower rates of lean tissue deposition might be limiting in protein at higher levels of consumption (Jackson and Wootton 1990 ). At any stage of recovery, there is the need for an adequate intake of energy, protein and other nutrients, and as all subjects consumed generous supplements of minerals and vitamins there is no reason to consider that any vitamin or trace mineral such as zinc was limiting. The malnourished state is characterized by infection and specific nutrient deficiencies, which separately or together lead to loss of appetite and weight loss. Once these problems are corrected, a profound desire to eat expresses itself as a voracious appetite (Ashworth 1974 ) that seeks to satisfy the metabolic requirements for energy and protein (Waterlow 1961 ) and which is sustained until some sense of near normal weight or body composition is achieved (Ashworth 1974 ). This sense of normality might be related in part to the repletion of lean body mass or the correction of sarcopaenia (Ashworth and Millward 1986 , Millward 1995 ), but also includes other more complex signals as appetite is reduced before the deficit in lean body mass is fully corrected (Jackson and Wootton 1990 ).

Energy consumption in excess of the requirements for maintenance promotes net tissue deposition with relatively small increases, resulting in substantial rates of weight deposition (Ashworth 1980 , Jackson and Wootton 1990 , Kerr et al. 1973 ). Available amino acids are preferentially utilized for net protein deposition, but they might be utilized with less efficiency if the pattern available is only a poor match for the composition of the proteins being formed. Under this circumstance, excess amino acids will be oxidized with an increase in urea formation (Harper et al. 1970 ). With only limited availability of amino acids, not all the competitive demands for the synthesis of individual or specific proteins can be satisfied (Jackson 1985 ). Therefore, there is the need to differentiate the relative contributions of factors acting either independently or together on different aspects of weight gain and urea kinetics at different stages of recovery.

Effect of stage of recovery.

Children were hospitalized for an average of 2 mo which did not differ between the two dietary groups. There were similar increments in height and weight between the two groups, and over the admission the subjects maintained a normal rate of growth in height and had significant catch-up growth in weight, to correct all wasting. These patterns of growth did not differ in relation to the initial diagnosis, and in gross terms both the LP and HP diets supported successful recovery. However, there were important differences between the groups. In malnourished children, the consumption of protein, 0.6 g · kg^-1 · d^-1, together with the energy consumption of 418 kJ · kg^-1 · d^-1 (100 kcal · kg^-1 · d^-1), enables weight and nitrogen balance to be maintained (Ashworth 1974 , Chan and Waterlow 1966 ). A higher consumption of energy, 460 kJ · kg^-1 · d^-1 (110 kcal · kg^-1 · d^-1), is required to maintain weight and nitrogen balance following recovery (Ashworth 1974 ), in part a reflection of the process of reductive adaptation and energy conservation associated with malnutrition. The weight gain at REC in the HP group, about 1 g/kg/d, approximated that expected for a normal child of similar age while in the LP group protein consumption appeared limiting for weight gain. By contrast during MAL there was consistent weight gain in the HP group, which was substantially greater than seen in the same group at REC, or in the LP group during either MAL or REC. The difference in weight gain can be attributed directly to the difference in the protein content of the diet and indirectly to the consumption of energy. There is a clearly defined interaction between the dietary energy and protein, and for a fixed consumption of protein an increase in the consumption of energy improves nitrogen balance and vice versa (Calloway 1981 ). Thus, the HP group consumed about 2.3 g · kg^-1 · d^-1 more protein than the LP group which enabled the deposition of 7 g · kg^-1 body weight · d^-1. The energy required to achieve this would vary, depending on the composition of the tissue being formed, but if the energy required to deposit tissue were 21 kJ/g tissue (5 kcal/g), energy consumption would have to be 146 kJ · kg^-1 · d^-1 (35 kcal · kg^-1 · d^-1), assuming an efficiency of protein deposition of about 50%. This would imply that in the HP group during MAL, either the requirement of energy for maintenance was only 314 kJ · kg^-1 · d^-1 (75 kcal · kg^-1 · d^-1), or body composition was changing with lean tissue being deposited in preference to adipose tissue, or a combination of the two. Either option is possible, with a reduction in the energy needs for maintenance, reflecting the increased efficiency of energy utilization when a nutrient deficiency is corrected (Kleiber 1945 ), and the mobilization of energy from adipose tissue representing the response to a diet limiting in energy (Coyer et al. 1987 , Kennedy et al. 1990 ). Whichever mechanism operates in practice, the practical implication is that if the higher protein diet is provided the dietary provision of energy should be less if weight is to be maintained during the early period of treatment. The difference in the rate of growth between MAL and REC in the HP group, despite similar intakes of protein and energy, indicates that during MAL there is a considerable potential together with a substantial drive toward the deposition of lean tissue (Millward 1995 ). With the correction of the depletion, both the potential and the drive appear to be lessened, or incapable of being satisfied. These data demonstrate the difficulty in defining with any precision the maintenance requirements for energy and protein under different metabolic conditions when there are variable changes taking place in a number of factors, which might include changes in adaptation, physical activity and growth. Dietary energy is utilized with greater efficiency in the malnourished state, at the cost of reductive adaptation, shown as decreases in resting energy expenditure of up to 30%, the thermic response to food and in protein turnover (Brooke and Cocks 1974 , Jackson 1985 , Waterlow 1992 ). The adaptive mechanisms which underlie this drive to increased efficiency in the metabolism of protein and energy can be brought into play in children who were previously exposed to marginal intakes of energy, enabling weight to be gained despite the consumption of diets which would be expected to be marginally inadequate in energy (Kennedy et al. 1990 ).

Effect of differences in energy intake.

In the malnourished state, the degree of tissue depletion is a major factor contributing to setting the metabolic demand and the drive toward greater consumption of energy and nutrients. As a step toward obtaining an understanding of the interactions, a comparison can be drawn between the LP group during RWG and the HP group during MAL and REC (Fig. 1) . For each study period, the protein consumed was virtually identical, but there were substantial differences in energy consumption and the metabolic state. The rate of weight gain was significantly greater during RWG than during MAL and during MAL than during REC. A comparison of the rate of weight gain between MAL and RWG shows that the drive to replete tissue was constrained by the availability of energy. A comparison between MAL and REC indicates the greater efficiency with which protein and energy can be used in response to the drive for tissue deposition, especially lean tissue deposition, with a 30% increase in crude nitrogen balance during MAL than REC. Compared with REC, during MAL and RWG amino acids were channelled to tissue deposition, and urea production was reduced by 40%.

Effect of differences in protein intake.

By comparing the HP and LP groups at each stage of recovery, the effect of differences in protein intake can be determined. During MAL, when the metabolic demand for tissue deposition is very high in the face of limited energy and protein availability, the importance of the interaction between energy and protein metabolism is made very clear. In the LP group the consumption of a maintenance level of energy and protein in the diet does not allow for net tissue deposition. Similar children consuming a diet providing less energy lose weight (Jackson et al. 1983 , Spady et al. 1976 ). However, in the HP group with increased consumption of protein, there was significantly greater rate of weight gain and crude nitrogen balance. In the HP group, proportionately more of the ingested nitrogen was partitioned to tissue formation and less to urea production (Table 5) . A similar effect of nitrogen balance and urea kinetics is seen during RWG with lean tissue comprising relatively more of the weight gained. The rate of weight gain during RWG in the HP group was about 25% less than in the LP group, in part a consequence of the consumption of energy being limited to 648 kJ · kg^-1 · d^-1 (155 kcal · kg^-1 · d^-1) compared with 690 kJ · kg^-1 · d^-1 (165 kcal · kg^-1 · d^-1). A number of factors might contribute to explaining the differences. Firstly, whereas in the LP group the formula was energy and nutrient dense, making it possible for the subjects to consume more energy and nutrients without any increase in the volume ingested, to reach the same level of energy consumption in the HP group required the ingestion of twice the volume. The stomach capacity of some of the subjects might have been exceeded (Ashworth 1974 , Brown et al. 1995 ). Secondly, a metabolic constraint might have limited the ability to handle the ingested nutrients. Given the very high rate of lean tissue deposition in the HP group, the formulation might have been limited in a specific nutrient (Rudman et al. 1975 ), or the higher rates of tissue deposition during MAL might have reduced the anabolic drive to tissue deposition during REC. Thirdly, when large amounts of protein are ingested there are recognized toxic effects. An aminostatic satiety mechanism may play a part in the stimulation of appetite during catch-up growth, with appetite being stimulated when the maximal rate at which amino acids are removed, either for tissue repletion or oxidation, exceeds the intake from protein (Millward 1996 ). If intake exceeds the maximal rate of removal, then appetite is reduced. Any substantial mismatch between the balance of amino acids available and that needed by the body would limit the flow of amino acids to tissue repletion. A reduction of intake might therefore be found when there is the need to handle and excrete a toxic excess of one or another essential amino acid, or when the availability of an essential amino acid is limited, or when the rate of formation of a nonessential amino acid is limited. The possibilities are not mutually exclusive, and to an extent each will reinforce the other.

Effect of reduced protein in REC.

At REC, crude nitrogen balance for the HP group was greater than for the LP group, with greater rates of urea production, excretion and hydrolysis. Despite the absolute increase in urea production in the HP group, urea utilization relative to urea production was significantly less in the HP than the LP group (Table 5) . The factors which exert a major influence over urea production and salvage have been clarified for normal adults when fasting (Hibbert and Jackson 1995 ), or consuming diets in which the protein content varies from 35 to 200 g/d (Child et al. 1997 ). Across this very wide range of intakes, urea production varies by less than 15% and is on average 180 mg N · kg^-1 · d^-1. In childhood the same relationships do not apply and this may be because of the demands associated with growth. In pregnancy (Forrester et al. 1994 , McClelland et al. 1997 ), early infancy (Steinbrecher et al. 1996 ), or during catch-up growth (Doherty and Jackson 1992 , Jackson et al. 1990 , Picou and Phillips 1972 ) when there is the need to economize on amino acids and nitrogen, the salvage of urea-nitrogen is increased. When consuming diets high in protein, urea production is not increased proportionately, and there are presumably other routes through which nitrogen can be lost, including increased fecal excretion (Child et al. 1997 ). In children recovering from malnutrition, variations in stool losses may make a critical difference to the achievement of N balance (Kennedy et al. 1990 ). There were no collections of feces in the present study, and it is not possible to identify the extent to which changes in the composition of stool might have made an important difference.

Effect of metabolic state: stage of recovery.

The control of urea kinetics is associated with reabsorption of urea along the collecting duct of the kidney, and through changes in the rate of hydrolysis of urea in the colon (Jackson 1995 , Schmidt-Nielsen 1970 ). Both are influenced by changes in the activity of the arginine-vasopressin sensitive urea transporter (You et al. 1993 ) with possible coordinate control accounting for enhanced retention and utilization of urea on low protein diets. We neither know the age at which these systems mature nor whether the set points to which they operate are similar in children. During malnutrition the combined effects of a reduced intake of nutrients and the presence of frequent infection exert an influence on all tissues. The gastrointestinal tract is obviously damaged in diarrhoeal disease, and there is impairment in renal function and hepatic function (Waterlow 1992 ). When compared with REC, there were clear differences between the two dietary groups during MAL for crude balance and rate of weight pain which have been interpreted as most likely interactions of energy and protein (see above). For urea kinetics, urea production was increased in the HP group and decreased in the LP group, with similar patterns for urea excretion and hydrolysis. The appearance was that the LP group was less able than the HP group to utilize the protein in the diet during MAL, but much better able to utilize the available dietary protein when recovered. For both groups, urinary excretion of 5-L-oxoproline during REC was 35 to 40% less than during MAL. The higher excretion of 5-L-oxoproline in urine in the HP than the LP group was significant during RWG. Figure 1 indicates that when protein consumption was equivalent urinary 5-L-oxoproline during MAL was twice that in REC or RWG, implying that any constraint on the availability of glycine was greatest during MAL.

Implications for management of malnutrition.

The present study showed that recovery from severe malnutrition can be achieved by subjects consuming diets which provide a wide range of protein content. However, all the studies were carried out at a time when the acute, life-threatening problems had been brought under control. Therefore, the results are of relevance to an understanding of dietary formulations which modify weight, body composition and urea kinetics and provide no information on the suitability of one or other diet for acute, initial treatment. For malnourished subjects, with the consumption of 418 kJ · kg^-1 · d^-1 (100 kcal · kg^-1 · d^-1) and 0.6 g protein/kg/d (LP) as a specially prepared formula, weight is maintained, but significant weight is gained when the same energy is consumed as a standard formula (HP). Thus, when consuming the LP formula, the protein content is limiting for weight gain, and when consuming the HP formula the requirement for energy to maintain weight is less than 418 kJ · kg^-1 · d^-1 (100 kcal · kg^-1 · d^-1).

It has been our practice for 30 y to feed a specially prepared, high-energy milk formula to reduce the volume required to achieve high rates of weight gain during catch-up growth (Ashworth 1969 , Waterlow 1961 ). The present study shows that the consumption of an unmodified infant formula (HP) of relatively lower energy density (2.8 MJ/kg feed, 670 kcal/kg feed) enables rapid catch-up growth with the deposition of relatively more lean and less adipose tissue. When consuming the standard formula (HP), the rate of weight gain might have been limited by the amount of formula ingested, which could have been a bulk effect or a reflection of the metabolic capacity of the subject having been reached.

At all stages, while consuming the standard formula (HP), there was a very high rate of urea production, hydrolysis and salvage of urea-nitrogen, suggesting that the pattern of amino acids in the formula was not a good match for the pattern of amino acids required by the body. This, together with the higher 5-L-oxoproline excretion on the standard formula, suggests the possibility of a specific problem in forming adequate amounts of nonessential amino acids, especially glycine, when consuming the HP formula. We found in adults consuming high-protein diets that there is an increase in the rate of urea-nitrogen salvage and 5-L-oxoproline excretion (Child et al. 1997 ) and in women consuming a low-protein diet given an oral load of L-methionine there was an increase in the rate of 5-L-oxoproline excretion (Meakins et al. 1998 ). It may be therefore that with higher levels of protein consumption, the ability to detoxify excess essential amino acids, which are not needed for tissue deposition, leads to an increased need for the formation of nonessential amino acids, such as glycine (Jackson 1999 ).

At all stages, about 25% the nitrogen intake was excreted as urea, indicating that the greater proportion of the protein consumed was retained in the body. However, nitrogen excretion was about 40 to 50% of urea production showing substantial retention of urea-nitrogen following hydrolysis. Only about 15% of the nitrogen derived from urea hydrolysis was returned to urea synthesis, indicating the nitrogen had been incorporated in metabolic products. The absolute amount of salvaged urea-nitrogen was most substantial during growth, when retention or urea-nitrogen was equivalent to 1 to 1.5 g protein · kg^-1 · d^-1.

The results of the present work indicate that a standard infant formula can be used to successfully rehabilitate malnourished subjects. However, there are important considerations which need further exploration. The HP group gained weight during MAL, which is not desirable when the acute problems of infection, metabolic disturbances, edema mobilization and fluid the electrolyte imbalances are being treated. Therefore, there is the need to determine the amount of formula that should be consumed to maintain weight under this circumstance, and the effect of resuscitation. The higher rate of lean tissue deposition during catch-up growth and the possible improved height gain are important benefits, but increased rates of urea-nitrogen salvage and a relative increase in 5-L-oxoproline excretion also implies limited availability of nonessential nitrogen. The possibility of toxic, excess consumption of essential amino acids needs to be looked at critically. The present study shows that in relation to lean tissue growth rehabilitation may be more effectively achieved on the HP formula and therefore, the possibility that this group experienced greater metabolic stress or other adverse effects needs to be explored with some care in greater detail.

	ACKNOWLEDGMENTS

 
We thank Akil G. Jackson for technical assistance.

	FOOTNOTES

 
¹ Supported by a research grant from the Nestlé Foundation.

³ Abbreviations used: ECG, energy cost of growth; EI, energy intake; EE, energy expenditure; H/A, height for age; HP, higher protein formula infant feed, unmodified commercial infant formula; LP, lower protein formula, modified commercial infant formula; MAL, stage of rehabilitation of a malnourished child; REC, stage after recovery of a malnourished child; RWG, stage of rapid weight gain of a malnourished child; W/A, weight for age; W/H, weight for height; relative to the median for age or height.

Manuscript received May 11, 1998. Initial review completed August 31, 1998. Revision accepted February 8, 1999.

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