Protein Metabolism in Human Neonates: Nitrogen-Balance ......Body protein metabolism is more intense in species of small mammals than in larger ones, 485 . 486 P. B. Pencharz et al.

Clinical Science and Molecular Medicine (1977) 52,485498.

Protein metabolism in human neonates: nitrogen-balance studies, estimated obligatory losses of

nitrogen and whole-body turnover of nitrogen

P. B. PENCHARZ,* W. P. STEFFEE,? W. COCHRAN, N. S. SCRIMSHAW,

Department of Nutrition and Food Science, Massachusetts Institute of Technology, Cambridge, Massachusetts, U.S.A., and Boston Hospital for Women, Boston, Massachusetts, U.S.A.

w. M. RAND AND V. R. YOUNG

(Received 3 March 1976; accepted 30 December 1976)

SummarY

1. Aspects of nitrogen metabolism in the human neonate were assessed in one full-term infant and six premature infants by means of nitrogen-balance measurements, estimates of obligatory nitrogen losses and determinations of whole-body nitrogen turnover.

2. Our data indicate that the mean protein requirement for maintenance is 1.1 g of protein day-' kg-' and that 3.8 g of protein day-' kg-' should be sufficient for adequate growth in healthy premature babies. 3. The mean obligatory urinary, faecal and

total nitrogen losses were estimated to be 24, 106 and 145 mg day-' kg-' respectively. These figures are compared with published values for older infants, and the possible metabolic basis for changes in nitrogen losses during growth and development is discussed. 4. Mean values for whole-body protein

synthesis and breakdown were 26.3 k 7.0 and 23.8 +7-4 g of protein day-' kg-I respectively. Dietary nitrogen intake accounted for 6 1 8 % of the nitrogen flux through the metabolic pool; urea excretion accounted for 2% of the nitrogem flux.

* Present address: The Montreal Children's Hospital, Department of Metabolism, Montreal, Que. H3H IP3, Canada.

t Present address: Boston University Medical Center, 75 East Newton Street, Boston, Mass. 02118, U S A .

Correspondence: Publications Unit, 16-334A, Depart- ment of Nutrition and Food Science, Massachusetts Institute of Technology, Cambridge, Mass. 02139, U S A .

5. The net protein gain, estimated from nitrogen-balance data, accounted for 9.6% of total daily protein synthesis.

6. These results are discussed in relation to published estimates of whole-body protein synthesis and breakdown at various ages. Their possible significance in the assessment of a 'maintenance' requirement for protein and amino acids during the period of rapid growth and development is also considered.

Key words: amino acids, growth, neonates, nitrogen balance, protein.

Introduction

Relatively few studies have examined the human neonate's requirement for dietary protein and the characteristics of its protein metabolism (Davidson, Levine, Gauer & Dann, 1967; Babson & Bramhall, 1969; Nicholson, 1970; Irwin & Hegsted, 1971; Raiha, 1974). Although the protein needs of premature babies may be higher than those for full-term infants, this greater requirement is neither well characterized nor explained metabolically. For these reasons, we have carried out a series of studies in premature infants within 1-45 days after their births, in order to characterize this group's nitrogen metabolism by measurements of nitrogen balance and estimates of obligatory nitrogen losses.

Body protein metabolism is more intense in species of small mammals than in larger ones,

485

486 P. B. Pencharz et al.

including man (Munro, 1969). Furthermore, it decreases with normal growth and development (Waterlow & Stephen, 1967; Picou & Taylor- Roberts, 1969; Steffee, Goldsmith, Pencharz, Scrimshaw & Young, 1976). As part of the present study, we estimated the rate of body nitrogen turnover in premature neonates, using the approach proposed by Picou & Taylor- Roberts (1969). We have previously applied this method in our studies on young adults (Steffee et al., 1976) and on elderly subjects (Winterer, Steffee, Perera, Uauy, Scrimshaw & Young, 1976). Our findings show that the intensity of nitrogen metabolism, per unit of body weight, is approximately eight times higher in neonates than in young adult subjects.

Methods

Subjects and diet One full-term (C.C.) and six premature in-

fants were studied; Table 1 presents the anthro- pometric and gestational characteristics of these seven infants. One baby (S.H.) was studied on three different occasions and two (L.C. and B.M.) were each studied twice (L.C.l, L.C.2; B.M.l, B.M.,). We have excluded the nitrogen-balance data from the first study with infant B.M. because this child received an exchange transfusion 11 h after the start of the balance period.

The birth weights ranged from 1120 to 1758 g for the premature babies, and their gestational ages were estimated to be 30-36 weeks.

Infant J.B. was small for gestational age (Usher, McLean & Scott, 1966). Infant C.C., who was less than 2 days old, lost weight during the study; this weight loss was probably caused by a change in body water content (Smith, 1946), since the baby was in positive nitrogen balance at the time of the study.

All the babies except M.N. consumed a commercial infant milk formula (Similac) ; infant M.N. was fed with expressed human breast milk. In addition, during the first two studies with infant S.H., 0.5 ml of medium- chain triglyceride was included in each feeding. All babies were fed every 3 h throughout the study period, except S.H. who was fed at 2 h intervals during the first study.

As shown in Table 2, the babies' total protein intakes varied from 1.5 to 4.4 g day-' kg-l and their energy intakes varied from 255 to 732 kJ day-' kg-l, equal to approximately 170 kJ/g of consumed protein (except for infant S.H., who received the medium-chain triglyceride supplement ; his energy intakes were 230 and 194 kJ/g of protein during the first and second nitrogen-balance studies respectively).

Procedures The experiments received the administrative

approval of the MIT Committee on the Use of Humans as Experimental Subjects, the Executive and Policy Committees of the MIT Clinical Research Center and the Research Advisory Committee of the Boston Hospital for Women. Written informed consent was obtained from

TABLE 1. Anthroponietric and gestational data on secen neonates studied for characteristics of body nitrogen metabolism Surface area was calculated from Wt.0'425 x Ht.0.725 x 71.84, where Wt. = weight in kg and Ht. = height in cm

(surface area cm2).

Subject and Birth wt. Age during Birth Surface Wt. gain study no. wt. during study study length area Gestation during study

(B) (g) (cm) (m2) (weeks) (g day-' kg-')

w . c . 1 J.B.1 M.N.1 L.C.1 L.C.2 B.M.l B.M.2 c.c.1

S.H.2 S.H.,

S.H.S

1729 1503 1129

1758 1673 1673 3473 I120 I I20 1120

1758

2218 24 days 1998 24 days I836 14 days 1772 44 hours 2055 14 days 1612 29 hours 1807 13 days 3345 45 hours 1091 16 days 1495 31 days 1935 45 days

44.4 45.5 45.7 42.0 42.0 41.9 41.9 48.5 38.1 38.1 38.1

0157 0154 0.148 0.138 0.146 0.132 0140 0200 0.104 0.128 0.147

30 34-35 32-33 35-36 35-36 34 34 42 31 31 31

19.2 26.1 12.6 15.9 17.3

-11.7 15.6 - 2.7

10.9 19.1 22.0

TABLE

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3.4

3.7

2.8

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575

625

462

420

615

38 1

735

258

672

620

605

548

596

444

400

585

361

70 1

242

464

508

576

156

134 71

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143

122

166 62

76

113

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97

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166 97

95

100 42

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253

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260

127

379

422 73

27

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71

66

45

54

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63

68

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37

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the parents, and permission was obtained from the infants’ pediatricians.

The technique used to determine nitrogen balance was a modification of that described by Hepner & Lubchenco (1960). Each nitrogen- balance period lasted 30-36 h. Plastic bags for urine were attached to the skin around the baby’s anus in order to collect each stool separately. After a stool had passed, the bag was removed and the faeces were wiped from the infant’s skin with cotton balls, which were included with the stools for analysis. Use of Carmine Red as a faecal marker facilitated the preparation of pooled samples (Fomon, 1974b). The apparent nitrogen balance was calculated as the difference between nitrogen intake and the sum of faecal and urinary nitrogen losses. We corrected this calculation for integumental and miscellaneous losses of nitrogen, as discussed below, in order to estimate the protein requirements for nitrogen equilibrium.

Gestational age was assessed in two ways: from an evaluation of (a) the infant’s anthropo- metric characteristics (length, weight and head circumference) and (b) his external characteristics, as described by Usher et al. (1966). All babies were examined for gestational age by one of us and then independently by the baby’s pediatrician, with essentially the same results. When a discrepancy existed between the clinical assessment and the obstetrical data, the clinical evaluation was used to estimate gestational age. We measured each infant’s body length from crown to heel, ensuring full extension without pelvic tilt. All body weights were recorded daily between 07.30 and 08.00 hours. Each infant’s weight gain was calculated as the weight change over a 3 days period, including the period of nitrogen-balance measurement.

Experinrental model To study dynamic aspects of whole-body

nitrogen metabolism, we modified slightly the approach described by Picou & Taylor- Roberts (1969), which involves the administration of [ 5N]glycine at a constant rate in order to achieve a steady state of 15N enrichment in the metabolic nitrogen pool. This method overcomes some of the difficulties (e.g. Water- low, 1969) inherent in the single-isotope-dose method of San Pietro & Rittenberg (1953).

The Picou & Taylor-Roberts method allows estimates to be made of the nitrogen flux (Q) through the metabolic pool, i.e. the nitrogen disposal rate (Shipley & Clark, 1972), and the rates of whole-body protein synthesis (S) and breakdown (C). The assumptions inherent in this approach to the study of whole-body protein turnover have been discussed and tested experimentally (Picou & Taylor-Roberts, 1969; Steffee et al., 1976).

Isotope administration We obtained [15N]glycine (95 atoms %

excess) from the Stohler Chemical Corp. (Waltham, Mass., U.S.A.). The enrichment of the purchased material was confirmed by mass- spectrographic analysis. In all of the studies, the [15N]glycine was administered at a known rate to supply about 0.6 mg of 15N 24 h-’ kg - l body weight.

Picou & Taylor-Roberts (1969) administered the labelled amino acid by constant intravenous or intragastric infusion. In our studies with healthy young adults given an adequate diet (Steffee et al., 1976), we found that [15N]-

glycine given orally every 3 h for 60 h allowed a satisfactory concentration of urinary [ l 5N]urea to be obtained. Furthermore, in the same study, we noted that estimates of body nitrogen flux were similar whether the subjects were given [ 5N]glycine orally or intravenously. Therefore, in this study with neonates, the [‘5NN]glycine was mixed with the bottle feeds, which the infants received every 2 or 3 h for a total of 30-36 h.

Infant B.M. received an exchange transfusion (2 volumes) with whole blood 11 h after the start of the first study. The exchange was performed for hyperbilirubinaemia, secondary to her being the recipient in a twin-transfusion syndrome. For these reasons the nitrogen- balance data from this study were excluded. However, we determined that the exchange caused a nitrogen loss of 539 mg and that this loss was mainly associated with erythrocytes. In addition, the time-course of urinary [ * 5N]- urea enrichment resembled that found with the other infants. We decided therefore to include the B.M.I results on body nitrogen turnover in the present analysis.

Analyses Food, urine and faeces were analysed for

total nitrogen; in addition, urea and creatinines

Nitrogen metabolism in neonates 489

were measured in the urine (Young, Taylor, Rand & Scrimshaw, 1973). After pretreating the urine with Permutit (Folin & Bell, 1917), we isolated the urinary urea nitrogen by a modification of the Conway diffusion method (Hawk, Oser & Summerson, 1954). The I5N enrichment of urinary urea was determined with the aid of a dual-collector, isotope-ratio mass spectrometer (Vacumetrics model MS11, Waltham, Mass., U.S.A.) (Steffee et al., 1976). The 15N enrichments of the administered isotope and of faeces were determined after digestion of the faecal samples (Munro & Fleck, 1969) and steam-distillation of the resulting ammonia (Rittenberg, 1946). Samples for mass-spectrographic analysis were measured in duplicate. The reproducibility of measurements of excess I5N was within 5 % ; the mass- spectrographic analysis has been previously described (Steffee et al., 1976).

Data analysis We calculated the SN enrichment of urinary

urea according to the method described by Rittenberg (1946). Enrichment was corrected for background values that were determined from urine samples collected over a 3 h period preceding each tracer study.

The plateau value of 5N enrichment of urea was estimated by two methods. For the analysis of the whole-body nitrogen turnover results described below, plateau enrichment was calculated by averaging the values for the last three data points of each turnover study, provided they did not differ by more than 25%.

Because the urinary I5N urea enrichment, which occurred after the beginning of isotope administration, usually represented a smooth, curvilinear change, we also predicted, by the use of asymptotic regression analysis, that plateau enrichment would occur. The midpoint of each urine collection was used as the time for each sample. The least-squares method was used to fit the individual data sets to an ex- ponential equation: y = C (l-e-kL). Total- body nitrogen flux was calculated by estimating the asymptote, y (a) = C, as the plateau value. The fitting was carried out by means of a non-linear program of a computer program package (Dixon, 1973). The program uses a modified Gauss-Newton technique (Hartley, 1961; Jennrich & Sampson, 1968) to search until five successive iterations do not change the total error mean square by more than 0.001 %. In addition, the program calculates the standard deviation of the estimates.

Statistical evaluations were performed with the Student's t-test, regression and correlation analysis (Dixon & Massey, 1969).

Results

Nitrogen balance Table 2 summarizes the nitrogen-balance

data for the seven infants. These results were based on stool and urine collections made during a 30-36 h period of [lsN]glycine administration. All infants were in positive apparent nitrogen balance, with values ranging from +85 to +438mgday-'kg-'bodyweight

TABLE 3. An estimate of the protein content of weight gain as calculated from nitrogen-balance data in six neonates

Subject and Wt. gain N balance Protein content study no. (g day-, kg-') (mg day-' kg-I) of weight gain

(%)

w.c.1 19.2 J.B.1 26.1 M.N., 12.6

L.C.2 17.3 B.M., 15.6 S.H.1 10.9

L.C.1 15.9

S.H.2 19.1 S.H.3 22.0

Mean +SD 17.6+ 4.6

253 40 1 260 127 379 422 270 335 384

314+95

8.2 9.6

12.9 8.0

13.6 16.9 15.5 11.0 10.9

11*8+3.1

490

500

-- 400 m

O*m-

s 200-

x = loo-

v m' E - c 0

P. B. Pencharz et al.

C

-

I 1 I

TABLE 4. Results of regression analysis ofprotein intake on nitrogen balance, weight gain and ihe relationship between absorbed nitrogen and retained nitrogen

Regression Equation

(Y = a+ bx) s b r ~~

(1) Apparent N balance (y ) on N intake (x ) * y = - 131+0,86x 0.12 0.93

(3) Apparent N balance (y ) on N absorbed (x)* y = - 24.2+0.78x 0 9 8 (4) N balance ( y ) on N absorbed (x)* y = - 3 8 . 9 + 0 7 8 ~ 0.048 0.99 ( 5 ) Weight gain (y)? on protein intake (x)j y = - 8.2+ 7 . 5 ~ 2.22 0.77

(2) N balance ( y ) on N intake (x)* y = - 1 4 5 + 0 . 8 6 ~ 0 1 2 0 9 3 0.050

* x and y expressed as mg of N day-' kg-'. N+faecal N). N balance = apparent N balance

t y expressed as g day-' kg-'. x expressed as g of protein day-' kg-'.

Apparent N balance = N intake-(urinary - integumental N loss.

In order to estimate the protein requirements for nitrogen equilibrium, we needed to include sweat and other miscellaneous losses of nitrogen in our calculations. Because these losses have not been determined for infants, we used data obtained in adults. Based on a review of published data, the FAO/WHO (1973) Committee chose a value of 5 mg of nitrogen day-' kg-' to cover the integumental and other minor routes for nitrogen loss in young men. Assuming a surface area of 1-75 mz for a young adult subject, we can calculate the unmeasured nitrogen losses at about 200 mg/m2. Applying this figure on a surface-area basis, we have estimated the miscellaneous nitrogen losses for the seven infants (Table 2). From these calculations, mean integumental and other unmeasured nitrogen losses are estimated to be 15.2 mg day-' kg-' in premature infants.

The regression analysis indicates that a mean intake of 170 mg of nitrogen day-' kg-' is required to replace urinary, faecal and other losses. Hence, a protein intake (N x 6.25) of 1.06 g day-' kg-I would be required, on average, to compensate for the major and minor routes of body loss of nitrogen. The value for the y-intercept, 145 mg day- kg-', provides a prediction of the mean total obligatory nitrogen loss for the group studied (Table 4, eqn. 2). Finally, the slope of the regression line is an estimate of the efficiency of dietary nitrogen utilization, and this value was found to be 86% for the range of protein intake given to the seven infants.

We also obtained a significant positive relationship (r = 0.77; P i 0.01) between dietary protein intake (g day-' kg-') and body weight gain (g day-' kg-') (Fig. 2). The regression

24 T Nitrogen metabo

* /

/

FIG. 2. Relationship between protein intake and body weight gain in human neonates. The continuous line depicts the regression equation y = -8.2+7.5x, where y = weight gain (g day-' kg-') and x = protein intake (g day-' kg-I).

500 t

I I I I 0 200 400 600

h b e d N (mg day-' kg-') Fro. 3. Relationship between retained N and absorbed N in human neonates. The continuous line depicts the regression equation y = -38*9+078x, where y = N retention (mg of N day-' kg-') and x = N absorption (mg of N day-' kg-').

equation was y = -8*2+73x, where y = weight gain (g day-' kg-I) and x = protein intake (g day-I kg-I). Hence, according to these calculations, a baby must have a mean intake of 1.09 g of protein day-l kg-' for weight maintenance alone, and this value is comparable to that calculated for body nitrogen maintenance (Table 4, eqn. 5).

Finally, we examined the relationship of retained nitrogen to absorbed nitrogen (Fig. 3); these values showed a significant linear correlation (r = 0.99; P < 0.001). The y-intercept provides an estimate of mean obligatory urinary N plus integumental and other minor losses; it was determined to be 38.9 mg of N day kg-' (Table 4, eqn. 4). The slope of the regression line indicates that absorbed nitrogen is utilized

ilism in neonates 491

with an efficiency of 78%, or only slightly less than the value derived from the regression of nitrogen balance on total intake of nitrogen (Fig. 1).

Whole-body nitrogen turnover

Table 5 shows the ['5N]urea enrichment during the final phase of the I5N turnover period and also shows the plateau values of enrichment based on methods described earlier. Fig. 4 depicts, for a representative case, the predicted and observed change in I5N enrichment of urinary urea during a 36 h period of I5N administration. By means of regression analysis, we estimated a plateau (asymptote) value for [15N]urea enrichment for each tracer study (Table 5). Only a small fraction of the ingested lSN was excreted in the infants' stools (Table 5). In calculating parameters of body nitrogen metabolism, we have assumed that all of the administered dose entered the metabolic pool. For one infant (L.C.'), the time-course of [15N]urea enrichment appeared to change linearly throughout and, in this case, it was impossible to predict a plateau value.

There was a wide range of variability in the precision with which it was possible to predict the plateau value by regression analysis. The estimated standard deviations of the plateau ranged from 5 to 76%. We therefore decided not to use this procedure for calculation of nitrogen flux, but rather to estimate the plateau from the last three data points if they varied by no more than 25%. With this criterion, we were able to utilize data from eight of the eleven turnover studies; the mean difference in the last three data points in these eight subjects was 15% (range 3-24%). In the remaining three subjects, the differences were 95% (M.N.'), 42% (C.C.1) and 81 % (S.H.J.

Table 6 summarizes the estimates for body nitrogen flux and the rates of whole-body protein synthesis and breakdown for the seven neonates. The mean flux was equivalent to 181 mg of N h-' kg-', which can be compared with the mean value of 25.7 mg of N h-I kg-' for healthy young adults (Steffee et al., 1976). In the present series of studies we did not obtain a statistically significant change in the nitrogen flux with age, although the data do suggest a trend towards lower values with increasing age. The mean rates of protein (N x 6.25) synthesis

D


TABLE 5. ouiput in faeces, urinary urea enrichmeni and plateau values for urea ' N , obtained by visual inspection and by non-linear regression analysis, in seven neonates given

['5N]glycine during 30-36 h Urine collections were usually made every 3 h ; times given are the midpoint of each collection. Plateau I5N enrichment was calculated by determination of the mean value for the last three collection periods. If the values of the final three collections differed by more than 25%, a

plateau estimate was not made. The predictive method is described in the text.

10-3x Plateau [I5N]urea Subject Time of IOd3x I5N enrichment (atoms % excess) I5N in

and study sample (atoms % excess) faeces* no. (h) Inspected Predicted (%)

M.N.,

L.C.,

L.C.2

B.M. ,

U.M.2

C.C.,

S.H.,

S . H q

S.H.3

W.C., 0 21.04 23.79 29.84

J.B. , 0 27.95 30.82 34.02 0

16.58 19.75 31.75 0

28.46 31.12 34.21 0

29.25 32.16 34.83 0

28.15 31.79 34.40 0

29.12 32.75 35.12 0

2408 28.26 34.25 0

18.16 22.25 33.74 0

2846 31.62 34.25 0

28.96 32.12 34.61

0.1 11.8 11.7 13.7 - 0.8 15.1 15.2 15.5 0.7 5.0 3.9 7.6 1 .o

16.3 18.3 19.8

1.7 20.7 17.7 22.0 0.5 8.3 8.8

10.3

0.4 16.4 17.6 17.6

1.5 9.4 9.3

13.2 1.4 6.4 9.0

11.6 - 0.8 11.3 10.0 1 0 6

0.5 12.0 11.7 13.0

12.3 2 1.3+_ 2.7 4.0

16.1 42.7+ 31.0 0.4

- lZ.O+ 7.2 I .2

17.1 -

18.4 3 1 . 2 2 7.2 1.2

8.6 15.9+ 3.9 2.6

16.8 20.9+ 1.0 0.8

- 17.3+ 13.2 I .o

- 21.3f8.6 4.6

11.4 13.4k2.3 0.3

11.7 27.4+_ 8.2 0.7

* Expressed as the percentage of the administered dose that appeared in the faeces passed during the I5N tracer study.


["N]Glycine administroton ( h 1

FIG. 4. Time-course of I5N enrichment of urinary urea N after oral doses of [lsN]glycine given every 3 h to subject B.M.2: 0, predicted; 0, observed.

and breakdown were 26.3 and 23.8 g day-l kg-' respectively. These two values are approximately eight times the rates that occur in young adults (Steffee et al., 1976) and about four times the values reported by Picou 8t Taylor-Roberts (1969) for infants of about 1 year of age.

The entry of nitrogen from food into the metabolic pool accounted for 6-18% (mean 12%) of the total nitrogen flux; urea nitrogen excretion constituted about 2% of the total nitrogen flux through the metabolic pool.

The daily protein gain, as calculated from the nitrogen-balance data, amounted to 11.8%

of total body-weight gain (Table 3). Thus, from the net body protein gain and the total body protein synthesis rate, we estimate that on average the protein gain accounted for only 9.6% of total protein synthesis per day (Table 7). These data indicate that the greater propor- tion of protein synthesis and, thus, of amino acid utilization is associated in the neonate with a rapid turnover of existing protein, rather than with net protein synthesis.

The results in Table 6 reveal considerable individual variation in estimated rates of whole- body protein synthesis and breakdown. Because dietary protein intakes varied among the infants,cwe examined the relationships between protein and energy intakes and the parameters of whole-body nitrogen metabolism. For this purpose, the parameters were expressed per unit body weight and per unit surface area; Table 8 shows the correlation matrices. No statistically significant correlation existed between protein or energy intakes and the rates of synthesis, breakdown, nitrogen flux, or the fraction of nitrogen flux that was excreted as urea. However, both protein and energy intakes were correlated significantly with synthesis and catabolism when they were expressed as fractions of whole-body nitrogen flux, except for energy intake (kJ day-' kg-') in relation to the term S/Q.

Discussion

Nitrogen balance Nitrogen-balance determinations among any

age group are difficult to carry out with pre-

TABLE 6. Whole-body nitrogen flux and rates of whole-body protein synihesis and breakdown in five neonates

Subject and study

no.

W.C.' J.B., L.C.1 L.C.2 B.M.1 B.M.2 S.H.2 S.H.3 Mean f s ~

Whole-body

(g day-' kg-')

4.18 3.51 3.60 3.19 6.38 3.58 4.99 5.34

435* 1.12

N flux (Q) Whole-body protein

synthesis (S) (g day-' kg-')

Whole-body protein breakdown (C)

(g day-' kg-')

25-2 21.1 21.9 19.0 39.1 21.3 305 32.5

26.3f 1.0

22.1 18.2 20.0 16.3 37.6 18.0 28.0 29.8

23,8+ 7.4

494 P . B. Pencharz et al.

TABLE 7. Relationship between body protein gain and whole-body protein synthesis in five neonates

Subject Estimated (Protein gainlprotein and study protein gain* synthesis) x 100

no. (g day-' kg-')

W.C., 2.26 J.B.1 3.08 L.C.' 1.88 L.C.2 2.04 B.M.2 1.84 S.H.2 2.25 S.H., 2.60

Mean ~ S D 2.28+ 0.43

9.0 14.6 8.6

10.7 8.6 7.4 8.0

9.6k 2.4

* 11.8'A of weight gain was estimated to be protein (Table 3); protein gain = weight gain x 0.1 18.

TABLE 8 . Statistical correlations ( r ) in neonates between dietary protein and energy intake andparariieters of whole-body protein turnover expressedper unit body weight and per unit body surface area

Q = N flux; S = whole-body protein synthesis; C = whole-body protein breakdown. * P<O.O5; ** P< 0.01.

Parameter of whole-body N metabolism

Dietary intake Q S C sl Q Cl Q

(g day-' kg-') Protein (g day-' kg-') - 0.52 - 0.53 - 0.58 - 0,80* - 0'84** Energy (kJ day-' kg-') - 0.47 - 0.48 - 0.53 - 0.69 - 0.79*

(g day-' m-') Protein (g day-' m-2) - 0.49 -0.51 - 0.58 - 0.87** - 0.87** Energy (kJ day-' m-*J - 0.49 -0.51 - 0.58 - 0.82* - 0.85**

cision (Wallace, 1959; Fomon & Owen, 1962). This study had the additional limitation of the short nitrogen-balance periods, which were necessary b e a c e the.study was part of an experiment to assess dynamic aspects of nitrogen metabolism in the neonate.

Davidson et al. (1967), in a double-blind prospective study of over 400 low-birth-weight infants, concluded that a protein intake of 4 g of cow's milk protein day-' kg-I was sufficient for satisfactory growth, but that 2 g day-' kg- ' was not. If the mean optimum intrauterine weight gain is assumed to be 20 g day-' kg - (Lubchenco, Hansman, Dresler & Boyd, 1963; Usher & McLean, 1969), then our data show that a milk protein intake of 3.8 g day-' kg-1 should be sutlicient for adequate growth in premature babies. This estimate may be com-

pared with the FAO/WHO (1973) recom- mendation, based largely on the studies of Fomon (1974a), that 2.4 g day-' kg-I is adequate for the first 3 months of life in full- term babies.

Chan & Waterlow (1966), in their studies of 1-year-old infants who were recovering from protein-calorie malnutrition, examined the regression of nitrogen balance on nitrogen intake. They estimated the maintenance requirements to be 100 mg of nitrogen day- l kg-' for this age group; however, they did not adjust the balance data for unmeasured losses of nitrogen. Kaye, Caughey & McCrory (1954), studying a group of 4- to 12-month-old babies, determined that infants of this age require 110 mg of nitrogen day-' kg-' for apparent nitrogen maintenance.


Our two estimates of maintenance (g day - kg-'), one for nitrogen equilibrium (1.06 g) and the other for body-weight maintenance (1.09 g), are in close agreement. This similarity suggests that, in spite of a baby's changing body composition during the latter part of gestation and during its early neonatal period (Widdow- son, 1968), its requirements for maintenance of nitrogen may be adequately predicted by either method.

We can estimate the obligatory losses of nitrogen from the regression of nitrogen balance and nitrogen intake, assuming that it is valid to extrapolate to a protein-free intake for this purpose. Approached in this way, the total obligatory nitrogen loss (from urine, faeces and other routes) amounts to 145 mg day-' kg-' (Table 4, eqn. 2). This figure could be an underestimate of the total obligatory nitrogen losses in stool and urine if the slope of the nitrogen balance-response curve is less at intakes of protein that approach requirements than at much lower intakes. This phenomenon has been shown to occur in rats given diets of low quality protein (Said & Hegsted, 1969) and in adult human subjects (Inoue, Fujita & Niiyama, 1973; Young, 1975).

Snyderman, Boyer & Holt (1961) studied premature infants whose mean age was greater than that in our study and suggested that their nitrogen-balance data agreed with those of Waterlow & Wills (1960) obtained with 3- to 24-month-old infants. These latter workers estimated obligatory nitrogen losses to be 33 mg day-' kg-' for faeces and 37 mg day-' kg-' for urine, with a total obligatory loss of 70 mg of nitrogen day-' kg-I. Chan & Waterlow (1966) also used regression analysis to determine total obligatory loss of nitrogen in infants of about 1 year old. The loss amounted to 97 mg of nitrogen day-' kg-'. Fomon, Demaeyer & Owen (1965) gave protein- deficient diets to 4- to 6-month-old infants and then directly estimated the obligatory nitrogen losses to be 37 and 20 mg day-' kg-l for urine and faeces respectively. In all these cases, however, the investigators did not take into account integumental and miscellaneous nitrogen loss; moreover, the subjects were develop- mentally more advanced than the seven infants studied here. Our regression analysis estimated the com-

bined urinary sweat and miscellaneous nitrogen

losses to be 38.9 mg day-' kg-I (Table 4, eqn. 4). Assuming the mean losses in sweat and miscellaneous losses to be 15.2 mg of nitrogen day-' kg-' (Table 2), we calculated the obligatory urinary nitrogen loss at 23-7 mg day-' kg-I. This estimate comes to about two-thirds of the values obtained with older infants (Waterlow & Wills, 1960; Fomon et ul., 1965) and presumably reflects the high capacity of the neonate to conserve body nitrogen, as discussed by McCance & Strangeways (1954) and studied in the neonatal rat by Czajka- Narins, Miller & Browning (1973).

The obligatory faecal nitrogen loss, estimated from the regression analysis (Table 4, eqn. 2 and eqn. 4), has been calculated to be 106 mg day-' kg - I. This value is markedly higher than those reported for older infants (Fomon et al., 1965) and adults (Young & Scrimshaw, 1968; Calloway & Margen, 1971 ; Scrimshaw, Hussein, Murray, Rand & Young, 1972). One explana- tion for this higher loss may be that it reflects the immaturity of the gastrointestinal tract in infants at this early period of development (Younoszai, 1974).

Whole-body nitrogen turnover

The representation of body protein metabolism by a single metabolic nitrogen pool and a single tissue protein pool is a necessary over- simplification of the complex mechanisms of body protein metabolism. Thus the measures of whole-body nitrogen turnover shown here represent a weighted average for protein synthesis and breakdown rates in the various body organs. Our study of nitrogen metabolism is therefore analogous to the assessment of energy metabolism by measurement of the basal metabolic rate (Waterlow, 1970). The mean rates of protein synthesis and breakdown were found to be 26.3 and 23.8 g of protein day-' kg-' respectively for the neonates in this study.

A plateau of [15N]urea enrichment was achieved in infant J.B. 30-36 h after he received a tracer dose of [15N]glycine. However, owing to the lack of precision in predicting plateau values of [ 5N]urea enrichment (Table 5), we chose to follow the simpler method of determining plateau enrichment by inspection, which is the method followed by Picou & Taylor-Roberts (1969). Although we do not


have any data from turnover studies lasting longer than 30-36 h, we suspect that recycling of I5N from the body protein pool would probably become a serious problem if the turnover studies were significantly lengthened. From studies in rats, Aub & Waterlow (1970) calculated that return of isotope via tissue protein breakdown would result in about a 2% increase of isotope in the metabolic nitrogen pool after 6 h of continuous isotope infusion. Whole-body protein synthesis in their rats was approximately 30 g of protein day-' kg-', or of the same order as we have obtained in human neonates. A significant degree of isotope recycling would give rise to an underestimate in the whole-body nitrogen flux.

The mean rates of whole-body protein synthesis and breakdown determined from the mathematical prediction of plateau values of urinary [I5N]urea enrichment were 17.8 and 15-5 g of protein day-' kg-' respectively, or about 70% of the rates estimated by inspection of the ['sN]urea enrichment values.

To avoid some of the problems inherent in the measurement of whole-body protein synthesis rates by methods that depend on administration of a single dose of labelled amino acid, Waterlow and his collaborators refined the technique of constant isotope infusion (Waterlow, 1969). This general approach has been used with rats (Waterlow & Stephen, 1967, 1968), newborn lambs (SoltCsz, Joyce & Young, 1973), human infants (Picou & Taylor- Roberts, 1969) and adults (Waterlow, 1967; Steffee et al., 1976; O'Keefe, Sender & James, 1974) and elderly people (Young, Steffee, Pencharz, Winterer & Scrimshaw, 1975). Picou & Taylor-Roberts (1969) estimated the mean rate of total protein synthesis in infants (aged 10-20 months) to be about 6 g day-' kg-' body weight. This value was the same whether or not the infants had an adequate (1.2 g/kg) or high (5.2 g/kg) intake of protein.

Using the less satisfactory approach of San Pietro & Rittenberg (1953), Nicholson (1970) estimated rates of whole-body protein synthesis to be 11-15 g day-' kg-' in three premature infants (aged 29-68 days), or somewhat lower than our estimates. Because the rates of protein synthesis in neonates were apparently comparable with those for adults (when expressed on a body surface area basis), Nicholson (1970) speculated that the infant achieves rapid growth

by synthesizing protein with half-lives that are longer than those in an adult. Although we looked for a possible effect of age and weight upon protein synthesis and breakdown, our results only suggest a decline in nitrogen flux with age and increased body weight; the differences observed with age were not statistically significant.

However, it is noteworthy that our data may reveal an effect of a stressful stimulus on total body protein metabolism. Infant B.M.,, who underwent an exchange transfusion during the first study, exhibited the highest total nitrogen flux. The exchange was performed, however, in an isothermal environment, and, as judged from her general behaviour and her heart and respiratory rates, the infant maintained a stable condition during and after the exchange.

Our data indicate that only 9.6% of the protein synthesized by the premature infant appears as a net gain in body protein. Thus the change in total body protein content represents a relatively small fraction of the total daily protein synthesis. Similarly, Hommes, Drost, Geraets & Reijena (1975) have calculated that a full- term, 3-week-old baby needs only 2.2% of its total energy intake to cover the energy cost of its net tissue gain during normal growth. Thus it would seem that the highenergy and protein intakes required for normal growth are more a function of the high rates of tissue protein turnover than of growth per se. This fact may not be surprising, since the body rapidly utilizes ATP equivalents for protein synthesis and then dissipates the free energy during protein breakdown (Blaxter, 1971).

Finally, an assumption is implicit in com- putations of human protein and amino acid requirements by the factorial method (FAO/ WHO, 1973; El Lozy & Hegsted, 1975), i.e. that protein maintenance in the young is metabolically equivalent to maintenance in the adult. Our findings suggest that this assumption is not justified and that tissue protein maintenance in the young represents a more active state of protein metabolism than in an adult individual. If this inference is correct, our results may help to explain why the concentration of essential amino acids in dietary protein required for infants and children is higher than that apparently required by adults (FAO/WHO, 1973), even though the amino acid composition of the protein gained is probably similar to

Nitrogen metabolism in neonales 491

that of existing tissue protein (Bunce & King, 1969).

Acknowledgments We thank Mrs Joyce Sylvestre, Head Nurse of the Special Care Nursery, Boston Hospital for Women, for her help and that of her staff in carrying out these studies. This work was supported in part by NIH grant no. AM 15856.

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