The current goal of nutritional management of preterm infants is to provide sufficient nutrients to allow resumption of growth and accretion of nutrients at intrauterine rates. Currently available preterm formulas, when fed at energy levels of 120-130 kcal · kg-1 · d-1, promote weight gain and support calcium and phosphorus retention rates at or above intrauterine accretion rates (1). However, the evidence from nutrient balance studies suggests that these formulas may promote deposition of considerably more body fat than occurs in utero (2-5). While increased fat deposition may improve thermoregulation and have other benefits for prematurely born infants, excess fat gain also might predispose infants to later obesity and its associated health problems (6). Thus, it is important to determine whether rates of fat-free mass (FFM) and fat mass (FM) deposition proportional to those of the fetus can be achieved without adverse affects on the rate of weight gain.
The studies of Kashyap et al. (5,7,8), summarized by Heird (9), show that nitrogen retention is related directly to protein intake, up to a protein intake of 3.6 g/kg-1/d-1, whereas fat deposition is related primarily to energy intake. Thus, the composition of newly deposited tissue appears to be a function of the relative proportions of protein and energy intake. If so, one strategy to decrease the ratio of FM:FFM deposition, which in most studies of formula-fed preterm infants exceeds the intrauterine rate, is to increase the protein:energy (P:E) ratio of the intake. However, higher concentrations of protein in preterm formulas may affect the absorption and utilization of other nutrients. In adults and animals, for example, high protein intakes have been shown to increase renal calcium loss (10). Although there is no evidence in preterm infants that protein intakes in the range of 3-4 g/kg-1/d-1 affect urinary calcium losses (7,11) and although high-protein diets have generally been shown to enhance the absorption of copper and zinc because of the formation of copper or zinc-amino complexes (12), studies addressing these issues are limited.
The nutrient retention data reported here were obtained as part of a larger study to test whether accretion rates of fat and fat-free mass closer to those of the fetus can be achieved by feeding a preterm infant formula with a higher P:E ratio. We hypothesized that a higher P:E formula would promote not only a higher retention rate of nitrogen but also higher retention rates of minerals present in lean tissue.
MATERIALS AND METHODS
Healthy, low-birth-weight (LBW) preterm infants were assigned randomly to be fed isocalorically for 3 weeks with one of two preterm formulas differing only in their P:E ratio (2.6 g/100 kcal or 3.2 g/100 kcal). As part of a larger study, we present a descriptive analysis of 72-h energy and nutrient balance studies performed in a subset of the subjects (15/30) at the end of the 3-week study period.
Preterm infants admitted to the neonatal nurseries of Texas Children's Hospital were selected on the basis of following criteria: (a) 28-32 weeks gestation, determined by either maternal dates or early ultrasound and confirmed by physical exam; (b) birth weight between the 10th and 90th percentiles (13); (c) absence of major congenital anomalies; (d) absence of ventilatory requirement at the time of enrollment; and (e) tolerance of full enteral feeds (150 ml/kg-1/d-1) by 4 weeks of age.
The 15 infants enrolled for balance studies were randomly assigned to study groups. The group assigned to the formula with the lower P:E ratio (n = 7) consisted of three boys and four girls, of whom four were Caucasian, two were black, and one was Asian. Mean (±SD) birth weight and gestational age, respectively, were 1411 ± 87 g and 29.8 ± 0.9 wk. The group assigned to the higher P:E ratio formula (n = 8) consisted of six boys and two girls; two were Caucasian, two were Hispanic, and four were black. Mean birth weight and gestational age, respectively, were 1268 ± 142 g and 29.1 ± 1.1 wk.
Once enrolled, the infants were transferred to the Neonatal Clinical Research Unit of Texas Children's Hospital. Weight was measured daily with an electronic scale (Sartorius, Gottingen, Germany). Length and head circumference were measured weekly with a pediatric length board (Holtain Ltd., Crymych, U.K.) and a metal measuring tape, respectively. Serum calcium, phosphorus, alkaline phosphatase, albumin, and prealbumin as well as blood urea nitrogen concentrations were measured just before and at the end of the 3-week study period.
Enteral feeding with a standard preterm formula was initiated at the discretion of the attending physician. Volume was advanced as tolerated to a total volume of 150 ml/kg-1/d-1. Once the desired intake (150 ml/kg-1/d-1) was achieved, the infant was assigned randomly to one of two formulas: a commercially available preterm formula (Similac Special Care; Ross Products Division, Abbott Laboratories, Columbus, OH, U.S.A.) providing 2.6 g protein/100 kcal or the same formula with 3.2 g protein/100 kcal. The latter formula was specially formulated for the study by Ross Products Division, Abbott Laboratories. Formula intake was controlled at 120 kcal/kg-1/d-1 throughout the study. The formulas had similar concentrations of calcium (1.46 vs. 1.51 g/L in the lower and higher P:E preterm formulas, respectively), phosphorus (0.96 vs. 0.96 g/L), magnesium (0.12 vs. 0.14 g/L), sodium (0.35 vs. 0.37 g/L), potassium (1.06 vs. 1.28 g/L), copper (2.6 vs. 2.1 mg/L), iron (5.7 vs. 5.7 mg/L), and zinc (13.9 vs. 13.0 mg/L). All infants received 400 IU of vitamin D daily in addition to the 180 IU/kg-1/d-1 received from formula. All but one received an elemental iron supplement of 2 mg/kg-1/d-1.
After 3 weeks of the assigned formula, a 72-h energy and nutrient balance determination was performed in the infant's incubator under thermoneutral conditions. Infants were fed every 3 h by orogastric tube or by nipple with a Volu-Feed (Ross Products Division, Abbott Laboratories). All formula was weighed on a Sartorius scale and shaken prior to feeding. An aliquot of each formula lot was saved for analysis. Vitamin and elemental iron preparations were analyzed for mineral content and accounted for in the balance. A preweighed cotton wipe was placed under the infant's head during the feeding and interfeeding interval to catch any regurgitation. Urine was collected for three consecutive 24-h periods using plastic urine bags adherent to stoma adhesive placed on the perineum. The bags were connected by acid-washed tubing to a roller pump that allowed collection of the urine into acid-washed plastic bottles containing 3 ml 6N HCl. The urine was collected on ice and refrigerated until the end of the balance period, when an aliquot was frozen at -20°C. Stools were collected between carmine red markers (100 mg/kg) given 72 h apart onto 100% cotton wipes placed in the diaper. When the urine collection was complete, urine bags were placed on the buttocks to collect the remaining stool. Stools were frozen at -20°C until the end of the balance period, at which time they were transferred to a -70°C freezer. All samples were handled with powder-free plastic gloves to avoid trace mineral contamination.
Total energy expenditure was measured for 6 h with a whole body respiration calorimeter/incubator (14). Measurements included all sleep and awake states, since the infant remained in the calorimeter throughout the 6 h with only minor interruptions for nursing care and feeding. The temperature in the calorimeter was set at the same level as the infant's incubator. Heart rate (90601A Space Labs, Redmond, WA, U.S.A.) and skin temperature at the axilla, back, and foot (thermistor probes 729 and 709A, Yellow Springs Instruments, Yellow Springs, OH, U.S.A.) were monitored continuously. Daily energy expenditure was taken as four times the energy expended in 6 h (15).
Net nutrient absorption was calculated as the difference between dietary intake of the nutrients, including vitamin and iron medications, and their fecal excretion during the 72-h balance period. Net nutrient retention was calculated as the difference between dietary intake and the sum of fecal and urinary excretion. Dermal losses were not taken into account. Metabolizable energy was defined as the difference between gross energy intake and the sum of energy losses in urine and stool. Energy storage was computed as metabolizable energy minus total daily expenditure. Energy storage as protein (nitrogen times 6.25) was estimated from protein retention at 5.4 kcal/g protein (2). Fat storage was computed from nonprotein energy storage divided by 9.3 kcal/g.
After the wipes was soaked overnight in 0.1 N HCl, feces were extracted from the contaminated portion of the wipes with a laboratory blender (Stomacher 80, Seward Medical, London, U.K.). If any fecal matter was left, the wipes were resoaked in 0.1N HCl and the procedure was repeated. The fecal-acid extract was transferred to an acid-washed container and freeze-dried to a smaller volume. The efficacy of the fecal extraction procedure was verified with a series of recovery studies on a fecal homogenate of known nitrogen, fat, and mineral content. The fecal homogenate was applied to the cotton wipes and then extracted and analyzed. Blank wipes were also processed. Mean recovery of fecal nitrogen, fat, and minerals ranged from 94 to 103%.
Formula, urine, and the fecal extracts were analyzed for energy content by bomb calorimetry (Parr Instruments, Moline, IL, U.S.A.), for fat by a modification of the Jeejeebhoy method (16), and for nitrogen by the Kjeldahl method (Kjeltec automatic analyzer 1030, Tecator, Hoganas, Sweden).
For mineral analysis, the formula and fecal extracts were dry-ashed in a muffle furnace (30400 Thermolyne Corp., Dubuque, IA, U.S.A.) at 440°C for 14 h. For sodium and potassium analyses, microwave digestion was used (MDS2000, CEM Corp., Matthews, NC, U.S.A.). Reference controls, Lab-trol (American Dade, Miami, FL, U.S.A.), U.C.C. and SeraChem (Fisher Scientific Orangeburg, NY, U.S.A.), and water blanks were subjected to the ashing procedure. Urine was analyzed directly.
Calcium, magnesium, sodium, potassium, and zinc concentrations were determined by atomic absorption spectrophotometry (3030B, Perkin Elmer Corp., Norwalk, CT, U.S.A.). Copper concentration was measured by graphite furnace atomic absorption spectrophotometry (Zeeman 3030, Perkin-Elmer Corp.). Phosphorus concentration was determined by a modification of the Fiske method (17).
Serum calcium, phosphorus, alkaline phosphatase, albumin, and prealbumin concentrations, as well as blood urea nitrogen concentration, were determined by automatic analyzer techniques (COBAS FARA II: Roche Diagnostic Systems, Inc., Montclair, NJ, U.S.A.).
Minitab Statistical Software (release 10.5X, 1995, Minitab Inc., State College, PA, U.S.A.) was used for data reduction and statistical analysis. Student's t tests were used to compare variables between the two groups. Correlations among nutrient balance indices were determined to assess possible interactions among nutrients. Analysis of variance with repeated measures was used to test for group (lower vs. higher P:E formula) and time (before and after the study interval) differences in biochemical indices (BMDP2V, 1993, BMDP Statistical Software, Inc., Los Angeles, CA, U.S.A.). Data are expressed as mean ± SD. Results were considered significant at p < 0.05.
The study was approved by the Institutional Review Boards of Baylor College of Medicine and Affiliated Hospitals. Informed consent was obtained from at least one parent of each infant prior to enrollment.
At the time of the nutrient balance study, the two groups of infants did not differ in postnatal age, weight, length, head circumference, or growth rate (Table 1). The mean rate of weight gain of both groups during the study period exceeded the intrauterine rate of gain (1).
Serum Biochemical Indices
Mean serum calcium concentrations differed significantly between groups at the onset as well as the end of the study (p = 0.04) (Table 2). Phosphorus and alkaline phosphatase values did not differ significantly before or after the period of study. Serum albumin concentration was significantly lower in the lower P:E formula group before as well as at the end of the study period (p = 0.05); albumin concentration did not change over the 3 weeks of study in either group. There were no differences in blood urea nitrogen concentration between groups at the beginning of the controlled feeding period, but after 3 weeks of the assigned formula, blood urea nitrogen concentration was significantly higher in the higher P:E formula group (p = 0.03).
Despite the higher intake and net absorption of nitrogen in the higher P:E group, net nitrogen retention did not differ significantly between groups (Table 3). Thus, the percentage of nitrogen intake retained by the higher P:E group was less than that retained by the lower P:E group. This, of course, was secondary to the higher urinary nitrogen losses of the higher P:E group. The absolute amount of nitrogen absorbed was positively correlated with nitrogen intake (r = 0.91; p = 0.001), but not the amount retained. The percentage of nitrogen retained was negatively correlated with nitrogen intake (r = -0.59; p = 0.02). Nitrogen retention was not correlated significantly with the retention of other nutrients or with serum albumin concentrations, serum prealbumin concentration, or blood urea nitrogen.
Energy and Fat Balances
Despite the somewhat higher fat intake of the higher P:E group, gross energy intake and metabolizable energy intake of the two groups did not differ significantly (Tables 3 and 4). Rates of energy expenditure also did not differ between groups, nor did energy stored as protein or fat.
Calcium, Phosphorus, and Magnesium Balances
There were no differences between feeding groups in intake, absorption, or retention of either calcium or phosphorus (Table 5). The higher magnesium intake of the higher P:E group was associated with greater fecal losses, resulting in similar net rates of absorption and retention. There were no significant correlations between nitrogen intake and calcium, magnesium, or phosphorus retention. Calcium retention was positively associated with the retention of phosphorus, magnesium, potassium, copper, and zinc (r = 0.56-0.82, p = 0.03-0.001). Phosphorus retention was positively correlated with the retention of magnesium, copper, and zinc (r = 0.73-0.87, p = 0.003-0.001). Magnesium retention was positively correlated with copper and zinc retention (r = 0.72-0.76, p = 0.003-0.001). No significant correlations were detected between the retention of calcium, phosphorus, or magnesium and serum concentrations of calcium, phosphorus, or alkaline phosphatase.
Sodium and Potassium Balances
Mean net absorption of both sodium and potassium was greater than 95% and did not differ between groups. Because of the higher potassium content of the higher P:E formula, the intake and net absorption of potassium were greater in this group; however, net retention did not differ between groups. Nitrogen intake was not significantly correlated with retention of sodium or potassium.
Copper and Zinc Balances
Neither intake, absorption, or retention of copper or zinc differed between groups (Table 6); nor was the retention of these minerals related to nitrogen intake. Copper retention (r = 0.70, p = 0.004) was positively correlated with zinc retention.
Contrary to our hypothesis, the above data demonstrate that the higher P:E preterm formula provided no apparent benefit in terms of nitrogen or lean tissue accretion in these preterm infants, who were born at 27-31 weeks of gestation. Although intake and absorption of nitrogen were higher in the higher P:E formula group, renal excretion also was higher; therefore, nitrogen retention did not differ between the two groups. Nitrogen retention rates observed for both groups were slightly higher than the fetal accretion rate of 295 mg/kg-1/d-1 at 32-33 weeks of gestation (18).
As expected, rates of fat accretion (2.0-2.2 g/kg-1/d-1) did not differ between groups. However, contrary to the findings of other studies (2-5), rates of fat accretion were only minimally higher than the fetal rate of 1.8 g/kg-1/d-1. The ratio of fat to lean tissue accretion observed in both groups (0.14 and 0.12, respectively) approach the ratio of 0.15 estimated for the reference fetus of comparable gestational age (18). The difference between the findings reported here and those of other studies is attributable to the comparatively higher rates of energy expenditure observed by us versus the 24-h rates observed by most other investigators (76 vs. 56-78 kcal/kg-1/d-1) (15,19-24). This highlights the difficulty of predicting rates of fat accretion from energy balance studies. An error in energy balance of 9.3 kcal/kg-1/d-1 results in an error in the estimated rate of fat accretion of 1 g/kg-1/d-1, which in this study translates into a 50% error in fat accretion. Clearly, body composition studies are needed to clarify the relationship between nutrient intake and composition of weight gain in preterm infants.
On the basis of previous studies of protein intake in preterm infants, we expected that a major proportion of the difference in protein intake between groups would be retained. Rather, despite a difference in mean nitrogen intake between groups of almost 100 mg/kg-1/d-1, the mean difference in nitrogen retention between groups was <20 mg/kg-1/d-1. The fact that this small difference was not statistically significant can possibly be attributed to limited sample size. Regardless of statistical significance, however, the observed difference represents a difference in protein deposition of only 0.12 g/kg-1/d-1, which is of insufficient clinical significance to warrant a study of the sample size required to detect a statistically significant difference (i.e., 40 infants in each group).
Serum albumin and prealbumin concentrations in the two groups did not differ after 3 weeks on the study formulas. However, the mean blood urea nitrogen concentration of the higher P:E group was higher than that of the lower P:E group. This suggests that the higher protein intake was not as completely utilized as the lower protein intake. Possible reasons for the poorer utilization of the higher protein intake include a difference in the clinical characteristics of the two groups, limited intake of another nutrient (i.e., intracellular minerals) required for optimal protein utilization, or a limited concomitant energy intake for the level of protein intake.
There was no indication that the clinical characteristics of the two groups differed. Both groups were clinically stable and grew at reasonable rates during the study. It also is unlikely that either group received an inadequate intake of any mineral required for optimal protein utilization. As discussed below, rates of retention of all minerals equalled or exceeded intrauterine accretion rates. Furthermore, the ratios of the retention of phosphorus, magnesium, sodium, and potassium to the retention of nitrogen were similar in both groups (0.26, 0.018, 0.080, and 0.13, respectively) and similar to those of the developing fetus (18). Thus, the most likely reason for the lower utilization of the higher protein intake is that the concomitant energy intake was limiting with respect to utilization of this protein intake.
The effect of concomitant energy intake on protein utilization of formula-fed LBW infants has not been studied extensively. Kashyap et al. (7) found only a small (statistically insignificant) difference in nitrogen retention between infants receiving a protein intake of 3.5-3.6 g/kg-1/d-1 and a concomitant energy intake of 149 vs. 115 kcal/kg-1/d-1 (77.5 vs. 74.4% of intake, respectively). However, in a subsequent study (8), these investigators found that infants receiving a slightly higher protein intake (3.8-3.9 g/kg-1/d-1) retained significantly more nitrogen with a concomitant energy intake of 142 vs. 120 kcal/kg-1/d-1 (77.3 vs. 70.4% of intake, respectively). In both studies, the higher energy intakes were accompanied by greater retention of energy, most likely in the form of body fat as indicated by the significantly greater skinfold thicknesses.
We did not expect the higher protein intake in the present study (3.7 g/kg-1/d-1) to be retained as completely at the same energy intake as the lower protein intake (3.1 g/kg-1/d-1). However, on the basis of the relationship between urinary nitrogen excretion (Nu) and the P:E ratio of the intake in 185 infants studied by Kashyap et al. (25), ie. Nu = 56.9 P:E - 51.1, we expected urinary nitrogen excretion in the higher P:E formula group to be ≈34 mg/kg-1/d-1 greater, on average, than that in the lower P:E formula group. Instead, urinary nitrogen excretion was 63 mg/kg-1/d-1 higher. While this amount is within the confidence intervals of the Kashyap et al. equation (25), it was of sufficient magnitude to result in a virtual lack of difference in nitrogen retention between the two groups. If this lack of difference is secondary to inadequate concomitant energy intake, as seems likely, it will not be possible to increase the rate of lean tissue accretion substantially by increasing only protein intake.
As stated above, retention rates of all minerals equalled or exceeded estimated intrauterine accretion rates (1,18,26) in both groups of infants. Calcium absorption and retention rates in both groups of infants are within the range of values reported by others in formula-fed LBW infants, i.e., net fractional retention rates of calcium ranging from 34 to 74% (11,27-33). There was concern that increased urinary sulfate resulting from protein catabolism on the higher P:E formula would interfere with renal calcium reabsorption, as seen in adults and animals (10). In this study, urinary excretion of calcium did not differ between groups. In another study to evaluate the effect of varying protein intake on renal calcium excretion in preterm infants, higher-protein formulas (3 and 2.7 vs. 2.2 g/100 kcal) resulted in lower urinary excretion of calcium (34). The assumed mechanism was enhanced growth and bone mineralization in those fed the higher protein intakes and, hence, a lower renal excretion of calcium. Absorption and retention of phosphorus and magnesium also are consistent with other reports (32,35). Trace mineral balances were all positive, in contrast to markedly negative balances for iron, copper, and zinc observed in term infants during the neonatal period (36) and in preterm infants during the first few days of life (37).
In summary, these nutrient retention data show that a preterm infant formula with a P:E ratio of 3.2 g/100 kcal provides no apparent benefit over a formula with a P:E ratio of 2.6 g/100 kcal in terms of lean tissue accretion. Contrary to the findings of other studies (2-5), the rates of fat accretion were only minimally higher than the fetal rate, resulting in a proportion of fat to lean tissue accretion close to the intrauterine ratio. Studies of actual changes in body composition of preterm infants incident to different diets are needed to resolve the discrepant findings from nutrient balance studies.
Acknowledgment: This work is a publication of the USDA/ARS Children's Nutrition Research Center, Department of Pediatrics, Baylor College of Medicine, Houston, TX and has been funded in part with federal funds from the U.S. Department of Agriculture, Agricultural Research Service, under Cooperative Agreement Number 58-6250-1-003. The contents of this publication do not necessarily reflect the views or policies of the U.S. Department of Agriculture, nor does mention of trade names, commercial products, or organizations imply endorsement by the U.S. government. This work was supported also by National Institutes of Health (M01 RR-00188-30) and by the generous contribution of formula by Ross Products Division, Abbott Laboratories.
The authors thank L. Schwartz for nursing supervision; C. Boutte and M. Puyau for technical support; K. Fraley for data management; L. Loddeke for editorial review; and I. Tapper of manuscript preparation. Written permission has been obtained from all persons named in the Acknowledgment.
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