Cholesterol is an important dietary component of human milk being an essential constituent of mammalian cell membranes and a metabolic precursor of steroid hormones and bile acids (1). Yet, cholesterol content of most American bovine-milk-based preterm formulas (PFs) is lower than that of human milk (less than 0.03 g/L compared with 0.12 to 0.18 g/L) (2,3). Although there are no recommendations on cholesterol requirements in premature neonates, cholesterol supplementation of preterm formulas could be of interest because it would tend to mimic the fat blend of human milk (4,5). Such a supplementation seems to be innocuous, but a prudent attitude should prevail.
Most European PFs are supplemented with long-chain polyunsaturated fatty acids derived from fish oils, algae, or egg phospholipids (3,6–9). When the latter source is used, it is derived from egg yolk that has a high cholesterol content (10). Consequently, these enriched PFs contain relatively high amounts of cholesterol (up to 0.30 g/L) (2) whose usage by the body in very young subjects is still unknown in detail.
One of the first steps involved in cholesterol metabolism and bile acid synthesis is hydroxylation of cholesterol at C-27 (11). This reaction is catalyzed by sterol 27-hydroxylase, which appears to be a mitochondrial cytochrome P-450 enzyme (CYP27). It has also been shown that CYP27 that is present in rat, rabbit, and human liver mitochondria, is the same enzyme that catalyzes 25-hydroxylation of vitamin D3 to yield 25-hydroxy-vitamin D3 (12). Two cell sites are available for hepatic vitamin D 25-hydroxylation. The first one, a high-affinity/low-capacity site, is located in the rough endoplasmic reticulum. The second one, a low-affinity/high-capacity site, is catalyzed by the mitochondrial CYP27 (12,13). Because cholesterol brought to the liver would possibly compete with vitamin D for CYP27, we made the assumption that dietary cholesterol supplementation could induce a decrease in plasma 25-hydroxy-vitamin D concentration. In a preliminary study, we observed that the mean plasma 25-hydroxy-vitamin D concentration was significantly lower in preterm infants fed a PF supplemented with polyunsaturated fatty acids derived from egg phospholipids (47 ± 22 nmol/L) than in infants fed a standard PF (77 nmol/L) (P < 0.001). Therefore, we performed a double-blind randomized study whose main objective was to evaluate the effect of dietary cholesterol on vitamin D status in preterm neonates.
Besides, biosynthesis of cholesterol in the liver is suppressed by dietary cholesterol. This is caused by depression of the biosynthesis of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase, the rate-limiting enzyme of cholesterol biosynthesis in the liver. Therefore, we monitored serum cholesterol concentrations as well as HMG-CoA reductase mRNA gene expression in an attempt to clarify the outcome of dietary cholesterol in very young subjects.
MATERIALS AND METHODS
A single tertiary neonatal center participated in the study (Edouard Herriot Hospital, University of Lyon). Only appropriate-for-gestational-age premature neonates were included after parental consent was obtained. Exclusion criteria were persistent oxygen needs at time of inclusion, major congenital malformations, breast-feeding after the second week of life, or absence of full enteral feeding at day 15 of life.
Indices of vitamin D metabolism were measured in preterm neonates fed preterm formulas with different cholesterol content, using a prospective, double-blind, randomized protocol. All subjects were previously fed banked human milk. At the end of the second week of life, human milk was changed for a PF whenever the mother was not able or did not want to breast feed her child. Thirty subjects (10 per group) were randomly assigned, by means of cards in sealed envelopes, to be fed one of the three experimental study formulas according to a pre-established randomization schedule. The macronutrient composition of these formulas was the same except for cholesterol: cholesterol concentration was lower than (PF1: < 0.03 g/L), similar to (PF2: 0.15 g/L) or higher than (PF3: 0.30 g/L) that of human milk (Table 1). These formulas were supplemented with long-chain polyunsaturated fatty acids from fish oils (PF1 and PF2) or egg yolk (PF3). The fat composition of PF1 and PF2 was identical, based on vegetable oil and fish oil, but free cholesterol was added in PF2 to get a final cholesterol concentration of 0.15 g/L. In PF3, all of the cholesterol was added as the egg yolk source of polyunsaturated fatty acids. The subjects were regularly fed these formulas from inclusion to the end of the study period, when they reached an equivalent age to theoretical term (i.e., 40 weeks post-conceptional age). All subjects received a daily oral vitamin D supplement (Uvesterol ADEC®, Crinex, Montrouge, France) according to a schedule previously established in our unit (1500 IU/d until 1500 g body weight, 1000 IU/d subsequently) to compensate for low cord-blood 25-hydroxy-vitamin D concentrations usually observed in France (14,15).
The main outcome variables were plasma 25 hydroxy-vitamin D (25OHD) and 1,25 dihydroxy-vitamin D (1,25OH2D) concentrations at the end of the study period (theoretical term). These concentrations were measured as previously described by Salle et al. (16). The secondary outcomes were serum calcium, serum alkaline phosphatase, and anthropometric data at theoretical term. Serum calcium and alkaline phosphatase were assayed by a biochemical procedure using commercial kits (Boehringer Mannheim, Germany). Additional variables were anthropometric data at birth, time of inclusion and discharge as well as milk intake, vitamin D intake, serum calcium, serum alkaline phosphatase, and plasma vitamin D metabolites (25OHD and 1,25OH2D) at time of inclusion.
To monitor cholesterol usage, HMG-CoA reductase gene expression and serum cholesterol were measured in a subgroup of 14 subjects (five PF1, five PF2, and four PF3 subjects) whose characteristics were not statistically different from those of the whole group. Because it has been shown that mononuclear cells' HMG-CoA reductase mRNA reflects that of liver cells (17), mononuclear cells from venous blood were immediately isolated by centrifugation of whole blood on a Ficoll gradient (18). The isolated cell population consisted of 85 to 95% lymphocytes. Total RNA was prepared as previously described by Boucher et al. (18). The number of HMG-CoA reductase gene mRNA copies was determined by competitive reverse transcriptase-polymerase chain reaction according to the method described by Powell et al. (19). The results were expressed as copy number per μg of total cell RNA. Serum concentrations of total cholesterol were measured by an enzymatic procedure using commercial kits (Boehringer Mannheim, Montreal, Canada) as previously described by Levy et al. (20).
All anthropometric variables (body weight, crown-heel length, and head circumference) were measured by the same person (BR).
We tested the null hypothesis that vitamin D metabolism is not influenced by dietary cholesterol supplementation using linear regression analysis with cholesterol content of formulas as independent variable. On the basis of preliminary observations, we have calculated that 7 subjects per feeding group were necessary to detect a 30 nmol/L difference in 25OHD concentrations assuming a power of 80% and a significance level of 5% (two-tailed test). Each variable was expressed as mean ± SD when the distribution was normal or as median [with the 25th and the 75th percentiles] otherwise. The t test was used to compare continuous normally distributed data. Kruskall-Wallis and Mann-Whitney U tests were used to compare non-parametric continuous data. A linear multiple regression analysis was used to investigate possible associations between the main outcome variables (plasma 25OHD and 1,25OH2D) and the following variables: gestational age and anthropometric data at birth; postnatal age, anthropometric data, plasma 25OHD, and 1,25OH2D at entry; postnatal age, gestational age, and anthropometric data at theoretical term; total milk and vitamin D intakes. For statistical calculations, we used StatView software SE V1.04 (Abacus Concepts, Berkeley, CA).
The study was approved by the Ethical Committee of Claude Bernard University (Lyon) and by the French Health Ministry in accord with the Helsinki declaration of 1975 revised in 1983. Informed written parental consent was obtained in all cases.
From November 1997 to December 1998, 36 subjects were eligible to participate in the study. Six were not included because of parental refusal. The 30 included subjects underwent the complete course of the study.
Gestational age, body weight, crown-heel length, and head circumference at birth and at time of inclusion were not significantly different between the three feeding groups (Table 2). Subjects fed PF3 were included at a younger age than the two other groups, but the final evaluation was performed at the same postnatal and gestational ages. During the study period, subjects from the three feeding groups ingested comparable amounts of milk. Although the total vitamin D intake (milk and supplementation) was close to 1300 IU/d in the three groups, subjects fed PF1 had a higher intake than those fed PF3 (Table 3).
At entry into the study, there were no significant differences in serum 25OHD or 1,25OH2D concentrations between the three groups (Table 4). We found no correlation between dietary cholesterol concentrations and the main outcome parameters. At the end of the study period, there were no significant differences in serum 25OHD, 1,25OH2D, phosphorus, alkaline phosphatase concentrations between the three feeding groups (data not shown). Adjustment for body weight, postnatal age, plasma 25OHD, and 1,25OH2D at time of inclusion; for body weight, postnatal age, and gestational age at theoretical term; and for milk and vitamin D intakes had no effect on plasma 25OHD and 1,25OH2D differences between the three groups.
There was no augmentation of serum cholesterol concentration on increasing dietary cholesterol during study period (r = 0.055, P = 0.515) (Table 4). Furthermore, at the end of the study, there was no statistical difference in mononuclear cell HMG-CoA reductase mRNA copy number (x104 per μg of total mRNA): 643 [409, 721] in the group fed PF1, 500 [319, 840] in the group fed PF2, and 778 [469, 1466] in the group fed PF3 (P = 0.613).
At the end of the study, body weight and length were lower in subjects fed standard preterm formula than in those fed formulas containing cholesterol, but these variables were significantly different only between PF1 and PF2 (Table 3). Weight gain during the study period was lower in subjects fed PF1 than in those fed PF2 but this difference did not reach a statistical significance.
In a previous non-randomized study (unpublished data), we have shown a decrease in vitamin D status in preterm infants fed a cholesterol-enriched formula (0.3 g/L). Because of the clinical relevance of this preliminary finding, we had to test the hypothesis of a negative effect of dietary cholesterol on vitamin D status in preterm neonates with this double-blind randomized study. Our results do not support this hypothesis because subjects fed formulas with medium or high-cholesterol content did not present a decrease in serum 25OHD or 1,25OH2D concentrations compared with subjects fed formulas with much lower cholesterol quantities.
Furthermore, to explain our finding, we postulated a possible competition for CYP27 that catalyzes 27-hydroxylation of C-triols as well as 25-hydroxylation of vitamin D3 (12). The current study did not give a clue as to the competition for CYP27. The lack of effect of cholesterol intake on vitamin D status might then be explained by the existence of another 25OHD synthesis pathway that involves a reconstituted enzyme containing adrenodoxin and NADPH-adrenodoxin reductase. This alternative pathway is thought to be involved in the synthesis of 25OHD in patients with cerebrotendinous xanthomatosis who have a deficiency in sterol 27-hydroxylase (12). We also speculate that 25OHD synthesis might occur in other organs that the liver (such as the kidneys) where cytochrome P-450 is expressed as well and has a higher affinity for vitamin D3 than C-triols.
Unexpectedly, and contrary to the results obtained in full-term neonates (3), we did not observe an increase in plasma total cholesterol, despite the dietary cholesterol load. Plasma cholesterol concentration was not higher in the group fed PF3 in comparison with the two other groups. That lack of differences in serum cholesterol concentrations could be due to an impaired intestinal absorption of cholesterol in immature preterm neonates. Actually, in the present study we cannot assure that cholesterol was well absorbed because it has been demonstrated that, like in adults (21), 30 to 40% of ingested cholesterol is absorbed in formula-fed preterm neonates (2). We did not observe changes in HMG-CoA reductase mRNA copy numbers in a relatively small subgroup of patients. Data from animal studies (22) and studies performed in adults, have shown a statistically significant reduction in mononuclear cell HMG-CoA reductase mRNA copies in subjects fed cholesterol-enriched diets, which reflects a lower rate of endogenous cholesterol synthesis (18). Our findings suggest that, in contrast to results obtained in older children, cholesterol intake did not modify the endogenous cholesterol synthesis in preterm infants (23,24). Our results are in agreement with those obtained in four-month-old mature babies (25). In sum, the absence of effect of dietary cholesterol on plasma cholesterol concentrations and on the endogenous cholesterol synthesis could be explained by an impaired intestinal absorption of cholesterol, but we speculate that this finding could be explained by a relative immaturity of the cholesterol biosynthetic pathway in preterm infants, though further studies are needed to address this issue.
The rapid growth phase that these newborns experience may yet be another explanation for the apparent absence of effect of ingested cholesterol on serum cholesterol concentrations and may suggest that extra cholesterol was used to build new tissues including the central nervous system. This hypothesis is supported by the fact that we observed a five-g/d difference in weight gain during the study period between subjects fed formulas that differ only by their cholesterol content (PF1 and PF2). This difference in growth could also be related to the difference in formula intake between PF1 and PF2. However, we cannot conclude from our study that there is a causal relationship between added cholesterol and growth.
In conclusion, dietary cholesterol had no deleterious effects on vitamin D status in preterm infants when cholesterol is provided in amounts equal or greater than those present in human milk. Besides, it had no effect either on serum cholesterol concentration or on HMG-CoA reductase mRNA copies in mononuclear cells. The latter findings suggest a different regulation of cholesterol metabolism in preterm infants compared with older infants and children. Further studies are needed to assess long-term effects of cholesterol supplementation on growth and on lipid or lipoprotein metabolism.
We are indebted to Geraint Jones, Jean Iwaz, and René Ecochard for editorial assistance. We also thank Milupa Research Center (Friedrichsdorf, Germany) for providing study formulas and the financial support of laboratory analysis.
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