Main

It has been well documented that infants fed human milk have higher plasma total- and LDL-Ch concentrations than those fed formula(1–8). This difference has been attributed to the greater Ch content of human milk compared with commercial formulas(8–10). Human milk contains significant quantities of Ch (0.26-0.28 mmol/L, 10-11 mg/dL). In contrast, cow's milk-based formulas possess lesser amounts (0.08-0.13 mmol/L, 3-5 mg/dL) of Ch, whereas soya-based formulations have none. In infants, a low Ch intake (0.09 mmol/L, 3.5 mg/dL) appears to up-regulate endogenous Ch synthesis(5,6). Infants fed human milk, with typically 3-fold higher Ch content compared with cow's milk-based commercial formulas currently available, appear to down-regulate cholesterogenesis(5,6).

The long-term implications of manipulating Ch synthesis and Ch transported by lipoproteins in plasma, in developing human infants, have not been established. Barker's retrospective studies(11–13) of men born in Hertfordshire, England, to identify environmental risk factors for cardiovascular disease, suggest a relationship(11) between type and duration of infant feeding, adult serum Ch concentration, and mortality from ischemic heart disease. Some authors(14,15) suggest that differences in plasma Ch concentrations in BF and formula-fed infants are transient in nature and do not manifest themselves past the age of 12 mo. However, others have reported lower serum Ch levels at ages 7-12 y(16) in children previously fed a low Ch formula as infants, and conversely, lower concentrations in women 30-50 y of age(17) previously BF as infants. In theory, smaller central pools of Ch later in life may contribute to reduced LDL-Ch concentrations, thereby potentially reducing cardiovascular disease risk.

Several studies of Ch supplementation of infant formulas, but using lower amounts of Ch than that found in human milk have reported higher plasma Ch concentrations(5–7,18–20) and lower cholesterogenesis(5,6) rates than their unsupplemented counterparts. One study from Finland(21) reported that infants fed formula supplemented with taurine + Ch had lower serum Ch concentrations when compared with those fed unsupplemented formula or BF, even though the formula contained as much Ch as did human milk. However, Ch supplementation of infant formula alone, in amounts identical to that found in human breast milk, have not been previously studied in terms of its effect on cholesterogenesis. Whether the differences in Ch content between breast milk and cow's milk-based formula are responsible for the dissimilar plasma lipid profile and FSR observed between BF and formula-fed infants have not been established.

We therefore conducted a prospective partially randomized controlled study evaluating the effects of Ch fortification of cow's milk-based infant formula, to human milk levels, on circulating Ch concentration and Ch synthesis rates to determine whether Ch synthesis rates and circulating Ch concentrations are influenced by dietary Ch intake when fatty acid composition is identical. We hypothesized that, at 4 mo of age, endogenous Ch FSR of infants fed RF+Ch, modified to contain Ch similar to amounts found in human milk, will be similar to the FSR of infants who are BF and will be less than the FSR of infants fed RF [schematically (FSR) BF ≈ RF+Ch < RF]. As an ancillary hypothesis, we theorized that, at 4 mo of age, plasma Ch concentrations of infants fed RF+Ch or BF will be similar, and both will be greater than those of infants fed RF.

METHODS

Study population and protocol. Thirty-seven healthy infants(19 male, 18 female) who were full-term, appropriate for gestational age, and had no parental history of hypercholesterolemia or hypertriglyceridemia were recruited from the normal newborn nurseries of the University Hospital of Cincinnati and other area hospitals. Fourteen infants exclusively BF for the first 4 mo of life comprised the human milk-fed group. Twenty-three were randomized according to a computer-generated random numbers table to receive RF (SMA® ready to serve iron fortified, 0.85 mmol Ch/L, 33 mg Ch/L; Wyeth-Ayerst Laboratories, Philadelphia, PA) (n = 11) or RF+Ch(SMA® ready to serve iron fortified, 3.44 mmol Ch/L, 133 mg/L Ch; Wyeth-Ayerst Laboratories) (n = 12). Crystalline-free Ch powder was dissolved in components oils, forming an emulsion, before combining macronutrient ingredients. Analysis of a random batch of RF+Ch was made before distribution to the infants to assure that the Ch contents were within 13 ± 2 mg/dL. The nutrient compositions of formulas are summarized in Table 1. All infants received their formula within the first 3-7 d of life. Formula was provided free to the formula-fed infants for the entire duration of the study to ensure compliance. The study was approved by the Institutional Review Boards of all involved hospitals, and informed consent was obtained from parents before enrollment of the infants. Thirty-two infants completed the study. There was insufficient blood drawn to determine FSR in one infant from the BF group, two from the RF group, and two from the RF+C group.

Table 1 Composition of types of formula and breast milk used in the study*
Full size table

Mothers from the three groups kept 3-d diet diaries for each month until the testing period at 4 mo. Mothers recorded information regarding the frequency of breast-feeding, the volume of supplemental RF per day, and the volume of formula per day in the formula groups. The diets were analyzed by a registered dietitian experienced in the analysis of these diaries, to determine whether any significant disparity occurred among the formula groups that may have confounded the outcome variable of FSR. By design, other forms of nutrition such as cereal were not allowed, except for multivitamins.

Plasma lipid analysis. Determinations of serum Ch and lipid profiles were performed at the Medical Research Laboratories in Cincinnati, OH, with enzymatic techniques validated by the National Institutes of Health Lipid Research Clinics. In the presence of Mn2+ and heparin, chylomicrons, VLDL, and LDL were selectively precipitated, leaving only HDL in solution. The precipitated lipoproteins were sedimented by centrifugation, leaving the clear supernate containing HDL-Ch that was then analyzed enzymatically. Triglycerides were also determined enzymatically. LDL-Ch concentrations were calculated from serum total Ch concentrations with the equation formulated by Friedewald et al.(22), used previously by Cruz et al.(6) with infants.

Cholesterol biosynthesis measurements. Ch synthesis rates were determined at 4 mo of age. This age was chosen because infants in the first 4 mo of life generally receive exclusively human milk or formula(23), so little potential existed for interference from other diets. Thus, differences in FSR and lipid profiles could be specifically attributed to the type of milk consumed. Measurements were performed over 48 h. On d 1, 8 mL of blood were obtained to determine baseline body water deuterium enrichment. Infants were then orally given 500 mg/kg body weight of deuterium oxide (D2O, 99.96% deuterium; Isotec Inc., Miamisburg, OH). On d 2, 8 mL of blood were obtained for deuterium enrichment measurement, after which 50 mg/kg body weight of D2O was given orally to maintain constant body water deuterium enrichment. On d 3, a final 8-mL blood sample was obtained. All blood samples were drawn between 0900 and 1100 h.

Ch FSRs were determined as the rate of incorporation of deuterium into the red blood cell membrane(24). Erythrocyte lipids were extracted by organic solvent and dried under nitrogen. Free Ch was isolated by thin-layer chromatography, then eluted from thin-layer chromatography silica scrapings and quantitatively transferred to Pyrex combustion tubes containing CuO and a silver wire. Tubes were flame-sealed under vacuum, and Ch was combusted completely at 520°C to CO2 and water. Combustion water was cryogenically separated from CO2 by vacuum distillation into Pyrex tubes containing 50 mg of zinc. These were flame-sealed under vacuum, and the water was reduced to H2 at 520°C. Deuterium enrichment of the resultant gas was measured by dual inlet isotope ratio mass spectrometry (VG Isogas 903D). Plasma-water enrichment was measured after dilution of 24- and 48-h plasma samples with water of known isotopic abundance to bring the enrichment into the working range of the International Atomic Energy Agency (Vienna) mass spectrometer calibration standards.

Erythrocyte Ch deuterium enrichment values at 24 and 48 h were expressed relative to the corresponding mean plasma water sample enrichment at each time point, after correction for the deuterium-protium ratio in Ch, to yield FSRs (pool/day) for the free Ch pool. The FSR index represents that fraction of the free portion of the rapidly turning over central Ch pool that is synthesized in 24 h as per the formula(24): FSR(%/day) = (δch/δplasma) × 0.478 × 100 where δ refers to deuterium enrichment above baseline over 24 h. The factor of 0.5 is used to yield a daily FSR from each respective 48-h time period.

Statistical analysis. One-way ANOVA was used to test differences among groups(25). The Tukey-Kramer method for multiple comparisons was used to determine differences between pairs of groups(25). General linear models were used to determine correlations between outcome variable (FSR) and the independent variables. Statistical significance was considered for p values less than 0.05. Results are presented as mean ± SEM.

RESULTS

Demographic data are summarized in Table 2. There were no differences in birth weight and length, weight, and ponderal index at 4 mo across treatment groups. Nor were differences observed in volume of milk intake among the formula groups as determined from dietary records.

Table 2 Characteristics of study population*
Full size table

ANOVA indicated that plasma total-Ch (p < 0.02) and LDL-Ch concentrations were higher (p < 0.04) in BF (4.31 ± 0.21, 2.27 ± 0.23 mmol/L), compared with RF-fed (3.39 ± 0.23, 1.21 ± 0.18 mmol/L) groups (Fig. 1; Table 3). There was an intermediate response in plasma total-Ch and LDL-Ch concentrations (3.83 ± 0.60, 1.78 ± 0.22 mmol/L) for infants fed RF+Ch. The difference between the RF+Ch-fed and BF, or RF-fed groups did not reach significance. Total/HDL-Ch ratios were higher(p < 0.02) in infants BF (3.79 ± 0.24) and higher(p < 0.05) in infants fed RF+Ch (3.52 ± 0.24), compared with those fed RF (2.83 ± 0.23) (Fig. 2); whereas not significantly different between BF and RF+Ch-fed infants. Similarly, LDL/HDL-Ch ratios were higher (p < 0.002) in infants BF (1.99 ± 0.23), and higher (p < 0.03) in infants fed RF+Ch (1.63 ± 0.24), compared with those fed RF (1.02 ± 0.23)(Fig. 3); whereas not significantly different between BF and RF+Ch-fed infants. No differences were observed in serum HDL-Ch and triglyceride concentrations among groups.

Figure 1
figure 1

Plasma lipoprotein concentrations at 4 mo of age in the three groups, X ± SEM. Significantly different from the BF group (*p < 0.02; τp< 0.04). Plasma total- and LDL-Ch concentrations of the RF group were not significantly different from BF or RF+Ch groups.

Full size image
Table 3 Serum lipid profile*
Full size table
Figure 2
figure 2

Plasma total/HDL-Ch concentrations at 4 mo of age in the three groups, X ± SEM. *BF significantly higher (p < 0.02) and RF+Ch-fed significantly higher (p < 0.05) than RF-fed groups; whereas not different between BF and RF+Ch-fed groups.

Full size image
Figure 3
figure 3

Plasma LDL/HDL-Ch concentrations at 4 mo of age in the three groups, X ± SEM. *BF significantly higher (p < 0.002) and RF+Ch-fed significantly higher (p < 0.03) than RF-fed groups; whereas not different between BF and RF+Ch-fed groups.

Full size image

Deuterium enrichments of body water and erythrocyte Ch are shown in Figure 4. Deuterium enrichment in body water was similar across treatment groups. Erythrocyte Ch deuterium enrichment was consistently lower in BF compared with formula-fed groups (Fig. 4). No difference in erythrocyte Ch enrichment was observed between RF and RF+Ch-fed groups.

Figure 4
figure 4

Deuterium enrichments of plasma water(outset) (BF infants, ○ RF-fed infants, ♦; RF+Ch-fed infants,▴;) and erythrocyte Ch (inset) (BF infants, ○; RF-fed infants,♦; RF+Ch-fed infants, ▴). Dotted line as determined previously by Jones et al.(24).

Full size image

Ch FSR of RF (8.58 ± 0.27%/d), and RF+Ch (8.29 ± 0.37%/d), were elevated (p < 0.0001) compared with BF infants (2.19± 0.29%/d), whereas FSR did not differ significantly between RF and RF+Ch groups (Fig. 5).

Figure 5
figure 5

FSR of BF and formula-fed infants at 4 mo of age, X ± SEM. FSR (%/d) for infants was 2.19 ± 0.29, ·, BF; 8.58 ± 0.27, ▴, RF+Ch (SMA, 3.43 mmol Ch/L); and 8.29 ± 0.37, ♦, RF (SMA, 0.85 mmol Ch/L).*Significantly different from the BF group, p < 0.0001.

Full size image

DISCUSSION

The main objective of the present work was to examine the effects of Ch fortification of cow's milk-based formula, in amounts similar to that found in human milk, upon circulating Ch concentrations and in vivo synthesis during early infancy. We demonstrated increased cholesterogenesis and a modest response in circulating plasma Ch concentrations in formula-fed infants compared with BF infants, as a result of dietary Ch fortification to levels found in breast milk.

The issue of whether dietary Ch in human milk is beneficial, benign, or detrimental to infants has been a topic of speculation for several decades(1–21). Progress in this area has been hindered by the difficulty in separating the effects of dietary Ch feeding from those of dietary fatty acids when a BF group is used as a control group. Because individual fatty acids may have significant direct effects on Ch metabolism(27,28), and the fatty acid composition of formula cannot be made identical to the fatty acid composition of human milk, no direct inferences with regard to the effect of fatty acids on Ch metabolism can be made in the present study.

Feedback inhibition of Ch synthesis mediated by changes in the activity of the rate-limiting step of Ch biosynthesis, hepatic hydroxymethylglutaryl CoA reductase, has been demonstrated by dietary Ch feeding in guinea pigs, hamsters, and pigs(29–31). In humans, the inhibitory effect of dietary Ch feeding on cholesterogenesis has been variable, with studies reporting both alterations(32–34) or no effect(35–37). The variable results of these former studies may be attributed to several factors, including the type and amount of dietary Ch and fat included in the diet and the efficacy of the methodologies used.

To our knowledge, only two previous studies examining Ch synthesis as a function of dietary Ch intake have been reported in infants(5,6). Using deuterium incorporation techniques, previous investigators(5,6) have estimated that endogenous Ch synthesis ranges from 2 to 11%/d in human infants BF or fed formula with varying concentrations of Ch and disparate fatty acid profiles. Both research groups attributed the differences in FSR to quantity of dietary Ch, reporting quantity of Ch intake to be significantly and negatively associated with FSR (p > 0.0001), although acknowledging the role that fatty acids, phytosterols, phytoesterogens, and hormones may have in mediating serum lipid concentration and endogenous Ch metabolism. These previous studies(5,6) did not specifically supplement Ch intake in formula-fed infants to the level found in breast milk. We conducted the study in a population of 4-mo-old infants fed identical formula, with 0.85 mmol Ch/L (33 mg Chl/L) or 3.44 mmol Ch/L (133 mg Ch/L). The effect of dietary Ch on endogenous Ch synthesis rates was specifically determined by eliminating confounding dietary variables; energy, protein, lactose, fat, and in particular linoleic acid consumption were not different between the formula groups.

In the present study, similar plasma total- and LDL-Ch concentrations and higher FSRs occurred in formula-fed infants, regardless of dietary Ch fortification, compared with BF infants. Wong et al.(5) and Cruz et al.(6) found lower serum total- and LDL-C concentrations and higher FSRs in formula-fed infants, that was dietary Ch dose-dependent, compared with BF infants. Moreover, infants fed soy formula supplemented with Ch to the level found in cow's milk-based formula had lower FSRs than those fed unsupplemented soy formula(6), suggesting the Ch component did suppress endogenous Ch synthesis. In the present study, infants fed cow's milk-based formula supplemented with Ch to the level found in human milk had FSRs that were not different from those fed regular cow's milk-based formula, suggesting that other factors may explain the differences between cholesterogenesis in breast- and formula-fed infants. Moreover it appears, from the plasma lipid results reported, that compared with infants BF or RF-fed, infants fed RF+Ch have an intermediate response to Ch fortification, suggestive of a modest effect.

In the present study small blood volumes precluded more extensive kinetic analysis, such as absorption, inter- and intra-pool transfer rates, absolute synthetic rates, and catabolic rates. Correspondingly, as FSR was the main outcome variable in the present study, fecal Ch excretion was not measured. However, increased Ch efflux appears as the most likely mechanistic hypothesis to explain the insignificant rise in plasma Ch despite increased dietary Ch in the RF+Ch-fed group. Fecal Ch excretion was measured in preterm infants(7) who received standard preterm formula with 30 mg of Ch/dL, compared with those fed the same formula (5.5 mg/dL Ch) or fortified breast milk (mean Ch content 15.3 mg/dL). The group fed the higher Ch formula had higher Ch excretion and Ch balance (35.5 mmol kg-1 d-1; +21.8 mg kg-1 d-1) than in the groups fed breast milk (20.1 mmol kg-1 d-1; +8.6) or the standard formula (18.2 mmol kg-1 d-1; 7.7 mg kg-1 day-1). The process enabling preterm infants to regulate a higher Ch intake than during breast feeding by increasing fecal Ch excretion, as suggested by Boehm et al.(7), may be operating similarly in the RF+Ch-fed infants in the present study, explaining the similar rates of cholesterogenesis observed compared with those RF-fed.

Our results and those previously reported(6) may also be explained in part by differences in quantity of Ch supplementation and Ch solubility in the respective formulations based on fatty acid profile. Differential intestinal absorption of Ch based on dietary fatty acid type and position in triacylglycerol for fats ingested concomitantly has been observed in rats(38–41), hamsters(42), and baboons(43) with no effect in rabbits(44), whereas similar work in humans is lacking. From work in baboons(43), it is has been suggested that Ch absorption and hepatic Ch concentration regulate plasma Ch responses to diet, but by different mechanisms. Furthermore, dietary lipid induced changes in intestinal morphology and nutrient uptake may or may not be reversible, affecting the ability of the intestine to adapt to an altered nutrient intake in later life(41). Similar to previous work(6) we controlled for the effects of fatty acids on Ch biosynthesis by feeding identical formula and varying only the concentrations of Ch.

The possibility that Ch added to the formula in our study was not absorbed cannot be ruled out. Absorption of supplemental Ch in the form administered in the diet may have direct bearing on the rates of cholesterogenesis reported in this study. Plasma Ch concentration comprises less than 9% of total body Ch(45). Ch absorption from the gastrointestinal tract is an integral component of whole body homeostasis. Enterohepatic recirculation of endogenous Ch readily mixes with dietary Ch to form a single pool of intestinal Ch(46). Gastrointestinal absorption of dietary Ch by adults is about 45-50%(47–50). Samuel and McNamara(48), comparing the absorption of endogenous and exogenous Ch in adults, found endogenous Ch absorption to be 46 ± 15%, and exogenous 34 ± 8%. Similar to those investigations, the current study used only nonesterified Ch. It should be noted that endogenous(biliary) Ch is entirely non-esterified Ch. So far, data describing Ch absorption in human infants have not been reported.

Nonesterified microcrystalline Ch added to commercial formula is ingested as a component of an emulsion, whereas dietary Ch in breast milk is unique, being ingested as a physiologic structural component. Thus, different transport medium for dietary Ch may have some bearing on bioavailability. Furthermore, Van Lier et al.(51) cautions that Ch will oxidize rather rapidly if an aqueous phase is present and that the stability of Ch in an emulsion may be suspect. The majority of Ch in human milk is nonesterified; 80% of this Ch is found in the outer milk fat globule membrane, which is formed predominantly from the plasma membrane of epithelial cells in the breast(52). The central core of the milk fat globule membrane contains 15-20% of total Ch as esters(53). It is necessary for Ch esters to be hydrolyzed before absorption, and only nonesterified Ch is absorbed, whereas the intact esters are not. Pancreatic Ch esterase catalyzes the hydrolysis. It does not seem reasonable that a Ch ester would be better absorbed than would nonesterified Ch, unless the exogenous Ch exceeds the solubility limit of the dietary oil. It is unlikely that 15-20% of total Ch in breast milk as esters would explain the Ch synthesis and plasma lipoprotein Ch differences in the current study.

The Ch content of breast milk during infancy is relatively constant across populations when age of development is considered(52). Similar to FSRs that were previously reported, 2.1%/d by Wong et al.(5), 2.62%/d by Cruz et al.(6) for BF infants at 4 mo of age, the FSR of BF infants in our study was 2.19%/d. Lipoprotein Ch concentrations we report are also similar to those previously reported(5,6) in BF infants. Composition of breast milk varies with stage of lactation and among mothers. Breast milk Ch concentration was 2.82-4.52 mmol/L (n = 6) for the Wong et al. report(5) and 2.59-3.88 mmol/L (n = 13) for the Cruz et al.(6) study. These similar results in two different groups of BF infants, coupled with our results in infants fed identical formula supplemented with Ch in amounts similar to that in breast milk, provide strong evidence that dietary Ch appears to be a factor responsible for the differences in circulating Ch concentrations, but does not fully explain the variations in cholesterogenesis observed(5,6) when infants are fed formula. The results of our study appear not to support the theory that adaptive regulatory mechanisms in early infancy enable human infants to respond to changes in Ch intakes within the physiologic range. The teleologic argument, that such homeostatic mechanisms would prevent excess Ch accumulation during breast-feeding or, conversely, provide for an increase in Ch availability during instances of lower or negligible intake, during formula feeding, is not wholly consistent with the results observed during our study. No evidence to date would disprove the assumption that breast milk is the gold standard for infant nutrition, and that values for FSR in BF infants are considered to be the norm. The apparent need for Ch in early infancy, a period rapid of growth, is reflected by the almost 4-fold difference in FSR for supplemented and unsupplemented infants compared with BF infants. This increase, however, appears to be irrespective of direct Ch supplementation. A possible interpretation would be that crystalline-free, food grade Ch supplementation of infant formula has a modest effect on circulating lipoprotein concentrations, and no effect on Ch synthesis in human infants at 4 mo of age. This interpretation assumes no differences in Ch absorption among the three feeding groups.

Methods for endogenous Ch synthesis determinations based on deuterium incorporation have been well established(24,54–56), offering the advantage of being direct, short-term, and noninvasive over intake balance methods(57–60) or isotopic kinetic decay analyses(45,61–63). The efficacy of using erythrocyte Ch deuterium enrichment to study human lipid metabolism was first reported by London and Schwartz(64). Subsequently, use of deuterium incorporation into newly synthesized erythrocyte membrane free Ch, based on the tritiated water uptake methodology developed in animals(65), was further refined and developed for measurement of short-term Ch synthesis in humans(24,66). The interpretation of results depends upon three primary assumptions that have been well described previously by Jones(24,66). The first is that a constant fraction of deuterium atoms in free Ch synthesized de novo originates from plasma water. Because water freely and rapidly diffuses across cell membranes, plasma water deuterium concentration provides a measure of precursor enrichment, and deuterium enrichment of the newly synthesized Ch molecules corresponds to enrichment of the central pool, which includes the liver, plasma, and intestine. A second assumption is that, within the central pool, rapid movement of newly synthesized Ch from the liver to plasma lipoproteins and then to cell membranes occurs, and therefore the deuterium content of erythrocyte membranes reflects newly synthesized Ch. The third assumption is that deuterium uptake by cell membranes represents synthesis from the central pool only, and it does not represent influx of newly formed Ch from other pools or synthesis by erythrocytes.

Recently Ch synthesis was quantified using two independent methods. Daily whole body Ch balance average over several days agreed with incorporation of deuterium oxide into newly synthesized Ch over a 24-h period(67). The correlation between the two methods was r = 0.745 (p < 0.001). Deuterium incorporation into erythrocyte membrane Ch is predicted to provide a reasonable measure of FSR, predicated upon extensive simulations of the free Ch central pool defined by other investigators(62,68).

Previous approaches to determining Ch FSR used both linear(54,69) and monoexponential(28,55,70) constrained-fit models. An assumption of these models is that, at constant precursor enrichment, as seen in Figure 4, body-Ch deuterium-enrichment plateaus at about one-half of the body-water deuterium-enrichment concentration(71). To avoid complications of recycled Ch and mixing between the rapidly miscible and the other two slower turning over pools, enrichment data from earlier linear phase time points provide the most reliable indexes of uptake for FSR calculation. Enrichment patterns are linear over the initial portion, 0-48 h, of this curve(5,6,70) as evidenced by our erythrocyte deuterium-enrichment data in Figure 4. Similar infant feeding studies(5,6) show a relatively linear erythrocyte deuterium-enrichment pattern with an asymptote in some subjects after 40 h.

The lower FSR in the BF group could potentially be viewed as an artifact resulting from a dilution effect. The higher dietary Ch intakes and plasma Ch concentrations of BF infants might possibly cause a higher Ch content in the central pool, generating a dilution effect on calculations of synthesis rates. This is unlikely, given that the central pool of BF infants is probably 15-20% larger compared with formula-fed infants, based on previous piglet studies(72). Moreover, as demonstrated in adults(55,70) and in infants fed either human milk or formula(73,74), erythrocyte membrane Ch is fairly constant regardless of diet. The 15-20% difference in the central pool between BF and formula-fed groups and the 15% difference in plasma Ch concentrations are therefore neither sufficient to explain nor proportionate to the almost 4-fold difference in FSR rates among groups. It has been theorized that approximately half the difference in FSR between the BF and formula-fed groups can be explained by an expanded central pool(5). The remaining difference in FSR might be due to down-regulation of hepatic hydroxymethylglutaryl-CoA reductase and Ch synthesis. It has also been suggested that expansion of the central pool is probably due to the increased absorption of dietary Ch in the BF infants coupled with a down-regulation of LDL-receptor activity in the liver(75,76). Our results showing no difference in FSR and circulating Ch concentrations between RF and RF+Ch formula-fed infants do not support the contention of an expanded central pool attributable to dietary Ch intake.

In summary, we have examined for the first time the in vivo endogenous Ch synthesis rates of human infants fed identical formula with and without crystalline food grade free Ch in amounts similar to human milk, and have established that Ch synthesis rates are not affected by this form of Ch supplementation.

We conclude that crystalline food grade-free Ch supplementation of infant formula affects lipid profiles modestly, but not Ch synthesis rates. These findings support the view that dietary Ch is not the sole component of human breast milk causing feedback inhibition of Ch biosynthesis in human infants. From these findings we speculate also that Ch added to cow's milk formula may not be well absorbed.

Acknowledgments. The authors thank Kathryn Pramuk, M.S., R.D., Wyeth-Ayerst, for aid in this study.