Atherosclerotic cardiovascular disease has a high prevalence in the Western world. It is now widely accepted that lowering blood cholesterol concentrations is associated with reduction in rates of cardiovascular morbidity and mortality. Because this illness is thought to originate in childhood, investigation into the effects of early nutrition on levels of plasma lipids is warranted (1). Long-term consequences of the manipulation by diet of the plasma lipid profile in early infancy are not known yet. However, exploration of the short-term influence is necessary to optimize the prevention and treatment of coronary vascular disease.
Lipid metabolism is influenced by the type of dietary protein through unknown mechanisms (2). Results in many studies have demonstrated that animal proteins, particularly casein, have a hypercholesterolemic effect (3,4). In contrast, evidence has been accumulated of the cholesterol-lowering properties of dietary protein of plant origin, particularly those of soybean proteins (5).
According to results in previous studies, the casein-to-whey ratio in infant formula influences levels of plasma lipids. Plasma cholesterol levels were highest in infants fed formulas with the highest casein-to-whey ratio (6). The objective of the current study was to assess whether different methods of deionizing whey proteins in infant formula, without alterations in the ratio of casein to whey, influence the plasma lipid profile.
MATERIALS AND METHODS
Full-term, healthy infants were studied prospectively from birth to 8 weeks of age, in a randomized, double-blind trial. Only infants with a history of normal gestation and normal birth were included. Those with no evidence of respiratory, neurologic, renal, hepatic, cardiovascular, gastrointestinal, or congenital anomaly were enrolled.
The study was approved by the Institutional Review Board of the Soroka Medical Center. Informed, written consent was obtained from both parents.
Whey-predominant (60%:40%) infant formula (Materna Plus, Maabarot Products, Maabarot, Israel) containing either ultrafiltrated or electrodialyzed whey was fed to the infants from birth to 8 weeks of age. Both formulas were otherwise identical in composition (Table 1). Infants were randomly assigned to receive one of the two types of formula. Until the study began, infants were breast fed. Parental interviews to determine possible side effects and physical examinations including anthropometric measures (weight, length, and head circumference), were performed at 0, 30, and 60 days during the study.
Blood samples were taken into EDTA-containing tubes, just before feeding (2-3 hours after previous feeding) at 8 weeks of age. Plasma was collected, and the high-density lipoprotein (HDL) fraction was separated from the other lipoproteins by the addition of a precipitating reagent (phosphotungstic acid, magnesium chloride) to a plasma sample (7). All plasma samples and HDL fractions were then stored at -20 °C for subsequent analysis.
Plasma samples were analyzed for triglycerides, total cholesterol and HDL cholesterol levels. Plasma triglycerides levels were determined by the enzymatic-colorimetric method, using lipoprotein lipase-glycerokinase, glycerophosphate oxidase, and a chromogen, as described elsewhere (8). Total plasma cholesterol and lipoprotein cholesterol contents were determined by the enzymatic-colorimetric method, using cholesterol esterase, cholesterol oxidase, peroxidase, and a chromogen (9). HDL was separated from low-density lipoprotein (LDL) and very-low-density lipoprotein (VLDL) fractions by precipitating apolipoprotein B with phosphotungstic acid, and cholesterol concentration was determined in the supernatant (7). VLDL cholesterol was calculated as triglycerides divided by 5. The LDL fraction was then calculated using the equation of Friedwald et al. (10)
Because of technical difficulties, we did not have our own control group of breast-fed infants. Therefore, current data were compared with plasma levels of breast-fed infants reported in the literature (11). The same analytical methods were used in both studies.
Data are presented as mean ± standard deviation. Statistical analysis was performed using the nonpaired t-test. The level of statistical significance was set at 0.05.
Thirty-five infants were enrolled in the study, 18 in group A, receiving formula deionized by ultrafiltration, and 17 in group B, receiving formula deionized by electrodialysis. Five infants failed to complete the study: poor compliance (1 in group A, 2 in group B), allergy to cow's milk (1 in group B), and febrile illness (1 in group A). Clinical data from both groups are presented in Table 2. There were no significant differences between groups in age, gender, birth weight, gestational age, age at study day 1, weight and length gain, and meal volume. Fasting plasma levels of the various lipids after 60 days are presented in Table 3. Infants in group A demonstrated significantly higher plasma levels of total cholesterol (p < 0.001) and of LDL cholesterol (p < 0.001), compared with levels in infants in group B. Values in group A were very similar to normal levels of plasma lipids in breast-fed infants reported in the literature (11).
The results of the current study illustrate an important aspect of dietary protein's effect on the plasma lipid profile in infancy. Plasma levels of total cholesterol and LDL cholesterol were responsive to variations in the method of whey deionization. The mechanisms responsible for these effects are unknown and were not sought in the current study.
Although we do not have plasma lipid values at baseline, before the infants began receiving the test formulas, we assumed that lipid levels in both groups were not different, because subjects were selected at random and had the same ethnic origin. The two formulas were identical in protein concentration and in the casein-to-whey ratio, and differed only in the method of whey deionization. In the electrodialysis process an electric potential drives only the ions from the whey concentrate. The ultrafiltration method is a membrane-filtration technique. In this process only small molecules (minerals, lactose, nonprotein nitrogen) and water can pass through a polymeric membrane (12). To the best of our knowledge, there are no data in the literature describing the possible different effects of these two methods on the formula's composition.
The quantity and characteristics of fat in both formulas were also identical, indicating that differences in values between groups cannot be ascribed to differences in dietary fat.
Lipid metabolism is well known to be influenced by the type of dietary protein, although the mechanism by which this occurs is unknown at present. Casein has proved to be hypercholesterolemic when compared with plant proteins (soybean) in both animal and human studies (13,14). Recently, it has been shown that a diet substituting soybean protein for animal protein has a more beneficial short-term effect on total cholesterol and LDL cholesterol levels in children with hypercholesterolemia than does a standard low-fat diet (15).
The mechanisms underlying these effects are not yet clear; several hypotheses have been proposed. Results in some studies suggest that alterations in bile acid or in cholesterol absorption may contribute to altered cholesterol homeostasis. However, no differences in the fecal excretion of bile acids or sterols were found (16). Another theory attributes the cholesterol-lowering effect of soy proteins to soy estrogens (17). This might also be the mechanism for the lower synthesis rate of fractional cholesterol recently reported in infants fed a modified-soy formula (18).
Although there is general agreement among investigators that pattern and proportionality of intake of amino acids can elicit changes in the plasma lipid profile, there is controversy regarding which individual amino acids are involved. In animal experiments, the amino acid composition of the diet affects serum cholesterol levels. Because similar variations in the plasma lipid profile occur when amino acids patterned after proteins are fed as occurs when the intact protein is fed, the influence of dietary proteins on plasma lipids is thought to be a result of the composition of constituent amino acids (19). Animals fed purified diets with high arginine content demonstrate a decrease in serum cholesterol (16), whereas an increase of dietary methionine over cystine causes hypercholesterolemia (20).
The low cystine content of caseins results in a high methionine:cystine ratio in casein-predominant formulas. A low ratio is seen in vegetable proteins and in whey-predominant infant formulas (21). We assume that such changes in whey proteins and in their constituent amino acids may occur during different deionization processes and may affect the blood lipid profile. We intend to examine this effect in future research.
Another potential mechanism that could explain our results is the removal of a hypocholesterolemic factor during the ultrafiltration process. Nonprotein compounds in soybean (isoflavones and saponins) were considered as potentially active components in cholesterol reduction. Some manufacturers use industrial procedures that remove a substantial portion of these active ingredients (5). Similarly, industrial whey deionization methods (ultrafiltration) may remove factors with lipid-lowering properties. Future investigation is needed to elucidate this question, as well.
The higher total cholesterol and LDL cholesterol levels observed in infants fed ultrafiltrated whey were within the normal range for this age group. Moreover, the levels were comparable to those reported in healthy breast-fed infants. Human milk contains higher levels of cholesterol and breast-fed infants generally have higher plasma cholesterol levels than do formula-fed infants (11). Because breast milk should be considered the “normal” feeding for this age group, further research is needed to explore whether “lower” plasma cholesterol levels in infants fed an electrodialyzed whey formula have any negative effects on growth and development.
According to our present knowledge the changes observed in the current results do not seem to enhance the risk of developing cardiovascular disease in adulthood. There is general agreement that dietary fat and cholesterol are important components of infant diets. The American Academy of Pediatrics has recently affirmed its position that lipid intakes should not be restricted in infants less than 2 years old, unless there is a family history of premature cardiovascular disease (22). This position is similar to recommendations given by the ESPGAN Committee on Nutrition, and in the United States by the American Heart Association, the American Health Foundation and the National Institutes of Health's Consensus Panel on Lowering Blood Cholesterol (23-26). Committees in Britain, Australia, Canada, and New Zealand have independently reached the same conclusions (1).
Nevertheless, further investigation of the impact of dietary protein on the development of the blood lipid profile in infancy is warranted. Such research may have significant long-term implications for the prevention and treatment of atherosclerotic cardiovascular disease.
Acknowledgment: This study was supported in part by Maabarot Products, Maabarot, Israel. It was presented at the 28th Annual Meeting of the European Society for Pediatric Gastroenterology and Nutrition, May, 1995, Jerusalem, Israel.
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