Journal of Pediatric Gastroenterology & Nutrition:
Original Articles: Hepatology and Nutrition
Oral Vitamin A, E and D Supplementation of Pre-Term Newborns either Breast-Fed or Formula-Fed: a 3-Month Longitudinal Study
Delvin, Edgard E.*†; Salle, Bernard L.†; Claris, Olivier†; Putet, Guy‡; Hascoet, Jean-Michel¶; Desnoulez, Laure§; Messai, Sam∥; Lévy, Émile‡
*Département de Biochimie Clinique, †Centre de recherche, Hôpital Ste-Justine, Montréal, Canada; Services de Réanimation et de Néonatologie: ‡Hôpitaux Édouard Herriot, §Hôpital-Nord, Lyon; ¶Maternité Régionale Universitaire-Nancy, France; ∥Laboratoires Crinex, Montrouge, France
Address all correspondence and reprint requests to Dr. Edgard E. Delvin, Département de Biochimie Clinique, Hôpital Ste-Justine, 3175 Côte Ste-Catherine, Montréal, Québec, Canada, H3T 1C5 (e-mail: firstname.lastname@example.org).
Background: In contrast to the studies of vitamin A and E status in children, adolescents and adults, information on preterm infants is scarce. In the present investigation we examined the vitamin A, D and E status of pre-term infants at birth, and verified whether, at 1 and 3 months, breast or formula feeding affected the plasma concentration of those vitamins while being supplemented with Uvesterol ADEC.
Patients and Methods: In this prospective study, 2 groups of consecutively recruited preterm newborns fed either breast milk or formula received 3000 IU of vitamin A, 5 IU of vitamin E and 1000 IU of vitamin D daily. Vitamin A and E were measured by high performance liquid chromatography and spectrophotometry. 25-hydroxyvitamin D, a surrogate marker for vitamin D status, was measured by radioimmunoassay, and retinol binding-protein concentration was measured by immunonephelometry.
Results: At birth, formula-fed and breast-milk fed infants had similar plasma concentrations of vitamin A (0.75 ± 0.20 and 0.64 ± 0.21 m mol/L, ns), 25-hydroxyvitamin D (34.4 ± 25.6 and 47.5 ± 26.7 nmol/L, ns) and vitamin E (9.5 ± 3.2 and 8.4 ± 3.3 μmol/L, ns). Vitamins A and E, and retinol binding-protein concentrations steadily increased with time in both groups of infants without attaining, at 3 months, values considered normal in term infants and in young children. At 3 months of age, concentrations of 25-hydroxyvitamin D reached values comparable to those observed in term infants.
Conclusion: Plasma concentrations of vitamins A and E and of retinol binding-protein steadily increased during the the study without reaching full repletion values. At the conclusion of the study, the type of nutrition did not affect plasma vitamin concentrations.
Optimal nutrition early in life is a general concern as inadequate food intake may have an impact on the development of diseases in later life. This is particularly important in preterm infants who are at risk of being subjected to oxidative stress (1). Vitamin A (retinol) and vitamin E (α-tocopherol) are two potent antioxidant nutrients. Beyond their beneficial effects in ischemic heart disease in adults (2-4), epidemiologic studies have shown that these vitamins have a significant role in immune function (5,6). Furthermore, vitamin A is not only involved in the regulation and promotion of growth and differentiation of many cell types such as those synthesizing the visual pigments in the retina (7,8) but it also plays a major role in maintaining the integrity of the epithelial cells of the respiratory tract (9,10).
Although the biologic roles of vitamin E are numerous, they are poorly understood. While its hydrophobic character favours its integration into the bi-layer cell membrane, the conjugated isoprene side-chain gives it an anti-oxidant property (11,12). Kohlschutter et al. (13) have suggested that during vitamin E deficiency states, the histologic degeneration of the central nervous system that precedes the fall of plasma vitamin E concentration, may be linked to impaired neuronal protection against lipid peroxidation. More recent reports indicate that not only the vitamin E status should be considered in the pathophysiology of this condition, but also the levels and the type of long-chain unsaturated fatty acids in the diet (14-16). Surprisingly, in contrast to the numerous studies on vitamin A and E status in children, adolescents and adults, studies in preterm infants are scarce.
In the present investigation we examined the vitamin A, D and E nutritional status of pre-term infants at birth and verified the hypothesis that supplementation with Uvesterol ADEC results in similar plasma concentrations of these vitamins in breast-fed and formula fed infants at 1 and 3 months of age.
PATIENTS AND METHODS
The protocol was approved by the "Comité Consultatif de la Recherche Biomédicale sur les Personnes, Lyon-A" for the 3 University-affiliated hospitals involved in the study. Since supplementation offers a potential therapeutic benefit for the infants included in such a study, ethical considerations forbid the presence of a control group. One hundred and thirty-five women, (114 singleton, 14 twin and 7 triplet pregnancies) were interviewed in chronological order. Seventy-six mothers (61 singleton, 10 twin and 5 triplet pregnancies) initially agreed to be included in the prospective study. Those who agreed were aged between 20 and 37 years (mean, 28.5; SD, 4.4) and were then asked whether or not they planned to breast-feed. To be included in the study, once their choice was made they had to accept either exclusive breast-feeding or formula feeding for a period of 3 months. None of the mothers received vitamin supplements during the last 3 months of pregnancy, and did they did not display signs of gestational diabetes or toxemia.
The mean gestational age of the infants included in the study was 33.5 weeks (SD, 1.0). None of the premature infants had bronchopulmonary dysplasia, necrotizing enterocolitis or chronic digestive disturbances. None received a vitamin D load in the first days of life. Babies with parenteral alimentation for more than 8 days were excluded. At each visit infants were examined, and length, weight and cranial circumferences were recorded. Formula feeding was standardised for the 3 Centres. Prenidal™ (Nestlé, Switzerland), containing 80 IU of vitamin D, 72 μg of vitamin A and 2 mg of vitamin E per dl was selected. All infants received 1 ml of Uvesterol ADEC® per day orally that contained 3000 IU (909 μg or 3.2 mol) of vitamin A in the form of retinol acetate, 5 IU (5 mg or 11.6 μmol) of vitamin E in the form of (DL) tocopherol acetate, and 1000 IU of vitamin D and 50 mg of vitamin C as recommended by Ricour et al. (17). Estimating a 50% patient loss at the conclusion of the study, it was calculated that a minimum of 50 infants would have to be enrolled to have an α error level of 0.05 and a β error level of 0.20 if a 20% mean increase in plasma vitamin A and E concentrations was accepted as significant.
Weight, to the nearest 5 g, was measured with an electronic scale, length with a stadiometer, and cranial circumference with a metric tape, both with a precision of 0.1 cm. Samples were serially collected in heparin coated tubes at entry into the study (D3), and after 30 (D30) and 90 (D90) days. Samples were immediately centrifuged and plasma was snap frozen until used. Samples were protected from UV light by wrapping tubes in aluminium foil during the processing. At the time of analysis, samples were thawed; tocopherol linoleate was added as internal standard (5μmol/L) and extracted with dichloro-methane. Vitamins A and E were measured by reverse phase high performance liquid chromatography on a C18 Spectrasyl® column as already described (18). Vitamins were quantified by UV spectrophotometry. 25-hydroxyvitamin D was measured by a radio-immunoassay following extraction in dichloromethane (IDS Laboratories, UK) and retinol binding protein (RBP) by immunonephelometry on the Immage (Beckman-Coulter, Brea, CA) using Dako monoclonal antibodies.
The significance of the difference in vitamins A and E concentrations between the breast-fed and formula-fed supplemented groups at D3, D30 and D90 was calculated with the two-tailed Student t test for unpaired variables with the Welsh's correction for unequal variances when appropriate. The difference in vitamin concentrations at the time of inclusion (D3), 30 and 90 days within the same group was assessed by an ANOVA repeated measures followed by Tuckey's multicomparison test. Descriptive statistics included mean and standard deviation.
Sixty-eight women (55 singleton, 9 twin and 4 triplet pregnancies) completed the study, leaving 62 formula-fed (FM) and 23 breast-fed (MM) infants. As shown in Table 1, weight and length at the time of entry were similar in both groups. Both weight and length were statistically higher in formula-fed infants at the conclusion of the study (P < 0.001). Sixty-six samples (19 from breast-fed and 47 from formula-fed infants) were available for vitamin A, E, 25 OHD and retinol binding-protein (RBP) measurement. Table 2 summarizes the data obtained. At birth, plasma vitamin A was at a concentration reflecting deficiency and was not statistically different between the 2 groups. The plasma concentration increased similarly in the course of the study in both groups of infants reaching mean values of 1.2 μmol/L and 1.1 μmol/L in the FM and the MM groups at 90 days, respectively. The initial plasma α-tocopherol concentration was also low in both groups and equally increased from the time of inclusion to 3 months, attaining mean values of 23.3 μmol/L and 22.7 μmol/L for the FM and MM groups, respectively. By 90 days of supplementation, only 9 infants in the FM group and 1 in the MM group reached the vitamin A repletion threshold level, (1.5 μmol/L) suggested by the Expert Panel on Vitamin A Nutrition (19). This contrasts with the vitamin E data for which only one infant in the MM group had a vitamin E concentration below the repletion threshold of 16.5 μmol/L (20). At entry, plasma RBP concentrations in the FM and MM groups were low and not different. They increased at the same rate to 0.85 ± 0.45 and 0.64 ± 0.25 at 30 days and to 1.05 ± 0.32 and 1.01 ± 0.38 μmol/L at 90 days. At birth, plasma 25(OH)D concentrations were comparable for the FM and MM groups and increased during the study reaching similar mean values at 90 days.
Figure 1 depicts the correlation between the plasma vitamin A and RBP concentrations at the 3 times of blood sampling. There is a significant relationship between the 2 variables, vitamin A accounting for 33% of the variation in RBP concentration.
There is a paucity of published longitudinal multicentre studies on circulating levels of vitamins A and E and RBP concentrations in pre-term infants. Our study provides such data up to 3 months of life.
At 90 days of life, both weight and length were slightly but statistically higher in the formula-fed infants than in the breast-fed infants. This observation has been reported before. Butte et al. (21) have shown, in a prospective study, a higher weight gain velocity accompanied by a higher fat-free mass gain in formula-fed than in breast-fed term infants. Putet et al. (22) have shown that weight and length gains were greater in very-low-birth-weight infants fed preterm formula milk than in those fed pooled pasteurized human milk.
Vitamins A, E and D are three very important nutrients in infancy. This is particularly true in early infancy, at a time of rapid growth. At birth, plasma vitamin A and E concentrations were at levels that, in children and adults, are felt to represent clinical deficiency (19,20,23). These observations agree with those of Shenai et al. (24) who have reported that 82% of preterm infants (<36 wks of gestation) studied had plasma retinol concentrations below the minimum adequate level (200 μg/L) and retinol-binding protein also below the adequate level (30 mg/L or 1.5 μmol/L). In the present group of premature newborn infants, plasma 25(OH)D concentration was within the normal range, reflecting the active vitamin D supplementation program during pregnancy that has resulted from prior studies and recommendations (25-28).
This longitudinal study demonstrates that oral supplementation increased plasma vitamins A, E and 25(OH)D concentrations. However, supplementation for 90 days with 3000 IU (3.2 μmol) of vitamin A and 5 mg (11.6 μmol) of vitamin E per day did not achieve plasma vitamin A and E concentrations characteristic of the repletion status. These observations contrast with our previous study, in which we reported that a similar supplementation protocol was sufficient to bring plasma vitamin A and E concentrations within the repletion range (29). This difference may be explained by the fact that the former study involved a group of term babies whereas the present one concerns pre-term infants. The failure to achieve full repletion vitamin A concentrations may, in part, be explained by the incomplete maturity of the fat digestion mechanisms at birth in premature infants, which develop within 3 to 4 months of life. This hypothesis derives from the report of Manson et al. (30) who have shown, by stable isotope breath-tests that the capacity to digest fat was incomplete in term and preterm infants and improved during the first months of life. They also showed that the maturation process was delayed in preterm infants. Picaud et al. (31), who evaluated the effect of dietary cholesterol on vitamin D metabolism in preterm neonates, reached a similar conclusion. Lipolysis of complex lipids appears to be the limiting step in digestion and absorption by the enterocytes (32). Although we have shown in ex-vivo studies on human fetal explants that lipid uptake at the apical membrane and secretion at the basolateral membrane are functional early in the foetal life (33-35), these results do not preclude the hypothesis that the efficiency of the process is not optimal in in vivo situations.
The pattern of plasma RBP results corroborates that of vitamin A. Indeed, its concentration increased in parallel with that of vitamin A, without reaching levels that are considered normal in the pediatric population (1.5 μmol/L). The fact that RBP, a protein that insures the transport of vitamin A to the peripheral target tissues (36), increased with vitamin A repletion supports the contention that the premature intestine absorbs this lipid-soluble vitamin and that the liver appropriately responds to the supplementation. This situation is similar to that with vitamin D (37-39). Our data thus underline that the gut absorption and secretion processes, in infants of less than 34 weeks of gestation, are functional and that I.V. or I.M. administration of vitamin is not necessary to improve the vitamin status.
Our results differ from those of Wardle et al. (40) who have shown that a 28-day supplementation of pre-term infants with vitamin A by orogastric gavage was insufficient to achieve serum retinol concentrations different from the placebo-control group. It must be stressed that their group of infants had a much lower birth weight (<1000 g) that the subjects of our study.
The biology of vitamin E, whose most abundant and active form is α-tocopherol, is complex. It is accepted that α-tocopherol must be located in membrane sites well exposed to reactive oxygen species (ROS), to efficiently act as a free radical scavenger (12). Vitamin E requirements of premature infants are unclear because the assessment of vitamin status is difficult. Some investigators have suggested that preterm infants fed breast milk may not need vitamin E supplementation. However, because vitamin E stores are low at birth, particularly in pre-term infants, and because of the increased risk of oxidative stresses in preterms, it is reasonable to supplement with vitamin E especially as breast-milk content in vitamin E decreases with time (41).
The premature infants in this study were not vitamin D deficient at birth, in contrast to former reports (37-39). This reflects the supplementation policy in pregnant women that is now enforced in France. Plasma 25OHD increased from the time of entry into the study to 3 months to values compatible with vitamin repletion without reaching levels that are considered as toxic (200 nmol/L).
There are no data available on the long-term effects of marginal vitamin A and E deficiency. This is most probably due to the difficulty of clinical assessment in view of the lack of reliable indices. We are therefore unable at this stage to predict the clinical importance of our findings in these two groups of patients. This uncertainty contrasts with vitamin D for which clinical outcome has been well documented by Zamora et al. (42). They have shown, in a retrospective study involving 149 pre-pubertal Caucasian girls, that vitamin D supplementation in infancy was associated with greater bone mineral content and increased bone mineral density.
In conclusion, we have shown that vitamin A and vitamin E administered at physiological levels according to the recommendations of ESPGAN (43) and the expert panel on dietary antioxidants and related compounds (19) for a period of up to 3 months was associated with an increase in plasma vitamin A and E without reaching full repletion values. We have also shown that the type of nutrition did not affect the plasma vitamin concentrations. Although our results suggest that a longer supplementation period in Vitamin A and E might be beneficial to preterm infants, a firm conclusion cannot be drawn, since the structure of the present study does not allow us to distinguish the effect of supplementation from the influence of time and maturity.
Acknowledgments: The authors wish to thank Ms. Chantal Dagenais for her expert secretarial contribution, Dr. Claire Dupuis and M. Denis Lemoyne for their help in the analysis of the vitamins. This work has been funded in part by an unrestricted grant from les Laboratoires Crinex, Montrouge, France and by the Hôpital Ste-Justine Research Centre.
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