Effect of vitamin A status at the end of term pregnancy on the saturation of retinol binding protein with retinol1–3 Vincent Sapin, Marie C Alexandre, Samira Chaïb, Jean A Bournazeau, Patrick Sauvant, Patrick Borel, Bernard Jacquetin, Pascal Grolier, Didier Lémery, Bernard Dastugue, and Véronique Azaïs-Braesco

KEY WORDS Retinol, human, pregnancy, retinol binding protein, RBP, transthyretin, b-carotene, vitamin E, placenta, cord blood, holo-RBP, apo-RBP, saturation, France

(5–7) or hypervitaminosis A (8). In addition, long-term hypovitaminosis A affects female and male reproductive tracts, resulting in reversible sterility (9, 10). Women with low dietary vitamin A intakes have difficulty becoming pregnant, mainly because of the effect of vitamin A deficiency on their health status (11–13). In addition, their infants, who have low concentrations of vitamin A at birth, have a higher risk of morbidity, possibly because of an impaired immune status (14). In addition, human obstetric disease states (IUGR and placental disruptions) may be associated with abnormal maternal blood retinol concentrations (15, 16). Acute hypervitaminosis A during the embryogenic period of human pregnancy may result in teratologic effects (8, 17, 18). These considerations suggest that there should be a thorough regulation of vitamin A metabolism between the mother, the placenta, and the fetus during pregnancy. Vitamin A status and metabolism have been poorly investigated in well-nourished women with normal pregnancies compared with the large number of studies in pathologic and malnourished pregnant women (19–24). However, healthy pregnant women undergo important physiologic adaptations that might influence retinol concentrations, especially an increase in blood volume (25). The aim of the present study was to determine the concentrations and circulating quantities of retinol and b-carotene (the main provitamin A carotenoid) in maternal blood and in arterial and venous cord blood (a classic reflection of fetal status) at term in normal pregnancy and to compare them with values from nonpregnant women. Because retinol circulates in blood as a ternary complex associated with retinol binding protein (RBP) and transthyretin, the concentrations of these proteins were measured, as was the extent of saturation of RBP with retinol.

INTRODUCTION Vitamin A (retinol) compounds are fat-soluble micronutrients that are critical for many functions, including vision, reproduction, growth, and regulation of cell proliferation and differentiation (1, 2). Therefore, vitamin A is essential for successful gestation and is supplied by the mother to her fetus (3, 4). Obstetric abnormalities [fetal malformations, intrauterine growth retardation (IUGR), and spontaneous abortion] were observed during gestation in animals subjected to hypovitaminosis A

1 From INSERM U-384, Laboratoire de Biochimie, Faculté de Médecine; INRA UMMM, Equipe Vitamines, CRNH; and Unité de Médecine MaternoFoetale, Maternité de l’Hôtel-Dieu, Clermont-Ferrand, France. 2 Supported by an INSERM grant (to VS). 3 Address reprint requests to INSERM U-384, Faculté de Médecine, 28, Place Henri Dunant, BP 38, 63001 Clermont-Ferrand, France. E-mail: vincent. [email protected]. Received September 16, 1998. Accepted for publication July 13, 1999.

ABSTRACT Background: Vitamin A (retinol), which is required for normal fetal development and successful gestation, circulates in the blood bound to a specific protein, the retinol binding protein (RBP). Little is known about the transport and metabolism of this complex protein or about retinol status during normal human pregnancy. Objective: The aim of this study was to assess retinol status and transport modalities of retinol in well-nourished women with normal pregnancies, a population poorly investigated compared with pathologic and malnourished pregnant women. Design: The maternal blood and cord blood concentrations of retinol, vitamin E, b-carotene, RBP, and transthyretin of pregnant French women at term (n = 27) were measured and compared with values from a nonpregnant control group (n = 27). In addition, holo-RBP (retinol bound), apo-RBP (retinol free), and total protein were assessed in both groups to enable the hemodilution occurring during pregnancy to be taken into consideration and to evaluate the extent of saturation of RBP with retinol. Results: Healthy pregnant women at term had normal serum circulatory amounts of retinol, vitamin E, binding proteins, and b-carotene. However, they had less binding of retinol to RBP (holo-RBP: 49.9% in pregnant women, 54.0% in cord blood, and 77.5% in the control group). Conclusion: The results of this study suggest that retinol homeostasis and transport are modified during normal human pregnancy. Am J Clin Nutr 2000;71:537–43.

Am J Clin Nutr 2000;71:537–43. Printed in USA. © 2000 American Society for Clinical Nutrition

537

538

SAPIN ET AL

SUBJECTS AND METHODS

Vitamin A, vitamin E, and b-carotene assays

Subjects

Vitamin A (all-trans retinol and retinyl palmitate), vitamin E (a-tocopherol), and all-trans b-carotene were determined from assays of 0.5 mL thawed serum or 1 g placental tissue, as described previously, with some minor modifications (28, 29). An equal volume of ethanol containing internal standards (retinyl laurate, tocopheryl acetate, and echinenone for retinol, vitamin E, and b-carotene, respectively) was added to each sample. The samples were then extracted twice with 2 volumes of hexane. After evaporation to dryness, the extract was dissolved in 250 mL of a mixture of dichloromethane and methanol (35:65, by vol) and dispatched equally into 2 injection vials. The compounds were analyzed by reversed-phase HPLC on a Waters (Milford, MA) apparatus equipped with a 600 pump, a 710 automatic injector, and a 996 diode-array detector and controlled by MILLENNIUM 2.1 (Millipore Waters Chromatography, Millipore, France). For retinol and a-tocopherol (vitamin E) measurements, the samples were eluted on a Nucleosil 250 3 4.6-mm C18 column (Interchim, Montlucon, France) by using pure methanol as the mobile phase (2 mL/min); the detection was performed at 325 and 292 nm. b-Carotene was separated after elution of the sample on a Zorbax 250 3 4.6-mm C18 column (Interchim) with a mixture (1.8 mL/min) of acetonitrile, dichloromethane, and methanol (70:20:10, by vol) and detected at 450 nm. Identification was based on coelution with authentic standards and ultraviolet light–spectrum comparisons. Quantification involved internal standardization and dose-response curves established with authentic standards.

Thirty pregnant women were recruited from the Department of Obstetrics and Gynecology of Hotel-Dieu Hospital, Clermont-Ferrand, France, when they arrived for normal delivery. All subjects gave their written, informed consent before participation in accordance with the ethical standards of the responsible institutional committee on human experimentation. Exclusion criteria included multiple pregnancies, disease states during pregnancy (preeclampsia, fetal malformation, IUGR, infection, and hepatic or digestive disease), preterm labor, and vitamin A supplementation. Twentyseven healthy, nonpregnant women who had undergone analytic screening constituted the control group. These women had volunteered for various experimental protocols at the Human Nutrition Research Center, Auvergne. The control group (29.5 ± 3.2 y) was age-matched with the pregnant group (29.7 ± 5.4 y). The control group did not take supplements and their smoking habits were similar to those of the pregnant group: 9 pregnant women smoked and 8 control women smoked. The absence of pregnancy was certified by immunologic measurement of b-chorionic gonadotropin. The women in both groups were of the same socioeconomic background (middle class) and race (white). Dietary intake of vitamin A during pregnancy To assess the dietary intake of vitamin A and carotenoids, we used a self-administered, semiquantitative food-frequency questionnaire derived from the one described by Russel-Briefel et al (26). The pregnant and control groups were asked weekly to indicate the average frequency of consumption of 39 food items and of vitamin supplements. Information about the typical serving size of each food item was obtained by using pictures of servings for which corresponding weights had been validated (27). The 39 food items collectively accounted for > 95% of the vitamin A and carotenoid intake in the French population (J Ireland-Ripert, Centre Informatique sur la Qualité des Aliments, Paris, personal communication, 1991). The questionnaire was given to the women on their admission to the hospital and was completed before discharge from the hospital. We took care that women in the pregnant group had not eaten unusual foods (ie, items not included on the list of 39 items) that may be major sources of vitamin A. Women in the control group filled in this questionnaire when they visted the research center to have their blood sampled. The daily dietary intake of vitamin A was calculated by using GENI (Micro 6, Nancy, France). Sample collection and treatment When each woman arrived at the hospital for delivery, 5 mL venous maternal blood was collected before active labor began. The women had been fasting for > 11 h, except for 3 who had eaten a light meal 3–5 h earlier. After delivery, a piece of placental tissue was immediately frozen at 280 8C. Arterial and venous cord vessels were located by trained midwives using morphologic and color indicators, and arterial and venous cord blood were collected separately. The serum was prepared by clotting red cells for 4 h in the dark (2 h at room temperature and 2 h at 4 8C) and then centrifuging the samples at 1000 3 g for 10 min at 4 8C. Serum was separated into aliquots and frozen at 280 8C until analyzed. The control group provided blood samples after fasting overnight and the samples were processed as described for the pregnant group.

RBP, transthyretin, and total blood protein measurements Total RBP and transthyretin were measured by using nephelometric kits on a nephelometer (model BN 100; Behring SA, Marburg, Germany). Total blood proteins were measured by using the Biuret method (Hitachi 717; Boehringer Mannheim Diagnostics, Mannheim, Germany) (30). Retinol, b-carotene, vitamin E, RBP, and transthyretin concentrations were divided by their paired total protein concentrations to account for hemodilution. Holo-RBP and apo-RBP measurements Holo-RBP (retinol bound) and apo-RBP (retinol free) in serum were assessed by using polyacrylamide gel electrophoresis (PAGE) immunoblotting analysis. Whole serum was subjected to vertical-slab nondenaturing PAGE (7.5% acrylamide), in which the release of retinol from holo-RBP was shown to be insignificant (< 2%) (31). The proteins were separated according to their electrophoretic mobilities (with respect to their net charge and molecular weight) and were transferred onto a nitrocellulose sheet. Both holo- and apo-RBP immunoreactive bands were visualized by using a rabbit anti-human RBP serum diluted to 1:200 (Behring) and biotinylated goat anti-rabbit immunoglobulins, avidin-bound peroxidase (ABC Reagents; Vector Laboratories, Burlingame, CA), and diaminobenzidine (Sigma-Aldrich Corp, Saint Quentin Fallavier, France) as substrate. The holo- and apo-RBP percentages were determined after densitometry of the membrane with a CD8 camera (Sony, Kyoto, Japan) and were analyzed with PICLAB software (Rage, Marseille, France). The delipidized serum (containing only apo-RBP) was prepared by mixing 1 volume of human serum with 2 volumes of 1-butanol and diisopropyl ether (80:20, by volume) for 4 h at room temperature. The

VITAMIN A HOMEOSTASIS IN NORMAL PREGNANCY mixture was then centrifuged at 1000 3 g for 10 min at 48C and the organic solvent layer was discarded.

TABLE 1 Characteristics of the pregnant women and their newborns1

Statistical analysis

Characteristic

Mean values for arterial and venous cord blood were calculated and compared with the paired values. Mean (± SD) values were calculated for all variables. Group comparisons were made by using Student’s t test, either paired (to compare maternal with arterial or venous cord serum and arterial with venous cord serum) or unpaired (to compare serum from the control group with that from the pregnant group). Spearman’s rank-order correlation test was used to assess the relations among the variables. Statistical procedures were performed by using STATVIEW (Abacus Concepts, Inc, Berkeley, CA). For all of the studies, the criterion for significance was P < 0.05.

RESULTS Newborns and dietary intake during pregnancy Three of the 30 pregnant women recruited to the study were excluded from the final analysis (Table 1): 2 because they had hypotrophic newborns (birth weight < 10th percentile) (32) and 1 because she took vitamin A supplements during pregnancy [1200 retinol equivalents (RE)/d]. All of the newborns from the remaining mothers were healthy and their morphometric values were within the standard range for French newborns (32). Growth, development, and postpartum changes were normal for all mothers and newborns on the basis of a medical examination that occurred in the 7 d after delivery. The dietary intake of vitamin A (preformed retinol plus provitamin A carotenoids) was not significantly different between the pregnant group and the control group (2150 ± 1170 compared with 1691 ± 1110 RE/d, respectively) and was well above the French daily recommended intake (1000 RE) (33). Maternal and cord blood retinol, RBP, transthyretin, b-carotene, and vitamin E concentrations at term Maternal and cord blood concentrations of retinol, b-carotene, and vitamin E are shown in Table 2. Retinyl palmitate was not detectable in the cord blood of any of the women and was measurable in the serum of only 2 women, in whom it reached 46 and 34 mmol/L. These women had eaten a meal in the 5 h before the blood sampling; thus, retinyl palmitate likely came from the newly absorbed vitamin A. The women did not show any peculiarity in any other variables measured. There were no significant differences between arterial and venous cord blood in any of the variables measured. Therefore, data on arterial and venous cord blood were combined into a single group called “cord blood.” We found significant differences in concentrations of retinol, vitamin E, and b-carotene between cord blood and maternal blood, which were 2-fold, 5-fold, and 14-fold higher, respectively, in maternal blood than in cord blood. Maternal serum retinol and vitamin E concentrations were significantly lower in the pregnant group than in the control group. It is well established that blood volume increases by > 20% in pregnant women during the second half of pregnancy. The magnitude of this phenomenon can be quantified by the dilution of stable and pregnancy-independent serum constituents such as total proteins (25). We observed that the mean concentration of total proteins was 3 Newborns Sex Male Female Birth weight (g) Placenta weight (g) Percentile of body weight Length (cm) Anthropometry Head circumference (cm) Thoracic circumference (cm)

539

Value 29.7 ± 5.42 276 ± 9 14.2 ± 4.2 10 17 18 9

16 11 3357 ± 412 570 ± 96 53.3 ± 25.2 50.8 ± 4.2 35.2 ± 1.3 33.3 ± 1.7

1 All data for the mothers were collected during the last routine antenatal medical examination (eighth month); all data for the newborns were collected immediately after birth. 2– x ± SD.

pregnant group than in that of the control group. When the retinol, b-carotene, and vitamin E concentrations in both groups were expressed on the basis of the circulating protein amounts, there was no significant difference between the groups. Retinol is a hydrophobic molecule transported in blood, bound to its specific carrier protein (RBP), which itself forms a soluble ternary complex with transthyretin. Both of these proteins were measured in maternal, cord, and control blood (Table 2). As with the other variables, maternal RBP and transthyretin concentrations were significantly different from their cord blood counterparts and from the control data. When we expressed the values on the basis of circulating proteins, however, these differences between the pregnant group and the control group disappeared. Mean retinol and b-carotene concentrations in the placenta were 0.023 ± 0.008 and 0.004 ± 0.001 mmol/g tissue, respectively. Retinyl palmitate was detected in placental tissue in only one sample. Retinol saturation coefficient of RBP in maternal and cord blood at term There were no significant differences in molar ratios of retinol to RBP between the pregnant group and the control group or between arterial and venous cord blood. However, cord blood values were significantly different from maternal values (Table 2). In addition, we studied the retinol saturation coefficient of RBP by measuring the percentages of holo- and apo-RBP (Figure 1). As expected, holo-RBP was the major form of RBP in the control group (Table 2). No degraded forms of RBP were observed in the control group or in the pregnant group (Figure 1). There were major differences in the percentages of holo- and apoRBP between the pregnant group (49.9% and 46.5%, respectively) and the control group (77.5% and 19.7%, respectively).

540

SAPIN ET AL

TABLE 2 Vitamin A (retinol) status, vitamin E status, and total protein concentrations in nonpregnant women (control group) and in maternal, arterial, and venous cord blood at full term in pregnant women1 Control group

Maternal blood

Pregnant group Arterial cord blood

Venous cord blood

Retinol (mmol/L) 1.65 ± 0.50 1.27 ± 0.382,3 0.69 ± 0.39 0.74 ± 0.35 (mmol/g protein) 0.023 ± 0.007 0.021 ± 0.005 — — Vitamin E (mmol/L) 41.77 ± 11.26 36.10 ± 10.843,4 7.04 ± 4.09 7.06 ± 3.44 (mmol/g protein) 0.572 ± 0.154 0.535 ± 0.154 — — b-carotene (mmol/L) 1.13 ± 0.52 0.98 ± 0.463 0.069 ± 0.093 0.058 ± 0.084 (mmol/g protein) 0.015 ± 0.007 0.014 ± 0.006 — — RBP (mmol/L) 2.03 ± 0.55 1.49 ± 0.382,3 1.13 ± 0.42 1.07 ± 0.25 (mmol/g protein) 0.028 ± 0.007 0.024 ± 0.005 — — Transthyretin (mmol/L) 4.01 ± 0.88 3.23 ± 0.592,3 1.75 ± 0.58 1.73 ± 0.44 (mmol/g protein) 0.055 ± 0.012 0.048 ± 0.008 — — Retinol:RBP 0.811 ± 0.117 0.848 ± 0.1653 0.672 ± 0.227 0.744 ± 0.167 54.0 ± 15.8 53.6 ± 13.0 holo-RBP (%) 77.48 ± 12.40 49.9 ± 15.12 apo-RBP (%) 19.70 ± 11.30 46.5 ± 15.52 43.0 ± 15.8 40.9 ± 14.2 Total protein (g/L) 73.0 ± 4.4 62.4 ± 4.02 ND ND 1– x ± SD. RBP, retinol binding protein; ND, not determined. Values expressed per gram protein account for the hemodilution in the vascular compartment during pregnancy. 2,4 Significantly different from control group (Student’s t test): 2 P = 0.0001, 4 P = 0.01. 3 Significantly different from arterial and venous cord blood, P < 0.05 (Student’s t test).

Percentages of holo- and apo-RBP in maternal and cord blood were not significantly different. Relations among the different biological variables of vitamin A status at the end of pregnancy We found no correlation between vitamin A concentrations and anthropometric variables of mothers or newborns (Table 3). Retinol, b-carotene, RBP, and transthyretin concentrations; the molar ratio of retinol to RBP; and holo- and apo-RBP in arterial cord blood were correlated with their counterparts in venous cord blood. In addition, we observed that the maternal b-carotene concentration was correlated with the cord blood concentration and the placental tissue b-carotene concentration (r = 0.62, P < 0.05). Such correlations were not found for preformed retinol. As expected, RBP and transthyretin concentrations were correlated with each other in maternal and cord blood and retinol concentrations were correlated with RBP concentrations in all types of blood samples.

DISCUSSION In the present study, we measured several variables linked to vitamin A status in maternal and cord blood of French women with normal pregnancies and adequate vitamin A status. Generally, we found retinol and b-carotene concentrations equal to or higher than those found in malnourished pregnant women or women with disease states during pregnancy (5, 6, 22–24, 34–38). Any differences could be explained by differences in socioeconomic conditions or dietary practices. However, our data are consistent with data from the few studies performed in healthy, well-nourished pregnant women (39).

As described previously (40), retinol concentrations were lower in the pregnant group than in the control group of nonpregnant women. However, plasma volume expansion is known to occur during pregnancy (25), resulting in decreased concentrations of stable markers such as total blood proteins. This hemodilution phenomenon was cited recently as one of the explanations for the decrease in maternal concentrations of retinol during pregnancy (40). By correcting the retinol values by using the paired protein concentrations, we established that the corrected amounts of retinol were not significantly different between the 2 groups. This implies that the same quantities of retinol were present in the blood of pregnant and nonpregnant women, suggesting that the same absolute amounts were circulating in the pregnant group but at a lower concentration than in the control group. Similarly, vitamin E, RBP, and transthyretin concentrations were lower in the pregnant group in this study and others (41–43). The biological consequences of a lower concentration of the same quantity of vitamins or binding proteins are unknown. It might be that some steps of the metabolism or transfer of these compounds, such as the binding to a receptor or to an active enzymatic site, are conditioned by the concentration more than by the total quantity in the whole blood. The corrected amounts of total RBP in maternal blood at term were not significantly different from the raw amounts in control blood. However, the percentages of holo- and apo-RBP in maternal and cord blood were significantly different at term, reflecting a modification in retinol transport during gestation. In our control group, 19.7% of RBP was present in serum as apo-RBP. In the pregnant group, apo-RBP was 46.5%. Apo-RBP was rarely measured in human and animal studies and increased percentages of serum apo-RBP were described in

VITAMIN A HOMEOSTASIS IN NORMAL PREGNANCY

541

FIGURE 1. Polyacrylamide gel electrophoresis immunoblotting analysis of apo- and holo-retinol binding protein (RBP) in serum of pregnant women and a control (nonpregnant) group. Samples (10 mL) were subjected to an immunoblotting technique (nondenaturing conditions). Band intensity was read by densitometry. Lane 1: control blood; lane 2: delipidized control blood (showing only apo-RBP); lane 3: maternal blood; lane 4: arterial cord blood; lane 5: venous cord blood.

only 2 cases. First, during vitamin A deficiency in rodents (44), an increase in serum apo-RBP (with a decrease in total RBP) was reported when the serum retinol concentration was low as a result of a vitamin A–deficient diet. In our study, the pregnant group had normal vitamin A intakes and the amounts of retinol and total RBP in their serum, after correction for hemodilution, were similar to those of the control group; therefore, these women were not considered to be vitamin A deficient. Second, increased apo-RBP was reported in human chronic renal failure (45–47), together with an increase in serum total RBP and retinol, whereas serum transthyretin remained normal. According to the authors of these reports, this increase in serum total RBP and retinol was due to a decrease in the glomerular filtration rate (GFR) and an impairment in tubular

reabsorption. In contrast, in the pregnant group, corrected total RBP, transthyretin, and retinol in serum were similar to those of the control group, whereas serum apo-RBP was higher. In addition, clinical and biological data for the pregnant group in the present study did not indicate the occurrence of proteinuria. However, we cannot exclude the possibility that some pregnant women could have had incipient and biologically nondetectable microalbuminuria. Furthermore, the GFR increases dramatically during normal pregnancy (48), whereas it decreases during chronic renal failure. Consequently, the increased apo-RBP in the pregnant group cannot be explained by renal failure during pregnancy or by vitamin A deficiency. Thus, our results suggest that pregnancy is a physiologic situation in which apo-RBP concentrations can increase.

TABLE 3 Spearman’s rank correlations for study variables1

Rol MB Rol CB RBP MB RBP CB Holo MB Apo MB Holo CB Apo CB Rt MB Rt CB TTR MB TTR CB b-Car MB b-Car CB

Rol MB

Rol CB

RBP MB

RBP CB

Holo MB

Apo MB

Holo CB

Apo CB

Rt MB

Rt CB

TTR MB

TTR CB

b-Car MB

b-Car CB

1

NS 1

0.77 NS 1

NS 0.64 0.51 1

NS NS NS NS 1

NS NS NS NS NS 1

NS NS NS NS NS NS 1

NS NS NS NS NS NS NS 1

0.59 NS NS NS 0.7 NS NS NS 1

NS 0.7 NS NS NS NS NS NS NS 1

NS 0.53 0.66 NS NS NS NS NS NS NS 1

NS 0.68 NS 0.79 NS NS NS NS NS NS NS 1

NS NS 0.41 NS 0.51 NS NS NS NS NS NS NS 1

NS NS NS NS NS NS NS NS NS NS NS NS 0.73 1

1 As reported previously, no significant differences were detected between arterial and venous cord blood for any of the variables. Therefore, arterial and venous data were combined into a single group called “cord blood.” Only correlations > 0.4 (P < 0.05) are reported. Rol, retinol; MB, maternal blood; CB, cord blood; RBP, retinol binding protein; Holo, percentage of holo-RBP; Apo, percentage of apo-RBP; Rt, retinol-RBP molar ratio; TTR, transthyretin; b-Car, b-carotene.

542

SAPIN ET AL

In some studies, it was reported that abnormal quantities of total RBP (ie, 60–2380 mg/L) are excreted in the urine of pregnant women (49, 50) because of the increase in the GFR during pregnancy (48). It is also known that after holo-RBP delivers its retinol to target tissues, the resulting RBP—devoid of retinol (apo-RBP)— shows a lower affinity for transthyretin and is rapidly excreted in the urine (51). In our study, we showed that the percentage of apoRBP in serum was higher in the pregnant group than in the control group, which agrees with the findings of other studies. Using apo- and holo-RBP, we estimated that retinol was actually bound to only 0.0156 g RBP/L (49.9% of 0.031 g/L) in the pregnant group compared with 0.032 g/L (77.5% of 0.042 g/L) in the control group. Similar analyses were made for cord blood samples. This result implies that the molar ratio of retinol to holo-RBP was 1.05, 1.34, and 1.72 for control blood, cord blood, and maternal blood, respectively. The fact that one molecule of RBP binds only one molecule of retinol correlates well with the molar ratio found in our control group (1.05). In contrast, the ratios of 1.34 and 1.72 could not be explained by our current understanding of RBP. For example, these ratios imply that 0.53 mmol retinol/L was circulating in the maternal blood not bound to RBP. The results of solubility studies indicate that retinol cannot be free in serum at a concentration > 0.06 mmol/L (52). Moreover, an increase in free retinol would result in a higher urinary loss of retinol and predispose the woman to a vitamin A deficiency at term, a situation that has not been reported under conditions similar to those of our study. Two main hypotheses might explain our findings of higher apoRBP concentrations and retinol–holo-RBP molar ratios in the cord blood of the pregnant group than in the blood of the control group in the face of the solubility and renal-filtration results. The first is that retinol might be bound to another still-uncharacterized protein, as suggested previously by Sklan et al (53, 54). The second is that pregnancy and in utero life alter the affinity of RBP for retinol. This lower affinity, possibly due to the known higher GFRs in pregnant women than in nonpregnant women or to an interaction of RBP with placental factors in both the mothers and the neonates, could lead to an easier dissociation of the retinol from its binding site on RBP under in vitro conditions during the analytic procedures of sample preparation and electrophoretic separation. The fact that vitamin E and b-carotene were 5- and 14-fold more concentrated, respectively, in maternal blood than in cord blood may have been due to lower tocopherol and b-carotene transport capacity in newborns than in their mothers, as suggested previously for tocopherol (39, 55). Although no specific membrane receptor has been found for these compounds, the transfer of the compounds could occur by passive diffusion or binding to lipoproteins, for which a receptor is present on placental membranes (56, 57). In contrast, as described by others (21), retinol was only twice as concentrated in the maternal blood as in the cord blood. Using perfused placentas, Dancis et al (58) showed that retinol (bound to RBP) was transferred across the placenta quickly, totally, and without metabolism. It is now established that placental membranes possess a specific receptor for RBP involved in the retinol transfer through membranes (59). These placental characteristics, combined with the importance of vitamin A in fetal development, could explain the relatively high transfer rate for retinol between maternal and fetal blood. As reported previously, no significant differences were found between arterial and venous cord blood concentrations for any of

the variables studied, suggesting a balance between placental and fetal vitamin A homeostasis (41). However, we measured concentrations that may not reflect the dynamic exchanges potentially occurring in the fetal blood or in the placental circulation. In conclusion, our findings show that well-nourished, healthy pregnant women have a normal vitamin A status at term. However, changes in the saturation rate of RBP strongly suggest a physiologic adaptation of vitamin A metabolism during pregnancy, which could be related to the delivery of vitamin A to the fetus via the placenta. An understanding of the mechanisms of this transfer is needed so that the involvement of vitamin A in obstetric pathologies such as IUGR can be investigated further. We thank Larry W Robertson and R Taylor for critically reading the manuscript; all the midwives for their availability, professional competence, and kindness, which made this study possible; and Marion Brandolini, Anne Partier, and Beatrice Chauveau for their help.

REFERENCES 1. Blomhoff RB, Green MT, Green JB, Berg T, Norum KR. Vitamin A metabolism: new perspectives on absorption, transport and storage. Physiol Rev 1991;71:951–90. 2. Underwood BA. Vitamin A in human nutrition: public health. In: Sporn MB, Roberts AB, Goodman DS, eds. The retinoids: biology, chemistry and medicine. 2nd ed. New York: Raven Press, 1994:211–29. 3. Chambon P. The retinoid signaling pathway: molecular and genetic analyses. Semin Cell Biol 1994;5:115–25. 4. Morriss-Kay G, Sokolova N. Embryonic development and pattern formation. FASEB J 1996;10:961–8. 5. Shah RS, Rajalakshmi R. Vitamin A status of the newborn in relation to gestational age, body weight, and maternal nutritional status. Am J Clin Nutr 1984;40:794–800. 6. Shah RS, Rajalakshmi R, Bhatt RV, et al. Liver stores of vitamin A in human fetuses in relation to gestational age, fetal size and maternal nutritional status. Br J Nutr 1987;58:181–9. 7. Neela J, Raman L. The relationship between maternal nutritional status and spontaneous abortion. Natl Med J India 1997;10:15–6. 8. Rosa F. International experience with retinoic acid embryopathy. Teratology 1991;43:419–25. 9. Eskild W, Hansson V. Vitamin A functions in the reproductive organs. In: Blomhoff RB, ed. Vitamin A health and disease. New York: Marcel Dekker, 1994:531–59. 10. Van Pelt AMM, Van Dissel-Emiliani FMF, Gaemers IC, Van den Burg M, Tanke HJ, De Rooij DG. Characteristics of aspermatogonia and preleptotene spermatocytes in the vitamin A deficient rats testis. Biol Reprod 1995;53:570–8. 11. Uotila J, Tuimala R, Pyykko K, Ahotupa M. Pregnancy-induced hypertension is associated with changes in maternal and umbilical blood antioxidants. Gynecol Obstet Invest 1993;36:153–7. 12. Neel NR, Alvarez JO. Chronic fetal malnutrition and vitamin A in cord serum. Eur J Clin Nutr 1990;44:207–12. 13. Simsek M, Naziroglu M, Simsek H, Cay M, Aksakal M, Kumru S. Blood plasma levels of lipoperoxides, glutathione peroxidase, beta carotene, vitamin A and E in women with habitual abortion. Cell Biochem Funct 1998;16:227–31. 14. Humphrey JH, West KP, Sommer A. Vitamin A deficiency and attributable mortality among under 5-year-olds. Bull World Health Organ 1992;70:225–32. 15. Noback CR, Takahashi YI. Micromorphology of the placenta of rats reared on marginal vitamin-A-deficient diet. Acta Anat (Basel) 1978;102:195–202. 16. Sharma SC, Bonnar J, Dostalova L. Comparison of blood levels of vitamin A, beta-carotene and vitamin E in abruptio placentae with normal pregnancy. Int J Vitam Nutr Res 1986;56:3–9.

VITAMIN A HOMEOSTASIS IN NORMAL PREGNANCY 17. Rothman KJ, Moore LL, Singer MR, Nguyen NSDT, Mannino S, Milunsky A. Teratogenicity of high vitamin A intake. N Engl J Med 1995;333:1369–73. 18. Shaw GM, Velie EM, Schaeffer D, Lammer EJ. Periconceptional intake of vitamin A among women and risk of neural tube defectaffected pregancies. Teratology 1997;55:132–5. 19. Butte NF, Calloway DH. Proteins, vitamin A, carotene, folacin, ferritin and zinc in Navajo maternal and cord blood. Biol Neonate 1982;41:273–8. 20. Hussein L, El-Shawarby O, Elnaggar B, Abdelmegid A. Serum vitamin A and carotene concentrations among Egyptian fullterm neonates in relation to maternal status. Int J Vitam Nutr Res 1988;58: 139–45. 21. Rondo PHC, Abbott R, Rodrigues LC, Tomkins AM. Vitamin A, folate, and iron concentration in cord and maternal blood of intrauterine growth retarded and appropriate birth weight babies. Eur J Clin Invest 1995;49:391–9. 22. Dimenstein R, Trugo NMF, Donangelo CM, Trugo LC, Anastacio AS. Effect of subadequate maternal vitamin A status on placental transfer of retinol and beta-carotene to the human fetus. Biol Neonate 1996;12:230–4. 23. Ziari SA, Mireles VL, Cantu CG. Serum vitamin A, vitamin E and beta-carotene levels in preeclamptic women in northern Nigeria. Am J Perinatol 1996;13:287–91. 24. Tamura T, Goldenberg RL, Johnston KE, Cliver SP, Hoffman HJ. Serum concentrations of zinc, folate, vitamins A and E, and proteins and their relationships to pregnancy outcome. Acta Obstet Gynecol Scand Suppl 1997;165:63–70. 25. Blackburn ST, Loper DL. The hematologic and hemostatic systems. In: Eoyang T, ed. Maternal, fetal and neonatal physiology: a clinical perspective. Philadelphia: WB Saunders, 1992:159–201. 26. Russell-Briefel R, Caggiula AW, Kuller LH. A comparison of three dietary methods for estimating vitamin A intake. Am J Epidemiol 1985;122:628–36. 27. Le Moullec N, Deheeger M, Preziosi P, et al. Validation du manuel photos utilisé pour l’enquête alimentaire de l’étude SU.VI.MAX. (Validation of photographic document used to estimate the amounts of foods eaten by subjects in the Suvimax study.) Cah Nutr Diét 1996;31:158–64 (in French). 28. Borel P, Tyssandier V, Mekki N, et al. Chylomicron b-carotene and retinyl palmitate responses are dramatically diminished when men ingest b-carotene with medium-chain rather than long-chain triglycerides. J Nutr 1998;128:1361–7. 29. Borel P, Mekki N, Boirie Y, et al. Postprandial chylomicron and plasma vitamin E responses in healthy older subjects compared with younger ones. Eur J Clin Invest 1997;27:812–21. 30. Camara PD, Wright C, Dextraze P, Griffiths WC. Comparison of a commercial method for total protein with a candidate reference method. Ann Clin Lab Sci 1991;21:335–9. 31. Siegenthaler G, Saurat JH. A slab gel electrophoresis technique for measurement of plasma retinol-binding protein, cellular retinolbinding and retinoic-acid-binding proteins in human skin. Eur J Biochem 1987;166:209–14. 32. Leroy B, Lefort F. A propos du poids et de la taille des nouveauxnés à la naissance. (The weight and size of newborn infants at birth.) Rev Fr Gynecol Obstet 1971;66:391–6 (in French). 33. Dupin H, Ahaham J, Giachetti I. Apports nutritionnels conseilles par la population Francaise. (French nutritional recommended daily intakes.) Paris: Elsevier, 1992:52–5 (in French). 34. Ibrahim K, Hassan TJ, Jafarey SN. Plasma vitamin A and carotene in maternal and cord blood. Asia Oceania J Obstet Gynecol 1991; 17:159–64. 35. Dison PJ, Lockitch G, Halstead AC, Pendray MR, Macnab A, Wittmann BK. Influence of maternal factors on cord and neonatal plasma micronutrients levels. Am J Perinatol 1993;10:30–5. 36. Bougle D, Voirin J, Mouhadjer M, Herou M, Laniece M, Duhamel JF. Vitamin A status in full-term and premature infants. Pediatrie 1970;45:715–9.

543

37. Chan V, Greenough A, Cheeseman P, Gamsu HR. Vitamin A status in preterm and term infants at birth. J Perinat Med 1993;21:59–62. 38. Karakilcik AZ, Aksakal M, Baydas G, Sozen R, Ayata A, Simsek M. Plasma beta-carotene concentrations in pregnancies, newborn infants and their mothers. JPMA J Pak Med Assoc 1996;46:77–80. 39. Yeum K-J, Ferland G, Patry J, Russell RM. Relationship of plasma carotenoids, retinol and tocopherols in mothers and newborn infants. J Am Coll Nutr 1998;17:442–7. 40. Christian P, West KP, Khatry SK, et al. Night blindness of pregnancy in rural Nepal—nutritional and health risks. Int J Epidemiol 1998;27:213–37. 41. Sasanow SR, Spitzer AR, Pereira GR, Heaf L, Watkins P. Effect of gestational age upon prealbumin and retinol-binding protein in preterm and term infants. J Pediatr Gastroenterol Nutr 1986;5:111–5. 42. Mukono IS, Matsuo M, Komura M, Nakamura H. Higher serum retinol-binding protein concentration in Indonesian neonates in Surabaya than Japanese neonates in Kobe. Biol Neonat 1991;60:163–7. 43. Jain SK, Shah M, Ransonet L, Wise R, Bocchini JA Jr. Maternal and neonatal plasma transthyretin (prealbumin) concentrations and birth weight of newborn infants. Biol Neonat 1995;68:10–4. 44. Lespine A, Periquet B, Jaconi S, et al. Decreases in retinol and retinol-binding protein during total parenteral nutrition in rats are not due to a vitamin A deficiency. J Lipid Res 1996;37:2492–501. 45. Lillehoj EP, Poulik MD. Normal and abnormal aspects of proteinuria. I: Mechanisms, characteristics and analyses of urinary protein. II: Clinical considerations. Exp Pathol 1986;29:1–28. 46. Mogielnicki RP, Waldmann TA, Strober W. Renal handling of low molecular weight proteins. I. L-Chain metabolism in experimental renal disease. J Clin Invest 1971;50:901–9. 47. Smith FR, Goodman DW. The effects of diseases of the liver, thyroid, and kidneys on the transport of vitamin A in human plasma. J Clin Invest 1971;50:2426–36. 48. Dunlop W. Serial changes in renal haemodynamics during normal human pregnancy. Br J Obstet Gynaecol 1981;88:1–9. 49. Cheung CK, Lao T, Swaminathan R. Urinary excretion of some proteins and enzymes during normal pregnancy. Clin Chem 1989; 35:1978–80. 50. Beetham R, Dawnay A, Menabawy M, Silver A. Urinary excretion of albumin and retinol-binding protein during normal pregnancy. J Clin Pathol 1988;41:1089–92. 51. Goodman DS. Plasma retinol-binding protein. In: Sporn MB, Roberts AB, Goodman DS, eds. The retinoids. Vol 2. New York: Academic Press, 1984:41–88. 52. Szuts EZ, Harosi FI. Solubility of retinoids in water. Arch Biochem Biophys 1991;237:297–304. 53. Donoghue S, Richardson DW, Sklan D, Kronfeld DS. Placental transport of retinol in sheep. J Nutr 1982;112:2197–203. 54. Sklan D, Shalit I, Lasebnik N, Spirer Z, Weisman Y. Retinol transport proteins and concentrations in human amniotic fluid, placenta, and fetal and maternal serums. Br J Nutr 1985;54:577–83. 55. Léger CL, Dumontier C, Fouret G, Boulot P, Descomps B. A short term supplementation of pregnant women before delivery does not improve significantly the vitamin E status of neonates—low efficiency of the vitamin E placental transfer. Int J Vitam Nutr Res 1998;68:293–9. 56. Bonet B, Chait A, Gown AM, Knopp RH. Metabolism of modified LDL by cultured human placental cells. Atherosclerosis 1995; 112:125–36. 57. Wittmaack FM, Gafvels ME, Bronner M, et al. Localization and regulation of the human very low density lipoprotein/apolipoprotein-E receptor: trophoblast expression predicts a role for the receptor in placental lipid transport. Endocrinology 1995;136:340–8. 58. Dancis J, Levitz M, Katz J, et al. Transfer and metabolism of retinol by the perfused human placenta. Pediatr Res 1992;32:195–9. 59. Sundaram M, Sivaprasadarao A, DeSousa MM, Findlay JB. The transfer of retinol from serum retinol-binding protein to cellular retinol-binding protein is mediated by a membrane receptor. J Biol Chem 1998;273:3336–42.