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,3
* The Huck Institutes of the Life Sciences,
Department of Nutritional Sciences, The Pennsylvania State University, University Park, PA;
Boston College, Boston, MA;
** Mel and Enid Zuckerman Arizona College of Public Health, University of Arizona, Tucson, AZ;
Tribhuvan University, Kathmandu, Nepal; and
National Eye Hospital, Kathmandu, Nepal
3To whom correspondence should be addressed. E-mail: fxr5{at}psu.edu.
ABSTRACT
Free holo-retinol binding protein (RBP) [i.e., unbound to transthyretin (TTR)] plays a role in transporting vitamin A across the placenta during pregnancy. In a cross-sectional study of clinically healthy urban women, we assessed the association among clinical and biochemical factors on estimated concentrations of free holo-RBP during the last trimester of pregnancy. Serum samples obtained from a subsample of women (n = 259), who had participated in the Night Vision Threshold Test study in Nepal, were analyzed for determinations of retinol by HPLC, and RBP, TTR, and 
-1 acid glycoprotein by radial immunodiffusion. Free holo-RBP concentrations were calculated using dissociation constants for free holo- and apo-RBP. Among these women, 30% were vitamin A deficient based on either the RBP:TTR index 
0.36 or serum retinol < 1.05 µmol/L. Using stepwise regression analyses, the RBP:TTR index explained 75% of the variance in free holo-RBP concentrations, whereas retinol explained only 14%. Women were classified as vitamin A sufficient (n = 185) or deficient (n = 74) using the RBP:TTR index and were stratified into 3 gestational groups (I: 24–28 wk, II: 29–33 wk, III: >33 wk). Concentrations of free holo-RBP were higher in vitamin A–sufficient women than in vitamin A–deficient women (mean ± SEM, 48.1 ± 1.2 vs. 27.6 ± 0.8 nmol/L; P < 0.001), and in a 3 x 2 factorial analysis, the interaction between gestational group and vitamin A status was significant. These results demonstrate that the RBP:TTR index is a useful proxy for free holo-RBP concentration and that vitamin A status affects its distribution.
KEY WORDS: • retinol • retinol binding protein • RBP:TTR index • transthyretin • vitamin A
Vitamin A (VA)4 has a critical role in reproduction in addition to its essential roles in vision, growth, and immune function. It is required for embryogenesis through its active metabolite, retinoic acid, which is a morphogen (1); later in gestation, it helps to build up fetal VA stores. The transfer of VA from mother to fetus is carefully regulated in such a way that it allows VA levels in the fetus to remain unaffected by alterations in maternal VA status except in conditions of deficiency and excess (2).
Experimental studies in animals (3,4) and observational studies in humans (5,6) demonstrated that the increase in fetal VA stores occurs during the last weeks of gestation, and it is from the mother’s hepatic VA stores that most if not all of these fetal stores are derived. In part, this is due to enhanced transfer of retinol, alone or bound to retinol binding protein (RBP), across the placenta (7,8) and to less utilization of retinyl esters from chylomicra and other lipoproteins (9) by the placenta, unless in extreme conditions as shown in RBP knockout (–/–) mice (10). Although building up of hepatic VA stores is considered the main outcome, extrahepatic tissues such as the lungs are also dependent on the transfer of VA from the mother to the fetus. In neonatal rats, it was demonstrated that hepatic VA stores represent only one-third of the total body VA content (3). Additionally, maternal stores supply the placenta’s VA content, which increases by 6-fold in the last trimester of pregnancy (3). Together, these observations indicate a substantial effect on the mother’s VA stores in late gestation. For example in pregnant lambs, 
0.88 µmol/L of retinol is transferred daily from ewes to fetal lambs with only 20% being recycled back to the ewe (8). Moreover, in populations in which VA deficiency is a public health problem, night blindness is more likely to occur in the last trimester of pregnancy, suggesting a demand on the mother’s VA stores (11).
During the last trimester of gestation, holo-RBP unbound to transthyretin (TTR) plays an important role in transporting VA from mother to fetus (12,13). This was corroborated by studies showing the appearance of 2 major peaks in gel filtration chromatography representing retinol bound to the RBP-TTR complex and retinol bound to RBP alone and not to TTR (free holo-RBP) in the plasma of pregnant women in the 3rd trimester (14). In addition, a recent study reported that in pregnant women, the distribution of retinol bound to the RBP-TTR complex decreases (15). These findings indicate that unlike nonpregnant individuals, the proportion of free holo-RBP increases in pregnant women (14).
The measurement of free holo-RBP is intricate and logistically difficult (14,16–18). A simple method suggested by Fex et al. (19,20) is to calculate its concentration based on dissociation constants for apo-RBP (i.e., RBP without retinol) and free holo-RBP, and measured concentrations of serum retinol, RBP, and TTR. These dissociation constants describe the relation between total serum RBP, TTR, and retinol concentrations, and thus the formation of free holo-RBP and the retinol-RBP-TTR complex (20). In the present study, we calculated free holo-RBP in pregnant women and assessed clinical and biochemical factors associated with its concentration during the last trimester of pregnancy.
SUBJECTS AND METHODS
Subjects. This was a cross-sectional study of a subsample of pregnant women participating in the Night Vision Threshold Test (NVTT) study at the Prashuti Griha Royal Maternity Hospital, Kathmandu, Nepal (21). The purpose of the NVTT was to determine the rate of night blindness in pregnant women (21). The study was approved by the Institutional Review Board Human Subjects Committee at the University of Arizona, by the Nepal Health Research Council in Kathmandu, Nepal, and by the Hospital Director at the Prashuti Griha Royal Maternity Hospital. A total of 1401 clinically healthy pregnant women attending the prenatal clinic of the hospital were enrolled in the study if they were >24 wk pregnant. Their demographics and health data were obtained through a questionnaire at the time of enrollment. Weeks of gestation were based on the recall history of last menstrual cycle, and blood samples were collected from a nonfasting subsample of women enrolled in this study (n = 350) (21). Among women who provided blood samples, 259 (74%) samples were available for the present study.
Blood samples.
Blood samples were obtained using standard venipuncture procedures and were clotted in the dark. Serum samples were prepared within 2 h of blood collection and were placed in a Dewar flask under liquid nitrogen at –70°C and stored at –20°C until transported to the CDC in Atlanta, Georgia, for retinol analysis (21). Serum aliquots were later sent to The Pennsylvania State University for analysis of RBP, TTR, -1 acid glycoprotein (AGP), and serum total protein concentrations. Determination of RBP and TTR concentrations and the index were under a charge-out agreement between The Pennsylvania State University, Dr. Rosales’ laboratory, and the University of Arizona.
Biochemical analysis.
Serum retinol was analyzed using HPLC as previously reported (22). Serum total protein concentration was measured using the Bio-Rad protein assay method (23). Serum concentrations of RBP, TTR, and AGP were measured by radial immunodiffusion using a previously described method (24). Antiserum for RBP was from Accurate Chemical and Scientific, and TTR and AGP were from Dako. Calibrators and reference standards for TTR, AGP, and serum total protein were from Dako and Seronorm (Accurate Chemical and Scientific), respectively, and those for RBP from Cortex Biochem. The between-plates precision (CV) of RBP was 7.5% and that for TTR was 7.9%. Based on the external reference, the accuracy of RBP was 5.5 ± 1.5% (mean ± SEM) and that of TTR was 8.02 ± 2.0%. The RBP:TTR index was calculated by taking the ratio of the molar concentrations of serum RBP to TTR. The VA status was determined using the RBP:TTR index [VA deficiency 0.36 (25)] or using serum retinol [VA deficiency <1.05 µmol/L (26)].
Calculation of free holo-RBP. The concentration of free holo-RBP was calculated using dissociation constants for apo-RBP (k1 = 0.33 µmol/L) and free holo-RBP (k2 = 0.075 µmol/L) derived from 2-phase partition experiments by Fex et al. [Fig. 1, (20)]. According to Fex et al. (20), these dissociation constants remain fixed under normal physiologic conditions of pH and ionic strength. Equations for the concentrations of total retinol ([retinol]), RBP ([RBPtotal]), and TTR ([TTRtotal]) were also used for the calculation of the concentration of free holo-RBP (19). Total retinol concentration represents mainly the concentrations of free holo-RBP and of the retinol-RBP-TTR complex (Fig. 1, Eq. a) because free retinol is considered negligible. The concentration of RBPtotal (Fig. 1, Eq. b) is equivalent to the sum of the concentrations of apo-RBP, free holo-RBP, apo-RBP-TTR, and the retinol-RBP-TTR complex. Similarly, the concentration of TTRtotal (Fig. 1, Eq. c) is determined by adding the concentrations of free TTR, apo-RBP-TTR, and the retinol-RBP-TTR complex.
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 | (f) |
Statistical analysis.
Data were analyzed using SPSS version 11.5. Descriptive statistics were calculated and the data were normally distributed. Exploratory data analyses using measures of central tendency and scatter plots were used to assess the association between the various analytes and other variables. Comparisons between groups (i.e., VA-deficient vs. VA-sufficient) were made with a t test. Pearson correlation coefficients (r) were used to assess the linear association between the various variables and free holo-RBP. Variables whose correlations with free holo-RBP were at P 0.15 were selected for the initial regression model. Backward stepwise regression analysis (27) was used to determine the final model with the smallest number of predictors of free holo-RBP concentration (F-statistics, P < 0.05). Finally, using forward stepwise regression analysis, the predictor variable that explained most of the variability in free holo-RBP concentration was determined. A categorical variable, "Gestational group" was created based on weeks of gestation (i.e., group I = 24–28 wk, II = 29–33 wk, and III = >33 wk), and a 3 x 2 factorial design was used to assess the main effects and interaction between gestational group and VA status on free holo-RBP. A 2-way ANOVA under the general linear model was used. This was a mixed-effects model, in which gestational group was chosen as the fixed variable, and VA status as the random variable with free holo-RBP as the dependent variable. Data are expressed as means ± SEM unless otherwise indicated and a P-value < 0.05 was considered to be significant.
RESULTS
Mothers enrolled in this study were clinically healthy (Table 1). The mean age of these women was 22.9 ± 0.2 y; half of them were primiparous (57%), and they did not report any signs or symptoms of abnormal pregnancy. None were taking any VA supplements. The majority of women (92%) had AGP < 1 g/L, indicating a low risk of inflammation. The mean and median values of the RBP:TTR index were 0.44 and 0.41, respectively, indicating that the majority of these women were VA sufficient (25). The mean and median concentrations of retinol were 1.22 and 1.20 µmol/L, respectively, corroborating VA sufficiency in most of the women. However, one-third of these women were VA deficient as measured by the RBP:TTR index (VA deficiency 0.36) or serum retinol [VA deficiency < 1.05 µmol/L, (26)] (Table 1).
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DISCUSSION
In this study, we assessed the clinical and biochemical factors associated with the concentration of free holo-RBP in the last trimester of pregnancy. We found that the RBP:TTR index explained most of the variability of free holo-RBP concentrations (75%) compared with other predictors, and showed that VA-deficient women (i.e., RBP:TTR index 0.36) had lower concentrations of free holo-RBP in the 3rd trimester than VA-sufficient women, among whom free holo-RBP was higher in the later weeks than in the earlier weeks of the 3rd trimester (Table 4). Although these women were clinically healthy and their mean serum retinol concentration was not different from that reported in French pregnant women at the end of gestation (15), 30% of the women in this study had VA deficiency as measured using serum retinol < 1.05 µmol/L or as a RBP:TTR index 0.36. The concentration of serum total protein in these women was 57 ± 6.4 g/L (mean ± SD), which is within the limits of that reported by others in pregnant women during the 3rd trimester (29). Although this concentration is indicative of hemodilution during pregnancy, plasma or serum retinol did not seem to be affected markedly by hemodilution. Lockitch (30) demonstrated a nonsignificant effect on plasma retinol concentrations due to hemodilution in pregnancy. Thus, the concentration of serum retinol in the present study represented the effect of underlying VA deficiency as others have shown using a cutoff concentration < 1.05 µmol/L during pregnancy (26).
There is strong evidence that free holo-RBP plays an important role in the delivery of retinol to the placenta. Studies in rats showed that in early gestation, maternal RBP is transported across the placenta and delivers retinol, whereas in late gestation, a different mechanism appears to be operating because the fetal liver is capable of synthesizing RBP (9). In nonhuman primates, radiolabeled retinol is transported to the fetal plasma at a higher rate than radiolabeled RBP suggesting a dissociation of retinol from RBP, probably at the maternal/fetal side of the placenta (31). This has been corroborated by studies in vitro in which radiolabeled retinol from RBP was taken up by the placenta and converted into retinyl esters (32). Accordingly, in vivo studies showed that maternal RBP does not cross the placental membrane barrier in the last trimester of gestation (33,34). In addition, studies employing RBP knockout (–/–) mice, which express human RBP, demonstrated that retinol bound to RBP in the maternal circulation has to dissociate from the maternal RBP before it can be transported across the placenta (10). The major functional components of the human placenta are the villi, which project into the intervillous space, where they are bathed in maternal blood (35). It is within the intervillous space that the mixing of nutrients coming from the mother and waste from the fetus takes place, and this provides a milieu highly susceptible to changes in ionic strength (36). This is important because the release of retinol from the RBP:TTR complex as well as from free holo-RBP is influenced by ionic strength and to some extent by variations in pH (37). Sivaprasadarao and Findlay (33) showed that placental brush border uptake of retinol from the RBP-TTR complex was 50% slower than that from free holo-RBP. Noy and Xu (38) noted that this difference in rate constants represents the differences between affinity of retinol to RBP and the RBP-TTR complex. These studies suggest that in the intervillous space, conditions are favorable for the rapid exchange of retinol between its binding protein and syncytiotrophoblast membrane, which is the transporting epithelium of the human placenta. Thus, the concentration of free holo-RBP in maternal blood might reflect the extent of an increase in free holo-RBP concentration in the intervillous space. A similar relation was demonstrated for amino acid concentrations between maternal plasma and the intervillous space (39).
The concentration of free holo-RBP represents a small proportion of total RBP in clinically healthy populations. Studies using the dissociation of apo-RBP or free holo-RBP from TTR indicated that 5% of the total amount of RBP occurs unbound in plasma, and 10–20% of this is free holo-RBP (20). Fex et al. (19) showed that the free holo-RBP concentration was 3% of total RBP concentration in clinically healthy men. However, conditions such as renal failure can increase the concentration of free holo-RBP. Burri et al. (40), using molecular-exclusion HPLC, measured free holo-RBP concentrations in patients with renal failure and determined that free holo-RBP represented 11.3% of total RBP concentrations.
Moreover, pregnancy can increase the concentration of free holo-RBP. In female Japanese quail (Coturnix coturnix japonica) in which free holo-RBP was measured, a 2-to 3-fold increase in free holo-RBP during spring months reflected an increased VA requirement of birds during reproduction, and the additional demand for the transfer of vitamin A into the eggs (41). In humans, Sklan et al. (14) using gel filtration chromatography found an increase in free holo-RBP in maternal blood, which increased with gestation, compared with blood from nonpregnant women. Recently, Sapin et al. (15) reported that during pregnancy, there is a significant change in the distribution between retinol bound to RBP (free holo-RBP) and retinol bound to RBP-TTR complex. In the present study, using stepwise regression analysis, we showed that the RBP:TTR index explained 75% the variability in free holo-RBP. This is noteworthy because previous reports demonstrated in pregnant women and their fetuses that RBP and TTR concentrations are good predictors of maternal nutritional status (29) and gestational age of the fetus (42,43). In tandem, the RBP:TTR index has high sensitivity in predicting low hepatic VA stores (25), and recent studies demonstrated its usefulness in assessing VA status. Zimmermann et al. (44) found a significant increase in the RBP:TTR index in children receiving salt fortified with iodine, iron, and VA, compared with those receiving salt fortified with iodine alone. In addition, Zago et al. (45) using population reference distributions and receiver operating characteristic curve analyses, determined that a cutoff value 0.37 for the RBP:TTR index had high sensitivity and specificity in assessing VA status. Although these studies found the index useful in assessing VA deficiency, others have not. In the study by Donnen et al. (46), malnourished children received VA-rich foods (palm oil, fish, and sweet potato) as part of their rehabilitation treatment for malnutrition regardless of being allocated to VA supplementation or placebo groups; this could have confounded the assessment of the RBP:TTR index. In the study by Filteau et al. (47), children who had acutely ingested kerosene had "renal failure" and an enhanced renal excretion of retinol and RBP. Thus, it is quite possible that in "renal failure," there is a disproportional urinary excretion of RBP compared with TTR, resulting in a decrease in the RBP:TTR index. However, the mild inflammations in these children (i.e., mean C-reactive protein concentration of 5 mg/L) accompanied by a 33% reduction in RBP suggests other pathology such as VA deficiency, which after 3 mo might have lessened, and thus biased the sensitivity and specificity analysis of the RBP:TTR index.
Nonetheless, there was a high correlation between the RBP:TTR index and free holo-RBP concentrations. This association did not represent an adjustment for hemodilution because correcting serum RBP or retinol concentrations for serum total protein concentration did not enhance retinol or RBP correlation to free holo-RBP (data not shown). In addition, this correlation did not appear to result from using serum RBP and TTR concentrations in calculating both the RBP:TTR index and free holo-RBP concentrations. For example, there was no correlation between the molar ratio of retinol:RBP and free holo-RBP concentrations (Fig. 2, panel B) despite the fact that serum retinol and RBP concentrations were used in calculating these variables. Moreover, the correlation between free holo-RBP and retinol (r = 0.32) or RBP (r = 0.49) was enhanced after adjusting each for serum TTR concentrations. The RBP:TTR index and the molar ratio of retinol:TTR showed high correlations with free holo-RBP (r = 0.86 in both cases). However, the best linear predictor of free holo-RBP was the RBP:TTR index (Fig. 2) as demonstrated by the scatter plot of standardized predicted values against studentized residuals for each of these correlations (data not shown).
Finally, the factorial analysis in which every level of one factor is paired with every level of the other factor showed a significant ordinal interaction between gestational group and VA status on free holo-RBP concentrations (Table 4). Although this was a cross-sectional study and gestational weeks were determined on the basis of recalled history, this analysis was unbiased because there was no prior knowledge of the VA status in the selection of these women or in eliciting their history of last menses. Rather, the factorial analysis helped in understanding how free holo-RBP concentrations differed by VA status and weeks of gestation in the 3rd trimester when using the RBP:TTR index compared with serum retinol. In the future, the public health implications of these results should be assessed. It would be important to demonstrate that an increase in free holo-RBP concentration is associated with a reduced risk of maternal night blindness or a reduced risk of neonatal mortality.
ACKNOWLEDGMENTS
We thank Sin H. Gieng for the analytical determination of serum total protein.
FOOTNOTES
1 Presented in part at Experimental Biology 04, April 2004, Washington, DC [Sankaranarayanan S, Suárez M, Taren D, Genaro-Wolf D, Pfeiffer C, Sowell A, Rosales, FJ. Vitamin A status affects the concentration of free holo-retinol binding protein in pregnant women from Nepal (abstract). FASEB J. 2004;18:#358.7]. 
2 Supported by a grant from the United States Centers for Disease Control and Prevention and the Task Force Sight and Life.
4 Abbreviations used: AGP, -1 acid glycoprotein; NVTT, Night Vision Threshold Test study; RBP, retinol binding protein; TTR, transthyretin; VA, vitamin A.
Manuscript received 23 May 2005. Initial review completed 12 July 2005. Revision accepted 25 August 2005.
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