the Science of the Total Environment The Science of the Total Environment 159 (1995) 119-127
Inter-individual
variation of selenium in maternal plasma, cord plasma and placenta
Anne M. Lee*a, Guy Huel”, Jean Godin”, Georgette Hellier a, Josiane Sahuquillo”, Thierry Moreaua, Phillipe Blotb aEpidemiological
Research bClinique
Unit, National ob&icale
Institute of Health and Medical Research, 16 avenue Paul Vaillant-Couturier, 94807 Wejuif Ceder, France Baudelocque, 123, Bouleuard de Port Royal, 75014 Paris, France
Received 21 October 1993; accepted 18 November 1993
Abstract Selenium (Se) in high doses has been known to cause injury to the fetus and newborn. The major difficulty in assessing the effects of seleniumon human reproduction stemsfrom the need for a suitable meansof estimating maternal and fetal exposure.The present investigation, therefore, examinesthe respectivereliability of maternal plasma,cord plasmaand placenta as epidemiologicalindicators as well as inter-individual variation of this trace element.An unselectedpopulation of 128pregnancieswasstudied.Obstetrical characteristicswere noted. Selenium concentrationswere determined for maternal plasma,cord plasma,and placental tissueby fluorometric analysis. Maternal plasma seleniumconcentrations (Se-Bm) were significantly greater than fetal concentrations (Se-Bc). Placental selenium(Se-PI) levels were four times that of fetal levels. Variability of Se-Bc is best explained by placental concentrations.Maternal weight and ethnic origin are significantlycorrelated with Se-Bc. Femalenewborn have higher seleniumlevelsthan male newborn. The present study demonstratesthe significanceof the placenta as an indicator of fetal seleniumexposure. Keywords:
Selenium;Placenta; Plasma;Newborn;
Human
1. Introduction Selenium (Se) is an essential trace element for man and is known for its role in regulating growth and development of the fetus and newborn [1,2]. The level of Se in blood is dependent on the quantity absorbed 131and recent studies in humans have shown a linear correlation between plasma and erythrocyte Se concentrations. A
*Corresponding
author.
paradoxical property of Se is that identical biological abnormalities arise from both Se insufficiency and excess[4]. In order to better understand what placentalselenium levels (Se-PI) represent as an indicator of fetal environmental pollution, it is important to study the influence of factors which might contribute noticeably to the inter-individual variations hitherto established. To date, blood has served as the biological material most commonly used in monitoring population exposure to selenium. Selenium re-
0048-9697/95/$09.50 0 1995 Elsevier Science BV. All rights reserved. SSDZ 0048-9697(95)04123-Q
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mains in the blood only briefly, hence, blood samples taken at birth are not necessarily representative of the environmental history of the fetus. In order to better evaluate changes in selenium levels over time, the human placenta may be a more appropriate tool. Placental concentrations of toxic substances reflect environmental contamination from the very early stages of gestation. The placenta is present in the uterus for several months and during this time represents the organ of maternal-fetal exchange [5]. The placenta is of added value as it provides pertinent information concerning the toxicological behaviour and dynamics of this biological interchange. It follows then, that the placenta should better reflect an average or integrated environmental exposure and provide a highly informative indicator of fetal selenium status as constituted by the mother’s body. In the present study, we hoped, firstly, to evaluate the usefulness and reliability of the placenta over blood as an environmental indicator, secondly, to investigate possible factors governing inter-individual variation of selenium levels in maternal plasma, cord plasma and placenta and thirdly, taking into account variation factors, to demonstrate that birth characteristics such as birthweight may be related to maternal exposure. 2. Materials
and methods
2.1. Population Mothers included in this study were under obstetrical care at the Baudeloque Maternity ward in Paris, France. Samples were obtained from 128 unselected pregnancies (mother and newborn pairs) and from 114 placentae. Tissue anomalies occurred in 14 placental samples. These abnormal specimens were sent to the anatomopathology department for diagnostic examination and were, therefore, unavailable for selenium analysis. All mothers were free of any pathological disease or ailment. Informed consent was obtained from all individuals. Information concerning maternal age, weight, height, parity, gestational age, ethnic origin, as well as family, social and obstetrical histories was obtained from all participants and recorded within
1.59 (1995)
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the framework of the Baudelocque perinatal study. Data regarding smoking habits, alcohol consumption and diet were obtained by direct interview conducted 3 days following delivery. The majority of the mothers studied were European (71.19%) while 28.9% of the mothers were of diverse ethnic origin and included African (17.2%), Asian (3.9%), and Antillian (3.9%) backgrounds. For the purpose of our analyses, mothers were divided into two classes, European and non-European. With regard to parity, subjects were categorized as either primiparous or multiparous. Subjects were also distinguished with regard to smoking habits and alcohol consumption. Mothers were classified as smoker (n = 22) if they smoked more than one cigarette daily during either the first, second and/or third trimester of pregnancy. They were classified as passive smokers (n = 42) if they were non-smokers during the pregnancy and more than two cigarettes per day were smoked in their presence throughout the entire pregnancy. Mothers were considered nonsmokers (n = 64) if they were not included in ‘stopped the smoker or passive groups. The smoking’ category consisted of mothers who smoked during the first trimester of pregnancy and who quit smoking before the last trimester of pregnancy was established. None of the participants began to smoke during the pregnancy. It should be noted that the total number of subjects is equivalent to the number of individuals which form the non-smoker, smoker and passive smoker groups. All other groupings are subdivisions of these main categories. With regard to alcohol, respondents who stated during the interview that they consumed more than 1 glass of alcohol weekly on a regular basis throughout the pregnancy were classified as consumers. All others were classified as non-consumers. Sex and birthweight of the newborn at delivery were noted and recorded. 2.2. Sample collection Maternal plasma samples (Se-Bm) were collected from the arm vein just prior to delivery. Cord plasma (Se-Bcl and placental (Se-Pl) samples, collected immediately after birth from the
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umbilical cord vein and placental tissue, respectively, were placed in polystyrene containers. Placental specimens of 4 X 10 g were cut from the dorsal (fetal) side of each placenta. Amounts of 0.5 ml of plasma and between 0.25 and 0.50 g of placental tissue were analyzed. All samples were frozen and stored at -80°C. 2.3. Chemical procedure
At the time of analysis, mineralization was achieved with 4 ml of an acidic mixture (nitric and perchloric acids). Samples were then placed in an electrothermic heater for 2 h at 100°C and overnight at 190°C. The following morning, samples were cooled and mixed with 1 ml of HCl and heated at 150°C for 30 min in order to reduce Se(W) to Se(W). Selenium levels were determined by the fluorometric method which involves the reaction of 2,3-diaminonapthalene (DAN) with Se(N) to form a fluorescent Se/DAN piazselenol. The validity of this method is discussed elsewhere [6]. The spectrofluorimeter was set at an excitation wavelength of 364 nm and emission wavelength of 523 nm. Selenium concentrations were measured against standard stock solutions. Plasma and placental selenium levels reported here are in units of rig/ml and rig/g, respectively. 2.4. Statistical analyses
Statistical analyses of the data were performed using classical statistical techniques; t-test, Pearson correlation coefficients and multiple regression analysis. Differences between mothers and newborn were tested by paired statistics. As the selenium distribution in all three compartments was skewed (Figs. 1, 2 and 31, average values are expressed as geometric means. Birth weight was adjusted for gestational age, sex, and maternal weight and height. The usefulness of such an adjustment has been described previously [7-lo].
parous; 55.5% of the births were male and 44.5% were female. 3.2. Descriptive characteristics of seleniumin maternal plasma, cord plasma and placenta
Histograms of the data reveal a consistent tendency towards log normality of the distribution, as illustrated in Figs. 1, 2 and 3. Table 1 lists the descriptive characteristics of Se-Bm, Se-Bc and Se-PI. Analysis of the results reveals that the mean of differences for selenium between mother and newborn is both positive and significant (paired t-test; P < 0.001). Selenium levels (geometric means) were highest in the placenta with concentrations four times that of the newborn and three times that of the mother. Such comparisons, however, remain suggestive as we are dealing with different biological tissues. 3.3. Relationship between Se levels in the mother, newborn and placenta
Table 2 summarizes the interrelationships between Se-Bm, Se-Bc and Se-PI. Highly significant correlations of 0.39 (P < 0.001) between Se-Bm and Se-Bc and of 0.49 (P < 0.001) between Se-Bm and Se-PI were observed. A highly significant correlation of 0.52 (P < 0.001) also exists between Se-Bc and Se-PI. To determine the actual contribution of Se-Bm and of Se-PI to the variability of Se-Bc status, a multiple regression was perNumber
Maternal Selenium
3. Results 3.1. Sample characteristics
The average age of the study population was 28.7 years (S.D. = +5.04). Of the mothers studied, 54.4% were primiparous, 45.6% were multi-
Fig. 1. Frequency distribution levels (in rig/ml).
of maternal plasma selenium
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A.M. Lee et al. /Sci. Total Emiron. 159 (1995) 119-127 Table 1 Average values of selenium concentrations in maternal plasma (Se-Bm), cord plasma (Se-Bc) and placenta (Se-PI)
Number
Cord Selenium
Parameter
Se-Bm (rig/ml)
Se-Bc (rig/ml)
Se-PI Wg)
Geometric means 5th-95th percentiles
68.6 49.5-95.6 128
50.1 38.3-68.5 128
209.1 172.9-262.2 114
; < O.OOOla aPaired t-test.
Fig. 2. Frequency distribution levels (in rig/g).
of placental tissue selenium
formed on the data (Table 3). According to the results, the relationship between Se-Bc and Se-P1 remains highly significant (P < 0.01) while the significance of the relationship between Se-Bc and Se-Bm decreases, yet nevertheless, remains significant (P < 0.05). 3.4. Relationship with quantitative maternal characteristics Table 4 demonstrates a statistically significant Number
cental enium els g/g)
Fig. 3. Frequency distribution levels (in rig/g>.
of placental tissue selenium
correlation between Se-Bc and maternal weight (r = +0.26; P < 0.01). Similar associations were not observed for Se-Bm and Se-P1 concentrations. No significant correlation was found for the variables: age at delivery, and maternal height. Table 5 compares mean selenium concentrations with parity. No statistically significant difference in selenium levels was noted for multiparous as opposed to primiparous mothers. 3.5. Relationship with qualitative maternal characteristics As shown in Table 6, a strong association exists between ethnic@ and Se-PI and Se-Bc levels (ttest, P < 0.0001) and to a lesser extent with Se-Bm (t-test, P < 0.05). Mean selenium values were consistently lower for Europeans vs. nonEuropeans. No statistically significant relationship was determined for smoking habits during pregnancy. With regard to alcohol consumption during pregnancy, mean Se-Bm concentrations were significantly statistically higher (t-test, P < 0.05) for mothers who consumed alcohol on a regular basis. 3.6. Relationship with newborn characteristics According to Table 7, increased Se-Bc and Se-PI levels were obtained for female newborn (52.5 rig/ml vs. 48.9 rig/ml in cord plasma for female and male newborn, respectively and 214 rig/g vs. 205 rig/g for the placenta of female and male newborn, respectively). These differences were statistically significant. No statistically significant relationships were noted for Se-Bm lev-
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Table 2 Correlation coefficients between selenium concentrations in maternal plasma (Se-Bm), cord plasma (Se-Bc) and placenta (Se-PI) Se-Bm
Parameter
Se-Bc
Table 3 Regression coefficients for selenium cord plasma concentrations (Se-Bc) as a function of maternal plasma (Se-Bm) and placental (Se-PI) selenium concentrations Dependant variable
Se-Bc
Pearson correlation coefficient (r) P-value n
0.39
Regression coefficient P-value n
Se-PI
n
els. Significant for gestational newborn.
Independant variables Se-PI
Se-Bm
0.65 < 0.01 114
0.21 < 0.05 114
Se-Bc
< 0.0001 128
Pearson correlation coefficient (r 1 P-value
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159 (1995) 119-127
0.49
0.52
< 0.0001 114
< 0.0001 128
relationships were not observed age and adjusted weight of the
4. Discussion
The present investigation reveals a strong correlation between selenium levels in human maternal, newborn and placental tissues, which implies that selenium passes readily across the placenta [ll]. Our results confirm findings published by Baglan et al. [12] in an earlier study in which human Se-PI, Se-Bc and Se-Bm levels were highly
correlated. Schramel et al. [13] have also determined a positive association between human Se-Bm and Se-Bc levels. However, according to their report, the correlation only seems to exist for low maternal selenium pools, perhaps an indication of saturation limits with regard to selenium uptake. Bostedt and Schramel [14] state that selenium uptake by the fetus in cattle appears to be selective, depending on the amount available. In contrast, Korpela et al. [15] argue against a statistically significant correlation between Se-Bm and Se-Bc (21 parturients and newborn). Correlations were not performed for all three compartments. BouglC et al. [16] found no correlation between Se-Bm and Se-Bc levels (38 parturients
Table 4 Correlation coefficients between selenium concentrations in maternal plasma (Se-Bm), cord plasma (Se-Bc), placenta (Se-PI), maternal age at delivery, maternal weight and maternal height Parameter Maternal
Se-Bm” - 0.02 NS
- 0.08 NS
- 0.09 NS
weight
Pearson correlation coefficient (r) t-test Maternal
Se-Plb
age at delivery
Pearson correlation coefficient (r ) t-test Maternal
Se-Bc”
0.12 NS
0.29 P < 0.001
0.09 NS
height
Pearson correlation coefficient Cr ) t-test NS. not significant. an = 128. ‘n = 114.
0.05 NS
0.04 NS
0.04 NS
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Table 5 Comparison of average selenium concentrations of maternal plasma (Se-Bm), cord plasma (Se-Bc) and placenta (Se-PI) with parity Parity
Se-Bm (rig/ml)
Se& (rig/ml)
Se-PI (rig/g)
67.2 47.3-86.3 72 NS”
50.1 34.9-66.1 12 NS”
208.9 167.1-249 62 NV
70.3 53.5-101.7 56
50.9 31.9-69.7 56
209.3 173.8-266.6 52
Primiparous
Geometric mean 5th-95th percentiles n Multiparous
Geometric mean 5th-95th percentiles n
“Comparison between primiparous and multiparous t-test: not significant.
and 39 newborn). Such discrepancies with our results may be attributed to the small sample sizes employed in both reports [15,16] and, therefore, to the lack of statistical confidence of the analyses performed. Our study established greater amounts of SeBm than Se-Bc. We estimated that the mother had approximately 30% more plasma selenium than the fetal compartment. It appears then, that the placenta may act as a barrier limiting the amount of selenium received by the fetus. Our findings are in agreement with those of BougM et al. [16] who determined both a positive and significant difference between Se-Bm and Se-Bc levels. Korpela et al. [15] on the other hand, cite no difference between Se-Bm and Se-Bc. The results from their paper agree with previous studies [17,18]. Our analysis implies that selenium concentrates in the placenta. Although it is difficult to compare placental and plasma1 concentrations, as previously mentioned, we are nevertheless in agreement with BouglC et al. [16] who have found that ‘placental levels were 3-fold higher than in both maternal and fetal plasma levels’. This suggests a certain retention of selenium in the placenta. It has been determined in sheep that placental transfer is bi-directional, an indication that the placenta is permeable to selenium on both sides. This bi-directional movement may have a bearing on the net retention of selenium in maternal, fetal and neonatal tissues [19]. It is thought that high doses of selenium may have toxic effects on
placental transfer mechanisms [20]. The strong correlation between selenium levels in all three compartments and its retention in the placenta, may argue for a transport process perhaps more complex than that of simple passive transport. In addition, the extent of transplacental transfer depends on the selenium compound involved as various forms cross the placenta with varying ease [21]. As our analysis is observational in nature and not experimental, this latter characteristic could not be verified. Differences with regard to maternal weight and ethnic origin may be explained by variations in diet since the selenium intake of the study population was mainly by accumulation through ingestion. In general, food is the primary route of selenium exposure and substantial variations in dietary intake exist in various parts of the world [22]. BouglC et al. [16] report a significant correlation between birthweight and Se-Bc in a small sample size (n = 39). As with our findings, other investigations do not confirm their results. Nevertheless, experimental studies have shown that long-term prenatal exposure to selenium in rodents decreases fetal weight [23]. That gestational age does not appear to influence selenium levels has been previously established [24,25]. Perhaps the most salient feature of our study is the unexpected difference in selenium status with respect to sex of the newborn. Female newborn exhibit higher amounts of plasma selenium than male newborn. Similarly, Brown and Burke [26] demonstrated that female rats show significantly
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159 (1995)
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Table 6 Comparison of selenium concentrations in maternal plasma (Se-Bm), cord plasma (Se-Bc) and placenta (Se-PI) with ethnicity, smoking habits and alcohol consumption Parameter
Se-Bm (rig/ml)
Se-Bc (rig/ml)
Se-PI (q/g)
67.1 49.5-91.6 92 P < 0.05
48.4 35.9-69.7 92 P < 0.0001
202.9 166.3-244.3 81 P < 0.0001
72.6 48.2-101.3 36
56.4 42.2-76.5 36
225.1 187.4-278.0 33
68.3 49.3-97.2 64
50.1 38.9-69.1 64
210.1 174.0-262.3 57
67.5 49.2-88.5 22 NSa
51.2 35.7-100.3 22 NY
215.7 177.3-296.3 21 NY
68.1 48.3-79.5 14 NS
52.8 40.3-109.4 14 NS
218.4 189.9-302.9 13 NS
67.1 55.0-89.9 11 NS
49.7 34.9-59s 11 NS
213.4 175.9-243.0 11 NS
69.4 46.3-107.5 42 NS
50.6 30.3-68.6 42 NS
203.8 153.6-267.1 36 NS
66.8 49.3-89.7 81 P < 0.05b
51.0 36.6-69.1 81 NSb
208.6 164.5-258.7 72 NSb
71.6 48.3-98.9 47
49.5 38.8-63.5 47
210.0 179.9-265.5 42
Ethnic@ European
Geometric mean 5th-95th percentiles n Non-European
Geometric mean .5th-95th percentiles n Smoking habits Non-smokers
Geometric mean 5th-95th percentiles n Smoker
(1st himester)
Geometric mean 5th-95th percentiles n Smoker
(2nd and 3rd trimester)
Geometric mean 5th-95th percentiles n Bopped
smoking
Geometric mean 5th-95th percentiles n Passiw
non-smoker
Geometric mean 5th-95th percentiles n Alcohol Non-consumer
Geometric mean 5th-95th percentiles n Consumer
Geometric mean 5th-95th percentiles n
aComparison between smoker and non-smoker; t-test: NS, not significant. bComparison between alcohol consumer and non-consumer; t-test: NS, not significant.
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Table I Comparison of selenium concentrations in maternal plasma (Se-Bm), cord plasma (Se-Bc) and placenta (Se-PI) with sex Sex Male Geometric mean 5th-95th percentiles n Female Geometric mean 5th-95th percentiles n
Se-Bm (rig/ml)
Se-Bc (rig/ml)
Se-PI Wg)
68.4 49.2-91.8 71 NV
48.9 35.8-65.0 71 P < 0.05a
205.2 168.4-250.9 64 P = 0.05a
68.7 49.5-98.7 57
52.5 38.8-69.6 57
214.3 172.1-278.5 50
aComparison between male and female; t-test: NS, not significant.
higher selenium retention in all tissues except the brain. It is apparent from the present study that sex differences in selenium concentrations begin during in utero life. Further investigation is necessary in order to determine whether such a difference can be attributed to varying metabolic requirements and development absorptivity. A chromosomal or hormonal influence may alter selenium levels in the sexes [27]. 5. Conclusions The use of human placenta as a biological indicator of fetal environmental exposure could be envisaged due to, in part, the strong correlations between Se-PI, Se-Bm and Se-Bc (Table 2) and the fact that the placenta best explains the variability of Se-Bc (Table 3). These factors along with the apparent selenium retention by the placenta suggest that the placenta may act as a barrier or regulator of fetal selenium transfer. Potential confounding factors such as sex of the newborn, maternal weight and ethnic@ must be taken into consideration in future epidemiological studies. Our analyses provide baseline data for further investigation in our understanding of maternal, fetal and placental selenium metabolism and toxicology.
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