孕期重金属元素和微量元素水平与子代先天性心脏病相关性的前瞻性队列研究北大核心CSCD

目的探讨孕期重金属元素和微量元素水平与子代先天性心脏病(简称先心病)的相关性,并建立孕期重金属元素和微量元素水平与子代先心病发生概率的预测模型。方法基于2010~2012年在甘肃省妇幼保健院开展的前瞻性出生队列研究,采用巢式病例对照研究方法,对14359名孕妇进行随访观察,以随访观察期内其子代确诊先心病的97名孕妇为先心病组,从队列人群中以1∶2的比例匹配194名子代未患先心病的孕妇为对照组。采用电感耦合等离子体质谱测定孕母孕20~24周时血液样本和胎儿脐血样本中的重金属元素和微量元素水平。采用多因素logistic回归分析评估重金属元素和微量元素水平与子代先心病之间的关联,并构建孕期重金属元素和微量元素水平与子代先心病发生概率的列线图预测模型。结果与对照组相比,先心病组孕妇血液样本中铝(aluminium,Al)、钠(natrium,Na)、钙(calcium,Ca)、钛(titanium,Ti)、硒(selenium,Se)、锶(strontium,Sr)、锡(stannum,Sn)、锑(stibium,Sb)、钡(barium,Ba)、钍(thorium,Th)水平较高,差异有统计学意义(P<0.05);先心病组脐血样本中Al、锌(zinc,Zn)、镁(magnesium,Mg)、钾(kalium,K)、Ca、Ti、铬(chromium,Cr)、铜(copper,Cu)、砷(arsenic,As)、Se、Sr、银(argentum,Ag)、镉(cadmium,Cd)、Sn、铅(plumbum,Pb)水平显著高于对照组(P<0.05)。多因素logistic回归分析显示,孕妇血液样本中Sb水平升高,其子代患先心病的风险显著增加(^(a)OR=4.81,P=0.004);而在脐血样本中,高浓度水平的Al(^(a)OR=4.22,P=0.013)、Mg(^(a)OR=8.00,P=0.014)、Pb(^(a)OR=3.82,P=0.049)与先心病的发生风险增加存在关联。孕期重金属元素和微量元素水平与子代先心病的预测模型中纳入的变量有:孕妇血Al、Th、Sb水平和脐血Al、Mg、Pb水平,绘制的列线图预测模型的校正曲线趋近于理想曲线。结论孕期Al、Th、Sb、Mg、Pb水平升高提示子代罹患先心病的风险增加,联合以上指标构建的列线图预测模型可以预测子代先心病的发生概率。     

Bile secretion of trace elements in rats with a congenital defect in hepatobiliary transport of glutathione

Bile secretion of trace elements, analyzed by proton-induced x-ray emission, was studied in rats with a congenital defect in hepatobiliary transport of organic anions [Groningen Yellow (GY) rats], in which the process of bile secretion resembles that of the neonatal period. Bile flow (-41%) and biliary glutathione secretion (-99%) were drastically impaired in GY rats compared with controls. Plasma concentrations of all detectable trace elements (Fe, Cu, Zn, Mo, Br, and Se), as well as that of simultaneously determined Ca, were similar in GY and age-matched control Wistar rats. Bile concentrations of Fe, Mo, Br, and Ca were also similar in both groups, resulting in a approximately 40% reduction of their secretion rates in GY rats. The concentrations of Zn (-62%) and Mn (-64%) were significantly lower in GY rats in contrast to that of Cu, which was 50% higher. Se could not be detected in bile of either group. Recovery in bile (% dose/3 h) after i.v. injection of MnCl2, CuSO4, or SeO2 (1 mg metal/kg) was lower in GY rats than in controls: Mn, 26 and 35%; Cu, 2.6 and 5%; and Se, 1.5 and 5%, respectively. Injection of ZnSO4 did not lead to increased Zn secretion in GY rats, and only 1.1% of the dose was recovered in controls. Thus, the hepatic handling of different endogenous and exogenously administered trace metals is affected to a variable extent in the GY rat. For a number of metals (e.g. Fe, Mo), this may be related to the reduced bile flow; for others (e.g. Zn, Mn, Cu), other regulatory factors appear to be responsible.     

Amniotic fluid levels of selected trace elements and heavy metals in pregnancies complicated with neural tube defects

The aims of this study were to determine the levels of trace elements and heavy metals, namely aluminum (Al), chromium (Cr), manganese (Mn), cobalt (Co), nickel (Ni), copper (Cu), zinc (Zn), arsenic (As), molybdenum (Mo), cadmium (Cd), tin (Sn), antimony (Sb), mercury (Hg), and lead (Pb), in the amniotic fluid of pregnant women, and to investigate their relationship with neural tube defects (NTDs). The study included 36 pregnant women whose fetuses were complicated with NTDs (study group) and 39 pregnant women with unaffected healthy fetuses (control group), who were matched for body mass index and gestational weeks. The amniotic fluid levels of trace elements and heavy metals were measured using inductively coupled plasma-mass spectrometry and compared between the two groups. Significantly lower mean levels of Zn and Mo and significantly higher levels of Al, Sn, Sb, and Hg in the study group than in the healthy control group were observed, which implied that these elements are possibly correlated with risk factors for the occurrence of NTDs. In contrast, there were no significant differences in the levels of Cr, Mn, Co, Ni, Cu, As, Cd, and Pb between the groups (P ≥ .05).     

Detection of HCMV DNA in placenta, amniotic fluid and fetuses of seropositive women by nested PCR

Human cytomegalovirus (HCMV) is the most common viral cause of intrauterine infection throughout the world. Its distribution patterns in different clinical samples are poorly understood. This study was performed to determine the frequency of CMV DNA positivity in maternal/fetus sera, placentas and amniotic fluid, together with maternal/fetus serology. Clinical specimens were obtained from 92 pregnant women who delivered by cesarean section. 98% of women and their neonates were HCMV IgG positive and 5.4% of these mothers were IgM positive, while no IgM was detected in neonates of IgM positive mothers. Among the IgG positive mothers, IgM was detected in 3.3% of their fetuses. 5.4% and 3.3% of maternal and fetal sera were HCMV DNA positive, respectively. The three neonates who were positive for HCMV DNA in sera were also positive for HCMV IgM and the PCR of their amnions was positive (p < 0.0001). 9.8% of placenta samples and 4.3% of amniotic fluid specimens were positive for HCMV DNA while among these placenta samples, two amnions were PCR positive (p = 0.046). Our results showed that there is not always a correlation between placenta and amnion infections. This may be due to reactivation of HCMV leading to placenta infection, as all affected placentas do not pass infection to fetuses and amniotic fluids. Detection of HCMV DNA in amnion and fetus plasma and the existence of fetus IgM against HCMV can also occur without clinical symptoms.     

Materno-fetal transport of creatine in the rat

The transfer of 14C-creatine to the rat fetus was studied following continuous i.v. infusion into the mother. In the presence of a relatively constant maternal plasma 14C-creatine concentration, creatine was accumulated by the chorioallantoic placenta and visceral yolk sac to concentrations higher than that found in maternal or fetal plasma. The ability of the extraembryonic membranes to accumulate creatine changed during gestation; nevertheless, these membranes concentrated creatine against a gradient throughout the period studied (14-22 days of gestation). Neither 14C-creatine nor 14C-urea were concentrated in the placentae or fetal plasma when compared to maternal plasma. Simultaneous infusion of beta-guanidinopropionic acid with 14C-creatine reduced both movement and accumulation of creatine into the fetoplacental unit. It is concluded that the accumulation of creatine by the chorioallantoic placenta and by the visceral yolk sac is an active process with creatine diffusing down its concentration gradient into the fetal circulation.     

Elemental Analysis of Dried Milks Used in Infant Feeding

Dried milks available for infant feeding in Hobart, Tasmania, were analysed by flame emission and atomic absorption spectrometry for the major elements Na, K, Ca, and Mg as well as the trace elements Fe, Cu, Zn, Ni, Cr, Sn, Cd and Pb. Protein was determined by analysis for nitrogen by the Kjeldahl method. Results are expressed in terms of weight of powder and normal dilution in water as used for infant feeding. Several of the milks varied in composition from the manufacturers' specifications. Although toxic trace elements were present in the dried milks, their concentrations were well within normally acceptable limits of safety. Results for random samples of cow's and human breast milk are given for comparison.     

The pharmacokinetics of phenytoin in perfused human placenta

In vivo phenytoin (PHT) and its metabolites are present in the fetal plasma. In order to determine the transfer kinetics and biotransformation characteristics for phenytoin in the human placenta, a dual recirculating perfused human placental lobule preparation was utilized. A single bolus of 14C-PHT with or without tritiated water (3H2O) was injected into the maternal reservoir. Peak concentrations of 3H2O and PHT appeared simultaneously in the maternal artery at 4 minutes and in the maternal vein at 9 minutes, whereas the peak concentrations of 14C-PHT and 3H2O in the fetal vein were at 9 and 7 minutes, respectively. After 4 hours of perfusion, the total phenytoin concentration in the fetal perfusate was only 73% of the maternal perfusate level. Protein-bound PHT was 37 +/- 8% and 7 +/- 3% of the total phenytoin in the maternal and fetal perfusate. The concentrations of free phenytoin in the maternal and fetal compartments were identical by 60 minutes and were maintained for the remainder of the experiment. The concentration of total PHT in perfused placenta was more than 3.5 times greater than the total maternal perfusate levels. In the placenta, PHT concentration was highest in the cytosolic fraction, and 57% of the placental PHT was in the free form. There was no evidence of parahydroxylation, oxidation, or conjugation of PHT. Phenytoin was also concentrated in nonperfused placental tissue. Thus the transfer of PHT by the human placenta is rapid and dependent upon protein binding and flow in both maternal and fetal circulations. Even though the human placenta does concentrate PHT, it does not metabolize PHT under these conditions.     

Transfer of riboflavin by the perfused human placenta

The transfer of radioactive riboflavin across the human term placenta has been studied in an in vitro perfusion system. The clearance index (clearance riboflavin:clearance antipyrine) toward the fetus averaged 0.69 and the transfer index (clearance riboflavin:clearance L-glucose) averaged 3.40. The respective indices in the reverse direction were 0.25 and 0.87. Stepwise increases in the concentration of riboflavin in the maternal perfusate were associated with parallel increases in transfer rates, expressed as ng/min, up to concentrations approximating 100 ng/ml. Above that concentration, the transfer rates continued to increase at a slower rate. Concurrently, there was a reduction of the transfer index from 2.7 to 1.1 at 1000 ng/ml. With the fetal circulation closed, the placenta established a gradient toward the fetus over a period of 150 min of 1.7. The transferred radioactivity was identified as riboflavin by high-performance liquid chromatography, whereas that retained within the placenta was metabolized to flavin mononucleotide (33-75%). The observations indicate a very effective active transport system directed toward the fetus which is limited in capacity to low concentrations of riboflavin.     

Transfer and metabolism of carnitine and carnitine esters in the in vitro perfused human placenta

The transfer and metabolism of L-carnitine, L-acetylcarnitine, and L-palmitoylcarnitine were studied in the human placenta at term by means of in vitro dual perfusion of a placental lobe. L-Carnitine transfer was 20% that of the freely diffusing antipyrine and 40% that of L-lysine. The transfer of L-acetylcarnitine was similar to that of L-carnitine, but no placental transfer of L-palmitoylcarnitine was found. In contrast to L-lysine, L-carnitine, and L-acetylcarnitine were not actively transported from the maternal to the fetal circulation. No stereospecific transfer of carnitine across the placenta was found. However, there was stereospecific uptake of carnitine by placental tissue. The placenta exhibited an active carnitine metabolism by esterifying free carnitine and hydrolyzing carnitine esters taken up from the perfusion medium and releasing the metabolites into the fetal and maternal circulations.     

Placental handling of zinc in the guinea pig

The distribution of zinc between the mother and the fetoplacental unit, and its placental transfer, were studied using stable and isotopic zinc in unanaesthetized pregnant guinea pigs and an in situ perfusion preparation. The concentration of stable zinc in fetal plasma and skeletal muscle was higher than that in the maternal tissues: 2.0 compared with 1.4 micrograms/ml and 84 with 49 ng/mg dry weight, respectively. The placenta and maternal and fetal liver had similar zinc concentrations: 90, 75 and 88 ng/mg dry weight, respectively. The ability of the placenta to concentrate 65Zn, measured 1 h after a single intravenous injection into the unanaesthetized mother, was comparable with that of the maternal liver. Maternal-fetal mass transfer of zinc was directly related to maternal plasma zinc concentrations from 0.7 to 24.1 micrograms/ml (b = 2 ng X min-1 X g-1 X microgram-1, r = 0.92). At physiological plasma levels, the calculated transfer would supply the fetus with 0.12 mg zinc/day, similar to the accretion rate over the last trimester. Placental transfer of zinc was not influenced by the concentration of zinc in the placental perfusate. Extraction of zinc from the perfusate was also slow, and partly by absorption. Maternofetal transfer of zinc was directly related to both uterine and umbilical blood flows. The high concentration of zinc in the syncytium, relative to both maternal and fetal plasma levels, suggests active uptake at the maternal surface, combined with a slow release into the fetus, down a concentration gradient.     

Clearance of calcium across in situ perfused placentas of intrauterine growth-retarded rat fetuses

Maternofetal clearance of 45Ca and 51Cr-EDTA (diffusional marker) were simultaneously measured across in situ perfused placentas of intrauterine growth-retarded (IUGR) and control rat fetuses on d 20 of gestation. IUGR was induced by uterine artery and vein ligation on d 17 of gestation. Control fetuses and their placentas were taken from sham-operated dams. We hypothesized that calcium transfer would be impaired across placentas of IUGR fetuses. The mean body wt of IUGR fetuses was 42% lower, and the mean nose-anus length was 16% lower than those of control fetuses. The mean total calcium content of IUGR fetuses was significantly lower than that of control fetuses, but not when it was normalized to body wt. The mean maternal whole blood ionized calcium concentration was not significantly different in the two groups. The materno-fetal clearance of 45Ca across IUGR placentas was significantly lower than that across control placentas (IUGR = 35.2 +/- 1.9 micro L/min/g placenta, mean +/- SEM; control = 93.1 +/- 12 microliters/min/g placenta, p less than 0.002). In contrast, the maternofetal clearance of 51Cr-EDTA, the reference diffusional marker, was not significantly different across IUGR and control placentas. We conclude that maternofetal transfer of calcium is reduced across placentas of IUGR rat fetuses.     

Pathogenesis of fetal hypomineralization in diabetic rats: evidence for delayed bone maturation

There is some evidence that fetuses of diabetic rats (FDR) are hypomineralized. To explore the pathogenic role of decreased maternal duodenal Ca absorption, fetal hypotrophy, and decreased placental calbindin-D9K, respectively, spontaneously diabetic rats fed a 1.0% Ca diet were compared with diabetic rats treated with 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] (15 ng/ 100 g) during week 3 of pregnancy, which restored duodenal calbindin-D9K concentrations to normal; with nondiabetic rats semistarved during week 3, which resulted in similar fetal hypotrophy; and with nondiabetic rats fed high cation diets (1.5% Ca-1.5% Sr and 1.5% Ca-3.5% Sr) during week 3, the latter of which repressed duodenal and placental calbindin-D9K toward concentrations measured in diabetic rats. In addition, fetal tibiae were studied histologically. Ca content was lower in 21.5-d-old FDR than in control fetuses. FDR had lower plasma osteocalcin (OC) levels and, on histomorphometry, increased hypertrophic cartilage width, indicating retarded bone maturation. Maternal 1,25(OH)2D3 treatment did not change Ca content and hypertrophic cartilage width in FDR. Fetuses of semistarved rats had plasma OC levels and hypertrophic cartilage width comparable to those of control fetuses. Fetuses of rats fed the 1.5% Ca-3.5% Sr diet were more severely hypomineralized than FDR but had higher plasma OC than both FDR and control fetuses, compatible with fetal Ca deficiency. Whereas diabetic placentas showed weak but homogeneous staining of calbindin-D9K in the labyrinth on immunohistology, degenerative zones were present in placentas of rats fed the 1.5% Ca-3.5% Sr diet. Thus, there is no mineralization defect in FDR caused by disturbed maternal duodenal Ca absorption or transplacental Ca transport, but a delay in bone maturation that is unexplained by their lower body weight.     

Serum immunoglobulins in multiple pregnancy

The concentrations of immunoglobulins (Ig) G.A.M. and E were determined in paired umbilical cord and maternal sera in 50 twin pregnancies. Mean IgG levels were higher in cord than maternal sera and in most cases the cord IgG level related more closely to that of the other twin than to either maternal level or birthweight, and was in the range for singletons of the same gestational age. The three cases of fetofetal transfusion syndrome were exceptional in the large difference between IgG concentrations in recipient and donor twins. The discrepancy was much greater than that found between the levels of proteins produced by the fetus, suggesting a disturbance in maternofetal placental transfer. IgM was detected in all cord sera, with one exception, and the level was not related to order of birth. IgA was detected in 16% of cord sera, 13% in sera from first borns. IgE was detected in only 8% of cord sera and there was no evidence of placental transfer.     

Bio-Element Status in Children With Acute Rheumatic Fever: Before Treatment and After Clinical Improvement

Acute rheumatic fever (ARF) is an autoimmune multisystem disease. Bio-elements are required in different quantities by an organism to maintain its physiologic function. Monitoring the status of bio-elements is critical in human health. This study aimed to determine possible changes in levels of bio-elements in children with ARF before and after treatment. Levels of trace and major elements in children with ARF were investigated. The study included 33 children with ARF (17 boys and 16 girls) and 20 healthy control children (11 boys and 9 girls). The ages ranged from 5 to 16 years (mean 11.4 ± 3.82 years) in the study group and from 6 to 15 years (mean, 10.7 ± 3.22 years) in the control group. Trace and major element concentrations (total of 14 elements) in the serum were measured by inductively coupled plasma-optical emission spectroscopy. Before treatment, the levels of the major elements potassium (K) and magnesium (Mg) in children with ARF were higher than in the control group, whereas the calcium (Ca) level was lower. Before treatment, the levels of trace elements iron (Fe), selenium (Se), zinc (Zn), aluminum (Al), and barium (Ba) were lower, whereas the copper (Cu), beryllium (Be), cadmium (Cd), chromium (Cr), gallium (Ga), and strontium (Sr) levels were higher in the serum of the patients with ARF than in the control patients. The major findings show that the homeostasis of some trace and major elements were altered in the children with ARF and that these alterations may be a contributing factor in the pathogenesis of this disease.     

Serum immunoglobulins in multiple pregnancy.

The concentrations of immunoglobulins (Ig) G.A.M. and E were determined in paired umbilical cord and maternal sera in 50 twin pregnancies. Mean IgG levels were higher in cord than maternal sera and in most cases the cord IgG level related more closely to that of the other twin than to either maternal level or birthweight, and was in the range for singletons of the same gestational age. The three cases of fetofetal transfusion syndrome were exceptional in the large difference between IgG concentrations in recipient and donor twins. The discrepancy was much greater than that found between the levels of proteins produced by the fetus, suggesting a disturbance in maternofetal placental transfer. IgM was detected in all cord sera, with one exception, and the level was not related to order of birth. IgA was detected in 16% of cord sera, 13% in sera from first borns. IgE was detected in only 8% of cord sera and there was no evidence of placental transfer.     

Free amino acid distribution inside the first trimester human gestational sac

The trophoblast functions of nutrient transport and protein synthesis generate high concentrations of amino acids in the placenta and in fetal blood during the second half of pregnancy, but little is known about these metabolic processes in embryonic and early fetal periods. The aim of this study is to compare the distribution of amino acids inside the first trimester gestational sac. Free amino acid concentrations were measured in homogenates of placental villi, in samples of coelomic and amniotic fluid, and in the maternal serum from 17 normal pregnancies between 7 and 11 weeks of gestation. Significant positive relationships between maternal serum and placental tissue were found for 10 amino acids, indicating that active amino acid transport and accumulation by the human syncytiotrophoblast occurs as early as 7 weeks of gestation. The transplacental flux of most amino acid transport from maternal blood to the exocoelomic cavity was against a concentration gradient. The highest placental amino acid concentrations were found for taurine, glutamic acid, glycine and alanine. The amniotic fluid contained lower mean concentration of all amino acids than coelomic fluid and maternal serum. The concentration distribution of individual amino acids in coelomic and amniotic fluid were related indicating a passive transfer through the amniotic membrane. A coelomic-maternal gradient was observed in 19 out of 24 amino acids measured and positive correlations were found between maternal serum and coelomic fluid for concentrations of α-aminobutyric acid, tyrosine and histidine, suggesting that these amino acids are only partially retained and/or transferred more rapidly by the early placenta.     

Transfer across perfused human placenta. IV. Effect of protein binding on free fatty acids.

The effect of protein binding on the rate of placental transfer of hexanoic (C 6) and decanoic (C 10) acids was investigated in an in vitro perfusion system of human placenta. As much as 30% of transferred C 6 was converted to more polar compounds, so that the observations related to the combined effects on transfer and metabolism. Less than 10% of C 10 was similarly metabolized. Both fatty acids are soluble in buffered salt solutions at the concentrations used (40 muM) and both are bound to serum albumin, C-10 having higher association constants (K' for C 6, 1.48 X 10(4); for C 10, 1.03 X 10(5). When the placenta is perfused with buffered salt solution, the transfer of C 6 is 22% more rapid than that of C 10. It is suggested that binding within the placenta retards C 10 more than C 6. The addition of 1 g/100 ml bovine serum albumin to the maternal perfusate reduces the transfer rate of C 10 by 80%, whereas 2 g/100 ml serum albumin has a more moderate effect on C 6 (a reduction of 50%). The addition of 1 g/100 ml serum albumin to the fetal perfusate increases transfer rate of both free fatty acids (FFA), C 6 by 25% and C 10 by about 250%. With equivalent concentrations of serum albumin in maternal and fetal perfusates, the transfer rate of C 10 was reduced by 65%, whereas there was no detectable effect on transfer of C 6 in two of three experiments. The transfer rate of FFA increase logarithmically with progressive shortening of the carbon chain from C 16 to C 8 when maternal and fetal perfusates contain serum albumin. Protein binding is apparently the determining factor. The rate of transfer falls off at C 6 and C 4, 4ven though protein-binding continues to decrease. The determining factor may be the hydrophilic nature of these molecules.     

Placental transfer favours high avidity IgG antibodies

The aim of the present study was to evaluate if maternal-foetal antibody placental transfer may be affected by antibody avidity. We compared the avidity index (AI) of IgG antibodies to tetanus toxoid (TT) and to type 3 pneumococcal antigen (Pn) in cord blood of 10 healthy term and 8 preterm infants and in their mothers' sera at delivery. In order to evaluate whether a heavier antigenic exposure may influence the placental transfer, we also studied 15 Pakistani maternal sera and cord blood pairs. TT- and Pn-specific antibody AI was significantly higher in Italian and Pakistani term infants than in their mothers, while a significant difference in specific TT antibody AI, but not in specific Pn antibody AI was observed between preterm infants and their mothers. Italian and Pakistani cord blood/maternal serum pairs showed comparable values of AI. Our data suggest that high avidity antibodies preferentially cross the placenta; this seems to start early during gestation and appears to be related to the nature of the antigen to which the antibodies are directed, but not to the degree of antigenic exposure.     

Transfer and metabolism of retinol by the perfused human placenta.

The transfer and metabolism of retinol by human placenta was investigated using an in vitro perfusion system with independent maternal and fetal circulations. 3H-retinol bound to albumin added to the maternal perfusate was rapidly taken up and concentrated by the placenta to levels 16.5 +/- 5.28 times the maternal perfusate. Approximately 8% of the retinol retained in the placenta was esterified. No metabolites were detected in the perfusates. Perfusion of placenta with retinol bound to retinol-binding protein (RBP) reduced the placental concentration to 4.4 +/- 1.72 times the maternal concentration and eliminated evidence of metabolism. The transfer rate of RBP:3H-retinol was less than that of albumin:14C-retinol when measured concurrently in three experiments (clearances 0.11 versus 0.75 mL/min, 0.21 versus 1.7 mL/min, and 0.29 versus 0.48 mL/min, respectively). Transfer of the radioactive retinol was more rapid than 125I-RBP or albumin, indicating that retinol was transferred independently of the proteins. The transfer index of retinol (clearance retinol:clearance L-glucose) was 0.73 +/- 0.085 compared to 2.1 +/- 0.36 for thiamin and 3.4 +/- 0.95 for riboflavin, both water-soluble vitamins with active transport systems. The retinol transferred to the fetal perfusate is not bound to RBP, as demonstrated by gel filtration chromatography and chromatography on a transthyretin affinity column, despite the availability of RBP in the cord serum added to the perfusate. The endogenous retinol in the cord serum is bound to RBP.(ABSTRACT TRUNCATED AT 250 WORDS)