Enantioselectivity in the steady-state pharmacokinetics and transplacental distribution of pindolol at delivery in pregnancy-induced hypertension

Nine patients taking oral doses of 10 mg/12 h rac-pindolol as part of their treatment for hypertension in pregnancy were recruited for the study. Maternal and fetal gestational age ranged from 20-38 years and 28-41 weeks, respectively. Blood was collected from the umbilical cord vein and from the mother from zero to 12 h after drug administration. Urine was collected for 12 h after rac-pindolol administration at the following intervals: 0-3, 3-6, 6-9, and 9-12 h. Plasma and urine concentrations of the pindolol enantiomers were determined by HPLC using a Chiralpak AD chiral column and fluorescence detection. The data were fitted to a one-compartment model and differences between (+)-R and (-)-S enantiomers were compared by the paired t-test (P < 0.05). Mean results are reported. The disposition of pindolol in maternal plasma was stereoselective, with higher AUC(SS)0-12 (84.34 vs. 95.69 ng.h/ml) and Cl(R) values (9.16 vs. 10.85 L/h) and lower Vd/f (251.38 vs. 225.17 L) and Cl/f (62.48 vs. 55.74 L/h) for the (+)-R pindolol. The transplacental distribution of pindolol was not stereoselective. Cord, plasma, and presumably fetal, concentrations of the pindolol enantiomers were 56% of the maternal plasma concentrations up to 6 h after the last dose.     

Rapid measurement of 8-oxo-7,8-dihydro-2'-deoxyguanosine in human biological matrices using ultra-high-performance liquid chromatography-tandem mass spectrometry

Interaction of reactive oxygen species with DNA results in a variety of modifications, including 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodG), which has been extensively studied as a biomarker of oxidative stress. Oxidative stress is implicated in a number of pathophysiological processes relevant to obstetrics and gynecology; however, there is a lack of understanding as to the precise role of oxidative stress in these processes. We aimed to develop a rapid, validated assay for the accurate quantification of 8-oxodG in human urine using solid-phase extraction and ultra-high-performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) and then investigate the levels of 8-oxodG in several fluids of interest to obstetrics and gynecology. Using UHPLC-MS/MS, 8-oxodG eluted after 3.94 min with an RSD for 15 injections of 0.07%. The method was linear between 0.95 and 95 nmol/L with LOD and LOQ of 5 and 25 fmol on-column, respectively. Accuracy and precision were 98.7-101.0 and <10%, respectively, over three concentrations of 8-oxodG. Recovery from urine was 88% with intra- and interday variations of 4.0 and 10.2%, respectively. LOQ from urine was 0.9 pmol/ml. Rank order from the greatest to lowest 8-oxodG concentration was urine>seminal plasma>amniotic fluid>plasma>serum>peritoneal fluid, and it was not detected in saliva. Urine concentrations normalized to creatinine (n=15) ranged between 0.55 and 1.95 pmol/μmol creatinine. We describe, for the first time, 8-oxodG concentrations in human seminal plasma, peritoneal fluid, amniotic fluid, and breast milk, as well as in urine, plasma, and serum, using a rapid UHPLC-MS/MS method that will further facilitate biomonitoring of oxidative stress.     

Determination of pindolol enantiomers in human plasma and urine by simple liquid–liquid extraction and high-performance liquid chromatography

A simple method for the measurement of pindolol enantiomers by HPLC is presented. Alkalinized serum or urine is extracted with ethyl acetate and the residue remaining after evaporation of the organic layer is then derivatised with (S)-(−)-α-methylbenzyl isocyanate. The diastereoisomers of derivatised pindolol and metoprolol (internal standard) are separated by high-performance liquid chromatography (HPLC) using a C₁₈ silica column and detected using fluorescence (excitation λ: 215 nm, emission λ: 320 nm). The assay displays reproducible linearity for pindolol enantiomers with a correlation coefficient of r²≥0.998 over the concentration range 8–100 ng ml⁻¹ for plasma and 0.1–2.5 μg ml⁻¹ for urine. The coefficient of variation for accuracy and precision of the quality control samples for both plasma and urine are consistently <10%. Assay parameters are similar to those of previously published assays for pindolol enantiomers, however this assay is significantly easier and cheaper to run. Clinically relevant concentrations of each pindolol enantiomer can readily be measured.     

Size heterogeneity of epidermal growth factor in human body fluids

We measured the concentration of immunoreactive (IR) hEGF in various body fluids by radioimmunoassay (RIA) and evaluated its size heterogeneity by size exclusion high performance liquid chromatography combined with RIA or with time-resolved immunoflurometric assay (TR-IFMA). Mean concentration was 80 ng/ml in urine, 65 ng/ml in milk, 50 ng/ml in seminal plasma, 25 ng/ml in armpit sweat, 1 ng/ml in breast sweat, 0.3 ng/ml in third-trimester amniotic fluid, 3 ng/ml in saliva, 1.5 ng/ml in tears and 0.3 ng/ml in gastric juice.

All the fluids except armpit sweat and gastric juice contained two to five molecular sizes of IR-hEGF. As well as the 6200-dalton (6.2kDa) hEGF we found at least four other different molecular sizes with approximate weights of 300, 150, 70 and 20kDa. The authentic 6.2kDa form made up >90% of the total IR-hEGF in all except the amniotic fluid where its proportion was 71%, and the seminal plasma where the proportion could not be determined.     

Regulation of Substances in Allantoic and Amniotic Fluid of the Chicken Embryo

Traditionally, the avian allantois has been considered a respiratory organ and a dumping ground for metabolic wastes. We tested the hypothesis that the allantoic fluid is also a depot for free amino acids and related compounds. To gain further insight in the specific role of the allantoic fluid, we included plasma and the amniotic fluid in this study. The work was carried out in 13- and 14-day-old chicken embryos. Using an HPLC-fluorometric technique, 40 of the 41 amino acids and related compounds investigated were detected. The amniotic fluid contained 32 compounds, while plasma and allantoic fluid contained 38 and 39 compounds, respectively. The glucose concentration was determined with a hexokinase technique. It was highest in plasma and lowest in the amniotic fluid. We identified three barriers that hyper- and hyporegulate a number of compounds: (1) a blood/allantois barrier, (2) a blood/amnion barrier, and (3) an allantois/amnion barrier. Compared with plasma and allantoic fluid, the amniotic fluid is a mostly hyporegulated environment.     

Catecholamines and uterine activity rhythms in the pregnant rhesus macaque.

This study was designed to examine the relationship between uterine contractile rhythms with maternal plasma and amniotic fluid catecholamine concentrations in the pregnant rhesus macaque. Six chronically catheterized rhesus macaques were maintained in a vest and tether system and exposed to a 12L:12D cycle. Continuous uterine activity recordings demonstrated a contractile pattern with peak activity at 2200 h (p less than 0.05). Paired maternal plasma and amniotic fluid samples were collected at 3-h intervals for 24 h between Days 131 and 148 of gestation. Samples were analyzed for norepinephrine, epinephrine, and dopamine by HPLC. Maximum plasma concentrations across the 24-h periods for norepinephrine (633 +/- 230; mean pg/ml +/- SEM) and dopamine (378 +/- 110) were observed at 2100 h and epinephrine (408 +/- 95) at 1200 h, but these values were not significant. The maximum amniotic fluid values were 378 +/- 126, 267 +/- 190, and 556 +/- 87 pg/ml for norepinephrine, epinephrine and dopamine, respectively. However, concentrations across 24 h did not differ. Neither maternal plasma nor amniotic fluid catecholamine concentrations were correlated with uterine activity rhythms. Therefore, we conclude that the nocturnal uterine activity in the rhesus macaque is not related to maternal arterial or amniotic fluid catecholamine concentrations.     

Comparative studies on amniotic fluid and plasma fibronectins.

Human fibronectin was isolated from second-trimester amniotic fluid, from amniotic fluid obtained at term and from adult plasma. The amniotic-fluid fibronectins had a slightly higher apparent molecular weight on sodium dodecyl sulphate/polyacrylamide-gel electrophoresis than the plasma fibronectin. Early- and late-amniotic-fluid fibronectin had 9.5 and 9.6% carbohydrate respectively, whereas plasma fibronectin had 5.8%. The amniotic-fluid fibronectins had similar mannose and sialic acid contents to plasma fibronectin, but greater amounts of glucosamine, galactosamine, galactose and fucose. There were no detectable differences in the amino-acid composition of amniotic-fluid and plasma fibronectins, and the patterns of peptides obtained after tryptic digestion of fibronectin from the two sources showed extensive similarities. Fibronectins from plasma and amniotic fluid were equally active in promoting cell attachment and were immunologically indistinguishable. These results show that fibronectin from amniotic fluid is more heavily glycosylated than plasma fibronectin or previously analysed fibronectins from cultured fibroblasts. The observed differences in glycosylation may be related to cell type and/or stage of development.     

Nevirapine,Sodium Concentration and HIV-1 RNA in Breast Milk and Plasma among HIV-Infected Women Receiving Short-Course Antiretroviral Prophylaxis

Introduction

Risk factors for breast milk transmission of HIV-1 from mother to child include high plasma and breast milk viral load, low maternal CD4 count and breast pathology such as mastitis.

Objective

To determine the impact of nevirapine and subclinical mastitis on HIV-1 RNA in maternal plasma and breast milk after intrapartum single-dose nevirapine combined with either 1-week tail of Combivir (zidovudine/lamivudine) or single-dose Truvada (tenofovir/emtricitabine).

Methods

Maternal plasma and bilateral breast milk samples were collected between April 2008 and April 2011 at 1, 4 and 6 weeks postpartum from HIV-infected Tanzanian women. Moreover, plasma samples were collected at delivery from mother and infant.

Results

HIV-1 RNA was quantified in 1,212 breast milk samples from 273 women. At delivery, 96% of the women and 99% of the infants had detectable nevirapine in plasma with a median (interquartile range, IQR) of 1.5 μg/mL (0.75–2.20 μg/mL) and 1.04 μg/mL (0.39–1.71 μg/mL), respectively (P < 0.001). At 1 week postpartum, 93% and 98% of the women had detectable nevirapine in plasma and breast milk, with a median (IQR) of 0.13 μg/mL (0.13–0.39 μg/mL) and 0.22 μg/mL (0.13–0.34 μg/mL), respectively. Maternal plasma and breast milk HIV-1 RNA correlated at all visits (R = 0.48, R = 0.7, R = 0.59; all P = 0.01). Subclinical mastitis was detected in 67% of the women at some time during 6 weeks, and in 38% of the breast milk samples. Breast milk samples with subclinical mastitis had significantly higher HIV-1 RNA at 1, 4 and 6 weeks (all P < 0.05).

Conclusion

After short-course antiretroviral prophylaxis, nevirapine was detectable in most infant cord blood samples and the concentration in maternal plasma and breast milk was high through week 1 accompanied by suppressed HIV-1 RNA in plasma and breast milk.     

Determination of cyclophosphamide enantiomers in plasma by LC-MS/MS: Application to pharmacokinetics in breast cancer and lupus nephritis patients

This article describes the enantioselective analysis of cyclophosphamide (CPA) in human plasma using LC-MS/MS. CPA enantiomers were extracted from plasma using a mixture of ethyl acetate and chloroform (75:25, v/v). The enantiomers were separated on a Chiralcel(R) OD-R column, with the mobile phase consisting of a mixture of acetonitrile and water (75:25, v/v) plus 0.2% formic acid. The protonated ions and their respective product ions were monitored using two functions, 261 > 141 for CPA enantiomers and 189 > 104 for the internal standard (antipyrine). Recovery rates were higher than 95% and the quantification limit was 2.5-ng/ml plasma for both enantiomers. The coefficients of variation and the relative errors obtained for the validation of intra- and interassay precision and accuracy were less than 10%. The method was applied for the investigation of the enantioselective pharmacokinetics of CPA in a lupus nephritis patient treated with 1 g CPA infused over 2 h and in a breast cancer patient treated with 0.9 g infused over 1 h. No stereoselectivity in the pharmacokinetic parameters was observed for either patient. Clearance values of 2.63 and 2.93 l/h and of 3.36 and 3.61 l/h for (-)-(S) and (+)-(R)-CPA were obtained for the breast cancer and lupus nephritis patient, respectively.     

Developmental changes in the glycosylation and binding properties of human fibronectins. Characterization of the glycan structures and ligand binding of human fibronectins from adult plasma, cord blood and amniotic fluid

Fibronectins from human adult plasma, fetal plasma and from amniotic fluid obtained during early and late gestation were compared with respect to (i) their reactivity with lectins, (ii) their binding to the physiological ligands gelatin and heparin, and (iii) the role of the carbohydrate residues in the binding to these two ligands. The two fibronectin isoforms displayed distinct developmental differences in both glycosylation and binding properties: (i) Proportions of tri/tetraantennary complex glycans compared to the fraction of biantennary structures, as inferred from the reactivity with concanavalin A, were highest in amniotic fluid fibronectin from late pregnancy, lower in amniotic fluid fibronectin from early gestation, and even lower in fetal and adult plasma fibronectins. Likewise, fucose (alpha 1-6) linked to the innermost N-acetylglucosamine of the chitobiosyl core, defined by reactivity with Lens culinaris agglutinin (LCA), was present primarily in amniotic fluid fibronectin, and decreased in content during gestation from the 2nd. to the 3rd. trimenon. Both fetal and adult plasma fibronectins were only weakly reactive with LCA, indicating a low content of (alpha 1-6) linked fucose residues. After prior treatment with sialidase, both plasma and amniotic fluid fibronectins strongly reacted with erythrocyte phytohaemagglutinin (E-PHA), indicating that both fibronectin isoforms contain bisecting (beta 1-4) N-acetylglucosamine residues. Amniotic fluid fibronectins showed much greater reactivity than adult and fetal plasma fibronectins with wheat germ agglutinin; binding of this lectin to amnion fluid fibronectins was not decreased by desialylation indicating the presence of poly(N-acetyllactosamine) units. Whereas amniotic fluid fibronectins were strongly reactive with peanut agglutinin, neither adult nor fetal plasma fibronectins did bind to this lectin unless after prior desialylation. Hence, both fibronectin isoforms contain O-glycan residues that are fully sialylated in fetal and adult plasma fibronectins, but only partly sialylated in amniotic fluid fibronectins. According to these differences, glycosylation of plasma and amniotic fluid fibronectins is under developmental regulation. (ii) Amniotic fluid fibronectins had a significantly lower binding activity for both heparin and gelatin than plasma fibronectins. Moreover, amnion fibronectin from late gestation displayed a significantly lower binding to these two ligands than amnion fibronectin from early gestation. Fetal plasma fibronectins had a lower binding activity for gelatin than adult plasma fibronectin. (iii) Treatment of fibronectins with sialidase, fucosidase and removal of N-glycans with endoglycosidases H and F did not affect binding to gelatin and heparin, indicating that the interaction of plasma and amnion fibronectin with these two ligands is not influenced by their oligosaccharide moieties.     

Stereoselective disposition of fenoprofen in plasma and synovial fluid of patients with rheumatoid arthritis

The simultaneous disposition of fenoprofen enantiomers in synovial fluid and plasma was studied in 11 patients with arthritis and chronic knee effusions treated with a single oral dose of 600 mg rac-fenoprofen. A plasma sample and a synovial fluid sample were collected simultaneously from each patient up to 16 h after the administration of fenoprofen. A stereospecific assay for fenoprofen using LC-MS-MS was developed and applied successfully to the analysis of the enantiomers in plasma (LOQ = 10 ng of each enantiomer/ml) and synovial fluid (LOQ = 25 ng of each enantiomer/ml). The values of the area under the curve (AUC) for the S-(+)-fenoprofen eutomer were approximately 2.5 times higher in plasma than in synovial fluid (256 vs 104 microg h/ml), while the values for the R-(-)-fenoprofen distomer were about four times higher in plasma than in synovial fluid (42.5 vs 10.5 microg h/ml). These data demonstrate accumulation of the S-(+)-fenoprofen eutomer in plasma and in synovial fluid, with concentrations versus time AUC (+)/(-) ratios of 6.0 in plasma and 9.9 in synovial fluid, suggesting a greater accumulation of the eutomer at the active site represented by synovial fluid than in plasma. This result demonstrates the importance of enantioselective methods and of analysis of synovial fluid rather than plasma in studies of the pharmacokinetics-pharmacodynamics of fenoprofen.     

Fetal-maternal transfer of [H3]-6 beta-hydroxycortisol in the pregnant ewe

Distribution and fetomaternal transfer of 6 beta-hydroxycortisol (6 beta-OHF) was studied using serial sampling following injection of tritium labelled 6 beta-OHF into various fluid compartments in the chronically cannulated unaesthesized pregnant ewe. There was a rapid transfer of 6 beta-OHF from the fetal circulation into amniotic fluid and maternal blood. In contrast, the maternal----fetal transfer of this steroid metabolite was considerably less. The sequence of appearance of 6 beta-OHF in fetal blood and amniotic fluid following injection into maternal blood suggests that this steroid is first transferred across the placenta to fetal blood before gaining entry into the amniotic fluid space. The half-lives of 6 beta-OHF after initial equilibration in maternal plasma, fetal plasma and amniotic fluid were 2.0 h, 5.1 h and 8.9 h respectively. The amniotic sac appears to contain a relatively static pool of 6 beta-OHF and may act as a "trap" for 6 beta-OHF in the ovine conceptus.     

Epidermal growth factor-like proteins in breast fluid and human milk

Epidermal growth factor (EGF), and the transforming growth factor-alpha (TGF-alpha) family of proteins, which also bind to the EGF receptor, have been associated with human breast cancer. The total EGF-like proteins were determined by a radioreceptor assay, and TGF-alpha by radioimmunoassay, in human milk and breast fluid samples. The breast fluids were collected by nipple aspiration from healthy premenopausal women. Both the 24 milks and 18 breast fluids assayed contained EGF-like proteins, at concentrations ranging from 32-600 ng/ml (median, 140 ng/ml), and 62-654 ng/ml (median, 205 ng/ml) respectively. Immunoreactive TGF-alpha proteins were detected at higher levels in 21 breast fluids (range, 0-50.0; median 5.1 ng/ml) than in 24 milk samples (range, 0-8.4; median, 0.8 ng/ml).     

Simultaneous enantioselective quantification of methadone and of 2-ethylidene-1,5-dimethyl-3,3-diphenyl-pyrrolidine in oral fluid using capillary electrophoresis

A capillary electrophoresis method was developed for the enantioselective quantification of methadone (MTD) and its main metabolite, 2-ethylidene-1,5-dimethyl-3,3-diphenyl-pyrrolidine (EDDP). The enantiomers of MTD and EDDP were resolved by CE in 5min using 0.2% highly sulphated gamma-cyclodextrins as chiral selectors and a 50mM phosphate solution at pH 4.5 as background electrolyte. The optimized method was applied and validated for oral fluid testing. Linear relationships were obtained for MTD enantiomers in the range of 8.1-625ng/mL and in the range of 7.6-500ng/mL for EDDP enantiomers. The detection limits ranged from 2.3 to 2.4ng/mL, whereas the limits of quantification ranged from 7.6 to 8.1ng/mL. Intra- and inter-assay precision and accuracy were acceptable, respectively. The method was applied to the analyses of 60 oral fluid specimens obtained from patients enrolled in a MTD maintenance programme. Our data pointed out that higher concentrations of (R)-MTD and the enantioselective excess of (S)-EDDP in OF may reflect the free fraction of MTD and EDDP enantiomers in plasma.     

Distribution of alpha 1-fetoprotein in fetal plasma, allantoic fluid, amniotic fluid and maternal plasma of cows.

Maximal concentrations of AFP, measured by RIA, were obtained in fetal plasma and amniotic and allantoic fluid between the 3rd and 4th month of gestation, with levels declining thereafter until term. AFP values in maternal plasma were unchanged. Throughout gestation, AFP values were higher in allantoic than in amniotic fluid and the ratio of allantoic fluid/amniotic fluid AFP was significantly correlated with gestational age.     

Motor, mental and behavioral developments in infancy are associated with fatty acid pattern in breast milk and plasma of premature infants

The objective of this study was to investigate any association between infants' early development and PUFA concentrations in early breast milk and infants' plasma phospholipids at 44 weeks gestational age. Fifty-one premature infants were included. The quality of general movement was assessed at 3 months, and motor, mental and behavioral development at 3, 6, 10 and 18 months corrected age using Bayley's Scales of Infant Development (BSID-II). Linoleic acid, the major n-6/n-3 FA ratios, Mead acid and the EFA deficiency index in early breast milk were negatively associated with development up to 18 months of age. DHA and AA, respectively, in infants' plasma phospholipids was positively, but the AA/DHA ratio negatively, associated with development from 6 to 18 months of age. Our data suggest that the commonly found high n-6 concentration in breast milk is associated with less favorable motor, mental and behavioral development up to 18 months of age.     

Amniotic fluid testosterone and testosterone glucuronide levels in the determination of foetal sex

Unconjugated testosterone levels were assayed in 351 amniotic fluid samples obtained at 15-19 weeks gestation. The median values for unconjugated testosterone in the 166 female foetuses and 185 male foetuses were 137 and 712 pmol/l respectively. Sixteen amniotic fluid samples from male foetuses had unconjugated testosterone levels lower than the highest female unconjugated testosterone value (361 pmol/l). Testosterone glucuronide was measured in amniotic fluid from 48 female and 55 male foetuses. There was a significant sex difference in the median values of testosterone glucuronide between female (median 160 pmol/l, range 64-465 pmol/l) and male (median 817 pmol/l, range 68-3707 pmol/l) amniotic fluid specimens (P less than 0.001). Of the sixteen male foetuses with amniotic fluid unconjugated testosterone levels in the female range, 12 had amniotic fluid testosterone glucuronide levels within the male testosterone glucuronide range of values. Hence used in conjunction with unconjugated testosterone, testosterone glucuronide increased the predictive accuracy of foetal sexing from 95.4 to 98.9%. Testosterone sulphate was measured in 24 female and 25 male amniotic fluid samples. There was no Testosterone sulphate was measured in 24 female and 25 male amniotic fluid samples. There was no significant difference between female (median 2591 pmol/l) and male (median 2964 pmol/l) testosterone sulphate levels.     

Fetomaternal adrenomedullin levels in diabetic pregnancy.

We investigated whether maternal and fetoplacental adrenomedullin, a newly discovered hypotensive peptide involved in the insulin regulatory system, is modified in diabetic pregnancy. We studied its correlation with pregnancy complications associated with this disease. Thirty-six pregnant women with diabetes (13 with type I and 23 with gestational diabetes mellitus) and in 40 uncomplicated pregnancies were included. 10 out of 36 diabetic pregnancies were complicated by gestational hypertension. In each woman, adrenomedullin concentration in maternal and fetal plasma and in amniotic fluid was assessed by specific radioimmunoassay. We found that overall mean amniotic fluid adrenomedullin concentration was higher (p < 0.05) in diabetic (14.7 +/- 1.6 fmol/ml) than in uncomplicated pregnancies (10.8 +/- 0.9 fmol/ml), whereas no differences were present in maternal and fetal plasma adrenomedullin levels between diabetic and uncomplicated pregnant women. High levels of amniotic fluid adrenomedullin were found in both type I and gestational diabetes mellitus pregnancies (13.7 +/- 1.4 and 15.6 +/- 2.2 fmol/ml, respectively). Diabetic pregnancies complicated by gestational hypertension showed lower (p < 0.05) amniotic fluid adrenomedullin concentrations than normotensive diabetic patients. These findings suggest that placental adrenomedullin production is upregulated in diabetic pregnancy, and it may be important to prevent excessive vasoconstriction of placental vessels.     

Comparison of the specific binding of cortisol in human amnion, amniotic fluid, and plasma

The purpose of this study was to compare the specific cortisol-binding protein found associated with human amnion with specific cortisol binding in human amniotic fluid and plasma. The electrophoretic mobility on polyacrylamide gels of the specific cortisol binding in amnion, amniotic fluid, and maternal plasma was identical. The influence of pH on cortisol binding activity was similar in all tissues and the cortisol binding was immunoprecipitable by a polyclonal antibody raised against human corticosteroid-binding globulin. The interaction of the cortisol binding protein with concanavalin A was studied in preterm amniotic fluid, term amniotic fluid, term amnion, and plasma from pregnant women at term and women under oral contraceptive treatment. Binding to concanavalin A was similar in term amnion and term amniotic fluid but was less than that found with both preterm amniotic fluid and term plasma. These results indicate that the cortisol binding protein associated with human amnion has similar characteristics to plasma corticosteroid-binding globulin, but that its state of glycosylation appears to be more like that of the cortisol binding protein in term amniotic fluid rather than in plasma.