Determination of lamivudine in plasma, amniotic fluid, and rat tissues by liquid chromatography

An HPLC method for the quantification of lamivudine (3TC) in rat plasma, amniotic fluid, placental and fetal tissues has been developed, validated and applied to the study of the placental transport of this drug in the pregnant rat. Placental and fetal tissues were processed using liquid-liquid extraction enhanced by salting out the sample using a saturated solution of ammonium sulfate. Plasma and amniotic fluid samples were processed by protein precipitation using 2 M perchloric acid. Reverse phase chromatography was performed using a phenyl column (5 microm, 150 mm x 2 mm i.d.) under a flow rate of 0.2 ml/min. The mobile phase consisted of 5% methanol in 20 mM dibasic phosphate buffer (pH 6). The method was validated over the range from 0.1 to 50 microg/ml for plasma and amniotic fluid and 0.2-50 microg/ml for the placental and fetal tissues.     

Simultaneous determination of abacavir and zidovudine from rat tissues using HPLC with ultraviolet detection

A simple high-performance liquid chromatography (HPLC) method has been developed and validated for the simultaneous determination of abacavir and zidovudine (AZT) in rat plasma, amniotic fluid, fetal, and placental tissues. Extraction of abacavir, AZT, and the internal standard, azidouridine (AZDU) in amniotic fluid was carried out by protein precipitation. Extraction from plasma, fetal and placental homogenates was achieved by using a salting out technique. Chromatographic separation was performed using a C8 column (150 mm x 4.6 mm, 5 microm). The mobile phase consisted of 12% acetonitrile in 25 mM sodium phosphate buffer (adjusted to pH 7 with sodium hydroxide) for the fetus, placenta, plasma and amniotic fluid samples at a flow rate of 0.8 mL/min. The method was validated over the range from 0.05 to 50 microg/mL for both abacavir and AZT in the four biological matrices. The absolute recovery of abacavir ranged from 79 to 94%, while AZT recoveries ranged from 79 to 90% in the different biological matrices. The internal standard recovery ranged from 90 to 92%. Acceptable intra- and inter-day assay precision (<10% R.S.D.) and accuracy (<10% error) were observed over 0.05-50 microg/mL for all four matrices.     

Simultaneous determination of 3'-azido-2',3'-dideoxyuridine and novel prodrugs in rat plasma by liquid chromatography

3'-Azido-2',3'-dideoxyuridine (AZDU) is a nucleoside analog structurally similar to zidovudine (AZT) with proven activity against human immunodeficiency virus (HIV). The purpose of this study was to develop and validate a high-performance liquid chromatographic (HPLC) method to quantitatively determine AZDU and its novel prodrugs in rat plasma simultaneously. A reversed-phase gradient elution HPLC method was developed to quantitate AZDU and its prodrugs, N3-pivaloyloxymethyl-3'-azido-2',3'-dideoxyuridine (I), 5'-pivaloyloxymethyl-3'-azido-2',3'-dideoxyuridine (II), 5'-O-valinyl-3'-azido-2',3'-dideoxyuridine hydrochloride (III) and 5'-O-phenylalanyl-3'-azido-2',3'-dideoxyuridine hydrochloride (IV), in rat plasma. AZDU and its prodrugs were analyzed using an octadecyl silane column with a mobile phase consisting of 0.04 microM sodium acetate buffer, pH 5.0, and acetonitrile, running in a segmented gradient manner at a flow rate of 2 ml/min. Acetonitrile was increased from 10 to 50% during the first 8 min by 5% per min, followed by 10% per min until it reached 90% acetonitrile. 3'-Azido-2',3'-dideoxy-5-ethyluridine (CS-85) was used as an internal standard (25 microg/ml). Compounds were detected by UV absorption at 261 nm. Extraction recoveries for all compounds were greater than 80%. Retention times of AZDU, CS-85, prodrugs I, II, III and IV were 3.3, 5.2, 9.1, 8.8, 6.3 and 7.3 min, respectively. Calibration plots were linear over the range of 0.25-100 microg/ml for AZDU and prodrugs II, III, and IV and 0.5-100 microg/ml for prodrug I. The limit of quantitation was 0.25 microg/ml for prodrugs II, III and IV and 0.5 microg/ml for prodrug I. The intra- and inter-day variations were less than 10% and accuracies were greater than 90%. This method is rapid, sensitive and reproducible for the determination of AZDU and prodrugs in rat plasma.     

Determination of 3′-azido-2′,3′-dideoxyuridine in maternal plasma, amniotic fluid, fetal and placental tissues by high-performance liquid chromatography

3′-Azido-2′,3′-dideoxyuridine (AZDU, Azddu, CS-87) is a nucleoside analog of 3′-azido-3′-deoxythymidine (zidovudine, AZT) that has been shown to inhibit human immunodeficiency virus (HIV-1). AZDU is a potential candidate for treatment of pregnant mothers to prevent prenatal transmission of HIV/AIDS to their unborn children. A rapid and efficient high-performance liquid chromatography (HPLC) method for the determination of AZDU concentrations in rat maternal plasma, amniotic fluid, placental and fetal tissue samples has been developed and validated. Tissue samples were homogenized in distilled water, protein precipitated and extracted using a C-18 solid-phase extraction (SPE) method prior to analysis. Plasma and amniotic fluid samples were protein precipitated with 2 M perchloric acid prior to analysis. Baseline resolution was achieved using a 4.5% acetonitrile in 40 mM sodium acetate (pH 7) buffer mobile phase for amniotic fluid, placenta and fetus samples and with a 5.5% acetonitrile in buffer solution for plasma at flow-rates of 2.0 ml/min. The HPLC system consists of a Hypersil ODS column (150×4.6 mm) with a Nova-Pak C-18 guard column with detection at 263 nm. The method yields retention times of 6.2 and 12.2 min for AZDU and AZT in plasma and 8.3 and 17.6 min for AZDU and AZT in amniotic fluid, fetal and placental tissues. Limits of detection ranged from 0.01 to 0.075 μg/ml. Recoveries ranged from 81 to 96% for AZDU and from 82 to 96% for AZT in the different matrices. Intra-day (n=6) and inter-day (n=9) precision (% RSD) and accuracy (% Error) ranged from 1.48 to 6.25% and from 0.50 to 10.07%, respectively.     

Liquid chromatography–tandem mass spectrometry analysis of cocaine and its metabolites from blood, amniotic fluid, placental and fetal tissues: study of the metabolism and distribution of cocaine in pregnant rats

The ability to simultaneously quantitate cocaine and its 12 metabolites from pregnant rat blood, amniotic fluid, placental and fetal tissue homogenates aids in elucidating the metabolism and distribution of cocaine. An efficient extraction method was developed to simultaneously recover these 13 components using underivatized silica solid-phase extraction (SPE) cartridges. The overall recoveries for cocaine and its metabolites were studied from pregnant rat blood (47–100%), amniotic fluid (61–100%), placental homogenate (31–83%), and fetal homogenate (39–87%). Extraction of the samples using silica is not classical SPE, but rather allows for the concentration of the sample into a small volume prior to injection and the removal of the proteins due to their strong interaction with the active silica surface. A positive ion mode electrospray ionization liquid chromatography–tandem mass spectrometry (LC–MS–MS) method was used and validated to simultaneously quantitate cocaine and 12 metabolites from these four biological matrices. A gradient elution method with a Zorbax XDB C₈ reversed-phase column was used to separate the components. Multiple reaction monitoring (MRM) of a product ion arising from the corresponding precursor ion was used in order to enhance the selectivity and sensitivity of the method. Low background noise was observed from the complex biological matrices due to efficient SPE and the selectivity of the MRM mode. Linear calibration curves were generated from 0.01 to 2.50 ppm. The method also showed high intra-day (n=3) and inter-day (n=9) precision (% RSD) and accuracy (% error) for all components. The limits of detection (LODs) for the method ranged from 0.15 to 10 ppb. The LODs of cocaine and its major metabolites were less than 1 ppb from all four biological matrices. This method was applied to the study of the metabolism and distribution of cocaine in pregnant rats following intravenous infusion to a steady state plasma drug concentration. The following results were observed in the pregnant rat study: (1) the observations correlated strongly with the previous literature data on cocaine metabolism and distribution, (2) cocaine and norcocaine accumulated in the placenta, (3) arylhydroxylation of cocaine was a major metabolic pathway, (4) para-arylhydroxylation of cocaine was favored over meta-arylhydroxylation in rats and (5) accumulation of cocaine and its major metabolites was observed in the amniotic fluid.     

Measurement of the anti-cancer agent gemcitabine in human plasma by high-performance liquid chromatography

A reversed-phase HPLC assay has been developed to determine the concentration of the anti-metabolite 2',2'-difluorodeoxycytidine (gemcitabine, dFdC) in human plasma over the concentration range of 0.5-150 microM (0.13-39.44 microg/ml), and 2',2'-difluorodeoxyuridine (dFdU), the deaminated, inactive metabolite, over the range of 1.0-227 microM (0.26-60 microg/ml). After the addition of 20 nmol 2'-fluorodeoxycytidine (FdC) as an internal standard, 0.5-ml samples of plasma were subjected to acetonitrile precipitation, followed by analysis using a gradient reversed-phase HPLC assay with UV detection. A Phenomenex Columbus C(18) column, 5 microm, 150 x 4.6 mm, and a Waters C(18), 4 microm, Nova-Pak Sentry guard column were used to achieve separation. FdC, dFdC and dFdU were monitored at 282, 269 and 258 nm, respectively, on a Waters 996 photodiode array detector. The mobile phase, run at a total flow-rate of 1.5 ml/min, was composed of two solvents: 50 mM ammonium acetate pH 5.0 in either 2% (solvent A) or 10% methanol (solvent B, v/v); 100% solvent A was run for 17 min, followed by a linear gradient to 100% solvent B over 14 min. FdC, dFdC and dFdU were resolved from endogenous compounds and had retention times of 13.6+/-0.5, 18.1+/-1.1 and 29.0+/-0.6 min, respectively. The assay was useful in measuring the plasma levels of both analytes in samples obtained from adult cancer patients participating in a Phase I trial of gemcitabine given as either a 1- or 2-h infusion weekly for 3 of 4 weeks.     

Determination of acyclovir in maternal plasma,amniotic fluid,fetal and placental tissues by high-performance liquid chromatography

Acyclovir [9-[(2-hydroxyethoxy)-methyl]-guanosine, Zovirax, ACV] is a synthetic purine nucleoside analog active against herpes simplex virus types 1 (HSV-1), 2 (HSV-2), and varicella zoster virus. Acyclovir has frequently been used in HSV-2 seropositive mothers to prevent prenatal transmission of herpes virus to their unborn children. A fast and reproducible HPLC method for the determination of the highly polar acyclvoir in maternal rat plasma, amniotic fluid, placental tissue, and fetal tissue has been developed and validated. Plasma and amniotic fluid samples were prepared by protein precipitation using 2 M perchloric acid and syringe filtering. Tissue samples were homogenized in distilled water, centrifuged, and extracted using a C(18) solid-phase extraction method prior to analysis. Baseline resolution was achieved for acyclovir and the internal standard gancyclovir, an anti-viral of similar structure to acyclovir, using an Agilent Eclipse XDB C(8) column (150 x 2.1 mm, 5 microm). The mobile phase used for the plasma and amniotic fluid was 10 mM acetate/citrate buffer-3.7 mM aqueous octanesulfonic acid (87.5:12.5, v/v) at a flow-rate of 0.2 ml/min. The mobile phase used for the tissue samples was 30 mM acetate/citrate buffer with 5 mM octanesulfonic acid-acetonitrile (99:1, v/v). Both aqueous mobile phase portions were pH adjusted to 3.08. All separations were done using an Agilent 1100 Series HPLC system with UV detection of 254 nm. The assay was validated for each matrix over a range of 0.25-100 microg/ml over 3 days using five replicates of three spiked concentrations. The relative standard deviation and percent error for each validation data set was <15% for middle and high quality control (QC) points and <20% for all low QC points. All calibration curves showed good linearity with an R(2)>0.99. The extraction efficiency for recovery of acyclovir from all matrices was >80%.     

Transplacental pharmacokinetics and fetal distribution of 2', 3'-didehydro-3'-deoxythymidine (d4T) and its metabolites in late-term rhesus macaques

BACKGROUND: The overall goal of human immunodeficiency virus (HIV) therapy during pregnancy is to maintain maternal health and reduce the probability of vertical transmission during gestation and delivery, while keeping toxicity risks low. Azidothymidine (AZT) is currently recommended for pregnant women infected with HIV; however, many pregnant women are unable to tolerate AZT because of toxicity. In the present study, the placental transfer and fetal accumulation of the anti-HIV compound 2',3'-didehydro-3'-deoxythymidine (d4T) and its active (triphosphorylated) and inactive (thymine and beta-aminoisobutyric acid) metabolites were examined at steady state in late-term rhesus macaques. METHODS: On the day of the hysterotomy, the mother was administered an intravenous loading dose of d4T, followed by a 3-hr steady-state intravenous infusion that also included [(3)H]d4T as a tracer. After 3 hr of infusion, the fetus was delivered by cesarean section under halothane/N(2)O anesthesia. Plasma, amniotic fluid, and tissues were analyzed for d4T and its inactive metabolites by HPLC; tissue samples were analyzed for d4T and active (phosphorylated) metabolites by strong anion-exchange HPLC. RESULTS: Maternal steady-state plasma concentrations of d4T were 1-2 microg/ml, with a fetal-to-maternal plasma ratio of 0.85 +/- 0.09. The fetal tissue distribution of radioactivity was highest in the kidney and lowest in the brain. D4T, thymine, and beta-aminoisobutyric acid were detected in all fetal tissues examined. CONCLUSIONS: Our data indicate that d4T readily crosses the placenta and is present in the fetus as parent compound or its inactive metabolites after maternal infusion. Although fetal plasma concentrations of d4T were similar to clinical d4T concentrations, no phosphorylated metabolites were detected. Teratology 62:93-99, 2000. Published 2000 Wiley-Liss, Inc.     

Maternal DDAVP-induced hyponatremia preserves fetal urine flow during acute fetal hemorrhage

Maternal administration of DDAVP induces maternal and fetal plasma hyponatremia, accentuates fetal urine flow, and increases amniotic fluid volume. Fetal hemorrhage represents an acute stress that results in fetal AVP secretion and reduced urine flow rate. In view of the potential therapeutic use of DDAVP for pregnancies with reduced amniotic fluid volume, we sought to examine the impact of maternal hypotonicity during acute fetal hemorrhage. Chronically catheterized pregnant ewes (130 +/- 2 days) were allocated to control or to DDAVP-induced hyponatremia groups. In the latter group, tap water (2,000 ml) was administered intragastrically to the ewe followed by DDAVP (20 microg bolus, 4 microg/h) and a maintenance intravenous infusion of 5% dextrose water for 4 h to achieve maternal hyponatremia of 10-12 meq/l. Thereafter, ovine fetuses from both groups were continuously hemorrhaged to 30% of estimated blood volume over a 60-min period. DDAVP caused similar degree of reductions in plasma sodium and osmolality in pregnant ewes and their fetuses. In response to hemorrhage, DDAVP fetuses showed greater reduction in hematocrit than control fetuses (14 vs. 10%). Both groups of fetuses demonstrated similar increases in plasma AVP concentration. However, the AVP-hemorrhage threshold was greater in DDAVP fetuses (22.5%) than in control (17.5%). Hemorrhage had no significant impact on plasma osmolality, electrolyte levels, or cardiovascular responses in either group of fetuses. Despite similar increases in plasma AVP, DDAVP fetuses preserved fetal urine flow rates, with values threefold those of control fetuses. These results suggest that under conditions of acute fetal stress of hemorrhage, maternal DDAVP may preserve fetal urine flow and amniotic fluid volume.     

The relationships between fetal and maternal placental blood flows.

Individual maternal and fetal flows to 706 placental cotyledons obtained from 9 chronically catheterized pregnant ewes and their fetuses (gestation age 123-141 days) were measured. The larger the cotyledon the greater the maternal and fetal blood flow to it. Both fetal and maternal flows to larger cotyledons, however, tended to be lower when corrected for the weight of the cotyledon perfused. Changes in fetal placental flow (dfgc, ml/min/g) occurring within 15 min of administration of 15 mg i.v. of captopril to the ewe were dependent on changes in fetal placental vascular resistance (dcotfr) and maternal flow (dmgc) according to the equation dfgc = 0.123 + 0.185 dmgc - 0.026 dcotfr. Changes in maternal placental flow occurring within 15 min of administration of 15 mg i.v. of captopril to the ewe were dependent on changes in maternal placental vascular resistance (dcotmr) and changes in fetal flow according to the equation dmgc = 0.483 + 0.496 dfgc - 0.0198 dcotmr. The changes in fetal flow over the next 1.5h of treatment with captopril at 6 mg/h were dependent on neither changes in fetal placental vascular resistance nor maternal placental flow. changes in maternal placental flows over the same time were no longer related to changes in fetal flow and depended only to a minimal extent on changes in maternal placental resistance. These analyses suggest that treatment of the pregnant ewe with captopril may have disturbed the normal relationships between maternal and fetal placental circulations.     

Gestational profiles of rat placental lactogen-II (rPL-II) and growth hormone (GH) in maternal and fetal serum, amniotic fluid, and placental tissue.

Rat placental lactogen-II (rPL-II) and growth hormone (rGH) in maternal and fetal serum, amniotic fluid, and placental tissue were measured by a homologous radioimmunoassay during the last half of pregnancy. rPL-II appeared first in maternal circulation and the placental tissue on day 11 of pregnancy. The maternal serum rPL-II concentration increased progressively and reached the peak value (684 +/- 76 ng/ml) on day 19, and declined thereafter up to term. rPL-II content in the tissue had a similar pattern to the maternal serum profile of rPL-II, while its concentration in the tissue increased dramatically on day 12 and remained high until day 19. Fetal serum rPL-II was detected on days 17 and 18, though its concentration was much lower (ranged between 3-10 ng/ml) than that of maternal serum. rPL-II in amniotic fluid was also detectable only on days 12-14 of pregnancy, and the peak value on day 13 was 22% of the maternal serum rPL-II concentration. The rGH concentration increased gradually as pregnancy advanced with a decline on the day before parturition. Although rGH in fetal serum increased on day 20 with a decline on the following day, it was slightly detectable in amniotic fluid on the last two days of pregnancy. The molecular profile of rPL-II in amniotic fluid and maternal serum of day 13 pregnant rats were examined by Western blotting. Anti-rPL-II serum detected two proteins with molecular weights (mol wt) of 19.5K and 20.5K in amniotic fluid and one protein with a mol wt of 20.5K in maternal serum under nonreducing conditions.(ABSTRACT TRUNCATED AT 250 WORDS)     

Simultaneous determination of N-butyramide-tacrine and tacrine in mouse plasma and brain homogenate by high-performance liquid chromatography with a simple gradient solvent system

A novel reversed-phase HPLC method was developed for the simultaneous determination of tacrine (THA) and the newly synthesized prodrug (N-butyramide-THA, BTHA) in mouse plasma and brain homogenate. The assay involves deproteinisation and subsequent detection at 240 nm with a gradient solvent system. Retention times were 18.5 and 9.3 min for BTHA and THA, respectively. Average recoveries for the analytes were 80.7% (BTHA) and 76.6% (THA) from plasma, and 75.0% (BTHA) and 68.4% (THA) from brain homogenate. Linear responses were observed over a wide range (0.25-20 microg/ml for BTHA in plasma and in brain homogenate, 0.025-20 microg/ml for THA in both matrices). Both BTHA and THA degraded from the prodrug can be detected even 12 h after intravenous administration of BTHA, indicating that BTHA is a promising prodrug for brain targeting.     

Technique validation by liquid chromatography for the determination of acyclovir in plasma

In this research project, a high-performance liquid chromatography (HPLC) method was developed for the determination of acyclovir (ACV) in plasma. The plasma samples, recharged with acyclovir and in presence of 5'-N-methylcarboxyamidoadenosine (MECA) as an internal standard, were purified using a solid-phase extraction technique with Waters Oasis HLB columns. The separation of the components from the extract was carried out in a LiChrospher 100 RP-18 column for further ultraviolet detection at a wavelength range of 250-260 nm. The mobile phase composition was 18% acetonitrile, sodium dodecylsulphate 5 mM and phosphate buffer at pH 2.6 with an analysis time of 13 min per sample. The average retention time for acyclovir was of 5.0 min and for the internal standard 11.2 min. The calibration curve was linear ranging between 0.05 and 1.80 microg/ml. The detection limit was 0.006 microg/ml with a quantification limit of 0.020 microg/ml. The ACV recuperation percentage for 250 microl of plasma was between 94.7 and 109.7% with a coefficient of variation not higher than 5.2%. This method was developed and validated for use in bioavailability and bioequivalence studies.     

Studies on the growth of the fetal sheep. Effects of surgical reduction in placental size, or experimental manipulation of uterine blood flow on plasma sulphation promoting activity and on the concentration of insulin-like growth factors I and II

The effect of long- and short-term manipulations of uterine blood flow on fetal plasma levels of IGF-I and -II have been studied in sheep at days 125-139 of pregnancy and compared with those in near term rats and guinea pig. The primary objective is to show that both long- and short-term reduction of uterine blood flow is associated with increase in the fetal plasma concentration of IGF-II while that of IGF-I falls. In the pregnant sheep long-term depression of utero-placental blood flow was caused by surgical reduction in placental mass (carunclectomy) prior to conception. This reduced fetal weight to 2.42 +/- 0.49 kg (SD) compared with 3.41 +/- 0.46 in controls; the respective values for uterine blood flow being 1694 +/- 558 and 913 +/- 324 ml/min respectively. This was associated with a fall in fetal plasma IGF-I concentration from 22.6 +/- 3.4 ng/ml to 14.9 +/- 1.31 ng/ml and a rise in IGF-II from 1952 +/- 284 ng/ml to 3360 +/- 914 ng/ml respectively. Similar changes in the plasma concentrations of IGF peptides were observed in fetal rats and guinea pigs in response to uterine artery ligation. Short-term reduction (60 min) of the uterine blood flow was caused either by compression of the common uterine artery to depress flow from 1491 +/- 375 to 648 +/- 216 ml/min or through intraarterial infusion of adrenaline at 35 ug/min to lower flow from 1628 +/- 339 to 1195 +/- 128 ml/min. Such falls in uterine blood flow had no significant effect on fetal plasma IGF-I levels but increased IGF-II levels by 30 to 60%.     

Assay of leucine aminopeptidase activity in vitro using large-pore reversed-phase chromatography with fluorescence detection

A chromatographic method for determination of leucine aminopeptidase (LAP) activity in complex matrices is described. L-Leucine-beta-naphthylamide was used as the substrate and its hydrolytic product, beta-naphthylamine, was monitored by fluorescence at 280 nm excitation and 400 nm emission wavelengths. Under optimized conditions, the components in the incubation mixture were baseline separated and eluted out of a large-pore (300 angstroms) reversed-phase C4 column (RPC4) within 15 min with a non-linear gradient elution of methanol (0.05% (v/v) trifluoroacetic acid additive). The detection limit of the hydrolytic product reached 0.35 pmol at three time signal-to-noise (S/N) ratio with 5 microl sample injection. The method showed a wide dynamic range for quantitation of both the hydrolytic product (10 ng/ml to 80 microg/ml) and LAP (0.1-46.0 microg/ml) with correlation coefficient larger than 0.998 and reproducibility <3 and 10% R.S.D. (n=3), respectively. A fairly broad range of incubation time could be selected within 1 h. The LAP activities and concentrations in rabbit serum, tears, and mouse lens homogenates were determined to be 41.8 (0.3 mg/ml), 2.8 (40.0 microg/ml), and 1.6 pmol/(microl min) (17.5 microg/ml), respectively, with reproducibility of 2-9% R.S.D. (n=3) and intra- and inter-day variation for the retention time of the hydrolytic product being <1% R.S.D. (n=3). The results indicate that the present method is rapid and sensitive as compared to the conventional one.     

Arachidonic acid metabolism by bovine placental tissue during the last month of pregnancy

Conversion of tritiated arachidonic acid (AA) into metabolites of the cyclo- and lipoxygenase pathways by bovine fetal placental tissue (200 mg) and fetal plus maternal placental tissue (400 mg) of Days 255, 265, 275 of gestation and at parturition (n = 5) during a 30 min incubation was measured using reverse-phase high pressure liquid chromatography. Fetal placental tissue produced 13,14-dihydro-15-keto-prostaglandin E2 (PGEM) as the major metabolite, the synthesis of which increased from Day 265 to Day 275 and parturition by 150% and 475%, respectively. In tissues collected at parturition, PGE2 synthesis was also detected. On Day 275 and at parturition fetal placental tissue synthesized the metabolite 12-hydroxyheptadecatrienoic acid (HHT), and throughout the experimental period the lipoxygenase product 15-HETE was detected with synthesis rates increasing over time of gestation. In addition, an unidentified metabolite was regularly found in the radiochromatograms which eluted at 1 h and 1 min (U101), between HHT and 15-HETE. The synthesis of this metabolite decreased as pregnancy progressed. Furthermore, various other polar and nonpolar metabolites pooled under the heading UNID were eluted, the production of which increased over time of gestation. The presence of maternal placental tissue did not influence the synthesis of PGEM, 15-HETE and U101, but the production of HHT was decreased when maternal tissue was present. Also, as pregnancy progressed, maternal placental tissue seemed to contribute to the pool of unidentified metabolites. In conclusion, fetal placental tissue seems to be the major source of the AA metabolites when compared with maternal placental tissue, and AA metabolism by bovine placental tissue is markedly increased throughout the last month of pregnancy, suggesting a role for AA metabolites in mechanisms controlling parturition.     

Fetal insulin and placental 3-O-methyl glucose clearance in near-term sheep

It is difficult, if not impossible, to measure the placental transfer of glucose directly because of placental glucose consumption and the low A-V glucose difference across the sheep placenta. We have approached the problem of quantifying placental hexose transfer by using a nonmetabolized glucose analogue (3-O-methyl glucose) which shares the glucose transport system. We have measured the clearance by using a multisample technique permitting least squares linear computing to avoid the errors implicit in the Fick principle. The placental clearance of 3-O-methyl glucose was measured in the control condition and after the administration of insulin to the fetal circulation. A glucose clamp technique was used to maintain constant transplacental glucose concentrations throughout the duration of the experiment. A control series was performed in which the only intervention was the infusion of normal saline. In these experiments the maternal and fetal glucose concentrations remained constant as did the volume of distribution of 3-O-methyl glucose in the fetus. The maternal insulin concentration remained constant and fetal insulin concentration changed from 11 +/- 2 microU/ml to 355 +/- 51 microU/ml (P less than 0.01). In the face of this large increase in fetal plasma insulin, there was no change in the placental clearance of 3-O-methyl glucose. In the control condition the clearance was 14.1 +/- 1.0 ml/min per kg and this was 13.8 +/- 1.0 ml/min per kg in the high insulin condition. Fetal insulin may change placental glucose flux by decreasing fetal plasma glucose concentrations but does not do so by changing the activity of the glucose transport system.     

Prp-c and Prp-Sc at the fetal-maternal interface

Scrapie is a naturally occurring prion (PrP) disease causing a fatal neurodegenerative disorder in sheep and goats. Previous studies suggest that scrapie is transmitted naturally through exposure to the scrapie agent in wasted placentas of infected ewes. This study determined the distribution and biochemical properties of PrP cellular (PrP-C) and the distribution of PrP scrapie (PrP-Sc) in reproductive, placental, and selected fetal tissues and fetal fluids in sheep. Glycosylated, N-terminally truncated, proteinase K-sensitive PrP-C with apparent molecular masses of 23-37 kDa was present in reproductive, placental, and fetal tissues and fetal fluids. PrP-C was low or undetectable in intercotyledonary chorioallantois, amnion, urachus, amniotic fluid, and fetal urine. In pregnant ewes, cotyledonary chorioallantois, allantoic fluid, and caruncular endometrium contained higher levels of PrP-C than did intercaruncular endometrium, myometrium, oviduct, ovary, fetal bladder, or fetal kidney. Caruncular endometrial PrP-C was up-regulated during pregnancy. Despite the wide distribution of PrP-C in reproductive, placental, and selected fetal tissues and fetal fluid, PrP-Sc was detected only in caruncular endometrium and cotyledonary chorioallantois of pregnant scrapie-infected ewes. The embryo/fetus may not be exposed to scrapie in utero because it is separated physically from PrP-positive allantois and chorioallantois by PrP-negative amnion.     

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.