Determination of Dabigatran Concentration in Human Plasma and Breast Milk.

Venous thromboembolism (VTE) is an important cause of death following childbirth. Dabigatran etexilate can be a useful prophylaxis in susceptible women during the postpartum period. However, it is not clear whether dabigatran is excreted into breast milk in amounts which can be harmful to the suckling baby. We have developed an accurate, sensitive, and specific assay for the quantitation of dabigatran in both human plasma and breast milk. This is particularly useful for the determination of the extent by which dabigatran is secreted into breast milk in relation to its systemic availability. Dabigatran was enriched from both matrices using solid-phase extraction prior to separation on a C8-RPLC column and detection using SRM on a QqTrap mass spectrometer. The assay was validated for specificity, sensitivity, linearity, precision, accuracy, and stability of the analyte in human plasma and breast milk. The lower limit of detection for dabigatran was 20 pg/ml in plasma and 75 pg/ml in breast milk. This assay will aid future studies for the measurement of dabigatran concentrations in human breast milk to help determine if dabigatran etexilate can safely be administered to breast-feeding women.

Intrapartum cardiotocograph interpretation by midwives and trainee obstetricians using a modified definition of a fetal heart rate deceleration.

The aim of this study was to assess cardiotocograph (CTG) interpretation by midwives and trainee obstetricians using the standard and a modified definition of fetal heart rate deceleration compared with consultant interpretation as the Gold Standard. A randomised survey using the online tool at: www.surveymonkey.com between 4 January and 24 April 2009, was conducted at a tertiary obstetric unit, UK. A total of 104 (∼54%) health professionals responded, providing 1,118 responses with respect to the presence of decelerations on 13 anonymised CTGs. Five obstetric consultants (62.5%) provided expert opinion. Midwives and trainee obstetricians were more likely to concur with Consultant opinion when using the modified definition of fetal heart rate deceleration compared with the standard definition. Larger scale studies may be needed to further evaluate the usefulness of the modified definition.

Efficacy of follow-up and contact tracing of women who test positive for genital tract chlamydia trachomatis prior to pregnancy termination.

We examined the efficacy of follow-up, contact tracing and the need for retreatment in women who were screen-positive for genital tract Chlamydia trachomatis prior to termination of pregnancy. Eighty-six of 1363 (6.3%) women screened positive. These women were significantly younger than those who screened negative (P < 0.0001). The genitourinary medicine (GUM) clinic was notified of 73 (84.9%) screen-positive women and 41 (47.7%) attended for follow-up. Contact tracing was undertaken in 38 (92.7%) women who attended and 29 (70.7%) women who attended required retreatment for Chlamydia. The median duration between pregnancy termination and GUM clinic attendance was significantly longer in women who required retreatment compared to those who did not require retreatment (P = 0.003). In conclusion, follow-up and contact-tracing of women who screen positive for genital tract C. trachomatis was incomplete. This may substantially compromise the cost-effectiveness of a screen-and-treat programme.

L-Arginine transport across the basal plasma membrane of the syncytiotrophoblast of the human placenta from normal and preeclamptic pregnancies.

Cord blood levels of nitrate/nitrite, as a measure of nitric oxide (NO), are generally increased in preeclampsia. As L-arginine is the precursor for NO synthesis, we hypothesized that L-arginine transport across the syncytiotrophoblast basal plasma membrane (BM) of placentas from preeclamptic patients is also increased. Glutamine-sensitive and -insensitive [(3)H]L-arginine uptakes into BM vesicles were measured and expressed as femtomoles per milligram of protein per minute. Total L-arginine uptake was 418 +/- 15 (mean +/- SEM; n = 9) in BM from control placentas (CBM) and 495 +/- 27 (n = 7) in BM from preeclamptic placentas (PE BM; P < 0.05, by two-tailed t test). Glutamine insensitive (system y(+)) uptake was 45 +/- 3 (n = 6) in CBM, with a significantly higher uptake of 97 +/- 23 (n = 5) into PE BM (P < 0.05, by two-tailed t test). There was no significant difference in glutamine-sensitive uptake between the two groups. The expression of mRNA for human cationic amino acid transporter (hCAT) 1, 2, and 4 (system y(+) genes) and 4F2hc (heavy chain of system y(+)L) was not different in homogenates of whole placenta from the two groups. Western blotting data showed that hCAT-1 protein expression in PE BM was higher than that in CBM. These data suggest increased activity of the BM system y(+) cationic amino acid transporter in preeclampsia. If reflected in vivo, a similar increase in transporter activity could alter the delivery of L-arginine to syncytiotrophoblast eNOS.

Cationic amino acid transporter activity in the syncytiotrophoblast microvillous plasma membrane and oxygenation of the uteroplacental unit.

The purpose of this study was to determine whether there is any relationship between the activity of cationic amino acid transporters in the microvillous plasma membrane (MVM) of the syncytiotrophoblast and the oxygenation of the uteroplacental unit. Oxygenation data were obtained at the time of caesarean section from the uterine veins, the maternal radial artery and the umbilical vessels of 7 normal (AGA) and 13 intrauterine growth restricted (IUGR) pregnancies. Microvillous plasma membranes were isolated from the same placentas and the activity of the system y(+) and y(+)L cationic amino acid transporters determined by measuring (3)H- l -arginine uptake in the presence and absence of l -glutamine. In IUGR pregnancies uterine venous Po(2) was significantly higher (AGA=44.7+/-8.0 mmHg; IUGR=57.2+/-2.3 mmHg, P< 0.05) and umbilical venous Po(2) was significantly lower (AGA=33.4+/-3.0 mmHg; IUGR=25.1+/-2.0 mmHg, P< 0.05) than in AGA pregnancies. System y(+)L activity, but not system y(+) activity, was inversely correlated with uterine venous Po(2) (P< 0.01; r(2)=0.4) in AGA and IUGR pregnancies. In IUGR pregnancies without associated maternal pre-eclampsia, y(+)L activity, but not y(+) activity, was also directly related to the umbilical O(2) content difference (P< 0.01; r(2)=0.9). A significant negative correlation was found between system y(+) and the umbilical O(2) content difference in AGA pregnancies (P< 0.01; r(2)=0.9). Our data are consistent with the hypothesis that in IUGR fetuses uterine oxygenation is not reduced and can be increased. The inverse correlation between system y(+)L activity and uterine venous Po(2) and the correlations with umbilical venous-arterial O(2) content difference suggest a relationship between cationic amino acid transporter activity and oxygen tension in the uteroplacental unit.

L-arginine transport by the microvillous plasma membrane of the syncytiotrophoblast from human placenta in relation to nitric oxide production: effects of gestation, preeclampsia, and intrauterine growth restriction.

Nitric oxide (NO) is an important regulator of placental perfusion, and its production is dependent on the activity of substrate (L-arginine) transporters. In the light of evidence for altered NO production in the feto-placental unit in preeclampsia and intrauterine growth restriction (IUGR), we investigated gestational changes in human placental L-arginine transport by systems y(+) and y(+)L in purified microvillous plasma membrane vesicles. We also examined the effect of preeclampsia and IUGR on the activity of these transport systems and the relationship between transporter activity and NO production (nitrate/nitrite concentrations) in the feto-placental unit. Between first trimester and term, there was a significant positive correlation between system y(+) activity and gestational age (r = 0.36; P = 0.013; n = 47), but a significant negative correlation between system y(+)L activity and gestational age (r = -0.6; P < 0.0001; n = 47). The activity of these transport systems was not altered in preeclampsia or IUGR. In placentas from normal term pregnancies, there was no correlation between the activity of microvillous plasma membrane L-arginine transporters and nitrate/nitrite concentrations in umbilical venous plasma or placental homogenate.

Development and polarization of cationic amino acid transporters and regulators in the human placenta.

We have investigated L-arginine transport systems in the human placental syncytiotrophoblast across gestation using purified microvillous (MVM) and basal (BM) plasma membrane vesicles. In MVM from first-trimester and term placentas, L-arginine transport was by systems y(+) and y(+)L. In BM (term placentas), however, there was evidence for system y(+)L only. The Michaelis constant of system y(+)L was significantly lower (P < 0.05) in first-trimester compared with term MVM and lower in term MVM compared with BM (P < 0.05). There was no functional evidence for system b(0+) in term MVM or BM. Cationic amino acid transporter (CAT) 1, CAT 4, and 4F2hc were detected using RT-PCR in placentas throughout gestation. rBAT was not detected in term placentas. An approximately 85-kDa and an approximately 135-kDa protein was detected by Western blotting in MVM under reducing and nonreducing conditions, respectively, consistent with the 4F2hc monomer and the 4F2hc-light chain dimer, and their expression was significantly higher (P < 0.05) in term compared with first-trimester MVM. These proteins were not detected in BM despite functional evidence for system y(+)L. These data suggest different roles for 4F2hc in the development and polarization of cationic amino acid transporters in the syncytiotrophoblast.

Management of intra-abdominal abscesses in Crohn's disease.

Over a 5-year period, 54 intra-abdominal abscesses were observed in 40 (20.8%) of 192 patients with Crohn's disease. The median age was 39 years (range 17-76 years); median interval from diagnosis, 7.5 years (range 0-24 years) and the median number of surgical operations was 2 (range 0-7). Forty abscesses (74.1%) were spontaneous and 14 (25.9%) were postoperative. Thirty abscesses were initially managed by laparotomy, 14 by percutaneous drainage, nine by incision and drainage and in one case the abscess drained spontaneously. Intra-abdominal abscesses were managed successfully by laparotomy in 23 (76.7%) of 30 patients, with a 93% success rate (13 of 14) for spontaneous abscesses managed by resection and primary anastomosis. Three of 8 (37.5%) spontaneous abscesses were managed successfully by percutaneous drainage, a temporising effect being achieved in a further two cases. There was no significant difference in sepsis score or duration of hospital stay for patients managed initially by laparotomy and those managed by drainage. However, patients with stricturing or fistulating Crohn's disease were much more likely to have initial management by laparotomy and in these patients surgical intervention was found to be an effective initial strategy.

Synthesis and evaluation of amino analogues of valproic acid.

Valproic acid, an antiepileptic drug, is extensively metabolized in humans. Two putative metabolites, 2-n-propyl-3-aminopentanoic acid (3-aminovalproic acid, 3-amino-VPA; 2a) and 2-n-propyl-4-aminopentanoic acid (4-amino-valproic acid, 4-amino-VPA; 4a), which may result from the transamination of the respective keto acids 1a and 3a may explain the unusual extended seizure protection elicited by valproic acid. The title compounds were synthesized as their diasteriomeric ethyl esters 2b and 4b and submitted for anticonvulsant evaluation by the Antiepileptic Drug Development Program of the National Institute of Neurological and Communicative Disorders and Stroke. The results verified our hypothesis, as 4b was active in the subcutaneous pentylenetetrazol (scMet) evaluation at 30 mg/kg. Both compounds were highly toxic at 300 mg/kg.