Measurement of rest and activity in newborn lambs using actigraphy: studies in term and preterm lambs.

The present study used actigraphy to monitor rest-activity cycles in lambs. We employed an Actiwatch Activity Monitor, which was secured on the lamb's neck in 13 term lambs and six preterm lambs. Activity measurements began on the day of delivery and lasted for 7.3+/-0.7 days. All lambs exhibited bouts of activity, lasting from approximately 2 to 60 min, separated by periods of inactivity of about equal duration. There was a progressive increase in the frequency and intensity of activity bouts with age, and a decrease in duration. In relation to postnatal age, preterm lambs had a significantly lower frequency and intensity of activity bouts compared with term lambs and significantly longer mean active bout duration. However, in relation to post-conceptual age, preterm animals were less active at birth, but thereafter the trajectory for activity development was steeper compared with the term lambs. These differences between term and preterm lambs may be due to several factors including differences in: (1) the lengths of time the two groups spent in utero and as neonates as a proportion of the perinatal period, which could influence the rate of muscle and bone growth; (2) prenatal and postnatal hormonal profiles; and (3) maternal care. We also found differences in postnatal motility in male and female lambs, with the trajectory of activity increasing in males at Days 4-5, which could be due, in part at least, to sex differences in both prenatal and postnatal hormonal profiles.

Dose-dependent pharmacokinetics and metabolism of valproic acid in newborn lambs and adult sheep.

Dose-dependent pharmacokinetics and metabolism of valproic acid (VPA) were studied in newborn and adult sheep to assess age-related differences in plasma protein binding and metabolic elimination. Newborn lambs received either a 10- (n = 8), 50- (n = 5), 100- (n = 4), or 250-mg/kg (n = 4) VPA i.v. bolus. Individual adult sheep (n = 5) received all four doses in a random order with an appropriate washout period between experiments. Unbound or metabolic clearance of VPA was significantly higher in adult sheep at the two lower doses when compared with lambs, and similar to the lambs at the two higher doses. Plasma protein binding was nonlinear at all doses. Estimates of binding capacity (B(max1)) at the saturable site were higher in adults (91.8 microg/ml) when compared with lambs (44.9 microg/ml), whereas the opposite trend was observed for binding affinity [K(d1) = 9.6 microg/ml (adult) versus 3.2 microg/ml (lambs)]. Characterization of developmental differences in overall VPA metabolic elimination involved fitting of unbound VPA plasma concentration data to a two-compartment model with Michaelis-Menten elimination. This resulted in similar in vivo estimates of apparent V(max) [445.0 microg/min/kg (adult) versus 429.9 microg/min/kg (lambs)]. However, apparent K(m) estimates appeared to be higher in lambs [30.0 microg/ml (adult) versus 69.6 microg/ml (lambs)]. Similar findings were obtained from in vivo estimates of V(max) and K(m) for VPA glucuronidation obtained from VPA-glucuronide metabolite urinary excretion data. Thus, it appears that age-related differences in metabolic clearance may be related to differences in the apparent in vivo K(m) as opposed to V(max) of VPA glucuronidation.

Pharmacokinetics and renal excretion of diphenhydramine and its metabolites, diphenylmethoxyacetic acid and diphenhydramine-N-oxide, in developing lambs.

The developmental disposition of diphenhydramine (DPHM) and its metabolites, diphenylmethoxyacetic acid (DPMA) and DPHM-N-oxide (DPHMNO), was investigated in postnatal lambs. Lambs received a DPHM intravenous (iv) bolus 15 days (Group A; n = 5) or 2 months (Group B; n = 6) after birth. Total body clearance of DPHM in postnatal lambs (Group A = 138.7 +/- 80.5 mL/min/kg; Group B = 165.7 +/- 51.3 mL/min/kg) was similar to the nonplacental clearance values (i.e., the component of fetal total body clearance that is not due to elimination via the placenta) estimated for fetal lamb (116.3 +/- 49. 6 mL/min/kg), and significantly greater than estimates in adult sheep (38.5 +/- 12.3 mL/min/kg). In addition, Group A DPHM renal clearance (CL(r), >1.80 +/- 1.24 mL/min/kg) was similar to that of the fetus (2.06 +/- 0.24 mL/min/kg), and significantly greater than that for Group B (0.26 +/- 0.17 mL/min/kg) and the adult (0.012 +/- 0.005 mL/min/kg). In contrast, similar to the fetal situation, postnatal DPMA CL(r) (Group A = 0.02 +/- 0.02 mL/min/kg; Group B = 0.05 +/- 0.01 mL/min/kg) was significantly less than adult values (0. 53 +/- 0.19 mL/min/kg). Because DPMA is not sequentially metabolized in sheep, the lower CL(r) in postnatal lambs results in longer apparent elimination half-lives of this metabolite (Group A = 90.4 +/- 32.2 h; Group B = 13.13 +/- 11.0 h) compared with that in the adult (2.9 +/- 1.6 h). No age-related difference in DPHMNO CL(R) was observed. Alterations in the CL(r) of DPHM and DPMA are likely related to differences in the rate of development of mechanisms (i.e. , tubular secretion and reabsorption and glomerular filtration rate) involved in the urinary drug excretion of organic acids and bases.

Ontogeny of valproic acid disposition and metabolism: a developmental study in postnatal lambs and adult sheep.

The ontogeny of valproic acid (VPA) disposition and metabolism was investigated in developing lambs and adult sheep (Dorset or Suffolk breed). Specifically, we wished to investigate the role of glucuronidation and beta-oxidation on VPA elimination during development. Catheters were implanted in a carotid artery, a jugular vein, and the urinary bladder in 10-day-old (10 d; n = 8), 1-month-old (1 M; n = 4), and 2-month-old lambs (2 M; n = 5). In adult sheep (n = 5), catheters were implanted in a femoral artery and vein. After the administration of a 10 mg/kg VPA i.v. bolus, serial blood samples and cumulative urine samples were collected for 36 h in the adult ewes and for 72 h in the lambs. Due to saturable protein binding, age-related differences in VPA clearance were more obvious when examining the total body clearance of unbound drug (Cl(u)tb). Mean Cl(u)tb increased significantly with age up to 2 months (10 d = 2.65 +/- 1.16 ml/min/kg; 1 M = 5.11 +/- 2.49 ml/min/kg; 2 M = 12.84 +/- 3.88 ml/min/kg) before decreasing to adult levels (7.73 +/- 2.64 ml/min/kg). Similarly, the urinary recovery of the major metabolite, VPA-glucuronide, was significantly less in 10 d lambs (29.2 +/- 16.0% of the dose) when compared with the adult and 2 M groups (both approximately 74% of the dose). No differences with age were observed in the portion of the dose excreted as the beta-oxidation metabolite, 2-n-propyl-3-oxopentanoic acid. The results suggest that alterations in Cl(u)tb with age may be attributable to postnatal development of enzymes involved in VPA glucuronidation.

Disposition of valproic acid in maternal, fetal, and newborn sheep. I: placental transfer, plasma protein binding, and clearance.

Separate 24-h maternal and fetal infusions of valproic acid (VPA) were administered to five pregnant sheep at 125 to 138 days gestation (term approximately 145 days) to determine maternal-fetal disposition. The pharmacokinetics of VPA were also investigated in five newborn 1-day-old lambs after a 6-h drug infusion. Plasma, urine, and amniotic and fetal tracheal fluid samples were analyzed for VPA using gas chromatography-mass spectrometry. During maternal drug infusion, the average steady-state fetal/maternal unbound VPA plasma concentration ratio was 0.81 +/- 0.09. Unbound maternal-to-fetal VPA placental clearance (69.0 +/- 20.2 ml/min/kg) was similar to that in the other direction (61.9 +/- 24.2 ml/min/kg); this indicates passive placental diffusion and intermediate placental permeability of VPA in sheep. Newborn unbound VPA clearance (0.66 +/- 0.28 ml/min/kg) was much lower than in the mother (5.4 +/- 2.7 ml/min/kg) or the fetus (62.1 +/- 22.4 ml/min/kg), and exhibited pronounced Michaelis-Menten characteristics. The elimination half-life of the drug was much longer in the newborn (18.6 +/- 2.6 h) relative to the mother (5.6 +/- 1.4 h) and the fetus (4.6 +/- 1.9 h). Thus, VPA elimination in newborn lambs is much slower as compared with adult sheep, a situation similar to that in humans. Plasma protein binding of VPA was saturable, with similar VPA binding capacities and affinities in maternal and fetal plasma. VPA was extensively displaced from binding sites in the newborn lamb during the first 1 to 2 days of life, possibly because of increased plasma free fatty acid concentrations at birth. Thereafter, newborn plasma appeared to have a similar VPA binding capacity but lower affinity compared with the mother and the fetus.

Disposition of valproic acid in maternal, fetal, and newborn sheep. II: metabolism and renal elimination.

Metabolism and renal excretion of valproic acid (VPA) were examined in maternal, fetal, and newborn sheep to identify the underlying reasons for the previously observed reduced VPA clearance in newborn lambs. Plasma and urine from VPA infusion studies in maternal, fetal, and newborn sheep were analyzed for VPA and its metabolites [VPA-glucuronide; beta-oxidation products: (E)-2-ene, (E)-3-ene, and 3-keto VPA; hydroxylated metabolites: 3-hydroxy, 4-hydroxy, and 5-hydroxy VPA (5-OH VPA); and 4-ene VPA, 4-keto VPA, 2-propylglutaric acid, and 2-propylsuccinic acid] using gas chromatography-mass spectrometry. All measured metabolites were detectable in maternal and fetal plasma, with 3-keto and 5-OH VPA being at higher concentrations in the fetus. Plasma concentrations of (E)-2-ene, (E)-3-ene, 3-keto, and 5-OH VPA were higher in the newborn compared with the mother, whereas those of the other metabolites were similar. A smaller percentage of the dose was excreted as VPA-glucuronide in newborn lamb urine (28.3 +/- 12.0%) compared with the mother (77.0 +/- 7.8%). Similarly, a lower fraction of the dose was excreted unchanged in newborn urine (11.0 +/- 5.8%) relative to the urine of the mother (19.3 +/- 5.8%); however, significantly larger percentages were excreted as (E)-2-ene (0.11 +/- 0.04 versus 0.02 +/- 0.01%), 3-keto (11.6 +/- 3.5 versus 1. 6 +/- 0.8%), 4-hydroxy (6.1 +/- 3.2 versus 2.3 +/- 1.3%), and 5-OH VPA (2.2 +/- 0.6 versus. 0.8 +/- 0.6%). The major reason for the reduced VPA elimination in newborn lambs appears to be impaired renal excretion and glucuronidation capacity. As a result, a larger fraction of the dose is channeled to beta-oxidation and hydroxylation pathways. The beta-oxidation activities are high at birth; this may explain the high plasma concentrations of (E)-2-ene and 3-keto VPA observed in newborn lambs and human newborns exposed to VPA.

Diphenhydramine disposition in the sheep maternal-placental-fetal unit: gestational age, plasma drug protein binding, and umbilical blood flow effects on clearance.

The objective of this study was to examine the interrelationships between maternal and fetal plasma drug protein binding, umbilical blood flow (Q(um)), gestational age (GA), and maternal-fetal diphenhydramine (DPHM) clearances in chronically instrumented pregnant sheep. Maternal and fetal DPHM placental (CL(mf) and CL(fm), respectively) and nonplacental (CL(mo) and CL(fo), respectively) clearances and steady-state plasma protein binding were determined in 18 pregnant sheep at 124 to 140 days' gestation (term, approximately 145 days). The data demonstrated a highly significant fall of approximately 66% in CL(fm) and a decreasing trend in CL(fo) ( approximately 47%) over the GA range studied. However, no such relationships existed between GA and CL(mf) or CL(mo). Concomitant with this was a decrease in fetal DPHM plasma unbound fraction with GA, with no such change being evident in the mother. Both CL(mo) and CL(fo) were related to the respective DPHM plasma unbound fraction. A strong relationship also existed between fetal plasma unbound fraction and CL(fm). Thus, the decrease in fetal unbound fraction of DPHM during gestation could contribute to the fall in CL(fm), and possibly CL(fo). However, over the GA range studied, fetal DPHM free fraction decreased by approximately 47%, whereas CL(fm) fell by approximately 66%. Because fetal unbound fraction and CL(fm) are linearly related, the GA-associated fall in unbound fraction appears to be insufficient to account for the entire decline in CL(fm). In separate studies in pregnant sheep, we observed a approximately 40% fall in weight-normalized Q(um) between 125 and 137 days' gestation. Because CL(fm) for DPHM is similar to that of flow-limited compounds (e.g., ethanol, antipyrine), this decrease in Q(um) may also contribute to the GA-related fall in CL(fm).

Diphenhydramine disposition in the sheep maternal-placental-fetal unit: determinants of plasma drug concentrations in the mother and the fetus.

The objective of this study was to identify the important factors that determine plasma concentrations of diphenhydramine (DPHM) in the mother and the fetus after maternal as well as fetal steady-state drug administration. Inter-relationships were evaluated between maternal and fetal placental and nonplacental clearances, plasma protein binding, and steady-state plasma concentrations of DPHM among data obtained from 18 pregnant sheep during late gestation. The major determinant of plasma DPHM concentrations in the mother after maternal as well as fetal administration appears to be maternal plasma protein binding and maternal nonplacental clearance. In contrast, the major determinant of fetal plasma DPHM concentrations after maternal drug administration was the extent of fetal first-pass hepatic drug uptake from the umbilical vein. However, after fetal drug administration, the fetal plasma concentrations were related to the extent of fetal plasma protein binding and fetal placental and nonplacental clearances. The index of fetal-to-maternal placental drug transfer after fetal drug administration (steady-state maternal-to-fetal plasma concentration ratio) was related to steady-state fetal plasma unbound fraction and fetal placental and nonplacental clearance. However, this index was not related to the magnitude of the factors operating on the maternal side of the placenta such as maternal plasma protein binding and maternal nonplacental clearance. This might indicate a lack of complete equilibration of the unbound drug concentrations on the two sides of the placenta at the exchange site.

Comparative formation, distribution, and elimination kinetics of diphenylmethoxyacetic acid (a diphenhydramine metabolite) in maternal and fetal sheep.

Deamination to diphenylmethoxyacetic acid (DPMA) is the major route of diphenhydramine (DPHM) clearance in many species. In this study, we assessed the contribution of this pathway to nonplacental DPHM elimination and disposition of DPMA in maternal and fetal sheep. Paired maternal-fetal experiments were conducted in five chronically catheterized pregnant sheep (124-140 days gestation) with an appropriate washout period in between. Both maternal and fetal dosing experiments involved administration of an i.v bolus of deuterium-labeled DPMA ([2H10]-DPMA) combined with a 6-h infusion of DPHM (or a bolus of unlabeled DPMA with an infusion of deuterium-labeled DPHM). Maternal and fetal arterial plasma and urine samples were collected and analyzed for DPMA, [2H10]-DPMA, DPHM, and deuterium-labeled DPHM concentrations using gas chromatography-mass spectrometry. The preformed DPMA (or [2H10]-DPMA) had a substantially lower clearance (maternal: 0.55 +/- 0.18 versus 40.9 +/- 14.0 ml/min/kg; fetal: 0.37 +/- 0.11 versus 285. 6 +/- 122.2 ml/min/kg) and steady-state volume of distribution (Vdss, maternal: 0.10 +/- 0.02 versus 2.1 +/- 1.1 l/kg; fetal: 0.40 +/- 0. 06 versus 13.1 +/- 3.1 l/kg) as compared with the parent drug. The contribution of DPMA formation to maternal and fetal DPHM nonplacental clearance in vivo was 1.78 +/- 2.12% and 0.87 +/- 0.56%, respectively, indicating that DPMA formation is not a major route of DPHM clearance in fetal or maternal sheep. The recoveries of DPMA (or [2H10]-DPMA) in maternal urine were 88.0 +/- 6.5 and 92.1 +/- 7. 4% of the administered dose during maternal and fetal dosing experiments, respectively. Thus, this metabolite does not appear to be secondarily metabolized in fetal or maternal sheep. These findings are in contrast to other species (dog, rhesus monkey, human) where DPMA and its conjugates constitute approximately 40 to 60% of the total DPHM metabolites.

Role of the liver and gut in systemic diphenhydramine clearance in adult nonpregnant sheep.

We investigated the contribution of the liver and gut to systemic diphenhydramine (DPHM) clearance in adult nonpregnant sheep in two separate studies. In the first study, a simultaneous 50-mg bolus each of DPHM and its deuterium-labeled analog ([2H10]DPHM) was administered to five sheep via the femoral (i.v.) and the portal venous (p.v.) routes in a randomized manner. Arterial plasma concentrations of DPHM, [2H10]DPHM, and their deaminated metabolites, DPMA (diphenylmethoxyacetic acid) and [2H10]DPMA, were measured using gas chromatography-mass spectrometry. The hepatic first-pass extraction of DPHM after p.v. administration was 94.2 +/- 3.7%. However, the area under the plasma concentration versus time profile of the metabolite after i.v. dosing was only 32.5 +/- 14.0% relative to that after p.v. administration. Thus, only approximately 32.5% of the i.v. dose is metabolized in the liver and a significant extrahepatic systemic clearance component is evident. Using the calculated total hepatic blood flow values, it was found that 98.6 +/- 9.2% of the i.v. dose eventually was delivered to the "hepatoportal" system. Because the drug delivered to the hepatoportal system is almost completely eliminated in a single pass (hepatic extraction approximately 94%), this indicates a lack of any significant pulmonary drug uptake. Also, because only approximately 32.5% of the i.v. dose is metabolized in liver, the gut is most likely responsible for the clearance of the remainder. This gut contribution to systemic DPHM clearance was confirmed in a separate direct study in four sheep where the steady-state DPHM gut extraction ratio was 49.0 +/- 3.0%. Thus, gut accounts for a significant proportion (>/=50%) of DPHM systemic clearance in sheep in spite of a very high hepatic drug extraction efficiency.

Simultaneous determination of diphenhydramine, its N-oxide metabolite and their deuterium-labeled analogues in ovine plasma and urine using liquid chromatography/electrospray tandem mass spectrometry.

Our studies on drug disposition in chronically instrumented pregnant sheep involve simultaneous administration of the antihistamine diphenhydramine (DPHM), its deuterated analogue ([2H10]DPHM) and their metabolites to the mother or the fetus via various routes. Such studies require sensitive and selective mass spectrometric methods for quantitation of these labeled and unlabeled compounds in order to assess comparative maternal and fetal drug metabolism. The objective of this study was to develop and validate a liquid chromatographic/tandem mass spectrometric (LC/MS/MS) method for the simultaneous quantitation of DPHM, its N-oxide metabolite and their deuterium-labeled analogues in ovine plasma and urine. Samples spiked with the analytes and the internal standard, orphenadrine, were processed using liquid-liquid extraction. The extract was chromatographed on a propylamino LC column and MS/MS detection was performed in the positive ion electrospray mode using multiple reaction monitoring. The linear concentration ranges of the calibration curves for the N-oxides and the parent amines were 0.4-100.0 and 0.2-250.0 ng ml-1, respectively. In validation tests, the assay exhibited acceptable variability (< or = 15% at analyte concentrations below 2.0 ng ml-1 and < 10% at all other concentrations) and bias (< 15% at all concentrations), and the analytes were stable under a variety of sample handling conditions. Using this method, the labeled and unlabeled N-oxide metabolite was identified in fetal plasma after DPHM and [2H10]DPHM administration. This method will be used further to examine the comparative metabolism of diphenhydramine to its N-oxide metabolite in the mother and the fetus.

Estimation of transplacental and nonplacental diphenhydramine clearances in the fetal lamb: the impact of fetal first-pass hepatic drug uptake.

Previous estimates of maternal and fetal placental and nonplacental clearances in pregnant sheep using a two-compartment open model have revealed higher values of fetal placental clearance (CLfm) compared to the maternal placental clearance (CLmf) for most drugs. This includes the antihistamine diphenhydramine (DPHM), which also has the highest weight-corrected fetal nonplacental clearance (CLfo) among the drugs studied. This study was designed to determine the reasons for this CLfm - CLmf difference and to identify the sites of high CLfo for DPHM. DPHM and a stable isotope-labeled analog, [2H(10)]DPHM, were simultaneously infused to steady state to the mother and fetus, respectively, in five pregnant sheep. CLmf, CLfm, CLmo and CLfo averaged 50.3 +/- 13.2, 214.4 +/- 30.8, 36.6 +/- 1.9 and 109.8 +/- 22.3 ml/min(-1)/kg(-1), respectively. By measuring diphenylmethoxyacetic acid and [2H(10)]diphenylmethoxyacetic acid levels in samples obtained from our previous study of fetal hepatic first-pass DPHM uptake, the hepatic first-pass extraction ratio of the drug from umbilical venous blood was estimated to be 0.44 +/- 0.05. This can account for virtually all of CLfo. Fetal hepatic first-pass uptake of maternally derived DPHM in the paired infusion study reduces the fetal/maternal plasma DPHM concentration ratio and results in significant underestimation of CLmf. When the CLmf estimate is corrected for this factor and for maternal-fetal DPHM plasma protein binding differences, its value approaches CLfm. Fetal hepatic first-pass uptake may also be a factor in the underestimation of CLmf for most of the other drugs. Conversely, a lower value of CLmf compared with CLfm provides evidence for significant fetal hepatic uptake of these compounds.

Use of an automated fluorescent microsphere method to measure regional blood flow in the fetal lamb.

We have developed a method for measuring regional blood flow by means of fluorescent microspheres in all organs and tissues of the fetal lamb, including brain, heart, lung, liver, gut, spleen, kidney, adrenal, brown fat, skin, muscle, bone, and placenta. Five different fluorescent-labeled microspheres were used: blue (B), yellow-green (Y), orange (O), red (R), and crimson (C). An automated, 96-well microplate fluorescent reader (bottom reading) was chosen for the assay because of the rapidity and high throughput that it offers. Tissue samples were digested by 4 M ethanolic KOH. The sedimentation method and dye extraction with Cellosolve acetate, as previously reported by others, were used for the sample processing. The bones were crushed and allowed to directly soak in Cellosolve acetate to extract the dye. The relationship between microsphere number and fluorescent intensity was linear over a broad range of microsphere numbers (80-20,000/mL). The coefficients of variation of within-run and between-run precision were 3.39 +/- 1.10% and 4.54 +/- 1.10%, respectively. Recovery of microspheres from tissues and blood averaged 94.3 +/- 2.5% and was not dependent on microsphere number. The spillover of the fluorescent signals into adjacent colors was 4.0 +/- 0.1% for O to Y, 8.1 +/- 0.4% for O to R, and 9.1 +/- 0.5% for R to C, and these values were constant over a wide range in concentrations of the microsphere pairs. No evidence was obtained for quenching of the emission of one fluorophore via photon absorption by another fluorophore. The measurements of regional blood flow obtained with fluorescent microspheres in three chronically instrumented fetal lambs at approximately 140 days gestation were similar to the flow estimates obtained using radioactive microspheres in four other fetal lambs at the same gestational age. The fluorescent method is thus a viable alternative to the radioactive technique for the measurement of regional blood flow to all fetal organs and tissues, particularly when an automated fluorescent microplate reader is employed to reduce analysis time.

Hepatic first-pass uptake of diphenhydramine. A comparative study between fetal and adult sheep.

In this study, two factors that could affect fetal drug exposure were examined: 1) the extent of elimination of drug delivered to the fetal liver from the placenta via the umbilical vein; and 2) the degree to which there is preferential distribution of drug in umbilical venous blood to the fetal upper body, as is the case with oxygen and other endogenous substances. Studies were conducted with the histamine H1 antagonist, diphenhydramine (DPHM), in chronically instrumented nonpregnant and pregnant sheep (115-138 days gestation). Hepatic presystemic DPHM elimination was assessed using simultaneous and separate administration of DPHM and stable isotope labeled DPHM ([2H10]DPHM) via the umbilical vein (test route) and abdominal inferior vena cava (control route) in fetal lambs by either bolus injection (N = 6) or 90-min infusion (N = 5), and in nonpregnant sheep (N = 5), by simultaneous and separate bolus injections of the two forms of the drug via the mesenteric vein (test route) and abdominal inferior vena cava (control route). With the bolus injection protocol, hepatic presystemic elimination was estimated by the ratio of the area under the arterial drug concentration vs. time curve for the test and control routes of drug administration, whereas with the fetal infusion study it was calculated as the difference in fetal DPHM clearance values for the test and control routes of administration. To test for isotope effects in the disposition of [2H10]DPHM in the ewe or fetus, both DPHM and [2H10]DPHM were simultaneously injected via the inferior vena cava; however, no isotope effects were noted. In the ewe, there was extensive (93.2 +/- 1.4%) presystemic elimination of DPHM (F 0.068 +/- 0.014). However, in the fetus, this did not occur with bolus drug injection (F = 1.10 +/- 0.07), nor were there differences in the fetal DPHM clearance values estimated from the tarsal and umbilical venous infusions. During the latter experiments, DPHM levels were higher in the femoral artery than in carotid artery, with both umbilical venous and inferior vena caval drug infusion, and this was opposite of the differences in the concentrations of glucose, lactate, and oxygen between these two vessels. Thus, there is extensive hepatic presystemic elimination of DPHM in adult sheep, but no evidence for this phenomenon in the fetus. Furthermore, the preferential distribution of umbilical venous blood to the upper body of the fetal lamb does not result in higher drug levels in ascending aortic blood.

The pharmacokinetics of valproic acid in pregnant sheep after maternal and fetal intravenous bolus administration.

The pharmacokinetics and disposition of valproic acid (VPA) have been assessed in pregnant sheep after both maternal and fetal iv bolus administration. The time course of VPA and 16 of its metabolites was followed in maternal and fetal arterial blood, amniotic fluid, and fetal tracheal fluid for 48 hr after administration. Fetal blood gas, acid-base, metabolic, cardiovascular, and fetal breathing activity parameters were also monitored. The disposition of VPA in maternal serum is best described by a biexponential function with a terminal elimination half-life of 2.13 +/- 0.49 hr and volume of distribution of 0.242 +/- 0.036 liter/kg. VPA transfer to fetal serum and other fetal fluids was rapid after drug administration. There was significant fetal exposure to VPA after maternal dosing (mean AUCinfinityFA/AUCinfinityMA = 0.410 +/- 0.118). Similarly, the disposition of VPA in fetal serum after fetal dosing is best described by a biexponential decay with a terminal elimination half-life of 3.37 +/- 1.37 hr. Once again, VPA transfer to other fluids was rapid. However, unlike basic compounds studied previously, VPA did not accumulate extensively in either amniotic or fetal tracheal fluid. The following metabolites were detected after drug administration in these experiments: (E)- and (Z)-2-ene VPA, (E)- and (Z)-3-ene VPA, 4-ene VPA, 3-keto VPA, 4-keto VPA, 3-OH VPA, 4-OH VPA, 5-OH VPA, and 2-PGA. Both maternal and fetal bolus administration of VPA elicited a significant reduction in fetal breathing movements, which may be attributed to the drug's action on gamma-aminobutyric acid dynamics in the central nervous system (CNS). This suggests that the significant fetal exposure to VPA may produce further CNS-related effects in utero.

Sensitive fused-silica capillary gas chromatographic assay using electron-capture detection for indomethacin in ovine fetal fluids.

A sensitive gas chromatographic (GC) method with electron-capture detection (ECD) has been developed to quantitate indomethacin (IND) in plasma, urine, amniotic, and tracheal fluids obtained from the pregnant sheep model. IND and the internal standard, alpha-methylindomethacin (alpha-Me-IND) are extracted by a simple liquid-liquid extraction procedure using ethyl acetate and derivatized with N-methyl-N-(tert.-butyldimethyl-silyl)trifluoroacetamide (MTBSTFA) at 60 degrees C for 50 min. The limit of quantitation (LOQ) is 1 ng/ml with a C.V. < 10% and signal-to-noise ratio > 10. Recoveries from all fluids were greater than 80%. Calibration curves were linear over the range of 1-32 ng/ml with a coefficient of determination (r2) > 0.999. Inter- and intra-day coefficients of variation were < 10% at concentrations of 2-32 ng/ml, and < 20% at the LOQ. Applicability of the developed method is demonstrated for a pharmacokinetic study of IND samples collected following long-term infusion of IND in a chronically instrumented ovine fetus.

Blood volume restitution and growth in fetal lambs after acute hemorrhage.

The effects of 15-50% fetal hemorrhage (at approximately 1%/min) were studied in 13 pregnant ewes at 130-135 days of gestation for up to 5 days posthemorrhage. The upper limit of acute blood loss appears to be approximately 45%, and the rate of restoration of blood volume decreases with the severity of hemorrhage, particularly with hemorrhage > 30-40%. The restoration of fetal blood volume was due primarily to the restoration of plasma volume; in the animals subject to 40-45% blood loss (n = 9), red cell mass was still only 69.1 +/- 3.9% of the prehemorrhage value at day 5 posthemorrhage. There appear to be two phases in the restoration of plasma: 1) plasma volume was restored by 2 h posthemorrhage and 2) the restoration of plasma protein mass occurred primarily from 2 to 24 h. There was a significant correlation between blood volume and plasma protein mass. However, the regression line for the posthemorrhage days was shifted significantly upward in relation to that for the hemorrhage day because of a significant rise of plasma protein concentration. This may be important for the maintenance of blood volume after hemorrhage. Finally, fetal growth rate was determined by comparing fetal weight estimated in utero (from blood volume) with birth weight in 12 nonhemorrhaged control fetuses and in the 9 fetuses subject to 40-45% hemorrhage. The average rate of growth per day was 1.57 +/- 0.34% and -1.82 +/- 1.02%, respectively. The latter value is not significantly different from zero, suggesting that the acute blood loss impaired fetal growth.

Oxygen consumption, acid-base status, and behavior during and after acute, severe hemorrhage in fetal lambs.

The metabolic and behavioral effects of 40-45% hemorrhage (at approximately 1%/min) were studied in nine fetal lambs in utero (130-135 days of gestation) until 2 days posthemorrhage. Umbilical blood flow (Qum) fell from 192 +/- 14 to 100 +/- 9 ml.min-1.kg-1 at the end of blood loss, and on the day of hemorrhage it was linearly related to blood volume. However, on the posthemorrhage days, Qum was restored even though blood volume was still reduced. Fetal O2 delivery (DO2) fell with the decrease in Qum and later due to anemia (decrease in hematocrit from 33.4 +/- 1.2 to 23.6 +/- 1.2%), from 946 +/- 81 to 494 +/- 47 mumol.min-1.kg-1, but the decrease was lessened because of a rise in umbilical venous PO2 (31.7 +/- 2.6 to 45.9 +/- 2.8 mmHg). Fetal O2 consumption was reduced during and for 2 h after hemorrhage (262 +/- 17 to 190 +/- 16 mumol.min-1.kg-1), and this was associated with modest lactic acidemia (1.25 +/- 0.11 to 2.91 +/- 0.43 mM). There was also a temporary reduction in fetal breathing activity, low voltage electrocortical state, and rapid eye movements, but this was not associated with hypoxemia. It is concluded that reductions in fetal DO2 achieved via fetal blood loss are better tolerated than during hypoxemia and that the associated depression in fetal biophysical activities involves mechanisms other than systemic hypoxia.

Determination of valproic acid and its metabolites using gas chromatography with mass-selective detection: application to serum and urine samples from sheep.

An improved method for the quantitative determination of valproic acid (VPA) and sixteen of its metabolites has been developed using gas chromatography-mass spectrometry with selected-ion monitoring. The method is applicable to serum or urine and all metabolites are measured in a single chromatographic run of 29.5 min. Ions selected for quantitative purposes were the characteristic [M-57]+ ions of the tert.-butyldimethylsilyl (tBDMS) derivatives. The method utilizes heptadeuterated VPA as well as six heptadeuterated metabolites as internal standards [i.e. 2-[2H7]propyl-2-pentenoic acid (2-ene[2H7]VPA), 2-[2H7]propyl-4-pentenoic acid (4-ene[2H7]VPA), 2-[2H7]propyl-3-oxopentanoic acid (3-keto[2H7]VPA), 2-[2H7]propyl-4-oxopentanoic acid (4-keto[2H7]VPA), 2-[2H7]propyl-3-hydroxypentanoic acid (3-OH[2H7]VPA), 2-[2H7]propyl-5-hydroxypentanoic acid (5-OH[2H7]VPA)]. The method demonstrates very good accuracy and precision over a large range of concentrations for VPA and all metabolites measured in both human and sheep biological fluids. The assay was applied to the analysis of VPA and metabolites in serum and urine samples collected from three non-pregnant ewes following intravenous bolus administration of a mixture of VPA and [13C4]VPA. Sheep were observed to produce measurable quantities of the majority of metabolites found in humans, with the notable exception of the di-unsaturated compounds (i.e. 2,3'-diene VPA and 2,4-diene VPA). The pharmacokinetics and metabolism of VPA and [13C4]VPA appear to be equivalent in the sheep model. The elimination half-life of VPA and [13C4]VPA in the ewe were estimated to be approximately 3.5 +/- 0.4 and 3.2 +/- 0.4 h, respectively.

Simultaneous analysis of diphenylmethoxyacetic acid, a metabolite of diphenhydramine, and its deuterium-labeled stable isotope analog in ovine plasma and urine.

Diphenylmethoxyacetic acid (DPMA) is a major metabolite of diphenhydramine in monkeys, dogs, and humans. The metabolic fate of diphenhydramine (DPHM) in sheep is not yet well understood; however, preliminary studies have demonstrated the presence of DPMA in the plasma and urine of sheep following an intravenous bolus of DPHM. Our current studies employ the simultaneous intravenous co-administration of DPHM and the stable isotope analog of DPHM to investigate the pharmacokinetics of DPHM in sheep. In these studies, in order to investigate the pharmacokinetics of the DPMA metabolite, measurement of both unlabeled and stable-isotope labeled DPMA is required. Thus, a stable isotope analog of DPMA ([2H10]DPMA) was synthesized, characterized, and purified for use as an analytical standard. The quantitative method for the gas chromatography-electron-impact mass spectrometry (GC-EI-MS) analysis of DPMA and [2H10]DPMA used a single step liquid-liquid extraction procedure using toluene for sample cleanup. The samples were derivatized with N-methyl-N-(tert.-butyldimethylsilyl) trifluoroacetamide. A 1.0-microliter aliquot of the prepared sample was injected into the GC-MS system and quantitated using selected-ion monitoring (SIM). One ion was monitored for each compound, namely, m/z 165 for the internal standard diphenylacetic acid, m/z 183 for DPMA, and m/z 177 for [2H10]DPMA. The ion chromatograms were free from chromatographic peaks co-eluting with the compound of interest. The calibration curve was linear from 2.5 ng/ml (limit of quantitation) to 250.0 ng/ml in both urine and plasma. The intra-day and inter-day variabilities of this assay method were within acceptable limits (below 20% at the limit of quantitation and below 10% at all other concentrations). This method was used to measure the concentration of DPMA and [2H10]DPMA in plasma and urine samples from a ewe in which equimolar amounts of DPHM and [2H10]DPHM were administered by an intravenous bolus dose via the femoral vein. DPMA appeared to persist longer in the plasma and the urine as compared to DPHM. This method is robust and reliable for the quantitation of DPMA and [2H10]DPMA in biological samples obtained from sheep (e.g. plasma and urine).