Religious beliefs affect grieving after pregnancy loss.

Religious beliefs and practices may aid in coping with bereavement and grief after pregnancy loss. Data from 103 women enrolled in the original Lehigh Valley Perinatal Loss Project, and who were followed-up for at least 1 year, were evaluated for the impact of initial religious practices and beliefs on the course and severity of grief. Religious practices corresponding to standard scales of religiosity and agreement with specific beliefs were rated by the women on a Likert scale of 1-5. Neither agreement with statements corresponding to extrinsic and intrinsic religiosity or to positive religious coping, nor frequency of religious service attendance was predictive of follow-up scores on the Perinatal Grief Scale. Religious struggle, agreement with statements classified as negative religious coping, and continued attachment to the baby were all associated with more severe grief.

Digital lock-in algorithm for biomedical spectroscopy and imaging instruments with multiple modulated sources.

Digital lock-in detection provides spectroscopic and imaging instruments a means of measuring physical quantities with improved signal to noise ratios compared to analogue detection schemes. We introduce a digital lock-in detection algorithm for measuring the amplitude and phase of multiple amplitude modulated signals simultaneously by using particular modulation and sampling constraints and averaging filters. The technique exhibits exceptional reduction in both noise and inter-source distortion. It is shown that the digital lock-in technique can be performed as a simple matrix multiplication in order to reduce computation time. The digital lock-in algorithm is described and analyzed under certain sampling and modulation conditions. Results are shown for experimental data.

Three-dimensional optical tomographic brain imaging in small animals, part 1: hypercapnia.

In this study, we explore the potential of diffuse optical tomography for brain oximetry. While several groups have already reported on the sensitivity of optical measurements to changes in oxyhemoglobin, deoxyhemoglobin, and blood volume, these studies were often limited to single source-detector geometries or topographic maps, where signals obtained from within the brain are projected onto 2-D surface maps. In this two-part study, we report on our efforts toward developing a volumetric optical imaging system that allows one to spatially resolve 3-D hemodynamic effects in rat brains. In part 1, we describe the instrumentation, optical probe design, and the model-based iterative image reconstruction algorithm employed in this work. Consideration of how a priori anatomical knowledge can be incorporated in the reconstruction process is presented. This system is then used to monitor global hemodynamic changes that occur in the brain under various degrees of hypercapnia. The physiologic cerebral response to hypercapnia is well known and therefore allows an initial performance assessment of the imaging system. As expected, we observe global changes in blood volume and oxygenation, which vary linearly as a function of the concentration of the inspired carbon dioxide. Furthermore, experiments are designed to determine the sensitivity of the reconstructions of only 1 mm to inaccurate probe positioning. We determine that shifts can significantly influence the reconstructions. In part 2 we focus on more local hemodynamic changes that occur during unilateral carotid occlusion performed at lower-than-normal systemic blood pressure. In this case, the occlusion leads to a predominantly monohemispherically localized effect, which is well described in the literature. Having explored the system with a well-characterized physiologic effect, we investigate and discuss the complex compensatory cerebrovascular hemodynamics that occur at normotensive blood pressure. Overall, these studies demonstrate the potential and limitations of our diffuse optical imager for visualizing global and focal hemodynamic phenomenon three dimensionally in the brains of small animals.

Three-dimensional optical tomographic brain imaging in small animals, part 2: unilateral carotid occlusion.

This is the second part of a two-part study that explores the feasibility of 3-D, volumetric brain imaging in small animals by optical tomographic techniques. In part 1, we demonstrated the ability to visualize global hemodynamic changes in the rat head in response to elevated levels of CO(2) using a continuous-wave instrument and model-based iterative image reconstruction (MOBIIR) algorithm. Now we focus on lateralized, monohemispherically localized hemodynamic effects generated by unilateral common carotid artery (CCA) occlusion. This illustrates the capability of our optical tomographic system to localize and distinguish hemodynamic responses in different parts of the brain. Unilateral carotid occlusions are performed in ten rodents under two experimental conditions. In the first set of experiments the normal systemic blood pressure is lowered to 50 mmHg, and on unilateral carotid occlusion, we observe an ipsilateral monohemispheric global decrease in blood volume and oxygenation. This finding is consistent with the known physiologic response to cerebral ischemia. In a second set of experiments designed to observe the spatial-temporal dynamics of CCA occlusion at normotensive blood pressure, more complex phenomena are observed. We find three different types of responses, which can be categorized as compensation, overcompensation, and noncompensation.

Near-infrared diffuse optical tomography.

Diffuse optical tomography (DOT) is emerging as a viable new biomedical imaging modality. Using near-infrared (NIR) light, this technique probes absorption as well as scattering properties of biological tissues. First commercial instruments are now available that allow users to obtain cross-sectional and volumetric views of various body parts. Currently, the main applications are brain, breast, limb, joint, and fluorescence/bioluminescence imaging. Although the spatial resolution is limited when compared with other imaging modalities, such as magnetic resonance imaging (MRI) or X-ray computerized tomography (CT), DOT provides access to a variety of physiological parameters that otherwise are not accessible, including sub-second imaging of hemodynamics and other fast-changing processes. Furthermore, DOT can be realized in compact, portable instrumentation that allows for bedside monitoring at relatively low cost. In this paper, we present an overview of current state-of-the -art technology, including hardware and image-reconstruction algorithms, and focus on applications in brain and joint imaging. In addition, we present recent results of work on optical tomographic imaging in small animals.

Remarkable spectator ligand effect on the rate constant of ligand substitution of (aqua)ruthenium(II) complexes.

The influence of two different di(1-pyrazolyl)alkane ligands on the rate constant of aqua ligand substitution of ruthenium(II) complexes with the formula [Ru(H2O)(L2)(tpmm)]2+ (L2 = di(1-pyrazolyl)methane (DPMet) or 2,2-di(1-pyrazolyl)propane (DPPro)) was investigated. A 9.4 x 10(5)-fold increase in the rate constant of ligand substitution at pH = 6.86 was observed when DPMet was replaced with DPPro. This remarkable increase was unexpected, considering that these bidentate ligands appear quite similar. To help lend insight into this dramatic spectator ligand effect, the activation parameters for the ligand substitution reactions were determined, and single-crystal X-ray data were collected on the structurally analogous (chloro)ruthenium(II) complexes, [Ru(Cl)(L2)(tpmm)]+. These results are discussed in the context of a heteroscorpionate effect exerted by the DPPro ligand.

International comparison of studies using the perinatal grief scale: a decade of research on pregnancy loss.

The Perinatal Grief Scale (PGS) has been used in many studies of loss in pregnancy, including miscarriage, stillbirth, induced abortion, neonatal death, and relinquishment for adoption. This article describes 22 studies from 4 countries that used the PGS with a total of 2485 participants. Studies that report Cronbach's alpha for their own samples give evidence of very high internal consistency reliability. Evidence for the validity of the PGS is also reviewed, such as convergent validity seen in its association with measures of mental health, social support, and marital satisfaction. The standard errors of the means for the total scale and for the subscales reveal fairly consistent scores, in spite of very different samples and types of loss; computation of means and standard deviations for the studies as a whole permits us to establish normal score ranges. Significantly higher scores were found in studies that recruited participants from support groups and self-selected populations rather than from medical sources, and from U.S. studies compared with those in Europe.

Expression of glutathione-dependent enzymes and cytochrome P450s in freshly isolated and primary cultures of proximal tubular cells from human kidney.

The expression of glutathione (GSH)-dependent enzymes and cytochrome P450 (P450) proteins in freshly isolated proximal tubular cells from human kidney (hPT), and the effect of primary culture on these enzymes, were determined. Freshly isolated hPT cells had relatively high activities of gamma-glutamyltransferase, gamma-glutamylcysteine synthetase, glutathione S-transferase (GST), glutathione disulfide reductase, and GSH peroxidase. Cytochrome P450 4A11 was detected in freshly isolated hPT cells, whereas CYP2E1 was not. Freshly isolated hPT cells also expressed GSTA, GSTP, and GSTT but not GSTM. Primary cultures of hPT cells maintained their epithelial-like nature and diploid status, based on measurements of morphology, cytokeratin expression, and flow cytometric analysis. hPT cells retained GSH-dependent enzyme activities during primary culture, whereas cells that had undergone subsequent passage exhibited a loss of activities of most GSH-dependent enzymes and no longer expressed P450s or GSTs. CYP4A11 expression in primary cultures of hPT cells was significantly increased after treatment for 48 h with either ethanol (50 mM) or dexamethasone (7 nM). GSTA, GSTP, and GSTT contents, although still detectable, were decreased compared with those of freshly isolated hPT cells. Our data show that hPT cells express enzymes involved in xenobiotic disposition, and that they thus provide a model suitable for studies of human renal drug metabolism. Furthermore, primary cultures of hPT cells may afford the opportunity to study factors regulating P450 enzyme expression in human kidney.

Formation of 20-hydroxyeicosatetraenoic acid, a vasoactive and natriuretic eicosanoid, in human kidney. Role of Cyp4F2 and Cyp4A11.

20-hydroxyeicosatetraenoic acid (20-HETE), an omega-hydroxylated arachidonic acid (AA) metabolite, elicits specific effects on kidney vascular and tubular function that, in turn, influence blood pressure control. The human kidney's capacity to convert AA to 20-HETE is unclear, however, as is the underlying P450 catalyst. Microsomes from human kidney cortex were found to convert AA to a single major product, namely 20-HETE, but failed to catalyze AA epoxygenation and midchain hydroxylation. Despite the monophasic nature of renal AA omega-hydroxylation kinetics, immunochemical studies revealed participation of two P450s, CYP4F2 and CYP4A11, since antibodies to these enzymes inhibited 20-HETE formation by 65. 9 +/- 17 and 32.5 +/- 14%, respectively. Western blotting confirmed abundant expression of these CYP4 proteins in human kidney and revealed that other AA-oxidizing P450s, including CYP2C8, CYP2C9, and CYP2E1, were not expressed. Immunocytochemistry showed CYP4F2 and CYP4A11 expression in only the S2 and S3 segments of proximal tubules in cortex and outer medulla. Our results demonstrate that CYP4F2 and CYP4A11 underlie conversion of AA to 20-HETE, a natriuretic and vasoactive eicosanoid, in human kidney. Considering their proximal tubular localization, these P450 enzymes may partake in pivotal renal functions, including the regulation of salt and water balance, and arterial blood pressure itself.

CYP2C19 participates in tolbutamide hydroxylation by human liver microsomes.

Tolbutamide is a sulfonylurea-type oral hypoglycemic agent whose action is terminated by hydroxylation of the tolylsulfonyl methyl moiety catalyzed by cytochrome P-450 (CYP) enzymes of the human CYP2C subfamily. Although most studies have implicated CYP2C9 as the exclusive catalyst of hepatic tolbutamide hydroxylation in humans, there is evidence that other CYP2C enzymes (e.g., CYP2C19) may also participate. To that end, we used an immunochemical approach to assess the role of individual CYP2Cs in microsomal tolbutamide metabolism. Polyclonal antibodies were raised to CYP2C9 purified from human liver, and were then back-adsorbed against recombinant CYP2C19 coupled to a solid-phase support. Western blotting revealed that the absorbed anti-human CYP2C9 preparation reacted with only recombinant CYP2C9 and the corresponding native protein in hepatic microsomes, and no longer recognized CYP2C19 and CYP2C8. Monospecific anti-CYP2C9 not only retained the ability to inhibit CYP2C9-catalyzed reactions, as evidenced by its marked (90%) inhibition of diclofenac 4'-hydroxylation by purified CYP2C9 and by human liver microsomes, but also exhibited metabolic specificity, as indicated by its negligible (<15%) inhibitory effect on S-mephenytoin 4'-hydroxylation by purified CYP2C19 or hepatic microsomes containing CYP2C19. Monospecific anti-CYP2C9 was also found to inhibit rates of tolbutamide hydroxylation by 93 +/- 4 and 78 +/- 6% in CYP2C19-deficient and CYP2C19-containing human liver microsomes, respectively. Taken together, our results indicate that both CYP2C9 and CYP2C19 are involved in tolbutamide hydroxylation by human liver microsomes, and that CYP2C19 underlies at least 14 to 22% of tolbutamide metabolism. Although expression of CYP2C19 in human liver is less than that of CYP2C9, it may play an important role in tolbutamide disposition in subjects expressing either high levels of CYP2C19 or a catalytically deficient CYP2C9 enzyme.

Role of human CYP4F2 in hepatic catabolism of the proinflammatory agent leukotriene B4.

Leukotriene B4 (LTB4), an arachidonic acid derivative, is a potent proinflammatory agent whose actions are terminated by catabolism via a microsomal omega-hydroxylation pathway. Although the liver serves as the principal site for LTB4 clearance from the systemic circulation, the attributes of hepatic LTB4 metabolism are ill defined in humans. Thus, we examined metabolism of LTB4 to its omega-hydroxylated metabolite 20-hydroxyleukotriene B4 (20-OH LTB4) by human liver microsomes and also purified the hepatic P450 enzyme underlying this reaction. Liver microsomes from 10 different subjects converted LTB4 to 20-OH LTB4 at similar rates (1.06 +/- 0.3 nmol/min/nmol P450; 0.25 +/- 0.1 nmol/min/mg protein). Analysis of the microsomal LTB4 20-hydroxylation reaction revealed kinetic parameters (apparent Km of 74.8 microM with a VMAX of 2.42 nmol/min/nmol P450) consistent with catalysis by a single P450 enzyme. Conventional chromatography combined with immunochemical screening with rat CYP4A1 antibodies was then used to isolate a P450 enzyme from human liver microsomes with a molecular weight of 57,000 and an NH2-terminal amino acid sequence 94% homologous (12Trp --> 12Gly) over the first 17 residues with the human CYP4F2 cDNA-derived sequence. Upon reconstitution with P450 reductase and phospholipid, CYP4F2 converted LTB4 to 20-OH LTB4 at a turnover rate of 392 pmol/min/nmol P450, whereas the other human liver P450s tested, including CYP4A11, exhibited neglible LTB4 omega-hydroxylase activity. Polyclonal antibodies to CYP4F2 were found to markedly inhibit (91.9 +/- 5%; n = 5) LTB4 20-hydroxylation by human liver microsomes. Microsomal 20-OH LTB4 formation was also inhibited 30% by arachidonic acid, a known CYP4F2 substrate, and 50% by prostaglandin A1 but was unaffected by lauric acid, palmitic acid, and PGF2alpha. Finally, a strong correlation (r = 0.86; P < 0.002; n = 10) was observed between CYP4F2 content and LTB4 20-hydroxylase activity in the human liver samples. Our results indicate that CYP4F2 is the principle LTB4 omega-hydroxylating enzyme expressed in human liver and, as such, may play an important role in regulating circulating as well as hepatic levels of this powerful proinflammatory eicosanoid.

Metabolism of arachidonic acid to 20-hydroxy-5,8,11, 14-eicosatetraenoic acid by P450 enzymes in human liver: involvement of CYP4F2 and CYP4A11.

20-Hydroxy-5,8,11,14-eicosatetraenoic acid (20-HETE) is a principal arachidonic acid (AA) metabolite formed via P450-dependent oxidation in hepatic and renal microsomes. Although 20-HETE plays an important role in the regulation of cell and/or organ physiology, the P450 enzyme(s) catalyzing its formation in humans remain undefined. In this study, we have characterized AA omega-hydroxylation to 20-HETE by human hepatic microsomes and identified the underlying P450s. Analysis of microsomal AA omega-hydroxylation revealed biphasic kinetics (KM1 and VMAX1 = 23 microM and 5.5 min-1; KM2 and VMAX2 = 144 microM and 18.8 min-1) consistent with catalysis by at least two enzymes. Of the human P450s examined, CYP4A11 and CYP4F2 were both potent AA omega-hydroxylases, exhibiting rates of 15.6 and 6.8 nmol 20-HETE formed/min/nmol P450, respectively. Kinetic parameters of 20-HETE formation by CYP4F2 (KM = 24 microM; VMAX = 7.4 min-1) and CYP4A11 (KM = 228 microM; VMAX = 49.1 min-1) resembled the low and high KM components, respectively, found in liver microsomes. Antibodies to CYP4F2 markedly inhibited (93.4 +/- 6%; n = 5) formation of 20-HETE by hepatic microsomes, whereas antibodies to CYP4A11 were much less inhibitory (13.0 +/- 9%; n = 5). Moreover, a strong correlation (r = 0.78; P < .02) was found between microsomal CYP4F2 content and AA omega-hydroxylation among nine subjects. The correlation (r = 0.76; P < .02) also noted between CYP4A11 content and 20-HETE formation stemmed from the relationship (r = 0.83; P < . 02) between hepatic CYP4A11 and CYP4F2 levels in the subjects. Finally, immunoblot analysis revealed that in addition to liver, both P450s also were expressed in human kidney. Our results indicate that AA omega-hydroxylation in human liver is catalyzed by two enzymes of the CYP4 gene family, namely CYP4F2 and CYP4A11, and that CYP4F2 underlies most 20-HETE formation occurring at relevant AA concentrations.

Characterization of CYP2C19 and CYP2C9 from human liver: respective roles in microsomal tolbutamide, S-mephenytoin, and omeprazole hydroxylations.

Individuals with drug metabolism polymorphisms involving CYP2C enzymes exhibit deficient oxidation of important therapeutic agents, including S-mephenytoin, omeprazole, warfarin, tolbutamide, and nonsteroidal anti-inflammatory drugs. While recombinant CYP2C19 and CYP2C9 proteins expressed in yeast or Escherichia coli have been shown to oxidize these agents, the capacity of the corresponding native P450s isolated from human liver to do so is ill defined. To that end, we purified CYP2C19, CYP2C9, and CYP2C8 from human liver samples using conventional chromatographic techniques and examined their capacity to oxidize S-mephenytoin, omeprazole, and tolbutamide. Upon reconstitution, CYP2C19 metabolized S-mephenytoin and omeprazole at rates that were 11- and 8-fold higher, respectively, than those of intact liver microsomes, whereas neither CYP2C9 nor CYP2C8 displayed appreciable metabolic activity with these substrates. CYP2C19 also proved an efficient catalyst of tolbutamide metabolism, exhibiting a turnover rate similar to CYP2C9 preparations (2.0-6.4 vs 2.4-4.3 nmol hydroxytolbutamide formed/min/nmol P450). The kinetic parameters of CYP2C19-mediated tolbutamide hydroxylation (Km = 650 microM, Vmax = 3.71 min-1) somewhat resembled those of the CYP2C9-catalyzed reaction (Km = 178-407 microM, Vmax = 2.95-7.08 min-1). Polyclonal CYP2C19 antibodies markedly decreased S-mephenytoin 4'-hydroxylation (98% inhibition) and omeprazole 5-hydroxylation (85% inhibition) by human liver microsomes. CYP2C19 antibodies also potently inhibited (>90%) microsomal tolbutamide hydroxylation, which was similar to the inhibition (>85%) observed with antibodies to CYP2C9. Moreover, excellent correlations were found between immunoreactive CYP2C19 content, S-mephenytoin 4'-hydroxylase activity (r = 0.912; P < 0. 001), and omeprazole 5-hydroxylase activity (r = 0.906; P < 0.001) in liver samples from 13-17 different subjects. A significant relationship was likewise observed between microsomal tolbutamide hydroxylation and CYP2C9 content (r = 0.664; P < 0.02) but not with CYP2C19 content (r = 0.393; P = 0.184). Finally, immunoquantitation revealed that in these human liver samples, expression of CYP2C9 (88. 5 +/- 36 nmol/mg) was 5-fold higher than that of CYP2C19 (17.8 +/- 14 nmol/mg) and nearly 8-fold higher than that of CYP2C8 (11.5 +/- 12 nmol/mg). Our results, like those obtained with recombinant CYP2C enzymes, indicate that CYP2C19 is a primary determinant of S-mephenytoin 4'-hydroxylation and low-Km omeprazole 5-hydroxylation in human liver. Despite its tolbutamide hydroxylase activity, the low levels of hepatic CYP2C19 expression (relative to CYP2C9) may preclude an important role for this enzyme in hepatic tolbutamide metabolism and any polymorphisms thereof.

Role of human liver P450s and cytochrome b5 in the reductive metabolism of 3'-azido-3'-deoxythymidine (AZT) to 3'-amino-3'-deoxythymidine.

Our laboratory has shown that human liver microsomes metabolize the anti-HIV drug 3'-azido-3'-deoxythymidine (AZT) via a P450-type reductive reaction to a toxic metabolite 3'-amino-3'-deoxythymidine (AMT). In the present study, we examined the role of specific human P450s and other microsomal enzymes in AZT reduction. Under anaerobic conditions in the presence of NADPH, human liver microsomes converted AZT to AMT with kinetics indicative of two enzymatic components, one with a low Km (58-74 microM) and Vmax (107-142 pmol AMT formed/min/mg protein) and the other with a high Km (4.33-5.88 mM) and Vmax (1804-2607 pmol AMT formed/min/mg). Involvement of a specific P450 enzyme in AZT reduction was not detected by using human P450 substrates and inhibitors. Antibodies to human CYP2E1, CYP3A4, CYP2C8, CYP2C9, CYP2C19, and CYP2A6 were also without effect on this reaction. NADH was as effective as NADPH in promoting microsomal AZT reduction, raising the possibility of cytochrome b5 (b5) involvement. Indeed, AZT reduction among six human liver samples correlated strongly with microsomal b5 content (r2 = 0.96) as well as with aggregate P450 content (r2 = 0.97). Upon reconstitution, human liver b5 plus NADH:b5 reductase and CYP2C9 plus NADPH:P450 reductase were both effective catalysts of AZT reduction, which was also supported when CYP2A6 or CYP2E1 was substituted for CYP2C9. Kinetic analysis revealed an AZT Km of 54 microM and Vmax of 301 pmol/min for b5 plus NADH:b5 reductase and an AZT Km of 103 microM and Vmax of 397 pmol/min for CYP2C9 plus NADPH:P450 reductase. Our results indicate that AZT reduction to AMT by human liver microsomes involves both b5 and P450 enzymes plus their corresponding reductases. The capacity of these proteins and b5 to reduce AZT may be a function of their heme prothestic groups.

The effect of a psychological intervention program on the risk of a new coronary event after angioplasty: a feasibility study.

Restenosis after successful percutaneous coronary angioplasty (PTCA) is a major problem because it occurs in 25% to 35% of all patients. Because psychological factors, especially anger and vital exhaustion, have been found to increase the risk of new cardiac events after PTCA, a behavioral intervention might contribute to the reduction of the risk of restenosis. To investigate the operational and methodological aspects of a behavioral intervention, and to estimate the effect size of the risk reduction, we did a feasibility study of angioplasty patients who remained exhausted after PTCA. Breathing therapy was used as the main method for intervention. Thirty patients who participated in the intervention program and 65 controls were followed during an average period of 16 and 18 months, respectively. It was observed that the intervention resulted in a significant decrease of the mean exhaustion scores and reduced the risk of a new coronary event (cardiac death, coronary artery bypass grafting, myocardial infarction, rePTCA, restenosis) by 50% (chi = 2.19; p = 0.13). These results indicate that a clinical trial to test the hypothesis that a reduction of vital exhaustion and hostility reduces the risk of a new cardiac event after PTCA, is feasible and merits the efforts required.

Identification of CYP4A11 as the major lauric acid omega-hydroxylase in human liver microsomes.

Human liver microsomes are capable of oxidizing lauric acid (laurate), a model medium-chain fatty acid, at both the omega- and omega-1 positions to form 12- and 11-hydroxylaurate, respectively. These laurate hydroxylation reactions are apparently catalyzed by distinct P450 enzymes. While the P450 responsible for microsomal laurate omega-1 hydroxylation in human liver has been identified as CYP2E1, the enzyme catalyzing omega-hydroxylation remains poorly defined. To that end, we employed conventional purification and immunochemical techniques to characterize the major hepatic laurate omega-hydroxylase in humans. Western blotting with rat CYP4A1 antibodies was used to monitor a cross-reactive P450 protein (M(r) = 52 kDa) during its isolation from human liver microsomes. The purified enzyme (7.4 nmol P450/mg protein) had an NH2-terminal amino acid sequence identical to that predicted from the human CYP4A11 cDNA over the first 20 residues found. Upon reconstitution with P450 reductase and cytochrome b5, CYP4A11 proved to be a potent laurate omega-hydroxylase, exhibiting a turnover rate of 45.7 nmol 12-hydroxylaurate formed/min/nmol P450 (12-fold greater than intact microsomes), while catalyzing the omega-1 hydroxylation reaction at much lower rates (5.4 nmol 11-hydroxylaurate formed/min/nmol P450). Analysis of the laurate omega-hydroxylation reaction in human liver microsomes revealed kinetic parameters (a lone Km of 48.9 microM with a VMAX of 3.72 nmol 12-hydroxylaurate formed/min/nmol P450) consistent with catalysis by CYP4A11. In fact, incubation of human liver microsomes with antibodies raised to CYP4A11 resulted in nearly 85% inhibition of laurate omega-hydroxylase activity while omega-1 hydroxylase activity remained unaffected. Furthermore, a strong correlation (r = 0.89; P < 0.001) was found between immunochemically determined CYP4A11 content and laurate omega-hydroxylase activity in liver samples from 11 different subjects. From the foregoing, it appears that CYP4A11 is the principle laurate omega-hydroxylating enzyme expressed in human liver.

Human CYP2C19 is a major omeprazole 5-hydroxylase, as demonstrated with recombinant cytochrome P450 enzymes.

Omeprazole (OP) is a potent antiulcer drug that is metabolized by liver cytochrome P450 (P450) enzymes. However, the identities of the P450 isoforms responsible for its metabolism have been controversial. 5-Hydroxyomeprazole (5OH-OP) formation cosegregates with the polymorphism of (S)-mephenytoin 4'-hydroxylation in humans, which is now known to be mediated by CYP2C19. Previous in vitro studies have indicated that liver microsomal 50H-OP formation correlates with both (S)-mephenytoin 4'-hydroxylase and CYP3A content. Inhibitor and CYP2C antibody studies also suggested that both enzymes may be involved in the 5-hydroxylation of OP, whereas CYP3A appears to be the predominant enzyme involved in OP sulfone (OP-S) formation. The present studies assessed the contribution of various CYP2C and CYP3A4 enzymes to OP metabolism by using recombinant human enzymes. CYP2C19, CYP2C8, CYP2C18, and CYP2C9 formed a single metabolite with an HPLC retention time identical to that of 5OH-OP. The turnover number for CYP2C19 was 13.4 +/- 1.4 nmol/min/nmol of P450, whereas those for CYP2C8, CYP2C18, and CYP2C9 were 2.2 +/- 0.1, 1.5 +/- 0.1, and approximately equal to 0.5 nmol/min/nmol of P450, respectively. Recombinant human CYP3A4 formed 5OH-OP and OP-S with turnover numbers of 5.7 +/- 1.1 and 7.4 +/- 0.9 nmol/min/nmol of P450, respectively, and formed a minor unidentified metabolite. CYP2C19 had a substantially lower KM for 5OH-OP formation than did CYP3A4, CYP2C8, or CYP2C18. Antibody to CYP2C proteins inhibited approximately equal to 70% of OP 5-hydroxylation at low substrate concentrations, comparable to those that may be encountered at therapeutically relevant doses, whereas antibody to CYP3A4 inhibited approximately equal to 30% of the activity. At high substrate concentrations, the contributions of the two enzymes to OP hydroxylation were roughly comparable (40-50%). In contrast, OP-S formation was completely inhibited by antibody to CYP3A4 proteins. The present study provides the first direct confirmation, using human recombinant P450 enzymes and selective antibody inhibition, that CYP2C19 is a major high affinity OP 5-hydroxylase and CYP3A4 is a low affinity OP-hydroxylating enzyme. The current work also shows, for the first time, that other CYP2C enzymes (CYP2C8, CYP2C9, and CYP2C18) may contribute to OP hydroxylation at high substrate concentrations. In contrast, OP-S was formed principally by CYP3A4.

Metabolism of fentanyl, a synthetic opioid analgesic, by human liver microsomes. Role of CYP3A4.

The microsomal metabolism of fentanyl, a synthetic opioid commonly used in anesthesia, was investigated in human liver. Incubation of fentanyl with human hepatic microsomes fortified with NADPH resulted in the formation of a single major metabolite, namely norfentanyl, as determined by GC/MS. No evidence was obtained for the formation of either desproprionylfentanyl or N-phenylpropionamide, the latter arising via N-dealkylation of the fentanyl amide nitrogen. Kinetic analysis of microsomal fentanyl oxidation revealed a single K(m) of 117 microM and a Vmax of 3.86 nmol of norfentanyl formed/min/nmol of cytochrome P450 (P450). Studies using chemical inhibitors of human P450 enzymes revealed that only agents known to inhibit CYP3A4 (e.g. ketoconazole and erythromycin) were capable of strongly inhibiting (> or = 90%) microsomal fentanyl oxidation. Marked inhibition (> 90%) of norfentanyl formation by liver microsomes was also observed with polyclonal antibodies to CYP3A4, whereas antibodies to other human P450s were without effect. Furthermore, rates of norfentanyl production by 10 individual human liver samples were highly correlated (r2 = 0.876, F = 56.46 p < 0.001) with immunochemically determined levels of CYP3A4 present in the samples but not with levels of CYP2C8, CYP2C9, CYP2C19, or CYP2E1. Our results indicate that CYP3A4 is the major catalyst involved in fentanyl oxidation to norfentanyl in human liver. Alterations in CYP3A4 levels or activity, as well as the concomitant administration of other therapeutic agents metabolized by this P450 enzyme, could lead to marked perturbations in fentanyl disposition and, hence, analgesic response.

1-Hydroxyethyl radical formation during NADPH- and NADH-dependent oxidation of ethanol by human liver microsomes.

Ethanol can be oxidized to the 1-hydroxyethyl radical (HER) by rat and deer mice liver microsomal systems. Experiments were carried out to evaluate the ability of human liver microsomes to catalyze this reaction, compare the effectiveness of NADH with that of NADPH, and assess the possible role of cytochrome b5 in HER formation. HER was detected as the alpha-(4-pyridly-1 -oxide)-N-t-butylnitrone/HER adduct. Human liver microsomes catalyzed HER formation with either NADPH or NADH as cofactor; rates with NADH were approximately 50% those found with NADPH. Chelex-100 treatment of the reaction mixture produced marked inhibition of HER formation, suggesting that a transition metal, such as iron, was required to catalyze the reaction. The addition of ferric chloride restore HER formation. Catalase (2600 units/ml) and superoxide dismutases (500 units/ml) nearly completely inhibited the reaction with either NADPH or NADH. The NADH-dependent rates of superoxide production, detected as 5,5-dimethyl-1-pyrroline-N-oxide-O2H, were approximately 50% the NADPH-dependent rates, which is consistent with the rates of HER formation. Anti-cytochrome b5 IgG decreased NADPH- and NADH-dependent HER formation, and this was associated with inhibition of superoxide formation with both reductants. These results indicate that human liver microsomes can catalyze the oxidation of ethanol of HER with either NADPH or NADH as reductant. The effectiveness of NADH may be significant in view of the increased NADH/NAD+ redox ratio in the liver as a consequence of ethanol oxidation by alcohol dehydrogenase. HER formation by human liver microsomes seems to be catalyzed by an oxidant derived from the interaction of iron with superoxide or H2O2, and a close association exists between HER formation and superoxide production. Cytochrome b5 seems to play a role in HER formation, most likely due to its effect on superoxide production.

Patterns of grief reaction after pregnancy loss.

Analysis of three waves of Perinatal Grief Scale scores for 194 bereaved subjects over the course of two years revealed patterns of change different from those commonly noted in the literature. Less than half the sample matched the "normal" model; the rest exhibited non-normal patterns that did not fit the alternative psychological models. Demographic variables and pregnancy history, both before and after the loss, help explain some of the differences in direction of the grief response.