Plasma and acrosomal membrane lipid content of saltwater crocodile spermatozoa.

This study describes the chemical lipid composition of the sperm plasma and acrosomal membranes of the saltwater crocodile Crocodylus porosus with the aim of providing new insights into sperm physiology, particularly that associated with their preservation ex vivo. The specific fatty acid composition of the sperm plasma and acrosomal membranes is documented. The mean (±s.d.) ratio of unsaturated to saturated membrane fatty acids within the plasma membrane was 2.57±0.50, and was determined to be higher than a similar analysis of the lipids found in the acrosomal membrane (0.70±0.10). The saltwater crocodile sperm plasma membrane also contained remarkably high levels of cholesterol (mean (±s.d.) 40.7±4.5 nmol per 106 sperm cells) compared with the spermatozoa of other amniote species that have so far been documented. We suggest that this high cholesterol content could be conferring stability to the crocodile sperm membrane, allowing it to tolerate extreme osmotic fluxes and rapid changes in temperature. Our descriptive analysis now provides those interested in reptile and comparative sperm physiology an improved baseline database for interpreting biochemical changes associated with preservation pathology (e.g. cold shock and cryoinjury), epididymal sperm maturation and capacitation/acrosome reaction.

Examining Differences in Recovery Outcomes between Male and Female Hip Fracture Patients: Design and Baseline Results of a Prospective Cohort Study from the Baltimore Hip Studies.

BACKGROUND: Incidence of hip fractures in men is expected to increase, yet little is known about consequences of hip fracture in men compared to women. It is important to investigate differences at time of fracture using the newest technologies and methodology regarding metabolic, physiologic, neuromuscular, functional, and clinical outcomes, with attention to design issues for recruiting frail older adults across numerous settings. OBJECTIVES: To determine whether at least moderately-sized sex differences exist across several key outcomes after a hip fracture. DESIGN, SETTING, AND PARTICIPANTS: This prospective cohort study (Baltimore Hip Studies 7th cohort [BHS-7]) was designed to include equal numbers of male and female hip fracture patients to assess sex differences across various outcomes post-hip fracture. Participants were recruited from eight hospitals in the Baltimore metropolitan area within 15 days of admission and were assessed at baseline, 2, 6 and 12 months post-admission. MEASUREMENTS: Assessments included questionnaire, functional performance evaluation, cognitive testing, measures of body composition, and phlebotomy. RESULTS: Of 1709 hip fracture patients screened from May 2006 through June 2011, 917 (54%) were eligible and 39% (n=362) provided informed consent. The final analytic sample was 339 (168 men and 171 women). At time of fracture, men were sicker (mean Charlson score= 2.4 vs. 1.6; p<0.001) and had worse cognition (3MS score= 82.3 vs. 86.2; p<0.05), and prior to fracture were less likely to be on bisphosphonates (8% vs. 39%; p<0.001) and less physically active (2426 kilocalories/week vs. 3625; p<0.001). CONCLUSIONS: This paper provides the study design and methodology for recruiting and assessing hip fracture patients and evidence of baseline and pre-injury sex differences which may affect eventual recovery one year later.

Multi-site and nasal swabbing for carriage of Staphylococcus aureus: what does a single nose swab predict?

BACKGROUND: Carriage of Staphylococcus aureus is a risk for infections. Targeted decolonization reduces postoperative infections but depends on accurate screening. AIM: To compare detection of S. aureus carriage in healthy individuals between anatomical sites and nurse- versus self-swabbing; also to determine whether a single nasal swab predicted carriage over four weeks. METHODS: Healthy individuals were recruited via general practices. After consent, nurses performed multi-site swabbing (nose, throat, and axilla). Participants performed nasal swabbing twice-weekly for four weeks. Swabs were returned by mail and cultured for S. aureus. All S. aureus isolates underwent spa typing. Persistent carriage in individuals returning more than three self-swabs was defined as culture of S. aureus from all or all but one self-swabs. FINDINGS: In all, 102 individuals underwent multi-site swabbing; S. aureus carriage was detected from at least one site from 40 individuals (39%). There was no difference between nose (29/102, 28%) and throat (28/102, 27%) isolation rates: the combination increased total detection rate by 10%. Ninety-nine patients returned any self-swab, and 96 returned more than three. Nasal carriage detection was not significantly different on nurse or self-swab [28/99 (74%) vs 26/99 (72%); χ2: P=0.75]. Twenty-two out of 25 participants with first self-swab positive were persistent carriers and 69/71 with first self-swab negative were not, giving high positive predictive value (88%), and very high negative predictive value (97%). CONCLUSION: Nasal swabs detected the majority of carriage; throat swabs increased detection by 10%. Self-taken nasal swabs were equivalent to nurse-taken swabs and predicted persistent nasal carriage over four weeks.

Multiple-strain colonization in nasal carriers of Staphylococcus aureus.

Staphylococcus aureus is a commensal that can also cause invasive infection. Reports suggest that nasal cocolonization occurs rarely, but the resources required to sequence multiple colonies have precluded its large-scale investigation. A staged protocol was developed to maximize detection of mixed-spa-type colonization while minimizing laboratory resources using 3,197 S. aureus-positive samples from a longitudinal study of healthy individuals in Oxfordshire, United Kingdom. Initial typing of pooled material from each sample identified a single unambiguous strain in 89.6% of samples. Twelve single-colony isolates were typed from samples producing ambiguous initial results. All samples could be resolved into one or more spa types using the protocol. Cocolonization point prevalence was 3.4 to 5.8% over 24 months of follow-up in 360 recruitment-positives. However, 18% were cocolonized at least once, most only transiently. Cocolonizing spa types were completely unrelated in 56% of samples. Of 272 recruitment-positives returning ≥12 swabs, 166 (61%) carried S. aureus continuously but only 106 (39%) carried the same single spa type without any cocolonization; 31 (11%) switched spa type and 29 (11%) had transient cocarriage. S. aureus colonization is dynamic even in long-term carriers. New unrelated cocolonizing strains could increase invasive disease risk, and ongoing within-host evolution could increase invasive potential, possibilities that future studies should explore.

Flying-fox (Pteropus spp.) sperm membrane fatty acid composition, its relationship to cold shock injury and implications for cryopreservation success.

The very large acrosome of Pteropus species spermatozoa is prone to damage during cooling procedures. Cryogenic succuss has been linked to membrane composition, therefore the lipid composition of five Pteropus species sperm acrosomal and plasma membranes were investigated to provide insight into reasons for cold shock susceptibility. Rapid chilling and re-warming of spermatozoa from three Pteropus species resulted in a decrease (P<0.05) in acrosomal integrity. Biochemical analysis of lipids revealed that stearic acid (18:0) was the predominant saturated fatty acid and oleic acid (18:1, n-9) the predominant unsaturated fatty acid in both acrosomal and plasma membranes. Linolenic acid (18:3, n-3) was only detected in plasma membranes of Pteropus hypomelanus and was detected in acrosomal membranes of all Pteropus spp. studied (except Pteropus giganteus). Although detected in both plasma and acrosomal membranes of Pteropus vampyrus, docosahexaenoic acid (22:6) was not detected at all in Pteropus poliocephalus, only in trace levels in the acrosomal and plasma membranes of P. giganteus and P. hypomelanus and not in acrosomal membranes of Pteropus rodricensis. No difference was seen in the levels of polyunsaturated fatty acids (PUFAs) within plasma membranes, however PUFAs were lower (P<0.05) in acrosomal membranes of P. giganteus compared with P. vampyrus. Pteropus spp. spermatozoa have a very low ratio of unsaturated/saturated membrane fatty acids (<0.5). Membranes containing more PUFAs are more fluid, so the use of cryogenic media which improves membrane fluidity should improve Pteropus spp. spermatozoal viability post-thaw.

USA300 methicillin-resistant S. aureus (USA300 MRSA) colonization and the risk of MRSA infection in residents of extended-care facilities.

To examine the pathogenesis of USA300 MRSA infection in long-term care residents, we performed a retrospective cohort study of 1691 adult residents of two extended-care facilities from 2003 to 2007 to assess whether the risk of subsequent MRSA infection is higher in USA300 MRSA-colonized residents compared to non-colonized residents or non-USA300 MRSA colonized residents. Six per cent of residents were colonized with USA300 MRSA; 12% of residents were colonized with non-USA300 MRSA; and 101 residents developed MRSA infection. The risk of infection was twofold higher in residents colonized with USA300 MRSA compared to residents not colonized with MRSA [adjusted hazard ratio 2·3, 95% confidence interval (CI) 1·1-4·5]. The risk of infection in USA300 MRSA-colonized residents was similar to USA300 MRSA non-colonized residents (relative risk 1·1, 95% CI 0·5-2·3). Our findings show that colonization with USA300 MRSA increases the risk of MRSA infection suggesting a similar pathogenesis.

The hormonal profile of hip fracture female patients differs from community-dwelling peers over a 1-year follow-up period.

UNLABELLED: Hormone levels were compared over a 1-year period between elderly women who had sustained a hip fracture and women of similar age and functional ability. Our study suggests progressive hormonal changes that may contribute to severe bone loss during the year following hip fracture. INTRODUCTION: Alterations in hormones affecting the musculoskeletal system may increase risk of hip fracture or poor post-fracture recovery in postmenopausal women. Most studies lack appropriate reference groups, and thus cannot assess the extent to which these alterations are attributable to hip fracture. METHODS: Women aged ≥65 years hospitalized for an acute hip fracture (Baltimore Hip Studies, BHS-3; n = 162) were age-matched to 324 women enrolled in the Women's Health and Aging Study I, a Baltimore-based cohort with similar functional status to the pre-fracture status of BHS-3 women. Both studies enrolled participants from 1992 to 1995. Insulin-like growth hormone-1 (IGF-1), parathyroid hormone (PTH), 1,25 dihydroxyvitamin D [1,25(OH)2D], and osteocalcin were evaluated at baseline and 2, 6, and 12 months post-fracture, and at baseline and 12 months in the comparison group. Between-group differences in trajectories of each hormone were examined. RESULTS: Baseline mean IGF-1 levels were significantly lower in hip fracture patients than the comparison group (75.0 vs. 110.5 µg/dL; p < 0.001). Levels increased by 2 months post-fracture, but remained significantly lower than those in the comparison group throughout the 12-month follow-up (p < 0.01). Levels of PTH and osteocalcin were similar between groups at baseline, but rose during the year post-fracture to significantly differ from the comparison women (p < 0.001). 1,25(OH)2D levels did not differ between the hip fracture and comparison women at any time. CONCLUSIONS: Older women who have sustained a hip fracture have progressive changes in hormonal milieu that exceed those of women of similar health status during the year following fracture.

Assessing and minimizing time-dependent inhibition of cytochrome P450 3A in drug discovery: a case study with melanocortin-4 receptor agonists.

1-[(2R)-2-([[(1S,2S)-1-amino-1,2,3,4-tetrahydronaphthalen-2-yl]carbonyl]amino)-3-(4-chlorophenyl)propanoyl]-N-(tert-butyl)-4-cyclohexylpiperidine-4-carboxamide (1) is a potent melanocortin-4 receptor agonist that exhibited time-dependent inhibition of cytochrome P450 (P450) 3A in incubations with human liver microsomes. In incubations fortified with potassium cyanide, a cyano adduct was identified by liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis as a cyanonitrosotetrahydronaphthalenyl derivative. The detection of this adduct suggested that a nitroso species was involved in the formation of a metabolite intermediate (MI) complex that led to the observed P450 inactivation. Further evidence supporting this hypothesis derived from incubations of 1 with recombinant P450 3A4, which exhibited a lambda(max) at approximately 450 nm. The species responsible for this absorbance required the presence of beta-nicotinamide adenine dinucleotide phosphate reduced form (NADPH), increased with increasing incubation time and decreased following the addition of potassium ferricyanide to the incubation mixture, suggestive of an MI complex. Similar results were obtained with rat liver microsomes and with recombinant P450 3A1. When rats were dosed with indinavir as a P450 3A probe substrate, plasma exposure to indinavir increased three-fold following pretreatment with 1, consistent with drug-drug interaction projections based on the k(inact) and K(I) parameters for 1 in rat liver microsomes. A similar approach was used to predict the magnitude of the corresponding drug-drug interaction potential in humans dosed with a drug metabolized predominantly by P450 3A, and the forecast area under the curve (AUC) increase ranged from four- to ten-fold. These data prompted a decision to terminate further evaluation of 1 as a development candidate, and led to the synthesis of the methyl analogue 2. Methyl substitution alpha to the amino group in 2 was designed to reduce the propensity for formation of a nitroso intermediate and, indeed, 2 failed to exhibit time-dependent inhibition of P450 3A in human liver microsomal incubations. This case study highlights the importance of mechanistic studies in support of drug-discovery and decision-making processes.

Within and between breed differences in freezing tolerance and plasma membrane fatty acid composition of boar sperm.

The response of sperm to cryopreservation and the fertility of frozen-thawed semen varies between species. Besides species differences in sperm physiology, structure and biochemistry, factors such as sperm transport and female reproductive tract anatomy will affect fertility of frozen-thawed semen. Therefore, studying differences in sperm cryotolerance between breeds and individuals instead of between species may reveal sources of variability in sperm cryotolerance. In the present study, the effect of cooling, re-warming and freezing and thawing on plasma membrane and acrosome integrity of sperm within and between Norwegian Landrace and Duroc breeds was studied. Furthermore, the relation between post-thaw survival rate and fatty acid composition of the sperm plasma membranes was investigated. Flow cytometry assessments of plasma membrane and acrosome integrity revealed no significant differences between breeds; however there were significant male-to-male variations within breeds in post-thaw percentages of live sperm (plasma membrane intact). The most abundant fatty acids in the plasma membranes from both breeds were palmitic acid (16:0), stearic acid (18:0), oleic acid (18:1, n-9), docosapentaenoic acid (22:5, n-6) and docosahexaenoic acid (22:6, n-3). The ratio of sigma operator 22:5, n-6 and 22:6, n-3/ sigma operator all other membrane fatty acids was significantly related to survival rate (plasma membrane integrity) of sperm for both Norwegian Landrace (correlation coefficient (r(s)) = 0.64, P < 0.05) and Duroc (r(s) = 0.67, P < 0.05) boars. In conclusion, male-to-male differences in sperm survival rate after freezing and thawing may be partly related to the amount of long-chain polyunsaturated fatty acids in the sperm plasma membranes.

Comparative aspects of sperm membrane fatty acid composition in silver (Vulpes vulpes) and blue (Alopex lagopus) foxes, and their relationship to cell cryopreservation.

Cryogenic protocols have been developed for the storage of farmed silver fox (Vulpes vulpes) spermatozoa. However, these same protocols and modifications of these protocols have failed to satisfactorily preserve spermatozoa collected from farmed blue foxes (Alopex lagopus). Because cryogenic success has been linked to membrane composition, the plasma membrane lipid composition of farmed blue fox and silver fox spermatozoa was studied. Silver fox spermatozoal membranes have significantly higher levels of docosapentaenoic acid (DPA; 22:5, n-6) compared to blue fox spermatozoa, and blue fox spermatozoal membranes have significantly higher levels of stearic acid (18:0). Silver fox spermatozoal membranes not only have a higher ratio of unsaturated/saturated membrane fatty acids, but also higher levels of membrane desmosterol and cholesterol.

Sperm membrane fatty acid composition in the Eastern grey kangaroo (Macropus giganteus), koala (Phascolarctos cinereus), and common wombat (Vombatus ursinus) and its relationship to cold shock injury and cryopreservation success.

Marsupial spermatozoa tolerate cold shock well, but differ in cryopreservation tolerance. In an attempt to explain these phenomena, the fatty acid composition of the sperm membrane from caput and cauda epididymides of the Eastern grey kangaroo, koala, and common wombat was measured and membrane sterol levels were measured in cauda epididymidal spermatozoa. While species-related differences in the levels of linolenic acid (18:3, n-6) and arachidonic acid (20:4, n-6) were observed in caput epididymal spermatozoa, these differences failed to significantly alter the ratio of unsaturated/saturated membrane fatty acids. However in cauda epididymidal spermatozoa, the ratio of unsaturated/saturated membrane fatty acids in koala and kangaroo spermatozoa was approximately 7.6 and 5.2, respectively; substantially higher than any other mammalian species so far described. Koala spermatozoal membranes had a higher ratio of unsaturated/saturated membrane fatty acids than that of wombat spermatozoa (t = 3.81; df = 4; p < or = 0.02); however, there was no significant difference between wombat and kangaroo spermatozoa. The highest proportions of DHA (22:6, n-3), the predominant membrane fatty acid in cauda epididymidal spermatozoa, were found in wombat and koala spermatozoa. While species-related differences in membrane sterol levels (cholesterol and desmosterol) were observed in cauda epididymidal spermatozoa, marsupial membrane sterol levels are very low. Marsupial spermatozoal membrane analyses do not support the hypothesis that a high ratio of saturated/unsaturated membrane fatty acids and low membrane sterol levels predisposes spermatozoa to cold shock damage. Instead, cryogenic tolerance appears related to DHA levels.

Stem-loop SL4 of the HIV-1 psi RNA packaging signal exhibits weak affinity for the nucleocapsid protein. structural studies and implications for genome recognition.

Encapsidation of the genome of the human immunodeficiency virus type-1 (HIV-1) during retrovirus assembly is mediated by interactions between the nucleocapsid (NC) domains of assembling Gag polyproteins and a approximately 110 nucleotide segment of the genome known as the Psi-site. The HIV-1 Psi-site contains four stem-loops (SL1 through SL4), all of which are important for genome packaging. Recent isothermal titration calorimetry (ITC) studies have demonstrated that SL2 and SL3 are capable of binding NC with high affinity (K(d) approximately 140 nM), consistent with proposals for protein-interactive functions during packaging. To determine if SL4 may have a similar function, NC-interactive studies were conducted by NMR and gel-shift methods. In contrast to previous reports, we find that SL4 binds weakly to NC (K(d)=(+/-14 microM), suggesting an alternative function. NMR studies indicate that the GAGA tetraloop of SL4 adopts a classical GNRA-type fold (R=purine, N=G, C, A or U), a motif that stabilizes RNA tertiary structures in other systems. In combination with previously reported gel mobility studies of Psi-site deletion mutants, these findings suggest that SL4 functions in genome recognition not by binding to Gag, but by stabilizing the structure of the Psi-site. Differences in the affinities of NC for SL2, SL3 and SL4 stem-loops can now be rationalized in terms of the different structural properties of stem loops that contain GGNG (SL2 and SL3) and GNRA (SL4) sequences.

Ethanol- and nicotine-induced membrane changes in embryonic and neonatal chick brains.

In order to study the effects of EtOH and/or nicotine on brain membrane fatty acid composition, various concentrations of EtOH and/or nicotine were injected into the air sac of chicken eggs at 0 days of incubation. Controls were injected with saline. Experimental groups were injected with either 200 micromol EtOH/kg egg, 100 micromol nicotine/kg egg, 200 micromol nicotine/kg egg, 200 micromol EtOH/kg and 100 micromol nicotine/kg egg, or 200 micromol EtOH/kg and 200 micromol nicotine/kg egg. In all experimental groups, EtOH- and nicotine-induced decreases in brain long-chain polyunsaturated membrane fatty acids were observed in stage 44 embryos, stage 45 embryos, and neonatal chicks. These EtOH- and nicotine-induced decreases in brain membrane polyunsaturated fatty acids correlated with elevated levels of brain lipid hydroperoxides and reduced brain acetylcholinesterase (AChE; EC. 3.1.1.7) activities.

PCR-based ELISA technique for malaria diagnosis of specimens from Thailand.

We performed a field evaluation of polymerase chain reaction (PCR)-based enzyme-linked immuno-sorbent assays (ELISA) for the diagnosis of malaria. A commercially available PCR-ELISA microplate hybridization (MPH) assay was used. Blood specimens were collected from 300 volunteers seeking care at malaria clinics in Thailand. Examination of 200 high power fields by Giemsa-stained thick and thin smear (GTTS) revealed 51 P. falciparum (Pf), 45 P. vivax (Pv), seven mixed Pf-Pv infections. These plus a random sample of 48 GTTS-negative specimens were selected for this study. All 151 specimens were processed for parasite DNA extraction and assayed by PCR-MPH. The target DNA sequence of the 18S small subunit ribosomal RNA (SSUrRNA) gene was amplified by PCR and hybridized with species-specific probes for Pf, Pv, P. malariae (Pm) and P. ovale (Po) immobilized in the wells of the microtiter plate and detected by colorimetric assay. Colour development was assessed at an optical density (OD) of 405 nm. An absorbance reading of > or = 0.1 was used as a positive cut-off. In comparison with GTTS results, PCR-MPH sensitivity was 91.4% (53/58, 95% CI 84.2-98.6) for Pf, 94.2% (49/52, 87.9-100) for Pv and specificity was 95.8% (46/48, 95% CI 90.2-100). There was statistically significant positive correlation between parasite densities < or = 7000/microl blood and absorbance reading, suggestive of PCR-MPH being semiquantitative. PCR-MPH also detected additional Pf and Pv cases as well as Pm and Po.

Cues trained apart compete for behavioral control in rats: convergence with the associative interference literature.

Contemporary theories of associative learning require cues be trained in compound for cue competition (interference) to occur. That is, Cues A and X should compete for behavioral control only if training consists of AX-outcome (O) trials and not if each cue is separately paired with O (i.e., X-O and A-O). Research with humans challenges this view by showing that A-O trials interpolated between training and testing of a X-O association impair responding to X (i.e., retroactive interference). In six conditioned suppression studies with rats, the authors demonstrate that two cues trained apart can each interfere with the potential of the other to predict the outcome. The authors conclude that this type of interference (a) reflects a failure to retrieve the target association due to priming at test of the interfering association and (b) is attenuated if the outcome is of high biological significance. These findings parallel previous reports in verbal learning research and suggest that a similar associative structure underlies some types of associations in nonverbal subjects.

Conditioned inhibition produced by extinction-mediated recovery from the relative stimulus validity effect: a test of acquisition and performance models of empirical retrospective revaluation.

Empirical retrospective revaluation is a phenomenon of Pavlovian conditioning and human causal judgment in which posttraining changes in the conditioned response (Pavlovian task) or causal rating (causal judgment task) of a cue occurs in the absence of further training with that cue. Two experiments tested the contrasting predictions made by 2 families of models concerning retrospective revaluation effects. In a conditioned lick-suppression task, rats were given relative stimulus validity training, consisting of reinforcing a compound of conditioned stimuli (CSs) A and X and nonreinforcement of a compound of CSs B and X, which resulted in low conditioned responding to CS X. Massive posttraining extinction of CS A not only enhanced excitatory responding to CS X, but caused CS B to pass both summation (Experiment 1) and retardation (Experiment 2) tests for conditioned inhibition. The inhibitory status of CS B is predicted by the performance-focused extended comparator hypothesis (J. C. Denniston, H. I. Savastano, & R. R. Miller, 2001), but not by acquisition-focused models of empirical retrospective revaluation (e.g., A. Dickinson & J. Burke, 1996; L. J. Van Hamme & E. A. Wasserman, 1994).

Conditions favoring retroactive interference between antecedent events (cue competition) and between subsequent events (outcome competition).

Retrieval of a target association (A-B) is often impaired if training of a similar association is interpolated between target training and testing; this is known as retroactive interference. Two experiments, in which rats were used as subjects in a sensory preconditioning preparation, studied the associative nature of retroactive interference between antecedent events (i.e., A and C in the A-B, C-B paradigm) and between subsequent events (i.e., B and C in the A-B, A-C paradigm). With the present preparation, retroactive interference was equally strong between antecedent events and between subsequent events. Moreover, interference occurred only if (1) an association was trained in the interpolated phase and (2) the target and interpolated associations had a common element in a common temporal location.

Discovery of a potent, orally bioavailable beta(3) adrenergic receptor agonist, (R)-N-[4-[2-[[2-hydroxy-2-(3-pyridinyl)ethyl]amino]ethyl]phenyl]-4-[4 -[4-(trifluoromethyl)phenyl]thiazol-2-yl]benzenesulfonamide.

As part of our investigation into the development of orally bioavailable beta(3) adrenergic receptor agonists, we have identified a series of pyridylethanolamine analogues possessing a substituted thiazole benzenesulfonamide pharmacophore that are potent human beta(3) agonists with excellent selectivity against other human beta receptor subtypes. Several of these compounds also exhibited an improved pharmacokinetic profile in dogs. For example, thiazole sulfonamide 2e (R = 4-F(3)C-C(6)H(4)) is a potent full beta(3) agonist (EC(50) = 3.6 nM, 94% activation) with >600-fold selectivity over the human beta(1) and beta(2) receptors, which also displays good oral bioavailability in several mammalian species, as well as an extended duration of action.

Cytochrome P450 3A4-mediated interaction of diclofenac and quinidine.

The metabolism of diclofenac to its 5-hydroxylated derivative in humans is catalyzed by cytochrome P450 (CYP)3A4. We report herein that in vitro this biotransformation pathway is stimulated by quinidine. When diclofenac was incubated with human liver microsomes in the presence of quinidine, the formation of 5-hydroxydiclofenac increased approximately 6-fold relative to controls. Similar phenomena were observed with diastereoisomers of quinidine, including quinine and the threo epimers, which produced an enhancement in the formation of 5-hydroxydiclofenac in the order of 6- to 9-fold. This stimulation of diclofenac metabolism was diminished when human liver microsomes were pretreated with a monoclonal inhibitory antibody against CYP3A4. In contrast, neither cytochrome b(5) nor CYP oxidoreductase appeared to mediate the stimulation of diclofenac metabolism by quinidine, suggesting that the effect of quinidine is mediated through CYP3A4 protein. Further kinetic analyses indicated that V(max) values for the conversion of diclofenac to its 5-hydroxy derivative increased 4.5-fold from 13.2 to 57.6 nmol/min/nmol of CYP with little change in K(m) (71-56 microM) over a quinidine concentration range of 0 to 30 microM. Conversely, the metabolism of quinidine was not affected by the presence of diclofenac; the K(m) value estimated for the formation of 3-hydroxyquinidine was approximately 1.5 microM, similar to the quinidine concentration required to produce 50% of the maximum stimulatory effect on diclofenac metabolism. It appears that the enhancement of diclofenac metabolism does not interfere with quinidine's access to the ferriheme-oxygen complex, implicating the presence of both compounds in the active site of CYP3A4 at the same time. Finally, a approximately 4-fold increase in 5-hydroxydiclofenac formation was observed in human hepatocyte suspensions containing diclofenac and quinidine, demonstrating that this type of drug-drug interaction occurs in intact cells.