Effect of P450 oxidoreductase variants on the metabolism of model substrates mediated by CYP2C9.1, CYP2C9.2, and CYP2C9.3.

OBJECTIVES: CYP2C9 is a microsomal cytochrome P450 that receives electrons from P450 oxidoreductase (POR) to metabolize about 15% of clinically used drugs. Similar to many P450 enzymes, CYP2C9 is polymorphic, with the hypomorphic *2 and *3 variants accounting for about 20% of White alleles. POR is also polymorphic, with the amino acid sequence variant A503V accounting for 19-37% of alleles in different populations. We aimed to understand how polymorphisms in these two interacting proteins might affect drug metabolism. METHODS: We assayed the activities of CYP2C9.1, CYP2C9.2, and CYP2C9.3 to metabolize diclofenac, flurbiprofen, and tolbutamide using a wild type or one of four POR variants (Q153R, A287P, R457H, and A503V). Human CYP2C9 and POR variants were expressed in bacteria, purified, and reconstituted in vitro and the Michaelis constant and maximum velocity were measured with each CYP2C9/POR combination and each substrate. RESULTS: With wild-type POR, the CYP2C9 activities were CYP2C9.1>CYP2C9.2>>CYP2C9.3 with all three substrates. Both the common A503V polymorphism and the rare Q153R variant showed modest increases in activity with all three CYP2C9 isoforms and all three substrates. This is in contrast to previous studies in which A503V showed a modest loss of function with CYP1A2, CYP2C19, CYP2D6, CYP3A4, and CYP17A1. The disease-causing POR variants A287P and R457H had a very low or unmeasurable activity with all CYP2C9 isoforms and all substrates, which is consistent with their low activities with other CYPs. CONCLUSION: POR variants affect CYP2C9 activities. The impact of a POR variant on catalysis varies with the isoform of CYP2C9 and the assay substrate.

Do Preoperative Nasal Antiseptic Swabs Reduce the Rate of Surgical Site Infections After Adult Thoracolumbar Spine Surgery?

INTRODUCTION: Surgical site infection (SSI) remains a major complication after adult spinal surgery. We investigated whether adding preoperative nasal decontamination by antiseptic swab (skin and nasal antiseptic povidone-iodine, SNA-PI) to our antimicrobial protocol reduces the SSI rate among our patients undergoing thoracolumbar spinal surgery. METHODS: We retrospectively reviewed all adult thoracolumbar spinal surgeries performed between June 2015 and May 2017 at a single hospital. Patients were divided into those who received nasal decontamination (SNA-PI+) and those who did not (SNA-PI-). SSI rates and responsible pathogens were compared between the cohorts. RESULTS: A total of 1,555 surgeries with nasal decontamination (SNA-PI+) and 1,423 surgeries without (SNA-PI-) were included. The SSI rate in the SNA-PI+ group was 13 of 1,555 (0.8%) versus 10 of 1,423 (0.7%) for SNA-PI- group (P = 0.68). The infection rate was the highest among posterior instrumented fusions in the SNA-PI+ group (1.4%). Methicillin-sensitive Staphylococcus aureus was responsible for 70% of infections in the SNA-PI- group and 38% in the SNA-PI+ group (P = 0.13). CONCLUSIONS: Routine nasal antiseptic swab before spine surgery did not affect the overall rate of SSI in thoracolumbar spinal surgeries. The incidence of methicillin-sensitive S aureus was lower in patients who received nasal decontamination (5/1,555, 0.3%) compared with those who did not (7/1,423, 0.5%); however, this result was not statistically significant (P = 0.57).

Effects of genetic variants of human P450 oxidoreductase on catalysis by CYP2D6 in vitro.

OBJECTIVES: Cytochrome P450 (P450) oxidoreductase (POR) donates electrons to all microsomal cytochrome P450s, including drug-metabolizing and steroidogenic enzymes. Severe POR mutations cause skeletal malformations and disordered steroidogenesis. The POR polymorphism A503V is found on approximately 28% of human alleles and decreases activities of CYP3A4 and steroidogenic CYP17, but not the activities of steroidogenic CYP21 or drug-metabolizing CYP1A2 and CYP2C19. CYP2D6 metabolizes about 25% of clinically used drugs; we assessed the capacity of POR variants to support the activities of human CYP2D6. METHODS: N-27 forms of wildtype (WT), Q153R, A287P, R457H and A503V POR, and WT CYP2D6 were expressed in Escherichia coli. POR proteins in bacterial membranes were reconstituted with purified CYP2D6. Support of CYP2D6 was measured by metabolism of EOMCC (2H-1-benzopyran-3-carbonitrile,7-(ethoxy-methoxy)-2-oxo-(9Cl)), dextromethorphan and bufuralol. Michaelis constant (K(m)) and maximum velocity (V(max)) were determined in three triplicate experiments for each reaction; catalytic efficiency is expressed as V(max)/K(m). RESULTS: Compared with WT POR, disease-causing POR mutants A287P and R457H supported no detectable CYP2D6 activity with EOMCC, but A287P supported approximately 25% activity with dextromethorphan and bufuralol. Q153R had increased function with CYP2D6 (128% with EOMCC, 198% with dextromethorphan, 153% with bufuralol). A503V supported decreased CYP2D6 activity: 85% with EOMCC, 62% with dextromethorphan and 53% with bufuralol. CONCLUSION: POR variants have different effects depending on the substrate metabolized. Disease-causing POR mutations R457H and A287P had poor activities, suggesting that diminished drug metabolism should be considered in affected patients. The common A503V polymorphism impaired CYP2D6 activities with two commonly used drugs by 40-50%, potentially explaining some genetic variation in drug metabolism.

CYP2C9-CYP3A4 protein-protein interactions: role of the hydrophobic N terminus.

Cytochromes P450 (P450s) interact with redox transfer proteins, including P450 reductase (CPR) and cytochrome b(5) (b5), all being membrane-bound. In multiple in vitro systems, P450-P450 interactions also have been observed, resulting in alterations in enzymatic activity. The current work investigated the effects and mechanisms of interaction between CYP2C9 and CYP3A4 in a reconstituted system. CYP2C9-mediated metabolism of S-naproxen and S-flurbiprofen was inhibited up to 80% by coincubation with CYP3A4, although K(m) values were unchanged. Increasing CYP3A4 concentrations increased the degree of inhibition, whereas increasing CPR concentrations resulted in less inhibition. Addition of b5 only marginally affected the magnitude of inhibition. In contrast, CYP2C9 did not alter the CYP3A4-mediated metabolism of testosterone. The potential role of the hydrophobic N terminus on these interactions was assessed by incubating truncated CYP2C9 with full-length CYP3A4, and vice versa. In both cases, the inhibition was fully abolished, indicating an important role for hydrophobic forces in CYP2C9-CYP3A4 interactions. Finally, a CYP2C9/CYP3A4 heteromer complex was isolated by coimmunoprecipitation techniques, confirming the physical interaction of the proteins. These results show that the N-terminal membrane binding domains of CYP2C9 and CYP3A4 are involved in heteromer complex formation and that at least one consequence is a reduction in CYP2C9 activity.

Posterolateral Versus Transforaminal Interbody L4/5 Fusion: Correlation With Subsequent Surgery.

STUDY DESIGN: This is a retrospective cohort study. OBJECTIVE: To compare posterolateral versus transforaminal interbody fusion (PLF vs. PLF+TLIF) of the L4/5 segment regarding rates of subsequent surgery, clinical and radiographic parameters, and patient satisfaction. SUMMARY OF BACKGROUND DATA: Surgical treatment of lumbar stenosis, decompression with or without fusion, is an efficacious treatment in select patients. Reoperation is thought to be a problem after lumbar fusion. Despite multiple studies, the fusion method that minimizes the need for subsequent surgery has yet to be determined. MATERIALS AND METHODS: A retrospective cohort study was conducted on 89 patients who had an isolated L4/5 decompression and fusion, from January 2006 to 2012. All patients had stenosis and degenerative spondylolisthesis at the L4/5 level. All surgeries were performed at a single center, using either PLF (31 patients) or PLF+TLIF (58 patients) techniques. Preoperative and postoperative patient-reported outcome measures (Oswestry disability index, visual analog scale back pain, visual analog scale leg pain) and radiographic parameters (L4/5 lordosis and overall lumbar lordosis) were measured. Patient satisfaction was acquired via a questionnaire. Chart reviews and patient questionnaires were used to determine the incidence of subsequent lumbar surgery over a minimum follow-up of 5 years. RESULTS: At an average of 8.7 years follow-up, 2 of 31 patients in the PLF group had subsequent lumbar surgery, compared with 16 of 58 patients in the PLF+TLIF group (6% vs. 28%; P=0.02). There were no significant differences between groups with respect to sex, age, body mass index, tobacco, perioperative measures, patient-reported outcomes, or radiographic parameters (P>0.05). CONCLUSIONS: Both PLF and PLF+TLIF are effective fusion methods for L4/5 stenosis and spondylolisthesis. In this study, patients treated with PLF were less likely to undergo a subsequent lumbar surgery. More research is needed to determine which factors influence whether PLF or PLF+TLIF should be used in these patients. LEVEL OF EVIDENCE: Level III.

Weight Loss Mediated Reduction in Xanthine Oxidase Activity and Uric Acid Clearance in Adolescents with Severe Obesity.

BACKGROUND: Increased xanthine oxidase (XO) activity and uric acid levels are known to be associated with obesity and hypertension; however, it is not known if obesity is directly responsible for these associations in youth. This study investigated the effect of weight loss on XO activity, uric acid, and their relationship to blood pressure change in obese youth to provide greater insight on how obesity increases cardiovascular risk. METHODS: This was an ancillary study in which 16 adolescents (mean age 15 ± 2 years) received meal replacement therapy over a period of four weeks. Outcomes measured at baseline and after intervention included weight, blood pressure, XO activity, plasma uric acid, uric acid clearance, and creatinine clearance. RESULTS: After the meal replacement intervention, participants experienced reductions in body weight (109.2 ± 16 kg vs. 105.2 ± 14 kg, p < 0.0001) and BMI (38.7 ± 4 kg vs. 37.4 ± 3 kg, p < 0.0001). Plasma XO activity was reduced by 9.8% (p = 0.016). Uric acid clearance was decreased by 39% (p = 0.006). SBP (systolic blood pressure) and plasma uric acid concentrations were reduced but did not achieve statistical significance (p = 0.34 and 0.38, respectively). DBP (diastolic blood pressure) was unchanged (p = 0.86). No significant relationships were found between changes in blood pressure and changes in either XO activity or plasma uric acid levels. CONCLUSION: Weight loss led to decreases in uric acid production by lowering XO activity and decreases in uric acid clearance by reducing glomerular filtration (GF) and increasing reabsorption. Changes in XO activity and uric acid levels did not correlate with changes in blood pressure.

Xanthine oxidase and cardiovascular risk in obese children.

BACKGROUND: Pathological mechanisms of how childhood obesity leads to increased risk of cardiovascular disease (CVD) are not fully characterized. Oxidative-stress-related enzymes, such as xanthine oxidase (XO), have been linked to obesity, endothelial dysfunction, and CVD in adults, but little is known about this pathway in children. The aim of this study was to determine whether differential XO activity is associated with endothelial dysfunction, CVD risk factors, or cytokine levels. METHODS: Fasting plasma samples were obtained from obese (BMI ≥ 95th percentile; n = 20) and age- and gender-matched healthy weight (BMI > 5th and < 85th percentile; n = 22) children and adolescents (mean age, 12 ± 3 years) to quantify XO activity. In addition, fasting cholesterol, insulin, glucose, blood pressure, endothelial function, and cytokine levels were assessed. RESULTS: We observed a 3.8-fold increase in plasma XO activity in obese, compared to healthy weight, children (118 ± 21 vs. 31 ± 9 nU/mg of protein; p < 0.001). Plasma XO activity was correlated with BMI z-score (r = 0.41), waist circumference (r = 0.41), high-density lipoprotein cholesterol (r = -0.32), oxidized low-density lipoprotein (r = 0.57), adiponectin (r = -0.53), and monocyte chemotactic protein-1 (r = -0.59). CONCLUSION: XO activity is highly elevated in obese children and correlates with CVD risk factors, suggesting that XO may play a role in increasing cardiovascular risk early in life in the context of obesity.