Michael V. Miles

High-fructose, medium chain trans fat diet induces liver fibrosis and elevates plasma coenzyme Q9 in a novel murine model of obesity and nonalcoholic steatohepatitis†

Diets high in saturated fat and fructose have been implicated in the development of obesity and nonalcoholic steatohepatitis (NASH) in humans. We hypothesized that mice exposed to a similar diet would develop NASH with fibrosis associated with increased hepatic oxidative stress that would be further reflected by increased plasma levels of the respiratory chain component, oxidized coenzyme Q9 (oxCoQ9). Adult male C57Bl/6 mice were randomly assigned to chow, high‐fat (HF), or high‐fat high‐carbohydrate (HFHC) diets for 16 weeks. The chow and HF mice had free access to pure water, whereas the HFHC group received water with 55% fructose and 45% sucrose (wt/vol). The HFHC and HF groups had increased body weight, body fat mass, fasting glucose, and were insulin‐resistant compared with chow mice. HF and HFHC consumed similar calories. Hepatic triglyceride content, plasma alanine aminotransferase, and liver weight were significantly increased in HF and HFHC mice compared with chow mice. Plasma cholesterol (P < 0.001), histological hepatic fibrosis, liver hydroxyproline content (P = 0.006), collagen 1 messenger RNA (P = 0.003), CD11b‐F4/80+Gr1+ monocytes (P < 0.0001), transforming growth factor β1 mRNA (P = 0.04), and α‐smooth muscle actin messenger RNA (P = 0.001) levels were significantly increased in HFHC mice. Hepatic oxidative stress, as indicated by liver superoxide expression (P = 0.002), 4‐hydroxynonenal, and plasma oxCoQ9 (P < 0.001) levels, was highest in HFHC mice. Conclusion: These findings demonstrate that nongenetically modified mice maintained on an HFHC diet in addition to developing obesity have increased hepatic ROS and a NASH‐like phenotype with significant fibrosis. Plasma oxCoQ9 correlated with fibrosis progression. The mechanism of fibrosis may involve fructose inducing increased ROS associated with CD11b+F4/80+Gr1+ hepatic macrophage aggregation, resulting in transforming growth factor β1–signaled collagen deposition and histologically visible hepatic fibrosis. (HEPATOLOGY 2010)

Bioequivalence of coenzyme Q10 from over-the-counter supplements

Abstract The objective of this study was to compare the relative bioavailability of two new products with solubilized and non-solubilized over-the-counter (OTC) coenzyme Q 10 products. Nine healthy adults were given single 180 mg doses of each coenzyme Q 10 formulation at two week intervals. A commercially-marketed, non-solubilized Q 10 powder formulation (product D) was only minimally absorbed, and was excluded from the analysis of data. ANOVA comparison of maximum plasma concentrations (C max ), time of maximum concentrations (t max ), areas under the concentration-time curves from times zero to 144 hours post dose (AUC 0-144h ), and areas under the concentration-time curves from times zero to infinity (AUC 0-∞ ) were not significantly different ( P > 0.05) between test products A (LiQ-10™) and B (Q-Nol™) and the reference product C (UbiQGel®). The upper limits of the 90% confidence intervals of the log-transformed ratios (A:C and B:C) of C max, AUC 0-144h, and AUC 0-∞ were >1.25 for both test products, but significant ( P 0-144h. The results of this study indicate that LiQ-10™ has increased bioequivalence compared to the reference product, but did not reach statistical significance. Q-Nol™ has increased bioavailability compared to the reference product ( P

Topiramate Pharmacokinetics in Infants

Summary: Purpose: This studys goal was to provide preliminary data on the pharmacokinetics of topiramate (TPM) in a cohort of infants (younger than 4 years) participating in an open‐label trial of TPM in refractory infantile spasms.

Automated microanalysis of gabapentin in human serum by high-performance liquid chromatography with fluorometric detection.

An automated high-performance liquid chromatographic method for the determination of gabapentin, 1-(amino-methyl)cyclohexaneacetic acid, in serum is described. The procedure involves protein precipitation with methanol followed by using a robotized derivatization with o-phthaldialdehyde reagent and automated high-performance liquid chromatography. The analog of gabapentin, 1-(aminomethyl)cycloheptaneacetic acid, was used as the internal standard. Blank serum was fortified with gabapentin (0.1-10.0 microg/ml) and internal standard. Separation was achieved on a Waters 5-microm reversed-phase column (10 cmx4.6 mm) with mobile phase consisting of 0.02 M phosphate buffer (pH 4.5)-acetonitrile (50:50, v/v). Eluents were monitored by fluorescence spectroscopy with excitation and emission wavelengths of 230 and 420 nm, respectively. The calibration curve for gabapentin in serum was linear (r=0.999) over the concentration range 0.1-10.0 microg/ml. The inter- and intraassay variations for three different gabapentin concentrations were < or =10% throughout. The lower limit of quantitation was found to be 0.1 microg/ml. Chromatography was unaffected by a range of commonly employed antiepileptic drugs or selected amino acids.

Plasma coenzyme Q10 reference intervals, but not redox status, are affected by gender and race in self-reported healthy adults.

BACKGROUND Abnormal concentrations of coenzyme Q(10) have been reported in many patient groups, including certain cardiovascular, neurological, hematological, neoplastic, renal, and metabolic diseases. However, controls in these studies are often limited in number, poorly screened, and inadequately evaluated statistically. The purpose of this study is to determine the reference intervals of plasma concentrations of ubiquinone-10, ubiquinol-10, and total coenzyme Q(10) for self-reported healthy adults. METHODS Adults (n=148), who were participants in the Princeton Prevalence Follow-up Study, were identified as healthy by questionnaire. Lipid profiles, ubiquinone-10, ubiquinol-10, and total coenzyme Q(10) concentrations were measured in plasma. The method used to determine the reference intervals is a procedure incorporating outlier detection followed by robust point estimates of the appropriate quantiles. RESULTS Significant differences between males and females were present for ubiquinol-10 and total coenzyme Q(10). Blacks had significantly higher Q(10) measures than whites in all cases except for the ubiquinol-10/total Q(10) fraction. CONCLUSIONS The fraction of ubiquinol-10/total coenzyme Q(10) is a tightly regulated measure in self-reported healthy adults, and is independent of sex and racial differences. Different reference intervals for certain coenzyme Q(10) measures may need to be established based upon sex and racial characteristics.

An improved gas chromatography assay for topiramate monitoring in pediatric patients.

An improved micromethod involving capillary gas chromatographic assay with liquid-liquid extraction and nitrogen phosphorus detection (GC/NPD) was developed and validated for the determination of topiramate (TPM) in human body fluids. The galactopyranose analog of TPM was used as the internal standard. Capillary gas chromatographic conditions yielded typical retention times of 6.8 min for TPM and 7.2 min for the internal standard. Calibrations were linear between 1.0 and 32 microg/mL. Between-day precision (n = 17) for three serum controls (3.0, 10, and 24.5 microg/mL) resulted in coefficients of variation of 6.9%, 7.3%, and 4.9%, respectively. The limit of detection was 0.42 microg/mL. There was an excellent linear correlation between the fluorescence-polarization immunoassay (FPIA) and GC/NPD determinations of 56 patient specimens (r2 = 0.981). Chromatograms showed no interfering peaks with the respective blank human samples or from many commonly prescribed drugs. Because of improved specificity and decreased sample volume requirements, this micromethod should be particularly useful for monitoring TPM therapy in pediatric patients, for patients with impaired renal function, and for research studies.

Valproate metabolites and hepatotoxicity in an epileptic population.

Summary: Idiosyncratic hepatotoxicity, although rare, is of major concern when one is treating patients with valproate (VPA). Several clinical criteria are associated with an increased risk of developing this complication, but more specific predictors are needed. It has been postulated that 4‐en‐ VPA or one of its further metabolites may be responsible for the hepatic toxicity and that under certain conditions the metabolism of VPA is shifted to this product. We postulated that measurement of serum concentrations of 4‐en‐VPA or another metabolite might be a simple technique that would be predictive of risk for developing idiosyncratic hepatotoxicity. Because this complication is rare, we chose to analyze our data by a multiple linear regression model, exploring associations between VPA or three of its metabolites and clinical risk factors for hepatotoxicity. 4‐en‐VPA correlated with older age and absence of encephalopathy. 4‐en‐VPA was only seen in patients receiving polytherapy; all patients were also receiving CBZ. 2‐en‐VPA correlated with poor nutritional status. We conclude that routine measurement of serum 4‐en‐VPA is unlikely to be a useful predictor of risk for developing fatal hepatotoxicity. Serum concentrations of 4‐en‐VPA may not reflect presence or effects in the liver as it may be metabolized to further intermediates or be bound to tissue. Thus, serum levels of 4‐en‐VPA do not reflect its important role in the pathogenesis of hepatotoxicity. This metabolite was détécted only in patients receiving polytherapy, a potent risk factor for developing this rare complication.

Intraindividual variability of carbamazepine, phenobarbital, and phenytoin concentrations in saliva.

Summary: The intraindividual variability of salivary carbamazepine (CBZ), phenobarbital (PB), and phenytoin (PHT) concentrations was studied in six healthy male volunteers. During three consecutive 1-week phases, subjects took one of the antiepileptic drugs (AED) for 5 days. Nine saliva and nine blood samples were collected simultaneously over the last 4 days of each phase. Salivary, unbound serum, and total serum CBZ, PB, and PHT concentrations were determined by fluorescence polarization immunoassay. Individual mean saliva (Cs)/total serum (Ct), unbound serum (Cu/Ct, and Cs/Cu concentration ratios for CBZ, PB, and PHT were similar to those reported previously. The intraindividual mean (SD) coefficients of variation (%) of the Cs/Ct, Cu/Ct, and Cs/Cu ratios for CBZ were 4.8 ± 2.2%, 4.4 ± 1.8%, and 5.0 ± 1.9%, respectively; for PB they were 5.1 ± 0.8%, 3.6 ± 0.9%, and 6.4 ± 1.0%, respectively; and for PHT they were 7.4 ± 3.1%, 5.5 ± 1.2%, and 8.3 ± 3.2%, respectively. The mean Cs/Ct and Cu/Ct ratios for CBZ and PHT were not significantly different, but they were different for PB (p = 0.01). However, the range of individual coefficients of variation of the Cs/Ct ratio for PB was 4.0–6.2%, which is acceptable for clinical monitoring. We conclude that the intraindividual variability of CBZ, PB, and PHT concentrations in saliva, based upon the saliva/serum concentration ratios of each AED, is not a factor that should dissuade clinicians from using saliva for the therapeutic monitoring of these agents.

Evaluation of the ames seralyzer for the determination of carbamazepine, phenobarbital, and phenytoin concentrations in saliva

The performance of the dry-phase apoenzyme reactivation immunoassay system (ARIS) for the measurement of carbamazepine (CBZ), phenobarbital (PB), and phenytoin (PHT) concentrations in saliva was compared with fluorescence polarization immunoassay (FPIA). Blood and saliva samples were collected from 163 adult and pediatric epilepsy patients, then analyzed using both methods. Regressions between ARIS saliva CBZ, PB, and PHT concentrations, and FPIA unbound and total serum concentrations were highly correlated, but the ARIS technique was somewhat less precise than the FPIA. Valproic acid co-medication did not affect the relationships between ARIS and FPIA saliva concentrations and unbound serum concentrations of PHT, but did disrupt the relationship between ARIS and FPIA saliva PHT and total serum PHT. The sensitivity, specificity, predicted value positive (PV+) of a therapeutic concentration, and predicted value negative (PV-) of a concentration outside the therapeutic range for the ARIS saliva technique compared very well with FPIA for CBZ, PB, and PHT. The ARIS technique for CBZ, PB, and PHT saliva determination provides acceptable accuracy, precision, and sensitivity for therapeutic monitoring. In practice, the benefits of the ARIS saliva technique, including ease of collection, safety, patient/parent acceptance, and short analysis time, are striking.

Topiramate concentration in saliva: an alternative to serum monitoring.

This study examines the relationship between serum and saliva topiramate concentrations, and attempts to determine if saliva may be a useful alternative to serum for therapeutic monitoring. Saliva and blood specimens were collected from 31 epilepsy patients (mean age 10.5 +/- 6.0 years; range 2.5 years to 24.8 years), and topiramate concentrations were determined by fluorescence polarization immunoassay. One patients results were omitted because the saliva concentration was below the limit of quantitation of the assay. A strong correlation exists between serum and saliva topiramate concentrations (adjusted r(2) = 0.97, n = 30, P < 0.0001). The mean fraction of saliva to serum concentration is 89.8% +/- 12.1% (range 62.9% to 112.7%). The results of this study support the use of saliva as a viable alternative to serum for monitoring topiramate therapy. Topiramate concentration in saliva: an alternative to serum monitoring.