Donald A. Wiebe

Purple Grape Juice Improves Endothelial Function and Reduces the Susceptibility of LDL Cholesterol to Oxidation in Patients With Coronary Artery Disease

BACKGROUND In vitro, the flavonoid components of red wine and purple grape juice are powerful antioxidants that induce endothelium-dependent vasodilation of vascular rings derived from rat aortas and human coronary arteries. Although improved endothelial function and inhibition of LDL oxidation may be potential mechanisms by which red wine and flavonoids reduce cardiovascular risk, the in vivo effects of grape products on endothelial function and LDL oxidation have not been investigated. This study assessed the effects of ingesting purple grape juice on endothelial function and LDL susceptibility to oxidation in patients with coronary artery disease (CAD). METHODS AND RESULTS Fifteen adults with angiographically documented CAD ingested 7.7+/-1.2 mL. kg(-1). d(-1) of purple grape juice for 14 days. Flow-mediated vasodilation (FMD) was measured using high-resolution brachial artery ultrasonography. Susceptibility of LDL particles to oxidation was determined from the rate of conjugated diene formation after exposure to copper chloride. At baseline, FMD was impaired (2.2+/-2. 9%). After ingestion of grape juice, FMD increased to 6.4+/-4.7% (P=0.003). In a linear regression model that included age, artery diameter, lipid values, and use of lipid-lowering and antioxidant therapies, the effect of grape juice on FMD remained significant (mean change 4.2+/-4.4%, P<0.001). After ingestion of grape juice, lag time increased by 34.5% (P=0.015). CONCLUSIONS Short-term ingestion of purple grape juice improves FMD and reduces LDL susceptibility to oxidation in CAD patients. Improved endothelium-dependent vasodilation and prevention of LDL oxidation are potential mechanisms by which flavonoids in purple grape products may prevent cardiovascular events, independent of alcohol content.

Use of Human Immunodeficiency Virus-1 Protease Inhibitors Is Associated With Atherogenic Lipoprotein Changes and Endothelial Dysfunction

Background—Human immunodeficiency virus protease inhibitors (HIV PIs) are associated with hyperlipidemia, hyperglycemia, and obesity; however, it is not known whether they increase risk of atherosclerotic vascular disease. The purposes of this study were to characterize the lipoprotein abnormalities associated with use of HIV PIs in individuals with HIV infection and to determine the pathophysiological significance of these changes by assessing their effect on endothelial dysfunction. Methods and Results—This was a cross-sectional study of 37 adults with HIV-1 infection who were receiving antiretroviral therapy. Twenty-two were taking HIV PIs (group 1); 15 were not (group 2). Lipids and lipoproteins were measured by enzymatic techniques and nuclear magnetic resonance spectroscopic analysis. Flow-mediated vasodilation (FMD) of the brachial artery was measured by high-resolution ultrasound. Subjects in both groups were similar in regard to age, time since diagnosis of HIV infection, and CD4 cell count. Group 1 subjects had higher total cholesterol (5.68 versus 4.42 mmol/L, P =0.007) and triglyceride (4.43 versus 1.98 mmol/L, P =0.009) levels, characterized by elevated levels of IDL and VLDL. Subjects in group 1 had impaired FMD (2.6±4.6%), indicative of significant endothelial dysfunction. Group 2 subjects had normal FMD (8.1±6.7%, P =0.005). In group 1, chylomicron, VLDL, IDL, and HDL cholesterol levels predicted FMD. Conclusions—Use of HIV PIs is associated with atherogenic lipoprotein changes and endothelial dysfunction. Because these metabolic and vascular changes predispose to atherosclerosis, monitoring and treatment of dyslipidemia in patients taking these medications is warranted.

Effect of ingestion of purple grape juice on endothelial function in patients with coronary heart disease

T “French Paradox” refers to the observation that the coronary heart disease mortality rate is lower in France than in other industrialized countries with similar prevalences of coronary risk factors.1,2 This paradox has been attributed to frequent consumption of alcohol-containing beverages, which increase highdensity lipoprotein (HDL) cholesterol levels and inhibit platelet function.1–7 Several epidemiologic studies suggest that ingestion of red wine, which contains several hundred different types of flavonoids, is more cardioprotective than beer or spirits.1–4 Indeed, the flavonoids found in red wine and purple grape juice (GJ) also inhibit platelet aggregation, and in 1 study, were shown to be powerful antioxidants that improved endothelial function.8–10 In that study, however, most subjects were taking vitamin E, so it is unclear if the observed results were due solely to the flavonoids in GJ or a combination of vitamin E and GJ.10 Furthermore, a high dose of GJ was administered (approximately 8 ml/kg/day) for only 2 weeks. The purpose of this study was to assess the endothelial function and antioxidant effects of 2 doses of purple GJ alone and in combination with vitamin E for 8 weeks. • • • The institutional review board of the University of Wisconsin Medical School approved this study. All subjects provided informed consent before participation. Twenty-two adults with angiographically documented coronary artery disease were recruited for this study. Subjects were not allowed to take vitamin supplements for 4 weeks before enrollment or during this study, except as prescribed by the research protocol. Subjects also were prohibited from consuming fruit products, tea, or alcoholic beverages during this study. Medications could not be changed during the study. All subjects ingested purple GJ (Welch’s 100% Concord Grape, Concord, Massachusetts) for 56 days. The first 11 subjects were instructed to drink 8.0 ml/kg of GJ, twice daily. For an average 80-kg person, this was approximately 640 ml/day (21 ounces) of GJ, which contained approximately 112 g of carbohydrate. The next 11 subjects were instructed to drink 4.0 ml/kg of purple GJ, once daily (low-dose group). After 28 days, subjects added vitamin E (d,l-a-tocopheryl) 400 IU to their daily intake of GJ. Subjects’ diaries indicated .90% compliance with GJ and vitamin E. Endothelial function was evaluated by measuring flow-mediated vasodilation (FMD) of the brachial artery using B-mode ultrasound. Studies were performed at baseline and at subsequent visits on the morning of phlebotomy, after a 12-hour fast. Subjects were instructed to drink their daily dose of GJ at least 2 hours before testing. Brachial artery diameters and blood flows were measured with a 7.5-MHz linear array vascular ultrasound transducer and an Agilent Technologies 5500 Sonos ultrasound system (Palo Alto, California). Increased forearm blood flow was induced by inflating a blood pressure tourniquet around the widest part of the forearm to a systolic blood pressure of 250 mm Hg for 4.5 minutes. Repeat brachial artery diameter and blood flow scans were obtained immediately and 1 minute after deflation of the tourniquet. Resting brachial artery diameter and blood flow scans were repeated 15 minutes later. Sublingual nitroglycerin (400 mg) was administered and final scans were performed after 3 minutes. The brachial artery was imaged in longitudinal sections 2 to 15 cm above the elbow. Images were recorded using the digital storage and retrieval software of the ultrasound system. Vessel diameters were measured in triplicate using digital calipers (Freeland Systems, Westfield, Indiana). Measurements were performed and interpreted by investigators who were blinded to subject information and study date. The brachial artery diameter was measured at end-diastole, using intima-media interfaces, or if they could not be visualized, media-adventitia interfaces, as landmarks. FMD was calculated as the ratio of the brachial artery diameter after reactive hyperemia to the baseline diameter, expressed as a percent change. Nitroglycerinmediated vasodilation was calculated in an analogous fashion. In this laboratory, intraobserver reliability for measurement of the brachial artery diameter is 0.987, reflecting an interclass correlation coefficient across all readings and conditions.10 Lipid and glucose levels were measured using enzymatic techniques on a Hitachi 747 analyzer (Tokyo, Japan) using standard reagents (Roche, Mannheim, Germany). Insulin levels were measured by radioimmunoassay. Low-density lipoprotein (LDL) particles were isolated from serum by sequential density ultracentrifugation between densities of 1.006 and 1.063 g/ml using a Beckman Optima ultracentrifuge (Fullerton, California) at 100,000 rpm (.400,000 g). The LDL-containing fraction was desalted with a 2-ml column of preswollen 12% cellulose and 0.1 mol/L From the University of Wisconsin Medical School, Madison, Wisconsin. This study was supported by an unrestricted grant from Welch’s Foods, Inc., Concord, Massachusetts. Dr. Stein’s address is: Section of Cardiovascular Medicine, University of Wisconsin Medical School, 600 Highland Avenue, H6/315 CSC (MC 3248), Madison, Wisconsin 53792. E-mail: jhs@medicine.wisc.edu. Manuscript received February 21, 2001; revised manuscript received and accepted April 10, 2001.

The C-3 Epimer of 25-Hydroxyvitamin D3 Is Present in Adult Serum

CONTEXT Epimers have identical molecular structure but differ in stereochemical configuration. It is widely believed that the C-3 epimer of 25-hydroxyvitamin D(3) [3-epi-25(OH)D(3)] is found only in neonates. However, this epimer was recently detected in a limited number of adults. The physiological importance of 3-epi-25(OH)D(3) is uncertain but might affect 25-hydroxyvitamin D test results and thereby reliability of the 25-hydroxyvitamin D(3) [25(OH)D(3)] measurement. OBJECTIVE This project describes development of a highly sensitive method for 3-epi-25(OH)D(3) measurement and establishes the prevalence of this epimer in adult clinical serum specimens. DESIGN, SETTING, PARTICIPANTS, AND MAIN OUTCOME MEASURE: Serum 25(OH)D(3), 3-epi-25(OH)D(3), and 25(OH)D(2) concentrations were determined in a cohort of patients (n = 214; age neonate to 80+ yr). High-performance liquid chromatography with ultraviolet detection and high-performance liquid chromatography tandem mass spectrometry with atmospheric pressure chemical ionization equipped with cyanopropyl analytical columns were used to baseline separate and quantitate 25(OH)D(3), 3 epi-25(OH)D(3), and 25(OH)D(2). RESULTS The C-3 epimer was detected in 212 of 214 (99%) of samples. Concentrations ranged from 1 to 93 ng/ml for 25(OH)D(3) and 0.1 to 23.7 ng/ml for 3-epi-25(OH)D(3). The relative amounts of epimer to 25(OH)D(3) ranged from 0 to 25.5% (mean 4.75%). The epimer amount increased as 25(OH)D(3) increased in a nonlinear mode. In sera with approximately the same 25(OH)D(3) concentration, the ratio of epimer to 25(OH)D(3) varied, e.g. at 25(OH)D(3) values of 20-22 ng/ml, the ratio varied from 2-8.5%. CONCLUSION 3-Epi-25(OH)D(3) is present in the majority of human serum specimens. Although this concentration is generally low, further work must investigate the impact of 3-epi-25(OH)D(3) on the various 25-hydroxyvitamin D assays and ultimately what information, if any, C-3 epimer measurement can provide clinically.

NHANES Monitoring of Serum 25-Hydroxyvitamin D: A Roundtable Summary

A roundtable to discuss monitoring of serum 25-hydroxyvitamin D [25(OH)D] in the NHANES was held in late July 2009. Topics included the following: 1) options for dealing with assay fluctuations in serum 25(OH)D in the NHANES conducted between 1988 and 2006; 2) approaches for transitioning between the RIA used in the NHANES between 1988 and 2006 to the liquid chromatography tandem MS (LC-MS/MS) measurement procedure to be used in NHANES 2007 and later; 3) approaches for integrating the recently available standard reference material for vitamin D in human serum (SRM 972) from the National Institute of Standards and Technology (NIST) into the NHANES; 4) questions regarding whether the C-3 epimer of 25-hydroxyvitamin D3 [3-epi-25(OH)D3] should be measured in NHANES 2007 and later; and 5) identification of research and educational needs. The roundtable experts agreed that the NHANES data needed to be adjusted to control for assay fluctuations and offered several options for addressing this issue. The experts suggested that the LC-MS/MS measurement procedure developed by NIST could serve as a higher order reference measurement procedure. They noted the need for a commutability study for the recently released NIST SRM 972 across a range of measurement procedures. They suggested that federal agencies and professional organizations work with manufacturers to improve the quality and comparability of measurement procedures across all laboratories. The experts noted the preliminary nature of the evidence of the 3-epi-25(OH)D3 but felt that it should be measured in 2007 NHANES and later.

Current Status of Clinical 25-hydroxyvitamin D Measurement: An Assessment of Between-Laboratory Agreement

BACKGROUND Historically, methodological differences and lack of standardization led to between-laboratory variability in 25(OH)D results. Recent observations raised concern about persisting variability. This quality assurance exercise investigated 25(OH)D result comparability between laboratories. METHODS Serum pools (n=25) were prepared to contain endogenous 25(OH)D(2) and 25(OH)D(3) at 25(OH)D concentrations from ~12 to 150 nmol/l (5-60 ng/ml). Aliquots were sent to 8 laboratories utilizing various 25(OH)D assay methods including high performance liquid chromatography with ultraviolet detection (LC-UV), LC with tandem mass spectroscopy detection (LC-MS/MS) or an automated immunoassay (Diasorin Liaison). The LC-UV results were selected as a referent to which all others were compared using linear regression and Bland-Altman analysis. RESULTS Good correlation (R(2)=0.87 to 0.97) was observed for all laboratories. Modest systematic bias was observed for some laboratories ranging from a positive mean bias of 10.5 nmol/l (4.2 ng/ml) to a negative mean bias of 3.5 nmol/l (1.4 ng/ml). For the laboratory with the greatest bias, 22/25 results were numerically higher (mean +15.7%) than LC-UV results. For Liaison, the primary error was likely random, whereas the major LC-MS/MS assay error source was biases likely due to calibration issues. CONCLUSIONS Modest inter-laboratory variability persists in serum 25(OH)D measurement. The National Institute of Standards and Technology 25(OH)D Standard Reference and calibration materials will further improve between-laboratory agreement for chromatography-based assays.

Optimized high-performance liquid chromatographic method for determination of lamotrigine in serum with concomitant determination of phenytoin, carbamazepine, and carbamazepine epoxide.

Lamotrigine (LG), phenytoin (PY), carbamazepine (CM), and carbamazepine epoxide (CE) are measured with an optimized procedure that uses thin sorbent extraction disks and a highly selective, sterically protected bonded silica high-performance liquid chromatography (HPLC) column. Routinely, serum (200 microliters at pH 6.8 with cyheptamide as internal standard) is applied to an Empore octyl (C8) solid-phase extraction disk to isolate the drugs. a water wash removes interferences, and the retained drugs are eluted with a small volume of solvent. The eluate is directly injected onto a Zorbax Stable Bond cyanopropyl HPLC column with quantification at 214 nm. Evaporation-concentration steps are unnecessary. Overall, for all drugs, between-run precision coefficients of variation (n = 16 each) ranged from 2.1% to 4.9% at concentrations from 0.75 to 20.5 mg/l; extraction recoveries fell within a range of 96% to 110% at concentrations of 2, 10, and 30 mg/l tested for each drug; the lowest limit of detection was 0.15 to 0.35 mg/l. The analytical response was linear for each drug > 80 mg/l (LG) and > 50 mg/l (PY, CM, and CE). Optimization graphs are presented to illustrate the rationale for selection of test parameters for a robust method. In addition, a comparison study between two commercial laboratories demonstrates accuracy problems associated with LG testing.

Optimized method for determination of gabapentin in serum by high-performance liquid chromatography

The anticonvulsant drug gabapentin and its heptaneacetic acid analog—used here as an internal standard—are isolated from serum (pH 9) with an octyldecyl (C-18) solid-phase sorbent column. To enhance analytical detection, trinitrobenzene derivatives of these extracted compounds are prepared quickly within 10 min. To furthur improve chromatographic selectivity, the derivatives are concentrated on a thin C-18 solid-phase membrane and interferences are washed away. The retained purified derivatives are eluted from the membrane with a small volume of solvent and the eluate is directly injected onto an Ultrasphere C-18 high-performance liquid chromatography column with quantification at 340 nm. No evaporation-concentration steps are necessary. Recoveries (extraction) of gabapentin and the internal standard are 94.2 ± 2.9% and 98 ± 2.0%, respectively. Analytical responses are linear from lower limit of sensitivity of 0.05 mg/L up to at least 10 mg/L. Between-run coefficients of variation (CV) range from 2.3 to 2.9% through the concentration range 0.5–4.0 mg/L. To illustrate the rationale for selection of test parameters for a robust method, we present optimization graphs for these processes. Moreover, we discuss the advantage of the packed cartridge and membrane sorbents as companion extraction devices.

Development of a sensitive LC/MS/MS method for vitamin D metabolites: 1,25 Dihydroxyvitamin D2&3 measurement using a novel derivatization agent.

Active vitamin D metabolites 1,25-dihydroxyvitamin D2 [1,25-(OH)2-D2; derived from ergocalciferol] and D3 [1,25-(OH)2-D3; derived from cholecalciferol] are found in low levels in the circulation and require a very sensitive method for measurement. Radioimmunoassay (RIA) has been the method of choice, but it lacks the specificity needed to distinguish between 1,25-(OH)2-D2 and -D3, whereas liquid chromatography-tandem mass spectrometry (LC/MS/MS) methods have the advantage of high specificity and sensitivity. Here, we compare a new derivative for ionizing 1,25-(OH)2-D to enhance the signal and provide the most sensitive assay for measuring vitamin D. We used the Amplifex diene method of derivatizing prior to LC/MS/MS and compared it to the standard RIA method and the 4-phenyl-1,2,4-triazole-3,5-dione (PTAD) method of derivatizing prior to LC/MS/MS. In the evaluation of 20 human serum samples, all methods correlated strongly across the upper levels of the standard 1,25-(OH)2-D2 and -D3 ranges (Amplifex and RIA, pc=0.97; Amplifex and PTAD, pc=0.96) but less strongly on the lower levels of the standard range (Amplifex and RIA, pc=0.81; Amplifex and PTAD, pc=0.65) suggesting differences in the sensitivities between the assays. The Amplifex method was determined to be more sensitive than the PTAD method, as peak areas were significantly higher for the Amplifex method and provided for a 10 fold higher signal-to-noise ratio than PTAD. Therefore, the Amplifex LC/MS/MS method is the most sensitive and specific method available for measuring 1,25-(OH)2-D2 and -D3 while using the smallest sample volume.

Plasma lipoprotein changes in humans induced by β-interferon

Abstract Two groups of patients were administered either 4.5 × 10 6 U or 90 × 10 6 U each of recombinant DNA-derived interferon-β serine (IFN-β ser ) i.v. daily for 10 days. IFN-β ser affected lipoprotein lipids of patients in a dose dependent fashion. A decrease in plasma total cholesterol concentration occurred 24 h after therapy was initiated, regardless of dose. A dose-related decrease in plasma cholesterol concentration of 9% and 23% for patients on the low dose and high dose respectively occurred after 9 days of therapy. The plasma total cholesterol concentration decrease resulted primarily from a decrease in LDL cholesterol of 28% and 50% for patients on low and high doses respectively of IFN-β ser . HDL-cholesterol was not significantly affected by IFN-β ser administration. A dose-related increase in plasma triglyceride concentration occurred during IFN-β ser , increasing 74% for patients on low dose and 136% for patients on high doses. This increase was only observed after 9 days on IFN-β ser . Cholesterol reduction and triglyceride increases followed different time courses indicating different mechanisms may be involved.