Beverley Huet

The Effect of α-Tocopherol Supplementation on LDL Oxidation A Dose-Response Study

Abstract Because much data have accrued to support the concept that oxidatively modified LDL (Ox-LDL) can promote atherogenesis, the role of antioxidants in decreasing LDL oxidation has assumed great importance. High-dose α-tocopherol supplementation in humans decreases the susceptibility of LDL to oxidation. Hence, the aim of the present study was to ascertain the minimum dose of α-tocopherol that would decrease the susceptibility of LDL to oxidation. The effect of α-tocopherol in doses of 60, 200, 400, 800, and 1200 IU/d on copper-catalyzed LDL oxidation was tested in a randomized placebo-controlled study over 8 weeks. There were eight subjects in each group. Oxidation of LDL was monitored by measuring the formation of conjugated dienes and lipid peroxides by the thiobarbituric acid–reacting substances (TBARS) assay over an 8-hour time course at baseline and after 8 weeks of supplementation. Neither placebo nor any of the doses of α-tocopherol resulted in any side effects or exerted an adverse effect on the plasma lipoprotein profile. However, there was a dose-dependent increase in plasma and lipid-standardized α-tocopherol levels with increasing doses of α-tocopherol supplementation. LDL α-tocopherol appeared to follow a similar trend. When the time-course curves of LDL oxidation and the kinetics of LDL oxidation were examined, there was no significant effect at 8 weeks compared with baseline in the groups that received placebo or α-tocopherol 60 or 200 IU/d. However, in the groups that received at least 400 IU/d α-tocopherol, there was a decreased susceptibility of LDL to oxidation, as shown by the mean levels in the time-course curves, prolongation in the lag phase, and a decrease in the oxidation rate. Furthermore, both plasma and LDL α-tocopherol correlated significantly with the lag phase of oxidation and inversely with the oxidation rate. The results of the present study show that the minimum dose of α-tocopherol needed to significantly decrease the susceptibility of LDL to oxidation is 400 IU/d.

Increased Toll-Like Receptor Activity in Patients With Metabolic Syndrome

OBJECTIVE The metabolic syndrome (MetS) is highly prevalent and confers an increased risk for diabetes and cardiovascular disease (CVD). While MetS is a proinflammatory state, there is a paucity of data on cellular inflammation in MetS. Toll-like receptors (TLRs) are classical pattern recognition receptors of the innate immune response. RESEARCH DESIGN AND METHODS The aim of this study was to examine monocyte TLR2 and TLR4 in MetS patients without diabetes or CVD and control subjects since both of the receptors have been implicated in atherosclerosis and insulin resistance. Fasting blood was obtained for TLR expression and activity. RESULTS Circulating levels of high-sensitivity C-reactive protein, interleukin (IL)-1β, IL-6, IL-8, and soluble tumor necrosis factor receptor 1 (sTNFR1) were significantly increased in MetS versus control subjects following adjustment for waist circumference. There was a significant increase in both TLR2 and TLR4 surface expression and mRNA on monocytes after adjustment for waist circumference. In addition to increased nuclear factor-κB nuclear binding, there was significantly increased release of IL-1β, IL-6, and IL-8 in MetS versus control subjects following priming of the monocytes with lipopolysaccharides. While both plasma free fatty acids and endotoxin were increased in MetS, they correlated significantly with TLR4 only. CONCLUSIONS In conclusion, we make the novel observation that both TLR2 and TLR4 expression and activity are increased in the monocytes of patients with MetS and could contribute to increased risk for diabetes and CVD.

Relationship between insulin sensitivity, hyperinsulinemia, and insulin-mediated sympathetic activation in normotensive and hypertensive subjects

The adrenergic response to high physiological hyperinsulinemia was studied in 39 hypertensive subjects (28 men and 11 women) and 25 normal volunteers (15 men and 10 women), using the euglycemic clamp technique. Control studies using 0.45% saline infusions (sham studies) were also performed. Before and during the clamp procedure, plasma norepinephrine (NE) and epinephrine (E) were measured using a high performance liquid chromatographic method (HPLC). The association between the increment in NE and E levels and insulin sensitivity, steady-state insulin level during the clamps, waist to hip ratio (WHR), baseline NE levels and gender was studied. NE levels increased during the hyperinsulinemic period (mean increase 46 +/- 6 pmol P < .001 upsilon baseline and P < .01 upsilon sham studies). E levels did not differ between the insulin clamps and the sham studies. Insulin sensitivity was not significantly associated with the increment in NE. Hypertensive subjects had a higher NE increase than the normotensive individuals (55 +/- 7 upsilon 30 +/- 10 pmol, P = .03), but also had higher insulin levels during the clamps (839 +/- 43 upsilon 522 +/- 38 pmol, P < .001). Insulin levels accounted for most of the differences in NE increase between the normotensive and hypertensive groups. Gender, adiposity and WHR were also associated with NE increment. We conclude that the insulin mediated sympathetic activation is not affected in the presence of decreased insulin sensitivity for glucose utilization. The greater degree of sympathetic activation observed in hypertensive subjects is a function of the level of insulinemia obtained during the clamps.

Effects of increasing doses of alpha-tocopherol in providing protection of low-density lipoprotein from oxidation

In this study, we tested whether 1,200 IU/day of alpha-tocopherol was more potent than 400 and 800 IU of alpha-tocopherol in decreasing low-density lipoprotein (LDL) oxidative susceptibility in a 2-month study. The decrease in LDL oxidation was significantly greater with 1,200 IU/day than 400 IU/day.

Defining variation in urinary oxalate in hyperoxaluric stone formers.

BACKGROUND AND PURPOSE The development of effective preventive therapy for renal calculi in patients with secondary hyperoxaluria (2°HO) relies on establishing the pattern of normal variation in urinary oxalate (uOx) and attempting to reduce it. Therefore, we evaluated uOx at baseline and at subsequent time points in stone formers with 2°HO. METHODS We reviewed the charts of 201 recurrent stone formers with 2°HO (uOx ≥ 40 mg/day). The 24-hour urine collections at baseline and after initiation of clinician-directed therapies were analyzed. Mixed models were constructed to analyze uOx over time for individual patients and as a group. Subgroup analyses were performed for enteric and idiopathic 2°HO. Coefficients of variation were computed using the root mean square error from linear models. RESULTS The etiology of 2°HO was enteric in 17.9% and idiopathic in 82.1% of patients. Among the 943 urine collections analyzed, 196 oxalate values were derived from the enteric group and 747 from the idiopathic group. The median number of uOx values measured per person was four. The median 24-hour uOx (mg/day) was significantly higher for the enteric group than for the idiopathic group at the time of diagnosis: 64.4 (interquartile range [IQR]=48-90) vs 46.0 (IQR=38-56), P<0.001) and during follow-up (58.2 [IQR=46-86] vs 44.2 [IQR=35-53], P<0.001). Over a median follow-up of 22.5 months, 44.4% of the enteric and 61.8% of the idiopathic patients had at least one normal uOx value (P=0.06). The coefficients of variation for the enteric and idiopathic groups were 40.8% and 27.3%, respectively, with variation randomly displayed in either direction for both groups. CONCLUSIONS Among patients with 2°HO, uOx demonstrates significant random variation over time even with the incorporation of standard treatments, with enteric HO demonstrating higher values and greater variance than idiopathic HO.

Variability in urinary oxalate measurements between six international laboratories

BACKGROUND Hyperoxaluria is a major risk factor for kidney stone formation. Although urinary oxalate measurement is part of all basic stone risk assessment, there is no standardized method for this measurement. METHODS Urine samples from 24-h urine collection covering a broad range of oxalate concentrations were aliquoted and sent, in duplicates, to six blinded international laboratories for oxalate, sodium and creatinine measurement. In a second set of experiments, ten pairs of native urine and urine spiked with 10 mg/L of oxalate were sent for oxalate measurement. Three laboratories used a commercially available oxalate oxidase kit, two laboratories used a high-performance liquid chromatography (HPLC)-based method and one laboratory used both methods. RESULTS Intra-laboratory reliability for oxalate measurement expressed as intraclass correlation coefficient (ICC) varied between 0.808 [95% confidence interval (CI): 0.427-0.948] and 0.998 (95% CI: 0.994-1.000), with lower values for HPLC-based methods. Acidification of urine samples prior to analysis led to significantly higher oxalate concentrations. ICC for inter-laboratory reliability varied between 0.745 (95% CI: 0.468-0.890) and 0.986 (95% CI: 0.967-0.995). Recovery of the 10 mg/L oxalate-spiked samples varied between 8.7 ± 2.3 and 10.7 ± 0.5 mg/L. Overall, HPLC-based methods showed more variability compared to the oxalate oxidase kit-based methods. CONCLUSIONS Significant variability was noted in the quantification of urinary oxalate concentration by different laboratories, which may partially explain the differences of hyperoxaluria prevalence reported in the literature. Our data stress the need for a standardization of the method of oxalate measurement.

Changes to trimethylamine-N-oxide and its precursors in nascent metabolic syndrome

Abstract Background Metabolic syndrome (MetS), a cardio-metabolic cluster afflicting 35% of American adults, increases cardiovascular disease (CVD) and type-2 diabetes (T2DM) risk. Increased levels of trimethylamine N-oxide (TMAO), a metabolite derived from choline and L-carnitine, correlates with CVD and T2DM. However, the precise role of TMAO and its precursors in MetS remains unclear. We tested the hypothesis that choline, L-carnitine and TMAO in MetS patients without CVD or T2DM would be altered and correlate with inflammatory markers. Materials and methods This was an exploratory study of 30 patients with nascent MetS (without CVD or T2DM) and 20 matched controls. MetS was defined by the Adult Treatment Panel III criteria. TMAO and its precursors were evaluated from each patient’s frozen early morning urine samples and quantified using liquid chromatography/mass spectrometry (LC-MS). These amines were correlated with a detailed repertoire of biomarkers of inflammation and adipokines. Results L-carnitine was significantly increased (p = 0.0002) compared to controls. There was a trend for a significant increase in TMAO levels (p = 0.08). Choline was not significantly altered in MetS. L-carnitine correlated significantly with soluble tumor necrosis factor 1 (sTNFR1) and leptin, and inversely to adiponectin. TMAO correlated with IL-6, endotoxin and chemerin. Neither choline, nor L-carnitine significantly correlated with TMAO. Conclusion L-carnitine is directly correlated with markers of inflammation in nascent MetS. Cellular L-carnitine could be a biomediator or marker of inflammation in the pathogenesis of MetS, and the sequelae of CVD and T2DM.

MP90-14 DOES SATURATION INDEX PREDICT STONE ACTIVITY IN PATIENTS WITH CALCIUM OXALATE NEPHROLITHIASIS?

INTRODUCTION AND OBJECTIVES: Urinary magnesium has been shown previously to inhibit kidney stone formation in chemical models however, when applied to in vivo human models the results have been conflicting. The purpose of this study is to investigate the timing of magnesium supplementation on the inhibitory effect on nephrolithiasis. We hypothesize that if magnesium is taken with meals, more will be absorbed in the small intestine and excreted in the kidney to allow for better inhibitory effect, specifically by reducing oxalate excretion. METHODS: We prospectively enrolled known calcium oxalate stone formers with isolated hyperoxaluria identified on 24 hour stone risk testing. Patients were then randomized to take magnesium supplementation either fasting or with food. An initial 24 hour urine collection was obtained on enrollment and then repeated after 7 days of magnesium supplementation to determine the effect on urinary excretion of oxalate. Participants were given a controlled diet during the 7 days of intervention which included adequate fluid intake, low oxalate, low salt, moderate animal protein, and normal calcium intake e the standard dietary treatment for hyperoxaluric kidney stone patients. RESULTS: Seven patients were enrolled with 3 patients randomized to each arm of magnesium supplementation. Those taking it with food experienced 41.2mg/d increase in urinary magnesium and a 25.2mg/d decrease in their urinary oxalate over the course of 7 days as compared to a 14.3mg/d increase in magnesium and 13.7mg/d decrease in citrate for those taking magnesium while fasting. There were only modest decreases in calcium oxalate supersaturation and calcium but profound increases in stone protective factors like citrate (Table). Secondary endpoints including sodium (decrease 53mg/d with food vs 84mg/d fasting) also showed improvement with little difference between groups. CONCLUSIONS: Those taking magnesium supplementation with food experienced a more than 3 fold increase in urinary magnesium and twice the reduction in urinary oxalate as those who took it while fasting. Additionally, secondary endpoints like citrate and sodium showed improvement with modest differences between groups. Our pilot study supports the need for further investigation with a larger sample to establish the significance of these trends.

MP90-13 24 HOUR URINE UTILIZATION IN NEPHROLITHIASIS TREATMENT: RESULTS FROM M-STONE (MULTI-CENTER COLLABORATION TO STUDY TREATMENT OUTCOMES IN NEPHROLITHIASIS EVALUATION)

INTRODUCTION AND OBJECTIVES: Urinary magnesium has been shown previously to inhibit kidney stone formation in chemical models however, when applied to in vivo human models the results have been conflicting. The purpose of this study is to investigate the timing of magnesium supplementation on the inhibitory effect on nephrolithiasis. We hypothesize that if magnesium is taken with meals, more will be absorbed in the small intestine and excreted in the kidney to allow for better inhibitory effect, specifically by reducing oxalate excretion. METHODS: We prospectively enrolled known calcium oxalate stone formers with isolated hyperoxaluria identified on 24 hour stone risk testing. Patients were then randomized to take magnesium supplementation either fasting or with food. An initial 24 hour urine collection was obtained on enrollment and then repeated after 7 days of magnesium supplementation to determine the effect on urinary excretion of oxalate. Participants were given a controlled diet during the 7 days of intervention which included adequate fluid intake, low oxalate, low salt, moderate animal protein, and normal calcium intake e the standard dietary treatment for hyperoxaluric kidney stone patients. RESULTS: Seven patients were enrolled with 3 patients randomized to each arm of magnesium supplementation. Those taking it with food experienced 41.2mg/d increase in urinary magnesium and a 25.2mg/d decrease in their urinary oxalate over the course of 7 days as compared to a 14.3mg/d increase in magnesium and 13.7mg/d decrease in citrate for those taking magnesium while fasting. There were only modest decreases in calcium oxalate supersaturation and calcium but profound increases in stone protective factors like citrate (Table). Secondary endpoints including sodium (decrease 53mg/d with food vs 84mg/d fasting) also showed improvement with little difference between groups. CONCLUSIONS: Those taking magnesium supplementation with food experienced a more than 3 fold increase in urinary magnesium and twice the reduction in urinary oxalate as those who took it while fasting. Additionally, secondary endpoints like citrate and sodium showed improvement with modest differences between groups. Our pilot study supports the need for further investigation with a larger sample to establish the significance of these trends.