Samar Basu

Use of Multiple Biomarkers to Improve the Prediction of Death from Cardiovascular Causes

BACKGROUND The incremental usefulness of adding multiple biomarkers from different disease pathways for predicting the risk of death from cardiovascular causes has not, to our knowledge, been evaluated among the elderly. METHODS We used data from the Uppsala Longitudinal Study of Adult Men (ULSAM), a community-based cohort of elderly men, to investigate whether a combination of biomarkers that reflect myocardial cell damage, left ventricular dysfunction, renal failure, and inflammation (troponin I, N-terminal pro-brain natriuretic peptide, cystatin C, and C-reactive protein, respectively) improved the risk stratification of a person beyond an assessment that was based on the established risk factors for cardiovascular disease (age, systolic blood pressure, use or nonuse of antihypertensive treatment, total cholesterol, high-density lipoprotein cholesterol, use or nonuse of lipid-lowering treatment, presence or absence of diabetes, smoking status, and body-mass index). RESULTS During follow-up (median, 10.0 years), 315 of the 1135 participants in our study (mean age, 71 years at baseline) died; 136 deaths were the result of cardiovascular disease. In Cox proportional-hazards models adjusted for established risk factors, all of the biomarkers significantly predicted the risk of death from cardiovascular causes. The C statistic increased significantly when the four biomarkers were incorporated into a model with established risk factors, both in the whole cohort (C statistic with biomarkers vs. without biomarkers, 0.766 vs. 0.664; P<0.001) and in the group of 661 participants who did not have cardiovascular disease at baseline (0.748 vs. 0.688, P=0.03). The improvement in risk assessment remained strong when it was estimated by other statistical measures of model discrimination, calibration, and global fit. CONCLUSIONS Our data suggest that in elderly men with or without prevalent cardiovascular disease, the simultaneous addition of several biomarkers of cardiovascular and renal abnormalities substantially improves the risk stratification for death from cardiovascular causes beyond that of a model that is based only on established risk factors.

Carbon tetrachloride-induced lipid peroxidation: eicosanoid formation and their regulation by antioxidant nutrients

Hepatic injury through carbon tetrachloride (CCl(4)) induced lipid peroxidation is well known and has been extensively used in the experimental models to understand the cellular mechanisms behind oxidative damage and further to evaluate the therapeutic potential of drugs and dietary antioxidants. Many of these methods that have often been used to study free-radical induced lipid peroxidation suffer methodological discrepancies when considering the measurements in vivo. Recent discovery of isoprostanes, non-enzymatically derived prostaglandin F(2)-like compounds, unfolded a new era of determination of oxidant stress in vivo. Cyclooxygenase (COX) catalysed prostaglandins formation from arachidonic acid and their involvement in inflammation is well known. This review mainly focuses on the formation of non-enzymatically and enzymatically catalysed eicosanoids, namely the isoprostanes and prostaglandin F(2alpha) (PGF(2alpha)), following CCl(4) treatment in animals and their regulation by antioxidants. Both eicosanoids are increased dramatically in the peripheral plasma, urine and/or liver tissues but with diverse kinetics of formation, release and excretion pattern. Free radical and COX-mediated oxidation of arachidonic acid products are intimately associated with experimental hepatotoxicity. Studies suggest that there is a link between initial involvement of oxidative stress and subsequent induction of the COX mediated inflammatory process, which may have an eminent role in the pathogenesis of liver diseases. Antioxidant nutrients have been shown to affect both the formation of isoprostanes and prostaglandin metabolite but the therapeutic values and exact mechanisms of action remain unclear.

Supplementation With Conjugated Linoleic Acid Causes Isomer-Dependent Oxidative Stress and Elevated C-Reactive Protein: A Potential Link to Fatty Acid-Induced Insulin Resistance.

Background—Conjugated linoleic acids (CLAs), a group of fatty acids shown to have beneficial effects in animals, are also used as weight loss supplements. Recently, we reported that the t 10 c 12 CLA-isomer caused insulin resistance in abdominally obese men via unknown mechanisms. The aim of the present study was to examine whether CLA has isomer-specific effects on oxidative stress or inflammatory biomarkers and to investigate the relationship between these factors and induced insulin resistance. Methods and Results—In a double-blind placebo-controlled trial, 60 men with metabolic syndrome were randomized to one of 3 groups receiving t 10 c 12 CLA, a CLA mixture, or placebo for 12 weeks. Insulin sensitivity (euglycemic clamp), serum lipids, in vivo lipid peroxidation (determined as urinary 8-iso-PGF2&agr; [F2-isoprostanes]), 15-ketodihydro PGF2&agr;, plasma vitamin E, plasma C-reactive protein, tumor necrosis factor-&agr;, and interleukin-6 were assessed before and after treatment. Supplementation with t 10 c 12 CLA markedly increased 8-iso-PGF2&agr; (578%) and C-reactive protein (110%) compared with placebo (P <0.0001 and P <0.01, respectively) and independent of changes in hyperglycemia or dyslipidemia. The increases in 8-iso-PGF2&agr;, but not in C-reactive protein, were significantly and independently related to aggravated insulin resistance. Oxidative stress was related to increased vitamin E levels, suggesting a compensatory mechanism. Conclusions—t 10 c 12 CLA supplementation increases oxidative stress and inflammatory biomarkers in obese men. The oxidative stress seems closely related to induced insulin resistance, suggesting a link between the fatty acid-induced lipid peroxidation seen in the present study and insulin resistance. These unfavorable effects of t 10 c 12 CLA might be of clinical importance with regard to cardiovascular disease, in consideration of the widespread use of dietary supplements containing this fatty acid.

Activation of Peroxisome Proliferator–Activated Receptor (PPAR)δ Promotes Reversal of Multiple Metabolic Abnormalities, Reduces Oxidative Stress, and Increases Fatty Acid Oxidation in Moderately Obese Men

OBJECTIVE— Pharmacological use of peroxisome proliferator–activated receptor (PPAR)δ agonists and transgenic overexpression of PPARδ in mice suggest amelioration of features of the metabolic syndrome through enhanced fat oxidation in skeletal muscle. We hypothesize a similar mechanism operates in humans. RESEARCH DESIGN AND METHODS— The PPARδ agonist (10 mg o.d. GW501516), a comparator PPARα agonist (20 μg o.d. GW590735), and placebo were given in a double-blind, randomized, three-parallel group, 2-week study to six healthy moderately overweight subjects in each group. Metabolic evaluation was made before and after treatment including liver fat quantification, fasting blood samples, a 6-h meal tolerance test with stable isotope fatty acids, skeletal muscle biopsy for gene expression, and urinary isoprostanes for global oxidative stress. RESULTS— Treatment with GW501516 showed statistically significant reductions in fasting plasma triglycerides (−30%), apolipoprotein B (−26%), LDL cholesterol (−23%), and insulin (−11%), whereas HDL cholesterol was unchanged. A 20% reduction in liver fat content (P < 0.05) and 30% reduction in urinary isoprostanes (P = 0.01) were also observed. Except for a lowering of triglycerides (−30%, P < 0.05), none of these changes were observed in response to GW590735. The relative proportion of exhaled CO2 directly originating from the fat content of the meal was increased (P < 0.05) in response to GW501516, and skeletal muscle expression of carnitine palmitoyl-transferase 1b (CPT1b) was also significantly increased. CONCLUSIONS— The PPARδ agonist GW501516 reverses multiple abnormalities associated with the metabolic syndrome without increasing oxidative stress. The effect is probably caused by increased fat oxidation in skeletal muscle.

Effects of n−6 PUFAs compared with SFAs on liver fat, lipoproteins, and inflammation in abdominal obesity: a randomized controlled trial

BACKGROUND Replacing SFAs with vegetable PUFAs has cardiometabolic benefits, but the effects on liver fat are unknown. Increased dietary n-6 PUFAs have, however, also been proposed to promote inflammation-a yet unproven theory. OBJECTIVE We investigated the effects of PUFAs on liver fat, systemic inflammation, and metabolic disorders. DESIGN We randomly assigned 67 abdominally obese subjects (15% had type 2 diabetes) to a 10-wk isocaloric diet high in vegetable n-6 PUFA (PUFA diet) or SFA mainly from butter (SFA diet), without altering the macronutrient intake. Liver fat was assessed by MRI and magnetic resonance proton (1H) spectroscopy (MRS). Proprotein convertase subtilisin/kexin type-9 (PCSK9, a hepatic LDL-receptor regulator), inflammation, and adipose tissue expression of inflammatory and lipogenic genes were determined. RESULTS A total of 61 subjects completed the study. Body weight modestly increased but was not different between groups. Liver fat was lower during the PUFA diet than during the SFA diet [between-group difference in relative change from baseline; 16% (MRI; P < 0.001), 34% (MRS; P = 0.02)]. PCSK9 (P = 0.001), TNF receptor-2 (P < 0.01), and IL-1 receptor antagonist (P = 0.02) concentrations were lower during the PUFA diet, whereas insulin (P = 0.06) tended to be higher during the SFA diet. In compliant subjects (defined as change in serum linoleic acid), insulin, total/HDL-cholesterol ratio, LDL cholesterol, and triglycerides were lower during the PUFA diet than during the SFA diet (P < 0.05). Adipose tissue gene expression was unchanged. CONCLUSIONS Compared with SFA intake, n-6 PUFAs reduce liver fat and modestly improve metabolic status, without weight loss. A high n-6 PUFA intake does not cause any signs of inflammation or oxidative stress. Downregulation of PCSK9 could be a novel mechanism behind the cholesterol-lowering effects of PUFAs. This trial was registered at clinicaltrials.gov as NCT01038102.

Supplementation with vitamins C and E inhibits the release of interleukin‐6 from contracting human skeletal muscle

Contracting human skeletal muscle is a major contributor to the exercise‐induced increase of plasma interleukin‐6 (IL‐6). Although antioxidants have been shown to attenuate the exercise‐induced increase of plasma IL‐6, it is unknown whether antioxidants inhibit transcription, translation or translocation of IL‐6 within contracting human skeletal muscle. Using a single‐blind placebo‐controlled design with randomization, young healthy men received an oral supplementation with either a combination of ascorbic acid (500 mg day−1) and RRR‐α‐tocopherol (400 i.u. day−1) (Treatment, n= 7), or placebo (Control, n= 7). After 28 days of supplementation, the subjects performed 3 h of dynamic two‐legged knee‐extensor exercise at 50% of their individual maximal power output. Muscle biopsies from vastus lateralis were obtained at rest (0 h), immediately post exercise (3 h) and after 3 h of recovery (6 h). Leg blood flow was measured using Doppler ultrasonography. Plasma IL‐6 concentration was measured in blood sampled from the femoral artery and vein. The net release of IL‐6 was calculated using Ficks principle. Plasma vitamin C and E concentrations were elevated in Treatment compared to Control. Plasma 8‐iso‐prostaglandin F2α, a marker of lipid peroxidation, increased in response to exercise in Control, but not in Treatment. In both Control and Treatment, skeletal muscle IL‐6 mRNA and protein levels increased between 0 and 3 h. In contrast, the net release of IL‐6 from the leg, which increased during exercise with a peak at 3.5 h in Control, was completely blunted during exercise in Treatment. The arterial plasma IL‐6 concentration from 3 to 4 h, when the arterial IL‐6 levels peaked in both groups, was ∼50% lower in the Treatment group compared to Control (Treatment versus Control: 7.9 pg ml−1, 95% confidence interval (CI) 6.0–10.7 pg ml−1, versus 19.7 pg ml−1, CI 13.8–29.4 pg ml−1, at 3.5 h, P < 0.05 between groups). Moreover, plasma interleukin‐1 receptor antagonist (IL‐1ra), C‐reactive protein and cortisol levels all increased after the exercise in Control, but not in Treatment. In conclusion, our results show that supplementation with vitamins C and E attenuated the systemic IL‐6 response to exercise primarily via inhibition of the IL‐6 protein release from the contracting skeletal muscle per se.

Experimental exposure to wood-smoke particles in healthy humans : Effects on markers of inflammation, coagulation, and lipid peroxidation

Particulate air pollution is known to increase cardiovascular morbidity and mortality. Proposed mechanisms underlying this increase include effects on inflammation, coagulation factors, and oxidative stress, which could increase the risk of coronary events and atherosclerosis. The aim of this study was to examine whether short-term exposure to wood smoke affects markers of inflammation, blood hemostasis, and lipid peroxidation in healthy humans. Thirteen subjects were exposed to wood smoke and clean air in a chamber during two 4-h sessions, 1 wk apart. The mass concentrations of fine particles at wood smoke exposure were 240–280 μg/m3, and number concentrations were 95,000–180,000/cm3. About half of the particles were ultrafine (< 100 nm). Blood and urine samples were taken before and after the experiment. Exposure to wood smoke increased the levels of serum amyloid A, a cardiovascular risk factor, as well as factor VIII in plasma and the factor VIII/von Willebrand factor ratio, indicating a slight effect on the balance of coagulation factors. Moreover, there was an increased urinary excretion of free 8-iso-prostaglandin2α, a major F2-isoprostane, though this was based on nine subjects only, indicating a temporary increase in free radical-mediated lipid peroxidation. Thus, wood-smoke particles at levels that can be found in smoky indoor environments seem to affect inflammation, coagulation, and possibly lipid peroxidation. These factors may be involved in the mechanisms whereby particulate air pollution affects cardiovascular morbidity and mortality. The exposure setup could be used to establish which particle characteristics are critical for the effects.

F2-Isoprostanes in Human Health and Diseases: From Molecular Mechanisms to Clinical Implications

Oxidative stress is implicated as one of the major underlying mechanisms behind many acute and chronic diseases, and involved in normal aging. However, the measurement of free radicals or their end products is complicated. Thus, proof of association of free radicals in pathologic conditions has been absent. Isoprostanes are prostaglandin-like bioactive compounds that are biosynthesized in vivo independent of cyclooxygenases, principally through free-radical catalyzation of arachidonic acid. Isoprostanes are now considered to be reliable biomarkers of oxidative stress, as evidenced by an autonomous study organized recently by the National Institutes of Health (NIH) in the United States. A number of these compounds have potent biologic activities such as vasoconstrictive and certain inflammatory properties. Isoprostanes are involved in many human diseases. Additionally, elevated levels of F(2)-isoprostanes have been seen in normal human pregnancy and after intake of some fatty acids, but their physiologic assignments have not yet been distinctive. This evidence indicates that measurement of bioactive F(2)-isoprostanes in body fluids offers a unique noninvasive analytic utensil to study the role of free radicals in physiology, oxidative stress-related diseases, experimental acute or chronic inflammatory conditions, and also in the assessment of various antioxidants, radical scavengers, and drugs.

Relation of Body Mass Index and Insulin Resistance to Cardiovascular Risk Factors, Inflammatory Factors, and Oxidative Stress During Adolescence

Background—This study assessed the relation of fatness and insulin resistance and their interaction with cardiovascular risk factors, inflammatory factors, and oxidative stress in thin and heavy adolescents. Methods and Results—Euglycemic insulin clamp studies were performed on 295 (169 male, 126 female) adolescents (mean±SE age, 15±0.1 years). Comparisons were made between (1) heavy and thin adolescents; (2) insulin-sensitive and insulin-resistant adolescents; and (3) thin insulin-sensitive (T-IS), thin insulin-resistant (T-IR), heavy insulin-sensitive (H-IS), and heavy insulin-resistant (H-IR) adolescents. Summed z scores were used to determine clustering of risk factors (fasting insulin, triglycerides, HDL-C, and systolic blood pressure [SBP]) among the groups. SBP, triglycerides, and fasting insulin were significantly higher and HDL-C significantly lower in the heavy adolescents. Fasting insulin and triglycerides were significantly higher and HDL-C significantly lower in the insulin-resistant adolescents. Among the 4 groups, the risk factors and cluster score followed a pattern of risk as follows: T-IS<T-IR<H-IS<H-IR, with H-IR significantly greater than the other groups and showing an interaction between fatness and insulin resistance. Conclusions—These results show the significant association of both fatness and insulin resistance and their significant interaction with cardiovascular risk factors in adolescence. The finding that insulin resistance may be acting interactively with fatness suggests that interventions directed at insulin resistance in addition to weight loss may be required to alter early development of cardiovascular risk.

Association of Type 2 Diabetes With Cyclooxygenase-Mediated Inflammation and Oxidative Stress in an Elderly Population

Background—Involvement of cyclooxygenase (COX)-mediated inflammation in type 2 diabetes has not been studied, and the association between cytokine-mediated inflammation and diabetes is not fully clarified. Methods and Results—15-Keto-dihydro-prostaglandin F2&agr; (a metabolite of prostaglandin F2&agr; and an indicator of COX-mediated inflammation), high-sensitivity C-reactive protein (CRP), serum amyloid protein A (SAA), 8-iso-PGF2&agr; (a nonenzymatic, free radical product of arachidonic acid and an indicator of oxidative stress), and &agr;-tocopherol were measured in a population-based sample of 77-year-old men (n=765), in which 112 men had type 2 diabetes. The inflammatory indicators were increased in men with diabetes (urinary 15-keto-dihydro-PGF2&agr;, P <0.001, CRP and SAA, P <0.05). However, when adjusted for body mass index, waist circumference, or fasting insulin, no association was found between diabetes and CRP or SAA. The oxidative stress indicator 8-iso-PGF2&agr; in urine was increased (P <0.01) in men with diabetes. Patients who were newly diagnosed with diabetes (<7 years since diagnosis) had increased urinary 15-keto-dihydro-PGF2&agr; and decreased &agr;-tocopherol, but 8-iso-PGF2&agr; was unaltered. Conclusions—This is the first study to show that type 2 diabetes in elderly men is related to COX-mediated inflammation, reflected by enhanced prostaglandin formation. The high levels of cytokine-mediated acute-phase proteins observed in men with diabetes appear to be related to obesity and increased fasting insulin. The results further suggest that the appearance of chronic inflammation is an early process in the pathogenesis of diabetes, whereas oxidative injury may be a later process, possibly related to inflammation.