On the antioxidant properties of erythropoietin and its association with the oxidative-nitrosative stress response to hypoxia in humans.

AIM: The aim of this study was to examine if erythropoietin (EPO) has the potential to act as a biological antioxidant and determine the underlying mechanisms. METHODS: The rate at which its recombinant form (rHuEPO) reacts with hydroxyl (HO˙), 2,2-diphenyl-1-picrylhydrazyl (DPPH˙) and peroxyl (ROO˙) radicals was evaluated in-vitro. The relationship between the erythopoietic and oxidative-nitrosative stress response to poikilocapneic hypoxia was determined separately in-vivo by sampling arterial blood from eleven males in normoxia and following 12 h exposure to 13% oxygen. Electron paramagnetic resonance spectroscopy, ELISA and ozone-based chemiluminescence were employed for direct detection of ascorbate (A(˙-) ) and N-tert-butyl-α-phenylnitrone spin-trapped alkoxyl (PBN-OR) radicals, 3-nitrotyrosine (3-NT) and nitrite (NO2-). RESULTS: We found rHuEPO to be a potent scavenger of HO˙ (kr = 1.03-1.66 × 10(11) m(-1) s(-1) ) with the capacity to inhibit Fenton chemistry through catalytic iron chelation. Its ability to scavenge DPPH˙ and ROO˙ was also superior compared to other more conventional antioxidants. Hypoxia was associated with a rise in arterial EPO and free radical-mediated reduction in nitric oxide, indicative of oxidative-nitrosative stress. The latter was confirmed by an increased systemic formation of A˙(-) , PBN-OR, 3-NT and corresponding loss of NO2- (P < 0.05 vs. normoxia). The erythropoietic and oxidative-nitrosative stress responses were consistently related (r = -0.52 to 0.68, P < 0.05). CONCLUSION: These findings demonstrate that EPO has the capacity to act as a biological antioxidant and provide a mechanistic basis for its reported cytoprotective benefits within the clinical setting.

Polycystic ovary syndrome influences the level of serum amyloid A and activity of phospholipid transfer protein in HDL2 and HDL3.

STUDY QUESTION: Is polycystic ovary syndrome (PCOS) associated with altered levels of pro-inflammatory high-density lipoproteins (HDL) and activity of HDL-associated enzymes? SUMMARY ANSWER: In PCOS, HDL contained increased levels of the inflammatory marker serum amyloid A (SAA) and altered functioning of HDL-associated phospholipid transfer protein (PLTP), with these changes being independent of BMI, body fat and insulin resistance (IR). WHAT IS KNOWN ALREADY: PCOS is associated with adipocyte-derived inflammation, which potentially increases the risk of cardiovascular disease and diabetes. SAA is an inflammatory marker that is released from hypertrophic adipocytes and interacts with HDL, reducing their anti-atherogenic properties. No studies have previously investigated if SAA-associated HDL influences the HDL-associated enzymes namely, PLTP and cholesterol ester transfer protein (CETP) in women with PCOS. PARTICIPANTS/MATERIALS, SETTINGS, METHODS: Obese women with PCOS were matched with controls for BMI and percentage body fat (n = 100/group; cohort-1); a subset of these women (n = 64/group; cohort-2) were further matched for IR. HDL in blood samples was subfractionated into HDL2 and HDL3 by rapid ultracentrifugation. SAA was measured in serum, HDL2 and HDL3 by an enzyme-linked immunosorbent assay and the activities of PLTP and CETP were measured in HDL2 and HDL3 by fluorimetric assays. MAIN RESULTS AND THE ROLE OF CHANCE: In the PCOS women from cohort-1, SAA was increased in serum, HDL2 and HDL3 (P = 0.038, 0.008 and 0.001 versus control, respectively), as was the activity of PLTP in HDL2 and HDL3 (P = 0.006 and 0.009 versus controls, respectively). In the PCOS women from cohort-2, SAA was increased in serum, HDL2 and HDL3, although only significantly in HDL3 (P = 0.083, 0.120 and 0.034 versus controls, respectively), as was the activity of PLTP in HDL2 and HDL3, although this was only significant in HDL2 (P = 0.045 and 0.070 versus controls, respectively). LIMITATIONS, REASONS FOR CAUTION: First, insulin sensitivity was not determined by the euglycaemic-hyperinsulinaemic clamp. Secondly, the method used to estimate body fat was not able to discriminate between visceral and peripheral fat. Thirdly, larger study groups would be required to confirm if PCOS independently contributed to SAA-related HDL and functional changes to this lipoprotein, independent of BMI, percentage body fat and IR. WIDER IMPLICATIONS OF THE FINDINGS: This is the first study to highlight the usefulness of HDL-associated SAA as a marker to identify increased inflammation in women with PCOS. This study also identified that the functioning of HDL was altered in women with PCOS. These findings illustrate a mechanism through which cardiovascular disease may increase in PCOS. STUDY FUNDING/COMPETING INTERESTS: Funded by the Irish Endocrinology Society. No competing interests. CLINICAL TRIAL REGISTRATION NUMBER: NCT001195168.

Disordered vascular compliance in haemochromatosis.

BACKGROUND: A relationship may exist between body iron stores, endothelial dysfunction and overall cardiovascular risk. AIMS: To compare vascular compliance, biochemical endothelial function and antioxidant status between patients with homozygous hereditary haemochromatosis and healthy controls. METHODS: Haemochromatosis patients and healthy controls were recruited. Measures of vascular compliance were assessed by applanation tonometry. Serological markers of endothelial function (plasma lipid hydroperoxides, cell adhesion molecules), antioxidant levels (ascorbate, lipid soluble antioxidants) and high-sensitivity C-reactive protein (CRP) were also measured. RESULTS: Thirty-five hereditary haemochromatosis patients (ten females, mean age 54.6) and 36 controls (27 female, mean age 54.0) were recruited. Haemochromatosis patients had significantly higher systolic and diastolic blood pressures. Pulse wave velocity (PWV) was significantly higher in male haemochromatosis patients (9.90 vs. 8.65 m/s, p = 0.048). Following adjustment for age and blood pressure, male haemochromatosis patients continued to have a trend for higher PWVs (+1.37 m/s, p = 0.058). Haemochromatosis patients had significantly lower levels of ascorbate (46.11 vs. 72.68 µmol/L, p = 0.011), retinol (1.17 vs. 1.81 µmol/L, p = 0.001) and g-tocopherol (2.51 vs. 3.14 µmol/L, p = 0.011). However, there was no difference in lipid hydroperoxides (0.46 vs. 0.47 nmol/L, p = 0.94), cell adhesion molecule levels (ICAM: 348.12 vs. 308.03 ng/mL, p = 0.32 and VCAM: 472.78 vs. 461.31 ng/mL, p = 0.79) or high-sensitivity CRP (225.01 vs. 207.13 mg/L, p = 0.32). CONCLUSIONS: Haemochromatosis is associated with higher PWVs in males and diminished antioxidants across the sexes but no evidence of endothelial dysfunction or increased lipid peroxidation.

Adiponectin multimers, body weight and markers of cardiovascular risk in adolescence: Northern Ireland Young Hearts Project.

BACKGROUND: Research examining the relationship between adiponectin (AN) isoforms, body weight and cardiovascular (CV) risk factors is limited, particularly in younger populations. OBJECTIVES: To investigate the inter-relationships between AN isoforms and CV risk factors, and their dependence on body weight status, in adolescents. DESIGN: Blood samples from 92 obese, 92 overweight and 92 normal weight age- and sex-matched adolescents were analysed for traditional cardiovascular disease (CVD) risk biomarkers and also total, high molecular weight (HMW), medium and low molecular weight (LMW) AN. RESULTS: A significant inverse association was observed between total and HMW AN and waist-hip ratio (P=0.015, P=0.006, respectively), triglycerides (P=0.003, P=0.003, respectively) and systolic blood pressure (P=0.012, P=0.024, respectively) and a significant positive association with high-density lipoprotein (P<0.001, P<0.001, respectively) in multi-adjusted analyses. There was no evidence of a relationship between multimeric AN and high-sensitivity C-reactive protein. There was also little evidence of a relationship between LMW AN and CVD risk factors. There was a strong, body mass index (BMI)-independent, association between AN, CVD biomarkers and the hypertriglyceridemic waist phenotype. CONCLUSION: Prominent, BMI-independent associations between total and HMW AN, but not LMW AN, and CVD risk factors were already evident in this young population. This research in adolescents supports the contention that AN subfractions may have different biological actions. These associations in apparently healthy adolescents suggest an important role for AN and its subfractions in the pathogenesis of metabolic syndrome traits and indicate that the potential for total or HMW AN to act as early universal biomarkers of CV risk warrants further study.

Does a diet high or low in fat influence the oxidation potential of VLDL, LDL and HDL subfractions?

BACKGROUND AND AIMS: High-fat diets have become increasingly popular for weight-loss, but their effect on the oxidation potential of lipoprotein subfractions has not been studied. Therefore, this study compared the effects of high-fat vs. low-fat weight reduction diets on this parameter. METHODS AND RESULTS: Very-low, low- and high-density lipoprotein (VLDL, LDL & HDL) subfractions were isolated by rapid ultracentrifugation from 24-overweight/obese subjects randomised to a high- or low-fat diet. The lipoprotein subfractions were assessed for oxidation potential by measuring conjugated diene (CD) production and time at half maximum. We found a significant between-group difference in oxidation potential. Specifically, a high-fat diet led to increased CD production in VLDL(A-D) and HDL(2&3), and a prolongation of time at half maximum. Within-group differences found that CDs increased in VLDL(A&D), LDL(I-III) and HDL(2&3) in the high-fat group and fell in VLDL(A-C) and HDL(2&3) and increased in LDL(I&II), in the low-fat group. Furthermore, following both diets all lipoprotein subfractions, except LDL(II) in the low-fat group, were protected against oxidation. CONCLUSION: These results demonstrate that at first glance, a high-fat diet may be indicative of having heart-protective properties. However, this may be erroneous, as although the time for oxidation to occur was prolonged, once this occurred these lipoproteins had the potential to produce significantly more oxidised substrate. Conversely, a low-fat diet may be considered anti-atherogenic, as these subfractions were protected against oxidation and mainly contained fewer oxidised substrate. Thus, increased fat intake may, by increasing the oxidation product within lipoprotein subfractions, increase cardiovascular disease.

High-altitude pulmonary hypertension is associated with a free radical-mediated reduction in pulmonary nitric oxide bioavailability.

High altitude (HA)-induced pulmonary hypertension may be due to a free radical-mediated reduction in pulmonary nitric oxide (NO) bioavailability. We hypothesised that the increase in pulmonary artery systolic pressure (PASP) at HA would be associated with a net transpulmonary output of free radicals and corresponding loss of bioactive NO metabolites. Twenty-six mountaineers provided central venous and radial arterial samples at low altitude (LA) and following active ascent to 4559 m (HA). PASP was determined by Doppler echocardiography, pulmonary blood flow by inert gas re-breathing, and vasoactive exchange via the Fick principle. Acute mountain sickness (AMS) and high-altitude pulmonary oedema (HAPE) were diagnosed using clinical questionnaires and chest radiography. Electron paramagnetic resonance spectroscopy, ozone-based chemiluminescence and ELISA were employed for plasma detection of the ascorbate free radical (A(·-)), NO metabolites and 3-nitrotyrosine (3-NT). Fourteen subjects were diagnosed with AMS and three of four HAPE-susceptible subjects developed HAPE. Ascent decreased the arterio-central venous concentration difference (a-cv(D)) resulting in a net transpulmonary loss of ascorbate, α-tocopherol and bioactive NO metabolites (P < 0.05 vs. LA). This was accompanied by an increased a-cv(D) and net output of A(·-) and lipid hydroperoxides (P < 0.05 vs. sea level, SL) that correlated against the rise in PASP (r = 0.56-0.62, P < 0.05) and arterial 3-NT (r = 0.48-0.63, P < 0.05) that was more pronounced in HAPE. These findings suggest that increased PASP and vascular resistance observed at HA are associated with a free radical-mediated reduction in pulmonary NO bioavailability.

Investigation of a multimarker approach to the initial assessment of patients with acute chest pain.

Early identification of acute coronary syndrome (ACS) is important to guide therapy at a time when it is most likely to be of value. In addition, predicting future risk helps identify those most likely to benefit from ongoing therapy. Cardiac troponin T (cTnT) is useful for both purposes although cannot reliably rule out ACS until 12 hours after pain onset and does not fully define future risk. In this review article we summarize our previously published research, which assessed the value of myocyte injury, vascular inflammation, hemostatic, and neurohormonal markers in the early diagnosis of ACS and risk stratification of patients with ACS. In addition to cTnT, we measured heart fatty acid binding protein (H-FABP), glycogen phosphorylase-BB, high-sensitivity C-reactive protein, myeloperoxidase, matrix metalloproteinase 9, pregnancy-associated plasma protein-A, D-dimer, soluble CD40 ligand, and N-terminal pro-brain natriuretic peptide (NT-proBNP). Of the 664 patients enrolled, 415 met inclusion criteria for the early diagnosis of acute myocardial infarction (MI) analysis; 555 were included in the risk stratification analysis and were followed for 1 year from admission. In patients presenting <4 hours from pain onset, initial H-FABP had higher sensitivity for acute MI than cTnT (73% vs. 55%; P=0.043) but was of no benefit beyond 4 hours when compared to cTnT. On multivariate analysis, H-FABP, NT-proBNP, and peak cTnT were independent predictors of 1-year death/MI. Our research demonstrated that, in patients presenting within 4 hours from pain onset, H-FABP may improve detection of ACS. Measuring H-FABP and proBNP may help improve long-term risk stratification.

Altered free radical metabolism in acute mountain sickness: implications for dynamic cerebral autoregulation and blood-brain barrier function.

We tested the hypothesis that dynamic cerebral autoregulation (CA) and blood-brain barrier (BBB) function would be compromised in acute mountain sickness (AMS) subsequent to a hypoxia-mediated alteration in systemic free radical metabolism. Eighteen male lowlanders were examined in normoxia (21% O(2)) and following 6 h passive exposure to hypoxia (12% O(2)). Blood flow velocity in the middle cerebral artery (MCAv) and mean arterial blood pressure (MAP) were measured for determination of CA following calculation of transfer function analysis and rate of regulation (RoR). Nine subjects developed clinical AMS (AMS+) and were more hypoxaemic relative to subjects without AMS (AMS-). A more marked increase in the venous concentration of the ascorbate radical (A(*-)), lipid hydroperoxides (LOOH) and increased susceptibility of low-density lipoprotein (LDL) to oxidation was observed during hypoxia in AMS+ (P < 0.05 versus AMS-). Despite a general decline in total nitric oxide (NO) in hypoxia (P < 0.05 versus normoxia), the normoxic baseline plasma and red blood cell (RBC) NO metabolite pool was lower in AMS+ with normalization observed during hypoxia (P < 0.05 versus AMS-). CA was selectively impaired in AMS+ as indicated both by an increase in the low-frequency (0.07-0.20 Hz) transfer function gain and decrease in RoR (P < 0.05 versus AMS-). However, there was no evidence for cerebral hyper-perfusion, BBB disruption or neuronal-parenchymal damage as indicated by a lack of change in MCAv, S100beta and neuron-specific enolase. In conclusion, these findings suggest that AMS is associated with altered redox homeostasis and disordered CA independent of barrier disruption.

The effects of cilostazol on exercise-induced ischaemia-reperfusion injury in patients with peripheral arterial disease.

OBJECTIVES: Cilostazol improves walking distance in peripheral arterial disease (PAD) patients. The study objectives were to assess the effects of cilostazol on walking distance, followed by the additional assessment of cilostazol on exercise-induced ischaemia-reperfusion injury in such patients. METHODS: PAD patients were prospectively recruited to a double-blinded, placebo-controlled trial. Patients were randomised to receive either cilostazol 100mg or placebo twice a day. The primary end-point was an improvement in walking distance. Secondary end-points included the assessment of oxygen-derived free-radical generation, antioxidant consumption and other markers of the inflammatory cascade. Initial and absolute claudication distances (ICDs and ACDs, respectively) were measured on a treadmill. Inflammatory response was assessed before and 30 min post-exercise by measuring lipid hydroperoxide, ascorbate, alpha-tocopherol, beta-carotene, P-selectin, intracellular and vascular cell-adhesion molecules (I-CAM and V-CAM), thromboxane B(2) (TXB(2)), interleukin-6, interleukin-10, high-sensitive C-reactive protein (hsCRP), albumin-creatinine ratio (ACR) and urinary levels of p75TNF receptor. All tests were performed at baseline and 6 and 24 weeks. RESULTS: One hundred and six PAD patients (of whom 73 were males) were recruited and successfully randomised from December 2004 to January 2006. Patients who received cilostazol demonstrated a more significant improvement in the mean percentage change from baseline in ACD (77.2% vs. 26.6% at 6 weeks, p=0.026 and 161.7% vs. 79.0% at 24 weeks, p=0.048) as compared to the placebo. Cilostazol reduced lipid hydroperoxide levels compared to a placebo-related increase before and after exercise (6 weeks: pre-exercise: -11.8% vs. +5.8%, p=0.003 and post-exercise: -12.3% vs. +13.9%, p=0.007 and 24 weeks: pre-exercise -15.5% vs. +12.0%, p=0.025 and post-exercise: -9.2% vs. +1.9%, p=0.028). beta-Carotene levels were significantly increased in the cilostazol group, compared to placebo, before exercise at 6 and 24 weeks (6 weeks: 34.5% vs. -7.4%, p=0.028; 24 weeks: 34.3% vs. 17.7%, p=0.048). Cilostazol also significantly reduced P-selectin, I-CAM and V-CAM levels at 24 weeks as compared to baseline (p<0.05). There was no difference between treatment groups for ascorbate, alpha-tocopherol, interleukin-6 and -10, hsCRP and p75TNF receptor levels. CONCLUSIONS: Cilostazol significantly improves ACD, in addition to attenuating exercise-induced ischaemia-reperfusion injury, in PAD patients.

Molecular detection of exercise-induced free radicals following ascorbate prophylaxis in type 1 diabetes mellitus: a randomised controlled trial.

AIMS/HYPOTHESIS: Patients with type 1 diabetes mellitus are more susceptible than healthy individuals to exercise-induced oxidative stress and vascular endothelial dysfunction, which has important implications for the progression of disease. Thus, in the present study, we designed a randomised double-blind, placebo-controlled trial to test the original hypothesis that oral prophylaxis with vitamin C attenuates rest and exercise-induced free radical-mediated lipid peroxidation in type 1 diabetes mellitus. METHODS: All data were collected from hospitalised diabetic patients. The electron paramagnetic resonance spectroscopic detection of spin-trapped alpha-phenyl-tert-butylnitrone (PBN) adducts was combined with the use of supporting markers of lipid peroxidation and non-enzymatic antioxidants to assess exercise-induced oxidative stress in male patients with type 1 diabetes (HbA(1c) 7.9 +/- 1%, n = 12) and healthy controls (HbA(1c) 4.6 +/- 0.5%, n = 14). Following participant randomisation using numbers in a sealed envelope, venous blood samples were obtained at rest, after a maximal exercise challenge and before and 2 h after oral ingestion of 1 g ascorbate or placebo. Participants and lead investigators were blinded to the administration of either placebo or ascorbate treatments. Primary outcome was the difference in changes in free radicals following ascorbate ingestion. RESULTS: Six diabetic patients and seven healthy control participants were randomised to each of the placebo and ascorbate groups. Diabetic patients (n = 12) exhibited an elevated concentration of PBN adducts (p < 0.05 vs healthy, n = 14), which were confirmed as secondary, lipid-derived oxygen-centred alkoxyl (RO.) radicals (a(nitrogen) = 1.37 mT and abeta(hydrogen) = 0.18 mT). Lipid hydroperoxides were also selectively elevated and associated with a depression of retinol and lycopene (p < 0.05 vs healthy). Vitamin C supplementation increased plasma vitamin C concentration to a similar degree in both groups (p < 0.05 vs pre-supplementation) and attenuated the exercise-induced oxidative stress response (p < 0.05 vs healthy). There were no selective treatment differences between groups in the primary outcome variable. CONCLUSIONS/INTERPRETATION: These findings are the first to suggest that oral vitamin C supplementation provides an effective prophylaxis against exercise-induced free radical-mediated lipid peroxidation in human diabetic blood. CLINICAL TRIALS REGISTRATION NUMBER: ISRCTN96164937.

LDL particle size in habitual exercisers, lean sedentary men and abdominally obese sedentary men.

Habitual exercisers enjoy considerable protection from coronary heart disease (CHD). Often, however, only modest differences in traditional CHD risk factors are apparent between habitual exercisers and their sedentary counterparts. For this reason, there is increasing interest in novel predictors of CHD, such as a preponderance of small, dense low-density lipoprotein (LDL) particles. Polyacrylamide gel electrophoresis was used to separate lipoprotein subfractions in 32 lean exercisers, 36 lean sedentary men and 21 obese sedentary men aged 30 - 45 years. Well-validated equations were used to determine LDL concentration and peak particle diameter. Waist girth was used to identify lean (< 100 cm) and obese (>or= 100 cm) individuals. LDL concentration was lower in lean exercisers than in lean sedentary men (2.64 +/- 0.44 vs. 3.76 +/- 0.79 mmol . l (-1), p < 0.001), suggesting that habitual exercise influences this risk factor. In contrast, there were no significant differences in LDL peak particle diameter between lean exercisers, lean sedentary men and obese sedentary men (27.92 +/- 0.67, 28.09 +/- 0.62 and 27.77 +/- 0.77 nm, respectively). In multiple linear regression analysis, triglyceride concentration was the only significant predictor of LDL PPD. These data suggest that habitual exercise influences LDL concentration but does not influence LDL particle size in men aged 30 - 45 years.

Oxidative modification of triacylglycerol-rich lipoproteins.

Lipoprotein oxidation is thought to play a pivotal role in the evolution of atherosclerosis. Low-density lipoprotein (LDL) is the main source of oxidized lipid in the arterial wall. Oxidation of LDL alters its properties in a number of ways, making it more atherogenic, but oxidation of other lipoprotein classes may also be important. Common mechanisms are likely to contribute to the oxidation of all lipoprotein classes, with enzyme-mediated oxidation likely to be most important. Antioxidant content, fatty acid composition, particle size and the presence of seeding hydroperoxides also influence oxidative reactions. Larger triglyceride-rich lipoproteins are less likely to enter the arterial wall than LDL, but when oxidized will deliver a greater oxidant load to the arterial wall.

Effects of GH on lipid peroxidation and neutrophil superoxide anion-generating capacity in hypopituitary adults with GH deficiency.

OBJECTIVES: Untreated growth hormone deficiency (GHD) is implicated in the increased cardiovascular risk associated with adult hypopituitarism. Oxidative stress, predisposing to lipid peroxidation, may be an important mediator of endothelial dysfunction, a pro-atherogenic state associated with adult GHD. DESIGN AND PATIENTS: In a randomized, double-blind, placebo-controlled study we investigated the effects of GH replacement on low-density lipoprotein (LDL) oxidation and neutrophil superoxide (O(-)(2)) generating capacity in 32 GHD adults (19 males, 13 females; age range 19-64 years) over 3 months. Thirty age- and sex-matched healthy controls were also studied. MEASUREMENTS: Lipid hydroperoxides (HPOs) in plasma were measured using the ferrous oxidation with xylenol orange (FOX) assay. The susceptibility of LDL to oxidation was assessed by the copper-catalysed lag phase of LDL oxidation. Neutrophil O(-)(2)- generating capacity was assessed by a lucigenin-based chemiluminescent assay of NADPH oxidase activity. Body composition was assessed using bioelectrical impedance analysis. RESULTS: Compared to controls, GHD subjects had higher LDL cholesterol (4.0 +/- 0.8 vs. 3.5 +/- 0.9 mmol/l, P < 0.01) and higher triglyceride concentrations (2.3 +/- 1.5 vs. 1.1 +/- 0.7 mmol/l, P < 0.001) but lower HDL cholesterol (1.1 +/- 0.3 mmol/l vs. 1.4 +/- 0.4 mmol/l, P < 0.01), lower levels of HPOs (0.72 +/- 0.35 vs. 0.92 +/- 0.20 microm, P < 0.01) and lower basal (2.5 +/- 1.5 vs. 4.5 +/- 2.3 mV/5 x 10(5) neutrophils, P < 0.01) and peak post-activation levels (23.2 +/- 11.1 vs. 34.4 +/- 15.6 mV/5 x 10(5) neutrophils, P < 0.01) of neutrophil O(-)(2)- generation. GH replacement resulted in an increase in HPOs from 0.70 +/- 0.39 to 0.86 +/- 0.19 microm (P < 0.05), although there was no change in the lag time of LDL oxidation. Neutrophil O(-)(2)- generating capacity was enhanced with a rise in basal O(-)(2)- generation from 2.8 +/- 1.4 to 5.4 +/- 4.6 mV/5 x 10(5) neutrophils (P < 0.05) and in peak post-activation O(-)(2)- generation from 21.9 +/- 9.5 to 35.8 +/- 21.7 mV/5 x 10(5) neutrophils (P < 0.05). LDL cholesterol was reduced from 4.1 +/- 0.8 mmol/l to 3.5 +/- 0.8 mmol/l (P < 0.01). No significant changes in measured parameters occurred in the placebo group. CONCLUSIONS: Adult GHD is associated with reduced lipid peroxidation and impaired neutrophil O(-)(2)- generating capacity, both of which are reversible with GH replacement. Our data suggest that: (i) that oxidative stress is not a major feature of the pro-atherogenic state in hypopituitary adults with GHD and (ii) a role for GH in modulating neutrophil function and leucocyte-lipoprotein interactions.

Lipoprotein oxidation and atherosclerosis.

Lipoprotein oxidation is a key early stage in the development of atherosclerosis. Oxidation of low-density lipoprotein (LDL) is initiated by both enzyme-mediated and non-enzymic mechanisms in vivo, and oxidized LDL has many atherogenic properties. Oxidation of LDL in vivo is likely to be influenced by local environmental factors, such as pH. The composition of LDL is also important, including such factors as antioxidant content, fatty acid composition and particle size.

Reperfusion injury is greater with delayed restoration of venous outflow in concurrent arterial and venous limb injury.

BACKGROUND: Complex limb trauma often involves combined arterial and venous injury, and the resultant ischaemia-reperfusion injury (IRI) causes both local and remote organ injury. This study assessed the influence of the timing of restoration of venous drainage on IRI. METHODS: Male New Zealand white rabbits (n = 36) were randomized into six groups: sham operation (group 1) and unilateral hind limb arterial and venous occlusion for 1 h followed by no reflow for 2 h (group 2), arterial and venous reflow for 2 h (group 3), arterial reflow alone for 2 h (group 4), arterial reflow alone for 1 h followed by arterial and venous (delayed) reflow for a further 1 h (group 5), and pretreatment with an enteral combination antioxidant before occlusion of both artery and vein and delayed venous reflow (group 6). Plasma hydroperoxide (HPO) and glutathione peroxidase concentration, hind limb skeletal muscle and lung tissue wet : dry weight ratios and myeloperoxidase (MPO) concentration were measured. RESULTS: The plasma HPO level in the femoral vein effluent was significantly greater after delayed venous reflow (mean(s.e.m.) 2. 02(0.54) micromol/l) than in control animals (0.98(0.10) micromol/l) (P < 0.05). There was also a significantly greater tissue wet : dry weight ratio after delayed venous reflow than in controls, in skeletal muscle (mean(s.e.m.) 6.89(0.14) versus 5.34(0.54); P < 0. 05) and lung (9.20(1.14) versus 7.23(0.38); P < 0.05) tissue. Lung tissue MPO activity was significantly greater after delayed venous reflow compared with controls (3.20(0.28) versus 1.86(0.14) units/g; P < 0.005), and also in comparison to simultaneous arterial and venous reflow (2.40(0.24) units/g; P < 0.05). In the antioxidant pretreatment group there was no significant increase in plasma HPO concentration, tissue MPO level or tissue wet : dry weight ratio compared with the control group. CONCLUSION: In combined major arterial and venous injury of the limb, delayed restoration of venous drainage leads to significantly greater local skeletal muscle injury and remote neutrophil-mediated lung injury. These results support the clinical rationale for early restoration not only of arterial inflow but also venous drainage by means of intraluminal shunts.

Very low density lipoprotein subfractions in Type II diabetes mellitus: alterations in composition and susceptibility to oxidation.

AIMS/HYPOTHESIS: Type II (non-insulin-dependent) diabetes mellitus is associated with raised triglycerides and increased very low density lipoprotein cholesterol. The aim of this study was to assess if very low density lipoprotein subfraction composition and potential to oxidise were altered in this condition. METHODS: Very low density lipoprotein was separated into four subfractions (A-->D) by a novel, rapid ultracentrifugation procedure. Analysis of each subfraction included lipid and fatty acid composition. Preformed peroxides were measured spectrophotometrically and conjugated dienes were used as an indicator of in vitro lipid oxidation. RESULTS: In all results we compared patient and control subfractions. Mean fasting plasma glucose was 8.9 +/- 2.0 mmol/l in patients vs 5.1 +/- 0.4 mmol/l in control subjects (p < 0.001); patient HbA1c was 7.6 +/- 1.4%. Patient total lipid standardised for apo B was higher than controls in subfractions A, B and C; A, 201 vs 60; B, 191 vs 40; C, 63 vs 21; D, 29 vs 34 micromol lipid per mg apo B (p < 0.05). Preformed peroxides were higher in all patient subfractions compared with controls: A, 340 vs 48; B, 346 vs 42; C, 262 vs 28; D, 54 vs 16 nmol per mg apo B (p < 0.001). Patient subfractions A and D were more susceptible to in vitro oxidation. Monounsaturated fatty acids were lower in patients subfractions, 35.2 vs 36.7; B, 35.1 vs 38.7; C, 34.4 vs 36.5; D, 33.0 vs 35.5 as per cent total (p < 0.05). CONCLUSIONS/INTERPRETATION: These results indicate abnormalities in very low density lipoprotein subfraction composition and oxidation profile in Type II diabetic subjects, which are characteristic of more atherogenic particles and that may contribute to the development of cardiovascular disease in these patients.

Assessment of susceptibility of low-density lipoprotein to oxidation.

It is now recognized that oxidation of low-density lipoprotein (LDL) is a key event in the development of atherosclerosis (1). In vivo, oxidation is believed to occur primarily in the arterial wall. In early atherosclerotic lesions oxidation may be initiated by enzymes, including myeloperoxidase and 15-lipoxygenase, or by reactive nitrogen species, while in more advanced lesions transition metals including copper play a role (2). Oxidized LDL has a number of atherogenic properties (3). It is taken up via the macrophage scavenger receptor and promotes the formation of foam cells. It is chemotatic for circulating monocytes and inhibits the migration of tissue macrophages out of the arterial wall. In addition, oxidized LDL promotes platelet aggregation, is directly toxic to cells in the arterial wall, promotes the synthesis of a range of cytokines and growth factors, and inhibits nitric oxide mediated arterial dilation.

HDL composition and HDL antioxidant capacity in patients on regular haemodialysis.

Recent evidence suggests that HDL can directly inhibit LDL oxidation, a key early stage in atherogenesis. Patients with chronic renal failure are at increased cardiovascular risk, have reduced HDL levels and altered HDL composition. We have therefore investigated whether compositional changes in HDL lead to decreased HDL antioxidant capacity in these patients. In comparison to control subject HDL, patient HDL contained less total cholesterol, cholesterol esters, phospholipids and alpha-tocopherol. LDL, HDL and LDL + HDL were standardised for protein and oxidised in the presence of Cu2+. The rate of propagation during HDL oxidation was reduced in the patient group (3.28+/-0.65 x 10(-5) vs. 4.60+/-0.97 x 10(-5) abs. U/min, P < 0.01). Lipid peroxide generation in patient HDL was decreased: 6.56+4.4 versus 13.42+/-7.0 nmol malondialdehyde (MDA)/mg HDL protein after 90 min and 14.45+/-3.8 versus 20.11+/-7.8 nmol MDA/mg HDL protein after 180 min. This is attributable to reduced HDL polyunsaturated fatty acid content in patients (0.53+/-0.12 vs. 0.72+/-0.16 mmol/g HDL, P < 0.01). The inhibitory effect of HDL on LDL oxidation was similar: 71 and 33% for patient HDL compared to 68 and 31% for control HDL, after 90 and 180 min, respectively. Compositional changes of HDL in patients on haemodialysis did not affect the antioxidant capacity of HDL after standardisation for HDL protein. However, reduced HDL levels in vivo may result in reduced HDL antioxidant capacity in these patients.

Absorption and antioxidant effects of quercetin from onions, in man.

OBJECTIVE: To investigate the in vivo effects of quercetin following the ingestion of fried onions. DESIGN: Five healthy volunteers, three males and two females aged between 25 and 39 y, ingested 225 g of fried onions after an overnight fast and peripheral venous blood was collected 0, 2, 4, 24 and 48 h after consumption. Quercetin in the plasma, total antioxidant capacity and susceptibility of low density lipoproteins (LDL) to oxidation were measured. RESULTS: Following the onion meal, quercetin levels increased from baseline values (28.4 +/- 1.9 ng/ml) to peak after 2 h (248.4 +/- 103.9 ng/ml), decreasing to baseline again after 24 h (P > 0.05). This was accompanied by an increase in the total antioxidant activity of the plasma from baseline (1.70 +/- 0.04 mmol/l trolox equivalents) to 1.75 +/- 0.10 mmol/l trolox equivalents after 2 h and 1.76 +/- 0.08 mmol/l trolox equivalents after 4 h. There was no significant change in the susceptibility of the plasma or the isolated LDL to oxidation over the 48 h period after consumption of the fried onions. In view of these negative findings, we isolated LDL and other lipoproteins from plasma at each time point. Quercetin was not detected in either LDL or VLDL, but was present in the HDL fraction, although this fraction also contains other proteins including albumin. CONCLUSIONS: Quercetin can be absorbed in humans from dietary sources to high enough concentrations to increase the overall antioxidant activity of the plasma. Quercetin, however, has a strong affinity for protein and provides no direct protective effect during LDL oxidation.

A simple method for assessing copper-mediated oxidation of very-low-density lipoprotein isolated by rapid ultracentrifugation.

The association of very-low-density lipoprotein (VLDL) with atherosclerosis remains controversial. However, studies have shown that oxidative modification of VLDL can promote foam cell formation, leading to the development of atherosclerosis. A rapid method is described which will allow the significance of VLDL oxidation to be assessed in clinical studies. VLDL was isolated from heparinized plasma by a 1-h, single spin ultracentrifugation. Total protein was standardized to 25 mg/L. Oxidation was promoted by the addition of copper ions (17.5 mumol/L, final concentration) incubated at 37 degrees C. Conjugated diene production was followed at 234 nm. Total assay preparation time was 2 h. Urate greatly inhibited the oxidation of VLDL and was successfully removed by size exclusion chromatography. VLDL isolated from frozen plasma (-70 degrees C) was stable for 15 weeks. This simple, rapid method for the isolation of VLDL may be applied to assess the significance of VLDL oxidation in disease.