Increased oxidative stress is associated with type-2 diabetes and related cardiovascular diseases but oxidative modification of LDL has been partially characterized. Our aim was to compare the lipid and fatty acid composition as well as the redox status of LDL from diabetic patients and healthy subjects. First, to ensure that isolation of LDL by sequential ultracentrifugation did not result in lipid modifications, lipid composition and peroxide content were determined in LDL isolated either by ultracentrifugation or fast-protein liquid chromatography. Both methods resulted in similar concentrations of lipids, fatty acids, hydroxy-octadecadienoic acid (HODE) and malondialdehyde (MDA). Then, LDL were isolated by ultracentrifugation from 8 type-2 diabetic patients and 8 control subjects. Compared to control LDL, diabetic LDL contained decreased cholesteryl esters and increased triglyceride concentrations. Ethanolamine plasmalogens decreased by 49%. Proportions of linoleic acid decreased in all lipid classes while proportions of arachidonic acid increased in cholesteryl esters. Total HODE concentrations increased by 56%, 12- and 15-hydroxy-eicosatetraenoicacid by 161 and 86%, respectively, and MDA levels increased by 2-fold. α-tocopherol concentrations, expressed relative to triglycerides, were lower in LDL from patients compared to controls while γ-tocopherol did not differ. Overall, LDL from type-2 diabetic patients displayed increased oxidative stress. Determination of hydroxylated fatty acids and ethanolamine plasmalogen depletion could be especially relevant in diabetes.
diabetes; fatty acids; peroxidation; plasmalogens; ultracentrifugation; fast-protein liquid chromatography
The fatty acid composition of cholesterol (cholesteryl) ester, triglyceride, and lecithin has been investigated in whole plasma and individual lipoproteins of healthy control subjects, of patients with malabsorption, and also of patients without malabsorption. The results show a decreased proportion of the essential fatty acid linoleic acid in all three lipid classes in both groups of patients as compared with the healthy controls. This abnormality was more marked in the malabsorbers, especially those with steatorrhoea secondary to intestinal resection. Unequivocal biochemical evidence of essential fatty acid deficiency, as indicated by the appearance of 5, 8, 11 eicosatrienoic acid in plasma lecithin, was observed in two patients, both of whom had undergone major intestinal resections. The results suggest that intestinal resection predisposes to the development of essential fatty acid deficiency.
This study was aimed at assessing oxidative stress in LDL from obese patients with metabolic syndrome (MetS) compared with LDL from type 2 diabetic patients or control volunteers, and determining their effects on platelets.
The profiles of lipids, fatty acids and fatty acid oxidation products were determined in LDL isolated from plasma of MetS patients, type 2 diabetic patients and volunteers (n=10 per group). The effects of LDL isolated from these participants on platelet arachidonic acid signaling cascade and aggregation were investigated.
Compared with LDL from control volunteers, LDL from obese MetS and type 2 diabetic patients contained lower cholesteryl esters, higher triacylglycerols and lower ethanolamine plasmalogens levels. Proportions of linoleic acid were decreased in phosphatidylcholine and cholesteryl esters in patients’ LDL. Among the markers of lipid peroxidation, oxidation products of linoleic acid (hydroxy-octadecadienoic acids) and malondialdehyde were increased by 59% and 2-fold, respectively in LDL from MetS patients and to the same extent in LDL from type 2 diabetic patients. LDL from MetS patients were as potent as LDL from type 2 diabetic patients in activating platelet arachidonic acid signaling cascade through increased phosphorylation of p38 MAPK and cytosolic phospholipase A2, and increased thromboxane B2 formation. LDL from patients with MetS and type 2 diabetes potentiated 3-fold and 3.5-fold respectively platelet aggregation whereas control LDL had no activating effects on platelets.
MetS in obese patients, without or with diabetes, is associated with increased oxidative stress in LDL, which trigger platelet activation.
The protocol is registered in ClinicalTrials.gov as NCT00932087.
Adult; Aged; Arachidonic Acid; metabolism; Biological Markers; blood; Blood Platelets; enzymology; metabolism; Diabetes Mellitus, Type 2; blood; complications; metabolism; Humans; Lipid Peroxidation; Lipids; blood; Lipoproteins, LDL; blood; chemistry; metabolism; Male; Metabolic Syndrome X; complications; Middle Aged; Obesity; blood; complications; metabolism; Oxidative Stress; Phospholipases A2, Secretory; blood; metabolism; Platelet Activation; Signal Transduction; Lipid peroxidation; Fatty acids; LDL; Oxidized LDL; Platelets; Metabolic syndrome; Type 2 diabetes; Obesity
Nonalcoholic fatty liver disease (NAFLD) is characterized by the accumulation of excess liver triacylglycerol (TAG), inflammation, and liver damage. The goal of the present study was to directly quantify the biological sources of hepatic and plasma lipoprotein TAG in NAFLD. Patients (5 male and 4 female; 44 ± 10 years of age) scheduled for a medically indicated liver biopsy were infused with and orally fed stable isotopes for 4 days to label and track serum nonesterified fatty acids (NEFAs), dietary fatty acids, and those derived from the de novo lipogenesis (DNL) pathway, present in liver tissue and lipoprotein TAG. Hepatic and lipoprotein TAG fatty acids were analyzed by gas chromatography/mass spectrometry. NAFLD patients were obese, with fasting hypertriglyceridemia and hyperinsulinemia. Of the TAG accounted for in liver, 59.0% ± 9.9% of TAG arose from NEFAs; 26.1% ± 6.7%, from DNL; and 14.9% ± 7.0%, from the diet. The pattern of labeling in VLDL was similar to that in liver, and throughout the 4 days of labeling, the liver demonstrated reciprocal use of adipose and dietary fatty acids. DNL was elevated in the fasting state and demonstrated no diurnal variation. These quantitative metabolic data document that both elevated peripheral fatty acids and DNL contribute to the accumulation of hepatic and lipoprotein fat in NAFLD.
Tissue injury due to hypoxia and/or free radicals is common in a variety of disease processes. This cross-sectional study aimed to investigate effect of chronic kidney diseases (CKD) and hemodialysis (HD) on hypoxia and oxidative stress biomarkers.
Forty pediatric patients with CKD on HD and 20 healthy children were recruited. Plasma hypoxia induced factor-1α (HIF-1α), vascular endothelial growth factor (VEGF) were measured by specific ELISA kits while, total antioxidant capacity (TAC), total peroxide (TPX), pyruvate and lactate by enzymatic/chemical colorimetric methods. Oxidative stress index (OSI) and lactate/pyruvate (L/P) ratio were calculated.
TAC was significantly lower while TPX, OSI and VEGF were higher in patients at before- and after-dialysis session than controls. Lactate and HIF-1α levels were significantly higher at before-dialysis session than controls. Before dialysis, TAC and L/P ratio were lower than after-dialysis. In before-dialysis session, VEGF correlated positively with pyruvate, HIF-1α and OSI correlated positively with TPX, but, negatively with TAC. In after-dialysis session, HIF-1α correlated negatively with TPX and OSI; while, OSI correlated positively with TPX.
CKD patients succumb considerable tissue hypoxia with oxidative stress. Hemodialysis ameliorated hypoxia but lowered antioxidants as evidenced by decreased levels of HIF-1α and TAC at before- compared to after-dialysis levels.
Oxidant stress is widely believed to participate in cardiovascular disease pathogenesis. However, progress in defining appropriate systemic anti-oxidant targetted therapies has been hindered by uncertainty in defining clinically relevant systemic oxidant stress measures. In a case control study, 50 subjects with CAD (> 50% stenosis in one or more major coronary vessels) and 54 without CAD (< 30% stenosis in all major coronary vessels) were tested. Plasma was isolated and stored under conditions designed to prevent artificial lipid peroxidation. Systemic levels of multiple (n=9) specific fatty acid oxidation products including individual hydroxy-octadecadienoic acids (HODEs), hydroxy-eicosatetraenoic acids (HETEs), and F2-Isoprostanes, were simultaneously measured by high performance liquid chromatography (HPLC) with on-line tandem mass spectrometry, along with traditional risk factors and C-reactive protein (CRP) levels. Of the markers monitored, only 9-HETE and F2-Isoprostanes, both products of free radical-mediated arachidonic acid oxidation, were significantly elevated in patients with angiographically defined CAD (9-HETE, 8.7 ± 4 vs. 6.8 ± 4 umol/mol arachidonate, p = 0.011; and F2-Isoprostanes, 9.4 ± 5 vs. 6.2 ± 3umol/mol arachidonate, p < 0.001). In multivariable analyses with simultaneous adjustment for Framingham Risk Score and C-reactive protein, 9-HETE (4th quartile OR=4.8, 95% CI=1.3 to 17.1; P = 0.016) and F2-Isoprostanes (4th quartile OR=9.7, 95% CI=2.56 to 36.9; P < 0.001) remained strong and independent predictors of CAD risk. Systemic levels of 9-HETE and F2-Isoprostanes are independently associated with angiographic evidence of CAD and appear superior to other specific oxidation products of arachidonic and linoleic acids as predictors of the presence of angiographically evident coronary artery disease.
Coronary artery disease; Oxidation; Oxidation products; F2-Isoprostanes
The prevalence of atherosclerotic cardiovascular disease in chronic hemodialysis (HD) patients has been demonstrated to be higher than in healthy people. Severe liver fibrosis is strongly associated with early carotid atherosclerosis and it might reduce the survival of patients who undergo both renal replacement therapy and transplantation. We wanted to assess whether nonalcoholic fatty liver disease (NAFLD) was associated with altered intima-media thickness (IMT) in HD patients as an independent marker of subclinical atherosclerosis. We enrolled 42 patients undergoing HD and 48 patients with normal renal function, all of them with high levels of aminotransferases and an ultrasonographic diagnosis of liver steatosis. The control group consisted of 60 healthy subjects. Laboratory tests for inflammatory and oxidative markers, ultrasonographic liver evaluation, carotid IMT measurement, and liver biopsy were performed. Different degrees of fibrosis were detected in our study cohort. Worse liver histopathological scores and higher plasmatic levels of C-reactive protein, reactive oxygen species, and vascular cell adhesion molecule-1 were found in HD patients. Carotid IMT was significantly higher (p < 0.005) in patients with histological steatosis. HD patients may develop active and progressive chronic hepatitis faster than patients with normal renal function and the thickness of their carotid intima-media might be markedly increased. These two conditions seem to be independent on classical risk factors and on metabolic syndrome. They might be related to the high levels of oxidants and to the inflammatory state, which are typical of patients undergoing HD. Independently related with the traditional risk factors for cardiovascular disease, nonspecific inflammation and oxide-reductive imbalance may play an important role in the progression of NAFLD and atherosclerotic disease in HD patients.
Chronic hepatitis; nonalcoholic steatohepatitis; oxidative stress; fibrosis; intima-media thickness; hemodialysis
Uremic state and hemobioincompatibility are implicated in subclinical inflammation and oxidative stress and progression of atherosclerosis in the hemodialysis (HD) population. To what extent different dialysis membranes contribute to oxidative stress induced by a dialysis procedure per se is still a subject of debate. Fifteen HD patients and 15 healthy controls were enrolled in this study. Patients received two index HD sessions with a cuprophane and polysulfone membrane two weeks apart. Blood samples were collected at baseline and then before and after HD sessions. We determined serum thiobarbituric acid, protein carbonyl, and serum nitrite/nitrate levels as markers of oxidative damage. We also measured erythrocyte glutathione level, catalase, superoxide dismutase and glutathione peroxidase activity, and serum vitamin C and E levels as antioxidant markers. At baseline, HD patients, in comparison with normal controls, had a trend towards increased oxidant state and depletion of antioxidants. Cuprophane dialysis induced a higher increase in production of oxidants, along with a lower compensatory increase of antioxidants when compared with polysulfone dialysis. In conclusion, a single HD session, even when conducted with a biocompatible membrane, appears to play an important role in the imbalance between ROS production and antioxidant defense, but to a milder extent than cuprophane dialysis.
oxidative stress; antioxidants; hemodialysis; biocompatibility; membranes
Patients with end-stage renal disease (ESRD) undergoing hemodialysis (HD) are apparently exposed to enhanced oxidative stress and to inflammation. It was the aim of this study to characterize the state of systemic oxidative stress of ESRD patients before and following HD using highly specific biomarkers, F2-isoprostanes and 4-hydroxynonenal (HNE). Furthermore the question should be answered, if there are associations between inflammation and systemic oxidative stress and/or between systemic oxidative stress and renal anemia, which is more or less typical for HD patients.
Patients and methods
Concentrations of F2-isoprostanes, HNE, C-reactive protein (CRP) as marker of inflammation, and hemoglobin were measured in serum samples of patients with ESRD before and after HD and of healthy control persons for comparison. Total (esterified plus free) F2-isoprostanes were quantified by highly sensitive gas chromatography/mass spectrometry technique, HNE by thin layer chromatography and HPLC/UV detection, CRP by immunoturbidimetry and hemoglobin by clinico-chemical routine assay.
1. HD patients showed significantly higher serum concentrations of F2-isoprostanes and HNE than healthy human control subjects. 2. Total (esterified plus free) F2-isoprostane levels before HD were not significantly different from those after HD, whereas HNE levels were significantly decreased in patients after HD. 3. F2-isoprostane concentrations in HD patients correlated with the levels of CRP, whereas HNE concentrations inversely correlated with the content of hemoglobin.
Both, F2-isoprostanes and HNE serum concentrations are useful oxidative stress parameters in ESRD patients undergoing HD. Whereas HNE strongly correlates with the severity of renal anemia, leading to left heart insufficiency, F2-isoprostanes (sum of free plus esterified) highly correlate with the degree of inflammation.
F2-isoprostanes; end-stage renal disease (ESRD); hemodialysis patients; C-reactive protein; 4-hydroxynonenal; renal anemia
We examined skin autofluorescence (sAF) in chronic kidney disease children (CKD) in relation to renal function and dialysis modality.
Twenty children on hemodialysis (HD), 20 on peritoneal dialysis (PD), 36 treated conservatively, and 26 healthy subjects were enrolled into the study. In all children sAF, pulse-wave velocity indexed to height (PWV/ht), left ventricular mass index (LVMI), blood pressure (BP), serum lipid profile, phosphate (P), calcium (Ca), and homocysteine were measured.
sAF was significantly elevated in CKD groups vs. controls and was significantly associated with PWV/ht, LVMI, BP, P, Ca × P product and homocysteine. sAF in HD and PD groups was positively correlated with dialysis vintage, and in the predialysis group negatively correlated with glomerular filtration rate (eGFR). Multiple regression analysis showed significant association of sAF with LVMI and P in the CKD patient group, and with dialysis treatment duration and BP in dialyzed children.
In CKD children, tissue accumulation of advanced glycation end-products (AGEs) was observed. This was aggravated as eGFR declined and was related to early cardiovascular changes and some biochemical cardiovascular disease (CVD) risk markers. sAF as a non-invasive method may be a useful tool for identification of a clinical risk factors of cardiovascular disease in CKD children.
Cardiovascular risk factors; Children; Dialysis; Nephrology
Oxidative stress is related to several diseases, including chronic renal insufficiency. The disequilibrium in the oxidant-antioxidant balance is the result of several metabolic changes. The majority of studies to-date have evaluated the grade of oxidative stress with a single biomarker, or a very limited number of them.
The present study used several important biomarkers to establish a score relating to oxidative stress status (glutathione S-transferase, superoxide dismutase, catalase, glutathione peroxidase, glutathione reductase, reduced and oxidized glutathione, thiobarbituric acid reactive substances and hemolysis test). The score of oxidative stress (SOS) was then applied to a group of patients with renal insufficiency not on hemodialysis, and compared to healthy control individuals.
The score for patients with chronic renal insufficiency was significantly different from that of the healthy control group (0.62 ± 1.41 vs. -0.05 ± 0.94; p < 0.001). The comparison between patients with chronic renal insufficiency and control individuals showed significant differences with respect to changes in the enzymatic antioxidant systems (glutathione S-transferase, glutathione reductase), non-enzymatic antioxidant system (oxidized glutathione) and oxidizability (hemolysis test) indicating significant oxidative stress associated with chronic renal insufficiency.
Patients with chronic renal insufficiency not on hemodialysis are susceptible to oxidative stress. The mechanisms that underlie this status are the consequence of changes in glutathione and related enzymes. The SOS scoring system is a useful biochemical parameter to evaluate the influence of oxidative stress on the clinical status of these patients.
Cardiovascular disease (CVD) is responsible for the majority of deaths in chronic renal failure (CRF). Oxidative stress plays a key role in pathogenesis of atherosclerosis and CVD, which is promoted by the production of reactive oxygen species (ROS) and impaired antioxidant enzymes. These ROS react with nitric oxide (NO) to produce cytotoxic reactive nitrogen species that cause oxidative injury to the endothelium. This study evaluated biomarkers of oxidative stress, NOx (total NO2 and NO3), and superoxide dismutase (SOD) enzyme in normal control and CRF patients as case group and correlated their association with CVD. This cross sectional study involved 173 CRF patients on different modes of treatment (hemodialysis, continuous ambulatory peritoneal dialysis (CAPD), and predialysis). Of these, 74 had CVD. The control group consisted of 33 healthy subjects who had no history of CRF and CVD. Both NOx and SOD levels were significantly lower (P<0.05, P<0.001, respectively) in the case group. Comparing between CRF patients with and without CVD, SOD level was found to be significantly lower in CRF patients with CVD (P<0.05). Logistic regression analysis showed significant association of CVD event with age, male gender, diabetes, SOD level, and lipid profile in CRF patients. Oxidative stress occurs in the CRF patients with or without CVD. This study found that NOx and SOD levels were reduced in all CRF patients with or without CVD. However, it was noted that the levels of these biomarkers of oxidative stress were significantly lower in CRF patients with CVD compared with CRF patients without CVD. Therefore, these oxidative stress markers maybe contributing factors in the pathogenesis of CVD in patients with CRF.
Chronic renal failure; CVD; oxidative stress
Patients with chronic kidney disease (CKD) have signs of genomic instability and, as a consequence, extensive genetic damage, possibly due to accumulation of uraemic toxins, oxidative stress mediators and other endogenous substances with genotoxic properties. We explored factors associated with the presence and background levels of genetic damage in CKD. A cross-sectional study was performed in 91 CKD patients including pre-dialysis (CKD patients; n = 23) and patients undergoing peritoneal dialysis (PD; n = 33) or haemodialysis (HD; n = 35) and with 61 healthy subjects, divided into two subgroups with the older group being in the age range of the patients, serving as controls. Alkaline comet assay and cytokinesis-block micronucleus assay in peripheral blood lymphocytes were used to determine DNA and chromosome damage, respectively, present in CKD. Markers of oxidative stress [malondialdehyde (MDA), advanced glycation end products (AGEs), thiols, advanced oxidation protein products and 8-hydroxy-2′-deoxyguanosine] and markers of inflammation (C-reactive protein, interleukin-6 and tumour necrosis factor alpha) were also measured. Micronucleus (MN) frequency was significantly higher (P < 0.05) in the CKD group (46±4‰) when compared with the older control (oC) group (27.7±14). A significant increase in MN frequency (P < 0.05) was also seen in PD patients (41.9±14‰) versus the oC group. There was no statistically significant difference for the HD group (29.7±15.6‰; P = NS) versus the oC group. Comet assay data showed a significant increase (P < 0.001) of tail DNA intensity in cells of patients with CKD (15.6±7%) with respect to the total control (TC) group (11±1%). PD patients (14.8±7%) also have a significant increase (P < 0.001) versus the TC group. Again, there was no statistically significant difference for the HD group (12.5±3%) compared with the TC group. Patients with MN values in the upper quartile had increased cholesterol, triglycerides, AGEs and MDA levels and lower albumin levels. Multiple logistic regression analysis showed that male gender, diabetes and treatment modality were independently associated with higher levels of DNA damage. Our results suggest that oxidative stress, diabetes, gender and dialysis modality in CKD patients increased DNA and chromosome damage. To confirm these data, prospective clinical trials need to be performed.
Hypertriglyceridemia is a common metabolic complication of chronic kidney disease (CKD) and an important risk factor for coronary heart disease in this patient population. The mechanisms responsible for the development of hypertriglyceridemia in subjects with CKD are not clear.
We studied very low density lipoprotein triglyceride (VLDL-TG) and VLDL-apolipoprotein B-100 (VLDL-apoB-100) kinetics in vivo in 6 subjects with non-dialysis-dependent CKD (CKD-ND), 6 subjects with CKD treated with peritoneal dialysis (CKD-PD) and 24 sex-, age- and body mass index-matched control subjects with normal renal function (12 control subjects each matched with the CKD-ND and CKD-PD group, respectively).
The secretion rates of VLDL-TG and VLDL-apoB-100 into plasma were not different between CKD-ND or CKD-PD and their respective control groups. The mean residence times of VLDL-TG and VLDL-apoB-100 in plasma, which represents the time VLDL-TG and VLDL-apoB-100 spend in the circulation after secretion by the liver, tended to be greater in subjects with CKD-ND than in control subjects (222 ± 38 vs. 143 ± 21 min, p = 0.07, and 352 ± 102 vs. 200 ± 20 min, p = 0.06, respectively) and were about two-fold greater in subjects with CKD-PD compared with their control group (248 ± 51 vs. 143 ± 21 min and 526 ± 116 vs. 182 ± 16 min, respectively; both p ≤ 0.01).
Impaired plasma clearance of VLDL-TG and VLDL-apoB-100 is the major abnormality associated with hypertriglyceridemia in patients with either CKD-ND or CKD-PD.
Isotope tracer; Lipoprotein; Metabolism; Renal failure
Data from studies in patients with nonalcoholic steatohepatitis (NASH) suggest an increased hepatic fatty acid oxidation. We have previously shown higher fasting plasma bile acid concentrations in patients with NASH. In-vivo and in-vitro studies suggest that bile acids by binding to peroxisome proliferator-activated receptor α activate fibroblast growth factor 21 (FGF21) and increase hepatic fatty acid oxidation.
Plasma bile acid levels were quantified in healthy controls (n = 38) and patients with biopsy-proven NASH (n = 36). Plasma concentration of fatty acids, β-hydroxybutyrate, insulin, glucose, leptin, alanine aminotransferase, FGF21, and 8-hydroxydeoxyguanosine, a measure of oxidative stress, were measured in 16 healthy controls and 10 patients with NASH in the fasted state and in response to 3 h of infusion of intralipid. In a subgroup of these patients (n = 6 each), plasma ceramide subspecies were quantified.
Fasting plasma bile acids, FGF21, and leptin concentrations were significantly higher in patients with NASH. In response to intralipid infusion there was an increase in plasma β-hydroxybutyrate and free fatty acid levels in both controls and NASH; however, the ratio of β-hydroxybutyrate/free fatty acid was higher in NASH (P = 0.02). Plasma FGF21 concentration increased in response to intralipid in patients with NASH only (P < 0.01). Plasma leptin, insulin, glucose, and alanine transferase concentrations did not change in either group after infusion of intralipid. Increase in total ceramides in response to intralipid was greater in NASH.
Elevated bile acids and FGF21 may be responsible for the higher hepatic fatty acid oxidation in NASH.
bile acids; fatty acid oxidation; fibroblast growth factor 21; nonalcoholic steatohepatitis
Sulfobutylether-beta-cyclodextrin (SBECD), a large cyclic oligosaccharide that is used to solubilize voriconazole (VRC) for intravenous administration, is eliminated mainly by renal excretion. The pharmacokinetics of SBECD and voriconazole in patients undergoing extracorporeal renal replacement therapies are not well defined. We performed a three-period randomized crossover study of 15 patients with end-stage renal failure during 6-hour treatment with Genius dialysis, standard hemodialysis, or hemodiafiltration using a high-flux polysulfone membrane. At the start of renal replacement therapy, the patients received a single 2-h infusion of voriconazole (4 mg per kg of body weight) solubilized with SBECD. SBECD, voriconazole, and voriconazole-N-oxide concentrations were quantified in plasma and dialysate samples by high-performance liquid chromatography (HPLC) and by HPLC coupled to tandem mass spectrometry (LC-MS-MS) and analyzed by noncompartmental methods. Nonparametric repeated-measures analysis of variance (ANOVA) was used to analyze differences between treatment phases. SBECD and voriconazole recoveries in dialysate samples were 67% and 10% of the administered doses. SBECD concentrations declined with a half-life ranging from 2.6 ± 0.6 h (Genius dialysis) to 2.4 ± 0.9 h (hemodialysis) and 2.0 ± 0.6 h (hemodiafiltration) (P < 0.01 for Genius dialysis versus hemodiafiltration). Prediction of steady-state conditions indicated that even with daily hemodialysis, SBECD will still exceed SBECD exposure of patients with normal renal function by a factor of 6.2. SBECD was effectively eliminated during 6 h of renal replacement therapy by all methods, using high-flux polysulfone membranes, whereas elimination of voriconazole was quantitatively insignificant. The SBECD half-life during renal replacement therapy was nearly normalized, but the average SBECD exposure during repeated administration is expected to be still increased.
In mice, 4F, an apolipoprotein A-I mimetic peptide that restores HDL function, prevents diabetes-induced atherosclerosis. We sought to determine whether HDL function is impaired in type 2 diabetic (T2D) patients and whether 4F treatment improves HDL function in T2D patient plasma in vitro.
RESEARCH DESIGN AND METHODS
HDL anti-inflammatory function was determined in 93 T2D patients and 31 control subjects as the ability of test HDLs to inhibit LDL-induced monocyte chemotactic activity in human aortic endothelial cell monolayers. The HDL antioxidant properties were measured using a cell-free assay that uses dichlorofluorescein diacetate. Oxidized fatty acids in HDLs were measured by liquid chromatography–tandem mass spectrometry. In subgroups of patients and control subjects, the HDL inflammatory index was repeated after incubation with L-4F.
The HDL inflammatory index was 1.42 ± 0.29 in T2D patients and 0.70 ± 0.19 in control subjects (P < 0.001). The cell-free assay was impaired in T2D patients compared with control subjects (2.03 ± 1.35 vs. 1.60 ± 0.80, P < 0.05), and also HDL intrinsic oxidation (cell-free assay without LDL) was higher in T2D patients (1,708 ± 739 vs. 1,233 ± 601 relative fluorescence units, P < 0.001). All measured oxidized fatty acids were significantly higher in the HDLs of T2D patients. There was a significant correlation between the cell-free assay values and the content of oxidized fatty acids in HDL fractions. L-4F treatment restored the HDL inflammatory index in diabetic plasma samples (from 1.26 ± 0.17 to 0.71 ± 0.11, P < 0.001) and marginally affected it in healthy subjects (from 0.81 ± 0.16 to 0.66 ± 0.10, P < 0.05).
In patients with T2D, the content of oxidized fatty acids is increased and the anti-inflammatory and antioxidant activities of HDLs are impaired.
Chronic kidney disease (CKD) is a major public health problem and can result in end-stage renal disease with need for dialysis or transplantation. In Europe up to 12% of the adult population had some renal impairment, while in the United States the end stage of CKD has increased dramatically from 209.000 in 1991 to 472.000 in 2004. Diabetes and hypertension are major causes of kidney pathology. Infection, particularly ascending infection, is more common with increasing age, as both immune function declines and associated pathology predisposing to infection, such as obstructive uropathy, becomes more common. Most pathological changes in the kidney appear to be initiated by oxidative stress, followed by an inflammatory reaction. Oxidative stress results from an imbalance between free radicals and their detoxification by endogenous and exogenous scavengers, including polyunsatured fatty acids (PUFA). Recent studies showed that PUFA supplementation slowed the rate of loss of renal function in patients with IgA nephropathy. Then, studies of omega-3 supplementation in dialysis patients describe salutary effects on triglyceride levels and dialysis access patency. We examined the relationship between total plasma PUFA levels and change in creatinine clearance over a three-year follow-up in the older persons enrolled in the InCHIANTI study, a population-based epidemiology study conducted in Tuscany, Italy. This study showed that older adults with low total plasma PUFA levels have a greater decline in creatinine clearance over three years of follow-up. These findings suggest that a higher dietary intake of PUFA may be protective against progression to chronic kidney disease.
Polyunsaturated fatty acids; renal function; chronic kidney disease; chronic renal failure
Evidence for increased oxidant stress has been reported in human atherosclerosis. However, no information is available about the importance of in situ oxidant stress in relation to plaque stability. This information is relevant because the morbidity and mortality of atherosclerosis are essentially the consequences of acute ischemic syndromes due to unstable plaques. We studied 30 carotid atherosclerotic plaques retrieved by endarterectomy from 18 asymptomatic (stable plaques) and 12 symptomatic patients (unstable plaques). Four normal arteries served as controls. After lipid extraction and ester hydrolysis, quantitation of different indices of oxidant stress were analyzed, including hydroxyeicosatetraenoic acids (HETEs), epoxyeicosatetraenoic acids (EETs), ketoeicosatetraenoic acids (oxo-ETEs), and F2-isoprostanes using online reverse-phase high-performance liquid chromatography tandem mass spectrometry (LC/MS/MS). All measurements were carried out in a strictly double-blind procedure. We found elevated levels of the different compounds in atherosclerotic plaques. Levels of HETEs were 24 times higher than EETs, oxo-ETEs, or F2-isoprostanes. Levels of HETEs, but not those of EETs, oxo-ETEs or F2-isoprostanes, were significantly elevated in plaques retrieved from symptomatic patients compared with those retrieved from asymptomatic patients (1,738 ± 274 vs. 1,002 ± 107 pmol/μmol lipid phosphorous, respectively; P < 0.01). One monooxygenated arachidonate species, 9-HETE, which cannot be derived from known enzymatic reactions, was the most abundant and significant compound observed in plaques, suggesting that nonenzymatic lipid peroxidation predominates in advanced atherosclerosis and may promote plaque instability.
Urinary liver-type fatty acid–binding protein (L-FABP) is a promising indicator of tubular but not glomerular damage. The aim of this study was to evaluate the clinical usefulness of urinary L-FABP as a prognostic biomarker in impaired diabetic nephropathy in type 2 diabetes.
RESEARCH DESIGN AND METHODS
This investigation involved a cross-sectional and longitudinal analysis of the relationship between urinary L-FABP levels and progressive nephropathy. Urinary L-FABP was measured with enzyme-linked immunosorbent assay. In the cross-sectional analysis, the association of urinary L-FABP, with the severity of diabetic nephropathy, was investigated in 140 patients with type 2 diabetes and in 412 healthy control subjects. Of the patients in the former study, 104 have been followed for 4 years. The progression of diabetic nephropathy was defined as progressive albuminuria, end-stage renal disease, or induction of hemodialysis.
Urinary L-FABP levels were progressively increased in subjects with normo-, micro-, or macroalbuminuria and further increased in patients with end-stage renal disease. In the longitudinal analysis, high urinary L-FABP levels were associated with the increase in albuminuria, progression to end-stage renal disease, or induction of hemodialysis. This was particularly demonstrated in the subgroup of patients without renal dysfunction (n = 59), where high urinary L-FABP levels were associated with the progression of diabetic nephropathy.
Urinary L-FABP accurately reflected the severity of diabetic nephropathy in type 2 diabetes, and its level was high in the patients with normoalbuminuria. Moreover, higher urinary L-FABP was a risk factor for progression of diabetic nephropathy.
Arginine deficiency and/or increased levels of circulating nitric oxide (NO) synthesis (NOS) inhibitors can cause reduced NOS, which may contribute to hypertension in patients with end-stage renal disease (ESRD). To test these hypotheses, NO oxidation products (NO2 + NO3 = NOx) and cyclic guanosine monophosphate (cGMP), the vasodilatory second messenger of NO, were measured in the blood, urine, and dialysate effluent of hemodialysis (HD) patients and compared with the blood and urine of healthy subjects. The subjects ate a controlled low-nitrate diet (∼330 μmol/d) for 48 hours before and during blood, dialysis effluent, and 24-hour urine collection. NOx output was significantly reduced in HD patients versus controls (552 ± 51 v 824 ± 96 μmol/24 h; P < 0.001), whereas cGMP output was not low versus controls. Plasma arginine level was normal and plasma levels of citrulline and the endogenous NOS inhibitor, asymmetric dimethylarginine (ADMA), were markedly elevated in patients with ESRD versus controls. Systolic blood pressure was greater in HD patients compared with controls despite concurrent antihypertensive therapy in most patients with ESRD. These studies suggest NO production is low in patients with ESRD undergoing HD, possibly because of the increased ratio of plasma ADMA to arginine.
End-stage renal disease; hypertension; arginine; cyclic guanosine monophosphate (cGMP); endogenous nitric oxide synthases (NOS) inhibitors
The objective of this study was to determine if children with phenylketonuria (PKU) have lower fatty acid concentrations in total erythrocyte lipid due to the phenylalanine restricted diet therapy compared to healthy control subjects. Dietary intake and fatty acid concentrations in total erythrocyte lipid were measured in twenty-one subjects (≤6 years of age) with PKU and twenty-three control children. Subjects with PKU had significantly lower protein and significantly higher polyunsaturated fat intake compared to controls. Subjects with PKU had significantly lower concentrations in total erythrocyte lipid of the sum of the ω-3,ω-6, saturated and polyunsaturated fatty acids. Concentrations of fatty acids among subjects with PKU were lower than control subjects but no subject with PKU exhibited any signs or symptoms suggestive of essential fatty acid deficiency, thereby suggesting that subjects with PKU in this cohort have normal and adequate essential fatty acid concentrations in total erythrocyte lipid.
Phenylketonuria; PKU; Fatty Acids; Lipids
Preeclampsia is accompanied by an extensive remodeling of the extracellular matrix of umbilical cord. It is associated with an increase in collagen content in the umbilical cord artery. Furthermore, preeclampsia distinctly reduces proteolytic and gelatinolytic activity, especially after activation with various agents.
We decided to develop a method for separation and determination of fatty acids from different tissues by high-performance liquid chromatography. That method allowed us to determine cholesteryl ester composition and content in umbilical cord arteries. Studies were performed on the umbilical cord arteries taken from 10 newborns delivered by healthy mothers and 10 newborns delivered by mothers with preeclampsia. Cholesteryl esters were isolated by thin layer chromatography. Fatty acids were liberated by basic hydrolysis and analyzed by HPLC of their p-bromophenacyl derivatives using detection at 254 nm. It was found that saturated fatty acids were the main group of fatty acids incorporated to cholesteryl esters in all control and preeclamptic umbilical cord arteries. Preeclampsia caused a significant increase in cholesteryl ester content in the umbilical cord arteries. An increase of neutral lipid content in vessel walls of newborns delivered by mothers with preeclampsia may be one of the factors that evoke the initiation of hypertension in utero and its amplification throughout childhood and adult life. The described method reduces time and cost consumption and allows us to determine almost all fatty acids forming cholesteryl esters contained in the tissue sample.
Cholesteryl Esters; Fatty Acids; HPLC; Preeclampsia; Umbilical Cord Artery
We recently cloned monoclonal IgM autoantibodies which bind to epitopes of oxidized low-density lipoprotein (OxLDL) from apoE-deficient mice (EO– autoantibodies). We now demonstrate that those EO– autoantibodies that were originally selected for binding to copper-oxidized low-density lipoproteins (CuOx-LDL), also bound both to the oxidized protein and to the oxidized lipid moieties of CuOx-LDL. The same EO– autoantibodies showed specific binding to products of oxidized 1-palmitoyl-2-arachidonoyl-phosphatidylcholine (OxPAPC) and to the specific oxidized phospholipid, 1-palmitoyl-2-(5-oxovaleroyl)-phosphatidyl-choline (POVPC), whereas oxidation of fatty acids (linoleic or arachidonic acid) or cholesteryl esters (cholesteryl-oleate or cholesteryl-linoleate) did not yield any binding activity. Those EO– autoantibodies that bound to oxidized phospholipids (e.g., EO6) inhibited the binding and degradation of CuOx-LDL by mouse peritoneal macrophages up to 91%, whereas other IgM EO– autoantibodies, selected for binding to malondialdehyde (MDA)-LDL, had no influence on binding of either CuOx-LDL or MDA-LDL by macrophages. F(ab′)2 fragments of EO6 were equally effective as the intact EO6 in preventing the binding of CuOx-LDL by macrophages. The molar ratios of IgM to LDL needed to maximally inhibit the binding varied from ∼8 to 25 with different CuOx-LDL preparations. Finally, a POVPC–bovine serum albumin (BSA) adduct also inhibited CuOx-LDL uptake by macrophages. These data suggest that oxidized phospholipid epitopes, present either as lipids or as lipid-protein adducts, represent one class of ligands involved in the recognition of OxLDL by macrophages, and that apoE-deficient mice have IgM autoantibodies that can bind to these neoepitopes and inhibit OxLDL uptake.
Cells acquire cholesterol in part by endocytosis of cholesteryl ester containing lipoproteins. In endosomes and lysosomes cholesteryl ester is hydrolyzed by acidic cholesteryl ester hydrolase producing cholesterol and fatty acids. Under certain pathological conditions, however, such as in atherosclerosis, excessive levels of cholesteryl ester accumulate in lysosomes for reasons that are poorly understood. Here, we have studied endosomal and lysosomal cholesteryl ester metabolism in cultured mouse macrophages and with cell-free extracts. We show that net hydrolysis of cholesteryl ester is coupled to the transfer of cholesterol to membranes. When membrane cholesterol levels are low, absorption of cholesterol effectively drives cholesteryl ester hydrolysis. When cholesterol levels in acceptor membranes approach saturation or when cholesterol export is blocked, cholesterol is re-esterified in endosomes. These results reveal a new facet of cellular cholesterol homeostasis and provide a potential explanation for cholesteryl ester accumulation in lysosomes of atherosclerotic cells.