Johannes M. Roob

Multicentric Validation of Proteomic Biomarkers in Urine Specific for Diabetic Nephropathy

Background Urine proteome analysis is rapidly emerging as a tool for diagnosis and prognosis in disease states. For diagnosis of diabetic nephropathy (DN), urinary proteome analysis was successfully applied in a pilot study. The validity of the previously established proteomic biomarkers with respect to the diagnostic and prognostic potential was assessed on a separate set of patients recruited at three different European centers. In this case-control study of 148 Caucasian patients with diabetes mellitus type 2 and duration ≥5 years, cases of DN were defined as albuminuria >300 mg/d and diabetic retinopathy (n = 66). Controls were matched for gender and diabetes duration (n = 82). Methodology/Principal Findings Proteome analysis was performed blinded using high-resolution capillary electrophoresis coupled with mass spectrometry (CE-MS). Data were evaluated employing the previously developed model for DN. Upon unblinding, the model for DN showed 93.8% sensitivity and 91.4% specificity, with an AUC of 0.948 (95% CI 0.898-0.978). Of 65 previously identified peptides, 60 were significantly different between cases and controls of this study. In <10% of cases and controls classification by proteome analysis not entirely resulted in the expected clinical outcome. Analysis of patients subsequent clinical course revealed later progression to DN in some of the false positive classified DN control patients. Conclusions These data provide the first independent confirmation that profiling of the urinary proteome by CE-MS can adequately identify subjects with DN, supporting the generalizability of this approach. The data further establish urinary collagen fragments as biomarkers for diabetes-induced renal damage that may serve as earlier and more specific biomarkers than the currently used urinary albumin.

Vitamin E kinetics in smokers and nonsmokers

Does cigarette smoking increase vitamin E utilization in vivo? A trial was carried out in 6 smokers and 5 nonsmokers of comparable ages and serum lipids. Subjects consumed 75 mg each d(3)-RRR and d(6)-all rac-alpha-tocopheryl acetates (natural and synthetic vitamin E, respectively) daily for 7 d with a standardized breakfast. Fasting blood samples were drawn on days -7, -6, -5, -4, -3, -2, -1, 0, 1, 2, 3, 4, 5, 6, 7, 9, 14, 21 (negative days indicate supplementation). In both groups, plasma d(3)-alpha-tocopherol concentrations were approximately double of d(6)-alpha-tocopherol. At day 0, the %d(3) alpha-tocopherols (d(3)-alpha-tocopherol/total-alpha-tocopherol x 100) were similar in both smokers and nonsmokers. Subsequently, there was a trend toward a faster exponential disappearance of the plasma %d(3) alpha-tocopherol in smokers compared with nonsmokers (0.30 +/- 0.04 compared with 0.24 +/- 0.05, p =.0565). The calculated %d(3) half-lives were 55.6 +/- 7.4 h in smokers and 72.1 +/- 17.3 h in nonsmokers (p =.0630). By day 21, the %d(3) in smokers had decreased to 1.4% +/- 0.3% while it was 2.2% +/- 0.7% (p =.0418) in the nonsmokers. These data suggest that smoking increases plasma vitamin E disappearance, but further studies are needed to confirm this finding and to assess its cause.

Urinary collagen fragments are significantly altered in diabetes: a link to pathophysiology.

Background The pathogenesis of diabetes mellitus (DM) is variable, comprising different inflammatory and immune responses. Proteome analysis holds the promise of delivering insight into the pathophysiological changes associated with diabetes. Recently, we identified and validated urinary proteomics biomarkers for diabetes. Based on these initial findings, we aimed to further validate urinary proteomics biomarkers specific for diabetes in general, and particularity associated with either type 1 (T1D) or type 2 diabetes (T2D). Methodology/Principal Findings Therefore, the low-molecular-weight urinary proteome of 902 subjects from 10 different centers, 315 controls and 587 patients with T1D (n = 299) or T2D (n = 288), was analyzed using capillary-electrophoresis mass-spectrometry. The 261 urinary biomarkers (100 were sequenced) previously discovered in 205 subjects were validated in an additional 697 subjects to distinguish DM subjects (n = 382) from control subjects (n = 315) with 94% (95% CI: 92–95) accuracy in this study. To identify biomarkers that differentiate T1D from T2D, a subset of normoalbuminuric patients with T1D (n = 68) and T2D (n = 42) was employed, enabling identification of 131 biomarker candidates (40 were sequenced) differentially regulated between T1D and T2D. These biomarkers distinguished T1D from T2D in an independent validation set of normoalbuminuric patients (n = 108) with 88% (95% CI: 81–94%) accuracy, and in patients with impaired renal function (n = 369) with 85% (95% CI: 81–88%) accuracy. Specific collagen fragments were associated with diabetes and type of diabetes indicating changes in collagen turnover and extracellular matrix as one hallmark of the molecular pathophysiology of diabetes. Additional biomarkers including inflammatory processes and pro-thrombotic alterations were observed. Conclusions/Significance These findings, based on the largest proteomic study performed to date on subjects with DM, validate the previously described biomarkers for DM, and pinpoint differences in the urinary proteome of T1D and T2D, indicating significant differences in extracellular matrix remodeling.

Increased concentrations of circulating vitamin E in carriers of the apolipoprotein A5 gene −1131T>C variant and associations with plasma lipids and lipid peroxidation

The aim of this study was to investigate the effects of the apolipoprotein A5 (APOA5) 1131T>C gene variant on vitamin E status and lipid profile. The gene variant was determined in 297 healthy nonsmoking men aged 20–75 years and recruited in the VITAGE Project. Effects of the genotype on vitamin E in plasma, LDL, and buccal mucosa cells (BMC) as well as on cholesterol and triglyceride (TG) concentrations in plasma and apolipoprotein A-I (apoA-I), apoB, apoE, apoC-III, and plasma fatty acids were determined. Plasma malondialdehyde concentrations as a marker of in vivo lipid peroxidation were determined. C allele carriers showed significantly higher TG, VLDL, and LDL in plasma, higher cholesterol in VLDL and intermediate density lipoprotein, and higher plasma fatty acids. Plasma α-tocopherol (but not γ-tocopherol, LDL α- and γ-tocopherol, or BMC total vitamin E) was increased significantly in C allele carriers compared with homozygote T allele carriers (P = 0.02), but not after adjustment for cholesterol or TG. Plasma malondialdehyde concentrations did not differ between genotypes. In conclusion, higher plasma lipids in the TC+CC genotype are efficiently protected against lipid peroxidation by higher α-tocopherol concentrations. Lipid-standardized vitamin E should be used to reliably assess vitamin E status in genetic association studies.

Effects of vitamin E depletion/repletion on biomarkers of oxidative stress in healthy aging.

Abstract: The effects on ex vivo LDL resistance to oxidation and biomarkers of in vivo oxidative stress in response to 3‐month dietary vitamin E restriction to 25% of recommended intake and 2‐month unrestricted dietary intake and supplementation with 800 IU/d were studied in 100 healthy, nonsmoking 20‐75‐year‐old volunteers. Significant changes in vitamin E status were associated with decreases and increases, respectively, in LDL resistance to oxidation in the depletion and supplementation period and with decreases in lipid peroxidation and oxidative DNA modification in the supplementation period. Healthy aging was not associated with enhanced susceptibility to oxidation in the depletion period.

Increased DNA dicarbonyl glycation and oxidation markers in patients with type 2 diabetes and link to diabetic nephropathy

Aim. The aim of this study was to assess the changes of markers of DNA damage by glycation and oxidation in patients with type 2 diabetes and the association with diabetic nephropathy. Methodology. DNA oxidation and glycation adducts were analysed in plasma and urine by stable isotopic dilution analysis liquid chromatography-tandem mass spectrometry. DNA markers analysed were as follows: the oxidation adduct 7,8-dihydro-8-oxo-2′-deoxyguanosine (8-OxodG) and glycation adducts of glyoxal and methylglyoxal—imidazopurinones GdG, MGdG, and N2-(1,R/S-carboxyethyl)deoxyguanosine (CEdG). Results. Plasma 8-OxodG and GdG were increased 2-fold and 6-fold, respectively, in patients with type 2 diabetes, with respect to healthy volunteers. Median urinary excretion rates of 8-OxodG, GdG, MGdG, and CEdG were increased 28-fold, 10-fold, 2-fold, and 2-fold, respectively, in patients with type 2 diabetes with respect to healthy controls. In patients with type 2 diabetes, nephropathy was associated with increased plasma 8-OxodG and increased urinary GdG and CEdG. In a multiple logistic regression model for diabetic nephropathy, diabetic nephropathy was linked to systolic blood pressure and urinary CEdG. Conclusion. DNA oxidative and glycation damage-derived nucleoside adducts are increased in plasma and urine of patients with type 2 diabetes and further increased in patients with diabetic nephropathy.

EFFECTS OF A PRE-DIALYSIS PATIENT EDUCATION PROGRAM ON THE RELATIVE FREQUENCIES OF DIALYSIS MODALITIES

♦ Background: Pre-dialysis education can guide the choice of the dialysis modality best tailored to meet the needs and preferences of individual patients with chronic kidney disease. ♦ Methods: In a retrospective single-center cohort study, we evaluated the impact of a pre-dialysis education program on the incidence rates of patients using hemodialysis (HD) and peritoneal dialysis (PD) in our unit. The frequency distribution of dialysis modalities between people attending our education program and people not attending the program (control group) was analyzed for the 4-year period 2004 - 2008. ♦ Results: From among all the incident chronic kidney disease 5D patients presenting during the 4-year period, we analyzed 227 who started dialysis either with an arteriovenous fistula or a PD catheter. In that cohort, 70 patients (30.8%) took part in the education program, and 157 (69.2%) did not receive structured pre-dialysis counseling. In the group receiving education, 38 patients (54.3%) started with PD, and 32 (45.7%), with HD. In the standard-care group not receiving education, 44 patients (28%) started with PD, and 113 (72%), with HD (p < 0.001). ♦ Conclusions: Our multidisciplinary pre-dialysis program had a significant impact on the frequency distribution of dialysis modalities, increasing the proportion of patients initiating dialysis with PD.

The Effect of Age on Vitamin E Status, Metabolism, and Function: Metabolism as Assessed by Labeled Tocopherols

Abstract: The effects of age on vitamin E metabolism were studied in 97 healthy 20‐75‐year‐old male nonsmoking Austrian volunteers of the VITAGE project. After a single oral intake of 30 mg d6‐RRR‐α‐ and d2‐RRR‐γ‐tocopheryl acetate, blood and 24‐hour urine was collected. Deuterated tocopherols in plasma and deuterated urinary metabolites were analyzed by GC‐MS. A first evaluation revealed a similar uptake of d6‐α‐ and d2‐γ‐tocopherol during the first 6 hours, and then d2‐γ‐tocopherol started to decrease. Urinary d2‐γ‐ carboxyethyl hydroxychroman metabolites (CEHCs) exceeded those of d6‐α‐CEHCs by about 10 times. There was no effect of age. Thus, there might be no need for a higher vitamin E intake for healthy elderly nonsmoking men.

Circulating leptin and NF‐κB activation in peripheral blood mononuclear cells across the menstrual cycle

Using the menstrual cycle as a model, this study focused on longitudinal changes and associations within a physiological network known to play a role in female fertility, including, as biologically active nodes, NF-κB, leptin and adiponectin, β-carotene, adipose tissue, and progesterone. In 28 women, leptin, adiponectin, β-carotene, and progesterone concentrations, NF-κB p65 and p50 activation in peripheral blood mononuclear cells (known to possess estrogen, progesterone and leptin receptors), total body fat (TBF) and subcutaneous adipose tissue (SAT) mass were determined at early (T1) and late follicular (T2) and mid (T3) and late (T4) luteal phase. Leptin and adiponectin concentrations were higher, while NF-κB p65 activation was lower at T3 compared with T1. NF-κB p65 activation was inversely related to leptin concentrations at T1, T3, and T4. β-Carotene was inversely related to leptin (T1,T2,T4) and SAT (T1,T3,T4). NF-κB p50 activation was inversely related to TBF (T4) and SAT (T3,T4), and leptin was positively related to TBF and SAT (T1-T4). Progesterone was inversely related to leptin (T2,T3), adiponectin (T3), TBF (T3,T4), and SAT (T2,T3,T4). By providing evidence of luteal phase-specific reduced NF-κB p65 activation in women under physiological conditions, this study bridges the gap between existing evidence of a Th1-Th2 immune response shift induced by reduced NF-κB p65 activation and a Th1-Th2 shift previously observed at luteal phase. For the first time, inverse regressions suggest inhibitory effects of leptin on NF-κB p65 activation at luteal phase, along with inhibitory effects of leptin as well as adiponectin on progesterone production in corpus luteum.

The Decrease in γ‐Tocopherol in Plasma and Lipoprotein Fractions Levels Off within Two Days of Vitamin E Supplementation

Abstract: The effects of vitamin E supplementation on α‐ and γ‐tocopherol concentrations were studied in plasma and lipoprotein fractions of five healthy volunteers taking 1000 IU/day of RRR α‐tocopherol for 4 days. Although plasma α‐tocopherol increased, γ‐tocopherol decreased. Compared with baseline, γ‐/α‐tocopherol ratios decreased from 48 h onward (P < 0.001). They all leveled off within 48 h. From 12 h onward, γ‐/α‐tocopherol ratios were higher in VLDL and IDL than in LDL and HDL, indicating that γ‐tocopherol is better maintained in triglyceride‐rich lipoprotein fractions. These data suggest that vitamin E supplementation exceeding 2 days does not further decrease γ‐tocopherol concentrations.