Katrin Borucki

B Vitamins and the Risk of Total Mortality and Cardiovascular Disease in End-Stage Renal Disease Results of a Randomized Controlled Trial

Background— In observational studies, hyperhomocysteinemia has been found to be a risk factor for total mortality and cardiovascular events in patients with end-stage renal disease. These patients have grossly elevated homocysteine levels that can be lowered by supplementation with folic acid and vitamin B12. We conducted a randomized clinical trial with B vitamins to reduce homocysteine levels and therefore cardiovascular events and total mortality. Methods and Results— This randomized, double-blind multicenter study was conducted in 33 dialysis centers in north and east Germany between July 2002 and July 2008. We randomly assigned 650 patients with end-stage renal disease who were undergoing hemodialysis to 2 postdialysis treatments: 5 mg folic acid, 50 &mgr;g vitamin B12, and 20 mg vitamin B6 (active treatment) or 0.2 mg folic acid, 4 &mgr;g vitamin B12, and 1.0 mg vitamin B6 (placebo) given 3 times per week for an average of 2 years. The primary outcome was total mortality; the secondary outcome was fatal and nonfatal cardiovascular events. The primary outcome occurred in 102 patients (31%) receiving the active treatment and in 92 (28%) receiving placebo (hazard ratio, 1.13; 95% confidence interval, 0.85 to 1.50; P=0.51). The secondary outcome occurred in 83 patients (25%) receiving the active treatment and in 98 (30%) receiving placebo (hazard ratio, 0.80; 95% confidence interval, 0.60 to 1.07; P=0.13). Conclusions— Increased intake of folic acid, vitamin B12, and vitamin B6 did not reduce total mortality and had no significant effect on the risk of cardiovascular events in patients with end-stage renal disease. Clinical Trial Registration— URL: www.anzctr.org.au. Unique identifier: ACTRN12609000911291. URL: www.cochrane-renal.org. Unique identifier: CRG010600027.

Detection of Recent Ethanol Intake With New Markers: Comparison of Fatty Acid Ethyl Esters in Serum and of Ethyl Glucuronide and the Ratio of 5-Hydroxytryptophol to 5-Hydroxyindole Acetic Acid in Urine

BACKGROUND At present, recent ethanol consumption can be routinely detected with certainty only by direct measurement of ethanol concentration in blood or urine. Because ethanol is rapidly eliminated from the circulation, however, the time span for this detection is in the range of hours. Several new markers have been proposed to extend the detection interval, but their characteristics have not yet justified their use in routine clinical practice. We therefore investigated three new markers and compared their kinetics and sensitivities: (1) fatty acid ethyl esters (FAEEs) in serum, (2) ethyl glucuronide (EtG) in urine, and (3) the ratio of 5-hydroxytryptophol to 5-hydroxyindole acetic acid (5-HTOL/5-HIAA) in urine. METHODS Seventeen healthy men participated in a drinking experiment. Blood and urine samples were collected twice daily on three consecutive days and once daily on days 4 and 5. Ethanol concentration was determined by gas chromatography, FAEE levels, by gas chromatography with mass spectrometry, EtG concentration, by liquid chromatography-tandem mass spectrometry, and 5-HTOL/5-HIAA ratio, by high-performance liquid chromatography. RESULTS The peak serum ethanol concentrations of the subjects ranged from 5.4 to 44.7 mmol/liter (mean +/- SD, 30.1 +/- 9.1 mmol/liter). In the case of the serum ethanol determination, 100% sensitivity was reached only immediately after the end of the drinking experiment, and in the case of FAEE levels and 5-HTOL/5-HIAA ratio, it tested for 6.7 hr after the end of the ethanol intake. Thereafter, these latter parameters declined until 15.3 hr (FAEEs) and 29.4 hr (5-HTOL/5-HIAA), subsequently remaining in a stable range until 78.5 hr without further decrease. In contrast, EtG concentration showed 100% sensitivity until 39.3 hr and thereafter decreased, falling to below the limit of quantification of 0.1 mg/liter at 102.5 hr. CONCLUSION After moderate drinking, EtG in the urine proved to be a superior marker of recent ethanol consumption in healthy subjects. This is because EtG is a direct ethanol metabolite, it occurs in the urine only when ethanol has been consumed, and its sensitivity remains at the level of 100% for 39.3 hr.

Rapid immunomagnetic negative enrichment of neutrophil granulocytes from murine bone marrow for functional studies in vitro and in vivo.

Polymorphonuclear neutrophils (PMN) mediate early immunity to infection but can also cause host damage if their effector functions are not controlled. Their lack or dysfunction is associated with severe health problems and thus the analysis of PMN physiology is a central issue. One prerequisite for PMN analysis is the availability of purified cells from primary organs. While human PMN are easily isolated from peripheral blood, this approach is less suitable for mice due to limited availability of blood. Instead, bone marrow (BM) is an easily available reservoir of murine PMN, but methods to obtain pure cells from BM are limited. We have developed a novel protocol allowing the isolation of highly pure untouched PMN from murine BM by negative immunomagnetic isolation using a complex antibody cocktail. The protocol is simple and fast (∼1 h), has a high yield (5–10*106 PMN per animal) and provides a purity of cells equivalent to positive selection (>80%). Most importantly, cells obtained by this method are non-activated and remain fully functional in vitro or after adoptive transfer into recipient animals. This method should thus greatly facilitate the study of primary murine PMN in vitro and in vivo.

Haplotypes in the UGT1A1 gene and their role as genetic determinants of bilirubin concentration in healthy German volunteers

BACKGROUND Genetic variations of UDP-glucuronyltransferase 1A1 (UGT1A1) influence the concentration of serum bilirubin. We investigated the association of four common polymorphisms including UGT1A1-53(TA)(n), and common haplotypes of the UGT1A1 gene with bilirubin levels in 218 Caucasian volunteers. METHODS Total bilirubin was measured in serum of 218 healthy Caucasian volunteers. Genotyping of four genetic variants was performed: UGT1A1-53(TA)(6/7), UGT1A1c.-3279T>G, UGT1A1c.-3156G>A, and UGT1A1c.211G>A. The association between polymorphisms/haplotypes and bilirubin levels were determined. RESULTS Minor allele frequencies were 0.36 for UGT1A1-53(TA)(7), 0.47 for c.-3279G, 0.33 for c.-3156A and 0.006 for c.211A. The three promoter polymorphisms were in close linkage disequilibrium. Common haplotypes were: -53(TA)(6)/c.-3279T/c.211G (frequency 0.530), -53(TA)(7)/c.-3279G/c.211G (frequency 0.365), and -53(TA)(6)/c.-3279G/c.211G (frequency 0.099). Male sex, UGT1A1-53(TA)(6/7) and the c.-3279GG variant were significantly associated with higher bilirubin concentrations. CONCLUSIONS Two UGT1A1 promoter polymorphisms (-53(TA)(6/7) and c.-3279T>G) and a common haplotype of the UGT1A1 gene are associated with serum bilirubin concentrations in Caucasians.

Addition of 2.5 g L-arginine in a fatty meal prevents the lipemia-induced endothelial dysfunction in healthy volunteers

BACKGROUND Endothelial function as determined by flow-mediated dilation (FMD) deteriorates during postprandial lipemia. The impairment can be neutralized by adding 50 g casein to the fatty meal. The objective of this study was to see which of the casein constituents is responsible for this neutralization effect. DESIGN A crossover design was used in 15 healthy volunteers, who were given 3 ml cream/kg body weight with and without 2.5 g L-arginine. Lipids and FMD were then determined hourly over a period of 6h. In substudies this design was repeated with 2.5 g phenylalanine and 5 g leucine in 11 subjects. The effect of 2.5 g L-arginine on FMD in the absence of cream was tested in seven subjects. RESULTS Without L-arginine the FMD deteriorated during 4h after the fat loading. Addition of L-arginine prevented this deterioration at 3 and 4h. Phenylalanine and leucine, in contrast, did not prevent the lipemic endothelial dysfunction. L-Arginine alone similarly had no acute effect on the FMD. CONCLUSION 2.5 g L-arginine, but not phenyalanine or leucine, is able to prevent the lipemic dysfunction of the endothelium.

In Heavy Drinkers Fatty Acid Ethyl Esters in the Serum Are Increased for 44 hr After Ethanol Consumption

BACKGROUND Fatty acid ethyl esters (FAEEs) have been proposed as a marker of ethanol consumption because they can be detected for up to 24 hr after a moderate intake of ethanol, even though blood ethanol remains increased for only 8 hr. Therefore, this study investigated whether FAEEs can be found during a time period exceeding 24 hr in a group of patients who were hospitalized for ethanol detoxification. A second aim was to study the distribution of FAEEs between lipoproteins during that time. METHODS Serum samples of 12 patients with acute ethanol intoxication were assayed for FAEEs. Blood samples were drawn 8.2, 20.2, 32.2, and 44.2 hr after hospitalization. FAEEs were quantified by gas chromatography-mass spectrometry. RESULTS Ethanol was no longer detectable after 20.2 hr from hospitalization, whereas FAEEs were still found after 32.2 and 44.2 hr. These late FAEEs were significantly higher than the FAEEs in 15 different healthy men who had abstained from ethanol for 4.5 days (p < 0.001 and p = 0.001). FAEEs were associated mainly with lipid-free serum but tended to accumulate in very-low-density lipoprotein in patients with moderate hypertriglyceridemia. CONCLUSIONS In heavy drinkers, the FAEEs were increased after ethanol consumption for at least 44 hr. It remains to be studied whether they originate from a single ethanol intake or, in addition, from a slow release out of body storage compartments.

A20 expression in dendritic cells protects mice from LPS‐induced mortality

DCs contribute to immune homeostasis under physiological conditions and regulate the immune activation during infection. The deubiquitinase A20 inhibits the activation of NF‐κB‐dependent immune reactions, and prevents the hyperactivation of DCs under steady‐state conditions. However, the role of DC‐specific A20 under pathological conditions is unknown. Here, we demonstrate that upon injection of low‐dose LPS, mice with DC‐specific A20 deletion (CD11c‐Cre A20fl/fl) died within 6 h, whereas A20fl/fl controls survived. LPS‐induced mortality in CD11c‐Cre A20fl/fl mice was characterized by increased serum levels of IL‐2, IL‐10, IL‐12, IFN‐γ, and TNF. Upon LPS stimulation, the activation of NF‐κB and ERK‐NFATc3 pathways were enhanced in A20‐deficient DCs, resulting in an increased production of IL‐2, IL‐12, and TNF both in vitro and in vivo. Targeted inhibition of ERK in A20‐deficient DCs abolished the increased production of IL‐2. A20‐deficient DCs failed to induce LPS tolerance, which was independent of T cells and the intestinal flora, since T‐cell depletion and decolonization of CD11c‐Cre A20fl/fl mice could not prevent death of LPS‐challenged CD11c‐Cre A20fl/fl mice. In conclusion, these findings show that DC‐specific A20 preserves immune homeostasis in steady‐state conditions and is also required for LPS tolerance.

Serum bile acids and GLP-1 decrease following telemetric induced weight loss: Results of a randomized controlled trial

Bile acids (BAs) are increasingly recognised as metabolic regulators, potentially improving insulin sensitivity following bariatric surgery. However, physiological relevance of such observations remains unknown. Hence, we analysed serum BA composition and associated gut-derived hormone levels following lifestyle-induced weight loss in individuals with metabolic syndrome (MetS). 74 non-smoking men (45–55 yr) with MetS were randomised to a lifestyle-induced weight loss program (supervision via telemonitoring) or to a control arm. Before and after a 6 months intervention period clinical and laboratory parameters, body composition, serum BA profile, FGF-19, and GLP-1 concentrations were determined in fasting blood samples. 30 participants in the control and 33 participants in the treatment arm completed the study and were included in the data analysis. In participants of the treatment arm lifestyle-induced weight loss resulted in markedly improved insulin sensitivity. Serum levels of BA species and total GLP-1 decreased, while FGF-19 remained stable. Serum BA composition changed towards an increased 12α-hydroxylated/non-12α-hydroxylated ratio. None of these parameters changed in participants of the control arm. Our results demonstrate that improved metabolic control by lifestyle modifications lowers serum levels of BAs and GLP-1 and changes serum BA composition towards an increased 12α/non-12α ratio (ICTRP Trial Number: U1111-1158-3672).

Decrease of serum S100B during an oral glucose tolerance test correlates inversely with the insulin response

Increased S100B serum levels have been considered as a marker of glial pathology, brain damage, and blood-brain-barrier impairment. However, S100B expression has also been detected outside the nervous system, suggesting that altered S100B serum levels may not exclusively reflect brain-specific pathologies. Notably, S100B secretion in adipocytes seems to be down-regulated by insulin, and up-regulated by stress and fasting. Therefore, we assumed that dynamic changes of S100B could be observed by challenging healthy subjects with an oral glucose tolerance test (OGTT). OGTT was performed in 17 healthy adult test persons (9 male and 8 female). Apart from S100B, glucose, free fatty acids, insulin, C-peptide, and cortisol were determined in all samples after an overnight fast (0 h), as well as 1h and 2h after ingestion of 75 g glucose. Mean S100B concentrations decreased about 20% during the first hour after glucose ingestion (P<0.001). This decrease of S100B levels was not related to the declining morning peak of cortisol. However, the decrease of serum-S100B 1h after glucose ingestion correlated inversely with the respective changes of serum-insulin (r = -0.484, P=0.049) and serum-C-peptide (r = -0.570, P = 0.017). Our study suggests an inverse correlation between insulin secretion and S100B release after a standardized OGTT. Additional experiments, including the administration of insulin and the measurement of other food intake-related factors are important to ascertain an insulin-regulated S100B release in vivo. To improve comparability between clinical studies assessing conditions with rather mild changes of serum S100B, blood should be taken in a more standardized way (e.g., after fasting overnight).

Glucose homeostasis in major depression and schizophrenia: a comparison among drug-naïve first-episode patients

There is evidence for insulin resistance in drug-naïve first-episode schizophrenia (Sz) patients. We have tested whether impaired insulin homeostasis is also present in first-episode patients with major depression (MD) and if this can be discerned from stress-related and medication effects. Homeostatic model assessment of insulin resistance (HOMA-IR) was determined in a cross-sectional cohort study of acute first-episode drug-naïve patients with MD (n = 18) or Sz (n = 24), and healthy controls (C, n = 43). Morning cortisol and catecholamine metabolites were assessed to control for hormonal stress axis activation. Subjects were matched for sex, age, body mass index and waist–hip ratio to exclude the possibility that overweight and visceral adiposity were potential confounding factors. HOMA-IR did not differ between MD and controls, but was increased in Sz compared to MD (p = 0.002) and controls (p = 0.012). Catecholamine metabolites were elevated in both patient groups, indicating presence of hormonal stress axis activation. However, diagnosis-related changes of HOMA-IR were independent from this. Impaired insulin sensitivity was absent in MD, but specifically related to the early disease course of Sz. Thus, considering previous studies in this field, MD may be related to impaired glucose/insulin homeostasis in the long-term but not in early disease stages.