Christof Ulrich

The relationships of markers of cholesterol homeostasis with carotid intima-media thickness.

Background The relationship of cholesterol homeostasis and carotid intima-media thickness (cIMT) is unknown. To address this, we assessed markers of cholesterol homeostasis (serum plant sterols and cholesterol precursor concentrations as surrogate measures of cholesterol absorption and synthesis, respectively) and cIMT in a middle-aged, statin-naive population. Methods In this prospective study of primary prevention cIMT was measured by ultrasound in 583 hospital employees aged 25–60 years without prevalent cardiovascular disease or lipid-modifying medication. The serum concentrations of plant sterols (as markers of cholesterol absorption) were measured by gas-liquid chromatography. Lathosterol serum concentrations were quantitated to assess hepatic cholesterol synthesis. Results cIMT correlated positively with serum cholesterol (r = 0.22, P<0.0005) and lathosterol-to-cholesterol (r = 0.18, P<0.001). In contrast, plant sterols, as markers of cholesterol absorption, showed a weak negative correlation to cIMT measurements (r = −0.18; P<0.001 for campesterol-to-cholesterol). Stratifying subjects by serum sterol levels, we found that cIMT increased continuously over quintiles of serum cholesterol (P<0.0005) and was positively associated to serum lathosterol-to-cholesterol levels (P = 0.007), on the other hand, plant sterol levels showed a weak negative association to cIMT (P<0.001 for campesterol-to-cholesterol). Conclusions In this population without prevalent cardiovascular diseases or lipid-modifying medication, markers of increased endogenous cholesterol synthesis correlated positively with cIMT, while markers of cholesterol absorption showed a weakly negative correlation. These data suggest that not only total serum cholesterol levels but also differences in cholesterol homeostasis are associated with cIMT.

Extending the Spectrum of α-Dicarbonyl Compounds in Vivo

Background: α-Dicarbonyls are central intermediates in the formation of advanced glycation end products (AGEs). Results: A quantitation method for the complete spectrum relevant in vivo was established. Conclusion: Non-enzymatic chemistry of glucose and l-ascorbic acid as precursors and α-dicarbonyl intermediates play an important role in vivo. Significance: Knowledge of plasma levels of α-dicarbonyls is crucial to understand the complex pathways of AGE formation in vivo. Maillard α-dicarbonyl compounds are known as central intermediates in advanced glycation end product (AGE) formation. Glucose is the primary source of energy for the human body, whereas l-threo-ascorbic acid (vitamin C) is an essential nutrient, involved in a variety of enzymatic reactions. Thus, the Maillard degradation of glucose and ascorbic acid is of major importance in vivo. To understand the complex mechanistic pathways of AGE formation, it is crucial to extend the knowledge on plasma concentrations of reactive key α-dicarbonyl compounds (e.g. 1-deoxyglucosone). With the present work, we introduce a highly sensitive LC-MS/MS multimethod for human blood plasma based on derivatization with o-phenylenediamine under acidic conditions. The impact of workup and reaction conditions, particularly of pH, was thoroughly evaluated. A comprehensive validation provided the limit of detection, limit of quantitation, coefficients of variation, and recovery rates. The method includes the α-dicarbonyls 1-deoxyglucosone, 3-deoxyglucosone, glucosone, Lederers glucosone, dehydroascorbic acid, 2,3-diketogulonic acid, 1-deoxypentosone, 3-deoxypentosone, 3,4-dideoxypentosone, pentosone, 1-deoxythreosone, 3-deoxythreosone, threosone, methylglyoxal, glyoxal; the α-keto-carboxylic acids pyruvic acid and glyoxylic acid; and the dicarboxylic acid oxalic acid. The method was then applied to the analyses of 15 healthy subjects and 24 uremic patients undergoing hemodialysis. The comparison of the results revealed a clear shift in the product spectrum. In most cases, the plasma levels of target analytes were significantly higher. Thus, this is the first time that a complete spectrum of α-dicarbonyl compounds relevant in vivo has been established. The results provide further insights into the chemistry of AGE formation and will be helpful to find specific markers to differentiate between the various precursors of glycation.

Monocyte Angiotensin Converting Enzyme Expression May Be Associated with Atherosclerosis Rather than Arteriosclerosis in Hemodialysis Patients

BACKGROUND AND OBJECTIVES Circulating monocytes can be divided into functionally distinct subpopulations according to their surface expression of CD14 and CD16. Monocytes with high-level expression of both antigens (CD14(++)CD16(+), Mo2 cells) are associated with cardiovascular morbidity and mortality in hemodialysis patients. These cells express angiotensin converting enzyme (ACE) on their surface. They are involved in the association of chronic inflammation and cardiovascular disease in kidney patients. Cardiovascular morbidity results from atherosclerosis (plaque-forming, vessel occluding disease) and arteriosclerosis (loss of arterial dampening function). It is unknown whether ACE-expressing proinflammatory monocytes are related to atherosclerosis, arteriosclerosis, or both. DESIGN, SETTING, PARTICIPANTS, & MEASUREMENTS During baseline examination for a prospective study on monocyte ACE expression and mortality, 60 chronic hemodialysis patients of an academic outpatient center were screened for atherosclerosis by carotid artery ultrasound, for arteriosclerosis by pulse pressure measurement, and for ACE expression on Mo2 cells by flow cytometry. RESULTS ACE expression on Mo2 monocytes was significantly higher in patients with severe compared with those with little or no carotid atherosclerosis. Mo2 ACE correlated with a score to semiquantify atherosclerosis and remained a significant predictor of carotid plaques in multivariate analysis including the other univariately associated variables of age, hemoglobin A1c, and albumin. Mo2 ACE was not related to pulse pressure. CONCLUSIONS ACE expression on Mo2, although being a known predictor of mortality and cardiovascular disease in end-stage renal disease patients, may act via enhancement of atherosclerosis rather than arteriosclerosis.

Differential effects on inhibition of cholesterol absorption by plant stanol and plant sterol esters in apoE−/− mice

Aims ‘Functional foods’ supplemented with plant sterol esters (PSE) and plant stanol esters (PSA) are therapeutic options for the management of hypercholesterolaemia. However, their effects on blood monocytes, endothelial function, atherogenesis, and sterol tissue concentrations are poorly understood. Methods and results Male apoE−/− mice (n= 30) were randomized to three different diets for 6 weeks (n= 10 per group): high-cholesterol (1.25%) western-type diet (WTD), WTD + 2% PSE, and WTD + 2% PSA. Both supplements reduced serum cholesterol. WTD + PSE resulted in increased plant sterol serum concentrations and increased inflammatory Ly-6C(high) monocyte numbers. WTD + PSA increased plant stanol serum concentrations and Ly-6C-monocyte numbers, but decreased vascular superoxide release, lipid hydroperoxides, and inflammatory cytokines in aortic tissue, in plasma, and in circulating monocytes. Despite reduced serum cholesterol concentrations, both supplements impaired endothelial vasodilation compared with WTD. WTD + PSA reduced the development of atherosclerotic lesions compared with WTD alone (12.7 ± 3.7 vs. 28.3 ± 3.5%), and WTD + PSE was less effective (17.5 ± 3.7%). WTD + PSE and WTD + PSA reduced the cholesterol content in the liver, but not in the brain. However, WTD + PSE and WTD + PSA increased plant sterol and plant stanol concentrations in the liver as well as in the brain. Conclusion PSE and PSA supplementation reduced serum cholesterol, but increased plant sterol and plant stanol concentrations. Elevated levels of PSE and PSA were associated with endothelial dysfunction and increased central nervous system depositions. Atherosclerotic lesion retardation was more pronounced in WTD + PSA, coinciding with higher regenerative monocyte numbers, decreased oxidative stress, and decreased inflammatory cytokines compared with WTD + PSE.

Is AV Fistula Patency Associated with Angiotensin-Converting Enzyme (ACE) Polymorphism and ACE Inhibitor Intake?

Background/Aims: Hemodialysis treatment requires a well-functioning vascular access. Access patency is limited by the development of venous intimal hyperplasia, which predisposes to fistula stenosis and subsequent thrombosis. In animal models, the renin-angiotensin system has a major role in the development of intimal hyperplasia. We investigated the association of the insertion/deletion polymorphism of the angiotensin-converting enzyme (ACE) and arteriovenous fistula patency in hemodialysis patients. Methods: In a longitudinal study, 137 hemodialysis patients who had undergone creation of a primary AV fistula were genotyped. The main study endpoint was unassisted access patency (time from fistula placement to the first episode of access failure). In addition, the intake of drugs blocking the renin-angiotensin system was assessed. Results: Fistula patency 12 months after fistula creation was 72% (DD patients), 65% (ID patients), and 73% (II patients; p = 0.40). Long-term intake of ACE inhibitors or AT-1 antagonists failed to increase fistula patency (p = 0.33). Conclusions: We suggest that pharmacological inhibition of the renin-angiotensin system is of limited value for prevention of arteriovenous fistula stenosis. Alternative strategies to prolong fistula patency should be studied.

Molecular Basis of Maillard Amide-Advanced Glycation End Product (AGE) Formation in Vivo

Background: Advanced glycation end products (AGEs) play a prominent role in various pathophysiologies. Results: A new class of lysine amide modifications was established in vivo. Conclusion: Non-enzymatic Maillard mechanisms participate on amide-AGE formation pathways in vivo. Significance: Plasma levels of the amide-AGEs were in the same range similar to other established lysine modifications and suggest a comparable impact of non-enzymatic biochemistry on pathophysiologies in the human organism. The Maillard reaction in vivo entails alteration of proteins or free amino acids by non-enzymatic glycation or glycoxidation. The resulting modifications are called advanced glycation end products (AGEs) and play a prominent role in various pathologies, including normoglycemic uremia. Recently, we established a new class of lysine amide modifications in vitro. Now, human plasma levels of the novel amide-AGEs N6-acetyl lysine, N6-formyl lysine, N6-lactoyl lysine, and N6-glycerinyl lysine were determined by means of LC-MS/MS. They were significantly higher in uremic patients undergoing hemodialysis than in healthy subjects. Model reactions with N1-t-butoxycarbonyl-lysine under physiological conditions confirmed 1-deoxy-d-erythro-hexo-2,3-diulose as an immediate precursor. Because formation of N6-formyl lysine from glucose responded considerably to the presence of oxygen, glucosone was identified as another precursor. Comparison of the in vivo results with the model experiments enabled us to elucidate possible formation pathways linked to Maillard chemistry. The results strongly suggest a major participation of non-enzymatic Maillard mechanisms on amide-AGE formation pathways in vivo, which, in the case of N6-acetyl lysine, parallels enzymatic processes.

Gene Polymorphism Association Studies in Dialysis: Vascular Access

Complications of the vascular access site for hemodialysis are a major cause of morbidity, suboptimal dialysis, and hospitalization. Vascular access for dialysis that is achieved by central venous catheters is associated with complications such as infection and thrombosis. Arteriovenous fistulas and grafts are also at risk for infectious complications. Further, proliferation of the venous wall with secondary thrombosis is a common pathophysiological process that leads to vascular access dysfunction. Genetic polymorphisms that contribute to vascular access failure are found among factors of the coagulation cascade, and host mediators that induce endothelial dysfunction as well as vessel wall proliferation. The two most common mutations of coagulation factors seem to influence the risk of central venous catheter and fistula thrombosis. Indeed, both the single nucleotide polymorphism of the factor V gene at amino acid position 506 (factor V Leiden mutation) and the prothrombin 20210 polymorphism have been associated with thrombotic complications of the vascular access. Among the endothelium‐directed factors, a polymorphism of the methylene tetrahydrofolate reductase gene coding for an enzyme that degrades the endothelium toxic product homocysteine, has been associated with fistula failure. While the angiotensin converting enzyme polymorphism does not seem to be associated with vascular access complications, polymorphisms of the profibrogenic cytokine transforming growth factor‐β1 are associated with the prognosis of native arteriovenous fistulae. The role of pro‐ and anti‐inflammatory cytokine gene polymorphisms as prognostic factors for vascular access is yet to be clearly defined.

Influence of Cholecalciferol Supplementation in Hemodialysis Patients on Monocyte Subsets: A Randomized, Double-Blind, Placebo-Controlled Clinical Trial

Background/Aims: Although most hemodialysis patients share a significant 25-hydroxyvitamin D [25(OH)D] deficiency, supplementation is controversially discussed. A potential influence on monocyte and T lymphocyte dysfunction advocates blood level-adapted supplementation as recommended by K/DOQI guidelines. This was a prospective double-blind randomized placebo controlled trial examining immune effects of 12 weeks of cholecalciferol supplementation. Methods: We initiated serum level-adapted de novo cholecalciferol supplementation in 38 hemodialysis patients. Outcome measures were: monocyte subset cell counts (CD14+CD16++ vs. CD14++CD16+ vs. CD14++CD16-), lymphocyte Th1/Th2 differentiation frequencies, serum inflammatory proteins CRP and TNFα, parathyroid hormone (PTH), FGF-23, and α-Klotho. Results: At baseline, the mean 25(OH)D serum level in the study population was 31.7 ± 14.3 nmol/l, and only 3% of patients had levels within the normal range. At 12 weeks, 25(OH)D levels were normalized in the verum group (87.8 ± 22.3 vs. placebo 24.6 ± 8.0 nmol/l, p < 0.0001). In parallel, 1,25(OH)2D levels increased in the verum group. Monocyte subset cell counts as well as Th1 and Th2 lymphocyte frequencies did not change significantly after 12 weeks of cholecalciferol supplementation. There was also no significant difference in PTH, alkaline phosphatase, calcium, phosphate, TNFα, FGF-23, α-Klotho and CRP levels. Conclusions: Oral cholecalciferol supplementation according to the K/DOQI recommendations normalizes 25(OH)D levels without relevant side effects such as hyperphosphatemia or hypercalcemia. However, beneficial pleiotropic effects on monocyte subset cell counts, T cell differentiation, or cytokine production could not be confirmed at least at the used dosage. PTH and FGF23 levels were not affected during cholecalciferol administration.

High cut-off dialysis in chronic haemodialysis patients

Haemodialysis patients suffer from chronic systemic inflammation and high incidence of cardiovascular disease. One cause for this may be the failure of diseased kidneys to eliminate immune mediators. Current haemodialysis treatment achieves insufficient elimination of proteins in the molecular weight range 15–45 kD. Thus, high cut‐off dialysis might improve the inflammatory state.

Medium Cut-Off (MCO) Membranes Reduce Inflammation in Chronic Dialysis Patients—A Randomized Controlled Clinical Trial

Background To increase the removal of middle-sized uremic toxins a new membrane with enhanced permeability and selectivity, called Medium Cut-Off membrane (MCO-Ci) has been developed that at the same time ensures the retention of albumin. Because many middle-sized substances may contribute to micro-inflammation we hypothesized that the use of MCO-Ci influences the inflammatory state in hemodialysis patients. Methods The randomized crossover trial in 48 patients compared MCO-Ci dialysis to High-flux dialysis of 4 weeks duration each plus 8 weeks extension phase. Primary endpoint was the gene expression of TNF-α and IL-6 in peripheral blood mononuclear cells (PBMCs), secondary endpoints were plasma levels of specified inflammatory mediators and cytokines. Results After four weeks of MCO-Ci the expression of TNF-α mRNA (Relative quantification (RQ) from 0.92 ± 0.34 to 0.75 ± 0.31, -18.5%, p<0.001)-α and IL-6 mRNA (RQ from 0.78 ± 0.80 to 0.60 ± 0.43, -23.1%, p<0.01) was reduced to a significantly greater extent than with High-flux dialyzers (TNF mRNA-RQ: -14.3%; IL-6 mRNA-RQ: -3.5%). After retransformation of logarithmically transformed data, measurements after MCO were reduced to 82% of those after HF (95% CI 74%–91%). 4 weeks use of MCO-Ci resulted in long-lasting change in plasma levels of several cytokines and other substances with a significant decrease for sTNFR1, kappa and lambda free light chains, urea and an increase for Lp-PLA2 (PLA2G7) compared to High-flux. Albumin levels dropped significantly after 4 weeks of MCO dialysis but increased after additional 8 weeks of MCO dialysis. Twelve weeks treatment with MCO-Ci was well tolerated regarding the number of (S)AEs. In the extension period levels of CRP, TNF-α-mRNA and IL-6 mRNA remained stable in High-flux as well as in MCO-Ci. Conclusions MCO-Ci dialyzers modulate inflammation in chronic HD patients to a greater extent compared to High-flux dialyzers. Transcription of pro-inflammatory cytokines in peripheral leukocytes is markedly reduced and removal of soluble mediators is enhanced with MCO dialysis. Serum albumin concentrations stabilize after an initial drop. These results encourage further trials with longer treatment periods and clinical endpoints.