Christian Götting

Vitamin D supplementation enhances the beneficial effects of weight loss on cardiovascular disease risk markers

BACKGROUND High blood concentrations of parathyroid hormone and low concentrations of the vitamin D metabolites 25-hydroxyvitamin D [25(OH)D] and calcitriol are considered new cardiovascular disease risk markers. However, there is also evidence that calcitriol increases lipogenesis and decreases lipolysis. OBJECTIVE We investigated the effect of vitamin D on weight loss and traditional and nontraditional cardiovascular disease risk markers in overweight subjects. DESIGN Healthy overweight subjects (n = 200) with mean 25(OH)D concentrations of 30 nmol/L (12 ng/mL) received vitamin D (83 microg/d) or placebo in a double-blind manner for 12 mo while participating in a weight-reduction program. RESULTS Weight loss was not affected significantly by vitamin D supplementation (-5.7 +/- 5.8 kg) or placebo (-6.4 +/- 5.6 kg). However, mean 25(OH)D and calcitriol concentrations increased by 55.5 nmol/L and 40.0 pmol/L, respectively, in the vitamin D group but by only 11.8 nmol/L and 9.3 pmol/L, respectively, in the placebo group (P < 0.001), whereas a more pronounced decrease occurred in the vitamin D group than in the placebo group in blood concentrations of parathyroid hormone (-26.5% compared with -18.7%; P = 0.014), triglycerides (-13.5% compared with +3.0%; P < 0.001), and the inflammation marker tumor necrosis factor-alpha (-10.2% compared with -3.2%; P = 0.049). The beneficial biochemical effects were independent of the loss in body weight, fat mass, and sex. However, compared with placebo, vitamin D supplementation also increased LDL-cholesterol concentrations (+5.4% compared with -2.5%; P < 0.001). CONCLUSIONS The results indicate that a vitamin D supplement of 83 microg/d does not adversely affect weight loss and is able to significantly improve several cardiovascular disease risk markers in overweight subjects with inadequate vitamin D status participating in a weight-reduction program. This trial was registered at clinicaltrials.gov as NCT00493012.

Poor outcome in end‐stage heart failure patients with low circulating calcitriol levels

Vitamin D receptor knockout mice develop typical signs of congestive heart failure (CHF). In approximately 20% of stable CHF patients, frankly low concentrations of the vitamin D hormone calcitriol are found.

Effects of n–3 fatty acids on macro- and microvascular function in subjects with type 2 diabetes mellitus

BACKGROUND Recent evidence supports the protective effects of n-3 (omega-3) fatty acids (n-3 FAs), such as eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), on vascular function. OBJECTIVE We investigated the effects of EPA and DHA on postprandial vascular function in subjects with type 2 diabetes mellitus. DESIGN In a double-blind, placebo-controlled, randomized, crossover manner, 34 subjects with type 2 diabetes mellitus received daily either 2 g purified EPA/DHA (termed n-3 FAs) or olive oil (placebo) for 6 wk. At the end of this period, we measured macrovascular (brachial ultrasound of flow-mediated dilatation; FMD) and microvascular [laser-Doppler measurements of reactive hyperemia (RH) of the hand] function at fasting and 2, 4, and 6 h after a high-fat meal (600 kcal, 21 g protein, 41 g carbohydrates, 40 g fat). RESULTS Fasting vascular function remained unchanged after n-3 FAs and placebo. Postprandial FMD decreased from fasting after placebo, with a maximum decrease (38%) at 4 h-an effect that was significantly reduced (P = 0.03 for time x treatment interaction) by n-3 FA supplementation (maximum decrease in FMD was at 4 h: 13%). RH remained unchanged after placebo, whereas it improved significantly (P = 0.04 for time x treatment interaction) after n-3 FA supplementation (maximum increase was at 2 h: 27%). CONCLUSIONS In subjects with type 2 diabetes mellitus, 6 wk of supplementation with n-3 FAs reduced the postprandial decrease in macrovascular function relative to placebo. Moreover, n-3 FA supplementation improved postprandial microvascular function. These observations suggest a protective vascular effect of n-3 FAs.

First Isolation of Human UDP-d-Xylose: Proteoglycan Core Protein β-d-Xylosyltransferase Secreted from Cultured JAR Choriocarcinoma Cells

Human UDP-d-xylose:proteoglycan core protein β-d-xylosyltransferase (EC 2.4.2.26, XT) initiates the biosynthesis of glycosaminoglycan lateral chains in proteoglycans by transfer of xylose from UDP-xylose to specific serine residues of the core protein. In this study, we report the first isolation of the XT and present the first partial amino acid sequence of this enzyme. We purified XT 4,700-fold with 1% yield from serum-free JAR choriocarcinoma cell culture supernatant. The isolation procedure included a combination of ammonium sulfate precipitation, heparin affinity chromatography, ion exchange chromatography, and protamine affinity chromatography. Among other proteins an unknown protein was detected by matrix-assisted laser desorption ionization mass spectrometry-time of flight analysis in the purified sample. The molecular mass of this protein was determined as 120 kDa by SDS-polyacrylamide gel electrophoresis. The isolated protein was enzymatically cleaved by trypsin and endoproteinase Lys-C. Eleven peptide fragments were sequenced by Edman degradation. Searches with the amino acid sequences in protein and EST data bases showed no homology to known sequences. XT was enriched by immunoaffinity chromatography with an immobilized antibody against a synthetic peptide deduced from the sequenced peptide fragments and was specifically eluted with the antigen. In addition, XT was purified alternatively with an aprotinin affinity chromatography and was detected by Western blot analysis in the enzyme-containing fraction.

Autologous stem cell therapy in the treatment of limb ischaemia induced chronic tissue ulcers of diabetic foot patients.

Background and aim:  Despite improvements in surgical revascularisation, limitations like anatomical factors or atherosclerosis limit the success of revascularisation in diabetic patients with critical limb ischaemia. Stem cells were shown to improve microcirculation in published studies. The aim of this study was to evaluate safety, feasibility and efficacy of transplantation of bone marrow derived cellular products regarding improvement in microcirculation and lowering of amputation rate.

Human Xylosyltransferase II Is Involved in the Biosynthesis of the Uniform Tetrasaccharide Linkage Region in Chondroitin Sulfate and Heparan Sulfate Proteoglycans

Human xylosyltransferase I (XT-I) initiates the biosynthesis of the glycosaminoglycan (GAG) linkage tetrasaccharide in proteoglycans. Xylosyltransferase II (XT-II) is a protein homologous to XT-I but with hitherto unknown activity or physiological function. Here, we report the enzymatic activity of XT-II and provide evidence that XT-II initiates the biosynthesis of both heparan sulfate and chondroitin sulfate GAGs. Transfection of the xylosyltransferase-deficient Chinese hamster ovary mutant pgsA-745 with XT-I or XT-II coding cDNA completely restored GAG biosynthesis. GAG disaccharide analysis revealed that XT-I- and XT-II-transfected pgsA-745 cells produced similar amounts of chondroitin sulfate and heparan sulfate. Furthermore, a high xylosyltransferase activity was measured after transfection with cDNAs encoding either isozyme. Analysis of the enzyme activity revealed that XT-II catalyzes the transfer of xylose to similar peptide acceptors as XT-I but with different efficiency. The optimal XT-II acceptor was observed using a bikunin-related peptide (Km 5.2 μm). Analysis of XT-I and XT-II mRNA expression in murine tissues showed a differential expression pattern for both enzymes. In particular, XT-II is highly expressed in liver tissue, where XT-I transcripts were not detected. This is the first report on the enzyme activity of XT-II and its involvement in chondroitin sulfate and heparan sulfate biosynthesis.

A randomized controlled trial on the efficacy of carbohydrate-reduced or fat-reduced diets in patients attending a telemedically guided weight loss program

BackgroundWe investigated whether macronutrient composition of energy-restricted diets influences the efficacy of a telemedically guided weight loss program.MethodsTwo hundred overweight subjects were randomly assigned to a conventional low-fat diet and a low-carbohydrate diet group (target carbohydrate content: >55% energy and <40% energy, respectively). Both groups attended a weekly nutrition education program and dietary counselling by telephone, and had to transfer actual body weight data to our clinic weekly with added Bluetooth® technology by mobile phone. Various fatness and fat distribution parameters, energy and macronutrient intake, and various biochemical risk markers were measured at baseline and after 6, and 12 months.ResultsIn both groups, energy intake decreased by 400 kcal/d compared to baseline values within the first 6 months and slightly increased again within the second 6 months. Macronutrient composition differed significantly between the groups from the beginning to month 12. At study termination, weight loss was 5.8 kg (SD: 6.1 kg) in the low-carbohydrate group and 4.3 kg (SD: 5.1 kg) in the low-fat group (p = 0.065). In the low-carbohydrate group, triglyceride and HDL-cholesterol levels were lower at month 6 and waist circumference and systolic blood pressure were lower at month 12 compared with the low-fat group (P = 0.005–0.037). Other risk markers improved to a similar extent in both groups.ConclusionDespite favourable effects of both diets on weight loss, the carbohydrate-reduced diet was more beneficial with respect to cardiovascular risk factors compared to the fat-reduced diet. Nevertheless, compliance with a weight loss program appears to be even a more important factor for success in prevention and treatment of obesity than the composition of the diet.Trial registrationClinicaltrials.gov as NCT00868387

Cloning and recombinant expression of active full-length xylosyltransferase I (XT-I) and characterization of subcellular localization of XT-I and XT-II.

Xylosyltransferase I (XT-I) catalyzes the transfer of xylose from UDP-xylose to serine residues in proteoglycan core proteins. This is the first and apparently rate-limiting step in the biosynthesis of the tetrasaccharide linkage region in glycosaminoglycan-containing proteoglycans. The XYLT-II gene codes for a highly homologous protein, but its physiological function is not yet known. Here we present for the first time the construction of a vector encoding the full-length GFP-tagged human XT-I and the recombinant expression of the active enzyme in mammalian cells. We expressed XT-I-GFP and various GFP-tagged XT-I and XT-II mutants with C-terminal truncations and deletions in HEK-293 and SaOS-2 cells in order to investigate the intracellular localization of XT-I and XT-II. Immunofluorescence analysis showed a distinct perinuclear pattern of XT-I-GFP and XT-II-GFP similar to that of α-mannosidase II, which is a known enzyme of the Golgi cisternae. Furthermore, a co-localization of native human XT-I and α-mannosidase II could also be demonstrated in untransfected cells. Using brefeldin A, we could also show that both xylosyltransferases are resident in the early cisternae of the Golgi apparatus. For its complete Golgi retention, XT-I requires the N-terminal 214 amino acids. Unlike XT-I, for XT-II, the first 45 amino acids are sufficient to target and retain the GFP reporter in the Golgi compartment. Here we show evidence that the stem regions were indispensable for Golgi localization of XT-I and XT-II.

Good Manufacturing Practice-Compliant Expansion of Marrow-Derived Stem and Progenitor Cells for Cell Therapy

Ex vivo expansion is being used to increase the number of stem and progenitor cells for autologous cell therapy. Initiation of pivotal clinical trials testing the efficacy of these cells for tissue repair has been hampered by the challenge of assuring safe and high-quality cell production. A strategy is described here for clinical-scale expansion of bone marrow (BM)-derived stem cells within a mixed cell population in a completely closed process from cell collection through postculture processing using sterile connectable devices. Human BM mononuclear cells (BMMNC) were isolated, cultured for 12 days, and washed postharvest using either standard open procedures in laminar flow hoods or using automated closed systems. Conditions for these studies were similar to long-term BM cultures in which hematopoietic and stromal components are cultured together. Expansion of marrow-derived stem and progenitor cells was then assessed. Cell yield, number of colony forming units (CFU), phenotype, stability, and multilineage differentiation capacity were compared from the single pass perfusion bioreactor and standard flask cultures. Purification of BMMNC using a closed Ficoll gradient process led to depletion of 98% erythrocytes and 87% granulocytes, compared to 100% and 70%, respectively, for manual processing. After closed system culture, mesenchymal progenitors, measured as CD105+CD166+CD14–CD45– and fibroblastic CFU, expanded 317- and 364-fold, respectively, while CD34+ hematopoietic progenitors were depleted 10-fold compared to starting BMMNC. Cultured cells exhibited multilineage differentiation by displaying adipogenic, osteogenic, and endothelial characteristics in vitro. No significant difference was observed between manual and bioreactor cultures. Automated culture and washing of the cell product resulted in 181 × 106 total cells that were viable and contained fibroblastic CFU for at least 24 h of storage. A combination of closed, automated technologies enabled production of good manufacturing practice (GMP)-compliant cell therapeutics, ready for use within a clinical setting, with minimal risk of microbial contamination.

Calcitriol Deficiency and 1-year Mortality in Cardiac Transplant Recipients

Background. Administration of the vitamin D hormone calcitriol improves survival in solid-organ transplanted experimental animals. We investigated whether lower serum calcitriol concentrations are associated with increased 1-year mortality in cardiac transplant recipients. Methods. We prospectively recruited 171 patients who underwent cardiac transplantation at out institution between May 2004 and April 2006. We assessed calciotropic hormones, inflammation markers, and renal function preoperatively and on postoperative days 6 (t1) and 21 (t2). Results. Serum creatinine and C-reactive protein increased, whereas calcitriol decreased significantly after transplantation (P<0.001). As determined by multivariable Cox regression analysis, the calcitriol level at t2 was an independent predictor of 1-year mortality. One-year mortality was 3.7 per 100 person-years in the tertile with the highest calcitriol concentrations at t2 (>18 pg/mL), 13.2 per 100 person-years in the intermediate tertile (11–18 pg/mL), and 32.1 per 100 person-years in the tertile with the lowest calcitriol concentrations at t2 (<11 pg/mL) (P<0.001). 25-hydroxyvitamin D deficiency (serum concentrations below 10 ng/mL), renal insufficiency (serum creatinine ≥1.6 mg/dL), and high serum concentrations of the inflammation markers C-reactive peptide and tumor necrosis factor-&agr; were predictors of a serum calcitriol concentration below 11 pg/mL (P=0.037–0.001). Conclusions. Low postoperative calcitriol concentrations are independently associated with high 1-year mortality in cardiac transplant recipients. A causal relationship has yet to be proven by intervention trials using active vitamin D.