Efficacy of Antihypertensive Drugs of Different Classes After Renal Denervation in Spontaneously Hypertensive Rats.

BACKGROUND: Renal denervation (RDN) lowers blood pressure (BP) in patients with uncontrolled hypertension. Limited data exist on the effectiveness of different antihypertensive medications following RDN on BP and maladaptive cardiac phenotypes. METHODS: Eighty-nine male spontaneously hypertensive rats with continuous BP recording underwent RDN or sham operation. Ten days postsurgery, spontaneously hypertensive rats were randomized to receive no antihypertensive medication, amlodipine, olmesartan, hydrochlorothiazide, bisoprolol, doxazosin, or moxonidine for 28 days. Cardiac remodeling was determined histologically, and activation of the renin-angiotensin-aldosterone system was explored. RESULTS: Before initiation of antihypertensive drugs, RDN reduced mean arterial pressure (-12.6 mm Hg [95% CI, -14.4 to -10.8]; P<0.001). At study end, mean arterial pressure was lower in RDN compared with sham operation in drug-naïve controls (P=0.006), olmesartan (P=0.002), amlodipine (P=0.0004), hydrochlorothiazide (P=0.006), doxazosin (P=0.001), and bisoprolol (P=0.039) but not in animals receiving moxonidine (P=0.122). Compared with pooled BP change of all other drug classes, mean arterial pressure change was largest for olmesartan (-15.9 mm Hg [95% CI, -18.6 to -13.2]; P<0.001) and amlodipine (-12.0 mm Hg [95% CI, -14.7 to -9.3]; P<0.001). In drug-naïve controls, RDN reduced plasma renin activity (-5.6%¸ P=0.03) and aldosterone concentration (-53.0%; P=0.005). In the presence of antihypertensive medication, plasma renin activity and aldosterone remained unchanged after RDN. Cardiac remodeling was not affected by RDN alone. In animals receiving olmesartan after RDN, cardiac perivascular fibrosis was attenuated. Amlodipine and bisoprolol following RDN reduced cardiomyocyte diameter. CONCLUSIONS: Following RDN, treatment with amlodipine and olmesartan resulted in the largest BP reduction. Antihypertensive medications mediated heterogeneous effects on renin-angiotensin-aldosterone system activity and cardiac remodeling.

Renal Denervation Prevents Atrial Arrhythmogenic Substrate Development in CKD.

BACKGROUND: In patients with chronic kidney disease (CKD), atrial fibrillation (AF) is highly prevalent and represents a major risk factor for stroke and death. CKD is associated with atrial proarrhythmic remodeling and activation of the sympathetic nervous system. Whether reduction of the sympathetic nerve activity by renal denervation (RDN) inhibits AF vulnerability in CKD is unknown. METHODS: Left atrial (LA) fibrosis was analyzed in samples from patients with AF and concomitant CKD (estimated glomerular filtration rate [eGFR], <60 mL/min per 1.73 m2) using picrosirius red and compared with AF patients without CKD and patients with sinus rhythm with and without CKD. In a translational approach, male Sprague Dawley rats were fed with 0.25% adenine (AD)-containing chow for 16 weeks to induce CKD. At week 5, AD-fed rats underwent RDN or sham operation (AD). Rats on normal chow served as control. After 16 weeks, cardiac function and AF susceptibility were assessed by echocardiography, radiotelemetry, electrophysiological mapping, and burst stimulation, respectively. LA tissue was histologically analyzed for sympathetic innervation using tyrosine hydroxylase staining, and LA fibrosis was determined using picrosirius red. RESULTS: Sirius red staining demonstrated significantly increased LA fibrosis in patients with AF+CKD compared with AF without CKD or sinus rhythm. In rats, AD demonstrated LA structural changes with enhanced sympathetic innervation compared with control. In AD, LA enlargement was associated with prolonged duration of induced AF episodes, impaired LA conduction latency, and increased absolute conduction inhomogeneity. RDN treatment improved LA remodeling and reduced LA diameter compared with sham-operated AD. Furthermore, RDN decreased AF susceptibility and ameliorated LA conduction latency and absolute conduction inhomogeneity, independent of blood pressure reduction and renal function. CONCLUSIONS: In an experimental rat model of CKD, RDN inhibited progression of atrial structural and electrophysiological remodeling. Therefore, RDN represents a potential therapeutic tool to reduce the risk of AF in CKD, independent of changes in renal function and blood pressure.

Antibiotic therapeutic drug monitoring in intensive care patients treated with different modalities of extracorporeal membrane oxygenation (ECMO) and renal replacement therapy: a prospective, observational single-center study.

BACKGROUND: Effective antimicrobial treatment is key to reduce mortality associated with bacterial sepsis in patients on intensive care units (ICUs). Dose adjustments are often necessary to account for pathophysiological changes or renal replacement therapy. Extracorporeal membrane oxygenation (ECMO) is increasingly being used for the treatment of respiratory and/or cardiac failure. However, it remains unclear whether dose adjustments are necessary to avoid subtherapeutic drug levels in septic patients on ECMO support. Here, we aimed to evaluate and comparatively assess serum concentrations of continuously applied antibiotics in intensive care patients being treated with and without ECMO. METHODS: Between October 2018 and December 2019, we prospectively enrolled patients on a pneumological ICU in southwest Germany who received antibiotic treatment with piperacillin/tazobactam, ceftazidime, meropenem, or linezolid. All antibiotics were applied using continuous infusion, and therapeutic drug monitoring of serum concentrations (expressed as mg/L) was carried out using high-performance liquid chromatography. Target concentrations were defined as fourfold above the minimal inhibitory concentration (MIC) of susceptible bacterial isolates, according to EUCAST breakpoints. RESULTS: The final cohort comprised 105 ICU patients, of whom 30 were treated with ECMO. ECMO patients were significantly younger (mean age: 47.7 vs. 61.2 years; p < 0.001), required renal replacement therapy more frequently (53.3% vs. 32.0%; p = 0.048) and had an elevated ICU mortality (60.0% vs. 22.7%; p < 0.001). Data on antibiotic serum concentrations derived from 112 measurements among ECMO and 186 measurements from non-ECMO patients showed significantly lower median serum concentrations for piperacillin (32.3 vs. 52.9; p = 0.029) and standard-dose meropenem (15.0 vs. 17.8; p = 0.020) in the ECMO group. We found high rates of insufficient antibiotic serum concentrations below the pre-specified MIC target among ECMO patients (piperacillin: 48% vs. 13% in non-ECMO; linezolid: 35% vs. 15% in non-ECMO), whereas no such difference was observed for ceftazidime and meropenem. CONCLUSIONS: ECMO treatment was associated with significantly reduced serum concentrations of specific antibiotics. Future studies are needed to assess the pharmacokinetic characteristics of antibiotics in ICU patients on ECMO support.

Glucose and Fat Tolerance Tests Induce Differential Responses in Plasma Choline Metabolites in Healthy Subjects.

Plasma choline shows associations with plasma glucose and lipids. We studied changes of choline metabolites after oral glucose tolerance test (OGTT) and fat tolerance test (OFTT). Eighteen healthy subjects (mean age 54.3 years; BMI 26.8 kg/m²) underwent 2 tests. First, OFTT (80 g fat) was applied and blood was collected at baseline and 4 h after OFTT. Seven days later, 75 g glucose was applied and blood was collected at baseline and 2 h after OGTT. Plasma concentrations of choline, betaine, trimethylamine N-oxide (TMAO), dimethylglycine, S-adenosylmethionine (SAM), lipids and glucose were measured. After OFTT, plasma choline declined (10.6 to 9.2 µmol/L; p = 0.004), betaine declined (33.4 to 31.7 µmol/L; p = 0.003), TMAO slightly increased (4.1 to 5.6 µmol/L; p = 0.105), glucose declined (5.39 to 4.98 mmol/L; p < 0.001), and triglycerides increased (1.27 to 2.53 mmol/L; p < 0.001). After OGTT, plasma choline increased (10.1 to 11.1 µmol/L; p < 0.001), TMAO declined (4.0 to 3.5 µmol/L; p = 0.029), dimethylglycine declined (2.0 to 1.7 µmol/L; p = 0.005), SAM declined (103 to 96 nmol/L; p = 0.041), but betaine, glucose, and SAM were unchanged. In conclusion, OFTT lowered plasma betaine and choline and caused heterogeneous changes in plasma TMAO. OGTT reduced the flow of methyl groups and plasma TMAO.

One-carbon metabolites and telomere length in a prospective and randomized study of B- and/or D-vitamin supplementation.

BACKGROUND: Vitamin B deficiency is common in elderly people and has been associated with an increased risk of developing age-related diseases. B-vitamins are essential for the synthesis and stability of DNA. Telomers are the end caps of chromosomes that shorten progressively with age, and short telomers are associated with DNA instability. OBJECTIVE: In the present randomized intervention study, we investigated whether the one-carbon metabolism is related to telomere length, a surrogate marker for cellular aging. DESIGN: Sixty-five subjects (>54 years) were randomly assigned to receive either a daily combination of vitamin D3 (1200 IU), folic acid (0.5 mg), vitamin B12 (0.5 mg), vitamin B6 (50 mg) and calcium carbonate (456 mg) (group A) or vitamin D3 and calcium carbonate alone (group B). Blood testing was performed at baseline and after 1 year of supplementation. The concentrations of several metabolites of the one-carbon pathway, as well as relative telomere length (RTL) and 5,10-methylenetetrahydrofolate reductase C677T genotype, were analyzed. RESULTS: At baseline, age- and gender-adjusted RTL correlated with total folate and 5-methyltetrahydrofolate (5-methylTHF). Subjects with RTL above the median had higher concentrations of total folate and 5-methylTHF compared to subjects below the median. At study end, gender- and age-adjusted RTL correlated in group A with methylmalonic acid (MMA; r = -0.460, p = 0.0012) and choline (r = 0.434, p = 0.0021) and in group B with 5,10-methenyltetrahydrofolate (r = 0.455, p = 0.026) and dimethylglycine (DMG; r = -0.386, p = 0.047). Subjects in the group A with RTL above the median had lower MMA and higher choline compared to subjects below the median. CONCLUSIONS: The present pilot study suggests a functional relationship between one-carbon metabolism and telomere length. This conclusion is supported by several correlations that were modified by B-vitamin supplementation. In agreement with our hypothesis, the availability of nucleotides and methylation groups seems to impact telomere length. Due to the small sample size and the limitations of the study, further studies should confirm the present results in a larger cohort.

Plasma Amyloid Beta 1-42 and DNA Methylation Pattern Predict Accelerated Aging in Young Subjects with Down Syndrome.

Gene methylation is an age-related dynamic process that influences diseases. Premature aging and disturbed methylation are components of Down syndrome (DS). We studied blood biomarkers and DNA methylation (DNAm) of three CpG sites (ASPA, ITGA2B, and PDE4C) in 60 elderly subjects (mean age = 68 years), 31 subjects with DS (12.1 years) and 44 controls (12.8 years). Plasma concentrations of amyloid beta (Aß) 1-42 and biomarkers of methylation were measured in the young groups. Subjects with DS had significantly higher concentrations of plasma S-adenosylhomocysteine (SAH) and Aß and reduced S-adenosylmethionine/SAH ratio compared with the controls. Methylations (%) of ASPA and ITGA2B were lower in DS [mean difference; 95 % confidence intervals = -2.2 (-4.5, 0.1) for ASPA and -5.0 (-8.9, -1.1) for ITGA2B]. Methylation of PDE4C did not differ between the groups. The sum of z-scores for methylations of ASPA and ITGA2B, both of which declined with age, was significantly lower in DS [-1.01 (-1.93, -0.20), p = 0.017]. Subjects with DS were found to be 3.1 (1.5-4.6) years older than their predicted age based on a regression model of the controls. Elevated SAH levels predicted lower DNAm of ASPA and ITGA2B in stepwise regression analysis. Therefore, methylation of three CpGs combined with plasma Aß has shown a 3-year accelerated aging in subjects with DS at the age of 12 years. Disorders in the methylation cycle explained pathoepigenetic modifications in subjects with DS. The influence of modifications in the methylation cycle on epigenetic markers of aging warrants further investigations.

Prospective study of telomere length and LINE-1 methylation in peripheral blood cells: the role of B vitamins supplementation.

PURPOSE: Deficiencies of folate, vitamins B12 and D are common age-related conditions. Vitamin B12 and folate are necessary for DNA methylation. Telomeres appear to be regulated by DNA methylation. Here, we study the effect of B vitamins supplementation on telomere length and global DNA methylation in a prospective study. METHODS: In total, 60 elderly subjects were supplemented for 1 year with either vitamin B12, B6, folate, vitamin D and calcium (group A n = 31) or only vitamin D and calcium (group B n = 29). LINE-1 methylation, relative telomere length (T/S), vitamin B12, folate, homocysteine (tHcy) , 5-methyltetrahydrofolate (5-methylTHF), S-adenosylhomocysteine (SAH), S-adenosylmethionine (SAM), cystathionine and vitamin D were quantified before and after supplementation. RESULTS: At baseline, tHcy was high, vitamin D was low, and T/S did not differ between groups A and B. Vitamin supplementation increased LINE-1 methylation in group A at site 317 but reduced LINE-1 methylation in group B at site 327. There was no correlation between T/S and LINE-1 methylation at baseline. Multiple backward regression analysis revealed baseline tHcy and 5-methylTHF are significant predictors of T/S. After supplementation in group B but not in group A, LINE-1 methylation correlated inversely with T/S, and LINE-1 methylation variation was an independent predictor of T/S variation. B vitamins decreased tHcy significantly in group A. Multiple backward regression analysis showed 5-methylTHF in group A and tHcy in group B were significant predictors for LINE-1 methylation. At baseline, the lower LINE-1 methylation observed in subjects with 5-methylTHF >10 nmol/l was in agreement with a reduced methyl group transfer due to a lower SAM formation. In group B, an increase in telomere length was correlated with lower LINE-1 methylation. Subjects with hyperhomocysteinemia >12 µmol/L had compared to those with normal tHcy a reduced LINE-1 methylation accompanied by a higher SAM and SAH (that inhibits demethylation of SAM) as well as lower 5-methylTHF. Additionally, subjects with tHcy > 12 µmol/L had longer telomeres when compared with subjects having tHcy < 12 µmol/L. CONCLUSIONS: The results suggest a possible effect of B vitamins for telomere biology in blood cells. Suboptimal B vitamins status and hyperhomocysteinemia are associated with altered DNA methylation and telomere length. These data have to be confirmed in future studies.

Pneumatic tube-transported blood samples in lithium heparinate gel separator tubes may be more susceptible to haemolysis than blood samples in serum tubes.

Pneumatic tube systems are widely used in hospitals. Advantages are high speed and rapid availability of the samples. However, the transportation by pneumatic tube promotes haemolysis. Haemolysis interferes with many spectrophotometric assays and is a common problem in clinical laboratories. The haemolysis index (HI) as a semi-quantitative representation of the level of haemolysis was compared in unpaired tube-transported and hand-delivered routine lithium heparinate plasma samples (n = 1368 and n = 837, respectively). Additionally, the HI distribution was measured in lithium heparinate plasma samples with a HI above the threshold value of 20 and in paired serum samples after transportation by pneumatic tube system. HI values above 20 can interfere with the selected assays: Creatine kinase (CK), creatine kinase-MB (CK-MB) and alanine aminotransferase (ALT) activities. These parameters were determined to demonstrate how haemolysis affects the results. 17.5% of the tube-transported plasma samples and 2.6% of the hand-delivered plasma samples had a HI above 20. The median HI in pneumatic tube-transported lithium heparinate plasma was 85 and 33 in the paired serum samples. The median HI difference between paired plasma and serum was 46. Blood samples in lithium heparinate tubes may be substantially more susceptible to haemolysis by pneumatic tube transportation than serum tube samples. Although our results cannot be universally applied to laboratories with different pneumatic tube systems, it is recommended that each laboratory evaluate carefully the degree of haemolysis after the transportation by the own pneumatic tube system and in terms of the sample type.

Effect of 1 year B and D vitamin supplementation on LINE-1 repetitive element methylation in older subjects.

BACKGROUND: Disturbed DNA methylation is causally related to chronic diseases like cancer and atherosclerosis. B vitamins are cofactors required for methyl group synthesis and may therefore affect DNA methylation. Vitamin D has epigenetic effects. We tested if B and D vitamin supplementation has an effect on genomic long interspersed nuclear element-1 (LINE-1) methylation and the metabolites S-adenosylmethionine (SAM) and S-adenosylhomocysteine (SAH). METHODS: Fifty subjects (median age 68.0 years) were supplemented with a daily oral dose of B vitamins (500 µg folic acid, 500 µg vitamin B12 and 50 mg vitamin B6), 1200 IU vitamin D and 456 mg calcium. Fasting blood samples were collected before and after 1 year of supplementation. LINE-1 methylation was determined in genomic DNA from blood cells as a surrogate for whole genome methylation. In addition, SAM, SAH and total homocysteine (tHcy) were measured in plasma samples. RESULTS: Plasma homocysteine decreased significantly after supplementation (12.8 vs. 9.1 µmol/L; p<0.05), whereas SAM, SAH, the SAM/SAH ratio and LINE-1 methylation did not change significantly. LINE-1 methylation was not significantly correlated with SAH, homocysteine or B vitamins. CONCLUSIONS: Long-term vitamin B supplementation had no effect on LINE-1 methylation in blood cells nor on plasma levels of SAM and SAH. Vitamin B and D supplementation seems to have no effect on DNA methylation, especially in cases where no severe deficiency exists.

The effect of a pneumatic tube transport system on platelet aggregation using optical aggregometry and the PFA-100.

BACKGROUND: Pneumatic tube sample transport is widely used in hospitals because it is convenient and reduces turnaround times. However, the preanalytical effects of pneumatic tube transport of blood samples on platelet function analysis are not fully understood. METHODS: Blood samples from 15 healthy subjects were collected before and after treatment with acetylsalicylic acid. Sample tubes were transported by pneumatic tube transport while the corresponding sample tubes were hand-delivered. Platelet function analysis was performed in platelet rich plasma by optical aggregometry and in whole blood by the PFA-100. RESULTS: Using the collagen-induced optical aggregometry a significant decrease of the aggregation amplitude (n = 30) was observed in tubed samples in comparison to the corresponding hand-delivered samples (low collagen concentration: 52.5% vs 56.1%, p = 0.006; and high collagen concentration respectively: 63.9% vs 67.1%, p = 0.011). Additionally, a slight prolongation of the PFA-100TM closure time for the epinephrine/collagen and the ADP/collagen stimulation was found in the tubed samples compared to the hand-delivered samples. CONCLUSIONS: The results indicate that the pneumatic tube sample transport impairs the platelet aggregation. Therefore, we recommend the manual transport of whole blood samples which are collected for optical aggregometry or PFA-100 analysis.

Enhanced bone metabolism in vegetarians--the role of vitamin B12 deficiency.

BACKGROUND: Vitamin B12 deficiency and bone fractures are common in vegetarians. However, a direct relationship between vitamin B12 status and bone metabolism in vegetarians has not been tested sufficiently. METHODS: Our study included 96 vegetarians (23 German vegans, and 54 German and 19 Indian lacto-, lacto-ovo-vegetarians) and 89 omnivores (Germans and Asian-Indian immigrants in Oman). Blood concentrations of total vitamin B12, holotranscobalamin (holoTC), 25OH-vitamin D (25(OH)D), total homocysteine (tHcy), methylmalonic acid (MMA), and the bone turnover markers (BTMs) bone alkaline phosphatase (BAP), osteocalcin (OC), pro-collagen type I N-terminal peptide (PINP) and C-terminal telopeptides of collagen I (CTx) were measured. RESULTS: Vegetarians from both population groups exhibited significantly higher concentrations of tHcy, MMA, folate, and BAP, but lower concentrations of holoTC and cobalamin compared with omnivores from the same population. Additionally, German vegetarians had higher circulating activities of BAP as well as higher CTx, OC, and PINP compared with their omnivorous controls. HoloTC and MMA were correlated with OC, CTx and BAP. Subjects with low vitamin B12 status (holoTC < or =35 pmol/L and MMA >271 nmol/L) had significantly lower serum concentrations of 25(OH)D, but higher tHcy and the BTMs P1NP, BAP, OC, and CTx, compared with subjects with normal vitamin B12 status. Multiple regression analysis showed that the association between BTMs and markers of vitamin B12 status was independent from the association with 25(OH)D. Approximately 12%-14% of the variation in the concentration of BTMs was explained by a regression model including holoTC, MMA and 25(OH)D. The strictness of the diet was not related to the magnitude of change in BTMs. CONCLUSIONS: Low vitamin B12 status is related to increased bone turnover in vegetarians which is independent from vitamin D status.

The cardiac effects of prolonged vitamin B12 and folate deficiency in rats.

In the recent past, hyperhomocysteinemia (HHCY) has been linked to chronic heart failure. Folate and vitamin B12 deficiencies are the common causes of HHCY. The impact of these vitamins on cardiac function and morphology has scarcely been investigated. The aim of this study was to conduct an analysis of the cardiac effect of folate and vitamin B12 deficiency in vivo. Two groups of rats, a control (Co, n = 10) and a vitamin-deficient group (VitDef, n = 10), were fed for 12 weeks with a folate and vitamin B12-free diet or an equicaloric control diet. Plasma and tissue concentrations of HCY, S-adenosyl-homocysteine (SAH), S-adenosyl-methionine (SAM), and brain natriuretic peptide (BNP) were measured. Moreover, echocardiographic and histomorphometric analyses were performed. VitDef animals developed a significant HHCY (Co vs VitDef: 6.8 +/- 2.7 vs 61.1 +/- 12.8 micromol/l, P < 0.001). Fractional shortening, left ventricular dimension at end-diastole and end-systole, posterior wall thickness, perivascular collagen, mast cell number, and BNP tissue levels were comparable in VitDef and Co animals. Interstitial collagen (Co vs VitDef: 6.8 +/- 3.0 vs 4.5 +/- 2.1%, P < 0.05), plasma BNP (Co vs VitDef: 180 +/- 80 vs 70 +/- 60 ng/l, P < 0.05), and tissue HCY (Co vs VitDef: 0.13 +/- 0.07 vs 0.07 +/- 0.04 micromol/g protein, P < 0.05) were lower in VitDef animals. Folate and vitamin B12 deficiency do not affect cardiac function and morphology.

Experimental folate and vitamin B12 deficiency does not alter bone quality in rats.

Hyperhomocysteinemia (HHCY) has been linked to fragility fractures and osteoporosis. Folate and vitamin B(12) deficiencies are among the main causes of HHCY. However, the impact of these vitamins on bone health has been poorly studied. This study analyzed the effect of folate and vitamin B(12) deficiency on bone in rats. We used two groups of rats: a control group (Co, n = 10) and a vitamin-deficient group (VitDef, n = 10). VitDef animals were fed for 12 wk with a folate- and vitamin B(12)-free diet. Co animals received an equicaloric control diet. Tissue and plasma concentrations of homocysteine (HCY), S-adenosyl-homocysteine (SAH), and S-adenosyl-methionine (SAM) were measured. Bone quality was assessed by biomechanical testing (maximum force of an axial compression test; F(max)), histomorphometry (bone area/total area; B.Ar./T.Ar.], and the measurement of biochemical bone turnover markers (osteocalcin, collagen I C-terminal cross-laps [CTX]). VitDef animals developed significant HHCY (Co versus VitDef: 6.8 +/- 2.7 versus 61.1 +/- 12.8 microM, p < 0.001) that was accompanied by a high plasma concentration of SAH (Co versus VitDef: 24.1 +/- 5.9 versus 86.4 +/- 44.3 nM, p < 0.001). However, bone tissue concentrations of HCY, SAH, and SAM were similar in the two groups. Fmax, B.Ar./T.Ar., OC, and CTX did not differ between VitDef and Co animals, indicating that bone quality was not affected. Folate and vitamin B(12) deficiency induces distinct HHCY but has no effect on bone health in otherwise healthy adult rats. The unchanged HCY metabolism in bone is the most probable explanation for the missing effect of the vitamin-free diet on bone.

Hyperhomocysteinemia induces a tissue specific accumulation of homocysteine in bone by collagen binding and adversely affects bone.

BACKGROUND: Recently, hyperhomocysteinemia (HHCY) has been suggested to have adverse effects on bone. This study investigated if an experimental HHCY in rats induces an accumulation of homocysteine (HCY) in bone tissue that is accompanied by bone loss and reduced bone strength. MATERIAL AND METHODS: HHCY was induced in healthy rats by either a methionine (Meth)- or a homocystine (Homo)-enriched diet and compared with controls. Homocystine is the product of two disulfide linked HCY molecules. Tissue and plasma concentrations of HCY, S-adenosylhomocysteine (SAH) and S-adenosylmethionine (SAM) were measured. Bones were assessed by biomechanical testing, histomorphometry, microCT and the measurement of biochemical bone turnover markers in plasma. RESULTS: Meth and Homo animals developed a significant HHCY that was accompanied by a tissue specific accumulation of HCY (1300 to 2000% vs. controls). 65% of HCY in bone was bound to collagen of the extracellular matrix. The SAH / SAM-ratio in bone and plasma of Meth and Homo animals exhibited a tissue specific increase indicating a reduced methylation capacity. Accumulation of HCY in bone was characterized by a distinct reduction of cancellous bone (proximal femur: -25 to -35%; distal femur -56 to -58%, proximal tibia: -28 to -43%). Accordingly, bone strength was significantly reduced (-9 to -12%). CONCLUSION: A tissue specific accumulation of HCY in bone may be a promising mechanism explaining adverse effects of HHCY on bone. A reduced methylation capacity of bone cells might be another relevant pathomechanism.

Low serum vitamin B12 is associated with recurrent pregnancy loss in Syrian women.

BACKGROUND: Hyperhomocysteinemia and B-vitamin deficiency are associated with recurrent abortion. Recent studies have not investigated functional markers of vitamin B12 deficiency, such as methylmalonic acid. METHODS: A total of 43 consecutive Syrian women with unexplained recurrent abortion and 32 pregnant controls were enrolled in the study. Serum folate, vitamin B12, methylmalonic acid and plasma homocysteine were determined. RESULTS: Vitamin B12 was significantly decreased in patients with recurrent abortion compared to controls (mean concentrations 197 vs. 300 pg/mL, p=0.004). The lowest mean serum vitamin B12 (172 pg/mL) was observed in primary aborters. Homocysteine was elevated in aborters in comparison to controls (8.3 vs. 7.1 micromol/L, p=0.093). Folate and methylmalonic acid did not differ significantly between the study groups. A highly significant correlation between homocysteine and methylmalonic acid and vitamin B12 was observed only in patients but not in controls (p<0.001 and p=0.002, respectively). In the logistic regression model, only serum vitamin B12 emerged with a significant odds ratio. CONCLUSIONS: The results confirm low serum vitamin B12 in recurrent abortion patients. However, methylmalonic acid did not support that functional vitamin B12 plays a role in this group. This unexpected result might be due to a decrease of the metabolically inert vitamin B12 fraction (holohaptocorrin) or confounding factors. Further studies are necessary to investigate the role of vitamin B12 deficiency in recurrent abortion.

Decreased p66Shc promoter methylation in patients with end-stage renal disease.

BACKGROUND: p66Shc is a stress response protein and partially regulated by epigenetic modifications. Mice lacking p66Shc have reduced atherosclerosis, increased resistance to oxidative stress and a prolonged life time. The aim of the present study was to compare promoter methylation of the p66Shc gene between healthy controls and patients with end-stage renal disease (ESRD). There are two reasons for studying patients with ESRD. First, patients with ESRD have a disturbed homocysteine metabolism, and second an increased risk of morbidity and mortality from cardiovascular disease is a constant finding in these patients. METHODS: In our study, we measured fasting levels of homocysteine, S-adenosylmethionine (SAM), S-adenosylhomocysteine (SAH) and 8-isoprostane in 22 patients and in 26 healthy, age- and sex-matched controls. The methylation of the p66Shc promoter and Line-1, as surrogate marker of whole genome methylation was quantified in peripheral blood mononuclear cells. RESULTS: In comparison to the control group, homocysteine, SAM, SAH, 8-isoprostane and whole genome methylation were significantly elevated in ESRD patients, while the p66Shc promoter methylation was significantly reduced. A significant correlation was found between SAH and p66Shc promoter methylation in the patient group. This observation underlines the role of SAH as a potent inhibitor of methyltransferases. Using backward regression analysis, we demonstrated that 8-isoprostane has a significant influence on p66Shc promoter methylation. In the control group and in patients with ESRD, increasing 8-isoprostane levels were linked to an elevated promoter methylation. CONCLUSIONS: Under physiological conditions, based on the results of the control group, the p66Shc expression is more silenced through epigenetic modifications. The atherosclerotic risk is dramatically increased in ESRD patients; therefore, our experimental results of methylation are in accordance with the clinical situation.

Experimental hyperhomocysteinemia reduces bone quality in rats.

BACKGROUND: Recently, hyperhomocysteinemia (HHCY) has been suggested as a new risk factor for osteoporosis. This study investigated if HHCY is a causal osteoporotic factor in vivo. METHODS: We used 3 groups of rats: a control group (n = 20), a moderate HHCY group (induced by a 2.4% methionine-enriched diet, n = 10), and an intermediate HHCY group (induced by a 2% homocystine-enriched diet, n = 10). We measured bone fragility [maximum force of an axial compression test (F(max))], bone area as percentage of total area (BAr/TAr, histomorphometry), and biochemical bone turnover markers [osteocalcin (OC) and collagen I C-terminal crosslaps (CTx)]. RESULTS: Compared with controls, 3 months of moderate or intermediate HHCY increased mean (SD) bone fragility at the femoral neck by 18% (6%) in methionine-fed (P = 0.001) and 36% (13%) in homocystine-fed rats (P <0.001). Mean (SD) BAr/TAr at the distal femur in methionine and homocystine groups was decreased by 45% (21%; P = 0.001) and 93% (9%; P = 0.001), respectively. At the femoral neck, BAr/TAr was decreased by 19% (11%; P <0.001) and 55% (19%; P <0.001). At the lumbar spine, the reduction of BAr/TAr was 17% (23%; P = 0.099) and 44% (19%; P <0.001). Plasma OC (bone formation marker) was decreased by 23% (20%; P = 0.006) and 34% (21%; P <0.001). Plasma CTx (bone resorption marker) did not differ between groups. CONCLUSION: Bone quality is consistently decreased in the presence of increased circulating homocysteine. The results provide evidence that HHCY is a causal osteoporotic factor.

Hyperhomocysteinemia and myocardial expression of brain natriuretic peptide in rats.

BACKGROUND: Hyperhomocysteinemia (HHcy) has been linked to impaired left ventricular function and clinical class in patients with chronic heart failure. We hypothesized that HHcy stimulates myocardial brain natriuretic peptide (BNP) expression and induces adverse left ventricular remodeling. METHODS: We randomized 50 rats into 5 groups. Groups Co1 and Co2 (controls) received a typical diet. Groups Meth, Hcy1, and Hcy2 were fed the same diet supplemented with 2.4% methionine, 1% homocystine, and 2% homocystine, respectively. After 12 weeks, we measured total plasma homocysteine (tHcy) and BNP in plasma and tissue, and we performed histomorphometric analyses. RESULTS: All animals had comparable baseline body weight [mean (SD) 234 (26) g] and total circulating Hcy [4.7 (1.7) micromol/L]. After 12 weeks of treatment, total circulating Hcy increased in Meth, Hcy1, and Hcy2 [27.3 (8.8), 40.6 (7.0), and 54.0 (46.0) micromol/L, respectively] and remained unchanged in Co1 and Co2. Serum BNP significantly increased in 1 of 10 animals in Meth, 3 of 10 animals in Hcy1, and 3 of 10 animals in Hcy2. Median (25th-75th percentile) BNP tissue concentrations in Hcy1 and Hcy2 were 55% higher than in the corresponding controls [Co1 vs Hcy1, 225 (186-263) vs 338 (262-410) pg/mg protein, P = 0.05; Co2 vs Hcy2, 179 (107-261) vs 308 (192-429) pg/mg protein, P = 0.12]. In the Meth group, BNP expression was comparable to that of controls [200 (159-235) vs 225 (186-263) pg/mg protein, P = 0.32]. The percentage of perivascular and interstitial collagen and mast cell infiltration were comparable in all groups, indicating no adverse cardiac remodeling. CONCLUSION: Three months of intermediate HHcy stimulated increased cardiac BNP expression that was not accompanied by adverse cardiac remodeling.

Hyperhomocysteinemia--the biochemical link between a weak heart and brittle bones?

Osteoporosis, chronic heart failure (CHF) and mild to moderate hyperhomocysteinemia (HHCY) can frequently be found in elderly individuals and often occur in the same individual. Due to demographic changes in the number of elderly people the total number of individuals suffering from osteoporosis and/or CHF, and hence the cost to society, will increase dramatically over the next 50 years. Thus, prevention of these diseases by identifying and modifying risk factors is a major issue. Recent large population-based prospective studies suggested HHCY as an independent risk factor for CHF and osteoporosis. However, the mechanisms that link HHCY to CHF and osteoporosis are almost unknown. Moreover, until now both diseases have been considered as independent diseases. The finding that heart and bones share a common biochemical risk factor raises the question if there is a biochemical link between these two diseases? This manuscript reviews the existing literature about HHCY and osteoporosis, about HHCY and CHF, and about possible mechanisms that link HHCY to both diseases. Existing data suggest that HHCY could be a useful paradigm with most interesting therapeutic implications, because HCY lowering therapy could favourably influence the course in CHF and osteoporotic patients.