Background and Aims Several systematic reviews/meta-analyses of observational studies have demonstrated associations between circulating trimethylamine-N-oxide (TMAO) and cardiovascular (CVD), cerebral, and renal diseases, including mortality in observational studies. However, causal roles for TMAO in these diseases are controversial. Interventions are lacking to show whether lowering TMAO in clinical trials could reduce the risks of these diseases. TMAO could still serve as a prognostic marker for the mentioned outcomes, but investigating this potential role requires high-quality methodologies. We conducted a systematic search and critical evaluation of published systematic reviews/meta-analyses in the field. Results We identified 27 systematic reviews/meta-analyses on the association between TMAO and stroke (n = 7), cardiovascular events including cause-specific and/or all-cause mortality (n = 14), and other outcomes (n = 6). The majority of the systematic reviews/meta-analyses found higher blood TMAO concentrations in patients who were positive for the outcomes. Primary studies included populations with multiple risk factors for the given outcomes and did not sufficiently adjust for potential confounders. Prospective studies examining associations between baseline TMAO and subsequent disease outcomes in healthy populations were entirely absent. Furthermore, we identified serious flaws in methods, conduct and reporting in the majority of the published systematic reviews/meta-analyses, thus leading to critically low confidence in the results. Conclusions High quality systematic reviews/meta-analyses examining the associations between TMAO and vascular or cerebral disease are needed to examine potential causal and/or predictive roles of TMAO in these diseases. This study is registered at the International Prospective Register of Systematic Reviews (PROSPERO) (CRD42024534940).

To define an intake threshold of vitamin B12 from food supplements that is sufficient to maintain normal body functions, but it does not cause pharmacological effects. We used studies on the amount of B12 absorbed following oral B12 application and non-comparative case-series studies to synthesize evidence on pharmacological effects of oral B12 (between < 10 µg and 3000 µg) in people with manifested deficiency. There is a dose-dependent intestinal absorption of B12 and in the same time effects on body metabolism and functions. Food supplements providing ≤ 20 µg B12 daily are unlikely to cause pharmacological effects, while 50 µg might correct abnormal biochemical markers in some deficient patients. Foods for special medical purposes for people who cannot absorb B12 may contain 100 µg to 150 µg B12. This dose may ensure 1–4 µg of the vitamin reaching the circulation on a daily basis independent of intrinsic factor. Dosages ≥ 200 µg/d should be considered as drugs that can correct anemia, metabolic markers and clinical symptoms. The content of vitamin B12 in food supplements should not exceed 20 µg. In addition, people with deficiency should receive appropriate medical treatment with high dose B12.

Objectives: Trimethylamine N-oxide (TMAO) is a gut bacteria-mediated liver metabolite of dietary betaine, choline, and carnitine, which is excreted by glomerular filtration. We studied whether TMAO is excreted by cardiovascular disease (CVD) in patients with chronic kidney disease (CKD). Methods: Among 478 patients with CKD stage G2 (n = 104), G3a (n = 163), G3b (n = 123), and G4 (n = 88), we studied the association between fasting plasma concentrations of TMAO, choline, or betaine at baseline and kidney function, prevalent CVD, and future renal outcomes during a mean follow-up of 5.1 years. Results: Decreased glomerular filtration rate was associated with higher plasma concentrations of TMAO, choline, and betaine. Baseline concentrations of TMAO were higher in participants with preexisting CVD compared to those without CVD (8.4 [10.1] vs. 7.8 [8.0] mmol/L; P = .047), but the difference was not significant after adjusting for confounders. During the follow-up, 147 participants experienced CVD or died, and 144 reached the predefined renal endpoint. In the adjusted regression analyses, TMAO or choline concentrations in the upper three quartiles (vs. the lowest quartile) were not associated with any of the study's clinical endpoints. In contrast, the adjusted hazard ratio of plasma betaine in the highest quartile versus the lowest quartile was 2.14 (1.32, 3.47) for the CVD endpoint and 1.64 (1.00, 2.67) for the renal endpoint. Conclusions: Elevated plasma TMAO concentrations were explained by impaired kidney function. Elevated plasma concentrations of betaine, but not those of TMAO or choline, constituted a risk factor for adverse outcomes. TMAO might not be an appropriate target to reduce CVD or renal outcomes in patients with preexisting CKD.

IntroductionHigh-mobility group box 1 (HMGB1) protein is a critical mediator of neutrophil extracellular trap (NET) formation (NETosis). Myeloperoxidase (MPO)-DNA complexes, a biomarker of NETs, and HMGB1 have been associated with delayed cerebral ischemia (DCI) after aneurysmal subarachnoid hemorrhage (aSAH). Additional mechanistic NET-related biomarkers and their role in the neuroinflammatory cascade surrounding DCI remain to be explored.MethodsA post-hoc analysis of a prospective, blinded, single-center biomarker observational study was performed. De novo measurements of serum citrullinated histone H3-DNA complexes (H3Cit-DNA), peptidylarginine deiminase 4 (PAD4), cell-free DNA (cf-DNA), and DNase 1 activity were conducted on admission (D0) and day 4 (D4). Delayed cerebral infarction (DCI) was defined as new cerebral infarction on CT head not present on the post-treatment scan.ResultsH3Cit-DNA, PAD4, cf-DNA, and DNase 1 activity were within quantifiable ranges in all serum samples analyzed at D0 and D4. Admission biomarker levels were not associated with DCI development. From D0 to D4, in both the DCI and the non-DCI groups, H3Cit-DNA levels significantly decreased, cf-DNA levels significantly increased, and PAD4 levels remained stable. In contrast, DNase 1 activity significantly decreased from D0 to D4 in the DCI group (p < 0.001) but not in the non-DCI group.ConclusionThis exploratory analysis demonstrated NET-related biomarkers such as H3Cit-DNA, PAD4, cf-DNA, and DNase 1 activity in all aSAH patients. A decline of systemic DNase 1 activity in the early phase might increase the risk of delayed cerebral ischemia.

Introduction: Rhabdomyolysis is characterized by destruction of muscle fibers by various causes and is diagnosed by increased creatine kinase concentrations in the blood. Myoglobin released into the blood may cause acute kidney injury. In this randomized controlled study, we hypothesized that myoglobin elimination would be faster when a hemoadsorber was added to a continuous veno-venous hemodialysis (CVVHD). Methods: Four patients in the control group received CVVHD with a high cut-off hemofilter using high blood and dialysate flows for 48 h. Four patients in the CytoSorb group received the same treatment, but in addition, the hemoadsorber CytoSorb® was inserted in front of the hemofilter and replaced once after 24 h. Blood samples were drawn simultaneously before (pre) and after (post) the hemofilter or else the hemoadsorber, after 5 and 30 min, as well as after 2, 4, 8, and 24 h. All measurements were repeated the next day after the hemoadsorber had been renewed in the CytoSorb group. Primary outcome was the area under the curve (AUC) of the relative myoglobin concentrations as percent of baseline. To evaluate the efficacy of myoglobin removal, relative reductions in myoglobin concentrations during one passage through each device at each time point were calculated. Results: Patients in the CytoSorb group had a significantly lower AUC during the first 24 h (42 ± 10% vs. 63 ± 6%, p = 0.029) as well as during the observation period of 48 h (26 ± 7% vs. 51 ± 12%, p = 0.029). The relative reductions for myoglobin were considerably higher in the CytoSorb group compared to the control group during the first 8 h. Conclusion: Myoglobin concentrations declined considerably faster when CytoSorb was added to a CVVHD. When compared to a high-cut-off hemofilter, efficacy of CytoSorb in myoglobin elimination was much better. Because of saturation after 8–12 h an exchange may be necessary.

Abstract Objectives Concentrations of pyridoxal 5′-phosphate (PLP) in serum and whole blood are routinely measured. The suitability of these markers in capturing vitamin B6 insufficiency is not well studied. Methods In 212 subjects, concentrations of PLP and thiamine pyrophosphate (TPP) were simultaneously measured in EDTA-whole blood using Chromsystems® (52052) method on HPLC devices. The whole blood PLP concentrations were compared to serum PLP concentrations measured using reagents from Immundiagnostik® (KC 2100). The whole blood TPP concentrations measured with the Chromsystems® (52052) were compared to those measured by the Chromsystems® (35000) method. Concentrations of homocysteine (tHcy) and cystathionine (Cys) were measured and used to judge the PLP methods. Results Serum PLP concentrations were on average 41% lower than whole blood PLP [mean (SD)=55.4 (83.0) vs. 131 (217) nmol/L]. Serum and whole blood PLP showed a strong correlation [Pearson correlation coefficient=0.724, p<0.001, n=204]. Eighty-five samples showed discrepant results for PLP status (serum PLP ≤30 nmol/L, but whole blood PLP >51 nmol/L) while 102 samples showed coherent results (reference group). The discrepancy group showed higher odds ratio for elevated tHcy >12.0 μmol/L compared to the reference group [OR (95% confidence intervals, CI)=2.1 (1.2–4.0)]. The OR (95% CI) of elevated Cys >300 nmol/L was 1.9 (1.0–3.5) in the discrepancy group compared to the reference group. TPP concentrations were 6% lower when using the Chromsystems®, 52052 compared to levels measured with Chromsystems®, 35000. Conclusions Serum and whole blood PLP concentrations disagree in a substantial number of samples. Serum PLP was better in reflecting elevated tHcy and Cys compared to whole blood PLP. Whole blood PLP underestimates the prevalence of vitamin B6 insufficiency. Methods of measuring TPP concentrations in whole blood were exchangeable.

Background: Early prognostication of COVID-19 severity will potentially improve patient care. Biomarkers, such as TNF-related apoptosis-inducing ligand (TRAIL), interferon gamma-induced protein 10 (IP-10), and C-reactive protein (CRP), might represent possible tools for point-of-care testing and severity prediction. Methods: In this prospective cohort study, we analyzed serum levels of TRAIL, IP-10, and CRP in patients with COVID-19, compared them with control subjects, and investigated the association with disease severity. Results: A total of 899 measurements were performed in 132 patients (mean age 64 years, 40.2% females). Among patients with COVID-19, TRAIL levels were lower (49.5 vs 87 pg/ml, P = 0.0142), whereas IP-10 and CRP showed higher levels (667.5 vs 127 pg/ml, P < 0.001; 75.3 vs 1.6 mg/l, P < 0.001) than healthy controls. TRAIL yielded an inverse correlation with length of hospital and intensive care unit (ICU) stay, Simplified Acute Physiology Score II, and National Early Warning Score, and IP-10 showed a positive cor- relation with disease severity. Multivariable regression revealed that obesity (adjusted odds ratio [aOR] 5.434, 95% confidence interval [CI] 1.005-29.38), CRP (aOR 1.014, 95% CI 1.002-1.027), and peak IP-10 (aOR 1.001, 95% CI 1.00-1.002) were independent predictors of in-ICU mortality. Conclusions: We demonstrated a correlation between COVID-19 severity and TRAIL, IP-10, and CRP. Multi- variable regression showed a role for IP-10 in predicting unfavourable outcomes, such as in-ICU mortality. registration:

IMPORTANCE: Myeloperoxidase (MPO)-DNA complexes, biomarkers of neutrophil extracellular traps (NETs), have been associated with arterial and venous thrombosis. Their role in aneurysmal subarachnoid hemorrhage (aSAH) is unknown. OBJECTIVES: To assess whether serum MPO-DNA complexes are present in patients with aSAH and whether they are associated with delayed cerebral ischemia (DCI). DESIGN, SETTING, AND PARTICIPANTS: Post-hoc analysis of a prospective, observational single-center study, with de novo serum biomarker measurements in consecutive patients with aSAH between July 2018 and September 2020, admitted to a tertiary care neuroscience ICU. MAIN OUTCOMES AND MEASURES: We analyzed serum obtained at admission and hospital day 4 for concentrations of MPO-DNA complexes. The primary outcome was DCI, defined as new infarction on brain CT. The secondary outcome was clinical vasospasm, a composite of clinical and transcranial Doppler parameters. We used Wilcoxon signed-rank-test to assess for differences between paired measures. RESULTS: Among 100 patients with spontaneous subarachnoid hemorrhage, mean age 59 years ( sd ± 13 yr), 55% women, 78 had confirmed aSAH. Among these, 29 (37%) developed DCI. MPO-DNA complexes were detected in all samples. The median MPO-DNA level was 33 ng/mL (interquartile range [IQR], 18–43 ng/mL) at admission, and 22 ng/mL (IQR, 11–31 ng/mL) on day 4 (unpaired test; p = 0.015). We found a significant reduction in MPO-DNA levels from admission to day 4 in patients with DCI (paired test; p = 0.036) but not in those without DCI ( p = 0.17). There was a similar reduction in MPO-DNA levels between admission and day 4 in patients with ( p = 0.006) but not in those without clinical vasospasm ( p = 0.47). CONCLUSIONS AND RELEVANCE: This is the first study to detect the NET biomarkers MPO-DNA complexes in peripheral serum of patients with aSAH and to associate them with DCI. A pronounced reduction in MPO-DNA levels might serve as an early marker of DCI. This diagnostic potential of MPO-DNA complexes and their role as potential therapeutic targets in aSAH should be explored further.

SCOPE:Probiotics exert immunomodulatory effects and may influence tryptophan metabolism in the host. Deficiency of nutrients related to C1 metabolism might stimulate inflammation by enhancing the kynurenine pathway. This study used Sprague Dawley rats to investigate whether a methyl-deficient diet (MDD) may influence tryptophan/kynurenine pathways and cytokines and whether probiotics can mitigate these effects.METHODS AND RESULTS:Rats are fed a control or MDD diet. Animals on the MDD diet received vehicle, probiotics (L. helveticus R0052 and B. longum R0175), choline, or probiotics + choline for 10 weeks (n = 10 per group). Concentrations of plasma kynurenine metabolites and the methylation and inflammatory markers in plasma and liver are measured.RESULTS:MDD animals (vs controls) show upregulation of plasma kynurenine, kynurenic acid, xanthurenic acid, 3-hydroxyxanthranilic acid, quinolinic acid, nicotinic acid, and nicotinamide (all p < 0.05). In the MDD rats, the probiotics (vs vehicle) cause lower anthranilic acid and a trend towards lower kynurenic acid and picolinic acid. Compared to probiotics alone, probiotics + choline is associated with a reduced enrichment of the bacterial strains in cecum. The interventions have no effect on inflammatory markers.CONCLUSIONS:Probiotics counterbalance the effect of MDD diet and downregulate downstream metabolites of the kynurenine pathway.

(1) Background: Chorea-acanthocytosis and McLeod syndrome are the core diseases among the group of rare neurodegenerative disorders called neuroacanthocytosis syndromes (NASs). NAS patients have a variable number of irregularly spiky erythrocytes, so-called acanthocytes. Their detection is a crucial but error-prone parameter in the diagnosis of NASs, often leading to misdiagnoses. (2) Methods: We measured the standard Westergren erythrocyte sedimentation rate (ESR) of various blood samples from NAS patients and healthy controls. Furthermore, we manipulated the ESR by swapping the erythrocytes and plasma of different individuals, as well as replacing plasma with dextran. These measurements were complemented by clinical laboratory data and single-cell adhesion force measurements. Additionally, we followed theoretical modeling approaches. (3) Results: We show that the acanthocyte sedimentation rate (ASR) with a two-hour read-out is significantly prolonged in chorea-acanthocytosis and McLeod syndrome without overlap compared to the ESR of the controls. Mechanistically, through modern colloidal physics, we show that acanthocyte aggregation and plasma fibrinogen levels slow down the sedimentation. Moreover, the inverse of ASR correlates with the number of acanthocytes (R2=0.61, p=0.004). (4) Conclusions: The ASR/ESR is a clear, robust and easily obtainable diagnostic marker. Independently of NASs, we also regard this study as a hallmark of the physical view of erythrocyte sedimentation by describing anticoagulated blood in stasis as a percolating gel, allowing the application of colloidal physics theory.

BACKGROUND:Peripheral neuropathy (PN) is common in patients with diseases that are in turn associated with deficiency of the B-vitamins, and vitamin treatment has shown mixed results.METHODS:This systematic review and meta-analysis studied the association between PN/pain and B-vitamin biomarkers and investigated whether vitamin treatment can ameliorate the symptoms. PubMed and Web of Science were searched according to the study protocol.RESULTS:A total of 46 observational and seven interventional studies were identified and included in the data synthesis. The presence of PN was associated with lowered B12 levels (pooled estimate [95% CIs] = 1.51 [1.23-1.84], n = 34, Cochran Q Test I2  = 43.3%, p = 0.003) and elevated methylmalonic acid (2.53 [1.39-4.60], n = 9, I2  = 63.8%, p = 0.005) and homocysteine (3.48 [2.01-6.04], n = 15, I2  = 70.6%, p < 0.001). B12 treatment (vs. the comparators) showed a non-significant association with symptom improvement (1.36 (0.66-2.79), n = 4, I2  = 28.9%). Treatment with B1 was associated with a significant improvement in symptoms (5.34 [1.87-15.19], n = 3, I2  = 64.6%, p = 0.059). Analysis of seven trials combined showed a non-significant higher odds ratio for improvement under treatment with the B-vitamins (2.58 [0.98-6.79], I2  = 80.0%, p < 0.001).CONCLUSIONS:PN is associated with lowered plasma vitamin B12 and elevated methylmalonic acid and homocysteine. Overall, interventional studies have suggested that B-vitamins could improve symptoms of PN. Available trials have limitations and generally did not investigate vitamin status prior to treatment. Well-designed studies, especially in non-diabetes PN, are needed. This meta-analysis is registered at PROSPERO (ID: CRD42020144917).

ABSTRACT Chorea-acanthocytosis (ChAc) and McLeod syndrome (MLS) are the core diseases among the group of rare neurodegenerative disorders that comprise neuroacanthocytosis syndrome (NAS). Both ChAc and MLS patients present with an increased number of irregularly spiky erythrocytes, so-called acanthocytes. The detection of acanthocytes is often a crucial parameter in the diagnosis of NAS. However, this approach is error-prone and not very reliable, typically explaining the misdiagnosis of NAS patients. Based on the standard Westergren method, we show that compared with that in healthy controls, the erythrocyte sedimentation rate (ESR) with a two-hour read-out is significantly prolonged in ChAc and MLS with no overlap. Thus, the ESR is a clear, robust and easily obtained diagnostic marker. Mechanistically, by applying modern colloidal physics, we show that acanthocyte aggregation and plasma fibrinogen levels slow the sedimentation process. Apart from its diagnostic value, ESR may also be the first biomarker for monitoring treatments for NAS patients. Further studies are required to test whether the ESR may also detect other NASs. In addition to medical progress, this study is also a hallmark of the physical view of the erythrocyte sedimentation process by describing anticoagulated blood in stasis as a percolating gel, allowing the application of colloidal physics theory.

Angiotensin-converting enzyme (ACE) 2 is known to be a functional receptor for SARS-CoV-2 in the current pandemic. Soluble ACE2 (sACE2) concentrations are elevated in patients with various cardiovascular disorders including heart failure. In a total of 182 consecutive adult patients with complex congenital heart disease (CHD) and 63 healthy controls, sACE2 concentrations were measured in serum using the Human ACE2® assay by Cloud-Clone Corporation and associated with clinical, laboratory and echocardiographic parameters. Median sACE2 levels were increased in patients with complex CHD as compared to healthy controls (761.9 pg/ml vs 365.2 pg/ml, p < 0.001). Moreover, sACE2 concentrations were significantly elevated in patients with a higher NYHA class ≥ III (1856.2 pg/ml vs 714.5 pg/ml in patients with NYHA class I/II, p < 0.001). Using linear regression analysis, higher sACE2 levels were associated with a higher NYHA class ≥ III, more severe CHD, a morphological left systemic ventricle, higher creatinine and the use of mineralocorticoid receptor antagonists (MRA) in the univariable model. The use of ACE inhibitors or angiotensin receptor blockers (ARB) was associated with lower sACE2 levels. In the multivariable model, higher sACE2 levels were independently associated with a higher NYHA class ≥ III (p = 0.002) and lower sACE2 levels with the use of ACE inhibitors or ARB (p = 0.001). Soluble ACE2 concentrations were significantly increased in all types of complex CHD with highest levels found in patients with NYHA class ≥ III. Moreover, a higher NYHA class ≥ III was the most significant determinant that was independently associated with elevated sACE2 concentrations.

Background: Vascular diseases are multifactorial and several risk factors may have synergetic effect on the global vascular risk. Among patients with diabetes, we investigated whether vitamin B6 species differ according to global cardiovascular risk. Methods: The present observational study included 122 patients with type 2 diabetes (mean (SD) age = 69.9 (9.1) years; 50% men). Concentrations of vitamin B6 vitamers were measured. Classical blood biomarkers and risk factors were used to compute a multivariate risk score. Results: Plasma concentrations of 4-pyridoxic acid were higher in patients with high risk versus those with low risk scores (48.2 (63.7) vs. 31.9 (15.0) nmol/L; p = 0.031). Plasma pyridoxine was significantly lowered in patients at high risk (2.8 (28.4) vs. 38.1 (127.8) nmol/L; p = 0.003). PAr index (4-pyridoxic acid/pyridoxal + pyridoxal 5′-phosphate) (1.05 (0.07) vs. 0.84 (0.06); p = 0.017) and the ratio of 4-pyridoxic acid/pyridoxine (7.0 (4.8) vs. 3.9 (3.2); p < 0.001) were higher in patients at high risk. After adjustment for cystatin C and C-reactive protein, only pyridoxine and 4-pyridoxic acid/pyridoxine ratio remained significantly different according to vascular risk scores. 4-Pyridoxic acid/pyridoxine ratio was the best marker to discriminate between patients according to their risk scores—area under the curve (AUC) (95% confidence intervals (CI)) = 0.72 (0.62–0.81). 4-Pyridoxic acid/pyridoxine ratio was directly related to plasma levels of soluble vascular cell adhesion molecule 1. Conclusion: Vitamin B6 metabolism was shifted in patients with multiple vascular risk factors. The catabolism to 4-pyridoxic acid was enhanced, whereas the catabolism to pyridoxine was lowered. High 4-Pyridoxic acid/pyridoxine ratio is independently associated with global cardiovascular risk.

Depression is associated with dysregulation of methyl group metabolism such as low S-adenosylmethionine (SAM). We previously reported that Flinders Sensitive Line (FSL) rats, an animal model of depression, had lower concentrations of liver SAM than the control rats, Flinders Resistant Line (FRL) rats. The present study investigated if SAM supplementation may correct liver SAM and behavioral abnormalities in this model. Moreover, we compared one-carbon (C1) metabolites, neurotransmitters, and gastrointestinal (GI) transit in SAM-treated versus imipramine (IMI)-treated animals. FSL rats received vehicle, IMI, SAM, or IMI + SAM (n = 9-10 per group) once daily through oral gavage for 4 weeks; FRL rats received vehicle. Behavior was assessed using standard tests for locomotion, cognition, and depressive-like behavior. Monoamine neurotransmitters and Cl metabolites were measured using UHPLC-ECD and UPLC-MS/MS, respectively. Compared to FRL rats, FSLs had lower liver SAM, higher plasma serotonin, lower hippocampal dopamine and serotonin turnover, and faster GI transit. Behaviorally, FSL rats showed impaired cognitive performance as well as increased depressive-like behavior compared to FRLs. Coadministration of IMI and SAM seemed to have adverse effects on spatial memory. SAM or IMI administration did not reverse Cl metabolites, neurotransmitters, or GI transit in FSLs. Despite low liver SAM in FSL rats, orally administered SAM did not show antidepressant effects in this specific animal model of depression.

Scope Probiotics may influence one‐carbon (C1) metabolism, neurotransmitters, liver function markers, or behavior. Methods and results Male adult Flinders Sensitive Line rats (model of depression, FSL; n = 22) received Lactobacillus helveticus R0052 and Bifidobacterium longum R0175 (109 or 1010 colony‐forming units per day) or vehicle for 10 weeks. The controls, Flinders Resistant Line rats (FRL, n = 8), only received vehicle. C1‐related metabolites were measured in plasma, urine, and different tissues. Monoamine concentrations were measured in plasma, hippocampus, and prefrontal cortex. Vehicle‐treated FSL rats had higher plasma concentrations of betaine, choline, and dimethylglycine, but lower plasma homocysteine and liver S‐adenosylmethionine (SAM) than FRLs. FSL rats receiving high‐dose probiotics had lower plasma betaine and higher liver SAM compared to vehicle‐treated FSL rats. FSLs had higher concentrations of norepinephrine, dopamine, and serotonin than FRLs across various brain regions. Probiotics decreased plasma dopamine in FSLs in a dose‐dependent manner. There were no detectable changes in liver function markers or behavior. Conclusions Probiotics reduced the flow of methyl groups via betaine, increased liver SAM, and decreased plasma dopamine and norepinephrine. Since these changes in methylation and catecholamine pathways are known to be involved in several diseases, future investigation of the effect of probiotics is warranted.

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.

Background: Folate is required for synthesis of methyl groups and DNA in growing cells. The association between folate and prostate cancer (PCa) is not conclusive. Methods: We investigated concentrations of folate vitamers, S-adenosylhomocysteine (SAH) and S-adenosylmethionine (SAM) in blood of men with PCa (n = 129) or benign prostatic hyperplasia (BPH) (n = 73) who were recruited just after the first diagnosis. Results: In younger subjects < 65 years, concentrations of (6S)-5-CH3-H(4)folate (15.3 vs. 17.7 nmol/L) or total folate (UPLC-MS/MS) (18.7 vs. 23.0 nmol/L) did not differ between men with BPH and those with PCa, while SAM was higher in the controls (128 vs. 116 nmol/L). Younger patients with low-and high grade cancer did not differ in (6S)-5-CH3-H4folate (17.8 vs. 17.3 nmol/L)or total folate (UPLC-MS/MS) (22.9 vs. 23.3 nmol/L), but SAM was lower in patients with low grade PCa (111 vs. 126 nmol/L). In the older group = 65 years, (6S)-5-CH3-H(4)wfolate (18.4 vs. 18.2 nmol/L) and total folate (UPLC-MS/ MS) (22.5 vs. 22.1 nmol/L) did not differ between BPH and PCa. Older patients with advanced tumors had lower (6S)-5-CH3-H4folate compared with those with low grade tumor (12.8 vs. 20.0 nmol/L: p=0.013). Plasma SAM was not different between older patients and controls and was not related to PCa grade. Conclusions: Lowered serum methyl folate measured at the time of diagnosis in older patients with advanced PCa, and lowered plasma SAM in younger patients with low grade PCa suggest differential folate metabolism that may have mechanistic, prognostic or predictive values.