Choline Pathway Research Articles

Prenatal choline supplementation enhances metabolic outcomes with differential impact on DNA methylation in Wistar rat offspring and dams

Choline is an essential nutrient required for proper functioning of organs and serves as a methyl donor. In liver where choline metabolism primarily occurs, glucose homeostasis is regulated through insulin receptor substrates (IRS) 1 and 2. The objective of this research was to determine the role of prenatal choline as a modulator of metabolic health and DNA methylation in liver of offspring and dams. Pregnant Wistar rat dams were fed an AIN-93G diet and received drinking water either with supplemented 0.25% choline (w/w) as choline bitartrate or untreated control. All offspring were weaned to a high-fat diet for 12 weeks. Prenatal choline supplementation led to higher insulin sensitivity in female offspring at weaning as well as lower body weight and food intake and higher insulin sensitivity in female and male adult offspring compared to offspring from untreated dams. Higher hepatic betaine concentrations were observed in dams and female offspring of choline-supplemented dams at weaning and higher glycerophosphocholine in female and male offspring at postweaning compared to the untreated control, suggestive of sustaining different choline pathways. Hepatic gene expression of Irs2 was higher in dams at weaning and female offspring at weaning and postweaning, whereas Irs1 was lower in male offspring at postweaning. Gene-specific DNA methylation of Irs2 was lower in female offspring at postweaning and Irs1 methylation was higher in male offspring at postweaning that exhibited an inverse relationship between methylation and gene expression. In conclusion, prenatal choline supplementation contributes to improved parameters of insulin signaling but these effects varied across time and offspring sex.

An NMR-based metabolic signature to identify clinically significant prostate cancer in patients undergoing biopsy.

Despite clinical suspicion of prostate cancer (PCa), 20-25% of patients exhibit a tumor-negative biopsy result. To assess the serum metabolic profile of clinically significant (cs) compared to clinically insignificant (ci) PCa or benign (Be) patients. 1078 serum samples were analyzed. Nuclear magnetic resonance (NMR) spectroscopy was used to quantify 73 metabolites, Random Forest for model algorithm. We identified a 22-metabolite panel, which discriminated csPCa (ISUP 2-5, n=328) from ciPCa (ISUP 1, n=101) and benign patients (negative biopsy, n=649) with a higher performance when combined with the standard clinical parameters age, prostate specific antigen (PSA), and percentage free PSA (%fPSA) (AUC 0.84) than the clinical parameters alone (AUC 0.73). Our study further revealed significant dysregulations of the urea cycle and the choline pathway along with changes in tricarboxylic acid (TCA) cycle, cholesterol metabolism, and a significant increase of the inflammation marker GlycB in csPCa patients. In particular, ornithine and dimethylglycine were the 2 most important features to discriminate csPCa from Be+ciPCa with significantly higher ornithine and lower dimethylglycine levels in patients with csPCa (ornithine: 63.7 ± 26.5 µmol/l, dimethylglycine: 12.6 ± 6.3 µmol/l, p<0.001) compared to Be+ciPCa patients (ornithine: 50.3 ± 31.6 µmol/l, dimethylglycine: 14.9 ± 7.7 µmol/l). This study discovered a 22-metabolite panel to discriminate patients with csPCa from Be+ciPCa patients when combined with age, PSA, and %fPSA. It may therefore be used as supportive biomarker to reduce the number of unnecessary biopsies and also to identify novel therapeutic targets in the future.

Mediterranean Diet Prior to Ischemic Stroke and Potential Circulating Mediators of Favorable Outcomes.

Background/Objectives. A Mediterranean diet (MD) has been associated with neuroprotective effects. We aimed to assess the MD's association with stroke prognosis and the potential mediators involved. Methods. Seventy patients with acute anterior circulation ischemic stroke were included. Dietary patterns were evaluated using the MEDAS scale, a food-frequency questionnaire, and a 24 h recall. Circulating biomarkers including insulin resistance (HOMA index), adipokines (resistin, adiponectin, leptin), choline pathway metabolites (TMAO, betaine, choline), and endothelial progenitor cells (EPCs) were measured. Early neurological improvement (ENI) at 24 h, final infarct volume, and functional outcome at 3 months were assessed. Results. Adherence to MD and olive oil consumption were associated with a lower prevalence of diabetes and atherothrombotic stroke, and with lower levels of fasting glycemia, hemoglobinA1C, insulin resistance, and TMAO levels. Monounsaturated fatty acids and oleic acid consumption correlated with lower resistin levels, while olive oil consumption was significantly associated with EPC mobilization. Multivariate analysis showed that higher MD adherence was independently associated with ENI and good functional prognosis at 3 months. EPC mobilization, lower HOMA levels, and lower resistin levels were associated with ENI, a smaller infarct volume, and good functional outcome. Conclusions. MD was associated with better prognosis after ischemic stroke, potentially mediated by lower insulin resistance, increased EPC mobilization, and lower resistin levels, among other factors.

The follicular fluid metabolome in infertile individuals between polycystic ovary syndrome and diminished ovarian reserve

Follicular fluid is the microenvironment of oocytes that plays a crucial role in oocyte development. This study intended to explore the follicular fluid metabolomics in diminished ovarian reserve (DOR), polycystic ovarian syndrome (PCOS), and normal ovary response (NOR) groups. For metabolomic analysis, we collected the follicular fluid samples from 28 patients with DOR, 28 patients with NOR, and 28 patients with PCOS. The identified metabolites were annotated using KEGG to determine the metabolic pathway disturbances in PCOS and DOR. Based on the regression model, we conducted ROC analysis to identify PCOS and DOR biomarkers in the follicular fluid. The present results identified that the DOR and NOR groups' differential metabolites were primarily enriched in the choline pathway. The concentrations of pregnanediol-3-glucuronide and 2-hydroxyestrone sulfate in the DOR and NOR groups were substantially different. The metabolites in the purine metabolism pathway were mainly enriched in the PCOS and NOR groups. N-Acetyl-S-(N-methylcarbamoyl) cysteine and 3,4-dehydrothiomorpholine in the PCOS and NOR groups were substantially different. We also identified metabolic alterations in PCOS and DOR follicular fluid, which provides novel ways for PCOS and DOR diagnosis and therapy.

Circulating choline pathway nutrients and risk of moyamoya disease

BackgroundCirculating choline pathway nutrients play a critical role in first stroke and recurrent stroke. However, there is limited information available on the effects of choline pathway nutrients on the risk of moyamoya disease (MMD) and its subtypes. We investigated the association between circulating choline and betaine and the incident risk of MMD and its subtypes.MethodsThe case-control study enrolled 385 patients with MMD [i.e., 110 transient ischemic attack (TIA)-type MMD, 157 infarction-type MMD, and 118 hemorrhagic-type MMD] and 89 matched healthy controls.ResultsSerum choline and betaine were inversely related to the risk of MMD and its subtypes. The risk of MMD was decreased with each increment in choline level [per 1 μmol increase: odds ratio (OR), 0.756; 95% CI, 0.678–0.843] and betaine level (per 1 μmol increase: OR, 0.952; 95% CI, 0.932–0.972), respectively. When choline and betaine were assessed as quartiles, compared with the lowest quartile of serum choline and betaine levels, those in the highest quartile had a significantly decreased risk of MMD (choline, Q4 vs. Q1: OR, 0.023; 95% CI, 0.005–0.118; betaine, Q4 vs. Q1: OR, 0.058; 95% CI, 0.018–0.184).ConclusionsSerum choline and betaine were associated with the decreased risk of MMD and its subtypes.

Abstract P161: Plasma Betaine, But Not Trimethylamine N-oxide Or Choline, Associated With 15-year Incident Diabetes: Coronary Artery Risk Development In Young Adults Study

Objective: Data from animal models supports a role for metabolites in the choline pathway in glycemic control and diabetes. Cross-sectional and case-control studies have documented positive associations between trimethylamine N-oxide (TMAO) and prevalent diabetes, but these findings have not been replicated in prospective analysis. Circulating choline has been positively, and betaine negatively, associated with diabetes in some, but not all, prospective data. We tested associations between three metabolites in the choline pathway (TMAO, choline, betaine) and 15-year incident diabetes in a diverse cohort of early-middle-aged adults. Methods: Study participants were from the Coronary Artery Risk Development in Young Adults (CARDIA) Study, a multicenter cohort of Black and White Americans established in 1985/86 (baseline n=5,115; ages 18-30). Plasma concentrations of TMAO, choline, and betaine were measured from Year 15 (2000-01) stored plasma (-70°C) with liquid chromatography mass spectrometry (LC-MS). Logistic regression was used to quantify associations between plasma metabolites and diabetes, adjusting for sociodemographic, behavioral, and clinical covariates. Results: Among 3,317 participants without diabetes at Year 15, n=373 developed diabetes over 15 years of follow-up (2000-2016). Plasma TMAO and choline were not significantly associated with incident diabetes in all multivariable-adjusted models, while betaine was negatively associated with incident diabetes in all models (Table). Conclusion: In this population-based cohort of early-middle-aged US adults, plasma TMAO and choline were not associated with incident diabetes over 15 years of follow-up, while plasma betaine was negatively associated with incident diabetes.

The gut microbiome and microbial metabolites in acute myocardial infarction

Emerging evidence has highlighted the role of gut microbiome in human health. However, the integrative role of gut microbiome and microbial metabolites in acute myocardial infarction (AMI) remains unclear. The current study profiles the microbial community through 16S rRNA gene sequencing and shotgun metagenomic sequencing and measures fecal short-chain fatty acids and circulating choline pathway metabolites among 117 new-onset AMI cases and 78 controls. Significant microbial alternations are observed in AMI patients compared with controls (P = 0.001). The abundances of nine species (e.g., Streptococcus salivarius and Klebsiella pneumoniae) are positively associated, and one species (Roseburia hominis) is inversely associated with AMI status and severity. A gut microbial score at disease onset is associated with the risk of major adverse cardiovascular events in 3.2 years (hazard ratio [95% CI]: 2.01 [1.04–4.24]) in AMI patients. The molar proportions of fecal acetate and butyrate are higher, and the circulating levels of choline and carnitine are lower in AMI patients than in controls. In addition, disease classifiers show that AMI cases and controls have a more distinct pattern in taxonomical composition than in pathways or metabolites. Our findings suggest that microbial composition and functional potentials are associated with AMI status and severity.

Plasma choline and betaine and risks of cardiovascular events and recurrent stroke after ischemic stroke

Choline and betaine have been suggested to play a pivotal role in neurotransmitter synthesis, cell membrane integrity, and methyl-group metabolism, exerting neuroprotective effects in patients with various neurological disorders. However, population-based evidence on choline and betaine with subsequent cardiovascular events after stroke is rare. We aimed to prospectively investigate the relationships of circulating choline and betaine with cardiovascular events and recurrent stroke in patients with ischemic stroke. We performed a nested case-control study within the China Antihypertensive Trial in Acute Ischemic Stroke. A total of 323 cardiovascular events (including 264 recurrent strokes) and 323 controls (free of recurrent cardiovascular events) matched for age (±1 y), sex, and treatment group were included. The primary endpoint was a composite of cardiovascular events after ischemic stroke. Plasma choline and betaine were measured at baseline by ultra-high-performance LC-MS/MS. Conditional logistic regression models were applied, and discrimination, reclassification, and calibration of models with choline pathway metabolites were evaluated. Plasma choline and betaine were inversely associated with cardiovascular events and recurrent stroke after ischemic stroke. Specifically, in fully adjusted models, each additional SD of choline and betaine was associated with 35% (95% CI: 20%-48%) and 30% (95% CI: 14%-43%) decreased risks of subsequent cardiovascular events, respectively, and 34% (95% CI: 16%-48%) and 29% (95% CI: 12%-43%) decreased risks of recurrent stroke, respectively. In addition, both choline and betaine offered substantial risk discrimination and reclassification improvement for cardiovascular events and recurrent stroke beyond traditional risk factors, as evidenced by an increase in C statistics, the net reclassification index, and integrated discrimination improvement. Plasma choline pathway metabolites, including choline and betaine, were associated with decreased risks of cardiovascular events and recurrent stroke and provided incremental value in risk discrimination and stratification in patients with ischemic stroke. This nested case-control study was based on the China Antihypertensive Trial in Acute Ischemic Stroke, which is registered at clinicaltrials.gov as NCT01840072.

Choline Pathway Nutrients and Metabolites and Cognitive Impairment After Acute Ischemic Stroke.

Choline metabolism was suggested to play pathophysiological roles in nervous system and atherosclerosis development. However, little is known about the impacts of choline pathway nutrients and metabolites on poststroke cognitive impairment. We aimed to prospectively investigate the relationships between circulating choline, betaine, and trimethylamine N-oxide with cognitive impairment among acute ischemic stroke patients. We derived data from CATIS (China Antihypertensive Trial in Acute Ischemic Stroke). Plasma choline, betaine, and trimethylamine N-oxide concentrations at baseline were measured in 617 participants. Cognitive impairment was evaluated using the Mini-Mental State Examination and the Montreal Cognitive Assessment. Reclassification and calibration of models with choline-related biomarkers were evaluated. Plasma choline and betaine were inversely associated with cognitive impairment. Compared with the lowest tertile, adjusted odds ratios of Mini-Mental State Examination-defined cognitive impairment for participants in the highest tertiles of choline and betaine were 0.59 (95% CI, 0.39-0.90) and 0.60 (95% CI, 0.39-0.92), respectively. In addition, both choline and betaine offered incremental predictive ability over the basic model with established risk factors, shown by increase in net reclassification improvement and integrated discrimination improvement. There were similar significant relationships between choline and betaine with cognitive impairment as defined by the Montreal Cognitive Assessment. However, plasma trimethylamine N-oxide was only associated with cognitive impairment evaluated using the Mini-Mental State Examination; the adjusted odds ratio was 1.33 (95% CI, 1.04-1.72) for each 1-SD increment of trimethylamine N-oxide. Patients with higher choline and betaine levels had lower risk of cognitive impairment after ischemic stroke, supporting promising prognostic roles of choline pathway nutrients for poststroke cognitive impairment.

Abstract 3740: Role of phosphatidylcholine in β-catenin-driven hepatocellular carcinoma

Abstract Introduction: This study aimed to clarify the role of phosphatidylcholine (PC) and choline metabolites in β-catenin-driven hepatocellular carcinoma (HCC) using human HCC cell lines and transgenic zebrafish expressing hepatocyte-specific activated β-catenin. Background: β-catenin is widely believed to be a major oncogene in HCC. PC is one of the major constituents of biological membranes and is synthesized via the phosphatidylethanolamine N-methyltransferase (PEMT) and cytidine diphosphate (CDP)-choline pathways in the liver. Several studies have reported that changes in gene expression and enzyme activity lead to altered PC synthesis in cancer, which contributes to malignant growth. However, the role of altered PC synthesis in HCC and the contribution of activated β-catenin to changes in PC metabolism have not been fully explored. Methods and Results: Transgenic zebrafish expressing hepatocyte-specific activated β-catenin develop HCC as adults and show significant liver enlargement during larval development due to increased cell proliferation. We measured PC levels in transgenic zebrafish HCC and non-transgenic control livers using liquid chromatography-mass spectrometry (LC-MS). PC levels in transgenic zebrafish HCC were significantly higher than in controls. Using a colorimetric assay kit, we found that PC levels were significantly higher in human HCC cell lines (SNU398, C3A and PLC/PRF/5) than in immortalized human hepatocytes (THLE2). To clarify the role of PC and choline metabolites in β-catenin-driven liver overgrowth, we treated larval zebrafish with bezafibrate (PEMT inhibitor) or meclofenoxate (CPT/CEPT inhibitor). We found that both of these inhibitors of PC lipid synthesis reduced larval liver size and decreased the number of EdU-labeled proliferative cells in transgenic zebrafish. Histologically, nonalcoholic steatohepatitis (NASH)-like changes, such as steatosis, ballooning, inflammation, and hepatocellular necrosis, were detected in the livers of transgenic larvae treated with bezafibrate or meclofenoxate. To characterize the role of PC in human HCC cell lines, we treated SNU398, C3A and PLC/PRF/5 cells with bezafibrate or meclofenoxate and measured PC levels by colorimetric assay and viability by CellTiter-Glo Assay. We found that both drugs decreased PC levels and viability in these HCC cell lines. Furthermore, SNU398 cells overexpressing activated β-catenin using a Tet-on system demonstrated higher PC levels and greater viability than SNU398 control cells. Conclusion: Our results suggest that increased PC synthesis is associated with cell proliferation, zebrafish larval liver overgrowth, and hepatocarcinogenesis. These results also suggest that activated β-catenin may regulate choline metabolites and contribute to an increase in PC production. Citation Format: Junko Kuramoto, Richard Smith, Sharanya M. Kalasekar, Alexis Fulbright, Gregory S. Ducker, Kimberley J. Evason. Role of phosphatidylcholine in β-catenin-driven hepatocellular carcinoma [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 3740.

Circulating metabolites from the choline pathway and acute coronary syndromes in a Chinese case-control study

BackgroundAccumulating evidence shows that circulating levels of trimethylamine N-oxide, which is generated from the metabolism of dietary choline, may predict cardiovascular disease among Caucasians. Acute coronary syndrome (ACS), one common presentation of cardiovascular disease, is a spectrum of signs and symptoms due to acute decreased blood flow in the coronary arteries. The relationship between the metabolites from choline pathway and ACS remains unclear. We aimed to assess the associations of circulating metabolites from the choline pathway with ACS among a Chinese population, who consume a different dietary pattern than their Western counterparts.MethodsWe recruited 501 participants who were admitted to the Department of Cardiology, Zhongshan Hospital,Shanghai China between March 2017 and June 2018, including 254 ACS cases and 247 controls. Liquid chromatography-tandem mass spectrometry was used to measure circulating concentrations of metabolites in the choline pathway, including betaine, choline, trimethylamine, and trimethylamine N-oxide. A composite metabolite score using a weighted sum of these four metabolites, and the betaine/choline ratio were calculated. Multivariable logistic regressions were applied to estimate the association of metabolites with ACS, with adjustment of age, sex, body mass index, smoking index, history of diseases, and kidney function.ResultsAfter adjusting for traditional risk factors, per 1-standard deviation (SD) increment in choline was positively associated with the odds of ACS [odds ratio (OR), 95% confidence interval (CI), 1.77(1.44–2.18)], and the other metabolites were not associated with ACS at a statistical significance level. Compared with participants in the lowest quartile of the metabolite score, those in the highest quartile had higher odds of ACS [OR (95% CI), 3.18(1.85–5.54), p < 0.001 for trend]. Per 1-SD increment in metabolite score was positively associated with higher odds of ACS [OR (95% CI), 1.80 (1.37–2.40)], and per 1-SD increment in the betaine/choline ratio was inversely associated with the odds of ACS [OR (95% CI), 0.49 (0.39–0.60)].ConclusionsAmong our Chinese participants, trimethylamine N-oxide was not associated with ACS, while a composite metabolite score of metabolites from the choline pathway was associated with increased odds of ACS. The choline pathway metabolites may be related to the pathophysiology of ACS among Chinese.

Metabolomic Profiling Reveals Cellular Reprogramming of B-Cell Lymphoma by a Lysine Deacetylase Inhibitor through the Choline Pathway

Despite the proven clinical antineoplastic activity of histone deacetylase inhibitors (HDACI), their effect has been reported to be lower than expected in B-cell lymphomas. Traditionally considered as “epigenetic drugs”, HDACI modify the acetylation status of an extensive proteome, acting as general lysine deacetylase inhibitors (KDACI), and thus potentially impacting various branches of cellular metabolism. Here, we demonstrate through metabolomic profiling of patient plasma and cell lines that the KDACI panobinostat alters lipid metabolism and downstream survival signaling in diffuse large B-cell lymphomas (DLBCL). Specifically, panobinostat induces metabolic adaptations resulting in newly acquired dependency on the choline pathway and activation of PI3K signaling. This metabolic reprogramming decreased the antineoplastic effect of panobinostat. Conversely, inhibition of these metabolic adaptations resulted in superior anti-lymphoma effect as demonstrated by the combination of panobinostat with a choline pathway inhibitor. In conclusion, our study demonstrates the power of metabolomics in identifying unknown effects of KDACI, and emphasizes the need for a better understanding of these drugs in order to achieve successful clinical implementation.

Abnormal liver phosphatidylcholine synthesis revealed in patients with acute respiratory distress syndrome

Acute respiratory distress syndrome (ARDS) is associated with a severe pro-inflammatory response; although decreased plasma cholesterol concentration has been linked to systemic inflammation, any association of phospholipid metabolic pathways with ARDS has not been characterized. Plasma phosphatidylcholine (PC), the major phospholipid of circulating lipoproteins, is synthesized in human liver by two biologically diverse pathways: the cytidine diphosphocholine (CDP):choline and phosphatidylethanolamine N-methyltransferase (PEMT) pathways. Here, we used ESI-MS/MS both to characterize plasma PC compositions and to quantify metabolic fluxes of both pathways using stable isotopes in patients with severe ARDS and in healthy controls. Direct incorporation of methyl-D9-choline estimated CDP:choline pathway flux, while PEMT flux was determined from incorporations of one and two methyl-D3 groups derived from methyl-D9-choline. The results of MS/MS analysis showed significant alterations in plasma PC composition in patients with ARDS versus healthy controls. In particular, the increased overall methyl-D9-PC enrichment and, most importantly, the much lower methyl-D3-PC and methyl-D6-PC enrichments suggest increased flux through the CDP:choline pathway and reduced flux through the PEMT pathway in ARDS. To our knowledge, this study is the first to demonstrate significant plasma PC molecular compositional changes combined with associated alterations in the dynamics of PC synthetic pathways in patients with ARDS.

Eggs early in complementary feeding increase choline pathway biomarkers and DHA: a randomized controlled trial in Ecuador

Background: Choline status has been associated with stunting among young children. Findings from this study showed that an egg intervention improved linear growth by a length-for-age z score of 0.63.Objective: We aimed to test the efficacy of eggs introduced early in complementary feeding on plasma concentrations of biomarkers in choline pathways, vitamins B-12 and A, and essential fatty acids.Design: A randomized controlled trial, the Lulun (“egg” in Kichwa) Project, was conducted in a rural indigenous population of Ecuador. Infants aged 6–9 mo were randomly assigned to treatment (1 egg/d for 6 mo; n = 80) and control (no intervention; n = 83) groups. Socioeconomic data, anthropometric measures, and blood samples were collected at baseline and endline. Household visits were made weekly for morbidity surveillance. We tested vitamin B-12 plasma concentrations by using chemiluminescent competitive immunoassay and plasma concentrations of choline, betaine, dimethylglycine, retinol, essential fatty acids, methionine, dimethylamine (DMA), trimethylamine, and trimethylamine-N-oxide (TMAO) with the use of liquid chromatography–tandem mass spectrometry.Results: Socioeconomic factors and biomarker concentrations were comparable at baseline. Of infants, 11.4% were vitamin B-12 deficient and 31.7% marginally deficient at baseline. In adjusted generalized linear regression modeling, the egg intervention increased plasma concentrations compared with control by the following effect sizes: choline, 0.35 (95% CI: 0.12, 0.57); betaine, 0.29 (95% CI: 0.01, 0.58); methionine, 0.31 (95% CI: 0.03, 0.60); docosahexaenoic acid, 0.43 (95% CI: 0.13, 0.73); DMA, 0.37 (95% CI: 0.37, 0.69); and TMAO, 0.33 (95% CI: 0.08, 0.58). No significant group differences were found for vitamin B-12, retinol, linoleic acid (LA), α-linolenic acid (ALA), or ratios of betaine to choline and LA to ALA.Conclusion: The findings supported our hypothesis that early introduction of eggs significantly improved choline and other markers in its methyl group metabolism pathway. This trial was registered at clinicaltrials.gov as NCT02446873.

Metabolomics and genomics: revealing the mechanism of corydalis alkaloid on anti-inflammation in vivo and in vitro

As one of the major active components of Qizhiweitong (QZWT) prescription, corydalis alkaloid (CA) has been reported to have noticeable effect of anti-inflammation in pharmacological studies. However, the anti-inflammatory function and potential mechanism of CA in anti-inflammation remain to be clarified. Hence, the aim of this study was to investigate the anti-inflammation efficacy and potential mechanism of CA in anti-inflammation by metabonomic approach coupled with related gene analyzes in vivo and in vitro. Mice acute inflammation was induced by subcutaneous injection of formalin in hind paws, and evaluated the anti-inflammatory effect of CA via detecting the index of paw edema. The results indicated that CA has an anti-inflammatory effect on mice through alleviating the paw edema significantly. Moreover, metabolic profiling was performed by high performance liquid chromatography quadrupole-time-of-flight mass spectrometer (HPLC-QTOF-MS) combined with multivariate data analysis, such as principal component analysis (PCA) and partial least-squares discriminant analysis (PLS-DA). Thirteen metabolic targets (including lysophosphatidylcholine (LysoPC), choline, arachidonic acid, phosphodimethylethanolamine) and related pathways of the cholinergic anti-inflammatory and arachidonic acid (AA) metabolism were identified, Meanwhile, the relevant target genes, like iNOS, NF-κB, TNF-α, were detected and verified in vitro. The results indicated that CA has a good pharmacological effect of anti-inflammation through regulating multiple disordered targets and metabolic networks, which provides a theoretical basis for further research on CA. Furthermore, there is a great potential for the development of CA to be a new anti-inflammatory drug with high effective activities, little side effects and weak drug resistance.

Plasma Metabolites From Choline Pathway and Risk of Cardiovascular Disease in the PREDIMED (Prevention With Mediterranean Diet) Study.

BackgroundThe relationship between plasma concentrations of betaine and choline metabolism and major cardiovascular disease (CVD) end points remains unclear. We have evaluated the association between metabolites from the choline pathway and risk of incident CVD and the potential modifying effect of Mediterranean diet interventions.Methods and ResultsWe designed a case‐cohort study nested within the PREDIMED (Prevention With Mediterranean Diet) trial, including 229 incident CVD cases and 751 randomly selected participants at baseline, followed up for 4.8 years. We used liquid chromatography–tandem mass spectrometry to measure, at baseline and at 1 year of follow‐up, plasma concentrations of 5 metabolites in the choline pathway: trimethylamine N‐oxide, betaine, choline, phosphocholine, and α‐glycerophosphocholine. We have calculated a choline metabolite score using a weighted sum of these 5 metabolites. We used weighted Cox regression models to estimate CVD risk. The multivariable hazard ratios (95% confidence intervals) per 1‐SD increase in choline and α‐glycerophosphocholine metabolites were 1.24 (1.05–1.46) and 1.24 (1.03–1.50), respectively. The baseline betaine/choline ratio was inversely associated with CVD. The baseline choline metabolite score was associated with a 2.21‐fold higher risk of CVD across extreme quartiles (95% confidence interval, 1.36–3.59; P<0.001 for trend) and a 2.27‐fold higher risk of stroke (95% confidence interval, 1.24–4.16; P<0.001 for trend). Participants in the higher quartiles of the score who were randomly assigned to the control group had a higher risk of CVD compared with participants in the lower quartile and assigned to the Mediterranean diet groups (P=0.05 for interaction). No significant associations were observed for 1‐year changes in individual plasma metabolites and CVD.ConclusionsA metabolite score combining plasma metabolites from the choline pathway was associated with an increased risk of CVD in a Mediterranean population at high cardiovascular risk.Clinical Trial Registration URL: http://www.controlled-trials.com. Unique identifier: ISRCTN35739639.

From masochistic enzymology to mechanistic physiology and disease

The pioneering work of Eugene Kennedy in the 1950s established the choline pathway for phosphatidylcholine (PC) biosynthesis. However, the regulation of PC biosynthesis was poorly understood at that time. When I started my lab at the University of British Columbia in the 1970s, this was the focus of my research. This article provides my reflections on these studies that began with enzymology and the use of cultured mammalian cells, and progressed to utilize the techniques of molecular biology and gene-targeted mice. The research in my lab and others demonstrated that the regulated and rate-limiting step in the choline pathway for PC biosynthesis was catalyzed by CTP:phosphocholine cytidylyltransferase. This enzyme is regulated by its movement from a soluble form (largely in the nucleus) to a membrane-associated form where the enzyme becomes activated. Gene targeting in mice subsequently demonstrated that this gene is essential for development of mouse embryos. The other mammalian pathway for PC biosynthesis is catalyzed by phosphatidylethanolamine N-methyltransferase (PEMT) that converts phosphatidylethanolamine to PC. Understanding of the regulation and function of the integral membrane protein PEMT was improved when the enzyme was purified (a masochistic endeavor) in 1987, leading to the cloning of the Pemt cDNA. Generation of knock-out mice that lacked PEMT showed that they were protected from atherosclerosis, diet-induced obesity, and insulin resistance. The protection from atherosclerosis appears to be due to decreased secretion of lipoproteins from the liver. We continue to investigate the mechanism(s) by which Pemt-/- mice are protected from weight gain and insulin resistance.

Alterations in metabolic pathways in stomach of mice infected with Helicobacter pylori.

Numerous studies of Helicobacter pylori (H.pylori) have been performed, but few studies have evaluated the effects of H.pylori infections using metabolome analysis, which involves the comprehensive study of low molecular weight metabolites. In this study, the metabolites in the stomach tissue of mice that had been infected with H.pylori SS1 for 1, 3, or 6 months were analyzed, and then evaluations of various metabolic pathways were performed to gain novel understandings of H.pylori infections. As a result, it was found that the glycolytic pathway, the tricarboxylic acid cycle, and the choline pathway tended to be upregulated at 1 month after the H.pylori SS1 infection. The urea cycle tended to be downregulated at 6 months after the infection. High levels of some amino acids were observed in the stomach tissue of the H.pylori SS1-infected mice at 1 month after the infection, whereas low levels of many amino acids were detected at 3 and 6 months after the infection. These results suggest that H.pylori infection causes various metabolic alterations at lesional sites, and these alterations might be linked to the crosstalk between H.pylori and the host leading to transition of disease conditions.

Polymorphic variants of genes involved in choline pathway and the risk of intrauterine fetal death.

Choline and folate metabolism disturbances may be involved in the occurrence of intrauterine fetal death (IUFD). The proper activity of this metabolism could be determined by genetic variants involved in choline pathway e.g. CHKA (gene encoding choline kinase α), PCYT1A (gene encoding CCTα) and CHDH (gene encoding choline dehydrogenase). Our study aimed at determining the genotype and allele frequencies of CHKA rs7928739, PCYT1A rs712012, PCYT1A rs7639752, CHDH rs893363 and CHDH rs2289205 polymorphisms in mothers with IUFD occurrence. The study involved 76 mothers with IUFD occurrence and 215 mothers of healthy children. Genetic analysis was performed with the use of PCR/RFLP method. The frequency of genotypes and alleles of studied polymorphisms was similar in both groups. The study revealed no association of PCYT1A, CHKA and CHDH polymorphisms in analysed groups of women. While evaluating the co-existence of analysed polymorphisms statistically significant correlation was revealed. Co-existence of CHKA rs7928739 AC/CHDH rs2289205 AA genotypes was observed statistically more frequently in the study group than in the control group (p = 0,031). There is no correlation between single CHKA rs7928739, PCYT1A rs712012, PCYT1A rs7639752, CHDH rs893363 and CHDH rs2289205 polymorphisms and the incidence of intrauterine fetal death. However, revealed statistically significant difference between co-existence of CHKA rs7928739 AC/CHDH rs2289205 AA genotypes between study groups suggest the need of further analysis.

Near infrared fluorescent imaging of choline kinase alpha expression and inhibition in breast tumors.

Choline kinase alpha (ChoKα) overexpression is associated with an aggressive tumor phenotype. ChoKα inhibitors induce apoptosis in tumors, however validation of their specificity is difficult in vivo. We report the use of optical imaging to assess ChoKα status in cells and in vivo using JAS239, a carbocyanine-based ChoKα inhibitor with inherent near infrared fluorescence. JAS239 attenuated choline phosphorylation and viability in a panel of human breast cancer cell lines. Antibody blockade prevented cellular retention of JAS239 indicating direct interaction with ChoKα independent of the choline transporters and catabolic choline pathways. In mice bearing orthotopic MCF7 breast xenografts, optical imaging with JAS239 distinguished tumors overexpressing ChoKα from their empty vector counterparts and delineated tumor margins. Pharmacological inhibition of ChoK by the established inhibitor MN58b led to a growth inhibition in 4175-Luc+ tumors that was accompanied by concomitant reduction in JAS239 uptake and decreased total choline metabolite levels as measured using magnetic resonance spectroscopy. At higher therapeutic doses, JAS239 was as effective as MN58b at arresting tumor growth and inducing apoptosis in MDA-MB-231 tumors, significantly reducing tumor choline below baseline levels without observable systemic toxicity. These data introduce a new method to monitor therapeutically effective inhibitors of choline metabolism in breast cancer using a small molecule companion diagnostic.