Plasma choline and betaine correlate with serum folate, plasma S-adenosyl-methionine and S-adenosyl-homocysteine in healthy volunteers.

BACKGROUND: Choline is essential for mammalian cell function. It plays a critical role in cell membrane integrity, neurotransmission, cell signaling and lipid metabolism. Moreover, choline is involved in methylation in two ways: a) its synthesis requires methyl groups donated by S-adenosyl-methionine (AdoMet); and b) choline oxidation product betaine methylates homocysteine (Hcy) to methionine (Met) and produces dimethylglycine. This later donates one carbon units to tetrahydrofolate (THF). METHODS: To evaluate the correlations of choline and betaine with folate, AdoMet, S-anenosyl-homocysteine (AdoHcy), total homocysteine (tHcy), and DNA methylation, choline, betaine and dimethylglycine were measured by LC-MS/MS in plasma of 109 healthy volunteers, in whom folate, AdoMet, AdoHcy, tHcy, and DNA methylation have previously been reported. RESULTS: Using a bivariate model, choline and betaine showed strong positive correlations with folate (r = 0.346 and r = 0.226), AdoHcy (r = 0.468 and r = 0.296), and correlated negatively with AdoMet/AdoHcy ratio (r = ­ 0.246 and r = ­ 0.379). Only choline was positively correlated with AdoMet (r = 0.453). Using a multivariate linear regression model, choline correlated strongly with folate ( ß = 17.416), AdoMet ( ß = 61.272), and AdoHcy ( ß = 9.215). Betaine correlated positively with folate ( ß = 0.133) and negatively with tHcy ( ß = ­ 0.194) ratio. Choline is an integral part of folate and methylation pathways. CONCLUSIONS: Our data highlight the importance of integrating choline in studies concerning addressing pathological conditions related to folate, homocysteine and methylation metabolism.

Methylation metabolites in amniotic fluid depend on gestational age.

OBJECTIVE: Methylation metabolism is essential for fetus development. However, normative data for amniotic fluid (AF) concentrations of methylation metabolites at different gestational ages are lacking. We aimed to determine in AF reference values of 14 intermediates involved in methylation. METHODS: Two hundred sixty-eight AFs sampled between 14 and 39 weeks of gestation were retrospectively selected in our AF bank. Next, we measured methionine (Met)-cycle intermediates [S-adenosyl Met (AdoMet), S-adenosyl-l-homocysteine (AdoHcy), total Hcy, Met, and methyl malonic acid] and methyl donors and methyl acceptors (betaine, dimethylglycine, sarcosine, free and total choline, free and total ethanolamine, creatine, and guanidinoacetate) by liquid chromatography coupled with tandem mass spectrometry. RESULTS: Reference ranges according to gestational age were determined for each parameter. Strong correlations between metabolites directly connected in their metabolic pathway and between total Hcy and betaine were observed. CONCLUSION: Methionine, an essential amino acid required for protein synthesis, is the only parameter that dramatically decreases with gestational age. The AdoMet/AdoHcy ratio exponentially increases from 25 weeks of gestation, which could reflect increasing methylation capacities. The negative correlation between betaine and total Hcy together with a constant betaine to dimethylglycine ratio during gestation suggests that betaine may be used as a methyl donor during fetal life.

Newborn screening for Cerebrotendinous Xanthomatosis: A retrospective biomarker study using both flow-injection and UPLC-MS/MS analysis in 20,000 newborns.

BACKGROUND AND AIMS: Cerebrotendinous Xanthomatosis (CTX) is a treatable disorder of bile acid synthesis caused by deficiency of 27-sterol hydroxylase -encoded by CYP27A1- leading to gastrointestinal and progressive neuropsychiatric symptoms. Biochemically, CTX is characterized by accumulation of the bile alcohol cholestanetetrol glucuronide (GlcA-tetrol) and the deficiency of tauro-chenodeoxycholic acid (t-CDCA) and tauro-trihydroxycholestanoic acid (t-THCA). MATERIALS AND METHODS: To ascertain the feasibility of CTX newborn screening (NBS) we performed a study with deidentified Dutch dried blood spots using reagents and equipment that is frequently used in NBS laboratories. 20,076 deidentified newborn blood spots were subjected to flow-injection (FIA)-MS/MS and UPLC-MS/MS analysis to determine the concentration of GlcA-tetrol and calculate the GlcA-tetrol/t-CDCA and t-THCA/GlcA-tetrol ratios. RESULTS: Using UPLC-MS/MS analysis both GlcA-tetrol concentration and/or metabolite ratios GlcA-tetrol/t-CDCA proved to be informative biomarkers; newborn DBS results did not overlap with those of the CTX patients. For FIA-MS/MS, GlcA-tetrol also was an excellent marker but when using the combination of the GlcA-tetrol/t-CDCA and t-THCA/GlcA-tetrol ratios also did not yield any screen positives. CONCLUSION: Newborn screening for CTX using only metabolite ratios following the measurement of three CTX biomarkers is possible using either FIA-MS/MS or UPLC-MS/MS, which paves the way for introduction of CTX NBS.

Is impaired energy production a novel insight into the pathogenesis of pyridoxine-dependent epilepsy due to biallelic variants in ALDH7A1?

BACKGROUND: Pyridoxine-dependent epilepsy (PDE) is due to biallelic variants in ALDH7A1 (PDE-ALDH7A1). ALDH7A1 encodes α-aminoadipic semialdehyde dehydrogenase in lysine catabolism. We investigated the gamma aminobutyric acid (GABA) metabolism and energy production pathways in human PDE-ALDH7A1 and its knock-out aldh7a1 zebrafish model. METHODS: We measured GABA pathway, and tricarboxylic acid cycle metabolites and electron transport chain activities in patients with PDE-ALDH7A1 and in knock-out aldh7a1 zebrafish. RESULTS: We report results of three patients with PDE-ALDH7A1: low paired complex I+II and complex II+III and individual complex IV activities in muscle biopsy in patient 1 (likely more severe phenotype); significantly elevated CSF glutamate in the GABA pathway and elevated CSF citrate, succinate, isocitrate and α-ketoglutarate in the TCA cycle in patient 3 (likely more severe phenotype); and normal CSF GABA pathway and TCA cycle metabolites on long-term pyridoxine therapy in patient 2 (likely milder phenotype). All GABA pathway metabolites (γ-hydroxybutyrate, glutamine, glutamate, total GABA, succinic semialdehyde) and TCA cycle metabolites (citrate, malate, fumarate, isocitrate, lactate) were significantly low in the homozygous knock-out aldh7a1 zebrafish compared to the wildtype zebrafish. Homozygous knock-out aldh7a1 zebrafish had decreased electron transport chain enzyme activities compared to wildtype zebrafish. DISCUSSION: We report impaired electron transport chain function, accumulation of glutamate in the central nervous system and TCA cycle dysfunction in human PDE-ALDH7A1 and abnormal GABA pathway, TCA cycle and electron transport chain in knock-out aldh7a1 zebrafish. Central nervous system glutamate toxicity and impaired energy production may play important roles in the disease neuropathogenesis and severity in human PDE-ALDH7A1.

Methylglyoxal and methylglyoxal-arginine adducts do not directly inhibit endothelial nitric oxide synthase.

Increased formation of the reactive dicarbonyl compound methylglyoxal (MGO) and MGO-derived advanced glycation end products (AGEs) seems to be implicated in endothelial dysfunction and the development of diabetic vascular complications. MGO reacts with arginine residues in proteins to generate the major glycated adducts 5-hydro-5-methylimidazolone (MG-H1) and argpyrimidine (AP). We investigated whether the free forms of these adducts contribute to vascular cell dysfunction by inhibition of endothelial nitric oxide synthase (eNOS). MG-H1 and AP were synthesized and purified by reversed-phase chromatography, and the conversion of labeled L-arginine to L-citrulline was used to monitor eNOS activity. In contrast to the endogenous eNOS inhibitor asymmetric dimethylarginine (half maximal inhibitory concentration, approximately 5 micromol/L), pathophysiological concentrations of MGO and MG-H1 and AP did not inhibit eNOS activity. Although MGO-derived AGEs are implicated in the development of diabetic vascular complications, this study indicates that this is not mediated via direct inhibition of eNOS activity.

Influence of different haemodialysis modalities on AGE peptide levels: intradialytic versus long-term results.

BACKGROUND: Peptide-linked degradation products of advanced glycation end products (AGE peptides) accumulate in chronic haemodialysis (HD) patients and may contribute to a number of HD-related long-term complications, such as accelerated atherosclerosis. METHODS: The influence of a single HD session versus long-term HD on serum AGE peptides was determined. The patients were randomized to HD with a low-flux polysulfone (PS; F 6HPS), a high-flux PS (F 60S), a superflux PS (F 500S), or a superflux cellulose triacetate (CTA; Tricea 150G) dialyzer. RESULTS: During a single HD session, both AGE peptides and reference peptides decreased significantly (AGE peptides: Tricea 150G -37.0 +/- 2.9%; F 6HPS -35.5 +/- 2.4%; F 60S -39.5 +/- 4.7%, and F 500S -43.3 +/- 2.1%, p = 0.005; reference peptides: Tricea 150G -73.2 +/- 8.8%; F 6HPS -73.2 +/- 7.9%; F 60S -72.5 +/- 8.2%, and F 500S -74.1 +/- 7.3%, p = 0.005). After 12 weeks of HD with the superflux CTA, the AGE peptide levels decreased significantly (week 1: 2.7 +/- 1.1 arbitrary units, week 12: 2.5 +/- 1.2 arbitrary units, decrease 7.4%; p = 0.01), whereas the AGE peptide levels remained unchanged after HD with each of the other three modalities. The reference peptide levels did not change after 12 weeks of HD. CONCLUSION: Although AGE peptides can be effectively removed during a single HD session, superflux CTA seems to be the only modality capable of reducing AGE peptides in the long term.

Improved glycemic control induced by both metformin and repaglinide is associated with a reduction in blood levels of 3-deoxyglucosone in nonobese patients with type 2 diabetes.

OBJECTIVE: Metformin has been reported to reduce α-dicarbonyls, which are known to contribute to diabetic complications. It is unclear whether this is due to direct quenching of α-dicarbonyls or to an improvement in glycemic control. We therefore compared the effects of metformin versus repaglinide, an antihyperglycemic agent with an insulin-secreting mechanism, on the levels of the α-dicarbonyl 3-deoxyglucosone (3DG). METHODS: We conducted a single-center, double-masked, double-dummy, crossover study involving 96 nonobese patients with type 2 diabetes. After a 1-month run-in on diet-only treatment, patients were randomized to either repaglinide (6 mg daily) followed by metformin (2 g daily) or vice versa each during 4 months with a 1-month washout between interventions. RESULTS: 3DG levels decreased after both metformin (-19.3% (95% confidence interval (CI): -23.5, -14.8)) and repaglinide (-20.8% (95% CI: -24.9, -16.3)) treatments, but no difference was found between treatments (1.8% (95% CI: -3.8, 7.8)). Regardless of the treatment, changes in glycemic variables were associated with changes in 3DG. Specifically, 3DG decreased by 22.7% (95% CI: 19.0, 26.5) per s.d. decrease in fasting plasma glucose (PG), by 20.0% (95% CI: 16.2, 23.9) per s.d. decrease in seven-point mean plasma glucose, by 22.5% (95% CI: 18.6, 26.6) per s.d. decrease in area under the curve for PG, by 17.2% (95% CI: 13.8, 20.6) per s.d. decrease in HbAlc, and by 10.9% (95% CI: 6.4, 15.5) per s.d. decrease in Amadori albumin. In addition, decreases in 3DG were associated with decreases in advanced glycation endproducts and endothelial markers. CONCLUSION: Improved glycemic control induced by both metformin and repaglinide is associated with a reduction in 3DG levels in nonobese individuals with type 2 diabetes. This may constitute a shared metabolic pathway through which both treatments have a beneficial impact on the cardiovascular risk.

Fasting and postprandial glycoxidative and lipoxidative stress are increased in women with type 2 diabetes.

OBJECTIVE: We studied acute changes in markers of glycoxidative and lipoxidative stress, including oxidized LDL, N(epsilon)-(carboxyethyl)-lysine (CEL), N(epsilon)-(carboxymethyl)-lysine (CML), and 3-deoxyglucosone (3DG), following two consecutive meals. RESEARCH DESIGN AND METHODS: Postmenopausal women (27 with normal glucose metabolism [NGM], 26 with type 2 diabetes) received two consecutive fat-rich meals and two consecutive carbohydrate-rich meals on two occasions. Glucose and triglyceride concentrations were measured at baseline and 1, 2, 4, 6, and 8 h following breakfast; lunch was given at 4 h. Oxidized LDL-to-LDL cholesterol ratio, CEL, CML, and 3DG were measured at baseline and at 8 h. RESULTS: Fasting oxidized LDL-to-LDL cholesterol ratio, 3DG, and CML were higher in women with type 2 diabetes compared with women with NGM and were comparable to the postprandial values at 8 h in NGM. Postprandial rises in the oxidized LDL-to-LDL cholesterol ratio and 3DG were similar in both groups. However, the oxidized LDL-to-LDL cholesterol ratio increased more after the fat-rich meals, whereas CML and 3DG increased more after the carbohydrate-rich meals. After the fat-rich meals, the increase in the oxidized LDL-to-LDL cholesterol ratio correlated with postprandial triglycerides, whereas the increase in 3DG was correlated with postprandial glucose. CONCLUSIONS: The acute changes in markers of glycoxidative and lipoxidative stress in both type 2 diabetes and NGM suggest that postabsorptive oxidative stress may partly underlie the association of postprandial derangements and cardiovascular risk.

Global DNA methylation measured by liquid chromatography-tandem mass spectrometry: analytical technique, reference values and determinants in healthy subjects.

BACKGROUND: Alterations in global DNA methylation are implicated in various pathobiological processes. We describe a liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MS/MS) method for determination of cytosine and 5-methylcytosine in DNA. METHODS: DNA was hydrolyzed using formic acid. Cytosine and 5-methylcytosine were separated by gradient-elution reversed-phase chromatography with a mobile phase containing nonafluoropentanoic acid (NFPA) as ion-pairing reagent and quantified using stable isotope dilution LC-ESI-MS/MS. The method was applied to DNA isolated from leukocytes of healthy volunteers. RESULTS: Linear calibration curves were obtained in the range 0.111-4.422 ng/microL [mean correlation co-efficient 0.9983 (SD=0.0011), n=9] for cytosine and 0.0048-0.1936 ng/microL [mean correlation coefficient 0.9991 (SD=0.0010), n=9] for 5-methylcytosine. The intra- and inter-assay CVs for the 5-methylcytosine/total cytosine ratio (mCyt/tCyt) was 1.7% (n=9) and 3.5% (n=8) for calf thymus DNA (mean mCyt/tCyt ratio 6.5%), and 4.5% (n=6) and 6.5% (n=14), respectively for pBR322 DNA (mean mCyt/tCyt ratio 0.48%). The limit of detection (signal-to-noise ratio 3) was 2 pg on-column for cytosine and 5-methylcytosine. In healthy subjects (n=109), the mCyt/tCyt ratio varied from 2.6% to 4.8% (median 4.1%). DNA methylation was negatively correlated to age, but only in subjects with the methylenetetrahydrofolate reductase (MTHFR) 677 TT genotype (p=0.046). No association with B-vitamin status was observed. CONCLUSIONS: This LC-ESI-MS/MS method is easy to perform and offers reproducibility, selectivity and sensitivity for studying DNA methylation. The method allows a sample throughput of approximately 200 samples/week. The MTHFR C677T genotype influences age-related changes in DNA methylation.

Measurement of Nepsilon-(carboxymethyl)lysine and Nepsilon-(carboxyethyl)lysine in human plasma protein by stable-isotope-dilution tandem mass spectrometry.

BACKGROUND: N(epsilon)-(Carboxymethyl)lysine (CML) and N(epsilon)-(carboxyethyl)lysine (CEL) are two stable, nonenzymatic chemical modifications of protein lysine residues resulting from glycation and oxidation reactions. We developed a tandem mass spectrometric method for their simultaneous measurement in hydrolysates of plasma proteins. METHODS: CML and CEL were liberated from plasma proteins by acid hydrolysis after addition of deuterated CML and CEL as internal standards. Chromatographic separation was performed by gradient-elution reversed-phase chromatography with a mobile phase containing 5 mmol/L nonafluoropentanoic acid as ion-pairing agent. Mass transitions of 205.1-->84.1 and 219.1-->84.1 for CML and CEL, respectively, and 209.1-->88.1 and 223.1-->88.1 for their respective internal standards were monitored in positive-ion mode. RESULTS: CML and CEL were separated with baseline resolution with a total analysis time of 21 min. The lower limit of quantification was 0.02 micromol/L for both compounds. Mean recoveries from plasma samples to which CML and CEL had been added were 92% for CML and 98% for CEL. Within-day CVs were <7.2% for CML and <8.2% for CEL, and between-day CVs were <8.5% for CML and <9.0% for CEL. In healthy individuals (n = 10), mean (SD) plasma concentrations of CML and CEL were 2.80 (0.40) micromol/L (range, 2.1-3.4 micromol/L) and 0.82 (0.21) micromol/L (range, 0.5-1.2 micromol/L), respectively. In hemodialysis (n = 17) and peritoneal dialysis (n = 9) patients, plasma concentrations of CML and CEL were increased two- to threefold compared with controls, without significant differences between dialysis modes [7.26 (1.36) vs 8.01 (3.80) micromol/L (P = 0.89) for CML, and 1.84 (0.39) vs 1.71 (0.42) micromol/L (P = 0.53) for CEL]. CONCLUSIONS: This stable-isotope-dilution tandem mass spectrometry method is suitable for simultaneous analysis of CML and CEL in hydrolysates of plasma proteins. Its robustness makes it suitable for assessing the value of these compounds as biomarkers of oxidative stress resulting from sugar and lipid oxidation.

Increased levels of N(epsilon)-(carboxymethyl)lysine and N(epsilon)-(carboxyethyl)lysine in type 1 diabetic patients with impaired renal function: correlation with markers of endothelial dysfunction.

BACKGROUND: Diabetic and non-diabetic patients with renal failure have an increased risk for cardiovascular disease, which may be the result of uraemic toxins, including advanced glycation end-products (AGEs). The aim of the study was to investigate the levels of well-characterized AGEs, N(epsilon)-(carboxymethyl)lysine (CML) and N(epsilon)-(carboxyethyl)lysine (CEL) in relation to kidney function and to study the relationship of these AGEs to endothelial function and inflammation in type 1 diabetic patients. METHODS: Plasma levels of CML and CEL were measured in 60 type 1 diabetic patients categorized as having normal glomerular filtration rate (GFR) (>80 ml/min, n = 31) or decreased GFR (<80 ml/min, n = 29) as estimated by the Cockcroft-Gault formula. To assess the relationship of these AGEs to endothelial function and inflammation, markers of endothelial function von Willebrand factor (vWf), soluble vascular cellular adhesion molecule-1 (sVCAM-1), soluble E-selectin (sE-selectin), soluble thrombomodulin (sTM), tissue type-specific plasminogen activator (tPA) and plasminogen activator inhibitor-1 (PAI-1), and C-reactive protein (CRP), a marker of inflammatory activity, were determined by enzyme-linked immunosorbent assays. RESULTS: Plasma levels of CML and CEL were increased in diabetic patients with decreased GFR as compared with patients with normal GFR [CML 4.9 (2-12.6) vs 2.9 (1.7-4.4) micromol/l, P<0.000; and CEL 1.7 (0.9-3.3) vs 1.2 (1.7-4.4) micromol/l, P = 0.004, respectively). Independently of the GFR, the plasma levels of CML and CEL were significantly associated with sVCAM-1, vWf and sTM. CONCLUSIONS: Plasma levels of CML and CEL rise with deterioration of GFR. Furthermore, CML and CEL levels are associated with markers of endothelial activation independently of renal function. This suggests an involvement of these AGEs in the acceleration of cardiovascular complications in patients with renal impairment.