Since the 1960s, it has been known that elevated levels of homocysteine (Hcy), 1 resulting from mutations in genes encoding Hcy-metabolizing enzymes, are harmful to humans (1, 2). During the past decade it has been established that even a mild increase in Hcy level is a risk factor for cardiovascular disease and stroke in humans (3, 4) and predicts mortality independently of traditional risk factors in patients with coronary artery disease (5). Plasma Hcy is also a risk factor for neurodegenerative disorders, such as dementia and Alzheimer's disease (6). In tissue cultures, Hcy does not support growth and induces apoptotic death in human endothelial cells (7). Animal and cell culture studies have shown that Hcy induces cell death and potentiates amyloid ␤-peptide toxicity in neurons (8).In humans, Hcy, formed from dietary methionine as a byproduct of cellular methylation reactions, is detoxified by folic acid-and vitamin B 12 -dependent re-methylation to methionine (2) or vitamin B 6 -dependent trans-sulfuration to cysteine (1). Whereas Hcy is formed in all human organs, most of its detoxification occurs in the liver and kidneys. Detoxification of Hcy in human vascular tissues and skin occurs only by re-methylation; enzymes of the trans-sulfuration pathway are not expressed in these tissues (9).Hcy is perhaps the most reactive amino acid in biological systems (1, 2). In addition to re-methylation to methionine or trans-sulfuration to cysteine (via cystathionine), Hcy is also metabolically converted to Hcy-thiolactone, S-nitroso-Hcy, AdoHcy, Hcy-containing disulfides, or homocysteic acid, each of which has been implicated in the pathology of hyperhomocystinemia (1, 10).Because of its similarity to the protein amino acid methionine, Hcy can exert its biological effects by interfering with protein biosynthesis (10 -18). For example, Hcy is metabolized to Hcy-thiolactone by methionyl-tRNA synthetase in a two-step reaction (19,20). In the first step (Reaction 1), methionyl-tRNA synthetase catalyzes activation of Hcy with ATP, which yields methionyl-tRNA synthetase (MetRS)-bound homocysteinyl adenylate.The second step (Reaction 2), in which the side chain thiolate of Hcy reacts with the activated carboxyl group of Hcy, yields Hcy-thiolactone.The energy of the anhydride bond in HcyϳAMP is conserved in the thioester bond in Hcy-thiolactone. Because of this, Hcythiolactone reacts with proteins, forming Hcy-containing adducts, in which the carboxyl group of Hcy is linked by an amide bond with ⑀-amino group of a protein lysine residue (21-23).Originally discovered in cultured cells (21-23), protein Nhomocysteinylation is now known to occur in humans (10,(12)(13)(14)(15)(16)(17)(18). Both Hcy-thiolactone (13,24,25) and N-linked proteinHcy (12,16,26,27) have been demonstrated in human blood. About 70% of circulating Hcy is N-linked to blood proteins, mostly hemoglobin and albumin (26). A protective mechanism against protein N-homocysteinylation appears to exist in humans (28,29).Two major forms of albumin exist in circulatio...

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BackgroundThis study investigated the effects of long term betaine supplementation on body composition, performance, and homocysteine thiolactone (HCTL) in experienced strength trained men.MethodsTwenty-three subjects were matched for training experience (4.8 ± 2.3 years) and body fat percentage (BF%: 16.9 ± 8.0%), randomly assigned to either a placebo (PL; n = 12) or betaine group (BET; n = 11; 2.5 g/day), and completed a 6 week periodized training program consisting of 3 two-week micro-cycles. Bench press and back squat training volumes were recorded and changes in training volume were assessed at each micro-cycle. Fasting urine was collected at baseline (BL), weeks 2, 4 and 6, and assayed for HCTL. Subjects were tested prior to and following 6 weeks of treatment. Arm and thigh cross sectional area (CSA) was estimated via girth and skin fold measurements. Body density was estimated via skin fold calipers and used to estimate BF%, fat mass (FM), and lean body mass (LBM). Performance was assessed via vertical jump (VJ), bench press 1 RM (BP), and back squat 1 RM (BS).ResultsArm CSA increased significantly (p < .05) in BET but not PL. No differences existed between group and time for changes in thigh CSA. Back squat training volume increased significantly (p < .05) for both groups throughout training. Bench press training volume was significantly (p < .05) improved for BET compared to PL at microcycles one and three. Body composition (BF%, FM, LBM) improved significantly (p < .05) in BET but not PL. No differences were found in performance variables (BP, BS, VJ) between groups, except there was a trend (p = .07) for increased VJ power in BET versus PL. A significant interaction (p < .05) existed for HCTL, with increases from BL to week 2 in PL, but not BET. Additionally, HCTL remained elevated at week 4 in PL, but not BET.ConclusionSix-weeks of betaine supplementation improved body composition, arm size, bench press work capacity, attenuated the rise in urinary HCTL, and tended to improve power (p = .07) but not strength.

A new analytical method is proposed for simultaneous determination, by liquid chromatography, of the three main urinary thiols-cysteine, cysteinylglycine, and homocysteine. To measure the total amount of these thiols urine is reduced with sodium borohydride, to convert disulfides to thiols which are then derivatized with 2-chloro-1-methylquinolinium tetrafluoroborate. Separation and quantitation of the 2-S-quinolinium thiol derivatives formed were achieved by high-performance liquid chromatography with detection at 355 nm. Validation showed the method enabled reliable simultaneous determination of these aminothiols in urine. The calibration graphs for each analyte, obtained by use of normal urine spiked with increasing amounts of cysteine, cysteinylglycine, and homocysteine, were linear (R2 > or = 0.997) over the range covering most practical situations. The recovery of the assay was 98-100% and sensitivity was 0.12-0.25 micromol L(-1). The method was applied to 91 different samples of normal urine to establish reference values for the aminothiols, normalized on creatinine.

A new, highly reactive, and thiol specific derivatization reagent for liquid chromatographic analysis has been developed. The reagent, 2-chloro-1-methylquinolinium tetrafluoroborate, reacts instantaneously with hydrophilic thiols in water under mild conditions. The 2-S-quinolinium derivatives, resulting from this reaction, are stable thioethers exhibiting well defined absorption maximum at 348 nm and molar absorptivity coefficient about 2 x 10 4 L mol -1 cm -1 . It is shown that under reversed-phase high performance liquid chromatography conditions with gradient elution, six thiols possessing different functional groups, can be baseline separated and quantified within 4 min in one analytical run.The lower limits of detection and quantitation of the analytes (20 µL injection volume) are within 0.3-1.5 pmol and 0.5-4.0 Downloaded by [University of Colorado at Boulder Libraries] at 11:45 22 December 2014 pmol, respectively. The assays are linear in the range of 0.025-8 nmol/mL with correlation coefficients values close to 0.9999.Synthesis of 2-chloro-1-methylquinolinium tetrafluoroborate, as well as isolation of the 2-S-quinolinium derivative of one model thiol, are described.

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