High-throughput capillary electrophoresis method for plasma cysteinylglycine measurement: evidences for a clinical application

Summary. Increased levels in plasma homocysteine and cysteine, and more recently, decreased levels in cysteinylglycine have been indicated as a risk factor for vascular diseases. Most assays focused their attention only on homocysteine determination and when also other thiols were measured, analytical times drastically increased. By modifying our previous method for thiols detection, we set up a rapid capillary electrophoresis method for the selective quantification of plasma cysteinylglycine, cutting the analysis time of about 50%. Samples were treated with tri-n-butylphosphine as reducing agent, proteins were precipitated with trichloroacetic acid and released thiols were successively derivatized by the selective thiol laser-induced fluorescence-labeling agent 5-iodoacetamidofluorescein and separated by capillary electrophoresis. A baseline separation between peaks was obtained in about 2 min using 3 mmol/L sodium phosphate/2.5 mmol/L boric acid as electrolyte solution with 75 mmol/L N-methyl-D-glucamine at pH 11.25 in a 47 cm long capillary with a cartridge temperature of 45 °C. The method application was checked by measuring plasma Cys-Gly levels in a group of patients affected by retinal vein occlusion (RVO), an important cause of visual loss in the elderly. The low levels of Cys-Gly found in the RVO patients suggest that these small thiols may have importance in the disease development. Authors’ addresses: Dr. Angelo Zinellu, Dr. Ciriaco Carru, Department Biomedical Sciences, Chair of Clinical Biochemistry, University of Sassari, Viale San Pietro 43/B, 07100 Sassari, Italy     

A highly selective phosphorescent chemodosimeter for cysteine and homocysteine based on platinum(II) complexes

A platinum(II) complex Pt(phen)CCC₆H₄CHO has been demonstrated as a highly selective phosphorescent chemodosimeter for cysteine and homocysteine. Upon addition of homocysteine or cysteine to its acetonitrile-water solution, an evident luminescent color variation from green to orange was observed. This red-shift of phosphorescence emission could be attributed to formation of a thiazinane through a selective reaction of aldehyde group of Pt(phen)CCC₆H₄CHO with cysteine/homocysteine, which was confirmed by ¹H NMR investigation and density functional theory (DFT) calculations. The further selectivity investigation indicates that it displays high selectivity for homocysteine and cysteine, and therefore can be utilized as cysteine/homocysteine phosphorescent chemodosimeter.     

A novel biosensor based on l-homocysteine desulfhydrase enzyme immobilized in eggshell membrane

A novel biosensor for homocysteine determination has been developed. The biosensor was fabricated with l-homocysteine desulfhydrase immobilized on the ammonium selective electrode by means of eggshell membrane. The measurement principle is based on determination of ammonia due to the enzymatic reaction in the medium by ammonium selective electrode. The effects of enzyme loading, glutaraldehyde concentration, pH, buffer concentration, temperature, dithiotreitol (DTT) concentration and ionic strength adjustment buffer (ISA) on the biosensor response were investigated in detail. The linear detection range and limit of detection (LOD) for homocysteine were found to be 0.15–1.8 mM and 55 μM, respectively. Finally, the homocysteine biosensor has been applied to plasma samples for determination of total homocysteine contents.     

Inhibition of Na(+),K(+)-ATPase activity in hippocampus of rats subjected to acute administration of homocysteine is prevented by vitamins E and C treatment

In the present study we evaluated the effect of acute homocysteine (Hcy) administration on Na⁺,K⁺-ATPase activity, as well as on some parameters of oxidative stress such as total radical-trapping antioxidant potential (TRAP) and on activities of antioxidant enzymes catalase (CAT), superoxide dismutase and glutathione peroxidase in rat hippocampus. Results showed that Hcy significantly decreased TRAP, Na⁺,K⁺-ATPase and CAT activities, without affecting the activities of superoxide dismutase and glutathione peroxidase. We also verified the effect of chronic pretreatment with vitamins E and C on the reduction of TRAP, Na⁺,K⁺-ATPase and CAT activities caused by Hcy. Vitamins E and C per se did not alter these parameters, but prevented the reduction of TRAP, Na⁺,K⁺-ATPase and CAT activities caused by Hcy. Our results indicate that oxidative stress is probably involved in the pathogenesis of homocystinuria and that reduction of Na⁺,K⁺-ATPase activity may be related to the neuronal dysfunction found in homocystinuric patients.     

Anti-angiogenic effects of homocysteine on cultured endothelial cells

High levels of homocysteine induce a sustained injury on arterial endothelial cells which accelerates the development of thrombosis and atherosclerosis. Some of the described effects of homocysteine on endothelial cells are features shared with an anti-angiogenic response. Therefore, we studied the effects of homocysteine on key steps of angiogenesis using bovine aorta endothelial cells as a model. Homocysteine decreased proliferation and induced differentiation. Furthermore, 5 mM homocysteine produced strong inhibitions of matrix metalloproteinase-2 and urokinase, two proteolytic activities that play a key role in extracellular matrix re-modeling, and decreased migration and invasion, other two key steps of angiogenesis. This study demonstrates that homocysteine can inhibit several steps of the angiogenic process.     

Fenofibrate induces a selective increase of protein-bound homocysteine in rodents: a PPARalpha-mediated effect

Elevated levels of plasma homocysteine (Hcy) are associated with increased risk of cardiovascular disease though it is uncertain whether increases in Hcy represent a cause or a consequence of the disease process. Plasma Hcy exists in reduced, free oxidized, and protein-bound forms, that together comprise total Hcy (tHcy). Free reduced Hcy is thought to be the atherogenic, though minor, sub-fraction of tHcy. Recent reports have indicated that fenofibrate and other fibrates are capable of moderately increasing plasma tHcy. As many of the effects of fibrates are known to be mediated by the nuclear receptor PPARalpha, we determined the effect of fenofibrate on tHcy in PPARalpha-deficient mice. We further examined the effect of fenofibrate and fenofibrate plus folate supplementation on total as well as protein-bound Hcy in rats. Fenofibrate significantly increased serum tHcy in wild-type mice but not in PPARalpha deficient mice. In rats, fenofibrate increased serum tHcy by 69%, while the co-administration of folate with fenofibrate increased tHcy by only 7%. In spite of the above increase in tHcy in rats, only the protein-bound fraction of Hcy was increased. In a further study, fenofibrate also induced a significant increase in tHcy, while in spite of this, ex vivo peroxidation of VLDL+LDL was beneficially lowered and the lag time prolonged. In summary, fenofibrate increases serum tHcy in rodents in a PPARalpha-dependent manner. The increase in rats is solely due to protein-bound Hcy as atherogenic, reduced Hcy was unchanged. While awaiting corroboration in human, our results suggest that the extent and mechanism of the increase in total Hcy in patients treated with fenofibrate should not a priori be associated with relevant risk.     

Quantitative methylation-sensitive arbitrarily primed PCR method to determine differential genomic DNA methylation in Down Syndrome

Relative levels of DNA hypermethylation were quantified in DS individuals using a new method based on a combination of methylation-sensitive arbitrarily primed polymerase chain reaction (MS-AP-PCR) and quantification of DNA fragments with the Agilent 2100 bioanalyzer. Four of the DS individuals had low plasma total homocysteine (tHcy) level (4.3 +/- 0.3 micromol/l) and 4 other had high-tHcy level (14.1 +/- 0.9 micromol/l). Eight healthy control individuals were matched to the DS cases for age, sex, and tHcy levels. We have identified and quantified six hypermethylated fragments. Their sizes ranged from 230-bp to 700-bp. In cases and controls, low-tHcy did not affect methylation level of identified fragments, mean methylation values were 68.0 +/- 39.7% and 52.1 +/- 40.3%, respectively. DNA methylation in DS individuals did not change significantly (59.7+/-34.5%) in response to high-tHcy level in contrast to controls (23.4 +/- 17.7%, P = 0.02). Further, the quantitative MS-AP-PCR using this microfludic system is a useful method for determining differential genomic DNA methylation.     

Redox regulation and reaction mechanism of human cystathionine-beta-synthase: a PLP-dependent hemesensor protein

Cystathionine beta-synthase in mammals lies at a pivotal crossroad in methionine metabolism directing flux toward cysteine synthesis and catabolism. The enzyme exhibits a modular organization and complex regulation. It catalyzes the beta-replacement of the hydroxyl group of serine with the thiolate of homocysteine and is unique in being the only known pyridoxal phosphate-dependent enzyme that also contains heme b as a cofactor. The heme functions as a sensor and modulates enzyme activity in response to redox change and to CO binding. Mutations in this enzyme are the single most common cause of hereditary hyperhomocysteinemia. Elucidation of the crystal structure of a truncated and highly active form of the human enzyme containing the heme- and pyridoxal phosphate binding domains has afforded a structural perspective on mechanistic and mutation analysis studies. The C-terminal regulatory domain containing two CBS motifs exerts intrasteric regulation and binds the allosteric activator, S-adenosylmethionine. Studies with mammalian cells in culture as well as with animal models have unraveled multiple layers of regulation of cystathionine beta-synthase in response to redox perturbations and reveal the important role of this enzyme in glutathione-dependent redox homestasis. This review discusses the recent advances in our understanding of the structure, mechanism, and regulation of cystathionine beta-synthase from the perspective of its physiological function, focusing on the clinically relevant human enzyme.     

Homocysteine in the context of cobalamin metabolism and deficiency states

It is becoming increasingly clear that serum vitamin B12 (cobalamin) concentration is a dubious indicator of functional B12 status and, in contrast to long-standing convention, correlates poorly with haematological indices. This, in turn, has led to poorly defined reference intervals for serum B12. Patients presenting with neurological disturbance due to B12 deficiency are at risk of not being diagnosed if total reliance is placed on serum B12 levels and haematological parameters. Plasma homocysteine remethylation is uniquely placed at the metabolic end-point of B12 metabolism such that plasma total homocysteine is proving to be a sensitive marker of functional B12 status. Studies also show that plasma homocysteine correlates better with holotranscobalamin than serum B12. It is suggested that clinicians should cease to be guided by surrogate haematological markers when more specific tests of B12 deficiency, such as holotranscobalamin and total homocysteine, exist. These tests demand greater prevalence in routine diagnostic use.