Homocysteine (Hcy)-thiolactone is toxic, induces epileptic seizures in rodents, and has been implicated in Alzheimer’s disease. Paraoxonase 1 (Pon1), a component of high-density lipoprotein, hydrolyzes Hcy-thiolactone in vitro. Whether this reflects a physiological function and whether Pon1 can protect against Hcy-thiolactone toxicity was unknown. Here we show that Hcy-thiolactone was elevated in brains of Pon1−/− mice (1.5-fold, p = 0.047) and that Pon1−/− mice excrete more Hcy-thiolactone than wild type animals (2.4-fold, p = 0.047). The frequency of seizures induced by intraperitoneal injections of L-Hcy-thiolactone was significantly higher in Pon1−/− mice compared with wild type animals (52.8% versus 29.5%, p = 0.042); the latency of seizures was lower in Pon1−/− mice than in wild type animals (31.8 min versus 41.2 min, p = 0.019). Using the Pon1 null mice, we provide the first direct evidence that a specific Hcy metabolite, Hcy-thiolactone, rather than Hcy itself is neurotoxic in vivo. Our findings indicate that Pon1 protects mice against Hcy-thiolactone neurotoxicity by hydrolyzing it in the brain, and suggest a mechanism by which Pon1 can protect against neurodegeneration associated with hyperhomocysteinemia and Alzheimer’s disease.

Genetic or nutritional disorders in homocysteine (Hcy) metabolism elevate Hcy-thiolactone and cause heart and brain diseases. Hcy-thiolactone has been implicated in these diseases because it has the ability to modify protein lysine residues and generate toxic N-Hcy-proteins with auto-immunogenic, pro-thrombotic, and amyloidogenic properties. Bleomycin hydrolase (Blmh) has the ability to hydrolyze L-Hcy-thiolactone (but not D-Hcy-thiolactone) to Hcy in vitro, but whether this reflects a physiological function has been unknown. Here, we show that Blmh (-/-) mice excreted in urine 1.8-fold more Hcy-thiolactone than wild-type Blmh (+/+) animals (P = 0.02). Hcy-thiolactone was elevated 2.3-fold in brains (P = 0.004) and 2.0-fold in kidneys (P = 0.047) of Blmh (-/-) mice relative to Blmh (+/+) animals. Plasma N-Hcy-protein was elevated in Blmh (-/-) mice fed a normal (2.3-fold, P < 0.001) or hyperhomocysteinemic diet (1.5-fold, P < 0.001), compared with Blmh (+/+) animals. More intraperitoneally injected L-Hcy-thiolactone was recovered in plasma in Blmh (-/-) mice than in wild-type Blmh (+/+) animals (83.1 vs. 39.3 μM, P < 0.0001). In Blmh (+/+) mice injected intraperitoneally with D-Hcy-thiolactone, D,L-Hcy-thiolactone, or L-Hcy-thiolactone, 88, 47, or 6.3%, respectively, of the injected dose was recovered in plasma. The incidence of seizures induced by L-Hcy-thiolactone injections (3,700 nmol/g body weight) was higher in Blmh (-/-) than in Blmh (+/+) mice (93.8 vs. 29.5%, P < 0.001). Using the Blmh null mice, we provide the first direct evidence that a specific Hcy metabolite, Hcy-thiolactone, rather than Hcy itself, is neurotoxic in vivo. Taken together, our findings indicate that Blmh protects mice against L-Hcy-thiolactone toxicity by metabolizing it to Hcy and suggest a mechanism by which Blmh might protect against neurodegeneration associated with hyperhomocysteinemia and Alzheimer's disease.

Hcy-thiolactone/creatinine ratio is a novel AMI risk predictor in patients with suspected CAD, independent of traditional risk factors and tHcy, but modified by vitamin B catabolism. These findings lend a support to the hypothesis that Hcy-thiolactone is mechanistically involved in cardiovascular disease.

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A simple and rapid assay using pyridoxal 5'-phosphate (PLP) as a derivatizing reagent was developed for the simultaneous determination of homocysteine (Hcy) and cysteine (Cys) in human plasma. Derivatization with PLP affords UV-absorbing tetrahydrothiazine and thiazolidine derivatives of Hcy and Cys, respectively. Separation of these derivatives was achieved in 5 min using a hydrophilic interaction liquid chromatography, followed by UV detection at 330 nm. Linearity in detector response was observed over the range of 0.25-20 μM for Hcy and 10-300 μM for Cys. The limit of quantification (LOQ) values for Hcy and Cys were 0.25 and 2.5 μM, respectively. The method was successfully applied to plasma samples donated by apparently healthy volunteers.

Genetic or nutritional deficiencies in 1 carbon and homocysteine (Hcy) metabolism elevate Hcy-thiolactone levels and are associated with cardiovascular and neurologic diseases. Hcy-thiolactone causes protein damage, cellular toxicity, and proatherogenic changes in gene expression in human cells and tissues. A polymorphic cardio-protective enzyme, paraoxonase 1 (PON1), hydrolyzes Hcy-thiolactone in vitro. However, whether Hcy-thiolactone hydrolysis is a physiologic function of the PON1 protein and whether polymorphisms in the PON1 gene affect Hcy-thiolactone levels in humans was unknown. Here we show that the PON1-192 genotype, which affects the enzymatic activity of the PON1 protein, also affected urinary Hcy-thiolactone levels, normalized to creatinine. Carriers of the PON1-192R allele had significantly lower Hcy-thiolactone/creatinine levels than individuals carrying the PON1-192Q allele. Individuals with low serum PON1 paraoxonase activity had significantly higher Hcy-thiolactone/creatinine levels compared with individuals with high paraoxonase activity. In contrast, Hcy-thiolactone/creatinine levels were unaffected by serum PON1 arylesterase activity or by PON1 protein levels. Taken together, these findings suggest that PON1 hydrolyzes Hcy-thiolactone in humans and that the interindividual variations in PON1 genotype/activity can modulate the pathology of hyperhomocysteinemia.-Perła-Kaján, J., Borowczyk, K., Głowacki, R., Nygård, O., Jakubowski, H. Paraoxonase 1 Q192r genotype and activity affect homocysteine thiolactone levels in humans.

Growing human head hair contains a history of keratin and provides a unique model for studies of protein damage. Here, we examined mechanism of homocysteine (Hcy) accumulation and keratin damage in human hair. We found that the content of Hcy-keratin increased along the hair fiber, with levels 5–10-fold higher levels in older sections at the hair’s tip than in younger sections at hair’s base. The accumulation of Hcy led to a complete loss of keratin solubility in sodium dodecyl sulfate. The increase in Hcy-keratin was accompanied by a decrease in methionine-keratin. Levels of Hcy-keratin were correlated with hair copper and iron in older hair. These relationships were recapitulated in model experiments in vitro, in which Hcy generation from Met exhibited a similar dependence on copper or iron. Taken together, these findings suggest that Hcy-keratin accumulation is due to copper/iron-catalyzed demethylation of methionine residues and contributes to keratin damage in human hair.