Thiolactonase Activity Research Articles

A fluorometric method for measuring homocysteine thiolactonase activity

The enzyme homocysteine thiolactonase (HTase) is involved in the metabolism of homocysteine, an amino acid. As a byproduct of the metabolism of methionine, a secondary amino acid, homocysteine (Hcy), a sulfur-containing amino acid, is produced. Hcy levels in the blood are linked to higher risks of heart disease, stroke, and other disorders. To create Hcy, HTase catalyzes the hydrolysis of homocysteine thiolactone (HTL). This process helps control the body's homocysteine levels and prevents the accumulation of homocysteine in the blood.This essay describes a precise, easy-to-follow procedure for measuring homocysteine thiolactonase (HTase) activity. This protocol (NAM-HTase) determines HTase activity via incubation with γ-thiobutyrolactone, an enzyme substrate, in a pH-appropriate solution at 37 °C for five minutes. It then uses thiol fluorometry to monitor the produced thiol groups. The newly formed thiol groups are reacted with the fluorescent probe N-(9-acridinyl)maleimide (NAM) and quantified using fluorometric intensity at an Ex/Em of 360/432 nm to evaluate HTase activity. The method is linear for 4-mercaptobutyric acid and homocysteine, from 0.005 ± 0.001 to 6 ± 0.04 µM. By comparing the HTase activity in matched samples to that of a standard method, this new protocol was found suitable for clinical applications. Its accuracy compared to other methodologies was shown by a correlation of 0.9995 between its values and those of a standard method. Using the H+ ion releasing method in matched samples, analysis of variance was used to verify the novel method against HTase activity. In conclusion, the proposed NAM-HTase method successfully measured HTase activity with high reliability.

A quantitative method for the assessment of homocysteine thiolactonase enzyme activity

This assay elucidates an accurate, simple, and precise protocol to quantify the activity of homocysteine thiolactonase (HTase). To establish HTase activity, the enzyme samples were incubated with a 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) buffer, which contained suitable concentrations of the homocysteine thiolactone as a substrate. To stop the enzyme's reaction, the CUPRAC reagent (Cu(Nc)22+) was added after a suitable incubation time. The reduction of Cu(II)-neocuproine complex (Cu(Nc)22+) to highly coloured Cu(I)-neocuproine complex (Cu(Nc)2+) by the produced homocysteine was quantified spectrophotometrically at 450 nm (CUPRAC method). The increase in the absorbance of the coloured Cu(I)-neocuproine complex (Cu(Nc)2+) was correlated directly to the activity of HTase. ANOVA analysis was utilised to validate the new method against homocysteine thiolactonase activity using the H+ ions liberating method in matched samples. In conclusion, according to the obtained correlation coefficient (0.9995) from the comparison of the current method with the reference method, the current method is effective in assay HTase activity with high reliability.

Homocysteine thiolactonase activity in coronary atherosclerosis

Homocysteine thiolactonase activity in coronary atherosclerosis

Evaluation of Paraoxonase, Arylesterase, and Homocysteine Thiolactonase Activities in Patients with Diabetes and Incipient Diabetes Nephropathy.

SummaryBackgroundThe aim of this study is to examine the relationship among the changes in activities of paraoxonase (PON), arylesterase (ARE) and homocysteine thiolactonase (HTLase) enzyme having antioxidant properties and the development of diabetic nephropathy (DN), one of the most common complications of diabetes.MethodsNormoalbuminuric type-2 diabetic patients (Group II, n=100), microalbuminuric type 2 diabetic patients (Group III, n=100) and the control group (Group I, n=100) were included in the study. The age and gender of the patient groups matched with the age and gender of the control group. HTLase, PON and ARE enzyme activities were measured by the spectrophotometric method using a g-thiobutyrinolactone, paraoxon, and phenylacetate substrates respectively. In this study, an autoanalyzer application was developed in order to measure HTLase enzyme activity for the first time.ResultsSerum HTLase, ARE and PON activities of Group III and Group II were significantly low compared to HTLase, ARE and PON results of Group I (p<0.05).ConclusionsBased on our results, PON, ARE and HTLase enzyme activities were found to be decreased due to the increase in the degree of DN.

Interaction between MTHFR 677C>T, PON1 192Q>R and PON1 55L>M polymorphisms and its effect on non-recurrent spontaneous abortion in Mexican women

MTHFR is a key enzyme in folate metabolism. Some genetic polymorphisms code for a less efficient enzyme, increasing serum concentrations of homocysteine. This has been associated with inadequate feto-maternal circulation and increased risk of spontaneous abortion. Paroxonase 1 (PON1) is a multifunctional enzyme that can detoxify homocysteine through its homocysteine thiolactonase activity. We evaluate the association between MTHFR 677 C>T polymorphisms and non-recurrent spontaneous abortion and its interaction with PON1 polymorphisms involved in homocysteine metabolism in women living in floricultural areas in Mexico. Sociodemographic, reproductive history, folic acid consumption during pregnancy and environmental exposure data of 264 women who had been pregnant sometime during the 10 years prior to study enrolment were collected. MTHFR 677 C>T, PON1 192Q>R and PON1 55L>M genotypes were determined by PCR amplification. Information on pregnancy outcome and maternal genotypes was obtained for 484 pregnancies: 34 non-recurrent spontaneous abortions (gestational age < 20 weeks) and 450 controls. GEE models were used to evaluate the association between MTHFR polymorphism and non-recurrent spontaneous abortion, and its interaction with PON1 polymorphisms.After adjusting for potential confounders, no significant association was found between the MTHFR 677 C>T maternal polymorphism and non-recurrent spontaneous abortion (OR CT vs CC= 0.39, 95% CI: 0.14-1.05; OR TT vs CC = 0.63, 95% CI: 1.22–1.80). No interactions with PON1 192Q>R or PON1 55L>M polymorphisms were identified (p for interaction = 0.88 and 0.41, respectively). PON1 55L>M maternal polymorphism was associated with higher risk of spontaneous abortion (OR LM/MM vs LL = 4.14, 95% CI: 1.49–11.54).Our results do not demonstrate an interaction between the MTHFR 677 C>T and PON1 192Q>R or PON1 55L>M maternal polymorphisms neither an independent association of MTHFR 677 C>T polymorphism with non-recurrent spontaneous abortion, whereas PON1 55LM/MM maternal genotype increase the odds of this event.

Ocular distribution of antioxidant enzyme paraoxonase & its alteration in cataractous lens & diabetic retina.

Background & objectives:The enzyme paraoxonase (PON), an antioxidant enzyme that has both arylesterase and thiolactonase activity, is well studied in cardiovascular diseases. Although a few studies have shown altered PON activity in ocular diseases such as age-related macular degeneration and diabetic retinopathy, but the tissue-wise expression of PON in its three gene forms has not been studied. This study was conducted to see the ocular distribution of PON for any altered expression in ocular pathologies such as in cataract and diabetes mellitus.Methods:Immunohistochemistry (IHC) of the ocular tissues was done for localizing all three forms of the PON in the human donor eyeballs. The PON arylesterase (PON-AREase) and thiolactonase (PON-HCTLase) activities were determined by spectrophotometry in kinetic mode, and the mRNA expression of the PON genes (PON1-3) was determined by reverse transcription-polymerase chain reaction.Results:IHC showed the presence of both PON1 and 2 in all the ocular tissues and PON3 was seen only in retina. The mRNA expression analysis showed that PON2 and PON3 were present in all the tissues, whereas PON1 was seen only in ciliary and retina. Both the PON-AREase and PON-HCTLase activities were detected in all ocular tissues and was in the order of lens>retina>choroid>ciliary body>iris. The expression and activity were studied in cataractous lens and in diabetic retina of the donor eyes. A significant decrease in PON-AREase activity was seen in cataractous lens (P<0.05) but not in diabetic retina, and there was an increase in PON- HCTLase activity (P<0.05) only in diabetic retina. Bioinformatic studies and in vitro experiments indicated that advanced glycation end products (AGE) such as carboxymethyl -lysine might decrease the PON- AREase activity of the PON.Interpretation & conclusions:Distribution of PON enzyme and its activity in ocular tissues is reported here. The study revealed maximal PON activity in lens and retina, which are prone to higher oxidative stress. Differential activities of PON were observed in the lens and retinal tissues from cataractous and diabetic patients, respectively.

Prognostic impact of homocysteine levels and homocysteine thiolactonase activity on long-term clinical outcomes in patients undergoing percutaneous coronary intervention

BackgroundNumerous studies have reported the relationship between elevated homocysteine (Hcy) levels and the risk of coronary artery disease. However, there is insufficient information about the effects of Hcy levels on long-term clinical outcomes in patients with coronary artery disease undergoing percutaneous coronary intervention (PCI). MethodsIn the Juntendo-registry cohort from 2003 to 2004, pre-procedural Hcy levels and Hcy thiolactonase activity (HTlase) were measured in 315 consecutive all-comer patients who underwent PCI for stable coronary artery disease or acute coronary syndrome (ACS). Receiver-operating characteristic (ROC) curves were created to assess the optimal cut-off values of Hcy and HTlase. Multivariable Cox proportional hazard regression analysis was used to identify the predictors of clinical outcome. The primary endpoint was all-cause mortality. ResultsThe patients’ mean age was 66±9 years, and 82.5% were males. The median follow-up period was 10.5 years, and overall mortality was 24.5% (73 deaths). On ROC analysis, the optimal cut-off values of Hcy and HTlase were 13.5μmol/L and 230IU/L, respectively. Kaplan–Meier survival analysis showed associations of both higher Hcy levels and lower HTlase activity with worse prognosis (both log-rank p<0.001). On multivariate Cox proportional hazard regression analysis, higher Hcy was strongly associated with the primary outcome, and the adjusted hazard ratio was 3.3 (95% confidence interval, 1.8–5.6; p<0.001). ConclusionsPre-procedural high Hcy levels and low HTlase activity were associated with worse long-term mortality in Japanese patients undergoing PCI. Moreover, Hcy levels are strongly predictive for mortality, independent of traditional risk factors. This may have implications for risk stratification and the therapeutic approach in this PCI era.

Serum Paraoxonase activity in relation to lipid profile in Age-related Macular Degeneration patients

Age-related Macular Degeneration (AMD) is a multifactorial disease causing visual impairment in old age. Oxidative stress is one of the main contributors for the disease progression. Paraoxonase (PON), a HDL-resident antioxidant enzyme which removes oxidized low density lipoprotein (oxLDL), which is not studied much in AMD. This study assesses the PON activities in relation to the lipid status and genetic variants in AMD patients. In this prospective case-control study, a total of 48 AMD patients and 30 unrelated healthy controls were recruited. The serum oxLDL and Plasma Homocysteine (Hcy) levels were estimated by ELISA. Plasma Homocysteine thiolactone (HCTL) was estimated by HPLC. Serum PON activities were estimated by spectrophotometry. PON gene expression was assessed by qPCR and protein expression by western blot, immunofluorescence and FACS analysis. Two known single nucleotide polymorphisms (SNPs) in the coding region of PON1, Q192R and L55M variants were checked in the AMD patients and controls and their association with PON activity and lipid levels were determined. Serum paraoxonase (PONase) and thiolactonase (PON-HCTLase) activities were significantly elevated in AMD patients than in controls apart from elevated serum levels of total cholesterol (TC), triglycerides (TG), oxLDL. While serum LDL levels in AMD patients correlate positively with PON HCTLase activity, the serum high density lipoprotein (HDL) correlates with both PONase and PON-HCTLase activities. However, multiple regression analysis showed that, amongst the parameters, only serum TG was a significant risk factor for AMD, after adjusting for demographic parameters as well as cataract. PON2 was significantly increased at the level of gene expression (p = 0.03) as seen in circulating peripheral blood mononuclear cells (PBMC) of AMD patients possibly mediated by the transcription factor SP1, that showed 2-fold increase. PON1 and 2 protein expressions also showed significant increase in the PBMC of AMD patients. At serum level, PON1 protein was significantly increased in AMD patients. Cholesterol transporters such as CD36, SR-B1 and ABCA1 gene expressions were also found to be higher (1.5, 1.9 and 2.4-fold respectively) in AMD, though not statistically significant. While the wet AMD (CNV) was found to be associated with increase in oxLDL and serum PONase activity, the dry AMD was associated with increased HDL and serum PON-HCTLase activity. The genotype and allele frequencies of Q192R & L55M were not significantly different between AMD patients and controls. However, altered lipid status and PON activities were associated with the genotype in AMD patients. A higher enzyme activity was observed for the RR genotype of Q192R in the cohort, irrespective of case and control. Thus the PON genotype and phenotype seem to play a role in the pathogenesis of AMD.

The effect of HDL-bound and free PON1 on copper-induced LDL oxidation

Oxidative modification of LDL plays an important role in the development of atherosclerosis. High-density lipoprotein (HDL) confers protection against atherosclerosis and the antioxidative properties of paraoxonase 1 (PON1) has been suggested to contribute to this effect of HDL. The PON1 exist in two major polymorphic forms (Q and R), which regulate the concentration and activity of the enzyme and alter its ability to prevent lipid oxidation. However, the association of Q192R polymorphism with PON1's capacity to protect against LDL lipoperoxidation is controversial. The aim of this study was to evaluate the effects of the purified PON1 Q192R and the partially purified HDL-bound PON1 Q192R isoenzymes (HDL-PON1 Q192R) on LDL oxidation, with respect to their arylesterase/homocysteine thiolactonase (HTLase) activities. Cupric ion-induced LDL oxidation was reduced up to 48% by purified PON1 Q192, but only 33% by an equivalent activity of PON1 R192. HDL-PON1 Q192 isoenzyme caused a 65% reduction, whereas HDL-PON1 R192 isoenzyme caused only 46% reduction in copper ion-induced LDL oxidation. These findings reflect the fact that PON1 Q and PON1 R allozymes may have different protective characteristics against LDL oxidation.The protection against LDL oxidation provided by HDL-PON1 Q192R isoenzymes is more prominent than the purified soluble enzymes. Inhibition of the Ca+2-dependent PON1 Q192R arylesterase/HTLase by the metal chelator EDTA, did not alter PON1's ability to inhibit LDL oxidation. These studies indicate that the active site involvement of the purified enzyme is not similar to the HDL-bound one, in terms of both PON1 arylesterase/HTLase activity and the protection of LDL from copper ion-induced oxidation. Moreover, PON1's ability to protect LDL from oxidation does not seem to require calcium.

Protective Role of Dietary Curcumin in the Prevention of the Oxidative Stress Induced by Chronic Alcohol with respect to Hepatic Injury and Antiatherogenic Markers.

Curcumin, an antioxidant compound found in Asian spices, was evaluated for its protective effects against ethanol-induced hepatosteatosis, liver injury, antiatherogenic markers, and antioxidant status in rats fed with Lieber-deCarli low menhaden (2.7% of total calories from ω-3 polyunsaturated fatty acids (PUFA)) and Lieber-deCarli high menhaden (13.8% of total calories from ω-3 PUFA) alcohol-liquid (5%) diets supplemented with or without curcumin (150 mg/kg/day) for 8 weeks. Treatment with curcumin protected against high ω-3 PUFA and ethanol-induced hepatosteatosis and increase in liver injury markers, alanine aminotransferase, and aspartate aminotransferase. Curcumin upregulated paraoxonase 1 (PON1) mRNA and caused significant increase in serum PON1 and homocysteine thiolactonase activities as compared to high ω-3 PUFA and ethanol group. Moreover, treatment with curcumin protected against ethanol-induced oxidative stress by increasing the antioxidant glutathione and decreasing the lipid peroxidation adduct 4-hydroxynonenal. These results strongly suggest that chronic ethanol in combination with high ω-3 PUFA exacerbated hepatosteatosis and liver injury and adversely decreases antiatherogenic markers due to increased oxidative stress and depletion of glutathione. Curcumin supplementation significantly prevented these deleterious actions of chronic ethanol and high ω-3 PUFA. Therefore, we conclude that curcumin may have therapeutic potential to protect against chronic alcohol-induced liver injury and atherosclerosis.

0143: Serum thiolactonase activity and paraoxonase gene polymorphism in coronary artery Tunisian patients with or without diabetes

Serum homocysteine thiolactonase activity is closely related to homocysteine and ox-LDL levels in coronary artery disease patients. Homocysteine thiolactonase activities are the result of environmental factors and genetic polymorphisms of paroxosonase. The relevance of gene polymorphism in coronary patients with or without diabetes was investigated in a case-control study. The population included 140 control subjects, 47 coronary artery disease patient without diabetes and 44 patients with type 2 diabetes. Serum thiolactonase activity decreased significantly in coronary artery disease patients: 318.9±156 U/l those without diabetes and 388.8±180 in the diabetic ones compared with the controls: 568.8±250U/l (p<0.001). These activities were associated with increased plasma homocysteine and ox-LDL levels (p<0.001). The paroxonase allele frequency is significantly higher in controls than coronary artery patients (p<0.001). The frequency of R high activity allele was also higher in diabetic coronary patients (11.3%), than in those without diabetes (4.2%). No significant association was found between paroxonase LN gene polymorphisms and diabetes in coronary patients. The difference in allele frequency for the paroxonase R gene polymorphism may be the cause of the high thiolactonase activity observed in coronary patients with type 2 diabetes mellitus. Further studies are needed to be conducted to elucidate the role of the enzyme in the development of vascular complications in these patients.

Relationship Between Serum Paraoxonase and Homocysteine Thiolactonase Activity, Adipokines, and Asymmetric Dimethyl Arginine Concentrations in Renal Transplant Patients

Paraoxonase lactonase activity protects against homocysteinylation; therefore, it can be a potential contributing factor to prevent atherosclerosis. We aimed to determine paraoxonase and HTLase activities and to clarify the relationship between HTLase activity and some cardiovascular risk factors, such as homocysteine, cystatin C asymmetric dimethylarginine (ADMA), and adipokines both in hemo dialyzed and transplanted patients. Among 114 hemodialyzed, 80 transplanted and 64 healthy control subjects, we investigated body mass index (BMI) as well as fasting serum contents of urea, uric acid, creatinine, cystatin C, homocysteine, glucose, lipids, total protein and albumin. Serum paraoxonase (PON 1) and HTLase activities were measured spectrophotometrically. ADMA, ADPN adiponectin, leptin (LEP) levels was determined with a sandwich enzyme-linked immunosorbent assay method. Dyslipidemic patients showed hypercholesterolemia, and high low-density lipoprotein (LDL); parallel with improved renal function, they displayed decreased cystatin C and homocysteine levels (P < .001). There was a significant negative correlation between PON 1 activity and cystatin C and homocysteine concentrations (P < .05). Obese patients revealed significantly higher LDL (P < .05) and leptin concentrations (P < .01). There was a significant positive correlation between PON 1 activity and adiponectin levels (P = .0276). Both dialyzed and transplanted patients displayed significantly lower HTLase activities compared to the control group (P < .001), particularly lower HTLase and PON 1 activities in dialyzed subjects compared with the transplanted group (P < .05). HTLase activity showed significant negative correlations with ADMA levels among the whole study population (P < .001), whereas positive associations were noted between PON 1 and HTLase activities (P < .001). HTLase activity may be a new predictor of cardiovascular risk in renal failure although it is modulated by other risk factors.

Connection Between Serum Homocysteine Thiolactonase and Paraoxonase Activity in Renal Transplanted Patients

Connection Between Serum Homocysteine Thiolactonase and Paraoxonase Activity in Renal Transplanted Patients

Paraoxonase lactonase activity (PON-HTLase), asymmetric dimethylarginine (ADMA) and platelet activating factor-acetylhydrolase (PAF-AH) activity in non-obese women with PCOS

Objective: Paraoxonase1 (PON1), exhibits both esterase activity (PON1-AREase) and homocysteine thiolactonase activity (PON1-HTLase) which respectively prevent LDL oxidation and detoxify homocysteine thiolactone (HTL). Platelet-activating factor-acetylhydrolase (PAF-AH) is an antioxidant enzyme preventing LDL oxidation by hydrolysis of oxidized phospholipids. Both of these enzymes exhibit a proatherogenic role. ADMA is an endogenous inhibitor of nitric oxide (NO) synthesis causing endothelial dysfunction. The aim was to compare non-obese PCOS patients with a BMI matched control group using the following characteristics: serum PON1-HTLase, ADMA, PAF-AH, and lipid and hormonal parameters. Results: 77 women with PCOS and 25 healthy subject were recruited for this study, The controls were non-obese BMI and age matched with the patients. There were no significant differences with respect to age, BMI, FSH, free testosterone, DHEA, androstenadion, total cholesterol, triglycerides, HDL, LDL, VLDL, fasting glucose/insulin ratio and HOMA-IR among the groups (p > 0.05). However, total testosterone and fasting glucose levels were significantly higher in the PCOS group (p < 0.05). On the other hand, PON1-HTLase levels (39.6 ± 5.77 vs. 33.8 ± 8.2, p = 0.02) were significantly lower in the PCOS group while ADMA levels (1.14 ± 0.6 vs. 3.37 ± 6.4, p = 0.004) were significantly higher in the PCOS group. However, there was no significant difference in PAF-AH activity among the groups Conclusions: Decreased PON1-HTLase and increased ADMA levels might be a relevant marker for the development of future atherosclerotic heart disease (AHD) in non-obese PCOS patients. Further studies are needed to confirm our results.

Decreased paraoxonase 1 (PON1) lactonase activity in hemodialyzed and renal transplanted patients. A novel cardiovascular biomarker in end-stage renal disease

Human paraoxonase-1 (PON1) has also been described as a lactonase. Decreased PON1 lactonase activity was found to be a predictor of cardiovascular disease. Homocysteine thiolactonase activity may prevent proteins from homocysteinylation and is thought to be a protective factor against the progression of atherosclerosis. Previous studies have demonstrated decreased PON1 paraoxonase activity in hemodialyzed (HD) and renal transplant (TRX) patients; however, lactonase activity has not been investigated. We aimed to determine the paraoxonase and lactonase activities and to clarify the relationship between lactonase activity and a set of cardiovascular risk factors, such as homocysteine, cystatin C and asymmetric dimethylarginine (ADMA) levels, in HD and TRX patients and in healthy controls. One hundred and eight HD and 78 TRX patients and 63 healthy controls were involved in the study. Paraoxonase and lactonase activities (paraoxon and gamma-thiobutyrolactone as substrates) were measured spectrophotometrically. ADMA level was determined with sandwich enzyme-linked immunosorbent assay. Both HD and TRX patients had significantly lower lactonase activities compared to the control group (P<0.05). Significantly lower paraoxonase activities were found in HD patients compared to the TRX group (P<0.05). Significant negative correlation was found between lactonase activity and ADMA level in the whole study population (P<0.001), while paraoxonase and lactonase activities showed significant positive correlation (P<0.001). Multiple regression analysis identified paraoxonase activity and homocysteine level as independent predictors of lactonase activity. Lactonase activity is a potential new predictor of cardiovascular risk in renal failure. Measurement of lactonase activity is recommended in future studies on HD and TRX patients.

065 Mediterranean diet decrease homocysteine levels and increase thiolactonase activities in elderly patients at high risk of cardiovascular disease

065 Mediterranean diet decrease homocysteine levels and increase thiolactonase activities in elderly patients at high risk of cardiovascular disease

Serum PON-1 Activity but not Q192R Polymorphism is Related to the Extent of Atherosclerosis

Paraoxonase-1 (PON1) is an antioxidant enzyme located in high density lipoprotein (HDL). PON1 was defined as a protective factor against atherosclerosis. The aim of this study was to investigate the possible relationship between serum paraoxonase (PONase), homocysteine thiolactonase (HTase) activities and PON1 Q192R polymorphism, and the extent and severity of atherosclerosis. Blood specimens were collected from 142 individuals who had no coronary artery lesions angiographically (control group) and 128 individuals who had angiographically documented coronary artery disease of several degrees (patient group). The extent and severity of arterial lesions were evaluated by the Gensini scoring system. PONase and HTase activities were measured in serum using a spectrophotometric method. PON1 Q192R polymorphism was evaluated using PCR-RFLP after DNA isolation from blood. Serum PONase and HTase activities were significantly lower in the patient group than in healthy controls (135.7±56.0U/mL vs 153.8±62.0U/mL, p< 0.05; 36.0±6.1 U/mL vs 43.0±4.04 U/mL, p< 0.01; respectively). In the patient group, there was a negative correlation between PONase, HTase activities and the Gensini score (r=-0.168, p= 0.039; r=-0.164, p= 0.006, respectively). In both groups, there was no significant difference in the distribution of PON1 Q192R polymorphism. In the patient group, the distribution of Gensini scores according to genotypes was not significant. It has been concluded that serum PONase and HTase activities might be a more relevant marker than PON1 genotype in evaluating the extent and severity of atherosclerosis.

263 Mediterranean diet decrease homocysteine levels and increase thiolactonase activities in elderly patients at high risk of cardiovascular disease

Food items might have a synergistic and antagonistic effect on health. The Mediterranean diet (MD) has long been associated with lower incidence of cardiovascular disease and cancer. Elevated blood homocysteine is a risk factor for cardiovascular disease. A 5-mmol/L increase is associated with an approximately 70% increase in relative risk of cardiovascular disease in adults. For patients with established risk factors, this risk is likely even greater. Thiolactonase; the antioxidant enzymatic component of HDL plays a crucial role in metabolizing homocysteine thiolactone and reducing homocysteine endothelial damages. A total of 53 elderly coronary artery disease patient swere recruited and divided into diabetic patient (n = 27; age = 68.3 years) and non diabetics (n = 26; age = 68.7 years). We evaluated plasma homocysteine levels (tHcy); thiolactonase activity (HTase) and studied the effects of adherence to a Mediterranean diet on them. Plasmatic tHcy was determined by capillaryGC-MS; HTLase activity was estimated by a kit immunoassay. Dietary intakes were evaluated by a validated food frequency questionnaire and transformed into a score traducing adherence to MD. Significantly higher median tHcy levels were found in diabetic patients as compared to the non diabetic ones (18.5 μmol/L vs. 5.8; p = 0.057) associated to lower HTase activities (266.8U/L vs. 327.4; p = 0.058). Moreover, in the diabetic patients tHcy levels were negatively associated with thiolactonase activities (r= −0.637; p 0.00). In this group, MD reduced diastolic blood pressure (r= −0.558; p = 0.014). In the non diabetic group, tHcy levels were negatively correlated with HTase activities (r = 0.759; p = 0.00) and diet score (r = −0.0706; p = 0.013) while HTase activities were positively correlated with diet score (r = 0.0759; p = 0.00). Elevated homocysteine in diabetic patients may partly be explained by the diminished thiolactonase activities and could be considered as an additional risk factor for cardiovascular events. Adherence to MD could be the first efficient step to prevent these complications.

Association of the C677T MTHFR Polymorphism With Homocysteine, Ox-LDL Levels, and Thiolactonase Activities in the Severity of Coronary Syndrome

Coronary artery diseases (CAD) are influenced by multiple genes of modest effect as the methylenetetrahydrofolate reductase (MTHFR) C677T gene polymorphism, related to MTHFR activity and total plasma homocysteine (tHcy) concentration. This study was designed to evaluate tHcy, oxidized low-density lipoprotein (LDL) (ox-LDL), high-sensibility C-reactive protein (Hs CRP) levels, and homocysteine thiolactonase (HTase) activities as new risk factors for CAD and to investigate an association between MTHFR polymorphism tHcy concentrations and coronary syndrome severity. Our results showed significantly higher levels of tHcy and ox-LDL in patients associated with lower HTase activities. These levels increased proportionally to disease severity. Total plasma Hcy levels were negatively correlated to HTase activities in patients where the TT genotype was significantly more frequent. In a multivariate analysis, tHcy level was the only independent factor affecting the coronary syndrome severity. High tHcy levels are associated with coronary syndrome severity and may be explained either by the elevated prevalence of TT genotype or by the diminished HTase activities.

Betaine Protects Chronic Alcohol and ω‐3 PUFA‐Mediated Down‐Regulations of PON1 Gene, Serum PON1 and Homocysteine Thiolactonase Activities With Restoration of Liver GSH

Paraoxonase (PON1) is an antioxidant enzyme that prevents LDL oxidation as well as detoxifies homocysteine thiolactone (HCTL), both of which can cause atherosclerosis. Chronic alcohol (ETOH) and high omega-3 polyunsaturated fatty acids (omega-3 PUFA) consumption may affect PON1 status presumably via reactive oxygen species by depleting liver glutathione (GSH), whereas betaine may counter their effects. Therefore, we investigated the influence of ETOH, omega-3 PUFA, and betaine on liver GSH, PON1 expression, lipid score, as well as serum PON1 and HCTLase activities. Experimental rats belonging to various dietary groups were pair-fed with Lieber-DeCarli low (2.8% the dietary calories as omega3-fatty acids) and high (13.8% the dietary calories as omega3-fatty acids) menhaden fish alcohol-liquid diets with and without betaine (10 g/l diet) for 8 weeks after which liver PON1 mRNA, GSH, lipid score, and serum PON1, HCTLase, and ALT activities were measured. High omega-3 PUFA decreased liver PON1 mRNA expression, serum PON1, and HCTLase activity by 23% (p < 0.01), 20% (p < 0.05), and 28% (p < 0.05), respectively compared to the low omega-3 PUFA group. ETOH decreased PON1 mRNA expression by 25 and 30% (p < 0.01) with concomitant 27% (p < 0.05) and 38% (p < 0.01), decrease in liver GSH levels in low and high omega-3 PUFA groups, respectively. Correspondingly, serum PON1 activity decreased by 23% (p < 0.05) and 58% (p < 0.01) while serum HCTLase activity decreased by 25% (p < 0.05) and 59% (p < 0.01) in the low and high omega-3 PUFA ETOH groups, respectively. Betaine restored liver PON1 mRNA expressions in low and high omega-3 PUFA ETOH groups with parallel restorations of PON1 activity and liver GSH. Concomitantly, betaine reduced hepatosteatosis accompanied by alleviation of liver injury caused by chronic alcohol and high omega-3 PUFA. Based on these results, we conclude that dietary betaine not only atheroprotective by restoring liver GSH that quenches free radicals, but also may alleviate liver injury by reducing hepatosteatosis.