Roberto Accinni

High-performance liquid chromatographic determination of total plasma homocysteine with or without internal standards.

Hyperhomocysteinemia is an independent risk factor for atherosclerosis and vascular occlusive disease. Assessment of total plasma concentration of homocysteine (tHcys) requires accurate and reproducible measurements. The aim of this study was to test a rapid isocratic HPLC method for tHcys analysis with an internal standard (I.S.) of alpha-mercaptopropionylglycine (MPG), 2-mercaptoethylamine (ME), or N-acetylcysteine (NAC) or without I.S., and to verify whether the use of an I.S. improves the precision. The method without I.S. showed an excellent linearity (y = 1.59x - 0.15, r = 1), recovery (100%) and a within-assay relative standard deviation (R.S.D.) of 1.2%. Instead, in our hands, the presence of I.S.s decreased the reproducibility (within-assay R.S.D. ranged from 4.5 to 6.5%) and lengthened the chromatogram by up to four to five times. In conclusion, HPLC measurement of plasma tHcys without I.S. improves accuracy with respect to determination with I.S.; moreover, this approach allows to routinely process larger amounts of plasma samples.

Screening of homocysteine from newborn blood spots by high-performance liquid chromatography with coulometric array detection

Homocystinuria, due to a deficiency of cystationine-beta-synthase, refers to the rare inborn error of the metabolism of homocysteine. The identification and prompt treatment of homocystinuria during the neonatal period can prevent or greatly reduce the severity of the clinical consequences. We report a new method for homocystinuria diagnosis from dried blood spots on newborn screening cards, based on high-performance liquid chromatography with electrochemical coulometric array detection. This method shows an excellent linearity (y=10.36x+0.04; r=0.999), precision (RSDs ranged from 2.7 to 5.8%), recovery (87%) and appears to be a cost-effective approach, being simple, rapid, sensitive and cheap.

Analytical performance and method comparison study of the total homocysteine fluorescence polarization immunoassay (FPIA) on the AxSYM analyzer

Abstract A fluorescence polarization immunoassay (FPIA) has been commercially released for routine large-scale testing of total homocysteine (tHcy) on the AxSYM analyzer. We evaluated the analytical performance of the AxSYM tHcy FPIA and compared it with the well established high-performance liquid chromatography (HPLC) and IMx tHcy FPIA methods. Homocysteine concentrations were measured by AxSYM and IMx tHcy FPIA and by a rapid isocratic HPLC method with fluorescence detection. Coefficient of variation (CV) of total imprecision for AxSYM tHcy was ≤5%, mean dilution recovery 102%, analytical sensitivity 0.70 μmol/l and linearity was good up to 1:8 dilution. Spearman rank correlations, rho, were 0.83 (p<0.0001) for AxSYM vs. HPLC, 0.97 (p<0.0001) for AxSYM vs. IMx and 0.83 (p <0.0001) for IMx vs. HPLC. Passing and Bablok regression Y-intercepts and slopes were: 2.944/0.937 (AxSYM vs. HPLC), −0.367/1.142 (AxSYM vs. IMx) and 2.632/0.805 (IMx vs. HPLC). Corresponding mean differences (AxSYM-Comparison Assay) recorded over a 5–50 μmol/l measured range were 1.80, −0.73 and 2.53 μmol/l. AxSYM tHcy FPIAs first rate precision, supported by the complete automation of the AxSYM analyzer, makes it fit for routine use and suitable for laboratories requiring homocysteine high-throughput testing capabilities.

Newborn screening of homocystinuria: quantitative analysis of total homocyst(e)ine on dried blood spot by liquid chromatography with fluorimetric detection

Identification of homocystinuric newborns is hindered by the pitfalls of neonatal screening programs. We propose a fluorimetric HPLC method with a rapid pre-analytical step for homocysteine determination from neonatal dried blood spot cards. Homocysteine in blood spots sampled among 2000 healthy newborns on living day 4, averaged 2.92+/-2.07 microM (range 0.4-7.5). In eight homocystinuric control children, mean values were 61.71+/-52.84 microM (range 18.9-145.7). The method showed a good linearity (r=0.999), precision (RSD<7%) and recovery (95%). The correlation between blood spots and plasma samples was r=0.90. This method has all the essential features for a homocystinuria screening program: an easy and rapid pre-analytical step combined with method linearity and precision.

8 Determination of Plasma Homocysteine

INTRODUCTION A number of epidemiological and clinical studies have linked elevated plasma homocysteine (Hcy) to atherosclerotic vascular disease affecting coronary, carotid, and peripheral vessels. Plasma Hcy can be considered a marker of methionine metabolic efficiency, mainly affected by dietary intake of vitamins, especially folate, vitamin B(6), and B(12), as well as by genetic mutations of key metabolic enzymes and renal elimination.

Oxidative Stress Assessment in Response to Ultraendurance Exercise: Thiols Redox Status and ROS Production according to Duration of a Competitive Race

Purpose. Response to an ultraendurance competitive race on thiols redox status, reactive oxygen species (ROS) production, and oxidative stress (OxS) was investigated according to duration. Methods. Twenty-four elite runners were examined: six completed 50 km and eighteen 100 km. Blood and urine samples were collected before and immediately after the race. Erythrocytes and plasma aminothiols by high-performance liquid chromatography, total antioxidant capacity (TAC), and OxS biomarkers (protein carbonyl (PC), thiobarbituric acid-reactive substances (TBARS), 8-isoprostane (8-iso-PGF2α), and 8-OH-2-deoxyguanosine (8-OH-dG)) by immunoenzymatic assays and ROS production by Electron Paramagnetic Resonance were assessed. Results. Significant increases (P between <0.05 and <0.0001) were recorded in plasma total and oxidized aminothiols concentration and TAC (P < 0.0001) only after 100 km: plasmatic (ROS production (+12 versus +29%), PC (+54 versus +115%), and TBARS (+28 versus +55%)) and urinary (8-OH-dG.creatinine−1 (+71 versus +158%) and 8-iso-PGF2α.creatinine−1 (+43 versus +135%)) concentrations for 50 and 100 km (duration 4 h 3′ versus 8 h 42′), respectively. Conclusion. Very prolonged ultraendurance exercise causes an increase in ROS production and OxS depending on specific biomarker examined but always linearly and directly related to exercise duration. Redox status of erythrocytes was preserved. A relationship between running performance and both prerace ROS production and antioxidant-redox status was found in 100 km race.

Determination of different forms of aminothiols in red blood cells without washing erythrocytes.

Detection and quantification of different aminothiols forms (reduced and total) in biological fluids are important for the investigation of oxidative stress-related diseases and cell homeostasis study. The aim of this study was to optimize a HPLC method in order to determine both reduced and total thiol forms in red blood cells (RBC) at low temperature without washing erythrocytes. Analytical recoveries for total and reduced thiols were 91.6-98.5 and 94.9-98.2% respectively. The relative standard deviations intra-assay for total and reduced thiols were 1.14-3.64 and 0.83-2.3% respectively and the relative standard deviations inter-assay for total and reduced thiols were 1.12-3.54 and 0.84-2.03%, respectively. This method allows specific analysis of the aminothiol state inside the RBC, as a model of intracellular metabolism functioning.

Morphine or its withdrawal affects plasma malondialdehyde, vitamin E levels and absence or presence of abstinence signs in rats.

Objectives Various experimental observations show that morphine treatment generates reactive oxygen species, and that its discontinuation leads to signs of withdrawal. We therefore investigated plasma malondialdehyde and vitamin E levels under both conditions to verify the occurrence of any alterations in oxidative metabolism, and whether these are associated with behavioural changes.

Efficacy of a Standardized Extract of Prunus mume in Liver Protection and Redox Homeostasis: A Randomized, Double-Blind, Placebo-Controlled Study

The antioxidant, anti‐inflammatory and hepatoprotective effects of Prunus mume (PM) have previously been demonstrated. This double‐blind, placebo‐controlled study was designed to evaluate the influence of two doses of a food supplement, made of 150 mg of a standardized PM extract on liver transaminases, lipid profile, glycemia, neopterin and reduced and oxidized thiols in plasma and erythrocytes, during a 3‐month treatment period, in healthy subjects with transaminases levels between 20 and 40 UI/L. Forty‐five subjects (56.0 ± 11.6 years) were enrolled. The results showed a beneficial and statistically significant effect versus placebo of PM extract on liver function, with a decrease versus baseline in alanine aminotransferase (47%), aspartate aminotransferase (7%), gamma‐glutamyl transpeptidase (15%) and glycemia (11%). The lipid profile modification was also positive with an increase versus baseline in HDL cholesterol (13%), and a decrease in LDL/HDL ratio (12%) and triglycerides (8%). The antioxidant action of PM translated into a decrease in oxidized glutathione, reduced/oxidized cysteine‐glycine, oxidized cysteine (intracellular pro‐oxidant) and neopterin (inflammation biomarker), was associated with an increase in reduced glutathione. These results are in favor of the use of a standardized extract of P. mume for the support of liver health and prevention of common metabolic and inflammation‐based diseases. Copyright