High-performance liquid chromatographic detection of lipid peroxidation in human seminal plasma and its application to male infertility

BACKGROUND: A simple and reliable high-performance liquid chromatographic (HPLC) method has been developed and validated for the analysis of malondialdehyde (MDA) in human seminal plasma. METHODS: After human seminal plasma is hydrolyzed, MDA is reacted with thiobarbituric acid (TBA) to form MDA(TBA)(2), a red-colored adduct with maximum absorbance at 532 nm. HPLC separation of the adduct in human seminal plasma was performed on a Lichrospher C(18) column. RESULTS: A mobile phase composed of 0.025 mol/l KH(2)PO(4) (pH 6.2)--methanol in the ratio 58:42 (v/v) was found to be the most suitable for this separation. Under the chromatographic conditions described, the MDA-TBA adduct had a retention time of approximately 4 min and good separation and detectability of MDA in human seminal plasma sample was obtained. The method proved to be linear calibration in the range of MDA from 0.10 to 2.50 micromol/l. The relative standard deviations of within- and between-assay for MDA analysis were 3.1% and 3.8%, respectively. The average recovery was 90.0-98.8% for the human seminal plasma samples. The method has been successfully applied to the study of the lipid peroxidation levels in the seminal plasma of male infertility. Semen samples were obtained from healthy volunteers and infertile males. Ejaculates were classified into studied subgroups and defined as: obstructive azoospermia, non-obstructive azoospermia, oligozoospermia, asthenozoospermia, oligoasthenozoospermia and oligoasthenoteratozoospermia. With the exception of obstructive azoospermic group, MDA concentrations of seminal plasma in control group had very significant difference with those in other infertile groups (P < 0.01). CONCLUSIONS: This indicated that lipid peroxidation could be harmful to male sperm and reproductive system, which may lead to male infertility.     

HPLC for simultaneous quantification of total ceramide, glucosylceramide, and ceramide trihexoside concentrations in plasma

BACKGROUND: Simple, reproducible assays are needed for the quantification of sphingolipids, ceramide (Cer), and sphingoid bases. We developed an HPLC method for simultaneous quantification of total plasma concentrations of Cer, glucosylceramide (GlcCer), and ceramide trihexoside (CTH). METHODS: After addition of sphinganine as internal calibrator, we extracted lipids from 50 microL plasma. We deacylated Cer and glycosphingolipids by use of microwave-assisted hydrolysis in methanolic NaOH, followed by derivatization of the liberated amino-group with o-phthaldialdehyde. We separated the derivatized sphingoid bases and lysoglycosphingolipids by HPLC on a C18 reversed-phase column with a methanol/water mobile phase (88:12, vol/vol) and quantified them by use of a fluorescence detector at lambda(ex) 340 nm and lambda(em) 435 nm. RESULTS: Optimal conditions in the Solids/Moisture System SAM-155 microwave oven (CEM Corp.) for the complete deacylation of Cer and neutral glycosphingolipids without decomposition were 60 min at 85% power, fan setting 7. Intra- and interassay CVs were <4% and <14%, respectively, and recovery rates were 87%-113%. The limit of quantification was 2 pmol (0.1 pmol on column), and the method was linear over the interval of 2-200 microL plasma. In samples from 40 healthy individuals, mean (SD) concentrations were 9.0 (2.3) micromol/L for Cer, 6.3 (1.9) micromol/L for GlcCer, and 1.7 (0.5) micromol/L for CTH. Plasma concentrations of GlcCer were higher in Gaucher disease patient samples and of CTH in Fabry disease patient samples. CONCLUSIONS: HPLC enables quantification of total Cer, GlcCer, and CTH in plasma and is useful for the follow-up of patients on therapy for Gaucher or Fabry disease.     

Low density lipoprotein immunoapheresis does not increase plasma lipid peroxidation products in vivo.

Extracorporeal elimination of low density lipoprotein (LDL) is frequently used in drug-resistant hypercholesterolemia. LDL-immunoapheresis selectively removes LDL and lipoprotein(a) [Lp(a)] from plasma. Lipid peroxidation is one unwanted side effect, that occurs during extracorporeal plasma treatment. The purpose of this study was to investigate the effect of LDL immunoapheresis on lipid peroxidation. Before and after a single LDL-immunoapheresis treatment, plasma concentrations of lipid hydroperoxides, determined with two different spectophotometric assays, thiobarbituric acid-reacting substances (TBARS), determined spectrophotometrically and malondialdehyde (MDA), determined by an MDA-TBA/HPLC method, were measured in 13 hypercholesterolemic patients. In addition MDA was also determined in the eluate of the apheresis column. Before treatment, plasma cholesterol and LDL cholesterol concentrations were significantly higher in patients than in healthy control subjects, as were the lipid peroxidation products. LDL-immunoapheresis treatment of the patients led to significant decreases in total cholesterol (69+/-8%), LDL-cholesterol (79+/-7%), HDL-cholesterol (35+/-17%), triglycerides (38+/-21%), apolipoprotein-B (77+/-6%), apolipoprotein-A1 (25+/-5%) and Lp(a) concentrations (76+/-10%). Changes in plasma lipid peroxide concentrations (17+/-8 nmol/l before vs. 14+/-5 nmol/l after treatment) were not significant, neither were those in TBARS (3. 0+/-2.6 micromol/l vs. 2.3+/-1.3 micromol/l) or MDA concentrations (1.03+/-0.17 micromol/l vs. 1.0+/-0.20 micromol/l). Patients with high baseline values showed a decrease, whereas others did not. MDA was present (0.57+/-0.13 micromol/l) in the eluate of the apheresis column, suggesting that, along with LDL, lipid peroxidation products are also removed. From these results we conclude that a single LDL-immunoapheresis treatment effectively reduces LDL and Lp(a) in the absence of increases in plasma lipid peroxidation products.     

A simple HPLC method for monitoring mycophenolic acid and its glucuronidated metabolite in transplant recipients.

Mycophenolic acid (MPA) is nowadays in broad clinical use as a substitute for azathioprine. An immunoassay for MPA recently received approval for clinical applications. The high performance liquid chromatography (HPLC) assay for measuring MPA and its glucuronide conjugate (MPAG) we describe here is not only rapid and simple but also extremely sensitive at plasma levels obtained during standard immunosuppressive regimens. The determination of MPAG is possible without any change of the chromatographic conditions (detection wavelength of 214 nm, mobile phase: acetonitrile and 50 mmol/l o-phosphoric acid (50:50, V/V), run time: 15 min). The required equipment is a standard HPLC system including a simple UV-detector. Sample volume of 400 microl is required for both determinations. Detection limit is 0.25 micromol/l for MPA and 5 micromol/l for MPAG. Linearity is excellent for serial dilutions (0.5-25 micromol/l for MPA, 25-500 micromol/l for MPAG) and high accuracies favour the method described. More than 2000 plasma samples tested for MPA in patients after heart transplantation within one year and more than 500 samples for MPAG underline the clinical applicability of this assay.     

Quantification of glutaric and 3-hydroxyglutaric acids in urine of glutaric acidemia type I patients by HPLC with intramolecular excimer-forming fluorescence derivatization

BACKGROUND: Glutaric aciduria type I (GA1) is an autosomal recessive disorder that usually causes neurological damage. Early diagnosis of the disease prior to the appearance of clinical symptoms can lead to better outcomes. METHODS: We describe a simple and selective HPLC method with intramolecular excimer-forming fluorescence derivatization to diagnose GA1. Glutaric acid (GA) and 3-hydroxyglutaric acid (3HGA) in urine and an internal standard were derivatized with 1-pyrenebutyric hydrazide (PBH). The derivatives were separated on a C18 column and fluorometrically detected at 475 nm (excitation of 345 nm) with a run time of 18 min. RESULTS: Excellent linearity over a wide range, reproducibility (coefficient of variation < or =14.5%), and sensitivity (limit of detection 0.4 micromol/l 3HGA and 0.2 micromol/l GA) were obtained. A retrospective study on previously diagnosed GA1 patients' urine from our laboratory archives between 1999 and 2004 was performed by analysts blinded to the study. CONCLUSIONS: The method enabled us to differentiate GA1 cases (n=36) from controls (n=99), regardless of the years of urine storage. The method is valuable for both retrospective and prospective diagnoses of GA1.     

Total plasma malondialdehyde levels in 16 Taiwanese college students determined by various thiobarbituric acid tests and an improved high-performance liquid chromatography-based method

Objectives: In determining the plasma malondialdehyde MDA levels in some Taiwanese college students, we found rather different results by using different thiobarbituric acid TBA tests, even by the high-performance liquid chromatography HPLC-based methods. Here, we re-evaluated four commonly used TBA tests and improved the HPLC-based test.

Design and Methods: We used the blood plasma of 16 college volunteers to determine plasma MDA by using four methods: a spectrophotometric measurement of thiobarbituric acid-reactive substances (TBARS) in the TCA-supernatant of plasma (Method A); a fluorescence measurement of plasma lipid peroxides (Method B); and two different HPLC-based measurements of MDA with either 532-nm measurement (Method C, HPLC/532 nm) or fluorescence measurement (Method D, HPLC/fluor.).

Results: The levels of MDA or TBA reactive substances obtained from the four methods differed substantially (0.39 ± 0.15; 2.14 ± 0.73; 0.75 ± 0.22; and 0.38 ± 0.15 μM for Methods A, B, C, and D, respectively). The results were positively correlated between Methods A and B (r = 0.740, p < 0.02) and between Methods C and D (r = 0.516, p < 0.05). However, results were negatively correlated between Methods B and D (r = −0.548, p < 0.05). Because most plasma MDA is bound to proteins, we modified the HPLC-based methods (C and D) by adding an alkaline hydrolysis step, and the plasma TBA-MDA adduct detected by HPLC/532 nm was referred to as total MDA. Results show that alkaline hydrolysis was a critical step for measurement of total MDA in plasma because this treatment led to release of MDA from plasma proteins. We also adapted the potassium iodide (KI) treatment of plasma from Method D to reduce lipid hydroperoxides. Our modified method gave a total MDA level in the 16 volunteers of 1.5 μM, which was equal to protein-bound MDA plus free MDA. This total MDA level was positively (p < 0.05) correlated with the level of TBA reactive substances obtained from Methods C (r = 0.63, p < 0.05) and D (r = 0.48, p < 0.07), but was not correlated with those from Methods A and B. The recovery (84105%), precision (within-assay coefficient of variation: 2.4%, between-assay coefficient of variation: 48%) and sensitivity of the modified procedure were comparable to other HPLC-based methods.

Conclusion: By using a validated modification of HPLC-based TBA method, the total plasma MDA in 16 Taiwanese college students was found to be 1.54 μM, which was relatively high compared to those obtained by other HPLC-based method, primarily due to the release of protein-bound MDA by alkaline hydrolysis. This level equaled the sum of protein-bound MDA and free MDA in plasma, confirming that this level represents total plasma MDA.     

Determination of free L-carnitine in human seminal plasma by high performance liquid chromatography with pre-column ultraviolet derivatization and its clinical application in male infertility

BACKGROUND: To develop and validate a simple and reliable high performance liquid chromatographic (HPLC) method for the analysis of free L-carnitine in human seminal plasma and to investigate its clinical significance as a potentially additional means of evaluating the infertile male. METHODS: After proteins in seminal plasma are precipitated with a mixture of acetonitrile and methanol (9:1; v/v), free L-carnitine in seminal plasma was derivatized to form its UV-absorbing ester. HPLC separation of the sample solution was performed on a Lichrospher SiO2 column and detected by ultraviolet absorbance at 260 nm. A mobile phase composed of acetonitrile-citric acid buffer (containing 12 mmol/L triethanolamine, pH 5.0) was found to be the most suitable for this separation at a flow rate of 1.2 mL/min and enabled the baseline separation of the free L-carnitine from interferences with isocratic elution. The free L-carnitine levels in seminal plasma were studied in both 30 control subjects and 87 patients with infertility. Ejaculates were classified into studied subgroups and defined as: asthenozoospermia (n=29), oligozoospermia (n=19) and oligoasthenoteratozoospermia (n=39). RESULTS: Under the chromatographic conditions described, the free L-carnitine derivative had a retention time of approximately 13 min. Good separation and detectability of free L-carnitine in human seminal plasma sample were obtained. The method proved to be linear in the range of L-carnitine from 0 micromol/L to 1000 micromol/L. The relative standard deviations of within- and between-assay for free L-carnitine analysis were 1.23 and 1.36 %, respectively. The recoveries were 91.6-96.5% for the human seminal plasma samples. Free L-carnitine concentrations in the populations were 392.66+/-107.18 micromol/L in the fertile group (n=30), 270.00+/-83.92 micromol/L in asthenozoospermia group, 187.97+/-43.90 micromol/L in oligozoospermia group and 175.65+/-67.07 micromol/L in oligoasthenozoospermia group. The large difference (P<0.01) between the fertile and infertile populations is evident and the difference between the subdivided groups in the infertile group is not significant (P>0.05). CONCLUSION: The determination of free L-carnitine level in seminal plasma may prove useful as a potentially biochemical marker of fertility and this is a useful guidance for the clinic therapy and the mechanismic study on the male reproduction.     

A high performance liquid chromatographic method for the measurement of total carnitine in human plasma and urine

Total carnitine in plasma and urine can be measured by high performance liquid chromatography (HPLC) using the novel fluorescent derivatisation reagents 6'-methoxynaphthacyl trifluoromethanesulfonate and 2'-phenanthrenacyl trifluoromethanesulfonate. Sample preparation for total carnitine analysis involves: extraction of plasma and urine in methanol, the optional addition of serine betaine as an internal standard, saponification of acyl carnitines with calcium hydroxide, followed by derivatisation with 6'-methoxynaphthacyl trifluoromethanesulfonate or 2'-phenanthrenacyl trifluoromethanesulfonate. The derivatives were separated using an alumina column and measured by fluorescence detection. The coefficient of variation was below 5% using internal standard calibration, and recoveries of acyl carnitines after saponification were over 90%. The total carnitine method was shown to be linear at biological levels for plasma (over the range 30-130 micromol/l) and urine (over the range 80-180 micromol/l). Advantages of this method include good precision, accuracy and linearity, the use of fluorescence to gain sensitivity, the small sample volume required and a relatively low sample preparation time.     

Comparison of high-performance liquid chromatographic and microbiological methods for determination of voriconazole levels in plasma

A new selective high-performance liquid chromatography (HPLC) method with UV detection for the determination of the investigational triazole voriconazole in human plasma by using acetonitrile precipitation followed by reverse-phase HPLC on a C(18) column was compared with a simple agar well diffusion bioassay method with Candida kefyr ATCC 46764 as the assay organism. Pooled plasma was used to prepare standard and control samples for both methods. The results of analyses with spiked serum samples (run as unknowns) were concordant by the bioassay and HPLC methods, with expected values being obtained. HPLC demonstrated an improved precision (3.47 versus 12.12%) and accuracy (0.81 versus 1.28%) compared to those of the bioassay method. The range of linearity obtained by both methods (from 0.2 to 10 microg/ml for HPLC and from 0.25 to 20 microg/ml for the bioassay) includes the range of concentrations of voriconazole (from 1.2 to 4.7 microg/ml) which are considered clinically relevant. Although either methodology could be used for the monitoring of patient therapy, the smaller variability observed with HPLC compared to that observed with the bioassay favors the use of HPLC for pharmacokinetic studies.     

Plasmatic antioxidant capacity due to ascorbate using TEMPO scavenging and electron spin resonance

BACKGROUND: Ascorbate is the most effective water-soluble antioxidant and its plasma concentration is usually measured by different methods including colorimetric assays, HPLC or capillary electrophoresis. Plasma antioxidant capacity is determined by indexes such as total reactive antioxidant potential, total antioxidant reactivity, oxygen radical absorbance capacity, etc. We developed an alternative method for the evaluation of the plasma antioxidant status due to ascorbate. METHODS: TEMPO kinetics scavenging analyzed by ESR spectroscopy was performed on plasma samples in different antioxidant situations. Plasma ascorbate concentrations were determined by capillary electrophoresis. Ascorbyl radical levels were measured by ESR. RESULTS: Plasma reactivity with TEMPO (PR-T) reflected plasma ascorbate levels. Average PR-T for normal plasmas resulted 85+/-27 micromol/l (n=43). PR-T during ascorbic acid intake (1 g/day) increased to an average value of 130+/-20 micromol/l (p<0.001, n=20). PR-T correlated with the plasmatic ascorbate levels determined by capillary electrophoresis (r=0.92), presenting as an advantage the avoiding of the deproteination step. Plasma ascorbyl radical levels increase from 16+/-2 to 24+/-3 nmol/l (p<0.005, n=14) after ascorbate intake. CONCLUSIONS: PR-T could be considered as a measure of the plasmatic antioxidant capacity due to the plasma ascorbate levels and could be useful to investigate different antioxidant situations.     

Validation of a rapid and sensitive liquid chromatography-tandem mass spectrometry method for free and total mycophenolic acid

BACKGROUND: Because mycophenolic acid (MPA) is highly protein bound and because the free fraction is the pharmacologically active portion, a rapid, reliable, and sensitive procedure is required to study the relationship between free MPA and treatment efficacy/toxicity. Liquid chromatography-tandem mass spectrometry is ideally suited for such a method. METHODS: Free MPA was isolated from plasma by ultrafiltration. An online extraction cartridge with a column-switching technique, analytical liquid chromatography over an Aqua Perfect C(18) column, and electrospray tandem mass spectrometry was used to quantify free and total MPA. To investigate ion suppression, a continuous infusion of MPA was introduced into the effluent from the HPLC column, and different ultrafiltrates and extracted plasma samples were injected on the column. RESULTS: A chromatographic run time of 4 min separated MPA from metabolites and internal standard, thereby avoiding interference from in-source fragmentation. Ion suppression occurred well before elution of MPA and internal standard. The lower limit of quantification for free MPA was 0.5 microg/L, and the method was linear to 1000 microg/L. Interassay imprecision (CV) was <10% for free MPA (0.5-333 microg/L). Agreement was good for free MPA (n = 52) and total MPA (n = 106) between the proposed method and a validated HPLC method with ultraviolet detection. The Passing-Bablok regression line was: y = 0.95x + 0.27 microg/L for free MPA and y = 0.98x + 0.03 mg/L for total MPA. CONCLUSIONS: The presented method allows the accurate, precise, and rapid determination of free and total MPA in plasma over a wide analytical range covering the concentrations relevant to pharmacokinetic studies and routine monitoring of this drug.     

Simultaneous analysis of D- and L-serine in cerebrospinal fluid by use of HPLC

BACKGROUND: D-Serine is a coagonist for the glycine-binding site of the N-methyl-D-aspartate receptors and has been implicated in various neuropsychiatric functions such as learning, memory, and nociception, as well as schizophrenia and Alzheimer disease. We developed an HPLC method for D- and L-serine in cerebrospinal fluid (CSF). METHODS: The dabsylated racemic serine peak, automatically collected using a previously reported HPLC separation process for CSF amino acids, was desalted and subjected to a chiral resolution HPLC step with a Sumichiral column using an ultraviolet-visible detector. RESULTS: The limits of quantification (signal-to-noise ratio = 10) for D- and L-serine were 0.8 and 1.3 micromol/L, respectively. The mean imprecision values (CVs) for within-day measurements of D- and L-serine were 2.1% and 1.8%, respectively, and for between-day were 6.2% and 6.6%. Mean recovery of CSF serine (sum of D-serine + L-serine) applied to the Sumichiral column was 87%. The mean (SD) d-serine concentrations in 45 CSF samples obtained from 16 patients with chronic pain due to degenerative osteoarthritis of the knees, 16 with postherpetic neuralgia, and 13 with no pain were, respectively, 3.97 (0.44), 1.85 (0.21), and 2.72 (0.32) micromol/L. CONCLUSION: D- and L-serine can be quantified with ultraviolet-visible detection of dabsyl derivatives. The dabsyl derivatives are stable and allow duplicate analysis of CSF samples in multisample runs.     

Comparison of Abbott IMx total homocysteine assay with a high pressure liquid chromatography method for the measurement of total homocysteine in plasma and serum from a Norwegian population.

In this study 189 samples were analysed for the measurement of homocysteine by the Abbott IMx homocysteine assay and an HPLC method. A strong correlation was obtained between the homocysteine measurements performed by the Abbott IMx homocysteine assay and the HPLC method (coefficient of correlation, r2 = 0.947, p < 0.0001). The plot of the difference for the homocysteine measurements between the two methods against the average of the two measurements resulted a mean difference of 0.80 +/- 6.66 (mean +/- 2SD), and 0.008 +/- 0.126 (mean +/- 2SD) for log converted values of homocysteine. The concentrations of homocysteine measured in all the samples by the two methods were not significantly different. However, the Abbott IMx homocysteine assay measured the concentrations of total homocysteine in hyperhomocysteinemia as significantly higher than the HPLC method (25.00 micromol/l vs. 23.12 micromol/l, p < 0.0001). More studies may be required to explore factors that may influence measurements of homocysteine by the Abbott IMx homocysteine assay and the HPLC method.     

Separation of plasma carotenoids and quantitation of beta-carotene using HPLC

A method was developed for the extraction and separation of human plasma carotenoids and quantitation of beta-carotene. Carotenoids were extracted from plasma with ethanol: hexane and separated by C18 reversed phase HPLC using spherical 3 micron packing. beta-Carotene was identified and quantitated using an external standard. The within-run precision of three different plasma pools ranged from 3.53-5.72% relative standard deviation (RSD). The between-run precision was 7.34% RSD. The method was linear to 500 micrograms/l with a statistical detection limit of 3.80 micrograms/l. Recovery of added beta-carotene was from 90.41-100.37%. This method was compared to a spectrophotometric 'total carotene' method. The mean plasma concentrations of 25 male and 25 female human volunteers for the 'total carotene' were 1,549 micrograms/l for all samples, 1,487 micrograms/l for males and 1,611 micrograms/l for females. The corresponding true beta-carotene concentrations obtained by HPLC analysis were 134.8, 115.9 and 153.7 micrograms/l, respectively. The true beta-carotene concentrations were on the average only 8.76% (8.07% for males and 9.46% for females) of the concentrations obtained by the spectrophotometric 'total carotene' method. Correlation between the methods had an r = 0.6107. The poor correlation is due to the difference in the measured components. Total carotene methods measure all solvent extractable moieties having absorbance in the 430-460 nm region, while the HPLC method quantitates true beta-carotene after chromatographic separation from other carotenoids. Reference intervals were established for plasma beta-carotene using REFVAL, an IFCC computer program for determining statistical reference intervals. The reference interval for all samples is 40 to 344 micrograms/l.     

Drug testing in blood: validated negative-ion chemical ionization gas chromatographic-mass spectrometric assay for enantioselective measurement of the designer drugs MDEA, MDMA, and MDA and its application to samples from a controlled study with MDMA

BACKGROUND: The enantiomers of the designer drugs 3,4-methylenedioxyamphetamine (MDA), 3,4-methylenedioxymethamphetamine (MDMA), and 3,4-methylenedioxyethylamphetamine (MDEA) differ in their pharmacologic and toxicologic potency. The aim of this study was to develop an assay for measuring these enantiomers in small plasma volumes and to analyze samples from a controlled study with MDMA. METHODS: The analytes were extracted from < or = 0.2 mL of plasma by mixed-mode solid-phase extraction. After derivatization with S-(-)-heptafluorobutyrylprolyl chloride, the resulting diastereomers were separated by gas chromatography (HP-5MS) within 17 min and detected by mass spectrometry in the negative-ion chemical ionization mode. The method was fully validated and applied to samples from a controlled study in which a single dose of racemic MDMA (75 mg) was administered. RESULTS: The derivatized enantiomers were well separated and detected with good sensitivity. The assay was linear (per enantiomer) at 1-50 microg/L for MDA and 5-250 microg/L for MDMA and MDEA. Analytical recovery, accuracy, repeatability, and intermediate precision data were within required limits. Extraction yields were 82.1%-95.3%. In the study samples, concentrations of R-(-)-MDMA significantly exceeded those of S-(+)-MDMA. Their ratios (R vs S) were always >1.0 and increased over time. Concentrations of S-(+)-MDA exceeded those of R-(-)-MDA, their ratios (R vs S) also increasing over time but remaining <1.0. CONCLUSIONS: This assay enables sensitive, reliable, and fast enantioselective measurement of MDA, MDMA, and MDEA in small volumes of plasma. The controlled study data confirm previous findings of MDMA and MDA enantiomer ratios (R vs S) increasing over time after ingestion of racemic MDMA.     

Comparison of three methods for total homocysteine plasma determination

Hyperhomocysteinemia is a well established risk factor for atherothrombotic disease, and the request for homocysteine determinations and the number of laboratories that need to perform this assay to assess individual risk profile is increasing. Different methods to evaluate homocysteine plasma levels are at present available. In the present study three methods, an in-house high-pressure liquid chromatographic (HPLC) method (considered as reference method) and two commercial immunoassays, an enzyme-linked immunoassay (EIA) and an automated fluorescence polarization immunoassay (FPIA), were used to measure homocysteine plasma levels in 100 samples. The median of homocysteine plasma levels obtained by HPLC was 9.0 micromol/L (range 4.2-23.0); the median of values obtained by EIA and FPIA were 10.6 micromol/L (range 3.3-21.5) and 9.6 micromol/L (4.8-20.2), respectively. The FPIA method showed the lowest within-run and between-run coefficients of variation (3.6% and 4.1%, respectively). There was a significant correlation between EIA and HPLC (r=0.81; p<0.0001), and between FPIA and HPLC (r=0.85; p<0.0001). The Bland-Altman analysis showed that FPIA agreed best with HPLC; EIA displayed a relatively wide scatter of difference data points. The present results indicate that the technological characteristics of the FPIA assay make this method suitable for the determination of Hcy in clinical laboratories.     

Speciation and quantification of thiols by reversed-phase chromatography coupled with on-line chemical vapor generation and atomic fluorescence spectrometric detection: method validation and preliminary application for glutathione measurements in human whole blood

BACKGROUND: We developed a sensitive, specific method for the low-molecular-mass thiols cysteine, cysteinylglycine, glutathione, and homocysteine and validated the method for measurement of glutathione in blood. METHODS: The technique was based on reversed-phase chromatography (RPC) coupled on line with cold vapor generation atomic fluorescence spectrometry (CVGAFS). Thiols were derivatized before introduction on the column by use of a p-hydroxymercuribenzoate (PHMB) mercurial probe and separated as thiol-PHMB complexes on a Vydac C4 column. Postcolumn on-line reaction of derivatized thiols with bromine allowed rapid conversion of the thiol-PHMB complexes to inorganic mercury with recovery of 100 (2)% of the sample. HgII was selectively detected by atomic fluorescence spectrometry in an Ar/H2 miniaturized flame after sodium borohydride reduction to Hg0. RESULTS: The relationship between thiol-PHMB complex concentration and peak area (CVGAFS signal) was linear over the concentration range 0.01-1400 micromol/L (injected). The detection limits were 1, 1, 0.6, and 0.8 nmol/L for cysteine, cysteinylglycine, homocysteine, and glutathione in the injected sample, respectively. The CVs for thiols were 1.5%-2.2% for calibrator solutions and 2.1% and 3.0% for real samples. The RPC-CVGAFS method allowed speciation of glutathione (reduced and oxidized) in human whole blood from healthy donors and from the coronary sinus of patients with idiopathic dilated cardiomyopathy during and after chronotropic stress. CONCLUSION: The RPC-CVGAFS method could be used to measure reduced and oxidized glutathione in human whole blood as disease biomarkers.     

A new enzymatic cycling method for ammonia assay using NAD synthetase

BACKGROUND: Ammonia is an important marker for liver diseases such as hepatitis and hepatic cirrhosis. Several methods have been developed for ammonia analysis. In particular, the enzymatic assay using glutamate dehydrogenase has been widely used. However, this method is not necessarily high in sensitivity and accuracy due to inhibition by interferences in plasma and instability over long-term storage. METHODS: We developed an ammonia assay using a system consisting of three enzymes, NAD synthetase (NADS; EC 6.3.1.5), glucose dehydrogenase (GlcDH; EC 1.1.1.47), and diaphorase (DI; EC 1.6.99.2). RESULTS: The calibration curve for ammonia with the cycling method was linear (r=0.999) up to 300 micromol/l. The within-run CVs of 10 and 20 micromol/l NH4Cl solutions and 24.1 micromol/l ammonia in human plasma were 2.3%, 1.5%, and 2.8%, respectively. The between-run CVs of them were 4.5%, 3.1%, and 2.8%, respectively. The recovery was between 96.3% and 105%, and the limit of detection was 2.4 micromol/l. No significant interference was observed with addition of the following components: hemoglobin, bilirubin, chyle, EDTA, heparin, and sodium citrate. Due to the high degree of specificity of NAD synthetase to ammonia, no amino compounds exhibited any effect on the ammonia assay. A high correlation was obtained between results of the present method (y) and a conventional glutamate dehydrogenase method in regression analysis; y=0.944x-6.160 with r=0.993 (n=125). However, an addition error was observed from Bland-Altman analysis (the 95% limits of agreement between the two methods; 9.51+/-5.92 micromol/l). CONCLUSION: This new enzymatic method is more sensitive, precise, and accurate than the conventional method. In particular, accurate assay for ammonia can be performed without interference in the presence of various compounds.     

In vivo total antioxidant capacity: comparison of two different analytical methods.

Several methods to assess the total antioxidant capacity (TAC) are available. However, the final value of measured TAC in the sample depends on the procedure used in every specific assay. This makes crucial the comparison of different analytical methods. The aim of our study was to evaluate analytical characteristics and laboratory reliability of two different assays: the ferric-reducing ability (FRAP) assay and a new spectrophotometric test (OXY-adsorbent test, Diacron, Italy). Unselected outpatients referred to the Institute of Clinical Physiology were studied (n = 187, 58 females, 129 males, mean age: 65 +/- 13 years). All blood samples were maintained on ice, centrifuged within 15 minutes after blood collection and then stored at -80 degrees C until performance of assay procedures. OXY assay: The lower limit of sensitivity was 6 micromol HClO/ml. The assay was found to be linear up to 440 micromol HClO/ml (r = -0.99, p < 0.001). Absorbance was linear over a wide concentration range with solutions containing uric acid in purified form (0-1000 micromol/l, r = -0.996, p < 0.001), serum (r = -0.99, p < 0.01) or plasma serially diluted (r = -0.99, p < 0.01). Mean value in plasma samples accounted for 366.2 +/- 7.2 micromol HClO/ml. Mean OXY value in females (353.4 +/- 13.2 micromol HClO/ml) was not different from that detected in males (372 +/- 8.6 micromol HClO/ml). A significant difference was observed between subjects without and with hypertension in serum OXY levels (344.8 +/- 9.9 and 383.2 +/- 10 micromol HClO/ml, p < 0.01, respectively). FRAP assay: The lower limit of sensitivity was 15 micromol/l. Linearity was observed up to 1000 micromol/l (r = 0.998, p < 0.001). Absorbance was linear over a wide concentration range with solutions containing uric acid in purified form (0-1000 micromol/l, r = 0.997, p < 0.001), serum (r = 0.99, p < 0.01) or plasma serially diluted (r = 0.99, p < 0.01). FRAP mean value in plasma samples, evaluated in 102 patients, accounted for 514.1 +/- 19.1 micromol/l. Mean FRAP in females (469 +/- 22.5 micromol/l) was not different from that detected in males (535 +/- 25.6 micromol/l). FRAP vs. OXY: A significant direct relationship was observed when comparing FRAP with OXY levels in the whole population (r = 0.22, p < 0.05). Neither of the methods are expensive and they are speedy and simple to perform. Values are reproducible and linearly correlated to the concentration of antioxidants present in the samples. For this reason, these methods may be considered practicable indicators of total antioxidant capacity, for routinely potential use in every laboratory and useful in all the studies concerning the evaluation of oxidative stress.     

Highly specific, simple and rapid method for the determination of malondialdehyde in blood using high-performance liquid chromatography.

A novel, highly specific, simple and rapid method for the determination of malondialdehyde (MDA), the routinely used marker for free radical generation in body fluids has been developed and evaluated. Serum samples from 30 healthy volunteers in heparin and 1,4-dithiothreitol-containing tubes stored at -80 degrees C were analyzed. The MDA-thiobarbituric acid complex was separated from interfering substances using HPLC. For the separation, reverse phase column MAC (4 x 250 mm, Biospher SI 120 PSI C18, particle size 7 microm) was used. The mixture of methanol and 8.3 mmol/l phosphate buffer, pH= 7.2, (35:65, v/v) was used as mobile phase. The volume of serum samples injected on the column was 50 microl. The analyte was detected at 532 nm. Retention time of MDA-thiobarbituric acid complex was 4.9+/-0.1 min at the flow rate 0.7 ml/min. Excellent linearity was achieved. The intra- and interassay coefficient of variation was 7.3% and 8.8%, respectively. The recovery was 95.6% and the detection limit was 0.1 micromol/l. The validity of this method was proved by comparison with the spectrophotometric determination of MDA-thiobarbituric acid complex by the method of Yagi at three different wavelengths (485, 532 and 560 nm) with Allen's correction.