Assays for total homocysteine and other thiols by capillary electrophoresis-laser-induced fluorescence detection. I. Preanalytical condition studies.

In recent papers, we presented a new analytical method for thiol quantification in serum. It is based on the use of capillary electrophoresis and laser-induced fluorescence to analyze thiol 6-iodoacetamidofluoresceine (IAF) derivatives. Quantitative results of homocysteine, glutathione, cysteine-glycin, and cysteine were shown (Clin. Chem. 45 (1999) 412). A comprehensive comparison of the quantitation of homocysteine in serum, using high-performance liquid chromatography/conventional fluorescence detection and fluorescence polarization immunoassay was also used (E. Caussé et al., Electrophoresis 21 (2000) 2074). Sample preparation prior to derivatization with IAF had never been investigated. In this work we present the results of quantitation of thiols in serum and plasma with three different anticoagulants widely used: ethylenediaminetetraacetic acid (EDTA), heparin, and sodium citrate. We show that serum and EDTA plasma gave the same results. Then serum protein precipitations by acetonitrile, acetone, sulfosalicylic acid, perchloric acid and trichloracetic acid, prior to derivatization by IAF, were also investigated. Their influence on the concentrations of the thiols were determined. Sulfosalicylic acid and acetonitrile precipitations are well adapted, whereas acetone cannot be used.

Detection and incidence of drug-induced liver injuries in hospital: a prospective analysis from laboratory signals.

AIMS: Liver damage remains the most frequent type of adverse drug reaction (ADRs) that can lead to the withdrawal of a drug from the market. The abnormal laboratory data identified by computerized hospital information systems can be used in order to improve the detection of ADRs. Our objectives were to assess the detection and incidence of drug-induced liver abnormalities in a university hospital inpatient population and to evaluate the underreporting rate of drug-induced liver injury. METHODS: We conducted a prospective study performed 1 week per month from June to October 1997. We selected patients by a computerized process using biochemistry laboratory data, based on serum enzyme values, alanine aminotransferase (over 2 fold normal) and alkaline phosphatase (over 1.5 fold normal). RESULTS: Among 1976 ALT and 1814 AP assays performed during the period of the study, 156 (7.9%) and 159 (8.8%) tests, respectively, fell into the selected criteria. These concerned 147 patients. Among these patients, 13 (8.8%) cases of drug-induced liver injuries were suspected. Seven cases were asymptomatic. Six cases were classified as serious by these criteria: hospitalization to investigate the cause of health status impairment (4 patients), prolongation of hospitalization (1 patient) and life-threathening (1 patient). Using the hospitalization database, the incidence of drug-induced liver injuries was estimated as 6.6 per 1000 inpatients a week. Only 1 case was reported by physicians in the same period. CONCLUSIONS: Computerization of biochemical data would allow the development of systems to improve detection of drug-induced injury. Moreover, underreporting remains important for such potentially serious ADRs, even in a university hospital.

Plasma total homocysteine and other thiols analyzed by capillary electrophoresis/laser-induced fluorescence detection: comparison with two other methods.

We present a new analytical method for thiol quantification in plasma, based on the use of capillary electrophoresis (CE) and laser-induced fluorescence (LIF) to analyze 6-iodoacetamidofluorescein derivatives. Quantitative results of homocysteine, glutathione, cysteinylglycine, and cystationine are presented. A comparison of the quantitation of homocysteine in plasma, using high performance liquid chromatography/fluorescence detection and fluorescence polarization immunoassay is proposed. The results indicate that these techniques for plasma total homocysteine (tHcy) determination can be used interchangeably. The major advantage of CE-LIF is that it can quantitate the thiols in one run while keeping the price of consumables reasonable.

Determination of asymmetrical dimethylarginine by capillary electrophoresis-laser-induced fluorescence.

Asymmetric dimethyl-L-arginine (ADMA) is a naturally occurring analogue of L-arginine (L-Arg), the substrate of nitric oxide synthase (NOS). ADMA is a potent endogenous inhibitor of NOS and accumulates in the plasma of patients with renal failure, with peripheral arterial occlusive disease or with clinically asymptomatic hypercholesterolemia. We measured circulating concentrations of L-arginine, symmetric and asymmetric dimethylarginine (SDMA and ADMA, respectively) in human serum. We developed a new method for the rapid determination of these molecules using capillary electrophoresis and laser-induced fluorescence (CE-LIF). All methylated arginines were labeled with fluorescein isothiocyanate (FITC) prior to analysis. Under the capillary electrophoresis (CE) conditions used, methylated arginine derivatives were well separated, with a migration time of around 10 min. These migration times were smaller than the ones of other amino acids which do not have the same charge at pH 10. Consequently, such basic amino acids were well separated from most of the other amines or amino acids. Moreover, CE allowed one to separate all the analogues of fluorescein thiocarbamyl-arginine. The results indicated that CE-LIF is useful as a selective, rapid, cheap and sensitive tool for the determination of methylated arginine products. This new technology might appreciate the endogenous substrate for NO synthase and facilitate the knowledge of the physiological and pathophysiological regulation of NO synthesis.

Lower alkaline phosphatase activity and occurrence of an abnormal hybrid intestinal/tissue non-specific isoform in Down's syndrome amniotic fluids.

Quantitative and qualitative analysis of alkaline phosphatases (AP) was performed on amniotic fluid in 59 normal pregnancies and 14 Down's syndrome (DS) pregnancies at 16, 18 and 19 weeks of gestation. In DS cases, intestinal and placental isoenzyme levels were significantly reduced (P<0.001) and the AP electrophoretic pattern was seen to be modified on polyacrylamide gel electrophoresis. A unique component was detected. After extraction and purification of the abnormal isoenzyme, peptide fragments obtained after cyanogen bromide cleavage indicated a hybrid heterodimeric AP composed of intestinal and tissue non-specific subunits, as evaluated by SDS polyacrylamide gel electrophoresis.

[Hyperinsulinism, heart rate variability and circadian variation of arterial pressure in obese hypertensive patients]. / Hyperinsulinisme, variabilité de la fréquence cardiaque et variation circadienne de la pression artérielle chez les sujets obèses hypertendus.

AIMS: During insulin resistance, sympathetic nerve activity is increased. However insulin resistance is a common feature of obesity and essential hypertension, it is unclear if chronic hyperinsulinemia per se contributes to sympathetic overactivation. The purpose of our study was to explore++ the relationships between chronic hyperinsulinemia and heart rate variability (HRV), a non-intensive tool to assess autonomic function, in obese and hypertensive subjects. METHODS: 24 hours Holter ECG for HRV time and frequency domain analysis was performed in 77 patients, mean age 53 +/- 10 years, 52 men and 25 women, free of diabetes, without beta-blockers, divided in four groups according to three parameters, body mass index (BMI > 27 kg/m2 in man and > 25 kg/m2 in woman defined obesity), arterial pressure and insulinemia (fasting insulinemia > 25 mUI/L defined hyperinsulinemia): 27 patients obese, hypertensive, with hyperinsulinemia; 28 patients obese, hypertensive, without hyperinsulinemia; 12 patients non obese, hypertensive, without hyperinsulinemia; 10 patients obese, normotensive, without hyperinsulinemia. RESULTS: In comparison with the three other groups, patients with hyperinsulinemia showed a significant decrease (p < 0.05) of SDNN and the power of total spectrum (0.01-1 Hz) band, which are indexes of global HRV, and a significant decrease (p < 0.005) of SD and the normalized power of the low frequency (0.04-0.15 Hz) band, both indexes reflecting sympathetic modulation of HRV. In contrast, no significant difference was observed between the four groups for indexes of HRV reflecting parasympathetic tone. These relations were independent of mean RR. Fasting insulinemia was significantly (p < 0.0001) related with HRV in time domain (SDNN; r = -0.43; SD: r = -0.49) and spectral domain (total spectrum: r = -0.49; low frequency: r = -0.52). CONCLUSION: Chronic hyperinsulinemia appears to be an important determinant of HRV, particularly for the indexes reflecting sympathetic influence, independent of obesity and hypertension.

Study of fluidity of low density lipoproteins from liver cirrhotic patients.

BACKGROUND AND AIM: Liver disease is accompanied by major quantitative and qualitative modifications in plasma lipoprotein metabolism. Alterations in plasma lipoprotein composition and a lower susceptibility to in vitro peroxidation of low density lipoprotein (LDL) and erythrocyte membranes have been observed in liver cirrhosis. The main objective of the present work was to investigate LDL chemical composition and fluidity in liver cirrhosis using the fluorescence polarization (Pf) of the 1,6-diphenyl-1,3,5-hexatriene (DPH) probe. METHODS AND RESULTS: The chemical composition of LDL was studied in 12 cirrhotic patients and 22 controls by conventional methods and its fatty acid composition by gas chromatography. LDL fluidity was determined by measuring the DPH Pf values. A decrease in molecular order was demonstrated by the significant (p < 0.05) decrease in Pf values in the cirrhotics. Modifications in LDL fluidity are correlated with its composition. A significant increase in triglyceride content (p < 0.05), and significant increases in triglyceride/protein and triglyceride/phospholipid ratios were observed in the cirrhotics. CONCLUSIONS: Our results demonstrate that the higher LDL fluidity of cirrhotic patients may be due to an increased triglyceride content.

Effect of nicotine and cotinine on the susceptibility to in vitro oxidation of LDL in healthy non smokers and smokers.

Cigarette smoke of which the major component is nicotine plays an important role in the development of cardiovascular diseases. To study the effect of in vitro incubation of LDL with nicotine and its metabolite, cotinine on a copper-induced peroxidation, we monitored the formation of conjugated dienes, hydroperoxides and thiobarbituric acid-reactive substances production. The LDL studied were taken from six non-smokers (aged 41.5 years) and six smokers who consumed at least ten cigarettes per day (40.7 years). LDL oxidation with CuSO4 showed that cigarette smoking promotes LDL susceptibility to peroxidative modification. During the peroxidation of LDL with nicotine (O to 5 mmol/1) and CuSO4 (5 micromol/l), the formation of hydroperoxides decreased when nicotine concentrations increased and the production of TBARS increased in a concomitant manner. The results showed that the presence of nicotine destabilized the production of hydroperoxides in LDL and increased the formation of secondary oxidation products. On the other hand, cotinine had no effect on LDL oxidative susceptibility in smokers and non-smokers.

Quantitation of homocysteine in human plasma by capillary electrophoresis and laser-induced fluorescence detection.

Homocysteine (Hcy) represents a branching point between the transsulfuration and transmethylation pathway of methionine. A large increase of plasma concentration of Hcy is observed in patients with inherited hyperhomocysteinemia. A moderated increase (above 10 microM) is also observed in various pathological conditions, such as arterial occlusion, hypertension, hyperlipidemia and chronic renal failure. While amino acids were largely studied using capillary electrophoresis with UV or laser-induced fluorescence detection (LIF), thiol-amino acids were not. In this work we present a new approach for testing homocysteine in human plasma using CE-LIF and fluorescein isothiocyanate. The low fluorescence yield of the fluorescein thiocarbamyl (FTC) thiol-amino acids limits, probably, the sensitivity of the detection to 8 x 10(-10) M (instead of 10(-12) M for FTC-arginine).

Physico-chemical properties of copper-oxidized very low density lipoproteins.

The aim of our study was to investigate the effect of Cu2+ catalyzed oxidation on VLDL physico-chemical properties and secondary structure of apo B-100. Incubation of very low density lipoproteins with copper ions resulted in a decrease in tryptophan and lysine residues parallel to lipid peroxidation products, conjugated dienes and TBARS. Fluorescence polarization showed an increase in the molecular order at the lipoprotein surface of VLDL, as demonstrated by the increase in Pf values of DPH. The secondary structure of apo B-100 was investigated by infrared spectroscopy. Increased order and structural changes, as observed after oxidative stress on VLDL, could be of relevance in the abnormal interactions between lipoproteins and cell membranes.

Role and use of expert systems within the clinical laboratory.

Artificial intelligence can be used, mainly in the form of knowledge based systems, for different parts of clinical laboratory activity. Most of the iterative tasks for control and validation, or for maintenance or repair of automated instruments can be assisted by specialized software. There are now specific programmes (i) for control and validation of the request, where the goal is to modify the requesting behaviour of the physician, using protocol driven requests and immediate feedback, incorporating simultaneously a check of test request redundancy, (ii) for helping in equipment trouble-shooting, (iii) for technical and biological validation because too many QC data are issued from large equipment to be checked and used properly by the technologist whilst the pathologist can be assisted for the final revision of reports and (iv) for assistance in the interpretation of laboratory reports, one of the most valuable aspects of the pathologist's participation in the patient care process.

Quantitation of RT-PCR products of bFGF-mRNA by capillary electrophoresis and laser-induced fluorescence.

The numerous challenges of modern biology and medical science require the development of analytical methodologies of extreme sensitivity and resolving power. Because it deals with the extraordinary complexity of biological mixtures and minute samples, capillary electrophoresis combined with laser-induced fluorescence (LIF) is now one of the most popular analytical biotechniques. Using polymer matrices and very high voltages, it allows the separation of very low quantities of microsamples and the selective detection of fluorescent species at a level of a few thousand molecules. This method of analysis has been used to quantitate reverse transcription-polymerase chain reaction (RT-PCR) products. In the authors' laboratory, they were looking for a means of analyzing mRNAs of cytokines. In the first attempt, they chose to quantitate RT-PCR products of basic fibroblast growth factor (bFGF)-mRNA, which could be involved in atherosclerosis. The results on two patients confirm the lower quantity of bFGF expressed in internal mammary athery (IMA) biopsies compared to human aortic cells. This study shows the usefulness of CE-LIF to identify and quantitate RT-PCR products and its ability to be used in clinical studies, to find very low levels of bFGF expression in atheroma biopsies.

Pancreatic cancer cell regulation by lipids and by basic fibroblast growth factor expression.

High fat intake is a risk factor for pancreatic cancer. Lipids may act either directly or in cooperation with growth-promoting polypeptides. In this study, the role of serum lipids, and mainly the often expressed intracellular basic fibroblast growth factor (bFGF) isoforms in cancer cells, was analyzed in pancreatic tumor cell proliferation. Serum lipids alone induced a 1.9-fold increase of human pancreatic cancer cell growth (p < 0.001). Treatment with bFGF had a weak mitogenic effect (1.2- to 1.3-fold increase) compared with those of insulin and transferrin (1.7- to 1.6-fold increase, respectively). The bFGF expression by a rat pancreatic cancer cell line that was transfected with bFGF cDNAs modified cell lipid contents and induced a higher proliferation rate than that found with the exogenous bFGF. Combined extra- and intracellular bFGFs increased cell growth by two to three times (p < 0.001), regardless the presence of extracellular lipids. The results obtained reflect the direct mitogenic effect of serum lipids and suggest that the endogenous bFGF of high molecular weight may be implicated in pancreatic cancer cell growth. By modifying cell lipids, bFGFs may interfere with other cell functions, like signal transduction.