Simultaneous quantitation of homocysteine, cysteine and methionine in plasma and urine by liquid chromatography-tandem mass spectrometry.

BACKGROUND: A method utilizing liquid chromatography-electrospray tandem mass spectrometry (LC-MS/MS) has been developed and evaluated for the determination of total homocysteine, cysteine and methionine in plasma and urine. The simultaneous measurement of homocysteine and methionine concentrations may help explain the underlying mechanism responsible for hyperhomo-cysteinaemia. METHODS: Samples were prepared by simple protein precipitation after reduction of disulphides by dithiothreitol. Reduced analyte signal caused by ionization suppression effects, seen with plasma samples, was compensated for with matrix-matched standards, and the use of isotopically labelled internal standards. Recovery for each analyte was better than 94%. RESULTS: Concentrations of plasma homocysteine determined by LC-MS/MS were compared with those obtained by two automated commercially available FDA-approved procedures: (i) high-performance liquid chromatography (HPLC) with pre-column derivatization and fluorescence detection and (ii) by fluorescence polarization immunoassay (FPIA). Agreement with the LC-MS/MS method is given by the Deming regression equations LC-MS/MS = 1.062 HPLC-0.01 and LC-MS/MS = 1.104 FPIA-0.43. CONCLUSION: Low reagent costs together with the relative simplicity of sample preparation make the LC-MS/MS method well suited, not only for research work but also in those laboratories with a tandem mass spectrometer, for the measurement of routine clinical samples.

Evaluation of serum neopterin, high-sensitivity C-reactive protein and thiobarbituric acid reactive substances in Egyptian patients with acute coronary syndromes.

The present study evaluated serum neopterin, high-sensitivity C-reactive protein (hs-CRP) and thiobarbituric acid reactive substances (TBARS) in Egyptian patients with acute coronary artery disease. Thirty-six patients with unstable angina aged (mean +/- SD) 61.3+/-9.4 years, 29 patients with myocardial infarction aged 58.2+/-8.7 years and 24 sex- and age-matched control subjects were included in the study. Neopterin levels were significantly higher in patients with myocardial infarction and those with unstable angina than in the healthy control group (P<0.001). The serum level of neopterin in the control group (median [range]) was 3.25 nmol/L (1.25 nmol/L to 5.4 nmol/L), whereas in patients with unstable angina and those with myocardial infarction, neopterin levels were 10.4 nmol/L (3.5 nmol/L to 15.2 nmol/L) and 12.6 nmol/L (3.25 nmol/L to 17.8 nmol/L), respectively. Levels of hs-CRP and TBARS were also significantly higher in patients with unstable angina and those with myocardial infarction than in the healthy control group (P<0.01). The medians (ranges) of hs-CRP were 4.8 mg/L (2.5 mg/L to 9.9 mg/L), 12.0 mg/L (4.6 mg/L to 31.0 mg/L) and 12.3 mg/L (7.5 mg/L to 32.1 mg/L) in the control group, patients with unstable angina and those with myocardial infarction, respectively. The means +/- SD of TBARS in the control group, patients with unstable angina and those with myocardial infarction were 0.64+/-0.17 mumol/L, 1.17+/-0.31 mumol/L and 1.17+/-0.49 mumol/L, respectively. TBARS positively correlated with hs-CRP and neopterin levels. Furthermore, when both patients and controls were classified according to their smoking status, significantly higher levels of neopterin and TBARS were found in the smokers of each subgroup than in the nonsmokers.In conclusion, the present study found a higher level of neopterin, hs-CRP and TBARS in patients with coronary artery disease. Serum neopterin and hs-CRP positively correlated with the level of TBARS. The authors suggest that triggering factors (eg, smoking, high cholesterol, elevated body mass index or raised blood pressure) may lead to increased oxidative stress, which induces an inflammatory insult leading to higher levels of inflammatory markers such as neopterin and hs-CRP.

Direct measurement of low density lipoprotein in whole blood by silver-enhanced gold-labelled immunoassay.

A competitive silver-enhanced gold-labelled immunoassay has been developed for the direct measurement of low density lipoprotein (LDL) in whole blood. Immobilized LDL and sample LDL compete for added antibody. Quantitation of the bound antibody/antigen complex is achieved by the addition of gold-labelled anti-immunoglobulin G followed by enhancement of absorbance by addition of silver ions. Whole-blood samples from fasting patients were assayed directly for LDL by the procedure and the corresponding plasma samples were assayed for total cholesterol, high density lipoprotein and triglycerides followed by the indirect calculation of LDL cholesterol. The correlation between the two methods was good (r = 0.82) and the SEGLISA exhibited good precision.

Multiple releasable fluorescein labels for immunoassay: the principle illustrated by an immunoassay for antibodies to the human immunodeficiency virus.

Attempts to increase the sensitivity of fluorescein-based fluorescence immunoassays by using multiple labelling have generally been unsuccessful because of concentration quenching. We have labelled antibodies to human immunoglobulin G with multiple fluorescein fluorophores attached by means of a disulphide linkage: this linkage can be rapidly and easily broken by treatment with dithiothreitol, allowing fluorescein to be released from the antibody and measured in free solution. Application of this technique to a fluorescence labelled immunosorbent assay for antibodies to the human immunodeficiency virus gave an approximately 20-fold increase in signal compared with an equivalent assay using fluorescein isothiocyanate.

A silver enhanced, gold labelled, immunosorbent assay for detecting antibodies to rubella virus.

A silver enhanced, gold labelled, immunosorbent assay (SEGLISA) for the detection of IgG antibodies to the rubella virus in human serum was developed. Pre-coated microtitre wells are used as the immobilised base of rubella antigens on to which any rubella antibodies from patient samples will bind. This antigen/antibody complex is then visualised firstly by gold labelled anti-immunoglobulin G, which binds to any human IgG that may be present, and then by silver amplification, resulting in a black permanent deposit on the microtitre well surface. Patient samples (n = 121) were screened using a commercially available enzyme linked immunosorbent assay (ELISA) and an equivalent SEGLISA. Results were comparable but the SEGLISA does not have the disadvantages associated with enzyme labels. The silver deposit may also be read visually or the dried plate may be stored for future reference.

Use of a silver-enhanced gold-labelled immunoassay for detection of antibodies to the human immunodeficiency virus in whole blood samples.

A silver-enhanced gold-labelled immunosorbent assay (SEGLISA) for the detection of antibodies to the immunodeficiency virus (HIV) in whole-blood samples is described. This new non-isotopic, non-enzymic immunoassay incorporates use of solid phase viral antigens which bind any HIV antibodies present in the test sample. The antigen/antibody complex is then detected by gold-labelled anti human immunoglobulin G (IgG) followed by silver amplification. We found that whole blood samples give false positives when using a horseradish peroxidase label, whereas the SEGLISA correctly identified 50 HIV antibody positive samples and 50 HIV antibody negative samples when using whole blood. The use of whole blood collected on filter paper is also described. The SEGLISA has good precision (CV = 7.5%) and sensitivity.

Detection of antibodies to the human immunodeficiency virus by a silver-enhanced gold-labelled immunosorbent assay.

We describe a new immunoassay for the detection of antibodies to the human immunodeficiency virus. The method is based on a silver enhanced gold-labelled immunosorbent assay (SEGLISA). Test sera are incubated in microtitre wells on which antigens have been coated. If present in the test sera, antibodies to the human immunodeficiency virus bind to the solid-phase antigens. Bound antibodies are quantitated with anti-human immunoglobulin labelled with gold. Positive specimens produce a faint pink deposit which is better visualised by silver enhancement which gives an intense black colour. The intensity of the colour is proportional to the bound antibody concentration. All the reagents are stable and the silver enhancement takes place under ambient light conditions. The assay has many of the advantages of micro enzyme-linked immunosorbent assays but does not suffer from the drawbacks associated with the use of an enzyme label. It requires fewer manipulations and is quicker to carry out than an equivalent enzyme-linked test. As the silver layer is permanent dried wells may be stored for future reading or checking.

Multiple labelling of immunoglobulin G, albumin and testosterone with a fluorescent terbium chelate for fluorescence immunoassays.

Human immunoglobulin G, human serum albumin and testosterone were labelled with the 4-aminosalicylic acid derivative of diethylenetriaminepentaacetic acid complexed with terbium ions. An exceptionally large amount of label, of the order of a few hundred moles of complex per mole of analyte, could be conjugated to the compounds tested by the use of poly-L-lysine. Self-quenching appears to be minimal, even with this high local concentration of fluorophores. The tracers were stable at 4 degrees C, and gave competitive calibration graphs at physiological concentrations.

Detection of antibodies to the human immunodeficiency virus by a rapid fluorescence-labelled immunosorbent assay.

The development and assessment of a fluorescence-labelled immunosorbent assay for the detection of antibodies to the human immunodeficiency virus is described. Test serum is incubated in microtitre wells on which antigens have been coated. If present in the test serum, antibodies to the human immunodeficiency virus bind to the solid-phase antigens. In turn the antibodies are quantified with anti-human immunoglobulin labelled with fluorescein. Positive samples produce an intense fluorescence which is measured in a spectrofluorimeter. When used to test a panel consisting of normal serum and antibody-positive serum from infected patients the assay proved to be 100% specific and to have a sensitivity of 100%. The assay has many of the advantages of micro enzyme-linked immunosorbent assays, but does not suffer from the drawbacks associated with the use of an enzyme label. It requires fewer manipulations and is quicker to carry out than an equivalent enzyme-linked test.

Determination of plasma cytidine deaminase activity by HPLC.

Cytidine deaminase is an enzyme of nucleic acid metabolism, the measurement of which has been proposed as a useful test for the early detection of pre-eclamptic toxaemia in pregnancy. The enzyme converts the nucleoside cytidine to uridine, with the release of ammonia, and it is the measurement of this latter compound that forms the basis of the conventional methods for the assay of cytidine deaminase. The low activity of the enzyme requires long incubation times, which in turn increase the possibility of contamination by exogenous ammonia. We have developed a new method for determining cytidine deaminase activity, utilising high performance liquid chromatography to measure the production of uridine. This method uses much shorter incubation times and is unaffected by ammonia contamination. This paper describes the development of the method and its comparison with the established assay. The relative merits of each are discussed. Finally, the adaptation of incubation and chromatographic conditions, in order to measure other enzymes of nucleic acid metabolism which are of clinical interest, is briefly mentioned.

On the use of fluorescent labels in immunoassay.

The principles and use of fluorescent labels in immunoassay are reviewed. Comparison is made with radio- and chemiluminescent immunoassays. Possible fluorescent labels are listed. The following types of assay are described: separation fluoroimmunoassay, immunofluorometric assay, fluorescence enhancement and fluorescence quenching assay, fluorescence polarization immunoassay, fluorescence energy transfer immunoassay, fluorescence protection immunoassay, alternative binding immunoassay, release fluoroimmunoassay and time-resolved fluorimetry and phosphorimetry.

Magnesium and the premenstrual syndrome.

Plasma and erythrocyte magnesium were measured in 105 patients with premenstrual syndrome (PMS) using a simple atomic absorption spectroscopy method. The erythrocyte magnesium concentration for the patients with PMS was significantly lower than that of a normal population. The plasma magnesium did not show this difference. The significance of this apparent cellular deficiency of magnesium is discussed.

Whole blood osmolality.

The osmolality of plasma and heparinised whole blood samples collected from hospital patients was estimated using measurement of the depression of freezing point. There was no clinically significant difference between osmolality measurement made on either whole blood, or plasma taken from the same patient. Neither cell volume nor haemolysis was found to affect the measurement. The reproducibility of whole blood measurements was similar to that for determinations carried out on plasma. Measurement of osmolality on whole blood is quicker and cheaper and needs a smaller specimen than if serum or plasma is used.

Hypomagnesium in the elderly.

The range of plasma concentrations in 177 and erythrocyte magnesium concentrations in 104 elderly patients was found to be similar to that for healthy adults. No significant difference was observed between the sexes or between patients taking diuretics and those not receiving diuretic therapy. The latter finding is contrary to some previously reported studies.