Role of leucocytes in free radical production during myocardial revascularisation.

OBJECTIVE: To evaluate the role of leucocytes in free radical production in patients with depressed or normal ejection fraction undergoing coronary bypass. DESIGN: Two randomised control trials. SETTING: Tertiary care centre. PATIENTS AND INTERVENTIONS: In the first study, 22 patients with ejection fractions of < or = 40% received blood cardioplegic reperfusion with (n = 11) or without (n = 11) leucocyte depletion. In the second study, 22 patients with ejection fractions > or = 45% received either leucocyte depleted (n = 11) or blood cardioplegia (n = 11). MAIN OUTCOME MEASURES: Glutathione, hypoxanthine, and lipid peroxidation products were measured in coronary sinus blood and plasma before aortic cross clamping and at 0, 15, and 30 minutes after unclamping. Haemodynamic variables and creatine kinase MB isoenzymes were monitored on the first postoperative day. Comparison between treatments was performed on difference (delta) between measurements at time 0 and at baseline, and on slopes obtained by fitting measurements after unclamping with a linear regression model. RESULTS: At unclamping no difference in delta for plasma glutathione redox ratio (oxidised/total glutathione, %) was observed between treated and control groups with low ejection fraction (delta = 16 (SD 8.39) and 24 (7.0) redox ratio %, respectively). Baseline value recovery rate (redox ratio %/min) was significantly faster in treated v control patients (slope -0.912 (0.380) v -0.158 (0.200), P < 0.005, respectively). Cardiac index showed a trend to greater improvement in the treated group (slope 0.04 (0.03) v 0.003 (0.002) 1/min/m2/h, P < 0.02, treated v controls, respectively). In patients with normal ejection fraction, leucocyte depletion did not result in significant improvement v controls. CONCLUSIONS: Leucocyte depletion seems to provide benefit only in patients with left ventricular dysfunction.

Relation between left ventricular function and oxidative stress in patients undergoing bypass surgery.

OBJECTIVE: To determine whether preoperative left ventricular ejection fraction (LVEF) is related to the degree of myocardial oxidative stress during bypass surgery in man. DESIGN: Observational study. SETTING: Tertiary care centre. PATIENTS AND INTERVENTIONS: 31 patients (LVEF range was 20% to 68%) undergoing elective coronary bypass surgery with blood cardioplegic reperfusion were studied. Arterial and coronary sinus blood was collected before aortic cross clamping (T0) and at 0 (T1), 15 (T2), and 30 (T3) minutes after unclamping. Transmural left ventricular biopsies were also obtained from 15 patients at T0 and at T1. MAIN OUTCOME MEASURES: Glutathione and adenine nucleotides were measured in myocardial biopsies, while coronary sinus-artery differences for glutathione, nucleotides, and products of lipid peroxidation were calculated from blood specimens. Creatine kinase (myocardial band; CK-MB) was measured in plasma at four and 12 hours after operation. RESULTS: Myocardial glutathione and adenine nucleotides were correlated (p < 0.02) with preoperative LVEF both at T0 (r = 0.909 and 0.672) and T1 (r = 0.603 and 0.605). Oxidised glutathione released from the heart during reperfusion was inversely correlated with LVEF (r = -0.448, -0.466, and -0461 at T1, T2, and T3, p < 0.01), while reduced glutathione (r = 0.519 and 0.640 at T1 and T2) and glutathione redox ratio (r = 0.647, 0.714, 0.645, and 0.702 at T0, T1, T2, and T3) showed a direct correlation (p < 0.01). Lipid peroxidation at T1 was negatively related to LVEF (r = -0.492). CK-MB was also negatively related to LVEF (r = -0.440 at 4 h and -0.462 at 12 h). CONCLUSIONS: The capacity to counterbalance oxidative burst following ischaemia and reperfusion appears to be related to the functional ability of the heart.

Measurement of erythrocyte acetylcholinesterase and plasma cholinesterase activity by a differential pH technique.

We describe a new electrochemical method for the determination of erythrocyte acetylcholinesterase activity (EC 3.1.1.7) and plasma cholinesterase (EC 3.1.1.8) activity, based on the measurements of pH variation due to release of acetic acid from acetylcholine. The major advantages of the differential pH procedure are simplicity, high reproducibility, no need for pre-treatment of samples, automatic correction of sample blanks, and speed and direct measurement of enzymatic reaction. The proposed methods are linear up to 7400 U/L at 30 degrees C and correlate well with the manual spectrophotometric method of Ellman for plasma cholinesterase and for washed erythrocytes. We adapted the same technique for the determination of erythrocyte cholinesterase using whole blood as sample and quinidine sulphate as inhibitor of pseudocholinesterase.

Tumor-associated trypsin inhibitor as a possible marker in male infertility.

The concentrations of tumor-associated trypsin inhibitor (TATI) in the seminal plasma of infertile males was studied. The TATI levels in seminal plasma were not correlated with either sperm count or ejaculate volume. High levels were observed in some men with unexplained infertility and high or normal sperm counts, whereas normal levels were observed in males with antisperm antibodies. The concentrations in seminal plasma were stable in the same subjects. These results suggest that TATI may be an important marker of reproductive pathology in men.

Tumor-associated trypsin inhibitor in induced and acquired immunodeficiency. Studies on transplanted and HIV-infected patients.

A new tumor marker, tumor-associated trypsin inhibitor (TATI), was studied in 5 patients who received successful kidney or pancreas grafts and in 30 subjects with antibodies against human immunodeficiency virus. Serum TATI concentrations were very high during the four first days after transplantation. Thereafter the serum levels decreased when the peptide was eliminated through the kidney. Consequently, the urine values were very high. The TATI concentrations of HIV positive subjects were compared with serum levels of HIV antigen and antibody, by Western blotting and determination of peripheral T-lymphocyte subpopulations. The occurrence of high concentrations of TATI in some HIV positive subjects and especially in AIDS patients, suggests that TATI could be useful in exploring physiopathological aspects of severe immunodeficiencies even if TATI levels were not correlated with the commonly used markers of the immune system status. The increased levels of TATI in immunological disorders suggests its possible use in assessing the immune response against cancer.

Determination of serum cholesterol on the Reflotron (BBR) dry chemistry analyser. Assessment of accuracy.

This investigation is aimed at assessing the accuracy of S-cholesterol determination by means of the Reflotron, an analytical system based upon the dry chemistry approach. Two sets of sera, one of them including specimens with elevated concentrations of urea, creatinine, glucose, urate or bilirubin, were assayed in parallel with the Refloton system, with a class-A "reference" method (including alkaline hydrolysis and extraction) and with a routine liquid chemistry enzymatic method. Results for both sets of sera indicated good agreement of the tested system with the routine method, whilst a small proportional negative bias was observed in comparison with the "reference" method. The use of the reference method as a basis for comparison allows more conclusive statements about accuracy to be made.

Detection of cryoglobulins by Coulter Counter model S-Plus IV/D.

Cryoglobulins may produce interference in results from electronic particle counters. Depending on their size, cold-precipitable globulin crystals can be counted as leucocytes and/or platelets. The Coulter Counter model S-Plus IV/D permits detection of this interference and its associated spurious counts by producing a characteristic WBC histogram accompanied by a leucocyte 'flag'. Using this instrument over a 1-year period, 10 cases of cryoglobulinaemia were detected, four from patients with lymphoproliferative disease and one from a patient with autoimmune disease. The remaining five were classified as essential mixed cryoglobulinaemia. The reproducibility of this instrument anomaly permits identification of the presence of cryoglobulins.

High-performance liquid chromatographic method for measuring hydroxylysine glycosides and their ratio in urine as a possible marker of human bone collagen breakdown.

Glucosyl-galactosyl-hydroxylysine (GGHYL) and galactosyl-hydroxylysine (GHYL) are constituents of collagen protein. The ratio of the two hydroxylysine glycosides varies with the collagen type and, moreover, for a given collagen type, it also varies according to the connective tissue. For example, in type I collagen (the most abundant in the body), the GGHYL/GHYL ratio tends to be greater in soft connective tissues and lower in bone. The hydroxylysine glycosides are not recycled during collagen turnover and are excreted in the urine. Therefore, the urinary GGHYL/GHYL ratio, which reflects the proportion of the two metabolites in the various collagens, may indicate the type of connective tissue affected by pathological turnover, and may thus be a promising marker of bone metabolism. In this paper a method is described for the measurement of urinary hydroxylysine glycosides by reversed-phase liquid chromatography after purification of the sample by solid-phase extraction. The method presented is analytically reliable and suitable for routine use in a clinical laboratory.

Determination of creatinine in serum and urine by a rapid liquid-chromatographic method.

We describe an HPLC ion-pair procedure for rapid and specific evaluation of creatinine in serum and urine. We used a 15 cm X 4.6 mm ODS column with a 50/50 (by vol) mixture of sodium decanesulfonic acid (10 mmol/L, pH 3.2) and methanol and measured absorbance at 236 nm. Serum (100 microL) or 30-fold-diluted urine (100 microL) was added to 400 microL of acetone. After centrifugation, the supernates (300 microL) were dried, reconstituted with the mobile phase, and injected into the HPLC. Assay precision was tested for concentrations of 10, 29, and 130 mg/L and yielded, respectively, 3.1%, 2.1%, and 1.1% for within-day CV and 2.8%, 2.1%, and 2.2% for total CV. Analytical recovery was 102 (+/- 6.7%). Linearity was demonstrated in the 0-200 mg/L range for serum and 0-3.5 g/L range for urine (r greater than or equal to 0.999). The detection limit for creatinine (signal-to-noise ratio = 3) was 0.5 mg/L. We used cimetidine for internal standardization. Correlation was good between this procedure and the Jaffé kinetic, the enzymatic (creatinine amidohydrolase), and the Fuller's earth alkaline picrate methods.

[Alloalbuminemia associated with myeloma]. / Alloalbuminemia associata a mieloma.

Alloalbuminemia occurring together with paraproteinemia in a case of proven myelomatosis is described. The clinical and laboratory findings of this case are listed. The importance of the reported case is emphasized.

Ethylene glycol-stabilized haemolysates for glycated haemoglobin assay. Further analytical investigations and applications to quality control programmes.

The direct effect of the ethylene glycol on the quantitation of glycated haemoglobins by ion-exchange minicolumn chromatography, affinity chromatography and high performance liquid chromatography was investigated. No evident effect was found for ethylene glycol concentrations between 0.0 and 0.5 (volume fraction). The ethylene glycol-stabilised haemolysates were also analysed by HPLC: the elution profiles were similar to those of haemolysates fresly prepared from whole blood, and they remained unchanged, even after 18 months of storage at -20 degrees C. A preliminary inter-laboratory trial among 15 Italian laboratories was organized, using these ethylene glycol-stabilised haemolysates and, for comparison, a lyophilized commercial preparation. From the data obtained in this trial we confirmed the validity of using ethylene glycol-stabilized haemolysates as control materials, both for intra- and inter-laboratory standardization of glycated haemoglobin measurements.

EDTA-dependent pseudothrombocytopaenia: a 12-month epidemiological study.

The phenomenon of in vitro platelet clumping and consequent pseudothrombocytopaenia in the presence of EDTA has been studied in 33,623 subjects referring to a general hospital in a 1-year period. The observed frequency was 0.13%. EDTA-dependent pseudothrombocytopaenia (PTP) was suspected when a routine blood counting by the Coulter S-Plus IV/D showed a peculiar leucocyte histogram and pseudoleucocytosis. Confirmation was obtained by the manual count and by the finding of platelet aggregates in a stained blood smear. EDTA-dependent PTP was diagnosed when the platelet number and the morphological examination of blood anticoagulated with sodium citrate from the same patient were normal. EDTA-dependent PTP was found in 23 subjects aged from 19 to 79 years (0.068% of the study population): 17 were patients suffering from miscellaneous diseases, while six were apparently healthy. As a rule, platelet clumping was evident within 60 minutes from blood collection, but a longer latency (2-3 h) was observed in a few cases. EDTA-dependent PTP is a rare, but misleading phenomenon, the recognition of which is important in order to avoid expensive and potentially harmful procedures.