Platelet thromboxane formation and bleeding time is influenced by ethanol withdrawal but not by cigarette smoking.

Platelet count, mean volume, aggregation and associated thromboxane (TXB2) formation, circulating platelet aggregates and bleeding time were examined in 19 noncirrhotic male alcoholic cigarette smokers for four weeks following cessation of prolonged heavy drinking, and in 24 nonalcoholic healthy male volunteers (10 smokers and 14 nonsmokers). The alcoholics showed a 9-fold increase (p less than 0.001) in ADP-stimulated platelet thromboxane formation one to two weeks after ethanol withdrawal. The effect was transient and coincided with a significant (p less than 0.01) shortening of skin bleeding time and a slight increase in circulating platelet aggregates suggesting proneness to thrombosis. No differences were seen between the smoking and nonsmoking healthy volunteers. We conclude that the recovery phase after prolonged heavy drinking is characterized by a transient increase in platelet reactivity which may lead to increased spontaneous formation of circulating platelet aggregates and shortening of bleeding time.

Circadian rhythm of serotonin transport in human platelets.

The circadian rhythm of serotonin active transport in human platelets was investigated in ten healthy men, aged 27-35 years. Blood was collected at 08.00, 14.00, 20.00, 02.00 and 08.00 hours the next morning. Simultaneous evaluation of the mean platelet volume, platelet distribution width, platelet distribution skewness and platelet number in whole blood was performed. Km and Vmax for serotonin transport varied considerably among individuals over 24 h. However, the mean values and distribution of these kinetic parameters were reduced at 02.00 hours. All platelet size or number parameters were stable and normal over 24 h; therefore, the reduction in mean Km and Vmax values at 02.00 h is not related to morphological platelet differences but either to platelet intrinsic factors or plasmatic variables. Knowledge of the affinity and capacity of serotonin transport throughout the diurnal cycle is important for future comparisons with depressed patients as well as other hormonal rhythms in patients and healthy humans.

Effects of different anticoagulants on human platelet size distribution and serotonin (5-HT) induced shape change and uptake kinetics.

The effect of collecting blood with disodium ethylene diamine tetraacetate (EDTA), citrate (NAC) or acid sodium citrate-dextrose (ACD) as anticoagulants on platelet count and size distribution was investigated. No difference between the three preparations regarding platelet count was found in whole blood. Preparation of platelet-rich plasma (PRP) significantly reduced the platelet count in NAC-PRP (p less than 0.01) to a value of 288 X 10(9)/l compared to those of 365 X 10(9)/l and 368 X 10(9)/l in EDTA and ACD blood respectively. A significant shift in the platelet size in EDTA-PRP towards larger cell volumes was observed. There were no differences in the size distribution pattern between NAC-PRP and ACD-PRP in spite of the differences in platelet count. Platelet 5-HT uptake kinetics in EDTA-PRP showed a 50 per cent reduction in both Km and Vmax compared to that in ACD-PRP. The study shows that the receptor mediating 5-HT induced shape change has a direct opposite pH dependence than that of the 5-HT carrier. Interference of receptor-mediated responses in 5-HT uptake studies in human platelets is clearly minimized at a lowered pH. The finding is probably of importance in disorders associated with platelet hyperaggregability.

Platelet uptake of serotonin (5-HT) during ethanol withdrawal in male alcoholics.

Changes in the kinetic variables of the platelet serotonin uptake, Km and Vmax, were studied in 7 male alcoholics, admitted for detoxification and in sex- and age-matched volunteers. On admission the alcoholics had lower Km values than reference subjects (p less than 0.05). During detoxification the Km values normalized. Vmax was normal throughout the study in spite of the changes in platelet count. The results of the study suggest that the affinity of serotonin to its uptake receptor is transiently increased after a period of heavy drinking.

Effects on cyclo-oxygenase of low and high dose aspirin.

The study was undertaken to develop clinically applicable methods to detect the influence of low dose treatment with acetylsalicylic acid (ASA) on the platelet function. The cyclooxygenase activity of the platelets was measured by determination of the metabolite 12-HHT after challenge with arachidonic acid. Two healthy subjects received 600 mg ASA in a single dose. Two hours after intake the salicylate concentration level in plasma was 600 nmol/ml, with a 90 per cent reduction after 24 hours. The platelet cyclooxygenase activity (12-HHT) was completely and irreversibly inhibited within two hours after ASA intake. Five subjects received 75 mg ASA for eight consecutive days. The salicylate concentrations two hours after the intake of the first and last tablet were within 65 and 71 nmol/ml plasma, indicating that no accumulation of salicylate occurred during the treatment period. The decrease in cyclooxygenase activity during low dose treatment varied in the different subjects between 60 and 90 per cent. The rate of recovery of cyclooxygenase activity after termination of the low dose ASA treatment was faster than after the large single dose.

Immediate effects of heparin and LMW heparin on some platelet and endothelial derived factors.

Heparin and a low molecular heparin fragment, injected intravenously in volunteers, increased the plasma concentrations of platelet factor 4, but did not induce platelet activation as judged from excretion of 2,3-dinor-TxB2 (a major thromboxane A2 metabolite) and beta-thromboglobulin (btg) in urine and from btg levels in plasma. Heparin prolonged, within the normal range, the bleeding time in all six subjects. Platelet aggregation in platelet rich plasma was potentiated by both heparins, but platelet number, mean platelet volume and platelet distribution width were not affected. No evidence for endothelial release of prostacyclin was obtained as judged from urinary excretion of 2,3-dinor-6-keto-PGF1 alpha (a major prostacyclin metabolite), and plasma concentrations of tissue plasminogen activator, its inhibitor (PAI-1) and the von Willebrand-factor were unchanged.

Inter-individual variations of the effect of low dose aspirin regime on platelet cyclooxygenase activity.

Thirteen healthy men (age range 24-59 years) received three single doses (30, 75, and 150 mg/day) of aspirin for seven days, followed by a wash-out period of three weeks, in a randomized order. The arachidonic acid metabolite 12-L-5,8,10-heptadecatrienoic acid (12-HHT) was taken as a measure of platelet cyclooxygenase activity. There was a large inter-individual variation in 12-HHT production prior to and during aspirin treatment. After one week of treatment the mean reduction was 69, 72 and 83% for the doses 30, 75 and 150 mg/day respectively. When the degree of cyclooxygenase inhibition was expressed per microgram aspirin administered per kg bw, a positive correlation was established to the activity before medication. It was found that doses exceeding 1500 micrograms per kg bw is required to achieve a predictable reduction in cyclooxygenase activity. Thus, by determining the pre-treatment cyclooxygenase activity in an individual it should be possible to adjust the enzyme activity to any desired level below 40% of its initial value. 150 mg aspirin/day for one week had a stimulating effect on the platelet basal production of 12-HHT when measured three weeks after the cessation of treatment. This rebound phenomenon was also observed up to six weeks after a single dose of 600 or 1200 mg of aspirin.

Abnormally high platelet activity after discontinuation of acetylsalicylic acid treatment.

Production of 12-L-hydroxy-5, 8, 10-heptadecatrienoic acid (12-HHT) from platelets and bleeding times were studied in 32 males during acetylsalicylic acid (ASA) treatment and 1 and 2 weeks after withdrawal. All patients (age 42-77 years) had ASA treatment because of angina pectoris. The metabolite 12-HHT is formed in the same amount as the proaggregatory and vasoactive metabolite thromboxane A2. Initially the daily ASA dose was 75 mg (n = 15), 160 mg (n = 12) or 250-300 mg (n = 5). In all patients, median 12-HHT level increased from 40 to 240g/750 x 10(6) platelets (P < 0.001) 1 week after withdrawal of ASA, and four patients had abnormally high values. Median bleeding time decreased from 312 to 268 s (P = 0.003) in the 75 mg group and from 315 to 235 s in the 160 mg group (P = 0.01). Two weeks after withdrawal of ASA, median 12-HHT was 390g/750 x 10(6) platelets and eight patients (25%) had abnormally high values. One patient still had a prolonged bleeding time. Wide interindividual variations were observed in all groups. Our results indicate that rapid withdrawal of ASA, may cause abnormally high 12-HHT levels reflecting increase of thromboxane A2 with possible hazardous effects in patients with cardiovascular disease.

Electrochemical treatment of cancer. II: Effect of electrophoretic influence on adriamycin.

Electrochemical treatment of cancer with DC electrodes changes the microenvironment of the cancer cells by electrophoresis. They may deteriorate and resorb. In larger neoplasms, the effects can be enhanced by electrophoresis of a chemotherapeutic agent. Adriamycin delivered into an electropositive neoplasm electrode will move outwards in the neoplastic area in a high concentration. When given intravenously and the neoplasm electrode is electronegative, the systemic effects may be diminished. The agent is instead accumulated in the neoplastic region in a high concentration. This principle may be used to reduce systemic effects of charged chemotherapeutic compounds. In this preliminary study of cancer in 14 patients, incurable with surgery, radiation treatment, or chemotherapy, beneficial effects were obtained after combined electrochemical treatment and the use of Adriamycin.

Electrochemical treatment of cancer. III: Plasma pharmacokinetics of adriamycin after intraneoplastic administration.

Plasma pharmacokinetics of Adriamycin (doxorubicin) has been studied after intraneoplastic administration during electrochemical treatment to patients with lung cancer that is noncurable with radiotherapy, surgery, or chemotherapy. Intraneoplastic administration of Adriamycin via the anode resulted in a dramatic change of the pharmacokinetic pattern in plasma as compared with what has been previously observed after intravenous administration. A fivefold reduction of the area under the plasma concentration time curve and a 25-fold reduction of the maximum plasma concentration was observed.

Electrochemical treatment of cancer. IV: Leukocyte and platelet counts in peripheral blood after electrochemical treatment of solitary lung neoplasms.

The effects on hematological parameters in peripheral blood was measured by electronic cell counting after electrochemical treatment (n = 11), after electrochemical treatment with concomitant intraneoplastic injection of the chemotherapeutic agent doxorubicin (Adriamycin) (n = 13), and after electrochemical treatment with concomitant intravenous infusion of Adriamycin (n = 2). After treatment, a reduction in lymphocyte count (p less than 0.01) and an increase in total leukocyte count (p less than 0.01) was observed. There was a trend that during treatment the platelet count was reduced.

Increased uptake of serotonin in platelets from car painters occupationally exposed to mixtures of solvents and organic isocyanates.

Twelve car painters occupationally exposed to mixtures of solvents and organic isocyanates were investigated concerning serotonin uptake in platelets. The data from the exposed workers were compared to data from a reference group consisting of 50 nonexposed volunteers. The mean platelet count in whole blood of the exposed workers was markedly lower than the corresponding value of the reference group. Three workers had values below the lower limit of the 95% tolerance interval of the referents. In both groups the serotonin transport strictly obeyed Michaelis-Menten's simple saturation kinetics. Nine of the twelve exposed workers had uptake rates which were significantly higher (p = 0.05) than those of the referents. The obtained data suggest that the present method may be used to reveal early organ damage, prior to the appearance of clinical symptoms, due to intermittent exposure to organic solvents and isocyanates.

Effect on the uptake kinetics of serotonin (5-hydroxytryptamine) in platelets from workers with long-term exposure to organic solvents. A pilot study.

Six workers from a paint industry were examined concerning the concentration of solvents in the breathing zone, the platelet count and kinetic uptake of serotonin [5-hydroxytryptamine (5-HT)], the serum concentration of calcium, potassium and sodium, and pH. The degree of exposure was found to be moderate. The results showed a decrease in platelet count in whole blood and platelet-rich plasma. The maximum uptake rate for serotonin in platelets (Vmax) was significantly increased compared to that of nonexposed subjects. No changes in the ionic concentration and pH of serum were observed. One worker was reexamined after a six-week period of nonexposure. Though the measured platelet variables were still aberrant, the values approached normal.

Dielectric spectroscopy of human blood.

Impedance measurement is an established technique for studying the passive electric properties of cell membranes. Dispersions can be detected by studying the electric properties (capacitance and conductance) in the radio-frequency range (kHz-mHz). The theoretical interpretation is based on the Maxwell-Wagner effect at the interface between the cytoplasm and the cell membrane. The specific electric variables of the membrane, the cytoplasm, as well as the surrounding plasma (medium) are estimated by non-linear regression fitting and appropriate equations. Using a four-electrode technique, we have measured the impedance with a commercial instrument working in the frequency range of 0.2-10 mHz interfaced to a computer. Differences were found in conductivity and capacitance of blood from 1) persons exposed to organic solvents, 2) patients with metal exposure, and 3) patients with cardiovascular disease. The effects of plasma components and haematocrit are crucial when undertaking measurements on whole blood. The results are difficult to interpret but we consider perturbations in the erythrocyte membrane to be involved. Potential clinical applications will be promoted by the development of the software.

Calcium- and lead-activated morphological changes in human erythrocytes: a spin label study of the cytoplasm.

Stimulation of the human red blood cell (RBC; erythrocyte) by Ca2+ or Pb2+ ions induced potassium efflux (the so-called Gárdos effect). Time-dependent changes in the volume and shape of the RBCs were monitored on the basis of mean cell volume and light scattering. Changes in the RBC cytoplasm were followed using electron paramagnetic resonance (EPR), after labeling of the cytoplasmic glutathione with 2,2,6,6-tetramethyl-4-maleimidopiperidine-N-oxyl. The RBC could still be activated in vitro with Ca2+ (plus ionophore A23187) or Pb2+ ions. Pb2+ retained its stimulatory-inhibitory activity. No linear relationship between the reciprocal of cell volume and the mobility (correlation time) of the probe was found over the morphological range covered upon Ca2+ activation. The response of the probe is considered to be mainly governed by the microviscosity due to hemoglobin and any influence by cytoskeleton products could not be discerned. Pretreatment of the RBC with N-ethylmaleimide (1 mM for 30 min) at room temperature prevented spin labeling. However, the RBC still responded to the ion activation, whereas this effect was eliminated after pretreatment at 37 degrees C. EPR of spin-labeled cells, before and after reaching the sphero-echinocytic state, resulted in similar final spectra. Spectra were also observed from RBCs fixed in various states with glutaraldehyde.

Increased erythrocyte volume in car repair painters and car mechanics.

The biological effect of occupational long term low level exposure to organic solvents was studied in 17 car repair painters and 28 car mechanics, and compared with a control group consisting of 46 healthy men not exposed to organic solvents. The erythrocyte count (RBC) was significantly decreased for both the painters and the mechanics compared with the control group, and a significant increase in the mean erythrocyte volume (MCV) was seen in painters. The mode in size distribution of the erythrocytes (MAXRBC) was shifted towards larger cell volumes for both painters and mechanics. When data from painters and mechanics were combined, the exposed group showed a significantly reduced erythrocyte count (RBC), an increased mean erythrocyte volume (MVC), and an increased mean platelet volume (MPV) compared with the controls. Exposure measurements in combination with analysis of haematological parameters may be a tool for early detection of cellular changes in the blood caused by exposure to solvents, before the appearance of clinical symptoms.

Increased isotransferrin ratio and reduced erythrocyte and platelet volumes in blood from thermoplastic industry workers.

Ten women (aged 31-61 years) and five men (aged 20-59 years) occupationally exposed to welding fumes of polyacetate containing diethylphthalate in a thermoplastic industry were studied. They had been employed 1-33 years (median: 11 years). Seven women (aged 35-55) and eight men (aged 26-73) acted as unexposed controls. The exposed persons showed increased isotransferrin ratio in blood serum and reduced volumes of erythrocytes and platelets in blood.

Study of the electrical impedance of blood from house painters.

Using a four-electrode technique the electrical impedance was measured at 150 kHz (100 microA) on whole blood samples from 13 male house painters (age 19-49 years; median 35 years) exposed occupationally to vapors from alkyd paints containing organic solvents and from a control group of 7 healthy unexposed men (age 27-53 years; median 37 years). The blood impedance of the two groups was monitored prior to and 9 days after an initial administration of acetylsalicylic (600 mg). The resistivity calculated from the impedance of whole blood from the painters remained significantly higher than the corresponding values for the controls. The hematological values for the two groups were within normal ranges, although the painters had significantly higher platelet counts than the controls. Effects of long-term exposure of the painters to volatile solvents are discussed.

Haematological changes in house painters using epoxy paints.

Haematological parameters, iso-transferrin ratio in plasma and serotonin uptake in platelets were studied in 10 men (age range 21-54 years) with occupational long-term, low level exposure to vapours from epoxy paints. The control group consisted of 10 healthy men (age range 20-48 years) not occupationally exposed to chemicals or organic solvents. The mean cellular volume of erythrocytes was significantly higher for the house painters than the controls (p less than 0.05). The plasma concentration of iso-transferrin with isoelectric point 5.7 (Tf5.7) and the ratio between Tf5.7 and total transferrin (Tftot) were significantly higher in the exposed group (p less than 0.05). The uptake of serotonin in platelets (Vmax) from the exposed workers was significantly lower than the values for the controls (p less than 0.01). The results indicate an association between the observed biological effects and the chemical exposure, and we speculate that this is caused by changes in structure and function of the cell membranes.