The interaction of ibuprofen and diclofenac with aspirin in healthy volunteers.

BACKGROUND AND PURPOSE: Aspirin reduces the risk of myocardial infarction and stroke by inhibiting thromboxane production in platelets. This inhibition can be competitively antagonized by some non-steroidal anti-inflammatory drugs (NSAIDs). EXPERIMENTAL APPROACH: By measuring thromboxane B(2) production in healthy volunteers, we investigated whether ibuprofen (800 mg three times daily for 7 days) or diclofenac (50 mg three times daily for 7 days) taken concurrently with aspirin 80 mg (once daily for 7 days) influenced the inhibitory effect of aspirin. The effects were compared with aspirin 30 mg (once daily for 7 days), which is the lowest dose of aspirin with a proven thromboprophylactic effect. KEY RESULTS: The median percentage inhibition of thromboxane B(2) levels by 30 mg or 80 mg aspirin was 90.3% (range 83.1-96.0%) and 98.0% (range 96.8-99.2%) respectively. The inhibition by concurrent administration of slow release diclofenac and 80 mg aspirin was 98.1% (range 97.2-98.9%), indicating no interference between aspirin and diclofenac. The inhibition decreased significantly by concurrent administration of immediate release ibuprofen and 80 mg aspirin (86.6%; range 77.6-95.1%) to a level less than 30 mg aspirin. CONCLUSIONS AND IMPLICATIONS: As alternatives are easily available, NSAIDs such as diclofenac should be preferred to ibuprofen for combined use with aspirin.

Genetic polymorphism in UDP-glucuronosyltransferase 2B7 and colorectal cancer risk.

Colorectal cancer (CRC) is one of the most common malignancies in the Western world. CRC is strongly associated with lifestyle factors. Susceptibility to CRC may be partly due to deficient detoxification capacity in the gastrointestinal tract. Genetic polymorphisms in detoxification enzymes result in variations in detoxification activities, which might influence the levels of carcinogens in the gastrointestinal tract, influencing the risk for CRC. To determine whether a genetic polymorphism in the detoxification enzyme UDP-glucuronosyltransferase 2B7 (UGT2B7) predisposes to CRC, 411 Caucasian patients with sporadic CRC and 600 Caucasian controls recruited from the same geographic area were genotyped for the functional UGT2B7 H268Y polymorphism. DNA was isolated and tested by a dual-color real-time polymerase chain reaction assay. Overall, no differences in genotype distributions between patients with CRC and controls were observed. When analyzed with respect to tumor location, a shift from the UGT2B7*I *2 into the UGT2B7*2*2 genotype was seen in patients with proximal CRC (OR 1.80, 95% CI 1.11-2.89). In the male patient subpopulation an even stronger association was observed (*1*1 + *1*2 vs. *2*2: OR 2.17, 95% CI 1.11-4.04; *1*2 vs. *2*2: OR 2.19, 95% CI 1.10-4.37). No associations with respect to tumor stage were seen. In conclusion, the frequency of the UGT2B7*2*2 genotype is higher in CRC patients with proximal location of the tumor, especially in males, which suggests that this genotype is associated with an increased risk for proximal CRC.

Bacteria in blood smears: overwhelming sepsis or trivial contamination.

It is unusual to find microorganisms in peripheral blood smears, and their presence is frequently associated with overwhelming sepsis and consequently a poor prognosis. In this report, we demonstrate 4 cases with bacteria in blood smears. Two of them had a fatal outcome, but the other 2 were caused by a contamination either via the central venous catheter or in vitro, both without dramatic outcome. The finding of bacteria in blood smears has to be interpreted carefully, and thorough examination of peripheral blood smears may be of great importance in the early diagnosis of bacteremia; however, in vitro contamination must be excluded.

A comparison of the effects of nabumetone vs meloxicam on serum thromboxane B2 and platelet function in healthy volunteers.

AIMS: To compare the effects of nabumetone and meloxicam, two cyclo-oxygenase-2 (COX-2) preferential nonsteroidal anti-inflammatory drugs (NSAIDs), on platelet COX-1 activity and platelet function. METHODS: Twelve healthy volunteers (3 male, 9 female, median age 22 years) participated in an open, randomized, cross-over trial of nabumetone 1000 mg twice daily vs meloxicam 7.5 mg twice daily during 1 week with 2 weeks wash-out. After a second 2 week wash-out period, one dose of indomethacin 50 mg was given as a positive control to check for NSAID induced inhibition of platelet function. COX-1 inhibition was measured as percentage inhibition of serum TXB2 generation in clotting whole blood, and as closure time with use of the platelet function analyser PFA-100. Data are reported as median with range. Paired variables were analysed using Wilcoxons signed rank test. RESULTS: TXB2 levels decreased significantly after all three medications, but percentage inhibition after nabumetone and indomethacin (88% and 97%, respectively) was significantly higher than after meloxicam (63%) (P<0.05). Closure times increased significantly after administration of all three medications (P<0.05). Increases in closure time after administration did not differ between nabumetone and meloxicam (24% and 14%, respectively), but were significantly larger after indomethacin administration (63%) (P<0.01). CONCLUSIONS: In the maximum registered dosage, nabumetone inhibits thromboxane production much more than meloxicam, signifying less COX-2 selectivity of the former. However, both nabumetone and meloxicam cause only minor impairment in platelet function in comparison with indomethacin and the difference between them is not significant.

Meloxicam, 15 mg/day, spares platelet function in healthy volunteers.

OBJECTIVE: To study the influence of meloxicam, a cyclooxygenase-2 (COX-2) preferential nonsteroidal anti-inflammatory drug, on serum thromboxane and platelet function in healthy volunteers with use of the maximum recommended daily dosage of 15 mg/day. METHODS: This study used an open, randomized crossover design. Indomethacin (INN, indometacin) was given as a positive control for nonsteroidal anti-inflammatory drug-induced inhibition of platelet function. The following variables were recorded: thromboxane B2 serum concentrations by radioimmunoassay, platelet aggregation by whole blood aggregometry in response to collagen 1.1 microg/L and to arachidonic acid 0.35 mmol/L, and closure time with use of the PFA-100. RESULTS: Serum thromboxane B2 at baseline was 535+/-233 nmol/L (mean +/- SD) and was reduced for 95% by indomethacin to 26+/-19 nmol/L (P < .001) and for 66% by meloxicam to 183+/-62 nmol/L (P < .001). Maximal platelet aggregation in response to collagen at baseline was 18.7+/-1.6 ohms (ohms). It was reduced by indomethacin to 7.3+/-4.5 ohms (P < .001), but not by meloxicam (19+/-2.5 ohms). Platelet aggregation in response to arachidonic acid at baseline was 12.2+/-2.0 ohms. It was reduced by indomethacin in all subjects to 0 ohms, but not by meloxicam (11+/-2.4 ohms). Closure time at baseline was 128+/-24 seconds and was prolonged by indomethacin to 286+/-38 seconds (P < .001). Meloxicam caused a minor prolongation of the closure time (141+/-32 seconds; P < .05). CONCLUSION: Meloxicam, 15 mg/day caused a major reduction of maximum thromboxane production but no reduction in collagen- or arachidonic acid-induced platelet aggregation and only minor increase of the closure time.

Reflex sympathetic dystrophy: is the immune system involved?

OBJECTIVE: Evaluation of immune system function in patients with reflex sympathetic dystrophy (RSD). DESIGN: Survey on blood samples obtained from RSD patients and from a randomly selected control group. The lymphocyte populations (T, B, NK cells), and the activated T cells (CD25, and HLA-Dr-positive CD4 and CD8 cells) were analyzed by flow cytometry with dual-color direct immunofluorescence after whole-blood lysis. Clinical chemistry parameters were analyzed in additional serum samples. SETTING: Tertiary care center (outpatient rehabilitation clinic). SUBJECTS: Thirteen patients (nine women) with RSD and a control group of 21 healthy individuals. MAIN OUTCOME MEASURES: The results of the flow cytometry analysis of RSD patients were related to those of the control subjects. Means were analyzed, and confidence intervals for differences of the means were calculated. The means of the clinical chemical analysis were related to local reference values. RESULTS: The flow cytometry analysis did not differ between RSD patients and healthy controls. Although in some patients an individual parameter of clinical chemical analysis differed from its reference value, all of the mean values were within reference limits. Stratification on medications with immunomodulatory effects and on probability of a definite diagnosis of RSD had no influence on the results. CONCLUSION: No association between immunologic indices and RSD was found. This finding is relevant, because recent theories stress that it is not the sympathetic nervous system but a local inflammatory reaction that is fundamental in the pathogenesis of RSD. The results of this study do not support this theory.

Reliability of five rapid D-dimer assays compared to ELISA in the exclusion of deep venous thrombosis.

Studies measuring the fibrin degradation product D-Dimer (DD) using enzyme-linked immunosorbent assays (ELISA) in patients with venographically proven deep venous thrombosis (DVT) suggest that it is possible to exclude DVT when DD level is below a certain cut-off level. However, ELISA methods are time-consuming and not available in all laboratories. Different rapid latex-agglutination assays have been investigated, but their sensitivity is considerably lower. In the present study we compared the value of four novel latex DD tests (Tinaquant, Minutex, Ortho and SimpliRed) and one rapid ELISA (VIDAS) to a classical ELISA DD assay (Organon Mab Y18) in 132 patients suspected of DVT. The VIDAS, a new quantitative automated ELISA, had a sensitivity of 100% and a negative predictive value of 100% for both proximal and distal DVT at a cut-off level of 500 ng/ml. The Tinaquant assay, a new quantitative latex method, had a sensitivity of 99% and a negative predictive value of 93% for both proximal and distal DVT at a cut-off level of 500 ng/ml. For proximal DVT only, both assays had a sensitivity and negative predictive value of 100%. VIDAS and Tinaquant correlated well with ELISA (correlation of r = 0.96 and r = 0.98 respectively). Sensitivities of the semi-quantitative latex assays Minutex, Ortho and SimpliRed were considerably lower (77%, 51% and 61% respectively). These results suggest that VIDAS and Tinaquant may be used instead of ELISA DD in the exclusion of DVT. Tinaquant can be performed within 20 min and VIDAS within 35 min. Both assays might be used as a routine screening test and should be evaluated in large clinical management studies.

The analysis of erythrocyte morphologic characteristics in urine using a hematologic flow cytometer and microscopic methods.

Three methods for the examination of erythrocyte morphology in urine are described: phase contrast microscopy, microscopy of cytocentrifuged and stained preparations, and erythrocyte analysis with the Technicon H1. Analysis with the H1 has not been described until now. All methods can be used to discriminate between dysmorphic and isomorphic erythrocytes. The red cell distribution width was the best H1 parameter for this discrimination. The authors have found a good correlation between the microscopic methods. The clinical impact of the three methods was studied with urine samples from patients with a confirmed diagnosis. The discrimination between renal and nonrenal hematuria is similar with phase contrast microscopy and cytocentrifuged preparations. The use of the H1 for this discrimination is not recommended.

Measurement of liver iron content in paraffin-embedded biopsies.

A reliable method for the determination of total liver iron in formalin-fixed, paraffin-embedded tissue is presented. The correlation with total liver iron in fresh tissue is good (r = 0.92). Material, which is processed routinely in the pathological anatomical department and stored in the archives, can be used for a quantitative iron determination for clinical or research purposes.

Accumulation of iron and iron compounds in liver tissue. A comparative study of the histological and chemical estimation of liver iron.

Unsatisfactory results obtained by histological evaluation of liver tissue in iron loading diseases prompted us to study the distribution of the total liver iron, haem iron and ferritin iron in post mortem human liver tissue from two different sites of the same liver. The total liver iron content was measured by flameless atomic absorption spectroscopy in native liver homogenates and in acid digested liver tissue from 60 consecutive autopsies, and the results from the two methods were compared. From the standard deviation of the duplicate analyses, it was deduced that the liver iron is possibly inhomogeneously distributed. The CVduplo (22%) of total iron, measured in acid digested tissue was higher than the CVduplo (14%) of total iron in homogenates from liver tissue from which non-homogenized tissue e.g. vessel walls, fibrotic tissue, had been removed. The CVduplo of ferritin iron and haem iron in liver homogenate was 14% and 30% respectively. The ferritin iron increased with an increasing total iron content until saturation of ferritin iron appeared to be reached at 2.5 micrograms ferritin iron per mg liver protein. When the results of total non-heam liver iron measurements are expressed properly (amount of iron per amount of homogenized liver protein), the distribution of iron is found to be homogeneous in both normal and pathological liver tissues. It was concluded that the estimation of liver iron content by visual microscopic evaluation is unsatisfactory, and that more reliable results are obtained by atomic absorption spectrophotometry.

Effect of a moderate fish intake on blood pressure, bleeding time, hematology, and clinical chemistry in healthy males.

This paper describes the outline and first results of an international study to investigate the effect of a reasonable amount of dietary fish on some aspects of cardiovascular risk. In Maastricht and Zeist, The Netherlands, and Tromsø, Norway, healthy male volunteers were given a dietary supplement consisting of 100 g/d of mackerel or meat for a 6-wk period. Compliance was monitored on the basis of the urinary excretion of lithium, which was added to the supplements. Average compliance was approximately 80% and this decreased slightly in time. Systolic blood pressure decreased in both groups to a comparable degree; consequently no specific effect of the fish supplement was observed. The fish supplement significantly prolonged bleeding times. Hematology was hardly affected but platelet counts decreased significantly. No indications were obtained for adverse effects of the fish supplement.

Use of a centrifugal analyzer for a chromogenic prothrombin time, a chromogenic activated partial thromboplastin time and a kinetic fibrinogen assay in a routine hospital laboratory.

A Cobas Bio centrifugal analyzer was used in a clinical laboratory for the performance of chromogenic clotting assays. Three commercially available photometric clotting tests--prothrombin time (PT), activated partial thromboplastin time (aPTT) and fibrinogen--were compared with the traditional clotting assays during 3 months. No great discrepancies were found between the traditional assays and the new photometric assays. The chromogenic PT could replace the traditional thrombotest, PT and Normotest, because it was sensitive and accurate over a broad range of clotting factor activity. Furthermore the chromogenic PT could be used to discriminate between a decreased clotting activity due to vitamin K deficiency or to a decreased protein synthesis by the liver. A decreased protein synthesis was confirmed by measuring a decrease in the serum cholinesterase activity. The chromogenic aPTT could be used for the assay of heparin concentrations in the therapeutic range and turned out to be more sensitive for deficiencies of factor VIII and factor IX than a traditional clotting aPTT. We conclude that the accuracy and practicability of clotting assays are improved by the new assays without diminishing the clinical value of the results.

Mechanism of regulation of calcium concentration by platelet membranes.

Calcium is recognised as an important messenger in the platelet activation. Both the external and internal membranes are considered to play an important role in the Ca2+ homeostasis of the cell. The aim of this review is to try to understand the mechanisms of regulation of the cytoplasmic free Ca2+ concentration by both kinds of membranes and mainly by internal membranes.

Simultaneous isolation of two platelet membrane fractions: biochemical, immunological and functional characterization.

Simultaneous isolation of two platelet membrane subfractions was achieved by centrifugation on 40% sucrose from a 100.000 g crude membrane fraction. Characterization of both types of membranes was carried out by different biochemical and immunological markers. Using a surface label, 3H Concanavalin A (3HCon A), a marker enzyme, phosphodiesterase, and lipid analysis, one of the fraction has been identified as external or plasma membranes, the other consists of intracellular membranes. Further two specific antibodies directed against external membrane antigens (LeKa and IgG L) react almost exclusively with the external membranes. Finally both kinds of membranes were able to uptake calcium but the affinity for this cation was higher for the internal than for the external membranes. This suggests that both membranes are implicated in the regulation of the cytoplasmic calcium concentration and that the internal membranes (dense tubular system) play the major part in this regulation.

The Ca2+ uptake and the hydrolysis of various nucleotide triphosphates by human platelet membranes.

Several nucleotide triphosphates (NTPs) were tested as energy source for the Ca2+ uptake by human platelet membrane vesicles. The Ca2+ uptake by these membranes was driven by ATP, GTP, ITP, UTP and CTP. The steady-state level of accumulated Ca2+ was equal with the different NTPs. The highest uptake velocity was found with ATP, but about 40-80% of the velocity with ATP could be accomplished with the other nucleotides. The highest affinity was also found with ATP (Km apparent = 15 microM). The liberation of Pi from the various NTPs was measured simultaneously with the Ca2+ uptake. The coupling ratio (moles of Ca2+ taken up/moles of Pi liberated) varied from 0.4 for ATP to 2.3 for UTP and was almost independent of the NTP concentration. The enzyme activity with ATP as substrate is strongly dependent on the Ca2+ concentration in contrast to the activity with GTP, ITP, UTP or CTP.

The phospholipid requirement of the (Ca2+ + Mg2+)-ATPase from human platelets.

The phospholipid requirement of the (Ca2+ + Mg2+)-ATPase present in a membrane fraction from human platelets was studied using various purified phospholipases. Only those phospholipases, which hydrolyse the negatively charged phospholipids, inhibited the (Ca2+ + Mg2+)-ATPase activity. The ATPase activity could be restored by adding mixed micelles of Triton X-100 and phosphatidylserine or phosphatidylinositol. Micelles with phosphatidic acid, phosphatidylcholine, phosphatidylethanolamine or sphingomyelin could not be used for reconstitution and inhibited the activity of the native enzyme.

Stimulation of the vitamin K-dependent carboxylase from bovine liver.

Vitamin K-dependent carboxylase from bovine liver is stimulated not only by reducing agents and bivalent metal ions (especially Mn2+), but also by several organic solvents (dimethyl sulphoxide, ketones and acetonitrile). The organic solvents stimulated both the carboxylation of glutamic acid residues and the formation of vitamin K epoxide. This stimulation by organic solvents was independent of the physical state of the phospholipid; it was highest at low temperatures and could only be demonstrated with vitamin K1 and not with 3-DTT-MK-O (the thioether adduct of menadione and dithiothreitol) or t-butyl hydroperoxide, which normally can substitute for vitamin K. We suggest that organic solvents exert their effect by changing the mobility of the isoprenoid side chain of vitamin K1 within the carboxylase complex.