Assessment of platelet-derived thrombogenicity with the total thrombus-formation analysis system in coronary artery disease patients receiving antiplatelet therapy.

BACKGROUND: Accurate evaluation of thrombogenicity helps to prevent thrombosis and excessive bleeding. The total thrombus-formation analysis system (T-TAS) was developed for quantitative analysis of platelet thrombus formation by the use of microchips with thrombogenic surfaces (collagen, platelet chip [PL-chip]; collagen plus tissue factor, atherome chip [AR-chip]). We examined the utility of the T-TAS in the assessment of the efficacy of antiplatelet therapy in patients with coronary artery disease (CAD). METHODS AND RESULTS: In this cross-sectional study, 372 consecutive patients admitted to the cardiovascular department were divided into three groups: patients not receiving any antiplatelet therapy (control, n = 56), patients receiving aspirin only (n = 69), and patients receiving aspirin and clopidogrel (n = 149). Blood samples were used for the T-TAS to measure the platelet thrombus-formation area under the curve (AUC) at various shear rates (1500 s(-1) [PL18 -AUC10 ] and 2000 s(-1) [PL24 -AUC10 ] for the PL-chip; 300 s(-1) [AR10 -AUC30 ] for the AR-chip). The on-clopidogrel platelet aggregation was measured by the use of P2Y12 reaction units (PRUs) with the VerifyNow system. The mean PL24 -AUC10 levels were 358 ± 111 (± standard deviation) (95% confidence interval [CI] 328.9-387.1) in the control group, 256 ± 108 (95% CI 230.5-281.5) in the aspirin group, and 113 ± 91 (95% CI 98.4-127.6) in the aspirin/clopidogrel group. In the aspirin/clopidogrel group, the PL24 -AUC10 was higher in poor metabolizers (PMs) with cytochrome P450 2C19(CYP2C19) polymorphisms (152 ± 112, 95% CI 103.4-200.6) than in the non-PM group (87 ± 74, 95% CI 73.8-100.2). CONCLUSIONS: Our findings suggest that the PL24 -AUC10 level measured by the T-TAS is a potentially suitable index for the assessment of antiplatelet therapy in CAD patients.

Liquid chromatographic-atmospheric pressure chemical ionization mass spectrometric analysis of opiates and metabolites in rat urine after inhalation of opium.

To examine the urinary excretion of opiates and their metabolites following inhalation exposure of rats to opium, analytical procedures for the simultaneous determination of the compounds in opium, the vapor derived by the volatilization of opium and the urine of rats exposed to the opium vapor were developed using liquid chromatography-atmospheric pressure chemical ionization mass spectrometry (LC-APCI-MS). Seven compounds were determined in the opium, namely morphine, codeine, thebaine, noscapine, papaverine, meconic acid and meconin. All seven were extracted with 2.5% acetic acid solution and subjected to LC-APCI-MS analysis. The separation was performed on an ODS column in acetonitrile-50 mM ammonium formate buffer (pH 3.0) using a linear gradient program and quantitative analysis was carried out in the selected ion monitoring mode ([M+H](+)). For the analysis of the volatilization of opium, the opium (1 g) was added to a glass pipe, which was then heated at 300 degrees C for 20 min. Negative pressure (air flow-rate; 300 ml/min) was used to draw the vapor through a series of glass wool and methanol traps. The total amount of each compound in the vapor was estimated by measurement of the compounds trapped in the glass wool and methanol. Wister rats (n=3) were exposed to the vapor derived from the volatilization system and the urinary amounts (0-72 h) of the six opiates and metabolites including morphine-3-grucronide (M3G) and morphine-6-grucronide (M6G) were measured after solid-phase extraction. The calibration curves for those compounds in the rat urine were linear over the concentration range 10-500 ng/ml. The recoveries for each analyte from the rat urine sample spiked with standard solution were generally greater than 80%, and the relative standard deviation for the analytical procedure was less than 8% with the exception of meconin. After inhalation exposure of rats to opium, M3G (5.45-14.38 micro g), morphine (2.27-4.65 micro g), meconin (0.54-1.85 micro g), codeine (0.54-1.85 micro g), noscapine (0.34-0.40 micro g) and papaverine (0.01-0.04 micro g) were detected in the urine over 72 h. However, only trace levels of thebaine were observed despite it being one of the major alkaloids found in the opium. On the other hand, a relatively large amount of meconin was detected in the vapor and the urine as compared with the opium. It is suggested that the presence of meconin in biological fluids could be indicative of opium ingestion by inhalation.

The study of the applicability of content uniformity and weight variation test--the state of commercial tablets and capsules in Japan.

This study intends to determine the rational criteria (e.g., threshold value) for applying the weight variation test and to investigate the adequacy of the acceptance value for existing commercial products in Japan. The studied products were 489 lots (3 lots x 163 products) of compressed tablets (plain, film-coated, sugar-coated) and 42 lots (3 lots x 14 products) of hard capsules marketed in Japan. The individual drug content and the weight of 10 units in a lot were determined for each product and the acceptance values were calculated according to the Japanese Pharmacopoeia thirteenth edition (JP13) Content Uniformity Test (M=100.0, k=2.2). Product-specific intra-lot relative standard deviation of content (RSDD), weight (RSDW) and concentration (RSDC) were calculated by analysis of variance (ANOVA) using three lots of data per product. The RSDD and RSDC tended to increase with the decrease of the label strength for plain tablets, but not for film-coated and sugar-coated tablets, and hard capsules. A good correlation was found between RSDD and RSDC but not between RSDD and RSDW. These findings indicate that 1) it is difficult to rationally set the threshold level for weight variation, especially regarding the dosage forms except for plain tablets, 2) the application of weight variation tests should, in principle, be decided on the mixing homogeneity that is RSDC. 3) Most (99.6%) of the tablets and all the capsules investigated met the requirement of content uniformity test of JP13. Therefore the criteria of the JP13 content uniformity test are considered acceptable from the viewpoint of manufacturing capability.

Assessment of gastric acidity of Japanese subjects over the last 15 years.

The gastric acidity of young to elderly Japanese subjects from 1989 to 1999 was assessed and compared with that obtained in 1984, using GA-Test capsules containing acid-dissolving granules of riboflavin. The percentage of achlorhydric subjects increased with age as observed before, however, an over all decrease in all age categories year by year was noted. The percentage of achlorhydric subjects aged 50 years in 1995-1999 was about 40%, which was lower than that (60%) in 1984. However, such a chronological change was not observed when the percentage of achlorhydric subjects was determined according to birth year, indicating that it is related to the birth year of subjects. The percentage of achlorhydric subjects correlated with infection by Helicobacter pylori. Considering the high percentage of achlorhydric elderly, bioavailability and bioequivalence studies should be performed taking into consideration the effects of gastric acidity on the in vivo performance of drug products.

Urinary schistosomiasis in southern Ghana: 1. Prevalence and morbidity assessment in three (defined) rural areas drained by the Densu river.

Epidemiological studies on urinary schistosomiasis were carried out in eight villages in the Ga and Akuapem South districts in Ghana. Single urine samples were collected from individuals aged 5 years and above between 10.00 and 14.00 h. The samples were examined for the presence of Schistosoma haematobium eggs using a filtration technique. Indirect morbidity was determined as the presence of microhaematuria and proteinuria using reagent strips, and macrohaematuria was recorded with the naked eye. Out of the study population of 3912 subjects, 2562 (65.5%) submitted urine samples. The prevalence of a Schistosoma haematobium infection ranged between 54.8 and 60.0%. Infection rates increased by age with a peak in the 10-19 years category, and decreased with increasing age. Disease prevalence was higher in males aged 15 years and above in Areas 2 (Ntoaso and Sansami Amanfro) and 3 (Dom Faase, Papase, Chento and Gidi Kope), whereas it was higher among males aged 10 years and above in Area 1 (Ayikai Doblo and Akramaman). The intensity of infection was highest among children aged 10-14 years in most of the villages. More than half of egg-positive children in this age group had a heavy infection (100 eggs and above in 10 ml of urine). Although both egg-positive and egg-negative individuals manifested variable degrees of macro- or micro-haematuria, microhaematuria was more prevalent among egg-positives (chi(2)=918.5, d.f.=1, P<0.01). The degree of microhaematuria and proteinuria were significantly associated with the intensity of the infection. These results indicate a high transmission of disease in the study area.

Activation of the right inferior frontal cortex during assessment of facial emotion.

We measured regional cerebral blood flow (rCBF) using positron emission tomography (PET) to determine which brain regions are involved in the assessment of facial emotion. We asked right-handed normal subjects to assess the signalers' emotional state based on facial gestures and to assess the facial attractiveness, as well as to discriminate the background color of the facial stimuli, and compared the activity produced by each condition. The right inferior frontal cortex showed significant activation during the assessment of facial emotion in comparison with the other two tests. The activated area was located within a triangular area of the inferior frontal cortex in the right cerebral hemisphere. These results, together with those of previous imaging and clinical studies, suggest that the right inferior frontal cortex processes emotional communicative signals that could be visual or auditory and that there is a hemispheric asymmetry in the inferior frontal cortex in relation to the processing of emotional communicative signals.

Neuroanatomical correlates of the assessment of facial attractiveness.

Frontal cortical damage can lead to changes in affective aspects of personality. However, the difficulty of dissociating such abnormalities from cognitive disorders has overshadowed most previous findings. Regional cerebral blood flow (rCBF) was measured with positron emission tomography (PET) while normal subjects were assessing facial attractiveness. Two left frontal regions showed a significant increase in rCBF while assessing facial attractiveness. The increased rCBF in the left anterior frontal cortex correlated with the overall percentage of assessments of a face as unattractive, while that in the left fronto-temporal junction correlated with the percentage of assessments of a face as attractive. These findings provide direct evidence that the left frontal regions are engaged in the assessment of facial attractiveness.

[Assessment of stability variation among batches, packaging, and formulations--application of matrixing and bracketing].

Assessment of shelf-life equivalence among batches, packaging or formulations of pharmaceutical products, which was based on the range of shelf-life estimates, was proposed as an alternative method to an analysis of variance (ANOVA). The power of this analysis was not significantly affected by assay error, whereas that of ANOVA decreased markedly as assay error increased. Thus, ANOVA exhibites a tendency to overlook the stability variation from stability data of a larger assay error, but this is not the case for the proposed method.

Neoglycoproteins with the synthetic complex biantennary nonasaccharide or its alpha 2,3/alpha 2,6-sialylated derivatives: their preparation, assessment of their ligand properties for purified lectins, for tumor cells in vitro, and in tissue sections, and their biodistribution in tumor-bearing mice.

Neoglycoproteins were prepared with chemoenzymatically synthesized complex biantennary N-glycan derivatives the nonreducing ends of which bear typical sequences found in glycoproteins. A chemically obtained biantennary heptasaccharide-azide was reduced and acylated with a 6-aminohexanoyl spacer. Elongation of the deprotected heptasaccharide using glycosyltransferases yielded a biantennary nonasaccharide with terminal galactose residues and two undecasaccharides terminating with alpha 2,6- or alpha 2,3-linked sialic acid. The free amino group of the spacer of these oligosaccharides was converted into an isothiocyanate. Its subsequent coupling to bovine serum albumin gave neoglycoproteins with a yield of 2.4-3.6 glycan chains per carrier molecule. This versatile synthetic pathway allows employment of a wide variety of complex-type glycans, which can be introduced to various test systems in vitro and in vivo to evaluate potential biomedical applications. Solid-phase assays with biotinylated sugar receptors revealed discriminatory binding properties of the three neoglycoproteins, especially for the mistletoe lectin. This direct assay system is preferable to the measurement of inhibitory capacities with respect to model ligands. Ligand type- and cell type-dependent quantitative differences in the binding properties of the probes were detected by FACScan analyses with a panel of tumor cell lines and by monitoring of staining in tissue sections for small cell and non-small-cell lung cancer and mesotheliomas. Biodistribution of iodinated neoglycoproteins in mice gave a prolonged presence of the sialylated probes in serum. Relative to the nonasaccharide, the uptake, especially of the iodinated neoglycoprotein with alpha 2,3-sialylated ligand chains, was clearly elevated in mice for kidneys and Ehrlich tumors. On the basis of the documented feasibility of these applications, it is concluded that the further elaboration of glycan chain variants by the described synthetic approach in combination with the given test panel is warranted to evaluate the potential of complex glycan chain-carrying neoglycoproteins for diagnostic and therapeutic purposes.

Assessment of the severity of coronary artery disease by thallium-201 washout rate after dipyridamole infusion--a coronary hemodynamic and metabolic study.

This study examined the relationship between the myocardial washout rate (WR) of thallium-201 (201Tl) in dipyridamole scintigraphy and both coronary flow reserve (CFR) and myocardial lactate extraction rate (LER) after dipyridamole infusion in 31 patients with coronary artery disease (CAD) without myocardial infarction and 16 control patients. Patients with CAD demonstrated significantly lower WR (21 +/- 17 vs 43 +/- 10%, p < 0.001), lower CFR (128 +/- 82 vs 242 +/- 89%, p < 0.001) and lower LER (-2 +/- 20 vs 10 +/- 10%, p < 0.05) than did the control patients. WR was significantly correlated with CFR (r = 0.50, p < 0.001) and LER (r = 0.41, p < 0.01) in all of the patients. CAD patients with dipyridamole-induced chest pain demonstrated significantly lower WR (14 +/- 20 vs 27 +/- 12%, p < 0.05), lower CFR (97 +/- 71 vs 162 +/- 82%, p < 0.05) and lower LER (-13 +/- 21 vs 11 +/- 9%, p < 0.001) than did CAD patients without chest pain. CAD patients with dipyridamole-induced ST depression demonstrated significantly lower WR (14 +/- 20 vs 29 +/- 8%, p < 0.05), lower CFR (105 +/- 79 vs 170 +/- 73%, p < 0.05) and lower LER (-8 +/- 21 vs 11 +/- 10%, p < 0.01) than did CAD patients without ST depression. These results suggest that the myocardial washout rate of 201Tl after dipyridamole infusion reflects the severity of coronary artery disease as assessed by coronary hemodynamics, myocardial metabolism, symptoms and electrocardiography.

Minimal residual disease status in pre-B acute lymphoblastic leukemia patients after chemotherapy and bone marrow transplantation: assessment of the anti-leukemic effects of chemotherapy and BMT.

We prospectively analyzed minimal residual disease (MRD) in four patients with B-cell precursor acute lymphoblastic leukemia who had been in complete remission for more than one year after chemotherapy and allogenic or autologous bone marrow transplantation (BMT). MRD was quantitatively estimated using polymerase chain reaction amplification to detect the complementarity-determining region III of the immunoglobulin heavy chain gene at limiting dilution DNA samples. Our study showed that remission induction chemotherapy reduced at most 2-logs of leukemia cells, and that subsequent consolidation chemotherapy induced further reduction of leukemia cells. In two cases, 10(-5) levels of MRD were detected two months after BMT. However, no MRD was detected four months after BMT. We also showed the effectiveness of ex vivo purging with anti-CALLA monoclonal antibodies which eliminated at least 2-logs of leukemia cells in autologous BMT. Our results suggest that this detection system is useful for assessing the reduction of the original leukemia clone, and that the presence of MRD within three months after BMT is not related to clinical outcome.

Determination of anaerobic threshold for assessment of functional state in patients with chronic heart failure.

The use of anaerobic threshold in assessment of aerobic capacity was evaluated in 34 normal subjects and 47 patients with various kinds of chronic heart disease. Anaerobic threshold was determined as the oxygen consumption (VO2) at which a linear relationship between pulmonary ventilation (VE) and VO2 was lost during progressive treadmill exercise. Anaerobic threshold determined in this manner was validated with that determined by blood lactate measurements in eight normal subjects and nine cardiac patients (r = .962, p less than .001). Thereafter, anaerobic threshold was determined only by respiratory measurements. In symptom-limited, maximal exercise, anaerobic threshold was reached well before maximal effort and corresponded to 70% of maximal VO2 both in normal subjects and cardiac patients. Anaerobic threshold decreased as age progressed in normal subjects (r = - .70, p less than .001). Anaerobic threshold in cardiac patients was lower than that in the normal subjects and decreased progressively as New York Heart Association functional classification advanced (normal, 32.95 +/- 6.17 ml/min/kg; class I, 22.78 +/- 3.74; class II, 16.99 +/- 3.66; class III, 12.97 +/- 2.76; p less than .01 between each group other than between class II and class III). Anaerobic threshold in cardiac patients correlated poorly with other objective indices, e.g., cardiomegaly (r = -.54, p less than .001) and rise in pulmonary wedge pressure (r = -.64, p less than .001). At anaerobic threshold, cardiac patients subjectively graded the work load as light (13%), light-to-moderate (27%), moderate (30%), and moderate-to-heavy (28%). Thus determination of anaerobic threshold by respiratory measurements is a safe, accurate, and objective method to measure aerobic capacity in cardiac patients and in normal subjects.