A comprehensive ovine model of blood transfusion.

BACKGROUND: The growing awareness of transfusion-associated morbidity and mortality necessitates investigations into the underlying mechanisms. Small animals have been the dominant transfusion model but have associated limitations. This study aimed to develop a comprehensive large animal (ovine) model of transfusion encompassing: blood collection, processing and storage, compatibility testing right through to post-transfusion outcomes. MATERIALS AND METHODS: Two units of blood were collected from each of 12 adult male Merino sheep and processed into 24 ovine-packed red blood cell (PRBC) units. Baseline haematological parameters of ovine blood and PRBC cells were analysed. Biochemical changes in ovine PRBCs were characterized during the 42-day storage period. Immunological compatibility of the blood was confirmed with sera from potential recipient sheep, using a saline and albumin agglutination cross-match. Following confirmation of compatibility, each recipient sheep (n = 12) was transfused with two units of ovine PRBC. RESULTS: Procedures for collecting, processing, cross-matching and transfusing ovine blood were established. Although ovine red blood cells are smaller and higher in number, their mean cell haemoglobin concentration is similar to human red blood cells. Ovine PRBC showed improved storage properties in saline-adenine-glucose-mannitol (SAG-M) compared with previous human PRBC studies. Seventy-six compatibility tests were performed and 17·1% were incompatible. Only cross-match compatible ovine PRBC were transfused and no adverse reactions were observed. CONCLUSION: These findings demonstrate the utility of the ovine model for future blood transfusion studies and highlight the importance of compatibility testing in animal models involving homologous transfusions.

Stored blood transfusion induces transient pulmonary arterial hypertension without impairing coagulation in an ovine model of nontraumatic haemorrhage.

BACKGROUND AND OBJECTIVES: Transfusion of blood products in particular older products is associated with patient morbidity. Previously, we demonstrated a higher incidence of acute lung injury in lipopolysaccharide-treated sheep transfused with stored blood products. As transfusion following haemorrhage is more common, we aimed to determine whether a 'first hit' of isolated haemorrhage would precipitate similar detrimental effects following transfusion and also disrupt haemostasis. MATERIALS AND METHODS: Anaesthetized sheep had 33% of their total blood volume collected into Leukotrap bags (Pall Medical), which were processed into packed red blood cells and cross-matched for transfusion into other sheep. After 30 mins, the sheep were resuscitated with either: fresh (<5 days old) or stored (35-42 days old) ovine blood followed by 4% albumin to replacement volume, albumin alone or normal saline alone and monitored for 4 h. RESULTS: The first hit of haemorrhage precipitated substantial decreases in mean arterial pressure however haemostasis was preserved. Transfusion of stored ovine blood induced (1) transient pulmonary arterial hypertension but no oedema and (2) reduced fibrinogen levels more than fresh blood, but neither induced coagulopathy. Thus, transfusion of stored blood affected pulmonary function even in the absence of overt organ injury. CONCLUSION: The fact that stored blood transfusions: (1) did not induce acute lung injury in contrast to previous lipopolysaccharide-primed animal models identifies the 'first hit' as an important determinant of the severity of transfusion-mediated injury; (2) impaired pulmonary dynamics verifies the sensitivity and vulnerability of the pulmonary system to injury.

Whole blood coagulation and platelet activation in the athlete: a comparison of marathon, triathlon and long distance cycling.

INTRODUCTION: Serious thrombembolic events occur in otherwise healthy marathon athletes during competition. We tested the hypothesis that during heavy endurance sports coagulation and platelets are activated depending on the type of endurance sport with respect to its running fraction. MATERIALS AND METHODS: 68 healthy athletes participating in marathon (MAR, running 42 km, n = 24), triathlon (TRI, swimming 2.5 km + cycling 90 km + running 21 km, n = 22), and long distance cycling (CYC, 151 km, n = 22) were included in the study. Blood samples were taken before and immediately after completion of competition to perform rotational thrombelastometry. We assessed coagulation time (CT), maximum clot firmness (MCF) after intrinsically activation and fibrin polymerization (FIBTEM). Furthermore, platelet aggregation was tested after activation with ADP and thrombin activating peptide 6 (TRAP) by using multiple platelet function analyzer. RESULTS: Complete data sets were obtained in 58 athletes (MAR: n = 20, TRI: n = 19, CYC: n = 19). CT significantly decreased in all groups (MAR -9.9%, TRI -8.3%, CYC -7.4%) without differences between groups. In parallel, MCF (MAR +7.4%, TRI +6.1%, CYC +8.3%) and fibrin polymerization (MAR +14.7%, TRI +6.1%, CYC +8.3%) were significantly increased in all groups. However, platelets were only activated during MAR and TRI as indicated by increased AUC during TRAP-activation (MAR +15.8%) and increased AUC during ADP-activation in MAR (+50.3%) and TRI (+57.5%). DISCUSSION: While coagulation is activated during physical activity irrespective of type we observed significant platelet activation only during marathon and to a lesser extent during triathlon. We speculate that prolonged running may increase platelet activity, possibly, due to mechanical alteration. Thus, particularly prolonged running may increase the risk of thrombembolic incidents in running athletes.

Potential of mid-infrared spectroscopy to aid the triage of patients with acute chest pain.

A total of 1,429 serum samples from 389 consecutive patients with acute chest pain were analyzed with the goal to aid the rapid diagnosis of acute myocardial infarction. To the best of our knowledge this is the largest and most comprehensive study on mid-infrared spectroscopy in cardiology. We were able to identify those signatures in the mid-infrared spectra of the samples, which were specific to either acute myocardial infarction or chest pain of other origin (angina pectoris, oesophagitis, etc). These characteristic spectral differences were used to distinguish between the cause of the donor's acute chest pain using robust linear discriminant analysis. A sensitivity of 88.5% and a specificity of 85.1% were achieved in a blind validation. The area under the receiver operating characteristics curve amounts to 0.921, which is comparable to the performance of routine cardiac laboratory markers within the same study population. The biochemical interpretation of the spectral signatures points towards an important role of carbohydrates and potentially glycation. Our studies indicate that the "Diagnostic Pattern Recognition (DPR)" method presented here has the potential to aid the diagnostic procedure as early as within the first 6 hours after the onset of chest pain.

Evaluation of caffeine as a test drug for CYP1A2, NAT2 and CYP2E1 phenotyping in man by in vivo versus in vitro correlations.

Caffeine is used to phenotype subjects in vivo for the cytochrome P450 isoforms CYP1A2 and CYP2E1, and for N-acetyltransferase type 2 (NAT2). However, how much of the variation in phenotyping parameters may be attributed to variations in CYP1A2 and CYP2E1 activities has not been determined. Therefore, this study intraindividually compared enzyme activities and/or content in liver samples with pharmacokinetic parameters of caffeine in vivo after administration of a test dose in 25 patients undergoing hepatectomy. Parameters measured in vitro were the high affinity components of caffeine 3-demethylation and phenacetin 0-deethylation, microsomal CYP1A2 and CYP2E1 immunoreactivity, and cytosolic sulfamethazine N-acetylation. Caffeine parameters in vivo included caffeine clearance from plasma and/or saliva, paraxanthine/caffeine ratios in plasma and saliva, plasma theophylline/caffeine ratio, and several metabolite ratios from spot urine sampled 6 h postdose. Correlations between parameters were determined using weighted linear regression analyses. Caffeine clearance and paraxanthine/caffeine ratios correlated most highly to intrinsic clearance of caffeine 3-demethylation and to CYP1A2 immunoreactivity (r= 0.584-0.82), whereas urinary CYP1A2 ratios correlated less strongly with CYP1A2 parameters in vitro. Assignment of acetylator phenotype by urinary NAT2 ratios was concordant with sulfamethazine-N-acetylation in vitro. In contrast to CYP1A2 parameters in vitro, CYP2E1 immunoreactivity was not related to the theophylline/caffeine plasma ratio. CYP1A2 activity, thus, is the major determinant of caffeine clearance and the paraxanthine/caffeine ratios in vivo, of which the saliva ratio 6 h postdose appears as the most advantageous parameter. The results confirm that phenotyping using caffeine provides valid estimates of CYP1A2 and NAT2 activity.

Absorption differences of ciprofloxacin along the human gastrointestinal tract determined using a remote-control drug delivery device (HF-capsule).

The single-dose absorption kinetics of ciprofloxacin in different regions of the human gastrointestinal tract were investigated using a remote-control drug delivery device (HF-capsule). Doses of 180 to 200 mg ciprofloxacin (as a lactic acid solution) were placed in the HF-capsule and administered to four healthy male adults. The position of the HF-capsule in the gastrointestinal tract was checked via radiographic examination. The release of the solution from the HF-capsule was induced by a radio signal. In each volunteer, the solution was released into five different regions of the gastrointestinal tract: the stomach (B), jejunum (C1), ileum (C2), ascending colon (D1), and descending colon (D2). Two control runs (A1, A2), involving oral administration of the solution, were used as a reference for calculation of area under the curve. The oral administration of a conventional 250-mg tablet (A3) was also studied. The plasma concentration of ciprofloxacin and urine concentrations of ciprofloxacin, desethylene- (M1), sulfo- (M2), and oxociprofloxacin (M3) were determined fluorimetrically by high-performance liquid chromatography. Intraindividual comparisons indicated a progressive decrease in the amount of ciprofloxacin absorbed (100 percent = mean of AUCA1 and AUCA2) from the jejunum (-61 percent, median), ileum (-75 percent), colon ascendens (-90 percent), and colon descendens (-95 percent). Absolute amounts of renally excreted ciprofloxacin and metabolites decreased due to the reduced absorption of ciprofloxacin, but the metabolite pattern was unchanged. It is concluded that the main absorption site for ciprofloxacin is the upper part of the intestinal tract (duodenum, jejunum).

Ciprofloxacin-caffeine: a drug interaction established using in vivo and in vitro investigations.

The inhibitory effects of ciprofloxacin and other quinolone derivatives on the hepatic cytochrome P450-dependent metabolism of caffeine have been investigated in humans. In vivo studies involved an intraindividual comparison of the single-dose kinetics of caffeine before and during quinolone administration in 12 healthy men. Changes of enzymatic caffeine degradation by the quinolones were studied in vitro using human liver microsomes from three donors. Enoxacin and pipemidic acid markedly prolonged caffeine elimination in vivo. A positive correlation exists between the doses of enoxacin or ciprofloxacin and the prolongation (increases) in the caffeine elimination half-life. Decreases in caffeine elimination, using doses of ciprofloxacin in the upper part of the recommended dose range, were approximately 1.5-fold in comparison with untreated control subjects, whereas in the case of enoxacin there was a sixfold change. In vitro results with enoxacin, ofloxacin, ciprofloxacin, and pipemidic acid show a competitive inhibition (Dixon plots) of caffeine 3-demethylation. Ciprofloxacin and enoxacin showed the strongest inhibitory effects in vitro, whereas ofloxacin had the lowest inhibitory effect. These results are qualitatively reflected in the in vivo results; however, the clinical effects may be dependent on pharmacokinetic disposition of the quinolone and this could explain the weak inhibitory action of ciprofloxacin in vivo.

Interaction between quinolones and caffeine.

The effects of multiple doses of ofloxacin 200 mg, ciprofloxacin 250 mg or enoxacin 400 mg (all twice daily) on the pharmacokinetic properties of single doses of caffeine (220 to 230 mg) were investigated in 12 healthy volunteers. Intraindividual comparisons showed that ciprofloxacin and enoxacin significantly inhibited the elimination of caffeine. Ofloxacin, however, did not affect any of the measured pharmacokinetic properties of caffeine. Thus, caffeine should be avoided in patients with liver disorders, cardiac arrhythmias, latent epilepsy or in intensive care while undergoing treatment with enoxacin or ciprofloxacin.

Biotransformation of caffeine and theophylline in mammalian cell lines genetically engineered for expression of single cytochrome P450 isoforms.

Primary steps in the metabolism of caffeine and theophylline are cleavage of methyl groups and/or hydroxylation at position 8, mediated by cytochromes P450. V79 Chinese hamster cells genetically engineered for stable expression of single forms of rat cytochromes P450IA1, P450IA2 and P450IIBI and human P450IA2 and rat liver epithelial cells expressing murine P450IA2 were used to overcome problems arising in the proper allocation of metabolic pathways to specific isoforms by conventional techniques. These cell lines were exposed to caffeine and/or theophylline, and concentrations of metabolites formed in the medium were determined by HPLC. Caffeine was metabolized by human, rat and murine P450IA2, resulting in the formation of four primary demethylated and hydroxylated metabolites. However, there were differences in the relative amounts of the metabolites. The human and the mouse P450IA2 isoforms predominantly mediated 3-demethylation of caffeine. The rat cytochrome P450IA2 mediated both 3-demethylation and 1-demethylation of caffeine to a similar extent. Theophylline was metabolized mainly via 8-hydroxylation. All cell lines tested were able to carry out this reaction, with highest activities in cell lines expressing rat or human P450IA2, or rat P450IA1. These results support the hypothesis that caffeine plasma clearance is a specific in vivo probe for determining human P450IA2 activity.

Disease pattern recognition testing for rheumatoid arthritis using infrared spectra of human serum.

BACKGROUND: In view of the importance of the diagnosis of rheumatoid arthritis, a novel diagnostic method based on spectroscopic pattern recognition in combination with laboratory parameters such as the rheumatoid factor is described in the paper. Results of a diagnostic study of rheumatoid arthritis employing this method are presented. METHOD: The method uses classification of infrared (IR) spectra of serum samples by means of discriminant analysis. The spectroscopic pattern yielding the highest discriminatory power is found through a complex optimization procedure. In the study, IR spectra of 384 serum samples have been analyzed in this fashion with the objective of differentiating between rheumatoid arthritis and healthy subjects. In addition, the method integrates results from the classification with levels of the rheumatoid factor in the sample by optimized classifier weighting, in order to enhance classification accuracy, i.e. sensitivity and specificity. RESULTS: In independent validation, sensitivity and specificity of 84% and 88%, respectively, have been obtained purely on the basis of spectra classification employing a classifier designed specifically to provide robustness. Sensitivity and specificity are improved by 1% and 6%, respectively, upon inclusion of rheumatoid factor levels. Results for less robust methods are also presented and compared to the above numbers. CONCLUSION: The discrimination between RA and healthy by means of the pattern recognition approach presented here is feasible for IR spectra of serum samples. The method is sufficiently robust to be used in a clinical setting. A particular advantage of the method is its potential use in RA diagnosis at early stages of the disease.

Biotransformation of methylxanthines in mammalian cell lines genetically engineered for expression of single cytochrome P450 isoforms. Allocation of metabolic pathways to isoforms and inhibitory effects of quinolones.

V79 Chinese hamster cells genetically engineered for stable expression of single forms of rat cytochromes P450IA1, P450IA2, P450IIB1, human P450IA2, and rat liver epithelial cells expressing murine P450IA2 were used to allocate metabolic pathways of methylxanthines to specific isoforms and to test the suitability of such cell lines for investigations on drug interactions occurring at the cytochrome expressed. The cell lines were exposed to caffeine and/or theophylline and concentrations of metabolites formed in the medium were determined by HPLC. Caffeine was metabolized by human, rat and murine P450IA2, resulting in the formation of four primary demethylated and hydroxylated metabolites. However, there were differences in the relative amounts of the metabolites. The human and the mouse P450IA2 isoforms predominantly mediated 3-demethylation of caffeine. The rat cytochrome P450IA2 mediated both 3-demethylation and 1-demethylation of caffeine to a similar extent. The results support the hypothesis that caffeine plasma clearance is a specific in vivo probe for determining human P450IA2 activity. Addition of the quinolone antibiotic agents pipemidic acid or pefloxacin, both known to inhibit caffeine metabolism in vivo and in human liver microsomes, reduced formation rates of all metabolites of caffeine in cells expressing rat and human P450IA2. Theophylline was mainly metabolized via 8-hydroxylation. All cell lines tested were able to carry out this reaction, with highest activities in cell lines expressing rat or human P450IA2, or rat P450IA1.

Variation of CYP1A2-dependent caffeine metabolism during menstrual cycle in healthy women.

BACKGROUND AND OBJECTIVES: The activity of the human cytochrome P450 CYP1A2 is decreased by female sex hormones during pregnancy or treatment with oral contraceptives. However, the influence of menstrual cycle on CYP 1A2 activity is not clear. METHODS: CYP1A2 activity was monitored in 15 women (13 with confirmed ovulatory cycles, 2 smokers, age (mean +/- SD) 27.8 +/- 3.8 years, body mass index 23.8 +/- 3.8 kg x m-2) using the specific substrate caffeine (mean doses 149 mg). After a run-in period started one week prior to expected onset of menses, daily saliva samples were taken 7.3 +/- 0.7 hours after caffeine intake throughout the cycle, and caffeine clearance was estimated from the paraxanthine to caffeine ratio therein. Ovulation was confirmed by progesterone serum concentration above 3 ng/ml in the second half of the cycle. RESULTS: Initial (day 2) caffeine clearance (n = 15, geometric mean) was 1.37 ml/min/kg body weight (coefficient of variation (CV) 48%). The ratio of caffeine clearance for the luteal (day -9 to -4 prior to onset of the next menses) to the follicular phase (days 5-10) was (n = 13, point estimate) 1.03 (90% CI 0.95-1.12), indicating that there was no difference in CYP1A2 activity between these cycle phases. The median intraindividual CV in ovulatory cycles (n = 13) was 23% (range 11% to 39%). As an additional finding, there was evidence for long-term fluctuations of CYP1A2 activity in most individuals. CONCLUSIONS: A dose adaptation according to the phase of menstrual cycle based on pharmacokinetics is not required for CYP1A2 substrates.

Lacking effect of grapefruit juice on theophylline pharmacokinetics.

Grapefruit juice inhibits the biotransformation of several drugs, including caffeine (23% clearance reduction), which is metabolized by the cytochrome P450 isoform CYP1A2. Since CYP1A2 also participates in theophylline biotransformation, a randomized change-over study on a possible interaction between grapefruit juice and theophylline was conducted. Twelve healthy young male nonsmokers were included (median 26 (range 23-30) years, weight 73 (65-85) kg). Theophylline was given as a single dose of 200 mg in solution (Euphyllin 200), diluted by 100 ml of either water or grapefruit juice (751 mg/l naringin). Subsequently, additional fractionated 0.91 of water or juice were administered until 16 hours postdose. Theophylline concentrations in plasma withdrawn up to 24 hours postdose were measured by HPLC, and its pharmacokinetics were estimated using compartment model independent methods. To compare between the 2 treatments, ANOVA based point estimates and 90% confidence intervals (given in parentheses) were calculated for the test (= grapefruit coadministration) to reference (= water coadministration) ratios (Tmax: differences). These were: Cmax 0.90 (0.81-1.00), AUC 1.02 (0.95-1.11), Cmax/AUC 0.88 (0.81-0.95), T 1/2el 1.03 (0.98-1.09), Tmax 0.15 h (-0.11h-0.41 h). Thus, no pharmacokinetic interaction between grapefruit juice and theophylline was observed. This finding is in contrast to the effect of grapefruit juice reported on caffeine metabolism and may be due to the contribution of enzymes other than CYP1A2 to primary theophylline metabolism or to differences in naringin and/or naringenin kinetics between studies.

Grapefruit juice increases oral nimodipine bioavailability.

The bioavailability of dihydropyridine calcium channel blockers following oral administration was shown to be increased by concomitant intake of grapefruit juice for all drugs of this class tested up to now. Here we report a randomized crossover interaction study on the effects of grapefruit juice on the pharmacokinetics of nimodipine and its metabolites. Eight healthy young men (4 smokers/4 nonsmokers) were included. Nimodipine was given as a single 30 mg tablet (Nimotop) with either 250 ml of water or 250 ml of grapefruit juice (751 mg naringin/l). Drug concentrations in plasma withdrawn up to 24 hours postdose were measured by GC-ECD, and model-independent pharmacokinetic parameters were estimated. The study was handled as an equivalence problem. Point estimators and ANOVA based 90% confidence intervals (CI) were calculated for the test (= grapefruit juice period) to reference (= water period) ratios using dose-normalized concentrations. The absence of a relevant interaction was assumed if the CIs were within the 0.67-1.50 range. Cmax for nimodipine reached 124% of the reference period (90% CI 0.76-2.01), AUC was increased to 151% (90% CI 114%-200%), respectively. The null hypothesis "relevant interaction" thus could not be rejected for the primary pharmacokinetic parameters AUC and Cmax. The ratios of metabolite AUC to parent drug AUC were slightly reduced with grapefruit juice intake. Additionally, there was evidence for a more pronounced hemodynamic response in the grapefruit juice period. To avoid the interaction, nimodipine should not be taken with grapefruit juice.

[Pharmacokinetics of a theophylline effervescent tablet]. / Pharmakokinetik einer Theophyllin Brausetablette.

The theophylline effervescent tablet (Euphyllin quick 200) provides a new acute medication for the treatment of pulmonary obstructive lung diseases. Its pharmacokinetics were determined in 12 healthy male volunteers and compared in a single-dose crossover study to those after administration of an oral solution (Euphyllin 200 ampoule). Based on the primary pharmacokinetic characteristics for rate and extent of absorption, Cmax and AUC, both formulations were bioequivalent.