There is evidence that reactive oxygen species (ROSs) are generated in extracorporeal circuits. Free radical scavenging enzymes (FRSEs) such as glutathione reductase (GSSG-R), glutathione peroxidase (GSH-Px), and superoxide dismutase (SOD) protect against the damaging effect of ROSs. The influence of extracorporeal treatment on FRSE activity was investigated in the plasma and red blood cells (RBCs) of 21 patients undergoing regular low-density lipoprotein (LDL) apheresis. The FRSEs GSSG-R, GSH-Px, and SOD were measured. Determinations were made before and after a single treatment. Because all apheresis patients suffered from coronary heart disease (CHD), 201 CHD patients and 90 individuals without CHD, neither group undergoing apheresis, served as controls. In apheresis patients, GSH-Px (33.9+/-8.2 U/g Hb) and GSSG-R (7.6+/-0.9 U/g Hb) activities were increased whereas SOD activity (5.4+/-1.5 U/g Hb) was decreased in RBCs before a single treatment compared to controls. Plasma FRSEs of apheresis patients were not different from those of controls. There was no effect of a single treatment on FRSEs in RBCs. However, a significant decrease in plasma GSH-Px activity (209.9+/-24.9 U/ml) due to the extracorporeal treatment was observed. These data show that long-term extracorporeal therapy with LDL apheresis modulates the activity of antioxidant enzymes in RBCs whereas a single treatment was without major effect on FRSE activity in RBCs and plasma, except for plasma GSH-Px.

Adult↗

PubMed1998

Superiority of combined CK-MB and troponin I measurements for the early risk stratification of unselected patients presenting with acute chest pain.

BACKGROUND: Recent studies have suggested that positive troponin I tests are associated with an increased risk of cardiac death during short-term follow-up. However, it is unknown if troponin I tests alone or in addition to CK-MB measurements are superior to predict unfavorable outcome during long-term follow-up. PATIENTS AND METHODS: In a prospective, double-blind study we assessed the prevalence and prognostic value of combined troponin I and CK-MB tests in an unselected cohort of patients (n = 292) admitted to the emergency department for acute chest discomfort. Patients were grouped according to the diagnosis on discharge in those with acute myocardial infarction (1), unstable angina (2), and noncardiac chest pain (3). Six months after enrollment, death rates were obtained and follow-up interviews were performed with respect to survival, recurrence of chest pain, and myocardial infarction. RESULTS: In patients with evidence of coronary heart disease, the mortality rate for abnormal troponin I and normal CK-MB levels was 5.0%. Baseline troponin I and elevated CK-MB levels were associated with a mortality rate of 4.0%. However, the mortality rate was significantly higher (11.1%) in patients presenting with elevated troponin I and CK-MB values. In patients without myocardial infarction on admission, 10.5% with positive troponin I tests died compared to 1.6% with negative tests. The mortality rate in patients without myocardial infarction was 2.7% for patients with elevated CK-MB but normal troponin I values. In patients with both markers elevated a significantly higher mortality rate (16.7%) was found, representing a 6-fold increase in the death event rate. With the additional knowledge of troponin I values, it could be demonstrated that certain cases were misclassified as having noncardiac chest pain. At least some of the latter patients with above-normal values of troponin I were retrospectively to be reclassified as unstable angina. Acute non-Q-wave myocardial infarctions were occasionally misdiagnosed as either angina pectoris or nonischemic chest pain. CONCLUSIONS: Our data suggest the superiority of combined CK-MB and troponin I measurements in clinical practice for the early risk stratification of patients presenting with acute chest pain. In nonmyocardial infarctions, both CK-MB and troponin I convey independent prognostic information with regard to fatal outcome. Troponin I tests in addition to CK-MB measurements contribute to a lower rate of misdiagnoses.

Aged↗

PubMed1998

Pharmacokinetics of mycophenolic acid (MPA) and determinants of MPA free fraction in pediatric and adult renal transplant recipients. German Study group on Mycophenolate Mofetil Therapy in Pediatric Renal Transplant Recipients.

Dosage guidelines for mycophenolate mofetil (MMF), an ester prodrug of the immunosuppressant mycophenolic acid (MPA), are still preliminary in children. This study compares the pharmacokinetics of MPA and its major metabolite MPA glucuronide (MPAG) in pediatric renal transplant recipients receiving 600 mg MMF/m2 body surface area twice a day to those of adults on the currently recommended oral dose of 1 g of MMF twice a day. Concentration-time profiles of 18 children (age, 10.7+/-0.72 yr; range, 5.9 to 15.3 yr) and 10 adults were investigated 1 and 3 wk after transplantation. Plasma concentrations of MPA and MPAG were measured by reverse-phase HPLC. Because MPA is extensively bound to serum albumin and only the free fraction is presumed to be pharmacologically active, the MPA free fraction was also analyzed by HPLC after separation through ultrafiltration. The areas under the concentration-time curves (AUC0-12) of total and free MPA throughout the 12-h dosing interval in children were, in general, comparable to the corresponding data in adult patients. The mean AUC0-12 of MPA and free MPA did not change significantly over the first 3 wk after transplantation, but there was substantial intra- and interindividual variation. MPAG-AUC0-12 values in children with primary renal transplant dysfunction were threefold higher than in those with functioning transplants. Renal impairment had no consistent effect on total MPA-AUC0-12 values, but the MPA free fraction in children (median, 1.65%; range, 0.40 to 13.8%) was significantly (r2=0.46) modulated by renal transplant function and serum albumin levels. In conclusion, concentration-time profiles of pediatric renal transplant recipients administered 600 mg MMF/m2 body surface area twice a day are comparable to those in adults on 1 g MMF twice a day in the first 3 wk after transplantation. Renal impairment and decreased serum albumin levels led to an increase in the free fraction of MPA and the free MPA-AUC0-12 values. Because the pharmacologic activity of MPA is a function of unbound drug concentration, these findings might be relevant for the pharmacodynamic effects of MPA.

Adolescent↗

PubMed1998

Clinical outcome and economic impact of aminoglycoside peak concentrations in febrile immunocompromised patients with hematologic malignancies.

The aim of this study was to investigate the clinical and economic significance of aminoglycoside peak concentrations in febrile neutropenic patients with hematologic malignancies. Sixty-one patients were treated according to protocol II of the Paul-Ehrlich-Gesellschaft: initial application of gentamicin or tobramycin in combination with a cephalosporin or ureidopenicillin and, after 3 days, a potential change of antibiosis to be decided in case of nonresponse. At the same time, samples were collected by an independent controller. We found a significant dependence of clinical outcome on aminoglycoside peak concentrations (P = 0.004). Twelve of 17 patients with peak concentrations > 4.8 mg/L, but only 13 of 44 patients with concentrations < or = 4.8 mg/L, responded to initial therapy. Average infection-related costs per patient with peak values > 4.8 mg/L were US$1429, $1790, and $1701 for nursing, diagnostics, and therapeutics, respectively (total $4920). Expenses for patients with peak concentrations < or = 4.8 mg/L were approximately 1.8-fold higher (average total $8718). If all 61 patients had achieved peaks > 4.8 mg/L, the potential savings would have totalled $167,112. We conclude that neutropenic patients form a target group for successful pharmacokinetic intervention and cost saving.

Adult↗

PubMed1998

Determination of monoethylglycinexylidide by fluorescence polarization immunoassay in highly icteric serum samples: modified precipitation procedure and HPLC compared.

Hyperbilirubinemia, which frequently occurs in severe liver disease, interferes with the fluorescence polarization immunoassay (FPIA) monoethylglycinexylidide (MEGX) assay manufactured by Abbott Diagnostics. Because the MEGX test is particularly helpful in this clinical situation, strategies have been developed to overcome this problem. Precipitation of serum with the Abbott Digoxin II precipitation reagent eliminates bilirubin. Therefore, we compared FPIA results after precipitation of 81 icteric samples from 27 MEGX tests to results obtained using a validated HPLC method. The precipitation did not substantially alter the performance characteristics of FPIA: detection limit, 8 microg/L; between-days imprecision, 5.3-6.2%; recovery, 102-104% (50-200 microg/L). This pretreatment of serum did not eliminate all interference, and only a poor correlation was observed between serum MEGX concentrations measured with HPLC or modified FPIA (r2 = 0.46; S(y/x) = 20.0 microg/L). In contrast, MEGX formation values calculated by subtraction of the prelidocaine MEGX concentration were in close agreement (r2 = 0.98; S(y/x) = 2.3 microg/L). Because only MEGX formation is clinically relevant, this modified FPIA procedure offers a simple and rapid alternative to HPLC.

Chemical Precipitation↗

PubMed1998

Simultaneous determination of mycophenolic acid and its glucuronide in human plasma using a simple high-performance liquid chromatography procedure.

We describe a reversed-phase HPLC method for determination of total mycophenolic acid (MPA), its free concentration (MPAf), and the glucuronide metabolite (MPAG), based on simple sample preparation and gradient elution chromatography. The compounds were quantified in parallel by absorbance at 254 nm and 215 nm in the internal standard mode. Linearity was verified up to 50 mg/L for MPA and up to 500 mg/L for MPAG (r >0.999). Detection limits at 215 and 254 nm were, respectively, 0.01 and 0.03 mg/L for MPA, and 0.03 and 0.1 mg/L for MPAG. The recovery of MPA was 95-106%; recovery of MPAG was 96-106%. The imprecision (CV) for MPA (0.2-25 mg/L) was <8.4% (254 nm) and <4.4% (215 nm) within day (n = 12) and <9.2% (254 nm) and <6.2% (215 nm) between days (n = 12). The imprecision for MPAG (10-250 mg/L) was <4.9% (254 nm) and <3.4% (215 nm) within day, and <6.1% (254 nm) and <5.9% (215 nm) between days. For quantification of MPAf, 100 microL of ultrafiltrate was applied directly to the column. The detection limit was 0.005 mg/L at 215 nm and 0.015 mg/L at 254 nm. In the range between 18-210 microg/L, the within-day CVs were <11.8% (n = 12) and the between-day CVs were <15.8% (n = 12).

Blood Proteins↗

PubMed1998

Cyclosporin whole blood immunoassays (AxSYM, CEDIA, and Emit): a critical overview of performance characteristics and comparison with HPLC.

Assays with different specificity are used for cyclosporin monitoring in clinical transplantation. A recent survey of 35 centers showed that 86% used immunoassays for cyclosporin A (CsA). In consensus documents the following performance criteria were recommended: (a) imprecision < or = 10% at 50 microg/L and < or = 5% at 300 microg/L; and (b) comparison with the reference method (HPLC) should yield a slope of 0.9-1.1, an intercept of -15 to 15 microg/L, and S(y/x) < or = 15 microg/L. The newly developed CsA assays for the AxSYM (Abbott) and the CEDIA (Boehringer Mannheim) as well as the Emit assay (Behring Diagnostica) were evaluated. Results from samples of heart, kidney, and liver recipients (100 specimens each) were compared with a validated HPLC-ultraviolet detection method. Between-series imprecision (CV) with commercial controls was 5.8% and 1.7% for AxSYM (70 and 300 microg/L), 11% and 5.5% for CEDIA (90 and 200 microg/L), and 8.1% and 4.5% for Emit (63 and 172 microg/L). In the presence of 300 microg/L parent CsA, cross-reactivities were (for AxSYM, CEDIA, and Emit, respectively) 7%, 4%, and none for AM1 (1 mg/L) and 12.6%, 25%, and 6% for AM9 (0.5 mg/L). Comparison with HPLC showed in heart and kidney recipients an average overestimation with the Emit and the CEDIA of approximately 22%, with overestimation in the AxSYM of 32%. In liver recipients, the most challenging patient group, the CEDIA and the AxSYM showed a mean overestimation of 43% and 47%, respectively, and the Emit differed by 31% compared with HPLC. None of the immunoassays fully satisfied the performance criteria recommended in the consensus documents. In terms of specificity, Emit ranks before CEDIA, which ranks before AxSYM. Regarding imprecision, the ranking is AxSYM < Emit < CEDIA. These limitations must be considered when using these assays for therapeutic drug monitoring of CsA in clinical transplantation.

Chromatography, High Pressure Liquid↗

PubMed1998

Modified pentamer formation assay for measurement of tacrolimus and its active metabolites: comparison with liquid chromatography-tandem mass spectrometry and microparticle enzyme-linked immunoassay (MEIA-II).

A modified pentamer formation assay (PFA) for quantification of tacrolimus and active metabolites after extraction from whole blood is described. The lower limit of detection was 2 microg/L. Intraassay precision (CV) was 5.7-13.7%, and the interassay CV was 6. 1-14.9%. Tacrolimus trough concentrations in 104 whole blood specimens from liver and kidney transplant recipients were compared with results from HPLC-tandem mass spectrometry (LC/MS/MS) and microparticle enzyme immunoassay (MEIA-II). Data were analyzed by difference plots and are presented as median (95% confidence intervals) of the method differences. MEIA-II results were on average 2.00 microg/L (range, -0.08 to 5.17 microg/L) higher than LC/MS/MS, whereas PFA results were only 1.07 microg/L (range, -2.62 to 5.33 microg/L) higher. Of 104 specimens tested, 25 displayed differences >/=3 microg/L between MEIA-II and PFA: median difference, 4.65 microg/L (range, 3.01-8.79 microg/L). The corresponding median difference between PFA and LC/MS/MS was -0.91 microg/L (range, -4.11 to 0.85 microg/L), and the difference between MEIA-II and LC/MS/MS was 3.67 microg/L (range, 1.88-6.34 microg/L), suggesting the presence of inactive metabolites that caused a positive bias in the immunoassay. In contrast, similar median differences were observed for the remaining 79 specimens: MEIA-II minus LC/MS/MS, 1.78 microg/L (range, -0.45 to 4.11 microg/L); PFA minus LC/MS/MS, 1.90 microg/L (range, -1.70 to 5.50 microg/L). Active tacrolimus metabolites may have contributed to the higher apparent tacrolimus concentrations in these specimens.

Chromatography, High Pressure Liquid↗

PubMed1997

Acceleration of hepatocellular energy by idebenone during early reperfusion after cold preservation ameliorates heat shock protein 70 gene expression in a pig liver model.

BACKGROUND: Heat shock proteins (HSPs) are induced in the liver after warm ischemia/reperfusion and are thought to be markers of hepatocellular injury and oxidative stress. METHODS: The influence of variable periods of cold storage followed by reperfusion on the expression of HSP70 was studied in the isolated perfused pig liver. Organs were harvested and stored in histidine-tryptophan-ketoglutarate solution at 4 degrees C and then perfused (210 min) in a closed water bath (38 degrees C), which subjects the liver to fluctuating outer pressure. The role of energy depletion, reactive oxygen intermediates, Kupffer cells, and circulating leukocytes in HSP70 expression was determined. RESULTS: HSP70 expression was not detectable in liver tissue before explantation or before reperfusion by Northern blot analysis using a pig HSP70 gene probe. HSP70 expression was observed after reperfusion depending on cold storage time. Kinetics of HSP70 expression monitored by reverse transcriptase polymerase chain reaction showed a rapid increase of mRNA within 1 hr, which was closely associated with delayed recovery of hepatocellular energy charge, as assessed by the ketone body ratio. The inactivation of Kupffer cells, the presence or absence of leukocytes, and the suppression of oxidative stress with the antioxidant idebenone, given during reperfusion, had no influence. However, feeding the animals with idebenone over 7 days before explantation led to a faster recovery of ketone body ratio, paralleled by a substantial suppression of HSP70 expression. CONCLUSIONS: Our data show that HSP70 expression during reperfusion is mainly dependent on the preceding cold storage time and the consecutive delayed recovery of the hepatocellular energy charge.

Animals↗

PubMed1997

Azathioprine-induced severe pancytopenia due to a homozygous two-point mutation of the thiopurine methyltransferase gene in a patient with juvenile HLA-B27-associated spondylarthritis.

Severe pancytopenia due to azathioprine (AZA) toxicity in patients with autoimmune diseases is not uncommon. We describe a 14-year-old girl with HLA-B27+ spondylarthritis who was treated with AZA 3 mg/kg/day and who suddenly developed severe pancytopenia in the seventh week of treatment. Analysis of the catabolic pathway of AZA revealed a homozygous deficiency of thiopurine methyltransferase (TPMT) on the basis of a combined 2-point mutation at nucleotide positions 460 and 719 in the gene for TPMT, causing a toxic level of the metabolic active 6-thioguanine nucleotides (6-TGN) (2,394 pmoles/8 x 10(8) red blood cells). The patient was transfusion dependent and finally recovered 8 weeks after the development of the pancytopenia. At that time, 6-TGN had already returned to normal therapeutic levels. Family studies revealed another homozygous deficiency in the mother, while the other family members were heterozygous.

Adolescent↗

PubMed1997

Suppression of leukocyte-enhanced cold ischemia/reperfusion injury of liver endothelium with the benzoquinone antioxidant idebenone.

OBJECTIVE: Despite the large body of evidence for a major role of neutrophils and oxidant stress, the exact pathogenesis of the early ischemia/reperfusion injury after cold preservation of the liver is not well understood. The potential benefit of an antioxidant on metabolic liver function during reperfusion has been demonstrated in several studies. MATERIALS AND METHODS: We describe a cold storage/reperfusion damage model with isolated perfused pig livers, where the effects of neutrophils and idebenone, a recently developed benzoquinone antioxidant were studied. The integrity of sinusoidal endothelial cells (SEC) was estimated by hyaluronic acid concentration in perfusate and the expression of endothelial constitutive nitric oxide synthase (ecNOS) after reperfusion and compared to lipid peroxidation and antioxidant content. RESULTS: Hyaluronic acid displayed the highest levels and ecNOS mRNA was most depressed in livers reperfused with neutrophils after 20 h cold storage; this was accompanied by an increase in lipid peroxidation (TBARS) and a breakdown of endogenous lipophilic antioxidants (alpha-tocopherol and coenzyme Q-10). These effects were attenuated, when neutrophils were excluded from reperfusion and almost completely abolished by the addition of 200 mumol/L idebenone. CONCLUSIONS: These data suggest that a leukocyte-mediated damage based on reactive oxygen species markedly contributes to the reperfusion injury of SEC after cold preservation of the liver. Therefore, the presence of effective antioxidants in the early reperfusion phase may be beneficial for liver graft integrity.

Animals↗

PubMed1997

Cyclosporin A trough levels correlate with serum lipoproteins and apolipoproteins: implications for therapeutic drug monitoring of cyclosporin A.

In a prospective study over 6 months, the relationship between serum lipid parameters and CsA whole blood trough concentrations was investigated in 39 renal transplant recipients receiving a triple immunosuppressive therapy with cyclosporin (CsA), azathioprine and prednisone. CsA trough concentrations were measured with a selective monoclonal immunoassay (Abbott TDx). Six months after transplantation, significant positive correlations were observed between the CsA trough concentration and serum concentrations of triglycerides (r = 0.448, p < 0.01), total cholesterol (r = 0.360, p < 0.05), and apoB (r = 0.418, p < 0.01). After exclusion of patients with over hypertriglyceridemia (> 400 mg/dl), however, the associations were no longer significant. HDL-cholesterol (HDL-C) and apo AI concentrations showed significant inverse correlations with the CsA trough level (HDL-C: r = -0.427, p < 0.01; apoAI: r = -0.350, p < 0.05); the correlations with the CsA trough level were still significant (HDL-C: r = -0.379, p < 0.05; apoAI: r = -0.354, p < 0.05) after exclusion of patients with triglyceride levels of > 400 mg/dl. As a result of these divergent effects on the plasma lipids and lipoproteins, there was a strong positive association (r = 0.633, p < 0.001) between the CsA trough concentration and the total cholesterol/HDL-C ratio. Consequently, elevated total cholesterol/HDL-C ratios that represent an increased atherogenic risk tended to be associated with higher CsA trough levels. In monitoring CsA therapy of renal transplant recipients on maintenance immunosuppressive therapy, it may well be advisable to adjust CsA dosages to obtain CsA trough levels within the lower therapeutic range for patients with an unfavorably high TC/HDL-C ratio.

Adult↗

PubMed1997

A simple and reliable reverse-phase high-performance liquid chromatographic procedure for determination of paclitaxel (taxol) in human serum.

A simple, fast, and reliable isocratic (mobile phase: acetonitrile/methanol/water [48/11/41], reverse-phase (C18 column) high-performance liquid chromatography method for the determination of paclitaxel concentration in human serum is presented. The procedure uses a new and convenient one-step sample-purification procedure that requires only 400 microliters of sample and uses N-heptylbenzamide as an internal standard. Paclitaxel is detected by UV absorbance measurement at 227 nm. The method has a broad linear range (0.01 to 10 mg/l, or 0.012 to 11.7 mumol/l; r > 0.999), and the detection limit is 0.01 mg/l (0.012 mumol/l). The deviation from target value is < or = 1.5%, and coefficients of variation are < or = 13.8% within runs and < or = 15.3% between runs. Recovery paclitaxel is > or = 92.6%. No interferences were observed from endogenous compounds or from more than 30 drugs that may be administered with paclitaxel. Docetaxel, which is not concurrently administered, coeluted with paclitaxel. Compared with previously published high-performance liquid chromatography procedures for the determination of paclitaxel, the particular advantage of the method presented here is its simple and rapid single-step sample-purification procedure, which makes a high recovery of paclitaxel from serum samples possible and results in a pure extract, avoiding interferences from endogenous compounds. The method is suitable for pharmacological studies and routine analysis.

Antineoplastic Agents, Phytogenic↗