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M Oellerich

Publications and source records attributed to M Oellerich.

At least 55 records · Page 3Linked to original sources
PubMed2000

Determination of the acyl glucuronide metabolite of mycophenolic acid in human plasma by HPLC and Emit.

BACKGROUND: The acyl glucuronide (AcMPAG) of mycophenolic acid (MPA) has been found to possess pharmacologic and potentially proinflammatory activity in vitro. To establish its pharmacologic and toxicologic relevance in vivo, a reversed-phase HPLC method was modified to simultaneously determine MPA, the phenolic MPA-glucuronide (7-O-MPAG), and AcMPAG. In addition, cross-reactivity of AcMPAG in the Emit assay for MPA was investigated. METHODS: The procedure used simple sample preparation, separation with a Zorbax Eclipse-XDB-C8 column, and gradient elution. AcMPAG was quantified as 7-O-MPAG-equivalents. RESULTS: The assay was linear up to 50 mg/L for MPA, 250 mg/L for 7-O-MPAG, and 10 mg/L for AcMPAG (r >0.999). Detection limits were 0.01, 0.03, and 0.04 mg/L for MPA, 7-O-MPAG, and AcMPAG, respectively. The recoveries were 99-103% for MPA, 95-103% for 7-O-MPAG, and 104-107% for AcMPAG. The within-day imprecision was <5.0% for MPA (0.2-25 mg/L), <4.4% for 7-O-MPAG (10-250 mg/L), and < or =14% for AcMPAG (0.1-5 mg/L). The between-day imprecision was <6.2%, <4.5%, and < or =14% for MPA, 7-O-MPAG, and AcMPAG, respectively. When isolated from microsomes, purified AcMPAG (1-10 mg/L) revealed a concentration-dependent cross-reactivity in an Emit assay for the determination of MPA ranging from 135% to 185%. This is in accordance with the bias between HPLC and Emit calculated in 270 samples from kidney transplant recipients receiving mycophenolate mofetil therapy, which was greater (median, 151.2%) than the respective AcMPAG concentrations determined by HPLC. AcMPAG was found to undergo hydrolysis when samples were stored up to 24 h at room temperature or up to 30 days at 4 degrees C or -20 degrees C. Acidified samples (pH 2.5) were stable up to 30 days at -20 degrees C. CONCLUSIONS: The HPLC and Emit methods for AcMPAG described here may allow investigation of its relevance for the immunosuppression and side effects associated with mycophenolate mofetil therapy.

Chromatography, High Pressure Liquid↗
PubMed2000

Genotyping of eight thiopurine methyltransferase mutations: three-color multiplexing, "two-color/shared" anchor, and fluorescence-quenching hybridization probe assays based on thermodynamic nearest-neighbor probe design.

BACKGROUND: The inherited deficiency of thiopurine methyltransferase (TPMT) leads to severe myelosuppression in homozygous patients treated with thiopurine derivatives. One in 300 Caucasians has a homozygous TPMT deficiency with no measurable enzyme activity. To date, eight single-point mutations have been characterized; one group (TPMT*3) accounts for 75% of these. METHODS: We used four LightCycler(TM) capillaries to investigate all eight mutations. The three mutations on exon 10 were detected in one capillary with a single "shared" anchor labeled 5' with Cy5.5 and 3' with fluorescein. A wild-type-compatible 3'-fluorescein-labeled probe 5' adjacent to the anchor covered the TPMT*7 mutation, and a 5'-LC-RED640-labeled probe 3' adjacent to the anchor covered the TPMT*3C mutation. For TPMT*4, the forward amplification primer was internally labeled with a fluorescence quencher [6-carboxytetramethylrhodamine (TAMRA)], and a 3'-fluorescein-labeled antisense wild-type-compatible probe was placed at the mutation. For TPMT*2 and TPMT*3D, located on exon 5, a shared anchor approach was chosen. TPMT*3B and TPMT*6 were detected in multiplex technique and TPMT*5 in conventional manner. Anchors and probes were designed using a thermodynamic nearest-neighbor model. RESULTS: All mutations were detected using four capillaries with one amplification protocol in 40 min. The concentrations of the shared anchors had to be decreased to reduce their intrinsic fluorescence resonance energy transfer signals. The quenching approach using TAMRA produced a very reproducible upside-down-shaped melting curve in channel 1 of the LightCycler. Deviations from wild type were easily detected because the smallest melting point shift for any possible mutation under the core of the probes was 1.5 degrees C. CONCLUSIONS: This total TPMT genotyping approach shows that it is possible to use double site-labeled anchor oligonucleotides, that channel 1 of the LightCycler can be used as detection channel for mutations using a quenching design, and that the designed probes enable detection of wild types with 100% likelihood.

Base Sequence↗
PubMed2000

DNA base bulge vs unmatched end formation in probe-based diagnostic insertion/deletion genotyping: genotyping the UGT1A1 (TA)(n) polymorphism by real-time fluorescence PCR.

BACKGROUND: Gilbert syndrome is a clinically inconsequential entity of mild unconjugated hyperbilirubinemia caused by an A(TA)(n)TAA insertion polymorphism (UGT1A1*28) in the promoter region of the gene coding for the enzyme UDP-glucuronosyltransferase 1 (EC 2.4.1. 17; UGT1A1). Present methods for genotyping this polymorphism are laborious. METHODS: Hybridization probes were designed complementary to the wild type (TA)(6) and to alleles with (TA)(7) and (TA)(8) repeats in the promoter region. Melting points were measured in samples representing all currently known alleles with (TA)(5) to (TA)(8) repeats. Probe melting points were predicted with a thermodynamic nearest-neighbor model for Watson-Crick paired probes. The dominant secondary structures resulting from probe hybridization were predicted by thermodynamic free energy calculations. Alternatively samples were genotyped based on amplicon size resolved by high-resolution polyacrylamide gel electrophoresis. RESULTS: Only short probes (22-24 bases) could be successfully used for genotyping this locus because of the very low stability of this TA repeat. Assays based on (TA)(7) or (TA)(8) genotype-compatible hybridization probes effectively discriminated five to eight TA repeats. The consecutive use of two different detection probes was necessary for better discrimination of some heterozygous genotypes. All results were in concordance with the alternative genotyping method. Of 100 investigated Caucasians (50 males, 50 females), 9 (9%) were homozygous for the (TA)(7) allele. CONCLUSIONS: The presented method for genotyping the (TA)(n) promoter polymorphism of the UGT1A1 gene with the LightCycler has the potential to genotype all currently known (TA)(n) repeats in a single assay and is sensitive toward possible new genotypes. Our findings also show that thermodynamic calculations are of practical value for the design of hybridization probe assays for the genotyping of insertion/deletion polymorphisms.

DNA Probes↗
PubMed1999

Assessment of liver function by the MEGX test in patients with schistosomiasis and cirrhosis.

OBJECTIVES: The aim of this study was to determine the effect of schistosomiasis infection on hepatic function in Egyptian patients with posthepatitic cirrhosis. DESIGN AND METHODS: Hepatic function, was assessed in 66 Egyptian patients, with (n = 30) and without (n = 36) schistosomal liver fibrosis due to Schistosoma mansoni and in 20 healthy controls, using the monoethylglycinexylidide (MEGX) test. Serum MEGX concentrations were measured before and 5, 15, 30, 60, 120, and 180 min after a lidocaine bolus. The sero-prevalence of antibodies to hepatitis C was also determined in the patients. RESULTS: MEGX test results were significantly lower in patients than in controls at all time points. MEGX test results declined with advancing Child Class. Receiver operating characteristic (ROC) curve analysis revealed the following areas under the ROC curves for discrimination of Child Class C from Child Classes A/B: 30 min, 0.762; 60 min, 0.743; 120 min, 0.731; 15 min, 0.728; 180 min, 0.728; 5 min, 0.602. Schistosomiasis infection had no influence on MEGX test results when cirrhotic patients with (Schisto+) and without (Schisto-) schistosomiasis were compared. While the prevalence of the hepatitis B surface antigen was only 16.7% (Schisto-) and 26.7% (Schisto+), there was an extremely high sero-prevalence of antibodies to hepatitis C (HCV) in both groups: 88.9% (Schisto-) and 73.3% (Schisto+). CONCLUSIONS: The association of schistosomal liver fibrosis with cirrhosis does not additionally influence MEGX formation. In addition, HCV rather than schistosomiasis infection must be considered as a major cause for the progressive liver disease in these patients.

Adult↗
PubMed1999

Identification of glucoside and carboxyl-linked glucuronide conjugates of mycophenolic acid in plasma of transplant recipients treated with mycophenolate mofetil.

1. Mycophenolic acid (MPA), is primarily metabolized in the liver to 7-O-MPA-beta-glucuronide (MPAG). Using RP-h.p.l.c. we observed three further MPA metabolites, M-1, M-2, M-3, in plasma of transplant recipients on MMF therapy. To obtain information on the structure and source of these metabolites: (A) h.p.l.c. fractions containing either metabolite or MPA were collected and analysed by tandem mass spectrometry; (B) the metabolism of MPA was studied in human liver microsomes in the presence of UDP-glucuronic acid, UDP-glucose or NADPH; (C) hydrolysis of metabolites was investigated using beta-glucosidase, beta-glucuronidase or NaOH; (D) cross-reactivity of each metabolite was tested in an immunoassay for MPA (EMIT). 2. Mass spectrometry of M-1, M-2, MPA and MPAG in the negative ion mode revealed molecular ions of m/z 481, m/z 495, m/z 319 and m/z 495 respectively. 3. Incubation of microsomes with MPA and UDP-glucose produced M-1, with MPA and UDP-glucuronic acid MPAG and M-2 were formed, while with MPA and NADPH, M-3 was observed. 4. Beta-Glucosidase hydrolysed M-1 completely. Beta-Glucuronidase treatment led to a complete disappearance of MPAG whereas the amount of M-2 was reduced by approximately 30%. Only M-2 was labile to alkaline treatment. 5. M-2 and MPA but not M-1 and MPAG cross-reacted in the EMIT assay. 6. These results suggest that: (i) M-1 is the 7-OH glucose conjugate of MPA; (ii) M-2 is the acyl glucuronide conjugate of MPA; (iii) M-3 is derived from the hepatic CYP450 system.

Chromatography, High Pressure Liquid↗
PubMed1999

Area under the plasma concentration-time curve for total, but not for free, mycophenolic acid increases in the stable phase after renal transplantation: a longitudinal study in pediatric patients. German Study Group on Mycophenolate Mofetil Therapy in Pediatric Renal Transplant Recipients.

Mycophenolate mofetil, an ester prodrug of the immunosuppressant mycophenolic acid (MPA), is widely used for maintenance immunosuppressive therapy in pediatric renal transplant recipients. However, little is known about the pharmacokinetics of MPA in this patient population in the stable transplant phase, and dosage guidelines are preliminary. The authors therefore compared the pharmacokinetics of MPA, free MPA, and the renal metabolite MPA glucuronide (MPAG) in the initial (sampling at 1 and 3 weeks) and stable phases (sampling at 3 and 6 months) posttransplant in 17 children (age, 12.0 +/- 0.77 years; range, 5.9 to 15.8 years), receiving the currently recommended dose of 600 mg MMF/m2 body surface area (BSA) twice a day. Plasma concentrations of MPA and MPAG were measured by reverse phase HPLC. Because MPA is extensively bound to serum albumin and only the free drug is presumed to be pharmacologically active, the authors also analyzed the MPA free fraction by HPLC after separation by ultrafiltration. The intraindividual variability of the area under the concentration-time curves (AUC0-12) of MPA throughout the 12-hour dosing interval was high in the immediate posttransplant period, but declined in the stable phase, whereas the interindividual variability remained unchanged. The median MPA-AUC0-12 values increased 2-fold from 32.4 (range, 13.9 to 57.0) mg x h/L at 3 weeks to 65.1 (range, 32.6 to 114) mg x h/L at 3 months after transplantation, whereas the median AUC0-12 values of free MPA did not significantly change over time. This discrepancy can be attributed to a 35% decline of the MPA free fraction from 1.4% in the initial phase posttransplant to 0.9% (p < 0.01) in the stable phase. In conclusion, pediatric renal transplant recipients given a fixed MMF dose exhibit a 2-fold increase of the AUC0-12 of total MPA in the stable phase posttransplant and a 35% decrease of the MPA free fraction, whereas the AUC0-12 of free MPA remains unchanged over time. Because the latter pharmacokinetic variable is theoretically best predictive of the clinical immunosuppressive efficacy of MMF, these findings may have consequences for the dosing recommendations of MMF in renal transplant recipients.

Adolescent↗
PubMed1999

Application of a thermodynamic nearest-neighbor model to estimate nucleic acid stability and optimize probe design: prediction of melting points of multiple mutations of apolipoprotein B-3500 and factor V with a hybridization probe genotyping assay on the LightCycler.

BACKGROUND: PCR-based mutation detection is prone to methodological errors, e.g., in restriction length fragment polymorphism (RFLP) and allele-specific amplification (ASA), false PCR results may occur because of technical faults or atypical new mutations. METHODS: We investigated the ability of a genotyping assay based on hybridization of labeled oligonucleotides to detect and discriminate known and as yet unknown mutations in the factor V and apolipoprotein B-100 genes. Expected melting points were calculated using a nearest-neighbor model for nucleic acid duplex stability and compared with experimental findings derived from LightCycler melting curves. A method for genotyping the apolipoprotein B-100 G10699A and C10698T mutations is presented. RESULTS: All mismatches tested for in the probed sequence could be detected with a single probe. The measured melting points were in good agreement with their values predicted using the nearest-neighbor model (r = 0.96; y = 0.98x + 1.18; S(y|x) = 0.96; n = 24). CONCLUSIONS: This procedure not only allows the identification of the mutation of interest but also enables the discrimination from other potential mutations in the vicinity of the former. The nearest-neighbor model is valid for hybridization probe assays on the LightCycler and should be of general value in setting up such assays. We have shown for two clinically relevant genotyping examples that the LightCycler mutation detection system has superior discriminatory performance compared with conventional RFLP or ASA PCR-based methods for molecular diagnostic purposes. With this method, in every hybridization probe assay, all mutations under a properly designed probe should be detectable, but they will not necessarily be discriminated from each other in all cases.

Apolipoproteins B↗
PubMed1999

Induction of inducible nitric oxide synthase is an essential part of tumor necrosis factor-alpha-induced apoptosis in MCF-7 and other epithelial tumor cells.

TNF-alpha-induced cytotoxicity is mediated by the intracellular "death domain" of the 55-kDa TNF-alpha receptor and has been demonstrated to be coupled with induction of inducible nitric oxide synthase (iNOS), leading to generation of nitric oxide radicals (NO*). Because it is still widely unknown, to what extent NO* participates in the execution of TNF-alpha-induced apoptosis, NO* production, iNOS expression, and enzyme activity in relation to TNF-alpha-induced apoptotic cell death were investigated in the human breast cancer cell line MCF-7 and various other malignant cell lines. Incubation with TNF-alpha led to induction of iNOS mRNA and protein as well as enhancement of NOS activity. Augmented synthesis of NO2, the stable end product of NO* generation, was significantly correlated with augmented rates of cell death. Measurement of TNF-alpha-triggered production of reactive oxygen species (ROS) suggested a major role for NO* within the generated oxygen radicals. Dying cells showed characteristic features of apoptosis. Addition of cycloheximide (CX) enhanced apoptotic cell death by increasing iNOS activity. L-Nitro-arginine-methylester (L-NAME), a competitive NOS-inhibitor, and iNOS antisense oligonucleotides effectively prevented NO2 generation and apoptosis. Evaluation of iNOS expression during TNF-alpha-induced cell death in various malignant cell lines demonstrated iNOS positivity for all TNF-alpha-sensitive cells. The only primarily resistant line was iNOS negative. In a resistant variant of MCF-7, iNOS mRNA was still detectable; however, treatment with TNF-alpha did not enhance NOS activity. TNF-alpha sensitivity and NO2 production were completely restored by the addition of CX. Taken together, iNOS induction plays an essential role in TNF-alpha-induced apoptosis of the investigated cell lines. Further studies are necessary to define the impact of NO* in relation to other specific effectors of apoptosis such as the caspases.

Apoptosis↗
PubMed1998

Increased serum levels of hyaluronic acid in pregnancies complicated by preeclampsia or hemolysis, elevated liver enzymes, and low platelets syndrome.

OBJECTIVE: Fifteen percent of patients who later have hemolysis, elevated liver enzymes, and low platelets syndrome develop initially have nonspecific symptoms. Early diagnosis could ensure adequate obstetric management; however, prognostic biochemical tests are lacking. We hypothesized that elevated hyaluronic acid serum levels might be an early indicator of hemolysis, elevated liver enzymes, and low platelets syndrome because it is known to be a sensitive marker of liver cell function. STUDY DESIGN: Hyaluronic acid in serum was measured in patients with normal pregnancies (n = 109) and in those patients with pregnancies complicated by preeclampsia (n = 14) or hemolysis, elevated liver enzymes, and low platelets syndrome (n = 11). RESULTS: A significant increase in hyaluronic acid serum concentrations was observed in patients with hemolysis, elevated liver enzymes, and low platelets syndrome or with preeclampsia (p < 0.05). The extent of hyaluronic acid serum levels in hemolysis, elevated liver enzymes, and low platelets syndrome correlated with the clinical severity of the individual course of disease as measured by intensive care unit time (r = 0.72; p < 0.02). CONCLUSIONS: Serum levels of hyaluronic acid in preeclampsia and hemolysis, elevated liver enzymes, and low platelets syndrome are significantly elevated and might play an important diagnostic and prognostic role in patients with hemolysis, elevated liver enzymes, and low platelets syndrome.

Adult↗