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Biomedical subjects

F L Kiechle

Publications and source records attributed to F L Kiechle.

At least 19 recordsLinked to original sources
PubMed1996

Counter modulation of adipocyte mitochondrial processes by insulin and S-oxalylglutathione.

Oxalyl thiolesters, a group of putative intracellular regulators, have been shown to be in vitro inhibitors of some cytosolic enzymes which are stimulated by insulin. In this study, the effects of insulin and oxalyl thiolesters on pyruvate dehydrogenase, beta-oxidation, and acyl-CoA hydrolase activities in mitochondria from rat epididymal adipocytes are compared. Using glutathione, CoASH, cysteine, and cysteamine as thiol sources, oxalyl thioesters were synthesized, purified, and quantitated. Mitochondria were isolated from rat epididymal adipocytes, some of which were incubated with or without insulin. Mitochondrial activities were determined by radioisotopic assay subsequent to control, insulin, or oxalyl thiolester incubation. Under the conditions used in this study, pyruvate dehydrogenase activity was increased 28% subsequent to 10-min incubation of adipocytes with 400 microU/ml insulin; in contrast, preincubation of adipocyte mitochondria with S-oxalylglutathione resulted in a dose-dependent 11-19% inhibition of pyruvate dehydrogenase. S-oxalylglutathione also attenuated the spermine-induced activation of pyruvate dehydrogenase. Insulin treatment resulted in a small but significant increase in beta-oxidation of palmitic acid while 100 microM S-oxalylglutathione mediated a 40% decrease in palmitate oxidation. Palmitoyl-CoA hydrolase activity was decreased 14% by insulin treatment; however, S-oxalylglutathione caused a 14-50% increase in hydrolase activity. The other oxalyl thiolesters were not as effective or as consistent as S-oxalylglutathione in modulation of the mitochondrial activities; free thiols and oxalic acid did not modulate the activities. In summary, pyruvate dehydrogenase, palmitate beta-oxidation, and palmitoyl-CoA hydrolase activities in adipocyte mitochondria were modulated in approximately equal but opposite directions by insulin and S-oxalylglutathione. These findings support the suggestion that oxalyl thiolesters may function as an intracellular signal recruited to return insulin to normal levels.

Adipocytes↗
PubMed1996

Specimen collection and storage for diagnostic molecular pathology investigation.

The success of the newest discipline in the diagnostic clinical pathology laboratory, molecular pathology, is dependent on proper collection and storage of both the original and processed (nucleic acid) specimen. This issue will grow in importance as test volumes increase in the diagnostic molecular pathology laboratory. This review is a distillation of a literature review by the Patient Preparation and Specimen Handling Committee of the College of American Pathologists. It describes specific collection, storage, and anticoagulant or preservative requirements based on the diagnostic molecular technique and/or specimen type used for analysis. This review serves as a guide for clinical laboratories interested in appropriate collection and storage of specimens to be used in nucleic acid-based analysis.

DNA↗
PubMed1996

Mitochondrial disorders. Methods and specimen selection for diagnostic molecular pathology.

Mitochondrial DNA is a circular double-stranded macromolecule. Each strand contains 16 569 base pairs. Mutations in mitochondrial DNA, including base substitutions in tRNA or rRNA genes, deletions, duplications, or base substitutions in genes for protein subunits, lead to specific diseases. The ratio of mutated to normal mitochondrial DNA may vary from tissue to tissue (heteroplasmy) in mitochondrial DNA diseases. Therefore, the source of the specimen is important in the evaluation of mitochondrial DNA mutations. Detection method selection is also critical. For example, single-strand conformation polymorphism is not as specific for tRNA mutations as is gene sequencing or amplification of a specific gene by polymerase chain reaction. Care in both specimen collection and analytic method are important in the successful evaluation of patients with a potential mitochondrial DNA disease.

DNA, Mitochondrial↗
PubMed1996

Diagnostic molecular pathology in the twenty-first century.

Diagnostic molecular pathology is expanding rapidly with the aid of the Human Genome Project and the development of potentially user-friendly molecular diagnostic methods. The diagnostic molecular pathology laboratory of the future must be prepared to purify DNA or RNA from a variety of sources and to investigate the sequence of the target genome of interest using automated amplification and hybridization detection systems. There will be a shift in the emphasis from phenotypic to genotypic diagnosis, and the diagnostic molecular pathology laboratory of the early twenty-first century will perform 5% to 10% of the volume of all laboratory testing.

Automation↗
PubMed1996

Specimen stability for DNA-based diagnostic testing.

The use of molecular diagnostic testing is increasing in the clinical setting; therefore, data regarding DNA stability in clinical specimens are essential for correct test performance and interpretation. This study was designed to determine DNA stability in peripheral blood and solid tissue under different storage conditions. DNA quality and yield were assayed by spectrophotometric absorbance, gel electrophoresis, and suitability for Southern hybridization and polymerase chain reaction (PCR), the most widely employed clinical DNA analyses. A second goal of the study was to evaluate DNA stability during storage at 4 degrees C for 1 month to 3 years. The data show that freezing or refrigeration of separated leukocytes is preferable for short- to intermediate-term storage and freezing is preferable for solid tissue. DNA degradation varying from slight to severe is seen inconsistently with such specimens, probably due to sampling of unevenly frozen-tissue areas. Depending on the degree of DNA degradation, analysis may still be possible by PCR and in some cases even by Southern hybridization. Once isolated, DNA was stable at 4 degrees C for at least 3 years. These results suggest a more flexible approach to specimen requirements for molecular pathology, as some samples that would routinely be rejected gave interpretable results.

Blood Cells↗
PubMed1995

Antibodies to carbonic anhydrase in patients with immune cholangiopathies.

BACKGROUND/AIMS: Bile duct epithelia contain an abundance of carbonic anhydrase. Antibodies to this enzyme have been described in autoimmune disorders. Serum from patients with immune-mediated liver diseases was studied to determine whether antibodies to carbonic anhydrase II and/or pyruvate dehydrogenase could distinguish autoimmune cholangitis as immunologically distinct from primary biliary cirrhosis. METHODS: Antibody assays to carbonic anhydrase II (Western blot) and pyruvate dehydrogenase (flow cytometry) were performed on the sera of patients with autoimmune cholangitis (6), primary biliary cirrhosis (12), primary sclerosing cholangitis (12), autoimmune hepatitis (12), and control (Gilbert syndrome; 8). RESULTS: Reactivity to carbonic anhydrase II was detected in 5 of 6 patients with autoimmune cholangitis, 1 of 12 patients with primary biliary cirrhosis, 1 of 12 patients with autoimmune hepatitis, and no other patients. Individuals with autoimmune cholangitis were more likely than the other patients to be reactive to carbonic anhydrase II (P < 0.001). Patients with primary biliary cirrhosis were more reactive to pyruvate dehydrogenase compared with all other groups (P < 0.001). CONCLUSIONS: An antibody to human carbonic anhydrase II is frequently detected in the sera of patients with autoimmune cholangitis and is uncommon or not present in other cholangiopathies. These data provide evidence that autoimmune cholangitis and primary biliary cirrhosis represent distinct entities with unique patterns of immunoreactivity.

Aged↗
PubMed1994

Carbonic anhydrase antibody in sera from patients with endometriosis.

Sera from 16 of 23 (69.6%) patients with endometriosis, a potential autoimmune disease, and 2 of 17 (11.8%) control individuals had autoantibodies against the bovine carbonic anhydrase (CA) molecular weight marker, as determined by the Western blot technique. The reactivity of these antibodies to purified human CA I, human CA II, and two preparations of bovine CA II were investigated. Of the 16 endometriosis patients who were reactive to the bovine CA molecular weight marker, 14 were reactive to at least one purified human CA isoenzyme tested, 8 were reactive to at least one purified bovine CA II, and 2 did not react with any of the CA isoenzymes tested. Variation in cross-reactivity between species and in the biochemical characteristics of various CA isoenzyme preparations may partially explain these findings. Autoantibodies to CA isoenzymes have recently been reported in other autoimmune diseases. Further investigation is required to determine the significance of CA autoantibody production in patients with endometriosis.

Adult↗
PubMed1993

Lactate oxidation for the detection of mitochondrial dysfunction in human skin fibroblasts.

To screen fibroblasts for defects in lactate/pyruvate oxidation, cells were grown to confluence in 25-cm2 flasks, rinsed, and incubated in glucose-free media containing 25 microM L-lactate and 0.1 microCi [D,L-1-14C]lactate. Lactate oxidation was measured as the amount of lactate oxidized in nmol of 14CO2 generated/mg protein/min. Fibroblasts from patients with mitochondrial or peroxisomal disorders had decreased lactate oxidation compared to the control (CON): CON, 1.9 +/- 0.13 nmol/mg/min; neonatal adrenoleukodystrophy (NALD), 0.45 +/- 0.01 (P < 0.001); rhizomelic chondrodysplasia punctata (RCDP), 0.13 +/- 0.002 (P < 0.001); mitochondrial defect of unknown etiology (MIT), 0.77 +/- 0.003 (P < 0.001); pyruvate dehydrogenase (PDH) deficiency, 0.98 +/- 0.02 (P < 0.001). This method is useful for screening fibroblasts for defects in lactate oxidation in patients with mitochondrial or peroxisomal disorders. Confirmation of the site of the defect may then be investigated with specific assays, e.g., PDH, in cellular homogenates: CON, 0.93 +/- 0.02 nmol/mg/min; NALD, 0.55 +/- 0.02; RCDP, 0.44 +/- 0.02; MIT, 0.53 +/- 0.03; PDH deficiency, 0.19 +/- 0.02.

Acidosis, Lactic↗
PubMed1993

Lactic acidosis and mitochondrial dysfunction in two children with peroxisomal disorders.

Mitochondrial myopathies and defects in oxidative phosphorylation have been described in some patients with peroxisomal disorders. Although peroxisomes and mitochondria play a role in the beta-oxidation of fatty acids, the metabolic interactions between the two are not well defined. Defects in peroxisomal beta-oxidation are associated with extracellular accumulation of very long-chain fatty acids and may be accompanied by alterations in the intracellular pool of fatty acyl-CoAs, which are known to alter mitochondrial function. This study was initiated to examine alterations in the intracellular pool of acyl-CoAs and mitochondrial function in two children with generalized disorders of peroxisomal function and clinical lactic/pyruvic acidaemia. Fibroblasts were cultured from skin biopsies obtained from one child with neonatal adrenoleukodystrophy (NALD) and another with rhizomelic chondrodysplasia punctata (RCDP). Fibroblast lactate oxidation was significantly inhibited in NALD by 76% and RCDP by 92% compared to control values of 1.9 +/- 0.1 nmol/min per mg protein. Pyruvate dehydrogenase (PDH) (mean +/- SEM; activity nmol/min per mg protein) was: NALD 0.55 +/- 0.02 (p < 0.01), RCDP 0.44 +/- 0.02 (P < 0.01), and controls 0.83 +/- 0.02. The acid-insoluble (long-chain and very long-chain) acyl-CoA levels (mean +/- SEM; pmol/mg protein) were: NALD 129 +/- 69 (p < 0.01), RCDP 65 +/- 15 (p < 0.05), and control 45 +/- 7. These two patients with generalized peroxisomal disorders exhibited an increase in intracellular acyl-CoA species accompanied by decreased PDH activity and clinical lactic/pyruvic acidaemia.

Acidosis, Lactic↗
PubMed1993

Comparison of the HemoCue beta-glucose photometer and reflotron for open heart surgery.

The HemoCue beta-glucose photometer (Angelholm, Sweden) was evaluated for use in monitoring blood glucose in both diabetic and nondiabetic patients undergoing open heart surgery. Because occasional discrepancies were noted in patients with low total proteins when the Reflotron (Boehringer Mannheim, Indianapolis, IN) was used for this purpose, the effects of protein and hematocrit on glucose results from both instruments were investigated and compared with plasma values from a Paramax 720 ZX (Baxter Healthcare, Irvine, CA). Linear-regression analysis of the HemoCue results (y) versus Paramax (x) yielded y = 0.956x + 0.35, r2 = 0.980, with Sy/x = 0.57 mmol/L (10.3 mg/dL). Results from the Reflotron (y) versus Paramax (x) yielded y = 1.075x - 0.10, r2 = 0.964, with Sy/x = 0.99 mmol/L (17.8 mg/dL). Bias plots of (HemoCue-Paramax) or (Reflotron-Paramax) versus glucose, hematocrit, or protein showed no effect of glucose on the results from either instrument and no effect of protein or hematocrit on the HemoCue findings. The Reflotron, however, showed a positive bias of up to 3.5 mmol/L (63 mg/dL) at protein concentrations between 30-40 g/L (3.0-4.0 g/dL) and a possible positive bias at low hematocrit levels.

Blood Chemical Analysis↗
PubMed1993

Nitric oxide. Biochemistry, pathophysiology, and detection.

Nitric oxide is generated from the terminal guanidino nitrogen of L-arginine yielding citrulline. This reaction is catalyzed by two major types of nitric oxide synthase: inducible and constitutive. Nitric oxide is a gaseous mediator responsible for a variety of physiologic phenomena. Its short half-life in biologic systems has created problems in its direct determination. Many experiments depend on the use of inhibitors of nitric oxide synthase to provide indirect evidence for the involvement of nitric oxide. Spectroscopic and electrochemical methods are the best for the direct measurement of nitric oxide; however, the advantages and disadvantages of each technique should be considered carefully before a specific method is selected.

Amino Acid Oxidoreductases↗
PubMed1992

Fasting induced alterations in mitochondrial palmitoyl-CoA metabolism may inhibit adipocyte pyruvate dehydrogenase activity.

1. Adipocytes from fed and fasted (24 hr) groups of rats were fractionated into mitochondria, microsomes and plasma membranes. 2. Fasting significantly decreased the mitochondrial activity of palmitoyl-CoA synthetase, palmitoyl-CoA hydrolase, beta-oxidation and pyruvate dehydrogenase. 3. Fasting elevated intramitochondrial long-chain acyl-CoA. 4. Pyruvate dehydrogenase was inhibited 50% by addition of 30 microM palmitoyl-CoA. 5. Fasting-induced changes in palmitoyl-CoA metabolism may modulate pyruvate dehydrogenase activity in adipocyte mitochondria.

Adipose Tissue↗
PubMed1992

A flow cytometric method to detect anti-pyruvate dehydrogenase antibody in primary biliary cirrhosis.

Primary biliary cirrhosis (PBC) is an autoimmune disease characterized by the presence of anti-mitochondrial antibodies specifically directed against the M2 group of mitochondrial antigens. Recently, the E-1, the E-2, and protein X components of pyruvate dehydrogenase enzyme complex have been identified as the major antigens within the M2 group of autoantigens. An immunoassay using pyruvate dehydrogenase enzyme complex as a specific antigen for the diagnosis of PBC was developed. Pyruvate dehydrogenase enzyme complex was attached to polystyrene microbeads, incubated with sera from PBC patients (n = 18), normal controls (n = 50), or patients with other autoimmune diseases (n = 26), followed by incubation with a second fluorescein isothiocyanate conjugated goat anti-human immunoglobulin and then analyzed by flow cytometry. High numbers of fluorescence channels (mean, 1,693 +/- 846) were obtained for all PBC sera except for two patients. Compared to the conventional anti-mitochondrial antibody assay, the assay had a sensitivity rate of 94% and a specificity rate of 100%. The reactive antibodies are predominantly of the immunoglobulin G3 subclass. Their levels could be correlated with the histopathologic stages of PBC. These results were corroborated by immunoblotting. Sera from patients with later stages of PBC strongly reacted with pyruvate dehydrogenase enzyme complex components, E1 alpha, and protein X.

Antibodies↗
PubMed1992

Quality improvement in the laboratory assessment of in vitro fertilization.

There has been a trend to use fewer laboratory tests during ovarian hyperstimulation prior to oocyte recovery, in vitro fertilization, and embryo transfer. Estradiol is routinely monitored during ovulation induction protocols. Estradiol rises steadily to supraphysiologic concentration during ovarian hyperstimulation. Review of the College of American Pathologists proficiency testing data from 1990 reveal that the within-method interlaboratory coefficient of variation meets the proposed maximum allowable analytical error of 11.8%. The luteinizing hormone level increases during ovarian hyperstimulation with a skewed distribution. Molecular variants exist that may bind with different affinities to monoclonal antibodies used in two-site sandwich assays. Polyclonal radioimmunoassays usually overestimate luteinizing hormone concentration. The College of American Pathologists proficiency testing data reveal that only three of eight methods with greater than 20 participants had a within-method interlaboratory coefficient of variation that met the proposed maximum allowable analytical error of 10% or less. International standardization of reference standards, antibodies, and labeling methods may improve the performance of this assay. The low pregnancy rate experienced by in vitro fertilization protocols suggests that additional laboratory tests need to be developed to monitor the receptivity of the endometrium for implantation and the quality of the oocyte and embryo.

Fertilization in Vitro↗
PubMed1991

Fluorometric determination of phosphatidylcholine as a measure of phospholipid methylation.

The successive methylation of phosphatidylethanolamine to phosphatidylcholine (phospholipid methylation) has been measured by the incorporation of S-[methyl-3H]adenosylmethionine or colorimetric assay of phosphatidylcholine extracted from adipocyte plasma membranes. A fluorometric assay for phosphatidylcholine was developed to measure phospholipid methylation. This assay is 10 times more sensitive than the colorimetric assay and demonstrates no significant interference with other methylated phospholipids. The fluorometric assay was used to determine a biphasic insulin dose response in adipocyte plasma membranes. This fluorometric assay for phosphatidylcholine represents an alternative method for monitoring phospholipid methylation, especially when increased sensitivity is required.

Adipose Tissue↗
PubMed1990

The effect of amino acids, monoamines and polyamines on pyruvate dehydrogenase activity in mitochondria from rat adipocytes.

The ability of polyamines and other cationic compounds including monoamines, amino acids, poly-L-arginine, poly-D-lysine and poly-L-lysine, to alter pyruvate dehydrogenase (PDH) activity in mitochondria from rat epididymal adipocytes was determined. PDH was assayed with the substrate [1-14C] pyruvate in the presence of 0.05 mM Ca2+ and Mg2+. Nine of the fourteen compounds tested at 0.1 mM caused a significant increase (procaine, 3-(beta-morpholinopropionyl) benzo [b]thiophene [VII], spermine, spermidine, putrescine, lysine and tryptophan) or decrease (poly-L-arginine, 3-(beta-piperidinopropionyl) benzo[b]thiophene) in PDH activity. None of these compounds nonenzymatically decarboxylated [1-14C] pyruvate to release 14CO2. NaF, a PDH phosphatase inhibitor, suppressed the stimulatory effects of those compounds tested: procaine, tryptophan, VII, spermine and spermidine. These results imply that these five compounds activate PDH activity through stimulation of the PDH phosphatase. When the Mg2+ concentration was increased from 0.05 to 4.5 mM, the stimulatory effect of spermine was increased, consistent with the finding by others that spermine lowers the Km of the enzyme for Mg2+. However, at Mg2+ concentrations greater than 0.3 mM, the stimulatory effect of VII was unaltered, procaine failed to alter PDH activity, lysine inhibited PDH activity, and poly-L-lysine stimulated PDH activity. Therefore, polyamines and other positively charged small molecules may be physiologic regulators of PDH activity.

Adipose Tissue↗
PubMed1990

A comparison of chromogen test strip (Chemstrip bG) and serum glucose values in newborns.

Although glucose oxidase-peroxidase chromogen test strips are frequently used to estimate serum glucose values in newborns, previous studies have not evaluated multiobserver variability of test strip readings and have included few infants with hypoglycemia. We compared values of 272 samples of serum glucose with values simultaneously obtained by chromogen test strips (Chemstrip bG) in newborns. The diagnostic sensitivity of a chromogen test strip less than 2.2 mmol/L for predicting a serum glucose level less than 1.9 mmol/L was 86% (95% confidence interval [CI], 75% to 94%), with 78% specificity (95% CI, 73% to 84%). The positive predictive value in our specimens, with a 21% prevalence of serum glucose levels less than 1.9 mmol/L, was 52% (95% CI, 41% to 62%), with a negative predictive value of 95% (95% CI, 91% to 100%). Fifty-eight of our serum glucose values were less than 1.9 mmol/L and the levels obtained by chromogen test strip were greater than or equal to 2.2 mmol/L in 8 of these cases. Review of these 8 cases showed that a delay in performing the laboratory glucose oxidase serum glucose could account for the discrepancy in 2 cases. Chromogen test strips are readily available and easy to use, but more sensitive, specific, accurate, and precise methods of serum glucose screening in newborns are needed.

Blood Glucose↗