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At least 613 records · Page 34Linked to original sources
PubMed1989

Isocratic high-performance liquid chromatographic determination of the concentration and specific radioactivity of phosphoenolpyruvate and uridine diphosphate glucose in tissue extracts.

A rapid and efficient isocratic high-performance liquid chromatographic method for studying the metabolism of phosphoenolpyruvate and uridine diphosphate glucose (UDPG) has been developed. For each compound this method can measure tissue concentrations in the range 0.1-1000 nmol/g of tissue and determine specific radioactivity. All measurements can be performed in 200 mg of tissue. The recoveries of uridine diphosphate [6-3H]glucose and phosphoenol[1-14C]pyruvate from liver tissue homogenates were 97 and 99%, respectively. Following intra-arterial infusion of [6-3H]glucose and [U-14C]lactate in conscious rat, the concentration and specific radioactivity of phosphoenolpyruvate and UDPG were determined in rat liver. The method may be applied to experimentation in small animals using radiolabelled precursors in order to quantitate in vivo the glycogenic and gluconeogenic fluxes.

Animals↗
PubMed1989

CA 125 in normal tissues and carcinomas of the uterine cervix, endometrium and Fallopian tube. II. Immunoradiometric determination in secretions, tissue extracts and serum.

The study deals with the occurrence of cancer antigen 125 (CA 125) in the normal and neoplastic uterine cervix, endometrium and fallopian tube and its applicability as a tumour marker. CA 125 concentrations were measured in 52 secretion specimens, in cytosol fractions of 97 tissue biopsies and in serum from 47 women with nonmalignant disorders and from 334 patients with carcinomas. High quantities of CA 125 (780-454860 U/ml) were detected in cervical mucus, intra-uterine and tubal fluid, exceeding those in the corresponding serum samples by factors of up to 2000. CA 125 concentrations were 9-53 fold higher in cytosol fractions of normal and neoplastic glandular epithelia of the endocervix and endometrium than in those of cervical squamous epithelia and the cervical wall. Despite similarly high antigen concentrations in normal glandular epithelia and adenocarcinomas serum levels elevated to above 65 U/ml were only found in patients with malignant tumours. The positivity rates in serum increased with tumour extent and were 0-43% for primary and 63-79% for recurrent cervical, endometrial and tubal adenocarcinomas. During long-term follow-up, CA 125 serum concentrations were concordant with the clinical course in 10 out of 11 patients with progressive carcinomas. According to these results, the release of CA 125 into the peripheral blood is apparently dependent on the infiltrative growth and the mass of the tumour rather than on the local tissue concentrations. The clinical use of CA 125 is limited to the detection of advanced adenocarcinomas of the Müllerian duct.

Adenocarcinoma↗
PubMed1985

Presence of the predicted C-flanking peptide of neuropeptide Y (CPON) in tissue extracts.

Isolation of the DNA sequence encoding neuropeptide Y (NPY) has suggested the presence of a precursor peptide consisting of 97 aminoacids. Three peptide products have been predicted from this precursor; an N terminal signal peptide, neuropeptide Y and a C terminal 30 amino acid flanking peptide. This study has demonstrated by a novel radioimmunoassay that this C-flanking peptide for NPY occurs naturally in tissues. The peptide is present in abundance throughout the central nervous system and qualitatively in both the brain and peripheral structures, it has the same distribution as NPY. Chromatographic analysis demonstrated that the measured CPON-immunoreactivity elutes in the position of the peptide synthesised according to the predicted sequence.

Adrenal Glands↗
PubMed1986

Measurement of free and esterified carnitine in tissue extracts by high-performance liquid chromatography.

A high-performance liquid chromatographic (HPLC) method for the determination of L-carnitine in clamped and frozen rat livers is described. L-carnitine + acetyl-CoA in equilibrium with acetyl-L-carnitine + CoASH Using the above enzymatic reaction, release of CoASH is stoichiometric with the L-carnitine added. The present method has made possible the determination of carnitine in liver tissues, which is difficult by the conventional enzymatic spectrophotometric method using 5,5'-dithiobis(2-nitrobenzoic acid), owing to acetyl-CoA hydrolysis during prolonged incubations at pH 7.8.

Acetyl Coenzyme A↗
PubMed1984

Immunological identification of seminalplasmin in tissue extracts of sex glands of bull.

Using immunoglobulin G (IgG) antibodies raised against highly purified, homogeneous seminalplasmin, an antimicrobial protein of bovine seminal plasma, it has been shown that bovine ampullae, gland vesicularis and corpus prostate, but not testes and epididymis, contain seminalplasmin. The content as estimated by radioimmunoassay employing 125I-seminalplasmin was: ampullae, 267 +/- 13; gland vesicularis, 275 +/- 14; and corpus prostate, 445 +/- 22 micrograms per g wet weight of the tissue. Seminalplasmin, as characterized by high-performance liquid chromatography and in vivo inhibition of RNA synthesis in E. coli, was isolated from gland vesicularis. The seminalplasmin content of bovine seminal plasma was shown to be 1%. A chymotryptic peptide of seminalplasmin comprising residues 1-13 from the amino terminus was found to compete with 125I-seminalplasmin for binding to anti-seminalplasmin IgG.

Animals↗
PubMed1992

Thin-layer chromatographic detection of zeranol and estradiol in fortified plasma and tissue extracts with Fast Corinth V.

In an attempt to improve sensitivity of thin-layer chromatographic (TLC) analysis and selectivity of visualizing agents for detection of estrogenic anabolic hormones, several dyes were screened for their chromogenic interactions with estrone, estradiol, diethylstilbestrol (DES), zeranol (zearalanol), zearalanone, and mycotoxins, zearalenone and zearalenol. Fast Corinth V salt was selected for its relatively high sensitivity. These anabolic compounds were separated by TLC and visualized with Corinth V and the results compared to iodine and starch visualization. Fortified bovine plasma and tissues (kidney, liver and muscle) and chicken muscles were analyzed after a clean-up procedure using solid-phase dual columns of alumina and anion-exchange resin. Iodine-starch clearly detected 4 ng of estradiol and DES while zeranol and zearalenone were detected at higher levels (10 ng). Fast Corinth V showed distinct spots with 2 ng of zeranol and 4 ng of zearalenone while faint spots were observed with estradiol and estrone standards. DES was not detectable at these levels. Less background interference was observed with Corinth V than with iodine-starch. The former confirmed spots detected by iodine-starch. This study suggests its selectivity for detection of zeranol and its metabolite, zearalanone, in the presence of steroidal compounds.

Animals↗
PubMed1989

Confirmation of D-aspartic acid in the novel dipeptide beta-aspartylglycine isolated from tissue extract of Aplysia kurodai.

A novel o-phthalaldehyde-reactive compound was found in the h.p.l.c. chromatogram of Aplysia kurodai extract. This compound was isolated by ion-exchange chromatography and preparative high-voltage paper electrophoresis. It was shown by optical-rotatory-dispersion spectrum and optical-resolution h.p.l.c. analysis that this compound consisted of equimolar amounts of D-aspartic acid and glycine. This compound resisted cleavage in the Edman reaction. This peptide was inferred to be beta-D-aspartylglycine, and this was confirmed by synthesis. beta-D-Aspartylglycine was detected in all tissues of Aplysia kurodai, with especially high concentrations in body wall (skin and muscle) and gill.

Animals↗
PubMed1995

Transfusion-associated graft-versus-host disease in a patient treated with Cladribine (2-chlorodeoxyadenosine): demonstration of exogenous DNA in various tissue extracts by PCR analysis.

Transfusion-associated graft-versus-host disease can occur in both immunocompetent and immunocompromised hosts. Cladribine is a synthetic analogue of adenine used in the treatment of lymphoid malignancies, commonly associated with a decrease in T lymphocytes. Cladribine was given for a low-grade non-Hodgkin's lymphoma with thrombocytopenia as the main side-effect. Six units of pooled non-irradiated platelets were transfused from six unrelated donors; 10 d later a clinical picture typical of graft-versus-host disease resulted. Polymerase chain reaction of the highly polymorphic DNA minisatellites and HLA-DR oligotyping were used to demonstrate the exogenous DNA. In the patient's blood and tissues, only the pattern of donor 5 was found. The patient (DRB1*0301/1101; DRB3*0101/02) and this donor (DRB1*0301/1104; DRB3*02) by chance shared a partial common haplotype. This complication highlights the sensitivity of DNA minisatellite analysis. It further raises the question of transfusion and of prophylactic irradiation of all blood products in immunosuppressed patients and those treated with cladribine. This case represents a previously unreported situation where an immunosuppressed patient was able to eliminate cells from five totally HLA-DR dissimilar donors but not from one heterozygous donor with strong HLA-DR similarity.

Aged↗
PubMed1993

[Comparison of hepatic tissue extraction rates of cytotoxic anticancer drugs during hepatic arterial chemotherapy--evaluation using direct hemoperfusion under hepatic venous isolation].

Hepatic extraction rates (ER) of anticancer drugs during hepatic arterial infusion were investigated with the aid of direct hemoperfusion (DHP) under hepatic venous isolation (HVI). Using mongrel dogs (n = 6), adriamycin (ADR), mitomycin C (MMC) and cisplatin (CDDP) were simultaneously administered to the hepatic artery at each dosage of 1 mg/kg in 10 minutes under HVI.DHP. Hepatic venous flow and plasma concentrations of each drug at the carotid artery, the inlet and outlet sides of DHP were periodically determined during HVI.DHP. Based on these data, drug adsorption and removal rates were estimated. In addition, hepatic tissue uptakes of each drug were calculated from the amounts of drug administered and leaked in the hepatic effluent. Subsequently, the percentage of tissue uptake of each drug to the amount of drug administered was determined as ER of each drug. Drug adsorption rates during the first 10 minutes after infusion showed no significant difference among three drugs. Drug removal rate of CDDP tended to be higher than those of other two drugs. ER of CDDP (54.8 +/- 18.3%) were significantly lower (p < 0.01) as compared to ADR (84.4 +/- 16.2%) and MMC (83.1 +/- 15.7%). These results indicate that ER of each drug should be taken into consideration to determine appropriate drug for hepatic arterial chemotherapy.

Adsorption↗
PubMed1989

A sensitive enzyme immunoassay system of rat epidermal growth factor in biological fluids and tissue extracts.

Rat epidermal growth factor was purified from rat submandibular glands to obtain specific antiserum for the establishment of an immunoassay system. Purified rat epidermal growth factor showed a single peak on reverse phase HPLC and a single band on sodium dodecyl sulphate polyacrylamide gel electrophoresis at mol wt 5100 in the presence of 2-mercaptoethanol and at mol wt 41,000 in the absence of 2-mercaptoethanol. Antibody against rat epidermal growth factor showed cross-reactivities with mouse and human epidermal growth factors on soft agar-double immunodiffusion test. The established sandwich enzyme immunoassay for rat epidermal growth factor had a high sensitivity (500 fg/tube), which made it possible to measure minute amounts of endogenous rat epidermal growth factor without pretreatment. Physiological concentrations of rat epidermal growth factor in rat biological fluids and tissues were determined. Species differences in physiological distributions of epidermal growth factor are discussed.

Animals↗
PubMed1990

Measurement of alpha-ketoglutarate dehydrogenase activity in tissue extracts and human platelets using reversed-phase high-performance liquid chromatography.

A new method for the determination of the activity of alpha-ketoglutarate dehydrogenase complex (KGDHC) in mouse brain and liver mitochondria and in human platelets using reversed-phase high-performance liquid chromatography is described. This method is based on the quantification of succinyl-CoA formed in the reaction catalyzed by KGDHC. Succinyl-CoA was separated using a YMC-Pack C8 column employing isocratic elution and detected spectrophotometrically at 254 nm. The detection limit of succinyl-CoA was 0.05 nmol. Succinyl-CoA in the supernatant of the assay mixture was stable for several hours at 4 degrees C and for a week when stored at -20 degrees C. The KGDHC assay showed good linearity with time and added protein, and all tissues demonstrated an absolute requirement for added alpha-ketoglutarate, nicotinamide dinucleotide, and coenzyme A and partial or no requirement for thiamine pyrophosphate, magnesium chloride, and dithiothreitol. The specific activities in liver and brain mitochondria and platelet homogenates determined by the present method were 19.2 +/- 0.9, 18.1 +/- 2.8, and 2.6 +/- 0.3 nmol/min/mg protein, respectively. In human platelets, the present method gives higher specific activity and lower blank values than a prior method using 14CO2 and may be useful in the diagnosis of KGDHC deficiency. This method is simple, rapid, and can be readily employed for the determination of KGDHC activity in various animal tissues and human platelets.

Acyl Coenzyme A↗
PubMed1983

Measurement of nucleoside kinases in crude tissue extracts.

The measurements of deoxyadenosine kinase, adenosine kinase, and deoxycytidine kinase were examined in human placental cytosol to achieve a valid and reliable assay linear with time and protein. Our studies confirm the need to inhibit deaminase enzymes, since deoxyadenosine and deoxycytidine undergo extensive deamination and phosphorolysis. The use of a uniformly labeled nucleoside substrate introduced an artifact because the chromatographic behavior of the deoxyribose-1-phosphate, formed during the assay, was difficult to distinguish from the deoxynucleoside phosphate product. Accurate product identification was also essential. Finally, the substitution of GTP in place of ATP as the phosphate donor, the addition of a sulfhydryl reducing agent and a monovalent cation need to be considered when an assay is optimized. The use of these methods have lead to valid assays in placental cytosol that are linear with time and protein. Consideration of these important principles are necessary when establishing a valid and reliable nucleoside kinase assay in a crude tissue preparation.

Adenosine Kinase↗