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Biomedical subjects

C W Gehrke

Publications and source records attributed to C W Gehrke.

At least 91 records · Page 5Linked to original sources
PubMed1977

Gas-liquid chromatography of fecal neutral steriods.

A method is described for the analysis of fecal neutral steriods with a dual-column gas-liquid chromatography (GLC) system. After saponification of the fecal slurry, the neutral steroids were extracted with hexane. The GLC separation of the compounds and quantitation were achieved by simultaneous injection of the derivatized and derivatized aliquots of the extract onto dual colmuns under identical conditions. The neutral steroids of interest were than identified by matching the retention times with those of known standards, and identification was confirmed by use of an interfaced GLC high-resolution mass spectrometry system. The detection limit was 0.003 mg of steroid/g of fecal slurry. The pricision of the method is illustrated by a relative standard diviation of 2-10% and a recovery of neutral steroids from 73-96%. The method was applied to the determination of fecal neutral steroids in a "High protein diet in colon cancer study". A considerably larger level of coprostanone than of coprostanol was observed. Data on neutral steroids in fecal samples from subjects on different diets are the subject of a separate publication.

Cholestanes↗
PubMed1977

Gas-liquid chromatography of nucleosides. Effect of silylating reagents and solvents.

The aims of this investigation were to study the completeness of silylation of nucleosides with three different reagents, bis(trimethylsilyl)trifluoroacetamide (BSTFA), bis(trimethylsilyl)acetamide (BSA) and trimethylsilylimidazole (TMSI), and to investigate the effect of different solvents (acetonitrile, pyridine, dimethylformamide, chloroform, methylene chloride, hexane, benzene, and toluene) on quantitation of derivatization. Closed-tube silylations of the nucleosides were performed with BSTFA, BSA, and TMSI, and for the most complete silylation, the optimal time, temperature, and molar excess of reagent were: for BSTFA, 150 degrees-15 min and 225 molar excess; for TMSI, 60 degrees-3 h and 1000 molar excess; and for BSA, 120 degrees-2 h and 250 molar excess. Also, silylations of seven major and minor nucleosides were carried out using a 1000 molar excess of BSTFA, BSA, and TMSI at 25 degrees with 5 min sonication, and at optimal silylation conditions as described above for the three reagents. The silylating strengths were determined by the increase in RWR (= weight response of nucleoside/weight response of pyrene) values, and are summarized for the amino group containing nucleosides silylated at room temperature as BSTFA greater than TMSI greater than BSA, and for silylation under optimal conditions as BSTFA greater than BSA greater than TMSI. The efficiency of silylation for the hydroxyl group-containing nucleosides silylated at room temperature was BSTFA greater than TMSI greater than BSA, and for silylation under optimal conditions BSTFA greater than TMSI = BSA...

Chromatography, Gas↗
PubMed1977

Characterization of a unique enzyme complex composed of S-adenosyl-L-methionine-tRNA-methyltransferase and aminoacyl-tRNA synthetase activities.

S-adenosyl-L-methionine-tRNA methyltransferases of a murine leukemia cell line were found to exist in a high molecular weight enzyme complex. Aminoacyl-tRNA synthetase activity always co-chromatographed and co-sedimented with methyltransferase activity in evidence of a unique association of these two groups of enzymes. Molecular weight studies showed a probable molecular weight of 9 X 10(5) daltsons for the intact complex which dissociates to complexes of 6 X 10(5) and 3 X 10(5) daltons. The complexes contain discrete polypeptides of 25,000-90,000 daltons as determined from SDS-gel electrophoresis. High resolution fatty acid analysis showed that only very small amounts of saponifiable lipids were associated with the purified enzyme complex. Similarly very little protein-bound sugars was found within the complex indicating that neither lipids nor sugars were involved in the protein-protein interactions of the complex. Analysis of tRNA methylated in vitro indicated the presence of most methyltransferase activities in the purified complex. Of note was the absence from the complex of the methyltransferase responsible for the production of ribo Tp.

Amino Acyl-tRNA Synthetases↗
PubMed1977

Fatty acid profile and cholesterol in skeletal muscle of trained and untrained men.

To compare the fatty acid distribution and cholesterol composition in trained and untrained isolated skeletal muscle membranes, a needle biopsy was performed on the vastus lateralis of the 10 distance runners and 10 sedentary men. The muscle sample was homogenized and centrifuged at 100,000 X g; the resulting pellet was analyzed by gas-liquid chromatography for individual fatty acids and cholesterol. The percentage of palmitic acid was significantly lower in the trained muscle tissue. Samples from the distance runners also tended to have a more frequent appearance of linolenic and eicosatrienoic acids, longer fatty acid hydrocarbon chains, and lower cholesterol concentration. It was concluded that trained muscles have an increased membrane fluidity which could beneficially affect the activity of membrane-bound enzymes and active transport. Longer chain length in the membrane lipids may be a means of producing an inner membrane cohesiveness in muscles of trained individuals.

Adult↗
PubMed1977

Composition, associated tissue methyltransferase activity, and catabolic end products of transfer RNA from carcinogen-induced hepatoma and normal monkey livers.

This investigation was designed to explore transfer RNA (TRNA) methyltransferase activity, urinary excretion levels of tRNA degradation products, and tRNA base composition in normal monkeys and in those with hepatocellular carcinomas induced by N-nitrosodiethylamine. After the development of the tumor, 24-hr urine specimens were collected, the monkeys were sacrificed, and the livers were removed for tRNA isolation and methyltransferase activity studies. The tRNA methyltransferase activity and capacity and the urinary excretion levels for selected tRNA degradation products (pseudouridine, N2,N2-dimethylguanosine, 1-methylinosine, 7-methylguanine, and beta-aminoisobutyric acid) were elevated for the hepatoma-bearing monkeys when compared to those with normal liver. The isolated tRNA pools were analyzed by high-resolution liquid chromatography, and similar base compositions were found for the hepatoma-bearing and normal monkeys. With the use of methyl-deficient Escherichia coli tRNA as the methyl receptor and the analytical procedure for tRNA anlysis, the methylating ability of the tRNA methyltransferases in hepatoma and normal liver extracts was determined. The hepatoma methyltransferase homogenates were found to produce increased levels of 7-methylguanine, N2,N2-dimethylguanine, and thymine, while the normal liver extracts gave higher levels of N2-methylguanine. These differences were not apparent in the base composition of the tRNA pools. The increased urinary excretion and higher methyltransferase activity of the hepatoma-bearing monkeys without an apparent increase in the methylated base content of their tRNA suggest increased tRNA tf individual isoaccepting tRNA's would be missed by analyzing the tRNA pools. The variations in the individual tRNA methyltransferase activities of the hepatoma and normal liver homogenates indicate a difference in the methlation of their tRNA's.

Animals↗
PubMed1977

High turnover rate of transfer RNA in tumor tissue.

Cancer patients and tumor-bearing animals excrete high levels of modified purines and pyrimidines some of which, e.g., N2,N2-dimethylguanosine, can originate only from transfer RNA (tRNA). Until recently, it could not be ascertained whether the high level of excretion of such compounds is due to cell death or specific tRNA turnover. However, an approach to this problem became feasible, with beta-aminoisobutyric acid as a probe. This compound is a terminal degradation product of thymine which is present in both DNA and tRNA. Since the pathway of synthesis of thymine is different in the two macromolecules, it and its end product, beta-aminoisobutyric acid can be differentially labeled with [14C]formate and [3H3]methylmethionine as precursors. Therefore the ratio of the two labels in the excreted beta-aminoisobutyric acid is a measure of the macromolecular origin of the degradation product. We have found from such analysis that tRNA's are not homogeneous in their turnover rate. There is a subpopulation that turns over much faster than the rest. The turnover rate of a subpopulation of tRNA's in tumor tissue exceeds the turnover rate of tRNA's in normal tissue. Such rapid degradation of tRNA's must be the source of the massive excretion of modified nucleosides by cancer patients which can be 10-fold higher than in normal subjects.

Aminoisobutyric Acids↗
PubMed1976

Gas-liquid chromatography of nucleosides. Derivatization and chromatography.

The aims of this investigation were to establish the optimum reaction conditions for silylation of nucleotides with bis(trimethylsilyl)trifluoroacetamide (BSTFA) and to investigate the chromatographic properties of the following nucleosides: adenosine, guanosine, cytidine, thymine, inosine, xanthosine, and uridine. Closed tube silylations were performed with a 1000 molar excess of BSTFA at 25, 75, 120, 150, and 175 degrees for 15, 30, 60, 120 and 240 min. The optimal time and temperature for derivatization were found to be 150 degrees and 15 min. Using these reaction conditions, samples were then silylated with 50, 100, 200, 500, and 1000 molar excess of BSTFA; a molar excess of 225 was best. The stability of the nucleoside derivatives on standing at room temperature for 1-7 days was investigated. Quantitative gas-liquid chromatography of trimethylsilyl (TMS) nucleosides can be performed if samples are analyzed within 48 h, after which time the relative weight response for the nucleosides decreased somewhat. Chromatographic column studies were made using various liquid phases and supports. With methylsiloxanes as the liquid phase, Supelcoport as support was found to be superior. Resolution of TMS guanosine from TMS cytidine was attempted at different column lengths using 3% (w/w) SE-30 on Supelcoport, different loadings (%, w/w) of SE-30 on Supelcoport, and different polarity liquid phases, 4% (w/w) OV-11 or 3% (w/w) OV-17 or 4% (w/w) Dexsil-300 on Supelcoport. A complete separation of the six ribonucleosides including guanosine and cytidine was obtained with 1 m x 4 mm I.D. glass columns of 4% (w/w) OV-11 and 3% (w/w) OV-17 on 100-120 mesh Supelcoport. It was observed that with 2'-deoxycytidine, one obtains the TMS cytosine peak (retention temperature 150 degrees) plus another peak with a retention temperature of 120 degrees, under all derivatization conditions with BSTFA and bis(trimethylsilyl)-acetamide. Similar formation of bases from other 2'-deoxyribonucleosides occurred when the molar excess of BSTFA was greater than 500. The minimal detectable amounts obtained for all the nucleosides ranged from 5 to 10 ng injected with a signal-to-noise ratio of 3. The relative standard deviations for all nucleosides and deoxynucleosides ranged from 1.2 to 4.8% on different methylsiloxanes on Supelcoport columns.

Adenosine↗
PubMed1976

Determination of trimethylsilyl methylated nucleic acid bases in urine by gas-liquid chromatography.

A method for the determination of the urinary excretion level of methylated nucleic acid bases by gas-liquid chromatography (GLC) has been developed. A clean-up procedure prior to GLC analysis consisted of hydrolysis, filtration, charcoal adsorption, and anion exchange. Studies to determine optimum derivatization conditions for conversion of the methylated bases to their trimethylsilyl derivatives and to evaluate GLC parameters and columns to obtain the best separation were conducted. Application of the method was shown by determining the excretion levels of methylated bases in urine of normal controls and of patients with various types of malignancy.

Adenine↗
PubMed1976

Beta-aminoaciduria in patients with Burkitt's lymphoma.

The massive amounts of beta-aminoisobutyric acid (beta-AIBA) in the urine of Burkitt's lymphoma patients were measured along with other alpha-amino acids and beta-alanine present in normal and decreased levels. The ratios of the amount of beta-AIBA to beta-alanine, in mumoles/kg urine collected in 24 hours, were elevated for all patients. The degree of elevation of beta-AIBA excretion and the ratio of the two beta-amino acids appeared to be related to the amount of tumor mass present. These analyses may have possible value in monitoring patients with Burkitt's lymphoma during their disease course.

Alanine↗
PubMed1975

Biological markers in breast carcinoma. I. Incidence of abnormalities of CEA, HCG, three polyamines, and three minor nucleosides.

Patients with breast carcinoma were screened for abnormal concentrations of CEA, HCG, putrescine, spermidine, spermine, pseudouridine, N2, N2-dimethylguanosine, and 1-methylinosine. Abnormal polyamine levels occurred in less than 15% of the patients. Among the nucleosides, N2, N2-dimethylguanosine was the most frequently abnormal, occurring in 57% of the patients with metastatic disease. CEA levels were abnormal in 30% of postoperative N+ patients and 74% of patients with metastatic disease, while HCG elevations were found in 45% and 50%, respectively. All the patients with one or more marker abnormalities could be detected by measuring only CEA, N2, N2-dimethylguanosine, and HCG. Among these three tests, a singular marker abnormality occurred in 35.8% of the patients, and all three tests were abnormal in 21.8% of the patients. The performance of these three tests in each patient revealed one or more abnormalities 97% of the patients with metastatic disease, and 67% of the postoperative N+ patients.

Breast Neoplasms↗
PubMed1975

The urinary excretion of nucleosides of ribonucleic acid by patients with advanced cancer.

By means of a sensitive and specific method utilizing gas-liquid chromatography, the excretion levels for three nucleosides, degradation minor base products of ribonucleic acid, primarily transfer ribonucleic acid, were determined in 24-hour urine specimens from over 200 patients with solid tumor malignancies. These nucleosides were N2,N2-dimethylguanosine, l-methylinosine, and pseudouridine. When compared to normal control values, elevated levels of these compounds were found for patients in each of several tumor types studied. Increases in pseudouridine excretion suggest increased tumor transfer ribonucleic acid turnover; in addition, for the methylated nucleosides, higher than normal values may reflect enhanced transfer ribonucleic acid methylase activity of the neoplastic cells.

Chromatography, Gas↗
PubMed1975

Search for amino acids in Apollo returned lunar soil.

The lunar samples from Apollo flights 11 through 17 provided the students of chemical evolution with an opportunity of examining extraterrestrial materials for evidence of early prebiological chemistry in the solar system. Our search was directed to water-extractable compounds with emphasis on amino acids. Gas chromatography, ion-exchange chromatography and gas chromatography combined with mass spectrometry were used for the analysis. It is our conclusion that amino acids are not present in the lunar regolith above the background levels of our investigations.

Amino Acids↗