Richard M. Halpern

Urinary excretion levels of unconjugated pterins in cancer patients and normal individuals.

Urinary excretion levels of seven unconjugated pterins in healthy individuals and in cancer patients, most of whom were undergoing chemotherapy, were measured utilizing a newly developed high-pressure liquid chromatographic system. Excretion of pterins in the control group appears to be under strict metabolic control as the values obtained were confined within a small range. When the mean excretion levels in control subjects were compared with those in cancer patients, we found a significant increase in the excretion of xanthopterin, neopterin and pterin and a significant decrease in isoxanthopterin by cancer patients. Biopterin levels, on the other hand, were found only slightly but not significantly increased, whereas pterin-6-carboxylic acid and 6-hydroxymethylpterin were found to be excreted in approximately equal amounts in both groups. Urinary excretion levels of pterins were monitored for a period of nine months in a patient being treated with chemotherapy for metastatic ovarian carcinomatosis. We found that the excretion pattern of pterins appeared to correlate with the clinical status of the patient. These results indicate that a definite imbalance in pterin, and possibly folate metabolism, is associated with the presence of malignant diseases.

N5-methyltetrahydrofolate: homocysteine methyltransferase activity in extracts from normal, malignant and embryonic tissue culture cells.

Abstract Malignant cells (J111, L1210, W-256) and human embryonic cells (FL) are unable to survive and grow when homocystine replaces methionine in tissue culture media containing excess vitamin B12 and folic acid. Extracts of these same cells when grown in media containing methionine and more than adequate vitamin B12 and folic acid have diminished N5-methyltetrahydrofolate: homocysteine methyltransferase activities in the absence of added cyanocobalamin when compared with extracts of normal cells (adult rat thymus and liver fibroblasts). Extracts of human monocytic leukemia (J111) and human amnion cells (FL) have normal enzymatic activity in the presence of added cyanocobalamin whereas the rodent malignant cells (W-256 and L1210) have abnormally low activity in the absence or presence of added vitamin B12.

A fluorimetric method for quantitation in the picomole range of N1-methylnicotinamide and nicotinamide in serum

Abstract A method is described for the fluorimetric determination of N 1 -methylnicotinamide in deproteinized serum extract and of nicotinamide after extraction into ethyl acetate from deproteinized serum extract and subsequent conversion to N 1 -methylnicotinamide. N 1 -methylnicotinamide is converted to fluorescent derivatives by treatment with acetophenone in alcoholic KOH followed by addition of 99% formic acid.

Nicotinamide: A natural inhibitor of tRNA methylase

Abstract A dialyzable tRNA methylase inhibitor has been isolated from rat liver. By chromatographic and electrophoretic data as well as ultraviolet and mass spectral data, the inhibitor is identified as nicotinamide. Kinetic evidence indicates that nicotinamide is an inhibitor of tRNA methylase.

Quantitative determination of pterins in biological fluids by high-performance liquid chromatography

During our continuing study of pteridine metabolism, the need arose for a more rapid and quantitative determination of pterins in biological fluids. By adopting and modifying previously developed techniques, we have obtained a rapid and sensitive method that allows the simultaneous determination of eight different pterins in human urine and blood. When examined over a 10-day period, the levels of pterins excreted by a normal individual averaged the following values expressed in picomoles per mg of creatinine: biopterin, 9104; neopterin, 6018; xanthopterin, 6561; pterin, 1136; isoxanthopterin, 636; pterin-6-carboxylate, 483; and 6-hydroxymethylpterin, 315. Moreover, 6-hydroxymethylpterin and pterin-6-carboxaldehyde were detected for the first time in the blood of normal individuals.

Separation of unconjugated pteridines by high-pressure cation-exchange liquid chromatography

In the course of determining the levels of unconjugated pteridines occurring in various biological fluids, such as urines, plasma and tissue culture media, a method has been developed for the separation and quantitative determination in the picomole range of ten 2-amino-4-hydroxy substituted pteridines. This method involves separation by high-pressure cation-exchange liquid chromatography and fluorescence detection of the eluted compounds at 450 nm. Optimal separation was obtained by isocratic elution with 3 mM phosphoric acid-7% methanol-1% acetonitrile at a flow-rate of 2 ml/min or with 1 mM ammonium dihydrogenphosphate pH 2.8-7% methanol-5% acetonitrile at a flow-rate of 1.5 ml/min. With either solvent, the order of elution of the compounds is: isoxanthopterin, pterin-6-carboxylic acid, xanthopterin, pterin-6-carboxaldehyde, D-erythro-neopterin, L-threo-neopterin, biopterin, 6-hydroxymethylpterin, pterin, 6-methylpterin. In addition, a systemic investigation of the effects of ammonium ion concentration and pH of the solvent as well as column temperature on the separation of these compounds was also conducted.

Phosphorylation on basic amino acids in myelin basic protein

Abstract Isolated rat brain myelin when incubated with γ 32 P labelled ATP yields proteins bearing acid labile, base stable phosphoryl groups. Phosphorylated myelin basic protein can be isolated and degraded with trypsin and pronase to yield principally phosphoarginine and phosphohistidine. Only a very small amount of phosphorerine survives the base treatment used in the isolation procedure.

Chapter 15. Chromosomal Protein Phosphorylation on Basic Amino Acids

Publisher Summary Phosphorylation of chromosomal proteins has generally been measured either after isolation of phosphorylated chromosomal proteins from the nucleus or after the action of a chromosomal protein kinase in an in vitro system. These procedures involve one or the other step carried out at an acidic pH value such that the only surviving phosphoryl linkage is found on the hydroxyl group of serine or threonine. The single most outstanding property of N -phosphorylated compounds is their extreme sensitivity to acidic pH and their relative stability under basic conditions. The procedure described in this chapter permits the detection of N -phosphoryl amino acids in chromosomal proteins based upon isolation of 32 P-labeled N -phosphoryl substances. This procedure has been used directly with rat liver preparations, rat mammary tumor carcinosarcoma, or directly from enzyme reaction mixtures.

Transfer RNA methylase inhibitors in neoplastic and normal rat tissue

Abstract With acid precipitation and dialysis, we show the presence of tRNA methylase inhibitors in normal adult rat liver. These inhibitors are virtually absent from the cortex of the highly malignant Walker-256 carcinoma from the rat. The tRNA methylase inhibitors of the rat liver are slowly dialyzable. It is proposed that the differences in the rate of tRNA methylase activity of normal and neoplastic tissues is in large part due to the difference in inhibitor content of each tissue.

A method for determination of methionine containing radioactivity in the thiomethyl moiety

Abstract A method is described for the determination of methionine containing a radioactive label in the thiomethyl moiety. Cyanogen bromide in aqueous acetic acid is used to convert the labeled thiomethyl moiety to labeled methylthiocyanate which is then extracted into toluene and counted by liquid scintillation.