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.

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.

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.

Malignant cells grown in culture excrete into their growth medium a folate catabolite that can be seen as a blue-fluorescent region on paper chromatograms of such media. This folate catabolite has now been identified by paper chromatography, thin-layer chromatography, and combined gas chromatography-mass spectrometry as 6- hydroxymethylpterin and not as pterin-6-carboxaldehyde as previously reported. Moreover, when pterin-6-carboxaldehyde was added to the growth medium of logarithmically growing malignant cells, it was primarily reduced to 6-hydroxymethylpterin. In contrast pterin-6-carboxylate was the principal product formed from added pterin-6-carboxaldehyde by normal established cell lines in culture. These results have been interpreted as indicative of a possible mechanism of folate catabolism in malignant cells. Folic acid or another folate derivative is oxidatively cleaved at the C-9-N-10 bond to yield pterin-6-carboxaldehyde as one of the products. This derivative is subsequently reduced to 6-hydroxymethylpterin, which is excreted into the growth medium.

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 32P-labeled N-phosphoryl substances. This procedure has been used directly with rat liver preparations, rat mammary tumor carcinosarcoma, or directly from enzyme reaction mixtures.

In studies of the phosphorylated proteins in rat liver and Walker-256, it was established that the ratio of various fractions of P-N linkages to P-O linkages varies from 0.6 to 3.1. In rat regenerating liver nuclei, the ratio of P-N and P-O varies with time after partial hepatectomy. Using [3H]-lysine and 32Pi, it is shown that phosphoryllysine forms in some new and, presumably, some preexisting H1 molecules. Using [3H]histidine and 32Pi, it is shown that phosphohistidine forms exclusively in preexisting H4. The half-life of H4 phosphohistidine appears to be about 2 h.

Isolated rat brain myelin when incubated with γ32P 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.

Levels of nicotinamide and N-1-methylnicotinamide in serum, liver, and kidney as well as renal clearances and 24-hr urine levels of N-1-methylnicotinamide were compared in normal rats and rats bearing Walker 256 tumors. There was no significant difference between normal and tumor-bearing rats with regard to nicotinamide levels. With regard to N-1-methylnicotinamide, tumor-bearing rats had significantly lower serum and liver levels and significantly higher 24-hr urine levels and renal clearances. Walker 256 tumor tissue and liver and kidney from a normal and a tumor-bearing rat were separately examined for S-adenosylmethionine:nicotinamide methyltransferase activity. The specific activity in tumor tissue extract was greater than that in each liver extract, which, in turn, was much greater than the specific activity in each tissue (liver and kidney) from the tumor-bearing rat was equal to the specific activity in the corresponding tissue of the normal rat. S-adenosylmethionine:nicotinamide methyltransferase was obtained with 18-fold purification from a tissue extract of Walker 256 tumor. The enzyme activity required activation by thiols, and maximal activity was observed at pH 8.6. The Km's for the substrates, S-adenosylmethionine and nicotinamide, were 7.0 x 10--3 mM and 0.50 mM respectively. The Ki's for the products, S-adenosylhomocysteine and N-1-methylnicotinamide, were respectively, 25 x 10--3 mM and greater than 5 mM.

The selective toxicity of antifolates for a variety of cancers can be improved, as illustrated by the combined administration of N5-methyltetrahydrofolate and methotrexate in tissue culture. When a variety of neoplastic cell types characterized by a deficiency of vitamin B12-dependent N5-methyltetrahydrofolate methyltransferase (5-methyltetrahydropteroyl-L-glutamate:L-homocysteine S-methyltransferase, EC 2.1.1.13) and normal adult cells are grown in media containing methotrexate and either N5-methyltetrahydrofolate or N5-formyltetrahydrofolate, not only is the selective toxicity of methotrexate demonstrated, but the advantage of using N5-methyltetrahydrofolate in place of N5-formyltetrahydrofolate is also revealed. The implications and applications of this particular combination in the treatment of human cancer are discussed.

A method is described for the fluorimetric determination of N1-methylnicotinamide in deproteinized serum extract and of nicotinamide after extraction into ethyl acetate from deproteinized serum extract and subsequent conversion to N1-methylnicotinamide. N1-methylnicotinamide is converted to fluorescent derivatives by treatment with acetophenone in alcoholic KOH followed by addition of 99% formic acid.

A new procedure for the fractionation of mucopolysaccharides based upon differences in their partition behavior in aqueous polymer two-phase systems has been devised. Systems containing dextran, poly(ethylene glycol), trimethylamino-poly(ethylene glycol), potassium bromide and sodium phosphate buffer were employed. Countercurrent distributions were performed with a miniature countercurrent distribution device designed especially for use with aqueous polymer two-phase systems. An advantage over the widely used procedures involving precipitation of mucopolysaccharides as their quaternary ammonium detergent complexes is that the countercurrent distribution pattern of a particular mucopolysaccharide is not affected by the simultaneous presence of other mucopolysaccharides. Preliminary distributions of labelled mucopolysaccharides isolated from the cells and culture medium of monolayer cultures of rat tumor cells demonstrate that the procedure is particularly well suited for the fractionation of very minute quantities of mucopolysaccharides.

When malignant W-256 rat breast carcinosarcoma cells are mixed with an equal number of normal adult rat liver fibroblasts and allowed to grow in a medium containing sufficientl-methionine and an excess of vitamin B12 and of folic acid, the malignant cells outgrow the normal cells, and within 2 weeks the tissue culture flasks contain only neoplastic cells. However, when ampledl-homocystine or homocysteine replaces methionine in the medium containing the same amount of vitamin B12 and folic acid, and seeded with the same type and number of malignant and normal cells, the malignant cells die and the normal cells thrive. Substantiating this concusion are the results of injections into rats of comparable numbers of cells from each group after 3 weeks of growth in tissue culture. Fatal malignancies are produced by the methionine-cultured cells, whereas no malignancies are produced by the homocystein-cultivated cells.

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.

ADVERTISEMENT RETURN TO ISSUEPREVArticleNEXTCharacterization of protein kinases forming acid-labile histone phosphates in Walker-256 carcinosarcoma cell nucleiDonald L. Smith, Chi-Ching Chen, Berndt B. Bruegger, Steven L. Holtz, Richard M. Halpern, and Roberts A. SmithCite this: Biochemistry 1974, 13, 18, 3780–3785Publication Date (Print):August 1, 1974Publication History Published online1 May 2002Published inissue 1 August 1974https://pubs.acs.org/doi/10.1021/bi00715a025https://doi.org/10.1021/bi00715a025research-articleACS PublicationsRequest reuse permissionsArticle Views164Altmetric-Citations74LEARN ABOUT THESE METRICSArticle Views are the COUNTER-compliant sum of full text article downloads since November 2008 (both PDF and HTML) across all institutions and individuals. These metrics are regularly updated to reflect usage leading up to the last few days.Citations are the number of other articles citing this article, calculated by Crossref and updated daily. Find more information about Crossref citation counts.The Altmetric Attention Score is a quantitative measure of the attention that a research article has received online. Clicking on the donut icon will load a page at altmetric.com with additional details about the score and the social media presence for the given article. Find more information on the Altmetric Attention Score and how the score is calculated. Share Add toView InAdd Full Text with ReferenceAdd Description ExportRISCitationCitation and abstractCitation and referencesMore Options Share onFacebookTwitterWechatLinked InRedditEmail Other access optionsGet e-Alertsclose Get e-Alerts

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.

In tissue cultures of normal adult and malignant mammalian cells, homocystine has been substituted for methionine in a medium rich in folic acid and cyanocobalamin. Normal adult cells thrive. Three highly malignant cell types from three different species, including man, die.