Quantification of 5'-deoxy-5'-methylthioadenosine in heat-treated natural rubber latex serum.

INTRODUCTION: 5'-Deoxy-5'-methylthioadenosine (MTA) is one of the biologically active components found in natural rubber latex (NRL) serum, a common waste product from rubber plantations. In this study the contents of MTA in heat-treated NRL serum were measured in order to assess the potential of the serum as an alternative source of MTA. OBJECTIVE: To devise an HPLC/UV-based quantitative analytical protocol for the determination of MTA, and to determine the effect of heat treatment on the content of MTA in NRL serum from various sources. METHODOLOGY: An HPLC/UV-based determination of MTA using an acidic eluant was devised and validated. In the heat treatment, the effect of refluxing times on MTA liberation was evaluated. RESULTS: The quantification protocol was validated with satisfying linearity, limits of detection and quantitation, precisions for peak areas and recovery percentages from intra- and inter-day operations. The amounts of MTA in the NRL sera from various sources increased with heat treatment to yield 5-12 µg MTA/mL of serum. CONCLUSION: The devised protocol was found to be satisfyingly applicable to the routine determination of MTA in NRL serum. The effect of heat treatment on the content of MTA also indicated another possible use for NRL serum, normally discarded in vast amounts by the rubber industry, as an alternative source of MTA.

Homozygous MTAP deletion in primary human glioblastoma is not associated with elevation of methylthioadenosine.

Homozygous deletion of methylthioadenosine phosphorylase (MTAP) in cancers such as glioblastoma represents a potentially targetable vulnerability. Homozygous MTAP-deleted cell lines in culture show elevation of MTAP's substrate metabolite, methylthioadenosine (MTA). High levels of MTA inhibit protein arginine methyltransferase 5 (PRMT5), which sensitizes MTAP-deleted cells to PRMT5 and methionine adenosyltransferase 2A (MAT2A) inhibition. While this concept has been extensively corroborated in vitro, the clinical relevance relies on exhibiting significant MTA accumulation in human glioblastoma. In this work, using comprehensive metabolomic profiling, we show that MTA secreted by MTAP-deleted cells in vitro results in high levels of extracellular MTA. We further demonstrate that homozygous MTAP-deleted primary glioblastoma tumors do not significantly accumulate MTA in vivo due to metabolism of MTA by MTAP-expressing stroma. These findings highlight metabolic discrepancies between in vitro models and primary human tumors that must be considered when developing strategies for precision therapies targeting glioblastoma with homozygous MTAP deletion.

Quantitative analysis of 5'-deoxy-5'-methylthioadenosine in melanoma cells by liquid chromatography-stable isotope ratio tandem mass spectrometry.

The frequent deletion of the human chromosomal region 9p21, including the methylthioadenosine phosphorylase (MTAP) gene, is hypothesized to lead to the intra- and/or extracellular accumulation of 5'-deoxy-5'-methylthioadenosine (MTA) in cancer cells and the subsequent promotion of tumor progression. The lack of sensitive methodology for the direct measurement of MTA in tumor cells has hampered the testing of this hypothesis to date. A liquid chromatography electrospray ionization tandem mass spectrometry method (LC-MS/MS) was developed for the absolute quantitative determination of MTA in cell culture media and cell extracts using stable isotope labeled MTA as an internal standard. Limit of detection (LOD) and lower limit of quantification (LLOQ) were 62.5 pM and 2 nM, respectively, and allowed the direct measurement of MTA in biological samples without prior enrichment. Average imprecision of MTA extraction from cells and cell media, as well as LC-MS/MS analysis were 9.7, 3.8 and 1.9%, respectively. The method enabled the demonstration of the accumulation of MTA in melanoma cell culture media reaching a steady-state level within 24h. Only a slight difference in extracellular MTA concentrations was observed between cells with and without MTAP expression. However, there was a fourfold increase in intracellular MTA concentration in melanoma cells lacking MTAP, thus confirming the hypothesized accumulation of MTA in human cancer cells harboring a chromosome 9p21 deletion.

Properties of L1210 cells resistant to alpha-difluoromethylornithine.

L1210 cells were selected for resistance to the ornithine decarboxylase (ODC) inhibitor, alpha-difluoromethylornithine. When grown in the absence of the inhibitor, these cells possessed very high ornithine decarboxylase levels. These represented about 1 part in 300 of the soluble protein, which is several hundred times greater than the maximal value found in the original L1210 cells. The resistant cells contained at least 100-fold higher levels of ODC mRNA but the half-life of ODC (about 45 min) was not altered significantly. The resistant cells had much higher putrescine and cadaverine levels than control cells, but there was no significant difference in cellular spermidine or spermine content or in production of 5'-methylthioadenosine, which is a measure of polyamine synthesis. Addition of putrescine to the control or resistant cells had no effect on their content of spermidine and spermine but addition of decarboxylated S-adenosylmethionine increased the content of spermidine and spermine. These results indicate that ornithine decarboxylase is not the rate-limiting step in polyamine synthesis in these L1210 cells. The growth of the alpha-difluoromethylornithine-resistant L1210 cells was inhibited when their ability to synthesize spermidine and spermine was blocked by the addition of the S-adenosylmethionine decarboxylase inhibitor, 5'-deoxy-5'-[N-methyl-N-(3-hydrazinopropyl)]aminoadenosine. Treatment with this compound produced a reduction of more than 85% in the production of 5'-methylthioadenosine and led to a large increase in the content of putrescine and a substantial decline in the content of spermidine and spermine. These results indicate the potential value of S-adenosylmethionine decarboxylase inhibitors as therapeutic agents in conditions where ODC inhibitors are ineffective.

Inhibition of promotion and persistent nodule growth by S-adenosyl-L-methionine in rat liver carcinogenesis: role of remodeling and apoptosis.

The resistant hepatocyte model (initiation/selection) and the triphasic model (initiation/selection followed by phenobarbital, for a maximum of 16 weeks) were compared for their ability to generate enzyme-altered foci (EAF) and nodules in the liver of Wistar rats initiated by diethylnitrosamine. The effects of S-adenosyl-L-methionine (SAM) on the development of preneoplastic tissue was tested in these experimental models. In the absence of phenobarbital (PB), EAF and early nodules (EN) went through a phase of rapid growth, between 4 and 9 weeks after initiation, to a phase in which progressive decrease in number and size occurred. By the 26th week only a few remodeling EAF and nodules were found. In PB-treated rats a rapid increase in the percentage of liver occupied by EAF and EN, up to the 9th week after initiation, was followed by a period of slow growth (from the 9th to the 20th week) and then, after PB withdrawal (20th week), by a drop in the number and size of EAF and EN. However, at the 26th week actively growing nodules with a low tendency to spontaneous remodeling (persistent nodules) developed. EAF and EN showed a high DNA synthesis 5 weeks after initiation. Thereafter, progressive decline in DNA synthesis, coupled with remodeling and decrease in number of biochemical markers, was seen both in the absence and, even though to a lesser extent, in the presence of PB, indicating that preneoplastic lesions became increasingly insensitive to PB. Relatively few apoptotic bodies could be observed in EAF and EN during PB treatment. After PB withdrawal, decrease in growth potential was coupled with increase in apoptotic bodies. In contrast, in persistent nodules relatively high apoptosis occurred which partially counterbalanced high DNA synthesis. Administration of SAM for a maximum of 16 weeks, starting at the 4th week after initiation, caused a great decrease in number and size of EAF and EN, associated with inhibition of DNA synthesis, high cell death by apoptosis, high remodeling, and loss of biochemical markers, in preneoplastic lesions of both PB-treated and untreated rats. A 1-8-week SAM treatment, started after the development of persistent nodules, caused a great regression of nodular lesions, coupled with a sharp fall in DNA synthesis and increase in apoptosis. It is suggested that inhibition by SAM of the development of preneoplastic tissue is linked to a shift of the equilibrium between cell production and cell death in favor of cell death.(ABSTRACT TRUNCATED AT 400 WORDS)

HPLC method development, validation, and impurity characterization of a potent antitumor nucleoside, T-dCyd (NSC 764276).

An HPLC method for the assay of an anticancer nucleoside, 4'-thio-2'-deoxycytidine (T-dCyd, NSC 764276), has been developed and validated. The stress testing of T-dCyd was carried out in accordance with ICH guidelines Q1A (R2) under acidic, alkaline, oxidative, thermolytic, and photolytic conditions. The separation of T-dCyd from its impurities and degradation products was achieved in 40min on a Luna® Phenyl-Hexyl column (150mm×4.6mm i.d., 3µm) with a gradient elution using ammonium phosphate buffer (pH 3.85) and methanol as the mobile phase. The gradient starts from 2% and ends at 80% of methanol. Detection is by UV at 282nm. LC-QTOF/MS was used to obtain mass data for characterization of impurities and degradation products. The proposed HPLC assay method was validated for specificity, linearity (concentration range 0.25-0.75mg/mL, r≥0.9998), accuracy (recovery 98.1-102.0%), precision (RSD≤1.5%), and sensitivity (LOD 0.1µg/mL). The developed method was suitable for the quality control and stability monitoring of the T-dCyd drug substance.

Liquid chromatography-mass spectrometry for measuring deoxythioguanosine in DNA from thiopurine-treated patients.

Adverse reactions and non-response are common in patients treated with thiopurine drugs. Current monitoring of drug metabolite levels for guiding treatment are limited to analysis of thioguanine nucleotides (TGNs) in erythrocytes after chemical derivatisation. Erythrocytes are not the target tissue and TGN levels show poor correlations with clinical response. We have developed a sensitive assay to quantify deoxythioguanosine (dTG) without derivatisation in the DNA of nucleated blood cells. Using liquid chromatography and detection by tandem mass spectrometry, an intra- and inter-assay variability below 7.8% and 17.0% respectively were achieved. The assay had a detection limit of 0.0003125ng (1.1 femtomoles) dTG and was quantified in DNA samples relative to endogenous deoxyadenosine (dA) in a small group of 20 patients with inflammatory bowel disease, all of whom had been established on azathioprine (AZA) therapy for more than 25 weeks. These patients had dTG levels of 20-1360mol dTG/10(6)mol dA; three patients who had not started therapy had no detectable dTG. This method, comparable to previous methods in sensitivity, enables the direct detection of a cytotoxic thiopurine metabolite without derivatisation in an easily obtainable, stable sample and will facilitate a better understanding of the mechanisms of action of these inexpensive yet effective drugs.

Thiolated nucleotides in yeast transfer RNA.

By culturing Saccharomyces cerevisiae in growth medium containing Mg35SO4, we have determined the extent and variation of tRNA thiolation in this yeast. We find that 5-methoxycarbonylmethyl-2-thiouridine (mcm5s2U)1 is the major, if not only, thiolated derivative in S. cerevisiae tRNA. In addition, a comparison of the chromatographic mobility of mcm5s2Up on cellulose thin layers with those reported for unknown uridine derivatives found in purified yeast tRNA digests, leads to the conclusion that at least two of these tRNAs contain this modification.

A comparative study of the reversed-phase HPLC retention behaviour of S-adenosyl-L-methionine and its related metabolites on Hypersil ODS and Supelcosil LC-ABZ stationary phases.

S-Adenosyl-L-methionine (SAM) and its metabolites S-adenosyl-L-homocysteine (SAH) and methyl-thioadenosine (MTA) are endogenous compounds that are heavily involved in a variety of biochemical processes, and have therefore been the target for several assays in body fluids and tissues. Reversed-phase chromatographic behaviour of SAM and its metabolites has been studied by using Supelcosil LC-ABZ column, specially designed for analysis of acidic, basic, zwitterionic and neutral compounds, and on a Hypersil ODS column as a function of mobile phase pH. The retentions of the compounds, expressed by the capacity ratio (k'), are measured on both column with mobile phases comprised of 10% acetonitrile and 10 mM ammonium formate buffer with pH values ranging from 2 to 9. Higher selectivity is observed on Supelcosil LC-ABZ within pH range 4-6. Different retention properties are observed at very low pH and seemed as if the Supelcosil LC-ABZ column reduced the effect of the mobile phase pH by about 1 pH unit. Whilst the Supelcosil column can be recommended for the routine analysis of SAM and its related metabolites in biological fluids by using mobile phase pH 5, the Hypersil ODS column may be suggested for use with mobile phase pH values of 3-4.

Analysis of thiopurines using aqueous normal phase chromatography.

The chromatography of several thiopurines is investigated using aqueous normal phase (ANP) conditions in conjunction with a silica hydride-based column. Both isocratic and gradient elution modes are tested. Detection of higher concentration samples is done by UV to demonstrate feasibility in this format while lower concentration samples utilize mass spectrometry (MS). Repeatability of successive runs is also tested with particular attention to gradient methods where the equilibration time of the stationary phase can be evaluated.

Pin-point chemical modification of RNA with diverse molecules through the functionality transfer reaction and the copper-catalyzed azide-alkyne cycloaddition reaction.

The internal modification of RNA has been successfully achieved by the functionality transfer reaction (FTR) and following click chemistry with diverse azide compounds. The benefits of the FTR have been demonstrated by its specificity, rapidity, broad applicability, and procedure simplicity.

Determination of mercaptopurine and its four metabolites by large-volume sample stacking with polarity switching in capillary electrophoresis.

This study describes approaches for stacking a large volume of sample solutions containing a mixture of mercaptopurine monohydrate, 6-methylmercaptopurine, thioguanine, thioguanosine, and thioxanthine in capillary electrophoresis (CE). After filling the run buffer (60 mM borate buffer, pH 8.5), a large sample volume was loaded by hydrodynamic injection (2.5 psi, 99.9 s), followed by the removal of the large plug of sample matrix from the capillary using polarity switching (-15 kV). Monitoring the current and reversing the polarity when 95% of current recovered, the separation of anionic analytes was performed in a run buffer < 20 kV. Around 44- to 90-fold improvement of sensitivity for five analytes was achieved by large-volume stacking with polarity switching when compared with CE without stacking. This method was feasible for determination of the analytes spiked in plasma. Removing most of electrolytes from plasma is a key step for performing large-volume sample stacking. Solid-phase extraction was used for pretreatment of biological samples. To our knowledge, this study is one of few applications showing the possibilities of this stacking procedure to analyze biological samples by large-volume sample stacking with polarity switching (LVSSPS) in CE.

Radioimmunoassays for the modified nucleosides N[9-(beta-D-ribofuranosyl)purin-6-ylcarbamoyl]-L-threonine and 2-methylthioadenosine.

Radioimmunoassays were established for the modified nucleosides N-[9-(beta-D-ribofuranosyl)purin-6-ylcarbamoyl]-L-threonine, t6A, and 2-methylthioadenosine, ms2A. The assays depended on the production of antisera specific for t6A and ms2A that have not been previously reported. The nitrocellulose membrane filtration and saturated ammonium sulfate RIA techniques were compared for efficiency. Various radioactive antigens were employed to establish which type of antigen would give the best binding. The tritium post-labeling procedure of Randerath and Randerath was used to obtain labeled nucleosides of high enough specific activity to be useful for RIAs when the labeled nucleoside was not available commerically. The specificity of the antibodies toward nucleosides and purified tRNAs is reported. Although the titer of the t6A antiserum was low, the specificity was very sharp. An interesting finding was that threonine, a major structural component of the side-chain modification of t6A, was completely infective as an inhibitor.

Presence of 2-methylthioribosyl-trans-zeatin in Azotobacter vinelandii tRNA.

Hydroxylated cytokinin, 2-methylthio-N6-(4-hydroxy-3-methylbut-2-enyl) adenosine, was found in the tRNA of Azotobacter vinelandii. This cytokinin had the trans configuration, unlike the cis configuration reported for that from other bacteria. Culture-condition-dependent changes in the content of this thiocytokinin and a few other thionucleosides in the tRNA of this bacterium have been observed.

[A new method of measuring 5'-methylthioadenosine with high pressure liquid chromatography]. / Un nuovo metodo di dosaggio della 5'-metiltioadenosina mediante cromatografia liquida ad alta pressione.

A rapid, sensitive and specific high performance liquid chromatographic method for the estimation of 5'-methylthioadenosine in biological samples has been developed. A double-step chromatographic on Dowex-50, H+ form, and Affi-Gel 601 were required before the chromatographic separation of sample on Partisil 10 SCX. The yield is quantitative and the procedure is simple and highly reproducible.

5-Methyl-2-thiouridine in the tRNA of Candida tropicalis and its localization in lysine tRNA.

35S incorporation studies showed that Candida tropicalis tRNA contained two thionucleosides, one of which was identified as 5-methyl-2-thiouridine. The other thionucleoside was alkali labile, and it appeared to be an ester. Pulse-chase experiments suggested that the two thionucleosides were structurally related. 5-Methyl-2-thiouridine was present in one of the lysine tRNAs. This is the first report of the presence of this nucleoside in a yeast tRNA.

Zeatin ribonucleosides in the transfer ribonucleic acid of Rhizobium leguminosarum, Agrobacterium tumefaciens, Corynebacterium fascians, and Erwinia amylovora.

Until recently, the presence in transfer ribonucleic acid (tRNA) of the hydroxylated cytokinin ribosylzeatin [N6-(4-hydroxy-3-methylbut-2-enyl)adenosine]was thought to be unique to higher plants. This extension of work from several laboratories indicates the presence of 2-methylthioribosylzeatin in the tRNA of the plant-associated bacteria Rhizobium leguminosarum, Agrobacterium tumefaciens, and Corynebacterium fascians, but not in that of Erwinia amylovora. This cytokinin has the cis configuration, as is normally found in the tRNA's of plants. The tRNA thionucleotide patterns in these bacteria are different from those of Escherichia coli, Bacillus subtilis, and Salmonella typhimurium, which contain the unhydroxylated analogs of ribosylzeatin or 2-methylthioribosylzeatin.