Determination of S-adenosylmethionine, S-adenosylhomocysteine, and methylthioadenosine in urine using solvent-modified micellar electrokinetic chromatography.

A new approach for direct determination of S-adenosylmethionine (SAM), S-adenosylhomocysteine (SAH), and methylthioadenosine (MTA) in urine was developed based on MEKC by using SDS modified with isobutanol in the presence of PEG-300. Analytes were first extracted with grafted phenylborononic acid. Using a 50 µm internal diameter silica capillary of 32 cm total length filled with 0.05 M SDS, 0.05 M H3 PO4 , 5% (v/v) isobutanol, and 10% (v/v) PEG-300, LOQ of 0.15 µM for SAM and SAH, and 0.2 µM for MTA was reached. Accuracy was 92% for MTA, 109% for SAH, and 105% for SAM, intra- and interday imprecision were <2.5 and ≤3%, respectively. The total time of analysis for one sample was 10 min. Analysis of 30 urine samples from healthy volunteers showed that the median SAM and SAH levels were 12.1 and 0.73 µM, respectively. MTA levels, which were determined in urine for the first time (according to our data), were 0.43 µM, and these values correlated well with the SAM level (r = 0.748, p < 0.01).

Quantitation of urinary nucleosides by high-performance liquid chromatography.

It is known that some modified, especially methylated, nucleosides originating from RNA degradation are excreted in abnormal levels in the urine of patients with malignant tumours and they have been proposed as tumour markers. Their measurement could provide a non-invasive diagnostic method, be helpful in the identification of different cancers and in the monitoring of therapeutic effects. In this study, we developed and optimized an analytical procedure to isolate and quantify normal and modified ribonucleosides. The extraction of urinary nucleosides was performed by affinity chromatography on a phenylboronic acid column prior to separation. The reversed-phase high-performance liquid chromatography method allowed a complete separation of sixteen urinary ribonucleosides. The recoveries for the different nucleosides ranged from 83 to 100%, except for xanthosine (66%) and pseudouridine (74%). In normal 24 h urine, the mean levels of thirteen nucleosides (in nmol of nucleoside/mumol of creatinine) were found to be as follows: dihydrouridine (6.37), pseudouridine (25.52), cytidine (0.07), uridine (0.21), 1-methyladenosine (2.19), inosine (0.30), guanosine (0.06), xanthosine (0.59), 3-methyluridine (0.11), 1-methylinosine (1.13). 1-methylguanosine (0.74), adenosine (0.21) and 5'-deoxy-5'-methylthioadenosine (0.12). The first results concerning two kinds of tumours, i.e. breast and floor of mouth tumours, showed some abnormal levels of ribonucleosides. Further experiments are now in progress to measure the modified nucleosides in urine of patients with different forms of cancer.

Disturbance in the metabolism of 5'-methylthioadenosine and adenine in patients with neoplastic diseases, and in those with a deficiency in adenine phosphoribosyltransferase.

5'-Methylthioadenosine (MTA) produced during the synthesis of polyamines is degraded to adenine by MTA phosphorylase. This pathway is considered to be the main source of endogenous adenine. We determined the concentrations of MTA and adenine in control subjects and in those with a pathological disorder. In patients with active leukemias, as well as with other types of malignancies, the concentrations of MTA and adenine in the urine were elevated. These changes seemed to be the result of an accelerated production of MTA due to an accelerated biosynthesis of polyamine. In patients with adenine phosphoribosyltransferase (APRT) deficiency, the concentrations of adenine in the urine were elevated, presumably due to a disturbance in the catabolism of adenine. Although adenine is a potent inhibitor of MTA phosphorylase, APRT-deficient patients did not excrete MTA into urine in concentrations significantly larger than noted for control subjects. However, the amount of MTA excreted positively correlated with that of adenine in these patients, hence that accumulated adenine probably had a slight, but positive, inhibitory effect on the degradation of MTA.

Chromatographic and radioimmunological methods for the determination of 5'-deoxy-5'-methylthioadenosine in biological fluids.

Two specific methods for the determination of 5'-deoxy-5'-methylthioadenosine (MTA) in biological samples have been developed. The chromatographic procedure requires a preliminary step on a phenylboronate column to remove non-cis-diol compounds. The sample is then analysed using a high-performance liquid chromatography system equipped with a reversed-phase column. 5'-Deoxy-5'-methyl-thio[2-3H]adenosine with high specific activity was synthesized and employed as an internal standard. An alternative radioimmunoassay (RIA) procedure has also been developed. The RIA method is based on competition between the unlabelled thio-ether and 3H-labelled MTA for the binding to a specific antiserum. Anti-MTA antibodies were obtained from rabbits immunized with the nucleoside covalently linked to carrier proteins. Both the chromatographic and RIA procedures gave identical results when employed to determine MTA in human urine.

Urinary excretion of methylthioadenosine in immunodeficient children.

A procedure is described for the separation and determination of methylthioadenosine in human urine. The procedure has been applied to urine from normal children, children with severe combined immunodeficiency and to children with other immunodeficiencies. Methylthioadenosine excretion in normal children was 0.16 +/- 0.03 nmol/mumol creatinine. Elevated urinary excretion was noted in six of seven children with severe combined immunodeficiency (0.41-5.2 nmol/mumol creatinine). A low excretion level (0.046 nmol/mumol creatinine) was noted in a child with severe combined immunodeficiency who was germ-free.

5'-Methylthioadenosine in urine from normal subjects and cancer patients.

In an attempt to study, in human body, the metabolism of 5'-methylthioadenosine, a byproduct of the polyamine biosynthesis, we examined whether this nucleoside is excreted into urine, and, if so, whether the amount increases when the synthesis of polyamines increases. We found that 5'-methylthioadenosine is a natural nucleoside in human urine as evidenced by two separate chromatography systems as well as the analysis of the acetylated compound by gas chromatography-mass spectrometry. Preliminary study did not show the elevation of urinary 5'-methylthioadenosine level in malignant patients, suggesting that the cleaving enzyme, 5'-methylthioadenosine phosphorylase, very efficiently removes this nucleoside in vivo.