Determination Of S-adenosylmethionine Research Articles

Determination of S-Adenosylmethionine and S-Adenosylhomocysteine in Human Urine by Ion Chromatography with Solid Phase Extraction Based on the Application of Micromolecule Ion-Pairing Agent

S-adenosylmethionine (SAM) and S-adenosylhomocysteine (SAH) play an important role in various biochemical reactions involving the maintenance of human health and disease prevention. In this study, an accurate, rapid, economical, and simple protocol is reported for the simultaneous determination of the SAM and SAH concentrations in human urine. 97% trifluoroacetic acid (0.05%) was used in the mobile phase as a micromolecule ion-pairing agent in order to enhance the formation of ion pairs and the protonation of the SAM and SAH analytes. Following solid phase extraction (SPE), the separation of SAM and SAH was performed by micromolecule ion pair chromatography using a C18 column. Three types of solid phase extraction columns were characterized for the purification of the urine samples, and their correlative mechanisms were evaluated in detail. Good linearity values were obtained across the concentration ranges from 0.5 to 60 μmol/L and 0.1 to 10 μmol/L for SAM and SAH, respectively. The limits of detection were equal to 0.006 and 0.01 μmol/L for SAM and SAH, respectively, while the limits of quantitation were 0.02 and 0.04 μmol/L for these analytes. The intra-day and inter-day precision values for the determined SAM and SAH species were less than 1.38. The recovery values of SAM and SAH in urine were between 83.65 and 98.41%. Thus, this method has demonstrated to be suitable for the determination of SAM and SAH in human urine.

Determination of S-adenosylmethionine and S-adenosylhomocysteine in blood plasma by UPLC with fluorescence detection

A validated approach to determine various methionine cycle metabolites (S-adenosylmethionine, S-adenosylhomocysteine, and methylthioadenosine) in human blood plasma is offered. The approach is based on solid-phase extraction (with grafted phenylboronic acid) and derivatization with chloroacetaldehyde followed by ultra-performance liquid chromatography with fluorescence detection. We used a 100 × 2.1 mm × 1.8 μm C18 column for the selective separation of analytes. Chromatographic separation was achieved with gradient elution of acetonitrile (flow rate 0.2 mL/min) from 2 to 20%. The eluent was initially composed of 10 mM KH2PO4 with 10 mM acetic acid and 25 μM heptafluorobutyric acid. The total analysis time was 11 min. Validation of the method included detection of the limit of detection (2 nM), limit of quantification (5 nM), accuracy (97.2–101%), and intra- and interday precision (2.2–9.0%). Analysis of plasma samples from healthy volunteers revealed that the average levels of S-adenosylmethionine, S-adenosylhomocysteine, and methylthioadenosine were 93.6, 20.9 and 14.8 nM, respectively.

Quantitation of S-Adenosylmethionine and S-Adenosylhomocysteine in Plasma Using Liquid Chromatography-Electrospray Tandem Mass Spectrometry.

We describe a simple stable isotope dilution method for accurate determination of S-adenosylmethionine (SAM) and S-adenosylhomocysteine (SAH) in plasma as a diagnostic test. SAM and SAH are key metabolic intermediates of methionine metabolism and the methylation cycle. Determination of SAM and SAH in plasma was performed by high performance liquid chromatography coupled with electrospray positive ionization tandem mass spectrometry (HPLC-ESI-MS/MS). Calibrators (SAM and SAH) and internal standards ((2)H3-SAM and (2)H4-SAH) were included in each analytical run for calibration. Sample preparation involved combining 20 μL sample with 180 μL of internal standard solution consisting of heavy isotope labeled internal standards in mobile phase A and filtering by ultracentrifugation through a 10 kd MW cutoff membrane. Sample filtrate (3 μL) was injected by a Shimadzu Nexera LC System interfaced with a 5500 QTRAP(®) (AB Sciex). Chromatographic separation was achieved on a 250 mm × 2.0 mm EA:faast column from Phenomenex. Samples were eluted at a flow rate of 0.20 mL/min with a binary gradient with a total run time of 10 min. The source operated in positive ion mode at an ion spray voltage of +5000 V. SAM and SAH resolved by a gradient to 100 % methanol with retention times of 6.0 and 5.7 min, respectively. The observed m/z values of the fragment ions were m/z 399 → 250 for SAM, m/z 385 → 136 for SAH, m/z 402 → 250 for (2)H3-SAM, m/z 203 → 46. The calibration curve was linear over the ranges of 12.5-5000 nmol/L for SAM and SAH.

Synthesis of a Novel Fluorescence Switch Conjugated Polymers and Its Discrimination Ability Toward S-Adenosylmethionine

Abstract A novel fluorescence switch was constructed based on poly 3-{[1-(2-hydrazino-2-oxoethyl) piperidin-4-ylidene]methyl} thiophene (PMTH) in the presence of Pb 2+ and S -adenosylmethionine (SAM). The fluorescence of PMTH solution can be efficiently quenched by Pb 2+ ions, resulting in a “fluorescence off” state. Upon the addition of SAM, a strong Pb 2+ chelator, to Pb 2+ -PMTH solution, fluorescence of PMTH can be restored, leading to fluorescence “on”. Based on this fluorescence switch characteristics of PMTH, a new method for recognition and determination of SAM was established. By investigating the fluorescence spectral characteristics of Pb 2+ -PMTH and Pb 2+ -SAM complexes in H 2 O/CH 3 CH 2 OH (4:1, V/V ) solution, we found this method offered advanced sensitivity and selectivity for detecting SAM in the presence of common amino acids and metal ions. Under optimum conditions, the relative fluorescence intensity exhibited a linear relationship with SAM concentration in the range of 1.0 × −8 –2.0 × 10 −6 M. The regression equation was Δ 68.51 + 72.32 C (μith a correlation coefficient r = 0.9982. The limit of detection was 8.72 × 10 −9 M. The method was successfully applied in the detection of SAM in real samples.

Determination of S-Adenosylmethionine and S-Adenosylhomocysteine by LC–MS/MS and evaluation of their stability in mice tissues

S-Adenosylmethionine (SAM) serves as a methyl donor in biological transmethylation reactions. S-Adenosylhomocysteine (SAH) is the product as well as the inhibitor of transmethylations and the ratio SAM/SAH is regarded as the measure of methylating capacity (“methylation index”). We present a rapid and sensitive LC–MS/MS method for SAM and SAH determination in mice tissues. The method is based on chromatographic separation on a Hypercarb column (30mm×2.1mm, 3μm particle size) filled with porous graphitic carbon stationary phase. Sufficient retention of SAM and SAH on the chromatographic packing allows simple sample preparation protocol avoiding solid phase extraction step. No significant matrix effects were observed by analysing the tissue extracts on LC–MS/MS. The intra-assay precision was less than 9%, the inter-assay precision was less than 13% and the accuracy was in the range 98–105% for both compounds. Stability of both metabolites during sample preparation and storage of tissue samples was studied: the SAM/SAH ratio in liver samples dropped by 34% and 48% after incubation of the tissues at 4°C for 5min and at 25°C for 2min, respectively. Storage of liver tissues at −80°C for 2 months resulted in decrease of SAM/SAH ratio by 40%. These results demonstrate that preanalytical steps are critical for obtaining valid data of SAM and SAH in tissues.

Determination of S-Adenosylmethionine and S-Adenosylhomocysteine from Human Blood Samples by HPLC-FL

S-adenosylmethionine plays an important role in many biochemical reactions, including human health maintenance and disease prevention. This paper proposes a simplified, rapid, and easy high performance liquid chromatography (HPLC) with fluorimetric detection method of S-adenosylmethionine and its product, S-adenosylhomocysteine. Determination of S-adenosylmethionine and S-adenosylhomocysteine was by HPLC with fluorimetric detection by the formation of the fluorescent 1,N 6-ethanoderivatives of these compounds, which showed a linear concentration range of 310−6–310−8 molL−1 for S-adenosylmethionine and 510−7–510−9for S-adenosylhomocysteine; the detection limits of the method ranged in the nanomolar domain. The method applied to real plasma samples lead to values of 60.1±19.6 for S-adenosylmethionine and 24±14.1 nmolL−1 for S-adenosylhomocysteine.

A novel method to analyze betaine in chicken liver: effect of dietary betaine and choline supplementation on the hepatic betaine concentration in broiler chicks.

Betaine was measured from liver tissue by a high-performance liquid chromatographic (HPLC) method developed for this study. The method involves homogenization of liver in acetate buffer at pH 6 and precipitation of protein with trichloroacetic acid, which was removed by diethyl ether extraction. Betaine was separated using a cation exchange column in Ca(2+) form and detected with a refractive index detector. This method also allows the determination of S-adenosylmethionine (S-AM) from the same liver extract but with different HPLC conditions. Broiler chicks were fed with experimental diets supplemented with four different doses of betaine or choline ranging from 0 to 5 mol equiv. After a 3 week feeding period, the livers were analyzed for betaine and S-AM. Dietary betaine was twice as efficient in increasing the hepatic betaine concentration as dietary choline. The hepatic S-AM concentrations were similar in all dietary treatments.

Determination of S-Adenosylmethionine and S-Adenosylhomocysteine in Plasma and Cerebrospinal Fluid by Stable-Isotope Dilution Tandem Mass Spectrometry

Available methods for the determination of nanomolar concentrations of S:-adenosylmethionine (SAM) and S:-adenosylhomocysteine (SAH) in plasma and cerebrospinal fluid (CSF) are time-consuming. We wished to develop a method for their rapid and simultaneous measurement. We used tandem mass spectrometry (MS/MS) for the simultaneous determination of SAM and SAH, with stable-isotope-labeled internal standards. The (13)C(5)-SAH internal standard was enzymatically prepared using SAH-hydrolase and [(13)C(5)]adenosine. The method comprises a weak anion-exchange solid-phase extraction procedure serving as clean-up step for the deproteinized plasma and CSF samples. After clean-up, samples were injected on a C(18) HPLC column, which was connected directly to the tandem mass spectrometer, operating in MS/MS mode. In plasma samples, the intraassay CVs for SAM and SAH were 4.2% and 4.0%, respectively, and the interassay CVs were 7.6% and 5. 9%, respectively. In CSF, the intraassay CVs for SAM and SAH were 6. 8% and 6.9%, respectively, and the interassay CVs were 4.2% and 5.5%, respectively. Mean recovery of SAM and SAH for both matrices at two concentrations was 93%. Detection limits for SAM and SAH in samples were 7.5 and 2.5 nmol/L, respectively. Concentrations of SAM and SAH in plasma from healthy subjects were within the previously reported ranges. In 10 CSF samples, the mean concentrations (range) were 248 (137-385) nmol/L for SAM and 11.3 (8.9-14.1) nmol/L for SAH. SAM and SAH can be analyzed by MS/MS, taking optimal advantage of the speed and high sensitivity and specificity of this relatively new analytical technique.

High Performance Liquid Chromatographic Analysis of S-Adenosylmethionine and S-Adenosylhomocysteine in Rat Liver

Abstract A sensitive and precise high performance liquid chromatographic method for the simultaneous quantitation of S-adenosylmethionine (SAMe) and S-adenosylhomocysteine (SAH) in rat liver is described. Liver is extracted by homogenization in 1.5 M PCA. SAMe and SAH are purified from contaminating substances by chromatography on Dowex 50 W × 2 and analyzed by HPLC on conventional styrene-type anion exchanger. Analytical chromatography, performed under i-socratic elution conditions, is achieved in 18 min. SAH is determined as such while SAMe as adenine after hydrolysis at 60°C, un der alkaline conditions. The sensitivity of the analytical proce dure allows the determination of SAMe and SAH in less than 200 mg of liver sample.

Assay of tissue levels of S-adenosylmethionine

Publisher Summary This chapter discusses the methods of preparation of S-Adenosylmethionine. S-Adenosylmethionine formed from methionine and ATP by an activating enzyme is the major methyl donor tot a variety of biological transmethylations. Significant levels of this intermediate are present in most mammalian tissues and can be determined with relative ease. The method is based on isotope dilution of S-adenosylmethionine (labeled with methyl- l4 C) by the unlabeled compound present in tissue extract and estimation of the specific activity of the diluted compound by the enzymatic formation of melatonin(methoxy- 14 C-acetyl- 3 H) from the methyl donor and N-acetylserotonin- 3 H of constant specific activity. Other potential methyl donors do not interfere with the determination of S-adenosylmethionine. This tissue extract does not contain any active methyltransferase; other possible substrates present in the tissue are not methylated by the highly specific hydroxyindole-O-methyltransferase. Normal tissue levels vary from about 9 μg/g in brain to about 50μg/g in adrenal glands. S-Adenosylethionine can transfer its ethyl group to N-acetylserotonin, but this compound is not normally present in tissues.