A rapid high-performance liquid chromatographic procedure for the simultaneous determination of methionine, ethionine, S-adenosylmethionine, S-adenosylethionine, and the natural polyamines in rat tissues

A method for the analysis of S-adenosyl-L-methionine (SAM) and S-adenosyl-L-ethionine (SAE) and their major metabolites by high-performance liquid chromatography is described. The procedure allows the simultaneous analysis of the natural polyamines, putrescine, spermidine, and spermine, and some of the major amino acids, methionine, tyrosine, and tryptophan. The uv absorbance at 254 nm is used for the determination of the SAM and SAE analogs, whereas the polyamines and amino acids are analyzed by fluorescence detection after postcolumn derivatization with o-phthalaldehyde. The method allows SAM and polyamine determinations by direct injection of the tissue extracts without prepurification. The procedure is applied to study the effects of DL-ethionine treatment on the SAM, SAE, methionine, and polyamine levels in various tissues of rats.     

Reversed-phase high-performance liquid chromatography procedure for the simultaneous determination of S-adenosyl-l-methionine and S-adenosyl-l-homocysteine in mouse liver and the effect of methionine on their concentrations

An improved reversed-phase high-performance liquid chromatography (HPLC) procedure with ultraviolet detection is described for the simultaneous determination of S-adenosyl-l-methionine (SAM) and S-adenosyl-l-homocysteine (SAH) in mouse tissue. The method provides rapid resolution of both compounds in a 25-μl perchloric acid extract of the tissue. The limits of detection in 25-μl injection volumes were 22 and 20 pmol for SAM and SAH, respectively. The limits of quantitation in 25-μl injection volumes were 55 and 50 pmol for SAM and SAH, respectively, with recovery consistently >98%. The assay was validated over linear ranges of 55–11 000 pmol for SAM and 50–10 000 pmol for SAH. The intra-day precision and accuracy were ≤6.4% relative standard deviation (RSD) and 99.9–100.0% for SAH and ≤6.7% RSD and 100.0–100.1% for SAM. The inter-day precision and accuracy were ≤5.9% RSD and 99.9–100.6% for SAH and ≤7.0% RSD and 99.5–100.1% for SAM. Compared to earlier procedures, the HPLC method demonstrated significantly better separation, detection limit and linear range for SAM and SAH determination. The assay demonstrated applicability to monitoring in mice the time-course of the effect of methionine on SAM and SAH levels in the liver. Administering methionine to mice increased by 10-fold the liver concentration of SAM and SAH within 2 h, which then rapidly decreased to the control levels by 8 h. This indicated that methionine was promptly converted to SAM and then rapidly catabolized into SAH. Thus, the metabolism of methionine to SAM should be considered in the supplementation of methionine to maintain SAM levels in the body.     

Polyamines: developmental alterations in regional disposition and metabolism in rat brain

The polyamines spermidine and spermine and the activity of the polyamine synthesizing enzyme, S-adenosyl-L-methionine (SAM) decarboxylase, were measured in regions of adult rat brains and during postnatal development. In the adult, although spermidine levels tended to correlate with the relative amounts of white matter in some areas, there were striking exceptions. SAM decarboxylase activity of the adult brain was higher than in most other mammalian tissues, although brain levels of polyamines were among the lowest. SAM decarboxylase activity appeared to be localized to cellular cytoplasm. Its activity increased with age in contrast to the levels of spermine, spermidine, DNA and RNA which decreased during postnatal development.     

A new method for isolation of polyamines from animal tissues

A new method for isolation of polyamines from tissues was developed and compared with the butanol extraction method which has been widely used for quantitative determination of polyamines. In the new method protein-free tissue extracts are applied to a small Dowex-50 column. The column is washed with appropriate buffers to remove ninhydrin-positive contaminants and the polyamines are eluted. With this method, the overall recovery of polyamines, after separation by paper electrophoresis and subsequent colorimetric determination with ninhydrin, is always over 90% (average 95%). This method is much better than the butanol method, which gives variable recoveries of 70–90%. The new method also has the advantages over the butanol method that the isolated polyamines are purer and the procedure is simpler.     

辅因子S-腺苷-L-甲硫氨酸甲基类似物及其应用

甲基化在生物学过程中发挥着重要作用。S-腺苷-L-甲硫氨酸(S-adenosyl-L-methionine, SAM)作为一种广泛存在于生命体中的辅因子,是大多数生物甲基化反应的甲基供体。SAM依赖型甲基转移酶(methyltransferases, MTase)通过将甲基从SAM分子特异性转移到底物,从而改变底物分子的各种理化性质和生物活性。近年来,许多具有替代甲基取代基的SAM类似物被合成并应用于甲基转移酶,以将不同修饰的基团特异性地转移到甲基转移酶的底物上,从而引入标记官能团或者新的烷基修饰。本文主要综述了近年来该领域不同SAM甲基类似物在合成和应用方面取得的进展,并对这一领域未来的研究方向进行展望。     

An improved and easy technique for polyamine determination in biological samples : Application to cell-free system from hypertrophied rat heart

An accurate, improved cation-exchange chromatographic method using o-phthalaldehyde and ultraviolet detection at 280 nm for the determination of free polyamines (putrescine, spermidine, spermine) has been developed. Different samples, such as the 105,000 g supernatant of reticulocyte or heart muscle, and KCl ribosomal wash containing initiation factors, can be analysed. The minor modification of reagents results in a good precision and sensitivity, which is demonstrated by a relative standard deviation of 5–9% and recoveries of 98%. This technique is of particular interest because it allows polyamine determination in biological samples with high concentrations of salt.     

Compensatory Aspects of the Biosynthesis of Spermidine in Tobacco Cells in Suspension Culture

The relationship between the biosynthesis of polyamines andethylene was examined in suspension cultures of Nicotiana tabacumL. cells. Aminooxyacetic acid (AOA), an inhibitor of 1-aminocyclopropane-1-carboxylicacid synthase, inhibited the production of ethylene and raisedlevels of spermidine by increasing the availability of S-adenosylmethionine(SAM) for the synthesis of polyamines. In contrast, methylglyoxalbis (guanylhydrazone) (MGBG), an inhibitor of S-adenosylmethioninedecarboxylase (SAMDC), an enzyme involved in the biosynthesisof polyamines, caused a slight increase in the rate of biosynthesisof ethylene. However MGBG did not decrease the rate of biosynthesisof polyamines in 10-day-old senescing cells. Although MGBG inhibitedthe conversion of L-[U-^l4C]methionine into labeled spermidinevia SAM both in 4-day-and in 10-day-cultured cells, it stimulatedthe conversion of L-[U-^l4C]aspartic acid into labeled spermidinein 10-day-cultured cells. In actively dividing 4-day-culturedcells, L-[U-¹⁴C]homo-serine was also converted into polyamines.In senescing cells, which produce large amounts of ethylene,the biosynthesis of spermidine from aspartic acid coincidedwith that from methionine. In actively growing cells, whichproduce large amounts of polyamines, the biosynthesis of spermidinefrom homoserine coincided with that from methionine. These resultsindicate that homoserine and aspartic acid can be both usedas precursors in the biosynthesis of polyamines and help tomaintain appropriate titers of polyamines, when SAMDC is inhibitedand the level of decarboxylated SAM becomes limiting. (Received May 14, 1990; Accepted March 11, 1991)     

S‐adenosyl‐l‐methionine usage during climacteric ripening of tomato in relation to ethylene and polyamine biosynthesis and transmethylation capacity

S‐adenosyl‐l ‐methionine (SAM) is the major methyl donor in cells and it is also used for the biosynthesis of polyamines and the plant hormone ethylene. During climacteric ripening of tomato (Solanum lycopersicum ‘Bonaparte’), ethylene production rises considerably which makes it an ideal object to study SAM involvement. We examined in ripening fruit how a 1‐MCP treatment affects SAM usage by the three major SAM‐associated pathways. The 1‐MCP treatment inhibited autocatalytic ethylene production but did not affect SAM levels. We also observed that 1‐(malonylamino)cyclopropane‐1‐carboxylic acid formation during ripening is ethylene dependent. SAM decarboxylase expression was also found to be upregulated by ethylene. Nonetheless polyamine content was higher in 1‐MCP‐treated fruit. This leads to the conclusion that the ethylene and polyamine pathway can operate simultaneously. We also observed a higher methylation capacity in 1‐MCP‐treated fruit. During fruit ripening substantial methylation reactions occur which are gradually inhibited by the methylation product S‐adenosyl‐l ‐homocysteine (SAH). SAH accumulation is caused by a drop in adenosine kinase expression, which is not observed in 1‐MCP‐treated fruit. We can conclude that tomato fruit possesses the capability to simultaneously consume SAM during ripening to ensure a high rate of ethylene and polyamine production and transmethylation reactions. SAM usage during ripening requires a complex cellular regulation mechanism in order to control SAM levels.     

Comparison of polyamine and S-adenosylmethionine contents of growing and encysted Acanthamoeba isolates

We have used High Performance Liquid Chromatography to determine metabolite characteristics of three recent isolates of Acanthamoeba which exhibit cultural characteristics consistent with those of established potential pathogens. Growing amoebae and dormant cysts of these isolates were explored in regard to their qualitative and quantitative intracellular levels of polyamine and S-adenosylmethionine metabolites. The polyamine found in the greatest concentration in the growing cells was 1,3-diaminopropane (DAP), followed by spermidine (SPD). A low level of putrescine was also found in the growing cells. These polyamines significantly decreased in concentration as the amoebae differentiated to cysts. N⁸-acetylspermidine and acetylspermine were found in both developmental stages while acetylcadaverine was found only in growing amoebae and N¹-acetylspermidine only in cysts. Acetylputrescine was present in both stages of two isolates but only in the growing amoebae of the third isolate. Spermine was not detected in any of the isolates.S-adenosylmethionine (SAM) and S-adenosylhomocysteine (SAH) were present in growing amoebae but SAM was undetectable or barely detectable in cysts. SAH also decreased in concentration during encystation of two of the isolates to a level comparable to that of the other isolate.The developmental transition from growing amoebae to dormant cysts is characterized metabolically by a threshold adjustment in concentration of SAM, SAH and of the polyamines (esp., DAP and SPD).     

High-performance liquid chromatographic procedure for the simultaneous determination of the natural polyamines and their monoacetyl derivatives

The separation of the natural polyamines and their monoacetyl derivatives by high-performance reversed-phase liquid chromatography is reported. Octane sulfonate was used to form ion pairs with the polycations and the o-phthalaldehyde method for post-column derivatization. The method allows polyamine and acetylspermidine determinations directly from tissue extracts and body fluids without pre-purification.     

Reversed-phase liquid chromatography with amperometric detection of lipophilic dopamine analogues and determination of brain and serum concentrations after sample clean-up on small sephadex G-10 columns

The liquid chromatographic determination of N-alkylated analogues of dopamine is described. The retention and separation of these compounds, ranging from dopamine to N,N-dibutyl-dopamine, was studied on four bonded-phase columns, of which Nucleosil 5 C₁, was chosen for routine use. The compounds were detected by a rotating disc amperometric detector. Samples of rat brain and serum were taken through a clean-up step on small Sephadex G-10 columns from which the dopamine analogues eluted in the same fraction as dopamine. The overall recovery was 70–90% from brain tissue and 60–70% from serum or plasma. The limit of detection for the catechol-containing compounds in tissue was 40–100 pg, for O-methylated ones 100–200 pg. The method is applied to the determination of dopamine analogues in rat brain after peripheral administration.     

Down-regulation of hypusine biosynthesis in plasmodium by inhibition of S-adenosyl-methionine-decarboxylase

An important issue facing global health today is the need for new, effective and affordable drugs against malaria, particularly in resource-poor countries. Moreover, the currently available antimalarials are limited by factors ranging from parasite resistance to safety, compliance, cost and the current lack of innovations in medicinal chemistry. Depletion of polyamines in the intraerythrocytic phase of P. falciparum is a promising strategy for the development of new antimalarials since intracellular levels of putrescine, spermidine and spermine are increased during cell proliferation. S-adenosyl-methionine-decarboxylase (AdoMETDC) is a key enzyme in the biosynthesis of spermidine. The AdoMETDC inhibitor CGP 48664A, known as SAM486A, inhibited the separately expressed plasmodial AdoMETDC domain with a Km _i of 3 μM resulting in depletion of spermidine. Spermidine is an important precursor in the biosynthesis of hypusine. This prompted us to investigate a downstream effect on hypusine biosynthesis after inhibition of AdoMETDC. Extracts from P. falciparum in vitro cultures that were treated with 10 μM SAM 486A showed suppression of eukaryotic initiation factor 5A (eIF-5A) in comparison to the untreated control in two-dimensional gel electrophoresis. Depletion of eIF-5A was also observed in Western blot analysis with crude protein extracts from the parasite after treatment with 10 μM SAM486A. A determination of the intracellular polyamine levels revealed an approximately 27% reduction of spemidine and a 75% decrease of spermine while putrescine levels increased to 36%. These data suggest that inhibition of AdoMetDc provides a novel strategy for eIF-5A suppression and the design of new antimalarials.     

Polyamine uptake by DUR3 and SAM3 in Saccharomyces cerevisiae

It has been reported that GAP1 and AGP2 catalyze the uptake of polyamines together with amino acids in Saccharomyces cerevisiae. We have looked for polyamine-preferential uptake proteins in S. cerevisiae. DUR3 catalyzed the uptake of polyamines together with urea, and SAM3 was found to catalyze the uptake of polyamines together with S-adenosylmethionine, glutamic acid, and lysine. Polyamine uptake was greatly decreased in both DUR3- and SAM3-deficient cells. The K(m) values for putrescine and spermidine of DUR3 were 479 and 21.2 mum, respectively, and those of SAM3 were 433 and 20.7 mum, respectively. Polyamine stimulation of cell growth of a polyamine requiring mutant, which is deficient in ornithine decarboxylase, was not influenced by the disruption of GAP1 and AGP2, but it was diminished by the disruption of DUR3 and SAM3. Furthermore, the polyamine stimulation of cell growth of a polyamine-requiring mutant was completely inhibited by the disruption of both DUR3 and SAM3. The results indicate that DUR3 and SAM3 are major polyamine uptake proteins in yeast. We previously reported that polyamine transport protein kinase 2 regulates polyamine transport. It was found that DUR3 (but not SAM3) was activated by phosphorylation of Thr(250), Ser(251), and Thr(684) by polyamine transport protein kinase 2.     

Fluorometric assay for polyamines in urine and tissues using electrophoresis on Titan III cellulose acetate

A simple, rapid assay method for polyamines (putrescine, spermidine, and spermine) in urine and tissues using electrophoresis on Titan III cellulose acetate was developed. In this procedure, polyamines are preliminarily extracted from a hydrolysate of urine or from supernatants of tissue homogenates by use of a Bio-Rex 70 minicolumn. After electrophoretic separation, polyamines are fluorometrically detected by the reaction with o-phthalaldehyde and 2-mercaptoethanol. Six extracts and two external standards of polyamines can be separated and detected in 11 min on a cellulose acetate strip. This method permits the determination of polyamines in a range of 0.1 mM (25 pmol) to 1.0 mM (250 pmol).     

Determination of polyamines in human saliva by high-performance liquid chromatography with fluorescence detection

A high-performance liquid chromatographic method for the determination of polyamines (spermine, spermidine and putrescine) in human saliva was developed. This method is based on pre-column derivatization with o-phthaldialdehyde (OPA). The derivatives were separated on a Nucleosil ODS column (250×4.6 mm I.D.; 5 μm). The gradient elution was performed with two mobile phases A (water) and B (methanol) at a flow rate of 0.8 ml/min. The column eluate was monitored by fluorescence detection (excitation, 360 nm; emission, 510 nm). The within- and between-assay coefficients of variation for all the compounds were below 5%. The detection limits for spermine, spermidine and putrescine were 0.04, 0.05 and 0.06 nmol/ml, respectively. The recovery was greater than 90%. Our analytical technique requires neither preliminary extraction with an organic solvent, nor long multi-step procedures. For saliva samples, this is a simple, rapid and highly reproducible method that can be easily applied to the routine determination of salivary polyamines, whose levels increase early in several pathological conditions.     

A sensitive, rapid, chemiluminescence-based method for the determination of diamines and polyamines

A new sensitive and rapid chemiluminescence-based method for the determination of diamines and polyamines is described. Phosphocellulose paper strips are used for the removal of neutral or negatively charged molecules from polyamine-containing fluid. The procedure is based on the determination of hydrogen peroxide, produced during the oxidation of polyamines, by a fairly specific serum amine oxidase. A plant diamine oxidase is used for the assay of diamines. This method permits the determination of diamines and polyamines in a range of 10 to 100 pmol and may be used for the assay of urinary polyamines.     

Overexpression of S‐adenosyl‐l‐methionine synthetase increased tomato tolerance to alkali stress through polyamine metabolism

S‐adenosyl‐l ‐methionine (SAM) synthetase is the key enzyme involved in the biosynthesis of SAM, which serves as a common precursor for polyamines (PAs) and ethylene. A SAM synthetase cDNA (SlSAMS1) was introduced into the tomato genome using the Agrobacterium tumefaciens transformation method. Transgenic plants overexpressing SlSAMS1 exhibited a significant increase in tolerance to alkali stress and maintained nutrient balance, higher photosynthetic capacity and lower oxidative stress compared with WT lines. Both in vivo and in vitro experiments indicated that the function of SlSAMS1 mainly depended on the accumulation of Spd and Spm in the transgenic lines. A grafting experiment showed that rootstocks from SlSAMS1‐overexpressing plants provided a stronger root system, increased PAs accumulation, essential elements absorption, and decreased Na⁺ absorption in the scions under alkali stress. As a result, fruit set and yield were significantly enhanced. To our knowledge, this is the first report to provide evidence that SlSAMS1 positively regulates tomato tolerance to alkali stress and plays a major role in modulating polyamine metabolism, resulting in maintainability of nutrient and ROS balance.     

A New Isolation Method of Plasmid Deoxyribonucleic Acid from Staphylococcus aureus Using a Lytic Enzyme of Achromobacter lyticus

An enzymatic procedure for the differential determination of polyamines, spermine and spermidine, has been established using beef plasma amine oxidase. This method was specific for these polyamines and required only one reaction system. Small amounts of polyamines (10µm to 80 µm of spermine and 10 µm to 100 µm of spermidine) were assayed by solving two simultaneous equations obtained from the rate assay method and the end point assay method. The calculated values were in good agreement with those obtained by other method.