Quantitative determination of underivatized polyamines by using isotope dilution RP-LC-ESI-MS/MS

A rapid, sensitive and selective method using LC-MS/MS was developed and validated for the simultaneous quantitative determination of five polyamines N(1),N(12)-diethylspermine (DESpm), N-ethylspermine (EtSpm), N(1)-ethylspermidine (EtSpd), spermidine (Spd) and N(1)-ethyl-1,3-diaminopropane (EtDAP) without any derivatization steps. The LC-MS/MS system was operated using the positive electrospray ionization (ESI) mode. The chromatographic separation only took 10min and was performed on a reversed phase C18 column with 0.1% heptafluorobutyric acid as the ion-pairing agent and acetonitrile gradient. Stable, deuterium labelled internal reference compounds of the five analytes were included in the quantification. The lower limit of quantification for all of the five analytes was 0.03muM and the method was linear for DESpm, EtSpd, Spd and EtDAP over the range of 0.03-60muM and for EtSpm over the range of 0.03-30muM. Correlation coefficients (R(2)) were always >0.995 for all the analytes. The precision of the overall method ranged from 0.2 to 9.7% as intra-day variability and from 0.9 to 6.8% as inter-day variability. The intra-day and inter-day accuracy of the assay ranged between 87.6-109.8% and 89.6-106.6%, respectively. The method has been applied successfully to quantify metabolites of DESpm as a substrate for recombinant human polyamine oxidase.     

Arcaine and MK-801 make recall state-dependent in rats

RATIONALE: Conditioned fear to context causes freezing and cardiovascular changes in rodents and has been used to measure anxiety. It also activates the dorsolateral column of the periaqueductal gray (dlPAG). Microinjections of cannabinoid agonists into the dlPAG produced anxiolytic-like effects in the elevated plus maze, but the effects of these treatments on fear conditioning remains unknown. OBJECTIVE: The objective of this study was to verify if intra-dlPAG injection of the CB1 cannabinoid receptor agonist anandamide (AEA) or the anandamide transport inhibitor AM404 would attenuate behavioral (freezing) and cardiovascular (increase of arterial pressure and heart rate) responses of rats submitted to a contextual fear-conditioning paradigm. MATERIALS AND METHODS: Male Wistar rats with cannulae aimed at the dlPAG were re-exposed to a chamber where they had received footshocks 48 h before. Fifteen minutes before the test, the animals received a first intra-dlPAG injection of vehicle or AM251, a CB1 receptor antagonist (100 pmol/200 nl), followed 5 min later by vehicle, AEA (5 pmol/200 nl) or AM404 (50 pmol/200 nl). Freezing and cardiovascular responses were recorded for 10 min. RESULTS: Freezing and cardiovascular responses were reduced by administration of either AEA or AM404 into the dlPAG before re-exposition to the aversively conditioned context. These effects were abolished when the animals were locally pretreated with AM251. The latter drug, even at a higher dose (300 pmol), was ineffective when administered alone into the dlPAG. CONCLUSION: The results suggest that facilitation of endocannabinoid-mediated neurotransmission in the dlPAG, through activation of local CB1 receptors, attenuates the expression of contextual fear responses.     

银屑病患者尿中四种多胺的检测

采用反向高效液相色谱分析法检测了60例银屑病患者的晨尿,正常人42例晨尿和40例24小时尿中多胺含量.结果发现正常人24小时尿与晨尿中多胺含量无显著性差异,患者晨尿中四种多胺均较对照组显著性上升.不同病期的腐胺.尸胺、精脒以及精脒/精胺比值有显著性差异;治疗后腐胺、尸胺,精眯显著性下降;地图状皮损尿腐胺显著性升高,炎症重者腐胺、精眯上升更明显.晨尿中多胺含量可作为银屑病病情判断和疗效观察的生化指标.     

多胺对人及哺乳动物生殖调控作用

多胺(polyamines)是带有正电荷的脂肪族化合物,通过结合DNA、RNA和蛋白质等,参与人及哺乳动物的基因表达调控、细胞信号传导等。多胺可影响下丘脑-垂体-性腺轴激素的分泌,进而影响生殖细胞质量、黄体形成及胚胎发育,对人及哺乳动物生殖过程发挥调控作用,但是其具体作用机制,迄今尚未阐明。笔者拟就多胺的生物合成,人及哺乳动物体内分布及其来源,对精子发生、卵子形成、囊胚着床及发育作用的最新研究进展进行综述,旨在为多胺用于辅助生殖技术(ART)中,改善妊娠结局提供依据。     

Neurotoxicity of reactive aldehydes: the concept of "aldehyde load" as demonstrated by neuroprotection with hydroxylamines

The concept of "oxidative stress" has become a mainstay in the field of neurodegeneration but has failed to differentiate critical events from epiphenomena and sequalae. Furthermore, the translation of current concepts of neurodegenerative mechanisms into effective therapeutics for neurodegenerative diseases has been meager and disappointing. A corollary of current concepts of "oxidative stress" is that of "aldehyde load". This relates to the production of reactive aldehydes that covalently modify proteins, nucleic acids, lipids and carbohydrates and activate apoptotic pathways. However, reactive aldehydes can also be generated by mechanisms other than "oxidative stress". We therefore hypothesized that agents that can chemically neutralize reactive aldehydes should demonstrate superior neuroprotective actions to those of free radical scavengers. To this end, we evaluated hydroxylamines as aldehyde-trapping agents in an in vitro model of neurodegeneration induced by the reactive aldehyde, 3-aminopropanal (3-AP), a product of polyamine oxidase metabolism of spermine and spermidine. In this model, the hydroxylamines N-benzylhydroxylamine, cyclohexylhydroxylamine and t-butylhydroxylamine were shown to protect, in a concentration-dependent manner, against 3-AP neurotoxicity. Additionally, a therapeutic window of 3 h was demonstrated for delayed administration of the hydroxylamines. In contrast, the free radical scavengers TEMPO and TEMPONE and the anti-oxidant ascorbic acid were ineffective in this model. Extending these tissue culture findings in vivo, we examined the actions of N-benzylhydroxylamine in the trimethyltin (TMT) rat model of hippocampal CA3 neurodegeneration. This model involves augmented polyamine metabolism resulting in the generation of reactive aldehydes that compromise mitochondrial integrity. In the rat TMT model, NBHA (50 mg/kg, sc, daily) provided 100% protection against neurodegeneration, as reflected by measurements of KCl-evoked glutamate release from hippocampal brain slices and septal high affinity glutamate uptake. In contrast, ascorbic acid (100 mg/kg, sc, daily) failed to protect CA3 neurons from TMT toxicity. In summary, our data support further evaluation of the concept of "aldehyde load" in neurodegeneration and the potential clinical investigation of agents that are effective traps for reactive aldehydes.     

Effect of methylglyoxal bisguanylhydrazone on polyamine biosynthesis,growth, and differentiation of cultured keratinocytes

Summary The putrescine, spermidine, and spermine content of subcultured human newborn foreskin keratinocytes was determined during growth and early plateau phase and found to be highest during growth. Exposure of the cells to methylglyoxal bis(guanylhydrazone) during growth phase caused a dose-dependent fall in intracellular spermidine and spermine levels and an increase in putrescine levels at higher concentrations. These effects reflect inhibition of S-adenosyl methionine decarboxylase by the drug. At 8×10^-6 M the drug reduced incorporation of leucine into protein, lowered or stopped the accumulation of DNA per dish, inhibited mitotic activity, and increased the histidine/leucine incorporation into protein. The last effect is regarded as induction of keratinization. All these effects were reversible if the use of the drug was discontinued after 3 days. Inhibition of the enzymes of polyamine biosynthesis may have value in psoriasis therapy.This work was performed during the tenure of a Society for Investigative Dermatology Fellowship (to M.S.P.).     

Polyamine reduced diet (PRD) nutrition therapy in hormone refractory prostate cancer patients

Background

Reducing polyamine uptake by selecting low polyamine-containing foodstuffs and reducing bacterial gut production can improve performance status and pain control in hormone refractory prostate cancer (HRPC) patients. Long term PRD observance and tolerance were assessed. Cancer specific survival was studied in function of PRD and time of PRD initiation.

Methods

Twenty-six volunteers, age: 68 ± 10 years with metastatic HRPC accepted a polyamine reduced diet and partial gut decontamination with oral neomycin or nifuroxazide (750 mg daily, one week out of two). Time from HRPC to PRD initiation was 10 ± 8 months. WHO performance status, EORTC pain scale, body weight, blood counts and serum proteins were regularly assessed. Sixteen other HRPC patients eating a normal diet served as “controls”.

Results

Mean diet observance is 25 ± 24 months. Tolerance is good. WHO performance status and EORTC pain scales were significantly improved respectively at 3 months (0.5 ± 0.7 vs 0.7 ± 0.9: p = 0.03) and 6 months (0.5 ± 0.8 vs 1 ± 1.3, p = 0.02) compared to initial values. Median cancer specific survival times after HRPC and PRD initiation are respectively 36 and 21 months. Eleven PRD patients started the diet before a 9 months cut-off period (after HRPC) and 15 patients after. Median cancer specific survival times for these two groups of patients are respectively 44 and 34 months, p = 0.014. Median cancer specific survival times (after HRPC) for PRD patients compared to controls are 36 vs 17 months (p = 0.004).

Conclusions

Polyamine-reduced diet is well observed and tolerated. It seems to improve and/or maintain quality of life for HRPC patients. Early PRD initiation in HRPC is promising and may impact favorably cancer specific survival. These results open a rationale for PRD in HRPC management and warrant further investigation.     

2型糖尿病患者多胺水平变化及其临床意义

目的探讨2型糖尿病(DM)患者血清多胺(腐胺、精胺、精脒)水平的变化及其临床意义.方法按照尿白蛋白的排泄率水平(AER)将DM分为3组.D1组AER＜20μg@min-1,D2组AER20～200μg@min-1,D3组AER＞200μg@min-1;按照视网膜病变(DR)的有无,将DM分为DR1组(无DR)、DR2组(非增殖性视网膜病变)、DR3组(增殖性视网膜病变).多胺水平采用高效液相色谱法(HPLC)测定,尿白蛋白采用RIA法测定,HbA1c采用亲合层析微柱法.结果DM患者多胺水平较正常对照组显著升高(P＜0.05),多胺水平与logAER呈显著正相关(P＜0.05),多胺水平在DR组显著升高(P＜0.01).结论2型DM患者血清多胺水平升高与DN及DR的微血管病变密切相关,多胺水平可能对DN及DR的发生发展起一定作用.