RESUMEN
Purine compounds are known to activate 5'-adenosine monophosphate-activated protein kinase (AMPK), which has important roles in treatments for renal cell carcinoma. The present study was aimed to investigate the effects of the purine analogue ENERGIF706 on the human renal carcinoma cell line 786O and the underlying mechanisms. The results revealed that ENERGIF706 (0.20.6 mg/ml) significantly decreased the cell viability to up to 36.4±2.4% of that of the control. Compared to 786O cells, ENERGIF706 exerted less suppressive effects on the viability of the human nontumorigenic renal cell line HK2. Flow cytometric analysis showed that ENERGIF706 contributed to cell cycle arrest at Sphase and triggered apoptosis of 786O cells. Immunoblot analysis revealed that antiapoptotic Bcell lymphoma 2 (Bcl2) levels were reduced and proapoptotic Bcl2associated X protein levels were diminished. In addition, activation of caspase9, caspase3 and poly(adenosine diphosphate ribose) polymerase (PARP) was promoted in 786O cells in response to ENERGIF706. Effects of ENERGIF706 on AMPK cascades were investigated and the results showed that ENERGIF706 enhanced phosphorylation of AMPKα (T172) and p53 (S15), a downstream target of AMPK. In addition, the AMPK activation, p53 (S15) phosphorylation, reduction of Bcl2, cleavage of caspase3 and PARP as well as suppressed cell viability induced by ENERGIF706 were reversed in the presence of AMPK inhibitor compound C (dorsomorphin). In conclusion, the findings of the present study revealed that ENERGIF706 significantly suppressed the viability of 786O cells via induction of cell cycle arrest and apoptosis, attributing to AMPK and p53 activation and subsequent cell cycle regulatory and apoptotic signaling. It was therefore indicated that ENERGIF706 may be suitable for the treatment of renal cell carcinoma.
Asunto(s)
Proteínas Quinasas Activadas por AMP/genética , Antineoplásicos Fitogénicos/farmacología , Bambusa/química , Células Epiteliales/efectos de los fármacos , Regulación Neoplásica de la Expresión Génica , Purinas/farmacología , Proteínas Quinasas Activadas por AMP/metabolismo , Antineoplásicos Fitogénicos/aislamiento & purificación , Apoptosis/efectos de los fármacos , Caspasa 3/genética , Caspasa 3/metabolismo , Caspasa 9/genética , Caspasa 9/metabolismo , Línea Celular , Línea Celular Tumoral , Activación Enzimática/efectos de los fármacos , Células Epiteliales/enzimología , Células Epiteliales/patología , Humanos , Riñón/efectos de los fármacos , Riñón/enzimología , Riñón/patología , Especificidad de Órganos , Extractos Vegetales/química , Poli(ADP-Ribosa) Polimerasas/genética , Poli(ADP-Ribosa) Polimerasas/metabolismo , Proteínas Proto-Oncogénicas c-bcl-2/genética , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , Purinas/aislamiento & purificación , Pirazoles/farmacología , Pirimidinas/farmacología , Puntos de Control de la Fase S del Ciclo Celular/efectos de los fármacos , Transducción de Señal , Proteína p53 Supresora de Tumor/genética , Proteína p53 Supresora de Tumor/metabolismo , Proteína X Asociada a bcl-2/genética , Proteína X Asociada a bcl-2/metabolismoRESUMEN
CONTEXT: Pinellia ternata (Thunb.) Berit., a perennial herb belonging to Araceae, is one of the few medicinal plants to produce purine alkaloids. It is speculated that endophytic bacteria from P. ternata may produce guanosine or inosine. However, there is no report about endophytic bacteria in P. ternata. OBJECTIVE: In this study, endophytic bacteria were isolated from P. ternata and examined for the first time. This study finds a novel way to increase the yield of P. ternata herb, and to provide some new alkaloid producers. MATERIALS AND METHODS: Plant material includes leaves, tubers, and roots of cultivated and wild P. ternata. The dilutions were smeared onto beef extract-peptone medium and cultured at 28 °C in darkness for 48-72 h. Co-culture treatments were prepared by inoculating 100 mL liquid 1/2 MS medium with bacterial culture broth at concentrations of 0 (control), 0.5%, and 1.5% (v/v). RESULTS: Of the 34 endophytic bacterial colonies isolated from P. ternata leaves, roots, and tubers, five strains were able to produce purine alkaloids. Results from 16s rDNA sequence analysis indicated that the bacteria belonged to Bacillus cereus, Aranicola proteolyticus, Serratia liquefaciens, Bacillus thuringiensis, and Bacillus licheniformis. Co-culture with living Serratia liquefaciens cells increased PLB growth by 58-71%. Co-culture with living Bacillus licheniformis cells increased PLB growth by 4-11%. CONCLUSION: This study provides a novel way for improving the yield of P. ternata herb, and for the production of purine alkaloids by the fermentation industry.
Asunto(s)
Alcaloides/aislamiento & purificación , Bacillus/aislamiento & purificación , Endófitos/aislamiento & purificación , Estiércol/microbiología , Pinellia , Serratia liquefaciens/aislamiento & purificación , Técnicas de Cocultivo , Hojas de la Planta , Raíces de Plantas , Tubérculos de la Planta , Purinas/aislamiento & purificaciónRESUMEN
We developed a method for the identification of 18 illegal adulterants in dietary supplements for erectile dysfunction by using high-performance liquid chromatography-mass spectrometry. The separation was achieved on a Cosmosil 3C18-EB column. The mobile phase consisted of 0.1% formic acid solution and 0.1% formic acid in acetonitrile, with gradient elution at a flow rate of 0.15 mL/min. The proposed method may be useful for the identification of illegal adulterants and for quality control of dietary supplements.
Asunto(s)
Cromatografía Líquida de Alta Presión/métodos , Suplementos Dietéticos/análisis , Contaminación de Alimentos/análisis , Contaminación de Alimentos/legislación & jurisprudencia , Espectrometría de Masas/métodos , Acetonitrilos , Benzodioxoles/aislamiento & purificación , Carbolinas/aislamiento & purificación , Formiatos , Inhibidores de Fosfodiesterasa 5/aislamiento & purificación , Piperazinas/aislamiento & purificación , Purinas/aislamiento & purificación , Control de Calidad , Citrato de Sildenafil , Soluciones , Sulfonas/aislamiento & purificación , Tadalafilo , Agentes Urológicos/aislamiento & purificación , Vasodilatadores/aislamiento & purificaciónRESUMEN
With the prefectural governments' aid of the purchase, the Division of Pharmacognosy, Phytochemistry and Narcotics, National Institute of Health Sciences (NIHS) successively has surveyed illegal constituents in health food products implicitly advertizing tonic or slimming effect since the fiscal year of 2002 (slimming type) or 2003 (tonic type). The average numbers of the analyzed products per year are about 100 (slimming type) and 150 (tonic type), respectively. We also continuously distribute standards of authentic samples of several illegal components such as N-nitrosofenfluramine (NFF) and sildenafil (SIL) to prefectural institutes and the average gross number per year is about 140. In the case of slimming type, the fact that the products containing NFF were widely sold in Japanese markets in 2002 is well known. In addition, phenolphthalein, fenfluramine, sibtramine, desdimethylsibtramine, orlistat, mazindol, Rhubarb, Senna Leaf, etc. have been found as illegal constituents. In the tonic type products, we have identified more than 20 synthetic compounds relating to the erectile dysfunction (ED) treatment drugs, SIL, vardenafil and tadalafil (TDF). Since 2005, their synthetic intermediates and the patented but non-approved PDE5 inhibitors also have been found. It should be noted that TDF was found in the shells of capsule in 2009 and that mutaprodenafil was found as pro-drug type illegal component in 2010. In this report identification method of these illegal constituents is briefly described and then analytical trend in this decade is reviewed.
Asunto(s)
Suplementos Dietéticos/análisis , Alimentos Orgánicos/análisis , Agencias Gubernamentales , Drogas Ilícitas/análisis , Fenfluramina/análogos & derivados , Fenfluramina/análisis , Fenfluramina/aislamiento & purificación , Análisis de los Alimentos , Japón , Piperazinas/análisis , Piperazinas/aislamiento & purificación , Purinas/análisis , Purinas/aislamiento & purificación , Citrato de Sildenafil , Sulfonas/análisis , Sulfonas/aislamiento & purificaciónRESUMEN
The successfully developed magnetic molecularly imprinted polymers (MMIPs) toward six synthetic phosphodiesterase type-5 (PDE-5) inhibitors were described. Sildenafil was used as template for the preparation of MMIPs using superparamagnetic core-shell nanoparticle as supporter. The obtained MMIPs were characterized using transmission electron microscope, Fourier transform infrared, X-ray diffraction, and vibrating sample magnetometer. High performance liquid chromatography (HPLC) with diode array detector (DAD) was used for the analysis of target analytes. The application of MMIPs as selective sorbent in the cleanup of herbal medicine samples prior to HPLC offered simple sample preparation. The adsorption capacity and selectivity of prepared MMIPs and magnetic non-molecularly imprinted polymers were investigated. The binding isotherms were obtained for sildenafil and fitted by Freundlich isotherm model. Structurally similar compound of sildenafil and a reference compound protocatechuic acid were used for investing the selective recognition of MMIPs.
Asunto(s)
Medicamentos Herbarios Chinos/química , Imidazoles/aislamiento & purificación , Inhibidores de Fosfodiesterasa 5/aislamiento & purificación , Piperazinas/aislamiento & purificación , Plantas Medicinales/química , Sulfonas/aislamiento & purificación , Cromatografía Líquida de Alta Presión , Hidroxibenzoatos/química , Imidazoles/química , Nanopartículas de Magnetita/química , Magnetometría , Imanes , Microscopía Electrónica de Transmisión , Impresión Molecular , Inhibidores de Fosfodiesterasa 5/química , Piperazinas/química , Purinas/química , Purinas/aislamiento & purificación , Citrato de Sildenafil , Extracción en Fase Sólida , Espectroscopía Infrarroja por Transformada de Fourier , Sulfonas/química , Triazinas/química , Triazinas/aislamiento & purificación , Diclorhidrato de VardenafilRESUMEN
We developed a method for the separation and identification of illegal adulterants (hydroxythiohomosildenafil, aminotadalafil, thiosildenafil, dimethylsildenafil, and thiodimethylsildenafil) from dietary supplements using high-performance liquid chromatography-mass spectrometry. The separation was achieved on a C18 column: the mobile phase consisted of 5 mM ammonium formate (pH 6.3)-acetonitrile (75 : 25, v/v) and acetonitrile, with gradient elution at a flow rate of 0.2 mL/min. The proposed method could also be used to separate vardenafil, homosildenafil, and dimethylsildenafil, all of which have the same molecular weight. Furthermore, the proposed method could simultaneously separate hydroxythiohomosildenafil, aminotadalafil, thiosildenafil, dimethylsildenafil, thiodimethylsildenafil, vardenafil, and homosildenafil. Thus, this method may be useful to identify medicinal ingredients for erectile dysfunction and their analogs and to control the quality of dietary supplements.
Asunto(s)
Benzodioxoles/aislamiento & purificación , Carbolinas/aislamiento & purificación , Cromatografía Líquida de Alta Presión/métodos , Suplementos Dietéticos/análisis , Contaminación de Medicamentos , Espectrometría de Masas/métodos , Inhibidores de Fosfodiesterasa 5/aislamiento & purificación , Pirimidinas/aislamiento & purificación , Sulfonas/aislamiento & purificación , Piperazinas/aislamiento & purificación , Purinas/aislamiento & purificación , Citrato de SildenafilRESUMEN
A sildenafil analogue was detected in a functional coffee sample labelled to have male sexual performance enhancement effects. This analogue was isolated and purified by flash chromatography and preparative high-performance liquid chromatography. Its structure was elucidated using high-resolution mass spectrometry; electrospray ionization-tandem mass spectrometry; and nuclear magnetic resonance spectroscopy, ultraviolet spectroscopy, and infrared spectroscopy. Compared with sildenafil, instead of an N-methylpiperazinyl moiety, ring opening of the piperazine ring with the loss of a carbon atom resulted in a substituted benzenesulfonamide. The chemical name of this analogue is N-[2-(dimethylamino)ethyl]-4-ethoxy-3-(1-methyl-7-oxo-3-propyl-6,7-dihydro-1H-pyrazolo[4,3-d]pyrimidin-5-yl)benzenesulfonamide. It is named descarbonsildenafil because it has one less carbon atom when compared with sildenafil.
Asunto(s)
Café/química , Piperazinas/química , Extractos Vegetales/química , Sulfonamidas/química , Sulfonas/química , Vasodilatadores/química , Cromatografía Líquida de Alta Presión , Espectrometría de Masas/métodos , Estructura Molecular , Piperazinas/aislamiento & purificación , Purinas/química , Purinas/aislamiento & purificación , Citrato de Sildenafil , Sulfonamidas/aislamiento & purificación , Sulfonas/aislamiento & purificación , Vasodilatadores/aislamiento & purificaciónRESUMEN
Two new phosphodiesterase-5 inhibitors (PDE-5) which consist of one sildenafil analogue and one thiosildenafil analogue have been found in heath supplements. The structural properties of these analogues have been elucidated by NMR, high resolution MS, MS(2), UV and IR spectroscopy. The sildenafil analogue is very similar to aildenafil and the thiosildenafil analogue is similar to thioaildenafil, except the ethoxy group bonded to phenyl ring is replaced by a propoxy group. Hence, the sildenafil analogue is named as propoxyphenyl aildenafil or propoxyphenyl methisosildenafil and the thiosildenafil analogue as propoxyphenyl thioaildenafil or propoxyphenyl thiomethisosildenafil.
Asunto(s)
Suplementos Dietéticos/análisis , Contaminación de Medicamentos , Inhibidores de Fosfodiesterasa 5/aislamiento & purificación , Piperazinas/aislamiento & purificación , Pirimidinas/aislamiento & purificación , Sulfonas/aislamiento & purificación , Cromatografía Líquida de Alta Presión , Espectroscopía de Resonancia Magnética , Estructura Molecular , Inhibidores de Fosfodiesterasa 5/química , Piperazinas/química , Purinas/química , Purinas/aislamiento & purificación , Pirimidinas/química , Citrato de Sildenafil , Espectrofotometría Ultravioleta , Espectroscopía Infrarroja por Transformada de Fourier , Sulfonas/química , Espectrometría de Masas en TándemRESUMEN
iErect, a new dietary supplement marketed as "100% natural" and sold over the Internet, was analyzed. It contains thiosildenafil, a sildenafil analogue already reported as an adulterant in herbal formulations, and a new compound whose structure was elucidated after isolation using NMR, MS and IR. It was named depiperazinothiosildenafil as it results from the hydrolytic cleavage of the S-N bond of the sulfonamide group of thiosildenafil. A capsule of iErect contains a very high amount (≈220mg) of thiosildenafil and ≈30mg of depiperazinothiosildenafil, which places consumers at risk for potentially serious side-effects.
Asunto(s)
Suplementos Dietéticos/análisis , Suplementos Dietéticos/normas , Contaminación de Medicamentos , Piperazinas/aislamiento & purificación , Preparaciones de Plantas/análisis , Preparaciones de Plantas/normas , Pirimidinas/aislamiento & purificación , Sulfonas/aislamiento & purificación , Tionas/aislamiento & purificación , Cápsulas , Cromatografía Líquida de Alta Presión , Contaminación de Medicamentos/legislación & jurisprudencia , Espectroscopía de Resonancia Magnética , Piperazinas/química , Purinas/química , Purinas/aislamiento & purificación , Pirimidinas/química , Citrato de Sildenafil , Espectroscopía Infrarroja por Transformada de Fourier , Sulfonas/química , Espectrometría de Masas en Tándem , Tionas/químicaRESUMEN
A sildenafil analogue was detected and isolated from a health supplement claimed for human use. The structure of this new analogue was elucidated using LC-UV, LC-Orbitrap-MS, IR spectroscopy, 1D and 2D NMR. It was characterized as dithio-desmethylcarbodenafil containing 2 thiocarbonyl groups instead of 2 carbonyl groups, and 4-methyl substitution, on the piperazine ring, rather than 4-ethyl substitution, when compared to sildenafil.
Asunto(s)
Suplementos Dietéticos/análisis , Contaminación de Medicamentos , Piperazinas/química , Sulfonas/química , Cromatografía Liquida/métodos , Humanos , Espectroscopía de Resonancia Magnética/métodos , Espectrometría de Masas/métodos , Piperazina , Piperazinas/aislamiento & purificación , Purinas/química , Purinas/aislamiento & purificación , Citrato de Sildenafil , Espectrofotometría Infrarroja/métodos , Espectrofotometría Ultravioleta/métodos , Sulfonas/aislamiento & purificaciónRESUMEN
The market success of the three approved synthetic phosphodiesterase type-5 (PDE-5) inhibitors for the treatment of erectile dysfunction has led to an explosion in counterfeit versions of these drugs. In parallel a large market has developed for herbal products claimed to be natural alternatives to these synthetic drugs. The herbal products are heavily advertised on the internet and are freely available to purchase without prescription. Furthermore, adulteration of these supposed natural medicines is a very common and serious phenomenon. Recent reports have shown that the adulteration has extended to the analogues of the three approved synthetic PDE-5 inhibitors. An Atmospheric Solids Analysis Probe (ASAP) was used for the direct analysis of the counterfeit pharmaceuticals and herbal products. Using the ASAP combined with time-of-flight mass spectrometry (TOF MS) it was possible to detect fraudulent counterfeit tablets. The physical appearance of the pills resembled the pills from the original manufacturer but contained the wrong active pharmaceutical ingredient (API). Detecting adulteration for five herbal supplements marketed as natural alternatives to PDE-5 inhibitors was also possible using the ASAP. Three types of adulteration were found in the five samples: adulteration with tadalafil or sildenafil, mixed adulteration (tadalafil and sildenafil), and adulteration with analogues of these drugs.
Asunto(s)
Medicamentos Falsificados/aislamiento & purificación , Inhibidores de Fosfodiesterasa 5/aislamiento & purificación , Preparaciones de Plantas/aislamiento & purificación , Extracción en Fase Sólida/métodos , Espectrometría de Masa por Ionización de Electrospray/métodos , Carbolinas/química , Carbolinas/aislamiento & purificación , Medicamentos Falsificados/química , Espectrometría de Masas/métodos , Inhibidores de Fosfodiesterasa 5/química , Piperazinas/química , Piperazinas/aislamiento & purificación , Preparaciones de Plantas/química , Purinas/química , Purinas/aislamiento & purificación , Citrato de Sildenafil , Sulfonas/química , Sulfonas/aislamiento & purificación , Tadalafilo , Factores de TiempoRESUMEN
In present study, a capillary electrophoresis-mass spectrometry (CE-MS) method was developed for the simultaneous analysis of 12 nucleosides and nucleobases including cytosine, adenine, guanine, cytidine, cordycepin, adenosine, hypoxanthine, guanosine, inosine, 2'-deoxyuridine, uridine and thymidine in natural and cultured Cordyceps using 5-chlorocytosine arabinoside as an internal standard (IS). The CE separation conditions and MS parameters were optimized systematically for achieving good CE resolution and MS response of the investigated compounds. The optimum CE electrolyte was 100 mM formic acid containing 10% (v/v) methanol. The optimum MS parameters were as follows: 75% (v/v) methanol containing 0.3% formic acid with a flow rate of 3 microL/min was selected as the sheath liquid; the flow rate and temperature of drying gas were 6 L/min and 350 degrees C, respectively. The optimized CE-MS method was successfully applied for the simultaneous determination of 12 nucleosides and nucleobases in natural and cultured Cordyceps. On the basis of quantitative results, the total content of nucleosides is much higher in cultured Cordyceps (9138+/-4823 microg/g for cultured C. sinensis; 3722+/-1446 microg/g for C. militaris) than in natural ones (2167+/-412 microg/g). However, the hypoxanthine (131+/-47 microg/g) and inosine (335+/-90 microg/g) are much higher in natural C. sinensis. Cordycepin, which is abundant in cultured C. militaris (2276.5+/-842.6 microg/g), is only found in natural C. sinensis with very low content and cannot be detected in the cultured ones.
Asunto(s)
Cordyceps/química , Nucleósidos/química , Extractos Vegetales/química , Electroforesis Capilar/métodos , Espectrometría de Masas/métodos , Nucleósidos/análisis , Nucleósidos/aislamiento & purificación , Extractos Vegetales/análisis , Purinas/análisis , Purinas/química , Purinas/aislamiento & purificaciónAsunto(s)
Contaminación de Medicamentos , Disfunción Eréctil/tratamiento farmacológico , Gliburida/efectos adversos , Hipoglucemia/inducido químicamente , Hipoglucemiantes/efectos adversos , Drogas Ilícitas/efectos adversos , Extractos Vegetales/química , Adulto , Anciano , Anciano de 80 o más Años , Carbolinas/química , Cromatografía Líquida de Alta Presión , Brotes de Enfermedades , Gliburida/aislamiento & purificación , Humanos , Hipoglucemia/epidemiología , Hipoglucemiantes/aislamiento & purificación , Masculino , Persona de Mediana Edad , Inhibidores de Fosfodiesterasa/química , Inhibidores de Fosfodiesterasa/aislamiento & purificación , Piperazinas/aislamiento & purificación , Extractos Vegetales/efectos adversos , Purinas/aislamiento & purificación , Citrato de Sildenafil , Singapur/epidemiología , Sulfonas/aislamiento & purificación , Tadalafilo , Adulto JovenRESUMEN
Two unknown compounds are detected and isolated from health supplements for the enhancement of sexual function. The structures of the unknown compounds are elucidated using high-resolution MS, ESI-MS/MS, NMR, UV and IR. One compound is identified as an analogue of sildenafil in which the oxygen atom is substituted with a sulfur atom in the pyrazolopyrimidine moiety, and an ethyl group instead of a methyl group is attached to the piperazinyl nitrogen. Hence, this compound is named thiohomosildenafil. Another compound is also a sildenafil analogue in which the oxygen atom is substituted with a sulfur atom in the pyrazolopyrimidine moiety. This compound is named thiosildenafil. Both the two compounds are first detected in health supplements. The UV, IR and completely assigned NMR data of thiohomosildenafil and thiosildenafil are first reported.
Asunto(s)
Suplementos Dietéticos/análisis , Contaminación de Alimentos , Inhibidores de Fosfodiesterasa/análisis , Inhibidores de Fosfodiesterasa/aislamiento & purificación , Piperazinas/análisis , Piperazinas/aislamiento & purificación , Sulfonas/análisis , Sulfonas/aislamiento & purificación , Humanos , Espectroscopía de Resonancia Magnética , Masculino , Espectrometría de Masas , Estructura Molecular , Inhibidores de Fosfodiesterasa/química , Piperazinas/química , Purinas/análisis , Purinas/química , Purinas/aislamiento & purificación , Citrato de Sildenafil , Espectrometría de Masa por Ionización de Electrospray , Espectrofotometría Infrarroja , Espectrofotometría Ultravioleta , Sulfonas/química , Espectrometría de Masas en TándemRESUMEN
Xanthine oxidase (XO) is a key enzyme which can catalyze xanthine to uric acid causing hyperuricemia in humans. By using the fractionation technique and inhibitory activity assay, an active compound that prevents XO from reacting with xanthine was isolated from wheat leaf. It was identified by the Mass and NMR as 6-aminopurine (adenine). A structure-activity study based on 6-aminopurine was conducted. The inhibition of XO activity by 6-aminopurine (IC(50)=10.89+/-0.13 microM) and its analogues was compared with that by allopurinol (IC(50)=7.82+/-0.12 microM). Among these analogues, 2-chloro-6(methylamino)purine (IC(50)=10.19+/-0.10 microM) and 4-aminopyrazolo[3,4-d] pyrimidine (IC(50)=30.26+/-0.23 microM) were found to be potent inhibitors of XO. Kinetics study showed that 2-chloro-6(methylamino)purine is non-competitive, while 4-aminopyrazolo[3,4-d]pyrimidine is competitive against XO.
Asunto(s)
Inhibidores Enzimáticos/síntesis química , Inhibidores Enzimáticos/farmacología , Purinas/síntesis química , Purinas/farmacología , Xantina Oxidasa/antagonistas & inhibidores , Alopurinol/síntesis química , Alopurinol/farmacología , Cromatografía Líquida de Alta Presión , Evaluación Preclínica de Medicamentos , Inhibidores Enzimáticos/aislamiento & purificación , Cinética , Espectroscopía de Resonancia Magnética , Espectrometría de Masas , Hojas de la Planta/química , Purinas/aislamiento & purificación , Espectrofotometría Ultravioleta , Relación Estructura-Actividad , Triticum/químicaRESUMEN
An analogue of acetildenafil was detected in an extract of pre-mixed bulk powder. To our knowledge, the powder was destined to be encapsulated and sold as a dietary supplement. The structure was identified by NMR, HR-ESI-MS, ESI-MSn and FTIR analyses. Owing to the inclusion of a hydroxyl group in acetildenafil, the detected compound was called 'hydroxyacetildenafil'. With increasing use of dietary supplements marketed for penile erectile dysfunction, the detection of analogues of sexual performance enhancers is important and timely.
Asunto(s)
Suplementos Dietéticos/análisis , Contaminación de Alimentos/análisis , Piperazinas/aislamiento & purificación , Sulfonas/aislamiento & purificación , Cromatografía Líquida de Alta Presión/métodos , Disfunción Eréctil/terapia , Análisis de los Alimentos/métodos , Humanos , Masculino , Inhibidores de Fosfodiesterasa/química , Inhibidores de Fosfodiesterasa/aislamiento & purificación , Piperazinas/química , Purinas/química , Purinas/aislamiento & purificación , Citrato de Sildenafil , Espectrometría de Masa por Ionización de Electrospray/métodos , Sulfonas/químicaRESUMEN
The objective of this study was to develop a practical method for the analysis of purine derivatives in urinary calculi using high-performance liquid chromatography (HPLC). The method presented herein includes extraction of purine derivatives from urinary stones, followed by chromatography on a reversed-phase column with UV detection. A simpler isocratic method was applied to quantitate 6 purines known to be components of urinary stones, namely uric acid, xanthine, hypoxanthine, 2,8-dihydroxyadenine, oxypurinol and allopurinol. Gradient method separated 10 additional peaks representing methyl derivatives of uric acid or xanthine (1-, 3-, 7-, and 9-methyluric acid, 1,3-,1,7-, and 3,7-dimethyluric acid, and 1-, 3-, and 7-methylxanthine) (Fig. 1). Detection limits for individual compounds ranged from 25 to 140 micrograms purine per g stone weight and precision (RSD%) was 0.5-2.4%. Both methods were next used to analyze purine derivatives in urinary calculi from 48 residents of Western Pomerania. Uric acid was the main component of 9 stones. All of the uric acid stones showed admixtures of 9 other purine derivatives: natural metabolites (hypoxanthine, xanthine, 2,8-dihydroxyadenine) and methyl derivatives of uric acid (1-,3-, and 7-methyluric acid, 1,3-dimethyluric acid, 3-, and 7-methylxanthine) originating from the metabolism of exogenous methylxanthines (caffeine, theophylline and theobromine) (Tab. 1,2). Methyl derivatives of uric acid and xanthine, with a maximal content in stones of 1.7%, have hitherto not been considered constituents of urinary calculi. Statistical analysis of the results revealed strong positive correlations between the level of uric acid and of other purine derivatives in stones (Fig. 2). Correlations were also found between levels of some purines and inorganic compounds (Tab. 3). The sensitivity and specificity of HPLC with UV detection satisfy the requirements of a reference method for the analysis of purines in urinary stones. Isocratic separation is simpler in terms of technique and equipment, and therefore more suitable for hospital laboratories. Examination of purine derivatives in stones may be very helpful for the diagnosis of abnormal purine metabolism and urolithiasis, particularly in dihydroxyadeninuria, xanthinuria and during treatment with allopurinol. Gradient separation requiring more sophisticated instrument seems useful for research purposes when the content of methyl derivatives of purines must be known. The present results indicate that urinary purines at concentrations lower than saturation point may nevertheless coprecipitate with oversaturated uric acid and appear as admixtures in urinary stones. The content of a purine derivative in stone depends on its average urinary excretion in the general population, similarity to the chemical structure of uric acid, and content of the latter in stone. These findings suggest that purines in stones represent a solid solution with uric acid as solvent. It is also plausible that methylxanthines, ubiquitous components of the diet and drugs, are involved in the pathogenesis of urolithiasis. Interpretation of results and practical significance of the determination of purine derivatives in stones is discussed, and future studies to assess the clinical importance of endo- and exogenous purine derivatives in urinary calculi are suggested.
Asunto(s)
Purinas/aislamiento & purificación , Cálculos Urinarios/química , Biomarcadores/análisis , Calcio/análisis , Cromatografía Líquida de Alta Presión/métodos , Humanos , Magnesio/análisis , Fósforo/análisis , Sensibilidad y Especificidad , Ácido Úrico/análisisRESUMEN
Cultures of Methanobacterium thermoautotrophicum were supplemented with 13C-labeled acetate or pyruvate, and the labeling pattern of the corrinoid, factor III, was established by 13C NMR spectroscopy. Complete 13C signal assignments were obtained by two-dimensional NMR experiments. The labeling pattern of factor III was analyzed by comparison with those of amino acids and nucleosides. The corrin ring system is derived from eight molecules of glutamate. The aminopropanol moiety is derived in a hitherto unknown pathway from pyruvate by reductive amination. The heterocyclic ring of hydroxybenzimidazole shares the labeling pattern of the imidazole ring of purines. The remaining four carbon atoms of the carbocyclic ring show the labeling signature of a carbohydrate with two of the carbons introduced from acetate and two from C-1 of pyruvate. However, erythrose can be ruled out as the specific precursor on the basis of a detailed investigation of aromatic amino acids indicating that erythrose 4-phosphate is obtained by reductive carboxylation of a triose precursor and not by the pentose phosphate cycle.