Desarrolla un método, altamente selectivo y específico, usando el equipo Cromatógrafo Líquido de Ultraperformance con detector Masa – Masa (UPLC – MS – MS) para la determinación de misoprostol éster a través de su metabolito misoprostol ácido en veinte muestras de orina de mujeres gestantes, provenientes de las Divisiones Médico Legales del Perú, que presumiblemente habrían ingerido tabletas de misoprostol. Se ensayan procedimientos de extracción líquido – líquido, cuyos resultados, en el presente estudio; no son reproducibles. Debido a ello se ensayan procedimientos de extracción en fase sólida (SPE) con cartuchos especiales HLB cuyos resultados son reproducibles. Este método logra cuantificar concentraciones hasta partes por billón (ppb), con una concentración promedio de 40,23 y demás valores que están dentro de los límites 11,5 y 81,6 ppb. El método es de gran utilidad para demostrar el uso de misoprostol como abortivo en investigaciones judiciales.

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Introduction: Severe ischemia-reperfusion injury (IRI) after liver transplantation are correlated with lower early and late graft and patient survival, higher infection rates, appearance of acute and chronic rejections and other complications. In this field, it has been demonstrated that the prostaglandin E1 (PGE1) presents cytoprotective, anti-inflammatory and vasodilator effects. Therefore, the PGE1 assembles most of the requirements necessary to prevent the development of the ischemia-reperfusion injury, especially if it is administered through via portal vein at full doses in the moment of graft reperfusion.Materials and methods: Female Largewhite-Landrace pigs (body weight between 30-35 kg) were used. The orthotopic liver transplantation were performed in 18 pairs of pigs. The study was divided into three groups as follows: Control group, 4 experiments of orthotopic liver transplantation with a cold preservation time of 3 hours in Wisconsin solution; Groups A and B, 7 experiments with 24 hours of cold preservation time in each group. Concerning intraportal infusion of medication, the Group B was infused with prostaglandin E1 at a dose of 0.15 ug/kg/min (Alprostadil 500ug) for 90 minutes in the portal reperfusion of the graft; and Group A was infused with saline solution for 90 minutes in the same time period. In the recipient surgery, biochemical (GOT, GPT, LDH, B-Galactosidase, Hialuronic Acid), hemodynamic and histological studies were carried out to evaluate the ischemia-reperfusion injury (IRI).Results: Survival rate in control group was 100 % and it presented a lower level of sinusoidal-hepatocellular injury and leukocyte activation than the groups A and B. From the beginning of the surgery until the moment of the PGE1 administration, both the group A and the group B were similar in relation to the histology, hepatocellular, endothelial and inflammatory damage markers. After the infusion of the medication (PGE1), the group B developed an increase of the total hepatic flow and a lower alteration of all the parameters of hepatocyte-sinusoidal injury and hepatic function, with an statistical significance in the great majority of them. The portal PGE1 infusion at full doses did not generate hemodynamic instability. Conclusions: The liver transplantation in pigs with 24 hours of cold ischemia of the graft produced a severe IRI that caused the death of all the animals in the groups A and B due to primary graft nonfunction. Nevertheless, with the intraportal PGE1infusion, the group B presented an increase of the total hepatic flow and a lower alteration of all the parameters of hepatocellular injury, sinusoidal damage and hepatic function. The portal PGE1 perfusion at full doses was well tolerated at hemodynamic level.

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No. of bitstreams: 1 cuervo_jco_me_bot.pdf: 1620368 bytes, checksum: d662e09785a2bb73aaa15aaf92df4d19 (MD5) Previous issue date: 2016-09-29 / Em ruminantes a luteólise natural é caraterizada pela liberação de vários pulsos de prostaglandina F2alfa (PGF) produzidos pelo útero. A PGF é o hormônio luteolítico, enquanto a prostaglandina E1 (PGE1) é considerada um mediador luteoprotetor. Em estudos anteriores, infusões com doses baixas de PGF no útero com intervalos de 6 horas (h) resultou em regressão do corpo luteo (CL). A proposta deste experimento é desenvolver um modelo para avaliar o efeito de baixas doses de PGE1, também infundidas no lúmen uterino sobre a resposta luteal à PGF intrauterina (IU). Vacas no dia 10 do ciclo estral receberam infusões IU de salina (0,1 ml de salina + 0,1 ml de DMSO), PGE (2 mg de PGE1 em 0,1ml de DMSO) ou PGF (0,25 mg of PGF em 0,1 ml de salina) em intervalos de 6 h em um desenho experimental 2 X 2. Portanto os animais foram agrupados em quatro tratamentos: SALINA (4 infusões de salina; n=5), PGE (4 infusões de PGE1; n=5), PGF (4 infusões de PGF; n=5) e PGE+PGF (4 infusões de PGE1+PGF; n=5). As concentrações plasmáticas de progesterona (P4) foram dosadas por radioimunoensaio e o volume luteal foi determinado por ultrassonografia transretal. As concentrações circulantes de PGFM e PGEM foram dosadas antes e 10 minutos após as primeiras duas infusões. Biopsias luteais foram coletadas de cada vaca 30 minutos após cada infusão para determiner a expressão de genes em resposta a cada tratamento. As concentrações circulantes de PGFM 10 minutos após as infusões foram maiores em vacas que receberam tratamentos com PGF e PGE+PGF em comparação com as vacas tratadas com salina e PGE. Da mesma forma, as concentrações de PGEM 10 minutos após cada infusão foram maiores em vacas tratadas com PGE e PGE+PGF em comparação com vacas dos grupos salina e PGF. As concentrações de P4 diminuiram no grupo PGF em comparação com o grupo Salina no tempo 12-h (48,9% do controle) após a primeira infusão de PGF, no tempo 24-h (20,2% do controle), e em todos tempos subsequentes (P < 0,05). Não foram encontradas diferenças nas concentrações circulantes de P4 entre os grupos Salina, PGE e PGF+PGE. Houve também uma diminuição do volume luteal entre o grupo PGF e os outros três grupos que foi observada nos tempos 24-h (56,4% do controle), 48-h (30,6% do controle), e 72-h (20,4% do controle) após o tratamento com PGF (P<0.05). Não houve diferenças no volume luteal entre os tratamentos salina, PGE e PGE+PGF. Por tanto, infusões IU simultâneas de baixas doses de PGE1 bloquearam a ação luteolitica de pulsos IU de PGF em vacas, como observado nas mudanças circulantes de P4 e volume luteal. Análises da expressão génica nas biopsias luteais coletadas após o terceiro pulso de PGF, indicam o padrão tipico de expressão de genes em resposta ao tratamento com PGF (FGF2, EGR1, FOS e FAS aumentaram; PTGFR, VEGFA, NR5A1 e STAR diminuiram) e o tratamento PGE+PGF bloqueou completamente as mudanças na expressão destes genes. Infusões IU de PGF e PGE1 parecem ser um excelente modelo para determiner o padrão de expressão de genes envolvidos no efeito luteoprotetor da PGE1. / In ruminants, natural luteolysis is characterized by the release of several pulses of prostaglandin F2alpha (PGF) produced by the uterus. Prostaglandin F2alpha is the luteolytic hormone, whereas prostaglandin E1 (PGE1) is considered to be a luteoprotective mediator. In previous studies, low doses of PGF infused into the uterus at 6 hour (h) intervals resulted in regression of the corpus luteum (CL). This study was designed to develop a model to study the effect of low doses of PGE1, also infused into the uterine lumen, on the luteal responses to intrauterine (IU) PGF. Cows on day 10 of the estrous cycle received IU infusions of saline (0,1 ml of saline + 0,1 ml of DMSO), PGE (2 mg of PGE1 in 0,1ml of DMSO) or PGF (0,25 mg of PGF in 0,1 ml of saline) at 6-h intervals in a 2 X 2 experimental design. Thus, there were four treatment groups: SALINE (4 saline infusions; n=5), PGE (4 PGE1 infusions; n=5), PGF (4 PGF infusions; n=5), and PGE+PGF (4 PGE1+PGF infusions; n=5). Radioimmunoassay was used to measure circulating progesterone (P4) concentrations and luteal volume was determined by transrectal ultrasonography. Circulating concentrations of PGFM and PGEM were measured before and 10 minutes after the first two infusions. A luteal biopsy was collected from each cow at 30 minutes after each infusion for later determination of gene expression in response to each treatment. Circulating concentrations of PGFM 10 minutes after infusions were greater in cows receiving treatments with PGF and PGE+PGF than in Saline or PGE-treated cows. In the same way, concentrations of PGEM 10 minutes after infusions, were greater in cows that were treated with PGE and PGE+PGF than in saline and PGF-treated cows. Concentrations of P4 in the PGF group decreased compared to those in the saline group by 12-h (48.9% of control) after first infusion of PGF, at 24-h (20,2% of control), and all subsequent time points (P < 0,05). No differences in circulating P4 concentrations were found between Saline, PGE, and PGF+PGE. There was also a decrease of luteal volume between the PGF group and the other three groups that was detectable at 24 (56,4% of control), 48 (30,6% of control), and 72 (20,4% of control) h after PGF treatment (P<0.05). There were no differences in luteal volume between Saline, PGE, or PGE+PGF. Thus, simultaneous IU infusion of a low dose of PGE1 blocked the luteolytic actions of IU PGF pulses in cattle, as measured by changes in circulating P4 and luteal volume. Analyses of gene expression in the luteal biopsy taken after the third PGF pulse indicate a typical pattern of gene expression in response to the PGF treatments (FGF2, EGR1, FOS and FAS increased; PTGFR, VEGFA, NR5A1 and STAR decreased) and that simultaneous PGE1 treatment completely blocked these gene expression changes. Thus, IU infusion of PGF and PGE1 seems to provide an excellent model for determining the patterns of gene expression involved in the luteoprotective effect of PGE1.