Validación de un método para determinar la concentración sérica de voriconazol por HPLC/UV / Validation of a method to determine the serum concentration of voriconazole by HPLC/UV-Vis

Antecedentes: El incremento de infecciones fúngicas invasivas ha incrementado el uso de voriconazol como profilaxis y tratamiento, siendo necesario monitorizar sus concentraciones séricas.Objetivo:Estandarizar y validar un método sencillo, con alta eficacia y especificidad para la determinación de voriconazol.Material y métodos:Para la cuantificación de voriconazol se empleó un equipo de cromatografía líquida de alta resolución Shimadzu, acoplado a un detector ultravioleta-visible diodo-array, realizando la separación cromatográfica con una columna Brisa LC2 C18. Las condiciones cromatográficas que se definieron fueron: temperatura de la columna, 35ºC; longitud de onda, 256 nm; volumen de inyección, 20 µl; flujo, 1,5 ml/min; tiempo de análisis, 9 min, fase móvil agua con ácido fórmico 0,5 % / acetonitrilo 65/35. Previo a la inyección cromatográfica, las muestras sufrieron un tratamiento consistente en la precipitación de proteínas con acetonitrilo y posterior centrifugación, inyectándose el sobrenadante. Se utilizó el programa estadístico SPSS v. 25, considerando una p<0,05 como estadísticamente significativa.Resultados:El método puesto a punto es selectivo y lineal (r2 =1), con un coeficiente de variación ≤5 %. En cuanto a la exactitud y la precisión los coeficientes de variación fueron ≤ 5 %, cumpliendo así con los requisitos establecidos para el rango de concentraciones 0,1 µg/ml-10 µg/ml.Conclusiones:La selectividad y la sencillez del tratamiento de muestra hacen de él un método eficaz, rápido y sencillo para la determinación de voriconazol en suero y con sensibilidad mayor al de los inmunoensayos utilizados. (AU)

Background: The high increase of invasive fungal infections has increased the use of voriconazole as prophylaxis and treatment, being necessary to monitor its serum concentrations.Objective:To standardize and validate a simple method with high efficacy and specificity for the determination of voriconazole.Method:For the quantification of voriconazole, a Shimadzu high performance liquid chromatography equipment was used, coupled to an ultraviolet-visible diode array detector, performing the chromatographic separation with a Brisa LC2 C18 column. The chromatographic conditions defined were: column temperature, 35ºC; wavelength, 256 nm; injection volume, 20 µl; flow rate, 1.5 ml/min; analysis time, 9 min, mobile phase water with formic acid 0.5 % / acetonitrile 65/35. Prior to chromatographic injection, the samples underwent a treatment consisting of protein precipitation with acetonitrile and subsequent centrifugation, and the supernatant was injected The SPSS v. 25 statistical program was used, considering a p<0.05 as statistically significant.Results:The method developed is selective and linear (r2 =1), with a coefficient of variation ≤ 5%. In terms of accuracy and precision, the coefficients of variation were ≤ 5 %, thus complying with the requirements established for the concentration range 0.1 µg/ml-10 µg/ml.Conclusion:The selectivity and the simplicity of the sample treatment make it an effective, fast and simple method for the determination of voriconazole in serum and with a higher sensitivity than the immunoassays used. (AU)

Development and validation of a method for simultaneous determination of pseudouridine and creatinine in human urine. Evaluation’s Index Pseudouridona/Creatinine in smokers and no-smokers

Introduction: The excretion of pseudouridine is increased in inflammatory processes related to a muscle mass loss found in patients with pulmonary involvement. Material and Methods: A rapid and sensitive method for cuantification and simultaneous determination of pseudouridine, a breakdown product of tRNA, and creatinine in human urine via HPLC was developed and validated. The mobile phase was 0.01 mol phosphate buffer (pH 6.1) containing 3 mmol octanesulphonic acid as ion pairing agent. Sample preparation is based on dilution and filtration. A LiCHros-pher® 100 RP-18 (5 μm) LiCHroCART® 250-4 (Merck) column with precolumn LiCHrospher® 100 RP-18 (5 μm) LiCHroCART® 50-4 (Merck) were used, flow rate of 1ml/min. Detection wavelength was set at 250 nm. Results: The analysis time was 17 min per sample. The calibration range of pseudouridine (Psu) and creatinine (Crea) were 0.23-22.5 and 11.45-1100 nmol/ml. The linearity of the method was r2 = 0.997 and 0.998 and the lower limit of quantification (LLOQ) was 0.175 and 8.59 nmol/ml respectively. The average recovery (%) was 95.55 for pseudouridine and 97.82 for creatinine by addition and 93.16 and 89.79 % by dilution. The estimation of the coefficients of variation were < 8% for all levels. Conclusions: A positive correlation was found between expected and observed values (Pearson correlation coefficient = 0.99 for pseudouridine and 0.99 for creatinine). A correlation was found between recovery of pseudouridine and recovery of creatinine (Pearson correlation coefficient = 0.86). This method was used to assess pseudouridine excretion in 30 healthy subjects (18 non-smokers and 12 smokers). There were no statistically differences between non-smokers and smokers (AU)

Introdución: La excreción de pseudouridina, está incrementada en procesos inflamatorios relacionados con pérdida de masa muscular encontradaen pacientes con afectación pulmonar. Material y Métodos: Se desarrolla y valida, un método, mediante HPLC, para la determinación simultanea de pseudouridina y creatinina en orina. Como fase estacionaria se utilizó una columna LiCHrospher® 100 RP-18 (5 μm) LiCHroCART® 250-4 (Merck). Fase móvil consistente en un tampón fosfato 0,01 M (pH = 6,1) conteniendo octanosulfónico 3mmol como agente de par iónico, y longitud de onda de 250 nm. La preparación de la muestra se basa en una dilución y filtración. La linealidad del método fue satisfactoria dentro del rango de concentración de 0,23-22,5 nmol/ml para pseudouridina y 1,45-1100 nmol/ml para creatinina, con límites de cuantificación de 0,175 y 8,59 nmol/ml, respectivamente. Resultados: La recuperación media fue del 95,55% para Pseudouridina y del 97,82% para Creatinina en la validación de adición de estándar interno y de 93,16 y 89,79% en la de dilución. Los coeficientes de variación fueron < del 8% en todos los niveles. Se encontró una correlación entre los valores encontrados y los esperados (coeficiente de correlación de Pearson de 0,99). Conclusiones: Existe correlación entre las recuperaciones de pesudouridina y creatinina, coeficiente de correlación de Pearson de 0,86. El método desarrollado, es rápido, sensible y selectivo para la simultanea determinación de pseudouridina y creatinina en orina humana. En este estudio preliminar con 30 voluntarios sanos, 18 no fumadores y 12 fumadores, no se han encontrado diferencias estadísticamente significativas entre ambos grupos con respecto a la excreción de pseudouridina (AU)

Catabolismo muscular en pacientes con enfermedad pulmonar obstructiva crónica / Muscle catabolism in patients with chronic obstructive pulmonary disease

Antecedentes y objetivos. La enfermedad pulmonar obstructiva crónica (EPOC) se acompaña de afectación muscular. La pseudouridina, catabolito del RNA, ha sido empleado en otras afecciones para evaluar el catabolismo muscular. Hemos examinado la excreción de pseudouridina en pacientes con diferentes estadios evolutivos de su EPOC. Sujetos y métodos. En cuatro grupos de sujetos (controles sanos y enfermos con bronquitis crónica, EPOC emergente y EPOC avanzada) determinamos la excreción urinaria de pseudouridina (orina aleatoria) mediante cromatografía líquida de alta resolución (HPLC). Resultados. La excreción de pseudouridina (cociente pseudouridina/creatinina [Psu/Crea]) en la población sana fue de (media ± DE) 19,9±6,6μmol/mmol y se halló muy incrementada en todos los enfermos con afección pulmonar: bronquitis crónica, 44,1±60,7μmol/mmol; EPOC emergente, 81,6±56,8μmol/mmol y EPOC avanzada, 140,1±68,0μmol/mmol (p<0,01 para todas las comparaciones con los sujetos normales y entre los pacientes con afección pulmonar). La edad y el sexo no influyeron en la excreción de pseudouridina. Conclusión. La excreción de pseudouridina está incrementada en pacientes con bronquitis crónica y EPOC. Se relaciona con el estadio de la enfermedad y su excreción es independiente de la edad y del sexo(AU)

Background and objectives. Chronic obstructive pulmonary disease (COPD) is accompanied by muscle involvement. Pseudouridine, a catabolite of RNA, has been used in other conditions to assess muscle catabolism. We have examined the excretion of pseudouridine in patients with different stages of COPDs evolution. Subjects and methods. We have defined four population groups: control group (without disease), chronic bronchitis group, emerging COPD group, and advanced COPD group. Pseudouridine was determined by high performance liquid chromatography, Results. Pseudouridine extraction (pseudouridine/creatinine ratio) was (mean + 19.9 (6.6) μmol/mmol in control group and was found to be very increased in all the patients with pulmonary condition: chronic bronchitis, 44.1 (60.75) μmol/mmol, 81.6 (56.8) μmol/mmol in emerging COPD group and 140.1 (68) μmol/mmol in advanced COPD for all the comparisons with normal subjects and among patients with lung disease). Age and gender did not affect pseudouridine excretion. Conclusions. The urinary excretion of pseudouridine is increased in chronic bronchitis and COPD and is related to disease stage. Its excretion is independent of age and gender(AU)

[Muscle catabolism in patients with chronic obstructive pulmonary disease]. / Catabolismo muscular en pacientes con enfermedad pulmonar obstructiva crónica.

BACKGROUND AND OBJECTIVES: Chronic obstructive pulmonary disease (COPD) is accompanied by muscle involvement. Pseudouridine, a catabolite of RNA, has been used in other conditions to assess muscle catabolism. We have examined the excretion of pseudouridine in patients with different stages of COPDs evolution. SUBJECTS AND METHODS: We have defined four population groups: control group (without disease), chronic bronchitis group, emerging COPD group, and advanced COPD group. Pseudouridine was determined by high performance liquid chromatography, RESULTS: Pseudouridine extraction (pseudouridine/creatinine ratio) was (mean+19.9 (6.6) µmol/mmol in control group and was found to be very increased in all the patients with pulmonary condition: chronic bronchitis, 44.1 (60.75) µmol/mmol, 81.6 (56.8) µmol/mmol in emerging COPD group and 140.1 (68) µmol/mmol in advanced COPD for all the comparisons with normal subjects and among patients with lung disease). Age and gender did not affect pseudouridine excretion. CONCLUSIONS: The urinary excretion of pseudouridine is increased in chronic bronchitis and COPD and is related to disease stage. Its excretion is independent of age and gender.

Seguimiento evolutivo de la concentración sérica de colesterol HDL en función del hábito tabáquico / Evolutive follow-up on cHDL serum concentration based on smoking habit

Objetivos: Estudiar las variaciones producidas a lo largo de 3 años en la concentración sérica de colesterol HDL (high density lipoprotein) en función del hábito tabáquico, en una población de varones sanos, así como la prevalencia del hábito en dicha población. Material y métodos: Estudio pareado en el que participan 346 pilotos que acuden a un reconocimiento médico laboral entre los años 2003 y 2006. Se determinan las modificaciones producidas en la concentración sérica de colesterol HDL en función de las diversas conductas respecto al hábito tabáquico. Resultados: El estudio cuenta con un total de 346 participantes con una edad media de 44 ± 9 años. Atendiendo a los cambios encontrados desde el punto de vista del hábito tabáquico, analizamos las variaciones producidas en las concentraciones de colesterol HDL a lo largo de estos tres años. Encontramos un descenso significativo en el grupo que se inicia en el consumo de tabaco (p = 0,01) de 16 mg/dl. En los individuos en los que no existen modificaciones desde el punto de vista del tabaco, encontramos un descenso de 2,62 mg/dl para los fumadores y de 3,32 mg/dl para los no fumadores, ambos estadísticamente significativos (p = 0,04 y p = 0,00 respectivamente). En los fumadores que abandonan el hábito, encontramos un aumento de 1,79 mg/dl, sin encontrar significación estadística (p = 0,4). No encontramos correlación entre las cifras de colesterol HDL y la carga tabáquica. En cuanto a las modificaciones producidas desde el punto de vista del hábito tabáquico, un total de 97 individuos (28%) se declaran fumadores activos durante el año 2003, abandonando el hábito a lo largo de los 3 años siguientes el 40,2% de los mismos. Por el contrario, un 16,7% de los no fumadores o ex fumadores en el año 2003 comienzan con el consumo de tabaco. Conclusiones: Existe un descenso importante en las cifras de colesterol HDL tras el inicio del hábito tabáquico. Tras el abandono del mismo, existe una ligera recuperación, sin que se haya podido demostrar significación estadística. La prevalencia del hábito tabáquico presenta cifras inferiores a la media nacional (AU)

Objective: To study the variations produced over a 3-year period in HDL serum concentration (high density lipoprotein cholesterol) based on the smoking habit, in a healthy male population and the prevalence of smoking in this population. Material and methods: A paired study in which 346 pilots who came for an occupation medical checkup in the years 2003 and 2006 participated. The changes produced in cHDL serum concentration based on different behaviors regarding the smoking habit were measured. Results: A total of 346 subjects, mean age 44 ± 9 years, participated in the study. Considering the changes found from the smoking habit point of view, we analyzed the variations produced in cHDL concentrations over these three years. We found a significant decrease in the group beginning with tobacco consumption (p = 0.01) of 16 mg /dl. In those individuals in whom no changes had occurred from the smoking point of view, we found a decrease of 2.62 mg/dl for the smokers and 3.32 mg/dl for the non-smokers, both statistically significant (p = 0.04 and p = 0.00, respectively). In the smokers who had quit smoking, we found an increase of 1.79 mg/dl, without finding statistical significance (p = 0.4). We did not find any correlation between the cHDL levels and the smoke load. In regards to the modifications produced from the smoking point of view, a total of 97 individuals (28%) declared they were active smokers during 2003, 40.2% of them quitting over the next 3 years. On the contrary, 16.7% of the non-smokers or ex-smokers in 2003 began to smoke. Conclusions: There is a significant decrease in cHDL levels after initiating the smoking habit. There is mild recovery after smoking cessation, but statistical significance could not be demonstrated. The prevalence of the smoking habit has lower levels than on the national level (AU)

Leucocitos, tabaquismo y enfermedad pulmonar obstructiva crónica / Leukocytes, tobacco use and chronic obstructive pulmonary disease

Antecedentes y objetivos: El tabaco provoca una reacción leucocitaria influida por factores como la edad, índice de masa corporal (IMC), presencia de enfermedad pulmonar obstructiva crónica (EPOC) y otras comorbilidades. Estudiamos la responsabilidad del tabaquismo en la respuesta leucocitaria, su relación con algunas comorbilidades y con la proteína C reactiva (PCR) como reactante de fase aguda. Población y método: Tres poblaciones con una edad media de 66,8 ± 8,4 años. Las dos primeras sin comorbilidades; una sin haber fumado nunca (n= 48), la otra fumadora (n= 51) y la tercera (n=63) con EPOC estable. Mediante la bioimpedancia eléctrica determinamos el IMC. La PCR se obtuvo por test de alta sensibilidad. Las comorbilidades se valoraron con el índice de Charlson y el índice de Charlson corregido. Resultados: Hubo un significativo aumento de los leucocitos en la población sana fumadora (7,9 ± 1,7 x103/mm3) respecto a la población sana no fumadora (6.4 ± 1,4 x103/mm3; p< 0,001). No hubo diferencia significativa entre el grupo EPOC, 7,4 ± 2,1 x103/mm3 y la población sana fumadora. La leucocitosis fue independiente de la carga tabáquica, edad, IMC, estadios GOLD y comorbilidades. La PCR se incrementa en la población fumadora y con presencia/diagnóstico de EPOC aunque sin relación estadística con la cifra de leucocitos. Conclusiones: El tabaquismo condiciona una repuesta leucocitaria que perdura en similar intensidad en la población con EPOC y una elevación independiente de la PCR. La enfermedad inflamatoria subclínica está ya presente en el fumador sano y se perpetúa con similar intensidad en la población con EPOC (AU)

Antecedents and objectives: tobacco produces a leukocyte reaction influenced by factors like age, body mass index (BMI), chronic obstructive pulmonary disease (COPD) and other comorbidities. We study the impact of tobacco use in the leukocyte reaction, its relationship with some comorbidities and with the C-reactive protein as a acute phase reactant. Population and method: three populations with a mean age of 66.8± 8.4 years. The first two populations without any comorbidities; one having never smoked (n=48), the other one smoking (n=51) and the third one (n=63) with a stable COPD. The BMI was determined through electrical bioimpedance. The C-reactive protein was determined by high-sensitivity CRP test. The comorbidities were assessed with the Charlson index and the corrected Charlson index. Results: the healthy smoking population presented a significant increase of leukocytes (7.9 ± 1.7 x103/mm3) in comparison with the healthy non-smoking population (6.4 ± 1.4 x103/mm3; p< 0,001). There was no significant difference between the COPD population (7.4 ± 2.1 x 103/mm3) and the healthy smoking population. Leukocytosis was independent of the tobacco load, age, BMI, GOLD stages and comorbidities. The CRP is augmented in the smoking population and with COPD present or diagnosed, although without any statistic relationship with leukocyte number. Conclusions: tobacco use determines a leukocyte response that lasts with similar intensity in the COPD population and with an independent increase of CRP. The subclinical inflammatory disease is already present in the healthy smoker and is perpetuated with similar intensity in the COPD population (AU)

Serum bilirubin levels on ICU admission are associated with ARDS development and mortality in sepsis.

BACKGROUND: Hyperbilirubinaemia is a common complication of sepsis. Elevated bilirubin may induce inflammation and apoptosis. It was hypothesised that increased serum bilirubin on Intensive Care Unit (ICU) admission contributes to sepsis-related acute respiratory distress syndrome (ARDS). METHODS: Serum bilirubin on ICU admission was measured in 1006 patients with sepsis. Serial serum bilirubin was analysed prospectively in patients with sepsis who had ARDS for a period of 28 days. The effects of clinical factors and variants of the UGT1A1 gene on serum bilirubin levels were determined. Outcomes were ARDS risk and mortality. RESULTS: During 60-day follow-up, 326 patients with sepsis developed ARDS, of whom 144 died from ARDS. The hyperbilirubinaemia (>or=2.0 mg/dl) rate in patients with ARDS (22.4%) was higher than in those without ARDS (14.1%, p = 0.002). For each 1.0 mg/dl increase in admission bilirubin, ARDS risk and 28- and 60-day ARDS mortalities were increased by 7% (OR = 1.07; p = 0.003), 20% (OR = 1.20; p = 0.002) and 18% (OR = 1.18; p = 0.004), respectively. Compared with subjects with bilirubin levels <2.0 mg/dl, patients with hyperbilirubinaemia had higher risks of ARDS (OR = 2.12; p = 0.0007) and 28-day (OR = 2.24; p = 0.020) and 60-day ARDS mortalities (OR = 2.09; p = 0.020). In sepsis-related ARDS, serial bilirubin levels in non-survivors were consistently higher than in survivors (p<0.0001). Clinical variables explained 29.5% of the interindividual variation in bilirubin levels, whereas genetic variants of UGT1A1 contributed 7.5%. CONCLUSION: In sepsis, a higher serum bilirubin level on ICU admission is associated with subsequent ARDS development and mortality.

Sex-specific association of epidermal growth factor gene polymorphisms with acute respiratory distress syndrome.

Epidermal growth factor (EGF) is involved in alveolar epithelial repair, lung fluid clearance and inflammation, and is regulated by sex hormones. An unmatched, nested case-control study was conducted to evaluate the associations of EGF variants with acute respiratory distress syndrome (ARDS) and the role of sex on the associations between EGF variants and ARDS. Patients with ARDS risk factors upon intensive care unit admission were enrolled. Cases were 416 Caucasians who developed ARDS and controls were 1,052 Caucasians who did not develop ARDS. Cases were followed for clinical outcomes and 60-day mortality. One functional single nucleotide polymorphism (SNP), rs4444903, and six haplotype-tagging SNPs spanning the entire EGF gene were genotyped. No individual SNP or haplotype was associated with ARDS risk or outcomes in all subjects. Sex-stratified analyses showed opposite effects of EGF variants on ARDS in males versus in females. SNPs rs4444903, rs2298991, rs7692976 and rs4698803, and haplotypes GGCGTC and ATCAAG were associated with ARDS risk in males. No associations were observed in females. Interaction analysis showed that rs4444903, rs2298991, rs7692976 and rs6533485 significantly interacted with sex for ARDS risk. The present study suggests that associations of epidermal growth factor gene variants with acute respiratory distress syndrome risk are modified by sex. The current findings should be replicated in other populations.

GNAS1 T393C polymorphism is associated with migraine.

Migraineurs have an interictal sympathetic nervous system (SNS) hypofunctionality and hypersensitivity to adrenergic amines. The GNAS1 T393C polymorphism has been associated with a distinct SNS sensitivity in healthy subjects. We tested GNAS1 T393C variant in two independent sets of subjects. In the case-control subset, 365 migraine patients [194 with aura (MA)] vs. 347 healthy controls were studied. A significant excess of the CC genotype was found in migraneurs (31.2%) as opposed to controls (20.2%; P=0.003). Using a logistic regression model corrected for sex, the CC genotype conferred a general risk for migraine twice [odds ratio (OR) 1.79, 95% confidence interval (CI) 1.27-2.53; P=0.001] higher than CT/TT genotypes. Using parents from 117 migraine families, a marginally significant trend for association could be observed (P=0.025), but the transmission disequilibrium test for alleles maternally transmitted failed to demonstrate familial association. In this subgroup, CC genotype conferred a risk for migraine over twice (OR 2.20; 95% CI 1.14-4.40; P=0.019) higher than TT/TC genotypes. In conclusion, the GNAS1 T393C variant is associated with migraine, which suggests a genetic basis for its higher SNS sensitivity.

One-step, PCR-mediated, gene disruption in the yeast Hansenula polymorpha.

Previous evidence based on the experience of our laboratory showed that one-step gene disruption in the yeast Hansenula polymorpha is not straightforward. A systematic study of several factors which could affect gene disruption frequency was carried out. We found that the more critical factor affecting one-step gene disruption in H. polymorpha is the length of the target gene region flanking the marker gene. Target gene regions of about 1 kb flanking the marker gene were necessary to obtain a disruption frequency of about 50%. However, the gene marker, either homologous or heterologous, the locus and the strain examined did not significantly affect the frequency of disruption; the highest disruption frequency obtained for the YNR1 gene was in the strain HMI39, using the Saccharomyces cerevisiae URA3 gene as a marker. Since long regions flanking the gene marker do not allow the easy PCR-mediated strategies, developed for S. cerevisiae, to obtain constructs to disrupt a given gene in H. polymorpha, an alternative PCR strategy was developed.