Three-dimensional microscale hanging drop arrays with geometric control for drug screening and live tissue imaging.

Existing three-dimensional (3D) culture techniques are limited by trade-offs between throughput, capacity for high-resolution imaging in living state, and geometric control. Here, we introduce a modular microscale hanging drop culture where simple design elements allow high replicates for drug screening, direct on-chip real-time or high-resolution confocal microscopy, and geometric control in 3D. Thousands of spheroids can be formed on our microchip in a single step and without any selective pressure from specific matrices. Microchip cultures from human LN229 glioblastoma and patient-derived mouse xenograft cells retained genomic alterations of originating tumors based on mate pair sequencing. We measured response to drugs over time with real-time microscopy on-chip. Last, by engineering droplets to form predetermined geometric shapes, we were able to manipulate the geometry of cultured cell masses. These outcomes can enable broad applications in advancing personalized medicine for cancer and drug discovery, tissue engineering, and stem cell research.

Frequency and levels of aflatoxin M1 in urine of children in Bogota, Colombia.

A study was conducted to investigate the frequency and levels of AFM1 and AFM2 in urine from children who attended the emergency service of a pediatric referral hospital in Bogota, Colombia. A survey on the consumption of foods likely to be a source of aflatoxins and on sociodemographic variables was conducted as well. The frequency of AFM1 in urine was found to be 41.7% with an average concentration in positive samples of 16 pg mL-1 ± 10.7 pg mL-1 (range > LOD-48.5 pg mL-1). The presence of AFM1 in the urine was related to the consumption of cereals likely to be contaminated with AFB1, especially corn and rice. No detectable levels of AFM2 were found in any sample. The results show that children's exposure to aflatoxins in Colombia is indeed a problem and should be one of the priorities of the health authorities. Continuous monitoring of aflatoxins in foods should be carried out, in compliance with Colombian regulations, using analytical methods that allow determination and quantification of aflatoxins in different biological and non-biological matrices at trace levels.

[Installed Capacity Perception Survey for the Training of Residents Program in Orthopedics and Care Needs in Acute and Chronic Musculoskeletal Pathology in Mexico]. / Encuesta de Percepción en Capacidad Instalada para la Formación de Médicos Residentes en Ortopedia (Traumatología) y Necesidades de la Atención en Patología Musculoesquelética Aguda y Crónica en México. Primera Parte.

INTRODUCTION: It is essential that orthopaedic resident physicians be highly proficient in all aspects, considering the balance between supply, demand, need and context. Fundamental to identify the capacity and quality installed for their training in Mexico. MATERIAL AND METHODS: Observational Study, transverse, non-probabilistic sampling-conglomerates, in two phases. The instrument has 8 domains, 57 variables and 4,867 items. 60 graduate professors of 20 states, 50 hospital sites, 22 university programs. RESULTS: 1,038 years of experience (collective intelligence), 17 years of experience/teacher (01 to 50 years). Identified: acute pathology 30 (2 to 90%), chronic pathology 30 (5 to 96%), patients 15 years, 10 (3 to 30%), patients between 15 and 65 years, 47 (2 to 78%), patients 65 years, 20 (2 to 60%), number of beds/seat 20 (2 to 510), number of clinics 3 (1 to 48), number of surgical procedures/headquarters per year at the national level, was 960 (50 to 24,650). The national average per resident doctor is 362 surgeries/year with 1,450 surgical times/year. CONCLUSIONS: The needs and resources for the training of physicians specializing in orthopedics/traumatology are highly heterogeneous, so it should be adapted to the epidemiological needs of the region of influence, in an area of epidemiological transition. 62.2% expressed not having or have bad academic and scientific infrastructure at its headquarters, more than 50% without rotation overseas and 90% without regular scientific production.

INTRODUCCIÓN: Es fundamental que los médicos residentes de ortopedia (traumatología) sean altamente competentes en todos los aspectos, considerando el equilibrio entre la oferta, demanda, necesidad y contexto. Es primordial identificar la capacidad y calidad instalada para su formación en México. MATERIAL Y MÉTODOS: Estudio observacional, transversal, muestreo no probabilístico-conglomerados, en dos fases. El instrumento tiene ocho dominios, 57 variables y 4,867 ítems. Sesenta profesores de postgrado de 20 estados, 50 sedes hospitalarias, 22 programas universitarios. RESULTADOS: 1,038 años de experiencia (inteligencia colectiva), 17 años de experiencia/profesor (01 a 50 años). Se identificó: patología aguda 30 (2 a 90%), patología crónica 30 (5 a 96%), pacientes 15 años, 10 (3 a 30%), pacientes entre 15 y 65 años, 47 (2 a 78%), pacientes 65 años, 20 (2 a 60%), número de camas/sede 20 (2 a 510), número de consultorios 3 (1 a 48), el número de procedimientos quirúrgicos/sede al año a nivel nacional fue de 960 (50 a 24,650). La media nacional por médico residente es de 362 cirugías/año con 1,450 momentos quirúrgicos/año. CONCLUSIONES: Las necesidades y recursos para la formación de médicos especialistas en ortopedia/traumatología son en alto grado heterogéneos, por lo cual se debería adaptar a las necesidades epidemiológicas de la región de influencia, en un ámbito de transición epidemiológica. Sesenta y dos punto dos por ciento expresó no tener o tener deficiente infraestructura académica y científica en su sede, más de 50% sin rotación al extranjero y 90% sin producción científica regular.

Evaluation of the supplementation of a feed additive as a potential protector against the adverse effects of 2. 5 ppm T-2 toxin on growing broiler chickens / Avaliação da suplementação de um aditivo alimentar como um protetor potencial contra os efeitos adversos de 2, 5ppm de toxina T-2 em frangos em crescimento

A trial was conducted to evaluate a feed additive containing epoxidase activity from a bacterium (Mycofix-S) as a potential protection against the adverse effects of 2.5 ppm dietary T-2 toxin in male growing broiler chickens. A total of 144 one-day-old Ross 308 male chicks were individually wing-banded and allotted into each of the four experimental groups. Group 1: negative control, no T-2 toxin or additive; group 2: Mycofix-S, 2.5 g/kg; group 3: positive control, 2.5 ppm T-2 toxin; group 4: 2.5 ppm T-2 toxin + 2.5 g/kg Mycofix-S. Feed and water were provided ad libitum for 28 days (days 1 to 28 of age). Each experimental treatment was replicated 6 times, with 6 birds per replicate pen. Response variables included performance parameters, serum activity of alkaline phosphatase (ALP) and amylase, relative weight of selected organs and histology of the upper digestive system. T-2 toxin at 2.5 ppm significantly (P = 0.016) decreased the 28-day body weight gain and cumulative feed intake without affecting feed conversion. The feed additive counteracted these adverse effects. Serum enzyme activities were not significantly (P>0.05) affected for the four experimental groups but when data from the groups receiving T-2 toxin was pooled and compared against the pooled data from groups without the toxin a significant decrease in amylase activity was observed in chickens receiving T-2 toxin. The histological examination of the upper digestive system revealed lesions in mouth, esophagus, proventriculus, gizzard and duodenum in the chickens fed T-2 toxin without the additive. Chickens fed T-2 toxin plus the additive showed lesions in the same tissues except in the duodenum. The results of the present study show that the addition of 2.5 g/kg of the feed additive tested protects against adverse effects on performance and also the integrity of the duodenal mucosa.(AU)

Foi realizado um experimento com o objetivo de avaliar um aditivo alimentar contendo atividade de epoxidase de uma bactéria (Mycofix-S) como proteção potencial contra os efeitos adversos de uma dieta com 2,5ppm de toxina T-2 em frangos de corte machos. Um total de 144 pintos machos Ross 308 de um dia de idade foram marcados na asa individualmente e alocados em um de quatro grupos experimentais: grupo 1: controle negativo, sem toxina T-2 ou aditivo; grupo 2: 2,5g/kg de Mycofix-S; grupo 3: controle positivo, 2,5ppm de toxina T-2; grupo 4: 2,5ppm de toxina T-2 + 2,5g/kg de Mycofix-S. Alimento e água foram fornecidos ad libitum por 28 dias (dias um a 28 de idade). Cada tratamento experimental foi replicado seis vezes, com seis pintos por gaiola de replicação. As variáveis de resposta incluíram parâmetros de desempenho, atividade sérica de fosfatase alcalina (ALP) e amilase, peso relativo de órgãos selecionados e histologia do sistema digestivo superior. A toxina T-2 a 2,5ppm diminuiu significativamente (P = 0.016) o ganho de peso corporal aos 28 dias e o consumo de alimento acumulado, sem afetar a conversão alimentar. O aditivo diminuiu os efeitos adversos. As atividades séricas das enzimas não foram afetadas significativamente (P>0.05) nos quatro grupos experimentais, porém, quando os dados dos grupos que receberam a toxina T-2 foram combinados e comparados com o pool de dados dos grupos sem toxina, foi observado um decréscimo significativo da atividade de amilase nos frangos que receberam a toxina T-2. O exame histológico do sistema digestivo superior revelou lesões em boca, esôfago, pró-ventrículo, moela e duodeno nos frangos alimentados com toxina T-2 sem aditivo. Frangos alimentados com toxina T-2 mais aditivo mostraram lesões nos mesmos tecidos, exceto no duodeno. Os resultados do presente estudo mostram que a adição de 2,5g/kg do aditivo alimentar testado protege contra os efeitos adversos sobre o desempenho e a integridade da mucosa duodenal.(AU)

FECTO DE LA RESTRICCIÓN ALIMENTICIA Y LA REALIMENTACIÓN SOBRE LA COMPOSICIÓN DEL MÚSCULO BLANCO DE Piaractus brachypomus / EFFECT OF FOOD DEPRIVATION AND RE-FEEDING ON MUSCLE WHITE COMPOSITION OF Piaractus brachypomus

Con el propósito de evaluar el efecto de la restricción alimenticia y la realimentación sobre la composición del músculo blanco de cachama blanca, se aplicaron dos protocolos de restricción alimenticia durante 84 días, cada uno dividido en un periodo de restricción alimenticia y un periodo de realimentación. En el primer protocolo, se aplicó una restricción alimenticia moderada (33,3%), y en el segundo, una restricción severa (50%); ambos grupos fueron comparados con un grupo control que recibió una ración alimenticia igual a la suministrada bajo condiciones de un cultivo comercial. Al finalizar el ensayo, ninguno de los tiempos de restricción alimenticia tuvo efectos significativos (p > 0,05) sobre el porcentaje de proteína del filete. Sin embargo, se observaron efectos significativos (p < 0,05) sobre los porcentajes de lípidos, cenizas y energía. Con respecto al porcentaje de cenizas musculares, se observó que la restricción alimenticia tendió a aumentar su valor, mientras que para el porcentaje de lípidos y los niveles de energía, se encontró el efecto contrario. No obstante, cuando los individuos finalizaron el periodo de realimentación, se observó un restablecimiento en los niveles de nutrientes comparados con los individuos no restringidos. En cuanto al perfil de ácidos grasos, el grupo control mostró con diferencias significativas (p < 0,05) mayor porcentaje de omega-3 (n-3). Estos resultados permiten concluir que los protocolos de alimentación aplicados estimularon, en Piaractus brachypomus, movilización de nutrientes musculares y su posterior restablecimiento, sin afectar la integridad del músculo blanco.

In order to evaluate the effect of food deprivation and re-feeding on the composition of cachama blanca white muscle, two restriction food protocols were used per 84 days, each one divided in a food restriction period and re-feeding period. In the first of them, it was used a moderate food restriction (33.3%) and in the second one, was a severe restriction (50%); both groups were compared with a control group receiving the samefood ration under commercial culture conditions. At the end of the trial, any of the times of food restriction had significant effects (p>0.05) on fillet protein percentage. However, significant differences (p<0.05) were observed on lipids, ash and energy percentages. In regards to muscle ash percentage, it was observed that food restriction tends to increase its value, and in contrast, was observed an opposite effect on lipids percentage and energy levels. However, when individuals completed the re-feeding period, a restoration in the levels of nutrients compared to non-restricted individuals was observed. As for the fatty acid profile, the control group showed with significant differences (p < 0,05) the highest percentage of omega-3 (n-3). These results suggest that restriction protocols applied in Piaractus brachypomus stimulated mobilization of nutrients muscle and its later restore without affecting the integrity of the white muscle.

Bioactivation of aflatoxin B1 by turkey liver microsomes: responsible cytochrome P450 enzymes.

1. A study was conducted to determine the cytochrome P450 enzymes responsible for the bioactivation of aflatoxin B1 into its epoxide form (AFBO) in turkey liver microsomes. 2. The strategies used included the measurement of prototype substrate activity for specific human P450s, use of selective inhibitors, determination of correlation between aflatoxin bioactivation and enzymatic activity of prototype substrates and the determination of immunoreactive proteins using antibodies against human P450s. 3. Enzymatic activity and immunoreactive proteins corresponding to the turkey orthologs CYP1A1, CYP1A2, CYP2A6 and CYP3A4 were detected, but not for the CYP2D6 ortholog. 4. The results of the inhibition and correlation studies strongly suggest that the turkey CYP2A6 ortholog and, to a lesser extent, the CYP1A1 ortholog, are involved in the bioactivation of aflatoxin B1 in turkey liver microsomes. 5. This is the first study reporting the role of CYP2A6 in the bioactivation of AFB1 in an avian species and the role of CYP1A1 in any species.

Cytochrome P450 enzymes involved in the metabolism of aflatoxin B1 in chickens and quail.

A study was conducted to identify the cytochrome P450 (CYP, CYP450) enzyme orthologs involved in the bioactivation of aflatoxin B(1) (AFB(1)) into the highly toxic metabolite known as aflatoxin-8,9-epoxide (AFBO) in quail and chicken hepatic microsomes. The strategies used included the use of specific CYP450 inhibitors and the correlation of prototype substrate activities with AFBO production. Additionally, the presence of the enzymes was qualitatively determined using an immunoblotting technique. The results showed that both quail and chicken microsomes have CYP1A1, CYP1A2, CYP2A6, and CYP3A4 enzymatic activity. A strong relationship between CYP1A1 and CYP2A6 activities and AFB(1) bioactivation was found in both species. Inhibition studies provided more evidence for the role of CYP2A6 in the bioactivation of AFB(1). The immunoblot results showed clear bands for the CYP2A6 and CYP3A4 orthologs in both species. The results of the present study indicate that CYP2A6 and, to a lesser extent, CYP1A1 are responsible for the bioactivation of AFB(1) into AFBO in both quail and chicken hepatic microsomes.

Evaluation of the ability of a feed additive to ameliorate the adverse effects of aflatoxins in turkey poults.

1. A study was conducted to evaluate the possible protective effect of a feed additive containing aluminosilicate and phytogenic substances against the adverse effects of aflatoxins in turkey poults. 2. Dietary treatments (6) were given to turkey poults from d 1 to d 42 of age. From d 1 to 21 the dietary treatments were as follows: 1, negative control, no aflatoxins or feed additive added; 2, feed additive control, 1 kg/t feed additive, no aflatoxins; 3, 250 ppb (microg/kg) aflatoxins, no feed additive; 4, 250 ppb aflatoxins + 1 kg/t feed additive; 5, 500 ppb aflatoxins, no feed additive; and 6, 500 ppb aflatoxins + 1 kg/t feed additive. From d 22 to 42, the dietary concentration of the feed additive was increased from 1 to 2 kg/t in all treatment groups receiving the feed additive (2, 4 and 6), while keeping constant the dietary concentrations of aflatoxins. 3. Aflatoxins at 250 ppb did not cause adverse effects on performance but affected certain toxicopathological parameters. At 500 ppb, adverse effects on performance and several toxicological parameters were observed. 4. Some of the adverse affects were partially or completely overcome by supplementation with the feed additive, including amelioration of the performance parameters, suppression of mortality and correction of the immunological alterations induced by the exposure to the aflatoxins.

Aflatoxicosis in chickens (Gallus gallus): an example of hormesis?

Poultry has commonly been considered highly susceptible to aflatoxins. However, among domestic fowl there is wide variability in specific species sensitivity to these mycotoxins. Comparative toxicological studies in avian species have shown that ducklings and turkey poults are the most sensitive species to aflatoxins, quails show intermediate sensitivity, whereas chickens are the most resistant. Hormesis is a dose-response phenomenon characterized by low-dose stimulation and high-dose inhibition. The low-dose stimulation is typically maximal at only about 30 to 60% greater than controls. Hormesis has been noted in regards to changes in body weight in numerous studies, including those performed for the US National Toxicology Program, with over 50 chemicals. The present paper assesses how relatively low levels of aflatoxin consumption in feed may affect the growth rate of chickens. In general, multiple independent investigations have shown that such aflatoxin consumption affects growth in a hormetic-like biphasic manner with a low dose stimulation and a high dose inhibition. Such observations were then generalized to other toxic agents and animal models, suggesting that low doses of stressor agents induce adaptive responses as reflected in accelerated growth rates. The implications of such hormetic dose responses are briefly discussed.

[Utility of chest x-ray like approach to etiology of community acquired pneumonia in children]. / Utilidad de la radiografía de tórax en niños como aproximación a la etiología de neumonía adquirida en la comunidad.

INTRODUCTION: Chest X-ray (CR) utility to predict etiology in community-acquired pneumonia (CAP), in children has been controversial. Nevertheless, some authors propose the use of well standardized radiological patterns. OBJECTIVE: To evaluate usefulness of modified Swischuk's radiological patterns (RaP) to determine probable etiology in children hospitalized with CAP. PATIENTS AND METHODS: Eighty children were studied using 6 standardized RaP. The RaP were blindly and individually analyzed by 9 pneumologists, who registered the results (stage 1). Thereafter, (stage 2) a second evaluation adding clinical information was performed. Then, the patients complementary examinations and clinical evolution were included (stage 3). The reference standard (RS) was generated from a blind consensus. Every result was compared with the RS using Student test. RESULTS: According to RS, children were classified as having a virus-like pneumonia in 63% of cases, bacterial in 13%, mixed in 16%, atypical in 5% and ADV in 3%. The agreement of stage 1 and 2 with RS was 64 and 77%, respectively. Virus and bacterial RaP agreement increased from 66 to 82 % (p < 0.001) and from 82 to 90% (p < 0.05), respectively after incorporating clinical parameters. CONCLUSION: Modified Swischuk's RaP used in association with clinical elements allows a quite satisfactory approach to etiologic diagnosis of CAP.

Utilidad de la radiografía de tórax en niños como aproximación a la etiología de neumonía adquirida en la comunidad / Utility of chest x-ray like approach to etiology of community acquired pneumonia in children

Introduction: Chest X-ray (CR) utility to predict etiology in community-acquired pneumonia (CAP), in children has been controversial. Nevertheless, some authors propose the use of well standardized radiological patterns. Objective: To evaluate usefulness of modified Swischuk's radiological patterns (RaP) to determine probable etiology in children hospitalized with CAP. Patients and Methods: Eighty children were studied using 6 standardized RaP. The RaP were blindly and individually analyzed by 9 pneumologists, who registered the results (stage 1). Thereafter, (stage 2) a second evaluation adding clinical information was performed. Then, the patients complementary examinations and clinical evolution were included (stage 3). The reference standard (RS) was generated from a blind consensus. Every result was compared with the RS using Student test. Results: According to RS, children were classified as having a virus-like pneumonia in 63 percent of cases, bacterial in 13 percent, mixed in 16 percent, atypical in 5 percent and ADV in 3 percent. The agreement of stage 1 and 2 with RS was 64 and 77 percent, respectively. Virus and bacterial RaP agreement increased from 66 to 82 percent (p < 0.001) and from 82 to 90 percent (p < 0.05), respectively after incorporating clinical parameters. Conclusion: Modified Swischuk's RaP used in association with clinical elements allows a quite satisfactory approach to etiologic diagnosis of CAP.

Introducción: La utilidad de la radiografía de tórax.(RT) para establecer etiología en niños con neumonía adquirida en la comunidad (NAC) es cuestionada. Objetivo: Evaluar la utilidad de los patrones radiológicos (PRa) de Swischuk modificados para determinar la probable etiología en niños hospitalizados con NAC. Pacientes y Método: Se estudió 80 niños mediante el uso de seis PRa estandarizados. Se procedió al análisis secuencial por nueve neumólogos; individualmente de manera ciega (etapa 1), se registró el PRa. Posteriormente (etapa 2) se agregó información clínica. En seguida se incluyó exámenes complementarios y la evolución de cada niño (etapa 3). El estándar de referencia (ER) fue generado mediante consenso ciego. Se comparó cada resultado con el ER mediante t de Student. Resultados: Según el ER, las etiologías identificadas fueron: viral (63 por ciento), bacteriana (13 por ciento), mixta (16 por ciento), atípica (5 por ciento) y ADV (3 por ciento). El rendimiento de la etapa 1 y 2 fue 64 y 77 por ciento>, respectivamente. El PRa viral y bacteriano aumentó de 66 a 82 por ciento> (p < 0,001) y de 82 a 90 por ciento (p < 0,05), respectivamente, luego de incorporar elementos clínicos. Conclusión: La utilización de PRa de Swischuk modificados, asociados a elementos clínicos, permitió un adecuado rendimiento como aproximación del diagnóstico etiológico en NAC

Proteomic analysis of ovomucoid hypersensitivity in mice by two-dimensional difference gel electrophoresis (2D-DIGE).

There is a need to develop reliable methods to assess the safety of genetically modified and other novel foods. The aim of this study was to identify protein biomarkers of food allergy in mice exposed to ovomucoid (OVM), a major food allergen found in chicken egg white. BALB/c mice were repeatedly sensitized by gavage with OVM and cholera toxin (CT) and control mice were exposed to a mixture of amino acids with CT. At the endpoint, all mice were challenged intraperitoneally with OVM and alum. Type-1 hypersensitivity was confirmed in OVM-sensitized mice by observation of clinical signs of anaphylaxis and elevated levels of plasma histamine, OVM-specific IgE and OVM-specific IgG by ELISA. Differential protein expression was assessed in albumin-depleted plasma as well as in mesenteric lymph node, liver, spleen, and ileum by two-dimensional difference gel electrophoresis (2D-DIGE). Differentially expressed proteins were identified by liquid chromatography with tandem mass spectrometry. Plasma proteins overexpressed in OVM-sensitized mice included haptoglobin (41-fold), serum amyloid A (19-fold) and peroxiredoxin-2 (1.9-fold). Further validation of these plasma proteins in other animal models of food allergy with different food allergens is required to assess their potential as candidate biomarkers for use in evaluating the allergenicity of novel foods.

Microsomal and cytosolic biotransformation of aflatoxin B1 in four poultry species.

1. This research evaluated differences in hepatic in vitro metabolism of aflatoxin B1 (AFB1) on selected avian species. 2. Microsomal and cytosolic liver fractions were obtained from chickens, ducks, quails and turkeys; eight males and eight females of each. 3. All microsomes studied produced AFB1-8,9-exo-epoxide (AFBO), a metabolite regarded as the active product of AFB1. Turkey microsomes produced 1.8 and 3.5 times more AFBO than quails and chickens microsomes, respectively. 4. Males from evaluated birds produced more AFBO than females, but statistically-significant differences between genders were observed only in ducks and turkeys. 5. The cytosolic fraction from all four species produced aflatoxicol (AFL). Turkey and duck hepatic cytosol produced more AFL than from quail and chickens. 6. It is known that turkeys are very sensitive to AFB1, quails are intermediate and chickens are particularly resistant; the differences in AFBO production shown in our study may help to explain the difference in vivo responses among turkeys, quail and chickens. 7. Moreover, AFL may be related to AFB1 toxicity; it was produced in larger amounts by hepatic cytosol from the more susceptible species. 8. Because AFBO production by microsomes in ducks was relatively low, it is possible that other toxicity mechanisms are involved in this highly susceptible species.

Occurrence of aflatoxin M1 in retail milk samples from Bogotá, Colombia.

A study was conducted to establish the occurrence and levels of contamination of aflatoxin M1 (AFM1) in retail milk from Bogotá, Colombia. A total of 241 samples were analysed during 2004 and 2005. Samples were cleaned up by an immunoaffinity column and AFM1 was quantified by liquid chromatography with fluorescence detection. A total of 69.2 and 79.4% of the samples analysed during 2004 and 2005, respectively, were found to contain levels of AFM1 above 10 ng l(-1). Levels of contamination ranged from 10.7 to 213.0 ng l(-1) in 2004, and from 10.6 to 288.9 ng l(-1) in 2005. Despite the high incidence of AFM1 found in the milk samples analysed, all samples complied with current local regulations, which allow AFM1 content in milk up to 400 ng l(-1). However, due to the high incidence of AFM1 in milk found in the present study, it is recommended that a permanent surveillance programme be established for milk consumed in Bogotá in order to prevent milk lots containing levels above the regulatory level entering the food chain.

Determination of ochratoxin A in green coffee by immunoaffinity column cleanup and liquid chomatography: collaborative study.

A collaborative study was conducted to evaluate a method using immunoaffinity column cleanup with liquid chromatography (LC) for the determination of ochratoxin A (OTA) in green coffee at levels that could be included in possible future regulations of the European Union. The test portion was extracted with methanol-3% aqueous sodium hydrogen carbonate solution (50 + 50, v/v). The extract was filtered, and the filtrate was diluted with phosphate-buffered saline and applied to an immunoaffinity column containing antibodies specific for OTA. After washing, the toxin was eluted from the column with methanol and quantified by LC with fluorescence detection. Pairs of 4 homogeneous noncontaminated and naturally contaminated materials (mean levels of < 0.12, 2.44, 5.15, and 13.46 ng/g) and blank samples (< 0.12 ng/g) for spiking were sent to 20 participant laboratories from 8 countries. The materials were analyzed according to the method description and all difficulties encountered in the analysis were reported. Statistical analysis was carried out according to the Harmonized Protocol of the International Union of Pure and Applied Chemistry. The relative standard deviation for repeatability (RSDr) ranged from 7.42 to 20.94%, and the relative standard deviation for reproducibility (RSDR) ranged from 16.34 to 29.17%. The method showed acceptable within-laboratory and between-laboratories precision for green coffee materials, as evidenced by HorRat values of < or = 0.85, at the studied range, for spiked and naturally contaminated materials. The mean recovery was 92.8% for green coffee material spiked with OTA at a level of 4.82 ng/g.

First colombian interlaboratory study for the determination of aflatoxin B1 in yellow corn.

An interlaboratory study for the determination of aflatoxin B1 in yellow corn was conducted with 16 laboratories that analyze for aflatoxins in Colombia. Naturally contaminated ground yellow corn (Monsanto DK 4004) with an assigned reference value of 26.3 µg/kg aflatoxin B1 was distributed as double blind duplicates in sachets of 55 g. z-Scores were computed for each of the results; repeatability of the two replicate analysis was also calculated. Four of the participating laboratories used HPLC, seven used TLC, one used fluorometry, one ELISA and three a semi quantitative analytical technique (Aflacard®). Only 10 of the 26 quantitative results (39%) had satisfactory z-scores, two scores were questionable (8%) and 14 of the 26 results had unsatisfactory z-scores (54%). A total of 8 laboratories had satisfactory repeatability (62%), and 5 had unsatisfactory repeatability (39%). The present study indicates that only about one third of the results for aflatoxin reported by Colombian laboratories have good accuracy (as measured by the z-score of the result), although satisfactory precision (measured as repeatability) is achieved by about two thirds of the laboratories. These results indicate that an improvement in quality assurance is needed in Colombian laboratories. The routine use of reference standards and reference materials is highly recommended.

Method validation for the determination of ochratoxin A in green and soluble coffee by immunoaffinity column cleanup and liquid chromatography.

A method was validated for the determination of ochratoxin A (OTA) in soluble and green coffee. Performance parameters evaluated included selectivity, accuracy, intermediate precision, linearity, limit of detection, limit of quantitation, and ruggedness. The method was found to be selective for OTA in both matrices tested. Recovery rates from soluble coffee samples ranged from 73.5 to 91.2%, and from green coffee samples from 68.7 to 84.5%. The intermediate precision (RSDr) was between 9.1 and 9.4% for soluble coffee and between 14.3 and 15.5% for green coffee analysis. The linearity of the standard calibration curve (r(2)) was <0.999 for OTA levels of 1.0-20.0 µg/kg in coffee samples. The limit of detection was determined to be 0.01 ng of OTA on column, while the limit of quantitation was found to be 0.03 ng on column. The limit of quantitation is equivalent to 0.6 µg/kg in soluble coffee samples and 0.3 µg/kg in green coffee samples. The results of the ruggedness trial showed two factors are critical for soluble coffee analysis: the extraction method, and the flow rate of the mobile phase. For green coffee analysis two critical factors detected were the extraction method and the storage temperature of the immunoaffinity column.Five samples of soluble coffee and 42 of green coffee were analysed using the validated method. All soluble coffee samples contained OTA at levels that ranged from 8.4 to 13.9 µg/kg. Six of the 42 green coffee samples analysed (14.3%) contained OTA at levels ranging from 0.9 to 19.4 µg/kg. The validated method can be used to monitor OTA levels in Colombian coffee for export or for local consumption.

Intoxication of Crotalaria pallida seeds to growing broiler chicks.

The adverse effects of feeding Crotalaria pallida (CP) seeds to chicks was investigated in a 21-day randomized trial of 4 dietary treatments (control, 1,2 and 3% ground CP seeds). Mortality rates in birds fed 0, 1, 2, and 3% dietary CP were 0, 2.1, 6.2, and 16.7%, respectively. Body weight gain and feed efficiency were adversely affected by all levels of inclusion of CP seeds, but feed intake was decreased only by dietary levels of 2 and 3%. Dietary CP of 2 and 3% increased the relative weight of lung, heart and spleen. Relative liver weight was increased by 2% dietary CP, but decreased by 3% CP. At day 14, serum GGT was increased by 2 and 3% dietary CP; serum ALT was significantly increased by 3% CP. No differences in ALT, AST or GGT were observed at day 21. Dietary levels equal to or greater than 1% CP are toxic for growing broiler chicks.

Phase II in vitro metabolism of 3-methylindole metabolites in porcine liver.

1. The Phase II in vitro metabolism of 3-methylindole (3MI) metabolites was investigated in pigs to determine the possible relationship between 3MI Phase II metabolism and 3MI accumulation in fat. Sulphation and glucuronidation of five of the seven major metabolites found to be produced by porcine microsomes was investigated using porcine cytosol and microsomes, respectively. The possible formation of glutathione conjugates was also investigated using microsomally activated 3MI intermediate(s). 2. No sulphation or glucuronidation was observed for metabolites 3-hydroxy-3-methyloxindole, 3-methyloxindole, indole-3-carbinol or 2-aminoacetophenone; however, 5-hydroxy-3-methylindole (5-OH-3MI) was conjugated with both sulphate and glucuronic acid. 3. The enzyme responsible for sulphation of 5-OH-3MI was identified as the thermostable form of phenol-sulphotransferase (TS-PST) based on its susceptibility to TS-PST inhibitors and the correlation between sulphation of 5-OH-3MI and sulphation of the prototype substrate p-nitrophenol (r = 0.94, p < 0.001). 4. A 3MI-glutathione adduct was identified in microsomal incubations containing 3MI and glutathione. 5. Sulphation of 5-OH-3MI was high in pigs with low levels of 3MI in fat. No relationship was observed between 3MI levels in fat and either glutathione transferase or glucuronidation activities in liver.