Toxicity profiling of the ethanolic extract of Citrullus lanatus seed in rats: behavioral, biochemical and histopathological aspects.

Citrullus lanatus (Cucurbitaceae) is conventionally used for the treatment of urinary tract infection, renal stones, hypertension, diabetes and diarrhoea. Current study evaluates acute and 28 days repeated toxicity ethanolic extract of C. lanatus seed (EECLS) in Wistar rats to measure its safety profile. The single dose (2000 mg/kg BW) of EECLS was administered while in 28 days repeated study 250, 500 and 1000 mg/kg BW were administered orally in rats. Parameters such as biochemical, haematological and histopathological were analysed in subacute toxicity study. During study, no apparent sign of toxicity, behavioural changes and mortality were detected in acutely exposed animals. In 28 days repeated toxicity study, rats did not show significant changes in behaviour, gross pathology, body weight, biochemical and haematological parameters. Abridged serum glucose and cholesterol levels during the study designate their roles in treatment of hyperglycaemic and hyperlipidaemic conditions. No significant difference was observed in histopathology of liver and kidneys of treated rats. The current investigation demonstrated that EECLS is non-toxic below 1000 mg/kg BW and provides protection to some body organs. The data propose that LD50 of EECLS was greater than 2000 mg/kg BW and the no observed adverse effect level (NOAEL) of EECLS was at the dose of 1000 mg/kg in rats. Taken together, our finding suggests that, EECLS is safe and provides some protection to body organs; also, its extract can be used for further preclinical and clinical evaluation for its therapeutic activity.

Evaluation of 5 methods for diagnosing failure of passive transfer in 160 Holstein calves.

BACKGROUND: Inadequate absorption of colostral IgG1 is termed failure of transfer of passive immunity (FTPI). Dairy calves with FTPI have increased mortality and morbidity in their first 6 months of life. OBJECTIVES: This study compared the clinical performance of 5 methods for diagnosing FTPI in Holstein calves. METHODS: An observational study was performed using 160 Holstein heifer calves. Serum was harvested at 48 hours of age, and FTPI was assessed using a digital Brix refractometer for total solids measurements, and digital refractometry and the biuret method to measure serum total protein (STP) concentrations. Serum gamma-glutamyltransferase activity was measured with an automated analyzer, and serum IgG was measured with the zinc sulfate turbidity test and an enzyme-linked immunosorbent assay. Diagnostic test performance was compared with that of the reference method (FTPI defined as a serum total IgG concentration <10 g/L). Test performance was evaluated using the area under the receiver operating characteristic curve, the sensitivity, the specificity, and the positive likelihood ratio at the optimal test cut point, and by calculating the kappa coefficient. RESULTS: A serum digital Brix percentage of <7.8% and an STP concentration of <52 g/L measured using digital refractometry were the best methods to identify calves with FTPI. The STP concentration measured with digital refractometry was 0.1 g/L lower than that measured with the biuret method. CONCLUSIONS: The digital Brix refractometer and the digital refractometer provide accurate and clinically useful methods for identifying dairy calves with FTPI. In this study, the excellent performance of the Brix refractometer was likely due to the use of a fixed sample volume (200 µL) and a uniform sample temperature at the time of measurement.

Analysis of the influence of the T393C polymorphism of the GNAS gene on the clinical expression of primary hyperparathyroidism / Análisis de la influencia del polimorfismo T393C del gen GNASen la expresión clínica del hiperparatiroidismo primario

Background: The receptor of parathyroid hormone and parathyroid hormone-related-protein (PTH/PTHrp) is located in the cell membrane of target tissues - kidney and osteoblasts. It is a G protein-coupled-receptor whose Gsα subunit is encoded by the GNAS gene. Our aim was to study whether the single nucleotide polymorphism (SNP) T393C of the GNAS gene is associated with renal stones, bone mineral density (BMD), or bone remodelling markers in primary hyperparathyroidism (PHPT).Methods: An analysis was made of clinical and biochemical parameters and densitometric values in three areas and their relationship with the T393C SNP of the GNAS gene in 261 patients with primary hyperparathyroidism and in 328 healthy controls. Genotyping was performed using the Custom Taqman(R) SNP Genotyping assay. Results: The genotype frequencies of GNAS T/C 393 were similar in the control and PHPT groups. No association was found between genotypes and clinical expression of PHPT (renal stones and bone fractures). A nonstatistically significant trend was seen to lower BMD in the lumbar spine, femoral neck, and total hip in both PHPT and control C homozygote subjects. Conclusion: Genetic susceptibility to PHPT related to the GNAS T393C polymorphism or a major influence in its development and clinical expression were found. A C allele-related susceptibility to lower BMD in trabecular bone in both PHPT and control subjects is not sufficient to suggest a more severe clinical expression of PHPT. This trend may be considered as a basis for further studies with larger sample sizes and complementary functional evaluation (AU)

Introducción: El receptor de la hormona paratiroidea y de la proteína relacionada con la hormona paratiroidea (PTH/PTHrp) está situado en la membrana celular de sus tejidos diana: riñón y osteoblastos. Se trata de un receptor unido a proteina G cuya subunidad Gsα está codificada por el gen GNAS. Nuestro objetivo fue estudiar si el polimorfismo de un sólo nucleótido (SNP) T393C del gen GNAS se asociaba con litiasis renal, densidad mineral ósea (DMO) o marcadores de remodelado óseo en el hiperparatiroidismo primario (HPTP). Métodos: Analizamos parámetros clínicos, bioquímicos y densitométricos en 3 zonas y su relación con el SNP T393C del gen GNAS en 261 pacientes con HPTP y en 328 controles sanos. El genotipado se realizó utilizando el ensayo Custom Taqman(R). Resultados: Las frecuencias genotípicas del SNP T/C 393 del GNAS fueron similares en ambos grupos control y HPTP. No encontramos ninguna asociación entre los genotipos y la expresión clínica del HPTP (litiasis renal y fracturas óseas). Encontramos una tendencia no estadísticamente significativa hacia una menor DMO en columna lumbar, cuello femoral y cadera en los sujetos control y HPTP portadores del alelo C. Conclusiones: No encontramos susceptibilidad genética para el desarrollo de PHPT relacionada con el polimorfismo T393C del gen GNAS ni influencia en su expression clínica. Sí hallamos una tendencia hacia niveles menores de DMO en el hueso trabecular relacionada con el alelo C en pacientes con PHPT y en sujetos control sin ser suficiente para sugerir una expresión clínica más grave. Estos resultados pueden ser considerados como un punto de partida para futuros estudios con mayor tamaño muestral y con evaluación funcional complementaria (AU)

Determination of CSF 5-methyltetrahydrofolate in children and its application for defects of folate transport and metabolism.

OBJECTIVE: To describe an assay of 5-methyltetrahydrofolate (5MTHF) in the cerebrospinal fluid (CSF) of children, to determine reference values, and to report the clinical significance of this assay in metabolic disorders affecting folate transport and metabolism. METHODS: CSF 5MTHF was determined by high-performance liquid chromatography with fluorescent detection in pediatric patients including one with FOLR1 gene mutation and one with methylenetetrahydrofolate reductase (MTHFR) deficiency. CSF total folate was measured using an automated analyzer. RESULTS: 5MTHF and total folate were determined in 188 and 93 CSF samples, respectively. CSF 5MTHF was high throughout the first six months of life and subsequently declined with age. Reference values of CSF 5MTHF and total folate were determined from 162 and 82 samples, respectively. The patient with FOLR1 gene mutation had extremely low CSF 5MTHF and total folate, though these values normalized after folinic acid supplementation. The patient with MTHFR deficiency had extremely low 5MTHF and moderately low total folate; these values were not associated and showed no significant change after folic acid supplementation. CONCLUSIONS: This 5MTHF assay is simple, rapid, sensitive, reliable, and cost-effective. It will aid in the diagnosis and therapeutic monitoring of metabolic disorders affecting folate transport and metabolism.

Analytical measurement and clinical relevance of vitamin D(3) C3-epimer.

With an ever-increasing clinical interest in vitamin D insufficiency, numerous automated immunoassays, protein binding assays, and in-house LC-MS/MS methods are being developed for the quantification of 25-hydroxyvitamin D(3) (25(OH)D(3)). Recently, LC-MS/MS methods have identified an epimeric form of 25(OH)D(3) that has been shown to contribute significantly to 25(OH)D(3) concentration, particularly in infant populations. This review describes the metabolic pathway and physiological functions of 3-epi-vitamin D, compares the capability of various 25(OH)D(3) methods to detect the epimer, and highlights recent publications quantifying 3-epi-25(OH)D(3) in infant, pediatric, and adult populations. In total, this review summarizes the information necessary for clinicians and laboratorians to decide whether or not to report/consider the C3-epimer in the analysis and clinical assessment of vitamin D status.

Influence of bilirubin photoisomers on unbound bilirubin measurement in clinical settings.

BACKGROUND: Measured unbound bilirubin concentration is influenced by bilirubin photoisomers. Bilirubin photoisomers are produced even with only a slight light exposure, and clinical samples are inevitably exposed to light. The objective of the study was to evaluate the influence of bilirubin photoisomers on the measurement of unbound bilirubin using serum of jaundiced neonates during blue light phototherapy. METHODS: Five neonates treated with phototherapy for hyperbilirubinaemia were enrolled. The samples were taken 12 h after initiation of phototherapy. Samples were processed by irradiation with blue light, by indoor ceiling light, by both blue light and indoor ceiling light or shaded. Bilirubin subfractions, total bilirubin and unbound bilirubin were measured. RESULTS: Compared with the non-irradiated samples, the (EZ)-cyclobilirubin concentration and (ZE)-bilirubin/(ZZ)-bilirubin ratio significantly increased in the blue light-irradiated samples, the (ZE)-bilirubin/(ZZ)-bilirubin ratio significantly increased in the indoor ceiling light-irradiated samples, and the (EZ)-cyclobilirubin, (EZ)-bilirubin and (ZE)-bilirubin/(ZZ)-bilirubin ratio significantly increased in the samples irradiated with both lights. No change was noted in unbound bilirubin in any group. CONCLUSIONS: We consider that changes in bilirubin photoisomers induced by light exposure during clinical practice do not influence the measured unbound bilirubin concentration.

Determination of the biological variation of S100ß and lactate dehydrogenase in disease-free patients with malignant melanoma.

BACKGROUND: Biological variation is important for determining analytical goals and for establishing the magnitude of change between two consecutive measurements. The aim of this study was to determine the biological variation for S100ß and lactate dehydrogenase in patients diagnosed with malignant melanoma but without evidence of disease recurrence. METHODS: The biological variation of S100ß and lactate dehydrogenase was estimated from a mean of four consecutive measurements in 32 patients diagnosed with malignant melanoma but without evidence of disease recurrence, 3 months after tumor resection or 4 months after finishing adjuvant treatment. The mean sampling interval was 3 months. RESULTS: Mean concentrations of S100ß and lactate dehydrogenase were 0.0557 µg/L and 6.3 µkat/L, respectively. Between-run analytical variation was 3.5% at 0.181 µg/L for S100ß and 3.5% at 2.83 µkat/L for lactate dehydrogenase. Biological variations obtained for S100ß and lactate dehydrogenase were 14.2% and 8.2%, respectively. The analytical goals (defined as 50% of biological variation) were 7.1% for S100ß and 4.1% for lactate dehydrogenase. CONCLUSIONS: The estimation of biological variation allows us to calculate analytical goals and reference change values. These are necessary tools for the correct interpretation of serial measurements in patient follow-up.

Fat malabsorption in cystic fibrosis: comparison of quantitative fat assay and a novel assay using fecal lauric/behenic acid.

OBJECTIVES: The gold standard for the diagnosis of fat malabsorption, the 72-hour fat balance study, requires a 3-day collection to generate a coefficient of fat absorption (CFA). We hypothesized that a new test using behenic acid (behenate test) as a nonabsorbable lipid marker may provide a facile means to assess fat absorption. The study proposed to answer 2 questions: first, whether the behenate test correlated with the gold standard and, second, whether the CFA improved when taking pancreatic enzymes during meals instead of taking them before meals. PATIENTS AND METHODS: The study compared the behenate test with the gold standard in 15 patients with cystic fibrosis during 3 arms that require 3- to 4-day hospitalization: first, taking pancreatic enzymes before meals; second, taking it during meals; and third, without taking it. RESULTS: The mean CFA was 78.3% when pancreatic enzymes were taken during meals and 80.4% when these enzymes were taken before meals. Correlation between the CFA and the behenate test for collections during all 3 arms was r = 0.219 (P = 0.001). CONCLUSIONS: Timing of ingestion of pancreatic enzymes does not significantly alter the CFA. Although the CFA correlates with the behenate test, the correlation is not robust enough to justify replacement of the gold standard by this test. It is unclear whether the poor correlation between tests relates to intermeal variability in fat excretion or other factors; however, the behenate test may be suitable as a screening test for the detection of fat malabsorption.

Multicentre evaluation of a new point-of-care test for the determination of CK-MB in whole blood.

BACKGROUND: The point-of-care (POC) test Roche CARDIAC CK-MB is a new assay which has been developed for the existing Roche Cardiac reader system. METHODS: We performed a multicentre evaluation at six sites to assess the analytical performance of the POC CK-MB assay and to compare it with a quantitative laboratory CK-MB assay. RESULTS: Within-series coefficients of variation (CV) resulting from 34 ten-fold measurements with patient samples ranged from 4.3% to 16.4%. Using quality control material, the mean CV values for day-to-day imprecision were 6.5% for the low level control and 8.4% for the high level control. Based upon 847 pairs of values, the mean relative bias of three independently calibrated lots of the POC CK-MB assay ranged from -6% to -11% in method comparisons with the lab CK-MB assay. The mean relative lot-to-lot differences of POC CK-MB were between -2% and +1%. No interference was observed with lipaemic blood (triglyceride concentrations up to 8.1 mmol/L), icteric blood (bilirubin concentrations up to 513 micromol/L), haemolytic blood (haemoglobin concentrations up to 0.12 mmol/L), biotin (up to 30 mg/L) and rheumatoid factor (up to 119 IU/mL), or with 53 standard or cardiological drugs even in toxic concentrations. There was no influence on the results by varying haematocrit values in the range from 21% to 54%. A slight interference with human anti-mouse antibodies type 2 was found. No significant influence on the results with POC CK-MB was found by using sample volumes between 135 and 165 microL. High CK-MB concentrations above the measuring range of POC CK-MB (1-40 microg/L) did not lead to false low results due to potential high-dose hook effect. No significant effect of sample age on recovery occurred up to a sample age of 24 h. No cross-reactivity was found between the POC CK-MB assay and either CK-MM or CK-BB. A substudy with healthy individuals confirmed the reference limits of 3.8 microg/L for females and 6.7 microg/L for males. CONCLUSIONS: The POC CK-MB assay showed a very good analytical performance with an excellent concordance with the calibration and reference laboratory method. It should be therefore suitable for its intended use in POC settings. Clin Chem Lab Med 2008;46:630-8.

Statistical methods for monitoring the relationship between the IFCC reference measurement procedure for hemoglobin A1c and the designated comparison methods in the United States, Japan, and Sweden.

BACKGROUND: The American Diabetes Association (ADA)/European Association for the Study of Diabetes (EASD)/International Diabetes Federation (IDF)/IFCC Consensus Statement on the worldwide standardization of HbA(1c) states that "... [HbA(1c)] results are to be reported world-wide in IFCC units ... and derived NGSP units ... , using the IFCC-NGSP master equation." METHODS: We describe statistical methods to evaluate and monitor the relationships as expressed in master equations (MEs) between the IFCC Reference Measurement procedure (IFCC-RM) and designated comparison methods (DCMs) [US National Glycohemoglobin Standardization Program (NGSP), Japanese Diabetes Society/Japanese Society for Clinical Chemistry (JDS/JSCC), and Mono-S in Sweden]. We applied these statistics, including uncertainty calculations, to 12 studies in which networks of reference laboratories participated, operating the IFCC-RM and DCMs. RESULTS: For NGSP and Mono-S, slope, intercept, and derived percentage HbA(1c) at the therapeutic target show compliance with the respective MEs in all 12 studies. For JDS/JSCC, a slight deviation is seen in slope and derived percentage HbA(1c) in 2 of the 12 studies. Using the MEs, the uncertainty in an assigned value increases from 0.42 mmol/mol HbA(1c) (IFCC-RM) to 0.47 (NGSP), 0.49 (JDS/JSCC), and 0.51 (Mono-S). CONCLUSIONS: We describe sound statistical methods for the investigation of relations between networks of reference laboratories. Application of these statistical methods to the relationship between the IFCC-RM and DCMs in the US, Japan, and Sweden shows that they are suitable for the purpose, and the results support the applicability of the ADA/EASD/IDF/IFCC Consensus Statement on HbA1c measurement.

Evaluation of analytical methods and workflow performance of the Architect ci8200 integrated serum/plasma analyzer system.

BACKGROUND: The Architect ci8200 is an integrated serum analyzer for photometric, electrochemical and immunological assays. Several assays of each category and the workflow performance of the system were compared with established laboratory procedures in two laboratories. METHODS: Measurements were compared with the ELECSYS 2010 (Roche Diagnostics) for CEA, PSA, FPSA, AFP, folate, vitamin B12, with the CENTAUR (Bayer) for TSH, T4, FT4, FSH and Estradiol, with the LIAISON (DiaSorin) for TSH, FT4 and FT3, with the Behring Nephelometer BN II (Dade-Behring) for ferritin, and with the INTEGRA 800 (Roche Diagnostics), and the AU640 (Olympus) for clinical chemistry assays. Workflow studies were performed to compare times of analysis required for defined analytical workloads. RESULTS: The coefficients of variation (CVs) for within-run imprecision were between 3% and 6% for CEA, PSA, FPSA, AFP and ferritin, and between 3% and 11% for TSH, FT4, FT3, folate and vitamin B12. The CVs for day-to-day imprecision for immunoassays were between 3% and 10%, except for vitamin B12 (CVs 11-13%) and FT4 (CV 10% -13%). For clinical chemistry tests corresponding CVs for within-run imprecision were < 1%, except for HDL, triglyceride, creatinine, ALT, LD and lipase (CVs<2%) and bicarbonate (CV 3%-6%) and magnesium (CV < 3%). The CVs for day-to-day imprecision for clinical chemistry tests were < 1%, except for sodium, CO(2), magnesium, phosphorus, glucose, uric acid, HDL, triglyceride, ALT, AST CK, lipase with CVs < 6% and for CO(2)<11%. Dilutional linearity testing of seven immunoassays and five clinical chemistry analytes resulted in recovery rates of 90-110%. Correlation studies with 15 immunoassays and 25 clinical chemistry tests showed acceptable agreements with established methods. Work flow analyses demonstrated a net gain in time of analysis up to 109 min depending on the size of the sample batch analyzed with the Architect ci8200 as the main analyzer as compared to the currently installed routine laboratory equipment. Median turn-around times were 7 and 30 min for chemistry assays and immunoassays, respectively, when ordered as STAT analyses, and 18 min when chemistry assays were ordered as routine determinations. CONCLUSIONS: Assays on the Architect ci8200 performed well, fulfilling quality control requirements as defined for instance by German quality control guidelines (RiliBAK). Method comparisons showed acceptable agreements with established assays. Workflow studies using the Architect ci8200 documented shorter times of analyses as compared with the conventionally established laboratory routine demonstrating the potential of integrated chemistry/immunoassay analyzers to provide faster and more efficient performance.

Quantitative determination of dithiocarbamates in human plasma, serum, erythrocytes and urine: pharmacokinetics of broccoli sprout isothiocyanates in humans.

BACKGROUND: Humans are exposed to substantial quantities of isothiocyanates and glucosinolates from vegetables. Since dietary isothiocyanates are widely regarded as potentially important chemoprotectors against cancer, reliable methods for measuring the plasma and tissue pharmacokinetics of isothiocyanates and their dithiocarbamate metabolites are essential for defining dosing regimens. METHODS: Isothiocyanates (ITC) and dithiocarbamates (DTC) react quantitatively with 1,2-benzenedithiol to produce 1,3-benzodithiole-2-thione that can be quantified spectroscopically. Although this cyclocondensation reaction has been highly useful for analyzing plant material and urine samples, the determination of DTC/ITC (the total quantity of DTC and ITC components in a sample that react in the cyclocondensation reaction) in blood and tissues has been hampered by their low levels and the high concentrations of proteins that interfere with the cyclocondensation reaction. The protein content of blood and tissues was reduced by the precipitation with polyethylene glycol (PEG) or ultrafiltration, and the sensitivity of the method was increased substantially by the solid phase extraction of the cyclocondensation product. RESULTS: Pharmacokinetic measurements were made in four human volunteers who received single doses of about 200 micromol of broccoli sprout isothiocyanates (largely sulforaphane, with lesser amounts of iberin and erucin). Isothiocyanates were absorbed rapidly, reached peak concentrations of 0.943-2.27 micromol/l in plasma, serum and erythrocytes at 1 h after feeding and declined with first-order kinetics (half-life of 1.77+/-0.13 h). The cumulative excretion at 8 h was 58.3+/-2.8% of the dose. Clearance was 369+/-53 ml/min, indicating active renal tubular secretion. CONCLUSION: A sensitive and specific method for quantifying DTC levels in human plasma, serum, and erythrocytes has been devised. Determinations of ITC/DTC levels are important because: (i) dietary isothiocyanates are of potential value in reducing the risk of cancer, and (ii) humans are extensively exposed to DTC as fungicides, insecticides, pesticides and rubber vulcanization accelerators.

Definições, conceitos e aspectos operacionais utilizados em farmacovigilância / Definitions, concepts and operational aspects utilized in drug surveillance

O artigo expõe definições e conceitos utilizados na área de farmacovigilância, métodos de pesquisa para a identificação de reações adversas a medicamentos, notificação e organização de sistemas de farmacovigilância. De modo geral, as ações de farmacovigilância assumem dois aspectos diversos conforme o objetivo a ser atingido: (i) ações dos profissionais de saúde, que mantêm contato direto com o paciente e (ii) organização de sistemas de coleta, análise, processamento de informaçao e disseminação dos resultados, que incluem os sistemas nacionais de farmacovigilância e o Centro Colaborativo de Monitorização Internacional de Medicamentos da Organização Mundial da Saúde. Para se estabelecer a verdadeira...