Comparison of urinary aflatoxin M1 and aflatoxin albumin adducts as biomarkers for assessing aflatoxin exposure in Tanzanian children

Purpose: To determine levels of urinary aflatoxin M1 (AFM1) in children and correlate the concentrations with previously reported aflatoxin albumin adduct (AF-alb) levels in these children.

Materials and methods: Matched urine and blood samples were collected from 84 Tanzanian children aged 6–14 months old. From 31 children in one village (Kigwa), samples were collected at three time points six months apart. Samples were collected from 31 and 22 children from two different regions at the second time point only. Urinary AFM1 was measured using a commercial enzyme-linked immunosorbent assay (ELISA) kit with a modified protocol to improve sensitivity. AF-alb was measured using an established ELISA method.

Results: The relative ranking of the three villages for exposure to aflatoxin based on either AFM₁ or AF-alb biomarker measurements was the same. In Kigwa village, both AFM1 and AF-alb levels were higher at six months post-harvest compared to baseline. However, at the next visit, the AFM1 levels dropped from a GM (interquartile range) of 71.0 (44.7, 112.6) at visit two to 49.3 (31.5, 77.3) pg/ml urine, whereas AF-alb levels increased from 47.3 (29.7, 75.2) to 52.7 (35.4, 78.3) pg/mg albumin between these two visits, reflecting the fact that AFM1 measures short-term exposure, whereas AF-alb measures longer term exposure. There was a correlation between AFB1 intake and AFM1 excretion (r=?0.442, p?≤?0.001).

Conclusions: Urinary AFM1 is a good biomarker for AFB1 exposure in Tanzanian children, reflecting geographical and temporal variations in exposure to this foodborne toxin.     

Qualitative and quantitative detection of aflatoxin B1 in poultry sera by enzyme-linked immunosorbent assay

An indirect competitive inhibition type enzyme-linked immunosorbent assay (ELISA) has been developed for the detection of aflatoxin B₁, in poultry sera. Preincubation of aflatoxin B₁, samples with the antibody prior to competition yielded better results in terms of higher sensitivity. After competition, amount of antibody bound to solid phase was measured by incubation with anti-rabbit immunoglobulins coupled with horse raddish peroxidase. Intensity of colour decreased as the amount of free aflatoxin B₁, increased. Final detection of aflatoxin B₁, was made by (i) visual comparison with standard aflatoxin B₁ using dot-ELISA (qualitative) and (ii) by plate-ELISA, where optical density was measured at 492 nm (quantitative). Plate-ELISA was more sensitive than dot-ELISA, with sensitivity limits being 100 fg and 1 pg per 10 μl, respectively. However, due to ease and speed of performance, dot-ELISA has greater potential as a test for the diagnosis of mycotoxicosis at the field level.     

Reduction in the urinary aflatoxin M1 biomarker as an early indicator of the efficacy of dietary interventions to reduce exposure to aflatoxins

Abstract

Aflatoxin B₁ is a persistent public health issue in Ghana. Assessment of AFB₁ intervention efficacy is currently dependent on long-term biomarkers. This study was designed to determine whether daily AFM₁ biomarker levels could be utilized as an early detection method for intervention efficacy. Participants were treated with a refined calcium montmorillonite clay (UPSN) or a placebo (calcium carbonate) in a crossover study. Urine samples were assessed for AFM₁ levels daily. UPSN treatment reduced AFM₁ biomarkers by 55% compared to the placebo. This is the first study to show that daily urinary AFM₁ levels can be used as a biomarker of internal aflatoxin B₁ exposure in short-term intervention trials to determine efficacy.     

Scanning electron microscopy of differentiated liver cells transformed in vitro by chemical carcinogens

A scanning electron microscopy study was carried out on differentiated liver cells transformed in vitro by three chemical carcinogens into cells that give rise to carcinomas. The results indicate that the transformed cells grow as a rule in tightly adherent monolayers but differ in topography. There is a tendency toward heterogeneity in cell shape compared to the normal and on the whole toward a larger number of surface microvilli in the malignant cell population. However, both in sparse and confluent cultures the topographic differences are often not striking enough to unequivocally distinguish single neoplastic cells from the normal.     

Quantification of D-amino acids in human urine using GC-MS and HPLC

Summary The relative amounts of free D-amino acids (D-AA) in the urine of seven healthy volunteers (age 27 to 49 years) were determined using chiral phase (Chirasil-L-Val) capillary gas chromatography in conjunction with selected ion monitoring mass spectrometry. The absolute amounts of free D-AA were determined by pre-column derivatization of the amino acids witho-phthaldialdehyde andN-isobutyryl-L-cysteine followed by high-performance liquid chromatographic separation and fluorescence detection of the isoindol derivatives formed. The following most abundant D-AA were found (highest and lowest absolute and relative amounts): D-Ser (379.8 — 30.1µMol/L; 56.5 — 19.0%), D-Ala (53.8 — 7.6µMol/L; 19.6 — 5.7%), D-Thr (5.8 — 0.25µMol/L; 3.4 — 1.0%), D-Val (3.7 — 0µMol/L; 4.2 — 0%), and D-Phe (3.5 — 0.35µMol/L; 4.8 — 1.4%).     

A study on human urine in a high-selenium area of China by 1H-NMR spectroscopy

In this study, high-resolution 600-MHz ¹H-NMR (nuclear magnetic resonance) spectroscopies were used to compare the urinary metabolic profiles of healthy humans and humans in a high-selenium area of China. NMR biomarkers for renal and liver lesions were observed by comparing the urine ¹H-NMR spectra. In urinary excretion, the concentrations in human urine samples of formate, lactate, acetate, hippurate, and alanine in overexposure to selenium were increased, whereas citrate, creatine, and TMAO excretion were decreased compared with that of the healthy human—some of them even disappeared. An interesting result was the appearance of formate in urine, which has previously been shown to lead to acidosis and chronic renal failure and interfere with the lumen and proximal tubular cells. The level of creatine was associated with the seminal activity. The changes of acetate and citrate may explain the disorder of the cellular energy metabolism caused by selenium, and the changes of other amino acids were a result of the reuptake of these compounds that had been blocked in the glomerulus and proximal tubule. The results elucidate the renal/liver lesion in humans in high-selenium area by ¹H-NMR spectroscopy and offer the molecular basic of selenium toxicity.     

Detection of Toxoplasma gondii-specific antibodies in pigs using an oral fluid-based commercial ELISA: Advantages and limitations

Toxoplasma gondii is a major food-borne parasite and undercooked meat of infected pigs represents an important source of infection for humans. Since infections in pigs are mostly subclinical, adequate diagnostic tests for use at the farm level are pursued. Oral fluid (OF) was shown to be a promising matrix for direct and indirect detection of infections with various pathogens in pigs. The objective of this study was to assess whether T. gondii infections in pigs could be diagnosed using an indirect ELISA kit adapted for OF samples (OF-ELISA). Routine serology and OF-immunoblot (IB) were used as standards for the comparison. For this, serial OF samples from sows (n = 8) and fatteners (n = 3) experimentally inoculated with T. gondii oocysts, individual field samples from potentially exposed sows (n = 9) and pooled OF samples from potentially exposed group-housed fatteners (n = 195 pig groups, including 2,248 animals) were analysed for antibodies against T. gondii by ELISA. For individual animals, OF-ELISA exhibited a relative diagnostic specificity of 97.3% and a relative diagnostic sensitivity of 78.8%. In experimentally infected animals, positive OF-ELISA results were observed from 1.5 weeks post inoculation (pi) until the end of the experimental setup (8 to 30 weeks pi); however, values below the estimated cut-off were occasionally observed in some animals despite constant seropositivity. In potentially exposed individual animals, OF- and serum-ELISA results showed 100% agreement. In group-housed fatteners, antibodies against T. gondii could be reliably detected by OF-ELISA in groups in which at least 25% of the animals were seropositive. This OF-ELISA, based on a commercially available serum-ELISA, may represent an interesting non-invasive screening tool for detecting pig groups with a high exposure to T. gondii at the farm level. The OF-ELISA may need further adjustments to consistently detect individual infected pigs, probably due to variations in OF antibody concentration over time.     

A rapid and validated HPLC method to quantify sphingosine 1-phosphate in human plasma using solid-phase extraction followed by derivatization with fluorescence detection

We describe the development and validation of analytical methodology for the determination of sphingosine 1-phosphate (S1P) in plasma. It uses solid-phase extraction (SPE) followed by an automated reversed-phase gradient HPLC column-switching system with a pre-column derivatization with o-phthalaldehyde (OPA) and fluorescence detection. The limit of quantification was determined at 100 ng/ml exogenous sphingosine 1-phosphate with a relative standard deviation for precision and accuracy <15%. The within- and between-day relative standard deviation for precision and accuracy were also less than 15%. This validated method should be suitable to quantify plasma concentration of sphingosine 1-phosphate in relatively large numbers of samples.     

Modification of the cytotoxicity of aflatoxin B1 in rat liver cells by steroids

The addition of steroids with aflatoxin B1 (AFB1) to rat liver cells in culture has been shown to increase the toxin's inhibitory action on growth and protein synthesis. In contrast the inhibition of RNA synthesis by AFB1 was unaffected. The steroid potentiates the direct action of AFB1 at initiation of translation.     

Aflatoxin Binders II: Reduction of aflatoxin M1 in milk by sequestering agents of cows consuming aflatoxin in feed

Sequestering agents bind dietary aflatoxin B1 (AFB1) and reduce absorption from an animal's gastrointestinal tract. As a result, they protect an animal from the toxic effects of AFB1 and reduce transfer of the metabolite, aflatoxin M1 (AFM1), into milk. Three experiments, using late-lactation Holstein cows fed AFB1-contaminated feed, were conducted to evaluate several potential sequestering agents for their abilities to prevent or reduce the transmission of AFM1 into milk. Six agents previously tested in our laboratory for AFB1 binding in vitro were evaluated in these experiments. These were: SA-20, an activated carbon (AC-A); Astra-Ben-20, a sodium bentonite (AB-20); MTB-100, an esterified glucomannan (MTB-100); Red Crown, a calcium bentonite (RC); Flow Guard, a sodium bentonite (FG); and Mycrosorb, a sodium bentonite (MS). Five of the six sequestering agents significantly (P < 0.01) reduced AFM1 contamination of milk (AB-20, 61%; FG, 65%; MS, 50%; MTB-100, 59%; and RC, 31%); whereas, AC-A, activated carbon, had no effect on AFM1 transmission at 0.25% of feed. By the first milking (1 day after cows consumed contaminated feed), AFM1 appeared in milk, then reached maximum levels after three days, and was absent from milk within four days after AFB1 was removed from the feed. Sodium bentonites at 1.2% of feed showed good potential as AFB1 binders; MTB-100, a yeast cell wall product, was equally effective at 0.05% in feed. Potential AFB1 binding agents should be evaluated experimentally to demonstrate efficacy. Our data show that sequestering agents can reduce AFM1 in milk of cows fed AFB1-contaminated feed.     

Detection of aggressive prostate cancer associated glycoproteins in urine using glycoproteomics and mass spectrometry

Clinical management of prostate cancer remains a significant challenge due to the lack of available tests for guiding treatment decisions. The blood prostate‐specific antigen test has facilitated early detection and intervention of prostate cancer. However, blood prostate‐specific antigen levels are less effective in distinguishing aggressive from indolent prostate cancers and other benign prostatic diseases. Thus, the development of novel approaches specific for prostate cancer that can differentiate aggressive from indolent disease remains an urgent medical need. In the current study, we evaluated urine specimens from prostate cancer patients using LC‐MS/MS, with the aim of identifying effective urinary prostate cancer biomarkers. Glycoproteins from urine samples of prostate cancer patients with different Gleason scores were characterized via solid phase extraction of N‐linked glycosite‐containing peptides and LC‐MS/MS. A total of 2923 unique glycosite‐containing peptides were identified. Glycoproteomic comparison on urine and tissues from aggressive and non‐aggressive prostate cancers as well as sera from prostate cancer patients revealed that the majority of AG prostate cancer associated glycoproteins were more readily detected in patient's urine than serum samples. Our data collectively indicate that urine provides a potential source for biomarker testing in patients with AG prostate cancer.     

A survey of aflatoxin B<Subscript>1</Subscript> and total aflatoxin contamination in baby food,peanut and corn products sold at retail in Indonesia analysed by ELISA and HPLC

Aflatoxin contamination has been well known as a world-wide health-threatening problem in tropical countries including Indonesia. This research was undertaken to determine the degree of aflatoxin contamination in different Indonesian foodstuffs. A preliminary survey was carried out to evaluate the level of total aflatoxin (AfT) and aflatoxin B₁ (AfB₁) contamination of baby foods, peanut products, and corn products, which were purchased from traditional markets and supermarkets in Indonesia during the year 2001-2002. Eighty two peanut products, 12 baby foods products, and 11 corn products from different brands were analysed for AfT and AfB₁ using the Enzyme-Linked Immunosorbent Assay (ELISA) method. The results indicate that, of the brands analysed, 35% of the peanut products were contaminated with aflatoxins at various levels (range 5 to 870 μg/kg). Peanut-chilli sauces had the highest percentage of AfT contamination 9/12 (75%), which was followed by traditional snacks 5/11 (45%), peanut butter 4/11 (40%), flour egg coated peanut 6/16 (37%), and peanut cake 3/10 (30%). Fried peanuts and roasted peanut were found to contain aflatoxin at relatively lower percentages of 9% and 8%, respectively. From the 12 analysed baby food samples, on the other hand, no sample was found to be contaminated with aflatoxins. Two of 11 samples (18%) of corn based products were contaminated with AfT, ranging between 5.8 and 12.4 μg/kg. Additionally, 30 selected samples in different concentration ranges were further analysed to verify the correlation between ELISA and HPLC techniques and results were compared.     

Detection of measles virus-induced apoptosis of human monocytic cell line (THP-1) by DNA fragmentation ELISA

Abstract Measles virus (wild strain, Toyoshima strain)-induced cell death is characterized by cell shrinkage, chromatin condensation, and nuclear fragmentation in a human monocytic cell line (THP-1). DNA fragmentation of measles virus-infected THP-1 cells was demonstrated by DNA agarose gel electrophoresis as well as by DNA fragmentation ELISA. When measles virus-infected THP-1 cells were cultured on monolayers of fibroblasts or human umbilical vein endothelial cells (HUVEC), the percentage of measles virus antigen-positive THP-1 cells and DNA fragmentation were significantly decreased. Addition of anti-intercellular adhesion molecule (ICAM)-1 (CD54) monoclonal antibody to culture of measles virus-infected THP-1 cells reduced significantly DNA fragmentation induced by measles virus. These findings suggest that inhibition of virus spread by fibroblasts and HUVEC reduces apoptosis, and ICAM-1 (CD54) may participate in the DNA fragmentation pathway.     

二乙基亚硝胺诱导大鼠肝癌组织CLDN1基因表达及其甲基化

目的探讨二乙基亚硝胺（diethylnitrosamine,DEN）诱导大鼠肝癌发生中肝癌组织CLDN1基因表达及其启动子甲基化的规律。方法65只雄性Wistar大鼠随机选择40只作为模型组,其余作为正常组。模型组在1-12周饮用含DEN80mg／L的饮水以诱癌（每日8mg／kg）,各组在造模过程的第4周、8周、12周、16周随机5只取肝,第20周剩余大鼠取肝,应用RT-PCR方法检测肝组织CLDN1mRNA的表达,应用MSP法检测肝组织CLDN1启动子甲基化和非甲基化。结果模型大鼠病死率为10％（4／40）,正常组无死亡。至第20周,成瘤率达到100％。RT—PCR显示,与正常组比较,模型组在16周和20周CLDN1 mRNA表达下调（P〈0．05）,其他各周两组差异不显著。MSP结果表明,模型组肝组织CLDN1甲基化率达77．78％,而正常肝组织甲基化率为24％,两者比较差异有显著性（P〈0．01）。结论CLDN1启动子甲基化及CLDN1基因表达下调与大鼠肝癌病变相关,对其机制值得进一步深入研究。     

Detection of antibody to hepatitis delta virus in human serum by double antigen sandwich ELISA

A simple rapid detection of antibody to hepatitis delta virus (anti-HDV) in human serum was developed by using double antigen sandwich ELISA. HDV gene fragment encoding HDAg was isolated from a Chinese patient infected with HDV by RT-PCR, and a high-efficient expression HD-PQE31 strain was constructed with the fragment. We obtained high titer and good quality hepatitis delta virus protein purified by Ni-NTA metal-affinity chromatography, which was identified by Western blot and ELISA, then we set up the double antigen sandwich ELISA for detection of anti-HDV in human serum, and the performance of the sandwich ELISA was evaluated in terms of specificity and sensitivity. Results were: 1) The purified HDAg protein's purity was 90%, and its ELISA titer was 1/100 000. 2) 42 anti-HDV positive sera were detected and showed that the sensitivity of sandwich ELISA was higher than that of competitive ELISA (t=2.44, p<0.01). 3) The inhibitory rates for 2 anti-HDV positive sera by the specific HDAg were 74% and 93% respectively. 4) For the assay of specificity, all 60 samples infected by other hepatitis viruses and 30 normal samples were negative for anti-HDV. These results suggested that the double antigen sandwich ELISA with purified recombinant HDAg showed higher specificity and sensitivity, It can be used in routine laboratories to diagnose the HDV infection.     

Comparison of hydrolysis and HPLC/MS/MS procedure with ELISA assay for the determination of S-phenylmercapturic acid as a biomarker of benzene exposure in human urine

The present study compared three methods for the determination of S-phenylmercapturic acid (S-PMA), a metabolite of benzene, in human urine: a HPLC/MS/MS technique with two different sample treatments (strong and partial hydrolysis) and a commercial assay based on anti-S-PMA monoclonal antibodies with chemiluminescence detection. Biological monitoring was done on 126 volunteers and the results were compared for the three methods and also with benzene exposure levels (range <3.0–592.5 μg/m³). The correlation between environmental monitoring data and S-PMA levels in non-smokers (n = 73) was highly significant (p < 0.0001, Student's t-test) for both HPLC/MS/MS methods (r = 0.65 both for strong acidic hydrolysis of the urine and hydrolysis at pH 2) but not for the immunoassay, which overestimated the S-PMA levels by about 8 μg/g creatinine (creat.). Therefore the immunoassay is only useful as a semiquantitative screening test, but quantitative results need to be confirmed by a more accurate method like HPLC/MS/MS. The HPLC/MS/MS procedure with strong acid hydrolysis led to a recovery of S-PMA about double that using pH 2 hydrolysis, giving more accurate results. The difference between the results with the two methods makes it difficult to compare the strong acidic hydrolysis data with the ACGIH BEI value of 25 μg/g creat. since the BEI^® documentation is based on data collected in pH conditions that were not always controlled, which may underestimate the true S-PMA concentration. Besides, as levels of benzene exposure were high, smoking was not considered a confounding factor. The BEI for S-PMA in end of shift urine samples could be reconsidered when sufficient data are available from studies where the analyses are carried out in comparable conditions of hydrolysis and monitoring only non-smoking subjects.     

A novel marker for assessment of liver matrix remodeling: An enzyme-linked immunosorbent assay (ELISA) detecting a MMP generated type I collagen neo-epitope (C1M)

A competitive enzyme-linked immunosorbent assay (ELISA) for detection of a type I collagen fragment generated by matrix metalloproteinases (MMP) -2, -9 and -13, was developed (CO1-764 or C1M). The biomarker was evaluated in two preclinical rat models of liver fibrosis: bile duct ligation (BDL) and carbon tetra chloride (CCL4)-treated rats. The assay was further evaluated in a clinical study of prostate-, lung- and breast-cancer patients stratified according to skeletal metastases. A technically robust ELISA assay specific for a MMP-2, -9 and -13 neo-epitope was produced and seen to be statistically elevated in BDL rats compared to baseline levels as well as significantly elevated in CCL4 rats stratified according to the amount of total collagen in the livers. CO1-764 levels also correlated significantly with total liver collagen and type I collagen mRNA expression in the livers. Finally, the CO1-764 marker was not correlated with skeletal involvement or number of bone metastases. This ELISA has the potential to assess the degree of liver fibrosis in a non-invasive manner.     

Improvement and validation the method using dispersive liquid-liquid microextraction with in situ derivatization followed by gas chromatography-mass spectrometry for determination of tricyclic antidepressants in human urine samples

A simple, rapid and sensitive method termed dispersive liquid-liquid microextraction (DLLME) combined with gas chromatography-mass spectrometry (GC/MS) was developed for the determination of tricyclic antidepressants (TCAs) in human urine sample. An appropriate mixture of methanol (disperser solvent), carbon tetrachloride (extraction solvent), and acetic anhydride (derivatization reagent) was injected rapidly into human urine sample. After extraction, the sedimented phase was analyzed by GC/MS. The calibration curves obtained with human urine were linear with a correlation coefficient of over 0.99 in the range of 2.0/5.0-100 ng mL(-1). Under the optimum conditions (carbon tetrachloride: 10 μL, methanol: 150 μL), the detection limits and the quantification limits of the tricyclic antidepressants were 0.5-2.0 ng mL(-1) and 2.0-5.0 ng mL(-1), respectively. The average recoveries of TCAs were 88.2-104.3%. Moreover, the inter- and intra-day precision and accuracy was acceptable at all concentrations. The results showed that DLLME is applicable to the determination of trace amounts of TCAs in human urine sample.