Metallothionein as a biomarker for mercury in tissues of rat fed orally with cinnabar

Cinnabar, as one of the most widely used mineral drugs in traditional Chinese medicines, has been proven to have prominent curative effects in clinical use for more than 2000 years. But the safety and toxicity of the drug has been under constant debate in clinic usage. Metallothionein (MT) contains about 30% of cysteine in the molecule, and plays an important detoxification role against heavy metals. In this study, it was used as a biomarker to assess mercurial accumulation in rats fed orally with cinnabar. After feeding rats with cinnabar by gastric gavage at different dosages and at different times, the distribution of heavy metals (including mercury, copper and zinc) and MT was investigated among rat tissues, including liver, kidney, heart, brain, testis and blood. Metals and MT determinations were carried out using inductively coupled plasma mass spectrometry (ICP‐MS) and a modified mercury saturation assay technique respectively. The results indicated that mercury was easily accumulated in the tissues of rats exposed to cinnabar, especially in kidney. For example: at a feeding dosage of 5 g kg^?1 (bw) for 4 weeks, the mercury concentrations in kidney were 13, 8.7, 21.6 and 26 times those in liver, testis, brain and heart respectively; and at 2.5 g kg^?1 (bw) for 2 weeks, the mercury concentrations in kidney were 21, 2.1, 3 and 21 times those in liver, testis, brain and heart respectively. In addition, mercury in kidney and liver of all cinnabar groups was significantly higher than that of the control group (P < 0.01). A high positive correlation observed between MT concentrations and mercury levels in both liver and kidney (R² = 0.9299, P < 0.02 for liver; R² = 0.9923, P < 0.0008 for kidney) indicated that MT could be used as a biomarker for mercury in tissues. Copyright © 2004 John Wiley & Sons, Ltd.     

Total Sulfur Concentration in Tissues of Control and Cadmium-Exposed Rats by Inductively Coupled Plasma Atomic Emission Spectrometry

Abstract

Total sulfur (S) concentration in biological samples was determined simultaneously with metal concentrations by inductively coupled plasma-atomic emission spectrometry (ICP).

A 0.2 g portion of liver and other tissues were wet-digested with 1.0 ml mixed acid (HNO₃ : HCLO₄ = 5 : 1, v/v) at 130 – 150 °C. The solution was concentrated to about 0.1 ml and then diluted to 5.0 ml with double distilled water. Concentration of S was determined by ICP using ammonium sulfate as a standard S compound. Sulfur and other element concentrations in an NBS standard reference material (Bovine Liver SRM 1577) were within the certified values by this method.

Concentrations of total S, cadmium (Cd), copper (Cu) and zinc (Zn) in the liver, kidney, spleen, lung, pancreas and blood serum were compared between the control and Cd-exposed rats. The three metal concentrations were increased significantly by Cd exposure. However, S concentration was not altered significantly in the liver and other tissues despite the extensive induction of metallothionein (MT) by the repeated Cd exposure. Metallothionein induced by the accumulated Cd (121 μg/g) and Zn (48 μg/g) in the liver was estimated to account for at maximum 7 % of the total S by assuming that the increased metals were all bound to MT. Concentration of S in blood serum was decreased significantly by Cd loading.     

Determination of bile acids in pig liver, pig kidney and bovine liver by gas chromatography-chemical ionization tandem mass spectrometry with total ion chromatograms and extraction ion chromatograms

An effective method has been developed for quantitative determination of six bile acids including lithocholic acid (LCA), deoxycholic acid (DCA), chenodeoxycholic acid (CDCA), hydodeoxycholic acid (HDCA), cholic acid (CA) and ursodeoxycholic acid (UDCA) in biological tissues including pig liver, pig kidney and bovine liver by gas chromatography-chemical ionization/tandem mass spectrometry (GC-CI/MS/MS). Camphor-10-sulphonic acid (CSA) was proposed as effective catalyst for bile acid derivatization. Reactions were accelerated ultrasonically. The effects of different catalysts and reaction times on derivatization efficiency were evaluated and optimized. Bile acids were determined as methyl ester-trimethylsilyl ether and methyl ester-acetate derivatives. The efficiency of trimethylsilylation and acetylation was evaluated. Trimethylsilylation was done with N,O-bis(trimethylsilyl)trifluoroacetamide (BSTFA) as the trimethylsilyl donating reagent in a ultrasonic bath for 20 min. Acetylation was done in pyridine with acetic anhydride at 40-45°C for 4 h. The former reaction was faster than the latter. Thus, trimethylsilylation was employed for the quantitative analysis. Negligible interferences from sterols in biological matrices were observed when the biological samples were treated with solid phase extraction before GC-CI/MS/MS. The linearity, reproducibility, detection limit and recovery were evaluated under the optimized conditions. Satisfactory results were obtained when bile acid derivatives of LCA, CDCA, HDCA, and UDCA were determined with total ion chromatograms (TIC) while DCA and CA were determined with extracted ion chromatograms (EIC), respectively. The detection limits (S/N=3) for six bile acids in biological tissues were ranging from 0.40 to 1.6 ng/mL and the recoveries indicated that the proposed method was feasible for the determination of trace bile acids in the biological samples studied. The experimental results for the animal tissues purchased from five different markets were compared. Interestingly, all of the six bile acids were present in pig liver while only the dihydroxy bile acids, DCA, CDCA and HDCA were found in pig kidney. In addition to DCA and CDCA, trihydroxy bile acid, CA, are the major bile acids in bovine liver.     

Ultra-trace analysis of platinum in human tissue samples

Background levels of platinum were determined in human autopsy tissues taken from five individuals. The investigated specimens were lung, liver and kidney. Sample preparation involved microwave digestion followed by an open vessel treatment. Inductively-coupled plasma sector field mass spectrometry (ICP-SFMS) was applied in combination with an ultrasonic nebulization/membrane desolvation system for sample introduction. Isotope dilution analysis was employed for accurate quantification of platinum. Excellent procedural detection limits (3 s validation) of 20, 20 and 34 pg g^–1 dry weight were obtained for lung, liver and kidney tissue, respectively. Due to the lack of appropriate biological reference material, road dust (BCR-723) was used for method validation. Platinum levels ranging between 0.03 and 1.42 ng g^–1 were determined in the investigated samples. The platinum concentrations observed in human lung tissue may reflect the increasing atmospheric background levels of platinum originating from car catalysts. The presence of platinum in kidney and liver tissue samples clearly indicates the bioavailability of the element.     

Searching for serum tumor markers for colorectal cancer using a 2‐D DIGE approach

Identification of specific protein markers for colorectal cancer (CRC) could provide a basis for its early diagnosis and detection, as well as clues to the molecular mechanisms governing cancer progression. In the present study, 2‐D DIGE coupled with MS was used to screen for biomarker candidates in the serum proteome of ten human CRC samples and ten healthy control samples. After pooling identical amounts of serum proteins (based on total protein concentration), albumin/IgG was depleted under partially denaturing conditions. Subsequently, the serum samples were labeled with three different CyDyes, and separated by 2‐D DIGE. After analysis with the biological variation analysis module of the DeCyder software, only three spots were found to be significantly elevated in all patient groups (with ratios from 1.52 to 9.08), whereas five spots were significantly down‐regulated in patients (with ratios from ?1.23 to ?10.21) (t‐test; p<0.05). Finally, two potential biomarkers, Transaldolase 1 and thyroid receptor interactor, were chosen for validation and analysis by ELISA with the serum of 30 CRC patients and 30 healthy controls. The serum levels of the two proteins correlated well with the 2‐D DIGE results. Thus, 2‐D DIGE approaches show great promise for biomarker discovery in CRC.     

Selenium determination in biological samples by X-ray emission spectroscopy

Determination of Se in blood serum by PIXE and XRF is presented. Two different sample preparation methods combined with two modes of sample excitation are compared. Both methods are shown to be suitable for Se determination in blood serum and in standard reference materials (horse kidney IAEA H-8 and bovine liver NBS 1577a).     

Metallothionein isoforms from horse, rabbit and rat separated by capillary zone electrophoresis at low pH

A comparative study of MT isoforms in rat liver and in commercial Sigma MT preparations from rabbit liver and horse kidney was performed using capillary zone electrophoresis (CZE). Electropherograms revealed the co-migration of MT forms from these species. A special form, the a-form (not binding Cd), occurred in various MT samples in different amounts, depending on the method used for MT purification. In the rabbit liver electropherogram a main form appeared (the b-form), which might be a modified MT form. A band of unknown composition, running ahead of the rat liver MT-I and -II forms on polyacrylamide gels, not having Cd binding affinity, probably had its counterpart in a yet unidentified CZE peak. CZE electropherograms of purified MT samples may contain main peaks that do not represent genuine and functional MT isoforms. Results are also presented which indicate that at low pH the MT-II form is more unstable than MT-I.     

Determination of 3-chloro-1,2-propanediol in biological samples by quick-easy-cheap-effective-rugged-and-safe extraction coupled with gas chromatography-mass spectrometry and its biodistribution in rats

3-Chloro-1,2-propanediol is a common food contaminant, but reports on its determination in biological tissues are lacking. In the present study, a method was developed to detect 3-chloro-1,2-propanediol contents in rat tissues by quick-easy-cheap-effective-rugged-and-safe extraction and gas chromatography-mass spectrometry analysis. Biological samples were extracted with ethyl acetate and purified with adsorbents. The optimized adsorbent for each sample was selected from 4–5 combinations of N-propylethylenediamine, octadecylsilane, graphitized carbon black, strong anion exchange, and florisil. Extracted 3-chloro-1,2-propanediol was derivatized with heptafluorobutyric anhydride and subjected to gas chromatography-mass spectrometry. This method had good linearity (correlation coefficients >0.99) in the range of 2–2000 ng/g for blood, kidney, liver, testis, and brain samples. The limits of detection were under 0.8 ng/g; the limits of quantification were 2 ng/g; the recovery rates were 85%–102%; and the matrix effects were 1.98%–7.67%. This method also had good precision. The dynamic changes in 3-chloro-1,2-propanediol in rats gavaged with 20 mg/kg b.w. for 24 h were detected using this method. The 3-chloro-1,2-propanediol content in each tissue sharply increased to a peak, rapidly decreased within 2 h, and stabilized at 12 h. 3-Chloro-1,2-propanediol persisted in the kidney, testis, and liver 24 h after gavage.     

Enzyme-linked immunosorbent assay and colloidal gold immunoassay for sulphamethazine residues in edible animal foods: investigation of the effects of the analytical conditions and the sample matrix on assay performance

To determine sulphamethazine (SMZ) residues in edible animal foods (pig muscle, chicken muscle, egg, fish, milk and liver), a competitive direct enzyme-linked immunosorbent assay (ELISA) and a colloidal gold immunoassay were established. The limits of detection of the ELISA and the colloidal gold immunoassay were 0.02 and 0.5 μg kg⁻¹, respectively. The specificity of the ELISA developed to the SMZ was high according to the results of cross-reactivity testing with 14 kinds of sulphonamides. To obtain a more sensitive immunoassay, buffer solution (30 mmol L⁻¹ phosphate-buffered saline with 0.05% Tween 20, pH 8.5) was optimized through the whole test procedure. A simple and efficient extraction method for the rapid detection of SMZ residues in foods was developed, with recoveries between 74 and 117.5%. Matrix effects can be avoided by 1:10 dilution of pig muscle, chicken muscle, egg, fish, milk and liver with optimal buffer. The detection limit of SMZ was 5 μg kg⁻¹ in liver and 2 μg kg⁻¹ in the other five samples. For the validation of the ELISA tests, sample extracts were analysed by ELISA and high-performance liquid chromatography. The results obtained by these two methods showed a good correlation (r ²) which was greater than 0.9. The colloidal gold immunoassay presented in this assay was successfully applied to determine SMZ in pig muscle, milk and fish below or equal to the maximum residue level (20 μg kg⁻¹).     

A rapid method for the determination of lasalocid in animal tissues and eggs by high performance liquid chromatography with fluorescence detection and confirmation by LC-MS-MS

A simple and rapid method has been developed for the extraction of lasalocid from chicken muscle, eggs and liver and kidney from chicken, pig, sheep and calf. This method allows the screening of a large number of samples, i.e. 30-40 within a working day, and has an overall analysis time of 90 min. Lasalocid standard solution can be detected at 1 ng ml-1 by both HPLC-fluorescence (HPLC-F) and LC-MS-MS; the limit of quantification in fortified samples by the described method is 1 ng g-1. Results show good repeatability and mean 'spiked' recoveries by HPLC-F in the range of 10 to 200 ng g-1 (ppb) of 103, 87, 107, 97, 97, 103, 93, 109 and 100% in chicken muscle, chicken liver, egg, pig liver, pig kidney, sheep liver, sheep kidney, calf liver and calf kidney, respectively. For concentrations between 1 and 6 ng g-1 of spiked lasalocid in eggs and chicken liver by LC-MS-MS, the average recoveries were 76 and 59%, respectively.     

Quantification of fish hepatic metallothioneins, naturally or artificially induced, by ELISA: A comparison with radioimmunoassay and differential pulse polarography

An ELISA has been developed for the quantification of metallothioneins (MT) in liver from different species of fish. MT has also been quantified by RIA and DPP to allow a comparison between the methods. The immunoassays were carried out with a polyclonal antibody raised against perch (Perca fluviatilis) hepatic MT [1] which cross-reacted with hepatic MT from dab (Limanda limanda), lemon-dab (Microstomus kitt), and cod (Gadus morhua). The ELISA was more sensitive for the detection of MT from the flatfish than RIA and DPP. The detection limits of MT by immunoassays were lower for fish caught in the field than for Cd-injected fish. The immunoreactivity differed between MT from different fish species, more markedly between fish MT and rabbit MT. MT values determined by the three methods have been compared and showed that the results were similar and significantly correlated. Advantages and disadvantages of the ELISA are discussed in relation to the other methods.     

Determination of cadmium and lead in biological samples by three ultrasonic-based samples treatment procedures followed by electrothermal atomic absorption spectrometry

The development of 3 different ultrasonic-based sample treatment methods, ultrasonic probe-assisted acid extraction, ultrasonic-assisted acid slurry, and ultrasonic-assisted acid pseudodigestion is presented. These methods were compared for the determination of Cd and Pb by electrothermal atomic absorption spectrometry in biological samples (blood and scalp hair) and validated by using certified materials BCR 397 human hair and BCR 185R bovine liver. The sample amounts chosen to perform the analysis were 100 mg and 0.5 mL for solids (human hair and bovine liver) and blood samples, respectively. An acid digestion induced by microwave energy was used to obtain the total metal concentrations and for comparative purposes. The best results were obtained with the ultrasonic-assisted acid pseudodigestion, with which it was possible to perform accurate and precise determination of the Cd and Pb contents in 2 certified reference materials and biological samples of 50 normal males of ages 25-40 years. The precision of the methods, together with their efficiency, rapidity, low cost, and environmental acceptability, make them good alternatives for the determination of trace metals from biological samples. The precision of the methods for accuracy evaluation, resulting in good agreement according to the t-test for a 95% confidence level, and the relative standard deviations were lower than 10% (n=10) for all determinations.     

Normalization using a tagged-internal standard assay for analysis of antibody arrays and the evaluation of serological biomarkers for liver disease

For minimizing systemic experimental variation in the analysis of antibody array data, we developed a novel median-centered/IgM-tagged-internal standard (TIS) assay normalization using median-centering and TIS assay-based determination of serum IgM concentrations. We evaluated five normalization methods by analyzing correlation coefficients and coefficients of variation for six serum proteins using human serum samples from normal controls (n = 25) and patients with liver cirrhosis (n = 25) or hepatocellular carcinoma (HCC; n = 29). Median-centered normalization improved correlation coefficients, while IgM-based normalizations improved coefficients of variation. The TIS assay was more efficient, economical, and reproducible for determining IgM concentrations than enzyme-linked immunosorbent assay. Additionally, we normalized antibody array data for six serum proteins using the median-centered/IgM-TIS assay, and evaluated serum biomarkers through distribution analysis of normalized fluorescence intensities and receiver operating characteristic analyses for the diagnosis of liver cirrhosis and HCC. Apolipoprotein A-1 and a combination of alpha-fetoprotein and C-reactive protein were determined to be potential serological biomarkers for liver cirrhosis and HCC, respectively. Thus, median-centered/IgM-TIS assay normalization is a useful approach for analyzing antibody array data and evaluating serological biomarkers for the diagnosis of liver disease or cancers.     

Potentiometric Biosensor System Based on Recombinant Urease and Creatinine Deiminase for Urea and Creatinine Determination in Blood Dialysate and Serum

A biosensor system for simultaneous determination of creatinine and urea in blood serum and dialysate samples was developed. It consisted of creatinine and urea biosensors based on a potentiometric transducers with two identical pH‐sensitive field‐effect transistors. In creatinine biosensor, creatinine deiminase immobilized via photopolymerization in PVA/SbQ polymer on one transistor served as a biorecognition element, while bovine serum albumin in PVA/SbQ polymer placed on the second transistor was used for reference. The urea biosensor was created in the same way but recombinant urease was used instead of creatinine deiminase. The linear ranges of creatinine and urea measurement were 0.02–2 mM and 0.5–15 mM, correspondingly, which allowed simultaneous determination of the metabolites. Response time of the biosensor system was 2–3 min; RSD of responses did not exceeded 5 %. The biosensors demonstrated absence of non‐selective response towards components of blood dialysate and serum. Urea and creatinine concentrations were determined in 20 samples of blood dialysate and serum. The results correlated well with traditional methods of analysis. Creatinine and urea biosensors were stable during five months of storage (during this time the responses decreased by about 10 %). The proposed biosensor system can be effectively used for analysis of serum samples and for hemodialysis control.     

生物样本中的甲基苯丙胺和氯胺酮含量的检测研究及其体内分布初探

建立了生物样本中甲基苯丙胺和氯胺酮的定量检验方法,该方法应用固相萃取与工作曲线气相色谱法(GC/NPD)对中毒者血、尿、胃内容、肝、脑等检材进行含量测定.通过实际案件,验证了该法具有样品处理简单、提取效率高、分析速度快等优点.案件数据表明,由口服引起的中毒途径,胃内容的含量最高,其次是尿、肝、脑、血等检材,进一步证实了...     

Suppression Of Ages Formation In Spontaneous Diabetic OLETF Rat By Organic Germanium Compound [Poly-trans-(2-carboxyethyl) Germasesquioxane] (Ge-132)

The model rat for the type 2 diabetes mellitus (NIDDM) in human were observed for 72 weeks after birth, administering an organic germanium compound [Bis(2-Carboxyethyl) germasesquioxane, Ge-132] perorally 100 mg/kg/day since 24 weeks old. Clinical examinations were followed throughout the observation period. Blood and urinary glucose in positive control OLETF rats tended to be higher than those treated with Ge-132. At the end of the 72nd week, animals were sacriificed to examine the pathological changes, specifically in pancreas, kidney and brain. Anti-AGE antibody stained proximal and distal tubles and basement membrane of glomerules in kidney, and accumulated AGE masses in cortex, hippocampus and cerebellum of OLETF rat's brain. Amyloid stains by basic congo red on kidney and brain revealed that the deposits of amyloid in kidney mesangium and in cortex, hippocampus and cerebellum were observable in OLETF rats. Ge-132 suppressed the deposition of amyloid tangles in kidneys and brains. Anti rat complement C'3 antibody reacted with AGE and amyloid tangles that were sensitive to anti-AGE antibody. AGE generated in vitro by incubating human serum, human gammaglobulin (HGG), or bovine serum albumin (BSA) with glucose activated complements, showing the consumption of complements in the hemolysis of hemolysin-coated sheep red blood cells. A novel device Quantum Resonance Spectrometer (QRS) could read the subtle bio-magnetism memorized in serum samples, demonstrating quantitative values reflecting the patho-physiology of OLETF rats.     

High‐sensitivity simultaneous quantification of tacrolimus and 13‐O‐demethyl tacrolimus in human whole blood using ultra‐performance liquid chromatography coupled to tandem mass spectrometry

Blood concentrations of tacrolimus show large variability among patients and the narrow therapeutic range is related to adverse effects. Therefore, therapeutic drug monitoring is needed for strict management. 13‐O‐Demethyl tacrolimus (13‐O‐DMT) was reported as the major metabolite formed by cytochrome P450 (CYP)3A such as CYP3A5. In previous studies, the best lower limit of quantification (LLOQ) was 0.1 ng/mL for both substances. However, this LLOQ may not be low enough now because the dosage of tacrolimus has decreased in recent years. The purpose of this study was to develop and validate a high‐sensitivity and high‐throughput assay for simultaneous quantification of tacrolimus and 13‐O‐DMT in human whole blood using ultra‐performance liquid chromatography with tandem mass spectrometry (UPLC–MS/MS). Thirty‐five stable kidney transplant recipients receiving tacrolimus were recruited in this study. The calibration curve range was 0.04–40 ng/mL. All calibration samples and quality control samples fulfilled the requirements of the US Food and Drug Administration and the European Medicines Agency guidelines for assay validation. Trough concentrations of tacrolimus and 13‐O‐DMT in 35 stable kidney transplant recipients receiving tacrolimus were within the range of the respective calibration curve. Our novel UPLC–MS/MS method is more sensitive than previous methods for quantification of tacrolimus and 13‐O‐DMT.     

Determination of 7,12-dimethylbenz[a]anthracene in orally treated rats by high-performance liquid chromatography and transfer stripping voltammetry

A number of polycyclic aromatic hydrocarbons (PAHs) have been shown to be toxicants, and induce carcinogenic and immunotoxic effects. As a model PAH agent, 7,12-dimethylbenz[a]anthracene (DMBA) was the strongest one tested in terms of its biological activities and biotransformation. A new and simple high-performance liquid chromatographic (HPLC) method with diode-array detection at 290 nm was developed and validated for monitoring of DMBA in different matrices (serum, liver and kidney) of rats orally treated with DMBA. Furthermore, the applicability of adsorptive transfer stripping voltammetry (AdTSV) on the pencil-lead graphite electrode to these samples was illustrated using our previously reported data for bulk aqueous solutions of DMBA. HPLC and AdTSV methods, which were compatible with each other, allowed DMBA to be detected down to the levels of 3.82x10-9 M (0.98 ppb) and 6.73x10-9 M (1.73 ppb), respectively. Olive oil solutions of DMBA in dose 50 mg/kg were orally administered. 60 days after a single dose of DMBA, its concentrations in these biological samples from rats were measured by means of both methods. Because of rapid biotransformation, DMBA could not be detected in serum. Only low levels of the compounds were deposited unchanged in kidney whereas its levels were considerably higher in liver. These methods were also applied to the assay whether there is an influence of the intake of aqueous extracts of Hypericum Perforatum L. plant on the parent DMBA levels accumulated in rat tissues.     

金属硫蛋白MT-2a与Cd(Ⅱ)和Cu(Ⅰ)络合的电喷雾质谱表征

采用电喷雾质谱法(ESI-MS)研究金属硫蛋白(MT,存在α和β两种结构域)-2a与金属离子Cd和Cu的络合作用.MT-2a由反相色谱分离纯化制得,并以电喷雾质谱鉴别.通过ESI-MS考察MT-2a与不同量的Cd和Cu的络合,结果表明,Cd能够优先与MT的α结构域络合,形成M2+4S11结构,并且该络合过程存在着明显的协同效应;Cd与MT的β结构域的络合形式为M2+4S9,其呈现出明显的随机松散络合特性;Cu优先与MT的β结构域络合,其络合形式由Cu4逐渐过渡到更高结合态;在Cu含量较高的条件下,Cu与MT呈现出多种络合方式.     

Capillary zone electrophoresis determination of oxytetracycline in pig tissue samples at maximum residue limits

Summary The determination of the antibiotic oxytetracycline (OTC), in pig tissues was investigated by capillary zone electrophoresis (CZE) with a prior solid-phase extraction (SPE) using alkyl-bonded silica and polymeric cartridges. The methodology developed allows determination of OTC in pig kidney, liver and muscle samples with detection limits below maximum residue limit values, and the procedures to extract OTC and clean-up the matrix are simple and reliable. The limit of detection for OTC was 160, 120 and 85 μg kg⁻¹ for kidney, liver and muscle samples, respectively. The average recoveries from spiked samples (200 μg kg⁻¹ and 1600 μg kg⁻¹) were in excess of 63% with coefficients of variation between 2.0 and 9.8%. This method would be useful for routine monitoring of oxytetracycline residues in pig tissues.