Metallothionein separation and analysis by reversed phase high performance liquid chromatography coupled with graphite furnace atomic absorption spectrometry

Abstract

The feasibility of using reversed-phase high performance liquid chromatography (RP-HPLC) for the separation of metallothioneins (MTs) and subsequent determination of cadmium in MTs by graphite furnace atomic absorption spectrometry (GFAAS) in rabbit kidney and liver has been studied. RP-HPLC was used to isolate, characterise and quantitate liver and kidney MT isoforms. The MTs were eluted from a radially compressed C18 column with a neutral sodium phosphate buffer and detected by UV absorbance at 254 nm. Rabbit liver MTs was found to be comprised of seven distinct isoforms with five of which were found to be subspecies of the MT-I isoform. Rabbit kidney MTs exhibited only two predominant isoforms. A standard calibration curve was constructed using purified rabbit kidney MT-I and MT-II which demonstrated excellent linear correlation between peak height and the quantity of MT injected into the column. Recovery of MT from RP-HPLC was found to exceed 90%. Kidney and liver tissues from rabbit by feeding low levels of cadmium in diets was assayed using the RP-HPLC analysis of cytosol samples. Feeding stable cadmium in the diet resulted in the deposition of MT in the kidney rather than in the liver. The cadmium content in MT isoforms was determined by GFAAS. Less than 10% of the total cadmium in kidney was associated with MTs.     

Electrothermal Atomic Absorption Spectrometry to Determine As,Cd, Cr,Cu, Hg,and Pb in Soils and Sediments: A Review and Perspectives

Environmental pollution from trace elements has been increasing in recent decades and has become an important concern for environmental agencies. The trace elements arsenic, cadmium, chromium, copper, mercury, and lead are among the elements that cause the greatest environmental impact and carry the highest risk to human health. Electrothermal atomic absorption spectrometry (ETAAS) has long been employed in trace element determination. In the last few years the main constraints of spectroscopy absorption methods have been overcome. These advances have increased the possibilities and the utility of ETAAS for trace element determination at μg L^?1 levels in difficult matrices such as soils and sediments, giving greater accuracy and precision, lower economic cost, and easier sample pretreatment than other methods. The main advances come from sample manipulation for matrix destruction and preconcentration, the use of new lab-on-valve FIA systems, the solid sampling, the use of new, more efficient modifiers and in situ trapping methods for analyte stabilization and pre-concentration, and the progress in the capacity to control the atomization temperature and to correct background spectral interferences. All of them have permitted an improvement in the sensitivity, decreasing the detection limits and manipulation process, and increasing the accuracy and precision of the analyses.

Moreover, the new technology in the optic and detector systems have given rise to high-resolution continuum source ETAAS (HR-CS ETAAS) spectrometers that solve most of the constraints presented by the more conventional line source ETAAS (LS ETAAS) spectrometers. HR-CS ETAAS enables a rapid detection of several elements at once, facilitates direct determination from solid sampling, and reduces the matrix interferences and background noise. Here we give an overview of the recent advances and the different possibilities of using ETAAS, drawing on studies from the last decade on methods to analyze As, Cd, Cu, Hg, and Pb in soils and sediments.     

Correlation of Toxicity with Lead Content in Root Tip Cells (Allium cepa L.)

The present study determines lead content in onion root tip cells (Allium cepa L.), correlating it with its toxicity. The treatment was carried at 25 ± 0.5°C using aqueous solutions of lead chloride at 0.1, 0.25, 0.50, 0.75, and 1 ppm for 12, 24, 48, and 72 h. For each treatment, a control where the lead solution was substituted by distilled water was included. After treatment, the meristems were fixed with a mixture of alcohol–acetic acid (3:1) and colored according to the technique of Feulgen. Lead content was quantified by graphite furnace absorption atomic spectrometry. The lead content in the roots ranged from 3.25 to 244.72 μg/g dry weight, with a direct relation with the concentration and time of exposure. A significant negative correlation was presented (r = −0.3629; p < 0.01) among lead content and root growth increment, and a positive correlation (r = 0.7750; p < 0.01) with the induction of chromosomic aberrations. In conclusion, lead is able to induce a toxic effect in the exposed roots, correlated with its content.     

Aluminium in human breast tissue

Aluminium is omnipresent in everyday life and increased exposure is resulting in a burgeoning body burden of this non-essential metal. Personal care products are potential contributors to the body burden of aluminium and recent evidence has linked breast cancer with aluminium-based antiperspirants. We have used graphite furnace atomic absorption spectrometry (GFAAS) to measure the aluminium content in breast biopsies obtained following mastectomies. The aluminium content of breast tissue and breast tissue fat were in the range 4-437 nmol/g dry wt. and 3-192 nmol/g oil, respectively. The aluminium content of breast tissue in the outer regions (axilla and lateral) was significantly higher (P=0.033) than the inner regions (middle and medial) of the breast. Whether differences in the regional distribution of aluminium in the breast are related to the known higher incidence of tumours in the outer upper quadrant of the breast remains to be ascertained.     

Direct determination of selenium in human blood plasma and seminal plasma by graphite furnace atomic absorption spectrophotometry and clinical application

Direct determination of selenium (Se) in body fluids by graphite furnace atomic absorption spectrophotometry (GFAAS) may suffer from problems like severe background, matrix effects, preatomization losses, and spectral interferences. In this study we evaluate critically the influence on the accuracy of the direct determination of Se in blood plasma and seminal plasma by GFAAS, and propose a simple, rapid, and accurate method, suitable for routine clinical analysis. The method for blood plasma is mainly based on studies by the use of matched matrix and a Pd-Ni modifier, but for seminal plasma only a Pd modifier is required. The method developed was also applied to study the Se distribution in plasma protein fractions of patients with hepatocellular carcinoma. The Se in plasma of patients was significantly lower than that of the controls. The distribution pattern of Se in blood plasma fractions of patients was also different from that of the controls.