Measuring Cation Transport by Na,K- and H,K-ATPase in Xenopus Oocytes by Atomic Absorption Spectrophotometry: An Alternative to Radioisotope Assays

Whereas cation transport by the electrogenic membrane transporter Na⁺,K⁺-ATPase can be measured by electrophysiology, the electroneutrally operating gastric H⁺,K⁺-ATPase is more difficult to investigate. Many transport assays utilize radioisotopes to achieve a sufficient signal-to-noise ratio, however, the necessary security measures impose severe restrictions regarding human exposure or assay design. Furthermore, ion transport across cell membranes is critically influenced by the membrane potential, which is not straightforwardly controlled in cell culture or in proteoliposome preparations. Here, we make use of the outstanding sensitivity of atomic absorption spectrophotometry (AAS) towards trace amounts of chemical elements to measure Rb⁺ or Li⁺ transport by Na⁺,K⁺- or gastric H⁺,K⁺-ATPase in single cells. Using Xenopus oocytes as expression system, we determine the amount of Rb⁺ (Li⁺) transported into the cells by measuring samples of single-oocyte homogenates in an AAS device equipped with a transversely heated graphite atomizer (THGA) furnace, which is loaded from an autosampler. Since the background of unspecific Rb⁺ uptake into control oocytes or during application of ATPase-specific inhibitors is very small, it is possible to implement complex kinetic assay schemes involving a large number of experimental conditions simultaneously, or to compare the transport capacity and kinetics of site-specifically mutated transporters with high precision. Furthermore, since cation uptake is determined on single cells, the flux experiments can be carried out in combination with two-electrode voltage-clamping (TEVC) to achieve accurate control of the membrane potential and current. This allowed e.g. to quantitatively determine the 3Na⁺/2K⁺ transport stoichiometry of the Na⁺,K⁺-ATPase and enabled for the first time to investigate the voltage dependence of cation transport by the electroneutrally operating gastric H⁺,K⁺-ATPase. In principle, the assay is not limited to K⁺-transporting membrane proteins, but it may work equally well to address the activity of heavy or transition metal transporters, or uptake of chemical elements by endocytotic processes.     

Screening of Trace Metals in the Plasma of Breast Cancer Patients in Comparison with a Healthy Population

The plasmas of breast cancer patients and healthy donors were analyzed for selected trace metals by a flame atomic absorption spectrophotometric method. In the plasma of breast cancer patients, mean concentrations of macronutrients/essential metals, Na, K, Ca, Mg, Fe, and Zn were 3584, 197.0, 30.80, 6.740, 5.266, and 6.170 ppm, respectively, while the mean metal levels in the plasma of healthy donors were 3908, 151.0, 72.40, 17.70, 6.613, and 2.461 ppm, respectively. Average concentrations of Cd, Cr, Cu, Mn, Ni, Pb, Sb, Sr, and Zn were noted to be significantly higher in the plasma of breast cancer patients compared with healthy donors. Very strong mutual correlations (r > 0.70) in the plasma of breast cancer patients were observed between Cd–Pb, Cr–Li, Li–K, Li–Cd, K–Cr, Li–Pb, Cr–Co, Cu–Ni, Co–K, Cd–K, and K–Pb, whereas, Al–Cr, Ca–Zn, Cd–Sb, Cd–Zn, Ca–Mg, Fe–Zn, and Na–Mn exhibited strong relationships (r > 0.60) in the plasma of healthy donors. The cluster analysis revealed considerably different apportionment of trace metals in the two groups of donors. The average metal concentrations of different age groups of the two donor categories were also evaluated, which showed the build-up of Al, Cd, Co, Cr, Mn, Li, Pb, Sb, and Zn in the plasma of breast cancer patients. The role of some trace metals in carcinogenesis is also discussed. The study indicated appreciably different patterns of metal distribution and correlation in the plasma of breast cancer patients in comparison with the healthy population.     

Uptake of zinc and copper by halophilic bacteria isolated from the dead sea shore,Jordan

Ten Gram-positive and gram-negative bacterial cultures were recovered from nine water, mud, and soil samples collected from the Dead Sea shore at Suwaymah. All bacterial cultures were able to grow at 10% NaCl and at 45°C. They were able to grow in nutrient media supplemented with 1250 ppm of Zn. Most of them, except cultures 2 and 8, were able to grow in nutrient medium supplemented with 1000 ppm of Cu. After 2 wk of incubation of these 10 cultures at different concentrations (5, 25, 100, and 500 ppm), stock solutions of both Zn and Cu elements, the maximum absorption using atomic absorption spectrometry for Zn was achieved by culture 7 at 11.2%, 1.0%, 38.4%, and 84.54%, respectively, from the previous stock solutions, whereas the maximum absorption of the same concentration of Cu was achieved by culture 3 at 6.2%, 55.56%, 85.66%, and 90.82%, respectively, of the different concentrations. After 3 wk of incubation, the estimated absorption for Zn was achieved by cultures 2, 9, and 10 at 19.2%, 16.68%, 42.92%, and 76.5%, 18.2%, 21.56%, 32.22%, and 77.43%, and 20.8%, 23.52%, 32.22%, and 82.84% of the previous stocks. The maximum absorption of the same concentration of Cu was achieved by culture 3 at 32.6%, 49.88%, 90.44%, and 91.86%, respectively. The accumulation of the absorbed metals was found to be maximum in the protoplast of all cultures. The accumulation at the cell wall was maximum for cultures 2 and 6 for Zn and Cu, respectively, and between the cell wall and the plasma membrane, it was maximum for cultures 2 and 8 for Zn and Cu, respectively.     

Characterization and Distribution of the Selected Metals in the Scalp Hair of Cancer Patients in Comparison with Normal Donors

Eighteen metals were estimated in the scalp hair samples from cancer patients (n = 111) and normal donors (n = 113). Nitric acid–perchloric acid wet digestion procedure was used for the quantification of the selected metals by flame atomic absorption spectrophotometry. In the scalp hair of cancer patients, highest average levels were found for Ca (861 μg/g), followed by Na (672 μg/g), Zn (411 μg/g), Mg (348 μg/g), Fe (154 μg/g), Sr (129 μg/g), and K (116 μg/g), whereas in comparison, the dominant metals in the scalp hair of normal donors were Ca (568 μg/g), Zn (177 μg/g), Mg (154 μg/g), Fe (110 μg/g), and Na (103 μg/g). The concentrations of Ca, Cd, Co, Cr, Fe, K, Mg, Mn, Na, Ni, Pb, Sb, Sr, and Zn were notably higher in the hair of cancer patients as compared with normal donors, which may lead to a number of physiological disorders. Strong positive correlations were found in Mn–Pb (0.83), Cd–Cr (0.82), Cd–Li (0.57), Fe–Pb (0.56), and Fe–Mn (0.55) in the hair of cancer patients whereas Na–Cd, Li–Cr, Li–Co, Co–Cd, Li–Cd, Na–Co, Na–Li, Ca–Mg and Na–Cr exhibited strong relationships (r > 0.50) in the hair of normal donors. Principal Component Analysis (PCA) of the data revealed seven PCs, both for cancer patients and normal donors, but with significantly different loadings. Cluster Analysis (CA) was also used to support the PCA results. The study evidenced significantly different pattern of metal distribution in the hair of cancer patients in comparison with normal donors. The role of trace metals in carcinogenesis was also discussed.     

Biosurfactant of marine origin exhibiting heavy metal remediation properties

The present study was aimed at elucidating the role of biosurfactant product isolated from a marine bacterium in removing heavy metals from heavy metal containing solutions. In this study, metal removal was biosurfactant-mediated. Efficiency of metal removal depended on the concentration of the metal as well as that of the biosurfactant. At a concentration 5×, the critical micelle concentration (CMC), almost complete removal of 100 ppm of lead and cadmium occurred. Atomic absorption spectroscopy (AAS) studies also showed metal removal at a concentration less than the CMC in contrast to earlier findings that only micelles are involved in metal removal. Fourier transform infrared spectroscopy (FTIR) and transmission electron microscopy (TEM) equipped with energy dispersive X-ray spectroscopy (EDS) further substantiated these findings.     

Total and speciation analyses of heavy metals in the sand fraction of Nigerian oil sands for human and ecological risk assessment

Total metal analysis and metal speciation of sand fraction of Nigerian oil sands were done to extract and partition heavy metals into six operationally defined fractions in order to assess environmental and health implications of the oil sand development. Soxhlet extraction of bitumen from the oil sand was done using toluene. Traces of water and extracting solvent were removed at 70°C from the sand fraction using oven. Elemental analysis was performed by Atomic Absorption Spectrophotometry. The heavy metals except Cu have low Pollution index values. Negative geo-accumulation index was obtained for the metals except Cu, indicating that the oil sands were uncontaminated with the metals. Strong and significant positive correlations existed between Fe/Pb, Zn/Cu, Cd/Pb, Ni/Cd, Ni/Pb, Cd/Fe, Ni/Fe and Cr/Zn, while strong and significant negative correlations existed between Mn/Pb and As/Mn, indicating common sources or chemical similarities and vice versa. T-test results indicated significant differences between the concentrations of the metals. Cross-plot analysis showed strong positive correlation between the sand fraction and Nigerian bitumen. Speciation analysis indicated highest and lowest indices of metal mobility for Zn and Pb, respectively. This study concluded that the sand fraction may not pose any environmental risks from elemental point of view.     

The aluminium content of breast tissue taken from women with breast cancer

The aetiology of breast cancer is multifactorial. While there are known genetic predispositions to the disease it is probable that environmental factors are also involved. Recent research has demonstrated a regionally specific distribution of aluminium in breast tissue mastectomies while other work has suggested mechanisms whereby breast tissue aluminium might contribute towards the aetiology of breast cancer. We have looked to develop microwave digestion combined with a new form of graphite furnace atomic absorption spectrometry as a precise, accurate and reproducible method for the measurement of aluminium in breast tissue biopsies. We have used this method to test the thesis that there is a regional distribution of aluminium across the breast in women with breast cancer. Microwave digestion of whole breast tissue samples resulted in clear homogenous digests perfectly suitable for the determination of aluminium by graphite furnace atomic absorption spectrometry. The instrument detection limit for the method was 0.48 μg/L. Method blanks were used to estimate background levels of contamination of 14.80 μg/L. The mean concentration of aluminium across all tissues was 0.39 μg Al/g tissue dry wt. There were no statistically significant regionally specific differences in the content of aluminium. We have developed a robust method for the precise and accurate measurement of aluminium in human breast tissue. There are very few such data currently available in the scientific literature and they will add substantially to our understanding of any putative role of aluminium in breast cancer. While we did not observe any statistically significant differences in aluminium content across the breast it has to be emphasised that herein we measured whole breast tissue and not defatted tissue where such a distribution was previously noted. We are very confident that the method developed herein could now be used to provide accurate and reproducible data on the aluminium content in defatted tissue and oil from such tissues and thereby contribute towards our knowledge on aluminium and any role in breast cancer.     

Ulltrastructural and trace metal studies on radiographers’ hair and nails

Scalp hair and fingernail samples of 42 medical radiographers and 42 nonradiographers (control) with matching age groups and food habits were collected for this study. Trace metal estimation by atomic absorption spectrometry (AAS) has indicated a significant increase (P < 0.001) in Zn, Cu, and Cd contents in the radiographers’ hair and nails. Scanning electron microscopy (SEM) reveled structural changes in the hair and nails of radiographers. Significant alterations in the Zn and Cd contents along with extensive structural damage in the hair and nails probably indicate that low-dose Χ-radiation imposes stress on these radiation workers.