Ultra-trace Cu isotope ratio measurements via multi-collector ICP-mass spectrometry using Ga as internal standard : an approach applicable to micro-samples

The capabilities of Cu isotope ratio measurements are often restricted by the small volumes of sample available and/or their low Cu concentration. In this work, an analytical approach was developed for performing Cu isotopic analysis via multi-collector ICP-mass spectrometry (MC-ICP-MS) at ultra-trace level using Ga as an internal standard for mass bias correction. The minimum concentration of Cu required for accurate and precise isotope ratio measurements was established to be 20 mu g L-1 with wet plasma conditions and 5 mu g L-1 with dry plasma conditions. The use of Ga as an internal standard for mass bias correction provided several advantages compared to Ni, i.e. improved internal precision on delta Cu-65 values and lower blank levels. Ga can also be used at a 4- fold lower concentration level than Ni. However, in wet plasma conditions, the signals of (ArO2H+)-Ar-36-O-16-H-1 and (ArNO+)-Ar-40-N-15-O-16 interfered with the signals of Ga-69(+) and Ga-71(+), respectively, while in dry plasma conditions, realized by the use of a desolvation unit, Ga-69(+) suffered from spectral interference from (ArN2H+)-Ar-40-N-14-H-1. These interferences were resolved by using medium mass resolution. For validation purposes, the approach was applied to commercially available blood and serum samples. The delta Cu-65 values for the samples measured at a concentration level of 5 mu g L-1 Cu and 5 mu g L-1 Ga using dry plasma conditions were in good agreement with those obtained for isotope ratio measurements at the "standard" concentration level of 200 mu g L-1 Cu and 200 mu g L-1 Ni using wet plasma conditions. In addition, the delta Cu-65 values obtained for micro-samples of serum/blood (volume of 100 mu L) were in good agreement with the corresponding ones obtained using the "standard" volume for isotopic analysis (500 mu L)

Cu isotopic signature in blood serum of liver transplant patients: a follow-up study

End-stage liver disease (ESLD) is life-threatening and liver transplantation (LTx) is the definitive treatment with good outcomes. Given the essential role of hepatocytes in Cu homeostasis, the potential of the serum Cu isotopic composition for monitoring a patient's condition post-LTx was evaluated. For this purpose, high-precision Cu isotopic analysis of blood serum of ESLD patients pre- and post-LTx was accomplished via multi-collector ICP-mass spectrometry (MC-ICP-MS). The Cu isotopic composition of the ESLD patients was fractionated in favour of the lighter isotope (by about -0.50 parts per thousand). Post-LTx, a generalized normalization of the Cu isotopic composition was observed for the patients with normal liver function, while it remained light when this condition was not reached. A strong decrease in the delta Cu-65 value a longer term post-LTx seems to indicate the recurrence of liver failure or cancer. The observed trend in favour of the heavier Cu isotopic composition post-LTx seems to be related with the restored biosynthetic capacity of the liver, the restored hepatic metabolism and/or the restored biliary secretion pathways. Thus, Cu isotopic analysis could be a valuable tool for the follow-up of liver transplant patients and for establishing the potential recurrence of liver failure

High-precision isotopic analysis of Cu in blood serum via multi-collector ICP-mass spectrometry for clinical investigation : steps towards improved robustness and higher sample throughput

High-precision isotopic analysis of Cu in blood serum via multi-collector ICP-mass spectrometry (MCICP-MS) has recently been shown valuable for diagnosis and prognosis of diseases affecting Cu metabolism. However, as a result of the low Cu concentration (#2 mg L-1) in blood serum compared to the high contents of concomitant matrix elements (e.g., similar to 3000 mg L-1 Na, similar to 1000 mg L-1 S, similar to 100 mg L-1 Ca and similar to 20 mg L-1 Mg) and the measurable effect that the matrix typically exerts on the extent of instrumental mass discrimination in MC-ICP-MS, an exhaustive sample preparation protocol is required. In this work, an alternative procedure for Cu isolation from serum prior to isotope ratio measurement by MC-ICP-MS was evaluated. This procedure involves removal of the alkali and earth alkaline metals using 1 mL of Chelex-100 chelating ion exchange resin, followed by further purification of the Cu-fraction using 250 mu L of AG MP1 anion exchange resin. The matrix elements were removed efficiently and Cu recoveries were quantitative in all cases. The alternative Cu isolation procedure showed several advantages compared to the conventional procedure, e.g., (i) a reduction in the amount of acids, cost and time required and (ii) lower blank levels. The effect of (remaining) concomitant matrix elements on the Cu isotope ratio accuracy and precision attainable was evaluated using both the standard interface (combination of standard cones and a large dry interface pump) and the high-transmission 'jet' interface (combination of 'jet' cones and a large dry interface pump). For standard solutions, the jet interface provided a superior internal precision (improved by a factor of 1.5) for the raw (uncorrected) Cu-65/Cu-63 ratio and a mitigated extent of instrumental mass discrimination. However, the influence of matrix elements on the Cu isotope ratio results was stronger with the jet interface than with the standard interface. The proposed procedure was validated using standards with known isotopic composition, a serum reference material and human serum samples. No statistical differences were obtained (at a 95% confidence interval) in the delta Cu-65 values using the proposed and the conventional (two column passes using 1 mL of AG MP1 resin) procedure. Finally, serum samples collected at different time points during a day from five volunteers were analysed in order to study diurnal variations in the serum Cu isotopic composition. No diurnal variations were established

Quantification and isotopic analysis of bulk and of exchangeable and ultrafiltrable serum copper in healthy and alcoholic cirrhosis subjects

Information on the Cu speciation in blood serum can be valuable for a better understanding of the metabolism of this essential transition metal, but Cu speciation analysis and, to an even larger extent, compound-specific high precision Cu isotopic analysis are challenging. In this work, quantification and isotopic analysis of Cu were carried out in bulk serum and in both its exchangeable + ultrafiltrable (EXCH + UF) Cu fraction and its non-exchangeable + non-ultrafiltrable (NEXCH + NUF) fraction using quadrupole and multi-collector ICP-mass spectrometry, respectively. The EXCH + UF serum Cu represents the labile Cu pool, ie. Cu loosely bound to proteins, such as albumin, alpha-2 macroglobulin and other low molecular weight compounds, while the NEXCH + NUF serum Cu contains the Cu firmly bound to ceruloplasmin (Cp). The method was evaluated using human, goat and fetal bovine serum and applied to serum samples from assumed healthy subjects and from patients with alcoholic liver cirrhosis (AC). The healthy subjects showed an isotopic composition of EXCH + UF serum Cu heavier (by on average + 0.4 parts per thousand) than that of their total serum Cu. In general, patients with AC showed higher EXCH + UF serum Cu concentrations and significantly lower delta Cu-65(EXCH) + up and delta Cu-65(serum) values than did healthy subjects. Within the AC population, delta Cu-65(EXCH+UF) values were comparable to or lower than the corresponding delta Cu-65 rum values, potentially reflecting the extent of labile Cu deregulation. As to be expected, the NEXCH + NUF serum Cu isotopic composition was similar to that of the total serum Cu, as most of the serum Cu is firmly bound to Cp

Isotopic analysis of Cu in blood serum by multi-collector ICP-mass spectrometry : a new approach for the diagnosis and prognosis of liver cirrhosis?

The isotopic composition of blood serum Cu has been investigated as a potential parameter for the diagnosis and prognosis of liver cirrhosis. Serum samples from supposedly healthy women (reference population) and from a group of female patients suffering from liver cirrhosis of different etiologies were analysed. The procedure for isolation of serum Cu and the measurement protocol for its isotopic analysis by multi-collector inductively coupled plasma-mass spectrometry (MC-ICP-MS) were evaluated. Significant differences in the isotopic composition of Cu were observed between the reference population and the patients. A wide spread in delta Cu-65 was observed within the cirrhosis population and delta Cu-65 seems to be linked to the severity of the disease. Patients with end-stage liver disease showed a significantly lighter serum Cu isotopic composition. Many clinical parameters used for the diagnosis and monitoring of liver diseases, i.e. the levels of aspartate aminotransferase, De Ritis ratio, prothrombin and international normalized ratio, albumin, bilirubin, Na and C-reactive protein, correlate well with the delta Cu-65 values, as did the ceruloplasmin level and the ceruloplasmin/Cu concentration ratio. The isotopic composition of serum Cu appears to reveal the synthetic and hepatocellular function of the liver synergistically with inflammation and fluid retention in the cohort studied. A relevant relationship was also observed between delta Cu-65 and scores of mortality risk, such as the Model for End-stage Liver Disease (MELD) and MELD-Na. Thus, the isotopic composition of serum Cu shows potential as a new approach for the prognosis of liver disease, and although further investigation is required, for evaluation of the mortality risk in end-stage liver disease and prioritization of liver transplant

Traditional fermentation of tef injera : impact on in vitro iron and zinc dialysability

Tef [Eragrostis tef (Zucc.) Trotter], an ancient cereal mainly produced in Ethiopia, is increasingly getting higher acceptance in the global market because it is gluten free and has high iron content. The aim of this study was to evaluate the in vitro dialysability of Fe and Zn in a backslop fermented gluten free flat bread known as injera. The traditional fermentation caused up to 49-66% reduction of phytic acid (PA). Molar ratios of PA:Fe and PA:Zn decreased from 14 to 1 and from 63 to 19, respectively, after 120 h of fermentation. The total soluble fractions of Fe and Zn ranged between 11 and 38% and between 11 and 29%, respectively, after 120 h of fermentation. The dialyzable Fe content of the white varieties ranged between 3 and 9% after 120 h fermentation while no effect was observed for the brown varieties. The dialyzable Zn ranged between 2 and 11%, with only a clear effect of fermentation in one white variety. Consumption of tef could be a good source of Fe and Zn, but may not provide the absolute recommended daily Fe and Zn intakes

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