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, i.e. 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‰) than that of their total serum Cu. In general, patients with AC showed higher EXCH + UF serum Cu concentrations and significantly lower δ65CuEXCH+UF and δ65Cuserum values than did healthy subjects. Within the AC population, δ65CuEXCH+UF values were comparable to or lower than the corresponding δ65Cuserum 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.

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 µg L-1 with wet plasma conditions and 5 µg 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 δ65Cu 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 36Ar16O21H+ and 40Ar15N16O+ interfered with the signals of 69Ga+ and 71Ga+, respectively, while in dry plasma conditions, realized by the use of a desolvation unit, 69Ga+ suffered from spectral interference from 40Ar14N21H+. These interferences were resolved by using medium mass resolution. For validation purposes, the approach was applied to commercially available blood and serum samples. The δ65Cu values for the samples measured at a concentration level of 5 µg L-1 Cu and 5 µg 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 µg L-1 Cu and 200 µg L-1 Ni using wet plasma conditions. In addition, the δ65Cu values obtained for micro-samples of serum/blood (volume of 100 µL) were in good agreement with the corresponding ones obtained using the "standard" volume for isotopic analysis (500 µL).

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 120h of fermentation. The total soluble fractions of Fe and Zn ranged between 11 and 38% and between 11 and 29%, respectively, after 120h of fermentation. The dialyzable Fe content of the white varieties ranged between 3 and 9% after 120h 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.

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‰). 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 δ(65)Cu 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.

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 δ(65)Cu was observed within the cirrhosis population and δ(65)Cu 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 δ(65)Cu 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 δ(65)Cu 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 transplants.