Bifunctionalized isotopologs as calibrants for standard-free quantitation of drug metabolites in plasma by liquid chromatography coupled with radiometry and mass spectrometry.

The reported method involves a novel workflow that eliminates the need for authentic reference standards for the quantitation of drug metabolites in biological samples using a single multi-isotopically labeled compound bearing both radio and stable isotopes. The resulting radio and stable bifunctionalized isotopolog (RADSTIL) of the parent drug is employed as a substrate for in vitro biotransformation to targeted RADSTILs of metabolites as calibrants. Inclusion of a radio label enables both radiometric and mass spectrometric detection. The addition of stable labels ensures the subsequent isotopic interference-free quantitation of unlabeled metabolites in preclinical and clinical samples. This affords a more accurate quantitation workflow compared with the current semi-quantitation method, which utilizes isotopic interfering radio isotopologs of metabolites alone as calibrants. The proof-of-concept is illustrated with (14 C,13 C2 )-acetaminophen where in vitro biotransformation produced (14 C,13 C2 )-sulfate and (14 C,13 C2 )-glucuronide calibrants. Absolute quantitation of the acetaminophen metabolites was then achieved by liquid chromatography coupled with radiometry and mass spectrometry. Quantitative data obtained by this method fell within 82-86% of the values from conventional LC-MS/MS method.

An isotope dilution liquid chromatography-tandem mass spectrometry candidate reference measurement procedure for aldosterone measurement in human plasma.

Accurate quantitation of aldosterone is clinically important in standardized testing for primary aldosteronism. The results are often variable when performed by clinical immunoassays. To standardize and ensure the accuracy of clinical systems, reference measurement procedures (RMPs) with higher metrological order are required. A simple and reliable isotope dilution LC-IDMS/MS-based measurement procedure for human plasma aldosterone has been developed. This method involved plasma spiked with a deuterium-labelled internal standard, equilibrated for 0.5 h, and extracted by liquid-liquid extraction (LLE) without derivatization. Aldosterone and its structural analogues were baseline separated with a C18-packed UHPLC column with gradient elution within 7 min. The signal intensity variability and measurement imprecision were reduced by bracketing calibration during plasma aldosterone value assignment. The limit of detection (LoD) was 19.4 pmol/L with a signal-to-noise ratio (S/N) > 3. The lowest limit of quantification (LLoQ) was 27.7 pmol/L (S/N > 10 and CV < 10.0%). LLE was performed with 1 mL of n-hexane/ethyl acetate (3:2, v/v), and the extraction recovery was determined to be 92.15 ± 3.54%. The imprecisions were ≤ 3.18% for samples at 124.8, 867.0, and 2628.5 pmol/L. The recoveries were 98.11-101.61%. The relative bias between this candidate RMP and the established RMP was 2.76-1.89%. The linearity response ranged from 27.7 to 2774.4 pmol/L with R2 = 0.999. The method performance met the requirements of RMPs (≤ 5% total CV and ≤ 3% bias). Furthermore, the developed method was applied to evaluate immunoassays through 41 patient sample comparisons. The calibration and measurement capability (CMC) of this method were also evaluated by measuring these samples. The candidate RMP can serve as an accurate reference baseline for routine methods and can be used for value assignment for reference materials. Selected ion chromatograms by LC-MS/MS using a C18 column for aldosterone and its structural analogues.

The blood copper isotopic composition is a prognostic indicator of the hepatic injury in Wilson disease.

Wilson disease (WD) is an autosomal recessive disorder of copper (Cu) metabolism. The gene responsible for WD, ATP7B, is involved in the cellular transport of Cu, and mutations in the ATP7B gene induce accumulation of Cu in the liver and ultimately in the brain. In a pilot study, the natural variations of copper stable isotope ratios (65Cu/63Cu) in the serum of WD patients have been shown to differ from that of healthy controls. In the present study, we challenged these first results by measuring the 65Cu/63Cu ratios in the blood of treated (n = 25), naïve patients (n = 11) and age matched healthy controls (n = 75). The results show that naïve patients and healthy controls exhibit undistinguishable 65Cu/63Cu ratios, implying that the Cu isotopic ratio cannot serve as a reliable diagnostic biomarker. The type of treatment (d-penicillamine vs. triethylenetetramine) does not affect the 65Cu/63Cu ratios in WD patients, which remain constant regardless of the type and duration of the treatment. In addition, the 65Cu/63Cu ratios do not vary in naïve patients after the onset of the treatment. However, the 65Cu/63Cu ratios decrease with the degree of liver fibrosis and the gradient of the phenotypic presentation, i.e. presymptomatic, hepatic and neurologic. To get insights into the mechanisms at work, we study the effects of the progress of the WD on the organism by measuring the Cu concentrations and the 65Cu/63Cu ratios in the liver, feces and plasma of 12 and 45 week old Atp7b-/- mice. The evolution of the 65Cu/63Cu ratios is marked by a decrease in all tissues. The results show that 63Cu accumulates in the liver preferentially to 65Cu due to the preferential cellular entry of 63Cu and the impairment of the 63Cu exit by ceruloplasmin. The hepatic accumulation of monovalent 63Cu+ is likely to fuel the production of free radicals, which is potentially an explanation of the pathogenicity of WD. Altogether, the results suggest that the blood 65Cu/63Cu ratio recapitulates WD progression and is a potential prognostic biomarker of WD.

Pharmacokinetics of 10B-p-boronophenylalanine (BPA) in the blood and tumors in human patients: A critical review with special reference to tumor-to-blood (T/B) ratios using resected tumor samples.

We reviewed 10B concentration kinetics in the blood and tumors in human patients administered with BPA. The 10B concentration in the blood peaked at the end of intravenous infusion of BPA, followed by a biphasic-decreasing curve with half-lives for the first and second components of the curve being 0.7-3.7 and 7.2-12.0 h, respectively. The mean tumor-to-blood (T/B) ratio obtained from resected tumor samples was 3.40 ± 0.83 for melanoma and the ratio ranged from 1.4 to 4.7 for glioblastoma.

Lighter serum copper isotopic composition in patients with early non-alcoholic fatty liver disease.

OBJECTIVE: The occurrence of non-alcoholic fatty liver disease (NAFLD) is globally increasing. To challenge the current incidence of NAFLD, non-invasive markers that could identify patients at risk or monitor disease progression are an important need. Copper intake and organ copper concentrations have earlier been linked to NAFLD progression, but serum copper does not adequately represent the disease state. Cu atoms occur under the form of two stable isotopes, 63Cu and 65Cu, and the ratio of both (expressed as δ65Cu, in  ‰) in blood serum has been shown to be altered in chronic liver disease. To assess whether the Cu isotope ratio might predict disease occurrence and progression of NAFLD, the serum Cu isotopic composition of patients with different stages of NAFLD was determined. RESULTS: Our results showed that serum δ65Cu values were lower in NAFLD patients, already at the level of simple steatosis, and remained stable during further disease progression. ROC analysis shows an almost perfect diagnostic ability of serum δ65Cu values for NAFLD, but no discrimination between different severity degrees could be made. Therefore, the serum Cu isotopic composition might show potential for early diagnosis of NAFLD patients.

Metabolic Framework for the Improvement of Autism Spectrum Disorders by a Modified Ketogenic Diet: A Pilot Study.

The ketogenic diet (KD) can improve the core features of autism spectrum disorders (ASD) in some children, but the effects on the overall metabolism remain unclear. This pilot study investigated the behavioral parameters in relation to blood metabolites and trace elements in a cohort of 10 typically developed controls (TC) and 17 children with ASD at baseline and following 3 months of treatment with a modified KD regimen. A nontargeted, multiplatform metabolomic approach was employed, including gas chromatography-mass spectrometry, 1H nuclear magnetic resonance spectroscopy, and inductively coupled plasma-mass spectrometry. The associations among plasma metabolites, trace elements, and behavior scores were investigated. Employing a combination of metabolomic platforms, 118 named metabolites and 73 trace elements were assessed. Relative to TC, a combination of glutamate, galactonate, and glycerol discriminated ASD with 88% accuracy. ASD had higher concentrations of galactose intermediates, gut microbe-derived trimethylamine N-oxide and N-acetylserotonin, and lower concentrations of 3-hydroxybutyrate and selenium at baseline. Following 3 months of KD intervention, the levels of circulating ketones and acetylcarnitine were increased. KD restored lower selenium levels in ASD to that of controls, and correlation analysis identified a novel negative correlation between the changes in selenium and behavior scores. Based on the different behavior responses to KD, we found that high responders had greater concentrations of 3-hydroxybutyrate and ornithine, with lower galactose. These findings enhance our current understanding of the metabolic derangements present in ASD and may be of utility in predicting favorable responses to KD intervention.

High-precision isotopic analysis of serum and whole blood Cu, Fe and Zn to assess possible homeostasis alterations due to bariatric surgery.

Bariatric surgery is an effective procedure to achieve weight loss in obese patients. However, homeostasis of essential metals may be disrupted as the main absorption site is bypassed. In this study, we determined Cu, Fe and Zn isotopic compositions in paired serum and whole blood samples of patients who underwent Roux-en-Y gastric bypass (RYGB) surgery for evaluation of longitudinal changes and their potential relation to mineral element concentrations and relevant clinical parameters used for monitoring the patient's condition. Samples from eight patients were collected pre-surgery and at 3, 6 and 12 months post-surgery. Multi-collector inductively coupled plasma-mass spectrometry (MC-ICP-MS) was used for high-precision isotope ratio measurements. Alterations in metal homeostasis related to bariatric surgery were reflected in the serum and whole blood Cu, Fe and Zn isotopic compositions. The serum and whole blood Cu became isotopically lighter (lower δ65Cu values) after bariatric surgery, reaching statistical significance at 6 months post-surgery (p < 0.05). The difference between the serum and the whole blood Zn isotopic composition increased after surgery, reaching significance from 6 months post-surgery onwards (p < 0.05). Those changes in Cu, Fe and Zn isotopic compositions were not accompanied by similar changes in their respective concentrations, making isotopic analysis more sensitive to physiological changes than elemental content. Furthermore, the Zn isotopic composition correlates with blood glycaemic and lipid parameters, while the Fe isotopic composition correlates with glycaemic parameters. Graphical Abstract.

BNCT induced immunomodulatory effects contribute to mammary tumor inhibition.

In the United States, breast cancer is one of the most common and the second leading cause of cancer-related death in women. Treatment modalities for mammary tumor are surgical removal of the tumor tissue followed by either chemotherapy or radiotherapy or both. Radiation therapy is a whole body irradiation regimen that suppresses the immune system leaving hosts susceptible to infection or secondary tumors. Boron neutron capture therapy (BNCT) in that regard is more selective, the cells that are mostly affected are those that are loaded with 109 or more 10B atoms. Previously, we have described that liposomal encapsulation of boron-rich compounds such as TAC and MAC deliver a high payload to the tumor tissue when injected intravenously. Here we report that liposome-mediated boron delivery to the tumor is inversely proportional to the size of the murine mammary (EMT-6) tumors. The plausible reason for the inverse ratio of boron and EMT-6 tumor size is the necrosis in these tumors, which is more prominent in the large tumors. The large tumors also have receding blood vessels contributing further to poor boron delivery to these tumors. We next report that the presence of boron in blood is essential for the effects of BNCT on EMT-6 tumor inhibition as direct injection of boron-rich liposomes did not provide any added advantage in inhibition of EMT-6 tumor in BALB/c mice following irradiation despite having a significantly higher amount of boron in the tumor tissue. BNCT reaction in PBMCs resulted in the modification of these cells to anti-tumor phenotype. In this study, we report the immunomodulatory effects of BNCT when boron-rich compounds are delivered systemically.

[Elemental and isotopic determination of lead in whole blood by ISIS-ICP-MS].

Objective: To establish a method for determination of lead and istope ratios in the blood by ISIS-ICP-MS. Methods: After wet digestion, the blood sample was on-line addition of thallium as internal standard and analyzed by ISIS-ICP-MS. Results: The limit of detection was 0.03 µg/L and the lower limit of quantification was 0.08 µg/L. The detection concentration was 0.45 µg/L and the minimum quantitative concentration was 1.49 µg/L. The relative standard deviations (RSD) were 0.3%~1.7%. The recovery was between 91.0% and 103.4%. The precision of the major lead isotope ratios was better than 0.3%. The calibrated isotope ratios of the standard liquid are close to the certificate. Conclusion: The method has a low detection limit, good precision and high accuracy, it is feasible for determination of lead concentration and isotope ratios in the bloune.

Serum and brain natural copper stable isotopes in a mouse model of Alzheimer's disease.

Alzheimer's disease is associated with the production of Cu rich aß fibrils. Because monitoring the changes in Cu level of organs has been proposed to follow the evolution of the disease, we analyzed the copper isotopic composition of serum and brain of APPswe/PSEN1dE9 transgenic mice, a model of Alzheimer's disease, and wild-type (WT) controls. Serum composition of 3, 6, 9 and 12-month-old mice, as well as the composition of 9 brains of 12-month-old mice are reported. In WT mice, brains were ~1‰ isotopically heavier than serum, and the Cu isotopic composition of the serum was isotopically different between males and females. We propose that this effect of sex on the Cu isotopic budget of the serum may be related to a difference of Cu speciation and relative abundance of Cu carriers. Brains of APPswe/PSEN1dE9 mice were slightly lighter than brains of WT mice, while not statistically significant. This trend may reflect an increase of Cu(I) associated with the formation of Aß fibrils. The Cu isotopic composition of the brains and serum were correlated, implying copper transport between these two reservoirs, in particular a transfer of Cu(I) from the brain to the serum. Altogether, these data suggest that Cu stable isotopic composition of body fluid may have the potential to be used as detection tools for the formation of Aß fibrils in the brain, but further work has to be done.

A new anion exchange purification method for Cu stable isotopes in blood samples.

The isotopic composition of iron, zinc, copper, and cadmium (δ56Fe, δ66Zn, δ65Cu, and δ114Cd) are novel and promising tools to study the metabolism and homeostasis of trace metals in the human body. Serum δ65Cu has been proposed as a potential tool for diagnosis of cancer in liquid biopsy, and other metals may have similar utility. However, accurate analysis of trace metal isotopes is challenging because of the difficulties in purifying the metals from biological samples. Here we developed a simple and rapid method for sequential purification of Cu, Fe, Zn, and Cd from a single blood plasma sample. By using a combination of 11 M acetic acid and 4 M HCl in the first steps of column chemistry on AG-MP1 resin, we dramatically improve the separation of Cu from matrix elements compared to previous methods which use concentrated HCl alone. Our new method achieves full recovery of Cu, Fe, Zn, and Cd to prevent column-induced isotope fractionation effects, and effectively separates analytes from the matrix in order to reduce polyatomic interferences during isotope analysis. Our methods were verified by the analysis of isotope standards, a whole blood reference material, and a preliminary sample set including five plasma samples from healthy individuals and five plasma samples from cancer patients. This new method simplifies preparation of blood samples for metal isotope analysis, accelerating multi-isotope approaches to medical studies and contributing to our understanding of the cycling of Fe, Zn, Cu, and Cd in the human body. Graphical abstract ᅟ.

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).

Simultaneous measurement of total estradiol and testosterone in human serum by isotope dilution liquid chromatography tandem mass spectrometry.

Reliable measurement of total testosterone and estradiol is critical for their use as biomarkers of hormone-related disorders in patient care and translational research. We developed and validated a mass spectrometry method to simultaneously quantify these analytes in human serum without chemical derivatization. Serum is equilibrated with isotopic internal standards and treated with acidic buffer to release hormones from their binding proteins. Lipids are isolated and polar impurities are removed by two serial liquid-liquid extraction steps. Total testosterone and estradiol are measured using liquid chromatography tandem mass spectrometry (LC-MS/MS) in combination of positive and negative electrospray ionization modes. The method shows broad analytical measurement range for both testosterone 0.03-48.5 nM (0.75-1400 ng/dL) and estradiol 11.0-5138 pM (2.99-1400 pg/mL) and excellent agreement with certified reference materials (mean bias less than 2.1% to SRM 971, BCR 576, 577, and 578) and a high order reference method (mean bias 1.25% for testosterone and -0.84% for estradiol). The high accuracy of the method was monitored and certified by CDC Hormone Standardization (HoSt) Program for 2 years with mean bias -0.7% (95% CI -1.6% to 0.2%) for testosterone and 0.1% (95% CI -2.2% to 2.3%) for estradiol. The method precision over a 2-year period for quality control pools at low, medium, and high concentrations was 2.7-2.9% for testosterone and 3.3-5.3% for estradiol. With the consistently excellent accuracy and precision, this method is readily applicable for high-throughput clinical and epidemiological studies.

Simultaneous quantification of busulfan, clofarabine and F-ARA-A using isotope labelled standards and standard addition in plasma by LC-MS/MS for exposure monitoring in hematopoietic cell transplantation conditioning.

In allogeneic hematopoietic cell transplantation (HCT) it has been shown that over- or underexposure to conditioning agents have an impact on patient outcomes. Conditioning regimens combining busulfan (Bu) and fludarabine (Flu) with or without clofarabine (Clo) are gaining interest worldwide in HCT. To evaluate and possibly adjust full conditioning exposure a simultaneous analysis of Bu, F-ARA-A (active metabolite of Flu) and Clo in one analytical run would be of great interest. However, this is a chromatographical challenge due to the large structural differences of Bu compared to F-ARA-A and Clo. Furthermore, for the bioanalysis of drugs it is common to use stable isotope labelled standards (SILS). However, when SILS are unavailable (in case of Clo and F-ARA-A) or very expensive, standard addition may serve as an alternative to correct for recovery and matrix effects. This study describes a fast analytical method for the simultaneous analysing of Bu, Clo and F-ARA-A with liquid chromatography-tandem mass spectrometry (LC-MS/MS) including standard addition methodology using 604 spiked samples. First, the analytical method was validated in accordance with European Medicines Agency guidelines. The lower limits of quantification (LLOQ) were for Bu 10µg/L and for Clo and F-ARA-A 1µg/L, respectively. Variation coefficients of LLOQ were within 20% and for low medium and high controls were all within 15%. Comparison of Bu, Clo and F-ARA-A standard addition results correspond with those obtained with calibration standards in calf serum. In addition for Bu, results obtained by this study were compared with historical data analysed within TDM. In conclusion, an efficient method for the simultaneous quantification of Bu, Clo and F-ARA-A in plasma was developed. In addition, a robust and cost-effective method to correct for matrix interference by standard addition was established.

Increasing the linear dynamic range in LC-MS: is it valid to use a less abundant isotopologue?

Liquid chromatography-mass spectrometry (LC-MS) has quickly become the analytical method of choice in forensic toxicology laboratories due to its ability to detect a very wide range of compounds in a single analysis. One of the major limitations of LC-MS however, is a relatively limited linear dynamic range for quantitation. A new approach to combating this problem is to use the [+1 M + H]+ isotope mass peak for quantitation, thereby reducing saturation at the detector and extending the linear range. This is particularly useful in full-scan applications, such as quadrupole-time-of-flight (QTOF) mass spectrometry, where the isotopic mass peaks are acquired as a matter of course. Due to the variation in abundance of naturally occurring isotopes for common elements, especially 13 C, this technique has the potential to lead to additional quantitative error. Through a review of published isotope ratio mass spectrometry data, we have assessed this potential for error and found that it is likely to be less than 2% and unlikely to be more than 4%, although this may not apply to compounds containing high numbers of nitrogen or sulphur atoms. This additional potential error must be considered before using this technique as it may not be appropriate for all applications. We have deemed it fit for purpose for our application and demonstrate the applicability of this technique to a quantitative LC-TOF method. © 2017 Commonwealth of Australia. Drug Testing and Analysis © 2017 John Wiley & Sons, Ltd.

Optimization of an acidic digestion method for the determination of total Pb concentration and its isotope ratios in human blood using ICP-QMS.

Adverse effects of lead (Pb) on human health are observed even at levels below 5 µg/dL, affecting principally the children population and suggesting that there is not a safe exposure level. The determination of Pb isotopic ratios (LIRs) in biological and environmental samples, is an appropriate tool to track and control the exposure sources, because LIRs constitutes the pollutant's isotopic signature and hence can be used to identify sources of Pb emission. This study proposes the optimization of a method in order to significantly reduce the biological samples' matrix interferences, and improves precision and accuracy in the measurements of LIRs. Four total blood digestion methods were evaluated and the results were subjected to statistical methods (ANOVA) determining the combination of HNO3:H2O2 (2:1 v v-1)/g from a sample on a hot plate as the best of them. For the method's validation, detection and quantification limits, linearity range, intermediate precision and recovery were evaluated. The total Pb (PbT) and LIRs were performed by ICP-QMS, defining the optimal value of detector dead time (DT), and correcting mass bias and instrumental drift for this matrix. LIRs based on 206Pb, 207Pb and 208Pb were determined at high precision (%RSD 0.03-0.49%), than those involving 204Pb (%RSD > 0.8). The optimized methodology can be used to identify pollution sources in blood and environmental samples using LIRs (206Pb/207Pb, 207Pb/208Pb, 208Pb/206Pb, etc.) in a trustworthy and simple way, with accurate results.

A Retinol Isotope Dilution Equation Predicts Both Group and Individual Total Body Vitamin A Stores in Adults Based on Data from an Early Postdosing Blood Sample.

BACKGROUND: Retinol isotope dilution (RID) is used to determine vitamin A total body stores (TBS) after an oral dose of a vitamin A stable isotope. The generally accepted prediction equation proposed by Olson's group in 1989 (Furr et al. Am J Clin Nutr 1989;49:713-6) includes factors related to dose absorption and retention, isotope equilibration in plasma compared with stores, catabolism during the mixing period, and the optimal time for measuring plasma isotope enrichment. OBJECTIVES: The objectives were 1) to develop a modified RID equation and identify an earlier sampling time for predicting TBS and 2) to improve prediction in individuals as well as groups. METHODS: To develop a modified RID equation, we used results of model-based compartmental analysis [the Simulation, Analysis and Modeling software (WinSAAM version 3.0.8; http://www.WinSAAM.org)] of plasma [13C10]retinol kinetic data from 32 previously studied, healthy young adults of European ancestry who had moderate vitamin A intakes and who ingested 2.95 µmol [13C10]retinyl acetate. RESULTS: We examined the time dependence of factors in the prediction equation related to absorption/retention (Fa) and isotope equilibration (S) and determined that 4 or 5 d postdosing was the optimal sampling time. TBS calculated by the equation TBS = Fa x S x (1/SAp), where SAp is plasma retinol specific activity (fraction of dose/µmol), were highly correlated with model-predicted TBS (r = 0.95 and 0.96 for 4 and 5 d, respectively; P < 0.001); predictions for individuals were also highly correlated (Rs = 0.94 and 0.94; P < 0.001). CONCLUSION: The equation TBS ≈ 0.5 × (1/SAp) accurately predicted vitamin A TBS in this group of 32 healthy young adults and its individual members with the use of data from 1 blood sample taken 4 d after isotope administration.

Medical applications of Cu, Zn, and S isotope effects.

This review examines recent applications of stable copper, zinc and sulfur isotopes to medical cases and notably cancer. The distribution of the natural stable isotopes of a particular element among coexisting molecular species varies as a function of the bond strength, the ionic charge, and the coordination, and it also changes with kinetics. Ab initio calculations show that compounds in which a metal binds to oxygen- (sulfate, phosphate, lactate) and nitrogen-bearing moieties (histidine) favor heavy isotopes, whereas bonds with sulfur (cysteine, methionine) favor light isotopes. Oxidized cations (e.g., Cu(ii)) and low coordination numbers are expected to favor heavy isotopes relative to their reduced counterparts (Cu(i)) and high coordination numbers. Here we discuss the first observations of Cu, Zn, and S isotopic variations, three elements closely related along multiple biological pathways, with emphasis on serum samples of healthy volunteers and of cancer patients. It was found that heavy isotopes of Zn and to an even greater extent Cu are enriched in erythrocytes relative to serum, while the difference is small for sulfur. Isotopic variations related to age and sex are relatively small. The 65Cu/63Cu ratio in the serum of patients with colon, breast, and liver cancer is conspicuously low relative to healthy subjects. The characteristic time over which Cu isotopes may change with disease progression (a few weeks) is consistent with both the turnover time of the element and albumin half-life. A parallel effect on sulfur isotopes is detected in a few un-medicated patients. Copper in liver tumor tissue is isotopically heavy. In contrast, Zn in breast cancer tumors is isotopically lighter than in healthy breast tissue. 66Zn/64Zn is very similar in the serum of cancer patients and in controls. Possible reasons for Cu isotope variations may be related to the cytosolic storage of Cu lactate (Warburg effect), release of intracellular copper from cysteine clusters (metallothionein), or the hepatocellular and biosynthetic dysfunction of the liver. We suggest that Cu isotope metallomics will help evaluate the homeostasis of this element during patient treatment, notably by chelates and blockers of Cu trafficking, and understand the many biochemical pathways in which this element is essential.

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.

Reliability of stable Pb isotopes to identify Pb sources and verifying biological fractionation of Pb isotopes in goats and chickens.

Stable Pb isotope ratios (Pb-IRs) have been recognized as an efficient tool for identifying sources. This study carried out at Kabwe mining area, Zambia, to elucidate the presence or absence of Pb isotope fractionation in goat and chicken, to evaluate the reliability of identifying Pb pollution sources via analysis of Pb-IRs, and to assess whether a threshold for blood Pb levels (Pb-B) for biological fractionation was present. The variation of Pb-IRs in goat decreased with an increase in Pb-B and were fixed at certain values close to those of the dominant source of Pb exposure at Pb-B > 5 µg/dL. However, chickens did not show a clear relationship for Pb-IRs against Pb-B, or a fractionation threshold. Given these, the biological fractionation of Pb isotopes should not occur in chickens but in goats, and the threshold for triggering biological fractionation is at around 5 µg/dL of Pb-B in goats.