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

Copper is an essential trace element required as cofactor in many enzymes. However, when it is present in excess, the high oxidative potential of free Cu ions can generate reactive free radicals with subsequent cellular damage. Thus, Cu homeostasis needs to be regulated strongly to maintain cellular Cu concentrations at levels at which both toxicity and deficiency are avoided1,2,3. The liver, as the principal storage site for Cu, plays a key role in Cu homeostasis. Dietary copper is absorbed in the intestine and transported to the liver via the portal blood, where it is taken up by hepatocytes. The copper taken up by the hepatocytes is incorporated into the copper-dependent ferroxidase ceruloplasmin (Cp) and subsequently secreted in the blood for distribution throughout the body. Total body Cu levels are controlled by excretion of excess Cu via the bile1,4,5. Cu status in the body is altered in liver disease, especially when there are obstructions in bile flow and/or impaired protein synthesis6,7. Studies with radioactive Cu have shown that Cu turnover in the liver increases from 20–30 days in healthy individuals to 600–700 days for patients with biliary cirrhosis8. Hepatic Cu overload has been observed in liver diseases that arise from etiologies such as Wilson’s disease (WD)9, viral hepatitis10, cholestasis9 and excessive alcohol consumption11. WD is a genetic disorder that leads to Cu accumulation, first in the liver, but ultimately also in the brain and other tissues12, and to low or normal serum Cu concentrations compared to controls. In contrast, high or normal Cu concentrations in blood serum can be observed in cirrhosis13 and hepatocellular carcinoma (HCC) patients. End-stage liver disease (ESLD) can arise from these etiologies and can be complicated by other life-threatening complications (e.g., hepatic encephalopathy, ascites, portal hypertension and hepatorenal syndrome)14,15. An elevated risk of HCC and extra-hepatic cancers has been established in patients with cirrhosis16. As a result of the above, Cu levels in serum of ESLD patients frequently overlap with those of the reference population and there is no clear relation between the Cu concentration and the clinical course of the disease. Recently however, the potential of the Cu isotopic composition in blood serum as a diagnostic tool for diseases affecting Cu metabolism has been suggested17. Patients with Wilson’s disease18, ESLD19 and HCC20 showed a lighter serum Cu isotopic composition than did the reference population. Due to the complexity of the liver, there is no single clinical test for assessing hepatic disorders21,22. A series of tests, for the determination of what is frequently termed “liver function parameters”, is performed for the management of liver diseases. These parameters are used for i) detection of hepatic injury, e.g., aspartate aminotransferase (AST) and alanine aminotransferase (ALT), ii) assessment of hepatic biosynthetic capacity, e.g., albumin (Alb), prothrombin time (PT) and international normalized ratio (INR), and iii) assessment of hepatic metabolism, e.g., bilirubin (Bili)22. Liver transplantation (LTx) is the definitive treatment with successful outcomes for patients with ESLD, for patients with HCC development and for patients with acute liver failure (ALF)23. In this context, the time on the liver transplant waiting list, the transplant itself and the recovery period are crucial factors determining the outcome of LTx. The aim of this research was to investigate the Cu isotopic signature in blood serum of patients with ESLD pre-LTx and its evolution post-LTx to evaluate the capability of this parameter for following up the patients and for establishing the potential recurrence of liver failure. For this, high-precision Cu isotopic analysis of ~100 blood serum samples from 32 liver transplant patients was carried out via multi-collector ICP-mass spectrometry (MC-ICP-MS), after digestion of the serum and chromatographic isolation of the target element. For each patient, blood serum samples collected pre- and post-LTx were analyzed. In addition, serum samples collected at several time points post-LTx (up to >1 year post-LTx for some patients) were included.