The aim of this work was to investigate the reappearance during liver neoplasia of bile acids (BAs) species, which are unusual in healthy adults, but common in fetuses. Serum and urine samples were collected from patients with hepatocellular carcinoma (HCC; n = 27), and for comparative purposes, with liver cirrhosis (n = 49), liver metastasis (n = 19), chronic viral hepatitis (n = 11) and healthy volunteer (control group; n = 26) groups. BAs were identified and measured by GC–MS. Hypercholanaemia was found in all groups of patients. In HCC, this was characterized by a marked increase in the chenodeoxycholate/cholate ratio in both serum and urine. Although increased levels of BAs, with hydroxylations at unusual positions, and oxo-BAs were found in HCC, these were not significantly different from those observed in other groups. However, BAs with a flat structure, i.e. Δ4-unsaturated- and 5α- or allo-BAs, which were almost absent in healthy subjects, were markedly increased in the serum and urine of HCC patients. They were also detected, although in much lower amounts, in liver metastasis and liver cirrhosis, but not in viral hepatitis. Flat-BAs were better detected in urine than in serum. Urinary Δ4-unsaturated-BA output was significantly lower in patients with small tumours (< 3 cm) compared with those with higher size tumours. No correlation between flat-BA output into urine and serum alpha-fetoprotein or total BAs was found. These results suggest that Δ4- and/or allo-BAs are particularly elevated in patients with HCC, which may be a potentially useful complementary, rather than alternative, marker for early detection of liver neoplasia.

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