Copper availability and NAFLD in epileptic patients treated with valproic acid 149 enzymes, or sequestering co-factors involved in this metabolic pathway, has been proposed as a possible mechanism for VPA-induced steatosis(4,5,8). Chronic treatment with enzyme-inducing antiep- ileptic drugs (carbamazepine, phenobarbital, or phe- nytoin) does not produce copper deﬁciency (9). However, the administration of VPA produces a signiﬁcant increase of copper biliary excretion in rats (10), and previously published results suggest that diminished copper availability is substantially prevalent among epileptic patients treated with VPA (11). In accordance with Aigner et al., a signiﬁcant proportion of non-alcoholic fatty liver disease patients should be considered copper-deﬁcient (12), and reduced copper availability may be involved in the development of non-alcoholic fatty liver (13), a hypothesis put forward at an earlier date by Geubel et al. (14). In our study, an investigation was carried out in epileptic patients treated with VPA on the possible relationship between copper availability and the serum activities of several liver enzymes, whose increase in non-alcoholic fatty liver disease is widely documented (15–22). As previously reported, copper deﬁciency was supposed in cases with a smaller ceruloplasmin-speciﬁc oxidase activity (activity per mass unit of enzyme protein) than the estimated lower limit of reference (11). Patients and methods A group of 101 epileptic out-patients (56 males and 45 females) with a mean age (±SEM) of 43.7 ± 2.5 years (range 17–84 years) and treated with VPA in monotherapy (n = 75) or polytherapy with associ- ated phenytoin, carbamazepine, and phenobarbital (n = 26) were studied. In all cases the daily drug administration was carried out in multiple doses. The blood samples were drawn before the breakfast and the morning dose of antiepileptic drugs, which had not been modiﬁed for at least 3 months prior to the study. Consequently the serum levels of VPA corre- spond to the trough steady-state concentrations (Css). None of the patients was receiving a copper dietary supplement, and pregnant women or those taking oral contraceptives were excluded. This study was carried out according to the good practice rules for investi- gation in humans of the Conselleria de Sanidade (Regional Ministry of Health) of the Xunta de Galicia, Spain. Serum copper concentration was determined by atomic absorption spectrometry (Perkin Elmer mod 5100PC, Waltham, USA) with carbon red atomiza- tion. Immunoreactive ceruloplasmin was determined in a BN ProSpec nephelometer (Siemens Health Care Diagnostics Inc., Newark, USA). The oxidase activity of ceruloplasmin was determined as previously described at 30 C using o-dianisidine dihydrochlor- ide as a substrate (23), and the ceruloplasmin- speciﬁc oxidase activity was calculated using the expression: speciﬁc oxidase activity (U/g) = 1000 oxidase activity (U/L)/immunoreactive ceruloplasmin (mg/L). Serum VPA concentrations were determined in a Dimension Xpand analyzer using reagents from Siemens Health Care Inc. Serum activities of alanine aminotransferase (ALT), aspartate aminotransferase (AST), gamma-glutamyltransferase (GGT), alkaline phosphatase (ALP),and butyrylcholinesterase (ChE), and concentrations of glucose, cholesterol, and tri- glycerides, were determined in an Advia 2400 Chem- istry System (Siemens Health Care Diagnostics Inc.). The platelet count, mean platelet volume (MPV), and platelet size distribution width (PDW) were measured in blood samples collected 2–3 hours beforehand in K3EDTA anticoagulated tubes using an Advia 2120 Hematology System (Siemens Health Care Diagnostics Inc.). High-sensitivity C-reactive protein (CRP) was determined in a BN II nephelometer (Siemens Health Care Diagnostics Inc.). The AST-to-platelet ratio index (APRI) was calculated in accordance with Wai et al. (24): APRI = (AST:URL/platelet count (109/L)) 100, where URL corresponds to the AST upper reference limits for men and women. The 4-variables liver ﬁbro- sis index (FIB-4) was calculated in accordance with Sterling et al. (25): FIB-4 = age AST/platelets (109/L) ALT1/2, where age is expressed in years and the AST and ALT activities in U/L. Statistical analysis of data was performed using the StatGraphics package, and the Kolmogorov- Smirnov test was applied to check for normality. Pearson’s correlation coefﬁcient was used when the data had a Gaussian distribution. Other- wise, Spearman’s correlation coefﬁcient was used. The results were expressed as mean ± SEM (median). Results The estimated lower reference limit for the speciﬁc oxidase activity of ceruloplasmin (2.5 percentile of the control group) was 333 U/g (11). Table I shows the results obtained for the different biochem- ical variables assayed in the patients treated with VPA in monotherapy, grouped according to their ceruloplasmin-speciﬁc oxidase activities (smaller or greater than 333 U/g). The differences between the mean (median) were not statistically signiﬁcant,