Valproic acid increases biliary copper excretion in the rat

doi:10.1016/S0920-1211(02)00155-9

Epilepsy Research

Volume 51, Issue 3, 1 October 2002, Pages 279-285

https://doi.org/10.1016/S0920-1211(02)00155-9 Get rights and content

Abstract

The effect of valproic acid (VPA) on the copper absorption and disposition in rat small intestine was investigated using an in situ recirculating perfusion method. Following addition of VPA (20 mg) to the perfusion of 30 ml of 0.9% sodium chloride solution (2 μg/ml copper as CuSO₄) there were no significant differences in copper decline during the perfusion. The absorption rate constant of copper (k_a) which was estimated from the copper decline in the perfusion was unchanged without and with VPA (0.19±0.02 vs. 0.17±0.03 1/h). These results indicate that VPA does not have an effect on copper absorption from the intestine. We also assessed biliary copper excretion by measuring bile flow and biliary copper concentrations. Addition of VPA markedly increased bile flow by 47% for the first hour of bile collection and 91% for the second hour and the biliary copper excretion closely followed the increase in bile flow (without VPA: 0.93±0.15 vs. with VPA: 1.44±0.21 mg for the first and without VPA: 0.98±0.13 mg vs. with VPA: 1.98±0.22 mg for the second hour of bile collection). The total mean value for the biliary copper excretion was increased by 79%. The serum VPA concentration after the perfusion was 31.1±3.2 μg/ml. This high excretion of copper induced by VPA into the bile may upset the homeostatic balance of copper and cause the abnormalities of serum copper concentration. Based on the present studies, we should pay attention to copper levels in patients with VPA treatment.

Introduction

Copper is an essential metal and is an integral cofactor for numerous enzymes, whereas copper accumulation leads to tissue damage. The regulatory mechanisms of copper concentration, including absorption, distribution, metabolism and excretion, are therefore important for protection against deficiency and accumulation. Several diseases and conditions influence copper homeostasis. Menkes' syndrome and Wilson's disease are well-known human inherited disorders caused by genetic defects in copper metabolism in which copper levels in the body were found to increase (Bull and Cox, 1994). Some diet and drugs have been shown to affect copper absorption (Cousins, 1985, Wapnir and Sia, 1996). One of these is antiepileptic drugs. Valproic acid (VPA) is widely used as a drug of the first choice in the treatment of epileptic patients with generalized and partial seizures. In patients administered VPA, abnormalities in the serum copper concentration have been reported and there is controversy over the concentration of copper in the serum of epileptic patients treated with VPA. Serum copper concentrations have been found to be decreased (Fichsel et al., 1983, Kaji et al., 1992), unchanged (Hurd et al., 1984, Verrotti et al., 2002), or increased (Kuzuya et al., 1993) in the presence of VPA.

During phenytoin treatment, a change in the blood concentration of zinc has been reported. Concerning these changes induced by antiepileptic drugs, Weismann et al. (1978) have proposed that the formation of chelate between zinc and phenytoin causes an increase in the absorption of zinc from the gastrointestinal tract in rats. The absorption of dietary copper from the gastrointestinal tract may be affected by treatment of VPA.

The biliary copper excretion is important for homeostatic regulation, because biliary excretion is quantitatively the major excretory route (Dijkstra et al., 1996). In animal experiments, VPA has an important pharmacological effect on copper homeostasis, since treatment with VPA increases bile flow (Dickinson et al., 1979, Watkins and Klaassen, 1981, Liu et al., 1992). From these points of view, changes in the excretion of copper from the hepatobiliary pathway by treatment with VPA should be also considered.

The purpose of the present study was first to determine if there were any changes in the absorption of copper from the gastrointestinal tract; and second, to assess the biliary excretion of copper with or without VPA treatment using an in situ recirculating perfusion method in rat small intestine.

Section snippets

Animal procedures

All experiments were performed in accordance with the Guidelines for Animal Experiments of Nagoya University School of Medicine.

Male Wistar rats (Japan SLC Inc., Shizuoka, Japan) weighing 280–310 g were housed in suspended stainless steel cages. The animals were allowed free access to food (Nihon Clea, Tokyo, Japan) and water, although food was withheld overnight before the experiment. Under sodium pentobarbital anesthesia (25 mg/kg i.p.), rats were placed on an operating plate. Anesthesia was

Results

The time courses of the changes in perfusate volume, perfusate copper concentration and copper amount are shown in Fig. 1. There was no significant change on addition of VPA (20 mg/30 ml) in either perfusate volume (P=0.8) (Fig. 1A) or perfusate copper concentration (P=0.7) (Fig. 1B) or copper amount (P=0.7) (Fig. 1C). The absorption rate constant adjusted for intestinal length (k_a) did not change significantly in the presence of VPA (Table 1). These results indicated that the abnormalities of

Discussion

In the present study, the decline in copper during the perfusion did not change in the presence of VPA, although VPA was absorbed into the systemic circulation (Fig. 1C and Table 1). Palm and Hallmans (1982) have shown that phenytoin treatment in epilepsy patients increases serum zinc and copper concentrations and they proposed that a chelate formation between the drug or its metabolites and the metal can increase the absorption of the metal in the intestine. But our results indicate that VPA

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How dose copper get into bile? new insights into the mechanism(s) of hepatobiliary copper transport

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Because PNNs are critical structural components of neocortical circuits containing fast-spiking, parvalbumin-expressing GABAergic projections to assemblies of pyramidal neurons, there is growing interest in the physicochemical basis of their probable pathological roles in at least some presentations of ASD [13–17]. Whereas valproic acid did not affect the absorption of copper from rat small intestine, which was measured using an in situ recirculation perfusion method, it did significantly increase bile flow and, thereby, increased biliary excretion of copper [20]. Serum concentrations of valproic acid of 31.1 ± 3.2 µg/ml measured at the end of the 2-h perfusion were associated with a total mean value for the increase in biliary copper excretion of 79% [20].
A 23 year old male diagnosed with autism spectrum disorder and treated with valproic acid presented with acute onset of hepatic failure; he died less than two months later. Laboratory studies led to a diagnosis of Wilson Disease. The case raised questions about a possible pathogenic role of disrupted copper homeostasis in ASD, and exacerbation of this disruption as a result of treatment with valproic acid. Screening for abnormalities of copper homeostasis may stimulate strategies for therapeutic targeting of a contributing etiological factor and avoidance of contraindicated medications.
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Normal copper balance is maintained by regulating its excretion, mainly by the hepatobiliary route, as the copper in bile is not available for reabsorption [2]. Valproic acid produces a significant increase of the bile flow [3,4] and biliary excretion of copper [4] in rats, and although previously published results for the serum levels of copper during valproic acid treatment are contradictory [5–8], a special focus on the possible copper deficiency in the patients treated with this drug has been recommended [4,5]. Hypoceruloplasminemia [9], and low liver copper concentrations [10,11], have been described in patients with non-alcoholic fatty liver disease, with a possible association between low copper stores and increased insulin resistance and other features of the metabolic syndrome [11].
In rats a significant increase of the copper biliary excretion is produced by valproic acid administration, however, a conclusive study on the possible appearance of copper deficiency in humans during treatment with this drug has still not been carried out.
In 101 adult epileptic patients treated in monotherapy (n = 75) and polytherapy (n = 26) with valproic acid, and 50 healthy controls, were determined serum copper, immunoreactive ceruloplasmin and its oxidase activity against o-dianisidine, in order to calculate the specific oxidase activity (activity per unit mass of enzyme protein) and copper/ceruloplasmin ratio.
Specific oxidase activity of ceruloplasmin and copper/ceruloplasmin ratio were significantly lower in the groups of patients treated with valproic acid than in the controls. Significant correlations were obtained between both biochemical variables (p < 0001). In 38% of the patients treated in mono or polytherapy, the specific oxidase activities of ceruloplasmin were smaller than the estimated lower limit of reference.
These results, possible due to a diminished copper content of the circulating ceruloplasmin, suggest that marginal or moderate copper deficiency may have a substantial prevalence among patients treated with valproic acid.
Valproate and copper accelerate TRH-like peptide synthesis in male rat pancreas and reproductive tissues
2006, Peptides
Treatment with valproate (Valp) facilitates the synthesis of TRH-like peptides (pGlu-X-Pro-NH₂) in rat brain where “X” can be any amino acid residue. Because high levels of TRH-like peptides occur in the pancreas and pGlu-Glu-Pro-NH₂ (Glu-TRH) has been shown to be a fertilization promoting peptide, we hypothesized that these peptides mediate some of the metabolic and reproductive side effects of Valp. Male WKY rats were treated with Valp acutely (AC), chronically (CHR) or chronically followed by a 2 day withdrawal (WD). AC, CHR and WD treatments significantly altered TRH and/or TRH-like peptide levels in pancreas and reproductive tissues. Glu-TRH was the predominant TRH-like peptide in epididymis, consistent with its fertilization promoting activity. Glu-TRH levels in the epididymis increased 3-fold with AC Valp. Phe-TRH, the most abundant TRH-like peptide in the pancreas, increased 4-fold with AC Valp. Phe-TRH inhibits both basal and TRH-stimulated insulin release. Large dense core vesicles (LDCV's) contain a copper-dependent enzyme responsible for the post-translational processing of precursors of TRH and TRH-like peptides. Copper (500 μM) increased the in vitro C-terminal amidation of TRH-like peptides by 8- and 4-fold during 24 °C incubation of homogenates of pancreas and testis, respectively. Valp (7 μM) accelerated 3-fold the processing of TRH and TRH-like peptide precursors in pancreatic LDCV's incubated at 24 °C. We conclude that copper, an essential cofactor for TRH and TRH-like peptide biosynthesis that is chelated by Valp, mediates some of the metabolic and reproductive effects of Valp treatment via acceleration of intravesicular synthesis and altered release of these peptides.
Assessment of copper status in epileptic patients treated with anticonvulsant drugs by measuring the specific oxidase activity of ceruloplasmin
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Significant increases in serum levels and decreases in hair copper levels have been previously described in epileptic patients treated with anticonvulsant drugs. A condition not directly related to copper nutriture, such as chronic treatment with these drugs, could increase the serum concentrations of copper and ceruloplasmin and would mask a possible copper deficiency produced by drug-increased biliary copper excretion. Serum immunoreactive ceruloplasmin concentration and its oxidase activity were determined in 90 adult epileptic patients treated with phenobarbital (n=60), phenytoin (n=70), carbamazepine (n=33) and valproic acid (n=8). The levels of ceruloplasmin and oxidase activity were significantly higher (P<0.001) than in an age and gender-matched control group (n=49). The significant correlations (P<0.01) between ceruloplasmin and the urinary excretion of d-glucaric acid, serum gamma-glutamyltransferase (GGT) and drug score in the patients group, would suggest that phenobarbital-type enzyme-inducing agents may increase the hepatic synthesis of ceruloplasmin. In 11 patients with a β-globulin migrating GGT isoform (GGT3), a sensitive marker of cholestasis, the levels of ceruloplasmin, oxidase activity and total GGT activity were significantly higher (P<0.05) than in the group of 79 patients without the GGT3 isoform; consequently, in some cases a drug-induced cholestasis may also contribute to the increase of serum copper and ceruloplasmin. The values obtained for the specific oxidase activity of ceruloplasmin (activity per unit mass of enzyme protein) suggest that in the most of the cases, chronic administration of phenobarbital, phenytoin, carbamazepine or valproic acid, does not produce marginal or moderate copper deficiency.
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PET with <sup>64</sup>Cu-histidine for noninvasive diagnosis of biliary copper excretion in long-Evans cinnamon rat model of Wilson disease
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Valproic acid increases biliary copper excretion in the rat

Abstract

Introduction

Section snippets

Animal procedures

Results

Discussion

Trends Genet.

J. Hepatol.

Hepatology

Toxicology

Toxicology

Epilepsy Res.

J. Invest. Dermatol.

Absorption, transport, and hepatic metabolism of copper and zinc: special reference to metallothionein and ceruloplasmin

Physiol. Rev.

Disposition of valproic acid in the rat: dose-dependent metabolism, distribution, enterohepatic recirculation and choleretic effect

J. Pharmacol. Exp. Ther.

How dose copper get into bile? new insights into the mechanism(s) of hepatobiliary copper transport

J. Hepatol.