Bile acids: the role of peroxisomes.

It is well established that peroxisomes play a crucial role in de novo bile acid synthesis. Studies in patients with a peroxisomal disorder have been indispensable for the elucidation of the precise role of peroxisomes. Several peroxisomal disorders are associated with distinct bile acid abnormalities and each disorder has a characteristic pattern of abnormal bile acids that accumulate, which is often used for diagnostic purposes. The patients have also been important for determining the pathophysiological consequences of defects in bile acid biosynthesis. In this review, we will discuss all the peroxisomal steps involved in bile acid synthesis and the bile acid abnormalities in patients with peroxisomal disorders. We will show the results of bile acid measurements in several tissues from patients, including brain, and we will discuss the toxicity and the pathological effects of the abnormal bile acids.

ids are transported out of the peroxisome and subsequently out of the hepatocyte and into the bile. Recently, bile acid transport activity has been detected and characterized by reconstitution of peroxisomal membrane proteins into liposomes; however, the identity of the transporter remains unknown ( 26 ). Conjugation of bile acids not only involves newly synthesized bile acids but also bile acids that have been deconjugated in the intestine and recycled via enterohepatic circulation ( 2 ). The deconjugated primary and secondary bile acids are reactivated to CoA thioesters at the cytosolic side of the endoplasmic reticulum by BACS ( 2 ); however, the question remains as to where they are reconjugated. By now, the peroxisomal localization of BAAT has been fi rmly established ( 25,27,28 ) but there is a longstanding dispute in the literature about a possible dual localization of BAAT, both in the peroxisome and in the cytosol (29)(30)(31)(32). A cytosolic localization would negate the need for recycled bile acids to be transported back into the peroxisome just to undergo conjugation. For this reason, further research is needed to fi rmly establish the precise localization of BAAT.

BILE ACID ABNORMALITIES IN PATIENTS WITH PEROXISOMAL DISORDERS
Investigation of the bile acid abnormalities in the various peroxisomal disorders has been of great value, not only for establishing that functional peroxisomes are necessary for bile acid biosynthesis, but also for determining which peroxisomal enzymes are involved in this pathway. Analysis of plasma from patients with a peroxisome defi ciency disorder (i.e., Zellweger syndrome, neonatal adrenoleukodystrophy, and infantile Refsum disease) reveals the presence of C 27 -bile acid intermediates but also a C 29dicarboxylic acid ( 33,34 ). This dicarboxylic acid (3 ␣ ,7 ␣ ,12 ␣ -trihydroxy-27-carboxymethyl-5 ␤ -cholestane-26oic acid) is thought to be formed by chain-elongation of THC-CoA ( 35,36 ). The extent of the accumulation of bile acid intermediates has been shown to vary greatly and correlates with the extent to which peroxisomes are deficient ( 34 ). In urine of patients with a peroxisome deficiency disorder, the C 27 -bile acid intermediates are also present, but predominantly as tetrahydroxy-5 ␤ -cholestanoic acid and sometimes even pentahydroxy-5 ␤ -cholestanoic acid (37)(38)(39). Hydroxylation of THCA (of the steroid nucleus mainly at C-1 and C-6 but also of the side chain at C-24, C-25 or C-26) is an important mechanism for increasing the polarity and thereby, the urinary excretion of the C 27 -bile acid intermediates. In addition, bile alcohol glucuronides are present in urine, which are formed via an alternative pathway for bile acid biosynthesis not requiring the participation of peroxisomes, i.e., the microsomal 25-hydroxylase pathway. The C 29 -dicarboxylic acid is poorly excreted and for this reason it is not always present in urine. The most striking bile acid abnormality in AMACR deficiency is the presence of only the (25R)-isomer of the C 27bile acid intermediates ( 40,41 ). In contrast, in other that has been shown to be capable of handling the C 27 -bile acid intermediates in expression studies is the very longchain acyl-CoA synthetase ( 2 ). It is expressed primarily in liver and kidney and is located in both the endoplasmic reticulum and in the peroxisome. In addition to the C 27bile acid intermediates, the enzyme has been shown to handle C24:0, C16:0, phytanic and pristanic acid as substrates, but is not capable of activating the primary C 24 -bile acids ( 2,4 ). To date, formation of DHC-CoA and THC-CoA has only been demonstrated at the endoplasmic reticulum and not in peroxisomes ( 5,6 ). The kinetic properties of the THC-CoA synthetase and choloyl-CoA synthetase activities at the endoplasmic reticulum have been shown to be different ( 6 ), which suggests that both BACS and very long-chain acyl-CoA synthetase are involved in the activation of the C 27 -and C 24 -bile acids at the endoplasmic reticulum. In agreement with a microsomal, and not peroxisomal, activation of the C 27 -bile acid intermediates is the demonstration of ATP-dependent transport of THC-CoA into the peroxisome ( 7 ). THCA and THC-CoA were incubated with isolated rat liver peroxisomes and the products of peroxisomal ␤ -oxidation were measured ( 7 ). The results of these experiments showed that THC-CoA is the preferred substrate for transport. However, it must be noted that peroxisomes isolated by differential centrifugation are leaky, which makes them not optimal for these kinds of experiments. For this reason, transport should ideally be studied by reconstitution of the transporter or peroxisomal membrane proteins in liposomes. The identity of the transporter for the C 27 -bile acyl-CoA's is at this moment still unknown.
The fi nal step in de novo bile acid synthesis is the conjugation/amidation of the C 24 -bile acids to glycine or taurine. This reaction is performed by the enzyme bile acyl-CoA: amino acid N -acyltransferase (BAAT), which converts the bile acyl-CoAs into tauro-or glyco-bile acids, thereby releasing free CoA (23)(24)(25). This reaction occurs in the peroxisome, after which the conjugated C 24 -bile ac-have not been well studied. It is important to gain insight into the accumulation of bile acids and their intermediates in tissues of patients because bile acid toxicity is postulated to play an important role in the underlying pathological mechanisms of these disorders, not only in liver but also in other organs such as the brain. For this reason, we analyzed bile acids by HPLC-negative ion electrospray tandem mass spectrometry ( 50,51 ) in several tissues of patients with different peroxisomal disorders. The results of these analyses have not been published previously. We studied liver, brain, kidney, heart, spleen, lung, pancreas, and bile of patients with a peroxisome defi ciency disorder (including patients with the severe Zellweger phenotype and patients with the milder neonatal adrenoleukodystrophy phenotype), patients with a defi ciency of DBP (including patients with a DBP defi ciency type I and type III) and one patient with an AMACR defi ciency. The results of the bile acid measurements in liver are presented in T able 1 and Fig. 2A . The main accumulating bile acids in the liver of patients with a peroxisome defi ciency disorder were t-THCA and THCA. Total C 27 -bile acid intermediates were signifi cantly increased and total C 24 -bile acids signifi cantly decreased compared with control livers. This resulted in clearly elevated C 27 /C 24 -bile acid ratios in all patient livers. The percentage of conjugated C 27 -bile acid intermediates was only 51.5% compared with 96.4% for the C 24 -bile acids. This shows that C 27 -bile acid intermediates are poor substrates for the conjugating enzyme BAAT. The fact that the C 24 -bile acids were almost fully conjugated (96.4% compared with 99% in control livers) suggests that BAAT could very well have a dual localization, not only in peroxisomes but also in the cytosol, or at least that BAAT is normally active and stable in the cytosol when peroxisomes are defi cient. Both in the livers with a peroxisome defi ciency and in the AMACR-defi cient liver, the C 29 -dicarboxylic acid (3 ␣ ,7 ␣ ,12 ␣ -trihydroxy-27carboxymethyl-5 ␤ -cholestane-26-oic acid) was present, whereas it was not detected in DBP-defi cient livers. In livers of patients with a DBP defi ciency, the main accumulating C 27 -bile acid intermediates were OH-THCA, t-THCA:1, and THCA, whereas those in the liver from the AMACRdefi cient patient were t-THCA and THCA. The bile acid measurements showed that two DBP-defi cient livers and the AMACR-defi cient liver were cholestatic but this did not change the fact that the C 27 /C 24 -bile acid ratios were elevated in all patient livers. As in the peroxisome-defi cient livers, the C 27 -bile acid intermediates were partly conjugated, confi rming that BAAT is poorly active with bile acids with an increased side chain length compared with C 24 -bile acids ( 29,52 ).
Bile of one patient with a peroxisome defi ciency and the AMACR-defi cient patient was analyzed in addition to the liver sample. For both patients, the C 27 /C 24 -bile acid ratio was higher in liver than in bile (2.58 vs. 1.66 in the peroxisome-defi cient patient) and the percentage of conjugated C 27 -bile acid intermediates was higher in bile than in liver (81% vs. 66% in the same patient). This suggests that C 27 -bile acid intermediates are less effi ciently transported across the canalicular membrane and that conju-peroxisomal disorders with a defect in bile acid biosynthesis, both the (25R)-and (25S)-isomers accumulate. Just like in patients with a peroxisome defi ciency disorder, many polyhydroxylated C 27 -bile acids are present in urine ( 41 ).
To date, no patients with a defi ciency of BCOX have been identifi ed but the bile acid abnormalities will most likely resemble those of peroxisome defi ciency disorders and AMACR defi ciency, although the main accumulating isomer will be the (25S)-isomer in BCOX defi ciency. The role of BCOX in the oxidation of bile acids has been established by in vitro studies with the purifi ed enzyme ( 42,43 ) and by the fact that patients with a defi ciency of the other peroxisomal acyl-CoA oxidase (straight-chain acyl-CoA oxidase) do not have a bile acid biosynthesis defect ( 44 ).
The bile acid abnormalities in DBP defi ciency are more complex because DBP displays two catalytic activities, resulting in multiple types of DBP defi ciency. In addition, the other peroxisomal bifunctional enzyme, L-bifunctional protein, also displays activity with certain isomers of the C 27 -bile acid intermediates. In all types of DBP defi ciency, DHCA, THCA, and the direct substrate of DBP, the 24enoyl of the C 27 -bile acid intermediates are present in plasma ( 45,46 ). When the hydratase activity is defi cient (DBP type I and II defi ciency, i.e., combined defi ciency of the hydratase and dehydrogenase activity and isolated hydratase defi ciency, respectively) (24S)-OH (25S), -THC-CoA is also present, being formed by L-bifunctional protein. This isomer is not a substrate for any of the two peroxisomal dehydrogenases and cannot be metabolized further. When only the dehydrogenase activity is defi cient (DBP type III defi ciency), (24S), -OH (25S), -THC-CoA is also present, but the main accumulating metabolite is (24R)-OH (25R), -THC-CoA, which is formed by the hydratase unit of DBP ( 45,46 ). A small portion is converted into (24R)-OH (25S), -THC-CoA by AMACR, which cannot be metabolized further ( 47 ). In urine, the presence of the taurine conjugate of hydroxylated 24-ene-THCA and dihydroxylated 24-ene-THCA is characteristic for patients with DBP defi ciency ( 22 ). As for peroxisome defi ciency disorders, the extent of the accumulation of the C 27 -bile acid intermediates varies between patients and this seems to correlate with the extent of the defi ciency of DBP. Bile acid abnormalities are not found in plasma in 26% of DBPdefi cient patients ( 48 ).
Although the bile acid abnormalities in plasma and urine of patients with a peroxisomal fatty acid oxidation disorder have been well characterized and can be used as diagnostic markers, bile acids in tissues of these patients intermediates or C 24 -bile acids levels. The only bile acids that were elevated were OH-THCA and DHCA. For two patients with a peroxisome defi ciency disorder, two DBPdefi cient patients, and four control subjects, tissue from different brain areas was analyzed, including frontal white and gray matter, parietal white and gray matter, and extra frontal and parietal cortex. No signifi cant difference between the bile acid content in the different brain areas was detected.

TOXICITY OF C 27 -BILE ACID INTERMEDIATES AND THE PATHOLOGICAL CONSEQUENCES OF A BILE ACID BIOSYNTHESIS DEFECT AT THE LEVEL OF THE PEROXISOME
The bile acid abnormalities in patients with a peroxisomal disorder have been shown to contribute to the liver disease associated with these disorders ( 53,54 ) and also have been hypothesized to have an effect on the developing nervous system ( 55 ). The C 27 -bile acid intermediates are more hydrophobic than their C 24 -products, and as shown by the bile acid measurements in tissues of patients described above, they are less effi ciently conjugated (augmenting to their hydrophobicity) and excreted into the bile. These factors will contribute to the retention of these intermediates in the liver, cholestasis, and, consequently, to hepatic injury in these patients. In addition, the primary bile acid defi ciency decreases the intestinal dietary lipid gated C 27 -bile acid intermediates are the preferred substrates for this transport. At the moment, it is not known which transporters in the canalicular membrane are specifi cally involved in this process.
In the analyzed peripheral tissues (kidney, heart, spleen, lung, pancreas) of the different peroxisomal patients, the C 27 -bile acid intermediates clearly accumulated and the C 27 /C 24 -bile acid ratio was comparable to the one found in liver ( Fig. 2B ). The pattern of accumulating metabolites was also comparable to liver except that the main accumulating C 27 -bile acid intermediates were mostly unconjugated (resulting in a percentage of conjugated C 27 -bile acid intermediates of around 20-25% in the different tissues), which again suggests that the conjugated C 27 -bile acid intermediates are more effi ciently excreted into the bile.
In brains of patients with a peroxisome defi ciency disorder, there were clear bile acid abnormalities ( T able 2 and Fig. 2C ). C 27 -bile acid intermediates were increased compared with control brains and C 24 -bile acids were decreased, resulting in a signifi cantly increased C 27 /C 24 -bile acid ratio. The accumulating C 27 -bile acid intermediates were THCA, DHCA, and OH-THCA, all unconjugated, consistent with a less effi cient passage of conjugated C 27bile acid intermediates of the blood-brain barrier. The C 29dicarboxylic acid was also detectable in brain. The brains of DBP-defi cient patients were much harder to distinguish from control brains based on the bile acid measurements. There were no signifi cant differences for total C 27 -bile acid Bile acids are expressed as nmol/g wet weight. BA, bile acids. CDCA and DCA cannot be distinguished with the method used for bile acid measurement. * P < 0.05; ** P < 0.005 ( t -test).
ates are more cytotoxic than mature C 24 -bile acids, with DHCA being clearly the most cytotoxic bile acid. In addition to the membrane-disruptive effects of bile acids due to their detergent properties, other mechanisms, like the induction of mitochondrial dysfunction, have been shown to be involved in the toxicity of bile acids ( 57 ). For this reason, the effect on the mitochondrial respiratory chain function was studied ( 56 ). It was shown that the different bile acids caused a dose-dependent decrease in ATP synthesis by isolated mitochondria oxidizing malate and glutamate (the order of potency of inhibition was: DHCA>THCA>CDCA>g-THCA>g-CDCA>t-THCA>CA). The unconjugated bile acids acted predominantly as uncouplers of the oxidative phophorylation whereas the conjugated bile acids (t-THCA, g-THCA and g-CDCA) primarily inhibited the respiratory chain. By inhibiting the respiratory chain, the conjugated bile acids t-THCA, g-THCA, and g-CDCA enhanced reactive oxygen species (ROS) production at the level of complex III. Increased ROS generation leads to depletion of the antioxidant defenses and can eventually lead to cell death. Interestingly, in the very fi rst report describing Zellweger syndrome, brain and liver mitochondria from a patient showed strongly reduced respiration with malate and succinate as substrates ( 58 ). In addition, in a mouse model with hepatocyte-selective elimination of peroxisomes, severely reduced activities of complex I, III, and V were measured ( 59 ) and in a Pex5 knockout mouse, a model for Zellweger syndrome, reduction in the activities of complex I and V were observed ( 60 ), supporting the physiological relevance of the in vitro fi ndings. The reduction of the activities of the respiratory chain complexes was accompanied by alterations of the mitochondrial inner membrane structure, which has also been observed in Zellweger patients ( 58,61,62 ).
Because the C 27 -bile acid intermediates are especially cytotoxic, reducing their levels could be benefi cial not only for the liver disease in peroxisomal patients but also to prevent potential injury to other organs by circulating C 27 -bile acid intermediates. C 27 -bile acid levels can be reduced by treatment with C 24 -bile acids, which downregulate bile acid biosynthesis via activation of the nuclear receptor farnesoid X receptor (FXR), resulting in reduced transcriptional activation of CYP7A1, the rate-limiting enzyme in bile acid biosynthesis. In addition, bile acid therapy will increase bile fl ow by replenishing the decreased levels of C 24 -bile acids and it will increase the intraluminal bile acid concentration, thereby facilitating the absorption of fats and fat soluble vitamins. Oral bile acid therapy has been tried with variable success in peroxisomal disorders ( 41,(63)(64)(65). The fi rst report of such a bile acid treatment was in a 6-month-old Zellweger patient who showed significant improvement of biochemical indices of liver function and a histological improvement in the extent of infl ammation and bile duct proliferation and disappearance of canalicular plugs ( 64 ). Steatorrhea improved and was accompanied by an improvement in growth. Plasma and urinary C 27 -bile acid intermediates decreased with treatment. Despite the observed improvements, the patient died at one year of age. Also, in subsequent studies it has been and lipid soluble vitamin absorption. Furthermore, we have shown that in brains of peroxisome defi ciency patients, C 27 -bile acid intermediates accumulate and that these were almost exclusively unconjugated, increasing their potential toxic effect. However, because peroxisomes are involved in multiple metabolic pathways, interpretation of the pathological consequences of the bile acid synthesis defect in patients is complex. For this reason, we have recently performed an in vitro study on the toxicity of the conjugated and unconjugated C 27 -bile acid intermediates. The effects on cell viability, mitochondrial respiratory chain function, and the production of oxygen radicals were examined in a rat hepatoma cell line and compared with the effect on these parameters of the mature C 24 -bile acids ( 56 ). These studies showed that C 27 -bile acids intermedi-

CONCLUDING REMARKS
The importance of proper peroxisomal function for the biosynthesis of bile acids has been fi rmly established. The peroxisomal enzymes involved in bile acid biosynthesis have all been identifi ed. Only the transporters required for the transport of the C 27 -bile acid intermediates into the peroxisome and for the conjugated C 24 -bile acids out of the peroxisome remain unidentifi ed. In addition, it is not clear yet whether deconjugated and recycled C 24 -bile acids need to reenter the peroxisome for reconjugation or whether this occurs in the cytosol. The bile acid abnormalities in patients with peroxisomal disorders have been well characterized and evidently play a role in the pathology of these disorders. The C 27 -bile acid intermediates have been shown to be more cytotoxic than the C 24 -bile acids, and the fi nding of primarily unconjugated C 27 -bile acid intermediates in brains from children with Zellweger syndrome suggests that the bile acid synthesis defect in these patients could also play a role in central nervous system pathology. Unfortunately, the clinical presentation of children with classical Zellweger syndrome is too severe due to the multiple metabolic derangements for a true positive effect of oral bile acid therapy in these patients but patients with a mild presentation (i.e., infantile Refsum disease) or a single enzyme defi ciency could very well benefi t from such a therapy or treatment with an FXR ligand to reduce the toxic C 27 -bile acid intermediates.
The authors thank Dr. Carl E. Clarke for providing patient material. Human tissue was obtained from the NICHD Brain and Tissue Bank for Developmental Disorders under contracts N01-HD-4-3368 and N01-HD-4-3383. We thank Henk Overmars for technical assistance.
shown that patients with Zellweger syndrome are too severely affected by the multiple metabolic derangements for bile acid therapy to have a positive effect on the outcome of the disease ( 63,65 ). However, in the Pex 2 knockout mouse, another model for Zellweger syndrome, positive effects of bile acid therapy were observed ( 55,66 ). Pex 2 knockout mice were fed a mixture of CA and ursodeoxycholic acid (UDCA), which resulted in a signifi cant improvement in postnatal survival. Increased body fat deposition was observed and improvement of the intestinal malabsorption. Compared with untreated mutant mice, bile acid treated Pex2 knockout mice were more active and less hypotonic but problems with balance and spasticity persisted. Interestingly, there was a signifi cant improvement in the extent of dendritic arborization of cerebellar Purkinje cells ( 55 ). Thus, bile acid therapy may improve some aspects of central nervous system development. In the liver, bile acid treatment alleviated intrahepatic cholestasis and prevented the development of severe steatohepatitis. However, the therapy exacerbated the degree of hepatic steatosis and worsened the already severe mitochondrial and cellular damage in the liver ( 66 ). These studies showed that peroxisome-defi cient hepatocytes are particularly sensitive to bile acid toxicity, indicating that bile acid therapy will be more benefi cial in patients with a relatively mild peroxisome biogenesis disorder (neonatal adrenoleukodystrophy or infantile Refsum disease) or patients with a single enzyme defect (AMACR, DBP, SCPx defi ciency). Indeed, a child with AMACR defi ciency has been reported to respond well to oral bile acid therapy for 7 years ( 41 ). On treatment, the C 27 -bile acids intermediates are virtually absent in urine, her liver enzymes have remained normal, and her fat-soluble vitamins have remained normal without supplementation. As an alternative to oral bile acid therapy, treatment with an artifi cial FXR ligand should be considered. These artifi cial FXR ligands are not harmful for the hepatocyte but they will downregu- Bile acids are expressed as nmol/g wet weight. BA, bile acids. CDCA and DCA cannot be distinguished with the method used for bile acid measurement. * P < 0.05; ** P < 0.005 ( t -test).