The pathway for the formation of the 2 primary bile acids from cholesterol was elucidated many decades ago after the close relationship between the chemical structure of the cholesterol nucleus and the basic structure of bile acids was realized. Thereafter, the complex multistep pathway by which the neutral sterol, cholesterol, is converted to the 2 principal acidic primary bile acids, cholic and chenodeoxycholic acids, became elucidated in detail (1). Each of the steps or reactions in the pathway for bile acid synthesis is catalyzed by at least 16 specific enzymes. The first genetic defect to be described in this complex pathway was the rare lipid-storage disease of cerebrotendinous xanthomatosis. This disease was clinically recognized in 1937 (2) but only recently has its biochemical and molecular basis been deciphered through elegant studies by Salen et al. (3-5) and Oftebro et al. (6). This disorder, caused by numerous different mutations in the sterol 27-hydroxylase enzyme (7,8) presents in adult life with symptoms of xanthomas, premature atherosclerosis and dementia. In 2000, through our screening program of cases of unexplained liver disease in infants and children we reported that the sterol 27-hydroxylase deficiency is also a cause of neonatal cholestatic liver disease that may spontaneously resolve in early life in some patients (9). This finding presented a "new face" to an "old disease" and was later corroborated by Clayton et al. (10). Early diagnosis of genetic defects in bile acid synthesis is crucial to successful therapy with primary bile acids, especially given the progressive devastating clinical course of a number of the defined defects.
DIAGNOSTIC ROLE OF MASS SPECTROMETRY
Our success in discovering bile acid synthetic defects as a cause of progressive cholestatic liver disease can be exclusively attributed to the application of mass spectrometry, and particularly the use of what in 1980 was a relatively new technique of fast atom bombardment ionization mass spectrometry (FAB-MS) (11). This new soft ionization technique, now considered obsolete with the subsequent introduction of electrospray ionization, revolutionized the way in which we were able to approach the difficulty of analyzing bile acids in biological samples. At that time, methods for qualitative and quantitative bile acid analysis by mass spectrometry required extensive preinstrumental workup of samples, steps involving extraction, purification, hydrolysis of the bile acid conjugates, chromatographic separation and purification and finally conversion to volatile derivatives to facilitate their vaporization for introduction to the mass spectrometer (12). Such procedures were time-consuming and, therefore, generally unsuitable for the routine screening of clinical samples for diagnosis-a factor that no doubt previously inhibited progress in identifying bile acid synthetic disorders. Whereas faster methods for bile acid analysis are available, including enzyme- and immuno-based assays (12), these do not provide any structural information and, therefore, are unhelpful in ascertaining metabolic defects in synthesis, and may even give misleading information.
In 1978, the new ionization technique of FAB-MS was shown to be applicable to the direct analysis of bile acids in biological samples. Bile acids were found amenable to soft ionization yielding intense negatively charged molecular ions with little fragmentation of the molecule. The procedure is relatively simple and rapid. A drop of urine or urine extract is spotted on the tip of the FAB probe containing a drop of glycerol as matrix, and this is inserted into the ion source where it is ionized by a beam of fast atoms generated from cesium or noble gas. Negative ions are generated from the bile acids present, and these are spluttered from the surface, focused and separated in the mass analyzer to generate a mass spectrum. In healthy adults and children, bile-acid excretion is relatively low (<20 μmol/L) (13), and FAB-MS is not sufficiently sensitive to detect bile acids (Fig. 1). However, in cholestatic liver disease, when urinary bile acid excretion increases significantly, bile acids are readily detected by FAB-MS. Series of ions (m/z 448, 464, 480, 498, 514, 528 and 530) correspond to the negatively charged molecular species of glycine, taurine and sulfate conjugates of dihydroxy-, trihydroxy- and tetrahydroxy-cholanoic acids (C24 bile acids), respectively. The inability of this ionization method to distinguish different positional or stereo-isomers means that more detailed gas chromatography-mass spectrometry (GC-MS) or electrospray ionization-mass spectrometry (ESI-MS) techniques are necessary to provide confirmatory evidence of a potential biochemical defect (12). On a practical point, a possible defect in primary bile acid synthesis may be overlooked if a patient is administered 3α,7β-dihydroxy-5β-cholanoic acid (UDCA) at the time of urine collection. UDCA and its metabolites give rise to the same ions as conjugated chenodeoxycholic (3α,7α-dihydroxy-5β-cholanoic acid) because both are dihydroxy-cholanoate structures. It is, therefore, recommended that therapy be temporarily stopped for at least 5 days to allow for wash-out of residual UDCA and its metabolites before performing this test. The presence of the primary bile acid conjugates in the urine indicates that pathways for normal primary bile acid synthesis are intact, and this rules out a genetic defect in the cholesterol-bile acid pathway as the cause of liver disease. This analytical breakthrough in bile acid analysis led a concerted effort to screen for possible defects in bile acid synthesis as a cause of idiopathic forms of liver disease in infants and children. The rationale for this approach was that any defect in bile acid synthesis would manifest as a condition of cholestasis caused by a loss of primary bile acids that provide the main stimulus for the promotion and secretion of bile and because of an accumulation of atypical bile acids that we proposed are potentially hepatotoxic leading to liver injury.
IDENTIFICATION OF GENETIC DEFECTS IN BILE ACID SYNTHESIS
The implementation of an international screening program for genetic causes of cholestatic liver disease at the Cincinnati Children's Hospital Medical Center led to the identification of 6 defects in bile acid synthesis in the last 20 years. These defects are clinically manifested as a spectrum of conditions including progressive cholestatic liver disease, fat-soluble vitamin malabsorption syndromes or neurological disease. These defects can be biochemically defined from the specific negative ion mass spectra, which yield characteristic ions (patterns) for the atypical metabolites resulting from the deficiency in activity of specific enzymes (Fig. 2).
Inborn errors in bile acid synthesis have now been identified in more than 100 patients from an estimated 5000 cases that we have been screened, accounting for about 2% of the cases of unexplained liver disease. Six bile acid synthetic defects have been definitively characterized and reported (Table 1), and mutations in the genes encoding the respective enzymes were described (Fig. 3). These are described in detail elsewhere (14).
A genetic defect, in the rate-limiting enzyme cholesterol 7α-hydroxylase was also recently reported in several patients based on the finding of mutations in the CYP7A1 gene (24). This condition does not seem to manifest as liver disease but rather a dyslipidemia syndrome that is unresponsive to statin therapy. In 1995, we described for the first time a defect in amidation of bile acids that presented as a syndrome of fat-malabsorption and mild liver disease (22). The phenotype of this metabolic defect was accurately postulated some years earlier by Hofmann and Strandvik (25), and we have recently established this to be due to a mutation in the gene encoding the bile acid-CoA:amino acid N-acyltransferase (unpublished data), the second enzyme involved in bile acid conjugation. This same defect has been reported to occur in a cohort of Amish patients who were found to have high concentrations of unconjugated bile acids in serum (23).
CLINICAL PRESENTATION AND THERAPEUTIC STRATEGY
The phenotype of bile acid synthetic defects is highly variable (14). Defects involving enzymes responsible for catalyzing reactions in the steroid nucleus, with the exception of the CYP7A1 deficiency (24), present with varying degrees of hyperbilirubinemia, elevations in serum transaminases and on clinical examination, hepatosplenomegaly. On the other hand, liver disease tends to be less severe in the defects that involve modifications to the cholesterol side-chain where these patients generally present with syndromes of fat-soluble vitamin malabsorption and/or neurological disease. Age at diagnosis of all of these diseases has been highly variable, and importantly bile acid synthetic defects should be considered as a possible explanation in cases of late-onset chronic cholestasis.
THERAPY FOR BILE ACID SYNTHETIC DEFECTS
Liver injury is a consequence of a failure to synthesize adequate concentrations of primary bile acids that are critical for bile acid-dependent bile flow, combined with the production of atypical metabolites, several of which have been shown to be intrinsically cholestatic and hepatotoxic (26). Long-term survival and clinical improvement of patients with bile acid synthetic defects is possible by downregulation of bile acid synthesis and provision of adequate levels of primary bile acids to provide a stimulus for generating bile flow. Both of these goals have been demonstrated with oral administration of the primary bile acid, cholic acid (3α,7α,12α-trihydroxy-5β-cholanoic acid) (27-30), now available only under an FDA Investigational New Drug Approval held by our group, and by chenodeoxycholic acid (3α,7α-dihydroxy-5β-cholanoic acid) (31,32) used for some time to treat patients with cerebrotendinous xanthomatosis (33,34). The dose administered orally is 10-15 mg/kg body weight per day, but this is titrated case by case against the biochemical response based on a reduction or disappearance of atypical metabolites in urine measured by FAB-MS analysis.
The efficacy of cholic acid in reducing the proportions of the atypical bile acid metabolites is greatest when cholic acid is given alone, rather than in combination with chenodeoxycholic or ursodeoxycholic acids, as was initially used in the first patients treated (28). Concomitant with disappearance of atypical bile acid metabolites, there is a consistent reduction and normalization in serum liver enzymes after initiating oral cholic acid administration. No adverse side effects have been associated with the use of cholic acid in any patient with treatment longer than 12 years in the longest treated patient. After our initial successful treatment of patients with primary enzyme defects in bile acids synthesis, we expanded cholic acid therapy to include patients with secondary bile acid synthetic defects (35), and specifically those with defects in peroxisomal β-oxidation of cholestanoic acid intermediates that have been found in animal studies to be hepatotoxic (36). Thus, 8 patients with peroxisomopathies are currently alive after treatment periods ranging 4.7-11 years duration. Of these, 4 patients had Refsum disease (37) whereas the remaining comprised cases of neonatal adrenoleukodystrophy or Zellweger syndrome (38). An additional 13 other patients with peroxisomal disorders were treated with cholic acid, but 10 died, or are presumed dead, and 3 were lost to follow-up. The treatment failures mostly included those patients with the more severe Zellweger syndrome where there was multiorgan disease. We conclude that this group will derive minimal benefit from this approach whereas those patients with single enzyme defects in peroxisomal function causing abnormal bile acid synthesis show greater responsiveness and benefit from oral cholic acid therapy.
The 2 primary bile acid defects that should not be treated with cholic acid are the oxysterol 7α-hydroxylase deficiency (20), because the CYP7B1 enzyme is unresponsive to feedback control by cholic acid and the amidation defect because these patients already produce vast amounts of unconjugated cholic acid (22,23). Glyco- or tauro-cholic acid is the likely treatment modality for conjugation defects but this has yet to be examined.
Finally, whereas UDCA because of its choleretic properties (39,40) may proved to be of temporary benefit in some patients with bile acid synthetic defects, specifically the 3β-hydroxyC27-steroid oxidoreductase deficiency (30), its long-term effectiveness will be limited by its failure to downregulate endogenous bile acid synthesis and prevent continued synthesis of atypical and potentially hepatotoxic bile acid intermediates and their metabolites.
We are grateful to the Food and Drug Administration for grant support from the Orphan Drug program (grant #FD-R-000995-02) and the National Institutes of Health, General Clinical Research Center grant M01 RR 08084. We also acknowledge many years of excellent technical support of our research personnel, Nancy O'Connell and Pinky Jha, and clinical/administrative support from Andrea Smith and Lona Pearson of the GCRC at the Cincinnati Children's Hospital Medical Center. Finally, we thank Dr. Falk Pharma Gmb, Freiburg, Germany, for supplying cholic acid to our patients over the last 20 years.
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