Molecular characterization of iodotyrosine dehalogenase deficiency in patients with hypothyroidism.

CONTEXT: The recent cloning of the human iodotyrosine deiodinase (IYD) gene enables the investigation of iodotyrosine dehalogenase deficiency, a form a primary hypothyroidism resulting from iodine wasting, at the molecular level. OBJECTIVE: In the current study, we identify the genetic basis of dehalogenase deficiency in a consanguineous family. RESULTS: Using HPLC tandem mass spectrometry, we developed a rapid, selective, and sensitive assay to detect 3-monoiodo-l-tyrosine and 3,5-diodo-l-tyrosine in urine and cell culture medium. Two subjects from a presumed dehalogenase-deficient family showed elevated urinary 3-monoiodo-l-tyrosine and 3,5-diodo-l-tyrosine levels compared with 57 normal subjects without thyroid disease. Subsequent analysis of IYD revealed a homozygous missense mutation in exon 4 (c.658G>A p.Ala220Thr) that co-segregates with the clinical phenotype in the family. Functional characterization of the mutant iodotyrosine dehalogenase protein showed that the mutation completely abolishes dehalogenase enzymatic activity. One of the heterozygous carriers for the inactivating mutation recently presented with overt hypothyroidism indicating dominant inheritance with incomplete penetration. Screening of 100 control alleles identified one allele positive for this mutation, suggesting that the c.658G>A nucleotide substitution might be a functional single nucleotide polymorphism. CONCLUSIONS: This study describes a functional mutation within IYD, demonstrating the molecular basis of the iodine wasting form of congenital hypothyroidism. This familial genetic defect shows a dominant pattern of inheritance with incomplete penetration.

Compromised intestinal lipid absorption in mice with a liver-specific deficiency of liver receptor homolog 1.

Bile acids (BAs) are water-soluble end products from cholesterol metabolism and are essential for efficient absorption of dietary lipids. By using targeted somatic mutagenesis of the nuclear receptor liver receptor homolog 1 (LRH-1) in mouse hepatocytes, we demonstrate here that LRH-1 critically regulates the physicochemical properties of BAs. The absence of LRH-1 and subsequent deficiency of Cyp8b1 eliminate the production of cholic acid and its amino acid conjugate taurocholic acid and increase the relative amounts of less amphipathic BA species. Intriguingly, while the expression of Cyp8b1 is almost extinguished in the livers of mice that lack LRH-1, the expression of the rate-limiting enzyme of BA synthesis, i.e., Cyp7a1, remains unchanged. The profound remodeling of the BA composition significantly reduces the efficacy of intestinal absorption of lipids and reuptake of BAs and facilitates the removal of lipids from the body. Our studies unequivocally demonstrate a pivotal role for LRH-1 in determining the composition of BAs, which, in turn has major consequences on whole-body lipid homeostasis.

Bile acid treatment alters hepatic disease and bile acid transport in peroxisome-deficient PEX2 Zellweger mice.

UNLABELLED: The marked deficiency of peroxisomal organelle assembly in the PEX2(-/-) mouse model for Zellweger syndrome provides a unique opportunity to developmentally and biochemically characterize hepatic disease progression and bile acid products. The postnatal survival of homozygous mutants enabled us to evaluate the response to bile acid replenishment in this disease state. PEX2 mutant liver has severe but transient intrahepatic cholestasis that abates in the early postnatal period and progresses to steatohepatitis by postnatal day 36. We confirmed the expected reduction of mature C24 bile acids, accumulation of C27-bile acid intermediates, and low total bile acid level in liver and bile from these mutant mice. Treating the PEX2(-/-) mice with bile acids prolonged postnatal survival, alleviated intrahepatic cholestasis and intestinal malabsorption, reduced C27-bile acid intermediate production, and prevented older mutants from developing severe steatohepatitis. However, this therapy exacerbated the degree of hepatic steatosis and worsened the already severe mitochondrial and cellular damage in peroxisome-deficient liver. Both untreated and bile acid-fed PEX2(-/-) mice accumulated high levels of predominantly unconjugated bile acids in plasma because of altered expression of hepatocyte bile acid transporters. Significant amounts of unconjugated bile acids were also found in the liver and bile of PEX2 mutants, indicating a generalized defect in bile acid conjugation. CONCLUSION: Peroxisome deficiency widely disturbs bile acid homeostasis and hepatic functioning in mice, and the high sensitivity of the peroxisome-deficient liver to bile acid toxicity limits the effectiveness of bile acid therapy for preventing hepatic disease.

Tissue carnitine homeostasis in very-long-chain acyl-CoA dehydrogenase-deficient mice.

Deficiency of very-long-chain acyl-CoA dehydrogenase (VLCAD) is the most common long-chain fatty acid oxidation defect and presents with heterogeneous clinical manifestations. Accumulation of long-chain acylcarnitines and deficiency of free carnitine have often been proposed to play an important role in disease pathogenesis. The VLCAD-deficient mouse exhibits similar clinical and biochemical phenotypes to those observed in humans and, therefore, represents an excellent model to study VLCAD deficiency. We measured carnitine and acylcarnitine profiles in liver, skeletal muscle (SkM), bile, and blood from VLCAD knock-out mice and controls under nonstressed and various stress conditions. Carnitine and acylcarnitines were extracted from body fluids with methanol and from tissues with acetonitrile, respectively, and were analyzed as their butyl esters using electrospray ionization tandem mass spectrometry. Fasting combined with a cold challenge for 8 h significantly induced liver long-chain acylcarnitine and free carnitine production. Acylcarnitines in SkM predominantly accumulated during exercise with a concomitant decrease of free carnitine. Changes in blood free carnitine did not correlate with carnitine homeostasis in liver and SkM. Our results demonstrate different tissue-specific long-chain acylcarnitine profiles in response to various stressors, which may be of importance with respect to the heterogeneous clinical manifestations of VLCAD deficiency in humans. Furthermore, we conclude that carnitine biosynthesis in the liver seems sufficiently active to maintain liver carnitine levels during increased demand. Our data suggest that carnitine supplementation in long-chain beta-oxidation defects may not be required, and blood carnitine concentrations do not reflect tissue carnitine homeostasis.

Developmental changes of bile acid composition and conjugation in L- and D-bifunctional protein single and double knockout mice.

Peroxisomal beta-oxidation is an essential step in bile acid synthesis, since it is required for shortening of C27-bile acid intermediates to produce mature C24-bile acids. D-Bifunctional protein (DBP) is responsible for the second and third step of this beta-oxidation process. However, both patients and mice with a DBP deficiency still produce C24-bile acids, although C27-intermediates accumulate. An alternative pathway for bile acid biosynthesis involving the peroxisomal L-bifunctional protein (LBP) has been proposed. We investigated the role of LBP and DBP in bile acid synthesis by analyzing bile acids in bile, liver, and plasma from LBP, DBP, and LBP:DBP double knock-out mice. Bile acid biosynthesis, estimated by the ratio of C27/C24-bile acids, was more severely affected in double knock-out mice as compared with DBP-/- mice but was normal in LBP-/- mice. Unexpectedly, trihydroxycholestanoyl-CoA oxidase was inactive in double knock-out mice due to a peroxisomal import defect, preventing us from drawing any firm conclusion about the potential role of LBP in an alternative bile acid biosynthesis pathway. Interestingly, the immature C27-bile acids in DBP and double knock-out mice remained unconjugated in juvenile mice, whereas they occurred as taurine conjugates after weaning, probably contributing to the minimal weight gain of the mice during the lactation period. This correlated with a marked induction of bile acyl-CoA:amino acid N-acyltransferase expression and enzyme activity between postnatal days 10 and 21, whereas the bile acyl-CoA synthetases increased gradually with age. The nuclear receptors hepatocyte nuclear factor-4alpha, farnesoid X receptor, and peroxisome proliferator receptor alpha did not appear to be involved in the up-regulation of the transferase.

Characterization of carnitine and fatty acid metabolism in the long-chain acyl-CoA dehydrogenase-deficient mouse.

In the present paper, we describe a novel method which enables the analysis of tissue acylcarnitines and carnitine biosynthesis intermediates in the same sample. This method was used to investigate the carnitine and fatty acid metabolism in wild-type and LCAD-/- (long-chain acyl-CoA dehydrogenase-deficient) mice. In agreement with previous results in plasma and bile, we found accumulation of the characteristic C14:1-acylcarnitine in all investigated tissues from LCAD-/- mice. Surprisingly, quantitatively relevant levels of 3-hydroxyacylcarnitines were found to be present in heart, muscle and brain in wild-type mice, suggesting that, in these tissues, long-chain 3-hydroxyacyl-CoA dehydrogenase is rate-limiting for mitochondrial beta-oxidation. The 3-hydroxyacylcarnitines were absent in LCAD-/- tissues, indicating that, in this situation, the beta-oxidation flux is limited by the LCAD deficiency. A profound deficiency of acetylcarnitine was observed in LCAD-/- hearts, which most likely corresponds with low cardiac levels of acetyl-CoA. Since there was no carnitine deficiency and only a marginal elevation of potentially cardiotoxic acylcarnitines, we conclude from these data that the cardiomyopathy in the LCAD-/- mouse is caused primarily by a severe energy deficiency in the heart, stressing the important role of LCAD in cardiac fatty acid metabolism in the mouse.

Method for the quantitative assay of fatty acid-bile acid conjugates by tandem mass spectrometry.

Fatty acid-bile acid conjugates and especially arachidyl amido cholic acid are synthetic molecules that were shown to prevent cholesterol gallstone formation in mice and hamsters as well as to dissolve pre-existing gallstones in mice. To measure these novel compounds we developed a liquid chromatography electrospray tandem mass spectrometry method based on the analysis of 100 microL of plasma with stearyl amido cholic acid (stamchol, 1.5 microM/L) added as internal standard. Repeatable calibrations between 0 and 50 microM/L exhibited consistent linearity and reproducibility. Inter- and intraassay C.V.s were 5.3-11.4% and 2.6-6.4%, respectively, at targeted concentrations of 0.1, 2.3 and 50 microM/L.

Cardiolipin deficiency in X-linked cardioskeletal myopathy and neutropenia (Barth syndrome, MIM 302060): a study in cultured skin fibroblasts.

We determined cardiolipin concentrations in cultured skin fibroblasts of 5 patients with X-linked cardioskeletal myopathy and neutropenia (Barth syndrome, MIM 302060) and in two groups of control patients. High-performance liquid chromatography-electrospray mass spectrometry was used to quantify total cardiolipin and subclasses of cardiolipin molecular species in cultured skin fibroblasts. Total cardiolipin and cardiolipin subclasses were decreased in patients with Barth syndrome as compared with normal control patients and disease control patients. Patients with Barth syndrome have a specific decrease of various cardiolipin molecular species, foremost tetralineoyl-cardiolipin. Therefore the analysis of cardiolipin in fibroblasts offers a specific biochemical approach to detect this disorder.

Quantitative and compositional study of cardiolipin in platelets by electrospray ionization mass spectrometry: application for the identification of Barth syndrome patients.

BACKGROUND: The concentration of cardiolipin (CL) in cultured skin fibroblasts is a useful indicator of Barth syndrome (BTHS; MIM 302060), but the sampling and culturing of fibroblasts are burdensome and time-consuming procedures. We investigated whether the analysis of CL in platelets might help to identify BTHS in patients suspected of having this condition. METHODS: We used HPLC and online electrospray ionization mass spectrometry (HPLC-ESI-MS) to quantify CL molecular species. The CL content of platelets was studied in blood samples of BTHS and non-BTHS patients. Control blood samples drawn from healthy adults were collected and analyzed within 24 h (n = 10) and 48 h (n = 10) to characterize any effect of sample shipping time on the CL content in platelets. Samples were collected from children 1-10 years of age who were not affected by BTHS (n = 6) and from BTHS patients (n = 4) and analyzed within 24 h. Results for all four groups were compared by a Student t-test for all individual analyses. RESULTS: We found different CL molecular species, e.g., (C18:2)(4)-CL. BTHS patients had a specific decrease of tetralinoleyl-CL concentrations in platelets (0.1-0.5 nmol/mg of protein; n = 4) compared with all control groups (2.3-5.5 nmol/mg of protein; n = 26). Only minor differences were observed among the different control groups. CONCLUSIONS: Quantitative and compositional analyses of CL in platelets by the proposed method allow identification of BTHS patients more rapidly than gene analysis or analysis of CL in cultured skin fibroblasts. The abnormality of CL may explain the abnormal mitochondrial function observed in BTHS. The differences between the control groups did not cause any complication.

Complete beta-oxidation of valproate: cleavage of 3-oxovalproyl-CoA by a mitochondrial 3-oxoacyl-CoA thiolase.

The beta-oxidation of valproic acid (VPA; 2-n-propylpentanoic acid) was investigated in vitro in intact rat liver mitochondria incubated with (3)H-labelled VPA. The metabolism of [4,5-(3)H(2)]VPA and [2-(3)H]VPA was studied by analysing the different acyl-CoA intermediates formed by reverse-phase HPLC with radiochemical detection. Valproyl-CoA, Delta(2(E))-valproyl-CoA,3-hydroxyvalproyl-CoA and 3-oxovalproyl-CoA (labelled and non-labelled) were determined using continuous on-line radiochemical and UV detection. The formation of these intermediates was investigated using the two tritiated precursors in respiratory states 3 and 4. Valproyl-CoA was present at highest concentrations under both conditions. Two distinct labelled peaks were found, which were identified as (3)H(2)O and [4,5-(3)H(2)]3-oxo-VPA. The formation of (3)H(2)O strongly suggested that VPA underwent complete beta-oxidation and that [4,5-(3)H(2)]3-oxo-VPA was formed by hydrolysis of the corresponding thioester. The hypothesis that 3-oxovalproyl-CoA undergoes thiolytic cleavage was investigated further. For this purpose a mito chondrial lysate was incubated with synthetic 3-oxovalproyl-CoA, carnitine and carnitine acetyltransferase for subsequent monitoring of the formation of propionylcarnitine and pentanoylcarnitine using electrospray ionization tandem MS. The detection of these compounds demonstrated unequivocally that the intermediate 3-oxovalproyl-CoA is a substrate of a mitochondrial thiolase, producing propionyl-CoA and pentanoyl-CoA, thus demonstrating the complete beta-oxidation of VPA in the mitochondrion. Our data should lead to a re-evaluation of the generally accepted concept that the biotransformation of VPA by mitochondrial beta-oxidation is incomplete.