Toward newborn screening of cerebrotendinous xanthomatosis: results of a biomarker research study using 32,000 newborn dried blood spots.

PURPOSE: Cerebrotendinous xanthomatosis (CTX) is a treatable hereditary disorder caused by the deficiency of sterol 27-hydroxylase, which is encoded by the CYP27A1 gene. Different newborn screening biomarkers for CTX have been described, including 7α,12α-dihydroxy-4-cholesten-3-one (7α12αC4), 5ß-cholestane-3α,7α,12α,25-tetrol glucuronide (GlcA-tetrol), the ratio of GlcA-tetrol to tauro-chenodeoxycholic acid (t-CDCA) (GlcA-tetrol/t-CDCA), and the ratio of tauro-trihydroxycholestanoic acid (t-THCA) to GlcA-tetrol (t-THCA/GlcA-tetrol). We set out to evaluate these screening methods in a research study using over 32,000 newborn dried blood spots (DBS). METHODS: Metabolites were extracted from DBS with methanol containing internal standard, which was then quantified by ultraperformance liquid chromatography tandem mass spectrometry (UPLC-MS/MS). RESULTS: The measurement of 7α12αC4 was complicated by isobaric interferences and was discontinued. A total of 32,737 newborns were screened based on the GlcA-tetrol concentration in DBS. GlcA-tetrol/t-CDCA and t-THCA/GlcA-tetrol ratios were also calculated. Newborns displaying both elevated GlcA-tetrol and GlcA-tetrol/t-CDCA ratio were considered to be screen positives. The t-THCA/GlcA-tetrol ratio was used to further distinguish CTX screen positives from Zellweger Spectrum Disorder (ZSD) screen positives. Only one newborn displayed both elevated GlcA-tetrol concentration in DBS and a typical CTX biochemical profile. This newborn was interpreted as a CTX-affected patient as CYP27A1 gene sequencing identified two known pathogenic variants. CONCLUSION: The results indicate that both GlcA-tetrol and the GlcA-tetrol/t-CDCA ratio are excellent CTX biomarkers suitable for newborn screening. By characterizing the relationship of GlcA-tetrol, t-CDCA, and t-THCA as secondary markers, 100% assay specificity can be achieved.

A newborn screening method for cerebrotendinous xanthomatosis using bile alcohol glucuronides and metabolite ratios.

Cerebrotendinous xanthomatosis (CTX) is a treatable neurodegenerative metabolic disorder of bile acid synthesis in which symptoms can be prevented if treatment with chenodeoxycholic acid supplementation is initiated early in life, making CTX an excellent candidate for newborn screening. We developed a new dried blood spot (DBS) screening assay for this disorder on the basis of different ratios between the accumulating cholestanetetrol glucuronide (tetrol) and specific bile acids/bile acid intermediates, without the need for derivatization. A quarter-inch DBS punch was extracted with methanol, internal standards were added, and after concentration the extract was injected into the tandem mass spectrometer using a 2 min flow injection analysis for which specific transitions were measured for cholestanetetrol glucuronide, taurochenodeoxycholic acid (t-CDCA), and taurotrihydroxycholestanoic acid (t-THCA). A proof-of-principle experiment was performed using 217 Guthrie cards from healthy term/preterm newborns, CTX patients, and Zellweger patients. Using two calculated biomarkers, tetrol:t-CDCA and t-THCA:tetrol, this straightforward method achieved an excellent separation between DBSs of CTX patients and those of controls, Zellweger patients, and newborns with cholestasis. The results of this small pilot study indicate that the tetrol:t-CDCA ratio is an excellent derived biomarker for CTX that has the potential to be used in neonatal screening programs.

Sodium taurocholate cotransporting polypeptide (SLC10A1) deficiency: conjugated hypercholanemia without a clear clinical phenotype.

UNLABELLED: The enterohepatic circulation of bile salts is an important physiological route to recycle bile salts and ensure intestinal absorption of dietary lipids. The Na(+)-taurocholate cotransporting polypeptide SLC10A1 (NTCP) plays a key role in this process as the major transporter of conjugated bile salts from the plasma compartment into the hepatocyte. Here we present the first patient with NTCP deficiency, who was clinically characterized by mild hypotonia, growth retardation, and delayed motor milestones. Total bile salts in plasma were extremely elevated (up to 1,500 µM, ref. <16.3) but there were no clinical signs of cholestatic jaundice, pruritis, or liver dysfunction. Bile salt synthesis and intestinal bile salt signaling were not affected, as evidenced by normal plasma 7α-hydroxy-4-cholesten-3-one (C4) and FGF19 levels. Importantly, the presence of secondary bile salts in the circulation suggested residual enterohepatic cycling of bile salts. Sequencing of the SLC10A1 gene revealed a single homozygous nonsynonymous point mutation in the coding sequence of the gene, resulting in an arginine to histidine substitution at position 252. Functional studies showed that this mutation resulted in a markedly reduced uptake activity of taurocholic acid. Immunofluorescence studies and surface biotinylation experiments demonstrated that the mutant protein is virtually absent from the plasma membrane. CONCLUSION: We describe the identification of NTCP deficiency as a new inborn error of metabolism with a relatively mild clinical phenotype. The identification of NTCP deficiency confirms that this transporter is the main import system for conjugated bile salts into the liver but also indicates that auxiliary transporters are able to sustain the enterohepatic cycle in its absence.

Principles and practice of lipidomics.

The technical advances in mass spectrometry, particularly the development of (ultra)-high-resolution/mass accuracy measurement capabilities in combination with refinement of soft ionization techniques, have increased the application and success of lipidomics to answer biological questions in relation to lipid metabolism. Together with other omics technologies, lipidomics has become an important tool to practice systems biology as lipids comprise a very significant part of the metabolome and play pleiotropic roles in cellular functions. As an increasing number of disorders are linked to lipid metabolism, lipidomics is used to search for biomarkers, understand disease mechanism and follow the efficacy of therapeutic options. This review provides a first introduction to the major methodological strategies currently used for mass spectrometry-based lipidomics and associated data pre-processing and analysis.

Newborn screening for Cerebrotendinous Xanthomatosis: A retrospective biomarker study using both flow-injection and UPLC-MS/MS analysis in 20,000 newborns.

BACKGROUND AND AIMS: Cerebrotendinous Xanthomatosis (CTX) is a treatable disorder of bile acid synthesis caused by deficiency of 27-sterol hydroxylase -encoded by CYP27A1- leading to gastrointestinal and progressive neuropsychiatric symptoms. Biochemically, CTX is characterized by accumulation of the bile alcohol cholestanetetrol glucuronide (GlcA-tetrol) and the deficiency of tauro-chenodeoxycholic acid (t-CDCA) and tauro-trihydroxycholestanoic acid (t-THCA). MATERIALS AND METHODS: To ascertain the feasibility of CTX newborn screening (NBS) we performed a study with deidentified Dutch dried blood spots using reagents and equipment that is frequently used in NBS laboratories. 20,076 deidentified newborn blood spots were subjected to flow-injection (FIA)-MS/MS and UPLC-MS/MS analysis to determine the concentration of GlcA-tetrol and calculate the GlcA-tetrol/t-CDCA and t-THCA/GlcA-tetrol ratios. RESULTS: Using UPLC-MS/MS analysis both GlcA-tetrol concentration and/or metabolite ratios GlcA-tetrol/t-CDCA proved to be informative biomarkers; newborn DBS results did not overlap with those of the CTX patients. For FIA-MS/MS, GlcA-tetrol also was an excellent marker but when using the combination of the GlcA-tetrol/t-CDCA and t-THCA/GlcA-tetrol ratios also did not yield any screen positives. CONCLUSION: Newborn screening for CTX using only metabolite ratios following the measurement of three CTX biomarkers is possible using either FIA-MS/MS or UPLC-MS/MS, which paves the way for introduction of CTX NBS.

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.

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.