Albert H. Bootsma

Quantitative plasma acylcarnitine analysis using electrospray tandem mass spectrometry for the diagnosis of organic acidaemias and fatty acid oxidation defects

Electrospray tandem mass spectrometry (ESI-MS/MS) of acylcarnitines has been successfully applied in newborn screening for defects in fatty acid oxidation and organic acidaemias (Millington et al 1990; Rashed et al 1995a, 1997). In addition, acylcarnitine analysis has also been applied for the selective screening for these disorders (Chace et al 1997; Van Hove et al 1993, 1995), for post-mortem diagnosis using bile fluid (Rashed et al 1995b) and for prenatal diagnosis of organic acidaemias and defects of fatty acid oxidation (Nada et al 1996; Shigematsu et al 1996). Since for selective screening for inborn errors of metabolism, apart from urine samples, mostly serum or plasma samples are sent in, we developed a quantitative ESI-MS/MS acylcarnitine analysis in plasma and included the analysis of free carnitine in the same assay. The results show that this analysis is highly sensitive and reproducible and therefore suitable for selective screening of fatty acid oxidation defects, organic acidaemias and secondary carnitine deficiency.

Rapid stable isotope dilution analysis of very-long-chain fatty acids, pristanic acid and phytanic acid using gas chromatography-electron impact mass spectrometry.

A common feature of most peroxisomal disorders is the accumulation of very-long-chain fatty acids (VLCFAs) and/or pristanic and phytanic acid in plasma. Previously described methods utilizing either gas chromatography alone or gas chromatography-mass spectrometry are, in general, time-consuming and unable to analyze VLCFAs, pristanic and phytanic acid within a single analysis. We describe a simple, reproducible and rapid method using gas chromatography/mass spectrometry with deuterated internal standards. The method was evaluated by analysing 30 control samples and samples from 35 patients with defined peroxisomal disorders and showed good discrimination between controls and patients. This method is suitable for routine screening for peroxisomal disorders.

Rapid analysis of conjugated bile acids in plasma using electrospray tandem mass spectrometry: application for selective screening of peroxisomal disorders

The final step in bile acid biosynthesis takes place in peroxisomes and involves oxidative cleavage of the side-chain of C 27 -β-cholestanoic acids, leading to the formation of the primary bile acids cholic and chenodeoxycholic acid. Therefore, in a number of peroxisomal disorders, including both peroxisomal biogenesis defects and isolated defects of peroxisomal β-oxidation, the accumulation of serum C 27 -5β-cholestanoic acids can be observed (Bjorkhem 1994; Clayton et al 1987; Libert et al 1991; Wanders et al 1995). Since routine gas chromatography mass-spectrometry (GC-MS) analysis of plasma bile acids requires laborious sample preparation including enzymatic and/or chemical deconjugation of plasma bile acids prior to derivatization and GC-MS analysis, we have developed a rapid HPLC electrospray tandem mass spectrometry (ESI-MS/MS) method for analysing taurine- and glycine-conjugated bile acids.

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.

Sodium Taurocholate Cotransporting Polypeptide (SLC10A1) Deficiency: Conjugated Hypercholanemia Without a Clear Clinical Phenotype

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. (Hepatology 2015;61:260–267)

Rapid diagnosis of organic acidemias and fatty-acid oxidation defects by quantitative electrospray tandem-MS acyl-carnitine analysis in plasma.

The analysis of circulating free carnitine and acyl-carnitines provides a powerful selective screening tool for genetic defects in mitochondrial fatty acid oxidation and defects in the catabolism of branched chain amino acids. Using electrospray tandem mass spectrometry (ESI/MS/MS) we developed a sensitive quantitative analysis of free carnitine and acyl-carnitines in plasma and/or serum. This method was evaluated by analyzing 250 control samples and 103 samples of patients suffering from twelve different defects in either mitochondrial fatty acid oxidation or the catabolism of branched chain amino acids. The reproducibility of the method was acceptable with a day-to-day coefficient of variation ranging from 6-15% for free carnitine and the different acylcarnitines. Except for one patient with a mild form of short chain acyl CoA dehydrogenase (SCAD) deficiency and a single sample from a patient with a mild form of multiple acyl CoA dehydrogenase (MAD) deficiency all patient samples were clearly abnormal under a wide variety of clinical conditions, illustrating the high sensitivity and specificity of the method.

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.

Oxidative breakdown and conversion of urocanic acid isomers by hydroxyl radical generating systems

cis-Urocanic acid (cis-UCA), formed from trans-urocanic acid (trans-UCA) by photoisomerization, has been shown to mimic suppressive effects of UV on the immune system. It is our hypothesis that UCA oxidation products in the skin play a role in the process of immunosuppression. Recently, both UCA isomers were found to be good hydroxyl radical scavengers and in this context we investigated the formation of products resulting from the interaction of hydroxyl radicals with UCA. Hydroxyl radicals were generated by (1) UV/H(2)O(2) (photooxidation), (2) ferrous ions/H(2)O(2) (Fenton oxidation) and (3) cupric ions/ascorbic acid. Oxidation products were identified by spectrometric methods and assessed by reversed-phase HPLC analysis. The photooxidation of UCA was induced by UV-B and UV-C, but not by UV-A radiation. Photooxidation and Fenton oxidation of trans-UCA, as well as of cis-UCA yielded comparable chromatographic patterns of UCA oxidation products. Several of the formed products were identified. The formation of three identified imidazoles was shown in UV-B exposed corneal layer samples, derived from human skin.

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.

The application of HPLC/ESI tandem mass spectrometry on urine-soaked filter-paper strips for the screening of disorders of purine and pyrimidine metabolism

Inherited disorders of purine and pyrimidine metabolism have a wide variety of clinical presentations that are often non-specific, and severity of individual cases ranges from fatal to asymptomatic. Because of this broad clinical spectrum, a simple, rapid and specific screening method is desired for the diagnosis of these disorders. Several high-performance liquid chromatography (HPLC) methods for screening for these disorders have been reported (Duran et al 1997; Sumi et al 1995), but so far these methods are time-consuming and non-specific. Connection of HPLC with tandem mass spectrometry (MS/MS) has the advantage of rapidity and specificity. We describe an analytical method for routinely screening disorders of purine and pyrimidine metabolism on urine-soaked filter-paper strips using reversed-phase HPLC coupled with electrospray ionization (ESI) MS/MS. This method enables easy sample collection, even from the patients home, mailing and storage (Blau et al 1998; McCann et al 1995).