Long chain base (LCB) is a unique building block found in sphingolipids. The initial step of LCB biosynthesis stems from serine:palmitoyl-CoA transferase enzyme, producing 3-ketodihydrosphingosine with multiple regulatory proteins including small subunit SPT a/b and orosomucoid-like protein1-3. 3-Ketodihydrosphingosine reductase and sphingolipid Δ4-desaturase, both of them poorly characterized mammalian enzymes, play key roles for neurological homeostasis based on their pathogenic mutation in humans. Ceramide synthase in mammals has six isoforms with distinct phenotype in each knockout mouse. In plants and fungi, sphingolipids also contain phytosphingosine due to sphingolipid C4-hydroxylase. In contrast to previous notion that dietary intake might be its major route in animals, emerging evidences suggested that phytosphingosine biosynthesis does occur in some tissues such as the skin by mammalian C4-hydroxylase activity of the DEGS2 gene. This short review summarizes LCB biosynthesis with their associating metabolic pathways in animals, plants and fungi.

Mucopolysaccharidosis (MPS) is a genetic disorder characterized by the accumulation of glycosaminoglycans in the body. Of the multiple MPS disease subtypes, several are caused by defects in sulfatases. Specifically, a defect in iduronate-2-sulfatase (ID2S) leads to MPS II, whereas N-acetylgalactosamine-6-sulfatase (GALN) and N-acetylgalactosamine-4-sulfatase (ARSB) defects relate to MPS IVA and MPS VI, respectively. A previous study reported a combined assay for these three disorders in a 96-well plate using a liquid chromatography-tandem mass spectrometry (LC-MS/MS)-based technique (Kumar et al., Clin Chem 2015 61(11):1363-1371). In our study, we applied this methodology to a Japanese population to examine the assay precision and the separation of populations between disease-affected individuals and controls for these three disorders. Within our assay conditions, the coefficient of variation (CV, %) values for an interday assay of ID2S, GALN, and ARSB were 9%, 18%, and 9%, respectively (n = 7). The average enzyme activities of ID2S, GALN, and ARSB in random neonates were 19.6 ± 5.8, 1.7 ± 0.7, and 13.4 ± 5.2 μmol/h/L (mean ± SD, n = 240), respectively. In contrast, the average enzyme activities of ID2S, GALN, and ARSB in disease-affected individuals were 0.5 ± 0.2 (n = 6), 0.3 ± 0.1 (n = 3), and 0.3 (n = 1) μmol/h/L, respectively. The representative analytical range values corresponding to ID2S, GALN, and ARSB were 39, 17, and 168, respectively. These results raise the possibility that the population of disease-affected individuals could be separated from that of healthy individuals using the LC-MS/MS-based technique.

AstractLysosomal storage disorders (LSDs) are characterized by the accumulation of lipids, glycolipids, oligosaccharides, mucopolysaccharides, and other biological substances because of the pathogenic deficiency of lysosomal enzymes. Such diseases are rare; thus, a multiplex assay for these disorders is effective for the identification of affected individuals during the presymptomatic period. Previous studies have demonstrated that such assays can be performed using liquid chromatography-tandem mass spectrometry (LC-MS/MS) with multiple reaction monitoring (MRM) detection. An assay procedure to quantify the activity of 11 enzymes associated with LSDs was provided. First, a validation study was performed using dried blood spot (DBS) samples with 100% and 5% enzyme activity for quality control (QC). Under the assay condition, the analytical range, defined as the ratio of the peak area of the enzyme reaction products from the DBS for QC with 100% enzyme activity to that from the filter paper blank sample, was between 14 for GALN and 4561 for GLA. Based on these results, the distribution of the enzyme activity for the 11 LSD enzymes was further examined. Consistent with the previous data, the enzyme activity exhibited a bell-shaped distribution with a single peak. The averaged enzyme activity for the healthy neonates was as follows: GLA, 3.80 ± 1.6; GAA, 10.6 ± 4.8; IDUA, 6.4 ± 2.3; ABG, 8.6 ± 3.1; ASM, 3.3 ± 1.1; GALC, 2.8 ± 1.3; ID2S, 16.7 ± 6.1; GALN, 1.2 ± 0.5; ARSB, 17.0 ± 8.7; NAGLU, 4.6 ± 1.5; and GUSB, 46.6 ± 19.0 μmol/h/L (mean ± SD, n = 200). In contrast, the enzyme activity in disease-affected individuals was lower than the minimum enzyme activity in healthy neonates. The results demonstrate that the population of disease-affected individuals was distinguished from that of healthy individuals by the use of LC-MS/MS.

Lysosomal storage disorders (LSDs) are characterized by an accumulation of various substances, such as sphingolipids, mucopolysaccharides, and oligosaccharides. The LSD enzymes responsible for the catabolism are active at acidic pH in the lysosomal compartment. In addition to the classically established lysosomal degradation biochemistry, recent data have suggested that lysosome plays a key role in the autophagy where the fusion of autophagosome and lysosome facilitates the degradation of amino acids. A failure in the lysosomal function leads to a variety of manifestations, including neurovisceral disorders. While affected individuals appear to be normal at birth, they gradually become symptomatic in childhood. Biomarkers for each condition have been well-documented and their proper selection helps to perform accurate clinical diagnoses. Based on the natural history of disorders, it is now evident that the existing treatment becomes most effective when initiated during presymptomatic period. Neonatal screening provides such a platform for inborn error of metabolism in general and is now expanding to LSDs as well. These are implemented in some areas and countries, including Taiwan and the U.S. In this short review, we will discuss several issues on some selected biomarkers for LSDs involving Fabry, Niemann–Pick disease type C, mucopolysaccharidosis, and oligosaccharidosis, with a focus on mass spectrometry application to biomarker discovery and detection.

Heparan sulfate (HS) is a type of glycosaminoglycan that plays a key role in a variety of biological functions in neurology, skeletal development, immunology, and tumor metastasis. Biosynthesis of HS is initiated by a link of xylose to Ser residue of HS proteoglycans, followed by the formation of a linker tetrasaccharide. Then, an extension reaction of HS disaccharide occurs through polymerization of many repetitive units consisting of iduronic acid and N-acetylglucosamine. Subsequently, several modification reactions take place to complete the maturation of HS. The sulfation positions of N-, 2-O-, 6-O-, and 3-O- are all mediated by specific enzymes that may have multiple isozymes. C5-epimerization is facilitated by the epimerase enzyme that converts glucuronic acid to iduronic acid. Once these enzymatic reactions have been completed, the desulfation reaction further modifies HS. Apart from HS biosynthesis, the degradation of HS is largely mediated by the lysosome, an intracellular organelle with acidic pH. Mucopolysaccharidosis is a genetic disorder characterized by an accumulation of glycosaminoglycans in the body associated with neuronal, skeletal, and visceral disorders. Genetically modified animal models have significantly contributed to the understanding of the in vivo role of these enzymes. Their role and potential link to diseases are also discussed.