IEM are individually rare but may be collectively as frequent as 1 affected individual in 500 newborn infants. They encompass many different single-gene disorders affecting many aspects of cellular metabolism . A significant portion relates to disorders of fatty acid, protein, and carbohydrate metabolism . Analysis of IEM relies on semiquantitative or quantitative measurement of characteristic small molecules in different body fluids including blood, plasma, cerebral spinal fluid, and urine [11,12]. Diagnosis must then be typically
confirmed using enzyme analysis in appropriate tissues and by genotyping. Some selected applications of MS for IEM are listed in Table 1. The complexity of a selected pathway is shown in Fig. 2 using the methionine-homocysteine cycle as example.
Homocysteine is markedly elevated in different inborn errors of homocysteine metabolism such as cystathionine ^-synthase, methionine synthase deficiencies,
Selected applications of mass spectrometry in IEM
Cholesterol and metabolites
Bile acid intermediates
Fatty acid oxidation defects, organic acidopathies Amino acidopathies (PKU, tyrosinemia type I)
SLOd and other defects of cholesterol biosynthesis Disorders of bile acid biosynthesis, peroxisomal disorders
Dried blood plasma
Dried blood plasma, urine
Dried blood plasma, urine Dried blood plasma Plasma
ESI-MS/MS: electrospray tandem mass spectrometry; GC-MS: gas chromatography-mass spectrometry; LC-MS/MS: liquid chromatography-tandem mass spectrometry.
a GM1 gangliosidosis, GM2 gangliosidosis, sialic acid storage disorder, sialidase/neuraminidase deficiency, galactosialidosis, I-cell disease, fucosidosis, and Pompe and Gaucher diseases. b Lysosomal storage disorder including Gaucher, Fabry, Niemann-Pick A/B, Krabbe, and Pompe diseases.
c Guanidinoacetate methyltransferase deficiency. d Smith-Lemli Opitz syndrome (defect of cholesterol biosynthesis).
and disorders of vitamin B12 metabolism affecting the conversion of Cbl-I to Cbl-II (Fig. 2). Although betaine-homocysteine methyltransferase deficiency in mice is known to cause hyperhomocysteinemia, this defect has not been reported in humans. In contrast, S-adenosyl homocysteine hydrolase deficiency may actually lead to low homocysteine levels. All compounds in this pathway can be analyzed although only few have clinical significance (homocysteine, methionine, arginine, ornithine, glycine, guanidinoacetate). Among the latter homocysteine may serve as an important biomarker to evaluate treatment efficacy and future risk for premature artherosclerosis. Analysis of homocysteine is readily made by LC-MS/ MS or ESI-MS/ MS . This approach allows fast sample turnover and consequently screening of at-risk populations.
Cysteine t t
Fig. 2. Methionine-homocysteine metabolism as an example for the complexity of intermediary metabolism. Most metabolites in the depicted pathways can be quantified by either GC-MS or ESI-MS/MS (AGAT: arginine-guanidinoacetate amidinotransferase; GAMT: guanidionacetate methyltransferase).
3.2. Analysis of organic acids including orotic acid
Organic acidemias, also known as organic acidurias, are a group of disorders characterized by increased excretion of organic acids in urine. They result primarily from deficiencies of specific enzymes in the breakdown pathways of amino acids or from enzyme deficiencies in ^-oxidation of fatty acids or carbohydrate metabolism. Organic acidemias can be classified into five categories including branched-chain organic acidemias, multiple carboxylase deficiency, including holocarboxylase synthetase deficiency and biotinidase deficiency, glutaric aciduria type I and related organic acidemias, fatty acid oxidation defects, and disorders of energy metabolism. For example, the diagnosis of methylmalonic aciduria (MMA) is made by measurement of organic acids in the urine using GC-MS. In MMAlarge amounts of methylmalonic acid, as well as methylcitrate, propionic acid, and 3-OH propionic acid, are present [14,15].
The application of ESI-MS/MS allows the identification and quantification of individual oligosaccharides for the diagnosis of glycoproteinoses (oligosaccharidurias)
such as GM1 gangliosidosis, GM2 gangliosidosis, sialic acid storage disorder, sialidase/ neuraminidase deficiency, galactosialidosis, I-cell disease, fucosidosis, and Pompe and Gaucher diseases . Recent work demonstrated the feasibility of this approach using 1-phenyl-3-methyl-5-pyrazolone derivatization and MS/MS precursor scan of m/z 175 in positive ion mode . This method has been adapted to high-throughput use allowing the application to management follow-up and eventually newborn screening for this group of disorders .
Similarly, a direct multiplex assay of lysosomal enzymes in dried blood spots has been developed for newborn screening . This approach is based on the incubation of dried blood spots at 37°C overnight with the appropriate substrates and stable isotopically labeled internal standards. If the enzyme was fully active, substrate was converted completely to the corresponding product which was quantified based on its relationship to the known concentration of the internal standard. Importantly, samples without dried blood spots ("blank") have to be used to adjust for background noise. Corresponding enzyme activities were calculated based on the assumption that 10 (xl of extraction solution contained 0.98 (xl of blood .
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