Stable-isotope dilution gas chromatography–mass spectrometric measurement of 3-hydroxyglutaric acid, glutaric acid and related metabolites in body fluids of patients with glutaric aciduria type 1 found in newborn screening

Abstract

We developed a simple and sensitive stable-isotope dilution method for the quantification of 3-hydroxyglutaric acid (3HGA) and glutaric acid (GA) in body fluids. In our method, tert-butyldimethylsilyl (tBDMS) derivatives of 3HGA and GA were measured with a conventional electron-impact ionization (EI) mode in gas chromatography-mass spectrometry (GC-MS). The control values for 3HGA in nmol/ml were 0.15+/-0.08 (serum; n=10) and 0.07+/-0.03 (CSF; n=10). In addition, glutarylcarnitine and free carnitine were quantified by electrospray tandem mass spectrometry. Using these methods, we monitored 3HGA, GA, and glutarylcarnitine in the body fluids of three patients with glutaric aciduria type 1 found during newborn screening. None of the patients had experienced neurological strokes, which are possibly caused by the accumulation of 3HGA, at 15-24 months of age under a disease-specific treatment, including carnitine supplementation. Our data showed that 3HGA levels were relatively high in some serum samples with lower glutarylcarnitine and carnitine levels, suggesting that carnitine supplementation may play a role in preventing the accumulation of 3HGA in patients with this disease.

Diagnosis and management of glutaric aciduria type I – revised recommendations

“…A positive MS/MS screening result should be confirmed by one or more alternative techniques, including a sensitive method for quantitative determination of GA and 3-OH-GA in urine or blood such as GC/MS (Al-Dirbashi et al 2005; Baric et al 1999; Shigematsu et al 2005), mutation analysis of the GCDH gene, and/or GCDH enzyme analysis (Christensen 1983; Goodman et al 1998). A normal urine or blood 3-OH-GA level will make the diagnosis unlikely, whereas an elevated urine or blood level of 3-OH-GA should be followed by mutation analysis and start of treatment.…”

ESI–MS/MS study of acylcarnitine profiles in urine from patients with organic acidemias and fatty acid oxidation disorders

“…MMA-emia and PPA-emia are the most common OAs, and are detectable through an elevation of C3, C3/C2 or C3/C16 in MS/MS screening using blood filter paper. However, diagnostic difficulty using MS/MS screening of dried blood spots has been reported in some cases of MMA (particularly for the mild form), and in such cases a confirmatory diagnosis can be made by GC/MS analysis of urine samples [5,13]. Our results also suggested that the mild form of MMA cannot be detected in MS/MS screening, although some cases might be detectable with urinary acylcarnitine analysis under carnitine loading.…”

“…MS/MS is a very sensitive, fast and powerful tool, and acylcarnitine screening by MS/MS requires only a small piece of filter paper containing dried blood. However, it is sometimes difficult to make a definite diagnosis by MS/MS analysis alone [6,13], and a confirmatory test such as urinary organic acid analysis using GC/MS [12], enzyme determination, or molecular analysis may be required [3]. Urinary organic acid analysis is useful for confirmation of diagnosis of many kinds of OAs, and often provides more detailed information on the condition and severity of OA patients; however, this technique is time-consuming and requires special skills for interpretation of data [12,14].…”

“…These cases included five of mild MMA-emia (cases [12][13][14][15][16], in which elevated excretion of relatively small amounts of methylmalonic acid and methylcitric acid were found in urinary organic acid analysis by GC/MS. Three of the 11 patients with severe MMAemia (cases 9, 10 and 11) and two patients with mild MMA-emia (cases 15 and 16) were receiving carnitine treatment.…”

Useful second‐tier tests in expanded newborn screening of isovaleric acidemia and methylmalonic aciduria

“…The extract solution collected from three wells for each individual was transferred to a glass test tube and dried under a nitrogen stream. The dried residue was derivatized using N-methyl-N-(tert-butyldimethylsilyl)-trifluoroacetamide (Shigematsu et al 2005). GC/MS analysis was performed on a model DSQ GC/MS (ThermoFisher, Tokyo, Japan) using a capillary column, SPB-50 (30 m × 0.25 mm inner diameter, film thickness 0.25 mm) (Supelco, Tokyo, Japan).…”