Quantitative acylcarnitine determination by UHPLC-MS/MS--Going beyond tandem MS acylcarnitine "profiles".

Tandem MS "profiling" of acylcarnitines and amino acids was conceived as a first-tier screening method, and its application to expanded newborn screening has been enormously successful. However, unlike amino acid screening (which uses amino acid analysis as its second-tier validation of screening results), acylcarnitine "profiling" also assumed the role of second-tier validation, due to the lack of a generally accepted second-tier acylcarnitine determination method. In this report, we present results from the application of our validated UHPLC-MS/MS second-tier method for the quantification of total carnitine, free carnitine, butyrobetaine, and acylcarnitines to patient samples with known diagnoses: malonic acidemia, short-chain acyl-CoA dehydrogenase deficiency (SCADD) or isobutyryl-CoA dehydrogenase deficiency (IBD), 3-methyl-crotonyl carboxylase deficiency (3-MCC) or ß-ketothiolase deficiency (BKT), and methylmalonic acidemia (MMA). We demonstrate the assay's ability to separate constitutional isomers and diastereomeric acylcarnitines and generate values with a high level of accuracy and precision. These capabilities are unavailable when using tandem MS "profiles". We also show examples of research interest, where separation of acylcarnitine species and accurate and precise acylcarnitine quantification is necessary.

The first case of mitochondrial acetoacetyl-CoA thiolase deficiency identified by expanded newborn metabolic screening in Italy: the importance of an integrated diagnostic approach.

A pilot expanded newborn screening programme to detect inherited metabolic disorders by means of liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) began in the Campania region, southern Italy, in 2007. By October 2009, >8,800 dried blood samples on filter paper from 11 hospitals had been screened. Within this screening programme, we identified a case of mitochondrial acetoacetyl-coenzyme A (CoA) thiolase deficiency [ß-ketothiolase (ß-KT) deficiency] by analysing the acylcarnitine profile from a dried blood spot with LC-MS/MS. Gas chromatography coupled with mass spectrometry analysis of urinary organic acids and LC-MS/MS analysis of urinary acylcarnitines were in line with this disorder. In fact, concentrations were well beyond the cut-off values of tiglyl carnitine, 3-hydroxybutyrylcarnitine and 2-methyl-3-hydroxybutyrylcarnitine, 2-methyl-3-hydroxybutyric acid and tiglyl glycine. The absence of 2-methylacetoacetic acid in urine may be attributed to: (i) the instability of this ß-ketoacid because it undergoes spontaneous decarboxylation to 2-butanone, which is highly volatile and thus difficult to detect, and (ii) the good health of the patient in the first days of life. ß-KT deficiency was subsequently diagnosed in the patient's older sister, who showed increased levels of the same metabolites but also small amounts of 2-methylacetoacetic acid, which is considered a key marker for ß-KT diagnosis. Genomic analysis revealed mutation c.1189C >G in exon 12 of the ACAT1 gene, which results in a severe defect because of the p.H397D amino acid change in both alleles of both patients.

NMR-based urinalysis for beta-ketothiolase deficiency.

BACKGROUND: Beta-ketothiolase deficiency is a rare inborn errors of metabolism (IEM) affecting the catabolism of isoleucine, characterized by severe ketoacidosis in children of 6 to 24months old. A prompt diagnosis is of paramount importance as the metabolic decompensation can be effectively reverted by glucose infusion and health outcomes are improved on a protein-restricted diet. Currently, majority of the laboratory diagnosis were made based on mass-spectrometry and molecular genetics while little is mentioned on the advancement of nuclear magnetic resonance (NMR) spectroscopy for the diagnosis of this condition. CASE: We report a case of beta-ketothiolase deficiency in a 1-y-old Chinese boy who presented with repeated vomiting, impaired consciousness and severe ketoacidosis. NMR urinalysis detected excessive amount of butanone (a disease specific marker of beta-ketothiolase deficiency), tiglylglycine, (intermediate of isoleucine catabolism) and ketones. Diagnosis of beta-ketothiolase deficiency was further established by molecular genetic studies of ACAT1 gene of the proband. CONCLUSIONS: This case illustrated that NMR-based urinalysis is complementary to organic acid analysis for diagnosis of beta-ketothiolase deficiency. The operation of NMR is simple and fast; sample preparation is a two-step procedure while the NMR acquisition is automatic and usually takes <15min. We envisage that NMR analysis will become more available in clinical laboratories and will play an important role in acute pediatric care.

Beta-ketothiolase deficiency brought with lethargy: case report.

Beta-ketothiolase deficiency is a rare autosomal recessive disorder of isoleucine and ketone body metabolism. This disorder is clinically characterized by ketoacidotic attacks. Ketoacidosis, vomiting, and dehydration, lethargy and coma may be seen during attacks. A 9-month-old girl was admitted to our hospital with acidosis and dehydration. The patient was lethargic. Ketoacidosis was suspected because of acetone odor on her breath. Her blood glucose level was 262 mg/dL and urine ketone was (++++). Branched chain amino acid levels were elevated in her blood sample. Organic acid analysis of urine revealed 2-methylacetoacetyl-CoA thiolase deficiency. This was reported because of rarity of the disease and we should consider it in the differential diagnosis of ketoacidotic episodes.