Assessment of methylcitrate and methylcitrate to citrate ratio in dried blood spots as biomarkers for inborn errors of propionate metabolism.

Deficiency of propionyl-CoA carboxylase causes propionic acidemia and deficiencies of methylmalonyl-CoA mutase or its cofactor adenosylcobalamin cause methylmalonic acidemia. These inherited disorders lead to pathological accumulation of propionyl-CoA which is converted in Krebs cycle to methylcitrate (MCA) in a reaction catalyzed by citrate synthase. In healthy individuals where no propionyl-CoA accumulation occurs, this enzyme drives the condensation of acetyl-CoA with oxaloacetate to produce citric acid (CA), a normal Krebs cycle intermediate. The competitive synthesis of CA and MCA through the same enzymatic mechanism implies that increase in MCA production is accompanied by decrease in CA levels. In this study, we assessed MCA concentration and the ratio of MCA/CA as plausible markers for propionic and methylmalonic acidemias. We measured MCA and CA in dried blood spots using liquid chromatography tandem mass spectrometry. The reference ranges of MCA, CA and MCA/CA in 123 healthy individuals were ≤0.63 µmol/L, 36.6-126.4 µmol/L and 0.0019-0.0074, respectively. In patients with propionic and methylmalnic acidemias (n = 7), MCA concentration ranged between 1.0-12.0 µmol/L whereas MCA/CA was between 0.012-0.279. This is the first report to describe the potential role of MCA and MCA/CA in dried blood spots as diagnostic and monitoring biomarkers for inherited disorders of propionyl-CoA metabolism.

Quantification of methylcitrate in dried urine spots by liquid chromatography tandem mass spectrometry for the diagnosis of propionic and methylmalonic acidemias.

Accumulation of methylcitrate is a biochemical hallmark of inborn errors of propionate metabolism, a group of disorders that include propionic acidemia, methylmalonic aciduria and cobalamin defects. In clinical laboratories, this analyte is measured without quantification by gas chromatography mass spectrometry as part of urine organic acids. Here we describe a simple, sensitive and specific method to quantify methylcitrate in dried urine spots by liquid chromatography tandem mass spectrometry. Methylcitrate is extracted and derivatized with 4-[2-(N,N-dimethylamino)ethylaminosulfonyl]-7-(2-aminoethylamino)-2,1,3-benzoxadiazole in a single step. A derivatization mixture was added to 3.2 mm disc of dried urine spots, incubated at 65 °C for 45 min and 4 µl of the reaction mixture were analyzed. Separation was achieved on C18 column with methylcitrate eluting at 3.8 min. Intraday and interday imprecision (n = 17) were ≤20.9%. The method was applied on dried urine spots from established patients and controls. In controls (n = 135), methylcitrate reference interval of 0.4-3.4 mmol/mol creatinine. In patients, methylcitrate ranged between 8.3 and 591 mmol/mol creatinine. Quantification of methylcitrate provides important diagnostic clues for propionic acidemia, methylmalonic aciduria and cobalamin disorders. The potential utilization of methylcitrate as monitoring biomarker of patients under treatment and whether it correlates with the clinical status has yet to be established.