The Stable Isotope Dilution Method for Measurement of Methylmalonic Acid: a Highly Accurate Approach to the Prenatal Diagnosis of Methylmalonic Acidemia

Abstract

SummaryWe report a stable isotope dilution method for the accurate measurement of methylmalonic acid (MMA) in amniotic fluid and its successful application to the prenatal diagnosis of methylmalonic acidemia. The stable isotopically-labeled analogue of MMA, (2-[2H3]methyl)malonic acid, was synthesized and used as an internal standard. Samples were extracted, methylated, and analyzed by chemical ionization gas chromatography-mass spectrometry (CI-GC-MS) operated in the selected ion monitoring (SIM) mode. The analytical method is rapid (8 h), ultrasensitive (lower limit -10 ng/ml), linear over three orders of magnitude, and reproducible. The concentration of MMA in amniotic fluids from normal pregnant women was 38 2 15 ng/ml (mean + 1 S.D.). This value is 20-fold less than our previous estimate by gas chromatographic analysis, indicating that interference by co-eluting materials was eliminated by use of SIM in the present method. Amniotic fluids from eight at-risk pregnancies were examined. MMA concentration in six of the pregnancies was in the normal range whereas the concentration in the remaining two pregnancies was elevated by greater than two orders of magnitude (125-250-fold). Analysis of urine from ten at-risk pregnancies showed a smaller (3%-9-fold) but significant increase in urinary MMA excretion in two women carrying affected fetuses. A control group of normal pregnant women had a mean urinary MMA excretion of 1.7 + 0.5 pg/mg creatinine, in excellent agreement with previous estimates. There was concordance in prenatal diagnosis of fetal status in all cases between amniotic fluid cell studies and MMA measurements in amniotic fluid and/or maternal urine. Amniotic fluid MMA determination by the stable isotope dilution technique provides a highly accurate technique for prenatal diagnosis of methylmalonic acidemia that is complementary to amniotic fluid cell studies, and may supplant such studies in selected instances. SpeculationThe stable isotope dilution method may be extended to prenatal diagnosis of other inborn errors of metabolism in which low molecular weight metabolites accumulate in amniotic fluid. The concentrations of metabolites, which accumulate in some of these disorders, are below the sensitivity limits of currently available analytical techniques and it is thus impossible to distinguish differences that may be present in pregnancies carrying affected and unaffected fetuses. The enhanced sensitivity of the stable isotope dilution method may permit the desired discrimination.The inherited methylmalonic acidemias are a group of metabolic disorders characterized clinicallv bv rotei in intolerance.

Stable Isotope Dilution Analysis of Isovalerylglycine in Amniotic Fluid and Urine and Its Application for the Prenatal Diagnosis of Isovaleric Acidemia

“…In each case, the protonated molecular ion (MH') is the base peak: these are the peaks at m/z 174 and 177 for IVG and D3-IVG, respectively. There are several other much smaller but ions are commonly observed in CI mass spectra using methane gas as reactant gas ( 13).…”

“…The stable isotope dilution method was employed since it provides the ultra-sensitivity necessary to accurately measure small amounts of IVG in normal amniotic fluids as well as those from affected pregnancies. It is fast, requiring only several hours (13). In our method presented here, we synthesized D3-IVG, (Fig.…”

Prenatal Diagnosis and Neonatal Monitoring of a Fetus with Glutaric Aciduria Type II Due to Electron Transfer Flavoprotein (β-Subunit) Deficiency

“…Prenatal diagnosis by amniotic fluid analysis using a stable isotope dilution method have been reported in several organic acidurias such as methylmalonic acidemia (22)(23)(24), isovaleric acidemia (25), and glutaric aciduria type I (26). In many of these cases, a substantial difference in concentration of the diagnostic compounds between normal controls and the affected fetus has been detectable.…”

Assessment of an electron‐impact GC‐MS method for organic acids and glycine conjugates in amniotic fluid

“…methylcitrate, glutarate, succinylacetone and glycine derivatives) are poorly quanti¢ed or even remain undetected. While sensitive and reliable methods have been described for the quantitative analysis of numbers of organic acids and glycine conjugates (Baric et al 1999;Gibson et al 1990Gibson et al , 1993Hine et al 1986;Inoue and Kuhara 2002;Jakobs et al 1984b^e, 1988Jakobs et al 1984b^e, , 1991Kelley and Stamas 1993;Kretschmer and Bachmann 1988;Naylor et al 1980;Rinaldo et al 1988;Schor et al 2002;Trefz et al 1981;Zinn et al 1982), these are usually devoted to one analyte only. Moreover, these methods involve fragmentation devices (chemical ionization or electron-capture negative-ionization modes) that have not yet been generalized and/or the use of isotopic internal standards.…”