Identification of a novel selenium metabolite, Se-methyl-N-acetylselenohexosamine, in rat urine by high-performance liquid chromatography--inductively coupled plasma mass spectrometry and--electrospray ionization tandem mass spectrometry

The major urinary metabolite of selenium (Se) in rats was identified by HPLC-inductively coupled argon plasma mass spectrometry (ICP-MS) and--electrospray tandem mass spectrometry (ESI-MS/MS). As the urine sample was rich in matrices such as sodium chloride and urea, it was partially purified to meet the requirements for ESI-MS. The group of signals corresponding to the Se isotope ratio was detected in both the positive and negative ion modes at m/z 300 ([M+H]+) and 358 ([M+CH3COO]-) for 80Se, respectively. These results suggested that the molecular mass of the Se metabolite was 299 Da for 80Se. The Se metabolite was deduced to contain one methylselenyl group, one acetyl group and at least two hydroxyl groups from the mass spectra of the fragment ions. The spectrum of the Se metabolite was completely identical to that of the synthetic selenosugar, 2-acetamide-1,2-dideoxy-beta-D-glucopyranosyl methylselenide. However, the chromatographic behavior of the Se metabolite was slightly different from that of the synthetic selenosugar. Thus, the major urinary Se metabolite was assigned as a diastereomer of a selenosugar, Se-methyl-N-acetyl-selenohexosamine.     

Screening urine of 3-week-old newborns: transient methylmalonic and hydroxyphenyllactic aciduria.

Urinary methylmalonic acid (MMA) and 4-hydroxyphenyllactic acid (HPL) have been determined in 3345 and 2498 3-week-old newborns, respectively. Urine was collected onto filter paper and assayed by a rapid gas chromatography-mass spectrometry method. Forty-six infants (1.7%) had elevated MMA levels (greater than 58.5 micrograms/mg creatinine, means + 5 SD) and 31 infants (1.2%) had elevated levels of HPL (greater than 87.7 micrograms/mg creatinine, means + 5 SD). Fifteen infants with elevated values of MMA were retested from one to several months after the first test. In 12 infants the MMA levels normalized, while in the remaining three, elevated methylmalonic acid persisted. Nine infants with elevated values of HPL were retested, and in all except one, HPL levels normalized. No access to clinical evaluation of the infants was available. Transient methylmalonic aciduria and transient tyrosyluria affect a substantial number of infants and the clinical significance of this phenomenon has yet to be determined.     

Two cases of benign methylmalonic aciduria detected during a pilot study of neonatal urine screening

Two cases of benign methylmalonic aciduria (MMAuria) were found among 9780 neonatal screenings using the previously described screening method consisting of urease digestion, ethanol deproteinization and gas chromatography-mass spectrometry. Combining this screening method with the stable isotope dilution technique showed very specific and sensitive measurements of methylmalonic acid in urine. The concentrations of urinary methylmalonic acid were measured at several ages. The levels of urinary methylmalonic acid in two patients varied from 0.27 to 3.04 mol/mol creatinine (control<0.01 mol/mol creatinine). Methylcitrate and homocystine were not increased in the patient's urine or blood. Blood propionylcarnitine was also at normal levels. The urinary methylmalonate excretions were decreased to the levels of about 50% of the start point after vitamin B12 treatment in one patient, but the other patient showed no change. No clinical abnormalities were observed during these periods.     

Identification of NAc-HCPC and NAc-beta-CEC, and qualitative analyses of sulphur amino acids in the urine of a patient with cystathioninuria using liquid chromatography/atmospheric pressure ionization mass spectrometry.

Standard sulphur amino acids and various cystathionine metabolites in the urine of a patient with cystathioninuria were analysed using liquid chromatography/mass spectrometry with an atmospheric pressure ionization interface system. Very intense quasi-molecular ions ([M + H]+) of synthetic cystathionine, N-monoacetylcystathionine, perhydro-1,4-thiazepine-3,5-dicarboxylic acid, S-(3-hydroxy-3-carboxy-n-propyl)cysteine, S-(2-carboxyethyl) cysteine, S-(2-hydroxy-2-carboxyethyl)homocysteine, S-(carboxymethyl)homocysteine, N-acetyl-S-(3-hydroxy-3-carboxy-n-propyl)cysteine and N-acetyl-S-(2-carboxyethyl)cysteine were observed by this method. Quasi-molecular ions ([M + H]+) of these sulphur amino acids were observed in the urine sample of the patient with cystathioninuria, and N-acetyl-HCPC and N-acetyl-beta-CEC as N-substituted sulphur amino acids were also identified in the urine of the same patient.     

Methylmalonic acid administration induces DNA damage in rat brain and kidney

Accumulation of methylmalonic acid (MMA) in tissues and biological fluids is the biochemical hallmark of methylmalonic aciduria. Affected patients present renal failure and severe neurological findings. Considering that the underlying pathomechanisms of tissue damage are not yet understood, in the present work we assessed the in vivo e in vitro effects of MMA on DNA damage in brain and kidney, as well as on p53 and caspase 3 levels, in the presence or absence of gentamicin (acute renal failure model). For in vitro studies, tissue prisms were incubated in the presence of different concentrations of MMA and/or gentamicin for one hour. For in vivo studies, animals received a single injection of gentamicin (70 mg/kg) and/or three injections of MMA (1.67 μmol/g; 11 h interval between injections). The animals were killed 1 h after the last MMA injection. Controls received saline in the same volumes. DNA damage was analyzed by the comet assay. We found that MMA and gentamicin alone or combined in vitro increased DNA damage in cerebral cortex and kidney of rats. Furthermore, MMA administration increased DNA damage in both brain and kidney. Gentamicin per se induced DNA damage only in kidney, and the association of MMA plus gentamicin also caused DNA damage in cerebral cortex and kidney. On the other hand, p53 and caspase 3 levels were not altered by the administration of MMA and/or gentamicin. Our findings provide evidence that DNA damage may contribute to the neurological and renal damage found in patients affected by methylmalonic aciduria.     

Simultaneous determination of asperosaponin VI and its active metabolite hederagenin in rat plasma by liquid chromatography-tandem mass spectrometry with positive/negative ion-switching electrospray ionization and its application in pharmacokinetic study

A new liquid chromatography-tandem mass spectrometry (LC-MS/MS) method operated in the positive/negative electrospray ionization (ESI) switching mode has been developed and validated for the simultaneous determination of asperosaponin VI and its active metabolite hederagenin in rat plasma. After addition of internal standards diazepam (for asperosaponin VI) and glycyrrhetic acid (for hederagenin), the plasma sample was deproteinized with acetonitrile, and separated on a reversed phase C18 column with a mobile phase of methanol (solvent A)-0.05% glacial acetic acid containing 10 mM ammonium acetate and 30 μM sodium acetate (solvent B) using gradient elution. The detection of target compounds was done in multiple reaction monitoring (MRM) mode using a tandem mass spectrometry equipped with positive/negative ion-switching ESI source. At the first segment, the MRM detection was operated in the positive ESI mode using the transitions of m/z 951.5 ([M+Na](+))→347.1 for asperosaponin VI and m/z 285.1 ([M+H](+))→193.1 for diazepam for 4 min, then switched to the negative ESI mode using the transitions of m/z 471.3 ([M-H](-))→471.3 for hederagenin and m/z 469.4 ([M-H](-))→425.4 for glycyrrhetic acid, respectively. The sodiated molecular ion [M+Na](+) at m/z 951.5 was selected as the precursor ion for asperosaponin VI, since it provided better sensitivity compared to the deprotonated and protonated molecular ions. Sodium acetate was added to the mobile phase to make sure that abundant amount of the sodiated molecular ion of asperosaponin VI could be produced, and more stable and intensive mass response of the product ion could be obtained. For the detection of hederagenin, since all of the mass responses of the fragment ions were very weak, the deprotonated molecular ion [M-H](-)m/z 471.3 was employed as both the precursor ion and the product ion. But the collision energy was still used for the MRM, in order to eliminate the influences induced by the interference substances from the rat plasma. The validated method was successfully applied to study the pharmacokinetics of asperosaponin VI and its active metabolite hederagenin in rat plasma after oral administration of asperosaponin VI at a dose of 90 mg/kg.     

Determination of serum methylmalonic acid by alkylative extraction and liquid chromatography coupled to tandem mass spectrometry

Despite the new advances in bioanalytical techniques, the analysis of low-molecular-weight organic acids in complex matrices is still a challenge. Although new strategies applying liquid chromatography-tandem mass spectrometry (LC-MS/MS) seem to be promising, sample preparation methodologies hamper its application in most clinical laboratories. The quantitation of methylmalonic acid (MMA) in biological matrices is an emblematic example due to its low concentration, the need for derivatization to increase its molecular weight, and the presence of the physiologically more abundant isomer succinic acid. Here we present a new strategy for rapid and sensitive MMA quantitation by combining alkylative extraction and LC-MS/MS. Alkylative extraction conditions were optimized to allow endogenous detection of MMA using only 50 μL of serum with a short sample preparation procedure. The formation of a unique ion from the MMA dipentafluorobenzyl derivative in negative atmospheric pressure chemical ionization (APCI) allowed its detection with high sensitivity and with no interference from succinic acid, a more abundant physiologically present isomer.     

Inhibition of mitochondrial creatine kinase activity from rat cerebral cortex by methylmalonic acid

Accumulation of methylmalonic acid (MMA) in tissues and biological fluids is the biochemical hallmark of patients affected by the neurometabolic disorder known as methylmalonic acidemia (MMAemia). Although this disease is predominantly characterized by severe neurological findings, the underlying mechanisms of brain injury are not totally established. In the present study, we investigated the effect of MMA, as well as propionic (PA) and tiglic (TA) acids, whose concentrations are also increased but to a lesser extend in MMAemia, on total (tCK), cytosolic (Cy-CK) and mitochondrial (Mi-CK) creatine kinase (CK) activities from cerebral cortex of 30-day-old Wistar rats. Total CK activity (tCK) was measured in whole cell homogenates, whereas Cy-CK and Mi-CK were determined, respectively, in cytosolic and mitochondrial preparations from rat cerebral cortex. We verified that tCK and Mi-CK activities were significantly inhibited by MMA at concentrations as low as 1 mM, in contrast to Cy-CK which was not affected by the presence of the acid in the incubation medium. Furthermore, PA and TA, at concentrations as high as 5 mM, did not alter CK activity. We also observed that the inhibitions provoked by MMA were fully prevented by pre-incubation of the homogenates with reduced glutathione, suggesting that the inhibitory effect of MMA was possibly mediated by oxidation of essential thiol groups of the enzyme. Considering the importance of CK for brain metabolism homeostasis, our results suggest that inhibition of this enzyme by increased levels of MMA may contribute to the neurodegeneration of patients affected by MMAemia and explain previous reports showing an impairment of brain energy metabolism and a reduction of brain phosphocreatine levels caused by MMA.     

Methylmalonic acid induces inflammatory response and redox homeostasis disruption in C6 astroglial cells: potential glioprotective roles of melatonin and resveratrol

Amino Acids - Methylmalonic acidemia is a neurometabolic disorder biochemically characterized by the accumulation of methylmalonic acid (MMA) in different tissues, including the central nervous...     

Isolation and characterization of eight lipid A precursors from a 3-deoxy-D-manno-octylosonic acid-deficient mutant of Salmonella typhimurium

Temperature-sensitive mutants of Salmonella typhimurium that are defective in the biosynthesis of 3-deoxy-D-manno-octulosonate are known to accumulate disaccharide precursor(s) of lipid A at 42 degrees C (Rick, P. D., Fung, L. W.-M., Ho, C., and Osborn, M. J. (1977) J. Biol. Chem. 252, 4904-4912). We have devised new methods for purifying this material by chromatography on DEAE-cellulose and silicic acid columns and have fractionated it into eight related anionic components that fall into four sets, as judged by their charge. Substances IA and IB have an apparent net charge of -1, IIA and IIB of -2, IIIA and IIIB of -3, and IVA and IVB of -4. Negative ion fast atom bombardment mass spectrometry reveals that the simplest component is IVA [( M - H]- at m/z 1404). Compound IVA is also the most abundant, representing 30-50% of the accumulated lipids after 3 h at 42 degrees C. Structural studies of IVA, including NMR spectroscopy described in the accompanying paper, reveal that it consists of O-(2-amino-2-deoxy-beta-D-glucopyranosyl)-(1----6)-2-amino-2-deoxy-alpha - D-glucose, acylated at positions 2, 3, 2', and 3' with beta-hydroxymyristoyl moieties and bearing phosphate groups at positions 1 and 4'. Compound IIIA ([M - H]- at m/z 1527) contains an additional phosphoethanolamine residue, while IIA ([M - H]- m/z 1535) bears an aminodeoxypentose substituent, presumably 4-amino-4-deoxy-L-arabinose. Compound IA ([M - H]- at m/z 1658) bears both a phosphoethanolamine and an aminodeoxypentose. The compounds of the less abundant B series are further derivatized with an ester-linked palmitoyl moiety. Our results demonstrate that these precursors are far more heterogeneous than previously suspected.     

Effect of propionic and methylmalonic acids on the high molecular weight neurofilament subunit (NF-H) in rat cerebral cortex

Propionic and methylmalonic acidemias are inherited neurometabolic disorders biochemically characterized by tissue accumulation of propionic (PA) and methylmalonic (MMA) acids, respectively. Neurofilaments (NF) are important cytoskeletal proteins and phosphorylation/dephosphorylation of NF is important to stabilize the cytoskeleton. We investigated the effects of PA and MMA on the high molecular weight neurofilament subunit associated with the cytoskeletal fraction of rat cerebral cortex along development. Cortical slices from 9- to 60-day-old rats were incubated with 2.5 mM PA or MMA. The cytoskeletal fraction was extracted and the immunoreactivity for phosphorylated or total NF-H was analyzed by immunoblotting using specific antibodies. Results showed that treatment of tissue slices with the acids induced an increased Triton-insoluble phosphorylated NF-H immunoreactivity in up to 17-day-old rats. Furthermore, treatments significantly increased the total amount of NF-H in 12-day-old rats. These findings indicate that PA and MMA alter the dynamic regulation of NF-H assembly in the cytoskeletal fraction.     

Identification of carpronium chloride and its metabolite in human urine by field desorption mass spectrometry using deuterium labelling

The identification of carpronium chloride and a metabolite in human urine has been performed by means of field desorption mass spectrometry using deuterium labelling. Essential points in this study are the simultaneous administration of a deuterium labelled drug ([2H9]carpronium chloride), a purification procedure by ion pair extraction with an iodine reagent, and the use of paper electrophoresis to examine the degree of clean-up. The field desorption mass spectra of the purified extracts obtained from sample urine gave a characteristic pattern resulting from the carpronium cation (m/z 160, m/z 169) and a metabolite of the N-(3-carbohydroxypropyl)trimethyl ammonium cation (m/z 146, m/z 155).     

Effects of arsenic exposure among semiconductor workers: a cautionary note on urinary 8-oxo-7,8-dihydro-2'-deoxyguanosine

Arsenic is a notorious environmental toxicant and was found to cause oxidative stress in cultured cells and animals. However, little work has been done in human studies, especially for the population occupationally exposed to arsenic. In order to investigate the effect of occupational exposure to arsenic in oxidative stress, we measured urinary 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodGuo) from 90 semiconductor workers including 50 exposed and 40 nonexposed subjects. A highly sensitive and specific isotope dilution LC-MS/MS method was used for quantification of 8-oxodGuo. The levels of inorganic arsenic (iAs3+, iAs5+), monomethylarsonic acid (MMA), and dimethylarsinic acid (DMA) in urine were determined by high-performance liquid chromatography-flow injection atomic absorption spectrometry (HPLC-FIAAS). Results showed that the mean urinary concentrations of total arsenic and 8-oxodGuo were significantly higher for exposed workers compared with the nonexposed workers. In addition, elevated urinary 8-oxodGuo concentrations of exposed workers were correlated with urinary levels of MMA (r = 0.44, P < 0.005) and the extent of primary methylation (the ratio of MMA to inorganic arsenic) (r = 0.40, P < 0.005). These findings suggested that occupational exposure to arsenic could result in the induction of oxidative stress. The presence and/or formation of MMA could play an important role in arsenic-involved injuries.     

Inhibition of the mitochondrial respiratory chain complex activities in rat cerebral cortex by methylmalonic acid

Propionic and methylmalonic acidemic patients have severe neurologic symptoms whose etiopathogeny is still obscure. Since increase of lactic acid is detected in the urine of these patients, especially during metabolic decompensation when high concentrations of methylmalonate (MMA) and propionate (PA) are produced, it is possible that cellular respiration may be impaired in these individuals. Therefore, we investigated the effects of MMA and PA (1, 2.5 and 5 mM), the principal metabolites which accumulate in these conditions, on the mitochondrial respiratory chain complex activities succinate: 2,6-dichloroindophenol (DCIP) oxireductase (complex II); succinate: cytochrome c oxireductase (complexII+CoQ+III); NADH: cytochrome c oxireductase (complex I+CoQ+complex III); and cytochrome c oxidase (COX) (complex IV) from cerebral cortex homogenates of young rats. The effect of MMA on ubiquinol: cytochrome c oxireductase (complex III) and NADH: ubiquinone oxireductase (complex I) activities was also tested. Control groups did not contain MMA and PA in the incubation medium. MMA significantly inhibited complex I+III (32–46%), complex I (61–72%), and complex II+III (15–26%), without affecting significantly the activities of complexes II, III and IV. However, by using 1 mM succinate in the assay instead of the usual 16 mM concentration, MMA was able to significantly inhibit complex II activity in the brain homogenates. In contrast, PA did not affect any of these mitochondrial enzyme activities. The effect of MMA and PA on succinate: phenazine oxireductase (soluble succinate dehydrogenase (SDH)) was also measured in mitochondrial preparations. The results showed significant inhibition of the soluble SDH activity by MMA (11–27%) in purified mitochondrial fractions. Thus, if the in vitro inhibition of the oxidative phosphorylation system is also expressed under in vivo conditions, a deficit of brain energy production might explain some of the neurological abnormalities found in patients with methylmalonic acidemia (MMAemia) and be responsible for the lactic acidemia/aciduria identified in some of them.     

Involvement of cinnamyl-alcohol dehydrogenase in the control of lignin formation in Sorghum bicolor L. Moench

The lignin structure and enzyme activities of normal and brown-midrib (BMR-6) mutant lines of Sorghum bicolor have been compared to identify the enzyme(s) involved in the reduction of the lignin content of the mutant. The results indicate that cinnamyl-alcohol dehydrogenase (CAD) and caffeic acid O-methyltransferase are depressed in the BMR-6 line, whereas the structural modifications correspond only to a reduction of CAD activity. Apparently, the change in the Sorghum lignin content, caused by depression of CAD activity, is accompanied by the incorporation of cinnamaldehydes into the core lignin.Abbreviations CAD cinnamyl-alcohol dehydrogenase - HPLC high-performance liquid chromatography - m/z mass number - OMT caffeic acid O-methyltransferase     

Development of Transgenic Mice Containing an Introduced Stop Codon on the Human Methylmalonyl-CoA Mutase Locus

The mutation R403stop was found in an individual with mut⁰ methylmalonic aciduria (MMA) which resulted from a single base change of C→T in exon 6 of the methylmalonyl-CoA mutase gene (producing a TGA stop codon). In order to accurately model the human MMA disorder we introduced this mutation onto the human methylmalonyl-CoA mutase locus of a bacterial artificial chromosome. A mouse model was developed using this construct.The transgene was found to be intact in the mouse model, with 7 copies integrated at a single site in chromosome 3. The phenotype of the hemizygous mouse was unchanged until crossed against a methylmalonyl-CoA mutase knockout mouse. Pups with no endogenous mouse methylmalonyl-CoA mutase and one copy of the transgene became ill and died within 24 hours. This severe phenotype could be partially rescued by the addition of a transgene carrying two copies of the normal human methylmalonyl-CoA mutase locus. The “humanized” mice were smaller than control litter mates and had high levels of methylmalonic acid in their blood and tissues.This new transgenic MMA stop codon model mimics (at both the phenotypic and genotypic levels) the key features of the human MMA disorder. It will allow the trialing of pharmacological and, cell and gene therapies for the treatment of MMA and other human metabolic disorders caused by stop codon mutations.     

Methylmalonic acid quantification in low serum volumes by UPLC-MS/MS

Methylmalonic acid (MMA) is a metabolic intermediate transformed to succinic acid (SA) by a vitamin B(12)-dependent catalytic step, and is broadly used as a clinical biomarker of functional vitamin B12 status. However, reported methods use between 100 and 1000 μL of serum or plasma making them sub-optimal for sample-limited studies, including those with neonates and infants. LC-MS/MS based protocols to measure MMA as n-butyl esters in the presence of tri-deuterated MMA (MMA-d(3)) were modified for use with 25 μL of human serum by scaling down sample processing volumes and analysis by UPLC-MS/MS. Plasma-based calibration solutions were found to be unnecessary, and chromatographic resolution and peak shape of SA and MMA was optimized in <4 min with isocratic 53:47 methanol/1.67 mM (pH 6.5) ammonium formate. Additionally, 1-cyclohexyl-urido-3-dodecanoic acid (CUDA) was included as internal standard allowing direct assessment of MMA recovery. Sample concentrations in the low normal range produced a signal:noise of >100:1. MMA intra- and inter-assay variability was under 10%. MMA-d(3) surrogate recovery averaged 93±14%. MMA stability exceeded three years in frozen samples and was unaffected by up to five freeze/thaw cycles. In conclusion, we report that methylmalonic acid can be measured with 25 μL of serum using water based standards. The assay signal:noise per concentration indicates that the method could perform as implemented with as little as 5 μL of serum. The reported method is applicable for studies of functional B12 status in sample limited experiments including investigations of nutritional status in neonates and in studies where low normal MMA levels are expected.     

Effect of hydroxycobalamin[c-lactam] on propionate and carnitine metabolism in the rat.

The administration in vivo of the cobalamin analogue hydroxycobalamin[c-lactam] inhibits hepatic L-methylmalonyl-CoA mutase activity. The current studies characterize in vivo and in vitro the hydroxycobalamin[c-lactam]-treated rat as a model of disordered propionate and methylmalonic acid metabolism. Treatment of rats with hydroxycobalamin[c-lactam] (2 micrograms/h by osmotic minipump) increased urinary methylmalonic acid excretion from 0.55 mumol/day to 390 mumol/day after 2 weeks. Hydroxycobalamin[c-lactam] treatment was associated with increased urinary propionylcarnitine excretion and increased short-chain acylcarnitine concentrations in plasma and liver. Hepatocytes isolated from cobalamin-analogue-treated rats metabolized propionate (1.0 mM) to CO2 and glucose at rates which were only 18% and 1% respectively of those observed in hepatocytes from control (saline-treated) rats. In contrast, rates of pyruvate and palmitate oxidation were higher than control in hepatocytes from the hydroxycobalamin[c-lactam]-treated rats. In hepatocytes from hydroxycobalamin[c-lactam]-treated rats, propionylcarnitine was the dominant product generated from propionate when carnitine (10 mM) was present. The addition of carnitine thus resulted in a 4-fold increase in total propionate utilization under these conditions. Hepatocytes from hydroxycobalamin[c-lactam]-treated rats were more sensitive than control hepatocytes to inhibition of palmitate oxidation by propionate. This inhibition of palmitate oxidation was partially reversed by addition of carnitine. Thus hydroxycobalamin[c-lactam] treatment in vivo rapidly causes a severe defect in propionate metabolism. The consequences of this metabolic defect in vivo and in vitro are those predicted on the basis of propionyl-CoA and methylmalonyl-CoA accumulation. The cobalamin-analogue-treated rat provides a useful model for studying metabolism under conditions of a metabolic defect causing acyl-CoA accretion.     

Mouse models for methylmalonic aciduria

Methylmalonic aciduria (MMA) is a disorder of organic acid metabolism resulting from a functional defect of methylmalonyl-CoA mutase (MCM). MMA is associated with significant morbidity and mortality, thus therapies are necessary to help improve quality of life and prevent renal and neurological complications. Transgenic mice carrying an intact human MCM locus have been produced. Four separate transgenic lines were established and characterised as carrying two, four, five or six copies of the transgene in a single integration site. Transgenic mice from the 2-copy line were crossed with heterozygous knockout MCM mice to generate mice hemizygous for the human transgene on a homozygous knockout background. Partial rescue of the uniform neonatal lethality seen in homozygous knockout mice was observed. These rescued mice were significantly smaller than control littermates (mice with mouse MCM gene). Biochemically, these partial rescue mice exhibited elevated methylmalonic acid levels in urine, plasma, kidney, liver and brain tissue. Acylcarnitine analysis of blood spots revealed elevated propionylcarnitine levels. Analysis of mRNA expression confirms the human transgene is expressed at higher levels than observed for the wild type, with highest expression in the kidney followed closely by brain and liver. Partial rescue mouse fibroblast cultures had only 20% of the wild type MCM enzyme activity. It is anticipated that this humanised partial rescue mouse model of MMA will enable evaluation of long-term pathophysiological effects of elevated methylmalonic acid levels and be a valuable model for the investigation of therapeutic strategies, such as cell transplantation.