LC-MS/MS Method for High-Throughput Analysis of Methylmalonic Acid in Serum, Plasma, and Urine: Method for Analyzing Isomers Without Chromatographic Separation.

Measurement of methylmalonic acid (MMA) plays an important role in the diagnosis of vitamin B12 deficiency. Vitamin B12 is an essential cofactor for the enzymatic carbon rearrangement of methylmalonyl-CoA (MMA-CoA) to succinyl-CoA (SA-CoA), and the lack of vitamin B12 leads to elevated concentrations of MMA. Measurement of MMA in biological samples is complicated because of the presence of succinic acid (SA), isomer of MMA. We developed a liquid chromatography tandem mass spectrometry (LC-MS/MS) method for MMA. The method utilizes derivatization and positive ion mode ionization, which is specific to polycarboxylic acids (MMA and SA are dicarboxylic acids), while derivatives of monocarboxylic acids at these conditions are not ionizable and not detectable. The only organic acid, other than MMA, that is detected in this method is SA. The described method does not require chromatographic resolution of the peaks of MMA and SA; quantitative measurement of MMA is performed using a deconvolution algorithm, which mathematically resolves signal corresponding to MMA, from the combined signal of MMA/SA. Because of the high selectivity of detection, this method utilizes isocratic chromatographic separation; reconditioning and re-equilibration of the chromatographic column between injections is unnecessary. The above features allow high-throughput analysis of MMA with injection-to-injection cycle time of approximately 1 minute.

Apoptotic PET Imaging of Rat Pulmonary Fibrosis with Small-Molecule Radiotracer.

PURPOSE: The purpose of this study was to assess the potential utility of small-molecule apoptotic radiotracer, 2-(5-[18F]fluoropentyl)-2-methyl malonic acid ([18F]ML-10), for positron emission tomography (PET)/computed tomography (CT) monitoring the progression of pulmonary fibrosis in a rat model. PROCEDURES: Male Sprague-Dawley rats were used to establish a rat model of pulmonary fibrosis by means of bleomycin (BLM) administration; control rats received saline (n = 12 per group). PET/CT with [18F]ML-10 and 2-deoxy-2-[18F]fluoro-D-glucose ([18F]FDG) was performed in two groups at different stages of pulmonary fibrosis. The fibrotic response and the cell apoptosis were assessed with histologic examination. Differences in the apoptosis rate, fibrotic activity, and the lung uptake of [18F]ML-10 and [18F]FDG between two groups were determined with Student t test. RESULTS: Compared with control group, BLM group showed a higher lung uptake of [18F]ML-10 at all imaging time points (all P < 0.001). During the fibrotic phase of this disease model (days 21 and 28), the lung uptake of [18F]ML-10 was higher than that of [18F]FDG in the BLM group (all P < 0.001). Moreover, accumulation of [18F]ML-10 in the lung tissues increased in proportion to the apoptosis rate (R2 = 0.9863, P < 0.0001) and fibrotic activity (R2 = 0.9631, P < 0.0001) of rat pulmonary fibrosis. Conversely, no correlation between [18F]FDG uptake and fibrotic activity was found. CONCLUSIONS: [18F]ML-10 PET/CT enabled monitoring the progression of rat pulmonary fibrosis, whereas [18F]FDG PET/CT could not. Implications for noninvasive diagnosis of pulmonary fibrosis, assessment of fibrotic activity, and evaluation of antifibrotic therapy are expected.

Predictive Value of [18F]ML-10 PET/CT in Early Response Evaluation of Combination Radiotherapy with Cetuximab on Nasopharyngeal Carcinoma.

PURPOSE: Apoptosis may be an indication of success therapy, and precise detection of apoptosis can provide instructional suggestions in the therapy management of malignant tumors. PROCEDURES: We used CNE-1 cell lines for in vitro experiments, and colony formation assay, CCK-8 assay, cell apoptosis analysis, and western blotting were performed. For in vivo experiments, subcutaneous xenotransplanted tumor models of CNE-1 in nude mice were established. Then, small animal positron emission tomography/X-ray computed tomography (PET/CT) images were acquired by tail intravenous injection of 2-(5-[18F]fluoropentyl)-2-methyl-malonic acid ([18F]ML-10) or 2-deoxy-2-[18F]fluoro-D-glucose ([18F]FDG) before and 24 h and 48 h after treatment. Moreover, expression of epidermal growth factor receptor (EGFR), Ki-67, Glut-1, and terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) was examined by immunohistochemical examination. Tumor volumes of mice were recorded every 2 days. RESULTS: In the presence of Cetuximab, the number of colonies of CNE-1 cells decreased significantly after irradiation at 1 and 2 Gy. In addition, Cetuximab increased the radiation-induced cytotoxicity and apoptosis of CNE-1 cells. Mechanistic studies demonstrated that Cetuximab enhanced radiosensitivity by suppressing the EGFR/PI3-K/AKT pathway. In PET/CT imaging, the tumors showed clear uptake of [18F]ML-10 at 24 h and 48 h after combined treatment, and the value of tumor/muscle (T/M) and SUVmax (the max of standard uptake value) was significantly higher than those of the other three groups. The T/M of [18F]ML-10 uptake showed a positive correlation of 0.926 with the apoptosis index (P < 0.001). However, the uptake of [18F]FDG in tumors exhibited no trend among the four groups. The T/M of [18F]FDG revealed a positive correlation of 0.926 with Glut-1 intensity (P < 0.001). CONCLUSIONS: Our work revealed that Cetuximab could increase the radiosensitivity of CNE-1 cells both in vitro and in vivo. Apoptosis imaging with [18F]ML-10 PET/CT is a promising modality for application in the response prediction of nasopharyngeal carcinoma.

Apoptotic PET Imaging of Rat Pulmonary Fibrosis With [18F]ML-8.

OBJECTIVE: To investigate the value of 2-(3-[18F]fluoropropyl)-2-methyl-malonic acid ([18F]ML-8) positron emission tomography (PET) imaging of rat pulmonary fibrosis. METHODS: Male Sprague-Dawley rats were divided into 2 groups, including pulmonary fibrosis model group and control group. The rat model was established by an intratracheal instillation of bleomycin (BLM). Control rats were treated with saline. Positron emission tomography/computed tomography (CT) with [18F]ML-8 or 18F-fluorodeoxyglucose ([18F]FDG) was performed on 2 groups. After PET/CT imaging, lung tissues were collected for histologic examination. Data were analyzed and comparisons between 2 groups were performed using Student t test. RESULTS: Bleomycin-treated rats showed a higher lung uptake of [18F]ML-8 than control rats ( P < .05). In BLM-treated rats, the lung to muscle relative uptake ratio of [18F]ML-8 was also higher than that of [18F]FDG ( P < .05). Pathological examination showed overproliferation of fibroblasts and deposition of collagen in lungs from BLM-treated rats. Compared to control rats, BLM-treated rats had higher lung hydroxyproline content ( P < .05). Immunofluorescence staining indicated more apoptotic cells in BLM-treated rats than those in control rats. Moreover, the apoptosis rate of lung tissues obtained from BLM-treated rats was higher than that from control rats ( P < .05). CONCLUSIONS: 2-(3-[18F]fluoropropyl)-2-methyl-malonic acid PET/CT could be used for noninvasive diagnosis of pulmonary fibrosis in a rat model.

Improved testing for vitamin B12 deficiency: correcting MMA for eGFR reduces the number of patients classified as vitamin B12 deficient.

Background Methylmalonic acid (MMA) can detect functional vitamin B12 deficiencies as it accumulates early when intracellular deficits arise. However, impaired clearance of MMA from blood due to decreased glomerular filtration rate (eGFR) also results in elevated plasma MMA concentrations. Alternative to clinical trials, a data mining approach was chosen to quantify and compensate for the effect of decreased eGFR on MMA concentration. Methods Comprehensive data on patient's vitamin B12, eGFR and MMA concentrations were collected ( n = 2906). The relationship between vitamin B12, renal function (eGFR) and MMA was modelled using weighted multiple linear regression. The obtained model was used to estimate the influence of decreased eGFR on MMA. Clinical impact was examined by comparing the number of patients labelled vitamin B12 deficient with and without adjustment in MMA. Results Adjusting measured MMA concentrations for eGFR in the group of patients with low-normal vitamin B12 concentrations (90-300 pmol/L) showed that the use of unadjusted MMA concentrations overestimates vitamin B12 deficiency by 40%. Conclusions Through a data mining approach, the influence of eGFR on the relation between MMA and vitamin B12 can be quantified and used to correct the measured MMA concentration for decreased eGFR. Especially in the elderly, eGFR-based correction of MMA may prevent over-diagnosis of vitamin B12 deficiency and corresponding treatment.

Efficient automated synthesis of 2-(5-[18F]fluoropentyl)-2-methylmalonic acid ([18F]ML-10) on a commercial available [18F]FDG synthesis module.

[18F]ML-10 (2-(5-[18F]fluoro-pentyl)-2-methylmalonic acid) is a small molecule positron emission tomography (PET) probe for apoptosis imaging. Automated synthesis of [18F]ML-10 was developed by using two different purification methods through a direct saponification procedure on a modified commercial [18F]Fluoro-2-Deoxyglucose ([18F]FDG) synthesizer. C18 purification method 1: The final [18F]ML-10 solution containing ethanol was obtained with radiochemical yields of 60±5% (n=5) at the end of bombardment (EOB) and radiochemical purity of 98% in 35min. Al2O3 and SCX purification method 2: To avoid possible side effects of a conventional ethanol-containing formulation, an new ethanol-free solution of [18F]ML-10 was also developed, the radiochemical yields was 50±5% (n=5, EOB) within 45min and the radiochemical purity was 98%.

Structural insights into the catalytic reaction trigger and inhibition of D-3-hydroxybutyrate dehydrogenase.

D-3-Hydroxybutyrate dehydrogenase catalyzes the reversible conversion of acetoacetate and D-3-hydroxybutyrate. These ketone bodies are both energy-storage forms of acetyl-CoA. In order to clarify the structural mechanisms of the catalytic reaction with the cognate substrate D-3-hydroxybutyrate and of the inhibition of the reaction by inhibitors, the enzyme from Alcaligenes faecalis has been analyzed by X-ray crystallography in liganded states with the substrate and with two types of inhibitor: malonate and methylmalonate. In each subunit of the tetrameric enzyme, the substrate is trapped on the nicotinamide plane of the bound NAD(+). An OMIT map definitively shows that the bound ligand is D-3-hydroxybutyrate and not acetoacetate. The two carboxylate O atoms form four hydrogen bonds to four conserved amino-acid residues. The methyl group is accommodated in the nearby hydrophobic pocket so that the formation of a hydrogen bond from the OH group of the substrate to the hydroxy group of Tyr155 at the active centre is facilitated. In this geometry, the H atom attached to the C(3) atom of the substrate in the sp(3) configuration is positioned at a distance of 3.1 Šfrom the nicotinamide C(4) atom in the direction normal to the plane. In addition, the donor-acceptor relationship of the hydrogen bonds suggests that the Tyr155 OH group is allowed to ionize by the two donations from the Ser142 OH group and the ribose OH group. A comparison of the protein structures with and without ligands indicates that the Gln196 residue of the small movable domain participates in the formation of additional hydrogen bonds. It is likely that this situation can facilitate H-atom movements as the trigger of the catalytic reaction. In the complexes with inhibitors, however, their principal carboxylate groups interact with the enzyme in a similar way, while the interactions of other groups are changed. The crucial determinant for inhibition is that the inhibitors have no active H atom at C(3). A second determinant is the Tyr155 OH group, which is perturbed by the inhibitors to donate its H atom for hydrogen-bond formation, losing its nucleophilicity.

Methylmalonic acid and neutrophil morphometric index in laboratory diagnosis of cobalamin deficiency without macrocytosis.

INTRODUCTION: This study aimed to investigate the utility of methylmalonic acid (MMA) and neutrophil Cell Population Data, available on the Beckman Coulter LH750 Analyser (Miami, FL, USA), in laboratory assessment of cobalamin status in patients at risk of cobalamin deficiency, without macrocytosis and inflammation. METHODS: The study group included 189 patients. Neutrophil Cell Population Data along with vitamin B12 and homocysteine were assessed in regard to MMA tertile groups. RESULTS: Statistically significant differences were between lower and upper MMA tertile in serum B12 (P ˂ 0.001), homocysteine (P = 0.001) and neutrophil morphometric index, NeS-DW (P = 0.029). Also, serum B12 concentrations were significantly different between lower and middle MMA tertile, P = 0.005. Receiver operating characteristic analysis of NeS-DW ability to detect MMA˃367 nmol/L revealed a significant area under the curve AUC = 0.761 P ˂ 0.001 95% CI 0.693-0.830. Optimal cut-off value was NeS-DW˃3.51% with sensitivity of 74.19% and specificity of 68.87%. CONCLUSION: In patients at risk of cobalamin deficiency and normal MCV, classification according to MMA revealed cobalamin status differences. Neutrophil morphometric index may be an indicator of early changes in neutrophil nucleus morphology caused by impaired cobalamin status.

High-Throughput Analysis of Methylmalonic Acid in Serum, Plasma, and Urine by LC-MS/MS. Method for Analyzing Isomers Without Chromatographic Separation.

Measurement of methylmalonic acid (MMA) plays an important role in the diagnosis of vitamin B12 deficiency. Vitamin B12 is an essential cofactor for the enzymatic carbon rearrangement of methylmalonyl-CoA (MMA-CoA) to succinyl-CoA (SA-CoA), and the lack of vitamin B12 leads to elevated concentrations of MMA. Presence of succinic acid (SA) complicates the analysis because mass spectra of MMA and SA are indistinguishable, when analyzed in negative ion mode and the peaks are difficult to resolve chromatographically. We developed a method for the selective analysis of MMA that exploits the significant difference in fragmentation patterns of di-butyl derivatives of the isomers MMA and SA in a tandem mass spectrometer when analyzed in positive ion mode. Tandem mass spectra of di-butyl derivatives of MMA and SA are very distinct; this allows selective analysis of MMA in the presence of SA. The instrumental analysis is performed using liquid chromatography-tandem mass spectrometry (LC-MS/MS) in positive ion mode, which is, in combination with selective extraction of acidic compounds, is highly selective for organic acids with multiple carboxyl groups (dicarboxylic, tricarboxylic, etc.). In this method organic acids with a single carboxyl group are virtually undetectable in the mass spectrometer; the only organic acid, other than MMA, that is detected by this method is its isomer, SA. Quantitative measurement of MMA in this method is performed using a deconvolution algorithm, which mathematically resolves the signal corresponding to MMA and does not require chromatographic resolution of the MMA and SA peaks. Because of its high selectivity, the method utilizes isocratic chromatographic separation; reconditioning and re-equilibration of the chromatographic column between injections is unnecessary. The above features of the method allow high-throughput analysis of MMA with analysis cycle time of 1 min.

Molecular PET Imaging of Cyclophosphamide Induced Apoptosis with 18F-ML-8.

In this paper, a novel small-molecular apoptotic PET imaging probe, (18)F-ML-8 with a malonate motif structure, is presented and discussed. After study, the small tracer that belongs to a member of ApoSense family is proved to be capable of imaging merely apoptotic regions in the CTX treated tumor-bearing mice. The experimental result is further confirmed by in vitro cell binding assays and TUNEL staining assay. As a result, (18)F-ML-8 could be used for noninvasive visualization of apoptosis induced by antitumor chemotherapy.

Use of LC-MS for the quality control of radiopharmaceuticals: example of [(18)F]ML10.

[(18)F]ML10 is a promising novel low molecular weight positron emission tomography probe for apoptosis. As part of the quality control to support clinical studies for cancer therapy monitoring in the GSK Clinical Imaging Centre, a simple and sensitive liquid chromatography mass spectrometry method has been developed and validated for the quantification of total ML10 and impurity content in the final product. Chromatographic separation of ML10 and its radiolabelling precursor and impurities was achieved. Mass curves were constructed from a concentration range of ML10 and known impurities and were linear. Quantification was achieved by comparison of the area under the curve for ML10 content (m/z = 205) and the mass curve. The method was validated over a concentration range of 0.1-1 µg/ml.

Automated radiosynthesis of [(18)F]ML-10, a PET radiotracer dedicated to apoptosis imaging, on a TRACERLab FX-FN module.

PURPOSE: [(18)F]ML-10 is the most advanced radiopharmaceutical for the clinical imaging of the apoptosis phenomenon by PET. The preparation of this radiopharmaceutical on a commercial radiosynthesis module and the requested quality controls for its release are presented herein. PROCEDURES: ML-10 as reference and its mesyloxy derivative as precursor for labelling with fluorine-18 were prepared. [(18)F]ML-10 was synthesized via a [(18)F]fluorine-de-mesyloxy aliphatic nucleophilic substitution via a GE TRACERLab® FX-FN module. Quality controls were performed. RESULTS: The labelling precursor was obtained in a four step synthesis in 28 % overall yield affording ML-10 in two steps (88 % yield). Pure [(18)F]ML-10 was obtained with a decay corrected yield of 39.8 % ± 8.4 % (n = 7) in 70 min and a specific activity of 235 ± 85 GBq/µmol at the end of synthesis. CONCLUSIONS: [(18)F]ML-10 was prepared on a widely available automated module and passed the quality control. A LC/MS method was developed to measure specific radioactivity.

Methylmalonate-semialdehyde dehydrogenase from Bacillus subtilis: substrate specificity and coenzyme A binding.

Methylmalonate-semialdehyde dehydrogenase (MSDH) belongs to the CoA-dependent aldehyde dehydrogenase subfamily. It catalyzes the NAD-dependent oxidation of methylmalonate semialdehyde (MMSA) to propionyl-CoA via the acylation and deacylation steps. MSDH is the only member of the aldehyde dehydrogenase superfamily that catalyzes a ß-decarboxylation process in the deacylation step. Recently, we demonstrated that the ß-decarboxylation is rate-limiting and occurs before CoA attack on the thiopropionyl enzyme intermediate. Thus, this prevented determination of the transthioesterification kinetic parameters. Here, we have addressed two key aspects of the mechanism as follows: 1) the molecular basis for recognition of the carboxylate of MMSA; and 2) how CoA binding modulates its reactivity. We substituted two invariant arginines, Arg-124 and Arg-301, by Leu. The second-order rate constant for the acylation step for both mutants was decreased by at least 50-fold, indicating that both arginines are essential for efficient MMSA binding through interactions with the carboxylate group. To gain insight into the transthioesterification, we substituted MMSA with propionaldehyde, as both substrates lead to the same thiopropionyl enzyme intermediate. This allowed us to show the following: 1) the pK(app) of CoA decreases by ∼3 units upon binding to MSDH in the deacylation step; and 2) the catalytic efficiency of the transthioesterification is increased by at least 10(4)-fold relative to a chemical model. Moreover, we observed binding of CoA to the acylation complex, supporting a CoA-binding site distinct from that of NAD(H).

Malonamide-functionalized gold nanoparticles for selective, colorimetric sensing of trivalent lanthanide ions.

A selective and sensitive molecular sensor for trivalent lanthanide (Ln(3+)) ions based upon a malonamide-functionalized gold nanoparticle was developed for colorimetric detection in water. A new synthetic approach permits nanoparticle synthesis, stabilization, and incorporation of a selective lanthanide binding site in a single, direct step. The design incorporates a specifically tailored dual function precursor ligand that bears a sodium thiosulfate (Bunte salt) group that links to the gold nanoparticle core and a tetramethylmalonamide (TMMA) group that serves as a selective Ln(3+) binding site. The sensor's colorimetric response to lanthanide ions is immediate, and it is sensitive down to approximately 50 nM for Eu(3+) and Sm(3+). This study demonstrates a general strategy for direct, convenient nanoparticle synthesis that enables the incorporation of analyte binding groups directly to the nanoparticle surface, allowing colorimetric sensors to be developed for widespread use. The one-step synthesis offers uniform surface ligand composition, reduces the volume of waste generated during nanoparticle synthesis and purification, produces functionalized gold nanoparticles that are stable in nonmodified aqueous environments, and offers colorimetric detection at ambient temperature.

From the Gla domain to a novel small-molecule detector of apoptosis.

Apoptosis plays a pivotal role in the etiology or pathogenesis of numerous medical disorders, and thus, targeting of apoptotic cells may substantially advance patient care. In our quest for novel low-molecular-weight probes for apoptosis, we focused on the uncommon amino acid gamma-carboxyglutamic acid (Gla), which plays a vital role in the binding of clotting factors to negatively charged phospholipid surfaces. Based on the alkyl-malonic acid motif of Gla, we have developed and now present ML-10 (2-(5-fluoro-pentyl)-2-methyl-malonic acid, MW=206 Da), the prototypical member of a novel family of small-molecule detectors of apoptosis. ML-10 was found to perform selective uptake and accumulation in apoptotic cells, while being excluded from either viable or necrotic cells. ML-10 uptake correlates with the apoptotic hallmarks of caspase activation, Annexin-V binding and disruption of mitochondrial membrane potential. The malonate moiety was found to be crucial for ML-10 function in apoptosis detection. ML-10 responds to a unique complex of features of the cell in early apoptosis, comprising irreversible loss of membrane potential, permanent acidification of cell membrane and cytoplasm, and preservation of membrane integrity. ML-10 is therefore the most compact apoptosis probe known to date. Due to its fluorine atom, ML-10 is amenable to radio-labeling with the (18)F isotope, towards its potential future use for clinical positron emission tomography imaging of apoptosis.

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 microL 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.

Fingerprints of damped quantum rotation observed in solid-state proton NMR spectra.

(1)H NMR spectra of the methyl group in an oriented crystal sample of methylmalonic acid with all three non-methyl protons replaced by deuterons are interpreted in terms of the damped quantum rotation (DQR) theory of NMR line shapes. The DQR approach offers a perfect theoretical reproduction of the observed spectra while the conventional Alexander-Binsch line-shape model shows evident defects in the present case. The temperature trends of the quantities characterizing the coherent and incoherent dynamics of the methyl group in the DQR approach (the effective tunnelling frequency and two coherence-damping rates) derived from the spectra are fairly reproduced using a model reported previously. The present findings provide further evidence of limitations to the validity of the common belief that molecular rate processes in condensed phases are necessarily classical.

Fumarase deficiency presenting with periventricular cysts.

A fumarase-deficient patient expressed a novel phenotype of congenital cerebral ventricular dilatation and periventricular cysts. The patient was a compound heterozygote for two mutations that are the only ones among the 12 published mutations that have been found in multiple, unrelated, fumarase-deficient patients.

Inversion of enantioselectivity of asymmetric biocatalytic decarboxylation by site-directed mutagenesis based on the reaction mechanism.

The introduction of two mutations (G74C/C188S) based on the estimated reaction mechanism resulted in the inversion of enantioselectivity of arylmalonate decarboxylase, which catalyses the asymmetric decarboxylation of arylmethylmalonate to give optically active arylpropionate.