Preclinical Evaluation of 18F-ML-10 to Determine Timing of Apoptotic Response to Chemotherapy in Solid Tumors.

PURPOSE: We investigated 2-(5-fluoro-pentyl)-2-methyl-malonic acid (18F-ML-10) positron emission tomography (PET) imaging of apoptosis posttherapy to determine optimal timing for predicting chemotherapy response in a mouse head/neck xenograft cancer model. PROCEDURES: BALB/c nude mice (4-8 weeks old) were implanted with UM-SCC-22B tumors. The treatment group received 2 doses of doxorubicin (10 mg/kg, days 0, 2). Small animal 18F-ML-10 PET/computed tomography was performed before and on days 1, 3, and 7 postchemotherapy. Using regions of interest around tumors, 18F-ML-10 uptake change was measured as %ID/g and uptake relative to liver. Terminal Uridine Nick-End Labeling (TUNEL) immunohistochemistry assay was performed using tumor samples of baseline and on days 1, 3, and 7 posttreatment. RESULTS: Treated mice demonstrated increased 18F-ML-10 uptake compared to baseline and controls, and 10 of 13 mice showed tumor volume decreases. All control mice showed tumor volume increases. Tumor-to-liver (T/L) ratios from the control group mice did not show significant change from baseline ( P > .05); however, T/L ratios of the treatment group showed significant 18F-ML-10 uptake differences from baseline compared to days 3 and 7 posttreatment ( P < .05), but no significant difference at 1 day posttreatment. CONCLUSION: 2-(5-Fluoro-pentyl)-2-methyl-malonic acid PET imaging has the potential for early assessment of treatment-induced apoptosis. Timing and image analysis strategies may require optimization, depending on the type of tumor and cancer treatment.

Prenatal diagnosis of methylmalonic aciduria by measuring methylmalonic acid in dried amniotic fluid on filter paper using gas chromatography-mass spectrometry.

Methylmalonic aciduria is a common inherited metabolic disorder. Methylmalonic acid (MMA), a key indicator of methylmalonic aciduria, increases in the amniotic fluid of affected fetuses. For prenatal diagnosis, the MMA in amniotic fluid can be measured by stable-isotope dilution gas chromatography-mass spectrometry. Here, we quantified the MMA in cell-free amniotic fluid samples that had been dried on filter paper and transported at ambient temperatures, and compared the results with data obtained from the original amniotic fluid. Our results indicated that the filter paper method was reproducible and accurate enough to be applied to clinical analysis. We also used the filter paper method to screen at-risk fetuses and obtained a clear diagnosis in each case. We conclude that our method enables the prenatal diagnosis of methylmalonic aciduria using practical procedures and a simplified method for transporting the samples.

Electrochemical sensing of vitamin B12 deficiency marker methylmalonic acid using PdAu-PPy tailored carbon fiber paper electrode.

Vitamin B12 is very important for human metabolism and its deficiency can cause anemia and the production of large red blood cells. An increased concentration of methylmalonic acid (MMA) is detected much before the transformation of blood cells, which thereby is an early indicator for mild or serious Vitamin B12 deficiency. A simple electrochemical sensor based on Palladium-Gold (PdAu) was developed by electrodeposition of PdAu nanoparticles on Polypyrrole (PPy) modified carbon fiber paper (CFP) electrode. The modified electrodes were characterized by High resolution transmission electron microscopy (HRTEM), Field emission scanning electron microscopy (FESEM) with energy-dispersive X-ray spectroscopy (EDS), X-ray diffraction (XRD), X-ray photoelectron spectroscopy (XPS) and electroanalytical techniques. Differential Pulse Voltammetric (DPV) studies have established that under optimum conditions, the developed sensor exhibits a broad linear dynamic range (4.01 pM - 52.5 nM) with a very low detection limit (1.32 pM). The proposed method was effectively applied in the non-enzymatic determination of MMA at an ultralow level in human blood serum and urine samples. The method displayed high selectivity toward MMA in the presence of other interfering substances.

Formation and metabolism of methylmalonyl coenzyme A in Corynebacterium glutamicum.

Genome sequence information suggests that B(12)-dependent mutases are present in a number of bacteria, including members of the suborder Corynebacterineae like Mycobacterium tuberculosis and Corynebacterium glutamicum. We here functionally identify a methylmalonyl coenzyme A (CoA) mutase in C. glutamicum that is retained in all of the members of the suborder Corynebacterineae and is encoded by NCgl1471, NCgl1472, and NCgl1470. In addition, we observe the presence of methylmalonate in C. glutamicum, reaching concentrations of up to 757 nmol g (dry weight)(-1) in propionate-grown cells, whereas in Escherichia coli no methylmalonate was detectable. As demonstrated with a mutase deletion mutant, the presence of methylmalonate in C. glutamicum is independent of mutase activity but possibly due to propionyl-CoA carboxylase activity. During growth on propionate, increased mutase activity has severe cellular consequences, resulting in growth arrest and excretion of succinate. The physiological context of the mutase present in members of the suborder Corynebacterineae is discussed.

Monitoring of tumor response to chemotherapy in vivo by a novel small-molecule detector of apoptosis.

Utilization of molecular imaging of apoptosis for clinical monitoring of tumor response to anti-cancer treatments in vivo is highly desirable. To address this need, we now present ML-9 (butyl-2-methyl-malonic acid; MW = 173), a rationally designed small-molecule detector of apoptosis, based on a novel alkyl-malonate motif. In proof-of-concept studies, induction of apoptosis in tumor cells by various triggers both in vitro and in vivo was associated with marked uptake of (3)H-ML-9 administered in vivo, in correlation with the apoptotic hallmarks of DNA fragmentation, caspase-3 activation and membrane phospholipid scrambling, and with correlative tumor regression. ML-9 uptake following chemotherapy was tumor-specific, with rapid clearance of the tracer from the blood and other non-target organs. Excess of non-labeled "cold" compound competitively blocked ML-9 tumor uptake, thus demonstrating the specificity of ML-9 binding. ML-9 may therefore serve as a platform for a novel class of small-molecule imaging agents for apoptosis, useful for assessment of tumor responsiveness to treatment.

[Appropriate implementation of automated reflex testing]. / Automatiserad reflexanalys måste införas på rätt sätt - Ny modell prövad i primärvården i Region Östergötland ­ aktiv uppföljning av beställningsmönster gav resultat.

The aim of automated reflex testing is to achieve more rational and cost-effective use of laboratory investigations in clinical practice. In order to make use of this cost-effectiveness, the new tests should have a short implementation phase and clear objective goals. To accomplish an equal health care, the usage of new tests should have minimal variations between different primary health care units. We started a new model in Ostergotland County regarding clinical usage of new tests by active follow-up in two levels, both primary health care unit level and individual physician level, during the implementation phase. This study shows that active follow-up and feedback resulted in both shorter implementation phase and minimal variation between different primary health care units.

Gas chromatographic-mass spectrometric analysis of biomarkers related to folate and cobalamin status in human serum after dimercaptopropanesulfonate reduction and heptafluorobutyl chloroformate derivatization.

Methylmalonic acid and total homocysteine belong to useful clinical indicators of cobalamin and folate status and are commonly measured separately. A sensitive and rapid method has been developed for simultaneous determination of both biomarkers and related metabolites in serum or plasma by isotope dilution gas chromatography-mass spectrometry (GC/MS). Thiols bound in disulfide bonds were released with 2,3-dimercapto-1-propanesulfonic acid (DMPS), and after deproteinization, they were phase-transfer derivatized with heptafluorobutyl chloroformate (HFBCF) in a single step. The reducing capability of the DMPS agent was comparable to that of the dithiothreitol, but exceeded the latter in much cleaner extracts obtained. The new method enabled GC/MS screening of amino acidic metabolites, including cystathionine and thiol-containing dipeptides, as their N(S,O)-heptafluorobutoxycarbonyl heptafluorobutyl ester derivatives in serum or plasma. Accurate quantitation of seven biomarkers was accomplished by using deuterated internal standards; the detection limits ranged from 7 to 20 nmol/L, the between-day precision from 1.5 to 8.8%, and the recoveries were between 83 and 103%. The results suggest that the new combined procedure of DMPS reduction and HFBCF derivatization make the method efficient for diagnostics of folate and cobalamin status as well as for screening of amino acidic metabolites in body fluids.

Mitochondrial encephalomyopathy with elevated methylmalonic acid is caused by SUCLA2 mutations.

We have identified 12 patients with autosomal recessive mitochondrial encephalomyopathy with elevated methylmalonic acid. The disorder has a high incidence of 1 in 1700 in the Faroe Islands due to a founder effect, and a carrier frequency of 1 in 33. The symptoms comprise hypotonia, muscle atrophy, hyperkinesia, severe hearing impairment and postnatal growth retardation. Neuroimaging showed demyelination and central and cortical atrophy, including atrophy of the basal ganglia, and some of the patients fulfilled the criteria for Leigh syndrome. Urine and plasma methylmalonic acid were elevated. Homozygosity mapping with the Affymetrix 10 K array revealed a homozygous region on chromosome 13q14 harbouring the SUCLA2 gene. Mutations in SUCLA2 were recently shown to cause a similar disorder in a small Israeli family. Mutation analysis identified a novel splice site mutation in SUCLA2, IVS4 + 1G --> A, leading to skipping of exon 4. The SUCLA2 gene encodes the ATP-forming beta subunit of the Krebs cycle enzyme succinyl-CoA ligase. The hallmark of the condition, elevated methylmalonic acid, can be explained by an accumulation of the substrate of the enzyme, succinyl-CoA, which in turn leads to elevated methylmalonic acid, because the conversion of methylmalonyl-CoA to succinyl-CoA is inhibited.

Assessing analytical specificity in quantitative analysis using tandem mass spectrometry.

OBJECTIVES: The necessity of confirmation of compound identity in quantitative analysis is well recognized for methods utilizing single mass spectrometry detection but is not commonly addressed for applications utilizing multiple-stage mass spectrometry (MSn). For MSn detection, no commonly accepted rules for assessment of analytical specificity in quantitative analyses have been established to date. METHODS: To assure compound identity, we evaluated approaches based on monitoring multiple mass transitions of a target compound followed by comparison of the branching ratios of the mass transitions. RESULTS: Monitoring multiple mass transitions along with evaluation of the ratio of their relative intensities allows the analyst to distinguish the target analyte from interferences in quantitative analysis. The strategy and the acceptance criteria are compound and method specific and should be established during the method development and validation. CONCLUSIONS: The certainty of analyte identity is very important in quantitative analysis using MSn detection; methods to verify analyte identity should be used in all critical applications.

Prenatal diagnosis for organic acid disorders using two mass spectrometric methods, gas chromatography mass spectrometry and tandem mass spectrometry.

We performed prenatal diagnosis of organic acid disorders using two mass spectrometric methods; gas chromatography mass spectrometry (GC/MS) and tandem mass spectrometry (ESI/MS/MS). Of 28 cases whose amniotic fluid was tested, 11 cases were diagnosed as "affected". All cases whose samples were diagnosed as "unaffected" were confirmed to have no symptoms or abnormalities in urinary organic acid analysis after birth. Of the 11 "affected" cases, two cases were missed by ESI/MS/MS but not by GC/MS. When the stability of metabolites in amniotic fluid was checked, it was found that acylcarnitines degraded in one week at room temperature, whereas organic acids such as methylmalonate or methylcitrate were stable for at least 14 days. Prenatal diagnosis by analysis using simultaneous two or more methods may be more reliable, though attention should be paid to sample transportation conditions.

A sensitive micromethod for the routine estimation of methylmalonic acid in body fluids and tissues using thin layer chromatography.

A routine method for the rapid estimation of methylmalonic acid (MMA) has been developed using thin-layer chromatography (TLC) on cellulose, locating the separated MMA zones by coupling with a diazo reagent and quantitating the results by densitometric scanning of a photocopy of the chromatogram on transparency film. The method can detect as little as 25 pg MMA in small volumes of urine, plasma and other body fluids or tissue homogenates, has potential for the simultaneous estimation of MMA, other organic acids, methylmalonyl coenzyme A and creatinine and should be of particular value in the investigation of occult cobalamin deficiency or suspected errors of vitamin B12 or intermediary metabolism.

Hydroxy acid metabolites of branched-chain amino acids in amniotic fluid.

A method is described in which ammonia chemical ionization gas chromatography-mass spectrometry was utilized in the selected ion monitoring mode to provide an accurate, selective approach to the quantification in amniotic fluid of a number of hydroxylated organic acids derived from the metabolism of the branched-chain amino acids. 2-Hydroxy-n-caproic acid was employed as an internal standard and the hydroxy acids were isolated from amniotic fluid by liquid partition chromatography and the trimethylsilyl derivatives were quantified. Normal values have been obtained for 2-hydroxyisovaleric acid, the sum of 2-hydroxyisocaproic acid and 2-hydroxy-3-methylvaleric acid, 2-methyl-3-hydroxybutyric acid, 3-hydroxyisovaleric acid and 2-ethyl-3-hydroxypropionic acid. The method also provides data on the concentration of methylmalonic acid. The concentration of 2-hydroxyisovaleric acid was not useful in the prenatal diagnosis of a fetus with maple syrup urine disease. Elevated concentrations of 2-methyl-3-hydroxybutyric acid as well as methylmalonic acid were found in the amniotic fluid of two fetuses with methylmalonic acidemia.

Microdetermination of methylmalonic acid and other short chain dicarboxylic acids by gas chromatography: use in prenatal diagnosis of methylmalonic acidemia and in studies of isovaleric acidemia.

We have developed a sensitive gas-chromatographic method for the determination of methylmalonic acid and other short chain dircarboxylic acids in biological samples. The method is based on the isolation of the short chain dicarboxylic acid fraction by Dowex 3 X 4 column chromatography followed by gas-chromatography analysis of these acids as methyl esters. 2-n-Pentyl-malonic acid is used as an internal standard. With this method, methylmalonic, succinic and methylsuccinic acids were consistently detected and accurately measured in urine and serum from normal subjects; the identity of these acids being verified by mass spectroscopy. The method's sensitivity permitted its used in the prenatal diagnosis of methylmalonic acidemia by measuring methylmalonic acid in urine and amniotic fluid from three pregnant heterozygous women at risk. One affected (vitamin B-12 responsive type) and two unaffected fetuses were correctly diagnosed prenatally as judged by postnatal investigations. The amount of methylmalonic acid in urine and amniotic fluid was distinctly increased (2 to 14 times normal) in the former and consistently normal in the latter two cases during the third trimester of pregnancy. Effect of prenatal therapy with large doses of vitamin B-12 was closely followed in the first case using analyses of multiple maternal urine specimens. Urinary methylsuccinic acid excretion was studied in two cases with isovaleric acidemia. It was normal in a sample from a patient in remission but was increased seven fold over control during an episode of ketoacidosis.

Methylmalonic acid in amniotic fluid and maternal urine as a marker for neural tube defects.

To evaluate the implication of methymalonic acid (MMA) in the early diagnosis of neural tube defects (NTD), a quantitative assay for MMA was established by using gas chromatography-mass spectrometry with stable isotope of MMA as an internal standard. Amniotic fluid and maternal urine MMA concentration, maternal serum folate, red blood cell folate and vitamin B12 levels were measured in the middle term of NTD-affected and normal pregnancies. Amniotic fluid and maternal urine MMA concentrations in the middle term of NTD affected pregnancies (1.4 +/- 0.9 micromol/L, and 22.1 +/- 12.6 nmol/micromol creatinine) were significantly higher than that of normal pregnancies (1.0 +/- 0. 4 micromol/L, and 2.5 +/- 1.1 nmol/micromol creatinine). There was no significant difference between normal and NTD pregnancies for serum folate, red blood cell folate and vitamin B12 levels. The results suggested that MMAs in amniotic fluid and maternal urine are sensitive markers for early diagnosis of NTD. Vitamin B12 is an active cofactor involved in the remethylation of homocycteine and its deficiency is an independent risk factor for NTD. MMA is a specific and sensitive marker for intracellular vitamin B12 deficiency. This study suggests that it is necessary to monitor the vitamin B12 deficiency and advocates vitamin B12 supplementation with folate prevention program.

[Connection between changing the vitamin and immune status and the character of the throat microflora in patients with chronic tonsillitis]. / Niedobór witamin oraz zaburzenia ukladu immunologicznego a charakter mikroflory gardla u chorych z przewleklym zapaleniem migdalków podniebiennych.

The author investigated 28 patients with chronic tonsillitis. The fungi sort Candida on the tonsils was discovered at 10 (35.72) patients, the bacterial flora was discovered at the other patients. There were more considerable decrease content of vitamins B2, C, methylnicotinamide and methylmalonic acid in the urine, vitamins B2 and methylnicotinamide in the saliva, vitamins A and B1 in the serum of the blood in the group of the patients with the fungus flora on the tonsils. It was discovered the decrease of numbers of lymphocytes CD8 and level of immunoglobulins G and M, disimmunoglobulinemia in this group. This decrease of the vitamins, cell and humoral immunity level leads to disbacteriosis, decompensation of the disease and development of the complications.

An audit of holotranscobalamin ("Active" B12) and methylmalonic acid assays for the assessment of vitamin B12 status: application in a mixed patient population.

BACKGROUND: Vitamin B12 insufficiency/deficiency is common in mixed patient populations. However there is no single marker which can reliably diagnose B12 insufficiency/deficiency. Elevated concentrations of methylmalonic acid (MMA) are considered the most representative marker of metabolic vitamin B12 insufficiency, but poor assay availability limits clinical utility. Low concentrations of serum vitamin B12 are often used to assess vitamin B12 status but this approach generates a high rate of false negative results. Emerging evidence indicates that holotranscobalamin (holoTC) may be a more reliable indicator of vitamin B12 status. AIMS AND METHODS: We substituted serum vitamin B12 measurement with holoTC, supported by MMA in patients referred for assessment of vitamin B12 status. A service evaluation was undertaken of the pattern of MMA values obtained for patients with holoTC 25-50 pmol/L (an indeterminate result). MMA cut-offs of 280 and 360 nmol/L were applied for patients ≤ 65 or >65 years respectively. RESULTS: A total of 4,175 consecutive patients were investigated and MMA was analysed for 19% of patients. The incidence of elevated MMA was 41% (holoTC, 25-29 pmol/L), 32% (30-34 pmol/L), 33% (35-39 pmol/L), 30% (40-44 pmol/L), and 26% (45-50 pmol/L). CONCLUSIONS: Our results indicate that in the clinical setting a holoTC between 25 and 50 pmol/L is a poor predictor for the concentration of MMA provided the goal is to identify patients with MMA values above the limits used in the present study. Further studies are needed to evaluate to what extent holoTC <25 and >50 pmol/L reflect circulatory MMA concentrations.

4-ethylphenyl-cobalamin impairs tissue uptake of vitamin B12 and causes vitamin B12 deficiency in mice.

Coß-4-ethylphenyl-cob(III) alamin (EtPhCbl) is an organometallic analogue of vitamin B12 (CNCbl) which binds to transcobalamin (TC), a plasma protein that facilitates the cellular uptake of cobalamin (Cbl). In vitro assays with key enzymes do not convert EtPhCbl to the active coenzyme forms of Cbl suggesting that administration of EtPhCbl may cause cellular Cbl deficiency. Here, we investigate the in vivo effect of EtPhCbl in mice and its ability, if any, to induce Cbl deficiency. We show that EtPhCbl binds to mouse TC and we examined mice that received 3.5 nmol/24h EtPhCbl (n=6), 3.5 nmol/24h CNCbl (n=7) or NaCl (control group) (n=5) through osmotic mini-pumps for four weeks. We analyzed plasma, urine, liver, spleen, submaxillary glands and spinal cord for Cbl and markers of Cbl deficiency including methylmalonic acid (MMA) and homocysteine (tHcy). Plasma MMA (mean±SEM) was elevated in animals treated with EtPhCbl (1.01±0.12 µmol/L) compared to controls (0.30±0.02 µmol/L) and CNCbl (0.29±0.01 µmol/L) treated animals. The same pattern was observed for tHcy. Plasma total Cbl concentration was higher in animals treated with EtPhCbl (128.82±1.87 nmol/L) than in CNCbl treated animals (87.64±0.93 nmol/L). However, the organ levels of total Cbl were significantly lower in animals treated with EtPhCbl compared to CNCbl treated animals or controls, notably in the liver (157.07±8.56 pmol/g vs. 603.85±20.02 pmol/g, and 443.09±12.32 pmol/g, respectively). Differences between the three groups was analysed using one-way ANOVA and, Bonferroni post-hoc test. EtPhCbl was present in all tissues, except the spinal cord, accounting for 35-90% of total Cbl. In conclusion, treatment with EtPhCbl induces biochemical evidence of Cbl deficiency. This may in part be caused by a compromised tissue accumulation of Cbl.