Promoterless, Nuclease-Free Genome Editing Confers a Growth Advantage for Corrected Hepatocytes in Mice With Methylmalonic Acidemia.

BACKGROUND AND AIMS: Adeno-associated viral (AAV) gene therapy has shown great promise as an alternative treatment for metabolic disorders managed using liver transplantation, but remains limited by transgene loss and genotoxicity. Our study aims to test an AAV vector with a promoterless integrating cassette, designed to provide sustained hepatic transgene expression and reduced toxicity in comparison to canonical AAV therapy. APPROACH AND RESULTS: Our AAV vector was designed to insert a methylmalonyl-CoA mutase (MMUT) transgene into the 3' end of the albumin locus and tested in mouse models of methylmalonic acidemia (MMA). After neonatal delivery, we longitudinally evaluated hepatic transgene expression, plasma levels of methylmalonate, and the MMA biomarker, fibroblast growth factor 21 (Fgf21), as well as integration of MMUT in the albumin locus. At necropsy, we surveyed for AAV-related hepatocellular carcinoma (HCC) in all treated MMA mice and control littermates. AAV-mediated genome editing of MMUT into the albumin locus resulted in permanent hepatic correction in MMA mouse models, which was accompanied by decreased levels of methylmalonate and Fgf21, and improved survival without HCC. With time, levels of transgene expression increased and methylmalonate progressively decreased, whereas the number of albumin-MMUT integrations and corrected hepatocytes in MMA mice increased, but not in similarly treated wild-type animals. Additionally, expression of MMUT in the setting of MMA conferred a selective growth advantage upon edited cells, which potentiates the therapeutic response. CONCLUSIONS: In conclusion, our findings demonstrate that AAV-mediated, promoterless, nuclease-free genome editing at the albumin locus provides safe and durable therapeutic benefit in neonatally treated MMA mice.

Identification of metabolite biomarkers in serum of rats exposed to chlorpyrifos and cadmium.

Chlorpyrifos (CPF) and cadmium (Cd) are widespread environmental pollutants, which are often present in drinking water and foods. However, the combined effects of CPF and Cd were not entirely clear at present. There was also no biomarker available to diagnose the poisoning of the two chemicals at low dose for long-term exposures. In this study, we investigated the change of serum metabolites of rats with subchronic exposure to CPF, Cd, and CPF plus Cd using gas chromatography-mass spectrometer-based metabolomics approach. We performed a stepwise optimization algorithm based on receiver operating characteristic to identify serum metabolite biomarkers for toxic diagnosis of the chemicals at different doses after 90-day exposure. We found that aminomalonic acid was the biomarker for the toxicity of Cd alone administration, and serine and propanoic acid were unique biomarkers for the toxicities of CPF plus Cd administrations. Our results suggest that subchronic exposure to CPF and Cd alone, or in combination at their low doses, could cause disturbance of energy and amino acid metabolism. Overall, we have shown that analysis of serum metabolomics can make exceptional contributions to the understanding of the toxic effects following long-term low-dose exposure of the organophosphorus pesticide and heavy metal.

Taurine, glutamic acid and ethylmalonic acid as important metabolites for detecting human breast cancer based on the targeted metabolomics.

BACKGROUND: This study investigated the use of serum amino acids and organic acids profiles as the novel metabolites for screening breast cancer (BC) patients. METHODS: A total of 116 subjects as training set were divided into the following three groups: BC patients (n= 34), benign (BE) patients (n= 38) and controls (n= 44). The amino acids profiles from three groups were measured using liquid chromatography-mass spectrometry and organic acids profiles in three groups were studied by gas chromatography-mass spectrometry. The resultant study data set was subjected to multivariate statistical analysis to identify important metabolites related with BC and construct the criteria for discriminating BC patients from BE subjects or controls. A test data set derived from 60 patients (30 BC and 30 BE subjects) and 30 controls was used to validate the stability of the different metabolites. RESULTS: The serum amino acids and organic acids profiles significantly differed between the BC patients, BE patients and the controls. Our results demonstrate that combinations of three candidate metabolites from taurine, glutamic acid and ethylmalonic acid were found to mirror tumour burden, with AUC values ranging from 0.751 to 0.834 when comparing BC patients to the controls. The areas under the curve from the taurine, glutamic acid and ethylmalonic acid in validated study were 0.901, 0.924 and 0.749, respectively. CONCLUSIONS: This study shows that amino acids and organic acids profiles will be a potential screening tool for BC patients. The dysregulated metabolism of amino acids and organic acids in breast cancer might be useful for the diagnosis, therapy, prognosis and understanding the pathogenesis of breast cancer.

Identification and Quantitation of Malonic Acid Biomarkers of In-Born Error Metabolism by Targeted Metabolomics.

Malonic acid (MA), methylmalonic acid (MMA), and ethylmalonic acid (EMA) metabolites are implicated in various non-cancer disorders that are associated with inborn-error metabolism. In this study, we have slightly modified the published 3-nitrophenylhydrazine (3NPH) derivatization method and applied it to derivatize MA, MMA, and EMA to their hydrazone derivatives, which were amenable for liquid chromatography- mass spectrometry (LC-MS) quantitation. 3NPH was used to derivatize MA, MMA, and EMA, and multiple reaction monitoring (MRM) transitions of the corresponding derivatives were determined by product-ion experiments. Data normalization and absolute quantitation were achieved by using 3NPH derivatized isotopic labeled compounds 13C2-MA, MMA-D3, and EMA-D3. The detection limits were found to be at nanomolar concentrations and a good linearity was achieved from nanomolar to millimolar concentrations. As a proof of concept study, we have investigated the levels of malonic acids in mouse plasma with malonyl-CoA decarboxylase deficiency (MCD-D), and we have successfully applied 3NPH method to identify and quantitate all three malonic acids in wild type (WT) and MCD-D plasma with high accuracy. The results of this method were compared with that of underivatized malonic acid standards experiments that were performed using hydrophilic interaction liquid chromatography (HILIC)-MRM. Compared with HILIC method, 3NPH derivatization strategy was found to be very efficient to identify these molecules as it greatly improved the sensitivity, quantitation accuracy, as well as peak shape and resolution. Furthermore, there was no matrix effect in LC-MS analysis and the derivatized metabolites were found to be very stable for longer time. Graphical Abstract ᅟ.

Estimation of the extent of in vivo formation of a mutagenic aromatic amine from a potent thyromimetic compound: correlation of in vitro and in vivo findings.

The development of compounds with the potential for genotoxicity poses significant safety risks as well as risks of attrition. Although genotoxicity evaluation of the parent molecule is routine and reasonably predictive, assessing the risk of commercialization when release of a genotoxic degradant and/or metabolite from a nongenotoxic parent molecule is suspected is much more challenging and resource intensive. Much of the risk of the formation of a genotoxic degradant/metabolite can be discharged with the conduct of carcinogenicity studies in models where the compound is formed, but this approach requires a great deal of time and resources. In this manuscript, we investigated the contribution of various factors (pH, serum instability, and hepatic metabolism) to the formation of a mutagenic aromatic amine from a potent and highly selective thyromimetic compound ([3-(3,5-dibromo-4-(4-hydroxy-3-isopropyl-5-methylphenoxy)-2-methylphenylamino)-3-oxopropanoic acid], compound 1), under in vitro conditions. The kinetic parameters obtained from in vitro experiments combined with the pharmacokinetics of 1in vivo (e.g., plasma concentration-time profile and clearance) were used to estimate the extent of in vivo formation of [4-(4-amino-2,6-dibromo-3-methylphenoxy)-2-isopropyl-6-methylphenol] (compound 2), in rats upon administration of a single oral dose of 1. The agreement between the predicted values (1.9% conversion of total administered dose) with the observed levels of 2 in rats (0.2%-2.2% of the 10 mg/kg dose, 10 mg/kg) further prompted the utilization of this approach to predict the extent of release of this mutagen in humans upon administration of 1. The projection of 0.13% conversion to 2 from an efficacious daily dose of 15 mg of 1 translated to the generation of 20 µg of 2 and provided the basis for the decision to terminate the development of 1.

Clinical, biochemical, magnetic resonance imaging (MRI) and proton magnetic resonance spectroscopy (1H MRS) findings in a fourth case of combined D- and L-2 hydroxyglutaric aciduria.

We report the fourth case of combined D-and L-2-hydroxyglutaric aciduria presenting with neonatal encephalopathy and subependymal cysts.

Pharmacokinetics, tissue distribution, and excretion of cis-malonato[(4R,5R)-4,5-bis(aminomethyl)-2-isopropyl-1,3- dioxolane]platinum(II) in dogs.

The pharmacokinetics, tissue distribution, and excretion of cis-malonato[(4R,5R)-4,5-bis(aminomethyl)-2-isopropyl-1,3- dioxolane]platinum(II) (SKI 2053R), a new potential anticancer agent, were investigated in dogs after a single intravenous administration of [14C]SKI 2053R (7 mg/kg, 100 microCi/kg). Total radioactivity in the plasma and ultrafiltrable plasma declined in a biexponential fashion with the initial half-lives of 0.63 +/- 0.05 hr (mean +/- SD) and 0.53 +/- 0.05 hr, and with the terminal half-lives of 51.08 +/- 3.26 hr and 15.19 +/- 3.75 hr, respectively. Radioactivity was well distributed into all tissues except the central nervous system. The majority of the radioactivity was found in the gastrointestinal contents, urine, and organs of elimination at all time points. The distribution pattern of [14C]SKI 2053R in the whole-body autoradiograms was consistent with that observed by the measurement of tissue concentrations. The 0-7 days cumulative urinary and fecal recoveries of total radioactivity were 87.30 +/- 2.93% and 8.68 +/- 1.30%, respectively, resulting in a total recovery of 95.98 +/- 1.61% of the administered dose. A large portion of [14C]SKI 2053R was distributed into the cellular fraction of mouse or rat blood, but was not into that of dog or human blood in vitro. The in vitro and in vivo binding of [14C]SKI 2053R to plasma protein was minimal to moderate.

Cor pulmonale as a complication of methylmalonic acidemia and homocystinuria (Cbl-C type).

We report an infant with a bronchiolitis-like illness and rapid deterioration who developed a cor pulmonale-like picture with a dilated right ventricle. Urinary organic acid assays established a probable diagnosis of Cbl-C-type methylmalonic aciduria, later confirmed by complementation studies. Despite medical intervention and cyanocobalamin treatment the patient died on his tenth hospital day. Postmortem examination showed the presence of thromboemboli in the pulmonary circulation. We hypothesize that acute cor pulmonale developed in this infant secondary to thromboembolism of his pulmonary circulation. A review of the literature shows that thromboembolism may be a part of this disease process.

[Composition of exhaled air, gas and energy metabolism and bio- chemical indicators of the blood and urine in humans after long- term exposure to hypercapnia and hypoxia]. / Sostav vydykhaemogo vozdukha, gazoénergoobmen i biokhimicheskie pokazateli krovi i mochi u cheloveka pri dlite'nom nakhozhdenii ego v usloviiakh giperkapnii i gipoksii.

Four volunteers were enclosed for 40 days in a hypercapnic environment. Their average age was 41-59 years, body weight, 66-90 kg, and height 173-182 cm. During the study the ambient temperature was 19-23 degrees C, relative humidity, 50 +/- 20%; pO2, 19-19.5%; and pCO2, 1.3%. On test days 21-22 and 38-39 pCO2 was increased to 4% and pO2 was decreased to 17%. The time, within which pCO2 was increased to 4% on test days 38-39 when compared to test days 21-22, grew 1.5-fold and amounted to 40 hours. The subjects had three meals a day, consuming canned foodstuffs, the caloric value of which was 2982 kcal/day. In the study the following parameters were measured: malonic dialdehyde in venous blood; catalase, lactate, pyruvate, urea, acid-base content, gases in capillary blood; total nitrogen, ammonia, urea, creatinine and uric acid in 24-hour urine samples. Nitrogen balance and protein nutrition index were calculated. Results were processed using Student's t-test. During exposure lipid peroxidation increased and catalase decreased; malonic dialdehyde in blood increased, being correlated with lower hydrocarbons in exhaled air; gas and energy turnover during hypercapnic intervals enhanced. The above changes in the exhaled air composition, gas and energy turnover, biochemical blood and urine parameters remained within adaptation norm. By the second week of the recovery period the above parameters, except for nitrogen metabolism which remained slightly inhibited, returned to the normal.

Heterogeneous alleles and expression of methylmalonyl CoA mutase in mut methylmalonic acidemia.

Methylmalonic acidemia (MMA) can be caused by mutations in the gene coding for the methylmalonyl CoA mutase (MCM) apoenzyme or by mutations in genes required for provision of its adenosylcobalamin cofactor. We have characterized MCM activity, gene structure, and expression in a series of primary fibroblast cell lines derived from patients with MCM apoenzyme deficiency. Southern blot analysis reveals normal HindIII and TaqI polymorphisms but no gross insertions, deletions, rearrangements, or point mutations at restriction endonuclease recognition sequences. Northern blot analysis demonstrates that several cell lines have specifically decreased steady-state levels of MCM mRNA. At least six independent alleles can be delineated by a haplotype of HindIII and TaqI polymorphisms, the level of mRNA expression, and the biochemical phenotype of the cells. These studies confirm the wide phenotypic spectrum of MMA and provide molecular genetic evidence for a variety of independent alleles underlying this disorder.

[Changes of creatine phosphokinase and malondialdehyde in the serum and clinical use of large doses of vitamin C following open heart surgery].

The protective effect of a large dose Vit. C on ischemic myocardium was studied on 10 patients who underwent open heart operation and received 250 mg/kg Vit. C before the start of ECC (Group II). Another 10 similar patients receiving no Vit. C served as a control (Group I). Blood samples were taken from the brachial artery at different time intervals for determination of CPK-MB and MDA in both groups. Considerable increases of serum CPK-MB and MDA in both group I and II were noted. The differences of CPK-MB and MDA concentration between Group I and II were highly significant from 2 to 12 hours postoperatively. The effect of Vit. C in the reduction of the release of CPK-MB and MDA after open heart operation was attributed to its action as an oxygen free radical scavenger, thus decreasing the peroxidation of the lipids present in the cell membrane.

Increased plasma levels of glutathione and malondialdehyde after acute ethanol ingestion in humans.

The effect of acute ethanol consumption on plasma glutathione (GSH) and malondialdehyde (MDA) concentrations was studied in two groups of healthy male subjects. The first group (n = 15) received an acute dose of ethanol (1.5 g/kg p.o. over a period of 3 h); in the control group (n = 15), ethanol was replaced isocalorically with carbohydrates. Blood samples were taken at 0 time (ethanol/carbohydrates ingestion) and every 60 min for 6 h. A significant increase in plasma MDA concentration as well as in plasma GSH values were observed in subjects receiving ethanol compared to controls. The enhancement of plasma GSH was accompanied by a concomitant increase of oxidized glutathione (GSSG). These data support the hypothesis of an increase of lipid peroxidation as a possible mechanism of acute ethanol toxicity. The enhancement of plasma GSH and GSSG may reflect an increased utilization and loss of the tripeptide from the liver induced by ethanol.

Chronic renal failure in methylmalonic acidaemia.

The renal function of 12 patients with non vitamin B12 responsive methylmalonic acidaemia has been investigated. Eight patients had reduced glomerular filtration rates, but the plasma creatinine concentration was only raised in those with values of less than 40 ml/min per 1.73 m2 surface area. The reduction in glomerular filtration was a function of the age and the severity of the disease. Plasma urate concentrations were increased in four patients but this may be secondary to the renal disease rather than its cause.

Therapeutic approaches to cobalamin-C methylmalonic acidemia and homocystinuria.

The use of hydroxocobalamin (OH-B12), betaine, carnitine, and folinic acid were studied in two children with the cobalamin C form of methylmalonic acidemia and homocystinuria. When daily injections of 1 mg OH-B12 were discontinued for 3 weeks, there was no significant change in total plasma homocysteine or methionine levels and only a modest increase in methylmalonate. Orally administered OH-B12 1 mg/d in one patient was associated with an increase in plasma homocystine and a decrease in methionine within 1 month. Withdrawal of betaine 250 mg/kg/d was also associated with a rise in plasma homocystine and a fall in methionine levels. Carnitine 100 mg/kg/d lead to an increase in urinary excretion of propionylcarnitine, but did not affect plasma methylmalonate levels. No beneficial biochemical effect of folinic acid could be documented at a dose of 25 mg/d. Our results suggest that daily injections of OH-B12 are not necessary to maintain metabolic control and that orally administered OH-B12 is unlikely to be effective. Betaine appears to act synergistically with OH-B12 and should be part of the treatment regimen. Although there are theoretical reasons for using L-carnitine and folinic acid, we could not document their effectiveness in these two patients.

Application of a new test for vitamin E deficiency to cystic fibrosis.

Cystic fibrosis patients with pancreatic insufficiency are at risk for the development of vitamin E deficiency. We report here the outcome of screening 13 cystic fibrosis patients with conventional descriptive measures of vitamin E status and a new functional test. The results were compared with those from age appropriate controls. Nine patients were found to be vitamin E sufficient based upon normal plasma vitamin E levels, the ratio of plasma vitamin E to total plasma lipids, and normal levels of in vitro erythrocyte malondialdehyde formation, the new functional measure of vitamin E status. Four patients considered vitamin E deficient, based upon low plasma vitamin E levels and plasma vitamin E to total plasma lipid ratios, demonstrated increased erythrocyte malondialdehyde formation in vitro when compared to age-matched controls. Since limited reference data in children are available to define normal plasma vitamin E levels and plasma vitamin E to total plasma lipid ratios, we suggest that for cystic fibrosis patients the functional in vitro malondialdehyde formation test may be a better measure of vitamin E status than static plasma levels.

Gas chromatographic analysis of phenylethylmalonamide in human plasma.

A method is described for the analysis of phenylethylmalonamide in human plasma. Analysis of plasma requires only 200 microliter of sample which is extracted with dichloroethane. After filtration and evaporation of the solvent the residue is reconstituted in 50 microliter of chloroform and 5 microliter are injected onto the gas chromatograph. The column used is a mixture of CDMS/WG11 coated on Chromosorb W HP 100-120 mesh. The method is suitable for use in single-dose pharmacokinetic studies.

Serum malanodialdehyde (TBA reactive substance) levels in cigarette smokers.

Serum malanodialdehyde (MDA) levels in 40 smokers and 23 non-smokers belonging to different age groups were estimated. The MDA levels were high in smokers of all age groups having a history of smoking for less than 10 years. MDA levels, however, were not elevated in smokers with a history of smoking for more than 10 years. These results are discussed and are interpreted as suggestive that MDA might alter the LDL and lead to deposition of cholesterol in arterial wall macrophages explaining thereby the risk of ischaemic heart disease in cigarette smokers.