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.

Serum metabolite profiling of ST-segment elevation myocardial infarction using liquid chromatography quadrupole time-of-flight mass spectrometry.

ST segment elevation myocardial infarction (STEMI) is one of the most common global causes of cardiovascular disease-related death. Several metabolites may change during STEMI. Hence, analysis of metabolites in body fluid may be considered as a rapid and accurate test for initial diagnosis. This study has therefore attempted to determine the variation in metabolites identified in the serum of STEMI patients (n = 20) and 15 controls. Samples collected from the Cardiology Department, Medical Faculty, Ataturk University, were extracted by liquid-liquid extraction and analysed using liquid chromatography quadrupole time-of-flight mass spectrometry. The METLIN database was used for the identification and characterization of metabolites. According to Q-TOF/MS measurements, 231 m/z values, which were significantly different between groups (P < 0.01 and fold analysis >1.5) were detected. Metabolite identification was achieved via the Human Metabolome database. According to the multivariate data analysis, leucine, isoleucine, l-proline, l-alanine, glycine, fumaric acid, citrate, succinate and carnitine levels were decreased, whereas levels of propionic acid, maleic acid, butyric acid, urea, oleic acid, palmitic acid, lysoPC [18:2(9Z)], glycerol, phoshpatidylethanolamine, caffeine and l-lactic acid were increased in STEMI patients compared with controls. In conclusion, malonic acid, maleic acid, fumaric acid and palmitic acid can be used as biomarkers for early risk stratification of patients with STEMI.

[Clinical and genetic analysis of a case with atypical ethyl malonate encephalopathy].

OBJECTIVE: To delineate the clinical and genetic characteristics of a girl featuring motor retardation, language retardation and regression, and light persisting diarrhea. METHODS: The patient was clinically examined and tested by tandem mass spectrometry and next generation sequencing. RESULTS: The proband could not stand and walk alone, and had light persisting diarrhea. She manifested language development retardation and regression. Laboratory tests were all normal, but the screening of metabolic disorders for urine and blood showed deficiency of short chain coenzyme A dehydrogenase due to elevated ethylmalonic acid and butyryl carnitine. By next generation sequencing, two compound heterozygous mutations of the ETHE1 gene, c.2T>A and c.488G>A, were discovered in the proband, which were respectively inherited from her father and mother. Bioinformatics analysis predicted both mutations to be pathogenic. The patient was diagnosed with ethylmalonic encephalopathy. Vitamin B1, B2, Coenzyme Q10, and L-carnitine were prescribed. The patient deteriorated and required liver transplantation at 4-year-1-month. CONCLUSION: Based on the clinical and genetic analysis, the proband was diagnosed with ethylmalonic encephalopathy caused by ETHE1 gene mutation. Next generation sequencing has provided a powerful tool for the diagnosis of such disorders.

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.

Biochemical, molecular and outcome analysis of eight chinese asymptomatic individuals with methyl malonic acidemia detected through newborn screening.

Methyl malonic academia (MMA) is characterized by abnormal accumulation of methyl malonic acid in body fluids. Patients usually have a variety of clinical symptoms including recurrent vomiting, metabolic acidosis, developmental delay, seizure, or death. However, a few cases where the patients have no symptom are also reported. Here, we conducted clinical, biochemical, and molecular analysis of eight Chinese patients identified through newborn screening between 2003 and 2013. All the patients had significantly higher blood propionylcarnitine (C3) concentrations, ratio of propionylcarnitine/acetylcarnitine (C3/C2); and their urine methyl malonic acid and methylcitric acid (MCA) excretions were remarkably higher than normal at diagnosis and during follow-ups. In addition, five different known mutations were identified in seven of the eight patients in either MUT or MMACHC. All these mutations were expected to produce defective proteins that would result in decreased or even total loss of methyl malonyl-CoA mutase activity. However, normal outcomes were found in all patients in physical growth, intellectual performance and cerebral MRI analysis at diagnosis (range, 14-53 days) and during follow-ups (range, 1.8-10 years). Our study is the first report of Chinese MMA patients with increased secretion of methyl malonic acid and molecular defects in MUT or MMACHC yet remain asymptomatic.

Dynamic metabolites profile of cerebral ischemia/reperfusion revealed by (1)H NMR-based metabolomics contributes to potential biomarkers.

Current metabolomic studies of ischemic brain mainly attach importance on a certain ischemic period, are lack of data about dynamic metabolites in ischemic stroke process, especially early period. Thus, in this study, (1)H NMR spectroscopy was used to investigate biochemical changes in the early stages of rats' focal cerebral ischemia reperfusion (I/R) injury. Serum samples of 0, 0.5, 1, 3, 6, 12, 24 h of reperfusion, based on multivariate data analyses, were tested to analyze the changing of metabolites during the early disease process. Partial least squares-discriminant analysis scores plots of the (1)H NMR data revealed clear differences among the experiment groups. Combination the results of loading plot and t-test, we found that 13 metabolites were changed significantly. Among that, malonic acid and glycine are the most noticeable variable metabolites. Dramatic changed malonic acid and glycine most probably served as biomarkers in this study. These findings help us understand the biochemical metabolite changes in early ischemic stroke stages, especially different periods. That may be conducive to distinguish at-risk individuals, benefit early diagnosis and understand the dynamic pathogenesis of early cerebral ischemia.

Structure-activity relationships and blood distribution of antiplasmodial aminopeptidase-1 inhibitors.

Malaria is a severe infectious disease that causes between 655,000 and 1.2 million deaths annually. To overcome the resistance to current drugs, new biological targets are needed for drug development. Aminopeptidase M1 (PfAM1), a zinc metalloprotease, has been proposed as a new drug target to fight malaria. Herein, we disclosed the structure-activity relationships of a selective family of hydroxamate PfAM1 inhibitors based on the malonic template. In particular, we performed a "fluoro-scanning" around hit 1 that enlightened the key positions of the halogen for activity. The docking of the best inhibitor 2 is consistent with in vitro results. The stability of 2 was evaluated in microsomes, in plasma, and toward glutathione. The in vivo distribution study performed with the nanomolar hydroxamate inhibitor 2 (BDM14471) revealed that it reaches its site of action. However, it fails to kill the parasite at concentrations relevant to the enzymatic inhibitory potency, suggesting that killing the parasite remains a challenge for potent and druglike catalytic-site binding PfAM1 inhibitors. In all, this study provides important insights for the design of inhibitors of PfAM1 and the validity of this target.

[Plasma metabonomics study of ischemic cerebral apoplexy rats treated with Tongsaimai pellets].

OBJECTIVE: To observe abnormal metabolic changes caused by ischemic cerebral apoplexy and the regulating action of Tongsaimai pellets on abnormal metabolism by analyzing the change of small molecules in plasma of ischemic cerebral apoplexy rat. To find the potential biomarkers, and to explore metabolic mechanisms of Tongsaimai pellets. METHOD: Rat models of middle cerebral artery occlusion was established with electric coagulation, and rats were divided into 4 groups, model group, sham-operation group, Tongsaimai pellets group and positive control group. Tongsaimai pellets and positive control group were orally administrated by 13.2 g x kg(-1) x d(-1) of crude drugs and 32 mg x kg(-1) x d(-1) of Nimodipine respectively, m odel and sham-operation group by equal volume of distilled water for a week. Plasma of model and sham-operation group were collected, and plasma of Tongsaimai pellets and positive control group were collected on the 1st, 3rd , 7th day after administration. Endogenous metabolites of four groups were determined with GC-MS. Partial least squares discriminant analysis (PLS-DA) was applied to analyze multivariate data and set up model, and T-test was used in significant statistical analysis. RESULT: Compared with sham-operation group rats, pyruvic acid, taurine and hydroxyproline obviously increased in model group rats, while lactic acid, glyceric acid, aminomalonic acid, fructose, tryptophan and leucine significantly decreased, so these metabolites were potential metabolic biomarkers. These endogenous metabolites except taurine got restoration in Tongsaimai group rats. CONCLUSION: Abnormal metabolite level in plasma can be certainly recovered by Tongsaimai pellets, and the treatment of Tongsaimai pellets can be connected with the regulation of related metabolic pathways.

Biochemical, molecular, and clinical characteristics of children with short chain acyl-CoA dehydrogenase deficiency detected by newborn screening in California.

BACKGROUND: Short-chain acyl-CoA dehydrogenase deficiency (SCADD) is an autosomal recessive inborn error of mitochondrial fatty acid oxidation with highly variable biochemical, genetic, and clinical characteristics. SCADD has been associated with accumulation of butyryl-CoA byproducts, including butyrylcarnitine (C4), butyrylglycine, ethylmalonic acid (EMA), and methylsuccinic acid (MS) in body fluid and tissues. Differences in genotype frequencies have been shown between patients diagnosed clinically versus those diagnosed by newborn screening. Moreover, while patients diagnosed clinically have a variable clinical presentation including developmental delay, ketotic hypoglycemia, epilepsy and behavioral disorders, studies suggest patients diagnosed by newborn screening are largely asymptomatic. Scant information is published about the biochemical, genetic and clinical outcome of SCADD patients diagnosed by newborn screening. METHODS: We collected California newborn screening, follow-up biochemical levels, and ACADS mutation data from September, 2005 through April, 2010. We retrospectively reviewed available data on SCADD cases diagnosed by newborn screening for clinical outcomes. RESULTS: During the study period, 2,632,058 newborns were screened and 76 confirmed SCADD cases were identified. No correlations between initial C4 value and follow-up biochemical markers (C4, EMA or MS levels) were found in the 76 cases studied. We found significant correlation between urine EMA versus MS, and correlation between follow-up C4 versus urine EMA. Of 22 cases where ACADS gene sequencing was performed: 7 had two or more deleterious mutations; 8 were compound heterozygotes for a deleterious mutation and common variant; 7 were homozygous for the common variant c.625G>A; and 1 was heterozygous for c.625G>A. Significant increases in mean urine EMA and MS levels were noted in patients with two or more deleterious mutations versus mutation heterozygotes or common polymorphism homozygotes. Clinical outcome data was available in 31 patients with follow-up extending from 0.5 to 60 months. None developed epilepsy or behavioral disorders, and three patients had isolated speech delay. Hypoglycemia occurred in two patients, both in the neonatal period. The first patient had concomitant meconium aspiration; the other presented with central apnea, poor feeding, and hypotonia. The latter, a c.625G>A homozygote, has had persistent elevations in both short- and medium-chain acylcarnitines; diagnostic workup in this case is extensive and ongoing. CONCLUSIONS: This study examines the largest series to date of SCADD patients identified by newborn screening. Our results suggest that confirmatory tests may be useful to differentiate patients with common variants from those with deleterious mutations. This study also provides evidence to suggest that, even when associated with deleterious mutations, SCADD diagnosed by newborn screening presents largely as a benign condition.

Application of quantitative nuclear magnetic resonance spectroscopy for the determination ofamantadine and acyclovir in plasma and pharmaceutical samples.

Rapid, simple, and selective methods for determining amantadine HCI and acyclovir antiviral drugs in pharmaceutical and plasma samples were developed using 1H-NMR spectroscopy with dimethyl sulfoxide (DMSO-d6) as the solvent. Integrations of the 1H-NMR signals at 2.07 and 7.82 ppm were used, respectively, for quantifying the two drugs, with the malonic acid signal at 3.24 ppm as the internal reference signal. Average recoveries of 98.24-101.00 +/- 4.82% and 97.7-100.38 +/- 3.36% were obtained for amantadine HCI and acyclovir in pharmaceutical samples, respectively. Average recoveries of 97.36-103.68 +/- 2.99 and 93.81-99.80 +/- 2.93 were obtained, respectively, for both drugs in plasma samples. The statistical Student's t-test gave t-values < or = 1.41 for analyzed pharmaceutical samples and t-values < or = 0.29 for analyzed plasma samples. These values indicated insignificant difference between the real and measured contents at the 95% confidence level. Application of the statistical F-test for the analytical results of amantadine HCI gave F-values < or = 6.44 and 2.80 in pharmaceutical and plasma samples, respectively. F-values < or = 6.82 and 3.86 were obtained for acyclovir in pharmaceutical and plasma, respectively. These values indicated insignificant differences in precisions between the developed NMR methods and arbitrarily chosen HPLC methods reported for determining both drugs in pharmaceutical and plasma samples.

Physicochemical and pharmacokinetic characterization of a spray-dried malotilate emulsion.

Malotilate (MT) is a hepatoprotective drug administered orally. However, MT was found to be a poorly water-soluble drug with low oral bioavailability. In the present investigation, a novel spray-dried emulsion (SDE) loaded with MT was prepared, and its physicochemical properties were characterized by rheological evaluation, particle size measurement, in vitro release, and surface morphology. The pharmacokinetic study of SDE, in comparison to MT suspension with the pure MT powder homogeneously dispersed in 0.5% CMC-Na solution, was also performed in rats after a single oral dose. It was found that SDE exhibited a 2.9-fold higher peak plasma concentration (C(max)) and 2.3-fold higher area under the curve (AUC) than MT suspension.

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.

Malonylcarnitine in newborns with non-syndromic cleft lip with or without cleft palate.

AIM: Malonyl-CoA is regarded as a key signaling molecule in mammalian cells. It is converted to acetyl-CoA, and to a lesser extent, to malonyl acid and malonylcarnitine (C3DC). Availability of carnitine has been reported to be essential for the developing fetus. The objectives of the present study were to analyze associations of malonylcarnitine, acetylcarnitine (C2), and free carnitine (CO) in subjects with orofacial clefts. METHODOLOGY: We performed a retrospective analysis of carnitine concentration obtained from a newborn screening program carried out in our institution. Concentrations of whole blood malonylcarnitine, acetylcarnitine, and free carnitine were measured using tandem mass spectrometry. The study group consisted of 51 children with nonsyndromic cleft lip with or without cleft palate. In total, 106 healthy children without congenital anomalies served as controls. Cut-off points were established using likelihood ratio values. RESULTS: The mean concentration of malonylcarnitine in the cleft group was lower than that of the control group, 0.048 micromol x L(-1) vs. 0.058 micromol x L(-1), respectively (P = 0.009). In patients with orofacial cleft, low malonylcarnitine levels (< or = 0.047 micromol x L(-1)) were 1.7 times more predominant than in healthy individuals (P = -0.03). The mean concentration of acetylcarnitine was also lower in affected newborns in comparison to controls, 33.8 micromol x L(-1) vs. 37.8 micromol x L(-1), respectively (P = 0.026). After analysis of acetylcarnitine and free carnitine concentrations, the likelihood ratio test did not indicate valuable cut-offpoints. CONCLUSION: The study provides initial data indicating a potential association between decreased malonylcarnitine and abnormal palatogenesis.

Multiple sources of metabolic disturbance in ETHE1-related ethylmalonic encephalopathy.

Ethylmalonic encephalopathy (EE) is a rare metabolic disorder caused by dysfunction of ETHE1, a mitochondrial dioxygenase involved in hydrogen sulfide (H2S) detoxification. Patients present in infancy with psychomotor retardation, chronic diarrhea, orthostatic acrocyanosis and relapsing petechiae. High levels of lactic acid, ethymalonic acid (EMA) and methylsuccinic acid (MSA) are detected in body fluids. Several pathways may contribute to the pathophysiology, including isoleucine, methionine and fatty acid metabolism. We report on a 15-month-old male presenting with typical EE associated with a homozygous ETHE1 mutation. We investigated oral isoleucine (150 mg/kg), methionine (100 mg/kg), fatty acid loading tests and isoleucine-restricted diet (200 mg/day) for any effects on several metabolic parameters. Before loading tests or specific dietary interventions, EMA, C4-C5 acylcarnitines and most acylglycines were elevated, indicating functional deficiency of short chain acyl-CoA (SCAD) as well as all branched acyl-CoA dehydrogenases. Excretion of EMA and n-butyrylglycine increased following each of the loads, and isoleucine led to increased levels of derivative metabolites. An isoleucine-restricted diet for 8 days corrected some of the abnormalities but led to no obvious clinical improvement and only partial effects on EMA. A principal component analysis supports the inference that these dietary conditions have consistent effects on the global metabolic profile. Our results suggest that multiple pathways modulate EMA levels in EE. They might all interact with H2S toxicity. Prolonged dietary interventions involving the restriction for branched aminoacids, fatty acids and methionine could be discussed as auxiliary therapeutical strategies in EE.

Highly sensitive quantification of serum malonate, a possible marker for de novo lipogenesis, by LC-ESI-MS/MS.

We describe a new sensitive and specific method for the quantification of serum malonate (malonic acid, MA), which could be a new biomarker for de novo lipogenesis (fatty acid synthesis). This method is based upon a stable isotope-dilution technique using LC-MS/MS. MA from 50 microl of serum was derivatized into di-(1-methyl-3-piperidinyl)malonate (DMP-MA) and quantified by LC-MS/MS using the positive electrospray ionization mode. The detection limit of the DMP-MA was approximately 4.8 fmol (500 fg) (signal-to-noise ratio = 10), which was more than 100 times more sensitive compared with that of MA by LC-MS/MS using the negative electrospray ionization mode. The relative standard deviations between sample preparations and measurements made using the present method were 4.4% and 3.2%, respectively, by one-way ANOVA. Recovery experiments were performed using 50 microl aliquots of normal human serum spiked with 9.6 pmol (1 ng) to 28.8 pmol (3 ng) of MA and were validated by orthogonal regression analysis. The results showed that the estimated amount within a 95% confidence limit was 14.1 +/- 1.1 pmol, which was in complete agreement with the observed X(0) = 15.0 +/- 0.6 pmol, with a mean recovery of 96.0%. This method provides reliable and reproducible results for the quantification of MA in human serum.

Quantification of malonylcarnitine in dried blood spots by use of MS/MS varies by stable isotope internal standard composition.

BACKGROUND: The utilization of MS/MS for the analysis of amino acids and acylcarnitines from dried blood spots (DBS) is routine in many newborn screening (NBS) laboratories. Recently, malonylcarnitine (C3DC) was shown to be elevated in the DBS of affected infants with malonic acidemia. Quantitative features were unknown, so that its measurement was an approximation. Synthesis of malonylcarnitine enabled both a study in the analytical characteristics of C3DC and a survey of its measurement in NBS laboratories. METHODS: Malonylcarnitine was enriched in blood and spotted onto filter paper cards. The DBS were sent to several laboratories for analysis, and the results were returned to the Centers for Disease Control and Prevention (CDC) for evaluation. Reports included a description of the MS/MS method utilized. RESULTS: A pilot proficiency survey shows a bimodal distribution of data from 98 laboratories. Analysis of proficiency data reveals the use of different stable isotope internal standards for quantification. Analysis of standard, labeled or unlabelled ((2)H(3)-octanoylcarnitine (C8), glutarylcarnitine (C5DC) and malonylcarnitine (C3DC) revealed significantly different ion detection values. Quantification in laboratories is based on the ratio of the metabolite in question to a reference stable isotope standard. CONCLUSIONS: Quantification of metabolites depends upon the reference isotope standard utilized. Quantification requires describing the standards used for estimation of concentration (a pseudo-concentration) and a notation that includes a reference to the isotope standard used. This descriptive method will enable harmonization of data in screening laboratories.

[Simulation of the disease caused by Hantaan virus in white mice by a complex of virological, biochemical, and hematological parameters].

The essence of studies was that the disease is simulated in 12-day albino mice subcutaneously infected with Hantaan virus, strain 76-118 in a dose of 10 LD50. As an efficiency index, the study of drugs uses major (death protection coefficient, mean animal lifetime) and auxiliary (virological: pathogen accumulation in the brain tissues of deceased animals) parameters, biochemical (the levels of creatinine, urea, alanine aminotransferase, aspartate aminotransferase, malonic dialdehyde), hematological (count of leukocytes, leukogram) ones; as well as interferon status (the levels of circulatory interferon, leukocytic production of alpha- and gamma-interferons). The procedure for simulating the disease caused by Hantaan virus on an experimental animal is used to choose effective drugs against the pathogen of hemorrhagic nephrosonephritis.

Vitamin and mineral status in elderly patients with chronic obstructive pulmonary disease.

INTRODUCTION: Eating problems are common in patients with chronic obstructive pulmonary disease (COPD), and intake of micronutrients might be lower than recommended. OBJECTIVE: To study dietary intake, serum and urinary concentration of trace elements and vitamins in elderly underweight patients with established severe COPD. METHODS: Outpatients at a university clinic for lung medicine, with COPD, 70-85 years old, with no other serious disease, and with a body mass index (BMI) of