Organic Acid Analysis Research Articles

Gas chromatography-mass spectrometric determination of organic acids in fruit juices by multiwalled carbon nanotube-based ion-pair dispersive solid-phase extraction and in situ butylation.

Fruit juices are naturally acidic, and the acidity is due to the formation of various organic acids formed in several metabolic processes. The content of acids varies due to various processing parameters during the preparation of fruit juices and their packaging for commercialization. Quantitative determination of organic acids provides the necessary information leading to changes occurred during processing. The organic acids were extracted by ion-pair dispersive solid-phase extraction by multiwalled carbon nanotubes and analyzed using gas chromatography-mass spectrometry (GC-MS) and in situ butylation. The developed analytical method was validated, and the obtained results showed a linearity in the range of 0.5-5000 μg/L levels of the analytes with limit of detection and quantification values of 2-10 and 5-20 μg/L, respectively. The inter- and intra-day reproducibilities are less than 15% with 80%-98% recoveries and less than 20% matrix effect. The developed method was used for the quantitative determination of organic acids present in fresh and packaged apple, grape, orange, and pomegranate juice samples. The content of organic acids was observed in the range of 0.26-3793 μg/L. Pimelic acid was not detected in any of the analyzed fruit juices. Fumaric acid (FA) was observed to be a major organic diacid present in the natural fruit juices. The results indicated that the processing of fruit juices for packaging decreases the content of organic acids in fruits. The developed GC-MS-based analytical method for the analysis of organic acids has good sensitivity and reproducibility for the quantitative determination of various organic acids in fruit juices. FA was observed to be the major carboxylic acid present in fruits. The processed juice samples possess the lowest concentration of organic acids, suggesting that processing has a significant effect on the concentration of organic acids in fruits.

Biochemical and molecular features of Chinese patients with glutaric acidemia type 1 detected through newborn screening

BackgroundGlutaric acidemia type 1 (GA1) is a treatable disorder affecting cerebral organic acid metabolism caused by a defective glutaryl-CoA dehydrogenase (GCDH) gene. GA1 diagnosis reports following newborn screening (NBS) are scarce in the Chinese population. This study aimed to assess the acylcarnitine profiles and genetic characteristics of patients with GA1 identified through NBS.Results From January 2014 to September 2020, 517,484 newborns were screened by tandem mass spectrometry, 102 newborns with elevated glutarylcarnitine (C5DC) levels were called back. Thirteen patients were diagnosed with GA1, including 11 neonatal GA1 and two maternal GA1 patients. The incidence of GA1 in the Quanzhou region was estimated at 1 in 47,044 newborns. The initial NBS results showed that all but one of the patients had moderate to markedly increased C5DC levels. Notably, one neonatal patient with low free carnitine (C0) level suggest primary carnitine deficiency (PCD) but was ultimately diagnosed as GA1. Nine neonatal GA1 patients underwent urinary organic acid analyses: eight had elevated GA and 3HGA levels, and one was reported to be within the normal range. Ten distinct GCDH variants were identified. Eight were previously reported, and two were newly identified. In silico prediction tools and protein modeling analyses suggested that the newly identified variants were potentially pathogenic. The most common variant was c.1244-2 A>C, which had an allelic frequency of 54.55% (12/22), followed by c.1261G>A (p.Ala421Thr) at 9.09% (2/22).ConclusionsNeonatal GA1 patients with increased C5DC levels can be identified through NBS. Maternal GA1 patients can also be detected using NBS due to the low C0 levels in their infants. Few neonatal GA1 patients may have atypical acylcarnitine profiles that are easy to miss during NBS; therefore, multigene panel testing should be performed in newborns with low C0 levels. This study indicates that the GCDH variant spectra were heterogeneous in this southern Chinese cohort.

Development of Second-Tier Liquid Chromatography-Tandem Mass Spectrometry Analysis for Expanded Newborn Screening in Japan.

To minimize false-positive cases in newborn screening by tandem mass spectrometry in Japan, practical second-tier liquid chromatography-tandem mass spectrometry analyses have been developed using a multimode ODS column with a single set of mobile phases and different gradient elution programs specific to the analysis of acylcarnitines, acylglycines, amino acids, and organic acids. Most analyses were performed using underivatized samples, except for analysis of methylcitric acid, and careful conditioning of the column was necessary for analyses of organic acids. Our second-tier tests enabled us to measure many metabolites useful for detection of target disorders, including allo-isoleucine, homocysteine, methylmalonic acid, and methylcitric acid. We found that accumulation of 3-hydroxyglutaric acid was specific to glutaric acidemia type I and that the ratio of 3-hydroxyisovaleric acid to 3-hydroxyisovalerylcarnitine was useful to detect newborns of mothers with 3-methylcrotonyl-CoA carboxylase deficiency. Data from the analysis of short-chain acylcarnitine and acylglycine were useful for differential diagnosis in cases positive for C5-OH-acylcarnitine or C5-acylcarnitine.

Implementation of an enclosed ionization interface for the analysis of liquid sample streams with direct analysis in real time mass spectrometry.

The development of an interface to analyze liquid sample streams with direct analysis in real time mass spectrometry (DART-MS) is of great interest for coupling various analytical techniques, using non-volatile salts, with MS. Therefore, we devised an enclosed ionization interface and a sample introduction system for the versatile analysis of liquid samples with DART-MS. The sample introduction system consists of a nebulizer, a spray chamber and a transfer line, while the confined ionization interface is created by implementing a cross-shaped housing between ion source outlet and mass spectrometer inlet. Methodical studies of the effects of various setup parameters on signal intensity and peak shape were conducted, while its diverse applicability was demonstrated by coupling with high-performance liquid chromatography (HPLC) for the analysis of alcohols, organic acids and furanic compounds. The confinement of the ionization interface results in a robust setup design with a well-defined ionization region for focusing of the sprayed sample mist. Thereby, an increase in analyte signal intensity by three orders of magnitude and improved signal stability and reproducibility were obtained in comparison with a similar open ionization interface configuration. Additionally, the successful quantification of alcohols could be demonstrated as well as the compatibility of the setup with HPLC gradient elution. A versatile setup design for the analysis of liquid sample streams with DART-MS was devised for monitoring reactions or hyphenating analytics with MS. The design minimizes interferences from the laboratory surroundings as well as allows for safe handling of hazardous and toxic chemicals, which renders it suitable for a broad range of applications.

Progress in pretreatment and analysis of organic Acids: An update since 2010

Organic acids, as an important component of food, have great influence on the flavor, texture, freshness of food. By lowering the pH of food to bacteriostatic acidity, organic acids are also used as additives and preservatives. Because organic acids are crucial to predict and evaluate food maturity, production and quality control, the rapid and sensitive determination methods of organic acids are necessary. This review aims to summarize and update the progress of the determination of organic acids in food samples. Pretreatment methods include simple steps (e.g., “dilute and shoot,” protein precipitation, filtration, and centrifugation) and advanced microextraction methods (e.g., hollow fiber liquid phase microextraction, stir bar sorptive extraction and dispersive micro-solid phase extraction). Advances in novel materials (nanomaterial), solvents (ionic liquids and supercritical fluids) and hybrid methods are clearly displayed in detail. Continuous progress which has been made in electrochemical method, two-dimensional chromatography, high resolution mass is thoroughly illustrated.

The low excretor phenotype of glutaric acidemia type I is a source of false negative newborn screening results and challenging diagnoses.

BackgroundGlutaric acidemia type I (GA1) is an organic acidemia that is often unrecognized in the newborn period until patients suffer an acute encephalopathic crisis, which can be mistaken for nonaccidental trauma. Presymptomatic identification of GA1 patients is possible by newborn screening (NBS). However, the biochemical “low‐excretor” (LE) phenotype with nearly normal levels of disease metabolites can be overlooked, which may result in untreated disease and irreversible neurological sequelae. The LE phenotype is also a potential source of false negative (FN) NBS results that merits further investigation.MethodsSamples from six LE GA1 patients were analyzed by biochemical and molecular methods and newborn screen outcomes were retrospectively investigated.ResultsFive LE GA1 patients were identified that had normal NBS results and three of these presented clinically with GA1 symptoms. One additional symptomatic patient was identified who did not undergo screening. Semiquantitative urine organic acid analysis was consistent with a GA1 diagnosis in two (33%) of the six patients, while plasma glutarylcarnitine was elevated in four (67%) of the six and urine glutarylcarnitine was elevated in four (80%) of five patients. Five GCDH variants were identified in these patients; three of which have not been previously linked to the biochemical LE phenotype.ConclusionsThe data presented here raise awareness of potential FN NBS results for LE GA1 patients. The LE phenotype is not protective against adverse clinical outcomes, and the possibility of FN NBS results calls for high vigilance amongst clinicians, even in the setting of a normal NBS result.

Value of amniotic fluid homocysteine assay in prenatal diagnosis of combined methylmalonic acidemia and homocystinuria, cobalamin C type

BackgroundCombined methylmalonic acidemia and homocystinuria, cobalamin C type (cblC defect) is the most common inborn error of cobalamin metabolism, and different approaches have been applied to its prenatal diagnosis. To evaluate the reliability of biochemical method for the prenatal diagnosis of cblC defect, we conducted a retrospective study of our 10-year experience at a single center.Methods248 pregnancies whose probands were diagnosed as cblC defect were referred to our center for prenatal diagnosis from January 2010 to December 2019. Prenatal data of Hcy levels determined by enzymatic cycling assay, acylcarnitine analysis using liquid chromatography tandem mass spectrometry, organic acid analysis using gas chromatography mass spectrometry, and genetic analysis by direct sequencing of 248 at-risk fetuses were retrospectively reviewed.ResultsFor 2.0 and 16.0 μmol/L levels of Hcy AF samples, the relative errors were − 2.5% and 2.8%, respectively. The respective measurement uncertainties were 13.07% and 14.20%. For the 248 at-risk fetuses, 63 fetuses were affected and 185 fetuses were unaffected. Hcy level of 13.20 (6.62–43.30) μmol/L in 63 affected fetuses was significantly higher than that in 185 unaffected fetuses of 2.70 (0.00–5.80) μmol/L, and there was no overlap between the affected and unaffected groups. The diagnostic sensitivity and specificity of Hcy were 100% and 92.05%, respectively. The positive and negative predictive values of the combination of Hcy, propionylcarnitine (C3), ratio of C3 to acetylcarnitine (C2; C3/C2), methylmalonic acid (MMA), and methylcitric acid (MCA) were both 100%. Sixteen fetuses displayed inconclusive genetic results of MMACHC variants, in which seven fetuses were determined to be affected with elevated levels of Hcy, C3, C3/C2 and MMA, and their levels were 18.50 (6.70–43.30) μmol/L, 8.53(5.02–11.91) μmol/L, 0.77 (0.52–0.97), 8.96 (6.55–40.32) mmol/mol Cr, respectively. The remaining nine fetuses were considered unaffected based on a normal amniotic fluid metabolite profile.ConclusionsHcy appears to be another characteristic biomarker for the prenatal diagnosis of cblC defect. The combination of Hcy assay with acylcarnitine and organic acid analysis is a fast, sensitive, and reliable prenatal diagnostic biochemical approach. This approach could overcome the challenge of the lack of genetic analysis for families with at-risk cblC defect fetuses.

Isovaleric acidemia due to compound heterozygous variants of IVD gene in a case

To analyze the clinical features, biochemical characteristics and molecular pathogenesis of a girl with isovaleric acidemia. Clinical features, blood spot amino acid profiles and urinary organic acid profiles of the patient were analyzed. Targeted capture, next generation sequencing and Sanger sequencing were carried out to detect potential variant of the IVD gene. The patient presented with poor weight gain, poor feeding, lethargy, and a "sweaty feet" odor 10 days after birth. Biochemical test suggested hyperammonemia. Blood spot amino acid profiles displayed a dramatic increase in isovalerylcarnitine (C5: 3. 044, reference range 0.04 - 0.4 μmol/L). Organic acid analysis of her urine sample revealed a high level of isovaleric glycine (669. 53, reference range 0 - 0.5). The child was ultimately diagnosed with isovaleric acidemia, and was found to harbor a paternally derived heterozygous variant c.149G>A (p.R50H) and a maternally derived heterozygous variant c.1123G>A (p.G375S) of the IVD gene. Her elder brother was a heterozygous carrier of c.1123G>A (p.G375S) variant. The c.149G>A (p.R50H) was a known pathogenic variant, while the c.1123G>A (p.G375S) variant was previously unreported. The pathogenesis of the patient was delineated from the perspective of genetics, which has provided a basis for clinical diagnosis, treatment as well as genetic counseling.

Enhancement in Plant Growth and Zinc Biofortification of Chickpea (Cicer arietinum L.) by Bacillus altitudinis

Zinc-solubilizing bacteria (ZSB) are potential and ideal substitutes for Zn supplement. Currently, limited reports are available on the bacteria displaying capability to transform insoluble forms of zinc (Zn) to an available form. Therefore, attempts were made to identify and characterize Zn-solubilizing strains from chickpea rhizosphere for growth promotion and Zn biofortification. Sixty-one bacteria isolated from rhizosphere of chickpea from different parts of Indo-Gangetic plains (IGP) of India were screened for Zn-solubilizing capabilities by cultivating on BTG medium supplemented with ZnO, Zn3(PO4)2, and ZnCO3. Two potential ZSB (BT3 and CT8) were chosen based on their zinc solubilization index and zinc solubilization efficiency and later identified by 16S rRNA gene sequence analysis. In vitro assays were performed to determine their plant growth-promoting attributes. PCR assay was performed to confirm the presence of zinc uptake regulator (zur) gene in potential strains. Pot experiments were carried out to evaluate the effect of two potential ZSB (BT3 and CT8) on growth promotion and Zn biofortification in chickpea. Atomic absorption spectroscopy was employed to determine the Zn content in the plants. Out of twenty-four ZSB, only two strains (BT3 and CT8) were able to solubilize all the three Zn sources. Isolate, BT3 released maximum concentration of Zn from ZnCO3 (150.2 ± 1.1 mg l−1), while CT8 released maximum from ZnO (130.2 ± 1.2 mg l−1) and Zn3(PO4)2 (116.5 ± 1.2 mg l−1), respectively. The pH of medium was strongly acidic which ranged from 5.2 to 3.9, and the organic acid analysis revealed hexanoic acid, pentanoic acid, and mandelic acids as prime organic acids produced by isolates. The presence of zur genes indicated the involvement of zur-mediated molecular mechanisms in these strains. These isolates also showed plant growth-promoting traits such as production of indole acetic acid (IAA), ammonia, siderophores, and phosphate solubilization. The 16S rRNA sequence analysis revealed that both the isolates had close similarity with Bacillus altitudinis. Inoculation of isolates BT3 and CT8 improved the growth parameters of chickpea and increased the plant’s Zn uptake by 3.9–6.0%. B. altitudinis strains (BT3 and CT8) show excellent capabilities to solubilize insoluble Zn compounds like oxides, phosphates, and carbonates of Zn, making them valuable source for improving Zn uptake and growth of chickpea.

Antioxidant and organic acid evaluation of Geaster saccatum mushroom by chemical and electrochemical assay at carbon nanotube paste electrode

The study was aimed to investigate the antioxidant and organic acid composition of Geaster saccatum mushroom by chemical and electrochemical assay. The antioxidant activity was done by using DPPH and ABTS reagents while the cyclovoltametry (CV) and differential pulse voltamery (DPV) were used to determine its electrochemical behavior at single walled carbon nano tube paste electrode in 0.02 M acetate buffer (pH 5.0), to explore the adsorptive character of its antioxidant compounds at working electrode as a function of pH, scan rate and potential. The organic acid analysis of this mushroom was carried out by High performance liquid chromatography. In DPPH, the scavenging rate was 56.4 ± 0.78%, at 20 µg/ml, reached to 93.4 ± 0.93% at 100 µg/ml, with 61.01 μg/ml as its IC50. Similarly, in ABTS, at same concentrations, scavenging rates were 38.14 ± 0.54% and 95.16 ± 0.43%, with 58.03 μg/ml as its IC50, respectively. HPLC quantified eight organic acids with gallic acid at highest concentration (193.34 mg/kg dry wt) followed by ketoglutaric acid (181.16 mg/kg dry wt.) and oxallic acid (69.54 mg/kg dry wt.). The CV produced a clear irreversible anodic peak at Epa = 0.69 ± 0.01 V, while, ascorbic acid produced a single anodic peak at Epa = 0.29 ± 0.01 V and gallic acid produced two anodic peaks at Epa = 0.41 ± 0.01 V and second at Epa = 0.69 ± 0.01 V. However, only one anodic peak of mushroom was found in DPV at 0.56 ± 0.01 V, which increased proportionally at each increasing concentration of mushroom extract. The results declared this mushroom as one of the best source of organic acids which characterized it as a better antioxidant food as well as medicine.

Inherited metabolic disorders among Turkish children with intellectual disability: A single-center experience

Aim: We aimed to determine the frequency of inherited metabolic disorders (IMD) among Turkish children with intellectual disability using laboratory tests for IMD.Materials and Methods: Children older than 5 years of age with intellectual disability admitted to the Pediatric Neurology Outpatient Clinic for any neurological symptom between July 1, 2018, and December 31, 2018 were analyzed. Children with intellectual disability of unknown etiology at admission were included in the study. Complete blood count, serum biochemical analysis, thyroid function tests, serum ammonia, lactate, homocysteine, biotinidase activity, serum amino acids, acylcarnitine profile, and urine organic acid analysis results were evaluated.Results: A total of 163 patients (108 boys) were included in the study. The female to male ratio was 1: 2. The ages of the patients were between 5 and 17 years and the mean age was 9.4 ± 3.6 years. Four patients were diagnosed with IMD. Two of them had mitochondrial disease, 1 of them had isovaleric acidemia, and 1 of them had 3-Methylcrotonyl-CoA carboxylase deficiency. The accompanying symptoms of intellectual disability were psychiatric symptoms, dysarthria, ataxia, tremor and seizure in children diagnosed with IMD. We found that the percentage of IMD was 2.5% among Turkish children with intellectual disability.Conclusion: In our study, all IMDs identified in children with intellectual disability were treatable. That result emphasizes the significance of evaluating children for inherited metabolic disorders among children with intellectual disability.

Sensitive and Reproducible Mass Spectrometry-Compatible RP-UHPLC Analysis of Tricarboxylic Acid Cycle and Related Metabolites in Biological Fluids: Application to Human Urine

We describe a method for the analysis of organic acids, including those of the tricarboxylic acid cycle (TCA cycle), by mixed-mode reversed-phase chromatography, on a CSH Phenyl-Hexyl column, to accomplish mixed-mode anion-exchange separations, which results in increased retention for acids without the need for ion-pairing reagents or other mobile phase additives. The developed method exhibited good retention time reproducibility for over 650 injections or more than 5 days of continuous operation. Additionally, it showed excellent resolution of the critical pairs, isocitric acid and citric acid as well as malic acid and fumaric acid, among others. The use of hybrid organic-inorganic surface technology incorporated into the hardware of the column not only improved the mass spectral quality and subsequent database match scoring but also increased the recovery of the analytes, showing particular benefit for low concentrations of phosphorylated species. The method was applied to the comparative metabolomic analysis of urine samples from healthy controls and breast cancer positive subjects. Unsupervised PCA analysis showed distinct grouping of samples from healthy and diseased subjects, with excellent reproducibility of respective injection clusters. Finally, abundance plots of selected analytes from the tricarboxylic acid cycle revealed differences between healthy control and disease groups.

Quantitative analysis of organic acids in pomelo fruit using FT-NIR spectroscopy coupled with network kernel PLS regression

Pomelo is a high-class characteristic food in China. The fruit quality of pomelo is much important for people’s health and sense of taste. After being picked, the fruit quality deteriorates because of the spontaneous physiological reactions due to the metabolism and changes of organic acids (OA). The detection of OA concentration is essential for evaluating the pomelo fruit quality. In this paper, the OA concentrations in pomelo fruit samples were quantitatively determined by using Fourier transform near-infrared (FT-NIR) spectroscopy coupled with the kernel partial least square (kernel PLS) method. The network architectural kernel was proposed for extracting the spectral feature data in the machine learning manner. The number of network hidden nodes were tuned in combination of the optimally selection of the PLS latent variables. An error-feedback iteration mechanism was used for self-adaptively training the network linking weights. Model calibration results show that the most suitable network structure was identified with 130 hidden nodes and 20 output nodes. The optimal kernel improved the PLS model with 8 feature latent variables. The model training results were acquired with the root mean square error as 0.834 g/kg, and the correlation coefficient as 0.936. In comparison with common kernel functions, the network kernel performed better in the model training part and in the testing part. The model prediction results indicated that the proposed automatically training of the network architectural kernel is feasible to improve the PLS modeling effects for the FT-NIR rapid analysis of OA in pomelo fruit.

New suitable deprotonating matrices for the analysis of carboxylic acids and some acidic compounds by matrix-assisted laser desorption/ionization mass spectrometry in negative ion mode.

Direct non-derivatization analysis of organic acids and acidic compounds by matrix-assisted laser desorption/ionization (MALDI) mass spectrometry (MS) in positive ion mode is not always possible due to the low ionization efficiency of analytes. Some new efficient deprotonating matrices were suggested that allowed the production of negative ions from acidic compounds during MALDI-MS experiments. Various tested carboxyl-containing compounds as well as compounds with acidic properties were mixed with the suggested deprotonating matrices [4-dimethylaminobenzaldehyde (DMABA), N,N-dimethylamino-p-phenylenediamine or 3-aminoquinoline] and applied on a standard MALDI target followed by recording MALDI mass spectra in negative ion mode. All the tested acidic compounds mixed with the suggested deprotonating matrices produced abundant [M - H]- ions under MALDI conditions. DMABA produced the strongest signals reflecting greater sensitivity of analysis. The suggested deprotonating matrices are commercially available compounds and are good alternatives to well-known matrices of this kind and, in particular, the often used 9-aminoacridine. DMABA is the best of the tested potential matrices and is suitable for the detection of low molecular weight carboxyl-containing compounds, substituted phenols, and mixtures of naphthenic acids by (-)MALDI-MS.

Altered metabolism of chloroplastic NAD kinase-overexpressing Arabidopsis in response to magnesium sulfate supplementation

ABSTRACT Nicotinamide adenine dinucleotide (NAD)/NAD phosphate (NADPH) is essential for numerous redox reactions and serve as co-factors in multiple metabolic processes in all organisms. NAD kinase (NADK) is an enzyme involved in the synthesis of NADP+ from NAD+ and ATP. Arabidopsis NADK2 (AtNADK2) is a chloroplast-localizing enzyme that provides recipients of reducing power in photosynthetic electron transfer. When Arabidopsis plants were grown on MS medium supplemented with 5 mM MgSO4, an AtNADK2-overexpressing line exhibited higher glutathione and total sulfur accumulation than control plants. Metabolomic analysis of major amino acids and organic acids using capillary electrophoresis–mass spectrometry demonstrated that overexpression of AtNADK2 affected a range of metabolic processes in response to MgSO4 supplementation.

Analysis of Organic Acids and Antioxidant Activity in Vitro of Citrus medica Jiaosu during Fermentation Process

Changes in the composition of organic acids and in vitro antioxidant activity of Citrus medica Jiaosu were investigated. During fermentation,scavenging rate on superoxide radical,hydroxyl radical and ABTS radical showed an increasing trend generally. DPPH radical scavenging rate and reducing power initially increased,then decreased,and gradually increased afterwards. The main organic acids of Citrus medica Jiaosu were L-malic acid and citric acid during 5~15 d fermentation,while they were lactic acid,acetic acid and L-tartaric acid during 40~90 d. Meanwhile,shikimic acid,ascorbic acid,succinic acid and oxalic acid were found in Citrus medica Jiaosu. The amount of total organic acid increased firstly,then decreased and then increased again until reaching its peak of(9.600±0.18) mg/mL at 90 d,growing by 105.47% compared with the amount in Citrus medica Jiaosu at 5 d. The different time points and species of organic acids were classified into two and three groups respectively by cluster analysis when rescaled distance cluster was 15. Both superoxide radical and hydroxyl radical scavenging rate showed extremly(P<0.01)significant positive correlation with the contents of lactic acid(the spatial correlation coefficient was 0.885 and 0.680 respectively). The research results revealed the changes in the composition of organic acids and in vitro antioxidant activity of Citrus medica Jiaosu,and provided a theoretical reference for precision preparation of Citrus medica Jiaosu.

Histamine H2-Receptor Antagonists Improve Non-Steroidal Anti-Inflammatory Drug-Induced Intestinal Dysbiosis

Dysbiosis, an imbalance of intestinal flora, can cause serious conditions such as obesity, cancer, and psychoneurological disorders. One cause of dysbiosis is inflammation. Ulcerative enteritis is a side effect of non-steroidal anti-inflammatory drugs (NSAIDs). To counteract this side effect, we proposed the concurrent use of histamine H2 receptor antagonists (H2RA), and we examined the effect on the intestinal flora. We generated a murine model of NSAID-induced intestinal mucosal injury, and we administered oral H2RA to the mice. We collected stool samples, compared the composition of intestinal flora using terminal restriction fragment length polymorphism, and performed organic acid analysis using high-performance liquid chromatography. The intestinal flora analysis revealed that NSAID [indomethacin (IDM)] administration increased Erysipelotrichaceae and decreased Clostridiales but that both had improved with the concurrent administration of H2RA. Fecal levels of acetic, propionic, and n-butyric acids increased with IDM administration and decreased with the concurrent administration of H2RA. Although in NSAID-induced gastroenteritis the proportion of intestinal microorganisms changes, leading to the deterioration of the intestinal environment, concurrent administration of H2RA can normalize the intestinal flora.

Screening and clinical analysis of isovaleric acidemia newborn in Zhejiang province

To investigate the incidence,clinical,biochemical and genetic characteristics of isovaleric acidemia (IVA) in Zhejiang province. Between January 2009 and December 2019, a total of 3 510 004 newborns were screened for IVA using tandem mass spectrometry. Patients of IVA were confirmed by urine organic acid and IVD gene detection. IVA patients were given diet and life management, supplemented with L-carnitine and glycine treatment, long-term followed up to observe and evaluate the growth and intellectual development. A total of 15 patients with IVA were diagnosed, with an incidence of 1/234 000. Three patients had acute neonatal IVA, and the rest were asymptomatic. The isovalerylcarnitine (C5) levels were increased in all patients. Twelve children underwent urinary organic acid analysis, of which 11 cases had elevated isovalerylglycine levels, 4 cases with 3-hydroxyisovalerate increased simultaneously. Eleven IVA patients underwent genetic testing, 9 patients were compound heterozygous variants in IVD gene, one with homozygous variants in IVD gene, and one harbored one IVD variant. Nineteen IVD variants (14 missense mutations, 3 intron mutations, 1 code shift mutation, and 1 synonymous mutation) were identified, 11 of which were not reported. Among the 15 IVA patients, one patient died and two patients were followed up locally. The remaining patients had no obvious clinical symptoms during the follow-up (2-79 months). Three patients presented with growth and development delay, the remaining had normal physical and mental development. The clinical manifestations of IVA are non-specific, and the gene spectrum is scattered. Newborn patients screened by tandem mass spectrometry can receive early diagnosis and treatment, so as to correct metabolic defects and pathophysiological changes.

Rapid analysis and identification of flavonoid and organic acid metabolites in Hawthorn using an on-line flow injection assisted electrochemical microreactor combined with quadrupole time-of-flight tandem mass spectrometry

In the present study, an electrochemical microreactor (EM) technique was developed for the analysis of natural flavonoids and organic acids in edible fruit. The EM connected to quadrupole time-of-flight tandem mass spectrometry (MS) was applied to study the oxidation fate of hyperoside, isoquercitrin, chlorogenic acid and epicatechin in hawthorn. Simulation of the oxidative phase I and phase II metabolism was accomplished in a reactor cell equipped with a glassy carbon working electrode. The structures of the electrochemistry system-generated products were clarified on the basis of MS full scanning. The results obtained by EM-MS were compared with previously reported conventional metabolism in rats and humans. In addition, a method for incubating the target compounds in vitro with liver microsomes was established, and then, the target analytes and their metabolites were analyzed using a sensitive and fast ultrahigh performance liquid chromatography-MS procedure. The results of the in vitro incubation with liver microsomes are in good agreement with the electrochemical simulated oxidation. Consequently, the combination of EM and MS is a suitable method to simulate the oxidative metabolism of active compounds in food.

Ultra-performance liquid chromatography (UPLC) for the determination of organic acids – The intermediates of branched-chain amino acid biosynthesis in Escherichia coli strains

Simple and rapid reverse-phase ultra-performance liquid chromatography with UV-detection (RP-UPLC-UV) was developed and validated for the simultaneous analysis of 2-isopropylmalate (2-IPM), 3-isopropylmalate (3-IPM), ketoisovalerate (KIV), ketoisocaproate (KIC), and α-keto-β-methylvalerate (KMV) in the culture fluid (CF) of branched-chain amino acid (BCAA) Escherichia coli strain producers. The RP-UPLC-UV method was developed using a Waters Acquity BEH C18 column (100 mm × 2.1 mm, 1.7 μm) with a mobile phase consisting of phosphate monobasic salt adjusted to a pH of 3.5 and methanol solution. The flow rate of the mobile phase was 0.5 mL min−1 with gradient elution. The RP-UPLC-UV method was compared with reverse-phase high-performance liquid chromatography with UV-detection (RP-HPLC-UV), ion-exchange high-performance liquid chromatography with UV-detection (IE-HPLC-UV), and ion-exchange high-performance liquid chromatography with electrochemical detection (IE-HPLC-ECD) and a postcolumn buffer for organic acid analysis. The proposed RP-UPLC-UV method was highly sensitive (LOD for KIV was 2.5 × 10−3 mg mL−1, for 2-IPM – 4.5 × 10−3 mg mL−1, for 3-IPM – 4.4 × 10−3 mg mL−1, for KIC – 3.4 × 10−3 mg mL−1, and for KMV – 2.8 × 10−3 mg mL−1) and successfully applied for the analysis of organic acids in the CF of E. coli strain producers.