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Jin L, Han X, He F, Zhang C. Prevalence of methylmalonic acidemia among newborns and the clinical-suspected population: a meta-analyse. J Matern Fetal Neonatal Med 2021; 35:8952-8967. [PMID: 34847798 DOI: 10.1080/14767058.2021.2008351] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 10/19/2022]
Abstract
IMPORTANCE Knowing the scale of rare inborn errors is important for screening and resource allocation. Evidence on the prevalence of methylmalonic acidemia (MMA) among newborns and the clinical-suspected population from large-scale screening programs needs to be systematically synthesized. OBJECTIVE To estimate the worldwide prevalence of MMA for newborns and the clinical-suspected population and explore the differences in different regions, periods, and diagnostic technologies. DATA SOURCES MEDLINE, Embase, CRD, Cochrane Library, Scopus, CINAHL, and PROSPERO. Study Selection: All studies reporting the epidemiology characteristics of MMA were selected. DATA EXTRACTION AND SYNTHESIS Characteristics of study, subjects, and epidemiology were extracted, random-effect models were used for meta-analyses. MAIN OUTCOME AND MEASURE Pooled prevalence of MMA. RESULTS This study included 111 studies. The pooled prevalence of MMA worldwide was 1.14 per 100,000 newborns (1516/190,229,777 newborns, 95% CI: 0.99-1.29) and 652.11 per 100,000 clinical-suspected patients (1360/4,805,665 clinical-suspected individuals, CI: 544.14-760.07). Asia and Africa got a higher pooled prevalence of MMA. The prevalence of MMA in newborns increased through the years, while that in the clinical-suspected population decreased. Collecting blood ≥ 72 h after birth had a higher pooled prevalence of MMA than collecting during 24 h-72 h after birth. The combining-use of MS/MS and GC/MS had a higher pooled prevalence than the single-use of MS/MS or GC/MS. Prevalence of cbl C, mut, cbl B, cbl A, isolated MMA, combined MMA and homocystinuria, vitamin B12-responsive MMA was synthesized. CONCLUSIONS AND RELEVANCE Prevalence of MMA among newborns was extremely low, but considerably high in the clinical-suspected population, indicating the need for more efficient newborn screening strategies and closer monitoring of the high-risk population for the early signs of MMA. Asia and Africa should attach importance to the high prevalence of MMA. Further diagnostic tests were recommended for the combining-use vs single-use of MS/MS and GC/MS and for collecting blood after 72 h vs during 24-72 h after birth.
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Affiliation(s)
- Lizi Jin
- National Center for Clinical Laboratories, Institute of Geriatric Medicine, Chinese Academy of Medical Sciences, Beijing Hospital/National Center of Gerontology, Beijing, P. R. China.,Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing, P. R. China
| | - Xueyan Han
- Department of Medical Statistics, Peking University First Hospital, Beijing, P. R. China
| | - Falin He
- National Center for Clinical Laboratories, Institute of Geriatric Medicine, Chinese Academy of Medical Sciences, Beijing Hospital/National Center of Gerontology, Beijing, P. R. China.,Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing, P. R. China
| | - Chuanbao Zhang
- National Center for Clinical Laboratories, Institute of Geriatric Medicine, Chinese Academy of Medical Sciences, Beijing Hospital/National Center of Gerontology, Beijing, P. R. China.,Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing, P. R. China
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Yu J, Liu X, Guo J, Zhao J, Li Y, Sun C, Liu L. GC-MS analysis of organic acids in rat urine: A protocol of direct ultrasound-assisted derivatization. Biomed Chromatogr 2020; 34:e4765. [PMID: 31778577 DOI: 10.1002/bmc.4765] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/27/2019] [Revised: 11/15/2019] [Accepted: 11/24/2019] [Indexed: 03/31/2025]
Abstract
The aim of the present study was to develop a novel ultrasound-assisted derivatization method for analysis of urine that can be used for preliminary screening and monitoring of metabolic disorders. Here we describe an ultrasound-assisted derivatization method followed by GC-MS analysis to quantify 26 organic acids in urine. The optimum levels of the variables affecting the yield of derivatization were investigated, including urease doses, derivatization reagents and derivatization conditions (duration time, reaction temperature and sonic power). The method exhibited the best results with 80 μl urease. The optimal reaction conditions were 100 μl BSTFA, 80% ultrasound power, 70°C and 40 min. This method showed satisfactory linearity, good reproducibility and an acceptable limit of detection and accuracy. Therefore, it could potentially be used to as a standard method to enable comparisons between laboratories. Finally, we applied our method to urine samples from pregnant rats administered 2 or 10 mg/kg folic acid supplementation.
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Affiliation(s)
- Jiaying Yu
- Department of Nutrition and Food Hygiene, Public Health College, Harbin Medical University, Harbin, P. R. China
| | - Xiaowei Liu
- Department of Nutrition and Food Hygiene, Public Health College, Harbin Medical University, Harbin, P. R. China
| | - Jing Guo
- Department of Nutrition and Food Hygiene, Public Health College, Harbin Medical University, Harbin, P. R. China
| | - Jinhui Zhao
- Department of Nutrition and Food Hygiene, Public Health College, Harbin Medical University, Harbin, P. R. China
| | - Ying Li
- Department of Nutrition and Food Hygiene, Public Health College, Harbin Medical University, Harbin, P. R. China
| | - Changhao Sun
- Department of Nutrition and Food Hygiene, Public Health College, Harbin Medical University, Harbin, P. R. China
| | - Liyan Liu
- Department of Nutrition and Food Hygiene, Public Health College, Harbin Medical University, Harbin, P. R. China
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Palmas F, Mussap M, Fattuoni C. Urine metabolome analysis by gas chromatography-mass spectrometry (GC-MS): Standardization and optimization of protocols for urea removal and short-term sample storage. Clin Chim Acta 2018; 485:236-242. [PMID: 30008426 DOI: 10.1016/j.cca.2018.07.006] [Citation(s) in RCA: 15] [Impact Index Per Article: 2.1] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/12/2018] [Revised: 06/25/2018] [Accepted: 07/05/2018] [Indexed: 12/13/2022]
Abstract
BACKGROUND Before derivatization, urine analyzed by gas chromatography-mass spectrometry (GC-MS) requires the complete removal of urea to avoid interferences. We aimed at establishing the most effective sample pretreatment for urea removing; moreover, we explored the impact of two short-term sample storage conditions on urine metabolome. METHODS 92 aliquots were obtained from a single sample collected from a healthy adult; they were divided into 6 groups. Group 1 consisted of untreated aliquots while groups 2-6 differed from each other for the addition of various defined urease solution volumes combined with either 30 min or 1-hour sonication time. Urine sample storage was tested by comparing 20 fresh aliquots analyzed after collection with 20 aliquots frozen at -80 °C for 72 h. RESULTS the most effective protocol consisted of the combination between 200 μL urease solution with 1-h sonication time; urease solution volumes >200 μL increase the risk to underestimate metabolite peaks because of sample dilution. Short-term storage of samples at -80 °C pointed out significant changes in the urine metabolic profile compared with that of fresh samples. CONCLUSIONS our study confirms the importance of urea removal for a reliable recognition and quantitation of metabolites; urine short-term storage at -80 °C should be carefully reconsidered.
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Affiliation(s)
- Francesco Palmas
- Department of Chemical and Geological Sciences, University of Cagliari, I-09042, Italy
| | - Michele Mussap
- Department of Surgical Sciences, University of Cagliari, Italy.
| | - Claudia Fattuoni
- Department of Chemical and Geological Sciences, University of Cagliari, I-09042, Italy
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Kubinec R, Kotora P, Ferenczy V, Blaško J, Podolec P, Hengerics Szabó A, Behúlová D, Bierhanzl V, Čabala R, Stuchlík S, Filipiak W, Thắng NM. Simultaneous analysis of carbohydrates, polyols and amines in urine samples using chemical ionization gas chromatography with tandem mass spectrometry. J Sep Sci 2017; 41:449-458. [DOI: 10.1002/jssc.201700715] [Citation(s) in RCA: 8] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/28/2017] [Revised: 09/28/2017] [Accepted: 10/20/2017] [Indexed: 11/09/2022]
Affiliation(s)
- Róbert Kubinec
- Institute of Chemistry, Faculty of Natural Sciences; Comenius University in Bratislava; Bratislava Slovakia
| | - Peter Kotora
- Institute of Process Engineering, Faculty of Mechanical Engineering; Slovak University of Technology in Bratislava; Bratislava Slovakia
| | - Viktória Ferenczy
- Institute of Chemistry, Faculty of Natural Sciences; Comenius University in Bratislava; Bratislava Slovakia
- Department of Laboratory Medicine; University Children´s Hospital; Bratislava Slovakia
| | - Jaroslav Blaško
- Institute of Chemistry, Faculty of Natural Sciences; Comenius University in Bratislava; Bratislava Slovakia
| | | | - Alexandra Hengerics Szabó
- Institute of Chemistry, Faculty of Natural Sciences; Comenius University in Bratislava; Bratislava Slovakia
| | - Darina Behúlová
- Department of Laboratory Medicine; University Children´s Hospital; Bratislava Slovakia
| | - Václav Bierhanzl
- Department of Analytical Chemistry, Faculty of Natural Sciences; Charles University in Prague; Prague Czech Republic
| | - Radomír Čabala
- Department of Analytical Chemistry, Faculty of Natural Sciences; Charles University in Prague; Prague Czech Republic
- Institute of Forensic Medicine and Toxicology; General University Hospital in Prague; Prague Czech Republic
| | - Stanislav Stuchlík
- Department of Molecular Biology, Faculty of Natural Sciences; Comenius University in Bratislava; Bratislava Slovakia
| | - Wojciech Filipiak
- Department of Pharmacodynamics and Molecular Pharmacology, Faculty of Pharmacy; Collegium Medicum in Bydgoszcz at the Nicolaus Copernicus University in Torun; Bydgoszcz Poland
| | - Ngô Mạnh Thắng
- Department of Physical and Analytical Chemistry, Faculty of Chemical Engineering, HCMC; University of Technology; HCM City Vietnam
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Purevsuren J, Bolormaa B, Narantsetseg C, Batsolongo R, Enkhchimeg O, Bayalag M, Hasegawa Y, Shintaku H, SeijiYamaguchi. The first Mongolian cases of phenylketonuria in selective screening of inborn errors of metabolism. Mol Genet Metab Rep 2016; 9:71-74. [PMID: 27830119 PMCID: PMC5094263 DOI: 10.1016/j.ymgmr.2016.10.008] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/30/2016] [Revised: 10/21/2016] [Accepted: 10/21/2016] [Indexed: 12/01/2022] Open
Abstract
Background Inborn errors of metabolism (IEM) are rare genetic disorders in which a single gene defect causes a clinically significant block in a metabolic pathway. Clinical problems arise due to either accumulation of substrates that are toxic or interfere with normal function, or deficiency of the products that are used to synthesize essential compounds. There is no report of screening results or confirmed cases of IEM in Mongolia. Only pilot study of newborn screening for congenital hypothyroidism was implemented in Mongolia, where the incidence of congenital hypothyroidism is calculated to be 1:3057 in Mongolia. Methods Two hundred twenty-three Mongolian patients, who had developmental delay, psychomotor retardation with unknown cause, seizures, hypotonia or liver dysfunction, were studied. Urinary organic acid analysis was performed in all cases using gas chromatography mass spectrometric (GC/MS) analysis. Blood amino acids and acylcarnitines were checked in the patients who had abnormal GC/MS analyses. Mutation analysis was done in the patients, who were suspected having specific inborn errors of metabolism by mass spectrometric analysis. Results One hundred thirty-nine children had normal urinary organic acid analyses. Thirty one had metabolites of valproic acid, 17 had non- or hypoketotic dicarboxylic aciduria, 14 had tyrosiluria, 12 had ketosis, 4 had elevation of lactate and pyruvate, 3 had increased excretion of urinary glycerol or methylmalonic acids, respectively, and 2 had elevation of phenylacetate and phenyllactate. We checked blood amino acids and acylcarnitines in 38 patients, which revealed phenylketonuria (PKU) in 2 patients, and one with suspected citrin deficiency. Mutation analysis in PAH was done in 2 patients with PKU, and previously reported p.R243Q, p.Y356X, p.V399V, p.A403V mutations were detected. Discussion In conclusion, these were the first genetically confirmed cases of PKU in Mongolia, and the study suggested that the newborn screening program for PKU was significant because it enabled early treatment dietary restriction, specialized formulas and other medical management for prevention of neurological handicaps in these children.
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Affiliation(s)
- Jamiyan Purevsuren
- Chidlren's Hospital, National Center for Maternal and Child Health, Bayangol district, Ulaanbaatar 16060, Mongolia
| | - Baasandai Bolormaa
- Chidlren's Hospital, National Center for Maternal and Child Health, Bayangol district, Ulaanbaatar 16060, Mongolia
| | - Chogdon Narantsetseg
- Chidlren's Hospital, National Center for Maternal and Child Health, Bayangol district, Ulaanbaatar 16060, Mongolia
| | - Renchindorj Batsolongo
- Chidlren's Hospital, National Center for Maternal and Child Health, Bayangol district, Ulaanbaatar 16060, Mongolia
| | - Ochirbat Enkhchimeg
- Chidlren's Hospital, National Center for Maternal and Child Health, Bayangol district, Ulaanbaatar 16060, Mongolia
| | - Munkhuu Bayalag
- Chidlren's Hospital, National Center for Maternal and Child Health, Bayangol district, Ulaanbaatar 16060, Mongolia
| | - Yuki Hasegawa
- Department of Pediatrics, Shimane University School of Medicine, 89-1 Enya, Izumo, Shimane 693-8501, Japan
| | - Haruo Shintaku
- Department of Pediatrics, Osaka City University Graduate School of Medicine, 1-4-3 Asahi-machi, Abeno-ku, Osaka 545-8585, Japan
| | - SeijiYamaguchi
- Department of Pediatrics, Shimane University School of Medicine, 89-1 Enya, Izumo, Shimane 693-8501, Japan
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6
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Kuhara T. [Present status of expanded newborn screening project for inborn errors of metabolism by tandem mass spectrometry]. Nihon Eiseigaku Zasshi 2014; 69:60-74. [PMID: 24476596 DOI: 10.1265/jjh.69.60] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/09/2022]
Abstract
In Japan, screening for six diseases including four inborn errors of metabolism has been performed since 1977 for all neonates to prevent severe mental handicaps or death. A rapid screening procedure for analysis of several amino acids and acylcarnitines in blood spots by tandem mass spectrometry was developed by Millington DS et al. in the early 1990s. Although it is called expanded (or extended) newborn screening, the procedure is insufficiently sensitive to or specific for several diseases. Screening for all diseases that can be screened using this procedure is suggested to be cost-ineffective. Many European countries target only two diseases: medium-chain acyl-CoA dehydrogenase deficiency and phenylketonuria; their prevalence in Caucasian populations is very high, but some countries target more than twenty diseases and others an intermediate number. A pilot study targeting 22 diseases suggests that the combined incidence is one per 9,000 (0.01%) in Japan. This primary screening requires secondary screening to confirm the disease using urine, and either organic acids with solvent extraction or metabolome without fractionation are analyzed by gas chromatography-mass spectrometry. There is no need for primary or secondary screening tests to be performed at the same laboratory because the skills required are quite different. Understanding of the methodological problems of tandem mass screening and amelioration of variation and false positivity rate of this screening method among laboratories are critical to the success of the screening system in Japan. GC/MS-based urine metabolomics is expected to become one of the primary screening methodologies for neonates/infants who are already ill.
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7
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Kuehnbaum NL, Britz-McKibbin P. New Advances in Separation Science for Metabolomics: Resolving Chemical Diversity in a Post-Genomic Era. Chem Rev 2013; 113:2437-68. [DOI: 10.1021/cr300484s] [Citation(s) in RCA: 201] [Impact Index Per Article: 16.8] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/07/2023]
Affiliation(s)
- Naomi L. Kuehnbaum
- Department of Chemistry
and Chemical Biology, McMaster University, Hamilton, Canada
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8
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Yoon HR. Determination of plasma dibasic amino acids following trimethylsilyl–trifluoroacyl derivatization using gas chromatography–mass spectrometry. Arch Pharm Res 2013; 36:366-73. [DOI: 10.1007/s12272-013-0038-1] [Citation(s) in RCA: 7] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/09/2013] [Accepted: 02/07/2013] [Indexed: 10/27/2022]
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Wettersten HI, Weiss RH. Applications of metabolomics for kidney disease research: from biomarkers to therapeutic targets. Organogenesis 2013; 9:11-8. [PMID: 23538740 DOI: 10.4161/org.24322] [Citation(s) in RCA: 22] [Impact Index Per Article: 1.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/19/2022] Open
Abstract
Metabolomics is one of the relative newcomers of the omics techniques and is likely the one most closely related to actual real-time disease pathophysiology. Hence, it has the power to yield not only specific biomarkers but also insight into the pathophysiology of disease. Despite this power, metabolomics as applied to kidney disease is still in its early adolescence and has not yet reached the mature stage of clinical application, i.e., specific biomarker and therapeutic target discovery. On the other hand, the insight gained from hints into what makes these diseases tick, as is evident from the metabolomics pathways which have been found to be altered in kidney cancer, are now beginning to bear fruit in leading to potential therapeutic targets. It is quite likely that, with greater numbers of clinical materials and with more investigators jumping into the field, metabolomics may well change the course of kidney disease research.
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Affiliation(s)
- Hiromi I Wettersten
- Division of Nephrology, Department of Internal Medicine, University of California, Davis, CA, USA
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10
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Abstract
Metabolomics--the nontargeted measurement of all metabolites produced by the body--is beginning to show promise in both biomarker discovery and, in the form of pharmacometabolomics, in aiding the choice of therapy for patients with specific diseases. In its two basic forms (pattern recognition and metabolite identification), this developing field has been used to discover potential biomarkers in several renal diseases, including acute kidney injury (attributable to a variety of causes), autosomal dominant polycystic kidney disease and kidney cancer. NMR and gas chromatography or liquid chromatography, together with mass spectrometry, are generally used to separate and identify metabolites. Many hurdles need to be overcome in this field, such as achieving consistency in collection of biofluid samples, controlling for batch effects during the analysis and applying the most appropriate statistical analysis to extract the maximum amount of biological information from the data obtained. Pathway and network analyses have both been applied to metabolomic analysis, which vastly extends its clinical relevance and effects. In addition, pharmacometabolomics analyses, in which a metabolomic signature can be associated with a given therapeutic effect, are beginning to appear in the literature, which will lead to personalized therapies. Thus, metabolomics holds promise for early diagnosis, increased choice of therapy and the identification of new metabolic pathways that could potentially be targeted in kidney disease.
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Abstract
Metabolomics--the nontargeted measurement of all metabolites produced by the body--is beginning to show promise in both biomarker discovery and, in the form of pharmacometabolomics, in aiding the choice of therapy for patients with specific diseases. In its two basic forms (pattern recognition and metabolite identification), this developing field has been used to discover potential biomarkers in several renal diseases, including acute kidney injury (attributable to a variety of causes), autosomal dominant polycystic kidney disease and kidney cancer. NMR and gas chromatography or liquid chromatography, together with mass spectrometry, are generally used to separate and identify metabolites. Many hurdles need to be overcome in this field, such as achieving consistency in collection of biofluid samples, controlling for batch effects during the analysis and applying the most appropriate statistical analysis to extract the maximum amount of biological information from the data obtained. Pathway and network analyses have both been applied to metabolomic analysis, which vastly extends its clinical relevance and effects. In addition, pharmacometabolomics analyses, in which a metabolomic signature can be associated with a given therapeutic effect, are beginning to appear in the literature, which will lead to personalized therapies. Thus, metabolomics holds promise for early diagnosis, increased choice of therapy and the identification of new metabolic pathways that could potentially be targeted in kidney disease.
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12
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Shigematsu Y, Hata I, Tajima G. Useful second-tier tests in expanded newborn screening of isovaleric acidemia and methylmalonic aciduria. J Inherit Metab Dis 2010; 33:S283-8. [PMID: 20440648 DOI: 10.1007/s10545-010-9111-9] [Citation(s) in RCA: 26] [Impact Index Per Article: 1.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 02/08/2010] [Revised: 03/19/2010] [Accepted: 04/12/2010] [Indexed: 10/19/2022]
Abstract
Common use of pivalate-generating antibiotics in newborns in Japan and low cutoff value of C5-acylcarnitine (C5) to detect mild forms of isovaleric acidemia (IVA) led to 1,065 positive results from IVA screening among 146,000 newborns tested by tandem mass spectrometry over the last 3 years. Using our method to determine isovalerylglycine (IVG) levels in dried blood spots (DBS) as a second-tier test with IVG cutoff value of 0.5 nmol/ml in DBS, one patient with severe IVA was identified, and no recall of the second DBS was needed. Retrospective analysis revealed that most patients with moderate to severe forms of IVA have decreased free-carnitine levels shortly after birth and higher levels of IVG than those of C5, which suggests that this method is useful in evaluating the severity of IVA. Another second-tier test, to measure methylmalonic acid (MMA) levels in DBS by gas chromatography/mass spectrometry (GC/MS), has been developed to overcome difficulties in screening methylmalonic aciduria (MMAU) and propionic acidemia. Methanol extract from DBS was dried and derivatized using N-methyl-N-(tert-butyldimethylsilyl)-trifluoroacetamide. GC/MS was performed using splitless injection, electron-impact ionization, and selected ion monitoring for data recording. MMAU patients had much higher DBS concentrations of MMA (24.2-321.9 nmol/ml) than control newborns (0.34 ± 0.11 nmol/ml). MMA measurement in DBS was thought to provide useful information about the severity of MMAU, as MMAU patients with high levels of MMA had decreased levels of free carnitine and mildly increased levels of propionylcarnitine.
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Affiliation(s)
- Yosuke Shigematsu
- Department of Health Science, Faculty of Medical Sciences, University of Fukui, Eiheiji-cho, Fukui, Japan.
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Shoemaker JD. One-step metabolomics: carbohydrates, organic and amino acids quantified in a single procedure. J Vis Exp 2010:2014. [PMID: 20613709 PMCID: PMC3156063 DOI: 10.3791/2014] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 10/31/2022] Open
Abstract
Every infant born in the US is now screened for up to 42 rare genetic disorders called "inborn errors of metabolism". The screening method is based on tandem mass spectrometry and quantifies acylcarnitines as a screen for organic acidemias and also measures amino acids. All states also perform enzymatic testing for carbohydrate disorders such as galactosemia. Because the results can be non-specific, follow-up testing of positive results is required using a more definitive method. The present report describes the "urease" method of sample preparation for inborn error screening. Crystalline urease enzyme is used to remove urea from body fluids which permits most other water-soluble metabolites to be dehydrated and derivatized for gas chromatography in a single procedure. Dehydration by evaporation in a nitrogen stream is facilitated by adding acetonitrile and methylene chloride. Then, trimethylsilylation takes place in the presence of a unique catalyst, triethylammonium trifluoroacetate. Automated injection and chromatography is followed by macro-driven custom quantification of 192 metabolites and semi-quantification of every major component using specialized libraries of mass spectra of TMS derivatized biological compounds. The analysis may be performed on the widely-used Chemstation platform using the macros and libraries available from the author. In our laboratory, over 16,000 patient samples have been analyzed using the method with a diagnostic yield of about 17%--that is, 17% of the samples results reveal findings that should be acted upon by the ordering physician. Included in these are over 180 confirmed inborn errors, of which about 38% could not have been diagnosed using previous methods.
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Affiliation(s)
- James D Shoemaker
- Edward A. Doisy Department of Biochemistry and Molecular Biology, Saint Louis University School of Medicine, USA.
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Kind T, Tolstikov V, Fiehn O, Weiss RH. A comprehensive urinary metabolomic approach for identifying kidney cancerr. Anal Biochem 2007; 363:185-95. [PMID: 17316536 DOI: 10.1016/j.ab.2007.01.028] [Citation(s) in RCA: 336] [Impact Index Per Article: 18.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/28/2006] [Revised: 01/10/2007] [Accepted: 01/20/2007] [Indexed: 12/14/2022]
Abstract
The diagnosis of cancer by examination of the urine has the potential to improve patient outcomes by means of earlier detection. Due to the fact that the urine contains metabolic signatures of many biochemical pathways, this biofluid is ideally suited for metabolomic analysis, especially involving diseases of the kidney and urinary system. In this pilot study, we test three independent analytical techniques for suitability for detection of renal cell carcinoma (RCC) in urine of affected patients. Hydrophilic interaction chromatography (HILIC-LC-MS), reversed-phase ultra performance liquid chromatography (RP-UPLC-MS), and gas chromatography time-of-flight mass spectrometry (GC-TOF-MS) all were used as complementary separation techniques. The combination of these techniques is best suited to cover a very large part of the urine metabolome by enabling the detection of both lipophilic and hydrophilic metabolites present therein. In this study, it is demonstrated that sample pretreatment with urease dramatically alters the metabolome composition apart from removal of urea. Two new freely available peak alignment methods, MZmine and XCMS, are used for peak detection and retention time alignment. The results are analyzed by a feature selection algorithm with subsequent univariate analysis of variance (ANOVA) and a multivariate partial least squares (PLS) approach. From more than 2000 mass spectral features detected in the urine, we identify several significant components that lead to discrimination between RCC patients and controls despite the relatively small sample size. A feature selection process condensed the significant features to less than 30 components in each of the data sets. In future work, these potential biomarkers will be further validated with a larger patient cohort. Such investigation will likely lead to clinically applicable assays for earlier diagnosis of RCC, as well as other malignancies, and thereby improved patient prognosis.
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Affiliation(s)
- Tobias Kind
- Genome Center, University of California, Davis, CA 95616, USA
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Yang Y, Sun F, Song J, Hasegawa Y, Yamaguchi S, Zhang Y, Jiang Y, Qin J, Wu X. Clinical and biochemical studies on Chinese patients with methylmalonic aciduria. J Child Neurol 2006; 21:1020-4. [PMID: 17156691 DOI: 10.1177/7010.2006.00231] [Citation(s) in RCA: 13] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 11/16/2022]
Abstract
Methylmalonic aciduria is a common organic aciduria disease. Recently, gas chromatography-mass spectrometry has been used to diagnose methylmalonic aciduria in China. Often, however, the diagnosis of methylmalonic aciduria is delayed because of a lack of technical expertise and the limited experience of general clinicians in China. In this study, the natural history, clinical features, and outcome of 77 Chinese patients with methylmalonic aciduria were investigated. Of the 77 patients, 31 (40.3%) had isolated methylmalonic aciduria and 46 (59.7%) had methylmalonic aciduria combined with homocystinemia. Thus, we observed a higher rate of the combined disease than studies conducted in other countries, suggesting that it might be more common in China. Total plasma homocysteine measurement might enable differential diagnoses of methylmalonic aciduria to be distinguished. The clinical spectrum of these 77 patients with methylmalonic aciduria ranged from neonatal death and severe symptoms to benign asymptomatic organic aciduria. Neonatal and infantile onset, which was a characteristic of the majority of cases, was associated with a greater severity relative to later-onset cases. Among the 17 cases who had onset after 3 years of age, only 1 patient had isolated methylmalonic aciduria and 16 had combined methylmalonic aciduria and homocystinemia. Nine of the patients with combined methylmalonic aciduria and homocystinemia completely recovered and exhibited normal intelligence, whereas seven improved, with a mild handicap.
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Affiliation(s)
- Yanling Yang
- Department of Pediatrics, Peking University First Hospital, Xicheng District, Beijing, China.
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Inoue Y, Shinka T, Ohse M, Ikawa H, Kuhara T. Application of optical isomer analysis by diastereomer derivatization GC/MS to determine the condition of patients with short bowel syndrome. J Chromatogr B Analyt Technol Biomed Life Sci 2006; 838:37-42. [PMID: 16516567 DOI: 10.1016/j.jchromb.2006.02.019] [Citation(s) in RCA: 19] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/14/2005] [Revised: 02/08/2006] [Accepted: 02/09/2006] [Indexed: 10/24/2022]
Abstract
To establish a method for separating the optical isomers of lactic acid, we modified the derivatization steps in our procedure for urinary mass-screening for inborn errors of metabolism. For chiral recognition, we chose O-trifluoroacetyl-(-)-menthylation derivatization instead of our previous method, trimethylsilyl derivatization, and the samples were then analyzed under GC/MS by capillary gas chromatography on a DB-5MS column. This method can be used to follow-up the condition of a patient with short bowel syndrome and to prevent onset and/or seizure. d-Lactic acid was also isolated from the urine of healthy controls as one of the main peaks in the chromatogram.
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Affiliation(s)
- Yoshito Inoue
- Division of Human Genetics, Medical Research Institute, Kanazawa Medical University, 1-1 Daigaku, Uchinada, Ishikawa 9200293, Japan.
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17
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Shen X, Deng C, Wang B, Dong L. Quantification of trimethylsilyl derivatives of amino acid disease biomarkers in neonatal blood samples by gas chromatography-mass spectrometry. Anal Bioanal Chem 2005; 384:931-8. [PMID: 16385411 DOI: 10.1007/s00216-005-0241-0] [Citation(s) in RCA: 21] [Impact Index Per Article: 1.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/01/2005] [Revised: 11/12/2005] [Accepted: 11/14/2005] [Indexed: 10/25/2022]
Abstract
A novel analytical procedure was developed for the rapid determination of disease biomarkers of maple syrup urine disease (MSUD), L-valine, L-leucine, L-isoleucine, and L-phenylalanine in dried blood spots. Amino acids extracted from neonatal dried blood spots were rapidly derivatized with bis-(trimethylsilyl)trifluoroacetamide (BSTFA) and then analyzed by gas chromatography-mass spectrometry (GC-MS). Derivatization conditions and the method validation were studied: optimal derivatization conditions were acetonitrile as reaction solvent, a temperature of 100 degrees C, and a reaction time of 30 min. The proposed method provided a detection limit lower than 2.0 microM, recovery between 92% and 106%, and relative standard deviation less than 8.0%. The method was further tested in screening for neonatal MSUD by determination of L-valine, L-leucine, L-isoleucine, and L-phenylalanine in blood samples. The experimental results show that GC-MS following BSTFA derivatization is a rapid, simple, and sensitive method for the determination of amino acid disease biomarkers in blood samples, and is a potential tool for fast screening of MSUD.
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Affiliation(s)
- Xizhong Shen
- Zhongshan Hospital, Fudan University, Shanghai, 200032, PR China
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18
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Deng C, Wang B, Liu L. Fast Diagnosis of Neonatal Phenylketonuria by Gas Chromatography-Mass Spectrometry Following Microwave-Assisted Silylation. Chromatographia 2005. [DOI: 10.1365/s10337-005-0686-5] [Citation(s) in RCA: 8] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/05/2022]
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19
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Inoue Y, Shinka T, Ohse M, Kuhara T. Differential chemical diagnosis of primary hyperoxaluria type II. Highly sensitive analysis of optical isomers of glyceric acid by GC/MS as diastereoisomeric derivatives. J Chromatogr B Analyt Technol Biomed Life Sci 2005; 823:2-6. [PMID: 16055048 DOI: 10.1016/j.jchromb.2005.03.036] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/27/2004] [Revised: 03/07/2005] [Accepted: 03/11/2005] [Indexed: 11/29/2022]
Abstract
We established a separation method for the optical isomers of glyceric acid in urine by modifying the derivatization steps of the procedure used for the screening and diagnosis. The trimethylsilyl derivatization step in the mass screening procedure was replaced by O-acetyl-(+)-2-butylation, and the samples were analyzed under equivalent GC/MS conditions by capillary gas chromatography on a DB-5MS column. This method can be applied to cases that show a high urinary concentration of glyceric acid to obtain a differential diagnosis of primary hyperoxaluria type II and d-glyceric aciduria easily. l-Glyceric acid was also isolated from the urine of healthy controls as one of the main peaks.
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Affiliation(s)
- Yoshito Inoue
- Division of Human Genetics, Medical Research Institute, Kanazawa Medical University, 1-1 Daigaku, Uchinada, Ishikawa 920-0293, Japan.
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20
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Deng C, Yin X, Zhang L, Zhang X. Development of microwave-assisted derivatization followed by gas chromatography/mass spectrometry for fast determination of amino acids in neonatal blood samples. RAPID COMMUNICATIONS IN MASS SPECTROMETRY : RCM 2005; 19:2227-34. [PMID: 16015674 DOI: 10.1002/rcm.2052] [Citation(s) in RCA: 21] [Impact Index Per Article: 1.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 05/03/2023]
Abstract
Analysis of amino acids in blood samples is an important tool for the diagnosis of neonatal amino acid metabolism disorders. In the work, a novel, rapid and sensitive method was developed for the determination of amino acids in neonatal blood samples, which was based on microwave-assisted silylation followed by gas chromatography/mass spectrometry (GC/MS). The amino acids were derivatized with N,O-bis(trimethylsilyl)trifluoroacetamide (BSTFA) under microwave irradiation. The controlled reaction was carried out employing BSTFA under conventional heating at 120 degrees C for 30 min. Experimental results show that microwave irradiation can accelerate the derivatization reaction of amino acids with BSFTA, and much shorten analysis time. The method validations (linear range, detection limit, precision and recovery) were studied. Finally, the method was tested by determination of amino acids in neonatal blood by the measurement of their trimethylsilyl derivatives by GC/MS in electron impact (EI) mode. Two biomarkers of L-phenylalanine and L-tyrosine in phenylketonuria (PKU)-positive blood and control blood were quantitatively analyzed by the proposed method. The results demonstrated that microwave-assisted silylation followed by GC/MS is a rapid, simple and sensitive method for amino acid analysis and is also a potential tool for fast screening of neonatal aminoacidurias.
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Affiliation(s)
- Chunhui Deng
- Department of Chemistry, Fudan University, Shanghai 200433, China
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21
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Deng C, Ji J, Zhang L, Zhang X. Diagnosis of congenital adrenal hyperplasia by rapid determination of 17alpha-hydroxyprogesterone in dried blood spots by gas chromatography/mass spectrometry following microwave-assisted silylation. RAPID COMMUNICATIONS IN MASS SPECTROMETRY : RCM 2005; 19:2974-8. [PMID: 16178052 DOI: 10.1002/rcm.2163] [Citation(s) in RCA: 17] [Impact Index Per Article: 0.9] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 05/04/2023]
Abstract
17alpha-Hydroxyprogesterone (17OHP) is considered to be the biomarker of congential adrenal hyperplasia (CAH). Screening for CAH in newborns by measuring levels of the biomarker of 17OHP has become routine. In the work, a rapid, simple and sensitive technique was developed for the diagnosis of neonatal CAH by the quantitative analysis of 17OHP in neonatal blood spots. The technique was based on microwave-assisted silylation (MAS) followed by gas chromatography/mass spectrometry (GC/MS). In the method, fast derivatization of 17OHP with N,O-bis(trimethylsilyl)trifluoroacetamide was performed by using microwave irradiation, and the trimethylsilyl derivative thus formed was analyzed by GC/MS. The results of the experiment indicate that MAS followed by GC/MS analysis is a rapid, simple and sensitive method for the determination of 17OHP in blood samples. The proposed technique has been shown to have potential as a powerful tool for the rapid diagnosis of neonatal CAH.
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Affiliation(s)
- Chunhui Deng
- Department of Chemistry, Fudan University, Shanghai 200433, P.R.China
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22
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Strelkov S, von Elstermann M, Schomburg D. Comprehensive analysis of metabolites in Corynebacterium glutamicum by gas chromatography/mass spectrometry. Biol Chem 2004; 385:853-61. [PMID: 15493881 DOI: 10.1515/bc.2004.111] [Citation(s) in RCA: 104] [Impact Index Per Article: 5.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/15/2022]
Abstract
AbstractAn analytical method based on gas chromatography/mass spectrometry was developed for metabolome investigation ofCorynebacterium glutamicum. For the first time a fast method for metabolic screening that can be automated is described for this organism. More than 1000 compounds could be detected per experiment, ca. 330 of those showed a peak area significantly above background. Out of these 164 compounds were identified so far, representing derivatives of 121 different metabolites, which were quantified in one sample. In spite of the different chemical nature of metabolites and high matrix content, a measurement reproducibility in the range of 6% error was achieved. The application of this method for the analysis of the adaptation ofC. glutamicumto different growth conditions is demonstrated.
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Affiliation(s)
- Sergey Strelkov
- Institute of Biochemistry, University of Cologne, Zülpicher Str. 47, D-50674 Cologne, Germany
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23
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Inoue Y, Kuhara T. Rapid and sensitive screening for and chemical diagnosis of Canavan disease by gas chromatography–mass spectrometry. J Chromatogr B Analyt Technol Biomed Life Sci 2004; 806:33-9. [PMID: 15149608 DOI: 10.1016/j.jchromb.2004.03.018] [Citation(s) in RCA: 13] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/22/2022]
Abstract
The use of a rapid and sensitive assay for N-acetylaspartate (NAA) in urine or eluates from dried urine on filter paper to make a chemical diagnosis of Canavan disease (CD) is described. It involves a simplified urease pretreatment for sample preparation and gas chromatography-mass spectrometry (EI, scanning mode) with or without stable isotope dilution. Significant improvements in the recovery of NAA and the GC-MS data-handling device made the assay without stable isotope dilution sensitive and quantitative enough to diagnose CD: Its coefficient of variation (CV) was below 12%. The CV obtained with stable isotope dilution was below 9%. One patient with CD had an abnormal NAA level that was more than 6 S.D. above the mean of the age-matched controls. This diagnostic procedure is accurate for screening and for the chemical diagnosis of CD, with a good cost:benefit ratio. The urinary NAA levels of the healthy controls decreased significantly with age. This change should be considered in making a chemical diagnosis of this disease.
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Affiliation(s)
- Y Inoue
- Division of Human Genetics, Medical Research Institute, Kanazawa Medical University, 1-1 Daigaku, Uchinada, Ishikawa 9200293, Japan
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24
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Guillo C, Perrett D, Hanna-Brown M. Validation and Further Optimisation of a Cyclodextrin-Modified Micellar Electrokinetic Capillary Chromatography Method for Urine Profiling. Chromatographia 2004. [DOI: 10.1365/s10337-004-0218-8] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/05/2022]
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25
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Deng C, Li N, Zhang X. Rapid determination of amino acids in neonatal blood samples based on derivatization with isobutyl chloroformate followed by solid-phase microextraction and gas chromatography/mass spectrometry. RAPID COMMUNICATIONS IN MASS SPECTROMETRY : RCM 2004; 18:2558-2564. [PMID: 15468143 DOI: 10.1002/rcm.1660] [Citation(s) in RCA: 40] [Impact Index Per Article: 1.9] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 05/24/2023]
Abstract
The purpose of this study was to develop a simple, rapid and sensitive analytical method for determination of amino acids in neonatal blood samples. The developed method involves the employment of derivatization and a solid-phase microextraction (SPME) technique together with gas chromatography/mass spectrometry (GC/MS). Amino acids in blood samples were derivatized by a mixture of isobutyl chloroformate, methanol and pyridine, and the N(O,S)-alkoxycarbonyl alkyl esters thus formed were headspace extracted by a SPME fiber. Finally, the extracted analytes on the fiber were desorbed and detected by GC/MS in electron impact (EI) mode. L-Valine, L-leucine, L-isoleucine, L-phenylanaline and L-tyrosine in blood samples were quantitatively analyzed by measurement of the corresponding N(O,S)-alkoxycarbonyl alkyl esters using an external standard method. SPME conditions were optimized, and the method was validated. The method was applied to diagnosis of neonatal phenylkenuria (PKU) and maple syrup urine disease (MSUD) by the analyses of five amino acids in blood samples. The results showed that the proposed method is a potentially powerful tool for simultaneous screening for neonatal PKU and MSUD.
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Affiliation(s)
- Chunhui Deng
- Department of Chemistry, Fudan University, Shanghai 200433, P.R. China
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26
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Deng C, Deng Y. Diagnosis of maple syrup urine disease by determination of L-valine, L-isoleucine, L-leucine and L-phenylalanine in neonatal blood spots by gas chromatography-mass spectrometry. J Chromatogr B Analyt Technol Biomed Life Sci 2003; 792:261-8. [PMID: 12860033 DOI: 10.1016/s1570-0232(03)00270-8] [Citation(s) in RCA: 35] [Impact Index Per Article: 1.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/25/2022]
Abstract
A novel method was developed for the diagnosis of maple syrup urine disease (MSUD) by the determination of L-valine, L-leucine, L-isoleucine and L-phenylalanine in dried blood spots of newborns by gas chromatography-mass spectrometry (GC-MS). The four amino acids were extracted from blood samples by methanol and derivatized by n-butanol and trifluroacetic anhydride under optimum reaction conditions. The corresponding single derivatives of the four amino acids were obtained under the optimum conditions. Their contents in blood samples were analyzed quantitatively by measurement of their derivatives by GC-MS in selected ion monitoring mode. MSUD can be diagnosed on the basis of the ratio of the total content of L-leucine and L-isoleucine to that of L-phenylalanine. The present method only took a short time to perform and required minimal sample preparation, which provided low detection limits and a relative standard deviation of less than 5.0%. The derivatization reactions of the four amino acids, L-valine, L-isoleucine, L-leucine and L-phenylalanine, with n-butanol and trifluroacetic anhydride were investigated and the optimum reaction conditions, including reaction time and temperature, were obtained and used for the determination of the amino acids in plasma samples.
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Affiliation(s)
- Chunhui Deng
- Department of Chemistry, Fudan University, Shanghai 200433, China.
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27
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García A, Barbas C. Capillary electrophoresis for the determination of organic acidurias in body fluids: a review. Clin Chem Lab Med 2003; 41:755-61. [PMID: 12880138 DOI: 10.1515/cclm.2003.115] [Citation(s) in RCA: 11] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/15/2022]
Abstract
A systematic review of the literature on capillary electrophoresis applied to short chain organic acid analysis in body fluids has been conducted with special interest on those acids related to inborn errors of metabolism. The technique is briefly described, as well as the choice of the main analytical parameters: sample pre-treatment, polarity, capillary type, background electrolyte, and detection. The applications described in the literature are listed and the main features of the technique are discussed.
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Affiliation(s)
- Antonia García
- Facultad de CC Experimentales y de la Salud, Universidad San Pablo-CEU, Boadilla del Monte, Madrid, Spain
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28
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Galli V, García A, Saavedra L, Barbas C. Capillary electrophoresis for short-chain organic acids and inorganic anions in different samples. Electrophoresis 2003; 24:1951-1981. [PMID: 12858368 DOI: 10.1002/elps.200305473] [Citation(s) in RCA: 57] [Impact Index Per Article: 2.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/06/2022]
Abstract
This review article is a comprehensive survey of capillary electrophoresis methods developed for the measurement of short-chain organic acids and inorganic anions in a wide variety of matrices, such as food and beverages, environmental, industry, and other applications, as well as clinical applications in body fluids such as urine, plasma or cerebrospinal fluid. Details of sample pretreatment and of electrophoretic conditions have been collected in tables, arranged by the type of matrix. Strategies employed for method development for the analysis of these compounds by capillary electrophoresis in real samples are discussed.
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Affiliation(s)
- Verónica Galli
- Facultad de CC. Experimentales y dela Salud, Urbanización Montepríncipe, E-28668 Boadilla del Monte (Madrid), Spain
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29
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Kuhara T. Diagnosis and monitoring of inborn errors of metabolism using urease-pretreatment of urine, isotope dilution, and gas chromatography-mass spectrometry. J Chromatogr B Analyt Technol Biomed Life Sci 2002; 781:497-517. [PMID: 12450676 DOI: 10.1016/s1570-0232(02)00670-0] [Citation(s) in RCA: 56] [Impact Index Per Article: 2.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 10/27/2022]
Abstract
To diagnose inborn errors of metabolism, it would be desirable to simultaneously analyze and quantify organic acids, purines, pyrimidines, amino acids, sugars, polyols, and other compounds using a single-step fractionation; unfortunately, no such method currently exists. The present article will be concerned primarily with a practical yet comprehensive diagnostic procedure of inborn errors of metabolism (IEM). This procedure involves the use of urine or eluates from urine on filter paper, stable isotope dilution, and gas chromatography-mass spectrometry (GC-MS). This procedure not only offers reliable and quantitative evidence for diagnosing, understanding and monitoring the diseases, but also provides evidence for the diagnosis of new kinds of IEM. In this review, the differential diagnosis for hyperammonemia are described; deficiencies of ornithine carbamoyl transferase, argininosuccinate synthase (citrullinemia), argininosuccinate lyase and arginase, lysinuric protein intolerance, hyperammonemia-hyperornithinemia-homocitrullinemia syndrome, and citrullinemia type II. The diagnosis of IEM of purine and pyrimidine such as deficiencies of hypoxanthine-guanine phosphoribosyl transferase, adenine phosphoribosyl transferase, dihydropyrimidine dehydrogenase, dihydropyrimidinase and beta-ureidopropionase are described. During the pilot study for newborn screening, we found neonates with diseases at a rate of 1 per 1,400 including propionic acidemia, methylmalonic acidemia, orotic aciduria, beta-ureidopropionase deficiency, lactic aciduria and neuroblastoma. A rapid and reliable prenatal diagnosis for propionic acidemia is also described.
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Affiliation(s)
- Tomiko Kuhara
- Division of Human Genetics, Medical Research Institute, Kanazawa Medical University 1-1 Daigaku, Uchinada-machi, Kahoku-gun, Ishikawa 920-0293, Japan.
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30
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Deng C, Deng Y, Wang B, Yang X. Gas chromatography-mass spectrometry method for determination of phenylalanine and tyrosine in neonatal blood spots. J Chromatogr B Analyt Technol Biomed Life Sci 2002; 780:407-13. [PMID: 12401368 DOI: 10.1016/s1570-0232(02)00632-3] [Citation(s) in RCA: 86] [Impact Index Per Article: 3.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/21/2022]
Abstract
In this paper we developed a simple, rapid and sensitive method for the quantitative analysis of phenylalanine (Phe) and tyrosine (Tyr) in dried blood spots of newborns by gas chromatography-mass spectrometry (GC-MS). Phe and Tyr in blood samples were reacted with N-methyl-N-(tert.-butyldimethylsilyl)trifluoroacetamide at 120 degrees C for 30 min and their corresponding single derivatives were obtained. Phe and Tyr were determined by measurement of their derivatives by GC-MS in the selected ion monitoring mode. Contents of Phe and Tyr in blood spots were calculated by external standard method. The ratio of Phe to Tyr was used for neonatal screening for phenylketonuria. The present method only took a few minutes to perform and required minimal sample preparation. In addition it provided low detection limits of 1.2 micromol l(-1) for Phe and 1.6 micromol l(-1)for Tyr.
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Affiliation(s)
- Chunhui Deng
- Department of Chemistry, Fudan University, Shanghai 200433, China.
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31
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Ohse M, Matsuo M, Ishida A, Kuhara T. Screening and diagnosis of beta-ureidopropionase deficiency by gas chromatographic/mass spectrometric analysis of urine. JOURNAL OF MASS SPECTROMETRY : JMS 2002; 37:954-962. [PMID: 12271438 DOI: 10.1002/jms.354] [Citation(s) in RCA: 20] [Impact Index Per Article: 0.9] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 05/23/2023]
Abstract
Dihydropyrimidine dehydrogenase (DHPDase), dihydropyrimidinase (DHPase) and beta-ureidopropionase (betaUPase) are the enzymes that catalyze the first, second, and third steps of the degradation of pyrimidines, respectively. beta-Ureidopropionate (betaUP) and beta-ureidoisobutyrate (betaUIB) are increased in the urine of patients with betaUPase deficiency. The original case in which betaUPase deficiency was discovered by NMR spectroscopy was an 11-month-old patient who presented with hypotonia and dystonic movement. We detected a second but asymptomatic case during a pilot study of neonatal screening with filter-paper urine, urease pretreatment and gas chromatography/mass spectrometry (GC/MS). The urease pretreatment of urine without fractionation resulted in a high recovery of these polar ureide compounds and allowed the highly sensitive GC/MS detection and diagnosis of betaUPase deficiency. betaUP and betaUIB were identified using GC/MS techniques. In the urine of the neonate with betaUPase deficiency, betaUP and betaUIB were persistently increased. Thymine, 5,6-dihydrothymine and 5,6-dihydrouracil were increased only moderately but significantly. It is known that thymine and uracil increase markedly in DHPDase deficiency, and 5,6-dihydrothymine and 5,6-dihydrouracil increase in DHPase deficiency. Therefore, betaUPase deficiency can be differentially diagnosed from the first and second enzyme deficiencies. Application of this specific and sensitive diagnostic procedure will lead to an understanding of the clinical heterogeneity of betaUPase deficiency. Furthermore, the identification of patients with defects in pyrimidine metabolism will enable doctors to avoid cancer chemotherapy with pyrimidine analogues such as 5-fluorouracil, which could be dangerous for these patients.
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Affiliation(s)
- Morimasa Ohse
- Division of Human Genetics, Medical Research Institute, Kanazawa Medical University, 1-1 Daigaku, Uchinada-machi, Kahoku-gun, Ishikawa 920-0293, Japan
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32
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Shinka T, Inoue Y, Yoshino M, Kakinuma H, Takahashi H, Kuhara T. Two cases of benign methylmalonic aciduria detected during a pilot study of neonatal urine screening. J Chromatogr B Analyt Technol Biomed Life Sci 2002; 776:65-70. [PMID: 12127326 DOI: 10.1016/s1570-0232(02)00127-7] [Citation(s) in RCA: 6] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/21/2022]
Abstract
Two cases of benign methylmalonic aciduria (MMAuria) were found among 9780 neonatal screenings using the previously described screening method consisting of urease digestion, ethanol deproteinization and gas chromatography-mass spectrometry. Combining this screening method with the stable isotope dilution technique showed very specific and sensitive measurements of methylmalonic acid in urine. The concentrations of urinary methylmalonic acid were measured at several ages. The levels of urinary methylmalonic acid in two patients varied from 0.27 to 3.04 mol/mol creatinine (control<0.01 mol/mol creatinine). Methylcitrate and homocystine were not increased in the patient's urine or blood. Blood propionylcarnitine was also at normal levels. The urinary methylmalonate excretions were decreased to the levels of about 50% of the start point after vitamin B12 treatment in one patient, but the other patient showed no change. No clinical abnormalities were observed during these periods.
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Affiliation(s)
- Toshihiro Shinka
- Division of Human Genetics, Medical Research Institute, Kanazawa Medical, University, 1-1 Daigaku, Uchinada, Ishikawa 920-0293, Japan.
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33
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Namera A, Yashiki M, Nishida M, Kojima T. Direct extract derivatization for determination of amino acids in human urine by gas chromatography and mass spectrometry. J Chromatogr B Analyt Technol Biomed Life Sci 2002; 776:49-55. [PMID: 12127324 DOI: 10.1016/s1570-0232(02)00075-2] [Citation(s) in RCA: 60] [Impact Index Per Article: 2.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 10/27/2022]
Abstract
The purpose of this study was to develop a simple and accurate analytical method to determine amino acids in urine samples. The developed method involves the employment of an extract derivatization technique together with gas chromatography-mass spectrometry (GC-MS). Urine samples (300 microl) and an internal standard (10 microl) were placed in a screw tube. Ethylchloroformate (50 microl), methanol-pyridine (500 microl, 4:1, v/v) and chloroform (1 ml) were added to the tube. The organic layer (1 microl) was injected to a GC-MS system. In this proposed method, the amino acids in urine were derivatized during an extraction, and the analytes were then injected to GC-MS without an evaporation of the organic solvent extracted. Sample preparation was only required for ca. 5 min. The 15 amino acids (alanine, aspartic acid, cysteine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, tyrosine, tryptophan, valine) quantitatively determined in this proposed method. However, threonine, serine, asparagine, glutamine, arginine were not derivatized using any tested derivatizing reagent. The calibration curves showed linearity in the range of 1.0-300 microg/ml for each amino acid in urine. The correlation coefficients of the calibration curves of the tested amino acids were from 0.966 to 0.998. The limit of detection in urine was 0.5 microg/ml except for aspartic acid. This proposed method demonstrated substantial accuracy for detection of normal levels. This proposed method was limited for the determination of 15 amino acids in urine. However, the sample preparation was simple and rapid, and this method is suitable for a routine analysis of amino acids in urine.
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Affiliation(s)
- Akira Namera
- Department of Legal Medicine, Hiroshima University Faculty of Medicine, 1-2-3 Kasumi, Minami-ku, Hiroshima, Japan.
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34
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Guernion N, Ratcliffe NM, Spencer-Phillips PT, Howe RA. Identifying bacteria in human urine: current practice and the potential for rapid, near-patient diagnosis by sensing volatile organic compounds. Clin Chem Lab Med 2001; 39:893-906. [PMID: 11758602 DOI: 10.1515/cclm.2001.146] [Citation(s) in RCA: 34] [Impact Index Per Article: 1.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/15/2022]
Abstract
Urinary tract infection (UTI) represents a significant burden for the National Health Service. Extensive research has been directed towards rapid detection of UTI in the last thirty years. A wide range of microbiological and chemical techniques are now available to identify and quantify bacteria in urine. However, there is a clear and present need for near, rapid, sensitive, reliable analytical methods, preferably with low-running costs, that could allow early detection of UTI and other diseases in urine. Here we review the "state of the art" of current practice for the detection of bacteria in urine and describe the advantages of the recent "e-nose" technology as a potential tool for rapid, near-patient diagnosis of UTI, by sensing volatile organic compounds (VOCs).
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Affiliation(s)
- N Guernion
- Faculty of Applied Sciences, University of the West of England, Bristol, UK
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COE FREDRICL, CLARK CYNDI, PARKS JOANH, ASPLIN JOHNR. SOLID PHASE ASSAY OF URINE CYSTINE SUPERSATURATION IN THE PRESENCE OF CYSTINE BINDING DRUGS. J Urol 2001. [DOI: 10.1016/s0022-5347(05)66044-2] [Citation(s) in RCA: 8] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 10/25/2022]
Affiliation(s)
- FREDRIC L. COE
- From the University of Chicago and LithoLink Corp., Chicago, Illinois
| | - CYNDI CLARK
- From the University of Chicago and LithoLink Corp., Chicago, Illinois
| | - JOAN H. PARKS
- From the University of Chicago and LithoLink Corp., Chicago, Illinois
| | - JOHN R. ASPLIN
- From the University of Chicago and LithoLink Corp., Chicago, Illinois
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Kuhara T. Diagnosis of inborn errors of metabolism using filter paper urine, urease treatment, isotope dilution and gas chromatography-mass spectrometry. JOURNAL OF CHROMATOGRAPHY. B, BIOMEDICAL SCIENCES AND APPLICATIONS 2001; 758:3-25. [PMID: 11482733 DOI: 10.1016/s0378-4347(01)00138-4] [Citation(s) in RCA: 78] [Impact Index Per Article: 3.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 11/23/2022]
Abstract
This review will be concerned primarily with a practical yet comprehensive diagnostic procedure for the diagnosis or even mass screening of a variety of metabolic disorders. This rapid, highly sensitive procedure offers possibilities for clinical chemistry laboratories to extend their diagnostic capacity to new areas of metabolic disorders. The diagnostic procedure consists of the use of urine or filter paper urine, preincubation of urine with urease, stable isotope dilution, and gas chromatography-mass spectrometry. Sample preparation from urine or filter paper urine, creatinine determination, stable isotope-labeled compounds used, and GC-MS measurement conditions are described. Not only organic acids or polar ones but also amino acids, sugars, polyols, purines, pyrimidines and other compounds are simultaneously analyzed and quantified. In this review, a pilot study for screening of 22 target diseases in newborns we are conducting in Japan is described. A neonate with presymptomatic propionic acidemia was detected among 10,000 neonates in the pilot study. The metabolic profiles of patients with ornithine carbamoyl transferase deficiency, fructose-1,6-bisphosphatase deficiency or succinic semialdehyde dehydrogenase deficiency obtained by this method are presented as examples. They were compared to those obtained by the conventional solvent extraction methods or by the tandem mass spectrometric method currently done with dried filter blood spots. The highly sensitive, specific and comprehensive features of our procedure are also demonstrated by its use in establishing the chemical diagnosis of pyrimidine degradation defects in order to prevent side effects of pyrimidine analogs such as 5-flurouracil, and the differential diagnosis of three types of homocystinuria, orotic aciduria, uraciluria and other urea cycle disorders. Evaluation of the effects of liver transplantation or nutritional conditions such as folate deficiency in patients with inborn errors of metabolism is also described.
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Affiliation(s)
- T Kuhara
- Division of Human Genetics, Medical Research Institute, Kanazawa Medical University, Ishikawa, Japan.
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Kuhara T, Ohdoi C, Ohse M. Simple gas chromatographic-mass spectrometric procedure for diagnosing pyrimidine degradation defects for prevention of severe anticancer side effects. JOURNAL OF CHROMATOGRAPHY. B, BIOMEDICAL SCIENCES AND APPLICATIONS 2001; 758:61-74. [PMID: 11482736 DOI: 10.1016/s0378-4347(01)00143-8] [Citation(s) in RCA: 22] [Impact Index Per Article: 0.9] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 10/27/2022]
Abstract
Inborn errors of pyrimidine degradation, dihydropyrimidine dehydrogenase deficiency and dihydropyrimidinase deficiency, are less rare than has generally been assumed. Many asymptomatic cases have been reported, and in patients with symptoms, the clinical abnormalities are variable and nonspecific. Withdrawal of pyrimidine analogues such as 5-fluorouracil (5FU), a commonly used anticancer drug, from the cancer chemotherapy regimens of patients with pyrimidine degradation deficiencies, however, is critical because 5FU is degraded in vivo by pyrimidine-degradative enzymes. Patients with these deficiencies suffer from severe neurotoxicity, sometimes leading to death, following administration of 5FU, and even otherwise asymptomatic homozygotes or heterozygotes may develop severe clinical symptoms upon administration of such medication. Therefore, a rapid and specific method for identifying cancer patients with these enzyme deficiencies prior to treatment with 5FU is critical. To address this problem, we established methods for highly sensitive yet specific determinations of thymine, uracil, dihydrothymine, dihydrouracil, orotate and creatinine simultaneously in 0.1-ml liquid urine or filter-paper urine. This method involves stable isotope dilution, a simplified urease treatment previously described and gas chromatography-mass spectrometry without prior fractionation. The high recovery and low C.V. values were obtained and healthy control values were also determined for these metabolites. Using artificially prepared urine specimens simulating these disorders. the chemical diagnosis can be made clearly, and no further analysis appears to be required for differential chemical diagnosis.
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Affiliation(s)
- T Kuhara
- Division of Human Genetics, Medical Research Institute, Kanazawa Medical University, Ishikawa, Japan.
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Xia ZW, Inoue Y, Ohse M, Shinka T, Kuhara T. A study on α-ketoadipic aciduria by gas chromatographic-mass spectrometry. World J Gastroenterol 2000; 6:766-769. [PMID: 11819692 PMCID: PMC4688861 DOI: 10.3748/wjg.v6.i5.766] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.0] [Reference Citation Analysis] [Key Words] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 02/06/2023] Open
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