Name: one-step metabolomics: carbohydrates, organic and amino acids quantified in a single procedure
Uploaded: 2010-06-25T17:15:00
Duration: 9 min 28 s
Description: Watch this Scientific Journal Video about One-step Metabolomics: Carbohydrates, Organic and Amino Acids Quantified in a Single Procedure at JoVE.com

能够安东尼托马斯的技术援助表示感谢。

处理尿液样本的程序<ol><li>在37℃水浴解冻的尿液样本。到一个新的容器中倒出，如果原来是大打折扣。 </li><li>一个最高分装13毫升样品，并储存在-20 ° C，在标示的锥形离心管。 </li><li>测量和记录样品的光密度将尿液滴在折射仪夫妇。 </li><li>通过0.2微米的过滤器过滤的样品。 </li><li>根据光密度测量出下面的样本量<table border="0"><tbody><tr><td> 1.000-1.009 </td><td> 1.00毫升</td></tr><tr><td> 1.010-1.019 </td><td> 0.50毫升</td></tr><tr><td> 1.020-1.050 </td><td> 0.25毫升+ 0.25毫升H 2 O </td></tr></tbody></table></li><li>指定的卷，然后转移到一个Reactivial包含以下的内部标准：500纳摩尔（nmoles）3肌酸; 10 nmoles D 3甲基丙二酸; 100以下13 个 C 3乳酸nmoles，13 C 3丙酮酸，13 C 2 15 ñ甘氨酸，D 3，D 5苯丙氨酸丝氨酸，D 11 hexanoylglycine，15 N 2 orotate，D 4癸二酸，13 C 6葡萄糖肌醇和D，D 6 5色氨酸。 </li><li> 20微升（μL）7.5单位/μL的尿素溶液（Calzyme实验室目录。116A0100）被添加到的样品，然后刷新下的CO 2通过惰性隔密封。 </li><li>样本是在37℃30分钟，更多的二氧化碳气体，在15分钟的时间间隔有保有压。 </li><li>添加脲酶溶液20μL，小瓶二氧化碳刷新，和样品保持在37 ° C 15分钟。 </li><li> 500μL30:70丙酮：甲醇添加，更换的橡胶隔膜与聚四氟乙烯涂层隔，样品在-20℃冷冻15分钟。 </li><li>固体去除离心1500转× 10分钟，然后放入干净的2.0 CC Reactivial（Supelco公司/适马）转自</li><li>添加如下三乙的三氟乙酸（茶/ TFA），（Sigma公司）： <ul><li>为1.00毫升的样品20μL </li><li> 40μL0.5毫升，或更少，样品</li></ul></li><li>顶部用乙腈和地点下氮气流在70 ° C，直到实现定容（茶/ TFA仍将）（〜15分钟）。 </li><li>重复第13步，直到沉淀形式（〜10分钟）高达4倍。 </li><li>约2分钟，放凉。顶过二氯甲烷，小心煮沸过，干（〜4:00分钟）。 </li><li>重复步骤15。 </li><li> MSTFA（N -甲基- N - trimethylsilyltrifluoroacetamide）（热科学）添加下列税率： <ul><li>为1.00毫升的样品150μL </li><li> 0.50毫升，或更少，样品200μL </li></ul></li><li>下氮气气氛和孵化的第70 ° C为1小时。 </li><li>转移到microvials，在氮气气氛下，气相色谱/质谱仪分析。 </li><li>自动注射由安捷伦5975气相色谱/质谱广场microvials：仪器温度：喷油器200 ° C，接口250℃，烤箱80 ° C为1分钟;坡道在4 ° C /分钟80-130 ° C，坡道6 °彗星/分钟130-200 ° C，坡道为12 ° C /分钟200-285 ° C，保持10分钟。列：25米，320微米，0.5微米的薄膜厚度为DB - 5编号。质谱：源230℃，四150℃，2.46次/秒的扫描50-650阿木。溶剂延迟3.5分钟。 </li></ol>代表性的成果请<a href="http://www.jove.com/files/ftp_upload/2014/ShoemakerResultHawkinsinuria.pdf">点击这里</a>看到代表结果。

<ol>
	<li>Shoemaker, J. D., Elliott, W. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Automated+screening+of+urine+samples+for+carbohydrates,+organic+and+amino+acids+after+treatment+with+urease.">Automated screening of urine samples for carbohydrates, organic and amino acids after treatment with urease.</a> J. Chromatogr. 562 (1-2), 125-138 (1991).</li><li>Kuhara, T., Shinka, T., Inoue, Y., Ohse, M., Zhen-wei, X., Yoshida, I., Inokuchi, T., Yamaguchi, S., Takayanagi, M., Matsumoto, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pilot+study+of+gas+chromatographic-mass+spectrometric+screening+of+newborn+urine+for+inborn+errors+of+metabolism+after+treatment+with+urease.">Pilot study of gas chromatographic-mass spectrometric screening of newborn urine for inborn errors of metabolism after treatment with urease.</a> J Chromatogr B Biomed Sci Appl. 731 (1), 141-147 (1999).</li><li>Fu, X., Iga, M., Kimura, M., Yamaguchi, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Simplified+screening+for+organic+acidemia+using+GC/MS+and+dried+urine+filter+paper:+a+study+on+neonatal+mass+screening.">Simplified screening for organic acidemia using GC/MS and dried urine filter paper: a study on neonatal mass screening.</a> Early Hum Dev. 58 (1), 41-55 (2000).</li><li>Kuhara, T., Ohdoi, C., Ohse, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Simple+gas+chromatographic-mass+spectrometric+procedure+for+diagnosing+pyrimidine+degradation+defects+for+prevention+of+severe+anticancer+side+effects.">Simple gas chromatographic-mass spectrometric procedure for diagnosing pyrimidine degradation defects for prevention of severe anticancer side effects.</a> J Chromatogr B Biomed Sci Appl. 758 (1), 61-74 (2001).</li><li>Kuhara, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Diagnosis+of+inborn+errors+of+metabolism+using+filter+paper+urine,+urease+treatment,+isotope+dilution+and+gas+chromatography-mass+spectrometry.">Diagnosis of inborn errors of metabolism using filter paper urine, urease treatment, isotope dilution and gas chromatography-mass spectrometry.</a> J Chromatogr B Biomed Sci Appl. 758, 3-25 (2001).</li><li>Iga, M., Kimura, M., Ohura, T., Kikawa, Y., Yamaguchi, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid,+simplified+and+sensitive+method+for+screening+fructose-1,6-diphosphatase+deficiency+by+analyzing+urinary+metabolites+in+urease/direct+preparations+and+gas+chromatography-mass+spectrometry+in+the+selected-ion+monitoring+mode.">Rapid, simplified and sensitive method for screening fructose-1,6-diphosphatase deficiency by analyzing urinary metabolites in urease/direct preparations and gas chromatography-mass spectrometry in the selected-ion monitoring mode.</a> J Chromatogr B Biomed Sci Appl. 746 (1), 75-82 (2000).</li><li>Kuhara, T., Ohse, M., Ohdoi, C., Ishida, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differential+diagnosis+of+homocystinuria+by+urease+treatment,+isotope+dilution+and+gas+chromatography-mass+spectrometry.">Differential diagnosis of homocystinuria by urease treatment, isotope dilution and gas chromatography-mass spectrometry.</a> J Chromatogr B Biomed Sci Appl. 742 (1), 59-70 (2000).</li><li>Young, V., Lo, S., Shoemaker, J., Thomas, A., Rhead, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Validation+and+Extension+of+the+Urease+Method+for+Urine+Organic+and+Amino+Acid+Analysis.">Validation and Extension of the Urease Method for Urine Organic and Amino Acid Analysis.</a> , (2008).</li><li>Smith, E. M., Hoi, J. T., Eissenberg, J. C., Shoemaker, J. D., Neckameyer, W. S., Ilvarsonn, A. M., Harshman, L. G., Schlegel, V. L., Zempleni, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Feeding+Drosophila+a+biotin-deficient+diet+for+multiple+generations+increases+stress+resistance+and+lifespan+and+alters+gene+expression+and+histone+biotinylation+patterns.">Feeding Drosophila a biotin-deficient diet for multiple generations increases stress resistance and lifespan and alters gene expression and histone biotinylation patterns.</a> J Nutr. 137 (9), 2006-2012 (2007).</li><li>Brown, S. M., Shoemaker, J. D., Lodge, J. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Importance+of+NADP+-Dependent+Isocitrate+Dehydrogenase+in+Resistance+to+Nitrosative+Stress+and+the+Inessentiality+of+Glucose-6-Phosphate+Dehydrogenase+for+Oxidative+Stress+Resistance+in+Cryptococcus+neoformans.">The Importance of NADP+-Dependent Isocitrate Dehydrogenase in Resistance to Nitrosative Stress and the Inessentiality of Glucose-6-Phosphate Dehydrogenase for Oxidative Stress Resistance in Cryptococcus neoformans.</a> Eukaryotic Cell. , (2010).</li><li>Shoemaker, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nutritional+Screening+in+Humans+by+Gas+Chromatography-Mass+Spectrometry+of+Urine+Metabolites+After+Substrate+Loading.">Nutritional Screening in Humans by Gas Chromatography-Mass Spectrometry of Urine Metabolites After Substrate Loading.</a> , (1992).</li><li>Baggot, P. J., Kalamarides, J. A., Shoemaker, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Valproate-induced+biochemical+abnormalities+in+pregnancy+corrected+by+vitamins:+A+Case+Report.">Valproate-induced biochemical abnormalities in pregnancy corrected by vitamins: A Case Report.</a> Epilepsia. 40 (44), 512-515 (1999).</li><li>Baggot, P. J., Eliseo, A. J., Kalamarides, J. A., Shoemaker, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+folate-dependent+metabolite+in+amniotic+fluid+from+pregnancies+with+normal+or+trisomy+21+chromosomes.">A folate-dependent metabolite in amniotic fluid from pregnancies with normal or trisomy 21 chromosomes.</a> Fetal Diagn Ther. 21 (1), 148-152 (2006).</li><li>Baggot, P. J., Eliseo, A. J., DeNicola, N. G., Kalamarides, J. A., Shoemaker, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Organic+acid+concentrations+in+amniotic+fluid+found+in+normal+and+Down+syndrome+pregnancies.">Organic acid concentrations in amniotic fluid found in normal and Down syndrome pregnancies.</a> Fetal Diagn Ther. 23 (3), 245-248 (2008).</li><li>Baggot, P. J., Eliseo, A. J., DeNicola, N. G., Kalamarides, J. A., Shoemaker, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pyridoxine-related+metabolite+concentrations+in+normal+and+Down+syndrome+amniotic+fluid.">Pyridoxine-related metabolite concentrations in normal and Down syndrome amniotic fluid.</a> Fetal Diagn Ther. 23 (4), 254-257 (2008).</li></ol>

代谢筛查实验室是美国圣路易斯大学，一个非营利性的公司所拥有的CLIA执照的临床实验室。博士苏梅克不直接从实验室收入的利润，但可能会间接受益于实验室生产力。没有方法，技巧，结果，正常范围内，宏，软件，图书馆或结论被认为是专有和免费提供给有兴趣的人士。

尿素酶方法（1）已被引用62次在各种修改的医学文献。松本组（2,3）简化程序，为高通量的新生儿疾病筛查和报告16000例患者的结果。 Kuhara和其他人（4-7）报道与生俱来的错误诊断的几起案件中使用的方法和后续。 Rhead（8）也证实，为临床诊断方法的实用性和后续与生俱来的错误。该方法已应用于熊尿，基因敲除小鼠，大象和整个果蝇和它们的幼虫（9）匀浆。 隐球菌定点突变前后的文化媒体也分析，没有脲（10步） 。该方法已应用到人类的营养评估医学生唐氏综合征患者和老人痴呆症患者的退伍军人，加载后口服剂量的氨基酸色氨酸，蛋氨酸和异亮氨酸（11）科目。所有八个B族维生素评估量化的三个氨基酸，其中，要求在一些点在其降解所有8维生素的分解产物。补充维生素药品和缓解的毒副作用已经报道（12）。从正常和唐氏综合症妊娠的羊水样本进行了分析和报道（13-15）。

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		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>MSD 5975</td>
<td>&nbsp;</td>
<td>Agilent</td>
<td>&nbsp;</td>
<td>GC/MS/Computer</td>
</tr>
<tr>
<td>MSD 5973</td>
<td>&nbsp;</td>
<td>Agilent</td>
<td>&nbsp;</td>
<td>GC/MS/Computer</td>
</tr>
<tr>
<td>Urease</td>
<td>&nbsp;</td>
<td>Calzyme</td>
<td>116A0100</td>
<td>Also Sigma C3</td>
</tr>
<tr>
<td>Stable Isotope Standards</td>
<td>&nbsp;</td>
<td>CDN Isotopes, Isotec, Cambridge Isotope Lab</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Solvents</td>
<td>&nbsp;</td>
<td>Fisher</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Analytes</td>
<td>&nbsp;</td>
<td>Sigma, Universidad Autonoma de Madrid, Ernesto Brunet</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>GC Columns</td>
<td>&nbsp;</td>
<td>J&amp;W Scientific</td>
<td>123-5026</td>
<td>&nbsp;</td>
<tr>
<td>TEA/TFA</td>
<td>&nbsp;</td>
<td>Fluka/Sigma</td>
<td>09747</td>
<td>&nbsp;</td>
<tr>
<td>Vials</td>
<td>&nbsp;</td>
<td>Supelco/Sigma</td>
<td>Z115088/12EA</td>
<td>&nbsp;</td>
<tr>
<td>Merlin Microseal</td>
<td>&nbsp;</td>
<td>Agilent</td>
<td>5181-8815</td>
<td>&nbsp;</td>
<tr>
<td>MSTFA</td>
<td>&nbsp;</td>
<td>Thermal Scientific</td>
<td>48913</td>
<td>&nbsp;</td>		</tbody>
		</table>
		</div>

one-step metabolomics: carbohydrates, organic and amino acids quantified in a single procedure

在美国出生的每个婴儿现在筛选长达42罕见遗传性疾病，称为“先天代谢的错误”。基于串联质谱筛查方法和量化有机酸血症的屏幕acylcarnitines还措施氨基酸。所有国家也进行了测试碳水化合物的疾病，如半乳糖的酶。非特异性，后续检测的阳性结果，因为结果可以是使用一个更明确的方法的要求。本报告介绍了“脲”与生俱来的错误检查的样品制备方法。结晶脲酶是用来清除体液中的尿素，允许其他大多数水溶性代谢产物的气相色谱在一个单一的过程中脱水和衍生。在氮气流中蒸发脱水的便利，加入乙腈和二氯甲烷。然后，三甲基硅烷在一个独特的催化剂，三乙基三氟存在。其次是宏观驱动192的代谢物和半定量使用专门的TMS衍生物的生物化合物的质谱库的每一个主要组件的自定义量化的自动注入和色谱。分析可能是执行上广泛使用的化学工作站平台，采用从作者提供的宏和库。在我们的实验室，超过16,000名患者的样本进行了分析，使用了约17％的诊断率的方法 - 也就是说，17％的样品结果显示订货医生应该采取行动后的结果。 ，其中包括超过180确认与生俱来的错误，其中约38％不能使用前面的方法诊断。

Watch this Scientific Journal Video about One-step Metabolomics: Carbohydrates, Organic and Amino Acids Quantified in a Single Procedure at JoVE.com

One-step Metabolomics: Carbohydrates, Organic and Amino Acids Quantified in a Single Procedure

脲中介代谢物的GC / MS分析的样品制备方法是由它的发明者。该方法允许单步跟进串联质谱新生儿筛查先天的错误量化碳水化合物，有机和氨基酸都在一个单一的过程。

一步代谢：碳水化合物，在一个单一的过程量化的有机和氨基酸

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 ...

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