对于血液处理快速的方法，以提高血浆肽水平的人血产量
1Charité Center for Internal Medicine and Dermatology, Division General Internal and Psychosomatic Medicine,Charité Universitätsmedizin Berlin, Campus Benjamin Franklin, 2Department of Internal Medicine, Institute of Neurogastroenterology and Motility,Martin-Luther Hospital, Academic Teaching Institution of Charité Universitätsmedizin Berlin, 3Department of Hepatology and Gastroenterology, Molecular Cancer Research Center (MKFZ), Campus Virchow-Klinikum,Charité-Universitätsmedizin Berlin
Summary
快速血液处理方法，可以在人类中使用，并产生更高的肽水平以及允许对正确分子形式的评估。因此，这种方法将在肽研究的宝贵工具。
Abstract
研究食物摄入管制领域越来越重要。这通常包括肽调节食物摄入量的测量。为正确的测定的肽的浓度的，它应该是血处理过程中是稳定的。然而，这不是对几种肽它们很快被内源性肽酶降解的情况。最近，我们开发了一种血液处理方法采用ř得出的温度，A cidification，P rotease抑制， 我 sotopic外源控制和D ilution（RAPID）对大鼠使用。这里，我们已经建立了这种技术的使用在人类和调查恢复，分子形式和食物摄取调节激素的循环浓度。的快速方法显著改善了125 I标记的生长抑素-28（+ 39％），高血糖素样肽-1（+ 35％）的回收，酰基生长素释放肽和胰高血糖素（+ 32％），胰岛素和亲吻促动素（+ 29％ ），注射nesfatin-1（+ 28％），瘦素（+ 21％）相比，标准处理（冰血EDTA，P <0.001），肽YY 3-36（+ 19％）。高效液相色谱表明内源性酰基生长素释放肽的洗脱在快速处理后的预期位置，而标准处理后的酰基生长素释放肽的62％被降解导致较早峰值可能表示desacyl生长素释放肽。快速加工后在正常体重受试者的血液酰基/ desacyl生长素释放比例为1：3相比1:23以下标准处理（p值= 0.03）。同时内生亲吻促动素水平的标准相比，处理后快速走高（+ 99％，P = 0.02）。快速血液处理方法，可以在人类中使用，产生较高的肽水平并且允许进行正确分子形式的评估。
Introduction
在肥胖1,2世界范围内日益流行的光，研究食物的摄入监管领域越来越重要。而迄今为止，只有一种肽，已知是外周产生并作用于中枢的刺激食物摄入，即生长素释放肽3，在过去的几十年中，范围广泛的肽已发现减少食物摄取， 例如 。瘦素，肽YY（PYY）和也胰高血糖素样肽-1（GLP-1）和胰岛素4，因此，在研究调查饥饿和饱满感肽水平的调节机制经常评估，并在同一时间，假设所研究的肽是稳定的，并在过程中等离子体形成高产量恢复。可是，很多时候不是这种情况下，由于内源性迅速击穿如之前如 。生长素释放肽是从酰基降解为desacyl生长素释放肽5。因此，我们最近描述了血液PROCES的快速方法唱采用ř得出的温度，A cidification老鼠，P rotease抑制， 我 sotopic外源控制和D ilution 6。这种方法改善了11 12的肽测试，并允许正确的循环分子形式的测定标准相比，血液处理（EDTA血冰）6复苏。这种方法已在几个随后的研究7-12被用于检测循环的生长素释放肽以及促肾上腺皮质激素释放因子13的。因此，该方法已被证明为在啮齿类动物的肽的研究是有用的。然而，由于啮齿类动物的研究并不总是平移到另一个物种，该方法应被用于在人血液中的用途确定为好。
本研究的目的是测试在人类中用于血液处理的快速方法相比标准血液处理中，在冰上EDTA采血管，这是广泛推荐14和频繁ūsed的临床和研究环境。我们测试的选择125参与食物摄入，包括设立的肽，以及新的候选的规则I标记的肽的回收最近建议发挥摄食调节作用（对食物摄入的影响在表1中示出）以下的处理用这两种方法。还选择激素来代表不同的长度和电荷（ 表2）的肽。此外，生长素我们调查的分子形式（S）按照标准和快速的方法。最后，我们评估内源性生长激素释放肽（酰基和desacyl生长素释放肽），以及亲吻促动素水平，肽也最近建议发挥食物摄入15,16的下面的快速或标准加工的调节作用。此外，我们还调查了受试者群体，这些肽水平具有广泛的身体质量指数（从10.2-67.6公斤/米2）研究possib的涉及到长期改变体重乐的差异。
诊断，评估和计划：
研究参与者
所有的研究参与者新住院患者（包含了内入院两天）心身医学科在查理特-Universitätsmedizin柏林，并签署知情同意书。为了避免唯一的女患者被纳入性别产生任何影响。总共有42科目参加了这项研究，并随机分为3组：正常体重（BMI 18.5-25公斤/米2，N = 12），神经性厌食症（BMI <17.5公斤/米2，N = 15）和肥胖（BMI“30公斤/米2，N = 15）。厌食症和肥胖患者根据疾病-10的国际分类诊断和住院体重增加（神经性厌食），或体重减少（肥胖），分别。所有正常体重的患者因躯体症状无相关的躯体疾病住院独占。有胃肠道症状，躯体或胃肠道手术史患者被排除在外。排除标准也包含了<18岁，目前在怀孕和未治疗精神性疾病。以增加或减少体重，分别接收饮食治疗前入院后第2天或3进行采血。人体测量参数进行了评估在同一天。
Protocol
（/十分之一百十四协议号EA1）的方案经当地伦理委员会对人类的研究。 1.血液处理根据标准程序或快速的方法，从前臂静脉和流程收集07:00至上午08时00分之间的静脉血后，隔夜快。指示受试者抽血前不能行使或烟雾。 对于标准处理，收集冷冻含有EDTA的试管血，离心10 min内的3000 XG在4℃下10分钟。收集上清，并保持在-80°C，直到进一步的处理用放射免疫法。 为快速处理?…
Representative Results
快速血液处理增加在人体血液中的125 I放射性标记的肽的标准相比，血液处理的产率。 标准血液处理（在冰上的EDTA血）后，放射性标记的肽的回收在9/9肽（ – K范围从48-68％， 图1A）为〜60％。快速处理提高了产率在所有125 I标记的肽，即在促生长素抑制素-28（+ 39％， 图1A），胰高血糖素样肽-1（+ 35％， 图1B?…
Discussion
我们之前报道，对于血液处理的快速方法，改善了11/12肽复苏相比，大鼠6个标准的血液处理。在本研究中，我们已经表明，这种方法也适合于在人体中使用。以下快速处理，测试了9 9 125 I标记的肽的回收率相比标准血液处理（在冰上EDTA采血管）的改善。所观察到的改善，从19-39％，这可能是相关的，特别是当该肽的产率是低，这可能被掩蔽时只有细微的差别预期的条件下不等。 </…
Divulgazioni
The authors have nothing to disclose.
Acknowledgements
这项工作是由德国研究基金会STE 1765 / 3-1（AS）和德国联邦教育与研究03IPT614A（CG）的支持。我们感谢莱因哈德隆梅尔和佩特拉布斯的出色的技术支持，以及卡琳·约翰逊和克里斯蒂娜Hentzschel与组织和人体测量的执行帮助
Materials
| diprotin A | Peptides International, Louisville, KY, USA | IDP-4132 | |
| E-64-d | Peptides International, Louisville, KY, USA | IED-4321-v | |
| antipain | Peptides International, Louisville, KY, USA | IAP-4062 | |
| leupeptin | Peptides International, Louisville, KY, USA | ILP-4041 | |
| chymostatin | Peptides International, Louisville, KY, USA | ICY-4063 | |
| Sep-Pak C18 cartridges | Waters Corporation, Milford, MA, USA | WAT051910 | 360 mg, 55-105 µm |
| acyl-ghrelin | Millipore, Billerica, MA, USA | 9088-HK | Radioactive |
| GLP-1 | Millipore, Billerica, MA, USA | 9035-HK | Radioactive |
| glucagon | Millipore, Billerica, MA, USA | 9030 | Radioactive |
| insulin | Millipore, Billerica, MA, USA | 9011S | Radioactive |
| leptin | Millipore, Billerica, MA, USA | 9081-HK | Radioactive |
| kisspeptin-10 | Phoenix Pharmaceuticals, Burlingame, CA, USA | T-048-56 | Radioactive |
| nesfatin-1 | Phoenix Pharmaceuticals, Burlingame, CA, USA | T-003-26 | Radioactive |
| PYY3-36 | Phoenix Pharmaceuticals, Burlingame, CA, USA | T-059-02 | Radioactive |
| somatostatin-28 | Phoenix Pharmaceuticals, Burlingame, CA, USA | T-060-16 | Radioactive |
| ZORBAX Rapid Resolution HT SB-C18 column | Agilent Technologies, Santa Clara, CA, USA | 822700-902 | 2.1 x 50 mm, 1.8 µm |
| Agilent 1200 LC | Agilent Technologies, Santa Clara, CA, USA | HPLC, several components, therefore no single catalog number | |
| Kisspeptin RIA | Phoenix Pharmaceuticals, Burlingame, CA, USA | # RK-048-56 | Radioactive |
| Total ghrelin RIA | Millipore, Billerica, MA, USA | # GHRT-89HK | Radioactive |
| Active ghrelin RIA | Millipore, Billerica, MA, USA | # GHRA-88HK | Radioactive |
| SigmaStat 3.1 | Systat Software, San Jose, CA, USA | online download |
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Citazione di questo articolo
Teuffel, P., Goebel-Stengel, M., Hofmann, T., Prinz, P., Scharner, S., Körner, J. L., Grötzinger, C., Rose, M., Klapp, B. F., Stengel, A. A RAPID Method for Blood Processing to Increase the Yield of Plasma Peptide Levels in Human Blood. J. Vis. Exp. (110), e53959, doi:10.3791/53959 (2016).