Tezenas du Montcel C, Duriez P, Lebrun N, Grouselle D, de Grimaudet B, Dardennes R, Epelbaum J, Cuenca M, Viltart O, Gorwood P, Tolle V
Université Paris Cité, UMR-S 1266 INSERM, Institut de Psychiatrie et Neuroscience de Paris (IPNP), F-75014, Paris, France.
Clinique des Maladies Mentales et de L'Encéphale, GHU Paris Psychiatrie et Neurosciences, Hôpital Sainte-Anne, F-75014, Paris, France.
Compr Psychoneuroendocrinol. 2022 May 20;11:100140. doi: 10.1016/j.cpnec.2022.100140. eCollection 2022 Aug.
The growing interest concerning the role of metabolic sensors in various eating disorders requires the implementation of a strict methodology to collect, store and process blood samples in clinical studies. In particular, measurement of isoforms of the appetite-stimulating hormone, ghrelin, has been challenging in clinical settings. Indeed the acyl ghrelin (AG) isoform is rapidly degraded into desacyl ghrelin (DAG) by blood esterases, thus optimal conditions for the conservation of AG and accurate determination of AG/DAG ratio should be used. Here, we compared different protease inhibitors (Aprotinin, PHMB, AEBSF) during blood collection, increasing delays (0-180 min) before centrifugation, plasma supplementation with various HCl concentrations, storage durations of frozen plasma (8 and 447 days) and immunoenzyme-assay procedures (one-step sequential) in healthy subjects. Optimal conditions were obtained by collecting blood with aprotinin and supplementation of plasma with 0.1 N HCl with subsequent freezing for at least 8 days and using one-step assay. Under such conditions, different patterns of secretion of ghrelin isoforms were characterized in patients with restrictive-type anorexia nervosa (AN-R) before and after nutritional recovery. We illustrate the pulsatile variations of ghrelin isoforms according to the time around a meal and hunger rates in 3 patients with AN-R. This study offers a comprehensive comparison of various conditions using selective and specific immunoassays for both ghrelin isoforms in order to optimize assay sensitivity and consistency among procedures. These assay conditions could therefore be widely used to elucidate precisely the role of ghrelin isoforms on eating behavior in physiological and pathological situations.
对代谢传感器在各种饮食失调中作用的兴趣日益浓厚，这就要求在临床研究中实施严格的方法来采集、储存和处理血样。特别是，在临床环境中，食欲刺激激素胃饥饿素异构体的测量一直具有挑战性。事实上，酰基胃饥饿素（AG）异构体可被血液酯酶迅速降解为去酰基胃饥饿素（DAG），因此应采用保存AG和准确测定AG/DAG比值的最佳条件。在此，我们比较了健康受试者在采血过程中使用不同的蛋白酶抑制剂（抑肽酶、聚六亚甲基双胍、AEBSF）、增加离心前的延迟时间（0 - 180分钟）、用不同盐酸浓度补充血浆、冷冻血浆的储存时间（8天和447天）以及免疫酶测定程序（一步序贯法）的情况。通过使用抑肽酶采血、用0.1 N盐酸补充血浆、随后冷冻至少8天并采用一步法测定，可获得最佳条件。在这种条件下，对限制型神经性厌食症（AN - R）患者营养恢复前后胃饥饿素异构体的不同分泌模式进行了表征。我们展示了3例AN - R患者中胃饥饿素异构体根据进食时间和饥饿率的脉动变化。本研究使用针对两种胃饥饿素异构体的选择性和特异性免疫测定法，对各种条件进行了全面比较，以优化测定灵敏度和程序间的一致性。因此，这些测定条件可广泛用于精确阐明胃饥饿素异构体在生理和病理情况下对饮食行为的作用。
