Bouhlel Jihéne, Ratel Jérémy, Abouelkaram Said, Mercier Frédéric, Travel Angelique, Baéza Elisabeth, Jondreville Catherine, Dervilly-Pinel Gaud, Marchand Philippe, Le Bizec Bruno, Dubreil Estelle, Mompelat Sophie, Verdon Eric, Inthavong Chanthadary, Guérin Thierry, Rutledge Douglas N, Engel Erwan
INRA, UR370 QuaPA, MASS laboratory, Saint-Genès-Champanelle, France; INRA, UMR GENIAL, AgroParisTech, Université Paris-Saclay, Massy, France.
INRA, UR370 QuaPA, MASS laboratory, Saint-Genès-Champanelle, France.
J Chromatogr A. 2017 May 12;1497:9-18. doi: 10.1016/j.chroma.2017.03.008. Epub 2017 Mar 7.
Starting from a critical analysis of a first "proof of concept" study on the utility of the liver volatolome for detecting livestock exposure to environmental micropollutants (Berge et al., 2011), the primary aim of this paper is to improve extraction conditions so as to obtain more representative extracts by using an extraction temperature closer to livestock physiological conditions while minimizing analytical variability and maximizing Volatile Organic Compound (VOC) abundancies. Levers related to extraction conditions and sample preparation were assessed in the light of both abundance and coefficient of variation of 22 candidate VOC markers identified in earlier volatolomic studies. Starting with a CAR/PDMS fiber and a 30min extraction, the reduction of SPME temperature to 40°C resulted in a significant decrease in the area of 14 candidate VOC markers (p<0.05), mainly carbonyls and alcohols but also a reduction in the coefficient of variation for 17 of them. In order to restore VOC abundances and to minimize variability, two approaches dealing with sample preparation were investigated. By increasing sample defrosting time at 4°C from 0 to 24h yielded higher abundances and lower variabilities for 15 and 13 compounds, respectively. Lastly, by using additives favouring the release of VOCs (1.2g of NaCl) the sensitivity of the analysis was improved with a significant increase in VOC abundances of more than 50% for 13 out of the 22 candidate markers. The modified SPME parameters significantly enhanced the abundances while decreasing the analytical variability for most candidate VOC markers. The second step was to validate the ability of the revised SPME protocol to discriminate intentionally contaminated broiler chickens from controls, under case/control animal testing conditions. After verification of the contamination levels of the animals by national reference laboratories, data analysis by a multivariate chemometric method (Common Components and Specific Weights Analysis - ComDim) showed that the liver volatolome could reveal dietary exposure of broilers to a group of environmental pollutants (PCBs), a veterinary treatment (monensin), and a pesticide (deltamethrin), thus confirming the usefulness of this analytical set-up.
本文从对一项关于肝脏挥发物组用于检测家畜暴露于环境微污染物的效用的首个“概念验证”研究（Berge等人，2011年）的批判性分析出发，主要目的是改进提取条件，以便通过使用更接近家畜生理条件的提取温度来获得更具代表性的提取物，同时将分析变异性降至最低并使挥发性有机化合物（VOC）丰度最大化。根据早期挥发物组学研究中确定的22种候选VOC标记物的丰度和变异系数，对与提取条件和样品制备相关的因素进行了评估。从使用CAR/PDMS纤维和30分钟提取开始，将固相微萃取（SPME）温度降至40°C导致14种候选VOC标记物的面积显著减少（p<0.05），主要是羰基化合物和醇类，并且其中17种的变异系数也有所降低。为了恢复VOC丰度并使变异性最小化，研究了两种处理样品制备的方法。将4°C下的样品解冻时间从0小时增加到24小时，分别使15种和13种化合物的丰度更高且变异性更低。最后，通过使用有利于VOC释放的添加剂（1.2克氯化钠），分析的灵敏度得到提高，22种候选标记物中有13种的VOC丰度显著增加超过50%。改良后的SPME参数显著提高了大多数候选VOC标记物的丰度，同时降低了分析变异性。第二步是在病例/对照动物测试条件下，验证修订后的SPME方案区分故意污染的肉鸡和对照的能力。在国家参考实验室核实动物的污染水平后，通过多元化学计量学方法（共同成分和特定权重分析 - ComDim）进行数据分析表明，肝脏挥发物组可以揭示肉鸡对一组环境污染物（多氯联苯）、一种兽药治疗（莫能菌素）和一种农药（溴氰菊酯）的膳食暴露，从而证实了这种分析方法的实用性。
