Lafrenière Nelson M, Watterson James H
Forensic Toxicology Research Laboratory, Department of Forensic Science, Laurentian University, 935 Ramsey Lake Rd., Sudbury, Ontario, Canada P3E 2C6.
Forensic Sci Int. 2009 Mar 10;185(1-3):100-6. doi: 10.1016/j.forsciint.2008.12.019. Epub 2009 Feb 7.
The detection of acute fentanyl exposure in fresh and decomposed skeletal tissues (marrow and bone), by automated enzyme-linked immunosorbent assay (ELISA) is described. Rats (n=15) were administered fentanyl acutely at a dose of 0 (n=3), 15 (n=3), 30 (n=3) or 60 microg/kg (n=6) by intraperitoneal injection, and euthanized within 20 min. Femora and tibiae were extracted from the fresh corpses and marrow was isolated from the femoral and tibial medullary cavities. The remains were then allowed to decompose outdoors to the point of complete skeletonization, and vertebrae and pelvi were recovered for analysis. In all cases, bones were cleaned in alkaline solution and then ground into a fine powder. Marrow was homogenized in alkaline solution. Fentanyl was extracted from ground bone by methanolic extraction. Extracts were adjusted to pH 6 and analyzed by ELISA. Perimortem heart blood was also collected and diluted in phosphate buffer prior to screening by ELISA. The effect of tissue type on ELISA response was examined through determination of binary classification test sensitivity and the relative decrease in absorbance (%DA, drug-positive tissues vs drug-free controls) in each tissue type. Overall, the %DA varied significantly between extracts from different skeletal tissues under a given dose condition, according to the general order of marrow>vertebrae approximately pelvi>epiphyseal bone approximately diaphyseal bone. Binary classification test sensitivity values for fentanyl in marrow, fresh epiphyseal (femoral and tibial) bone, fresh diaphyseal (femoral and tibial) bone, decomposed vertebrae and decomposed pelvic bone were 100%, 16-33%, 0-16%, 0-33% and 66-100%, respectively, at the 60 microg/kg dose level. While mean %DA values showed a strong positive correlation with those in marrow and blood measurements and the administered dose (r=0.997 and 0.986), such a correlation was not observed in assays of decomposed tissues (r=-0.157 and -0.315). These results suggest that the type of skeletal tissue sampled and position within a given bone may be important considerations in the choice of substrate for fentanyl screening in skeletal tissues, but that quantitative analysis of drugs in decomposed bones may be of limited interpretive value.
本文描述了通过自动化酶联免疫吸附测定法（ELISA）检测新鲜和腐败骨骼组织（骨髓和骨骼）中急性芬太尼暴露情况。将15只大鼠通过腹腔注射分别给予剂量为0（n = 3）、15（n = 3）、30（n = 3）或60微克/千克（n = 6）的芬太尼，并在20分钟内实施安乐死。从新鲜尸体中取出股骨和胫骨，从股骨和胫骨骨髓腔中分离出骨髓。然后将遗体置于户外使其分解至完全白骨化，回收椎骨和骨盆进行分析。在所有情况下，骨骼均在碱性溶液中清洗，然后研磨成细粉。骨髓在碱性溶液中匀浆。通过甲醇提取法从研磨后的骨骼中提取芬太尼。将提取物调节至pH 6并通过ELISA进行分析。还收集了濒死期心脏血液，并用磷酸盐缓冲液稀释后通过ELISA进行筛查。通过测定二元分类试验敏感性以及每种组织类型中吸光度的相对下降率（%DA，药物阳性组织与无药物对照）来检查组织类型对ELISA反应的影响。总体而言，在给定剂量条件下，不同骨骼组织提取物的%DA差异显著，大致顺序为骨髓>椎骨≈骨盆>骨骺骨≈骨干骨。在60微克/千克剂量水平下，骨髓、新鲜骨骺（股骨和胫骨）骨、新鲜骨干（股骨和胫骨）骨、腐败椎骨和腐败骨盆骨中芬太尼的二元分类试验敏感性值分别为100%、16 - 33%、0 - 16%、0 - 33%和66 - 100%。虽然平均%DA值与骨髓和血液测量值以及给药剂量呈强正相关（r = 0.997和0.986），但在腐败组织检测中未观察到这种相关性（r = -0.157和 -0.315）。这些结果表明，在选择用于骨骼组织中芬太尼筛查的底物时，所采集的骨骼组织类型和给定骨骼内的位置可能是重要的考虑因素，但对腐败骨骼中药物的定量分析可能具有有限的解释价值。
