title: GC/MS detection of central nervous tissue as specified BSE risk material in meat products and meat and bone meals: thermal stability of markers in comparison with immunochemistry and RT-PCR.
abstract: Methods for the detection of central nervous tissue (CNT) are urgently needed in food control as a means for controlling strict adherence to both food labeling and banning of specified BSE risk material. Here, we report data on heat stability of the CNT markers neuron-specific enolase (NSE) in western blotting, glial fibrillary acidic protein (GFAP) in an enzyme linked immunoassay, mRNA(GFAP) in a real-time PCR assay, and several fatty acids (C22:6, C24:0-OH, C24:1ω9/ω7, C24:1ω9-OH/ω7-OH, and C24:0) in gas chromatography mass spectrometry (GC/MS). The sample matrix, a standard material of emulsion-type sausage with varied contents of CNT (brain), was heat-treated in three studies: (1) routine meat technological heat treatment with low (85 °C, 30 min), medium (115 °C, 30 min), and high (133 °C, 30 min, 3 bar) heating of 72 anonymous samples from a blind trial; (2) heat treatment under experimental conditions (100, 110, …, 200 °C, 45 min); and (3) fractionized heating of central nervous system (up to three times) under moderate routine technological conditions (85, 100, and 115 °C, 30 min). The markers of the immunochemical methods showed a low GFAP or very low NSE temperature stability at medium and high temperature conditions. The real-time PCR assay gave inconsistent, non-quantitative results, which indicated an uncontrollable matrix effect. The relevant GC/MS markers (C24:0-OH, C24:1ω9/ω7, and C24:1ω9-OH/ω7-OH) proved to be extremely stable. Neither meat and bone meal conditions (133 °C) nor experimental heating (up to and above 140 °C) showed any reduction of GC/MS CNT quantification. On the contrary, a slight but significant increase was noted over a certain temperature range (120-140 °C) for most fatty acids, possibly due to an improved extractability of the fatty acids. We conclude that a quantitative approach is highly unreliable when using immunochemical methods; moreover, these methods might be basically prone to false-negative results depending on heat treatment and matrix composition. Therefore, antibodies with higher affinity to heat-treated CNT marker epitopes are needed. Relevant amounts of CNT (≥0.5%) in low- and medium-heated products would still be reliably detectable by the GFAP ELISA, which justifies its use as a screening method in official food control. The results obtained by the real-time PCR assay were contradictory to recently published data, indicating a need for further protocol optimization and collaborative trials. Up to date, the analytical approach using GC/MS is the only valid procedure as pertaining to heat stability and quantitative analysis; consequently, it should be recommended as the reference procedure in official food control for CNT detection in heat-treated meat products.
authors.affiliation: Institut für Lebensmittelhygiene, Universität Leipzig, An den Tierkliniken 1, 04103, Leipzig, Germany. luecker@vmf.uni-leipzig.de
authors.forename: Ernst
authors.initials: E
authors.lastname: Lücker
authors.forename: Wolfgang
authors.initials: W
authors.lastname: Biedermann
authors.forename: Thomas
authors.initials: T
authors.lastname: Alter
authors.forename: Andreas
authors.initials: A
authors.lastname: Hensel
mesh_terms: D000818:Animals; D001717:Birds; D001923:Brain Chemistry; D002417:Cattle; D016643:Encephalopathy, Bovine Spongiform / Q000635:transmission*; D008401:Gas Chromatography-Mass Spectrometry / Q000379:methods*; D005904:Glial Fibrillary Acidic Protein / Q000032:analysis; D006358:Hot Temperature; D007120:Immunochemistry; D008460:Meat / Q000032:analysis*; D008461:Meat Products / Q000032:analysis; D010751:Phosphopyruvate Hydratase / Q000032:analysis; D055550:Protein Stability; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012756:Sheep; D013552:Swine
chemical_list: D005904:Glial Fibrillary Acidic Protein; D010751:Phosphopyruvate Hydratase