Lipid biomarkerek HPLC-HRMS vizsgálata történeti embertani minták tbc diagnosztikája során

Abstract

Tuberculosis (TB) is not only an infectious disease but one of the top 10 causes of death, spreading mainly with aerosol transmission and accompanying the history of humankind for several millennia. TB is caused in humans and animals by members of the Mycobacterium tuberculosis complex. For better understanding of the disease and the evolutional background of its causative agent, involvement of palaeopathological investigations is surpassingly important. Traditionally, palaeopathology is using a broad variety of markers, which are observable by macroscopic investigations. These markers are mainly related to different extra-pulmonary forms of TB. However, these manifestations develop only in a few cases. Moreover, many markers are not pathognomonic of TB. Therefore, to avoid underestimation of TB prevalence in paleopathological studies, the diagnosis on archaeological material requires a multidisciplinary approach. For better estimation of TB incidence in past populations, an array of specific biomarkers can be brought into play to confirm initial, macromorphology-based skeletal diagnoses, namely aDNA and lipid biomarkers. The three lipid biomarker groups, which are mainly involved in such investigations are the mycolic acids (MAs), the mycocerosic acids (MCs) and the C27 mycolipenic acid (ML). These unique lipids can be located in the lipid-rich mycobacterial cell wall. The application of lipid-based TB diagnostic approach has been proved to be robust and reliable through many examples. A variety of analytical methods have been employed for mycobacterial lipid biomarker profiling. Fluorescence HPLC is well-developed for the analysis of mycolic acids and phthiocerols. Furthermore, one isolated case of direct mass spectrometric detection of M. tuberculosis free mycolic acids has been also introduced. Our aim was to establish a lipid-biomarker-based HPLC-MS method for TB diagnosis in historical human samples, as this instrumentation is available in many laboratories, has the potential of a quick and sensitive and at the same time an affordable measurement protocol. We successfully developed and optimised a method, which is capable to separate and detect MAs and MCs. Moreover, we optimised the sample pre-treatment process. We started to build an MA and MC lipid profile library, which can serve as a comparison to diagnose TB. For the verification of new methods and the estimation of reliability of new markers in macroscopic analysis, palaeopathologists usually use well-documented collections of skeletons and mummies from the pre-antibiotic era. For this purpose, we chose to test our method on the Vác Mummy Collection. In the case of four out of six mummified individuals, MC profiles of characteristic M. tuberculosis MCs were recorded. The HPLC-ESI-MS method, developed for the detection of MCs, opens a new avenue for the detection of ancient mycobacterial disease, encompassing both tuberculosis, leprosy and joint cases.