Mycocerosic acid biomarkers for the diagnosis of tuberculosis in the Coimbra Skeletal Collection

Tuberculosis has been a scourge of humans over many millennia, but questions remain regarding its evolution and epidemiology. Fossil biomarkers, such as DNA and long-chain mycolic acids, can be detected in ancient skeletal and other materials. The phthiocerol dimycocerosate waxes are also robust biomarkers for tuberculosis and sensitive methods are available for the detection of their mycocerosic acid components. The presence of mycocerosic acids was investigated in 49 individuals from the 1837–1936 Coimbra Identified Skeletal Collection (Portugal), half with documentary data indicating tuberculosis as a cause of death. Samples were hydrolysed, acidic components converted to pentafluorobenzyl esters, the non-hydroxylated long-chain esters isolated, and this fraction separated into multimethyl-branched and other esters by normal phase high performance liquid chromatography. Negative ion chemical ionisation gas chromatography mass spectrometry was used to detect diagnostic C29, C30 and C32 mycocerosic acids. Mycocerosic acids were detected in archaeological material for the first time, illustrating that they are valuable biomarkers for the diagnosis of ancient tuberculosis. A 72% correlation with the Coimbra burial record supported TB as the major cause of death. In addition, 30% of the skeletons, positive for mycocerosates, showed the presence of related long-chain mycolipenic acids.