5-Methylcytosine (5 mC) in genomic DNA has important epigenetic functions in 
embryonic development and tumor biology. 5-Hydroxymethylcytosine (5 hmC) is 
generated from 5 mC by the action of the TET (Ten-Eleven-Translocation) enzymes 
and may be an intermediate to further oxidation and finally demethylation of 5 
mC. We have used immunohistochemistry (IHC) and isotope-based liquid 
chromatography mass spectrometry (LC-MS) to investigate the presence and 
distribution of 5 hmC in human brain and brain tumors. In the normal adult 
brain, IHC identified 61.5% 5 hmC positive cells in the cortex and 32.4% 5 hmC 
in white matter (WM) areas. In tumors, positive staining of cells ranged from 
1.1% in glioblastomas (GBMs) (WHO Grade IV) to 8.9% in Grade I gliomas 
(pilocytic astrocytomas). In the normal adult human brain, LC-MS also showed 
highest values in cortical areas (1.17% 5 hmC/dG [deoxyguanosine]), in the 
cerebral WM we measured around 0.70% 5 hmC/dG. levels were related to tumor 
differentiation, ranging from lowest values of 0.078% 5 hmC/dG in GBMs (WHO 
Grade IV) to 0.24% 5 hmC/dG in WHO Grade II diffuse astrocytomas. 5 hmC 
measurements were unrelated to 5 mC values. We find that the number of 5 hmC 
positive cells and the amount of 5 hmC/dG in the genome that has been proposed 
to be related to pluripotency and lineage commitment in embryonic stem cells is 
also associated with brain tumor differentiation and anaplasia.