An efficient extraction method for quantitation of adenosine triphosphate in mammalian tissues and cells

Firefly bioluminescence is widely used in the measurement of adenosine 5′-triphosphate (ATP) levels in biological materials. For such assays in tissues and cells, ATP must be extracted away from protein in the initial step and extraction efficacy is the main determinant of the assay accuracy. Extraction reagents recommended in the commercially available ATP assay kits are chaotropic reagents, trichloroacetic acid (TCA), perchloric acid (PCA), and ethylene glycol (EG), which extract nucleotides through protein precipitation and/or nucleotidase inactivation. We found that these reagents are particularly useful for measuring ATP levels in materials with relatively low protein concentrations such as blood cells, cultured cells, and bacteria. However, these methods are not suitable for ATP extraction from tissues with high protein concentrations, because some ATP may be co-precipitated with the insolubilized protein during homogenization and extraction, and it could also be precipitated by neutralization in the acid extracts. Here we found that a phenol-based extraction method markedly increased the ATP and other nucleotides extracted from tissues. In addition, phenol extraction does not require neutralization before the luciferin–luciferase assay step. ATP levels analyzed by luciferase assay in various tissues extracted by Tris–EDTA–saturated phenol (phenol–TE) were over 17.8-fold higher than those extracted by TCA and over 550-fold higher than those in EG extracts. Here we report a simple, rapid, and reliable phenol–TE extraction procedure for ATP measurement in tissues and cells by luciferase assay.

This study showed that phenol–TE extraction is suitable for measurement of ATP levels in various mammalian tissues (B, brain; H, heart; L, liver; S, spleen; M, muscle) with high protein concentrations and cells, and provided better and more efficient extraction than trichloroacetic acid (TCA) and ethylene glycol (EG), which are recommended in the commercially available ATP assay kits. The ATP levels extracted by phenol–TE were over 17.8-fold higher (depending on tissues) than those in TCA extracts and over 550-fold higher than those in EG extracts. We recommend this method for assay of ATP from mammalian tissues and blood.Figure optionsDownload as PowerPoint slideHighlights
► An extraction method for ATP and other nucleotides from mammalian tissues and blood.
► Phenol-based extraction provided better ATP extraction than chaotropic reagents.
► Phenol-based extraction does not require neutralization before the luciferase assay.