HPLC Methods for Analysis of Porphyrins in Biological Media

Abstract

Changes in porphyrin concentrations in biological media may serve as biological indicators of exposure and toxicity from a wide variety of drugs and chemical agents. This unit describes procedures for quantitative extraction of porphyrins from urine, feces, blood, and biological tissues as well as their separation and analysis by HAPLY spectrofluorometrc techniques.

Heme dynamics and trafficking factors revealed by genetically encoded fluorescent heme sensors

“…The genes for mKATE2 and CG6 used to construct HS1 were purchased from GENESCRIPT and codon-optimized for expression in E. coli and S. cerevisiae or humans. Standard published methods for protein expression and purification (18), heme binding studies (20,27), fluorimetry, UV/visible spectroscopy, microscopy, flow cytometry, immunoblotting (41), catalase activity (34), oxygen consumption (41), total heme analysis (42), and cell growth are outlined in SI Appendix, SI Materials and Methods.…”

From Synthesis to Utilization: The Ins and Outs of Mitochondrial Heme

“…Extracted heme can be chromatographed by high performance liquid chromatography (HPLC) using a reverse-phase C18 column coupled to an in-line UV-vis detector for quantification [231,232]. This method allows for differentiating heme b from other heme types, including heme o or heme a [233]. Alternatively, extracted heme or heme from soluble lysates or isolated hemoproteins may be quantified using the pyridine hemochromagen assay, which is based upon the distinct spectral features of the bis-pyridine ferrous heme complex [234][235][236][237].…”

“…The quartz cuvette contains a reflective coating that effectively increases the path length, making the instrument much more sensitive to absorption of light in turbid samples [237,238]. Finally, the measurement of porphyrin fluorescence is a very sensitive technique for the quantification of heme, with a limit of detection of 10 −9 M [232,233]. While heme itself is non-fluorescent, de-metallation of the heme iron in boiling oxalic acid results in the formation of fluorescent PP IX free base (λ ex = 400 nm; λ em = 662 nm) [232,233].…”

“…Finally, the measurement of porphyrin fluorescence is a very sensitive technique for the quantification of heme, with a limit of detection of 10 −9 M [232,233]. While heme itself is non-fluorescent, de-metallation of the heme iron in boiling oxalic acid results in the formation of fluorescent PP IX free base (λ ex = 400 nm; λ em = 662 nm) [232,233]. Although the aforementioned methods serve as powerful tools for measuring heme, they suffer from a number of drawbacks.…”

Ambient Light Promotes Selective Subcellular Proteotoxicity after Endogenous and Exogenous Porphyrinogenic Stress

“…A reverse-phase octadecyl silica (C18) Waters XSelect HSS T3 column (100-Å pore size, 3.5-m particle size, 4.6 ϫ 150-mm dimension) HPLC column was used. The fluorescence detector was set at 402 nm (excitation) and 625 nm (emission) (15). The column (equilibrated for 10 min) was eluted at a flow rate of 1.0 ml/min with linear gradients of solvents A and B (solvent A, 0.05 mM monobasic sodium phosphate, pH 3.5, in water; solvent B, methanol).…”