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Abstract
AIM: To validate a spectrofluorometric method for measuring chlorophyll a metabolites, specifically phytoporphyrin (= phylloerythrin), as well as the chlorins, pheophorbide a and pyropheophorbide a, in the blood of photosensitive cattle and sheep.
METHODS: Standard methanolic solutions of pheophorbide a (25 µM), pyropheophorbide a (25 µM), and phytoporphyrin (<3.7 µM) were prepared. Serum and plasma samples were obtained from cattle (n=5), sheep (n=3), and one alpaca, with clinical facial eczema (i.e. photosensitive), as well as from clinically normal (n=2 of each) adult cows, recently weaned calves, and sheep (controls). Standard solutions of the three metabolites were characterised using high-performance liquid chromatography (HPLC), with mass spectrometry, in conjunction with absorption and emission spectral data, and were compared with sera from photosensitive animals. In the latter, phytoporphyrin was the only metabolite detected. Calibration curves were prepared by adding different ratios of methanol and standard solutions of phytoporphyrin in methanol to diluted serum from control animals. Peak areas of fluorescence spectra were determined in samples from photosensitive animals.
RESULTS: Pheophorbide a and pyropheophorbide a produced typical chlorin spectra, and had excitation/emission maxima of 408/669 nm and 409/669 nm, respectively. Phytoporphyrin showed a typical porphyrin fluorescence spectrum, with excitation/emission maxima of 425/644 nm. Pyropheophorbide a and phytoporphyrin had very similar chromatographic retention times, the same chemical formula and same mass, but were distinguishable by differences in their absorption spectra. In sera from photosensitive animals, the fluorescence emission at 644 nm was shown to arise solely from phytoporphyrin and not from any other chlorophyll a metabolites. Calibration curves using sera and plasma from control animals gave reliable measurements of phytoporphyrin in the range 0.4–6 µM. The sera of facial eczema-affected cattle and sheep had concentrations of phytoporphyrin ranging from 0.4 to 1.8 and 0.9 to 2.8 µM, respectively. Haemolysed serum samples were not suitable for determination of phytoporphyrin with this method.
CONCLUSIONS AND CLINICAL RELEVANCE: A spectrofluorometric method for the quantification of phytoporphyrin in the blood of photosensitive animals has been validated, and can be applied to the measurement of other chlorophyll a metabolites in blood. This will be a useful tool in the further investigation of the cause and pathogenesis of idiopathic photosensitivities of farm animals.
Acknowledgements
Financial support from the Institute of Veterinary, Animal and Biomedical Sciences (Massey University); the McGeorge, Lewis Fitch and Massey University Research Funds; as well as the Tertiary Education Commission's BRCSRA fund, is gratefully acknowledged. We are indebted to Michael Spencer for the SciFinder Scholar information regarding the correct molecular structure of phytoporphyrin, David Officer and Shannon Bullock for their expertise in porphyrins and preliminary investigations, Mark Patchett for information on conjugation and phytoporphyrin, Peter Dobbie and David Lun for valuable technical discussions, Sue Pannifer of New Zealand Veterinary Pathology Ltd for the clinical chemistry and serum and plasma separations, Quentin Roper for the graphics, Mike Hogan for technical assistance, and Keith Thompson for reviewing the manuscript.