The Detection Of Keto-carotenoids, Metabolic Studies And The Role Of Uv-induced Damage In Hacat Cells

ObjectiveThe separation of keto-carotenoid by HPLC equipped with PDA on both C18 and C30 columns; the assessment of five species of shrimps and lobsters from the local market of Beijing for astaxanthin amount and composition;the investigation into accumulation in different organs and the configuration variation of keto-carotenoid molecule in the metabolism of SD rats;the influence of keto-carotenoid on activity of LDH and proliferation after irradiated by ultraviolet A and ultraviolet B in human HaCaT cells.MethodsBy HPLC equipped with PDA keto-carotenoid was readily separated on a C18 column under following conditions:a Diamonsil^TM column;mobile phase A=acidic acetonitrile-water;mobile phase B=ethyl acetate;linear gradient;monitoring wavelength=480nm;the wavelength range of PDA=280-580nm;sample injection volume=20μl.The conditions of C30-HPLC-PDA was a Waters YMC Carotenoid S-5 column;mobile phase A=acidic acetonitrile-water;mobile phase B=MTBE;linear gradient;monitoring wavelength=480nm;the wavelength range of PDA=250-600nm;sample injection volume=20μl.Keto-carotenoid fraction was extracted from serum,organs and dung after fed into SD rat and the geometrical isomer composition of keto-carotenoid was assessed by C30-HPLC-PDA.Human HaCaT cell irradiated by UVB/UVA was treated with keto-carotenoid and cultured for 24 hours.The LDH level was detected and the cell proliferation was observed by MTT essay.ResultBy HPLC-PDA,keto-carotenoid was readily separated on both C18 and C30 columns acidic acetonitrile mobile phase.This result is going to form the possibility of their quantization on C30-HPLC-PDA.Astaxanthin monomers accumulated in spleen and serum of SD rat.The geometrical isomer composition of canthaxanthin was assessed by C30-HPLC-PDA.No significant increase in the Z-isomer ratio of canthaxanthin in vivo was observed during its absorption,transportation and metabolism in liver.Keto-carotenoid increases the proliferation of HaCaT cells and relieves the LDH level of supernatant after UVB/UVA irradiation.ConclusionAccording to the comparison of its chromatographic behavior and spectral characters with those of reference samples,keto-carotenoid was identified.Its quantification was therefore able to be considered by C18-HPLC-PDA.It was very possible to assess the geometrical isomer composition of keto-carotenoid by C30-HPLC-PDA.The present results indicate that keto-carotenoid relieve damage of HaCaT cells irradiated by UVA or UVB.