Enzymatic Metabolism of Ergosterol by Cytochrome P450scc to Biologically Active 17α,24-Dihydroxyergosterol

SummaryWe demonstrate the metabolism of ergosterol by cytochrome P450scc in either a reconstituted system or isolated adrenal mitochondria. The major reaction product was identified as 17α,24-dihydroxyergosterol. Purified P450scc also generated hydroxyergosterol as a minor product, which is probably an intermediate in the synthesis of 17α,24-dihydroxyergosterol. In contrast to cholesterol and 7-dehydrocholesterol, cleavage of the ergosterol side chain was not observed. NMR analysis clearly located one hydroxyl group to C24, with evidence that the second hydroxyl group is at C17. 17α,24-Dihydroxyergosterol inhibited cell proliferation of HaCaT keratinocytes and melanoma cells. Thus, in comparison with cholesterol and 7-dehydrocholesterol, the 24-methyl group and the C22-C23 double bond of ergosterol prevent side chain cleavage by P450scc and change the enzyme’s hydroxylase activity from C22 and C20, to C24 and C17, generating bioactive product

Sequential Metabolism of 7-Dehydrocholesterol to Steroidal 5,7-Dienes in Adrenal Glands and Its Biological Implication in the Skin

Since P450scc transforms 7-dehydrocholesterol (7DHC) to 7-dehydropregnenolone (7DHP) in vitro, we investigated sequential 7DHC metabolism by adrenal glands ex vivo. There was a rapid, time- and dose-dependent metabolism of 7DHC by adrenals from rats, pigs, rabbits and dogs with production of more polar 5,7-dienes as detected by RP-HPLC. Based on retention time (RT), UV spectra and mass spectrometry, we identified the major products common to all tested species as 7DHP, 22-hydroxy-7DHC and 20,22-dihydroxy-7DHC. The involvement of P450scc in adrenal metabolic transformation was confirmed by the inhibition of this process by DL-aminoglutethimide. The metabolism of 7DHC with subsequent production of 7DHP was stimulated by forscolin indicating involvement of cAMP dependent pathways. Additional minor products of 7DHC metabolism that were more polar than 7DHP were identified as 17-hydroxy-7DHP (in pig adrenals but not those of rats) and as pregna-4,7-diene-3,20-dione (7-dehydroprogesterone). Both products represented the major identifiable products of 7DHP metabolism in adrenal glands. Studies with purified enzymes show that StAR protein likely transports 7DHC to the inner mitochondrial membrane, that 7DHC can compete effectively with cholesterol for the substrate binding site on P450scc and that the catalytic efficiency of 3βHSD for 7DHP (Vm/Km) is 40% of that for pregnenolone. Skin mitochondria are capable of transforming 7DHC to 7DHP and the 7DHP is metabolized further by skin extracts. Finally, 7DHP, its photoderivative 20-oxopregnacalciferol, and pregnenolone exhibited biological activity in skin cells including inhibition of proliferation of epidermal keratinocytes and melanocytes, and melanoma cells. These findings define a novel steroidogenic pathway: 7DHC→22(OH)7DHC→20,22(OH)27DHC→7DHP, with potential further metabolism of 7DHP mediated by 3βHSD or CYP17, depending on mammalian species. The 5–7 dienal intermediates of the pathway can be a source of biologically active vitamin D3 derivatives after delivery to or production in the skin, an organ intermittently exposed to solar radiation

Surface Enhanced Raman Spectroscopy of Lactoferrin Adsorbed on Silvered Porous Silicon Covered with Graphene

We registered surface enhanced Raman scattering (SERS) spectra of the human lactoferrin molecules adsorbed on a silvered porous silicon (por-Si) from 106–1018 M solutions. It was found that the por-Si template causes a negative surface potential of silver particles and their chemical resistivity to oxidation. These properties provided to attract positively charged lactoferrin molecules and prevent their interaction with metallic particles upon 473 nm laser excitation. The SERS spectra of lactoferrin adsorbed from 106 M solution were rather weak but a decrease of the concentration to 10-10 M led to an enormous growth of the SERS signal. This effect took place as oligomers of lactoferrin were broken down to monomeric units while its concentration was reduced. Oligomers are too large for a uniform overlap with electromagnetic field from silver particles. They cannot provide an intensive SERS signal from the top part of the molecules in contrast to monomers that can be completely covered by the electromagnetic field. The SERS spectra of lactoferrin at the 10-14 and 10-16 M concentrations were less intensive and started to change due to increasing contribution from the laser burned molecules. To prevent overheating the analyte molecules on the silvered por-Si were protected with graphene, which allowed the detection of lactoferrin adsorbed from the 10-18 M solution

SEC analysis of poly(acrylic acid) and poly(methacrylic acid)

The accurate characterization of molar-mass distributions of poly(acrylic acid) (PAA) and poly(methacrylic acid) (PMAA) by size-exclusion chromatography (SEC) is addressed. Two methods are employed: direct aqueous-phase SEC on P(M)AA and THF-based SEC after esterification of P(M)AA to the associated methyl esters, P(M)MA. P(M)AA calibration standards, P(M)AA samples prepared by pulsed-laser polymerization (PLP), and PAA samples prepared by reversible addition-fragmentation chain transfer (RAFT) are characterized in a joint initiative of seven laboratories, with satisfactory agreement achieved between the institutions. Both SEC methods provide reliable results for PMAA. In the case of PAA, close agreement between the two SEC methods is only observed for samples prepared by RAFT polymerization with weightaverage molar mass between 80 000 and 145 000 g mol-1 and for standards with peak molar masses below 20 000 g mol-1. For standards with higher molar masses and for PLP-prepared PAA, the values from THF-based SEC are as much as 40% below the molar masses determined by aqueous-phase SEC. This discrepancy may be due to branching or degradation of branched PAA during methylation. While both SEC methods can be recommended for PMAA, aqueous-phase SEC should be used for molar-mass analysis of PAA unless the sample is not branched. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

Local Melatoninergic System as the Protector of Skin Integrity

The human skin is not only a target for the protective actions of melatonin, but also a site of melatonin synthesis and metabolism, suggesting an important role for a local melatoninergic system in protection against ultraviolet radiation (UVR) induced damages. While melatonin exerts many effects on cell physiology and tissue homeostasis via membrane bound melatonin receptors, the strong protective effects of melatonin against the UVR-induced skin damage including DNA repair/protection seen at its high (pharmocological) concentrations indicate that these are mainly mediated through receptor-independent mechanisms or perhaps through activation of putative melatonin nuclear receptors. The destructive effects of the UVR are significantly counteracted or modulated by melatonin in the context of a complex intracutaneous melatoninergic anti-oxidative system with UVR-enhanced or UVR-independent melatonin metabolites. Therefore, endogenous intracutaneous melatonin production, together with topically-applied exogenous melatonin or metabolites would be expected to represent one of the most potent anti-oxidative defense systems against the UV-induced damage to the skin. In summary, we propose that melatonin can be exploited therapeutically as a protective agent or as a survival factor with anti-genotoxic properties or as a “guardian” of the genome and cellular integrity with clinical applications in UVR-induced pathology that includes carcinogenesis and skin aging