Structural requirements of the human sodium-dependent bile acid transporter (hASBT): role of 3- and 7-OH moieties on binding and translocation of bile acids.

Bile acids (BAs) are the end products of cholesterol metabolism. One of the critical steps in their biosynthesis involves the isomerization of the 3ß-hydroxyl (-OH) group on the cholestane ring to the common 3α-configuration on BAs. BAs are actively recaptured from the small intestine by the human Apical Sodium-dependent Bile Acid Transporter (hASBT) with high affinity and capacity. Previous studies have suggested that no particular hydroxyl group on BAs is critical for binding or transport by hASBT, even though 3ß-hydroxylated BAs were not examined. The aim of this study was to elucidate the role of the 3α-OH group on BAs binding and translocation by hASBT. Ten 3ß-hydroxylated BAs (Iso-bile acids, iBAs) were synthesized, characterized, and subjected to hASBT inhibition and uptake studies. hASBT inhibition and uptake kinetics of iBAs were compared to that of native 3α-OH BAs. Glycine conjugates of native and isomeric BAs were subjected to molecular dynamics simulations to identify topological descriptors related to binding and translocation by hASBT. Iso-BAs bound to hASBT with lower affinity and exhibited reduced translocation than their respective 3α-epimers. Kinetic data suggests that, in contrast to native BAs where hASBT binding is the rate-limiting step, iBAs transport was rate-limited by translocation and not binding. Remarkably, 7-dehydroxylated iBAs were not hASBT substrates, highlighting the critical role of 7-OH group on BA translocation by hASBT, especially for iBAs. Conformational analysis of gly-iBAs and native BAs identified topological features for optimal binding as: concave steroidal nucleus, 3-OH "on-" or below-steroidal plane, 7-OH below-plane, and 12-OH moiety toward-plane. Our results emphasize the relevance of the 3α-OH group on BAs for proper hASBT binding and transport and revealed the critical role of 7-OH group on BA translocation, particularly in the absence of a 3α-OH group. Results have implications for BA prodrug design.

Neuromelanins in various regions of human brain are associated with native and oxidized isoprenoid lipids.

Neuromelanin (NM) isolated from seven regions of the human brain is found to contain series of natural and oxidized isoprenoid lipids. Specifically, dolichols (dol) and dolichoic acids (dol-CA) with 14-22 and 14-21 isoprene units are identified. Standards of nor-dolichol and nor-dolichoic acid were used to determine the relative amounts of dol and dol-CA compared to the total lipids present in NM for each region. The cerebellum, putamen, globus pallidus, and premotor cortex contained similar amounts of dol, comprising approximately 8-9.5% of the total lipid weight. Interestingly, the corpus callosum contains substantially lower quantities of both dol and dol-CA compared to the other regions-less than 4% dol relative to the total lipid weight. Oxidized and reduced dolichol-related species were identified and determined to be region-dependent.

New melanic pigments in the human brain that accumulate in aging and block environmental toxic metals.

Neuronal pigments of melanic type were identified in the putamen, cortex, cerebellum, and other major regions of human brain. These pigments consist of granules 30 nm in size, contained in organelles together with lipid droplets, and they accumulate in aging, reaching concentrations as high as 1.5-2.6 microg/mg tissue in major brain regions. These pigments, which we term neuromelanins, contain melanic, lipid, and peptide components. The melanic component is aromatic in structure, contains a stable free radical, and is synthesized from the precursor molecule cysteinyl-3,4-dihydroxyphenylalanine. This contrasts with neuromelanin of the substantia nigra, where the melanic precursor is cysteinyl-dopamine. These neuronal pigments have some structural similarities to the melanin found in skin. The precursors of lipid components of the neuromelanins are the polyunsaturated lipids present in the surrounding organelles. The synthesis of neuromelanins in the various regions of the human brain is an important protective process because the melanic component is generated through the removal of reactive/toxic quinones that would otherwise cause neurotoxicity. Furthermore, the resulting melanic component serves an additional protective role through its ability to chelate and accumulate metals, including environmentally toxic metals such as mercury and lead.

Quantification of naturally occurring pyrrole acids in melanosomes.

Three naturally occurring pyrrole acids were found in Sepia, human black hair, and bovine choroid and iris melanosomes using high-performance liquid chromatography and mass spectrometry--pyrrole-2,3-dicarboxylic acid (PDCA), pyrrole-2,3,5-tricarboxylic acid (PTCA) and pyrrole-2,3,4,5-tetracarboxylic acid (PTeCA). PDCA and PTCA are common markers quantified from oxidative degradation of eumelanins. Using standards, the amounts of naturally occurring PDCA and PTCA were determined and compared to those obtained following peroxide oxidation of the same samples. Because the naturally occurring acids are water soluble, these results indicate that care must be exercised when comparing PDCA and PTCA yields from the degradation analyses of melanins isolated and prepared by different methods. This work also establishes that PTeCA is a naturally occurring pyrrole acid in melanosomes.

Identification and quantification of dolichol and dolichoic acid in neuromelanin from substantia nigra of the human brain.

Neuromelanin (NM) isolated from the substantia nigra of the human brain is found to contain a series of dolichoic acids (dol-CA) containing 14-20 isoprene units. This is the first observation of dol-CA in a natural system. Using internally spiked nor-dolichol and nor-dolichoic acid standards, the concentrations of dolichol (dol) and dol-CA present in NM were determined. Remarkably, dol was only four times as abundant as dol-CA in NM. The distribution of dol-CA chains lengths in NM also differed from that of dol, suggesting that the enzyme(s) responsible for the conversion of dol to dol-CA prefer a dolichol substrate containing 19 isoprene units.

The differing embryonic origins of retinal and uveal (iris/ciliary body and choroid) melanosomes are mirrored by their phospholipid composition.

The phospholipids present in uveal (iris/ciliary body and choroid) and retinal bovine ocular melanosomes were identified using mass spectrometry. Similar phospholipid content is found for the two types of uveal melanosome, with sphingomyelin being the major species. Significant differences are found between the uveal and retinal melanosome. Glycerophosphoethanolamine (GPEtn) is the major species in the retinal pigment epithelium (RPE); 93% of the GPEtn contain polyunsaturated fatty acids, notably docosahexanoic acid and arachidonic acid, in the sn-2 position. RPE melanosomes also contain detectable quantities of glycerophosphoserine and glycerophosphate; these species were not detected in the uveal samples. While the structural and functional roles of melanosomal lipids largely remain to be determined, these different lipid compositions reported herein offer new insights into the roles of melanosomes in the different ocular tissues.