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

Abstract

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. 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. dolichol dolichoic acid liquid chromatography-mass spectrometry neuromelanin substantia nigra The potential interplay between neuromelanin (NM) and the loss of pigmented neurons in Parkinson's disease has prompted a significant effort to understand its structure and interactions. Although NM is commonly referred to as a “lysosome-related organelle”, it is a complex entity with melanic components bound to metals, peptides, and lipids, the structures of which are still unknown (1.Zecca L. Costi P. Mecacci C. Ito S. Terreni M. Sonnino S. Interaction of human substantia nigra neuromelanin with lipids and peptides.J. Neurochem. 2000; 74: 1758-1765Crossref PubMed Scopus (88) Google Scholar, 2.Fedorow H. Pickford R. Hook J.M. Double K.L. Halliday G.M. Gerlach M. Riederer P. Garner B. Dolichol is the major lipid component of human substantia nigra neuromelanin.J. Neurochem. 2005; 92: 990-995Crossref PubMed Scopus (56) Google Scholar, 3.Aime S. Fasano M. Bergamasco B. Lopiano L. Valente G. Evidence for a glycidic-lipidic matrix in human neuromelanin, potentially responsible for the enhanced iron sequestering ability of substantia-nigra.J. Neurochem. 1994; 62: 369-371Crossref PubMed Scopus (42) Google Scholar, 4.Zecca L. Swartz H.M. Total and paramagnetic metals in human substantia-nigra and its neuromelanin.J. Neural Transm. Park. Dis. Dement. Sect. 1993; 5: 203-213Crossref PubMed Scopus (102) Google Scholar, 5.Zecca L. Mecacci C. Seraglia R. Parati E. The chemical characterization of melanin contained in substantia-nigra of human brain.Biochim. Biophys. Acta. 1992; 1138: 6-10Crossref PubMed Scopus (88) Google Scholar, 6.Zecca L. Shima T. Stroppolo A. Goj C. Battiston G.A. Gerbasi R. Sarna T. Swartz H.M. Interaction of neuromelanin and iron in substantia nigra and other areas of human brain.Neuroscience. 1996; 73: 407-415Crossref PubMed Scopus (163) Google Scholar, 7.Tribl F. Gerlach M. Marcus K. Asan E. Tatschner T. Arzberger T. Meyer H.E. Bringmann G. Riederer P. “Subcellular proteomics” of neuromelanin granules isolated from the human brain.Mol. Cell. Proteomics. 2005; 4: 945-957Abstract Full Text Full Text PDF PubMed Scopus (86) Google Scholar). While there have been many studies examining the properties of the melanic pigment, less is known about the lipids and proteins present. Dolichol (dol) reportedly accounts for 14% of the dry weight of NM pigment (2.Fedorow H. Pickford R. Hook J.M. Double K.L. Halliday G.M. Gerlach M. Riederer P. Garner B. Dolichol is the major lipid component of human substantia nigra neuromelanin.J. Neurochem. 2005; 92: 990-995Crossref PubMed Scopus (56) Google Scholar). Yet to date, the quantification of the distribution of dol present in NM has not been reported, and it is not known whether the lipid distribution in NM is the same as that in the surrounding midbrain tissue. Determining such information could be important to developing a better understanding of potential link between NM, brain aging, and Parkinson's disease. Dolichols are polyisoprenoid α-saturated alcohols occurring in species-specific series, typically 14–22 residues in human cells. Dol are present in all cellular membranes and can accumulate in some tissues. Accumulation of dol in the brain is characteristic of ageing (8.Edlund C. Soderberg M. Kristensson K. Isoprenoids in aging and neurodegeneration.Neurochem. Int. 1994; 25: 35-38Crossref PubMed Scopus (22) Google Scholar). Increased dol levels are also observed in hepatocarcinogenesis and ceroid lipofuscinosis (9.Ericsson J. Appelkvist E.L. Runquist M. Dallner G. Biosynthesis of dolichol and cholesterol in rat-liver peroxisomes.Biochimie. 1993; 75: 167-173Crossref PubMed Scopus (25) Google Scholar). On the other hand, dol concentrations in brains of Alzheimer's disease patients are drastically decreased (8.Edlund C. Soderberg M. Kristensson K. Isoprenoids in aging and neurodegeneration.Neurochem. Int. 1994; 25: 35-38Crossref PubMed Scopus (22) Google Scholar). Roles for membrane-associated dol have been postulated; an increase in the membrane dol may alter membrane structure, fluidity, and functionality (10.Andersson M. Appelkvist E.L. Kristensson K. Dallner G. Distribution of dolichol and dolichyl phosphate in human-brain.J. Neurochem. 1987; 49: 685-691Crossref PubMed Scopus (27) Google Scholar). The significance of the separate dol pool located in the lysomal luminal compartment remains to be determined (11.Low P. Peterson E. Edlund C. Brunk U. Appelkvist E.L. Nonmembrane associated dolichol in rat-liver.Lipids. 1992; 27: 1-9Crossref PubMed Scopus (16) Google Scholar). Luminal dol is believed to be a possible source of increased dol in ageing (12.Edlund C. Soderberg M. Kristensson K. Dallner G. Ubiquinone, dolichol, and cholesterol metabolism in aging and Alzheimer's Disease.Biochem. Cell Biol. 1992; 70: 422-428Crossref PubMed Scopus (62) Google Scholar) and may be the source of dolichol seen in NM. A correlation between the amount of lipofuscin and the concentration of dol indicates the two may be related (13.Kril J.J. Pullarkat R.K. Pullarkat P.S. Harper C.G. Brain and liver dolichol in chronic-alcoholism - a necropsy study.Alcohol. Clin. Exp. Res. 1990; 14: 528-530Crossref PubMed Scopus (4) Google Scholar, 14.Pullarkat R.K. Reha H. Accumulation of dolichols in brains of elderly.J. Biol. Chem. 1982; 257: 5991-5993Abstract Full Text PDF PubMed Google Scholar). Herein, we report the characterization by mass spectrometry of dol and its oxidized derivative, dolichoic acid (dol-CA), in NM granules from human substantia nigra (SN). The concentrations of specific dol and dol-CA molecular species in NM were determined to be within about a factor of four of each other. Only very small amounts of dol-CA were detected in total pig brain lipids, requiring that the dol-CA be separated from the more abundant dol by ion exchange chromatography prior to mass spectrometry. This is the first report of dol-CA as a natural product in mammalian tissues. This study was approved by the Institutional Review Board and performed in accordance with the ethics committee of Duke University Medical Center. NM was isolated from the SN pars compacta region of the human midbrain of neurologically normal adult individuals within 48 h after death and immediately frozen at −80°C as described previously (15.Double K.L. Ben-Shachar D. Youdim M.B.H. Zecca L. Riederer P. Gerlach M. Influence of neuromelanin on oxidative pathways within the human substantia nigra.Neurotoxicol. Teratol. 2002; 24: 621-628Crossref PubMed Scopus (80) Google Scholar, 16.Zecca L. Fariello R. Riederer P. Sulzer D. Gatti A. Tampellini D. The absolute concentration of nigral neuromelanin, assayed by a new sensitive method, increases throughout the life and is dramatically decreased in Parkinson's disease.FEBS Lett. 2002; 510: 216-220Crossref PubMed Scopus (200) Google Scholar). The dissection of the SN from the frozen human midbrain was performed on a cold plate at −10°C, homogenized in distilled water (0.03 g/ml) in a glass-Teflon homogenizer, followed by centrifugation at 12,000 g for 10 min. The pellets were washed twice with 30 ml of phosphate buffer (0.05 M, pH 7.4)/g of tissue. The SN pellets were next incubated at 37°C for 3 h with 20 ml Tris buffer (0.050 M, pH 7.5) containing SDS (5 mg/ml)/g of tissue, as standard protocol for the isolation of NM granules. The suspension was centrifuged at 18,000 g for 20 min at 20°C. The supernatant was removed, and 20 ml/g of tissue of the previously described incubating solution containing 4 mg of proteinase K/g tissue was added. The sample was incubated for 3 h at 37°C. The NM pigment was separated by centrifugation at 18,000 g, and washed/centrifuged (12,000 g) twice with 5 ml of NaCl (9 mg/ml). The NM was then washed/centrifuged (12,000 g) with 5 ml of water. After dialysis against water (to remove low molecular weight compounds and remaining salts), the NM was suspended in 1 ml of methanol, sonicated for 5 min, and centrifuged (12,000 g, 30 min, 20°C). The supernatant containing the lipid component was aspirated. The precipitate was resuspended in 1 ml of hexane and centrifuged identically to the methanol fraction. The solvent extracts were combined and evaporated under nitrogen. Next, the 1.2 mg dried lipid fraction was redissolved in 200 μl chloroform. Then 25 μl was transferred and diluted to 100 μl in chloroform:dimethyl sulfoxide (DMSO) (1:1, v/v). A nor-dolichol internal standard synthesized by Avanti Polar Lipids (Alabaster, AL) with n = 13–21 (see supplementary Scheme I) was added to give a final concentration of 100 ng/μl, and 5 μl of the final mixture was injected onto a reversed-phase column for liquid chromatography-mass spectrometry (LC/MS) analysis as described below. The levels of dol species in NM granules were estimated by comparing the monoisotopic ion abundance of the naturally occurring dol with those of the nor-dol standard. A more rigorous quantitation was not pursued due to sample limitations and the lack of a stable isotope-labeled dol standard. Because there is no standard for dol-CA commercially available, the concentration of dol-CA relative to dol in NM was estimated by measuring the signal response ratio of the nor-dol-CA (Avanti Polar Lipids) (see supplementary Scheme II) to the nor-dol standards, When analyzed by LC/MS, equal weight quantities (50 ng) (about equal molar) of nor-dol and nor-dol-CA yielded [M+Ac]− to [M-H]− monoisotopic ion abundance ratio of 1.7 (see supplementary Figs. I and II for 1H-NMR of nor-dol and nor-dol-CA). A 2.5 mg dol mixture (Avanti Polar Lipids) was dissolved into 1 ml acetone. Jones reagent was prepared using 6.7 ml water, 3.35 g CrO3, and 2.9 ml H2SO4 (18 M). Water was added until no precipitate remained (approximately 2 ml). Next, 8 μl of Jones reagent were added drop-wise to the dol mixture. The liquid portion, including the lipids, was aspirated; the salt precipitate was rinsed twice with 100 μl acetone to ensure complete extraction of lipids, and the rinses were added to the original supernatant. The solution was neutralized by adding NaHCO3 until reaching pH ∼7, filtered, and centrifuged. Three ml of aqueous NaCl (saturated) were added to the solution. The lipids were then extracted three times with 3 ml portions of ethyl acetate. The organic layers were combined and dried down. Half of the final product was redissolved in 100 μl of isopropyl alcohol for LC/MS analysis. LC/MS analysis was performed using a Shimadzu LC system (comprising a solvent degasser, two LC-10A pumps and a SCL-10A system controller) coupled to a QSTAR XL high-resolution quadrupole time-of-flight mass spectrometer (Applied Biosystems, Foster City, CA). The negative ion TOF mode of Q-Star XL mass spectrometer was calibrated using PPG3000 (Applied Biosystems). LC was operated at a flow rate of 200 μl/min with a linear gradient as follows: 100% A was held isocratically for 2 min and then linearly increased to 100% B over 14 min and held at 100% B for 4 min. Mobile phase A consisted of methanol/acetonitrile/aqueous 1 mM ammonium acetate (60/20/20, v/v/v). Mobile phase B consisted of 100% ethanol containing 1 mM ammonium acetate. Five μl of the lipid sample dissolved in 50/50 chloroform/DMSO were injected onto a Zorbax SB-C8 reversed-phase column (5 μm, 2.1 × 50 mm) obtained from Agilent (Palo Alto, CA). The postcolumn split diverted ∼10% of the LC flow to the ESI source of the mass spectrometer. The anion-exchange fractionation of porcine brain lipids (Avanti Polar Lipids, Part number: 131101) was performed using an Agilent 1200 HPLC system. The DEAE column (DEAE-5PW, 10 μm, 7.5 mm × 7.5 cm) was from Sigma. The LC flow rate was 2 ml/min. Solvent A consisted of chloroform/methanol/water (2/3/1, v/v/v). Solvent B consisted of chloroform/methanol/480 mM ammonium acetate (2/3/1, v/v/v). Lipid fractionation was carried out by step-elution: after loading 100 mg porcine brain total lipids in 1.5 ml of chloroform/methanol/water (2/3/1, v/v/v) onto the DEAE column, the lipids were eluted with 16-ml steps of chloroform/methanol/aqueous ammonium acetate (2:3:1, v/v), with ammonium acetate concentrations of 0, 30, 60, 120, 240, and 480 mM successively in the aqueous component. Each fraction was then converted to a two-phase Bligh-Dyer system consisting of chloroform/methanol/water (2/2/1.8, v/v/v) by adding appropriate volumes of chloroform and water. The lower phases were dried under a stream of nitrogen. For LC/MS analysis, the dried lipids were dissolved in 100 μl chloroform, followed by adding 100 μl DMSO. Typically, 10 μl of the sample solution was injected onto a C8 reversed-phase column for LC/MS analysis as described above. Figure 1A shows the total ion chromatogram of the LC-MS of the lipid extract of NM isolated from the human SN. The major lipid species, as identified by MS/MS, include phospholipids, sulfatides, sphingolipids, as well as prenol lipids, which are eluted off the reversed-phase (C8) column in later part the gradient. Figure 1B is the averaged mass spectrum showing the NM lipid species with m/z 1180–1440 that elute from 15.4–16.8 min. The series of peaks near m/z 1236, 1304, 1372, and 1440 are interpreted as the acetate adducts [M+Ac]− of dol species with chain lengths of n = 17, 18, 19, and 20, respectively (n denotes the number of isoprene units). The peaks near m/z 1258, 1326 and 1394 represent an unknown series of polyisoprenoid derivatives, not reported in previous studies (17.Pennock J.F. Hemming F.W. Morton R.A. Dolichol: A naturally occurring isoprenoid alcohol.Nature. 1960; 186: 470-472Crossref PubMed Scopus (48) Google Scholar, 18.Behrens N.H. Leloir L.F. Dolichol monophosphate glucose: An intermediate in glucose transfer in liver.Proc. Natl. Acad. Sci. USA. 1970; 66: 153-159Crossref PubMed Scopus (178) Google Scholar, 19.Chojnacki T. Dallner G. The biological role of dolichol.Biochem. J. 1988; 251: 1-9Crossref PubMed Scopus (241) Google Scholar). To show their relative ion abundance, extracted ion chromatograms near m/z 1372 and 1326 corresponding to [Dol-19+Ac]− and the major starred peak are given in that the ion in the averaged mass spectrum of 1B not the relative abundance of dol and unknown series of polyisoprenoid To unknown species, was is the spectrum of the ion near m/z from the human NM on from and mass at m/z the structure of n = dol-CA was for the m/z species mass of the [M-H]− ion is To the dol-CA structure, we synthesized dol-CA from dol as described in the Figure is the spectrum of [M-H]− of the n = dol-CA standard. Although the mass of are within 20 the mass of the m/z ion at at is and remains to be the of the m/z ion is the of the nor-dol-CA standard (Avanti Polar Lipids, to which for the corresponding ion at m/z at at supplementary for dol-CA (n = Figure The for the naturally occurring and the standard are that the peak near m/z is the starred peaks in 1B near m/z 1258, and 1394 are to [M-H]− of a series of dol-CA species with n = 18, 19, and 20, dol and dol-CA for and of the dry weight of NM, respectively in 1 also the absolute amount of dol and with n = 14–22 isoprene present mg of NM, and the of each chain present. The dol species is n = of dol and dol-CA in NM from the SN of the human midbrain using nor-dol and nor-dol-CA as internal chain species distribution of dol from of human determined by peak at dol mg total dol-CA mg total species distribution of dol from of human determined by peak at R.K. Reha H. Accumulation of dolichols in brains of elderly.J. Biol. Chem. 1982; 257: 5991-5993Abstract Full Text PDF PubMed Google Scholar). in a new dol-CA ion were observed total lipid of brains by mass spectrometry not pig brain it remained possible that dol-CA species be present at very low levels in the total brain lipid extract and of performed anion-exchange fractionation on 1 g of porcine brain lipids (Avanti Polar Lipids) prior to LC/MS analysis. in dol-CA species are detected in the lipid fraction eluted from DEAE with 30 mM ammonium acetate as the aqueous dol in the DEAE The of dol-CA in the 30 mM fraction is with the negative associated with DEAE only dol species (n = are detected as negative acetate by LC/MS dol is a mixture of molecular species, consisting of isoprene T. Dallner G. The biological role of dolichol.Biochem. J. 1988; 251: 1-9Crossref PubMed Scopus (241) Google Scholar). with are the of the for those from the in some the have the A for understanding structure and in 5: Scopus (48) Google Scholar). The responsible for the of to dol, which isoprene using its the A for understanding structure and in 5: Scopus (48) Google Scholar). Although dol is the prenol lipid present in dol is as the B. F. The and of dolichyl phosphate and in the PubMed Scopus Google Scholar). The is then to and dol B. F. The and of dolichyl phosphate and in the PubMed Scopus Google Scholar). that can dol to B. F. The and of dolichyl phosphate and in the PubMed Scopus Google Scholar). is less abundant in is dol not is converted to important include and the for B. F. The and of dolichyl phosphate and in the PubMed Scopus Google Scholar, L. S. from to A human Chem. Int. PubMed Scopus Google Scholar). are less abundant While is the first isolation and analysis by mass spectrometry of dol-CA as a natural product from tissue, of has been using an in system G. A. of the in conversion of dolichol to in Biophys. Acta. 1993; PubMed Scopus Google Scholar). conversion of dol to dol-CA in extracts was G. A. of the in conversion of dolichol to in Biophys. Acta. 1993; PubMed Scopus Google no was showing the of in on of the of the it may be that the first in dol is the of dol to The biological of dol-CA could the system for of dolichol, dolichoic acid and acid in Biophys. Acta. PubMed Scopus Google Scholar). studies of with dol-CA that the acid is which is oxidized to of dolichol, dolichoic acid and acid in Biophys. Acta. PubMed Scopus Google Scholar). the the study that there is a for the metabolism of dol-CA it were to be naturally within tissues. The distribution of dol in NM is of it is of the dol distribution in and in the human brain (10.Andersson M. Appelkvist E.L. Kristensson K. Dallner G. Distribution of dolichol and dolichyl phosphate in human-brain.J. Neurochem. 1987; 49: 685-691Crossref PubMed Scopus (27) Google Scholar, 14.Pullarkat R.K. Reha H. Accumulation of dolichols in brains of elderly.J. Biol. Chem. 1982; 257: 5991-5993Abstract Full Text PDF PubMed Google Scholar). NM from the dol 18, 19, and 20 show relative in R.K. Reha H. Accumulation of dolichols in brains of elderly.J. Biol. Chem. 1982; 257: 5991-5993Abstract Full Text PDF PubMed Google Scholar) and by of 1.5 and respectively all of human is the species E. and Lipid Res. 2005; PubMed Scopus Google the lipid distribution in NM to be in of lengths and a distribution the distribution of dol-CA species is more 19 isoprene suggesting that the that dol-CA from dol prefer over the other molecular previous studies of the of human and dol-CA was not observed in high-resolution mass spectrometry was not to the This and analysis of total pig brain lipids that the NM granules are in dol-CA and may contain the enzyme(s) responsible for its in we were to dol-CA in porcine brain lipids only were to on an exchange to remove the major lipids, which signal of components dol-CA mass spectrometry. remains in the for dol-CA in NM and its and within the The of a in for the conversion of dol to dol-CA in brain be a first After of the the dol and and the of and Duke University for The mass spectrometry in the of at the Duke University Medical and were by the number from the of and the for for of The for the of liquid chromatography for lipid analysis, and Avanti Polar Lipids, for nor-dol-CA and nor-dol with