1-O-Alkyl-2-(ωω-oxo)acyl-sn-glycerols from Shark Oil and
`Human Milk Fat Are Potential Precursors
`of PAF Mimics and GHB
`Karsten Hartvigsena,b Amir Ravandib, Richard Harkewiczc, Hiroshi Kamidod, Klaus
`Bukhavea, Gunhild Hølmera, and Arnis Kuksisb,*
`aBiocentrum-DTU, Biochemistry and Nutrition, Centre for Advanced Food Studies, Technical University of Denmark, DK-
`2800 Lyngby, Denmark, bBanting and Best Department of Medical Research, University of Toronto, Toronto, ON M5G 1L6,
`Canada, cDepartment of Chemistry and Biochemistry, University of California, La Jolla, California 92093-0601,
`and dDepartment of Medicine, Kurume University, Kurume, Fukuoka, Japan
`
`ABSTRACT: This study examines the feasibility that peroxidation
`and lipolysis of 1-O-alkyl-2,3-diacyl-sn-glycerols (DAGE) found in
`shark liver oil and human milk fat constitutes a potential source of
`dietary precursors of platelet activating factor (PAF) mimics and of
`gamma-hydroxybutyrate (GHB). Purified DAGE were converted
`into 1-O-alkyl-2-acyl-sn-glycerols by pancreatic lipase, without
`isomerization, and transformed into 1-O-alkyl-2-oxoacyl-sn-glyc-
`erols by mild autooxidation. The various core aldehydes without
`derivatization, as well as the corresponding dinitrophenylhydra-
`zones, were characterized by chromatographic retention time and
`diagnostic ions by online electrospray mass spectrometry. Core
`aldehydes of oxidized shark liver oil yielded 23 molecular species
`of 1-O-alkyl-sn-glycerols with short-chain sn-2 oxoacyl groups,
`ranging from 4 to 13 carbons, some unsaturated. Autooxidation of
`human milk fat yielded 1-O-octadecyl-2-(9-oxo)nonanoyl-sn-glyc-
`erol, as the major core aldehyde. Because diradylglycerols with
`short fatty chains are absorbed in the intestine and react with cyti-
`dine diphosphate-choline in the enterocytes, it is concluded that
`formation of such PAF mimics as 1-O-alkyl-2-(ω-oxo)acyl-sn-glyc-
`erophosphocholine from unsaturated dietary DAGE is a realistic
`possibility. Likewise, a C4 core alcohol produced by aldol-keto re-
`duction of a C4 core aldehyde constitutes a dietary precursor of the
`neuromodulator and recreational drug GHB, which has not been
`previously pointed out.
`Paper no. L9953 in Lipids 41, 679–693 (July 2006).
`
`Platelet-activating factor (PAF), 1-O-alkyl-2-acetyl-sn-glyc-
`erophosphocholine (GroPCho), is a biologically active phospho-
`
`*To whom correspondence should be addressed at Banting and Best Depart-
`ment of Medical Research, University of Toronto, 112 College Street,
`Toronto, ON M5G 1L6, Canada. E-mail: arnis.kuksis@utoronto.ca
`Present address of first author: Department of Medicine, University of Cali-
`fornia, La Jolla, CA 92093-0682. Present address of fourth author: Midori
`Health Care Foundation, 3-22-5 Tarumi-cho, Suita, Osaka 564-0062, Japan.
`Present address of fifth author: Department of Human Nutrition, The Royal
`Veterinary and Agricultural University, DK-1958, Frederiksberg, Denmark.
`Abbreviations: CapEx, capillary exit; CDP, cytidine diphosphate; DAGE, 1-
`O-alkyl-2,3-diacyl-sn-glycerol; DNPH, 2,4-dinitrophenylhydrazine; GE, 1-
`O-alkyl-sn-glycerol; GHB, gamma-hydroxybutyric acid; GroPCho, glyc-
`erophosphocholine; LC/ESI-MS, LC/electrospray ionization/MS; 2-MAGE,
`1-O-alkyl-2-acyl-sn-glycerol; 3-MAGE, 1-O-alkyl-3-acyl-sn-glycerol; PAF,
`platelet-activating factor; PtdCho, phosphatidylcholines (PtdCho);Rf, rela-
`tive retention factor; RT, retention time.
`
`lipid with diverse physiological and pathological effects in a va-
`riety of cells and tissues (1). Gamma-hydroxybutyric acid (GHB)
`is a simple four-carbon FA with an extraordinary range of physi-
`ological and pharmacological effects (2). PAF is known to be en-
`zymatically synthesized by either the remodeling or the de novo
`pathways. PAF mimics, however, are generated by secondary
`peroxidation of unsaturated 1,2-diacyl-sn-GroPCho in cell mem-
`branes, which retain a short-chain residue esterified at the sn-2
`position (3). This short-chain residue may contain either a ω-
`methyl, ω-aldehyde, ω-alcohol, or ω-carboxyl group for PAF-
`like activity. Investigations of the biological activities by multi-
`ple assays have shown that PAF-like lipids containing an sn-1
`alkyl ether linkage are more effective than the corresponding sn-
`1 acyl derivatives, and that, in general, the shorter the sn-2 chain
`residue the more active the PAF mimic (4,5). Although it has
`been suggested (2) that GHB may also arise via lipid peroxida-
`tion, the exact mechanism has not been established.
`In the present report, we demonstrate the feasibility of meta-
`bolic transformation of 1-O-alkyl-2,3-diacyl-sn-glycerols
`(DAGE) from shark liver oil and human milk into the corre-
`sponding core aldehydes, 1-O-alkyl-2-(ω-oxo)acyl-sn-glycerols,
`by mild autooxidation and lipolysis. We have previously shown
`that short-chain 1,2-diradyl-sn-glycerols are absorbed intact in
`the intestine (6,7) and that exogenous 1,2-diradyl-sn-glycerols
`are incorporated intact into the phosphatidylcholines (PtdCho)
`(8). We have shown elsewhere (9) that such PAF mimics pre-
`pared synthetically induce platelet aggregation and inhibit en-
`dothelium-dependent arterial relaxation. We postulate that the
`C4 core aldehydes, either as glycerolipids or glycerophospho-
`lipids, are reduced to the corresponding C4 core alcohols by en-
`dogenous aldol-keto reductases (10) before release into circula-
`tion as GHB. There has been no previous work on the core alde-
`hydes arising from oxidation of alkyldiacylglycerols, although
`the nonvolatile oxidation products of triacylglycerols have been
`previously discussed (11–16).
`
`EXPERIMENTAL PROCEDURES
`
`Materials. Crude deep-sea shark liver oil was a gift from Baldur
`Hjaltason, LYSI Ltd., Reykjavik, Iceland. The lyophilized
`
`Copyright © 2006 by AOCS Press
`
`679
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`680
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`K. HARTVIGSEN ET AL.
`
`human milk sample was a gift from Dr. J. Cerbulis of the East-
`ern Regional Research Center, USDA, Philadelphia, PA. It was
`one of six milk samples obtained from nursing mothers in the
`Philadelphia area and used in a collaborative investigation of
`chloropropanediol diesters in human milk samples (17). The
`alkyldiacylglycerol composition of these samples ranged from
`0.5 to 5 mol%. Linoleic acid, 4-dimethylaminopyridine, and
`N,N′-dicyclohexylcarbodiimide were obtained from Sigma-
`Aldrich (St. Louis, MO). 1-O-Octadecyl-sn-glycerol was ob-
`tained from Fluka (Ronkonkoma, NY). All solvents used were
`of analytical or HPLC grade.
`Preparative TLC. Several preparative TLC systems were
`employed to purify the various transformation products. All
`TLC plates were prepared in the laboratory (200 × 200 × 0.25
`mm) and activated for 2 h at 110°C before use. System A con-
`sisted of silica gel H, developed in hexane/diethyl ether (90:10,
`vol/vol). System B consisted of silica gel G containing 5%
`boric acid, developed in hexane/isopropyl ether/acetic acid
`(50:50:4, by vol). System C consisted of silica gel H, devel-
`oped in hexane/diethyl ether/acetic acid (80:20:2, by vol).
`Lipids were visualized under UV light after spraying with 0.2%
`2,7-dichlorofluorescein in ethanol (18), whereas the core alde-
`hydes were visualized as purple areas after spraying with the
`Schiff base reagent (19). Migration of a component is given as
`the relative retention factor (Rf). Lipids and core aldehydes
`were recovered from the TLC plates by scraping off the gel,
`extracting it with chloroform/methanol (2:1, vol/vol), washing
`with water, drying over anhydrous sodium sulfate, evaporating
`under nitrogen, and dissolving in chloroform/methanol (2:1,
`vol/vol). The 2,7-dichlorofluorescein was removed with 1%
`ammonium hydroxide.
`GLC. Injections were made at 100°C, and after 30 s the oven
`temperature was programmed at 20°C/min to either 130°C
`(FAME) or 180°C (diacetyl-derivatized 1-O-alkyl-sn-glycerols
`(GE)), and then to 240°C at 5°C/min (18). The GLC system
`consisted of a polar capillary column (SP 2380, 15 m × 0.32
`mm i.d., Supelco, Mississauga, ON) installed in a Hewlett-
`Packard (Palo Alto, CA) Model 5880 gas chromatograph
`equipped with a flame ionization detector. Hydrogen was used
`as carrier gas at 3 psi. FAME and diacetyl-GE were identified
`on the basis of retention times (RT) compared with commer-
`cially available external reference compounds.
`HPLC. Reversed-phase HPLC was performed with a
`Hewlett-Packard Model 1090 liquid chromatograph (Palo Alto,
`CA) using an HP ODS Hypersil C18 column (5 µm; 200 × 2.1
`mm i.d.; Hewlett-Packard, Palo Alto, CA) and eluted isocrati-
`cally with 100% Solvent A (methanol/water/30% ammonium
`hydroxide, 88:12:0.5, by vol) for 3 min, followed by a linear
`gradient to 100% Solvent B (methanol/hexane/30% ammo-
`nium hydroxide, 88:12:0.5, by vol) in 25 min, which was kept
`for another 6 min (20). Kim et al. (20) washed the HPLC col-
`umn with 0.1 M ammonium acetate at 0.5 mL/min for 5 min at
`the end of each run and did not observe any ill effects on the
`performance of column or the quality of the mass spectra.
`When 2,4-dinitrophenylhydrazine (DNPH) derivatives were
`analyzed, the effluent was led through a UV detector (358 nm)
`
`Lipids, Vol. 41, no. 7 (2006)
`
`installed before the mass spectrometer. The flow was 0.4
`mL/min.
`Electrospray ionization MS (ESI-MS). Reversed-phase
`HPLC with online electrospray ionization MS (LC/ESI-MS)
`was performed by admitting the entire HPLC column effluent
`into a Hewlett-Packard Model 5988B quadrupole mass spec-
`trometer (Palo Alto, CA) equipped with a nebulizer-assisted
`electrospray interface (Hewlett-Packard Model 59987A, Palo
`Alto, CA) as previously described (19). Nitrogen was used as
`both nebulizing (60 psi) and drying gas (60 psi, 270°C). Capil-
`lary voltage was set at 4 kV, the endplate voltage was 3.5 kV,
`and the cylinder voltage was 5 kV in the positive mode of ion-
`ization. In the negative mode, the values were −3.5 kV, −3 kV,
`and −3.5 kV, respectively. Both negative and positive ESI spec-
`tra were taken in the mass range 300–1100 amu. The capillary
`exit (CapEx) was set at 120 and −120 V in the positive and neg-
`ative ion mode, respectively.
`Preparation of DAGE from shark liver oil and human milk.
`A total lipid extract of freeze-dried human milk was prepared
`as previously described (21). DAGE was recovered from the
`human milk lipid extract and the shark liver oil by preparative
`double one-dimensional TLC (system A). The purified DAGE
`was subjected to regiospecific analysis to reveal the sn-1-O-
`alkyl-, sn-2 acyl-, and sn-3 acyl-chain composition and distrib-
`ution. The complete procedure is outlined in Scheme 1.
`Hydrolysis with pancreatic lipase and Grignard degrada-
`tion. Purified DAGE of shark liver oil and human milk were
`hydrolyzed by digestion with diethyl ether pre-extracted pan-
`creatic lipase (22). The digestion was performed in the pres-
`ence of gum arabic for 30 min, and the digestion products were
`extracted with diethyl ether. Alternatively, the purified DAGE
`were deacylated by Grignard degradation (23) in order to ver-
`ify the results obtained from pancreatic lipase digestion. The
`degradation products were resolved and recovered by TLC
`(system B).
`Preparation of FAME and diacetyl-GE. Purified fractions of
`DAGE, 2-MAGE, and 3-MAGE originating from shark liver
`oil were treated with 6% H2SO4 in methanol for 2 h at 80°C to
`produce FAME and GE. After the reaction, the lipids were ex-
`tracted twice with chloroform. GE and FAME were resolved
`and recovered by preparative TLC (system C). Purified GE was
`derivatized to diacetyl-GE for 30 min at 80°C with acetic an-
`hydride/pyridine (1:1, vol/vol; 75 µL). The profiles of FAME
`and diacetyl-GE were determined by GLC.
`Autooxidation of 2-MAGE. Mild peroxidation was per-
`formed by flushing the purified 2-MAGE from either shark
`liver oil or human milk in a tube with oxygen, capping, and
`heating at 80°C for 3 h. The peroxidized 2-MAGE was ana-
`lyzed by reversed-phase LC/ESI-MS.
`Preparation of DNPH derivatives. Aldehyde preparations
`of oxidized 2-MAGE were derivatized by reaction with DNPH
`in the dark (0.5 mg in 1 mL 1 N HCl) for 2 h at room tempera-
`ture and 1 h at 4°C (24). The DNPH derivatives were extracted
`with chloroform/methanol (2:1, vol/vol), dried over anhydrous
`sodium sulfate, evaporated under a stream of nitrogen, dis-
`solved in chloroform/methanol (2:1, vol/vol), and analyzed by
`
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`PRECURSORS OF PAF MIMICS AND GHB
`
`681
`
`SCHEME 1. Flow sheet for the regiospecific analysis of DAGE from shark liver oil and the procedure for isolation
`and characterization of core aldehydes from oxidized and digested DAGE.
`
`reversed-phase LC/ESI-MS. Derivatization with DNPH, there-
`fore, increased the detection limit for the core aldehydes by
`MS, and further provided additional ions for characterization
`of the alkyl ether core aldehydes, as well as an opportunity to
`monitor the components by UV detection at 358 nm.
`Preparation of
`reference 1-O-octadecyl-2-(9-oxo)-
`nonanoyl-sn-glycerol. The esterfication of 1-O-octadecyl-sn-
`glycerol with linoleic acid was performed by the carbodiimide-
`mediated process (25). Linoleic acid (75 µmol), 1-O-octadecyl-
`sn-glycerol (100 µmol), and 4-dimethyl-aminopyridine (10
`µmol) were dissolved in dry n-hexane. This solution was added
`to a suspension of N,N′-dicyclohexylcarbodiimide (100 µmol)
`in dry n-hexane and shaken vigorously for 17 h at room tem-
`perature. After filtration, solvent was evaporated under nitro-
`gen, and the residue was purified by preparative TLC (system
`B). We have previously reported the LC/ESI-MS analysis of
`this and other related synthetic neutral ether lipids (26).
`The synthesized and purified 1-O-octadecyl-2-octadeca-
`dienoyl-sn-glycerol was subjected to triphenylphosphine re-
`ductive ozonization as previously described (19). The resulting
`reference core aldehyde, 1-O-octadecyl-2-(9-oxo)nonanoyl-sn-
`
`glycerol (Rf = 0.11), was purified by preparative TLC (system
`B) and analyzed by reversed-phase LC/ESI-MS, and its iden-
`tity was established on basis of RT, averaged mass spectrum,
`and fragmentation pattern.
`
`RESULTS
`
`Isolation of DAGE. Preparative TLC resolved the crude shark
`liver oil into six bands, which corresponded to monoacyl (mono-
`radyl) glycerols (Rf = 0.01), free cholesterol (Rf = 0.13), triacyl-
`glycerol (Rf =0.37), DAGE (Rf = 0.55), cholesteryl esters (Rf =
`0.93), and squalene (Rf = 0.97). The TLC bands were scraped
`off the plate and analyzed by high-temperature GLC, which in-
`dicated that the DAGE made up 55% of the shark liver oil. Re-
`versed-phase LC/ESI-MS indicated that DAGE was composed
`of at least 50 species eluting between 10 and 37 min (chromato-
`gram not shown). Similarly, the DAGE content of human milk
`was estimated to be approximately 1% of total fat and was made
`up of numerous species, of which only a few were abundant.
`Regiospecific analysis of DAGE (26). The purified DAGE
`were subjected to a regiospecific analysis to reveal the sn-1-O-
`
`Lipids, Vol. 41, no. 7 (2006)
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`AKER EXHIBIT 2010 Page 3
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`

`682
`
`K. HARTVIGSEN ET AL.
`
`alkyl, sn-2-acyl, and sn-3-acyl chain composition and distribu-
`tion, which were determined by TLC (system B) and GLC, fol-
`lowing pancreatic lipase hydrolysis and Grignard degradation.
`Pancreatic lipase hydrolysis of the DAGE yielded 1-O-
`alkyl-2-acyl-sn-glycerols (2-MAGE, Rf = 0.32) as the major
`product (97%) and 1-O-alkyl-3-acyl-sn-glycerol (3-MAGE, Rf
`= 0.41) as the minor product (3%), along with GE (Rf = 0.05),
`free FA (Rf = 0.66), and original DAGE (Rf = 0.90).
`The high recovery of 2-MAGE compared with 3-MAGE is
`consistent with the resistance of the sn-1 ether linkage to the
`action of most enzymes, and furthermore indicates a very low
`rate of isomerization and a low affinity of the pancreatic lipase
`for the sn-2-position of DAGE. The non-selective Grignard
`degradation yielded the 2-MAGE and 3-MAGE in equal
`amounts together with GE and the free FA as the tertiary alco-
`hols (Grignard reaction products). The DAGE, 2-MAGE, and
`3-MAGE fractions recovered from the pancreatic lipase diges-
`tion and Grignard degradation were treated with sulfuric
`acid/methanol to produce FAME and glyceryl ethers (GE),
`which were resolved by preparative TLC (system C). The puri-
`fied GE were converted into the diacetyl GE by reaction with
`acetic anhydride and pyridine, and the FAME and the GE ac-
`etates were identified and quantified by GLC. Reversed-phase
`LC/ESI-MS analysis of 2-MAGE isolated from the shark liver
`oil following pancreatic lipolysis of DAGE showed a total of
`49 species, of which 20 species were abundant, eluting between
`10 and 26 min (chromatogram not shown).
`Table 1 gives the regiospecific distribution of the fatty
`
`chains of shark liver oil DAGE as determined by GLC analysis
`of products of pancreatic lipolysis and Grignard degradation.
`The predominant sn-1-O-alkyl fatty chains were the monoun-
`saturated alcohols (18:1n-9, 54.9%, and 16:1n-7, 12.2%) and
`saturated alcohols (16:0, 11.2%), with much smaller amounts
`of a diunsaturated alcohol (18:2n-6, 1%). Small amounts of
`odd-carbon saturated and monounsaturated fatty alcohols were
`also detected. Pancreatic lipase digestion and Grignard degra-
`dation gave similar FA profiles and selectivity for the sn-2- and
`sn-3-positions. The most abundant sn-2-FA were 16:0, 16:1n-
`7, 18:1n-9, 20:1n-9, 22:1n-11/13, 22:5n-3, and 22:6n-3, with
`18:1n-9 accounting for more than 50% of the total. The most
`abundant sn-3-FA were 16:0, 16:1n-7, 18:0, 18:1n-9, 20:1n-9,
`22:1n-11/13, and 22:4n-3. The FA 18:1n-9, 22:5n-3, and 22:6n-
`3 were preferentially associated with the sn-2-position, whereas
`18:0, 20:1n-9, 22:1n-11/13, and 22:4n-3 were mostly in the sn-
`3-position.
`LC/ESI-MS characterization of reference core aldehydes.
`The identities of all synthetic neutral ether lipids were estab-
`lished by combined TLC and LC/ESI-MS analysis. The 1-O-
`octadecyl-2-(9-oxo)nonanoyl-sn-glycerol was produced in
`high yield and purity by reductive ozonization of 1-O-octa-
`decyl-2-(9-cis,12-cis)-octadecadienoyl-sn-glycerol. Figure 1A
`shows the total LC/ESI-MS positive ion current profile (CapEx
`+120 V) of synthetic 1-O-octadecyl-2-(9-oxo)nonanoyl-sn-
`glycerol (RT = 14.8 min). Figure 1B shows the full mass spec-
`trum averaged over the entire peak with eight major ions being
`observed. The assignments for the observed ions, their corre-
`
`TABLE 1
`Regiospecific Distribution (mol%) of FA of Shark Liver Oil DAGE as Determined
`by GLC-FID After Pancreatic Lipase Hydrolysis or Grignard Degradationa
`sn-2 Acyl
`Grignard
`nd
`1.4
`0.3
`18.8
`4.9
`0.7
`nd
`nd
`1.7
`48.9
`0.3
`0.8
`0.4
`0.2
`nd
`5.5
`0.2
`0.1
`0.3
`3.4
`0.2
`1.4
`3.3
`0.9
`
`Lipase
`sn-1 Alkyl
`Fatty chain
`nd
`1.0
`12:0
`1.0
`2.3
`14:0
`0.3
`0.4
`15:0
`18.4
`11.2
`16:0
`4.7
`12.2
`16:1n-7
`0.7
`nd
`16:2n-4
`nd
`0.6
`17:0
`nd
`2.2
`17:1
`1.2
`2.9
`18:0
`51.8
`54.9
`18:1n-9
`nd
`4.6
`18:1n-7
`0.8
`1.0
`18:2n-6
`0.3
`nd
`18:3n-3
`0.2
`nd
`18:4n-3
`nd
`0.6
`19:0
`6.1
`2.6
`20:1n-9
`0.1
`nd
`20:2n-6
`nd
`nd
`20:4n-6
`0.1
`nd
`20:5n-3
`3.7
`0.2
`22:1n-11/13
`0.2
`nd
`22:4n-3
`1.6
`nd
`22:5n-3
`4.1
`nd
`22:6n-3
`0.7
`nd
`24:1n-3
`aAverage of two determinations. nd, not detected.
`
`sn-3 Acyl
`Lipase
`Grignard
`nd
`nd
`0.7
`0.8
`0.1
`0.2
`15.9
`19.5
`3.2
`3.2
`0.9
`1.0
`nd
`nd
`nd
`nd
`4.6
`3.7
`21.3
`16.9
`5.8
`6.6
`0.7
`0.2
`0.4
`0.3
`0.2
`0.1
`nd
`nd
`11.1
`12.4
`0.3
`0.3
`nd
`nd
`0.5
`0.2
`18.0
`21.0
`5.9
`7.4
`0.5
`0.4
`1.5
`0.8
`0.3
`0.2
`
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`PRECURSORS OF PAF MIMICS AND GHB
`
`683
`
`FIG. 1. Reversed-phase LC/ESI-MS analysis of synthetic 1-O-octadecyl-2-(9-oxo)nonanoyl-sn-
`glycerol. (A) Total positive ion current profile. (B) Full mass spectrum averaged over the entire
`peak, zoomed to m/z 450–600, in (A). All of the ions detected in the spectrum were assigned
`to the original reference compound. Note the high abundance for the m/z 499 ion relative to
`m/z 498, suggesting that the m/z 499 ion is not entirely composed of the 13C isotopic ion of
`m/z 498. The LC/ESI-MS analysis showed that the optimal diagnostic ion of the 1-O-alkyl-2-
`(ω-oxo)acyl-sn-glycerols is the [M – 17]+ ion, which in (B) corresponds to m/z 481. Additional
`details are given in Table 2, Scheme 2, and the text.
`
`sponding 13C isotopic components, and their relative abun-
`dances are presented in Table 2. The proposed molecular struc-
`tures for these assigned ions are shown in Scheme 2. Sodium
`and ammonium adducts are common features of ESI, and it is
`also known that aldehydes in methanol solutions, as encoun-
`tered in the mobile phase, are converted to the corresponding
`neutral hemi-acetal form (a methanol adduct).
`The presence of an ion at m/z 498.70 corresponding to the
`[M]+ ion was unexpected and only observed in the samples with
`the underivatized monoalkylglycerols containing a free aldehyde
`ester group. We confirmed this observation with the correspond-
`ing 3-isomer reference compound, 1-O-octadecyl-3-(9-
`oxo)nonanoyl-sn-glycerol (data not shown). It would be ex-
`pected that the observed ion would have m/z 499.75 correspond-
`ing to the protonated molecular ion [M + H]+. As indicated in
`Table 2 and Scheme 2, we propose that the [M]+ ion at m/z
`
`498.70 arises from a dehydrated ammonium adduct, [M + NH4
`− H2O]+, and not directly from the ionization as a radical cation,
`[M]+·. Another unexpected observation was the relatively high
`abundance (59%) of the m/z 499.75 ion compared with the m/z
`498.70 ion, which is much higher than the calculated contribu-
`tion of the 13C isotope (32%) for this compound. The other ions
`have the 13C contribution showing the expected ~32% relative
`abundance (Table 2). This suggests that the peak at m/z 499.75 is
`actually composed of two different species, [(M + 13C1) + NH4
`– H2O]+ and the protonated molecular ion, [M + H]+. As indi-
`cated in Table 2 and Scheme 2, we propose that the [M + H]+ ion
`can also arise indirectly from the neutral loss of ammonia from
`the molecular ammonium adduct, [M + NH4 – NH3]+.
`Due to the lengthy and complicated ion assignment and the
`multiple possible pathways of ion formation, we have decided
`to describe the ions as mass difference from the exact mass of
`the 1-O-alkyl-2-(ω-oxo)-sn-glycerol, that is, [M ± X]+.
`
`Lipids, Vol. 41, no. 7 (2006)
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`

`684
`
`K. HARTVIGSEN ET AL.
`
`TABLE 2
`Mass Spectrometric Analysis of Synthetic 1-O-Octadecyl-2-(9-Oxo)Nonanoyl-sn-Glycerol: Assignment
`of Detected Ions Including Corresponding 13C Isotopic Ions and Their Relative Abundancea
`
`Ion ID
`[M – 17]+
`
`[M]+
`
`[M + 1]+
`
`[M + 18]+
`
`[M + 23]+
`
`[M + 32]+
`
`[M + 50]+
`
`Ion (m/z)
`(Rel. Ab.)b
`481.65
`(100%)
`
`13C Ion (m/z)
`+1
`
`498.70
`(80%)
`
`499.75
`(21%)d
`
`516.70
`(9%)
`
`521.70
`(24%)
`
`530.70
`(2%)
`
`548.70
`(9%)
`
`+1
`
`+1
`
`+1
`
`+1
`
`(Rel. Ab.)
`482.65
`(33%)
`
`499.75
`(32%)c
`
`500.75
`(24%)e
`
`522.70
`(33%)
`
`549.80
`(31%)
`
`Ion assignment
`[M + H – H2O]+ and/or
`[M + NH4 – NH3 – H2O]+
`
`[M + NH4 – H2O]+
`
`[M + H]+ and/or [M + NH4 – NH3]+
`
`[M + NH4]+
`
`[M + Na]+
`
`[M + CH3OH + NH4 – H2O]+
`
`[M + CH3OH + NH4]+
`
`[M + 55]+
`
`+1
`
`553.85
`(91%)
`aAverage of four mass spectra.
`bRel. Ab., relative ion abundance.
`cCalculated Rel. Ab. based on expected 13C contribution and the average of the other detected 13C isotopic ions.
`dCalculated Rel. Ab. as the total ion abundance for m/z 499.75 subtracted the contribution from the 13C isotopic ion of m/z
`498.70; calculated Rel. Ab. based on the calculated ion abundance. Additional details are given in Figure 1, Scheme 2,
`and the text.
`eCalculated Rel. Ab. based on the calculated ion abundance for m/z 499.75 [M+1]+, see d.
`
`554.75
`(31%)
`
`[M + CH3OH + Na]+
`
`SCHEME 2. Proposed ionization and formation of various ions in the spectrum of synthetic 1-O-octadecyl-2-(9-
`oxo)nonanoyl-sn-glycerol. Additional details are given in Figure 1, Table 2, and the text.
`
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`

`PRECURSORS OF PAF MIMICS AND GHB
`
`685
`
`FIG. 2. Reversed-phase LC/ESI-MS analysis of synthetic and DNPH derivatized 1-O-octadecyl-
`2-(9-oxo)nonanoyl-sn-glycerol. (A) Total negative ion current profile. (B) Reconstructed single-
`ion chromatogram of the m/z 677 [M – 1]− ion. (C) Full mass spectrum averaged over the en-
`tire peak, zoomed to m/z 300–800, in (A). The diagnostic ion of 1-O-alkyl-2-(DNPH-oxo)acyl-
`sn-glycerols is thus the [M – 1]− ion.
`
`The best diagnostic ion of 1-O-alkyl-2-oxoacyl-sn-glycerols
`under our experimental LC/ESI-MS conditions was the [M −
`17]+ ion at m/z 481.65, which we propose arises from the de-
`hydration of the m/z 498.70 [M + 1]+ ion (Fig. 1B, Table 2, and
`Scheme 2).
`Further identification of 1-O-octadecyl-2-(9-oxo)nonanoyl-
`sn-glycerol was performed by derivatization with DNPH. Fig-
`ure 2 shows the total LC/ESI-MS negative ion current profile
`with CapEx at −120 V of the DNPH-derivatized 1-O-octa-
`decyl-2-(9-oxo)nonanoyl-sn-glycerol (RT 19.0 min, Fig. 2A)
`along with the reconstructed single-ion chromatogram (Fig.
`2B) for the [M – 1]− ion, and the full mass spectrum (Fig. 2C)
`averaged over the entire peak. As demonstrated in Figure 2C,
`1-O-octadecyl-2-(DNPH-9-oxo)nonanoyl-sn-glycerol was
`characterized by the deprotonated molecular ion, [M − 1]− at
`m/z 677, which was used as the diagnostic ion.
`
`Characterization of 2-MAGE core aldehydes in autooxi-
`dized shark liver oil. Figure 3 shows the total positive ion cur-
`rent profile of the 1-O-alkyl-2-acyl-sn-glycerols before autoox-
`idation (Fig. 3A) and after 3 h autooxidation (Fig. 3B) of shark
`liver oil 2-MAGE. The resulting ether core aldehydes (i.e., 1-
`O-alkyl-2-(ω-oxo)acyl-sn-glycerols) eluted with retention
`times ranging from 9 to 15 min, the hydroperoxides (i.e., 1-O-
`alkyl-2-(hydroperoxy)acyl-sn-glycerols) and other oxidation
`products with retention times ranging from 5 to 25 min, and the
`non-oxidized 1-O-alkyl-2-acyl-sn-glycerols with retention
`times from 25 to 35 min. The core aldehydes made up about
`5%, and the other oxidation products about 40%, of the total
`peak area (Fig. 3B). Figure 3 also shows the reconstructed sin-
`gle-ion chromatograms for the [M − 17]+ ion of 1-O-octade-
`cenyl-2-(4-oxo)butyroyl-sn-glycerol (Fig. 3C) and 1-O-octade-
`cenyl-2-(9-oxo)nonanoyl-sn-glycerol (Fig. 3D). Several 1-O-
`
`Lipids, Vol. 41, no. 7 (2006)
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`AKER EXHIBIT 2010 Page 7
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`

`686
`
`K. HARTVIGSEN ET AL.
`
`FIG. 3. Reversed-phase LC/ESI-MS analysis of 1-O-alkyl-2-acyl-sn-glycerols isolated from shark
`liver oil before and following oxidation. (A) and (B) Total positive ion current profiles of non-
`oxidized and autooxidized (80°C for 3 h in oxygen atmosphere) 1-O-alkyl-2-acyl-sn-glycerols,
`respectively. (C) and (D) reconstructed single-ion chromatograms of the m/z 409 and 479 [M −
`17]+ diagnostic ion of 1-O-octadecenyl-2-(4-oxo)butyroyl-sn-glycerol and 1-O-octadecenyl-2-
`(9-oxo)nonanoyl-sn-glycerol, respectively.
`
`alkyl-2-oxoacyl-sn-glycerols were identified on the basis of the
`[M − 17]+ and [M]+ ions (Table 3). The major ether core alde-
`hydes corresponded to 18:1-4:0Ald (20%), 16:1-9:0Ald (18:1-
`7:0Ald) (10%), 16:0-9:0Ald (2%), 16:0-10:1 (17:1-9:0Ald)
`(2%), 18:1-9:0Ald (20%), and 18:1-10:1Ald (7%).
`Figure 4 shows the total LC/ESI-MS negative ion current pro-
`file (Fig. 4A) of DNPH-derivatized and purified 1-O-alkyl-2-
`oxoacyl-sn-glycerol, derived from shark liver oil DAGE, along
`with selected, reconstructed single-ion mass chromatograms for
`the [M – 1]− ion of 1-O-hexadecenyl-2-(DNPH-4-oxo)butyroyl-
`
`sn-glycerol (Fig. 4B), 1-O-hexadecyl-2-(DNPH-4-oxo)butyroyl-
`sn-glycerol (Fig. 4C), 1-O-octadecenyl-2-(DNPH-4-oxo)buty-
`royl-sn-glycerol (Fig. 4D), 1-O-octadecenyl-2-(DNPH-7-
`oxo)heptanoyl-sn-glycerol (Fig. 4E), 1-O-octadecenyl-2-
`(DNPH-9-oxo)nonanoyl-sn-glycerol (Fig. 4F), and 1-O-octa-
`decyl-2-(DNPH-9-oxo)nonanoyl-sn-glycerol (Fig. 4G), which
`all were eluted over the time period of 10 to 20 min.
`Table 4 lists the identified molecular species along with the
`uncorrected peak areas attributed to them. The [M − 1]− ion
`provides only the molecular weight of the compound. Further
`
`Lipids, Vol. 41, no. 7 (2006)
`
`AKER EXHIBIT 2010 Page 8
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`

`PRECURSORS OF PAF MIMICS AND GHB
`
`687
`
`TABLE 3
`Identification of Molecular Species of 1-O-Alkyl-2-Oxoacyl-sn-Glycerols,
`Produced by Autooxidation of Pancreatic Lipase Treated Shark Liver Oil
`DAGE and Estimated by LC/ESI-MSa
`
`1-O-Alkyl-2-oxoacyl-sn-glycerol
`[M]+
`[M – 17]+
`(m/z)
`(m/z)
`426
`409
`
`RT
`(min)
`9.2
`
`Molecular species
`Carbon
`number
`22:1 Ald
`
`25:1 Ald
`
`25:0 Ald
`
`26:1 Ald
`
`Tentative
`identity
`18:1-4:0 Ald
`
`16:1-9:0 Ald
`18:1-7:0 Ald
`
`16:0-9:0 Ald
`
`16:0-10:1 Ald
`17:1-9:0 Ald
`
`27:1 Ald
`
`18:1-9:0 Ald
`
`11.0
`
`11.3
`
`11.7
`
`12.6
`
`nd
`
`nd
`
`nd
`
`496
`
`508
`
`451
`
`453
`
`465
`
`479
`
`491
`
`13.2
`18:1-10:1 Ald
`28:2 Ald
`aTwo analyses. RT, retention time; Ald, aldehyde; nd, not detected.
`
`confirmation of the structure was obtained from the linear cor-
`relation (R = 0.982) obtained between retention time and mo-
`lecular mass among the 1-O-alkyl-2-DNPH-oxoacyl-sn-glyc-
`erols, when the latter was calculated as the total carbon number
`minus one carbon per double bond (Table 4). This elution fac-
`tor makes it possible to calculate the relative retention times of
`unknowns with considerable accuracy, which helped to choose
`among likely structures represented by the same molecular
`mass within the reversed-phase LC/ESI-MS profile. Further
`characterization of the molecular species was obtained from
`knowledge of the possible formation of esterified aldehyde
`residues and the ratios of the corresponding sn-1 and sn-2 fatty
`chain moieties (Tables 1 and 4). For example, the masses at m/z
`577 and 579 ([M – 1]− ions) can only represent the 1-O-hexa-
`decenyl- and 1-O-hexadecyl-2-(DNPH-4-oxo)butyroyl-sn-
`glycerols (Figs. 4B and 4C), respectively. The ion at m/z 605
`[M – 1]−, however, could represent both the 1-O-octadecenyl-
`2-(DNPH-4-oxo)butyroyl-sn-glycerol and the 1-O-pentadecyl-
`2-(DNPH-7-oxo)heptanoyl-sn-glycerols (Table 4 and Fig. 4D),
`but on the basis of the much higher abundance of sn-1 alkyl
`18:1n-9 in comparison with 15:0 (Table 1), it is obvious that
`the peak area mainly represents the 1-O-octadecenyl-2-
`(DNPH-4-oxo)butyroyl-sn-glycerol. The alternative identities
`of the species are written in parentheses in Table 4.
`Characterization of 2-MAGE core aldehydes in autooxidized
`human milk. The small DAGE fraction isolated from human milk
`fat yielded a 1-O-hexadecylglycerol and oleic acid from the sn-2-
`and sn-3-positions as the major fatty chains. Figure 5 shows the
`total LC/ESI-MS negative ion current profile (Fig. 5A) of DNPH-
`derivatized 1-O-alkyl-2-oxoacyl-sn-glycerols, derived from
`human milk DAGE, along with the reconstructed single-ion mass
`chromatogram (Fig. 5B) for the [M − 1]− ion of 1-O-octadecyl-2-
`(DNPH-9-oxo)nonanoyl-sn-glycerol (RT = 18.8 min), and the
`full mass spectrum (Fig. 5C) averaged over the entire peak. This
`was the only ether core aldehyde characterized from human milk.
`
`DISCUSSION
`
`The major source of PAF-like lipids (or mimics/analogues) is
`the unregulated oxidative modification of cellular and plasma
`phospholipids. There have been numerous reports on isolation
`of the PAF-like lipids containing a long-chain fatty ester in the
`sn-1-position and a short-chain (ω-oxo)acyl group in the sn-2-
`position of PtdCho (27). The immediate precursors of these
`mimics are the 1,2-diacyl- and 1-O-alkyl-2-acyl-sn-GroPCho
`with saturated acyl and alkyl chains in the sn-1-position.
`Among these, the most frequently reported are 1-hexade-
`canoyl- and 1-octadecanoyl-2-(5-oxo)pentanoyl-sn-GroPCho,
`which are generated from the corresponding arachidonates
`(27). The corresponding sn-1 alkyl ether derivative, which is
`structurally more closely related to PAF, has been studied less
`frequently due to the more limited supply of potential precur-
`sors, although it was the first PAF analogue identified (27) and
`both DAGE and 1-O-alkyl-2-acyl-sn-glycerophospholipids are
`present in significant amounts of tissue lipids (28). The present
`study demonstrates the feasibility of generating PAF analogue
`precursors in the intestine by lipolysis and peroxidation of the
`DAGE, which constitute a significant proportion of dietary fats
`such as shark liver oil and milk fat. In addition, this study has
`recognized certain unusual features of 1-O-alkyl-2-(ω-
`oxo)acyl-sn-glycerols as precursors of PAF analogues. The 1,2-
`diradyl-sn-glycerol moieties generated from DAGE of shark
`liver oil were characterized by the presence of sites of unsatu-
`ration in both fatty chains. The peroxidation would, therefore,
`be expected largely to affect the 18:2n-6, 18:3n-3, and espe-
`cially the 22:5n-3 and 22:6n-3, which would be anticipated to
`yield C4 to C9 core aldehydes in combination with both satu-
`rated and monounsaturated alkyl chains in the sn-1-position.
`The present results confirm that peroxidation of the monoun-
`saturated