Nutrient Metabolism
`
`in the American Diet
`Meats and Fish Consumed
`Contain Substantial Amounts
`of Ether-Linked
`Phospholipids1
`
`MERLE L BLANK, EDGAR A. CRESS,
`Z1GRIDA L SMITH AND FRED SHYDER2
`
`Medical Sciences Division, Oak Ridge Associated universities,
`Oak Ridge, TN 37831-0117
`
`ABSTRACT The primary goal of this study was to de
`termine the amounts
`of ether-containing
`phospholipids,
`along with their concentration
`of certain polyunsaturated
`acyl groups,
`from selected,
`commonly consumed foods
`of animal origin (salmon, catfish, pork, beef,
`turkey and
`chicken).
`Levels of ether-linked
`glycerolipids
`in the
`samples were of particular
`interest, because ingestion of
`ether lipids could contribute-to the production of platelet-
`activating
`factor
`(PAF;
`l-alkyl-Z-acetyl-sn-glycero-S-
`phosphocholine),
`one of the most potent biological medi
`ators
`known.
`Alkylacyl-sn-glycero-3-phosphocholine
`was found in all of the meats, with pork loin having the
`highest
`levels (0.9 umol/g tissue)
`and chicken breasts
`the lowest
`(0.1 umol/g tissue). Although choline plas-
`malogens were
`not
`as
`evident
`as
`the
`ubiquitous
`ethanolamine
`plasmalogens,
`substantial
`amounts
`(1.0
`umol/g
`tissue)
`of
`alk-l-enylacyl-sn-glycero-3-phos-
`phocholine were found in tissues
`from beef and turkey.
`Triacylglycerols
`contained greater proportions
`of satu
`rated fatty acids
`than phospholipids,
`and the ether-
`linked phospholipids were generally more unsaturated
`than diacyl species of the same phospholipid. Our data
`indicate that
`in addition to the phospholipid fraction of
`commonly eaten animal
`tissues
`supplying substantial
`amounts of polyunsaturated
`fatty acids,
`they are also a
`rich source of ether-linked
`lipids. Dietary ether-linked
`phospholipids
`could influence the lipid composition
`of
`host
`tissues
`to the extent
`that biological
`responses
`produced by ether
`lipid mediators would be affected.
`J.
`Nutr. 122: 1656-1661,
`1992.
`
`INDEXING KEY WORDS:
`
`•dietary glycerylethers
`•plasmalogens
`fatty acids
`•polyunsaturated
`•ether-containing phospholipids
`•edible animal
`tissues
`
`factor
`Platelet-activating
`sn-glycero-3-phosphocholine;
`hibits many diverse biological
`those
`involving
`inflammatory
`
`l-O-alkyl-2-acetyl-
`(PAF;
`alkylacetyl-GPC)3
`ex
`activities,
`ranging from
`and allergic
`reactions
`
`to those of a physio
`responses
`and antihypertensive
`logical nature
`such as those
`related
`to reproduction,
`fetal
`development
`and
`parturition
`(Snyder
`1990).
`Upon agonist
`stimulation,
`PAF can be produced
`by a
`number
`of different
`cell
`types via remodeling
`of the
`biologically
`inactive
`precursor,
`l-O-alkyl-2-acyl-sn-
`glycero-3-phosphocholine
`(alkylacyl-GPC)
`(Snyder
`1990). The ability
`to produce PAF is attenuated
`in
`cells that
`are deficient
`in arachidonic
`acid, but PAF
`biosynthesis
`can be restored by repletion with either
`arachidonic
`acid (Ramesha
`and Pickett
`1986, Suga et
`al. 1990) or certain other polyunsaturated
`fatty acids
`(Suga et al. 1990). Therefore,
`polyunsaturated
`fatty
`acyl groups
`are important
`not only as precursors
`of
`eicosanoids
`but also for the optimum production
`of
`PAF. The
`cells or
`tissues
`that
`are responsible
`for
`increasing the circulating
`levels of PAF, under
`specific
`conditions
`of stimulation,
`have not been unequivo
`cally
`established.
`Because
`of
`the
`high
`biological
`potency
`of PAF [levels as low as 2 nmol/L can ac
`tivate
`human
`platelets
`(Nunez
`et al. 1989)], even
`small
`increases
`in cellular
`levels of alkylacyl-GPC
`could potentiate
`the production
`of PAF. Platelet-ac
`tivating
`factor
`can also be biosynthesized
`by a de
`novo pathway
`(Snyder
`1990) beginning with
`alkyl-
`glycerols
`that
`could be produced
`from dietary
`lipids
`containing
`the basic
`1-O-alkyl-sn-glycerol
`structure
`as a part of more
`complex
`lipid structures.
`
`by the Office of Energy Research, U.S. Department
`'Supported
`of Energy (contract
`no. DE-AC05-760R00033),
`and the National
`Institute
`of Diabetes and Digestive and Kidney Diseases
`(grant ROÃ(cid:141)
`DK42804-01A1).
`should be addressed.
`2To whom correspondence
`GPE,
`sii-glycero-3-phosphocholine;
`3Abbreviations
`used: GPC,
`GPI,
`sn-glycero-3-phos-
`sfl-glycero-3-phosphoethanolamine;
`phoinositol;
`GPS,
`sn-glycero-3-phosphoserine;
`PAF, platelet-ac
`tivating
`factor, alkylacetyl-GPC.
`
`0022-3166/92
`
`$3.00 ©1992 American
`
`Institute
`
`of Nutrition.
`
`Received 11 November
`1656
`
`1991. Accepted
`
`19 March 1992.
`
`Downloaded from https://academic.oup.com/jn/article-abstract/122/8/1656/4769556
`by jmjones@casimirjones.com
`on 24 August 2018
`
`AKER EXHIBIT 2009 Page 1
`
`

`

`DIETARY ETHER-LINKED LIPIDS
`
`1657
`
`alkylacyl-GPC
`that
`shown
`previously
`We have
`tissues
`can be increased with
`dietary
`levels
`in rat
`of alkylglycerol
`diacetates
`(Blank et al.
`supplements
`1991). However,
`the
`contribution
`of naturally
`oc
`curring
`alkylglycerolipids
`in the American
`diet
`to
`tissue levels of alkylacyl-GPC are presently unknown.
`This report
`represents
`the first step in understanding
`this
`process,
`through
`the
`determination
`of ether-
`content
`and composition
`of some
`commonly
`con
`sumed meats
`and fish in the American
`diet.
`
`MATERIALS AND METHODS
`
`area of pond-
`from the middorsal
`tissues
`Muscle
`beef
`ribeye
`raised
`catfish
`and Norwegian
`salmon,
`steaks,
`pork loin chops,
`and breasts
`of chicken
`and
`turkey were used for lipid analyses. Approximately
`20
`g of each food sample
`(purchased
`fresh from a local
`supermarket) was excised, with care taken to exclude
`any gross areas of adipose
`tissue. Each sample was
`obtained
`from three different
`animals of each species
`and analyzed
`separately. After measuring
`the wet
`weight,
`the tissues were placed into 100 mL of chlo
`roform and homogenized
`using a Polytron
`homoge-
`nizer
`(Brinkman
`Instruments,
`Westbury,
`NY);
`an
`equal volume
`(100 mL) of methanol was then added
`and the samples were again homogenized.
`The chlo-
`roform-methanol
`homogenates were then shaken for
`20 min,
`centrifuged
`(560 x g for 10 min at
`room
`temperature),
`and the
`supernatants
`containing
`the
`lipid fractions were removed. The protein pellets were
`extracted
`once more
`using
`100 mL of chloroform-
`methanol
`(1:1, v/v) and, after centrifugation,
`this ex
`tract was pooled with the first supernatant. One-half
`volume
`of water was
`added
`to
`the
`chloroform-
`methanol
`solution
`of lipids and, after centrifugation,
`the lower chloroform layer containing
`the lipids was
`recovered. This
`extraction
`technique was based on
`the principle
`originally
`described
`by Bligh and Dyer
`(1959). The total
`lipids were weighed
`and then dis
`solved in 3 mL of chloroform and stored at
`-20°C
`until
`analyzed
`(usually within
`1 wk).
`lipids
`Approximately
`250 ug of each sample of total
`was analyzed by TLC on 250-um layers of Silica Gel
`G using a solvent
`system of hexane-diethyl
`ether-
`acetic
`acid (80:20:1 by volume);
`the developed TLC
`plates were
`then
`sprayed with
`sulfuric
`acid
`and
`charred at 180°Cfor 1 h. Samples were visually com
`pared with a known
`amount
`of palmitic
`acid, chro-
`matographed
`in an adjacent
`lane, and only extracts
`that were estimated
`to contain <2% free fatty acids by
`weight were
`used. Based
`on
`addition
`of known
`amounts
`of hexadecyldipalmitoylglycerol
`to 250 ug of
`total
`lipids from the beef steak, we would also be able
`to detect
`levels of >1% alkyldiacylglycerols
`in the
`total
`lipids with this TLC technique.
`Phospholipids
`were separated by TLC on layers of Silica Gel H in a
`Downloaded from https://academic.oup.com/jn/article-abstract/122/8/1656/4769556
`by jmjones@casimirjones.com
`on 24 August 2018
`
`TABLE 1
`
`Lipid concentration of muscle tissue from selected fish, meat
`and poultry products consumed by humans^
`
`Animal
`
`Total
`
`lipids
`
`Total phospholipids
`
`wet32.8
`SalmonCatfishPorkBeefChickenTurkeymg/g
`
`±7.218.1
`±0.354.37
`
`
`±3.238.5
`
`±0.453.68
`
`±17.233.1
`
`±0.164.86
`
`±4.627.0
`
`±0.355.85
`
`±2.420.0
`
`±0.175.10
`± 4.5wt5.76
`±0.04
`of each species.
`are means ±SEMfrom three animals
`'Values
`The milligrams
`of total phospholipids were calculated
`by mul
`tiplying the milligrams
`of phospholipid
`phosphorus
`by 25. Total
`lipids were determined
`gravimetrically.
`
`acid-
`system of chloroform-methanol-acetic
`solvent
`(50:25:6:2 by volume). After Chromatographie
`water
`development,
`the plates were either
`sprayed with sul
`furic
`acid and charred
`for determination
`of phos
`phorus
`(Rouser et al. 1966) or (with preparative TLC
`plates)
`the layers were exposed to ammonia
`vapor
`for
`~3 min before spraying with 2,7-dichlorofluorescein
`(0.1% in ethanol)
`to locate the separated phospholipid
`bands under UV light
`for their
`subsequent
`extraction
`by the method of Bligh and Dyer
`(1959). Subclasses of
`diradyl-GPC
`and
`diradyl-GPE
`were
`quantified
`as
`diradylglycerobenzoate
`derivatives
`by normal-phase
`HPLC as previously
`described
`(Blank et al. 1987).
`Diradylglycerobenzoate
`derivatives
`from the GPC-
`and GPE-containing
`phospholipids
`were
`also sepa
`rated into subclasses
`by preparative
`TLC and then
`subjected
`to methanolysis
`for
`the
`analysis
`of acyl
`groups as their methyl
`ester derivatives
`by gas-liquid
`chromatography
`(Blank et al. 1984). Composition
`of
`the alkyl and alk-1-enyl
`ether chains was determined
`by reverse-phase HPLC of the dibenzoate
`derivatives
`of
`the
`alkyl-
`and alk-1-enyl-glycerols
`(Blank et al.
`1983).
`in the data were
`differences
`significant
`Statistically
`for unpaired
`variables;
`P
`based on Student's
`t
`test
`values > 0.05 were not considered
`statistically
`signif
`icant.
`
`RESULTS
`
`there were
`errors,
`standard
`by the
`As indicated
`in the amount
`of total
`lipid per gram
`large variations
`the meat
`from catfish and turkey con
`of wet
`tissue;
`tained
`the
`lowest
`amounts
`of
`lipid
`(Table
`1). No
`neutral
`ether
`lipids
`(e.g., alkyldiacylglycerols)
`were
`detected
`in any of the tissue
`lipids when they were
`examined
`by TLC using the nonpolar
`solvent
`system
`consisting
`of hexane-diethyl
`ether-acetic
`acid (80:20:1
`
`AKER EXHIBIT 2009 Page 2
`
`

`

`1658
`
`BLANK ET AL.
`
`TABLE2
`Distribution of acyl groups in triacylglycerols from selected fish, meat and poultry products consumed by humans1
`
`
`
`Acylgroup16:018:018:l(n-9)18:2(n-6)
`
`groups27.8
`±0.89.1
`
`
`±0.313.6
`±0.45.3
`
`±0.55.3
`
`
`±0.744.7
`
`±0.134.3
`
`±1.051.3
`
`±0.429.4
`
`±1.61.6
`
`±1.323.2
`
`±1.013.0
`
`±0.94.7
`±0.5Turkey23.7
`±1.1
`±0.7
`±1.4
`20:4(n-6)
`0.9 ±0.7
`0.4 ±0.1
`0.7 ±0.1
`20:5(n-3)
`0.1 ±0.1
`0.4 ±0.1
`4.8 ±1.1
`22:5(n-3)
`1.3 ±1.1
`0.2 ±0.1
`2.4 ±0.8
`22:6|n-3)Salmon20.6
`±0.1
`0.6 ±0.4Chicken24.0
`0.8 ±0.5Porkg/100
`8.5 ±2.4Catfish18.6
`'Values represent means ±SEMfrom three animals of each species. Dashes indicate the absence of that particular acyl group.
`
`g28.1
`±1.910.9
`
`
`±1.148.7
`
`±2.33.0
`±0.1
`0.1 ±0.0Beefacyl
`
`±0.54.3
`
`
`±0.439.3
`
`±0.518.1
`±0.2
`
`0.3 ±0.20.1
`
`that were
`by volume). Levels of alkyldiacylglycerols
`<1% of the total
`lipid weight would probably not be
`detected
`by this technique. Triacylglycerols
`were the
`major
`lipid class of all
`samples
`analyzed,
`and the
`distribution
`of selected acyl groups
`from this fraction
`is summarized
`in Table 2. Of the samples
`analyzed,
`triacylglycerols
`from salmon
`had the
`highest
`per
`centages
`of polyunsaturated
`(>2 double bonds)
`acyl
`groups.
`Not
`unexpectedly,
`triacylglycerols
`from
`muscle
`tissues of the other animals
`contained
`either
`small
`(e.g., catfish and turkey) or <0.5% of fatty acids
`with four or more double bonds. Significantly
`greater
`amounts
`of
`linoleic
`acid were
`present
`in triacyl
`glycerols
`from catfish
`(13%),
`chicken
`(18%)
`and
`turkey
`(23%) compared with
`the other
`three
`food
`stuffs
`(P < 0.01). Triacylglycerols
`from pork and beef
`were
`the most
`saturated,
`containing
`only
`small
`amounts
`of linoleic
`acid and virtually
`no other poly
`unsaturated
`acyl groups.
`the
`in
`There was
`less
`variation
`content
`per gram of wet
`tissue within
`
`phospholipid
`an animal
`
`found with
`(Table 1 and Table 3) than was
`species
`because
`the
`total
`lipid amounts.
`This
`is probably
`component
`of
`triacylglycerols,
`which
`are the major
`the total
`lipids, act as cellular
`lipid storage sites that
`are more responsive
`to the general nutritional
`status
`of the
`animal
`than
`the phospholipids,
`which
`have
`both structural
`and functional
`roles. Diacyl-GPC was
`the major phospholipid
`subclass
`in every sample ana
`lyzed and was highest
`in salmon
`tissue
`(Table 3).
`Whether
`expressed
`as the percentage
`of the choline
`phosphatides
`or per gram of wet weight, meat
`from
`pork had the highest
`levels of alkylacyl-GPC. Nearly
`one-third
`of the diradyl-GPC fraction from beef tissue
`was in the choline plasmalogens
`(the alk-1-enylacyl-
`GPC subclass). High levels of alk-1-enylacyl-GPC
`are
`also found in bovine heart muscle
`(Horrocks
`1972),
`and
`greater
`amounts
`of
`choline
`plasmalogens
`(Davenport
`1964) seem to be a general
`characteristic
`of bovine muscle
`tissue. The
`concentration
`of alk-
`1-enylacyl-GPC
`was
`also high
`in the diradyl-GPC
`
`TABLE3
`Phospholipid composition of muscle tissue from selected fish, meat and poultry products consumed by humans1
`
`Catfish
`
`Pork
`
`Beef
`
`Turkey
`
`Chicken
`
`\Lmol/g
`
`Salmon
`Phospholipid
`tissueSphingomyelinDiradyl-GPI/GPSAlk-
`±0.020.62
`
`±0.040.47
`
`±0.040.51
`
`±0.040.80
`
`
`±0.050.96
`±0.070.48
`
`
`±0.040.20
`±0.060.03
`
`1-enylacyl-GPCAlkylacyl-GPCDiacyl-GPCAlk-1-enylacyl-GPEAlkylacyl-GPEDiacyl-GPE0.16
`±0.010.13
`
`±0.160.17
`±0.090.25
`
`±0.080.89
`
`
`±0.040.37
`±0.000.40
`
`
`±0.023.55
`
`±0.022.66
`
`±0.032.48
`
`±0.121.35
`
`±0.063.12
`
`±0.074.51
`
`±0.071.00
`
`±0.080.79
`±0.160.98
`
`±0.230.57
`
`±0.340.64
`
`±0.320.23
`
`±0.040.04
`
`±0.040.03
`
`±0.120.02
`
`±0.020.07
`
`±0.050.10
`
`±0.020.01
`
`±0.001.15
`
`±0.000.71
`
`±0.000.56
`
`±0.000.23
`
`±0.060.56
`
`±0.001.26
`±0.050.53
`±0.05
`±0.040.39
`±0.010.39
`±0.220.35
`±0.060.10
`'Values are means ±SEMfrom three animals of each species. Phospholipids were separated by TLC and phosphorus was determined as
`described in Materials and Methods. Abbreviations used: GPC, sn-glycero-3-phosphocholine; GPE, sn-glycero-3-phosphoethanolamine; GPI,
`sn-glycero-3-phosphoinositol; GPS, sn-glycero-3-phosphoserine.
`
`
`
`±0.010.64
`
`
`±0.201.03
`
`±0.030.90
`
`
`±0.050.22
`
`Downloaded from https://academic.oup.com/jn/article-abstract/122/8/1656/4769556
`by jmjones@casimirjones.com
`on 24 August 2018
`
`AKER EXHIBIT 2009 Page 3
`
`

`

`DIETARY ETHER-LINKED LIPIDS
`
`1659
`
`Distribution
`
`subclasses
`in ether-linked
`of alkyl and alk-1-enyl groups
`from selected fish, meat and poultry
`products
`
`and cboline glycerophospholipids
`of ethanolamine
`consumed
`by humans1
`
`TABLE 4
`
`group16:116:018:118:016:116:018:118:016:116:018:118:016:116:018:118:016:116:018:118:016:116:018:118:0EthanolamineAlk-1-enyl1.6
`AnimalSalmonCatfishPorkBeefTurkeyChickenEther
`
`gNANANANA12.6
`
`groupsNANANANA4.8
`
`±0.363.6
`
`
`±2.318.8
`
`±0.89.5
`
`±1.35.2
`
`±0.221.0
`
`±1.154.2
`
`±0.714.8
`
`±0.60.8
`
`±0.133.6
`
`±1.920.2
`
`±0.438.8
`
`±1.1trace41.6
`
`±0.015.2
`
`
`±0.164.7
`
`±0.33.9
`
`±0.00.6
`
`±0.144.5
`
`±0.127.2
`
`±1.424.8
`
`±4.0NANANANA0.8
`
`±0.181.4
`
`
`±2.06.6
`
`±1.11.2
`
`±0.17.5
`
`±0.324.8
`
`±0.651.2
`
`±1.35.9
`
`±0.11.3
`
`±0.263.9
`
`±1.521.7
`
`±0.77.3
`
`±0.8trace69.4
`
`±0.135.2
`
`
`±0.745.8
`
`±0.67.4
`
`±0.20.9
`
`±0.167.4
`
`±2.017.4
`
`±1.49.0
`
`±0.50.1
`
`±0.073.1
`
`±2.65.2
`±0.19.0
`
`±1.27.3
`
`
`±0.241.5
`
`±0.39.3
`
`±1.28.2
`
`±1.80.7
`
`±1.80.6
`
`±1.21.2
`
`±0.153.2
`±0.259.4
`
`
`±0.077.8
`
`±0.072.0
`
`±3.313.4
`
`±1.47.2
`
`±1.114.4
`
`±0.29.9
`
`±0.619.8
`
`±0.48.2
`
`±0.77.1
`
`±0.529.2
`
`±0.70.8
`
`±0.00.4
`
`±1.20.6
`
`±0.61.6
`
`±0.158.5
`
`±0.158.1
`
`±0.282.4
`
`±0.373.0
`
`±0.119.1
`
`±3.07.8
`
`±0.212.5
`
`±0.918.3
`
`±0.924.8
`
`±0.219.0
`
`±0.87.8
`
`±2.34.9
`±0.3Alkylg/100
`±1.1CholineAlk-1-enylether
`±2.0Alkyl1.1
`±1.5
`the sample was
`from the means of samples
`from two different animals of each species; NA signifies that
`'Values are means ±variations
`not analyzed because insufficient material was available. Other ether groups were also present
`(e.g., 14:0, 15:0 and 17:0], but none represented
`>5.0% of the total.
`
`the diradyl-GPC
`fraction from turkey breasts. Unlike
`phospholipids,
`the diradyl-GPE class contained
`only
`low levels of the alkylacyl-GPE
`subclass
`(0.01 to 0.10
`(¿mol/g)in all
`tissues. As with many
`other
`animal
`tissues
`(Horrocks
`1972), ethanolamine
`plasmalogens
`represented
`a large, and often the major
`(catfish, pork,
`beef and turkey),
`subclass
`of the diradyl-GPE phos
`pholipids.
`showed
`chain composition
`of the ether
`Analysis
`the bulk
`that 16:0, 18:0 and 18:1 moieties
`composed
`phospho
`of ether groups present
`in the ether-linked
`lipids
`from these
`foodstuffs
`(Table 4). This
`is not
`surprising
`because
`these are the main components
`of
`the ether groups
`found in most animal
`species previ
`ously analyzed (Horrocks
`1972). There was a striking
`similarity
`in the composition
`of ether chains
`from the
`two avian species. Ether
`lipids from catfish contained
`the highest
`amounts
`of the 16:1 and 18:1 alk-1-enyl
`and alkyl groups.
`acyl groups, with at
`of selected
`The distribution
`least
`two double bonds per molecule,
`in subclasses
`of
`diradyl-GPC and diradyl-GPE from the six different
`edible tissues
`are shown in Table 5. These phospho-
`Downloaded from https://academic.oup.com/jn/article-abstract/122/8/1656/4769556
`by jmjones@casimirjones.com
`on 24 August 2018
`
`with
`compared
`from fish, when
`subclasses
`lipid
`of the other
`animals,
`contained
`from most
`tissues
`levels of 20:5 and 22:6 acyl moi
`significantly
`higher
`eties, with 22:6 being the most prominent
`of the two
`acyl groups. Although
`18:2 and 20:4 were virtually
`absent
`in the phospholipids
`from salmon,
`phospha-
`tides from catfish possessed
`reasonably
`high levels of
`these
`two fatty acids. This difference
`in acyl com
`position
`between
`salmon
`and catfish
`likely
`reflects
`the different
`food chains present
`in the environments
`(saltwater
`vs.
`freshwater)
`of
`the
`two
`species. All
`tissues
`from the warm-blooded
`animals
`contained
`high levels of 18:2 and 20:4 and lesser amounts
`of 20:
`5 and 22:6 in the
`subclasses
`of diradyl-GPE
`and
`diradyl-GPC.
`Significantly
`greater quantities
`of 22:6
`were present
`in ethanolamine
`and choline
`phospho
`lipids
`from turkey
`and chicken
`breasts
`than in the
`meat
`from beef and pork (P < 0.001). Phospholipid
`subclasses
`of all animal
`tissues
`analyzed had a much
`higher content of polyunsaturated
`fatty acids (Table 5)
`than triacylglycerols
`(Table 2) from the same cut of
`meat. Except
`in salmon,
`the ether-linked
`subclasses
`generally
`contained
`a greater proportion
`of 22:6 than
`
`AKER EXHIBIT 2009 Page 4
`
`

`

`1660
`
`BLANK ET AL.
`
`TABLE 5
`
`Distribution of selected polyunsaturated acyl groups in subclasses of ethanolamine and choline pbospbolipids
`meat and poultry products consumed by humans1
`
`from selected fish,
`
`enyl-acyl0.3
`
`AnimalSalmonCatfishPorkBeefTurkeyChickenAcylgroup18:2(n-6)20:4(n-6)20:5|n-3)22:6(n-3|18:2(n-6)20:4(n-6|20:5(n-3|22:6(n-3|18:2(n-6|20:4(n-6)20:5(n-3)22:6(n-3|18:2(n-6)20:4(n-6)20:5(n-3)22:6|n-3)18:2(n-6)20:4(n-6)20:5|n-3)22:6(n-3)18:2(n-6)20:4|n-6)20:5(n-3)22:6(n-3)Alk-1-
`
`±0.12.0
`
`
`±0.25.7
`
`±0.549.9
`
`±5.62.4
`
`±0.48.0
`
`±0.22.8
`
`±0.342.6
`
`±2.423.1
`
`±0.725.0
`
`±3.30.6
`
`±0.0ab_ab23.4
`
`g1.7
`±0.41.4
`
`
`±0.35.3
`
`±1.046.0
`
`±2.810.5
`±0.54.8
`
`
`±0.68.6
`
`±0.62.9
`
`±0.53.5
`
`±1.414.5
`
`±0.642.9
`
`±3.212.0
`
`±0.728.6
`
`±1.48.0
`
`±1.519.5
`
`±1.2tra_ab19.4
`
`±5.00.5
`
`±0.2b_bNANANANA9.2
`
`groupsNANANANA4.7
`±0.00.9
`
`
`±0.35.0
`
`±1.071.3
`
`±0.75.0
`
`±0.211.3
`
`±1.08.4
`
`±1.432.6
`
`±1.964.2
`
`±1.48.6
`
`±2.2_ab_ab32.8
`
`±0.610.8
`
`
`±0.55.2
`
`±0.125.6
`
`±2.441.4
`
`±2.78.2
`
`±0.9_b_b34.4
`
`±0.21.1
`
`
`±0.47.5
`
`±1.639.6
`
`±3.514.7
`
`±1.23.4
`
`±1.02.0
`
`±0.75.2
`
`±0.925.5
`
`±3.21.5
`
`±0.6_ab_ab19.7
`
`±2.614.1
`
`
`±2.40.9
`
`±0.1atrab16.6
`
`±3.15.7
`
`
`±1.40.8
`
`±0.4ab_ab9.0
`
`±9.56.6
`
`±1.818.3
`
`
`±3.32.0
`
`±3.22.0
`
`±0.4a0.8
`
`±1.50.3
`
`±0.3b7.9
`
`±0.8ab3.1
`±2.014.3
`
`
`±0.722.3
`±0.221.8
`
`
`±0.222.2
`±0.68.8
`
`
`±1.00.5
`
`±1.01.5
`
`±1.02.1
`
`±1.81.4
`
`±0.70.6
`
`±0.0a2.7
`
`±O.lb7.5
`
`±0.2b7.5
`
`±0.2b7.4
`
`±0.2ab16.9
`
`±0.4b10.1
`
`±0.1ab16.1
`
`±0.2b11.1
`
`±0.5ab14.8
`
`±0.7ab7.1
`
`±1.210.1
`
`±1.29.0
`
`±1.47.7
`
`±1.021.3
`
`±0.710.3
`
`±2.21.3
`
`±0.60.7
`
`±0.31.4
`
`±1.41.2
`
`±1.31.1
`±0.4b3.2
`
`
`±0.3ab1.1
`
`±O.lab1.9
`
`±O.lab12.3
`
`±0.3b4.4
`±0.5bDiradyl-GPCAlkvl-acyl0.3
`±0.6abDiacyl0.6
`±0.4abAlk-1-enyl-acylacyl
`±1.3abDiradyl-GPEAlkyl-acylNANANANA2.9
`±1.0bDiacylg/100
`
`±6.02.9
`
`
`±1.20.4
`
`±0.4a0.2
`
`±0.2ab22.5
`
`±1.04.7
`
`±0.7trab1.3
`
`±0.2ab23.3
`
`
`±0.53.1
`±0.3_ab_ab
`
`'Values are means ±SEMfrom three animals of each species. Dashes indicate the acyl group was not observed, and tr means trace amounts
`(e.g., <0.2%) were present; NA signifies
`that
`the sample was not analyzed because insufficient material precluded an acceptable
`accuracy.
`Acyl moieties
`from ether-linked
`subclasses
`represent only the sn-2 position; acyl groups from the diacyl subclasses
`are from both the sn-1 and
`sn-2 positions, meaning that
`if these unsaturated
`fatty acids are located exclusively
`at the sn-2 position of the diacyl subclasses,
`then the
`values for diacyls should be multiplied
`by two to be directly comparable with values of the ether subclasses.
`aValues are significantly
`lower
`than in the corresponding
`subclass
`from salmon (P < 0.05). Walues
`are significantly
`lower than the corresponding
`subclass
`from catfish (P <
`0.05). Abbreviations
`used: GPC, sn-glycero-3-phosphocholine;
`GPE, sn-glycero-3-phosphoethanolamine.
`
`diacyl subclasses. The
`was found in the corresponding
`alk-1-enylacyl-GPC
`subclass
`from pork (P < 0.05), and
`the alk-1-enylacyl-
`and alkylacyl-GPC
`subclasses
`of
`turkey (P < 0.01) and chicken
`[P < 0.02), were signifi
`cantly enriched
`in arachidonate when compared with
`their diacyl
`counterparts.
`
`DISCUSSION
`
`the amounts
`The aim of this study was to quantify
`subclasses
`of
`of ether-linked
`lipids,
`including
`the
`diradyl-GPE
`and diradyl-GPC,
`in edible meats
`and
`fish, because such information
`is not available for this
`possibly
`important
`source
`of potent
`biologically
`active mediators.
`The
`amount
`of alkylacyl-GPC
`in
`the meats was of particular
`interest because
`(based on
`limited
`experimental
`results)
`the
`basic
`structure,
`alkyllyso-GPC
`(lyso-PAF), would
`likely be produced
`
`(Tso 1985). Platelet-activating
`processes
`by digestive
`factor could be produced directly by enzymatic
`acety-
`lation
`of the lyso-PAF, or, alternatively,
`subsequent
`reacylation
`of
`lyso-PAF
`in tissues would
`produce
`alkylacyl-GPC,
`a precursor
`of PAF and a very rich
`source of eicosanoid
`precursors
`such as arachidonic
`acid. Ether-linked
`phospholipids
`were
`present
`in
`every animal
`tissue
`that we analyzed;
`however,
`no
`ether-linked
`neutral
`lipids were
`detected.
`Ether
`groups
`in
`the
`ethanolamine
`and
`choline
`glycerophosphatides
`were composed primarily
`of mix
`tures
`of 16:0, 18:1 and 18:0 moieties.
`These
`same
`three groups are also the major alkyl chains
`found in
`the PAF produced
`by stimulated
`rabbit
`and human
`neutrophils
`(Mueller et al. 1984). The largest amount
`of alkylacyl-GPC was found in pork,
`in which it was
`equivalent
`to -700 mg/kg meat. Whether
`and to what
`extent
`these levels of dietary ether
`lipids would affect
`the
`production
`of,
`and
`subsequent
`biological
`
`Downloaded from https://academic.oup.com/jn/article-abstract/122/8/1656/4769556
`by jmjones@casimirjones.com
`on 24 August 2018
`
`AKER EXHIBIT 2009 Page 5
`
`

`

`DIETARY ETHER-LINKED LIPIDS
`
`1661
`
`of pigeon and ox skeletal
`
`by high-performance
`benzoates
`and alk-1-enyl-glycerol
`alkyl-
`133: 430-436.
`liquid chromatography. Anal. Biochem.
`Blank, M. L, Cress, E. A., Smith, Z. L. & Snyder, F. (1991) Dietary
`supplementation
`with ether-linked
`lipids and tissue lipid com
`position. Lipids 26: 166-169.
`and
`Blank, M. L., Cress, E. A. & Snyder, F. (1987) Separation
`quantitation
`of phospholipid
`subclasses
`as
`their
`diradyl-
`glycerobenzoate
`derivatives
`by normal-phase
`high performance
`liquid chromatography.
`J. Chromatogr.
`392: 421-425.
`Blank, M. L., Robinson, M., Fitzgerald, V. & Snyder, F. (1984) Novel
`quantitative method for determination
`of molecular
`species of
`phospholipids
`and diglycerides.
`J. Chromatogr.
`298: 473-482.
`Bligh, E. G. & Dyer, W. J. (1959) A rapid method of total
`lipide
`extraction
`and purification.
`Can.
`J. Biochem.
`Physiol.
`37:
`911-917.
`]. B. (1964) The phospholipids
`Davenport,
`muscle. Biochem.
`J. 90: 116-122.
`and metabolism of
`Horrocks, L. A. (1972) Content,
`composition,
`mammalian
`and avian lipids that contain ether groups. In: Ether
`Lipids (Snyder, F., ed.), chap.
`IX. Academic Press, New York,
`NY.
`(1984) The
`J. T. & Wykle, R. L.
`Mueller, H. W., O'Flaherty,
`factor syn
`molecular
`species distribution
`of platelet-activating
`thesized by rabbit and human neutrophils.
`J. Biol. Chem. 259:
`14554-14559.
`of
`Inhibition
`Nunez, D., Kumar, R. & Hanahan, D. J. (1989)
`[3H]platelet
`activating
`factor
`(PAF) binding by Zn: a possible
`explanation
`for
`its
`specific
`antiaggregating
`effects
`in human
`platelets. Arch. Biochem. Biophys. 272: 466-475.
`Pickett, W. C. & Ramesha, C. S. (1987) Ether phospholipids
`control
`and 20:4-depleted
`rat PMN: additional
`evidence
`l-O-alkyl-2-20:4-sn-glycerol-3-phosphocholine
`specific
`pholipase A2- Agents Actions 21: 390-392.
`factor
`Ramesha, C. S. & Pickett, W. C. (1986) Platelet-activating
`and leukotriene
`biosynthesis
`is inhibited in polymorphonuclear
`leukocytes
`depleted
`of arachidonic
`acid.
`J. Biol. Chem.
`261:
`7592-7595.
`Rouser, G., Siakotos, A. N. & Fleischer, S. (1966) Quantitative
`analysis
`of phospholipids
`by thin-layer
`chromatography
`and
`phosphorus
`analysis of the spots. Lipids 1: 85-86.
`Snyder, F. (1990) Platelet
`activating
`factor and related acetylated
`lipids as potent biologically
`active cellular mediators. Am. J.
`Physiol. 259: C697-C708.
`Suga, K., Kawasaki, T., Blank, M. L. & Snyder, F. (1990) An
`arachidonoyl
`(polyenoic)-specific
`phospholipase
`A2 activity
`regulates
`the synthesis
`of platelet-activating
`factor
`in granulo-
`cytic HL-60 cells.
`J. Biol. Chem. 265: 12363-12371.
`Tso, P. (1985) Gastrointestinal
`digestion and absorption
`Adv. Lipid Res. 21: 143-186.
`
`in
`for a
`phos-
`
`of lipid.
`
`responses induced by, PAF in humans are presently
`unknown. However, our analyses demonstrate
`that
`ether-linked glycerolipids occur in the meat, fish and
`poultry consumed in the American diet at levels suffi
`cient to justify the continuation of our animal studies
`designed to assess the role of dietary ether-linked
`glycerolipid supplementation
`on the production of
`PAF and on associated biological alterations
`(e.g.,
`blood pressure and inflammatory responses).
`The phospholipid fractions represented from 10%
`(pork) to 25% (turkey) of the total lipid weight in the
`foodstuffs analyzed. Because the phospholipids, and
`especially the ether-linked subclasses, contain much
`more unsaturated
`acyl groupings than the triacyl-
`glycerols, they would be expected to be major dietary
`sources of not only ether-linked lipids but also poly-
`unsaturated fatty acids. It has already been demon
`strated that cellular polyunsaturated acyl groups are
`required for maximum generation of PAF by cells
`(Pickett and Ramesha 1987, Ramesha and Pickett
`1986, Suga et al. 1990), and the polyunsaturated fatty
`acids released from the phospholipids by digestive
`processes would provide another dietary source of
`these important
`acyl groups. Therefore,
`the rela
`tionship between dietary ether-linked glycerolipids
`and polyunsaturated fatty acids, relative to the potent
`synergistic biological metabolites both can generate,
`is important
`to consider in nutritional approaches to
`treating diseases involving PAF and eicosanoid medi
`ators.
`
`ACKNOWLEDGMENT
`
`to Shirley Poston for the
`The authors are grateful
`expert secretarial assistance in preparing this manu
`script.
`
`LITERATURE CITED
`
`Blank, M. L., Cress, E. A., Lee, T-c., Stephens, N., Piantadosi, C. &
`Snyder, F. (1983| Quantitative
`analysis of ether-linked
`lipids as
`
`Downloaded from https://academic.oup.com/jn/article-abstract/122/8/1656/4769556
`by jmjones@casimirjones.com
`on 24 August 2018
`
`AKER EXHIBIT 2009 Page 6
`
`