(cid:19)(cid:19)(cid:19)(cid:19)(cid:19)(cid:20)
`
`Petition for Inter Partes Review
`Of U.S. Patent 8,278,351
`Exhibit
`ENZYMOTEC - 1025
`
`

`

`620
`
`COMMUNICATIONS
`
`was monitored at 420 nm. Fluorescence polarization (P)
`was calculated from the equation, P = IW — I‘m/lvv +
`Ivh ~ Z, in which Ivv and Ivh are the fluorescence inten-
`sities measured with emission analyzer parallel or perpen-
`dicular, respectively, to the polarization of the detection
`system for vertically and horizontally polarized light.
`
`RESULTS AND DISCUSSION
`
`Figure 1 presents a Vant Hoff’ s representation of 1,6-
`diphenyl 1,3,5-hexatriene (DPH) fluorescence polariza-
`tion, P, of vesicles of phospholipids prepared from marine
`calanoid copepods collected either in the North Atlantic
`or in tropic seas. Lower P-values represent more fluid
`structures. From these data, it may be inferred that the
`most fluid membranes were present in C. finmarchicus
`collected in the spring and the most rigid ones in the
`copepods inhabiting the tropic seas. C. finmarchicus
`sampled in early fall revealed values in between these two
`extremes. In addition, there are two distinct breaks in the
`curves on the P vs llT plots. The break at the higher
`temperature indicates onset, while that at the lower
`temperature indicates completion of phase separation of
`these phospholipids. The sea water temperature at the
`
`0.20
`
`0.15
`
`0.1
`
`005
`
`‘C
`
`32
`3.3
`3.4
`3.5
`3.6
`3.7 + 03x
`
`FIG. 1. Temperature dependency (IIT) of DPH fluorescence polariza-
`tion (P) in phospholipids of marine calanoid copepods. Calculus app.
`were collected at Dona Paula, Goa (——V—) and Bombay (—A—) at
`the Southwest coast of India, while C. finmarchicus were collected
`at the West coast of Norway in the fall (—D—) and in the spring
`(—O—), respectively.
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`LIPIDS, Vol. 23, No. 6 (1988)
`
`time of collecting C. finmarchicus was about 10 C but
`varied between 2 and 20 C during the year. The tempera-
`ture in the tropic seas was about 25—26 C at the time of
`sample collecting and varied from 25 to 30—31 C during
`the year. The temperature at the onset of phase separa-
`tion of phospholipids from each sample was close to the
`temperature at which the organism lived, except in the
`C. finmarchicus collected in the fall. However, it should
`be remembered that the above values for phospholipid
`vesicles might be modulated if other membrane consti-
`tuents (proteins, sterols, etc.) also were present.
`The observation that the temperature at onset of the
`phase separation of the membrane lipids coincides with
`growth temperature has been made with other poikilo-
`therms (10,18-20), but it is not documented as a general
`phenomenon (7.21). Cossins and Prosser (18) reported that
`the onset of phase separation of phospholipids from
`synaptosomal membranes of arctic sculpin adapted to 0 C
`occurred at 5 C while that for goldfish acclimated at 5 C
`occurred at 10 C. It can be inferred from Figure 1 of
`Prosser and Cossin’s paper that phospholipids of synap-
`tosomal membranes of goldfish adapted at 25 C show
`phase separation around the growth temperature (18). In
`a current study of liver phospholipids of the carp,
`Cypn'nus carpio L., we found that the onset of phase
`separation of phospholipids of summer-adapted fish oc-
`curred around 25 C, while that of winter-adapted fish oc-
`curred around 6 C (unpublished observations). In earlier
`work on the freshwater copepod Cyclops vicinus, we also
`found that phase separation temperatures were similarly
`related to the actual growth temperature (10). Although
`the water temperature at the time of collecting C. fin-
`marchicus was the same in the fall and the spring (10 C),
`the higher polarization value and phase separation tem-
`perature of phospholipids of the fall sample may suggest
`fihat the fall specimens retained a “summer” state in their
`'pids.
`Whether marine species similar to C. vicinus can
`regulate the physical state of their phospholipids accord-
`ing to the temperature or whether they lack this prop-
`erty, like the freshwater crustacean Daphnia magna, re-
`quires further investigation. Some freshwater crustaceans
`and fish are exposed to fluctuation in their environmen-
`tal temperature. In cases of tropic seas, this is less pro-
`nounced. Judging from the temperature range at which
`the phase separation occurs, it may be inferred that C.
`finmarchicus can tolerate less changes in the water tem-
`perature than the copepods in the tropic seas.
`Spring-collected C. flnmarchicus did not survive ex-
`posure to 20 C longer than two hr, and copepods collected
`at Dona Paula lost their swimming activity but did not
`die when exposed to 17 C for six hr (unpublished obser-
`vations). Because the former can be regarded as a cold
`stenothermic and the latter as a warm stenothermic
`species, it is tempting to speculate that this is at least
`partially related to the phase behavior of
`their
`membranes.
`Table 1 shows that the above differences in the phase
`behavior are not easily explained by the fatty acid
`composition of the phospholipids. An inverse relationship
`between environmental temperature and fatty acid un-
`saturation also was observed in this case. This was due
`mainly to a higher level of docosahexaenoic acid in phos-
`pholipids of C. finmarchicus. Despite these differences.
`
`

`

`621
`
`TABLE 1
`
`COMMUNICATIONS
`
`TABLE 2
`
`Fatty Acid Composition (mol %) in Phospholipids
`of Calanoid Copepods
`
`Composition of Phospholipids (% wt) in Calanoid Copepods
`
`Species
`
`Calanus spp.
`
`C. finmarchius
`
`Origin
`India“
`lndiab Norwayc Norwayd
`Water temperature (°C):
`25
`26
`10
`10
`
`
`2.4
`3.4
`3.9
`4.7
`14:0
`0.1
`0.5
`0.1
`0.2
`14:1
`0.2
`0.7
`0.3
`0.2
`15:0
`tr
`0.1
`tr
`0.2
`15:1
`15.2
`16.5
`16.0
`19.6
`16:0
`0.7
`0.3
`1.6
`1.4
`16.1
`0.3
`0.1
`0.3
`0.9
`16:2
`0.1
`0.7
`0.2
`1.3
`16:3
`0.8
`2.0
`4.0
`8.7
`18:0
`4.5
`2.9
`3.7
`4.5
`18:1
`0.6
`0.8
`1.5
`1.5
`18:2
`tr
`0.7
`1.8
`1.7
`18:3
`0.2
`1.9
`2.5
`0.3
`18:4
`1.2
`0.1
`0.2
`tr
`20:1
`1.9
`1.2
`4.5
`2.5
`20:4
`30.2
`24.1
`25.2
`15.7
`20:5
`0.2
`0.2
`0.2
`0.5
`22:3
`0.3
`0.2
`0.1
`0.2
`22:4
`0.5
`0.4
`2.3
`2.4
`22:5
`40.0
`42.9
`31.2
`33.4
`22:6
`Sat/unsat
`0.49
`0.31
`0.29
`0.22
`Total polyen(%)
`55.0
`66.1
`71.0
`73.8
`
`
`“Bombay.
`bDona Paula, Goa.
`”Spring.
`dFall.
`
`the spring-collected C’. finmarchicus and the copepods col—
`lected at Dona Paula showed similar saturated to un-
`saturated ratios but great differences in the phase be-
`havior of their phospholipids. Moreover, the two samples
`of copepods from the tropic seas showed differences in
`phospholipid fatty acid compositions as well as in the
`saturated to unsaturated fatty acid ratios, although the
`P-values and the phase separation temperatures were
`almost identical (Fig. 1). Thus, it is highly probable that
`control occurs at a level beyond the overall distribution
`of fatty acids in phospholipids. Table 2 shows that the
`phospholipids of copepods from tropic seas were poorer
`in sphingomyelin and phosphatidic acid. and richer in
`phosphatidylethanolamine than those in C. finmarchicus.
`Phosphatidylcholines in spring-collected C. finmarchicus
`contained more polyunsaturated acids (82% vs 62%) and
`had a lower samrated-tounsamrated fatty acid ratio (0.15
`vs 0.38) than those of the tropic copepods (Table 3). Even
`though phospholipids were not separated according to
`molecular-species composition, one could expect that di-
`unsaturated phospholipids would be present whenever the
`level of total unsaturated fatty acids exceeds 50 mol %.
`As shown in Table 3, the phosphatidylcholines and phos-
`phatidylethanolamines were richer in diunsaturated phos-
`pholipids than were the phospholipids of copepods in the
`tropic seas (32% vs 12% and 17% vs 11%, respectively),
`and this could explain the observed differences in the P-
`values (Fig. 1). Because the phase separation temperature
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`
`Species:
`Origin:
`
`Calanus spp.
`India“
`
`Calanus finmarchius
`Norwayb
`
`3.6
`3.1
`3.9
`—
`
`6.8
`3.9
`35.6
`
`28.9
`6.8
`
`10.1
`8.9
`6.4
`5.2
`
`1.1
`7.6
`29.3
`
`22.3
`9.8
`
`Phosphatidic acid
`Phosphatidylserine
`Phoaphatidylinositol
`Lysophosphatidyl—
`ethanolamine
`Lysophosphatidyl-
`choline
`Sphingomyelin
`Phosphatidylcholine
`Phosphatidyl-
`ethanolamine
`Cardiolipin
`
`“Dona Paula. Goa.
`bSpring.
`
`TABLE 3
`
`Fatty Acid Compodtion (mol %) of Phosphatidylcholines
`and Phosphatidylethanolamines in Calenoid Copepods
`
`Phospholipid
`
`Phosphatidyl-
`choline
`
`Phosphatidyl-
`ethanolamine
`
`Origin
`
`India“
`
`Norwayb
`
`India“
`
`Norwayb
`
`14:0
`14:1
`15:0
`16:0
`16:1
`18:0
`18:1
`18:2
`18:3
`18:4
`20:3
`20:4
`20:5
`22:4
`22:5
`22:6
`Sat/unset
`Total polyen(%)
`
`3.6
`tr
`0.4
`20.9
`1.0
`2.6
`6.4
`1.8
`2.0
`2.4
`tr
`4.9
`21.9
`0.7
`2.2
`28.2
`0.38
`62.0
`
`2.5
`tr
`0.1
`10.2
`1.1
`0.2
`2.6
`0.7
`0.2
`1.7
`tr
`5.0
`42.8
`tr
`0.5
`32.0
`0.15
`82.0
`
`1.0
`tr
`tr
`20.4
`0.4
`12.4
`2.6
`1.3
`0.9
`0.5
`tr
`6.0
`14.3
`0.3
`3.0
`36.7
`0.51
`61.0
`
`“Calanus ssp., Dona Paula, Goa
`bC. finmarchicus, spring.
`
`0.5
`tr
`tr
`24.6
`0.1
`2.2
`4.3
`1.3
`0.3
`1.5
`tr
`3.3
`9.8
`0.7
`1.6
`50.00
`0.38
`67.0
`
`of 1-palmitoyl,2-docosahexaenoyl phosphatidylcholine is
`about - 10 C (22) and that of diunsaturated molecules is
`even lower, phospholipids of marine copepods should ex-
`hibit lower phase-separation temperatures than those
`observed. Phaspholipids of these copepods behave simi-
`larly to those of the b0vine retinal rod outer segment
`membranes. Although the latter are as rich in polyenes
`as the phospholipids of copepods investigated here, they
`too contain fair amounts of supraenes (dipolyunsaturated
`phospholipids)
`(23,24) and exhibit phase-separation
`temperature between 15 and 5 C (25).
`
`LIPIDS, Vol. 23, No. 6 (1988)
`
`

`

`622
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`COMMUNICATIONS
`
`It has been proposed that this results from a precise
`balance between disaturated and diunsaturated phospho-
`lipid molecules (25). The differences demonstrated in
`physical parameters of phospholipid vesicles indicate an
`adaptation of membrane physical states to temperatures.
`
`ACKNOWLEDGMENTS
`
`We thank S. K. Chakraborty, Central Institute of Marine Fisheries,
`Bombay, India, for making possible the collecting of marine
`oopepods. Thanks go also to E. Lehoaki (Jozsef A. University,
`Swged, Hungary) for help in fluorescence polarization measurements.
`
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`
`@999pr
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`"I
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`(1983)
`
`[Received January 26. 1987; Revision accepted February 16. 1988]
`
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`LHEVdfifiaémm
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