U.S.S.N. 95/001,774
`Declaration of Dr. Jacek Jaczynski
`
`Appendix K
`
`Bottino et al., “Resistance of Certain
`Longchain Polyunsaturated Fatty Acids of
`Marine Oils to Pancreatic Lipase
`Hydrolysis” Lipids 2, 489-93 (1967)
`
`°°°°°‘ 14
`
`AKBM 1053 Part 3
`
`00000114
`
`

`
`433
`
`'
`
`I. G. CoNini.1o,'F. B. Curr AND A. GOSWAMI
`REFERENCES
`
`_
`_
`_
`synthesis cannot be specified.
`Many organs had significant “C activity in
`one or more fatty acids having retention times
`equal to those for 2025, 2212» 2213. 334» 22-'5
`and 22:5.
`In the case of tatty acids from
`lungs and from brain, two of these were shown
`to be 22:2 and 22:4.
`In brain tissue about
`40% of the MC activity was in fatty acids
`other than 20:4 and of this about half was
`in a fraction tentatively identified as 18:2.
`Though the chemical
`identity of
`this Com-
`pound was not established further, it ma)’ be
`A3-11 octadecadienoic acid, 5Yfl‘h95lZ°d mm A9
`ml, or A” octadecadienoic acid. The pres-
`ence of the latter isomer in Pig brain “55‘_’°
`has been reported hi’ Ki5him°t° fmd,Radin
`(15). Formation of A-9 octadecadienoic acid
`from 14C.°1ejc acid by rat
`liver mtcrosomes
`was shown by Hoflotrllayfl? 31- (1‘5,)(-m::‘) 1::
`fatty acid (presuma y
`e same is
`_
`shown to be synthesized from l-“C-acefyl C°-A
`by subcellular particles of rat livenby Harlan
`and wakij
`(17). We have previously also
`observed biosynthesis of
`this isomer b? '3‘
`liver microsomes
`incubated with “C-acelY1
`COA (18).
`
`ACKNOWLEDGMENTS
`
`11,3, cm was supported by Grant No. on-5114 from
`the National Science Foundation and lnslltytional 5'3"‘
`No, IN.25l-I from the American Cancer Society.
`
`1. F.. Vitamins‘ Hormones 22, 797-795
`1, Mueller,
`(1964).
`2. Witteri, P. W., and R. T. Holman, Arch. Biochem
`Biophys. 41, 266-273 (I952).
`3. Klrschman, I. C., and I. G. Conlglio, 1. Biol. Chem,
`236, 2200-2103 (1961).
`4. Hirsch, 1., and E. H. Ahrcns, Jr., Ibid. 213, 311-321)
`(1958).
`5. Metcalte, L. D., and A. A. Sehmitz. Analyt. Chan
`33, 363864 (1961)-
`6. Goswami, A. l(., and J. G. Coniglio, J. Nutr. 89, 210-
`216 (1966).
`7. Bennett, M.. and E. Coon, J. Lipid Res. 7, 448449
`(1966).
`a_ Fguquhar, 1. W., W. lrisull, Jr., P. Rosen, W. Sluflel
`and 1;, pt, Ahrens, Jr., Nutr. Rev. (Suppl) 17, no. 8, pt,
`
`2'i,3°Ei1§x9viiir>', M. F. S., and R. 1-I. S. Thompson, lilo-
`chem.
`.7. 53, 340-347 (1953)-
`_
`.
`10. Clilt, F. P. and R. P. Cook,
`Ibtd. 26, 1800-1803
`2 .
`“iii Amador, 5., and W. E. C. Waclter, Clin. Chem. 9,
`343-350 (1962).
`_
`1?. Conislio. J. 6.. J. T. Davis and 5- Aylward, J.
`Nutr. 84, 255-271 (1964).
`_
`-
`p
`13. Kjrsehman, J. C.. Ph.D. Thesis, Vanderbilt Univer-
`
`s“i’3,p'al¢(;),i,°;i_19_t6,l)I{., J. L. Eeare,‘G. H. Beaten, E. F.
`Caldwell, G. Ozawa and E, W. Mei-laxity. 1- Biol Chem.
`
`and N. s. Radin, J. Lipid Res, 5,
`20:'5.”l5€ii52|i1l.ilii!9l)S::
`98-102 (1964)
`15_ Honowgy,
`17, W., R. Peloflo and S.
`I. want,
`Bioclicrn. Blophys Res. Communs. 12, zoom (1963).
`17. Harlan, W. R., Jr., and S. I. Wakil. J. Biol. Chem.
`238, 3216-3223 (1963).
`_
`18, Goswami, A., and J. G. Conigho, Vilth Iritcrna.
`tional Congress of Nutrition.
`in Dress.
`
`[Received April 19, 1967]
`
`Resistance of Certain Long-Chain Polyunsaturated Fatty Acids
`of Marine Oils to Pancreatic Lipase Hydrolysis
`NESTOR R. BOTTINU, GLORIA A. VANDENBURG and RAYMOND REISER, Department of
`' Biochemistry and Biophysics, Texas A&M University, College Station, Texas
`
`ABSTRACT
`
`When whale oil triglycerides were sub-
`jected to pancreatic lipase hydrolysis,
`eicosnpentaenoic
`and
`docosahexaenoic
`acids were found mainly in the di- and
`triglyceride products, suggesting that they
`are in the 1,3-positions but resistant to the
`action of the lipase. Their presence in the
`1,3-positions was confirmed. Their resist-
`ance to pancreatic lipase hydrolysis was
`demonstrated by analysis of the products
`of the enzyme action on: (a) a concen-
`trate of highly unsaturated whale oil tri-
`glycerides;
`(b)
`the latter after random-
`ization; and (c)
`synthetic
`l,2-di-octa-
`decency]-3-eicosapentaenoyl glycerol.
`Docosapentaenoic acid was also shown
`to be present in the 1,3-position of whale
`oil triglycerides but was not lipase resist-
`ant. It is postulated that the presence of
`a double bond near the carhoxyl group
`exercises an inhibitory effect, or that the
`location of the double bonds in the resist-
`ant acids places
`their
`terminal methyl
`groups close to the carboxyl, producing a
`steric hindrance efiect.
`
`INTRODUCTION
`
`In A STUDY on THE srnucrunr. of marine
`mammal oils by the use of pancreatic lipase,
`the distribution of fatty acids in the hydrolytic
`products of whale oil suggested that eicosapen-
`taenoic (20:5) and docosahexaenoic (22:6)
`acids, but not docosapentaenoic (22:S) acid,
`are resistant
`to the action of that hydrolytic
`enzyme. The results of the present study con-
`firm the resistance of those acids to pancreatic
`lipase action, even though the acids are located
`in the 1,3-positions of whale oil triglycerides.
`A preliminary report of this work has been
`presented (1).
`
`EXPERIMENTAL
`
`The location of the 20:5, 22:5, and 22:6
`acids
`in the whale glyceride molecules and
`
`‘One of the samples of whale on was from the Arista
`Company, New York. The other was obtained through
`the courtesy or XL 5. Oleott.
`
`the resistance of these acids to the activity of
`pancreatic lipase were determined by analyses
`of the products of the enzyme action on:
`(a)
`unmodified whale oil;
`(b) a concentrate of
`highly unsaturated whale oil triglycerides; (c)
`the latter after randomization; and (d) syn.
`thetic 1,2-di- octadecenoyI- 3 -eicosapentaenoyl
`glycerol.
`'
`Methods
`
`The triglycerides of two samples of whale
`oil‘ were purified by preparative thin-layer
`chromatography (TLC). A highly unsaturated
`fraction was prepared from one of them by
`crystallization at —60C (2). Menhaden Oil was
`provided by the Department of Oceanography,
`Texas A&M University. Lipase (EC 3.1.1.3)
`from hog pancreas, PI.-III, was purchased from
`Worthington Biochemical Corporation, Free-
`hold, N. J. Lipase hydrolyses were performed
`in vitro by the procedure of Luddy et al. (3),
`including the determination of the fatty acid
`composition of the free fatty acids and of the
`mono-, di-. and triglyceride products.
`Randomization of
`the highly unsaturated
`concentrate of whale oil was achieved by treat-
`ment with 0.l M lithium secondary butylate
`in dimethyl formamide (4). The reaction mix-
`ture was kept under nitrogen at room tempera-
`ture for 3 days. The rearranged triglycerides
`were purified by preparative TLC.
`Purification of triglycerides by TLC was
`achieved on 0.25-mm thick layers of silica gel ”
`(Adsorbosil-1, Applied Science Laboratories,
`State College, Pa.) on 20 x 20 cm glass plates.
`The developing solvent system was a mixture of
`petroleum ether (30-60C hp)-ethyl ether-ace-
`tic acid (60:40:l.6, v/v/V).
`Gas—liquid chromatography (GLC) was per-
`formed in a Research Specialties Model 600
`gas cbromatograph (Warner-Chilcott Labora-
`tories Division, Richmond, Calif). The
`chromatograph was equipped with an argon
`ionization detector and a6 ft x 1/4 in. column
`packed with 15% diethylene glycol succinate
`on 6080 mesh Chromosorb W. The column
`was operated isothermally at l95C. The identi-
`ties of the quantitatively more important peaks
`were ascertained by comparing their relative
`retention times with those of known standards.
`
`489
`
`Lirins, VoL. 2, N0. 6
`
`00000115
`
`lr
`IIs
`Ii
`
`ii
`
`00000115
`
`

`
`490
`
`Infrared spectra were obtained in a IR8
`Beckman infrared spectrophotometer between
`sodium chloride pellets.
`l,2-Di-octadecenoyi3-eicosapentaenoyl glyc-
`erol was
`synthesized from 1,2-dioleto and
`eicosapentaenoyl chloride and purified by TLC.
`A manuscript describing this synthesis is in
`preparation. Eicosapentaenoic acid, 91% pure,
`isolated from menhaden oil, was purchased
`from the Hormel Institute, Austin, Minn.
`
`RESULTS AND oiscussiou
`
`NESTOR R. B07-nuo, GLORIA A. VANDENBURG AND RAYMOND REISER
`TABLE 1
`Major Fatty Acid Components of Whale Oil Tliglymfldgg
`and Its Lipase Hydrolysis Products
` —§
`Whale all:
`'
`P d
`1
`ro uctso liydrolysisi
`Original
`Sample
`AcidI
`FA MG
`DG
`To
`TG
`..:...._#___.j_:_3.__
`Dercentage.
`14:0.
`7.1
`9.3
`9.2
`3.1
`8.7
`1»
`2.1
`7.7
`43.9
`3.4
`4.6
`11
`16:0
`125
`55
`9.9
`20.1
`14.8
`I
`7.;
`10.0
`7.9
`21.1
`14.9
`11
`16:1
`120
`213.9
`17.8
`11.3
`16.7
`1
`3.5
`24.1
`16.6
`9.2
`14.4
`11
`18:1
`3.7
`40.3
`29.4
`37.5
`322
`1
`19.0
`45.0
`30.1
`38.7
`33.6
`11
`20.-1
`1.9
`0.3
`1.1
`5.3
`2.6
`1
`2.8
`1.1
`2.1
`2.3
`2.1
`11
`20;:
`118
`2.1
`11.8
`2.0
`6.6
`1
`26.0
`2.4
`17.1
`3.0
`8.1
`it
`72:5
`20
`0.5
`2.2
`2.9
`3.9
`1
`6.8
`tr
`3.9
`5.9
`5.2
`tr
`22:6
`10.1
`0.3
`5.3
`2.0
`4.9
`1
`
`
`
`
`tr 3.13.95.811 17.3
`
`
`"
`-Chain length: number of double bonds.
`‘PA = Fatty Acids; M -= Morioglycei-ides; D6 =
`Diglyccrides‘, T6 = Triglycerides. '
`cAve1age of duplicate analyses.
`
`I Evidences of Resistance
`After 50% pancreatic lipase hydrolysis of
`the whale oil
`triglycerides,
`the concentrations
`of the 20:5 and 22:6 acids were lower in both
`the fatty acid and the monoglyceride fractions.
`but higher in the diglyceride and triglyceride
`fractions of the resultant mixture than in the
`original oil (Table I). This suggests that these
`two polyunsaturated fatty acids are in the l-
`and 3- positions but are resistant to the action
`of the lipase. That the 20:5, 22:5, and 22:6
`acids of the whale oil are in the 1,3-positions
`to the 20:5 and 22:6 acids, susceptible to the
`has been reported by Broclterhoff and Hoyle
`action of pancreatic lipase.
`(5). The accumulation of longchain polyun-
`Since the concentration of some of the poly-
`saturated fatty acids in the diglycerides after
`unsaturated acids were low in the original
`lipase hydrolysis of marine oils has also been
`whale oil, a highly unsaturated concentrate
`reported by others (4, 6, 7).
`was obtained by removal of the more saturated
`Not all
`the polyunsaturated acids of whale
`glyceiides by crystallization from acetone at
`oil behave as the 20:5 and 22:6 acids. The
`-500 (2). The concentrate was then subject-
`22:5 acid was present in the free fatty acids
`ed to pancreatic lipase hydrolysis. The results
`and was not enriched in the di- and triglyc-
`are presented in Table II~A.
`It can be seen
`erides, although like the 20:5 and 22:6 acids
`that, as compared to a level of about 22% in
`it was in low concentration in the monoglYC*
`the concentrate, there were only 7% and 8%
`eride products of hydrolysis (Table I). There-
`of the 20:5 acid in tlie free fatty acid and
`fore, the 22:5 acid must be considered as also
`monoglyceride fractions,
`respectively. There
`present
`in the 1,3-positions; but.
`In 00111135‘
`TABLE 11
`Th: Efloct of Randomization on the Products at Pancreatic Lipase Action
`on a Highly Unsaturated 1-‘raction.from Whale Oil Triglycerides
`(major tarry acids only)
`__+...__._.j.-————.——~
` <é—*—jj
`(C) Recalculation of (B) oniittins
`20:5 and 22:6
`
`—————-:—-———'-*“*‘
`(A) Whale DI] highly unsaturated ro=
`Concen-
`um
`(omml
`TG)
`
`Acid»
`
`4.9
`2.4
`15.6
`25.5
`3.5
`22.3
`4.6
`12.6
`
`§§§5E§§§mmu*—~ca
`
`_
`hd ‘
`Products of yruysls
`339
`TC’
`FA M0
`parentage
`6.3
`4.1
`1.1
`1.3
`34.5
`20.3
`27.0
`20.0
`3.2
`3.6
`8.2
`26.4
`0.9
`3.1
`2.0
`11.4
`
`4.1
`4.6
`16.9
`38.0
`2.3
`7.0
`8.0
`3.5
`
`4.7
`2.4
`12.1
`19.4
`3.1
`30.1
`4.3
`15.9
`
`unsaturated TG
`(B) Randomized whale on highly
`.—j— —
`Products of hydrolysis
`indRandom.
`Products of |1YdI01Y555
`MG DG
`'I‘G
`'l'G
`PA MG DG
`TG
`F‘
`percentage
`P¢t'€9I'll38=
`7.3
`4.6
`9.5
`7.7
`3.0
`2.0
`3.9
`3.4
`18.6
`14.6
`24.2
`14.5
`16.8
`17.1
`34.9
`28.7
`2.7
`4.5
`3.5
`7.6
`16.4
`29.7
`45
`52
`5.9
`8.7
`6.8
`10.8
`
`Random-
`ized
`7°
`
`4.6
`2.3
`14.1
`25.3
`3.1
`22.4
`4.8
`11.3
`
`8-5
`5.1
`24.0
`35.1
`1.5
`3.2
`3.6
`2.8
`
`4.3
`2.0
`12.9
`17.8
`3.9
`29.6
`5.1
`13.9
`
`6.9
`3.5
`21.3
`38.2
`4.7
`7.2
`
`9.0
`5-4
`25.5
`37.3
`1.6
`3.8
`
`7-5
`3-5
`21-3
`31-5
`6-9
`9-0
`
`RESISTANCE or MARINE Fnm Acios TO Pltuciuamc LIPASE Hvnnorvsis
`
`491
`
`TABLE 111
`Major Fatty Acid Components ofMenhade11 Oil Triglyc-
`erides and Its Lipase Hydrolysis Products
`
`TG
`
`Acid-
`
`ofigmi
`T6
`
`Products or hydrolysis”
`MG
`DG
`l-‘A
`percentage
`6.3
`7.7
`11.0
`14.2
`11.1
`14:0
`17.4
`14.7
`17.2
`24.9
`19.4
`16:0
`8.1
`9.!
`17.6
`13.6
`16.1
`16:1
`3.7
`3.4
`7.6
`3.2
`56
`18:0
`5.0
`4.7
`10.1
`5.7
`16.2
`16:1
`6.0
`6.6
`1.6
`2.8
`3.8
`20: I
`15.5
`22.3
`2.0
`11.4
`10.5
`20:5
`tr
`2.2
`0.6
`2.5
`1.4
`22.5
`
`
`
`
`1.5 15.1 15.07.322:6 16.0
`
`‘Chain lentzthznumber of double bond:
`5 FA -= Fatty Acids; MG = Monoglycerides; D6 = Di-
`glycerldes; TG - Triglycerides.
`
`were 26% in the diglycerides and 30% in the
`triglycerides. The results from the concentrate
`thus reinforce previous indicadons of resist-
`ance. The distribution of the 22:6 acid in the
`hydrolysis products also indicates
`resistance
`but to a somewhat lesser degree. The 22:5 acid
`was hydrolzed normally as shown by its rela-
`tively high level in the fatty acid fraction.
`In order to rule out position in the triglyc-
`eride molecule as the determining factor in the
`low degree of hydrolysis of the 20:5 and 22:6
`acids, an aliquot of the highly unsaturated
`concentrate was randomized by chemical treat-
`ment. Whale 011 offers unusual resistance to
`rearrangement by the use of standard proced-
`ures. Several combinations of catalysts, sol-
`vents and different
`times of treatment were
`tested before satisfactory results could be ob-
`tained. Sodium methoxide in methanol solu-
`tion produced methyl esters ditficult to separate
`from the randomized triglycerides. A xylene
`suspension of the same catalyst (8) was only
`partially effective. Lithium secondary butylate
`in dimethyl formamide solution (4) was found
`to be effective when the reaction period was
`prolonged for 3 days at room temperature.
`This procedure was therefore used. The ran-
`domized triglyceride products, purified by TLC,
`were analyzed by ‘GLC and subjected to pan-
`creatic lipase hydrolysis. The results are pre-
`sented in Table IIB. Since the fatty acid com-
`positions of the four products of hydrolysis
`are not similar, one might conclude that
`the
`randomization is
`incomplete. However,
`this
`criterion would only be valid if all the acids
`were equally susceptible to the lipase, a condi-
`tion which is not met due to the presence of
`the resistant 20:5 and 22:6 acids.
`If the data
`are recalculated omitting the 20:5 and 22:6
`acids or, in other words, making the n0nresist-
`
`ant acids equal to 100%, the figures shown in
`Table IIC are obtained. The quite similar con-
`centrations of the six major acids in all four
`fractions indicates effective randomization.
`The presence of significant amounts of 20:5,
`22:5, and 22:6 acids in the monoglycerides
`after, but not before randomization (Table
`IIB).
`indicates that
`they were not originally
`located in the 2-position in the whale oil
`tri-
`glycerides. Finally, the very low levels of 20:5
`and 22:6 acids in the free fatty acid fraction
`of the pancreatic lipase hydrolysis products of
`the randomized oil
`indicate that
`the reduced
`degree of hydrolysis of those acids is not due
`to the positional specificity of the enzyme, but
`is due to a characteristic of the fatty acid
`molecule itself.
`
`‘
`
`In order to compare the behavior of the 20:5
`and 22:6 acids in the pancreatic lipase hy-
`drolysis of whale oil with their behavior when
`located mainly in the 2-position as in fish oils,
`menhaden oil
`triglycerides were subjected to
`pancreatic lipase hydrolysis (Table III). The
`distribution of the 20:5 and 22:6 acids in the
`hydrolysis products of menhaden oil
`is dif-
`forent from that in whale oil products (Table
`1), although their concentrations in the two
`oils are quite similar. This is further evidence
`that the distribution of these acids in the two
`oils is different and that in whale oil hydrolysis
`their
`resistance to pancreatic lipase is
`inde-
`pendent of their position.
`It required about 2 min to attain 50% hy-
`drolysis of the untreated whale oils under the
`conditions used. An extended reaction time
`should increase the general degree of hydrolysis
`but leave higher concentrations of the resistant
`20:5 and 22:6 acids in the unhydrolyzed di-
`or
`triglycerides. This was found to be true
`only for 20:5, whose concentrations after 2,
`3, and 5 min of hydrolysis were 11.8, 14,4
`and 18.2% respectively in diglycerides and
`13.8, 13.3, and 19.7% respectively in lriglyc.
`erides. The concentration of 22:6 after 2, 3,
`and 5 min of hydrolysis was 5.9, 5.3, and 5.4%
`respectively in diglycerides and 11.0, 7.2, and
`6.7% respectively in triglycerides. The lack
`of increase in percentage of 22:6 in the di.
`and triglycerides with time might be due [0 its
`having approached maximum levels at the 2.
`min period.
`It was also found that the rate of hydrolysis
`decreased appreciably after half the triglyceride
`acids were released. This is a logical conse-
`quence of distribution in the 1,3-position of
`the 20:5 and 22:6 acids,
`their resistance to
`hydrolysis, and the reported presence of the
`Lirins, VOL. 2, No. 5
`
`. FA = pmy Acids; M6 = Monoglyoerides; D6 =- Dislycerides; TG= TliElY¢¢'ld¢5-
`s ci,..inlengtl1:numhe: or double bonds.
`-
`LIPIDS, VOL. 2, No. 6
`
`00000116
`
`00000116
`
`

`
`492
`
`Nesron R. BOTITND. Gtoiuit A. Vanoansuno AND Ramona REISER
`
`Rssrsritncia on MARINE Fitmr Acms T0 Pancnsxrrc Lmsn HYDROLYSIS
`
`493
`
`C2, and C“ acids in only 50% of whale oil
`triglycerides (2).
`Proof of Resistance
`the 20:5 acid
`Proof of the resistance of
`(and by inference of the 22:6 acid) was ob-
`tained by study of the action of pancreatic
`lipase on synthetic 1,2-di-octadecenoyl-3-eico
`sapentaenoyl glycerol. The results are present-
`ed in Table IV. The fatty acid compositions
`of the triglycerides before lipase hydrolysis and
`of the monoglyceride and triglyceride products
`of hydrolysis show that
`the substance synthe-
`sized is,
`in fact, 1,2-di-octadecenoyl-3-eico
`sapentaenoyl glycerol, with some contamina-
`tion due to impurities in the starting materials.
`The experimental values for the composition
`of the fatty acid and diglyceride fractions are
`closer to the values calculated on the assump-
`tion of resistance than on the assumption of
`nonresistance. The small amount of mono-
`giycerides produced is another indication of
`resistance. The presence of 17% 20:5 acid
`in the fatty acid fraction indicates that some
`hydrolysis of that acid took place. This could
`be due to the resistance to the enzyme D0‘
`being absolute,
`to the presence of a hydrolyz-
`able isomer of the 20:5 acid in the starting
`material, or to an alteration in the structure of
`the all as 20:5 acid during the chemical syn-
`thesis of the triglyceride. Analyses of the start-
`ing material showed that there were 9% im-
`purities as ascertained by GLC and that only
`75% of the theoretical amount of glutaric acid
`was produced by KMn0, oxidation in acetic
`acid medium (9). Examination of the original
`20:5 acid and the 1,2-di-octadecenoyl-3-eico
`sapentaenoyl glycerol by infrared spectrom-
`etry showed that only traces of trans isomer-
`ization occurred during the synthesis.
`Mechanism of Resistance
`It is evident that in spite of being located at
`the’l,3-positions of the whale oil triglycerides,
`the 20:5 and 22:6 acids resist pancreatic lipase
`hydrolysis while the 22:5 acid is hydrolyzed
`without difficulty. The explanation for
`this
`phenomenon may lie in differences in their
`molecular structures:
`
`20:5 cH,cH,(cn=cHcH.), — cH,cH,
`coon
`22:5 cn.cH,(cn:cHcH,), ~ CI-i,CH,
`cH._crr,cooH
`,
`22:6 Cl-l.CH,_’(CI~{=—_CHCl-l,), ~ CH=CH
`cn.cH.coaoH
`In view of the evidence presented by others
`(10)
`the «:3 structure is assumed for these
`IJPIDS, VOL. 2, No. 6
`
`Acid“
`
`TABLE TV
`Products of the Action of Pancreatic Lipase on
`l,2-Di—octadeccnoyl-3-cicosapentaenoyl Glycerol
` ._eé_T‘
`original
`Products of hydrolysis:
`TG
`Flt
`MG
`DG E
`Mole pcrcenl=
`Theoretical (nonreslsunce)
`59,
`69.7
`54.5
`100
`77.7
`13:1 + impur.“
`30:,
`10.:
`45.4
`o
`12.3
`20:5
`Theoretical (absolute resistance)
`597
`69.7
`100
`I
`54.5
`18:1 + impur.
`m’, ~
`20:;
`o
`-
`45.5
`20:5
`Experimental
`730
`71.5
`33.0
`99.1’
`60.8
`lllzi + impur.
`
`
`23.5 17.0 0.9! 39.220:5 pf.)i
`
`
`
`“FA = Fatty Acids; MG =- Monoglyccrides; Dq ,
`Diglycerides; ‘T6 = Triglycerides.
`l7Chain lengthzdouble bond.
`<'l‘he detector response to the 20:5 acid was found to
`be 0.83 times that of the 18.1. However, no correction was
`applied since it would have had no significant cifcct on
`the conclusions.
`‘The preparation of 20:5 acid used had 8.9% impurities
`of other _fatty acids. Since they are not expected to be
`an .
`lipase resistant, their percentages are added to that of cm
`-No MG should be obtained
`‘Very small amount of MG obtained.
`
`three acids. Since their terminal 17 carbon
`chains are identical, any differences
`in be-
`havior must be assumed to be caused by dif-
`ferenccs in their structure at thecarboxyl end
`of the chain. The responsible factor could be
`the proximity of the double bond to the car-
`boxyi group, since the first double bond of the
`resistant 20:5 and 22:6 acids lies closer
`to
`the carboxyl group than does that of the non-
`resistant 22:5 acid. This view is strengthened
`by the demonstration by Kieiman et ai. (ii)
`that the trans-3-enoic acids of Grindelia oxy-
`Iepis seed oil are also resistant
`to lipase hy-
`drolysis. The presence of methyl groups in a
`position close to the carboxyl end has also
`been shown to hinder hydrolysis by the lipase
`(12).
`Another difference in structure between the
`resistant and the susceptible polyunsaturated
`acids lies in the space relations of their term-
`inal methyl to their carboxyl groups. As shown
`in the photographs of the molecular models
`(Figure 1)
`the terminal methyl groups of the
`resistant acids lie close to their carboxyl groups.
`This proximity may cause a stcric hindrance
`effect on the hydrolysis by the lipase.
`Metabolic implications
`The resistance of some of the polyunsat-
`urated fatty acids of whale oil to pancreatic
`lipase hydrolysis provides an explanation for
`the finding by Garton et al. (13) that Whalfi
`
`
`
`A
`
`Fro.
`
`1. Molecular models of the 20:5 (A), 22:6 (B), and 22:5 (C) acids of marine oils.
`
`oil can be crystallized almost unchanged from
`the depot tissues of pigs fed high doses of the
`oil for a prolonged period of time.
`in pre-
`liminary experiments in this laboratory, how-
`ever, neither the triglycerides, nor the phos-
`pholipids of thoracic duct lymph of rats admin-
`istered by stomach tube one dose of the highly
`unsaturated concentrate of whale oil, contained
`the marine long-chain polyunsaturated acids.
`The presence of whale glycerides in the tissues
`of Garton’s pigs may have been the product
`of a low degree of intestinal absorption over
`along period of ingestion.
`ACKNOWLEDGMENT
`Supported in part by
`a grant
`from the National
`institute of Health (AM-06011).
`
`REFERENCES
`1. Bottlno. N. R., G. Vmdcrbitrgh and Raymond Raiser,
`Federation Pine 25, Jill
`(1966).
`
`1>., and L. Maddison_ 1, 50¢ chm,
`2 Hilditch, r.
`Ind. (Trans.) 61, 159-173 (1942); Ibid. 67, 253.257 (1943).
`3- muddy. F. E. K A. Barford. s. 1-: Herb, r. Magm-
`
`i046-l(l5l
`
`IAOCS 42,
`
`1(1;2;1;4)and R. W. Riememchneider, JAocs 41, 593.595
`4. Erockerhofl.
`l-l., Arch Biochem. Biophys. 110, 536-
`592 (I965).
`5. Brockerhofi. R, and R. I. Hoyle, Arch. Btochern.
`Biophys. 102, 452-455 (1963).
`6. Dolev, A., and H. S. Olcott.
`(1965).
`7. Yurkowski, M.. and H. Erocl(erhofl_ Biochini. lliophys,
`Acta l25, 55-59 (1966).
`ll. Ecitey, E. W., ind. Eng. Chem. 40. H83-l I96 (194%).
`9. Rain, P.
`l(., personal communication.
`10. Acionan, R. G., C. A. Eaton and P. M. Jangaard,
`Can. I. Biochcrn. 43. l5l3-1520 (1965).
`ll. Klciman, R..
`I-'. R. Ecarle and I. A. Woifl (Ab-
`stract) MOCS 42, WA (1965).
`12. Blornstrand. R., N. Tryding and G. Wesriiti, Acta
`Physiol. Scand. 37, 91-96 (I956).
`'13. Garton, G. A.,
`'1'. P. llflyditch and M. L. Meal-a_
`Biochem. J. 50, 5l7-524 (1952).
`
`[Received lune I2, 1967]
`
`Lrrros, VOL. 2, No. 6
`
`00000117
`
`00000117
`
`

`
`U.S.S.N. 95/001,774
`
`Declaration of Dr. Jacek Jaczynski
`
`Appendix L
`
`Hernell et 211., “Does the Bile Salt-
`Stimulated Lipase of Human Milk Have a
`Role in the Use of the Milk Long-Chain
`Polyunsaturated Fatty Acids?” J Pediatr 0
`Gastroenterol Nutr 16: 426-31(1993)
`
`00000118
`
`00000118
`
`

`
`Journal of Pediatric Gastroenterology and Nutrition
`16:426—43l © 1993 Raven Press, Ltd., New York
`
`Does the Bile Salt-Stimulated Lipase of Human Milk Have
`a Role in the Use of the Milk Long—Chain Polyunsaturated
`Fatty Acids?
`
`Olle Hernell, *Lars Blackberg, ’rQi Chen, TBerit Sternby, and TAICC Nilsson
`
`Departments of Pediatrics and *Medical Biochemistry and Biophysics, University of Umezi, Umed; and TDepartment
`of Medicine, University of Lund, Lund, Sweden
`'
`
`
`
`arachidonic acid and eicosapentaenoic acid (20:5 n-3) re-
`Summary: Long-chain polyunsaturated (LCP) fatty acids
`lease rates with either colipase-dependent lipase or
`derived from linoleic (18:2 n-6) and oz-linolenic 08:3 n-3)
`BSSL, albeit the release was more rapid with the milk
`acids are considered essential nutrients in preterm in-
`enzyme than with colipase-dependent lipase. Again, the
`fants. The efficiency by which such fatty acids are re-
`most efficient release as absorbable free fatty acids was
`leased as absorbable products from triacylglycerol was
`achieved when the two lipases operated together. The
`explored in vitro using rat chylomicron triacylglycerol as
`relative resistance to hydrolysis of arachidonic acid and
`‘ substrate. When incubated with purified human pancre-
`eicosapentaenoic acid by colipase-dependent lipase was
`atic colipase-dependent lipase and colipase, arachidonic
`best explained by the localization of the first double bond
`acid (20:4 n-6) was released less efficiently than linoleic
`to the 8-5 position of the respective fatty acid. The results
`acid from such triacylglycerol. This difference was not
`obtained suggest that BSSL is of importance for the effi-
`seen when purified human milk bile salt-stimulated lipase
`cient use of human milk LCP fatty acids. Key Words: Bile
`_(BSSL) was incubated with the triacylglycerol substrate,
`salt-stimulated lipase»-—Colipase-dependent 1ipase———Long-
`and it was almost abolished when colipase-dependent li-
`chain polyunsaturated fatty acids-——Fat digestion--Breast-
`pase (with colipase) and BSSL acted simultaneously, as
`fed infant.
`they do in breast-fed infants. There was no difference in
`
`
`In recent years there has been an increasing
`awareness that long-chain polyunsaturated (LCP)
`fatty acids may be an essential nutrient for preterm
`infants. The reason is that preterm infants have a
`relatively low capacity to synthesize these deriva-
`tives from the precursor fatty acids, that is, linoleic
`acid (l8:2 n-6) of the n-6 series and Ot-llIlOlC1’llC acid
`(18:3 n-3) of the n-3 series (1,2).
`Some LCP fatty acids, e.g., dihomo-7-linolenic
`acid (20:3 n-6) and arachidonic acid (20:4 n-6), both
`derived from linoleic acid, and eicosapentaenoic
`acid (20:5 n-3), derived from on-linolenic acid, are
`precursors of the biologically active eicosanoids.
`
`
`Address correspondence and reprint requests to Dr. 0.
`Hernell, Department of Pediatrics, University of Umea, S-901 87
`Umeé, Sweden.
`Manuscript received April 9, 1992; revision received July 13,
`1992; accepted January 4, 1993.
`
`LCP fatty acids, particularly arachidonic acid and
`docosahexaenoic acid (2226 n-3), are also qualita-
`tively important constituents of membrane phos-
`pholipids (3), the proportion being particularly high
`in the gray matter of the brain and in the photore-
`ceptor cells of the retina (4-6). Hence, the require-
`ment for these fatty acids is particularly high during
`the period of rapid development of the central ner-
`vous system, that is, the last trimester of pregnancy
`through early infancy. Calculations have shown.
`that in fully breast-fed preterm infants the content
`of these LCP fatty acids in human milk suffice to
`meet the requirements for tissue accretion (1,7). Re-
`cently it has been recommended to supplement in-
`fant formulas intended for preterm infants with LCP
`fatty acids approximating the amount and composi-
`tion of human milk (8,9). Supplementation with
`only n-3 LCP fatty acids causes depletion of n-6
`
`426
`
`00000119
`
`00000119
`
`

`
`BSSL AND USE OF LCP FATTY ACIDS
`
`427
`
`LCP fatty acids, that is, arachidonic acid (8,l0,
`11).
`There is still limited information about the path-
`ways by which these fatty acids are used when oc-
`curring in human milk, or in infant formulas supple-
`mented with lipids containing such fatty acids. In
`human milk, LCP fatty acids are enriched in the
`phospholipid fraction. Because triacylglycerol con-
`stitutes >98% of the milk lipids, most of the milk
`LCP fatty acids are carried by this lipid fraction
`(12). In the present article we have used chylomi-
`cron triacylglycerol containing labeled linoleic and
`arachidonic acids, or labeled arachidonic and eicos-
`apentaenoic acids, as a model substrate for incuba-
`tions in vitro with purified human pancreatic coli-
`pase-dependent lipase and bile salt—stimulated milk
`lipase, the two lipases involved in intestinal fat di-
`gestion in breast-fed infants (13,14). We have thus
`compared the relative rates of release of these fatty
`acids as absorbable products when incubated with
`either lipase alone or with the two enzymes in com-
`bination.
`
`‘
`
`MATERIALS AND METHODS
`
`Preparation of Enzymes and
`Radioactive Chylomicrons
`
`Human pancreatic colipase-dependent lipase (15)
`and colipase (16) were purified according to Sternby
`and Borgstrom and bile salt-stimulated lipase
`(BSSL) was purified from human milk as described
`by Blackberg and Hernell (17). [1-MC]-labeled 18:2
`n-6 (52.6 mCi/mmol), [5,6,8,9,1l,12,14,'15-3H]-
`labeled 20:4 n-6 (83.8 Ci/mmol), and [5,6,8,9,11,
`12,14,15,17,18-3H]-labeled 20:5 n-3 (79.0 Ci/mmol)
`' were purchased from New England Nuclear.
`In the first series of experiments, chylomicrons
`were obtained by feeding a mesenteric lymph duct
`cannulated rat (18) 50 p.Ci [”C]18:2 n-6 and 50 p.Ci
`[3H]20:4 n-6 dispersed in 1 ml 20% Intralipid (Kabi
`Vitrum AB, Stockholm, Sweden) through a gastric
`fistula 24 h after cannulation as described (19). In a
`second set of experiments, 50 p.Ci [”C]20:4 n-6 and
`50 p.Ci [3H]20:5 n-3 were mixed with 1 ml of a chlo-
`roform solution containing 1' mg egg phosphatidyl-
`choline/ml. The solvent was evaporated under a
`stream of N2 and immediately dispersed in 0.9%
`NaCl by buzzing. The dispersion was then mixed
`with 10% (vol/vol) fish oil triacylglycerol (MAX-
`EPA, Naturprodukter, Orebro, Sweden) emulsion
`prepared by ultrasonication of 1% gum arabic in
`
`0.9% NaCl for 2 min. The fish oil contained as ma-
`jor n-3 fatty acids 20:5 n-3 (16.5%) and 22:6 n-3
`(12.3%). The fish oil emulsion thus prepared (2 ml)
`was fed via a gastric fistula to another lymph duct
`cannulated rat. After feeding the radioactive com-
`pounds, chyle was collected on ice. Na; EDTA was
`added to a final concentration of 2 mM, and the
`chyle was then stored at 4°C. After the chyle had
`been diluted with 1.1% NaCl containing 2 mM
`EDTA, chylomicrons were-isolated by ultracentrif-
`ugation at 25,000 rpm for 2 h at 4°C using a Beck-
`man SW 41 swinging bucket rotor (19).
`
`Incubation Conditions and Lipid Analysis
`
`The incubation mixture was composed of 10 mM
`Hepes buffer (pH 7.4), 2.5 mM CaCl2, and 0.12 M
`NaCl containing 2.0 mM sodium taurodeoxycholate
`(NaTDC) and 1.5 mM sodium taurocholate (NaTC).
`Incubations were performed at 37°C in a total vol-
`ume of 3 ml with purified human colipase-
`dependent lipase and colipase, with purified BSSL,
`or with the two lipases in combination. The amount
`of each enzyme added is given in Figs.
`1 and 2.
`After preincubating the medium with the enzyme(s)
`for 5 min, the labeled chylomicrons were added.
`During the following 60 min of incubation, aliquots
`were withdrawn at various time intervals as indi-
`
`cated in Figs. 1 and 2. The lipids were immediately
`extracted with 7-8 vol of chloroform/methanol (1 : 1,
`vol/vol) containing 0.005% butylated hydroxytolu-
`ene. The lipid fractions were separated by thin-
`layer chromatography,
`the spots visualized by
`staining with iodine, the different lipid fractions (tri-
`acylglycerol, diacylglycerol, monoacylglycerol, and
`free fatty acid) transferred into counting vials, and
`the radioactivity determined as described (20). The
`triacylglycerol content of the chylomicron sub-
`strates was determined by a colorometric enzymat-
`ic kit method (Boehringer-Mannheim GmBH).
`Data are expressed as percentage of total radio-
`activity (“C and 3H, respectively) present in each
`lipid class. All values are means of duplicate sam-
`ples. Although radioactivity also was present in po-
`lar lipids, this fraction remained stable during the
`incubations (data not shown).
`
`RESULTS
`
`Hydrolysis of [3H]arachidonic and [“C]linoleic
`Acid-Labeled Chylomicrons
`
`When colipase-dependent lipase (together with
`colipase) was incubated with [”C]18:2 n-6- and
`
`J Pediarr Gastroenterol Nutr, Vol. 16, Na. 4, 1993
`
`00000120
`
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`

`
`428
`
`0. HERNELL ET AL.
`
`TG(.)FFA(V)
`
`(%) DG(0)MG(V) g
`DlSTRlBU-“ONOFRADIOACTIVI-lY
`
`30
`
`but not 18:2, accumulated in the diacylglycerol frac-
`tion; >25% as compared with <10% after 30 min of
`incubation and still a more than twofold higher con-
`centration after 60 min (Fig. 1d). No obvious differ-
`ence in accumulation in the diacylglycerol fraction
`was observed when the two lipases opera