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On the regulation of lipolysis in an insect egg observations in vitro
BiachOnica et Biophysicu Acta, g Eisevier Scientific Publishing
296 ( I 973) 466-470 Company, Amsterdam
- Printed
in The Netherlands
BBA 56214
ON THE REGULATION
OF LIPOLYSIS
OBSERVATIONS
IN VITRO*
JAMES
and PAUL
L. KRYSAN
October
znd,
EGG
L. CUSS
Northern Grain Insects Research Laboratory, Agriculture, Brookings S.D. 57006 (U.S.A.) (Received
1N AN INSECT
Agriculturul
Research
Service,
U.S.
Department
of
I 972)
SUMMARY
The triglycerides of eggs of the western corn rootworm (Diabvotica virgfira LeConte, Coleoptera: Chrysomelidae) are unavailable to an apparently freely active triglyceride lipase (glycerol ester hydrolase, EC 3. I. I .3) in isotonic homogenates. Homogenization of the eggs in a hypotonic medium or treatment of isotonic homogenates with freeze-thaw or sonication will release the triglycerides permitting hydrolysis by endogenous lipase. The observations suggest that a case of structure-linked substrate latency has been observed.
INTRODUCTION
The insect egg is a closed system (respiration and water exchange excepted) which is rich in yolk to supply the energy and structural metabolites required for embryogenesis. In most insect species, most energy used for embryogenesis is derived from triglycerides and the pattern of lipid metabolism has been determined for the eggs of several insects (reviewed by Agreil’, Kinsella’, Gilbert3, and Fast4). A dearth of information exists concerning the mechanisms of mobilization of insect-egg triglycerides. Lipolysis is presumably an early step in the process and indeed Fodor’ observed fipase activity in grasshopper eggs, and several workers have ascribed to lipase the hydrolysis of short chain triglycerides by insect egg preparations6-‘. We have recently reported the presence of lipase in eggs of the western corn rootworm (Diahrofica virgtfera LeConte)‘. This report describes experiments done in vitro which suggest that in western corn rootworm eggs the triglyceride is latent, i.e. it is unavailable to an otherwise active lipase; presumably this triglyceride latency is related to the regulation of lipolysis irr viva. * In cooperation
with the South
Dakota
Agricultural
Experiment
Station.
LIPOLYSIS
IN INSECT
METHODS
AND
EGG
467
MATERIALS
Western corn rootworm eggs (obtained as described by Howe and George”) were homogenized using all glass tissue grinders. Generally, 50 eggs were homogenized in 1 ml of homogenization medium ; 0.8 ml of the homogenate was transferred to a test tube and incubated at 25 “C for 30 min in a shaking device oscillating at 140 cycles/s, 2 cm/cycle. Following incubation, the reaction was stopped and the amount of free fatty acids present determined by previously described methods (ref. I I). Thinlayer chromatography of the reaction products (extracted by the method of Bligh and Dyer’ ‘) was done by the method of Guss’ 3. Freeze-thaw steps were done by immersing the test tubes containing the homogenates in a mixture of solid CO, and acetone. Sonication was done in the homogenizer mortar (after homogenization) using a Bronwill sonifier equipped with a microprobe set at minimal power. The sample was held in an ice-water bath during the 20-s sonifying period. Homogenates treated by sonication or freeze-thaw, or homogenized in a hypotonic medium, are called disrupted, a term descriptive of what presumably happens to the egg structures. Radioactively labeled triolein (obtained from Applied Sciences Laboratories) and the label distribution determined by thin-layer chromatography and liquid scintillation spectrometry to be 97. I 8 ‘A triglyceride, 0.73 ‘A diglyceride, 0.37 % monoglyceride, I .7 I % free fatty acid) was added to the system by placing a hexane solution of glyceryl tri[ I-“C]oIeate into the homogenizer mortar and evaporating the hexane with N,. Following this loo eggs and 2 ml of isotonic medium were added, the eggs were homogenized, but the homogenate was left in the mortar during the incubation which was carried out as described above. Aliquots of the reaction medium were extracted by the method of Bligh and DyerI’. From each reaction tube, one aliquot was subjected to thin-layer chromatography, the lipid spots visualized with I, vapor, the plate marked and, following sublimation of the I,, the individual spots were scraped into scintillation vials. 2 ml of methanol were added followed by the addition of scintillation solution 0.5 h later after which the radioactivity was determined by liquid scintillation spectrometry. Another aliquot of each sample was placed directly into a scintillation vial, the solvent evaporated, and prepared for counting as above. This latter sample served as the control to determine the efficiency of recovery of radioactivity from the silica scrapings following thin-layer chromatography. From 82 to 91 “i;‘,of the radioactivity was recovered. The hypotonic medium was 0.001 M Tris-maleate (pH 7.0). Our so-called isotonic medium consisted of I I g NaCl and I .4 g KC1 dissolved in I 1 of 0.001 M Tris-maleate buffer (pH 7.0). For convenience, we call it isotonic because it was derived from an insect Ringer’s formula; however, we do not know and cannot readily determine whether it is precisely isotonic with reference to the lipid vesicles. RESULTS
AND
DISCUSSION
The basis of our experimental system was the hydrolysis of endogenous triglycerides by endogenous Iipase in egg hemogenates. The extent of Iipolysis in a homogenate depended on the tonicity of the homogenizing medium and on the manner in which the homogenate was treated. Eggs boiled before homogenization, incubation,
J. L. KRYSAN,
468 TABLE
I
EFFECT OF DISRUPTIVE TREATMENTS GLYCERIDES __~_ Experimental conditions* Expt
_~~~___~ 1 2
3 4 5
P. L. CUSS
ON LIPOLYSIS
EGG
TRI-
Free fbtty acids released in 30 mitt (pmoles)
~~~
Isotonic homogenate Hypotonic homogenate Isotonic homogenate; disrupted by sonication Isotonic homogenate; disrupted by freezing Isotonic homogenate; 3o-min delay before disruption by freezing * See text for experimental
OF ENDOGENOUS
Mean
S.D.
(3 exptsl ____ 0.041 0.310
0.007 0.042
0.363 0.337
0.037 0.014
0.265
0.017
~~
details.
and extraction yielded no measurable free fatty acids. When eggs were homogenized in an isotonic solution, little lipolysis occurred (Table I, Expt I, and Fig. I, Lane 3). When the solution was hypotonic, considerable lipolysis occurred (Table I, Expt 2, and Fig. I, Lane 4). Incidentally, one can conclude from the observations in Fig. I
Fig. 1. Thin-layer chromatographic separation of the lipids of disrupted and non-disrupted egg homogenates. I, mixture of reference compounds; (a) sterol esters, (b) triglycerides, (c) free fatty acids, (d) 1.3,-diglycerides, (e) 1,2,-diglycerides and free sterols, (f) monoglycerides, (g) unresolved polar lipids; 2, lipids from eggs homogenized in an acidified isotonic medium and extracted immediately; 3, lipid extract from eggs homogenized in an isotonic medium and incubated for 30 min at 25 “C prior to extraction; 4. lipid extract from eggs homogenized in a hypotonic medium and incubated for 30 min at 25 “C prior to extraction; 5, lipid extract from eggs homogenized in an isotonic medium, sonified for 20 s, incubated for 30 min at 25 “C. and extracted. Fig. 2. The effect of freeze-thaw treatment on the appearance of free fatty acids in isotonic egg homogenates. Western corn rootworm eggs (500 per homogenate) were homogenized in IO ml of isotonic medium; one-half of the homogenate was incubated at 25 “C (- -); the other one-half was frozen in a solid CO,-acetone mixture, thawed, and then incubated (&). At the indicated time intervals a o.8-ml aliquot was withdrawn, the reaction stopped, and the free fatty acids determined. Each point is the mean of four such experiments. The vertical bars represent the S.D.
LIPOLYSIS
IN INSECT
EGG
469
that the free fatty acids observed quantitatively (Table I) were in fact derived from triglycerides. The results of the previously described experiments suggested that isotonic conditions protected a structure from rupture and therefore blocked lipolysis, but the isotonic medium could have prevented lipolysis. This latter explanation was ruled out by the observation that lipolysis occurred under isotonic conditions following either sonication or freeze-thaw (Table I, Expts 3 and 4, and Fig. I, Lane 5). The relationship between time and the appearance of free fatty acids in homogenates prepared in the isotonic medium, with and without a freeze-thaw step, is illustrated in Fig. 2. Even after I h of incubation, very little free fatty acid appeared in the non-disrupted homogenate. In artificial substrate systems, rootworm egg lipase requires a protective agent to avoid denaturation’. Conceivably the lack of lipolysis in the non-disrupted isotonic medium was due to denaturation of the enzyme. To test this hypothesis, we homogenized eggs and incubated the homogenate for 30 min before tissue disruption and subsequent incubation to permit lipolysis. The results (Table I, Expt 5) show that lipolysis occurred and therefore lack of lipolysis under isotonic conditions was not due to enzyme denaturation. The previously described experiments established that the endogenous lipase would not hydrolyze endogenous triglyceride in a non-disrupted isotonic homogenate and therefore we concluded that a structural barrier prevented lipolysis. We conducted a series of experiments to test the hypothesis that either the enzyme, or an enzyme activator, was held in an inactive form by a membraneous structure. Negative results in those experiments (unpublished results) led us to evaluate the availability of the endogenous triglyceride. We added radioactively labeled triolein (27.3 Ci/mole) to a homogenate prepared in the isotonic medium. In the 3o-min incubation period, less than 5 % of the radioactivity recovered was triolein (Table II).
TABLE
II
LIP0LYS.S OF EXOGENOUS 30 min INCUBATION FrUCti#n
TRIOLEIN
IN
Percent
dpm in each fraction
Heated
control*
Triglyceride Diglyceride
96. to
Monoglyceride Free fatty acid
ISOTONIC
Experimental 6.28 5.84 7.80 82.62
EGG
HOMOGENATES
AFTER
S. D. 2.10
0.96 0.73 5.64
* Eggs heated to IOO “C for IO min before homogenization in presence of radioactive and subjection to further treatment as described in Methods and Materials section.
triolein
The endogenous triglyceride was not hydrolyzed; the thin-layer chromatographic pattern of the bulk lipid in this experiment as seen by I, staining is typified by the chromatogram shown in Fig. I, Lane 3. We conclude from this last experiment that in our homogenate system the enzyme is freely active and the substrate is latent. The substrate latency is probably membrane-based because the factors (hypotonicity, freeze-thaw and sonication) that
410
J. L. KRYSAN.
P. L. CUSS
permit lipolysis can disrupt membranes. We suggest that the barrier surrounding the triglyceride, as we have observed it in vitro, plays a role in the regulation of lipolysis in vivo. That suggestion is consistent with the speculations of Lui and Davis14. Based upon ultrastructural observations they speculated that the specific structure of lipid droplets in black fly tissues function in the regulation of lipid utilization. REFERENCES I Agrell, I. (1964) in The Physiology of Insecta (Rockstein, M., ed.), Vol. I, pp. 91-148, Academic Press, New York and London 2 Kinsella, J. E. (1966) Camp. Biochem. Physiol. 19, 291-304 3 Gilbert, L. I. (1967) in Advances in Insect Physiology (Beament, J. W. L., Treherne, J. E. and Wigglesworth, V. B., eds), Vol. 4, pp. 69-21 I, Academic Press, London and New York 4 Fast, P. G. (1970) in Progress in the Chemistry of Fats and other Lipids (Holman, R. T., ed.), Vol. 11 (Part 2), pp. 179-244, PergamOn PreSS, Oxford 5 Fodor, P. J. (I 947) Enzymologia I 2, 343-349 6 Gaeta, 1. and Zappanico, A. (1959) Ric. Sci. 29, 788-791 7 Carlson, L. D. (1941) Biol. Bul/. 81, 375-387 8 Urbani, E. (1962) in Advances in Morphogenesis (Abercrombie, M. and Brachet, J., eds), Vol. 2, pp. 61-108, Academic Press, New York and London 9 Guss, P. L. and Krysan, J. L. (1972) J. Insect Physio/. 18, 1181-1195 IO Howe, W. L. and George, B. W. (1966) in Insect Colonization and Moss Production (Smith. C. N.. ed.), pp. 367-383, Academic Press, New York and London I I Krysan, J. L. and Guss, P. L. (1971) Biochim. Biophys. Acta 239, 349-352 12 Bligh, E. G. and Dyer, W. J. (1959) Can. J. Biochem. Physiol. 37. 911~917 13 Guss, P. L. (1969) Ann. Entornol. Sot. Am. 62, 675-676 14 Lui,T. P.andDavies, D. M.(r97z)J. LipidRes. 13, 115-118
296 ( I 973) 466-470 Company, Amsterdam
- Printed
in The Netherlands
BBA 56214
ON THE REGULATION
OF LIPOLYSIS
OBSERVATIONS
IN VITRO*
JAMES
and PAUL
L. KRYSAN
October
znd,
EGG
L. CUSS
Northern Grain Insects Research Laboratory, Agriculture, Brookings S.D. 57006 (U.S.A.) (Received
1N AN INSECT
Agriculturul
Research
Service,
U.S.
Department
of
I 972)
SUMMARY
The triglycerides of eggs of the western corn rootworm (Diabvotica virgfira LeConte, Coleoptera: Chrysomelidae) are unavailable to an apparently freely active triglyceride lipase (glycerol ester hydrolase, EC 3. I. I .3) in isotonic homogenates. Homogenization of the eggs in a hypotonic medium or treatment of isotonic homogenates with freeze-thaw or sonication will release the triglycerides permitting hydrolysis by endogenous lipase. The observations suggest that a case of structure-linked substrate latency has been observed.
INTRODUCTION
The insect egg is a closed system (respiration and water exchange excepted) which is rich in yolk to supply the energy and structural metabolites required for embryogenesis. In most insect species, most energy used for embryogenesis is derived from triglycerides and the pattern of lipid metabolism has been determined for the eggs of several insects (reviewed by Agreil’, Kinsella’, Gilbert3, and Fast4). A dearth of information exists concerning the mechanisms of mobilization of insect-egg triglycerides. Lipolysis is presumably an early step in the process and indeed Fodor’ observed fipase activity in grasshopper eggs, and several workers have ascribed to lipase the hydrolysis of short chain triglycerides by insect egg preparations6-‘. We have recently reported the presence of lipase in eggs of the western corn rootworm (Diahrofica virgtfera LeConte)‘. This report describes experiments done in vitro which suggest that in western corn rootworm eggs the triglyceride is latent, i.e. it is unavailable to an otherwise active lipase; presumably this triglyceride latency is related to the regulation of lipolysis irr viva. * In cooperation
with the South
Dakota
Agricultural
Experiment
Station.
LIPOLYSIS
IN INSECT
METHODS
AND
EGG
467
MATERIALS
Western corn rootworm eggs (obtained as described by Howe and George”) were homogenized using all glass tissue grinders. Generally, 50 eggs were homogenized in 1 ml of homogenization medium ; 0.8 ml of the homogenate was transferred to a test tube and incubated at 25 “C for 30 min in a shaking device oscillating at 140 cycles/s, 2 cm/cycle. Following incubation, the reaction was stopped and the amount of free fatty acids present determined by previously described methods (ref. I I). Thinlayer chromatography of the reaction products (extracted by the method of Bligh and Dyer’ ‘) was done by the method of Guss’ 3. Freeze-thaw steps were done by immersing the test tubes containing the homogenates in a mixture of solid CO, and acetone. Sonication was done in the homogenizer mortar (after homogenization) using a Bronwill sonifier equipped with a microprobe set at minimal power. The sample was held in an ice-water bath during the 20-s sonifying period. Homogenates treated by sonication or freeze-thaw, or homogenized in a hypotonic medium, are called disrupted, a term descriptive of what presumably happens to the egg structures. Radioactively labeled triolein (obtained from Applied Sciences Laboratories) and the label distribution determined by thin-layer chromatography and liquid scintillation spectrometry to be 97. I 8 ‘A triglyceride, 0.73 ‘A diglyceride, 0.37 % monoglyceride, I .7 I % free fatty acid) was added to the system by placing a hexane solution of glyceryl tri[ I-“C]oIeate into the homogenizer mortar and evaporating the hexane with N,. Following this loo eggs and 2 ml of isotonic medium were added, the eggs were homogenized, but the homogenate was left in the mortar during the incubation which was carried out as described above. Aliquots of the reaction medium were extracted by the method of Bligh and DyerI’. From each reaction tube, one aliquot was subjected to thin-layer chromatography, the lipid spots visualized with I, vapor, the plate marked and, following sublimation of the I,, the individual spots were scraped into scintillation vials. 2 ml of methanol were added followed by the addition of scintillation solution 0.5 h later after which the radioactivity was determined by liquid scintillation spectrometry. Another aliquot of each sample was placed directly into a scintillation vial, the solvent evaporated, and prepared for counting as above. This latter sample served as the control to determine the efficiency of recovery of radioactivity from the silica scrapings following thin-layer chromatography. From 82 to 91 “i;‘,of the radioactivity was recovered. The hypotonic medium was 0.001 M Tris-maleate (pH 7.0). Our so-called isotonic medium consisted of I I g NaCl and I .4 g KC1 dissolved in I 1 of 0.001 M Tris-maleate buffer (pH 7.0). For convenience, we call it isotonic because it was derived from an insect Ringer’s formula; however, we do not know and cannot readily determine whether it is precisely isotonic with reference to the lipid vesicles. RESULTS
AND
DISCUSSION
The basis of our experimental system was the hydrolysis of endogenous triglycerides by endogenous Iipase in egg hemogenates. The extent of Iipolysis in a homogenate depended on the tonicity of the homogenizing medium and on the manner in which the homogenate was treated. Eggs boiled before homogenization, incubation,
J. L. KRYSAN,
468 TABLE
I
EFFECT OF DISRUPTIVE TREATMENTS GLYCERIDES __~_ Experimental conditions* Expt
_~~~___~ 1 2
3 4 5
P. L. CUSS
ON LIPOLYSIS
EGG
TRI-
Free fbtty acids released in 30 mitt (pmoles)
~~~
Isotonic homogenate Hypotonic homogenate Isotonic homogenate; disrupted by sonication Isotonic homogenate; disrupted by freezing Isotonic homogenate; 3o-min delay before disruption by freezing * See text for experimental
OF ENDOGENOUS
Mean
S.D.
(3 exptsl ____ 0.041 0.310
0.007 0.042
0.363 0.337
0.037 0.014
0.265
0.017
~~
details.
and extraction yielded no measurable free fatty acids. When eggs were homogenized in an isotonic solution, little lipolysis occurred (Table I, Expt I, and Fig. I, Lane 3). When the solution was hypotonic, considerable lipolysis occurred (Table I, Expt 2, and Fig. I, Lane 4). Incidentally, one can conclude from the observations in Fig. I
Fig. 1. Thin-layer chromatographic separation of the lipids of disrupted and non-disrupted egg homogenates. I, mixture of reference compounds; (a) sterol esters, (b) triglycerides, (c) free fatty acids, (d) 1.3,-diglycerides, (e) 1,2,-diglycerides and free sterols, (f) monoglycerides, (g) unresolved polar lipids; 2, lipids from eggs homogenized in an acidified isotonic medium and extracted immediately; 3, lipid extract from eggs homogenized in an isotonic medium and incubated for 30 min at 25 “C prior to extraction; 4. lipid extract from eggs homogenized in a hypotonic medium and incubated for 30 min at 25 “C prior to extraction; 5, lipid extract from eggs homogenized in an isotonic medium, sonified for 20 s, incubated for 30 min at 25 “C. and extracted. Fig. 2. The effect of freeze-thaw treatment on the appearance of free fatty acids in isotonic egg homogenates. Western corn rootworm eggs (500 per homogenate) were homogenized in IO ml of isotonic medium; one-half of the homogenate was incubated at 25 “C (- -); the other one-half was frozen in a solid CO,-acetone mixture, thawed, and then incubated (&). At the indicated time intervals a o.8-ml aliquot was withdrawn, the reaction stopped, and the free fatty acids determined. Each point is the mean of four such experiments. The vertical bars represent the S.D.
LIPOLYSIS
IN INSECT
EGG
469
that the free fatty acids observed quantitatively (Table I) were in fact derived from triglycerides. The results of the previously described experiments suggested that isotonic conditions protected a structure from rupture and therefore blocked lipolysis, but the isotonic medium could have prevented lipolysis. This latter explanation was ruled out by the observation that lipolysis occurred under isotonic conditions following either sonication or freeze-thaw (Table I, Expts 3 and 4, and Fig. I, Lane 5). The relationship between time and the appearance of free fatty acids in homogenates prepared in the isotonic medium, with and without a freeze-thaw step, is illustrated in Fig. 2. Even after I h of incubation, very little free fatty acid appeared in the non-disrupted homogenate. In artificial substrate systems, rootworm egg lipase requires a protective agent to avoid denaturation’. Conceivably the lack of lipolysis in the non-disrupted isotonic medium was due to denaturation of the enzyme. To test this hypothesis, we homogenized eggs and incubated the homogenate for 30 min before tissue disruption and subsequent incubation to permit lipolysis. The results (Table I, Expt 5) show that lipolysis occurred and therefore lack of lipolysis under isotonic conditions was not due to enzyme denaturation. The previously described experiments established that the endogenous lipase would not hydrolyze endogenous triglyceride in a non-disrupted isotonic homogenate and therefore we concluded that a structural barrier prevented lipolysis. We conducted a series of experiments to test the hypothesis that either the enzyme, or an enzyme activator, was held in an inactive form by a membraneous structure. Negative results in those experiments (unpublished results) led us to evaluate the availability of the endogenous triglyceride. We added radioactively labeled triolein (27.3 Ci/mole) to a homogenate prepared in the isotonic medium. In the 3o-min incubation period, less than 5 % of the radioactivity recovered was triolein (Table II).
TABLE
II
LIP0LYS.S OF EXOGENOUS 30 min INCUBATION FrUCti#n
TRIOLEIN
IN
Percent
dpm in each fraction
Heated
control*
Triglyceride Diglyceride
96. to
Monoglyceride Free fatty acid
ISOTONIC
Experimental 6.28 5.84 7.80 82.62
EGG
HOMOGENATES
AFTER
S. D. 2.10
0.96 0.73 5.64
* Eggs heated to IOO “C for IO min before homogenization in presence of radioactive and subjection to further treatment as described in Methods and Materials section.
triolein
The endogenous triglyceride was not hydrolyzed; the thin-layer chromatographic pattern of the bulk lipid in this experiment as seen by I, staining is typified by the chromatogram shown in Fig. I, Lane 3. We conclude from this last experiment that in our homogenate system the enzyme is freely active and the substrate is latent. The substrate latency is probably membrane-based because the factors (hypotonicity, freeze-thaw and sonication) that
410
J. L. KRYSAN.
P. L. CUSS
permit lipolysis can disrupt membranes. We suggest that the barrier surrounding the triglyceride, as we have observed it in vitro, plays a role in the regulation of lipolysis in vivo. That suggestion is consistent with the speculations of Lui and Davis14. Based upon ultrastructural observations they speculated that the specific structure of lipid droplets in black fly tissues function in the regulation of lipid utilization. REFERENCES I Agrell, I. (1964) in The Physiology of Insecta (Rockstein, M., ed.), Vol. I, pp. 91-148, Academic Press, New York and London 2 Kinsella, J. E. (1966) Camp. Biochem. Physiol. 19, 291-304 3 Gilbert, L. I. (1967) in Advances in Insect Physiology (Beament, J. W. L., Treherne, J. E. and Wigglesworth, V. B., eds), Vol. 4, pp. 69-21 I, Academic Press, London and New York 4 Fast, P. G. (1970) in Progress in the Chemistry of Fats and other Lipids (Holman, R. T., ed.), Vol. 11 (Part 2), pp. 179-244, PergamOn PreSS, Oxford 5 Fodor, P. J. (I 947) Enzymologia I 2, 343-349 6 Gaeta, 1. and Zappanico, A. (1959) Ric. Sci. 29, 788-791 7 Carlson, L. D. (1941) Biol. Bul/. 81, 375-387 8 Urbani, E. (1962) in Advances in Morphogenesis (Abercrombie, M. and Brachet, J., eds), Vol. 2, pp. 61-108, Academic Press, New York and London 9 Guss, P. L. and Krysan, J. L. (1972) J. Insect Physio/. 18, 1181-1195 IO Howe, W. L. and George, B. W. (1966) in Insect Colonization and Moss Production (Smith. C. N.. ed.), pp. 367-383, Academic Press, New York and London I I Krysan, J. L. and Guss, P. L. (1971) Biochim. Biophys. Acta 239, 349-352 12 Bligh, E. G. and Dyer, W. J. (1959) Can. J. Biochem. Physiol. 37. 911~917 13 Guss, P. L. (1969) Ann. Entornol. Sot. Am. 62, 675-676 14 Lui,T. P.andDavies, D. M.(r97z)J. LipidRes. 13, 115-118