Interrelationship Between Serum 8-Lipoprotein and Cholinesterase

K. bf. KVTTY,~ G. RQWDEN, AND A. R. CQX Department of Laboratories, The Dr. Charles A. Janeway Child Health Centre, and

Department of Pathology and Medicirte, Faculty of Medicine, Memorial University of Newfoundland, St. John", Newfourzdland

Received November 2 1, 1972

KUTW, K. M., ROWBEN, G., and Cox, A. R. Interrelationship between serum p-lipoprotein and cholinesterase. Can. J. Biochem. 51,883-887 (1973 ).

The possible association between serum p-lipoproteins and cholinesterase was investigated in vivo after artificial induction of hyprlipidemia in rabbits, resulting from the injection of the lipopolysaccharide of Escherichia coli (E. coli endotoxin), and in vitro by following the action of phospholipase D upon isolated human serum &lipoprotein.

A marked increase in serum pre-&lipoproteins (VLDL) was found to occur within 24 h after the injection of E. coli endotoxin in rabbits. In the next 48-72 h, a marked reduction in pre-& (VLDL) concomitant with a marked increase in B- (LDL) and a- (HDL) lipoproteins was observed. A sharp increase in cholinesterase isoenzyme activities was visualized (starch gel zymogram) with the development of hyper-pre-beta-lipoproteinemia and decrease with hyper-beta-lipoproteinemia.

When isolated serum p-lipoproteins were incubated with phospholipase D for 24 h at 37 "C, release of cholinesterase associated with a decrease in P-lipoprotein concentrations along with the formation of a lipoprotein with a pre-beta mobility was noticed.

These observations suggest that cholinesterase is intimately associated with the structure of @-lipoprotein and the point of association of the enzyme is likely to be at the phosphoryl choline site of the lipoprotein molecule.

K u m , K. M., RQWBEN, G., et Cox, A. R. Interrelationship between serum p-lipoprotein and cholinesterase. Can. J. Biochem. 51,883-887 (1973).

L'association possible entre les p-lipoprotkines et la cholinestCrase est recherchCe in vivo, aprbs induction expirimentale de I'hyperlipidCmie chez les lapins, par injection du lipopoly- saccharide d'Escherichiu coli (endotoxine d'E. coli) et irt virro, en suivant l'action de la phospholipase D sur les p-lipoprotkines isolCes du sCrum humain.

Chez les lapins, 24 h apr8s l'injection, l'endotoxine d'E. coli provoque une augmentation marquCe des pr6-p-1ipoprotCines (VLDL) sCriques. De 48 B 72 h aprbs, il se produit une rCduction importante des prC p- (VLDL), concornitante B une augmentation marqu6e des p- (LDL) et des a-1ipoprotCines (HDL). L'activitC des isoenzymes de la cholinestkrase augmente de f a ~ o n prononcCe (zymogramme sur gel d'amidon) avec l'installation de 1'hyperprC-p-1ipoprotCinCmie et elle diminue avec l'hyper-8-1ipoprotCinCmie.

Quand les p-1ipoprotCines sCriques isolCes sont incubCes en prCsence de phospholipase D durant 24 h B 37 "C, il y a 1ibCration de cholinestCrase associCe B une diminution des p-lipo- protCines avec formation d'une 1ipoprotCine qui migre B la fason d'une prBbeta.

Ces rCsultats suggbrent que la cholinestkrase est Ctroitement associCe B la structure de la p- 1ipoprotCine et le lieu de cette association se situerait au site phosphorylcholine de la molCcule IipoprotCique. nraduit par le journal]

Introduction A relationship between serum chdinesterase and P-lipoprote~ms had been suggested ( 3 ) on

In the preliminary study ( ), it was 'IBerved the basis of immu~oe~ectrophoretic, histochemi- that the concentrations of both serum cholines- cd, and ultrasonication studies. Dubbs (4) terase and low lipoproteins rose demonstrated an increased release of cholinester- in rabbits, 24 h after the injection of E. coli ase isoenzymes when human serum was ultra- endotoxin. A similar increase in serum cholin- esterase activity had been demonstrated in hyper- sonicated and he also suggested that P-lipopro-

lipemic patients ( I , 2). tein is able to sequester cholinesterase. The results of a further in vitro study using cholines-

'Request for reprints should be addressed to Dr. terase inhibitors (5) indicated that serum cholin-

K. M. K ~ ~ ~ ~ , D ~ . Charles A. Janeway Child Health esterase is involved in maintaining the structural Centre, St. John's, Newfoundland. stability of P-lipoproteins.

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884 CAN. B. BIOGWEM. VOL. 51, 19'93

The purpose of this sta~dy was to analyse fur- ther the relationship between semm ckolinester- me m d P-lipproteins in vivo me% to obtain a basic understanding of the link between the enzyme and the lipoprotein.

MateMs and Methods Seven albino rabbits (four females and three males)

weighing between 2 and 3 kg were used in these experi- ments. The animals had free access to food and water. Each animal was injected with the E. csli endotoxin as described earlier ( I ) , and b%o& was collected at 0,24,48, and 72 h after the injection.

Semm samples from each s f the seven rabbits obtained at identical times were pooled and prepared for electron microscopy of lipoproteins as detailed earlier ( 6 ) (but using 2% ammonium rnolybdate instead of phmphotungstate and a pH sf 6.81, and the preparations were photographed immediately. The magnification of the microscope was calibrated by means sf a. shadowed carbon replica of diffraction gating with a. cross-line spacing of 54 860 lines per inch. Methds of improving the reproducibility s f magnification of the rmPicroscope were followed through- out the study (7> 8). Measurement of the particles was taken from prints by means of an XI0 magnifier with calibrated graticule.

The same semm pools as described above were used for separation of lipoprotein by electrophoresis on ceIlulose acetate (9) and sf ckholinesterase iso- enzymes by starch gel electrophoresis ( 1 ) .

Total low density lipoprotein was measured by the dextram precipitation method ( 10). 7 5 s includes all the lipoproteins sf density <1.063 and hence these measured serum Hipoproteins we wow term as dextrm- precipitabb lipoproteins (DPL). TrigBymrides and cholesterol were quantitated by an autoanalyzer pro- cedure (11, 1%).

Hn VItro Experiments With PhospBzolipwse D Human semm p-lipoproteins prepmared by ultra-

centrifugation ( 13 ) were adjusted to pH 7 and incu- bated with HPfacaspholipase D V I O mg/ml of the lipsoprotein) for 24h at 37 OC. SimuItmeously, a control was run without the enzyme. At the end of the incubation period, the lipoprotein pattern was monitored by electrophoresis (9) and that of cholin- esterase by starch gel electrophoresis ( 1 ) .

The electron micrescopic examination of the negatively stained preparations of the serum re- veded several classes of lipoproteins. The four major groups of serum lipoproteins have been dasssed previously ( 14 ) . 'The &mensions of the particles measured are as folbws: (1 ) chyIomi- crons with diameters in excess sf 1287 A, (2)

'Phospholipase D (Sigma No. B-7'758) from cab- bage, ~ Y P 1) *

very Isw density lipoproteins (VEDL) from 35 7 to 858 A, ( 3 ) low density lipoproteins (LDL) from 2 14 to 356 A, and (4 ) high density lipo- proteins (HDL) up to 143 A. The VLDL are larger and more varied in their dimensions than m y other three groups and they often have an electron translucent core surrounded by a characteristic dark hdo. The characteristic ap- pearance of the lipproteins is iHustrated in Fig. 1 and the number of particles of the four classes of lipoproteins present in each of the samples is expressed as a percentage of the total measured particles (Fig. 2).

An increase in the numbers of VLDL in the 24 h specimen is apparent from the measure- ments. The slight rise in the percentage sf chylo- microns may reflect a red increase or may result from diBculties in distinguishing between larger VLDL particles and small chylomicrons. An in- crease in the LDL and HDL and decrease in the VLDL in 48- a d 72-h specimens is also appar- ent. These changes observed in the electron micrographs parallelled closely with lipoprotein patterns on the cellulose acetate membranes (Fig. 1) . Elution of the individual bands from the cellulose acetate strips after electrsphsresis gave particles of diameters within the range of the previously stated classification in whole serum, i.c. beta corresponded to LDL, pre-beta to VLDL, m d alpha to HDL.

Cholinesterase isoenzyme bands 1, 2, 3, and 4 (Fig. 1 ) were found to be markedly increased on the starch gel zymograms in 24 h along with the formation of pre-p-lipoproteins. This was followed by a gradual to marked reduction of the isoenzymes in 48-72 h along with the fomatisn sf p-lipoprotein and disappearance of the pre-/3- Iipoproteins.

A significant rise in triglycerides (Tablie 1 ) occurred in 24 h followed by a reduction in 48- 72 h. Cholesterol was also found to be signifi- cantly increased in 24 h with only a moderate reduction thereafter. DPL were also markedly elevated in 24 h. The concentration of DPL, 21- though found to be lower in 48-72 h compared with that of the 24 h specimens, were signifi- cantly higher than were these sf the 0 h semm specimens. These results indicate, both in terms of ratio of triglyceride to cholesterol and DPL concentrations, that hyper-pre-beta-lipoprotein- emia proceeds to hyper-beta-Bipproteinemia in 24-48 h after the endotoxin administration.

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KUITY ET AL.: SERUM 8-LIPOPROTEIN AND CHOLINESTEWASE 885

FIG. 1. Electron micrographs illustrating the various serum lipoproteins obtained at 0, 24, 48, and 72 h after treat- ment. Negative-stained preparations (x 70 000). Gels a-d represent the cholinesterase patterns for samples run from &a2 h. The various bands of activity are numbered 1-4. Gels @-it similarly show the distribution of lipoproteins from 0-92 h. I, MDL; 2, LDL; and 3, VLDL. Vertical arrows indicate the directions in which the gels were run and horizontal arrows the point of application of the sample. (The numbering system for cholinesterase isoenzyrnes according to Harris eb al. (19) is in the reverse order, i.e. the band with the greatest mobility is 1 .)

TABLE 1. Serum concentrations of triglycerides, cholesterol, and dextran-precipikble lipoprotein (DPL) in rabbits before (6 h) and 24, 48, and 72 h after the injection of E. coli endotoxin

Class of compounds 6% ?a Oh 24 h 48 h 72 h

Triglycerides 80 _+ %7* 727 + 2157 354 f 49f 319 +_ 327 Cholesterol 64 5 5.7 339 & 43.27 276 f 27.9f 25% f 11.9f DPL 183 _+ 25 1020 f 219* 636 k 467 701 + 39%

*Meran standard deviation. tSbtistically significant compared with the O h values ( P < 0.05).

Phospkolipase D treatment of the human serum /3-lipprotein was found to release cholin- esterase (Fig. 3 ) and was associated with the complete disappearance of P-lipoprotein along with the formation of a lipoprotein with pre-beta mobility.

Discussion The most interesting feature of this study is

the enhancement of cholinesterase isoenzyme activities dong with the development of hyper-

pre-beta-lipproteinemia and dmost the com- plete disappearance of the isoenzymes with the production of hyper-beta-lip~pcoteinemia~

These observations demonstrate further the relationship between cholinesterase and P-lipo- proteins. Such a relationship between cholines- terase and P-lipoproteins has been suggested on the basis of immuraoelectrophoreeic, histocksemi- cal, and ultrasonication experiments using puri- fied human serum /3-lipoprotein ( 3 ) and whole human serum (4). However, the manner in

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CAN. J. BBWKEM. VOL. 51. 1973

f l M E ( R ) FIG. 2. Variations in the numbers of diRerent types sf

lipoproteins present within samples obtained at 0-72 h. Results are expressed as percentages of the total measured particles.

FIG. 3. Cellulose acetate eiectropkoretic patterns s f lipoproteins (A and B) and starch gel electrophoretic patterns of cholkesterase (C and D) before and after treatment sf the purified human serum @-lipoprotein with phospholipase D. I, @-lipoprotein ; 2, pre-0-lipoprotein ; anad 3, cholinesterase.

which such an association between the enzyme and the lipoprotein takes place is not explained. On the other h a d , in our earlier studies (5 1, it was shown that when semm cholinesterase was inhibited with dm@ like eserine or isoniazid, semm p-lipoproteins were either partially or completely degraded dong with the formation of a lipoprotein with a pre-beta mobility. Similar changes in /3-lipoproteins were noticed concomi- tant with the release of cholinesterase when serum was ultrasonicated. Furthermore, both human and rabbit serum chslinesterases were found to increase the stability of /3-lipoproteins. Those observations indicate that j3-lipprotein not only needs chsginesterase for its stability, but whew the enzyme is either inhibited or released from it, then it acquires a pre-beta appearance.

The next important question is where such an interaction between cholinesterase and P-lipo-

protein could take place. It could be hypothe- sized that the most likely p i n t of association must be at the phssphoq1choline site of lecithin, an important component of lipoproteins. This could be explained on the following bases. ( 1 ) There is a dose stmctursal similarity between acetylcholine which is a naturd substrate for cholinesterases, and phosphoryl chofine. (2 ) Organophosphorous compounds are well recog- nized cholinesterase inhibitors; therefore the presence of a phosphorous group along with a quarternary ammonium group (choline in le- cithin) could have a strong affinity for cholin- esterase and at the same time block the catalytic activity of the enzyme.

This hypothesis was tested experimentally by studying the action of phospholipase D upon purified semm P-lipoproteins. It was very evi- dent that this enzyme released the cholinesterase from P-lipoprotein and at the same time the lipo- protein acquired a pre-beta mobility. Since chsl- inesterase, according to our hypothesis, is bound at the ghosphoryl choline site, splitting of the link between phosphoric acid and choline should cause the release of the enzyme. This is the reason why we chose phospholipase D in this studv.

.I

Further studies in this direction using different systems may provide a better understanding of the structure and metabolism of serum lipopro- tein, m d probably of plasma membranes. For example, increased fragility of the erythrocytes has been demmstrated (15) when treated with eserine. It is possible, as has been suggested ( 16 1, that this may be due to the instability of the lipoprotein of the erythrocyte membranes.

The results of our present study also pose the question as to whether LDL (beta) could be derived from VLDL (pre-beta) . Previous sbser- vations indicate such a possibility. A partial breakdown of VEDE into EDE has been demon- strated by Levy et aH. ( 1 7 ) using Bm%I-labelled peptides. It has also been suggested (1 8 ) that all of the LDL is obtained from VLDL metabolism.

A growing body of evidence suggests that VLDL is broken down by a process involving the hydrolysis of triglycerides with resultant con- version into smaller lipoproteins ( 1 7 ) . The en- zyme that is involved in such a reaction is thought to be a lipoprotein lipase. Therefore what is left behind after the action of this enzyme is a VLDL with depleted triglyeerides. This

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KUTTY ET AL.: SERUM 8-LIPOPWOTEHN A N D GWOEHNESTERASE 887

coplad be the precursor of LBL, and when this combines with cholinesterase a true LBL might be formed. A hypothetical formula could be presented showing this relationship.

Enzyme? ~ L D L ---------- + Pre-LDL + Cholinesterase

-----em--- + LDL

This scheme, although it has limitations, might help us in explaining some of our present sbser- vations such as the increase in chslinesterase activity with the appearance of VLDL and de- crease with the disappearance of VLDL and appearance of LDL.

We thank Drs. Peterson and Hutton for their in- valuable help during the preparation of this paper. We also thank Messrs. B. Josephson, A. Howell, and A. Acharya for their excellent technical help and Misses N. Wellman and P. Candow for their secretarial help.

These studies were supported by a grant from the Canadian Heart Foundation.

Part of this paper was presented at the FEBS Special Meeting, April 1973, in Dublin, Ireland.

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