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Biochimica et Biophysics Acta, 531 (1978) 233-236 0 Elsevier/North-Holland Biomedical Press

BBA Report

BBA 51231

THE ACTIVITY OF AN ESTERIFIED CHOLESTEROL TRANSFERRING FACTOR IN HUMAN AND RAT SERUM

P.J. BARTER* and J.I. LALLY

Clinical Biochemistry Unit, School of Medicine, The Flinders University of South Australia, Bedford Park, South Australia 5042 (Australia)

(Received May 29th, 1978)

Summary

In incubations containing mixtures of lipoproteins isolated either from humans or from rats, the addition of human lipoprotein-free serum (the dialysed 1.25 g/ml infranatant solution) was more than five times more effec- tive than that of the rat in promoting the transfer of esterified [ 3H] cholesterol from high density lipoproteins to very low density lipoproteins, regardless of the species origin of the lipoproteins, implying the existence of an esterified cholesterol transferring factor of much greater activity in human than in rat serum.

Introduction

The rat has frequently been used as an experimental model in studies of plasma lipoprotein metabolism, including studies of the esterified cholesterol moiety [ 11. Yet in terms of plasma esterified cholesterol, the rat differs in a number of respects from other species. The major cholesteryl ester fatty acid of rat high density lipoproteins (HDL) is arachidonic acid [2], while in humans it is linoleic acid [ 31. In humans the cholesteryl ester fatty acid composition is essentially identical in all postabsorptive lipoprotein fractions [ 31, whereas in the rat the composition in the very low density lipoproteins (VLDL) is similar to that in the liver and quite different from that in other plasma lipoproteins [2]. In contrast to the rat [ 1,2], it has recently been reported that the esterified cholesterol in rabbit plasma may exchange between the different lipoprotein fractions in a process requiring the presence of a component in the lipoprotein-free fraction of serum [4] ; a similar phenomenon has also been ob-

*To whom all correspondence should be addressed. Abbreviations: HDL, high density lipoprotein p = 1.07-1.20 g/ml; VLDL, very low dens& lipoproteins p < 1.006 g/ml (see also ref.‘5).

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served in human plasma (Barter, P.J. and Lally, J.I., unpublished data). The possibility that rat serum may lack such an esterified cholesterol transferring factor has been examined in this report in which comparative studies of humans and rats are described.

Blood was collected from 24-h fasted, 250-g male Porton-Wistar rats and from 16-h fasted normal human males, aged 18-25 years. The blood was im- mediately defibrinated and the serum separated at 4°C.

Labelled lipoproteins. An aliquot of serum was incubated with [ 1,2-3H] - cholesterol (52 Ci/mmol, New England Nuclear Corp., Boston, Mass.), (1 &i in 10 ~1 ethanol per 10 ml serum) in a stoppered tube for 6 h in a 37°C shaking water-bath. The HDL was subsequently isolated using a single 100 000 X g ultracentrifuge spin at p = 1.070 g/ml for 24 h and two separate 100 000 X g ultracentrifuge spins at p = 1.20 g/ml for 48 h each [ 51. The HDL was sub- sequently dialysed against 0.02 M phosphate buffer (pH 7.4) containing 0.9% NaCl. Esterified [3H]cholestero1 accounted for a mean of 55 and 44% of the total [ 3H] cholesterol in rat and human HDL, respectively.

Unlabelled lipoproteins. VLDL were isolated by ultracentrifugation at 100 000 X g at p = 1.006 g/ml for 16 h and washed by a further 16-h spin at the same density.

Lipoprotein-free serum. After subjecting serum to a single 100 000 X g ultracentrifugation at p = 1.25 g/ml for 48 h, the infranatant fraction was re- covered and dialysed against the phosphate-buffered saline.

Incubations. HDL isolated from 0.3 ml serum, VLDL isolated from 1 ml human or 2 ml rat serum and the 1.25 g/ml infranatant fraction isolated from 1 ml serum were made up to a final volume of 1.4 ml and incubated in a lo-ml stoppered tube for 6 h in a 37°C shaking water-bath. All tubes contained 0.002 M p-chloromercuriphenylsulfonate to inhibit 1ecithin:cholesterol acyl- transferase activity [6] so that the results would not be complicated by ester- ification of the free [3H]cholesterol present. Incubations were stopped by placing the tubes in ice.

The VLDL and HDL were separated by a single 18-h, 100 000 X g ultra- centrifugation at p = 1.006 g/ml at 4°C. The supernatant and infranatant frac- tion were both extracted with a solution of isopropanol/n-heptane/0.5 M H, SO, (40 : 10 : 1, v/v) [ 71 and the lipids were subsequently separated by thin-layer silicic acid chromatography using hexaneldiethyl ether/methanol/acetic acid (90 : 20 : 3 : 2, v/v) as solvent. The esterified and free cholesterol bands were eluted with diethyl ether and assayed for 3H activity [8] and mass [9].

Transfers of esterified cholesterol between plasma lipoprotein fractions have previously been well documented, whether as a net mass transfer from human HDL to VLDL [lo] or as an exchange, without net mass transfer, between rabbit VLDL and LDL [ 41. By using labelled esterified cholesterol in these present studies, rather than relying on simple mass measurements, it was ensured that any transfers at all, whether reversible or irreversible, would be ob- served and that any species differences would be maximized.

It was found that when incubations of either human or rat lipoproteins were performed in buffer alone, without the addition of the 1.25 g/ml infra- natant fraction of either species, very little (3-10’S) of the HDL esterified [ 3H] cholesterol was recovered in VLDL after 6 h incubation; this observation

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was in accord with earlier studies in the rabbit in which the reversible transfers of esterified cholesterol between VLDL and LDL were dependent on the presence of some component in the 1.25 g/ml infranatant fraction [4]. While a similar requirement for the 1.25 g/ml infranatant fraction was found in these present studies, there was a marked difference in its activity in the human and rat.

Regardless of whether the incubation mixture, contained lipoproteins isolated from humans or from rats, the addition of the human serum 1.25 g/ml infranatant fraction was more than five times more effective than that of the rat in promoting transfers of esterified [ 3H] cholesterol from HDL to VLDL (Table I), a finding that could not be explained in terms of differences in 1ecithin:cholesterol acyltransferase activity which had, in any case, been al- most totally inhibited by p-chloromercuriphenylsulfonate (less than 5% of the free [ 3H] cholesterol was esterified during any of the 6 h incubations).

TABLE I

TRANSFERS OF ESTERIFIED [3H]CHOLESTEROL FROM HDL TO VLDL

Each value represents mean f S.E. of 8 separate incubations. Numbers in parentheses represent the percentage of incubation esterified [3H]cholesterol recovered in VLDL. Two separate preparations of both HDL (labelled) and VLDL (unlabelled) were each isolated from serum of donor humans and rats. and each mixture of HDL and VLDL (two each for both human and rat lipoprotein) was incubated in quadruplicate with both human (4 different subjects) and rat (pooled from at least 4 different animals) 1.25 g/ml infranatant fraction. Each incubation tube contained an amount of labelled HDL isolated from 0.3 ml serum (mean 0.32 and 0.35 ~mol cholesterol esterified in human and rat HDL, respectively), an amount of unlabelled VLDL isolated from 1 ml human and 2 ml rat serum (mean 0.22 and 0.26 /ano1 esterified cholesterol in human and rat VLDL, respectively) and an amount of the dialysed 1.25 g/ml infranatant fraction, isolated from 1 ml human or rat serum in a final incubation volume of 1.4 ml. Total radioactivity (in esterified plus free cholesterol) ranged from 130 000-350 000 dpm/ml in the different HDL preparations; for presentation here, all values have been standardized to a total radio- activity of 10 000 dpm per incubation. Incubations were performed in the presence of 0.002 Mp-chloro- mercuriphenylsulfonate in a 37’C shaking water-bath and stopped by placing the tubes in ice.

Source of 1.25 g/ml Esterified [‘HIcholesterol distribution after 6 h (dpm per incubation) infranatant fraction

Human lipoproteins Rat lipoproteins

HDL VLDL HDL VLDL

HLlnX+Il 325Oil90 1250f200 2240f350 3100f230 (28%) (58%)

Rat 4260 f 220 21Oi 25 4870 f 670 620 f 55 (5%) (11%)

The finding of a low activity of an esterified cholesterol transferring factor in rat serum is consistent with the previously reported lack of exchange of esterified cholesterol between rat lipoproteins [ 1,2], and may explain why, in contrast to the human [ 31, the fatty acid composition of cholesteryl esters differs markedly in the different lipoprotein fractions of rat plasma [2].

In conclusion, while this species difference casts further doubt on the validity of the rat as a model for studying human plasma esterified cholesterol transport, it may well be possible to exploit the difference when trying to as- sess the physiological importance of an esterified cholesterol transferring factor in human serum.

This work was supported by grants from the National Heart Foundation of Australia and from the National Health and Medical Research Council of Australia.

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