Kinetics of LDL subfractions

After injecting 1261-or l%labeled lipoproteins, plasma was subjected to discontinuous density gradient centrifugation, which separates low-denstty lipoprotdnr (LOL) Mto three subfractions. Analysis of changes In apoiipoprotein B-specific activity with time showed that light LOL is normally the product of Intermediate-density lipoprotein and the precursor of heavy LOL, which is subeequentiy converted to heavier LOL. In hyperapobetailpoproteinemla these relationships are maintained but there is overproduction of light and heavy LOL secondary to Increased synthesis of very low-density lipoprotein. in famiiiai hyperchoiesteroiemla, light LOL is produced norm&y but Its conversion into heavy LOL is reduced and independent synthesis of the latter is apparent. These observations suggest that the LOL receptor normaHy plays a role in the conversion of light into heavy LOL. They also provide an expianation for previously documented differences in LOL composition between hyperapobetaiipoproteinemia, in which there is a relative increase in choierteroi-depleted heavy and heavier subfractions, and famjiiai hyperchoiesteroiemla, in which there is a relative increase in the cholesterol-enriched light subfraction. (AM HEART J 1987;113:514.)

Gilbert R. Thompson, M.D., Babie Teng, M.Sc., and Alan D. Sniderman, M.D. London,

England,

and Montreal,

Quebec, Canada.

Insights into physiologic mechanisms often have beengained in the past by studying the alterations induced by inherited disease,and we have usedthis approachto investigate the kinetics of low-density lipoprotein (LDL) subfractions. However, before discussing our findings we must first define and describethe two disordersof LDL metabolism that we have studied on this account. Increasedlevels of LDL often result in hypercholesterolemia and an enhanced risk of premature coronaryheart disease(CHD). This increasein LDL cholesterolwas originally termed type II hyperlipoproteinemia,’ the risk of CHD being greatest in persons in whom this abnormality is dominantly inherited, otherwiseknown a8familial hypercholesterolemia (FH). An increasednumber of LDL particles in plasma, as gaugedby immunochemical measurement of the concentration of the apoprotein B moiety of LDL, can also occur without obvious hyparcholesterolemia. This syndrome has been termed hyperapobetalipoproteinemia(hyperapoB), and it also tends to be familial and aseociatedwith an increased risk of CHD.2 Hypertriglyceridemia, usually with a type IV phenotype, is a common accompaniment.3 From the Medical Research Council Lipoprotein Team, Hammersmith Hospital, London, and the Cardiovascular Research Unit, Royal Victoria Hospital, Montreal. Reprint requests: G. R. Thompson, M.D., MRC Lipoprotein Team, Hammersmith Hospital, Ducane RD., London WI2 OHS.

514

CONTRASTING HYPERAPO B

FEATURES

OF LOL IN FH AN0

The contrastinglipid profiles of FH and hyperapo B are exemplified in Table I. A more marked increase in cholesterol than in apoprotein B is responsiblefor the raised cholesterol: protein ratio that characterizesLDL in FH, whereasin hyperapo B the increaseis greater in apoprotein B than in cholesterol,which results in a reducedLDL choleeterol:protein ratio. Quantitative estimates of very low-density lipoprotein (VLDL) and LDL turnover show that increasedLDL levels in FH result from a combination of increasedLDL-apoprotein B synthesis and reducedcatabolism,much of the increaElein synthesis being via a VLDL-independent pathway. In hyperapo B there is also oversynthesisof LDLapoprotein B, but this is largely secondary to increasedsynthesis of VLDL-apoprotein B and fs not accompaniedby any impairment of LDL cataboli8m.4 COMPOSITION

OF LOL SUBFRACTIONS

Various authors using various methods of separation had previously described between three and five subfractions of LDL in normal subjects and patient8 with hypertriglyceridemia. The particular method of separationwe have used, namely a fivestep discontinuous density gradient, has been describedin detail elsewhere6and involves 40 hours of ultracentrifugation in a Beckman SW 50.1 rotor (Beckman Instruments, Palo Alto, Calif.) at 10’ C.

V0lunl0 113 Number 2, Part 2

LDL kinetics

515

I. Cholesterol: apoprotein B ratios of LDL subfractions (mean k SD)

Table

Subject

Control

Light

(1)

Heavy (2)

Heavier (3)

1.55 rf: 0.14

1.23 +- 0.12

0.85

-+ 0.13 (n = 4)

Hyperapo B

1.47 rt: 0.16

0.98 -+ O.ll*

0.80 k 0.11 (n = 1)

(n = 6) FH (n = 5)

2.05

1.37 2 0.16

1.11 * 0.15

(n = 6)

-c 0.23t

*p < 0.01 “ersus control. tp < 0.001 versus control.

At the end of this period usually two, but sometimes three, fractions of LDL can be visualized.These can be recoveredvia a tube piercerand assayedfor their content of lipid and protein. In normal LDL, just under one third of the apoprotein B is in the light fraction 1, just over two thirds is in the heavy fraction 2, and ~5% is in the heavier fraction 3. A similar distribution was evident in hyperapo B, except that almost 10% of the apoprotein B was recoveredin fraction 3, whereasin FH >40% was in light LDL, 55% in heavy LDL, and <2% in fraction 3. The explanation for the marked differences in cholesterol:protein ratio of LDL in FH and hyperapo B noted abovebecomesobvious on inspection of Table I, which showsthe composition of the subfractions. Comparedwith control subjects,patients with FH have an abnormal form of light LDL, which is cholesterolenriched, whereassubjects with hyperapo B havean abnormal form of heavyLDL, which is cholesterol depleted. Similar trends are evident in the heavier fraction 3. TURNOVER

OF APOPROTEIN

B IN LDL SUBFRACTIONS

To investigate the mechanism for the differing distribution and composition of the three subfractions of LDL, a series of apoprotein B turnover studies was undertaken in the three groups of subjectswhosebiochemical data are shown in Table I. A full description of their clinical details, the methods used, and the results of these studies has been published elsewhere.4 After lnjectlon of ‘2sl-VLDL. Generally there was a reasonableprecursor-productrelationship between the specific activity-time curvesof VLDL and intermediate-density lipoprotein (IDL) in all three groups of subjects, although in some instancesthe VLDL and IDL curves intersected several hours before the peak of IDL-specific activity had been reached. In all instances there was a precursorproduct relationship between IDL and light LDL, and in most control subjects and patients with

I

I

I

I

1

2

3

4

DAYS

1. Specific activity of LDL subfractions (light, A; heavy, o ; heavier, l ) in a patient with hyperapo B after an injection of 13’1-LDL.

Fig.

hyperapo B there was an analogousrelationship betweenlight and heavy LDL. However,there was a notable lack of any such relationship betweenlight and heavy LDL in patients with FH, which suggests the existence of VLDL-independent synthesis of heavy LDL in this disorder. After Injection of 1251-lDL. To further investigatethe apparent lack of a precursor-product relationship betweenlight and heavy LDL in FH, two heterozygotes were given labeled “broad-spectrum” IDL (Svedbergflotation unit 12 to 60 rather than the more conventional Sr 12 to 20). The results were similar to those obtained by injecting VLDL and confirmed that heavy LDL is derived only partially and rather slowly from light LDL in FH. However, even in a homozygote, some conversion of IDLderived light LDL into heavy LDL did take place. After InJecttin of ‘5’l-LDL. After injection of labeled whole LDL, plasma was obtained for isolation of

516

Thompson,

February 1987 American Heart Journal

Teng, and Sniderman

Fig. 2. Proposed model for LDL metabolism. Turnover rates of light and heavy LDL are determined independently and the proportion of light LDL converted into heavy LDL is measured by deconvolution analysis. The rate of synthesis of total LDL is derived as synthesis of light LDL plus direct synthesis of heavy LDL, the latter being calculated as synthesis of heavy LDL minus amount of light LDL converted to heavy LDL.’

Table II. Quantitation of turnover of apoprotein B in light and heavy LDL L-LDL

Subjects Control (n = 2) Hyperapo B (n = 2) FH (n = 2)

H-LDL

Pool (mg)

FCR (/day)

ACR (mdkglday)

Pool

(mgl

FCR f/day)

ACR h&kgldaY)

591 1216 2689

0.71 1.10 0.23

5.9 17.1 9.1

1885 3301 3676

0.50 0.40 0.26

12.8 17.3 14.1

L = light; H = heavy; ACR = absolute

catabolic

(%,I

75 89 44

rate.

LDL subfractions and analysis of apoprotein Bspecific activity in a control subject and patients with hyperapo B and heteroxygous FH. In each instance the turnover of light LDL was initially faster than that of heavy LDL, this difference being exaggerated in hyperapo B but much less marked in FH. Int&estingly; stimulation of receptor-mediated LDL catabolism in an FH heteroxygote by cholestyramine and mevinolin resulted in partial restoration of the normal differential between the removal rates of liiht and heavy LDL.6 The proportion of apoprotein B found in heavier LDL (+&on 3) was highest in patients with hyperapo B, and data that suggest that this slowly turning over fraotion is the produot of heavy LDL metabolism are shown in Fig. 1. After injection of labeled LDL subfrectkms.

In cm-

trol +ibjects and patients with hyperapo B, injection of ‘I-light LDL gave rise to heavy LDL with a precursor-product reIationahip similar to that observed when labeled VLDL had been injected, but there was no evidence that heavy LDL gave rise to light LDL after injeotion of i311-heavy LDL. However, the reverse seems to apply in FH, with no real precursor-prtiuct relationship between light and heavy LDL but some eviciwme4.#w~ heavy LDL may be eonv~

L-H conversion

slowly back to l&&t LDL.

By separately analyzing the specMc activity-time curves of light and heavy LDL and quantitating the

proportion of liiht LDL converted to heavy LDL, by de.convolution analysis, and by measuring the sixes of the two intravasqular pools of LDL it is possible to qua&it&e the rates of turnover of each fraction (Table II). The results show that light LDL turns over faster and is largely converted into heavy LDL in control subjects and patients with hyperapo B. In contrast, the fractional catabolic rates @‘CR) of both subfractions are reduced in FH and there is <50% conversion of light into heavy LDL during the course of 10 days. This results in twice as much expansion of the light LDL pool in FH as occurs in hyperapo B, whereas the heavy LDL pool is inererteed to a similar extent in both disorders. STUDIES IN RABBITS

In view of the defective conversion of light to heavy LDL in FH, the possible role of LDL receptors in this process was investigated in both norme,l and homozygous Watanabe Heritable Hyperlipidemic (WHEEL) rabbits, which laek LDL receptors. Diierentially labeled native and cyclohexanedioneblocked preparations of human light LDL and similar preparations of heavy LDL were injected and their FCR determined. Differences between the FCR of native and cyclohexanedione-blocked light LDL were greater than between~the FCR of native and cyclohexanedione-blocked heavy LDL in nor-

Voluma 113

LDL kinetics

Numbor 2, Part 2

mal rabbits, but the FCR of all four preparations were similar in WHHL rabbits. These findings suggestthat the LDL receptorplays a greaterrole in the hepatic uptake of light than of heavy LDL. Direct evidence in support of this conclusion was obtained by measurement of liver radioactivity.’ However, the relevance of these findings to the human situation is uncertain in that no conversion of human light to heavy LDL could be documented in normal rabbits. IN VITRO UPTAKE

The possibility that light LDL might be preferentially metabolized by the LDL receptor wasinvestigatedin cultured human fibroblasts. However,similar rates of high-athnity binding and degradation wereobservedirrespective of whether light or heavy LDL was the ligand. Similarly, no difference in uptake or degradationwas observedwhen light and heavy LDL were incubated with human monocyte macrophages.sThese data, which suggestthat light and heavy LDL have similar affinities for both the LDL receptor and also for the scavengerpathway, are difficult to reconcile with the data in rabbits. However, it was recently suggestedthat there are important differencesbetweenexpressionof receptors for LDL in human liver and in cultured fibroblasts9 Conceivably, reversible binding of LDL in the liver, reversibility seemingly being a feature of the hepatic receptor(s),could help modulate conversion of light to heavy LDL and thus explain the decreasedconversionobservedin FH. CONCLUSIONS

These data clearly indicate the existenceof subspeciesof LDL and their interconversion.The precise physiologic processesinvolved remain to be determined but a facilitatory role for the LDL receptor is proposed on the basis of the defective conversion of light to heavy LDL observedin FH and its improvement by measuresknown to stimulate receptor-mediated LDL catabolism. The respectiveroles of cholesterolester transfer protein in the movement of cholesterolester from light LDL to VLDL in exchangefor triglyceride and of hepatic lipase in the subsequenthydrolysis of the latter to generateheavy LDL remain to be defined. In contrast with FH, conversionof light to heavyLDL is, if anything, supranormal in hyperapo B and there is significant conversionof heavy to heavier LDL. Our findings not only account for the differences

517

of LDL composition in FH and hyperapoB but also support Berman’s contentiorP that the conventional two-pool model of calculating LDL turnover is inadequate.Instead we proposethe model shown in Fig. 2, which is a twin two-pool model allowing for independent input into each intravascular pool, as wellas conversionof light into heavy LDL. Calculation of total LDL turnover by sucha model results in higher values for FCR than the simple two-pool model.4Although light LDL is normally converted to heavyLDL, the reversemay alsooccur,especially in FH. For this and other reasonscomputer modeling will probably prove to be the most accurate method of determining LDL turnover, as it has already for VLDL turnover. REFERENCES

1. Fredrickson DS, Levy RI, Lees RS. Fat transport in lipoproteins-an integrated approach to mechanisms and disorders. N Engl J Med 1967;276:148. 2. Sniderman A, Shapiro S, Marpole D, Skinner B, Teng B, Kwiterovich PO. Association of coronary atherosclerosis with hyperapobetalipoproteinemia (increased protein but normal cholesterol levels in human low density lipoproteins). Proc Nat1 Acad Sci USA 1980;77:694. 3. Sniderman AD, Wolfson C, Teng B, Franklin FA, Bachorik PS, Kwiterovich PO. Association of hyperapobetalipoproteinemia with endogenous hypertriglyceridemia and atherosclerosis. Ann Intern Med 1982:97:833. 4. Teng B, Sniderman AD, S&tar AK, Thompson GR. Metabolic basis of hyperapobetalipoproteinemia. Turnover of apolipo protein B in low density lipoprotein and its precursors and subfractions compared with normal and familial hypercholesterolemia. J Clin Invest 1986,‘77:663. 5. Teng B, Thompson CR, Sniderman AD, Forte TM, Krauss RM, Kwiterovich PO. Composition and distribution of low density lipoprotein fractions in hyperapobetalipoproteinemia, normulipidemia and familial hypercholesterolemia. Proc Nat1 Acad Sci USA 1983;80:6662. 6. Thompson GR, Teng B, Sniderman AD. Effects of stimulating receptor-mediated LDL catabolism on turnover of LDL subfractions in familial hwercholesterolemia. In: Barbara L, Hermon Dowling R, Hof;nann AF, Roda E, eds. Recent advances in bile acid research. New York: Raven Press, 19%X199. 7. Kano M, Koizumi J, Jadhav A, Thompson GR. Turnover of light and heavy LDL in normal and WHHL rabbits. In: Greten H, Windler E, Beisiegel U, eds. Receptor-mediated uptake in the liver. Berlin: Springer-Verlag, 1986% 8. Knight BL, Thompson GR, Soutar AK. Binding and degradation of heavy and light subfractions of low density lipoprotein by cultured fibroblasta and macrophages. Atherosclerosis 1986$9:301. 9. Hoeg JM, Demosky SJ, Lackner KJ, Osborne JC, Oliver C, Brewer HB. The expressed human hepatic receptor for low density lipoprotein differs from the fibroblasts low density lioonrotein receptor. Biochem Biophys Acta 1986;876:13. 10. &r&n M. Kinetic analysis and modelling: theory and applications to lipoproteins. In: Berman M, Grundy SM, Howard BV, eds. Lipoprotein kinetics and modelling. New York: Academic Press Inc, 1982:3.