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Saturday 1 November 1986

METABOLIC EPIDEMIOLOGY OF PLASMACHOLESTEROL

Mechanisms of VARIATION of PLASMACHOLESTEROL within POPULATIONS and

between POPULATIONS

INTERNATIONAL COLLABORATIVE STUDY GROUP*

Summary Plasma cholesterol concentrations varywidely within and between populations,

underlying a broad range of coronary heart disease

mortality. Since the metabolic bases of this variation arepoorly understood, low-density lipoprotein (LDL)metabolism was investigated, kinetically and in vitro, in fivepopulation samples from nutritionally disparate countries;109 men with LDL cholesterol 1·0-6·7 mmol/l werestudied. Slow production rate and rapid fractional

catabolism, possibly due to high LDL receptor activity,underlay the lowest LDL cholesterol levels within eachpopulation; highest levels were largely maintained by rapidproduction rate. Differences in production rate explainedmore of the variation in LDL cholesterol than differences infractional catabolic rate. Very low LDL levels in the Africansubjects were due to slow production rate; differences infractional catabolic rate and production rate interacted indetermining levels in the European populations. Directrelations were present between saturated fatty acid intakeand production rate, and between monounsaturated fattyacid intake and fractional catabolic rate, and may contributeto differences in LDL metabolism between populations.

*UK.—B. Lewis, P. R. Turner, J. Revill, R. Konarska, A. La Ville, M.Shaikh, J. Charlton, United Medical and Dental Schools, St Thomas’Hospital, London

South Africa.—J. E. Rossouw, M. J. Weight, B. R. Dando, P. L. Jooste,National Research Institute for Nutritional Diseases, Medical ResearchCouncil of South Africa, Tygerberg

Spain.—L. Masana, R. Sola, P. Sarda, A. Escobar, J. Salas, Facultad deMedicina, Reus, Universidad de Barcelona, Barcelona

Italy.—M. Mancini, G. Marotta, E. Farinaro, A. Postiglione, ClinicaMedica, Universita di Napoli, Naples

Finland.—Y. A. Kesaniemi, T. A. Miettinen, Second Department ofMedicine, University of Helsinki, Helsinki

Introduction

THE metabolic determinants of plasma cholesterol andlow-density lipoprotein (LDL) concentrations are ofinterest because of the strong and causal association betweenthese and the risk of coronary heart disease (CHD).1, Therate of progression of atherosclerosis is directly related toplasma LDL cholesterol.3 Plasma cholesterol and LDLcholesterol vary greatly within populations’ and theirdistributions show striking diversity among different

populations.5,6 Since this within-population and between-population variation in cholesterol underlies a several-foldrange of CHD risk,,8 we have investigated its metabolicbases in normal men in five countries. We describe kineticstudies of LDL metabolism and observations on catabolismof this lipoprotein in vitro; a standard procedure wasadapted for epidemiological use.9 To measure cell mediatedcatabolism of LDL directly we chose blood mononuclearcells, which are readily accessible and may be harvested inadequate numbers for study in their native state-ie,without modifications resulting from cell culture or

derepression. In this paper we extend our previous report9on variation of plasma cholesterol within a single Britishpopulation, to encompass five communities with a widerange of CHD mortality rates and plasma cholesterolconcentrations.

Subjects and Methods

Population SamplingRandom samples of men aged 35-49 were identified in the five

participating centres. Criteria for exclusion were a body-mass indexof less than 20 or more than 27, major illness, recent minor febrileillness, use of therapeutic diets, use of drugs affecting lipidmetabolism, and evidence (eg, presence of xanthomas) of severefamilial hyperlipidaemia. In Helsinki, men were identified from apopulation register. In Baix-Camp, an agricultural region nearBarcelona, men were selected from the population of ten villages. InCape Town and Naples, the staff of single large industries weresampled; in Cape Town, African employees were selected, in viewof the particularly low mean plasma cholesterol values 10,11 and CHDmortality" in this ethnic group, to extend the range of cholesterol

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distributions. In Sheffield, the sample was selected randomly fromthe age-sex register of a group general practice.

Initial plasma cholesterol and LDL cholesterol measurementswere made. The distribution and the limits of the lowest, modal,and highest deciles of LDL cholesterol were defined for eachpopulation sample. Subjects were then recalled for repeat lipid andlipoprotein analyses in the fasted state, and those with LDL

cholesterol values within the respective deciles were entered into thestudy; they continued with their habitual patterns of food intakethroughout. All investigations were conducted in the autumn orwinter. The numbers of subjects are shown in table i. Dietaryconsumption of major nutrients was assessed in each populationsample. 7-day written food consumption records were analysed bymeans of national food composition tables.

Laboratory ProceduresAll methods have been described previously.9 In brief, plasma

lipids were measured by automated enzymatic procedures commonto the five centres, all of which used internal and external qualitycontrols. Each centre exchanged samples with the London

laboratory to ensure standardisation of lipid and lipoprotein assays.Lipoproteins were separated by stepwise preparativeultracentrifugation13 and differential precipitation.14 To ensure acommon protocol and to minimise interlaboratory variation, oneworker (P. R. T.) participated in the investigations in all the centres.All critical solutions were prepared in the London laboratory.

For kinetic studies autologous LDL (d =1 1-019-1-063 g/ml) waslabelled with ’1251 under sterile conditions15 according to a commonprotocol, and 5 Ci (1 Ci= 3-7x10° Bq) was injectedintravenously. Fractional catabolic rate of LDL apolipoprotein B(apo B) was estimated from the urine:plasma radioactivity ratio onthe 7th day after injection, as validated and described previously,9and LDL apo B production rate was calculated as the product offractional catabolic rate and pool size. All lipoprotein lipids andLDL apo B concentrations for estimation of pool sizes weremeasured in London within 48 h of isolation; shipment of plasmasamples was at 0-4°C. Urinary recovery of orally administeredp-aminobenzoic acid was measured to verify quantitative urinecollection;16 tests in which recoveries were less than 85% wererejected.To assess the catabolism of LDL in vitro, we used freshly isolated

blood mononuclear cells to provide an index of ambient LDLreceptor activity. In cells prepared thus, the catabolism of LDL islargely mediated by the LDL receptor pathway (ref 17 and P. R.Turner, personal communication).The degradation of 1-LDL by freshly isolated blood

mononuclear cells (approximately 90% lymphocytes) was

measured in laboratories in Spain, South Africa, Finland, andBritain. We performed quintuplicate- assays as previouslydescribed,9,17 omitting preliminary derepression of receptoractivity.

All variables were adjusted for age and body-mass index. Weexamined the relation between LDL cholesterol values, metabolicvariables, and diet by regression analysis using the GLIM

package.18 Production rate and fractional catabolic rate of LDL apoB, stratum (ie, highest, modal, or lowest decile), and country wereanalysed as the explanatory variables; in a separate analysisdegradation of LDL by blood mononuclear cells was substituted forfractional catabolic rate. The aim was to show whether the

regression coefficients for the metabolic variables differed betweenstrata and between countries.Ethical approval was given by the regional hospital or generalpractice ethical committees.

Results

Plasma lipid and lipoprotein concentrations are shown intable 1. Mean values of plasma cholesterol and of LDLcholesterol were ranked in the order UK > Finland > Italy> Spain > South Africa; mean LDL cholesterol was lowestin South African men (21 mmol/1) and highest in Britishmen (4-2 mmol/1).- and individual values ranged from 1 0 to

TABLE I-LIPID AND LIPOPROTEIN VALUES (mmol/1) ANDBIOMETRIC DATA* *

HDL = high-density lipoprotein; BMI = body-mass index. *Means, SEMs.

6-7 mmol/1. Cholesterol concentrations in plasma andin its LDL fraction were highly correlated (r = 0-96). LDLcholesterol values were related positively to body-massindex (p < 0-02). Although LDL in the pooled data was notrelated significantly to age, correlations with age variedwithin the five populations. Hence LDL cholesterolconcentrations and metabolic data have been adjusted forage and body-mass index in the following analyses.

Table II shows the relations between LDL cholesterol,the kinetic findings, and LDL degradation in vitro in thepooled adjusted data. Within each population sample and inthe pooled data (n = 109) LDL cholesterol varied directlywith the production rate of LDL apo B (figure, A). Therewas an inverse relation between LDL cholesterol and LDL

apo B fractional catabolic rate in the pooled data and withineach population sample (figure, B); the findings werecongruent for the relation between LDL cholesterol andLDL degradation by mononuclear cells in vitro (figure, C).Regression models were fitted predicting LDL cholesterolas a linear or logarithmic function of production rate,fractional catabolic rate, and both metabolic variables.Because there was little difference when linear or

logarithmic terms were used, linear terms were chosen. Therelations between LDL cholesterol and the metabolic datawere similar in all five population samples-ie, the

regression coefficients showed no consistent differences.LDL cholesterol varied directly with the production rate ofLDL apo B in the lowest, modal, and highest deciles(regression coefficient for all three strata was 032; SEM0-025, p < 0001). There was an inverse relation (p < 0-001)between LDL cholesterol values and LDL apo B fractionalcatabolic rate; the regression coefficients varied betweenstrata (p<0-01) and were -4-75 (lowest decile), -693(modal decile), and - 7-93 (top decile).On the basis of the regression analysis, we estimated the

proportion of the variance in LDL cholesterol withinpopulations and within strata explained by the metabolicdata. Differences in production rate considered aloneaccounted for 65% of this variance and differences in

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TABLE II-LDL CHOLESTEROL VALUES, LDL APO B KINETIC DATA AND LDL DEGRADATION BY MONONUCLEAR CELLS*

*All data are adjusted for age and body-mass index.

Relations between LDL cholesterol and LDL apo B production rate (A) and fractional catabolic rate (B), and LDL catabolism in vitro byblood mononuclear cells (C).Vertical bars represent the means and SEM of metabolic data in the deciles of each population sample; heavy vertical bars show the means and SEM for the

modal deciles. U = United Kingdom; F = Finland; I = Italy; S = Spain; A = South Africa.

fractional catabolic rate considered alone accounted for14%. The two metabolic determinants of LDL cholesterolin combination accounted for 84%-ie, for substantiallymore of its variance than either considered separately.

Differences between the five population samples in thedistributions of LDL cholesterol and LDL apo B valueswere also associated with variations both in production rateand in fractional catabolic rate of LDL apo B. Comparisonof these metabolic data in the modal deciles of the populationsamples (table n) shows that the lowest concentrations(found in the African participants) were attributable to avery low production rate which outweighed their lowfractional catabolism. The somewhat higher LDL apo Band LDL cholesterol concentrations in the Spanish subjectswere maintained by the interaction of a greater productionrate with a considerably higher rate of fractional catabolism.Differences in LDL cholesterol and LDL apo B betweenthe African, Spanish, and Italian modal samples wereassociated with congruent differences in production rate;variation in fractional catabolic rate did not contribute tothese interpopulation differences. However, differences inLDL cholesterol and LDL apo B between the populationsamples with the highest values of these variables (UK,Finland, and Italy) were not explained by differences inproduction rate; they were attributable to variation in

fractional catabolic rate, to which they were inverselyrelated. The rate of degradation of LDL by bloodmononuclear cells in vitro showed differences congruentwith those in LDL apo B fractional catabolism in vivo, beinglowest in the South African participants and showing aninverse relation with LDL cholesterol and LDL apo Bvalues in the British, Finnish, and Spanish populationsstudied.

Dietary assessment showed considerable interpopulationand interindividual differences in intakes of saturated fattyacids and of monounsaturated fatty acids, but only modestdifferences in intakes of polyunsaturated fatty acids. Nosubstantial or consistent differences in nutrient intake werenoted between subjects in the top, modal, or lowest decileswithin each population. In the pooled data, the significantpositive correlations were between intake of saturated fattyacids and LDL apo B production rate (r = 0,367, p < 0’001),between intake of monounsaturated fatty acids and LDLapo B fractional catabolic rate (r=0-303, p<0’01), andbetween LDL degradation by mononuclear cells in vitroand intake of monounsaturated fatty acids (r=0-365,p < 0-001). LDL degradation was inversely related to intakeof saturated fatty acids (r = &mdash; 0 344, p < 0-001).There was a significant correlation between the two

independent measures of LDL catabolism, LDL apo B

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fractional catabolic rate and LDL degradation by bloodmononuclear cells (r=0-49, p<0-001 in the unadjusteddata).

Discussion

The purpose of this study was to seek metabolic bases forthe extensive variation of plasma cholesterol level bothwithin populations and between different populations.LDL cholesterol is the major component of plasmacholesterol, hence the metabolism of this lipoprotein classwas investigated.The rate of production and rate of fractional catabolism of

LDL were measured by studying the kinetics of apo B, itsstructural peptide. This extensively used procedure’5,1920was adapted for epidemiological use by measurement of theurine/plasma radioactivity ratio after injection of a low doseof autologous radiolabelled LDL.9 The contribution ofmononuclear cells to whole-body catabolism of LDL issmall, the major site being the liver Zlz2 Nevertheless, it wasnoteworthy in the present study that the rate of catabolism ofLDL by freshly isolated underepressed mononuclear cellsin vitro correlated significantly with an independentmeasure of LDL fractional catabolic rate in vivo and that thetwo variables showed congruent differences within

populations and between populations.The metabolic findings associated with within-

population variation in LDL cholesterol were consistent inall centres. LDL cholesterol was directly and linearly relatedto the production rate of LDL apo B. By contrast, theinverse relation between LDL cholesterol and the fractionalcatabolic rate of LDL apo B seemed to be curvilinear;subjects in the lowest decile of LDL cholesterol had

considerably higher fractional catabolic rates than those inthe modal decile (table II), whereas the differences infractional catabolism between the modal and highest decileswere small. Hence production rate influenced LDLcholesterol values throughout their distribution, althoughthe effect of fractional catabolic rate was most pronounced inthe lowest part of the LDL cholesterol distribution.

Throughout the range of LDL cholesterol values,production rate and fractional catabolic rate interacted indetermining these values; however, when the two kineticvariables were analysed separately production rate explaineda considerably greater proportion of the variance of LDLcholesterol than did fractional catabolic rate. The regressioncoefficient for LDL apo B production rate (0-32) representsa stronger relation to LDL cholesterol than the coefficientsfor LDL apo B fractional catabolic rate (47, 69, and 7-9),since production rate has a far wider numerical range thanhas fractional catabolism. In earlier clinical investigationsoverproduction of LDL apo B was the main kineticabnormality accounting for high LDL in patients withnon-familial, presumably polygenic, hypercholesterol-aemia.23,24

Although it is possible, in theory, to attribute the highfractional catabolic rate of LDL in the lowest decile to itssmaller pool size, this has not been proven experimentally.25Further, this notion is unlikely to be correct since ourfindings of a greater rate of LDL catabolism in vitro cannotbe thus explained.We may therefore conclude that within five nutritionally

disparate randomly selected population samples, the highestconcentrations of plasma cholesterol and LDL cholesterolresult largely from greater production of LDL, thoughslower catabolism contributes to their maintenance. Thuscommon ("polygenic") hypercholesterolaemia may be

attributed to these metabolic differences. On the other hand,low plasma cholesterol and LDL cholesterol result bothfrom slower production of LDL and from its more rapidcatabolism.No single metabolic mechanism explained differences

in LDL cholesterol between populations. The Africanparticipants had the lowest LDL cholesterol and achievedthis entirely by slow rates of LDL apo B production, despiteunexpectedly low rates of fractional catabolism of LDL. Inan earlier comparison of LDL metabolism in ten AmericanIndians and five Caucasians the lower LDL cholesterol inthe Indians was also attributed to lower production of thislipoprotein.26 There were comparably low rates of LDLcatabolism by blood mononuclear cells. In Spain, the lowLDL cholesterol reflected an interaction between relativelyslow production rate and rapid catabolism, as measuredkinetically and in vitro. The highest modal LDL cholesterolvalues, seen in Finland, Italy, and the UK, were the result ofslower fractional catabolism, and there was an inverserelation between LDL cholesterol and its fractionalcatabolism in these population samples.The relation beween dietary intake of fatty acids and

LDL kinetics may underlie these associations. Differencesin plasma cholesterol distribution between populations arelargely attributable to nutritional patterns: variation inintake of saturated fatty acids seems to be the most powerfulsingle dietary determinant of serum cholesterol. The notionthat diet has a primary role is supported by crossover feedingexperiments.27 Some workers have proposed that a highintake of dietary cholesterol represses hepatic LDL receptoractivity in man,28 as it does in laboratory animals,29 therebyraising plasma cholesterol concentration. LDL receptoractivity in human blood mononuclear cells is profoundlyreduced by a high cholesterol diet.3oWe found significant associations between LDL

metabolism and dietary intake of two classes of fatty acids.LDL production was directly related to intake of saturatedfatty acids, a finding previously suggested by the results ofcontrolled feeding experiments on the metabolic ward. 31Thus the high mean serum cholesterol seen in populationsin which the habitual diet is rich in saturated fat may be

explained in part by greater rates of LDL production. Thefractional catabolism of LDL, and also the catabolism ofLDL by isolated mononuclear cells, were directly related tointake of monounsaturated fatty acids. Since the intake ofthis class of fatty acids in several Mediterranean countries ishigh by virtue of the widespread use of olive oil, in which themajor fatty acid is oleic acid, the low mean serum cholesterolvalues in these countries may be related in part to high ratesof fractional catabolism.The metabolic findings underlying the interpopulation

differences in serum cholesterol and LDL cholesterol maybe considered in the light of these nutritional relations. Theextremely low values characteristic of the African

population sample are associated with a very limited intakeof all forms of fat (17% of dietary energy); the low rates ofproduction and fractional catabolism observed in theAfrican participants may be related to their restricted intakesof, respectively, saturated and monounsaturated fatty acids(4% and 8-6% of dietary energy, respectively). The lowserum cholesterol in the Spanish and Italian subjects,maintained by intermediate to high production rates andhigh fractional catabolic rates, may reflect moderate intakeof saturated fatty acids (12-3% of energy in Spanish and17 3% in Italian subjects) and particularly highconsumption of monounsaturated fatty acids (194% in

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Spanish and 23% in Italian subjects). In the northernEuropean countries the even higher concentrations of serumcholesterol may result from high production and slowfractional catabolism of LDL, due in turn to their highintake of saturated fatty acids (17 -5 % in Finnish and 15 -1 1 %in British subjects) and lower intake of monounsaturatedfatty acids (15’4% in Finnish and 11’5% in British subjects).A unifying hypothesis follows from investigations of a

genetically hyperlipidaemic rabbit strain in which LDLreceptors are deficient:32 it is that low hepatic receptoractivity not only impairs catabolism of LDL but also, byreducing the catabolism of intermediate-densitylipoprotein, allows a greater proportion of this lipoprotein tobe converted to LDL. Hence low hepatic LDL receptoractivity could underlie both decreased catabolism andincreased production of LDL. Thus differences in LDLcholesterol distributions between the four Europeanpopulations could be attributed to differences in LDLreceptor activity, accounting for contributions made bydifferences both in fractional catabolism and in productionrate to differences in LDL cholesterol. However, the kineticfindings in the African participants cannot be reconciledwith this explanation; LDL production was low despiteevidence of low LDL receptor activity. An alternativemechanism for the low rate of LDL production seems to beoperative; possibly the intake of saturated fat determines thefraction of very low-density lipoprotein that is converted toits metabolic product LDL.31 A genetic explanation for thisethnic difference in serum cholesterol and lipoproteinmetabolism is unlikely, since crossover feeding experimentshave shown that differences in cholesterol values are largelyenvironmentally determined.27 On the basis of this

hypothesis, differences in LDL receptor activity may alsocontribute to variation of LDL cholesterol within

populations, providing a partial explanation of differences inplasma cholesterol and risk of CHD. Within populations theregression analysis showed that a larger proportion of theobserved variance was attributable to differences in

production rate than to differences in catabolic variables andimplies that variation in production rate is determined inpart by mechanisms that do not influence fractionalcatabolism. However, high rates of fractional catabolism aswell as low production contribute to maintenance of LDLcholesterol values in the lowest decile, and a primary role ofhigh LDL receptor activity may underlie both kineticmechanisms.

Certain genetic hyperlipidaemiasz3 are associated withoverproduction of LDL; the genetically determinedphenotypes of apolipoprotein E also determine LDL

cholesterol,33,34 people with the Ez/Ez phenotype having lowvalues associated with reduced conversion of intermediate-density lipoprotein to LDL.35 Among genetic factors,several mutants are know to cause hypercholesterolaemiadue to major impairment of receptor function,36 and there isevidence of polygenic control of the LDL receptor innormal subjects.37.38

In Finland the study was carried out under a contract with the Finish LifeInsurance Companies and was supported by the Finnish State Council forMedical Research. In Spain the study was supported by CAICYT.

Correspondence should be addressed to Professor B. Lewis, Departmentof Chemical Pathology and Metabolic Disorders, United Medical and DentalSchools, St Thomas’ Hospital, London SEl 7EH.

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2. Lipid Research Clinics Program. The LRC coronary primary prevention trial results.II. The relationship of reduction in incidence of coronary heart disease tocholesterol lowering. JAMA 1984; 251: 365-74.

3. Duffield RGM, Lewis B, Miller NE, Jamieson CW, Brunt JNH, Colchester ACF.Treatment of hyperlipidaemia retards progression of symptomatic femoralatherosclerosis. Lancet 1983; ii: 639-42.

4. Lewis B, Chait A, Wootton IDP, et al. Frequency of risk factors for ischaemicheart-disease in a healthy British population with particular reference to

serum-lipoprotein levels. Lancet 1974; i: 141-46.5. Castelli WP, Cooper GR, Doyle JT, et al. Distribution of triglyceride and total, LDL

and HDL cholesterol in several populations: a cooperative lipoprotein phenotypingstudy. J Chron Dis 1977; 30: 147-69.

6. Lewis B, Chait A, Sigurdsson G, et al. Serum lipoproteins in four Europeancommunities: a quantitative comparison. Eur J Clin Invest 1978; 8: 165-73.

7. Keys A. Seven countries. Cambridge, Mass: Harvard University Press, 1980.8. Neaton JD, Kuller LM, Wentworth D, Borhani NO. Total and cardiovascular

mortality in relation to cigarette smoking, serum cholesterol concentration anddiastolic blood pressure among black and white males followed for five years. AmHeart J 1984; 108: 759-70.

9. Turner PR, Konarska R, Revill J, et al. Metabolic study of variation in plasmacholesterol level in normal men. Lancet 1984, ii: 663-65.

10. Thandroyen FT, Asmal AC, Leary WP, Mitha AS. Comparative study of plasmalipids, carbohydrate tolerance and coronary angiography in three racial groups.S Afr Med J 1980; 57: 533-36.

11. Rossouw JE, van Staden DA, Benade AJS, et al. Is it normal for serum cholesterol torise with age? In: Nestel PJ, ed. Atherosclerosis VII. Amsterdam: Elsevier (inpress).

12. Wyndham CH. Mortality from cardiovascular disease in the various populationgroups in the Republic of South Africa. S Afr Med J 1979; 56: 1023-30.

13. Havel RJ, Eder MA, Bragdon JH. The distribution and chemical composition ofultracentrifugally separated lipoproteins in human serum. J Clin Invest 1955; 34:1345-53.

14. Wamick GR, Albers JJ. HDL cholesterol quantitation. Comparison of six

precipitation methods and ultracentrifugation. In: Report of HDL methodologyworkshop. US Department of Health Education and Welfare. Bethesda: NationalInstitutes of Health, 1979.

15. Janus ED, Nicoll A, Wootton R, Turner PR, Magill PJ, Lewis B. Quantitative studiesof very low density lipoprotein conversion to low density lipoprotein in normalcontrols and primary hyperlipidaemic states. Eur J Clin Invest 1980; 10: 149-59.

16. Bingham S, Cummings JH. The use of 4-aminobenzoic acid as a marker to validate thecompleteness of 24 h urine collections in man. Clin Sci 1983; 64: 629-35.

17. Bilheimer DW, Ho YK, Brown MS, Anderson RGW, Goldstein J. Genetics of thelow density lipoprotein receptor. Diminished receptor activity in lymphocytes fromheterozygotes with familial hypercholesterolemia. J Clin Invest 1978; 61: 678-96.

18. Baker RJ, Nelder JA. The GLIM System, Release 3. Oxford: Numerical AlgorithmsGroup, 1978.

19. Langer T, Strober W, Levy RI. The metabolism of low density lipoprotein in familialtype II hyperlipoproteinemia. J Clin Invest 1972; 51: 1528-36.

20. Thompson GR, Myant NB. Low density lipoprotein turnover in familial

hypercholesterolaemia after plasma exchange. Atherosclerosis 1976; 23: 371-77.21. Spady DK, Billheimer DW, Diestschy JM. Rates of receptor-dependent and

-independent low density lipoprotein uptake in the hamster. Proc Natl Acad SciUSA 1983; 80: 3499-503.

22. Pittman RC, Steinberg D. Sites and mechanisms of uptake and degradation of highdensity and low density lipoproteins. J Lipid Res 1984; 25: 1577-85.

23. Janus ED, Nicoll AM, Turner PR, Magill P, Lewis B. Kinetic bases of the primaryhyperlipidaemias: studies of apolipoprotein B turnover in genetically-definedsubjects. Eur J Clin Invest 1980; 10: 161-71.

24. Kesaniemi YA, Grundy SM. Significance of low density lipoprotein production in theregulation of plasma cholesterol level in man. J Clin Invest 1982; 70: 13-22.

25. Thompson GR, Spinks T, Ranicar A, Myant HB Non-steady-state studies oflow-density lipoprotein turnover in familial hypercholesterolaemia. Clin Sci 1977;52: 361-69.

26. Garnick MB, Bennett PH, Langer T. Low density lipoprotein metabolism andlipoprotein cholesterol content in southwestern Amencan Indians. J Lipid Res1979; 20: 31-39.

27. Antonis A, Bersohn I. The influence of diet on serum lipids in South African white andBantu prisoners. Am J Clin Nutr 1962; 10: 484-99.

28. Goldstein JL, Kita T, Brown MS. Defective lipoprotein receptors and atherosclerosis.N Engl J Med 1983; 309: 288-96.

29. Hui DY, Innerarity TL, Mahley RW. Lipoprotein binding to canine hepaticmembranes. metabolically distinct apo-E and apo-B, E receptors. J Biol Chem1981; 256: 5646-55.

30. Mistry P, Miller NE, Laker M, Hazzard WR, Lewis B. Individual variation in theeffects of dietary cholesterol on plasma lipoproteins and cellular cholesterolhomeostasis in man. J Clin Invest 1981, 67: 493-502.

31. Cortese C, Levy Y, Janus ED, et al. Modes of action of lipid-lowering diets in man:studies of apolipoprotein B kinetics in relation to fat consumption and dietary fattyadd composition. Eur J Clin Invest 1983; 13: 79-85.

32. Kita T, Brown MS, Bilheimer DW, Goldstein JL. Delayed clearance of very lowdensity and intermediate density lipoproteins with enhanced conversion to lowdensity lipoprotein in WHHL rabbits. Proc Natl Acad Sci USA 1982; 79:5693-97.

33. Utermann G, Prim N, Steinberg A. Polymorphism of apolipoprotein E.III. Effect of asingle polymorphic gene locus on plasma lipid levels in man. Clin Genet 1979; 15:63-72.

34. Wandell MR, Suckling PA, Janus ED. Genetic variation in human apolipoprotein E.J Lipid Res 1982; 23: 1174-82

35. Turner PR, Cortese C, Wootton R, Marenah CB, Miller N, Lewis B. Plasma

apolipoprotein B metabolism in familial type III dysbetalipoproteienaemia Eur JClin Invest 1985; 15: 100-12.

36. Goldstein JL, Brown MS. Progress in understanding the LDL receptor and HMGCoA reductase, two membrane proteins that regulate the plasma cholesterol. JLipid Res 1984; 25: 1450-61.

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37. Weight M, Cortese C, Sule U, Miller N, Lewis B. Heritability df the low densitylipoprotein receptor activity of human blood mononuclear cells. Clin Sci 1982; 62:397-402.

38. Maartman-Moe K, Maques P, Borresen A, Berk K Low density lipoprotein activityin cultured fibroblasts from subjects with or without ischaemic heart disease. ClinGenet 1981; 20: 337-45.

LOCAL RECURRENCE OF RECTALADENOCARCINOMA DUE TO INADEQUATE

SURGICAL RESECTION

Histopathological Study of Lateral Tumour Spreadand Surgical Excision

P. QUIRKEM. F. DIXON

P. DURDEY*N. S. WILLIAMS

Departments of Pathology and Surgery, University of Leeds,Leeds LS2 9JT

Summary In 52 patients with rectal adenocarcinomawhole-mount sections of the entire

operative specimen were examined by transverse slicing.There was spread to the lateral resection margin in 14 of 52(27%) patients and 12 of these proceeded to local pelvicrecurrence. The specificity, sensitivity, and positivepredictive values were 92%, 95%, and 85%, respectively.In a retrospective stage-matched and grade-matchedcontrol group there was local recurrence in the same

proportion of patients, but in this series no patient had beenshown by routine sampling to have lateral spread. In rectaladenocarcinoma, local recurrence is mainly due to lateralspread of the tumour and has previously beenunderestimated.

Introduction

IN different hands the incidence of local recurrence afterresection of rectal adenocarcinoma varies from 4 to 50%.1-7Possible explanations include inadequate resection/,6,7suture implantation of tumour cells from the lumen oradjacent lymphatics,9 and the development of a

metachronous anastomotic tumour;7 however, the maincause remains unproven, especially in patients who haveundergone curative resections. 2

In this study we have assessed the degree of lateral spreadby scrupulous histopathological study of the resectedtumour and related this prospectively to short-termrecurrence rates.

Patients and Methods

Patients

36 men and 16 women with biopsy-proven rectaladenocarcinoma were investigated prospectively, their operationstaking place between 1983 and 1985. The majority of patients whounderwent low sphincter-saving resections came from a unit with aspecial interest in preservation of the anal sphincter, whereas mostof those who underwent abdominoperineal excisions came fromother units. The nature of the procedure-curative or palliative-was recorded at the time of operation. Curative procedures weredefined as those in which the surgeon was confident that all

macroscopic tumour had been removed and in which there was noevidence of metastatic spread at operation or on preoperativeultrasound or computerised tomography of the liver. Palliativeoperations were defmed as those in which there was evidence ofresidual tumour determined at the time of operation or from thepreoperative tests mentioned above. Local tumour recurrence was

*Present address: Surgical Unit, The London Hospital, London E1.

Fig 1-Dissection method for rectal carcinomas.

After transverse slicing (top) the slice showing maximal lateral spread wasdivided into blocks for routine processing (black slice). The remaining slices(dotted lines) were totally embedded for whole mount sectioning (lower left).Lower right of figure is an H & E stain in a patient with no recurrence whodied after 6 months because of liver metastases.

identified clinically with a tissue diagnosis where possible or bycomputerised tomography of the pelvis.1O Median follow-up was 23months, range 9-29. 25 patients underwent an abdominoperinealresection, 26 a sphincter-saving operation, and 1 a Hartmann’s

operation.

Histopathological TechniquesThe resected specimen was received fresh, opened anteriorly,

pinned on to a cork board, and fixed in 10% formalin. Dissectionwas performed by one pathologist (P. Q.) and consisted of serial5-10 mm slicing of the whole tumour and the surroundingmesorectum in the transverse plane, in contrast to routine samplingmethods in which a variable number of blocks are taken only fromthe luminal surface. The slice containing the most lateral spread wasidentified as the "primary slice" and was selected for division intomultiple blocks which were all embedded and then routinelyprocessed for haematoxylin and eosin (H & E) staining in 5 tJll1sections for immediate reporting (fig 1). The remaining slices werewholly embedded for single large-mount sections of the tumour andmesorectum, cut at 10 pxn on a sledge microtome, and stained withH & E. The specimen was carefully searched for lymph nodes andproximal and distal resection margins were inspected. Anyinvolvement of adjacent organs was noted. The sections wereexamined microscopically for lateral resection margin involvement.If none was found, the extent of extramural spread was measuredfrom the muscularis propria to the outermost part of the tumour; ifmesorectal or lymph node deposits were present the measurementwas made to the outer border of the deposit. The distance from theoutermost part of the tumour or tumour deposit to the resectionmargin (as shown in fig 2), the pattern of invasion whether pushingor infiltrating," the grade of the tumour, 12 and Dukes stage werealso noted.

Fig 2-Diagram to show measurements made on each slice.

(1) Outer aspect of the muscular layer to the lateral limit of the tumour. (2)Outer limit of the tumour defined as the most lateral penetration of themesorectum by tumour whether lymphatic (2A) or direct tumour spread(2B).