Oxidised LDL and progression of atherosclerosis

234

LETTERS to the EDITOR

Oxidised LDL and progression of atherosclerosis SiR,-Professor Salonen and colleagues (April 11, p 883) claim that autoantibodies against oxidised low-density lipoprotein (LDL) predict progression of carotid atherosclerosis. We doubt whether this conclusion can be drawn on the basis of the ultrasound method used and data presented. We were surprised by the description of the ultrasound assessment of atherosclerosis, especially the results of the repeated measurements of intima-media thickness from video-recordings on 30 cases and 30 controls. Intima-media thickness was measured originally from video-recordings by the scanning physician Dr Riitta Salonen (R.S.). Three measurements were done of the far wall of the right and left common carotid artery, at the site of the greatest intima-media thickness, and the mean of these six measurements was used to represent the degree of atherosclerosis. The increase in intima-media thickness was taken as the difference between two-year and baseline values. Because of concerns over the accuracy of this method, the Medical Research Council of the Academy of Finland (which has been supporting this research) initiated in January, 1992, investigations into the reliability of the ultrasound approach. In February, 1992, R. S. agreed to remeasure baseline and two-year follow-up thicknesses on 40 individuals selected from the 128 men on whom previous articles1,2were based and also data for the 30 cases and 30 controls in the Lancet article. The re-measurements on these video-recordings were done by R. S. blind to the subject’s identity or to whether the recording was a baseline or a follow-up. Two of us (M. L. and K. Poikolainen.) then analysed these re-measurements and data on intima-media thickness. In the sample of 40 men the Pearson correlation coefficient between progression of atherosclerosis (two-year increase in intima-media thickness) based on the original data and on the re-measurements by R. S. under controlled conditions was

only 0 We

18. were

surprised to read the repeated intima-media thickness done on video-recordings for the 30 cases and 30

measurements

controls in the Lancet article. Salonen et al state that the same observer repeated the measurements blind to the original result and to risk factors. The two-year increase "ranged from 0-05 mm to 1 ’00 mm among cases and from about 0-08 mm to 0-03 mm among controls" and Salonen et al conclude that "all cases and controls were classified identically as in the original categorisation". The range for atherosclerosis progression given in table i is from 0-06-0-73 mm for cases and - 0-05 to 0-02 mm for controls. When we applied these limits to the 40 men for whom the original measurements had been given to us and for whom re-measurements had been done by R. S. in February, 1992, we found that 9 of the 11I men (82%) originally classified as cases (two-year increase 0 060-73 mm) would have remained cases but none of the controls (- 0-05 to 0-02 mm) would have remained controls-indeed, 15 of them would have been categorised as cases (the other 2 had a progression of 0-04 mm, which lies between the lower limit for cases and the upper limit for controls). These results are in sharp contrast

the claim that on re-measurement all cases and controls remained in the original categorisation. Only 0-04 mm differentiates cases from controls (see figure, p 885). This means that the precision of the evaluation of intima-media thicknesses has to be under that limit if it is to differentiate cases from controls. Salonen’s group has previously reported1,2 that the axial resolution of the B-mode in the ATL UM4 duplex ultrasound system used in the present study is 0-3 mm-so how could they classify controls and cases correctly? Discussing inter and intra observer variability in their ultrasound measurements Salonen and colleagues cite (as they did before1) a study they did using different ultrasound equipment (Biosound Phase 2 real-time scanner).3 Furthermore, baseline and follow-up videotapes were not read in random order (as reported): according to the report of R. S. to the Academy of Finland, baseline videotapes were always read first and the subject’s identity and whether she was reading baseline or follow-up recordings was not hidden. We do not deny the usefulness of ultrasound methods in the assessment of atherosclerosis. Our comments specifically relate to the reliability of the numbers reported by Salonen and his co-workers. to

Department of Medicine, Kuopio University

MARKKU LAAKSO

National Public Health Institute, Helsinki

KARI POIKOLAINEN

Department of Medicine, Kuopio Univesity, 70210 Kuopio, Finland

KALEVI PYÖRÄLÄ

1. Salonen JT, Salonen R, Seppanen K, Kantola M, Suntioinen S, Korpela H. Interactions of serum copper, selenium, and low density lipoprotein cholesterol in atherogenesis. Br Med J 1991; 302: 756-60 2. Salonen R, Salonen JT. Progression of carotid atherosclerosis and its determinants: a

population-based ultrasonography study. Atherosclerosis 1990; 81: 33-40 R, Haapanen A, Salonen JT. Measurement of intima-media thickness of common carotid arteries with high-resolution B-mode ultrasonography: inter- and intraobserver variability Ultrasound Med Biol 1991; 17: 225-30.

3. Salonen

SIR,-Although Professor Salonen and colleagues’ results are very intriguing, I would raise some points. Firstly, the intima media thickness (IMT) is only one of the indirect ultrasonographic criteria of carotid atherosclerosis severity. In fact, to increase the chance of detecting an atherosclerotic plaque both non-parallelism of the ultrasonic interfaces and the focalisation of the thickening has to be present. It seems that only the IMT criteria were used in this study. Furthermore, looking at the baseline IMT (table i) it is unlikely that most patients had atherosclerotic plaques in the common carotid arteries. In fact, the range of IMT is between 0-62 and 1-5 mm (combining cases and controls). In the largest ultrasound-based epidemiological study-ie, the Atherosclerosis Risk in Community (ARIC), including a randomly selected cohort of about 16 000 Americans-the 90th percentile for the combined far-wall common carotid IMT is 1-6 mm.1 Secondly, 30 cases and 30 controls, showing the highest and lowest 2 year IMT progression rate, respectively, were selected from the participants to the Kuopio ischaemic heart disease risk factor (KIHD) study. In previous

235

KIHD publications/,3 the upper progression rate was 0-9 mm, which in the present analysis is 0-73 mm. Therefore it seems that at least 1 of the patients of the original cohort was excluded, without explanation. Similarly, the patient with the highest baseline IMT-ie, 2 25 mm, was also excluded by the investigators. Furthermore, the progression rate range for the 30 controls is 0-05 per 0-002 mm, although it was previously reported that only 17 patients showed no or negative IMT changes. Thirdly, although the correlation coefficients demonstrate an impressive consistency of association between the IMT progression rate and the antibody titre, the means and SD of the two groups overlap significantly. Furthermore, the plot (p 885) suggests that two subgroups can be identified, one showing progression of 0-3 mm or more, and the second, including the original control group and some of the cases, showing no definitive IMT changes. The plot also suggests that the correlation is probably driven by extreme observations and that the antibody titre does not seem to be either sensitive or specific in differentiating cases and controls. -

Bowman

Gray School of Medicine,

Winston-Salem, North Carolina 27103, USA

a

chelating adsorbent,

serum

iron level in

a

dog with

an

iron

overload could reduced.6 Department of Psychiatry and Behavioral Sciences, Mental Sciences Institute, University of Texas Health Science Center at Houston, Houston, Texas 77030, USA

MEIR STRAHILEVITZ

M, Possible utilization of extracorporeal hemoperfusion in the treatment of coronary artery disease. Atherosclerosis 1977; 26: 373-77. 2. Saal SD, Parker TS, Gordon BR, et al. Removal of low-density lipoprotein in patients by extracorporeal immunoadsorption. Am J Med 1986; 80: 583-89. 3. Boberg H, Gackowski A, Hombach V, et al. Treatment of familial hypercholesterolemia by means of specific immunoadsorption. J Clin Apheresis 1988; 4: 59-65. 4. Parker TS, Gordon BR, Saal SD, et al. Plasma high density lipoprotein is increased in man when low density lipoprotein (LDL) is lowered by LDL-pheresis. Proc Natl Acad Sci USA 1986; 83: 777-81. 5. Palmer A, Gjorstrup P, Severm A, et al. Treatment of systemic lupus erythematosus by extracorporeal immunoadsorption. Lancet 1988; ii: 272. 6. Ambrus CM, Anthone S, Deshpande G, et al. Extracorporeal removal of iron with immobilized desferrioxamine. ASAIO-Trans 1987; 33 (3): 749-53. 1. Strahilevitz

MICHELE MERCURI

G, Sharret AR, Barnes R, et al. Carotid atherosclerosis measured by B mode ultrasound in populations: associations with cardiovascular risk factors in the ARIC 1991; 134: 250-56. study. Am Epidemiol J 2. Salonen R, Salonen JT. Progression of carotid atherosclerosis and its determinants: a population-based ultrasonography study. Atherosclerosis 1990; 81: 33-40. 3. Salonen JT, Salonen R, Seppanen K, Kantola M, Suntioinen S, Korpela H. Interactions of serum copper, selenium, and low density lipoprotein cholesterol in atherogenesis. BMJ 1991; 302: 756-60. 1. Heiss

SIR,-Professor Salonen and colleagues’ demonstration (April 11, p 883) that titre of antibodies to oxidised low-density lipoprotein

(LDL) is a predictor of the progression of carotid atherosclerosis should, among other things, encourage evaluation of treatment

strategies that may reduce oxidised LDL. One such strategy you discuss in your accompanying editorial is the use of antioxidants, such as vitamin E. Another interesting possible therapeutic intervention to effectively reduce oxidised LDL in patients with disease is severe hypercholesterolaemia and atherosclerotic modified extracorporeal immunoadsorption of LDL.1 This affmity adsorption treatment, which uses specific immunoadsorption of LDL on columns containing antibodies to human apolipoprotein B in an extracorporeal circuit, is associated with striking reduction in LDL, clinical improvement, and regression of skin xanthomata2,3 and coronary stenosis.3 The procedure greatly reduces plasma LDL without much reducing HDL (one study reports an increase in HDL after the procedure4). Although the therapeutic effect on atherosclerosis is assumed to be the result of selective removal of native LDL, there is no information about the possible reduction of oxidised LDL in the atherosclerotic lesion. Such a reduction could result from "desorption" of oxidised LDL from the atherosclerotic lesion into plasma, because of a substantial lowering of native LDL in plasma and the subsequent adsorption of the oxidised LDL to the LDL antibodies in the immunoadsorbent. Another mechanism that could lead to a decrease in oxidised LDL in the atherosclerotic lesion would be the reduction in production of oxidised LDL after substantial fall in native LDL in blood and body stores, including the atherosclerotic lesion. If the first mechanism operates, then it may be possible to increase the efficacy of extracorporeal immunoadsorption treatment by use of an immunoadsorbent that contains antibodies to oxidised LDL. You point out in your editorial that autoantibodies are probably not relevant to the initiation of the atherosclerotic lesion. Nonetheless, it is possible that removal of antibodies to oxidised LDL from plasma and body stores, including the atherosclerotic lesion, would be therapeutic. Such a goal can be achieved by incorporation of oxidised LDL, such as malondialdehyde-LDL in the extracorporeal immunoadsorption colomn. Alternatively, autoantibodies can be removed if protein A is used as the immunoadsorbent.’’ A chelating agent adsorbent can be bound to the extracorporeal column matrix, to reduce oxidant levels in blood and body stores. With an extracorporeal device that incorporated desferrioxamine as

SIR,-Professor Salonen and colleagues have demonstrated a predictive value of the antibody titre to malondialdehyde (MDA) modified lysine residues in low-density lipoprotein (LDL) and the development of carotid atherosclerosis. Although this is clearly important as a marker of disease, antibody formation may be a secondary occurrence,! and oxidation of lipoproteins does not account for the varying prevalence of atherosclerosis associated with changes in the lipoprotein profile. We suggest that inhibition of oxidation may be a less effective target for preventing atherosclerosis than modulation of apolipoprotein metabolism. LDL is modified by macrophages in the intimal microenvironment, rather than in plasma, with MDA derived from lipid peroxidation binding to apo B lysine residues and allowing recognition and internalisation by the scavenger receptor. There are no reports of modification of circulating plasma LDL, which increases its uptake by scavenger receptors, even in patients with a higher prevalence of atherosclerosis such as those with uraemia, diabetes, reduced plasma vitamin E, hyperlipoprotein [a], familial hypercholesterolaemia, or hypolipoprotein AI. By contrast, Salonen et al review data showing oxidised LDL located within the macrophage-derived foam cells of atheromatous plaques. These findings suggest that the critical processes involved in promoting atheroma are related to variations in the lipoprotein profiles that alter the delivery and distribution of lipids, rather than to oxidative modification in the intima, which may be the final pathway common to all factors promoting atherogenesis. The amount of intimal lipid peroxidation will be dependent mainly on the availability of the substrate, which is determined by delivery and removal rates, and intracellular processing routes regulated by lipoproteins and dietary and genetic factors. Reduced delivery of substrate to macrophages might account for the protective effect of truncated forms of apo B and hypocholesterolaemia.2 Conversely, other mutations of apo B result in reduced binding to native LDL receptors, hypercholesterolaemia, and increased atherosclerosis.3 Variations on the removal side would explain the substantial protection against atherosclerosis by increased lipoprotein AI expression (resulting in increased HDL) in transgenic mince,4 and in man,s and by higher HDL-cholesterol concentrations protecting against atherosclerosisand restenosis after angioplasty.7 Thus although oxidation of LDL is clearly important in the final generation of atherosclerosis, the clinical importance of the many other mechanisms involved may be greater because they may allow for effective therapeutic intervention. Wide variations in mortality from atherosclerosis are explained solely on the basis of altered plasma lipoprotein profiles. Department of Nephrology, Royal London Hospital and Medical College,

SIMON ROSELAAR

London E1 1BB, UK

JOHN CUNNINGHAM

1. Editorial. Antibodies to oxidized LDL in atherosclerosis. Lancet 1992; 339: 889-900. 2. Krul ES, Kinoshita M, Talmud P, et al. Two distinct truncated apolipoprotein B species in a kindred with bypobetalipoproteinaemia. Arteriosclerosis 1989; 9:

856-68.

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3. Tybjaerg-Hansen

A, Gallagher J, Vincent J, et al. Familial defective apolipoprotein B100: detection in the United Kingdom and Scandinavia, and clinical characterisrics often cases. Atherosclerosis 1990; 80: 235-42. 4. Rubin E, Krauss RM, Spangler E, Verstuyft JG, Clift SM. Inhibition of early atherogenesis in transgenic mice by human apolipoprotein AI Nature 1991; 353: 265-67. 5. Tall AR. Plasma high density lipoproteins: metabolism and relationship to atherogenesis. J Clin Invest 1990; 86: 379-84. 6. Gordon DJ, Rifkind BM. High density lipoprotein-the clinical implication of recent studies. N Engl J Med 1989; 321: 1311-16 7. Shah PK, Amin J. Low high density lipoprotein level is associated with increased restenosis rate after coronary angioplasty. Circulation 1992; 85: 1279-85.

*** These letters have been show to Professor Salonen and colleagues, whose reply follows.-ED. L. SIR,-As a result of the criticism initiated by Dr Pyorala and co-workers, the Finnish Academy reviewed our ultrasonographic assessment of carotid atherosclerosis. Consequently, we undertook several reassessments of the measurements of carotid intima-media thickness as measured from the videotapes of the ultrasound measurements. The result of the reassessment for the 60 subjects reported in this study was given in the section "Assessment of atherosclerosis" reported in our Lancet article (April 11, p 883), and these data demonstrate the reproducibility of the original data that are reported in the body of the text. We are in part to blame for the continued misunderstanding of our data by Dr Laakso and colleagues because there was a mistake in our description of the study sample: in the last sentence of the section "Study design" we stated that no other selection criteria were used. However, before the selection of the cases and controls of this study, subjects from the larger cohort (n = 128) with severe symptomatic ischaemic heart disease (n = 24) and 1 with missing data for serum low-density lipoprotein (LDL) cholesterol were excluded. This explains the discrepancy that both Laakso and others and Mercuri note, compared with previous descriptions of the cohort. In the reproducibility study, Dr Riitta Salonen did re-measurements of the videotapes of all 60 subjects in this report, blinded with respect to the original values and to all risk-factor values (as we note in the text). Unfortunately, a typographical error has led to a further important misunderstanding. On the third line of the section "Assessment of atherosclerosis" the text should have read - 0-08 mm instead of 0-08. Thus, in these re-measurements, all cases and controls (n 60) of our report were classified as in the original categorisation. Furthermore, all 128 video-recordings of both baseline and two-year follow-up ultrasound scannings of the original cohort have also been re-measured. The Pearson correlation coefficient for the two-year change in intima-media thickness between the original and repeat measurements in the entire data set was 0-71, varying between 0-12 (for a non-random subsample selected by Markku Laakso) and 0-97 (for the 60 subjects of our case-control study) in subsamples according to the time of re-measurements. The mean of absolute values of differences between the original and the repeat measurements (as estimate of the empirical measurement precision) was 0-1 mm. Obviously there was some random measurement error in the quantitative assessment of the intima-media thickness of carotid arteries in the larger cohort from which subjects of this study were drawn. However, there was probably little misclassification of the cases and controls in the subjects of this report, since they were deliberately selected from the extreme ends of the distribution of intima-media thickness values. The cases and controls were defined before measurement of antibody titre, and this was done blindly. Even if some cases/controls were assigned erroneously, this would most likely be independent of the antibody titre, and, if anything, such non-differential misclassification would have weakened any observed relation. We agree with Mercuri that the intima-media thickness is only one way to estimate the severity of carotid atherosclerosis. However, as we emphasise repeatedly in our paper, because we were investigating early carotid disease, we used intima-media thickness, being the most commonly used measure of the severity of early atherosclerosis.’ Because there were no previous studies of this nature, our pilot study was aimed at looking for a relation between antibody titre in cases and controls at the extreme ends of the spectrum of carotid atherosclerosis. Further investigations are =

needed to confirm this initial study and to determine the strength of the association and sensitivity/specificity of antibody measurements, since they may relate to severity and location of atherosclerotic disease. Dr Strahilevitz’s speculations about extracorporeal removal of oxidised LDL or antibodies to epitopes of oxidised LDL are interesting but await the demonstration that oxidised LDL occurs in plasma (as opposed to the arterial lesion) and to an understanding of whether antibodies to oxidised LDL have any pathological role, or are merely markers for the oxidation process. Finally, we agree fully with Dr Roseloor and Cunningham that the pathogenesis of atherosclerosis is indeed complex and that the plasma concentration of LDL and its rate of delivery to the arterial wall is crucially important. Even if oxidation of LDL were the "whole story" (which, of course, it is not), it is obvious that if there were fewer LDL particles to be oxidised, there would be less atherosclerosis. JUKKA T. SALONEN HEIKKI KORPELA Research Institute of Public Health, RIITTA SALONEN University of Kuopio, 70211 Kuopio, Finland KRISTIINA NYYSSÖNEN Department of Biomedical Sciences, University of Tampere

SEPPO YLA-HERTTUALA

Department of Medicine, University of California, San Diego, California, USA

ROBERT YAMAMATO SUSAN BUTLER WULF PALINSKI JOSEPH L. WITZTUM

1. Wikstrand

science Quantitative humans. Arterioscler Thromb

J, Wiklund O. Frontiers in cardiovascular

measurements

of atherosclerotic manifestations

in

1992; 12: 114-19

Air emboli in human donor liver SiR,—Venous air embolism is a dangerous complication that has also been reported during liver transplantation with the use of a venovenous bypass or after improper unclamping of the infrahepatic vena cava.1,2 Intrahepatic air collections, although suspected, have never been demonstrated to cause obstruction of liver vessels. During donor hepatectomy in an 18-year-old man who died from a road accident, in-situ perfusion with preservation solution showed abnormal discolouration of the liver surface and the organ was not used for transplantation. Magnetic resonance imaging (MRI) of the discarded liver showed air in peripheral venous and arterial branches (figure). Apparently, the air had prevented adequate entry of preservation solution into the vessels. MRI also demonstrated air collections in 12 out of 36 isolated donor livers that were subsequently transplanted (figure). The amount of air ranged from some bubbles in the central parts of the hepatic and portal veins (10 cases) to major accumulations of air in the more peripheral portal vein branches in the right lobe (2 cases) in which the air extended to the edge of the liver, a distribution pattern that reflects the centrifugal flow in the portal vein. In 1 of the 2 livers, air was also found in concomitant hepatic artery branches.

MRI sections.

Left= discarded donor liver, arrows indicate vessels with air (dark) blocking preservative (bright). Right= pretransplant donor liver, large arrow indicates air in portal vein branch and small arrows indicate air bubbles in hepatic artery branch. UW=plastic bag filled with University of Wisconsin organ preservative