The Journals of Gerontology Series A: Biological Sciences and Medical Sciences 59:M62-M67 (2004)
© 2004 The Gerontological Society of America
Long-Term Effects of Lipoprotein(a) on Carotid Atherosclerosis in Elderly Japanese
Toshihiko Iwamoto,
Sara Fukuda,
Souichirou Shimizu and
Masaru Takasaki
Department of Geriatric Medicine, Tokyo Medical University Hospital, Japan.
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Abstract
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Background. Serum level of lipoprotein(a) [Lp(a)] is hereditarily constant throughout life within an individual, but the relationship between Lp(a) and atherosclerosis in elderly people is still controversial.
Methods. Serum Lp(a) levels were studied in 208 elderly Japanese participants aged 80 years with a variety of diseases, using carotid ultrasonography (US), brain computerized tomography (CT), electrocardiography (ECG), and ankle brachial pressure index (ABPI). Carotid plaque lesions were divided into 3 types based on the US echogenicity assessed by a computer-assisted system: L type (hypoechoic plaque), H type (hyperechoic plaque), and M type (heterogeneous plaque).
Results. The frequency of the L type and occlusion was significantly higher in the high Lp(a) group (n = 38) than in the normal Lp(a) group (n = 170). The mean height of the plaque was also greater in the high Lp(a) group than in the normal Lp(a) group. There was no difference in CT findings between the two groups. Myocardial ischemia on ECG and low ABPI (<0.9) were both frequently, but not significantly, seen in the high Lp(a) group. Among factors influencing US findings, multiple regression analysis showed that high Lp(a) correlated markedly to L type and cigarette smoking correlated to M type.
Conclusions. These findings indicate that, in Japanese elderly patients aged 80, serum Lp(a) strongly correlates with hypoechoic carotid lesions, which correspond histologically to lipid-rich, unstable atherosclerosis. This suggested that Lp(a) could promote the formation of atherosclerosis throughout life, and play a role as an independent risk factor for circulatory disturbance of the organ later in life.
IT is known that a variety of genetic and environmental factors promote atherothrombotic disorders as well as focal factors at the site of the vessel wall (1). In the atherothrombotic process due to these factors, the longer the period of exposure to these factors, the more the atherothrombotic changes progress. Therefore, ageing, a risk factor for arteriosclerosis, is always associated with these factors. However, it has been unclear how long these factors affect the entire atherothrombotic process since environmental factors such as hyperlipidemia are also usually influenced by lifestyle. From the viewpoint of hyperlipidemia, lipoprotein(a) [Lp(a)] is a unique lipoprotein that is hereditarily constant throughout life within an individual (2,3). Lp(a) has been considered to be an independent risk factor for atherothrombosis (2–21), but no evidence of association between Lp(a) and risk for cardiovascular events was reported in a prospective study (22). Disagreement on the role of Lp(a) in atherothrombotic disorders may be attributable to the age of the participants enrolled in the studies, and further study is necessary in elderly people.
Furthermore, carotid artery atherosclerosis has been considered to reflect systemic atherosclerosis, and recent advances of imaging equipment such as digitized and computer-assisted ultrasonography (US) have made histological diagnosis of carotid artery atherosclerosis possible (23,24). To estimate the influence of Lp(a) on the vessel wall during an 80-year period, 208 elderly patients aged exactly 80 years were studied by determination of serum Lp(a) concentration and carotid US.
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MATERIALS AND METHODS
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A total of 208 Japanese participants aged 80 years, who had various symptoms, vascular risk factors, and atherosclerotic disorders, were enrolled in a cross-sectional study. They underwent US, brain computed tomography (CT), electrocardiography (ECG), and ankle-brachial pressure index (ABPI) simultaneously with determination of serum Lp(a) levels. Criteria for exclusion were cardioembolic stroke and cerebral hemorrhage, which could affect CT findings, in addition to malignancy, severe hepatic or renal failure, acute diseases, and alcoholism as well as any therapy known to affect serum Lp(a) levels. Clinical diagnoses revealed 63 cases of ischemic stroke and/or transient ischemic attacks confirmed by both history and CT, 13 cases of old myocardial infarction documented by both history and ECG, 22 cases of arteriosclerosis obliterans, 15 cases of Alzheimer's disease, 17 cases with dizziness alone, and 78 cases with one of the various risk factors. This study was performed in accord with the Helsinki Declaration of 1975 as revised in 1983.
US Findings
Extracranial carotids of all patients, lying in a supine position with the head turned slightly from the sonographer, were examined bilaterally with high-resolution ultrasonography (Yokogawa Medical System U-sonic RT4600, Tokyo, Japan) using a 7.5-MHz probe. The vessel wall changes of the carotid arteries were investigated on transverse, lateral, antero-oblique, and postero-oblique views. On US findings, occlusion and plaque were both considered to be carotid lesions regardless of the number. Plaque, defined as a localized intima-media thickening (IMT) more than 2.1 mm in thickness, was measured in terms of its height at the point of maximal thickness and the length of lesions with a thickness of 2.1 mm or more along the vessel wall. This definition was adopted because maximum IMT was within 2.0 mm in the normal population, although it is increasing with age (25). Occlusion was defined as a severe stenosis of 90% or more of the arterial lumen, in which the degree of stenosis was obtained by dividing the diameter of the residual lumen at the point of maximal stenosis by the true vessel diameter.
All images were recorded on photographic paper and reviewed: those showing maximum plaque information (thickness) were used for computer analysis. The plaque images on photographic paper were scanned using an Epson GT9500 scanner (Seiko Epson Ltd., Suwa, Nagano, Japan) and the images were transferred to a Power Macintosh computer (Apple Computer, Inc., Tokyo, Japan) using the Adobe Photoshop 6.0 program (Adobe Systems, Inc., San Jose, CA). Each plaque was outlined, and its gray scale content (from 0, which denotes black, to 255, which denotes white) was analyzed for the mean, standard deviation (SD), median, and total pixel count. Comparison of the plaque with the muscular tissue allowed normalization across the patient population to account for variations in gain settings and intertransducer variability (24). A histogram showing the pixel count on the y-axis and the gray scale on the x-axis was produced for each lesion, as shown in Figure 1.
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Figure 1. Representative ultrasound findings by computer analysis. Pixels included in the lesion were delineated (dotted line) and displayed as a histogram on the basis of echogenicity from 0 (black) to 255 (white). A histogram shows pixel count on the y-axis and gray scale on the x-axis. In this case, a histogram of 18,994 pixels shows a distribution curve with each parameter on the echogenicity of the M-type lesion (heterogeneous plaque)
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Based on the shape of the histogram, the lesions were classified into 3 types. The hyperechoic (H) and echolucent (L) types showed a monopeak or skewed distribution curve, but the curve did not reach the abscissa on either the left or right side. The mixed (M) type had a curve that extended to the abscissae on both sides (24). The H and M types have an adjusted mean echogenic value greater than the mean plus 1 SD of muscular tissue.
In this study, the plaque ratio was defined as the rate of numbers of plaque to arteries observed in a group. Furthermore, a case with more than one type of plaque was expressed as a combined case, and a single type of plaque was classified as that particular type, regardless of the amount of plaques. US findings were also categorized as showing either unilateral or bilateral carotid lesions.
Brain CT, ECG, and ABPI Findings
Low-density areas (LDA) on CT images obtained with a Shimadzu SCT-5000T (Tokyo, Japan) were classified into 3 groups according to the distribution: basal ganglionic type (LDA localized in the perforating artery territory), cortical type (LDA localized in the cortices), and leuko-araiosis type (26) (ill-defined, patchy, or diffuse, LDA in the deep white matter) alone without any localized LDA. Myocardial ischemia was defined as horizontal depressions of 1 mm or more in ST segment or presence of abnormal Q wave on ECG at rest. Low ABPI was defined where the measurement was less than 0.9.
Determination of Lp(a)
Serum samples were obtained from fasting venous blood by centrifugation at 3000 g for 10 minutes at room temperature and storage at -80°C until assay. Lp(a) was determined by mean of a commercially available LA kit [Immunotickles AutoLp(a), A & T Co., Tokyo, Japan]. After adding the buffer solution for 4 minutes and 40 seconds and the anti-Lp(a) antibody-fixed latex for 5 minutes to 0.5 ml of sample serum at 37°C, Lp(a) concentration was measured spectrophotometrically at 570 nm according to the standard curve. The detection limit was 2 mg/dL for Lp(a). Serum Lp(a) was stratified into two levels: the normal Lp(a) level was established at 40 mg/dL or less; high levels were defined as those higher than 40 mg/dL.
Assessment of Cardiovascular Risk Factors
Cases of hypertension were defined as those with a casual blood pressure 
160/95 or patients taking antihypertensive drugs. Diabetes mellitus was defined as fasting blood glucose concentration 126 mg/dl or patients receiving nutrition therapy and antidiabetic medication. Hyperlipidemia was defined as a fasting blood cholesterol concentration 220 or cases receiving diet therapy and cholesterol-lowering medication. Cigarette smoking was defined as a smoking index of 200 or more (cigarettes/day x years smoking).
Statistical Methods
To detect the Lp(a) effects on clinical data, subjects were divided into two groups according to the Lp(a) levels: normal Lp(a) and high Lp(a) groups. Since Lp(a) levels showed skewed distribution, they were log-normalized to produce an approximate but not strictly normal distribution. One-way analysis of variance (ANOVA) and Fisher's protected least significant difference or Student's t test were used to assess differences among the various group classifications for these parameters. Fisher's exact probability test and the chi-square test were also applied to analyze the incidence of each stratum of variables and risk factors. Finally, multiple and stepwise regression analyses were performed to identify the factors influencing the US findings. These calculations were done with the StatView program on a Macintosh personal computer. A value of p <.05 was considered to indicate a statistically significant difference.
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RESULTS
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Background Factors of Two Groups by Lp(a) Levels
Males comprised 48.2% of the normal Lp(a) group (n = 170), which was similar to the 44.7% of the high Lp(a) group (n = 38) (Table 1). There was no significant difference in gender, risks, and chief diseases except for the prevalence of Alzheimer's disease (Table 1).
US Findings in Relation to Lp(a) Levels
The incidence of carotid lesions tended to be higher in the high Lp(a) group than in the normal Lp(a) group (Table 2). L type and occlusion were significantly more frequent in the high Lp(a) group. Among plaque ratios from 160 plaques in the normal Lp(a) group and 51 plaques in the high Lp(a) group, the L-type plaque ratio was higher in the high Lp(a) group than in the normal Lp(a) group. The average height of the plaque in the high Lp(a) group was significantly greater than that of the normal Lp(a) group. The mean serum level of Lp(a) was significantly higher in cases with bilateral carotid lesion, L type and M type, compared with that in cases without carotid lesions (Table 3).
CT, ECG, and ABPI Findings of Two Group by Lp(a) Levels
The distribution of CT findings in the normal Lp(a) group was similar to those in the high Lp(a) group (Tables 4). The mean Lp(a) level in cases with leuko-araiosis alone was significantly higher than that in cases without LDA (Table 5). Myocardial ischemia and low ABPI were both frequently but not significantly seen in the high Lp(a) group.
Factors Influencing Carotid Lesion Types
Using gender, hypertension, diabetes, hypercholesterolemia, cigarette smoking, and Lp(a) as variables, Lp(a) and cigarette smoking were adopted by stepwise regression analysis (Table 6). Multiple regression analysis showed that high Lp(a) was markedly correlated with L-type carotid lesions and cigarette smoking correlated with M-type carotid lesions.
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DISCUSSION
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Recent studies have revealed a strong correlation between high Lp(a) levels in coronary artery disease, and histopathological studies have confirmed the presence of Lp(a) deposition in atherosclerotic plaque (4,5). Lp(a) has been considered to be a risk factor for atherosclerosis independent of serum cholesterol and high-density lipoprotein (HDL) levels (6–21). Ridker and colleagues, who prospectively followed a cohort of white, healthy, middle-aged men for 7.5 years, however, identified no evidence linking baseline plasma Lp(a) concentration and risk of future thromboembolic stroke (22). These findings suggested that the results could be different according to age since the atherothrombotic process would correlate to exposing periods of Lp(a). Therefore, this study was conducted to clarify the relationship of Lp(a) to atherothrombosis in elderly people aged 80 years.
Carotid atherosclerosis, which usually indicates systemic atherosclerosis, can be easily depicted with US, and even histological assessment is possible by the echogenicity and texture as follows: Hypoechogenicity reveals lipid deposition, intraplaque hemorrhage, and/or superimposed thrombi, while hyperechogenicity indicates mainly fibrosis and calcification (27–29). The assessment has become increasingly objective with the use of computer-assisted systems, resulting in high interobserver reliability (24). However, there are few studies on the relationship between plaque morphology and Lp(a) levels. Most previously published papers have reported that carotid atherosclerosis, detected by US and quantified by plaque-scoring systems, significantly and independently correlate with Lp(a) levels, although it is modified by hypercholesterolemia, fibrinogen, and antithrombin III (6–8). The grade of carotid stenosis measured by US was also reported to correlate positively with Lp(a) levels (9). Furthermore, Lp(a) has been considered a risk factor for IMT even in individuals free of prevalent cardiovascular disease (10).
Költringer and colleagues reported that cases with plaque, which were divided into smooth-surface plaque and ulcerations, were both associated with high Lp(a) levels (11). In our study, plaques were classified into three types on the basis of echogenicity and texture assessed by a computer-assisted system (24). US findings showed that, in the high Lp(a) group, L-type lesions and occlusions were not only more frequent, but the height of the plaque was significantly greater. Multiple regression analysis also showed that L-type lesions strongly and independently correlated with a high Lp(a) level, while M-type lesions correlated with cigarette smoking. These results indicated that hypoechoic carotid lesions seen in US corresponded histologically to lipid deposition, intraplaque hemorrhage, and/or superimposed thrombi, and that they were promoted by high Lp(a) over long periods.
Pathological examination has revealed two extremes of plaques: lipid-rich plaques, which consist of a lipid core with a fibrous cap that tends to rupture or bleed within it, and fibrous plaques (30). The mechanism of atherogenicity, however, may be explained partly by the high affinity of apolipoprotein(a) [apo(a)] to intimal components, by the greater oxidizability of Lp(a) than low density lipoproteins, allowing smooth muscle cells to proliferate due to Lp(a) interfering with transforming growth factor-ß activity, and by an interaction through the formation of circulating immune complexes containing Chlamydia pneumoniae-specific IgG (immunoglobulin G) antibodies (2,3,31). A high Lp(a) level also promotes not only atherosclerosis with increasing age, but also accelerates thrombosis by means of inhibiting the tissue-type plasminogen activator and blocking plasminogen binding to platelets, monocytes, endothelial cells, fibrinogen, fibrin, and 
2 antiplasmin (2,3).
Since hypoechoic carotid plaques are considered to be unstable plaques predisposing to circulatory disturbances (29,32,33), elderly populations with high Lp(a) could be at risk for atherothrombotic infarction, myocardial infarction, and peripheral artery disease. No significant differences were found in our study, regarding basal ganglionic and cortical infarctions, and leuko-araiosis alone, although Lp(a) levels were increased in cases with isolated leuko-araiosis. Myocardial ischemia and low ABPI were both frequent in the high-Lp(a) group, but no significant differences were recorded between the two groups, presumably due to the small sample size.
Although uniformly accepted standards of Lp(a) are lacking, an apparent threshold for coronary risk was recognized at Lp(a) levels of 30 to 40 mg/dL or even at 20 mg/dL (2). Murai and coworkers reported that, dividing Japanese participants into two groups with a cutoff at 17 mg/dl determined by the double-diffusion test (Ouchterlony), a significant association between high plasma level of Lp(a) and either coronary heart disease or cortical cerebral infarction was found (16). In our study using latex immunoturbidity assay, the high Lp(a) level group (more than 40 mg/dL) was separated from the normal group. The results of the present study suggest that high Lp(a) levels could accelerate systemic atherosclerosis, presumably contributing to formation of lipid-rich plaques throughout life, resulting in circulatory disturbances later in life. Elderly people usually have a variety of vascular risk factors for a long period of time, and they seem susceptible to atherothrombosis due to both high Lp(a) levels and underlying advanced vascular lesions. However, selection bias should be taken into account: the population in our study was hospital based, and the participants did not reflect the general Japanese elderly population aged 80. In addition, the participants had survived 80 years, but more participants with high Lp(a) levels might have died earlier due to vascular events. Further longitudinal studies on the role of Lp(a) in the prevalence of cardiovascular disease are warranted.
Our findings indicate that high Lp(a) levels are independent risk factors for carotid hypoechoic lesions in elderly people because of the atherogenic and thrombogenic potential of Lp(a).
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Acknowledgments
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The authors are grateful to Professor J. Patrick Barron of the International Medical Communications Center of Tokyo Medical University for reviewing this manuscript.
Address correspondence to Toshihiko Iwamoto, MD, Department of Geriatric Medicine, Tokyo Medical University Hospital, 6-7-1 Nishishinjuku, Shinjuku-ku, Tokyo, 160-0023, Japan. E-mail: i-wam{at}ma.kcom.ne.jp
Received October 28, 2002
Accepted January 13, 2003
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Abstract
Materials and Methods
