Serum lipoprotein(a) profiles in a singaporean population

Pathology (1999) 31, pp. 225 ± 229

SERUM LIPOPROTEIN(A) PROFILES IN A SINGAPOREAN POPULATION M O H -S I M W O N G *, W I L L I A M L. S. C H E W ²

AND

TA R -C H O O N AW *

Department of Labora tory Medicine, National University Hospital* and Department of Medicine, Alexandra Hospital², Singapore

Summary Lipoprotein(a) [Lp(a)] is formed when apolipoprotein(a) is linked to low density lipoprotein (LDL)-cholesterol via a single disulfide bond. It is an independent risk factor for myocardial infarction and raised concentrations are associated with an increased risk of developing coronary artery disease. Singapore has a multi-racial population of 77% Chinese, 14% Malays and 7% Indians. Studies have shown that the Indians have significantly higher standardised mortality ratios (SMR) compared to the Chinese and the Malays. We measured serum Lp(a) concentrations in 803 healthy individuals recruited from the Multiphasic Health Screening Programme, using the Macra Lp(a) sandwich enzyme immunoassay kit (Strategics Diagnostics, Delaware, USA). Lp(a) concentrations were skewed in all three groups. Our population mean was 9.0 mg/dl, with 50th, 75th and 95th percentile values of 10.2, 19.8 and 43.1 mg/dl, respectively, which are lower than values reported from Caucasian populations (15.0, 29.0 and 60.0 mg/dl, respectively). Males had lower Lp(a) concentrations than females (P < 0.05). The Indian group had significantly higher concentrations (median 12.3 mg/dl) compared to their Chinese (median 9.6 mg/dl) and Malay (median 8.4 mg/dl) counterparts (P < 0.05). This could partly account for the higher SMR seen in the Indian population in Singapore. As serum Lp(a) concentrations are method ± and population-dependent, we recommend that laboratories determine their own reference ranges by their method to avoid misclassification of the coronary heart disease (CHD) risk of patients. Key words: Cholesterol, coronary heart disease, ethnic variation, lipoprotein(a). Abbreviations: CHD, coronary heart disease; Lp(a), Lipoprotein(a); SMR, standardised mortality ratios. Accepted 12 February 1999

INTRODUCTION Coronary heart disease (CHD) is a leading cause of mortality and morbidity in both developed and less developed countries.1 In Singapore, it is the second leading cause of death, accounting for over 3,500 deaths (23.6% of total deaths) in 1995. W hile there has been a decline in CHD mortality in western developed countries, the CHD mortality rate in Singapore has rem ained fairly constant (24.1% of total deaths in 1994, 23.6% in 1995, and 25.6% in 1996).2

Lipoprotein (a) [Lp(a)] was first reported by Berg in 1963. 3 Each Lp(a) particle has one or two m olecules of apo(a) linked to one molecule of apo B100 by a single disulfide bond. Lp(a) differs from low density lipoprotein (LDL) in m olecular weight (it is found predominantly in the 1.0 to 1.2 g/ml density range), electrophoretic m obility and protein to lipid ratio. Lp(a) exhibits structural polymorphism, due principally to genetically determined apolipoprotein(a) [apo(a)] size heterogeneity. M ost populations exhibit a markedly skewed distribution, with most individuals having low concentrations. There is m arked interindividual variation, but intra-individual variation is remarkably constant.4 High plasma concentrations of Lp(a) have been associated with an increased incidence of CHD.5 ±7 Lp(a) has been found in atherosclerotic plaques; it accumulates in plaques following its binding to macrophages via highaffinity receptors, and its concentration in plaques is proportional to its plasma concentration.8 ,9 Lp(a) particles are capable of binding other apo B100 lipoprotein particles through an interaction between the kringle 4 domains on apo(a) and proline residues on apo B, thus facilitating Lp(a) and LDL aggregate formation, prolonging their intimal residence time and increasing their potential for oxidation and macrophage uptake. 10 Lp(a) exhibits sequence homology with plasminogen, and interferes with plasminogen binding and activation in the coagulation and fibrinolytic pathway. The GRIPS study 11 confirm ed Lp(a) as an important risk factor for myocardial infarction (M I), ranking it fifth behind LDL, family history of M I, plasma fibrinogen and high density lipoprotein (HDL) (inverse relation). Bostom and colleagues 1 2 also state that an elevated plasma Lp(a) is an independent risk factor for the developm ent of premature CHD in males, comparable in magnitude and prevalence to a total cholesterol level of greater than or equal to 6.2 mm ol/l, or a HDL level of less than or equal to 0.9 m mol/l. Studies have shown ethnic differences in the susceptibility to CHD in Singapore. The Indians had significantly higher standardised m ortality ratios (SM R) for CHD compared to the Chinese and the M alays. 13 ± 1 5 This could be secondary to genetic and/or environm ental differences in their lipid profile. In this study, we aim to determ ine the population distribution and reference range for Lp(a) in Singapore, to assess if there are ethnic differences in Lp(a) concentrations and, if so, whether these differences could explain the ethnic differences in CHD SM R.

ISSN 0031±3025 printed/ISSN 1465-3931 online/99/030225 ± 05  1999 Royal College of Pathologists of Australasia

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WONG et al.

MATERIALS AND METHODS Fasting blood samples were collected in preservative-free tubes from individuals on their first visit to the National University Hospital’s Multiphasic Health Screening Programme over a 15 mol period. Sera were stored frozen at ±70ÊC until analysis, were not subjected to greater than 1 freeze-thaw cycle and were assayed within 60 d of collection. All analyses were carried out in the Department of Laboratory M edicine at the National University Hospital. Serum Lp(a) was analysed by the M acra Lp(a) sandwich enzyme immunoassay kit (Strategic Diagnostics, Newark, DE, USA). The kit utilises two antibodies, viz. an immobilised mouse monoclonal anti-Lp(a) antibody and a goat polyclonal Lp(a) antibody conjugated with horseradish peroxidase. The absorbance of the final product is read at 492 nm and is directly proportional to the Lp(a) concentration. A new standard curve was constructed for each kit. The intra and inter-assay CVs are less than 6 and 11%, respectively. The assay range is 0 ± 80 mg/dl. Samples were assayed in duplicate, values were then extrapolated from the standard curve and the mean values were reported as the final concentration. Samples with intraassay CVs > 10% were reassayed. Samples with concentrations greater than the highest standard were diluted and reassayed.

STATISTICAL ANALYSIS Box-and-whisker plots were used to determine the population distribution of Lp(a). Rank percentiles are used to determine Lp(a) reference ranges. Non-parametric methods were used to compare the various groups.

TA B L E 1

Characteristics of study population (n = 803).

n Mean age (age range) (yrs) Chinese (n ) M ean age (yrs) Indians (n) M ean age (yrs) M alays (n) M ean age (yrs) Mean systolic BP (mmHg) Mean diastolic BP (mmHg) BM I (kg/m 2 )

Males

Females

352 44 ´2 (18 ±79) 181 43.3 95 45.9 77 44.0 127 81 23.7

451 42 ´5 (16 ±75) 172 44.0 164 41.8 115 41.1 123 77 23.0

that males had significantly lower Lp(a) values than fem ales (p < 0.05). There was no significant difference in Lp(a) values between pre-m enopausal and post-menopausal wom en. There was also no significant difference between the Chinese and the M alay groups, but a statistically significant difference was seen between the Chinese and the Indian groups (P < 0.05), and between the M alay and Indian groups (p < 0.05) when computed by the Kruskal ± Wallis and the Tukey’s tests. Indian wom en had the highest Lp(a) levels among the groups [mean Lp(a) 13.2 m g/dl]. There was no significant age-related difference in our study population.

RESULTS A total of 803 individuals were included in the study. None of these individuals had risk factors for CHD or an increased a priori risk of developing CHD. The characteristics of the study population are described in Table 1. The distribution of Lp(a) in the study population by sex and ethnic group is shown in Fig. 1. Table 2 shows the reference ranges for Lp(a) in our study population, categorised by ethnic group and sex. As the distributions are skewed, percentiles are quoted in determining the reference range instead of limits based on calculation using mean and standard deviation. The Newman ± Keuls procedure showed

DISCUSSION In the last decade, Lp(a) has become established as an independent risk factor for premature CHD. 11 ± 1 2,1 6 ±1 7 Lp(a) concentrations are population-dependent; most populations exhibit skewed distributions, with the majority of individuals having low concentrations. Our study shows this to be the case in the Singapore population both as a whole and when divided by sex and ethnic group. Our population mean for Lp(a) was 9.0 m g/dl, with 50th, 75th and 95th percentile

Fig. 1 Box-and-whisker plots illustrating serum Lp(a) distribution in the study population, categorised by sex and ethnic group. CM , Chinese men; CW, Chinese women; IM, Indian men; IW, Indian women; M M, M alay men; MW, Malay women; TM, Total men; TW, Total women.

227

SERUM LIPOPROTEIN (A) PROFILE S

TA B L E 2

Serum Lp(a) concentrations (mg/dl) in the study population, categorised by sex and ethnic group.

n

Mean sSDd

5% percentile

25% percentile

50% percentile

75% percentile

95% percentile

Men Women P* Total

352 451 < 0.05 803

7.74 (2.83) 10.18 (2.81)

1.07 1.56

4.08 4.91

8.60 11.14

17.47 22.15

34.50 46.73

9.04 (2.85)

1.33

4.58

10.17

19.81

43.07

Chinese

Men Women P* Total

180 172 < 0.05 352

7.62 (2.81) 10.27 (2.81)

1.06 1.83

3.83 4.94

7.82 11.17

16.69 20.38

34.46 48.18

8.63 (2.89)

1.30

4.58

9.50

18.86

42.37

Malays

Men Women P* Total

77 115 < 0.05 192

6.71 (2.69) 9.15 (2.60)

1.54 1.99

3.60 4.77

7.00 9.2

16.18 19.86

38.17 39.23

8.46 (2.69)

1.62

4.18

8.85

17.92

40.15

Indians

Men Women P* Total

95 164 < 0.05 259

8.62 (2.80) 11.22 (2.93)

0.95 1.14

4.46 5.60

10.85 13.23

19.28 25.75

34.59 47.04

10.08 (2.89)

1.10

5.17

12.28

22.43

43.87

Total

* The Newman± Keuls procedure is used here to test the difference between the means of two groups. Using the critical value of 0.05, we found a statistical difference between the means in the men and the women in the total study population as well as in the three ethnic groups.

values of 10.2, 19.8 and 43.1 m g/dl; these values are considerably lower than values reported from Caucasian populations (15.0, 29.0 and 60.0 mg/dl) and other Asian populations. 18 ± 2 0 The considerable variation in Lp(a) levels am ong different populations (Table 3) may reflect true population differences, or may be due to different methodologies used and may be further compounde d by the lack of a prim ary standard for Lp(a).

Lp(a) concentrations greater than 30 mg/dl (found in approximately 20% of the Caucasian population) are reported to be associated with a two-fold increased risk of developing CHD. Moreover, a serum Lp(a) concentration greater than or equal to 30 m g/dl is a risk factor for restenosis following percutaneous transluminal coronary angioplasty. 21 If we adopted the cut-off value of 30 mg/dl, then only 12% of our study population would be considered

TA B L E 3 Population studies on serum Lp(a), listed by number of subjects, mean and median Lp(a) concentrations, P values obtained (where available) and method used.

Population

n

Mean Lp(a) (mg/dl)

Caucasian Danes Chinese, Han nationality

466 102 controls 105 CHD cases

East Greenland Eskimos West Greenland Eskimos European, Caerphilly European, M anchester European, Oxfordshire Finnish

78 100 1808M 137 (100M, 37F) 227 (161M, 66F) 575 (286M, 289F)

Hungarian Hungarian Icelandic Indian, Punjab Indian, West London Japanese

202 190 184 117 (65M, 52F) 376 (194M, 182F) 2997 (1235M, 1762F)

Malaysian Sri Lankan

123 667M controls 80M CHD cases

12.9

Sudanese, Karthum Swedish

105 4646 (2702M, 1944F)

45.7

Turkish

248 (127M, 121F)

Tyrolean, Innsbruck

279

Median Lp(a) (mg/dl)

P value

Method

Klausen, et al.4 4 Qin, et al.1 9

6.3 14.5 25.7

p < 0.001

Klausen, et al.4 4

11.9 7.8 10.0 8.2 12.4 M: 19.0 F: 16.9 10.5 8.3 13.5 18.5 18.1 M: 17.2 F: 21.8

M: 8.6 F: 8.5

M: 15.3 F: 16.0

NS

IRM A IRM A IRM A RIA

Bhatnagar, et al.3 1 Bhatnagar, et al.3 1 Bhatnagar, et al.3 1 Leinc, et al.4 5

p < 0.001

EID EID IRM A IRM A ELISA

Csaszar4 6 Sandholzer, et al.2 0 Sandholzer, et al.2 0 Bhatnagar, et al.3 1 Bhatnagar, et al.3 1 Nago, et al.1 8

EID Turbidimetric

Sandholzer, et al.2 0 Jungner 4 7

EID Turbidimetric

Sandholzer, et al.2 0 Jungner 4 7

ELISA

Orem, et al.4 8

EID

Sandholzer, et al.2 0

6.0 9.0

M: 21.1 F: 21.6

M: 15.0 F: 18.0

p < 0.001

M: 15.3 F: 17.5

NS

14.1

EID = electroimmunodiffusion, IRM A = immunoradiometric assay, NS = not significant.

Reference

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WONG et al.

as having an increased risk of developing CHD. How ever, if a cut-off value of 22.8 mg/dl were used, then this would then correspond to 20% of our population. Studies have shown that the Indian population in Singapore has higher SM Rs for CHD than the Chinese and the M alay populations. Data from the Coronary Artery Disease in Asian Indians (CADI) study 22 also shows that Asian Indians have the highest CHD rates of any ethnic group studied, despite the low incidence of classical risk factors and the fact that nearly half of this group were lifelong vegetarians. Studies of different populations also report an increased CHD mortality in Indian ethnic groups.2 3 ± 26 The age-standardised CHD mortality in the United Kingdo m among men and wom en of Asian origin is approximately 40% higher than in the general population.26 Our study shows that the Indians had significantly higher Lp(a) levels than the Chinese and the M alays, and this finding is also seen in the Indians of South India.2 7 to whom our cohort of Indians are related. This similarity of data shows a comm on genetic predisposition to having higher levels of Lp(a), with environmental influences playing a secondary role. Sethi et al.2 8 also showed that while newborns in Singapore have relatively anti-atherogenic lipid profiles, the Indian neonates had higher Lp(a) concentrations than the Chinese and M alay neonates, and that this difference was statistically significant. Such a genetic predisposition is not too surprising in view of the fact that the apo(a) gene is the major gene controlling apo(a) isoforms,2 9 and the size of the apo(a) isoforms accounts for 23% of Lp(a) concentration.3 0 Bhatnagar and colleagues 3 1 also presented data which showed that serum Lp(a) concentrations in people from the Indian subcontinent who were living in West London were sim ilar to those of their siblings living in India hence that Lp(a) levels were not affected by m igration. The mean Lp(a) levels in both Indian populations were also significantly higher than in white European populations in the United Kingdom (P < 0.01). Lp(a) concentration rises in early childhood and remains stable in most subjects throughout adult life.3 2 How ever, its concentration can be affected by many m etabolic and therapeutic factors, such as diet 33 and weight.3 4 ,3 5 The Indian diet in Singapore is high in saturated fatty acids and cholesterol and this could be partly responsible. This is also reflected in the higher serum triglyceride levels seen am ong the Indian males when com pared to the Chinese and M alay males (P < 0.05). Hughes et al.3 6 reported that the Indian men and wom en in Singapore had significantly lower HDL concentrations than their Chinese and M alay counterparts, and attributed this to less efficient clearance. Our study also shows this to be the case, with mean HDL values of 0.8 and 1.0 mmol/l in Indian men and wom en, respectively. Hughes also concluded that the higher SM R in Indians could not be explained by major risk factors of cigarette sm oking, blood pressure and serum cholesterol. It is likely that the cause for the higher SM R in the Singaporian Indian population is multifactorial, encompassing both genetic and environmental influences, and that it cannot be explained on the basis of a raised Lp(a) value alone. Lp(a) concentration is currently measured using a range of m ethodologies. These include immunon ephelometric, immunoturb idimetric, EIA, ELISA, RIA and IRMA. Disagreement within and between methods has been reported.3 2,37 ,38 Factors responsible for discrepancies include differences in calibration, differences in antisera or in

combinations of antisera, the variable degree of crossreactivity with plasm inogen, size heterogeneity of apo(a) isoforms and storage and sample conditions. The International Federation of Clinical Chemistry (IFCC) has initiated an international standardisation project in search of a secondary reference m aterial for Lp(a) m easurement. 39 Phase 1 assessment results showed that a significant number of pre-existing com mercial and research Lp(a) assays perform ed sub-optimally for precision, linearity and parallelism, and that this issue should be addressed before any standardisation effort will succeed. Current Lp(a) values and reference ranges are method-dependent, thus we recom mend that each laboratory determ ine their ow n reference range using their particular method so as to avoid misclassificaton of CHD risk in patients. Lp(a) is an acute phase reactant4 0,4 1 and to avoid misinterpretation this should be taken into account when raised values are encountered. The value of a raised Lp(a) concentration must also be taken within the context of other risk factors for CHD. M aher et al.4 2 showed that while the best correlate of baseline CHD severity was Lp(a), persistent Lp(a) elevations were no longer atherogenic or clinically life-threatening in men with CHD and raised LDL and Lp(a) levels if their LDL levels were substantially reduced (by > 10% ). This was echoed by Thompson et al. 43 who showed that decreasing Lp(a) was unnecessary if LDL concentration was reduced to 3.4 mmol/l or less. Currently, no specific treatment is instituted for the lowering of Lp(a) levels. The prevention of atherosclerosis and the developm ent of CHD has been to lower LDL to levels of 3.5 m mol/l or lower. This lowering of LDL appears to mitigate against the contribution of Lp(a) in the developm ent of atherosclerosis. A C K N O W L E D G E M E N T S The authors wish to thank the staff from the Clinical Chemistry and Laboratory Information System divisions of the Department of Laboratory M edicine for their invaluable assistance in the study, Dr Dong Fang (Department of Biostatistics, National University of Singapore) for statistical advice, and Associate Professor Sunil Sethi (Department of Laboratory M edicine, National University Hospital) for his invaluable suggestions. Address for correspondence: M -S.W., Department of Laboratory Medicine, National University Hospital, 5 Lower Kent Ridge Road, Singapore 119074. E-mail: [email protected]

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