Lipoprotein(a) serum concentration in familial combined hyperlipidemia

Clinica

ELSEVIER

Chimica

Acta 223 (1993) 121-127

Lipoprotein(a) serum concentration in familial combined hyperlipidemia Maurizio Fonda, Paolo G. Da Col, Rosa La Verde, Claudia Battello, Maurizio Fisicaro, Maurizio Tonizzo, Luigi Cattin Atherosclerosis Research Unit, Institute of Clinica Medica Generale, University of Trieste. Cattinara Hospital, Trieste. Italy (Received

18 May 1993; revision

received

1 I August

1993; accepted

6 September

1993)

Abstract The lipoprotein(a) (Lp(a)) concentrations in serum were measured by an ELISA technique in 53 subjects affected by familial combined hyperlipidemia (FCHL) and in 347 healthy individuals. Lp(a) geometric means did not differ significantly between the two groups despite the different distributions. In hyperlipidemic subjects, the distribution was markedly shifted to the right (median 17 mg/dl) while in controls it was highly skewed to the left (median = 11 mg/dl). In FCHL, Lp(a) serum levels did not differ between patients with or without coronary heart disease (CHD). It was concluded that, differently from familial hypercholesterolemia (FH), in FCHL Lp(a) may not be elevated in comparison with an adequate control population. Key words: Lp(a) serum concentration; disease

Familial combined hyperlipidemia;

Coronary heart

1. Introduction Lipoprotein(a) (Lp(a)) has been recognized as a genetically determined, low density lipoprotein (LDL)-like particle in which a single copy of apoprotein B-100 (apo B- 100) is linked by a disullide bridge to apoprotein(a) [ 1,2], the specific glycoprotein marker of Lp(a). This additional glycoprotein moiety confers the pre-/3 mobility * Corresponding

author.

0 1993 Elsevier Science Publishers 0009-8981/93/$06.00 SSDI 0009-898 1(93)05697-D

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observed on agarose gel electrophoresis [3]. Lp(a) is inherited as a quantitative genetic trait and its serum concentration varies considerably from individual to individual. According to epidemiological studies, it has been proposed that the pathogenicity of Lp(a) with respect to atherosclerotic cardiovascular disease should start at Lp(a) levels above 30 mg/dl [4]. The cardiovascular risk of Lp(a) increases in the presence of high plasma levels of LDL [5]. However, the correlation of Lp(a) and hypertriglyceridemic states has received relatively less attention. Familial combined hyperlipidemia (FCHL) is a relatively common lipid disorder inherited as an autosomal dominant trait, associated with an increased risk of coronary heart disease. Multiple lipoprotein defects have been described, several of which may be atherogenic: small very low density lipoprotein (VLDL), apo B-rich VLDL, unesterified cholesterol-rich VLDL, elevated intermediate density lipoprotein (IDL), small and dense LDL, low CT/ape B LDL ratio [6-81. However, the relative atherogenicity of these metabolic disorders is unknown, but, as a hypothesis, an elevated Lp(a) serum level could explain the increased coronary heart disease (CHD) risk in some FCHL subjects. In the present study, we have evaluated Lp(a) serum distribution in a group of FCHL subjects. 2. Materials and methods 2.1. Subjects The study was performed in 53 unrelated subjects, 34 men, 19 women (age 25-78 years, mean 54), followed as out-patients in our Lipid Clinic (Table 1). Each subject was classified as having FCHL [6]. To diagnose FCHL, lipid and lipoprotein levels were obtained on first-degree relatives of prospective patients after a dietetic washout period of at least 4 weeks. Families were defined as having FCHL, present as multiple lipoprotein phenotypes, when half the family members had elevation of cholesterol and/or triglycerides more than the 90th percentile of the reference population [9] and when the proband or at least one of the first-degree relatives had a history of premature atherosclerotic heart disease (onset < 55 years). Most FCHL subjects were mildly or moderately overweight. Subjects with type III hyperlipidemia were excluded. All lipid-lowering medications were discontinued at least 1 month before the study. None of the subjects suffered from acute illness nor was taking

Table 1 Clinical characteristics

of subjects

Subjects

with FCHL N=53

Age (years, mean f SD.) Sex (M/F)

54 f 12 34119

Body mass index (kg/m2) Smoking (%) Hypertension (%) Systolic BP (mmHg) Diastolic BP (mmHg)

27 f 3.5 28% 40% 148 + 22 94 f 10

M. Fondu et ul. IClin.

Chim. Actu 223 (1993)

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123

drugs that might affect Lp(a) levels. Lipid and Lp(af levels of FCHL subjects were compared with values obtained in 347 age- and sex-matched subjects consuming an unrestricted diet isolated from a randomly selected population living in the same area and enrolled in an epidemiological survey [9]. 2.2. method Blood samples for lipid and lipoproteins analysis were drawn after a 12-h overnight fast. Serum was separated within l-2 h by low speed centrifugation and analysed enzymatically for total cholesterol and triglycerides (Boehringer-Biochemia Mannheim, FRG). Cholesterol in high density lipoproteins (HDLc) was determined enzymatically in the supernatant after Mg-phosphotungstate precipitation to remove apo B-containing lipoproteins [lo]. LDL cholesterol was then calculated by Friedewald’s formula [ll]. In 6 cases serum triglycerides were above 400 mgidl (namely, 418,#8,461, 583, 617, 635 mg!dl) and therefore VLDL was isolated by preparative ultracentrifugation (d I 1.006 g/ml), according to Have1 et al. [12]. LDL cholesterol was then measured in the bottom fraction after HDL precipitation by Mg-phosphotungstate. Lp(a) levels were measured on sera stored at -80°C for < 3 months by means of an ELISA kit (Immunozym Lot. No. 000137, Immuno AG Vienna, Austria) [13]. This is a double-antibody kit, with polyclonal sheep anti-human Lp(a) as a catching antibody and sheep anti-human Lp(a) conjugated to horseradish peroxidase as a detecting antibody. Secondary reference standards provided with the kit at five different concentrations (0, 7, 14, 32,63 mgidl) were used for establishing the standard curve. The coefficients of variation, inter- and intra-assay, were 4.5 and 5.5%, respectively. Serum lipoproteins were separated by agarose gel electrophoresis and precipitated with phosphotungstate-MgCl? [14], in order to exclude broad-0 band. In addition, type III was excluded on the basis of the cholesterol/triglycerides ratio in VLDL (~0.42) 1121. The diagnosis of angina was based on Rose-Blackburn criteria (151. The diagnosis of myocardial infarction was based on electrocardiographic criteria, cardiac-enzyme changes and clinical history. 2.3. Statistical analyses The results are expressed as mean f S.D. and were analysed by STATVIEW II, a standard statistical package for Macintosh [16]. Data were compared by the Mann-jitney test. Since the data were not always normally distributed, Spearman rank correlation coefficients were calculated. Log-transformed Lp(a) values were employed because the distribution of Lp(a) clearly deviated from normality. 3. Results Table 2 exhibits the means and standard deviations of lipid parameters in serum. Total cholesterol, triglycerides, HDL cholesterol and LDL cholesterol differed significantly between FCHL subjects and controls (P I 0.001). Although Lp(a) geometric means did not differ between the two groups, the frequency distribution of the lipoprotein were very different, as shown in Fig. 1. Lp(a)

Table 2 Lipid and Lp(a) concentrations (mean f S.D.) in serum in FCHL and age-sex matched controls

Cholesterol (mgidl) Triglycerides (mg/dl) HDL cholesterol (mgidl) LDL cholesterol (mg/dl)

Controls (n = 341)

FCHL (n = 53)

226 f 44 126 f 76 57 ZJE16

282 zt 311 f 39 f 181 f

142f 40 10.5 (3.09-35.48)~

Lp(Gb(m&W

48” 99” 8” 43”

11.6 (4.36-30,2fC

%ignificantly different from controls (P zz 0.001). bGeometric mean. CTransformed mean f S.D. of the log Lp(a) values.

concentrations in controls were distributed in the typical highly skewed manner (skewness = 1.322) while the distribution of Lp(a) in FCHL subjects was shifted to the right (skewness = -0.066). Lp(a) concentration was not >35 mg/dl in FCHL subjects but 50% of subjects had Lp(a) values > 17 mg/dl. No correlation between Lp(a) concentration and serum lipoproteins was found either in controls or in FCHL subjects. No differences in Lp(a) serum concentrations were found between FCHL subjects with or without CHID, as well as in other lipoproteins (Table 3).

Frequency (%)

I

Controls n = 347 Median = 1 1 mg/dl

30

50 , 40

FCHL subrects n = 53 Median = 17 mg/dl

3a 20 ia -. 01 0

I 0

10

20

30 40 Lpfa) mgidl

50

60

I 70

80

Fig. 1. Serum Lp(a) distribution in FCHL subjects and in controls.

Lipid and Lp(a) concentrations (mean f SD.) in serum in CHD + and CHD - FCHL subjects CHD +

Cholesterol (mg/dl) Triglycerides (mgidl) HDL cholesterol (mgdl) LDL cholesterol (mgidl) Lp(a)”(mgidl)

(n= 11)

CHD fn = 42)

214 f 40 301 l 67 37 l 1 177 f 43 11.7 (3.98-34.3)b

284 f 51 314 f IO6 39 f 9 182 f 44 11.5 (4.42-30.1 jh

The term CHD includes subjects with angina or myocardial infarction. aGeometric mean. “Transformed mean f S.D. of the log Lp(a) values.

4. Discussion Several studies have shown that high plasma levels of Lp(a) represent an independent risk factor for CHD [4,17-201. The distribution curve of this lipoprotein in plasma from Caucasians is continuous but highly skewed. Thus, plasma Lpfa) can vary from barely detectable levels to 80 mgidl and it is difficult to discriminate between normal and pathological values. One may consider as normal Lp(a) levels 17 mg/dl [2]. A previous report suggested that in normolipidemic subjects Lp(a) levels higher than 30 mg/dl may signify 1.75 times the normal risk for myocardial infarction 141.On the other hand, Lp(a) >20 mgldl may discriminate between control subjects and myocardial infarction survivors, as the distribution of Lpfa) in these patients is shifted to the right [l’?]. Nevertheless, black subjects have Lp(a) levels higher than whites with a bell-shaped distribution of this lipoprotein, but show less propensity for atherosclerosis [2 11. In our study the Lp(a) median value in FCHL was higher than that of controls, but without statistical significance. In a previous report, on the contrary, II B hyperlipidemic subjects (not classified as having FCHL) had lower Lp(a) values than in controls with a highly skewed distribution curve of this lipoprotein [22]. In our study, the comparison of the distribution curve of Lp(a) values in FCHL and controls deserves further comments: first, in FCHL the right part of the curve was truncated at 35 mg/dl; second, the absence of FCHL subjects with very high levels of Lpfa) was associated with a relative increase of subjects with medium-high values, so that the curve appears to be shifted to the right. These differences are probably due to a different level of phenotypes but we cannot exclude a bias in the selection of FCHL cases. The lack of correlation between Lp(a) and LDL cholesterol might suggest that, in FCHL, Lp(a) and LDL are under a separate metabolic control. On the contrary, in healthy normolipidemic individuals plasma levels of Lp(a) seem to be modulated by the LDL-receptor activity 1231.Considering the clinical impact of the peculiar distribution of Lp(a), we compared CHD-positive and CHD-negative FCHL patients. Lp(a) serum levels did not discriminate between the two subsets of

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223 I I993

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patients. On the contrary, it is known that Lp(a) is a strong risk factor for CHD in patients with familial hypercholesterolemia [5]. The different pathogenesis of these two hyperlipidemias can explain this behaviour. Probably, other multiple apo Bcontaining lipoprotein defects play an essential role in atherogenicity of FCHL. 5. References I 2 3 4 5

6 I 8

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14

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