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Thyroid hormone (fT4) reduces lipoprotein(a) plasma levels
ATHEROSCLEROSIS
ELSEVIER
Atherosclerosis 115 (1995) 65-71
Thyroid hormone (fr4) reduces lipoprotein(a) plasma levels Fritz H o p p i c h l e r *a, C h r i s t o p h S a n d h o l z e r b, R o y M o n c a y o c, G e r d U t e r m a n n b, Hans Georg Kraft b ~Department of Internal Medicine, University of Innsbruck, Anichstr. 35, A-6020 Innsbruck, Austria blnstitute of Medical Biology and Human Genetics, University of Innsbruek, Innsbruek, Austria CDepartment of Nuclear Medicine, University of Innsbruck, Innsbruck, Austria
Received 27 June 1994; revision received 28 November 1994; accepted 13 December 1994
Abstract
To study the influence of thyroid hormone on Lp(a) plasma concentration we measured Lp(a), total cholesterol, LDL-C, HDL-C, triglycerides and fr4 levels and determined apo(a) phenotypes in 26 patients with hyperthyroidism in a follow-up study before and after thyreostatic treatment. The pretreatment values of total cholesterol (TC), LDL-C, and Lp(a) were significantly reduced as compared with those of healthy controls. The reduced mean Lp(a) concentrations could not be explained by a difference of apo(a) 'size allele' frequencies between patients and controls. During thyreostatic treatment mean concentrations of TC, LDL-C, and HDL-C increased significantly. The mean Lp(a) value was not changed after 4 weeks of treatment. The individual changes of Lp(a), however, correlated significantly with those of LDL-C levels (R = 0.40, P = 0.04). Eighty-one per cent of the patients showed an increase of Lp(a) or no change of the Lp(a) level and 19% reacted with a decrease upon thyreostatic treatment. The observed lipid and lipoprotein changes were not different in patients with Graves disease or multifocal toxic goiter. The results indicate that Lp(a) plasma levels are decreased in the hyperthyroid state irrespective of the pathogenic mechanism. Keywords: Lipoprotein(a); Hyperthyroidism; Lipoprotein(a) plasma concentration; Apolipoprotein(a) phenotypes; Lipoprotein(a) metabolism
1. Introduction
Lipoprotein(a) (Lp(a)) is a complex macromolecular particle in human plasma that consists of a low density lipoprotein (LDL) and a large glycoprotein designated apolipoprotein(a) (apo(a)). Numerous case-control studies have demonstrated that Lp(a) concentrations in plasma * Corresponding author.
are elevated in patients with coronary heart disease (CHD) and stroke [1-4]. Lp(a) plasma concentrations are almost completely genetically determined [5,6] and Lp(a) can thus be described as a genetically determined independent risk factor for atherosclerosis [7]. Lp(a) levels are not significantly affected by environmental factors, or by age, sex and many lipid-lowering drugs [8-11]. Turnover studies have shown that Lp(a) plasma concentrations are largely determined by its syn-
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F. Hoppichler et al./ Atherosclerosis 115 (1995) 65-71
thetic rate [12,13], rather than by its catabolism. The major site of apo(a) synthesis is the liver [14]. The sites of Lp(a) degradation, especially the significance of the LDL receptor for Lp(a) catabolism, are still unclear although Lp(a) contains apo B-100, one of the two principal ligands for the LDL-receptor. In vitro studies on binding and uptake of Lp(a) by the LDL-receptor have yielded conflicting results [15-17]. Studies on the effect of HMG-CoA-reductase-inhibitors on Lp(a) levels [11] could not demonstrate a contribution of the LDL-receptor mediated route of Lp(a) degradation in vivo. In contrast, in studies with transgenic mice it was shown that the removal of Lp(a) from plasma is increased when human LDL receptors are overexpressed [18]. Hyperthyroidism is known to be associated with decreased plasma LDL-C concentrations, which can be explained by two mechanisms: (i) increased catabolism due to upregulated LDL receptor activity [19,20], and/or (ii) a reduction of apo B-100 synthesis, which was demonstrated in rats with hyperthyroidism [21,22]. Little is known about the influence of thyroid function on Lp(a) concentration [23-25]. While DeBruin et al. [23] and Engler et al. [25] reported a decrease of Lp(a) levels during thyroid hormone substitution, suggesting that the LDL receptor might be of importance for Lp(a) catabolism, another study [24] could not find such an effect. We measured fr4, Lp(a), total cholesterol, LDL-C, HDL-C and triglycerides in 26 hyperthyreotic patients before and after thyreostatic treatment. Furthermore, we categorized the patients according to the pathogenic background of their disease into immunogenic and non-immunogenic hyperthyroidism, i.e. Graves' disease and multinodular toxic goiter, respectively. 2. Subjects and methods Patients with suspected hyperthyroidism were referred to the thyroid out-patient unit of our hospital. From these patients we selected 26 untreated hyperthyroid patients (mean age 49.1 years, range 15-89 years) into the study during spring and summer 1992. None of the participating patients had evidence of malignant neoplasms
or renal, liver or vascular disease. Postmenopausal women with osteoporosis undergoing hormone substitution, as well as patients with diabetes mellitus or familiar hypercholesterolemia, were excluded. A 99 mTc scintigram wascarried out routinely. A standard dose of 37 MBq was given orally. In addition a sonographic investigation was done using a linear 7.5 MHz scanner (Sonoline SL1, Siemens, Erlangen, Germany). Patients presenting diffuse goiter, homogeneous Tcuptake, reduced echogenicity and elevated TSH-receptor antibody levels were classified as having immunogenic hyperthyroidism, i.e. Graves' disease (n= 16). Patients presenting nodular goiter (n = 10), hot nodules on the Tcscintigraphy, and normal levels of TSH-receptor antibodies were classified as non-immunogenic hyperthyroidism. The study group comprised 20 females and 6 males who were from the province of Tyrol. No dietary restrictions were enforced. None of the patients was taking any medication except as described below. Venous blood samples were taken in the morning following an overnight fasting period. After 4 weeks all patients were sampled again and all samples were immediately frozen at -70°C. The quantification of all lipid parameters was performed on the same plate (within the same assay) for pretherapeutic and posttherapeutic samples. Additionally, control samples were taken from 26 euthyroid subjects (without medication) before and after a 4 weeks time period in the same way as described above in order to study the intraindividual variance of Lp(a) levels.
2. I. Medication At the time the diagnosis was established a treatment with 60 mg methiamazole/day and 60 mg propranolol/day was instituted. The dose was tapered off according to the levels of the thyroid hormones during subsequent controls. Thyroid hormone levels were controlled every week. According to these levels in all patients the dosage of methiamazole could be reduced to 40 or 20 mg/ day during the fourth week of treatment. 2.2. Lipoprotein(a) quantification Lp(a) was quantified in human plasma by a sandwich enzyme-linked immunoabsorbent assay
F. Hoppichler et al. / Atherosclerosis 115 (1995) 65-71
(ELISA) essentially as described in [26]. Briefly, afffinity-purified polyclonal anti-human apo(a) antibody (5 /zg/ml) was bound to the wells of a microtiter plate. The blocking buffer used was 0.1% casein in PBS (pH 7.3). Appropriate dilutions (ranging from 1/10 to 1/1000) of plasma were added. The apo(a) antigen was detected with the anti-(a) monoclonal antibody 1A2 conjugated with horseradish peroxidase. This antibody does not cross-react with plasminogen [26]. A commercially available Lp(a) standard (Immuno, Vienna, Austria) was used throughout. Concentrations were expressed as Lp(a) mass in mg/dl. Pretreatment and posttreatment samples were applied on the same ELISA plate and therefore only the very low intra-assay coefficient of variance (CV) = 1.94% [26] had to be considered. This very low CV was valid for low ( < 5 mg/dl) as well as for high ( > 30 mg/dl) Lp(a) concentrations. The inter-assay CV of the Lp(a) assay was 4.95%. The sensitivity of the assay was 6 ng Lp(a) mass/ well.
2.3. Apo(a) phenotyping Apo(a) phenotypes were determined in 26 patients by immunoblotting exactly as described in [27] using the monoclonal antibody 1A2 as the first and a horseradish peroxidase-conjugated anti-mouse IgG (Dakopatts, Glostrup, Denmark) as the second antibody. Isoforms were binned and designated according to our original nomenclature as described in [27]. 2.4. Lipid measurement Total cholesterol, HDL cholesterol and triglycerides were determined by commercially available enzymatic colorimetric assays (Boehringer Mannheim, Germany). LDL cholesterol plasma concentration was calculated according to the formula of Friedewald. 2. 5. Parameters of thyroid function The concentration of free T4 (fT4) was determined using a radioimmunoassay (Amerlex-MAB FT4, Amersham, UK). The normal reference levels for fT4 were 11.8 to 23.4 pmol/1, fr3 was measured by RIA (RIA-Gnost - - fT3, Behring; reference values 3.5-6.2 pmol/1). The serum levels
67
of TSH were also determined using an RIA system (RIA-Gnost hTSH, Behring). Patients classified as hyperthyroid presented an elevation of iT4 while at the same time TSH levels were suppressed. Antibodies directed against the TSHreceptor (TSHR-Abs) were determined using a radioreceptorassay (TRAK-Assay, Henning, Berlin, Germany). Normal values for the TRAK test are < 15 U/1.
2.6. Statistics Either the non-parametric paired Wilcoxon rank sum test (Lp(a), triglycerides, T4) or the paired t-test (other variables) was applied to test for significance of differences between pretreatment and posttreatment levels. Correlation analysis was done by linear regression analysis. Comparison of phenotype frequencies between patients and a control-group [7] was performed by chi-square analysis. The two groups were dichotomized according to the presence of small versus large apo(a) isoforms as outlined previously [7]. Briefly, the apo(a) phenotypes associated with high Lp(a) levels were combined into one group, whereas the second category included all subjects with phenotypes correlating with low Lp(a) concentrations. The SPSS statistical package (SPSS Inc., Chicago, IL) was used. 3. Results
We measured iT4, Lp(a), LDL, HDL, total cholesterol and triglycerides in 26 hyperthyroid patients with either Graves' disease (n = 16) or multifocal autonomy (n = 10) (Tables 1 and 2). As a result of the thyreostatic treatment the mean Table 1 Serum IT4 and lipid levels before and during thyreostatic therapy in 26 hyperthyroid patients (mean _ S.D.)
IT4 (U/l) Lp(a) (mg/dl) TC (mg/dl) LDL-C (mg/dl) HDL-C (mg/dl) TG (mg/dl)
Before
During
P value
60.10 7.60 194.8 122.1 42.7 118.0
18.37 7.73 226.9 146.8 51.6 144.6
0.000 0.287 0.007 0.002 0.013 0.082
_+ 30.19 + 7.66 _+ 54.2 +_ 48.6 __+9.04 _+ 62.4
+_ 10.39 _+ 7.13 + 68.0 _ 56.0 + 21.2 + 83.6
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F. Hoppiehler et al. / Atherosclerosis 115 (1995) 65-71
Table 2 fT4, Lp(a) and LDL-C levels before (I) and after (II) treatment in patients with Graves' disease and multifocal goiter (mean _+
Table 3 Percentage changes of LDL and Lp(a) concentrations upon thyreostatic treatment (4 weeks)
S.D.) I Graves disease (n = 16) fT4 (U/l) 64.2 _+ 31.4 Lp(a) (mg/dl) 6.41_+ 6.59 LDL-C (mg/dl) 107 _+ 31.6 TC (mg/dl) 183 _+45.4
II
15.0 7.02 133 214
P value
_+ 8.96 _ 6.37 _+ 33.5 _+45.7
0.001 0.221 0.002 0.026
Hyperthyroid multinodular goiter (n = I0) fI'4 (U/l) 53.60 _+ 28.5 23.75 _+ 10.7 Lp(a) (mg/dl) 9.59 _+ 9.15 8.60 _+ 8.48 LDL-C (mg/dl) 144.5 _+ 61.8 167.8 _+ 76.2 TC (mg/dl) 214.3 _+ 63.6 246.8 _+ 93.0
0.007 0.859 0.185 0.203
levels of fF4 decreased from 60.2 pmol/1 ( _+30.2) to 18.4 pmol/1 ( _+ 10.4). Thus after four weeks an euthyroid state was obtained (Table 1). None of the patients presented a T3 hyperthyroidism. Plasma LDL-C levels were low at the beginning of the study and increasedsignificantly during therapy (from 122.1 + 48.6 mg/dl to 146.8 _+ 56.3 mg/dl; P = 0.002). Concordantly, total cholesterol increased from 194.8 mg/dl (_+ 54.2 mg/dl) to 226.9 mg/dl (_+68 mg/dl; P = 0.007). A similar elevation was observed for HDL-C (42.7 _ 9 mg/dl to 51.6 _+ 21.2 mg/dl; P = 0.013). The levels of triglyerides also showed a tendency to increase during the observation period, but the difference was not statistically significant (118 _+ 62.4 mg/dl to 144.6 _+ 83.6 mg/dl; P=0.082) (Table 1). Mean baseline levels of Lp(a) in hyperthyroid patients were significantly lower in the patient group compared with a healthy euthyroid control population from the same area (patients 7.60 + 7.66 mg/dl vs. 17.03 + 18.40 mg/dl; P < 0.001). Lp(a) basal values were not different in the two groups of hyperthyroid patients (Table 2). No change in mean Lp(a) levels (7.60 mg/dl _+ 7.66 mg/dl before and 7.73 mg/dl _+ 7.13 mg/dl after) was found in the total group of our study subjects after a 4 weeks treatment period (Table 1). Before treatment the range of observed Lp(a) values was 0.25-25.4 mg/dl. Under treatment the range was 0.50-28.3 mg/dl. Although the mean Lp(a) value
Lp(a) concentration range (mg/dl)
N
A% LDL
A% Lp(a)
0-5 5-10 10-15 > 15
12 6 4 4
34.8 _+ 40.6 18.8_+33.1 14.9___16.0 8.9 + 29.7
103.5 + 105.4 -13.6+26.0 -6.2_+11.5 - 10.0 _+ 31.4
Mean
24.1% (+34.5)
42.1% (+92.3)
R (between A% LDL and A% Lp(a))= 0.40 (P = 0.041).
after treatment was not different from baseline, this did not reflect the individual changes of Lp(a) concentration. When the percentage changes of Lp(a) levels were compared with those of LDL-C values, a significant correlation was detected. In Table 3 the patients are grouped according to their pretreatment Lp(a) level, and the mean percentage changes of Lp(a) and LDL-C are listed. The mean percentage change of Lp(a) values (42.1% _+ 92.3%) was even higher than that of LDL-C (24.1% _+ 34.5%). The percentage changes of Lp(a) levels were also determined in a group of 26 healthy controls. In this group the mean percentual change after a period of 4 weeks was -5.94% _+ 28.2%. When the controls were grouped as in Table 3 according to their basal Lp(a) concentration, the mean percentage changes were - 18.8%, - 0.143%, 14.1%, and - 0.475% for the 0-5 mg/dl, 5-10 mg/dl, 10-15 mg/dl, and > 15 mg/dl group, respectively. Looking at the individual changes of Lp(a) concentration, an inhomogeneous picture was obtained: 15 patients showed an elevation ( > 5%) of Lp(a) levels, 6 patients had a decrease of Lp(a) levels, and in 5 patients the Lp(a) plasma concentration was not changed. Patients with an increase of Lp(a) levels during treatment are designated as responders and the others as non-responders. Apo(a) isoforms were determined by immunoblotting. The frequency distribution of the apo(a) phenotypes, i.e. the ratio of small to large isoforms, was not different from that of the general Tyrolean population [7] (Table 4). The low Lp(a) baseline levels in hyperthyroid patients
F. Hoppichler et al. / Atherosclerosis 115 (1995) 65-71
Table 4 Distribution of apo(a) phenotypes in hyperthyroid patients and in a control group
Phenotypes with at least one large apo(a) isoform (O, $3, $3/$4, $4) Phenotypes with at least one small apo(a) isoform (B, $2/$3, $2, $2/$4)
Hyperthyroid patients
Control group
n
(%)
n
21
(80.8)
310 (73.1)
5 (19.2)
114 (26.9)
(%)
Z 2= 0.740 (d.f. = 1, N.S.).
could not be explained by a higher frequency of large apo(a) isoforms in the patient-group. The frequencies of large versus small apo(a) isoforms were not different between responders and non-responders. They were also not different in the two groups of hyperthyroid patients. Nine patients with Graves' disease (56%) and 6 patients with multifocal toxic goiter (60%) were responders. Also, the Lp(a) baseline values were similar in responders and non-responders (data not shown). 4. Discussion
The aim of our study on 26 patients with hyperthyroidism was (i) to investigate whether baseline Lp(a) plasma concentrations are affected by hyperthyroidism similar to LDL plasma concentrations, (ii) to test if Lp(a) levels increase during treatment of hyperthyroidism comparably to LDL, and (iii) to determine if the pathogenic mechanism has any influence on these parameters. In previous studies [23-25] Lp(a) levels were found to be elevated in hypothyroid patients and reduced in hyperthyroid patients, but the influence of apo(a) size alleles had not been completely excluded. In agreement with these studies, reduced baseline Lp(a) plasma concentrations were detected also in our hyperthyroid patients. Despite the different populations that were studied the mean Lp(a) values were surprisingly similar (7.5 mg/dl in the Netherlands [23], 7.3 mg/dl in
69
Switzerland [25] and 7.6 mg/dl in Tyrol (this study)). Because of the well known inverse correlation between Lp(a) concentration and apo(a) isoform size [5,27], it is necessary to exclude differences in the apo(a) isoform frequencies between patients and controls. An overrepresentation of large isoforms for example would also result in a reduced mean Lp(a) concentration. We have excluded such an effect of different apo(a) isoform frequencies by comparing the frequencies of large and small apo(a) isoforms between hyperthyroid patients and a normal Tyrolean population [7]. We did not detect a significant difference between the apo(a) phenotype frequencies of the two groups and therefore the low mean Lp(a) concentration in our patients is a true finding and not biased by apo(a) type frequency differences (Table 4). Two mechanisms can be envisaged by which low Lp(a) baseline levels are caused by hyperthyroidism. First, LDL-receptor status: it is well known that LDL levels are reduced in hyperthyroid patients. This reduction can be explained by increased LDL catabolism through upregulation of the LDL receptor. If the LDL receptor plays a significant role also in Lp(a) catabolism, one would expect low Lp(a) plasma concentrations in patients with hyperthyroidism. Second, Lp(a) synthesis: in vivo studies in rats report a reduction of apo B-100 synthesis after induction of hyperthyroidism. Thyroid hormones in pharmacologic doses were also shown to increase the editing efficacy of apo B mRNA, leading to a > 90% content of mRNA for apo B-48 in rat liver [21,22]. Both effects lead to decreased LDL-C concentration in the hyperthyroid state. Since Lp(a) contains apo B-100, both cited effects could also lead to a decreased Lp(a) concentration. Because the Lp(a) plasma concentration is influenced more by the synthetic rate than by its catabolism [12,13], the reduction of apo B synthesis might be the major mechanism by which hyperthyroidism affects Lp(a) levels. Although the mean Lp(a) level was reduced in hyperthyroid patients, after 4 weeks of thyreostatic therapy no treatment effect on the mean Lp(a) level was found in the whole group, while
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F. Hoppichler et al. / Atherosclerosis 115 (1995) 65-71
total cholesterol and LDL-C increased significantly during this time (Table 1). However, when we compared the relative changes of Lp(a) and LDL-C, a notable correlation between the two was found (R = 0.40, P = 0.04). This is comparable with the results of Engler and Riesen [25], who found a similar correlation (R = 0.43). In our cohort the huge variance was most likely responsible for an overall zero change of the mean Lp(a) concentration. Also in the Swiss study [25] a large variation was noted (Fig. 2 of [25]), but an increase of the mean Lp(a) level was found. This discrepancy might be due to the different medication and/or the different length of the treatment periods (our study, 28 + 0 days; Engler et al. [25], 186 + 142 days). It could be that changes in mean Lp(a) concentration take longer to become significant because of the slow synthetic rate of Lp(a) [13]. Looking at the individual changes of Lp(a) concentration upon treatment we observed a differentiated picture with large differences in individual responses. These results resemble those observed during treatment of hypercholesterolemic patients with HMG-CoA-reductase-inhibitors [11]. These patients showed different individual changes of Lp(a) plasma concentration and the overall change of Lp(a) levels was zero. These studies and our results in hyperthyroid patients indicate that Lp(a) is not consistently altered by the LDL-receptor status. There might be a considerable interindividual variability in the significance of the LDL-receptor activity on Lp(a) plasma levels. From in vitro binding studies it is known that Lp(a) binds with lower affinity to the LDL-receptor than LDL. From this difference it can be expected that the LDL-receptor pathway plays a role in Lp(a) degradation only in subjects with low LDL-C levels. Consistent with this hypothesis, Engler et al. [25] found significant changes of Lp(a) concentration only in hyperthyroid patients who had low LDL levels. Also in our study the changes of Lp(a) concentration during therapy were most pronounced in subjects belonging to the lowest LDL quartile (mean A% Lp(a) = 67.6%) and much smaller in those belonging to the highest LDL quartile (mean A% Lp(a) = 16.1%).
The significance of the percentage changes of Lp(a) concentrations was substantiated by two facts. Firstly, pretreatment and posttreatment samples were all frozen and then assayed on the same ELISA plate. Thus only the very low intraassay CV had to be considered and therefore also the changes of the subjects with low Lp(a) levels were real. Secondly, in a control group of euthyroid subjects Lp(a) levels were measured before and after 4 weeks. The mean percentage change of Lp(a) in this control group was only -5.94%, indicating that the 42.1% found during treatment was a significant finding. Moreover, in the group with low Lp(a) levels (0-5 mg/dl) the difference of mean percentage Lp(a) changes between hyperthyroid and euthyroid subjects was even more pronounced (103.5% in hyperthyroid versus - 18.8% in euthyroid subjects). The different pathogenic mechanisms leading to hyperthyroidism influenced neither the low baseline Lp(a) values nor Lp(a) concentration during thyreostatic treatment (Table 2). This indicates that the thyroid status itself effects low baseline Lp(a) levels in patients with Graves' disease and with multifocal goiter, and not the different pathologic mechanism of hyperthyroidism. Our results show that some patients respond to thyreostatic treatment with an increase of both Lp(a) and LDL plasma concentrations to hyperlipemic levels. Since patients with increased LDL and Lp(a) levels have about a 6-fold [3] increased risk for coronary heart disease, it is important to measure Lp(a) and LDL in patients under thyreostatic treatment.
Acknowledgments The excellent technical assistance of Gerlinde Obendorf and Linda Fineder is gratefully acknowledged. The work was supported by grant $4610 of the Austrian Science Foundation (Fonds zur F6rderung der wissenschaftlichen Forschung) to G.U.
References [1] Rhoads GG, Dahlen G, Berg K, Morton NE, Dannenberg AL. Lp(a) lipoprotein as a risk factor for myocardial infarction. J Am Med Assoc 1986;246:2540.
F. Hoppichler et al./ Atherosclerosis 115 (1995) 65-71 [2] Hoefler G, Harnoncourt F, Paschke E, Mirtl W, Pfeiffer KH, Kostner GM. Lipoprotein(a). A risk factor for myocardial infarction. Arteriosclerosis 1988;8:398. [3] Armstrong VW, Cremer P, Eberle E, Manke A, Schulze F, Wieland H, Kreuzer H, Seidel D. The association between serum Lp(a) concentrations and angiographically assessed coronary atherosclerosis - - dependence on serum LDL-levels. Atherosclerosis 1986;62:249. [4] Seed M, Hoppichler F, Reaveley D, McCarthy S, Thompson GR, Boerwinkle E, Utermann G. Relation of serum lipoprotein(a) concentration and apolipoprotein(a) phenotype to coronary heart disease in patients with familial hypercholesterolemia. N Engl J Med 1990;322:1494. [5] Utermann G, Kraft HG, Menzel H J, Hopferwieser T, Seitz C. Genetics of the quantitative Lp(a) lipoprotein trait. I. Relation of Lp(a) glycoprotein phenotypes to Lp(a) lipoprotein concentrations in plasma. Hum Genet 1988;78:41. [6] Kraft HG, K6chl S, Menzel H J, Sandholzer C, Utermann G. The apolipoprotein(a) gene: a transcribed hypervariable locus controlling plasma lipoprotein(a) concentration. Hum Genet 1992;90:220. [7] Sandholzer C, Saha N, Kark JD, Rees A, Jaross W, Dieplinger H, Hoppichler F, Boerwinkle E, Utermann G. Apo(a) isoforms predict risk for coronary heart disease - - a study in 6 populations. Arteriosclerosis Thromb 1992;12:1214. [8] Utermann G. The mysteries of lipoprotein(a). Science 1989;246:904. [9] Gurakar A, Hoeg JM, Kostner GM, Papadopoulos NM, Brewer HB Jr. Levels of lipoprotein Lp(a) decline with neomycin and niacin treatment. Atherosclerosis 1985;57:293. [10] Vessby B, Kostner G, Lithell H, Thomis J. Diverging effects of cholestyramine on apolipoprotein B and lipoprotein Lp(a). Atherosclerosis 1982;44:61. [11] Kostner GM, Gavish D, Leopold B, Bolzano K, Weintraub MS, Breslow JL. HMG CoA reductase inhibitors lower LDL cholesterol without reducing Lp(a) levels. Circulation 1982;80:1313. [12] Krempler F, Kostner GM, Bolzano K, Sandhofer F. Turnover of lipoprotein(a) in man. J Clin Invest 1980;65:1483. [13] Rader DJ, Cain W, Zech LA, Usher D, Brewer HB Jr. Variation in lipoprotein(a) concentration among individuals with the same apolipoprotein(a) isoform is determined by the rate of lipoprotein(a) production. J Clin Invest 1993;91:443. [14] Kraft HG, Menzel HJ, Hoppichler F, Vogel W, Utermann G. Changes of genetic apolipoprotein phenotypes caused by liver transplantation - - implications for apolipoprotein synthesis. J Clin Invest 1989;83:137.
I
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[15] Krempler F, Kostner GM, Roscher A, Haslauer F, Bolzano K. Studies on the role of specific cell surface receptors in the removal of lipoprotein(a) in man. J Clin Invest 1983;71:1431. [16] Steyrer E, Kostner GM. Interaction of lipoprotein Lp(a) with the B/E receptor: a study using isolated bovine adrenal cortex and human fibroblast receptors. J Lipid Res 1990;31:1247. [17] Maartmann-Moe K, Berg K. Lp(a) lipoproteins enter cultured fibroblasts independently of the plasma membrane low density lipoprotein receptor. Clin Gen 1981;20:352. [18] Hofmann SL, Eaton DL, Brown MS, McConathy JW, Goldstein JL, Hammer RE. Overexpression of human low density lipoprotein receptors leads to accelerated catabolism of Lp(a) lipoprotein in transgenic mice. J Clin Invest 1990;85:1542. [19] Staels B, van Tol A, Chan L, Will H, Verhoeven G, Auwerx J. Alterations in thyroid status modulate apolipoprotein, hepatic triglyceride lipase and low density lipoprotein receptor in rats. Endocrinology 1990;127:1144. [20] Chait A, Bierman EL, Albers JJ. Regulatory role of triiodothyronine in the degradation of low density lipoprotein by cultured human skin fibroblasts. J Clin Endocrinol Metab 1979;48:887. [21] Davidson N, Carlos R, Drewek M, Parmer T. Apolipoprotein gene expression in the rat is regulated in a tissuespecific manner by thyroid hormone. J Lipid Res 1988;29:1511. [22] Davidson N, Powell L, Wassis S, Scott J. Thyroid hormone modulates the introduction of a stop codon in rat liver apolipoprotein B messenger RNA. J Biol Chem 1988;263:13482. [23] De Bruin TWA, Barlingen H, Linde-Sibenius Trip, Vuurst AR, Akveld M J, Erkelens DW. Lipoprotein(a) and apolipoprotein B plasma concentrations in hypothyroid, euthyroid and hyperthyroid subjects. J Clin Endocrin Metab 1993;76:121. [24] Klausen IC, Nielsen FE, Hegediis L, Gerdes LU, Charles P, Faergeman O. Treatment of hypothyroidism reduces low-density-lipoprotein but not lipoprotein(a). Metabolism 1992;4:911. [25] Engler H, Riesen WF. Effect of thyroid function on concentrations of lipoprotein(a). Clin Chem 1993;39:2466. [26] Kronenberg F, Lobentanz E, K6nig P, Utermann G, Dieplinger H. Effect of sample storage on the measurement of lipoprotein(a), apolipoproteins B and A-IV, total and high density lipoprotein cholesterol and triglycerides. J Lipid Res 1994;35:1318. [27] Utermann G, Menzel H J, Kraft HG, Duba HC, Kemmler HG, Seitz C. Lp(a) glycoprotein phenotypes, inheritance and relation to Lp(a) lipoprotein concentration in plasma. J Clin Invest 1987;80:458.
ELSEVIER
Atherosclerosis 115 (1995) 65-71
Thyroid hormone (fr4) reduces lipoprotein(a) plasma levels Fritz H o p p i c h l e r *a, C h r i s t o p h S a n d h o l z e r b, R o y M o n c a y o c, G e r d U t e r m a n n b, Hans Georg Kraft b ~Department of Internal Medicine, University of Innsbruck, Anichstr. 35, A-6020 Innsbruck, Austria blnstitute of Medical Biology and Human Genetics, University of Innsbruek, Innsbruek, Austria CDepartment of Nuclear Medicine, University of Innsbruck, Innsbruck, Austria
Received 27 June 1994; revision received 28 November 1994; accepted 13 December 1994
Abstract
To study the influence of thyroid hormone on Lp(a) plasma concentration we measured Lp(a), total cholesterol, LDL-C, HDL-C, triglycerides and fr4 levels and determined apo(a) phenotypes in 26 patients with hyperthyroidism in a follow-up study before and after thyreostatic treatment. The pretreatment values of total cholesterol (TC), LDL-C, and Lp(a) were significantly reduced as compared with those of healthy controls. The reduced mean Lp(a) concentrations could not be explained by a difference of apo(a) 'size allele' frequencies between patients and controls. During thyreostatic treatment mean concentrations of TC, LDL-C, and HDL-C increased significantly. The mean Lp(a) value was not changed after 4 weeks of treatment. The individual changes of Lp(a), however, correlated significantly with those of LDL-C levels (R = 0.40, P = 0.04). Eighty-one per cent of the patients showed an increase of Lp(a) or no change of the Lp(a) level and 19% reacted with a decrease upon thyreostatic treatment. The observed lipid and lipoprotein changes were not different in patients with Graves disease or multifocal toxic goiter. The results indicate that Lp(a) plasma levels are decreased in the hyperthyroid state irrespective of the pathogenic mechanism. Keywords: Lipoprotein(a); Hyperthyroidism; Lipoprotein(a) plasma concentration; Apolipoprotein(a) phenotypes; Lipoprotein(a) metabolism
1. Introduction
Lipoprotein(a) (Lp(a)) is a complex macromolecular particle in human plasma that consists of a low density lipoprotein (LDL) and a large glycoprotein designated apolipoprotein(a) (apo(a)). Numerous case-control studies have demonstrated that Lp(a) concentrations in plasma * Corresponding author.
are elevated in patients with coronary heart disease (CHD) and stroke [1-4]. Lp(a) plasma concentrations are almost completely genetically determined [5,6] and Lp(a) can thus be described as a genetically determined independent risk factor for atherosclerosis [7]. Lp(a) levels are not significantly affected by environmental factors, or by age, sex and many lipid-lowering drugs [8-11]. Turnover studies have shown that Lp(a) plasma concentrations are largely determined by its syn-
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thetic rate [12,13], rather than by its catabolism. The major site of apo(a) synthesis is the liver [14]. The sites of Lp(a) degradation, especially the significance of the LDL receptor for Lp(a) catabolism, are still unclear although Lp(a) contains apo B-100, one of the two principal ligands for the LDL-receptor. In vitro studies on binding and uptake of Lp(a) by the LDL-receptor have yielded conflicting results [15-17]. Studies on the effect of HMG-CoA-reductase-inhibitors on Lp(a) levels [11] could not demonstrate a contribution of the LDL-receptor mediated route of Lp(a) degradation in vivo. In contrast, in studies with transgenic mice it was shown that the removal of Lp(a) from plasma is increased when human LDL receptors are overexpressed [18]. Hyperthyroidism is known to be associated with decreased plasma LDL-C concentrations, which can be explained by two mechanisms: (i) increased catabolism due to upregulated LDL receptor activity [19,20], and/or (ii) a reduction of apo B-100 synthesis, which was demonstrated in rats with hyperthyroidism [21,22]. Little is known about the influence of thyroid function on Lp(a) concentration [23-25]. While DeBruin et al. [23] and Engler et al. [25] reported a decrease of Lp(a) levels during thyroid hormone substitution, suggesting that the LDL receptor might be of importance for Lp(a) catabolism, another study [24] could not find such an effect. We measured fr4, Lp(a), total cholesterol, LDL-C, HDL-C and triglycerides in 26 hyperthyreotic patients before and after thyreostatic treatment. Furthermore, we categorized the patients according to the pathogenic background of their disease into immunogenic and non-immunogenic hyperthyroidism, i.e. Graves' disease and multinodular toxic goiter, respectively. 2. Subjects and methods Patients with suspected hyperthyroidism were referred to the thyroid out-patient unit of our hospital. From these patients we selected 26 untreated hyperthyroid patients (mean age 49.1 years, range 15-89 years) into the study during spring and summer 1992. None of the participating patients had evidence of malignant neoplasms
or renal, liver or vascular disease. Postmenopausal women with osteoporosis undergoing hormone substitution, as well as patients with diabetes mellitus or familiar hypercholesterolemia, were excluded. A 99 mTc scintigram wascarried out routinely. A standard dose of 37 MBq was given orally. In addition a sonographic investigation was done using a linear 7.5 MHz scanner (Sonoline SL1, Siemens, Erlangen, Germany). Patients presenting diffuse goiter, homogeneous Tcuptake, reduced echogenicity and elevated TSH-receptor antibody levels were classified as having immunogenic hyperthyroidism, i.e. Graves' disease (n= 16). Patients presenting nodular goiter (n = 10), hot nodules on the Tcscintigraphy, and normal levels of TSH-receptor antibodies were classified as non-immunogenic hyperthyroidism. The study group comprised 20 females and 6 males who were from the province of Tyrol. No dietary restrictions were enforced. None of the patients was taking any medication except as described below. Venous blood samples were taken in the morning following an overnight fasting period. After 4 weeks all patients were sampled again and all samples were immediately frozen at -70°C. The quantification of all lipid parameters was performed on the same plate (within the same assay) for pretherapeutic and posttherapeutic samples. Additionally, control samples were taken from 26 euthyroid subjects (without medication) before and after a 4 weeks time period in the same way as described above in order to study the intraindividual variance of Lp(a) levels.
2. I. Medication At the time the diagnosis was established a treatment with 60 mg methiamazole/day and 60 mg propranolol/day was instituted. The dose was tapered off according to the levels of the thyroid hormones during subsequent controls. Thyroid hormone levels were controlled every week. According to these levels in all patients the dosage of methiamazole could be reduced to 40 or 20 mg/ day during the fourth week of treatment. 2.2. Lipoprotein(a) quantification Lp(a) was quantified in human plasma by a sandwich enzyme-linked immunoabsorbent assay
F. Hoppichler et al. / Atherosclerosis 115 (1995) 65-71
(ELISA) essentially as described in [26]. Briefly, afffinity-purified polyclonal anti-human apo(a) antibody (5 /zg/ml) was bound to the wells of a microtiter plate. The blocking buffer used was 0.1% casein in PBS (pH 7.3). Appropriate dilutions (ranging from 1/10 to 1/1000) of plasma were added. The apo(a) antigen was detected with the anti-(a) monoclonal antibody 1A2 conjugated with horseradish peroxidase. This antibody does not cross-react with plasminogen [26]. A commercially available Lp(a) standard (Immuno, Vienna, Austria) was used throughout. Concentrations were expressed as Lp(a) mass in mg/dl. Pretreatment and posttreatment samples were applied on the same ELISA plate and therefore only the very low intra-assay coefficient of variance (CV) = 1.94% [26] had to be considered. This very low CV was valid for low ( < 5 mg/dl) as well as for high ( > 30 mg/dl) Lp(a) concentrations. The inter-assay CV of the Lp(a) assay was 4.95%. The sensitivity of the assay was 6 ng Lp(a) mass/ well.
2.3. Apo(a) phenotyping Apo(a) phenotypes were determined in 26 patients by immunoblotting exactly as described in [27] using the monoclonal antibody 1A2 as the first and a horseradish peroxidase-conjugated anti-mouse IgG (Dakopatts, Glostrup, Denmark) as the second antibody. Isoforms were binned and designated according to our original nomenclature as described in [27]. 2.4. Lipid measurement Total cholesterol, HDL cholesterol and triglycerides were determined by commercially available enzymatic colorimetric assays (Boehringer Mannheim, Germany). LDL cholesterol plasma concentration was calculated according to the formula of Friedewald. 2. 5. Parameters of thyroid function The concentration of free T4 (fT4) was determined using a radioimmunoassay (Amerlex-MAB FT4, Amersham, UK). The normal reference levels for fT4 were 11.8 to 23.4 pmol/1, fr3 was measured by RIA (RIA-Gnost - - fT3, Behring; reference values 3.5-6.2 pmol/1). The serum levels
67
of TSH were also determined using an RIA system (RIA-Gnost hTSH, Behring). Patients classified as hyperthyroid presented an elevation of iT4 while at the same time TSH levels were suppressed. Antibodies directed against the TSHreceptor (TSHR-Abs) were determined using a radioreceptorassay (TRAK-Assay, Henning, Berlin, Germany). Normal values for the TRAK test are < 15 U/1.
2.6. Statistics Either the non-parametric paired Wilcoxon rank sum test (Lp(a), triglycerides, T4) or the paired t-test (other variables) was applied to test for significance of differences between pretreatment and posttreatment levels. Correlation analysis was done by linear regression analysis. Comparison of phenotype frequencies between patients and a control-group [7] was performed by chi-square analysis. The two groups were dichotomized according to the presence of small versus large apo(a) isoforms as outlined previously [7]. Briefly, the apo(a) phenotypes associated with high Lp(a) levels were combined into one group, whereas the second category included all subjects with phenotypes correlating with low Lp(a) concentrations. The SPSS statistical package (SPSS Inc., Chicago, IL) was used. 3. Results
We measured iT4, Lp(a), LDL, HDL, total cholesterol and triglycerides in 26 hyperthyroid patients with either Graves' disease (n = 16) or multifocal autonomy (n = 10) (Tables 1 and 2). As a result of the thyreostatic treatment the mean Table 1 Serum IT4 and lipid levels before and during thyreostatic therapy in 26 hyperthyroid patients (mean _ S.D.)
IT4 (U/l) Lp(a) (mg/dl) TC (mg/dl) LDL-C (mg/dl) HDL-C (mg/dl) TG (mg/dl)
Before
During
P value
60.10 7.60 194.8 122.1 42.7 118.0
18.37 7.73 226.9 146.8 51.6 144.6
0.000 0.287 0.007 0.002 0.013 0.082
_+ 30.19 + 7.66 _+ 54.2 +_ 48.6 __+9.04 _+ 62.4
+_ 10.39 _+ 7.13 + 68.0 _ 56.0 + 21.2 + 83.6
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F. Hoppiehler et al. / Atherosclerosis 115 (1995) 65-71
Table 2 fT4, Lp(a) and LDL-C levels before (I) and after (II) treatment in patients with Graves' disease and multifocal goiter (mean _+
Table 3 Percentage changes of LDL and Lp(a) concentrations upon thyreostatic treatment (4 weeks)
S.D.) I Graves disease (n = 16) fT4 (U/l) 64.2 _+ 31.4 Lp(a) (mg/dl) 6.41_+ 6.59 LDL-C (mg/dl) 107 _+ 31.6 TC (mg/dl) 183 _+45.4
II
15.0 7.02 133 214
P value
_+ 8.96 _ 6.37 _+ 33.5 _+45.7
0.001 0.221 0.002 0.026
Hyperthyroid multinodular goiter (n = I0) fI'4 (U/l) 53.60 _+ 28.5 23.75 _+ 10.7 Lp(a) (mg/dl) 9.59 _+ 9.15 8.60 _+ 8.48 LDL-C (mg/dl) 144.5 _+ 61.8 167.8 _+ 76.2 TC (mg/dl) 214.3 _+ 63.6 246.8 _+ 93.0
0.007 0.859 0.185 0.203
levels of fF4 decreased from 60.2 pmol/1 ( _+30.2) to 18.4 pmol/1 ( _+ 10.4). Thus after four weeks an euthyroid state was obtained (Table 1). None of the patients presented a T3 hyperthyroidism. Plasma LDL-C levels were low at the beginning of the study and increasedsignificantly during therapy (from 122.1 + 48.6 mg/dl to 146.8 _+ 56.3 mg/dl; P = 0.002). Concordantly, total cholesterol increased from 194.8 mg/dl (_+ 54.2 mg/dl) to 226.9 mg/dl (_+68 mg/dl; P = 0.007). A similar elevation was observed for HDL-C (42.7 _ 9 mg/dl to 51.6 _+ 21.2 mg/dl; P = 0.013). The levels of triglyerides also showed a tendency to increase during the observation period, but the difference was not statistically significant (118 _+ 62.4 mg/dl to 144.6 _+ 83.6 mg/dl; P=0.082) (Table 1). Mean baseline levels of Lp(a) in hyperthyroid patients were significantly lower in the patient group compared with a healthy euthyroid control population from the same area (patients 7.60 + 7.66 mg/dl vs. 17.03 + 18.40 mg/dl; P < 0.001). Lp(a) basal values were not different in the two groups of hyperthyroid patients (Table 2). No change in mean Lp(a) levels (7.60 mg/dl _+ 7.66 mg/dl before and 7.73 mg/dl _+ 7.13 mg/dl after) was found in the total group of our study subjects after a 4 weeks treatment period (Table 1). Before treatment the range of observed Lp(a) values was 0.25-25.4 mg/dl. Under treatment the range was 0.50-28.3 mg/dl. Although the mean Lp(a) value
Lp(a) concentration range (mg/dl)
N
A% LDL
A% Lp(a)
0-5 5-10 10-15 > 15
12 6 4 4
34.8 _+ 40.6 18.8_+33.1 14.9___16.0 8.9 + 29.7
103.5 + 105.4 -13.6+26.0 -6.2_+11.5 - 10.0 _+ 31.4
Mean
24.1% (+34.5)
42.1% (+92.3)
R (between A% LDL and A% Lp(a))= 0.40 (P = 0.041).
after treatment was not different from baseline, this did not reflect the individual changes of Lp(a) concentration. When the percentage changes of Lp(a) levels were compared with those of LDL-C values, a significant correlation was detected. In Table 3 the patients are grouped according to their pretreatment Lp(a) level, and the mean percentage changes of Lp(a) and LDL-C are listed. The mean percentage change of Lp(a) values (42.1% _+ 92.3%) was even higher than that of LDL-C (24.1% _+ 34.5%). The percentage changes of Lp(a) levels were also determined in a group of 26 healthy controls. In this group the mean percentual change after a period of 4 weeks was -5.94% _+ 28.2%. When the controls were grouped as in Table 3 according to their basal Lp(a) concentration, the mean percentage changes were - 18.8%, - 0.143%, 14.1%, and - 0.475% for the 0-5 mg/dl, 5-10 mg/dl, 10-15 mg/dl, and > 15 mg/dl group, respectively. Looking at the individual changes of Lp(a) concentration, an inhomogeneous picture was obtained: 15 patients showed an elevation ( > 5%) of Lp(a) levels, 6 patients had a decrease of Lp(a) levels, and in 5 patients the Lp(a) plasma concentration was not changed. Patients with an increase of Lp(a) levels during treatment are designated as responders and the others as non-responders. Apo(a) isoforms were determined by immunoblotting. The frequency distribution of the apo(a) phenotypes, i.e. the ratio of small to large isoforms, was not different from that of the general Tyrolean population [7] (Table 4). The low Lp(a) baseline levels in hyperthyroid patients
F. Hoppichler et al. / Atherosclerosis 115 (1995) 65-71
Table 4 Distribution of apo(a) phenotypes in hyperthyroid patients and in a control group
Phenotypes with at least one large apo(a) isoform (O, $3, $3/$4, $4) Phenotypes with at least one small apo(a) isoform (B, $2/$3, $2, $2/$4)
Hyperthyroid patients
Control group
n
(%)
n
21
(80.8)
310 (73.1)
5 (19.2)
114 (26.9)
(%)
Z 2= 0.740 (d.f. = 1, N.S.).
could not be explained by a higher frequency of large apo(a) isoforms in the patient-group. The frequencies of large versus small apo(a) isoforms were not different between responders and non-responders. They were also not different in the two groups of hyperthyroid patients. Nine patients with Graves' disease (56%) and 6 patients with multifocal toxic goiter (60%) were responders. Also, the Lp(a) baseline values were similar in responders and non-responders (data not shown). 4. Discussion
The aim of our study on 26 patients with hyperthyroidism was (i) to investigate whether baseline Lp(a) plasma concentrations are affected by hyperthyroidism similar to LDL plasma concentrations, (ii) to test if Lp(a) levels increase during treatment of hyperthyroidism comparably to LDL, and (iii) to determine if the pathogenic mechanism has any influence on these parameters. In previous studies [23-25] Lp(a) levels were found to be elevated in hypothyroid patients and reduced in hyperthyroid patients, but the influence of apo(a) size alleles had not been completely excluded. In agreement with these studies, reduced baseline Lp(a) plasma concentrations were detected also in our hyperthyroid patients. Despite the different populations that were studied the mean Lp(a) values were surprisingly similar (7.5 mg/dl in the Netherlands [23], 7.3 mg/dl in
69
Switzerland [25] and 7.6 mg/dl in Tyrol (this study)). Because of the well known inverse correlation between Lp(a) concentration and apo(a) isoform size [5,27], it is necessary to exclude differences in the apo(a) isoform frequencies between patients and controls. An overrepresentation of large isoforms for example would also result in a reduced mean Lp(a) concentration. We have excluded such an effect of different apo(a) isoform frequencies by comparing the frequencies of large and small apo(a) isoforms between hyperthyroid patients and a normal Tyrolean population [7]. We did not detect a significant difference between the apo(a) phenotype frequencies of the two groups and therefore the low mean Lp(a) concentration in our patients is a true finding and not biased by apo(a) type frequency differences (Table 4). Two mechanisms can be envisaged by which low Lp(a) baseline levels are caused by hyperthyroidism. First, LDL-receptor status: it is well known that LDL levels are reduced in hyperthyroid patients. This reduction can be explained by increased LDL catabolism through upregulation of the LDL receptor. If the LDL receptor plays a significant role also in Lp(a) catabolism, one would expect low Lp(a) plasma concentrations in patients with hyperthyroidism. Second, Lp(a) synthesis: in vivo studies in rats report a reduction of apo B-100 synthesis after induction of hyperthyroidism. Thyroid hormones in pharmacologic doses were also shown to increase the editing efficacy of apo B mRNA, leading to a > 90% content of mRNA for apo B-48 in rat liver [21,22]. Both effects lead to decreased LDL-C concentration in the hyperthyroid state. Since Lp(a) contains apo B-100, both cited effects could also lead to a decreased Lp(a) concentration. Because the Lp(a) plasma concentration is influenced more by the synthetic rate than by its catabolism [12,13], the reduction of apo B synthesis might be the major mechanism by which hyperthyroidism affects Lp(a) levels. Although the mean Lp(a) level was reduced in hyperthyroid patients, after 4 weeks of thyreostatic therapy no treatment effect on the mean Lp(a) level was found in the whole group, while
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F. Hoppichler et al. / Atherosclerosis 115 (1995) 65-71
total cholesterol and LDL-C increased significantly during this time (Table 1). However, when we compared the relative changes of Lp(a) and LDL-C, a notable correlation between the two was found (R = 0.40, P = 0.04). This is comparable with the results of Engler and Riesen [25], who found a similar correlation (R = 0.43). In our cohort the huge variance was most likely responsible for an overall zero change of the mean Lp(a) concentration. Also in the Swiss study [25] a large variation was noted (Fig. 2 of [25]), but an increase of the mean Lp(a) level was found. This discrepancy might be due to the different medication and/or the different length of the treatment periods (our study, 28 + 0 days; Engler et al. [25], 186 + 142 days). It could be that changes in mean Lp(a) concentration take longer to become significant because of the slow synthetic rate of Lp(a) [13]. Looking at the individual changes of Lp(a) concentration upon treatment we observed a differentiated picture with large differences in individual responses. These results resemble those observed during treatment of hypercholesterolemic patients with HMG-CoA-reductase-inhibitors [11]. These patients showed different individual changes of Lp(a) plasma concentration and the overall change of Lp(a) levels was zero. These studies and our results in hyperthyroid patients indicate that Lp(a) is not consistently altered by the LDL-receptor status. There might be a considerable interindividual variability in the significance of the LDL-receptor activity on Lp(a) plasma levels. From in vitro binding studies it is known that Lp(a) binds with lower affinity to the LDL-receptor than LDL. From this difference it can be expected that the LDL-receptor pathway plays a role in Lp(a) degradation only in subjects with low LDL-C levels. Consistent with this hypothesis, Engler et al. [25] found significant changes of Lp(a) concentration only in hyperthyroid patients who had low LDL levels. Also in our study the changes of Lp(a) concentration during therapy were most pronounced in subjects belonging to the lowest LDL quartile (mean A% Lp(a) = 67.6%) and much smaller in those belonging to the highest LDL quartile (mean A% Lp(a) = 16.1%).
The significance of the percentage changes of Lp(a) concentrations was substantiated by two facts. Firstly, pretreatment and posttreatment samples were all frozen and then assayed on the same ELISA plate. Thus only the very low intraassay CV had to be considered and therefore also the changes of the subjects with low Lp(a) levels were real. Secondly, in a control group of euthyroid subjects Lp(a) levels were measured before and after 4 weeks. The mean percentage change of Lp(a) in this control group was only -5.94%, indicating that the 42.1% found during treatment was a significant finding. Moreover, in the group with low Lp(a) levels (0-5 mg/dl) the difference of mean percentage Lp(a) changes between hyperthyroid and euthyroid subjects was even more pronounced (103.5% in hyperthyroid versus - 18.8% in euthyroid subjects). The different pathogenic mechanisms leading to hyperthyroidism influenced neither the low baseline Lp(a) values nor Lp(a) concentration during thyreostatic treatment (Table 2). This indicates that the thyroid status itself effects low baseline Lp(a) levels in patients with Graves' disease and with multifocal goiter, and not the different pathologic mechanism of hyperthyroidism. Our results show that some patients respond to thyreostatic treatment with an increase of both Lp(a) and LDL plasma concentrations to hyperlipemic levels. Since patients with increased LDL and Lp(a) levels have about a 6-fold [3] increased risk for coronary heart disease, it is important to measure Lp(a) and LDL in patients under thyreostatic treatment.
Acknowledgments The excellent technical assistance of Gerlinde Obendorf and Linda Fineder is gratefully acknowledged. The work was supported by grant $4610 of the Austrian Science Foundation (Fonds zur F6rderung der wissenschaftlichen Forschung) to G.U.
References [1] Rhoads GG, Dahlen G, Berg K, Morton NE, Dannenberg AL. Lp(a) lipoprotein as a risk factor for myocardial infarction. J Am Med Assoc 1986;246:2540.
F. Hoppichler et al./ Atherosclerosis 115 (1995) 65-71 [2] Hoefler G, Harnoncourt F, Paschke E, Mirtl W, Pfeiffer KH, Kostner GM. Lipoprotein(a). A risk factor for myocardial infarction. Arteriosclerosis 1988;8:398. [3] Armstrong VW, Cremer P, Eberle E, Manke A, Schulze F, Wieland H, Kreuzer H, Seidel D. The association between serum Lp(a) concentrations and angiographically assessed coronary atherosclerosis - - dependence on serum LDL-levels. Atherosclerosis 1986;62:249. [4] Seed M, Hoppichler F, Reaveley D, McCarthy S, Thompson GR, Boerwinkle E, Utermann G. Relation of serum lipoprotein(a) concentration and apolipoprotein(a) phenotype to coronary heart disease in patients with familial hypercholesterolemia. N Engl J Med 1990;322:1494. [5] Utermann G, Kraft HG, Menzel H J, Hopferwieser T, Seitz C. Genetics of the quantitative Lp(a) lipoprotein trait. I. Relation of Lp(a) glycoprotein phenotypes to Lp(a) lipoprotein concentrations in plasma. Hum Genet 1988;78:41. [6] Kraft HG, K6chl S, Menzel H J, Sandholzer C, Utermann G. The apolipoprotein(a) gene: a transcribed hypervariable locus controlling plasma lipoprotein(a) concentration. Hum Genet 1992;90:220. [7] Sandholzer C, Saha N, Kark JD, Rees A, Jaross W, Dieplinger H, Hoppichler F, Boerwinkle E, Utermann G. Apo(a) isoforms predict risk for coronary heart disease - - a study in 6 populations. Arteriosclerosis Thromb 1992;12:1214. [8] Utermann G. The mysteries of lipoprotein(a). Science 1989;246:904. [9] Gurakar A, Hoeg JM, Kostner GM, Papadopoulos NM, Brewer HB Jr. Levels of lipoprotein Lp(a) decline with neomycin and niacin treatment. Atherosclerosis 1985;57:293. [10] Vessby B, Kostner G, Lithell H, Thomis J. Diverging effects of cholestyramine on apolipoprotein B and lipoprotein Lp(a). Atherosclerosis 1982;44:61. [11] Kostner GM, Gavish D, Leopold B, Bolzano K, Weintraub MS, Breslow JL. HMG CoA reductase inhibitors lower LDL cholesterol without reducing Lp(a) levels. Circulation 1982;80:1313. [12] Krempler F, Kostner GM, Bolzano K, Sandhofer F. Turnover of lipoprotein(a) in man. J Clin Invest 1980;65:1483. [13] Rader DJ, Cain W, Zech LA, Usher D, Brewer HB Jr. Variation in lipoprotein(a) concentration among individuals with the same apolipoprotein(a) isoform is determined by the rate of lipoprotein(a) production. J Clin Invest 1993;91:443. [14] Kraft HG, Menzel HJ, Hoppichler F, Vogel W, Utermann G. Changes of genetic apolipoprotein phenotypes caused by liver transplantation - - implications for apolipoprotein synthesis. J Clin Invest 1989;83:137.
I
71
[15] Krempler F, Kostner GM, Roscher A, Haslauer F, Bolzano K. Studies on the role of specific cell surface receptors in the removal of lipoprotein(a) in man. J Clin Invest 1983;71:1431. [16] Steyrer E, Kostner GM. Interaction of lipoprotein Lp(a) with the B/E receptor: a study using isolated bovine adrenal cortex and human fibroblast receptors. J Lipid Res 1990;31:1247. [17] Maartmann-Moe K, Berg K. Lp(a) lipoproteins enter cultured fibroblasts independently of the plasma membrane low density lipoprotein receptor. Clin Gen 1981;20:352. [18] Hofmann SL, Eaton DL, Brown MS, McConathy JW, Goldstein JL, Hammer RE. Overexpression of human low density lipoprotein receptors leads to accelerated catabolism of Lp(a) lipoprotein in transgenic mice. J Clin Invest 1990;85:1542. [19] Staels B, van Tol A, Chan L, Will H, Verhoeven G, Auwerx J. Alterations in thyroid status modulate apolipoprotein, hepatic triglyceride lipase and low density lipoprotein receptor in rats. Endocrinology 1990;127:1144. [20] Chait A, Bierman EL, Albers JJ. Regulatory role of triiodothyronine in the degradation of low density lipoprotein by cultured human skin fibroblasts. J Clin Endocrinol Metab 1979;48:887. [21] Davidson N, Carlos R, Drewek M, Parmer T. Apolipoprotein gene expression in the rat is regulated in a tissuespecific manner by thyroid hormone. J Lipid Res 1988;29:1511. [22] Davidson N, Powell L, Wassis S, Scott J. Thyroid hormone modulates the introduction of a stop codon in rat liver apolipoprotein B messenger RNA. J Biol Chem 1988;263:13482. [23] De Bruin TWA, Barlingen H, Linde-Sibenius Trip, Vuurst AR, Akveld M J, Erkelens DW. Lipoprotein(a) and apolipoprotein B plasma concentrations in hypothyroid, euthyroid and hyperthyroid subjects. J Clin Endocrin Metab 1993;76:121. [24] Klausen IC, Nielsen FE, Hegediis L, Gerdes LU, Charles P, Faergeman O. Treatment of hypothyroidism reduces low-density-lipoprotein but not lipoprotein(a). Metabolism 1992;4:911. [25] Engler H, Riesen WF. Effect of thyroid function on concentrations of lipoprotein(a). Clin Chem 1993;39:2466. [26] Kronenberg F, Lobentanz E, K6nig P, Utermann G, Dieplinger H. Effect of sample storage on the measurement of lipoprotein(a), apolipoproteins B and A-IV, total and high density lipoprotein cholesterol and triglycerides. J Lipid Res 1994;35:1318. [27] Utermann G, Menzel H J, Kraft HG, Duba HC, Kemmler HG, Seitz C. Lp(a) glycoprotein phenotypes, inheritance and relation to Lp(a) lipoprotein concentration in plasma. J Clin Invest 1987;80:458.