Short- and long-term effects of LDL-apheresis on lipoprotein (a) serum levels

9

Clinica Chimica Acta, 195 (1990) 9-16

Elsevier

CCA 04894

Short- and long-term effects of LDL-apheresis lipoprotein (a) serum levels

on

Michael Matthias Ritter, Klaus Siihler, Werner Richter and Peter Schwandt Medical Department II, Klinikum Grosshadern. Munich (FRG)

(Received 1 February 1990; revision received 29 August 1990; accepted 5 October 1990) Key words:

LDL-apheresis; Lipoprotein (a); Immunabsorption; precipitation

Heparin-induced extracorporeal LDL-

Summary

The effect of two extracorporeal elimination procedures for LDL on lipoprotein (a) concentrations in serum was studied in patients with heterozygous familial hypercholesterolemia. During a single apheresis serum lipoprotein (a) levels fell by 45% and 58%. Over a long-term period with weekly elimination lipoprotein (a) concentrations were lowered significantly by 43% (after lo-50 treatments) and 30% (after 51-99 treatments) compared to pre-treatment values. The rise during the week following apheresis was comparable to the corresponding reincrease of LDL-cholesterol and apolipoprotein B. We conclude that both apheresis techniques are very effective in reducing lipoprotein (a) serum levels.

Introduction

In 1963, Berg [l] described a lipoprotein particle - designated lipoprotein (a) (Lp (a)) - which has pre-/Ii mobility on lipoprotein electrophoresis but which floats in a high density range between 1.05 to 1.10 g/ml during ultracentrifugation. Therefore it was also named ‘sinking pre-/? lipoprotein’. Its protein moiety consists of a specific apolipoprotein (a) which is bound by disulfide bridges to apolipoprotein B 100. Despite similar physicochemical properties and clearance from serum by similar mechanisms as LDL [2], the formation and secretion of Lp (a) is independent of the metabolism of the other apolipoprotein B containing lipoproteins [3,4]. With the possible exception of patients with abetalipoproteinemia Lp (a) can be detected in every indiCorrespondence and requests for reprints to: Dr. M. Ritter, II. Medical Department, Klinikum Grosshadern, Marchioninistrasse, D-8000 Munich 70, FRG. OOO9-8981/90/S03.50 fi 1990 Elsevier Scientific Publishers B.V. fBiomedica1 Division)

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vidual [5]. Regarding the composition of lipoprotein (a) there is a striking similarity between apo (a) and plasminogen with an unusual 37-fold repetition of the plasminogen kringle 4-like domain [6]. Lipoprotein (a) has gained much interest since raised concentrations in serum have been considered as an additional and independent risk factor for coronary artery disease and myocardial infarction in most [5,7-141 but not all studies [ 151.Furthermore, elevated serum levels are associated with ischemic cerebrovascular disease [ 13, 16181 and serve as a predictor of vein graft stenosis [19]. Lp (a) levels in serum are genetically determined [8, 201 and exhibit much greater variation between individuals than the other lipoproteins. Both dietary and pharmacological interventions are usually not successful in reducing Lp (a) levels in serum [21]. Extracorporeal LDL elimination is an effective way of lowering LDL-cholesterol levels and is under investigation for secondary prevention in patients with coronary heart disease and severe hypercholesterolemia after failure of conventional therapy. Because of the similar physicochemical properties we wondered whether the two most specific of these techniques also influence Lp (a) serum levels and could therefore result in a long-term change of another risk factor for atherosclerosis. Patients 13 patients were treated on a regular weekly basis by extracorporeal LDL-elimination procedures. All were heterozygous for familial hypercholesterolemia. Each patient had advanced coronary heart disease as shown by coronary angiography and failed to respond sufficiently to maximally tolerated combined drug therapy (with the exception of HMG-CoA-Reductase inhibitors, which were not available at the time apheresis therapy started). Eight patients (mean age: 44 years; range: 35-61) were treated by immunabsorption [22]. Briefly, for this procedure blood is withdrawn from an antecubital vein. After separation from the blood cells by a continuous blood flow cell separator, the plasma is directed over columns containing polyclonal apolipoprotein B antibodies coupled to a Sepharose Cl-4B gel. The LDL-cleared plasma is finally returned to the patient together with the blood cells by a second venous access. Five further patients (mean age: 46 years; range: 40-51) were treated by heparininduced extracorporeal LDL-precipitation (HELP) [23]. Hereby, in short, separation of blood cells and plasma is performed by a polycarbonate membrane filter. Subsequently LDL are precipitated at an acidic pH in the presence of high concentrations of heparin. After removal of the LDL by a precipitation filter the excess heparin is retained by heparin-adsorbers and a bicarbonate dialysis is performed to restore the physiological pH. Finally, the plasma and blood cells are returned. All patients adhered strictly to a low-fat and low-cholesterol diet. During the ongoing study they did not take any lipid-lowering drugs but continued with their other medication. These studies were carried out according to the principles of the second Declaration of Helsinki and the protocol of both studies was approved by our ethics committee. The efficiency of each procedure for Lp (a) elimination was determined two to six times in each patient using sera prepared from blood drawn without anticoagulant

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immediately before and after apheresis. It was allowed to clot and was then centrifuged with 2 500 x g for 10 minutes (temperature 4°C). To evaluate the long term efficiency all patients were studied at various times during their course of apheresis treatments, which varied between 55 and 130 treatments by now. Values for Lp (a) before the first apheresis treatment were available for 8 patients (4 for each procedure). The rise in Lp (a) during a random week following apheresis was studied in 11 patients (7 with immunabsorption and 4 with HELP). Methods Lipoprotein (a) was determined using a two-site anti-apo (a) immunoradiometric assay (Pharmacia Diagnostics AB, Uppsala, Sweden). Our intra- and inter-assayvariance was < 5% and < 9%, respectively (determined at 100 and 250 U/l). Apolipoprotein B was measured by immun-nephelometry (Behring-Werke, Marburg, FRG). LDL-cholesterol was calculated according to the formula of Friedewald [24] after enzymatic determination (Boehringer, Mannheim, FRG) of total- and HDL-cholesterol as well as serum triglycerides (analyzer: EPOS Eppendorf, Hamburg, FRG). All results are expressed as mean values + SEM. Statistical analysis was performed with the Wilcoxon’s matched pair signed-rank test. Results As shown in Table I lipoprotein (a), apolipoprotein B and LDL-cholesterol were reduced after a single treatment by between 45 and 5 1% using HELP and by between 58 and 68% with immunabsorption. This difference between both procedures was statistically significant. The fall in lipoprotein (a), apolipoprotein B and LDL-cholesterol was related in a highly significant way (Lp (a) vs. ApoB: r=0.960, PC 0.001; Lp (a) vs. LDL: r = 0.732, P < 0.005). Further measurements directly before and after the glass column (containing the sepharose) and the precipitation filter showed a reduction in Lp (a) of 6&95$, thereby demonstrating that Lp (a) is eliminated specifically by the applied procedure.

TABLE I Elimination of lipoprotein (a), apolipoprotein during each apheresis

Lipoprotein (a) Apolipoprotein B LDL-cholesterol

B and LDL-cholesterol by HELP and immunabsorption

HELP (n=5)

Immunabsorption

45 (4.5)s 51 (2.8)s 50 (4.3)s

58 (3.9)%* 61 (2.9)%* 68 (2.4)%*

x (SEM), *P
(n = 8)

Figure 1 shows the decline in Lp (a) levels in eight patients after 10 to 50 treatments with a mean decrease of 43% compared to pretreatment values (P < 0.05). The greatest absolute reduction was achieved in the two patients with the highest pre-treatment values of 880 and 760 U/l. Continuing apheresis for a further 50 treatments led to a slight increase, but the mean decrease of 30°F;compared to the pre-treatment value is still significant (PC 0.05) (mean absolute values of Lp (a) (n = 8): pre-apheresis 348 U/l @EM: 109), after 10-50 treatments 180 U/l (SEM: 44) and after 51-99 treatments 219 U/l (SEM: 60)). The inclusion of Lp (a) levels of the other five patients for whom pre-treatment values are not available - gives the same result: there is a small, but not significant increase of the mean Lp (a) serum level from the first to the second year of treatment. Figure 2 demonstrates the rise in Lp (a), apolipoprotein B and LDL-cholesterol during the week following treatment. Discussion These results show that Lp (a) elimination by extracorporeal apheresis is of the same magnitude as the elimination of LDL-cholesterol. Immunabsorption is superior to HELP when comparing the elimination characteristics for Lp (a), apolipoprotein B and LDL-cholesterol. The ongoing studies of both procedures - especially with a repeated coronary angiography - will have to show, whether this difference can be sustained and is of clinical importance. Our results do also show, that the pre-treatment values of Lp (a) are lowered by approximately 40% (maybe even more for those patients with very high levels) and that once the patients are in a steady-state condition lipoprotein (a) is not lowered after another year of treatment (as is also true for LDL). Also the reincrease during the week after elimination closely resembles that of LDL. Therefore, despite the independent and separate metabolic pathways of both

Lipoprotein 1000 1

(a) (U/l)

0 treatments

apheresis -

lmmunabsorption

Fig. 1. Long-term

decrease

(n=4)

C-C HELP

of lipoprotein

(a) during

(n=4)

LDL-apheresis.

13

pmol/f~~

v

1.0 -

LDL-Cholesterol

mmol/l 5.0

-

4.0

-

3.0

w lmmunabsorptien SF-ZHELP (n= 4)

o+

before after treatment

VW ’

Fig. 2. Reincrease of lipoprotein (a), apoli~protein

day2

day4

(n= 7)

day7

B and LDL-cholesterol during the week after apheresis.

apoprotein B containing lipoproteins they behave very similar concerning extracorporeal elimination with immunabsorption or HELP. Another apoprotein B containing lipoprotein, VLDL, is cleared differently by both procedures, the measurement of VLDL-t~glycerides directly before and after the glass column and the precipitation filter shows a reduction of 64% for HELP but none for immunabsorption [25]. Therefore, the behavior of apoprotein B containing lipoproteins during apheresis concerning HELP and immunabsorption is not fully predictable and this underscores the relevance of direct measurements of Lp (a). Despite major changes in other lipoprotein serum concentrations Lp (a) serum levels do not change in response to dietary and pharmacological interventions, including 16 g/d cholestyramine, 600 mg/d benzafibrate or 500 mg/d nicotinic acid [5, 26-281. Concerning HMG-CoA-reductase-inhibitors either no [29] or even detrimental changes [30] have been reported. There is one report of a slight reduction in Lp (a) serum levels by a combination of neomycin (2 g/d) and niacin (3 g/d) treatment [31], but the authors did not observe a consistent effect but rather interindividual differ-

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ences [21]. High levels of alcohol intake [32] as well as the anaboiic steroid stanazol [33] are known to lower Lp (a) values but this is without clinical relevance. Plasmapheresis and replacement by albumin solution reduced Lp (a) serum concentrations by 26% [34], the same authors demonstrated a short-term reduction of Lp (a) by a different immunabsorption technique between 6.5 and 14.4% in one patient and a reduction of 20 and 29% by HELP in two patients. Altogether, only extracorporeal elimination procedures effectively and consistently eliminate Lp (a) and are so far the only known possibility to reduce Lp (a) serum levels. If the knowledge of Lp (a) as an additional risk factor increases, selected patients with advanced coronary heart disease might profit from apheresis although their LDL levels could be lowered enough by conventional treatment.

This work was supported by the Bundesminister fur Forschung und Technologie (grants 01 2585027 and 01 2585014). References I Berg K. A new serum type system in man - the Lp system. Acta Pathol Microbial Stand 1963;59:369 382. 2 Krempler F, Kostner GM, Roscher A, Haslauer F, Bolzano K, Sandhofer F. Studies on the role of specific ceil surface receptors in the removal of lipoprotein (a) in man. J Clin Invest 1983;71:1431-144 I. 3 Krempler F, Kostner G, Boizano K, Sandhofer F. Lipoprotein (a) is not a metabolic product of other Lipoproteins containing apolipoprotein B. Biochim Biophys Acta 197~575:63-70. 4 Krempler F, Kostner GM, Bolzano K, Sandhofer F. Turnover of lipoprotein (a) in man. J Clin Invest 1980;65:1483-.1490. 5 Albers JJ, Adolphson JL, Hazard WR. Radioimmunoassay of human plasma Lp (a) lipoprotein. J Lipid Res 1977;18:331-338. 6 McLean JW, Tomlinson JE, Kuang W-J et al. cDNA sequence of human apolipoprot~in (a) is homologous to plasminogen. Nature 1987;300:132-137. 7 Dahlen G, Berg K, Gillniis T, Ericson C. Lp (a) lipoprotein/pre$, lipoprotein in Swedish middle-aged males and in patients with coronary heart disease. Clin Genet 1975;7:334-341. 8 Dahlen G, Berg K, Frick MH. Lp (a) lipoprotein/pre-~-l-lipoprotein, serum lipids and atherosclerotic disease. Clin Genet t 976;9:558-566. 9 Frick MH, Dahlen G, Berg K, Valle M, Hekali P. Serum lipids in angiographically assessed coronary atherosclerosis. Chest 1978;73:62265. 10 Kostner GM, Avogaro P, Cazzolato G. Marth E, Bittolo-in G. Qunici GB. Lipoprotein Lp (a) and the risk for myocardial infarction. Atherosclerosis 1981;38:51-61. 11 Armstrong VW, Cremer P, Eberle E. et al. The association between serum Lp (a) concentrations and angiographically assessed coronary atherosclerosis. Dependence on serum LDL levels. Atherosclerosis 1986;62:249-257. 12 Dahlin GH, Guyton JR, Attar M, Farmer JA, Kautz JA, Gotto AM. Association of levels of lipoprotein Lp (a), plasma lipids, and other lipoproteins with coronary artery disease documented by angiography. Circulation 198&74:X8-765. 13 Murai A, Miyahara T, Fujimoto N, Matsuda M, Kameyama M. Lp (a) Lipoprotein as a risk factor for coronary heart disease and cerebral infarction. Atherosclerosis 1986;.59:199-204. 14 Rhoads GG, Dahlen G, Berg K, Morton NE, Dannenberg AL. Lp (a) Lipoprotein as a risk factor for myocardiat infarction. JAMA 1986;256:2540-2544.

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