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Extracorporeal immunoadsorption for the specific removal of lipoprotein (a) (Lp(a) apheresis): preliminary clinical data
ELSEVIER SCIENCE IRELAND
Chemistry and Physics of Lipids 67/68 (1994) 323-330
Chemistry and Physics of LIPID$
Extracorporeal immunoadsorption for the specific removal of lipoprotein (a) (Lp(a) apheresis): preliminary clinical data ,a • a Sergei N. Pokrovsky , A n d r e i V. S u s s e k o v b, O l g a I. A f a n a s l e v a , I r i n a Y. A d a m o v a a, A n a t o l y A . L y a k i s h e v b, V a l e r y V. K u k h a r c h u k b alnstitute of Experimental Cardiology, blnstitute of Clinical Cardiology. Cardiology Research Center. Third Cherepkovskaya Street 15A, Moscow 121552. Russia
(Accepted 12 November 1992)
Abstract
The extracorporeal procedure for the specific removal of lipoprotein (a) (Lp(a)) from human plasma - - Lp(a) apheresis - - was applied to the treatment of three patients with coronary artery disease documented by angiography. Their initial lipid levels were as follows: total cholesterol, 210-230 mg/dl; low-density lipoprotein (LDL) cholesterol, 140-160 mg/dl; Lp(a), 90-120 mg/dl. The patients underwent a total of 168 procedures without significant side effects. Lp(a) apheresis reduced the Lp(a) level by removing up to 88% of Lp(a). Other plasma compounds, including LDL and plasminogen, remained practically unchanged. Lp(a) apheresis appears to be a unique, effective and specific method for lowering the Lp(a) level. Additional trials are needed to evaluate the clinical effect of this treatment. Key words." Lipoprotein (a); Apolipoprotein (a); Atherosclerosis; Apheresis; Immunosorbent
1. Introduction
Numerous studies have confirmed that elevated Lp(a) levels correlate with an increased risk of atherosclerosis, myocardial infarction and stroke (Dahlen, 1986; Armstrong et al., 1986; Rhoads, 1986; Zenker et al., 1986; Murai et al., 1986; Kostner et al., 1987). In addition to increasing the risk of developing atherosclerosis in native vessels, elevated levels of Lp(a) also impart a risk of stenosis in vein grafts after coronary artery bypass
* Corresponding author.
surgery (Hoff, 1988). These observations led the investigators to the conclusion that Lp(a) is an independent genetic risk factor for atherosclerotic vascular disease. According to the epidemiological studies, pathogenicity of Lp(a) would start at Lp(a) levels above 30-35 mg/dl. It may be noted that this value varies among populations and different studies (Dahlen, 1990). Currently little is known about the role played by Lp(a) in the majority (over 80%) of subjects having a normal plasma Lp(a) level. The attractive hypothesis concerning the normal physiological role of Lp(a) is that Lp(a) might be able to assist with wound healing and is thought to
0009-3084/94/$07.00 © 1994 Elsevier Science Ireland Ltd. All rights reserved. SSDI 0009-3084(93)02222-D
324
S.N. Pokrovsky et al./ Chem. Phys. Lipids 67/68 (1994) 323-330
promote atherosclerosis when present in excess (Lawn, 1992). Given the relationship between Lp(a) and atherosclerotic vascular disease and the pathological association of Lp(a) with atherothrombotic lesions, the lowering of elevated levels of Lp(a) is a reasonable treatment for patients with cardiovascular disease. However, there have been no studies to demonstrate the beneficial effect obtained by the reduction of an elevated Lp(a) level. The underlying problem with this study is the absence of effective means of lowering the Lp(a) level. The Lp(a) concentration in human plasma is insensitive to diet and to common hypolipidemic agents. Some beneficial, although not universal, effect was reported for neomycin and niacin administration (Guraker et al., 1985). The anabolic steroid stanozolol has also been shown to reduce in part plasma Lp(a) concentration (Albers et al., 1984). A hypothesis about the possible effect of vitamin C on Lp(a) level was reported (Rath and Pauling, 1990). Some reduction of Lp(a) level was achieved during the different extracorporeal procedures used for the treatment of patients with familial hypercholesterolemia (FH) (Armstrong et al., 1989; Oette et al., 1990; Richter et al., 1990). However, such procedures are designed for LDL elimination in patients with high LDL level, and their effect on Lp(a) concentration is not specific. We developed an immunosorbent for the specific removal of Lp(a) from human plasma (Pokrovsky et al., 1991). Here we report the first results of Lp(a) apheresis in the treatment of three patients with coronary artery disease.
2. Materials and methods 2.1. Patients
Three patients were included in this study (Table 1). One of the selected patients (LN) has heterozygous FH and was under lipid-lowering therapy with lovastatin (20-80 mg/day) for 6 months before the study. Therapy with lovastatin for this patient was continued during the treatment by Lp(a) apheresis. No lipid-lowering drugs were prescribed to the other two patients. At the beginning of the Lp(a) apheresis treatment all patients had high Lp(a) levels and no pronounced abnormalites in the levels of other lipids (Table 1). Patient FI had a myocardial infarction 5 years before the study. All patients suffered from stable angina pectoris (0-8 attacks per day) and took nifidepine (30 mg/day). Patient FI also took obsidan (40 mg/day) and patient LN nitrosorbid (30 mg/day). The pre-treatment angiography examination revealed severe coronary stenosis in all cases. Written consent was obtained from all individuals prior to the commencement of the study, which was approved by the Ethical Committee of the Cardiology Research Center, Russian Academy of Medical Science, Moscow. 2.2. Columns
Monospecific polyclonal antibody to human Lp(a) was obtained from immune sheep serum. Immunosorbent was prepared by immobilization of antibody to Sepharose CL-4B as described above (Pokrovsky et al., 1991). Each column was
Table 1 General characteristics of patients treated by Lp(a) apheresis Subjects
ZE LN FI
Age, sex
53, M 39, M 52, M
Diagnosis
Pre-treatment value (mg/dl) TC
LDL-C
HDL-C
Lp(a)
210 230 217
140 160 140
45 38 34
120 110 90
CAD, angina FH, CAD, angina CAD, angina, MI (1986)
CAD, coronary artery disease; FH, familial hypercholesterolemia; MI, myocardial infarction.
S.N. Pokrovsky et aL / Chem. Phys. Lipids 67/68 (1994) 323-330
filled with 400 ml of sorbent and tested for sterility and pyrogenicity. Anti-Lp(a) immunoadsorbtion columns are reusable. Between the treatments the columns were stored at 4°C in storage solution, which was washed prior to each Lp(a) apheresis procedure. Two personal columns were assigned to each patient.
Lp(a) apheresis procedure Lp(a) apheresis was carried out according to the routine experience of LDL apheresis treatment with immunosorbent (Borberg et al., 1988; Kukharchuk et al., 1988; Richter et al., 1992). Each patient was treated with two plasma exchanges to define a tolerance to extracorporeal procedure. 1 1 of patient plasma was passed through the immunoadsorption columns, which subsequently were used only for that patient. After the second plasma exchange the last 50 ml of plasma passed through each column was injected into the patient as a test dose (Saal et al., 1986). If no reaction to these plasma samples was observed, the third treatment as a standard Lp(a) apheresis procedure was carried out, as only in the third procedure was the whole volume of plasma passed through the column being returned to the patient. Lp(a) apheresis was performed by blood cell
325
centrifuge separator Cobe Spectra ('Cobe', USA) (Fig. 1). Access through the antecubital vein provided the blood flow rate 100-120 ml/min. The plasma perfusion rate through the column during the procedure was 40 ± 5 ml/min. Anticoagulation was achieved by the infusion of 12 00020 000 units of heparin solution. The duration of the procedure was 150-180 min, and 5.2-8.0 1 of plasma was treated during one procedure. Each column was used once per procedure. Regeneration was achieved by sequential washes of saline (1 1) 0,2 M glycine-HCl buffer (1 1), phosphate buffer (1 1) and saline (1 1).
2.3. Placebo procedure During Lp(a) apheresis treatment two placebo procedures were performed. The anti-Lp(a) immunoadsorption columns were replaced with columns filled with Sepharose CL-4B gel without antibodies. The conditions of column preparation and immunoapheresis procedure were the same as for standart Lp(a) apheresis described above.
2.4. Clinical and laboratory assays Blood samples for analysis were taken directly before and after each Lp(a) apheresis or placebo procedure.
COLUMN CONTROL UNIT
~t~~~
,.~~[~9
LLS"!~ TOWASTE
Fig. 1. General scheme of Lp(a) apheresis.
326
Total cholesterol (TC) and triglycerides (TG) were determined by enzymatic test kits (Boehringer Mannheim, Germany). High-density lipoprotein cholesterol (HDL-C) concentrations were determined by the phosphotungstate/MgCl2 procedure (Boehringer Mannheim, Germany). Lowdensity lipoprotein cholesterol (LDL-C) was calculated as the difference between TC and (HDL - C + TG / 5). The LDL cholesterol value obtained by this method also includes cholesterol contained in Lp(a). The Lowry method was used for total protein determination. Plasminogen and fibrinogen were determined by reaction with chromogenic substrate using kits (Behring, Germany). Apo B was quantified by immunoturbodimetric assay with sheep monospecific polyclonal antibody to human apoB, developed in our laboratory. Lp(a) was quantified by a non-competitive enzyme-linked immunosorbent assay (ELISA). A sheep polyclonal antibody to human Lp(a) was obtained as previously described (Pokrovsky, 1991). Microtitre plates ('Linbro', UK) were coated with anti-Lp(a) antibody (1.10 -3 mg protein/well) in an overnight incubation at 4°C. After washing and blocking for 2 h with 1% bovine albumin, 0.1 ml of appropriately diluted Lp(a) standards or serum samples were incubated in the wells for 1 h at room temperature. The wells were then washed, and peroxidase-labelled anti-Lp(a) antibody (0.5 × 10-3 mg/protein/well) was added to each well and incubation continued for a further 1 h at room temperature. Finally, a freshly prepared solution of orthophenilendiamine and 0.01% H202 was added; colour development was stopped after 15 min with an equal volume of 1 N H2SO4 and the plate read at 492 nm. Crossreactivity with plasminogen was <0.01% using this method. To control the presence of sheep antibodies in patients' plasma, the ELISA method was performed. Microtitre plates (Linbro, UK) were coated with sheep immunoglobulins purified from normal non-immune serum (1 x 10-3 mg/well). After overnight incubation at 4°C the plate was treated with 1% bovine serum albumin. Each plasma sample was diluted 1:25 and was added to four wells
S.N. Pokrovsky et aL / Chem. Phys. Lipids 67/68 (1994) 323-330
of the plate. The pool of newborn plasma (5 persons) and pool of healthy donor (8 persons) were used as negative control. Positive control was not available. The time of incubation was 1 h at room temperature. After washing, the plate was incubated with peroxidase-labeled anti-human sheep antibody (Sigma, USA). The interaction was revealed with a freshly prepared solution of phenilendiamin and 0.01% H202. At 492 nm the optical density was measured after the addition of 1 N H2SO4. 3. Results
A typical elution profile of TC, apo B and Lp(a) from the immunoadsorption column after the Lp(a) apheresis procedure is shown in Fig. 2. The content of TC and apo B in this material was 56% and 13%, respectively, corresponding to the composition of Lp(a) particles isolated in vitro from the plasma of this patient The effects of a single Lp(a) apheresis procedure on plasma concentration of lipids Lp(a), total protein, plasminogen and fibrinogen for all patients are shown in Table 2. According to the change in total protein concentration before and after procedure, the plasma dilution effect is 19% on average. The Lp(a) level decreased by 74% on average during the procedure. The reduction of plasminogen and HDL-C concentrations was 18%. A more pronounced decrease in plasma TC and LDL-C concentrations was observed ( 2 4 % and 21%, respectively). The TG and fibrinogen levels were significantly lower after the procedure (51% and 40%, respectively). In order to evaluate the specific effect of the Lp(a) apheresis procedure on the level of plasma parameters, the placebo procedures were applied to patients LN and FI. For ethical reasons, only one placebo procedure was done for each of these patients. The reduction of total protein level during the placebo procedure proved that a decrease of over 20% in concentration of plasma components was due to a plasma dilution effect. The decrease of plasminogen and HDL-C levels was the same as those for Lp(a) apheresis (18%). A 20% and 18% reductions of TC and LDL-C levels were observed. The placebo procedures also
S.N. Pokrovsky et al. / Chem. Phys. Lipids 67/68 (1994) 323-330
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150
60
PBS
gl~cin
line
40
100 E
lo
d
o Lp(a)
t-.
• apoB z~ TC
c
~
5(?
v
20
o
a
e~
1.0
2.0 VOLUME OF ELUTION (L)
I5.0~4£~ib-'qlb-~::'-~> ,
Fig. 2. Profile of i m m u n o a b s o r p t i o n column regeneration after Lp(a) apheresis procedure.
significantly affected the plasma TG and fibrinogen levels (65% and 39% of reduction, respectively). The Lp(a) apheresis treatment continued at weekly intervals with the exception of patients' holidays and illness. A graphic picture of the pretreatment and post-treatment Lp(a) level observed under long-term Lp(a) apheresis is shown in Fig. 3. We take the average of the ten last pre-treatment
values of Lp(a) concentration and compare it with the initial Lp(a) level before the course of treatment by Lp(a) apheresis. According to this estimation, the reduction of Lp(a) level was 19% for patient LN (after 80 procedures), 21% for patient ZI (after 40 procedures) and 10% for patient FI (after 48 procedures). There are no statistically significant changes in plasma TC, LDL-C and HDLC levels.
Table 2 Plasma Lp(a), lipids and total protein before and after Lp(a) apheresis procedure Parameters
Patients LN
ZE
Before Lp(a) (mg/dl) T C (mg/dl) L D L - C (mg/dl) H D L - C (mg/dl) TG Total protein (g/l)
Plasminogen Fibrinogen
91 240 192 36 81 75 91 1.9
± ± ± ± ± ± ± ±
6 40 5 0 0 7 5 0
FI
After
Before
After
Before
After
27 192 148 30 45 61 84 1.3
93 ± 16 194+4 132 ± 6 40 ± 0 180 ± 20 67 -4- 6 118 ± 7 2.4 ± 0
28 148 108 33 108 59 109 1.5
76+ 2 208 ± 9 144± 5 37 ± 0 230 ± 13 69 ± 7 100 ± 8 2.5 ± 0
13±6 160 ± 5 120 q- 4 30+0 72±0 51±4 92±5 1.5 q- 0
± 3 ± 4 ± 8 ± 0 ± 0 ± 6 -4- 4 ± 0
Data represent mean ± S.D. for 20 treatments.
+2 +4 ±6 ± 0 ± 20 ± 6 ± 5 ±0
328
S.N. Pokrovsky et al./ Chem. Phys. Lipids 67/68 (1994) 323-330 180 I
/
i
144
L
108
--
/
•
"6" d
36
t
I
0 0 17.06.91
92
184
276
Time (days)
.368
460 1.10.92
Fig. 3. Lp(a) level of patient FI during long-term Lp(a) apheresis procedure.
Three patients received a total of 168 Lp(a) apheresis procedures up to November 1992. Lp(a) apheresis was generally well tolerated by the patients. Eight side effects, such as chills without a temperature rise (2 cases) and arterial pressure drops (6 cases, all for patient ZI), were observed. No significant decline of hemoglobin and hematocrit levels were found. As sheep protein is present as the ligand in the immunoabsorption columns, the patients' plasma was monitored for the presence of human antibody against sheep protein. The patients' plasma
during all long-term Lp(a) apheresis treatment was collected for this testing. The analysis of all tested samples was performed on the same microtitre plate. The test used was not quantitative, because of the absence of a positive control. All plasma samples except the plasma of newborns have positive responses in this test (Fig. 4). During the longterm Lp(a) apheresis treatment one patient has shown a moderate rise in anti-sheep IgG levels, while the anti-sheep IgG levels of others have remained within the pre-treatment range. 4. Discussion
~ 1,0 befoTe ( w i t h o u t )
,,o=o0,.
-
g "o 0,5
•
_o o
©
0
5
10
15
20
25
Time (months)
Fig. 4. Dynamic of human anti-sheep antibody level of patients treated by Lp(a) apheresis. O, patient LN; O, patient ZE; II, patient FI. Controls: A, healthy donors; A, newborns.
The association between plasma cholesterol and atherosclerosis, recognized for many years, led to the development of several approaches in lipidlowering therapy. The fact that lipid-lowering therapy reduces the risk of atherosclerosis is proved by many studies. The very successful treatment of elevated plasma cholesterol levels, especially in the case of FH patients, is extracorporeal LDL elimination: LDL apheresis. Now the atherogenic role of Lp(a) is postulated and, by analogy with other risk reduction studies, it is reasonable to conclude that a similar beneficial effect may be realized with the reduction of Lp(a) level. A study demonstrating that the lowering of elevated Lp(a) levels can reduce risk is necessary to prove this suggestion. However, until
S.N. Pokrovsky et al. / Chem. Phys. Lipids 67/68 (1994) 323-330
now there has not been any simple means to effect significantly elevated Lp(a) levels. The results of our study confirm that extracorporeal removal of Lp(a) from human plasma - Lp(a) apheresis - - is an effective and safe method for reducing elevated Lp(a) levels. In contrast with drugs and other procedures, it is very specific for decreasing Lp(a) concentration in human plasma. All other plasma components, including LDL and plasminogen, are not affected. The reduction in plasma fibrinogen levels during the Lp(a) apheresis procedure is unlikely to be due to plasma dilution, as it is significantly greater. It may be secondary to the formation of fibrin clots, which we observed in the plasma separation system. To test the possibility of Lp(a) interaction with fibrinogen, reported by Harpel (1992) we performed in vitro experiments with a small immunoadsorption column. No significant decrease of fibrinogen levels in the in vitro Lp(a) apheresis procedure was found. The decrease of fibrinogen levels during the in vivo placebo procedure also confirms the non-specificity of this effect. The non-specific elimination of TG was also observed in Lp(a) apheresis and the placebo procedure. The possible explanation for this is the impact of anticoagulation therapy on the activation of tryglyceride lipase (Wang et al., 1992). The two phenomena of fibrinogen and TG lowering will be the subject of our future investigation. Three patients with coronary artery disease documented by angiography were treated by Lp(a) apheresis for 1.5 years. At the beginning of the study all patients had elevated Lp(a) levels but no abnormalities in the values of other lipids. No other marked risk factors were present, excluding patient LN, whose TC level was easily normalized by drug therapy with lovastatin. The goal of each Lp(a) apheresis procedure was the reduction of Lp(a) levels to a value considered to be normal. This was achieved using two immunoadsorbtion columns worked once per procedure. The technique of the immunoapheresis procedure permits a greater reduction of Lp(a) per single procedure by using two columns in rotation in more then two chromatography cycles. It is known that the guideline for post-treatment values under long-term LDL apheresis was established
329
after 6 years' evperience (Oette and Borberg, 1988). We suppose that additional trials are needed to determine the optimal strategy and clinical benefit of Lp(a) apheresis. We hope that the Lp(a) apheresis procedure can be effectively applied in future for the correction of hyper Lp(a) level in human blood. We also suggest that Lp(a) apheresis can give us the unique possibility of investigating the role of Lp(a) particles in the development of atherosclerotic plaque formation. This technique may be used for the study of metabolism of Lp(a) isoforms, and it will be one of our major subjects for the future. The international multicenter clinical trial of Lp(a) apheresis can help to obtain the answers to many open questions about Lp(a). Everyone with an interest in participating in such a trial is invited to take part. 5. References Alberts, J., Taggart, H.M., Appelbaum-Bowden, O., Haffner, S., Chesnut, C.H., and Hazzard, W.R., 1984, Reduction of lecithin-cholesterol acyltransferase, apolipoprotein D and Lp(a) lipoprotein with the anabolic steroid stanozolol. Biochim. Biophys. Acta 759, 293-296. Armstrong, V.W., Cremer, P., Eberle, E., et al., 1986, The association between Lp(a) concentration and angiographically assessed coronary atherosclerosis. Atherosclerosis 62, 249-257. Armstrong, V.W., Schleef, J., Thiery, J., Muche, R., et al., 1989, Effect of HELP-LDL-apheresis on serum concentration of human lipoprotein (a): kinetic analysis of the posttreatment return to baseline levels. Eur. J. Clin. Invest. 19, 235-240. Dahlen, G., 1990, Incidence of Lp(a) lipoprotein among populations, in: A.H. Scanu (Ed.), Academic Press, New York, pp. 151-173. Dahlen, G.H., Guyton, J.R., Attar, H., Farmer, J.A., Kautz, J.A., and Gotto, A.M.., 1986, Association of levels of lipoprotein Lp(a), plasma lipids and other lipoproteins with coronary artery disease documented by angiography. Circulation 74, 758-765. Guraker, A., Hoeg, J.M., Kostner, G., Paradopoulos, N.M., and Brewer, H.B., 1985, Levels of lipoprotein (a) decline with neomycin and niacin treatment. Atherosclerosis 57, 293-301. Harpel, P., 1992, Lipoprotein (a), thrombosis and atherosclerosis [abstract], in: International Conference on Lipoprotein, Thrombosis and Atherosclerosis, October 1992, Shizuoka, Japan. Hoff, H.F., Beck, G.J., Skibinski, C.I., et al., 1988, Lp(a) level as a predictor of vein graft stenosis after coronary artery bypass surgery in patients. Circulation 77, 1238-1244.
330 Kostner, G.M., Avogaro, P., Cazzolato, G., Marth, E., BittoloBon, G., and Qunici, G.B., 1987, Lipoprotein Lp(a) and the risk of myocardial infarction. Atherosclerosis 38, 51-61. Kukharchuk, V.V., Konovalov, G.A., Vedernikov, A.V., et al., 1988, Long-term application of three types of sorbent for LDL-apheresis. Plasma Ther. Transfus. Technol. 9, 45-47. Lawn, R., 1992, Lipoprotein (a) in heart disease. Sci. Am. 266, 26-32. Murai, A., Miyahara, T., Fujimoto, N., Matsuda, M., and Kameyama, M., 1986, Lp(a) lipoprotein as a risk factor for coronary heart disease and cerebral infarction. Atherosclerosis 59, 199-204. Oette, K., and Borberg, H., 1988, Variables involved in regression of atherosclerosis in familial hypercholesterolemia patients under long-term LDL-apheresis. Plasma Ther. Transfus. Technol. 9, 17-23. Oette, K., Borberg. H., Godehardt, E., Kadar, J., and Hombach, V., 1990, Extracorporeal immunospecific LDL elimination in severe hypercholesterolemia: effects on plasma lipoproteins and atherosclerosis, in: A.M. Gotto, M. Mancini, W.O. Richter and P. Schwaundt (Eds.), Treatment of Severe Hypercholesterolemia in the Prevention of Coronary Heart Disease, Vol. 2, Karger, Basel, pp. 175-182. Pokrovsky, S.N., Adamova, I.Y., Afanasieva, O.Y., and Benevolenskaya, G.F., 1991, Immunosorbent for selective
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removal of lipoprotein (a) from human plasma: in vitro study. Artif. Organs 15, 136-139. Rath, M., and Pauling, L., 1990, Hypothesis: lipoprotein (a) is a surrogate for ascorbate. Proc. Natl. Acad. Sci. USA 97, 6204-6207. Rhoads, G.G., Dahlen, G., Berg, K., Morton, N.E., and Dannenberg, A.L., 1986, Lp(a) lipoprotein as a risk factor for myocardial infarction. J. Am. Med. Assoc. 256, 2540-2544. Richter, W.O., Suhler, K., and Schwandt, P., 1990, Extracorporeal LDL elimination by immunoabsorption: side effects and influences on other serum lipoproteins and serum parameters, in: A.M. Gotto, M. Mancini, W.O. Richter and P. Schwaundt (Eds.), Treatment of Severe Hypercholesterolemia in the Prevention of Coronary Heart Disease, Vol. 2, Karger, Basel, pp. 183-187. Saal, S.D., Parker, T.S., Gordon, B.R., et al., 1986, Removal of low-density lipoproteins in patients by extracorporeal immunoadsorption. Am. J. Med. 80, 583-589. Wang, C.-S., Hartsuck, Y., and McConathy, W.Y., 1992, Structure and properties of lipoprotein lipase. Biochim. Biophys. Acta 1123, 1-17. Zenker, G., Koltringer, P., Bone, G., Niederkorn, K., Pfeifler, K., and Jiirgens, G., 1986, Lipoprotein (a) as a strong indicator for cerebrovascular disease. Stroke 17, 942-945.
Chemistry and Physics of Lipids 67/68 (1994) 323-330
Chemistry and Physics of LIPID$
Extracorporeal immunoadsorption for the specific removal of lipoprotein (a) (Lp(a) apheresis): preliminary clinical data ,a • a Sergei N. Pokrovsky , A n d r e i V. S u s s e k o v b, O l g a I. A f a n a s l e v a , I r i n a Y. A d a m o v a a, A n a t o l y A . L y a k i s h e v b, V a l e r y V. K u k h a r c h u k b alnstitute of Experimental Cardiology, blnstitute of Clinical Cardiology. Cardiology Research Center. Third Cherepkovskaya Street 15A, Moscow 121552. Russia
(Accepted 12 November 1992)
Abstract
The extracorporeal procedure for the specific removal of lipoprotein (a) (Lp(a)) from human plasma - - Lp(a) apheresis - - was applied to the treatment of three patients with coronary artery disease documented by angiography. Their initial lipid levels were as follows: total cholesterol, 210-230 mg/dl; low-density lipoprotein (LDL) cholesterol, 140-160 mg/dl; Lp(a), 90-120 mg/dl. The patients underwent a total of 168 procedures without significant side effects. Lp(a) apheresis reduced the Lp(a) level by removing up to 88% of Lp(a). Other plasma compounds, including LDL and plasminogen, remained practically unchanged. Lp(a) apheresis appears to be a unique, effective and specific method for lowering the Lp(a) level. Additional trials are needed to evaluate the clinical effect of this treatment. Key words." Lipoprotein (a); Apolipoprotein (a); Atherosclerosis; Apheresis; Immunosorbent
1. Introduction
Numerous studies have confirmed that elevated Lp(a) levels correlate with an increased risk of atherosclerosis, myocardial infarction and stroke (Dahlen, 1986; Armstrong et al., 1986; Rhoads, 1986; Zenker et al., 1986; Murai et al., 1986; Kostner et al., 1987). In addition to increasing the risk of developing atherosclerosis in native vessels, elevated levels of Lp(a) also impart a risk of stenosis in vein grafts after coronary artery bypass
* Corresponding author.
surgery (Hoff, 1988). These observations led the investigators to the conclusion that Lp(a) is an independent genetic risk factor for atherosclerotic vascular disease. According to the epidemiological studies, pathogenicity of Lp(a) would start at Lp(a) levels above 30-35 mg/dl. It may be noted that this value varies among populations and different studies (Dahlen, 1990). Currently little is known about the role played by Lp(a) in the majority (over 80%) of subjects having a normal plasma Lp(a) level. The attractive hypothesis concerning the normal physiological role of Lp(a) is that Lp(a) might be able to assist with wound healing and is thought to
0009-3084/94/$07.00 © 1994 Elsevier Science Ireland Ltd. All rights reserved. SSDI 0009-3084(93)02222-D
324
S.N. Pokrovsky et al./ Chem. Phys. Lipids 67/68 (1994) 323-330
promote atherosclerosis when present in excess (Lawn, 1992). Given the relationship between Lp(a) and atherosclerotic vascular disease and the pathological association of Lp(a) with atherothrombotic lesions, the lowering of elevated levels of Lp(a) is a reasonable treatment for patients with cardiovascular disease. However, there have been no studies to demonstrate the beneficial effect obtained by the reduction of an elevated Lp(a) level. The underlying problem with this study is the absence of effective means of lowering the Lp(a) level. The Lp(a) concentration in human plasma is insensitive to diet and to common hypolipidemic agents. Some beneficial, although not universal, effect was reported for neomycin and niacin administration (Guraker et al., 1985). The anabolic steroid stanozolol has also been shown to reduce in part plasma Lp(a) concentration (Albers et al., 1984). A hypothesis about the possible effect of vitamin C on Lp(a) level was reported (Rath and Pauling, 1990). Some reduction of Lp(a) level was achieved during the different extracorporeal procedures used for the treatment of patients with familial hypercholesterolemia (FH) (Armstrong et al., 1989; Oette et al., 1990; Richter et al., 1990). However, such procedures are designed for LDL elimination in patients with high LDL level, and their effect on Lp(a) concentration is not specific. We developed an immunosorbent for the specific removal of Lp(a) from human plasma (Pokrovsky et al., 1991). Here we report the first results of Lp(a) apheresis in the treatment of three patients with coronary artery disease.
2. Materials and methods 2.1. Patients
Three patients were included in this study (Table 1). One of the selected patients (LN) has heterozygous FH and was under lipid-lowering therapy with lovastatin (20-80 mg/day) for 6 months before the study. Therapy with lovastatin for this patient was continued during the treatment by Lp(a) apheresis. No lipid-lowering drugs were prescribed to the other two patients. At the beginning of the Lp(a) apheresis treatment all patients had high Lp(a) levels and no pronounced abnormalites in the levels of other lipids (Table 1). Patient FI had a myocardial infarction 5 years before the study. All patients suffered from stable angina pectoris (0-8 attacks per day) and took nifidepine (30 mg/day). Patient FI also took obsidan (40 mg/day) and patient LN nitrosorbid (30 mg/day). The pre-treatment angiography examination revealed severe coronary stenosis in all cases. Written consent was obtained from all individuals prior to the commencement of the study, which was approved by the Ethical Committee of the Cardiology Research Center, Russian Academy of Medical Science, Moscow. 2.2. Columns
Monospecific polyclonal antibody to human Lp(a) was obtained from immune sheep serum. Immunosorbent was prepared by immobilization of antibody to Sepharose CL-4B as described above (Pokrovsky et al., 1991). Each column was
Table 1 General characteristics of patients treated by Lp(a) apheresis Subjects
ZE LN FI
Age, sex
53, M 39, M 52, M
Diagnosis
Pre-treatment value (mg/dl) TC
LDL-C
HDL-C
Lp(a)
210 230 217
140 160 140
45 38 34
120 110 90
CAD, angina FH, CAD, angina CAD, angina, MI (1986)
CAD, coronary artery disease; FH, familial hypercholesterolemia; MI, myocardial infarction.
S.N. Pokrovsky et aL / Chem. Phys. Lipids 67/68 (1994) 323-330
filled with 400 ml of sorbent and tested for sterility and pyrogenicity. Anti-Lp(a) immunoadsorbtion columns are reusable. Between the treatments the columns were stored at 4°C in storage solution, which was washed prior to each Lp(a) apheresis procedure. Two personal columns were assigned to each patient.
Lp(a) apheresis procedure Lp(a) apheresis was carried out according to the routine experience of LDL apheresis treatment with immunosorbent (Borberg et al., 1988; Kukharchuk et al., 1988; Richter et al., 1992). Each patient was treated with two plasma exchanges to define a tolerance to extracorporeal procedure. 1 1 of patient plasma was passed through the immunoadsorption columns, which subsequently were used only for that patient. After the second plasma exchange the last 50 ml of plasma passed through each column was injected into the patient as a test dose (Saal et al., 1986). If no reaction to these plasma samples was observed, the third treatment as a standard Lp(a) apheresis procedure was carried out, as only in the third procedure was the whole volume of plasma passed through the column being returned to the patient. Lp(a) apheresis was performed by blood cell
325
centrifuge separator Cobe Spectra ('Cobe', USA) (Fig. 1). Access through the antecubital vein provided the blood flow rate 100-120 ml/min. The plasma perfusion rate through the column during the procedure was 40 ± 5 ml/min. Anticoagulation was achieved by the infusion of 12 00020 000 units of heparin solution. The duration of the procedure was 150-180 min, and 5.2-8.0 1 of plasma was treated during one procedure. Each column was used once per procedure. Regeneration was achieved by sequential washes of saline (1 1) 0,2 M glycine-HCl buffer (1 1), phosphate buffer (1 1) and saline (1 1).
2.3. Placebo procedure During Lp(a) apheresis treatment two placebo procedures were performed. The anti-Lp(a) immunoadsorption columns were replaced with columns filled with Sepharose CL-4B gel without antibodies. The conditions of column preparation and immunoapheresis procedure were the same as for standart Lp(a) apheresis described above.
2.4. Clinical and laboratory assays Blood samples for analysis were taken directly before and after each Lp(a) apheresis or placebo procedure.
COLUMN CONTROL UNIT
~t~~~
,.~~[~9
LLS"!~ TOWASTE
Fig. 1. General scheme of Lp(a) apheresis.
326
Total cholesterol (TC) and triglycerides (TG) were determined by enzymatic test kits (Boehringer Mannheim, Germany). High-density lipoprotein cholesterol (HDL-C) concentrations were determined by the phosphotungstate/MgCl2 procedure (Boehringer Mannheim, Germany). Lowdensity lipoprotein cholesterol (LDL-C) was calculated as the difference between TC and (HDL - C + TG / 5). The LDL cholesterol value obtained by this method also includes cholesterol contained in Lp(a). The Lowry method was used for total protein determination. Plasminogen and fibrinogen were determined by reaction with chromogenic substrate using kits (Behring, Germany). Apo B was quantified by immunoturbodimetric assay with sheep monospecific polyclonal antibody to human apoB, developed in our laboratory. Lp(a) was quantified by a non-competitive enzyme-linked immunosorbent assay (ELISA). A sheep polyclonal antibody to human Lp(a) was obtained as previously described (Pokrovsky, 1991). Microtitre plates ('Linbro', UK) were coated with anti-Lp(a) antibody (1.10 -3 mg protein/well) in an overnight incubation at 4°C. After washing and blocking for 2 h with 1% bovine albumin, 0.1 ml of appropriately diluted Lp(a) standards or serum samples were incubated in the wells for 1 h at room temperature. The wells were then washed, and peroxidase-labelled anti-Lp(a) antibody (0.5 × 10-3 mg/protein/well) was added to each well and incubation continued for a further 1 h at room temperature. Finally, a freshly prepared solution of orthophenilendiamine and 0.01% H202 was added; colour development was stopped after 15 min with an equal volume of 1 N H2SO4 and the plate read at 492 nm. Crossreactivity with plasminogen was <0.01% using this method. To control the presence of sheep antibodies in patients' plasma, the ELISA method was performed. Microtitre plates (Linbro, UK) were coated with sheep immunoglobulins purified from normal non-immune serum (1 x 10-3 mg/well). After overnight incubation at 4°C the plate was treated with 1% bovine serum albumin. Each plasma sample was diluted 1:25 and was added to four wells
S.N. Pokrovsky et aL / Chem. Phys. Lipids 67/68 (1994) 323-330
of the plate. The pool of newborn plasma (5 persons) and pool of healthy donor (8 persons) were used as negative control. Positive control was not available. The time of incubation was 1 h at room temperature. After washing, the plate was incubated with peroxidase-labeled anti-human sheep antibody (Sigma, USA). The interaction was revealed with a freshly prepared solution of phenilendiamin and 0.01% H202. At 492 nm the optical density was measured after the addition of 1 N H2SO4. 3. Results
A typical elution profile of TC, apo B and Lp(a) from the immunoadsorption column after the Lp(a) apheresis procedure is shown in Fig. 2. The content of TC and apo B in this material was 56% and 13%, respectively, corresponding to the composition of Lp(a) particles isolated in vitro from the plasma of this patient The effects of a single Lp(a) apheresis procedure on plasma concentration of lipids Lp(a), total protein, plasminogen and fibrinogen for all patients are shown in Table 2. According to the change in total protein concentration before and after procedure, the plasma dilution effect is 19% on average. The Lp(a) level decreased by 74% on average during the procedure. The reduction of plasminogen and HDL-C concentrations was 18%. A more pronounced decrease in plasma TC and LDL-C concentrations was observed ( 2 4 % and 21%, respectively). The TG and fibrinogen levels were significantly lower after the procedure (51% and 40%, respectively). In order to evaluate the specific effect of the Lp(a) apheresis procedure on the level of plasma parameters, the placebo procedures were applied to patients LN and FI. For ethical reasons, only one placebo procedure was done for each of these patients. The reduction of total protein level during the placebo procedure proved that a decrease of over 20% in concentration of plasma components was due to a plasma dilution effect. The decrease of plasminogen and HDL-C levels was the same as those for Lp(a) apheresis (18%). A 20% and 18% reductions of TC and LDL-C levels were observed. The placebo procedures also
S.N. Pokrovsky et al. / Chem. Phys. Lipids 67/68 (1994) 323-330
"
327
150
60
PBS
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line
40
100 E
lo
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o Lp(a)
t-.
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c
~
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v
20
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Fig. 2. Profile of i m m u n o a b s o r p t i o n column regeneration after Lp(a) apheresis procedure.
significantly affected the plasma TG and fibrinogen levels (65% and 39% of reduction, respectively). The Lp(a) apheresis treatment continued at weekly intervals with the exception of patients' holidays and illness. A graphic picture of the pretreatment and post-treatment Lp(a) level observed under long-term Lp(a) apheresis is shown in Fig. 3. We take the average of the ten last pre-treatment
values of Lp(a) concentration and compare it with the initial Lp(a) level before the course of treatment by Lp(a) apheresis. According to this estimation, the reduction of Lp(a) level was 19% for patient LN (after 80 procedures), 21% for patient ZI (after 40 procedures) and 10% for patient FI (after 48 procedures). There are no statistically significant changes in plasma TC, LDL-C and HDLC levels.
Table 2 Plasma Lp(a), lipids and total protein before and after Lp(a) apheresis procedure Parameters
Patients LN
ZE
Before Lp(a) (mg/dl) T C (mg/dl) L D L - C (mg/dl) H D L - C (mg/dl) TG Total protein (g/l)
Plasminogen Fibrinogen
91 240 192 36 81 75 91 1.9
± ± ± ± ± ± ± ±
6 40 5 0 0 7 5 0
FI
After
Before
After
Before
After
27 192 148 30 45 61 84 1.3
93 ± 16 194+4 132 ± 6 40 ± 0 180 ± 20 67 -4- 6 118 ± 7 2.4 ± 0
28 148 108 33 108 59 109 1.5
76+ 2 208 ± 9 144± 5 37 ± 0 230 ± 13 69 ± 7 100 ± 8 2.5 ± 0
13±6 160 ± 5 120 q- 4 30+0 72±0 51±4 92±5 1.5 q- 0
± 3 ± 4 ± 8 ± 0 ± 0 ± 6 -4- 4 ± 0
Data represent mean ± S.D. for 20 treatments.
+2 +4 ±6 ± 0 ± 20 ± 6 ± 5 ±0
328
S.N. Pokrovsky et al./ Chem. Phys. Lipids 67/68 (1994) 323-330 180 I
/
i
144
L
108
--
/
•
"6" d
36
t
I
0 0 17.06.91
92
184
276
Time (days)
.368
460 1.10.92
Fig. 3. Lp(a) level of patient FI during long-term Lp(a) apheresis procedure.
Three patients received a total of 168 Lp(a) apheresis procedures up to November 1992. Lp(a) apheresis was generally well tolerated by the patients. Eight side effects, such as chills without a temperature rise (2 cases) and arterial pressure drops (6 cases, all for patient ZI), were observed. No significant decline of hemoglobin and hematocrit levels were found. As sheep protein is present as the ligand in the immunoabsorption columns, the patients' plasma was monitored for the presence of human antibody against sheep protein. The patients' plasma
during all long-term Lp(a) apheresis treatment was collected for this testing. The analysis of all tested samples was performed on the same microtitre plate. The test used was not quantitative, because of the absence of a positive control. All plasma samples except the plasma of newborns have positive responses in this test (Fig. 4). During the longterm Lp(a) apheresis treatment one patient has shown a moderate rise in anti-sheep IgG levels, while the anti-sheep IgG levels of others have remained within the pre-treatment range. 4. Discussion
~ 1,0 befoTe ( w i t h o u t )
,,o=o0,.
-
g "o 0,5
•
_o o
©
0
5
10
15
20
25
Time (months)
Fig. 4. Dynamic of human anti-sheep antibody level of patients treated by Lp(a) apheresis. O, patient LN; O, patient ZE; II, patient FI. Controls: A, healthy donors; A, newborns.
The association between plasma cholesterol and atherosclerosis, recognized for many years, led to the development of several approaches in lipidlowering therapy. The fact that lipid-lowering therapy reduces the risk of atherosclerosis is proved by many studies. The very successful treatment of elevated plasma cholesterol levels, especially in the case of FH patients, is extracorporeal LDL elimination: LDL apheresis. Now the atherogenic role of Lp(a) is postulated and, by analogy with other risk reduction studies, it is reasonable to conclude that a similar beneficial effect may be realized with the reduction of Lp(a) level. A study demonstrating that the lowering of elevated Lp(a) levels can reduce risk is necessary to prove this suggestion. However, until
S.N. Pokrovsky et al. / Chem. Phys. Lipids 67/68 (1994) 323-330
now there has not been any simple means to effect significantly elevated Lp(a) levels. The results of our study confirm that extracorporeal removal of Lp(a) from human plasma - Lp(a) apheresis - - is an effective and safe method for reducing elevated Lp(a) levels. In contrast with drugs and other procedures, it is very specific for decreasing Lp(a) concentration in human plasma. All other plasma components, including LDL and plasminogen, are not affected. The reduction in plasma fibrinogen levels during the Lp(a) apheresis procedure is unlikely to be due to plasma dilution, as it is significantly greater. It may be secondary to the formation of fibrin clots, which we observed in the plasma separation system. To test the possibility of Lp(a) interaction with fibrinogen, reported by Harpel (1992) we performed in vitro experiments with a small immunoadsorption column. No significant decrease of fibrinogen levels in the in vitro Lp(a) apheresis procedure was found. The decrease of fibrinogen levels during the in vivo placebo procedure also confirms the non-specificity of this effect. The non-specific elimination of TG was also observed in Lp(a) apheresis and the placebo procedure. The possible explanation for this is the impact of anticoagulation therapy on the activation of tryglyceride lipase (Wang et al., 1992). The two phenomena of fibrinogen and TG lowering will be the subject of our future investigation. Three patients with coronary artery disease documented by angiography were treated by Lp(a) apheresis for 1.5 years. At the beginning of the study all patients had elevated Lp(a) levels but no abnormalities in the values of other lipids. No other marked risk factors were present, excluding patient LN, whose TC level was easily normalized by drug therapy with lovastatin. The goal of each Lp(a) apheresis procedure was the reduction of Lp(a) levels to a value considered to be normal. This was achieved using two immunoadsorbtion columns worked once per procedure. The technique of the immunoapheresis procedure permits a greater reduction of Lp(a) per single procedure by using two columns in rotation in more then two chromatography cycles. It is known that the guideline for post-treatment values under long-term LDL apheresis was established
329
after 6 years' evperience (Oette and Borberg, 1988). We suppose that additional trials are needed to determine the optimal strategy and clinical benefit of Lp(a) apheresis. We hope that the Lp(a) apheresis procedure can be effectively applied in future for the correction of hyper Lp(a) level in human blood. We also suggest that Lp(a) apheresis can give us the unique possibility of investigating the role of Lp(a) particles in the development of atherosclerotic plaque formation. This technique may be used for the study of metabolism of Lp(a) isoforms, and it will be one of our major subjects for the future. The international multicenter clinical trial of Lp(a) apheresis can help to obtain the answers to many open questions about Lp(a). Everyone with an interest in participating in such a trial is invited to take part. 5. References Alberts, J., Taggart, H.M., Appelbaum-Bowden, O., Haffner, S., Chesnut, C.H., and Hazzard, W.R., 1984, Reduction of lecithin-cholesterol acyltransferase, apolipoprotein D and Lp(a) lipoprotein with the anabolic steroid stanozolol. Biochim. Biophys. Acta 759, 293-296. Armstrong, V.W., Cremer, P., Eberle, E., et al., 1986, The association between Lp(a) concentration and angiographically assessed coronary atherosclerosis. Atherosclerosis 62, 249-257. Armstrong, V.W., Schleef, J., Thiery, J., Muche, R., et al., 1989, Effect of HELP-LDL-apheresis on serum concentration of human lipoprotein (a): kinetic analysis of the posttreatment return to baseline levels. Eur. J. Clin. Invest. 19, 235-240. Dahlen, G., 1990, Incidence of Lp(a) lipoprotein among populations, in: A.H. Scanu (Ed.), Academic Press, New York, pp. 151-173. Dahlen, G.H., Guyton, J.R., Attar, H., Farmer, J.A., Kautz, J.A., and Gotto, A.M.., 1986, Association of levels of lipoprotein Lp(a), plasma lipids and other lipoproteins with coronary artery disease documented by angiography. Circulation 74, 758-765. Guraker, A., Hoeg, J.M., Kostner, G., Paradopoulos, N.M., and Brewer, H.B., 1985, Levels of lipoprotein (a) decline with neomycin and niacin treatment. Atherosclerosis 57, 293-301. Harpel, P., 1992, Lipoprotein (a), thrombosis and atherosclerosis [abstract], in: International Conference on Lipoprotein, Thrombosis and Atherosclerosis, October 1992, Shizuoka, Japan. Hoff, H.F., Beck, G.J., Skibinski, C.I., et al., 1988, Lp(a) level as a predictor of vein graft stenosis after coronary artery bypass surgery in patients. Circulation 77, 1238-1244.
330 Kostner, G.M., Avogaro, P., Cazzolato, G., Marth, E., BittoloBon, G., and Qunici, G.B., 1987, Lipoprotein Lp(a) and the risk of myocardial infarction. Atherosclerosis 38, 51-61. Kukharchuk, V.V., Konovalov, G.A., Vedernikov, A.V., et al., 1988, Long-term application of three types of sorbent for LDL-apheresis. Plasma Ther. Transfus. Technol. 9, 45-47. Lawn, R., 1992, Lipoprotein (a) in heart disease. Sci. Am. 266, 26-32. Murai, A., Miyahara, T., Fujimoto, N., Matsuda, M., and Kameyama, M., 1986, Lp(a) lipoprotein as a risk factor for coronary heart disease and cerebral infarction. Atherosclerosis 59, 199-204. Oette, K., and Borberg, H., 1988, Variables involved in regression of atherosclerosis in familial hypercholesterolemia patients under long-term LDL-apheresis. Plasma Ther. Transfus. Technol. 9, 17-23. Oette, K., Borberg. H., Godehardt, E., Kadar, J., and Hombach, V., 1990, Extracorporeal immunospecific LDL elimination in severe hypercholesterolemia: effects on plasma lipoproteins and atherosclerosis, in: A.M. Gotto, M. Mancini, W.O. Richter and P. Schwaundt (Eds.), Treatment of Severe Hypercholesterolemia in the Prevention of Coronary Heart Disease, Vol. 2, Karger, Basel, pp. 175-182. Pokrovsky, S.N., Adamova, I.Y., Afanasieva, O.Y., and Benevolenskaya, G.F., 1991, Immunosorbent for selective
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removal of lipoprotein (a) from human plasma: in vitro study. Artif. Organs 15, 136-139. Rath, M., and Pauling, L., 1990, Hypothesis: lipoprotein (a) is a surrogate for ascorbate. Proc. Natl. Acad. Sci. USA 97, 6204-6207. Rhoads, G.G., Dahlen, G., Berg, K., Morton, N.E., and Dannenberg, A.L., 1986, Lp(a) lipoprotein as a risk factor for myocardial infarction. J. Am. Med. Assoc. 256, 2540-2544. Richter, W.O., Suhler, K., and Schwandt, P., 1990, Extracorporeal LDL elimination by immunoabsorption: side effects and influences on other serum lipoproteins and serum parameters, in: A.M. Gotto, M. Mancini, W.O. Richter and P. Schwaundt (Eds.), Treatment of Severe Hypercholesterolemia in the Prevention of Coronary Heart Disease, Vol. 2, Karger, Basel, pp. 183-187. Saal, S.D., Parker, T.S., Gordon, B.R., et al., 1986, Removal of low-density lipoproteins in patients by extracorporeal immunoadsorption. Am. J. Med. 80, 583-589. Wang, C.-S., Hartsuck, Y., and McConathy, W.Y., 1992, Structure and properties of lipoprotein lipase. Biochim. Biophys. Acta 1123, 1-17. Zenker, G., Koltringer, P., Bone, G., Niederkorn, K., Pfeifler, K., and Jiirgens, G., 1986, Lipoprotein (a) as a strong indicator for cerebrovascular disease. Stroke 17, 942-945.