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A new low density lipoprotein apheresis system using two dextran sulfate cellulose columns in an automated column regenerating unit (LDL continuous apheresis)
Atherosclerosis,
19
68 (1987) 19-25
Elsevier Scientific Publishers Ireland, Ltd. ATH 04018
A new low density lipoprotein apheresis system using two dextran sulfate cellulose columns in an automated column regenerating unit (LDL continuous apheresis) Hiroshi Mabuchi, Ichiro Michishita, Mitsuaki Takeda, Hajime Fujita, Junji Koizumi, Ryoyu Takeda, Satoshi Takada and Michikazu Oonishi Second Department of Internal Medicine, Kanazawa University School of Medicine, Kanazawa and Kanegafuchi Chemical Industry Central Research L.&oratory, Kobe (Japan)
(Japan).
(Received 23 February, 1987) (Revised, received 2 April, 1987) (Accepted 10 April, 1987)
We describe a new low density lipoprotein (LDL) apheresis system using dextran sulfate cellulose column in an automated column regenerating unit (LDL continuous apheresis). Two columns containing 150 ml of dextran sulfate cellulose were used, and the whole extracorporeal circulation was about 400 ml in volume. After 600 ml of plasma was adsorbed into the first column, the second column was used as an adsorbent and meanwhile the first column was regenerated. Thus, the 2 columns were used alternately without losing the potency of the columns. As the apparatus was automatically controlled by a computerized unit, no extra manipulation is necessary compared with the conventional single-column method. By treating 4-5 liters of plasma, non-high density lipoprotein (HDL)-cholesterol levels decreased by 63-71%, and HDL-cholesterol levels remained unchanged. Thus, this new method of LDL apheresis can safely reduce LDL-cholesterol to any desired level and will be applicable for the treatment of child and adult familial hypercholesterolemic patients with severe coronary heart disease.
Key words: LDL continuous apheresis; Familial hypercholesterolemia
Introduction As homozygous patients with familial hypercholesterolemia (FH) are highly resistant to Correspondence to: Dr. H. Mabuchi, 2nd Department of Internal Medicine, Kanazawa University School of Medicine, Takara-machi 13-l. Kanazawa, Ishikawa 920, Japan.
0021-9150/87/$03.50
cholesterol-lowering drugs [1,2], other forms of therapy have been sought for treating FH homozygotes. These include liver transplantation [3], partial ileal bypass [4], biliary diversion [5], portacaval shunt [6], hyperalimentation [7] and plasmapheresis [S]. Thompson et al. [g] treated 2 FH homozygotes by repeated plasma exchange, using a continuous-flow blood cell separator.
0 1987 Elsevier Scientific Publishers Ireland, Ltd.
20 Thereafter, several papers reported the effectiveness of extracorporeal perfusion in the treatment of homozygotes [8-121. Recently, procedures for selective removal of LDL, such as heparin-agarose affinity column chromatography [13-151, immunoadsorption by the anti-low density lipoprotein (LDL) sepharose column [16], the dextransulfate cellulose column method [17,18] and double-filtration plasmapheresis [l&19] have been applied to the treatment of FH. LDL apheresis using a dextran sulfate cellulose column is highly effective and specific in removing the LDL fraction [17]. However, the adsorption capacity of the dextran sulfate cellulose beads is limited. Because a single column of 400 ml becomes saturated and the LDL fraction may pass through the column, 2 or more columns will be necessary for the treatment of severe hypercholesterolemic patients [16,20,21]. A larger extracorporeal circuit might cause hypovolemic symptoms in child FH homozygotes and in adult FH patients with severely deteriorated cardiac function. Stoffel et al. have developed a system of 2 columns used in rotation; while LDL was being desorbed from one column (out of the circuit) the other (in circuit) served as immunoadsorbent [16,20]. We describe here a new LDL apheresis system (LDL continuous apheresis) using dextran sulfate cellulose beads in 2 smaller columns, which can be regenerated automatically and used repeatedly during the procedure. Patients and methods Two patients with FH were studied after informed consent and approval by the Committee on New Drugs and Equipment at Kanazawa University Hospital. Patient 1 (SO.) is heterozygous and patient 2 (Y.Y.) is homozygous. Their initial
clinical data are shown in Table 1. An arteriovenous shunt was constructed between the radial artery and cephalic vein of the patients. Y.Y. had been treated with several kinds of plasmapheresis for more than 6 years [19,21]. S.O. had been treated by conventional LDL apheresis using a dextran sulfate cellulose column for more than 1 year [21]. The LDL continuous apheresis system is shown in Fig. 1. The circuit was mounted on an MA-01 system (Kanegafuchi Chemical Industrial Company Ltd., Osaka). Two columns each containing 150 ml of cellulose beads covalently bound to dextran sulfate [17] (Liposorber, Kanegafuchi Chemical Industrial Company Ltd.) were used as the specific adsorbent of apolipoprotein B-containing lipoproteins. The plasma separator consisted of polysulfone hollow fibers (Sulflux, Kanegafuchi Chemical Industrial Company Ltd.) with an average pore diameter of 0.2 pm and an effective surface area of 0.5 m2 in a 140 ml chamber. The blood was mixed continuously with heparin (20 units per min), and plasma separated from the blood cells by passing blood through the hollow fibers. Then the plasma was passed through the first column of Liposorber. After passing through this column, the plasma was recombined with the cell-rich stream retained by the hollow fibers, and then infused into an antecubital vein. Polyvinylchloride tubes were used for the circuit. The dead volume of the entire system, consisting of the plasma separator, tubing, and column volumes, was about 400 ml, and was first filled with physiological saline containing heparin (5 units per ml). The blood flow rate was adjusted to SO-100 ml/min, and the plasma flow rate during treatment was adjusted to approximately 20-30 ml/mm. After 600 ml of plasma was adsorbed into the
TABLE 1 INITIAL CLINICAL AND LABORATORY DATA IN THE 2 PATIENTS WITH FAMILIAL HYPERCHOLESTEROLEMIA Patient
Age (yrs)
Diagnosis
Sex
1 SO. 2 Y.Y.
51 M 37 M
Heterozygous Homozygous
Height
Weight
b-4
W
Cholesterol (mg/dI)
Triglyoxide (mg/dI)
Phospholipid (mg/dI)
Achilles tendon thickness ’ (mm)
168 169
12 57
327 613
205 102
215 382
23.5 42.0
a Normal value in Japanese subjects, 6.3 f 0.2 mm (mean f SEM).
21 mWdl 300
z 200 cti
,
I
0 t
‘r
‘.
Inlet
$ E
100
0
0
0.5 Treated
1
3 Liter
2 Plasma
Volume
Fig. 2. Cholesterol concentrations before and after the single dextran-sulfate column in 2 treatments (solid lines and broken tines) of patient S.O. Cholesterol including HDL cholesterol at the outlet does not increase until the treatment of 1.5 liter of plasma.
first column, the second column was used for the treatment of the next 600 ml of plasma. While the second column was used as an adsorbent, the first column was rinsed with regeneration fluids (0.7 M NaCI) and refilled with physiological saline contaming heparin. While the first column was used as an adsorbent, the second column was regenerated. All these regeneration procedures were operated by an automatically-controlled apparatus (MA-01). These procedures were repeated 7-9 times in each treatment of 4-5 liters of plasma. The columns were discarded after each treatment. All treatments were carried out in the outpatient clinic. Each treatment lasted on average 3-4 h after which the patients were released. Vital signs were monitored at regular intervals
Fig. 1. LDL-apheresis system using two dextran-sulfate columns in an automated column regenerating unit. MS, membrane filter for plasma separator. Pl, pump for blood withdrawal. P2, pump for plasma separation. P3, pump for regenerating columns. Sl, Physiological saline containing heparin. S2; regenerating fluid. Cl and C2, dextran-sulfate
columns. VI-VIO, valves. (A) Cl is used as an adsorbent for plasma, while C2 is filled with Sl. Vl, V3, V6, V7 and VlO are open, while V2, V4, VS, V8 and V9 are closed. (B) Physiological saline (Sl) replaces the Cl saturated with LDL without losing plasma, while the plasma replaces the C2 filled with Sl. Vl, V4, V5, V7 and VlO are open, while V2, V3, V6, V8 and V9 are closed. (C) C2 is used as an adsorbent of plasma, while Cl is regenerated by S2. V2, V4, V5, V8 and V9 are open. while Vl, V3, V6, V7 and VlO are closed. After regeneration Cl will be filled with Sl. The procedure will be repeated from (A) to (C) using Cl and C2 in rotation.
22 ReSldtS
throughout the extracorporeal treatment. The treatment was repeated once every week for patient SO., and twice every week for patient Y.Y. Plasma total cholesterol [22] and triglyceride [23] concentrations were determined by enzymatic analysis. All apolipoproteins were determined by the single radial immunodiffusion method (Daiichi Chemical Co. Ltd., Tokyo) with an agarose gel plate containing 3% goat antiserum against purified human apolipoproteins [24,25]. The concentration of HDL-cholesterol was measured in plasma after precipitation of VLDL and LDL with phosphotungstic acid [26]. The concentration of non-HDL-cholesterol was calculated as the difference between the total cholesterol and the HDL cholesterol levels.
When one column of 400 ml of Liposorber was used for the treatment of patient S.O., plasma cholesterol with the exception of HDL-cholesterol would not pass through the column until the treatment of 1 liter of plasma was administered. Thereafter, the column was saturated and the plasma cholesterol in addition to HDL-cholesterol passed through the column (Fig. 2). When the LDL continuous apheresis system of 150 ml of adsorbent was applied to patients with plasma cholesterol levels of 220-250 mg/dl, the small amount of the plasma cholesterol including HDL-cholesterol passing through the column did not increase even after 3-4 repeated uses (Fig. 3).
m%il 200
Plasm.
Cholesterol
et Inlet
Plasm. Cholerrer,,l at Outlet
-X-I HDL Cholesterol
0
0.5
i
i
heated
Plasma Volume
.i
i
5 Liter
i
0:s
;
Treated
i
;
4 Liter
Plasma Volume
Fig. 3. Cholesterol concentrations before and after the 2 columns in the automated column regenerating unit. (A) patient S.O.; (B) patient Y.Y. Non-HDL-cholesterol concentrations at the outlet does not increase after 7-9 repeated uses of the columns.
23
Mean (* SEM) reductions of total cholesterol in patients 1 and 2 were 54% (from 215 f 6 to 98 f 2 mg/dl) and 54% (from 239 f 7 to 110 f 2 mg/dl), respectively. Reductions of non-HDL-cholesterol (total cholesterol minus HDL-cholesterol) in patients 1 and 2 were 63% (from 185 + 5 to 69 f 2 mg/dl) and 71% (from 182 + 6 to 53 f 2 mg/dl), respectively. Fig. 4 shows decays of plasma non-HDLcholesterol during the procedure. The value of the trapping coefficiency f was calculated as: f = 1 - C,“,/C,” where C, and C,,,, are the concentrations of plasma non-HDL-cholesterol at the inlet and outlet of the column. If the removal of the plasma component simply depends on the f value, the decay of the non-HDL cholesterol (C) should be expressed as an exponential function of the plasma that passed through the column: C/C,, = D x exp( - f x V/V,)
0.1-L
,
0
where C, is the initial concentration of the nonHDL-cholesterol, and V,, is the total intra- and extracorporeal circulation volume, and expressed as P (total plasma volume) + 230 ml in this system; V is the volume of treated plasma; D is the diluting factor of plasma by the circuit and is expressed as P/(P + 230). Assuming that the total circulation volume is 8% of body weight, we can calculate the value of P using the patient’s hema-
0.5
I
1 Treated
I
0
I
2
3
4
Plasma
1 Lifer
Volume
Fig. 4. Decay of plasma non-HDL-cholesterol during LDL apheresis in the automated column regenerating unit. (A) patient SO.; (B) patient Y.Y. According to this plot, the f values can be calculated from the slopes, and the y intercepts give D values for this system.
TABLE 2 PLASMA LIPIDS AND APOLIPOPROTEIN Patient
1 O.S. 2Y.Y.
No. of determinations
Cholesterol
9 10
239k7 215*6
Before
After 110+2 98*2
Before
After
57fl 30*1
57*1 29+1
APHERESIS
Apo A-I
PhosphoIipid
Triglyceride
HDL-cholesterol
Before
After
Before
After
Before
After
129+11 158k17
80*7 72+9
257*7 202k7
1so*4 126*4
169&2 120+4
‘165+3 113*3
Apo C-II
APO-B
Apo A-II
LEVELS BEFORE AND AFTER LOW DENSITY LIPOPROTEIN
Apo E
Apo C-III
Before
After
Before
After
Before
After
Before
After
Before
After
34+1 29+1
34*1 29+1
147*4 139*4
37k2 51k2
4.3 * 0.2 4.2 f 0.2
3.1 f 0.1 2.3kO.l
8.7kO.3 7.OkO.6
6.4kO.2 3.7*0.3
4.3 + 0.2 5.2kO.4
1.9*0.1 2.2*0.2
All values are mg per dl and are given as mean f SEM
24 tocrit value. As the data in Fig. 4 were plotted on semilogarithmic paper, the slopes of these straight lines gave f values, which were calculated as 0.860 and 0.852 in patients SO. and Y.Y., respectively. Thus, f should be 0.85 in this column system. Thus, C/C, = P/(P + 230) x exp[ -0.85 X V/(P + 230)] If C, and P are given, C will be predicted from the volume (V) of the treated plasma. Plasma cholesterol, triglyceride and phospholipid levels decreased significantly by this treatment, while HDL-cholesterol levels did not change (Table 2). Apolipoproteins B, C-II, C-III and E decreased significantly, while apolipoproteins A-I and A-II barely decreased (Table 2). Plasma protein and peripheral blood cell counts did not change significantly. This treatment was well tolerated by the patients. No chills occurred during or after these procedures. There were no changes in vital signs. Discussion LDL apheresis has been proved to be highly effective in homozygous and heterozygous patients with FH [13-171. If large volumes of plasma in severe hypercholesterolemic patients are treated, the dextran sulfate cellulose column will be saturated and a second column will be necessary for further removal of LDL [17,20,21]. If the volume of the column is increased, hypovolemic reactions may occur, especially in patients with small body size or severely damaged hearts. For safe and effective treatment through the LDL apheresis system, a smaller and more effective column is necessary. If the column can be regenerated immediately during the procedure, 2 columns can be used alternately without losing the efficiency of the column [16]. Our procedure consists of a smaller extracorporeal unit which can be used repeatedly without losing the potency of the column. As the apparatus was automatically controlled by a computerized unit, no extra manipulation is necessary compared with the conventional single column
method. As the efficiency is preserved even after 5 repeats of the procedure, LDL-cholesterol will become theoretically less than 90% of the initial levels if the procedures are repeated more than 10 times and the plasma volume treated is above 8 liters. Normalization of LDL-cholesterol levels in homozygous FH patients can be achieved only by increasing the treatment frequency or increasing the volume of plasma treated at each procedure [27]. If our method of LDL apheresis is repeated twice every week in homozygous patients, the plasma cholesterol levels can be safely reduced to less than 250 mg/dl before and less than 100 mg/dl after each procedure without concomitant drug therapy. As LDL apheresis removes LDLcholesterol and leaves HDL-cholesterol in the plasma, long-term LDL apheresis is expected to stop and reverse atherosclerosis [27,29]. No adverse reactions related to the extracorporeal volume and cell separator were seen in our patients with severe coronary heart disease. Extracorporeal immunoadsorption has been proved to be effective in the treatment of patients with FH and an automatic regeneration system was applied to the procedure [16,20]. As this column is not used as a disposable apparatus, patients experienced episodes of chills [20], although results of cultures for microorganisms and tests for pyrogens were negative. Thus, this new method of LDL apheresis can safely reduce LDL-cholesterol to any desired level and will be applicable for the treatment of child and adult FH patients with severe coronary heart disease. Acknowledgements We are indebted to the Kanegafuchi Chemical Industrial Company Ltd. for providing the Liposorber, Sulflux and MA-01 system, and to Miss Ikeda for her outstanding technical assistance. This work was supported by the Scientific Research Grants of the Education Ministry of Japan (No. 59480198) and the grants for Primary Hyperlipidemia Research Projects of the Welfare Ministry of Japan.
25 References 1 Goldstein, J.L. and Brown, M.S., Familial hypercholesterolemia. In: Stanbury J.B., Wyngaarden, J.B., Fredrickson. D.S., Goldstein, J.L. and Brown, M.S. (eds.), The Metabolic Basis of Inherited Disease, 5th edn., McGraw-Hill Book Co., New York, 1983, p. 672. 2 Kane, J.P. and Have], R.J., Treatment of hypercholesterolemia, Ann”. Rev. Med., 37 (1986) 427. 3 Starzl. T.E.. Bilheimer, D.W., Bahson, H.T., Shaw, B.W.. Jr., Hardesty, R.L., Griffith, B.P., Iwatsuki, S., Zitelli, B.J., Gartner, J.C. Jr., Malatack, J.J. and Urbach, A.H., Heartliver transplantation in a patient with familial hypercholesterolemia, Lancet, 1 (1984) 1382. 4 Buchwald, H. and Varco, R.L., Partial ileal bypass for hypercholesterolemia and atherosclerosis. Surg. Gynecol. Obstet., 124 (1967) 1231. 5 Deckelbaum, R.J., Lees, R.S., Small, D.M., Hedberg, SE. and Grundy, S.M., Failure of complete bile diversion and oral bile acid therapy in the treatment of homozygous familial hypercholesterolemia, New Engl. J. Med., 296 (1977) 465. 6 Starzl. T.E., Chase, H.P., Putnam, C.W. and Porter, K.A.. Potacaval shunt in hyperlipoproteinemia, Lancet, 2 (1973) 940. 7 Torvik, H., Feldman, H.A., Fischer, J.E. and Lees, R.S.. Effects of intravenous hyperalimentation on plasma lipoproteins in severe familial hypercholesterolemia, Lancet, 1 (1975) 601. 8 Thompson, G.R., Lowenthal R. and Myant. N.B.. Plasma-exchange in the management of homozygous familial hypercholesterolemia, Lancet, 1 (1975) 1208. 9 Berger, G.M.B., Miller, J.R., Bonnici, F., Joffe, H.S. and Dubovsky, D.W., Continuous flow plasma exchange in the treatment of homozygous familial hypercholesterolemia. Am. J. Med., 65 (1978) 243. 10 King. M.E.W., Breslow, J.L. and Lees, R.S., Plasma-exchange therapy of homozygous familial hypercholesterolemia, New Engi. J. Med., 302 (1980) 1457. 11 Apstein, C.S.. Zilversmit, D.B., Lees, R.S. and George, P.K., Effect of intensive plasmapheresis on the plasma cholesterol concentration of patients with familial hypercholesterolemia, Atherosclerosis, 31 (1978) 105. 12 Keller, C., Hailer. S., Demant, T., Wolfram, G. and Zollner, N., Effect of plasma exchange with and without concomitant drug treatment on lipids and lipoproteins in patients with familial hypercholesterolemia confirmed by tissue culture, Atherosclerosis. 57 (1985) 225. 13 Lupien, P.J., Moorjani, S. and Awad, J.. A new approach in the management of familial hypercholesterolemia: removal of plasma-cholesterol based on the principle of affinity chromatography, Lancet, 1 (1976) 1261. 14 Lupien, P.J., Moorjani, S., Lon. M., Brun, D. and Gagne, C.L., Removal of cholesterol from blood by affinity binding to heparin agarose: evaluation on treatment in homozygous familial hypercholesterolemia, Pediatr. Res., 14 (1980) 113. 15 Graisely, B., Cloarec, M., Salmon, S., Polonovski, J.. Polonovski, Cl. Delacotte, J.M., Gardent, J., Cavalier, J.,
16
17
18
19
20
21
22
23
24
25
26
27 28
29
Vergoz, D. and Salmon, Ch., Extracorporeal plasma therapy for homozygous familial hypercholesterolemia, Lancet. 2 (1980) 1147. Stoffel, W., Boberg, H. and Greve, V., Application of specific extracorporeal removal of low density lipoprotein in familial hypercholesterolemia, Lancet, 2 (1981) 1005. Yokoyama, S., Hayashi, R., Kikkawa, T., Tani, N., Takada. S., Hatanaka, K. and Yamamoto, A., Specific sobent of apolipoprotein B-containing lipoproteins for plasmapheresis. Characterization and experimental use in hypercholesterolemic rabbits, Arteriosclerosis, 4 (1984) 276. Yokoyama, S., Hayashi, R., Satani. M. and Yamamoto, A.. Selective removal of low density lipoprotein by plasmapheresis in familial hypercholesterolemia, Arteriosclerosis 5 (1985) 613. Mabuchi, H., Michishita, I., Sakai, T., Sakai, Y., Watanabe, A., Wakasugi, T. and Takeda, R., Treatment of homozygous patients with familial hypercholesterolemia by double-filtration plasmapheresis, Atherosclerosis, 61 (1986) 135. Saal, S.D., Parker, T.S., Gordon, B.R., Studebaker, J.. Hudgins, L, Ahrens, E.H., Jr. and Rubin, A.L., Removal of low-density lipoproteins in patients by extracorporeal immunoadsorption, Am. J. Med.. 80 (1986) 583. Mabuchi, H., Michishita, I., Fujita, H., Takeda, M., Kajinami. K., Itoh, H., Wakasugi, T.. Kobaladze. AS.. Watanabe, A., Oota, M.. Ueda, K.. Takegoshi, T.. Miyamoto, S. and Takeda, R.. Treatment of familial hypercholesterolemia by selective removal of apolipoprotein B-containing lipoproteins through dextran sulfate column, submitted for publication. Allain C.C., Poon, L.S.. Chan, C.S.G., Richmond, W. and Fu, P.C., Enzymatic determination of total serum cholesterol Clin. Chem., 20 (1974) 470. Sugiura, M., Oikawa, T., Hirano, K. et al.. A simple colorimetric method for determination of serum triglycerides with lipoprotein lipase and glycerol dehydrogenase, Chn. Chim. Acta, 81 (1977) 152. Sakai, Y., Itakura, K., Kanada, T. et al.. Quantitation of apolipoprotein A-I in pooled human serum by single radial immunodiffusion and sodium dodecyl sulfate-polyacrylamide gel electrophoresis, Anal. B&hem.. 137 (1984) 1. Goto, Y.. Akanuma, Y., Harano, Y. et al., Determination of serum apohpoproteins A-I, A-II, B. C-II, C-III and E in normolipidemic healthy Japanese subjects; determined by SRID method, J. Clin. B&hem. Nutr.. 1 (1986) 73. Burstein. M., Scholonick. H.R. and Morfin. R., Rapid method for isolation of lipoproteins from serum by precipitation with polyanions, J. Lipid Res., 11 (1970) 583. Thompson, G.R., Plasma exchange for hypercholesterolemia, Lancet, 1 (1981) 1246. Thompson, G.R., Myant, N.B., Kilpatrick, D.. Oakley. C.M., Raphael, M.J. and Steiner, R.E., Assessment of long-term plasma exchange for familial hypercholesterolemia, Br. Heart J., 43 (1980) 680. Thompson, G.R., Miller, J.P. and Breslow, J.L., Improved survival of patients with homozygous familial hypercholesterolemia treated with plasma exchange, Brit. Med. J., 291 (1985) 1671.
19
68 (1987) 19-25
Elsevier Scientific Publishers Ireland, Ltd. ATH 04018
A new low density lipoprotein apheresis system using two dextran sulfate cellulose columns in an automated column regenerating unit (LDL continuous apheresis) Hiroshi Mabuchi, Ichiro Michishita, Mitsuaki Takeda, Hajime Fujita, Junji Koizumi, Ryoyu Takeda, Satoshi Takada and Michikazu Oonishi Second Department of Internal Medicine, Kanazawa University School of Medicine, Kanazawa and Kanegafuchi Chemical Industry Central Research L.&oratory, Kobe (Japan)
(Japan).
(Received 23 February, 1987) (Revised, received 2 April, 1987) (Accepted 10 April, 1987)
We describe a new low density lipoprotein (LDL) apheresis system using dextran sulfate cellulose column in an automated column regenerating unit (LDL continuous apheresis). Two columns containing 150 ml of dextran sulfate cellulose were used, and the whole extracorporeal circulation was about 400 ml in volume. After 600 ml of plasma was adsorbed into the first column, the second column was used as an adsorbent and meanwhile the first column was regenerated. Thus, the 2 columns were used alternately without losing the potency of the columns. As the apparatus was automatically controlled by a computerized unit, no extra manipulation is necessary compared with the conventional single-column method. By treating 4-5 liters of plasma, non-high density lipoprotein (HDL)-cholesterol levels decreased by 63-71%, and HDL-cholesterol levels remained unchanged. Thus, this new method of LDL apheresis can safely reduce LDL-cholesterol to any desired level and will be applicable for the treatment of child and adult familial hypercholesterolemic patients with severe coronary heart disease.
Key words: LDL continuous apheresis; Familial hypercholesterolemia
Introduction As homozygous patients with familial hypercholesterolemia (FH) are highly resistant to Correspondence to: Dr. H. Mabuchi, 2nd Department of Internal Medicine, Kanazawa University School of Medicine, Takara-machi 13-l. Kanazawa, Ishikawa 920, Japan.
0021-9150/87/$03.50
cholesterol-lowering drugs [1,2], other forms of therapy have been sought for treating FH homozygotes. These include liver transplantation [3], partial ileal bypass [4], biliary diversion [5], portacaval shunt [6], hyperalimentation [7] and plasmapheresis [S]. Thompson et al. [g] treated 2 FH homozygotes by repeated plasma exchange, using a continuous-flow blood cell separator.
0 1987 Elsevier Scientific Publishers Ireland, Ltd.
20 Thereafter, several papers reported the effectiveness of extracorporeal perfusion in the treatment of homozygotes [8-121. Recently, procedures for selective removal of LDL, such as heparin-agarose affinity column chromatography [13-151, immunoadsorption by the anti-low density lipoprotein (LDL) sepharose column [16], the dextransulfate cellulose column method [17,18] and double-filtration plasmapheresis [l&19] have been applied to the treatment of FH. LDL apheresis using a dextran sulfate cellulose column is highly effective and specific in removing the LDL fraction [17]. However, the adsorption capacity of the dextran sulfate cellulose beads is limited. Because a single column of 400 ml becomes saturated and the LDL fraction may pass through the column, 2 or more columns will be necessary for the treatment of severe hypercholesterolemic patients [16,20,21]. A larger extracorporeal circuit might cause hypovolemic symptoms in child FH homozygotes and in adult FH patients with severely deteriorated cardiac function. Stoffel et al. have developed a system of 2 columns used in rotation; while LDL was being desorbed from one column (out of the circuit) the other (in circuit) served as immunoadsorbent [16,20]. We describe here a new LDL apheresis system (LDL continuous apheresis) using dextran sulfate cellulose beads in 2 smaller columns, which can be regenerated automatically and used repeatedly during the procedure. Patients and methods Two patients with FH were studied after informed consent and approval by the Committee on New Drugs and Equipment at Kanazawa University Hospital. Patient 1 (SO.) is heterozygous and patient 2 (Y.Y.) is homozygous. Their initial
clinical data are shown in Table 1. An arteriovenous shunt was constructed between the radial artery and cephalic vein of the patients. Y.Y. had been treated with several kinds of plasmapheresis for more than 6 years [19,21]. S.O. had been treated by conventional LDL apheresis using a dextran sulfate cellulose column for more than 1 year [21]. The LDL continuous apheresis system is shown in Fig. 1. The circuit was mounted on an MA-01 system (Kanegafuchi Chemical Industrial Company Ltd., Osaka). Two columns each containing 150 ml of cellulose beads covalently bound to dextran sulfate [17] (Liposorber, Kanegafuchi Chemical Industrial Company Ltd.) were used as the specific adsorbent of apolipoprotein B-containing lipoproteins. The plasma separator consisted of polysulfone hollow fibers (Sulflux, Kanegafuchi Chemical Industrial Company Ltd.) with an average pore diameter of 0.2 pm and an effective surface area of 0.5 m2 in a 140 ml chamber. The blood was mixed continuously with heparin (20 units per min), and plasma separated from the blood cells by passing blood through the hollow fibers. Then the plasma was passed through the first column of Liposorber. After passing through this column, the plasma was recombined with the cell-rich stream retained by the hollow fibers, and then infused into an antecubital vein. Polyvinylchloride tubes were used for the circuit. The dead volume of the entire system, consisting of the plasma separator, tubing, and column volumes, was about 400 ml, and was first filled with physiological saline containing heparin (5 units per ml). The blood flow rate was adjusted to SO-100 ml/min, and the plasma flow rate during treatment was adjusted to approximately 20-30 ml/mm. After 600 ml of plasma was adsorbed into the
TABLE 1 INITIAL CLINICAL AND LABORATORY DATA IN THE 2 PATIENTS WITH FAMILIAL HYPERCHOLESTEROLEMIA Patient
Age (yrs)
Diagnosis
Sex
1 SO. 2 Y.Y.
51 M 37 M
Heterozygous Homozygous
Height
Weight
b-4
W
Cholesterol (mg/dI)
Triglyoxide (mg/dI)
Phospholipid (mg/dI)
Achilles tendon thickness ’ (mm)
168 169
12 57
327 613
205 102
215 382
23.5 42.0
a Normal value in Japanese subjects, 6.3 f 0.2 mm (mean f SEM).
21 mWdl 300
z 200 cti
,
I
0 t
‘r
‘.
Inlet
$ E
100
0
0
0.5 Treated
1
3 Liter
2 Plasma
Volume
Fig. 2. Cholesterol concentrations before and after the single dextran-sulfate column in 2 treatments (solid lines and broken tines) of patient S.O. Cholesterol including HDL cholesterol at the outlet does not increase until the treatment of 1.5 liter of plasma.
first column, the second column was used for the treatment of the next 600 ml of plasma. While the second column was used as an adsorbent, the first column was rinsed with regeneration fluids (0.7 M NaCI) and refilled with physiological saline contaming heparin. While the first column was used as an adsorbent, the second column was regenerated. All these regeneration procedures were operated by an automatically-controlled apparatus (MA-01). These procedures were repeated 7-9 times in each treatment of 4-5 liters of plasma. The columns were discarded after each treatment. All treatments were carried out in the outpatient clinic. Each treatment lasted on average 3-4 h after which the patients were released. Vital signs were monitored at regular intervals
Fig. 1. LDL-apheresis system using two dextran-sulfate columns in an automated column regenerating unit. MS, membrane filter for plasma separator. Pl, pump for blood withdrawal. P2, pump for plasma separation. P3, pump for regenerating columns. Sl, Physiological saline containing heparin. S2; regenerating fluid. Cl and C2, dextran-sulfate
columns. VI-VIO, valves. (A) Cl is used as an adsorbent for plasma, while C2 is filled with Sl. Vl, V3, V6, V7 and VlO are open, while V2, V4, VS, V8 and V9 are closed. (B) Physiological saline (Sl) replaces the Cl saturated with LDL without losing plasma, while the plasma replaces the C2 filled with Sl. Vl, V4, V5, V7 and VlO are open, while V2, V3, V6, V8 and V9 are closed. (C) C2 is used as an adsorbent of plasma, while Cl is regenerated by S2. V2, V4, V5, V8 and V9 are open. while Vl, V3, V6, V7 and VlO are closed. After regeneration Cl will be filled with Sl. The procedure will be repeated from (A) to (C) using Cl and C2 in rotation.
22 ReSldtS
throughout the extracorporeal treatment. The treatment was repeated once every week for patient SO., and twice every week for patient Y.Y. Plasma total cholesterol [22] and triglyceride [23] concentrations were determined by enzymatic analysis. All apolipoproteins were determined by the single radial immunodiffusion method (Daiichi Chemical Co. Ltd., Tokyo) with an agarose gel plate containing 3% goat antiserum against purified human apolipoproteins [24,25]. The concentration of HDL-cholesterol was measured in plasma after precipitation of VLDL and LDL with phosphotungstic acid [26]. The concentration of non-HDL-cholesterol was calculated as the difference between the total cholesterol and the HDL cholesterol levels.
When one column of 400 ml of Liposorber was used for the treatment of patient S.O., plasma cholesterol with the exception of HDL-cholesterol would not pass through the column until the treatment of 1 liter of plasma was administered. Thereafter, the column was saturated and the plasma cholesterol in addition to HDL-cholesterol passed through the column (Fig. 2). When the LDL continuous apheresis system of 150 ml of adsorbent was applied to patients with plasma cholesterol levels of 220-250 mg/dl, the small amount of the plasma cholesterol including HDL-cholesterol passing through the column did not increase even after 3-4 repeated uses (Fig. 3).
m%il 200
Plasm.
Cholesterol
et Inlet
Plasm. Cholerrer,,l at Outlet
-X-I HDL Cholesterol
0
0.5
i
i
heated
Plasma Volume
.i
i
5 Liter
i
0:s
;
Treated
i
;
4 Liter
Plasma Volume
Fig. 3. Cholesterol concentrations before and after the 2 columns in the automated column regenerating unit. (A) patient S.O.; (B) patient Y.Y. Non-HDL-cholesterol concentrations at the outlet does not increase after 7-9 repeated uses of the columns.
23
Mean (* SEM) reductions of total cholesterol in patients 1 and 2 were 54% (from 215 f 6 to 98 f 2 mg/dl) and 54% (from 239 f 7 to 110 f 2 mg/dl), respectively. Reductions of non-HDL-cholesterol (total cholesterol minus HDL-cholesterol) in patients 1 and 2 were 63% (from 185 + 5 to 69 f 2 mg/dl) and 71% (from 182 + 6 to 53 f 2 mg/dl), respectively. Fig. 4 shows decays of plasma non-HDLcholesterol during the procedure. The value of the trapping coefficiency f was calculated as: f = 1 - C,“,/C,” where C, and C,,,, are the concentrations of plasma non-HDL-cholesterol at the inlet and outlet of the column. If the removal of the plasma component simply depends on the f value, the decay of the non-HDL cholesterol (C) should be expressed as an exponential function of the plasma that passed through the column: C/C,, = D x exp( - f x V/V,)
0.1-L
,
0
where C, is the initial concentration of the nonHDL-cholesterol, and V,, is the total intra- and extracorporeal circulation volume, and expressed as P (total plasma volume) + 230 ml in this system; V is the volume of treated plasma; D is the diluting factor of plasma by the circuit and is expressed as P/(P + 230). Assuming that the total circulation volume is 8% of body weight, we can calculate the value of P using the patient’s hema-
0.5
I
1 Treated
I
0
I
2
3
4
Plasma
1 Lifer
Volume
Fig. 4. Decay of plasma non-HDL-cholesterol during LDL apheresis in the automated column regenerating unit. (A) patient SO.; (B) patient Y.Y. According to this plot, the f values can be calculated from the slopes, and the y intercepts give D values for this system.
TABLE 2 PLASMA LIPIDS AND APOLIPOPROTEIN Patient
1 O.S. 2Y.Y.
No. of determinations
Cholesterol
9 10
239k7 215*6
Before
After 110+2 98*2
Before
After
57fl 30*1
57*1 29+1
APHERESIS
Apo A-I
PhosphoIipid
Triglyceride
HDL-cholesterol
Before
After
Before
After
Before
After
129+11 158k17
80*7 72+9
257*7 202k7
1so*4 126*4
169&2 120+4
‘165+3 113*3
Apo C-II
APO-B
Apo A-II
LEVELS BEFORE AND AFTER LOW DENSITY LIPOPROTEIN
Apo E
Apo C-III
Before
After
Before
After
Before
After
Before
After
Before
After
34+1 29+1
34*1 29+1
147*4 139*4
37k2 51k2
4.3 * 0.2 4.2 f 0.2
3.1 f 0.1 2.3kO.l
8.7kO.3 7.OkO.6
6.4kO.2 3.7*0.3
4.3 + 0.2 5.2kO.4
1.9*0.1 2.2*0.2
All values are mg per dl and are given as mean f SEM
24 tocrit value. As the data in Fig. 4 were plotted on semilogarithmic paper, the slopes of these straight lines gave f values, which were calculated as 0.860 and 0.852 in patients SO. and Y.Y., respectively. Thus, f should be 0.85 in this column system. Thus, C/C, = P/(P + 230) x exp[ -0.85 X V/(P + 230)] If C, and P are given, C will be predicted from the volume (V) of the treated plasma. Plasma cholesterol, triglyceride and phospholipid levels decreased significantly by this treatment, while HDL-cholesterol levels did not change (Table 2). Apolipoproteins B, C-II, C-III and E decreased significantly, while apolipoproteins A-I and A-II barely decreased (Table 2). Plasma protein and peripheral blood cell counts did not change significantly. This treatment was well tolerated by the patients. No chills occurred during or after these procedures. There were no changes in vital signs. Discussion LDL apheresis has been proved to be highly effective in homozygous and heterozygous patients with FH [13-171. If large volumes of plasma in severe hypercholesterolemic patients are treated, the dextran sulfate cellulose column will be saturated and a second column will be necessary for further removal of LDL [17,20,21]. If the volume of the column is increased, hypovolemic reactions may occur, especially in patients with small body size or severely damaged hearts. For safe and effective treatment through the LDL apheresis system, a smaller and more effective column is necessary. If the column can be regenerated immediately during the procedure, 2 columns can be used alternately without losing the efficiency of the column [16]. Our procedure consists of a smaller extracorporeal unit which can be used repeatedly without losing the potency of the column. As the apparatus was automatically controlled by a computerized unit, no extra manipulation is necessary compared with the conventional single column
method. As the efficiency is preserved even after 5 repeats of the procedure, LDL-cholesterol will become theoretically less than 90% of the initial levels if the procedures are repeated more than 10 times and the plasma volume treated is above 8 liters. Normalization of LDL-cholesterol levels in homozygous FH patients can be achieved only by increasing the treatment frequency or increasing the volume of plasma treated at each procedure [27]. If our method of LDL apheresis is repeated twice every week in homozygous patients, the plasma cholesterol levels can be safely reduced to less than 250 mg/dl before and less than 100 mg/dl after each procedure without concomitant drug therapy. As LDL apheresis removes LDLcholesterol and leaves HDL-cholesterol in the plasma, long-term LDL apheresis is expected to stop and reverse atherosclerosis [27,29]. No adverse reactions related to the extracorporeal volume and cell separator were seen in our patients with severe coronary heart disease. Extracorporeal immunoadsorption has been proved to be effective in the treatment of patients with FH and an automatic regeneration system was applied to the procedure [16,20]. As this column is not used as a disposable apparatus, patients experienced episodes of chills [20], although results of cultures for microorganisms and tests for pyrogens were negative. Thus, this new method of LDL apheresis can safely reduce LDL-cholesterol to any desired level and will be applicable for the treatment of child and adult FH patients with severe coronary heart disease. Acknowledgements We are indebted to the Kanegafuchi Chemical Industrial Company Ltd. for providing the Liposorber, Sulflux and MA-01 system, and to Miss Ikeda for her outstanding technical assistance. This work was supported by the Scientific Research Grants of the Education Ministry of Japan (No. 59480198) and the grants for Primary Hyperlipidemia Research Projects of the Welfare Ministry of Japan.
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