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Treatment of homozygous patients with familial hypercholesterolemia by double-filtration plasmapheresis
Atherosclerosis, 61 (1986) 135-140 Elsevier Scientific Publishers Ireland,
135 Ltd.
ATH 03803
Treatment of Homozygous Patients with Familial Hypercholesterolemia by Double-filtration Plasmapheresis Hiroshi Mabuchi, 2nd Department
Ichiro Michishita, Takeshi Sakai, Yasuyuki Sakai, Akira Watanabe, Takanobu Wakasugi and Ryoyu Takeda of Internal Medicine, Kanarawa University School of Medicine, Kanatawa,
Ishikawa
920 (Japan)
(Received 12 March, 1986) (Accepted 7 April, 1986)
Summary Two homozygous patients with familial hypercholesterolemia were treated by double-filtration plasmapheresis. The plasma separated by the first filter was subsequently led to the second filter of ethylene vinylalcohol co-polymer hollow fibers, which trap very low density lipoprotein (VLDL), intermediate density lipoprotein (IDL) and low density lipoprotein (LDL) preferentially to other plasma constituents. Serum, VLDL, IDL, LDL cholesterol levels decreased by 55, 68, 59 and 55% respectively. HDL cholesterol levels decreased by 39%. Immunoglobulins and fibrinogen levels decreased significantly. Cutaneous and tendinous xanthomas became smaller.
Key words:
Double-filtration
plasmapheresis
- Homozygous
Introduction Homozygous patients with familial hypercholesterolemia show exceptionally high plasma cholesterol levels, exceeding 600 mg/dl, which reflect extreme increases in low density lipoprotein (LDL) concentration [1,2]. For homozygotes, the risk of death from coronary artery disease 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. Address reprint requests to Hiroshi Mabuchi, M.D., 2nd Department of Internal medicine, Kanazawa University School of Medicine, Takara-machi 13-1, Kanazawa, Ishikawa 920, Japan. OOZl-9150/86/$03.50
0 1986 Elsevier Scientific
Publishers
Ireland,
familial hypercholesterolemia
(CAD) or aortic stenosis before adult life is very high [1,3]. Several drugs are known to reduce LDL concentrations in heterozygotes, and reduction of LDL cholesterol levels by cholestyramine has been proved to be effective in decreasing the incidence of CAD [4,5]. However, these drugs are usually not effective in the homozygotes [6]. Thus, other forms of therapy have been attempted. These include liver transplantation [7], partial ileal by-pass [8], biliary diversion [9], portacaval shunt [lo], hyperalimentation [ll] and plasmapheresis [12-171. Thompson et al. [12] treated two homozygotes by repeated plasma exchange, using a continuous-flow blood-cell separator. Thereafter several papers reported the effectiveness of extracorporeal perfusion in the Ltd
136
treatment of homozygotes [12-171. Here, we describe a new approach to the management of homozygous familial hypercholesterolemia by double-filtration plasmapheresis.
sizes. The first filter consisted of polyvinylalcohol hollow fibers with an average pore diameter of 0.2 pm (PLASMACURE, Kuraray Co., Ltd., Japan). The first filter works as a plasma separator, which filters and separates the plasma component from whole blood during extracorporeal circulation. Separated plasma was subsequently led to the second filter of ethylene vinylalcohol co-polymer (EVAL, 4~, Kuraray Co., Ltd., Japan) hollow fibers with an average pore diameter of 0.03 pm. The filtrate from the second filter was mixed with cell-rich blood and returned via an antecubital vein, while approximately 300 ml of the rest in the second filter was discarded. Plasmapheresis was performed by KM-8500 (Kuraray Co., Ltd., Japan). The extracorporeal circulation device was first filled with physiological saline containing heparin (5 units/ml). The blood flow-rate during treatment was adjusted to approximately 100 ml/ min. The total amount of plasma treated by each procedure was 3 liters. Thirty grams of human albumin solution were infused during the procedure. The treatment was carried out in the in-patient clinic in case Y.Y., and in the out-patient clinic in case M.T. Blood pressure, ECG and pulse rate were monitored at regular intervals throughout the extracorporeal treatment. The procedure was repeated every 2 weeks, and later in case Y.Y. once every week.
Materials and Methods Two homozygous patients with familial hypercholesterolemia were studied after informed consent and approval by the Committee of New Drugs and Equipment at Kanazawa University Hospital. Homozygous patients were diagnosed when they had juvenile xanthomatosis and their cholesterol levels were about twice those of the heterozygous parents. Pedigrees of the two homozygotes were shown in our previous paper, in which patient 1 is case Y.Y. of family No. 2, and patient 2 is case M.T. of family No. 1. Their LDL receptor activities studied by cultured skin fibroblasts showed receptor-defective type [18]. Their clinical and laboratory data are shown in Table 1. Case Y.Y. has been treated by 1000 mg/day of probucol 1191. Serum lipoprotein lipid and other laboratory analyses were performed before and after each procedure. Blood samples were allowed to clot at room temperature. Methods of lipoprotein fractionations and lipid determinations were described in our previous paper [20]. Statistical calculations were performed with Student’s paired t-test.
Results
Double-filtration plasmapheresis An arteriovenous shunt was constructed between the radial artery and cephalic vein of the patients. The principle of double-filtration plasmapheresis reported by Agishi et al. [21] was adopted for the present study. This procedure utilizes 2 hollow-fiber filters with different pore
Changes in serum cholesterol levels are shown in Fig. 1. Changes in lipoprotein cholesterol, serum triglyceride and phospholipid levels are shown in Table 2. With treatment of 2-week intervals serum cholesterol in patients Y.Y. and M.T. decreased from 360 rtc 15 (mean+ SEM) to 160 k 6 (a de-
TABLE 1 CLINICAL AND LABORATORY DATA IN 2 HOMOZYGOUS PATIENTS WITH FAMILIAL HYPERCHOLESTEROLEMIA Patient
Age (yr) Sex
Height
weight
(cm)
(kg)
Cholesterol
Triglyceride
Phospholipid
(mg/dl)
(mg/dl)
(mg/dl)
Achilles tendon thickness (mm)
Y.Y.
M.T. ’ Normal
37 M 24 M value in Japanese
169 175 subjects,
57 71
613 566
6.3 f 0.2 mm (mean f SEM).
102 231
382 394
42.0 12.0
a
137
.
0
’
lb1 M.T.
,....,...‘1’.“,....,...,,,,,,,,,,,,,,,,,,,.,,..,-~.,,,,,,,,,.,~.~~,~~~~,~
0
5
10
15
10
15
30
55
40
.5
50
55
60
65
70
75 weeks
Fig, 1. Changes
of serum cholesterol
concentration
before and after double-filtration
plasma
pheresis.
a = patient
Y.Y., b = patient
M.T
crease of 55.6%) and from 423 -t_ 17 to 201 * 13 mg/dl (a decrease of 52.5%), respectively. With treatment at l-week interval, serum cholesterol in patient Y.Y. decreased from 299 A 5 to 134 t_ 2 mg/dl (a decrease of 55.2%). Thus, the average reduction of serum cholesterol by, this treatment was 54.7%. Very low density lipoprotein (VLDL), intermediate density lipoprotein (IDL) and LDL cholesterol levels decreased by 67.9, 58.6 and 54.6%, respectively, while high density lipoprotein (HDL) cholesterol decreased by 39.4%. Serum triglyceride and phospholipid decreased by 68.5 and 48.1%, respectively. Other laboratory data before and after doublefiltration plasmapheresis are as follows. Erythrocyte count, platelet count, hemoglobin and hematocrit showed no significant changes. Leukocyte count increased transiently. Total protein and albumin decreased significantly (P < 0.001) in spite of supplements with human albumin solution. Immunoglobulins, C3, C4 and fibrinogen decreased significantly (P < 0.001). Urea nitrogen (BUN), uric acid, creatinine showed no significant changes. Aspartate aminotransferase (SGOT), alanine amniotransferase (SGPT), lactic dehydrogenase (SLDH), alkaline phosphatase (P-ase), creatine kinase (SCPK) showed slight reductions (P < 0.001). Na, K and Cl showed no significant changes.
Cutaneous and tendinous xanthomas in case Y.Y. showed marked decrease in size. The procedure was well tolerated in both patients. Discussion Several methods of extracorporeal plasma treatment have been applied for the treatment of homozygous patients with familial hypercholesterolemia. More than 45 patients (21 homozygotes and 24 heterozygotes) with familial hypercholesterolemia have records of treatment by plasma exchange. The method of exchanging plasma with 2-4 liters of plasma protein fractions at intervals of l-2 weeks safely and effectively controlled hypercholesterolemia and sometimes induced regression of xanthomas and atherosclerosis in the patients [12-171. However, plasma exchange reduces both atherogenic (VLDL, IDL and LDL) [22,23] and antiatherogenic (HDL) lipoproteins [24], and removes other plasma constituents, such as albumin, immunoglobulins, and others. Recently, procedures for selective removal of LDL, such as heparin-agarose affinity-column chromatography [25-271, immunoadsorption by the anti-LDL sepharose column [28] and dextransulfate cellulose column [29-311, have been applied to the treatment of familial hypercholesterolemia.
Total
M.T.
Y.Y.
Patient
2 1 2
(week)
Interval
322+
75
7
360+15 299+ 5 423+17
Before
146*
4
160+ 6 134* 2 2Olk-13
After
Cholesterol Serum
6 58 11
No. of determination
28kl
19*5 29*1 29*4
Before
VLDL
29+1
36*4 27+1 34*5
6*0.1 9+0.6 10+2 9+0.6
Before
IDL
12*0.6
13+3 llkO.5 17*3
After
205k
6
24Ok14 187k 4 287kll
Before
LDL
93*3
llOk8 84k2 135*9
After
33+1
31k6 32*1 41*2
Before
HDL
20+0.6
15+1 2OkO.6 25kl
After
LEVELS BEFORE AND AFTER DOUBLE-FILTRATION
After
CHOLESTEROL
All values are mg/dI and are given as means f SEM.
SERUM LIPID LEVELS AND LIPOPROTEIN
TABLE 2
149*
6
147k13 147k 7 153k18
Before
47+
2
59+12 55*11 49+ 5
After
Triglyceride
258*
5
242+16 246+ 3 326+13
Before
134& 3
120+10 128& 2 172k13
After
Phospholipid
PLASMAPHERESIS
139
Double filtration plasmapheresis has been devised for selective removal of large molecules in plasma that are relevant to immune disease and high viscosity disease without the expensive centrifugation apparatus required for plasma separation [21]. The clinical effectiveness of this method has been ascertained in patients with collagen disease, autoimmune disease, and others [21]. As the molecular weights of VLDL and LDL are bigger than those of HDL [32], albumin and immunoglobulins, the second filter can selectively filter out the bigger proteins. Yokoyama et al. reported the effects of this method in the treatment of familial hypercholesterolemia [30]. In the present study both serum cholesterol and LDL cholesterol levels decreased by 55%, while HDL cholesterol decreased by 39% and albumin decreased by 8-11s with supplements of 30 g of albumin solution. IgG and IgA decreased by 28-44%, while IgM and fibrinogen decreased by 50-66s. Thus, the second filter removes LDL and fibrinogen and IgM more efficiently than HDL and other plasma constituents. These changes of lipoprotein cholesterol may be favorable for the prevention of atherosclerosis. Reductions of serum triglyceride, VLDL cholesterol and IDL cholesterol, which also have been thought to be atherogenic [22,23], decreased by 68, 59 and 68%, respectively. As the fibrinogen level plays an important part in the development of stroke and myocardial infarction [33], a decrement of fibrinogen levels may be valuable for the prevention of atherosclerosis. But, reductions of other protein constituents except LDL and fibrinogens may not have favorable effects. Although several methods of removing LDL selectively have been developed [25-311, the method described in the present work is one of the most convenient, safe and inexpensive extracorporeal methods if albumin, immunoglobulins and HDL can be supplemented sufficiently. As this treatment has been proved to be safe in these two homozygotes, long-term treatment may be expected to reverse atherogenic changes in coronary arteries and other peripheral arteries. Acknowledgement We are indebted to the Kuraray Co. Ltd. for providing the membrane filters (PLASMACURE and EVAL
4~).
References 1 Goldstein, J.L. and Brown, M.S., Familial hypercholesterolemia. In: J.B. Stanbury, J.B. Wyngaarden, D.S. Fredrickson, J.L. Goldstein and MS. Brown (Eds.), The Metabolic Basis of Inherited Disease, McGraw-Hill, New York, 1983, p. 612. 2 Mabuchi, H., Tatami, R., Ueda, K., Ueda, R., Haba, T., Kametani, T., Watanabe, A., Wakasugi, T., Ito, S., Koizumi, J., Ohta, M., Miyamoto, S. and Takeda, R., Serum lipid and lipoprotein levels in Japanese patients with familial hypercholesterolemia, Atherosclerosis, 32 (1979) 435. 3 Mabuchi, H., Tatami, R., Haba, T., Ueda, K., Ueda, R.. Kametani, T., Itoh, S., Koizumi, J., Oota, M.. Miyamoto. S., Takeda, R. and Takeshita, H., Homozygous familial hypercholesterolemia in Japan, Amer. J. Med., 65 (1978) 290. 4 Lipid Research Clinics Program, The Lipid Research Clinics Coronary Primary Prevention Trial Results, Part 1 (Reduction in incidence of coronary heart disease), J. Amer. Med. Ass., 251 (1984) 351. 5 Lipid Research Clinics Program, The Lipid Research Clinics Coronary Primary Prevention Trial Results, Part 2 (The relationship of reduction in incidence of coronary heart disease to cholesterol lowering), J. Amer. Med. Ass., 251 (1984) 365. A.K., Cholestyramine therapy in patients 6 Khachadurian, homozygous for familial hypercholesterolemia (familial hypercholesterolemic xanthomatosis), J. Atheroscler. Res., 8 (1968) 177. I Starzl, T.E., Bilheimer, D.W., Bahnson, H.T., Shaw, Jr., B.W., Hardesty, R.L., Griffith, B.P., Iwatsuki, S., Zitelli, B.J., Gartner, Jr., J.C., Malatack, J.J. and Urbach, A.H., Heart-liver transplantation in a patient with familial hypercholesterolemia, Lancet, i (1984) 1382. 8 Buchwald, H., Moore, R.B. and Varco, R.L., Surgical treatment of hyperlipidemias, Part 1 (Apologia), Part 2 (The laboratory experience), Part 3 (Clinical status of the partial ileal bypass operation), Circulation, 49 (Suppl. 1) (1974) 1. 9 Deckelbaum, R.J., Lees, R.S., Small, D.M., Hedberg, S.E. and Gnmdy, SM., Failure of complete bile diversion and oral bile acid therapy in the treatment of homozygous familial hypercholesterolemia, N. Engl. J. Med., 296 (1977) 465. 10 Starzl, T.E., Chase, H.P., Putnam, C.W. and Porter, K.A., Portacaval shunt in hyperlipoproteinemia, Lancet, ii (1973) 940. 11 Torvik, H., Feldman, H.A., Fisher, J.E. and Lees, R.S., Effects of intravenous hyperalimentation on plasma lipoproteins in severe familial hypercholesterolemia, Lancet, i (1975) 601. 12 Thompson, G.R., Lowenthal, R. and Myant, N.B., Plasmaexchange in the management of homozygous familial hypercholesterolemia, Lancet, i (1975) 1208. 13 Berger, G.M.B., Miller, J.R., Bonmci, F., Joffe, H.S. and Dubovsky, D.W., Continuous flow plasma exchange in the treatment of homozygous familial hypercholesterolemia, Amer. J. Med., 65 (1978) 243.
140
14 King, M.E.E., Breslow, J.L. and Lees, R.S., Plasma-exchange therapy of homozygous familial hypercholesterolemia, N. Engl. J. Med., 302 (1980) 1457. 15 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. 16 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. 17 Van de Wiel, A., Kruiswijk, T., De Gruijter, A.J., Hart, H.C. and Imhof, J.W., Plasma exchange in the treatment of heterozygous familial hypercholesterolemia, Acta Med. Stand., 218 (1985) 233. 18 Haba, T., Mabuchi, H., Yoshimura, A., Watanabe, A., Wakasugi, T., Tatami, R., Ueda, K., Ueda, R., Kametani, T., Koizumi, J., Miyamoto, S., Takeda, R. and Takeshita, H., Effects of ML236B(compactin) on sterol synthesis and low density lipoprotein receptor activities in fibroblasts of patients with homozygous familial hypercholesterolemia, J. Clin. Invest., 67 (1981) 1532. 19 LeLorier, J., DuBreuil-Quidoz, S., Lussier-Cacan, S., Huang, T.S. and Davignon, J., Diet and probucol in lowering cholesterol concentrations - Additive effects on plasma cholesterol concentrations in patients with familial type II hyperlipoproteinemia, Arch. Intern. Med., 137 (1977) 1429. 20 Mabuchi, H., Haba, T., Tatami, R., Miyamoto, S., Sakai, Y., Wakasugi, T., Watanabe, A., Koizumi, J. and Takeda, R., Effects of an inhibitor of 3-hydroxy-3-methylglutaryl coenzyme A reductase on serum lipoproteins and ubiquinone-10 levels in patients with familial hypercholesterolemia, N. Engl. J. Med., 305 (1981) 478. 21 Agishi, T., Kaneko, I., Hasuo, Y., Hayasaka, Y., Sanada, T., Ota, K., Amemiya, H., Sugino, N., Abe, M., Ono, T., Kawai, S. and Yamane, T., Double-filtration plasmapheresis, Trans. Amer. Sot. Artif. Intern. Organs, 26 (1980) 406. 22 Mahley, R.W., Atherogenic hyperlipoproteinemia, Med. Clin. N. Amer., 66 (1982) 375. 23 Tatami, R., Mabuchi, H., Ueda, K., Ueda, R., Haba, T., Kametani, T., Ito, S., Koizumi, J., Ohta, M., Miyamoto, S., Nakayama, A., Kanaya, H., Oiwake, H., Genda, A. and Takeda, R., Intermediate-density lipoprotein and cholesterol-rich very low density lipoprotein in angiographically
24
25
26
27
28
29
30
31
32
33
determined coronary artery disease, Circulation, 64 (1981) 1174. Gordon, T., Castelli, W.P., Hjartland, M.C., Kannel, W.B. and Dawber, T.R., High density lipoprotein as a protective factor against coronary heart disease, Amer. J. Med., 62 (1977) 707. 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, i (1976) 1261. 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, Pediat. Res., 14 (1980) 113. Graisley, B., Cloarec, M., Salmon, S., Polonovski, Cl., Delacotte, J.M., Gardent, J., Cavalier, J., Vergoz, D. and Salmon, Ch., Extracorporeal plasma therapy for homozygous familial hypercholesterolemia, Lancet, ii (1980) 1147. Stoffel, W., Boberg, H. and Greve, V., Application of specific extracorporeal removal of low density lipoprotein in familial hypercholesterolemia, Lancet, ii (1981) 1005. Yokoyama, S., Hayashi, R., Kikkawa, T., Tani, N., Takada, S., Hatanaka, K. and Yamamoto, A., Specific sorbent of apolipoprotein B-containing lipoproteins for plasmapheresis Characterization and experimental use in hypercholesterolemic rabbits, Arteriosclerosis, 4 (1984) 276. Yokoyama, S., Hayasbi, 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., Takeda, M., Kajinami, K., Itoh, H., Wakasugi, T., Kobaladze, AS., Ueda, K. and Takeda, R., Selective removal of apolipoprotein B-containing lipoproteins by dextran sulfate column in homozygous and heterozygous patients with familial hypercholesterolemia, Submitted for publication. Have], R.J., Goldstein, J.L. and Brown, M.S., Lipoproteins and lipid transport. In: P.K. Bondy and L.E. Rosenberg (Eds.), Metabolic Control and Disease, 8th edition, W.B. Saunders Co., Philadelphia, 1980, p. 393. Wihelmsen, L., Svardsudd, K., Korsan-Bengtsen, K., Larsson, B., Welin, L. and Tibblin, G., Fibrinogen as a risk factor for stroke and myocardial infarction, N. Engl. J. Med., 311 (1984) 501.
135 Ltd.
ATH 03803
Treatment of Homozygous Patients with Familial Hypercholesterolemia by Double-filtration Plasmapheresis Hiroshi Mabuchi, 2nd Department
Ichiro Michishita, Takeshi Sakai, Yasuyuki Sakai, Akira Watanabe, Takanobu Wakasugi and Ryoyu Takeda of Internal Medicine, Kanarawa University School of Medicine, Kanatawa,
Ishikawa
920 (Japan)
(Received 12 March, 1986) (Accepted 7 April, 1986)
Summary Two homozygous patients with familial hypercholesterolemia were treated by double-filtration plasmapheresis. The plasma separated by the first filter was subsequently led to the second filter of ethylene vinylalcohol co-polymer hollow fibers, which trap very low density lipoprotein (VLDL), intermediate density lipoprotein (IDL) and low density lipoprotein (LDL) preferentially to other plasma constituents. Serum, VLDL, IDL, LDL cholesterol levels decreased by 55, 68, 59 and 55% respectively. HDL cholesterol levels decreased by 39%. Immunoglobulins and fibrinogen levels decreased significantly. Cutaneous and tendinous xanthomas became smaller.
Key words:
Double-filtration
plasmapheresis
- Homozygous
Introduction Homozygous patients with familial hypercholesterolemia show exceptionally high plasma cholesterol levels, exceeding 600 mg/dl, which reflect extreme increases in low density lipoprotein (LDL) concentration [1,2]. For homozygotes, the risk of death from coronary artery disease 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. Address reprint requests to Hiroshi Mabuchi, M.D., 2nd Department of Internal medicine, Kanazawa University School of Medicine, Takara-machi 13-1, Kanazawa, Ishikawa 920, Japan. OOZl-9150/86/$03.50
0 1986 Elsevier Scientific
Publishers
Ireland,
familial hypercholesterolemia
(CAD) or aortic stenosis before adult life is very high [1,3]. Several drugs are known to reduce LDL concentrations in heterozygotes, and reduction of LDL cholesterol levels by cholestyramine has been proved to be effective in decreasing the incidence of CAD [4,5]. However, these drugs are usually not effective in the homozygotes [6]. Thus, other forms of therapy have been attempted. These include liver transplantation [7], partial ileal by-pass [8], biliary diversion [9], portacaval shunt [lo], hyperalimentation [ll] and plasmapheresis [12-171. Thompson et al. [12] treated two homozygotes by repeated plasma exchange, using a continuous-flow blood-cell separator. Thereafter several papers reported the effectiveness of extracorporeal perfusion in the Ltd
136
treatment of homozygotes [12-171. Here, we describe a new approach to the management of homozygous familial hypercholesterolemia by double-filtration plasmapheresis.
sizes. The first filter consisted of polyvinylalcohol hollow fibers with an average pore diameter of 0.2 pm (PLASMACURE, Kuraray Co., Ltd., Japan). The first filter works as a plasma separator, which filters and separates the plasma component from whole blood during extracorporeal circulation. Separated plasma was subsequently led to the second filter of ethylene vinylalcohol co-polymer (EVAL, 4~, Kuraray Co., Ltd., Japan) hollow fibers with an average pore diameter of 0.03 pm. The filtrate from the second filter was mixed with cell-rich blood and returned via an antecubital vein, while approximately 300 ml of the rest in the second filter was discarded. Plasmapheresis was performed by KM-8500 (Kuraray Co., Ltd., Japan). The extracorporeal circulation device was first filled with physiological saline containing heparin (5 units/ml). The blood flow-rate during treatment was adjusted to approximately 100 ml/ min. The total amount of plasma treated by each procedure was 3 liters. Thirty grams of human albumin solution were infused during the procedure. The treatment was carried out in the in-patient clinic in case Y.Y., and in the out-patient clinic in case M.T. Blood pressure, ECG and pulse rate were monitored at regular intervals throughout the extracorporeal treatment. The procedure was repeated every 2 weeks, and later in case Y.Y. once every week.
Materials and Methods Two homozygous patients with familial hypercholesterolemia were studied after informed consent and approval by the Committee of New Drugs and Equipment at Kanazawa University Hospital. Homozygous patients were diagnosed when they had juvenile xanthomatosis and their cholesterol levels were about twice those of the heterozygous parents. Pedigrees of the two homozygotes were shown in our previous paper, in which patient 1 is case Y.Y. of family No. 2, and patient 2 is case M.T. of family No. 1. Their LDL receptor activities studied by cultured skin fibroblasts showed receptor-defective type [18]. Their clinical and laboratory data are shown in Table 1. Case Y.Y. has been treated by 1000 mg/day of probucol 1191. Serum lipoprotein lipid and other laboratory analyses were performed before and after each procedure. Blood samples were allowed to clot at room temperature. Methods of lipoprotein fractionations and lipid determinations were described in our previous paper [20]. Statistical calculations were performed with Student’s paired t-test.
Results
Double-filtration plasmapheresis An arteriovenous shunt was constructed between the radial artery and cephalic vein of the patients. The principle of double-filtration plasmapheresis reported by Agishi et al. [21] was adopted for the present study. This procedure utilizes 2 hollow-fiber filters with different pore
Changes in serum cholesterol levels are shown in Fig. 1. Changes in lipoprotein cholesterol, serum triglyceride and phospholipid levels are shown in Table 2. With treatment of 2-week intervals serum cholesterol in patients Y.Y. and M.T. decreased from 360 rtc 15 (mean+ SEM) to 160 k 6 (a de-
TABLE 1 CLINICAL AND LABORATORY DATA IN 2 HOMOZYGOUS PATIENTS WITH FAMILIAL HYPERCHOLESTEROLEMIA Patient
Age (yr) Sex
Height
weight
(cm)
(kg)
Cholesterol
Triglyceride
Phospholipid
(mg/dl)
(mg/dl)
(mg/dl)
Achilles tendon thickness (mm)
Y.Y.
M.T. ’ Normal
37 M 24 M value in Japanese
169 175 subjects,
57 71
613 566
6.3 f 0.2 mm (mean f SEM).
102 231
382 394
42.0 12.0
a
137
.
0
’
lb1 M.T.
,....,...‘1’.“,....,...,,,,,,,,,,,,,,,,,,,.,,..,-~.,,,,,,,,,.,~.~~,~~~~,~
0
5
10
15
10
15
30
55
40
.5
50
55
60
65
70
75 weeks
Fig, 1. Changes
of serum cholesterol
concentration
before and after double-filtration
plasma
pheresis.
a = patient
Y.Y., b = patient
M.T
crease of 55.6%) and from 423 -t_ 17 to 201 * 13 mg/dl (a decrease of 52.5%), respectively. With treatment at l-week interval, serum cholesterol in patient Y.Y. decreased from 299 A 5 to 134 t_ 2 mg/dl (a decrease of 55.2%). Thus, the average reduction of serum cholesterol by, this treatment was 54.7%. Very low density lipoprotein (VLDL), intermediate density lipoprotein (IDL) and LDL cholesterol levels decreased by 67.9, 58.6 and 54.6%, respectively, while high density lipoprotein (HDL) cholesterol decreased by 39.4%. Serum triglyceride and phospholipid decreased by 68.5 and 48.1%, respectively. Other laboratory data before and after doublefiltration plasmapheresis are as follows. Erythrocyte count, platelet count, hemoglobin and hematocrit showed no significant changes. Leukocyte count increased transiently. Total protein and albumin decreased significantly (P < 0.001) in spite of supplements with human albumin solution. Immunoglobulins, C3, C4 and fibrinogen decreased significantly (P < 0.001). Urea nitrogen (BUN), uric acid, creatinine showed no significant changes. Aspartate aminotransferase (SGOT), alanine amniotransferase (SGPT), lactic dehydrogenase (SLDH), alkaline phosphatase (P-ase), creatine kinase (SCPK) showed slight reductions (P < 0.001). Na, K and Cl showed no significant changes.
Cutaneous and tendinous xanthomas in case Y.Y. showed marked decrease in size. The procedure was well tolerated in both patients. Discussion Several methods of extracorporeal plasma treatment have been applied for the treatment of homozygous patients with familial hypercholesterolemia. More than 45 patients (21 homozygotes and 24 heterozygotes) with familial hypercholesterolemia have records of treatment by plasma exchange. The method of exchanging plasma with 2-4 liters of plasma protein fractions at intervals of l-2 weeks safely and effectively controlled hypercholesterolemia and sometimes induced regression of xanthomas and atherosclerosis in the patients [12-171. However, plasma exchange reduces both atherogenic (VLDL, IDL and LDL) [22,23] and antiatherogenic (HDL) lipoproteins [24], and removes other plasma constituents, such as albumin, immunoglobulins, and others. Recently, procedures for selective removal of LDL, such as heparin-agarose affinity-column chromatography [25-271, immunoadsorption by the anti-LDL sepharose column [28] and dextransulfate cellulose column [29-311, have been applied to the treatment of familial hypercholesterolemia.
Total
M.T.
Y.Y.
Patient
2 1 2
(week)
Interval
322+
75
7
360+15 299+ 5 423+17
Before
146*
4
160+ 6 134* 2 2Olk-13
After
Cholesterol Serum
6 58 11
No. of determination
28kl
19*5 29*1 29*4
Before
VLDL
29+1
36*4 27+1 34*5
6*0.1 9+0.6 10+2 9+0.6
Before
IDL
12*0.6
13+3 llkO.5 17*3
After
205k
6
24Ok14 187k 4 287kll
Before
LDL
93*3
llOk8 84k2 135*9
After
33+1
31k6 32*1 41*2
Before
HDL
20+0.6
15+1 2OkO.6 25kl
After
LEVELS BEFORE AND AFTER DOUBLE-FILTRATION
After
CHOLESTEROL
All values are mg/dI and are given as means f SEM.
SERUM LIPID LEVELS AND LIPOPROTEIN
TABLE 2
149*
6
147k13 147k 7 153k18
Before
47+
2
59+12 55*11 49+ 5
After
Triglyceride
258*
5
242+16 246+ 3 326+13
Before
134& 3
120+10 128& 2 172k13
After
Phospholipid
PLASMAPHERESIS
139
Double filtration plasmapheresis has been devised for selective removal of large molecules in plasma that are relevant to immune disease and high viscosity disease without the expensive centrifugation apparatus required for plasma separation [21]. The clinical effectiveness of this method has been ascertained in patients with collagen disease, autoimmune disease, and others [21]. As the molecular weights of VLDL and LDL are bigger than those of HDL [32], albumin and immunoglobulins, the second filter can selectively filter out the bigger proteins. Yokoyama et al. reported the effects of this method in the treatment of familial hypercholesterolemia [30]. In the present study both serum cholesterol and LDL cholesterol levels decreased by 55%, while HDL cholesterol decreased by 39% and albumin decreased by 8-11s with supplements of 30 g of albumin solution. IgG and IgA decreased by 28-44%, while IgM and fibrinogen decreased by 50-66s. Thus, the second filter removes LDL and fibrinogen and IgM more efficiently than HDL and other plasma constituents. These changes of lipoprotein cholesterol may be favorable for the prevention of atherosclerosis. Reductions of serum triglyceride, VLDL cholesterol and IDL cholesterol, which also have been thought to be atherogenic [22,23], decreased by 68, 59 and 68%, respectively. As the fibrinogen level plays an important part in the development of stroke and myocardial infarction [33], a decrement of fibrinogen levels may be valuable for the prevention of atherosclerosis. But, reductions of other protein constituents except LDL and fibrinogens may not have favorable effects. Although several methods of removing LDL selectively have been developed [25-311, the method described in the present work is one of the most convenient, safe and inexpensive extracorporeal methods if albumin, immunoglobulins and HDL can be supplemented sufficiently. As this treatment has been proved to be safe in these two homozygotes, long-term treatment may be expected to reverse atherogenic changes in coronary arteries and other peripheral arteries. Acknowledgement We are indebted to the Kuraray Co. Ltd. for providing the membrane filters (PLASMACURE and EVAL
4~).
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