chapter 1 therapeutic apheresis in dyslipidemia

2 3www.avidscience.com

Advances in Dyslipidemia Advances in Dyslipidemia

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AbstractIn the last decades, LDL apheresis was introduced

as an extracorporeal treatment option for patients with homozygous or severe heterozygous familial hypercho-lesterolemia. The prognosis of patients suffering from severe dyslipidemia, sometimes combined with elevated lipoprotein (a) levels, and coronary heart disease refrac-tory to diet and lipid-lowering drugs is poor. For such patients, regular treatment with low-density lipoprotein apheresis is the therapeutic option. There are six different LDL apheresis systems available today: cascade filtration or lipid filtration, immunoadsorption, heparin-induced LDL precipitation, dextran sulfate LDL adsorption, direct adsorption of lipoproteins, and Liposorber D. There is a strong correlation between dyslipidemia and atheroscle-rosis. Besides the elimination of other risk factors, in se-vere dyslipidemia therapeutic strategies focus on a drastic reduction of serum lipoproteins. Despite maximum thera-py with a combination of different kinds of lipid-lowering drugs, sometimes the goal of therapy cannot be reached. In such patients LDL apheresis is indicated. Technical and clinical aspects of these six different LDL apheresis meth-ods are shown here. There were no significant differences with respect to all cholesterols, or triglyceride observed. The different published data clearly demonstrate that treatment with LDL apheresis in patients suffering from severe dyslipidemia refractory to conservative therapy is effective and safe in long application. A disadvantage is the high costs and the expensive technologies of the dif-

Chapter 1

Therapeutic Apheresis in Dyslipidemia

Rolf Bambauer¹*, Ralf Schiel² and Reinhard Latza³

1Formerly Institute for Blood Purification, Germany2IInselklinik Heringsdorf GmbH, Germany3ILaboratorium of Medicine, Germany

*Corresponding Author: Rolf Bambauer, Frank-enstrasse4, 66424 Homburg, Germany, Tel: 0049/(0)6841/68500; Fax: 0049/(0)6841/68561; Email: [email protected]

Copyright: © 2015 Rolf Bambauer et al.

This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source.



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ferent artificial methods.

KeywordsDyslipidemia; Coronary Heart Disease; LDL Apher-

esis; Cascade Filtration/Lipid Filtration; Immunoadsorp-tion; Heparin-induced LDL Precipitation; Dextran Sulfate LDL Adsorption; Direct Adsorption of Lipoproteins; Li-posorber D.

IntroductionFamilial hypercholesterolemia (FH) is an autosomal

disorder associated with well-characterized mutations of hepatocyte apolipoprotein-B (apo-B) receptors resulting in decreased low-density lipoprotein (LDL) removal by the liver. FH exhibits a gene dosage effect [1]. Homozy-gotes may have cholesterol in the range of 650 – 1,000 mg/dL, xanthoma in the range of 250 – 550 mg/dL, xanthoma by the age of 20 years, and atherosclerosis by the age of 30 [2]. Through numerous epidemiological examinations, the importance of cholesterol - and of LDL in particular – in the development of coronary sclerosis has not only been qualitatively substantiated, but also a continuing re-lationship between cholesterol levels and coronary mor-bidity has been established [3]. The LDL concentration in the blood is particularly significant in the development of atherosclerosis and especially of coronary heart disease (CHD).

In patients with FH, the LDL-receptor is changed

such that the LDL particles can no longer be recognized. As a result, their adsorption can no longer be mediated. This means an accumulation of LDL in the blood. Fur-thermore, a surplus of cholesterol also blocks the enzyme 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase which normally inhibits the cholesterol synthe-sis rate. Brown and Goldstein also discovered the structure of the LDL receptor, and they found that the structure of this receptor was defective in many patients with FH [4]. The receptors do not exist at all in patients with homozy-gous FH. Thus FH was the first metabolic disease which could be related to the mutation of a receptor gene. The standard therapy of patients with homozygous and hete-rozygous FH has been diet and lipid-lowering drugs. In all severe cases in which these therapies failed, LDL apheresis is indicated. LDL apheresis eliminates cholesterols and all lipoproteins very effectively and reduces the increased inflammatory activity of the atherosclerotic vessel wall in patients with FH and overt atherosclerosis [5].

In 2003 guidelines and indications for the implemen-tation of LDL apheresis were laid for Germany (Table 1) [6]. In the United States the Clinical Application Commit-tee (AAC) of the American Society for Apheresis (ASFA) established guidelines and indications for diseases which were treated with therapeutic apheresis (TA) which were reviewed every 7 years [2,7,8].

The innovation in this medical field was aimed on one hand at metabolizing LDL intravascular through medica-



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tion or at inhibiting cholesterol synthesis and on the other hand at elimination of cholesterol from the intravascular spaces. Here only the various extracorporeal elimination of cholesterol methods are discussed by the authors, which are listed in Table 2. Six different artificial extracorporeal methods for LDL-cholesterol elimination are mentioned, which had influenced the prognosis of the primary and secondary dyslipoproteinemia tremendously and are used today.

Table 1: Indications for LDL apheresis [6].

All the techniques described here are effective and well tolerated. Based on an average drop in cholesterol of 50- 60 percent per session, a treatment interval of 7–14 days is advisable and described here [9]. The constant re-duction of cholesterol is meant, above all, to prevent the progression or the development of atherosclerosis. By lowering the cholesterol from 400 mg/dL to 200mg/dL

treatment can almost double a patient´s life expectancy, according to at least one study. A large disadvantage is the high costs of the different artificial methods therefore the LDL apheresis treatments are most performed in the in-dustrialized nations [10-19].

Table 2: Extracorporeal methods for elimination of cholesterol [9,20].

• HomozygousformofFH(frequency1:1,000,000inGermany),absoluteindication(LDL:600-1000mg/dL).

• Heterozygous,severeformsofFHwithcholesterolvaluesbetween300-600mg/dL,when,despitemaximumtherapy(24-32g/dayionexchangercom-binedwith40-80mgHMG-CoA-reductaseinhibitors)orintoleranceofthistherapy,LDLcannotbeconstantlyheldbelow200mg/dL.

• Severeformsofpolygenichypercholesterolemia,ifthetargetvalueof200mg/dLLDLhasnotbeenreachedafteroneyearofmaximumtreatment.

• Inpatientsover60yearsofage,LDLapheresisshouldonlybeimplementedinexceptionalcircumstances.

Year Authors Method Advantage Disadvantage1967 DeGennesetal.

[10]Plasmapheresis Quickandwelltol-

eratedeliminationofpathologicsubstances

Unselectively,dan-gerofinfections,

bleeding,andrisksofsubstitutionsolutions

1980 Agishietal.[11] Cascadefiltration Semi-selectivity Dangerofinfections,Loweffectiveness

1981 Stoffeletal.[12] Immunoadsorp-tion

Selectivity,effective-ness,regeneration,and

reusability

Expensivetech-nology

1983 Borbergetal.[13] Immunoadsorp-tion

Selectivity,effec-tiveness


1983 Wielandetal.[14] Heparin-inducedLDLprecipitation

(HELP)

Selectivity,effectiveness


1985 Noséetal.[15] Thermofiltration Selectivity,effec-tiveness

Outdatedtechnology,behaviourofmacro-moleculesunderheatunknown,notavail-

able1985 Antwilleretal.[16] Dextransul-

fate-inducedLDLprecipitation


Expensivetech-nology,

notavailable1987 Mabuchietal.[17] Dextransulfate

LDLadsorption(Liposorber-LA15)



1993 Boschetal.[18] LDLhemoper-fusion(DALI)

Selectivity,effectiveness,

simpletechnology

Unknown

2003 Ottoetal.[19] LDLhemoper-fusion

(LiposorberD)


simpletechnology

Unknown



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Epidemiology and PathophysiologyFH is one of the most common inherited disorders;

there are 10,000,000 people with FH worldwide, mainly heterozygous. The most common FH cause is mutations along the entire gene that encode for LDL receptor pro-tein, but it has been also described that mutations in apoli-poprotein B and protein converts subtilisin/hexin type 9 genes produce this phenotype [20,21]. An increased level of cholesterol is almost always due to an increase in cir-culating LDL, usually with simultaneous increase in very low-density lipoprotein (VLDL) and decrease in high den-sity lipoprotein (HDL). This constellation accelerates the development of arteriosclerosis and, in particular, CHD. Heterozygous FH occurs with a frequency of 1: 500 and the homozygous form with a frequency of 1: 1,000,000. Patients with homozygous, familial hypercholesterolemia nearly all die before the age of 30.

Despite substantial progress in diagnosis, drug ther-apy and cardio surgical procedures, atherosclerosis with myocardial infarction, stroke, and peripheral vascular dis-ease still maintain its position at the top of morbidity and mortality statistics in industrialized nations [22]. Estab-lished risk factors widely accepted are smoking, arterial hypertension, diabetes mellitus, and central obesity. The role of cholesterol-bearing lipoproteins in pathogenesis is well established. It is suggested that elevated lipid con-centrations in the serum lead to their accumulation in the

intima of arteries that results in the development of ath-erogenic plaques. These alterations seem to be accompa-nied by changes in vessel tone and endothelial regulation [23,24].

It has been demonstrated that dyslipoproteinemia plays a key role in the pathogenesis of atherosclerosis and CHD. There is a strong correlation between hyperlipi-demia and atherosclerosis [21,25]. Various authors have investigated the effects of hyperlipidemia on endothelial function [3,25-27]. Vascular endothelium is considered to be the largest endocrine, paracrine, and autocrine partici-pant in the regulation of numerous homeostatic vascular functions [28]. Endothelial cells sense changes in hemo-dynamic forces such as pressure and shear stress as well as circulating and locally formed vasoactive substances released by blood cells. In response to these stimuli, en-dothelial cells synthesize and release biologically active substances such as nitride oxide (NO), prostacyclin, en-dothelium-derived hyperpolarizing factor, endothelin, prostaglandin H2, thromboxane A2, heparin sulfate, trans-forming growth factor, vascular endothelial growth factor, basic fibroblast growth factor, platelet-derived growth fac-tor, tissue plasminogen activator, plasminogen activator inhibitor-1, oxygen free radicals, and others [29]. These substances modulate vascular tone through their relax-ing and contracting actions as well as vascular structure through production of growth promoting and growth-inhibiting factors. In hypercholesterolemia patients, in-



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travenous reconstituted HDL infusion rapidly normalizes endothelium-dependent vasodilation by increasing NO bioavailability. This may in part explain the protective effect of HDL on coronary heart disease and illustrates the potential therapeutic benefit of increasing HDL in patients at risk from atherosclerosis [30]. The endothe-lium also regulates hemostasis and thrombosis through its antiplatelet, anticoagulant, and fibrinolytic functions as well as inflammation through the expression of chemo tactic and adhesion molecules [31]. Endothelium plays a key role in vascular homeostasis. The endothelium is in a strategic location between the blood and vascular smooth muscle; thus it is a primary target for injury from mechan-ical forces and processes related to cardiovascular risk fac-tors [29,32].

FH may also be caused by mutations in the gene en-coding apo-B, the proprotein convertase/kexin type 9 (PCSK9) gene, or rare mutations in the LDLRAP1 gene [33]. Apo-B is important for lipid metabolism because se-rum lipoproteins, including LDL and VLDL, contain apo-B as a structural component. The level of apo-B correlates with cardiovascular risk [34]. Apo-B is also necessary for transport of VLDL from the liver into the plasma [35]. PCSK9 is a convertase enzyme that mediates the degrada-tion of the LDL receptor [36]. The importance of (PCSK9) is shown by significantly lower LDL levels and incidence of CHD in patients with nonsense mutations in the PCSK9 gene [37]. The LDLRAP1 gene encodes an adaptor protein for trafficking of LDL receptors in cells in the liver, and

mutations have been identified in patients with either FH an autosomal dominant disorder or an autosomal reces-sive hypercholesterolemia [38].

A positive correlation between elevated triglyceride blood levels and heart attacks has been established in nu-merous studies [3,39]. Hypertriglyceridemia is prevalent in 18.6 percent of men and 4.2 percent of women between the ages of 16 and 65. Of particular importance is that in-creased triglycerides (TG) are often accompanied by low-HDL cholesterol blood levels. Elevated TG represent a use-ful marker for risk of CHD, particularly when HDL levels are low [39]. The strong association between the ratio of TG/HDL and the risk of CHD suggests a metabolic inter-action between the TG and cholesterol ester-rich lipopro-teins in increasing risk of CHD [40]. Dyslipoproteinemia in combination with diabetes mellitus causes a cumulative insult to the vasculature resulting in more severe disease which occurs at an earlier age in large and small vessels as well as capillaries. The most common clinical conditions resulting from this combination are myocardial infarction and lower extremity vascular disease. Ceriello et al. show an independent and cumulative effect of postprandial hypertriglyceridemia and hyperglycemia on endothelial function, suggesting oxidative stress as common media-tor of such effect [41]. Patients with severe heterozygous FH and clinical CAD or homozygous FH are at very high CHD risk and require intensive LDL-lowering therapy and LDL apheresis [42].



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As an anti-atherogenic factor, HDL cholesterol cor-relates inversely to the extent of postprandial- lipemia. A high concentration of HDL is a sign that triglyceride-rich particles are quickly decomposed in the postprandial phase of lipemia. Conversely, with a low HDL concentra-tion this decomposition is delayed. Thus, excessively high triglyceride concentrations are accompanied by very low HDL counts. This combination has also been associated with an increased risk of pancreatitis [23]. The LDL-HDL ratio is one of the highest strongest predictors of prema-ture CHD events. In patients with a ratio of >5.0 with high triglyceride concentrations the risk of coronary events is to those with normal triglyceride concentrations four times higher [43].

The importance of Lp(a) as an atherogenic substance is very similar to LDL. But it also contains Apo(a), which is very similar to plasminogen, enabling Lp(a) to bind to fibrin clots. Binding of plasminogen is prevented and fi-brinolytic obstructed. Thrombi are integrated into the walls of the arteries and become plaque components. Thus, many studies show that high Lp(a) concentrations are associated with an early occurrence of CHD and apo-plectic insult [44].

Uttermann found six different Lp(a) phenotypes: S4, S3, S2, S1, B, and F. They investigated the influences of these phenotypes on the Lp(a) levels and found that phe-notypes S1, S2, and B were associated with high, and phe-notypes S4 and S3 with low Lp(a) concentrations (45).

It has not yet been determined whether CAD is mainly primarily associated with Lp(a) levels or with the pheno-types. It can be concluded that high levels of Lp(a) are as-sociated with CHD; the isoforms S2, S1, B, F are linked to CHD; and patients with premature CHD showed the highest Lp(a) levels as well as the is forms S2, S1, B, and F. Grainger et al. showed that Lp(a) and Apo A enhance proliferation of human smooth muscle cells in culture by inhibiting the activation of plasminogen to plasmid, thus blocking the proteolysis activation of transforming growth factor-β (TGF-β), an autocrine inhibitor of human vascular smooth muscle cells. The activation of TGF-β is inhibited in the aortic wall and serum of mice expressing Apo A as a consequence of Apo (a) inhibition [46].

In recent years, different studies have shown that Lp(a) is a major independent risk factor for atheroscle-rosis, increasing cardiovascular morbidity and mortality at a younger age [45,47,48]. Gaubatz et al. observed that the metabolic fate of the Lp(a)-triglyceride rich lipopro-tein complex, which is more abundant in hypertriglyceri-demia, may be different from that of conventional Lp(a) and may contribute uniquely to the progression or se-verity of cardiovascular disease [47]. Komai et al. dem-onstrated that oxidized Lp(a) is more potent than native Lp(a) in stimulating vascular smooth cells. Oxidized Lp(a) may play an important role in the pathogenesis of vascular disease [48]. Elevated Lp(a) levels are correlated with the extent of CHD and the mortality of these patients. Ducas



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et al. reported of an acquired Lp(a) excess in patients with renal disease as a marker for cardiovascular risk [49]. The elevation of plasma Lp(a) concentrations in patients with renal diseases appears to be related to proteinuria and is, therefore, amenable to treatment. High Lp(a) levels in re-nal disease suggest an important role of the kidneys in the metabolism [50]. Among older adults in the United States, an elevated level of Lp(a) lipoprotein is an independent predictor of stroke, death from vascular disease, and death from any cause in men, but not in women [51].

Thus far, no sufficiently drug therapy has been avail-able to decrease high Lp(a) levels. N-acetylcysteine has been shown to induce a dose-dependent reduction in Lp(a) levels about seven percent by causing dissociation of the Apo A by cleavage of disulfide bonds [52]. In recent years several studies using monoclonal antibody inhibi-tion of the protein convertase PCSK9 have demonstrated reductions in Lp(a) blood levels, but the studies have been of short duration with small numbers of patients [53]. Further studies must show the effectivity in long-term use. Very high Lp(a) levels can only be normalized by LDL apheresis [52]. The lowering absolute levels of Lp(a) should be the main objective of any Lp(a) – lowering strat-egy for prevention of CHD in addition to modification of conventional risk factors [54].

Another strong risk factor for accelerated partheno-genesis, which must be mentioned here, are the wide-spread high homocysteine levels found in dialysis patients.

This risk factor is independent of classic risk factors such as high cholesterol and LDL levels, smoking, hyperten-sion, and obesity, and much more predictive of coronary events in dialysis patients than are these better-known factors. Homocysteine is a sulfur amino acid produced in the metabolism of methionine [55]. Under normal condi-tions, about 50 percent of homocysteine is remethylated to methionine and the remaining via the transsulfuration pathway [20]. Vitamins are important cofactors for the enzymes in the methionine metabolism (folic acid and vi-tamin B12 for the remethylation pathway and vitamin B6, or pyridoxine, for the transsulfuration pathway). The kid-ney is an important metabolic site for removal (up to 70 percent) of plasma homocysteine [56]. In many patients, the therapy with vitamins B6 and B12 and folic acid is suf-ficient.

Defining hyperhomocysteinemia as levels greater than the 90th percentile of controls and elevated Lp(a) level as greater than 30mg/dL, the frequency of the combi-nation increased with declining renal function. Fifty-eight percent of patients with a GFR less than 10 mL/min had both hyperhomocysteinemia and elevated Lp(a) levels, and even in patients with mild renal impairment, 20 per-cent of patients had both risk factors present [57].

LDL-Apheresis TherapyCHD remains one of the main causes of death in the

mortality statistics of the industrial nations, despite con-



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siderable progress in diagnostics, development of new medications, such as HMG-CoA-reductase inhibitors as well as cardiosurgical measures. Cholesterol concentra-tions of over 200 mg/dL represent an increased coronary risk. This risk is double at cholesterol values between 200–250 mg/dL and fourfold at values of 250–300 mg/dL [20]. In addition to familial disposition, other risk factors that contribute to coronary heart disease are smoking, adi-posity, diabetes mellitus, stress, reduced HDL, increased Lp(a), and fibrinogen.

Usually the severe forms of hypercholesterolemia are due to a relative or absolute reduction of LDL receptors in the liver resulting in a decreased plasma clearance of lipids. For these patients, reduction of intake dietary fats is advised. Depending on type of condition, various medi-cations are available, such as colestyramin, colestipol, β-fibrates, fenofibrate, nicotinic acid, β-pyridylcarbinol, probucol, and D-thyroxine. Since the introduction of HMGCoA-reductase inhibitors, which can also be com-bined with other lipid-lowering drugs, LDL reduction up to 50 percent of the original concentration can be achieved. In many cases, this appears to be sufficient. Nu-merous studies based on large numbers of patients have investigated the affectivity and safety of the various HMG-CoA-reductase inhibitors [58,59]. During the testing pe-riod, numerous side effects like diarrhea, obstipation, other gastrointestinal diseases, myositis, rhabdomyolysis, and others were observed [60]. With the introduction of

selective and semiselective extracorporeal elimination methods for cholesterol, LDL, Lp(a), and triglycerides, all forms of previous therapy-resistant hypercholesterolemia can now be effectively treated [61].

Severe heterozygous forms of familial hypercholes-terolemia or other forms of dyslipoproteinemia with cho-lesterol values between 250 and 600 mg/dL are also to be allocated to the therapy group for LDL-apheresis. Prin-cipally, these forms first require maximum dietetic and medicational therapy, for example with, 24–32 g ion ex-changer in combination with 40–80 mg CSE inhibitors. If, despite this maximum therapy or due to therapy in-tolerance, LDL cannot be constantly held below 200 mg/dL, then LDL-apheresis is indicated. Only in cases of ex-ceptional circumstances should patients over 60 years of age be given LDL-apheresis treatment; however, diagnosis should be supported by corresponding examinations, and the patient should be a non-smoker. All patients should be placed under cardiologic observation with ECG under ex-ercise, thallium scintigraphy, and, possibly, coronary an-giography, to register reduced progression or the desired regression of the coronary heart condition.

The advantages can, however, be estimated by differ-ent studies. The quotient relevant for cost-effective assess-ment: [cost of treatment—costs saved]: [improvement in life quality] cannot be exactly calculated at present. To calculate it, detailed information is required about the ex-penses saved through illnesses avoided (heart attack, an-



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gina pectoris, and premature coronary death). The stand-ard therapy for FH besides diet is, lipid-lowering drugs and the LDL-apheresis. Up to now only cascade filtration, immunoadsorption, heparin-induced LDL precipitation, LDL adsorption through dextran sulfate, the DALI he-moperfusion system, and the Liposorber D system have been of clinical relevance. The requirement that the origi-nal level of cholesterol is to be reduced by at least 60 per-cent is fulfilled by all the systems listed in Table 3.

Cascade FiltrationCascade filtration (CF), membrane differential filtra-

tion (MDF) or double filtration plasmapheresis seems to be superior to conventional plasmapheresis but less effec-tive than adsorption or precipitation techniques [20,62]. The CF was developed by Agishi et al. in Japan and was the first semiselective technique used for treating hypercho-lesterolemia [63]. The secondary membrane in cascade fil-tration has a cut off of approximately one million daltons. LDL cholesterol has a molecular weight of approximately 2,300,000 daltons and is thus retained by this membrane. All other molecules, which are larger than one million dal-tons are also retained, while plasma components smaller than one million daltons pass through the membrane and are returned to the patient. Thus, with plasma separation of 2,500–3,000 mL, total cholesterol can be reduced by ap-proximately 35–50 percent of the original value and LDL cholesterol by approximately 30–45 percent thereof (Table

3) [9,20].

Table 3: Effectiveness of the various LDL apheresis methods (reduction in percent of original concentration

[9,20].

Cascade filtration

(2,500-3,000 ml plasma)

Immuno-Adsorption(4,000-5,000 ml plasma)

Heparin- induced LDLprecipitation

(HELP)(2,500-3,000ml plasma)

LDL-adsorp-tion

(dextran-sulfate)

(Liposorber)(2,500-3,000ml plasma)

LDL hemo-

perfusion(DALI)

(1.6 blood Volume)

LDL hemo-

perfusion(Lipo-

sorber D)(1.5 bloodVolume)

Reductioninpercentoforiginalconcentration

% % % % % %

Cholesterol 35–50 30 50 45 60 55LDL 30–45 35 45 30–40 60-75 60-75HDL 35–50 20 10-20 --- 16-29 5-13Lp(a) 60–70 60 46 60 60-75 60-75Triglycerides 60 60 60 70 ca44 ca66Fibrinogen 50 10–20 50 30 16 20-40IgM 35 10–20 --- --- 21 14IgA 55 10-20 --- --- --- ---FactorVIII --- 10-20 10-20 20 --- ---C3 --- --- 50 --- --- ---C4 --- --- 50 --- --- ---Plasminogen --- --- 50 --- --- ---

Due to irregular pore distribution with different di-ameters in the secondary membrane, plasma components with smaller molecular weight can also be retained, such as fibrinogen (MW:Å 340,000), HDL (MW: Å 400,000), and IgM (MW: Å 1,000,000). Given the cardiovascular risk factor, it appears beneficial to reduce fibrinogen, while de-creasing HDL, a protective factor against atherosclerosis, appears to be harmful [63]. Geiss et al. found that MDF



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is an effective method to lower elevated concentrations of atherogenic lipoproteins. The concomitant loss of other macromolecules transiently improves hemorrheology but demands a close monitoring of immunoglobulin concen-trations as a safety parameter [64].

With the new synthetic secondary membranes such as the lipidfilter EC-50 (Asahi, Japan), and new types of machines, better effectiveness and selectivity in the sep-aration of the blood components can be reached in the treatment of hypercholesterolemia. This cascade filtration system is called lipid filtration. By treating a higher mean plasma volume of 3,370 mL, lipid filtration led to an in-creased reduction rate particularly for LDL cholesterol. Fibrinogen and Lp(a), with pre-treatment levels of HDL cholesterol, total protein, and immunoglobulins remained unchanged and were not significantly different from the values before the last apheresis [65]. MDF or lipid filtra-tion is as safe and effective as the HELP-system with re-spect to the extracorporeal removal of LDL-cholesterol, Lp(a), fibrinogen, by treating identical plasma volumes.

A new term rheopheresis was created for special method of cascade filtration designed to reduce blood viscosity in the management of diseases with impaired microcirculation, like age-related macular degeneration, diabetes mellitus, coronary artery disease, peripheral arterial occlusive disease, cerebro-vascular stroke, and sudden deafness. Especially in the treatment of diabetic

retinopathy and of the age-related macular degeneration, the results with the rheopheresis are very promising [66]. Terai et al. observed that changes in retinal vascular di-ameter seem to be associated with the systemic effect of a single LDL-apheresis. Vasodilatation of the arterioles and the venules improved after LDL-apheresis, indicating an improvement of ocular perfusion in patients with hyper-cholesterolemia [67]. Fully automated apheresis machines have been developed so that continous manual steering of flows and blood pressures is no longer required (Octo Nova/Diamed, Monet/Fresenius, both Germany). The de-velopment of new membranes with various cut offs will probably allow for safe and effective double or triple filtra-tion in the near future.

Immunoadsorption (IA)This method was first described by Stoffel et al. in

1981 and by Borberg et al. in 1983 as LDL-apheresis with anti-LDL sepharose columns [68,69]. After primary sepa-ration, the plasma is perfused through sepharose columns coated with LDL antibodies, or other antibodies. The LDL molecules in the plasma are adsorbed onto the antibodies on the columns. This is a reversible antigen-antibody bond accord based on the principle of affinity chromatography [20]. Antibodies against the protein component in hu-man LDL cholesterol (apolipoprotein B 100) gained from sheep are covalently bound to sepharose particles. These are heteroclonal sheep antibodies against apo protein B,



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which are bound to sepharose after cyanogens bromide activation. In one column, 3 grams of LDL cholesterol can be adsorbed. Both columns contain 300–320mL of se-pharose particles. Before the column is saturated with the absorbed lipoproteins (600–800mL plasma), the plasma flow is switched to the other column; while one column is used for adsorption, the off-line column is generated with neutral saline buffer solution, lysine buffer (pH 2.4), and neutral buffer again. The treated plasma is then mixed with the cellular components of the blood and returned to the patient. The entire procedure takes 2.5–3 hours via a computerized apheresis monitor. After the treatment, the columns are rinsed, and after the same procedure, filled with sterile solution. The immunoadsorption columns can be used for a minimum of 40 treatments in the same patient.

The antigen-antibody bound is reversed by using a mixture of lysine and hydrochloric acid with a pH of 2.8. The pH is then increased to 7.4 using a sodium chloride solution, buffered with phosphate, and rinsed with physi-ological common salt solution. This procedure restores the binding capacity of the columns and prepares them for use again. In this way, any required volume of plasma can be perfused in one treatment session (3–10 L). The advantage of this method is the high selectivity, effective-ness for all apo-B-containing lipoproteins, and regenerat-ing capacity of the columns. The disadvantage is the high expenditure required not only for the treatment itself, but

also for the regeneration process. The immunoadsorption columns are approved for regular and continued use [68]. Given the high cost of the columns, implementation of this system is only viable on a long-term basis that is to say, at least 40 times per patient. At a perfusion volume of 3–6 litters per session, the LDL cholesterol is reduced to 30–40 percent of the original level. HDL, serum proteins, immunoglobulins, and fibrinogen, and so forth drop by approximately 15–20 percent and return to their normal level, however, after approximately 24 hours.

Two different systems are currently available, the LDL Therasorb system (Miltenyi Biotec, Germany) and the LDL and Lp(a)-Excorim system (Fresenius, Germany). The matrix sepharose is coupled with specific antihuman apolipoprotein B-100 or antihuman Lp(a) sheep antibod-ies. Both systems are safe and effective in clinical use, even in long-term treatment. Indications for the extracorporeal elimination of LDL cholesterol are primary and second-ary dyslipoproteinemia, and for the Lp(a) IA the solitary familial Lp(a) elevation. Table 4 shows a compilation of some of the results of IA. The efficacy of the LDL-apher-esis IA columns did not decrease after 60 treatments ses-sions. The columns selectivity also remained unchanged. This treatment showed a beneficial effect of long-term LDL-apheresis [76,77].



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Table 4: Immunoadsorption in hypercholesterolemia (selection of literature).

Year Authors Patients (n) Therapy dura-tion (years)

Reduction of LDL (%)

1988 Borbergetal.[70] 5 3–5 52–671988 OetteandBorberg[71] 10 5–8 711990 Richteretal.[72] 8 1–3 561993 Bambaueretal.[73] 4 1–2 55–581996 Richteretal.[74] 18 8.6 60–702000 Schambergeretal.[75] 14 2 61

Lipoprotein (a)-ApheresisLipoprotein (a) (Lp(a)) represents a class of lipopro-

tein particles which have lipid composition similar to LDL and a protein moiety, apo-B 100, covalently linked to apo(a), a glycoprotein with striking structural similari-ties to plasminogen [78]. High plasma levels of Lp(a) are associated with an increased risk for atherosclerotic coro-nary heart disease (CHD) by a mechanism yet to be de-termined. Because of its structural properties, Lp(a) can have both atherogenic and thrombopenia potentials. The means for correcting the high plasma levels of Lp(a) are still limited in effectiveness. All drug therapies tried thus far have failed. Individuals in the top fifth of Lp(a) had more than a twofold higher risk of CAD compared with those in the bottom fifth [54]. The most effective thera-peutic methods in lowering Lp(a) are the LDL-apheresis methods. Since 1993, special immunoadsorption poly-clonal antibody columns (Pocard, Russia) containing

sepharose bound anti-Lp(a) have been available for the treatment of patients with elevated Lp(a) serum concen-trations [78,79].

Monospecific polyclonal antibody to human Lp(a) was obtained from immune sheep serum. Pokrovsky et al. described preparing immunosorbent by immobilizing of antibodies to Sepharose CL-4B [78]. For the treatment, two personal columns are assigned to each patient. Each column is filled with 400 mL of sorbent tested for steril-ity and pyrogenicity. Anti-Lp(a) immunoadsorption col-umns are reusable. Between the treatments, the columns are stored at 40C in storage solution, which is rinsed prior to each Lp(a)-apheresis procedure [79].

The numerous studies provide the epidemiological evidence that Lp(a) is an independent risk factor in the pathogenesis of coronary heart disease and artheriosclero-sis. The individual plasma level is genetically determined. Diet or available drugs do not influence the plasma level of Lp(a). Until today there is only one device available on the market which allows the specific removal of Lp(a) from plasma (Lipopak, Pocard, Russia). As with LDL-apheresis in homozygotes-specific Lp(a) is a life-saving therapy in severe cases with elevated Lp(a) as the sole risk factor.



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Heparin-Induced LDL Precipitation (HELP)

In 1982, Seidel and Wieland reported on a new meth-od of extracorporeal elimination of low-density lipo-proteins [80]. This method was abbreviated HELP. After primary separation, the plasma is mixed in a ratio of 1:1 with an acetate-acetic acid buffer (pH 4.85), so that the pH of this mixture is 5.1. Then, 100,000 U heparin per liter are added to the buffer. After the plasma has been mixed thoroughly with the acetate-acetic acid buffer and hepa-rin, LDL cholesterol precipitates in the acid environment together with fibrinogen and heparin to form insoluble precipitates. These precipitates are then removed from the plasma by means of a polycarbonate membrane. The re-maining free heparin is almost completely removed by a heparin absorber (DEAE cellulose). The acidulous plasma is returned to a physiological pH value using bicarbonate dialysis, and the plasma, free of LDL, is returned to the patient with the blood cells. Although the method is tech-nically complicated, it is reliable and effective. It is, how-ever, non-selective for in addition to the cholesterols, C3, C4, fibrinogen, plasminogen, and factor VIII, and so forth are also eliminated. HDL reaches its original level after 24 hours, while the fibrinogen concentration only increases gradually; the amount of plasma should, therefore, be lim-ited to 3 three litres [80].

More recently, a compact unit has been designed that

somewhat reduces the cost of the equipment. The Plasmat Futura (B. Braun, Germany) is easy to use and safe in han-dling. The priming rinsing and reinfusion are fully auto-mated. The entire treatment uses only disposable material, the machine does not need descaling or disinfection, and no piped water supply or reverse osmosis is required. The user is safely guided through all treatment steps and sup-ported with message prompts and warnings.

Studies on patients undergoing regular extracorpor-eal LDL-elimination indicate that the incidence of adverse cardiovascular clinical events can be reduced much ear-lier than by drug therapy alone. These immediate clinical benefits, which take place directly after the apheresis, can-not be due, however, to an improvement in coronary mor-phology (i.e., regression of atherosclerotic lesions), since such improvement can only be observed after several months of treatments [81]. The improved myocardial per-fusion and clinical symptoms after LDL-apheresis is likely due to an improvement in rheology as well as producing an immediate positive influence on endothelium function and an increase in the volume of vasodilatory, anti aggres-sive nitrogen monoxide released or a reduction in the vol-ume of available endothelin [82]. A more than 60 percent reduction of LDL at weekly intervals is clearly associated with an early regression of lipid-rich vascular lesions. LDL apheresis reduces the shear-stress of the flowing blood on vulnerable plaques either by its effect on plasma viscosity and/or on the vasomotoric reserve, thus leading to a lower



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peripheral arterial resistance. Furthermore, LDL-apheresis eliminates oxidized LDL, which might counteract plaque stabilization by its inflammatory effects. LDL-apheresis to with coagulation factors normalizes hypercoagulatory states, thus preventing atherothrombotic events at the site of vulnerable or erosive plaques [82].

The safety and long-term applicability of the HELP system has been proved in more than 120,000 treatments. Serious complications have never been observed [83], and the technology of the equipment has been improved over time. Many authors have shown that there is clear clinical evidence that a drastic lowering of LDL concentrations by HELP reduces significantly the rate of total and coronary mortality as well as the incidence of cardiovascular events in high-risk hypercholesterolemic patients [84,85]. Wang suggested that simultaneous reduction of pro-inflamma-tory and prothrombotic factors with atherogenic lipopro-teins by HELP apheresis may contribute to improvement of endothelial dysfunction and thereby inhibit progres-sion of atherosclerotic lesions and stabilize the existing plague [86]. Otto et al. found that LDL apheresis slightly, but significantly reduced CRP concentrations in patients with CHD on statin therapy, which may contribute to the stabilization of atherosclerosis in hypercholesterolemic patients treated with LDL apheresis [87]. These results are even more impressive when known age-related increase in CRP over treatment period is taken into account [86,87]. Zannetti et al. found that Pentraxin 3 (PTX3) a key com-

ponent of the humeral arm of instate immunity possibly aiming at tuning arterial activation associated with dam-age vascular was acutely reduced by HELP apheresis. Lowering PTX3 levels in high risk patients is associated with disease regression of cardiovascular events [88].

All HELP treatments have demonstrated successful secondary prevention for patients with familial hyper-cholesterolemia, coronary artery disease, cardiac bypass, heart transplantation, or acute cerebral infarction (stroke) [89]. Elimination of fibrinogen and other substances also has an influence on blood viscosity, rheology, and eryth-rocyte aggregation; thus, the microcirculatory situation as a whole can be significantly improved. A selection of lit-erature and LDL elimination has been compiled in Table 5. Severe side effects are rare: so far, the only side effects reported have been transient shivering and hypotension.

Dextran Sulfate Low-Density Lipo-protein (Liposorber)

Mabuchi et al. reported in 1987 on LDL absorption with dextran sulphate (Liposorber LA-15, Kaneka, Japan) [96]. Low-molecular dextran sulfate (MW 4500) can se-lectively absorb all substances containing apolipoprotein B. Dextran sulfate is covalently bound to cellulose parti-cles. The dextran sulfate was selected as an affinity legend of LDL adsorbent for its high affinity and low toxicity. The binding mechanism is the direct interaction between



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dextran sulfate and the positively charged surface of apoli-poprotein B-containing lipoproteins (LDL, VLDL, and Lp(a)). The dextran sulfate has a structure similar to that of the LDL receptor and seems to act as a type of pseudo receptor [97]. Approximately 2.5 grams LDL can be bound per column. After primary separation, the plasma is per today through the columns, where all material containing Apo-B such as cholesterol, LDL, VLDL, and triglycerides is absorbed. Free of cholesterol, the plasma is returned to the patient. After 500 mL of plasma, the columns are satu-rated and require regeneration with 4.1 percent NaCl so-lution. After rinsing with Ringer’s solution, they are ready for use again. The effectiveness of this treatment is good, and cholesterol is eliminated selectively (Table 3). Occa-sionally, with a perfusion volume of more than four lit-ers, a marked drop in the Quick level can occur, probably caused by the absorption of factor VIII [97].

This method has also found widespread clinical use in recent years. Side effects are rare and of a minor na-ture such as hypotension, nausea, hypoglycemia, and light allergic reactions. In a retrospective study we con-ducted with 32 patients and 955 LDL-apheresis sessions, we observed that these side effects were observed in 12 percent of all treatment sessions. A shock situation only arose in 0.4 percent, and in 0.2 percent allergical reactions occurred, which were easily treated [98]. The dextran-in-duced allergical reactions have not been observed so far. Low-molecular dextran sulfate is much less allergenic than

the forms of dextran which are normally implemented as a plasma expander and have a higher molecular weight of 40,000 to 80,000. The advantage of the Liposorber system is the selectivity by elimination of all apo B-containing li-poproteins and the high effectiveness. A disadvantage is the labour intensive technology.

Table 5: Clinical results with the HELP-apheresis

system. (B.Braun, Germany) (selection of literature).

ESRD: end-stage renal failure; HD: hemodialysis; HTX: heart transplantation.

Year Authors Diagnosis Patients(n)

Drop out(n)

Therapy duration(years)

Side effects

(%)

Im-proved

(n)1987 Eisenhauer

etal.[90]FH,CHD 13 --- 0.5–1.3 ? 13

1991 Seideletal.[91]

FH,CHD 51 5 1.0 2.9 46

1993 Boschetal.[92]

FH,CHD,ESRD

3(HD) --- 1.5 13.0 3

1994 Schuff-Werneretal.[81]

FH,CHD 51 12 2.0 2.8 39

1997 Jägeretal.[84]

FH,CHD,HTX

15 --- 3.6 --- 5

1998 Mellwigetal.[93]

FH,CHD 9 --- ? ---- 8

1999 Donneretal.[94]

FH 4 --- ? ? 4

2000 Schettleretal.[82]

FH,CHD 18 --- >0.5 --- 18

2001 Moriartyetal.[95]

FH 4 --- 0.5 --- 4

2008 Wangetal.[86]

FH,CHD 22 --- 1.0 --- 22

2012 vanBuurenetal.[43]

FH 27 --- 1.3–16.6 3.6 24

2014 Zanettietal.[88]

FH 19 --- 1.0 --- 19



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More than 60 percent reduction of the pre-treatment cholesterol values can be achieved by one treatment with the Liposorber system. The effectiveness of therapy has also been observed over time in several long-term clinical studies (Table 6). In more than 75 percent of cases, pa-tients improved or reached regression of coronary athero-sclerosis. The observed side effects were between 0.5 and 4 percent [98,100,101,106]. The Liposorber system is safe and effective, even in high-risk hypercholesterolemia pa-tients. In children, the Liposorber system has proved to be safe and effective, too [109].

Table 6: LDL adsorption with dextran sulfate (Li-posorber LA-15, Kaneka, Japan) in hypercholesterolemia

(selection of literature).

Two clinical reports described excellent long-term follow-up results for patients with coronary artery disease who had been treated with LDL-apheresis using dextran sulfate cellulose columns plus adjunctive cholesterol low-ering drug therapy [110,111]. Intensive cholesterol-lower-ing therapy with LDL-apheresis and lipid-lowering drugs can achieve a substantial decrease in LDL-cholesterol lev-els to induce regression of coronary artery disease [99].

Complement activation takes place with essentially all adsorbers; however, activated complement is removed by binding to specific proteins such as C4 binding protein. In the presence of anticoagulating heparin, bradykinin is formed, but the production of bradykinin is low and nor-mally not clinically significant [20]. However, drugs that inhibit angiotensin converting enzyme (ACE inhibitors) can exacerbate the clinical effect of this amount of brady-kinin to the point of serious adverse effect including ana-phylaxis [112].

Olbricht et al. reported on a anaphylactic reaction in a patient during ACE inhibition therapy and LDL-apher-esis [112]. He observed reactions similar to those which frequently occur in ACE inhibition therapy in combina-tion with ANG9-high-flux dialysis as well as with reusable polysulphone membranes in intermittent hemodialysis [113]. The functioning mechanism of these anaphylac-tic reactions has still not been finally clarified, although they are presumably induced by the increased release of

Year Authors Diag-nosis

Patients(n)

Drop out(n)

Therapyduration(years)

Side effects

(%)

Im-proved

(%)1988 Thompsonet

al.[99]FH,CHD

20 --- 2.1 0.5 19

1992 Gordonetal.[100]

FH 54 --- 0.25 --- 54

1994 Daidaetal.[101]

FH,CHD

66 --- 1.0 --- 45

1996 Kroonetal.[102]

FH,CHD

21 --- 2.0 1.3 21

1997 Gordonetal.[103]

FH,CHD

45 4 0.5 4.0 41

1997 Bambaueretal.[97]

FH,CHD

120 35 6.0 2.2 85

1998 Mabuchietal.[104]

FH,CHD

130 --- 6.0 --- 94

1999 Nishimuraetal.[105]

FH 30 5 2.3 --- 4

1999 Richteretal.[106]

FH,CHD

8 --- 3.0-5.0 --- 6

2003 Bambaueretal.[98]

FH,CHD

32 --- 8.0 0.3 31

2010 Moriartyetal.[107]

FH,CHD

10 --- 0.5 --- 10

2015 Juliusetal.[108]

FH 24 --- 7.25 --- 24



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bradykinin. Bradykinin is formed through activation of the contact activation system, which consists of the com-ponents: high-molecular-weight kininogen, prekallikrein, Hagemann’s factor, and coagulating factor XI. When plas-ma comes into contact with very high negatively charged dextran sulfate, the concentration of bradykinin in the plasma increases considerably [113]. Bradykinin is quick-ly decomposed through the activity of kininase I and II; thus patients undergoing lipid apheresis are not normally affected.

Kininase II is identical to angiotensin-converting enzyme and is blocked by the administration of ACE in-hibitors. This results in an increase of bradykinin in the bloodstream and to an anaphylactic reaction. These ana-phylactic reactions are not specific to a particular mem-brane or surface type, but can always occur in ACE in-hibition when blood or plasma comes into contact with contact-activating surfaces. The reactions are independ-ent of the type of ACE inhibitor; the presence of dialysate is not necessary, for they also develop in cell-free plasma. The varying degrees of severity are presumably connected to the expression of the ACE-coding gene being subject to strong individual fluctuations [9,20]. There is a con-traindication for ACE inhibitors by LDL-apheresis. ACE inhibitors must not be administered to patients undergo-ing LDLapheresis [114].

Low-Density Lipoprotein Hemoper-fusion

In 1993, Bosch et al. first described the direct adsorp-tion of lipoproteins (DALI, Fresenius, Germany) the low density lipoprotein hemoperfusion [115]. The new ad-sorber, which is compatible with human whole blood, uses a matrix of polyacrylate beads. In the DALI system, blood is perfused through the adsorber, which contains 480 mL of polyacrylate-coated polyacrylamide, without regeneration. The column has a capacity of more than 1.5–2.0 blood volumes for effective adsorption of cholesterol, LDL, Lp(a), and triglycerides. Regeneration is not neces-sary because the column is used for only one treatment [116]. In a very simple extracorporeal circuit, the blood is pumped through the LDL adsorber. The elimination of LDL, Lp(a) particles, and other lipoproteins from whole blood is performed by adsorption onto polyacrylate-coat-ed polyacrylamide beads. The small porous beads with a diameter of 150–200 μm are immobilized in the adsorber with the aid of two sieves. The beads have a porous struc-ture which exploits the principle of size exclusion chro-matography. The sponge-like structure of the beads offers a very large inner and outer surface for adsorption (more than 99 percent of the overall surface of over 1,000 m² is located within the beads).

The adsorption of LDL and Lp(a) and other lipopro-teins occurs by polyacrylate ligands covalently binding to



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the polyacrylamide surface. Like the LDL-receptor, poly-acrylate, consists of polyanions, with negatively charged carboxylate groups [116]. The polyanions interacts selec-tively with the cationic groups in the apoprotein B moiety of LDL and Lp(a). Due to this electrochemical interaction, the lipoproteins are immobilized on the beads. By flowing, the whole blood past the beads affects only a minor inter-action between the blood cells and the similarly small out-er surface of the beads. The smaller lipoproteins can easily penetrate the inner sponge-like structure of the beads via the pores. HDL can also penetrate the beads, but because the apo A-coated HDL is not attracted to the ligand, it is not affected by the adsorber and cannot be eliminated. Monitoring of this simple extracorporeal blood circula-tion system is carried out by measuring the blood pressure in the afferent and efferent blood lines and at the adsorber inlet. Anticoagulation is carried out by first applying a heparin bolus, then by a continuous ACD-A solution in-fusion into the blood line as it exits the patient’s vein.

Besides lipoproteins, the DALI system also adsorbs the positively charged ions calcium and magnesium. Therefore, the columns have to be prerinsed with 4–6 lit-ers of a priming solution containing these electrolytes. The adsorber is thereby saturated with these cations, thus preventing hypocalcemia and hypomagnesia during the treatment [117]. The DALI system can be run at three dif-ferent adsorber sizes (DALI 500, 750, and 1000mL adsorb-ers). After passing the adsorber, the blood depleted of all

apoB-containing lipoproteins is put back into the patient. The advantages are good selectivity, high effectiveness, and a simple technology. The potential for possible micro particle release from the columns as with all adsorbers can be avoided prevented by more and better rinsing of the columns and a careful handling [118]. A second leukocyte type in line filter can be added after column to further re-duce the possibility of micro particle release. Such a filter is recommended to maximize safety. In a five-year follow-up, long-term therapy with DALI was safe, effective, and selective as LDL and Lp(a) could be reduced by >60 per-cent per session in approximately 100 minutes treatment time, while decrease and the incidence of side effects were low [119]. The DALI system has proven to be safe, effec-tive, and simple to perform.

Another whole blood lipoprotein apheresis system (Liposorber D, Kaneka Japan) is commercially available too. The Liposorber D system is the second whole blood perfusion type LDL apheresis system developed on the basis of the technology of the dextran sulfate Liposorber LA-15 system. Liposorber D adsorbs positively charged LDL, VLDL, and Lp(a) particles from whole blood us-ing negatively charged polyanions. Liposorber D contains negatively charged dextran sulfate covalently bound to cel-lulose [120]. The negatively charged surfaces activate the intrinsic coagulation pathway; prolongation of a PTT and shortening of PT have been observed in the LDL apher-esis with the dextran sulfate column. Coagulation factors



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such as factors XI and XII were reduced by dextran sulfate adsorption, but those coagulation factors returned to nor-mal range within one or two days after the treatment. A Japanese multicenter clinical trial found a significantly re-duce in LDL, Lp(a), and triglycerides by using Liposorber D [121]. Adverse events, which were observed, were hy-pocalcemia during treatment caused by ACD-A solutions, the symptoms disappeared by administration of calcium, and slight hypotension.

From a technical point of view, the Liposorber D whole blood adsorption column has clear advantages over the usual LDL-apheresis systems that require plas-ma separation. The system is simpler and easier to handle because no plasma separation procedure is necessary. In vitro evaluations have shown that the adsorbent efficiently adsorbs LDL and Lp(a) with a good biocompatibility. The clinical results of this technology are very encouraging. The advantages are good selectivity, effectiveness, and a simple technology [121]. Possible columns configurations DL 50, DL 75, and DL 100 are available.

A treatment performed with the Liposorber D and the new developed machine DX-21 reduces the apoB li-poproteins without having great influence on HDL, other important plasma components or blood cells. Liposorber D chemically is identical to that for Liposorber LA-15 with a modification of the size of the adsorbent beads suit-able for whole blood processing. The Liposorber D system

is comparable with the DALI system. The handling with the DX- 21 machine is easy, safe, and the user is guided through all treatment steps. The developed whole blood perfusion LDL-apheresis system Liposorber D is a safe and simple apheresis system and thus a useful modality to remove LDL and Lp(a) from whole blood in hypercholes-terolemic patients.

Gene Therapy and New DrugsGiven the recent disappointments in the hypercholes-

terolemia there is intensified interest in new approaches for reducing LDL cholesterol, Lp(a) etc. [122]. Transla-tional investigators have refocused there attention on both homozygous and heterozygous FH as human model sys-tems of extreme LDL elevations.

For example the PCSK9 inhibitors are mentioned. In recent years, several studies using monoclonal antibody inhibition of the protein convertase subtilisin/kexin type 9 (PCSK9) have demonstrated reductions in Lp(a) blood levels, but the studies have been of short duration with small numbers of subjects, and relationship to dose, sex, and background lipid therapy have not fully established [123,124]. Although percentage reductions from base-line were greater in those with lower starting Lp(a) lev-els, the absolute reductions were substantially greater in those considered at higher risk, with baseline Lp(a) >125 nmol/L [123]. But further larger studies are necessary to



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show if the inhibition of PCSK9 with human monoclonal antibodies is as effective as the regularly weekly or two-weekly Lp(a)-apheresis. Comparable for both therapeutic methods as Lp(a)-apheresis and monoclonal antibodies were the higher decreases of pre-treatment levels over the time in older patients with initial higher Lp(a) levels than in younger patients with lower Lp(a) levels and lower risk [123,125].

Other examples are lomitapide and mipomersen [125]. But even today, the gene therapy, is still no real al-ternative to regular lipid apheresis treatment [126,127]. The aim of gene therapy in FH is the over expression of the LDL receptor by insertion of receptor-encoding transgene with the help of a suitable vector. So far, the adenovirus or adeno-associated virus vectors have been found par-ticularly suitable. They infect both resting and dividing cells and remain episomal in the cytoplasma – not in the genome. They are casily to be manipulated at the molec-ular level so that they can have a high immunogenicity, i.e., a cellular and humeral immuno response against the foreign protein leads to the elimination of hepatocytes in-fected by the vector.

So far, there is only one trial for the treatment of ho-mozygous FH in humans. The hepatocytes were isolated, cultured, and then infected with one of the LDL recep-tor gene-encoding retroviruses. The liver cells were then reinfused into the portal vein. In some of the patients in this pilot study, this treatment lowered the LDL by 6–25

% [128].

Conclusion of LDL-ApheresisAll of the extracorporeal techniques described above

are effective and well tolerated. With weekly or biweekly treatment, the average LDL cholesterol concentration can be reduced to approximately 50–60 percent of the origi-nal levels. LDL concentration increases again after each apheresis session, but does not return to the original level. After a few sessions, it balances out. The increase after apheresis can be slowed down by lipid lowering drugs. By lowering the cholesterol from 400 mg/dL to 200 mg/dL, treatment can almost double a patient’s life expectancy. The LDL-apheresis treatment must be repeated after the above-mentioned treatment procedure in homozygous and severe heterozygous FH life-long or until other better therapy technologies are available.

LDL-apheresis decreases not only LDL mass but also improves the patient’s life expectancy. LDL-apheresis per-formed with different techniques decreases the suscepti-bility of LDL to oxidation. This decrease may be related to a temporary mass imbalance between freshly produced and older LDL particles. Furthermore, the baseline fatty acid pattern influences pre-treatment and post-treatment susceptibility to oxidation [94].

Streicher et al. observed that despite drug therapy, LDL apheresis significantly stimulates the residual LDL-receptor expression in FH via the reduction of available



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extracellular cholesterol resulting in delayed reappearance of hypercholesterolemia in between treatments [129]. The acute effect of lipid apheresis on serum lipidome could be predominantly attributed to lipoprotein changes, while blood cell damages during this procedure caused addi-tional, less-pronounced changes. The importance of spe-cific changes in particular lipid species remains to be es-tablished [130].

The techniques vary somewhat in selectivity. Cascade filtration reduces HDL concentration, which probably has an atherogenous effect in the long term. CF, HELP, and dextran sulfate adsorption to a lesser extent, and whole blood hemoperfusion systems reduce the average fibrino-gen concentration. This reduction can prove advantageous as the viscosity of the blood is reduced and the rheological characteristics improved. Moreover, fibrinogen is an inde-pendent risk factor in the development of CHD. The vari-ation in viscosity is directly related to vascular resistance, which can profoundly influence CHD and atherosclerosis [61]. Mediators of blood viscosity (in addition to hema-tocrit, shear forces, and temperature) include red blood cell (RBC) deformability, RBC aggregation, and plasma viscosity. A single LDL apheresis treatment reduces blood viscosity by more than 20 percent and maintains this re-duction for at least 7 days [131]. Fibrinogen is responsible for 20 percent of plasma viscosity.

The primary aim in reducing cholesterol concentra-tion is to prevent the development and progression of ath-

erosclerosis. There are sufficient data that this therapeutic aim can be achieved. There are many reports on decrease and slower progression of atherosclerotic changes in coro-nary vessels and carotids after patients have been treated for one or more years with lipid apheresis. Selective LDL elimination through LDL-apheresis represents a decisive breakthrough in the treatment of high-risk patients with hypercholesterolemia, whose treatment has, up to now, been inadequate, despite strict diets and lipid-reducing medication.

The Apheresis Applications Committee of the ASFA summarized the LDL-apheresis in familial hypercholes-terolemia as follows. The goal of LDL-apheresis is to re-duce the time-averaged total cholesterol levels by 45–55 percent, the LDL levels by 40–60 percent, and the Lp(a) by 40–60% [8]. FDA approved indications for patients with FH unresponsive to pharmacologic and dietary manage-ment are:

1. Functional homozygote´s with a LDL cholesterol >500 mg/dL,

2. Functional heterozygote’s with no known cardio-vascular disease but a LDL cholesterol >300 mg/dL,

3. Functional heterozygote’s with known cardiovas-cular disease and LDL cholesterol >200 mg/dL [8].



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Patients without FH but with high LDL or Lp(a) cho-lesterol who cannot tolerate or whose conditions are un-responsive to conventional therapy can also be treated [8]. During pregnancy, LDL cholesterol levels in individuals affected by FH can rise to extreme levels that can compro-mise uteroplacental perfusion. There are case reports of the use of LDL-apheresis in these indications to allow for successful completion of pregnancy. TPE can be effective but because of the availability of the selective removal sys-tems and their enhanced efficiency of cholesterol removal, the use of TPE to treat FH is uncommon. It may, however, be the only option in small children [8].

A reduction in costs is a valid demand in view of the scarce resources available in the healthcare system. Com-missions, consisting of physicians, administration spe-cialists and representatives of the health insurance funds and others, nowadays decide at a “round table” who will be granted medical facilities and who will not, this is a clinical routine adopted only in Germany. Physicians are committed to helping all the patients entrusted to them to the best of their knowledge, and this means that medi-cal treatment—and particularly the apheresis processes—must become affordable. This demand represents a great challenge to physicians, politicians, health organisations, and, above all, to the manufacturers. Industry constantly justifies the high costs with the extensive research and de-velopment required. All those involved in the healthcare system must intensify their cooperation in this respect.

Nevertheless, medical progress is advancing and will not be stopped. Since the introduction of hollow fiber membranes, exceptional efforts in research and develop-ment have been undertaken in the apheresis sector alone, enabling, for example, the introduction of selective sepa-ration techniques into every day clinical practice—tech-niques which were a thought of at the beginning of the eighties. This is reflected in the numerous national and international specialist congresses, which take place each year.

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chapter 1 therapeutic apheresis in dyslipidemia

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