Differential effects of lipoprotein apheresis by lipidfiltration or dextran sulfate adsorption on lipidomic profile

Atherosclerosis Supplements 14 (2013) 151e155 www.elsevier.com/locate/atherosclerosis

Differential effects of lipoprotein apheresis by lipidfiltration or dextran sulfate adsorption on lipidomic profile J. Gra¨ßler a,*, S. Kopprasch a, J. Passauer a, S. Fischer a, K. Schuhmann a,b, S. Bergmann c, G. Siegert c, A. Shevchenko b, S.R. Bornstein a, U. Julius a a

Department of Internal Medicine III, University Hospital Carl Gustav Carus Dresden, Germany b Max Planck Institute of Molecular Cell Biology and Genetics, Dresden, Germany c Institute of Clinical Chemistry and Laboratory Medicine, University Hospital Carl Gustav Carus Dresden, Germany

Abstract Objective and methods: Acute modification of plasma lipidomic profile was assessed by top-down shotgun profiling on a LTQ Orbitrap hybrid mass spectrometer in 14 patients treated with two different apheresis techniques: plasma lipidfiltration (LF) and whole blood dextran sulfate adsorption (DSA). Results: Patients treated with DSA revealed a significantly more pronounced reduction of LDL-cholesterol (LDL-C), a diminished decrease of HDL-cholesterol (HDL-C) and triglycerides (TG), and a similar reduction in lipoprotein (a) (Lp(a)) level. Against the overall tendency of reduction of lipid metabolites of all lipid classes in post-apheresis plasma, independent of apheresis technology applied, a highly significant increase of phosphatidylethanolamines (PE) in response to DSA was observed. Conclusion: These data indicate that DSA technology may be associated with an activation or damage of blood cells at contact surface which subsequently leads to a massive liberation of cellular and membrane PE’s. Pathophysiological consequences, especially with respect to coagulation system and oxidative stress, have to be further elucidated. Ó 2012 Elsevier Ireland Ltd. All rights reserved. Keywords: Whole blood and plasma lipoprotein apheresis; Lipidomics; Coagulation; Oxidative stress; Phosphatidylethanolamines; Membrane lipids

1. Introduction Lipoprotein apheresis is the “ultima ratio” treatment in severely hyperlipidemic patients, including patients with homozygous familial hypercholesterolemia and those with heterozygous familial hypercholesterolemia resistant or intolerant to statin therapy [1e3]. Lipoprotein apheresis is also highly efficient in the treatment of patients with elevated Lp(a). In a longitudinal study Jaeger et al. [4] * Corresponding author. University Hospital Carl Gustav Carus, Department of Internal Medicine III, Division Pathological Biochemistry, Fetscherstrasse 74, D-01307 Dresden, Germany. Tel.: þ49 351 4583230; fax: þ49 351 4585330. E-mail address: [email protected] (J. Gra¨ßler). 1567-5688/$ - see front matter Ó 2012 Elsevier Ireland Ltd. All rights reserved. http://dx.doi.org/10.1016/j.atherosclerosissup.2012.10.006

demonstrated a significant reduction of cardiovascular risk in high Lp(a) patients under apheresis treatment. Various lipoprotein apheresis techniques are currently used which acutely reduce LDL-C and Lp(a) levels up to 75% [1,3]. In general, lipoprotein apheresis today is a safe technology. The rate of adverse events like nausea, flushing, headaches, angina or hypotension is below five percent [1,3]. Although very rare, there are a number of severe adverse events, like anaphylactoid reactions in patients with ACE inhibitors, which may limit the application of this procedure [5]. Furthermore, each of the technologies has its specific pleiotropic effects on coagulation system, inflammatory activity, and oxidative stress, thereby modulating the individual atherosclerotic risk [1,6e8]. Up to now objectifiable selection criteria of the optimal lipoprotein

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apheresis technology for an individual patient are lacking. Therefore, there is a need to identify new biomarkers which better allow to define the best suitable apheresis technology for the individual patient. New technologies, like lipidomics technology, which provides information about hundreds of lipid metabolites of all lipid classes in plasma, present a valuable possibility to characterize specific features of each lipoprotein apheresis technology [9,10]. In the present study, we therefore aimed at analyzing acute effects of two lipoprotein apheresis techniques e (1) plasma lipidfiltration (LF, Diamed, Cologne, Germany, Octo Nova) and (2) whole blood dextran sulfate adsorption (DSA, LiposorberÒ D, Kaneka Pharma Europe N.V., Wiesbaden, Germany) on plasma lipidomic profile. We were able to show that DSA in contrast to LF was associated with a significant postapheresis increase of several PE metabolites probably indicating a massive contact-triggered cell activation or damage. 2. Methods 2.1. Patients All subjects gave written consent to the study, which was approved by the local Ethics Committee (EK199052011). All patients were under ongoing apheresis therapy. Routine medication was maintained. Group 1: LF: 4 men/3 woman; age: 55e80 years; BMI: 21.4e36.8 kg/m2; mean treated plasma volume: 3750  270 ml.

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Group 2: DSA: 4 men/3 woman; age: 48e73 years; BMI: 23.4e34.0 kg/m2; mean treated blood volume: 7442  480 ml. Comorbidities, like hypertension and type-2 diabetes mellitus, were equally distributed in both groups. Blood samples from each subject were obtained immediately before and immediately after two apheresis sessions. Samples were shock frozen with liquid nitrogen and stored at 80  C until analysis. 2.2. Integral blood lipid indices LDL-C, HDL-C, total cholesterol (TC), and TG levels were measured using the Roche automated clinical chemistry analyzer MODULAR (Roche Diagnostics GmbH, Mannheim, Germany). 2.3. Lipidomics Plasma lipidome was screened by top-down shotgun profiling on a LTQ Orbitrap hybrid mass spectrometer as previously described in Ref. [9]. High resolution spectra of 178 lipid species in 12 lipid classes were analyzed. Lipid classes, which have been analyzed: cholesterol esters (CE), triacylglycerols (TAG), diacylglycerols (DAG), lysophosphatidylcholines (LPC), lysophosphatidylethanolamines (LPE), phosphatidylcholines (PC), ether phosphatidylcholines (PCO), phosphatidylethanolamines (PE), ether phosphatidylethanolamines (PEO), phosphatidylinositol (PI), sphingomyelins (SM), and ceramides (Cer).

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l l l l ) rs es des nes nes nes ers nes ers ito lins des ro ro ro ides p(a te i i i i id i i h h te ste ste L os ye am er es cer cer hol lam hol et et lam l n es e e c i l l l m e o ly gly o lc no lc line no yl er in ho Cho Cho igly er y y g g d t C l l i d d a a C n l ti am hat Tr ho eth es acy acy ati eth hi LLta ol i ol ha ylc l p Sp LD HD Di sph dyl to Tr sp tid tidy than hos Ch i t o o a P ph pha Ph ph pha yle d s so os os ati h Ly pho h h P P p so os Ly Ph

Fig. 1. Apheresis-induced acute changes in integral lipid parameters and lipidomic profile after LF and DSA; *p  0.05, **p  0.01, ***p  0.001 LF vs. DSA; data as means  SEM.

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2.4. Statistical analysis

3. Results

Differences between LF and DSA apheresis techniques were assessed by one-way analysis of variance (ANOVA). All analyses were conducted using the PASW Statistics 18 (SPSS) software.

DSA resulted in a significantly more pronounced reduction of LDL-C (74.5  1.1% vs. 66.2  1.8%), but a smaller reduction of HDL-C (12.8  1.6% vs. 18.7  1.3%) and triglycerides (38.7  7.6% vs.

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] ] ] ] ] ] ] ] ] ] ] G :2 :4 :2 :2 :1 :3 :3 :2 :2 :1 :1 TA [46 [48 [48 [50 [50 [50 [51 [51 [51 [52 [52 m G AG AG AG AG Su TAG TAG TAG TAG TAG TAG TA T T T T

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Fig. 2. Apheresis-induced acute changes of triacylglycerides (TAG) and cholesterol esters (CE) after LF and DSA; #p  0.1, *p  0.05 LF vs. DSA; data as means  SEM.

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associated with a significantly less pronounced reduction of selected CE and TAG species (Fig. 2). In contrast, the class of PE increased significantly after DSA (Fig. 1). In accordance with this finding eight PE species were found to increase after DSA (Fig. 3). For PEO species comparable effects could not be demonstrated (Fig. 3). Furthermore, three DAG species revealed a significant post-apheresis increase after DSA, whilst the whole DAG class showed a tendency to decrease (Fig. 4).

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4. Discussion

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DSA (n=7) ] ] ] ] ] ] ] PE 6:1 6:2 6:3 6:4 8:5 8:6 0:6 3 3 3 3 3 3 4 m [ [ [ [ [ [ [ Su PE PE PE PE PE PE PE

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] ] ] ] ] ] ] O :8 :7 :5 :5 :4 :3 :3 PE [34 [36 [36 [36 [38 [38 [40 m O EO EO EO EO EO EO Su PE P P P P P P Fig. 3. Apheresis-induced acute changes of phosphatidylethanolamines (PE) and phosphatidylethanolamine ethers (PEO) after LF and DSA; *p  0.05, ***p  0.001 LF vs. DSA; data as means  SEM.

57.5  4.2%); (Fig. 1). In general, smaller decreases of HDL in comparison to LDL are able to favorably affect lipid metabolism. The decrease of Lp(a) by about 73% was comparable for both apheresis technologies (Fig. 1). Except phosphatidylethanolamines all lipid classes decreased after both methods of lipoprotein apheresis (Fig. 1). As a rule, a tendency of a more pronounced reduction of all lipid classes, except PI, could be observed after LF, which were statistically significant for LPC, PC, PCO, SM, and Cer (Fig. 1). Although not reaching statistical significance for the lipid classes of CE and TAG as a whole, DSA was also

Preliminary studies have shown that general changes of serum lipidomic profile induced by lipoprotein apheresis were mainly determined by lipid profile and content of lipoprotein particles, which were removed by the particular procedure [10,11]. Therefore, the degree of reduction of nearly all lipid metabolites was closely correlated with either the reduction of LDL- or HDL-cholesterol [10]. Now, in clear contrast, we found significantly higher values of eight different PE metabolites (PE [36:1], [36:2], [36:3], [36:4], [38:5], [38:6], [40:6]) resulting in a higher concentration of the whole PE lipid class in plasma after DSA. Lipoprotein apheresis by DSA has shown to be highly effective in removing LDL-C and Lp(a) [12]. The affinity ligand, dextran sulfate, has been found to exhibit a high affinity to LDL and a low toxicity [13]. The whole blood dextran sulfate adsorption system consists of one lipoprotein adsorption column with negatively charged dextran sulfate cellulose beads in order to selectively remove positively charged apo B containing lipoproteins, like VLDL, LDL, and Lp(a) from plasma. The system has been widely used and its safety and efficacy have been established in several clinical investigations [12,14]. Nevertheless, our finding of increased post-apheresis PE levels, achieved on the basis of a new sophisticated lipidomics technology, should inspire a clinical long-term reevaluation of the DSA technology. As established by Leidl et al. [15] all types of blood cells, like monocytes, lymphocytes, granulocytes, platelets, and erythrocytes, come into consideration as a source of PE. Recent studies indicate that different cell types activated by pathophysiological agonists respond via formation of eicosanoids, which are bound to PE’s. Thus, a new family of oxidized phospholipids was identified in thrombin-activated human platelets [16]. Furthermore, activated platelets and monocytes have shown to generate hydroxyphosphatidylethanolamines via lipoxygenase [17]. In summary, this study shows that lipoprotein apheresis with DSA technology was associated with an exclusive increase of post-apheresis PE levels, indicating the possibility of cell activation or damage during this procedure. The pathophysiological significance of this observation remains to be established.

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l l l ] ] ] ] ] ] ] ) G :5 :4 :1 :2 :1 :2 :1 ro ro ides p(a ro t e s te s t e DA [32 [34 [34 [36 [36 [36 [38 r L s e e e e m c G G G G G G G ol ol ol ly Su DA DA DA DA DA DA DA Ch -Ch -Ch Trig l L L ta LD HD to Fig. 4. Apheresis-induced acute changes of diacylglycerols (DAG) after LF and DSA; *p  0.05, **p  0.01, ***p  0.001 LF vs. DSA; data as means  SEM.

Conflicts of interest In the last 3 years, U. J. was reimbursed travel expenses by Diamed, Fresenius, and B. Braun. He was paid honoraria for lectures by MSD and Fresenius as well as for lipidologic evaluations by Fresenius. The other authors state that they do not have anything to declare. Acknowledgments We thank Martina Kohl, Sigrid Nitzsche, and Eva Schubert for the excellent technical support. We are grateful for the excellent work of the nurses working in the apheresis unit: Kerstin Johne, Franziska Meister, Elke Neubert, and Birgit Lippold. References [1] Julius U, Frind A, Tselmin S, Kopprasch S, Poberschin I, Siegert G. Comparison of different LDL apheresis methods. Expert Rev Cardiovasc Ther 2008;6:629e39. [2] Julius U, Tselmin S, Fischer S, Passauer J, Bornstein SR. The Dresden Apheresis Center e experience with LDL apheresis and immunoadsorption. Atheroscler Suppl 2009;10:12e6. [3] Thompson GR. Recommendations for the use of LDL apheresis. Atherosclerosis 2008;198:247e55. [4] Jaeger BR, Richter Y, Nagel D, et al. Longitudinal cohort study on the effectiveness of lipid apheresis treatment to reduce high lipoprotein(a) levels and prevent major adverse coronary events. Nat Clin Pract Cardiovasc Med 2009;6:229e39. [5] Kroon AA, Mol MJ, Stalenhoef AF. ACE inhibitors and LDLapheresis with dextran sulphate adsorption. Lancet 1992;340:1476. [6] Kopprasch S, Graessler J, Bornstein SR, et al. Beyond lowering circulating LDL: apheresis-induced changes of systemic oxidative stress markers by four different techniques. Atheroscler Suppl 2009; 10:34e8.

[7] Kopprasch S, Julius U, Gromeier S, Kuhne H, Graessler J. Distinct effects of LDL apheresis by hemoperfusion (DALI) and heparininduced extracorporeal precipitation (HELP) on leukocyte respiratory burst activity of patients with familial hypercholesterolemia. J Clin Apher 2000;15:249e55. [8] Ramunni A, Burzo M, Verno L, Brescia P. Pleiotropic effects of LDL apheresis. Atheroscler Suppl 2009;10:53e5. [9] Graessler J, Schwudke D, Schwarz PE, Herzog R, Shevchenko A, Bornstein SR. Top-down lipidomics reveals ether lipid deficiency in blood plasma of hypertensive patients. PLoS One 2009;4:e6261. [10] Tselmin S, Schmitz G, Julius U, Bornstein SR, Barthel A, Graessler J. Acute effects of lipid apheresis on human serum lipidome. Atheroscler Suppl 2009;10:27e33. [11] Wiesner P, Leidl K, Boettcher A, Schmitz G, Liebisch G. Lipid profiling of FPLC-separated lipoprotein fractions by electrospray ionization tandem mass spectrometry. J Lipid Res 2009;50: 574e85. [12] Julius U, Parhofer KG, Heibges A, Kurz S, Klingel R, Geiss HC. Dextran-sulfate-adsorption of atherosclerotic lipoproteins from whole blood or separated plasma for lipid-apheresis e comparison of performance characteristics with DALI and Lipidfiltration. J Clin Apher 2007;22:215e23. [13] Tani N. Development of selective low-density lipoprotein (LDL) apheresis system: immobilized polyanion as LDL-specific adsorption for LDL apheresis system. Ther Apher 2000;4:135e41. [14] Yamamoto A, Kojima S, Shiba-Harada M, Kawaguchi A, Hatanaka K. Assessment of the biocompatibility and long-term effect of LDL-apheresis by dextran sulfate-cellulose column. Artif Organs 1992;16:177e81. [15] Leidl K, Liebisch G, Richter D, Schmitz G. Mass spectrometric analysis of lipid species of human circulating blood cells. Biochim Biophys Acta 2008;1781:655e64. [16] Morgan LT, Thomas CP, Kuhn H, O’Donnell VB. Thrombin-activated human platelets acutely generate oxidized docosahexaenoicacid-containing phospholipids via 12-lipoxygenase. Biochem J 2012;431:141e8. [17] Maskrey BH, Bermudez-Fajardo A, Morgan AH, et al. Activated platelets and monocytes generate four hydroxyphosphatidylethanolamines via lipoxygenase. J Biol Chem 2007;282:20151e63.