Lovastatin Alters Blood Rheology in Primary Hyperlipoproteinemia: Dependence on Lipoprotein(a)? Wolfgang Koenig, MD, Reinhard Hehr, MSc, Herwig H. Ditschuneit, MD, Klaus Kuhn, MD, Edzard Ernst, MD, Julian Rosenthal, MD, a n d Vinzenz Hornbach, MD
As part of a randomized, single-blind, comparative study evaluating the eficacy oflovas-
tatin and bezafibrate retard in the treatment of primary hypercholesterolemia, hemorheologic parameters (whole blood viscosity, hematocrit, plasma viscosity, red blood cell aggregation and deformability, and fibrinogen) were studied in 35 patients. Whole blood viscosity and plasma viscosity improved significantly after 3 months of treatment with lovastatin, whereas other hemorheologic variables remained unchanged. Stratifying 24 patients by their lipoprotein Lp(a) levels showed that in those with lowLp(a) (525 mg/dL) high-density lipoprotein cholesterol increased and red blood cell aggregation as well as deformability decreased considerably, whereas in the group with high Lp(a) levels (>25 mg/dL), the opposite behavior was observed. Treatment of primary hypercholesterolemia with lovastatin may not only reduce the risk for atherosclerotic complications by its pronounced decrease of low-density lipoprotein cholesterol, but also may favorably alter blood rheology, and may decrease insudation of plasmatic components into the arterial wall and improve tissue perfusion, in particular on the microcirculatory level. The possible relevance of Lp(a) levels for the hemorheologic effects of lovastatin remains to be elucidated.
pidemiologic and clinical studies indicate that E hyperlipoproteinemias are associated with alterations in hemostasis and hemorheologic parameters.
which is largely determined by fibrinogen) and total cholesterol, as well as apo-protein B.5 Lowe et a1.6observed elevated alpha-2 antiplasmin levels and fibrinogen values in patients with hyperliIn the Northwick Park Heart Study,' a correlation poproteinemia type 11, whereas other hemostatic between factor VII, fibrinogen, and total cholesterol variables such as alpha-2 macroglobulin and factor could be shown. The Gothenburg Study's3 reported a VIII c showed a tendency to increased values, which, statistically significant positive relationship between however, was not statistically significant from agefibrinogen and total cholesterol, and a negative assoand sex-matched controls. In their study, blood visciation between fibrinolytic activity and triglyceride levels. More recently, in the Framingham ~ o h o r t , ~ cosity at different shear rates, PV, and hematocrit also were elevated in hyperlipidemic patients, high total cholesterol levels also were found to be whereas red blood cell (RBC) deformability was not related to elevated fibrinogen values. On analyzing different from controls. The same group also redata from a large population sample, we found a close correlation between plasma viscosity (PV; ported increased plasma fibrinogen and increased platelet aggregation in type I1 hyperlip~proteinemia.~ Miller et al.' demonstrated a positive correlation From the Department of Internal Medicine IV and Section of Pharmacobetween dietary fat intake and factor VII c levels on therapy (Drs. Koenig, Hehr, Ditschuneit, Kuhn, Rosenthal, and Homthe following day. Patients who showed increased bach), Ulm University Medical Center, Ulm, Federal Republic of Gertriglyceride levels 8 hours after a standardized meal many; and the Department of Physical Medicine and Rehabilitation also presented with increased factor VII c levels as (Dr. Ernst), University of Vienna, AKH. Vienna, Austria. Address for compared with patients with normal triglyceride mereprints: Wolfgang Koenig. MD. Department of Internal Medicine IV, t a b o l i ~ m .A ~ carbo-hydrate-reduced, fat-modified University of Ulm, Robert-Koch-Str. 8, D-7900 Ulm/Federal Republic diet in men with hyperlipidemia resulted in a signifiof Germany.
J Clin Pharmacol 1992;32:539-545
from the SAGE Social Science Collections. All Rights Resewed.
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KOENIG ET AL
cant fall in mean levels of clotting factors VII, VIII, and X, and a rise in fibrinolytic activity.'O Other researchers found elevated PV in various forms of hyperlip~proteinemias."-'~ Weihmayr, Ernst, and Matrail4 confirmed blood viscosity to be increased in hyperlipidemic patients and also found raised RBC aggregation. Thus, hemostasis or blood rheology seems to be pathologically altered in hyperlipidemic patients. This abnormality may increase their risk for arterial vascular complications through an as yet unknown interaction of these factors in the pathogenesis of atherosclerosis. By improving hemostatic and hemorheologic parameters through lipid-lowering drugs, a clinically favorable effect may occur. This report deals with the rheologic effects of lovastatin, a hydroxymethylglutaryl coenzyme A (HMG-CoA) reductase inhibitor, in patients with primary hyperlipoproteinemia.
METHODS Patients
Thirty-five patients with primary hyperlipoproteinemia, 1 7 women and 18 men, mean age 47.4 years, were included in the study after all lipid-lowering drugs had been discontinued and the patients were observed for 4 weeks on a standard lipid-lowering diet [American Heart Association diet recommended) including the administration of placebo tablets. Minimum lipid criteria was total cholesterol of at least 250 mg/dL. Exclusion criteria were: women of child-bearing age, age > 70 or 4 8 years, plasma triglycerides > 260 mg/dL or hyperlipidemias type I, 111, IV, or V, history of alcohol abuse, impaired liver function, unstable angina, myocardial infarction or coronary bypass surgery within the previous 4 months, malignant ventricular arrhythmias, diabetes mellitus, secondary hypercholesterolemia, partial ileal bypass, biliary obstruction, or symptoms or history of gallstones and finally known hypersensitivity to HMG-CoA reductase inhibitors. No other lipid-lowering drugs or hemorheologically active compounds were allowed. All patients gave informed consent. Twenty-six patients had hyperlipoproteinemia type IIa according to Fredrickson, and nine were type IIb. Four patients were regular smokers and 11 had a history of coronary artery disease. The average body mass index was 23.9 kg/m2 in women and 27.0 kg/m2 in men. Study Design
The study was part of a single-blind, randomized, comparative trial to evaluate the safety, tolerability,
540
0
J Clin Pharmacol 1992;32:539-545
and efficacy of lovastatin and bezafibrate sustained release in the treatment of hypercholesterolemia. Before initiation of drug treatment and in 4-week intervals, lipids, lipoproteins, safety parameters, and rheologic variables were determined. The initial dose was 20 mg lovastatin, given once daily. If necessary, dosage was increased stepwise up to a minimum of 80 mg/day. The median daily dose was 40 mg. Comparisons were made between baseline, and 3 months after therapy. The results of the lovastatin subgroup are reported. Blood Sampling
Blood was sampled according to the recommendations of the International Committee for Standardization in Haernatol~gy.'~ Blood was drawn by an experienced nurse with a 19-gauge cannula after patients had been sitting for at least 10 minutes. Only shortterm venous occlusion and minimal suction were applied. Samples for hemorheologic determinations were anticoagulated with 1.5 mg/mL ethylenediaminetetra-acetic acid. Analytical Procedures
Total cholesterol and triglycerides were determined enzymatically (Boehringer Mannheim Kit). Highdensity lipoprotein (HDL) cholesterol was determined by the PTA/MgCl, precipitation method (Boehringer Mannheim). Low-density lipoprotein (LDL) cholesterol was calculated by Friedewald's formula (LDL-C = TC - TG/5 - HDL-C)." Apolipoproteins Al, A2, and B were quantified by Mancini's and Carbonara's radial immunodiffusion method" using plates with polyclonal antibodies (IMMUNO, Wien, Austria). Lipoprotein(a) also was assessed according to Mancini's method followed by electrophoresis." Within 2 hours after blood collection, the following rheologic measurements were assessed in triplicate: whole blood viscosity (BV) at three different shear stresses and 37OC, firstly at native hematocrit, and secondly at 45% hematocrit using a rotational viscometer (Controlled Shear Stress Rheometer, Carrimed, Dorking, England), PV by a rolling-ball viscometer, at 37°C (Haake microviscometer, KPrlsruhe, FRG), RBC aggregation photometrically ,483C;A; MA 01 aggisgometer, Myrenne, Roetgen, FRG) and RBC deformability using the St. George's Filtrometer (Carrimed, Dorking England). Five-micron polycarbonate filters of the same batch (Nuclepore Corp., Pleasanton, USA), 20% hematocrit. and a constant pressure head of 5 cm H,O were used for filtration. The RBC transit time was taken as a mea-
BLOOD RHEOLOGY AFTER LOVASTATIN
sure of RBC deformability. All studies were done at room temperature if not indicated otherwise. Coefficients of variation for repeated measurements were 6.7%. 3.3%, 2.1% for BV, 1.4% for Hct, 0.7% for PV, 10.3% for RBCA, and 5.9% for RBC transit time. Statistics Results are expressed as means and their standard errors. Paired Students t tests were used for statistical analysis of the differences between baseline values and after 3 months of treatment. Unpaired t tests for unequal variances were used for subgroup analyses. All calculations were done with the statistical program BMDP (Statistical Software, Inc., Los Angeles. CA). RESULTS Changes in Plasma Lipids and Lipoproteins Table I presents plasma lipids and lipoproteins before and after 3 months of therapy with lovastatin. The treatment led to a substantial decrease in total cholesterol (-25%) and an even more pronounced decrease in LDL cholesterol (-36%). Triglyceride levels decreased moderately by -12% (P = .09). Highdensity lipoprotein cholesterol showed a small trend to raised levels (+7%). Apoprotein B decreased markedly (-21%), and apoprotein A2 was lowered slightly by -9% (P = .0526). Apoprotein A1 values remained essentially unchanged. In 24 patients, lipoprotein Lp(a) was measured. During therapy with lovastatin there was no significant change. On stratifying patients by their Lp(a) levels (cut-point 25 mg/dL), the following results were obtained: in both subgroups, total cholesterol
decreased approximately to the same extent. Lowdensity lipoprotein cholesterol decreased more markedly in those with low Lp(a) levels (-35%) compared with the group with high levels (-28%). This difference was not statistically significant, however. Highdensity lipoprotein cholesterol increased in patients with low Lp(a) by 12% but decreased in the group with high Lp(a) levels by 15% (difference: P = .0885). Triglycerides were lowered to the same degree in both groups. Apoprotein A1 remained essentially unchanged, apoprotein A2 decreased by lo%, and apoprotein B decreased more pronouncedly (-20%) in patients with high Lp(a) than in those with low Lp(a) (-15%) (difference: P = .2057). Changes in Hemorheologic Parameters Table I1 presents hemorheologic parameters in the total study population before and after therapy with lovastatin. Plasma viscosity decreased significantly by 4%, whereas fibrinogen remained unchanged. Whole blood viscosity at three different shear stresses decreased moderately (-TO%, -7%, -6%). Red blood cell aggregation and deformability showed no significant changes during therapy. Table 111 presents the hemorheologic changes in patients with high Lp(a) (>25 mg/dL) and in those with low Lp(a) levels (125 mg/dL). The decrease of whole blood viscosity at low and medium shear stresses was slightly more pronounced in patients with low Lp(a) as compared with those with high levels. The difference, however, was not statistically significant. Plasma viscosity decreased in both subgroups, but statistical significance was only attained in those with high Lp(a) levels. Contrasting effects were observed for RBC aggregation and deformability. In patients with high Lp(a),these parameters de-
Lipids/Lipoproteins Before and After Treatment with Lovastatin (Mean, SEM) Variable
Mean Before
Mean After
Mean Difference
SE Mean Difference
Total cholesterol (mg/dL) HDL-cholesterol (mg/d L) LDL-cholesterol (mg/dL) Trig1ycerides (mg/d L) Apoprotein A 1 (mg/dL) Apoprotein A 2 (mg/dL) Apoprotein B (mg/dL) Lipoprotein Lp (a)* (rng/dL)
326 2 12 50+ 4 2 4 8 2 15 1552 9 1702 8 65? 4 122? 6 9.5
244 2 8 54+ 4 1592 8 1362 9 1 7 6 2 11 59+ 3 962 4 6
-82 +4 - 89 - 19 +6 +6 -26 0
8 4 12 9 10
* Measured in 24 patients only; median
SEM
=
3 4
P
% Change
0.0000 0.3066 0.0000 0.0900 0.543 1 0.0526 0.0010
-25 +7 -36 -12 +4
-9 -21
Standard error of the mean.
~~~
ARTERIOSCLEROSIS AND LIPID-LOWERING DRUGS
54 1
KOENIG ET AL
TABLE II Blood Rheology Before and After Treatment with Lovastatin (Mean, SEM, N Variable
BV 168.6(mPa.s) BV 421.5(mPa-s) BV 1686.6(mPa-s) Hct (%) PV (mPa.s) EA (units) RCTT (units) Fibrinogen (g/L) ~. --
Mean Before
Mean After
Mean Difference
SE Mean Difference
8.0 f 0.3 5.6 t 0.2 4.5 f 0.1 46.6 t 1.1 1.34t 0.01 3.3 t 0.2 14.4 t 0.7 2.5 k 0.2
7.2 f 0.3 5.2 f 0.1 4.2 f 0.1 47.6 f 1.2 1.29 0.01 3.2 t 0.2 13.7 t 0.4 2.5 2 0.1
0.8 -0.4 -0.3 t-1.0 -0.05 -0.1 -0.7
0.4 0.2 0.1 1.4 0.01 0.3 0.8
-
*
* BV Whole blood viscosity at 45% hematocrit; three different shear stresses; Hct cell transit time SEM Standard error of the mean
teriorated during therapy, whereas in those with low I,p(a), a substantial improvement was observed. Because of decreasing numbers in the subgroup with high Lp(a) (n = 8),differences did not reach a formal level of significance (P = .1199 and .1326, respect ivel y ) .
DISCUSSION Hydroxymethylglutaryl coenzyme A reductase inhibitors are the most potent cholesterol-lowering
~
hematocrit. PV
~
plasma viscos#ty.€A
=
35)
P
0.0552 0.0710 0.0574 0.4823 0.0004 0.7391 0.3836 0.8580 ~
% Change
--lo -- 7 -6 +2 -4 -3 -5
erythrozyte-aggregation.RCTT
=
red
drugs known today. The decrease of plasma lipids and lipoproteins after 3 months of treatment with lovastatin was comparable to data from other ~tudies.'~-~~ In accordance with several a u t h o r ~ ,we ~ ~demon-~~ strated a consistent decrease of PV. The extent of the decrease was of the same magnitude as that reported by these authors. The hematocrit standardized whole blood viscosity showed a decrease at all shear stresses that was of borderline statistical significance. No relevant change was noted for hematocrit.
TABLE 111 Hemorheological Parameters Stratified by Lipoprotein Lp(a) >25 mg/dl (n (n = 16, Below) (Mean, SEM, N = 24) Variable
BV 168.6(mPa-s) BV 421.5(mPa-s) BV 1686.6(mPa-s) Hct (%)
-
PV (mPa s) EA (units) RCTT (units) Fibrinogen (g/L) SEM
542
=
0
=
8, Above) and Lp(a) ~ 2 mg/dl 5
Mean Before
Mean After
Mean Difference
SE Mean Difference
8.4 f 0.8 7.8 f 0.5 5.8 f 0.4 5.6 f 0.3 4.5 f 0.2 4.4 0.2 44.2 f 0.8 47.5 f 2.2 1.35 0.02 1.34f 0.02 3.5 f 0.3 3.4 f 0.3 13.0 1.4 15.2 5 1.3 2.5 f 0.3 2.9 f 0.4
7.9 k 0.4 6.9 f 0.3 5.6 0.2 5.1 f 0.1 4.3 f 0.1 4.2 f 0.1 41.6 f 0.5 49.0 f 1.7 1.26f 0.02 1.29? 0.02 4.1 f 0.7 2.8 f 0.2 15.1 ? 1.0 13.7 f 0.5 2.5 ? 0.3 2.7 f 0.3
-0.5 -0.9 -0.3 -0.4 -0.2 -0.2 -2.6 +1.5 -0.09 -0.05 +0.6 -0.6 +2.1 -1.5
0.9 0.7 0.5 0.4 0.2 0.2 0.7 2.5 0.03 0.02 0.8 0.4
-0.2
* *
*
*
Standard error of the mean; for other explanations see Table /I.
J Clin Pharrnacol 1992:32:539-545
P
% Change
-6 -11 -4 -8 -5 -5 -15 +6
1.4
0.6105 0.2087 0.6155 0.2686 0.1771 0.3847 0.0558 0.5457 0.011 1 0.0902 0.4714 0.0957 0.0890 0.3162
0.3
0.5383
-6
1 .o
-7 -3 +17 - 19
+ 16 - 10
BLOOD RHEOLOGY AFTER LOVASTATIN
RBC aggregation, deformability, and for fibrinogen, when the data of the total study population were analyzed. On stratifying 24 patients for their Lp(a) levels, a slightly more pronounced decrease of whole blood viscosity was seen in patients with low Lp(a) as compared with those with high levels of Lp(a). Plasma viscosity was reduced in both groups during therapy. Red blood cell aggregation and deformability decreased substantially in patients with low Lp(a) and presented with a pronounced increase in those with high levels of Lp(a). These data are intriguing because studies investigating hemorheologic effects of lipid-lowering drugs in relation to Lp(a) levels, to our knowledge, have not been published so far. Lipoprotein Lp(a) is a strong, genetically deterand therapeutically frequently refractoryz8marker of premature atherosclerosis. The lipid composition of Lp(a) is similar to LDL, and both contain a molecule of apoprotein B. Lipoprotein Lp(a), however, consists of a further protein, apoprotein (a). Lipoprotein Lp(a) is synthesized by the liver"; so far its metabolism has not been elucidated. Krempler et al.30demonstrated that the metabolism of Lp(a) is independent of other lipoproteins. The physiologic relevance of Lp(a) remains to be determined.26Apoprotein (a) modifies the structural properties of LDL particles to which they are linked.26 Possible atherogenic effects of Lp(a) may consist in the interference of apoprotein (a) with the uptake of particles containing apoprotein B 100 by the LDL receptor or through homology of apoprotein (a) with plasminogen. Furthermore, plasminogen specifically binds to human endothelial cells.31Because plasminogen induces fibrinolysis by binding to fibrin, it is suggested that Lp(a)also binds to fibrin by nature of the homology of apo (a) with plasminogen. This binding may lead to inhibition of fibrinolysis by competitive displacement of plasminogen. Lipoprotein Lp(a) is able to bind to plasminogen receptors of the endothelial ce11.32.33 Conceivably, Lp(a) represents the link between lipid metabolism and the hemostatic system. The beneficial effects of lovastatin on blood fluidity found in the entire study population were a reduction of both PV and whole blood viscosity. Theoretically, an improvement of the membrane properties of RBCs might have been expected because of the pronounced decrease of plasma lipids and the possible change of the cholesterol/phospholipid ratio of the membrane lipoproteins. Levy et al.34 showed just such changes for the platelet membrane. The effects of lovastatin on RBC deformability, however, have been reported c o n t r o ~ e r s i a l l y .In ~ ~this *~~ study we only demonstrated a slightly positive effect
ARTERIOSCLEROSIS AND LIPID-LOWERING DRUGS
on RBC fluidity in patients with low Lp(a).The same was true for RBC aggregation. Other lipid-lowering drugs investigated for their hemorheologic effects include f e n ~ f i b r a t e , ~beza~,~' fibrate37'38and gernfibro~il.~'The results of these studies are partly controversial, most probably because of a lack of comparability of the study populations and differences in hemorheologic methods. In most of these studies, however, favorable effects on blood rheology were found. In the present study, the decrease of PV in the presence of unchanged fibrinogen could be attributed to a direct effect of the decreased plasma lipids/lipoprotein levels. A concentration-dependent increase of PV has been shown for triglycerides and chylomicrons" and for very low-density lipoprotein (VLDL) and LDL chole~tero1."~~~ Although HMG-CoA reductase inhibitors decrease LDL cholesterol more markedly than fibrates, both compounds lead to a comparable decrease in PV. This can be explained by the concomitant reduction of fibrinogen by fib rate^.^^,^^ Clearly, our results have to be confirmed in further controlled trials. In particular, the possible relevance of Lp(a) levels for the hemorheologic effects of HMGCoA reductase inhibitors remains to be elucidated. A partial explanation of this phenomenon may be attributed to the behavior of HDL cholesterol, which increased in patients with low Lp(a)but decreased in those with high values of LP(a). Ruhenstroth et al.41 recently reported a highly significant negative correlation between the erythrocyte aggregation value and serum concentrations of HDL cholesterol. Interactions of Lp(a) with fibr~nectin,~' a high-molecular weight, rheologically active glycoprotein, or with p r o t e ~ g l y c a n s might , ~ ~ further influence the endothelial cell surface behavior. Finally, its interference with the specific binding of LDL to cell surface receptors, in the presence of markedly decreased LDL plasma levels during therapy with HMG-CoA reductase inhibitors, might be of importance for the pathophysiologic meaning of Lp(a) in this context. In summary, treatment of hyperlipoproteinemia with lovastatin may not only reduce the atherosclerotic risk by its pronounced decrease of LDL cholester01,~ but ~ also could alter favorably blood rheology. On the microcirculatory level, this might decrease insudation of plasmatic components into the arterial wall. REFERENCES 1. Meade TW, Brozovic M, Chakrabarti RR, Haines AP, Imeson JD, Mellows S, Miller GI. North WRS, Stirling Y. Thompson SG: Haemostatic function and ischaemic heart disease: Principal results of the Northwick Park Heart Study. Lancet 1986;ii:533-537.
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BLOOD RHEOLOGY AFTER LOVASTATIN Sarno A: Blood rheology changes during bezafibrate treatment. Br 1 C h Pracl 1988:42:456-458. 38. Niort G. Bulgarelli A. Cassader M. Pagano G: Effect of shortterm treatment with bezafibrate on plasma fibrinogen, fibrinopeptide A, platelet activation and blood filterability in atherosclerotic hyperfibrinogenemic patients. Atherosclerosis 1988; 71:113-119. 39. Ciuffetti G . Orecchini C. Siepi D. Lupattelli G. Ventura A: llcniorheological aclivitv of gernfihrozil in primary hyperlipidcmias. in: Paoletti K (ed.): I h g s Affccfing Lipid Metabolism. Berlin: Springer-\’erlag. 1987:372-375. 40. Schuff-Werner P. Schutz E. Seidel D:LD1.-cholesterol, an underestimated determinant of plasma viscosity? Clin Haemorheol 1989:9:525. 41. Ruhenstroth-Bauer G, Mossmer G, Ottl J. Koenig-Erich S. Heinemann G: Highly significant negative correlations between
ARTERIOSCLEROSIS AND LIPID-LOWERING DRUGS
erythrocyte aggregation value and serum concentration of high density lipoprotein cholesterol in a sample from a normal population and in patients with coronary heart disease. Eur 1 Clin Invest 1987: 17:275-279. 42. Ehnholm C, Jauhiainen M, Metso J: Interaction of lipoprotein(a) with fibronectin and its potential role in atherogenesis. Eur Heart j199O;ll(Suppl E):190-195. 43. Kostner GM. Bihari-Varga M: Is the atherogenecity of Lp(a) caused by its reactivity with proteoglycans? Eur Heart 1990: 1l(Supp1 E):184-189. 44. Brown G, Albers JJ. Fischer LD. Schaefer SM. I.in I-T. Kaplan Ch. Zhao X-Q. Bisson BD. Fitzpatrick VF. Dodge HT: Kegression of coronary artery disease as a result of intensive lipid-lowering therapy in men with high levels of apolipoprotein B. N Engl j Med 1990:323:1289-1 298.
545
As part of a randomized, single-blind, comparative study evaluating the eficacy oflovas-
tatin and bezafibrate retard in the treatment of primary hypercholesterolemia, hemorheologic parameters (whole blood viscosity, hematocrit, plasma viscosity, red blood cell aggregation and deformability, and fibrinogen) were studied in 35 patients. Whole blood viscosity and plasma viscosity improved significantly after 3 months of treatment with lovastatin, whereas other hemorheologic variables remained unchanged. Stratifying 24 patients by their lipoprotein Lp(a) levels showed that in those with lowLp(a) (525 mg/dL) high-density lipoprotein cholesterol increased and red blood cell aggregation as well as deformability decreased considerably, whereas in the group with high Lp(a) levels (>25 mg/dL), the opposite behavior was observed. Treatment of primary hypercholesterolemia with lovastatin may not only reduce the risk for atherosclerotic complications by its pronounced decrease of low-density lipoprotein cholesterol, but also may favorably alter blood rheology, and may decrease insudation of plasmatic components into the arterial wall and improve tissue perfusion, in particular on the microcirculatory level. The possible relevance of Lp(a) levels for the hemorheologic effects of lovastatin remains to be elucidated.
pidemiologic and clinical studies indicate that E hyperlipoproteinemias are associated with alterations in hemostasis and hemorheologic parameters.
which is largely determined by fibrinogen) and total cholesterol, as well as apo-protein B.5 Lowe et a1.6observed elevated alpha-2 antiplasmin levels and fibrinogen values in patients with hyperliIn the Northwick Park Heart Study,' a correlation poproteinemia type 11, whereas other hemostatic between factor VII, fibrinogen, and total cholesterol variables such as alpha-2 macroglobulin and factor could be shown. The Gothenburg Study's3 reported a VIII c showed a tendency to increased values, which, statistically significant positive relationship between however, was not statistically significant from agefibrinogen and total cholesterol, and a negative assoand sex-matched controls. In their study, blood visciation between fibrinolytic activity and triglyceride levels. More recently, in the Framingham ~ o h o r t , ~ cosity at different shear rates, PV, and hematocrit also were elevated in hyperlipidemic patients, high total cholesterol levels also were found to be whereas red blood cell (RBC) deformability was not related to elevated fibrinogen values. On analyzing different from controls. The same group also redata from a large population sample, we found a close correlation between plasma viscosity (PV; ported increased plasma fibrinogen and increased platelet aggregation in type I1 hyperlip~proteinemia.~ Miller et al.' demonstrated a positive correlation From the Department of Internal Medicine IV and Section of Pharmacobetween dietary fat intake and factor VII c levels on therapy (Drs. Koenig, Hehr, Ditschuneit, Kuhn, Rosenthal, and Homthe following day. Patients who showed increased bach), Ulm University Medical Center, Ulm, Federal Republic of Gertriglyceride levels 8 hours after a standardized meal many; and the Department of Physical Medicine and Rehabilitation also presented with increased factor VII c levels as (Dr. Ernst), University of Vienna, AKH. Vienna, Austria. Address for compared with patients with normal triglyceride mereprints: Wolfgang Koenig. MD. Department of Internal Medicine IV, t a b o l i ~ m .A ~ carbo-hydrate-reduced, fat-modified University of Ulm, Robert-Koch-Str. 8, D-7900 Ulm/Federal Republic diet in men with hyperlipidemia resulted in a signifiof Germany.
J Clin Pharmacol 1992;32:539-545
from the SAGE Social Science Collections. All Rights Resewed.
539
KOENIG ET AL
cant fall in mean levels of clotting factors VII, VIII, and X, and a rise in fibrinolytic activity.'O Other researchers found elevated PV in various forms of hyperlip~proteinemias."-'~ Weihmayr, Ernst, and Matrail4 confirmed blood viscosity to be increased in hyperlipidemic patients and also found raised RBC aggregation. Thus, hemostasis or blood rheology seems to be pathologically altered in hyperlipidemic patients. This abnormality may increase their risk for arterial vascular complications through an as yet unknown interaction of these factors in the pathogenesis of atherosclerosis. By improving hemostatic and hemorheologic parameters through lipid-lowering drugs, a clinically favorable effect may occur. This report deals with the rheologic effects of lovastatin, a hydroxymethylglutaryl coenzyme A (HMG-CoA) reductase inhibitor, in patients with primary hyperlipoproteinemia.
METHODS Patients
Thirty-five patients with primary hyperlipoproteinemia, 1 7 women and 18 men, mean age 47.4 years, were included in the study after all lipid-lowering drugs had been discontinued and the patients were observed for 4 weeks on a standard lipid-lowering diet [American Heart Association diet recommended) including the administration of placebo tablets. Minimum lipid criteria was total cholesterol of at least 250 mg/dL. Exclusion criteria were: women of child-bearing age, age > 70 or 4 8 years, plasma triglycerides > 260 mg/dL or hyperlipidemias type I, 111, IV, or V, history of alcohol abuse, impaired liver function, unstable angina, myocardial infarction or coronary bypass surgery within the previous 4 months, malignant ventricular arrhythmias, diabetes mellitus, secondary hypercholesterolemia, partial ileal bypass, biliary obstruction, or symptoms or history of gallstones and finally known hypersensitivity to HMG-CoA reductase inhibitors. No other lipid-lowering drugs or hemorheologically active compounds were allowed. All patients gave informed consent. Twenty-six patients had hyperlipoproteinemia type IIa according to Fredrickson, and nine were type IIb. Four patients were regular smokers and 11 had a history of coronary artery disease. The average body mass index was 23.9 kg/m2 in women and 27.0 kg/m2 in men. Study Design
The study was part of a single-blind, randomized, comparative trial to evaluate the safety, tolerability,
540
0
J Clin Pharmacol 1992;32:539-545
and efficacy of lovastatin and bezafibrate sustained release in the treatment of hypercholesterolemia. Before initiation of drug treatment and in 4-week intervals, lipids, lipoproteins, safety parameters, and rheologic variables were determined. The initial dose was 20 mg lovastatin, given once daily. If necessary, dosage was increased stepwise up to a minimum of 80 mg/day. The median daily dose was 40 mg. Comparisons were made between baseline, and 3 months after therapy. The results of the lovastatin subgroup are reported. Blood Sampling
Blood was sampled according to the recommendations of the International Committee for Standardization in Haernatol~gy.'~ Blood was drawn by an experienced nurse with a 19-gauge cannula after patients had been sitting for at least 10 minutes. Only shortterm venous occlusion and minimal suction were applied. Samples for hemorheologic determinations were anticoagulated with 1.5 mg/mL ethylenediaminetetra-acetic acid. Analytical Procedures
Total cholesterol and triglycerides were determined enzymatically (Boehringer Mannheim Kit). Highdensity lipoprotein (HDL) cholesterol was determined by the PTA/MgCl, precipitation method (Boehringer Mannheim). Low-density lipoprotein (LDL) cholesterol was calculated by Friedewald's formula (LDL-C = TC - TG/5 - HDL-C)." Apolipoproteins Al, A2, and B were quantified by Mancini's and Carbonara's radial immunodiffusion method" using plates with polyclonal antibodies (IMMUNO, Wien, Austria). Lipoprotein(a) also was assessed according to Mancini's method followed by electrophoresis." Within 2 hours after blood collection, the following rheologic measurements were assessed in triplicate: whole blood viscosity (BV) at three different shear stresses and 37OC, firstly at native hematocrit, and secondly at 45% hematocrit using a rotational viscometer (Controlled Shear Stress Rheometer, Carrimed, Dorking, England), PV by a rolling-ball viscometer, at 37°C (Haake microviscometer, KPrlsruhe, FRG), RBC aggregation photometrically ,483C;A; MA 01 aggisgometer, Myrenne, Roetgen, FRG) and RBC deformability using the St. George's Filtrometer (Carrimed, Dorking England). Five-micron polycarbonate filters of the same batch (Nuclepore Corp., Pleasanton, USA), 20% hematocrit. and a constant pressure head of 5 cm H,O were used for filtration. The RBC transit time was taken as a mea-
BLOOD RHEOLOGY AFTER LOVASTATIN
sure of RBC deformability. All studies were done at room temperature if not indicated otherwise. Coefficients of variation for repeated measurements were 6.7%. 3.3%, 2.1% for BV, 1.4% for Hct, 0.7% for PV, 10.3% for RBCA, and 5.9% for RBC transit time. Statistics Results are expressed as means and their standard errors. Paired Students t tests were used for statistical analysis of the differences between baseline values and after 3 months of treatment. Unpaired t tests for unequal variances were used for subgroup analyses. All calculations were done with the statistical program BMDP (Statistical Software, Inc., Los Angeles. CA). RESULTS Changes in Plasma Lipids and Lipoproteins Table I presents plasma lipids and lipoproteins before and after 3 months of therapy with lovastatin. The treatment led to a substantial decrease in total cholesterol (-25%) and an even more pronounced decrease in LDL cholesterol (-36%). Triglyceride levels decreased moderately by -12% (P = .09). Highdensity lipoprotein cholesterol showed a small trend to raised levels (+7%). Apoprotein B decreased markedly (-21%), and apoprotein A2 was lowered slightly by -9% (P = .0526). Apoprotein A1 values remained essentially unchanged. In 24 patients, lipoprotein Lp(a) was measured. During therapy with lovastatin there was no significant change. On stratifying patients by their Lp(a) levels (cut-point 25 mg/dL), the following results were obtained: in both subgroups, total cholesterol
decreased approximately to the same extent. Lowdensity lipoprotein cholesterol decreased more markedly in those with low Lp(a) levels (-35%) compared with the group with high levels (-28%). This difference was not statistically significant, however. Highdensity lipoprotein cholesterol increased in patients with low Lp(a) by 12% but decreased in the group with high Lp(a) levels by 15% (difference: P = .0885). Triglycerides were lowered to the same degree in both groups. Apoprotein A1 remained essentially unchanged, apoprotein A2 decreased by lo%, and apoprotein B decreased more pronouncedly (-20%) in patients with high Lp(a) than in those with low Lp(a) (-15%) (difference: P = .2057). Changes in Hemorheologic Parameters Table I1 presents hemorheologic parameters in the total study population before and after therapy with lovastatin. Plasma viscosity decreased significantly by 4%, whereas fibrinogen remained unchanged. Whole blood viscosity at three different shear stresses decreased moderately (-TO%, -7%, -6%). Red blood cell aggregation and deformability showed no significant changes during therapy. Table 111 presents the hemorheologic changes in patients with high Lp(a) (>25 mg/dL) and in those with low Lp(a) levels (125 mg/dL). The decrease of whole blood viscosity at low and medium shear stresses was slightly more pronounced in patients with low Lp(a) as compared with those with high levels. The difference, however, was not statistically significant. Plasma viscosity decreased in both subgroups, but statistical significance was only attained in those with high Lp(a) levels. Contrasting effects were observed for RBC aggregation and deformability. In patients with high Lp(a),these parameters de-
Lipids/Lipoproteins Before and After Treatment with Lovastatin (Mean, SEM) Variable
Mean Before
Mean After
Mean Difference
SE Mean Difference
Total cholesterol (mg/dL) HDL-cholesterol (mg/d L) LDL-cholesterol (mg/dL) Trig1ycerides (mg/d L) Apoprotein A 1 (mg/dL) Apoprotein A 2 (mg/dL) Apoprotein B (mg/dL) Lipoprotein Lp (a)* (rng/dL)
326 2 12 50+ 4 2 4 8 2 15 1552 9 1702 8 65? 4 122? 6 9.5
244 2 8 54+ 4 1592 8 1362 9 1 7 6 2 11 59+ 3 962 4 6
-82 +4 - 89 - 19 +6 +6 -26 0
8 4 12 9 10
* Measured in 24 patients only; median
SEM
=
3 4
P
% Change
0.0000 0.3066 0.0000 0.0900 0.543 1 0.0526 0.0010
-25 +7 -36 -12 +4
-9 -21
Standard error of the mean.
~~~
ARTERIOSCLEROSIS AND LIPID-LOWERING DRUGS
54 1
KOENIG ET AL
TABLE II Blood Rheology Before and After Treatment with Lovastatin (Mean, SEM, N Variable
BV 168.6(mPa.s) BV 421.5(mPa-s) BV 1686.6(mPa-s) Hct (%) PV (mPa.s) EA (units) RCTT (units) Fibrinogen (g/L) ~. --
Mean Before
Mean After
Mean Difference
SE Mean Difference
8.0 f 0.3 5.6 t 0.2 4.5 f 0.1 46.6 t 1.1 1.34t 0.01 3.3 t 0.2 14.4 t 0.7 2.5 k 0.2
7.2 f 0.3 5.2 f 0.1 4.2 f 0.1 47.6 f 1.2 1.29 0.01 3.2 t 0.2 13.7 t 0.4 2.5 2 0.1
0.8 -0.4 -0.3 t-1.0 -0.05 -0.1 -0.7
0.4 0.2 0.1 1.4 0.01 0.3 0.8
-
*
* BV Whole blood viscosity at 45% hematocrit; three different shear stresses; Hct cell transit time SEM Standard error of the mean
teriorated during therapy, whereas in those with low I,p(a), a substantial improvement was observed. Because of decreasing numbers in the subgroup with high Lp(a) (n = 8),differences did not reach a formal level of significance (P = .1199 and .1326, respect ivel y ) .
DISCUSSION Hydroxymethylglutaryl coenzyme A reductase inhibitors are the most potent cholesterol-lowering
~
hematocrit. PV
~
plasma viscos#ty.€A
=
35)
P
0.0552 0.0710 0.0574 0.4823 0.0004 0.7391 0.3836 0.8580 ~
% Change
--lo -- 7 -6 +2 -4 -3 -5
erythrozyte-aggregation.RCTT
=
red
drugs known today. The decrease of plasma lipids and lipoproteins after 3 months of treatment with lovastatin was comparable to data from other ~tudies.'~-~~ In accordance with several a u t h o r ~ ,we ~ ~demon-~~ strated a consistent decrease of PV. The extent of the decrease was of the same magnitude as that reported by these authors. The hematocrit standardized whole blood viscosity showed a decrease at all shear stresses that was of borderline statistical significance. No relevant change was noted for hematocrit.
TABLE 111 Hemorheological Parameters Stratified by Lipoprotein Lp(a) >25 mg/dl (n (n = 16, Below) (Mean, SEM, N = 24) Variable
BV 168.6(mPa-s) BV 421.5(mPa-s) BV 1686.6(mPa-s) Hct (%)
-
PV (mPa s) EA (units) RCTT (units) Fibrinogen (g/L) SEM
542
=
0
=
8, Above) and Lp(a) ~ 2 mg/dl 5
Mean Before
Mean After
Mean Difference
SE Mean Difference
8.4 f 0.8 7.8 f 0.5 5.8 f 0.4 5.6 f 0.3 4.5 f 0.2 4.4 0.2 44.2 f 0.8 47.5 f 2.2 1.35 0.02 1.34f 0.02 3.5 f 0.3 3.4 f 0.3 13.0 1.4 15.2 5 1.3 2.5 f 0.3 2.9 f 0.4
7.9 k 0.4 6.9 f 0.3 5.6 0.2 5.1 f 0.1 4.3 f 0.1 4.2 f 0.1 41.6 f 0.5 49.0 f 1.7 1.26f 0.02 1.29? 0.02 4.1 f 0.7 2.8 f 0.2 15.1 ? 1.0 13.7 f 0.5 2.5 ? 0.3 2.7 f 0.3
-0.5 -0.9 -0.3 -0.4 -0.2 -0.2 -2.6 +1.5 -0.09 -0.05 +0.6 -0.6 +2.1 -1.5
0.9 0.7 0.5 0.4 0.2 0.2 0.7 2.5 0.03 0.02 0.8 0.4
-0.2
* *
*
*
Standard error of the mean; for other explanations see Table /I.
J Clin Pharrnacol 1992:32:539-545
P
% Change
-6 -11 -4 -8 -5 -5 -15 +6
1.4
0.6105 0.2087 0.6155 0.2686 0.1771 0.3847 0.0558 0.5457 0.011 1 0.0902 0.4714 0.0957 0.0890 0.3162
0.3
0.5383
-6
1 .o
-7 -3 +17 - 19
+ 16 - 10
BLOOD RHEOLOGY AFTER LOVASTATIN
RBC aggregation, deformability, and for fibrinogen, when the data of the total study population were analyzed. On stratifying 24 patients for their Lp(a) levels, a slightly more pronounced decrease of whole blood viscosity was seen in patients with low Lp(a) as compared with those with high levels of Lp(a). Plasma viscosity was reduced in both groups during therapy. Red blood cell aggregation and deformability decreased substantially in patients with low Lp(a) and presented with a pronounced increase in those with high levels of Lp(a). These data are intriguing because studies investigating hemorheologic effects of lipid-lowering drugs in relation to Lp(a) levels, to our knowledge, have not been published so far. Lipoprotein Lp(a) is a strong, genetically deterand therapeutically frequently refractoryz8marker of premature atherosclerosis. The lipid composition of Lp(a) is similar to LDL, and both contain a molecule of apoprotein B. Lipoprotein Lp(a), however, consists of a further protein, apoprotein (a). Lipoprotein Lp(a) is synthesized by the liver"; so far its metabolism has not been elucidated. Krempler et al.30demonstrated that the metabolism of Lp(a) is independent of other lipoproteins. The physiologic relevance of Lp(a) remains to be determined.26Apoprotein (a) modifies the structural properties of LDL particles to which they are linked.26 Possible atherogenic effects of Lp(a) may consist in the interference of apoprotein (a) with the uptake of particles containing apoprotein B 100 by the LDL receptor or through homology of apoprotein (a) with plasminogen. Furthermore, plasminogen specifically binds to human endothelial cells.31Because plasminogen induces fibrinolysis by binding to fibrin, it is suggested that Lp(a)also binds to fibrin by nature of the homology of apo (a) with plasminogen. This binding may lead to inhibition of fibrinolysis by competitive displacement of plasminogen. Lipoprotein Lp(a) is able to bind to plasminogen receptors of the endothelial ce11.32.33 Conceivably, Lp(a) represents the link between lipid metabolism and the hemostatic system. The beneficial effects of lovastatin on blood fluidity found in the entire study population were a reduction of both PV and whole blood viscosity. Theoretically, an improvement of the membrane properties of RBCs might have been expected because of the pronounced decrease of plasma lipids and the possible change of the cholesterol/phospholipid ratio of the membrane lipoproteins. Levy et al.34 showed just such changes for the platelet membrane. The effects of lovastatin on RBC deformability, however, have been reported c o n t r o ~ e r s i a l l y .In ~ ~this *~~ study we only demonstrated a slightly positive effect
ARTERIOSCLEROSIS AND LIPID-LOWERING DRUGS
on RBC fluidity in patients with low Lp(a).The same was true for RBC aggregation. Other lipid-lowering drugs investigated for their hemorheologic effects include f e n ~ f i b r a t e , ~beza~,~' fibrate37'38and gernfibro~il.~'The results of these studies are partly controversial, most probably because of a lack of comparability of the study populations and differences in hemorheologic methods. In most of these studies, however, favorable effects on blood rheology were found. In the present study, the decrease of PV in the presence of unchanged fibrinogen could be attributed to a direct effect of the decreased plasma lipids/lipoprotein levels. A concentration-dependent increase of PV has been shown for triglycerides and chylomicrons" and for very low-density lipoprotein (VLDL) and LDL chole~tero1."~~~ Although HMG-CoA reductase inhibitors decrease LDL cholesterol more markedly than fibrates, both compounds lead to a comparable decrease in PV. This can be explained by the concomitant reduction of fibrinogen by fib rate^.^^,^^ Clearly, our results have to be confirmed in further controlled trials. In particular, the possible relevance of Lp(a) levels for the hemorheologic effects of HMGCoA reductase inhibitors remains to be elucidated. A partial explanation of this phenomenon may be attributed to the behavior of HDL cholesterol, which increased in patients with low Lp(a)but decreased in those with high values of LP(a). Ruhenstroth et al.41 recently reported a highly significant negative correlation between the erythrocyte aggregation value and serum concentrations of HDL cholesterol. Interactions of Lp(a) with fibr~nectin,~' a high-molecular weight, rheologically active glycoprotein, or with p r o t e ~ g l y c a n s might , ~ ~ further influence the endothelial cell surface behavior. Finally, its interference with the specific binding of LDL to cell surface receptors, in the presence of markedly decreased LDL plasma levels during therapy with HMG-CoA reductase inhibitors, might be of importance for the pathophysiologic meaning of Lp(a) in this context. In summary, treatment of hyperlipoproteinemia with lovastatin may not only reduce the atherosclerotic risk by its pronounced decrease of LDL cholester01,~ but ~ also could alter favorably blood rheology. On the microcirculatory level, this might decrease insudation of plasmatic components into the arterial wall. REFERENCES 1. Meade TW, Brozovic M, Chakrabarti RR, Haines AP, Imeson JD, Mellows S, Miller GI. North WRS, Stirling Y. Thompson SG: Haemostatic function and ischaemic heart disease: Principal results of the Northwick Park Heart Study. Lancet 1986;ii:533-537.
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