Hs-CRP may be associated with white blood cell count in metabolic syndrome patients treated with Ginkgo biloba

Atherosclerosis 218 (2011) 250–252

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Letter to the Editor Hs-CRP may be associated with white blood cell count in metabolic syndrome patients treated with Ginkgo biloba Keywords: Metabolic syndrome patients High-sensitivity C-reactive protein (hs-CRP) Monocytes Eosinophils Ginkgo biloba

To the Editor, In several clinical trials, it has been demonstrated that eosinophil cationic protein (ECP), a sensitive marker of eosinophil activation, is associated with major adverse cardiac events (MACEs) (cardiac death, non-fatal myocardial infarction, in-stent restenosis) after bare metal stent (BMS) [1] and drug-eluting stent (DES) implantation [2], and is implicated in risk prediction for clinical outcome. These findings suggested that allergic inflammation may play a key role in these adverse reactions occurring after stent implantation [1–5]. Furthermore, eosinophils are directly involved in coronary atherosclerosis [1,6] and thus, ECP has been established as a new biomarker [6]. Since we had observed a decrease in nanoplaque formation and size as well as in biomarkers of oxidative stress, plaque formation, stability and progression, and inflammation in a clinical pilot study in metabolic syndrome patients treated with Ginkgo biloba, we revisited these laboratory parameters [7–10]. Thereby, special attention was directed to the number of white blood cells (WBC). A preventional, randomized, 3-month study comprising a 1month dietary run-in phase followed by a study treatment period of 2 months was conducted in the Phase I–II study clinic of the UMHAPT “Zaritza Johanna” University Hospital, Sofia, Bulgaria. The project has been reviewed and approved by the local Ethics Committee and the Bulgarian Drug Agency. Eleven patients (2 male, 9 female) with metabolic syndrome aged 26–48 years were recruited, provided that they fell within the additional inclusion criteria smoking (all 11 patients were smokers) and blood lipoprotein(a) [Lp(a)] concentration >30 mg/dL (9 patients). The inclusion criterion smoking was enclosed to clearly demonstrate the antioxidative effect of ginkgo, and Lp(a) > 30 mg/dL was enclosed to confirm the Lp(a) lowering by 23.4% (p < 0.0234) from the preceding ginkgo study in aortocoronary bypass patients (8 patients) [11]. The standard therapy of the patients was 2 × 120 mg/d G. biloba special extract EGb 761 (Rökan novo® : Spitzner Arzneimittel, Ettlingen; Tebonin® : Schwabe Pharmaceuticals, Karlsruhe, Germany) over 2 months. No statins, no calcium antagonists and no nitrate compounds were given. No adverse events occurred and all the patients felt well during and after ginkgo intake [7]. Nanotechnologic biosensor ellipsometry, photometric methods, commercial ELISAs and EIAs were applied [7,8]. Hematology was 0021-9150/$ – see front matter © 2011 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.atherosclerosis.2011.05.007

elaborated by means of a CELL-DYN® 3700 instrument (Abbott, Wiesbaden, Germany). This apparatus uses combined impedance and MAPSSTM (multi-angle-polarized-scatter-separation) flow cytometry with a 5 mW helium neon laser. Statistical significance of the blood parameter changes was calculated by the paired, twosided Student’s t-test and the Wilcoxon signed-rank test, which is more sensitive than a t-test when the number of values is small. Changes were considered statistically significant at p < 0.05. For linear correlation analysis we calculated Pearson’s correlation coefficient r. Statistical significance was obtained from Student’s  t-distribution using t = r (n − 2)/(1 − r 2 ), where n is the number of data points and n − 2 the degree of freedom. A correlation between two data sets is significant if the two-sided probability p < 0.05 [7–10]. Briefly, the results of biomarker pattern analysis in the blood plasma of the patients shall be summarized as follows: nanoplaque formation was reduced by 14.3% (p < 0.0077), nanoplaque size by 23.4% (p < 0.0004), oxLDL/LDL by 21.0% (p < 0.002), 8-iso-PGF2␣ by 39.8% (p < 0.0027), MPO by 29.6% (p < 0.0137), IL-6 by 12.9% (p < 0.0407), Lp(a) by 26.3% (p < 0.001), MMP-9 by 32.9% (p < 0.042), whereas SOD was augmented by 17.7% (p < 0.0095), GPx by 11.6% (p < 0.001), cAMP by 43.5% (p < 0.001), and cGMP by 32.9% (p < 0.001) [7–10]. Detailed data for lowering in high-sensitivity Creactive protein (hs-CRP), WBC, monocytes (MON) and eosinophils (EOS) are presented in Table 1. As important potential confounders of the reported associations, also age/gender, body mass index (BMI), fasting glucose and triglycerides (TG) are added. The inflammatory status was characterized by hs-CRP, myeloperoxidase (MPO), tumor necrosis factor ␣ (TNF␣), and transforming growth factor ␤1 (TGF␤1 ) [12,13]. Furthermore, matrix metalloproteinase9 (MMP-9) was measured as a relatively new marker to assess plaque stability [14]. MPO contributes to the enzymatic modification of LDL particles in monocytes [15] and is associated with the formation of MMP-9. On the other hand, MMP-9 in patients suffering from an acute coronary syndrome seems to be directly correlated to hs-CRP [16,17]. Thus, the metabolic syndrome status may be characterized by an interconnective network of biomarkers preferably dominated by oxidative stress (ROS) [11,18]. Since ginkgo extract is a strong oxygen free radical scavenger [19,20], the lowering in hs-CRP by 39.3% and consequently in WBC by 7.5% [21,22] in these metabolic syndrome patients after a 2month ginkgo intake was not surprising. The decrease in WBC apparently touched MON and EOS specifically, while lymphocytes (LYM) and granulocytes (GRAN) (not shown) were not affected, an unexpected finding (Table 1). Through multiple correlations between changes in MON and EOS counts with changes in the biomarkers, we tried to unravel a mechanistic explanation. The striking direct correlations to the hs-CRP values are depicted in Fig. 1. It is remarkable that patients 7 and 13 who show the highest baseline hs-CRP values (or rather CRP values) and the highest hs-CRP reductions also demonstrate the highest reductions in

Table 1 Hs-CRP concentration and WBC, MON, EOS and LYM blood cell count in 11 patients with metabolic syndrome before and after 2 months of ginkgo therapy. In addition, baseline values of age/gender, BMI, fasting glucose and TGs are specified. Patient

Age [years]/ gender

BMI [kg/m2 ]

Glucose [mg/dL]

TG [mg/dL]

hs-CRP [mg/L]

WBC [l/nL]

MON [l/nL]

EOS [l/nL]

LYM [l/nL]

Baseline values

Before

After

Change [%]

Before

After

Change [%]

Before

After

Change [%]

Before

After

Change [%]

Before

After

Change [%]

26♀ 47♀ 35♀ 39♀ 43♀ 28♂ 43♀ 48♀ 48♂ 48♀ 32♀

37.52 27.11 39.51 38.23 37.40 45.03 25.93 36.80 28.47 44.15 34.82

98.9 90.3 88.1 114.0 89.2 105.4 81.1 98.4 94.9 103.1 74.9

48 198 118 232 125 126 68 120 402 344 84

1.60 5.35 3.15 4.78 5.47 46.60 3.39 4.93 1.82 0.39 19.87

0.64 2.13 1.55 3.06 1.60 29.29 2.79 4.40 0.74 0.52 7.41

−60.0 −60.2 −50.8 −36.1 −70.8 −37.2 −17.7 −10.8 −59.6 +33.3 −62.7

9.5 7.5 7.8 7.0 5.4 8.5 5.0 13.0 8.5 7.5 7.0

8.9 7.5 6.2 7.1 4.5 7.7 4.6 9.8 8.5 7.6 7.0

−6.3 0.0 −20.5 +1.4 −16.7 −9.4 −8.0 −24.6 0.0 +1.3 0.0

0.190 0.375 0.390 0.350 0.162 0.595 0.246 0.650 0.400 0.300 0.490

0.089 0.150 0.124 0.142 0.045 0.154 0.046 0.098 0.170 0.152 0.243

−53.2 −60.0 −68.2 −59.4 −72.2 −74.1 −81.3 −84.9 −57.5 −49.3 −50.4

0.095 0.075 0.234 0.210 0.054 0.340 0.123 0.260 0.160 0.225 0.280

0.089 0.075 0.124 0.071 0.045 0.077 0.046 0.098 0.170 0.076 0.081

−6.3 0.0 −47.0 −66.2 −16.7 −77.4 −62.6 −62.3 +6.3 −66.3 −71.1

4.1 2.6 2.3 2.1 1.6 2.5 1.3 2.5 2.7 2.9 2.6

3.5 2.7 2.0 2.3 1.5 2.0 1.8 2.9 2.3 3.0 2.7

−14.6 +3.9 −13.0 +9.5 −6.3 −20.0 +38.5 +16.0 −14.8 +3.5 +3.9

Mean (11) ±SEM Median p

39.7 2.5 43

35.91 1.93 37.40

94.4 3.4 94.9

170 34 125

−64.6 3.7 −60.0 <0.001

0.187 0.087 0.028 0.011 0.210 0.077 <0.0046

−42.7 9.6 −62.3 <0.0076

2.5 0.2

2.4 0.2

+0.6 5.1 +3.5 n.s.

8.85 4.92 4.09 2.51 4.78 2.13 <0.0436

−39.3 9.3 −50.8 <0.0049

−7.5 2.8 −6.3 <0.0238

7.9 7.2 0.6 0.5 7.5 7.5 <0.0518

n.s.

A

-16

251

EOS [l/nL]

4

4

MON [l/nL]

r = 0.76 p < 0.0112

0

r = 0.63 p < 0.0383

0

r = 0.63; p < 0.0383 r = 0.60; p < 0.0649 r = −0.65; p < 0.0417 r = −0.62; p < 0.0564 r = −0.64; p < 0.0464 r = 0.79; p < 0.0326

-4

-4

r = 0.76; p < 0.0112 r = 0.72; p < 0.0133 r = −0.63; p < 0.0684 r = −0.65; p < 0.0566 r = −0.61; p < 0.0585 r = 0.71; p < 0.0504

-8

Δ hs-CRP [mg/L]

-12

-8

Δ hs-CRP [mg/L]

-12

Table 2 Linear correlation analysis between changes in the blood concentration of hs-CRP, TNF␣, IL-6, MMP-9, SOD, MPO, and MON and EOS blood cell count. Pearson’s correlation coefficient r and error probability p were calculated.

-20

B

hs-CRP [mg/L] TNF␣ [pg/mL] IL-6 [pg/mL] MMP-9 [ng/mL] SOD [U/mL] MPO [ng/mL]

0.0

-0.1

-0.2

-0.3

-0.4

-0.5

0.0

-0.1

-0.2

-0.3 -16

MON and EOS. Weaker correlations resulted for some further biomarkers of oxidative stress and inflammation (Table 2). Hence, the hs-CRP lowering plays the decisive role. Niccoli et al. [1] could not demonstrate a predictive value of hs-CRP or common predictors for MACEs on clinical outcome. The linkage between baseline hs-CRP and MACEs in BMS patients has

Δ MON [1/nL]

-20

Fig. 1. Significant correlations between changes in MON (A) and EOS blood cell count (B) and changes in hs-CRP concentration. For linear correlation analysis, Pearson’s correlation coefficient r was calculated.

Δ EOS [1/nL]

0.377 0.128 0.047 0.017 0.375 0.142 <0.0001

Letter to the Editor / Atherosclerosis 218 (2011) 250–252

Baseline values 1 2 3 4 6 7 8 9 10 12 13

252

Letter to the Editor / Atherosclerosis 218 (2011) 250–252

been controversially discussed [23,24]. Hs-CRP may have failed to predict MACEs due to concomitant statin therapy [25]. In Niccoli’s study [1], all the patients had received statins, in our metabolic syndrome study, however, not. Furthermore, the changes of MON (reference 0–0.8 nL−1 ) and EOS (reference 0–0.45 nL−1 ) after 2month ginkgo therapy occurred solely within the physiological range of variation. This also holds largely for the correlating cytokines and biomarkers. Just as hs-CRP, TNF␣ and MPO are positively correlated with MON and EOS, even though in part only borderline significant (Table 2). The anti-inflammatory and anti-atherosclerotic effect of ginkgo is thus underlined. The negative correlation with IL-6 and MMP-9, again in the physiological range, is to be seen within the frame of the regulation in cytokine balance [26,27]. More specifically, IL-6 is implicated in the regulation of Lp(a) [28]. In our study, Lp(a) is also negatively correlated to MON (r = −0.30; n.s.) and EOS (r = −0.65; p < 0.0402). Eliminating the dependent variables MON/EOS from the negative correlations MON/EOS – IL-6 and MON/EOS – Lp(a), and consequently plotting the relation Lp(a) – IL-6, then the latter is positively correlated. This means, when the IL-6 lowering is high, also the decrease in Lp(a) is high and vice versa. Thus, the finding raised by Lippi et al. [29] is confirmed that the variation in plasma Lp(a) concentration is accounted for by inherited sequences within or closely linked to the apo(a) gene and this gene has IL-6 responsive elements within its sequence. Thus, a lowering of Lp(a) is explainable via down-regulation of IL-6 [18]. The matrix metalloproteinase MMP-9 effects a degradation of extracellular connective tissue structures. A high decrease of MMP-9 (extracellular matrix stabilizing) matters an only slight reduction in MON/EOS (negative correlations). Similar to MMP-9 also TGF␤1 (r = −0.81; p < 0.008) is negatively correlated to MON, both of which can thus be interpreted as a beneficial, plaque-stabilizing and fibrous cap-strengthening effect. Finally, SOD is inversely correlated with MON and EOS. That means the higher the SOD-activity the lower is the ROS concentration and thus MON and EOS count in consequence. Although our study allows only limited statements due to the small number of patients enrolled, nevertheless some conclusions can cautiously be drawn. Taking into account the direct association between MON and EOS to atherosclerosis as well as of EOS to allergic inflammation, it can be concluded that ginkgo may be used as complementary drug with potentially preventive character after percutaneous coronary intervention, stent implantation and coronary-artery bypass graft. Conflict of interest

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G. Siegel a,b,∗ Charité-University Clinic Berlin, Institute of Physiology, Thielallee 71, D-14195 Berlin, Germany b University of Uppsala Biomedical Center, SE-751 23 Uppsala, Sweden a

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E. Ermilov Charité-University Clinic Berlin, Institute of Physiology, Thielallee 71, D-14195 Berlin, Germany ∗ Corresponding

author at: Charité-University Clinic Berlin, Institute of Physiology, Thielallee 71, D-14195 Berlin, Germany. Tel.: +49 30 450 52 8521; fax: +49 30 450 52 8930. E-mail address: [email protected] (G. Siegel) 17 March 2011 Available online 14 May 2011