## 4 Discussion  
The current study determined the effect of physiological concentrations of caffeine and 5-CQA on the LPS-induced cytokine production in whole blood cultures. The main findings were that caffeine induces lower TNF-\(\alpha\) in vitro concentrations. These effects were observed for caffeine concentrations that can be attained after ingestion of two to three cups of coffee and suggest a limited anti-inflammatory effect of caffeine. On the other hand, 5-CQA at physiological concentrations did not influence in vitro cytokine-stimulated  
Figure 1: Effects of caffeine and LPS-stimulation on culture cAMP concentrations. Values are expressed as means \(\pm\) SD. # indicates significant differences vs. control. cAMP: Adenosine-3′,5′-cyclic monophosphate. \begin{table}
\begin{tabular}{c c c c c c c c} \hline  & \multicolumn{6}{c}{**5-CQA Concentrations**} & \multicolumn{3}{c}{**ANOVA**} \\ \hline  & Control & 5 nM & 20 nM & 200 nM & 2 uM & 20 uM & \(p\) (\(\eta^{2}\)) \\ \hline IL-10 (pg-10\({}^{-3}\) cells) & 21.2 \(\pm\) 4.5 & 20.8 \(\pm\) 6.6 & 20.2 \(\pm\) 5.7 & 19.2 \(\pm\) 6.8 & 17.4 \(\pm\) 6.2 & 17.8 \(\pm\) 6.5 & 0.438 (0.083) \\ IL-6 (pg-10\({}^{-3}\) cells) & 385.2 \(\pm\) 92.4 & 351.2 \(\pm\) 83.1 & 326.5 \(\pm\) 93.9 & 291.9 \(\pm\) 86.1 & 252.0 \(\pm\) 85.8 \# & 202.2 \(\pm\) 82.5 \# & 0.002 \({}^{*}\) (0.286) \\ TNF-\(\alpha\) (pg-10\({}^{-3}\) cells) & 58.2 \(\pm\) 9.8 & 55.1 \(\pm\) 10.1 & 55.4 \(\pm\) 8.0 & 49.2 \(\pm\) 9.9 & 46.9 \(\pm\) 7.3 & 44.7 \(\pm\) 8.5 & 0.068 (0.169) \\ IL-12 (pg-10\({}^{-3}\) cells) & 4.88 \(\pm\) 0.10 & 4.22 \(\pm\) 0.97 & 4.53 \(\pm\) 0.12 & 4.24 \(\pm\) 0.14 & 4.05 \(\pm\) 0.98 & 3.75 \(\pm\) 0.10 & 0.650 (0.058) \\ IL-1\(\beta\) (pg-10\({}^{-3}\) cells) & 16.2 \(\pm\) 4.0 & 18.5 \(\pm\) 5.0 & 17.8 \(\pm\) 5.3 & 18.4 \(\pm\) 5.8 & 15.8 \(\pm\) 7.7 & 14.8 \(\pm\) 5.7 & 0.571 (0.067) \\ MD\(\alpha\) (nM) & 0.61 \(\pm\) 0.11 & 0.65 \(\pm\) 0.14 & 0.70 \(\pm\) 0.14 & 0.68 \(\pm\) 0.14 & 0.70 \(\pm\) 0.11 & 0.65 \(\pm\) 0.10 & 0.259 (0.111) \\ \hline \end{tabular} Values are expressed as means \(\pm\) SD\({}^{*}\), \(p\) < 0.05 indicates significant differences (ANOVA one-way test). # Indicates significant differences vs. control. \(\eta^{2}\) value is provided as a measure of effect size.  
\end{table} 
Table 3: Effects of 5-CQA on LPS-stimulated cytokine production and MDA concentration. production. This is the first known study using human blood-derived cells, to analyze the anti-inflammatory effect of chlorogenic acid, 5-CQA. A diluted whole-blood method was applied for the determination of cytokine-stimulated production. When whole blood, rather than isolated cells, is used, the natural cell-cell interactions are preserved avoiding artifacts from preparation, whereas the methods used to isolate peripheral blood mononuclear cells modify the lymphocyte to monocyte ratio and remove endogenous immunomododulators. Therefore, in vivo conditions are more accurately represented using whole-blood culture methods [29]. In addition, physiological concentrations of both caffeine and 5-CQA were used. In this regard, two studies performed by our research group have shown plasma concentrations around 40-50 uM after ingesting 6 mg caffeine/kg body weight [26; 30], the caffeine content of three to four cups of coffee. Furthermore, we found that plasma caffeine concentrations after ingesting one cup of coffee are around 10 uM (unpublished results). These observations fit with results reported by others, with peak concentrations of 1-10 uM after ingesting a cup of coffee, and 20-50 uM following the ingestion of two to three cups of coffee or an equivalent caffeine amount [2; 31; 32]. These observations lead us to propose that caffeine concentrations higher than 50 uM are not relevant and, therefore, we focused the present study on a range of concentrations relevant to habitual levels of caffeine consumption (0-50 uM). Considering the caffeine ingestion needed to achieve values close to 50 uM, these figures would not be commonly attained.