Based on the premise of a concentration equilibrium of CGA and QA between the systemic circulation and urine (Chereson, 1996) we determined the influence of increasing CGA and QA doses on renal excretion. Due to a lack of references (such as CGA sulfates and glucuronides) we achieved semi-quantitative data (see chapter 7.3.1.5.2.1).
5.2.3.1 Effect of an increasing amount of CGA and QA consumed by coffee on renal excretion
Renal excretions correlated negatively with increasing CGA doses ingested and high inter-individual variations (Table 10-10; Appendix). We presume a relationship between GI - TT and CGA bioavailability. More specifically, one volunteer showed a much longer GI - TT (Ileo 5) and therefore the renal excretion was twice as high as for the other subjects. The relationship between GI – TT and renal excretion of each volunteer shown in Figure 5-2 gives a hint that a decelerated passage of CGA in the gastrointestinal tract leads to a higher absorption rate.
0 2 4 6
Tmax CQA ileal excretion [h]
Figure 5-2: Relationship of total renal excretion of consumed CGA in %, with maximum of ileal excretion (Tmax CQA) in hours of each ileostomist given for all doses (HIGH; MEDIUM; LOW). Linear fit: R2 = 0.64; y = 2.8x + 6.2.
Due to the extensive biotransformation that CGA undergo in the human body it is not possible to calculate recovery rates of individual CGA. Therefore, total renal excretion of CGA is reported here. It was determined between 8.0 ± 4.9% for the high dose and 14.6 ± 6.8% for the low dose. Other research groups detected similar renal amounts being renally excreted in ileostomists of 8% after ingestion of 385 µmol CGA from coffee (with a comparable semi-quantitative analytical method as used in our study) (Stalmach et al., 2010) and 11% after administration of 2,800 µmol pure CA (5a) (Olthof et al., 2001) (enzyme treatment was used as analytical method) (see Table 2-14, State of knowledge). In healthy volunteers the total renal recovery of CGA after coffee consumption was reported to be about 3 times higher in contrast to ileostomists. This was reported by Stalmach et al., 2009 and Rio et al., 2010 who used a comparable analytical method to our study (Stalmach et al., 2009; Rio et al., 2010).
In previous bioavailability studies healthy probands consumed CGA and considerable lower total excretions of CGA were observed (see Table 2-13, State of knowledge).
However, these groups used different analytical methods (mostly with enzymatic cleavage of CGA metabolites), different CGA dosage forms such as encapsulated
green coffee and different CGA compositions in ingested food such as artichoke leaf extract (see Table 2-13, State of knowledge). Due to this, the reported total renal CGA excretions determined in these studies (Rechner et al., 2001; Ito et al., 2005;
Wittemer et al., 2005; Farah et al., 2008) are not further discussed.
As previously reported, we found unmetabolized CGA in urine (Olthof et al., 2001;
Stalmach et al., 2009; Stalmach et al., 2010; Kahle et al., 2011). Hereby, especially 3-FQA (3a) and 3-CQA (1b) of these two CGA subgroups were predominant in urine, independent of the administered dose (Table 10-10, Appendix, Figure 5-3), whereas in plasma the 4-acyl compounds were predominant after enzyme hydrolysis (Figure 5-3, Table 10-13, Appendix). The 5-acyl compounds (5-FQA (3c), 5-CQA (1d)) were detected in both compartments only in minor amounts. The group of Renouf et al.
determined after coffee consumption and enzymatic hydrolysis of plasma in terms of apparent plasma appearance the following order: 3-FQA > 5-FQA > 4-CQA > 4-FQA
> 5-CQA > 3-CQA (Renouf et al., 2013). The differences to our observations (see Figure 5-3) might be caused due to the fact that healthy volunteers (with colon) consumed the coffee in this study.
Interesterification reactions at the physiological plasma pH could be a reason for this.
Such interesterification reactions were also observed in the ex vivo incubation experiments with CGA and ileal effluents, see Table 10-2, Appendix. Especially, the 3-acyl compounds (3-FQA (3a), 3-CQA (1b)) showed high recoveries in these ex vivo experiments as well as in urine and plasma after coffee consumption (see Figure 5-3).
Furthermore, the elimination of CGA and metabolites via urine could be affected by the various phase II conjugation reactions. The SULT enzyme family seems to prefer the 3- and 4-acyl CQA and FQA, similar to the UGT enzyme family, which additionally showed a high affinity for 4-CQA and 4-IFQA (see Figure 5-3, Table 10-10, Appendix). Both enzyme families showed an inhibition for 5-CQA (1d) or 5-FQA (3c) conjugation.
Figure 5-3: Representive kinetics of caffeoylquinic acids (CQA), feruloylquinic acids (FQA), isoferuloylquinic acids (IFQA) and metabolites in urine (µmol*mg-1 creatinine) and plasma (after enzyme hydrolysis, in nM) of ileostomist no. 2 after the consumption of coffee (HIGH CGA dose, 4,525 µmol). Sulf = sulfate, GlucA = glucuronide.
0 4 8 24 48
Despite unmetabolized CGA, we were not able to identify any diCQA (4a-c) in plasma or in urine. Caffeoylquinides (CQL) (2a-b) were highly absorbed; particularly its sulfated metabolites (CQL-Sulf) were detectable in urine (Table 10-10, Appendix).
In comparison to CGA the QA (10a) moiety of CQL (2a-b) have an intra-molecular ester bridge which decreases the polarity. Specifically, the calculated log D(pH6.0) for CQL was + 0.4 which is more suitable for passive absorption than the log D(pH 6.0) for CQA of – 2.9 (calculated with MARVIN SKETCH 5.3.1).
As early as 1964 one study reported QA (10a) in urine (Halpern, 1964), whereas previous bioavailability studies described its colonic metabolites in urine at its most, e.g. hippuric acid (Rechner et al., 2001; Olthof et al., 2003; Kahle, 2008).
In 1970 Adamson and coworkers orally administered 12 g [14C] QA to Rhesus monkeys. This group detected QA being aromatized by the gut flora to hippuric acid and found 32% of the radioactivity as hippuric acid in urine (Adamson et al., 1970).
But, in 2011 Pero and Lund observed millimolar concentrations of QA in urine after consumption of a nutritional supplement (AIO + AC-11®) (Pero and Lund, 2011).
However, the renal dose dependent recovery of QA was determined here for the first time. Recovery rates were nearly similar in all trials with about 15%. Hence, the recovery of QA in urine was not affected by the consumed dose or GI-TT. But a potential hydrolysis of CGA into QA and HCA was indicated by measurable free HCA in our studies; and in previous work (Kahle et al., 2007). Taking the potential hydrolysis of CGA into account the actual recovery rates of free QA consumed with the coffee brews under study was estimated to be less than 15%.
5.2.3.2 Effect of increasing CGA dose consumption on CGA metabolite formation
The total percentage of CGA metabolites (conjugates or hydrolytic products) in urine were not affected by the doses consumed, whereas the ratios of sulfation : glucuronidation (sulf : glucA) in urine showed a dose-dependency similar to the formation of metabolites excreted via ileal effluents. The sulf : glucA ratio changed from 0.7 : 1 (HIGH), 1 : 1 (MEDIUM), up to 1.3 : 1 (LOW). In a previous study with ileostomy volunteers (Stalmach et al., 2010), the urinary sulf : glucA ratio was 5.6 : 1. In this study CGA consumption via coffee compared to our lowest coffee dose (LOW) was 2.7 times lower. This could explain the higher amount of sulfation in
this study (Stalmach et al., 2010). We conclude that sulfation is limited by increasing dose conceivably due to enzymatic saturation, limitation of substrates, limited transport capacities of the enterocyte (influx and efflux) or an influence of the GI - TT.
Our findings of a regioselectivity preferences of conjugating enzymes at the hydroxyl position 3´ (Figure 2-1, Sate of knowledge) confirmed the findings of recently published studies (Stalmach et al., 2010; Wong et al., 2010).
Thereupon, the conjugation enzymes being involved in renal and ileal excretions are regulated by the substitutes at the aromatic ring of the hydroxycinnamic acid moieties, dose unaffected. For instance, we determined a sulfation preference for CA (5a) and DHCA (6a) both molecules have two hydroxyl groups at position 3´ and 4´
(see Figure 4-13, Results). Furthermore, a strong glucuronidation affinity for IFA (7g) and IFQA (3i, 3j), both molecules with a methoxy group at position 4´ was observed (Figure 4-14, Results).
Moreover, for the first time we described metabolites of the intact CGA and corresponding metabolic profile in urine (such as CQA-Sulf and CQA-GlucA), in spite of sulfated CQL (2e-f) which were already reported by (Stalmach et al., 2009;
Stalmach et al., 2010). We were able to show that conjugating enzymes involved in renal excretion are also regulated by the hydroxycinnamic acid moiety. For instance, whereas the CQL (2a-b), a CA with quinide moiety, showed a strong affinity for sulfation (Figure 4-13, Results), the CQA (1a-d) a CA with quinic acid moiety seemed to hinder the preferred sulfation with the consequence of higher affinity to the UGT enzyme family (Figure 4-14, Results).