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24 May 2019

31 Pages

Impact of Cocoa Products Intake on Plasma and Urine Metabolites: A Review of Targeted and Non-Targeted Studies in Humans

and
1
Centro Nacional de Ciencia y Tecnología de Alimentos, Universidad de Costa Rica, San Pedro 11501-2060, Costa Rica
2
Escuela de Tecnología de Alimentos, Universidad de Costa Rica, San Pedro 11501-2060, Costa Rica
*
Author to whom correspondence should be addressed.
Nutrients2019, 11(5), 1163;https://doi.org/10.3390/nu11051163
This article belongs to the Special Issue Cocoa, Chocolate and Human Health
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Abstract

Cocoa is continuously drawing attention due to growing scientific evidence suggesting its effects on health. Flavanols and methylxanthines are some of the most important bioactive compounds present in cocoa. Other important bioactives, such as phenolic acids and lactones, are derived from microbial metabolism. The identification of the metabolites produced after cocoa intake is a first step to understand the overall effect on human health. In general, after cocoa intake, methylxanthines show high absorption and elimination efficiencies. Catechins are transformed mainly into sulfate and glucuronide conjugates. Metabolism of procyanidins is highly influenced by the polymerization degree, which hinders their absorption. The polymerization degree over three units leads to biotransformation by the colonic microbiota, resulting in valerolactones and phenolic acids, with higher excretion times. Long term intervention studies, as well as untargeted metabolomic approaches, are scarce. Contradictory results have been reported concerning matrix effects and health impact, and there are still scientific gaps that have to be addresed to understand the influence of cocoa intake on health. This review addresses different cocoa clinical studies, summarizes the different methodologies employed as well as the metabolites that have been identified in plasma and urine after cocoa intake.

1. Introduction

1.1. Theobroma cacao L. composition

Cocoa (Theobroma cacao L.) is a tree from the Malvaceae family. Its seeds are covered by a sweet and sour mucilage which contains approximately 11% of sugars, mainly sucrose, and an acidic environment with a pH of about 3.5–3.8. Citric acid is the main organic acid present in the pulp. Others such as oxalic, phosphoric, malic and tartaric acid are also present [1,2,3,4,5,6].
Fat is the main component of cocoa beans: it accounts to almost 50% of the cotyledon dry weight, where 98% corresponds to neutral lipids, and 2% to polar lipids, mainly phospholipids and glycolipids. It has been reported that the major cocoa fatty acids are palmitic, stearic and oleic [7,8].
Proteins represent 17–20% of the dried bean [9,10]. Vicilin-like globulins and albumins are the two most abundant proteins and are key factors in quality development during fermentation. Concerning the amino acid profile, glutamic acid and aspartic acid showed the highest contents while cysteine showed the lowest ones, in both fermented and non-fermented cocoa derived products [11].
From a functional point of view, cocoa has been considered an important source of different bioactive compounds. The more important groups of compounds present in cacao are flavonoids, mainly flavan-3-ols, phenolic acids, methylxanthines, peptides, N-phenylpropenoyl-L-amino acids, and stilbenes [5,12,13,14,15,16,17,18,19,20].
Polyphenols represent approximately 13% of the dried unfermented cocoa beans [21], where proanthocyanidins can reach 58% of the total polyphenol content, followed by catechins and anthocyanins with 37% and 4%, respectively [22]. Proanthocyanidins are usually (epi) catechin-based, i.e., procyanidins, and the main anthocyanins are cyanidin-3-galactoside and cyanidin-3-arabinoside [21,23,24].
The main methylxanthines are theobromine, caffeine, theophylline, and 7-methylxanthine. Theobromine is the most concentrated methylxanthine, and it could be present in concentration levels of 1–2% (dry basis) in cocoa seeds; caffeine also has an important contribution from 0–2% (dry basis) [5].
Additionally, N-phenylpropenoyl-L-amino acids have been identified in cocoa and described as polyphenol-amino acid conjugates, some of which have been related to antioxidant mechanisms, inducers of mitochondrial activity and inhibitors of pathogen adhesion in stomach tissues [20].
Cocoa and cocoa derived products suffer significant changes throughout the processing, and this has to be taken into account when studying the effects of cocoa intake on human health.
Traditionally, cocoa undergoes different processing steps after harvesting, some of which are fermentation, drying, roasting [6], and size reduction steps.
During fermentation and drying cocoa beans have extensive proteolysis, oxidation, and polymerization reactions, among others, resulting in a decrease on total flavonoids and the emergence of new metabolites [5,25,26,27,28].
Also, during roasting, high temperatures lead to glucose and sugar depletion, and to the formation of pyrazines, pyrroles, quinoxalines, pyrones, phenylalk-2-enals, lactones, diketopiperazines, and derivatives of phenylalanine, furanones and others. Compounds, such as triacylglycerols, alcohols, esters, and acids, do not go through significant changes [5]. On the other hand, flavanols and procyanidins can be affected by different temperature dependent reactions, as for example epimerization [3,29,30,31].
Roasted beans [3,5] are the starting point for chocolate and cocoa powder production, which are the cocoa derived products most commonly consumed. In both cases, formulation steps are needed, which increase the diversity of compounds present in these matrices and their interactions.

1.2. Cocoa and Health: General Aspects

A variety of health benefits have been associated to the intake of cocoa and its derived products, many of them attributed to the intake of polyphenols, particularly flavonoids [32,33,34]: improvement in insulin resistance by lowering serum insulin and in flow-mediated dilatation [35], as well as improvement in blood pressure, maintenance of normal endothelium-dependent vasodilation, vascular and platelet function, increased cerebral blood flow, potential cancer prevention, and anti-inflammatory and antioxidant activity [36,37,38,39,40]. Nonetheless, contradictory evidence has been reported indicating that more research is still needed [41,42,43,44,45,46].
It has been proposed that cocoa polyphenols can also have an influence on the gut microbiota, promoting the development of microorganisms as Lactobacillus spp. and Bifidobacterium spp. Microbiota can metabolize polyphenols into different bioactives, as valerolactones, phenylpropionic and phenylacetic derivatives some of which could activate anti-inflammatory pathways [33,47,48].
Other bioactive compounds, like methylxanthines, have shown possible beneficial effects. For example, caffeine may improve exercise performance, but more research is needed to understand the mechanisms behind this statement [49].
To understand the effects of cocoa on human health, it is fundamental to know which metabolites are produced after cocoa intake, as this can help understand the absorption, distribution, metabolism, and excretion mechanisms [47,50]. The development of different types of clinical trials contributes to this knowledge; and, in relation to cocoa, there is a particular need to promote long term research.
This review collects the results of different studies that traced several metabolites in urine and plasma after the acute or chronic cocoa intake.

2. Methodological Considerations

Data collection was done by searching individual and compound keywords in different databases, such as: cocoa, chocolate, humans, randomized, acute, health, untargeted, non-targeted, metabolomics, urine, plasma, pharmacokinetics, polyphenols, (epi) catechin, procyanidin, methylxanthines, and theobromine. The main databases used were: Google Scholar, ScienceDirect, ACS Publications, and Springer Link. The searches were done in between August 2016 and February 2019 and included studies from 1999 to February 2019.

3. Results

Table 1 and Table 2 summarize several studies that analyze the effects of the intake of different cocoa derivatives, such as chocolate (Table 1) and cocoa powder drinks or extracts (Table 2) in human plasma and urine samples using a targeted methodology. Meanwhile Table 3 summarizes non-targeted researches, including chocolate or cocoa powder drink intake studies, as well as one cross sectional study.
Table 1. Summary of the main aspects of targeted cocoa studies, involving chocolate intake.
Table 2. Summary of the main aspects of targeted cocoa studies, involving cocoa powder or cocoa extracts-based drinks intake.
Table 3. Summary of the main aspects of untargeted cocoa intervention studies.
The methodological aspects are detailed, including the main objective of the studies, the experimental design applied [51,52,53,54], information regarding the subjects, matrices and dose employed, the biological samples studied, the analytical instrumental technique used to measure the metabolites, and the statistics applied to process data.
A total of 34 studies were reviewed, 30 of them used a targeted approach, and 4 of them were untargeted. The clinical studies employed different cocoa derived matrices. Nine of these studies included chocolate intake intervention, 24 used cocoa powders or cocoa extracts beverages, and 2 of them had a cross-sectional experimental design which did not apply dietary interventions [55,56].
Most researches followed acute single dose interventions, nonetheless only 2 of them evaluated the effect of a daily cocoa-derived products intake after 4 weeks [57,58]. Additionally, 10 studies included a non-cocoa-derived control intake in their experimental design.
Regarding the type of samples analyzed, most studies (n) investigated plasma (n = 8), urine (n = 14) or both plasma and urine (n = 12). Additionally, one study analyzed saliva [59] and another studied feces [60].
The number of volunteers (n) that finalized clinical studies was varied. In five studies only 5 volunteers participated, in ten studies 5 < n ≤ 10 were part of the clinical trial, eight studies included 10 < n ≤ 20 and twelve studies recruited and selected volunteers in a range from 21 to 80 volunteers.
Most clinical studies investigated samples from healthy non-smokers. Only two trials obtained samples from smokers [61] and from volunteers with high cardiovascular risk [57]. Ten studies investigated only men, 18 studies men and women, and one study investigated children [56].
LC-UV/Vis (liquid chromatography couple to ultraviolet or visible detectors) and FLD (fluorescence detectors), and LC-MS (liquid chromatography couple to mass spectrometers) were the predominant analytical platforms reported. In particular, when using an untargeted approach, LC-MS was present in all studies; and one study also applied NMR (nuclear magnetic resonance) [20].
Further on, Table 4, Table 5, Table 6, Table 7 and Table 8 were used to classify the metabolites identified in urine and plasma, in different studies with different approaches.
Table 4. Metabolites analyzed in plasma after different acute cocoa intake interventions, by targeted methodologies.
Table 5. Metabolites detected in plasma samples, that discriminated the baseline from treatments (enriched dark chocolate, dark chocolate and white chocolate), and times after intake where increase in measured signal was the highest, in an untargeted study [84].
Table 6. Metabolites analyzed in urine after acute cocoa interventions and studied with targeted methodologies.
Table 7. Metabolites analyzed in urine after acute cocoa intervention studied with non-targeted methodologies.
Table 8. Metabolites analyzed in urine and plasma after acute, short-term, or regular cocoa intake interventions, studied with non-targeted and targeted methodologies.
In particular, studies that mentioned acute intake of cocoa are summarized in the following tables: (a) Table 4 and Table 6 include information of different metabolites as concentrations measured in the biological samples and the corresponding sampling periods. The type of matrix analyzed is also indicated. Both tables summarize information of targeted studies, the first focused on plasma (Table 4) and the other in urine (Table 6) analysis; (b) Table 5 shows the time after intake of different chocolates, where the highest signals of the different metabolites were detected or expected in plasma samples, (c) Table 7 focuses on the main metabolites found in urine that discriminated between different periods of time after intake, with an untargeted approach.
On the other hand, discriminating metabolites in between regular/chronic consumers and non-regular/chronic consumers observed in urine and plasma are listed on Table 8. This covers both targeted and untargeted studies.
Metabolites were organized by means of the chemical group, mainly methylxanthines, flavonoids and conjugates, phenolic acids, and lignans. As for the data collected from untargeted studies, data organization was done in a way that respected as much as possible the authors´ original classification. Additionally, in each category metabolites were organized alphabetically.

4. Discussion

4.1. Methylxanthines

Theobromine showed some of the higher concentrations in plasma and urine after cocoa intake in different human intervention trials (Table 4 and Table 5). One study [81] showed that unmetabolized theobromine in urine was close to 1.5 times higher when consuming methylxanthine enriched powder compared to the non-enriched cocoa powder, which indicates that renal clearance could vary in function of exposure. Due to a possible saturation of the pathway, a lack of enzymes and/or cofactors to process the metabolite may have had affected [81,88].
There is evidence that suggests that excretion also depends on gender and age. After consumption of chocolate in a cross-over study, theobromine excretion showed by women almost doubled man´s levels and was also higher for volunteers younger than 29 years, and lower compared to the ones older than 54 years [84].
A variety of methylxanthines metabolites were also observed in non-targeted studies (Table 7), which follow characteristic metabolic pathways [89]. Some of them have been proposed as possible biomarkers of regular cocoa intake as AMMU, 3-methyluric acid, 7-methylxanthine, 3-methylxanthine, theobromine, and 3,7-dimethyluric acid [55].

4.2. Polyphenols

Polyphenols such as (epi) catechin, and the corresponding glucuronides, sulfates, with or without methylations were the most frequently analyzed metabolites.
One example is a study that evaluated plasma and urine samples after drinking a cocoa beverage. The most abundant flavanol metabolites were (-)-epicatechin-3´-β-D-glucuronide, (-)-epicatechin-3´-sulfate, 3´-O-methyl-(-)-epicatechin-5-sulfate, and 3´-O-methyl-(-)-epicatechin-7-sulfate which represented close to 94% of the total flavanol metabolites that were measured in plasma [82].
This is consequence of the metabolic pathways that flavanols and phenolic acids follow. When absorbed, these metabolites are transferred to the liver or other tissues where phase I and phase II reactions occur. In phase I, cytochrome enzymes lead to hydroxylations, oxidations and reductions; meanwhile, phase II produces conjugates by action of glucuronosyl transferases, sulfotransferases, and catechol-O-methyl transferases [75,90,91,92].
In general terms these metabolites reached maximum concentrations in plasma in less than 4 hours after intake (Table 4). One exception was (-)-epicatechin-7´-β-D-glucuronide, that in one study required 12.8 ± 4.8 h after cocoa intake to reach maximal concentrations [66].
When these metabolites are present in plasma, they could exert particular effects. For example, one study analyzed the impact of chocolate intake on oxidative damage, and observed an increase in plasma antioxidant activity and plasma lipid oxidation products decrease as epicatechin plasma levels increased [63]. Additionally, elimination rates have an influence on the probabilities of accumulation in blood or tissues, and the subsequent effect on health [93].
In this matter, when studying urine samples, the required time to reach maximal concentration was found to be largely variable. While (epi) catechin-O-sulfate isomer required a time in between 0–2 h after cocoa powder in milk intake to reach the higher concentration [77], (-)-epicatechin-7-β-D-glucuronide required a time in between 10–24 h after dark chocolate consumption [66].
Influence of age on metabolism was analyzed in an intervention that evaluated two cocoa powder drinks. Non-significant differences or small differences were observed for the different pharmacokinetic parameters, in the renal clearance, total excreted metabolites, and apparent volume of distribution in two age groups: 18–35 years and 65–80 years. Some of the small differences observed were associated with changes in renal function due to normal aging processes [82].
Studies converge in that increasing polymerization degree (n > 2) of procyanidins hinders their absorption. Procyanidin B2 dimer has been detected in plasma after consuming a cocoa beverage, showing a maximum concentration in the period between 0.5–2 h (Table 5). In this same study, B5 dimer was not detected [68]. Similar results were observed in another study where volunteers drank samples with cocoa extracts that contained procyanidins from 2–10 units. In this study, B2 dimer was detected in plasma in approximately 80% of the volunteers, indicating maximum concentrations at 2 h after intake, and with concentrations close to the limit of detection. Additionally, B5 dimer was not detected in any of the plasma samples [80].
Polyphenol absorption and excretion can depend on the food matrix, but contradictory information exists. For example, excretion patterns of microbial phenolic acids metabolites measured in urine differed depending on the liquid vehicle of cocoa powder consumed: water or milk [78]. One randomized study compared the levels of (-)-epicatechin glucuronide in plasma after the intake of cocoa powder with milk or water, and reported no statistical differences in the results from each group [73]. Also, milk protein did not affect the bioavailability of polyphenols present in a chocolate drink [74]. In another study the excretion of different flavan-3-ol metabolites within 24 h after cocoa intake was close to 20% of the ingested dose if cocoa was provided as a water-based beverage, but only 10% if ingested as a milk-based cocoa drink [77]. When consuming two cocoa beverages, either prepared with water or with milk, the total excretion was not affected, but kinetics was different. In the same period of time after intake (-)-epicatechin glucuronide was the most concentrated metabolite when water was the vehicle—doubling the concentration of total excreted (-)-epicatechin sulfates- but when milk was used, glucuronides and total sulfates reached similar concentrations in urine [75]. No significant differences in excretion patterns were found when comparing a variety of metabolites in urine after consuming cocoa powder in milk or in water [86]. It is suggested that polyphenol absorption and metabolism might be affected because of the interactions between milk polypeptides and polyphenols, which can decrease the bioaccessibility [94,95]. In addition, polyphenol absorption could also be affected by carbohydrates, as it has been reported that flavanol uptake could increase when simultaneously consuming these macronutrients [50].
Flavanol absorption and metabolism has also been demonstrated to be dependent on the stereochemistry. Differences in absorption and metabolism were detected after the consumption of equal amounts of (-)-epicatechin, (-)-catechin, (+)-catechin, and (+)-epicatechin in a cocoa based milk drink [79].
Non-absorbed flavonoids and phenolic acids could follow metabolic pathways that are carried out by the colonic microbiota [66,90,91,92]. Some epicatechin metabolites can return to small intestine including non-absorbed epicatechin pass to the colon, which can account a total close to 70 % of the total ingested [96].
Microbial metabolism can lead to the production of phenolic acids, sulfated and glucuronidated valerolactones [57]. Some examples are ferulic acid, phenylacetic acid, hippuric acid (Table 6), 4-hydroxy-5-(dihydroxyphenyl)-valeric acid glucuronide, and methoxyhydroxyphenyl valerolactone (Table 6 and Table 7).
In general terms, these types of metabolites require longer times to reach mean concentrations in urine and plasma than the metabolites following phase II metabolism (Table 4 and Table 6, Table 7 and Table 8). To illustrate, ferulic and m-hydroxyphenyl acetic acids showed maximum concentrations in between 24 and 48 h after chocolate intake, and vanillic acid was the only metabolite that returned to basal level concentrations in the 48 h of observation [65].
Research suggests that consumption of cocoa and derivatives could modulate human metabolism. A randomized controlled cross-over intervention trial analyzed the acute consumption of three types of chocolates in 42 healthy volunteers and concluded that several endogenous metabolites were affected (Table 5 and Table 7). In particular, several amino acids, organic acids, creatinine, lactate, and N1-methylnicotinamide decreased meanwhile pyruvate, tyrosine, and p-hydroxyphenylacetate increased after the intervention. Flavan-3-ols, methylxanthines and their metabolites were suggested to modulate endogenous and colonic microbial metabolism [84].
Studies with longer periods of intervention detected additional metabolome changes due to regular cocoa consumption (Table 8). For example, different glucuronide and sulfate conjugates of (-)-epicatechin, O-methyl-epicatechin, dihydroxyphenyl valerolactones, and methoxy hydroxyphenyl valerolactones increased their levels in plasma after regular consumption of cocoa powder in a non-healthy group of volunteers. Hydroxyphenylacetic acids, AMMU isomers, 5-(3´,4´-dihydroxyphenyl)-γ-valerolactone, and its glucuronides and sulfates have been proposed as biomarkers of regular consumption of cocoa [55,57]. Further on, one study reported that methylglutaryl carnitine, derived from the acylcarnitine pathway, decreased after a long-term cocoa intake [55].
More recent studies have supported this information as 5-(3´,4´-dihidroxyphenyl)-γ-valerolactone has also been proposed as a specific biomarker of flavan-3-ols consumption, deriving only from (epi) catechin-based mono and oligomeric cocoa flavanols [83].
There is also evidence that polyphenol consumption from cocoa products might change the gut microbiota, exerting prebiotic effects, and which could be related to the activation of anti-inflammatory pathways with benefits in the host and alter the obtained profile of metabolites. One study observed an increase of Lactobacillus spp. and Bifidobacterium spp. in contrast with a decrease of Clostridium spp. populations after daily consumption during one month of a flavanol-rich cocoa beverage, compared to a low flavanol cocoa beverage [33,97,98].

4.3. Other Metabolites

Metabolites different than methylxanthines and polyphenols metabolism have also been detected after cocoa intake. For example, trigonelline (N-methylnicotinic acid) and hydroxynicotinic acid may be a result of nicotinic acid metabolism. They were detected in urine only within 6 h after cocoa powder consumption. Also, diketopiperazines such as cyclo(ser-tyr) and cyclo(pro-pro) were reported for the first time associated to cocoa powder intake and are considered flavor and taste metabolites [86].
N-phenylpropenoyl-L-amino acids have also been metabolites of interest as there is some preliminary evidence of their biological activity. Thirteen of them have been reported in urine after cocoa intake [20].
Additionally, significant changes in plasma metabolites after 6 h of chocolate intake have been reported for β-hydroxybutyrate, acetone, acetoacetate, and aspartate. (Table 5) [84].
One study observed that daily dark chocolate intake for 2 weeks modulated energy and hormone metabolism of humans with low and high anxiety traits, and also modulated their microbial gut metabolism. Urine analysis showed a reduction of stress hormone metabolites, and other stress-related metabolites presented a trend to change towards the low anxiety profile. Some of these metabolites are glycine, citrate, trans-aconitate, proline, DOPA, β-alanine, hippurate, and p-cresol sulfate [99]. This can eventually help understand the impact of cocoa intake on health.

5. Conclusions

A diversity of metabolites has been identified in urine and plasma after consumption of cocoa or cocoa derived products. Theobromine and caffeine have been some of the main methylxanthines identified together with their metabolites. Polyphenolic phase I and phase II metabolism leads mainly to (epi)catechin sulphates, glucuronides, and sulfoglucuronides. Non absorbed polyphenols are transformed by colonic microorganisms yielding a diversity of metabolites that range from phenolic acids to different valerolactones, and more complex metabolites.
Biomarkers of consumption of cocoa have been proposed: AMMU, 3-methyluric acid, 7-methylxanthine, 3-methylxanthine, theobromine, 3,7-dimethyluric acid, hydroxyphenylacetic acid, and 5-(3´,4´-dihydroxyphenyl)-γ-valerolactone; the latter with its correspondant glucuronides and sulfates.
Evidence shows that absorption, metabolism, and excretion of cocoa metabolites depend on the food matrix, the dose, age, gender, overall health status and other factors such as the polymerization degree (e.g., procyanidins), and stereochemistry (e.g., flavanols).
The development of clinical studies is fundamental to understand the metabolic pathways of different metabolites present in cocoa and further on the effects on health. Special attention must be given to properly design the experiments, and instrumental methodologies for the extraction and quantification of metabolites in biological samples.

Author Contributions

Writing—original draft preparation: A.L.M.-G.; review and editing: P.E.

Funding

This research was done within the project 735-B5-050 “Valorización de matrices alimentarias mediante la evaluación del impacto del procesamiento sobre el perfil metabolómico”, funded by the University of Costa Rica, Costa Rica.

Conflicts of Interest

The authors declare no conflict of interest.

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