Abstract
Background:
Coffee is one of the most frequently consumed beverages worldwide. Research on effects of coffee drinking has focused on caffeine; however, coffee contains myriad biochemicals that are chemically unrelated to caffeine, including 3,4-dihydroxyphenyl compounds (catechols) such as caffeic acid and dihydrocaffeic acid (DHCA).
Objective:
This prospective within-subjects study examined effects of drinking caffeinated or decaffeinated coffee on plasma free (unconjugated) catechols measured by liquid chromatography with series electrochemical detection (LCED) after batch alumina extraction. To confirm coffee-related chromatographic peaks represented catechols, plasma was incubated with catechol-O-methyltransferase and S-adenosylmethionine before the alumina extraction; reductions in peak heights would identify catechols.
Methods:
Ten healthy volunteers drank 2 cups each of caffeinated and decaffeinated coffee on separate days after fasting overnight. With subjects supine, blood was drawn through an intravenous catheter up to 240 minutes after coffee ingestion and the plasma assayed by alumina extraction followed by LCED.
Results:
Within 15 minutes of drinking coffee of either type, >20 additional peaks were noted in chromatographs from the alumina eluates. Most of the coffee-related peaks corresponded to free catechols. Plasma levels of the catecholamines epinephrine and dopamine increased with both caffeinated and decaffeinated coffee. Levels of other endogenous catechols were unaffected. Plasma DHCA increased bi-phasically, in contrast with other coffee-related free catechols.
Interpretation:
Drinking coffee—whether caffeinated or decaffeinated—results in the rapid appearance of numerous free catechols in the plasma. These might affect the disposition of circulating catecholamines. The bi-phasic increase in plasma DHCA is consistent with production by gut bacteria.
Keywords: Coffee, Catechols, Dihydrocaffeic Acid, Caffeine, Decaffeinated, Catecholamines
1. INTRODUCTION
Coffee is one of the most frequently imbibed beverages worldwide. Health effects of coffee ingestion have been the subject of active attention by academicians and the media for many years.
Research on effects of coffee drinking has focused mainly on caffeine; however, coffee contains many 3,4-dihydroxyphenyl compounds (catechols) that are chemically distinct from caffeine (Figure 1) and are present even in decaffeinated coffee. In particular, caffeic acid and dihydrocaffeic acid (DHCA)—as their names imply—are abundant in coffee.
Figure 1: Molecular structures of catechols in coffee and endogenous catechols.
Catechols are defined by adjacent hydroxyl groups on a benzene ring. Coffee-related catechols are chemically unrelated to caffeine (inset). The endogenous catecholamine epinephrine (EPI) was noted in decaffeinated coffee (see Table 1).
Catechols are efficiently metabolized by sulfoconjugation catalyzed by monoamine-preferring phenolsulfotransferase and O-methylation catalyzed by catechol-O-methyltransferase (COMT), and the preponderance of coffee-related phenolic acids in plasma are conjugated or O-methylated [1]. Caffeic acid, ferulic acid, and DHCA have been detected in free (unconjugated) form after coffee drinking [2–5]. Whether other free catechols are found in human plasma after drinking caffeinated or decaffeinated coffee has been unknown.
Effects of drinking caffeinated vs. decaffeinated coffee on plasma levels of endogenous catecholamines are poorly understood. Coffee ingestion elevates the urinary excretion rate of the endogenous catecholamine epinephrine (EPI) [6], an effect thought to result from adrenomedullary stimulation evoked by caffeine [7]; however, previous studies have not compared caffeinated vs. decaffeinated coffee in terms of effects on plasma EPI concentrations. Effects of drinking caffeinated or decaffeinated coffee on free dopamine (DA) and norepinephrine (NE) levels have also not been described. Coffee drinking might increase plasma plasma NE via effects on central neural systems that regulate sympathetic noradrenergic outflows. Thus, drinking coffee, whether caffeinated or decaffeinated, increases skeletal muscle sympathetic activity [8].
Coffee ingestion rapidly increases plasma DHCA levels [2, 9, 10]. In addition, gut bacteria efficiently metabolize chlorogenic acid and caffeic acid to form DHCA [11–13]. Drinking coffee, whether caffeinated or decaffeinated, might therefore result in a sustained increase in plasma DHCA levels [14].
This prospective, within-subjects, observational study had the purpose of describing the profile of plasma catechols after healthy volunteers drank caffeinated vs. decaffeinated coffee. Plasma was assayed for free catechols by liquid chromatography with series electrochemical detection (LCED) after batch alumina extraction [15]. The alumina extraction procedure efficiently purifies catechols, and the employed LCED approach separates and enables sensitive detection of reversibly oxidizable small molecules. The resulting coffee-related chromatographic peaks would therefore be expected to represent free catechols. To confirm this, plasma samples after caffeinated or decaffeinated coffee ingestion were incubated with COMT and the methyl group donor S-adenosylmethionine (SAMe) before the alumina extraction. Enzymatic O-methylation would decrease the heights of chromatographic peaks corresponding to catechols.
2. METHODS
2.1. Subjects
The subjects were healthy volunteers who were studied as outpatients at the NIH Clinical Center. Each participant gave written informed consent to be studied under an IRB-approved protocol before the research procedures were done. The protocol (NIH Clinical Protocol 06N0047) was about dietary or environmental factors that might influence reference values for plasma levels of catechols.
The subjects were at least 18 years old and were not pregnant or lactating. A medical history and physical examination and routine screening clinical pathology tests were done to confirm health status before admission to the clinical study. There was no recruitment by advertisement, and the study was open to eligible subjects regardless of age, sex, race, ethnicity, or habit of coffee drinking. Participants were reimbursed for their time and inconvenience as called for in the protocol.
2.2. Experimental procedures
The experimental procedures were carried out at the NIH Clinical Center between August, 2015 and September, 2019. The study used a prospective, within-subjects design to compare effects of caffeinated vs. decaffeinated coffee ingested at the same time of day on separate days. The sequence of the type of coffee was not systematic.
The study size was based on a power calculation approved by the Scientific Review Committee of the National Institute of Neurological Disorders and Stroke (NINDS). Sample size calculations indicated that 10 subjects would be adequate to detect differences of 25% or greater using repeated measure general linear models with Bonferroni adjustment for responses of multiple endogenous plasma catechols.
2.2.1. Setup
Each participant reported to a dedicated patient testing room in the morning after having fasted (except for water) overnight. Room temperature was 72–77 degrees Fahrenheit. An intravenous catheter (IV) was placed in an arm vein.
2.2.2. Physiological variables
Electrocardiographic leads were placed to monitor the heart rate and rhythm. Blood pressure was tracked continuously using an automated finger cuff device (Nexfin, bmEye, Amsterdam, The Netherlands or Finapres Medical Systems, Amsterdam, The Netherlands). Also measured were forearm blood flow by impedance plethysmography (Hokanson, Issaquah, WA), fingertip microcirculatory flow using a laser-Doppler flowmeter (ADInstruments, Colorado Springs, CO), and fingertip skin temperature using a thermistor (ADInstruments). The physiological signals were recorded using a PowerLab system (ADInstruments) and analyzed by LabChart software bundled with the PowerLab system. Forearm vascular resistance (FVR) was calculated from the mean arterial pressure recorded using the finger cuff device divided by the concurrent forearm blood flow.
2.2.3. Coffee drinking and blood sampling
After at least 15 minutes with the subject at rest supine, a baseline blood sample (about 5 mL) was drawn through the IV. Each subject then drank 2 cups of coffee (caffeinated or decaffeinated Folger’s K-cups™) on separate days, without a set sequence) over about 1–2 minutes while sitting or semi-recumbent. The subject was then returned to the supine position. Blood was drawn through the IV at 15, 30, 45, 60, 120, 180, and 240 minutes, with the subject supine for at least 15 minutes before each sample was drawn.
2.2.4. Neurochemical variables
Plasma levels of catechols were assayed by batch alumina extraction followed by liquid chromatography with series electrochemical detection [15]. Batch alumina extraction partially purifies catechols [16]. Under basic conditions alumina adsorbs catechols. The alumina is washed, and after each wash the alumina with adsorbed catechols is centrifuged and the supernatant discarded. In the final step of the alumina extraction, an acid is added to the alumina. The acidification causes the catechols into desorb into the supernate. An aliquot of the supernate is then injected into the LCED system.
The chromatographic separation was by reverse phase, ion pairing liquid chromatography with series electrochemical detection. For post-column electrochemical detection there were 3 electrodes in series, with the first set at an oxidizing potential and the third at a reducing potential. Signals from the third potential were recorded, providing a measure of reversibly oxidized species [17].
The assay was modified in that additional standards (DHCA, 5-S-cysteinyldopa (Cys-DOPA), and 5-S-cysteinyldopamine (Cys-DA) were included, and 2 different volumes of the same specimen could be assayed to ensure chromatographic peaks were on scale. The extracted standards were (in order of elution from the chromatographic column) 3,4-dihydroxyphenylglycol (DHPG), norepinephrine (NE), 3,4-dihydroxyphenylalanine (DOPA), epinephrine (EPI), the internal standard, 3,4-dihydroxybenzylamine (DHBA), 3,4-dihydroxyphenylethanol (DOPET), dopamine (DA), 3,4-dihydroxyphenylacetic acid (DOPAC), DHCA, and caffeic acid (Sigma-Aldrich, St. Louis, MO). Cys-DA and Cys-DOPA were provided by the National Institute of Mental Health.
2.2.5. Identification of catechols by incubation of plasma with COMT+SAMe
Combining COMT with tracer-labelled SAMe was the basis for radioenzymatic assays that preceded LCED for measuring levels of catecholamines in biological fluids [18, 19]. With this background we designed the COMT+SAMe experiments reported here.
Reagents used for the incubation of plasma after coffee drinking with COMT+SAMe were COMT (110.75 U/μL, Calzyme, San Luis Obispo, CA), SAMe (10 mM, Sigma, Ronkonkoma, NY), MgCL2 (50 mM, Quality Biological, Inc., Gaithersburg, MD), and PBS 10X phosphate buffer (50 mM, pH 7.4, KDmedical, Columbia, MD).
For incubations of plasma with COMT+SAMe, 500 μL of plasma were combined with 40 μL of COMT, 60 μL SAMe, 30 μL phosphate buffer, and 30 μL of MgCL2 (total 660 μL) and incubated at 37 oC for 2 hours before being assayed by the alumina extraction procedure.
Chromatographic peaks were determined to correspond to catechols based on decreases in the peak heights by COMT+SAMe incubation of the plasma before the alumina extraction. To confirm that the decreases in chromatographic peak heights after COMT+SAMe incubation were due to the enzyme activity (indicating the presence of catechols) and not to degradation of heat-sensitive non-catechol compounds, the same plasma samples with added SAMe but without added COMT were incubated at 37 °C for 2 hours.
2.3. Coffee caffeine contents
The manufacturers of the caffeinated and decaffeinated coffee were contacted to confirm the ranges of caffeine in the administered coffee. According to the website for the Center for Science in the Public interest (https://deploymentpsych.org/system/files/member_resource/Caffeine%20content%20list.pdf), Folger’s Classic Roast K-cup™ coffee contains 75–150 mg caffeine per 8 ounces. The USDA National Nutrient Database states there are 2 mg of caffeine in each 6 oz. or 8 oz serving of Folger’s decaffeinated coffee. Folger’s K-cup™ decaffeinated coffee contains 2–5 mg per 8 ounces.
2.4. Efforts to limit bias
Except for the Research Nurse, who prepared the coffee using a Keurig™ brewing system (Keurig Dr Pepper, Burlington, MA) and entered data into the research database, all the research personnel in this study were blinded as to the treatment with caffeinated or decaffeinated coffee until the data were tabulated. The Research Nurse did not participate in assaying plasma samples or analyzing the data.
2.5. Data analysis and statistics
Mean values were expressed ± 1 standard error of the mean (SEM). Data for plasma levels of catechols were analyzed by 2-factor analyses of variance (ANOVAs) using GraphPad Prism (GraphPad Software, San Diego, CA), to assess differences as a function of time (within subjects), differences as a function of coffee type, and differences between caffeinated and decaffeinated as a function of time. Changes from baseline were examined using Dunnett’s post-hoc test. When there were missing data from interfering co-chromatographing peaks, a mixed model was used. A p value less than 0.05 defined statistical significance.
3. RESULTS
3.1. Subjects
The subjects were 10 healthy adult volunteers (mean age 48 ± 5 (SEM) years, 8 women (7 Caucasian, 1 African-American) and 2 men (1 Caucasian, 1 Asian-American)). Of the 10, 6 reported consuming 1–4 cups of caffeinated or decaffeinated coffee per day, 2 had stopped drinking coffee at least 2 months prior to testing, 1 drank 2 cups of tea per day, and 1 drank only decaffeinated herbal teas.
3.2. LCED assay of coffee-related catechols
The LCED assay method resolved and quantified several catechols of interest—DHPG, NE, DOPA, EPI, Cys-DOPA, DA, DOPET, Cys-DA, DOPAC, and DHCA (Figure 2A), as well the internal standards, which were DHBA or isoproterenol. In order to detect caffeic acid along with these catechols, the chromatographic run time had to be about 240 minutes. Standards for other catechols known to occur in coffee (e.g., chlorogenic acid) had retention times exceeding 240 minutes and were not measured.
Figure 2: Chromatographic recordings of (A) extracted standards, (B) plasma catechols at baseline in a healthy volunteer, (C) catechols 15 minutes after drinking 2 cups of caffeinated coffee in the same individual, and (D) catechols 15 minutes after drinking 2 cups of decaffeinated coffee in the same individual on a different day.
Inset in (A) shows relatively rapidly-eluting chromatographic peaks corresponding to 3,4-dihydroxyphenylglycol (DHPG), norepinephrine (NE), 3,4-dihydroxyphenylalanine (DOPA), epinephrine (EPI), 5-S-cysteinyldopa (Cys-DOPA), dopamine (DA), 3,4-dihydroxyphenylethanol (DOPET), 5-S-cysteinyldopamine (Cys-DA), and 3,4-dihydroxyphenylacetic acid (DOPAC). Dihydrocaffeic acid (DHCA) elutes relatively late and is the last eluting standard. The internal standard (IS) was 3,4-dihydroxybenzylamine. Note that after drinking coffee, at least 20 additional chromatographic peaks are present and that the results are nearly identical after caffeinated and decaffeinated coffee.
Assaying decaffeinated coffee by LCED after alumina extraction identified several catechols (Table 1). The main catechols were DHCA and caffeic acid. EPI, DOPA, DOPAC, and DHPG were also detected.
Table 1:
Catechol contents of decaffeinated coffee.
| Analyte | MW | μM |
|---|---|---|
| (daltons) | ||
| DHCA | 182 | 21.08 |
| Caffeic acid | 180 | 20.05 |
| EPI | 183 | 1.55 |
| DOPA | 197 | 0.90 |
| DOPAC | 168 | 0.82 |
| DHPG | 170 | 0.21 |
| NE | 169 | 0.00 |
| DA | 153 | 0.00 |
Notes: Sanka™ decaffeinated coffee was assayed. Abbreviations: DA=dopamine; DHCA=dihydrocaffeic acid; DHPG=3,4-dihydroxyphenylglycol; DOPA=3,4-dihydroxyphenylalanine; DOPAC=3,4-dihydroxyphenylacetic acid; EPI=epinephrine; NE=norepinephrine.
3.4. Coffee-related catechols in plasma
In all subjects, after coffee drinking there were at least 20 additional chromatographic peaks that did not correspond with known endogenous catechols. Representative chromatographs are in Figure 2. The chromatographs after drinking caffeinated coffee were remarkably similar to those after drinking decaffeinated coffee (Figures 2C and 2D).
Figure 3 shows chromatographs from a participant at baseline (Figure 3A) and at 120 minutes (Figure 3B) and 240 minutes (Figure 3C) after drinking caffeinated coffee. Between 120’ and 240’ the heights of all the coffee-related peaks decreased or stayed the same, with the exception of DHCA, the peak height for which increased substantially between 120’ and 240’.
Figure 3: Chromatographic recordings of alumina eluates from a healthy volunteer at (A) baseline, (B) 120 minutes after, and (C) 240 minutes after drinking 2 cups of caffeinated coffee.
Dashed lines indicate coffee-related peaks. Plasma DHCA increased substantially between 120 and 240 minutes (B, C), in contrast with other coffee-related peaks.
3.5. COMT+SAMe
Plasma from 2 subjects was assayed with or without incubation of the plasma with COMT+SAMe before the alumina extraction. In both participants, COMT+SAMe decreased the heights of or eliminated most of the additional coffee-related peaks. A representative chromatograph shows the reductions in heights of late-retaining peaks in Figure 4 and early-retaining peaks in Figure 5, for both caffeinated and decaffeinated coffee.
Figure 4: Chromatographic recordings (240 minutes duration) of extracted plasma 45 minutes after drinking caffeinated or decaffeinated coffee in a healthy volunteer, without or with incubation of the plasma with catechol-O-methyltransferase (COMT) and S-adenosylmethionine (SAMe) before the alumina extraction.
(A) Caffeinated coffee, without COMT+SAMe incubation; (B) Caffeinated coffee, with COMT+SAMe incubation; (C) Decaffeinated coffee, no COMT+SAMe incubation; (D) Decaffeinated coffee, with COMT+SAMe incubation. The internal standard was isoproterenol (ISO). Dashed lines placed to emphasize reduction or elimination of several chromatographic peaks by COMT+SAMe, indicating the peaks correspond to catechols. Brown circles placed to highlight disappearance of the dihydrocaffeic acid (DHCA) peak and blue circles disappearance of the caffeic acid peak after incubation of plasma with COMT+SAMe before the alumina extraction.
Figure 5: Early portions (first 20 minutes) of the same chromatographic recordings as in Figure 4.
(A) Caffeinated coffee, without COMT+SAMe incubation; (B) Caffeinated coffee, with COMT+SAMe incubation; (C) Decaffeinated coffee, no COMT+SAMe incubation; (D) Decaffeinated coffee, with COMT+SAMe incubation. Dashed lines placed to emphasize reduction or elimination of several early-retaining peaks by COMT+SAMe, indicating the peaks correspond to catechols. These include the peaks for the endogenous catechols 3,4-dihydroxyphenylglycol (DHPG), norepinephrine (NE), 3,4-dihydroxyphenylalanine (DOPA), and epinephrine (EPI).
3.6. Plasma catecholamines and endogenous catechols
Plasma levels of EPI and DA increased rapidly after drinking either caffeinated or decaffeinated coffee (Figure 6A, 6B). For statistical testing a mixed model was used for plasma EPI and DA levels, due to missing data as a result of co-chromatographic interfering peaks.
Figure 6: Mean (± SEM) values for plasma levels of dihydrocaffeic acid (DHCA) and endogenous catecholamines as a function of time after ingestion of 2 cups of caffeinated (black) or decaffeinated (gray) coffee.
(A) Dihydrocaffeic acid (DHCA); (B) Epinephrine (EPI); (C) Dopamine (DA); (D) Norepinephrine (NE). There were rapid, variable increases in plasma EPI and DA and slow, small increases in NE after coffee drinking either caffeinated or decaffeinated coffee.
For plasma EPI the interaction effect of coffee type X time was not statistically significant, meaning that the time-related trends in EPI levels did not depend on the type of coffee. At 15’ plasma EPI was already increased from baseline (p<0.0001 for caffeinated, p=0.016 for decaffeinated by Dunnett’s test). Mean plasma EPI for caffeinated coffee peaked at 0.38 pmol/mL at 45’. Thereafter, plasma EPI decreased slowly but was still above baseline at 240’ (p=0.039). Mean plasma EPI for decaffeinated coffee peaked at 0.28 pmol/mL at 45’. Thereafter, plasma EPI decreased and was no longer significantly above baseline.
Plasma DA responses to coffee ingestion were quite variable across individuals. Due to this variability, mean plasma DA did not change significantly as a function of time. Time-related trends in plasma DA levels did not depend on the coffee type.
Plasma NE levels increased relatively slowly and to a small extent after drinking caffeinated or decaffeinated coffee (Figure 6C). Although the time main effect was statistically significant (p=0.0088), at all time points the increases from baseline were not significant for either coffee type.
Plasma levels of other endogenous catechols were unchanged after drinking caffeinated or decaffeinated coffee (Table 2).
Table 2: Plasma catechols (means ± SEM) after drinking caffeinated or decaffeinated coffee.
Times are in minutes after drinking 2 cups of coffee. Concentrations are in units of pmol/mL (nM). Data for the main catechols of interest are in the Figures.
| CAFFEINATED | |||||
| TIME | DOPA | DHPG | DOPAC | Cys-DOPA | Cys-DA |
| 0 | 8.69 ± 0.76 | 5.80 ± 0.81 | 14.1 ± 5.1 | 3.80 ± 0.31 | 0.170 ± 0.063 |
| 15 | 6.74 ± 0.67 | 4.90 ± 0.77 | 15.4 ± 5.6 | 6.00 ± 2.22 | 0.072 ± 0.027 |
| 30 | 6.71 ± 0.54 | 4.70 ± 0.69 | 15.30 ± 5.0 | 6.00 ± 1.93 | 0.078 ± 0.023 |
| 45 | 7.20 ± 0.50 | 4.79 ± 0.61 | 14.5 ± 4.8 | 5.60 ± 1.58 | 0.074 ± 0.021 |
| 60 | 7.01 ± 0.59 | 4.58 ± 0.51 | 13.8 ± 4.6 | 5.01 ± 1.15 | 0.124 ± 0.048 |
| 120 | 7.89 ± 0.62 | 5.67 ± 0.63 | 12.9 ± 4.7 | 4.03 ± 0.68 | 0.090 ± 0.029 |
| 180 | 7.00 ± 0.80 | 6.27± 0.67 | 13.0 ± 5.0 | 3.64 ± 0.49 | 0.074 ± 0.030 |
| 240 | 8.22 ± 1.07 | 6.94 ± 0.82 | 12.9 ± 5.3 | 3.59 ± 0.67 | 0.063 ± 0.026 |
| DECAFFEINATED | |||||
| TIME | DOPA | DHPG | DOPAC | Cys-DOPA | Cys-DA |
| 0 | 9.03 ± 0.92 | 5.29 ± 0.777 | 17.1 ± 5.20 | 4.64 ± 0.90 | 0.112 ± 0.053 |
| 15 | 8.36 ± 0.60 | 4.85 ± 0.65 | 18.6 ± 5.1 | 5.44 ± 1.93 | 0.072 ± 0.036 |
| 30 | 7.95 ± 0.87 | 5.05 ± 0.73 | 18.6 ± 5.0 | 5.19 ± 1.60 | 0.075 ± 0.031 |
| 45 | 8.34 ± 0.89 | 5.01 ± 0.69 | 17.7 ± 4.5 | 5.43 ± 1.55 | 0.081 ± 0.030 |
| 60 | 8.75 ± 1.16 | 5.17 ± 0.61 | 16.5 ± 4.1 | 5.15 ± 1.41 | 0.085 ± 0.031 |
| 120 | 8.91 ± 1.04 | 5.71 ± 0.78 | 15.5 ± 4.0 | 4.30 ± 1.32 | 0.083 ± 0.026 |
| 180 | 8.83 ± 0.99 | 6.33 ± 0.57 | 15.13 ± 4.6 | 4.03 ± 0.769 | 0.076 ± 0.025 |
| 240 | 8.72 ± 1.19 | 5.84 ± 0.59 | 14.7 ± 4.8 | 3.85 ± 0.93 | 0.064 ± 0.019 |
Abbreviations: DOPA=3,4-dihydroxyphenylalanine; DHPG=3,4-dihydroxyphenylglycol; DOPAC=3,4-dihydroxyphenylacetic acid; Cys-DOPA=5-S-cysteinyldopa; Cys-DA=5-S-cysteinyldopamine.
3.7. Plasma DHCA
DHCA was the only identified coffee-related catechol that was detected consistently in the LCED chromatographs. Plasma DHCA varied as a function of time (p=0.020) but not coffee type, and the interaction effect of coffee type X time was non-significant. In all the participants plasma DHCA levels increased rapidly after drinking coffee, regardless of type (Figure 7A and 7B). At 15’ mean plasma DHCA was already increased from baseline (p=0.0091 by Dunnett’s test; Figure 7C). At 30’, 45’, and 60’ plasma DHCA was highly significantly above baseline (p<0.0001). The nadir mean value (9.2 pmol/mL) occurred at 120’. Subsequently, plasma DHCA increased in a second phase, with the peak mean value at 240’ above the baseline mean value (p=0.012). There was substantial variability in responses of plasma DHCA levels, which was especially notable at 15’ and 240’ (Figure 7A, 7B).
Figure 7: Individual values for plasma dihydrocaffeic acid (DHCA) after drinking (A) caffeinated or (B) decaffeinated coffee in 10 healthy volunteers.
Note that in all participants there are rapid increases in plasma DHCA levels after drinking either type of coffee. The increases are generally sustained for 240 minutes. (C) Mean (± SEM) values for plasma DHCA after drinking caffeinated (black circles) or decaffeinated (gray circles) coffee. Plasma DHCA increased bi-phasically.
3.8. Physiological variables
Finger systolic and diastolic blood pressures tended to increase after coffee drinking (p=0.060 for systolic; p=0.069 for diastolic; Figure 8). The increase in systolic pressure depended on the coffee type, since the interaction effect of coffee type X time was significant (p=0.039). The increase in diastolic pressure did not depend on the coffee type.
Figure 8: Physiological indices (means ± SEM) after drinking coffee (black=caffeinated, gray=decaffeinated).
(A) Finger systolic blood pressure (BPs); (B) Finger diastolic blood pressure (BPd); (C) Heart rate (HR); (D) Forearm vascular resistance (FVR); (E) Laser-Doppler (L-D) flux on the pad of the thumb, an index of cutaneous microcirculatory flow; (F) Skin temperature (°C), from a thermistor applied to the fifth finger. Drinking caffeinated coffee increased BPs. For both caffeinated and decaffeinated coffee cutaneous microcirculatory flow and skin temperature decreased.
For both caffeinated and decaffeinated coffee heart rate decreased slightly (p=0.0139 for the time effect, non-significant coffee type X time interaction effect). Cutaneous microcirculatory flow measured by laser-Doppler fluximetry decreased after coffee drinking (Figure 8F; p=0.0017 for the time effect, non-significant coffee type X time interaction effect). Skin temperature also decreased (Figure 8G; p<0.0001 for the time effect, non-significant coffee type X time interaction effect).
4. DISCUSSION
We report that drinking coffee, whether caffeinated or decaffeinated, results in the rapid appearance of numerous free (unconjugated) catechols in the plasma. Comparisons of chromatographs from samples before vs. after coffee drinking demonstrated at least 20 additional peaks.
The alumina extraction partially purifies catechols [16], the reverse phase chromatographic setup separates small organic amphipathic molecules, and the series arrangement of post-column electrodes quantifies reversibly oxidizable species [17]. This combination results in highly sensitive and specific detection of catechols. Reduction or elimination of the chromatographic peaks by incubation of the plasma with COMT+SAMe before the alumina extraction confirmed that the additional coffee-related peaks represented catechols.
The increases in plasma EPI and DA levels after ingestion of decaffeinated coffee were unexpected. It has been presumed that EPI responses to coffee ingestion reflect effects of caffeine [7]. A potential explanation for the concurrent increases in plasma EPI and DA levels is decreased plasma clearance of these catecholamines because of competition with coffee-related catechols for enzymes or transmembrane transporters. In addition, about 99% of circulating DA is in the form of DA sulfate [20]. Even a small decrease in DA sulfoconjugation could increase plasma DA levels, due to either competition for or inhibition of phenolsulfotransferases by coffee-related phenolic acids [21, 22].
We detected DHCA in the plasma of all but one subject at baseline, despite all the subjects having fasted overnight. Baseline DHCA varied by about 50-fold across individuals, without any obvious relationship to the habit of coffee drinking. Since many foodstuffs such as fruits and green vegetables contain catechol acids, sustained plasma DHCA responses to common dietary constituents may account for DHCA in plasma even after several hours of fasting.
4.1. Limitations
There was no untreated control group to assess physiological changes as a function of time under the testing conditions.
The number of subjects was too small to assess whether individual variability in plasma catechol responses to coffee drinking depend on age, sex, race, ethnicity, or habitual coffee intake.
Coffee-related chromatographic peaks sometimes interfered with EPI or DA peaks, resulting in incomplete datasets. In this situation a mixed model rather than ANOVA was used.
The plasma bioavailability of DHCA in coffee could not be estimated in the present study because of the likelihood of other sources of circulating DHCA in coffee, such as chlorogenic acid and caffeic acid.
Except for DHCA and caffeic acid, the coffee-related peaks did not correspond to standards for catechols in coffee. LC with post-column time-of-flight mass spectroscopy might identify the coffee-related catechols seen in the LCED chromatographs; however, based on the chromatographic peak heights the concentrations may be in the femtomolar range.
4.2. Perspective
Several reports have suggested that coffee drinkers have a decreased risk of developing Parkinson disease (PD) [23–27]. Most research about potential mechanisms of this protective effect has emphasized caffeine [28]; however, caffeine itself does not benefit patients with PD [29]. A potential alternative explanation is based on the catechols that are present even in decaffeinated coffee [11, 30–35]. In fruit flies, decaffeinated coffee mitigates dopaminergic neuronal loss in two PD models—alpha-synuclein transgenic and PRKN null mutants—whereas caffeine is ineffective [33]. A recent review focused on the outlook for a role of decaffeinated coffee in neurodegenerative diseases [30].
Since gut bacteria convert chlorogenic acid and caffeic acid to DHCA [13, 36], the late increase in plasma DHCA may reflect individually variable production of DHCA from phenol acid precursors in the enteric microbiome [3, 5]. Assessment of plasma DHCA responses to coffee ingestion might provide a novel means to test for altered microbiomic metabolism [37].
4.3. Conclusions
Within 15 minutes of drinking caffeinated or decaffeinated coffee, the plasma of healthy volunteers contains >20 additional compounds, most of which are still present at 240 minutes. Reductions in LCED peaks by incubating plasma with COMT+SAMe confirmed the coffee-related peaks correspond to catechols. Unexpectedly, caffeinated and decaffeinated coffee exert similar effects on plasma levels of endogenous catecholamines. Unlike other catechols, DHCA in plasma increases bi-phasically after ingestion of either caffeinated or decaffeinated coffee.
Supplementary Material
HIGHLIGHTS.
Coffee contains numerous 3,4-dihydroxyphenyl compounds (catechols).
The catechols are present even in decaffeinated coffee.
Alumina+liquid chromatography+series electrochemical detection detects catechols.
After drinking caffeinated or decaffeinated coffee, >20 catechols are present in the plasma.
Drinking either type of coffee increases plasma epinephrine levels.
5. ACKNOWLEDGEMENTS
5.1. Financial support
The research reported here was supported by the Intramural Research Program of the NIH, NINDS.
Funding source: Division of Intramural Research, NINDS, NIH.
Abbreviations:
- ANOVA
analysis of variance
- COMT
catechol-O-methyltransferase
- Cys-DA
5-S-cysteinyldopamine
- Cys-DOPA
5-S-cysteinylDOPA
- DA
dopamine
- DHCA
dihydrocaffeic acid
- DHPG
3,4-dihydroxyphenylglycol
- DOPA
3,4-dihydroxyphenylalanine
- DOPAC
3,4-dihydroxyphenylacetic acid
- EPI
epinephrine
- FVR
forearm vascular resistance
- IV
intravenous catheter
- LCED
liquid chromatography with electrochemical detection
- NE
norepinephrine
- NIH
National Institutes of Health
- NINDS
National Institute of Neurological Disorders and Stroke
- PD
Parkinson disease
- SAMe
S-adenosylmethionine
Footnotes
Financial Disclosure/Conflict of Interest: The authors have no conflicts of interest to disclose.
Declaration of interests
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
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