Identification of Urinary and Salivary Biomarkers for Coffee Consumption

Downloaded by UNIV OF CALIFORNIA SAN FRANCISCO on December 13, 2014 | http://pubs.acs.org Publication Date (Web): May 29, 2012 | doi: 10.1021/bk-2012-1098.ch002

Chapter 2

Identification of Urinary and Salivary Biomarkers for Coffee Consumption Roman Lang,* Anika Wahl, Timo Stark, and Thomas Hofmann Chair of Food Chemistry and Molecular Sensory Science, Technical University of Munich, Lise-Meitner-Str 34, 85354 Freising, Germany *E-mail: [email protected]

To evaluate putatively beneficial effects of coffee on human health, intervention studies are conducted. To correlate possible short term effects of consumption, the subjects need to run through washout periods prior to the coffee intervention to serve as their own control. The coffee constituents trigonelline and N-methylpyridinium (NMP) were identified as compounds contributing most to the dissimilarities between the urine of coffee drinkers and non-coffee drinkers. Application of a developed stable isotope dilution assay in a coffee intervention study revealed significantly higher values of trigonelline and NMP (normalized to creatinine) in coffee drinker urine for up to 48 h and 72 h, respectively, when compared to non-coffee drinkers, proposing these two compounds as indicators for coffee consumption. Further investigations demonstrated, that trigonelline and NMP can be detected in human salivary fluid for ~16 h. According to acquired food data, roast coffee appears to be the predominating source for trigonelline and NMP in human diet.

Introduction Food is meant to taste good and provide sufficient energy and nutrients to the body to meet the metabolic requirements. However, nutrition has a fundamental impact on human health and plays an important role in the development of chronic diseases.

© 2012 American Chemical Society In Recent Advances in the Analysis of Food and Flavors; Toth, S., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2012.


Evidence was found for an association of a diet rich in foods with high glycemic load and the development of coronary heart disease and diabetes type 2. Epidemiologic studies suggest “healthy” nutritional patterns rich in fruit, vegetables, fish and whole grain cereals are linked to lower rates of coronary heart disease, cancers and diabetes (1–5). The fact, that the instant impact of the diet may be small or even negligible, while the consumption over a life span may significantly affect health (6), severely complicates the investigations of the underlying mechanisms involved. In contrast to controlled studies with rigid dietary recommendations, data on nutritional habits in “free-living populations” are recorded by questionnaires. These, however, are prone to bias since they rely on the study participant recalling the consumed diet in rather great detail. Suitable markers could help objectify these data with respect to accuracy (7). The compounds in Figure 1 give an overview on some of the few food biomarkers developed and validated as objective measures to assess dietary intake.

Figure 1. Structures of proposed biomarkers indicating consumption of citrus fruit (A: proline betaine), wine (B: metabolites of resveratrol, e.g. resveratrol-3-O-glucuronide) and whole-grain rye (C: alkylresorcinols, e.g. 1,3-dihydroxy-5-nonadecylbenzene).

Determination of urinary proline betaine has been proposed as a quantitative marker for consumption of citrus fruit (7). Alkylresorcinols, which are present nearly exclusively in the outer parts of wheat and rye grains have been investigated as markers for consumption of whole grain cereals (8, 9). Metabolites of the stilbenoid resveratrol, present in e.g. berries and grapes, were found to be suitable biomarkers for wine consumption (10). A luxury much appreciated and habitually consumed by millions of people is roast coffee. Valued particularly for its stimulating effect and its pleasant flavor, moderate consumption of the brew is discussed in the context of putative beneficial side effects on human health, e.g. lowering the risk to develop diabetes type 2 or Alzheimer‘s disease (11–13). The underlying mechanisms, however, are unclear. In order to examine both short and long term effects of roast coffee on health by means of intervention studies, the intake of coffee needs to be recorded. A valid quantitative biomarker for coffee consumption could help to objectify and to add accuracy to the data from dietary questionnaires. Some compounds have 14 In Recent Advances in the Analysis of Food and Flavors; Toth, S., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2012.


been proposed for this purpose in the past, e.g. feruloylglycine, which is a major urinary metabolite of chlorogenic acids (14). However, chlorogenic acids are widely distributed in plants and therefore are not specific for coffee (15). The aim of the studies was to identify coffee specific compounds in human urine suitable to trace coffee consumption, develop a high throughput quantitation method and evaluate the feasibility of the markers in a pilot scale human intervention study.

Experimental Section Chemicals were obtained from Sigma-Aldrich (Steinheim, Germany). d3-creatinine was obtained from CDN (Dr. Ehrenstorfer, Augsburg, Germany). N-methylpyridinium iodide, d3-N-methylpyridinium iodide and d3-trigonelline hydroiodide were synthesized (16). The holistic screening of samples of coffee drinker urine and non-coffee drinker urine by HILIC-UPLC-Time-of-Flight-mass spectrometry, principal component analysis, validation of a stable isotope dilution assay (SIDA) for LC-MS/MS quantitation of N-methylpyridinium, trigonelline and creatinine in human urine, as well as the design of the human intervention study are published elsewhere (17). Quantitation of N-Methylpyridinium and Trigonelline in Human Urine For quantitation of N-methylpyridinum, trigonelline and creatinine, the urine sample (10 µL) was diluted with the internal standards (d3-NMP 1 µM, d3-trigonelline 1 µM and d3-creatinine 10 µM in acetonitrile/water 9/1, 1 mL), centrifuged (12,500 rpm, 5 min) and the supernatant injected into the LC-MS/MS system (1 µL). Quantitation of N-Methylpyridinium and Trigonelline in Human Saliva For quantitation of N-methylpyridinium (NMP) and trigonelline in human saliva, calibration standards were prepared by mixing aliquots of blank saliva (900 µL) with aqueous dilutions of NMP and trigonelline (0.200-500 µM, 100 µL) yielding saliva samples containing NMP and trigonelline in concentrations of 0.02-50 µM. To evaluate precision and accuracy, quality control samples were prepared by spiking blank saliva (900 µL) with solutions of NMP and trigonelline (100 µL) yielding samples with low (0.31 µM in saliva) and high (2.5 µM in saliva) addition levels. The lower limit of quantification (LLOQ) was defined as the lowest calibration standard with precision 80 %. The calibration standards, quality control samples and authentic saliva samples (200 µL) were mixed with the internal standard solution (d3-NMP and d3-trigonelline, 1 µM, 200 µL) and acetonitrile (600 µL) and vortexed briefly. The samples were centrifuged (12,500 rpm, 5 min) and the clear supernatant analysed by LC-MS/MS (17). 15 In Recent Advances in the Analysis of Food and Flavors; Toth, S., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2012.

Saliva samples were obtained from coffee drinkers (n=2) before and directly after coffee ingestion, as well as after extensive mouth rinsing with citric acid (0.5% in drinking water) and drinking water. Within the first 30 min, saliva samples were collected at 5 min intervals, and finally after 40, 50, 60, 90 and 120 min. Saliva samples were processed and analysed in triplicate (n=3).


Analysis of Trigonelline and NMP in Coffee Powder and Food Products The sample (200 mg) was mixed with aqueous ethanol (50/50, v/v, 5 mL) and heated in a closed reaction vessel (1 h, 100 °C). After cooling, the suspension was decanted into a 25 mL volumetric flask, filled with water and mixed. An aliquot (100 µL) was added to the internal standard solution (d3-trigonelline 1 mM and d3-NMP 0.25 mM, 100 µL) and incubated with light shaking (5 min). An aliquot of the mixture (100 µL) was applied on a 96 well plate SPE (100 mg, RP18) preconditioned with acetonitrile and water (200 µL each). The sample was sucked through the material and washed with water (2×100 µL). The combined filtrates were diluted with acetonitrile (1+9, v+v) and injected into the HPLC-MS/ MS system (2 µL). Each sample was extracted, processed and analysed in duplicate (n=2).

Results and Discussion Identification of Coffee Specific Compounds in Human Urine In order to locate coffee specific compounds in human urine, a holistic screening of individual as well as pooled urine samples of habitual coffee drinkers (n=9) and non-coffee drinkers (n=9) was performed by means of HILIC-UPLC-Time-of-Flight mass spectrometry. Each sample was injected in quadruplicate and from the exact mass-retention time data, a principal component analysis revealed a distinct differentiation of coffee drinker- and non-coffee drinker urine (17). Comparison of the pooled samples and visualization of the differences in an S-Plot forwarded three ions (see Figure 2) contributing most to the dissimilarities of the two samples. Calculation of the sum formulae based on the exact masses proposed C7H8NO2, C7H9N2O and C6H8N, fitting well to the compounds trigonelline (A), N-methylnicotinamide (B) and N-methylpyridinium (C). The identity of these compounds was verified by comparison of the retention times and the exact masses of reference compounds and authentic coffee-drinker urine samples as given in Figure 3. Trigonelline is a phytohormone widely distributed in plants and therefore present in many foods like tomatoes, barley corn and particularly legumes (18). In green coffee, it is the second most abundant alkaloid beside caffeine and accounts for roughly 0.3% to 1% dry weight basis (19). It is known that trigonelline degrades to volatile pyridines and pyrroles, nicotinic acid, N-methylpyridinium and other compounds during coffee roasting (20). Pyrolysis experiments with various salts of trigonelline demonstrated, that N-methylpyridinium becomes a major reaction product when temperatures at 245°C were applied (21). In 16 In Recent Advances in the Analysis of Food and Flavors; Toth, S., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2012.


contrast to trigonelline and N-methylpyridinium, N-methylnicotinamide is one of the major metabolites of niacin, a compound present in many different foods. We excluded N-methylnicotinamide as it was not a coffee specific substance, and focused on trigonelline and NMP as potential markers for coffee consumption.

Figure 2. Visualization of ions (m/z) contributing most to the dissimilarities between pooled samples of coffee drinker urine and non-coffee drinker urine by means of an S-Plot (Markerlynx™). The compounds at the bottom left area are the most specific markers for coffee drinker’s urine: A) trigonelline, B) N-methynicotinamide, C) N-methylpyridinium. Original data from (17).

Method Development for Quantitation of the Coffee Specific Compounds in Urine The quantitative analysis of trigonelline and N-methylpyridinium (NMP) in urine specimens was planned to be a high throughput stable isotope dilution analysis involving a minimum of sample preparation. 17 In Recent Advances in the Analysis of Food and Flavors; Toth, S., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2012.


Figure 3. Base peak Ion chromatograms (ESI+, m/z 50-600) of pooled coffee drinker urine (A), the extracted ion chromatograms of the most important markers m/z 138.0565 (B), 137.0724 (C), 94.0662 (D), and the Base Peak Ion chromatogram of a standard solution containing the reference compounds N-methylpyridinium (1), N-methylnicotinamide (2) and trigonelline (3) (E).

18 In Recent Advances in the Analysis of Food and Flavors; Toth, S., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2012.


The composition of the eluent was a mixture of aqueous acetonitrile containing formic acid and ammonium acetate, allowing baseline separation of creatinine, trigonelline and NMP under isocratic conditions. The mass spectrometer was operated in positive electrospray mode. Compound specific mass transitions were optimized for selective quantitation in MS/MS. Calibration standards of mixtures of analytes and deuterated internal standards were prepared in artificial urine since no urine matrix void of NMP, trigonelline and creatinine could be obtained. The area ratios of analyte and internal standard were plotted versus the concentration ratios, and linear regressions were calculated with 1/x weighing. In the final protocol, a small aliquot of sample was diluted in a solution of the labeled internal standards in acetonitrile. Occasionally occurring precipitates were removed by centrifugation, and the resulting solution could be directly injected into the LC-MS/MS system. Creatinine, trigonelline and NMP could be quantified in urine with a precision 79) and d3-trigonelline (m/z 141>97) are shaded, the analytes NMP (m/z 94>79) and trigonelline (m/z 138>94) are given in white. A) 120 min after coffee ingestion, B) 16 h after coffee ingestion (with enlargement 1.4 to 2.6 min), C) saliva of a non-coffee drinker.

To investigate the value of oral fluid as an alternative to urine for control of coffee ingestion, we validated the quantitation method for saliva matrix. Calibration of the standards and references was done in saliva with no detectable amounts of NMP and only small concentrations of trigonelline (56 pmol/mL) leading to an offset of the calibration (cf. Table 1). The precision and accuracy of the standards based on the back calculated data were

Identification of Urinary and Salivary Biomarkers for Coffee Consumption

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