Profile and Content of Betalains in Plasma and Urine of Volunteers

Article pubs.acs.org/JAFC

Cite This: J. Agric. Food Chem. XXXX, XXX, XXX−XXX

Profile and Content of Betalains in Plasma and Urine of Volunteers after Long-Term Exposure to Fermented Red Beet Juice Tomasz Sawicki,† Joanna Topolska,† Ewa Romaszko,‡ and Wiesław Wiczkowski*,† †

Institute of Animal Reproduction and Food Research, Polish Academy of Sciences in Olsztyn, Tuwima 10 Str., 10-748 Olsztyn, Poland ‡ NZOZ Atarax, 1 Maja 3 Str., 10-117 Olsztyn, Poland ABSTRACT: The aim of this study was to determine profile and content of betalains in volunteers’ plasma and urine after longterm exposure to fermented red beet juice. During 6 weeks, 24 healthy volunteers consumed juice with a dose of 0.7 mg betalains/kg body weight. Betalains were analyzed by means of micro-HPLC-MS/MS. Twelve betalain derivatives were found in blood plasma and urine after juice intake. The highest betalains level in blood plasma (87.65 ± 15.71 nmol/L) and urine (1.14 ± 0.12 μmol) was found after the first and second week of juice intake, respectively. During juice consumption, the contribution of betalain metabolites was higher than that of native betalains, and interindividual variability in profile and content of betalains was observed. Summarizing, it was observed that long-term and regular consumption of the juice causes stabilization of profile and content of betalains in physiological fluids of volunteers, which include native compounds and their decarboxylated and dehydrogenated metabolites. KEYWORDS: betalains, red beetroot, urine, plasma, absorption, betalain metabolites

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plasma after the intake of cactus pear. Similarly, Allegra et al.13 found the unchanged form of indicaxanthin in rat plasma after a single intake of cactus pear. In addition, after a single ingestion of red beetroot juice by a human, only unchanged forms of betanin and isobetanin were detected in the samples of urine.14−16 Unfortunately, the previous studies do not provide data on the profile of native betalains and their metabolites in blood plasma and urine both after a single intake and after longterm consumption of betalain-rich products. Therefore, the aspects related to betalains absorption, metabolism, and excretion have so far not been fully known. Furthermore, the literature available does not contain any information about the interindividual variability in the content and profile of betalains in physiological fluids. It is known that the absorption, metabolism, distribution, and excretion of bioactive compounds, and thereby their bioavailability, may be associated with dietary habits, age, genetic aspects, microbiota, disease state, drug intake, and physical activity, as well as alcohol and smoking habits.17 However, these processes are also significantly affected by a specific chemical structure of bioactive compounds. What is more, under longterm exposition to biologically active substances, differences/ variations in gut microbiota could also be a very important factor.18 The aim of this research was to determine the profile and concentration of betalains in volunteers’ blood plasma and urine after long-term exposure to fermented red beet juice. In addition, the interindividual variability in the profile and content of betalains in these physiological fluids was examined.

INTRODUCTION Nowadays, it is believed that fruits, vegetables, and whole grain products are a crucial part of the human diet, since upon intake their phytochemical compounds may prevent or postpone the development of some chronic diseases.1,2 Red beetroot (Beta vulgaris L. ssp. vulgaris) is one of the most popular vegetables considered as a valuable source of bioactive compounds.3,4 It is cultivated in many countries worldwide and regularly consumed as a component of the daily diet. The main bioactive compounds found in red beetroot are betalains. These bioactive substances consist of two groups: betacyanins (red− violet pigments) and betaxanthins (yellow−orange pigments).5,6 Betalains have a number of health-promoting properties, exhibiting a strong antioxidant, antiviral, anticancer, antilipidemic, and antibacterial activity.6−9 It was also found that the main compound of betalains - betanin - induced the transcription factor Nrf2 and resulted in an increase of heme oxygenase-1, paroxonase 1, and cellular GSH.10 What is more, these natural pigments are very often used for the coloring of many groceries such as ice cream, yogurt, jam, and sweets.3,11 Finally, they are legalized by the European Union and labeled as E-162.5 Despite the presence of betalain pigments in many food products, their profile and content in human biological fluids has not been well-known yet. This knowledge is necessary as it may help to determine potential prohealth effects of betalains on consumers’ health. Therefore, investigations focused on the characterization of betalains profile and content, as well as determination of their metabolites appearing in human physiological fluids, should be undertaken. There are only a few studies describing the absorption of betalains after a single consumption of products containing these pigments. In the study of Tesoriere et al.12 only two native compounds (indicaxanthin and betanin) were detected in human blood © XXXX American Chemical Society

Received: February 20, 2018 Revised: March 27, 2018 Accepted: April 9, 2018

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DOI: 10.1021/acs.jafc.8b00925 J. Agric. Food Chem. XXXX, XXX, XXX−XXX

Article

Journal of Agricultural and Food Chemistry

column (33 μm, Polymeric Reversed Phase, 200 mg/3 mL, Torrance, CA, USA). The first step consisted of a 2-fold dilution of samples in water with 0.05% formic acid, vortexing by 1 min, and centrifugation (Centrifuge 5427R, Eppendorf, Germany) for 10 min (14,000 × g, 4 °C). Then, after conditioning the Strata-X column with a mixture of methanol/water (0.5/0.5, v/v, 1 mL) and 0.05% formic acid aqueous solutions (1 mL), the diluted samples were loaded. Next, the column was washed with 2 mL of water with 0.05% formic acid, and betalains were eluted with 2 mL of 50% methanol. The eluent obtained was evaporated to dryness with a stream of nitrogen at 30 °C and dissolved in 100 μL of water containing 0.05% formic acid. Finally, before injection into the micro-HPLC-MS/MS, the solution of all samples was centrifuged (20 min, 14,000 × g, 4 °C). In the case of red beet juice, instead of extraction, before the analysis it was only diluted 50 times in the extraction mixture. Each sample was prepared in triplicate. The micro-HPLC system (LC200, Eksigent, Vaughan, ON, Canada) coupled with a mass spectrometer (QTRAP 5500, AB SCIEX, Vaughan, ON, Canada) consisting of a triple quadrupole, ion trap, and ion source of electrospray ionization (ESI) was used to perform the analysis of betalains. The chromatographic determinations were performed on the HALO C18 column (100 mm × 0.5 mm x 2.7 μm; Eksigent, Vaughan, ON, Canada) at 45 °C with the flow rate of 25 μL/min. The elution was conducted using a solvent gradient system consisting of solvent A (0.012% formic acid aqueous solution with 5 mM ammonium bicarbonate) and solvent B (0.012% formic acid and 10% water acetonitrile solution with 5 mM ammonium bicarbonate). Gradient was as follows: 0% B (0−1.0 min), 0−20% B (1.0−2.0 min), 20−90% B (2.0−3.0 min), 90−90% B (3.0−3.8 min), 90−0% B (3.8− 4.0 min), and 0% B (4.0−5.0 min). The qualitative and quantitative analyses were made using the Multiple Reaction Monitoring (MRM) method. The quantity of betalains was calculated from the microHPLC-MS/MS peak area against betanin, as described earlier by Sawicki et al.3 An optimal identification of compounds analyzed was achieved under the following conditions: positive ionization, curtain gas: 25 L/min, collision gas: ion-spray voltage, 5400 V, temperature: 350 °C, 1 ion source gas: 35 L/min, 2 ion source gas: 30 L/min, declustering potential: 180 V, entrance potential: 10 V, collision energy: 40 eV, and collision cell exit potential: 27 V. Statistical Analysis. The data are presented as mean ± the standard error of the mean (SEM). To measure the differences between means of measurement points, one-way analysis of variance (ANOVA) and Tukey’s post hoc test were applied. P < 0.05 was considered significant. The statistical analysis was performed using the Statistica software (version 10.0; Stat Soft Corp., USA). The coefficients of variation (CV) for the concentration of major compounds and for the total concentration of betalains detected in plasma and urine samples was calculated with the following formula: CV [%] = RSD × 100%, where “RSD” stands for relative standard deviation, with ≤10%, 10−25%, and ≥25% considered low, moderate, and extensive interindividual variation.

Fermented red beetroot juice was used in the experiment, being an excellent source of betalains and characterized by a richer profile of betalain compounds than fresh red beet juice.19

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MATERIALS AND METHODS

Chemicals, Reagents, and Study Material. All reagents, including methanol, acetonitrile, formic acid, water, and ammonium bicarbonate were purchased from Sigma Chemical Co. (St. Louis, MO, USA). Fermented red beetroot juice was obtained from Zakład Przetwórstwa Owocowo-Warzywnego VITAPOL Sp. J. M. T. Ciszkiewicz, Podkowa Leśna, Poland (eng. Fruit and Vegetable Processing Plant VITAPOL Sp. J. M. T. Ciszkiewicz). Subject and Study Design. The study was conducted in accordance with the ethical principles of the Declaration of Helsinki and the study protocol approved by the Bioethical Committee at the Faculty of Medical Science of the University of Warmia and Mazury in Olsztyn (Poland, No. 7/2015). All volunteers were fully informed about the study and signed an informed consent form. Twenty-four healthy volunteers (19 women and five men), ages between 25 and 40, took part in the experiment. The study was carried out under the medical supervision of ATARAX Clinic, Olsztyn, Poland. The volunteers enrolled in the study had to meet the following criteria: they had to be certified healthy at a medical interview; have a body mass index (BMI) under 30; have no gastrointestinal disturbances, including gastric and duodenal ulcers; they could not participate in other clinical trials within 90 days prior to the survey, take drugs, abuse alcohol, be pregnant and breast-feeding, or take any medications or vitamin supplements. The experiment was conducted for 7 weeks and was divided into two periods. The first period (wash-out phase) lasted 1 week, while the second period lasted 6 weeks. During the first period volunteers were on their daily diet deprived of products containing betalain pigments (such as red beetroot products, strawberry ice cream, strawberry yogurts, wines, fruit juices, and other products containing E162 dye) in order to wash out betalain compounds from their body. In the second period of the experiment, volunteers’ diet was enriched with fermented red beetroot juice (200 mL/60 kg of body weight, 41.8 ± 1.9 mg of betalains/200 mL of juice) as the only source of betalains. Volunteers were asked to consume the juice every morning after breakfast. Before and throughout the study (once a week for 7 weeks), fasting blood samples from the elbow or forearm vein were taken to heparinized vacutainers (8 samples from each volunteer). Next, blood was centrifuged (500 × g, 15 min, 1000 × g, 10 min, 4 °C, Centrifuge MPW-351R, MPW-Med Instrument, Poland), and the obtained plasma was frozen and stored at −80 °C until analysis. In addition, according to the above scheme (−1, 0, 1, 2, 3, 4, 5, and 6 week), urine samples were collected from the volunteers (8 samples from each volunteer). The samples obtained were immediately frozen and stored at −80 °C until analysis. The schematic course of the experiment is shown in Figure 1. Sample Preparation and Chromatographic Analysis. The extraction and analysis of betalains in fermented red beet juice and the collected blood plasma and urine were carried out as described previously by Sawicki et al.20 Briefly, extraction of betalains from blood plasma and urine samples was carried out with the use of the Strata-X

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RESULTS AND DISCUSSION To determine the biological activity of phytochemicals it is important to investigate their fate after absorption and metabolism, thereby discovering the forms in which these substances occur in human physiological fluids. This shall be done upon long-term exposure to bioactive compounds, as only then the effects of both host metabolism and microflora of the gastrointestinal tract (which requires adaptation to the diet consumed) may be determined. Since bioactive compounds are often naturally present or frequently added to many commercially available foodstuffs, humans are exposed to these compounds on a daily basis. Betalains - natural pigments are an interesting and important group of these bioactive substances. Despite a limited prevalence in the plant kingdom, these colorants are widely used in the food industry21 as natural compounds used for the coloring of a wide range of food products. What is more, there is evidence that in in vitro

Figure 1. Schematic outline of the procedure of long-term exposure of volunteers to fermented red beet juice. B


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Journal of Agricultural and Food Chemistry conditions betalains display strong biological activities. It is, therefore, necessary to investigate the fate of these compounds in human physiological.5,9,22 To the best of our knowledge, this study is the first that shows the profile and content of native betalains and their metabolites in blood plasma and urine of volunteers after a long-term consumption of a red beetroot product. To explore betalains fate, volunteers in our study were served with fermented red beet juice, characterized by a more rich profile of betalain compounds than fresh red beet juice,18 which consequently allowed for the observation of the biotransformation of a larger range of compounds. The profile and content of betalains found in the juice of fermented red beet are presented in Table 1. Micro-HPLC-MS/MS analysis revealed eight

Table 2. Betalains Identified in Fermented Red Beet Juice, Blood Plasma, and Urine no.

compounds vulgaxanthin I

Betaxanthins 0.61

2

betanin

Betacyanins 1.96

3

isobetanin

2.01

551/389

4

betanidin

2.05

389/345

5 6

isobetanidin 17-decarboxy-betanin

2.11 2.12

389/345 507/345, 507/301

7

17-decarboxy-isobetanin

2.20

0.4

8

17-decarboxy-neobetanin

2.26

18.1 11.3 39.9 26.8 1.9 1.4 0.2 41.75 ± 1.86a

9

neobetanin

2.35

507/345, 507/301 505/343, 505/297 549/387

10

2,17-bidecarboxyneobetanin 2,15,17-tridecarboxyneobetanin 2,17-bidecarboxy-betanin

2.70

2,15,17-tridecarboxybetanin 6′-O-feruloyl-betanin/ isobetanin 2,15,17-tridecarboxy-2,3dehydro-neobetanin

contribution [%]

Betacyanins 2 3 4 5 6 7 14 a

betanin isobetanin betanidin isobetanidin 17-decarboxy-betanin 17-decarboxy-isobetanin 6′-O-ferulyl-betanin/isobetanin total

MRM ion pairs [m/z]

vulgaxanthin I

Betaxanthins 1

retention time [min]

1

Table 1. Contribution of Betalains in Fermented Red Beet Juice no.

compounds

11 12 13

Value was expressed as mg of betalains/200 mL of juice.

14 15

betalains in the juice consumed, based on the comparison of their retention time and the presence of respective parent and daughter ion pairs (Table 2) with the previously published data.3,23 Among betalains found, seven compounds belonged to the betacyanins group (betanidin, isobetanidin, betanin, isobetanin, 17-decarboxy-betanin, 17-decarboxy-isobetanin, and 6′-O-ferulyl-betanin/isobetanin), while one belonged to the betaxanthins group (vulgaxanthin I). The main compound found in fermented red beet juice was betanidin (aglycone of betanin), which covered almost 40% of total betalains identified in this juice. A similar profile of betalains in fermented red beetroot juice was presented by Czyżewska et al.19 In the study cited, six betalain compounds were detected in fermented juice: five compounds belonged to the betacyanins group (betanin, isobetanin, betanidin, isobetanidin, and neobetanin), and one belonged to the betaxanthins group (vulgaxanthin I). A similar profile of betalain compounds in fermented juices may be associated with both similar conditions of the fermentation processes and the growth of the same bacterial strains (Lactobacillus sp.) responsible for lactic fermentation. In our study, the solid phase extraction and microhigh performance liquid chromatography coupled with mass spectrometry was used to determine the profile and content of red beet betalains and their metabolites in volunteers’ blood plasma and urine.20 As a result of the above procedure 12 betalains were found in the fluids collected after a long-term consumption of fermented red beet juice (Tables 2−4). In addition to five native betalains, their seven metabolites were detected. In contrast, the previous studies investigated only the fate of betalains after a single ingestion of the products rich in these compounds and showed the presence of only native

340/323, 340/277

juice

551/389

juice, plasma, urine juice, plasma, urine juice, plasma, juice juice, plasma, urine juice

2.78

461/299, 461/255 417/255

2.80

463/301

2.81

419/257

2.83

727/551, 727/389 415/253

3.06

sample

plasma, urine plasma, urine urine plasma, urine plasma, urine plasma, urine juice, urine plasma, urine

betalains in blood plasma or urine studied. Among them, the analysis of betanin and isobetanin in urine after the intake of red beet products was described in three studies.14−16 In turn, the study of Teoserie et al.12 pointed to the occurrence of only two compounds (indiaxathin and betanin) in human plasma after a single intake of cactus pears. In addition, Allegra et al.13 found unchanged indiaxathin in rats’ plasma after the administration of betaxanthin preparation. On the other hand, in the study of Clifford et al.24 betalains were not identified in human blood plasma samples. However, the interpretation of these findings by the authors is incomprehensible. It is indicated that before absorption, betalains were intensively metabolized to unknown compounds, and it was, therefore, impossible to measure betalains in plasma samples. If this had been the case, these pigments would not have been discovered in the urine of volunteers after the consumption of betalain-rich products. Meanwhile, the above cited studies14,15 show the presence of native betalains in the urine of volunteers after betalains intake. Therefore, other factors related to study design, sample treatment, and condition of analysis could determine the results obtained by Clifford et al.24 In turn, our study, which was aimed to develop a method for the qualitative and quantitative analysis of betalains in physiological fluids,20 demonstrated that samples of rat plasma collected after the administration of betalain preparation into the stomach contained eight native betalains as well as their two metabolites. The identification schema of 11 compounds, including betanin (no. 2), isobetanin (no. 3), betanidin (no. 4), C


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Figure 2. Suggested routes of betalains biotransformation after consumption (compound designation in Table 2). Glu = glucose, R = feruloyl.

Sawicki et al.3 The remaining three compounds found were identified for the first time. Compound no. 11, characterized by the pseudomolecular ion at m/z 417 [M + H]+ and the fragment ion at m/z 255 [M + H − 162]+, was described as 2,15,17-tridecarboxy-neobetanin. Compound no. 13 that gave

isobetanidin (no. 5), 17-decarboxy-betanin (no. 6), 17decarboxy-isobetanidin (no. 7), 17-decarboxy-neobetanin (no. 8), neobetanin (no. 9), 2,17-bidecarboxy-neobetanin (no. 10), 2,17-bidecarboxy-betanin (no. 12), and 6′-O-feruloyl-betanin/ isobetanin (no. 14), was carried out as described earlier by D


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place of absorption of these compounds (stomach only−rats,20 entire digestive tract−volunteers (this study)). Unfortunately, all previous studies do not provide any unambiguous information related to the places and mechanisms related to absorption and metabolic paths of betalains. Nevertheless, based on the knowledge of other biologically active substances, among other flavonoids,25,26 and based on the results obtained in this study, some suggestions on the processes betalains may undergo in a human organism after consumption may be presented. Since native forms of betalains were found in blood plasma of volunteers after ingestion, it may be assumed that red beet betalains are absorbed from both the stomach and the small intestine without any transformation. In the case of rats, the absorption of betalains from stomach after 60 min from the administration of betalain preparation was demonstrated;20 however, the mechanism of this process itself has not been described. Presumably the absorption process of these pigments may take place in two ways: passive and active. Betalains might be involved in the process of simple diffusion, as they are lowmolecular-weight substances with a high affinity for water and may, therefore, penetrate through the pores with water. Betalains might also be absorbed by means of an active transport that requires energy input and the presence of specific carriers. In this case it is possible to transfer betalains that occur in a glucosidic form through an intestinal cell wall by means of SGLT1 carrier - a glucose transporter dependent on Na+ concentration gradient. Moreover, as in the case of anthocyanins,27 an organic anion carrier, bilitranslocase (TC 2.A.65.1.1), present in the gastric and intestine epithelium, may be involved in the absorption of betalains. This process may be possible because, as indicated by Kanner et al.16 and Tesoriere et al.,12,28 in certain conditions betalains may exist in an ionic form and become molecules that can be transported through the biological membranes with the participation of bilitranslocase. The results obtained in our study revealed a very small amount of aglycones (betanidin, 0.9%) that occurred in volunteers’ blood plasma despite a significant contribution of betalain aglycones in red beet juice consumed (66.7%, Table 1). Such a very low percentage of betalain aglycones present in the total content of betalains (betanidin plus isobetanidin, 0.6%) was also found in blood plasma of rats.20 There are several possible explanations of this phenomenon. The data obtained may indicate that the free forms of betalains are absorbed at a considerably small level or are not absorbed at all. However, it is also possible that the aglycones of betalain are absorbed yet immediately degraded due to their poor stability.21 What is more, due to this low stability, betalain aglycones may be degraded already in the lumen of the digestive tract before absorption. On the other hand, the appearance of aglycones in blood plasma may result from two different processes that are not mutually exclusive. First, the slight amount of betalain aglycones present in blood plasma could have formed as a result of betanin (glucosides of betanidin) hydrolysis in cytosole of the small intestine mucosa cells by β-glycosidases present there. Second, it is also plausible that betalain aglycones originated in the large intestine, since betalains which had not been absorbed in the upper part of the digestive tract could reach the large intestine and get hydrolyzed by β-glucosidases produced by the colon bacteria and alkaline medium. Next, the aglycones created could be absorbed by the mucous membrane of the large intestine. On the other hand, most of the betalains found in blood plasma are their glucoside derivatives (99.1%),

the molecular ion at m/z 419 [M + H]+ with the fragment ion at m/z 257 [M + H − 162]+ was identified as 2,15,17tridecarboxy-betanin. MS/MS analysis of compound no. 15 revealed the pseudomolecular ion at m/z 415 [M + H]+ and the fragment ion at m/z 253 [M + H − 162]+. Taking these results into account, compound no. 15 was identified as 2,15,17tridecarboxy-2,3-dehydro-neobetanin. Based on betalain compounds identified in plasma and urine samples, the possible degradation/transformations routes of these compounds in human body were presented in Figure 2. Apart from five native betalains (betanin, isobetanin, betanidin, 17-decarboxy-betanin, and 6′-O-ferulyl-betanin/isobetanin), their seven metabolites, including decarboxylated and dehydrogenated derivatives, were found (neobetanin, 17-decarboxyneobetanin, 2,17-bidecarboxy-betanin, 2,17-bidecarboxy-neobetanin, 2,15,17-tridecarboxy-betanin, 2,15,17-tridecarboxyneobetanin, and 2,15,17-tridecarboxy-2,3-dehydro-neobetanin). Among betalains identified, nine compounds (betanin, isobetanin, 17-decarboxy-betanin, 17-decarboxy-neobetanin, neobetanin, 2,15,17-tridecarboxy-neobetanin, 2,17-bidecarboxy-betanin, 2,15,17-tridecarboxy-betanin, and 2,15,17-tridecarboxy-2,3-dehydro-neobetanin) were found in both blood plasma and urine. At the same time two other betalains (2,17bidecarboxy-neobetanin and 6′-O-feruloyl-betanin/isobetanin) were detected only in urine samples, while one derivative (betanidin) was detected only in blood plasma (Table 2). Summing up, the results obtained indicate that after the consumption of the experimental beverage red beet betalains were absorbed intact and transformed to decarboxylated and dehydrogenated forms (Figure 2). In addition, there were significant differences found between the profile of betalains detected in blood plasma of volunteers who followed a longterm diet with fermented red beet juice and the profile of these compounds in blood plasma of rats which were subjected to 60 min exposure to betalains via gastric administration.20 In the case of native compounds, among eight betalains present in fermented red beet juice consumed by volunteers (betanin, isobetanin, betanidin, isobetanidin, 17-decarboxy-betanin, 17decarboxy-isobetanin, 6′-O-feruloyl-betanin/isobetanin, and vulgaxanthin I), only four compounds were found in blood plasma of these volunteers (betanin, isobetanin, betanidin, and 17-decarboxy-betanin). In turn, all betalain compounds occurring in betalains preparation administrated into rats’ stomach were found in blood plasma of these rats (betanin, isobetanin, betanidin, isobetanidin, 17-decarboxy-betanin, 17decarboxy-isobetanin, 2-decarboxy-neobetanin, and neobetanin).20 Among native betalains, both in the plasma of volunteers and rats, isobetanin and 17-decarboxy-betanin were predominant. In the case of betalain metabolites in volunteers’ blood plasma, six derivatives of betalains were found (17-decarboxy-neobetanin, neobetanin, 2,15,17-tridecarboxyneobetanin, 2,17-bidecarboxy-betanin, 2,15,17-tridecarbrbxybetanin, and 2,15,17-tridecarboxy-2,3-dehydro-neobetanin), while only two in rats blood plasma (15-decarboxy-betanin and 2,17-bidecarboxy-betanin). However, it should be emphasized that only one metabolite, 2,17-bidecarboxy-betanin, occurred in both blood plasma of rats and humans. These results suggest that there are some differences in the biological fate of betalains in humans and rats. It is also plausible, without excluding the above argument, that such differences in the profile may result from the application of two different research schemes, including the length of exposure to betalains (60 min−rats,20 6 weeks−volunteers (this study)) and the potential E


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Figure 3. Plasma and urine betalains content in volunteers who consumed fermented red beet juice (0.7 mg of betalains per kg body weight). Values are means ± SEM, n = 24. Values with different letters are significantly different at P < 0.05.

Table 3. Proportion of Betalain Derivatives Detected in Blood Over Time proportion [%] week no.

compounds

2 betanin 3 isobetanin 4 betanidin 6 17-decarboxy-betanin native 8 17-decarboxy-neobetanin 9 neobetanin 11 2,15,17-tridecarboxy-neobetanin 12 2,17-bidecarboxy-betanin 13 2,15,17-tridecarboxy-betanin 15 2,15,17-tridecarboxy-2,3-dehydro-neobetanin metabolites

−1

0

1

2

3

4

5

6

av of 1−6

0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0

0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0

8.1 0.0 5.4 5.6 19.1 0.0 0.0 24.4 7.6 15.8 33.1 80.9

5.4 19.8 0.0 22.2 47.4 0.0 0.0 24.2 0.0 20.0 8.4 52.6

23.4 0.0 0.0 0.0 23.4 0.0 0.0 39.5 0.0 37.1 0.0 76.6

10.1 0.0 0.0 10.8 20.9 11.0 10.3 16.8 12.8 14.3 13.9 79.1

16.5 0.0 0.0 17.4 33.9 0.0 0.0 22.8 0.0 22.2 21.1 66.1

16.3 0.0 0.0 0.0 16.3 0.0 15.7 24.5 0.0 22.3 21.2 83.7

13.3 3.3 0.9 9.3 26.8 1.8 4.3 25.4 3.4 22.0 16.3 73.2

which may suggest that the absorption mechanisms present in rats and humans prioritize betalains present in the glucosidic form. Considering the above deliberations, further research is needed to determine which paths of permeation through biological membranes (simple diffusion, organic anion carrier, SGLT1 transporter) are responsible for the absorption mechanisms of betalains. Moreover, at the same time, vulgaxanthin I present in juice consumed was not detected in the samples of blood plasma and urine collected. It may be, therefore, concluded that this betaxanthin representative was not absorbed at any level. However, previous studies12,13 showed that native indiaxathin - another betaxanthin representative - present in cactus pears - is absorbed. Consequently, an alternative explanation of the lack of vulgaxanthin I in volunteers’ plasma and urine may be the fact that this substance is characterized by a high lability,29,30 which may cause an intensive degradation in the environment of the digestive tract after ingestion. Again, based on the knowledge of flavonoids, e.g. anthocyanins, which upon consumption occur in human urine and blood plasma in both native and glucuronided, methylated, and sulfated derivatives forms,31 it may be assumed that betalain pigments may undergo a similar metabolic transformation. However, in blood plasma and urine of volunteers collected during our study, glucuronided, methy-

lated, and sulfated derivatives of betalain were not found. On the other hand, the samples of blood plasma and urine collected revealed a significant amount of decarboxylated and dehydrogenated derivatives of betalain. The formation of these metabolites might have been triggered by two factors, namely exposure of native betalains to increased temperature of volunteers’ bodies (higher than in the case of plant material) and the changing acidity of the environment of the gastrointestinal tract. These factors together could have contributed to the biotransformation of native betalains into decarboxylated and dehydrogenated forms. It was previously observed in the in vitro study of Wybraniec,32 that increased temperature and different acidity of the environment facilitates the transformation of betalains. Since after the consumption of fermented red beet juice betalains were frequently exposed to the above-mentioned factors, such elucidation appears plausible. In addition, as in the case of polyphenolic compounds,33 decarboxylation and dehydrogenation of native betalains might be catalyzed by gut microbes. All above results obtained in this study indicate that betalain undergoes biotransformation upon ingestion, with decarboxylation and dehydrogenation being the main metabolic paths of these pigments in the human body. The changes in the profile and concentration of red beet betalains in human blood plasma and urine are shown in Figure 3 and Tables 3 and 4. Neither native betalains nor their F


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Journal of Agricultural and Food Chemistry Table 4. Proportion of Betalain Derivatives Detected in Urine Over Time proportion [%] week no.

compounds

2 betanin 3 isobetanin 6 17-decarboxy-betanin 14 6′-O-feruloyl-betanin/isobetanin native 8 17-decarboxy-neobetanin 9 neobetanin 10 2,17-bidecarboxy-neobetanin 11 2,15,17-tridecarboxy-neobetanin 12 2,17-bidecarboxy-betanin 13 2,15,17-tridecarbobxy-betanin 15 2,15,17-tridecarboxy-dehydro-neobetanin metabolites

−1

0

1

2

3

4

5

6

av of 1−6

0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0

0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0

0.8 0.6 7.0 11.7 20.1 18.2 0.0 14.5 30.3 0.0 16.5 0.4 79.9

23.2 0.0 5.5 4.7 33.4 16.0 0.0 1.2 21.3 16.2 11.9 0.0 66.6

32.2 0.0 3.6 0.0 35.8 19.1 1.2 25.8 8.3 7.0 2.8 0.0 64.2

44.0 0.0 5.3 3.6 52.9 15.5 0.0 29.6 2.0 0.0 0.0 0.0 47.1

33.3 0.0 4.6 0.0 37.9 18.1 0.0 36.4 7.6 0.0 0.0 0.0 62.1

37.0 0.0 0.0 0.0 37.0 22.3 0.0 40.7 0.0 0.0 0.0 0.0 63.0

28.5 0.1 4.3 3.3 36.2 18.2 0.2 24.7 11.6 3.8 5.2 0.1 63.8

found in blood plasma within the period of 1−6 weeks were 2,15,17-tridecarboxy-neobetanin (no. 11), 2,15,17-tridecarboxybetanin (no. 13), and 2,15,17-tridecarboxy-2,3-dehydro-neobetanin (no. 15), which covered more than 25%, 22.0%, and 16% of total betalains discovered, respectively. Such a fingerprint of the main betalain metabolites was determined in blood plasma collected within the period of 2−6 weeks after the consumption of experimental beverage. On the other hand, a different fingerprint of dominant betalain metabolites was found after the first week of the challenge. The highest percentage concentration was found in the case of 2,15,17tridecarboxy-2,3-dehydro-neobetanin (no. 15, 33.1%). The next were 2,15,17-tridecarboxy-neobetanin (no. 11, 24.4%) and 2,15,17-tridecarboxy-betanin (no. 13, 15.8%). In the case of urine, the average profile of betalain metabolites for the period of 1−6 weeks was dominated by 2,17-bidecarboxy-neobetanin (no. 10), which covered more than 24% of total betalains excreted. The next were 17-decarboxy-neobetanin (no. 8, 18.2%) and 2,15,17-tridecarboxy-neobetanin (no. 11, 11.6%). Similarly as in blood plasma, the fingerprint with 2,17bidecarboxy-neobetanin as the main betalain metabolite was found in urine samples collected within the period of 3−6 weeks after the intake of fermented juice. On the other hand, a different fingerprint of dominant betalain metabolites in urine was detected after the first and second week of a regular consumption of juice. The highest percentage concentration was found in the case of 2,15,17-tridecarboxy-neobetanin (no. 11, 30.3% and 21.3%, respectively). In the case of the first week, the next were 17-decarboxy-neobetanin (no. 8, 18.2%) and 2,15,17-tridecarboxy-betanin (no. 13, 16.5%), while after the second week, the next were 2,17-bidecarboxy-betanin (no. 12, 16.2%) and 17-decarboxy-neobetanin (no. 8, 16.0%). On average, in both physiological fluids studied (blood plasma and urine), betanin (no. 2, 13.3% and 28.5%, respectively) was the predominant compound among native betalains found. What is more, in our study the interindividual variability in betalains profile and content after long-term exposure to fermented red beet juice was evaluated. To explain the notion of individual variability, the coefficient of variation (CV) was used to inform about the differentiation of a group with respect to a certain characteristic. In this case, the CV for major compounds and total betalains in plasma and urine samples was calculated. Considerable interindividual variations in betalains profile and content were found. The CV for the concentration

metabolites were identified in blood plasma and urine samples collected before red beet juice consumption, indicating that the wash-out phase (1 week) with a strict betalains-free diet was effective (Tables 3 and 4, Figure 3). After starting a regular intake of fermented red beet juice, betalains appeared in volunteers’ blood plasma and urine and were present throughout the whole experiment (6 weeks). However, during the first 2 weeks of a daily consumption of juice, the total concentration of betalains in blood plasma and urine significantly fluctuated (Figure 3). The highest content of total betalains (87.65 ± 15.71 nmol/L) in blood plasma was found after the first week of juice consumption. In the case of urine, the highest content of total betalains (1.14 μmol) was observed 1 week later - after the second week of beverage intake. Having reached the maximum concentration in the following weeks, betalains content in both physiological fluids gradually decreased to stabilize after the third week of red beet juice consumption. The lowest level of total betalains in blood plasma (2.87 ± 0.09 nmol/L) and urine (0.48 ± 0.07 μmol) was detected after the third and fourth week of the experimental diet, respectively. It is characteristic that both the maximum and the minimum concentration of betalains occurred in the urine of volunteers with a one-week delay compared to blood plasma. This may result from the fact that, just as in the case of other phytochemicals,34 the process of adaptation of volunteers’ bodies toward betalains metabolism must take place. Betalains present in blood plasma and the urine after a regular intake of fermented red beet juice were mainly represented by metabolites. The average percentage concentration of betalain metabolites in blood plasma and urine was 73.2% (Table 3) and 63.8% (Table 4), respectively. At the same time, native betalains found in blood plasma and urine comprised only 26.8% and 36.2% of total betalain derivatives explored, respectively. However, in both physiological fluids, a similar contribution of native compounds and metabolites to the total concentration of betalains was observed. In the case of blood plasma, after the second week of juice consumption, the percentage concentration of native betalains (47.4%) approached the values found for betalain metabolites (52.6%). A similar observation was found for urine but after the fourth week of the challenge and with the predominance of native compounds (52.9% - native compounds versus 47.1% metabolites). On average, the main betalain metabolites G


Article


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of major compounds detected in plasma samples was between 4% and 140%, 4% and 116%, 5% and 51%, 12% and 79%, and 2% and 178% for betanin, 17-decarboxy-betanin, 2,15,17tridecarboxy-betanin, 2,15,17-tridecarboxy-neobetanin, and 2,15,17-tridecarboxy-2,3-dehydro-neobetanin, respectively. The CV for the dominant betalain compounds (betanin, 2,17-tridecarboxy-neobetanin, and 2,15,17-tridecarboxy-neobetanin) identified in urine samples was from 45% to 120%, from 47% to 109%, and from 59% to 464%, respectively. In the case of total betalains concentration, the CV for plasma samples was from 5% to 78%, while for urine it was from 40% to 67%. In the case of plasma samples, the CV calculated for the last three measurement points (weeks 4, 5, and 6) was under 10%, which suggests that there are no statistical differences between these points. In conclusion, to the best of our knowledge, this is the first time when the profile and content of betalain compounds have been determined in blood plasma and urine of volunteers after a long-term, regular intake of the fermented red beetroot juice. We have shown that after the consumption of fermented juice of red beet, native betalains and their deglucosylated, decarboxylated, and dehydrogenated metabolites are present in human physiological fluids. What is more, our study demonstrated that the metabolites of betalains dominated in both blood plasma (73.2%) and urine (63.8%). However, it was also indicated that aglycones of betalain were poorly absorbed, and, consequently, most of the betalains found in physiological fluids were glucoside derivatives of betalains which were mainly absorbed. Concluding, the results presented in this study may help to interpret the biological properties and the potential physiological functions of betalains upon consumption. Further studies are needed to determine the place of absorption and the metabolic path of betalains, as well as to demonstrate whether there are any strong arguments for health-promoting activities of red beet betalains.

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AUTHOR INFORMATION

Corresponding Author

*Phone: +48 89 5234604. Fax: +48 89 524012. E-mail: w. [email protected]. ORCID

Tomasz Sawicki: 0000-0002-4304-1625 Wiesław Wiczkowski: 0000-0001-6021-5589 Funding

This research was supported by the statutory funds of the Department of Chemistry and Biodynamics of Food of the Institute of Animal Reproduction and Food Research of the Polish Academy of Sciences in Olsztyn, Poland. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The authors wish to thank the volunteers who participated in the study. REFERENCES

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Profile and Content of Betalains in Plasma and Urine of Volunteers

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