effect of simultaneous consumption of milk and coffee on

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Article

Effect of Simultaneous Consumption of Milk and Coffeeon Chlorogenic Acids’ Bioavailability in Humans.

Giselle Silva Duarte, and Adriana FarahJ. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/jf201906p • Publication Date (Web): 31 May 2011

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1

Effect of Simultaneous Consumption of Milk and Coffee on Chlorogenic Acids’ 1

Bioavailability in Humans. 2

GISELLE S. DUARTE † and ADRIANA FARAH † ‡* 3

4

† Instituto de Química, Universidade Federal do Rio de Janeiro (UFRJ), Rio de Janeiro, 5

RJ, Brazil and ‡Instituto de Nutrição, Universidade Federal do Rio de Janeiro (UFRJ), 6

Rio de Janeiro, RJ, Brazil. 7

8

† ‡*Correspondent author: Adriana Farah. Instituto de Química. Av. Athos da Silveira 9

Ramos, 149, Ilha do Fundão, CT, Bloco A, 528A, Rio de Janeiro, RJ, 21941-909, 10

Brazil. Tel +55 21 2562 7352 Fax +55 21 25628213. E-mail: [email protected]. 11

12

Running title: CGA bioavailability after coffee and milk consumption 13

ACS Paragon Plus Environment

2

ABSTRACT 14

Different studies have shown that milk may interact with polyphenols and affect their 15

bioavailability in humans. The present study investigated the effect of the simultaneous 16

consumption of coffee and milk on the urinary excretion of chlorogenic acids (CGA) 17

and metabolites. Subjects were submitted to consumption of water, instant coffee (609 18

mmol of CGA) dissolved in water and instant coffee dissolved in whole milk. Urine was 19

collected for 24h after consumption of each treatment for analysis of CGA and 20

metabolites by HPLC/LC-MS. The amount of CGA and metabolites recovered after 21

consumption of combined coffee-milk (40 ± 27 %) was consistently lower in all 22

subjects compared to coffee alone (68 ± 20 %). Concluding, the simultaneous 23

consumption of milk and coffee may impair the bioavailability of coffee CGA in 24

humans. 25

26

Keywords: Chlorogenic acids, bioavailability, coffee, coffee and milk interaction, 27

polyphenols. 28

29

30

31


3

INTRODUCTION 32

In the last few years, coffee began to be considered by many as a functional 33

food, owing to its high content of bioactive compounds, mainly the chlorogenic acids 34

(CGA), which usually account for about 1-4% of roasted coffee composition [1]. These 35

phenolic compounds are esters of hydroxycinnamic acids and quinic acid. The main 36

subclasses in coffee are caffeoylquinic acids (CQA), dicaffeoylquinic acids (di-CQA) 37

and feruloylquinic acids (FQA) with at least 3 isomers per group [2]. Minor CGA 38

compounds include p-coumaroylquinic acids (p-CoQA) and mixed esters such as 39

caffeoylferuloylquinic acids and caffeoyltryptophan. A series of studies have shown that 40

these compounds possess antioxidant [3], antimicrobial [4], hepatoprotective [5], 41

immuno-stimulating [6] and hypoglycaemic properties [7], among others. CGA lactones 42

(CGL) have also shown to be bioactive in vivo [8]. As a consequence, various new 43

coffee based products are being created and massive research is performed in the intent 44

of combining the peculiar and appreciated flavour of coffee with its biological 45

properties [1]. 46

Today, coffee is the most consumed beverage in the world, with USA and Brazil 47

as the main consumer countries. Regarding consuming habits, while in USA adding 48

cream to coffee is a regular practice, in Brazil, whole, skimmed or semi-skimmed milk 49

accompany coffee very often, in different amounts, according to the population segment 50

or individual tastes. One of the most common consumption habits is adding a few grams 51

or milliliters of instant or brewed coffee to a cup of whole milk. 52

We have recently shown that CGA are considerably bioavailable in humans [9; 53

10]. However, previous studies have also shown that the bioavailability of polyphenols 54

may be affected by the interaction with dietetic constituents, especially proteins [11; 55

12]. Additionally, Muralidhara et al. [13] and Dupas et al. [14] showed using in vitro 56


4

assays that albumin and casein, respectively, were able to bind CGA by both covalent 57

and non-covalent interactions and suggested that the simultaneous consumption of 58

coffee and milk could result in a negative impact on CGA absorption. However, studies 59

investigating this hypothesis are scarce and inconclusive. Thus, the aim of the present 60

study was to evaluate the effect of the simultaneous consumption of coffee and milk on 61

the urinary excretion of CGA and metabolites. 62

63

SUBJECTS AND METHODS 64

Subjects. Five non-smoking subjects (3 female and 2 male), 24-35 years of age, 65

were recruited at Federal University of Rio de Janeiro (UFRJ). They were healthy as 66

judged by a medical questionnaire, with normal blood values for hemoglobin and 67

hematocrit and were not taking any medication or nutritional supplements at the time of 68

the study. The study protocol was approved by the Ethics Committee of Clementino 69

Fraga Filho Hospital (UFRJ) and fully explained to the subjects, who gave their written 70

informed consent prior to participation in the study. 71

Coffee beverages preparation. Samples of regular and instant coffee (C. 72

canephora cv. Conillon) were kindly provided by COCAM Company (São Paulo, 73

Brazil). The coffee beverage was prepared, by mixing 4 g of commercial medium 74

roasted instant coffee with 200 mL of freshly hot water (60-70ºC). The coffee-milk 75

beverage was prepared in the same way as for the coffee beverage, with replacement of 76

water by whole milk (200 mL) purchased in a local market and free of additives. The 77

milk doses offered to the subjects contained 9.6 g of carbohydrates, 6.7 g of proteins 78

and 7.1 g of lipids. Both drinks were prepared on each day of the study, immediately 79

before consumption. 80


5

Study design and sample collection. Subjects were instructed not to consume 81

phenolic-containing foods during the 48h prior to each experiment and during the 24h 82

of the day of the experiment. They were monitored to repeat the same diet for the three 83

treatments. All the treatments were performed in a randomized crossover design with a 84

minimum 7d interval between tests. On separated days, after 10h overnight fasting, a 85

standard amount of test beverage (200mL) was offered to each subject. Urinary samples 86

were collected for 4h before (baseline) and at intervals of 0-4h, 4-8h, 8-12h, 12-24h 87

after each treatment into appropriate plastic containers and aliquots for determination of 88

CGA and metabolites were acidified with HCL and kept frozen in -80ºC until analysis. 89

Blood hemoglobin and hematocrit analyses. Hemoglobin was mensured by the 90

cyanomethmioglobin method, using a commercial kit (Bioclin, Quibasa, Brazil). 91

Hematocrit was determined by conventional capillary centrifugation. 92

CGA extraction in brewed coffee. CGA in coffee beverages were extracted 93

according to Farah et al. [8], using methanol (40%) and Carrez solutions for 94

clarification. 95

CGA and metabolites extraction in urine samples. GGA and metabolites were 96

extracted in duplicates as described in detail by Monteiro et al. [9], using Helix pomatia 97

(Sigma-Aldrich, USA) extract containing �-glucoronidase and sulfatase activities for 98

deconjugation of glucuronic acid conjugates and sulfated forms. 99

Analyses of CGA and metabolites in coffee and urine. Analyses were performed 100

according to Farah et al. [8] for coffee and to Monteiro et al. [9] for urine, using a 101

HPLC-DAD gradient system (SPD-M10A SHIMADZU, Japan), with a C18 Kromasil 102

guard column and column (New York, USA) and a gradient with methanol and 0.3% 103

formic acid running at 1mL/min. Identification and quantification of CGA and 104

metabolites in coffee and urine were performed by comparison of the retention time of 105


6

investigated peaks with those of the respective standards. Peaks’ identities were 106

confirmed by LC-MS, according to Farah et al. [15]. 107

Standards were injected as a pool. A mixture of 3-CQA, 4-CQA, and 5-CQA 108

was prepared from 5-CQA using the isomerization method of Trugo and Macrae [16]. 109

For di-CQA, a mixture of 3,4-diCQA, 3,5-diCQA e 4,5-diCQA was kindly donated by 110

Professor Macrae (University of Reading, England). Standards of 5-CQA, caffeic acid, 111

ferulic acid, isoferulic acid, p-coumaric acid, dihydrocaffeic acid, vanillic acid, gallic 112

acid, syringic acid, sinapic acid, benzoic acid, 4-hydroxybenzoic acid, 2,4- 113

dihydroxybenzoic acid, trans-3-hydroxycinnamic acid, 3-(4’-hydroxyphenyl)propionic 114

acid, 3,4-dihydroxyphenylacetic acid and hippuric acid were purchased from Sigma-115

Aldrich (St. Louis, MO, USA). 116

Recovery determinations were made by considering the total number of 117

equivalent moieties of cinnamic and quinic acids consumed in the coffee extracts and 118

the total number of phenolic acid moieties recovered in urine collected for 24h after 119

each treatment. 120

Statistical analysis. Results are presented as means with corresponding SD. The 121

comparison between the urinary concentrations of CGA and metabolites was performed 122

using ANOVA factorial and one-way ANOVA, with post-test of Fisher. All statistical 123

analyses were performed using GraphPad Prism® software version 4.0 (USA). 124

Differences were considered significant at p < 0.05. 125

126

RESULTS 127

128

CGA in brewed coffee beverages. Nineteen major CGA compounds were quantified in 129


7

the 4g portion of instant coffee (in 200 mL of water or milk) offered to subjects in both 130

coffee treatments (Table 1). CQA represented most of CGA composition (77.5 %), 131

followed by FQA (~11 %), diCQA and CQL (~3.5 % each), caffeoyltryptophan (~3 %), 132

5-p-CoQA (~1 %) and caffeoylferuloylquinic acids (~0.5 %). The mean total amount of 133

CGA in the instant coffee portion was 0.61 mmol, which corresponded to 1.19 mmol in 134

equivalent moieties of cinnamic and quinic acids. 135

136

Subjects characterization. Three female and 2 male subjects participated in this study. 137

All hemoglobin and hematocrit values were normal compared to reference data. There 138

was no significant difference among the anthropometric and biochemical parameters 139

observed in all three treatments (Table 2). 140

141

Urinary excretion of CGA compounds and metabolites after water, coffee and coffee-142

milk consumption. Six CGA (3-CQA, 4-CQA, 5-CQA, 3,4-di-CQA, 3,5-diCQA e 4,5-143

diCQA) and 16 phenolic compounds (caffeic, vanillic, ferulic, isoferulic, p-coumaric, 144

gallic, 4-hydroxybenzoic, dihydrocaffeic, syringic, sinapic, 2,4-dihydroxybenzoic, 145

hippuric, 3,4-dihydroxyphenylacetic, 3-(4’-hydroxyphenyl)propionic, trans-3-146

hydroxycinnamic and benzoic acids) were identified in the baseline urine and in all 147

intervals for 24h after the consumption of all 3 treatments. 148

Baseline CGA values ranged from 46.5 to 922 μmol with variations from 1 to 9 149

fold for the same individual intra-day. Total phenolic acids ranged from 330.1 to 4823.2 150

μmol, with variations from one to fourteen fold for the same individual intra-day. 151

The total urinary excretion of phenolic compounds in the water treatment was 152

1.3 ± 0.8 mmol. Higher amounts of CGA and metabolites were excreted after coffee 153

(3.3 ± 1.4 mmol, p=0.004) and coffee-milk (2.2 ± 0.6 mmol, p=0.006) consumption. 154


8

While after water consumption the highest average excretion of phenolic compounds 155

occurred between 4-8h, after coffee and coffee-milk consumption, the highest average 156

excretion occurred between 8-12h (Figure 1). Although a large inter-individual 157

variation was observed in the urinary excretion of CGA and metabolites in all three 158

treatments, varying from 0.33 to 4.8 mmol, subjects showed a similar pattern of 159

excretion up to 24h after the consumption of the 3 treatments regarding the 160

predominance of compounds. Hippuric, 3,4-dihydroxyphenylacetic, dihydrocaffeic, 161

vanillic and gallic acids were the main compounds identified after the beverages 162

consumption, except for in water treatment in which 3,4-dihydroxyphenylacetic acid 163

was a minor compound. Together, these five compounds were responsible for about 164

96% and 97% of all compounds excreted in water and coffee treatments respectively. 165

Hippuric acid (N-benzoyl-glycine) excretion after water, coffee and coffee-milk 166

consumption represented respectively 88%, 80% and 80% of total phenolic compounds 167

identified up to 24h after consumption. Higher amounts of this compound were 168

observed in urine after coffee (2.5 ± 0.4 mmol) and coffee-milk (1.7 ± 0.3 mmol) 169

consumption when compared to water consumption (1.1 ± 0.4 mmol). 3,4-170

dihydroxyphenylacetic acid was the second major compound excreted up to 24h and 171

followed the same pattern as hippuric acid, with a higher excretion in coffee treatment 172

(0.5 ± 0.3 mmol), followed by coffee-milk (0.2 ± 0.1 mmol) and water treatments (0.01 173

± 0.02 mmol). High levels of dihydrocaffeic, vanillic and gallic acids were also present 174

in the urine of subjects after consumption of both coffee beverages, being together 175

responsible for 7 % of total urinary excretion of phenolic compounds (Figure 2). 176

In respect to minor phenolic compounds, as p-coumaric, syringic, sinapic, 177

benzoic, 4-hydroxybenzoic, 2,4-dihydroxybenzoic, trans-3-hydroxycinnamic and 178

isoferulic acids, together they contributed to less than 5 % of total urinary excretion 179


9

without significant differences among the three treatments. In respect to CGA 180

compounds, 3-CQA, 4-CQA and 5-CQA isomers were found in urine samples, resulting 181

in less than 1 % of total urinary excretion. Although a decrease of 23% was observed in 182

the urinary excretion of CQA in coffee and coffee-milk treatments (from 4.37 ± 3.9 183

μmol to 3.36 ± 2.02 μmol), it was not statistically significant. DiCQA were also 184

identified in the urine of subjects, but it was not possible to quantify them because the 185

values were below the detection limit (LOD = 0.002 μmol/L of 5-CQA). No FQA, p-186

CoQA, caffeoyltryptophan or CGL was identified in the evaluated urine samples. 187

Regarding the urinary recovery of phenolic metabolites and CGA up to 24h after 188

the consumption of coffee containing on average 0.61 mmol of CGA - which 189

corresponded to 1.20 mmol of equivalents of cinnamic and quinic acids - subjects 190

excreted from 0.41 to 1.1mmol (average of 68 %) of consumed CGA equivalents in 191

coffee treatment. On the other hand, after coffee-milk consumption only 0.31 to 0.64 192

mmol (average of 40 %) of ingested CGA equivalents was excreted. This represents an 193

average decrease of 28 % comparing to coffee alone. 194

Considering the total urinary excretion of intact CGA compounds, only from 0.3 195

% to 1.2 % (average of 0.7 %) of total CGA was recovered in urine after coffee 196

consumption, whereas from 0.2% to 0.7 % (average of 0.5 %) was recovered after 197

coffee-milk consumption, an average decrease of 19 % comparing to coffee alone. 198

199

DISCUSSION 200

The CGA contents in both plain coffee and coffee-milk beverages (200 mL) 201

used in the present study are in agreement with contents reported in the literature for 202

coffee brews [8; 15-17]. 203


10

This is the first study to quantify CGA and metabolites in human urine after the 204

consumption of water, coffee and instant coffee added to milk. The large inter 205

individual variability observed in the urinary excretion of CGA and metabolites in all 206

treatments has been commonly observed in studies evaluating the bioavailability of 207

polyphenols [9-11, 18, 19] and is probably due to differences on the subjects’ capacity 208

to absorb and metabolize these compounds, among other factors. 209

Regarding baseline and water treatment results, Nurmi et al. [18] have 210

previously identified 10 phenolic compounds (averaging 95μmol) in human’s baseline 211

urine samples after a 7-day phenolic free-diet. In the present study, although subjects 212

were monitored not to consume food sources of phenolic compounds during the 48h 213

prior to each experiment and throughout the 24h of the days of experiment, the presence 214

of phenolic compounds not only in baseline urine intervals, but during 24h after water 215

consumption is not surprising considering the results from Nurmi et al. [18] after 7-day 216

phenolic free-diet as well as the large amounts of CGA identified in freshly secreted 217

human digestive fluids after 12h fasting [17]. This reinforces the hypotheses of long 218

half-life of these compounds through enterohepatic recycling [17, 20] and less probably, 219

partial storage and slow release of CGA in the human body, earlier proposed by Booth 220

et al. [20]. Because the excretion patterns of phenolic compounds in urine after water 221

and coffee treatments were very different along the 24h collection, the water treatment 222

values were not used as a blank for coffee treatments. 223

Since studies evaluating the 24h urinary excretion of CGA and metabolites after 224

coffee consumption are scarce, it is difficult to make comparisons. Monteiro et al. [9] 225

observed that human subjects excreted about 0.12 mmol of CGA compounds up to 6h 226

after consumption of a decaffeinated coffee brew containing 3.4 mmol of CGA. In the 227

present study, although the CGA content in both coffee beverages offered to the 228


11

subjects was 6 times lower than the one used by Monteiro et al. [9], the total amount of 229

metabolites excreted by our subjects up to 8h after coffee consumption was 6.4 times 230

higher (757 mmol) than those reported by Monteiro et al. [9]. Moreover, Farah et al. 231

[10] reported excretion of 0.25 mmol of CGA compounds up to 8h after oral 232

administration of green coffee extract capsules containing in total 0.45 mmol of CGA, 3 233

times lower amount than the one observed in the present study. The higher amount of 234

CGA metabolites identified in the present study may be explained by the fact that 235

Monteiro et al. [9] and Farah et al. [10] identified 12 compounds in urine whereas 22 236

compounds (including a few major compounds) were quantified in the present study. 237

On the other hand, Olthof et al. [21], reported excretion of 9.3 mmol of phenolic 238

compounds in 24h urine after 7day oral administration of a supplement containing 5.5 239

mmol of 5-CQA. The higher CGA dose consumed by the subjects, the higher number of 240

identified compounds (60) and the longer period of intervention (7 consecutive days) 241

used by Olthof et al. [21] probably justify the higher excretion observed compared with 242

the present study. 243

The major hippuric acid excretion after the intake of dietary polyphenols in 244

humans has previously been reported [21-24]. This acid could partially derive from the 245

action of microorganisms in the colon, which are able to hydrolyze 5-CQA to caffeic 246

and quinic acids [24] or from hydrolysis and/or absorption of 5-CQA in the small 247

intestine [7, 9, 10]. According to the metabolic pathway proposed by Olthof et al. [24], 248

subsequently, the caffeic acid molecule would be absorbed and then �-oxidized to 249

benzoic acid, whereas the molecule of quinic acid would originate the cyclohexane 250

carboxylic acid, which would then be aromatized to benzoic acid by the action of 251

colonic microflora, and then absorbed by the tissues. In the kidney, benzoic acid would 252

be conjugated with glycine and then excreted in the urine as hippuric acid. Thus, each 253


12

molecule of 5-CQA would be able to give rise to two molecules of hippuric acid. 254

Additionally, this acid may also be formed from other food components such as the 255

aromatic amino acids tryptophan, tyrosine and phenyl-alanine [22], and benzoic acid 256

derivatives, widely used as food preservatives in industrial products like canned foods 257

[25]. 258

Differently from hippuric acid, 3,4-dihydroxyphenylacetic acid, the second 259

major compound excreted in the 24h urine of all subjects after coffee beverages 260

consumption, is considered to be a marker of polyphenols metabolism, more 261

specifically the class of flavonoids [20, 23, 25]. This compound is also known to derive 262

from the action of intestinal bacteria on the molecule of caffeic acid [18, 25] and, for the 263

first time, was identified in the urine of humans after ingestion of brewed coffee. 264

The major excretion of dihydrocaffeic and vanillic acids after coffee brew 265

consumption has already been reported in the literature [9; 10; 20; 21; 26]. The fact that 266

dihydrocaffeic acid is considered to be a primary metabolite of caffeic acid by Booth et 267

al. [20] and Farah et al. [10], among other studies, is consistent with a higher urinary 268

excretion of this compound in the first 12 hours after coffee consumption compared to 269

the period between 12-24h. Vanillic acid is, on the other hand, known as a secondary 270

metabolite of molecules of quinic and caffeic acids [10; 21; 26], which explains a higher 271

excretion of this compound in the period between 12-24h after consumption of both 272

coffee beverages. The major excretion of gallic acid after coffee consumption had 273

already been reported by Monteiro et al [10]. Its excretion has also been identified by 274

Olthoff [21, 24] after 5-CQA consumption. Like vanillic acid, this compound is also 275

known as a secondary metabolite of caffeic, ferulic and quinic acid molecules. 276

Different studies [9; 10; 27; 28] suggest that ferulic, isoferulic, vanillic and 277

dihydrocaffeic acids are the main metabolites of caffeic acid identified in plasma or 278


13

urine of humans. Additionally, the urinary excretion of 3-(4’-hydroxyphenyl) propionic, 279

3 trans-hydroxycinnamic and benzoic acids, identified for the first time in urine after 280

coffee consumption, has previously been reported in the literature after the consumption 281

of other polyphenols-rich foods or beverages [18; 20; 23; 27]. Figure 3 presents the 282

urinary metabolites identified in the present study as well as in previous studies 283

evaluating the metabolism of CGA and hydroxycinnamates. 284

The total percentage of CGA and metabolites, as well as the nature of the 285

metabolites recovered in urine for 24h after coffee consumption indicates that an 286

average of 68 ± 20 % of CGA ingested was absorbed in the whole digestive tract. This 287

includes absorption of intact CGA or primary CGA metabolites (such as caffeic and 288

ferulic acids) by stomach and small intestine, as reported in the literature [9, 10, 22, 23] 289

and absorption of primary and secondary metabolites in the large intestine after colonic 290

action, as previously reported in various studies evaluating the metabolism of phenolic 291

acids and CGA [18-31]. The lack of data on the urinary recovery of CGA and 292

metabolites after coffee consumption in the literature makes comparisons difficult. The 293

present results are in accordance with the recovery of 67% of Olthoff et al. [24] in 294

ileostomy fluids during 24h after consumption of 2.8 mmol of 5-CQA. Farah et al. [10] 295

reported that on average 26% of total CGA were recovered in the urine of subjects up to 296

8h after consumption of decaffeinated green coffee extract containing 451 �mol of 297

CGA, when expressed in absolute values. In the present study, when we calculate the 298

urinary recovery up to 8h after ingestion of coffee alone, the average recovery was 299

16.2% (13.7% - 19.1 %), lower than the percentage reported by Farah et al. [10]. This 300

lower percentage may be explained by the fact that the highest excretion in the present 301

study occurred from 8 to 24 hours after coffee consumption. Stalmach et al. [32] 302

reported an average recovery of 29% of glucuronated and sulfated forms of CGA and 303


14

primary metabolites in healthy subjects after consumption of 412 μmol of CGA. 304

Comparing the recoveries from Farah et al. [10] and Stalmach et al. [32] with the total 305

phenolic compounds recovered in the present study (68 ± 20 %), The higher urinary 306

recovery percentages obtained in the present study may be explained by the compounds 307

derived from the metabolism of colonic bacteria as well as other compounds which 308

were only accounted for in this study. Excluding from recovery calculations the 309

compounds 3-hydroxyphenylacetic, 3,4-dihydroxyphenylpropionic, 2,4-310

dihydroxybenzoic, and trans-3-hydroxycinnamic acids, which are known as exclusively 311

colonic metabolites, only 10.5% - 23% of total CGA consumed were recovered in urine 312

in the form of metabolites after coffee, which is in accordance with the literature [9, 10, 313

21, 24]. 314

Lower total recovery values (averaging 51 %) would be obtained if water 315

treatment excretion was used as a blank. However, there is no way to guarantee that the 316

amount of phenolic compounds excreted during fasting would still be excreted as a 317

baseline even after phenolic-containing food consumption. This is corroborated by the 318

different excretion profiles of water and coffee treatments cited above. 319

The range of urinary concentrations of CGA and metabolites recovered in the 320

present study is in the same order of magnitude (�mol) as those from different studies 321

evaluating the metabolism of other polyphenols [9,10,19,24], even though percent 322

recoveries varied considerably in such studies according to the metabolites accounted 323

for, analytical methodologies applied, study designs and so forth. For example, in a 324

review from Manach et al. [34] urinary recoveries of 1.1 % and 30.2 % of naringenin 325

from orange and grapefruit juice, respectively, were reported after consumption of 23 326

mg eq naringenin. On the same line, urinary recovery of 0.1% of metabolites was 327

observed after consumption of epigallocatechin gallate through green tea (2 mg/kg of 328


15

body weight), while 55 % recovery was observed after consumption of 2 g of pure 329

cathechin. Similar variations have been observed in studies investigating isoflavone 330

bioavailability. While Kano et al. [35] reported a urinary recovery of 6.8 % of total 331

isoflavones after consumption of 0.6 mg of daidzein and 1 mg of genistein from soy 332

beverage, Setchell et al. [36] reported a recovery of 54.5 % after consumption of 0.55 333

mg of daidzein and 0.15 mg of genistein from soy germ. 334

The low recovery of intact CGA compounds (less than 1%) is in accordance 335

with previous studies in humans evaluating the metabolism of CGA and other 336

hydroxycinamates [9; 10, 24, 29], supporting evidence that urine is not the preferential 337

excretion route of intact CGA compounds [29], which are excreted in free and 338

conjugated forms in digestive fluids and re-absorbed in the intestinal tract after 339

breakdown of conjugates by the intestinal microflora [7]. Exceptions are the two studies 340

from Stalmach et al. [32, 33] evaluating ileostomized and healthy patients in which 24h 341

urinary recovery of sulfated and glucuronated forms of CGA after coffee consumption 342

were equivalent to 8 – 10 % of the consumed CGA amount. Considering the different 343

analytical methodologies used as well as the lower amounts consumed in these studies 344

compared to the present study as well as other studies, comparisons are difficult. Dose-345

response studies using the same methodologies are required for further discussion. 346

Comparing the total 24h excretion of polyphenols after both coffee treatments, 347

as the subjects ingested the same amount of CGA in both of them, an average reduction 348

of 28% on the excretion of CGA and metabolites after the consumption of coffee-milk 349

treatment indicates that adding coffee to milk quantitatively altered the absorption and/ 350

or the metabolism of CGA from coffee in all subjects. This decrease was mainly driven 351

by the differences in the colonic metabolites hippuric acid and 3,4-352

dihydroxyphenylacetic acid. The lower excretion of these two compounds when 353


16

comparing coffee and coffee-milk treatments was also observed by Urpi-Sarda, et al. 354

[19] investigating the effect of simultaneous consumption of cocoa powder and milk in 355

humans. Although the 23% decrease in the urinary recovery of CGA compounds in 356

coffee-milk treatment comparing to coffee alone was not significant probably due to the 357

small number of subjects as well as the differences among them, the 28% decrease in 358

total urinary excretion demonstrates that adding large amounts of milk to coffee 359

decreased the bioavailability of these compounds. This was consistently observed in all 360

subjects. 361

It is noteworthy to mention that the amount of milk used in this study is 362

commonly suggested by instant coffee manufacturers in Brazil, but the proportion of 363

coffee and milk used by consumers may vary considerably according to cultural habits 364

and individual preferences around the world. It is possible that lower amounts of milk 365

added to coffee would not interfere in CGA absorption and/or metabolism. In fact, 366

Renoulf et al [37] reported that the addition of 20% of whole milk to coffee did not 367

affect CGA bioavailability in humans. 368

Studies investigating the effect of simultaneous consumption of coffee and other 369

dietary components on the absorption and metabolism of CGA in the human body are 370

scarce. Nonetheless, in addition to agreeing with the results from Urpi-Sarda [19] cited 371

above, the present results are consistent with the findings of Mullen et al [38], who 372

observed in humans a significant decrease in the urinary excretion of flavonoids 373

metabolites caused by the addition of milk to a cocoa drink. According to the authors, 374

this result seems to be a direct consequence of interactions between milk constituents 375

and flavonoids present in chocolate, which probably would have caused changes in the 376

mechanism of transport of these compounds through the intestinal wall into the 377

bloodstream. These findings, as well as those observed in the present study corroborate 378


17

the results from Dupas et al. [14] who observed that at least 40% of 5-CQA were 379

associated with milk proteins, especially casein in an in vitro digestion system. From 380

this amount, about 17% of 5-CQA remained complexed to milk proteins and peptides to 381

the end of digestion, modifying, hypothetically, its absorption. Also, in addition to 382

showing that ferulic, caffeic and gallic acid, as well as 5-CQA may interact with whey 383

proteins such as �-lactoglobulin, Rawel et al. [39] observed in an in vitro digestion 384

system that these complexes may adversely affect their susceptibility to proteolysis by 385

gastrointestinal enzymes such as trypsin, chymotrypsin, pepsin and pancreatin. This 386

event could, therefore, hinder the release of phenolic compounds from the protein 387

complex and consequently their absorption. 388

Additional studies have investigated the effect of the simultaneous consumption 389

of milk on polyphenols bioactivity. Serafini et al. [40, 41]; demonstrated that adding 390

26% of whole milk to black/green teas and blueberry juice, respectively, affected the 391

antioxidant status in human plasma. Similar results were observed by Reddy et al. [42] 392

in a study in which 20% of whole milk was associated to black tea. In line with these 393

results, Lorenz et al [43] demonstrated that adding 10% of whole milk to black tea 394

counteracted the favorable health effects of tea cathechins on vascular function. The 3 395

studies attributed the milk interference on the bioactivity of polyphenols to the negative 396

impact of its proteins on polyphenols absorption. On the other hand, 3 additional studies 397

evaluating the effect of simultaneous consumption of milk and polyphenols from green 398

tea [44, 45], black tea [44, 46] and blueberry juice [47] on plasma antioxidant activity 399

did not observe differences among treatments, although the authors acknowledged the 400

possibility of formation of polyphenol-protein complexes. 401

In conclusion, considering the results from the present study as well as from 402

previous studies, the simultaneous consumption of milk and coffee may produce a 403


18

negative effect on CGA bioavailability in humans. This effect seems to depend on the 404

proportion milk to coffee used. In addition to dose-response studies evaluating CGA 405

bioavailability, more studies are needed in order to determine the minimum proportion 406

of milk to coffee that will impair CGA bioavailability after coffee consumption. 407

408

AKNOWLEGMENTS 409

The authors would like to thank COCAM Company (São Paulo, Brazil) for 410

providing the instant coffee samples as well as the financial support of the Brazilian 411

National Research Council (CNPQ) and Fundação Carlos Chagas Filho de Amparo à 412

Pesquisa do Estado do Rio de Janeiro (FAPERJ). 413

414

LITERATURE CITED 415

416

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328. 603


27

Table of Figures. 604

605

Figure Figure Caption

1 Total of CGA and metabolites excreted in urine of subjects for 24h after water, coffee and coffee-milk consumption at following time intervals (0-4; 4-8; 8-12; 12-24h). Different letters in the same time interval indicate statistical difference (one-way-ANOVA, with Fisher post test, p< 0.05).

2 Major (A, B) and minor (C) phenolic compounds excreted for 24h after water, coffee and coffee-milk consumption. Different letters in the same time interval indicate statistical difference (one-way-ANOVA, with Fisher post test, p< 0.05).

3 Simplified scheme of metabolites of 5-caffeoylquinic acid (5-CQA) -excluding conjugated forms with glucuronic acid and sulfate -, as a representative of chlorogenic acids class based on results from previous studies as well as from the present study.

606


28

Table 1. Contents of the main chlorogenic acid compounds (CGA) in the instant coffee 607

portion (4g) offered to the subjects (n=5) in both coffee treatments1. 608

Compound Contents (�mol/ portion)

3-caffeoylquinic acid 151.2 ± 7.9



Total caffeoylquinic acids 472.9 ± 13.8 3,4-dicaffeoylquinic acid 9.3 ± 0.4

3,5-dicaffeoylquinic acid 6.3 ± 0.3

4,5-dicaffeoylquinic acid 5.7 ± 0.2

Total dicaffeoylquinic acids 21.2 ± 0.1 3-feruloylquinic acid 28.0 ± 1.6

4- feruloylquinic acid 21.2 ± 0.9

5- feruloylquinic acid 17.9 ± 1.2

Total feruloylquinic acids 67.1 ± 1.6 5-p-coumaroloylquinic acid 6.76 ± 0.6 Caffeoyferuloylquinic acids2 3.70 ± 0.2

4-caffeoylquinide acid 9.08 ± 1.3 5-caffeoylquinide acid 11.28 ± 2.5

Total caffeoylquinide acids 20.37 ± 1.2 Caffeoyltryptophan 17.50 ± 2.3

Total CGA 609.53 ± 12.8 1 Results are mean ± SD of triplicate extraction; 2 Total of 6 isomers. 609 610

Table 2. Average anthropometric and biochemical characterization of subjects (n=5) on 611

the days of water, coffee and coffee-milk treatments. 612

Mean ± SD Min-Máx Reference values

Age (years) 26,5 ± 0,71 24 -35 - BMI (kg/m2) 22,45 ± 3,18 19,32-27,70 18,5 – 25,01

Hematocrit (%) 44,0 ± 2,97 38,3-58,6 40 -45%2 Hemoglobin(g/dL) 12,16 ± 1,19 11,32-16,6 11,5-13,52

1World Health Organization (WHO); 2[48]. 613

614


29

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