Ihor
Member
- Joined
- Feb 25, 2018
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- 216
I used to hear that coffee may interfere with the digestion of fats in the gastric tract, but I have never tested it. Nevertheless, after my experiments with diet, I clearly found out that dietary fat is one of the problematic issues for me, although several years ago I did not recall these problems and at that time I almost never drank coffee in my life before. I started drinking coffee two years ago and for some time I remember, during this time I did not throw it for more than a week and on average consumed 2-3 coffee a day. One way or another, during all this time when my health problems started, I observed parts of undigested fat in my stool, and I also deal with frequent constipation. Of course, there is no direct correlation and many variables, but today I came across a study which says that methylxanthines such as caffeine, theobromine and theophylline are active pancreatic lipase inhibitors. Therefore, based on this study, will we not get, as a result of a long diet with a combination of dietary fat and coffee, undigested fat that will clog the entire length of the gastrointestinal tract?
Methylxanthines such as caffeine, theobromine and theophylline are intensively consumed as food components by large proportion of human population all over the world. This class of compounds show various biological activities and have been found to act as broad specificity inhibitors towards numerous enzymes. However, their action on digestive enzymes have not been yet investigated. In this paper we aimed to evaluate the effects of methylxanthines on the human pancreatic lipase activity in vitro. Emulsions of short- and long-chain triglycerides (tributyrin and tripalmitate, respectively) were used as substrates. The concentrations of methylxanthines in the reaction mixtures covered the range between 0.015 mmol/L to 15 mmol/L. We demonstrated that all three tested substances, caffeine, theophylline and theobromine inhibited the hydrolysis of tributyrin and tripalmitate catalysed by human pancreatic lipase in dose-dependent fashion. The highest lipase inhibition ratios during tripalmitate and tributyrin hydrolysis were 25.74% and 79.54% respectively in the presence of caffeine, 29.89% and 62.79% respectively with theophylline and 21.08% and 67.74% respectively in the presence of theobromine. All the tested methylxanthines exert stronger inhibition in the short-chain triglyceride lipolysis comparing to long-chain substrates. Their mechanism of action involves most likely the interaction with enzyme protein but not substrate emulgation. In case of tripalmitate lipolysis all the methylxanthines showed mixed type of inhibition. Interestingly, during tributyrin lipolysis theophylline behaved as classical noncompetitive inhibitor.
INTRODUCTION.
Human pancreatic lipase (HPL) is the most important lipolytic enzyme involved in the digestion of dietary triglycerides (TG). HPL is a 50 kDa polypeptide composed of 449 amino acid residues and N-glycosylated on Asn166 with a high mannose carbohydrate moiety [Miled et al., 2000]. The HPL molecule contains two main structural domains: a large N- -terminal domain with the α/β hydrolase motif and the active site with a catalytic triad formed by Ser 152, Asp 176, His 263, and a smaller C-terminal domain [Colin et al., 2008]. The active site is covered by a large surface loop called a ‘lid’ domain (Cys 237 - Cys 261) [Winkler et al., 1990; Ranaldi et al., 2010]. The HPL activity towards water-soluble substrates is low, but the enzyme is highly active when operating at the interface between water and hydrophobic triglyceride layer. This phenomenon is known as interfacial activation [Steiner & Williams, 2002]. Physiological concentrations of bile acids inhibit lipolysis by covering the oil-water interface and preventing the adsorption of HPL at that interface [Brockman, 2000]. This inhibition by bile salts can be overcome by the presence of colipase, a specifi c lipase-anchoring protein secreted by pancreas. Colipase forms a specifi c 1:1 complex with lipase and facilitates its adsorption at the bile acid layered lipid-water interface [Miled et al., 2000, Freie et al., 2006]. In vitro, the highest specifi c HPL activity in excess of colipase and with 4 mmol/L sodium deoxycholate, at pH 8.0 was recorded for tributyrin as a substrate [Miled et al., 2000].
Methylxanthines, including caffeine (1,3,7-trimethylxanthine), theobromine (3,7-dimethylxanthine) and theophylline (3,7-dimethylxanthine), are natural purine alkaloids produced by many plant species. These compounds are present in large quantities in a variety of food, beverages and pharmaceutical products. Caffeine is consumed every day by more than 80% of the world population [Papaioannou et al., 2005]. It is a constituent of coffee, tea [Mandel, 2002], cocoa beans, guarana, colas [Harland, 2000], and included in many medicines, particularly over-the-counter drug mixtures for cold and cough [Baselt, 2004] as well as in weight loss supporting products [Greenway et al., 2004]. Human body metabolises caffeine to theobromine and theophylline [Grosso & Bracken, 2005] and these derivatives are also present in cocoa, guarana, tea and chocolate products [Caudle & Bell, 2000]. Average daily consumption of theophylline and theobromine, accounted only for drinking tea and cocoa, is estimated to 0.51 mg and 39 mg, respectively [WHO, 1995]. Theophylline is also a frequently used bronchodilating agent with a narrow serum therapeutic range [Møller et al., 2000]. Theobromine, however, at the therapeutic dose of 500 mg/kg has been used in the treatment of cardiac oedema and angina pectoris [Eteng & Ettarh, 2000].
Methylxanthines exert a strong impact on the operation of central and peripheral nervous system [Mandel, 2002], cerebral blood fl ow and metabolism, cardiovascular system, respiratory and renal systems and gastrointestinal system [Greenway et al., 2004; Haller et al., 2004; Ruhl & Everhart, 2005; Westerterp-Plantenga et al., 2006]. There are several recent lines of evidence suggesting that methylxanthines stimulate thermogenesis, promote weight and fat loss [Greenway et al., 2004], as well as inhibit various enzymes, such as PDE, PLA2 [Mandel, 2002] and chitinase [Rao et al., 2005].
Nevertheless, neither of the three mentioned methylxanthines have been suspected to inhibit digestive enzymes, so far. Therefore, the aim of this study was to assess the potential inhibitory effects of caffeine, theophylline and theobromine on human pancreatic lipase, measured on short-chain and long-chain triglyceride substrates.
RESULTS AND DISCUSSION.
Dose-response inhibitory effect of methylxanthines on short-chain and long-chain triglycerides hydrolysis
The hydrolytic activity of human pancreatic lipase towards long-chain and short-chain triglycerides depended on the dose of methylxanthines added to the reaction mixture. For tripalmitate lipolysis, the lowest concentration (0.015 mmol/L) of caffeine, theophylline and theobromine inhibited the hydrolysis by 7.98%, 18.06% and 6.46%, respectively, and by 23.78%, 18.94% and 21.75% for tributyrin lipolysis. A thousand fold increase in methylxanthines concentration resulted in a severe drop in lipase activity. As a result, the lipase inhibition ratio of tripalmitate hydrolysis was 21.08% in the presence of theobromine, 25.74% for caffeine and 29.89% for theophylline. During tributyrin hydrolysis the value of the inhibition ratio varied from 62.79% for theophylline, 67.74% for theobromine, to 79.54% for caffeine (Figure 1). These results suggest that the inhibitory effect of methylxanthines on human pancreatic lipase may be put in the order as follows: 1,3-dimethylxanthine (theophylline) > 1,3,7-trimethylxantine (caffeine) > 3,7-dimethylxanthine (theobromine) for the long-chain triglyceride, and 1,3,7-trimethylxantine (caffeine) > 3,7-dimethylxanthine (theobromine) > 1,3-dimethylxanthine (theophylline) for the short- -chain triglyceride hydrolysis. It is likely that the inhibitory effi cacy of methylxanthines depends on the number of methyl groups on the purine ring and their different positions. The presence of –CH3 group at the N1 atom seems to facilitate the inhibition of the long-chain triglyceride hydrolysis, while methylation at the N7 site – to facilitate the short- -chain triglyceride hydrolysis inhibition. The importance of the methyl group position as an inhibitory effect determinant was observed by Sugawara et al. [2005], who showed that caffeine, theophylline and theobromine blocked human organic anion transporter 1 (hOAT1) in the following order: theophylline > theobromine > caffeine. Nafi si and his collaborators [Nafi si et al., 2002] reported that the blockage of the purine N7 atom by -CH3 group resulted in lower capability of caffeine for Mg(II) and Ca(II) cation chelation, as compared to theophylline.
Preincubation effects
The inhibitory effect of methylxanthines depended on the incubation system used (Table 1). All the methylxanthines showed the highest inhibitory effect when preincubated with the enzyme (incubation system 3). This indicates that methylxanthines are able to bind with the protein. This is consistent with the results of Sugawara et al. [2005] and Westerterp-Plantenga et al. [2006] who described the interaction of caffeine, theophylline and theobromine with human organic anion transporter 1 or with adenosine receptors, and with phosphodiesterases that hydrolyse cyclic AMP or cyclic GMP.
The lack of preincubation period of caffeine, theophylline or theobromine with the enzyme (incubation system 1) resulted in a signifi cant (p<0.05) decrease in the inhibitory effect of methylxanthines. This indicates that to inhibit lipase effi ciently, the methylxanthines have to interact directly with the protein, prior to the enzyme-substrate complex formation. Without preincubation with the enzyme, the affi nity of methylxanthines for pancreatic lipase restricts the enzyme-triglyceride interaction by about 30%. Furthermore, since the triglycerides are in excess in the reaction mixture, this would favour enzyme-substrate interactions rather than enzyme-methylxanthines interactions .
Fish & Thompson [1991] described similar observations in relation to tannic acid and red kidney bean lectin that have direct inhibitory effects on digestive enzymes. The authors reported the lack of signifi cant changes in the α-amylase activity when the enzyme was not preincubated with red kidney bean lectin or tanic acid. In our studies, the incubation of methylxanthines with the excessive amounts of substrates prior to exposure to lipase resulted in further reduction of the inhibition ratio (incubation system 2). In case of tributyrin hydrolysis, the decrease in the lipase activity was not higher than 25%, whereas in case of tripalmitate hydrolysis not higher than 10%. This suggests that caffeine, theophylline and theobromine interact with highly emulsifi ed substrates without exerting any detrimental effect on the emulsifi cation process. To our best knowledge, this is the fi rst report that shows no effects of purine alkaloids on the lipid emulsifi cation process.
Studies of inhibition types
The detailed analysis of the lypolysis kinetic parameters (Michaelis-Menten constant and maximum velocity), carried on in the presence or absence of alkaloids, demonstrated that the methylxanthines differed in the inhibitory activity as well as in the mechanism of action. Independently on the substrate used, caffeine and theobromine signifi cantly increased Km and decreased Vmax (Table 2). Therefore they were mixed type inhibitors of lipase, which means that they were able to bind both free enzyme and an enzyme- -substrate complex. The same conclusions were presented by Gu et al. [2011], who studied the effect of cocoa extract on lipase activity. However, the authors attributed the inhibitory effect to procyanidins contents only and did not comment the role of theobromine in this process. In our study, the effect of the third methylxanthine, i.e. theophylline, was dependent on the substrate hydrolysed by lipase. In the tripalmitate lipolysis reaction, it acted similarly to caffeine and theobromine and evoked mixed type inhibition. On the other hand, during the tributyrate lipolysis, theophylline reduced the maximal reaction velocity, but the Km values remained unchanged. This indicates that in short-chain substrate hydrolysis theophylline is a classic non-competitive inhibitor.
CONCLUSION.
Based on the analysis presented in this report, we show for the fi rst time ever that caffeine, theobromine and theophylline which are ubiquitous components of our diet can signifi cantly impede the lipid hydrolysis catalysed by the pancreatic lipase, even at low doses. The extent of inhibition depends not only on the methyloxanthine concentration but also on the lipid type. All the tested methylxanthines inhibit short-chain triacylglyceride lipolysis more effi ciently than the long-chain substrates, and the mechanism of their action relies on the interaction with protein but not on substrate emulsification.
Source:
Methylxanthine Drugs Are Human Pancreatic Lipase Inhibitors
"Methylxanthine Drugs Are Human Pancreatic Lipase Inhibitors"
ABSTRACT.
Methylxanthines such as caffeine, theobromine and theophylline are intensively consumed as food components by large proportion of human population all over the world. This class of compounds show various biological activities and have been found to act as broad specificity inhibitors towards numerous enzymes. However, their action on digestive enzymes have not been yet investigated. In this paper we aimed to evaluate the effects of methylxanthines on the human pancreatic lipase activity in vitro. Emulsions of short- and long-chain triglycerides (tributyrin and tripalmitate, respectively) were used as substrates. The concentrations of methylxanthines in the reaction mixtures covered the range between 0.015 mmol/L to 15 mmol/L. We demonstrated that all three tested substances, caffeine, theophylline and theobromine inhibited the hydrolysis of tributyrin and tripalmitate catalysed by human pancreatic lipase in dose-dependent fashion. The highest lipase inhibition ratios during tripalmitate and tributyrin hydrolysis were 25.74% and 79.54% respectively in the presence of caffeine, 29.89% and 62.79% respectively with theophylline and 21.08% and 67.74% respectively in the presence of theobromine. All the tested methylxanthines exert stronger inhibition in the short-chain triglyceride lipolysis comparing to long-chain substrates. Their mechanism of action involves most likely the interaction with enzyme protein but not substrate emulgation. In case of tripalmitate lipolysis all the methylxanthines showed mixed type of inhibition. Interestingly, during tributyrin lipolysis theophylline behaved as classical noncompetitive inhibitor.
INTRODUCTION.
Human pancreatic lipase (HPL) is the most important lipolytic enzyme involved in the digestion of dietary triglycerides (TG). HPL is a 50 kDa polypeptide composed of 449 amino acid residues and N-glycosylated on Asn166 with a high mannose carbohydrate moiety [Miled et al., 2000]. The HPL molecule contains two main structural domains: a large N- -terminal domain with the α/β hydrolase motif and the active site with a catalytic triad formed by Ser 152, Asp 176, His 263, and a smaller C-terminal domain [Colin et al., 2008]. The active site is covered by a large surface loop called a ‘lid’ domain (Cys 237 - Cys 261) [Winkler et al., 1990; Ranaldi et al., 2010]. The HPL activity towards water-soluble substrates is low, but the enzyme is highly active when operating at the interface between water and hydrophobic triglyceride layer. This phenomenon is known as interfacial activation [Steiner & Williams, 2002]. Physiological concentrations of bile acids inhibit lipolysis by covering the oil-water interface and preventing the adsorption of HPL at that interface [Brockman, 2000]. This inhibition by bile salts can be overcome by the presence of colipase, a specifi c lipase-anchoring protein secreted by pancreas. Colipase forms a specifi c 1:1 complex with lipase and facilitates its adsorption at the bile acid layered lipid-water interface [Miled et al., 2000, Freie et al., 2006]. In vitro, the highest specifi c HPL activity in excess of colipase and with 4 mmol/L sodium deoxycholate, at pH 8.0 was recorded for tributyrin as a substrate [Miled et al., 2000].
Methylxanthines, including caffeine (1,3,7-trimethylxanthine), theobromine (3,7-dimethylxanthine) and theophylline (3,7-dimethylxanthine), are natural purine alkaloids produced by many plant species. These compounds are present in large quantities in a variety of food, beverages and pharmaceutical products. Caffeine is consumed every day by more than 80% of the world population [Papaioannou et al., 2005]. It is a constituent of coffee, tea [Mandel, 2002], cocoa beans, guarana, colas [Harland, 2000], and included in many medicines, particularly over-the-counter drug mixtures for cold and cough [Baselt, 2004] as well as in weight loss supporting products [Greenway et al., 2004]. Human body metabolises caffeine to theobromine and theophylline [Grosso & Bracken, 2005] and these derivatives are also present in cocoa, guarana, tea and chocolate products [Caudle & Bell, 2000]. Average daily consumption of theophylline and theobromine, accounted only for drinking tea and cocoa, is estimated to 0.51 mg and 39 mg, respectively [WHO, 1995]. Theophylline is also a frequently used bronchodilating agent with a narrow serum therapeutic range [Møller et al., 2000]. Theobromine, however, at the therapeutic dose of 500 mg/kg has been used in the treatment of cardiac oedema and angina pectoris [Eteng & Ettarh, 2000].
Methylxanthines exert a strong impact on the operation of central and peripheral nervous system [Mandel, 2002], cerebral blood fl ow and metabolism, cardiovascular system, respiratory and renal systems and gastrointestinal system [Greenway et al., 2004; Haller et al., 2004; Ruhl & Everhart, 2005; Westerterp-Plantenga et al., 2006]. There are several recent lines of evidence suggesting that methylxanthines stimulate thermogenesis, promote weight and fat loss [Greenway et al., 2004], as well as inhibit various enzymes, such as PDE, PLA2 [Mandel, 2002] and chitinase [Rao et al., 2005].
Nevertheless, neither of the three mentioned methylxanthines have been suspected to inhibit digestive enzymes, so far. Therefore, the aim of this study was to assess the potential inhibitory effects of caffeine, theophylline and theobromine on human pancreatic lipase, measured on short-chain and long-chain triglyceride substrates.
RESULTS AND DISCUSSION.
Dose-response inhibitory effect of methylxanthines on short-chain and long-chain triglycerides hydrolysis
The hydrolytic activity of human pancreatic lipase towards long-chain and short-chain triglycerides depended on the dose of methylxanthines added to the reaction mixture. For tripalmitate lipolysis, the lowest concentration (0.015 mmol/L) of caffeine, theophylline and theobromine inhibited the hydrolysis by 7.98%, 18.06% and 6.46%, respectively, and by 23.78%, 18.94% and 21.75% for tributyrin lipolysis. A thousand fold increase in methylxanthines concentration resulted in a severe drop in lipase activity. As a result, the lipase inhibition ratio of tripalmitate hydrolysis was 21.08% in the presence of theobromine, 25.74% for caffeine and 29.89% for theophylline. During tributyrin hydrolysis the value of the inhibition ratio varied from 62.79% for theophylline, 67.74% for theobromine, to 79.54% for caffeine (Figure 1). These results suggest that the inhibitory effect of methylxanthines on human pancreatic lipase may be put in the order as follows: 1,3-dimethylxanthine (theophylline) > 1,3,7-trimethylxantine (caffeine) > 3,7-dimethylxanthine (theobromine) for the long-chain triglyceride, and 1,3,7-trimethylxantine (caffeine) > 3,7-dimethylxanthine (theobromine) > 1,3-dimethylxanthine (theophylline) for the short- -chain triglyceride hydrolysis. It is likely that the inhibitory effi cacy of methylxanthines depends on the number of methyl groups on the purine ring and their different positions. The presence of –CH3 group at the N1 atom seems to facilitate the inhibition of the long-chain triglyceride hydrolysis, while methylation at the N7 site – to facilitate the short- -chain triglyceride hydrolysis inhibition. The importance of the methyl group position as an inhibitory effect determinant was observed by Sugawara et al. [2005], who showed that caffeine, theophylline and theobromine blocked human organic anion transporter 1 (hOAT1) in the following order: theophylline > theobromine > caffeine. Nafi si and his collaborators [Nafi si et al., 2002] reported that the blockage of the purine N7 atom by -CH3 group resulted in lower capability of caffeine for Mg(II) and Ca(II) cation chelation, as compared to theophylline.
Preincubation effects
The inhibitory effect of methylxanthines depended on the incubation system used (Table 1). All the methylxanthines showed the highest inhibitory effect when preincubated with the enzyme (incubation system 3). This indicates that methylxanthines are able to bind with the protein. This is consistent with the results of Sugawara et al. [2005] and Westerterp-Plantenga et al. [2006] who described the interaction of caffeine, theophylline and theobromine with human organic anion transporter 1 or with adenosine receptors, and with phosphodiesterases that hydrolyse cyclic AMP or cyclic GMP.
The lack of preincubation period of caffeine, theophylline or theobromine with the enzyme (incubation system 1) resulted in a signifi cant (p<0.05) decrease in the inhibitory effect of methylxanthines. This indicates that to inhibit lipase effi ciently, the methylxanthines have to interact directly with the protein, prior to the enzyme-substrate complex formation. Without preincubation with the enzyme, the affi nity of methylxanthines for pancreatic lipase restricts the enzyme-triglyceride interaction by about 30%. Furthermore, since the triglycerides are in excess in the reaction mixture, this would favour enzyme-substrate interactions rather than enzyme-methylxanthines interactions .
Fish & Thompson [1991] described similar observations in relation to tannic acid and red kidney bean lectin that have direct inhibitory effects on digestive enzymes. The authors reported the lack of signifi cant changes in the α-amylase activity when the enzyme was not preincubated with red kidney bean lectin or tanic acid. In our studies, the incubation of methylxanthines with the excessive amounts of substrates prior to exposure to lipase resulted in further reduction of the inhibition ratio (incubation system 2). In case of tributyrin hydrolysis, the decrease in the lipase activity was not higher than 25%, whereas in case of tripalmitate hydrolysis not higher than 10%. This suggests that caffeine, theophylline and theobromine interact with highly emulsifi ed substrates without exerting any detrimental effect on the emulsifi cation process. To our best knowledge, this is the fi rst report that shows no effects of purine alkaloids on the lipid emulsifi cation process.
Studies of inhibition types
The detailed analysis of the lypolysis kinetic parameters (Michaelis-Menten constant and maximum velocity), carried on in the presence or absence of alkaloids, demonstrated that the methylxanthines differed in the inhibitory activity as well as in the mechanism of action. Independently on the substrate used, caffeine and theobromine signifi cantly increased Km and decreased Vmax (Table 2). Therefore they were mixed type inhibitors of lipase, which means that they were able to bind both free enzyme and an enzyme- -substrate complex. The same conclusions were presented by Gu et al. [2011], who studied the effect of cocoa extract on lipase activity. However, the authors attributed the inhibitory effect to procyanidins contents only and did not comment the role of theobromine in this process. In our study, the effect of the third methylxanthine, i.e. theophylline, was dependent on the substrate hydrolysed by lipase. In the tripalmitate lipolysis reaction, it acted similarly to caffeine and theobromine and evoked mixed type inhibition. On the other hand, during the tributyrate lipolysis, theophylline reduced the maximal reaction velocity, but the Km values remained unchanged. This indicates that in short-chain substrate hydrolysis theophylline is a classic non-competitive inhibitor.
CONCLUSION.
Based on the analysis presented in this report, we show for the fi rst time ever that caffeine, theobromine and theophylline which are ubiquitous components of our diet can signifi cantly impede the lipid hydrolysis catalysed by the pancreatic lipase, even at low doses. The extent of inhibition depends not only on the methyloxanthine concentration but also on the lipid type. All the tested methylxanthines inhibit short-chain triacylglyceride lipolysis more effi ciently than the long-chain substrates, and the mechanism of their action relies on the interaction with protein but not on substrate emulsification.
Source:
Methylxanthine Drugs Are Human Pancreatic Lipase Inhibitors
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