 |
|
Pea and whey in the control of high blood pressure
September 17, 2003Hypertension or high blood pressure is a major risk factor for the development of cardiovascular diseases, which are the leading causes of morbidity and mortality in western society. It is estimated that 20% of the world's adult population suffers from hypertension. Recently, some functional foods have received considerable attention for their effectiveness in both the prevention and the treatment of hypertension. This is among others due to the presence of food derived bioactive peptides with potential antihypertensive properties. Compared to antihypertensive drugs, these peptides, as part of a (functional) food or as nutraceutical, have no side effects and are less expensive. Moreover, as a more natural product, they appeal to the consumer.
Angiotensin I converting enzyme (ACE) inhibitory peptides exert an antihypertensive effect by inhibition of the angiotensin I converting enzyme in the cardiovascular system, hence preventing the formation of the potent vasoconstrictor angiotensin II and the degradation of the vasodilator bradykinin. In order to reduce the blood pressure after oral administration, ACE inhibitory peptides have to reach the bloodstream in an active form. Therefore, stability against human proteases and intestinal transport of active peptides is required. In the current field of ACE inhibitory peptide research, the maintenance of the activity in the oral delivery route is often not taken into account. ACE inhibitory peptides have mainly been isolated from milk proteins and have not yet been studied from pea protein.
The aim of this work was to investigate the release of ACE inhibitory activity from pea and whey protein. Moreover, whey protein, from which certain bioactive peptides already have been characterised, was compared to the unknown pea protein. Emphasis was placed on the in vitro study of the activity of ACE inhibitory peptides after oral administration in the human body, from the gastrointestinal tract to their site of action, the cardiovascular system. The maintenance of bioactivity in the oral delivery route is necessary to exert an antihypertensive effect.
A diagnostic assay to measure ACE activity was transformed into an ACE inhibition assay and subsequently optimised to obtain a more sensitive and less expensive test. This spectrophotometric method, where ACE inhibition was measured with the substrate FAPGG and rabbit lung acetone extract as ACE source, was validated by the antihypertensive drugs captopril, enalapril and its active derivative enalaprilat, and by the lactokinin Ala-Leu-Pro-Met-His-Ile-Arg. In addition, it indicated the release of ACE inhibitory activity after digestion of pea and whey protein. A more delicate and standardised assay was obtained by applying pure ACE from porcine kidney as ACE source. The ACE inhibitory activity of captopril and Ala-Leu-Pro-Met-His-Ile-Arg was also verified in this assay. The ACE inhibition assay presented a relatively simple and reliable tool to screen for ACE inhibitory peptides from food proteins.
The activity of the potent ACE inhibitory peptide Ala-Leu-Pro-Met-His-Ile-Arg, originally derived from a tryptic digest of b-lactoglobulin, was investigated in vitro in the oral delivery route. During in vitro gastrointestinal digestion, the heptapeptide was partially degraded by chymotrypsin, likely to Ala-Leu-Pro-Met and His-Ile-Arg, the latter also possessing ACE inhibitory activity. Nevertheless, after hydrolysis by stomach and pancreatic proteases, half of the initial peptide concentration and 70% of the initial ACE inhibitory activity could still be recovered. The lactokinin was rapidly broken down by rat intestinal tissue peptidases, while almost no degradation was observed after incubation with Caco-2 homogenates. The intestinal transport of 1 mM of the lactokinin was investigated in a Caco-2 Bbe cell monolayer mounted in an Ussing chamber. After 10 min of incubation at 37°C, substantial ACE inhibitory activity was found in the serosal compartment after threefold concentration of the samples. Concomitantly, MALDI-TOF spectrometry detected the heptapeptide in the serosal compartment. Under the observed experimental conditions, the ACE inhibitory peptide Ala-Leu-Pro-Met-His-Ile-Arg was transported intact through the Caco-2 Bbe cell monolayer, although at low concentrations. It seems that the degradation by intestinal peptidases constitutes the major barrier in the oral delivery route of this lactokinin.
Fermentation by different lactic acid bacteria was studied as a means to release ACE inhibitory activity from pea and whey protein. Fermentation of pea protein by Lactobacillus helveticus yielded the highest ACE inhibitory activity and was therefore selected for subsequent experimentation. Next, pea and whey protein were fermented by Lactobacillus helveticus and the yeast Saccharomyces cerevisiae in monoculture and in combination at 28 and 37°C. Fermentation was always followed by in vitro gastrointestinal digestion and the digests of non-fermented protein solutions served as controls. After fermentation, the ACE inhibitory activity (%) of a 2.73 mg/ml sample increased by 18 to 30% for all treatments, except for the fermentations of whey protein by Saccharomyces cerevisiae at 28°C, where no significant change was observed. After digestion, however, both fermented and non-fermented samples reached maximal ACE inhibitory activity (%). The degree of proteolysis showed only a minor increase after fermentation and augmented sharply after digestion. The whey (fermented) digests tended to have lower 50% inhibitory concentrations (IC50) (0.148-0.072 mg/ml), hence higher ACE inhibitory activity, than the pea (fermented) digests (0.183-0.093 mg/ml). The non-fermented digested samples were at least as ACE inhibitory active as the fermented ones. The non-fermented whey protein digest showed the lowest IC50 value and hence the highest ACE inhibitory activity of all. For pea protein, the non-fermented sample had the one but lowest IC50 value. These results suggest that in vitro gastrointestinal digestion was the predominant factor controlling the formation of ACE inhibitory activity, hence indicating its importance in the bioavailability of ACE inhibitory peptides.
Therefore, the formation of ACE inhibitory activity during in vitro gastrointestinal digestion of pea and whey protein was investigated more profoundly.
Firstly, the conditions during in vitro gastrointestinal digestion of pea and whey protein leading to maximal release of ACE inhibitory activity were characterised. In batch experiments, three in vitro gastrointestinal digestions varying in pH and incubation time in the stomach and the small intestine phase, were compared for pea and whey protein. The digestion simulating the physiological conditions of protein hydrolysis in the human body sufficed to achieve the highest ACE inhibitory activity, with IC50 values of 0.076 mg/ml for pea and 0.048 mg/ml for whey protein. The degree of proteolysis did not correlate with the ACE inhibitory activity and pea protein was more susceptible to hydrolysis by gastrointestinal proteases than whey protein. In a semi-continuous reactor model of gastrointestinal digestion (pre-SHIME), response surface methodology was used to investigate the influence of temperature, incubation time in the stomach phase and incubation time in the small intestine phase on the ACE inhibitory activity and the degree of proteolysis. For the pea protein, a central composite design could constitute a linear model for the degree of proteolysis and a quadratic model for the ACE inhibitory activity, expressed as log IC50. Within the model, maximal degree of proteolysis was observed at the highest temperature and the longest incubation time in the small intestine phase, while maximal ACE inhibitory activity was obtained at the longest incubation times in the stomach and the small intestine phase. For the whey protein, no significant model for both responses could be designed. Yet, the overall results demonstrate that the ACE inhibitory activity of pea and whey protein hydrolysates can be controlled by the conditions of the in vitro gastrointestinal digestion. This has implications in food processing, where optimal digestion conditions can be set, as well as in physiological digestion, where pathological defects can hamper release of bioactivity.
Next, the evolution of ACE inhibitory activity, degree of proteolysis and protein degradation was investigated during in vitro physiological digestion. A 2.73 mg/ml pea digest showed maximal ACE inhibitory activity (%) already in the early stomach phase and its IC50 further decreased in the small intestine phase. For whey digest, however, the level of 100% ACE inhibitory activity was only attained in the small intestine phase. Subsequent supplementation of a rat intestinal acetone powder, which simulated the digestion by brush border enzymes, resulted in an increase in IC50 value for both proteins. This decrease in ACE inhibitory activity was less pronounced for pea compared to whey. Yet, a substantial amount of peptides were still ACE inhibitory active after digestion of pea and whey protein by gastrointestinal and brush border enzymes and the final IC50 values were 0.093 mg/ml for pea and 0.128 mg/ml for whey. The degree of proteolysis presented a different evolution and augmented more for pea than for whey in both the stomach and small intestine phase, while the brush border peptidases caused a smaller increase in the degree of proteolysis of pea compared to whey. SDS-PAGE showed that the major part of the proteins in pea was already broken down in the stomach phase. The major whey protein b-lactoglobulin was only degraded from the small intestine phase onwards. The presence of known ACE inhibitory peptides in the pea and whey protein sequences was studied using a dedicated computer program. This program makes use of a database of about 500 reported ACE inhibitory sequences and their IC50 values, and produces a theoretical score, weighted on the total number of amino acids, for the potential of ACE inhibitory peptides in a given source protein. For pea, vicilin and albumin PA2 obtained scores as high as 10 on ACE inhibitory activity, compared to 16 for b-casein taken as a reference protein. b-lactoglobulin exceeded all other proteins with a score of 26. To our knowledge, this is the first described large ACE inhibitory peptide database that attributes scores for the potential of ACE inhibitory activity to protein sequences. In silico digestion of these proteins by pepsin, trypsin and chymotrypsin directly released two ACE inhibitory peptides from PA2, one from vicilin and one from b-lactoglobulin with an IC50 lower than 100 µM. The high ACE inhibitory peptide score of b-lactoglobulin and the fact that this protein resists digestion by pepsin are consistent with the substantial decrease in IC50 for the whey digest during the small intestine phase digestion. The database and in silico digestion facilitate the efficient release of ACE inhibitory peptides and the study of proteins on a large scale.
The ACE inhibitory activity of pea and whey hydrolysate, obtained by in vitro gastrointestinal digestion, with IC50 values of 0.070 mg P/ml and 0.041 mg P/ml, was further increased upon purification by ultrafiltration-centrifugation and subsequent fractionation of the permeate by RP-HPLC. The most active RP-HPLC fractions eluted at 24-28% acetonitrile and therefore contained short, hydrophobic peptides, which is in accordance with the structure-activity relationship of ACE inhibitory peptides. The IC50 values of these fractions amounted to 0.016 mg P/ml for pea and 0.003 mg P/ml for whey. In this way, the ACE inhibitory activity of the pea digest was more than 4 times enriched, while that of the whey digest was more than 13 times augmented. This may suggest that in whey hydrolysate very potent ACE inhibitory peptides were present next to low active peptides, while in pea digest all peptides had similar ACE inhibitory activity.
The intestinal transport of active compounds in the digests and permeates was investigated in Caco-2 cell monolayers. All samples retained relatively high ACE inhibitory activity after 2 h incubation in the presence of Caco-2 homogenates. Addition of 9 mg P/ml digests and 10 mg P/ml permeates in the apical compartments of the Caco-2 cell monolayers resulted in the detection of no or only little ACE inhibitory activity in the basolateral compartments after 1 h transport experiment, even after concentration of the sample. Some ACE inhibitory activity was observed in the transport experiments after addition of 45 mg P/ml digests and 50 mg P/ml permeates in the apical compartments, but these were associated with compromised cell monolayer integrity as indicated by decreased transepithelial electrical resistance and increased sodium fluorescein fluxes. Hence, not the degradation by intestinal peptidases, but the uptake by the cell monolayer seemed to be the decisive factor with respect to the transport of active compounds in the ACE inhibitory digests and permeates. As the Caco-2 model is tighter than intestinal mammalian tissue, transport of these peptides in substantial quantities might still take place in vivo. Overall, these observations suggest that the protein digests can reach the blood stream in an active form
After intravenous administration of a dose of 50 mg P/kg BW in spontaneously hypertensive rats, pea permeate exerted a transient, but strong antihypertensive effect of 44.4 mmHg. Whey permeate exerted no effect at a dose of 50 mg P/kg BW. Further research into potential positive effects in the prevention and treatment of hypertension, for example after daily administration over a longer period of time or after addition of a single higher dose, is necessary.
In conclusion, this work presented a profound investigation on the release of ACE inhibitory activity from pea and whey protein and the maintenance of this activity in the oral delivery route. For the first time, the formation of high ACE inhibitory activity from pea protein is described. This knowledge can be applied in functional foods for the prevention and treatment of hypertension.
References
* V. Vermeirssen, J. Van Camp, L. Devos & W. Verstraete (2003). Release of Angiotensin I Converting Enzyme (ACE) inhibitory activity during in vitro gastrointestinal digestion: from batch experiment to semi-continuous model. Journal of Agricultural and Food Chemistry, 51(19), 5680-5687.
* V. Vermeirssen, J. Van Camp, K. Decroos, L. Van Wijmelbeke & W. Verstraete (2003). The impact of fermentation and in vitro digestion on the formation of ACE inhibitory activity from pea and whey protein. Journal of Dairy Science, 86(2), 429-438.
* V. Vermeirssen, J. Van Camp & W. Verstraete (2002). Optimisation and validation of an angiotensin-converting enzyme inhibition assay for the screening of bioactive peptides. Journal of Biochemical and Biophysical Methods, 51(1), 75-87.
* V. Vermeirssen, B. Deplancke, K.A. Tappenden, J. Van Camp, H.R. Gaskins & W. Verstraete (2002). Intestinal transport of the lactokinin Ala-Leu-Pro-Met-His-Ile-Arg through a Caco-2 Bbe monolayer. Journal of Peptide Science, 8(3), 95-100.
Ghent University
Peptides Current Events and Peptides News RSSLeeds researchers reshape the future of drug discovery
Scientists in Leeds have devised a new way to create the next generation of man-made molecules in a breakthrough that could revolutionise drug development.
Conference report highlights new research into drug delivery to treat eye disease
Researchers are investigating microneedles, nanoparticles and polymer carriers as potential new techniques to combat the leading cause of visual impairment and blindness in the United States, according to a report from the Third Annual ARVO/Pfizer Ophthalmics Research Institute Conference.
Fatty diet during pregnancy makes new cells in fetal brain that cause early onset obesity
A study in rats shows that exposure to a high-fat diet during pregnancy produces permanent changes in the offspring's brain that lead to overeating and obesity early in life, according to new research by Rockefeller University scientists.
Lack of vitamin D linked to Parkinson's disease
A majority of Parkinson's disease patients had insufficient levels of vitamin D in a new study from Emory University School of Medicine.
Discovery of natural compounds that could slow blood vessel growth
Using computer models and live cell experiments, biomedical engineers at the Johns Hopkins University School of Medicine have discovered more than 100 human protein fragments that can slow or stop the growth of cells that make up new blood vessels.
Form of Crohn's disease traced to disabled gut cells
Scientists report online this week in Nature that they have linked the health of specialized gut immune cells to a gene associated with Crohn's disease, an often debilitating and increasingly prevalent inflammatory bowel disorder.
What can we do for prevention and therapy of anaerobe-associated infections?
Anaerobic microorganisms are important constituents of both human and animal intestinal microbiota. Infections caused by anaerobic bacteria are increasingly being recognized as major problem in clinical medicine.
Bio-imaging mass spectrometry techniques reveal molecular details about complex systems
Understanding biology at the systems level is difficult, especially when studying complex specimens like tissue slices or communities of organisms in a biofilm. Scientists must be able to identify, quantify and locate the molecules present in the samples.
Protein misprediction uncovered by new technique
A new bioinformatics tool is capable of identifying and correcting abnormal, incomplete and mispredicted protein annotations in public databases.
NIST membrane model may unlock secrets of early-stage Alzheimer's
Researchers at the National Institute of Standards and Technology (NIST) and three collaborating institutions are using a new laboratory model of the membrane surrounding neurons in the brain to study how a protein long suspected of a role in early-stage Alzheimer's disease actually impairs a neuron's structure and function.
More Peptides Current Events and Peptides News Articles
 | Chemistry of Peptide Synthesis by N. Leo Benoiton Chemistry of Peptide Synthesis is a complete overview of how peptides are synthesized and what techniques are likely to generate the most desirable reactions. Incorporating elements from the author’s role of Career Investigator of the Medical Research Council of Canada and his extensive teaching career, the book emphasizes learning rather than memorization. The text uses clear language and... |
 | Peptide Synthesis and Applications (Methods in Molecular Biology) Hands-on experts describe in step-by-step detail the key methodologies of contemporary peptide synthesis and illustrate their numerous applications. The techniques presented include protocols for chemical ligation, the synthesis of cyclic and phosphotyrosine-containing peptides, lipoamino acid- and sugar-conjugated peptides, and peptide purification and analyses. Additional chapters detail... |
 | Handbook of Biologically Active Peptides Peptides play a crucial role in many physiological processes including actions as neurotransmitters, hormones, and antibiotics. Research has shown their importance in such fields as neuroscience, immunology, pharmacology, and cell biology. The Handbook of Biologically Active Peptides presents, for the first time, this tremendous body of knowledge in the field of biologically active peptides in... |
 | Nutraceutical Proteins and Peptides in Health and Disease (Nutraceutical Science and Technology) This is the first book to cover bioactive proteins and peptides in the area of nutraceuticals and functional foods, providing state-of-the-art information. It examines recent advancements in the field and the biological activities associated with them. It explores the beneficial health effects of peptides, including antimicrobial and antioxidant properties, and discusses hypoallergenic foods such... |
 | Antimicrobial Peptides and Human Disease (Current Topics in Microbiology and Immunology) Microbes are in our midst soon after birth. Thankfully, the number of harmless (and often beneficial) microbes far out number those that would do us harm. Our ability to ward-off pathogens in our environment, including those that can colonize our exterior and/or interior surfaces, depends on the integrative action of the innate and adaptive immunity systems. This volume of CTMI, entitled... |
 | Peptide Synthesis Protocols (Methods in Molecular Biology) (Methods in Molecular Biology) by Michael W. Pennington, Ben M. Dunn In Peptide Synthesis Protocols leading authorities assemble in one volume a broad range of state-of-the-art methods for the preparation, purification, and synthesis of peptides. These powerful and useful protocols contain many innovations not previously described, including a compilation of chemical modification procedures and peptide synthesis improvements. The authors describe each procedure... |
 | HPLC of Peptides and Proteins: Methods and Protocols (Methods in Molecular Biology) (Methods in Molecular Biology) Monash Univ., Clayton, Victoria, Australia. Provides comprehensive coverage of all commonly used HPLC techniques with step-by-step instructions. Presents examples of a wide range of peptide and protein applications and discusses key separation parameters for achieving high resolution. For researchers. DNLM:... |
 | Mass Spectrometry of Proteins and Peptides: Methods and Protocols, Second Edition (Methods in Molecular Biology) When the last edition of this book was published in 2000, the field of proteomics was in its infancy. Now in its adolescence, proteomics is fundamentally transforming biological and medical research. Much of this transformation can be attributed to technological advancements, particularly in mass spectrometry. As a result of these rapid developments that have led to expanded areas of research and... |
 | Chemical Approaches to the Synthesis of Peptides and Proteins (New Directions in Organic & Biological Chemistry) by Paul Lloyd-Williams, Fernando Albericio, Ernest Giralt Organic chemists working on the synthesis of natural products have long found a special challenge in the preparation of peptides and proteins. However, more reliable, more efficient synthetic preparation methods have been developed in recent years. This reference evaluates the most important synthesis methods available today, and also considers methods that show promise for future... |
 | Amino Acid and Peptide Synthesis (Oxford Chemistry Primers, 7) by John Jones This is a thoroughly updated edition of one of the best selling titles in the Oxford Chemistry Primer series. John Jones provides an excellent, easy to read introduction to amino acid and peptide synthesis aimed at second and final year students. The text begins with a brief survey of the role and diversity of amino acids, peptides, and proteins in nature, and goes on to describe and explain the... |
| © 2008 BrightSurf.com |