1. Figs. 2A &2B: In vitro screening of SPH and RP-HPLC fractions for ACE and renin inhibition. Fig. 2C: IC50 determination. Fig. 2D: In vivo BP test in SHRs. 50 g Deboned Salmon I. Preparation of Salmon Protein Samples II. Evaluation of in vitro and in vivo ACE and renin inhibition properties. SF1 SF2 SF4 SF3 Freeze -dried Fractions Salmon Protein Hydrolysate (SPH) Methods Introduction Antihypertensive properties In vitro assays In vivo assay Blood pressure measurement in SHRs ACE Inhibition Renin Inhibition Angiotensin II Vasoconstriction Blood pressure Chymase ACE Renin from the Kidney Bradykinin Vasodilator Inactivates Bradykinin Increasing blood pressure Fig.1: Mechanism of blood pressure regulation in the Renin- Angiotensin System (RAS) Results Conclusions Future Directions References Acknowledgement. Mechanism of action of BAPs as antihypertensive agents. Objectives Hypotheses The objectives of this study were therefore :  To digest salmon protein precipitate using selected gastrointestinal enzymes.  To investigate the in vitro antihypertensive properties of the enzyme-derived salmon protein hydrolysates (SPH) and RP-HPLC fractions.  To determine the in vivo antihypertensive effects of the SPH and most potent RP-HPLC fraction(s) in spontaneously hypertensive rats (SHRs). 1.Lawes CM, Vander Hoorn S, Rodgers A. (2008). Global burden of blood pressure-related disease, 2001. Lancet, 371(9623):1513-1518. 2. Girgih, A., Udenigwe, C., Li, H., et al. (2011). Kinetics of enzyme inhibition and antihypertensive effects of hemp Seed (Cannabis sativa L.) protein hydrolysates. Journal of the American Oil Chemists' Society, 88(11):1767-1774.  Salmon protein hydrolysate (SPH) produced from enzymatic hydrolysis using a combination of gastrointestinal enzymes (pepsin, trypsin and chymotrypsin) and its RPHPLC peptide fraction (SF3) significantly (p<0.05) inhibited the activities of angiotensin I-converting enzyme (ACE) by 90 and 92% respectively, as well as the activities of renin (52 and 72% respectively).  In vivo blood pressure lowering tests using spontaneously hypertensive rats (SHRs) as a model indicated that SF3 significantly reduced systolic blood pressure by up to 42.1 ± 3.380 mmHg, a value slightly higher than the maximum lowering effect (40.45 ± 1.62 mmHg) attained by the drug captopril, which was used as a positive control.  By respectively inhibiting ACE and renin uncompetitively and non-competitively, salmon proteins offer promise as important components of future functional food formulations for the management of hypertension.  Simulated gastrointestinal digestion of salmon protein precipitate could yield protein hydrolysates and bioactive peptides with the capacity to inhibit the activities of ACE and renin, and thus potentially lower elevated BP.  Fractionation of such protein hydrolysates and BAPs could generate partially purified short peptide sequences with enhanced in vitro and in vivo blood pressure-lowering properties.  Value-added utilization of salmon muscle proteins could add functional value to otherwise waste food proteins thus reducing waste disposal-linked carbon footprints. Futhermore, the BAPs obtained could be used as tools for studying the effective management of hypertension thus boosting the prospects of their eventual incorporation into functional foods and nutraceuticals as health-promoting agents. This work was funded through a Discovery Grant from the Natural Sciences and Engineering Research Council of Canada (NSERC) to Dr R.E. Aluko, as well as financial support from the Manitoba Health Research Council/Research Manitoba to I.D. Nwachukwu. Every year, over 7 million fatalities are recorded as a result of hypertension, a risk factor for cardiovascular diseases, and a leading cause of death which affects 15-20% of all adults worldwide. (1) In North America alone, more than $300 billion is lost annually in physician services, hospital costs, lost wages and reduced productivity due to heart diseases and stroke. Therefore, strategies for maintaining normal blood pressure (BP) and/or reducing elevated BP are crucial for preventing the loss of millions of lives, lowering the incidence of associated debilitating disease conditions, and mitigating economic losses attributable to hypertension. Anti-hypertensive agents such as bioactive peptides (BAPs) have recently emerged as potent inhibitors of the activities of angiotensin-converting enzyme (ACE) and renin, two key enzymes in the renin-angiotensin pathway which regulates BP in humans. (2) This work evaluated the BP-lowering properties of BAPs from the muscles of salmon fish. Further studies will involve the purification, identification and characterization of amino acid sequences present in SF3 using HPLC and tandem mass spectrometry. Results Kinetics of enzyme inhibition and blood pressure-lowering effects of salmon (Salmo salar) protein-derived peptides in vitro and in spontaneously hypertensive rats. Ifeanyi D. Nwachukwu 1, Abraham T. Girgih 1, Tom A. Gill 2, and Rotimi E. Aluko 1 1 Department of Human Nutritional Sciences, University of Manitoba, Winnipeg, R3T 2N2, MB, Canada; 2 Department of Process Engineering and Applied Science, Dalhousie University, Halifax, B3H 4R2, NS, Canada. Intervention with BAPs Freeze-dry SPH and <1 kDa fractions 4 separate RP-HPLC fractions of SPH were freeze-dried after evaporation with a rotary evaporator, and subsequently screened for antihypertensive properties. 2A2B 2C 2D 3A 3B 3C 3D Figs. 3A& 3B: Lineweaver -Burk plots of the inhibition of angiotensin –I converting enzyme (ACE) by different concentrations of the most active in vitro antihypertensive salmon peptides SPH (3A), and most active RP-HPLC peptide fraction, SF3, (3B). Figs. 3C & 3D: Lineweaver -Burk plots of the inhibition of human recombinant renin by different concentrations of the most active in vitro antihypertensive salmon peptides SPH (3C), and most active RP-HPLC peptide fraction, SF3, (3D). SPH was prepared by sequentially hydrolyzing mechanically-deboned salmon muscle proteins using pepsin and a trypsin + chymotrypsin combination. Angiotensin I Angiotensinogen from the Liver Isoelectric precipitation Simulated gastrointestinal digestion of protein precipitate with pepsin, trypsin and chymotrypsin. Membrane Filtration IC 50 Inhibition ACE and Renin Kinetics Assay Peptide fraction elution monitored at 220 nm. Fractions automatically collected every 1 min. Fractions were eluted at a flow rate of 10 mL/min using a gradient of 0.1% TFA. SPH was dissolved in 0.1% TFA, filtered and injected into a RP-HPLC column. K m (mM) = Michaelis-Menten constant; V max (ΔA.min -1 or FIU.min -1 ) = maximum reaction velocity in the presence or absence of inhibitor; CE = catalytic efficiency; K i (mg/mL) = enzyme-inhibitor dissociation constant; Ki/Km = inhibition efficiency of the inhibitor.