Enhanced Lacto-Tri-Peptide Bio-Availability by Co-Ingestion of Macronutrients

Some food-derived peptides possess bioactive properties, and may affect health positively. For example, the C-terminal lacto-tri-peptides Ile-Pro-Pro (IPP), Leu-Pro-Pro (LPP) and Val-Pro-Pro (VPP) (together named here XPP) are described to lower blood pressure. The bioactivity depends on their availability at the site of action. Quantitative trans-organ availability/kinetic measurements will provide more insight in C-terminal tri-peptides behavior in the body. We hypothesize that the composition of the meal will modify their systemic availability. We studied trans-organ XPP fluxes in catheterized pigs (25 kg; n=10) to determine systemic and portal availability, as well as renal and hepatic uptake of a water-based single dose of synthetic XPP and a XPP containing protein matrix (casein hydrolyte, CasH). In a second experiment (n=10), we compared the CasH-containing protein matrix with a CasH-containing meal matrix and the modifying effects of macronutrients in a meal on the availability (high carbohydrates, low quality protein, high fat, and fiber). Portal availability of synthetic XPP was 0.08 ± 0.01% of intake and increased when a protein matrix was present (respectively 3.1, 1.8 and 83 times for IPP, LPP and VPP). Difference between individual XPP was probably due to release from longer peptides. CasH prolonged portal bioavailability with 18 min (absorption half-life, synthetic XPP: 15 ± 2 min, CasH: 33 ± 3 min, p<0.0001) and increased systemic elimination with 20 min (synthetic XPP: 12 ± 2 min; CasH: 32 ± 3 min, p<0.0001). Subsequent renal and hepatic uptake is about 75% of the portal release. A meal containing CasH, increased portal 1.8 and systemic bioavailability 1.2 times. Low protein quality and fiber increased XPP systemic bioavailability further (respectively 1.5 and 1.4 times). We conclude that the amount and quality of the protein, and the presence of fiber in a meal, are the main factors that increase the systemic bioavailability of food-derived XPP

Transorgan fluxes in a porcine model reveal a central role for liver in acylcarnitine metabolism

Acylcarnitines are derived from mitochondrial acyl-CoA metabolism and have been associated with diet-induced insulin resistance. However, plasma acylcarnitine profiles have been shown to poorly reflect whole body acylcarnitine metabolism. We aimed to clarify the individual role of different organ compartments in whole body acylcarnitine metabolism in a fasted and postprandial state in a porcine transorgan arteriovenous model. Twelve cross-bred pigs underwent surgery where intravascular catheters were positioned before and after the liver, gut, hindquarter muscle compartment, and kidney. Before and after a mixed meal, we measured acylcarnitine profiles at several time points and calculated net transorgan acylcarnitine fluxes. Fasting plasma acylcarnitine concentrations correlated with net hepatic transorgan fluxes of free and C2- and C16-carnitine. Transorgan acylcarnitine fluxes were small, except for a pronounced net hepatic C2-carnitine production. The peak of the postprandial acylcarnitine fluxes was between 60 and 90 min. Acylcarnitine production or release was seen in the gut and liver and consisted mostly of C2-carnitine. Acylcarnitines were extracted by the kidney. No significant net muscle acylcarnitine flux was observed. We conclude that liver has a key role in acylcarnitine metabolism, with high net fluxes of C2-carnitine both in the fasted and fed state, whereas the contribution of skeletal muscle is minor. These results further clarify the role of different organ compartments in the metabolism of different acylcarnitine specie

Organ Total Net balances of XPP peptides.

<p>Study 1. Post-prandial total net balances across organs over 90 min experimental period after <i>intra-gastric</i> administration of tri-peptide (XPP) mixtures: a synthetic dose of XPP (XPP), XPP containing casein hydrolyte (CasH) and spiked CasH (CasH + XPP). Organs: portal drained viscera (PDV), Liver, Splanchnic area (SPL), Kidneys. Positive balance is net release by an organ. Negative balance is net uptake by an organ. Values are mean ± SEM. Statistics: unpaired t-test for comparison of net balances of natural occurring XPP vs administrated XPP (Control vs XPP), water-based vs protein matrix (XPP vs CasH), and for comparison protein matrixes with vs without XPP spike (CasH vs CasH+XPP). Hooks: significance p<0.05. dotted hooks: tendency p<0.10.</p

Portal Drained Viscera fluxes of XPP—Effect of a protein matrix.

<p>Post-prandial portal drained viscera (PDV) fluxes after <i>intra-gastric</i> administration of tri-peptide (XPP) mixtures: control salt solution (Control), synthetic XPP’s (XPP), casein hydrolyte rich in XPP (CasH) or spiked CasH (CasH + XPP). A: Isoleucine-proline-proline (IPP). B: Leucine-proline-proline (LPP). C: Valine-proline-proline (VPP). Respective number of observations for Control, XPP, CasH and CasH +XPP are for graph A: n = 6, 9, 8 and 9; graph B: n = 6, 10, 9 and 9; graph C: n = 5, 9, 9 and 9. Values are mean ± SEM. Positive values is net release, negative values is net uptake. Statistics: repeated measures two-way ANOVA, mixed model, planned comparisons. All curves are significantly different from the XPP mixture: effect test mixture p<0.01; effect time p<0.01; interaction p<0.01</p

Composition of test mixtures—Study 1.

<p>¹⁾ Chemical purities of the IPP, LPP, and VPP synthetic products were 93.4, 95.0 and 98.7%, respectively (Bachem, Weil am Rhein, Switzerland).</p><p>²⁾ The given amounts of tri-peptides isoleucine-proline-proline (IPP), leucine-proline-proline (LPP), valine-proline-proline (VPP) in the casein hydrolysate (CasH, Casimax, DSM Food Specialties, Delft, The Netherlands). The casein hydrolysate contained 57% protein with 5.4, 16.5 and 0.3 mg/g protein of LPP, LPP and VPP, respectively.</p><p>³⁾ Total XPP = total amount of IPP, LPP and VPP.</p><p>Composition of test mixtures—Study 1.</p

Systemic levels of XPP peptides—Effect of protein matrix.

<p>Post-prandial arterial concentrations after intra-gastric administration of control salt solution (Control), synthetic XPP’s (XPP), casein hydrolysate rich in XPP (CasH) and spiked CasH (CasH + XPP). A: Isoleucine-proline-proline (IPP). B: Leucine-proline-proline (LPP). C: Valine-proline-proline (VPP). Respective number of observations for Control, XPP, CasH and CasH +XPP are for graph A: n = 9, 10, 9 and 10; graph B: n = 10, 10, 9 and 10; graph C: n = 8, 10, 9 and 10. Values are mean ± SEM. Statistics: repeated measures two-way ANOVA, mixed model, planned comparisons. All curves are significantly different from the XPP mixture: effect test mixture p<0.001; effect time p<0.001; interaction p<0.001.</p

Composition of test meals—Study 2.

<p>¹⁾: Indicated is the amount of protein supplied by the isolates.</p><p>²⁾: CHO: maize starch, sucrose and glucose in the weight ratio 2:1:1.</p><p>³⁾: Fat: soybean oil and sunflower oil in the weight ratio 4:1.</p><p>⁴⁾ The casein hydrolysate product (Casimax, DSM Food Specialties, Delft, The Netherlands) contained 57% protein with 5.4, 16.5 and 0.3 mg/g protein LPP, LPP and VPP, respectively.</p><p>⁵⁾ Fiber: modified citrus pectin.</p><p>Composition of test meals—Study 2.</p

Protein fractional synthesis rates within tissues of high- and low-active mice.

With the rise in physical inactivity and its related diseases, it is necessary to understand the mechanisms involved in physical activity regulation. Biological factors regulating physical activity are studied to establish a possible target for improving the physical activity level. However, little is known about the role metabolism plays in physical activity regulation. Therefore, we studied protein fractional synthesis rate (FSR) of multiple organ tissues of 12-week-old male mice that were previously established as inherently low-active (n = 15, C3H/HeJ strain) and high-active (n = 15, C57L/J strain). Total body water of each mouse was enriched to 5% deuterium oxide (D2O) via intraperitoneal injection and maintained with D2O enriched drinking water for about 24 h. Blood samples from the jugular vein and tissues (kidney, heart, lung, muscle, fat, jejunum, ileum, liver, brain, skin, and bone) were collected for enrichment analysis of alanine by LC-MS/MS. Protein FSR was calculated as -ln(1-enrichment). Data are mean±SE as fraction/day (unpaired t-test). Kidney protein FSR in the low-active mice was 7.82% higher than in high-active mice (low-active: 0.1863±0.0018, high-active: 0.1754±0.0028, p = 0.0030). No differences were found in any of the other measured organ tissues. However, all tissues resulted in a generally higher protein FSR in the low-activity mice compared to the high-activity mice (e.g. lung LA: 0.0711±0.0015, HA: 0.0643±0.0020, heart LA: 0.0649± 0.0013 HA: 0.0712±0.0073). Our observations suggest that high-active mice in most organ tissues are no more inherently equipped for metabolic adaptation than low-active mice, but there may be a connection between protein metabolism of kidney tissue and physical activity level. In addition, low-active mice have higher organ-specific baseline protein FSR possibly contributing to the inability to achieve higher physical activity levels

Activated whole-body arginine pathway in high-active mice.

Our previous studies suggest that physical activity (PA) levels are potentially regulated by endogenous metabolic mechanisms such as the vasodilatory roles of nitric oxide (NO) production via the precursor arginine (ARG) and ARG-related pathways. We assessed ARG metabolism and its precursors [citrulline (CIT), glutamine (GLN), glutamate (GLU), ornithine (ORN), and phenylalanine (PHE)] by measuring plasma concentration, whole-body production (WBP), de novo ARG and NO production, and clearance rates in previously classified low-active (LA) or high-active (HA) mice. We assessed LA (n = 23) and HA (n = 20) male mice by administering a stable isotope tracer pulse via jugular catheterization. We measured plasma enrichments via liquid chromatography tandem mass spectrometry (LC-MS/MS) and body compostion by echo-MRI. WBP, clearance rates, and de novo ARG and NO were calculated. Compared to LA mice, HA mice had lower plasma concentrations of GLU (71.1%; 36.8 ± 2.9 vs. 17.5 ± 1.7μM; p<0.0001), CIT (21%; 57.3 ± 2.3 vs. 46.4 ± 1.5μM; p = 0.0003), and ORN (40.1%; 55.4 ± 7.3 vs. 36.9 ± 2.6μM; p = 0.0241), but no differences for GLN, PHE, and ARG. However, HA mice had higher estimated NO production ratio (0.64 ± 0.08; p = 0.0197), higher WBP for CIT (21.8%, 8.6 ± 0.2 vs. 10.7 ± 0.3 nmol/g-lbm/min; p<0.0001), ARG (21.4%, 35.0 ± 0.6 vs. 43.4 ± 0.7 nmol/g-lbm/min; p<0.0001), PHE (7.6%, 23.8 ± 0.5 vs. 25.6 ± 0.5 nmol/g-lbm/min; p<0.0100), and lower GLU (78.5%; 9.4 ± 1.1 vs. 4.1 ± 1.6 nmol/g lbm/min; p = 0.0161). We observed no significant differences in WBP for GLN, ORN, PHE, or de novo ARG. We concluded that HA mice have an activated whole-body ARG pathway, which may be associated with regulating PA levels via increased NO production

Transhepatic bile acid kinetics in pigs and humans

Bile acids (BAs) play a key role in lipid uptake and metabolic signalling in different organs including gut, liver, muscle and brown adipose tissue. Portal and peripheral plasma BA concentrations increase after a meal. However, the exact kinetics of postprandial BA metabolism have never been described in great detail. We used a conscious porcine model to investigate postprandial plasma concentrations and transorgan fluxes of BAs, glucose and insulin using the para-aminohippuric acid dilution method. Eleven pigs with intravascular catheters received a standard mixed-meal while blood was sampled from different veins such as the portal vein, abdominal aorta and hepatic vein. To translate the data to humans, fasted venous and portal blood was sampled from non-diabetic obese patients during gastric by-pass surgery. The majority of the plasma bile acid pool and postprandial response consisted of glycine-conjugated forms of primary bile acids. Conjugated bile acids were more efficiently cleared by the liver than unconjugated forms. The timing and size of the postprandial response showed large interindividual variability for bile acids compared to glucose and insulin. The liver selectively extracts most BAs and BAs with highest affinity for the most important metabolic BA receptor, TGR5, are typically low in both porcine and human peripheral circulation. Our findings raise questions about the magnitude of a peripheral TGR5 signal and its ultimate clinical applicatio