Journal of Food Bioactives, ISSN 2637-8752 print, 2637-8779 online
Journal website www.isnff-jfb.com

Original Research

Volume 29, March 2025, pages 60-76

Presence of γ-glutamyl and β-aspartyl isopeptides, diketopiperazines, pyroglutamyl peptides, in addition to normal peptides in fish and soy sauces: Structures, contents and their bioavailability

Figures

Figure 1.
Figure 1. Mass spectrometry (MS) chromatograms of AccQ-derivatized compounds in the VFS. (a) Peaks were detected by precursor ion scan targeting the AccQ-derived product ion (m/z 171.1) in the positive mode across the m/z of 225–250, 250–275, 275–300, 300–350, 350–400, 400–500 and 500–600. Identified amino acids, peptides, and their metabolites are labelled using single-letter amino acid codes or full names. (b) Enlarged MS chromatogram at the scan range m/z 400–500. Other abbreviations: Hydroxyproline (O), β-alanine (*D), ethanolamine (*S), γ-aminobutyric acid (*E), isopropanolamine (*T), prolinamide (P-NH2), taurine (Ta), and citrulline (Cit). Lysine reacted with one and two AccQ are indicated as K1 and K2, respectively. Ornithine, putrescine, and cadaverine reacted with one AccQ are indicated as Or1, Pu1, and Ca1, respectively. Unlabelled peaks could not be assigned to amino acids, peptides, or their metabolites.
Figure 2.
Figure 2. MS chromatograms of non-derivatized compounds in the VFS. Peaks were detected by total ion scan in the positive mode across the m/z of 100–200, 200–225, 225–250, 250–275, 275–300, and 300–350. Peaks of amino acids are presented by single-letter amino acid codes. Peaks of modified amino acids with organic acids are indicated using prefixes such as acetyl-, propionyl-, and butyryl- and single-letter amino acid codes. Peaks with numbers were identified as peptides. Peaks with same numbers indicated presence of peptide isomers with same precursor and product ions. Unlabelled peaks could not be assigned to amino acids, peptides, or their metabolites. *Indicates the ion consisting of two pyroglutamic acids and proton.
Figure 3.
Figure 3. Representative MS chromatograms of two isomers of the underivatized α− and γ-glutamyl-dipeptides in the VFS. Samples and the synthetic standard peptides were analysed using RP-HPLC-MS/MS in MRM mode. αEM and γEM represent α-Glu-Met and γ-Glu-Met, respectively. For other glutamyl dipeptides are shown to be similar.
Figure 4.
Figure 4. Representative MS chromatograms of four isomers of underivatized aspartyl-dipeptide (Lα-Asp-X, Lβ-Asp-X, Dα-Asp-X, and Dβ-Asp-X) in the VFS. X is occupied by Val (V), Ile (I), Leu (L) and Phe (F). Samples and synthetic standard peptides were analysed using RP-HPLC-MS/MS in MRM mode.
Figure 5.
Figure 5. Structure of α- and γ-glutamyl dipeptides (a) and α- and β- L/D aspartyl dipeptides (b). X is occupied by amino acid residues.
Figure 6.
Figure 6. Heatmap of contents of all forms of 10 glutamyl dipeptides,16 aspartyl dipeptides, and 7 normal major peptides in the samples. Refer Figure 5 for structures of isomers.
Figure 7.
Figure 7. Heatmap of contents of 16 pyroglutamyl peptides and 12 diketopiperazines in the samples. pGlu represents pyroglutamyl residue. The Cyclo-Tyr-Pro is a diketopiperazine consisting of Tyr and Pro residues.

Tables

Table 1. Estimated sequences of peptides in the derivatized derived with AccQ based on m/z of product ions and precursor ions
 
SequencesRTPrecursor ion (m/z)MWProduct ion (m/z)
The derivatives were resolved by LC-MS/MS as shown in Figure 1a. Asterisks (*) represent immonium ions of amino acid. AccQ b1 ion was derived from AccQ moiety of the peptide derivatives. a, b, and c series and x, y and z series of product ions of peptides are present. M/Z is mass per charge. MW represents molecular weight of peptides without derivatization. RT is retention time.
Gly-Pro17.8134317230 (Gly*), 70.0 (Pro*), 116.0 (y1), 171.0 (AccQ, b1), 173.3 (y2), 227.8 (b2) & 343.3
Glu-Pro19.5041524484 (Glub), 116 (y1), 171.0 (AccQ, b1), 227 (z2), 272 (a2), 272 (x2), 300 (b2) & 415
Ala-Pro20.3535718644.2 (Ala*), 70.0 (Pro*), 116.1 (y1), 171.0 (AccQ, b1), 186.8 (y2), 242.2 (b2) & 357.0
Ser-Pro17.6537320260 (Ser*), 70 (Pro*), 116 (y2), 171 (AccQ, b1), 258 (b2), 203 (y2) & 373
Pro-Pro20.4638321270.4 (Pro*), 116.1 (y1), 171.1 (AccQ, b1), 212.8 (y2), 240 (a2), 240 (x2), 267.9 (b2) & 383.0
Val-Pro26.2238521472.3 (Val*), 70.2 (Pro*), 116.2 (y1), 171.1 (AccQ, b1), 215.1 (y2), 242.1 (a2), 269.9 (b2) & 385.0
Ile-Pro29.9539922870.2 (Pro*), 86.0 (Ile*), 116.4 (y1), 171.1 (AccQ, b1), 228.8 (y2), 256.3 (x2), 284.1 (b2) & 399.0
Leu-Pro30.4739922870.6 (Pro*), 86.2 (Leu*), 116.4 (y1), 171.1 (AccQ, b1), 228.8 (y2), 256.3 (x2), 284.1 (b2) & 399.0
Glu-Pro19.6341524470.4 (Pro*), 84 (Glub) 102.2 (Glu*), 116.0 (y1), 171.1 (AccQ, b1) 187 (c1), 272 (a2), 300 (b2) & 415
Gly-Pro-Ala17.5841424344 (Gly*), 127, 155, 171.1 (AccQ, b1), 187,200, 228, 244 & 414
Ser-Pro-Asp19.0048831760 (Ser*), 171.1 (AccQ, b1), 255 (z2), 318 (y3), 355 (b3) & 488
Gly-Pro-Thr20.3544427370 (Pro*), 74 (Thr*) 114, 127, 145, 171 (AccQ, b1), 200 (a2), 217 (y2), 228 (b2) & 444
Ala-Hyp16.2637320244 (Ala*), 86,3 (Hyp*), 132.1 (y1), 171.0 ( AccQ, b1), 203.0, (y2), 242.3 (b2) & 373.0
Ile-/Hyp25.5641524486.0 (Ile*), 86.0 (Hyp*), 132.4 (y1), 171.1 (AccQ, b1), 245.1 (y2), 256.1 (a2), 284.1(b2) & 415.0
Leu/Hyp26.0041524486.0 (Ile*), 86.0 (Hyp*), 132.4 (y1), 171.1 (AccQ, b1), 245.1 (y2), 256.1 (a2), 284.1(b2) & 415.0
Phe-Hyp21.59449278145, 171 (AccQ, b1), 279 (y2), 318 (b2) & 449
Gly-Pro-Hyp16.8045628570 (Pro*), 86 (Hyp*), 127 (a3), 132 (y1), 171 (AccQ, b1), 229 (y2), 286 (y3) & 456
Gly-Ile23.0035918830 (Gly*), 86 (Ile*), 128, 132 (y1), 145, 171 (AccQ, b1), 189 (y2), 200 (a2), 228 (b2), & 359
Gly-Leu23.5035918830 (Gly*), 86 (Ile*), 128, 132 (y1), 145, 171 (AccQ, b1), 189 (y2), 200 (a2), 228 (b2), & 359
Gly-Ile (Heavy type)23.0036018986/87 (Ile*), 171/172 (AccQ, b1), 190 (y2), 201 (a2), 229 (b2), & 359
Gly-Leu (Heavy type)23.5036018986/87 (Leu*), 171/172 (AccQ, b1), 190 (y2), 201 (a2), 229 (b2), & 359
Gly-His10.14383212110 (His*), 156 (y1), 171 (AccQ, b1), 200 (a1), 213 (y2), 338 (a3), 365 (b3) & 383
Pro-Glu13.7541524470 (Pro*), 102 (Glu*), 171.1 (AccQ, b1), 264.1, 244.2, 367.4 & 415
Gln-Pro13.5041424384 (Glnb), 116 (y1), 171 (AccQ, b1), 244 (y2) & 414
Glu-Met21.0044927884 (Glub), 171 (AccQ, b1), 272 (a2), 249 (x2), 279 (y2), 300 (b2) & 449
Asp-Ile21.5041724688 (Asp*), 132 (y1), 171 (AccQ, b1), 247 (y2), 286 (b2) & 417
Asp-Leu22.0041724688 (Asp*), 132 (y1), 171 (AccQ, b1), 247 (y2), 286 (b2) & 417
Ala-Ile24.6837320244 (Ala*), 86 (Ile*), 171 (AccQ, b1), 214 (a2), 242 (b2) & 373
Ala-Leu24.7837320244 (Ala*), 86 (Leu*), 171 (AccQ, b1), 214 (a2), 242 (b2) & 373
Asn-Glu15.2043226187 (Asn*), 70 (Asnb), 171 (AccQ, b1), 258 (a2), 286 (b2) & 433
Val-Gly20.3034517472 (Val*), 55 (Valb), 171 (AccQ, b1), 242 (a2), 270 (b2) & 345
Ala-Val12.1035918844 (Ala*), 171 (AccQ, b1), 189 (y2), 215 (a2) & 359
Gly-Gly-Gly11.10360189171 (AccQ, b1), 190 (y3), 200 (a2) & 360
Gly-Met19.70377206171 (AccQ, b1),186 (c1), 207 (y2), 228 (b2) & 377
Ala-Glu16.4038921844 (Ala*), 171 (AccQ, b1), 148 (y1), 219 (y2), 243 (b2) & 389
Gly-Phe20.3539322230 (Gly*), 120 (Phe*), 166 (y1), 171 (AccQ, b1), 201 (a2), 228 (b2) & 393
Gly-Gly-Pro15.9040022930 (Gly*), 70 (Pro*), 116 (y1), 171 (AccQ, b1), 200 (a2), 173 (y2), 227 (b2) & 400
Glu-Ile25.0043126084 (Glub), 86 (Ile*), 102 (Glu*), 132 (y1), 171 (AccQ, b1), 261 (y2), 300 (b2) & 431
Glu-Leu26.0043126084 (Glub), 86 (Leu*), 102 (Glu*), 132 (y1), 171 (AccQ, b1), 261 (y2), 300 (b2) & 431
Glu-Phe27.00465294120 (Phe*), 171 (AccQ, b1), 278 (z2), 278 (b2), 295 (y2) & 465
Glu-Val21.5046529472 (Val*), 102 (Glu*) 171 (AccQ, b1), 229 (z2) & 417
Ala-Glu-Asn25.5043126044 (Ala*), 171 (AccQ, b1), 261 (b3) & 431
Glu-Gly14.52375204102 (Glu*), 171 (AccQ, b1), 272 (a2) & 375

 

Table 2. Estimated sequences of peptides in the VFS without any derivatization
 
SequencesPeak markPrecursor ion (m/z)MWProduct ion (m/z)
The peptides were resolved by LC-MS/MS as shown in Figure 2. Adduct ions consisting of two molecules and a proton are marked with †. One asterisk (*) represents immonium ion of amino acids. Two asterisks (**) represent related ions generated by decomposition of amino acid residues, except for the immonium ion. pGlu represents pyroglutamyl residue. MW represents molecular weight.
pGlu-dipeptides
  pGlu-Gly118818784 (pGlu*), 30 (Gly*), 56 (pGlu**) & 76 (y1) (heavy type)
  pGlu-Ala220120084 (pGlu*), 44 (Ala*), 41, 56 (pGlu**), 90 (y1) & 155 (a2)
  pGlu-Ser321721684 (pGlu*), 60 (Ser*), 28, 41, 56 (pGlu**) & 106 (y1)
  pGlu-Asp424524484 (pGlu*), 28, 41, 56 (pGlu**), 134 (y1) & 227 (b2)
  pGlu-Pro522722684 (pGlu*), 70 (Pro*), 28, 41, 56 (pGlu**), 116 (y1), 181 (a2) & 208 (b2)
  pGlu-Glu625925884 (pGlu*), 102 (Glu*), 41, 56 (pGlu**), 84 (Glu**) 148 (y1) & 241 (b2)
  pGlu-Thr723123084 (pGlu*), 74 (Thr*), 28, 41, 56 (pGlu**), 120 (y1) & 213 (b2)
  pGlu-Met826126084 (pGlu*), 104 (Met*), 41, 56 (pGlu**), 61 (Met**), 150 (y1) 215 (a2) & 243 (b2)
  pGlu-Ile924324284 (pGlu*) 86 (Ile*), 41, 56 (pGlu**), 132 (y1), 129 (b2) & 197 (a2)
  pGlu-leu1024324284 (pGlu*), 86 (Ile*), 4, 56 (pGlu**), 44 (Ile**), 132 (y1), 197 (a2), 141 (z1) & 225 (b2)
  pGlu-Val1122922884 (pGlu*) 72 (Val*), 28, 41, 56 (pGlu**), 118 (y1), 183 (a2) & 211 (b2)
  pGlu-Phe1227727684 (pGlu*), 120 (Phe*), 56 (pGlu**), 166 (y1) & 231 (a2)
  pGlu1313113084 (pGlu*), 41, 56 (pGlu**) &130 (y1)
  Two pGlu13†25925884 (pGlu*), 28, 41, 56 (pGlu**) & 130 (y1)
Glu-dipeptides
  α and γ Glu-Met14279278102 (Glu*), 104 (Met*), 84 (Glu**), 61 (Met**) & 150 (y1)
  α and γ Glu-Val15247246102 (Glu*), 72 (Val*), 84 (Glu**), 41, 55 (Val**), 118 (y1) & 230 (z2)
  α and γ Glu-Ile16261260102 (Glu*), 86 (Ile*), 84 (Glu**), 132 (y1) & 244 (Z2)
  α and γ Glu-leu17261260102 (Glu*), 86 (Leu*), 84 (Glu**), 132 (y1) & 244 (Z2)
  α and γ Glu-Phe18295294102 (Glu*), 120 (Phe *), 84 (Glu**), 166(y1) & 278 (z2)
Asp-dipeptides
  α and β-L/D-Asp-Val1923323288 (Asp*), 72 (Val*), 70 (Asp**), 55 (Val**), 118 (y1) & 187 (a2)
  α and β-L/D-Asp-Ile/leu2024724688 (Asp*), 86 (Ile*), 70 (Asp**), 44 (Ile**), 132 (y1), 201 (a2) & 229 (b2)
  α and β-L/D-Asp-Phe2128128088 (Asp*), 120 (Phe*), 70 (Asp**), 166 (y1) & 235 (a2)
Cyclic dipeptides
  Cyclo-Gly-Pro2215515470 (Pro*), 30 (Asp*), 70 (Asp**), 98 (z1) & 195 (b2)
  Cyclo-Ala-Pro2316916870 (Pro*), 44 (Ala*) & 98 (z1)
  Cyclo-Asp-Pro2421321270 (Pro*), 88 (Asp*), 70 (Asp**) & 195 (b2)
  Cyclo-His-Pro2523523470 (Pro*), 110 (His*), 82, 121, 123 (His**) & 217 (b2)
  Cyclo-Thr-Pro2619919870 (Pro*), 74 (Thr*), 98 (z1), 153 (a2) & 281 (b2)
  Cyclo-Lys-Pro2722622570 (Pro*), 70, 84, 112 (Lys**), 98 (z1), 181 (a2) & 209 (b2)
  Cyclo-Glu-Pro2822722670 (Pro*), 84 (Glu**) 116 (y1), 181 (a2) & 209 (b2)
  Cyclo-Ser-Pro2918518470 (Pro*), 60 (Ser*) & 167 (b2)
  Cyclo-Asn-Pro3021221170 (Pro*), 70 (Asn**) & 195 (b2)
  Cyclo-Pro-Pro3119519470 (Pro*) & 98 (b1)
  Cyclo-Ile-Pro3221121070 (Pro*), 86 (Ile*) & 44 (Ile**)
  Cyclo-Leu-Pro3321121070 (Pro*), 86 (Ile*), 44 (Ile**) & 194 (z2)
  Cyclo-Val-Pro3419719670 (Pro*), 72 (Val*) & 55 (val**)
  Cyclo-Phe-Pro3524524470 (Pro*) & 116 (y1)
  Cyclo-Tyr-Pro3626126070 (Pro*), 136 (Tyr*), 91 & 107 (Tyr**)

 

Table 3. Contents of some normal major dipeptides in the inner content and washed tissue of small intestine and blood plasma after oral administration of vehicle and Vietnamese fish sauce (VFS)
 
Normal PeptidesInner content of small intestine (μM)Small intestinal tissue (nmol/Kg)Blood plasma (nM)
AnteriorPosteriorPortal bloodAbdominal blood
VehicleTreatment (60 mins)VehicleTreatment (60 mins)VehicleTreatment (60 mins)VehicleTreatment (60 mins)VehicleTreatment (60 mins)
The inner content of small intestine was eluted with 10 mL PBS. Data are shown as mean ± standard deviation (n = 3). Asterisks indicate significant differences between values of vehicle and VFS using t-test (†p < 0.10, *p < 0.05).
Normal major peptides
  Ala-Glu1.9 ± 0.42.1 ± 0.1764.7 ± 349.52,866.7 ± 114.1*571.3 ± 48.32,304.7 ± 136.5*21.0 ± 4.018.3 ± 5.713.7 ± 3.522.0 ± 10.4
  Gly-Phe1.6 ± 0.71.4 ± 0.4238.0 ± 66.1748.7 ± 426.7†247.3 ± 89.2776.0 ± 278.6*61.7 ± 4.561.7 ± 25.885.3 ± 13.881.7 ± 10.9
  Gly-Ile2.5 ± 1.32.6 ± 0.4101.3 ± 7.57294.0 ± 58.6*110.7 ± 15.0415.3 ± 52.5*6.0 ± 2.04.3 ± 1.26.0 ± 1.75.0 ± 1.0
  Gly-Leu7.3 ± 2.67.1 ± 2.2631.3 ± 136.52,149.3 ± 493.7*514.7 ± 209.11,895.3 ± 390.8*25.7 ± 17.229.7 ± 5.128.7 ± 22.528.0 ± 21.6
  Gly-Pro0.2 ± 0.10.2 ± 0.5118.7 ± 42.7184.0 ± 72.9144.7 ± 14.5422.0 ± 104.3*45.0 ± 7.047.7 ± 9.356.3 ± 49.354.0 ± 43.6
  Ala-Pro0.02 ± 0.00.02 ± 0.042.7 ± 15.042.7 ± 11.018.7 ± 9.046.0 ± 8.0*5.3 ± 1.54.3 ± 3.80.0 ± 0.00.0 ± 0.0
  Val-Pro0.1 ± 0.00.1 ± 0.00.0 ± 0.00.0 ± 0.00.0 ± 0.00.0 ± 0.00.0 ± 0.00.0 ± 0.00.0 ± 0.00.0 ± 0.0

 

Table 4. Contents of glutamyl dipeptides and aspartyl dipeptides in the inner content and washed tissue of small intestine and blood plasma after oral administration of vehicle and Vietnamese fish sauce (VFS)
 
Peptide IsomersInner content of small intestine (μM)Small intestinal tissue (nmol/Kg)Blood plasma (nM)
AnteriorPosteriorPortal bloodAbdominal blood
VehicleTreatment (60 mins)VehicleTreatment (60 mins)VehicleTreatment (60 mins)VehicleTreatment (60 mins)VehicleTreatment (60 mins)
The inner content of small intestine was eluted with 10 mL PBS. Data are shown as mean ± standard deviation (n = 3). Asterisks indicate significant differences between values of vehicle and VFS using t-test (†p < 0.10, *p < 0.05, **p < 0.01).
Glu-dipeptide isomers
  α-Glu-Met0.04 ± 0.010.1 ± 0.1†1,063.1 ± 502.01,920.9 ± 224.0*1,135.1 ± 478.02,454.7 ± 702.0*17.7 ± 1.616.6 ± 2.714.9 ± 1.711.9 ± 1.8
  α-Glu-Val0.2 ± 0.10.2 ± 0.04165.7 ± 78.3352.7 ± 91.6*172.4 ± 58.0553.7 ± 232.0*6.8 ± 1.35.8 ± 0.44.7 ± 0.33.6 ± 0.7
  α-Glu-Ile0.3 ± 0.20.5 ± 0.04192.1 ± 194.9630.3 ± 70.3*167.5 ± 117.6980.1 ± 510.0*8.9 ± 2.54.9 ± 1.44.9 ± 1.53.5 ± 0.8
  α-Glu-Leu1.2 ± 0.52.1 ± 0.3*653.7 ± 602.01,971.9 ± 572.0*636.9 ± 474.03,079.5 ± 1,486.0*8.9 ± 0.67.9 ± 1.54.9 ± 1.54.0 ± 1.1
  α-Glu-Phe0.5 ± 0.20.8 ± 0.2†277.0 ± 278.0875.1 ± 256.0*286.9 ± 192.01,411.5 ± 660.0*2.2 ± 1.31.7 ± 0.10.8 ± 1.41.1 ± 0.1
  γ-Glu-Met0.1 ± 0.020.4 ± 0.04**3,888.8 ± 2,422.07,210.0 ± 1,242.0†878.8 ± 166.71,402.4 ± 145.2**256.9 ± 194.0203.0 ± 90.2196.4 ± 47.9159.0 ± 40.1
  γ-Glu-Val0.1 ± 0.15.6 ± 3.5*1,424.5 ± 88.22,354.0 ± 594.0*623.7 ± 167.11,885.1 ± 778.8*167.0 ± 6.1228.3 ± 27.5*167.0 ± 6.1228.3 ± 27.5*
  γ-Glu-Ile0.3 ± 0.32.4 ± 1.8†5,638.9 ± 330.05,933.1 ± 1,556.0339.7 ± 32.7807.0 ± 292.1*86.9 ± 22.692.7 ± 13.073.2 ± 0.789.6 ± 10.0*
  γ-Glu-Leu0.2 ± 0.031.7 ± 0.9*5,124.7 ± 302.05,890.1 ± 1,590.03,555.7 ± 901.95,879.1 ± 1,198.1*306.7 ± 95.0230.7 ± 54.9212.8 ± 35.4277.0 ± 53.4
  γ-Glu-Phe0.04 ± 0.022.7 ± 1.6*6,763.6 ± 802.07,513.9 ± 1,760.02,320.0 ± 441.54,100.0 ± 2,331.3*266.3 ± 70.5251.7 ± 35.2205.7 ± 29.9270.6 ± 27.7*
Asp-dipeptide isomers
  Lα-Asp-Val0.1 ± 0.010.2 ± 0.03*83.5 ± 41.1448.7 ± 391.652.5 ± 14.2204.9 ± 98.5†0.3 ± 0.00.2 ± 0.20.5 ± 0.40.3 ± 0.4
  Dα-Asp-Val0.02 ± 0.010.1 ± 0.01*50.2 ± 8.3275.5 ± 66.4*34.2 ± 6.4116.9 ± 44.0*3.2 ± 2.32.5 ± 1.61.5 ± 0.13.9 ± 0.4**
  Lβ-Asp-Val0.1 ± 0.010.4 ± 0.14*11.7 ± 4.2171.5 ± 43.3**7.3 ± 0.0461.6 ± 2.6**3.8 ± 1.110.5 ± 3.1**2.0 ± 0.26.1 ± 1.7*
  Dβ-Asp-Val0.01 ± 0.00.03 ± 0.01*2.4 ± 0.515.9 ± 3.9**2.2 ± 0.47.2 ± 2.6*0.6 ± 0.43.7 ± 0.7*0.4 ± 0.43.7 ± 1.9*
  Lα-Asp-Ile0.04 ± 0.010.1 ± 0.01*58.1 ± 18.5118.2 ± 48.2†32.34 ± 3.747.1 ± 11.3†0.5 ± 0.30.3 ± 0.60.1 ± 0.20.3 ± 0.1
  Dα-Asp-Ile0.01 ± 0.00.01 ± 0.0*50.6 ± 2.589.7 ± 62.213.4 ± 4.440.6 ± 20.4†0.9 ± 0.21.0 ± 0.41.3 ± 0.02.2 ± 0.5*
  Lβ-Asp-Ile0.01 ± 0.00.1 ± 0.03**21.1 ± 2.181.4 ± 24.8**17.7 ± 6.364.9 ± 15.9**7.0 ± 0.91 0.2 ± 1.5*6.6 ± 0.29.9 ± 1.3*
  Dβ-Asp-Ile0.01 ± 0.00.01 ± 0.01*3.9 ± 0.620.8 ± 5.4**2.4 ± 0.56.2 ± 0.9**0.5 ± 0.02.1 ± 0.6**0.3 ± 0.02.9 ± 0.6**
  Lα-Asp-Leu0.0 ± 0.00.01 ± 0.0*8.2 ± 1.537.6 ± 9.7*4.8 ± 1.512.8 ± 1.2*0.0 ± 0.00.04 ± 0.10.1 ± 0.10.1 ± 0.1
  Dα-Asp-Leu0.01 ± 0.00.01 ± 0.0*8.7 ± 3.755.2 ± 71.94.7 ± 0.86.9 ± 2.94.7 ± 1.86.3 ± 1.56.9 ± 0.28.6 ± 0.7*
  Lβ-Asp-Leu0.02 ± 0.010.3 ± 0.2*107.2 ± 4.0210.9 ± 66.1*51.5 ± 18.9128.2 ± 24.7**4.9 ± 0.814.9 ± 1.9*4.0 ± 0.510.1 ± 2.5*
  Dβ-Asp-Leu0.01 ± 0.00.1 ± 0.04*25.7 ± 0.869.2 ± 27.5*14.3 ± 3.152.3 ± 19.4*7.1 ± 1.410.9 ± 1.6*8.0 ± 1.614.5 ± 3.9*
  Lα-Asp-Phe0.06 ± 0.010.1 ± 0.2*116.7 ± 55.7238.5 ± 33.2*101.5 ± 42.870.8 ± 13.40.7 ± 0.40.3 ± 0.20.2 ± 0.30.3 ± 0.2
  Dα-Asp-Phe0.01 ± 0.00.01 ± 0.0**9.5 ± 2.910.4 ± 1.83.8 ± 0.86.9 ± 2.2†0.3 ± 0.020.4 ± 0.0*0.0 ± 0.00.5 ± 0.1*
  Lβ-Asp-Phe0.02 ± 0.010.2 ± 0.1*16.6 ± 3.592.4 ± 29.6**26.5 ± 0.869.3 ± 27.5*3.4 ± 0.65.1 ± 0.8*2.1 ± 0.75.9 ± 2.3*
  Dβ-Asp-Phe0.01 ± 0.00.1 ± 0.03*8.9 ± 2.361.2 ± 11.9**11.9 ± 0.021.8 ± 0.4**4.6 ± 1.09.3 ± 1.5**4.6 ± 0.811.2 ± 4.1*

 

Table 5. Contents of diketopiperazines and pyroglutamyl dipeptides in the inner content and washed tissue of small intestine and blood plasma after oral administration of vehicle and Vietnamese fish sauce (VFS)
 
Modified peptidesInner content of small intestine (μM)Small intestinal tissue (nmol/Kg)Blood plasma (nM)
AnteriorPosteriorPortal bloodAbdominal blood
VehicleTreatment (60 mins)VehicleTreatment (60 mins)VehicleTreatment (60 mins)VehicleTreatment (60 mins)VehicleTreatment (60 mins)
The inner content of small intestine was eluted with 10 mL PBS. Data are shown as mean ± standard deviation (n = 3). Asterisks indicate significant differences between values of vehicle and VFS using t-test (†p < 0.10, *p < 0.05, **p < 0.01).
Diketopiperazines
  Cyclo-Tyr-Pro0.01 ± 0.00.02 ± 0.0*29.7 ± 11.9128.7 ± 10.1*7.4 ± 1.720.7 ± 4.9*459.3 ± 163.9414.7 ± 166.5368.7 ± 83.5398.7 ± 166.7
  Cyclo-Glu-Pro0.04 ± 0.02.6 ± 1.3*55.9 ± 34.6622.0 ± 315.7*136.5 ± 6.9954.7 ± 348.1*175.7 ± 48.0901.3 ± 221.8**108.0 ± 6.6424.0 ± 107.6**
  Cyclo-Asn-Pro0.04 ± 0.05.6 ± 3.6*114.1 ± 24.2972.8 ± 461.6*138.5 ± 20.23,015.9 ± 1,652.4*385.3 ± 53.71,359.7 ± 170.7**252.0 ± 47.6888.0 ± 217.2**
  Cyclo-Thr-Pro0.02 ± 0.00.4 ± 0.2*77.9 ± 19.0654.1 ± 183.4*99.4 ± 17.0547.2 ± 124.1*419.7 ± 94.45,175.3 ± 723.6**316.0 ± 64.43,666.7 ± 461.9**
  Cyclo-Pro-Pro0.01 ± 0.00.02 ± 0.0**20.0 ± 1.453.5 ± 5.1*25.5 ± 2.954.7 ± 6.2*175.0 ± 11.5619.3 ± 155.9**125.0 ± 22.5442.3 ± 57.6**
  Cyclo-Ser-Pro0.01 ± 0.011.5 ± 0.9*378.3 ± 19.11,242.3 ± 340.9*248.3 ± 41.51,571.3 ± 555.6**335.7 ± 53.54,514.7 ± 757.7**238.0 ± 54.33,233.3 ± 305.5**
  Cyclo-Ile-Pro0.01 ± 0.00.04 ± 0.01*22.7 ± 6.9143.9 ± 4.6**44.7 ± 8.8172.7 ± 36.7**97.0 ± 13.7860.3 ± 306.0**45.3 ± 10.0553.6 ± 115.5**
  Cyclo-Leu-Pro0.01 ± 0.00.03 ± 0.0**74.8 ± 7.7316.9 ± 94.3*101.1 ± 9.6216.5 ± 31.3**74.0 ± 10.6785.3 ± 192.8**46.0 ± 3.0598.0 ± 72.1**
  Cyclo-Val-Pro0.01 ± 0.00.1 ± 0.0**63.3 ± 10.3343.3 ± 6.5**48.7 ± 5.7229.8 ± 18.7**167.3 ± 12.72,366.7 ± 453.1**150.3 ± 45.91,900.0 ± 200.0**
  Cyclo-Phe-Pro0.002 ± 0.00.003 ± 0.033.8 ± 8.6192.9 ± 62.6*21.5 ± 7.667.5 ± 19.4*47.7 ± 5.5141.3 ± 45.2*28.3 ± 11.774.0 ± 24.8*
  Cyclo-Asp-Pro0.4 ± 0.039.3 ± 5.9*160.8 ± 10.7918.7 ± 464.3*205.6 ± 33.53,202.7 ± 1,791.0*224.3 ± 73.4869.3 ± 406.6*214.7 ± 68.4590.3 ± 149.9**
  Cyclo-Ala-Pro0.01 ± 0.00.1 ± 0.0*104.7 ± 8.4286.5 ± 3.2**58.5 ± 13.4215.7 ± 18.5**271.3 ± 10.12,064.0 ± 325.7**222.7 ± 46.11,866.7 ± 208.2**
Pyroglutamyl peptides
  pGlu-Phe-Gln0.0 ± 0.00.02 ± 0.0*0.5 ± 0.31.3 ± 0.32.4 ± 0.43.3 ± 1.80.0 ± 0.00.0 ± 0.00.0 ± 0.00.0 ± 0.0
  pGlu-Gln-Pro0.2 ± 0.20.1 ± 0.340.8 ± 38.127.2 ± 6.7156.5 ± 29.8198.2 ± 63.90.0 ± 0.00.0 ± 0.00.0 ± 0.00.0 ± 0.0
  pGlu-Val-Gln0.0 ± 0.00.0 ± 0.00.9 ± 0.80.8 ± 0.219.3 ± 1.518.4 ± 12.90.0 ± 0.00.0 ± 0.00.0 ± 0.00.0 ± 0.0
  pGlu-Gln-Leu-Leu0.0 ± 0.00.0 ± 0.00.3 ± 0.10.2 ± 0.10.7 ± 0.23.7 ± 5.30.0 ± 0.00.0 ± 0.00.0 ± 0.00.0 ± 0.0
  pGlu-Leu-Ser-Glu0.0 ± 0.00.0 ± 0.000.0 ± 00.000.0 ± 00.000.0 ± 00.000.0 ± 00.00.0 ± 0.00.0 ± 0.00.0 ± 0.00.0 ± 0.0
  pGlu-Leu-Ser0.0 ± 0.00.0 ± 0.01.04 ± 0.60.9 ± 0.321.5 ± 6.226.3 ± 5.30.0 ± 0.00.0 ± 0.00.0 ± 0.00.0 ± 0.0
  pGlu-Leu-Leu0.0 ± 0.00.00 ± 0.000.3 ± 0.20.4 ± 0.18.1 ± 5.412.5 ± 2.90.0 ± 0.00.0 ± 0.00.0 ± 0.00.0 ± 0.0
  pGlu-Pro0.1 ± 0.41.5 ± 1.2†15.8 ± 7.5306.6 ± 237.2†240.0 ± 35.6880.0 ± 66.8*28.6 ± 1.853.6 ± 6.2*22.6 ± 1.651.5 ± 18.2*
  pGlu-Gln0.1 ± 0.20.7 ± 0.2**25.8 ± 3.8130.3 ± 41.4**260.0 ± 50.5540.0 ± 117.4*0.0 ± 0.00.0 ± 0.00.0 ± 0.00.0 ± 0.0
  pGlu-Ala0.0 ± 0.00.9 ± 0.6*0.01 ± 0.0190.6 ± 121.2*280.0 ± 240.0760.0 ± 200.0†0.0 ± 0.00.0 ± 0.00.0 ± 0.00.0 ± 0.0
  pGlu-Leu0.1 ± 0.00.4 ± 0.2*12.4 ± 0.878.7 ± 42.4*344.0 ± 76.9822.0 ± 294.0*18.0 ± 2.317.3 ± 4.116.8 ± 0.717.9 ± 0.1*
  pGlu-Val0.02 ± 0.011.1 ± 0.8†3.8 ± 2.01225.4 ± 161.7†173.6 ± 102.61,448.0 ± 658.0*4.2 ± 7.214.6 ± 24.94.0 ± 6.914.5 ± 8.6
  pGlu-Tyr0.5 ± 0.10.9 ± 0.2†108.9 ± 22.4175.9 ± 43.9†318.0 ± 99.8384.0 ± 98.913.4 ± 7.510.0 ± 6.43.6 ± 0.63.5 ± 2.5
  pGlu-Ile0.03 ± 0.020.4 ± 0.3†4.9 ± 0.476.1 ± 57.8†87.2 ± 29.3358.0 ± 162.2*8.0 ± 1.99.3 ± 6.17.1 ± 2.49.20 ± 1.1
  pGlu-Phe0.02 ± 0.010.2 ± 0.1*4.4 ± 1.938.2 ± 21.8*91.0 ± 27.8310.0 ± 187.8†3.5 ± 0.56.0 ± 7.51.5 ± 0.43.7 ± 4.2