Improved in vivo Tracking of Orally Administered Collagen

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Improved in vivo Tracking of Orally Administered Collagen Hydrolysate Using Stable Isotope Labeling and LC–MS Techniques Yuki Taga, Yu Iwasaki, Yasutaka Shigemura, and Kazunori Mizuno J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.9b00571 • Publication Date (Web): 30 Mar 2019 Downloaded from http://pubs.acs.org on March 31, 2019

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Journal of Agricultural and Food Chemistry

Improved in vivo Tracking of Orally Administered Collagen Hydrolysate Using Stable Isotope Labeling and LC–MS Techniques

Yuki Taga,*,† Yu Iwasaki,‡ Yasutaka Shigemura,‡ and Kazunori Mizuno†

†Nippi

Research Institute of Biomatrix, 520-11 Kuwabara, Toride, Ibaraki 302-0017, Japan

‡Department

of Nutrition, Faculty of Domestic Science, Tokyo Kasei University, 1-18-1 Kaga,

Itabashi-ku, Tokyo 173-8602, Japan

*Corresponding Author Nippi Research Institute of Biomatrix, 520-11 Kuwabara, Toride, Ibaraki 302-0017, Japan Tel: +81-297-71-3046; Fax: +81-297-71-3041 E-mail: [email protected]

1

ACS Paragon Plus Environment


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ABSTRACT

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Collagen-derived hydroxyproline (Hyp)-containing oligopeptides, known to have various

3

physiological functions, are detected in blood at markedly higher concentrations after oral

4

ingestion of collagen hydrolysate. Monitoring the absorption and metabolism of the bioactive

5

peptides is essential to investigate the beneficial effects of collagen hydrolysate. We previously

6

developed an internal standard mixture by sequential protease digestion of stable isotope-labeled

7

collagen, which enabled highly accurate quantitation of collagen-derived oligopeptides by liquid

8

chromatography–mass spectrometry (LC–MS). However, the use of proteases caused a profound

9

imbalance in the generated peptides. Here we employed partial acid hydrolysis to achieve more

10

efficient and balanced peptide generation. Various stable isotope-labeled oligopeptides were

11

detected after 0.5 h acid hydrolysis, and marked enhancement of peptide generation compared

12

with the previous enzymatic method was observed, especially for Hyp-Gly (27.8 ± 0.6 ng/µg vs

13

0.231 ± 0.02 ng/µg). The acid hydrolysate was then heated to generate labeled cyclic dipeptides.

14

Using the novel internal standard mixture in LC–MS, we were able to simultaneously quantitate

15

23 collagen-derived oligopeptides in human plasma and urine after oral administration of

16

collagen hydrolysate.

17 18

Keywords: collagen hydrolysate, peptide, kinetics, stable isotope labeling, LC–MS

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INTRODUCTION

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Oral ingestion of collagen hydrolysate (also referred to as gelatin hydrolysate or collagen

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peptide) has been reported to have beneficial effects on bone,1 joint,2 skin,3 blood sugar,4 blood

22

pressure,5 lipid metabolism,6 immune system,7 and so on. Collagen contains a unique amino acid,

23

4-hydroxyproline (Hyp), which is post-translationally hydroxylated from Pro residues at the Yaa

24

position of collagenous Gly-Xaa-Yaa repeats (~100 residues/1000 amino acid residues). Hyp-

25

containing dipeptides and tripeptides are considered to be the main active ingredients because

26

these collagen-specific peptides appear in blood at markedly higher concentrations (µM level)

27

compared with other food protein-derived peptides (nM level) after the ingestion.8, 9 Pro-Hyp is

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the leading collagen-derived peptide in blood and has various biological activities, including

29

growth stimulation of skin fibroblasts,10,

30

improvement of skin barrier dysfunction,13 and modulation of immune response.7 Hyp-Gly, the

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second most abundant collagen-derived peptide in blood,14 shows similar bioactivities.7,

32

Various X-Hyp-Gly-type tripeptides are also detected in blood at high concentrations.15-17 This

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type of tripeptide promotes osteoblast differentiation, particularly Ala-Hyp-Gly and Leu-Hyp-

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Gly,18 and X-Hyp-Gly where X is branched-chain aliphatic amino acids strongly inhibits

35

angiotensin-converting enzyme.19 We recently reported that Hyp-containing cyclic dipeptides,

36

cyclo(X-Hyp), were efficiently transported into the blood after oral ingestion of collagen

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hydrolysate.20, 21 Cyclo(Pro-Hyp) effectively enhanced the growth rate of skin fibroblasts more

38

than Pro-Hyp,21 and several beneficial effects are reported for cyclo(Ser-Hyp).22-24

11

promotion of osteoblast differentiation,12

13, 14

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The presence of Hyp within the peptide sequence confers high peptidase/protease resistance to

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collagen-derived oligopeptides.19 Orally ingested collagen hydrolysate is sequentially hydrolyzed

41

in the gastrointestinal tract, but large amounts remain in the peptide form due to its high 3



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stability.9 The resulting Hyp-containing dipeptides and tripeptides are absorbed into the blood

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via peptide transporters on enterocytes.25,

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Pro-Hyp and Gly-Pro-Hyp were distributed to various tissues and cells at varying

45

concentrations.27,

46

detected in skin after oral ingestion of collagen hydrolysate, but the concentration did not

47

directly correlate with that in blood.29 The biological activities of collagen-derived oligopeptides

48

are considered to be exerted after being carried by the bloodstream to target tissues, and the

49

remaining unused peptides are excreted into the urine after being gradually degraded. However,

50

the detailed kinetics of collagen-derived peptides in the body after the ingestion of collagen

51

hydrolysate has not been fully elucidated.

28

26

Previous studies reported that orally administered

Yazaki et al. showed that various collagen-derived oligopeptides were

52

Liquid chromatography–mass spectrometry (LC–MS) in multiple reaction monitoring (MRM)

53

mode allows sensitive and selective quantitation by setting channels of precursor and fragment

54

ions specific to target analytes.30, 31 Many studies have used the mass spectrometric approach to

55

analyze collagen-derived peptides in blood and urine.15-17,

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study performed MRM analysis of collagen-derived di- and tripeptides in undiluted plasma and

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urine samples after trichloroacetic acid deproteinization without using internal standards.32

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However, matrix components potentially cause ionization suppression (or enhancement),

59

especially in the complex biological samples.33 The matrix effects critically impair the

60

quantitative accuracy and can vary between individuals and even within individuals at different

61

times. To overcome this problem, we previously developed an internal standard mixture of

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collagen-derived peptides, named SI-digest, prepared from stable isotope-labeled collagen (SI-

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collagen) in which Pro, Lys, Arg, and their post-translationally modified forms are all substituted

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with stable isotopically heavy ones.16, 34 SI-collagen was treated with trypsin/chymotrypsin and

20, 26, 29, 32

For example, a previous

4


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mouse plasma to mimic the protein degradation pathways in the body. Generated peptides having

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stable isotopically heavy Pro and/or Hyp are theoretically identical to the corresponding light

67

peptides in all respects except for the mass, which enables compensation for the matrix effects by

68

using them as internal standards. Using SI-digest, we achieved simultaneous quantitation of 21

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collagen-derived oligopeptides in plasma by LC–MS.17

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Although SI-digest contained numerous types of collagen-derived peptides labeled with stable

71

isotopes, there was an imbalance in the peptide generation.16 For example, the content of Hyp-

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Gly was only 0.04%, which was markedly lower than that of other peptides, such as Pro-Hyp

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(1.15%) and Gly-Pro-Hyp (2.18%). Hyp-Gly is abundantly detected in blood after the ingestion

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of collagen hydrolysate,14 but it was difficult to generate this peptide by the in vitro enzymatic

75

reaction, even with plasma proteases. On the other hand, only small peaks in SI-digest were

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detected for Gly-Pro-Ala and Gly-Ala-Hyp (data not reported), which are susceptible to

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enzymatic degradation.17 If we can produce internal standards of these peptides more efficiently,

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we can reduce the cost and expand the applicability of this method.

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In the present study, we employed partial acid hydrolysis to generate internal standard peptides

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from SI-collagen. The procedure hydrolyzes peptide bonds at random, and thus, an efficient and

81

balanced preparation of various types of oligopeptides was expected. We first estimated peptide

82

generation from SI-collagen by partial acid hydrolysis, and the quantitative performance of LC–

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MS analysis with the prepared internal standard mixture, named SI-oligo, was then evaluated.

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We finally performed comprehensive quantitation of collagen-derived oligopeptides in human

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plasma and urine obtained after oral administration of collagen hydrolysate using heat-treated SI-

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oligo.

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MATERIALS AND METHODS

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Chemicals. Standards of 4-Hyp and hydroxylysine (Hyl) were purchased from Sigma-Aldrich

90

(St. Louis, MO), and standards of Pro and Lys were purchased from Wako Chemicals (Osaka,

91

Japan). PicoTag sample tubes were purchased from Waters (Milford, MA). Gly-Pro, Pro-Hyp,

92

Hyp-Gly, Gly-Ala-Hyp, Gly-Pro-Ala, Gly-Pro-Arg, Gly-Pro-Hyp, and cyclo(Gly-Pro) were

93

purchased from Bachem (Bubendorf, Switzerland). Other peptides were custom synthesized by

94

AnyGen (Gwangju, Korea).

95

Preparation of SI-Oligo and Heat-Treated SI-Oligo. SI-collagen solution, which was 13C

6-Lys,

13C 15N -Arg, 6 4

96

prepared by culturing human embryonic lung fibroblasts with

and

97

13C 15N -Pro 5 1

98

evaporator CVE-3100 (EYELA, Tokyo, Japan) and then subjected to acid hydrolysis (6 N

99

HCl/1% phenol, 110°C in the gas phase under N2) for 0.5, 1, 2, or 4 h. The acid hydrolysate was

100

dissolved in distilled water and stored at −30°C until used for analysis. The hydrolysate prepared

101

by 0.5 h acid hydrolysis (SI-oligo) was further heated in 50 mM sodium acetate buffer (pH 4.8)

102

at 85°C for 1 h and stored at −30°C.

as described previously,34 was dried in PicoTag sample tubes using a centrifugal

103

The SI-oligo, heat-treated SI-oligo, and SI-digest, which was prepared by sequential protease

104

digestion of SI-collagen using trypsin/chymotrypsin and mouse plasma,16 were diluted with 0.1%

105

formic acid for LC–MS analysis in MRM mode. The samples were analyzed by a 3200 QTRAP

106

hybrid triple quadrupole/linear ion trap mass spectrometer (AB Sciex, Foster City, CA) coupled

107

to an Agilent 1200 Series HPLC system (Agilent Technologies, Palo Alto, CA) with or without

108

mixing with amino acid and peptide standards. Chromatographic separation was performed using

109

an Ascentis Express F5 HPLC column (5 µm particle size, L × I.D. 250 mm × 4.6 mm; Supelco,

110

Bellefonte, PA) at a flow rate of 400 µL/min and a column temperature of 30°C with a binary 6


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gradient as follows: 100% solvent A (0.1% formic acid) for 7.5 min, linear gradient of 0–90%

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solvent B (100% acetonitrile) for 12.5 min, 90% solvent B for 5 min, and 100% solvent A for 5

113

min. The MRM transitions of amino acids and oligopeptides are shown in Table S1.

114

Concentration of stable isotopically labeled amino acids and oligopeptides was determined by

115

the peak area ratio of the labeled analytes relative to the corresponding nonlabeled standards.

116

Human Study. The human study was performed according to the Helsinki Declaration under

117

the control of medical doctors. The experimental protocol was approved by the experimental

118

ethical committee of Tokyo Kasei University and Nippi Research Institute of Biomatrix. The

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volunteers were informed of the objectives of the study and the potential risks of ingestion of

120

collagen hydrolysate, such as diarrhea and abdominal pain. Before the experiment, five healthy

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volunteers (one male and four females, average age 25.4 years) fasted for 12 h before ingesting

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10 g of porcine skin collagen hydrolysate (Q'sai, Fukuoka, Japan) dissolved in 200 mL of water.

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Approximately 10 mL of venous blood and 30 mL of urine were collected from each volunteer

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before (0 h) and 1, 2, and 4 h after the ingestion. Plasma prepared from each blood and urine

125

were then deproteinized by adding three volumes of ethanol followed by centrifugation at 3000

126

rpm for 10 min at 10°C. The ethanol-soluble samples were stored at −30°C until analysis.

127

Creatinine concentration was measured according to the method of Zhiri et al. with slight

128

modifications.35 Creatinine in the ethanol-soluble plasma and urine samples were resolved on an

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Inertsil ODS-3 (5 µm particle size, L × I.D. 250 mm × 4.6 mm; GL Sciences, Tokyo, Japan)

130

using an LC-20 series HPLC system (Shimadzu, Kyoto, Japan). Binary gradient elution was

131

performed with 20 mM ammonium acetate and 100% acetonitrile as the mobile phases at a flow

132

rate of 1.0 mL/min. The column was maintained at 40°C, and the absorbance of the eluate was

133

monitored at 234 nm. 7



134

Analysis of Urine Samples with or without SI-Oligo. Two urine samples collected 1 h after

135

the oral administration of collagen hydrolysate were used for this experiment. One was “thick

136

urine” (creatinine concentration = 6.5 µmol/mL), and another was “thin urine” (creatinine

137

concentration = 2.6 µmol/mL). The ethanol-soluble fraction was diluted with 0.1% formic acid

138

5-, 20-, 100-, or 500-fold and mixed with SI-oligo. Hyp-Gly and Gly-Pro-Hyp in the samples

139

were analyzed by LC–MS as described above. Urinary concentration of the peptides was

140

determined based on the peak area using external calibration curves constructed in the range of

141

5–200 pmol/mL of standards with or without correction using the corresponding stable

142

isotopically labeled peptides derived from SI-oligo.

143

Analysis of Time-Course Plasma and Urine Samples. The ethanol-soluble fraction of

144

plasma (60 µL) was mixed with heat-treated SI-oligo and then dried using the centrifugal

145

evaporator. The sample was reconstituted with 60 µL of 0.1% formic acid. The ethanol-soluble

146

fraction of urine (6 µL) was diluted to 60 µL with 0.1% formic acid after adding heat-treated SI-

147

oligo. These samples were analyzed by LC–MS as described above. Plasma and urinary

148

concentration of collagen-derived amino acids and oligopeptides was calculated by the peak area

149

ratio of nonlabeled analytes relative to the corresponding stable isotopically labeled analytes

150

derived from heat-treated SI-oligo. The area under the concentration–time curve (AUC0–4 h) in

151

plasma and urine was calculated using the trapezoidal rule.

152 153

RESULTS AND DISCUSSION

154

Peptide Generation from SI-Collagen by Partial Acid Hydrolysis. Stable isotope-labeled

155

amino acids, dipeptides, and tripeptides generated from SI-collagen by acid hydrolysis (6 N HCl

156

at 110°C for 0.5–4 h) were measured by LC–MS in MRM mode (Figure 1). The reaction time 8


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was insufficient to completely hydrolyze the collagen to amino acids, and thus, a number of

158

dipeptides and tripeptides were detected in the acid hydrolysates. The generation of dipeptides

159

slightly increased from 0.5 to 1 h and then decreased with increasing reaction time (Figure 1B).

160

On the other hand, the amount of generated tripeptides peaked at 0.5 h and decreased after that in

161

a stepwise fashion leading to very low levels at 4 h (Figure 1C and D). In association with the

162

decrease in oligopeptides, the concentrations of amino acids consistently increased with the

163

reaction time (Figure 1A). We confirmed the generation of Hyl, which is another collagen-

164

specific amino acid, in addition to Hyp.

165

Compared with SI-digest prepared by protease digestion, the efficiency of oligopeptide

166

generation was markedly enhanced by the partial acid hydrolysis. The total amount of dipeptides

167

and tripeptides generated by the brief acid hydrolysis (0.5 h) was 2.5- and 5-fold higher,

168

respectively, than that in SI-digest. In particular, a critical effect was observed with respect to

169

Hyp-Gly, which was difficult to generate by the in vitro enzymatic digestion, leading to a 120-

170

fold increase (27.8 ± 0.6 ng/µg vs 0.231 ± 0.02 ng/µg). The enhanced efficiency of peptide

171

generation was also observed for Gly-Pro-Y- and Gly-X-Hyp-type tripeptides prone to

172

enzymatic degradation,17 except for Gly-Pro-Hyp, which was present in substantial amounts in

173

SI-digest probably due to its high resistance to protease digestion. The random peptide bond

174

cleavage by partial acid hydrolysis was shown to efficiently generate various types of

175

oligopeptides labeled with stable isotopes from SI-collagen. In addition, the reproducibility of

176

peptide generation was high as evidenced by the low standard deviation (SD) values (Figure 1).

177

We used the acid hydrolysate of SI-collagen prepared by 0.5 h acid hydrolysis, namely SI-oligo,

178

in the following experiments.

9



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To generate internal standards of cyclic dipeptides, cyclo(X-Hyp), which also appear in the

180

blood after the ingestion of collagen hydrolysate,20, 21 we further treated SI-oligo by heating at

181

85°C for 1 h. We previously reported that this treatment efficiently converts X-Hyp-Gly-type

182

tripeptides to the collagen-specific cyclic dipeptides.20 MRM chromatograms of stable

183

isotopically labeled amino acids, dipeptides, tripeptides, and cyclic dipeptides in SI-oligo and

184

heat-treated SI-oligo are shown in Figure 2. We used a pentafluorophenylpropyl column in the

185

reversed-phase mode for analysis of the diverse compounds, including polar ones, which enabled

186

a simultaneous measurement of the wide range of analytes without ion-pair reagents as reported

187

previously.16,

188

various cyclo(X-Hyp) appeared in heat-treated SI-oligo with decreases in X-Hyp-Gly (Figures 2

189

and S1). Cyclo(Gly-Pro) was also generated probably by thermal conversion of Gly-Pro-Y-type

190

tripeptides.20,

191

labeling index was 95.5%–99.9% (Table S2). The concentration of each labeled peptide in the

192

two types of internal standard mixture was determined by the heavy-to-light peak area ratio after

193

mixing with known concentrations of standards (Table S2). The total content of labeled peptides

194

was 25.2% (w/w, SI-oligo) and 21.3% (heat-treated SI-oligo) of the hydrolyzed SI-collagen,

195

which indicates the high efficiency of this method to prepare internal standards of collagen-

196

derived oligopeptides.

17, 20

36

While cyclo(X-Hyp) was almost undetectable in SI-oligo, intense peaks of

Only slight unlabeled peaks were detected for respective compounds, and the

197

Comparison of Quantitative Value Determined with or without SI-Oligo. To verify the

198

usefulness of SI-oligo for quantitative analysis of collagen-derived peptides using LC–MS, we

199

analyzed urine samples collected 1 h after oral administration of collagen hydrolysate. As shown

200

in Figure 3, without correction using internal standard peptides derived from SI-oligo, the

201

quantitative values of Hyp-Gly and Gly-Pro-Hyp dramatically decreased with lowering the 10


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dilution rate both in thick and thin urine samples. This indicates that ionization suppression

203

induced by matrix components critically impaired the quantitative accuracy in urine. In contrast,

204

the concentrations estimated with correction using SI-oligo were constant among all dilution

205

rates in the urine samples, demonstrating that those values were much more reliable. The urinary

206

concentrations estimated without SI-oligo matched with those of with SI-oligo when the samples

207

were diluted 500 times. However, Gly-Pro-Hyp in the thin urine was not able to be detected at

208

the high dilution. Since the density of urine widely varies from time to time, it is difficult to

209

ensure the quantitative accuracy without using internal standards. In addition, we previously

210

showed that the concentration of Hyp-containing peptides was markedly underestimated as a

211

result of ionization suppression in plasma.16 Our results indicate that we can perform highly

212

accurate quantitation of collagen-derived oligopeptides in these complex biological samples only

213

by adding SI-oligo before analysis.

214

Comprehensive Quantitation of Collagen-Derived Oligopeptides in Plasma and Urine

215

using Heat-Treated SI-Oligo. We used heat-treated SI-oligo as the internal standard mixture to

216

analyze collagen-derived oligopeptides, including cyclic dipeptides, in human plasma and urine

217

obtained up to 4 h after the administration of collagen hydrolysate. Since the concentration of

218

stable isotopically heavy peptides in heat-treated SI-oligo was predetermined (Table S2), we

219

were able to simply calculate the concentration of respective analytes in the samples based on the

220

light-to-heavy peak area ratio. We successfully detected two amino acids, nine dipeptides, seven

221

tripeptides, and seven cyclic dipeptides both in plasma and urine. The concentration of detected

222

oligopeptides in the time-course samples ranged from 0.610 pmol/mL (Phe-Hyp-Gly) to 17.2

223

nmol/mL (Pro-Hyp) in plasma and 9.32 pmol/mL (Phe-Hyp-Gly) to 760 nmol/mL (Pro-Hyp) in

224

urine. Figure S2 shows the plasma kinetic data. To compare the concentration–time curve 11



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between the two biological fluids, the results were normalized to creatinine concentration (Figure

226

4). We did not detect Gly-Pro-Y- and Gly-X-Hyp-type tripeptides listed in Figure 1D, except for

227

Gly-Pro-Hyp, probably due to their low resistance to peptidase/protease degradation.17 No peaks

228

of Lys, Hyl, and their respective labeled forms were detected here due to ionization suppression.

229

An adequate chromatographic separation is needed for analysis of these amino acids.

230

The AUC0–4 h, Cmax, and Tmax values are summarized in Table 1. Although the concentration of

231

free Hyp was significantly higher than that of oligopeptides in plasma, a large proportion of Hyp

232

excreted into the urine was in the peptide form, consistent with early observations.37, 38 Pro-Hyp

233

showed the highest concentration both in plasma and urine followed by Hyp-Gly in plasma and

234

Glu-Hyp in urine. Dipeptides and tripeptides in plasma reached maximum levels at

235

approximately 1 h after the administration of collagen hydrolysate (Tmax = 1.0–1.4 h). A delay in

236

appearance of the peak in urine, compared with that in plasma, was observed for almost all of the

237

dipeptides and tripeptides (Tmax = 1.4–2.0 h), except for Leu-Hyp-Gly and Phe-Hyp-Gly (Tmax =

238

1.0 h). The presence of hydrophobic amino acids within the peptide sequence seemed to enhance

239

the speed of peptide absorption and excretion. Comparing the creatinine-normalized peptide

240

concentration, the values of most peptides were significantly higher in plasma than in urine. In

241

contrast, the peptide concentration in urine was higher or comparable with that in plasma for

242

some peptides, such as Gly-Pro, Glu-Hyp, and Phe-Hyp-Gly. This difference suggests that the

243

efficiency of delivery to and consumption in tissues is different among respective peptides,

244

although we need to take account of differences in peptide degradation, which complicates our

245

understanding of peptide kinetics.

246

The kinetic profile of cyclic dipeptides was obviously different from that of other peptides. In

247

plasma, the time to the maximum concentration of cyclic dipeptides was slightly shifted 12


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backwards compared with that of linear dipeptides and tripeptides (Tmax = 1.0–2.0 h), and the

249

peptide concentration was maintained for a relatively long time, as we reported previously.20

250

Except for cyclo(Leu-Hyp) and cyclo(Phe-Hyp), this tendency was more marked in urine (Tmax =

251

2.0–4.0 h). Cyclic peptides are supposed to be directly absorbed and excreted into the urine

252

without undergoing enzymatic degradation due to their characteristic structures.39 Unlike linear

253

oligopeptides, cyclic peptides cannot be generated from other peptides by partial hydrolysis in

254

the gastrointestinal tract and blood. However, although the content of cyclic dipeptides in the

255

collagen hydrolysate used in this study was less than 0.1% (data not shown), substantial amounts

256

of collagen-derived cyclic dipeptides were detected in plasma and urine. This suggests that

257

peptide cyclization occurred in the body. Previous works reported that, although cyclo(Pro-Hyp)

258

was detected in urine, this cyclic dipeptide was regarded to be artificially converted from Pro-

259

Hyp during experimental procedures.38, 40 However, we recently demonstrated that Pro-Hyp was

260

not converted into cyclo(Pro-Hyp) during ethanol deproteinization and drying of the ethanol-

261

soluble fraction,21 which were the only procedures used here. Furthermore, the apparent

262

differences in the kinetic profile between cyclic dipeptides and their potential precursors,

263

including dipeptides and tripeptides, indicate that the cyclic dipeptides detected in plasma and

264

urine were not experimental by-products. Further work is needed to elucidate whether cyclic

265

peptides are possibly formed in the body.

266

In the present study, we developed an improved internal standard mixture, SI-oligo, for highly

267

accurate quantitation of collagen-derived oligopeptides by LC–MS. A total of 25 dipeptides and

268

tripeptides labeled with stable isotopes were generated by partial acid hydrolysis of SI-collagen,

269

and seven labeled cyclic dipeptides were prepared by a subsequent heat treatment. When

270

compared with the previous method using sequential protease digestion, there are many 13



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advantages, including in the efficiency of peptide generation (25.2% vs 8.0%, w/w), preparation

272

time (1 h vs 3 days), and ethical considerations (the sacrifice of mice in the protease digestion

273

method). The only disadvantage is that internal standards of Asn- or Gln-containing peptides

274

cannot be prepared due to deamidation of the side chain during acid hydrolysis. Using the novel

275

method, we can comprehensively track the absorption and excretion of collagen-derived peptides

276

after oral administration of collagen hydrolysate by analyzing blood and urine samples. We

277

demonstrated that collagen-derived oligopeptides were accurately quantitated, even in thick

278

urine. Although the kinetic profile in urine was somewhat different from that in plasma, analysis

279

of urine samples collected in a time course can be an alternative approach when a noninvasive

280

evaluation is preferred. Furthermore, in addition to the kinetic analysis of plasma and urine

281

samples, SI-oligo can be used as the internal standard for various analyses, such as Caco-2

282

permeability assay of collagen-derived peptides.

283

Lys residues lying at the Yaa position of the collagenous Gly-Xaa-Yaa sequence are

284

hydroxylated to Hyl. The hydroxylation occurs in 15%–90% of total Lys with varying with the

285

tissue, age, and collagen type,41 and the presence of the hydroxyl group hampers enzymatic

286

peptide bond hydrolysis.42 Therefore, similar to the Hyp-containing peptides, there may be Hyl-

287

containing oligopeptides in the blood, such as Hyl-Gly and X-Hyl-Gly. Although Hyp has been

288

used as an indicator of absorption and metabolism of orally ingested collagen hydrolysate,

289

evaluation using another marker of collagen would be helpful for the kinetic study. SI-oligo can

290

be used for quantitative analysis of Hyl and Hyl-containing peptides as the internal standard.

291 292

ABBREVIATIONS USED

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Hyp, hydroxyproline; LC–MS, liquid chromatography–mass spectrometry; MRM, multiple

294

reaction monitoring; SI-collagen, stable isotope-labeled collagen; Hyl, hydroxylysine; AUC, area

295

under the concentration–time curve; SD, standard deviation

296 297

Supporting Information. Figure S1: MRM chromatograms of stable isotope-labeled cyclic

298

dipeptides in SI-oligo and heat-treated SI-oligo. Figure S2: Plasma concentrations of collagen-

299

derived amino acids and oligopeptides after oral administration of collagen hydrolysate. Table

300

S1: MRM transitions of amino acids and oligopeptides. Table S2: Content and labeling index of

301

stable isotopically labeled amino acids and oligopeptides in SI-oligo and heat-treated SI-oligo.

302

This material is available free of charge via the Internet at http://pubs.acs.org.

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REFERENCES

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428

Figure captions

429

Figure 1. Generation of labeled amino acids and oligopeptides by partial acid hydrolysis of SI-

430

collagen. Contents of (A) amino acids, (B) dipeptides, (C) X-Hyp-Gly-type tripeptides, and (D)

431

other types of tripeptide in acid hydrolysates of SI-collagen hydrolyzed with 6 N HCl at 110°C

432

for 0.5, 1, 2, or 4 h and in SI-digest. Stable isotopically labeled amino acids are indicated by

433

underlining. The data represent the mean ± SD of three separate experiments.

434 435

Figure 2. MRM chromatograms of stable isotope-labeled amino acids and oligopeptides in (A)

436

SI-oligo and (B) heat-treated SI-oligo. Stable isotopically labeled amino acids are indicated by

437

underlining.

438 439

Figure 3. Quantitation of Hyp-Gly and Gly-Pro-Hyp in urine with or without SI-oligo. (A and B)

440

Thick urine and (C and D) thin urine samples collected 1 h after oral administration of collagen

441

hydrolysate were deproteinized with ethanol, diluted 5, 20, 100, or 500 times, and mixed with SI-

442

oligo. The concentration of Hyp-Gly and Gly-Pro-Hyp in the samples was estimated by LC–MS

443

with or without correction using SI-oligo. The data represent the mean ± SD of three separate

444

independent measurements.

445 446

Figure 4. Plasma and urinary levels of collagen-derived amino acids and oligopeptides after oral

447

administration of collagen hydrolysate. Plasma and urine samples collected before (0 h) and 1, 2,

448

and 4 h after the administration of collagen hydrolysate were analyzed by LC–MS with heat-

449

treated SI-oligo used as an internal standard. The data represent the mean ± SD (n = 5). Cr,

450

creatinine. 22


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Tables Table 1. AUC0-4 h, Cmax, and Tmax of amino acids and oligopeptides in urine and plasma after oral administration of collagen hydrolysate AUC0-4 h (mmol/mol Cr•h) Plasma Urine 8130 ± 1980 3.73 ± 2.47 2580 ± 880 12.5 ± 14.4

Cmax (mmol/mol Cr) Plasma Urine 2720 ± 850 1.50 ± 1.34 1000 ± 460 6.33 ± 8.56

Tmax (h) Plasma Urine 1.00 ± 0.00 1.80 ± 0.45 1.20 ± 0.45 2.20 ± 1.10

Gly-Pro Ala-Hyp Glu-Hyp Ile-Hyp Leu-Hyp Phe-Hyp Pro-Hyp Ser-Hyp Hyp-Gly

3.11 ± 0.53 26.1 ± 6.3 31.0 ± 9.0 12.4 ± 1.5 26.4 ± 7.0 10.5 ± 1.5 441 ± 173 19.9 ± 4.9 50.2 ± 33.2

5.48 ± 1.25 10.6 ± 2.9 31.6 ± 5.2 4.87 ± 0.44 9.35 ± 0.86 5.18 ± 0.80 293 ± 100 6.87 ± 2.40 13.6 ± 6.1

1.49 ± 0.17 15.0 ± 4.1 13.1 ± 5.4 7.89 ± 0.75 18.5 ± 4.3 6.19 ± 0.56 197 ± 107 10.1 ± 3.5 25.6 ± 14.7

1.99 ± 0.47 4.55 ± 1.36 12.6 ± 3.2 2.12 ± 0.34 4.36 ± 0.72 2.22 ± 0.55 116 ± 45 2.98 ± 1.46 5.21 ± 2.25

1.00 ± 0.00 1.00 ± 0.00 1.40 ± 0.55 1.00 ± 0.00 1.00 ± 0.00 1.00 ± 0.00 1.20 ± 0.45 1.20 ± 0.45 1.00 ± 0.00

1.60 ± 0.55 1.60 ± 0.55 2.00 ± 0.00 1.40 ± 0.55 1.40 ± 0.55 1.60 ± 0.55 2.00 ± 0.00 2.00 ± 0.00 1.80 ± 0.45

Gly-Pro-Hyp Ala-Hyp-Gly Glu-Hyp-Gly

6.09 ± 1.65 14.1 ± 4.9 14.4 ± 5.1

2.01 ± 0.49 1.40 ± 0.50 5.47 ± 1.81

2.97 ± 0.89 10.9 ± 4.2 8.79 ± 4.01

1.00 ± 0.00 1.00 ± 0.00 1.20 ± 0.45

2.00 ± 0.00 1.60 ± 0.55 2.00 ± 0.00

0.449 ± 0.224

0.108 ± 0.046

0.267 ± 0.168

1.00 ± 0.00

1.00 ± 0.00

0.115 ± 0.032

0.0847 ± 0.0371

1.00 ± 0.00

1.00 ± 0.00

Pro-Hyp-Gly Ser-Hyp-Gly

23.2 ± 11.5 19.3 ± 9.5

1.97 ± 1.00 1.62 ± 0.64

0.0644 ± 0.0366 15.6 ± 10.2 15.0 ± 9.6

0.804 ± 0.232 0.637 ± 0.254 2.73 ± 1.32 0.0621 ± 0.0331 0.0586 ± 0.0401 0.833 ± 0.403 0.782 ± 0.488

1.00 ± 0.00 1.20 ± 0.45

2.00 ± 0.00 1.80 ± 0.45

Cyclo(Gly-Pro) Cyclo(Ala-Hyp) Cyclo(Glu-Hyp) Cyclo(Leu-Hyp) Cyclo(Phe-Hyp) Cyclo(Pro-Hyp) Cyclo(Ser-Hyp)

33.9 ± 10.8 5.69 ± 2.17 1.03 ± 0.23 2.69 ± 0.66 0.899 ± 0.284 22.8 ± 8.2 5.10 ± 1.68

14.6 ± 3.3 2.57 ± 0.80 0.852 ± 0.153 2.14 ± 0.46 0.546 ± 0.147 5.82 ± 1.39 2.30 ± 0.77

12.0 ± 4.3 1.90 ± 0.79 0.374 ± 0.103 1.01 ± 0.28 0.414 ± 0.153 10.1 ± 4.7 1.73 ± 0.62

4.75 ± 0.99 0.992 ± 0.311 0.357 ± 0.071 0.714 ± 0.166 0.228 ± 0.079 2.32 ± 0.58 1.00 ± 0.42

1.20 ± 0.45 1.60 ± 0.55 1.20 ± 0.45 1.00 ± 0.00 1.00 ± 0.00 1.20 ± 0.45 2.00 ± 0.00

2.20 ± 1.10 2.80 ± 1.10 2.00 ± 0.00 1.60 ± 0.55 1.20 ± 0.45 2.00 ± 0.00 4.00 ± 0.00

　　 Pro Hyp

Leu-Hyp-Gly Phe-Hyp-Gly

The data represent the mean ± SD (n = 5).

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Figure Graphics Figure 1

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Figure 2

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Figure 3

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Figure 4

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GRAPHICS FOR TABLE OF CONTENTS

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Improved in vivo Tracking of Orally Administered Collagen

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