Hydroxylysyl-pyridinoline occurrence and chemical characteristics of collagen present in jumbo squid (Dosidicus gigas) tissues

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YJFCA 2599 1–8 Journal of Food Composition and Analysis xxx (2015) xxx–xxx

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Original Research Article

Hydroxylysyl-pyridinoline occurrence and chemical characteristics of collagen present in jumbo squid (Dosidicus gigas) tissues E. Ramı´rez-Guerra a, Miguel A. Mazorra-Manzano a, Josafat M. Ezquerra-Brauer b, Elizabeth Carvajal-Milla´n a, Ramo´n Pacheco-Aguilar a, Marı´a E. Lugo-Sa´nchez a, Juan C. Ramı´rez-Sua´rez a,*

Q1 Hugo

Q2 a Centro de Investigacio´n en Alimentacio´n y Desarrollo, A.C. Carretera a la Victoria Km 0.6, Apdo. Postal 1735, C.P. 83000 Hermosillo, Sonora, Mexico b

Departamento de Investigacio´n y Posgrado en Alimentos, Universidad de Sonora, Blvd. Luis Encinas y Rosales s/n, Apdo. Postal 1658, C.P. 83000 Col. Centro, Hermosillo, Sonora, Mexico

A R T I C L E I N F O

A B S T R A C T

Article history: Received 27 October 2014 Received in revised form 3 June 2015 Accepted 8 June 2015 Available online xxx

Hydroxylysyl-pyridinoline (HP) occurrence and chemical characteristics of collagen from jumbo squid tissues were investigated. Muscle collagen (MC) was higher in Glu, Arg and Gly, but lower in hydrophobic amino acids compared with skin collagen (SC). Lys hydroxylation (%) was higher (P < 0.05) in MC (46.9  4.01) than SC (23.4  1.70). Carbohydrate content (% dry wt.) was similar (P  0.05) among MC (16.6  0.53) and SC (15.2  1.12), showing arabinose (MC, 11.1  0.21 vs. SC, 11.7  0.91), glucose (MC, 3.3  0.10 vs. SC, 2.8  0.10, P < 0.05) and xylose (MC, 0.9  0.10 vs. SC, 0.7  0.11, P < 0.05); mannose (1.1  0.11) and galactose (0.2  0.01) were found only in MC. FT-IR analysis suggests major supraorganizational rearrangement in MC than SC, through presence of more stable triple-helix structures associated to Gly, Hyl, polar amino acids and carbohydrate contents. HP chemical nature and its tissuespecific distribution (MC, 4.6 mmol/mol collagen) indicate that specific Lys hydroxylation can be a critical regulatory step on cross-link formation. Chemical composition variations and HP distribution suggest squid collagens have quite different biomechanical requirements, i.e., muscle or skin collagen rigidity or elasticity. ß 2015 Published by Elsevier Inc.

Keywords: Hydroxylysyl-pyridinoline Collagen Jumbo squid FT-IR Carbohydrate content Physicochemical properties Food analysis Food composition

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1. Introduction

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Jumbo squid (Dosidicus gigas) supports a large local fishery in the central Gulf of California, Mexico. Compared with fish muscle, squid muscle preferences do not depend on taste as from texture; therefore, understanding of its main components physicochemical properties becomes crucial. Structurally, squid muscle shows a unique bundle of muscle fibers made of circumferential bands sandwiched between thinner radial bands that are covered from each side by two connective tissue sheets (Krieg and Mohseni, 2012). This connective tissue, mainly represented by collagen and elastin, constitutes a scaffold for myofibrillar proteins, and has a strong effect in textural properties of raw and/or cooked muscle (Ando et al., 1999). Differential scanning calorimetric studies of squid connective tissue have revealed transition temperatures over 100 8C,

* Corresponding author. Tel.: +52 662 289 2400x368; fax: +52 662 280 04 21 11. E-mail address: [email protected] (J.C. Ramı´rez-Sua´rez).

suggesting a thermo-stable protein system (Torres-Arreola et al., 2008; Valencia-Pe´rez et al., 2008). It has been suggested that the highly ordered fibrous structure of squid mantle collagen imparts this high thermal resistance and poor solubility properties (Ando et al., 2001; Torres-Arreola et al., 2008). This uncommon psychochemical behavior is consistent for collagen extracted from squid by-products, such as skin used for gelatin production (Go´mez-Guille´n et al., 2002). Physicochemical properties and stability of the triple-helix structure of collagen are mediated mainly by intramolecular forces such as hydrogen bonds and Van der Waals interactions (Shoulders and Raines, 2009). Besides, post-translational modifications such as hydroxylation or glycosylation, mainly of lysine and proline, increase dramatically the thermal stability of this triple helix (Shoulders and Raines, 2009). Lysine modification of collagen is a highly complicated sequential processes catalyzed by several groups of enzymes leading to the final step of this biosynthesis, the covalent intermolecular cross-linking (Yamauchi and Sricholpech, 2012). In the cell, specific lysine residues are hydroxylated to form hydroxylysine. Then, specific hydroxylysine residues located in the

http://dx.doi.org/10.1016/j.jfca.2015.06.003 0889-1575/ß 2015 Published by Elsevier Inc.

Please cite this article in press as: Ramı´rez-Guerra, H.E., et al., Hydroxylysyl-pyridinoline occurrence and chemical characteristics of collagen present in jumbo squid (Dosidicus gigas) tissues. J. Food Compos. Anal. (2015), http://dx.doi.org/10.1016/j.jfca.2015.06.003

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helical domain of the molecule are glycosylated by the addition of galactose or glucose-galactose to form galactosyl-hydroxylysine and/or glucosyl-galactosyl-hydroxylysine, respectively (Yamauchi and Sricholpech, 2012). Outside the cell, an enzymatic oxidative deamination occurs to the telopeptidyl lysine and hydroxylysine residues producing reactive aldehydic residues. These aldehydes initiate a series of nonenzymatic condensation reactions to form covalent intra- and intermolecular cross-links, which are critical for the biomechanical functions of the collagen fibrils. These covalent intermolecular cross-linkings result in the formation of pyridinolines, compounds related with the structural stability and psychochemical properties of collagen (Eyre, 1987). Two of the main representative pyridinium cross-links in collagen, lysylpyridinoline (LP) and hydroxylysyl-pyridinoline (HP) have origin during collagen biosynthesis and molecule self-assembly, and both are products of e-amino oxidation of lysine and hydroxylysine, respectively, by the lysyl oxidase (LOX) enzyme (Palamakumbura and Trackman, 2002). Pyridinium cross-links incidence in marine species has been scarcely studied; however, its presence in salmon (Salmo salar L.), red sea bream (Pagrus major), yellowtail (Seriola quinqueradiata) and tiger puffer (Fugu rubripes) muscular collagens has been detected (Ando et al., 2006; Li et al., 2005). While a considerable amount of HP predominates in heat-insoluble collagens from Asiatic squid species like long-finned squid, Japanese common squid, flying squid, cuttlefish and arrow squid (Ando et al., 2001), it seems that their presence and distribution in marine species and land mammal species could be influenced by environment factors and physiological status. Probably, the fast growing and short-life cycle of jumbo squid, combined with the capacity to migrate vertically and horizontally, and its rapid response to environmental conditions changes could be factors associated with a possible high amount of cross-linked collagen. Technological approach for squid collagen is limited due to insufficient knowledge about the presence of pyridinium cross-links in its structures and their psychochemical implications. Thus, the aim of the present study was to evaluate the hydroxylysyl-pyridinoline (HP) occurrence and chemical characteristics of collagen present in jumbo squid tissues.

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2. Material and methods

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2.1. Reagents

4-Chloro-7-nitrobenzofurazan (NBD-Cl) (99%), L-a-amino-n87 88 butyric acid (98%), trifluoroacetic acid (TFA) (99%), tetrahydro89 furan (THF) (99%), acetic acid (99%), L-amino acid standards 90 (98%), heptafluorobutyric acid (HBFA) (99%) and type I collagen 91 from bovine achilles tendon were purchased from Sigma Aldrich 92 (St. Louis, MO, USA). Potassium bromide (98%), methanol (99%), 93 Q3 hydrochloric acid (98%) and acetonitrile (99%) were purchased 94 from J.T. Baker (Center Valley, PA, USA). O-phthalaldehyde (OPA) 95 (99%) was purchased from Pierce (Dallas, TX, USA). Standard 96 hydroxylysyl-pyridinoline (HP) (99%) was purchased from Wako 97 Pure Chemicals Co. (Richmond, VA, USA) and CHROMABOND1 98 Crosslinks polypropylene columns were purchased from 99 Macherey-Nagel (Bethlehem, PA, USA). 100

2.2. Raw material

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Thirty fresh (less than 24 h postcapture) jumbo squid mantles (40–60 cm length) were obtained from a local fish retailer and stored at 80 8C before use. Partially thawed mantles were manually skinned to get both tissues for analysis. All recovered skin was separated in three batches of 200 g each. On the other hand, mantles were chopped-mixed to obtain three, 200 g batches.

All batches (skin and muscle) were used for collagen extraction as later described. This procedure was repeated three times (n = 3).

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2.3. Isolation of crude collagens

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Collagens were prepared from skin and mantle muscle according to the methodology of Sato et al. (1988). All operations were performed at 4 8C. Minced skin or muscle was stirred in 0.1 M NaOH overnight. The pellet was collected by centrifugation at 10,000  g for 30 min. This procedure was repeated three times to remove noncollagenous protein. A final wash was performed with distilled water in order to remove residual NaOH. The collagen obtained was freeze-dried and stored for further analysis.

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2.4. Amino acid analysis

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The amino acid content was determined following the procedure described by Va´zquez-Ortı´z et al. (1995). Briefly, two milligrams of lyophilized collagen was hydrolyzed in 6 M HCl at 150 8C for 6 h. Hydrolyzed sample was dried in an evaporator, and then re-dissolved in citrate buffer pH 2.2. Finally, samples were mixed with L-a-amino-N-butyric acid as internal standard (2 mg/ mL) and derived with an O-phthalaldehyde (OPA) solution before HPLC analysis. The chromatographic separation was carried out on a Series 1100 HPLC system (Hewlett Packard Co., Waldbrom, Germany) using a octadecylsilane (ODS) C18 column (4.6 mm ID  150 mm, Agilent Inc., Palo Alto, CA, USA). A gradient run was developed at a flow rate of 1.2 mL/min using Methanol 100% (solution A) and 0.1 M acetate buffer, pH 7.2 with tetrahydrofuran (990:10, v/v) (solution B) as the mobile phases. The gradient (v/v) program used was as follows: column equilibration for 15 min using 95% of solution A and 5% of solution B; 0–4 min, 70% A, 30% B; 4–6 min, 70% A, 30% B; 6–10 min 50% A, 50% B; 10–15 min, 50% A, 50% B; 15–18 min, 20% A, 80% B and 18–22 min 20% A, 80% B. Fluorescence was continuously monitored at lex = 350 nm and lem = 450 nm using a FLD Cell detector (Agilent Inc., Waldbrom, Germany). Proline and hydroxyproline were determined by the 4-chloro7-nitrobenzofurazan (NBD-Cl) derivative method described by Va´zquez-Ortı´z et al. (2005) with slight modifications. Briefly, 125 mL of hydrolyzed sample in citrate buffer (pH 2.2) was mixed with 500 mL borate buffer (pH 10.4). Then, 250 mL of this solution was mixed with NBD-Cl/methanol solution (1:1, v/v) and heated at 60 8C for 5 min. The derivative reaction was stopped by adding 50 mL of 1 M HCl. Samples filtered were analyzed using the same ODS C18 column described above. The Mobile phase was methanol 100% (solution A) and 0.1 M sodium acetate buffer (pH 7.2) with methanol and tetrahydrofuran (900:95:5, v/v/v) (solution C). Separation was performed as follows: column equilibration for 15 min using 20% of solution A and 80% of solution C. The elution was performed by using the following gradient (v/v) program: 0– 5 min, 20% A and 80% C; 5–6 min, 100% A; 6–8 min, 100% A. The eluate was monitored by fluorescence at lex = 460 nm and lem = 590 nm. For quantification, calibration curves were prepared using proline and hydroxyproline standards solutions in the range of concentrations from 40 to 800 mg per mL. The amino acid contents of dry collagens were reported as mass percentage per gram protein. Total protein of dry crude collagens was determined according to Woyewoda et al. (1986). Besides, in order to obtain the collagen content of crude collagen preparations, hydroxyproline content was multiplied by 7.14 protein conversion factor (Ando et al., 2006).

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2.4.1. Hydroxylation of proline and lysine Hydroxylation of proline (Pro) and lysine (Lys) was calculated from the amino acid composition according to the following

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Please cite this article in press as: Ramı´rez-Guerra, H.E., et al., Hydroxylysyl-pyridinoline occurrence and chemical characteristics of collagen present in jumbo squid (Dosidicus gigas) tissues. J. Food Compos. Anal. (2015), http://dx.doi.org/10.1016/j.jfca.2015.06.003

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formulas: Degree of Pro hydroxylation ð%Þ ¼

Hydroxyproline content  100% Hydroxyproline content þ Proline content

(1)

170 169 Degree of Lys hydroxylation ð%Þ ¼

Hydroxylysine content  100% Hydroxylysine content þ Lysine content

(2)

3

lex = 297 nm and lem = 395 nm. HP content was estimated as moles per mole of collagen. A factor of 7.14 was used to convert mass of hydroxyproline to mass of collagen, and HP concentration was calculated assuming that collagen had a molecular mass of 3.38  105 g/mol (Ando et al., 2006).

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2.7.1. Pyridinoline identification by HPLC-LC/MSD analysis In order to confirm the identity of pyridinoline, digested lyophilized collagen samples were analyzed by using a HPLC-LC/ MSD trap system model G2445D (Agilent, Palo Alto, CA, USA). Spectra were compared with the one obtained for HP standard. A C18-SORBAX 300SB nano-column was used (50 mm  75 mm, 3.5 mm particle size, 300 A˚ pore size, Agilent, Germany). The mobile phase was a mixture of deionized water/acetonitrile/formic acid (97:3:0.1, v/v) (solution A) and acetonitrile/deionized water/ formic acid (90:10:0.1, v/v) (solution B). The chromatographic separation was achieved at a flow rate of 0.3 mL/min using a gradient elution (v/v) program as follow: 0–10 min, 0% A, 3% B; 10–20 min, 65% A, 45% B; 20–33 min, 35% A, 65% B; 33–40 min, 97% A, 3% B,. Mass spectrometric detection was carried out in the positive ion mode with a scan range of 50–2200 m/z, 5 L/min dry gas, 235 8C dry temperature and capillary 1500 V.

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2.8. Statistical analysis

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2.5. Carbohydrate analysis

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Carbohydrate content in collagen samples was determined according to Carvajal-Millan et al. (2004). Sample was hydrolyzed with 2 N trifluoroacetic acid at 120 8C for 2 h. After hydrolysis, the reaction was stopped by cooling the tubes on ice and the solution was evaporated under airflow at 40 8C, rinsed twice with 200 mL of deionized (DI) water and re-dissolved with a final volume of 500 mL of DI water. Inositol was used as internal standard. Samples were filtered through a 0.2 mm Whatman filter paper before HPLC analysis. Chromatographic analysis was performed using a Supelcogel Pb column (300  7.8 mm; Supelco, Inc., Bellefont, PA, USA) with 5 mM H2SO4 solution as mobile phase at a flow rate of 0.6 mL/min and 50 8C. A Varian 9012 HPLC system with Varian 9040 refractive index detector (Varian, St. Helens, Australia) controlled via the Star Chromatography Workstation (version 5.50) was used.

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2.6. Fourier transform infrared spectroscopy (FT-IR)

3. Results and discussion

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Fourier transform infrared (FT-IR) spectra of lyophilized collagens were recorded on a Nicolet FT-IR spectrophotometer (Nicolet Instrument Corp., Madison, WI, USA) using KBr pellets (2 mg sample/200 mg KBr). Sample signals were obtained in transmission mode from 400 to 4000 cm1 at 4 cm1 resolution.

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2.7. Pyridinium cross-links determination

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One hundred milligrams of lyophilized collagen was hydrolyzed with 3 mL 6 M HCl at 150 8C for 6 h. Then, the hydrolyzed sample was pre-fractionated by solid phase extraction (SPE) using a Chromabond1 Cross-links column to remove interfering molecules. Briefly, 1.6 mL of hydrolyzed sample was diluted with an equal volume of 90% acetic acid and mixed with 2.5 mL of acetonitrile in a 5 mL glass tube. Then, the solution was transferred to a SPE column previously equilibrated with 2.5 mL of washing buffer prepared with acetonitrile, acetic acid and DI water (8:1:1, v/v/v). The column was extensively washed 4 times with 2.5 mL of washing buffer and a final wash with 400 mL of DI water. The column was drained and pyridinium cross-links were eluted with 200 mL of 1% Heptafluorobutyric acid (HBFA) directly on a HPLC micro-insert vial. Pyridinolines were separated using a Series 1100 HPLC system (Hewlett Packard Co., Waldbrom, Germany) coupled to a fluorescence detector. An automatic injection-system equipped with a 100 mL syringe was used to load 20 mL of each sample to an ODS C18 Microsorb-MV column (100 C18, 4.6 mm ID  250 mm, Microsorb, Rainin, CA, USA) and separation was performed at a flow rate of 1 mL/min at 40 8C. Mobile phase consisted of 0.12% HBFA in DI water (solution A) and 50% acetonitrile (solution B). The column was equilibrated with 20% of solvent B prior to use and chromatographic separation was achieved with a gradient elution from 20 to 30% of solvent B in 20 min. The pyridinolines were monitored for fluorescence at

The amino acid composition of skin and muscle collagens extracted from jumbo squid mantle is shown in Table 1. Glycine (Gly), aspartic acid (Asp), glutamic acid (Glu) and arginine (Arg) were the most abundant amino acids in both analyzed collagens. However, higher concentration (P < 0.05) of Glu, Arg and Gly were registered for muscle collagen. The main difference was noted in Gly content which was 1.5 times higher in muscle collagen than in skin collagen. On the other hand, hydrophobic amino acids content such as leucine (Leu), isoleucine (Ile), methionine (Met), phenylalanine (Phe) and valine (Val) was more pronounced (P < 0.05) in skin collagen than in muscle collagen. Skin collagen showed a slightly higher (P  0.05) content of imino acids (proline (Pro) + hydroxyproline (Hyp)), although the ratio between Pro and Hyp remained similar (0.5) for both collagen sources. Polar amino acids (Asp, Glu and Arg) content in collagen extracted from jumbo squid tissues was higher than those reported for other squid species (Ando et al., 2001). The skin collagen of marine species tends to contain lower amounts of polar amino acids than their terrestrial counterpart species; for example, scalloped hammerhead shark (SS, Sphyrna lewini), bigeye snapper (Priacanthus macracanthus), common horse mackerel (Trachurus japonicas), yellow sea bream (Dentex tumifroms) and tiger puffer (Takifugu rubripes) show minor polar amino acid contents than pig or bovine skin collagens (Jongjareonrak et al., 2005; Lin and Liu, 2006; Yata et al., 2001). However, jumbo squid skin collagen showed higher amounts of polar amino acids than bovine type I collagen (Table 1). At physiological conditions, the side chains of Asp and Glu acids contain a carboxyl group that gives a negative charge, while the guanidine group of Arg side chain is positively charged (basic). All these polar residues are able to interact with water or neighboring opposite charged residues from adjacent a-chains in squid collagen, thus, improving stability to triple helix structure. On the other hand, hydrophobic amino acid content

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Data analysis was performed using ANOVA one-way by 243 employing the statistical software package NCSS 2007. A Tukey Q4244 test was used when significant differences were detected 245 (P < 0.05) between the means. At least three replicates were 246 carried out for each analysis. 247

Please cite this article in press as: Ramı´rez-Guerra, H.E., et al., Hydroxylysyl-pyridinoline occurrence and chemical characteristics of collagen present in jumbo squid (Dosidicus gigas) tissues. J. Food Compos. Anal. (2015), http://dx.doi.org/10.1016/j.jfca.2015.06.003

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Table 1 Amino acid composition (% per gram protein) of collagens extracted from jumbo squid tissues. Amino acid

Muscle collagen Mean  S.D.

Aspartic acid Arginine Alanine Glutamic acid Glycine Histidine Hydroxylysine Hydroxyproline Isoleucine Leucine Lysine Methionine Phenylalanine Proline Serine Threonine Tyrosine Valine Imino acids* Total

7.60b  0.4 11.2c  0.3 6.50a  1.2 12.3c  0.2 27.3b  0.9 0.60a  0.1 1.40a  0.2 14.2b  1.1 0.90a  0.2 2.80a  0.4 1.50a  0.1 0.90a  0.1 0.50a  0.1 8.20a  0.6 1.50a  0.1 4.30b  1.9 0.80a  0.1 1.90a  0.2 22.4a  0.4

Bovine collageny

Skin collagen CV 0.05 0.02 0.18 0.02 0.03 0.16 0.14 0.07 0.22 0.14 0.06 0.11 0.20 0.07 0.06 0.44 0.12 0.10 0.02

100.0

Mean  S.D.

CV

7.20b  0.2 6.90b  0.4 4.20a  1.2 9.50b  0.4 18.3a  1.8 1.40b  0.2 1.20a  0.3 15.5b  2.2 2.60b  0.2 3.90b  0.2 4.00c  0.2 1.90b  0.1 2.30b  0.3 8.30a  0.9 3.60b  0.7 3.80b  0.1 2.30b  0.2 3.20b  0.2 23.80a  2.3

Mean  S.D.

CV

6.25a  0.0 3.16a  0.1 10.8b  0.8 6.42a  0.6 28.1b  0.6 3.25c  0.2 5.00b  1.3 6.74a  0.4 2.70b  0.7 3.80a  0.9 2.70b  0.9 1.40b  1.0 2.00b  0.5 11.4b  1.4 1.47a  1.1 0.84a  0.2 0.72a  0.1 2.90b  0.4 18.1b  0.9

0.03 0.06 0.28 0.04 0.10 0.14 0.25 0.14 0.08 0.05 0.05 0.05 0.13 0.11 0.20 0.03 0.09 0.06 0.10

100.0

0.00 0.03 0.07 0.09 0.02 0.06 0.26 0.06 0.26 0.24 0.33 0.71 0.25 0.12 0.75 0.23 0.14 0.14 0.05

100.0

Means  S.D. (n = 3). * Imino acids = Proline + Hydroxyproline. y Type I collagen from bovine achilles tendon was used as reference material. Limit of quantification = 0.05%. Different superscript in each row indicates significant difference (P < 0.05). Total protein of dry crude collagens was determined according to Woyewoda et al. (1986).

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comparison between skin collagen from fish and terrestrial species is more difficult to establish due to high variability. However, in this research, hydrophobic amino acids were clearly higher in squid skin collagen than in muscle collagen. In concordance with the chemical nature of hydrophobic amino acids, their presence in jumbo squid skin collagen probably helps to reduce the solubility of molecule in water. Besides, they could be contributing to form partial unfolding structures in the collagen molecule, probably due to the presence of hydrophobic amino acid rich domains. Native state, partial unfolding or random structures of a protein, like collagen, could define their mechanical and physicochemical properties, as well as their physiological functions; thus, it would not be strange that the flexibility requirements of squid skin could be associated with a flexible fibril-collagen structure. Furthermore, Gly content in squid skin collagen preparation was lower than muscle collagen; similar results have been reported by other species of squid skins and/or fish skin collagens (Jongjareonrak et al., 2005; Yan et al., 2009). This result could be explained by taking into consideration the biological role of skin, where its main function is to provide elasticity and/or external protection (as a physical barrier) against environmental factors. This type of collagen is not involved in biomechanical efforts, such as the muscle collagen. Besides, collagen composition can differ from species, and even among specimens of the same species. It is well known that high Gly, Pro and Hyp contents can impart a higher stability and thermal resistance to collagen’s triple helical structure, since this structure results from a repetitive Gly-X-Y sequence, where X and Y positions are frequently occupied by Pro and Hyp residues. This triple helical structure is maintained mainly by hydrogen bonds formed between the atoms that conform each helix (Shoulders and Raines, 2009). Nature adjusts thermal stability of collagen monomers to the body temperature in warm-blooded animals or to the environment temperature in cold-blooded animals by changing the hydroxyproline content in the protein (Leikina et al., 2002). The amino acid composition of jumbo squid collagens points out to a highly stable structure, consistent with the thermal behavior reported for this protein present in the mantle

of this and others cephalopods (Mizuta et al., 2009; TorresArreola et al., 2008; Valencia-Pe´rez et al., 2008). Proline hydroxylation in squid skin collagen was slightly higher (P < 0.05) than that found in muscle collagen (Table 2), with values of 65% and 63%, respectively. However, Pro hydroxylation of these squid collagens was higher than other marine skin collagens such as that for Atlantic salmon (Salmo salar L.) (34–40%) (Moreno et al., 2012), marine eel fish (Evenchelys macrura) (49%) (Veeruraj et al., 2013), Spanish mackerel (Scomberomorous niphonius) (39%) and catfish (Pangasianodon hypophthalmus) (41–42%) (Singh et al., 2011). On the other hand, the degree of Lys hydroxylation present in jumbo squid muscle collagen (Table 2) can be essential information to suggest that hydroxylysine (Hyl) has biological roles in self-assembly and stabilization of the molecule through pyridinium cross-links formation. Although, Hyl distribution in both collagens was very similar (Table 1), the hydroxylation degree of this amino acid in muscle collagen was twice higher (P < 0.05) than in skin collagen. The high Lys hydroxylation degree is frequently found in highly insoluble collagens with a high crosslinking degree. These cross-links are formed mainly by oxidative deamination of the e-amino group of specific Lys and/or Hyl present in the amino and carboxy-telopeptides by lysyl oxidase (LOX) (Eyre and Wu, 2005). The chemistry of these cross-links is dependent on both, the nature and age of the collagenous tissue (Sims et al., 2000). Thus, cross-link differences are due to the Table 2 Hydroxylation degrees of proline and lysine and hydroxylysyl-pyridinoline (HP) content in collagen from jumbo squid tissues. Tissue

Hydroxylation degree (%) Lysine

Proline

Muscle Skin

HP (mmol/mol collagen)

Mean  S.D.

CV

Mean  S.D.

CV

63.1  0.60a 65.2  0.21b

0.01 0.01

46.9  4.01a 23.4  1.70b

0.08 0.08

Mean  S.D.

CV

4.6  0.01 n.f.

0.01

Mean  S.D. (n = 3). HP limit of quantification = 50 pmol/mol collagen. Different superscript in each column indicates significant difference (P < 0.05).

Please cite this article in press as: Ramı´rez-Guerra, H.E., et al., Hydroxylysyl-pyridinoline occurrence and chemical characteristics of collagen present in jumbo squid (Dosidicus gigas) tissues. J. Food Compos. Anal. (2015), http://dx.doi.org/10.1016/j.jfca.2015.06.003

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degree of hydroxylation of both, the telopeptide and specific lysines found in the triple helix (Eyre and Wu, 2005). Pyridinium cross-link content in collagen extracted from skin and muscle of jumbo squid mantle is shown in Table 2. In order to express HP content as mol/mol of collagen in crude collagen preparations, it was necessary to estimate the collagen content in tissues by multiplying the hyp content by 7.14 protein conversion factor, as previously described in material and methods; in both cases, skin and muscle collagens had a content of >92%. Hydroxylysyl-pyridinoline (HP) content in muscle collagen from squid mantle was 4.6 mmol/mol of collagen. However, HP was not detected in skin collagen. The chromatographic analysis of HP in hydrolyzed sample after pretreatment on a SPE column is shown in Fig. 1a. It can be observed that HP, identified as a single peak on the sample, was detected at the same retention time of the HP standard (data no shown). A standard curve was constructed using four concentrations, ranging from 107 to 270 pmol of HP (Fig. 1b). Regression analyses for the standard curve (y = 2.0618x  169.02) gave a determination coefficient (r2) > 0.98 and a variation coefficient (CV) < 4% for all measures. Thus, accuracy of set points and linearity of calibration curve described a reliable analytical quantification. HP content in jumbo squid muscle collagen was lower than that reported by Ando et al. (2001) for soluble and insoluble collagen of several squid species after treatment by boiling water for 10 and 30 min. Regardless of species and/or heat-treatment condition, HP content was over 0.6 mol per mole of collagen. However, these authors used a different conversion factor and collagen mass to calculate the HP concentration in their collagens. On the other hand, the HP content in jumbo squid muscle collagen is comparable to that reported for red sea bream (Pagrus major) and half of that reported for Yellowtail (Seriola quinqueradiata) intramuscular collagen, with values of 3.44 and 8.80 mmol per mole of collagen, respectively (Ando et al., 2006). Our results indicated that HP crosslinking has a specific distribution in jumbo squid tissues, occurring only in the intramuscular collagen. The fact that HP was not found in skin collagen is consistent with other previous reports on bovine and human skin (Eyre, 1987). Variations in collagen cross-linking appear to be more tissue-specific than collagen type (Eyre et al., 2008). However, differences in Lys hydroxylation of squid muscle collagen suggest a possible relation with essential cross-linking regulation mechanisms. It has been proposed that the basic pathway of pyridinium cross-linking is regulated primarily by the hydroxylation pattern of the telopeptide and triple-helix domain lysine residues (Myllyla¨ et al., 2007); this could be related with HP occurrence only in squid muscle collagen. On the other hand, the neighboring sequences around the cross-linking of lysine residues can also affect the ensuing collagen chemistry. For example, the pitch of collagen molecules packed in skin collagen fibrils is thought to facilitate the histidine residue reaction in the formation of histidino-hydroxlysinonorleucine (HHL), a mature trivalent cross-link found in skin type I collagen (Sims et al., 2000). Based on amino acid analysis (Table 1), histidine content in squid skin collagen was more than double than that found in skin collagen, which could serve as substrate in HHL formation. However, the later needs to be confirmed.

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Fig. 1. Hydroxylysyl-pyridinoline (HP) quantification by HPLC chromatography: (a) hydrolysate of jumbo squid mantle collagen, (b) standard HP calibration curve.

It is well known that HP is the advanced stage of cross-link product from the Hyl oxidation route, which predominates in most connective tissues except in skin (Eyre and Wu, 2005). Additionally, it is well accepted that HP cross-link appears to be distributed in tissues subjected to mechanical stresses as it could be the case of jumbo squid mantle muscle; therefore it was expected to be found only in intramuscular collagen as shown in Table 2. The squid mantle muscle has a complex fibrillar structure of collagen which provides structural support and stores elastic potential energy to reduce reliance on muscle force during swimming (Krieg and Mohseni, 2012). Therefore, it is reasonable to find HP cross-link in collagen from this anatomical region of squid, providing mechanical resistance to the mantle structure during propulsive-jet swimming operations. Additionally, enzymatic studies of LOX from jumbo squid tentacles (Torres-Arreola et al., 2012) support our results about the natural HP occurrence in muscle and stabilization of squid muscle collagen by covalent cross-linking pathway, similar to its counterpart terrestrial species and human tissues. Carbohydrate analysis of skin and muscle collagen from jumbo squid mantle is shown in Table 3. Total carbohydrate content was similar (P  0.05) in both collagens. Arabinose, glucose and xylose were the carbohydrates found in both collagens while mannose and galactose were found only in the collagen from muscle. Arabinose was the monosaccharide found in the highest concentration in both tissues (P  0.05), showing values of 11.7 and 11.1% for muscle and skin, respectively. Furthermore, slight differences (P < 0.05) in glucose and xylose contents were found in collagens. Although these monosaccharides were also found in collagen from body walls of Japanese flying squid (Todarodes pacificus), and octopus (Octopus vulgaris), they were in lower concentration than jumbo squid collagen (only in the range of 2.89 and 3.96% of total carbohydrates) (Kimura, 1972). In addition, low carbohydrate contents (<1%) have been also reported for intramuscular

Table 3 Carbohydrate analysis (% per gram of dry wt.) of skin and muscle collagens from jumbo squid. Tissue

Muscle Skin

Total carbohydrate

Arabinose

Mean  S.D.

Mean  S.D.

a

16.6  0.50 15.2a  1.12

CV 0.03 0.07

a

11.1  0.21 11.7a  0.91

Glucose CV 0.02 0.08

Mean  S.D. a

3.3  0.10 2.8b  0.10

Galactose CV 0.03 0.03

Mean  S.D. 0.2  0.01 n.f.

Mannose CV 0.05

Mean  S.D. 1.1  0.11 n.f.

Xylose CV 0.10

Mean  S.D. a

0.9  0.10 0.7b  0.11

CV 0.11 0.16

Means  S.D. (n = 3). Different superscript in each column indicates significant difference (P < 0.05). Limit of quantification = 0.0001%.

Please cite this article in press as: Ramı´rez-Guerra, H.E., et al., Hydroxylysyl-pyridinoline occurrence and chemical characteristics of collagen present in jumbo squid (Dosidicus gigas) tissues. J. Food Compos. Anal. (2015), http://dx.doi.org/10.1016/j.jfca.2015.06.003

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connective tissues of marine crab (Scylla serrata) (Sivakumar et al., 2000) and farmed Atlantic salmon (Salmo salar L.) (Moreno et al., 2012). As shown, carbohydrate distribution in jumbo squid collagens differed slightly depending on tissue; however, this small difference can be associated with differences in biomechanical requirements between connective tissues, as well as on stability and/or ultimate physicochemical properties of its collagens. Carbohydrate occurrence and their specific distribution in collagen is not a casual event, it responds to a critical post-transductional modification implicated in biosynthesis, self-assembly, transporting and molecule deposition into extracellular matrix (ECM) (Shoulders and Raines, 2009). The most studied post-transductional glycosylation event in collagen is Hyl O-linked glycosylation, catalyzed by lysil-hydroxylase (LH3) (Myllyla¨ et al., 2007). LH3 has

been shown to possess three catalytic activities sequentially required to produce first Hyl, and then its glycosylated forms (GalHyl and Glc-Gal-Hyl), by the lysyl hydroxylase (LH), galactosyltransferase (GT), and finally the glucosyltransferase (GGT) activities, respectively (Myllyla¨ et al., 2007). This LH3-mediated glycosylation occurs at least at five specific sites in type I collagen, Q5 the preferred site being the one located at a1–87, which is the site involved in intermolecular pyridinium crosslinking formation (Sricholpech et al., 2012). Furthermore, some collagen subtypes contain specific carbohydrate side chains with an unknown function. However, there are some implications of carbohydrates in nonenzymatic cross-linking reactions. For example, aging is characterized by structural changes in the ECM in virtually every tissue and organ system where carbohydrate adducts may directly affect several physicochemical properties of collagen, including

Fig. 2. Fourier transform infrared spectra (FT-IR) of skin (a) and muscle (b) collagens from jumbo squid mantle.

Please cite this article in press as: Ramı´rez-Guerra, H.E., et al., Hydroxylysyl-pyridinoline occurrence and chemical characteristics of collagen present in jumbo squid (Dosidicus gigas) tissues. J. Food Compos. Anal. (2015), http://dx.doi.org/10.1016/j.jfca.2015.06.003

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Fig. 3. MS/MS spectrum of HP purified from jumbo squid mantle collagen. HP molecular masses are given in g/mol.

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conformation, ligand binding, LOX-mediated cross-linking, and interaction with other molecules, all of which undergo ageassociated changes (Reiser et al., 1992). A specific distribution of mannose in squid connective tissues becomes relevant since it has been associated with a collagen turnover mechanism, in which mannose recognition by lectin-like proteins is important to internalize collagen for lysosomal degradation (Ju¨rgensen et al., 2011). However, the specific biological role of mannose in jumbo squid muscle collagen, alternative to structural or thermal-related functions, remains to be elucidated. Interestingly, arabinose showed the highest carbohydrate content in squid collagens; however, further research also is needed in order to find its possible role in jumbo squid collagen. Definitely, particularities in amino acid composition and carbohydrate distribution could be implicated in the supraorganizational rearrangement of skin and muscle collagen of jumbo squid mantle. In this way, the organizational arrangement of squid collagens was investigated by FT-IR analysis, which was directly related to the amides regions (A, B, I, II and III) (Fig. 2). The comparison among FT-IR collagen spectra indicates slight changes in vibrational frequencies of amides A and B at 3400–3440 cm1 and 3070 cm1, respectively. The major spectrum changes were registered in the spectral range of 1000–1700 cm1, in which include amides I, II, III and carbohydrate vibrational frequencies. Amide I band (1600–1660 cm1) is associated with stretching vibrations of carbonyl groups (C5 5O) in peptides, this being the most important factor in order to elucidate the secondary structure of collagens: b-turn, 1660–1700 cm1; a-helix, 1645–1659 cm1; irregular structure, 1640–1644 cm1; b-sheet or extended structure, 1620–1640 cm1 (Li et al., 2013). Amide II (1550 cm1) is associated with NH bending and C–N stretching. Amide III (1220– 1320 cm1) is related with N–C–N stretching and N–H, and also involved with the triple helical structure. The triple helical structure of collagen would be estimated with the absorption ratio between amide III frequency and the 1452 cm1 band, which has to be approximately equal to 1.0 (Li et al., 2013). As well, amide III intensity absorption arises from wagging vibrations of –CH2 groups from the glycine backbone and proline side chains (Nagarajan et al., 2012). In native type I collagen, two distinct peaks are observed at 1082 and 1032 cm1, corresponding to total protein and carbohydrates signals, respectively. These two peaks were associated with C–O and C–O–C stretching absorption of the carbohydrate moieties (Belbachir et al., 2009). In collagen glycation studies, the computed intensity ratio of the 1032 cm1 band with respect to the amide I band at 1660 cm1 has been used to verify spectral modifications in this region, being a spectroscopic marker of glycation degree

(Guilbert et al., 2013). In this respect, typical carbohydrate frequency peaks were registered in FT-IR spectrums (Fig. 2) in both jumbo squid analyzed collagens, being a good indicator of carbohydrate presence that gives support to results obtained in HPLC carbohydrate analysis. In molecules with elements of symmetry, such as in the triple helical structure of collagen, only certain bands (i.e., carbohydrate functional groups) may be active in IR. FT-IR spectrums of skin and muscle collagens from jumbo squid mantle showed slight differences, although enough to make inferences in supra-organizational level of molecule. Changes in absorbance intensities and broadenings of amides A, B, I, II and III suggest a major occurrence of hydrogen bonds and carbonyl groups (that may be due to high polar amino acid contents) in squid muscle collagen (Fig. 2b) that it could be well related with a persistent triple helical structure. On the other hand, some authors proposed that the use of a 1660 cm1/1690 cm1 absorbance ratio instead of an area ratio would be more representative of the pyridinoline to dehydrodihydroxynorleucine (deH-DHLNL) ratio (de Campos Vidal and Mello, 2011). Fibrillogenesis (self-assembly) of collagen has been found to be associated with broadening and a slight shift to lower wave number of the amide A peak, an increase in intensity and slight shift to lower wave number of amide III peak, band broadening and shift of amide I to lower wave number and shift of amide II peak to lower wave number (de Campos Vidal and Mello, 2011). The above changes are therefore associated with increased intermolecular interactions (hydrogen bonds) in muscle collagen than the skin collagen, as shown in Fig. 2b. The MS/MS spectrum of HP purified from jumbo squid mantle collagen is shown in Fig. 3. HP was identified by their molecular mass deduced from their ions on electrospray mass spectrometry. A mass-to-charge ratio (m/z) of 428.7 was obtained for HP, which is comparable with the theoretical mass of 429 g/mol, previously reported for HP isolated from bone and urine samples (Vesper et al., 2003). Thus, MS/MS analysis of purified compound helped us to validate the identity of the HP cross-link in jumbo squid mantle muscle collagen.

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4. Conclusion

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The chemical composition differences between skin and muscle collagens from jumbo squid suggest a different organizational rearrangement of the molecule in these tissues. Mantle muscle collagen appears to be a bundle of fibrils stabilized through HP crosslinking. Certainly, this singularity of the muscle collagen could be associated with the particular biology of its mantle and their physiological requirements such as the biomechanical effort

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Please cite this article in press as: Ramı´rez-Guerra, H.E., et al., Hydroxylysyl-pyridinoline occurrence and chemical characteristics of collagen present in jumbo squid (Dosidicus gigas) tissues. J. Food Compos. Anal. (2015), http://dx.doi.org/10.1016/j.jfca.2015.06.003

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during swimming activities. Technological implications of HP and glycation degree of squid mantle muscle collagen remain uncertain; thus, their contents and its influence in collagen solubilization, thermal and mechanical properties need to be considered in future research.

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Acknowledgments

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The first author thanks CONACyT for the doctoral scholarship awarded and also to Dr. Ruggero Tenni, Monica Villegas-Ochoa and Karla Martinez-Robinson for their technical support during experimental procedure of this work.

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Please cite this article in press as: Ramı´rez-Guerra, H.E., et al., Hydroxylysyl-pyridinoline occurrence and chemical characteristics of collagen present in jumbo squid (Dosidicus gigas) tissues. J. Food Compos. Anal. (2015), http://dx.doi.org/10.1016/j.jfca.2015.06.003

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