- Email: [email protected]
The changes in crosslink contents in tissues after formalin fixation
ANALYTICAL BIOCHEMISTRY Analytical Biochemistry 318 (2003) 118–123 www.elsevier.com/locate/yabio
The changes in crosslink contents in tissues after formalin fixation Masashi Abe,a Masaaki Takahashi,a,* Kentaro Horiuchi,b and Akira Naganoa a
Department of Orthopaedic Surgery, Hamamatsu University School of Medicine, Shizuoka, Hamamatsu 431-3192, Japan b Department of Life Science, Hamamatsu University School of Medicine, Shizuoka, Hamamatsu 431-3192, Japan Received 3 February 2003
Abstract The aim of this study was to detect crosslinks of collagen and elastin in formalin-fixed tissue, to perform quantification of these crosslinks, and to investigate the effects of formalin fixation on crosslink contents in human yellow ligament and cartilage. Pyridinoline (Pyr) is a stable and nonreducible crosslink of collagen. Pentosidine (Pen) is a senescent crosslink formed between arginine and lysine in matrix proteins, including collagen. Desmosine (Des) and its isomer isodesmosine (Isodes) are crosslinks specifically found in elastin. It is useful to measure crosslink contents of collagen and elastin as a way of investigating the properties of various tissues or their pathological changes. If it is possible to evaluate crosslinks of collagen and elastin in formalin-fixed tissues, we can investigate crosslinks in a wide variety of tissues. We used HPLC to compare the concentrations of Pyr, Pen, Des, and Isodes in the formalin-fixed tissues with their concentrations in the frozen tissues. Pyr and Pen were detected in both the formalin-fixed yellow ligament and the cartilage, and their concentrations were not significantly affected by or related to the duration of formalin fixation. Des and Isodes were detected in the formalin-fixed yellow ligament but in significantly lower amounts compared to the frozen samples. We concluded that crosslinks of collagen were preserved in formalin, but crosslinks of elastin were not preserved in it. The reason for this might be that formalin did not fix elastin tissues sufficiently or it destroyed, masked, or altered elastin crosslinks. Ó 2003 Elsevier Science (USA). All rights reserved. Keywords: Crosslinks; Formalin; High-performance liquid chromatography; Yellow ligament; Cartilage
Collagen and elastin are prominent constituents of various tissues and organs for which tensile strength and resiliency are important functional parameters. The properties of collagen and elastin differ among various organs, depending on organ maturation and degeneration due to aging, the presence of connective tissue diseases, and organ abnormalities. There are many kinds of intra- and intermolecular crosslinks in collagen and elastin that have been used as biochemical markers reflecting their pathophysiological changes [1–5]. Pyridinoline (Pyr),1 which is present in many connective tissues, is a stable and nonreducible crosslink of collagen that is physiologically necessary to maintain the structure of the collagen fibril [6]. In addition, Pyr is the *
Corresponding author. Fax: +81-53-435-2296. E-mail address: [email protected] (M. Takahashi). 1 Abbreviations used: Pyr, Pyridinoline; Pen, pentosidine; AGE, advanced glycation end product; Des, desmosine; Isodes, idodesmosine.
major crosslink of mature bone and cartilage [7]. Pentosidine (Pen), a senescent crosslink formed between arginine and lysine in matrix proteins, including collagen, is one of the advanced glycation end products (AGEs) [8] and is a sensitive marker of proteins modified by AGEs in aging, diabetes, and uremia [8,9]. Desmosine (Des) and its isomer isodesmosine (Isodes) are crosslinks specifically found in elastin [10]. Des and Isodes, which are derived from the posttranslational modification of four lysyl residues, are responsible for the elastic property and insolubility of elastin, and these contents in tissues reflect elastin contents [11,12] and the changes due to aging [13]. It is useful to measure crosslink contents of collagen and elastin as a way of investigating the properties of various tissues or their pathological changes. Previously, we had developed a high-performance liquid chromatographic (HPLC) method to measure these crosslinks of collagen and elastin in the hydrolysates of human yellow ligament [14]. In this method, samples should be fresh and be stored in a deep
0003-2697/03/$ - see front matter Ó 2003 Elsevier Science (USA). All rights reserved. doi:10.1016/S0003-2697(03)00194-5
M. Abe et al. / Analytical Biochemistry 318 (2003) 118–123
freezer for the measurement of crosslinks. When the method is used this way, however, the number and amount of samples are limited, because samples are usually obtained only during autopsy or surgery. If we can use formalin-fixed tissue samples for a crosslink assay, the number and amount of samples could be increased. The aim of this study was to detect crosslinks of collagen and elastin in formalin-fixed tissue, to perform quantification of these crosslinks, and to investigate the effects of formalin fixation on crosslink contents in human yellow ligament and cartilage.
Materials and methods Specimens and preparation of samples Ten specimens of yellow ligament and 12 specimens of cartilage from hip joints were obtained during surgery performed at our department. All procedures were in accordance with the principles of the Declaration of Helsinki, 1975 (revised 1996). Each specimen of yellow ligament was separated into four pieces, and one of them was stored at )80 °C in a deep freezer while the remaining three pieces were preserved in formalin solutions (10% buffered, pH 7) for 1, 3, or 6 months. Each cartilage specimen was separated into two pieces, one of which was stored at )80 °C in a deep freezer and the other of which was preserved in formalin solutions for 1 year. For the assay, each sample was freeze-dried under vacuum, and samples of approximately 7 mg dry weight were hydrolyzed in 1.5 ml 6 N HCl at 110 °C for 24 h in screw-top hydrolysis glass tubes (Iwaki-Glass, Japan). The hydrolysates were divided and prepared separately for crosslink prefractionation. All crosslinks in the hydrolysate (100 ll) were eluted using an SP-Sephadex column (Hþ form, 0:8 1:0 cm; Pharmacia Fine Chemical AB, Uppsala, Sweden), by the previously described method, with some modifications [14]. The eluate was evaporated under vacuum, and the residue was dissolved in 100 ll of water for analysis of Pyr and Pen, and in 50 ll of water for HPLC analysis of Des and Isodes. For the analysis of Des and Isodes, the same prefractionation was done twice, and 50 ll of each obtained solution were mixed.
119
An ODS reversed-phase column (8 mm 10 cm) prepacked with Radial-Pak C18 of 10 lm particle size, Type 8C1810 l (Waters Associates, Inc.), was used. Two mobile phases of a mixture of 20 mM phosphate buffer containing 0.2% SDS (final pH 3.5) and MeCN (19:1, v/v) and the same mixture but containing SDS and MeCN in the proportions of 3:2 (v/v) were used. Chromatographic analysis was performed through binary gradient elution. For analytical and columncleaning purposes, flow conditions were programmed on the system controller. The flow rate was 1.0 ml/min at room temperature. We injected 10-ll samples to measure Pyr and Pen and 60-ll samples to measure Des and Isodes. The greater injection volume for measurement of Des and Isodes was needed because there were some samples from formalin-fixed yellow ligament in which they were not detected even when the sample volume was suitable for the measurement of Pyr and Pen by HPLC. For the detection of Pyr and Pen, we measured the fluorescence at 397 nm with excitation at 305 nm, and for Des and Isodes we measured UV absorbance at 275 nm. Using our protocol, the recoveries of standards using SP-Sephadex C-25 (n ¼ 8) were 86.4–98.3% for Pyr, 78.7–95.6% for Pen, 83.6–97.9% for Des, and 85.6– 99.3% for Isodes [14]. The hydroxyproline content was measured through an automated amino acid analyzer system (Model 835–50; Hitachi, Tokyo, Japan) using an aliquot of the hydrolysate of the samples. The concentrations of crosslinks in each yellow ligament and cartilage sample were expressed as millimoles per mole of hydroxyproline for Pyr and Pen and for Des and Isodes, and we compared the concentrations between the frozen and the formalin-fixed samples. Preparation of standard crosslinks Pyr was isolated from a human cortical bone (a 13year-old tibiae). The purification methods we used were described previously [15]. Pen was synthesized [16], and its concentration was calibrated with authentic Pen, which was a gift from Dr. V.M. Monnier (Institute of Pathology, School of Medicine, Case Western Reserve University, Cleveland, OH, USA). Standard Des and Isodes were purchased from Elastin Products (St. Louis, MO, USA).
HPLC analysis Statistical analysis The measurement of crosslinks was performed using the method described previously [14]. Briefly, the HPLC system consisted of a Model DP-8020 pump (TOSOH, Tokyo, Japan), a Waters Model 474 scanning fluorescence detector (Waters Associates, Inc., Milford, MA, USA), a model PD-8020 variable-wavelength detector (TOSOH), a Model AS-8010 autosampler (TOSOH), and a Model PX-8020 system controller (TOSOH).
The statistical significance of difference between two groups was determined using the Mann–Whitney U test. The correlation between the measurements and the parameters was determined using the Spearmann rank correlation test. Analysis was performed using StatView J 5.0 on a Macintosh computer. p values of less than 0.05 were considered statistically significant.
120
M. Abe et al. / Analytical Biochemistry 318 (2003) 118–123
Results The typical HPLC chromatograms of the hydrolysate of the frozen and formalin-fixed yellow ligament stored for 1, 3, and 6 months obtained from a 71-year-old man and those of the frozen and formalin-fixed cartilage stored for 1 year obtained from an 85-year-old woman are shown in Figs. 1–3. The chromatographic patterns of Pyr and Pen in the hydrolysate of the formalin-fixed yellow ligament and cartilage were similar to those of the frozen yellow ligament and cartilage at every time point (Figs. 1 and 2). We also detected chromatographic peaks of Des and Isodes in the hydrolysate of the formalin-fixed yellow ligament, and we found an additional peak between the peaks of Des and Isodes in the formalin-fixed yellow ligament that was not detected in the frozen yellow ligament (Fig. 3). Neither Des nor Isodes was detected in the hydrolysate of cartilage. We compared the concentrations of Pyr, Pen, Des, and Isodes in the formalin-fixed tissues with those in the frozen tissues (Table 1). We found that the mean proportions of Pyr and Pen in the yellow ligament formalin-fixed for 1, 3, and 6 months compared to the proportion in the frozen samples were 111 and 87%, 106 and 98%, and 123 and 100%, respectively. The mean proportion of Pyr and Pen in the cartilage formalin-fixed for 1 year compared to the proportion in the frozen samples were 102 and 114%, respectively, and the mean proportion of Des and Isodes in the yellow ligament formalin-fixed for 1, 3, and 6 months compared to the
proportion in the frozen samples were 48 and 46%, 46 and 47%, and 55 and 52%, respectively. These results suggest a significant decrease in Des and Isodes in the formalinfixed yellow ligament (Table 1). Table 2 shows the correlations of Pyr, Pen, Des, and Isodes between the formalin-fixed tissues and the frozen tissues. There was a significant correlation of Pyr and Pen between the yellow ligaments that were formalin-fixed for 1, 3, and 6 months and the frozen yellow ligaments and between the cartilages that were formalin-fixed for 1 year and the frozen cartilages. On the other hand, there was no correlation of Des or Isodes between the yellow ligaments that were formalin-fixed for 1 month and the frozen yellow ligaments (Des, r ¼ 0:399, p ¼ 0:1222, and Isodes, r ¼ 0:473, p ¼ 0:1967, N ¼ 10). However, there was a significant correlation of Des and Isodes between the yellow ligaments that were formalin-fixed for 3 and 6 months and the frozen yellow ligaments (formalin-fixed for 3 months: Des, r ¼ 0:629, p ¼ 0:0475; Isodes, r ¼ 0:757, p ¼ 0:0365, N ¼ 10; formalin-fixed for 6 months: Des, r ¼ 0:806, p ¼ 0:0141; Isodes, r ¼ 0:916, p ¼ 0:0048, N ¼ 10), and the correlation between the yellow ligament formalinfixed for 6 months and the frozen tissue was the highest.
Discussion Formalin is a highly reactive chemical and electrophilic species that reacts with various crosslinking
Fig. 1. HPLC chromatograms of the hydrolysate of frozen yellow ligament and yellow ligament formalin-fixed for 1, 3, and 6 months. Spectrofluorometer, emission/excitation wavelengths were 397/305 nm. Closed arrow, Pyr; open arrow, Pen.
M. Abe et al. / Analytical Biochemistry 318 (2003) 118–123
121
Fig. 2. HPLC chromatograms of the hydrolysate of frozen cartilage and cartilage formalin-fixed for 1 year. Spectrofluorometer, emission/excitation wavelengths were 397/305 nm. Closed arrow, Pyr; open arrow, Pen.
Fig. 3. HPLC chromatograms of the hydrolysate of frozen yellow ligament and yellow ligament formalin-fixed for 1, 3, and 6 months. UV detector, absorbance at 275 nm. Closed arrow, Des; open arrow, Isodes; closed triangle, another peak.
functional groups in tissue proteins, nucleic acid, and polysaccharides [17], and it makes methylene crosslinks that are stable and irreversible. Formalin-fixed tissues do not become hardened, and shrinkage is minimized [17]. When collagen and elastin structures in formalinfixed specimens were observed histochemically, researchers found that formalin-fixed elastin retained its elasticity [18], and therefore, it is assumed that collagen and elastin are retained in formalin. However, because formalin has the crosslinking function, it may mask, alter, or destroy natural crosslinks of collagen and elastin. Commonly, crosslinks of collagen and elastin are measured in the hydrolysate of fresh or frozen
samples using HPLC. If it is possible to evaluate crosslinks of collagen and elastin in formalin-fixed tissues, we can investigate crosslinks in a wide variety tissues and tissues of a specific disease stored in the past. Pyr is a stable mature crosslink of collagen, and it cannot be reduced by sodium borohydride. This crosslink plays an important role in maintaining the structure and function of collagen. On the other hand, the glycated proteins undergo many nonenzymatical reactions and result in advanced glycation end products. Pen is one such advanced glycation end product. In this study, Pyr and Pen were detected in both the formalin-fixed yellow ligament and cartilage. Concentrations of Pyr
122
M. Abe et al. / Analytical Biochemistry 318 (2003) 118–123
Table 1 Concentrations of Pyr, Pen, Des, and Isodes in formalin-fixed vs frozen tissues Yellow ligament
Cartilage Formalin-fixed
Frozen Pyr/Hypro (mmol/mol) Pen/Hypro (mmol/mol) Des/Hypro (mmol/mol) Isodes/Hypro (mmol/mol)
1 month
0:81 0:35 0:10 0:07 9:50 6:19 14:90 9:83
Formalin-fixed 3 months
0:88 0:35 0:08 0:04 4:45 2:98 6:43 4:43 111:2 22:7 87:2 18:3 48:4 21:2 45:6 24:4
Pyr/Hypro (% to frozen) Pen/Hypro (% to frozen) Des/Hypro (% to frozen) Isodes/Hypro (% to frozen)
6 months
0:81 0:29 0:09 0:03 4:69 4:25 7:80 7:80 106:0 29:0 97:7 20:9 46:2 28:4 46:6 30:4
0:96 0:34 0:09 0:06 5:19 3:23 7:80 4:83
Frozen
1 year
2:61 0:79 0:21 0:07
2:59 0:72 0:24 0:07
123:1 22:6 100:1 19:8 55:0 22:0 52:1 20:0
102:3 23:5 114:1 23:2
The mean concentrations SD of Pyr, Pen, Des, and Isodes in frozen and formalin-fixed yellow ligament and cartilage and the mean proportions SD of Pyr, Pen, Des, and Isodes in each period formalin-fixed yellow ligament to those in frozen tissue and the mean proportions SD of Pyr and Pen in cartilage formalin-fixed for 1 year to those in frozen tissue. Measured values of Pyr, Pen, Des, and Isodes were all normalized to measured values of hydroxyproline (Hypro). * p < 0:05 vs frozen.
Table 2 Correlations of Pyr, Pen, Des, and Isodes in each period formalin-fixed ligament and cartilage compared with frozen tissues Pyr
Pen
Des
Isodes
r
p
r
p
r
p
r
p
Yellow ligament Frozen vs formalin-fixed 1 month Frozen vs formalin-fixed 3 months Frozen vs formalin-fixed 6 months
0.780 0.711 0.852
0.0056 0.0187 0.0008
0.872 0.889 0.905
0.0004 0.0002 <0.0001
0.399 0.629 0.806
0.1222 0.0475 0.0141
0.473 0.757 0.916
0.1967 0.0365 0.0048
Cartilage Frozen vs formalin-fixed 1 year
0.575
0.0382
0.658
0.0125
Spearmann rank correlation test.
and Pen in the yellow ligament and cartilage were not significantly affected or related to the duration of formalin fixation, and there was a significant correlation between the concentrations of crosslinks in frozen yellow ligament and cartilage and those in formalin-fixed tissues. These results indicated that natural crosslinks of collagen were preserved in formalin, and formalin did not mask, alter, or destroy Pyr and Pen, so formalinfixed tissues can be used for investigating the contents of Pyr and Pen. Des and Isodes are crosslinking amino acids specifically located in elastin, and their concentration in some tissues can reflect the elastin contents [12]. In the present study, Des and Isodes were detected in the formalin-fixed yellow ligament, but the concentrations were significantly decreased compared with the concentrations found in frozen samples. Therefore, we concluded that crosslinks of elastin were not preserved in formalin. The reason for this might be that formalin did not fix elastin tissues sufficiently or it destroyed, masked, or altered crosslinks. We were not able to clarify the reason in this study. It has been reported that elastin was not significantly fixed by formalin, because its elastic recovery rate after stretching was only 50–70% [18]. This indicates that formalin cannot fix elastin
completely or that elastin is destroyed during formalin fixation. Although the mean concentrations of Des and Isodes decreased in the yellow ligament formalin-fixed for 1 month, they did not show further reduction in the ligaments fixed for 3 and 6 months. There was a significant correlation of Des and Isodes between the frozen yellow ligament and the yellow ligament formalin-fixed for 3 and 6 months. The reduced elastin crosslinks might be stable in formalin for a long period. An unidentified peak was found between the peaks of Des and Isodes on the chromatograms of only the formalin-fixed yellow ligaments. The appearance of an additional peak in formalin-fixed samples might indicate that some of the crosslinks of elastin, possibly Des or Isodes, were modified by formalin. The decrease in Des and Isodes might indicate that some amount of them was modified into another crosslinks or was broken by formalin fixation. This result showed that the longitudinal investigation of the contents of Des or Isodes in formalin-fixed tissues was not adequate and that the concentration of Des or Isodes in the formalin-fixed tissues did not correspond to that in frozen or fresh samples. However, measurement of the concentration of Des or Isodes in
M. Abe et al. / Analytical Biochemistry 318 (2003) 118–123
formalin-fixed tissues is useful when a comparison is made among formalin-fixed tissues.
Acknowledgment We thank Ms. Ayako Fujiwara for her technical help.
References [1] P. Garnero, J. Rousseau, P. Delmas, Molecular basis and clinical use of biochemical markers of bone, cartilage, and synovium in joint diseases, Arthritis Rheum. 43 (2000) 953–968. [2] S. Sakata, M. Takahashi, K. Kushida, M. Oikawa, H. Hoshino, M. Denda, T. Inoue, The relationship between pentosidine and hemodialysis-related connective tissue disorders, Nephron 78 (1998) 260–265. [3] M. Takahashi, K. Kushida, H. Hoshino, M. Suzuki, M. Sano, S. Miyamoto, T. Inoue, Concentrations of pyridinoline and deoxypyridinoline in joint tissues from patients with osteoarthritis or rheumatoid arthritis, Ann. Rheum. Dis. 55 (1996) 324–327. [4] P.J. Stone, Potential use of collagen and elastin degradation markers for monitoring liver fibrosis in schistosomiasis, Acta Tropica 77 (2000) 97–99. [5] D.C. Bode, E.D. Pagani, W.R. Cumiskey, R. von Roemeling, L. Hamel, P.J. Silver, Comparison of urinary desmosine excretion in patients with chronic obstructive pulmonary disease or cystic fibrosis, Pulm. Pharmacol. Ther. 13 (2000) 175–180. [6] D. Eyre, J. Wu, P. Woops, The cartilage collagens: structural and metabolic studies, J. Rheumatol. 18 (Suppl. 27) (1991) 49–51. [7] D. Eyre, H. Oguchi, The hydroxypyridinium crosslink of skeletal collagens: their measurement, properties and a proposed pathway of formation, Biochem. Biophys. Res. Commun. 92 (1980) 403–410.
123
[8] D.R. Sell, V.M. Monnier, Sructure elucidation of a senescence cross-links from human extracellular matrix, J. Biol. Chem. 264 (1989) 21597–21602. [9] V.M. Monnier, D.R. Shell, R. Nagaraj, S. Miyata, S. Grandhee, P. Odetti, S. Ibrahim, Maillard reaction-mediated molecular damage to extracellular matrix and other tissue proteins in diabetes, aging, and uremia, Diabetes 41 (Suppl. 2) (1992) 36–41. [10] J. Thomas, D.F. Elsden, S.M. Partridge, Degradation products from elastin: partial structure of two major degradation products from the cross-linkages in elastin, Nature 200 (1963) 651–652. [11] B.C. Starcher, Determination of the elastin content of tissues by measuring desmosine and isodesmosine, Anal. Biochem. 79 (1977) 11–15. [12] Y. Yamaguchi, J. Haginaka, M. Kunitomo, H. Yasuda, Y. Bando, High-performance liquid chromatographic determination of desmosine and isodesmosine in tissues and its application to studies of alteration of elastin induced by atherosclerosis, J. Chromatogr. 422 (1987) 53–59. [13] M. Watanabe, T. Sawai, H. Nagura, K. Suyama, Age-related alteration of cross-linking amino acids of elastin in human aorta. Tohoku, J. Exp. Med. 180 (1996) 115–130. [14] J.R. Chen, M. Takahashi, K. Kushida, M. Suzuki, K. Suzuki, K. Horiuchi, A. Nagano, Direct detection of crosslinks of collagen and elastin in the hydrolysates of human yellow ligament using single-column high performance liquid chromatography, Anal. Biochem. 278 (2000) 99–105. [15] D. Fujimoto, T. Moriguchi, Pyridinoline, a non-reducible crosslink of collagen Quantitative determination, distribution, and isolation of a crosslinked peptide, J. Biochem. 83 (1978) 863–867. [16] S.K. Grandhee, V.M. Monnier, Mechanism of formation of the maillard protein cross-link pentosidine, J. Biol. Chem. 266 (1991) 11649–11653. [17] C.H. Fox, F.B. Johnson, J. Whiting, P.P. Roller, Formaldehyde fixation, J. Histochem. Cytochem. 33 (1985) 845–853. [18] Y.C. Fung, S.S. Sobin, The retained elasticity of elastin under fixation agents, J. Biol. Eng. 103 (1981) 121–122.
The changes in crosslink contents in tissues after formalin fixation Masashi Abe,a Masaaki Takahashi,a,* Kentaro Horiuchi,b and Akira Naganoa a
Department of Orthopaedic Surgery, Hamamatsu University School of Medicine, Shizuoka, Hamamatsu 431-3192, Japan b Department of Life Science, Hamamatsu University School of Medicine, Shizuoka, Hamamatsu 431-3192, Japan Received 3 February 2003
Abstract The aim of this study was to detect crosslinks of collagen and elastin in formalin-fixed tissue, to perform quantification of these crosslinks, and to investigate the effects of formalin fixation on crosslink contents in human yellow ligament and cartilage. Pyridinoline (Pyr) is a stable and nonreducible crosslink of collagen. Pentosidine (Pen) is a senescent crosslink formed between arginine and lysine in matrix proteins, including collagen. Desmosine (Des) and its isomer isodesmosine (Isodes) are crosslinks specifically found in elastin. It is useful to measure crosslink contents of collagen and elastin as a way of investigating the properties of various tissues or their pathological changes. If it is possible to evaluate crosslinks of collagen and elastin in formalin-fixed tissues, we can investigate crosslinks in a wide variety of tissues. We used HPLC to compare the concentrations of Pyr, Pen, Des, and Isodes in the formalin-fixed tissues with their concentrations in the frozen tissues. Pyr and Pen were detected in both the formalin-fixed yellow ligament and the cartilage, and their concentrations were not significantly affected by or related to the duration of formalin fixation. Des and Isodes were detected in the formalin-fixed yellow ligament but in significantly lower amounts compared to the frozen samples. We concluded that crosslinks of collagen were preserved in formalin, but crosslinks of elastin were not preserved in it. The reason for this might be that formalin did not fix elastin tissues sufficiently or it destroyed, masked, or altered elastin crosslinks. Ó 2003 Elsevier Science (USA). All rights reserved. Keywords: Crosslinks; Formalin; High-performance liquid chromatography; Yellow ligament; Cartilage
Collagen and elastin are prominent constituents of various tissues and organs for which tensile strength and resiliency are important functional parameters. The properties of collagen and elastin differ among various organs, depending on organ maturation and degeneration due to aging, the presence of connective tissue diseases, and organ abnormalities. There are many kinds of intra- and intermolecular crosslinks in collagen and elastin that have been used as biochemical markers reflecting their pathophysiological changes [1–5]. Pyridinoline (Pyr),1 which is present in many connective tissues, is a stable and nonreducible crosslink of collagen that is physiologically necessary to maintain the structure of the collagen fibril [6]. In addition, Pyr is the *
Corresponding author. Fax: +81-53-435-2296. E-mail address: [email protected] (M. Takahashi). 1 Abbreviations used: Pyr, Pyridinoline; Pen, pentosidine; AGE, advanced glycation end product; Des, desmosine; Isodes, idodesmosine.
major crosslink of mature bone and cartilage [7]. Pentosidine (Pen), a senescent crosslink formed between arginine and lysine in matrix proteins, including collagen, is one of the advanced glycation end products (AGEs) [8] and is a sensitive marker of proteins modified by AGEs in aging, diabetes, and uremia [8,9]. Desmosine (Des) and its isomer isodesmosine (Isodes) are crosslinks specifically found in elastin [10]. Des and Isodes, which are derived from the posttranslational modification of four lysyl residues, are responsible for the elastic property and insolubility of elastin, and these contents in tissues reflect elastin contents [11,12] and the changes due to aging [13]. It is useful to measure crosslink contents of collagen and elastin as a way of investigating the properties of various tissues or their pathological changes. Previously, we had developed a high-performance liquid chromatographic (HPLC) method to measure these crosslinks of collagen and elastin in the hydrolysates of human yellow ligament [14]. In this method, samples should be fresh and be stored in a deep
0003-2697/03/$ - see front matter Ó 2003 Elsevier Science (USA). All rights reserved. doi:10.1016/S0003-2697(03)00194-5
M. Abe et al. / Analytical Biochemistry 318 (2003) 118–123
freezer for the measurement of crosslinks. When the method is used this way, however, the number and amount of samples are limited, because samples are usually obtained only during autopsy or surgery. If we can use formalin-fixed tissue samples for a crosslink assay, the number and amount of samples could be increased. The aim of this study was to detect crosslinks of collagen and elastin in formalin-fixed tissue, to perform quantification of these crosslinks, and to investigate the effects of formalin fixation on crosslink contents in human yellow ligament and cartilage.
Materials and methods Specimens and preparation of samples Ten specimens of yellow ligament and 12 specimens of cartilage from hip joints were obtained during surgery performed at our department. All procedures were in accordance with the principles of the Declaration of Helsinki, 1975 (revised 1996). Each specimen of yellow ligament was separated into four pieces, and one of them was stored at )80 °C in a deep freezer while the remaining three pieces were preserved in formalin solutions (10% buffered, pH 7) for 1, 3, or 6 months. Each cartilage specimen was separated into two pieces, one of which was stored at )80 °C in a deep freezer and the other of which was preserved in formalin solutions for 1 year. For the assay, each sample was freeze-dried under vacuum, and samples of approximately 7 mg dry weight were hydrolyzed in 1.5 ml 6 N HCl at 110 °C for 24 h in screw-top hydrolysis glass tubes (Iwaki-Glass, Japan). The hydrolysates were divided and prepared separately for crosslink prefractionation. All crosslinks in the hydrolysate (100 ll) were eluted using an SP-Sephadex column (Hþ form, 0:8 1:0 cm; Pharmacia Fine Chemical AB, Uppsala, Sweden), by the previously described method, with some modifications [14]. The eluate was evaporated under vacuum, and the residue was dissolved in 100 ll of water for analysis of Pyr and Pen, and in 50 ll of water for HPLC analysis of Des and Isodes. For the analysis of Des and Isodes, the same prefractionation was done twice, and 50 ll of each obtained solution were mixed.
119
An ODS reversed-phase column (8 mm 10 cm) prepacked with Radial-Pak C18 of 10 lm particle size, Type 8C1810 l (Waters Associates, Inc.), was used. Two mobile phases of a mixture of 20 mM phosphate buffer containing 0.2% SDS (final pH 3.5) and MeCN (19:1, v/v) and the same mixture but containing SDS and MeCN in the proportions of 3:2 (v/v) were used. Chromatographic analysis was performed through binary gradient elution. For analytical and columncleaning purposes, flow conditions were programmed on the system controller. The flow rate was 1.0 ml/min at room temperature. We injected 10-ll samples to measure Pyr and Pen and 60-ll samples to measure Des and Isodes. The greater injection volume for measurement of Des and Isodes was needed because there were some samples from formalin-fixed yellow ligament in which they were not detected even when the sample volume was suitable for the measurement of Pyr and Pen by HPLC. For the detection of Pyr and Pen, we measured the fluorescence at 397 nm with excitation at 305 nm, and for Des and Isodes we measured UV absorbance at 275 nm. Using our protocol, the recoveries of standards using SP-Sephadex C-25 (n ¼ 8) were 86.4–98.3% for Pyr, 78.7–95.6% for Pen, 83.6–97.9% for Des, and 85.6– 99.3% for Isodes [14]. The hydroxyproline content was measured through an automated amino acid analyzer system (Model 835–50; Hitachi, Tokyo, Japan) using an aliquot of the hydrolysate of the samples. The concentrations of crosslinks in each yellow ligament and cartilage sample were expressed as millimoles per mole of hydroxyproline for Pyr and Pen and for Des and Isodes, and we compared the concentrations between the frozen and the formalin-fixed samples. Preparation of standard crosslinks Pyr was isolated from a human cortical bone (a 13year-old tibiae). The purification methods we used were described previously [15]. Pen was synthesized [16], and its concentration was calibrated with authentic Pen, which was a gift from Dr. V.M. Monnier (Institute of Pathology, School of Medicine, Case Western Reserve University, Cleveland, OH, USA). Standard Des and Isodes were purchased from Elastin Products (St. Louis, MO, USA).
HPLC analysis Statistical analysis The measurement of crosslinks was performed using the method described previously [14]. Briefly, the HPLC system consisted of a Model DP-8020 pump (TOSOH, Tokyo, Japan), a Waters Model 474 scanning fluorescence detector (Waters Associates, Inc., Milford, MA, USA), a model PD-8020 variable-wavelength detector (TOSOH), a Model AS-8010 autosampler (TOSOH), and a Model PX-8020 system controller (TOSOH).
The statistical significance of difference between two groups was determined using the Mann–Whitney U test. The correlation between the measurements and the parameters was determined using the Spearmann rank correlation test. Analysis was performed using StatView J 5.0 on a Macintosh computer. p values of less than 0.05 were considered statistically significant.
120
M. Abe et al. / Analytical Biochemistry 318 (2003) 118–123
Results The typical HPLC chromatograms of the hydrolysate of the frozen and formalin-fixed yellow ligament stored for 1, 3, and 6 months obtained from a 71-year-old man and those of the frozen and formalin-fixed cartilage stored for 1 year obtained from an 85-year-old woman are shown in Figs. 1–3. The chromatographic patterns of Pyr and Pen in the hydrolysate of the formalin-fixed yellow ligament and cartilage were similar to those of the frozen yellow ligament and cartilage at every time point (Figs. 1 and 2). We also detected chromatographic peaks of Des and Isodes in the hydrolysate of the formalin-fixed yellow ligament, and we found an additional peak between the peaks of Des and Isodes in the formalin-fixed yellow ligament that was not detected in the frozen yellow ligament (Fig. 3). Neither Des nor Isodes was detected in the hydrolysate of cartilage. We compared the concentrations of Pyr, Pen, Des, and Isodes in the formalin-fixed tissues with those in the frozen tissues (Table 1). We found that the mean proportions of Pyr and Pen in the yellow ligament formalin-fixed for 1, 3, and 6 months compared to the proportion in the frozen samples were 111 and 87%, 106 and 98%, and 123 and 100%, respectively. The mean proportion of Pyr and Pen in the cartilage formalin-fixed for 1 year compared to the proportion in the frozen samples were 102 and 114%, respectively, and the mean proportion of Des and Isodes in the yellow ligament formalin-fixed for 1, 3, and 6 months compared to the
proportion in the frozen samples were 48 and 46%, 46 and 47%, and 55 and 52%, respectively. These results suggest a significant decrease in Des and Isodes in the formalinfixed yellow ligament (Table 1). Table 2 shows the correlations of Pyr, Pen, Des, and Isodes between the formalin-fixed tissues and the frozen tissues. There was a significant correlation of Pyr and Pen between the yellow ligaments that were formalin-fixed for 1, 3, and 6 months and the frozen yellow ligaments and between the cartilages that were formalin-fixed for 1 year and the frozen cartilages. On the other hand, there was no correlation of Des or Isodes between the yellow ligaments that were formalin-fixed for 1 month and the frozen yellow ligaments (Des, r ¼ 0:399, p ¼ 0:1222, and Isodes, r ¼ 0:473, p ¼ 0:1967, N ¼ 10). However, there was a significant correlation of Des and Isodes between the yellow ligaments that were formalin-fixed for 3 and 6 months and the frozen yellow ligaments (formalin-fixed for 3 months: Des, r ¼ 0:629, p ¼ 0:0475; Isodes, r ¼ 0:757, p ¼ 0:0365, N ¼ 10; formalin-fixed for 6 months: Des, r ¼ 0:806, p ¼ 0:0141; Isodes, r ¼ 0:916, p ¼ 0:0048, N ¼ 10), and the correlation between the yellow ligament formalinfixed for 6 months and the frozen tissue was the highest.
Discussion Formalin is a highly reactive chemical and electrophilic species that reacts with various crosslinking
Fig. 1. HPLC chromatograms of the hydrolysate of frozen yellow ligament and yellow ligament formalin-fixed for 1, 3, and 6 months. Spectrofluorometer, emission/excitation wavelengths were 397/305 nm. Closed arrow, Pyr; open arrow, Pen.
M. Abe et al. / Analytical Biochemistry 318 (2003) 118–123
121
Fig. 2. HPLC chromatograms of the hydrolysate of frozen cartilage and cartilage formalin-fixed for 1 year. Spectrofluorometer, emission/excitation wavelengths were 397/305 nm. Closed arrow, Pyr; open arrow, Pen.
Fig. 3. HPLC chromatograms of the hydrolysate of frozen yellow ligament and yellow ligament formalin-fixed for 1, 3, and 6 months. UV detector, absorbance at 275 nm. Closed arrow, Des; open arrow, Isodes; closed triangle, another peak.
functional groups in tissue proteins, nucleic acid, and polysaccharides [17], and it makes methylene crosslinks that are stable and irreversible. Formalin-fixed tissues do not become hardened, and shrinkage is minimized [17]. When collagen and elastin structures in formalinfixed specimens were observed histochemically, researchers found that formalin-fixed elastin retained its elasticity [18], and therefore, it is assumed that collagen and elastin are retained in formalin. However, because formalin has the crosslinking function, it may mask, alter, or destroy natural crosslinks of collagen and elastin. Commonly, crosslinks of collagen and elastin are measured in the hydrolysate of fresh or frozen
samples using HPLC. If it is possible to evaluate crosslinks of collagen and elastin in formalin-fixed tissues, we can investigate crosslinks in a wide variety tissues and tissues of a specific disease stored in the past. Pyr is a stable mature crosslink of collagen, and it cannot be reduced by sodium borohydride. This crosslink plays an important role in maintaining the structure and function of collagen. On the other hand, the glycated proteins undergo many nonenzymatical reactions and result in advanced glycation end products. Pen is one such advanced glycation end product. In this study, Pyr and Pen were detected in both the formalin-fixed yellow ligament and cartilage. Concentrations of Pyr
122
M. Abe et al. / Analytical Biochemistry 318 (2003) 118–123
Table 1 Concentrations of Pyr, Pen, Des, and Isodes in formalin-fixed vs frozen tissues Yellow ligament
Cartilage Formalin-fixed
Frozen Pyr/Hypro (mmol/mol) Pen/Hypro (mmol/mol) Des/Hypro (mmol/mol) Isodes/Hypro (mmol/mol)
1 month
0:81 0:35 0:10 0:07 9:50 6:19 14:90 9:83
Formalin-fixed 3 months
0:88 0:35 0:08 0:04 4:45 2:98 6:43 4:43 111:2 22:7 87:2 18:3 48:4 21:2 45:6 24:4
Pyr/Hypro (% to frozen) Pen/Hypro (% to frozen) Des/Hypro (% to frozen) Isodes/Hypro (% to frozen)
6 months
0:81 0:29 0:09 0:03 4:69 4:25 7:80 7:80 106:0 29:0 97:7 20:9 46:2 28:4 46:6 30:4
0:96 0:34 0:09 0:06 5:19 3:23 7:80 4:83
Frozen
1 year
2:61 0:79 0:21 0:07
2:59 0:72 0:24 0:07
123:1 22:6 100:1 19:8 55:0 22:0 52:1 20:0
102:3 23:5 114:1 23:2
The mean concentrations SD of Pyr, Pen, Des, and Isodes in frozen and formalin-fixed yellow ligament and cartilage and the mean proportions SD of Pyr, Pen, Des, and Isodes in each period formalin-fixed yellow ligament to those in frozen tissue and the mean proportions SD of Pyr and Pen in cartilage formalin-fixed for 1 year to those in frozen tissue. Measured values of Pyr, Pen, Des, and Isodes were all normalized to measured values of hydroxyproline (Hypro). * p < 0:05 vs frozen.
Table 2 Correlations of Pyr, Pen, Des, and Isodes in each period formalin-fixed ligament and cartilage compared with frozen tissues Pyr
Pen
Des
Isodes
r
p
r
p
r
p
r
p
Yellow ligament Frozen vs formalin-fixed 1 month Frozen vs formalin-fixed 3 months Frozen vs formalin-fixed 6 months
0.780 0.711 0.852
0.0056 0.0187 0.0008
0.872 0.889 0.905
0.0004 0.0002 <0.0001
0.399 0.629 0.806
0.1222 0.0475 0.0141
0.473 0.757 0.916
0.1967 0.0365 0.0048
Cartilage Frozen vs formalin-fixed 1 year
0.575
0.0382
0.658
0.0125
Spearmann rank correlation test.
and Pen in the yellow ligament and cartilage were not significantly affected or related to the duration of formalin fixation, and there was a significant correlation between the concentrations of crosslinks in frozen yellow ligament and cartilage and those in formalin-fixed tissues. These results indicated that natural crosslinks of collagen were preserved in formalin, and formalin did not mask, alter, or destroy Pyr and Pen, so formalinfixed tissues can be used for investigating the contents of Pyr and Pen. Des and Isodes are crosslinking amino acids specifically located in elastin, and their concentration in some tissues can reflect the elastin contents [12]. In the present study, Des and Isodes were detected in the formalin-fixed yellow ligament, but the concentrations were significantly decreased compared with the concentrations found in frozen samples. Therefore, we concluded that crosslinks of elastin were not preserved in formalin. The reason for this might be that formalin did not fix elastin tissues sufficiently or it destroyed, masked, or altered crosslinks. We were not able to clarify the reason in this study. It has been reported that elastin was not significantly fixed by formalin, because its elastic recovery rate after stretching was only 50–70% [18]. This indicates that formalin cannot fix elastin
completely or that elastin is destroyed during formalin fixation. Although the mean concentrations of Des and Isodes decreased in the yellow ligament formalin-fixed for 1 month, they did not show further reduction in the ligaments fixed for 3 and 6 months. There was a significant correlation of Des and Isodes between the frozen yellow ligament and the yellow ligament formalin-fixed for 3 and 6 months. The reduced elastin crosslinks might be stable in formalin for a long period. An unidentified peak was found between the peaks of Des and Isodes on the chromatograms of only the formalin-fixed yellow ligaments. The appearance of an additional peak in formalin-fixed samples might indicate that some of the crosslinks of elastin, possibly Des or Isodes, were modified by formalin. The decrease in Des and Isodes might indicate that some amount of them was modified into another crosslinks or was broken by formalin fixation. This result showed that the longitudinal investigation of the contents of Des or Isodes in formalin-fixed tissues was not adequate and that the concentration of Des or Isodes in the formalin-fixed tissues did not correspond to that in frozen or fresh samples. However, measurement of the concentration of Des or Isodes in
M. Abe et al. / Analytical Biochemistry 318 (2003) 118–123
formalin-fixed tissues is useful when a comparison is made among formalin-fixed tissues.
Acknowledgment We thank Ms. Ayako Fujiwara for her technical help.
References [1] P. Garnero, J. Rousseau, P. Delmas, Molecular basis and clinical use of biochemical markers of bone, cartilage, and synovium in joint diseases, Arthritis Rheum. 43 (2000) 953–968. [2] S. Sakata, M. Takahashi, K. Kushida, M. Oikawa, H. Hoshino, M. Denda, T. Inoue, The relationship between pentosidine and hemodialysis-related connective tissue disorders, Nephron 78 (1998) 260–265. [3] M. Takahashi, K. Kushida, H. Hoshino, M. Suzuki, M. Sano, S. Miyamoto, T. Inoue, Concentrations of pyridinoline and deoxypyridinoline in joint tissues from patients with osteoarthritis or rheumatoid arthritis, Ann. Rheum. Dis. 55 (1996) 324–327. [4] P.J. Stone, Potential use of collagen and elastin degradation markers for monitoring liver fibrosis in schistosomiasis, Acta Tropica 77 (2000) 97–99. [5] D.C. Bode, E.D. Pagani, W.R. Cumiskey, R. von Roemeling, L. Hamel, P.J. Silver, Comparison of urinary desmosine excretion in patients with chronic obstructive pulmonary disease or cystic fibrosis, Pulm. Pharmacol. Ther. 13 (2000) 175–180. [6] D. Eyre, J. Wu, P. Woops, The cartilage collagens: structural and metabolic studies, J. Rheumatol. 18 (Suppl. 27) (1991) 49–51. [7] D. Eyre, H. Oguchi, The hydroxypyridinium crosslink of skeletal collagens: their measurement, properties and a proposed pathway of formation, Biochem. Biophys. Res. Commun. 92 (1980) 403–410.
123
[8] D.R. Sell, V.M. Monnier, Sructure elucidation of a senescence cross-links from human extracellular matrix, J. Biol. Chem. 264 (1989) 21597–21602. [9] V.M. Monnier, D.R. Shell, R. Nagaraj, S. Miyata, S. Grandhee, P. Odetti, S. Ibrahim, Maillard reaction-mediated molecular damage to extracellular matrix and other tissue proteins in diabetes, aging, and uremia, Diabetes 41 (Suppl. 2) (1992) 36–41. [10] J. Thomas, D.F. Elsden, S.M. Partridge, Degradation products from elastin: partial structure of two major degradation products from the cross-linkages in elastin, Nature 200 (1963) 651–652. [11] B.C. Starcher, Determination of the elastin content of tissues by measuring desmosine and isodesmosine, Anal. Biochem. 79 (1977) 11–15. [12] Y. Yamaguchi, J. Haginaka, M. Kunitomo, H. Yasuda, Y. Bando, High-performance liquid chromatographic determination of desmosine and isodesmosine in tissues and its application to studies of alteration of elastin induced by atherosclerosis, J. Chromatogr. 422 (1987) 53–59. [13] M. Watanabe, T. Sawai, H. Nagura, K. Suyama, Age-related alteration of cross-linking amino acids of elastin in human aorta. Tohoku, J. Exp. Med. 180 (1996) 115–130. [14] J.R. Chen, M. Takahashi, K. Kushida, M. Suzuki, K. Suzuki, K. Horiuchi, A. Nagano, Direct detection of crosslinks of collagen and elastin in the hydrolysates of human yellow ligament using single-column high performance liquid chromatography, Anal. Biochem. 278 (2000) 99–105. [15] D. Fujimoto, T. Moriguchi, Pyridinoline, a non-reducible crosslink of collagen Quantitative determination, distribution, and isolation of a crosslinked peptide, J. Biochem. 83 (1978) 863–867. [16] S.K. Grandhee, V.M. Monnier, Mechanism of formation of the maillard protein cross-link pentosidine, J. Biol. Chem. 266 (1991) 11649–11653. [17] C.H. Fox, F.B. Johnson, J. Whiting, P.P. Roller, Formaldehyde fixation, J. Histochem. Cytochem. 33 (1985) 845–853. [18] Y.C. Fung, S.S. Sobin, The retained elasticity of elastin under fixation agents, J. Biol. Eng. 103 (1981) 121–122.