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Solubility of tissue hydroxyproline in experimental intestinal anastomoses
EXPERIMENTAL
AND
MOLECULAR
Solubility TH. HENDRIKS,
PATHOLOGY
43, 253-259 ( 1985)
of Tissue Hydroxyproline in Experimental Intestinal Anastomoses W. L. E. M. HESP, A. A. KLOMPMAKERS, E. J. C. LUBBERS, AND H. H. M. DE BOER
Department
of General
Surgery.
Received
March
St. Radborrd
University
15. 1985, and in revised
Hospital, form
Nijmegen, Jane
The Netherlands
7. 1985
Salt and acid solubility of collagen are thought to reflect its degree of crosslinking. In order to examine postoperative changes in crosslinking of intestinal collagen, which are of importance to the stability of the intestinal wall, we have investigated the solubility of hydroxyproline in rabbit ileum and colon, both in unwounded intestine and after construction of an anastomosis. Solubility in salt and dilute acid was increased by a sonication procedure. This way, 9% of total hydroxyproline in the unwounded colonic wall was salt soluble and 65% acid soluble. A similar distribution was observed in ileum. Three days after operation the salt-soluble fraction was significantly elevated in samples from the anastomotic area. In colon, this increase also persisted 7 days postoperatively. Comparison of anastomotic samples collected 3 and 7 days after surgery shows a significant decrease in the acid-soluble and a significant increase in the solid fraction. This phenomenon occurs both in ileum and colon. These results indicate that during the first days after operation the integrity of the intestinal wall is weakened not only by a loss of collagen. but also by a changed solubility of the remaining collagen. k 19x5 Academic PXSS. IK.
INTRODUCTION Anastomotic leakage is frequently seen after surgery of the large bowel. Since this complication is attended by high morbidity and mortality (Schrock et al., 1973) it is important to study the mechanisms involved in order to try to diminish its incidence rate. The structural integrity of the intestinal wall depends heavily on its connective tissue, particularly the submucosa (Fogdestam and Gottrup, 1980), of which collagen is the predominant constituent. Therefore, changes in collagen metabolism may be expected to affect the stability of the intestinal wall. It has been known for some time that the construction of experimental colonic anastomosis leads to a massive degradation of intestinal collagen, which is reflected by a decreased hydroxyproline concentration during the first days after surgery (Cronin of al., 1968; Jiborn et al., 1980). We have shown recently that a similar process occurs after ileal anastomosis in the rabbit (Hesp et al., 1984). Supposedly, old collagen is broken down and replaced by new collagen, which initially lacks the supportive strength of the original material, thus leading to a temporarily weakened intestinal segment around the anastomosis. This hypothesis is based upon examination of total hydroxyproline levels. Since the structure of old and new collagen may be expected to differ, particularly with respect to crosslinking (Tanzer, 1976; Bentley, 1979), further support might be obtained from investigation of the chemical behavior of the collagen fibers. As a first measure, we have investigated the salt and acid solubility of intestinal hydroxyproline before and after anastomosis.
Fifty-six
MATERIALS AND METHODS male New Zealand White rabbits with an average weight of 2351 ?
OOl4-480018.5 $3.00 Copyright 0 1985 by Academic Press, Inc. All rights of reproduction in any form reserved.
254
HENDRIKS
ET AL.
274 g (SD, II = 55) were used. The animals were divided into two groups, which received either an ileal or a colonic anastomosis. In both groups, 14 rabbits each were sacrificed on Day 3 and Day 7 after operation. Surgical procedures were performed in sterile conditions and have been published elsewhere (Hesp et al., 1984). Ileal anastomoses were constructed 10 cm proximal to the appendix tip and colonic anastomoses 3-5 cm above the Douglas pouch. During operation a 5-cm long segment was removed for determination of various parameters of unwounded intestine. Postoperatively, the animals were fed ad libitum. At the appropriate time they were sacrificed by means of an overdose of Pentothal and the intestinal segment containing the anastomosis (1 cm) was collected. Thus, in each animal, hydroxyproline levels in the anastomotic segment could be compared with those in control segments, collected at operation. Samples were frozen and stored in liquid N, immediately after collection. Subsequently, samples were pulverized in a Braun microdismembrator, lyophilized, and stored at -30°C. Solubilization of tissue hydroxyproline was effected by means of a sonication procedure. A lo-mg lyophilized sample was suspended (in a Kontes tissue grinder, glass on glass) in 1.5 ml 0.15 M NaCl and sonicated (150 W, amplitude 20km, in a MSE Soniprep 150) in ice for 5 x 1 min with lmin intervals. The salt-soluble fraction was collected by centrifugation for 20 min at 20,OOOg. The residue was resuspended in 1.5 ml 0.5 M HAc and sonicated the same way. The insoluble residue was separated from the acid-soluble fraction by centrifugation for 60 min at 20,OOOg. Samples were kept at 4°C during the entire procedure. Hydroxyproline was measured in the starting material (total hydroxyproline) and in both salt- and acid-soluble fractions. Hydroxyproline in the insoluble fraction was calculated by subtracting the amounts found in the latter fractions from that measured in the original lyophilized sample. Hydroxyproline was measured (in duplicate) essentially according to Prockop and Udenfriend (1960), using a hydroxyproline standard from Calbiochem (USA). For the determination of total hydroxyproline, lyophilized tissue (2-5 mg) was hydrolyzed in 1.5 ml 6 N HCl. The salt- and acid-soluble fractions were made up to 750 ~1 with water and hydrolyzed overnight after addition of 750 ~1 12 N HCI. Further procedure has been described elsewhere (Hesp et al., 1984). Protein was assayed using bovine serum albumin (Type V, Sigma) as a standard (Lowry et al., 1951). Methods used for statistical evaluation of the data are given together with the results. RESULTS Total hydroxyproline levels in the unwounded rabbit colonic wall were clearly higher than those in the ileal wall: average values (n = 28) 12.39 and 8.82 p.g/mg dry wt, respectively. Although the exact composition of intestinal collagen remains to be determined, the approximate collagen concentration may be calculated by multiplying by a factor 6.94 (Jackson and Clearly, 1967). This means that the collagen concentration approximated 61 and 86 Fg/rng dry wt in ileum and colon, respectively. Since average protein concentrations were 668 p,g and 698 pg/mg dry wt, 9.2% of protein in the ileal wall was collagen, while in the colonic wall collagen constituted 12.3% of total protein. Extraction of lyophilized samples with 0.15 M NaCl or 0.5 M HAc solubilized only a minor part of total hydroxyproline: 3 and I%, respectively. However, sonication greatly enhanced the amount of hydroxyproline which remained in
HYDROXYPROLINE
SOLUBILITY
AFTER
ANASTOMOSIS
255
80-
0
2
4 Time
6 8 ol smlcation
10 (min
1
FIG. 1. Effect of time of sonication on the solubility of intestinal wall hydroxyproline. Lyophilized samples of intestine from three different rabbits were sonicated for an increasing number of I-min periods-with I-min intervals-in successively 0.15 M NaCl (open symbols) and 0.5 M HAc (closed symbols).
solution after subsequent centrifugation. Figure 1 shows the effect of increased times of sonication on the salt and acid solubility of hydroxyproline in unwounded intestine from three different rabbits. This way, approximately 12% of total hydroxyproline could be solubilized by 0.15 M NaCl and over 80% by 0.5 M HAc. Acid-treated samples were routinely centrifuged at 20,OOOg for 60 min; centrifugation at 80,OOOg for 60 min did not significantly affect the amount of hydroxyproline in the supernatant. From these results, a sonication time of 5 min was chosen for subsequent analysis of hydroxyproline solubility after intestinal anastomosis. If applied to the samples used for Fig. 1, this procedure brought 56% of total protein into the salt-soluble fraction and a further 35% into the acid-soluble fraction. Table I shows the distribution of hydroxyproline over the various fractions in lyophilized tissue from both unwounded ileal and colonic wall. Although the absolute amount of hydroxyproline was clearly higher in colon, the relative distribution over the fractions was similar. Construction of an intestinal anastomosis induces a transient loss of hydroxyproline from the wound area. Three days after operation total hydroxyproline was significantly reduced in ileal anastomoses (Fig. 2). This change was caused mainly by a lowering of acid-soluble material. Seven days postoperatively total hydroxyproline was increased by almost 40% as compared to control values obtained at operation. A similar change was evident in all subfractions. Loss of hydroxyproline from colonic anastomosis was more extensive 3 days after operation (Fig. 3). Here both the salt-soluble and the acid-soluble fractions were significantly reduced also. After 7 days total hydroxyproline was back to the level measured in control segments. While the acid-soluble fraction was still slightly lowered, the salt-soluble fraction was significantly increased.
HENDRIKS
256
Solubility of Hydroxyproline
ET AL.
TABLE I in the Unwounded Intestinal Wall” Hydroxyproline @g/mg dry weight) Average
Ileum Total Salt soluble Acid soluble Insoluble
8.82 0.75 5.68 2.39
?z 0.19 2 0.03 ” 0.22 e 0.21
Colon Total Salt soluble Acid soluble Insoluble
12.39 1.18 8.38 3.37
-t t f f
Range 7.09-11.21 0.40-0.98 3.52-8.12 0.50-4.69
0.31 0.04 0.26 0.27
10.23-15.66 0.86-1.62 5.35-11.71 0.68-5.89
M
M
0 Lyophilized tissue was sonicated (5 min) in successively 0.15 NaCl and 0.5 HAc and hydroxyproline was measured in both supernatants. Results are expressed as average value and SEM (n = 28).
60
total
-20
salt
SOlUbk
acid
soluMe
insoluble
FIG. 2. Changes in solubility of intestinal wall hydroxyproline after ileal anastomosis. Hydroxyproline was measured in a control segment, removed at operation, and in the anastomotic segment collected 3 or 7 days after operation. Results are expressed as average percentage change after operation, calculated with respect to the control segment, with SEM (n = 14). The absolute (pg/mg dry weight) hydroxyproline levels in the different fractions of the control segments are given in Table I. The level of significance (P value) for the various differences is calculated with a paired-sample Student’s t test and represented in the following way: *O.Ol < P < 0.05, **O.OOl < P G 0.01, ***P s 0.001.
HYDROXYPROLINE
SOLUBILITY
AFTER
I Ik
ANASTOMOSIS
257
insoluble
FIG. 3. Changes in solubility of intestinal wall hydroxyprohne after colonic anastomosis. Hydroxyproline was measured in a control segment, removed at operation, and in the anastomotic segment collected 3 or 7 days after operation. Results are expressed as average percentage change after operation, calculated with respect to the control segment, with SEM (n = 14). The absolute (&mg dry weight) hydroxyproline levels in the different fractions of the control segments are given in Table I. The level of significance (P value) for the various differences is calculated with a paired-sample Student’s t test and represented in the following way: *O.Ol < P < 0.05, **O.OOl < P G 0.01, ***P 6 0.001.
Thus, the composition of total intestinal hydroxyproline changed considerably after anastomosis (Table II). In ileum, after 3 days, the salt-soluble fraction was significantly elevated. If the composition at 7 days after operation was compared with that 3 days after operation, both salt-soluble and acid-soluble fractions were significantly reduced and the insoluble fraction was significantly enhanced. Comparing the distribution measured at 7 days with that of unwounded tissue, the acid-soluble part appeared decreased and the insoluble fraction increased, though not significantly so. In colon, significant changes were apparent after 3 days both in salt-soluble and acid-soluble fractions, which were higher and lower, respectively, if compared to levels measured in control segments. Seven days after operation, the salt-soluble fraction remained significantly elevated with respect to the preoperative value. The acid-soluble fraction was significantly reduced, with respect to both control value and value measured 3 days postoperatively. Finally, the insoluble fraction was significantly elevated only if compared to the 3-day value. DISCUSSION The ability to form fibers with a high structural stability enables collagen to act as the major supporting framework in the intestinal wall. Covalent crosslinks between collagen molecules confer on the fibers the high tensile strength and resistance to chemical attack necessary for their function. A number of different crosslinks has been characterized for some time (Tanzer, 1976). Within one type of tissue variations may occur, for instance as the result of aging (Bentley, 1979) or a disease, either heritable or acquired (Kivirikko and Risteli, 1976).
258 Percentage
HENDRIKS
Distribution
of Hydroxyproline in the Anastomosis
TABLE II over the Various Fractions in Unwounded 3 and 7 Days after Operation”
Total (pg/mg dry weight) Ileum Unwounded tissue (n = 28) Anastomosis 3 days after operation (n = 14) Anastomosis 7 days after operation (n = 14) Colon Unwounded tissue (n = 28) Anastomosis 3 days after operation (n = 14) Anastomosis 7 days after operation (n = 14)
ET AL.
% Salt soluble
Intestine
and
% Acid soluble
% Insoluble
8.82 _f 0.19
8.5 _t 0.3
64.3 i 2.1
27.2 + 2.3
8.20 + 0.25**
10.3 2 0.4*
68.9 t 3.4
20.8 + 3.6
11.85 + 0.21***,tt+
8.0 2 0.3ttt
56.8 -c 2.2t:
35.2 i 2.2tt
12.35 z 0.31
9.2 f 0.3
65.1 -c 1.8
25.7 -t 1.9
9.39 t 0.35***
11.2 L 0.6**
62.2 t 2.2*
26.6 + 2.1
12.09 k 0.39ttt
11.5 k 0.8**
55.8 2 1.4*+
32.7 k 1.8
a LyophiIized tissue was sonicated (5 min) in successively 0.15 M NaC1 and 0.5 M HAc and hydroxyproline was measured in both supernatants. Absolute total hydroxyproline levels are given together with percentage distribution over the 3 subfractions (average with SEM). Results after operation are compared with those obtained preoperatively in the same rabbits and differences are tested for significance with a paired-sample Student’s t test (*). Differences between results obtained at 3 and 7 days postoperatively in different groups of rabbits are tested for significance by means of a f test for two random tests (t). * 0.01 < P c 0.05.
** 0.001 < P c 0.01. ***p c 0.001. t 0.01 S P 0.05. tt 0.001 S P 0.01. tttp s 0.001.
Intestinal anastomosis leads to a massive breakdown of collagen, measured as lowered hydroxyproline concentrations, both in ileum (Hesp et al., 1984) and in colon (Jiborn et al., 1980). This transient state of decreased collagen content could be a major contributing factor to the incidence of anastomotic leakage, particularly in the colon where this effect is more extensive than in the ileum (Hesp et al., 1984). However, next to the absolute amount of collagen present, its quality, in terms of number and degree of crosslinking, may also be important for determining the efficiency of its structural support. One way to investigate the degree of crosslinking, and thus presumably the stability, of collagen is by means of its solubility. Neutral salt solutions will solubilize only the least crosslinked molecules present (e.g., young collagen), while dilute acetic acid extracts the more crosslinked collagen molecules. Neither of these solvents would be expected to solubilize highly crosslinked (mature) collagen. The solubility of hydroxyproline in the intestinal wall is very low. Salt- and acid-soluble hydroxyproline constitutes only 0.4 and 4%, respectively, of total tissue hydroxyproline from rat intestine (Irvin and Hunt, 1974; Jiborn et al., 1980). These values are similar to those observed by us if lyophilized rabbit intestine was treated in the absence of sonication. Sampling of the proper anastomotic healing area severely restricts the amount of tissue available for analysis. If only such minor percentages of total hydroxyproline are solubilized, analysis of relatively small differences between samples is rendered impossible, because of biological variation and the intrinsic error of the hydroxyproline assay. Therefore, we have investigated various means to increase the solubility of hydroxyproline in the intestinal wall. Sonication of
HYDROXYPROLINE
SOLUBILITY
AFTER ANASTOMOSIS
259
lyophilized tissue proved to be the most efficient method of enhancing salt and acid solubility (Fig. 1). This procedure yields a means to distinguish three fractions, which contain enough hydroxyproline for assay in tissue samples from the same rabbit. In the sonication-solubilized fractions collagen is present not in the monomeric form but rather as aggregates which presumably still contain part of the crosslinks. Pepsin treatment of the sonicated material and subsequent electrophoresis in sodium dodecyl sulfate on polyacrylamide shows the presence of normal (Y, (I), CQ (I), and (Y, (III) chains (results not shown). The distribution of intestinal hydroxyproline over the three fractions is clearly affected by the construction of ar. anastomosis (Table II). Both in ileum and colon, the salt-soluble fraction is increased 3 days after operation. In colon, this effect is still significant after 7 days. Also, the acid-soluble fraction declines and the insoluble fraction increases from 3 to 7 days postoperatively. These results indicate that, next to the absolute concentration of hydroxyproline, its relative distribution over the various fractions also changes in the anastomotic area. During the first days after operation the integrity of the intestinal wall is weakened not only by a loss of collagen, but also by the increased salt solubility of the remaining collagen. The differences observed between the composition at 3 and 7 days postoperatively, indicate a shift from the acid-soluble to the insoluble fraction. Thus, 7 days after operation, when the original hydroxyproline concentration has been restored (colon) or even surpassed (ileum), its solubility has diminished again. Presumably, the chances for disturbance of the integrity of the connective tissue layer and subsequent anastomotic dehiscence have been reduced by then. ACKNOWLEDGMENTS The authors gratefully acknowledge Mr. P. Thissen for expert technical assistance and Dr. Ph. van Elteren (Department of Statistical Support, University of Nijmegen) for statistical analysis of the experimental data.
REFERENCES BENTLEY. J. P. (1979). Aging of collagen. J. Znvesr. Dermufol. 73, 80-83. CRONIN, K., JACKSON, D. S. and DUNPHY, J. E. (1968). Changing bursting strength and collagen content of the healing colon. Surg. Gynecol. Obster. 126, 747-753. FOGDESTAM, I. and GOTTRUP, F. (1980). Biomechanical methods in wound-healing research with special reference to ski? and gastrointestinal tract. In “Biology of Collagen” (A. Viidik and J. Vuust, eds.), Academic Press. pp 363-371. New York/London. HESP, F. L. E. M., HENDRIKS. T., LUBBERS, E. J. C., and DE BOER, H. H. M. (1984). Wound healing in the intestinal wall. A comparison between experimental ileal and colonic anastomoses. Dis. Colon Rectum 21, 99-104. IRVIN, T. T.. and HUNT. T. K. (1974). Reappraisal of the hdaling process of anastomosis of the colon. Sup. Gynecol. Obstet. 138, 741-746. JACKSON, D. S., and CLEARY, E. G. (1967). The determination of collagen and elastin. In “Methods of Biochemical Analysis” (D. Glick. ed.), Vol. XV, pp. 25-76. John Wiley & Sons, New York. JIBORN, H., AHONEN. J., and ZEDERFELDT, B. (1980). Healing of experimental colonic anastomoses. III. Collagen metabolism in the colon after left colon resection. Amer. J. Surg. 139, 398-405. KIVIRIKKO, K. I., and RISTELI, L. (1976). Biosynthesis of collagen and its alterations in pathological states. Med. Biol. 54, 159-186. LOWRY, 0. H., ROSEBROUGH,N. J., FARR, A. L.. and RANDALL, R. J. (1951). Protein measurement with the folin phenol reagent. J. Biol. Ctzem. 193, 265-275. PROCKOP, D. J. and UDENFRIEND, S. (1960). A specific method for the analysis of hydroxyproline in tissues and urine. Anal. Biochem. 1, 228-239. SCHROCK. T. R., DEVENEY. C. W., and DUNPHY. J. E. (1973). Factors contributing to leakage of colonic anastomosis. Ann. Surg. 177, 513-518. TANZER, M. L. (1976). Cross-linking. In “Biochemistry of Collagen” (G. N. Ramachandran and A. H. Reddy, eds.), pp. 137-162. Plenum, New York.
AND
MOLECULAR
Solubility TH. HENDRIKS,
PATHOLOGY
43, 253-259 ( 1985)
of Tissue Hydroxyproline in Experimental Intestinal Anastomoses W. L. E. M. HESP, A. A. KLOMPMAKERS, E. J. C. LUBBERS, AND H. H. M. DE BOER
Department
of General
Surgery.
Received
March
St. Radborrd
University
15. 1985, and in revised
Hospital, form
Nijmegen, Jane
The Netherlands
7. 1985
Salt and acid solubility of collagen are thought to reflect its degree of crosslinking. In order to examine postoperative changes in crosslinking of intestinal collagen, which are of importance to the stability of the intestinal wall, we have investigated the solubility of hydroxyproline in rabbit ileum and colon, both in unwounded intestine and after construction of an anastomosis. Solubility in salt and dilute acid was increased by a sonication procedure. This way, 9% of total hydroxyproline in the unwounded colonic wall was salt soluble and 65% acid soluble. A similar distribution was observed in ileum. Three days after operation the salt-soluble fraction was significantly elevated in samples from the anastomotic area. In colon, this increase also persisted 7 days postoperatively. Comparison of anastomotic samples collected 3 and 7 days after surgery shows a significant decrease in the acid-soluble and a significant increase in the solid fraction. This phenomenon occurs both in ileum and colon. These results indicate that during the first days after operation the integrity of the intestinal wall is weakened not only by a loss of collagen. but also by a changed solubility of the remaining collagen. k 19x5 Academic PXSS. IK.
INTRODUCTION Anastomotic leakage is frequently seen after surgery of the large bowel. Since this complication is attended by high morbidity and mortality (Schrock et al., 1973) it is important to study the mechanisms involved in order to try to diminish its incidence rate. The structural integrity of the intestinal wall depends heavily on its connective tissue, particularly the submucosa (Fogdestam and Gottrup, 1980), of which collagen is the predominant constituent. Therefore, changes in collagen metabolism may be expected to affect the stability of the intestinal wall. It has been known for some time that the construction of experimental colonic anastomosis leads to a massive degradation of intestinal collagen, which is reflected by a decreased hydroxyproline concentration during the first days after surgery (Cronin of al., 1968; Jiborn et al., 1980). We have shown recently that a similar process occurs after ileal anastomosis in the rabbit (Hesp et al., 1984). Supposedly, old collagen is broken down and replaced by new collagen, which initially lacks the supportive strength of the original material, thus leading to a temporarily weakened intestinal segment around the anastomosis. This hypothesis is based upon examination of total hydroxyproline levels. Since the structure of old and new collagen may be expected to differ, particularly with respect to crosslinking (Tanzer, 1976; Bentley, 1979), further support might be obtained from investigation of the chemical behavior of the collagen fibers. As a first measure, we have investigated the salt and acid solubility of intestinal hydroxyproline before and after anastomosis.
Fifty-six
MATERIALS AND METHODS male New Zealand White rabbits with an average weight of 2351 ?
OOl4-480018.5 $3.00 Copyright 0 1985 by Academic Press, Inc. All rights of reproduction in any form reserved.
254
HENDRIKS
ET AL.
274 g (SD, II = 55) were used. The animals were divided into two groups, which received either an ileal or a colonic anastomosis. In both groups, 14 rabbits each were sacrificed on Day 3 and Day 7 after operation. Surgical procedures were performed in sterile conditions and have been published elsewhere (Hesp et al., 1984). Ileal anastomoses were constructed 10 cm proximal to the appendix tip and colonic anastomoses 3-5 cm above the Douglas pouch. During operation a 5-cm long segment was removed for determination of various parameters of unwounded intestine. Postoperatively, the animals were fed ad libitum. At the appropriate time they were sacrificed by means of an overdose of Pentothal and the intestinal segment containing the anastomosis (1 cm) was collected. Thus, in each animal, hydroxyproline levels in the anastomotic segment could be compared with those in control segments, collected at operation. Samples were frozen and stored in liquid N, immediately after collection. Subsequently, samples were pulverized in a Braun microdismembrator, lyophilized, and stored at -30°C. Solubilization of tissue hydroxyproline was effected by means of a sonication procedure. A lo-mg lyophilized sample was suspended (in a Kontes tissue grinder, glass on glass) in 1.5 ml 0.15 M NaCl and sonicated (150 W, amplitude 20km, in a MSE Soniprep 150) in ice for 5 x 1 min with lmin intervals. The salt-soluble fraction was collected by centrifugation for 20 min at 20,OOOg. The residue was resuspended in 1.5 ml 0.5 M HAc and sonicated the same way. The insoluble residue was separated from the acid-soluble fraction by centrifugation for 60 min at 20,OOOg. Samples were kept at 4°C during the entire procedure. Hydroxyproline was measured in the starting material (total hydroxyproline) and in both salt- and acid-soluble fractions. Hydroxyproline in the insoluble fraction was calculated by subtracting the amounts found in the latter fractions from that measured in the original lyophilized sample. Hydroxyproline was measured (in duplicate) essentially according to Prockop and Udenfriend (1960), using a hydroxyproline standard from Calbiochem (USA). For the determination of total hydroxyproline, lyophilized tissue (2-5 mg) was hydrolyzed in 1.5 ml 6 N HCl. The salt- and acid-soluble fractions were made up to 750 ~1 with water and hydrolyzed overnight after addition of 750 ~1 12 N HCI. Further procedure has been described elsewhere (Hesp et al., 1984). Protein was assayed using bovine serum albumin (Type V, Sigma) as a standard (Lowry et al., 1951). Methods used for statistical evaluation of the data are given together with the results. RESULTS Total hydroxyproline levels in the unwounded rabbit colonic wall were clearly higher than those in the ileal wall: average values (n = 28) 12.39 and 8.82 p.g/mg dry wt, respectively. Although the exact composition of intestinal collagen remains to be determined, the approximate collagen concentration may be calculated by multiplying by a factor 6.94 (Jackson and Clearly, 1967). This means that the collagen concentration approximated 61 and 86 Fg/rng dry wt in ileum and colon, respectively. Since average protein concentrations were 668 p,g and 698 pg/mg dry wt, 9.2% of protein in the ileal wall was collagen, while in the colonic wall collagen constituted 12.3% of total protein. Extraction of lyophilized samples with 0.15 M NaCl or 0.5 M HAc solubilized only a minor part of total hydroxyproline: 3 and I%, respectively. However, sonication greatly enhanced the amount of hydroxyproline which remained in
HYDROXYPROLINE
SOLUBILITY
AFTER
ANASTOMOSIS
255
80-
0
2
4 Time
6 8 ol smlcation
10 (min
1
FIG. 1. Effect of time of sonication on the solubility of intestinal wall hydroxyproline. Lyophilized samples of intestine from three different rabbits were sonicated for an increasing number of I-min periods-with I-min intervals-in successively 0.15 M NaCl (open symbols) and 0.5 M HAc (closed symbols).
solution after subsequent centrifugation. Figure 1 shows the effect of increased times of sonication on the salt and acid solubility of hydroxyproline in unwounded intestine from three different rabbits. This way, approximately 12% of total hydroxyproline could be solubilized by 0.15 M NaCl and over 80% by 0.5 M HAc. Acid-treated samples were routinely centrifuged at 20,OOOg for 60 min; centrifugation at 80,OOOg for 60 min did not significantly affect the amount of hydroxyproline in the supernatant. From these results, a sonication time of 5 min was chosen for subsequent analysis of hydroxyproline solubility after intestinal anastomosis. If applied to the samples used for Fig. 1, this procedure brought 56% of total protein into the salt-soluble fraction and a further 35% into the acid-soluble fraction. Table I shows the distribution of hydroxyproline over the various fractions in lyophilized tissue from both unwounded ileal and colonic wall. Although the absolute amount of hydroxyproline was clearly higher in colon, the relative distribution over the fractions was similar. Construction of an intestinal anastomosis induces a transient loss of hydroxyproline from the wound area. Three days after operation total hydroxyproline was significantly reduced in ileal anastomoses (Fig. 2). This change was caused mainly by a lowering of acid-soluble material. Seven days postoperatively total hydroxyproline was increased by almost 40% as compared to control values obtained at operation. A similar change was evident in all subfractions. Loss of hydroxyproline from colonic anastomosis was more extensive 3 days after operation (Fig. 3). Here both the salt-soluble and the acid-soluble fractions were significantly reduced also. After 7 days total hydroxyproline was back to the level measured in control segments. While the acid-soluble fraction was still slightly lowered, the salt-soluble fraction was significantly increased.
HENDRIKS
256
Solubility of Hydroxyproline
ET AL.
TABLE I in the Unwounded Intestinal Wall” Hydroxyproline @g/mg dry weight) Average
Ileum Total Salt soluble Acid soluble Insoluble
8.82 0.75 5.68 2.39
?z 0.19 2 0.03 ” 0.22 e 0.21
Colon Total Salt soluble Acid soluble Insoluble
12.39 1.18 8.38 3.37
-t t f f
Range 7.09-11.21 0.40-0.98 3.52-8.12 0.50-4.69
0.31 0.04 0.26 0.27
10.23-15.66 0.86-1.62 5.35-11.71 0.68-5.89
M
M
0 Lyophilized tissue was sonicated (5 min) in successively 0.15 NaCl and 0.5 HAc and hydroxyproline was measured in both supernatants. Results are expressed as average value and SEM (n = 28).
60
total
-20
salt
SOlUbk
acid
soluMe
insoluble
FIG. 2. Changes in solubility of intestinal wall hydroxyproline after ileal anastomosis. Hydroxyproline was measured in a control segment, removed at operation, and in the anastomotic segment collected 3 or 7 days after operation. Results are expressed as average percentage change after operation, calculated with respect to the control segment, with SEM (n = 14). The absolute (pg/mg dry weight) hydroxyproline levels in the different fractions of the control segments are given in Table I. The level of significance (P value) for the various differences is calculated with a paired-sample Student’s t test and represented in the following way: *O.Ol < P < 0.05, **O.OOl < P G 0.01, ***P s 0.001.
HYDROXYPROLINE
SOLUBILITY
AFTER
I Ik
ANASTOMOSIS
257
insoluble
FIG. 3. Changes in solubility of intestinal wall hydroxyprohne after colonic anastomosis. Hydroxyproline was measured in a control segment, removed at operation, and in the anastomotic segment collected 3 or 7 days after operation. Results are expressed as average percentage change after operation, calculated with respect to the control segment, with SEM (n = 14). The absolute (&mg dry weight) hydroxyproline levels in the different fractions of the control segments are given in Table I. The level of significance (P value) for the various differences is calculated with a paired-sample Student’s t test and represented in the following way: *O.Ol < P < 0.05, **O.OOl < P G 0.01, ***P 6 0.001.
Thus, the composition of total intestinal hydroxyproline changed considerably after anastomosis (Table II). In ileum, after 3 days, the salt-soluble fraction was significantly elevated. If the composition at 7 days after operation was compared with that 3 days after operation, both salt-soluble and acid-soluble fractions were significantly reduced and the insoluble fraction was significantly enhanced. Comparing the distribution measured at 7 days with that of unwounded tissue, the acid-soluble part appeared decreased and the insoluble fraction increased, though not significantly so. In colon, significant changes were apparent after 3 days both in salt-soluble and acid-soluble fractions, which were higher and lower, respectively, if compared to levels measured in control segments. Seven days after operation, the salt-soluble fraction remained significantly elevated with respect to the preoperative value. The acid-soluble fraction was significantly reduced, with respect to both control value and value measured 3 days postoperatively. Finally, the insoluble fraction was significantly elevated only if compared to the 3-day value. DISCUSSION The ability to form fibers with a high structural stability enables collagen to act as the major supporting framework in the intestinal wall. Covalent crosslinks between collagen molecules confer on the fibers the high tensile strength and resistance to chemical attack necessary for their function. A number of different crosslinks has been characterized for some time (Tanzer, 1976). Within one type of tissue variations may occur, for instance as the result of aging (Bentley, 1979) or a disease, either heritable or acquired (Kivirikko and Risteli, 1976).
258 Percentage
HENDRIKS
Distribution
of Hydroxyproline in the Anastomosis
TABLE II over the Various Fractions in Unwounded 3 and 7 Days after Operation”
Total (pg/mg dry weight) Ileum Unwounded tissue (n = 28) Anastomosis 3 days after operation (n = 14) Anastomosis 7 days after operation (n = 14) Colon Unwounded tissue (n = 28) Anastomosis 3 days after operation (n = 14) Anastomosis 7 days after operation (n = 14)
ET AL.
% Salt soluble
Intestine
and
% Acid soluble
% Insoluble
8.82 _f 0.19
8.5 _t 0.3
64.3 i 2.1
27.2 + 2.3
8.20 + 0.25**
10.3 2 0.4*
68.9 t 3.4
20.8 + 3.6
11.85 + 0.21***,tt+
8.0 2 0.3ttt
56.8 -c 2.2t:
35.2 i 2.2tt
12.35 z 0.31
9.2 f 0.3
65.1 -c 1.8
25.7 -t 1.9
9.39 t 0.35***
11.2 L 0.6**
62.2 t 2.2*
26.6 + 2.1
12.09 k 0.39ttt
11.5 k 0.8**
55.8 2 1.4*+
32.7 k 1.8
a LyophiIized tissue was sonicated (5 min) in successively 0.15 M NaC1 and 0.5 M HAc and hydroxyproline was measured in both supernatants. Absolute total hydroxyproline levels are given together with percentage distribution over the 3 subfractions (average with SEM). Results after operation are compared with those obtained preoperatively in the same rabbits and differences are tested for significance with a paired-sample Student’s t test (*). Differences between results obtained at 3 and 7 days postoperatively in different groups of rabbits are tested for significance by means of a f test for two random tests (t). * 0.01 < P c 0.05.
** 0.001 < P c 0.01. ***p c 0.001. t 0.01 S P 0.05. tt 0.001 S P 0.01. tttp s 0.001.
Intestinal anastomosis leads to a massive breakdown of collagen, measured as lowered hydroxyproline concentrations, both in ileum (Hesp et al., 1984) and in colon (Jiborn et al., 1980). This transient state of decreased collagen content could be a major contributing factor to the incidence of anastomotic leakage, particularly in the colon where this effect is more extensive than in the ileum (Hesp et al., 1984). However, next to the absolute amount of collagen present, its quality, in terms of number and degree of crosslinking, may also be important for determining the efficiency of its structural support. One way to investigate the degree of crosslinking, and thus presumably the stability, of collagen is by means of its solubility. Neutral salt solutions will solubilize only the least crosslinked molecules present (e.g., young collagen), while dilute acetic acid extracts the more crosslinked collagen molecules. Neither of these solvents would be expected to solubilize highly crosslinked (mature) collagen. The solubility of hydroxyproline in the intestinal wall is very low. Salt- and acid-soluble hydroxyproline constitutes only 0.4 and 4%, respectively, of total tissue hydroxyproline from rat intestine (Irvin and Hunt, 1974; Jiborn et al., 1980). These values are similar to those observed by us if lyophilized rabbit intestine was treated in the absence of sonication. Sampling of the proper anastomotic healing area severely restricts the amount of tissue available for analysis. If only such minor percentages of total hydroxyproline are solubilized, analysis of relatively small differences between samples is rendered impossible, because of biological variation and the intrinsic error of the hydroxyproline assay. Therefore, we have investigated various means to increase the solubility of hydroxyproline in the intestinal wall. Sonication of
HYDROXYPROLINE
SOLUBILITY
AFTER ANASTOMOSIS
259
lyophilized tissue proved to be the most efficient method of enhancing salt and acid solubility (Fig. 1). This procedure yields a means to distinguish three fractions, which contain enough hydroxyproline for assay in tissue samples from the same rabbit. In the sonication-solubilized fractions collagen is present not in the monomeric form but rather as aggregates which presumably still contain part of the crosslinks. Pepsin treatment of the sonicated material and subsequent electrophoresis in sodium dodecyl sulfate on polyacrylamide shows the presence of normal (Y, (I), CQ (I), and (Y, (III) chains (results not shown). The distribution of intestinal hydroxyproline over the three fractions is clearly affected by the construction of ar. anastomosis (Table II). Both in ileum and colon, the salt-soluble fraction is increased 3 days after operation. In colon, this effect is still significant after 7 days. Also, the acid-soluble fraction declines and the insoluble fraction increases from 3 to 7 days postoperatively. These results indicate that, next to the absolute concentration of hydroxyproline, its relative distribution over the various fractions also changes in the anastomotic area. During the first days after operation the integrity of the intestinal wall is weakened not only by a loss of collagen, but also by the increased salt solubility of the remaining collagen. The differences observed between the composition at 3 and 7 days postoperatively, indicate a shift from the acid-soluble to the insoluble fraction. Thus, 7 days after operation, when the original hydroxyproline concentration has been restored (colon) or even surpassed (ileum), its solubility has diminished again. Presumably, the chances for disturbance of the integrity of the connective tissue layer and subsequent anastomotic dehiscence have been reduced by then. ACKNOWLEDGMENTS The authors gratefully acknowledge Mr. P. Thissen for expert technical assistance and Dr. Ph. van Elteren (Department of Statistical Support, University of Nijmegen) for statistical analysis of the experimental data.
REFERENCES BENTLEY. J. P. (1979). Aging of collagen. J. Znvesr. Dermufol. 73, 80-83. CRONIN, K., JACKSON, D. S. and DUNPHY, J. E. (1968). Changing bursting strength and collagen content of the healing colon. Surg. Gynecol. Obster. 126, 747-753. FOGDESTAM, I. and GOTTRUP, F. (1980). Biomechanical methods in wound-healing research with special reference to ski? and gastrointestinal tract. In “Biology of Collagen” (A. Viidik and J. Vuust, eds.), Academic Press. pp 363-371. New York/London. HESP, F. L. E. M., HENDRIKS. T., LUBBERS, E. J. C., and DE BOER, H. H. M. (1984). Wound healing in the intestinal wall. A comparison between experimental ileal and colonic anastomoses. Dis. Colon Rectum 21, 99-104. IRVIN, T. T.. and HUNT. T. K. (1974). Reappraisal of the hdaling process of anastomosis of the colon. Sup. Gynecol. Obstet. 138, 741-746. JACKSON, D. S., and CLEARY, E. G. (1967). The determination of collagen and elastin. In “Methods of Biochemical Analysis” (D. Glick. ed.), Vol. XV, pp. 25-76. John Wiley & Sons, New York. JIBORN, H., AHONEN. J., and ZEDERFELDT, B. (1980). Healing of experimental colonic anastomoses. III. Collagen metabolism in the colon after left colon resection. Amer. J. Surg. 139, 398-405. KIVIRIKKO, K. I., and RISTELI, L. (1976). Biosynthesis of collagen and its alterations in pathological states. Med. Biol. 54, 159-186. LOWRY, 0. H., ROSEBROUGH,N. J., FARR, A. L.. and RANDALL, R. J. (1951). Protein measurement with the folin phenol reagent. J. Biol. Ctzem. 193, 265-275. PROCKOP, D. J. and UDENFRIEND, S. (1960). A specific method for the analysis of hydroxyproline in tissues and urine. Anal. Biochem. 1, 228-239. SCHROCK. T. R., DEVENEY. C. W., and DUNPHY. J. E. (1973). Factors contributing to leakage of colonic anastomosis. Ann. Surg. 177, 513-518. TANZER, M. L. (1976). Cross-linking. In “Biochemistry of Collagen” (G. N. Ramachandran and A. H. Reddy, eds.), pp. 137-162. Plenum, New York.