Urinary excretion of hydroxyproline and proline during surgical stress

Urinary excretion of hydroxyproline and proline during surgical stress

Urinary Excretion of Hydroxyproline and Proline during Surgical Stress BoLfVAR PEREYRA. M.D., Caracas, From the Instituto de Medicine Experimental, d...

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Urinary Excretion of Hydroxyproline and Proline during Surgical Stress BoLfVAR PEREYRA. M.D., Caracas,

From the Instituto de Medicine Experimental, dad Central de Venezuela, Caracas, Venezuela.

Universi-

metabolism. It was also considered of interest to determine the urinary excretion of proline since the hydroxyproline found in collagen comes from the hydroxylation of proline. Daily urinary excretion of creatinine was determined too in order to detect possible changes in creatinine excretion or errors in urinary collections. Six male patients were studied for six days prior to and eight days after surgical intervention. Surgery included cure of hernia (three patients), subtotal gastrectomy (two patients), and cholecystectomy (one patient). For the entire study period the patients were submitted to the standard diet of the University Hospital, but food items containing hydroxyproline, even in small amounts, were excluded from the diet and were substituted by other foods rich in hydroxyproline-free proteins. Twenty-four hour urine specimens were collected using toluene as a preservative and aliquots of these specimens were frozen under toluene. All biochemical determinations were carried out in duplicate. Total hydroxyproline was assayed by the method of Prockop and Udenfriend [7]. For proline analysis the method of Troll and Lindsley [8] was followed, except that hydrolysis was carried out by autoclaving with hydrochloric acid in vacuum-sealed [email protected] ampules. The product of hydrolysis was evaporated to dryness under vacuum at room temperature to eliminate the hydrochloric acid. This was followed by the addition of distilled water to a final volume equal to ten times that of the urine sample. Total nitrogen was determined by the Kjeldahl micromethod. Creatinine was analyzed by the method of Bonsnes and Taussky [9], which was slightly modified. Statistical variations were assayed by use of the Student’s t test and p = 0.05 was considered as the upper limit of statistical significance.

the initial studies of Cuthbertson [1,2] it has been well established that for a few days after surgical intervention an increased protein catabolism takes place which is reflected in an increase of urinary nitrogen. For a long time it was believed that metabolically collagen was relatively inert, but in the past few years various authors have demonstrated that this protein undergoes significant changes under different physiologic conditions [3]. Moreover, the importance of collagen for the cicatrization process and the structural and metabolic changes which this protein undergoes during wound healing are also known [4]. Lately, important contributions to the study of collagen under various pathologic conditions have been made [3,5]. It was therefore considered of interest to investigate whether collagen participates in some way in the catabolic reactions of surgical stress. Since hydroxyproline is an amino acid which is found in the organism almost exclusively as part of the colragen molecule and since its urinary excretion is considered as an important index of the metabolism of this protein in z&o [6], it was decided to study the urinary excretion of hydroxyproline during the period of surgical stress. FTER

A

MATERIAL AND METHODS The six patients selected for this study had disorders known not to cause an increased urinary excretion of hydroxyproline. In addition to hydroxyproline, total nitrogen was also determined in order to establish whether there is a relationship between possible changes in hydroxyproline excretion and general changes in protein Vol. 115, June 1968

Venezuela

RESULTS In general terms, an increased urinary excretion of hydroxyproline, proline, and total nitro777

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FIG. 1. Urinary excretion of total hydroxyproline, 0 represents the day of the operation.

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American

Jownal

of Surgery

Urinary Excretion

of Hydroxyproline

779

TABLE I COMPARISON OF AVERAGE EXCRETORY VALUES OF TOTAL HYDROXYPROLINE, TOTAL PROLINE, TOTAL NITROGEN, AND CREATININE DURING FOUR DAY PREOPERATIVE PERIOD WITH MAXIMAL TWENTY-FOUR HOUR SINGLE VALUES DURING EIGHT DAY POSTOPERATIVE PERIOD* Values before Surgery Excretory

Values

(a)

Total hydroxyproline (mg./24 hr.) Total proline (mg./24 hr.) Total nitrogen (gm./24 hr.) Creatinine (pm./24 hr.) NOTE:

Maximal Excretion after Surgery

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35, 26 f

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1, 59 f

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0, 31 f

0, 08


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(a) (b)

Means f standard error of the average values obtained four days before surgery. Means =h standard error of the highest twenty-four hour excretion values observed postoperative period. (c) Means f standard error of the differences (b - a). * Six patients were studied. t Statistically significant.

gen was encountered in the present study of the catabolic reaction to surgical stress. In four of the patients there was a progressive decrease in the excretion of hydroxyproline, proline, and total nitrogen for the entire preoperative period. (Fig. 1.) In all of the patients there was an increase in the urinary excretion of hydroxyproline, proline, and total nitrogen during the eight days subsequent to surgery. The changes observed in the excretion of these substances were statistically significant when average amounts of each substance excreted during the four day pre-

0, 02t

in the eight day

operative period were compared with the highest postoperative values obtained. (Table I.) The changes in the excretion of only hydroxyproline and proline were statistically significant when the average excretion values for the four day preoperative period were compared with average values for the eight day postoperative period. (Table IL) COMMENTS

Stress is a biological phenomenon of great interest in medicine and the alterations produced in the organism as a consequence of stress are

TABLE II COMPARISON OF AVERAGE EXCRETORY VALUES OF TOTAL HYDROXYPROLINE, TOTAL PROLINE, TOTAL NITROGEN, AND CREATININE DURING THE FOUR DAY PREOPERATIVE PERIOD WITH THOSE OBTAINED DURING THE EIGHT DAY POSTOPERATIVE PERIOD* Values Excretory

NOTE:

hr.)

(a) Means f standard (b) Means f standard (c) Means f standard * Six patients were studied. t Statistically significant.

Vol. 115, June 1968

Values

after

Surgery

(a)

(b)

Values

Total hydroxyproline (mg./24 Total proline (mg./24 hr.) Total nitrogen (gm./24 hr.) Creatinine (gm./24 hr.)

before

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four days before surgery. during eight days after surgery.

Pereyra numerous. Cuthbertson demonstrated that in stress there is an increased protein catabolism which is reflected in a negative nitrogen balance and in an increase in the total urinary nitrogen excretion [1,2]. Discarding variations related to the dietary supply of hydroxyproline and to alterations in its reabsorption at the renal tubular level, modifications in the urinary excretion of hydroxyproline are commonly interpreted as due to changes in the metabolism of collagen, whether in its synthesis, its degradation, or both [IO]. Moreover, if the curves corresponding to the urinary excretion of hydroxyproline are similar to those produced by the urinary excretion of total nitrogen, a participation of collagen in the general protein changes which result from stress is indicated. The results obtained in the present work tend to suggest that in the days after surgical intervention collagen is really implicated in the catabolic reaction. It is known that structural hydroxyproline, that is, hydroxyproline which forms an integral part of collagen, comes from the hydroxylation of proline during protein synthesis at the ribosomal level [11].* The pronounced increase in proline excretion suggests therefore that this phenomenon, as observed during the postoperative period, may possibly be due, at least in part, to faulty proline hydroxylation. It is worth recalling, nevertheless, that Levenson and Watkin [lZ] showed by the use of N15-labelled glycine that the rate of incorporation of glycine is not affected by stress, which indicates that the cause of the negative nitrogen balance is not an anabolic defect. The increased urinary excretion of proline could also depend on increased catabolism of other proteins which contain proline since the distribution of this amino acid is much wider than that of hydroxyproline, which is found almost exclusively in collagen, as stated previously. It may be mentioned in this respect that considerable aminoaciduria has been demonstrated after stress [2,13]. The possible role of the kidney in the increased excretion of proline and hydroxyproline has not been evaluated in the present work. In 1954 Nardi [13] showed bymeans of paper chromatography an increase in the urinary excretion of various amino acids but could not * Recently Bhatnagar, Rosenbloom, Kivirikko, and Prockop (Biochim. et Biophys. Acta, 149: 273, 1967) have shown that hydroxylation of collagen precursors can take at postribosomal stages.

find an appreciable increase in the excretion of free proline and hydroxyproline. He did not discard the possibility that the origin of the aminoaciduria might reside in alterations of the mechanisms of tubular reabsorption. It is known that the mechanism for the reabsorption of free hydroxyproline is highly efficient (greater than 99 per cent) although it is less so for peptidic hydroxyproline (80 per cent) [6]. According to this, the increase noted by us probably stems from the presence of hydroxyproline-containing peptides favoring the hypothesis that the observed increase might be of a metabolic origin. The decrease in the excretion of hydroxyproline, proline, total nitrogen, and creatinine prior to the surgical intervention could be related to the type of diet to which these patients were submitted for the purpose of this study. SUMMARY

The urinary excretion of hydroxyproline, proline, total nitrogen, and creatinine in six patients was analyzed for a period of six days prior to and eight days after surgery. In the postoperative period, an increased amount of proline and hydroxyproline is excreted which could be related to the protein catabolic reaction which takes place during surgical stress. The results obtained suggest that collagen participates in the metabolic changes which are produced as a result of the aforementioned phenomenon. The increased urinary excretion of proline, the metabolic precursor of hydroxyproline, could be related to the increased urinary excretion of total amino nitrogen and at the same time to a defect in the hydroxylation of proline. Acknowledgment: I wish to express my gratitude to the personnel of the Department of Surgery I of the University Hospital, especially Dr. Guillermo Negrette de Windt, Head of the Department, for their collaboration. I would also like to pay homage to the late Dr. Miguel Perez Carrefio, previous Head of the Department, for his generous spirit o! cooperation. REFERENCES 1. CUTHBERTSON, D. P. The disturbance of metabolism produced by bony and non bony injury, with notes on certain abnormal conditions of bone. Biochem. J., 24: 1244, 1930. 2. CUTHBERTSON, D. P. The distribution of nitrogen and sulfur in urine during conditions of increased catabolism. Biochem. J., 25: 236, 1931. American

Jouvnal of Surgery

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of Hydroxyproline

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3 SMILEY, D. J. and ZIFF, M. Urinary hidroxyproline excretion and growth. Physiol. Rev., 44: 30,

8. TROLL, W. and LINDSLEY, J. A photometric method for the determination of proline. J. Biol. Chem.,

1964. 4. OGILVIE, R. R. and DOUGLAS, D. M. Collagen synthesis and preliminary wounding. &it. J. Surg., 51: 149, 1964. 5. HARRIS, E. D., JR. and SJOERLXMA, A. Collagen profile in various clinical conditions. Lancet, 2: 7466, 707, 1966. 6. SJOERDSMA,A., UNDENFRIEND,S., KEISER, H., and LEROY, E. C. Hydroxyproline and collagen metabolism. Ann. Int. Med., 63: 672, 1965. 7. PROCKOP, D. J. and UDENFRIEND, S. A specific method for the analysis of hydroxyproline in tissues and urine. Ann. Biochem. Q Exper. Med., 1: 228, 1960.

215: 655, 1955. 9. BONSNES, R. W. and TAUSSKY, H. H. On the colorimetric determination of creatinine by the Jaffe reaction. J. BioE. Chew., 158: 581, 1943. 10. ROBERTSON, W. V. B. Metabolism of collagen in mammalian tissues. Biophys. J., 4: 93, 1964. 11. UDENFRIEND, S. Formation of hydroxyproline in collagen. Science, 15: 1, 1966. 12. LEVENSON, S. M. and WATKIN, D. M. Protein requirements in injury and certain acute and chronic diseases. Fed. Proc., 18: 1155, 1960. 13. NARDI, G. L. “Essential” and “nonessential” amino acids in the urine of severely burned patients. J. Clin. Invest., 33: 847, 1954.

Vol.ll5,June 1968