The origin of the urinary peptide hydroxyproline in burns

CLINICA

THE

CHIMICA ACTA

ORIGIN

OF THE

97

URINARY

PEPTIDE

HYDROXYPROLINE

IN

BURNS*

S. H. JACKSON

AND

T. G. ELLIOTT

Division of Biochemistry Research, Research Institute, The Hospital for Sick Children, Toronto, and The Department of Pathological Chemistry, University of Toronto (Canada) (Received

October 4, 1968)

SUMMARY

A double labelling the origin of the increased

method, urinary

using c3H] and ;14Clproline, peptide hydroxyproline

an experimental burn. The excretion of total hydroxyproline elevation, elevation

has been used to study

excretion

follows a biphasic

in rats receiving

pattern

with an initial

a return to normal, and then a prolonged elevation. During the initial the hydroxyproline originates from a collagen “pool” with a rapid metab-

olism. During the second elevation

it comes from generalized

catabolism

of “mature”

collagen. There was no evidence that the peptide hydroxyproline came from resorption of collagen at the burn site itself. Burning did not stimulate the synthesis of new collagen.

A ten to fifty-fold proline (PHP)

increase

in the daily urinary

has been observed

in burn patients1-3.

eight to ten days after burning

excretion

of peptide

The excretion

and may remain elevated

hydroxy-

rises to a peak

for several weeks. The peak

and duration of the increased PHP appear to be related to the extent and severity of the burn, although attempts by some to establish a correlation have been relatively unsatisfactory233. This failure is largely a result of limitations in the clinical estimates of the degree of burn “which vary tremendously

with the patient,

the type of burn and the

observer”3. The fact that the majority of urinary peptides in the severely burned patient have originated from collagen1 does support the concept that PHP excretion reflects the extent of cutaneous thermal damage. All reports on this subject suggest that PHP in the urine arises as a result of the thermal denaturation of dermal collagen followed by proteolysis. However, there are several features which argue against this concept. First, the high excretion rates may persist long after one would expect much resorption of proteolysis products from the site of the burn. Second, the marked elevation of PHP excretion that occurs normally, coincident with the growth spurt of puberty shows that PHP may arise from increase in synthesis of protein. It is possible that the PHP excretion in burns may * Supported by Grant 9325-14 from the Defense Research Board, Canada. Clin. Chim.

Acta,

23 (1969) 97-103

98

JACKSON,

ELLIOTT

be related to a repair mechanism. Finally, the generalized protein catabolic effect of the burn4 may liberate PHP from collagen remote from the site of the burn. This study was designed to elucidate the contribution of these factors to PHP excretion governed

resulting from experimental thermal burns in rats. The following principles the experimental design. The hydroxyproline in collagen is derived by

oxidation of proline after the proline is incorporated in a “protocollagen” molecule5. of rat connective tissue derives from dietar?, Less than 0.10/i, of the hydroxyproline hydroxyproline6,‘. Thus, labelled proline will serve as a precursor to label the hydroxyproline in collagen. If labelled proline is administered to rats and the animals arc then kept for several weeks after the administration, the bulk of the labelled hydroxyproline will be in the mature, insoluble collagen, with little remaining in the new, soluble collagen because of the latter’s rapid rate of turnovers. Thus, mature collagen can be distinguished from new collagen. If rats that have been so treated with “H Iproline are given an experimental burn and, at the same time, are given ~l”C Iproline, the specific activity of the PHP in respect to each label will indicate whether it derived from mature

collagen

or collagen

freshly

synthesized

from amino

acids.

METHODS

The method Bergman

of analysis

of total urinary

hydroxyproline

was based on that of

and Loxleyg as modified by I
Reagents Ehzrlich’s reaged. 600,; perchloric

Add 33.3 g of $-dimethylaminobenzaldehyde

to roe ml of

acid.

Chlovamifze T. Make up a IO?, W/V solution in water each day. Dilute I part of this with 4 parts of buffer. R@ev pH 6.0. Dissolve 57 g of sodium acetate (3 H,O), 37.5 g of tri-sodium citrate

(2 H,O), 5.5 g of citric acid (H,O) and 385 ml of isopropanol

in water and dilute

to I 1 with water. Procedure Mix I ml of urine with I ml of 6 N HCl in a glass ampoule. Seal and heat at 100’ overnight. Evaporate the contents of the ampoule to dryness irz vacua. Dissolve in IO ml of water. Add 0.3 ml of this solution to each of two test tubes. .4dd 0.8 ml of isopropanol to each. To one add 0.6 ml of chloramine T solution. To the other add 0.6 ml of buffer. After 4 min add 3.9 ml of Ehrlich’s reagent that has been diluted 3 to 13 with isopropanol. Mix. Heat in a water bath at 60” for 25 min. Read in a Spectronic 20 (or similar) spectrophotometer at wave length 560 nm. Zero the instrument with the unoxidized blank. A series of standard hydroxyproline solutions containing up to 3 mgl), are run concurrently. Clin.

Chim.

Actu,

21) (1969) 97-103

HYD~O~YPROLI~E

IN BURNS

99

The specific activity of the PHP was determined by the method of Prockop et ~1.11incorporating the use of semicarbazide as suggested by LeRoy et aLIz. In this method the PHP, after hydrolysis and separation through a Dowex 50 column, is oxidized to parole-z-carboxylic acid. The solution is extracted with toluene several times to remove non-specific labelled material, including the oxidation product of proline. The last extract, after washing with 0.2 M semicarbazide solution, serves as a blank. The pyrrolecarboxylic acid is then converted to pyrrole by heating in a boiling water bath. Shaking with toluene now transfers the pyrrole to the toluene. After washing with the semicarbazide, a portion of the toluene solution is taken for calorimetric estimation with Ehrlich’s reagent. The radioacti~~ity of the remainder is determined by scintillation counting after addition of scintillation phosphors (POP and POPOP). The method of Hendler13 for the simultaneous counting of 3H and l*C with channels ratio quench correction was used. The entire 24-h urine collection from each rat, less the aliquot taken for that hydroxyproline determination, was used for specific activity estimation. Since the recovery of hydrox~7prolin~ by this procedure was poor (about 50:/o) a correction for each sample, based the total hydroxyproline estimation, was used in the calculation of the specific activity. This was expressed as decomposition per minute per ,ug (DPM/pg) of hydroxyproline. EXPERIMENTAL

Twenty male, white rats (about 200 g) were separated into two equal groups. Each rat was injected intraperitoneally with 250 PC of L-[3H]proline (U). Two and one-half weeks later they were transferred to individual metabolic cages and placed on a hydroxyproline-free diet. After two days, 24-h urine collections were commenced and continued for the duration of the experiment. One day later (three weeks after injection of the [3H]proline) each animal was injected intraperitoneally with 5 PC of L-[14Cjproline (V). Ten animals then received a standard burn. The entire dorsal surface was close-clipped with machine clippers from the tail to the base of the neck. Under ether anaesthesia, the animal was placed, back down, in a hair net and suspended so that the dorsal surface was immersed from the base of the neck to the tail and about half way up the sides in water at 85” and held in there for 15 sec. On four subsequent days all animals were injected with 4, 3, z and I ,& of [14Cjproline respectively. The experiment was terminated four weeks after the burn. The burn was sufficiently deep to affect the skin without involving the underlying musculature. In the first week the area became pale and inflexible. During the second week the burned skin split and came free from the body in places. ‘By the fourth week the injured skin was sloughing off in large flaps and a new growth of thin moist tissue was exposed. Determinations were made of the daily excretion of total hydroxyproline, and of its specific activity in respect to both 3H and 1%. RESULTS

The results are shown in Figs. I to 5. Fig. I illustrates the total hydroxyproline

excretion.

Since the free hydroxy-

Clin. Chim. .4&z, 23 (1969) 97-103

JACKSON, ELLIOTT

100

proline excretion is only about 3 “/oof the total, the total is effectively equal to the peptide hydroxyproline (PHP). There was a distinct biphasic response with an initial elevation in the first few days and a return to control levels during the four- to eightday period and then an increase to a plateau which was maintained for the remainder Toial

hydrcxyprcline

excretion

.8

.z .

.b

E” z

.4

.2 0

4

a Days

12

lb

after burn

Fig. I. Total hydroxyproline excretion.

of the experiment. tjCTehave observed a strong tendency toward biphasic response in human patients (unpublished), although not so distinct as in this experiment. The specific activity of the [SHlhydroxyproline is shown in Fig. 2. Initially, this was significantly lower in the burned animals than in the controls. At about the fourth day, when the total hydroxyproline excretion returned to the control level, there was a sharp increase in the specific activity of the burned animals so that it Specific

Fig.

2.

Activity

of pH]Hydroxyproline

Specific activity of [3H,h)-droxyprolinc.

significantly higher than the controls. This differential was maintained for the remainder of the experiment. The daily excretion of r3Hjhydroxyproline was calculated from the total hydroxyproline and its specific activity. This is shown in Fig. 3. With the exception of the

became

Clin. Claim. Acta,

23 (rgGg)

97-103

HYDROXYPROLINE

0

IN BURKS

4

a

101

12

I7

Doyroffer burn Fig. 3. Urinary excretion of [3H]hydroxyproline,

corrected

for loss of label in assay.

first day, there was a significantly greater excretion of the labelled by the burned rats than the controls throughout the experiment.

hydroxyproline

The results with 14C present a different picture. The specific activity of [WIhydroxyproline is shown in Fig. 4. This was higher in the control animals than in the

6

Fig. 4. Specific activity

of [‘K]hydroxyproline.

burned animals throughout. However, when urinary excretion of [14C]hydroxyproline was calculated from the specific activity and the total hydroxyproline (Fig. 5), we see there was no significant difference in the [%]hydroxyproline excreted by burned rats and controls after the first and second day. DISCUSSION

The hydroxyproline in urine is largely in the form of small peptides of such a size that they readily pass from the plasma into the glomerular filtrateI”. There is apparently no active tubular reabsorption. Free hydroxyproline is reabsorbed so that, although the concentration of free hydroxyproline is much higher than that of the peptides in the plasma the reverse is found in the urine. These peptides probably arise as catabolic fragments from the breakdown of collagen. C&z.

Chinz.

Acta,

23

(1969)

97-103

JACKSON,

102

/

lb

UrNnary

excretion

ELLIOTT

of C “-hydroxyproline

2800 2400 Burned

animals Controls

1600

Standard

error of meon

800

v

Fig.

0

5. Urinary

8 Days otter burn

4

excretion

The biphasic

12

17

of [14C]hydroxyproline.

response

of the urinary

hydroxyproline

to the burn

indicates

that two sources of the peptides are involved. This is further verified by the change in 3H specific activity. In the first phase there is increased catabolism of a collagen “pool” with low specific activity.

This would be the “soluble”

collagen which, because

of its rapid turnover, becomes depleted of 3H label during the three weeks between injection of the [3H]proline and administration of the burn*. As this “pool” decreases there is an increasing

catabolism of the “mature” collagen as indicated by the increase of specific activity. Thus, the second phase is dissolution of mature collagen, probably arising from the generalized catabolic effect of the burn. That there is such a general effect is indicated by loss of body weight of the burn rats despite a food consumption which, after the first few days, was equal to that of the controls (Table I).

248 264 243 274 244 246 208

‘54 259

The results with the 1°C label demonstrated that there was no increase in the rate of incorporation of proline into peptide hydroxyproline under the stress of the burn. Since hydroxyproline can only originate from proline as a result of the synthesis of collagen molecules, it follows that there was no stimulation of collagen synthesis by the burn. Thus, the generalized effect of a burn on collagen metabolism appears to be purely catabolic with no compensatory anabolic component. The lower l”C Clin.

Chim.

*4cta,

23 (1969)

9:-103

HYDROXYPROLINE

IN BURNS

103

specific activity of the hydroxyproline in the burned animals was the result of dilution by hydroxyproline from collagen synthesized prior to the burn (Fig. 4). With the exception of the first, and possibly the second day the total [lK]proline incorporated into peptide hydroxyproline was the same in both groups of animals (Fig. 5). There is little chance that any appreciable amount of the increased hydroxyproline excretion in the burned animals comes from resorption at the site of the burn. In the initial phase of increased excretion, when one might expect a maximum resorption, the 3H specific activity is low. It should be high if mature skin collagen is being resorbed. During the second period, when the 3H specific activity and the total excretion is high, the skin has formed into a hardened, avascular crust from which there could not be appreciable absorption. REFERENCES I S. H. JACKSON, Clin. Chim. Acta, 12 (1965) 389. 2 S. ESTES AND D. BLOCKER, Texas Rept. Biol. Med., 24 (1966) 54. 3 L. KLEIN AND J. H. DAVIS, Surg. Forum, 15 (1964) 465. 4 S. H. JACKSON, Clin. Chim. Acta, 22 (1968) 443. 5 S. UDENFRIEND, .%ie?~Ce,152 (1966) 1335. 6 D. PROCKOP, A. KAPLAN AND S. UDENFRIEND, Arch. B&hem. Biophys., IOI (1963) 499. 7 H. STEGEMANN, 2. Physiol. Chem., 311 (1958) 41. 8 L. KLEIN AND P. H. WEISS, B&hem. Biophys. Res. Commun., 21 (1965) 311. g G. BERGM.\N AND F. LOXLEY, Anal. Chem., 35 (1963) Ig6I. IO W. KOEVOET, Clin. Chim. Acta, 12 (1965) 232. I I D. PROCKOP, S. UDENFRIEND AND A. LINDSTEDT, 1. Biol. Chem., 236 (1961) 1395. 12 E. C. LEROY, E. D. HARRIS, Jr. AND A. SJOEDSM.~, Anal. B&hem., 17 (1966) 377. 13 R. \V. HENDLER, Anal. B&hem., 7 (1964) IIO. 14 A. C. KIBRICI~, G. KITAGAM~A, M. L. MASKALERIS, R. G.MNES, Jr. AND A. T. MILHOR.IT, Puoc. SOC. &ptt. Biol. Med., rrg (1965) 622.

Clin. Claim. Acta, 23 (1969) 97-103