Clinical Biochemistry, Vol. 29, No.

3, 225-229, 1996 Copyright 1996 The Canadian Society of Clinical Chemists Printed in the USA. All fights reserved 0009-9120/96 $15.00 + .00 ELSEVIER S0006-2952(96)00003-3

A Simplified Method for the Analysis of Hydroxyproline in Biological Tissues

G. KESAVA REDDY and CHUKUKA S. ENWEMEKA Department of Physical Therapy, University of Kansas Medical Center, 3901 Rainbow Blvd., Kansas City, KS 66160
A critical study of the diffen.fnt steps involved in previous procedure for hydroxyproline assay allows the direct measurement of collagen content in tissue homogenates without losing the advantages of the method. The procedure is based on alkaline hydrolysis of the tissue homogenate and subsequent determination of the free hydroxyproline in hydrolyzates. Chloramine-T was used to oxidize the free, hydroxyproline for the production of a pyrrole. The addition of Ehrlich's reagent resulted in the formation of a chromophon.~ that can be measured at 550 nm. Optimal assay conditions were determined using tissue homogenate and purified acid soluble collagen along with standard hydroxyproline. Critical parameters such as the amount of chloramine-T, sodium hydroxide, p-dimethylaminobenzaldehyde, pH of the reaction buffer, and length of oxidation time were examined to obtain satisfactory results. The method has been applied to samples of tissue homogenate and purified acid soluble collagen, with recovery of added hydroxyproline of 101 6.5 and 104 6.0 (SD) percent, respectively. The method is highly sensitive and reproducible when used to measure the imino acid in tissue homogenates. The modified hydroxyproline assay presented in this communication will be useful for routine measurement of collagen content in extracts of various tissue specimens. In addition, the modified method can be used for batch processing of column fractions to monitor the collagen concentrations during purification.

KEY WORDS: hydroxyproline; tissue hydrolyzate; collagen; Ehrlich's reagent.

Introduction

zyme prolyhydroxylase (EC.1.14.11.2) (1). The occurrence of this imino acid is thought to be confined almost exclusively to the connective tissue collagen, where it is present in 1:he Y position of the Gly-X-Y repeating tripeptide ('.2). Because of its restricted and unique distribution in connective tissue collagen, the metabolism of collagen and its regulation is conveniently studied by measuring the Hyp content

H uct of proline hydroxylation catalyzed by an en-

Ydroxyproline (Hyp) is a post-translational prod-

Correspondence: G.K. Reddy, Department of Physical Therapy, University of Kansas Medical Center, 3901 Rainbow Blvd., Kansas City, KS 66160. Manuscript received September 8, 1995; revised and accepted December 11, 1995.
C L I N I C A L B I O C H E M I S T R Y , V O L U M E 29, J U N E 1996

in a number of normal and clinical situations. There is increased utilization of this assay because of the need to monitor the amount of collagen in a variety of pathological conditions, including tumor invasion and metastasis, rheumatoid arthritis, diabetes mellitus, muscular dystrophy, and chronic ulcers. Considering the role of collagen and its significance in many clinically important connective tissue diseases, a great deal of attention has been directed toward the development of an assay capable of both detecting and measuring the collagen quantitatively. Measurement of Hyp in various tissues, plasma, and urine for the investigation of normal and pathological conditions of collagen metabolism is possible by colorimetric methods (3-6), high performance liquid chromatography (7,8), gas chromatography/ mass spectrometry (9), and enzymatic methods (10). The numerous assay procedures described for hydroxyproline indicate the limits of each one with regard to specificity, sensitivity, reproducibility, accuracy, and practical approach. In general, most of the procedures are laborious and involve many timeconsuming steps. Chromatography on ion-exchange resins or repeated extractions with organic solvents are steps recommended by several investigators (3,4). In 1980, Huszar et al. (11) described a simple procedure for determining Hyp in order to monitor collagen and its fragments. The method was successfully used with higher sensitivity by eliminating the time-consuming steps involved in the Hyp assay. However, the application of the method for the quantitation of Hyp in tissue homogenates has not been fully studied. Hence, in the present investigation, an attempt has been made to develop a simple assay for direct quantitation of Hyp in tissue homogenates. The method described herein is a modification of an assay reported by Huszar et al. (11) and originally introduced by Stegemann and Stadler (4). The modifications include a change in hydrolysis procedure, reduction of the sample volume, and omission of the neutralization step with citric acid. Since this
225

REDDY AND ENWEMEKA

analytical procedure is intended to be used to quantitate the tissue collagen, other minor modifications were incorporated to optimize the conditions for the measurement of Hyp in biological tissue samples. Materials and methods Chemicals: Chloramine-T, p-dimethylaminobenzaldehyde, acid soluble collagen, and L-hydroxyproline were purchased from Sigma Chemical Co., (St. Louis, U.S.A.). Sodium acetate, citric acid, perchloric acid, n-propanol, sodium hydroxide, and acetic acid were purchased from Fisher Scientific (St. Louis, U.S.A.). All other chemicals were of analytical grade.
PREPARATION OF REAGENTS

ASSAY PROCEDURE

For the assay, O'-ring screw-capped Nalgene high temperature polypropylene tubes of 2 mL capacity were used. As summarized in the flow chart shown below, aliquots of standard Hyp/test samples were hydrolyzed in alkali. The hydrolyzed samples were then mixed with a buffered chloramine-T reagent, and the oxidation was allowed to proceed for 25 min at room temperature. The chromophore was then developed with the addition of Ehrlich's reagent, and the absorbance of reddish purple complex was measured at 550 nm using a Gilford RESPONSE T M spectrophotometer. Absorbance values were plotted against the concentration of standard Hyp, and the presence of Hyp in unknown tissue extracts was determined from the standard curve. A flow chart of the hydroxyproline assay procedure follows: 1. Aliquots of standard Hyp (2-20 ~g) prepared from stock solution and test samples containing Hyp under 10 ~Lg/mL were mixed gently with sodium hydroxide (2N final concentration) in a total volume of 50 ~L. 2. The samples were hydrolyzed by autoclaving at 120 C for 20 min. 3. 450 ~L of chloramine-T was added to the hydrolyzate, mixed gently, and the oxidation was allowed to proceed for 25 min at room temperature. 4. 500 ~LL of Ehrlich's aldehyde reagent was added to each sample, mixed gently, and the chromophore was developed by incubating the samples at 65 C for 20 min. 5. Absorbance of each sample was read at 550 nm using a spectrophotometer. Results The optimal conditions for the Hyp assay in tissue hydrolyzates were evaluated using tissue homogenate and purified acid soluble collagen along with the standard Hyp. The calibration curve was initially established using standard Hyp. The results demonstrated that the absorbance is linearly related to the amount of Hyp over the range of 0-20 mg (Fig. 1). Since this procedure was originally intended to be used for quantitating collagen in tissue homogenates, we optimized the conditions by examining the effect of various critical parameters including the concentrations of aldehyde, chloramine-T and sodium hydroxide, pH of the buffer, and oxidation time of the chemical reaction. In the test sample, we used the tissue homogenate from rabbit Achilles tendon and purified acid soluble collagen (dissolved in 50 mM acetate buffer pH 3.5) for the measurement of Hyp.
EFFECTS OF ALKALI AND CHLOROMINE W CONCENTRATIONS, OXIDATION TIME, AND P H ON CHROMOPHORE FORMATION

Hydroxyproline stock
A solution containing 1 mg/mL of Hyp was prepared in distilled water.

Acetate-citrate buffer pH 6.5

The buffer was prepared by dissolving 120 g of sodium acetate trihydrate, 46 g of citric acid, 12 mL acetic acid, and 34 g of sodium hydroxide in distilled water; pH was adjusted to 6.5 and brought to one liter.

Chloromine T reagent (0.056M)

1.27 g of chloramine T was dissolved in 20 mL 50% n-propanol and brought to 100 mL with acetatecitrate buffer.

Ehrlich's reagent (1M)

15 g of p-dimethylaminobenzaldehyde was dissolved in n-propanol/perchloric acid (2:1 v/v) and brought to 100 mL. Since this reagent is not stable, it was prepared freshly before each assay.

Sample preparation
Since we were interested in studying the qualitative and quantitative changes of collagen during normal tissue repair process, a rabbit Achilles tendon specimen was chosen for the measurement of hydroxyproline. Soon after sacrificing rabbits, Achilles tendons were removed surgically, frozen in liquid nitrogen, and then lyophilized. The lyophilized tissue sample was homogenized thoroughly in distilled water using a polytron homogenizer. In addition to this fine tissue homogenate, purified acid-soluble collagen (dissolved in 50 mM acetate buffer, pH 3.5) was included as a test sample for the estimation of Hyp.
226

Initially we examined the effects of various concentrations of sodium hydroxide and chloramine-T
CLINICAL BIOCHEMISTRY, VOLUME 29, J U N E 1996

4-

Lt~

3"

2-

t~

///
i i i i

DETERMINATION OF TISSUE HYDROXYPROLINE

Figure 2D. A pH of the acetate-citrate buffer between 6.0 to 6.5 yielded maximal absorbance values for all samples. At pH values below 5, the formation of chromophore was found to be negligible; however, alkaline pH of the buffer did not influence the absorbance of standard Hyp except for those at pH 8.0. These observations further indicate that pH of the reaction media plays a critical role during the oxidation of Hyp.
EFFECT OF P-DIMETHYLAMINOBENZALDEHYDECONCENTRATION

<
i i

12

16

20

24

H y d r o x y p r o l i n e (~g)
Figure 1 - - Calibration curve for the assay ofhydroxyproline. A designated a m o u n t of s t a n d a r d hydroxyproline was pipetted into test tubes a n d assayed as described u n d e r

Materials and methods. on the development of chromophore and results are presented in Figure 2. The concentration of sodium hydroxide ranging 0.25N-8N (final concentration) was tested for the optimal hydrolysis. It is to be noted that 2N sodium hydroxide (final concentration) was found to be optimal for the hydrolysis of the sample (Fig. 2A). At a lower concentration of sodium hydroxide, the tbrmation of the chromophore was found to be lower ,clue to incomplete hydrolysis of the sample. However, higher concentrations of sodium hydroxide (>2N) did not influence the hydrolysis of the samples and, thus, no changes were detected in chromophore absorbance readings. Similarly, the influence of various amounts of chloramine-T (ranging 0.005M-0.06M) on the formation of pyrrol-2-carboxylic acid was examined, and the results are shown in Figure 2B. Absorbance values using 0.025M chloramine-T (final concentration) were found to be maximal for all samples (i.e., tissue homogenate, acid soluble collagen and standard Hyp). Although the absorbance values of standard Hyp were not altered appreciably at lower concentrations of the chloramine-T reagent, a considerable change was observed in the absorbance values for the tissue homogenate and acid soluble collagen. Either a slight decrease or no major change was observed in absorbance values at higher concentrations of chloramine-T (>0.025M). The results in Figure 2C indicate that the development of the chromophore is dependent on the function of oxidation time. An investigation of this relation indicated that optimal results are obtained in 20-25 min of incubation, and thereafter the extension of the oxidation time did not influence the formation of the chromophore. No differences were noticed in oxidation time between test samples and standard Hyp. The effects of the acetate-citrate buffer at various pH wLlues on the reactivity of chloromine-T during the oxidation process are shown in
CLINICAL BIOCHEMISTRY, VOLUME 29, JUNE 1996

We examined the effect of various concentrations of p-dimethylamino-benzaldehyde (ranging 0.1251.5M final concentration) in Ehrlich's reagent on the formation of the color complex using standard Hyp. As the final concentration of Ehrlich's reagent increased to 0.5M, the absorbance values increased in proportion. Thereafter, the absorbance of the color complex decreased significantly as the concentrations of aldehyde rose to 1.5M final concentration (results not shown). Moreover, at higher concentrations of aldehyde, the formation of chromophore shifted from reddish purple to pale greenish color. As reported earlier (12), the absorbance spectrum of the reaction product was found to be a well-defined peak at 550 nm (results not shown).
RECOVERY STUDIES

The recoveries documented in Table 1 are those obtained using tissue homogenate and purified acid soluble collagen after the addition of the designated amount of standard Hyp. Each value is the mean of duplicate measurements of individual sample. The results clearly demonstrate that the procedure developed under the conditions described above is precise, sensitive to Hyp, and highly reproducible with no loss of recovery. It may be noted that there might appear to be a lower recovery at a Hyp concentration of 20 ~g.
REPRODUCIBILITY OF THE ASSAY

The reproducibility of the assay was demonstrated by analyzing 10 identical samples of purified acid soluble collagen solution with a concentration of 50 ~g/mL of 50 mM acetic acid. The samples were found to have Hyp concentrations ranging 5.8 to 6.4 ~g/mL with a mean value of 6.1 ~g/mL and a standard deviation of 0.21 ~g/mL.
AP I A I N P LC TO S

The application of the method for other tissue samples was examined using tissue specimens such as heart, lung, kidney, liver, and testis obtained from rabbits. The tissues were excised from the rabbit after sacrificing them, frozen in liquid nitrogen, and stored at -70 C until further use. Two grams of frozen tissue sample was homogenized in 5 mL sa227

REDDY AND ENWEMEKA

1.0
0.6

A
,s'

B
,':" ~ A S C

=
v

0.8 o.6

TH
--" A S C

m
m

0.4
e~

0.4

......~.--o...~...,...~...~...,..,, TH
oO 1 Q"

0.2

...

............................

0.2
i I 2.0 ~ I 4,0 , I 6.0 i I 8.0

.<
0 0.0

0.0 0.00

0.02

0.04

0.06

NaOH Concentration
1.0

[N]
2.0

C h l o r a m i n e - T C o n c e n t r a t i o n [M]

E
0.8

C
.

D
," "A.

.. o . . . . . ."*

.........

~*

-,ASC TH

= 1.6 u~ 1.2 ,~ ~- 0.8

"

tt~ 0.6

~ / " /7 ' = " ~ .. p ~. /

-~'~'-'~

: ,,
/: / ' - ~ - ~ =
] ,
,

:"

.~-----"--~-----~--~'. "~
t

"~

0.4

t_ 0.4

ASC TH

...
0.2 0
I , ! h l , l ,

-<
0.0 10 20 30 40

: ,&
I .oAr

/ :

4.0

5.0

6.0

'7.0

8.0

9.0

T i m e (min)

pH

Figure 2 -- Optimization of various conditions for hydroxyproline assay. A: The influence of sodium hydroxide on sample hydrolysis; B: the effect of chloramine-T on the formation of pyrrol-2-carboxylic acid; C: the effect of oxidation time on the formation of chromophore; D: the influence of pH of the reaction mixture on the assay. Aliquots of tissue homogenate (TH), purified acid soluble collagen (ASC), and a known amount of standard hydroxyproline (SH) were pipetted into a series of test tubes. The effects of different variables were examined and a chromophore was developed as described under Materials and methods. line using a polytron homogenizer. The content of Hyp was quantified as previously described. Table 2 represent the results of an analysis of different tissues from rabbit, and an average of five different samples of each tissue type. The collagen values were calculated assuming 12.5% of collagen is Hyp (5).
Discussion

Collagen is the most a b u n d a n t connective tissue protein in vertebrates, representing approximately one third of body protein. The metabolism of collagen and its regulation are of vital interest in a number of clinically important diseases t h a t are characterized by an accumulation or loss of tissue collagen. Excessive production of collagen has been documented in proliferate disorders such as liver cirrhosis, lung fibrosis, sceleroderma, and tumor growth. 228

Loss of tissue collagen has been observed in certain disorders of connective tissue including rheumatoid arthritis and wound/ulcer damaged tissues. In other clinical situations, such as tissue repair and wound healing, overproduction and deposition of collagen are required to heal the damaged tissues. To understand the critical role of collagen in various pathophysiological conditions, several methodologies were developed for the determination of Hyp in various tissues and physiological fluids such as plasma and urine. All cases of these methods have two common steps: (a) hydrolysis of sample with either strong acid or alkali to liberate Hyp and (b) detection of free imino acid by either colorimetric, fluorimetric, or high-performance liquid chromatographic methods. The method described here is essentially based on the alkaline hydrolysis of tissue homogenate and subsequent quantitation of free Hyp in the hydrolyzates. The entire procedure comprises three steps: CLINICALBIOCHEMISTRY,VOLUME29, JUNE 1996

DETERMINATION OF TISSUE HYDROXYPROLINE

TABLE 1

Recovery of Hydroxyproline Added to the Test Samples Addition of SH (~g)

0

Percentage Recovery TH
--

TABLE2 Measured Hydroxyproline Content and the Calculated Collagen Content of Various Rabbit Tissues Hydroxyproline (g/100 g of Frozen Tissue) 0.023 0.037 0.034 0.019 0.032 Standard Deviation 0.002 0.003 0.003 0.002 0.002 Collagen (g/100 g of Frozen Tissue) 0.18 0.30 0.27 0.15 0.26

ASC Tissue
--

2 4 6 8 10 12 14 16 20

100 110 107 106 100 104 98 97.5 88 101 _+6.54*

105 108 110 109 108 108 100 100 92 104 _+5.96*

Heart Lung Kidney Liver Testis

Tissue hydrolyzate (TH) containing 1.5 ~g of Hyp in 25 ~mL and acid soluble collagen (ASC) containing 1 ~g of Hyp in 25 ~L were used iin this recovery study. An incremental amount of standard Hyp (SH) was added to examine the percentage of recovery by the proposed method. * Average percentage recovery (mean _+SD). (a) hydrolysis, (b) oxidation, and (c~) development of chromophore. The method is precise, simple, and specific for Hyp. In this method, we not only optimized conditions for the standard curve, but also for tissue homogenates as well as purified collagens. The results clearly demonstrate t h a t the developm e n t of chromophore with Hyp in tissue homogenate and purified collagen was similar to t h a t of standard Hyp, suggesting t h a t other proteins do not interfere in the reaction process. The interference of proline during the oxidation can be excluded based on earlier reports (6) t h a t the chromogen can be formed with Ehrlich's reagent only if proline is further oxidized with sodium periodate (6). Although the procedure presented here is somewhat similar to t h a t of the earlier method (11), several modifications were incorporated after optimizing the conditions for the m e a s u r e m e n t of tissue Hyp. Such modifications include the omission of drying the sample before hydrolysis and t r e a t m e n t of the sample with citric acid for neutralization after hydrolysis. Examination of the effect of various citric acid concentrations on the absorbance values revealed t h a t the deletion of citric acid from the reaction medium did not affect the accuracy or sensitivity of the assay (results not shown). Moreover, the concentrations of reagents used in the current protocol differ slightly from the earlier method (11). The total volume of the sample, including all reagents necessary for the reaction medium, was condensed to 1 mL with no loss of sensitivity and simplicity. With the retention of all advantages of the earlier procedure (11), the modified method described in this communication is highly applicable from the point of the view of a~]alysis of collagen content in

various tissues during pathophysiological conditions. Also, the procedure described here for the Hyp assay can be extended for the detection of collagen fragments in column fractions from various chromatographic techniques employed for the purification of collagen. In conclusion, a convenient and reliable method for the determination of Hyp in biological tissue samples has been established, which m a y provide a useful tool in clinical and biomedical research areas.
References

1. Kivirikko KI, Myllyla R. In: Cunninham LW, Frederiksen DW, editors. Methods in enzymology, Vol. 82. New York: Academic Press, 1982:245-319. 2. Nemethy G, Scheraga HA. Stabilization of collagen fibrils by hydroxyproline. Biochemistry 1986; 52: 3184-3188. 3. Neuman RE, Logan MA. The determination of hydroxyproline. J Biol Chem 1950; 184: 299-306. 4. Stegemann H, Stalder K. Determination of hydroxyproline. Clin Chim Acta 1967; 18: 267-273. 5. Edwards CA, O'Brien WD Jr. Modified assay for determination of hydroxyproline in a tissue hydrolyzate. Clin Chim Acta 1980; 104: 161-167. 6. Blumenkrantz N, Asboe-Hansen G. An assay for hydroxyproline and proline on one sample and a simplifled method for hydroxyproline. Anal Biochem 1967; 63: 331-340. 7. Stimler NP. High performance liquid chromatographic quantitation of collagen biosynthesis in explant cultures. Anal Biochem 1984; 142: 103-108. 8. Green GD, Reagan K. Determination of hydroxyproline by high pressure liquid chromatography. Anal Biochem 1992; 201: 265-269. 9. Tredget EE, Falk N, Scott PG, Hogg AM, Burke JF. Determination of 4-hydroxyproline in collagen by gas chromatography/mass spectrometry. Anal Biochem 1990; 190: 259-265. 10. Ito A, Uoji H, Mori Y. An enzymatic estimation of free hydroxyproline in tissue hydrolyzates. Anal Biochem 1985; 151: 510-514. 11. Huszar G, Maiocco J, Naftolin F. Monitoring of collagen and collagen fragments in chromatography of protein mixtures. Anal Biochem 1980; 105: 424-429. 12. Prockop DJ, Udenfriend S. A specific method for the analysis of hydroxyproline in tissues and urine. Anal Biochem 1960; 1: 228-239.

CLINICALBIOCHEMISTRY,VOLUME29, JUNE 1996

229