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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.

zyme prolyhydroxylase (EC. (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

and L-hydroxyproline were purchased from Sigma Chemical Co. In the test sample.REDDY AND ENWEMEKA analytical procedure is intended to be used to quantitate the tissue collagen. frozen in liquid nitrogen. In addition to this fine tissue homogenate. pH of the buffer. As summarized in the flow chart shown below.). Since this procedure was originally intended to be used for quantitating collagen in tissue homogenates. Louis. The results demonstrated that the absorbance is linearly related to the amount of Hyp over the range of 0-20 mg (Fig. and the oxidation was allowed to proceed for 25 min at room temperature. mixed gently. Acetate-citrate buffer pH 6. and acetic acid were purchased from Fisher Scientific (St. The samples were hydrolyzed by autoclaving at 120 °C for 20 min. Absorbance values were plotted against the concentration of standard Hyp. other minor modifications were incorporated to optimize the conditions for the measurement of Hyp in biological tissue samples.27 g of chloramine T was dissolved in 20 mL 50% n-propanol and brought to 100 mL with acetatecitrate buffer. p-dimethylaminobenzaldehyde. and the oxidation was allowed to proceed for 25 min at room temperature. 1). A flow chart of the hydroxyproline assay procedure follows: 1. and the absorbance of reddish purple complex was measured at 550 nm using a Gilford RESPONSE T M spectrophotometer. AND P H ON CHROMOPHORE FORMATION Hydroxyproline stock A solution containing 1 mg/mL of Hyp was prepared in distilled water.5) for the measurement of Hyp. Chloromine T reagent (0..056M) 1. and then lyophilized. 3. 12 mL acetic acid. chloramine-T and sodium hydroxide. 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. perchloric acid. and oxidation time of the chemical reaction. and 34 g of sodium hydroxide in distilled water. J U N E 1996 .S. Sample preparation Since we were interested in studying the qualitative and quantitative changes of collagen during normal tissue repair process. it was prepared freshly before each assay. 5. Absorbance of each sample was read at 550 nm using a spectrophotometer. U. All other chemicals were of analytical grade. Sodium acetate. we used the tissue homogenate from rabbit Achilles tendon and purified acid soluble collagen (dissolved in 50 mM acetate buffer pH 3. and the chromophore was developed by incubating the samples at 65 °C for 20 min. Materials and methods Chemicals: Chloramine-T. a rabbit Achilles tendon specimen was chosen for the measurement of hydroxyproline.5 and brought to one liter. The chromophore was then developed with the addition of Ehrlich's reagent. 2. acid soluble collagen. PREPARATION OF REAGENTS ASSAY PROCEDURE For the assay. The hydrolyzed samples were then mixed with a buffered chloramine-T reagent. Since this reagent is not stable. O'-ring screw-capped Nalgene high temperature polypropylene tubes of 2 mL capacity were used. sodium hydroxide. 226 Initially we examined the effects of various concentrations of sodium hydroxide and chloramine-T CLINICAL BIOCHEMISTRY.). 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. Soon after sacrificing rabbits. Louis. and the presence of Hyp in unknown tissue extracts was determined from the standard curve. pH was adjusted to 6. aliquots of standard Hyp/test samples were hydrolyzed in alkali.A. 46 g of citric acid.S.5 The buffer was prepared by dissolving 120 g of sodium acetate trihydrate. The lyophilized tissue sample was homogenized thoroughly in distilled water using a polytron homogenizer. citric acid. mixed gently.A. we optimized the conditions by examining the effect of various critical parameters including the concentrations of aldehyde.5) was included as a test sample for the estimation of Hyp. EFFECTS OF ALKALI AND CHLOROMINE W CONCENTRATIONS. (St. VOLUME 29. pH 3. U. 500 ~LL of Ehrlich's aldehyde reagent was added to each sample. n-propanol. Achilles tendons were removed surgically. 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. The calibration curve was initially established using standard Hyp. 450 ~L of chloramine-T was added to the hydrolyzate. purified acid-soluble collagen (dissolved in 50 mM acetate buffer. 4. OXIDATION TIME.

The samples were found to have Hyp concentrations ranging 5.. EFFECT OF P-DIMETHYLAMINOBENZALDEHYDECONCENTRATION < i i 0 4 8 12 16 20 24 H y d r o x y p r o l i n e (~g) Figure 1 . 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.025M). These observations further indicate that pH of the reaction media plays a critical role during the oxidation of Hyp. Either a slight decrease or no major change was observed in absorbance values at higher concentrations of chloramine-T (>0. A pH of the acetate-citrate buffer between 6. Thereafter. At pH values below 5.025M chloramine-T (final concentration) were found to be maximal for all samples (i. thus. the formation of chromophore shifted from reddish purple to pale greenish color. Although the absorbance values of standard Hyp were not altered appreciably at lower concentrations of the chloramine-T reagent..06M) on the formation of pyrrol-2-carboxylic acid was examined. and thereafter the extension of the oxidation time did not influence the formation of the chromophore.0 to 6. the tbrmation of the chromophore was found to be lower . and testis obtained from rabbits. on the development of chromophore and results are presented in Figure 2.21 ~g/mL. VOLUME 29. Moreover.1 ~g/mL and a standard deviation of 0.5 yielded maximal absorbance values for all samples. alkaline pH of the buffer did not influence the absorbance of standard Hyp except for those at pH 8. Similarly.005M-0. No differences were noticed in oxidation time between test samples and standard Hyp. and the results are shown in Figure 2B. 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 results clearly demonstrate that the procedure developed under the conditions described above is precise. frozen in liquid nitrogen. and highly reproducible with no loss of recovery. the influence of various amounts of chloramine-T (ranging 0.5M.5M final concentration (results not shown).Calibration curve for the assay ofhydroxyproline.8 to 6. Absorbance values using 0. An investigation of this relation indicated that optimal results are obtained in 20-25 min of incubation. kidney. However.4 ~g/mL with a mean value of 6. 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.25N-8N (final concentration) was tested for the optimal hydrolysis.clue to incomplete hydrolysis of the sample. and stored at -70 °C until further use.0. The tissues were excised from the rabbit after sacrificing them. It may be noted that there might appear to be a lower recovery at a Hyp concentration of 20 ~g. 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. The concentration of sodium hydroxide ranging 0. It is to be noted that 2N sodium hydroxide (final concentration) was found to be optimal for the hydrolysis of the sample (Fig. no changes were detected in chromophore absorbance readings. the absorbance spectrum of the reaction product was found to be a well-defined peak at 550 nm (results not shown).1251.e. acid soluble collagen and standard Hyp). As the final concentration of Ehrlich's reagent increased to 0. 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. tissue homogenate. the absorbance of the color complex decreased significantly as the concentrations of aldehyde rose to 1. at higher concentrations of aldehyde. however. As reported earlier (12). a considerable change was observed in the absorbance values for the tissue homogenate and acid soluble collagen. sensitive to Hyp. liver. Each value is the mean of duplicate measurements of individual sample. JUNE 1996 We examined the effect of various concentrations of p-dimethylamino-benzaldehyde (ranging 0. the formation of chromophore was found to be negligible. lung. The results in Figure 2C indicate that the development of the chromophore is dependent on the function of oxidation time. the absorbance values increased in proportion.5M final concentration) in Ehrlich's reagent on the formation of the color complex using standard Hyp.4- Lt~ 3" 2- t~ /// i i i i DETERMINATION OF TISSUE HYDROXYPROLINE Figure 2D. At a lower concentration of sodium hydroxide. 2A). Two grams of frozen tissue sample was homogenized in 5 mL sa227 . higher concentrations of sodium hydroxide (>2N) did not influence the hydrolysis of the samples and.

..0 6.. "~ t "~ 0... D: the influence of pH of the reaction mixture on the assay. sceleroderma... ~* -.Optimization of various conditions for hydroxyproline assay.8 " tt~ 0.REDDY AND ENWEMEKA 1.0 i I 8." "A. .--o.~. 228 Loss of tissue collagen has been observed in certain disorders of connective tissue including rheumatoid arthritis and wound/ulcer damaged tissues. ..0 . Discussion Collagen is the most a b u n d a n t connective tissue protein in vertebrates..0.~ = • ] .8 C . The method described here is essentially based on the alkaline hydrolysis of tissue homogenate and subsequent quantitation of free Hyp in the hydrolyzates.0 9.0 5.. several methodologies were developed for the determination of Hyp in various tissues and physiological fluids such as plasma and urine. fluorimetric.. 0.0 0..0 10 20 30 40 : . I i f i 0.~-----"--~-----~--~'.. / -~'~'-'~ : .. l ."* ° ..< 0 0..6 • ~ / " •/7 ' = " ~ .s' B . Excessive production of collagen has been documented in proliferate disorders such as liver cirrhosis.ASC TH = 1.. C: the effect of oxidation time on the formation of chromophore.0 8.... /: / ' .. and an average of five different samples of each tissue type. . .& I .. TH •oO• °1¢ Q" c 0. JUNE 1996 ..0 [N] 2.~.. The content of Hyp was quantified as previously described.. 0.. p ~. lung fibrosis. o .. B: the effect of chloramine-T on the formation of pyrrol-2-carboxylic acid. -< 0..0 C h l o r a m i n e ..0 '7... I 6... such as tissue repair and wound healing. The effects of different variables were examined and a chromophore was developed as described under Materials and methods. Aliquots of tissue homogenate (TH)....6 ° TH --" A S C m m 0. Table 2 represent the results of an analysis of different tissues from rabbit.~. To understand the critical role of collagen in various pathophysiological conditions.0 0.02 0.~. purified acid soluble collagen (ASC)..0 0. or high-performance liquid chromatographic methods..T C o n c e n t r a t i o n [M] E 0.. In other clinical situations. The collagen values were calculated assuming 12...5% of collagen is Hyp (5).. A: The influence of sodium hydroxide on sample hydrolysis. and tumor growth. line using a polytron homogenizer..6 u~ 1....... .... D . .6 A .':" ~ A S C m = v 0. representing approximately one third of body protein.06 NaOH Concentration 1.. overproduction and deposition of collagen are required to heal the damaged tissues.2 ..04 0.0 T i m e (min) pH Figure 2 -.4 t_ 0.2 .. 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.° ..8 o. ! h l .4 e~ 0....~ ~. The entire procedure comprises three steps: CLINICALBIOCHEMISTRY...0 ..2 i I 2.VOLUME29. 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.2 0 I .00 i I ...~ .0 ~ I 4.4 . and a known amount of standard hydroxyproline (SH) were pipetted into a series of test tubes.°oAr°° : / : 4. :" • . .4 ASC TH ..

Reagan K. 82. Neuman RE. Burke JF. Vol. Ito A. In this method.034 0. Maiocco J. 11.VOLUME29. 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. New York: Academic Press. suggesting t h a t other proteins do not interfere in the reaction process. Edwards CA. 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. several modifications were incorporated after optimizing the conditions for the m e a s u r e m e n t of tissue Hyp. Logan MA. An incremental amount of standard Hyp (SH) was added to examine the percentage of recovery by the proposed method. 6. 63: 331-340. Anal Biochem 1967. 5. Udenfriend S. Clin Chim Acta 1967. 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.037 0. which m a y provide a useful tool in clinical and biomedical research areas. 8. Uoji H. Although the procedure presented here is somewhat similar to t h a t of the earlier method (11). An enzymatic estimation of free hydroxyproline in tissue hydrolyzates.54* 105 108 110 109 108 108 100 100 92 104 _+5. Determination of hydroxyproline by high pressure liquid chromatography. CLINICALBIOCHEMISTRY.032 Standard Deviation 0. O'Brien WD Jr. (a) hydrolysis. Prockop DJ. Anal Biochem 1984. Frederiksen DW. (b) oxidation.003 0. 190: 259-265. Asboe-Hansen G. 142: 103-108. Determination of hydroxyproline. The determination of hydroxyproline. 9. * Average percentage recovery (mean _+SD). Determination of 4-hydroxyproline in collagen by gas chromatography/mass spectrometry. Hogg AM. simple. 151: 510-514. Anal Biochem 1992.26 ASC Tissue -- 2 4 6 8 10 12 14 16 20 100 110 107 106 100 104 98 97. we not only optimized conditions for the standard curve. Nemethy G. Huszar G. Stalder K. was condensed to 1 mL with no loss of sensitivity and simplicity. Green GD. Scheraga HA. Anal Biochem 1960. 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). References 1. Mori Y.5 88 101 _+6.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. 2. 1: 228-239. 10. Naftolin F. 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.27 0. Stegemann H.002 0. Myllyla R. 184: 299-306. 4. In conclusion. 1982:245-319. Also. 12. 104: 161-167. 18: 267-273. In: Cunninham LW. Stimler NP. Tredget EE. Modified assay for determination of hydroxyproline in a tissue hydrolyzate. Clin Chim Acta 1980. JUNE 1996 229 . 7. The total volume of the sample. Anal Biochem 1985. but also for tissue homogenates as well as purified collagens. Anal Biochem 1980.15 0. and (c~) development of chromophore. Stabilization of collagen fibrils by hydroxyproline.18 0.30 0. Methods in enzymology. 52: 3184-3188. editors. The method is precise. 3. and specific for Hyp.003 0. Moreover. Falk N.023 0. Biochemistry 1986.019 0. Kivirikko KI.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. High performance liquid chromatographic quantitation of collagen biosynthesis in explant cultures. including all reagents necessary for the reaction medium. Blumenkrantz N. An assay for hydroxyproline and proline on one sample and a simplifled method for hydroxyproline. 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).96* Heart Lung Kidney Liver Testis Tissue hydrolyzate (TH) containing 1. J Biol Chem 1950. 105: 424-429. the concentrations of reagents used in the current protocol differ slightly from the earlier method (11). Scott PG.002 Collagen (g/100 g of Frozen Tissue) 0. 201: 265-269.002 0. a convenient and reliable method for the determination of Hyp in biological tissue samples has been established. With the retention of all advantages of the earlier procedure (11). Anal Biochem 1990. A specific method for the analysis of hydroxyproline in tissues and urine. Monitoring of collagen and collagen fragments in chromatography of protein mixtures.