Chica Boris, Vl. 29, No 199% Prine inthe CSA. All rights reserved '$0006-2952(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 Bivd., Kansas City, KS 66160 ‘A critical study of the dite ‘cedure for hydroxyproline of collagen content in tissue homogenates without os ‘advantages of the method. The procedure is based on al hydrolysis of the tissue homogenate and subsequent determi- nation of the free hydroxyproline in hydrolyzates. Chloramine-T \Was used to oxidize the free hydroxyproline for the production of a pyrrole. The addition of Ehtlich’s reagent resulted in the formation of a chromophore that can be measured at 550 nm. ‘Optimal assay conditions were determined using tissue homog- fenate and purified acid soluble collagen along with standard hydroxyproline. Critical parameters such as the amount of chlo- ramine-T, sodium hydroxide, p-dimethylaminobenzaldehyde, pH of the reaction butter, and length of oxidation time were ‘examined to obtain satisfactory resulls. The method has been applied to samples of tissue homogenate and purified acid ‘soluble collagen, with recovery of added hydroxyproline of 107 += 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 rou- tine 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 c ‘gen concentrations during purification. KEY WORDS: hydroxyproline; tissue hydrolyzate; col lagen; Ehrlich’s reagent. Introduction Hey Prline iy is. a post-transtational prod- uct of proline hydroxylation catalyzed by an en- zyme prolyhydroxylase (EC.1.14,11.2) (1). The oc- currence of this imino acid is thought to be confined almost exclusively to the connective tissue collagen, where it is present in the Y position of the Gly-X-Y repeating tripeptide (2), Because of its restricted and unique distribution in connective tissue colla- gen, the metabolism of collagen and its regulation is conveniently studied by measuring the Hyp content Correspondence: G.K. Reddy, Department of Physical ‘Therapy. University of Kansas Medical Center, 3901 Rainbow Blvd., Kansas City, KS 66160. ‘Manuscript received September 8, 199% cepted December 11, 1995, revised and ac CLINICAL BIOCHEMISTRY, VOLUME 29, JUNE 1996 ina 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 va- riety of pathological conditions, including tumor in- vasion and metastasis, rheumatoid arthritis, diabe- tes mellitus, muscular dystrophy, and chronic ul- Considering the role of collagen and i significance in many clinically important connec- tive tissue diseases, a great deal of attention has been directed toward the development of an assay capable of both detecting and measuring the coll: gen quantitatively, Measurement of Hyp in various tissues, plasma, and urine for the investigation of normal and patho- logical conditions of collagen metabolism is possible by colorimetric methods (3-6), high performance liq- uid chromatography (7,8), gas chromatography/ mass spectrometry (9), and enzymatic methods (10), ‘The numerous assay procedures deseribed for hy- droxyproline indicate the limits of each one with re- gard to specificity, sensitivity, reproducibility, a racy, and practical approach. In general, most of the procedures are laborious and involve many time- consuming 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 success- fully 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 investi- gation, 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 origi- nally introduced by Stegemann and Stadler (4). The modifications include a change in hydrolysis proce- dure, reduction of the sample volume, and omission of the neutralization step with citric acid, Since thisREDDY AND ENWEMEKA, analytical procedure is intended to be used to quan- titate 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-dimethylaminobenz- aldehyde, acid soluble collagen, and L-hydroxy- proline were purchased from Sigma Chemical Co., (St. Louis, U.S.A.). Sodium acetate, citric acid, per- chloric acid, n-propanol, sodium hydroxide, and ace- tic acid were purchased from Fisher Scientific (St. Louis, U.S.A.). All other chemicals were of analytical grade. PREPARATION OF REAGENTS Hydroxyproline stock A solution containing 1 mg/mL of Hyp was pre- pared 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 acetate- citrate buffer. Ehrlich’s reagent (1M) 15 g of p-dimethylaminobenzaldehyde was dis- solved in n-propanol/perchlorie acid (2:1 viv) 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 qualita- tive and quantitative changes of collagen during normal tissue repair process, a rabbit Achilles ten- don specimen was chosen for the measurement of, hydroxyproline. Soon after sacrificing rabbits, Achil- les tendons were removed surgically, frozen in liquid nitrogen, and then lyophilized. The lyophilized tis- sue 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, 206 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™ spectrophotometer. Absorbance values were plotted against the concentration of standard Hyp, and the presence of Hyp in unknown tissue extracts was de- termined from the standard curve. A flow chart of the hydroxyproline assay procedure follows: 1. Aliquots of standard Hyp (2-20 ug) prepared from stock solution and test samples contain- ing Hyp under 10 yg/mL were mixed gently with sodium hydroxide (2N final concentra- tion) in a total volume of 50 L. 2, The samples were hydrolyzed by autoclaving at 120°C for 20 min. 3. 450 ul of chloramine-T was added to the hy- drolyzate, mixed gently, and the oxidation was allowed to proceed for 25 min at room tempera- ture. 4. 500 pL 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 homog- enate and purified acid soluble collagen along with the standard Hyp. The calibration curve was ini- tially 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. D. Since this procedure was originally intended to be used for quantitating collagen in tissue homog- enates, 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 measure- ment of Hyp. EFFECTS OF ALKALI AND CHLOROMINE T CONCENTRATIONS, OXIDATION TIME, AND PH ON CHROMOPHORE FORMATION, Initially we examined the effects of various con- centrations of sodium hydroxide and chloramine-T CLINICAL BIOCHEMISTRY, VOLUME 29, JUNE 1996TERMINATION OF TIS Absorbance at 550 nm 4 8 2 1 2 2 Hydroxyproline (ug) Figure 1 — Calibration curve for the assay of hydroxypro- line. A designated amount of standard hydroxyproline was pipetted into test tubes and assayed as deseribed under 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 concentra- tion) was found to be optimal for the hydrolysis of the sample (Fig. 2A), At a lower concentration of sodium hydroxide, the formation of the chromophore was found to be lower due to incomplete hydrolysis of the sample. However, higher concentrations of, sodium hydroxide (>2N) did not influence the hydro- lysis of the samples and, thus, no changes were detected in chromophore absorbance readings. Simi- larly, the influence of various amounts of chlora- mine-T (ranging 0.005M-0.06M) on the formation of pyrrol-2-carboxylic acid was examined, and the re- sults are shown in Figure 2B. Absorbance values using 0,025M chloramine-T (final concentration) were found to be maximal for all samples (ic, tissue homogenate, acid soluble collagen and standard Hyp). Although the absorbance values of standard Hyp were not altered appreciably at lower concen- trations of the chloramine-T reagent, a considerable change was observed in the absorbance values for the tissue homogenate and acid soluble collagen. Ei- ther a slight decrease or no major change was ob- served in absorbance values at higher concentr: tions of chloramine-T (>0.025M) The results in Figure 2C indicate that the devel- opment 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 ex- tension 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 values on the reactivity of chlo romine-T during the oxidation process are shown in cur NICAL BIOCHEMISTRY, VOLUME 29, JUNE 1996 Figure 2D. A pH of the acetate-citrate buffer be- ‘tween 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 ab- sorbance 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 oxi- dation of Hyp. EFFECT OF P-DIMETHYLAMINOBENZALDEHYDE CONCENTRATION We examined the effect of various concentrations of p-dimethylamino-benzaldehyde (ranging 0.125- 1.5M final concentration} in Ehrlich’s reagent on the formation of the color complex using standard Hyp. ‘As the final concentration of Ebrlich’s reagent in- creased to 0.5M, the absorbance values increased in proportion. Thereafter, the absorbance of the color complex decreased significantly as the concentra- tions of aldehyde rose to 1.5M final concentration (results not shown). Moreover, at higher concentra- tions 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 de- veloped under the conditions described above is pre- cise, 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 ug. REPRODUCIBILITY OF THE ASSAY ‘The reproducibility of the assay was demon- strated 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 ug/mL with a mean value of 6.1 wgimL and a stan- dard deviation of 0.21 ug/ml. APPLICATIONS ‘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 rab- bit 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 sa- aor