Determination of traces of proline in biological fluids

ANALYTICAL

BIOCHEMISTRY

Determination

226-233

19,

(1967)

of Traces D. KRUZE

of Proline

AND

in Biological

Fluids’

P. WIERZCHOWSKI

Department of General Chemistry, Krakow&i

School of Medicine, 26/28, Warsaw, Poland

Przedmkdcie

Received September

6, 1966

All the previous methods of determining proline without isolating it from the material analyzed are burdened with an error caused by the interference of other amino acids (l-3). Wren and Wigall (4) recently reported a modification of the method of Chinard (5) ; this modification also is not completely specific for proline, as it does not abolish the influence of hydroxproline and tyrosine on the color reaction. In the present work the estimation of proline is based on the color reaction with isatin after proline isolation by means of combined column-paper chromatography (6). Potato starch was used as absorbant in the columns as previously applied by Stein and Moore (7, 8) for separation of amino acids. This method is especially adjusted for determination of slight amounts of this compound in biological material, even in samples containing 2 pg of proline and a considerably higher amount of other amino acids. EXPERIMENTAL

Apparatus 1. Chromatographic columns 0.9 by 32 cm. 2. Containers, 50 ml in volume, connected with particular columns directly or by means of plastic tubing. 3. An automatic device (Fig. 1) for placing the eluate from the column on separate sheets of paper, moving continuously at the rate of 0.75 cm/hr. To evaporate the eluate deposited on the paper, a stream of warm air is blown under each column (6). 4. Glass tanks for ascending chromatography. Reagents and Adsorb&s 1. Isatin reagent, consisting of 0.2 gm of isatin dissolved in 96 ml of 80% (v/v) aqueous acetone solution with the addition of 4 ml of glacial ‘Presented at the Third Meeting of Federation Societies, April 47, 1966, Warsaw, Poland. 226

of European

Biochemical

DETERMINATION

OF

PROLINE

227

FIQ. 1. Diagram of apparatus for placing an eluate from the chromatographic column on a moving paper sheet: (a) sheet of paper; (b) column; (c) tube blowing warm air under the column outlet; (d) trace formed by the eluate which dropped and dried.

acetic. The solution can be stored in a capped flask in the refrigerator over 30 days. 2. Phenol saturated with water. Freshly distilled phenol is shaken with water and left until the phenol layer clarifies. The solution stored in a dark flask is stable at room temperature over 20 days. 3. Alcohol solutions: (a) 10% ( v / v ) a q ueous solution of n-propanol; (b) 80% (v,/v) aqueous solution of n-propanol; (c) 10% acidic solution of n-propanol (up to 90 ml of distilled water, 10 ml of n-propanol, and 2.0 ml of 1 N HCl). 4. Potato starch, thoroughly washed water, dried in air, ground in a mortar, and sieved. 5. Whatman No. 4 paper. 6. Sulfonated polystyrene resins Amberlite IR-120.

Preparation

of Mnterial.

9 vol of absolute ethanol.

The sample was deproteinized by adding The precipitated protein was centrifuged and

228

KRUZE

AND

WIERZCHOWSKI

a known volume of the cleared supernatant was diluted with an equal volume of water and desalted on cation-exchange resin Amberlite IR120 (9, 10). Amino acids bound on the ion exchanger were eluted with 6N ammonia solution; the eluate thus formed was evaporated to dryness. Preparation of Columns. A thick suspension of starch in 80% npropanol was poured into the column up the desired level (30 cm). The column filled with starch was washed with acidified 10% solution of n-propanol until an acidic eluate began to flow out; then the adsorbent was washed with 20 ml of 10% n-propanol. After chromatography, the column was regenerated by letting through it about 30 ml of acidified 10% propanol and washing with about 20 ml of 10% propanol. The regeneration #may be repeated and the column may be used an unlimited number of times within several months. Analytical Procedure. The deproteinized and desalted sample was dissolved in about 1 ‘ml of 10% propanol and placed in the column prepared in the manner described above. Immediately after the solution had soaked in, about 0.5 ml of 10% propanol was added to rinse the inner walls of the column and the column was connected to a container with 40 ml of 10% propanol. The solution should flow out of the column at a rate of about 1 ml/hr. This can be achieved by placing the container at a suitable height above the column. After 36 hr, proline and even the slower traveling amino acids flow out of the column. The sheet on which the substances were automatically placed in the form of a narrow band was subjected to ascending chromatography twice for 24 hr in 80% propanol. After chromatography, the paper was inbibed with isatin solution, partially dried at room temperature for 20 min, and subsequently developed in a chamber containing air saturated with water vapor at 70” for 25 min. Under such conditions proline formed a blue spot, which was cut on into narrow strips and placed in the tube. The yellow background was removed by twice rinsing out with 5 ml of absolute ethanol. After complete removal of the second portion of ethanol, the blue spot was eluted with 5 ml of phenol-saturated water. The process was carried out in darkness. In 10 min the content of the tube was gently mixed and within the next 5 min it was ‘mixed once more and measured directly in the photometer. The blank was prepared with slip of the paper with the yellow background in the sa’me way as the test proper. Maximum absorption of color solution was at wavelength 610 mp. A Pulfrich calorimeter with Elpho attachment (photoelectric cell) and I-58 filter was used. The adequate amount of proline for determining extinction was obtained from the standard curve prepared for various quantities of

DETERMINATION

OF

229

PROLINE

1.4 f

%

E 73 i3 .g 0”

IO

20

40

30 Proline

FIQ. 2. Relation

between

amount

50

(pg)

of proline

and optical

density.

standard solution according to the above-mentioned procedure. The calorimetric chambers used were 10 mm thick and the correlation between extinction and proline content was linear within 2.5 to 50 pg of proline in the sample (Fig. 2). RESULTS

Estimation of the error of the method taining 2.5 to 50 pg of proline. Table 1 method. The variation coefficient was samples below 5 pg was it higher. The not exceed 2%. In order to evaluate the

was performed for samples conshows the reproducibility of the lower than 5%; only for the error of the determination does losses of proline in the course of

TABLE 1 Estimation of Error of the Method in Determination of Proline Subjected to Column-Paper Chromatography and Color Reaction with &tine No. of determinations

2.5 5.0 10.0 25.0 50.0

2.55 5.02 10.14 24.80 49.78

5 5 5 5 5

Standard deviation.

0.21 0.24 0.40 0.40 1.08

pg

Relating

Accuracy

S.D.,

8.2 4.8 3.8 1.6 2.2

Irg

%

0.05 0.02 0.14 0.20 0.22

2.0 0.4 1.4 0.8 0.4

the column-paper chromatography, five replications of the analyses were carried out for each respective sample containing 2.5, 5.0, 10.0, and 25.0 pg of proline. Five other samples with the same amounts of proline were simultaneously placed on the paper and the determination was carried out under the same conditions without chromatography. The results

230

KRUZE

AND

WIERZCHOWSKI

TABLE 2 Losses of Proline in the Course of Column-Paper Without

1 2 3 4

chromatography, liB

2.5 5.0 10.0 25.0

After

Chromatography”

chromatography, lig

2.45 5.05 9.88 24.90

Recovery,

%

98 101 99 100

(1The values under the heading “Without chromatography” were obtained by placing on the paper appropriate amounts of proline dissolved in 0.1 ml of solution. Measurements were performed immediately after the color reaction with isatine.

obtained from two series of determinations differed means that there were no losses during chromatography Determination

in Biological

only by 2%. It (Table 2).

Fluids

Urine. The application of column-paper chromatography permits complete separation of proline from other chemical compounds in the urine.

Pm. 3. Chromatogram of 10 ml of deproteinized and desalted human urine of healthy adult individual: (I) direction of placing the eluate from the column; (II) amending chromatography. Pro = proline, Hy-pro = hydroxyproline ; (l-7) amino acids and other compounds giving a color reaction with isatin.

DETERMINATIOIC

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231

PROLIKE

Figure 3 represents a chromatogram developed by isatin obtained from 10 ml of urine. The first ammo acids flowing out from the column were’ proline and hydroxyproline, which were separated by ascending chromatography. As the result of both these processes proline is located in a place distant from the spots formed by other compounds. The results obtained for five different samples of urine from a healthy adult individual are shown in Table 3. The concentration of proline TABLE 3 Results of Proline Determinations in Five 10 ml Samples of Wrine and Recovery of Proline Added to Each Sample SaEllple 1

2 3 4 5

Proh$$on$.,

Proline added, Plc

4.2 7.4 5.6 10.5 6.0

Probe

5.0 5.0 5.0 5.0 5.0

Proline recovered

found, icEt

9.4 12.5 10.5 15.7 10.9

PS

%

5.2 5.1 4.9 5.2 4.9

104 102 98 104 98

for these samples ranged from 0.42 to 1.05 pg/ml. To each urine sample 5 pg of proline was added and the total amount of proline was determined. The deter’mination was repeated twice for each sample; Table 3 gives the averages obtained from two determinations. The recovery of the proline added was on the average 101% (Table 3). It means that there were no losses of proline during deproteinization and other manipulations of the material analyzed. In order to evaluate the accuracy and the precision of the determination of proline in urine, five simultaneous analyses were performed. Serum and Other Biological Fluids. Serum was deproteinized and desalted in the same way as the samples of urine and 0.5 ml samples of serum were analyzed. Proline on chromatograms gave a distinctly separated spot, as on a chromatogram of urine. The proline values for five different serum samples ranged from 15 to 24 pg./ml. The calculated variation coefficient for five samples of the same serum amounted to Error of the Method Material

Urine Plasma Cerebrospinal

fluid

TABLE 4 in Determinations of Proline in Biological

Fluids

Volume. ml

Found, pg

Relative standard deviation, ?JO

10 0.5 10

8.4 10.9 7.2

4.9 4.1 4.8

232

KRUZE

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WIERZCHOWSKI

4.1%. In addition, the method was applied to determine proline in the cerebrospinal fluid and in deproteinized hydrolyzates of urine. For proline determination 10 and 0.2 ml samples of cerebrospinal fluid and of hydrolyzed urine were taken, respectively. In both fluids analyzed the variation coefficient did not exceed 5% (Table 4).

The methods of proline determination used so far are not adapted for analysis of samples containing a few micrograms of proline in the presence of many times higher concentrations of other amino acids. For this reason most authors concluded the absence of proline in normal adult human urine or decided that its concentration was too low to be determined by accessible procedures (11, 12). The additional separation by paper chromatography makes possible a reliable isolation of proline from the material examined; and the sensitivity of the color reaction of proline with isatin on the paper allows a determination of small quantities of proline starting from 2 pg in the investigated samples; and the results are independent of the other amino acids present. Although the ion-exchange chromatographic method as developed by Stein, Moore, and Speckmann (13) is to date the most satisfactory method for amino acid analysis, the use of the isatin reagent. instead of ninhydrin in proline determination increases the sensitivity of the reaction. This method, which uses combined column-paper chromatography, is suitable for analysis of urine, blood, and protein hydrolyzates with a low proline content: For determining proline, 10 ml of urine was used. Analysis of smaller volumes increases the relative error with regard to trace proline quantities. When determining blood serum proline, it is not necessary to use just 0.5
DETERMINATION

OF

PROLINE

233

SUMMARY

A method for proline determination is described. The procedure war especially adjusted to determine small amounts of this amino acid in biological material. The first step of the analysis is the isolation of proline from other compounds by column-paper chromatography, and then the color reaction with isatin is performed. An amount of proline beginning with 2 pg in the sample may be determined by this method. REFERENCES 1.

AND LINDSLEY, J., J. Biol. Chem. 215, 655 (1955). M., Anal. Biochem. 2, 353 (1961). SUMMER, G. K., AND ROSZEL, N. O., Clin. Chem. 11, 455 (1965). WREN, J. J., AND WIGGAL, P. H., Biochem. J. 94, 216 (1965). CHINARD, F. P., J. Biol. Chem. 199, 91 (1952). WIERZCHOWSKI, P., AND KRUZJC,D., J. Chromatog. 14, 194 (1964). STEIN, W. H., AND MOORE, S., J. Biol. Chem. 176, 337 (1948). Moom, S., AND &IN, W. H., J. Bid. Chem. 178, 53 (1949). BOULANGER, P., AND BISERTE, G., Bull. Sot. Chim. Biol. 33, 1930 (1951). WIGGINS, L. F., AND WIGGINS, J. H., Nature 170, 279 (1952). KING, J. S., JR., in “Protides of Biological Fluids” (H. Peeters, ed.), p. 292. Elsevier, Amsterdam, 1965. STEIN, W. H., J. Biol. Chem. 201, 45 (1953). MOORE, S., SPACKMAN, D. H., AND STEIN, W. H., Anal. Chem. W, 1185 (1958). DRUZE, D., AND IWA~SKA, J., Chem. Anal. 10, 237 (1965). KRUZE, D., AND IWA~SKA, J., Chem. Anal. 11, 279 (1966). TROLL,

2. MESSER, 3.

4. 5. 6.

7. 8. 9.

10. 11. 12. 13. 14. 15.

W.,