Clin. Biochem. 7, 251-257 (1974)

A N A U T O M A T E D P R O C E D U R E F O R Q U A N T I T A T I V E D E T E R M I N A T I O N OF H Y D R O X Y P R O L I N E ,

NELLY BLUMENKRANTZ and GUSTAV ASBOE-HANSEN

Un~versiby of Copenhvbgen Depar~men~ of Dermatology and Connective Tissue Resea~'ch Laboratories, Rigshospital, Copenhagen, Denmark.

(Received February ~0, 197~)

CLBIA, 7, (3) : 251-257 (1974) Clan, Bioehem.

Blumenkrantz, Nelly and As boe-Hansen, Gustav

University of Copenhagen, Departmen~ of Derma~'ology and Connective Tissue Research Labora$ories, Rigshospital, Copenhagen, Denmark.

AN AUTOMATED PROCEDURE FOR QUANTITATIVE DETERMINATION OF HYDROXYPROLINE

1. An automated procedure for quantitative assay of hydroxyproline in urine including a comparison with manual assays in common use i s described. The procedure is based on the oxidation of hydroxyproline by chloramine T in aqueous solution. The oxidation product reacts with Ehrlich's reagent, and the chromogen obtained is registered in a recorder connected to the colori- meter.

2. The assay can be used to determine hydroxyproline in other biological ma- terials as well. I t includes other reagents and a simpler flow diagram than a previously reported method for determination of hydroxyproline in colla- gen and elastin. The method is simple, sensitive, reproducible, and specific. It is proposed for studies of collagen metabolism and recommended for routine medical chemistry laboratories.

AN AUTOMATED ADAPTATION OF A MANUAL PROCEDURE for the determination of h y d r o x y p r o l i n e in u r i n e 2 is de sc r i bed . D i f f e r e n c e s in r e a g e n t s a n d

f l o w d i a g r a m r e n d e r t h i s a u t o m a t e d a s s a y m o r e s ens i t i ve t h a n an a u t o - m a t e d p r o c e d u r e e l a b o r a t e d b y G r a n t 3 on t h e bas i s of S t e g e m a n n s m a n u a l a s s a y 1

MATERIAL

Citrate-Phosphate Buffer pH 6.0 was prepared as follows: 1) A 0.005 M solution of citric acid. 2) A 0.01 M solution of dibasic sodium phosphate. 17.9 ml of solution 1 was mixed with 32.1 ml of solution 2 and the total volume diluted with distilled water to 100 ml.

Chloramine T solution. A 0.0177 M solution in distilled water was prepared. When kept in the refrigerator in a dark, tightly capped bottle, the solution could be used for up to 15 days.

Correspondence to: Nelly Blumenkrantz, Ph.D.

252 B L U M E N K R A N T Z et al

Ehrlich's reagent. 7.0 g para-dimethylaminobenzaldehyde was dissolved in 15 ml of 70% perchloric acid and the solution adjusted to 100 ml with isopropyl alcohol.

Sodium hydroxyde solution. A 6 N NaOH solution in distilled water.

Standard solutions. Solutions of hydroxypro~ine (Hypro) containing 2, 4 and 6 ~g per ml buffer were prepared as indicated below. (See "preparat ion of samples"). Solutions of higher and lower concentrations were also used to determine minimum and max- imum sensitivity. Solutions of proline (Pr) 12, 18 and 24 ~g per ml buffer were also assayed.

Preparation of samples.

24-hour urines were collected from individuals on collagen-free diet. One ml of filtered urine was added to an equal volume of 12 N HCI and then hydrolysed at 110 ° for 16 hours or at 130 ° for 3 hours. Two different procedures were used af ter hydrolysis.

1. Evaporation of the HCI under vacuum at 65 ° followed by suspension in 2 ml buffer. 0.5 ml of the suspension was diluted to 10 ml with buffer. 2. Neutralization of 0.5 ml of the suspension with 0.5 ml of 6 N NaOH followed by addition of 9 ml buffer. In both cases, the diluted samples were centrifuged at 2900 rpm for 10 minutes, and the supernatant was used for Hypro determination. Standards were submitted to a similar procedure, i.e. heating with 6 N HCI followed b y evaporation or neutraliza- tion of the acid and fur ther dilution with the buffer. The Hypro standards with a final concentration of 2, 4 and 6 I~g/ml were used in every run.

For the determination of hydroxyproline in other biological materials, e.g. tibiae of chick embryos and skin, a similar procedure was followed.

Autoanalyzer

The instrument used for the the analysis was the Auto-analyzer produced by Tech- nicon Instruments Corporation, Chauncey, N.Y.

ASSAY

The flow diagram for the quantitative automated assay of Hypro is shown in Fig 1. Alternating cups of sample and water are placed in the sampler. The samples are fed into the machine at the rate of 60-2/1 sam- ples per hour, i .e. of the 60 seconds corresponding to each sample 40 sec- onds are used for delivery of the sample and 20 seconds for the water. Accordingly, the actual volume of the samples delivered is 2 /3 of that indicated in the flow diagram, i .e. 0.53 ml. Acidflex tubing is used for the intake and delivery of Ehrlich's reagent and for the waste flow, while in all the other steps clear Tygon tubing is used. The reaction is carried out as follows. Chloramine T is added to the sample whereafter they f low together throughout the stream to be mixed in a 14 turns' single coil. The oxidation is performed while the combined flow passes a time-delay coil of 10 turns (made from an original ~31/~ turn coil). After the oxida- tion, Ehrlich's reagent is added and the flow mixed in another single coil. The current is heated at 75 ° in a heating bath with a delay coil of 281//.o turns and then passed through a third single coil. The color produced is measured with a 555 nm interference filter in the colorimeter attached to

HYDROXYPROLINE DETERMINATION 253

FI,,QW DIAGRAM Proportionating pump

Oellvery ml/mm. I0 Iurns

OePoy coil 14 lurns

Single toll

q.l

_ ~ Ct

P Wosle = Acl Single COd I

t

Single coil AC: Acidflex lubing CI : Clear TycJon tubing

olorlmeter Recorder: filter 555 nm

Hea lincj bolh 75 °C

Air O.G 0

Chlorom~ne r 1.2 Sample 2 o

0 Somple 0.8 Woste~

O0 WaferE nrlich's reocjen I 2.01"2 ~°OoooO°

2.39 0

Fig. 1 - - Auto-analyzer flow diagram for hydroxyproline assay.

the machine. The calorimeter is fitted to the machine with a 1.5 mm f low cell. The transmissions are registered in the recorder connected to the calorimeter. In case low concentrations of hydroxyproline are measured, a Goerz-Servogor S RE 543 recorder is used. The transmissions are con- vetted into absorbances by using the ruler belonging to the machine.

When the run is finished, the tubing used for the intake of Ehrlich's reagent is washed with iso-propanol, while all the rest of the system is washed with 1 N HC1.

COMMENTS

Effect of pH of diluting buffer. Maximum color yields were obtained by using buffer pH 6.0. However, no difference in color yield was observed in case other buffers of the same molarity and pH were used. No signi- ficant differences were observed if samples or standards of pH 3 to 12 were analysed.

Effect of dilution of evaporated samples with distilled water. If the samples were diluted with distilled water instead of buffer pH 6, slightly lower color yields were obtained although the difference was not signi- ficant.

254 B L U M E N K R A N T Z e$ al

GTO0

0.600

0.500

0.40C

,q 0.30C

0.20(:

O.IOC

I 1 1 I

Hydroxyproline : 1.06 / ~

I I0

B I

G I

I I

I I

I I I

i m r & ~ A I

o / i / ; / $ ! ! o !

o ! 0 . / o

0

o !

o !

20 30 40 50 60 70 "80 90

T E M P E R A T U R E =(3

Fig. 2 - - E f fec t of temperature on the chromogen ~ v e n by the p ~ r o ] e der ivat ive of hydroxyprol ine and Ehrl ich 's reagent .

Effect of temperature and time of heating on color yield. Maximum color yields were obtained at 75 ° under the experimental conditions de- scribed (Fig. 2). A decrease in the duration of the heat ing step caused a decrease in the color yield (Table 1).

Effect of isopropanoI and n-propanol in Ehrlich's reagent. If isopropanol

H Y D R O X Y P R O L I N E D E T E R M I N A T I O N

T A B L E 1

COLOR YIELDS UNDER DIFFERENT HEATING CONDITION ¢~

255

Heating Jacketed coil Delay'coil

20 turns 1 double coil 2 jacketed cogs 2 double coils 28~ turns Hypro ~g 10 min. 11 rain 12 rain 12.5 min 16.5 rain

1.06 0.078 b 0.115 0.140 0.165 0.215 2.12 0.164 1.230 0.280 0.330 0.430 3.18 0.251 0.345 0.398 0.485 0.620

• )Times represent complete reaction periods b)Values are expressed as absorbances at 555 nm.

1.0 O C Z J I I I 1 I I I 1 I I l I , i

0.8

0.7

0.6 6 =

0.5

~,. 0.4 I,,.

~.1 0.3

.,,d

t~O.2

0.1

4

01$ I - ,

I I I 1 I

2

i !.3 I.I

0.8

0.4

15 ' 1.7

p.g HYDROXYPROLINE

I I I I I I I I I .

6

4

2 2

Fig . 3 - - A u t o m a t e d a s s a y f o r h y d r o x y p r o l i n e . S t a n d a r d curve .

T A B L E 2

U R I N A R Y EXCRETION OF HYDROXYPROLINE.

COMPARISON OF THE NEW AUTOMATED ASSAY WITH SOME MANUAL PROCEDURES.

Number of Hydroxyprol ine in urine samples rag/24 hrs.

Kivir ikko et al. Blumenkrantz and Blumenkrantz and New automated assay (4) Asboe-Hansen (5) Asboe-Hansen (6)

28 42.2 4- 2.4,) 40.1 -4- 6.0 44.5 4- 3.2 45 ~ 2

• )Figures represent mean ~ s tandard error of the mean.

256 B L U M E N K R A N T Z e$ al

o.9 Q8 0.7 6 6

pH 3.0 HzO O.

JA . . . . . :[ o. 4 4 pH 3.O HzO

~. 0.4

",J pH 3.0

~0.2

0.1

/~.g HYDROXYPROL INE

Fig. 4 - - E f fec t of pH on the au tomated assay. Compar i son of the proposed pro- cedure wi th G r a n t ' s procedure.

was used, the chromogen obtained was maximum and considerably strong- er than with n-propanol which was recommended by Grant s

Effect of solvent and concentration of chloramine T. A maximum color yield was obtained if 0.0177 M chloramine T in water was used as oxidant, whereas the chloramine T solution used by Grant was a 0.2 M solution in citrate buffer pH 6 and n-propanol, which, in our experiments, gave a considerably lower color yield.

Automated assay for hydroxyproline as related to other procedures. As shown in Table 2, no significant differences in the urinary hydroxyproline values were observed if compared to the most sensitive manual assays (4, 5, 6).

Sensitivity and specificity. The reaction was sensitive in the range of 0.2 to 6 ~g. (Fig. 3). No interference of proline was observed.

DIscussIOl~

In Grant's procedure 3, standards were adjusted to pH 3.0, while the samples hydrolysed in 6 N HC1 had a pH less than 1 after the addition of an equal volume of 1 N NaOH and dilution with water. The significance

HYDROXYPROLINE DETERMINATION 257

of this pH difference remains unexplained. In our assay, identical treat- ment of samples and standards ensured more constant results than hither- to obtained by manual and automated procedures.

Grant used a more complex flow diagram, i .e. perchloric acid was added to the sample-oxidant flow and mixed through 2 double coils, whereaf ter Ehrl ich 's reagent was introduced into the system as a separate reagent. Our adding of perchloric acid to the Ehrl ich reagent simplifies the pro- cedure. The substitution of the cooling coil by a single coil also makes the procedure more simple.

The higher temperature used during the reaction increases the sen- si t ivity of the method (Fig. 2).

In contradistinction to the Grant method 3, our modifications ensure a re turn to the base-line after the peaks (Fig. 4).

The procedure is quick, reproducible, sensitive and specific. I t is recom- mended for routine medical chemistry laboratories.

ACKNOWLEDGMENT

The expert technical assistance of Ole Christensen is acknowledged.

REFERENCES

1. Stegemann, H. (1958). Z. Physiol. Chem. 311, 41-45. 2. Stegemann, H. and Stalder, K. (1967). Clin. Chim. Ac ta 18, 267-273. 3. Grant, R. A. (1964). J. Clin. Path. 17, 685-686. 3a. Grant, R. A. Application of the auto-analyzer to connective tissue analyses.

The determination of hydroxyproline. Technicon Methodology Sheet. p. 1-4. 4. Kivirikko, K. I., Lait~nen, O. and Prockop, D. A. (1967). Anal. Biochs~r~ 19, 249-255. 5. Blumenkrantz, N. and Asboe-Hansen, G. (1973). Anal. Biochem~ 55, 288-291. 6. Blumenkrantz, N. and Asboe-Hansen, G. Assay for hydroxyproline and proline on

one sample. Anal. Biovhem (In press)