219

Clinica

Chimica

0 Elsevier

Acta,

Scientific

64 (1975)

Publishing

BRIEF TECHNICAL

219-221 Company,

Amsterdam

- Printed

in The Netherlands

NOTE

CGA 1304

A SIMPLIFIED METHOD FOR THE ESTIMATION TOTAL HYDROXYPROLINE

B.T. NOBBS, A.W. WALKER Department

(Received

OF URINARY

and T.J. DAVIES

of Clinical Biochemistry,

St. Luke’s

Hospital,

Guildford,

Surrey

(U.K.)

April 4, 1975)

Introduction A urinary hydroxyproline assay suitable for handling large numbers of samples was sought as part of a screening programme for the early detection of secondary malignancy in bone. A number of published techniques [l-5] were tried, including several automated procedures, but the most suitable was found to be a modification of a manual method [5] which uses a strong cation exchange resin to bind “free” and “peptide-bound” hydroxyproline and to catalyse the subsequent acid hydrolysis of the peptides. The original technique was in the form of a kit under the tradename “Hypronosticon” (Organon Laboratories Ltd), but this proved too cumbersome and uneconomic for large scale screening. The modification here outlined differs from the original with respect to hydrolysis time, composition of reagents and standardisation. Materials Resin

Amberlite CG-120 type 200 mesh cation exchange resin (BDH), converted from the Na’ to the H’ form by treatment with hydrochloric acid. Chloramine-T oxidant 0.7 g Chlorarnine-T (BDH) was dissolved pH 6.0; 12.65, plus 15 ml propan-2-01. Colour reagent 25 g p-Dimethylamino-benzaldehyde

acid and propan-2-01

in 35 ml citrate/acetate

(BDH) dissolved added to a final volume of 500 ml.

buffer

in 50 ml perchloric

220

Standard 0.5 mmol/l Hydrolysis

L-hydroxyproline

in water.

tubes (Sovirel,

Screw-capped 2.50 ml.

Levallois-Perret, France) borosilicate glass tubes calibrated

in the

laboratory

at

Specimens

Preliminary experiments indicated that interference by free hydroxyproline of dietary origin could be minimized by imposing a meat and gelatine free diet for only 24 hours prior to collection of a single morning, but not overnight, specimen of urine. Method 200-300 mg of resin were weighed into each hydrolysis tube and 0.5 ml urine added. A standard and a blank tube containing 0.5 ml standard or deionised water, respectively, were also set up. The resin was washed by adding 9 ml deionised water, centrifuging at 2000 X g for 5 minutes, and decanting off the clear supernatant. The tubes were then autoclaved at 121°C for 3 hours. After cooling, the hydroxyproline was eluted off under mildly alkaline conditions and the volume in each tube made up to 2.5 ml. To 0.5 ml of the supematant, 1.0 ml propan-Z-01 and 0.5 ml fresh chloramine-T oxidant were added, followed after 5 minutes by 5.0 ml colour reagent. After vigorous mixing, the tubes were incubated at 60°C for 20 minutes and then cooled to room temperature. The absorbance at 560 nm was measured against the blank in a Gilford 300-N spectrophotometer set to read concentration directly. The instrument was calibrated using the 0.5 mmol/l standard. The ratio of hydroxyproline to urinary creatinine was then calculated. Creatinine was estimated by reaction with alkaline picrate using Technicon AutoAnalyzer method N-11 b. Discussion The method was found to be linear between 0.02 and 1.5 mmol/l hydroxyproiine. No ~terference was observed from urea, creatinine, L&mine, L-glutamic acid, L-tryptophan, L-phenylalanine, ascorbic acid, porphobilinogen or delta-amino-laevulinic acid. Of the compounds investigated for interference with the assay, only L-proline produced an effect, 10 mmol/l proline increasing the apparent hydroxyproline concentration by 0.11 mmol/l. Recoveries of 96 to 104% were obtained, and analyses in duplicate on a total of 100 samples ranging in concentration from 0.02 to 0.36 mmol/l hydroxproline gave a standard deviation of 0.0045 about a mean of 0.084 mmol/l. Using this technique we confirmed the finding by Mautalen [6] of a circadian rhythm in the hydroxyproline : creatinine ratio, with the sample

221

collected between 08.00 and 12.00 approximating most closely to the mean daily value. The use of the hydroxyproline : creatinine ratio does not appear to result in any loss of sensitivity and indeed it has been claimed that this ratio is of greater predictive value for the presence of bony metastases than is the total hydroxyproline concentration alone [ 71. The adoption of a single morning sample is much more convenient than 24 hour collections for screening purposes and also halves the overall period of dietary restriction. References 1

D.J.

Prockop

and

2

R.A.

Grant,

J. Clin.

3

C.

Dreux

Brighton.

and

J.

1967,

4

C.A.

Pennock,

5

B.C.

Goverde

6

C.A.

Mautalen,

7

A.

Cuscbieri

S. Udenfriend, Pathol.,

17

Leymarie.

Anal.

Biochem.,

(1964)

1 (1960)

228

685

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in

Clinical

Chemistry.

Technicon

European

P. 151 G.R. and

Moore F.J.N.

Clin. and

R.A.

Sci.

and

M.D.

Hoyle.

Veenkamp,

Clin.

Mol.

46

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Med..

J. Med. Chin

(1974)

Br. J. Exp.

Lab.

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41

Technol.. (1972)

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27 29

(1970)

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Symposium,

A simplified method for the estimation of urinary total hydroxyproline.

219 Clinica Chimica 0 Elsevier Acta, Scientific 64 (1975) Publishing BRIEF TECHNICAL 219-221 Company, Amsterdam - Printed in The Netherlan...
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