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
Automation
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
F&ate,
Med..
J. Med. Chin
(1974)
Br. J. Exp.
Lab.
Acta,
41
Technol.. (1972)
419
Pathol..
53
(1972)
237
27 29
(1970)
302
Symposium,
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
Automation
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
F&ate,
Med..
J. Med. Chin
(1974)
Br. J. Exp.
Lab.
Acta,
41
Technol.. (1972)
419
Pathol..
53
(1972)
237
27 29
(1970)
302
Symposium,
