CLINICA CHIMICA ACTA

DETERMINATION

HERMXNN

267

OF HYDROXYPROLINE

STEGEMANN

lnstitut fCr Riochew&,

AND KARLHEINZ

Max-Plan&Institut

Biologische Bundesanstalt,
fiiv expevimentelle
Medizin,

(hceivcd

1967)

July jrd,

STALDER
351 Ham.
Miinden and
34 Gittingen (Germany)

A critical review is given of hydroxyproline

analysis. Our procedure published
previously-oxidation
of the imino acid by chloramine-T
in a buffer near neutrality
and coupling of the chromogen formed with Ehrlichs
aldehyde in strong perchloric acid-is
re-evaluated in the light of several modifications of this method applied by different investigators. It was possible to establish a procedure which is faster
and more sensitive than the former one and as reliable. The range is from 0.2 up to
6pg hydroxyproline
in a z-ml sample. A certain deviation from the standard procedure
will not appreciably influence the results.
For material containing little hydroxyproline
(urine, plant or animal tissue)
separation from interfering substances by ion exchange is advisable.

Among the methods for the determination

of hydroxyproline
the oxidation of
this imino acid to a compound reacting with p-dimethylaminobenzaldehyde
to form
a chromophore is regarded as a relatively specific and sensitive assay.
For the oxidation either Neuman and Logans hydrogen peroxide treatment in
strong alkaline solution is used or the chloramine-T
(sodium N-chloro-p-toluenesulfonamide) procedure in a buffer near neutrality introduced by us2. It has been shown
that neither pyrrole nor pyrrole- or hydroxypyrroline-carboxylic
acid act as chromogens and that hydroxyproline
or its derivatives behave similarly under both conditions of the analysi9. Closely related compounds interfere little or not at a116. Especially, tyrosine gives no color under our conditions and an excess of the oxidizing
agent, which will bleach the colored solution, is more conveniently
destroyed in the
case of chloramine-T
than in that of hydrogen peroxide.
Our method has been modified by several workerW9 or compared with other
methodsa. The present paper re-evaluates the procedure for hydroxyproline
in hydrolysates of tissues, of blood plasma and urine, since during recent years, formation and
metabolism of this imino acid have received increased attention and itsa mount often
serves as a diagnostic tool. The method as it is presented in this paper is faster and
more sensitive than our former procedure.
Clin. Chim.

Acta.

18 (1967)

267-273

268

STEGEMANN,

STAIDER

MATERIALS

Chloramine-T. 1.41 g chloramine-T (sodium N-chloro-$-toluene sulfonamide)

is dissolved in IO ml water, IO ml n-propanol is added followed by 80 ml of the buffer
pH 6 (stable for several weeks in a dark bottle at 4).
~~e~~~elpeyc~Zoy~cacid (stable for several hours and not more than 2 days,
sufficient for IOO determinations). 15.0 g of $-dimethyl-amino-benzaldehyde
in a
volumetric flask (IOO ml) are suspended in 60 ml n-propanol and 26 ml of perchloric
acid (60%, techn.) should be added slowly. The volume is made up with n-propanol.
Bugler pH 6 (ref. 2).
50.0 g citric acid *I II,0 (analytical grade),
IZ ml acetic acid (g6%),
IZO g sodium acetate.3
Hz0 (analytical grade) and
34 g sodium hydroxide are dissolved in water and brought to IOOO ml: Ten
drops of toluene were added as preservative.
(a) The buffer is used for the color reaction, by taking 500 ml and adding IOOml
of water and r50 ml of ti-propanol. This buffer is stable for several months.
(b) For the elution of the cation exchange columns in the pretreatment step
of urine, 50 ml of the stock buffer have to be diluted with 450 ml of water.
PROCEDURE

(A)

Hydrolysis of tissue
Hydrolysis is carried out in 6 N I-ICI (usually x ml/I0 mg tissue or protein) at
107 for 16 h in a test tube with ball joint. Adding charcoal in a IOOOfold excess to
hydroxyproline does not influence the recovery of the iminoacid. Most of the HCl in
the hydrolysate should be removed by evaporation to dryness.

Two ml of filtered urine (human or from laboratory animals) is neutralized and

heated for IO min at 80. Precipitated carbonates and proteins are centrifuged off,
the precipitate is washed once with destilled water. The supernatants are combined
and applied to a column of 0.8 cm diameter filled with IO ml Amberlite IRA 410
(OH-) IOO-zoo mesh. The column is first eluted with 35 ml of distilled water (fraction
I) then with 50 ml of I N HCl (fraction 2). Free hydroxyproline may be determined
in the unhydrolysed fraction 2. For the determination of the peptide-bound hydroxyproline both fractions are hydrolysed.
(C) P~etyeat~~e~tof plasma
5 ml plasma are autoclaved (3 h, 30 psi), Precipitated protein is removed by
filtration and washed several times with small amounts of water. Initial filtrate and
washings are combined and applied to a column with IO ml Amberlite IRA 410 (OH-,
100-200 mesh), as done with urine, see R. High molecular gelatin-like substances are
eluted with 35 ml of distilled water (fraction I). Smaller hydroxyproline peptides and
free hydroxyproline are then eluted with 50 ml I N HCl (fraction 2). For the determination of total hydroxyproline the fractions have to be hydrolysed at this stage;
see Table II also.
C&n. Cl&n. Acta, 18 (1967) 267-273

IIYDROXYPROLINE

(D) Hydrolysis

DETERMINATION

of peptide-bound

269

hydroxyproline

N HCl is added to make the solution 6 N. Hydrolysis is performed overnight

at 107. Thereafter the hydrochloric acid is removed by vacuum distillation using a
flash evaporator.The
hydrolysate is taken up in 5 ml of diluted acetate-citrate
buffer
pH 6 and passed through a column containing 3 ml Dowex 50 x 8 (H+) 200-400 mesh
equilibrated with the same buffer. The column is washed twice with 2 ml of buffer,
the effluent is collected in a ro-ml volumetric flask. After the column has run dry
the volumetric flask is brought to volume. I or 2 ml are usually sufficient for one
12

hydroxyproline

determination

as described

under E.

(E)

Determination
of hydroxyproline
The volume of the sample containing 0.2-6 pg of hydroxyproline
should be
(or brought to) 2 ml. The sample may have passed through the ion exchange column.
The effluents buffer capacity and pH should be in accordance with the pH of the
chloramine-T
reagent. The sample (2 ml) is mixed in a test tube with I ml chloramine-T solution both having a temperature of about 20. If one uses pipettes, handling of more than 40 tubes is not advisable. With a semiautomatic
pipette (like the
2 cc Mini-pet of the Manostat
Corp. N.Y., USA) 300 tubes may be analyzed in one
run. After 20 min I ml of the aldehyde/perchloric
acid solution is added and the mix-

ture shaken thoroughly. If no schlieren can be seen any more, the tubes are immersed
sed about 6 cm deep into a waterbath big enough to keep the temperature at 60 even
when a large number of tubes are handled. After 15 min, the rack is cooled under tapwater and the color is read in a photometer (e.g. Zeiss-Elk0 III) using a filter (550 nm)
within 45 min.-One
may shake the mixture with 2 ml of r,z-dichlorobenzene.
After
settling, the color of the lower layer is read (stable for at least 24 h). Sensitivity is increased about two times.
RESULTS AKD DISCUSSIOK

The hydrolysis of hydroxyproline-containing

proteins was recently investigated
by Pashley et aL9 and the resulting hydroxyprolines
analyzed. For procedures involving resolution on ion exchange columns it should be taken into account that allohydroxyproline
is formed during hydrolysis and that this compound is eluted from
acidic resins later. The degree of conversion in relation to time, concentration
of the
iminoacid and excess of HCl was investigated by Stalder et a1.5. For little conversion
to allo-hydroxyproline,
the excess of HCl should be smaller as recommended for best
stability of other hydroxy amino acids. Chloramine-T in a buffer at about pH6 was
first introduced by Stegemannz. Perchloric acid was substituted for sulfuric acid to
avoid precipitation of calcium during the analysis of bonelo. Most of the later modifications did not change these conditions too radically. Buffer of pH 8 has been employed7; different concentrations of reagents were applied39%7, or a higher concentration
of organic solvents introduced 3. The method was converted to a fully automatic
procedure in a Technicon analyzer.
Various hydroxyproline
determinations
using hydrogen peroxide to oxidize
this iminoacid were critically compared with the method using chloramine-T8,
and
it was found that the latter seemed to be especially reliable. Most of our work has
Clin. Chim. Acta, 18 (1967) 267473

STEGEMASN, STAI,I)EII

270

been done with our procedure described in 1958 and proved to be re~roduciblc.
How
ever, recently, we have reexamined
the following variables:
a. The concentration
of the oxidizing Chloramine-T
solution has been kept at
0.017 M2 as adopted by3 and6. Lower concentration-for
instance one seventh, as
preferred by7-will
improve somewhat the color yield in the upper range of the OHproline curve (see Table I, A I), however, the determination
is much more dependent
on the amount of other constituents
and their elimination
by extraction
or ion exchange becomes essential.
b. The pH-value should be near neutrality,
as recommended earliert. Between
pH 6 and the later modification to pH 8 (ref. 7) there is no difference in the slope of the
extinction curve (Table I, A 2). Usually, one has an acid hydrolysate.
After removing
hydrochloric
acid by evaporation,
the citrate buffer can accommodate
the rest of
the acid.
c. The time the chloramine-T
acts on hydroxyproline
is of little influence
(Table I, A 3) and a large number of tubes may be analyzed. With a senliautomatic
pipette, resistant to organic solvents as well as perchloric acid, we handle up to 300
tubes in one run.

--_.
_-_.pugHydroxyproline
---______
6
2
4

_.~._

~~
-.
,j. ChEoramine-T

Extinction

I. Concentration
0.5 standard units
I .o standard units
2.0 standard units
2. pH of the solution
pH 6 (standard)

pH 8*
pH 8
3, Reaction time
20 min (standard)
30 nun
45 nun

9.5
190
78s

390
380
368

580
565
525

r9o
199
190

380
384
3Q

565

r9o
192

3So
386
380

560
560
5.5

r9r
189
=go
187

355
355
375
374
376
374

527
.ir5**
5.57
554**
555
552**

r9o
r9o
194
195
193

376
375
381
378
37

545
555
5%
565
545

190

13. Ald~h~d~-~e~c~~~y~c acid mixture

I Concentration of the aldehyde
with 15.3% HClO, in the mixture
with IO:,& aldehyde in the mixture

180
180

with I 5f:/0 aldehyde in the mixture (standard)

with

20%

aldehyde in the mixture*

2. HClO, concentration
with 15% aldehyde in the mixture
with r 1.7% HCIO,
with 13.5%
with 15.396 (standard)
with x7.1%
,,
with 18.9%
,,
* Alteration of pH compensated
** Readings after I hour.

by calculated

10%

amounts of perchloric

j62
,358

._

acid.

HYDROXYPROLINE

values

DETERMINATION

271

d. An increased concentration
of the Ehrlich aldehyde has no effect on low
of hydroxyproline,
but improves both stability and yield of the resulting

chromophore at higher levels. 20% aldehydea, was compared with 10%~ and 15%
and the latter chosen for the standard procedure (Table I, B I). The tertiary amino
group was neutralized with the corresponding amount of perchloric acid in evaluating
the best aldehyde concentration.
For automatic procedures, lower levels are preferredll.
e. Combining the aldehyde with the perchloric acid3 speeds up the handling,
but decreases the stability of this reagent. The perchloric acid concentration
could be
varied in a wide range. For optimal results we take a mixture with 15.60/~ perchloric
acids (Table I, B 2).
f. As the organic solvent we introduced in 1958 methyl cellosolve for its high
boiling point and good dissolving properties. The glycol-monomethyl
ether was also
taken over by Prockop and Udenfriend7 as well as by Woessnere. Bergmana, on the
other hand, replaced it by propanol-2. The reported instability-after
a few weeksof reagents in cellosolve is probably due to the solvent not being carefully distilled.
TABLE

I1

READINGSOF HYDROXYPROLINEAFTER
(calculated

Treatment qf

the hydrolysate

None

4.6

4.8

Rat tail
tendon

Potato tuber
(dried)

Pulp
potato

II.2

0.013

0.2 I

II.2

0.009

0.21

from
tubers

III

INFLUENCE

Treatment

PRETREATMENTS

Rat skin

Passage through ion exchanger

TABLE

VARIOUS

as per cent of dry material, error ?I 3%)

OF

PRETREATMENT

ON

HYDROXYPROLINE

of the autoclaved plasma*

VALUES

IN

pg Hydroxyproline

Hydrolysis
Hydrolysis and passage through Dowex-50
As indicated in chapter (C)
fraction I
fraction 2

PLASMA

per milliliter

Normal
plasma

Plasma with plasma expander

Haemaccel
@ (gelatin
base)
corresponding
to 19.5 ,ug HYP

1.2
1.8

8.9
13.8

(40% recovered)
(62 o/0 recovered)

21.1

(979/o recovered)

2.2
2.6

3.0

* Same specimen of human serum throughout.

TABLE

IV

HYDROXYPROLINE
(mg

IN

RAT

URINE

excreted per day and rat)

Diet

Fract. I
0.31
0
0
Fract. 2
0.32
0.39
0.33
Diet A: Altromin (Altrogge, D 491 Lage).
Diet B: Pulp from potato tubers (mainly cell walls).
Diet C: Oats.
* Authentic

hydroxyproline

c*
0

0.61

added to urine sample C corresponding

to 0.300 mg per day

Clin. Chim. Acta,

18 (1967)

267-273

272

STEGEMANN,STALDER

(We checked both the chloramine-T

and the aldehyde solution of our former formula
after q-years standing (dark bottles, 20') and we still found 30% of the usual
color yield in hydroxyproline
determinations).
To avoid the inconvenient distillation
procedure for methyl cellosolve, we went back to n-propanol as used by Neuman and
Logan, Stegemann
and Griffin12, or for detection in thin-layer chromatographyl3.
It is more volatile than the ether, however, it does not need purification. Increased
concentration
of the organic solvent for higher readings in the hydroxyproline
determination, as proposed by Bergman and Loxley3, has been adopted. The volume of the
final mixture has been reduced from 5 to 4 ml, I ,ug of hydroxyproline
yielding an
O.D.,,, of about IOO per cm. By reducing the volumes further as did Glimcherl*,
0.01 pg hydroxyproline
will give a color twice as high as the corresponding blank. As
one can see from Table I, smaller errors in pipetting or timing do not influence the
results.
In spite of the fact that our original procedure can tolerate large amounts of
otherproteinconstituentsandanevenlargerexcessofcarbohydratesforroutineanalysis,
for best results it is advisable to remove interfering and unspecific chromogens or
inhibitors by extraction or by ion exchange procedures, if urine or tissues very low
in hydroxyproline
(less than I part in 1000) have to be analyzed. Trying both extraction and ion exchange procedures, the latter proved to be more reliable in our hands
and less time consuming. These results are in accordance with Firschein and Shills,
As reported in a previous communicationlB~17 we have adapted the chloramine-T
method for the determination
of hydroxyproline
in urine with an error of + 3%. Our
procedure includes passage through an anion exchange resin as the first step. Two
fractions are obtained. The first fraction from the direct effluent may contain precursors or breakdown products of collagen, whereas a second fraction obtained by elution
with I N HCl contains low-molecular
hydroxyproline
peptides and free hydroxyproline. This separation made it possible to differentiate
between the excretion of
larger or smaller subunits of gelatin. The same holds true for the excretion of a plasma
expander derived from gelatin and related breakdown products.
As shown in Table II, hydroxyproline
readings do not increase appreciably
after ion exchange treatment of the hydrolysate of crude collagen samples or hydroxyproline-containing
glycoproteins of other origin, e.g., pulp from potato tubers. (Values
from unpublished work of A. Rijpsch and H. Stegemann.)
However, if hydroxyproline content is very low, as in whole potato tubers, more accurate values will be
obtained after passage through ion exchange columns. For hydroxyproline
determination in plasma, a pretreatment
proved to be necessary (Table III).
Table IV demonstrates
the influence of feeding on hydroxyproline
values in
urine. Standard rat pellets may contain collagen products, therefore they contribute
to a higher hydroxyproline
excretion in urine. Hydroxyproline
as present in the pulp
of potato tubers does not increase the hydroxyproline
excretion. As further shown
in Table IV, authentic hydroxyproline
is recovered by the method described with an
error of f 3%.
REFERENCES
I R. E. NEUMANN
2 H. STEGEMANN,

AND M. A. LOGAN, J.Biol.Chem.,

Ho++e-Seylers

.< 1. BERGMAN AND R: iox~ku,

4 J. D. OGLE, R. B. ARLINCHAUS
Clin.

Chim.

Actn,

18 (1967)

267-273

184 (1950) 2gg.

Chem.. 311 (1958) 41.
35 (1963) 1961.
AND M. A. LOGAN, Arch.Biochem.Bioph~s.,

2. Physiol.
Anal. ihem.,

94 (1961)

85,

HYDROXYPROLINE

DETERMINATION

273

5 K. STALDER, H. STEGEMANN AND G. BERNHARD, Hoppe-Seylers

2. Phvsiol. Chenz., 337 (1964)

79.

6 J. F. WOESSNER, Arch. Biochem. Biophys., g3 (1961) 440.

7 D. J. PROCKOP AND S. UDENFRIEND, Anal. Biochem., I (1960) 228.
8 0. DAHL AND K. PERSSON, Acta Chem. Stand., 17 (1963) 2499.
Q D. H. PASHLEY. C. K. CLAYCOMB AND G. W. SUMMERS, Apzal. Biochem., 15 (1966) 154.
10 G. FUCHS, H. STEGEMANN AND W. EGER, Langenbecks Arch. Klin. Chir.,-3;;
(Ig6;)24o.
II Ii. A. GRANT, J. Clin. Pathol., 17 (1964) 685. Technikon-Symposium,
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13 H. STEGEMANN, K. HILLEBRECHT AND W. NIEN, Hop@-Seylers
Z. PIz.vsiol. Chew., 340 (1965)
12.

14
15
16
17

M.
H.
I(.
I<.

J. GLIMCHER, J. Ul/vnstruct. Res., 13 (1965) 163.

E. FIRSCHEIN AND J. I. SHILL, Anal. Biochem.. 14 (1966) 296.
STALDER, 2. Anal. Chew., 212 (1965) rgh.
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Z. Phvsiol. Chew., 348 (1967) 242

Clin. Chim. Acta, 18 (1967) 267-273