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A quick and specific assay for hydroxyproline
ANALYTIC.4L
55, 288-291 (1973)
BIOCHEMISTRY
A Quick NELLY
and
Specific
BLUMENKl
Assay AND
for
Hydroxyproline
GUSTAV
ASBOE-HANSEN
of Copc&rgerl, Depndmetlt of Demmtology (with Conneclive Lnbomtorie.s), Rigshospitctl, 2100 Copenhagerl, Dennmrk
I:t~ir~aily
Tissue
liesearch
Rccr%ivcd
December
26, 1972;
accepted
March
23, 1973
H,ydrosypl~olin~~ is oxidized by I)criodic arid and various periodates in strong acid medium, while proline is oxidized only in neutral medium (1). This phenomenon led us to ekbornting an assay method for quantitative dcterminntion of hydroxyproline in the prc’scnce of proline. The proccdnte and ik application for quantitatiw ussay of 11~dro~.vproline in isolntcd collagen and in skin and urine are rellortccl holom.
Culture tubes, ncrcw capped with Teflon liners (Kimax) 120 X 15 mm were used. Periodic ncid solution. A I-M solution of periodic acid (Fischer Scientific Co.! in 9 M H,,PC)., (E. Merck A. G., Darmstadt) was prepared and stored in brown bottles for up to 6 months in the dark. Citrate-phosphate bzlffer. The buffer was prepared by mixing 154 ml of 0.1.5M citric acid (Fischer Scientific Co.) with 346 ml 08.6M dibasic sodium phosphate (J. T. Baker Chemical Co.). The final pH of the buffer was 7.0. Extrnctio?z
sol&ion. The extraction solution was prepared by mixing 2.50 ml of toluene with 250 ml of isobutanol and 100 ml of n-propanol. Stcmlnrrl hydrosyproline solution. L-Hydroxyproline was prepared at ii concentration of 10 ,tg/ml. ~Otunclnrrl proline sollrtion,. L-Prolinc was prepared at two different concentrations, i.e., 50 ,,g,/ml and 200 ,~g/nil. p-~in~efhylanlinobensaldeh~~e~ (Ehrl’ch reagent) (analytical grade, E. hZerc,k A. G., Darmstadt). Fifteen milliliters of isobutanol was added to 4 g of p-dimethylaminobenzaldehyde in a beaker, and then 4.5 ml of perchloric acid (70%, E. Merck A. G., Darmst.adt) was added. The solution ins st,oretl for several weeks at, 4°C in a dark bottle. &w~~~ Pwcrcl/rre. Samples were hydrolyzed overnight in 6 N HCI in senlcd t,llbes at 118°C. The hydrolyzatcs were evaporated to dryness in I’~CROat 6.5”C. Evaporated samples were cdissolvcdin distilled water. 2.s Copyright All rights
0 1973 l,y Academic PWSS. Inc. of rc,production in any form rearrvcd.
IIYI)ROXYPROLINE
ASSAY
289
One milliliter of the snm~)le (containing 2.5-50 & hydroxyproline) WL.F acldcd to 6 ml buffer and 0.5 ml of I ;\;I periodic acid in 9 XI I-I,PO, anIl the solut8ion carefully mixed. Then 2.0 ml of the extraction solution wcrc added and the tubes stirred in a Vortes miser. TIw tubes were ~&HA in a test tube rack, and the rack was co~crcd with aluminum foil and shaken on an Equipoise horizont’nl shaking machine for 30 min. The tubes acre then ccntrifllgctl at. low q~c11 during 10 min in order t.0 sq)ar:ttc~ the acl~~ous and organic phase A semimicro or micro aclaptation oi’ the, assay proccdurc was lWrformei1 in case n low content of hydrozyproline was suspected. The only difference with t,he nbovementimed procetlurc were the ~ol\mxe~ of the samples and the reagents added. For t,he scmimicbro assay 3 ml bl8fcr, 0.5 ml periodic acid, and 1.75 ml of the extraction mistluc were added to 0.5 ml of the sample. For the microassay 1.5 ml buffer, 0.5 ml l,criodir acid. ancl 1.2 ml estraction rnisturc WL‘C added to 0.25 ml of the sanlplc. To 600 ,~l of the organic phase 150 /II of Ehrlich reagent were added. The tubes were stirred vigorously. The color was alloyed to develop through 15 min at room tempsrnture, ant1 the nl)sorbancc was read at 565 11111on :t Beckman I>IJ spc~trol~hotomctcr. The color w\;ns stable for 4 hr.
Assays of pure hydrosypraline stnndnrds indicated that the absorbance obtained at 565 nm was proportional to tlic content, of ligclrosyproline. The rcartion was sensitive oTer a broad r:~ngr, ant1 it followed the Im~~bcrt and Beer lag from “,-50 1~6 (Fig. 1) The micromethod allowed determinatjiou of liytlrosyl~roline from l-20 II.F;, while with the semimicro assay 2.5-40 ,~g c:m IX determined.
The specificity of the reaction was tesicd with known mixtures of proline and liydroxyproline, running parallel determinations by the method of Bivirikko et nl. (2) and our assay on aliquots of similar coml~osition. The results obtained with t,he two methods were in good agreement. The adrant’age of our assay method was its requiring approximately $4 of the time and fewer steps with the same specificity (100 assays can be performed within 4 hr), while, on the other hand, Kiviiikko et CL’S (2) metllocl is more sensitive. Urinary excretion of hydrosyproline was also measured with our assay method and with that of Kivirikko et al. (2). Comparable results were obtained (Table 1). h~say~ of hydrolyzed skin collagen with our method yielded values for t~ydroxyproline similar to thoec of the above-mentioned authors (2 1. The I-M periodic acid in 9 M H,.PO, used in the oxidative step, keeping for l)criotls of 6 or more months, offers an advantage over the chloramine T used by Kivirikko et CIZ. (2)) which has to be prepared daily. When sulfosnlicylic acid is used to deprot’einize urine and to detect free h~drosyproline in the supernntant fluid, our procedure needs no llrcvious neutralization, mhilr, according to our experience, the Kivirikko et (II. method (2) needs neutraliznt.ion to get optimum osicintion. AIo.>t. of the methods for qu:mt,itnt.ive assay of Iiydrosyproline are based on its oxidation to a l)yrrol derivative, which can react wit,h P-tlimetl~~lnmi~~obc~~zal~l~~l~ycle. I\Iany oxidants have been used in the diverse methods (hypochlorite, hydrogen peroxide, hypobromite, and chlornmine T) (2-6). Ron-ever, periodic acid has not been used with that purpose. Hydrosylysine and li~drosyl~roline, the two amino acids characteristic of collagen, can be detcrminetl on the same sample by utilizing the differc>nt capability of aqueous an(1 acid solutions of periodic acid for osidntion (7 1. Determination Sample
Nn.
Urine Skin
IS IO
of the HydrcqprtIline with the New
TAULF: 1 Content in Human Away PIZethoda
Hypro mg,‘% ,g!‘jmg
New hrs I>1 W
u Samples of urine and skin were hydrolyzed the formattion of a chromogen wiih Ehrlich’s in 9 hf H3P0,. The results are compared with * 1)1X: Dry defatted skin.
assay
2s.49 * 6.1 93 + 2
Urine
2nd Skin
Kivirikko
et al.
25.20 + 7.4 s7 + 2
and hydroxyproline was determined re:tgeni aft,er oxidation with periodic 6he Kivirikko rt (II. (2) met.hod.
by
acid
HYDROXYPROLlNE
ASSAX
291
A quick snd specific assay for hydrosyprolinc was developed on the basis of cliffcrentinl oxidation of this liydrosyimitto acid by periodic acid at, acid pH under conditions which do not allow prolinc or hyclrosylysine to he oxidized. The product of t,he oxidation reacts with p-dimethylRminob~nz:lltlclIpdc. The assay allows the determinntion of 2-50 !~.f: of hydrosyprolinc. By using lwrioclic acid in nqueotw and acid medium, ltydrosyl>-sine ant1 hydrosylxolinc can bc tloterminc~d scparntely on t,he enme sample.
55, 288-291 (1973)
BIOCHEMISTRY
A Quick NELLY
and
Specific
BLUMENKl
Assay AND
for
Hydroxyproline
GUSTAV
ASBOE-HANSEN
of Copc&rgerl, Depndmetlt of Demmtology (with Conneclive Lnbomtorie.s), Rigshospitctl, 2100 Copenhagerl, Dennmrk
I:t~ir~aily
Tissue
liesearch
Rccr%ivcd
December
26, 1972;
accepted
March
23, 1973
H,ydrosypl~olin~~ is oxidized by I)criodic arid and various periodates in strong acid medium, while proline is oxidized only in neutral medium (1). This phenomenon led us to ekbornting an assay method for quantitative dcterminntion of hydroxyproline in the prc’scnce of proline. The proccdnte and ik application for quantitatiw ussay of 11~dro~.vproline in isolntcd collagen and in skin and urine are rellortccl holom.
Culture tubes, ncrcw capped with Teflon liners (Kimax) 120 X 15 mm were used. Periodic ncid solution. A I-M solution of periodic acid (Fischer Scientific Co.! in 9 M H,,PC)., (E. Merck A. G., Darmstadt) was prepared and stored in brown bottles for up to 6 months in the dark. Citrate-phosphate bzlffer. The buffer was prepared by mixing 154 ml of 0.1.5M citric acid (Fischer Scientific Co.) with 346 ml 08.6M dibasic sodium phosphate (J. T. Baker Chemical Co.). The final pH of the buffer was 7.0. Extrnctio?z
sol&ion. The extraction solution was prepared by mixing 2.50 ml of toluene with 250 ml of isobutanol and 100 ml of n-propanol. Stcmlnrrl hydrosyproline solution. L-Hydroxyproline was prepared at ii concentration of 10 ,tg/ml. ~Otunclnrrl proline sollrtion,. L-Prolinc was prepared at two different concentrations, i.e., 50 ,,g,/ml and 200 ,~g/nil. p-~in~efhylanlinobensaldeh~~e~ (Ehrl’ch reagent) (analytical grade, E. hZerc,k A. G., Darmstadt). Fifteen milliliters of isobutanol was added to 4 g of p-dimethylaminobenzaldehyde in a beaker, and then 4.5 ml of perchloric acid (70%, E. Merck A. G., Darmst.adt) was added. The solution ins st,oretl for several weeks at, 4°C in a dark bottle. &w~~~ Pwcrcl/rre. Samples were hydrolyzed overnight in 6 N HCI in senlcd t,llbes at 118°C. The hydrolyzatcs were evaporated to dryness in I’~CROat 6.5”C. Evaporated samples were cdissolvcdin distilled water. 2.s Copyright All rights
0 1973 l,y Academic PWSS. Inc. of rc,production in any form rearrvcd.
IIYI)ROXYPROLINE
ASSAY
289
One milliliter of the snm~)le (containing 2.5-50 & hydroxyproline) WL.F acldcd to 6 ml buffer and 0.5 ml of I ;\;I periodic acid in 9 XI I-I,PO, anIl the solut8ion carefully mixed. Then 2.0 ml of the extraction solution wcrc added and the tubes stirred in a Vortes miser. TIw tubes were ~&HA in a test tube rack, and the rack was co~crcd with aluminum foil and shaken on an Equipoise horizont’nl shaking machine for 30 min. The tubes acre then ccntrifllgctl at. low q~c11 during 10 min in order t.0 sq)ar:ttc~ the acl~~ous and organic phase A semimicro or micro aclaptation oi’ the, assay proccdurc was lWrformei1 in case n low content of hydrozyproline was suspected. The only difference with t,he nbovementimed procetlurc were the ~ol\mxe~ of the samples and the reagents added. For t,he scmimicbro assay 3 ml bl8fcr, 0.5 ml periodic acid, and 1.75 ml of the extraction mistluc were added to 0.5 ml of the sample. For the microassay 1.5 ml buffer, 0.5 ml l,criodir acid. ancl 1.2 ml estraction rnisturc WL‘C added to 0.25 ml of the sanlplc. To 600 ,~l of the organic phase 150 /II of Ehrlich reagent were added. The tubes were stirred vigorously. The color was alloyed to develop through 15 min at room tempsrnture, ant1 the nl)sorbancc was read at 565 11111on :t Beckman I>IJ spc~trol~hotomctcr. The color w\;ns stable for 4 hr.
Assays of pure hydrosypraline stnndnrds indicated that the absorbance obtained at 565 nm was proportional to tlic content, of ligclrosyproline. The rcartion was sensitive oTer a broad r:~ngr, ant1 it followed the Im~~bcrt and Beer lag from “,-50 1~6 (Fig. 1) The micromethod allowed determinatjiou of liytlrosyl~roline from l-20 II.F;, while with the semimicro assay 2.5-40 ,~g c:m IX determined.
The specificity of the reaction was tesicd with known mixtures of proline and liydroxyproline, running parallel determinations by the method of Bivirikko et nl. (2) and our assay on aliquots of similar coml~osition. The results obtained with t,he two methods were in good agreement. The adrant’age of our assay method was its requiring approximately $4 of the time and fewer steps with the same specificity (100 assays can be performed within 4 hr), while, on the other hand, Kiviiikko et CL’S (2) metllocl is more sensitive. Urinary excretion of hydrosyproline was also measured with our assay method and with that of Kivirikko et al. (2). Comparable results were obtained (Table 1). h~say~ of hydrolyzed skin collagen with our method yielded values for t~ydroxyproline similar to thoec of the above-mentioned authors (2 1. The I-M periodic acid in 9 M H,.PO, used in the oxidative step, keeping for l)criotls of 6 or more months, offers an advantage over the chloramine T used by Kivirikko et CIZ. (2)) which has to be prepared daily. When sulfosnlicylic acid is used to deprot’einize urine and to detect free h~drosyproline in the supernntant fluid, our procedure needs no llrcvious neutralization, mhilr, according to our experience, the Kivirikko et (II. method (2) needs neutraliznt.ion to get optimum osicintion. AIo.>t. of the methods for qu:mt,itnt.ive assay of Iiydrosyproline are based on its oxidation to a l)yrrol derivative, which can react wit,h P-tlimetl~~lnmi~~obc~~zal~l~~l~ycle. I\Iany oxidants have been used in the diverse methods (hypochlorite, hydrogen peroxide, hypobromite, and chlornmine T) (2-6). Ron-ever, periodic acid has not been used with that purpose. Hydrosylysine and li~drosyl~roline, the two amino acids characteristic of collagen, can be detcrminetl on the same sample by utilizing the differc>nt capability of aqueous an(1 acid solutions of periodic acid for osidntion (7 1. Determination Sample
Nn.
Urine Skin
IS IO
of the HydrcqprtIline with the New
TAULF: 1 Content in Human Away PIZethoda
Hypro mg,‘% ,g!‘jmg
New hrs I>1 W
u Samples of urine and skin were hydrolyzed the formattion of a chromogen wiih Ehrlich’s in 9 hf H3P0,. The results are compared with * 1)1X: Dry defatted skin.
assay
2s.49 * 6.1 93 + 2
Urine
2nd Skin
Kivirikko
et al.
25.20 + 7.4 s7 + 2
and hydroxyproline was determined re:tgeni aft,er oxidation with periodic 6he Kivirikko rt (II. (2) met.hod.
by
acid
HYDROXYPROLlNE
ASSAX
291
A quick snd specific assay for hydrosyprolinc was developed on the basis of cliffcrentinl oxidation of this liydrosyimitto acid by periodic acid at, acid pH under conditions which do not allow prolinc or hyclrosylysine to he oxidized. The product of t,he oxidation reacts with p-dimethylRminob~nz:lltlclIpdc. The assay allows the determinntion of 2-50 !~.f: of hydrosyprolinc. By using lwrioclic acid in nqueotw and acid medium, ltydrosyl>-sine ant1 hydrosylxolinc can bc tloterminc~d scparntely on t,he enme sample.