Method and test composition for determination of lipid peroxide

A method for the determination of peroxide in a sample is disclosed which comprises reacting the peroxide with a chromogen represented by the general formula (I) or (II) ##STR1## wherein R.sub.1 and R.sub.3 represent amino, mono- or di- substituted amino, hydroxyl or hydroxyalkyl, R.sub.4 and R.sub.5 represent hydrogen, alkyl, alkylene, acyl, halogen, sulphone, nitro, carboxyl, hydroxyl or hydroxyalkyl, R.sub.2 represents hydrogen, ##STR2## wherein R.sub.6 represents hydrogen, alkyl, aralkyl, alkylene, aryl or mono- or di- substituted aryl, and --Z-- may change to --Z.dbd. by resonance and represents --S--, --O--, --N.dbd., ##STR3## wherein R.sub.7, R.sub.8, R.sub.9 and R.sub.10 have the same significance as R.sub.6 in the presence of heme compound, iodide or bromide and measuring the absorbancy of the reaction solution in the visible ray region. Also disclosed is a test composition for the determination of peroxide which comprises a chromogen as defined above and a compound selected from a heme compound, iodide and bromide.

The present invention relates to a method for the determination of 
peroxide. More particularly, it relates to a method for the determination 
of peroxide in a sample by reacting the peroxide with a compound which is 
converted to a pigment by oxidation (hereinafter referred to as 
"chromogen") in the presence of a heme compound, iodide or bromide and 
measuring the absorbancy of the reaction solution colored in the visible 
ray region. 
The determination of peroxide in vivo is recognized as important for the 
diagnosis of arteriosclerosis, diabetes mellitus, etc. 
As the methods for the determination of peroxide in a sample, direct 
methods such as iodide titration method, rohdan iron method, chromatograph 
method and ultraviolet absorption method and indirect method such as 
thiobarbituric acid method are known. However, these methods are not 
satisfactory with respect to sensitivity, and further they require the 
removal of the substance contained in the sample and affecting the 
determination. 
Recently, a method has been proposed wherein peroxide is determined by 
reacting peroxide with a chromogen in the presence of a kind of metal 
compound and by measuring the absorbancy of the reaction solution colored 
by the formation of pigment. (Japanese Published Unexamined Patent 
Application Nos. 92391/79 and 23401/80). A simple method which is 
excellent in sensitivity is in demand. 
To this end, studies have been made, and it has been found that peroxide 
and cumene hydroperoxide are determined by reaction with a chromogen 
represented by the general formula (I) or (II) below to form a pigment, 
followed by measurement of the absorbancy of the colored reaction solution 
in the visible ray region. 
##STR4## 
In the formulae, R.sub.1 and R.sub.3 represent amino, mono- or 
di-substituted amino, hydroxyl or hydroxyalkyl, R.sub.4 and R.sub.5 
represent hydrogen, alkyl, alkylene, acyl, halogen, sulphone, nitro, 
carboxyl, hydroxyl or hydroxyalkyl, R.sub.2 represents hydrogen, 
##STR5## 
wherein R.sub.6 represents hydrogen, alkyl, aralkyl, alkylene, aryl or 
mono- or di-substituted aryl, and --Z-- may change to --Z.dbd. by 
resonance and represents --S--, --O--, --N.dbd., 
##STR6## 
wherein R.sub.7, R.sub.8, R.sub.9 and R.sub.10 have the same significance 
as R.sub.6. When Z has three bonds, the position of the double bond of the 
compound represented by the general formula (II) may change. 
Substituents of substituted amino in R.sub.1 and R.sub.3 include alkyl, 
alkylene, hydroxyalkyl, acylaminoalkyl and acyl. As the substituted aryl 
in R.sub.6, substituted phenyl is exemplified and the substituents include 
halogen, alkyl, amino, acylamino, and alkoxycarbonylamino. Aryl includes 
phenyl. 
As an aralkyl, phenylaklyl such as benzyl and substituted phenylakyl such 
as substituted benzyl are exemplified. The substituents have the same 
significance as those in the substituted aryl mentioned above. 
In the above definition, alkyl includes an alkyl group having 1-6 carbon 
atoms and methyl, ethyl, propyl, butyl, pentyl, hexyl and cyclohexyl are 
exemplified. Acyl includes an acyl group having 2-5 carbon atoms, and 
acetyl and propionyl are exemplified. Alkoxy include an alkoxy group 
having 1-5 carbon atoms and methoxy, ethoxy, propoxy and butoxy are 
exemplified. 
These compounds are generally known and are easily prepared by the methods 
illustrated by the following reaction formulae. 
##STR7## 
The principal of the present invention is on the basis of the fact that the 
reaction of peroxide or cumene hydroperoxide with a chromogen in the 
presence of heme compound, iodide or bromide proceeds stoichiometrically 
to form a pigment and the amount of formed pigment is proportioal to the 
amount of peroxide or cumene hydroperoxide in the sample. 
The principle is illustrated as follows. 
##STR8## 
In the above formulae, R.sub.1, R.sub.2, R.sub.3, R.sub.4, R.sub.5 and Z 
have the same significance as defined above and X is I or Br. ROOR' and 
##STR9## 
represent a peroxide. 
As is apparent form the above equations, one molecule of the compound 
represented by the general formula (I') or (II') per one --O--O-- group is 
produced by the reaction and therefore the number of the --O--O-- groups 
in a sample is determined according to the present invention. 
The compounds represented by the general formula (I') or (II') are 
generally known pigments which exhibit a characteristic absorption at the 
wavelength between 500-800 nm and have a large value of molecular 
extinction coefficient. 
According to the present invention, the present method is applied to the 
determination of peroxide in a sample such as serum, blood, etc. 
In carrying out the present method, a sample is used as itself or after 
dilution with water, propanol, etc. and if necessary the solution is 
subjected to centrifugation to remove the substance which may interfere 
with the measurement and the supernatant is used as a test sample. Usually 
the sample is used in a concentration of 1-500 nmol/ml, preferably 30-200 
nmol/ml as --O--O-- group. 
The sample is added to the appropriate buffer solution, preferably, buffer 
having a pH of 2-10. Then to the solution are added, (1) heme compound, 
iodide or bromide (2) chromogen represented by the general formula (I) or 
(II) and if necessary, (3) surfactant for promoting the dissolution of 
peroxide, a chelating reagent such as EDTA for chelating the metal in the 
sample and sodium chloride for inhibiting the ceruloplasmin activity. 
The reaction is carried out at a temperature of 10.degree.-15.degree. C., 
preferably 30.degree.-40.degree. C. and usually completes in 5-30 minutes. 
After completio of the reaction, the absorbancy of the reaction solution 
(E.sub.S) is measured at the characteristic absorption wavelength of the 
pigment formed from chromogen. 
The diluent used for the dilution of sample and the standard compound such 
as cumene hydroperoxide are subjected to the same procedures as described 
above to obtain blank absorbancy (E.sub.B) and standard absorbancy 
(E.sub.STD). 
The concentration of peroxide (L.sub.p) is calculated by the following 
equation. 
##EQU1## 
A: the concentration of peroxide in standard solution 
As the heme compound used in the present invention, hemoglobin, myoglobin 
and iron chlorophyllin are exemplified. 
Iodide and bromide include alkaline metal salts such as potassium salt, 
sodium salt and lithium salt and alkali earth metal salts such as calcium 
salt, aluminum salt and balium salt of iodide or bromine. The heme 
compound is used in a concentration of 0.1 mg/120 g/l. Iodide and bromide 
are usually used in a concentration of 1-100 mg/ml. Surfactant, chelating 
agent and sodium chloride are used in a concentration of 0.001-10%. 
Chromogen is used in a concentration of 0.001-1 mg/ml. 
As buffers, phosphate buffer, tris-HCl buffer, succinate buffer, citrate 
buffer, acetate buffer, etc. may be used in a concentration of 0.005-2 
mol/l. 
Examples of the chromogen used in the present invention are shown in Table 
1. The symbols in Table 1 have the following meaning. 
__________________________________________________________________________ 
##STR10## 
__________________________________________________________________________ 
A: 
N(CH.sub.3).sub.2 
G: 
##STR11## Q: 
##STR12## 
B: 
N(C.sub.2 H.sub.5).sub.2 
K: 
##STR13## Y.sub.p : 
##STR14## 
D: 
##STR15## L: 
##STR16## Y.sub.m : 
##STR17## 
E: 
##STR18## M: 
##STR19## Y.sub.O : 
##STR20## 
F: 
##STR21## P: 
##STR22## Y.sub.B : 
##STR23## 
T: 
##STR24## 
__________________________________________________________________________ 
TABLE 1 
______________________________________ 
Compound 
number Formula R.sub.1 R.sub.2 
R.sub.3 
R.sub.4 
R.sub.5 
______________________________________ 
1 I A H A H H 
2 I NH.sub.2 
H A H H 
3 I A H A CH.sub.3 
CH.sub.3 
4 I A E B H H 
5 I D F D H H 
6 I D H D CH.sub.3 
CH.sub.3 
7 I A G OH CH.sub.3 
H 
8 I OH K OH CH.sub.3 
H 
9 I A L B H OH 
10 II A H A H H 
11 II A M A H H 
12 II B P D CH.sub.3 
H 
13 II D Q D H H 
14 II D Q OH CH.sub.3 
CH.sub.3 
15 II A Y.sub.P 
A H H 
16 II A Y.sub.O 
A H H 
17 II A Y.sub.m 
A H H 
18 II A Y.sub.B 
A H H 
19 II A E A H H 
20 II A T A H H 
______________________________________ 
The degree of color development, the stability of color and the influence 
of the components in serum on the determination value when Compound (I) is 
used as a chromgen, are illustrated by the following experiment. 
Experiment 1 
Compound Nos. 1-20 in the amount indicated in Table 2 are dissolved in 1 ml 
of dimethylformamide (hereinafter referred to as "DMF"). The test reagent 
is prepared by adding 0.1 g Triton X-100, DMF solution of Compound Nos. 
1-20, 1 g of EDTA and 6.7 mg of hemoglobin to 100 ml of 0.1 M phosphate 
buffer (pH 5.0). 
3 ml of the reagent solution is poured into a test tube for each compound 
and linolic acid (A) is added thereto. The reaction is carried out at 
37.degree. C. for 30 minutes and the absorbancy of the reaction solution 
(E.sub.S) is measured. The blank absorbancy (E.sub.B) is measured by 
repeating the above procedures without the addition of linolic acid. 
As a control, the absorbancies (E.sub.SC and E.sub.BC) are measured using 
4-amino antipyrine (hereinafter referred to as 4 AA) and 
m-methyl-(N-ethyl, N-acetoaminoethyl) aniline (hereinafter referred to as 
EMAE) as coloring reagent, and the degree of color development of test 
compound is calculated from the following equation defining the degree of 
color development of control as 100. 
##EQU2## 
The stability of color is determined as follows. The reaction solution is 
further incubated at 37.degree. C. for 30 minutes and E.sub.S -E.sub.B is 
calculated. "S.sub.0 " means that the value of E.sub.S -E.sub.B was not 
changed by this incubation and "S.sub.1 " means that the change of the 
value is 10% or less. The influence of bilirubin and vitamin C in the 
sample on the determination value is indicated by the value M. The value M 
is determined by repeating the above experiment using the sample 
containing 4 .mu.g/3 ml of bilirubin or 2 .mu.g/3 ml of vitamin C, 
measuring the absorbancy (E.sub.S') and calculating from the following 
equation: 
##EQU3## 
The symbol (-) means that the value (M) is 3% or less. The symbol (.+-.) 
means that the value is 3-6% and (+) for 6-20% and (++) for 20% or more. 
TABLE 2 
______________________________________ 
Degree of 
Chromogen color Inhibitor 
No. Amount mg development 
B.sub.i 
V.sub.c 
Stability 
______________________________________ 
1 10.0 538 - - S.sub.0 
2 10.0 313 - - S.sub.1 
3 12.0 277 - .+-. S.sub.0 
4 14.7 495 - .+-. S.sub.0 
5 23.4 532 - .+-. S.sub.0 
6 16.8 575 - - S.sub.0 
7 16.4 251 + .+-. S.sub.1 
8 14.3 304 + + S.sub.1 
9 19.2 527 - .+-. S.sub.0 
10 11.2 572 - .+-. S.sub.0 
11 15.0 470 - .+-. S.sub.0 
12 21.4 581 - .+-. S.sub.0 
13 24.8 519 - .+-. S.sub.0 
14 22.1 294 .+-. + S.sub.0 
15 3.1 506 - .+-. S.sub.0 
16 3.1 311 .+-. .+-. S.sub.0 
17 3.1 547 - .+-. S.sub.0 
18 10.0 523 - .+-. S.sub.0 
19 3.3 536 - .+-. S.sub.0 
20 3.3 478 - .+-. S.sub.0 
Control 
4 AA:6.7 100 - .+-. S.sub.0 
EMAE:26.6 
Compar- 
4 AA:6.7 43.4 ++ + S.sub.0 
ison 1 Phenol:33 
Compar- 
M-T method 210 ++ + S.sub.0 
ison 2 
______________________________________ 
B.sub.i : bilirubin 
V.sub.c : vitamin C 
MT: malon dialdehydethiobarbituric acid 
For comparison, 4 AA-phenol or malon dialdehydethiobarbituric acid is used 
as a chromogen and the results are shown in Table 2. 
Another aspect of the present invention is to provide a test composition 
for the determination of peroxide which comprises a chromogen represented 
by the general formula (I) or (II), a compound selected from heme 
compound, iodide and bromide and a buffer. The composition may contain a 
surfactant, chelating reagent and sodium chloride. 
The composition may be used in various forms. For example, the ingredients 
may be mixed in liquid form or powder form. 
Certain specific embodiments of the present invention are illustrated by 
the following representative examples.

EXAMPLE 1 
The test reagent is prepared by adding 0.1 g of Tricon X-100, 1 ml of DMF 
solution containing 10 mg of Compound 1, 5.6 mg of hemoglobin and 1 g of 
EDTA to 100 ml of 0.1 M phosphate buffer (pH 5.0). 
As the sample containing peroxide, 1 ml of linolic acid (A) and 1 ml of 
linolenic acid (B) are respectively diluted with isopropanol to make 100 
ml of a solution. 
20 .mu.l of the test sample is added to 3 ml of the test reagent and 
incubated at 37.degree. C. with stirring. The absorbancy of the reaction 
solution at 728 nm is monitored for 10 minutes from the start of the 
reaction. The absorbancy reaches equilibrium within about 5 minutes. 
The same procedures as described above are repeated except that cumene 
hydroperoxide is used as a standard compound and the absorbancy of the 
reaction solution at 728 nm is measured about 10 minutes from the start of 
the reaction. The standard curve between the absorbancy and the 
concentration of peroxide is prepared by repeating the above procedure 
varying the concentration of cumene hydroperoxide. 
The peroxide values for the samples containing linolic acid (A) and 
linolenic acid (B) are calculated from the standard curve to obtain 37 for 
(A) and 29.5 for (B). 
For comparison, peroxide values of the samples are determined according to 
the known iodide titration method to obtain 35.1 for (A) and 21.3 for (B). 
The present method and the known method described above are repeated five 
times for samples (A) and (B). The coefficient of variation by the present 
method is 0.1% for (A) and 0.15% for (B) and that by the known method is 
10.5% for (A) and 12.3% for (B). 
EXAMPLE 2 
The samples indicated in Table 3 are dissolved in water or isopropanol in a 
ratio of 10% (V/V). 50 .mu.l of each solution is added to 3 ml of the test 
reagent of Example 1. The reactionis carried out under the same conditions 
as in Example 1 and the absorbancy of the reaction solution is measured. 
The results are shown in Table 3. 
TABLE 3 
______________________________________ 
The amount of 
peroxide*1 
Sample 
Solvent (nmol/g) C.V (%).sup.2 
______________________________________ 
1 Distilled water 
390.7 0.31 
2 " 274.4 0.52 
3 " 135.9 0.86 
4 " 183.1 0.42 
5 Isopropanol 15.2 3.21 
______________________________________ 
1: Emulgen 404 (nonionic surfactant, product of Kao Atras Co., Ltd.) 
2: Emul 20T (anionic surfactant, product of Kao Atras Co., Ltd.) 
3: Quartamin 86P (cationic surfactant, product of Kao Atras Co., Ltd.) 
4: Tetrahydrofuran 
5: Ethylether 
*1: The value is the average of five measurements. 
*2: CV: coefficient of variation 
EXAMPLE 3 
In this example, 0.2 ml of normal and patient serum are added to 4 ml of 
isopropanol and the solutions are subjected to centrifugation at 2,000 
r.p.m. for 5 minutes. To 0.5 ml of the supernatant is added 3 ml of the 
test reagent of Exampole 1, and the mixture is incubated at 37.degree. C. 
for 10 minutes. The absorbancy of the reaction solution (E.sub.S) is 
measured at 728 nm. 
The same procedures as described above are repeated for 0.5 ml of 200 
nmol/ml cumene hydroperoxide isopropanol solution and 0.5 ml of 
isopropanol to obtain the absorbancies E.sub.STD and E.sub.B. The amount 
of peroxide (L.sub.P) in serum calculated from the following equation is 
15.2 nmol/ml for normal and 96.3 nmol/ml for patient. 
##EQU4## 
EXAMPLE 4 
The same procedures as described in Example 1 are repeated except that the 
compounds indicated in Table 4 instead of Compound 1 are used as chromogen 
and the peroxide values (PA) for sample (A) and (PB) for sample (B) are 
determined. The results are shown in Table 4. 
TABLE 4 
______________________________________ 
Reaction 
Wavelength time PA PB 
Chromogen 
(nm) (min.) (nmol/Kg) 
(nmol/Kg) 
______________________________________ 
2 700 &lt;5 38.5 30.4 
3 720 &lt;5 38.3 29.9 
4 720 15 36.9 25.1 
5 730 15 37.2 26.2 
6 730 &lt;5 38.9 28.9 
7 600 20 40.3 30.5 
8 600 20 36.1 26.4 
9 720 15 39.2 29.8 
10 665 &lt;5 38.5 28.7 
11 665 20 37.1 26.8 
12 670 15 37.2 27.0 
13 670 16 36.8 26.1 
14 620 20 35.9 26.5 
19 665 &lt;5 39.0 27.3 
______________________________________ 
EXAMPLE 5 
The same procedures as described in Example 1 are repeated except that 0.56 
mg of sodium salt or iron chlorophyllin or 5.6 mg of myoglobin (Sigma Co.) 
is used instead of hemoglobin. The peroxide value obtained using iron 
chlorophyllin is 38.2 for sample (A) and 29.3 for sample (B) and that 
obtained using myoglobin is 37.8 for sample (A) and 28.5 for sample (B). 
EXAMPLE 6 
In this example, 0.1 g of Triton X-100, 1 ml of DMF solution containing 10 
mg of Compound 1, 1 g of potassium iodide and 1 g of EDTA are dissolved in 
100 ml of 0.1 M phosphate buffer (pH 4.0) and the solution is used as test 
reagent. 
The same procedures as described in Example 1 are repeated using the test 
reagent for 50 .mu.l of sample (A) or (B). The peroxide value is 37.5 for 
sample (A) and 28.7 for sample (B). 
EXAMPLE 7 
In this example, as the standard peroxide, linolic acid is oxidized with 
air at 23.degree. C. for 72 hours according to the method described in 
Canad. J. Biochem. 47, 485 (1969). The oxidation product is subjected to 
extraction with a solvent system of petroleum ether/67% methanol--33% 
water. The layer of methanol--water is concentrated to obtain an oily 
matter. The oily matter is subjected to thin layer chromatography using 
silica gel and hexane--ether--acetic acid (60:40:1) as a developer. Silica 
gel showing an Rf value of 0.23 is subjected to elution using ethanol and 
the eluate is concentrated to obtaina racemic mixture of equimolar amounts 
of linolic acid having --OOH at the 9-position and linolic acid having 
--OOH at the 13-position. 310.4 mg of the obtained mixture is dissolved in 
1 l of isopropanol to obtain 1 .mu.mol/ml solution (hereinafter referred 
to as test solution). 
The test reagent is prepared by adding 0.1 g of Triton X-100, 1 ml of DMF 
solution containing 10 mg of Compound 20, 5.6 mg of hemoglobin and 1 g of 
EDTA to 100 ml of 0.1 M phosphate buffer (pH 5.0). 
The test solution is diluted ten-fold with isopropanol. 100 .mu.l of the 
diluted test solution is added to 3 ml of the test reagent in a test tube 
and 100 .mu.l of isopropanol is added to 3 ml of the test reagent in 
another test tube. The mixtures are incubated at 37.degree. C. for ten 
minutes and the absorbancies of the reaction solutions are measured at 666 
nm to obtain O.D. values of 0.308 and 0.085 respectively. The increase in 
absorbancy by the addition of oxidized linolic acid is calculated as 
0.223. 
Then, 391.9 mg of Methylene Blue is dissolved in 1 l of water and the 
solution is diluted ten-fold with water. 100 .mu.l of the blue colored 
solution is added to 0.1 M phosphate buffer (pH 5.0) and the absorbancy of 
the solution is measured to obtain a value of 0.225. 
Then, 100 .mu.l of the test solution having the concentration indicated in 
Table 5 is added to 3 ml of the test reagent and the mixture is incubated 
at 37.degree. C. for ten minutes. The absorbancy (E.sub.N) of the reaction 
solution is measured at 666 nm. As a blank, the same procedures as 
described above are repeated except using isopropanol instead of the test 
solution and the absorbancy (E.sub.B) is measured at 666 nm. The results 
are shown in Table 5. As is apparent from the table, the concentration of 
oxidized linolic acid is proportional to the value of E.sub.N -E.sub.B. 
TABLE 5 
______________________________________ 
Concentration of 
linolic acid (nmol/ml) 
0 25 50 100 150 
E.sub.N -E.sub.B 
0 0.057 0.113 0.227 
0.340 
______________________________________ 
EXAMPLE 8 
In this example, 100 .mu.l of isopropanol solution of cumene hydroperoxide 
having the concentration indicated in Table 6 is added to 3 ml of 50 mM 
citrate buffer (pH 4.7) containing 1 mg/ml Emulgen 106 (non-ionic 
surfactant, product of Kao Atras Co., Ltd.), 5 mg/dl hemoglobin and 2.5 
mg/dl Compound 18. 
The mixture is incubated at 37.degree. C. for 30 minutes. The absorbancy of 
the reaction solution (E.sub.N) is measured at 666 nm. 
As a blank test, the same procedures as described above are repeated except 
using isopropanol instead of cumene hydroperoxide solution and the 
absorbancy (E.sub.B) is measured. 
The results are shown in Table 6. As is apparent from the table, the 
concentration of cumene hydroperoxide is proportional to the value of 
E.sub.N -E.sub.B. 
TABLE 6 
__________________________________________________________________________ 
Concentration of 
cumene hydroperoxide 
0 25 50 l00 
150 
(nmol/ml) 
E.sub.N -E.sub.B 
0 0.056 
0.113 
0.225 
0.336 
__________________________________________________________________________ 
REFERENCE EXAMPLE 1 
In this example, 1 g of Methylene Blue is dissolved in 100 ml of water and 
1 g of sodium borohydride is added little by little to proceed the 
reduction. When the precipitate of leuco base is desposited and the 
solution is discolored, 20 ml of chloroform is added and the mixture is 
vigorously stirred to extract leuco base. 
The chloroform layer is filtered through filter paper, dehydrated and 
desalted. Then, 2 ml of phenyl isocyanate is added and the mixture is 
subjected to reaction at room temperature for 24 hours. 
After completion of the reaction, methanol is added to remove excess 
isocyanate and the mixture is stirred at room temperature for 3 hours. 
The mixture is subjected to column chromatography using silica gel having 
the size of 60-80 mesh (product of Kanto Kagaku Co., Ltd.) and using 
chloroform as a developer to obtain Compound 19 having a melting point of 
100.degree.-115.degree. C. 
REFERENCE EXAMPLE 2 
The same procedures as described in Reference Example 1 are repeated except 
that o-, m- or p-chlorophenyl uisocyanate or p-bromophenyl isocyanate is 
used instead of phenylisocyanate to obtain Compound 16 (oil form), 
Compound 17 (m.p. 73.degree.-77.degree. C.), Compound 15 (m.p. 
76.degree.-83.degree. C.) and Compound 18 (m.p. 80.degree.-90.degree. C.), 
respectively.