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Influence of non-protein nitrogen substances on the indophenol reaction
257
BRIEF NOTES
Influence of non-protein nitrogen substances on the indophenol reaction Testing the general application of a direct method for the determination of ammonia in blood’, attention has been drawn to the inhibition of the indophenol reaction by some non-protein nitrogen (npn) substance+4. Lorent suggested the following mechanism for this phenomenon. When free amino groups are present in considerable amount in the reaction mixture, these should be able to bind all or some of the available hypochlorite and thus interfere with the first step of the indophenol reaction, the formation of chloramine from ammonia and hypochlorite. All npn compounds capable of exchanging a proton for Cl+ (chloronium) should be able to interfere with the reaction. Using reagents as described previouslys, we have investigated the influence of a number of npn substances on the indophenol reaction, with and without an added ammonia standard (Tables I and II). Test substances were obtained from Merck, Darmstadt and from Sigma Medical Company. Determinations were performed in triplicate immediately after preparation of the aqueous solutions and the reaction was performed at 4’ up to the final calorimetric reaction, which was done in a dark waterTABLB
I
INDOPHENOL
REACTION
WITH
NON-PROTEIN
NITROGEN
COMPOUNDS
Deviations from the reagent blank reading expressed 3 ml, A 625 nm, d = I cm. Mel
npn substance +zmoles
wt.
NH,-NH,+
60 ‘I7 240 I21 I65 204 I79 89 I46 II5 95 II7 II9 I31 I3I 168 I32 75 II3 75 I46 I74 149 I32 I3I I47 I31 I58 I55 181
Urea L-Valine L-Cystine L-Cystein L-Phenylalanine L-Tryptophan Hippuric acid L-_llanine I_-Lysine L-Proline Guanidine-HCl Guanidoacetic acid L-Threonine Creatine Hvdroxyproline U;ic acid L-Asparagine Glycine Creatinine L-Serine L-Glutamine L-Arginine L-Methionine DL-.kSpartiC acid L-Isoleucine Glutamic acid L-Leucine Allantoin r-Histidine L-Tvrosine
All 30 compounds
25 000
added nmolrs
2500
5000 0
0
as nmoles NH,-NH,+.
500
1000
0
Reaction 50
100 0
0
0
volume
0
-2
-
-
-
-
4 4 I9 6 6 20
26 -3 6 9 9 I7 215 -3
-
4 2 : 7 24
4 7 7
-
-
-
4 6
II
4 4
-2 IO
9 I4 I65 I4
7 9 7
: 6 9
12
59 16 I3 -
-
IO
6 Clin.
Chim.
Acta,
33 (1971)
257-259
BRIEF NOTES
258 TABLE
II
INHIBITIOPiOF THE INDOPHEPZOL. REACTIONBY xoix-PROTEIN NITROGENSUBsTaNcEs Results expressed as percentage inhibition A 625 nm, d = I cm. _~___ Mol. NH,-NH,+ wvdcs 50 50 wt. npn compound nmoles z_jooo 5000 .60 Urea, “/b inhibition 146 L-Glutamine I32 L-Asparagine 158 Allantoin 240 L-Cystine Hippuric acid ‘79 L-Argininc 20 ‘74 89 L-Alanine 86 105 L-Serine 89 75 Glycine 25 147 Glutamic acid 40 149 Creatine IO 132 DL-Aspartic acid 60 117 Guanidoacetic acid L-Proline IIj 91 100 119 L-Threonine 85 146 L-Lysine 67 131 L-Leucine 60 113 Creatinine 82 Hydroxyprolinc 13’ Uric acid 168 78 IO0 L-Cystein 121 8j Guanidine-HCl 95 100 117 L-Valine 100 131 r-lsoleucinc 100 149 L-Methionine 100 165 L-Phenylalanine 100 155 L-Histidine IO0 L-Tyrosinc 181 L-Tryptophan 90 20-l All 30 compounds
IO0
s9
of the ammonia
50 2500
standard.
.____ 50 50 IO00 500
50 250
-
.i
8 26 63 23 25 15 40
22
82
20
77 58 27 26 68 54
9 7 12
9
16
-
-
6 9 ‘4 17
100
26 15 56 30 65 65 75 7.5 65 91
30 34 47 59 27 2‘t
100
20
18
100
79 100
97 JO0
48
91 64
43 57
18
I5
9
_.~ 7 14 ‘7 15 9 -
Reaction 50 IO0
-
volume
50 50
50 “5
3:ml. so 5
-
6
6
-
9
9
IO
-
-
bath
at 38”. Absorbance was read in a Zeiss PMQII spectrophotometer at 625 nm. With all thirty compounds together in solution, it is notable that there was no increased absorbance (Table I). The increase in absorbance, found with a number of the individual test substances was small in most cases. As to allantoin, there seems to be a direct relation between the amount of “ammonia” measured and the concentration of the test solution. Valine, methionine and histidine showed decreased absorbance (inhibition) in higher concentrations. Inhibition of the indophenol reaction (Table II) was not observed with concentrations of less than 2500 nmoles/3 ml reaction volume, when all substances were put together. Some of the individual amino acids, however, caused partial inhibition of the reaction in concentrations as low as 25 or 50 nmoles/3 ml (tyrosine, tryptophan). DISCUSSION
When using a direct method for the determination of ammonia in biological fluids with the indophenol reaction, the composition of these fluids with regard to proteins (which have to be precipitated or have to be withheld by dialysis of the fluid Cl;%. Chim. Acta,
33 (1971) 257-259
BRIEF
XOTES
259
as in some automated
procedures)
and non-protein
nitrogen
substances
should be
taken
into account. In the case of urine, where normally creatinine and uric acid will dominate with respect to inhibition, there are no problems. The ammonia content of urine is high permitting manifold dilution, the indophenol reaction being very sensitive5. There are more difficulties with blood or plasma, in which the ammonia content is relatively low. Here, however, one can take advantage of the inhibition of the indophenol reaction. In patients with so-called endogenous hepatic coma or praecoma, hyperaminoacidaemia-which is caused by massive liver cell necrosis-is found. In these cases, as a rule due to fulminant hepatitis or terminal cirrhosis, the ammonia content (when measured with a direct method using the Berthelot reaction) will be factitiously low or zero as a result of inhibition by the excessive amounts of plasma amino acids. In patients with exogenous hepatic coma (which is caused by portasystemic shunting of blood containing ammonia and other nitrogenous substances from the bowels, orin patients with cirrhosis-by electrolyte disturbances) no elevation of the plasma amino acids is present and the arterial ammonia level is found, to be increased. Thus, thanks to the interference of npn substances with the indophenol reaction, it is possible to differentiate between exo- and endogenous hepatic coma using the simple direct method for the determination of blood ammonia’96 (which, due to the peripheral metabolism of ammonia, is always to be performed on arterial blood): a zero “ammonia level” will point to a grave prognosis. Department University
of Internal Hosjdal,
Groniqen
(The
C. H.
Medicine, ANITA
GIPS
REITSEMA
Netherlands)
I K. KOXITZER AND S. VOIGT, Cl&. Chim. Acta, 8 (1963) 5. 2 H. OKUDA, S. FUJII AND Y. KAWASHIMA, Tokushima J. Exptl. Med., IZ (1965) II. 3 W.M~~LLER-BEISSENHIRTZ, Z. Anal.Chem., 212 (1965) 145. 4 K. LOREXTZ, Z. Klin. Chem. Klin. Biochem., 5 (1967) 296. 5 C. H. GIPS, h. KEITSEMA AND M. WIBBENS-ALBERTS, Clin.Chim. Acta, 29 (1970) 501. 6 C. H. GIPS AND M. WIBBENS-ALBERTS, Clin.Chiwz. Acta, zz (1968) 183.
Received
August
20,
1970. Clin.
Chim.
Acta,
33 (1971) 257-259
BRIEF NOTES
Influence of non-protein nitrogen substances on the indophenol reaction Testing the general application of a direct method for the determination of ammonia in blood’, attention has been drawn to the inhibition of the indophenol reaction by some non-protein nitrogen (npn) substance+4. Lorent suggested the following mechanism for this phenomenon. When free amino groups are present in considerable amount in the reaction mixture, these should be able to bind all or some of the available hypochlorite and thus interfere with the first step of the indophenol reaction, the formation of chloramine from ammonia and hypochlorite. All npn compounds capable of exchanging a proton for Cl+ (chloronium) should be able to interfere with the reaction. Using reagents as described previouslys, we have investigated the influence of a number of npn substances on the indophenol reaction, with and without an added ammonia standard (Tables I and II). Test substances were obtained from Merck, Darmstadt and from Sigma Medical Company. Determinations were performed in triplicate immediately after preparation of the aqueous solutions and the reaction was performed at 4’ up to the final calorimetric reaction, which was done in a dark waterTABLB
I
INDOPHENOL
REACTION
WITH
NON-PROTEIN
NITROGEN
COMPOUNDS
Deviations from the reagent blank reading expressed 3 ml, A 625 nm, d = I cm. Mel
npn substance +zmoles
wt.
NH,-NH,+
60 ‘I7 240 I21 I65 204 I79 89 I46 II5 95 II7 II9 I31 I3I 168 I32 75 II3 75 I46 I74 149 I32 I3I I47 I31 I58 I55 181
Urea L-Valine L-Cystine L-Cystein L-Phenylalanine L-Tryptophan Hippuric acid L-_llanine I_-Lysine L-Proline Guanidine-HCl Guanidoacetic acid L-Threonine Creatine Hvdroxyproline U;ic acid L-Asparagine Glycine Creatinine L-Serine L-Glutamine L-Arginine L-Methionine DL-.kSpartiC acid L-Isoleucine Glutamic acid L-Leucine Allantoin r-Histidine L-Tvrosine
All 30 compounds
25 000
added nmolrs
2500
5000 0
0
as nmoles NH,-NH,+.
500
1000
0
Reaction 50
100 0
0
0
volume
0
-2
-
-
-
-
4 4 I9 6 6 20
26 -3 6 9 9 I7 215 -3
-
4 2 : 7 24
4 7 7
-
-
-
4 6
II
4 4
-2 IO
9 I4 I65 I4
7 9 7
: 6 9
12
59 16 I3 -
-
IO
6 Clin.
Chim.
Acta,
33 (1971)
257-259
BRIEF NOTES
258 TABLE
II
INHIBITIOPiOF THE INDOPHEPZOL. REACTIONBY xoix-PROTEIN NITROGENSUBsTaNcEs Results expressed as percentage inhibition A 625 nm, d = I cm. _~___ Mol. NH,-NH,+ wvdcs 50 50 wt. npn compound nmoles z_jooo 5000 .60 Urea, “/b inhibition 146 L-Glutamine I32 L-Asparagine 158 Allantoin 240 L-Cystine Hippuric acid ‘79 L-Argininc 20 ‘74 89 L-Alanine 86 105 L-Serine 89 75 Glycine 25 147 Glutamic acid 40 149 Creatine IO 132 DL-Aspartic acid 60 117 Guanidoacetic acid L-Proline IIj 91 100 119 L-Threonine 85 146 L-Lysine 67 131 L-Leucine 60 113 Creatinine 82 Hydroxyprolinc 13’ Uric acid 168 78 IO0 L-Cystein 121 8j Guanidine-HCl 95 100 117 L-Valine 100 131 r-lsoleucinc 100 149 L-Methionine 100 165 L-Phenylalanine 100 155 L-Histidine IO0 L-Tyrosinc 181 L-Tryptophan 90 20-l All 30 compounds
IO0
s9
of the ammonia
50 2500
standard.
.____ 50 50 IO00 500
50 250
-
.i
8 26 63 23 25 15 40
22
82
20
77 58 27 26 68 54
9 7 12
9
16
-
-
6 9 ‘4 17
100
26 15 56 30 65 65 75 7.5 65 91
30 34 47 59 27 2‘t
100
20
18
100
79 100
97 JO0
48
91 64
43 57
18
I5
9
_.~ 7 14 ‘7 15 9 -
Reaction 50 IO0
-
volume
50 50
50 “5
3:ml. so 5
-
6
6
-
9
9
IO
-
-
bath
at 38”. Absorbance was read in a Zeiss PMQII spectrophotometer at 625 nm. With all thirty compounds together in solution, it is notable that there was no increased absorbance (Table I). The increase in absorbance, found with a number of the individual test substances was small in most cases. As to allantoin, there seems to be a direct relation between the amount of “ammonia” measured and the concentration of the test solution. Valine, methionine and histidine showed decreased absorbance (inhibition) in higher concentrations. Inhibition of the indophenol reaction (Table II) was not observed with concentrations of less than 2500 nmoles/3 ml reaction volume, when all substances were put together. Some of the individual amino acids, however, caused partial inhibition of the reaction in concentrations as low as 25 or 50 nmoles/3 ml (tyrosine, tryptophan). DISCUSSION
When using a direct method for the determination of ammonia in biological fluids with the indophenol reaction, the composition of these fluids with regard to proteins (which have to be precipitated or have to be withheld by dialysis of the fluid Cl;%. Chim. Acta,
33 (1971) 257-259
BRIEF
XOTES
259
as in some automated
procedures)
and non-protein
nitrogen
substances
should be
taken
into account. In the case of urine, where normally creatinine and uric acid will dominate with respect to inhibition, there are no problems. The ammonia content of urine is high permitting manifold dilution, the indophenol reaction being very sensitive5. There are more difficulties with blood or plasma, in which the ammonia content is relatively low. Here, however, one can take advantage of the inhibition of the indophenol reaction. In patients with so-called endogenous hepatic coma or praecoma, hyperaminoacidaemia-which is caused by massive liver cell necrosis-is found. In these cases, as a rule due to fulminant hepatitis or terminal cirrhosis, the ammonia content (when measured with a direct method using the Berthelot reaction) will be factitiously low or zero as a result of inhibition by the excessive amounts of plasma amino acids. In patients with exogenous hepatic coma (which is caused by portasystemic shunting of blood containing ammonia and other nitrogenous substances from the bowels, orin patients with cirrhosis-by electrolyte disturbances) no elevation of the plasma amino acids is present and the arterial ammonia level is found, to be increased. Thus, thanks to the interference of npn substances with the indophenol reaction, it is possible to differentiate between exo- and endogenous hepatic coma using the simple direct method for the determination of blood ammonia’96 (which, due to the peripheral metabolism of ammonia, is always to be performed on arterial blood): a zero “ammonia level” will point to a grave prognosis. Department University
of Internal Hosjdal,
Groniqen
(The
C. H.
Medicine, ANITA
GIPS
REITSEMA
Netherlands)
I K. KOXITZER AND S. VOIGT, Cl&. Chim. Acta, 8 (1963) 5. 2 H. OKUDA, S. FUJII AND Y. KAWASHIMA, Tokushima J. Exptl. Med., IZ (1965) II. 3 W.M~~LLER-BEISSENHIRTZ, Z. Anal.Chem., 212 (1965) 145. 4 K. LOREXTZ, Z. Klin. Chem. Klin. Biochem., 5 (1967) 296. 5 C. H. GIPS, h. KEITSEMA AND M. WIBBENS-ALBERTS, Clin.Chim. Acta, 29 (1970) 501. 6 C. H. GIPS AND M. WIBBENS-ALBERTS, Clin.Chiwz. Acta, zz (1968) 183.
Received
August
20,
1970. Clin.
Chim.
Acta,
33 (1971) 257-259