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Ammonia determination in a trichloroacetic acid blood filtrate after microdiffusion with the aid of the rubazonic acid reaction
55
CLINICA CHIMICA ACTA
AMMONIA
DETERMINATION
FILTRATE
AFTER
RUBAZONIC
WITH
ACID BLOOD
THE AID OF THE
ACID REACTION
A. F. K. BUYS BALLOT
AND
Clinical Chemistry Laboratory, Rotterdam (The Netherlands)
(Received July
IN A TRICHLOROACETIC
MICRODIFFUSION
zznd,
C. STEENDIJK Municipal
Bergweg Hospital,
1964)
SUMMARY A technique for the determination of blood ammonia based on the following steps is described: I. Preparation of a protein-free trichloroacetic acid filtrate of venous blood. 2. The diffusion of ammonia out of the alkaline filtrate. 3. The calorimetric determination of the NH,-N in an acid medium with the aid of the rubazonic acid reaction, Twelve determinations of a standard ammonia solution gave a mean value of 0.62 ,ug NH,-N per ml with a standard deviation of 0.02 l”g NH,-N per ml. The normal venous blood ammonia level, with this method, varies from 0.30 to 0.80 ,ug NH,-N per ml. Results of determinations of blood NH,-N of patients with liver diseases are described.
Ammonia is said to originate in the gastrointestinal tract by the action of intestinal bacteria on nitrogenous substrates. An increase in peripheral venous blood ammonia levels is found by impaired hepatic functions such as cirrhosis of the liver and portacaval shuntsl. After oral intake of, for instance, proteins, blood, amino acids, ammonium salts and urea, the peripheral venous blood ammonia levels increased in patients with cirrhosis of the liver or hepatitis and in patients with cancer of the liver 2. Because of the very low quantities of ammonia which normally appear in the peripheral blood, an accurate method for the determination is necessary. Nessler’s reagent is not sensitive enough. The ninhydrin reaction and the phenolate-hypochlorite reagent are sensitive and have found application in several methods3-6. Kala’ described a modification of Kruse and Mellon’s calorimetric ammonia determination8, which he called the rubazonic acid method. As it was described as being very sensitive, we tried to apply this method for the determination of blood C&n. Chim. Acta, 12 (1965) 55-62
ammonia. In a trichloroacetic acid blood filtrate, WC dcterniinc~d the amount (of ammonia with the aid of the rubazonic acid reaction after diffusion, as dcscribt~d by Seligson 9.
Reageds I. A zoo,& solution of trichloroacetic acid in distilled water. 2. Saturated potassium carbonate p.a. solution in distilled water. Dissolve IIO g potassium carbonate to IOO ml. The solution is kept several days in an exsiccator above I M citric acid at 50’ to remove all traces of ammonia. 3. +I N sulfuric acid p.a. Dilute 5 ml concentrated sulfuric acid p.a. with distilled water to IOO ml and add one drop of Sterox SE. 4. I M acetate buffer, pH 4.1. (130 ml I M sodium acetate + 370 ml I 12f acetic acid.) 5. zO,b Solution of chloramine-T (sodium paratoluenesulfonchloramide) in distilled water. To be prepared fresh each week and stored in a dark bottle. 6. Pyridine p.a. This liquid can only be used as long as it is colorless. If it turns yellowish brown, it has to be distilled before use. 7. Pyrazolon reagent. Mix equal parts of solution A and solution B. Solution A: 150 mg 3-methyl-phenyl-5-pyrazolon in 40 ml pyridine. Solution B: 25 mg bispyrazolon. (3,3’-dimethyl-I,I’-diphenyl-4,4’-bi-z-pyrazoline-5,5’-dion) in 40 ml pyridine. Dissolve by shaking. The solutions A and B ha\:e to be prepared fresh each day. S. 3o/o Solution of sodium thiosulfate in distilled water. 9. Standard ammonium sulfate solution (IO mg”/b NH,-N). Dissolve 47r.4 mg ammonium sulfate p.a. in I 1 distilled water. IO. Standard samples in IO:/, trichloroacetic acid p.a. Dilute reagent 9. with reagent I. and distilled water to obtain standard concentrations of 0.25; 0.50; 1.00 and 2.00 ,ug NH,-N per ml. Note: a. The blank, the standard samples and the 200,” solution of trichloroacetic acid must be prepared with the same distilled water. b. The glassware was cleaned by rinsing once with cont. HCl, three times with distilled water, once with a 2!0 solution of sodium carbonate in distilled water and five times with distilled water. Afterwards it was dried at about 60”.
Equifwnent
For diffusion 25-ml penicillin bottles are used. Each bottle is fitted with a rubber stopper through which a glass rod with a rounded end is placed. The rounded end of the glass rod should extend to about 20 mm from the bottom of the bottle. 2. An electric rotor as described by Seligson9. 3. Calibrated 12 ml centrifuge tubes. 4. A spectrophotometer with a r-cm glass cuvette or a calorimeter with an interference filter 550 mp. I.
Analytical
procedure
Draw approximately IO ml blood, without stasis, and bring 5 ml of this blood immediately over into a rz-ml calibrated centrifuge tube, containing 5 ml ice-cooled Clin.
Chim.
Acta,
IL (1965) 55-62
DETERMINATION
OF BLOOD
57
AMMONIA
20% trichloroacetic acid. The tube should be immediately capped with parafilm and mixed by shaking vigorously. The other 5 ml, like the first portion, are transferred into a tube which contains, besides the trichloroacetic acid, 5 lug NH,-N. These “additions” are further handled exactly the same way as the samples. Reimmerse the tubes in the ice bath and take them to the laboratory where they are centrifuged for IO min at approximately IOOOg (about 2500 rev./min). After centrifugation the tubes have reached approximately room temperature. After measuring the total volume and the volume of the precipitate, decant the clear supernatant into an ice-cooled test tube and cap with parafilm (further referred to as the filtrate). Transfer into the diffusion bottle, at room temperature, I ml saturated potassium carbonate and 2 ml of the filtrate. Close the bottle immediately with the rubber stopper containing the glass rod, which has been moistened with the I N sulfuric acid-Sterox solution. This wetting of the glass rod can best be done by immersing the rod up to 5 mm from the rubber stopper in the sulfuric acid and withdrawing it slowly so that no large drop should remain on the rod. Care must be taken to avoid any contact of the acidified rod with either the bottle or the contents. All determinations must be carried out at least in duplicate. A series of standards has to be determined to eliminate differences in experimental conditions.
Prepare for each determination or series of determinations: a. Blanks consisting of 2 ml 10% trichloroacetic acid. b. 2 ml standard samples in the following concentrations: 0.25; 0.50; 1.00 and 2.00 pg NH,-N per ml. c. Samples. d. Additions. Place the diffusion bottles on the rotor and rotate them 2 h at 50 rev./min. After the rotation period, the rubber stoppers containing the glass rods are removed from the bottles and washed with 2 ml acetate buffer, allowing the fluid to flow into a test tube in which the color reaction is further carried out. Add 0.2 ml chloramine-T solution to each test tube and mix; 90 set later 2.0 ml pyrazolon reagent from a burette are added, after which all the tubes are mixed. Then 0.2 ml sodium thiosulfate is added and all the tubes are mixed again. After the gas bubbles have disappeared the extinction can be measured at 550 m,u, using distilled water as a blank (for instance in a Beckman D.U. spectrophotometer with r-cm cuvettes). The concentrations are read on a standard curve. Calculation
The concentration of blood ammonia nitrogen is calculated by means of the following formula (3) : (NH,-N)moa
=
(NHa-N)rirtratex (Vtotar-0.1 Vprecipitate) (Vt,ta1-
Vtcaa)
V total V precipitate
= total volume in the centrifuge tube. = the volume of the precipitate in the centrifuge tube. = the volume of the trichloroacetic acid brought in the centrifuge tube. = ,ug ammonia nitrogen per ml blood. ;gzcN)bhmd (NH,-N)rntrate = lug ammonia nitrogen per ml filtrate.
Gin.
Chim. Acta, 12 (1965) 55-62
The calculated
value of the “additions”
The chloramiuc-7‘ reaction time F$‘lrcfound that chloramine-T
can 1,~ obtained
could react
from thy formula:
under our experimental
conditions
during I or z min in a mild acid environment without disturbing the color reaction. The color development decreased strongly with a longer reaction time as is shown in Table
I.
TABI,E
1
Reaction time of rhlovami~ze-Z (mix) _._ ~~
Extinction at 550 nzp in 1 -cm cuvettes
0.172
I 2
0.168 0.155 0.145
3 4
o.ogo
IO --
Iicaction mixture: 2 ml standard solution of ammonia, 0.5 ml sodium acetate solution (40 g sodium acetate 3 aq. to 100 ml with distilled water), 0.2 ml chloramine-T r”;, solution, 2 ml pprazolon reagent and 0.1 ml sodium thiosulfate .i”Cl solution.
Color stability after color development The extinction values can be determined rubazonic
acid reaction.
We ascertained
that
about 5 min after completion the extinction
values remained
of the quite
stable during 2 h and showed only very small differences, after having been corrected for the blank, with the values found after 5 min. See Table II. TABLE II STABILITY OF
THE
RUBAZONIC
ACID
COLOR
AT
ROOM
TEMPERATURE
Extinction at 550 mp after completion
XH,-N per ml standard solution
pg
of the color veactim after 5 WZZ~,
uftev 120
0.25
o.ozfz
0.044
0.50
o.ot(g
o.ogo
I.00
0.181
0.18L
milz
Accuracy A standard solution containing approximately o.G ,ug NH,-N per ml 10% trichloroacetic acid was determined twelve times. The following values were found: 0.62; 0.64; 0.63; 0.60; 0.61; 0.63; 0.65; 0.62; 0.58; 0.60; 0.59 and 0.64 pug NH,-N per ml, with a standard deviation of 0.02 pug NH,-N per ml from the mean value of 0.62. lug NH,-N per ml. Clin. Chim. Acta.
12
(1965)
55-62
DETERMINATION
59
OF BLOOD AMMONIA
~ate~ials ~h~ch can d~st~rb the color ~eact~on We found that several nitrogenous compounds can elevate the extinction values at 550 my when present in the filtrate. Tested were those compounds mentioned in Table III. z ml of a solution containing 0.5-0.6 mg of one of these compounds per ml in a 10% solution of trichloroacetic acid, was treated as described in Table I. TABLE SOME
III
NITROGENOUS
COMPOUNDS
Materials tested, 0.5-0.6 mg per ml 10% trichloroacetic acid Atanine Arginine Aspartic acid Asparagine Cystine Glutamic acid Glutamine Glycine Hydroxyproline Isoleucine Lysine Methionine Phenylalanine Proline Serine Tryptophan Tyrosine Acetyicholinc Urea Creatine Creatinine * Tryptophan
THAT
OCCUR
IN THE
TRICHLOROACETIC
ACID
BLOOD
FILTRATE
Calculated amounts of NH,-N in pg @er ml after determination of D:g 0.56 0.17
3.86 0.65 0.53 0.17 0.40 8.46 0.07 0.28
0.55 0.20
0.34 0.68 1.23 *
0.42 0.00 0.00 0.00 0.00
reacted
with chloramine-T,
giving a bluish purple colored precipitate.
The complete diffusion of the ammonia out of the standard samples was achieved after approximately 3 h as shown in Fig. I, nevertheless we obtained good results with a diffusion time of z h. Fig. I shows that even after 3 h of diffusion a small amount of ammonia is still liberated from the blood filtrate. Although it has been mentioned3 that during the diffusion no more ammonia develops in the trichloroacetic acid blood filtrate, we believe that under our experimental conditions a small part of the nitrogenous compounds present in the filtrate produces ammonia. We found that a freshly prepared relatively strong solution of glutamine (1x0 pg per ml) in a 10% solution of trichloroacetic acid gave a 0.15 ,~g per ml amount of NH,-N in our ~monia determination. The preservation of the trichloroacetic acid bloodjiltrate We determined the amount of ammonia in a trichloroacetic acid blood filtrate and found 0.62 ,ug NH8-N per ml filtrate. After storing the filtrate in a refrigerator at o-4” during 4+ and 24 h, the values were determined again and found to have inC&n. Chim, Ada.
12 (1965)
55-62
creased to 0.70 and 0.84 ,q NH,-N per ml filtrate. The same filtrates were also kept at room temperature and measured after 41 and 23 11. \Vc then found 0.82 ant1
1.65pg NH,-K
per ml filtrate. This shows that the ~leternlination
be done soon after the c~ntrifugation the filtrates
for prolonged
in the tr~ch~~~r~)aceti~.acid filtrate
and that it is not even recommended
should to keep
time in an ice bath.
Standard came A straight line was always obtained after z h of diffusion when we plotted the concentration of the standards and the corrected extinction values (= measured extinction value of the standard Fig. z for an example.
60
120
180
Diffusion time
tess measured
240
in minutes
0
extinction
a25 0.50 pg
NH,-N
value of the blank).
7.00 per
ml
See
2.00 standard
solution
Fig. I Diffusion of standard ammonia solutions in ro 0’0 trichloroacetic acid and of trichloroacetic acid blood filtrates. Diffusion vessels contain I ml saturated sodium carbonate and 2 ml standard solution or filtrate. Rotor 50 rev./min. Color reaction is carried out as described under “Analytical puce&are”. X = 2.~ ,ug NH,-N per ml 107~ trichloroacetic acid. R = Trichloroacetic acid filtrate of blood sample I. C = o.5o pg NH,-N per ml 10% trichloroacetic acid. D =: Trichloroacetic acid filtrate of blood sample Il. Fig.
2.
Standard curve after respectively
two hours (A) and one hour (B) of diffusion
Recoveries
of added ammoGa We added 5 ,ug NH,-N to the 5 ml trichloroacetic acid before separating the 5 ml blood in the centrifuge tube. We find an average recovery of 1070/b of the calculated value with a standard deviation of 5.3:/o, as is shown in Table IV. The blood ammonia levels were determined of 20 fasting individuals, or individuals who had breakfasted at least 2 h before venipuncture, aged from 25 to 76 years without clinical evidence of liver disorders. The values found were : 0.80; 0.54; Clin. Chim. Ada,
12 (1965) 55-62
DETERMINATION
TABLE
IV
RECOVERIES OF ADDED .___ _~ ~__...______~__
pg NH,-N ml blood
Subject
3 4 : ; 9
12
‘3 *4
Calculated pg NH,-N per ml “addition”
0.80
0.80 0.87
IO2
0.82
IO0
0.89
99 113 105
0.82 0.88 0.87
0.77 0.77 I .oG 0.83
0.81 I.19 0.92
0.96 1.35
1.80
0.71 I.43
O.GG
0.92
0.86
107%;
standard deviation
0/ORecovery
112 III
0.76 0.84 1.29
0.84
0.78 I.47
Mean recovery
~__
Retermined pg NH,-N per ml “addition” 0.89
0.50 0.53 I .52 0.33
Ii
Patient
__~_~_____.~._~~~_
per
0.50
IO
TABLE
ANMONIA
0.48 0.67 0.61 0.7’ 0.47 0.41 I .04
2
NH,-N
61
OF BLOOD AMMONI.4
III
“4 IO.5 II0
103 107
5.3%.
V VALUES
IN VENOUS
Condition
BLOOD
OF SOME
PATIENTS
.-
Regurgitation jaundice; fasting Regurgitation jaundice; fasting Hepatitis; fasting Cirrhosis of the liver ; fasting Cirrhosis of the liver; fasting Cirrhosis of the liver; fasting After a protein-rich meal Cirrhosis of the liver; subcomatose After treatment with neomycin Artificial portacaval shunt; fasting
,u,g NH,-N
per ml blood
0.33 0.78 0.66 0.35 0.76 I .02 1.89 2.19 0.95 I.80
0.38; 0.64; 0.57; 0.72; 0.66; 0.30; 0.53; 0.45; 0.48; 0.67; 0.61; 0.71; 0.49; 0.47; 0.41; 0.50; 0.50 and 0.53 ,ug NH,-N per ml blood giving a mean value of 0.53 ,~g NH,-N per ml blood with a standard deviation of 0.1~5 pg NH,-N per ml blood with values ranging from 0.30 to 0.80 pug NH,-N per ml blood. In conclusion we should like to state some of the values that we found in different patients. See Table V. Three fasting patients with jaundice (A and B had regurgitation jaundice and C had hepatitis), had values of respectively 0.33,0.78 and 0.66 ,ug NH,-N per ml blood. In two patients (D and E) with a cirrhosis of the liver, after fasting for at least 15 h, we found values of 0.35 and 0.76 ,ug NHtN per ml blood. We determined a value of 1.02 pg NH$-N per ml blood by a patient (F) with cirrhosis of the liver who had not breakfasted. One hour after he had eaten a protein-rich meal, the value increased to I.89 pg NH,-N per ml blood. A patient (G) with cirrhosis of the liver, in a subcomatose condition, had a value of 2.19 pug NH,-N per ml blood. After having been treated during ten days with neomycin we found a value of 0.95 pg NH,-N per ml blood, in spite of the protein-rich diet on which the patient had been put. Ctin. Claim. Acta, 12 (1965) 55-62
An ambulant patient (H) with an artificial portacaval shunt, \vhiclr 11~1 1~~1 introduced four years ago, had a fasting value of 1.80 jrg NH,-N 1”~ ml bl~~otl.
The determination of the blood ammonia values in a trichloroacetic acid blood proved to be a good method to reduce the autolysis of nitrogenous compounds. A diffusion time of I h can also be used instead of our z-h period. The same straight standard curve can be constructed, but with lower extinction values (S;CC Fig. 2). Instead of the r-h period we prefer a 2-h diffusion period because of tht greater accuracy in determining the low normal blood ammonia values, e\en though the possibility of an increase in the NH,-N value due to aspecific low-molecular nitrogenous compounds is somewhat greater. The calculated values, with a diffusion period of I h, for the NH,-N in blood filtrates are practically the same as the values found after 2 h of diffusion, thus the increase due to aspecific nitrogenous compounds is very slight. The actual color reaction is very simple but care should be taken that the P'H is first brought to 3.9 to 4.2 with a buffer (7) before adding the chloramine-T, and that it is not allowed to react longer than z min. We found no difference in the chosen diffusion time, in the color reaction or in the color intensity while using I N sulfuric acid or I M citric acid. The addition of Sterox SE to the sulfuric acid did not raise the values of the blank, but it helped to distribute the acid evenly over the rods. It is not recommended to store the standard solutions in polyethylene bottles which may alter the concentrations of the as this material can absorb ammonial”, solutions. In order to find out if a direct reaction was possible, without first letting the trichloroacetic acid blood filtrate diffuse, we brought 2 ml filtrate to the right PH with sodium acetate and performed the reaction in this solution. The standards were handled in exactly the same way. With this method we found for a normal health!. individual 1.58 ,ug NH,-N per ml blood, and with the diffusion technique 0.45 pg NH,-N per ml blood. This clearly shows that the direct reaction method is easily influenced by nitrogenous compounds other than ammonia, which are present in the filtrate, as was already demonstrated in Table III. A great advantage in our method is that the color with the rubazonic acid reaction is not only very stable, but it cannot be influenced by ammonia which ma! be absorbed by the reaction mixture after the addition and the reaction of the sodium thiosulfate. filtrate
REFERENCES I P. FABIANI, S. BOULU AND F. LamB, Presse Mid.. h6 (1958) 138 L V. D. PERU AND R. S. NELSON,CZ~~.C/Z~WZ. Acta, 9 (1964) 13. 3 D. G. NATHAN AND F. L. RODKEY, J. Lab. Clin. Med., 49 (1957) 779. 4 G. E. MILLER AND J. D. RICE, Am. J. Clin. Pathol., 39 (1963) 97. 5 Ii. KONITZER AND S. VOIGT, C&n. Chive. Acta, 8 (1963) 5. 6 1. C. H. FENTON. Clin.Chim. .4&a. 7 (1462) 161. u 7 k KALA, Phavnkzzie, 18 (1963) 29: ’ ’ _ ’ 8 J, M. KRUsE ANDM. G. MELLON, Anal. Chem., 25 (1953) 1188. 9 D. SELIGSON AND H. SELIGSON, J. Lab. Clint. Med., 38 (1951) 324 IO L. S. THEOBALD, Analyst, 84 (1959) 570. C&n. Chim.
Acta,
12 (1965)
55-62
CLINICA CHIMICA ACTA
AMMONIA
DETERMINATION
FILTRATE
AFTER
RUBAZONIC
WITH
ACID BLOOD
THE AID OF THE
ACID REACTION
A. F. K. BUYS BALLOT
AND
Clinical Chemistry Laboratory, Rotterdam (The Netherlands)
(Received July
IN A TRICHLOROACETIC
MICRODIFFUSION
zznd,
C. STEENDIJK Municipal
Bergweg Hospital,
1964)
SUMMARY A technique for the determination of blood ammonia based on the following steps is described: I. Preparation of a protein-free trichloroacetic acid filtrate of venous blood. 2. The diffusion of ammonia out of the alkaline filtrate. 3. The calorimetric determination of the NH,-N in an acid medium with the aid of the rubazonic acid reaction, Twelve determinations of a standard ammonia solution gave a mean value of 0.62 ,ug NH,-N per ml with a standard deviation of 0.02 l”g NH,-N per ml. The normal venous blood ammonia level, with this method, varies from 0.30 to 0.80 ,ug NH,-N per ml. Results of determinations of blood NH,-N of patients with liver diseases are described.
Ammonia is said to originate in the gastrointestinal tract by the action of intestinal bacteria on nitrogenous substrates. An increase in peripheral venous blood ammonia levels is found by impaired hepatic functions such as cirrhosis of the liver and portacaval shuntsl. After oral intake of, for instance, proteins, blood, amino acids, ammonium salts and urea, the peripheral venous blood ammonia levels increased in patients with cirrhosis of the liver or hepatitis and in patients with cancer of the liver 2. Because of the very low quantities of ammonia which normally appear in the peripheral blood, an accurate method for the determination is necessary. Nessler’s reagent is not sensitive enough. The ninhydrin reaction and the phenolate-hypochlorite reagent are sensitive and have found application in several methods3-6. Kala’ described a modification of Kruse and Mellon’s calorimetric ammonia determination8, which he called the rubazonic acid method. As it was described as being very sensitive, we tried to apply this method for the determination of blood C&n. Chim. Acta, 12 (1965) 55-62
ammonia. In a trichloroacetic acid blood filtrate, WC dcterniinc~d the amount (of ammonia with the aid of the rubazonic acid reaction after diffusion, as dcscribt~d by Seligson 9.
Reageds I. A zoo,& solution of trichloroacetic acid in distilled water. 2. Saturated potassium carbonate p.a. solution in distilled water. Dissolve IIO g potassium carbonate to IOO ml. The solution is kept several days in an exsiccator above I M citric acid at 50’ to remove all traces of ammonia. 3. +I N sulfuric acid p.a. Dilute 5 ml concentrated sulfuric acid p.a. with distilled water to IOO ml and add one drop of Sterox SE. 4. I M acetate buffer, pH 4.1. (130 ml I M sodium acetate + 370 ml I 12f acetic acid.) 5. zO,b Solution of chloramine-T (sodium paratoluenesulfonchloramide) in distilled water. To be prepared fresh each week and stored in a dark bottle. 6. Pyridine p.a. This liquid can only be used as long as it is colorless. If it turns yellowish brown, it has to be distilled before use. 7. Pyrazolon reagent. Mix equal parts of solution A and solution B. Solution A: 150 mg 3-methyl-phenyl-5-pyrazolon in 40 ml pyridine. Solution B: 25 mg bispyrazolon. (3,3’-dimethyl-I,I’-diphenyl-4,4’-bi-z-pyrazoline-5,5’-dion) in 40 ml pyridine. Dissolve by shaking. The solutions A and B ha\:e to be prepared fresh each day. S. 3o/o Solution of sodium thiosulfate in distilled water. 9. Standard ammonium sulfate solution (IO mg”/b NH,-N). Dissolve 47r.4 mg ammonium sulfate p.a. in I 1 distilled water. IO. Standard samples in IO:/, trichloroacetic acid p.a. Dilute reagent 9. with reagent I. and distilled water to obtain standard concentrations of 0.25; 0.50; 1.00 and 2.00 ,ug NH,-N per ml. Note: a. The blank, the standard samples and the 200,” solution of trichloroacetic acid must be prepared with the same distilled water. b. The glassware was cleaned by rinsing once with cont. HCl, three times with distilled water, once with a 2!0 solution of sodium carbonate in distilled water and five times with distilled water. Afterwards it was dried at about 60”.
Equifwnent
For diffusion 25-ml penicillin bottles are used. Each bottle is fitted with a rubber stopper through which a glass rod with a rounded end is placed. The rounded end of the glass rod should extend to about 20 mm from the bottom of the bottle. 2. An electric rotor as described by Seligson9. 3. Calibrated 12 ml centrifuge tubes. 4. A spectrophotometer with a r-cm glass cuvette or a calorimeter with an interference filter 550 mp. I.
Analytical
procedure
Draw approximately IO ml blood, without stasis, and bring 5 ml of this blood immediately over into a rz-ml calibrated centrifuge tube, containing 5 ml ice-cooled Clin.
Chim.
Acta,
IL (1965) 55-62
DETERMINATION
OF BLOOD
57
AMMONIA
20% trichloroacetic acid. The tube should be immediately capped with parafilm and mixed by shaking vigorously. The other 5 ml, like the first portion, are transferred into a tube which contains, besides the trichloroacetic acid, 5 lug NH,-N. These “additions” are further handled exactly the same way as the samples. Reimmerse the tubes in the ice bath and take them to the laboratory where they are centrifuged for IO min at approximately IOOOg (about 2500 rev./min). After centrifugation the tubes have reached approximately room temperature. After measuring the total volume and the volume of the precipitate, decant the clear supernatant into an ice-cooled test tube and cap with parafilm (further referred to as the filtrate). Transfer into the diffusion bottle, at room temperature, I ml saturated potassium carbonate and 2 ml of the filtrate. Close the bottle immediately with the rubber stopper containing the glass rod, which has been moistened with the I N sulfuric acid-Sterox solution. This wetting of the glass rod can best be done by immersing the rod up to 5 mm from the rubber stopper in the sulfuric acid and withdrawing it slowly so that no large drop should remain on the rod. Care must be taken to avoid any contact of the acidified rod with either the bottle or the contents. All determinations must be carried out at least in duplicate. A series of standards has to be determined to eliminate differences in experimental conditions.
Prepare for each determination or series of determinations: a. Blanks consisting of 2 ml 10% trichloroacetic acid. b. 2 ml standard samples in the following concentrations: 0.25; 0.50; 1.00 and 2.00 pg NH,-N per ml. c. Samples. d. Additions. Place the diffusion bottles on the rotor and rotate them 2 h at 50 rev./min. After the rotation period, the rubber stoppers containing the glass rods are removed from the bottles and washed with 2 ml acetate buffer, allowing the fluid to flow into a test tube in which the color reaction is further carried out. Add 0.2 ml chloramine-T solution to each test tube and mix; 90 set later 2.0 ml pyrazolon reagent from a burette are added, after which all the tubes are mixed. Then 0.2 ml sodium thiosulfate is added and all the tubes are mixed again. After the gas bubbles have disappeared the extinction can be measured at 550 m,u, using distilled water as a blank (for instance in a Beckman D.U. spectrophotometer with r-cm cuvettes). The concentrations are read on a standard curve. Calculation
The concentration of blood ammonia nitrogen is calculated by means of the following formula (3) : (NH,-N)moa
=
(NHa-N)rirtratex (Vtotar-0.1 Vprecipitate) (Vt,ta1-
Vtcaa)
V total V precipitate
= total volume in the centrifuge tube. = the volume of the precipitate in the centrifuge tube. = the volume of the trichloroacetic acid brought in the centrifuge tube. = ,ug ammonia nitrogen per ml blood. ;gzcN)bhmd (NH,-N)rntrate = lug ammonia nitrogen per ml filtrate.
Gin.
Chim. Acta, 12 (1965) 55-62
The calculated
value of the “additions”
The chloramiuc-7‘ reaction time F$‘lrcfound that chloramine-T
can 1,~ obtained
could react
from thy formula:
under our experimental
conditions
during I or z min in a mild acid environment without disturbing the color reaction. The color development decreased strongly with a longer reaction time as is shown in Table
I.
TABI,E
1
Reaction time of rhlovami~ze-Z (mix) _._ ~~
Extinction at 550 nzp in 1 -cm cuvettes
0.172
I 2
0.168 0.155 0.145
3 4
o.ogo
IO --
Iicaction mixture: 2 ml standard solution of ammonia, 0.5 ml sodium acetate solution (40 g sodium acetate 3 aq. to 100 ml with distilled water), 0.2 ml chloramine-T r”;, solution, 2 ml pprazolon reagent and 0.1 ml sodium thiosulfate .i”Cl solution.
Color stability after color development The extinction values can be determined rubazonic
acid reaction.
We ascertained
that
about 5 min after completion the extinction
values remained
of the quite
stable during 2 h and showed only very small differences, after having been corrected for the blank, with the values found after 5 min. See Table II. TABLE II STABILITY OF
THE
RUBAZONIC
ACID
COLOR
AT
ROOM
TEMPERATURE
Extinction at 550 mp after completion
XH,-N per ml standard solution
pg
of the color veactim after 5 WZZ~,
uftev 120
0.25
o.ozfz
0.044
0.50
o.ot(g
o.ogo
I.00
0.181
0.18L
milz
Accuracy A standard solution containing approximately o.G ,ug NH,-N per ml 10% trichloroacetic acid was determined twelve times. The following values were found: 0.62; 0.64; 0.63; 0.60; 0.61; 0.63; 0.65; 0.62; 0.58; 0.60; 0.59 and 0.64 pug NH,-N per ml, with a standard deviation of 0.02 pug NH,-N per ml from the mean value of 0.62. lug NH,-N per ml. Clin. Chim. Acta.
12
(1965)
55-62
DETERMINATION
59
OF BLOOD AMMONIA
~ate~ials ~h~ch can d~st~rb the color ~eact~on We found that several nitrogenous compounds can elevate the extinction values at 550 my when present in the filtrate. Tested were those compounds mentioned in Table III. z ml of a solution containing 0.5-0.6 mg of one of these compounds per ml in a 10% solution of trichloroacetic acid, was treated as described in Table I. TABLE SOME
III
NITROGENOUS
COMPOUNDS
Materials tested, 0.5-0.6 mg per ml 10% trichloroacetic acid Atanine Arginine Aspartic acid Asparagine Cystine Glutamic acid Glutamine Glycine Hydroxyproline Isoleucine Lysine Methionine Phenylalanine Proline Serine Tryptophan Tyrosine Acetyicholinc Urea Creatine Creatinine * Tryptophan
THAT
OCCUR
IN THE
TRICHLOROACETIC
ACID
BLOOD
FILTRATE
Calculated amounts of NH,-N in pg @er ml after determination of D:g 0.56 0.17
3.86 0.65 0.53 0.17 0.40 8.46 0.07 0.28
0.55 0.20
0.34 0.68 1.23 *
0.42 0.00 0.00 0.00 0.00
reacted
with chloramine-T,
giving a bluish purple colored precipitate.
The complete diffusion of the ammonia out of the standard samples was achieved after approximately 3 h as shown in Fig. I, nevertheless we obtained good results with a diffusion time of z h. Fig. I shows that even after 3 h of diffusion a small amount of ammonia is still liberated from the blood filtrate. Although it has been mentioned3 that during the diffusion no more ammonia develops in the trichloroacetic acid blood filtrate, we believe that under our experimental conditions a small part of the nitrogenous compounds present in the filtrate produces ammonia. We found that a freshly prepared relatively strong solution of glutamine (1x0 pg per ml) in a 10% solution of trichloroacetic acid gave a 0.15 ,~g per ml amount of NH,-N in our ~monia determination. The preservation of the trichloroacetic acid bloodjiltrate We determined the amount of ammonia in a trichloroacetic acid blood filtrate and found 0.62 ,ug NH8-N per ml filtrate. After storing the filtrate in a refrigerator at o-4” during 4+ and 24 h, the values were determined again and found to have inC&n. Chim, Ada.
12 (1965)
55-62
creased to 0.70 and 0.84 ,q NH,-N per ml filtrate. The same filtrates were also kept at room temperature and measured after 41 and 23 11. \Vc then found 0.82 ant1
1.65pg NH,-K
per ml filtrate. This shows that the ~leternlination
be done soon after the c~ntrifugation the filtrates
for prolonged
in the tr~ch~~~r~)aceti~.acid filtrate
and that it is not even recommended
should to keep
time in an ice bath.
Standard came A straight line was always obtained after z h of diffusion when we plotted the concentration of the standards and the corrected extinction values (= measured extinction value of the standard Fig. z for an example.
60
120
180
Diffusion time
tess measured
240
in minutes
0
extinction
a25 0.50 pg
NH,-N
value of the blank).
7.00 per
ml
See
2.00 standard
solution
Fig. I Diffusion of standard ammonia solutions in ro 0’0 trichloroacetic acid and of trichloroacetic acid blood filtrates. Diffusion vessels contain I ml saturated sodium carbonate and 2 ml standard solution or filtrate. Rotor 50 rev./min. Color reaction is carried out as described under “Analytical puce&are”. X = 2.~ ,ug NH,-N per ml 107~ trichloroacetic acid. R = Trichloroacetic acid filtrate of blood sample I. C = o.5o pg NH,-N per ml 10% trichloroacetic acid. D =: Trichloroacetic acid filtrate of blood sample Il. Fig.
2.
Standard curve after respectively
two hours (A) and one hour (B) of diffusion
Recoveries
of added ammoGa We added 5 ,ug NH,-N to the 5 ml trichloroacetic acid before separating the 5 ml blood in the centrifuge tube. We find an average recovery of 1070/b of the calculated value with a standard deviation of 5.3:/o, as is shown in Table IV. The blood ammonia levels were determined of 20 fasting individuals, or individuals who had breakfasted at least 2 h before venipuncture, aged from 25 to 76 years without clinical evidence of liver disorders. The values found were : 0.80; 0.54; Clin. Chim. Ada,
12 (1965) 55-62
DETERMINATION
TABLE
IV
RECOVERIES OF ADDED .___ _~ ~__...______~__
pg NH,-N ml blood
Subject
3 4 : ; 9
12
‘3 *4
Calculated pg NH,-N per ml “addition”
0.80
0.80 0.87
IO2
0.82
IO0
0.89
99 113 105
0.82 0.88 0.87
0.77 0.77 I .oG 0.83
0.81 I.19 0.92
0.96 1.35
1.80
0.71 I.43
O.GG
0.92
0.86
107%;
standard deviation
0/ORecovery
112 III
0.76 0.84 1.29
0.84
0.78 I.47
Mean recovery
~__
Retermined pg NH,-N per ml “addition” 0.89
0.50 0.53 I .52 0.33
Ii
Patient
__~_~_____.~._~~~_
per
0.50
IO
TABLE
ANMONIA
0.48 0.67 0.61 0.7’ 0.47 0.41 I .04
2
NH,-N
61
OF BLOOD AMMONI.4
III
“4 IO.5 II0
103 107
5.3%.
V VALUES
IN VENOUS
Condition
BLOOD
OF SOME
PATIENTS
.-
Regurgitation jaundice; fasting Regurgitation jaundice; fasting Hepatitis; fasting Cirrhosis of the liver ; fasting Cirrhosis of the liver; fasting Cirrhosis of the liver; fasting After a protein-rich meal Cirrhosis of the liver; subcomatose After treatment with neomycin Artificial portacaval shunt; fasting
,u,g NH,-N
per ml blood
0.33 0.78 0.66 0.35 0.76 I .02 1.89 2.19 0.95 I.80
0.38; 0.64; 0.57; 0.72; 0.66; 0.30; 0.53; 0.45; 0.48; 0.67; 0.61; 0.71; 0.49; 0.47; 0.41; 0.50; 0.50 and 0.53 ,ug NH,-N per ml blood giving a mean value of 0.53 ,~g NH,-N per ml blood with a standard deviation of 0.1~5 pg NH,-N per ml blood with values ranging from 0.30 to 0.80 pug NH,-N per ml blood. In conclusion we should like to state some of the values that we found in different patients. See Table V. Three fasting patients with jaundice (A and B had regurgitation jaundice and C had hepatitis), had values of respectively 0.33,0.78 and 0.66 ,ug NH,-N per ml blood. In two patients (D and E) with a cirrhosis of the liver, after fasting for at least 15 h, we found values of 0.35 and 0.76 ,ug NHtN per ml blood. We determined a value of 1.02 pg NH$-N per ml blood by a patient (F) with cirrhosis of the liver who had not breakfasted. One hour after he had eaten a protein-rich meal, the value increased to I.89 pg NH,-N per ml blood. A patient (G) with cirrhosis of the liver, in a subcomatose condition, had a value of 2.19 pug NH,-N per ml blood. After having been treated during ten days with neomycin we found a value of 0.95 pg NH,-N per ml blood, in spite of the protein-rich diet on which the patient had been put. Ctin. Claim. Acta, 12 (1965) 55-62
An ambulant patient (H) with an artificial portacaval shunt, \vhiclr 11~1 1~~1 introduced four years ago, had a fasting value of 1.80 jrg NH,-N 1”~ ml bl~~otl.
The determination of the blood ammonia values in a trichloroacetic acid blood proved to be a good method to reduce the autolysis of nitrogenous compounds. A diffusion time of I h can also be used instead of our z-h period. The same straight standard curve can be constructed, but with lower extinction values (S;CC Fig. 2). Instead of the r-h period we prefer a 2-h diffusion period because of tht greater accuracy in determining the low normal blood ammonia values, e\en though the possibility of an increase in the NH,-N value due to aspecific low-molecular nitrogenous compounds is somewhat greater. The calculated values, with a diffusion period of I h, for the NH,-N in blood filtrates are practically the same as the values found after 2 h of diffusion, thus the increase due to aspecific nitrogenous compounds is very slight. The actual color reaction is very simple but care should be taken that the P'H is first brought to 3.9 to 4.2 with a buffer (7) before adding the chloramine-T, and that it is not allowed to react longer than z min. We found no difference in the chosen diffusion time, in the color reaction or in the color intensity while using I N sulfuric acid or I M citric acid. The addition of Sterox SE to the sulfuric acid did not raise the values of the blank, but it helped to distribute the acid evenly over the rods. It is not recommended to store the standard solutions in polyethylene bottles which may alter the concentrations of the as this material can absorb ammonial”, solutions. In order to find out if a direct reaction was possible, without first letting the trichloroacetic acid blood filtrate diffuse, we brought 2 ml filtrate to the right PH with sodium acetate and performed the reaction in this solution. The standards were handled in exactly the same way. With this method we found for a normal health!. individual 1.58 ,ug NH,-N per ml blood, and with the diffusion technique 0.45 pg NH,-N per ml blood. This clearly shows that the direct reaction method is easily influenced by nitrogenous compounds other than ammonia, which are present in the filtrate, as was already demonstrated in Table III. A great advantage in our method is that the color with the rubazonic acid reaction is not only very stable, but it cannot be influenced by ammonia which ma! be absorbed by the reaction mixture after the addition and the reaction of the sodium thiosulfate. filtrate
REFERENCES I P. FABIANI, S. BOULU AND F. LamB, Presse Mid.. h6 (1958) 138 L V. D. PERU AND R. S. NELSON,CZ~~.C/Z~WZ. Acta, 9 (1964) 13. 3 D. G. NATHAN AND F. L. RODKEY, J. Lab. Clin. Med., 49 (1957) 779. 4 G. E. MILLER AND J. D. RICE, Am. J. Clin. Pathol., 39 (1963) 97. 5 Ii. KONITZER AND S. VOIGT, C&n. Chive. Acta, 8 (1963) 5. 6 1. C. H. FENTON. Clin.Chim. .4&a. 7 (1462) 161. u 7 k KALA, Phavnkzzie, 18 (1963) 29: ’ ’ _ ’ 8 J, M. KRUsE ANDM. G. MELLON, Anal. Chem., 25 (1953) 1188. 9 D. SELIGSON AND H. SELIGSON, J. Lab. Clint. Med., 38 (1951) 324 IO L. S. THEOBALD, Analyst, 84 (1959) 570. C&n. Chim.
Acta,
12 (1965)
55-62