A Simple and Rapid Procedure for the

Determination of Amino-nitrogen
and Ammonia in Urine

T. W. Clarkson and L. Ferraio

The ninhydrin-colorimetric procedure for the determination of urinary 2 amino-

nitrogen has been simplified by the use of the Conway microdiffusion unit to remove
ammonia from the sample. In this way, urinary ammonia is measured, and at the
same time an ammonia-free urine sample is ready for 2 amino-nitrogen analysis.
Values for total and free 2 amino-nitrogen in normal urine samples were found to be
similar to those previously reported by more complicated procedures.

THE METHOD described by Rubinstein and Pryce for the determination

of amino-nitrogen in tissue fluids requires that all traces of ammonia
be removed from the sample (1). In their method, this was achieved#{149}by
adding potassium carbonate to the sample and evaporating to dryness
in a vacuum desiccator, usually overnight. The sample is then dissolved
in distilled water and amino-nitrogen determined.
Their procedure may be simplified by the use of the Conway micro-
diffusion unit to remove the ammonia from the urine sample. In this
way, urinary ammonia is measured according to Conway (2), and at
the same time, an ammonia-free urine urine sample is ready for
amino-nitrogen analysis.
Method
Reagents and material for microdiffusion procedure are as described
by Conway except that 1.0 N NaOH was used in place of saturated
K2C03, and in some cases, 6 N HOSO4 was used in place of the borate
buffer to absorb ammonia.
Reagents and materials for colorimetric estimation of amino-
nitrogen are as described by Rubinstein and Pryce (1).

Prom the Department of Radiation Biology and Biophysics, University of Rochester School
of Medicine and Dentistry, Rochester, NY 14620.
This paper is based on work performed under contract with the US Atomic Energy Com-
mission at the University of Rochester Atomic Energy Project and is Report No. UR-49-996.
Received for publication March 7, 1968; accepted for publication Oct 11, 1968.
433
434 CLARKSON & FERRAIO Clinical Chemistry

Procedure
Free 2 Amino-nitrogen and Ammonia
Place 1 ml of boric acid-buffer indicator solution in the center well,
and 250 I of urine and 250 l of distilled water in the right hand side
of the outer compartment of the diffusion unit. Add quickly 1.0 ml of
1.0 N NaOH to the left hand side of the outer compartment and im-
mediately place the cover over the unit. Carefully tilt and rotate the
unit to mix the urine with the NaOH, and allow to stand at room
temperature for at least 180 mm. As described by Conway, titrate the
solution in the center well with 0.2 N sulfuric acid to determine the
amount of ammonia (2). Remove a 0.5-mi sample from the outer well
and dilute to 10 ml with distilled water. Remove 0.5 ml of this solution
for determination of amino-nitrogen according to Rubinstein and
Pryce (1). For routiiie determinations, a convenient standard solution
consists of 28.6 mmol/l (N114)2S04 (80 mg N per 100 ml) and 20 mmol/l
glutamic acid (14 mg amino nitrogen per 100 ml). Add 250 i and 500 l
of standard solution to the chamber of the diffusion unit and proceed
as described for urine samples.

Total of Amino-nitrogen
Add 1 ml of concentrated hydrochloric acid to 2 ml of urine c&ntained
in a Pyrex test tube (15 X 1.15 cm). Seal the tube in a gas-oxygen flame,
and place in an oven at 150#{176} overnight. Allow to cool. Open the tube,
and decant the fluid into a small beaker or plastic vial. Add sufficient
4 N NaOH to adjust the pH to approximately 7.0 (usually 2.6-2.8 ml
NaOH is needed). Transfer a 500-l sample to the outer vessel of the
diffusion unit and proceed as in the determination of free amino-
nitrogen as discussed above. The same standard solution may be used.

Results
The absorbance of the distilled water blank was 0.060 (SD =
± 0.006), when averaged over determinations performed on different
days. However, the standard deviation was only 0.001 for 10 determina-
tions made in the same colorimetric run.
The mean recoveries of ammonia, added as ammonium sulfate and
of amino-nitrogen added as glutamic acid, to 10 urine samples were
96% (SD = ± 2) and 98% (SD = ± 1), respectively.
Ten samples from the same urine specimen, analyzed according to
Rubinstein and Pryce, gave the following values expressed as milli-
grams of N per 100 ml ± SD: free amino-nitrogen, 140 ± 3, and
bound amino-nitrogen 220 ± 4. The corresponding values determined
Vol. 15. No. 6. 1969 a AMINO.NITROGEN 435

by the present method were 147 ± 3 and 220 ± 4. The urinary ammonia
measured according to Conway (2) had an average value of 50 ± 1
compared to 48 ± 1 by our method.
The standard deviations quoted above indicate that, in our hands,
the methods of Rubinstein and Pryce and of Conway gave the same
precision as our method.
When amino acids are determined in the presence of ammonium salts,
a small residual amount of ammonia remains in the solution in the
outer chamber of the Conway unit. The average ammonia nitrogen in
the normal urines reported in Table 1 was 54 mg/100 ml. If no correc-
tion is made for the ammonia interference, the 54 mg/100 ml normal
level would increase the amino-nitrogen readings by approximately
1.0 mg/100 ml. This would be equal to 7% of the average amino-
nitrogen concentration found in the urines reported in Table 1. If 6 N
ILSO4 is used in the central well of the Conway unit, the interference
due to ammonia is only 3.4%. However, because the concentration of
H2S04 is so high, it is impossible to measure the absorbed ammonia.
An exact correction can he made for the ammonia interference by
making blank runs on solutions containing known concentrations of
ammonium sulfate. A more convenient way is to add ammonium sulfate
to the amino-nitrogen standard in the proportion given in the method
section. When this standard solution is used along with the distilled
water blank, the error due to ammonia interference in the amino-
nitrogen readings was only 1.7% (SD = ± 1.2%), as determined for
the normal urines reported in Table 1.
The values reported in Table 1 for normal urinary excretion may he
compared with values in the literature. Jagenburg (3) in his review
of the literature noted that average values for free amino-nitrogen
determined by the ninhydrin-carbon dioxide method were in the range
between 101 and 165 mg N per 24 hr. Using ion-exchange chromatog-
raphy, followed by a photometric ninhydrin determination, Jagenburg
reported an average value of 124 ± 28 mg per 24 hr from 26 urine
samples from adult males. We find an average value of 160 ± 35 mg N

Table 1. URINARY a AMINO-NITROGEN AND AMMONIA NITROGEN IN

SPECIMENS FROM 20 MALE LABORATORY WORKERS

a Amino-nitrogen Ammonia nitrogen

mg/4 hr mg/kg/4 hr mg/g creatinine mg/4 hr mg’kg/E4 hr ng/g creatinine

Free 160 ± 35 2.19 ± 0.40 75 ± 19 - - -

Bound 254 ± 1,0 3.40 ± 1.6 125 ± 70 - - -

Total 414 ± 152 5.63 ± 1.5 203 ± 69 59S ± 202 5.0 ± 1.9 255 -- t9

All results are the means ± standard deviations.

436 CLARKSON & FERRAIO Clinical Chemistry

per 24 hr. When the excretion of free amino-nitrogen is expressed as

milligrams N per kilogram per 24 hr, the value reported in Table 1
agrees with previous findings (1, 3).
Some authors quote amino-nitrogen excretion relative to that of
creatinine. Using the specific ion-exchange photometric ninhydrin
method, Clarkson and Kench (4) and Jagenburg (3) report values of
52 ± 10 and 80 ± 21 mg N per gram of creatinine. These compare
with our value of 78 ± 19 mg N per gram of creatinine.
There are fewer reports in the literature concerning bound amino-
nitrogen. Rubinstein and Pryce (1) report an average from 12 male
adults of 167 ± 44 mg N per 24 hr, as compared to our finding of
254 ± 150.
It has been claimed by Kekki (5) that the determination of amino-
nitrogen as copper complexes measures both the free and the bound
amino-nitrogen. Using this method, Kekki reports mean values of
248 ± 104 mg/24 hr from 95 adults. All other workers report average
values in the range 179-500 mg N per 24 hr (6-9). Our total amino-
nitrogen value is 414 ± 152.
The ammonia excretion values reported in Table 1 lie in the normal
range of 0.4-1.0 g N per 24 hr (10).

Discussion
This method offers the advantage in time and simplicity in using the
Conway microdiffusion procedure for ammonia determinations to
remove ammonia from the urine sample prior to measurement of
amino-nitrogen.
NaOH, 1.0 N, was found to be just as effective, and required the same
diffusion times for the removal of added ammonia nitrogen from stand-
ard solutions on urine samples, as the high concentration (40% [w/vi
KOH) advised by Conway (2). Furthermore, 40% KOH gave high
readings for x amino-nitrogen in normal urine of about double the
reported normal values, suggesting some hydrolysis of bound amino-
nitrogen takes place. A diffusion time of 180 mm is an adequate period
in which to remove the ammonia, though longer times may be used, such
as overnight periods.
Since the colorimetric procedure is identical to that described by
Rubinstein and Pryce (1), their comments concerning color yield of
various amino acids and the small interference due to urea apply to
this method also.
References
1. Ruhiiistein, II. M., and Pryce, J. D., The eolorinwtrie estinIatiOI, of a-aminO-IIitrOgen in
tissue fluids. J. Clin. Pathol. 12, 80 (1959).
Vol. 15, No. 6, 1969 a AMINO-NITROGEN 437

2. Conway, E. J., Microdiffusion Analyss and Volumetric Error (ed. 3). Crosby Lockwood
and Son Ltd., London, 1950, p. 87.
3. Jagenburg, 0. H., The urinary excretion of free amino acids and other amino compounds
by the human. Scand. J. Clia. Lab. Invest. 11 (Suppl. 43), 1 (1959).
4. (‘larkson, T. W., and Kench, J. E., Urinary excretion of amino acids by ‘UCII absorbing
heavy metals. Biochenl. J. 62, 361 (1956).
5. Kekki, M., Microdeterinination of amino-nitrogen as copper complexes: A modification
for plasma and urine. Scand. J. Clin. Lab. Invest. 11, 311 (1959).
6. Albanese, A. A., Frankston, J., and Irby, V., The estimation of methionine in protein
hydrolysates and human urine. J. Biol. Chem. 156, 293 (1944).
7. Albanese, A. A., bit, L E., Frankston, J., and Irby, V., Estimation and characterization
of bound amino N of normal human urine. Fed. Proc. 5, 118 (1946).
8. Kalant, H., and Alucci, H., The influence of diet on Urinary amino nitrogen levels. J. Clin.
Endocrinol. 15, 481 (1955).
9. Geer, R. P., Hantman, R. K., and Swett, C. V., Quantitative chromatographic studies on
urinary amino acid excretion. Clin. Chem. 14, 12 (1968).
10. Diem, K., Documenta Geigy. Geigy Pharmaceutical Co., Manchester 1962, p. 528.