A COLORIMETRIC METHOD FOR THE DETERMINATION OF

METHYLAMINE IN URINE

BY ANDREW A. ORMSBY AND SHIRLEY JOHNSON

(From the Department of Biochemistry and Nutrition, and the Clinical Laboratory of
the John Scaly Hospital, University of Texas, Medical Branch, Galveston)

(Received for publication, July 14, 1950)

In the course of feeding experiments in which mono-, di-, and trimethyl-

amines were fed to dogs, it was desirable to determine the urinary excretion
of methylamine (monomethylamine). The available methods were found
to be cumbersome and not well adapted to the routine analysis of a large

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number of urines. Weber and Wilson (1) separated ammonia from the
amines by the use of yellow mercuric oxide and determined methylamine
in the filtrate by the nitrous acid reaction. Kapeller-Adler and Krael
(2) and Kapeller-Adler and Toda (3) determined methylamine in urine
by an involved method which required concentration of the urine, aeration
of amines into HCl, chloroform fractionation of the residue, and finally
a determination of alkimide groups by reaction with hydriodic acid. By
the use of this method Kapeller-Adler and Krael found relatively large
amounts of methylamine in normal urines, a finding which we have been
unable to confirm.
A reaction between methylamine and lactose in alkaline solution is
employed to give a red color (4, 5) for the detection of lactose in urine.
A study of the factors affecting this reaction has shown that, under the
proper conditions, it can be utilized as a simple procedure for the deter-
mination of methylamine.

EXPERIMENTAL

Preliminary experiments indicated that a satisfactory, reproducible color

could be obtained when the reaction mixture contained 0.5 ml. of 3 per cent
lactose and 0.2 ml. of 20 per cent NaOH. The sample and lactose were
mixed, made up to a volume of 7 ml., 0.2 ml. of 20 per cent NaOH was
added, and the mixture incubated at 56” for 30 minutes. After removal
from the bath, the intensity of color slowly increases over a period of several
hours. It was found, however, that readings of standards and unknowns
increased at the same rate and, since the major part of the color developed
during the 1st hour, all subsequent readings were made 1 hour after re-
moval of the tubes from the bath.
A greater concentration of lactose increased the intensity of color, but
the color of the blank became disproportionately large. The blank has a
711
712 DETERMINATION OF METHYLAMINE IN URINE

yellow color with an absorption maximum at 355 rnp; methylamine gives

a red color with maxima at 405 and 545 rnp (Fig. 1). Readings are best
taken at the 545 rnp maximum where absorption by the blank is negligible.
Although this is a rather weak maximum for the color given by methyl-
amine, it will be shown later that the intensity may be greatly increased
by the addition of ammonia. Subsequent readings were made with the
Klett-Summerson photoelectric calorimeter with the No. 54 filter.
E$ect of Ammonia-Although ammonia itself gives no color by this
reaction, it affects very markedly the color given by methylamine. The

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FIG. 1. Absorption spectra of color developed from methylamine (o), and of blank
(X). 0.1 mg. of MeNH2-N, 0.5 ml. of 3 per cent lactose, 0.2 ml. of 20 per cent NaOH,
no ammonia, total volume of 7 ml.; heated at 56” for 30 minutes. Blank prepared in
the same way except for the omission of methylamine. Readings were made on a
Cenco-Sheard spectrophotelometer with a 1 cm. cuvette. The methylamine was
read against the blank as a reference; the blank was read against the distilled water.

effect of ammonia depends upon the alkalinity of the reaction mixture

(Fig. 2). It is evident that on the addition of increasing amounts of am-
monia there is at first an intensification of the color, and then a rapid de-
crease in intensity. Greatest sensitivity is obtained by the addition of
7 mg. of NHB-N in the presence of 0.2 ml. of 20 per cent NaOH. The
disappearance of all color, noted when more than 5 mg. of NH,-N are added
in the presence of 0.1 ml. of 20 per cent NaOH, is coincident with complete
disappearance of the yellow color of the blank.
The shape of the absorption curve also changes in the presence of am-
monia (Fig. 3). As ammonia is added, the position of the 405 rnp maximum
 A. A. ORMSBY AND S. JOHNSON 713
3oc

.~'-*,0.2ml 2O%NaOH

200

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B
too

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I

2 4

FIG. 2. Effect of ammonia on the color developed from methylamine. 0.1 mg. of
nethylamine N, 0.5 ml. of 3 per cent lactose, varying amounts of NHs-N (added as a
reutralieed solution of (NH&SOI), 0.1 or 0.2 ml. of 20 per cent NaOH, final volume
)f 7 ml. Tubes incubated at 56” for 30 minutes, and read 1 hour after removal from
,he water bath. Readings taken on the Klett-Summerson photoelectric calorimeter
vith filter No. 54.

FIG. 3. Effect of ammonia on the absorption spectrum. All the samples contained
1 mg. of MeNHZ-N, 0.5 ml. of 3 per cent lactose, 0.2 ml. of 20 per cent NaOH, and
mmonia added as specified. Each sample was read against a blank containing the
ame amount of NH*.

3shifted to longer wave-lengths and its height decreases;the maximum at

45 rnp becomes more pronounced with a slight shift towards shorter
714 DETERMINATION OF METHYLAMINE IN URINE

wave-lengths. The latter maximum, in the presence of 7 mg. of NHS-N,

is at 540 m/l.
It is apparent that the presence of ammonia in urine must be considered
if methylamine is to be determined. Complete removal of ammonia by
the method of Weber and Wilson (1) was found impractical since the
ammonia-free filtrate gave turbid solutions in the calorimetric determina-
tion of methylamine. A procedure which is useful if the concentration of
methylamine is low is the determination of ammonia by nesslerization,
followed by the addition of enough ammonia to the sample to give a total
of 6 to 8 mg. of NHI-N. This method, suitable for the determination of
methylamine in normal urine, fails if the methylamine concentration is too
high, since the presence of more than 0.5 mg. of methylamine N per 100

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ml. of solution to be nesslerized causes a turbidity on nesslerization. In
feeding experiments with methylamine it was possible to ignore urinary
ammonia. The small samples used contained such small quantities of
ammonia that 7 mg. of NHI-N were added routinely in order to obtain
the optimum ammonia concentration (6 to 8 mg.). In general it was
found that if the ratio of NH,-N to MeNH2-N is less than 20: 1, ammonia
may be neglected; if this ratio is greater than 20: 1, ammonia may be deter-
mined and the amount in the sample adjusted to the optimum ammonia
concentration.
Di- and Trimethylamines-These amines, like ammonia, give no color
themselves but do affect the color given by methylamine. The effects,
of less significance than those of ammonia, consist mainly of an inhibition
of color when relatively large amounts of the amines are present (Table I).
Thus, under the conditions usually employed (about 0.05 mg. of MeNH2-N
and 7 mg. of NH3-N), the determination of methylamine would not be
seriously affected by the presence of 20 times as much dimethylamine or
40 times as much trimethylamine.

Standard Procedure
Reagents-
I. Standard methylamine solution. A stock standard is prepared con-
taining 20 gm. of methylamine hydrochloride (E. K.) per liter. The nitro-
gen content of this solution (approximately 4 mg. of N per ml.) is checked
by a Kjeldahl determination. A working standard, containing 0.1 mg.
of N per ml., is prepared by appropriate dilution of the stock solution-
Both stock and working standards are preserved by the addition of a few
drops of chloroform and appear to be stable indefinitely.
2. 3 per cent lactose, preserved with thymol.
3. Ammonia solution. 6.607 gm. of pure dry (NH&304 are dissolved
in water, the solution neutralized to litmus, and made up to a volume of
 A. A. ORMSBY AND S. JOHNSON 715

100 ml. Chloroform is added as a preservative. This solution contains

14 mg. of NHI-N per ml.
4. 80 per cent NaOH.
Preparation of Sample-In feeding experiments the methylamine con-
centration in urine was such that the amount needed for a determination
was on the order of 0.1 ml. In these instances the urine was used directly
since control experiments indicated that urinary pigments did not interfere
to any appreciable extent. Urine can be used directly if the methylamine
N concentration is 10 mg. per cent or more.
For methylamine N concentration between 1 and 10 mg. per 100 ml., a
distillate is employed. A Kirk micro-Kjeldahl distilling apparatus is con-

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venient for the steam distillation. 10 ml. of urine are used, the amines
being liberated by the addition of 5 ml. of 30 per cent NaOH. A few
drops of mineral oil prevent foaming. The receiving flask contains 10

TABLE I
E$ect of Di- and Trimethylamines on Determination of Monomethylamine

I Calorimeter readings
Addition
0.1 mg. MeNH,-N,
no ammonia added

None....................................... 82
lmg.Me~NHz-N........................... 92
2 ‘I ‘I . . .. .. . . . . ... ... ... ... .. 79
5 “ “ . . . ... ... ... .... .. .... ... .. 45
lmg.MeaN-N.............................. 104
2 I‘ “ . ... . . ... ... ... ... ... .. 91
5 “ “ . . 88

ml. of 0.2 N HCl. The pH of the contents of the receiving flask must be
checked (nitrazine paper) at intervals during the distillation, a few drops
of 2 N HCl being added if necessary to maintain an acid reaction. Dis-
tillation is carried out for 10 minutes after steam enters the condenser, then
for 1 minute with the acid in the receiving flask lowered below the con-
denser tip, and finally for 1 minute with the condenser emptied of water.
Control experiments showed complete recovery of methylamine under these
conditions. The distillate is neutralized (pH 6 to 7), transferred to a 50
ml. volumetric flask, and diluted to volume. 6 ml. samples (representing
1.2 ml. of original urine) are used for analysis.
If the methylamine concentration is less than 1 mg. of N per 100 ml., it
is necessary to concentrate the urine. 50 ml. of urine are acidified with
HCI and concentrated on a water bath, with the aid of an infra-red lamp,
to about 10 ml. The concentrate is transferred to the distilling apparatus
716 DETERMINATION OF METHYLAMINE IN URINE

and treated as described above, except that the receiving flask containc
10 ml. of 2 N HCl. In this manner a sample representing 6 ml. of the
original urine can be analyzed, provided the ammonia concentration i$
not too high.
Method of Procedure-Samples estimated to contain 0.01 to 0.15 mg. of
methylamine N are measured into test-tubes (maximum volume of sample,
6 ml.). Suitable amounts of the dilute standard (e.g., volumes containing
0.01, 0.05, and 0.1 mg.) are measured into similar tubes and an additional
tube, containing 6 ml. of water, is used as a blank. All tubes are.now ad-
justed to a volume of 6 ml. by the addition of water.

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300 -

200 -

.8

1
IO0 -

I I I
0 0.05 0.10 0.15
MiU&%-ams Mothyh&-N
FIG. 4. Standard curve. Readings made with the Klett-Summerson photoelectric
calorimeter, with filter No. 54.

To each tube add 0.5 ml. of ammonia solution (or, when necessary, the
calculated volume to bring the ammonia N content to 6 to 8 mg.), followed
by 0.5 ml. of 3 per cent lactose.
Add 0.2 ml. of 20 per cent NaOH to each tube and mix by inversion.
Violent shaking must be avoided since aeration seems to decrease the
intensity of the final color. Incubate the tubes, covered with glass bulbs
or marbles to prevent evaporation, in a 56” water bath for 30 minutes.
Remove the tubes from the bath and read the intensities of color after 1
hour at room temperature. We have made all readings on the Klett-
Summerson photoelectric calorimeter, using a No. 54 filter.
 A. A. ORMSBY AND S. JOHNSON 717

Results
There is a direct proportion between color and concentration over the
entire range covered (Fig. 4). Recoveries of methylamine added to normal
urine are shown in Table II.
The urine used in the recovery experiments had an original concentration
of 0.34 mg. of methylamine N per 100 ml., and this value was subtracted
in calculating the recoveries shown.
Methylamine of Normal Urine-Analyses of four 24 hour specimens of
dog urine gave values of 0.69 to 1.09 mg. of methylamine N per 24 hours.
Analyses of six 24 hour specimens of human urine gave a range of 1.11 to
4.40 mg. of methylamine N per 24 hours. Expressed as methylamine, the

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average daily excretion of the dogs was 1.84 mg., and of the human sub-

TABLE II
Recovery of Methylamine Added to Urine
I&N’&N added Found

mg. px 100 ml. mg. )I3 loo ml.

1 0.96
5 6.05
10 10.4
20 21.0
40 38.2
60 59.9
80 79.2

jects 5.71 mg. This is to be contrasted with the findings of Kapeller-

Adler and Krael (2), who reported a daily excretion of 30 mg. of methyl-
amine by dogs and 80 mg. by man.
SUMMARY

A method is presented by which methylamine can be determined by a

simple calorimetric procedure. The effect of ammonia on the reaction is
employed to increase the sensitivity of the determination. Di- and tri-
methylamines will interfere only if present in high concentration. Some
values are reported for the normal excretion of methylamine in dogs and
in man.
BIBLIOGRAPHY

1. Weber, F. C., and Wilson, J. B., J. Biol. Chem., 36,385 (1918).

2. Kapeller-Adler, R., and Krael, J., Biochem. Z., 236,394 (1931).
3. Kapeller-Adler, R., and Toda, K., Biochem. Z., 248, 403 (1932).
4. Fearon, W. R., Introduction to biochemistry, 3rd edition, New York, 123 (1947).
5. Ormsby, A. A., and Johnson, S., J. Lab. and Clin. Med., 34, 562 (1949).
A COLORIMETRIC METHOD FOR THE
DETERMINATION OF METHYLAMINE
IN URINE
Andrew A. Ormsby and Shirley Johnson
J. Biol. Chem. 1950, 187:711-717.

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