the filter paper was digested and the nitrogen contain estimated by the micro-Kjeldahl method. More than 100 samples of liver extracts were subjected to this test ...
Jan., 1955]
PROTEIN
ESTIMATION
OF
PROTEOSIB
EXTRACTS
PROTEIN-PROTEOSES
IN CRUDE LIVER EXTRACTS
FOR
PARENTERAL USE By N. K. IYENGAR M. D. CHAKRAVARTI and H. Iv. BISWAS
(Central Drugs Laboratory, 3, Kyd Street, Calcutta-16) The latest British Pharmacopoeia has not included any monograph on Liver Extract
injections, although they In the United States
are very widely used. Pharmacopoeia, there are
monographs on different kinds of liver extract injections. No standards for purity or toxicity have been laid down. In the United States, all liver extract preparations are to be approved by the Anti-anaemic Preparations Advisory Board. The purity, toxicity and potency are thereby safeguard for the public by this Board which carries out all the required tests after obtaining the required data from the manufacturers.
determined
by precipitating 1 cc. of liver sample with 1 cc. of 20% Trichloracetic acid. The precipitate was filtered and washed with 10% of the acid. The residue along with the filter paper was digested and the nitrogen contain estimated by the micro-Kjeldahl method. More than 100 samples of liver extracts were subjected to this test and the protein nitrogen content varied between nil and 0.08%. A limit of not more than 0.08% protein nitrogen content has been suggested as a standard.
was
extract
Many samples of liver extract were found appreciable amounts of proteoses as judged by Picric Acid precipitation of the filtrate from trichloracetic acid precipitation. It was felt that the higher proteose content may be a contributory factor in causing acute toxic to contain
country,
there is
such Board at
no
control the purity, toxicity and potency of liver extract injections put on the market. At present there are no rigid standards on the basis of which samples can be de-
present to
clared
acceptable
or
otherwise.
The
administration of some liver extracts have been received. An investigation of the probable
Method
for
the estimation
sent paper tests.
deals
with
some
of the chemical
The crude liver extracts for injection have been found to contain small amounts of proteins as judged by 10% Trichloracetic acid preIt is essential that this protein cipitation. content should be extremely small if not alabsent. The protein nitrogen content
together
in the
samples. desig-
combined
protein('parenteral).
1 cc. of the liver extract is diluted to 10 cc.
with water.
To 0.5 cc. of this are added 4.5 cc. clear saturated solution of picric acid. The resulting turbidity of the solution is measured of
a
in terms of
light using
content of the
important chemical, pharmacological and microbiological tests that could be applied to all liver extract (injection) samples. The pre-
of
proteoses in Liver extracts
gets safe and potent preparations. therefore been made to work out have Attempts some
protein present
This factor should therefore be strictly nated as combined proteins-proteoses.
Photometer
consumer
con-
This includes the
acid solution.
picric
small amounts of
of such toxic effects has been undertaken It was felt that some in this laboratory. standards for purity, toxicity and potency should be worked out so as to ensure that the causes
was
Before prescribing a limit sidered necessary. for the presence of proteoses, a simple method for its estimation had to be evolved. The test to be described in this paper is based on the turbidity produced on the addition of satu-
Central
Drugs Laboratory is called upon to give a definite opinion on liver extract (injections) samples, as regards their acceptabality. Reports of acute toxic effects on patients after the
A limit test for proteoses
reactions.
rated In this
IYENGAR AND OTHERS
:
5
mm
used
cell.
as
picric
in
5
a
Pulfrich
and a acid solution is
(S 51)
The transmission value is
measure
of
the
a
protein-proteose
sample.
More than 150
(parenteral)
No.
filter
The clear
blank.
comparative
transmission
samples of crude liver extracts foreign as well as Indian
both of
manufacture
were submitted to the above test. The transmission values varied between 14 to 100%. Most of the reputed samples were found
to have transmission values between 35 to 100. A number of well known liver extract samples gave transmission values of 75-100, indicating of insignificant amounts protein-proteose. gives the transmission values obatined for reputed samples and Table II for other samples not so well known. Table I
THE INDIAN MEDICAL GAZETTE Not
Reputed make
Transmission
percentage
(1)
49
(2)
68
(3)
50
make
Reputed
mission
(42)
make
percentage
(43)
75
(6)
100
(7) (8)
100
(9)
100
(10)
ioo
(11)
85
Transmission
so
make
percentage
(41)
97
(5)
Not
reputed
4G
percentage
(46)
15
Trans-
100
(4)
Trans-
mission
so
reputed
Table I
1955
[Jan.,
(14)
84
38
(47)
40
(48)
18.5
14
(49)
19.5
15
(50)
35
,
(44)
(15)
80
(16)
72
(17)
84
(18)
76
(19)
75
highest proteose
(20)
68
22
(21)
37.5
make.
(22)
32.5
(23)
39
corresponding to a transmission 32% determined by the method note,
allowance
.
(12)
100
(24)
04
(13)
100
(25)
33
(45)
From the
14
figures
.
in Table I it is apparent
that the lowest transmission
which
content is
belongs
appears
to
of
content
protein-proteose
of not less than described in this
be reasonable in
for the
amounts
category of repurted
to the
The limit of
corresponding to 32.5% in sample No.
presence
protein-proteose.
giving
an
of
unavoidable
A
transmission
value lower than 32 indicates abnormal amounts Table II Not
so
reputed make
Transmission
percentage
protein-proteose. Many well known makers liver extract contain insignificant amounts of
Not
Transmission
so
reputed make
percentage
43
(21)
24
(2)
19
(22)
53
(3)
15
(23)
42
(4)
45
(24)
28
(5)
44
(25)
42
(6)
42
(26)
17
(7)
42
(27)
19.5
mum.
(8)
42
(28)
22
tracts
of
(9)
18
(29)
32
should
be
17.5
(30)
38
(12)
18
(32)
42
(13)
18
(33)
44
(34)
40
14.5
(15)
14.5
(35)
38
(16)
17
(36)
40
(17)
18
(37)
42
(18)
24
(38)
41
(19)
22
(39)
42 '
(20)
3G.5
(40)
in
42
amounts
sible This
limit a
it may be
patients,
presumed
at least to be
or
of
protein-
a
to be respon-
factor.
contributory
be reduced to
should, therefore,
a
mini-
When this has been done in Liver
reputed made
liver
amounts of
(31)
abnormal
proteose is responsible for unpleasant reactions
produce
15
(14)
of
presence
42
(ID
of
protein-proteose. This should therefore be regarded as an important criterion of purity. Although it cannot be definitely stated that the
(1)
(10)
of
other
extracts
attempts
manufacturers
The test and the
in this paper may be utilized
to eliminate this
as
to
minimal
containing
protein-proteose.
proposed
guide
by
manufacturers,
ex-
far
as
as
possible.
pharmacological tests to control the purity and toxicity and quality are being worked out in the Pharmacology Department of the Central Drugs Laboratory. Further commuThe
