PROCESS FOR THE DETERMINATION OF DIABETES

PROCEDE DE DEPISTAGE DU DIABETE

English Abstract

ABSTRACT
There is described a process for the determination of
diabetes based on the hemoglobin species A1C. The hemo-
globin species A1C is determined in principle according to
the method of Gabbay, K.H. et al. in Diabetes Vol. 28,
1979, but the determination is effected in whole blood
and not in hemolysates of red blood cells. In addition,
the precipitation of the proteins and the color reaction
are performed simultaneously.

Claims

The embodiments of the invention in which an exclusive
property or privilege is claimed are defined as follows:
1. A method for the determination of the haemoglobin
species A1C according to the photometric method, which com-
prises incubating a whole blood sample at an elevated tempera-
ture in an acidic solution, simultaneously precipitating pro-
teins and carrying out a colour reaction and, after incubation
and separation of denatured proteins, carrying out a photo-
metric determination and comparing the value obtained with a
standard in a manner known per se.
2. A method according to claim 1, wherein the whole blood
sample is incubated at 95° - 105°C.
3. A method according to claim 1, wherein the acidic sol-
ution is oxalic acid solution.
4. A method according to claim 2, wherein the acidic sol-
ution is oxalic acid solution.
5. A method according to claim 3, wherein the acidic sol-
ution is 0.4N oxalic acid solution.
6. A method according to any one of claims 1 to 3, wherein
the proteins are precipitated using trichloroacetic acid and
simultaneously the colour reaction is carried out using thiobar-
bituric acid.
7. A method according to any one of claims 4 or 5, wherein
the proteins are precipitated using trichloroacetic acid and
simultaneously the colour reaction is carried out using thiobar-
bituric acid.
8. A method according to any one of claims 1 to 3, wherein

the proteins are precipitated using trichloroacetic acid and
simultaneously the colour reaction is carried out using thiobarbit-
turic acid, and wherein the trichloroacetic acid concentration
of the sample is adjusted to ca 6-10% with aqueous trichloro-
acetic acid which is almost saturated with thiobarbituric acid.
9. A method according to any one of claims 1 to 3, wherein
the proteins are precipitated using trichloroacetic acid and
simultaneously the colour reaction is carried out using thiobarbit-
turic acid, and wherein the trichloroacetic acid concentration
of the sample is adjusted to ca 8% with aqueous trichloroacetic
acid which is almost saturated with thiobarbituric acid.
10. A method according to any one of claims 1 to 3, wherein
the sample is incubated for about 30 minutes at 37°C before the
separation of the denatured proteins.
11. A method according to any one of claims 1 to 3, wherein
the denatured proteins are separated by centrifugation or fil-
tration.

Description

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RAN 4094/4
It is known that the erythrocytes of human beings
contain, besides the most representative haemoglobin
S species A (ca 90~) and A2 (ca 2.5%) as well as small
amounts of species F (ca 0.5%), further haemoglobin species.
These further haemoglobin species make up ca 5-8~ of the
total haemoglobin. Of these further haemoglobin species,
- species AlC is quantitatively the most significant, since
it makes up 4-6~ of the entire haemoglobin. It is known
that diabetics have high values of haemoglobin species
AlC [Rahbars et al. Biochem. Biophys. Res. Commun. 36, 838-
-843 (1969)]. Furthermore, it is known that haemoglobin
species AlC values do not reflect the actual blood sugar
values, but are to be evaluated as the integral of the
preceding four months values. Accordingly, the determination
of haemoglobin species AlC is of particular diagnostic
value.
Haemoglobin species AlC can be determined by cation-
-exchange chromatography of haemolysates of red blood cells
[Trivelli L.A. et al. N.Engl. J. Med. 824, 353-357 (1971)],
:
but this method is too expensive to be able to be used for
carrying out a series of investigations. Furthermore, it
is known that haemoglobin species AlC can be determined by
Klt/4.11.1980
, . ~

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incubating a haemolysate of red blood cells in acidic
solution, precipitating the proteins, carrying out a
colour reaction after separation of the proteins and
subsequently carrying out a photometric determination
after incubation. The value obtained is thereafter
compared with a standard in a manner known per se.
[Gabbay K. H. et al. Diabetes Vol. 28 (1979)].
In accordance with the present invention it has now
been found that the aforementioned photometric determination
method for haemoglobin species AlC can be substantially
simplified by on the one hand carrying out the determination
not with a haemolysate of red blood cells but in whole
blood and by on the other hand simultaneously carrying out
the precipitation of the proteins and the colour reaction.
,
The present invention is accordingly concerned with
a method for the determination of the haemoglobin species AlC according
to the photometric method,~which comprises incubating a
whole blood sample at an elevated temperature in acidic
solution, simultaneously precipitating the proteins and
carrying out the colour reaction and after incubation and
separation of the denatured proteins, carrying out a
photometric determination and comparing the value obtained
with a standard in a manner known per se.
: '

6'~
The first incubation of the whole blood sample is
preferably carried out at a temperature of 95-105C.
This incubation is preferably carried out for 1 to 5 hours,
particularly for 5 hours.
The acidic solution is conveniently a 0.2-lN oxalic
acid, sulphuric acid or hydrochloric acid solution. A 0.4N
oxalic acid solution is preferred.
.
The separation of the proteins is carried out using
a suitable protein-precipitation agent such as, for example,
trichloroacetic acid.
The colour reaction is carried out using an agent
which forms a colour complex. Thiobarbituxic acid is
especially suitable for thls purpose. ~ -
The agent which forms a colour complex should be
:
soluble in sufficient amounts in the protein-precipitation
agent. In accordance with an especlally preferred
embodiment there is used trichloroacetic acid which is
almost saturated with thiobarbituric acid.

'7~
Before the separation of the denatured proteins the
mixture is preferably incubated for about 30 minutes at
abou~ 37C.
The separation of the denatured proteins is carried
out according to customary methods; for example, by
centrifugation or filtration.
The photometric measurement is carried out at 443 nm.
The wavelength can vary when filters are used.

4~7~
~ 5 --
The following Examples illustrate the present
invention:
Example 1
0.1 ml blood samples are placed in test tubes.
1.4 ml of 0.2M (0.4N) oxalic acid are added thereto and
the tubes are closed. The tubes are incubated on a
heating block at 100C for 5 hours. Thereupon, the tubes
are cooled down to room temperature.
` l ml of a 0.025M solution of thiobarbituric acid
in 20~ aqueous trichloroacetic acid is added to each tube.
Since the thiobarbituric acid does not dissolve very well,
the tubes are warmed to 60C. The solution obtained can
; ~ be used immediately or can be stored for 7 days. The tubes
are incubated for 30 minutes at 37C and then left to stand
for 30 minutes at room temperature. The tubes are
centrifuged at 3000 rpm;for 5 minutesl whereupon the
supernatants are measured photometrically at 430 nm and a
layer thickness of l cm. At this wavelength the colour
~ complex formed by the thiobarbituric acid exhibits a
- 20 molar extinction coefficient of 26.
Example 2
Blood samples from diabetics and healthy test persons
~ ' ' , :
.

were investigated according to the method described in
Example 1. In this case the following haemoglobin species
Al~ values were ascertained:
Diabetic Healthy test person
Patient 1 13.5~ Test person 1 4.2
Patient 2 12.9% Test person 2 4.
Patient 3 12.3% Test person 3 4.6~
Patient 4 10.6~ Test person 4 4.9%
Patient 5 10.2~ Test person 5 5.2%
Patient 6 10.0~ Test person 6 5~3
From these values it is clearly evident that the
diabetics have high values of haemoglobin species AlC.
Exam~e 3
For the preparation of the standard, samples which
contain known, increasing amounts of haemoglobin species
; AlC are prepared in a manner known per se. The haemoglobin
species AlC content of these samples is determined
according to the method described in Example 1, whereupon a
standard curve is prepared in the usual manner.