Note : Les descriptions sont présentées dans la langue officielle dans laquelle elles ont été soumises.
5~5~
A Method of Determining Alcohol Consumption
The invention is concerned with the determination of an
individual person's alcohol consumption by means of
quantifying isotransferrin in an isotransferrin-containing
body fluid sample from the individual to be tested.
This method utilizes the finding that isotransferrins
of a certain pI can be correlated with an individual's
alcohol comsumption; the particular names (different
identities) of these isotransferrins are irrelevant and
thus do not impose any restrictions on the practicability
of the invention. On the basis of the correlation
established between isotransferrins and alcohol consump-
tion the invention can be a helpful tool in deciding
about an adequate treatment program in each specific
situation.
pH and pI values will vary with temperature and ion
concentration. The pH and pI values set forth in the
present specification and claims apply to a temperature
of 23 C and are measured in an aqueous solution of
piperazine (20 mM total) buffered with formic acid to
the pH or pH=pI in question. Thus the invention is not
to be construed as being restricted to the particular
values set forth: It comprises also any cases involving
corresponding pH and pI values at other -temperatures
and ion concentrations.
Two major methods ilave been proposed earlier for
determining -the con-tent of the aforesaid isotransferrins
in a serum sample. One method is based on isoelectric
focusing (IEF) (Stibler, H., Borg, S. and Allgulander
C. Acta Med. Scand. 206(1979), p. 275-81); it utilizes
the fact that different isotransferrins migrating in a
pH gradient under the action of an electric field will
cover different distances depending on the pI values of
the individual iso forms, these individually different
AL-HB/pb/sl
1985-02-05 *
pI values in turn being due to dissimilar contents of
ionizable groups. In our particular case, the difference
in pI values is believed to be due inter alia to
different contents of sialic acid groups; it appears
that isotransferrins have been discovered which have
from 0 up to 5 sialic acid groups and have isoelectric
points of 6.1, 5.9, 5.7, 5.5, 5.3 and 5.1, respectively.
These isotransferrins will be referred to below as
being, in that same order, asialotransferrin, monosialo
transferrin, disialotransferrin, trisialotransferrin,
tetrasialotransferrin and pentasialotransferrin,
respectively. It is possible that each of these iso-
transferrins will be found later on to actually consis-t
of a plurality of individual isotransferrins; however,
for the sake of simplicity they are here treated as
just one substance each.
The second one of the aforesaid two methods relies on
utilizing the phenomenon that different isotransferrins
have different types of terminal carbohydrate groups.
Thus for instance, a desialo form characteristically
lacks a sialyl group on at least one carbohydrate
chain. One of -the procedures employed with the framework
of this second method (Cervén, E. et.al., Upsala J.
Med. Sci. 86 xx (1981) p. 39 - 53) relies on the
assumption that galactose is present as a terminal
group of desialotransferrins. Cervén, E. et.al. there-
fore propose a procedure involving the use of a galactose-
binding lectin for obtaining a biospecific affinity
reaction between that lec-tin and the aforesaid assumed
terminal galactose. At first a serum sample is contacted
with an immunoadsorbent whereby -transferrin - irrespective
of its iso form - is caused to bind to said adsorbent.
Then the transferrin-loaded adsorbent is separated, and
the desialotransferrin attached thereto is to be
detected with the aid of labeled galactose-binding
lectin.
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.3
Both of these known methods involve heavy drawbacks.
The IEF method is unpractical and takes too much time
to be suitable for clinical routine work. In the course
of our experiments for further developlng a test
according to Cerven, E. et.al. we have found that there
is no correlation between the results obtained according
to the lectin method and those obtained according to
the IEF method. Obviously, therefore, these two methods
do not measure the same items. It is not possible
either, by means of the Cervén et.al. method, to bring
out a significant difference between addict-type
alcohol consumers and non-consumers of alcohol; the
reason for this is probably that desialotransferrins
lack not only sialic acid but also galactose - so the
very concept of measuring desialotransferrin with the
aid of a galactose-binding lectin is basically wrong.
It is a known fact, moreover, that the affinity between
lectin and sugar is weak. Also, the high background
level encountered in this method may be due to sugar
groups which are present on the transferrin adsorbent
employed and tend to interfere with the lectin.
It will thus be appreciated that there is a great need
for a simple and reliable method for determining
alcohol consumption by means of an isotransferrin assay
test. It is an object of the present invention to
provide such a method which is suitable for clinical
routine work and which measures specifically the
concentration of such isotransferrins as can be corre-
lated to the test alcohol person's consumption during
the last few weeks preceding the test.
According to the invention, this is achieved by quanti-
fyiny in a body fluid containing isotransferrin the
total concentration of certain combinations of isotrans-
ferrins having pI values exceediny 5.6. These combinations
are (A) isotransferrins of pI 5.7, 5.9 and 6.1; or (B)
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1985-02-05 *
isotransferrins of pI 5.9 and 6.1; or (C) isotransferrin
of pI 6.1. The total concentration of isotransferrins
according to combination (A) or (B) or (C) is a measure
of a person's alcohol consumption during the last few
weeks before the sample has been taken.
According to one embodiment of the invention, a sample
of the person's body fluid is contacted with an adsorbent
capable of separating the isotransferrin inter se so as
to give a fraction (I) containing at least one isotrans-
ferrin of pI greater than 5.6 and one or more fractions
containing the bulk (>95 ~) of the remaininy isotrans-
ferrins. After this separation the amount of fraction
(I) transferrin is determined in a known per se manner.
This amount is then a measure of the concentration of
those isotransferrins in the sample which are charac-
terized by a pI exceeding 5.6 and which have been
carried over into fraction (I).
Fraction (I) thus contains isotransferrin according to
any of the combinations (A), (B) or (C) above. Minor
amounts of isotransferrin having a pI lower than 5.6
may be present in this fraction. The other fraction(s)
contain(s) the isotransferrins that have not been
transferred to fraction (I), and actually may contain
some isotransferrins having a pI higher than 5.6. It is
imperative at any rate that the aforesaid separation
yielding the said fraction (I) be carried out in a
reproducible manner for samples which are to be compared
inter se.
Special embodiments are set forth in the appended
claims.
The sample may be a body fluid sample, e.g. a serum
sample, if desired diluted with a suitable buffer.
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5~
As regards the choice of adsorption methods, it is
quite feasible to employ any one of them provided it is
effective to bring about the aforesaid separation.
Among chromatographic methods may be mentioned so-called
batch methods and methods employing columns. For an
operator having an average skill in the art it is an
easy matter to find out, by some simple tests, whether
or not a method is suitable in each individual case. In
the present stage of the art the most advantageous
procedure is ion exchange chromatography but it is also
possible to employ other adsorption methods having a
high selectivity for the isotransferrins contemplated
here.
When proteins are subjected to ion exchange chromatography
their separation is normally achieved with the aid of
gradient or stepwise elution. In clinical routine work
these procedures are thwarted by practical difficulties;
but the separation can be carried out easily, in one
step and in a short time, if an anion exchanger is used
which has a high buffering capacity for a pH in a range
between the isoelectric points of trisialotransferrin
and asialotransferrin and whlch is set to a pH in that
range. Before the chromatographic procedure is performed
the sample should be diluted with a cation buffer
having about the same pH as that to which the ion
exchanger is set. Then the chromatographic procedure
can be carried out in that the diluted sample is added,
allowed to run through the ion exchanger, and then
collected. If required more buffer solution may be
added, but this is not necessary in the practice of the
present invention. This type of chromatography ls
termed "isocratic"; it is performed at constant pH and
constant ionic strength. Depending on the pH employed
one of the desired combinations (A), (B) or (C) will be
separated from the other known isotransferrins. In this
procedure the pH and the high buffering capacity of the
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1985-02-05 *
6 ~ s~
lon exchanger are utilized for obtaining the desired
separation. Isotransferrins having a pI lower than the
pH of the ion exchanger are adsorbed to the exchanger,
whereas the isotransferrins having a higher pI are
eluted.
In case the ion exchanger is a cation exchanger it
should be adjusted to a pH within the same range as
that set forth above for the anion exchanger, this
adjustment being achieved with the aid of an anion
buffer. The sample too is diluted with anion buffer.
The chromatographic procedure will then result in
adsorption of one of the desired combinations (A), (B)
or (C) onto the ion exchanger, depending on the pH. The
cation exchanger should have a high buffering capacity
within the same pH range as the aforesaid anion exchanger.
In its most preferred embodiment (pH = 5.65 + 0~02) the
invention gives a high degree of correlation with the
IEF method. In the 5.65 ~ 0.02 pH range the invention
also provides better discriminative detection of high
alcohol consumption than does the IEF method - probably
because the method of the invention measures not only
disialotransferrin but also monosialo- and asialotrans-
ferrins which too occur at increased levels in cases
where alcohol consumption has been high.
Isocratic chromatography in accordance with the invention
as compared to the IEF method has the great advantage
of requiring much shorter times for its analyses. The
time requirecl to obtain each result in accordance with
the IEF method will amount to about 5 days, as compared
to the about 1.5 hrs required when the method of the
present invention is employed.
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5~
The variables set forth below are important for carrying
out the separation by means of isocratic ion exchange
chromatography with an anion exchanger.
The most suitable pH for the separation is determined
by the isoelectric points of the isotransferrins to be
separated. The isotransferrins of clinical ln-terest are
disialo-, monosialo- and asialotransferrin of pI 5.7,
5.9 and 6.1 respectively. The other isotransferrins,
which have no clinical relevance in the context of
excessive alcohol consumption, are pentasialo-, tetra-
sialo- and trisialotransferrin of pI 5.1, 5.3 and 5.5
respectivel~. To attain a good reproducibility of the
separation procedure it is suitable to choose a pH for
the ion exchanger within any one of the following three
ranges: (A) pH 5.5 to 5.7, preferably 5.65 + 0.02; (B)
pH 5.7 to 5.9, preferably 5.80 + 0.02; (C) pH 5.9 to
6.1, preferably 6.00 + 0.02. In case of alternative (A)
the pH value chosen is intermediate between the pI
values of tri- and disialotransferrins respectively;
consequently the disialo, monosialo and asialo forms
will pass through the column and can be collected. In
case of alternative (B) monosialotransferrin and
asialotransferrin will pass through the ion exchanger,
and in case of alternative (C) asialotransferrin will
pass through. In providing these alternatives (A), (B)
and (C) the inven-tion in its most preferred embodiments
makes it possible to adequately take into account also
the potential presence of monosialo- and asialo-
transferrins.
In order for an anion exchanger to perform a fully
satisfactory separation it should have a very high and
uniform buffering capacity in the separation range
contemplated. This capacity should be higher than 3
milliequivalents per 100 ml of ion exchanger and pH
unit within -the pH range of from 5 to 6.5.
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1985-02-05 *
\
An example of a suitable ion exchanger is the polyion
exchanger PBE 94 (Pharmacia Fine Chemicals AB, Sweden).
This exchanger is based on a crosslinked agarose having
a large number of charged groups attached to its
monosaccharide units via ether bonds, these charyed
groups being selected specifically for giving a high
and uniform buffering capacity over the pH range
contemplated.
Before the sample solution is added the ion exchanger
should be conditioned to a pH in confirmity with one of
the aforesaid pH ranges (A), (B) or (C).
In practlcing the method an adsorbent volume of 50 to
150 /ul is suitable. The volume of the diluted sample
may be as large as 5 to 10 times the adsorbent volume
without affecting the pH of the ion exchanger or the
separation result.
The separation may be performed at temperatures within
a wide range - for example at 15-35 C, especially
21-25 C.
The buffer should be of the cation type in order to
prevent adsorption of buffering substances to the anion
exchanger. The term "cation type buffer" refers to a
buffer whose buffering component is poten-tially cationic
at the pH comtemplated. The pKa value of the buffer
substance should suitably be chosen so as to fall
within the pH range (pH 5 - 6.5) of the separation
procedure, preferably within one of -the aforesaid pH
ranges (A), (B) or (C). The buffering capacity of the
buffer should be high but is not a critical factor in
the method of this invention because the adequate
buffering is provided for by -the ion exchanger. The
temperature dependence of -the buffer should be similar
to that of the pI of the isotransferrins.
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1985-02-05 *
Ionic strength should be low so that it will not affect
the actual course of the separation which, for the
reasons explained above, is pH-dependent. A suitable
concentration of buffer substance is one within the
range of from 20 to 30 mM, the concentration of the
oppsitely charged ion then being 25 to 50 mM.
~mong the buffering systems studied up to now, the best
ones are found to be -the following: 20 mM piperazine
hexahydrate - formic acid, or 30 mM bis-(2-hydroxyethyl)-
piperazine - formic acid, adjusted with the aid of
formic acid to a desired pH the value of which depends
on the separation alternative desired. The piperazinium
ion pKa is 5.56 at 23.5 C (Handbook of Chemistrv and
Physics).
The volumetric amount of the sample is not a critical
factor for the separation procedure. If the amount if
ion exchanger is (say) 100 /ul it will be suitable to
use sample volumes of 10 - 50 /ul serum diluted with
buffer to a final volume of 200 - 1000 /ul which is
poured onto the column.
For 100 /ul of adsorbent, the sample volume currently
believed to be optimum amounts to 20 /ul of serum
diluted to a 500 /ul final volume with a suitable
buffer.
The statement above that the determination procedure
following after the separation is carried out "in a
known per se manner" implies that in this stage recourse
may be had to any method that is sufficiently sensitive
and specific. Many of the current and preferred methods
rely on an antigen-antibody reaction for this determi-
nation - that is, they belong to the large group of
so-called immunological determination methods. Numerous
techniques within this immunological field are known to
AL-HB/pb/sl
1985-02 05 *
the artisan. All of these me-thods are potentially
applicable to the invention even though some of them
are more suitable than others, in view of e.g. such
factors as specificity, sensitivity or selectivity
inherent in a particular technique. As an example may
be mentioned procedures utilizing at least one labeled
reactant. ~etermination methods of this type are
described elaborately in the literature, see for
example the specification and claims of GB-A-1552607
and GB-A-1548741. Alternative determination me-thods are
turbidimetric determination (light scattering immuno-
assays), (Price, C.P. et al. Am. Clin. Biochem. 20
(1983), p. 1-14) and "Sol Particle Immunoassay" - e.g.
agglutination of antibody-coated gold particles by
reaction with immunochemical bivalent or multivalent
antigens (transferrin) (Leuvering, J.H. et al. ~. Imm.
Meth. 62 (1983) p. 163-174).
In certain cases determination methods of generally the
aforesaid type may utilize biospecific affinity relation-
ships other than antigen-antibody affinity. These will
then work in an equivalent manner. Examples of pairs of
biochemical species having such other biospecific
affinities of some sorts are: Protein A - IgG; carbo-
hydrate - lectin; Clq - immunocomplex; RF factor -
immunocomplex; biotin - avidin; etc.
The invention will be further illustrated below by way
of a number of non-limitative working examples one of
which comprises a comparative study in relation to
previously known methods.
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Example 1
Determinatlon of isotransferrin pI
Transferrin was purified from (i) a serum pool from
addicted alcohol consumers and (ii) a serum pool from
blood donors. The transferrin was then separated into
its individual isotransferrins (pentasialo-, tetrasialo-,
trisialo-, disialo-, monosialo- and asialotransferrins)
in a FPLC system (Pharmacia Fine Chemicals AB, Sweden).
The ion exchanger was an anion exchanger column (Mono-Q,
Pharmacia Fine Chemicals AB, Sweden), and the elution
system employed was a linear pH gradient obtained with
the aid of successive mixes of two solutions, viz.,
solution A = 20 mM piperazine hexahydrate adjusted to
pH 6.8 with 5 M formic, and solution B = 20 mM piperazine
hexahydrate adjusted to pH 4.8 with 5 M formic acid.
These two solutions conjointly provide for a linear pH
gradient ranging from pH 6.8 to pH 4.8. The system is
tempered to 23 C.
The transferrin sample is applied to the column having
an initial pH of 6.8. At this pH all the isotransferrins
have a negative charge and therefore attach to the ion
exchanger. When the pH is lowered within the range of
the gradient the charge of each isotransferrin will
progressively change from negative to less negative and
finally to zero. The exact pH conditions under which an
isofransferrin will proceed to zero charge will vary
from isotransferrin to isotransferrin. When its charge
is zero the transferrin will be released from the ion
exchanger; it is then eluted through and from the
column by being entrained in the eluent. The "isoelectric
point" is this very point at which the charge of the
isotransferrin is zero. A spectrophotometer is used to
indicate when the isotransferrin leaves the column, and
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12
in this stage fractions are collected corresponding to
the spectrophotometer recordings for each of the
isotransferrins. In these fractions, which contain the
different zero charge isotransferrins, the pH is
measured; in each case this measured pH corresponds
directly to the isoelectric point (pI) of the iso-
transferrin in that fraction.
It will be seen that these experiments have been
carried out with the same buffer system (20 mM piperazine
hexahydrate ~ formic acid) and at the same temperature
(23 C) as in Example 2. The pI values obtained -therefore
can be used for prescribing the pEI for the separation
of the isotransferrins to be searched for.
The pI values of the individual isotransferrins as thus
determined according to the present Example are the
following:
Addicted
Blood alcohol
donors consumers
pentasialo 5.1 5.1
tetrasialo 5.3 5.3
trisialo 5.5 5.5
disialo 5.7 5.7
monosialo fnd 5.9
asialo fnd 6.1
"fnd" means "fraction not detectible" because its
concentra-tion is too low to be detected.
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13
Example 2
Isocratic chromatography for separation and determination
of the individual isotransferrins
Packing of gel in columns
100 ml of polyion exchanger PBE 94 obtained by allowing
a particle suspension to settle (Pharmacia Fine Chemicals AB,
Sweden) were mixed with 900 ml of 24 ~ ethanol tv/v in
water) to form a slurry. From this slurry 1 ml was
pipetted into a microcolumn. The gel was allowed to
settle in the column whereupon most of the ethanol was
decanted, so that then only the gel sediment was left
in the column.
The gel was then conditioned to the desired pH by means
of 1.5 ml of piperazine hexahydrate - formic acid
buffer, 0.020 M in piperazine adjusted with 5 M formic
acid to pH 5.65 and containing 0.1 ~ Tween~ 20 (poly-
oxyethylene sorbitan laurate, Atlas Chemical Ind. Inc.)
at 23 C. When all of the buffer solution had passed
through the column (about 45 min.) the gel was ready
for use.
Isotransferrin _eparation
20 /ul of a serum sample were diluted with 480 /ul of
the above piperazine buffer and added to the conditioned
microcolumn. The eluate ~500 /ul) was collected in a
test tube. When af-ter about 15 minutes the column had
ceased to drip it was discarded and the eluate was
recovered.
Fixing antibody to solid phase
Agarose (partlcle size 1 - 5 /um) was CNBr activated in
a manner analogous to that described in US-P-3645852.
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~5~
14
To 10 g of activated particles were added 5 ml of
sheep-antirabbi-t antibodies and 45 ml of 0.1 M NaHCO3,
pH 8. This ~ixture was incubated overnight at +4 C.
Thereafter the gel was centrifuged at 2000 g for 10
minutes whereupon it was washed at first with Tris
buffer (0.05 M tris-thydroxymethyl)-aminomethane
adjusted to pH 8.1 with hydrochloric acid) containing
1 M NaC1 and then with acetate buffer (0.1 M, adjusted
to pH 4.0 with hydrochloric acid) containing 1 M NaCl.
Residual active groups on the gel were blocked by means
of ethanolamine (1.0 M, adjusted to pH 9.0 with hydro-
chloric acid) for 60 minutes at room temperature.
Finally the gel was twice subjected to washes with Tris
buffer and acetate buffer in alternating succession,
whereupon it was diluted to a concentration of 10 mg/ml
with phosphate buffer (0.05 M, pH 7.4) containing
0.15 M NaCl, 0.02 ~ NaN3 and 0.1 ~ Tween~ 20.
Isotransferrin determination
To the eluate or to a test tube containing a known
amount of transferrin were pipetted, in the order as
stated,
100 /ul of 125I transferrin (22 ng/ml) (produced by
iodination of transferrin as described by Greenwood,
Hunter in Biochem. J. 89 (1963), p. 114),
100 /ul of rabbit anti-transferrin antibody solution
diluted 1:500 (DAKOpatts, Denmark),
2 ml of antibody (10 mg/ml) fixed to solid phase (as
described above).
Next the tube was incubated for 60 minutes at room
temperature without shaking, and this was followed by
centrifugation at 2000 g for 10 minutes. The supernatant
was then discarded by decanting (composition as set
forth above).
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~2~
Residual activity in the tube was measured bv means of
a gamma counter during 1 minute. This was done on a
number of serum samples obtained from non-consumers of
alcohol and from addicted alcohol consumers. The values
measured are listed in Table 1.
With the aid of the corresponding piperazine ~ formic
acid buffer set to pH 5.80 ~ 0.02 the corresponding
values of total mono- and asialotransferrins were
measured. These values too are listed in Table 1.
Any contents of the above-discussed isotransferrlns in
any unknown samples may be determined in this manner,
in that the measured values are compared with corre-
sponding values obtained from standard samples con-
taining known amounts of transferrins.
Example 3
Comparison of (i) the method of the present invention,
(ii) the IEF method and _(iii) the lectin method
The lectin method (Cervén, E. et al., Upsala J. Med.
Sci. 86 (1981) p. 39.52) and the IEF method (Stibler, H.,
Borg, S. and Allgulander, C., Acta Med. Scand. 206
(1979) p. 275-81) were carried out on serum samples
some of which came from the same persons and were taken
on the same occasion as -those in Example 1. The results
together with those obtained in accordance with Example l
are listed in Table 1.
E'rom this Table it will be seen
(l) that there is an unmistakahly clear correlation
between the IEF method and the present novel
method (correlation coefficient r = 0.7);
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1985-02-05 *
5~
16
(2) that there is no such clear correlation between
the lectin method on one hand and either the
present novel method or the IEF method on the
other hand;
(3) that the present novel method and the IEF method
give a degree of discrimination between non-consumers
of alcohol and addicted consumers which has to be
regarded as significant whereas the lectin method
does not give a significant degree of discrimination;
and
(4)` that the discrimination obtained with the present
novel method is better than that obtained with the
IEF method.
According to results obtained during the priority year
the correlation with alcohol comsumption can be improved
if the transferrin in sample is saturated with ferric
ions. This is accomplished by adding a few drops of
concentrated ferric citrate solution to the serum
sample or diluting a definite volume of serum in less
concentrated ferric citrate solution.
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17
Table 1
Percentage Bound radio- A~ount of di-, mono-,
of disialo- active lectin asialotransferrin
transferrin acc to lectin acc to present method
acc to IEF method (cpm) t= invention)
method (/ug/ml serum)
pH
5.65l) 5.802)
2 ND 47 11
3 ND 73 14
Non-consumers 5 ND 51 17
of alcohol 5 17 018 64 18
7 17 218 67 ND
3 18 739 93 ND
6 ND 134 ND
6 ND 115 ND
7 ND 194 ND
8 ND 129 ND
8 ND 137 ND
Addicted alcohol 9 ND 134 ND
- consumers 9 ND 197 ND
18 267 161 77
17 129 152 65
13 16 222 260 96
13 ND 163 94
16 17 304 ND ND
ND 16 931 162 ND
ND = not done
1) pH 5.65 is used for determining disialo-, monosialo-
and asialotransferrins
2) pH 5.80 is used for determining monosialo- and
asialotransferrins
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