Note: Descriptions are shown in the official language in which they were submitted.
1145737
BACKGROUND OF THE INVENTION:
The present invention relates to a novel filling compo-
sition for use in the column of a liquid chromatograph, and to
the liquid chromatography particularly suitable for clinical
examinations, using the filling composition.
It is extremely important for the diagnosis of
diseases and the establishment of the guiding principle of
treatments of the disease to obtain information on the morbid
state by analysing the properties and the components of the
patient's physiological specimens such as plasma, serum, cerebro-
:-~ spinal fluid, urine, etc.
Hitherto, such information has been obtained by
. various chemical and biochemical analytic means, howeve~r ln the
cases of elucidation of the morbid state of multifarious cases,
a:method of examination for obtaining exact information of the
morbid state is required.
~ . Particularly in cases of the diseases of the kidney
:~ and of the liverj according to the complicatedness of the morbid
statæ, ther.e:has been a keen request for the development of new
-~ 20 indications~ That is, as the indications conventionally utilized
: of the morbid state, various determined values are mentioned, for
: . ~ example in the renal disease, the values representing the renal
functions such as creatinine,uric acid and electrolytes in blood,
protein and sugar in urine and pH of urine, and in the hepatic
: disease, the values representing the hepatic functions such as
~hA . eiviti of bioenz~mes, ~ Instance, ~OT, GPT, LDP anG L~P,
. ,. :
. ~
~ .
: ~
;
11~5737
and`the components of bloods, for instance, protein and bilirubin.
These data of various determined values are playing
important roles and are actually utilized for establishing the dia-
gnosis and the guiding principles for treatment in their own ways.
However, recently it has been begun to point out that
there is an intimate relationship between the uremic toxins and
the morbid state of renal diseases, and the above-mentioned values
determined by the conventional methods are not able to confirm
the presence of the above-mentioned toxins, and moreover, no
simple methods for detecting and confirming the toxins have yet
been developed. Also in the hepatic diseases, in spite of the
suggestion of the presence of various substances by abnormal
metabolism and of the etiopathogenic substances, there are no
suitable methods for detecting and confirming such substances at
present.
In short, the present analytical methods of chemistry
and biochemistry are not sufficient, although they are useful.
And accordingly, the development of a suitable method for detecting
and confirming the above-mentioned substance for supplying newer
indications of the morbid state is keenly expected.
In consideration of the above-mentioned status quo, the
utilization of the liquid chromatography which is one of analyti-
cal means based on the different principles from those of chemical
and biochemical analytical methods has recently attracted the
specialists' attention. Even thermally and chemically unstable
~substances sr possibly determined by the liquid chromatography
I - 2 -
" ll'~i 73~
without being denaturation, and multiple components can be
determined with a relatively small amount of the specimen, and
accordingly, in principle, the applicability of the liquid
chromatography in the fields of medicine and clinic is large
enough. However, in order to put the liquid chromatography into
practical use in the above-mentioned fields, there have been
problems to be solved concerning the selection of the filling
material, the establishment of conditions for separation in the
chromatography and the pre-treatments adopted in accordance with
the necessity.
For instance, Chang et al.l) tried to detect the above-
mentioned toxins appearing in the blood of the patient suffering
from the renal disease by using the liquid chromatography with
columns filled with porous beads of cross-linked dextran.
As a result, Chang et al. found that in the-serum of
the patient suffering from the-renal disease different from the
normal serum there aré some substances which give specific peaks
in the chromatogram obtained by their liquid chromatography, and
suggested that the peaks include some harmful substances.
However, there are defects in the method of Chang et al.,
that is, the specific peaks obtained by the method are extremely
broad and the method necessitates a relatively large amount of
the serum specimen of 2 to 3 ml and moreover, it takes 4 to 7
hours in treating one specimen.
)T.M.S. Chang et al. "Trans. Amer. Artif. Int. Organs", Vol. XX,
page 364 (1974).
1145737
Accordingly, the method is not satisfactory as the
clinical method of examination.
On the other hand, Furst 2)proposes a method of analysis
of serum specimens by the high-speed liquid chromatography, how-
ever, although Furst's method was successful in shortening the
time period for analysis, there is a defect of appearing many
peaks over-lapping each other in the obtained chart of the
chromatography to make the separation and identification of each
component difficult. In addition, either of these methods has
not been tried with an intention of applying to clinical examina-
tion. That is, although the analysis of bio-specimens by liquid
chromatography has been tried, even if it is possible to detect
some specific peaks in the chromatogram of a specimen from the
patient suffering from a disease of the liver or the kidney, the
result does not provide information on the relationship between
the progress of the morbid state and the appearance of the peaks
in the chromatogram. Accordingly, both methods of Chang and
Furst have not been put into practical use.
We, in consideration of the above-mentioned status quo,
have studied the methods for effectively examining clincially the
physiological specimens from patients of, particularly, renal
diseases, etc., and as a result, we have found that on the appli-
cation of the liquid chromatography using a specified filling
)P. Furst, "Clinical Nephrology", Vol 5 (4), page 198 (1976)
~:14573;''
composition in its columns to such physiological specimen, it
is possible to separate and detect the peaks in the chromatogram,
which have relationship to the progress of the morbid state of
the above-mentioned diseases within a short time period with only
a small amount of the specimen.
Accordingly, the object of the present invention is
to offer a novel filling composition for use in the column used
for the liquld chromatography, particularly suitable for clinical
examinations.
;10 Another object of the present invention is to offer the
chromatographical technique using the column utilizing the above-
mentioned filling composition, particularly the technique of high-
speed li~uid chromatography. The other objects of the present
invention will be made clear from the following description.
¦ Thus, the present invention provides a filler composition
for use in a column for liquid chromatography used for clinical
examination, comprising a bead-like hydroxymethylated copolymer
of styrene and divinylbenzene having a serum protein absorbed or
adhered thereonto in an amount of 0.1 to 1% by weight of the bead-
like hydroxymethylated copolymer on the dry basis.
In another aspect, the invention includes a method of
~liquid ohromatography comprising using a column filled up with a
substance formed by adsorping or adhering of a serum protein on-
"''~! ~ to a bead-like hydroxymethylated copolymer of styrene and di-
~J vinylbenzene.
BRIEF EXPLANATION OF DRAWINGS:
l .
~ Figure 1 of the drawings shows chromatograms of serum".~
specimens obtained by the high-speed liquid chromatography in
~ Example l, and (a) is the chromatogram of normal serum, and
-~30~ ~ tb)~is the chromatogram of the serum of a patient suffering from
~ it~ ~
;f ~ renal failure.
~ ~ - 5 -
-- 1145737
Figure 2 of the drawings also shows chromatograms of
serum specimens obtained by the high-s?eed liquid chromatography
in Example 3, and (a) is the chromatogram of normal serum, and
(b) is the chromatogram of the serum of a patient suffering from
liver disorder, and (c) is the chromatogram of the same patient
', ~
-
- 5a -
;~ ,
~ 1145~73~
; after recovering from the liver disorder.
DETAILED DESCRIPTION OF THE INVENTION:
The filling composition for use in the column of the
liquid chromatography according to the present invention comprises
the bead-like substance consisting of a hydroxymethylated
copolymer of styrene and divinylbenzene, to which serum protein
has been adsorbed or is adhering.
The filling composition of tke present invention is
prepared as follows:
(a) The preparation of the bead-like substance comprising a
hydroxymethylated copolymer of styrene and divinylbenzene:
For instance, a monomeric mix~ure of styrene and
divinylbenzene is polymerized in suspension in a non-solvent, for
instance, in water in the presence of a polymerization initiator
to obtain a bead-like copolymer of styrene and divinylbenzene.
By bringing the bead-like copolymer into reaction with formal-
dehyde, a hydroxymethylated copolymer of styrene and divinyl-
benzene is obtained. The commercialized bead-like copolymer of
styrene and divinylbenzene hitherto available for use in the
column of the high-speed chromatography as the filler may be
applied as well as in the following step.
The particle size of the bead-like copolymer and its
degree of hydroxymethylation are possibly selected respectively in
accordance with the purpose of the liquid chromatography, however,
the former i acually S ~o 50 mioron in diameter and the latter
~ ~ : : ,
~ ~ - 6 -
,.'~ ,,
.
.
114S737
is usually 0.05 to O.S.
A (b) Adsorption ~ adherence of serum protein onto the bead-like
oxymethylated copolymer of styrene and divinylbenzene:
~7J~bG/~f~
A serum protein, for instance, albumin and- ~ lin,
obtained from a broad range of mammals such as mankind, cattle,
horse, dog or sheep is dissolved in a solvent mentioned as follows,
and the solution is brought into contact with the above-mentioned
bead-like hydroxymethylated copolymer under agitation and then the
system is separated into the liquid and the solid to obtain a
hydroxymethylated copolymer of styrene and benzene having a serum
protein adsorbed or adhering thereonto. The above-mentioned
contact process is èxecuted by immersing the above-mentioned hydr-
oxyme~hylated bead-like copolymer into the solution of serum
protein or by passing the solution of serum protein through a
column filled with the bead-like hydroxymethylated copolymer. In
addition, adsorption or adhesion is automatically completed only
by bringing the above-mentioned two substances into contact with
each other because of the adsorbing facility of the hydroxy-
methylated copolymer for the serum protein.
The above-mentioned solution of serum protein used for
adsorption or a &esion is obtained by dissolving the protein into
water, a buffer solution or a buffer solution containing a salt
such as sodium perchlorate or an organic solvent such as
methanol, ethanol, propanol and dioxan. In cases where the
concentration of the serum protein in the above~mentioned solution
is high, the adsorption or adhesion of the serum protein to the
1:~573~7
hydroxymethylated copolymer is completed within a short time
period, however, the state of the adsorption or adhesion tendsto be
non-uniform, while in cases where the above-mentioned concentra-
tion is low, it takes a long period of time for the completion of
adsorption or adhesion. In such circumstances, the concentration
of the serum protein in the above-~entioned solution is preferably
0.2 to 5% by weight.
As is mentioned above, after having the serum protein
adsorbed or adhering onto the bead-like hydroxymethylated copolymer
of styrene and divinylbenzene, the filllng composition of the
present invention is obtained by washing well the bead-like
copolymer thus treated. In order to apply the filling composition
into the liquid chromatography, the filling composition is filled
into the column for use in the chromatographical analysis.
However, since the interparticle coagulative tendency has been
raised by the adsorption of the serum protein, the uniform fill-
ing of the filling composition into the column has come to be
difficult. Care should be paid on this fact. In order to avoid
the troubles on the filling of the column with the filling
composition of the present invention, it is recommended that the
hydroxymethylated copolymer not yet having adsorbed the serum pro
tein is in advance filled into the column and after equipping the
filled column onto the conventional appz~atus of the liquid
chromatography, the solution of the serum protein is passed
through the column. According to the above-mentioned technique,
the time required for the serum protein to be adsorbed or to
114573~ 1
adhere onto the bead-like hydroxymethylated copolymer becomes
longer, while on the other hand there is a merit of simplification
of the preparatory operation for the analysis.
On the treatment for adsorption or adhesion of the serum
protein onto the bead-like hydroxymethylated copolymer, the tempera-
ture is kept at a degree at which denaturation of the serum
protein does not occur, that is, 5 to 70C, preferably at 20 to
40C.
The pH of the solution of the serum protein may be in
the range in which the denaturation ana coagulation of the serum
protein do not occur, and is usually selected suitably from the
range of 5 to 9, however, it is natural to avoid the isoelectric
point of the serum protein. In addition, on the preparation of
the solution of the serum protein, the use of a buffer solution
having the same pH value as the pH value of the moving phase used
for the liquid chromatography carried out by using the column
filled with the filling composition of the present invention
facilitates the maintenance of the steady state of the solution
of the serum protein during the operation of the examination and
is suitable for the purpose.
Accordingly, the adsorption or adhering of the serum
protein onto the bead-like hydroxymethyl2ted copolymeris preferably
carried out under the same conditions as far as possible to those
at the time of examination, in considering the stability of the
filling composition of the present invention.
The amount of the serum protein adsorbed or adhering
1145737
onto the bead-like hydroxymethylated copolymer of thepresent inven-
tion varies slightly corresponding to the kinds of the bead-like
hydroxymethylated copolymer and those of the serum protein for use
in the liquid chromatography, however, usually in the range of 0.1
to 1% by weight of dry matter, preferably 0.2 to 0.5~ by weight
of dry matter.
The column filled with the filling composition of the
present invention is possibly used for examination of various
physiological specimens after installing the column onto any
commercial apparatus for liquid chroma_ography or onta any other
apparatus having the same function as ~ove, and the thus instal-
led apparatus is able to separate and detect the components of the
specimen as the peaks which have relationships to the progress of
the morbid state. In addition, the physiological specimens
mentioned in the present invention include the-blood compone~ts
A - Iyp~Ph~ c~5e~fes~
such as serum and plasma, cérebrospinal fluid, lymph~; asai~
urine, etc.
In the next place, the method of examination utilizing
the filling composition of the present invention, particularly
the method of examination of physiological specimens by the high-
speed liquid chromatography can be executed under the following
conditions, however, the followings only illustrate the mode of
its execution referring to examples no, being limited in them-
selves.
As the moving phase for use in the column filled with
the filling composition of the present invention, water, a buffer
- 1~573~
solution or the buffer solution containing some salt component
such as sodium perchlorate or an organic solvent such as methanol,
ethanol, isopropyl alcohol, dioxan, etc. is preferable. Parti-
cularly preferable material as the moving phase is a phosphate
buffer, and by using the phosphate buffer as the moving phase,
extremely finely separated peaks are available in the chromato-
gram.
In addition, since the filling composition of the pre-
sent invention contains the protein and accordingly there is a
probability of suffering from undesira~ie transformations such
as the degradation by microorganisms d17~ing the long time period
of using the column filled with the filling composition of the
present invention, it is preferable in those cases to add a
minute amount of an anti-microbial agent such as sodium azide,
etc. to the moving phase.
The temperature at which the examination is carried out
by using the liquid chromatography utilizing the filling composi-
tion of the present invention is 20 to 40C. The amount of the
specimen required for carrying out the analysis by the above-
mentioned apparatus may be 1 to 15 microliter.
The liquid chromatography according to the present
invention is provided with a detector comprising an ultra-violet
spectrophotometer and an ordinary spect-ophotometer, however,
other than t'nose mentioned, a differential refractometer, a
fluorescence spectrophotometer, an infrared spectrophotometer,
a radiation detector, a polarograph or a conductometer may be
~ 1i~3~
optionally utilized after careful selection.
In addition, in order to quantify the obtained data,
the peak area on the chromatogram is possibly quantified by
connecting a data-treating machine to the above-mentioned detector.
The liquid chromatography utilizing the filling composi-
tion of the present invention is not only applicable to the
clinic examination but also, for instance, to separately collect-
ing fractions after filling into a larger column.
The present invention is explained as follows by refer-
ring to the Examples, however, the sco?e of the present invention
is not to be limited within Examples.
Example 1:
- After filling a hydroxymethylated bead-like (10 to 15
microns in diameter) copolymer of styrene and divinylbenzene
prepared by a well known process into a stainless-steel column of
4 mm in-diameter and 50 cm in length by an ordinary technique,
the column was installed onto a high-speed liquid chromatograph
provided with an ultraviolet detector.
Into the thus installed column, a phosphate buffer
of pH of 7.4 was introduced at a rate of 1.2 ml/min and after
confirming that the indications of the recorder and of the
integrating planimeter became stabilize~, an aqueous 10% solution
of human serum albumin was introduced l-=o the column from the
specimen-inlet. After repeating the introduction of the solution
of albumin to obtain the fixed height of the indication on the
recorder or the fixed value of the indication of area on the
11~S737
integrating planimeter, the treatment of the bead-like filler with
the above-mentioned solution of albumin was completed.
Then, using the above-mentioned high-speed liquid
chromatograph provided with the thus prepared column in which
the bead-like filling had ad'sorbed or a &ere~'to the human serum
albumin completely, the examination of the serum of a normal
person and the serum of a patient of chronic renal failure under
dialytic treatment was carried out.
As the results of the examina~ion which took 30 minutes,
lo the charts shown in Fig. 1 illustratins the peaks a, (b), (c), d,
e, (f) and (g) were obtained, the paren~hesized peaks appearing
only on the chart of the serum of the pztient of chronic renal
failure.
The mutualseparation of the peaks appearing only on the
chart taken on the serum of t~e patient of chronic renal failure
''and their ~uantificàtion suggest the application of the quantified
values of these peaks as the other values of clinical examination
than the conventional values of conventional clinical examination.
For comparison, the same procedure of the high-speed
liquid chromatography was carried out except that the filling of
the column was carried out using the same bead-like hydroxymethy-
lated copolymer of styrPne and divinylbenzene, however, without
having adsorbed or adhering the serum zl_umin, on the serum of the
patient of chronic renal failure under dialytic treatment.
The result showed that (1) the peak a was lower than
that o~taincd by using the filling composition of the present
- 13 -
'' "
,
:114573'7
invention and (2) the separation of the peaks b, _, d, etc. was
insufficient.
Table 1: Effluent Time and Relative Area of Peaks of
Normal Serum and Serum of Renal Failure in
Liquid Chromatography
Relative Area of Peaks of
Specimen ~ I l
a b c d e f g
.
Normal Serum 9895 _ _ 323 52 _
Serum of 8046 1301293 ~S-3 1001505 38
Effluent time 4.7 6.9 7.8 8.9 10.6 11.8 25.4
of each peak to
tmin~ _ 16.5
Example 2:
Using the same high-speed liquid chromatograph equipped
with the column filled with the filling composition obtained by
the procedures shown in Example 1, the transition of the morbid
state of a patient with the patient's history of from slight renal
failure through hospitalization to the treatment with dialysis was
: followed by the chromatographic examination of the patient's serum
as well as by the determination of BUN --d creatinine. The results
are shown Ln Table 2.
As is seen in Table 2, corresponding to the aggravation
~ 114~737
of the values of conventional clinical examination such as BUN
and creatinine in the serum, the number of the abnormal peaks
appearing in the chromatogram increased indicating the usefulness
of the method according to the present invention.
Table 2: Progress of Morbid State of a Patient of
Renal Failure and Results of Chromatography
of the Patient's Serum
Relative Area of Each Peak Values of
Days of clin cal examn. Clinical
.~ observation b c d e f g ~ (mg/dl) findings
0 72 _ a3a 47l _ _10l ~ ~
7 57 _ 736 446 _ _101 8.3
48 78 116 434 514 21 116 105 9.2 aggravation
56 95 214 534 618 70 191 104 9.9 hospitalization
63 104 234 770 626 170 168 102 10.8 preparation for
dialysis
144 243 730 61Q 191 388 102 10.8
77 151 328 588 589 222 545 103 10.5 _
" ____
: Example 3:
A female JCL-SD rat was made ,~ be a model of the liver
disorder by administratlon of 1,000 mg/kg of D-galactosamine and
: its serum was sampled before the administration tnormal stage),
during the morbid state and during its recovering period, and the
,~ ~ .
~ , '
~ - 15 -
-:
" ' ' ~
- .
. . . . .
1~73~
specimens of the serum were examined by the high-speed liquid
chromatograph shown in Example 1 provided with the column filled
with the filling composition of the present invention. As the
results, a chart shown in Fig. 2 and the relative values of area
of the peaks on the chromatogram shown in Table 3 were obtained.
Separately, biochemical analyses were carried out on GOT, GPT and
bilirubin, the results being shown also in Table 3.
As is shown in Fig. 2 and Table 3, in the serum of the
rat administered with D-galactosamine to be a model of the liver
disorder, the values of GOT, GPT and b:l;rubin were clearly
abnormal after administration as compared to the values before
the administration and after the recovery has begun. Just corre-
sponding to the transition of the above-mentioned values, each of
the peaks of more than 5 minutes of the effluent time showed a
conspicuous change.
" ..
Table 3: Result of Chromatography of Rat in Model Liver
Disorder with Results of Clinical Examination
SpecimenRelative Area of each Peak of Nos. Data of Clinical .
of serum Examirlation (m~/dl) .
1 2 3 4 5 6 7 8 9GOT GPT Bilirubin .
state34156 :37.37 132211 7~3 - 246128100 70 less than
.
At liver 35292 41155 30 202 38 1075 512 795 4560 2260 3.9
disorder
. .
At reco- l
vering379113i 122 651808 78 _ 56 113 540 520 0.5 .
.
Effluent
: time of 4.56.3 6.87.4 8.8 10.6 13.8 15.4 19.1
- ~14573~
As is possibly understood from the results of the above-
mentioned examination, by the application of the high-speed liquid
chromatography utilizing the filler composition of the present
invention, a chromatographic pattern corresponding to the transi-
tion of the morbid state is available also in the case of the
liver disorder. On the other hand, the examination of the above-
mentioned specimens of the serum by the same chromatography,
however, with the column filled with the bead-like oxymethylated
copolymer of styrene and divinylbenzene without adsorbed or adher-
ing the human serum albumin gave a char_ in which, as is shown in
the later part of Example 1, the peak 1 was lower in height and
the separation of the peaks 2, 3, 4.,7 a-nd 8 was insufficient.
,L
