Aqueous solution as an eluent used in liquid chromatography

Disclosed is an aqueous solution as an eluent used in liquid chromatography for analysis of a specimen obtained from a human living body, comprising a mixture of acetonitrile and an aqueous solution of a member selected from the group consisting of acetic acid, a polybasic organic acid, a salt of organic acid and ammonium carbonate.

BACKGROUND OF THE INVENTION 
The present invention relates to an aqueous solution as an eluent used in 
liquid chromatography for analysis of a specimen obtained from a human 
living body, comprising a mixture of acetonitrile and an aqueous solution 
of a member selected from the group consisting of acetic acid, a polybasic 
organic acid, a salt of organic acid and ammonium carbonate, and to a 
method for analyzing a specimen obtained from a human living body, 
comprising subjecting the specimen obtained therefrom to liquid 
chromatography while using, as an eluent, a mixture of acetonitrile and an 
aqueous solution of a member selected from acetic acid, a polybasic 
organic acid, a salt of organic acid and ammonium carbonate. More 
particularly, the present invention relates to an aqueous solution as 
eluent used in liquid chromatography for analysis of a specimen obtained 
from a human living body in order to detect a specific peak or a specific 
peak pattern intercorrelating to the hepatic morbid state, comprising a 
mixture of acetonitrile and an aqueous solution of a member selected from 
the group consisting of acetic acid, a polybasic organic acid, a salt of 
organic acid and ammonium carbonate, the volume ratio of acetonitrile to 
said aqueous solution of acetic acid or ammonium carbonate being 5:95 to 
25:75, preferably 5:95 to 15:85, and the volume ratio of acetonitrile to 
said aqueous solution of the polybasic organic acid or the salt of organic 
acid being 1:99 to 25:75, preferably 5:95 to 15:85, and to a method for 
analysing a specimen obtained from a human living body in order to 
detecting a specific peak or specific peak pattern intercorrelating to the 
hepatic morbid state, comprising subjecting the specimen obtained 
therefrom to liquid chromatography while using, as an eluent, a mixture of 
acetonitrile and an aqueous solution of a member selected from acetic 
acid, a polybasic organic acid, a salt of organic acid and ammonium 
carbonate, the volume ratio of acetonitrile to said aqueous solution of 
acetic acid or ammonium carbonate is 5:95 to 25:75, preferably 5:95 to 
15:85, and the volume ratio of acetonitrile to said aqueous solution of 
the polybasic organic acid or the salt of organic acid is 1:99 to 25:75, 
preferably 5:95 to 15:85. 
It is extremely important for the diagnosis and treatment of a disease of a 
patient to analyze the components and the properties of a specimen such as 
blood, plasma, serum, cerebrospinal fluid, urine etc. taken from the 
patient and to obtain the information(s) concerning the morbid state of 
the patient based on the thus obtained analytical data. Such informations 
have been hitherto obtained by subjecting such a specimen to various 
chemical or biochemical analytical means, however, for the purpose of 
elucidating the various morbid states in various cases or seizing the more 
exact morbid state, the development of analytical methods by which more 
accurate informations are available has been demanded. 
Particularly, in the cases of hepatic diseases and nephrotic diseases, 
because of the complicated morbid states thereof, the analytical methods 
which can provide new indices closely related to the morbid states of 
hepatic diseases and nephrotic diseases have been demanded. 
As the conventional index of the morbid states of the hepatic diseases, the 
activity values of enzymes in the living body, for instance, 
GOT(glutamic-oxaloacetic transaminase), GPT(glutamic-pyruvic 
transaminase), LDH (lactic acid dehydrogenase), LAP(leucine 
aminopeptidase), etc., the biochemical analytical values of the blood 
components such as protein, lipoprotein, neutral fat, bilirubin, 
cholesterol etc. may be used. 
However, it is difficult to trace precisely the change of the morbid state 
of a hepatic disease which causes the generation of very complicated 
factors in the patient suffering from the hepatic disease only by the 
utilization of the values obtained by the chemical analyses and 
biochemical analyses. 
Accordingly, a new trial for diagnosing the morbid state of hepatic 
diseases while utilizing antigen-antibody reaction has been carried out 
(refer to Gastr., 76, 665(1979)), and it was reported that chronic active 
hepatitis and chronic inactive hepatitis or liver cirrhosis can be 
diagnosed at the respective probabilities of 50 to 60% and 20 to 40%, 
however, the method is complicated in its operations and is not 
satisfactory from the viewpoint of the accuracy in judging. 
On the other hand, utilization of liquid chromatography is now attracting 
attention. Liquid chromatography is one of the analytical techniques based 
on the principles different from those on which the chemical or 
biochemical analytical method stands. 
Utilization of liquid chromatography in the medical and clinical fields has 
been greatly expected in principle from the viewpoints that even the 
thermally and/or chemically unstable substances can be isolated and 
detected by liquid chromatography without being denaturated and that 
liquid chromatography is able to analyse many components in one operation 
while using a relatively minute amount of specimen, and a number of trials 
have been carried out in utilizing liquid chromatography. 
However, in general, the peaks or the peak pattern in the chromatogram 
obtained by liquid chromatography are different to each other case by case 
where the eluents are different, and it changes according to the mode of 
combination of the eluent and the specimen, and accordingly, it is 
difficult to seize the peak, which directly correlates to the morbid state 
in a chromatogram. Particularly, in hepatic diseases, a method by which 
the morbid state of each of the various hepatic diseases including chronic 
hepatitis, liver cirrhosis etc. can be traced has not been established. 
As a result of the present inventor's studies for the object of finding a 
method of clinical analysis of the specimen obtained from the patient 
suffering from a hepatic disease, in which an extremely small amount of 
the specimen is subjected to liquid chromatography, thereby obtaining a 
fraction exhibiting a peak which correlates to the progress of morbid 
state or the degree of seriousness of the disease within a short time 
period in a simple manner and quantifying the peak to examine the change 
of the morbid state from the change of the thus quantified peak appearing 
in the fractions, the present inventor has found a method fulfilling the 
object, and has attained the present invention. 
SUMMARY OF THE INVENTION 
In a first aspect of the present invention, there is provided an aqueous 
solution as an eluent used in liquid chromatography for analysis of a 
specimen obtained from a human living body, comprising a mixture of 
acetonitrile and an aqueous solution of a member selected from the group 
consisting of acetic acid, a polybasic organic acid, a salt of organic 
acid and ammonium carbonate. 
In a second aspect of the present invention, there is provided a method for 
analyzing a specimen obtained from a human living body, comprising 
subjecting the specimen obtained therefrom to liquid chromatography while 
using, as an eluent, a mixture of acetonitrile and an aqueous solution of 
a member selected from acetic acid, a polybasic organic acid, a salt of 
organic acid and ammonium carbonate.

In FIG. 2, K-1 denotes healthy person, K-2 denotes patient suffering from 
acute hepatitis, K-3 denotes patient suffering from chronic hepatitis, K-4 
denotes patient suffering from liver cirrhosis, K-5 denotes patient 
suffering from a complication of liver cirrhosis and encephalosis K-6 
denotes patient suffering from a complication of liver cirrhosis and 
hepatoma, K-7 denotes patient suffering from a complication of liver 
cirrhosis, hepatoma and encephalosis, K-8 denotes patient suffering from 
fatty liver, K-9 denotes patient suffering from lupoid hepatitis, K-10 
denotes patient suffering from cholestasis, and K-11 denotes patient 
suffering from primary biliary cirrhosis. In each of FIGS. 8-1 to 8-10, 
respective abscissas show elution time (min.). 
DETAILED DESCRIPTION OF THE INVENTION 
The method for analyzing of the present invention comprises the steps of 
subjecting a specimen obtained from a human living body to liquid 
chromatography to analyze the specimen, to detect a specific peak or a 
specific peak pattern in a fractions obtained from the specimen by the use 
of a mixture, as an eluent, of acetonitrile and an aqueous solution of a 
member selected from the group consisting of acetic acid, a polybasic 
organic acid, a salt of organic acid and ammonium carbonate, and to use 
the thus detected peak or peak pattern as an index of the morbid state of 
a hepatic disease such as acute hepatitis, chronic hepatitis, liver 
cirrhosis, hepatoma, fulminant hepatitis, lupoid hepatitis, cholestasis, 
primary biliary cirrhosis, liver fibrosis, etc. 
According to the present invention, the peak intercorrelating to the 
hepatic morbid state is sharply formed and detected within 30 min of 
subjecting the specimen to liquid chromatography about 10 microliters of a 
specimen such as plasma, serum, cerebrospinal fluid, lymph, ascitic fluid, 
bile or urine as it is or the deproteinized specimen, and a hepatic 
disease can be diagnosed at a probability of almost 90 to 100% by the 
method according to the present invention. 
According to the present invention, chronic hepatitis, liver cirrhosis of 
which no abnormality could have been detected from the test value on 
hepatic function such as GOT, GPT etc. can be detected as the respective 
peaks. For instance, while the conventional test value shows a reduction 
with the aggravation of the morbid state from acute hepatitis to liver 
cirrhosis, the peak according to the present invention shows an increase 
in its intensity (as the area of the peak or the height of the peak). 
In addition, it has been recognized that the peak according to the present 
invention shows the increase of its intensity with the appearance of the 
sign of aggravation of liver cirrhosis, for instance, disturbance of 
consciousness, appearance of ascitic fluid, retention of ascitic fluid, 
etc., the aggravation of liver cirrhosis hitherto having been difficult to 
quantify From these facts, the present invention is an extremely useful 
means and provides new informations concerning the morbid state of hepatic 
diseases. 
The packing material used in the present invention may be those 
commercialized as the packing material for high-speed liquid 
chromatography, and among them, the silane-treated packing material 
derived from silica is preferable. Such a packing material can be obtained 
by a known method, for instance, by treating pulverized silica with a 
silane compound. The column prepared by filling the packing material is 
set to a commercialized liquid chromatographic apparatus or an optional 
apparatus provided with the same function as above and is placed at the 
service of analyzing the specimen taken from a human living body. 
The specimen obtained from a human living body is used as it is, is used 
after pre-treating with a known de-proteining reagent such as methanol, 
trichloroacetic acid, perchloric acid, etc. or is used after removal of 
high-molecular weight protein by a membrane for ultrafiltration (it is 
possible to operate the chromatographic apparatus by providing an 
apparatus for removing the high-molecular substances in front of the 
column). However, in order to improve the life of the packing material, 
the stability of the column (frequently hindered by clogging) and the 
analytical accuracy, the specimen is preferably pretreated for removal of 
proteins. 
The eluent used in the present invention is a mixture of acetonitrile and 
an aqueous solution of a member selected from the group consisting of 
acetic acid, a polybasic organic acid, a salt of organic acid and ammonium 
carbonate. The volume ratio of acetonitrile to the aqueous solution of 
acetic acid or ammonium carbonate in the mixture as the eluent is 5:95 to 
25:75, preferably 5:95 to 15:85, and the volume ratio of acetonitrile to 
the aqueous solution of the polybasic organic acid or the salt of organic 
acid in the mixture as the eluent is 1:99 to 25:75, preferably 5:95 to 
15:85. The concentration of acetic acid is 0.01 to 1.0% by weight, 
preferably 0.1 to 0.5% by weight, and the concentration of ammonium 
carbonate in the aqueous solution of ammonium carbonate is 0.1 to 10% by 
weight, preferably 0.3 to 3.0% by weight. 
Further, the concentration of the polybasic organic acid is 0.01 to 1.0% by 
weight, preferably 0.1 to 0.5% by weight, and the concentration of the 
salt of organic acid in the aqueous solution of the salt of organic acid 
is 0.01 to 10% by weight preferably 0.1 to 5.0% by weight. The salt of an 
organic acid is used after dissolving in distilled water or water treated 
by an ion-exchanging resin. 
As the salt of an organic acid, sodium salt, potassium salt, magnesium salt 
and ammonium salt of monobasic acid such as formic acid, acetic acid, 
propionic acid etc., those salts of dibasic acid such as oxalic acid, 
succinic acid etc. and those salts of tribasic acid such as citric acid, 
etc. may be used, and as polybasic organic acid, dibasic acid such as 
oxalic acid, succinic acid etc. and tribasic acid such as citric acid etc. 
may be used. Of course, an aqueous solution of the acid may be used after 
neutralizing with a basic substance such as sodium hydroxide, potassium 
hydroxide etc., and the salt may be a mixture of more than two salts. 
In addition, in the elution, the amount of acetonitrile relates to both the 
eluting speed of the components of the specimen for analysis and the 
separating efficiency of the peaks in chromatogram, and the concentration 
of the salt of an organic acid therein relates to the separation of the 
peaks. 
Furthermore, the eluent may be used after being added with an antiseptic 
such as sodium azide etc. 
The thus prepared eluent according to the present invention does not have a 
bad influence upon the packing material, and does not cause the clogging 
of the column even in the case of a specimen containing basic, acidic or 
amphoteric substance in a large amount, and makes the stabilized 
determination possible. 
The liquid chromatography is carried out at a temperature generally in a 
range of 5.degree. to 40.degree. C. with the specimen in an amount of 
about 5 to 20 microlitres. 
In the case where the eluent is the mixture of acetonitrile and the aqueous 
solution of salt of organic acid or polybasic organic acid, the separated 
fractions are detected by an ultra violet light in a range of 200 to 280 
nm, for instance, 270 nm to find out the peak, and for seizing the 
relationship to the morbid state more precisely, for instance, it is 
preferable to detect the peak by using two light waves, for instance, 220 
and 270 nm. Also, in the case where the eluent is the mixture of 
acetonitrile and the aqueous solution of acetic acid, the separate 
fractions are detected by an ultra violet light in a range of 200 ,to 280 
nm, and for seizing the relationship to the morbit state more precisely, 
for instance, it is preferable to detect the peak by using two light 
waves, for instance, 220 and 270 nm. Further, in the case where the eluent 
is the mixture of acetonitrile and the aqueous solution of ammonium 
carbonate, the separated fractions are detected by an ultra violet light 
in the rage of 200 to 280 nm, it is preferable to detect the peak by using 
two light waves, for instance, 220 and 270 nm. 
In the case where the eluent is a mixture of acetonitrile and the aqueous 
solution of acetic acid, salt of organic acid or acetic acid, when 
fluorescent analysis is carried out, a light 220 to 360 nm is used for 
excitation and a fluorescence of 280 to 520 nm, preferably, a light of 320 
to 350 nm is used for excitation and a fluorescent light of 450 to 470 nm 
is used. 
The present invention is not only utilizable to the clinical analysis but 
also utilizable to the analysis of the components of specimens taken from 
the living body, for instance, by packing a larger column with the carrier 
(packing material, in other word, the filler) to fractionally collect the 
component showing the peak(s). 
The present invention will be explained more in detail while referring to 
the following non-limitative examples: 
EXAMPLE 1 
After packing a stainless-steel column of 4 mm in diameter and 30 cm in 
length with a commercialized packing material (.mu.-Bondapak.RTM. phenyl, 
10 micrometers in particle diameter, made by Waters Co.), the thus packed 
column was set to a high-speed liquid chromatographic apparatus provided 
with a UV-detector and a mixture of acetonitrile and an aqueous 0.1% 
solution of sodium oxalate (volume ratio of acetonitrile to aqueous 
solution of 10:90) was poured into the thus packed column at a rate of 1.0 
ml/min to stabilize the UV-detector and the recorder. 
Into 500 microliters of a serum obtained from a healthy person or of a 
serum taken from a patient of complication of liver cirrhosis and 
encephalosis, 5 microliters of methanol was added, and after mixing them, 
the mixture was kept in a warm water bath at 60.degree. C. for 20 min to 
coagulate the proteinic components. Thereafter, the thus treated mixture 
was subjected to centrifugal separation at 5.degree. C. for 20 min and at 
3000 G to collect the supernatant liquid, which was dried to solid and 
dissolved in 500 microliters of methanol to obtain the specimen to be 
examined. 
Each 10 microliters of the thus prepared specimens was poured into the 
column of the high-speed liquid chromatographic apparatus to carry out the 
analysis under a UV-light of 270 nm at a sensitivity of 0.02. 
As a result of analysis for 20 min, the respective chromatograms, one for 
the healthy person shown in FIG. 1B and the other for the patient shown in 
FIG. 1A were obtained. In FIG. 1A, three unusual peaks 1, 2 and 3 were 
observed, which were not observed in FIG. 1B of the serum of the healthy 
person. 
EXAMPLE 2 
The respective sera taken from 51 patients suffering from various liver 
diseases were analyzed by high-speed liquid chromatography under the same 
conditions as in Example 1 and the results are shown in Table 1. 
TABLE 1 
______________________________________ 
Total Number of patients 
Rate of 
number showing the speci- 
detection 
Hepatic disease 
of patients 
fic peaks (%) 
______________________________________ 
Acute hepatitis 
3 3 100 
Chronic hepatitis 
11 11 100 
Lupoid hepatitis 
2 2 100 
Cirrhosis 20 20 100 
C.sup.+ with hepatoma 
2 2 100 
C.sup.+ with c.c..sup.++ 
5 5 100 
C.sup.+ with hepatoma 
2 2 100 
and c.c..sup.++ 
(Cirrhosis in 
29 29 100 
total) 
Primary choleic 
3 3 100 
cirrhosis 
Cholestasis 3 3 100 
(total) 51 51 100 
______________________________________ 
Notes: 
C.sup.+ means cirrhosis 
c.c..sup.++ means encephalosis 
As seen in Table 1, all the serumal specimens obtained from patients 
suffering from at least one hepatic disease showed at leat one specific 
unusual peak in the chromatographic pattern, the specific unusual peak 
being not observed in the chromatographic patern of the serum takne from 
healthy person. 
In addition, in the case where the patients were classified concerning the 
appearance and the insensity (area or height) of one of the specific 
unusual peaks, Peak No. 3 including the changes of the patients' morbid 
states, a result shown in FIG. 2 was obtained. 
In FIG. 2, when thepeak area was compared between the respective patient 
group, for instance those of acute hepatitis, those of chronic hepatitis, 
liver cirrhosis and liver cirrhosis with encephalosis, it is noticed that 
the peak area depends on the morbid state of the patients. Particularly, 
in the patient suffering from liver cirrhosis with a complication of 
encephalosis, the peak showed a specifically large area. 
EXAMPLE 3 
A plasma obtained from a patient suffering from cholestasis and treated as 
in Example 1 was subjected to highspeed chromatographic analysis in the 
same apparatus as in Example 1 under the same conditions as in Example 1 
except for using a mixture of acetonitrile and an aqueous 0.1% solution of 
sodium acetate (volume ratio of 10:90), a mixture of acetonitrile and an 
aqueous 0.1% solution of trisodium citrate (volume ratio of 10:90) or a 
mixture of acetonitrile and an aqueous 0.1% solution of sodium succinate 
instead of the mixture of acetonitrile and an aqueous 0.1% solution of 
sodium oxalate in Example 1. As a result, in every case, unusual peak(s) 
was observed in the chromatogram, which has never been observed in the 
plasma of healthy persons. 
EXAMPLE 4 
The same serum specimen as in Example 1 was divided into the two equal 
portions, and one of the portion was directly subjected to the high speed 
liquid chromatography under the same conditions as in Example 1, and the 
other portion was subjected to de-proteinization and subsequently 
subjected to the high speed liquid chromatography as above. The 
de-proteinization was carried as follows: 
After mixing the serum specimen with an aqueous 5% solution of 
trichloroacetic acid at a volume ratio of 1:1, the mixture was centifuged 
to precipitate the insoluble matter in the mixture, and the supernatant 
liquid was used for chromatography. 
As the result, both the thus treated specimen and the not-treated serum 
itself gave the respective chromatographic patterns and in each of the two 
patterns, unusual peak was recognized. Such peak did not appear in both 
the chromatographic patterns obtained from the un-treated serum and from 
the treated serum (de-proteinized) taken from a healthy person. 
The above-mentioned test was carried out under the different conditions of 
the high speed liquid chromatography while using a mixture of acetonitrile 
and an aqueous 0.1% solution of citric acid (volume ratio of 10:90) 
instead of the mixture of acetonitrile and an aqueous 0.1% solution of 
sodium oxalate as in Example 1. 
The results were the same as above. Namely, the specific unusual peaks 
appeared in both the chromatographic pattern on the de-proteinized serum 
of the patient and the chromatographic pattern on the not-de-proteinized 
serum of the patient. Such peak did not appear in both the chromatographic 
pattern on the de-proteinized serum of the healthy person and the 
chromatographic pattern on the not-de-proteinized serum of the healthy 
person. 
The result of the present Example is useful in simplifying the test 
procedures in the method according to the present invention. 
EXAMPLE 5 
A serum obtained from a patient suffering from acute liver failure or a 
serum taken from a healthy person was subjected to high-speed liquid 
chromatography while using the same apparatus used in Example 1 except for 
using a fluorescence monitor (made by HITACHI Works) at .lambda.ex of 220 
nm and .lambda.em of larger than 340 nm, under the same conditions as in 
Example 1 except for using a mixture of acetonitrile and an aqueous 0.3% 
solution of sodium acetate (volume ratio of 10:90) shown in FIG. 3A, a 
mixture of acetonitrile and the aqueous 0.3% solution of sodium acetate 
(volume ratio of 11.25:88.75) shown in FIG. 3B, a mixture of acetonitrile 
and the aqueous 0.3% solution of sodium acetate (volume ratio of 
12.5:87.5) shown in FIG. 3C or a mixture of acetonitrile and the aqueous 
0.3% solution of sodium acetate (volume ratio of 15:85) at a flow rate of 
2.0 ml/min instead of the mixture of acetonitrile and the aqueous solution 
at the flow rate of 1.0 ml/min in Example 1. The results are shown in 
FIGS. 3A to 3D. As seen in FIGS. 3A to 3D, at least 4 unusual peaks are 
observed in every chromatogram of the serum of the patient, however, 
almost any one of such unusual peak could not observed in the chromatogram 
of the healthy person's serum. In addition it was found that the volume 
ratio of acetonitrile to the aqueous solution of sodium acetate (0.3%) may 
have a correlation to the number of peaks in the chromatogram under the 
specified conditions. 
EXAMPLE 6 
The same serum of the same patient or the same serum of the healthy person 
used in Example 5 was subjected to high-speed liquid chromatography while 
using the same apparatus as in Example 5 under the same conditions as in 
Example 5 except for using a mixture of acetonitrile and an aqueous 0.1% 
solution of sodium acetate (volume ratio of 11.25:88.75), a mixture of 
acetonitrile and an aqueous 0.3% solution of sodium acetate (volume ratio 
of 11.25:88.75), a mixture of acetonitrile and an aqueous 1.0% solution of 
sodium acetate (volume ratio of 11 25:88.75) or a mixture of acetonitrile 
and an aqueous 3.0% solution of sodium acetate instead of the mixture of 
acetonitrile and the aqueous solution of sodium acetate in Example 5. 
The results are shown in FIGS. 4A to 4D, wherein each of the chromatograms 
of the healthy person's serum was piled on the corresponding chromatogram 
of the patient's serum. 
As seen in the figures, while unusual peaks (blackened in the figures) are 
clearly seen in the chromatograms of the patient's serum, almost any one 
of such peak could not be seen in any one of chromatograms of the healthy 
person. In addition, it was found that the concentration of sodium acetate 
in the aqueous solution of the mixture may correlate to the number of 
peaks appearing in the chromatogram of the patient's serum. 
EXAMPLE 7 
The serum or the ascitic fluid obtained from a rat suffering from liver 
cirrhosis due to artificial inhalation of carbon tetrachloride for a long 
term was subjected to high-speed liquid chromatography in the same 
procedures and conditions in Example 6 except for using a mixture of 
acetonitrile and an aqueous 0.3% solution of sodium oxalate (volume ratio 
of 11.25:88.75) instead of the mixture of acetonitrile and the aqueous 
solution of sodium salt in Example 6. 
As a result, two peaks were observed in both the chromatograms of the serum 
and of the ascitic fluid of the cirrhotic rat, which were almost the same 
as the peaks c and d in FIG. 3B. 
However, no such peaks was seen in the chromatogram of normal rat's serum 
or ascitic fluid obtained by the same procedures in the present high speed 
liquid chromatography. 
These findings suggest the probable use of the method according to the 
present invention in the test for evaluating the pharmaceutical effects 
while using experimental animals. 
EXAMPLE 8 
After having confirmed that the specific peak No. 3 appearing in the 
chromatogram of the patient's serum who was suffering from liver cirrhosis 
accompanying encephalosis in Example 1 according to the present invention 
also appears in every chromatogram of the respective sera of the three 
patients shown in Table 2 taken by the same procedures as in Example 1, 
the specific peak No. 3 was traced by the same techniques as in Example 1 
on the three patients, respectively together with the tracing by 
biochemical examination, the results being shown also in Table 2. 
TABLE 2 
______________________________________ 
Pa- Intensity Value of biochemi- 
tient of cal analysis 
No. Clinical finding 
Peak No. 3 
GPT (KU) 
GOT (KU) 
______________________________________ 
No. 1 degree of 9.3 .times. 10.sup.4 
112 52 
encephalosis, II 
No. 2 degree of 12.6 .times. 10.sup.4 
85 42 
ascites, .sup.++ 
No. 3 degree of 15.6 .times. 10.sup.4 
95 44 
encephalosis, I 
and degree of 
ascites, .sup.++ 
______________________________________ 
As seen in Table 2, while the index according to the present invention (the 
intensity of a specific peak in chromatogram prepared according to the 
present invention) corresponds very well to the clinical findings, the 
conventional value such as GPT and GOT do not necessarily correspond to 
the clinical findings. 
From these findings, it is clear that the present invention is able to 
offer extremely precise informations concerning the presence or absence of 
the complication and the degree of seriousness of the disease in the 
patient suffering from hepatic disease(s). 
EXAMPLE 9 
In Example 1, the height of the respective peaks appearing in the 
chromatogram of the patient according to the present invention was 
examined while changing the wave length of ultraviolet ray to 220, 230, 
240, 250, 260 and 280 nm in UV detector of the high-speed liquid 
chromatographic apparatus in Example 1, the results are shown in FIG. 5. 
As seen in FIG. 5, while the peak No. 1 and the peak No. 3 showed nearly 
the same tendency of height change corresponding to the change of wave 
length, only the peak No. 2 showed a different tendency of height change. 
EXAMPLE 10 
The serum of a healthy person or the serum of a patient suffering from 
cholestasis was treated as in Example 1 and subjected to the high-speed 
liquid chromatography as in Example 1 except for using a mixture of 
acetonitrile and an aqueous 3.0% solution of ammonium carbonate (volume 
ratio of 10:90) instead of the mixture of acetonitrile and the aqueous 
solution of sodium oxalate in Example 1 and pouring 20 microlitres of the 
de-proteinized specimen into the column, detection of the peaks being 
carried out while using two UV-lights, 220 and 270 nm in wave length 
simultaneously. As seen in FIGS. 6 and 7, while in the chromatogram of the 
patient's serum, four unusual peaks No. 1, No. 2, No. 3 and No. 4 were 
observed, none of such peaks could be observed in the chromatogram of the 
healthy person's serum. 
These findings suggest that the morbid state of patients suffering from 
hepatic disease(s) will be judged by the analysis according to the method 
of the present invention. 
EXAMPLE 11 
Each of the sera obtained from 51 patients under the treatment for hepatic 
disease(s) was subjected to the analysis by the high-speed liquid 
chromatography according to the present invention under the same 
conditions as in Example 10. The results of the analysis are shown in 
Table 3, and the typical chromatographic patterns are shown in FIGS. 8-1 
to 8-10. 
As seen in Table 3, all the chromatographic patterns show the unusual peaks 
as are seen in FIGS. 8-1 to 8-10, which has never observed in the 
serum-chromatographic pattern of healthy persons. 
TABLE 3 
______________________________________ 
Relationship between the peaks in the high-speed liquid 
chromatographic pattern and the hepatic diseases 
Number of patients 
Total showing Peaks of 
Hepatic number of No. No. No. No. 
disease patients 1 2 3 4 
______________________________________ 
Acute hepatitis 
3 3 2 1 2 
Chronic hepatitis 
10 9 6 1 8 
Alcoholic hepatitis 
1 1 1 1 1 
Lupoid hepatitis 
1 1 1 -- 1 
Liver cirrhosis 
20 18 17 4 18 
C* and hepatoma 
2 2 2 -- 2 
C* and C.C** 5 4 4 2 4 
C*, C.C** and hepatoma 
2 1 2 2 2 
(Cirrhosis total) 
29 25 25 8 26 
Primary biliary C* 
3 3 1 -- 2 
Cholestasis 3 3 1 3 3 
(grand total) 50 45 37 14 43 
______________________________________ 
Notes: 
C* means liver cirrhosis 
C.C** means encephalosis 
EXAMPLE 12 
The result of tracing the course of a woman of age of 44 diagnosed as acute 
hepatitis, chronic hepatitis and urticaria from the beginning of treatment 
as an outpatient, treatment as a hospitalized patient and 
de-hospitalization to treatment on relapse by the conventional biochemical 
examination and the method according to the present invention is shown in 
Table 4. In the method according to the present invention, the total area 
of the peaks Nos. 1 to 4 appearing in the high-speed liquid 
chromatographic pattern obtained by applying the same procedures as in 
Example 10 was used as the index. 
By continued treatment, the values of biochemical examination gradually 
approached to the normal values and at the time of de-hospitalization 
(after 110 days of hospitalization), the values were in the normal range. 
On the other hand, the area of the specific peaks appearing in the 
chromatographic pattern showed a tendency of reducing slowly, however, the 
peaks are still remaining and since such peaks are not observed in the 
chromatographic pattern of the healthy person's serum, the thus obtained 
results suggest the necessity of continuing the treatment after her 
dehospitalization. 
The above-mentioned situations tell that the method according to the 
present invention are able to watch the morbid state of hepatic diseases 
more precisely than the conventional biochemical methods for examination. 
TABLE 4 
______________________________________ 
Biochemical Values 
Total 
GOT GPT LDH .gamma.-GTP 
peak 
Treatment Course 
(KU) (KU) (IU/l) 
(U/l) area 
______________________________________ 
Yes time of 740 860 1198 1135 -- 
occurrence 
" 6 days after 
340 669 474 198 -- 
hospitaliza- 
tion 
" 19 days after 
111 159 279 226 35.5 .times. 
hospitaliza- 10.sup.4 
tion 
" 39 days after 
69 78 288 124 5.4 .times. 
hospitaliza- 10.sup.4 
tion 
" 65 days after 
38 70 218 136 4.5 .times. 
hospitaliza- 10.sup.4 
tion 
" 95 days after 
26 28 262 82 3.0 .times. 
hospitaliza- 10.sup.4 
tion 
" Time of 25 30 178 75 2.7 .times. 
dehospitaliza- 10.sup.4 
tion (110 days) 
No 10 days after 
67 68 427 77 10.5 .times. 
dehospitaliza- 10.sup.4 
tion 
" 21 days after 
76 97 620 158 25.1 .times. 
dehospitaliza- 10.sup.4 
tion (relapse) 
Normal value 
8-40 5-35 50-400 
0-60 nearly 
zero 
______________________________________ 
EXAMPLE 13 
The same serum specimen was subjected to the high-speed liquid 
chromatography under the same conditions as in Example 10 except for using 
a mixture of acetonitrile and an aqueous 0.3% solution of ammonium 
carbonate (volume ratio of 8:92) instead of the mixture of acetonitrile 
and the aqueous solution in Example 10 to obtain a chromatographic pattern 
showing the specific unusual peaks as that shown in FIG. 9, which appeared 
after 7 min of the beginning of chromatography and did not appear in the 
chromatogram of the healthy person's serum. The same peaks were observed 
in the chromatograms of the patients respectively suffering from various 
liver diseases. 
EXAMPLE 14 
The same serum as in Example 10 was treated by another procedure of adding 
500 microlitres of an aqueous 30% solution of trichloroacetic acid to 500 
microlitres of the serum at a room temperature, subjecting the thus 
treated mixture to centrifuge, pouring 40 microlitres of the thus obtained 
supernatant liquid into the column. As a result, unusual peaks were 
observed in the chromatographic pattern, which have never been observed in 
the chromatographic pattern of the healthy person's serum. 
In addition, the same serum taken from the patient was directly subjected 
without effecting de-proteinization to the high-speed chromatography as 
above. 
In this case, also nearly the same peaks were observed in the 
chromatographic pattern. 
EXAMPLE 15 
A serum obtained from a patient suffering from acute liver failure was 
subjected to the high-speed liquid chromatography under the same 
conditions as those in Example 10 except for using a mixture of 
acetonitrile and an aqueous 0.3% solution of ammonium carbonate (volume 
ratio of 7.5:92.5), a mixture of acetonitrile and the aqueous 0.3% 
solution of ammonium carbonate (volume ratio of 15:85) or a mixture of 
acetonitrile and the aqueous 0.3% solution of ammonium carbonate (volume 
ratio of 25:75) instead of the mixture of acetonitrile and the aqueous 
solution in Example 10, while using a fluorescence monitor of .lambda.ex 
of 220 nm and .lambda.em of larger than 340 nm. 
The results are respectively shown in FIG. 10 (the case of volume ratio of 
7.5:92.5), FIG. 11 (the case of volume ratio of 15:85) and FIG. 12 (the 
case of volume ratio of 25:75). 
As seen in the figures, the appearance and the number of the peaks depended 
on the volume ratio of acetonitrile to the aqueous solution of ammonium 
carbonate, however, such peak did not appear in the chromatographic 
pattern of the healthy person's serum. 
EXAMPLE 16 
The same procedures as in Example 13 were effected except for not carrying 
out the de-proteinizing of the serum and using the two columns connected 
in series. As a result, a peak corresponding to the peak No. 2 in FIG. 9 
of Example 13 was clearly recognized in the chromatographic pattern of the 
present serum. On the other hand, in the chromatographic pattern obtained 
by subjecting the not-deprotenized serum of a healthy person to the same 
high-speed liquid chromatographic apparatus, no such peak was recognized. 
COMATIVE EXAMPLE 1 
For the sake of comparison, the same serum of the patient suffering from 
cholestasis in Example 10 was pre-treated as in Example 10 and 
subsequently subjected to the high-speed liquid chromatography while using 
two columns connected in series and packed with a carrier (HG-3011, made 
by HITACHI Works) and a phosphoric acid buffer solution of pH of 7.4 as 
the eluent instead of the mixture of acetonitrile and the aqueous solution 
of ammonium carbonate in Example 10, under the same conditions as in 
Example 10. As a result, no unusual peak other than those of uric acid and 
creatinine could be detected in the chromatographic pattern. 
EXAMPLE 17 
A serum taken from a patient suffering from liver cirrhosis was subjected 
to the same high-speed liquid chromatography as in Example 10 except for 
using a mixture of acetonitrile and an aqueous 0.3% ammonium carbonate 
(volume ratio of 10:90) instead of the mixture of acetonitrile and the 
aqueous solution in Example 10. 
As a result, a chromatographic pattern shown in FIG. 13 was obtained and 
four unusual peaks, Nos. 1, 1', 2 and 2' were recognized therein. In the 
chromatographic pattern obtained from the serum of a healthy person, such 
peak was not detected. 
EXAMPLE 18 
Each one of sera obtained from four patients suffering from alcoholic fatty 
liver was subjected to the same high-speed liquid chromatography as in 
Example 17. The results are shown in Table 5 together with the values of 
biochemical examination of the same serum. 
From Table 5, it is recognized that the area of the peaks appearing in the 
chromatographic pattern increases with the aggravation of the morbid state 
of the liver disease, and that the method according to the present 
invention is applicable to clinical examination of the patients suffering 
from hepatic disease(s). 
TABLE 5 
______________________________________ 
Specimen from Patients Nos. 
Item 1 2 3 4 
______________________________________ 
GOT(KU) 62 44 40 123 
GPT(KU) 63 58 69 287 
Alkali-phosphatase 
7.5 9.8 10.2 12.9 
.gamma.-GTP (U/liter) 
45 138 123 693 
T.S.BA* 1.1 1.3 2.7 336 
(micromol/liter) 
GCA** 0 0 0 44 
(micromol/liter) 
Area of Peak 1.8 1.9 1.5 12.5 
No. 1'*** 
Area of Peak 3.65 3.5 5.1 30.6 
No. 2'*** 
______________________________________ 
Note: 
***Peaks Nos. 1' and 2' appearing in the chromatographic pattern of the 
respective sera from the patient, the peak being detected by UV of 270 nm 
and the area being divided by 10.sup.4. 
T.S.BA*: total serum bile acid 
GCA**: glycocholic acid. 
EXAMPLE 19 
Serum specimens were obtained from a patient suffering from chronic renal 
failure complicated with hepatitis, respectively, before treatment (refer 
to FIG. 14) and after 2 month-treatment (refer to FIG. 15), and subjected 
to the high-speed liquid chromatography according to Example 10 under the 
same conditions as in Example 13. 
The results are shown in FIGS. 14 and 15, wherein the peaks N and N' are 
recognized to be due to kidney failure and the area thereof clearly shows 
the reduction after treatment. In addition, the peaks H and H' are due to 
hepatitis, and no improvement due to the treatment was recognized. 
For reference, the values of biochemical examination before and after 
treatment are shown below: 
______________________________________ 
Creatinine BUN GOT Amount of 
(mg/dl) (mg/dl) (KU) dairy urine (ml) 
______________________________________ 
Before 14 160 40 600 
treatment 
After 10 120 45 1200 
treatment 
______________________________________ 
EXAMPLE 20 
Each of the two serum specimens used in Example 1 was treated by the same 
procedures as in Example 10 and subsequently subjected to the high-speed 
liquid chromatography under the same conditions as in Example 10 except 
for using a mixture of acetonitrile and an aqueous 0.1% solution of acetic 
acid (volume ratio of 10:90) and further using UV-lights of 210 and 280 nm 
at a sensitivity of 0.02 instead of the mixture of acetonitrile and an 
aqueous solution of ammonium carbonate and of UV-lights of 220 and 270 nm 
at a sensitivity of 0.04 in Example 10. Of the thus obtained two 
chromatographic patterns within 20 min (refer to FIGS. 16 and 17), the one 
taken on the patient's serum showed unusual peak (refer to FIG. 2) which 
was not detected in the pattern on the healthy person's serum (refer to 
FIG. 16). 
EXAMPLE 21 
Each one of the two serum specimens, respectively obtained from a patient 
suffering from liver cirrhosis accompanying encephalosis and a healthy 
person was pre-treated by the precedures of de-proteinization shown in 
Example 10 and subsequently subjected to chromatography under the same 
conditions as in Example 10 except for using a mixture of acetonitrile and 
an aqueous 0.01% solution of acetic acid (volume ratio of 10:90) under 
UV-lights of 220 nm at a sensitivity of 0.04 and 280 nm at a sensitivity 
of 0.02 instead of the mixture of acetonitrile and the aqueous solution 
under UV-lights of 220 nm at a sensitivity of 0.04 and 270 nm at a 
sensitivity of 0.04 to obtain the two chromatographic patterns (refer to 
FIGS. 18 and 19) within 20 min. In the chromatographic pattern (FIG. 19) 
taken on the patient's serum, an unusual peak was recognized, which was 
not recognizable in the chromatographic pattern taken on the healthy 
person's serum. 
EXAMPLE 22 
Serum specimens were obtained from a patient suffering from a complication 
of liver cirrhosis, hepatoma and encephalosis corresponding to the 
aggravation of his morbid state and after treating the specimen for 
de-proteinization as in Example 1, the thus treated specimen was subjected 
to high-speed liquid chromatography under the same conditions as in 
Example 10. The results are shown in Table 6 together with the values 
obtained by the conventional biochemical examination for reference. As 
seen in Table 6, in spite of the aggravation of the patient's morbid state 
from the degree of encephalosis of I to the degree of II, the values of 
biochemical examination did not show any remarkable change except for the 
value of NH.sub.3. On the other hand, the area of the peaks appearing in 
the chromatographic patterns on the patient's serum accurately reflects 
the change of the morbid state, and the applicability of the method 
according to the present invention has been thus elucidated. 
TABLE 6 
______________________________________ 
Serum taken at Stage of 
Deg. 
encephalo- 
Item(unit) sis.sup.1 I 
II Normal value 
______________________________________ 
GOT (KU) 67 68 8-40 
GPT (KU) 36 32 5-35 
Alkali Phos..sup.2 (U) 
22.6 25.9 2.7-10.0 
.gamma.-GTP (u/liter) 
24 12 0-60 
T-chol..sup.3 (mg/dl) 
111 61 130-230 
LDH (IU/liter) 
340 330 50-400 
NH.sub.3 (mcG/dl) 
124 251 0-120 
BUN (mg/dl) 17 14 10-20 
Area of the peak(s) in 
7.5 .times. 10.sup.4 
12.5 .times. 10.sup.4 
nearly 0 
the serum detected by 
the present invention 
under UV of 280 nm 
______________________________________ 
Notes: 
.sup.1 Degree of encephalosis 
.sup.2 Alkali phosphatase 
.sup.3 total cholesterol. 
EXAMPLE 23 
Each one of the serum specimens, respectively obtained from a patient 
suffering from liver cirrhosis with encephalosis and from a healthy person 
was de-proteinized as in Example 1 and subsequently subjected to the 
high-speed liquid chromatography under the same conditions as in Example 1 
except for using a mixture of acetonitrile and an aqueous 0.1% solution of 
citric acid (volume ratio of 10:90) instead of the mixture of acetonitrile 
and the aqueous solution in Example 1 to obtain the two chromatographic 
patterns within min, one of which corresponds to the patient (FIG. 20A), 
and the other of which corresponds to the healthy person (FIG. 20B). As 
seen in FIGS. 20A and 20B, an unusual peak was detected in the patient's 
serum, which was not recognized in the healthy person's serum. 
EXAMPLE 24 
Each one of the two plasmic specimens, respectively obtained from a patient 
suffering from cholestasis and from a healthy person was pre-treated by 
the same procedures as in Example 1 and subsequently subjected to 
high-speed liquid chromatography under the same conditions as in Example 1 
except for using a mixture of acetonitrile and an aqueous 0.1% solution of 
oxalic acid or succinic acid (volume ratio of 10:90) instead of the 
mixture of acetonitrile and the aqueous solution in Example 1. 
As the result, an unusual peak appeared in the chromatographic pattern on 
the patient's plasma regardless of the kinds of the solute in the aqueous 
solution (oxalic acid and succinic acid). However, such a peak was not 
detected in the pattern on the healthy person's plasma. 
EXAMPLE 25 
Each one of the same serum specimens as in Example 23 were treated by the 
same procedures as in Example 1 except for using a mixture of acetonitrile 
and an aqueous 0.5% solution of citric acid (volume ratio of 10:90) 
instead of the mixture of acetonitrile and the aqueous solution in Example 
1 to obtain the two chromatographic patterns, of which the one 
corresponding to the serum of the patient showed an unusual peak which was 
not present in the pattern corresponding to the healthy person. 
EXAMPLE 26 
Each one of the serum specimens, respectively obtained from a patient 
suffering from acute liver failure and from a healthy person was treated 
as in Example 1 except for using a mixture of acetonitrile and an aqueous 
0.1% solution of citric acid (volume ratio of 8:92, 12:88 or 15:85) under 
the same conditions as in Example 1 to obtain six chromatographic patterns 
of the respective serum specimens. As the result, an unusual peak was 
observed in the respective chromatographic patterns on the serum of the 
patient, however, such a peak was not detected in the three 
chromatographic patterns on the healthy person's serum. 
EXAMPLE 27 
The high-speed liquid chromatography in Example 1 was applied to the serum 
specimen obtained from a patient suffering from a complication of liver 
cirrhosis and hepatoma to whom encephalosis was recognized, for tracing 
the patient's morbid state. 
The results are shown in Table 7 together with the values of biochemical 
examination on the patient. 
TABLE 7 
______________________________________ 
Specimen taken in 
Item (unit) Stage A Stage B Normal value 
______________________________________ 
GOT (KU) 67 68 8-40 
GPT (KU) 36 32 5-35 
alkali (U) 22.6 25.9 2.7-10.0 
phosphatase 
.gamma.-GTP 
(u/liter) 24 12 0-60 
LDH (IU/liter) 
340 330 50-400 
T-chole- 
(mg/dl) 111 61 130-230 
sterol 
NH.sub.3 
(mcG/dl) 124 251 0-120 
BUN (mg/dl) 17 14 10-20 
Area of Peak 4.87 .times. 10.sup.4 
6.28 .times. 10.sup.4 
nearly 0 
detected by 270 nm 
Clinical finding on 
I II -- 
the degree of en- 
cephalosis 
______________________________________ 
EXAMPLE 28 
The morbid state of a hospitalized patient suffering from liver cirrhosis 
and being under treatment was traced by using the specified peak appearing 
in the chromatographic pattern obtained by the same procedures as in 
Example 23 and in addition by using the values of biochemical examination. 
The results are shown in Table 8 as follows: 
TABLE 8 
______________________________________ 
Intensity 
of the peak 
Biochemical value 
Clinical finding 
(count .times. 10.sup.4) 
GPT (KU) GOT (KU) 
______________________________________ 
Degree of encephalo- 
7.5 112 52 
sis II 
Degree of chole- 
9.98 85 42 
stasis.sup.++ 
Degree of encephalosis 
13.9 95 44 
I plus Degree of chole- 
stasis.sup.++ 
______________________________________ 
As seen in Table 8, the findings by the present invention corresponds 
favorably to the clinical findings, however, the conventional values of 
biochemical examination did not necessarily correspond to the clinical 
findings. These results clearly indicate that the present invention is 
able to supply an extremely precise information concerning the seriousness 
in a patient of a hepatic disease and the presence and absence of the 
complication in a hepatic disease.