JPS5928496A - Method for diagnosing hepatic disease - Google Patents

Abstract

PURPOSE:To diagnose easily hepatic diseases, by analyzing a peak or peak pattern unique to the hepatic diseases in a biological sample by the liquid chromatography. CONSTITUTION:In analyzing a biological sample by using the liquid chromatography with a packing for high-speed liquid chromatography, preferably a silica type packing trated with a silane, an aqueous solution of acetonitrile and ammonium carbonate, usually at (5/95)-(25/75), preferably (5/95)-(15/85), volume ratio between the acetonitrile and ammonium carbonate as a mobile phase to measure the separated peak at 5-40 deg.C, preferably a low temperature. The resultant specific peak or peak pattern is used as an index to the pathosis of the hepatic diseases.

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a method for diagnosing liver diseases using liquid chromatography. More specifically, it is a method for obtaining indicators of pathological conditions, in particular, pathological conditions such as liver damage and renal damage, by high-performance liquid chromatography, and further includes analyzing peaks or peak patterns specific to liver diseases in the obtained biological sample. In particular, it relates to a method for easily diagnosing liver disease.

It is extremely important for the diagnosis and treatment of pathological conditions to obtain information on pathological conditions by analyzing the components and properties of biological samples, such as collapsing fluids such as plasma and serum, brain, spinal fluid, or urine. Conventionally,
Although this information is obtained using many chemical or biochemical analysis methods, it is necessary to obtain more accurate information for the purpose of elucidating the pathological conditions of various cases or determining the pathological conditions more accurately. There is a strong need for the development of analytical methods that can provide accurate information.

In particular, since the pathological conditions of liver and kidney diseases are complex, there is a desire for the development of analytical methods that can provide new indicators closely related to the pathological conditions. Conventional pathological indicators include GOT (glutamic oxaloacetic transaminase), GPT (glutamic oxaloacetic transaminase), I, and DH (lactate dehydrogenase) for liver diseases.

Biological enzyme activity values such as LAP (leucine aminopeptidase), biochemical analysis values (test values) of collapsing fluid components such as the protein bilirubin, and in the case of kidney disease, blood chemical analysis values of creatinine, uric acid, and electrolytes. Test values include urine protein, sugar, and pH levels.

The above test values can be used as indicators for diagnosis and treatment.
It is actually used. However, with the current chemical or biochemical analysis methods, it is not possible to investigate the presence of uremic toxins, various abnormal substances, and pathogenic substances, which are said to be closely related to the disease state. This leaves people dissatisfied with the diagnosis and treatment.

In view of the above-mentioned current situation, the use of liquid chromatography, which is an analysis method based on a principle different from conventional chemical and biochemical analysis methods, has been attracting attention in recent years.

Liquid chromatography can separate and detect even thermally and chemically unstable substances without altering the substance, and it is also possible to analyze a large number of components with a relatively small amount of sample. In principle, there is hope for its use in the medical and clinical fields, and several attempts have been made.

However, in liquid chromatography, the peaks or peak patterns obtained even for the same sample vary depending on the measurement conditions such as the setting of separation conditions or the pretreatment of the specimen sample, and the detection peaks related to the measurement conditions and pathological conditions may differ. The relationship has not been fully grasped, and it has not necessarily been put to practical use as a clinical analysis method.

In particular, the pathology of liver diseases has complex developmental factors as described below, and therefore it is difficult to separate the components of a sample specimen. In fact, there are no known examples of detection of specific components during liver disease using liquid chromatography.

For example, Meyer et al. attempted to detect hepatocyte-specific antigens by liquid chromatography using cross-linked agarose-based porous beads as a packing material [C1In.

Exp, Immunol, 10.89 (1972)
). However, the method of Meyer et al. showed the same pattern as normal M0 serum and was unable to obtain a unique peak related to pathological conditions.

First, First proposed a method for analyzing serum samples by high-performance liquid chromatography [CIl+11 (!I
llNephrology, Vol 15i4119
8 (1976)], however, Ftir [lt
In the method described above, many peaks overlap, making it difficult to separate and identify each component.

In addition, there are examples of detecting various peaks regarding liver failure (notification, severity, and fulminant disease) using liquid chromatography (18th Japanese Liver J1M Society General Meeting TJ4 Abstract P59)
(S57. July 9-10)). However, as a result of the inventors' follow-up tests, the above method was found to be effective against acute hepatitis, chronic infusion liver moxibustion,
Regarding samples derived from liver cirrhosis and liver cancer, no abnormal peaks related to pathological conditions could be detected. Liver failure is a special pathological condition among liver diseases, and occurs in many types including p1 immune system to 11 immune system. Therefore, it is relatively easy to detect some of those components. On the other hand, the pathophysiology of liver diseases, which are classified into acute hepatitis, chronic hepatitis, cirrhosis, liver cancer, etc. depending on the degree of progression and severity, involves complex developmental factors.
It is presumed that the pathological condition-related component (spring element) exhibits more complex properties and cannot be separated and detected using the above analysis method.

In view of the above-mentioned circumstances, the present inventors have developed a method for clinically analyzing biological samples, particularly those associated with diseases such as liver and kidney disease, that allows for the simple and short time of separating peaks associated with the progression of pathological conditions using an extremely small amount of sample.
As a result of extensive research aimed at examining changes in liver and kidney function from changes in the peaks of the liver and kidneys, the present invention was achieved by discovering a method capable of achieving the above objectives.

The present invention is a method for analyzing biological samples by liquid chromatography, in which an acetonitrile/ammonium carbonate solution is transferred, a 1 m peak is measured as a phase, and the 4fJ6 peak or peak pattern is determined as a liver disease 7 (Ll) peak. Provided are lf and lr methods for diagnosing liver disease W, which are characterized in that they are used as indicators of pathological conditions.

As a method for diagnosing the pathology of liver diseases without using liquid chromatography, we have proposed a method using Hitoshi Kakamami 1 liver fetus antigen (Ga5tr, 76.665 (197
9)). Among them, 50 to 6 for chronic active hepatitis.
It has been reported that with a probability of 0%, it is possible to determine chronic incomprehensible hepatitis or cirrhosis with a probability of 20 to 40%. However, the above method requires complicated operations and is not satisfactory in terms of judgment accuracy.

On the other hand, the present invention uses high-performance liquid chromatography to obtain biological samples, such as serum, cerebral spinal fluid, lincoal fluid, ascites, bile, or urine, either directly or after protein removal treatment.
The system sharply separates and detects component peaks that correlate with pathological conditions in a short period of about 30 minutes, starting from a level of 0μ, and as described later, it is possible to diagnose the severity of liver disease with a probability of 90 to 100%. Considering the fact that current treatment methods are made to prevent progression to conditions with extremely high mortality rates such as liver cirrhosis and liver cancer because liver diseases are continuous, the present invention has extremely high accuracy and is suitable for use in the therapeutic field. The contribution to this can be said to be significant.

The present invention is applicable not only to fulminant hepatitis but also to acute hepatitis.

It can be used to treat the pathology of liver diseases such as chronic hepatitis, cirrhosis, hepatic 1θ, lupoid hepatitis, cholestasis, primary biliary cirrhosis, and liver fibrosis, as well as hepatorenal complications arising from renal diseases.
It can also be diagnosed in nephrosclerosis.

The present invention will be explained in detail below.

The filler used in the present invention may be one that is commercially available as a filler for high performance liquid chromatography, and among them, silane-treated silica-based fillers are preferred. The filler can be obtained by known methods, for example, by treating ground silica with a silane compound. The material filled with the filler is transferred to a commercially available liquid chromatography device Pt or any device having an equivalent function, and subjected to the biological assay aimed at in the present invention.

The mobile phase according to the invention is an acetonitrile/ammonium carbonate aqueous solution. The acetonitrile/ammonium carbonate aqueous solution usually has a volume ratio of 5/95 to 25/75 (volume ratio), preferably 5/95 to 15/85. Ammonium carbonate is distilled water.

water, neutral buffer, e.g. pit=7.4
Use by dissolving in phosphate buffer, etc. Normally, the concentration fy of ammonium carbonate in an aqueous ammonium carbonate solution is o, t=
to % by weight, preferably 03 to 3%. The mixing ratio of ammonium carbonate mW and acetonitrile can be arbitrarily selected within the above range.

Note that the mobile phase may be used with addition of a salt component such as sodium chloride or a preservative such as sodium azide, if necessary. The amount of acetonitrile is related to the elution rate of analyte sample components and the separation of peaks, and the degree of ammonium carbonate σ,' is related to separation of peaks.

For biological assay, the sample can be used as is, or after pretreatment with a known protein removal reagent such as methanol, trichloroacetic acid, or perchloric acid. Alternatively, high molecular weight substances may be removed using a filter membrane. To improve the analysis accuracy of 3'a agent life 2, pretreatment +! l! Samples that have undergone manipulation are preferred.

The analysis temperature is usually 5 to 40°C, and in particular, it is preferable that the temperature of the liquid C is low in order to prevent the release of free ammonia.

The biological sample may have a size of about 5 to 20 microns. A method of detecting the minute 1'jln peak at the same time in the ultraviolet region of 200 to 280 nm, preferably 220 and 270 rzn, is preferred from the viewpoint of relevance to pathological conditions. In addition, in fluorescence analysis, excitation wavelength 220-36onm + M light wavelength 280-520nm
m, preferably with an excitation wavelength of 320-350 nm H
4J light wavelength 45 (1-470 μm). By combining the mobile phase of the present invention and the above detection wavelength, it is possible to improve liver and kidney function.
It is possible to obtain peaks specific to the change in liver and kidney function, and it is possible to diagnose hepatic and renal function from the peaks and/or patterns. Note that by matching the wavelengths of the violet and fluorescent regions, the 4'iW IJt for diagnosis can be increased.

As a detection power method - C and other differential hiJ refractive index, Red Sun 1 ()
Luminous intensity, polarography, conductivity, etc. may also be used.

The diagnostic method of the present invention is extremely simple. In other words, in the measurement peaks obtained by the above method, as will be described later, there are no or no peaks specific to liver diseases. This is carried out by processing the Xk value and/or recognizing its peak pattern.

Furthermore, in order to convert the data into constants, it is also possible to connect a data processor to the detector and process the number of peak areas (+l^ or υ, Qi).

The present invention is not limited to the above-mentioned clinical analysis, but can also be used, for example, to fill a large column and separate each component.

Hereinafter, the present invention will be explained in more detail with reference to Examples.

Example 1 Commercially available filler μmJlondapak"'phe++y
1 (particle size ~10M: cron, manufactured by Waters) in a standard manner into a stainless steel column of 4 x 30 C1n, and set in a high performance liquid chromatography apparatus equipped with an ultraviolet detector. 3 of the columns formed as described above.
Add acetonitrile/35X ammonium carbonate aqueous solution (volume ratio: 10/90) to 1U tube. (lrnt/v) m, violet ray detector, record n1 to stabilize 〇 Take 500μ of each of normal human serum and cholestasis Jrn7K, add 5CC of methanol, and mix. Treated in a hot bath at 60°C for 20 minutes to coagulate the protein components, and centrifuged at 5°C and 3000G for 20 minutes. The supernatant was taken out. After drying, 50011 tons of methanol was added to remove the remaining pickles. It was dissolved and used as a sample specimen.

20 μtf for each sample! : The sample was injected and analyzed using the high performance liquid chromatography equipment (fiffl) described above. In addition, ultraviolet analysis): t22
o.

Simultaneous measurements were performed at 270 μm and a sensitivity of 0.04.

As a result, a chart as shown in FIG. 1 was obtained after an analysis time of 20 minutes. The analysis results of normal human serum are shown in FIG. 2. Abnormal peaks observed in the 7 squirrels in Figure 1 (peaks 1 and 2)
, 3.4) were not detected in the normal person shown in Figure 2. These results revealed that the pathology of liver disease can be determined by analyzing the serum of patients with liver disease using the method of the present invention.

Example 2 Patient 5 being treated for various liver diseases 7w under the same conditions as Example 1
Sera from 0 people were analyzed. The results are shown in Table 1 and a typical example is shown in FIG.

As is clear from this, abnormal peaks not seen in normal people were observed in all liver diseases.

Table 1) Relationship between iLC peak and liver disease Example A 44-year-old woman diagnosed with acute hepatitis, chronic hepatitis, and urticaria received medical treatment (hospitalization) and the course of treatment leading to re-treatment at a limited hospital. Table 2 shows the results of conventional biochemical tests and follow-up using the method of the present invention. In the method of the present invention, serum was analyzed in the same manner as in Example 1, and the area of the l-specific peak (peak Jg 1 + 2 + 3-1-4) was used as an index.

After 1M of treatment, biochemical analysis values gradually approached normal values, and at discharge point Jζ (110 days after hospital visit), almost normal range was maintained. i? The IiM value also showed a tendency to gradually decrease with treatment. However, since the peak viscosity value cannot be reduced by a normal person, the method of Xylophone 11
This suggests the need to continue treatment even after treatment.

From the above results, it can be said that the method of the present invention allows for more detailed monitoring of pathological conditions than current biochemical testing methods.

Table 2 Example 4 Since the mobile phase in Example 1 was acetonitrile 103% ammonium carbonate aqueous solution (volume IH ratio 8/92).
1. All serum samples were analyzed under the same conditions.

As a result, a chart as shown in Figure @4 was obtained, and after 7 minutes, liver disease and abnormal peak 75 were reported. :,
However, this was not the case in normal people. In addition, each fi1 liver disease patient is introduced in the same way, especially the peak (1, 1'; 2° 2') is thick.

Example 5 Contrary to the example procedure, the serum treatment conditions were as follows: serum 50% by drying at room temperature.
500 μt of 30% trichloroacetic acid aqueous solution for 0 μtK
was added, mixed, centrifuged, and the supernatant was used as a sample and 40μ was injected.
r, L presence (-absent abnormal peak could not be detected.

Example G In Example 1, serum was collected from a patient with acute R1 insufficiency nl'In''
f,: 8 mobile phase with acetonitrile 10.3% ammonium carbonate aqueous solution 11-ratio 7.5/92.5.1.5
/ 85 and 25/75. Flow F-2,0me/m
λ6 with a fluorescent monitor (manufactured by Hitachi) as a detector.
The same method was used except that x=220 nm +λam>340 nm.

The results are shown in Figure 5 (7,5/92.5) and Figure 6 (1
5/85) and Fig. 7 (25/75), abnormal peaks ranging from peaks ■ to 0 were observed in Keisha Kousei, whereas these were almost absent in normal subjects.

It was possible to detect various peaks by changing the valley/distance ratio of acetonitrile in this manner.

Example 7 In Example 4, the serum was not subjected to protein removal treatment, and its t1
The analysis was carried out under the same conditions except that two J-rings were connected in series. As a result, an abnormal peak corresponding to peak (2) in Fig. 4 was clearly detected, and no normal cases were detected.

Comparative Example For comparison, HG was used as a packing material in the same column as in the above example.
-3011 (manufactured by Hitachi) and a phosphate buffer (pl+ 7.4) as the mobile phase were carried out under the same conditions as in Example 4. Serum f/i: t, j: No unusual n peak could be detected.

Example 8 In Example 1, the transferred U-11 phase was mixed with acetonitrile 70
．． 3 ammonium carbonate aqueous solution (volume-acclimate ratio 10/90),
After changing the serum to liver cirrhosis patient serum ■, we analyzed it using the same method and obtained the chart shown in Figure 298, in which abnormal peaks (peaks ■, ■, ■', ■') were detected, but normal Detected by humans! I didn't.

Example 9 In Example 8, the patient serum was changed to one with alcohol and one with fatty liver, and the results of examining changes over time were 21'! It is shown in Table 3. For reference, biochemical test values are also shown.

It was observed that the number of abnormal peaks increased as disease H worsened, and it became clear that the method could be applied to clinical tests.

Table 3 Example 10 Serum from a patient undergoing treatment for chronic renal failure and hepatitis was used under the same conditions as in Example 4.

The results are shown in Figure 9 (before treatment) and Figure 10 (2 months treatment).
As shown in Biochemical test values before treatment were creati, =
BUN: 14 m (/ / di r blood urea (BUN)
): 160Ida/di, GOT: 401(U,
600CC bed test/day, creatinine after 2 months treatment: I Omy/di
.

G O'r: 45 KUT JIIl liquid urine'4
4i'd'ACB UN): 120 mqldl
, urine 14,200cc/day. Peaks N and N' in Figures 9M and 10 were recognized as peaks due to renal failure, and clearly decreased after treatment.

Note that 1-I and H' are peaks derived from I) flame, and no improvement was observed.

[Brief explanation of the drawing]

Figures 1 and 3 to 10 are diagrams showing liquid chromatography analysis patterns of blood from various liver diseases, and Figure 2 is a diagram showing liquid chromatography analysis patterns of normal human serum. It is. Figure 1 Figure 2 IJILJ'tiU:'4;111inna'l'U]
Tonneau Figure 3 Figure 3 Figure 4. Figure 5 Figure 6 Figure 7 Open during elution (8) Figure 8

Claims (1)

[Claims] (1) In a method of analyzing biological samples by liquid chromatography, the fractionated V peak is measured using an acetonitrile/ammonium carbonate solution as a mobile phase, and the 0.4% peak or peak pattern is used as an indicator of the pathology of liver disease. A diagnostic method for liver disease that involves making a judgment.