JPS58109849A - Novel analysis for separation of inositol - Google Patents

Abstract

PURPOSE:To determine myoinositol quickly and easily in good accuracy and to discriminate exactly the functions of the kidney, by separating the myoinositol in blood serum etc. from another material by using liquid chromatography and analyzing by using a differential refractometer. CONSTITUTION:In an analysis of myoinositol in body fluids (blood serum etc.), a sample is injected into a liquid chromatography column packed with a packing having a hydrophilic property (a polyvinyl alcohol group adsorbent or an adsorbent of silica gel hydrophilic radical etc.) and separation is carried out by using water as a solvent and then, it is determined by using a differential refractometer as a detector. It can be measured also by an ultraviolet spectrometer by adding a color producing reagent such as nitric acid, CaCl2 etc. The concentration of the myoinositol is known by a preliminarily found calibration curve. In this manner, the functions of the kidney is discriminated by measuring quickly and simply with high accuracy. Especially in the transplantation of the kidney, it is suitable for the discrimination of the functions quickly after the transplantation.

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a new method for the separation and separate quantification of myo-inositol.

Myo-inositol is widely distributed in living organisms, and in addition to being orally ingested by mammals, it is also biosynthesized from glucose. Myo-inositol exists in large amounts in the brain, kidneys, kidneys, etc. both when it exists alone (free myo-inositol) and as phosphoinositol bound to lipids. In the process of elucidating that myo-inositol has a close relationship with the functions of internal organs, the inventors came to invent a new method for separating and emplacement of myo-inositol.

That is, the conventional separation and emplacement method of myo-inositol is as follows:
One of them is gas chromatography. However, this method has the disadvantages that myo-inositol is a high boiling point substance, so it must be silylated and the measurement time is long. A chemical analysis method (shake method) using nitric acid or the like is also known, but even this method requires a long time for measurement. When determining the function of internal organs by quantifying myo-inositol, rapid measurement is often required. For example, in the case of kidney transplantation, analysis must be completed within several minutes after sampling to determine renal function. The present invention is based on this viewpoint.

The use of liquid chromatography is characterized by the ability to perform measurements more quickly than C2. This is a method for separating and quantifying myo-inositol. Myo-inositol as used in the present invention refers to free myo-inositol and substances in which myo-inositol and lipids are bound. What is a substance that combines myo-inositol and lipids?

Hydrochloric acid methanol decomposition (2) can quantitatively and easily free myo-inositol (=).

Sample C2 to be separated and quantified There are no particular limitations, but when the purpose is to determine the function of internal organs of mammals, serum or body fluids in organs can be used as samples.

Particularly when the purpose is to determine the function of the excised kidney, for example, an initial laminar exudate obtained by washing out an ischemic kidney graft may be used as a sample. Furthermore, a liquid extracted from part or all of the removed kidney can also be used. It is often easier to analyze these samples for myo-inositol by deproteinizing them with trichlorhydric acetic acid or the like. Furthermore, when it is desired to measure total myo-inositol, the above-mentioned sample after hydrochloric acid methanol decomposition may be used.

Examples of preferred analytical methods are described below. Water is most preferable as the developing solution due to the solubility of myo-inositol, and depending on the case C2, it may be mixed with an organic substance that is compatible with water. The packing material for the packed column used for separation may be of any kind, but it is more preferable to use one that has water affinity.1 For example, a polyvinyl alcohol-based adsorbent, an adsorbent prepared by introducing a hydrophilic substituent into silica gel, a cellulose thread adsorbent, Acrylamide, vinylpyrrolidone-based adsorbents, etc. can be used. As a detector, an RI-detector (differential refractometer) is superior in that it does not require pretreatment, but it is also possible to develop a color using a coloring agent such as nitric acid or calcium chloride and then detect using UV light. be.

In addition, the inventors have discovered that myo-inositol present in renal tissue leaks out in large quantities into the initial exudate after washing out an excised renal graft, particularly an ischemic renal graft. Based on this fact, the myo-inositol concentration in the initial effluent perfusite (hereinafter abbreviated as HIP) of the excised renal graft was determined by measuring the hot water content of free myo-inositol in the upper groove, and then
=We discovered that kidney function can be determined from this data. Conventional methods for measuring renal function include methods that utilize color reaction with tetrazolium salts, methods that measure pH on the kidney surface, and methods that use oxygen consumption rate C2%Na.
Ratio calculation methods are known for /, but these conventional methods require long measurement times, have problems with experimental operations, and are often unstable in their results or difficult to judge. It has the following drawbacks.

It has been found that a simple and accurate method for determining kidney function is possible by quantifying not only B111P described above but also myo-inositol, which is present in the blood.

To give a specific example using HIP,
Insert and fix a silicon catheter, etc., and sample the fluid that comes out when lactated Ringer's solution is injected from the renal artery. Collect one supernatant from this FIBP using a centrifugal separator, etc. Use as a sample for inositol analysis.

Kidney function can be judged by the survival rate after transplant surgery in animal experiments or the concentration of creatinine in serum.

In this way, the correlation between myo-inositol, the survival rate of animals after transplantation surgery, and the concentration of creatinine in the serum is determined.
It was found that animals transplanted with kidneys containing high levels of myo-inositol had lower survival rates and also had higher levels of creatinine.

Examples are shown below, but they do not limit the present invention.

Example/ A gel containing one polymer unit of vinyl alcohol and divinylbenzene was packed. Serum from 5 people in a liquid chromatograph equipped with a column of length jOaa, a differential refraction needle, a liquid pump, and a recorder/integration needle! μt was injected and chromatographic separation was performed using water as a solvent. The separation chromatogram when the liquid was pumped at /$ is shown in the @/ figure. (In the figure, RI [Jnii] is a unit that indicates the change in refractive index, and its value is proportional to the concentration of inositol at a low concentration C:.) From the calibration curve obtained in advance, the inositol concentration in this person's serum is 3. ．． It turned out to be rlB.

On the other hand, as a result of analyzing the same sample using the conventional method of silylation-gas chromatography, the result was still 3. A result of jq/ltl was obtained.

[Brief explanation of the drawing]

The fs1 diagram shows a separation a madogram when the liquid was fed with 71I4/&lIl in Example C:. Patent applicant Asahi Kasei Industries, Ltd.

Claims (1)

[Claims] / A method for separating myo-inositol from other substances, characterized by using liquid chromatography. A method characterized by separating myo-inositol from other substances and quantifying it by using liquid chromatography. A method 3 according to claim 1, wherein myo-inositol and other substances are substances that exist in body fluids. A method for separating and fixing according to a method for separating and quantifying according to claim 2, characterized in that myo-inositol and other substances are substances that exist in body fluids. j Separation and quantitative method according to claim 2, characterized in that a differential refractometer is used as a detector 6. Claim @ claimed in claim 2, characterized in that a filler having water affinity is used. Separation and emplacement method 2 A method for determining renal function characterized by quantifying myo-inositol using liquid chromatography