CN103913581A - Cyclophorase determination method for triglyceride in serum - Google Patents

Abstract

The invention discloses a cyclophorase determination method for triglyceride in serum, and belongs to a method for measuring materials by measuring color change generated by reactions. A technical solution lies in that a reagent I contains effective components of glycerol kinase, 3-phosphoglycerol dehydrogenase, phosphoglycerol oxidase, peroxidase, adenosine triphosphate, NADH, 4-aminoantipyrine and 2,4-dichlorophen; and a reagent II only contains an effective component of lipoprotein lipase. Free glycerol in the serum is subjected to circular reactions with adenosine triphosphate, NADH, 4-aminoantipyrine and 2,4-dichlorophen in the reagent I under catalysis of glycerol kinase, 3-phosphoglycerol dehydrogenase, phosphoglycerol oxidase and peroxidase to generate quinoneimine; and after the reagent II is added, triglyceride is hydrolyzed to generate glycerol, and then the circular reaction is carried out. The content of triglyceride is calculated by an instrument based on quinoneimine generated by reactions in the reagent II, by using quinoneimine generated by reactions in the reagent I as a blank.

Description

The cyclophorase assay method of Triglycerides in Serum

Technical field

The invention belongs to a kind of assay method that comprises enzyme; Or utilize visible ray, and carry out the method for test material by the result generation change color of test reaction, particularly relate to a kind of cyclophorase assay method that detects Triglycerides in Serum with Biochemical Analyzer.

Background technology

The assay method of triglyceride in serum (TG) generally can be divided into chemical method, enzyme process and the large class of chromatography 3.The present invention introduces the triglyceride in a kind of enzymatic cycling assay for detecting serum, triglyceride (TG) generates glycerine and fatty acid under lipoprotein lipase (LPL) catalysis, glycerine and ATP generate glycerol-3-phosphate (G-3P) under glycerokinase (GK) catalysis, be brought in circular response, be oxidized to dihydroxyacetone phosphate (DHAP) by GPO (GPO), DHAP is reduced to again G-3P by glycerine-3 phosphate dehydrogenase (G-3PDH), simultaneously with NADH to NAD ⁺conversion, in iterative cycles, the amount of G-3P and DHAP is constant, and product H ₂o ₂the amount of accumulation is decided by reaction time and cpm, and therefore, its sensitivity substantially exceeds general enzymatic analysis, detects product H ₂o ₂can coupling peroxidase (POD), can react colorimetric estimation by Trinder.

Its theoretical derivation is as follows:

Assaying reaction is as Fig. 2.

If SV=1.5 μ is l, R ₁=200 μ l, R ₂=50 μ l, t ₁=3min, t ₂=5min, triglyceride range of linearity 11.00mmol/L, in serum, the high value of dissociative glycerin pathology is 0.60mmol/L.

According to the derivation of equation:

$v=\frac{V}{1+\frac{K_n^{\mathrm{TG}}}{\left[\mathrm{TG}\right]}+\frac{K_m^{H_{2}O}}{\left[H_{2}O\right]}}$

$v=\frac{V}{1+\frac{K_m^{\mathrm{TG}}}{\left[\mathrm{TG}\right]}}$

$v=\frac{V\left[\mathrm{TG}\right]}{K_m^{\mathrm{TG}}+\left[\mathrm{TG}\right]}$

Known if the TG range of linearity is 11.0mmol/L, SV=1.5 μ l, R ₁=200 μ l, R ₂=50 μ l, t ₁=3min, t ₂=5min.

$t=4.606K_m^{\mathrm{TG}}/V_{\max }+\left[\mathrm{TG}\right]/V_{\max }$

V _max＝381.6U/L

The concentration of LPL in reagent I is: 643.9U/L × 251.5/50=1919.45U/L

$v=\frac{V}{1+\frac{K_m^{\mathrm{ATP}}}{\left[\mathrm{ATP}\right]}+\frac{K_m^{\mathrm{FG}}}{\left[\mathrm{FG}\right]}}$

Known: $K_m^{\mathrm{ATP}}=1.3\×{10}^{-5},$ $K_m^{\mathrm{FG}}=9.4\×{10}^{-5}$

In first step reaction:

[FG]＝11.6mmol/L×1.5/1.5+200+50＝65.606×10 ^-6mol/L

[ATP]＝2×200/1.5+200+50＝1.59×10 ^-3mol/L

be about 122 times

Formula can be changed into:

$v=\frac{V}{1+\frac{K_m^{\mathrm{FG}}}{\left[\mathrm{FG}\right]}}$

$v=\frac{V\left[\mathrm{FG}\right]}{K_m^{\mathrm{FG}}+\left[\mathrm{FG}\right]}$

According to formula: $t=4.606K_m^{\mathrm{TG}}/V_{\max }+\left[\mathrm{TG}\right]/V_{\max }$

V _max＝100.4U/L

The consumption of glycerokinase is: 100.4U/L × 251.5/200=126.29U/L

GPO consumption:

[G-3-P]＝[FG]＝65.606×10 ^-6mol/L

According to formula: $t=4.606K_m^{G-3-P}/V_{\max }+[G-3-P]/V_{\max }$

V _max＝1211U/L

The concentration of GPO in reagent I should be: 1211U/L × 251.5/200=1523U/L

The consumption of glycerol-3-phosphate:

[NADH] configuration concentration in reagent I is 1.0mmol/L, is 1.59 × 10 in the concentration of reactant liquor ^-3mol, much larger than formula can calculate by single substrate reactions, according to formula

$t=4.606K_m^{\mathrm{DHAP}}/V_{\max }+\left[\mathrm{DHAP}\right]/V_{\max }$

The consumption of glycerol-3-phosphate is: 428U/L

In reagent I, concentration is: 428U/L × 251.5/200=538.2U/L, in circulation enzyme process, for making DHAP be converted into rapidly glycerine-3 phosphoric acid, toolenzyme consumption should be 3 × 538.2U/L=1615U/L

The consumption of peroxidase is:

In reagent I 2, the configuration concentration of 4-Dichlorophenol is 2.0mmol/L, and the concentration of 4-AA is 2.0mmol/L, due in reaction system much larger than Michaelis constant, therefore above formula can be changed into:

$v=\frac{V}{1+\frac{K_m^{\left[H_2{O}_2\right]}}{\left[H_2{O}_2\right]}+\frac{{K_m}^{\mathrm{ATP}}}{\left[\mathrm{ATP}\right]}+\frac{{K_m}^{4-\mathrm{CP}}}{[4-\mathrm{CP}]}}$

$v=\frac{V}{1+\frac{K_m^{\left[H_2{O}_2\right]}}{\left[H_2{O}_2\right]}}.$

$v=\frac{V\left[H_2{O}_2\right]}{K_m^{\left[H_2{O}_2\right]}+\left[H_2{O}_2\right]}$

Known wherein [H ₂o ₂]=69.18 × 10 ^-6mol/L

be greater than [H ₂o ₂] measure for end-point method, the reaction time is 2min

$t=4.606K_m^{\left[H_2{O}_2\right]}/V_{\max }+\left[H_2{O}_2\right]/V_{\max }$

The consumption of toolenzyme peroxidase is:

V _max＝120.56μmol/min.L＝103.19U/L

In reagent I, concentration is: 103.19U/L × 251.5/200=389U/L, in circulation enzyme process for making H ₂o ₂be converted into rapidly quinones, should make toolenzyme consumption reach 3 times, so toolenzyme consumption should be 3 × 389U/L=1167U/L.

Calculate toolenzyme and substrate consumption according to reaction equation and enzyme kinetics equation, enzyme activator and antiseptic, the formula of this kit is: every liter of Tris-HCl damping fluid of reagent I includes Tris100mmol～300mmol, 2,4-Dichlorophenol 1.0mmol～3.0mmol, MgSO ₄9.0mmol～15.0mmol, sodium taurocholate 3.0mmol～5.0mmol, atriphos 1.5mmol～2.5mmol, glycerokinase 100U～160U, GPO 1200U～2000U, peroxidase 1000U～1400U, 4-AA 1.5mmol～2.5mmol, glycerol-3-phosphate dehydrogenase 1200U～2000U, NADH0.8mmol～1.2mmol, Proclin-300 antiseptic 100 μ l～300 μ l; Every liter of Tris-HCl damping fluid of reagent II includes Tris100mmol～300mmol, lipoprotein lipase 1800U～2100U, TritonX-1000.08g～0.16g, Proclin-300 antiseptic 100 μ l～300 μ l.

What in Biochemical Analyzer detection serum, TG adopted is enzyme process, generally adopts the method (GPO-PAP method) of the reagent such as LPL, GPO, POD, 4-AA and phenol.The present invention adopts glycerine circulation enzyme process to measure, first dissociative glycerin carries out glycerine circulation enzyme process and measures in serum, produce red quinone imines, add after the second reagent, lipoprotein lipase hydrolyzing triglyceride produces glycerine, cyclophorase and substrate reactions in glycerine and reaction system, in reasonable time, measure, the quinone imines producing taking the first step is as blank, the quinone imines producing taking second step reaction is as assaying reaction, calculate the concentration of triglyceride, technical characterstic of the present invention is to be more suitable for trace detection, affected by piarhemia, jaundice, haemolysis little.

Summary of the invention:

In order to solve in prior art the problem that in serum, the assay method of TG exists endogenous glycerine to disturb, the invention provides a kind of economical convenient and easy, the serum triglyceride assay method that accuracy is higher, can eliminate the impact of endogenous glycerine.

The technical scheme that solves this technical problem employing is: two chromogen substances and cyclophorase coexist in reagent I, and reagent II only contains lipoprotein lipase effective constituent; Its assay method is: serum is first bathed 3～5 minutes in 37 DEG C of temperature with reagent I, and in serum, dissociative glycerin reacts with reagent I and generates quinone imines, adds reagent II to bathe 4～7 minutes in 37 DEG C of temperature, after triglyceride hydrolysis, generates red quinone imines through series reaction.Instrument detects at 500nm wavelength place, and the quinone imines reacting taking reagent I is as blank, and the quinone imines being produced by reagent II reaction calculates the content of triglyceride.

Every liter of Tris-HCl damping fluid of mentioned reagent I includes Tris200mmol, 2,4-Dichlorophenol 2.0mmol, MgSO ₄12.0mmol, sodium taurocholate 4.0mmol, atriphos 2.0mmol, glycerokinase 130U, GPO 1600U, peroxidase 1200U, 4-AA 2.0mmol, glycerol-3-phosphate dehydrogenase 1600U, NADH1.0mmol, Proclin300 antiseptic 200 μ l; Every liter of Tris-HCl damping fluid of reagent II includes Tris200mmol, lipoprotein lipase 1950U, Triton X-1000.12g, Proclin300 antiseptic 200 μ l.

In mentioned reagent I and reagent II, the pH value of Tris-HCl damping fluid is 7.6 ± 0.2.

In said determination, the volume ratio of reactant is: sample: reagent I: reagent II=3: 300～500: 30～70.

In reagent I of the present invention, contain glycerokinase, glycerol-3-phosphate, GPO, peroxidase, atriphos, NADH, 4-AA, 2, the effective constituents such as 4-Dichlorophenol; Reagent II only has lipoprotein lipase effective constituent.Dissociative glycerin glycerokinase in reagent I in serum, glycerol-3-phosphate, GPO, under Catalyzed Synthesis By Peroxidase with atriphos, NADH, oxygen, 4-AA, 2, the circular responses such as 4-Dichlorophenol generate red quinone imines, add after reagent II, after triglyceride hydrolysis, generate glycerine, carry out again circular response and generate red quinone imines, instrument detects at 500～520nm wavelength place, the quinone imines producing taking reagent I reaction is as blank, and the quinone imines being produced by reagent II reaction calculates the content of triglyceride.Not affected by endogenous glycerine, its using method is identical with original enzyme process with scope, is a kind of triglyceride detection method of trace detection preferably.

The present invention compared with prior art has the following advantages: not affected by endogenous glycerine, the range of linearity can reach 11.0mmol/L.Its using method is identical with original enzyme process with scope, can not increase experimenter's burden, does not increase reagent cost, economical convenient and easy, affected by piarhemia, jaundice, haemolysis etc. little, is the TG detection method that a kind of accuracy is higher.

Brief description of the drawings

Accompanying drawing is the response curve figure that the present invention measures healthy human body TG.

Fig. 2 is that triglyceride decomposes and glycerine circular response figure.

Embodiment:

Below by embodiment and accompanying drawing, the present invention is described in further details.

Embodiment 1

The composition of reagent:

A. reagent I:

Every liter of Tris-HCl damping fluid of reagent I includes Tris200mmol, 2,4-Dichlorophenol 2.0mmol, MgSO ₄12.0mmol, sodium taurocholate 4.0mmol, atriphos 2.0mmol, glycerokinase 130U, GPO 1600U, peroxidase 1200U, 4-AA 2.0mmol, glycerol-3-phosphate dehydrogenase 1600U, NADH1.0mmol, Proclin300 antiseptic 200 μ l.

B. reagent II:

Every liter of Tris-HCl damping fluid of reagent II includes Tris200mmol, lipoprotein lipase 1950U, Triton X-1000.12g, Proclin300 antiseptic 200 μ l.

In mentioned reagent I and reagent II, the pH value of Tris-HCl damping fluid is 7.6 ± 0.2.

C. titer: 2.0mmol/L trioleate aqueous solution.

Wherein, MgSO ₄for the activator of glycerokinase, Triton X-100 is surfactant, and Proclin-300 is efficient liquid antiseptic, and sodium taurocholate is bacteriostatic agent.

Embodiment 2.

Mensuration program

On the full-automatic Biochemical Analyzer of Japanese OLYMPUS AU2700, instrument automatically joins 1.5 μ l samples in 200 μ l reagent I and mixes, hatch 3 minutes for 37 DEG C, add 50 μ l reagent II to mix, hatch 5 minutes for 37 DEG C, fully-automatic analyzer detects at 500nm wavelength place, and instrument calculates TG result automatically, specifically in table 1.

Table 1 robotization Biochemical Analyzer of the present invention test condition

Reaction OD _tGcalculated value=OD ₂-OD ₁× [(SV+R ₁v ₁)/(SV+R ₁v ₁+ R ₂v ₂)]

Triglyceride concentration=F × OD _tG

Wherein OD _tGit is the absorbance that triglyceride produces.OD ₁that sample adds the absorbance recording after reagent I reaction, OD ₂be that sample adds the absorbance recording after reagent II reaction, SV is the volume of serum, R ₁v ₁the volume of reagent I, R ₂v ₂it is the volume of reagent II.F is correction factor.

Add OD after reagent II ₁extension rate be [(SV+R ₁v ₁)/(SV+R ₁v ₁+ R ₂v ₂)], add the absorbance after reagent II to be OD ₁× [(SV+R ₁v ₁)/(SV+R ₁v ₁+ R ₂v ₂)].So the absorbance of the quinone imines being produced by triglyceride is: OD _tG=OD ₂-OD ₁× [(SV+R ₁v ₁)/(SV+R ₁v ₁+ R ₂v ₂)], i.e. OD _tG=OD ₂-(1.5+200)/(1.5+200+50) × OD ₁.As long as measure OD ₁and OD ₂can calculate the concentration of triglyceride.

Below by adopting three kinds of diverse ways to detect the glycerine recovery in serum, further illustrate good effect of the present invention.The glycerine recovery is the percent of the glycerine numerical value of measuring after reaction and the ratio of the glycerine numerical value adding, the geometric ratio product that glycerine is triglyceride in this reaction, glycerine is measured the recovery does not affect triglyceride determination as best taking the glycerine adding, be that the glycerine recovery is lower, illustrate that glycerine is lower on triglyceride determination impact, glycerine does not count in triglyceride determination value.

1. detected object: health examination personnel 86 people, the wherein male sex 52 people, 42.5 years old mean age; Women 34 people, 39.5 years old mean age, on an empty stomach blood sampling.

2. employing method and reagent

2.1 reagent:

(1) method A: every liter of Tris-HCl damping fluid of reagent I includes Tris200mmol, 2,4-Dichlorophenol 2.0mmol, MgSO ₄12.0mmol, sodium taurocholate 4.0mmol, atriphos 2.0mmol, glycerokinase 130U, GPO 1600U, peroxidase 1200U, 4-AA 2.0mmol, glycerol-3-phosphate dehydrogenase 1600U, NADH1.0mmol, Proclin300 antiseptic 200 μ l; Every liter of Tris-HCl damping fluid of reagent II includes Tris200mmol, lipoprotein lipase 1950U, Triton X-1000.12g, Proclin300 antiseptic 200 μ l.

(2) method B: reagent I and reagent II were configured to single agents by 4: 1 and measure, measures wavelength 500nm, reaction time 8.1min.

(3) method C: high performance liquid chromatography adopts Shimadzu LC-10A high performance liquid chromatograph, mobile phase is the normal hexane containing 1% isopropyl alcohol and 0.5% acetonitrile, flow velocity 1.2mL/min detects wavelength 230nm.

(4) glycerine (analyze pure, sigma company, molecular weight 92.09, density is 1.2613mg/dl, 25 DEG C).

2.2. instrument: Japanese Olympus AU2700 type automatic clinical chemistry analyzer; Shimadzu LC-10A is high

Effect liquid phase chromatogram instrument.

2.3. method

2.3.1 measure 86 parts of healthy population triglyceride levels by reagent A, B, tri-kinds of methods of C respectively,

And carry out result comparison.

2.3.2 above-mentioned serum is hybridly prepared into pooled serum, adds respectively 0,0.85,1.70,3.40mmol/L glycerine, measure respectively the relatively recovery tests of two kinds of methods by reagent A, two kinds of methods of B.

2.3.3 assay method

Method A: sample: reagent I: reagent II=1.5: 200: 50,37 DEG C, sample and reagent I temperature were bathed 3 minutes, add reagent II, react 5 minutes, and 500nm wavelength place end-point method is measured.

Reagent B: sample: reagent=1: 100,37 DEG C, in 8.1 minutes reaction time, 500nm wavelength place end-point method is measured.

Method C mobile phase is the normal hexane containing 1% isopropyl alcohol and 0.5% acetonitrile, and flow velocity 1.2ml/min detects wavelength 230nm.Normal hexane layer direct injected in sample thief preparation, sampling volume 5 μ l while measuring total glycerine (TTG), 10 μ l while measuring dissociative glycerin.The concentration of standard solution is carried out to linear regression to glycerine/interior mark peak area ratio, obtain regression equation, calculate for sample glycerol concentration, the difference of total glycerine and dissociative glycerin is the value of real triglyceride.

3. three kinds of methods are measured TG comparison:

Pass through statistical analysis: employing method A and C measurement result there was no significant difference, t=0.97, P=0.4215, correlativity is good, r=0.9701, Y _{method A}=1.2031+0.9620X _{method C}; Method A can eliminate dissociative glycerin and disturb, and method B can not eliminate dissociative glycerin impact, in table 2; Employing method A and B measurement result have significant difference, t=9.65, P=0.0031.

Table 2. recovery experiment tables of data

In the inventive method, in the first step preincubate phase, dissociative glycerin is converted into quinone imines, adding after reagent II, starting TG hydrolysis reaction, TG is converted into quinone imines, response curve is shown in Fig. 1, the red quinone imines that instrument produces first step reaction is blank, and the quinone imines producing with second step reaction calculates the content of TG, is real TG content.

In method B single agents, dissociative glycerin and TG simultaneous reactions generate quinone imines, and result of calculation is dissociative glycerin and TG sum.High performance liquid chromatography can determine respectively the value of total glycerine and dissociative glycerin, not affected by dissociative glycerin.

Relatively can find out through above, reagent II of the present invention only has lipoprotein lipase, and other is reagent I, the accuracy that can ensure that by changing the component of reagent I, II Triglycerides in Serum detects.

Claims (5)

1. a cyclophorase assay method for Triglycerides in Serum, is characterized in that cyclophorase and substrate coexist in reagent I, and reagent II only contains lipoprotein lipase effective constituent; Its assay method is: serum is first bathed 3～5 minutes in 37 DEG C of temperature with reagent I, in serum, dissociative glycerin and reagent I carry out circular response generation quinone imines, after adding reagent II, bathe 4～7 minutes in 37 DEG C of temperature, after triglyceride hydrolysis, produce glycerine and generate quinone imines through circular response again; Reaction reaches after certain hour, and instrument detects at 500～520nm wavelength place, and the quinone imines producing taking reagent I reaction is as blank, and the quinone imines being produced by reagent II reaction calculates the content of triglyceride, and computing formula is:

OD _TG＝OD ₂-OD ₁×[(SV+R ₁V ₁)/(SV+R ₁V ₁+R ₂V ₂)]

Triglyceride concentration=F × OD _tG

Wherein OD _tGthe absorbance that triglyceride produces, OD ₁that sample adds the absorbance recording after reagent I reaction, OD ₂be that sample adds the absorbance recording after reagent II reaction, SV is the volume of serum, R ₁v ₁the volume of reagent I, R ₂v ₂be the volume of reagent II, F is correction factor.

2. the cyclophorase assay method of Triglycerides in Serum according to claim 1, is characterized in that the first step and second step reaction all adopt enzymatic cycling assay for detecting glycerine, generate red quinone imines and measure.

3. the cyclophorase assay method of Triglycerides in Serum according to claim 1, is characterized in that every liter of Tris-HCl damping fluid of reagent I includes Tris100mmol～300mmol, 2,4-Dichlorophenol 1.0mmol～3.0mmol, MgSO ₄9.0mmol～15.0mmol, sodium taurocholate 3.0mmol～5.0mmol, atriphos 1.5mmol～2.5mmol, glycerokinase 100U～160U, GPO 1200U～2000U, peroxidase 1000U～1400U, 4-AA 1.5mmol～2.5mmol, glycerol-3-phosphate dehydrogenase 1200U～2000U, NADH0.8mmol～1.2mmol, Proclin-300 antiseptic 100 μ l～300 μ l; Every liter of Tris-HCl damping fluid of reagent II includes Tris100mmol～300mmol, lipoprotein lipase 1800U～2100U, Triton X-1000.08g～0.16g, Proclin-300 antiseptic 100 μ l～300 μ l.

4. the cyclophorase assay method of Triglycerides in Serum according to claim 3, the pH value that it is characterized in that Tris-HCl damping fluid in reagent I and reagent II is 7.6 ± 0.2.

5. the cyclophorase assay method of Triglycerides in Serum according to claim 3, is characterized in that the volume ratio of measuring is: sample: reagent I: reagent II=3: 300～500: 30～70.