CN102645414A

CN102645414A - Reagent kit capable of determining urine inositol

Info

Publication number: CN102645414A
Application number: CN2012100996297A
Authority: CN
Inventors: 李子樵; 朱雪青
Original assignee: SHANGHAI AILEX TECHNOLOGY Co Ltd
Current assignee: Ailex Technology Group Co ltd; Zhejiang Ailex Medical Co ltd
Priority date: 2012-04-06
Filing date: 2012-04-06
Publication date: 2012-08-22
Anticipated expiration: 2032-04-06
Also published as: CN102645414B

Abstract

The invention relates to a medicine reagent, in particular to a reagent kit capable of determining urine inositol. The reagent kit comprises a reagent R1 and a reagent R2, wherein the reagent R1 comprises buffer solution, 1.0 g/L of Thio-NAD+, 1.0 g/L of stabilizing agent and 0.5 g/L of preservative. The reagent R2 comprises buffer solution, 5-6 g/L of NADH, 50-100 KU/L of inositol dehydrogenase, 0.1-0.2 g/L of stabilizing agent and 0.5 g/L of preservative. Compared with the prior art, the reagent kit has the beneficial effects that the preparation is simple and convenient, no special instrument is required, the operation can be finished on a common fully-automatic or semi-automatic instrument in a clinical laboratory, the whole process only requires 10 minutes, the labor and the materials are greatly saved, and meanwhile, a large quantity of samples can be determined in the clinical.

Description

Measure the kit of urine inositol

[technical field]

The present invention relates to a kind of pharmaceutical reagent, be specifically related to a kind of kit of measuring the urine inositol.

[background technology]

Inositol in the human body mainly is present in (for tens of times in the blood) in the cell, is distributed widely in the human body with the form of free state or phosphatidylinositols constituent.Phosphatidylinositols decomposes through approach such as receptor activations, generates inositol-3-phosphoric acid, the effect of performance " second messenger " in cell.In addition, under diabetic disease states (hyperglycaemia), inositol content reduces (sorbierite increase) in the especially neural cell, finally can cause Na, the active decline of K-ATPase, causes nerve block.

Through the urine mysoinositol content behind the mensuration glucose load, can under AT state, detect the unusual of sugar tolerance.Had report in 1858 and show, exist the inositol of high concentration to drain (diabetic exceeds 10 times of normal persons) in diabetic's the urine, prompting can be used as one of index of sugar tolerance reduction.And; People's such as Hejin report shows; Before and after the 75g glucosieloading test, there is good correlativity in the blood glucose value behind the recruitment (Δ UMI) that the urine mysoinositol is drained and the glucose load, be can reflect on an empty stomach can't clear and definite glucose load after one of the good index of hyperglycemia state.In addition, the correlativity of inositol and diabetic keratopathy neurotrosis and nephrosis also more and more receives publicity.

At present, the detection method of inositol adopts high-efficient liquid phase technique (HPLC), vapor-phase chromatography (GC) or mass spectroscopy methods such as (MS) more, except that the expensive instrument of needs, and trivial operations, test speed is slow, and needs the professional and carry out operation.

[summary of the invention]

The present invention is in order to overcome the deficiency of prior art, and a kind of simple to operate, quick, sensitive, accurate kit of quantitative mensuration urine inositol is provided.

To achieve these goals, the present invention has designed a kind of kit of measuring the urine inositol, comprises reagent R1 and reagent R2, wherein:

Reagent R1 comprises damping fluid; Thio-NAD ⁺1.0g/L; Stabilizing agent 1.0g/L; Antiseptic 0.5g/L;

Reagent R2 comprises damping fluid; NADH 5-6g/L; Myo-Inositol dehydrogenase 50-100KU/L; Stabilizing agent 0.1-0.2g/L; Antiseptic 0.5g/L.

The pH value of said reagent R1 and reagent R2 is respectively 5-12.

Among the said reagent R1, damping fluid is Tris damping fluid 0.1mol/L or MES damping fluid 20g/L.

Among the said reagent R1, stabilizing agent is a kind of in bovine serum albumin(BSA), sweet mellow wine or the amino acid.

Among the said reagent R1, antiseptic is a kind of in sodium azide, thimerosal or the gentamicin.

Also comprise sequestrant 0.8g/L among the said reagent R2.

Among the said reagent R2, damping fluid is Tris damping fluid 0.1mol/L or GOOD buffering agent 10g/L.

Among the said reagent R2, stabilizing agent is a kind of in bovine serum albumin(BSA), sweet mellow wine or the amino acid.

Among the said reagent R2, antiseptic is a kind of in sodium azide, thimerosal or the gentamicin.

Described sequestrant is an ethylenediamine tetraacetic acid.

The present invention compares with prior art; Prepare simple, convenient; Need not specific apparatus, on the common full-automatic or semi-automatic biochemical analyzer of clinical labororatory, get final product complete operation, whole process only needs 10 minutes; Saved manpower and material resources significantly, making simultaneously clinically great amount of samples to be measured becomes possibility.The present invention is used to measure the enzyme circular response reagent of urinating mysoinositol, has advantages of higher stability, can be widely used in clinical diagnosis, and reduce the waste of reagent.

[description of drawings]

Fig. 1 measures the curvilinear trend figure of reagent absorbance after mixing for kit mysoinositol solution of the present invention and inositol.

Fig. 2 is kit measurement saccharification haemocyanin y=0.989x+0.726 of the present invention, R ²=0.992 o'clock the range of linearity.

Fig. 3 is kit measurement saccharification haemocyanin y=1.038x-1.634 of the present invention, R ²=0.991 o'clock the range of linearity.

[embodiment]

Below in conjunction with accompanying drawing the present invention is further described.

Embodiment 1:

Adopt the reagent of accomplishing after preparing to detect; Accuracy, precision and linear setting requirement have been reached; Consider stability and myo-Inositol dehydrogenase wherein, NADH instability under solution state that reagent stores; This product is processed the double reagent product, and reagent (R1) is 3:1 with the solution state volume ratio of reagent (R2), and concrete composition distribution and compound method are following:

Reagent R1:

Reagent R2 (during the freeze-dried powder state)

Reagent R2a (solution)

GOOD buffering agent 10g/L

Bovine serum albumin(BSA) 1.0g/L

NaN ₃ 0.2g/L

Reagent R2b (freeze-dried powder)

NADH 5g/L

Myo-Inositol dehydrogenase 50KU/L

Reagent R2 (during solution state)

After the composition of reagent (R1) and reagent (R2) distributed; Preparing according to the mode of liquid double reagent earlier, can obtain product, is freeze-dried powder like reagent (R2); Need reagent (R2a) is added in the reagent (R2b) before then using, form each constituent for setting reagent (R2) solution of concentration.The freeze-drying process of reagent (R2) is following:

Earlier solution is cooled to-40 ℃ from 25 ℃; This cooling was kept 4 hours; Guarantee that the solution temperature in the bottle is even, slowly be warmed up to 25 ℃ at (under the pressure of 20pa) under certain vacuum tightness then, this process need 3-4 hour; Under the vacuum tightness of 25 ℃ temperature and 20pa pressure, carry out drying then.Above process is controlled by the vacuum freeze-drying machine fully; Need about 37 hours, when the temperature of treating freeze-dried powder is consistent with the temperature of freeze dryer hothouse, represent that freeze-dried powder has reached abundant dry status; Dry run finishes; In vial, charge into nitrogen at last, cover bottle stopper, accomplish the freeze-drying of whole reagent R2.

Experiment one:

Equipment: Hitachi's 7170 type automatic biochemistry analyzers

Measure the reaction principle of reagent according to the urine inositol; Consider reagent sensitivity and inhale the influence relation and automatic clinical chemistry analyzer tankage and restriction at present between the appearance ratio, adopt sample: reagent (R1): reagent (R2)=12 μ L: 180 μ L: the ratio of 60 μ L makes an experiment.

At first adopt ultraviolet-visible pectrophotometer under the 405nm wavelength, to hatch following with 37 ℃; With certain density inositol with after the single reagent of preparation mixes according to reaction ratio; Carry out absorbance and change scanning; Can be observed inositol solution and after mixing, have tangible absorbance ascendant trend with inositol mensuration reagent, it is fixing that its slope keeps within a certain period of time, and the result is as shown in Figure 1.

Consider the surge time of mixed potential of hydrogen of double reagent and temperature, the absorbance of therefore on Hitachi's automatic biochemistry analyzer, having chosen the 6th minute to the 10th minute changes the content that detects inositol.

Below be the reaction system parameter of confirming:

Specimen types: human urine; Consumption: 12 μ L;

Reagent dosage: reagent (R1) 180 μ L, reagent (R2) 60 μ L;

Reaction conditions: urine 12 μ L; After mixing with reagent (R1), under 37 ℃, hatched 5 minutes, mixed and hatch 1 minute after adding reagent (R2) then; Next adopt the wavelength of 405nm to measure; METHOD FOR CONTINUOUS DETERMINATION 4 minutes was whenever measured 1 time at a distance from 1 minute, obtained absorbance pace of change (Δ A/min).

Calibration steps: the solution that the employing inositol is mixed with 100 μ mol/L is as calibration object.

When adopting the fresh patient of mentioned reagent mensuration to urinate the content of inositol, on Hitachi's 7170 automatic biochemistry analyzers, detect, as contrast, the result is as shown in Figure 2 with the HPLC method.

Among Fig. 2, result's regression curve is measured in the curve representative, and ordinate is mensuration result of the present invention, and horizontal ordinate is the mensuration result of HPLC method, and measurement unit is μ mol/L.

Statistics y=0.989x+0.726;

Measure routine number: 40 examples;

Coefficient R=0.9959, R ²=0.992;

Concrete parameter sees the following form:

The sample numbering	This method	HPLC
			1	69.52	68.15
2	63.09	61.18
			3	57.30	56.86
4	42.98	43.35
			5	48.02	49.28
6	49.90	51.90
			7	28.89	28.21
8	30.94	30.01
			9	45.05	44.47
10	70.44	71.06

11	37.65	36.99
			12	40.12	38.47
13	68.03	65.50
			14	55.48	53.30
15	47.00	48.14
			16	49.44	49.95
17	47.23	46.29
			18	31.89	31.03
19	40.45	41.68
			20	57.40	57.12
21	53.96	54.02
			22	58.47	58.80
23	41.51	40.12
			24	65.99	66.76
25	71.65	72.15
			26	39.60	39.98
27	45.65	45.37
			28	48.61	47.19
29	26.78	26.26
			30	57.31	56.76
31	23.52	22.92
			32	35.09	34.80
33	57.47	55.56
			34	30.02	31.01
35	48.06	46.57
			36	42.19	44.42
37	67.10	69.78
			38	32.31	32.15
39	62.64	63.33
			40	43.17	42.84

This shows, adopt enzyme round-robin method of the present invention to measure inositol, have good correlativity with the HPLC method.

Using inositol preparation mxm. is the linear standard items of 400 μ mol/L, and it is following to measure the result through mentioned reagent:

Linear standard solution (μ mol/L)	Measure result (μ mol/L)
		0.0	0.18
80.0	81.22
		160.0	159.27
240.0	245.88
		320.0	321.70

400.0

393.26

Among Fig. 3, ordinate is the concentration of linear standard solution of the present invention, the concentration value of horizontal ordinate for adopting this method to record, and measurement unit is μ mol/L, wherein R ²=0.991, this shows that adopt reagent of the present invention, the mensuration higher limit that can reach that the enzyme round-robin method is measured inositol is 400 μ mol/L.

Embodiment 2:

Reagent R1:

Reagent R2:

Use the assay method identical, result such as following table: (measurement unit: μ mol/L) with exemplifying embodiment 1

The sample numbering	This method	HPLC
			1	42.60	41.21
2	60.16	58.31
			3	47.60	46.85
4	32.25	33.44
			5	51.22	50.19
6	52.97	51.21
			7	45.29	47.35
8	67.11	65.17

9	27.68	27.16
			10	34.33	35.72
11	64.72	62.09
			12	30.15	32.88
13	71.26	69.92
			14	28.19	29.03
15	49.51	48.99
			16	58.29	59.10
17	52.32	52.85
			18	68.23	66.84
19	37.54	36.91
			20	51.70	52.57
21	58.25	58.59
			22	65.78	66.92
23	39.79	39.41
			24	48.17	47.10
25	57.21	56.55
			26	26.31	26.79
27	45.13	45.67
			28	72.89	71.67
29	41.96	40.78
			30	53.66	55.02
31	35.49	34.77
			32	30.01	31.60
33	42.71	44.29
			34	32.81	32.33
35	43.17	42.17
			36	62.09	60.12
37	60.22	61.55
			38	48.90	46.27
39	55.26	53.89
			40	23.57	24.90

Statistics: y=1.038x-1.634

Measure routine number n=40 example

Coefficient R=0.9956, R ²=0.9912.

Claims

1. a kit of measuring the urine inositol comprises reagent R1 and reagent R2, wherein:

2. the kit of mensuration urine inositol according to claim 1 is characterized in that: it is 5-12 in addition that the pH value of said reagent R1 and reagent R2 divides.

3. the kit of mensuration urine inositol according to claim 1 is characterized in that: among the said reagent R1, damping fluid is Tris damping fluid 0.1mol/L or MES damping fluid 20g/L.

4. the kit of mensuration urine inositol according to claim 1 is characterized in that: among the said reagent R1, stabilizing agent is a kind of in bovine serum albumin(BSA), sweet mellow wine or the amino acid.

5. the kit of mensuration urine inositol according to claim 1 is characterized in that: among the said reagent R1, antiseptic is a kind of in sodium azide, thimerosal or the gentamicin.

6. the kit of mensuration urine inositol according to claim 1 is characterized in that: also comprise sequestrant 0.8g/L among the said reagent R2.

7. the kit of mensuration urine inositol according to claim 1 is characterized in that: among the said reagent R2, damping fluid is Tris damping fluid 0.1mol/L or GOOD buffering agent 10g/L.

8. the kit of mensuration urine inositol according to claim 1 is characterized in that: among the said reagent R2, stabilizing agent is a kind of in bovine serum albumin(BSA), sweet mellow wine or the amino acid.

9. the kit of mensuration urine inositol according to claim 1 is characterized in that: among the said reagent R2, antiseptic is a kind of in sodium azide, thimerosal or the gentamicin.

10. the kit of mensuration urine inositol according to claim 6 is characterized in that: described sequestrant is an ethylenediamine tetraacetic acid.