CN1603822A

CN1603822A - Method for detecting phosphate radical in blood

Info

Publication number: CN1603822A
Application number: CN 200410088432
Authority: CN
Inventors: 阴彩霞; 杨频; 霍方俊
Original assignee: Shanxi University
Current assignee: Shanxi University
Priority date: 2004-11-03
Filing date: 2004-11-03
Publication date: 2005-04-06
Anticipated expiration: 2024-11-03
Also published as: CN1295509C

Abstract

This invention can qualitative and quantitative measure the phosphate radical content in blood by use of HEPES buffer solvent with PH value 6.5 to 7.5 and with pyrocatechol violet and ytterbium ion as color-developing agent. This method is free of interference of other contents and can be used in clinic.

Description

A kind of method that detects phosphate radical in the blood

Technical field:

The present invention relates to a kind of detection method of phosphate radical, specifically belong to a kind of method that detects phosphate radical in the blood.

Background technology:

Phosphorus is one of more element of content in human body, is only second to calcium.80 percent and calcium in conjunction with and be stored in bone and the tooth, be distributed in remaining 20 percent minute in the soft tissue such as nerve fiber, the inorganic phosphate in the blood only accounts for few part, constitutes the buffer system of blood.Containing the then possible situation of too much inorganic phosphate in the blood is: hypoparathyroidism, late period chronic nephritis, vitamin D picked-up too much, Huppert's disease or be in the union phase.Inorganic phosphate is low excessively in the blood, may have hyperparathyroidism, tubular degeneration pathology, rickets or diseases such as osteomalacia, parathyroid secondary hyperplasia, long-term diarrhea or malabsorption.Therefore, clinically, measure the conventional method of the index of inorganic phosphate in the blood as blood sample analysis.

About measuring the phosphate radical of blood, method both domestic and external in the past can reduce following several:

1, the use chromatography of ions detects the phosphate radical (L.Opliti, R.Chiaraluce, V.Consalvi, N.Cetulli and R.Scandurra, Clinica Chimica Acta, 184 (1989) 155-166) in the blood;

2, sequence injection immobilized enzyme method is measured the inorganic phosphate radical (M.D.Luque de Castro, RafaelQuiles.Juan M.Fern á ndez-Romero, and Ernesto Fern á ndez, Clin.Chem, 41/1 (1995) 99-102) in the blood;

3, use phosphate radical (Suzanne L.Tobey and Eric V.Anslyn, Organic Letter, Vol.5, No.12, (2003) 2029-2031) in the synthetic receptor determination blood;

But there are some problems respectively in these above methods:

1, chromatography of ions is measured the phosphate radical in the blood clinically because the narrow range of linearity needs dilute blood, just can go to measure.

2, sequence injection immobilized enzyme method is measured the inorganic phosphate radical in the blood, is subjected to reduce the interference of hydrogen peroxide material.

3, use phosphate radical in the synthetic receptor determination blood,, also can be subjected to the water miscible restriction of acceptor simultaneously, so bring many inconvenience to surveying work owing to need a large amount of synthetic works and need the screening acceptor.

Summary of the invention:

The object of the present invention is to provide a kind of simple, sensitive, fast, the method for phosphate radical in the detection by quantitative blood.

The invention provides a kind of method that detects phosphate radical in the blood, this method phosphate radical that to be developer based on ytterbium ion, with the pyrocatechol violet detect in pH is the HEPES buffer solution of 6.5-7.5 quantitatively in the blood, it is as follows that it specifically detects step:

1, get human blood sample, the centrifugal yellowish clear liquid in upper strata that gets was placed 2 hours on high speed tabletop centrifuge;

2, get the yellowish clear liquid in above-mentioned upper strata, change the ultrafiltration cup over to, in room temperature and 5 * 10 ⁵Use the PM-10 ultra filtration membrane ultrafilter ultrafiltration of molecular cut off under the condition of nitrogen gas of Pa, collect the colourless limpid liquid that leaches ,-17 ℃ to-20 ℃ preservations, for measurement as 10kD;

3, the HEPES buffer solution of preparation pH6.5-7.5, and prepare 2 * 10 ^-3The Yb of M ³⁺Solution and 2 * 10 ^-3The pyrocatechol violet solution of M;

4, the HEPES buffer solution of 2ml is added in the clean ultraviolet cuvette,, draws 2 * 10 with microsyringe as blank ^-3The pyrocatechol violet solution 10-100 μ l of M is added in this cuvette, and solution is detected on the UV, visible light spectrophotometer by colourless flavescence at this moment, and 443nm has absorption maximum;

5, in above-mentioned cuvette, add 2 times of molal quantitys to the Yb of pyrocatechol violet solution ³⁺Solution, solution is detected on the UV, visible light spectrophotometer by xanthochromia indigo plant at this moment, finds that maximum absorption band becomes 623nm by above-mentioned 443nm;

6, get above-mentioned colourless limpid liquid, be added in this cuvette with microsyringe gradually, application of sample limit, limit is detected on the UV, visible light spectrophotometer, adding along with limpid liquid, maximum absorption band is become again to 443nm gradually by 623nm again, and the color of solution is also returned yellow by blue stain gradually, when absorption peak reaches maximum at 443nm, during the complete flavescence of the color of solution, stop to add limpid liquid;

7, by [Yb ³⁺] * 2/V _BloodCalculate the content of phosphate radical in the blood.

Described Yb ³⁺Solution is YbCl ₃Solution or Yb (ClO ₄) ₃Solution.

Described Yb ³⁺Can use Nd ³⁺, Sm ³⁺, Gd ³⁺, Er ³⁺Or Ho ³⁺Replace.

The present invention has following advantage: 1, do not need synthetic any reagent, detect on the UV, visible light spectrophotometer, testing process is simple, and can with the naked eye obviously observe this effect; 2, our detection method has shown good selectivity to phosphate radical; 3, our detection is quantitative, the very big range of linearity is arranged, and error is very little; Obviously, this invention can be applied to the phosphate radical that detects clinically in the blood.

Description of drawings:

Fig. 1 to Fig. 8 is the UV, visible light absorption figure of phosphate radical in the detection blood of corresponding embodiment 1 to embodiment 8.

Embodiment:

Embodiment 1:

Get human blood sample, centrifugal that the yellowish clear liquid in upper strata was placed 2 hours on TGL-16B type high speed tabletop centrifuge; Get the yellowish clear liquid 6ml in above-mentioned upper strata, change 10ml ultrafiltration cup over to, in room temperature and 5 * 10 ⁵Use the PM-10 ultra filtration membrane of molecular cut off under the condition of nitrogen gas of Pa,, collect the colourless limpid liquid that leaches ,-17 ℃ to-20 ℃ preservations, for measurement with Amicon Model 8010 ultrafilter ultrafiltration as 10kD; HEPES (10mM) buffer solution of preparation pH7.0, and prepare 2 * 10 ^-3The YbCl of M ₃Solution and 2 * 10 ^-3The pyrocatechol violet solution of M; The HEPES buffer solution of 2ml is added in the clean ultraviolet cuvette,, draws 2 * 10 with microsyringe as blank ^-3The pyrocatechol violet solution 50 μ l of M are added in this cuvette, and solution is detected on the UV, visible light spectrophotometer by colourless flavescence at this moment, and 443nm has absorption maximum; In above-mentioned cuvette, add 2 * 10 ^-3The Yb of M ³⁺Solution 100 μ l, solution is detected on the UV, visible light spectrophotometer by xanthochromia indigo plant at this moment, finds that maximum absorption band becomes 623nm by above-mentioned 443nm; Get above-mentioned limpid liquid, be added in this cuvette with microsyringe gradually, application of sample limit, limit is detected on HP8453 UV, visible light spectrophotometer, adding along with limpid liquid, maximum absorption band is become again to 443nm gradually by 623nm again, and the color of solution is also returned yellow by blue stain gradually, when absorption peak reaches maximum at 443nm, during the complete flavescence of the color of solution, this moment, limpid liquid addition was 90 μ l; Calculate: 100 * 2/90, in the blood phosphate content be 2.22mmol/L, UV, visible light absorption figure sees Fig. 1.

Press embodiment 1 and survey 10 human bloods, in the blood phosphate content be 1.42-2.22mmol/L with the blood of statistics medically in inorganic phosphate content range (0.81-2.26mmol/L) surely close.

Embodiment 2: HEPES (10mM) buffer solution of preparation pH6.5, all the other are with embodiment 1, in the blood phosphate content be 2.22mmol/L, UV, visible light absorption figure sees Fig. 2.

Embodiment 3: with Yb (ClO ₄) ₃Solution, get another people's blood, all the other are with embodiment 1,100 * 2/125, in the blood phosphate content be 1.60mmol/L, UV, visible light absorption figure sees Fig. 3.

Embodiment 4: use ErCl ₃Solution, all the other all the other with embodiment 1,100 * 2/90, in the blood phosphate content be 2.22%, UV, visible light absorption figure sees Fig. 4.

Embodiment 5: use NdClO ₄) ₃Solution, all the other are with embodiment 1,100 * 2/90, in the blood phosphate content be 2.22mmol/L, UV, visible light absorption figure sees Fig. 5.

Embodiment 6: use SmClO ₄) ₃Solution, all the other are with embodiment 1,100 * 2/90, in the blood phosphate content be 2.22mmol/L, UV, visible light absorption figure sees Fig. 6.

Embodiment 7: use GdCl ₃Solution, all the other are with embodiment 1,100 * 2/90, in the blood phosphate content be 2.22mmol/L, UV, visible light absorption figure sees Fig. 7.

Embodiment 8: use HoCl ₃Solution, all the other are with embodiment 1,100 * 2/90, in the blood phosphate content be 2.22mmol/L, UV, visible light absorption figure sees Fig. 8.

Claims

1, a kind of method that detects phosphate radical in the blood is characterized in that, this method comprises following detection step:

(1) get human blood sample, the centrifugal yellowish clear liquid in upper strata that gets was placed 2 hours on high speed tabletop centrifuge;

(2) get the yellowish clear liquid in above-mentioned upper strata, change the ultrafiltration cup over to, in room temperature and 5 * 10 ⁵Use the PM-10 ultra filtration membrane ultrafilter ultrafiltration of molecular cut off under the condition of nitrogen gas of Pa, collect the colourless limpid liquid that leaches ,-17 ℃ to-20 ℃ preservations, for measurement as 10kD;

(3), the HEPES buffer solution of preparation pH6.5-7.5, and prepare 2 * 10 ^-3The Yb of M ³⁺Solution and 2 * 10 ^-3The pyrocatechol violet solution of M;

(4), the HEPES buffer solution of 2ml is added in the clean ultraviolet cuvette, as blank, with microsyringe absorption 2 * 10 ^-3The pyrocatechol violet solution 10-100 μ l of M is added in this cuvette, and solution is detected on the UV, visible light spectrophotometer by colourless flavescence at this moment, and 443nm has absorption maximum;

(5), in above-mentioned cuvette, add 2 times of molal quantitys to the Yb of pyrocatechol violet solution ³⁺Solution, solution is detected on the UV, visible light spectrophotometer by xanthochromia indigo plant at this moment, and maximum absorption band becomes 623nm by above-mentioned 443nm;

(6), get colourless limpid liquid, be added in this cuvette with microsyringe gradually, application of sample limit, limit is detected on the UV, visible light spectrophotometer, adding along with limpid liquid, maximum absorption band is become again to 443nm gradually by 623nm again, and the color of solution is also returned yellow by blue stain gradually, when absorption peak reaches maximum at 443nm, during the complete flavescence of the color of solution, stop to add colourless limpid liquid;

(7), by [Yb ³⁺] * 2/V _BloodCalculate the content of phosphate radical in the blood.

2, by the described a kind of method that detects phosphate radical in the blood of claim 1, it is characterized in that described Yb ³⁺Solution is YbCl ₃Solution or Yb (ClO ₄) ₃Solution.

3, by claim 1 or 2 described a kind of methods that detect phosphate radical in the blood, it is characterized in that described Yb ³⁺Can use Nd ³⁺, Sm ³⁺, Gd ³⁺, Er ³⁺Or Ho ³⁺Replace.