CN101514984B - Reagent for determining serum free carnitine - Google Patents

Abstract

The invention provides a reagent for determining serum free carnitine. The reagent is a buffer solution which comprises the following components according to the content: 0.001 to 10g/L of thionicotinamide adenine dinucleotide, 0.01 to 10g/L of reducing coenzymedihydrocoenzyme 1, and 0.5 to 500KU/L of carnitine dehydrogenase. The enzymatic cycling reaction reagent for determining free carnitine has higher stability, can be widely applied to clinical in vitro diagnosis in vitro, and reduce the waste of the reagents.

Description

Measure the reagent of serum free carnitine

Technical field

The present invention relates to the detection by quantitative FC method of (FREE CARNITINE is called for short FCar), more specifically, relate to the reagent of FCar content in detection by quantitative human plasma or serum.

Background technology

Carnitine is that mediation lipid acid enters by mitochondrial membrane the important cofactor that plastosome carries out β-oxidation generation confession muscle and other cellular metabolism institute energy requirement.It is naturally occurring biostearin nutritive substance in a kind of human body.Its major function is that transhipment fat enters plastosome, promotes lipid acid burning, for body provides energy.Generally, carnitine shortage can cause skeletal muscle and cardiac muscle disorder, because these two kinds of tissues that muscle is all high energy consumption.The content that detects carnitine can be for organizing relevant disease to diagnose and treat support with this.In addition the carnitine of ephrosis and dialysis patient there will be metabolism disorder situation, to carnitine monitor can to clinical measure take provide support, in addition, some clinical meanings about carnitine are also in discovery.

The measuring method of having reported at present has high performance liquid phase (HPLC) method and mass spectrum (MS) method etc., and most susceptibilitys are inadequate.What have need to use radio isotope, easily causes radiocontamination.Some methods is complicated, takes longlyer, can not adapt to routine clinical use.

Summary of the invention

The object of this invention is to provide a kind of reagent of measuring serum free carnitine, the defect existing to overcome prior art.

Know-why: the carnitine in sample is at β-sulphur Reduced nicotinamide-adenine dinucleotide (Thio-NAD ⁺) oxidized form exist under by the specific dehydrogenation carnitine that is oxidized to of carnitin dehydrogenases (CDH), β-sulphur Reduced nicotinamide-adenine dinucleotide (Thio-NAD simultaneously ⁺) oxidized form changes at β-sulphur Reduced nicotinamide-adenine dinucleotide (Thio-NADH) reduced form.And dehydrogenation carnitine under β-Reduced nicotinamide-adenine dinucleotide reduced form (NADH) exists by the specific carnitine that is reduced into of carnitin dehydrogenases (CDH), β-Reduced nicotinamide-adenine dinucleotide reduced form (NADH) also changes into β-Reduced nicotinamide-adenine dinucleotide oxidized form (NAD simultaneously ⁺).So carnitine and dehydrogenation carnitine are at Thio-NAD ⁺with under the existence of NADH, by CDH, carry out the continuous accumulation that enzyme circulating reaction causes Thio-NADH, reach after balance, the growing amount of Thio-NADH and the concentration of the carnitine in sample are certain proportion, thereby can by measuring the formation speed of Thio-NADH, to carry out the FC of sample quantitative.And, Thio-NAD ⁺can participate in above-mentioned circulating reaction with NADH until exhausted state, sensitivity is very high.

Reagent of the present invention, comprises the buffered soln of the component of following content:

Thio-NAD+ (Thio-NAD+) 0.001g/L～10g/L

NADH (NADH) 0.01g/L～10g/L

Carnitin dehydrogenases (CDH) 0.5KU/L～500KU/L

Said buffered soln is not particularly limited, and can be the conventional buffer reagents such as Tris, phosphoric acid salt or GOOD ' S, and the pH value of solution is 4～9;

Preferably, said reagent can also comprise buffer reagent, sanitas, sequestrant, stablizer, tensio-active agent or proteinase inhibitor etc.;

Said sanitas is selected from microbiotic or the common cationic tensio-active agents such as sodium azide, Thiomersalate, gentamicin, paraxin, as cetyl trimethyl ammonia bromide or tetradecyl trimethylammonium ammonia bromide; ;

Said stablizer can select bovine serum albumin (BSA), glycerine, amino acid, metal ion, sequestrant as ethylenediamine tetraacetic acid (EDTA) (EDTA), tensio-active agent or proteinase inhibitor etc., concentration is not particularly limited, only otherwise affect normal reaction assay, preferably concentration is 0.1～5g/L;

Preferably, reagent of the present invention can be double reagent, and this reagent is divided into reagent (R1) and reagent (R2), also all components can be fitted over and forms together single reagent, also can be made into 2 above parts and form many reagent, but take double reagent mode as good;

In said double reagent, the component of reagent (R1) is for content being:

Thio-NAD+ (Thio-NAD+) 0.001g/L～10g/L;

NADH (NADH) 0.01g/L～10g/L;

The pH value of solution is 4-7

The component of reagent (R2) is for content being:

Carnitin dehydrogenases (CDH) 0.5KU/L～500KU/L

The pH value of solution is 8-9;

Reagent can be liquid reagent, or is freeze-dried reagent, and before using, water or certain damping fluid re-use after dissolving.

Reagent of the present invention can adopt the method method of conventional physical mixed to be prepared, and freeze-dried reagent also can adopt conventional method, by the liquid preparation of said ratio, carries out after freeze-drying, obtains product;

Reagent of the present invention, can be for FCar content in detection by quantitative human plasma or serum.

Reagent of the present invention, preparation simply, fast, does not need specific apparatus, on the common full-automatic or semi-automatic biochemical analyzer of clinical labororatory, gets final product complete operation, and whole process only needs 10 minutes, greatly saved man power and material, made clinical a large amount of samples are measured and become possibility simultaneously.Of the present invention have higher stability for measuring the enzyme circulating reaction reagent of FC, can be widely used in clinical in vitro diagnosis in vitro, and reduce the waste of reagent.

Accompanying drawing explanation

Fig. 1 is the contrast of this law and HPLC method

Fig. 2 is that the reagent that utilizes present method to measure is linear.

Embodiment

Embodiment 1

According to following formulated reagent:

After having prepared, detect, the accuracy of setting, precision and linear requirement have been reached, consider stability that reagent stores and wherein carnitin dehydrogenases, NADH are unstable under solution state, this product is made to double reagent product, the volume ratio of R1 and R2 reagent is 2: 1, and concrete composition distribution and compound method are as follows:

Reagent R1:

Tris damping fluid 12g/L

Thio-NAD+ 1g/L

BSA 1g/L

NaN ₃ 0.5g/L

Reagent R2:

Tris damping fluid 12g/L

BSA 1g/L

NaN3 0.2g/L

R2-B dry powder:

CDH 300KU/l

NADH 6g/L

After the composition of R1 and R2 reagent is distributed, first according to the mode of liquid double reagent, carry out the preparation of FC reagent, CDH in R2-B and NADH have been added in R2-A lysate according to setting concentration, obtained product.Or adopt following method to carry out freeze-drying, prepare freeze-dried preparation:

First solution is cooled to-40 ℃ from 25 ℃, this is cooling maintains four hours, guarantee that the solution temperature in bottle is even, then under certain vacuum tightness, (under the pressure of 20pa) is slowly warmed up to 25 ℃, this process need 3-4 hour, then at 25 ℃ and under the vacuum tightness of the pressure of 20pa, be dried, this process is controlled by vacuum-freeze-dry machine completely, approximately needs 37 hours.Deng the temperature of dried frozen aquatic products and the temperature of Freeze Drying Equipment kiln, when consistent, represent that dried frozen aquatic products has reached abundant drying regime, drying process finishes, and finally in vial, pours nitrogen, covers stopper, completes the freeze-drying of whole 2-B.Obtain freeze-dried preparation.

Embodiment 2

Application Example:

Equipment: Hitachi's 7170 type automatic biochemistry analyzers:

According to the reaction principle of FC detection reagent, consider reagent sensitivity and inhale the relation that affects and requirement and the restriction of automatic clinical chemistry analyzer reaction tank capacity at present between sample ratio, adopt sample: reagent 1: reagent 2=12 μ L: 200 μ L: the ratio of 100 μ L is tested.

First adopt ultraviolet-visible pectrophotometer under 405nm wavelength and 37 ℃ hatch under, after the FC single reagent of certain density FC and preparation is mixed according to reaction ratio, carry out absorbancy and change scanning, finding after FC and FC detection reagent are mixed has very significantly absorbancy ascendant trend, its slope is fixed, See Figure 1.

Therefore consider the surge time of the mixed acidity of double reagent and temperature, on Hitachi's automatic biochemistry analyzer, chosen the content that the absorbancy of the 6th minute to the tenth minute changes to detect FC.

Definite reaction system parameter below.

Sample collection and consumption: human serum, consumption: serum 12 μ L.

Reagent dosage: R1200 μ L, R2100 μ L.

Reaction conditions: serum 12 μ L, and after R1 reagent mixes, hatching after 5 minutes for 37 ℃, hatch one minute after adding reagent R2 to mix, and then adopt 405nm wavelength to start to measure, and METHOD FOR CONTINUOUS DETERMINATION 4 minutes, measured once every one minute.Ask absorbancy changing value (Δ A/min).

Calibration steps: the solution that is 50 μ mol/L with commercially available carnitine compound concentration is as standard substance.

Mentioned reagent is measured the content of the FCar in fresh patients serum, and in Hitachi, 7170 automatic clinical chemistry analyzers detect, and in contrast, result is as Fig. 1 for HPLC method:

In Fig. 1, curve 1 representation theory curve, curve 2 represents the regression curve of measurement result, and ordinate zou is measurement result of the present invention, and X-coordinate is HPLC measurement result, and measure unit is μ mol/L;

Statistics: y=0.961x+1.33

Measure number of cases=20

Correlation coefficient r=0.990

As can be seen here, adopt enzyme circulation method mensuration FC of the present invention and HPLC to there is good dependency.

With commercially available carnitine preparation maximum, be the linear criterion product of 200 μ mol/L, as follows by mentioned reagent measurement result:

In Fig. 2, ordinate zou is the concentration of linear criterion liquid of the present invention, the concentration of X-coordinate for adopting this law to record, and measure unit is μ mol/L;

R wherein ²=0.9997

As can be seen here, adopt reagent of the present invention, it is 200 μ mol/L that enzyme circulation method mensuration FC can reach determination of the upper limit.

Embodiment 3

According to following formulated reagent:

Use the method identical with embodiment 1 to measure, result is as following table:

Statistics: y=0.986x+0.08

Measure number of cases=20

Correlation coefficient r=0.990.

Claims (2)

1. measure the reagent of serum free carnitine, it is characterized in that, this reagent is the buffered soln that comprises the component of following content:

Thio-NAD ⁺thio-NAD+ 0.00lg/L-10g/L

NADH NADH 0.01g/L-10g/L

Carnitin dehydrogenases CDH 0.5KU/L-500KU/L

And the double reagent that described reagent is comprised of reagent R1 and reagent R2,

And described double reagent is selected from following (a) and (b) or (c) group:

(a) component of reagent R1 and content are:

The component of reagent R2 and content are:

(b) component of reagent R1 and content are:

The component of reagent R2 and content are:

Carnitin dehydrogenases CDH 300KU/L

NADH 6g/L；

(c) component of reagent R1 and content are:

The component of reagent R2 and content are:

2. reagent according to claim 1, is characterized in that, reagent is freeze-dried reagent.

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