LT3876B - Method for the determination of triglycerides - Google Patents

Abstract

Triglyceride determination method is based on the determination of the concentration of triglycerides (neutral fats) by measuring the electrochemical parameters and may be used in medicine. The method is based on the hydrolytic, catalytic lipase reaction of triglycerides. The reaction product, the glycerol affected by the magnesium chloride and adenosine triphosphate containing a buffer solution of glycerokinase and glycerophosphate oxidation ferments immobilised in a lavsan membrane, undergoes phosphorylation and oxidation later at the same time evolving the hydrogen peroxide.

Description

The invention relates to the analysis of materials by means of enzymes which determine electrochemical parameters. Specifically, the invention relates to an improvement in the method of analyzing triglyceride neutral fat concentrations. The present invention may be used in medicine for diagnostic purposes (e.g., nephrotic syndrome or other disorders where triglyceride elevation is observed) / CnpaBonHHK. JIa6oparopHbie Mein ^ bi iiccaeųoBaniiH b KJiHHHKe.n.p. B.B. MeHbiHHKOBa. 1987, c. 246-247 /.

Triglycerides are a group of related glycerol esters consisting of glycerol and three fatty acid residues. The exact composition of fatty acid residues in natural fats can vary within certain limits. Therefore, the triglyceride assays available are based on glycerol determination, i. y. the triglyceride content obtained is not expressed in units of mass of the substance, but the number of triglyceride molecules is found by the amount of glycerol resolved by alkaline or enzymatic hydrolysis.

ClkOOCR,

I ^{2 1}

CIIOOCIC ch ₂ oocr ₃ ch ₂ oh

CHOH + fatty acids (Lh ₂ OH

These methods differ from each other in hydrolysis and glycerol determination methods.

An enzymatic method for the detection of triglycerides is known, which relies on phosphorylation of glycerol with phosphoenolpyruvic acid in the presence of ADP (adenosine diphosphate) and the enzymes glycerokinase and pyruvate kinase. The reaction produces pyruvic acid, which is quantitated spectrophotometrically by the amount of oxidized NADH (nicotinamide adenine dinucleotide) in the presence of lactate dehydrogenase / Cnp. JlačopaTopHbie Meroju.1 iiccjieųoBamiu b KJiHHHKe, n. p. B.B. MeHbniHKOBa. 1987. C. 246 - 247 /.

The disadvantage of this method is the use of three enzymes and three cofactors in their intake. during the process.

An enzymatic method is known in which a very specific enzyme, lipase, is used to hydrolyze triglycerides, and the amount of glycerol resolved is determined spectrophotometrically by the amount of oxidised dye produced during the next enzymatic reaction / Clin. Chem. 1982, v. 28, no. 10, p. 2077-2080 /.

Triglycerides Jd ► glycerol + fatty acids ^h 2 ^U

Glycerol + ATP gdcerokinase ^ i- _a -glycerophosphate + ADP MgCl ₂ glycerophosphatoxidase

1-a-Glycerophosphate + q -► dihydroacet- _TI 'Tonsphosphate ^{+ H} 2 ^U 2 ^ 2θ2 + dye Ρ ^£ -θ- · -— ^{az £} > oxidized dye + H ₂ O

Enzymes, substrates and sample are added to the measuring cell, so a certain amount of enzyme is used for each measurement.

The disadvantage of the method is that the assay must be performed in a transparent medium and also requires the use of high-cost enzymes.

Technologically, the closest is the enzymatic method in which the glycerol liberated by the enzyme lipase is determined by the polarographic / ana form of the NADH formed using a platinum anode. Letters, 1987, v. 20, no. 5, p. 783-790 /.

Triglycerides ^H 2 °

Glycerol + NAD * Glycerol + Fatty Acids + Glycerodehydrogenase Dihydroxy * Acetone + NADH + H ⁺

Add lipase and test sample dissolved in buffer to the measuring cell, and immobilize the glycerol dehydrogenase on a semipermeable halogen membrane by adding NAD ⁺ (nicotinamide adenine dinucleotide) and adjusting the concentration of NADH to the same as the triglyceride.

This method is quite sophisticated and does not exhibit high precision in the determination of triglycerides in biological fluids, and also consistently adds NAD ⁺ , a very expensive coenzyme for many enzymes, and determines triglycerides from the reduced amount of coenzyme NAD-H.

The object of the invention is to simplify the process and increase its sensitivity.

The essence of the way is revealed in the following diagram. Triglycerides are hydrolyzed by the enzyme lipase to glycerol, which is phosphorylated and further oxidized by the action of immobilized semi-permeable membrane glycerokinase and glycerophosphatoxidase enzymes in the presence of ATP (adenosine triphosphate) buffer. This results in H2O2 at a concentration proportional to the triglyceride content in the sample analyzed and can be determined from the anode current increase by oxidizing H2O2 at an anode potential of + 0.6 V:

Triglycerides ► Glycerol + fatty acids h ₂ o

Glycerol + ATP Glycerokinase * 1-a-Glycerophosphate + ADP

1-a-Glycerophosphate + o, glycerophosphatoxidase dihydroace-, _{γγ o} ° 2 tonphosphate ^{+ H} 2 ° 2

electrode + 0.6V> H ₂ O + 10 ₂ + 2e '

The assay is performed using an electrochemical hydrogen peroxide sensor consisting of a platinum anode coated with an enzymatic microporous lavash membrane with immobilized glycerokinase and glycerophosphatoxidase enzymes and a reference Ag / AgCI electrode. Introduce into the measuring cell a lipase and test sample dissolved in ATP and MgCl2 buffer solution. The anode current measurements are then carried out to deduce the triglyceride concentration.

The method is illustrated by examples.

Example I. Determination of triglycerides in olive oil.

The enzyme membrane is prepared as follows: 0.5 ml of 0.01 M phosphate buffer (pH 7.2) is dissolved in 2.5 units. glycerokinase, 3 pcs. glycerophosphatoxidase and 1.5 mg bovine serum albumin. 10 ml of 5% glutaric aldehyde are added to the resulting solution and the solution is poured onto a 4 cm ² microporous lavender membrane which is covered with an acetylated cellulose membrane.

The enzyme-immobilized polarographic transducer is placed in a 1-ml cell with 3 mM ATP and 3 mM MgCl 2 in buffer and the background electrode current is determined. Introduce 0.1 ml of olive oil suspension in phosphate buffered saline (0.01 M, pH 7.2) and 5 units. lipases. After 30 minutes incubation determines stationary electrode current. The results of the assays are shown in Table 1 and in the calibration graph 1, respectively. The triglyceride concentration in olive oil is subtracted from the encyclopedia / XHMHqecKHH 3nu, HKJionexuiqecKHH cjioBapb. 1983, c. 407 / Triglycerides (mainly trioleatglycerides, M = 885) containing 80% olive oil.

table

Of olive oil concentration in a cuvette, g / 1 0 0.18 0.34 0.7 1.10 1.38 1.66 2.8 Triglycerides concentration, mM 0 0.16 0.31 0.63 1.0 1.25 1.5 2.5 Stationary current, nA 0 0.44 1.54 4.4 7.26 9.9 11.22 12.54

As shown in Table 1 (calibration graph 1), the linear dependence of the electrode current is simply proportional to the triglyceride concentration up to 1.5 mM

The membrane with immobilized enzymes can be used for more than two weeks and refrigerated for up to 6 months.

The linear dependence of the electrode current is also proportional to the concentration of other materials, i. y. glycerol and ATP formed during reactions. Examples 2 and 3 confirm this.

Example II. Determination of glycerol in buffer.

The enzyme electrode and lattice are prepared according to Example I. The cell buffer is added to dissolved therein 3 mM ATP and 3 mM MgCl ₂ and sets the background electrode current. Introduce glycerol buffer into the cuvette and determine the steady state current of the electrode. The results are shown in Table 2 and Calibration Chart II, respectively.

table

Glycerin concentration in the cuvette, mM 0 0.05 0.15 0.2 0.25 0.3 0.35 0.4 0.45 0.5 0.55 0.7 Electrode current, nA 0 2.64 6.6 10.56 13.42 16.06 16.5 18.26 18.92 19.58 20.24 21.12

As shown in Table 2, the linear dependence of the electrode current is proportional to glycerol concentration up to 0.3 mM.

UI Example. Determination of ATP in buffer.

The enzyme electrode and lattice are prepared according to Example I. A buffer containing 3 mM MgCE and 2 mM glycerol was added to the well and background electrode current was determined. The ATP solution is then introduced into the lattice and the stationary current of the electrode is determined. The results are shown in Table 3 and the calibration graph ΠΙ, respectively.

table.

ATP concentration, mM 0 0.5 1 1.5 2 2.5 3 3.5 4 Electrode current, nA 0 5.5 8.58 10.78 12.98 13.42 14.52 14.96 13.86

As can be seen from the data, the linear dependence of steady-state electrode current on ATP concentration is simply proportional to ATP concentration up to 1.5 mM.

The advantage of this electrochemical triglyceride determination method is that it does not require the continuous addition of expensive NAD ⁺ and the triglyceride determination according to

NADH oxidation - reduction reaction. All NAD-dependent dehydrogenase systems present in the test biological media will not interfere with the assay. The method is much more accurate and faster.

Claims (1)

DEFINITION OF INVENTION The method for determining triglycerides is based on depositing the test sample with an enzyme lipase into an electrochemical lattice filled with a buffer containing a platinum anode with a semipermeable enzymatic membrane, then measuring the anode current to measure the triglyceride concentration, b. characterized by the addition of adenosine triphosphate (ATP) and MgCl 2 to the buffer solution, and the platinum anode is coated with a lavender membrane with immobilized glycerokinase and glycerophosphatoxidase enzymes.