EP1274858A1

EP1274858A1 - Method for determining the concentration of carnitine in fluids

Info

Publication number: EP1274858A1
Application number: EP01921009A
Authority: EP
Inventors: Fritz Pittner; Alfred Lohninger; Thomas Schalkhammer; Christian Mayer; Matthias Lepek; Claudia Micaela Dietrich-Ostry; Christian Vater; Alexander Piller
Original assignee: Befa Handelsgesellschaft mbH
Current assignee: Befa Handelsgesellschaft mbH
Priority date: 2000-04-17
Filing date: 2001-04-06
Publication date: 2003-01-15
Also published as: US20030162241A1; WO2001079530A1; AU2001248129A1

Abstract

The invention relates to a 'fluid injection analysis' (FIA) method for determining the concentration of carnitine in biological fluids. According to said method, the biological fluid containing carnitine is at least partially oxidized with a carnitine dehydrogenase (CDH) with the help of NAD+, at which point the concentration of at least one reaction product, in particular the concentration of NADH, is determined.

Description

Method for determining the concentration of carnitine in liquid substances

The invention relates to a method for determining the concentration of carnitine in biological liquids, in which the carnitine-containing biological liquid is at least partially oxidized with a carnitine dehydrogenase (CDH) with the aid of NAD ⁺ , whereupon the concentration of at least one reaction product, in particular the concentration of NADH, is determined.

Numerous clinical studies on carnitine metabolism suggest that secondary carnitine deficiency states are part of the clinical picture in numerous deficiency diseases. Lower carnitine levels can be found in every pregnancy, in premature babies, infections, stress and generally in a metabolism with increased lipid turnover. A simple method that can be carried out routinely and in which a corresponding analysis would be possible directly in routine laboratories or at a specialist has not been disclosed to date. Above all, the accompanying control during pregnancy could be made more efficient with such a simple methodology and, thanks to improved diagnostics, help to reduce the treatment costs in the area of neonatology and gynecology.

Carnitine (ß-hydroxy-γ-N-trimethylammoniumbutanoic acid) occurs ubiquitously in nature and is characterized by a number of essential functions in the intermediary metabolism. In particular, the carnitine system transports activated short, medium and long chain fatty acids in the entire cell area in the form of acyl carinitine esters. Long-chain fatty acids can only enter the mitochondria as carnitine esters, where they are oxidized for energy. Carnitine is also characterized by a regulatory role in controlling the ratio of acyl-coenzyme A to free CoASH. Fatty acid residues of coenzyme A are transferred through specific acyltransferases. Carnitine also plays an important role in detoxification, since poorly usable intermediates of the metabolism are accumulated as coenzyme A esters in the cells and can thus inhibit mitochondrial function. As carnitine esters, these compounds can be released by the cell and excreted by the kidneys. Finally, carnitine is of particular importance in the activation of immunocompetent cells and for the stability of many membranes as well as for membrane synthesis and repair, in particular the erythrocyte membrane.

The basic implementation of carnitine with a carnitine hydrogenase, in which an at least partial oxidation was carried out with the aid of NAD ⁺ and the reaction product, namely NADH, is subsequently determined, has already been described in the literature. In particular, reference is made to EP 437 373 AI. The corresponding scientific publications in CLIN. CHEM. 36/12, 2072-2076 (1990), the specific parameters to be observed for the determination can be found in detail and it can be seen in particular that the fluorometric determination with the required sensitivity can only be achieved in a known method if an amplifier is used , The reaction takes place with carnitine dehydrogenase and diaphorase, with resazurin being converted to resorufin in order to form a corresponding fluorescent dye with which the desired sensitivity is achieved. The proposed device for fluorometric measurement thus requires two reactors and, moreover, can only be addressed as linear in a narrow concentration range.

In addition to the additional effort for a second reactor, the additional reactants naturally represent further sources of interference in the determination. FR 2 596 865 A1 proposes a method for dosing carnitine, again using diaphorase and proposing an electrochemical determination. Electrochemical determinations are usually disturbed by a large number of redox-active substances, to which, for example, paracetamol, vitamin C or the like. count which are also found in the serum and can therefore impair the determination.

The invention now aims to provide a simple and rapid method by means of which quantitative determination of carnitine in the blood or in the serum is made possible with reduced expenditure on equipment. To achieve this object, the method according to the invention essentially consists in that the sample is injected into a carrier stream and is guided on the way to a detector via a CDH immobilized on a carrier, that aminosilanized glass bodies with controlled pore size (CPGs) before the immobilization of the CDH blocked with BSA at those sites where proteins can physisorb without activation, that the CPGs are rinsed with DTT solution before the immobilization of the CDH, that the CDH is stabilized by using a combination of DTT, NAD ⁺ and EDTA and that the Determination of the NADH concentration is carried out spectrophotometrically or directly by fluorescence measurement. The fact that the sample is only injected into a carrier stream suggests a simple measuring method which requires little equipment, the sensitivity being increased so far by the special measures in connection with the immobilization of the CDH and the preparation of the carrier can be that an immediate determination without the use of amplifiers or other auxiliary reagents succeed. In particular, the blockade with BSA that was carried out prior to the immobilization of the CDH leads to the immobilized enzyme being protected and stabilized against oxidation. BSA (Bovine serum albumin) is a protease and nuclease-free preparation of serum albumin. The addition of BSA on the one hand leads to a stabilization of the conformation of the CDH, but on the other hand both carboxyl and amino groups are made available by the BSA. The combination of CPG - Linker - BSA - CDH can increase the yield of the immobilization and thus more enzyme can be immobilized on the carrier, which enables the simple measurement method. The further in DTT (DiThioThreitol) used in the process according to the invention acts as a stabilizing reagent for proteins which carry SH groups. DTT reduces disulfide bridges and protects free SH groups. In the case of SH groups in the area of the active center, the addition of DTT has an effect above all by improving the catalytic activity. The catalytic activity improved in this way allows an immediate determination to be achieved in a particularly simple and rapid method, for example by spectrophotometric determination of the NADH concentration. If a fluorescence measurement is carried out within the scope of the method according to the invention, this can be carried out immediately with the aid of the improvement in sensitivity achieved and without the aid of further reactions.

L-carnitine dehydrogenase from Agrobacterium sp. Is preferred as the carnitine dehydrogenase. used, this L-carnitine dehydrogenase L-carnitine using NAD ⁺ to dehydro-camitin (ß-oxo-γ-N-trimethyl-ammoniurαbutanoic acid) oxidized. The reaction proceeds according to the following equation:

L- _C amitin ₊ NAD + Carnitindeh ^y dro ^q enase dehydrocamitin + NADH ₊ H ⁺

L-carnitine dehydrogenase requires NAD ⁺ as a coenzyme and has a molecular weight of 114 kD. The enzyme is highly specific for L-carnitine and NAD ⁺ and uses NADP ⁺ and the closely related substances D-carnitine, D, L-4-amino-3-hydroxybutyric acid, 3-hydroxybutyric acid, choline, tartrate, malate , Ethanol and isopropanol not. A weak (10%) side activity was only observed with D, L-Carnitinairdd.

There are 2 different forms of dehydrogenase, one with high and one with low affinity for carnitine. The K values for the forms with high and low affinity for L-carnitine are 0.29 and 6.1 mM (V _max 4.74 and 4.9 U / mol) and for NAD ⁺ 0.018 bwz. 0.042 mM. The enzyme is produced by detergents (cetyltrimethylammonium bromide and SDS) and by the heavy Metal ions Ag ⁺ , Hg ⁺ , Co ⁺ , Hg ²⁺ , Zn ²⁺ , Cu ⁺ , Mn ²⁺ , Ni ²⁺ , Pb ²⁺ as well as through D-carnitine, p-chloro mercuribenzonate, choline, hydroxy laminate, borate and Hydrazine hydrate inhibited and activated by magnesium and lithium. The active form of the enzyme is a dimer, which has an isoelectric point at 5.4. The forward reaction has an optimum around pH = 9, the reverse reaction at pH = 7. The enzyme is thermally stable over a long period of time up to 34 ° C, and has a temperature optimization of 45 ° C with a measuring time of 100 seconds. The NADH formed in the above reaction can be measured in a simple manner photometrically or directly flourimetrically, a significant increase in the absorption being observed. In principle, acetyl-carnitine can also be determined in the same way, provided that such acetyl-carnitine is first converted to carnitine. Carnitine transferases, esterases or lipases or basic hydrolysis can be used for the conversion of acetyl-carnitine to carnitine. In any case, the actual method of determination consists in measuring the concentration of NADH from the reaction of carnitine with carnitine dehydrogenase (CDH).

The NADH concentration is determined according to the invention by spectrophotometry or by fluorescence measurement. The spectrophotometric determination can preferably be carried out at about 340 nm, whereas the direct fluorescence measurement at wavelengths of 375 or 520 nm is particularly easy.

Compared to the previously known, relatively complex determination method in which acetyl-carnitine transferase, i. H. Acetyl-CoA-carnitine-O-acetyltransferase is used, the method according to the invention using CDH is characterized by a substantially smaller substrate spectrum of the enzyme and disorders such as those in the case of acetyl-carnitine transferases by the presence of choline or Tris-hydroxymethyl-methylglycine can be observed are avoided. CDH is therefore significantly superior for the specific determination of carnitine in the previously known methods with regard to the specific implementation of carnitine. The sensitivity can also can be increased significantly due to the specific enzyme, with significantly lower costs. In order to reduce continuous rinsing of the system with NAD ⁺ and the associated costs, the procedure is advantageously such that the sample is diluted with a measurement buffer containing NAD ⁺ , in particular Tris / HCl buffer, and brought into contact with CDH before the reaction with CDH becomes.

Another advantage of the method according to the invention, in addition to the higher specific sensitivity, is also the relatively short reaction time, so that reproducible signals can be achieved after a short time even if the conversion is not complete. While, for example, in "steady state" measurements, sample solutions are passed over the enzyme until a constant signal is obtained, according to the invention there is a much faster measurement method, which has become known as "fluid injection analysis" (FIA) , for use. Instead of an equilibrium measuring arrangement with a flow cell, an FIA measurement is therefore selected, in which it is no longer the entire area but only the height of the signal that provides direct information about the concentration. For this purpose, the procedure according to the invention is such that the sample is injected into a carrier stream and is guided on the way to a detector via a CDH immobilized on a carrier.

The use of CDH immobilized on a carrier represents a further simplification and improvement of the reproducibility of the method according to the invention, the immobilization of CDH being possible by observing special conditions. For this purpose, the procedure is such that the CDH is immobilized on aminosilanized glass, the immobilization of CDH on aminosilanized glass being accomplished in a particularly simple manner by means of 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide (EDC). Aminosilanized vitreous bodies with controlled pore size (CPGs) are used as carriers, which block those sites before the immobilization of the CDH with BSA on which proteins can physisorb without activation. The immobilization takes place by covalent binding of the enzyme, whereby the selected EDC coupling has proven to be a particularly easy to handle and efficient method of immobilization on the surfaces of aminosilanized glass with a controlled pore size. The vitreous with the immobilized enzyme can then be filled into columns and used in this way.

Carnitine dehydrogenase, like most enzymes, shows a decrease in activity over time. In order to keep the degradation of the carnitine hydrogenase activity as low as possible, it must first be ensured that the CDH is not already oxidized during the immobilization, for which purpose the CPGs are rinsed with DTT solution before the CDH is immobilized , Dithiotreitol (DTT) has also proven to be an excellent stabilizer for the enzyme CDH, and the procedure according to the invention is therefore such that the CDH is stabilized by using a combination of DTT, NAD ⁺ and EDTA.

In the context of the photospectrometric measurement or also the fluorescence measurement, the sensitivity can be increased further by adding dyes in the measuring cell of the detector.

In addition to the stabilizers DTT, NAD and EDTA (ethylenediaminetetraacetic acid) already described, the addition of protease inhibitors to stabilize the carnitine dehydrogenase activity has proven to be particularly advantageous.

Within the scope of the determination methods according to the invention, a precisely defined amount of the liquid sample is injected into a continuously flowing carrier stream and transported to a detector, the FIA method being used. In the drawing, the apparatus arrangement for implementing this methodology is explained in more detail. The sample first gets into one Autosampier 1 and is injected into a continuously flowing carrier stream and finally transported to a detector 2. On the way to the detector, the sample mixes with a flow buffer 3, whereby dye can also be added here. The use of a dye makes the measurement even more efficient and sensitive. The control of the sample or the carrier flow takes place via the pump 4 using injection valves 5, which are controlled fully automatically via software of a connected computer 6. After supplying NAD ⁺ buffer from the container 7, the sample reaches a reaction reactor 9 via the pump 8, which contains the CDH immobilized on a carrier. In the bioreactor 9, the carnitine is converted and a reaction product is formed, which can be measured. The conversion product NADH generates a signal in the spectrophotometer or detector 2, which can be made visible in the form of a peek on the monitor 10 of the computer 6. With fully automatic processing of the signals, the carnitine content can be displayed immediately.

With a reaction module, i.e. With a filling of vitreous bodies with immobilized CDH, almost a thousand determinations can be carried out, which corresponds to a service life of about 4 months in normal laboratory operation. The system can be re-calibrated at any time and can be operated without special knowledge after a short training period.

The term flow injection analysis (FIA) describes a technique in which a precisely defined amount of a liquid sample is injected into a continuously flowing carrier stream and is subsequently transported to a detector. The sample mixes on the way to the detector with the carrier solution surrounding it, the carrier solution either only serving to transport the sample to the detector or also containing reagents for converting the analyte. Depending on the requirements, the dispersion of the sample zone can be controlled by various parameters, and in particular by the type and size of a mixing loop, which is indicated by 11 in the drawing. is characterized, as well as by the diameter of the lines used or by changing the sample volume. In contrast to flow methods, the flow injection analysis produces reproducible signals in the form of peeks, although the mixing of the sample with the running buffer is not homogeneous and chemical reactions do not necessarily have to reach their equilibrium in the system sequence. The reproducibility of the FIA signals is based on the fact that the dwell time of the samples in the system and all parameters influencing the dispersion of the sample are kept constant.

Aminopropyl-methyldiethoxysilane in toluene was used for the aminosilanization of vitreous bodies with a controlled pore size. The immobilization on such aminosilanized CPG's, as mentioned at the beginning, is preferably carried out with l-ethyl-3- (3-dimethylaminopropyl) carbodiimide (EDC), which converts a carboxyl group into a very active ester intermediate, which then contains both amines (amide bond) and thiols (Thioester bond) react as well as can decompose again into a carboxyl group and inactive EDC by hydrolysis. Since CDH has proven to be relatively susceptible to oxidation, it is advantageous to largely rule out denaturing properties of the surface of the CPGs. Blocking the CPGs with BSA (Bovine Serum Albumin) and subsequent rinsing with DTT (dithiotreitol) are suitable for this purpose. Blocking occupies all of the CPGs' physisorbing sites with BSA, whereas the subsequent rinsing with DTT both washes away excess BSA and imparts reducing properties to each capillary. A protease inhibitor can also be used. With CPGs pretreated in this way, after immobilization of CDH within the scope of the measurement method according to the invention, reproducible measurements in the μmol / 1 range could be achieved after a relatively short lead time.

Claims

Claims:

1. A method for determining the concentration of carnitine in biological liquids, in which the carnitine-containing biological liquid is at least partially oxidized with a carnitine dehydrogenase (CDH) with the aid of NAD ⁺ , whereupon the concentration of at least one reaction product, in particular the concentration of NADH, is determined , characterized in that the sample is injected into a carrier stream and passed on the way to a detector via a CDH immobilized on a carrier, in that aminosilanized vitreous bodies with controlled pore size (CPGs) are blocked at those locations before the immobilization of the CDH with BSA, on which proteins can physisorb without activation, that the CPGs are rinsed with DTT solution before the immobilization of the CDH, that the CDH is stabilized by using a combination of DTT, NAD ⁺ and EDTA and that the determination of the NADH concentration is spectrophotometric or immediately by h fluorescence measurement is carried out.

2nd Method according to Claim 1, characterized in that the sample is diluted with a measurement buffer containing NAD ⁺ , in particular Tris / HCl buffer, before the reaction with CDH and brought into contact with CDH.

3. The method according to claim 1 or 2, characterized in that the immobilization of CDH on aminosilanized glass is carried out by means of EDC.

4. The method according to claim 2 or 3, characterized in that dyes are added in the measuring cell of the detector to increase the sensitivity.