KIT FOR THE DETERMINATION OF L-CARNITINE IN BIOLOGICAL FLUIDS AND TISSUES

Abstract: A kit is described for the determination of L-carnitine in biological fluids and tissues, consisting essentially of; a) a first container containing Reagent 1, said Reagent 1 comprising HEPES, EDTA, acetyl-coenzyme A and DTNB; b) a second container containing Reagent 2, said Reagent 2 consisting of carnitine acetyl transferase. Said kit is conveniently presented with the reagents in lyophilized form.

Description: Kit for the determination of L-carnitine in biological fluids and tissues The invention described herein relates to a kit for the determination of L-carnitine in biological fluids. Background to the invention L-carnitine plays a fundamental role in metabolism, being a key element in the oxidation of long-chain fatty acids and thus in the production of energy. There are many pathological states the underlying cause of which is a deficiency of L-carnitine, and L-carnitine determination is necessary in order to establish the precise aetiology of the related disease processes. There has been a growing awareness among clinicians over recent years of the importance of L-carnitine deficiencies (Bieber et al. Fed. Proc. 41 , 2858 (1982); Stanley, Adv. Pediatr. 34, 59 (1987). One of the first methods used for determining L-carnitine (hereinafter called carnitine for short) was described by Marquis and Fritz in 1964 (Journal of Lipid Research 5, 184- 187). Various other methods have been described, such as, for example, those by Marzo et al., J. Chromatogr. 527, 247 (1990); and Hoppel, in: Hommes (ed.), Techniques in Diagnostic Human Biochemical Genetics, New York, Wiley-Liss, 309-326 (1991). The most widespread methods are based on the reaction of the enzyme carnitine acetyl transferase (CAT; EC 2.3.1.7): CAT L-carnitine + acetyl-CoA -> acetyl L-carnitine + CoA-SH. One very widespread method for the indirect determination of carnitine is based on the reaction of acetyl-Coenzyme A, released by the preceding reaction with 5,5'-dithiobis-2-nitrobenzoate (DTNB), which in turn releases the thiophenylate ion, which is determined spectrophotometrically at 412 nm (see, in addition to the above- mentioned Marquis, Seccombe D.W., Clinical Chemistry, 22, No. 10, 1589- 1592, 1976; Pearson, Methods of Enzymatic Analysis, 4, 1758- 1771 , 2nd edition - Bergmeyer; Casillas E.R. Biochimica et Biophysica Acta, 184, 566-577, 1969; Cederblad G. Clinica Chimica Acta, 33, 1 17. 123, 1971 ; Carrier H.N., Muscle & Nerve July/Aug 326-328, 1980). This method determines free carnitine. For the determination of the short-chain acyl carnitines, and thus of total carnitines, the sample is subjected to alkaline hydrolysis, converting the acyl carnitines to free carnitine. The known methods entail elaborate preparation phases, or use techniques which are only poorly conducive to automation of the analyses. For a discussion of the subject, see US Patent 5,316,917, filed in the name of Duke University, and incorporated herein for reference purposes. This patent aims to solve the problem of the automation of the analyses. This need is strongly felt in clinical laboratories that have to carry out large numbers of determinations. The Duke University patent offers the solution of an automated spectrophotometric method, consisting in the following steps: 1) addition of a plurality of samples of deproteinized biological fluids to a set of wells of a centrifugal spectrophotometric analyzer; 2) addition to each sample of: a) acetyl-Coenzyme A in an amount sufficient to react essentially with all the free carnitine in the sample to produce acetyl- carnitine and free Coenzyme A; b) DTNB in an amount sufficient to convert essentially all the Coenzyme A produced in the previous reaction to thiophenylate, and then c) simultaneously combine CAT, in each sample, in an amount sufficient to set off the reaction between acetyl-Coenzyme A and free carnitine and bring it to completion, and then d) determine the amount of thiophenylate present in the sample simultaneously by spectrophotometry. The kit used to implement the method described in the patent cited includes: a) a first container containing a solution of DTNB at a concentration ranging from 0.27 to 27 mmol/L, at a pH ranging from 6.5 to 8.5, and b) a second container containing a solution consisting of acetyl-Coenzyme A at a concentration ranging from 1.2 to 120 mmol/L, where the two solutions can be mixed together prior to use to form a solution containing DTNB at a concentration ranging from 0.23 to 23 mmol/L and acetyl-Coenzyme A at a concentration ranging from 0.17 to 17 mmol/1. In actual fact, the kit must also include a third container containing from 4 to 40 kU/L of CAT, for example in the form of a solution at a concentration ranging from 1.72 to 172 kU/L. It is envisaged that the third container should contain CAT in lyophilized form. Also envisaged in the kit are containers with carnitine standards in aqueous solution from 0.1 to 10 mmol/L and of octanoyl L-carnitine from 0.1 to 10 mmol/L. Also provided in the kit is a fourth container containing a 3-[N- morpholine]propanesulphonic acid hydrochloric solution, from 0.1 to 10 mmol/L. The execution of the procedure entails the preparation of 4 solutions: 1. DTNB; HEPES; EDTA at pH 7.5 2. MOPS-HC1 3. KOH In fact, the procedure described in the patent is called "three reagent chemistry". It is known that the solutions envisaged in the kit described above must be stored at low temperature, from 0 to 4°C, as prescribed in the patent cited. US Patent 6,027,690, a divisional patent of US Patent 5,872,008, filed in the name of Bair and Shug, provides a kit for the diagnosis of premenstrual syndrome, based on the determination of free and total carnitine in the blood. Apart from the specific indication of the method, to implement the method the kit consists of: a) a first container containing a solution of acetyl-Coenzyme A at a concentration ranging from 1.2 to 120 mmol/L, and b) a second container containing a solution consisting of DTNB or N-(p-2(benzimidazolyl)phenyl)maleimide at a concentration ranging from 0.27 to 27 mmol/L at a pH ranging from 6.5 to 8.5, where the two solutions can be mixed together prior to use to form a solution consisting of DTNB or N-(p-2(benzimidazolyl)phenyl)maleimide at a concentration ranging from 0.23 to 23 mmol/L and acetyl-Coenzyme A at a concentration ranging from 0.17 to 17 mmol/L In the execution of the procedure according to US patent 6,072,690, the method described in US Patent 5,316,917 can be adopted amongst others. A Boehringer Mannheim kit is commercially available for the determination of L-carnitine in plasma, seminal fluid, and urine. The determination is based on the following reactions: CAT L-carnitine + acetyl- CoA -> acetyl L-carnitine + CoA-SH. ACS CoA-SH + ATP ÷acetate - acetyl- CoA + AMP + PR MK AMP + ATP -=> 2 ATP + 2 pyruvate LDH 2 pyruvate + 2 NADH +2 H + -» 2 L-lactate + 2 NAD + The amount of NADH consumed during the reaction is equivalent to half the amount of L-carnitine. NADH is determined by absorbance at 334 (Hg), 340 or 365 (Hg) nm. The kit comprises: 1) 3 vials (1) each containing 0.7 g of a Coenzyme/buffer mixture consisting of Tris buffer pH 7.0, NADH 5 mg, ATP 6 mg, acetyl- Coenzyme A 4 mg, PEP 3 mg, magnesium acetate, and stabilizing agents. 2) Vial (2) containing approximately 3 ml of enzyme suspension consisting of acetyl-CoA synthetase (ACS), 2U approx., myokinase (MK), 160 U approx., lactate dehydrogenase (LDH), 240 U approx., and pyruvate kinase (PK), 240 U approx. 3) Vial (3) containing 0.2 ml of carnitine acetyl transferase (CAT) enzyme suspension, 60 U approx. 4) Vial (4) containing L-carnitine standard. 5) Vial (5) containing detergent solution. Prior to use, the contents of one vial (1) are diluted with 10 ml of distilled water and 1 ml is added from vial (5) . The other vials are used as such. The procedure consists in the addition of the sample (or standard) to solution (1), then addition of suspension (2), measurement of absorbance, then addition of suspension (3) and subsequent measurement of absorbance. Abstract of the invention It has now been found that it is possible to provide a kit for the determination of L-carnitine or short-chain acyl L-carnitines consisting of only two vials complete with all the necessary reactants, with the exception of the standards. Conveniently, the kits contain the reactants in lyophilized form. One of the advantages afforded by the realization of the invention described herein consists in the possibility of determining carnitine over a broad range of concentrations. The kit according to the invention comprises: a) a first container containing Reagent 1 , said Reagent 1 consisting of HEPES, EDTA, acetyl-Coenzyme A and DTNB; b) a second container containing Reagent 2, said Reagent 2 consisting of carnitine acetyl transferase. Therefore, one subject of the invention described herein is a kit for the determination of L-carnitine in biological tissues and fluids, its use for the determination of L-carnitine in biological tissues and fluids and methods for the determination of L-carnitine in biological tissues and fluids using the kit. According to the invention described herein, the kit permits the determination of L-carnitine in biological fluids and tissues. Examples of biological fluids and tissues are blood, plasma and seminal fluid. When suitably treated according to conventional techniques, other tissues can be analyzed with the kit according to the invention. Detailed description of the invention In a first preferred form, Reagent 1 consists of: HEPES 0.1 M, pH 7.5 EDTA 50 mM, pH 7 acetyl-Coenzyme A DTNB 10, 1 mM Preferably, Reagent 1 has the following composition per ml: HEPES 0.1 M, pH 75 0.652 mL EDTA 50 mM, pH 7 0.174 mL acetyl-Coenzyme A 0.087 mL DTNB 10.1 mM 0.087 mL The Reagent 1 container preferably contains 3 mL of Reagent 1. In a first preferred form, Reagent 2 consists of: carnitine acetyl transferase 0.500 mL If deemed appropriate, the kit may also contain other elements such as L-carnitine standards, other reactants for the preparation of samples, e.g. plasma or seminal fluid. Reagents 1 and 2 may also essentially contain, in addition to the basic reactants necessary for the reactions, i.e. acetyl-Coenzyme A, DTNB and carnitine acetyl transferase, additional substances useful for the analysis, or alternative substances to those contained in the kit capable of producing the same effect, e.g. buffers, chelating agents, complexing agents, pH correctors, preservatives, and stabilizing agents. Any changes or improvements to the kit supplied on the basis of the invention described herein, which substantially lead to the same result and afford the same advantages fall within the compass of the invention described herein. The preferred form of the kit is that in which the reagents are in lyophilized form. This form permits prolonged storage of the reagents in normal ambient conditions, without particular precautions, with obvious advantages from the point of view of the marketing of the kit. The lyophilization is done according to the conventional techniques used in the field and requires no further description. The kit according to a first embodiment of the invention described herein (3 mL of Reagent 1 and 0.500 mL of Reagent 2) is sufficient for approximately 13 determinations, if the manual method is used, or for approximately 60 determinations, if the automatic method is used. Both methods are described in detail here below. A first preferred embodiment of the invention described herein relates to the determination of carnitine in plasma. The invention described herein is based on the reaction that takes place between L-carnitine and acetyl-CoA in the presence of the enzyme carnitine acetyl transferase (CAT). The Coenzyme A (CoA) produced by this reaction reacts in turn with 5.5'-dithiobis-2- nitrobenzoate (DTNB), releasing thiophenylate ions (TNB ) which absorb at a wavelength of 412 nm, in the case of the use of a manual spectrophotometer, or at a wavelength of 405 nm, in the case of the use of an automatic-type spectrophotometer. The reactions involved are all quantitative, and therefore the TNB- absorbance value can be used to determine the carnitine concentration in the sample. The invention is illustrated with reference to a preferred form of its embodiment, namely for the determination of carnitine in plasma. It is understood that the principles involved in the invention described herein are equally applicable to other tissues, which are treated according to their nature, using conventional procedures which are a matter of general knowledge to people with average experience in the field. The determination method comprises the following steps: a) deproteinizing of the plasma sample in an acid milieu; b) neutralization of the sample; c) incubation. It is in step c) that the enzymatic reactions occur using the kit according to the invention described herein: CAT L-carnitine + acetyl-CoA - acetyl L-carnitine + CoA-SH CoA-SH + DTNB --> CoA-S-NB + TNB- + H + The amount of TNB" ions forming in the latter reaction is equivalent to that of the L-carnitine initially present in the sample. Ambient pH weakly influences the reactions and therefore the result of the analysis; in fact, at pH above 8.5 CAT is inactivated, whereas at pH values below 7 the dissociation of DTNB is incomplete, and therefore the L-carnitine values will be underestimated. As an alternative to steps a) and b) described above, the sample can be deproteinized by simple filtration. EXAMPLE 1 Preparation of solutions Acetyl-CoA 12.35 mM: solubilize 86 mg of acetyl-CoA in 8.6 mL of bidistilled water. This solution will be used to prepare Reagent 1. Carnitine acetyl-transferase (CAT): dilute the CAT with bidistilled water (1 volume of CAT + 1 1 volumes of bidistilled water). This solution will be used to prepare Reagent 2. If not used immediately, the reagent can be stored at -20°C and defrosted at the time of use. EDTA 50 mM pH 7.0: solubilize 2.2 g of EDTA in 50 mL of bidistilled water in a 100 mL calibrated flask. Bring the pH to 7.0 with glacial acetic acid and top up to volume with bidistilled water. The solution is stable for 6 months at 4°C. DTNB 10 mM: solubilize 34.4 mg of DTNB with 8.6 ml of Hepes 0.1 M at pH 7.5. Hepes 0. 1 M at pH 7.5: solubilize 2.38 g of Hepes in 50 mL of bidistilled water in a 100 mL calibrated flask. Bring the pH to 7.5 with potash 1.25 M, and top up to volume with bidistilled water. The solution is stable for 6 months at 4°C. Perchloric acid 2.75 M: dilute 23.2 mL of 70% perchloric acid in bidistilled water in a 100 mL calibrated flask. The solution is stable for 6 months at ambient temperature. Potassium carbonate 1.2 M: solubilize 16.6 g of potassium carbonate with 80 mL of bidistilled water. Mix and top up to volume with bidistilled water. The solution is stable for 6 months at ambient temperature. Potassium hydroxide 1.25 M: solubilize 8.25 g of potassium hydroxide with 80 mL of bidistilled water a 100 mL calibrated flask. Mix and top up to volume with bidistilled water. The solution is stable for 6 months at ambient temperature. Reagent 1 : prepare 100 mL of reagent: to 65.5 mL of Hepes 0.1 M at pH 7.5 add 17.3 mL of EDTA 50 mM, pH 7.0, 8.6 mL of DTNB 10 mM and 8.6 mL of acetyl- CoA 12.35 mM. The solution is stable for 6 months at -20°C. Deproteinizing of plasma /serum and neutralization of sample Transfer 300 μL of plasma/ serum and 40 μL of perchloric acid 2.75 M into a 1.5 ml Eppendorf tube. Mix well and then centrifuge at 10,000 x g for 5 min at 4°C. Transfer 250 μL of the supernatant and add 40 μL of potassium carbonate 1.2 M in a 1.5 ml Eppendorf tube. Mix and then leave the samples to digest in ice for 10 min. Centrifuge again at 10,000 x g for 5 min at 4°C. Remove the supernatant for analysis. Deproteinizin of plasma /serum by filtration Load the sample of plasma/ serum on AMICON type filters with cut-off ranging from 5000 to 20000 NMW. Load the sample (400 μL approx.) in an Eppendorf tube containing the filter, to be centrifuged at 15000 x g for 30-40 minutes at 4°C. Remove the filtrate for analysis. Enzymatic analysis A) Manual type spectrophotometry. Varian DMS-80 UV/VIS spectrophotometer Wavelength: 412 nm Cuvette: single-use, disposable, 1 cm optical path Temperature: 20-25°C Measurement: against air. Incubation: transfer to cuvette Blank (μL) Sample (μL) Bidistilled water 1000 750 Sample - 250 Reagent 1 230 230 Stir. After 5 minutes measure the absorbance of the blank and the sample (Ai). On completion of reading, add 20 μL of CAT (Reagent 2) to each test tube and mix. After another 10 minutes repeat the measurement of the blank and the sample (A 2 ). The concentration is calculated as follows: ΔA C= x F x 1000 ε x d where: C = concentration of sample (nmol/mL) ΔA = (A2-Al) samp le - (A2-Aι)blank ε = coefficient of molar extinction of TNB- (412 nm = 13.6 L x μmoH x cm) d = optical path length (cm) F = dilution factor (6.55) 1000 = conversion factor to obtain the result in nmol/mL. Example of calculation: Blank 0.169 0.172 0.003 Sample 0.170 0.242 0.072 Applying the formula indicated above: C = 33.2 nmol/mL. B) Automatic type spectrophotometer Before starting the analysis, dilute Reagent 1 and Reagent 2 5 times with bidistilled water (e.g., to l mL of Reagent 1 add 4 mL of bidistilled water; to 1 mL of Reagent 2 add 4 mL of bidistilled water) . Experimental conditions Procedure for programming the Roche mod. Cobas-Mira S automatic analyzer (software used N* 8347) FREE CARNITINE SW 884*7 COBAS MIRA S GENERAL MEASUREMENT MODE ABSORB REACTION MODE D-R-S-SRI CALIBRATION MODE LIN REGR REAGENT BLANK REAG/ SOL CLEANER NO WAVELENGTH 405 nm DECIMAL POSITION 2 UNIT μmol/L ANALYSIS DILUTION NAME H 2 0 FACTOR NO TIME NO STD: MAIN DIRECT MAIN STD 200 μmol/L POST DIL FACTOR 3 CONC. FACTOR NO SAMPLE CYCLE VOLUME 50 μL DILUTION NAME H 0 VOLUME 10 μL REAGENT CYCLE 1 VOLUME 250 μL START Rl CYCLE 7 VOLUME 40 μL DILUTION NAME H 2 0 VOLUME 10 μL CALCULATION SAMPLE LIMIT 0.1 POINT TI REAC. DIRECTION INCREASE CHECK ON CONVERS. FACTOR 1 OFFSET 0 TEST RANGE LOW ON HIGH ON NORMAL RANGE LOW YES HIGH NO NUMBERS OF STEPS 1 CALC. STEP A ENDPOINT FIRST READING 6 LAST READING 30 CALIBRATION CALIB. INTERVAL ON REQUEST BLANK REAGENT RANGE LOW -0.0001 HIGH 0.1000 BLANK RANGE LOW -0.0050 HIGH 0.0050 STANDARD POS: 1 1 : 200 μmol/L 2: 100 μmol/L 3: 75 μmol/L 4: 50 μmol/L 5: 30 μmol/L 6: 20 μmol/L 7: 10 μmol/L 8: 0 μmol/L REPLICATE DUPLICATE DEVIATION 10% CORRECTION STD NO CONTROL CS1* POS: 11 LOW 90 μmol/L ASSIGN 100 μmol/L HIGH 1 10 μmol/L CS2* POS: 12 LOW 40 μmol/L ASSIGN 50 μmol/L HIGH 60 μmol/L CS3* POS: 13 LOW 12 μmol/L ASSIGN 20 μmol/L HIGH 28 μmol/L Note: the samples called CS 1 , CS2 e CS3 refer to the reference standards and are prepared by scalar dilution of the mother solution of L-carnitine inner salt at a concentration of 200 μmol/L. The calculation of the concentrations of the blanks is done automatically by the analyzer which extrapolates them from a straight calibration line to be prepared fresh for each analysis session (possibly in duplicate). The above-described sample preparation and analysis conditions being equal, it is possible to calculate the carnitine concentration for an unknown sample immediately by multiplying its absorbance value by the factor 1028.382. In this case neither the calibration line nor the control samples will be used. Whenever the unknown samples have been deproteinized by acidification and subsequent neutralization with the procedure described above, the value of the concentration obtained must be multiplied by the factor 1.3146. Given here below, by way of an example, is a table directly correlating the L-carnitine concentrations with the absorbance readings. CALCULATION OF L-CARNITINE CONCENTRATIONS USING THE FACTOR 481 62 Abs μmo /L Abs μmob/L Abs μmoh/L Abs μmoli/L Abs μmoli/L Abs μmoh/L 0001 0482 0051 24563 0101 48644 0151 72725 0201 96806 0251 12089 0002 0963 0052 25044 0102 49125 0152 73206 0202 97287 0252 12137 0003 1445 0053 25526 0103 49607 0153 73688 0203 97769 0253 12185 0004 1926 0054 26007 0104 50088 0154 74169 0204 98250 0254 12233 0005 2408 0055 26489 0105 50570 0155 74651 0205 98732 0255 12281 0006 2890 0056 26971 0106 51052 0156 75133 0206 99214 0256 12329 0007 3371 0057 27452 0107 51533 0157 75614 0207 99695 0257 12378 0008 3853 0058 27934 0108 52015 0158 76096 0208 10018 0258 12426 0009 4335 0059 28416 0109 52497 0159 76578 0209 10066 0259 12474 0010 4816 0060 28897 0110 52978 0160 77059 0210 101 14 0260 12522 0011 5298 0061 29379 0111 53460 0161 77541 0211 10162 0261 12570 0012 5779 0062 29860 0112 53941 0162 78022 0212 10210 0262 12618 0013 6261 0063 30-42 0113 54423 0163 78504 0213 10259 0263 12667 0014 6743 0064 30824 0114 54905 0164 78986 0214 10307 0264 12715 0015 7224 0065 31305 0115 55386 0165 79467 0215 10355 0265 12763 0016 7706 0066 31787 0116 55868 0166 79949 0216 10403 0266 12811 0017 8188 0067 32269 0117 56350 0167 80431 0217 10451 0267 12859 0018 8669 0068 32750 0118 56831 0168 80912 0218 10499 0268 12907 0019 9151 0069 33232 0119 57313 0169 81394 0219 10547 0269 12956 0020 9632 0070 33713 0120 57794 0170 81875 0220 10596 0270 13004 0021 10114 0071 34195 0121 58276 0171 82357 0221 10644 0271 • 13052 0022 10S96 0072 34677 0122 58758 0172 82839 0222 10692 0272 13100 0023 11077 0073 35158 0123 59239 0173 83320 0223 10740 0273 13148 0024 11559 0074 35640 0124 59721 0174 83802 0224 10788 0274 13196 0025 12041 0075 36122 0125 60203 0175 84254 0225 10836 0275 13245 0026 12522 0076 36603 0126 60684 0176 84765 0226 10885 0276 13293 0027 13004 0077 37085 0127 61166 0177 85247 0227 10933 0277 13341 D028 13485 0078 37566 0128 61647 0178 85728 0228 10981 0278 13389 0029 13967 0079 38048 0129 62129 0179 86210 0229 11029 0279 13437 0030 14449 0080 38530 0130 62611 0180 86692 0230 11077 0280 13485 0031 14930 0081 39011 0131 63092 0181 87173 0231 11125 0281 13534 0032 15412 0082 39493 0132 63574 0182 87655 0232 11174 0282 13582 0033 15893 0083 39974 0133 64055 0183 88136 0233 11222 0283 13630 0034 16375 0084 40456 0134 64537 0184 88618 0234 11270 0284 13678 0035 16857 0085 40938 0135 65019 0185 89100 0235 11318 0285 13726 0036 17338 0086 41419 0136 65500 0186 89581 0236 11366 0286 13774 0037 17820 0087 41901 0137 65982 0187 90063 0237 11414 0287 13822 0038 18302 0088 42383 0138 66464 0188 90545 0238 11463 0288 13871 0039 18783 0089 42864 0139 66945 0189 91026 0239 11511 0289 13919 0040 19265 0090 43346 0140 67427 0190 91508 0240 11559 0290 13967 0041 19746 0091 43827 0141 67908 0191 91989 0241 11607 0291 14015 0042 20228 0092 44309 0142 68390 0192 92471 0242 11655 0292 14063 0043 20710 0093 44791 0143 68872 0193 92953 0243 11703 0293 14111 0044 21191 0094 45272 0144 69353 0194 93434 0244 11752 0294 14160 0045 21673 0095 45754 0145 69835 0195 93916 0245 11800 0295 14208 0046 22155 0096 46236 0146 70317 0196 94398 0246 11848 0296 14256 0047 22636 0097 46717 0147 70798 0197 94879 0247 11896 13297 14304 0048 23118 0098 47199 0148 71280 0198 95361 0248 11944 0298 14352 0049 23599 0099 47680 0149 71761 0199 95842 0249 11992 0299 14400 0050 24081 0100 48162 0150 72243 0200 96324 0250 12041 0300 14449

Claims: CLAIMS 1. Kit for the determination of L-carnitine in biological fluids and tissues, consisting essentially of: a) a first container containing Reagent 1 , said Reagent 1 comprising HEPES, EDTA, acetyl-Coenzyme A and DTNB; b) a second container containing Reagent 2, said Reagent 2 comprising carnitine acetyl transferase. 2. Kit according to claim 1 , in which said Reagent 1 consists essentially of: HEPES 0.1 M, pH 7.5 EDTA 50 mM, pH 7 acetyl-Coenzyme A DTNB 10.1 mM 3. Kit according to claim 1 , in which the composition of said Reagent 1 is as follows: HEPES 0.1 M, pH 7.5 0.652 mL EDTA 50 mM, pH 7 0.174 mL acetyl-Coenzyme A 0.087 mL DTNB 10.1 mM 0.087 mL 4. Kit according to claim 3, in which said first container contains 3 ml of said Reagent 1. 5. Kit according to anyone of claims 1 or 3-4, in which said container contains 0.500 ml of said Reagent 2. 6. Kit according to anyone of claims 1-5, for the determination of carnitines in plasma. 7. Kit according to anyone of claims 1-5, for the determination of carnitines in seminal fluid. 8. Kit according to anyone of claims 1-5, for the determination of carnitines in urine. 9. Kit according to anyone of claims 1-8, which additionally comprises containers containing carnitine standards. 10. Kit according to any of the foregoing claims, in which the reagents are in lyophilized form. 11. Use of the kit according to claims 1- 10 for the determination of L-carnitine in biological fluids and tissues. 12. Use according to claim 11, in which said fluid is selected from the group consisting of plasma, seminal fluid and urine. 13. Method for the determination of L-carnitine in biological tissues and fluids, characterized in that it uses the kit according to claims 1- 10. 14. Method according to claim 13, in which said fluid is selected from the group consisting of plasma, seminal fluid and urine.

Inventors: PACE SILVIA, PACE, SILVIA
Applicants: PACE SILVIA, SIGMA TAU IND FARMACEUTI, PACE, SILVIA, SIGMA-TAU INDUSTRIE FARMACEUTICHE RIUNITE S.P.A.
Application year: 2001
Main classification: C12Q1
IPC codes: C12Q1