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L-Carnitine Enzymatic UV Test for the Determination of L-Carnitine in Research Samples from Seminal Plasma, Serum Or Urine

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L-Carnitine Enzymatic UV Test for the Determination of L-Carnitine in Research Samples from Seminal Plasma, Serum Or Urine For life science research only. Not for use in diagnostic procedures. L-Carnitine Enzymatic UV test for the determination of L-carnitine in research samples from seminal plasma, serum or urine. Cat. No. 11 242 008 001 y Version 20 Test-Combination for approx. 3 × 10 determinations Content version: February 2019 Store at +2 to +8° C 1. Product overview Application The test combination is intended as a research tool to help increase scientific knowledge about the physio- logical consequences of L-carnitine deficits. Kit Contents Bottle Contents Sample material Serum, urine and seminal plasma. 1 • 3 × 0.7 g coenzyme buffer/mixture. • lyophilisate Linearity The test is linear in the range of 5.6 - 112 ␮M L-carni- •consisting of tine in the test solution. • Tris buffer, pH 7.0; • NADH, 5 mg; Reference values • ATP, 6 mg; ␮ • Acetyl-coenzyme A, 4 mg; serum 6.9 mg/l or 43 M (3) • PEP, 3 mg; urine 28.2 mg/day or 175 ␮M/day (men), (5) • Magnesium acetate and stabilizers. 13.9 mg/day or 86 ␮M/day (women) 2a Enzyme Component A: ␮ • lyophilisate seminal 40-100 mg/l or 250-620 M (4,6,7, 8) • acetyl-CoA synthetase (ACS) > 2 U. fluid 2b Enzyme Blend B: • 3 ml enzyme suspension Storage and • The reagents are shipped at +15 to +25°C. • consisting of Stability • myokinase approx. 160 U; • Store at +2 to +8°C. The reagents are stable until the • lactate dehydrogenase approx. 240 U; expiration date printed on the label when stored • pyruvate kinase approx. 240 U. under these conditions. 3 • 0.2 ml enzyme suspension carnitine ace- Note: tyl transferase. • After reconstitution, the solution 1 is stable for 4 • approx. 60 U. days at +2 to +8°C. • Ready-to-use. 4 • L-carnitine standard solution. • Bring solution 1 to +15 to +25°C prior to use. • approx. 100 mg/l (exact concentration • After reconstitution, the suspension 2 is stable for 3 see bottle label). months at +2 to +8°C. • Ready-to-use. 5 • Detergent solution. • Ready-to-use. 2. Procedures and required material Principle (1) L-carnitine is acetylated to acetyl carnitine by acetyl coenzym A (acetyl CoA) in the presence of the enzyme 2.1 Before you begin carnitine acetyl transferase (CAT). The resulting coen- zyme A (CoA) is acetylated back to acetyl CoA in the Preparation of • To prepare solution 1, dissolve contents of one bot- presence of adenosine-5’-triphosphate (ATP) and ace- kit working tle 1 with 10 ml double dist. water. Add 1 ml from tate, catalyzed by the enzyme acetyl CoA synthetase solutions bottle 5 (detergent) and mix. (ACS). This results in the formation of adenosine-5’- • Before use keep solution 10 min at +15 to +25°C. monophosphate (AMP) and inorganic pyrophosphate • To prepare solution 2, dissolve ACS lyophilisate (PPi ). In the presence of ATP, supported by myokinase from bottle 2a with the enzyme suspension (bottle (MK), AMP forms twice the amount of adenosine-5’- 2b). diphosphate (ADP). This is converted in the following reaction with phosphoenol pyruvate (PEP) in the pres- Standard solution When analyzing the standard solution contained in the ence of pyruvate kinase (PK). The pyruvate formed is Test-Combination, the dilution factor F is 1. reduced to L-lactate by reduced nicotinamide adenine dinucleotide (NADH) in the presence of lactate dehy- The standard solution contains approx. 100 mg/l (exact drogenase (LDH). concentration see bottle label). The amount of NADH consumed during the reaction is equivalent to half the amount of L-carnitine. NADH is 2.2 Preparation of serum or plasma samples the parameter to be measured. It is determined on the basis of its absorption at 334 (Hg), 340 or 365 (Hg) nm. Additional For deproteinization of samples: CAT reagents • Perchloric acid, approx. 0.6 M, (1) L-carnitine + acetyl CoA acetyl carnitine required + CoA • Potassium carbonate solution, approx. 1.2 M (approx. 165 g K CO /l water). ACS 2 3 (2) CoA + ATP + acetate acetyl CoA + Procedure AMP + PPi Step Action MK 1 Remove blood from an uncongested vein and fill (3) AMP + ATP 2 ADP into a test tube which may contain EDTA. PK 2 Prepare serum or plasma in the usual way. (4) 2 ADP + 2 PEP 2 ATP + 2 pyruvate LDH (5) 2 pyruvate + 2 NADH 2 L-lactate + + 2 H+ 2 NAD+ 0219.11 268 341 001 : sigma-aldrich.com Deproteinization Serum or plasma has to be deproteinized as follows. 3. Standard protocol Step Action Additional equip- Glass cuvette: 1 cm path length. 1 Pipet into 10-ml centrifuge tubes: ment required Note: Disposable cuvettes may be used instead of • 1 ml Perchloric acid solution (0.6 M). glass cuvettes. • 1 ml ice-cooled serum or plasma sample. 2•Mix. Wavelength 340 nm, Hg 365 nm or Hg 334 nm. • Keep on ice for 10 min. Note: The absorption maximum of NADH is at 340 nm. • Centrifuge at 3000 × g (6000 rpm, r = 7 cm) With spectrophotometers, measurements are carried for 10 min. out at the absorption maximum; when working with 3 Pipet 1 ml of the supernatant into a new tube. spectral-line photometers equipped with a mercury Note: Supernatant might be slightly turbid. vapour lamp, measurements are carried out at 365 nm 4 Add 200 µl Potassium carbonate solution or 334 nm. (approx. 1.2 M). Protocol Measure against air (without a cuvette in the light 5 Mix and incubate on ice for 20 min. path) or against water. 6 Centrifuge at 3000 × g (6000 rpm, r = 7 cm) for 5 min. Temperature: +15 to +25°C Final volume: 2.205 ml 7 Transfer the supernatant into a new tube. Step Action Note: The separated supernatant is stable for 5 days at +2 to +8°C in a closed vial. 1 Pipet into cuvettes: Reagent Blank Sample Standard Dilution factor The calculated dilution factor is F = 2.34. (optional) ρ ( serum = 1.03 g/ml; liquid portion serum: 0.92). Solution 1 1 ml 1 ml 1 ml Sample -500 µl- (serum, 2.3 Preparation of urin samples plasma or untreated Protocol Use untreated urine for the test. urine)** Note: Urine samples stored at +2 to +8°C are stable Standard - - 100 ␮l for about 3 days. solution Step Action Suspension 100 ␮l 100 ␮l100 ␮l 1 Collect urine of 24 hours. 2 (bottle 2a + 2b) 2 Measure the total volume of the urine. Double dist. 1.1 ml 600 ␮l1 ml Dilutionfactor Urine is used undiluted, the dilution factor is F = 1.0. water **For deproteinized seminal plasma use 200 ␮l sample volume and add 900 µl double dist. 2.4 Preparation of seminal plasma samples water. Additional required • Perchloric acid, approx. 0.6 M. 2Mix. reagents • Potassium carbonate solution, approx. 1.2 M 3 Measure the absorbances of the solutions after 10 min (A ). (approx. 165 g K2CO3/l water). 1 4 Start the reaction by addition of 5 µl of Suspen- Protocol sion 3 (bottle 3). Step Action 5Mix. 1 Pipet into 10-ml centrifuge tubes: 6 Exactly 30 min after adding suspension 3 mea- • 0.5 ml Perchloric acid solution (0.6 M). sure the absorbances of the solutions in quick • 0.5 ml ice-cooled seminal plasma sample. succession (A2). 2•Mix. 7 Measure the absorbances again exactly 10 min • Keep on ice for 10 min later (A3). • Centrifuge at 3000 × g (6000 rpm, r = 7 cm) for 10 min. 3 Pipet 500 µl of the supernatant into a new tube. Absorbance Absorbance difference of the blank ⌬ Note: Supernatant, may be slightly turbid. difference = (A1 -A2)blank -3 × (A2 -A3)blank = ( Ablank) 4 Add 100 ␮l Potassium carbonate solution (approx. 1.2 M). Absorbance difference of the sample ⌬ 5 Mix and incubate on ice for 20 min. = (A1 -A2)sample - 3 × (A2 -A3)sample = ( Asample) 6 Centrifuge at 3000 × g (6000 rpm, r = 7 cm) for 5 min. Substract the absorbance difference of the blank from 7 Transfer the supernatant into a new tube. the absorbance difference of the sample to obtain ⌬A. ⌬ Note: The separated supernatant is stable for Note: Dilute the sample if Asample is higher than 5 days at +2 to +8° C in a closed vial. 1.100 or 0.600 (respectively measured at 340 nm, Hg 334 nm or Hg 365 nm). Calculation of the The deproteinization and neutralization procedures ␳ Because of the high L-carnitine concentration in semi- dilution factor F require a dilution factor F. The density ( = 1.035 g/ml) nal plasma, it is usually not necessary to increase the and the liquid portion of seminal plasma (98% = 0.98) sample volume. have to be taken into account in calculating this factor. If 0.5 ml seminal plasma, 0.5 ml perchloric acid solu- tion, 0.5 ml supernatant after deproteinization and 0.1 ml potassium carbonate solution are used, the dilution factor is: (0.5 × 1.035 × 0.98) + 0.5 0.6 F = × = 2.42 0.5 0.5 2 sigma-aldrich.com 4. Calculation Calculation of the For L-carnitine the concentration in food is: concentration c = 2.205 × 161.2 × ⌬A ⑀ × 1.0 × 0.1 × 2 General According to the general equation for reactions in which the amount of NADH consumed is equivalent to = 1777 × ⌬A half the amount of substrate, the concentration is cal- ⑀ culated by: c = V × MW × F × ⌬A (mg/l) = (mg L-carnitine/l sample solution) ⑀ × d × v × 2 If the sample has been diluted during sample preparation, the result must be multiplied by the V = final volume [ml] dilution factor F.
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