Determination Of Carnitine Research Articles

Quantification of Carnitine, Acetylcarnitine, and Total Carnitine in Tissues by High-Performance Liquid Chromatography: The Effect of Exercise on Carnitine Homeostasis in Man

A method for the quantitative determination of carnitine, acetylcarnitine, and total carnitine in tissue was developed for application to clinical research and diagnosis. Human skeletal muscle and heart specimens (10–20 mg) were homogenized in 1 ml of water. Aliquots of the resulting homogenates (50 μl) were extracted with 1.0 ml of acetonitrile:methanol (3:1) and the carnitine-related compounds were isolated using columns containing 300 mg of silica gel. Samples were then derivatized with 4′-bromophenacyl trifluoromethanesulfonate for spectrophotometric detection or 2-(2,3-naphthalimino)ethyl trifluoromethanesulfonate for fluorescence detection and quantified by high-performance liquid chromatography. Fluorometric detection of 2-(2,3-naphthalimino)ethyl ester derivatives afforded a 500-fold increase in sensitivity when compared to derivatization with 4′-bromophenacyl trifluoromethanesulfonate. This methodology permitted detection of acetylcarnitine in dilute human muscle homogenates at quantities of 790 fmol of acetylcarnitine injected. The method was applied to a series of human skeletal muscle biopsy samples obtained from subjects performing exercise at high work loads. The method permitted quantification of carnitine, acetylcarnitine, and total carnitine (sum of carnitine and all acylcarnitines) and demonstrated the specific redistribution of the carnitine pool from carnitine to acetylcarnitine with exercise above the lactate threshold. This HPLC method is facile, and provides a sensitive and specific approach for use in human biopsy specimens.

Disorders of mitochondrial long-chain fatty acid oxidation.

The oxidation of long-chain fatty acids requires a series of enzymes which are located in or on the mitochondrial membranes. These include carnitine palmitoyltransferases I and II, a carnitine-acylcarnitine translocase and, newly discovered, very long-chain acyl-CoA dehydrogenase and the mitochondrial trifunctional protein. These last two chain-shorten acyl-CoA esters to the point where they can be transferred to the more soluble medium- and short-chain-specific enzymes within the mitochondrial matrix. The disorders of long-chain fatty acid oxidation show a rather similar range of clinical and biochemical features, though with different emphasis in the different conditions. Patients with severe defects usually present early with acute attacks of hypoketotic hypoglycaemia and impaired liver function, or with cardiomyopathy or cardiac arrhythmia. In milder variants, skeletal myopathy with intermittent myoglobinuria develops later in life. 3-Hydroxyacyl-CoA dehydrogenase deficiency is unusual in producing peripheral neuropathy and retinitis pigmentosa. Treatment is based on the avoidance of fasting and replacement of normal dietary fat by medium-chain triglyceride, the medium-chain fatty acids entering the mitochondria in a carnitine-independent manner and bypassing the long-chain part of the spiral. Diagnosis must ultimately be based on direct assay of the enzyme involved, but preliminary indicators may come from determination of carnitine and intermediate metabolites in plasma, urinary organic acid profiling, and radioisotopic screening assays with lymphocytes or cultured fibroblasts.

Precolumn fluorescence derivatization of carnitine and acylcarnitines with 4-(2-aminoethylamino)-7-nitro-2,1,3-benzoxadiazole prior to high-performance liquid chromatography

4-(2-Aminoethylamino)-7-nitro-2,1,3-benzoxadiazole (NBD-ED) was synthesized as a precolumn fluorescence derivatization reagent for the high-performance liquid chromatographic determination of carnitine and acylcarnitines. Carnitine and acylcarnitines were reacted with NBD-ED (2.0 m M) and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (35 m M) in pyridine-dimethylformamide (1:4) at room temperature for 2 h. The NBD-ED derivatives of carnitine and acylcarnitines were separated by gradient elution with water-acetonitrile containing 10 m M trifluoroacetic acid on a reversed-phase column and the eluate was monitored with excitation at 470–485 nm and emission at 530–540 nm. Carnitine and sixteen acylcarnitines were determined within 45 min. The detection limits (signal-to-noise ratio = 3) for the carnitine-related compounds were 10–100 fmol. Four other NBD-alkyldiamines were also studied.

Determination of Carnitine and Saturated-Acyl Group Carnitines in Human Urine by High-Performance Liquid Chromatography with Fluorescence Detection

A high-performance liquid chromatographic method was developed for the determination of carnitine and 11 acylcarnitines (acetyl-, propionyl-, butyryl-, isovaleryl-, hexanoyl-, octanoyl-, decanoyl-, lauroyl-, myristoyl-, palmitoyl-, and stearoylcarnitine) in human urine. Urine samples were treated with a disposable cation-exchange column to extract carnitine and acylcarnitines using cyclopentanoylcarnitine as an internal standard. The extracted carnitine and acylcarnitines were reacted with 3-bromomethyl-6,7-dimethoxy-1-methyl-2(1H)-quinoxalinone as a fluorescence derivatization reagent and were separated and detected by high-performance liquid chromatography with gradient elution. Calibration curves gave good linearities at the individual assay range. Analytical recoveries were 70-76% for palmitoylcarnitine and stearoylcarnitine, and were more than 96% for carnitine and the 9 other acylcarnitines. The lower limits of quantitation were 103 nmol/ml for carnitine, 19 nmol/ml for acetylcarnitine, and 0.5-10.5 nmol/ml for other acylcarnitines. Precision and accuracy including within- and between-run were ≦ 19% and −17 ∼ +13% at the lowest concentrations of the individual assay ranges. The coefficient of correlation between this method and the radioenzymatic method was found to be 0.988 for the measured concentration of carnitine. We concluded that the present method can be used to measure carnitine and acylcarnitines in human urine.

Liquid chromatographic determination of carnitine by precolumn derivatization with pyrene-1-carbonyl cyanide

A method is described for the determination of carnitine chloride in pharmaceutical preparations by precolumn derivatization and high-performance liquid chromatography (HPLC). Carnitine chloride is derivatized by reaction with pyrene-1-carbonyl cyanide (PCC) in dimethyl sulphoxide. The PCC derivative of carnitine is then used for quantitative HPLC analysis on a cation-exchange column with fluorescence detection. The calibration graph was linear over a sample concentration range from 2.0 to 20 μg/ml; the limit of detection was 0.5 μg/ml under the conditions studied. The proposed method was applied satisfactorily to the determination of carnitine chloride in pharmaceutical preparations.

Fluorometric determination of carnitine in serum with immobilized carnitine dehydrogenase and diaphorase

A fluorometric flow-injection method for determining carnitine with use of immobilized enzymes carnitine dehydrogenase (EC 1.1.1.108) and diaphorase (EC 1.8.1.4) was developed and applied to the assay of carnitine in serum of patients treated with valproic acid. After fractionation and hydrolysis of carnitines in serum samples by perchloric acid and potassium hydroxide, liberated carnitine was converted to resorufin by immobilized carnitine dehydrogenase and diaphorase in the presence of beta-NAD+ (1.0 mmol/L), resazurin (12.5 mumol/L), and Tris acetate (0.6 mol/L, pH 9.0) at 37 degrees C. The fluorescence intensity of resorufin was monitored at lambda Ex 560 nm and lambda Em 580 nm. The calibration curve was linear for carnitine amounts from 0.1 to 1.0 nmol. Quantitative analytical recovery and satisfactory within- and between-run imprecision of carnitine in each carnitine fraction were obtained. Interference by bilirubin, serum albumin, and hemoglobin was negligible. Carnitine deficiencies were detected in about 20% of the valproic acid-treated patients (n = 198). The present method should be useful for monitoring carnitine deficiencies in clinical laboratories.

An improved high-performance liquid chromatographic assay for the determination of free and esterified carnitine in animal tissues.

A high-performance liquid chromatographic procedure for the determination of carnitine that is converted to coenzyme A(CoA) stoichiometrically by carnitine acetyltransferase is described. The chromatographic separation was accomplished by using Unisil QC8 column and a mixture of 190 mM KH2PO4 and methanol (87:13, v/v) as eluent. The present method is rapid, sensitive, and can be used for the routine analysis of animal tissues and plasma.

Use of anion-exchange resin in F - form in sample processing for determination of carnitine

Use of anion-exchange resin in F - form in sample processing for determination of carnitine

Gas chromatographic determination of carnitine in tissues

Carnitine [4-(N-trimethylamino)-3-hydroxybutyrate, (I)] is determined inhigh concentrations in skeletal musculature, heart, and epididymis, and in lower concentrations in the liver, brain kidneys, and blood [4, 7]. At the present time, it is known that (I) is an active carrier in the transport of fatty acids across the internal mitochondrial membranes to the sites of 8-oxidation [7]; (I) is also a most important factor in the conversion of acetyl CoA in the cells of the sex glands [5], and is a regulator of the catabolism of branched aminoacids in the skeletal musculature [3, i0]. Methods for the quantitative determination of (I) in the free and esterified form have been described [6], whereby the most sensitive and specific is the determination of the enzyme activity of carnitine acetyl transferase [8, 12] which requires work with the radioactive :~C-acetyl CoA. The gas chromatographic analysis of (I) is applied significantly less often [ii].

Determination of carnitine by high-performance liquid chromatography using 9-anthryldiazomethane

The high-performance liquid chromatographic determination of carnitine chloride was investigated by using 9-anthryldiazomethane (ADAM) as a pre-column derivatization reagent. Carnitine chloride and the internal standard N,N-dimethylglycine reacted with ADAM to give a stable ester derivative in the presence of sodium dodecyl sulphate (SDS) used to mask the basic function. The ADAM derivative of carnitine was separated from decomposition products of the reagent and related compounds such as amino acid derivatives on a silica gel column eluted with methanol-5% aqueous SDS-phosphoric acid (990:10:1). The calibration plot was linear over a sample concentration range from 0.02 to 100 ng per injection. The detection limit for carnitine chloride was about 1 pg per injection (signal to noise ratio = 4), by fluorometric detection.

Measurement of free and esterified carnitine in tissue extracts by high-performance liquid chromatography

A high-performance liquid chromatographic (HPLC) method for the determination of l-carnitine in clamped and frozen rat livers is described. L-carnitine + acetyl-CoA ⇆ acetyk-camitine + CoASH Using the above enzymatic reaction, release of CoASH is stoichiometric with the l-carnitine added. The present method has made possible the determination of carnitine in liver tissues, which is difficult by the conventional enzymatic spectrophotometric method using 5,5′-dithiobis(2-nitrobenzoic acid), owing to acetyl-CoA hydrolysis during prolonged incubations at pH 7.8.

Determination of carnitine, butyrobetaine, and betaine as 4′-bromophencyl ester derivatives by high-performance liquid chromatography

A method for determination of carnitine, 4-(N,N,N-trimethylammonio)butanoate (butyrobetaine), and 2-(N,N,N-trimethylammonio)acetate (betaine) is described. These ω-trimethylammonio carboxylates and the chemically analogous internal standards 4-(N,N-dimethyl-N-propylammonio)-3-hydroxybutanoate or 5-(N,N,N-trimethylammonio)hexanoate were derivatized by reaction wiht 4′-bromophenacyl triflate in the presence of N,N-diisopropylethylamine. The trialkylammonio carboxylate 4′-bromophenacyl ester derivatives were separated from other sample constituents by reversed-phase ion-pair high-performance liquid chromatography with spectrophotometric detection at 254 nm. Standard curves were linear over a sample concentration range of 10–100 nmol/ml. Quantities of 2.5 nmol of ω-trialkylammonio acid derivatives injected into the chromatography were detected with signal-to-noise ratios greater than 50.

Lipid storage myopathies. A review of metabolic defect and of treatment.

Various cases of lipid storage myopathies have been described. The biochemical defect could be determined in only some of these cases. The syndromes identified to date are as follows: carnitine deficiency (type I lipid storage myopathy), carnitine-palmityltransferase (CPT) deficiency and pyruvate-decarboxylase deficiency. In the last two diseases the vacuolization in muscle is not marked. The case of a 10 year old carnitine deficient patient with a history of insidious muscle weakness in the proximal limb and neck muscles is presented. The patient was treated with oral carnitine and a medium chain triglyceride diet for 18 months and her clinical status has remained improved. In other lipid storage patients prednisone treatment resulted in improvement. In cases of suspected lipid storage myopathy the following studies are indicated: 1) examination of ketone bodies in serum and urine during fasting, long chain and medium chain triglyceride diets; 2) serum triglyceride and serum carnitine; 3) study on fresh muscle and fibroblasts with labeled substrates, biochemical determination of carnitine and CPT in muscle.

Carnitine content of pea seedling cotyledons

Using a radioactive assay for the determination of carnitine, the amount in pea cotyledons was shown to vary with age.

Separation and determination of carnitine and its esters in human serum.

A colorimetric method for the determination of carnitine and its derivatives is described. Free carnitine, acetylcarnitine and palmitoylcarnitine were extracted with chloroform-methanol. After evaporation, the residue was dissolved in n-butanol and water; palmitoylcarnitine in the organic phase, and free carnitine and acetylcarnitine by passing the aqueous phase through a column of Amberlite CG-120 (H+). Separated carnitine derivatives were hydrolyzed to free carnitine which was followed by the Amberlite column chromatography. After eluting with 2N NH4OH solution, the samples were esterified and determined colorimetrically.

A method for the determination of carnitine in the picomole range

A method is described for the determination of L-carnitine in the range 20–2000 pmoles. Incubation of L-carnitine with [1- 14C]acetyl-coenzyme A and acetyl-CoA: carnitine O-acetyltransferase (EC 2.3.1.7) yields labeled acetylcarnitine. The formed acetylcarnitine is separated from labeled acetyl-coenzyme A by passing the reaction mixture through a column of the anion exchange resin Dowex 2-X8 and its isotope content determined in a liquid scintillation counter. Short-chain acyl derivatives of L-carnitine are substrates for the enzyme and will also be determined through exchange in the enzymic reaction. In 33 samples of human blood plasma the concentration of carnitine (+short-chain acyl carnitine) was 51 ± 10.5 μmoles/ l (mean ± S.D.). In biopsies of human skeletal muscle obtained at surgery the concentration was 12.8 ± 2.9 μmoles/g dry weight, in mouse skeletal muscle 1.73 ± 0.27 μmoles/g dry weight, and in mouse heart 4.66 ± 0.73 μmoles/g dry weight. The precision of the method expressed as relative standard deviation of duplicate analyses was 2–5%. The recovery of carnitine added to plasma or to perchloric acid extracts of muscle was 97 ± 12.5% and 103 ± 5.9%, respectively.

Excretion of l-carnitine in man

1. 1. The method for carnitine determination described by Marquis and Fritz has been adapted for determination of carnitine in human urine. 2. 2. The excretion of carnitine in normal men was 175 ± 80.7 μmoles per day (mean ± S.D.) and in women 86 ± 72.6 μmoles per day. The difference in carnitine excretion between men and women was statistically significant. 3. 3. Considerable inter- and intraindividual variations in carnitine excretion were noted. The carnitine excretion during the night was lower than during the day. 4. 4. After administration of carnitine (1520 μmoles of l-carnitine) to 3 subjects the additional excretion in urine over a 3-day period was 18, 43, and 66%.

Determination of carnitine in biological materials

A method has been developed whereby carnitine may be estimated quantitatively in the presence of a number of other compounds including choline and betaine. The method has been extended through the use of preparative procedures to the determination of carnitine in crude natural materials.