Aging skeletal muscle mitochondria in the rat: decreased uncoupling protein-3 content.

The goal of the present study was to discern the cellular mechanism(s) that contributes to the age-associated decrease in skeletal muscle aerobic capacity. Skeletal muscle mitochondrial content, a parameter of oxidative capacity, was significantly lower (25 and 20% calculated on the basis of citrate synthase and succinate dehydrogenase activities, respectively) in 24-mo-old Fischer 344 rats compared with 6-mo-old adult rats. Mitochondria isolated from skeletal muscle of both age groups had identical state 3 (ADP-stimulated) and ADP-stimulated maximal respiratory rates and phosphorylation potential (ADP-to-O ratios) with both nonlipid and lipid substrates. In contrast, mitochondria from 24-mo-old rats displayed significantly lower state 4 (ADP-limited) respiratory rates and, consequently, higher respiratory control ratios. Consistent with the tighter coupling, there was a 68% reduction in uncoupling protein-3 (UCP-3) abundance in mitochondria from elderly compared with adult rats. Congruent with the respiratory studies, there was no age-associated decrease in carnitine palmitoyltransferase I and carnitine palmitoyltransferase II activities in isolated skeletal muscle mitochondria. However, there was a small, significant decrease in tissue total carnitine content. It is concluded that the in vivo observed decrease in skeletal muscle aerobic capacity with advanced age is a consequence of the decreased mitochondrial density. On the basis of the dramatic reduction of UCP-3 content associated with decreased state 4 respiration of skeletal muscle mitochondria from elderly rats, we propose that an increased free radical production might contribute to the metabolic compromise in aging.

Myocardial ischemia selectively depletes cardiolipin in rabbit heart subsarcolemmal mitochondria.

Mitochondria contribute to myocyte injury during ischemia. After 30 and 45 min of ischemia in the isolated perfused rabbit heart, subsarcolemmal mitochondria (SSM), located beneath the plasma membrane, sustain a decrease in oxidative phosphorylation through cytochrome oxidase. In contrast, oxidation through cytochrome oxidase in interfibrillar mitochondria (IFM), located between the myofibrils, remains unaffected. Cytochrome oxidase activity in the intact membrane requires an inner mitochondrial membrane lipid environment enriched in cardiolipin. During ischemia, the content of cardiolipin decreased only in SSM, whereas the content of other phospholipids was preserved. Ischemia did not alter the composition of the cardiolipin that remained in SSM. Cardiolipin content was preserved in IFM during ischemia. Thus cardiolipin is a relatively early target of ischemic mitochondrial damage, leading to loss of oxidative phosphorylation through cytochrome oxidase in SSM.

Separation and quantitation of phospholipids and lysophospholipids by high-performance liquid chromatography.

We describe a comprehensive approach to the separation, quantitation, and characterization of phospholipids and lysophospholipids present in complex biological samples. The central feature is a normal-phase HPLC separation of individual phospholipid and lysophospholipid classes. In this single chromatographic step, phospholipids and lysophospholipids are separated and recovered for quantitation by organic phosphate assay and characterization by acyl-group composition. Recovery of phospholipids and lysophospholipids from HPLC averages 80-90%. Isolated phospholipid and lysophospholipid fractions are available for separation of individual molecular species by second-dimension reverse-phase HPLC and characterization of individual molecular species by mass spectrometry.

Simultaneous determination of O6-benzylguanine and 8-oxo-O6-benzylguanine in human plasma by reversed-phase high-performance liquid chromatography.

A high-performance liquid chromatographic assay for the quantification of O6-benzylguanine (O6BG) in human plasma was modified to include the metabolite, O6-benzyl-8-oxo-guanine (8-oxo-O6BG). O6-(p-Chlorobenzyl)guanine was used as the internal standard. Plasma samples were extracted with ethyl acetate and chromatographed on a C18 base-deactivated reversed-phase column. Separation was accomplished by gradient elution with mobile phases consisting of acetonitrile and phosphate buffer, pH 3.60. Eluted compounds were observed with diode array detection at 288 nm (O6BG) and 292 nm (8-oxo-O6BG). Standard curves were linear from 12.5 ng/ml to 1000 ng/ml, with an average regression coefficient of 0.999 (n=5) for both compounds. The lowest limit of quantitation was 25 ng/ml, with a signal-to-noise ratio of 8:1. The within-day relative standard deviations for O6BG quality control samples (n=18) with concentrations of 735 ng/ml, 305 ng/ml and 38 ng/ml were 2.4%, 4.2% and 5.3%, respectively. The within-day relative standard deviations for 8-oxo-O6BG quality control samples (n=18) at concentrations of 735 ng/ml, 420 ng/ml and 42 ng/ml were 2.2%, 4.0% and 7.1%, respectively. The day-to-day relative standard deviations for the same control specimens were 3.1%, 4.8% and 7.1% for O6BG, respectively, and 2.3%, 4.7% and 11.0% for 8-oxo-O6BG, respectively. This method was applied to plasma samples obtained from patients in a clinical trial of O6-benzylguanine. O6-Benzyl-8-oxo-guanine was identified in patient plasma specimens by liquid chromatography-electrospray mass spectrometry by comparison with spectral data acquired from reference material.

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 microliters) 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.

Quantification of free carnitine, individual short- and medium-chain acylcarnitines, and total carnitine in plasma by high-performance liquid chromatography.

This paper describes a method for the quantitative determination of free carnitine, acetylcarnitine, propionylcarnitine, hexanoylcarnitine, octanoylcarnitine, and total carnitine in plasma. Carnitine and acylcarnitines were extracted from 100 microliters of plasma with acetonitrile/methanol and isolated using 0.5-ml columns of silica gel. Samples were then derivatized with 4'-bromophenacyl trifluoromethanesulfonate and quantified by high-performance liquid chromatography with detection at 260 nm. Carnitine and acylcarnitines were quantified in normal human plasma and the plasma of patients diagnosed with methylmalonic aciduria, propionic acidemia, and medium-chain acyl-CoA dehydrogenase deficiency.

Quantification of carnitine and specific acylcarnitines by high-performance liquid chromatography: application to normal human urine and urine from patients with methylmalonic aciduria, isovaleric acidemia or medium-chain acyl-CoA dehydrogenase deficiency.

This paper describes the development of a high-performance liquid chromatographic method for the quantitation of free carnitine, total carnitine, acetylcarnitine, propionylcarnitine, isovalerylcarnitine, hexanoylcarnitine and octanoylcarnitine in human urine. Carnitine and acylcarnitines were isolated from 10 or 25 microliters of urine using 0.5-ml columns of silica gel, derivatized with 4'-bromophenacyl trifluoromethanesulfonate and separated by high-performance liquid chromatography. Using 4-(N,N-dimethyl-N-ethylammonio)-3-hydroxybutanoate ("e-carnitine") as the internal standard, standard curves (10-300 nmol/ml) were generated. Carnitine and acylcarnitines were quantified (when they were present) in normal human urine and the urine of patients diagnosed with one of three different disorders of organic acid metabolism: methylmalonic aciduria, isovaleric aciduria, isovaleric acidemia, and medium-chain acyl-CoA dehydrogenase deficiency.

Derivatization of isolated endogenous butyrobetaine with 4'-bromophenacyl trifluoromethanesulfonate followed by high-performance liquid chromatography.

A method for the isolation and chromatography of butyrobetaine from plasma, urine, and liver is described. The recovery of [3H-methyl]butyrobetaine from spiked biological samples was from 76-80%. Spiked samples then were derivatized with 4'-bromophenacyl trifluoromethanesulfonate and the butyrobetaine 4'-bromophenacyl ester was isolated by high-performance liquid chromatography (HPLC). Radioactivity eluted in a single peak which co-chromatographed with authentic butyrobetaine 4'-bromophenacyl ester. Two identical liver specimens were treated according to this isolation procedure. Prior to derivatization, one specimen was treated with butyrobetaine hydroxylase. After derivatization, there was no butyrobetaine 4'-bromophenacyl ester peak in the specimen treated with butyrobetaine hydroxylase. The HPLC detection sensitivity to butyrobetaine 4'-bromophenacyl ester was 1 pmol injected with a signal-to-noise greater than 2:1.

High-performance liquid chromatographic separation of acylcarnitines following derivatization with 4'-bromophenacyl trifluoromethanesulfonate.

A high-performance liquid chromatographic method for the separation of acylcarnitines after derivatization with 4'-bromophenacyl trifluoromethanesulfonate is presented. Derivatization of acylcarnitines was achieved at room temperature within 10 min. Separation of the acylcarnitine 4'-bromophenacyl esters was accomplished by high-performance liquid chromatography using as the analytical column a Resolve-PAK 5-microns C18 radially compressed cartridge eluted with a tertiary gradient containing varying proportions of water, acetonitrile, tetrahydrofuran, triethylamine, potassium phosphate, and phosphoric acid. Acylcarnitine 4'-bromophenacyl esters were detected spectrophotometrically at 254 nm. Baseline separation was obtained for a standard mixture (5 nmol of each injected) containing carnitine, acetyl-, propionyl-, butyryl-, valeryl-, hexanoyl-, heptanoyl-, octanoyl-, nonanoyl-, decanoyl-, lauroyl-, myristroyl-, palmitoyl-, and stearoylcarnitine. Nearly complete separation was obtained for a standard mixture containing butyryl-, isobutyryl-, isovaleryl-, and 2-methylbutyrylcarnitine. The method was applied to a normal human urine and then to this same urine spiked with the acylcarnitine standards. Urinary acylcarnitine profiles from patients having propionic acidemia, isovaleric acidemia, and medium-chain acyl-CoA dehydrogenase deficiency were performed. Urinary isovalerylcarnitine was quantified in the patient with isovaleric acidemia using heptanoylcarnitine as an internal standard.

Determination of ibuprofen and its major metabolites in human urine by high-performance liquid chromatography.

A sensitive and selective high-performance liquid chromatographic assay for free and total ibuprofen and its major metabolites in human urine is described. Urine is acidified, drug and metabolites are extracted into hexane-propanol, back-extracted into sodium bicarbonate, neutralized and chromatographed. Ibufenac (4-isobutylphenylacetic acid) and 2-phenylpropionic acid were employed as internal standards. The extraction efficiencies were 94-100% for all compounds. The two metabolites and their internal standard were separated using an isocratic chromatographic system, followed by an abrupt step gradient to a second eluent for separation of ibuprofen and its internal standard with a total run time of 18 min. Detection was by a fixed-wavelength detector (214 nm). Sample-to-sample and day-to-day reproducibility studies yielded coefficients of variability of less than 9% for all compounds. The sensitivity was sufficient to determine 2.5 micrograms/ml free ibuprofen in 100 microliters urine.

An enzymatic method for the determination of butyrobetaine via conversion to carnitine after isolation by high performance liquid chromatography.

An improved procedure for the determination of butyrobetaine [4-(N,N,N-trimethylammonio)butanoate] in plasma and tissue is described. Butyrobetaine was isolated by ion-exchange chromatography and high performance liquid chromatography. The isolation procedure was internally standardized with [3H]butyrobetaine. The recovery of butyrobetaine was greater than 90%. Following isolation butyrobetaine was enzymatically converted to carnitine using butyrobetaine hydroxylase and the resulting carnitine was assayed using carnitine acetyltransferase and [14C]acetylcoenzyme A. The conversion of butyrobetaine to carnitine and of carnitine to [14C]acetylcarnitine was greater than 98% as determined by high performance liquid chromatography. Using this method was analysed human sera (healthy controls) and tissues (autopsy) and found the following values: serum, 4.67 nmol/ml; kidney 17.6 nmol/g; liver, 26.5 nmol/g. The serum butyrobetaine values of twins suffering from carnitine deficiency were normal (3.78 and 3.87 nmol/ml), while the carnitine supplementation therapy caused an increase. Animal samples were analyzed and the values were 3-4 times higher than previously reported by others.

Rapid high-performance liquid chromatography of 3-methylhistidine in human urine.

An internally standardized method for the determination of 3-methylhistidine in human urine is presented. This methylated amino acid and the chemically analogous internal standard 3-ethylhistidine were isolated from human urine specimens using small columns of cation-exchange resin. Quantification was accomplished by high-performance liquid chromatography using post-column derivatization with o-phthalicdicarboxaldehyde-2-mercaptoethanol followed by fluorometric detection. Sample-to-sample and day-to-day reproducibility were shown to have respective relative standard deviations of 2 and 5% for a human urine specimen containing 250 nmol/ml 3-methylhistidine when using 250 microliter urine per analysis. The chromatographic separation was evaluated in terms of various peak descriptors (capacity factor and retention time) and "Chromatographic Figures of Merit" (peak symmetry and chromatographic efficiency). The utility of the method was demonstrated by its successful application to 1000 human urine specimens.

Improved high-performance liquid chromatographic method for the determination of 6-N,N,N-trimethyllysine in plasma and urine: biomedical application of chromatographic figures of merit and amine mobile phase modifiers.

An internally standardized method for the determination of 6-N,N,N-trimethyllysine in human plasma, human urine, rat plasma, rat urine and hydrolyzed rat urine is described. This methylated amino acid and the procedural internal standard 6-N,N,N-trimethyllysine were isolated from the sample matrices using short ion-exchange columns and detected following high-performance liquid chromatography using a postcolumn reaction (o-phthalic-dicarboxaldehyde-2-mercaptoethanol) and fluorometric detection. The reliable detection limit for 6-N,N,N-trimethyllysine was 0.2 nmol/ml in 200 microliters of human plasma. The chromatographic separation exploits the unique properties of a novel tertiary amine mobile phase modifier, 3-(N,N-dimethylamino)-1,2-propanediol. The capacity factor and "Chromatographic Figures of Merit" (including peak asymmetry and relative system efficiency) were calculated for the chromatographic peak representing 6-N,N,N-trimethyllysine in over 2200 injections made while evaluating 900 biological specimens.

Determination of carnitine, butyrobetaine, and betaine as 4'-bromophenacyl 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 omega-trimethylammonio carboxylates and the chemically analogous internal standards 4-(N,N-dimethyl-N-propylammonio)-3-hydroxybutanoate or 6-(N,N,N-trimethylammonio)hexanoate were derivatized by reaction with 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 omega-trialkylammonio acid derivatives injected into the chromatograph were detected with signal-to-noise ratios greater than 50.

Derivatization of carboxylic acids by reaction with 4'-bromophenacyl trifluoromethanesulfonate prior to determination by high-performance liquid chromatography.

The use of 4'-bromo-2-hydroxyacetophenone trifluoromethanesulfonate ester (4'-bromophenacyl triflate) in the preparation of carboxylic acid 4'-bromophenacyl ester derivatives for spectrophotometric detection in high-performance liquid chromatography is described. The reagent is prepared in 66% yield by the reaction of 4'-bromo-2-diazoacetophenone with trifluoromethanesulfonic acid in anhydrous sulfur dioxide and is stable for 3-6 months. Reactions of 10(-6) M carboxylate N,N-diisopropylethylammonium salts with this reagent in acetonitrile at room temperature proceed to completion in 1-5 min. Optimal rates of reaction are obtained with a 10-fold equivalent excess of alkylating agent and 5 equivalents of N,N-diisopropylethylamine present. The process has been applied successfully to mono-, di- and tricarboxylic and sterically hindered carboxylic acids.