Charles L. Hoppel

Biochemical differences between subsarcolemmal and interfibrillar mitochondria from rat cardiac muscle: Effects of procedural manipulations

Differences in oxidative metabolism between subsarcolemmal and interfibrillar heart mitochondria were investigated. Interfibrillar mitochondria oxidized substrates donating reducing equivalents at Complex I (NADH-CoQ reductase), Complex II (succinate-CoQ reductase), and Complex III (CoQH2-cytochrome c reductase) more rapidly than did subsarcolemmal mitochondria. There was no difference in oxidation of substrates entering the electron transport chain at Complex IV (cytochrome c oxidase). Differences expressed in normal-ionic-strength medium at Complexes II and III but not I were eliminated in low-ionic-strength medium. The concentrations of cytochromes and activities of NADH and cytochrome c oxidase were virtually the same in the two populations. In permeabilized mitochondria, activities of succinate-duroquinone and TMPD plus ascorbate oxidase were significantly lower in the subsarcolemmal mitochondria. Differences in membrane permeability between the populations were suggested by the greater permeability of subsarcolemmal mitochondria to exogenous NADH. The influence of isolation buffers and preparative procedures on the two classes of mitochondria were also examined. Characteristic biochemical and morphological properties of the two populations were unchanged by exposing each to the preparative procedure used to isolate the alternate population; the oxidative performance of the two populations cannot be equalized by experimental manipulation.

Inhibition of mitochondrial respiration by analogs of 4-phenylpyridine and 1-methyl-4-phenylpyridinium cation (MPP+), the neurotoxic metabolite of MPTP

In order to clarify the structural requirements associated with the inhibition of mitochondrial respiration by MPP+, the neurotoxic metabolites of the Parkinsonian agent MPTP, ten sets of pyridine/N-methylpyridinium pairs and a few miscellaneous compounds were evaluated on rat liver intact mitochondria (Mw) and on submitochondrial particles (SMP). The pyridinium partners were much more potent inhibitors on Mw than on SMP, indicating that they are concentrated inside mitochondria by the energy-dependent process previously reported for MPP+, probably as a consequence of non-specific passive transport across the mitochondrial inner membrane in response to the transmembrane potential. In the SMP assay, the neutral pyridines were stronger inhibitors than were the pyridinium cations, and the inhibitory potency varied little with structural changes. The N-methylated forms of beta-carbolines may act as endogenous MPP+-like agents.

Tetraphenylborate potentiates the respiratory inhibition by the dopaminergic neurotoxin MPP+ in both electron transport particles and intact mitochondria

The cytotoxicity of 1-methyl-4-phenylpyridinium (MPP+) is believed to arise as a consequence of its time- and energy-dependent accumulation inside mitochondria, followed by inhibition of electron transport at Complex I of the respiratory chain. Consistent with our proposal that the accumulation of MPP+ represents a passive Nernstian transport into mitochondria in response to the transmembrane electrochemical potential gradient, tetraphenylborate (TPB-) was found to accelerate the onset of the respiratory inhibition by MPP+ on intact mitochondria. Moreover, the ultimate level of inhibition reached was unexpectedly also increased. The latter is now explained by our finding that TPB- elicits a 12-fold enhancement of MPP+ inhibition of respiration in electron transport particles. It is suggested that TPB- facilitates access of MPP+ to its intramembrane site of inhibitory action in Complex I.

High-performance liquid chromatographic separation of acylcarnitines following derivatization with 4′-bromophenacyl trifluromethanesulfonate

Abstract 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-μm C 18 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.

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.

Determination of free trimethyllysine in plasma and tissue specimens by high-performance liquid chromatography

A method for the determination of 6-N-trimethyllysine in tissues and plasma is described. Trimethyllysine and the chemically analogous 6-N-triethyllysine (internal standard) were isolated from acid-soluble fractions of tissue homogenates or plasma by combined ion-exchange--ion-exclusion chromatography. Trimethyllysine and triethyllysine were separated from other sample constituents by reversed-phase ion-pair high-performance liquid chromatography, derivatized post-column by reaction with o-phthalicdicarboxaldehyde and 2-mercaptoethanol, and detected fluorometrically. Standard curves were linear over a sample concentration range of 0.5--4 nmol/ml. The detection limit corresponded with 25 pmol trimethyllysine injected into the chromatograph. The procedure was used for the determination of trimethyllysine in plasma, liver, kidney, and mixed skeletal muscle of rat.

Separation of acylcarnitines from biological samples using high-performance liquid chromatography

A reverse-phase high-performance liquid chromatography technique to separate carnitine and acylcarnitines from a biological matrix is described. The method utilizes a step gradient to provide baseline resolution of acylcarnitines (individually or by class) for subsequent quantification using a sensitive radioenzymatic assay. The method requires minimal sample preparation and prevents any contamination among groups of acylcarnitines. This technique has been applied to liver tissues of rats obtained under a variety of conditions. These studies demonstrate the validity and utility of the HPLC method while confirming the applicability of the perchloric acid fractionation of acylcarnitines by functional class. The present HPLC method permits resolution of long-chain acylcarnitines in the presence of large excess concentrations of carnitine and short-chain acylcarnitines (coelution of unesterified carnitine with long-chain acylcarnitines less than or equal to 0.05%). Thus, the method will be of use in the study of acylcarnitines in biological systems over a broad spectrum of metabolic conditions.

Trimethyllysine metabolism in lean and obese zucker rats during fasting

Abstract The effect of starvation upon the metabolism of trimethyllysine in lean and obese female Zucker rats was studied. All rats were fed a carnitine and trimethyllysine limiting diet for one to two weeks before starvation was initiated. Fed rats and rats starved for three, six, and nine days were used. Liver free and peptide-linked trimethyllysine, and urine total trimethyllysine were measured. Lean and obese Zucker rats had similar hepatic-free and peptide-linked trimethyllysine content when expressed per g protein or per mg DNA. Obese Zucker rats excreted more total trimethyllysine during starvation relative to lean rats. Starvation did not affect trimethyllysine excretion over time, although there were significant decreases in total carnitine excretion in both lean and obese starved rats. Both phenotypes demonstrated a high efficiency of entry of trimethyllysine into the carnitine biosynthetic pathway. We conclude that lean and obese female Zucker rats, similar to male Sprague-Dawley rats, are extremely efficient in the conversion of trimethyllysine into carnitine.