B-Lapachone, an o-naphthoquinone, induces a novel caspase-and p53-independent apoptotic pathway dependent on NAD(P)H:quinone oxidoreductase1 (NQO1). NQO1reduces B-lapachone to an unstable hydroquinone that rapidly undergoes a two-step oxidation back to the parent compound, perpetuating a futile redox cycle. A deficiency or inhibition of NQO1rendered cells resistant to B-lapachone. Thus, B-lapachone has great potential for the treatment of specific cancers with elevated NQO1levels (e.g., breast, non^small cell lung, pancreatic, colon, and prostate cancers). We report the development of mono(arylimino) derivatives of B-lapachone as potential prodrugs. These derivatives are relatively nontoxic and not substrates for NQO1when initially diluted in water. In solution, however, they undergo hydrolytic conversion to B-lapachone at rates dependent on the electron-withdrawing strength of their substituent groups and pH of the diluent. NQO1enzyme assays, UV-visible spectrophotometry, high-performance liquid chromatographyelectrospray ionization-mass spectrometry, and nuclear magnetic resonance analyses confirmed and monitored conversionof each derivative to B-lapachone. Once converted, B-lapachone derivatives caused NQO1-dependent, A-calpain-mediated cell death in human cancer cells identical to that caused by B-lapachone. Interestingly, coadministration of N-acetyl-L-cysteine prevented derivative-induced cytotoxicity but did not affect B-lapachone lethality. Nuclear magnetic resonance analyses indicated that prevention of B-lapachone derivative cytotoxicity was the result of direct modification of these derivatives by N-acetyl-L-cysteine, preventing their conversion to B-lapachone. The use of B-lapachone mono(arylimino) prodrug derivatives, or more specifically a derivative converted in a tumor-specific manner (i.e., in the acidic local environment of the tumor tissue), should reduce normal tissue toxicity while eliciting tumor-selective cell killing by NQO1 bioactivation.

A novel procedure for the quantitative isolation and purification of acyl-coenzyme A esters is presented. The procedure involves two steps: 1) tissue extraction using acetonitrile/2-propanol (3+1, v+v) followed by 0.1M potassium phosphate, pH 6.7, and 2) purification using 2-(2-pyridyl)ethyl functionalized silica gel. Recoveries determined by adding radiolabelled acetyl-, malonyl-, octanoyl-, oleoyl-, palmitoyl-or arachidonyl-coenzyme A to powdered rat liver varied from 93% to 104% for tissue extraction and 83% to 90% for solid phase extraction. The procedure described allows for isolation and purification, with high recoveries, of acyl-coenzyme A esters widely differing in chainlength and saturation.Published methods for the analysis of tissue acyl-coenzyme A content are focused on either short-[1,2], medium-[3] or long-chain acyl-coenzyme A esters [4,5], with the isolation of one acyl-coenzyme A subgroup to the exclusion of others. Recovery of acyl-coenzyme A esters from tissue specimens is often disappointing, with documented recoveries between 30 and 60% [1,5,6]. A general procedure for the isolation of a wide range of acyl-coenzyme A esters, with good documented recoveries from tissues, is presently unavailable.Despite their widely different polarities, we have shown that acylcarnitines (short-, medium-, and long-chain) can be isolated, in a single fraction, from biological samples using organic solvent extraction followed by ion-exchange solid phase extraction (SPE) [7]. This is a highly selective approach, since it combines two orthogonal procedures [8] of isolation: organic solvent extraction and ion-exchange. Extraction procedures for short-chain acyl-coenzyme A esters often use acid precipitation [1,2], but these methods would exclude long-chain acylcoenzyme A esters. Therefore, we investigated extraction procedures originally developed for long-chain acyl-coenzyme A esters, using a mixture of acetonitrile, isopropanol, and aqueous buffer [4]. An SPE anion-exchange column is needed that would be uncharged at pH 7, since elution at high pH would cause hydrolysis of acyl-coenzyme A esters. Finding none commercially available, we ordered custom SPE columns from Supelco, (Bellefonte, PA), packed with 100 mg of 2-(2-pyridyl)ethyl functionalized silica gel. The pKa of these SPE *Corresponding author. Fax: +1 216 368 5162. E-mail address: charles.hoppel@case.edu. Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. , we analyzed rat heart, skeletal muscle, and liver and we found that the recovery of malonyl-coenzyme A from liver was much worse than for skeletal musc...

BACKGROUND:Analysis of carnitine and acylcarnitines by tandem mass spectrometry (MS/MS) has limitations. First, preparation of butyl esters partially hydrolyzes acylcarnitines. Second, isobaric nonacylcarnitine compounds yield false-positive results in acylcarnitine tests. Third, acylcarnitine constitutional isomers cannot be distinguished.

A validated quantitative method for the determination of free and total carnitine, butyrobetaine, and acylcarnitines is presented. The versatile method has four components: (1) isolation using strong cation-exchange solid-phase extraction, (2) derivatization with pentafluorophenacyl trifluoromethanesulfonate, (3) sequential ion-exchange/reversed-phase (ultra) high-performance liquid chromatography [(U)HPLC] using a strong cation-exchange trap in series with a fused-core HPLC column, and (4) detection with electrospray ionization multiple reaction monitoring (MRM) mass spectrometry (MS). Standardized carnitine along with 65 synthesized, standardized acylcarnitines (including short-chain, medium-chain, long-chain, dicarboxylic, hydroxylated, and unsaturated acyl moieties) were used to construct multiple-point calibration curves, resulting in accurate and precise quantification. Separation of the 65 acylcarnitines was accomplished in a single chromatogram in as little as 14 min. Validation studies were performed showing a high level of accuracy, precision, and reproducibility. The method provides capabilities unavailable by tandem MS procedures, making it an ideal approach for confirmation of newborn screening results and for clinical and basic research projects, including treatment protocol studies, acylcarnitine biomarker studies, and metabolite studies using plasma, urine, tissue, or other sample matrixes.