Specific and rapid analysis of ubiquinones using Craven's reaction and HPLC with postcolumn derivatization

Shimada, Haruo; Kodjabachian, David; Ishida, Masami

doi:10.1194/jlr.d700006-jlr200

Cited by 7 publications

(6 citation statements)

References 31 publications

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“…To the 30 mg wet pellet of the E. tenella sporozoites, 600 μL of 2-propanol was added to extract quinones and quinols. To the 600 μL of supernatant, 6 μL of 2-propanol supplemented with 120 mM FeCl 3 was added to oxidize all the quinol to the quinone [23]. After the removal of insoluble debris by filtration through a 0.2 μm pore-size filter (Ultra-free, millipore), 4 μL of the filtrated extracts was injected to the LC-MS system, consisting of a pump (L-2100, Hitachi, Japan), column oven at 40 °C (L-2300, Hitachi), analytical column (Inertsil ODS-3, 2 μm, 2.1 mm i.d.…”

Section: Methodsmentioning

confidence: 99%

Novel Characteristics of Mitochondrial Electron Transport Chain from Eimeria tenella

Matsubayashi

Inaoka

Komatsuya

et al. 2019

Genes

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Eimeria tenella is an intracellular apicomplexan parasite, which infects cecal epithelial cells from chickens and causes hemorrhagic diarrhea and eventual death. We have previously reported the comparative RNA sequence analysis of the E. tenella sporozoite stage between virulent and precocious strains and showed that the expression of several genes involved in mitochondrial electron transport chain (ETC), such as type II NADH dehydrogenase (NDH-2), complex II (succinate:quinone oxidoreductase), malate:quinone oxidoreductase (MQO), and glycerol-3-phosphate dehydrogenase (G3PDH), were upregulated in virulent strain. To study E. tenella mitochondrial ETC in detail, we developed a reproducible method for preparation of mitochondria-rich fraction from sporozoites, which maintained high specific activities of dehydrogenases, such as NDH-2 followed by G3PDH, MQO, complex II, and dihydroorotate dehydrogenase (DHODH). Of particular importance, we showed that E. tenella sporozoite mitochondria possess an intrinsic ability to perform fumarate respiration (via complex II) in addition to the classical oxygen respiration (via complexes III and IV). Further analysis by high-resolution clear native electrophoresis, activity staining, and nano-liquid chromatography tandem-mass spectrometry (nano-LC-MS/MS) provided evidence of a mitochondrial complex II-III-IV supercomplex. Our analysis suggests that complex II from E. tenella has biochemical features distinct to known orthologues and is a potential target for the development of new anticoccidian drugs.

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Section: Methodsmentioning

confidence: 99%

Novel Characteristics of Mitochondrial Electron Transport Chain from Eimeria tenella

Matsubayashi

Inaoka

Komatsuya

et al. 2019

Genes

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“…Cells incubated under anaerobic conditions for 24 h were harvested by centrifugation, and 2‐propanol was added (20 μL·mg −1 wet weight of cells) to extract quinones and quinols. In total, 1 μL of 120 m m FeCl 3 solution dissolved in 2‐propanol was added to 100 μL of the extract to oxidize quinols prior to analysis , and this was used as a sample for measurement. Rhodoquinone‐9 (RQ 9 ) was prepared from Ascaris suum and was used as a standard for HPLC analysis in addition to ubiquinone‐10 (UQ 10 ), which was purchased from Wako Chemicals (Osaka, Japan).…”

Section: Methodsmentioning

confidence: 99%

Anaerobic respiration coupled with mitochondrial fatty acid synthesis in wax ester fermentation by Euglena gracilis

et al. 2018

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In Euglena gracilis , wax ester fermentation produces ATP during anaerobiosis. Here, we report that anaerobic wax ester production is suppressed when the mitochondrial electron transport chain complex I is inhibited by rotenone, whereas it is increased by the uncoupler carbonyl cyanide m ‐chlorophenylhydrazone ( CCCP ). The ADP / ATP ratio in anaerobic cells is elevated by treatment with either rotenone or CCCP . Gene silencing experiments indicate that acyl‐CoA dehydrogenase, electron transfer flavoprotein ( ETF ), and rhodoquinone ( RQ ) participate in wax ester production. These results suggest that fatty acids are synthesized in mitochondria by the reversal of β‐oxidation, where trans ‐2‐enoyl‐CoA is reduced mainly by acyl‐CoA dehydrogenase using the electrons provided by NADH via the electron transport chain complex I, RQ , and ETF , and that ATP production is highly supported by anaerobic respiration utilizing trans ‐2‐enoyl‐CoA as a terminal electron acceptor.

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“…[26][27][28][29] Moreover, Craven's reaction gives fluorescent products at a later stage of the reaction. It was reported that ubiquinone incubated at room temperature for 12-24 h in ethyl cyanoacetate with methanolic potassium hydroxide solution resulted in fluorescent products with excitation at 430 nm and emission at 530 nm.…”

Section: Resultsmentioning

confidence: 99%

Determination of Idebenone in Plasma by HPLC with Post-Column Fluorescence Derivatization Using 2-Cyanoacetamide

Nohara

Suzuki

Yamazaki

et al. 2012

CHEMICAL & PHARMACEUTICAL BULLETIN

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Idebenone is a benzoquinone analog that is used in the treatment of several neurological disorders including Friedreich's ataxia. It was found that the reaction of idebenone with 2-cyanoacetamide under alkaline conditions generates fluorescent products, and the reaction was considered to proceed via Craven's reaction. The reaction mixture from idebenone gave fluorescence with excitation and emission maximum wavelengths at 358 nm and 409 nm, respectively. It was adopted for HPLC with post-column fluorescence derivatization of idebenone. Idebenone in the plasma showed a linear response in the range of 0.5-32 ng (25-1600 ng/mL), and the quantitation limit (S/N 10) was 12.5 ng/mL. The detection limit (S/N 3) of the standard solution of idebenone was 0.1 ng. The HPLC system was applied to the human blood plasma obtained by finger prick. The plasma sample obtained by finger prick gave a similar chromatogram to that of venous blood obtained by venipuncture.Key words idebenone; finger prick; 2-cyanoacetamide; fluorescence derivatization; HPLC Idebenone is a synthetic analog of ubiquinone, 1) which is an essential component of the mitochondrial respiratory chain responsible for oxidative phosphorylation, and may prevent oxidative damage in the human body as an antioxidant. 2,3)Idebenone also has potent antioxidant activity and facilitates the flux of electrons along the mitochondrial electron transport chain, increasing the production of ATP, and has already been clinically investigated for the treatment of several neurological disorders, which include Friedreich's ataxia and Leber's hereditary optic neuropathy. 4,5) The pharmacokinetic properties of idebenone have been investigated by HPLC. Several procedures have been developed to analyze idebenone in biological samples by HPLC coupled with UV, electrochemical, or mass spectrometry detection. [6][7][8][9] HPLC methods employing mass spectrometry and electrochemical detectors have higher selectivity and sensitivity than those with a UV detector; however, these methods are not available in many laboratories because of a lack of specialized equipment. HPLC coupled with a fluorophotometric detector has been widely used in the determination of various substances in biological samples because of its high sensitivity and selectivity. However, idebenone needs fluorescence derivatization for the development of fluorimetric detection because it is not a fluorescent substance itself. 2-Cyanoacetamide has been used as a post-column derivatization reagent of fluorimetric HPLC for catecholamines, 3,4-dihydroxyphenylalanine, unsaturated disaccharides, and benzoquinones. [10][11][12][13][14][15] In the present study, 2-cyanoacetamide was tested for the fluorophotometric reagent of idebenone because idebenone is a benzoquinone compound. It was found that idebenone reacted with 2-cyanoacetamide under alkaline conditions with heating to form fluorescent products. This reaction was applied to HPLC with post-column fluorescence derivatization of idebenone.The salting out effect is useful a...

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Specific and rapid analysis of ubiquinones using Craven's reaction and HPLC with postcolumn derivatization

Cited by 7 publications

References 31 publications

Novel Characteristics of Mitochondrial Electron Transport Chain from Eimeria tenella

Novel Characteristics of Mitochondrial Electron Transport Chain from Eimeria tenella

Anaerobic respiration coupled with mitochondrial fatty acid synthesis in wax ester fermentation by Euglena gracilis

Determination of Idebenone in Plasma by HPLC with Post-Column Fluorescence Derivatization Using 2-Cyanoacetamide

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