Simultaneous assay of the activities of two key enzymes in cholesterol metabolism by gas chromatography—mass spectrometry

Honda, Akira; Shoda, Junichi; Tanaka, Naomi; Matsuzaki, Yasushi; Osuga, Toshiaki; Shigematsu, Nobuharu; Tohma, Masahiko; Miyazaki, Hiroshi

doi:10.1016/0378-4347(91)80370-r

Cited by 32 publications

(16 citation statements)

References 46 publications

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“…5␤-Cholestane-3␣,7␣,12␣,24R,25-pentol and 5␤-cholestane-3␣,7␣,12␣,24S,25-pentol were prepared from 5␤-cholestane-3␣,7␣,12␣,25-tetrol by the method of Hoshita (24). [25,26,26,26,27,27 (27) were prepared by previously described methods. Troleandomycin and 6␤-hydroxytestosterone were purchased from Sigma.…”

Section: Methodsmentioning

confidence: 99%

Side Chain Hydroxylations in Bile Acid Biosynthesis Catalyzed by CYP3A Are Markedly Up-regulated in Cyp27 Mice but Not in Cerebrotendinous Xanthomatosis

Honda¹,

Salen²,

Matsuzaki³

et al. 2001

Journal of Biological Chemistry

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The accumulation of various 25-hydroxylated C 27 -bile alcohols in blood and their excretion in urine are characteristic features of cerebrotendinous xanthomatosis (CTX) a recessively inherited inborn error of bile acid synthesis caused by mutations in the mitochondrial sterol 27-hydroxylase (CYP27) gene. These bile alcohols may be intermediates in the alternative cholic acid side chain cleavage pathway. The present study was undertaken to identify enzymes and reactions responsible for the formation of these bile alcohols and to explain why Cyp27 ؊/؊ mice do not show CTX-related abnormalities. Microsomal activities of 5␤-cholestane-3␣,7␣,12␣-triol 25-and 26-hydroxylases, 5␤-cholestane-3␣,7␣,12␣,25-tetrol 23R-, 24S-, and 27-hydroxylases and testosterone 6␤-hydroxylase, a marker enzyme for CYP3A, in Cyp27 ؊/؊ mice livers were markedly up-regulated (5.5-, 3.5-, 6.5-, 7.5-, 2.9-, and 5.4-fold, respectively). In contrast, these enzyme activities were not increased in CTX. The activities of 5␤-cholestane-3␣,7␣,12␣-triol 25-and 26-hydroxylases and 5␤-cholestane-3␣,7␣,12␣,25-tetrol 23R-, 24R-, 24S-, and 27-hydroxylases were strongly correlated with the activities of testosterone 6␤-hydroxylase in control human liver microsomes from eight unrelated donors. Troleandomycin, a specific inhibitor of CYP3A, markedly suppressed these microsomal side chain hydroxylations in both mouse and human livers in a dosedependent manner. In addition, experiments using recombinant overexpressed human CYP3A4 confirmed that these microsomal side chain hydroxylations were catalyzed by a single enzyme, CYP3A4. The results demonstrate that microsomal 25-and 26-hydroxylations of 5␤-cholestane-3␣,7␣,12␣-triol and microsomal 23R-, 24R-, 24S-, and 27-hydroxylations of 5␤-cholestane-3␣,7␣,12␣,25-tetrol are mainly catalyzed by CYP3A in both mice and humans. Unlike Cyp27 ؊/؊ mice, CYP3A activity was not up-regulated despite marked accumulation of 5␤-cholestane-3␣,7␣,12␣-triol in CTX.

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Section: Methodsmentioning

confidence: 99%

Side Chain Hydroxylations in Bile Acid Biosynthesis Catalyzed by CYP3A Are Markedly Up-regulated in Cyp27 Mice but Not in Cerebrotendinous Xanthomatosis

Honda¹,

Salen²,

Matsuzaki³

et al. 2001

Journal of Biological Chemistry

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“…Gas liquid chromatography-mass spectrometry (GC-MS) with selected-ion monitoring (SIM) was performed using a JMS-SX102 instrument equipped with a JMA DA-6000 data-processing system (JEOL, Tokyo, Japan). The accelerating voltage was 10 kV, the ionization energy was 70 eV, the trap current was 300 A, and the mass spectral resolution was about 10,000 (38). An Ultra Performance capillary column (25 m ϫ 0.32 mm id) coated with methylsilicone (Agilent Technologies, DE) was used at a flow rate of helium carrier gas of 1.0 ml/min.…”

Section: Oxysterol Analysismentioning

confidence: 99%

FXR-mediated down-regulation of CYP7A1 dominates LXRα in long-term cholesterol-fed NZW rabbits

Pan

et al. 2003

Journal of Lipid Research

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We investigated how cholesterol feeding regulates cholesterol 7 ␣ -hydroxylase (CYP7A1) via the nuclear receptors farnesoid X receptor (FXR) and liver X receptor ␣ (LXR ␣ ) in New Zealand white rabbits. After 1 day of 2% cholesterol feeding, when the bile acid pool size had not expanded, mRNA levels of the FXR target genes short-heterodimer partner (SHP) and sterol 12 ␣ -hydroxylase (CYP8B) were unchanged, indicating that FXR activation remained constant. In contrast, the mRNA levels of the LXR ␣ target genes ATP binding cassette transporter A1 (ABCA1) and cholesteryl ester transfer protein (CETP) increased 5-fold and 2.3-fold, respectively, associated with significant increases in hepatic concentrations of oxysterols. Activity and mRNA levels of CYP7A1 increased 2.4 times and 2.2 times, respectively. After 10 days of cholesterol feeding, the bile acid pool size increased nearly 2-fold. SHP mRNA levels increased 4.1-fold while CYP8B declined 64%. ABCA1 mRNA rose 8-fold and CETP mRNA remained elevated. Activity and mRNA of CYP7A1 decreased 60% and 90%, respectively. Feeding cholesterol for 1 day did not enlarge the ligand pool size or change FXR activation, while LXR ␣ was activated highly secondary to increased hepatic oxysterols. As a result, CYP7A1 was up-regulated. After 10 days of cholesterol feeding, the bile acid (FXR ligand) pool size increased, which activated FXR and inhibited CYP7A1 despite continued activation of LXR ␣ . Thus, in rabbits, when FXR and LXR ␣ are activated simultaneously, the inhibitory effect of FXR overrides the stimulatory effect of LXR ␣ to suppress CYP7A1 mRNA expression. It is well established that feeding cholesterol to rats upregulates cholesterol 7 ␣ -hydroxylase (CYP7A1), the ratelimiting enzyme for classic bile acid synthesis (1-4). As a result, hepatic cholesterol destined for circulating LDL is diverted to bile acid synthesis, and the accumulation of cholesterol in the plasma and liver is prevented. Unlike in rats, feeding cholesterol to New Zealand white (NZW) rabbits was associated with repressed CYP7A1 (5) and a tremendous increase in plasma cholesterol levels. Further studies suggested that expansion of the bile acid pool size was responsible for inhibition of CYP7A1 in the cholesterol-fed rabbits (6), and that the inhibition was not directly related to the accumulated cholesterol (7). However, the molecular mechanism(s) for these findings remains unknown.Recent studies have shown that the orphan nuclear receptors farnesoid X receptor (FXR) (8-10) and liver X receptor ␣ (LXR ␣ ) (11-13) are negative and positive regulators of CYP7A1 transcription. The potent ligands for FXR activation are the bile acids chenodeoxycholic, deoxycholic, and lithocholic acid (8), while the ligands for LXR ␣ activation are oxysterols (14,15). It was reported that 24 S ,25-epoxycholesterol (24 S ,25E), 24 S -hydroxycholesterol (24 S -OH), 22 R -hydroxycholesterol (22 R -OH) (16,17), and 27-hydroxycholesterol (27-OH) (18)

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“…of the microanalysis of endogenous substances in biological specimens by GC-MS. As described in our previous investigations (7,10,11), MVA was lactonized easily to MVL and the resulting MVL was converted to the corresponding alkylamides with the primary alkylamines under mild conditions without any catalysts. This amidation via MVL from MVA was a characteristic reaction for g-hydroxy fatty acids such as MVA, and the resulting alkylamides were further converted to the final derivatives suitable for the detection of ionization.…”

Section: Discussionmentioning

confidence: 90%

“…Different quantities (5,10,25,50, and 100 mg of protein) of normal rat liver microsomes were used for the enzyme assay, and extraction and quantification of MVL were conducted as described in Materials and Methods. As shown in Fig.…”

Section: Application To a Microscale Assaymentioning

confidence: 99%

Highly sensitive assay of HMG-CoA reductase activity by LC-ESI-MS/MS

Honda

Mizokami

Matsuzaki

et al. 2007

Journal of Lipid Research

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We have developed a new sensitive and specific nonradioisotope assay method to measure the activity of HMG-CoA reductase, the rate-controlling enzyme in the cholesterol biosynthetic pathway. This method was based upon a stable isotope dilution technique by liquid chromatographytandem mass spectrometry using electrospray ionization in positive mode. Mevalonic acid, the product of HMG-CoA reductase, was converted to mevalonolactone (MVL) in an incubation mixture, extracted by a salting-out procedure, derivatized into the mevalonyl-(2-pyrrolidin-1-yl-ethyl)-amide, and then purified using a disposable silica cartridge. The resulting mevalonylamide was quantified by selected reaction monitoring using the positive electrospray ionization mode. The detection limit of this mevalonylamide was found to be 240 amol (signal-to-noise ratio 5 3), ?833 times more sensitive than that of MVL measured by a conventional radioisotope (RI) method (200 fmol). The variances between sample preparations and between measurements by this method were analyzed by one-way layout and calculated to be 3.2% and 1.8%, respectively. The recovery experiments were performed using incubation mixtures spiked with 0.77-2.31 nmol MVL/mg protein and were validated by a polynomial equation. These results showed that the estimated concentration within a 95% confidence limit was 0.47 6 0.07 nmol/mg protein, which coincided completely with the observed X 0 nmol/mg protein with a mean recovery of 94.6%. This method made it possible to measure HMG-CoA reductase activity with a high degree of reproducibility and reliability, and especially with sensitivity superior to that of the conventional RI method.

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Simultaneous assay of the activities of two key enzymes in cholesterol metabolism by gas chromatography—mass spectrometry

Cited by 32 publications

References 46 publications

Side Chain Hydroxylations in Bile Acid Biosynthesis Catalyzed by CYP3A Are Markedly Up-regulated in Cyp27 Mice but Not in Cerebrotendinous Xanthomatosis

Side Chain Hydroxylations in Bile Acid Biosynthesis Catalyzed by CYP3A Are Markedly Up-regulated in Cyp27 Mice but Not in Cerebrotendinous Xanthomatosis

FXR-mediated down-regulation of CYP7A1 dominates LXRα in long-term cholesterol-fed NZW rabbits

Highly sensitive assay of HMG-CoA reductase activity by LC-ESI-MS/MS

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