Saori Nakagawa

High-performance liquid chromatography determination of ketone bodies in human plasma by precolumn derivatization with p-nitrobenzene diazonium fluoroborate

We developed and validated a sensitive and convenient high-performance liquid chromatography (HPLC) method for the specific determination of ketone bodies (acetoacetate and D-3-hydroxybutyrate) in human plasma. p-Nitrobenzene diazonium fluoroborate (diazo reagent) was used as a precolumn derivatization agent, and 3-(2-hydroxyphenyl) propionic acid was used as an internal standard. After the reaction, excess diazo reagent and plasma proteins were removed by passing through a solid-phase cartridge (C(18)). The derivatives retained on the cartridge were eluted with methanol, introduced into the HPLC system, and then detected with UV at 380 nm. A calibration curve for acetoacetate standard solution with a 20-microl injection volume showed good linearity in the range of 1 to 400 microM with a 0.9997 correlation coefficient. For the determination of D-3-hydroxybutyrate, it was converted to acetoacetate before reaction with the diazo reagent by an enzymatic coupling method using D-3-hydroxybutyrate dehydrogenase and lactate dehydrogenase. A calibration curve for D-3-hydroxybutyrate standard solution also showed good linearity in the range of 1.5 to 2000 microM with a 0.9988 correlation coefficient. Analytical recoveries of acetoacetate and D-3-hydroxybutyrate in human plasma were satisfactory. The method was successfully applied to samples from diabetic patients, and results were consistent with those obtained using the thio-NAD enzymatic cycling method used in clinical laboratories.

Oxysterol changes along with cholesterol and vitamin D changes in adult phenylketonuric patients diagnosed by newborn mass-screening

BACKGROUND Phenylketonuria (PKU) possibly leads to hypocholesterolemia and lowered vitamin D (VD) status. Metabolism of oxysterols linking with those of cholesterol and VD has never been examined in PKU. METHODS Blood oxysterols along with blood phenylalanine, lipids and VD were examined for 33 PKU adults aged 21-38 years and 20 age-matched healthy controls. RESULTS Total- and low-density cholesterols, and 25-hydroxy VD(3) were decreased significantly in the PKU group (cholesterols, 10% decrease; 25-hydroxy VD(3) 35% decrease vs. the control group). 24S-hydroxycholesterol (24S-OHC) eliminating brain cholesterol, and 27-OHC and 7α-hydroxycholesterol (7α-OHC) representing peripheral and hepatic cholesterol elimination, respectively, were significantly decreased in PKU group: 24S-OHC, 25% decrease, p<.01; 27-OHC and 7α-OHC, 35-40% decrease, p<.001. 7β-Hydroxycholesterol (7β-OHC) reflecting oxidative stress was increased significantly in PKU group (p<.05). 7α-OHC and 27-OHC levels in PKU group always showed similar values, regardless of other parameters while the 24S-OHC and 7β-OHC levels decreased and increased, respectively, showing significant correlations with phenylalanine level (p<.005). 27-OHC level showed a significant positive correlation with the 25-hydroxy VD(3) level in this group (p<.001). CONCLUSION Blood oxysterol changes predominate over blood cholesterol changes and influence on VD status in adult PKU patients.

Simultaneous determination of pregnenolone and 17α-hydroxypregnenolone by semi-micro high-performance liquid chromatography with an immobilized cholesterol oxidase as a pre-column reactor: Application to bovine adrenal fasciculata cells

A method for the simultaneous determination of pregnenolone and 17α-hydroxypregnenolone by high-performance liquid chromatography with an immobilized cholesterol oxidation enzyme reactor was developed. Pregnenolone and 17α-hydroxypregnenolone were converted to progesterone and 17α-hydroxyprogesterone, respectively, by the immobilized enzyme packed into the reactor column, and could thus be monitored by UV absorption at 240 nm. The calibration curves for pregnenolone and 17α-hydroxypregnenolone were linear in the range of 0.4-10 and 0.3-10 μg/ml with a correlation coefficient of 0.9993 and 0.9998, respectively. The detection limit at a signal-to-noise ratio of 3 was 0.12 and 0.08 μg/ml for pregnenolone and 17α-hydroxypregnenolone, respectively. The conversion rate of pregnenolone to progesterone and 17α-hydroxypregnenolone to 17α-hydroxyprogesterone was 90.6% and 99.3%, respectively. Intra-day and inter-day precision (in terms of percentage coefficient of variation) were less than 9.3%, with accuracy greater than 94.8%. This method was successfully applied to the simultaneous determination of pregnenolone and 17α-hydroxypregnenolone secreted into the culture medium of bovine adrenal fasciculata cells and of both analytes produced within the cells.

Inhibitory effect of protopanaxatriol ginseng metabolite M4 on the production of corticosteroids in ACTH-stimulated bovine adrenal fasciculata cells.

AIMS We investigated the pharmacological effects of saponins isolated from ginseng root and their metabolites, which occur by hydrolysis of the sugar moieties connecting the aglycone of saponins in the digestive tract, on the production of corticosteroids in bovine adrenal fasciculata cells in vitro. MAIN METHODS The levels of corticosteroids produced from adrenal corticotropic hormone (ACTH)-stimulated bovine adrenal fasciculata cells were determined under the presence or absence of ginseng saponins (ginsenosides) and their metabolites using fluorometry, gas-chromatography-mass spectrometry, and sweeping-micellar electrokinetic capillary chromatography. KEY FINDINGS An end metabolite of the protopanaxatriol saponins in ginseng, 20(s)-protopanaxatriol (M4), strongly reduced ACTH-stimulated cortisol production. M4 significantly inhibited the production of cortisol induced by different stimuli, alamethicin, dibutyryl cyclic AMP, forskolin, and 22(R)-hydroxycholesterol, a membrane-permeable cholesterol. However, it did not affect the production of cortisol by either pregnenolone, a precursor of cortisol synthesis, or cyclic AMP. Furthermore, M4 significantly inhibited the production of pregnenolone, progesterone, deoxycorticosterone, cortisol, and corticosterone in a dose-dependent manner. SIGNIFICANCE Results strongly suggest that protopanaxatriol saponins in ginseng are prodrugs metabolized in the digestive tract so that the end metabolite, M4, produces inhibitory activity of corticosteroid production in the adrenal fasciculata cells in vivo. The results also suggest that M4 inhibits the conversion from cholesterol to pregnenolone because the production of pregnenolone was reduced.

Ezetimibe decreases serum oxidized cholesterol without impairing bile acid synthesis in Japanese hypercholesterolemic patients

OBJECTIVE Cholesterol and diet-derived oxidized cholesterol are absorbed in the small intestine and eliminated by bile acids. We determined whether ezetimibe, a selective cholesterol absorption inhibitor, changes serum oxidized cholesterol levels. METHODS We measured levels of plant sterols, cholesterol precursors, and oxysterols by gas chromatography-mass spectrometry in 47 hypercholesterolemics and 32 controls. Twenty-four hypercholesterolemics received 10 mg ezetimibe/day for 4 weeks. RESULTS Plant sterols were 30-42% higher in hypercholesterolemics than in controls and positively correlated with low-density lipoprotein-cholesterol (LDL-C). Ezetimibe decreased plant sterols by 21-53%, but did not change bile acid synthesis markers. 7β-hydroxycholesterol, a marker for non-enzymatic oxidation of cholesterol, was 66% higher in hypercholesterolemics than controls. Ezetimibe decreased 7β-hydroxycholesterol levels by 15% regardless of LDL-C reduction. CONCLUSIONS Ezetimibe decreases serum oxidized cholesterol generated by non-enzymatic reactions without impairing bile acid synthesis. Ezetimibe may maintain cholesterol excretion into bile and alleviate the diet-derived oxidative burden.

Detection of cytochrome P450 2C19 gene polymorphism from noninvasive samples by cycling probe technology

Background The proportion of poor metabolizers (PMs) of cytochrome P450 (CYP) 2C19 is much higher in the Japanese population than in European populations. Cycling probe technology (CPT) is a simple signal amplification technique for targeting specific DNA sequences. CPT utilizes a chimeric DNA-RNA-DNA probe that is cleaved by the enzyme ribonuclease (RNase H). In this study, using CPT, we aimed to detect the CYP2C19 gene polymorphism from noninvasive samples to determine extensive metabolizers (EMs) and PMs of CYP2C19. Methods DNA samples were extracted from hair, buccal mucosa, and blood cells. Primers and cycling probes were designed specifically for region G636A for exon 4 and G681A for exon 5, reported to be gene polymorphisms of CYP2C19. Results DNA extracted from hair follicle cells and buccal epithelial cells was the same as that collected from invasive blood sampling. The genotype of CYP2C19 was successfully identified as either EM or PM in 71 samples, producing identical results to those for the TaqMan method, except in three samples. Conclusions We successfully detected the two gene polymorphisms of CYP2C19 from noninvasive samples using a simple DNA extraction method and CPT.

Reduction of lipid accumulation rescues Bietti’s crystalline dystrophy phenotypes

Significance Bietti’s crystalline dystrophy (BCD) is an autosomal recessive, progressive chorioretinal degenerative disease. Retinal pigment epithelium (RPE) cells are impaired in patients with BCD, but the underlying mechanisms of RPE cell damage have not yet been determined because cells from lesions cannot be readily acquired from patients with BCD. In the present study, we successfully generated a human in vitro model of BCD, BCD patient-specific iPSC-RPE cells, and demonstrated that the accumulation of free cholesterol caused RPE cell damage and subsequent cell death via the induction of lysosomal dysfunction and impairment of autophagy flux in BCD-affected cells. We believe these findings provide evidence of the possible therapeutic efficacy of reducing intracellular free cholesterol in BCD. Bietti’s crystalline dystrophy (BCD) is an intractable and progressive chorioretinal degenerative disease caused by mutations in the CYP4V2 gene, resulting in blindness in most patients. Although we and others have shown that retinal pigment epithelium (RPE) cells are primarily impaired in patients with BCD, the underlying mechanisms of RPE cell damage are still unclear because we lack access to appropriate disease models and to lesion-affected cells from patients with BCD. Here, we generated human RPE cells from induced pluripotent stem cells (iPSCs) derived from patients with BCD carrying a CYP4V2 mutation and successfully established an in vitro model of BCD, i.e., BCD patient-specific iPSC-RPE cells. In this model, RPE cells showed degenerative changes of vacuolated cytoplasm similar to those in postmortem specimens from patients with BCD. BCD iPSC-RPE cells exhibited lysosomal dysfunction and impairment of autophagy flux, followed by cell death. Lipidomic analyses revealed the accumulation of glucosylceramide and free cholesterol in BCD-affected cells. Notably, we found that reducing free cholesterol by cyclodextrins or δ-tocopherol in RPE cells rescued BCD phenotypes, whereas glucosylceramide reduction did not affect the BCD phenotype. Our data provide evidence that reducing intracellular free cholesterol may have therapeutic efficacy in patients with BCD.