In accordance with the KFSC 2018, vitamin contents, including vitamins C, B2, B12, D, and A, were determined by HPLC (Nanospace SI-1, Shiseido, Japan) equipped with a UV detector (UV 3200, Shiseido, Japan). The conditions utilized for the analysis of fat-soluble and water-soluble vitamins are shown in Tables S3 and S4 , respectively.
Nanospace si 1
Manufactured by Shiseido
Sourced in Japan
NANOSPACE SI-1 is a laboratory equipment designed for the analysis and characterization of nanomaterials. It utilizes a scanning probe microscopy technique to provide high-resolution imaging and measurement of surface topography and properties at the nanometer scale.
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5 protocols using nanospace si 1
1
Vitamin Content Analysis by HPLC
2
Quantification of Plasma and Erythrocyte α-Tocopherol
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Plasma and erythrocyte α-tocopherol was extracted using a protocol described previously [30 (link)]. The erythrocyte samples were homogenized with saline (sample: saline 1: 3, w/w). Plasma samples and homogenized erythrocyte samples samples were extracted by chloroform/methanol (2/1 in volume) containing 100 μM butylated hydroxytoluene (BHT). Thereafter, the extracts of these samples were centrifuged at 15,000 rpm at 4°C. The concentration of α-tocopherol was determined by using an HPLC-ECD system with an electrochemical detector (NANOSPACE SI-1; Shiseido; Tokyo, Japan). The analyte was eluted with methanol/NaClO4 (50 mM) at a flow rate of 0.7 mL/min in a Wakosil-2 5C18 RS column (Wako; Tokyo, Japan). The concentration of α-tocopherol was determined by comparing the area under the curve of the analyte with that of the standard. A standard curve was prepared by using serial dilutions (1 μM, 500 nM, and 100 nM) of α-tocopherol standard (Eisai Chemical Company; Tokyo, Japan).
3
HPLC-ECD Quantification of CoQ Homologs
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CoQ homologue concentrations were determined using an HPLC-ECD system, as reported previously,(12 (link)) with minor modifications. Briefly, samples were added to a 9-fold volume of HPLC-grade 2-propanol (Fisher Chemicals, Fairlawn, NJ), vigorously mixed, and centrifuged. Supernatants thus obtained were injected onto the HPLC-ECD system. Mobile phase: 50 mM NaClO4 in methanol/2-propanol (7/3, v/v); flow rate: 1.0 ml/min; analytical column: KANTO RP-18 (L) GP, 5 μm × 150 mm × 4.6 mm (Kanto Chemical, Tokyo, Japan); post-reduction column: RC-10, 15 mm × 4 mm (IRICA, Kyoto, Japan); detector: ECD (600 mV) NANOSPACE SI-1 (Shiseido, Tokyo, Japan).
4
Quantifying CoQ10 and Free Cholesterol in Cells
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Concentrations of CoQ10 and free cholesterol (FC) in cells were determined by HPLC as reported previously.(4 (link)) Briefly, cells were seeded in 24-well plates and incubated for various times. The cell media were removed and the cells were washed twice with ice cold PBS. Then, 400 µl of HPLC grade 2-propanol (Fisher Chemicals, Fairlawn, NJ) was added. Samples were collected and centrifuged. Extracts thus obtained were injected into the HPLC-ECD system. Mobile phase: 50 mM NaClO4 in methanol/2-propanol (7/3, v/v); flow rate: 1.0 ml/min; analytical column: KANTO RP-18 (L) GP, 5 µm × 150 mm × 4.6 mm (Kanto Chemical, Tokyo, Japan); post-reduction column: RC-10, 15 mm × 4 mm (IRICA, Kyoto, Japan). CoQ10 and FC concentrations were determined by an electrochemical detector (600 mV; NANOSPACE SI-1, Shiseido, Tokyo, Japan) and a UV detector (210 nm; SPD-10A, Shimadzu, Kyoto, Japan), respectively.
CoQ10 in sucrose gradient fractions was extracted by using a 5-volume quantity of methanol and a 10-volume quantity of hexane. Triolein was used as an internal standard. Hexane fractions thus obtained were dried under a nitrogen gas stream and redissolved in 2-propanol. The samples were directly injected into the HPLC system described above.
CoQ10 in sucrose gradient fractions was extracted by using a 5-volume quantity of methanol and a 10-volume quantity of hexane. Triolein was used as an internal standard. Hexane fractions thus obtained were dried under a nitrogen gas stream and redissolved in 2-propanol. The samples were directly injected into the HPLC system described above.
5
Quantifying CoQ10 and Free Cholesterol
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Concentrations of CoQ10 and free cholesterol (FC) in cells were determined by HPLC, as reported previously.(3 (link)) Briefly, cells were seeded in 24-well plates at a density of 30,000/cm2 and incubated for various times. The cell media were removed and the cells were washed with ice-cold PBS twice. Then, 400 µl of HPLC grade 2-propanol (Fisher Chemicals, Fairlawn, NJ) was added. Samples were collected and centrifuged. Extracts thus obtained were injected into the HPLC-ECD system. Mobile phase: 50 mM NaClO4 in methanol/2-propanol (7/3, v/v); flow rate: 1.0 ml/min; analytical column: KANTO RP-18 (L) GP, 5 µm × 150 mm × 4.6 mm (Kanto Chemical, Tokyo, Japan); post-reduction column: RC-10, 15 mm × 4 mm (IRICA, Kyoto, Japan). CoQ10 and FC concentrations were determined by an electrochemical detector (600 mV; NANOSPACE SI-1, Shiseido, Tokyo, Japan) and a UV detector (210 nm; SPD-10A, Shimadzu, Kyoto, Japan), respectively.
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