Journal: The Journal of Biological Chemistry

Article Title: Bifunctional DEGS2 has higher hydroxylase activity toward substrates with very-long-chain fatty acids in the production of phytosphingosine ceramides

doi: 10.1016/j.jbc.2023.104603

Figure Lengend Snippet: Greatly reduced but residual presence of PHS-containing CERs in the epidermis of Degs2 KO mice. Lipids were extracted from epidermis of postnatal day 0 WT (n = 3) and Degs2 KO (n = 3) mice, and CERs ( A and B ) and acyl-CERs ( C and D ) were measured via LC-MS/MS. Values presented are means + SD (∗∗ p < 0.01; Student’s t test) of the total quantities of PHS-CERs, SPH-CERs, and DHS-CERs ( A ); the quantity of each PHS-CER species containing the indicated FA moiety ( B ); the total quantities of acyl-PHS-CERs, acyl-SPH-CERs, and acyl-DHS-CERs ( C ); and the quantity of each acyl-PHS-CER species containing the indicated ω-hydroxy FA ( D ). CER, ceramide; DHS-CER, DHS containing CER; FA, fatty acid; PHS-CER, PHS-containing CER; SPH-CER, SPH-containing CER.

Article Snippet: To these samples, we added 375 μl of chloroform/methanol (1:2, v/v) and d 9 -labeled CER/acyl-CER standards ( d 9 -SPH-CER, 10 pmol; d 9 -DHS-CER, 5 pmol; d 9 -PHS-CER, 2.5 pmol; N -(26-oleoyloxy( d 9 ) hexacosanoyl) D- erythro -SPH [ d 9 -acyl-SPH-CER], 10 pmol [Avanti Polar Lipids]) and mixed vigorously.

Techniques: Liquid Chromatography with Mass Spectroscopy

Journal: The Journal of Biological Chemistry

Article Title: Bifunctional DEGS2 has higher hydroxylase activity toward substrates with very-long-chain fatty acids in the production of phytosphingosine ceramides

doi: 10.1016/j.jbc.2023.104603

Figure Lengend Snippet: Greatly reduced but residual presence of PHS-containing CERs in DEGS2 KO keratinocytes. A , DEGS2 KO keratinocytes were generated using the CRISPR/Cas9 system. The exon structure ( black , coding sequence; white , untranslated regions) of human DEGS2 and the nucleotide sequences of WT and DEGS2 KO keratinocytes (KO clones 1 and 2) around the guide RNA target sequences ( light blue ) and the protospacer-adjacent motif sequences ( magenta ) in exon 2 are shown. B – D , lipids were extracted from controls (controls 1 and 2) and DEGS2 KO keratinocytes (KO 1 and 2) differentiated for 14 days, and CERs and acyl-CERs were analyzed via LC-MS/MS. Values presented are means + SD (n = 3; ∗∗ p < 0.01; ∗ p < 0.05; Scheffé’s test) of the total quantities of PHS-CERs, SPH-CERs, DHS-CERs, acyl-PHS-CERs, acyl-SPH-CERs, and acyl-DHS-CERs ( B ); the quantity of each PHS-CER species containing the indicated FA moiety ( C ); and the quantity of each acyl-PHS-CER species containing the indicated ω-hydroxy FA ( D ). E , HEK 293T cells were transfected with pCE-puro 3× FLAG-1 (vector) or pCE-puro 3× FLAG-FA2H plasmid. After 24 h of transfection, lipids were extracted, and 2-hydroxy palmitic acid-containing SPH-CER (2-OH CER) and PHS-CERs were quantified via LC-MS/MS. Values presented are means + SD (n = 3; ∗∗ p < 0.01; ∗ p < 0.05; Student’s t test). CER, ceramide; DHS-CER, DHS containing CER; PHS-CER, PHS-containing CER; SPH-CER, SPH-containing CER.

Article Snippet: To these samples, we added 375 μl of chloroform/methanol (1:2, v/v) and d 9 -labeled CER/acyl-CER standards ( d 9 -SPH-CER, 10 pmol; d 9 -DHS-CER, 5 pmol; d 9 -PHS-CER, 2.5 pmol; N -(26-oleoyloxy( d 9 ) hexacosanoyl) D- erythro -SPH [ d 9 -acyl-SPH-CER], 10 pmol [Avanti Polar Lipids]) and mixed vigorously.

Techniques: Generated, CRISPR, Sequencing, Clone Assay, Liquid Chromatography with Mass Spectroscopy, Transfection, Plasmid Preparation

Journal: The Journal of Biological Chemistry

Article Title: Bifunctional DEGS2 has higher hydroxylase activity toward substrates with very-long-chain fatty acids in the production of phytosphingosine ceramides

doi: 10.1016/j.jbc.2023.104603

Figure Lengend Snippet: Preference for VLC substrates in the hydroxylation reaction by DEGS2. A and B , DEGS1 KO HAP1 cells were transfected with pEFh-3× FLAG-1 (vector), pEFh-3× FLAG-DEGS1, or pEFh-3× FLAG-DEGS2 plasmid. After 24 h, cells were labeled with 2 μM d 7 -DHS for 4 h. A , proteins were prepared and subjected to immunoblotting (IB) using anti-FLAG or anti-GAPDH (loading control) antibodies. B , lipids were extracted, and d 7 -labeled SPH-CERs, PHS-CERs, SPH-HexCERs, PHS-HexCERs, SPH-SMs, and PHS-SMs were quantified via LC-MS/MS. Values presented are means + SD of the respective lipids, with FA species color-coded (n = 3; ∗∗ p < 0.01; Dunnett’s test versus vector control). CER, ceramide; DHS-CER, DHS containing CER; FA, fatty acid; HexCER, monohexosylceramide; ND, not detected; PHS-CER, PHS-containing CER; SM, sphingomyelin; SPH-CER, SPH-containing CER.

Article Snippet: To these samples, we added 375 μl of chloroform/methanol (1:2, v/v) and d 9 -labeled CER/acyl-CER standards ( d 9 -SPH-CER, 10 pmol; d 9 -DHS-CER, 5 pmol; d 9 -PHS-CER, 2.5 pmol; N -(26-oleoyloxy( d 9 ) hexacosanoyl) D- erythro -SPH [ d 9 -acyl-SPH-CER], 10 pmol [Avanti Polar Lipids]) and mixed vigorously.

Techniques: Transfection, Plasmid Preparation, Labeling, Western Blot, Liquid Chromatography with Mass Spectroscopy

Journal: The Journal of Biological Chemistry

Article Title: Bifunctional DEGS2 has higher hydroxylase activity toward substrates with very-long-chain fatty acids in the production of phytosphingosine ceramides

doi: 10.1016/j.jbc.2023.104603

Figure Lengend Snippet: Tissue distribution of t20:0 PHS-CERs and their production by DEGS2. A and B , lipids were extracted from six tissues (kidney, esophagus, anterior stomach, posterior stomach, small intestine, and large intestine) of 2-month-old WT mice (n = 3), and the quantities of SPH-CERs and PHS-CERs containing LCBs with chain lengths from d/t16 to d/t26 were quantified via LC-MS/MS. Values presented are means + SD of the total quantities of SPH-CERs and PHS-CERs ( A ; LCB chain lengths are color coded) and the quantities of t18:0- or t20:0-containing PHS-CERs ( B ; FA chain lengths are color-coded). C , lipids were extracted from the esophagus and anterior stomach of six-month-old WT (n = 3) and Degs2 KO (n = 3) mice, and the quantities of t18:0 and t20:0 PHS-CERs were examined via LC-MS/MS. Values presented are means + SD of the total quantities of PHS-CERs, with FA species color coded. D and E , esophagus and anterior stomach of 6-month-old WT and Degs2 KO mice were prepared and subjected to hematoxylin/eosin staining ( D ) and lucifer yellow assay ( E ). Scale bars, 50 μm ( D ) and 10 μm ( E ). F , lipids were extracted from the esophagus and anterior stomach of 6-month-old WT (n = 3) and Degs2 KO (n = 3) mice, and PHS-CERs, SPH-CERs containing 2-hydroxy FA (2-OH CERs), and SPH-CERs containing 3-hydroxy FA (3-OH CERs) were quantified via LC-MS/MS. Values presented are means + SD of the total quantities of PHS-CERs, 2-OH CERs, and 3-OH CERs. G and H , DEGS1 KO HAP1 cells were transfected with pCE-puro 3× FLAG-SPTLC1, pCE-puro 3× FLAG-SPTSSB, and pCE-puro HA-SPTLC3 plasmids together with pEFh-3× FLAG-1 (vector), pEFh-3× FLAG-DEGS1, or pEFh-3× FLAG-DEGS2 plasmid, and cultured for 24 h. G , proteins were prepared and subjected to immunoblotting (IB) with anti-FLAG, anti-HA, or anti-GAPDH (loading control) antibodies. H , lipids were extracted, and d20:1 SPH-CERs and t20:0 PHS-CERs were analyzed via LC-MS/MS. Values presented are means + SD of the total quantities of the respective lipids, with FA chain length color coded (n = 3; ∗∗ p < 0.01; Dunnett’s test versus vector control for d20:1 SPH-CERs and Student’s t test for t20:0 PHS-CERs). CER, ceramide; DHS-CER, DHS containing CER; FA, fatty acid; PHS-CER, PHS-containing CER; SPH-CER, SPH-containing CER.

Article Snippet: To these samples, we added 375 μl of chloroform/methanol (1:2, v/v) and d 9 -labeled CER/acyl-CER standards ( d 9 -SPH-CER, 10 pmol; d 9 -DHS-CER, 5 pmol; d 9 -PHS-CER, 2.5 pmol; N -(26-oleoyloxy( d 9 ) hexacosanoyl) D- erythro -SPH [ d 9 -acyl-SPH-CER], 10 pmol [Avanti Polar Lipids]) and mixed vigorously.

Techniques: Liquid Chromatography with Mass Spectroscopy, Staining, Transfection, Plasmid Preparation, Cell Culture, Western Blot

Journal: ACS Omega

Article Title: LC-MS/MS-Based Method for the Multiplex Detection of 24 Fentanyl Analogues and Metabolites in Whole Blood at Sub ng mL –1 Concentrations

doi: 10.1021/acsomega.7b01536

Figure Lengend Snippet: LC-MS/MS ion chromatogram of a high calibrator. Each peak represents the quantitative transition ion (qualitative transition ion not shown). Fentanyl analogue and internal standard peak identities: (1) norfentanyl- d 5 , (2) norfentanyl, (3) furanyl norfentanyl, (4) remifentanil acid, (5) butyryl norfentanyl, (6) remifentanil, (7) acetyl fentanyl, (8) acetyl fentanyl- 13 C 6 , (9) alfentanil, (10) U-47700, (11) acetyl fentanyl 4-methylphenethyl, (12) acrylfentanyl, (13) fentanyl, (14) AH-7921, (15) fentanyl- d 5 , (16) 4-ANPP, (17) para -methoxyfentanyl, (18) furanyl fentanyl, (19) despropionyl para -fluorofentanyl, (20) (±)- cis -3-methyl fentanyl, (21) butyryl/isobutyryl fentanyl, (22) carfentanil, (23) para -fluorobutyryl/ para -fluoroisobutyryl fentanyl, (24) sufentanil, and (25) valeryl fentanyl. Separation between butyryl/isobutyryl and para -fluoroisobutyryl fentanyl/ para -fluorobutyryl fentanyl was not achieved due to isomerism.

Article Snippet: Internal standards were acetyl fentanyl- 13 C 6 , fentanyl- d 5 , and norfentanyl- d 5 from Cerilliant.

Techniques: Liquid Chromatography with Mass Spectroscopy

Journal: ACS Omega

Article Title: LC-MS/MS-Based Method for the Multiplex Detection of 24 Fentanyl Analogues and Metabolites in Whole Blood at Sub ng mL –1 Concentrations

doi: 10.1021/acsomega.7b01536

Figure Lengend Snippet: Quantitative ion chromatogram of an accidental overdose case from late 2016. Abbreviations are as follows: (1) norfentanyl- d 5 , (2) norfentanyl, (8) acetyl fentanyl 13 C 6 , (10) U-47700, (13) fentanyl, (15) fentanyl- d 5 , (16) 4-ANPP, (18) furanyl fentanyl, (19) despropionyl para -fluorofentanyl, (21) butyryl/isobutyryl fentanyl, and (23) para -fluorobutyryl/ para -fluoroisobutyryl fentanyl. Inset shows the low-response count region of IMF analogues.

Article Snippet: Internal standards were acetyl fentanyl- 13 C 6 , fentanyl- d 5 , and norfentanyl- d 5 from Cerilliant.

Techniques:

Journal: Journal of Exposure Science & Environmental Epidemiology

Article Title: Exposure to tobacco smoke and validation of smoking status during pregnancy in the MIREC study

doi: 10.1038/s41370-017-0011-z

Figure Lengend Snippet: Summary statistics of plasma cotinine in 1st (T1) and 3rd (T3) trimesters by self-reported smoking status in that trimester

Article Snippet: The following calibration standards: (−)-Cotinine, S(−)-Nicotine, (±)-Cotinine-d3 and (±)-Nicotine-d4 were acquired from Cerilliant, while trans-3′-Hydroxycotinine and trans-3′-Hydroxycotinine-d3 were acquired from Toronto Research Chemicals (Toronto, Ontario, Canada).

Techniques:

Journal: Journal of Exposure Science & Environmental Epidemiology

Article Title: Exposure to tobacco smoke and validation of smoking status during pregnancy in the MIREC study

doi: 10.1038/s41370-017-0011-z

Figure Lengend Snippet: Histogram and kernel density estimate for log cotinine concentration in first trimester, showing cut-point of 5.21 ng/mL and setting values below LOD to a constant LOD/2

Article Snippet: The following calibration standards: (−)-Cotinine, S(−)-Nicotine, (±)-Cotinine-d3 and (±)-Nicotine-d4 were acquired from Cerilliant, while trans-3′-Hydroxycotinine and trans-3′-Hydroxycotinine-d3 were acquired from Toronto Research Chemicals (Toronto, Ontario, Canada).

Techniques: Concentration Assay

Journal: Journal of Exposure Science & Environmental Epidemiology

Article Title: Exposure to tobacco smoke and validation of smoking status during pregnancy in the MIREC study

doi: 10.1038/s41370-017-0011-z

Figure Lengend Snippet: Multiple logistic regression model for plasma cotinine detected in 1st trimester of non-smokers

Article Snippet: The following calibration standards: (−)-Cotinine, S(−)-Nicotine, (±)-Cotinine-d3 and (±)-Nicotine-d4 were acquired from Cerilliant, while trans-3′-Hydroxycotinine and trans-3′-Hydroxycotinine-d3 were acquired from Toronto Research Chemicals (Toronto, Ontario, Canada).

Techniques:

Journal: Journal of Exposure Science & Environmental Epidemiology

Article Title: Exposure to tobacco smoke and validation of smoking status during pregnancy in the MIREC study

doi: 10.1038/s41370-017-0011-z

Figure Lengend Snippet: Multiple linear regression model for log cotinine in cord plasma (parametric method)

Article Snippet: The following calibration standards: (−)-Cotinine, S(−)-Nicotine, (±)-Cotinine-d3 and (±)-Nicotine-d4 were acquired from Cerilliant, while trans-3′-Hydroxycotinine and trans-3′-Hydroxycotinine-d3 were acquired from Toronto Research Chemicals (Toronto, Ontario, Canada).

Techniques:

Journal: Drug Metabolism and Disposition

Article Title: Interindividual Variation in CYP3A Activity Influences Lapatinib Bioactivation

doi: 10.1124/dmd.119.088823

Figure Lengend Snippet: Measurement of CYP3A activity by midazolam 1′-hydroxylation and CYP3A5-selective activity by T-5 N-oxidation in genotyped human liver microsomes. (A) Midazolam (2.5 μM) was incubated with single-donor human liver microsomes (0.03 mg protein/ml) supplemented with NADPH-generating system for 4 minutes. (B) T-5 (5 μM) was incubated with single-donor human liver microsomes (0.1 mg protein/ml) supplemented with NADPH-generating system for 15 minutes. Formation of 1′-hydroxymidazolam and T-5 N-oxide was measured by LC-MS/MS analysis. Each point is the mean of three experiments, each performed in triplicate. CYP3A5*3/*3 donors, n = 5; CYP3A5*1/*3 donors, n = 3; CYP3A5*1/*1 donors, n = 4. Metabolite formation was compared across CYP3A5 genotypes by one-way ANOVA using GraphPad Prism 7 software.

Article Snippet: Midazolam, 1′-hydroxymidazolam, and d 4 -1′-hydroxymidazolam were purchased from Cerilliant Corporation (Round Rock, TX).

Techniques: Activity Assay, Incubation, Liquid Chromatography with Mass Spectroscopy, Software

Journal: Drug Metabolism and Disposition

Article Title: Interindividual Variation in CYP3A Activity Influences Lapatinib Bioactivation

doi: 10.1124/dmd.119.088823

Figure Lengend Snippet: Midazolam 1′-hydroxylation, T-5 N-oxidation, and lapatinib O-debenzylation in pooled human hepatocytes. Genotyped human hepatocytes were from a pool of three donors each. (A) Midazolam (2.5 μM) and (B) T-5 (5 μM) were incubated with hepatocytes (0.5 × 106 cells/ml) in suspension for 30 minutes. (C) Lapatinib (10 μM) was incubated with hepatocytes (0.5 × 106 cells/ml) in suspension for 2 hours. Formation of 1′-hydroxymidazolam, T-5 N-oxide, and lapatinib M1 was measured by LC-MS/MS analysis. Relative levels of M1 were determined by the ratio of M1 to lapatinib peak areas and normalized to CYP3A5*1/*1 donors. Each point is from a single experiment performed in replicate (two to four). The mean is indicated by the line, and errors are the S.D.

Article Snippet: Midazolam, 1′-hydroxymidazolam, and d 4 -1′-hydroxymidazolam were purchased from Cerilliant Corporation (Round Rock, TX).

Techniques: Incubation, Liquid Chromatography with Mass Spectroscopy

Journal: Drug Metabolism and Disposition

Article Title: Interindividual Variation in CYP3A Activity Influences Lapatinib Bioactivation

doi: 10.1124/dmd.119.088823

Figure Lengend Snippet: CYP3A and CYP3A5-selective activity in genotyped single-donor human hepatocytes. (A) Midazolam (2.5 μM) and (B) T-5 (5 μM) were incubated with hepatocytes (0.5 × 106 cells/ml) in suspension for 30 minutes. Formation of 1′-hydroxymidazolam (A) and T-5 N-oxide (B) was measured by LC-MS/MS analysis. Results are the mean values for each donor from experiments performed in triplicate. CYP3A5*3/*3 donors, n = 8; CYP3A5*1/*3 donors, n = 5; CYP3A5*1/*1 donors, n = 2. Comparison one-way ANOVA (GraphPad Prism 7).

Article Snippet: Midazolam, 1′-hydroxymidazolam, and d 4 -1′-hydroxymidazolam were purchased from Cerilliant Corporation (Round Rock, TX).

Techniques: Activity Assay, Incubation, Liquid Chromatography with Mass Spectroscopy