Metabolism of lipid derived aldehyde, 4-hydroxynonenal in human lens epithelial cells and rat lens

Sanjeev Choudhary, Sanjay Srivastava, Tianlin Xiao, Usha P. Andley, Satish Srivastava, Naseem Ansari

Research output: Contribution to journalArticle

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Abstract

PURPOSE. An earlier study showed that 4-hydroxynonenal (HNE), formed as a result of increased lipid peroxidation in oxidative stress, causes loss of lens transparency. To determine how HNE is detoxified in ocular tissues, its metabolism in cultured human lens epithelial cells (HLECs) as well as rat lens was investigated. METHODS. Rat lens or HLECs were incubated with 30 nmol (5 × 105 cpm/μmol) of HNE in 2 mL Krebs-Hansleit buffer for 1 hour at 37°C. The medium, after ultrafiltration was analyzed by high performance liquid chromatography (HPLC), using a C-18 reversed-phase column. The metabolites were separated by using a gradient consisting of solvent A (0.1% aqueous trifluoroacetic acid) and solvent B (100% acetonitrile) at a flow rate of 1 mL/min. Fractions containing radioactivity were pooled and analyzed using electrospray ionization mass spectroscopy (ESI-MS) or gas chromatography-chemical ionization mass spectroscopy (GC/CI-MS). RESULTS. On HPLC, the incubation media from cultured lens and HLECs separated into three major radioactive peaks. Peak I of the HLECs and lens treated with HNE was identified to be a mixture of glutathione (GS) conjugates of HNE and 1,4-dihydroxy-2-nonene (DHN). The identity of the conjugates was confirmed by ESI-MS. Based on the retention times, peaks II, and III were assigned to 4-hydroxy-2-nonenoic acid (HNA) and unmetabolized HNE, respectively. The identities of HNA and HNE were confirmed by spiking the tissue extracts with synthetic metabolites and finally by GC/CI-MS. Sorbinil, an aldose reductase (AR) inhibitor, attenuated GS-DHN levels and cyanamide, an aldehyde dehydrogenase inhibitor, decreased formation of HNA. CONCLUSIONS. The results show that the major metabolic transformation of HNE in rat lens and HLECs involves conjugation with GS and oxidation to HNA. The GS-HNE conjugate is reduced to GS-DHN by AR. Thus, under normal physiological conditions, the lens has multiple routes to detoxify HNE. However, oxidative stress may overwhelm the metabolic capacity of the lens to detoxify HNE and lead to formation of cataract.

Original languageEnglish (US)
Pages (from-to)2675-2682
Number of pages8
JournalInvestigative Ophthalmology and Visual Science
Volume44
Issue number6
DOIs
StatePublished - Jun 1 2003

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Lipid Metabolism
Aldehydes
Lenses
Epithelial Cells
Mass Spectrometry
Aldehyde Reductase
Gas Chromatography
4-hydroxy-2-nonenal
Acids
Oxidative Stress
Cyanamide
High Pressure Liquid Chromatography
Trifluoroacetic Acid
Aldehyde Dehydrogenase
Tissue Extracts
Ultrafiltration
Cataract
Radioactivity
Lipid Peroxidation
Glutathione

ASJC Scopus subject areas

  • Ophthalmology

Cite this

Metabolism of lipid derived aldehyde, 4-hydroxynonenal in human lens epithelial cells and rat lens. / Choudhary, Sanjeev; Srivastava, Sanjay; Xiao, Tianlin; Andley, Usha P.; Srivastava, Satish; Ansari, Naseem.

In: Investigative Ophthalmology and Visual Science, Vol. 44, No. 6, 01.06.2003, p. 2675-2682.

Research output: Contribution to journalArticle

Choudhary, Sanjeev ; Srivastava, Sanjay ; Xiao, Tianlin ; Andley, Usha P. ; Srivastava, Satish ; Ansari, Naseem. / Metabolism of lipid derived aldehyde, 4-hydroxynonenal in human lens epithelial cells and rat lens. In: Investigative Ophthalmology and Visual Science. 2003 ; Vol. 44, No. 6. pp. 2675-2682.
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abstract = "PURPOSE. An earlier study showed that 4-hydroxynonenal (HNE), formed as a result of increased lipid peroxidation in oxidative stress, causes loss of lens transparency. To determine how HNE is detoxified in ocular tissues, its metabolism in cultured human lens epithelial cells (HLECs) as well as rat lens was investigated. METHODS. Rat lens or HLECs were incubated with 30 nmol (5 × 105 cpm/μmol) of HNE in 2 mL Krebs-Hansleit buffer for 1 hour at 37°C. The medium, after ultrafiltration was analyzed by high performance liquid chromatography (HPLC), using a C-18 reversed-phase column. The metabolites were separated by using a gradient consisting of solvent A (0.1{\%} aqueous trifluoroacetic acid) and solvent B (100{\%} acetonitrile) at a flow rate of 1 mL/min. Fractions containing radioactivity were pooled and analyzed using electrospray ionization mass spectroscopy (ESI-MS) or gas chromatography-chemical ionization mass spectroscopy (GC/CI-MS). RESULTS. On HPLC, the incubation media from cultured lens and HLECs separated into three major radioactive peaks. Peak I of the HLECs and lens treated with HNE was identified to be a mixture of glutathione (GS) conjugates of HNE and 1,4-dihydroxy-2-nonene (DHN). The identity of the conjugates was confirmed by ESI-MS. Based on the retention times, peaks II, and III were assigned to 4-hydroxy-2-nonenoic acid (HNA) and unmetabolized HNE, respectively. The identities of HNA and HNE were confirmed by spiking the tissue extracts with synthetic metabolites and finally by GC/CI-MS. Sorbinil, an aldose reductase (AR) inhibitor, attenuated GS-DHN levels and cyanamide, an aldehyde dehydrogenase inhibitor, decreased formation of HNA. CONCLUSIONS. The results show that the major metabolic transformation of HNE in rat lens and HLECs involves conjugation with GS and oxidation to HNA. The GS-HNE conjugate is reduced to GS-DHN by AR. Thus, under normal physiological conditions, the lens has multiple routes to detoxify HNE. However, oxidative stress may overwhelm the metabolic capacity of the lens to detoxify HNE and lead to formation of cataract.",
author = "Sanjeev Choudhary and Sanjay Srivastava and Tianlin Xiao and Andley, {Usha P.} and Satish Srivastava and Naseem Ansari",
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T1 - Metabolism of lipid derived aldehyde, 4-hydroxynonenal in human lens epithelial cells and rat lens

AU - Choudhary, Sanjeev

AU - Srivastava, Sanjay

AU - Xiao, Tianlin

AU - Andley, Usha P.

AU - Srivastava, Satish

AU - Ansari, Naseem

PY - 2003/6/1

Y1 - 2003/6/1

N2 - PURPOSE. An earlier study showed that 4-hydroxynonenal (HNE), formed as a result of increased lipid peroxidation in oxidative stress, causes loss of lens transparency. To determine how HNE is detoxified in ocular tissues, its metabolism in cultured human lens epithelial cells (HLECs) as well as rat lens was investigated. METHODS. Rat lens or HLECs were incubated with 30 nmol (5 × 105 cpm/μmol) of HNE in 2 mL Krebs-Hansleit buffer for 1 hour at 37°C. The medium, after ultrafiltration was analyzed by high performance liquid chromatography (HPLC), using a C-18 reversed-phase column. The metabolites were separated by using a gradient consisting of solvent A (0.1% aqueous trifluoroacetic acid) and solvent B (100% acetonitrile) at a flow rate of 1 mL/min. Fractions containing radioactivity were pooled and analyzed using electrospray ionization mass spectroscopy (ESI-MS) or gas chromatography-chemical ionization mass spectroscopy (GC/CI-MS). RESULTS. On HPLC, the incubation media from cultured lens and HLECs separated into three major radioactive peaks. Peak I of the HLECs and lens treated with HNE was identified to be a mixture of glutathione (GS) conjugates of HNE and 1,4-dihydroxy-2-nonene (DHN). The identity of the conjugates was confirmed by ESI-MS. Based on the retention times, peaks II, and III were assigned to 4-hydroxy-2-nonenoic acid (HNA) and unmetabolized HNE, respectively. The identities of HNA and HNE were confirmed by spiking the tissue extracts with synthetic metabolites and finally by GC/CI-MS. Sorbinil, an aldose reductase (AR) inhibitor, attenuated GS-DHN levels and cyanamide, an aldehyde dehydrogenase inhibitor, decreased formation of HNA. CONCLUSIONS. The results show that the major metabolic transformation of HNE in rat lens and HLECs involves conjugation with GS and oxidation to HNA. The GS-HNE conjugate is reduced to GS-DHN by AR. Thus, under normal physiological conditions, the lens has multiple routes to detoxify HNE. However, oxidative stress may overwhelm the metabolic capacity of the lens to detoxify HNE and lead to formation of cataract.

AB - PURPOSE. An earlier study showed that 4-hydroxynonenal (HNE), formed as a result of increased lipid peroxidation in oxidative stress, causes loss of lens transparency. To determine how HNE is detoxified in ocular tissues, its metabolism in cultured human lens epithelial cells (HLECs) as well as rat lens was investigated. METHODS. Rat lens or HLECs were incubated with 30 nmol (5 × 105 cpm/μmol) of HNE in 2 mL Krebs-Hansleit buffer for 1 hour at 37°C. The medium, after ultrafiltration was analyzed by high performance liquid chromatography (HPLC), using a C-18 reversed-phase column. The metabolites were separated by using a gradient consisting of solvent A (0.1% aqueous trifluoroacetic acid) and solvent B (100% acetonitrile) at a flow rate of 1 mL/min. Fractions containing radioactivity were pooled and analyzed using electrospray ionization mass spectroscopy (ESI-MS) or gas chromatography-chemical ionization mass spectroscopy (GC/CI-MS). RESULTS. On HPLC, the incubation media from cultured lens and HLECs separated into three major radioactive peaks. Peak I of the HLECs and lens treated with HNE was identified to be a mixture of glutathione (GS) conjugates of HNE and 1,4-dihydroxy-2-nonene (DHN). The identity of the conjugates was confirmed by ESI-MS. Based on the retention times, peaks II, and III were assigned to 4-hydroxy-2-nonenoic acid (HNA) and unmetabolized HNE, respectively. The identities of HNA and HNE were confirmed by spiking the tissue extracts with synthetic metabolites and finally by GC/CI-MS. Sorbinil, an aldose reductase (AR) inhibitor, attenuated GS-DHN levels and cyanamide, an aldehyde dehydrogenase inhibitor, decreased formation of HNA. CONCLUSIONS. The results show that the major metabolic transformation of HNE in rat lens and HLECs involves conjugation with GS and oxidation to HNA. The GS-HNE conjugate is reduced to GS-DHN by AR. Thus, under normal physiological conditions, the lens has multiple routes to detoxify HNE. However, oxidative stress may overwhelm the metabolic capacity of the lens to detoxify HNE and lead to formation of cataract.

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