Determination of NAD⁺ and NADH level in a Single Cell Under H₂O₂ Stress by Capillary Electrophoresis

Thesis/Dissertation · Tue Jan 01 04:00:00 EST 2008

DOI:https://doi.org/10.2172/939381· OSTI ID:939381

Xi, Wenjun ^[1]

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1. Iowa State Univ., Ames, IA (United States)

A capillary electrophoresis (CE) method is developed to determine both NAD⁺ and NADH levels in a single cell, based on an enzymatic cycling reaction. The detection limit can reach down to 0.2 amol NAD⁺ and 1 amol NADH on a home-made CE-LIF setup. The method showed good reproducibility and specificity. After an intact cell was injected into the inlet of a capillary and lysed using a Tesla coil, intracellular NAD⁺ and NADH were separated, incubated with the cycling buffer, and quantified by the amount of fluorescent product generated. NADH and NAD⁺ levels of single cells of three cell lines and primary astrocyte culture were determined using this method. Comparing cellular NAD⁺ and NADH levels with and without exposure to oxidative stress induced by H₂O₂, it was found that H9c2 cells respond to the stress by reducing both cellular NAD⁺ and NADH levels, while astrocytes respond by increasing cellular NADH/NAD⁺ ratio.

• Research Organization: Ames Lab., Ames, IA (United States)
• Sponsoring Organization: USDOE Office of Science (SC)
• DOE Contract Number: AC02-07CH11358
• OSTI ID: 939381
• Report Number(s): IS--T 2782
• Country of Publication: United States
• Language: English

Related Subjects

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CHEMISTRY
ELECTROPHORESIS
SENSITIVITY
SPECIFICITY