An ESR and HPLC-EC assay for the detection of alkyl radicals

Our plain-English summary of An ESR and HPLC-EC assay for the detection of alkyl radicals, by C.P. Novakov, D. Feierman, A.I. Cederbaum, et al (Chemical Research in Toxicology, 2001).

The study

• Authors: C.P. Novakov, D. Feierman, A.I. Cederbaum, D.A. Stoyanovsky
• Journal: Chemical Research in Toxicology
• Published: 2001;14(9):1239-46
• Cited by: 14 articles, as counted by NIH iCite
• Read it: PubMed · Publisher

Abstract

The correlation of lipid peroxidation with release of alkanes (RH) is considered a noninvasive method for the in vivo evaluation of oxidative stress. The formation of RH is believed to reflect a lipid hydroperoxide (LOOH)-dependent generation of alkoxyl radicals (LO*) that undergo beta-scission with release of alkyl radicals (R*). Alternatively, R* could be spin-trapped with a nitrone before the formation of RH and analyzed by ESR. Extracts from the liver and lung of CCl(4)- and asbestos-treated rats that were previously loaded with nitrones exhibited ESR spectra suggesting the formation of iso-propyl, n-butyl, ethyl, and pentyl radical-derived nitroxides. In biological systems, various nitroxides with indistinguishable ESR spectra could be formed. Hence, experiments with N-tert-butyl-alpha-phenylnitrone (PBN) for spin trapping of R* were carried out in which the nitroxides formed were separated and analyzed by HPLC with electrochemical detection (EC). The C(1-5) homologous series of PBN nitroxides and hydroxylamines were synthesized, characterized by ESR, GC-MS, and HPLC-EC, and used as HPLC standards. For in vivo generation and spin trapping of R*, rats were loaded with CCl(4) and PBN. The HPLC-EC chromatograms of liver extracts from CCl(4)-treated rats demonstrated the formation of both the nitroxide and hydroxylamine forms of PBN/*CCl(3), as well as the formation of a series of unidentified PBN nitroxides and hydroxylamines. However, formation of PBN adducts with retention times similar to these of the PBN/C(2-5) derivatives was not observed. In conclusion, we could not correlate the production of PBN-detectable alkyl radicals with the reported CCl(4)-dependent production of C(1-5) alkanes. We speculate that the major reason for this is the low steady-state concentrations of R* produced because only a small fraction of LO* undergo beta-scission to release R*.

In plain English

This was a laboratory chemistry study with an animal experiment. It did not study asbestos. The article mentions asbestos only in describing earlier reports by other researchers, in which liver and lung extracts from rats treated with carbon tetrachloride or asbestos gave signals attributed to alkyl radicals. Alkyl radicals are short-lived molecules thought to form when fats in cell membranes are damaged. The authors made reference versions of the products formed when the trapping chemical PBN captures one- to five-carbon alkyl radicals. They showed that liquid chromatography with electrochemical detection could separate products that looked the same by electron spin resonance. They then gave 9 rats, pretreated with pyrazole to induce the liver enzyme P450 2E1, carbon tetrachloride and PBN (5 to 200 mg per kg of body weight), and analysed liver extracts. They found PBN products of the trichloromethyl radical and several unidentified products. They found no evidence of ethyl, isopropyl, n-butyl, pentyl or isopentyl products. The authors concluded this was likely because alkyl radicals are present at low concentrations. They noted that other trapping chemicals might work better.

We wrote this plain-English summary from the full article with the help of AI, and a second AI model checked each sentence against the article. It is not the authors' wording; the abstract and the article are their own account.

What the study did and found

• Design: Laboratory method-development study (chemical synthesis and analysis) combined with an animal experiment in rats; not a study of asbestos
• Who was studied: Sprague-Dawley rats (9 animals, the article prints 'n ) 9'), weighing 120 to 150 g, pretreated with pyrazole; plus laboratory-made chemical standards
• Exposure: No asbestos exposure in this study. To induce the liver enzyme P450 2E1, rats were pretreated with pyrazole (200 mg/kg body weight per day for 3 days). They were then given the spin trap PBN (5 to 200 mg/kg body weight) and, 15 minutes later, carbon tetrachloride injected into the abdomen (1 mL of pure CCl4 per kg). Rats were killed 60 minutes after the carbon tetrachloride.
• Main results:
  • In the laboratory standards, the PBN products of one- to five-carbon alkyl radicals took longer to come off the chromatography column as the carbon chain got longer.
  • The propyl and pentyl PBN products could not be told apart by electron spin resonance, but chromatography with electrochemical detection separated both forms of each.
  • In liver extracts from carbon tetrachloride-treated rats, the nitroxide and hydroxylamine forms of the PBN product of the trichloromethyl radical were detected, together with an unidentified nitroxide.
  • The trichloromethyl PBN product peaks in rat liver extracts decreased considerably when lower doses of PBN were given.
  • No chromatography evidence was found in rat liver extracts for the ethyl, isopropyl, n-butyl, pentyl or isopentyl PBN products the authors had expected.
  • Additional PBN products were seen in the rat liver extracts, but their structure remained unknown and they did not match the one- to five-carbon alkyl products.
  • The authors also reported, without showing data, that they could not detect the alkyl PBN products in laboratory liver-microsome systems used to induce lipid peroxidation.
• The authors' conclusion: The authors concluded that they had established chromatography methods for analysing PBN-trapped radicals. They wrote that their attempt to trap alkyl radicals in living rats was unsuccessful, and that this was likely because alkyl radicals are present at low steady-state concentrations.
• Limitations the authors state:
  • The authors stated that the unstable nature of lipid-derived radicals and their many breakdown pathways reduce how efficiently alkyl radicals can be trapped. They called this a severe limitation of such indirect detection techniques.
  • The authors stated that their data do not rule out that other trapping chemicals (nitrones) could capture alkyl radicals more efficiently.
  • The authors noted that the electron spin resonance signal they attributed to the trichloromethyl product could also come from lipid- and alkyl-derived PBN products.

Topics

Medical terms in this study, explained in our Lexicon: Oxidative Stress, Retention TIme.

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