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Potential relationship between non-enzymatic antioxidants

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Potential relationship between non-enzymatic antioxidants

Postby ianjager » Thu Sep 18, 2008 5:08 pm

This is the first report regarding quantitation of enzymatic (eg. superoxide dismutase, glutathione reductase & glucose-6-phosphate dehydrogenase) and non-enzymatic (eg. glutathione) antioxidant levels in isolated Leydig cells that I can find in the scientific literature:

Mechanisms by which hypoxia augments Leydig cell viability and differentiated cell function in vitro (PhD Dissertation, 1993)

by Mark A. Kukucka, MS, DVM, PhD

Department of Biomedical Sciences
Virginia-Maryland Regional College of Veterinary Medicine
Virginia Polytechnic Institute & State University
Blacksburg, Virginia 24061-0442


ABSTRACT:

The 1980s heralded the discovery and identification of extra-pituitary sources of the neurohypophysial hormone oxytocin in non-neural tissues of several animal species. The presence, location and biosynthesis of significant amounts of oxytocin in the ovarian corpus luteum was followed by the immunocytochemical demonstration of an oxytocin-like peptide in the testicular interstitial cells. Leydig cells, which comprise up to 80% of the testicular intertubular cell population, are known to synthesize testosterone in situ. Indirect evidence indicated that an oxytocin-like peptide was also present in Leydig cells. The question arose whether this peptide was synthesized de novo by Leydig cells or was taken up and stored by the cells following biosynthesis at some other intra- and/or extra-gonadal source(s). Since luteinizing hormone (LH) and ascorbate are known to augment the production of oxytocin in ovarian granulose cells, varying concentrations of these two stimulants were used to monitor the biosynthesis of oxytocin from isolated Leydig cells in culture.

Highly enriched populations of guinea pig Leydig cells were isolated using a method that employed enzymatic dissociation and Percoll gradient centrifugation. Since ambient oxygen tensions are toxic to cultured Leydig cells leading to decreased steroidogenic capacity, the antioxidant defense system of isolated Leydig cells was discerned. Decreased levels of several antioxidants including superoxide dismutase, glutathione reductase, glucose-6-phosphate dehydrogenase and total glutathione were measured. Using the dichlorofluorescin (DCF-DA) assay, it was determined that isolated Leydig cells were capable of accumulating hydrogen peroxide (H2O2). Leydig cells maintained in an atmosphere composed of 19% oxygen produced H2O2 at a faster rate than similar cells incubated at 3% oxygen.

Using a polyclonal antibody (Ab)-based immunoaffinity column, oxytocin biosynthesis was monitored in Leydig cells incubated with a mildly stimulating dose (0.1 ng/ml) of ovine LH for 24, 48 and 72 hours in the presence of increasing concentrations of sodium ascorbate (1- 500 mM) under culture conditions of hypoxia and normoxia. Following solid phase extraction and immunoaffinity purification, sample supernatants were analyzed for both testosterone and oxytocin content as measured by radioimmunoassay (RIA) and high performance liquid chromatography-electrochemical detection (HPLC-ECD) respectively. Hypoxic culture conditions and low (1-10 mM) concentrations of sodium ascorbate augmented the production of oxytocin from Leydig cells in culture. Higher (50-500 mM) levels of ascorbate and normoxic culture conditions suppressed both testosterone and oxytocin production in isolated Leydig cells. Because oxytocin synthesis was found to be cycloheximide-sensitive, we conclude that Leydig cells possess the biosynthetic machinery necessary to manufacture oxytocin. The isolated oxytocin peptide was purified by HPLC with fraction collection followed by polyclonal-Ab immunoaffinity column chromatography. Comparison of the amino acid sequence of the isolated octapeptide with authentic oxytocin provides unequivocal evidence that Leydig cells synthesize oxytocin de novo. Considering the widespread use of vitamin C as a dietary supplement, the research reported yields valuable mechanistic information on the reproductive biologic role of vitamin C in gonadal steroid and peptide hormone metabolism.

http://scholar.lib.vt.edu/theses/availa ... 08-170416/


Vitamin C (aka ascorbate) is another non-enzymatic antioxidant found in significant levels within the testes. As an antioxidant, ascorbate's primary role is donate electrons to neutralize reactive species of oxygen including superoxide (to H2O2) and hydroxyl free radicals (to H2O). When ascorbate acts as a scavenger (by donating an electron to a free radical), ascorbate is oxidized in the process to the ascorbate free radical and dehydro-ascorbate. The ascorbate free radical and the dehydro-ascorbate are reduced back to ascorbate either by NADH catalyzed by semidehydroascorbate reductase (and forming NAD) or reduced glutathione (GSH) catalyzed by dehydroascorbate reductase (and forming oxidized glutathione (GSSG)).

n.b. Vitamin C also works along with glutathione peroxidase (a major free radical-fighting enzyme) to revitalize vitamin E.

Interestingly, Kukucka et. al. reported finding significant levels of oxytocin (a disulfide containing octapeptide) in isolated Leydig cells. Kukucka theorized in the introduction of his PhD dissertation (back in 1993) that open chain oxytoceine (the reduced form of oxytocin) may also act as a scavenger (by donating an electron to a free radical), oxytoceine may then be oxidized back to oxytocin. As noted above, the ascorbate free radical and the dehydro-ascorbate are reduced back to ascorbate either by NADH catalyzed by semidehydroascorbate reductase (and forming NAD) or reduced glutathione (GSH) catalyzed by dehydroascorbate reductase (and forming oxidized glutathione (GSSG)).... why couldn't the ascorbate free radical and dehydro-ascorbate be reduced back to ascorbate by reduced oxytoceine (forming closed-ring oxytocin)?

Thus, the redox potential of oxytocin <---> oxytoceine may drive ascorbate <---> dehydro-ascorbate or vice versa as part of the non-enzymatic antioxidant defense system.


Other publications by Dr. Mark A. Kukucka
http://www.nextbio.com/b/literature/lit ... MA+Kukucka
ianjager
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Postby ianjager » Sat Sep 20, 2008 6:27 pm

Could the redox potential of oxytoceine drive the formation of reduced ascorbate and oxytocin.... is this chemistry right?

Does anyone know the redox potentials for oxytoceine <----> oxytocin?

Does anyone know the redox potentials for ascorbate <----> dehydroascorbate?
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Re: Potential relationship between non-enzymatic antioxidants

Postby Hypoxia » Fri Sep 23, 2011 7:53 pm

We have some people using our C-Chambers to do this exact research right now. Feel free to follow the below link and ask a question to receive application notes on our Hypoxia Chamber http://www.biospherix.com/cell-culture-equipment/hypoxia-chamber-c-chamber.html.

This is a little old but it's worth a try, one of our customers.
https://qspace.library.queensu.ca/bitstream/1974/1394/1/Sullivan_Richard_M_200808_Phd.pdf

Don't Forget to mention Biology Online.

Thanks,
Brad
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