Validation of a Reversed‐Phase High Performance Liquid Chromatography Method for the Simultaneous Analysis of Cysteine and Reduced Glutathione in Mouse Organs

Brundu, Serena; Nencioni, Lucia; Celestino, Ignacio; Coluccio, Paolo; Palamara, Anna Teresa; Magnani, Mauro; Fraternale, Alessandra

doi:10.1155/2016/1746985

Cited by 28 publications

(25 citation statements)

References 17 publications

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“…Evident reductions, although not significant, were found in the spleen, while no changes were found in the brain ( Figure 1A). Quantification of cysteine was more difficult because of the previously reported low content of this aminoacid in BALB/c mice 14 ; however, we observed in pancreas of old mice, where the highest concentration of cysteine was measured, a marked decrease in the aminoacid (P = 0.06) ( Figure 1B).…”

Section: Old Mice Contain Low Quantity Of Gsh and Cysteine In Diffementioning

confidence: 76%

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Glutathione increase by the n‐butanoyl glutathione derivative (GSH‐C4) inhibits viral replication and induces a predominant Th1 immune profile in old mice infected with influenza virus

et al. 2019

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During aging, glutathione (GSH) content declines and the immune system undergoes a deficiency in the induction of Th1 response. Reduced secretion of Th1 cytokines, which is associated with GSH depletion, could weaken the host defenses against viral infections. We first evaluated the concentration of GSH and cysteine in organs of old mice; then, the effect of the administration of the N‐butanoyl GSH derivative (GSH‐C4) on the response of aged mice infected with influenza A PR8/H1N1 virus was studied through the determination of GSH concentration in organs, lung viral titer, IgA and IgG1/IgG2a production, and Th1/Th2 cytokine profile. Old mice had lower GSH than young mice in organs. Also the gene expression of endoplasmic reticulum (ER) stress markers involved in GSH metabolism and folding of proteins, that is, Nrf2 and PDI, was reduced. Following infection, GSH content remained low and neither infection nor GSH‐C4 treatment affected Nrf2 expression. In contrast, PDI expression was upregulated during infection and appeared counterbalanced by GSH‐C4. Moreover, the treatment with GSH‐C4 increased GSH content in organs, reduced viral replication and induced a predominant Th1 response. In conclusion, GSH‐C4 treatment could be used in the elderly to contrast influenza virus infection by inducing immune response, in particular the Th1 profile.

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Section: Old Mice Contain Low Quantity Of Gsh and Cysteine In Diffementioning

confidence: 76%

“…GSH and cysteine in the spleen, lymph nodes, lungs, pancreas, and brain of BALB/c mice were quantified following a HPLC method validated according to US and European standards and applied for determination of the thiol species in several mouse organs …”

Section: Methodsmentioning

confidence: 99%

Glutathione increase by the n‐butanoyl glutathione derivative (GSH‐C4) inhibits viral replication and induces a predominant Th1 immune profile in old mice infected with influenza virus

et al. 2019

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“…The thiol content in the spleen, lymph nodes, pancreas, and brain of uninfected or infected mice was determined at 2, 5, and 9 weeks after virus inoculation by a high-performance liquid chromatography (HPLC) method which has recently been validated according to U.S. and European standards (46). Briefly, a portion of each organ (10 to 20 mg) was immersed in 500 l of precipitating solution (100 ml containing 1.67 g of glacial meta-phosphoric acid, 0.2 g of disodium EDTA, 30 g of NaCl), and then it was homogenized and sonicated.…”

Section: Methodsmentioning

confidence: 99%

Glutathione Depletion Is Linked with Th2 Polarization in Mice with a Retrovirus-Induced Immunodeficiency Syndrome, Murine AIDS: Role of Proglutathione Molecules as Immunotherapeutics

Brundu

Palma

Picceri

et al. 2016

J Virol

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Injection of the LP-BM5 murine leukemia virus into mice causes murine AIDS, a disease characterized by many dysfunctions of immunocompetent cells. To establish whether the disease is characterized by glutathione imbalance, reduced glutathione (GSH) and cysteine were quantified in different organs. A marked redox imbalance, consisting of GSH and/or cysteine depletion, was found in the lymphoid organs, such as the spleen and lymph nodes. Moreover, a significant decrease in cysteine and GSH levels in the pancreas and brain, respectively, was measured at 5 weeks postinfection. The Th2 immune response was predominant at all times investigated, as revealed by the expression of Th1/Th2 cytokines. Furthermore, investigation of the activation status of peritoneal macrophages showed that the expression of genetic markers of alternative activation, namely, Fizz1, Ym1, and Arginase1, was induced. Conversely, expression of inducible nitric oxide synthase, a marker of classical activation of macrophages, was detected only when Th1 cytokines were expressed at high levels. In vitro studies revealed that during the very early phases of infection, GSH depletion and the downregulation of interleukin-12 (IL-12) p40 mRNA were correlated with the dose of LP-BM5 used to infect the macrophages. Treatment of LP-BM5-infected mice with N-(N-acetyl-L-cysteinyl)-S-acetylcysteamine (I-152),an N-acetyl-cysteine supplier, restored GSH/cysteine levels in the organs, reduced the expression of alternatively activated macrophage markers, and increased the level of gamma interferon production, while it decreased the levels of Th2 cytokines, such as IL-4 and IL-5. Our findings thus establish a link between GSH deficiency and Th1/Th2 disequilibrium in LP-BM5 infection and indicate that I-152 can be used to restore the GSH level and a balanced Th1/Th2 response in infected mice. IMPORTANCEThe first report of an association between Th2 polarization and alteration of the redox state in LP-BM5 infection is presented. Moreover, it provides evidence that LP-BM5 infection causes a decrease in the thiol content of peritoneal macrophages, which can influence IL-12 production. The restoration of GSH levels by GSH-replenishing molecules can represent a new therapeutic avenue to fight this retroviral infection, as it reestablishes the Th1/Th2 balance. Immunotherapy based on the use of pro-GSH molecules would permit LP-BM5 infection and probably all those viral infections characterized by GSH deficiency and a Th1/ Th2 imbalance to be more effectively combated. Infection with the LP-BM5 murine leukemia virus causes a profound and broad immunodeficiency in susceptible mouse strains, such as C57BL/6 (B6) mice. This disease is known as murine AIDS (MAIDS) and is characterized by early polyclonal T-and B-cell activation, splenomegaly, lymphadenopathy, hypergammaglobulinemia, decreased T-and B-cell responses, increased susceptibility to opportunistic pathogens, and the development of terminal B-cell lymphomas and neurological dysfunctions (1-4). MAIDS is charact...

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“…C57BL/6 mice were infected and treated with I-152 (30 μmol/mouse) three times a week, every other day, for a total of 9 weeks. At 2, 5 and 9 weeks after virus inoculation, GSH content in the lymph nodes was determined by HPLC [39,42]. Data are the mean ± S.D.…”

Section: Figurementioning

confidence: 99%

“…CD-1 mice were i.p. injected with I-152 (30 µmol/mouse), and the thiol content was determined by HPLC [39]. (+) detectable; (/) undetectable; (↑) increased, (↓) decreased and (=) unchanged compared to control levels.…”

Section: Figurementioning

confidence: 99%

Boosting GSH Using the Co-Drug Approach: I-152, a Conjugate of N-acetyl-cysteine and β-mercaptoethylamine

et al. 2019

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Glutathione (GSH) has poor pharmacokinetic properties; thus, several derivatives and biosynthetic precursors have been proposed as GSH-boosting drugs. I-152 is a conjugate of N-acetyl-cysteine (NAC) and S-acetyl-β-mercaptoethylamine (SMEA) designed to release the parent drugs (i.e., NAC and β-mercaptoethylamine or cysteamine, MEA). NAC is a precursor of L-cysteine, while MEA is an aminothiol able to increase GSH content; thus, I-152 represents the very first attempt to combine two pro-GSH molecules. In this review, the in-vitro and in-vivo metabolism, pro-GSH activity and antiviral and immunomodulatory properties of I-152 are discussed. Under physiological GSH conditions, low I-152 doses increase cellular GSH content; by contrast, high doses cause GSH depletion but yield a high content of NAC, MEA and I-152, which can be used to resynthesize GSH. Preliminary in-vivo studies suggest that the molecule reaches mouse organs, including the brain, where its metabolites, NAC and MEA, are detected. In cell cultures, I-152 replenishes experimentally depleted GSH levels. Moreover, administration of I-152 to C57BL/6 mice infected with the retroviral complex LP-BM5 is effective in contrasting virus-induced GSH depletion, exerting at the same time antiviral and immunomodulatory functions. I-152 acts as a pro-GSH agent; however, GSH derivatives and NAC cannot completely replicate its effects. The co-delivery of different thiol species may lead to unpredictable outcomes, which warrant further investigation.

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Validation of a Reversed‐Phase High Performance Liquid Chromatography Method for the Simultaneous Analysis of Cysteine and Reduced Glutathione in Mouse Organs

Cited by 28 publications

References 17 publications

Glutathione increase by the n‐butanoyl glutathione derivative (GSH‐C4) inhibits viral replication and induces a predominant Th1 immune profile in old mice infected with influenza virus

Glutathione increase by the n‐butanoyl glutathione derivative (GSH‐C4) inhibits viral replication and induces a predominant Th1 immune profile in old mice infected with influenza virus

Glutathione Depletion Is Linked with Th2 Polarization in Mice with a Retrovirus-Induced Immunodeficiency Syndrome, Murine AIDS: Role of Proglutathione Molecules as Immunotherapeutics

Boosting GSH Using the Co-Drug Approach: I-152, a Conjugate of N-acetyl-cysteine and β-mercaptoethylamine

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