Main Research Findings

1) Daily consumption of glutathione supplementation was found to increase compartment stores of glutathione within the body.

2) Administration of glutathione precursors was found to increase cellular stores of glutathione and ultimately protect the skin from oxidative and environmental stress.

Selected Data

1) This study performed by Richie et al employed a randomized, double-blinded, placebo-controlled design to investigate the long-term effects of oral glutathione (GSH) supplementation on body stores of GSH in healthy adults. To recruit participants, the study coordinator utilized a multifaceted approach, employing fliers, newspaper and radio advertisements, online announcements, and word-of-mouth referrals to reach potential subjects in the local Hershey/Harrisburg, PA area. Interested individuals were initially prescreened via telephone to assess their potential eligibility based on predetermined inclusion and exclusion criteria. Individuals who passed the initial screening were invited to visit the clinic, where they provided informed consent and underwent a comprehensive eligibility screening.

The inclusion criteria stipulated that participants be healthy male and female non-smokers, aged 30-79 years, who had not taken antioxidant supplements for at least one month prior to enrollment. Exclusion criteria encompassed a history of chronic disease or antioxidant GSH supplement usage within the past month. Eligible subjects were then randomly assigned to one of three treatment groups with equal probability: a low-dose GSH group receiving 250 mg GSH per day orally, a high-dose GSH group receiving 1,000 mg GSH per day orally, and a placebo group receiving an inactive substance.

The GSH and placebo capsules were formulated as follows: GSH 125 mg, (125 mg GSH, 360 mg cellulose); GSH 500 mg (500 mg GSH, 15 mg cellulose); and placebo (470 mg cellulose). Critically, both subjects and investigators were blinded to the group assignment, ensuring impartiality throughout the study. At baseline, trained nurse interviewers administered a structured questionnaire to each subject to collect comprehensive information on demographics, occupation, lifestyle habits, medical history, usage of medication and dietary supplements, alcohol consumption, and past cigarette smoking history. University pharmacists dispensed either active supplements or placebo at the baseline visit, and subjects were provided detailed instructions for supplement usage. Participants were instructed to continue taking the capsules for six months, maintain a daily pill diary, and return all unused capsules at each visit.

To assess the reversibility of any GSH-related changes, there was a one-month washout period between six and seven months. After one, three, and six months, subjects returned to the clinic to return their unused capsules and receive new capsules. At baseline and after one, three, six, and seven months, biological samples including blood, urine, and exfoliated buccal mucosal cells were collected. Outcome measures included GSH levels in whole blood, erythrocytes, plasma, and lymphocytes, as well as exfoliated buccal mucosal cells. Secondary outcomes included immune function activities, such as NK cell cytotoxicity and lymphocyte proliferation at baseline and three months, and respiratory burst and neutrophil phagocytosis at baseline and three and six months.

2) This study completed by the research team of Cui et al evaluated the protective effects of topical glutathione amino acid precursors on skin health when faced with environmental and oxidative stresses. The experimental design integrated in vitro cell culture assays, ex vivo skin models, and an in vivo clinical trial, providing a comprehensive understanding of the mechanisms and efficacy of glutathione amino acid precursors. The primary goal was to determine if glutathione amino acid precursors could enhance cellular glutathione (GSH) levels and provide skin protection against stressors like oxidative agents, UV radiation, and pollutants.

The in vitro studies formed the foundation of the investigation. Normal human epidermal keratinocytes were cultivated under controlled laboratory conditions. These cells were then exposed to various stress-inducing agents, including menadione to induce oxidative stress, blue light, and pollutants, mimicking real-world environmental challenges. A key aspect of the in vitro work was the meticulous quantification of GSH levels, as well as related markers such as glutathione disulfide, reactive oxygen species (ROS), and adenosine triphosphate (ATP). To assess glutathione amino acid precursors effectiveness, its performance was compared against ascorbic acid (AA), a well-known antioxidant often used in skincare. These comparisons used cell viability assays, reactive oxygen species assays, and specific quantification methods such as UPLC-MS/MS.

To move beyond basic cell culture, the study incorporated ex vivo skin models. These models, derived from discarded human skin, maintain some of the structural and functional complexity of native skin. By treating these ex vivo skin samples with glutathione amino acid precursors prototypes before exposing them to UVA or UVB radiation, researchers could evaluate glutathione amino acid precursor’s protective effects against UV-induced damage. The assessment of DNA damage using markers like cyclobutene pyrimidine dimers, and the analysis of barrier proteins filaggrin and loricrin, provided insights into how glutathione amino acid precursors may shield the skin from UV-related harm. These analyses were facilitated by immunohistochemical staining techniques, allowing for visualization and characterization of specific proteins and cellular structures.

The in vivo component of the study took the form of a randomized, double-blind, placebo-controlled clinical trial. This trial involved 21 healthy female participants with Fitzpatrick skin types III and IV. In this study, the effects of the investigational products were measured using D-Squame tape strip samples to quantify GSH and glutathione disulfide levels, offering valuable insights into the impact of glutathione amino acid precursors on skin’s endogenous antioxidant defense. The clinical trial was a key component of this study, as it allowed the researchers to translate their in vitro and ex vivo findings to a real-world scenario, providing more relevant information on how the studied compound behaves in situ.

Discussion

1) The results of the study conducted by Richie et al demonstrated that oral glutathione (GSH) supplementation can effectively increase body compartment stores of GSH in healthy adults. This conclusion is based on the findings from a six-month randomized, double-blinded, placebo-controlled trial involving 54 non-smoking adults who were assigned to receive either a placebo, 250 mg/day of GSH, or 1000 mg/day of GSH. The key outcome measures were GSH levels in various body compartments, including blood, erythrocytes, plasma, lymphocytes, and exfoliated buccal mucosal cells. The study also assessed secondary outcomes related to immune function in a subset of participants.

The results indicated that GSH levels in blood increased significantly after one, three, and six months compared to baseline in both the low-dose and high-dose GSH groups, while no such increase was observed in the placebo group. Notably, at six months, mean GSH levels in erythrocytes, plasma, and lymphocytes had increased by 30-35% in the high-dose group, which was significantly greater than the increases observed in the placebo group. In exfoliated buccal cells, a remarkable 260% increase in GSH levels was observed in the high-dose group at six months, again highlighting the effectiveness of GSH supplementation.

Figure 1: Effects of oral GSH supplementation on levels of GSH in the blood samples and specific erythrocyte samples after 1 month, 3 months, 6 months, and 7 months of treatment.

The low-dose GSH group also experienced significant increases in GSH levels, specifically showing a 17% increase in blood and a 29% increase in erythrocytes at six months compared to baseline. These increases in the low-dose group were statistically significant compared to baseline, although not to the same extent as the high-dose group. In most cases, the increases in GSH levels were dose- and time-dependent, indicating that the higher dose and longer duration of supplementation were associated with greater increases in GSH stores. However, it is essential to note that GSH levels in both dose groups generally returned to baseline levels after a one-month washout period, suggesting that continuous supplementation is necessary to maintain elevated GSH levels. One thing to note is that the 1,000 mg group levels remained above baseline levels after the wash out.

In addition to assessing GSH levels, the study examined the impact of GSH supplementation on oxidative stress. Results showed that in both GSH dose groups, there was a reduction in oxidative stress, as indicated by decreases in the ratio of oxidized to reduced glutathione in whole blood after six months. This finding suggests that GSH supplementation may help to shift the balance towards a more reduced environment, potentially protecting against oxidative damage. The supplementation also appeared to have a direct effect on immune function. Natural killer (NK) cell cytotoxicity increased significantly in the high-dose group compared to the placebo group at three months, indicating a potential enhancement of immune function with GSH supplementation.

Further analyses revealed that changes in blood GSH were highly correlated with those in erythrocytes, and changes in lymphocyte GSH were significantly correlated with those in both blood and erythrocytes. Interestingly, no associations were observed for GSH changes in buccal cells. No significant differences were observed between the groups for other immune parameters or GCL activity after supplementation. The observed results in the NK cell data could also be related to the few male participants. Overall, these results suggest that oral GSH supplementation can effectively increase body stores of GSH and may also reduce oxidative stress and enhance certain aspects of immune function, particularly NK cell cytotoxicity.

Figure 2: Effects of oral GSH supplementation on levels of GSH in plasma, lymphocytes, and buccal cells after 1 month, 3 months, 6 months, and 7 months of treatment.

2) The findings of the study performed by Cui et al provided evidence that topical application of glutathione amino acid precursors effectively enhances cellular glutathione (GSH) levels and protects the skin from oxidative and environmental stressors. Through a carefully designed series of experiments, the researchers demonstrated the benefits of glutathione amino acid precursors across in vitro, ex vivo, and in vivo models, highlighting its potential as a valuable ingredient in skincare formulations.

In vitro studies using normal human epidermal keratinocytes revealed that treatment with glutathione amino acid precursors significantly increased both GSH and glutathione disulfide levels, leading to an improved GSH/glutathione disulfide ratio, which is a key indicator of cellular redox balance. The protective effects of glutathione amino acid precursors were further underscored when cells were challenged with menadione, a known inducer of oxidative stress. Menadione treatment depleted cellular GSH and reduced the GSH/glutathione disulfide ratio, but glutathione amino acid precursor supplementation effectively restored GSH levels and improved the redox balance. This protective effect extended to other stressors, as glutathione amino acid precursors also shielded cells from blue light-induced ROS production and pollutant-induced ATP depletion, demonstrating a broad-spectrum protective action. Notably, glutathione amino acid precursor’s protective capabilities against menadione-induced oxidation were found to surpass those of ascorbic acid (AA), suggesting a superior antioxidant mechanism.

The researchers’ investigations also focused on the mechanism behind glutathione amino acid precursors efficacy. By comparing glutathione amino acid precursors to other antioxidants in vitro, such as ascorbic acid, the study showed that it was better than other comparable ingredients. This is because of the unique ability of glutathione amino acid precursors to protect cells and catalyze the elimination of xenobiotics. Through various measurements, the researchers also found that exposure to environmental stresses resulted in ATP level decline in the test cultures. But glutathione amino acid precursors mitigated this effect, making it more useful than standard antioxidants to protect skin against environmental pollutants.

Moving beyond cell-based assays, the study utilized ex vivo skin models to examine the UV-protective effects of glutathione amino acid precursors. Topical application of a glutathione amino acid precursor formulation to ex vivo skin samples prior to UVB irradiation led to a significant suppression of cyclobutene pyrimidine dimers (CPD), markers of DNA damage. Similarly, under UVA irradiation, glutathione amino acid precursor treatment resulted in lower levels of oxidative DNA lesions, as indicated by 8-OHdG staining. These findings suggest that glutathione amino acid precursor’s protective mechanism extends to shielding the skin from UV-induced DNA damage, a critical factor in preventing photoaging and skin cancer. Moreover, IHC analysis of filaggrin and loricrin, key proteins involved in epidermal barrier function, revealed that glutathione amino acid precursors treatment enhanced their levels in UVB-exposed skin, supporting the protective effects of glutathione amino acid precursors on barrier function.

In vivo investigations were conducted through a randomized, double-blind, placebo-controlled clinical study that enrolled 21 healthy subjects. The results confirmed the findings that glutathione amino acid precursors work to benefit the skin. In the clinical study, the glutathione amino acid precursor treatment restored GSH/glutathione disulfide levels after UV treatment, confirming its in vivo GSH-boosting effects. It’s important to note that in vitro models suggested that glutathione amino acid precursors are responsible for increasing skin thickness, but further research is required to better understand how topical application has any role in promoting skin structure, skin appearance and collagen creation.

In conclusion, this study supports the use of glutathione amino acid precursors in skincare formulations. Through experimentation, the researchers demonstrated that glutathione amino acid precursors effectively increase cellular GSH levels, protects skin cells from oxidative and environmental stressors, reduces UV-induced DNA damage, and supports epidermal barrier function. These findings provide a solid scientific basis for the development of skincare products that harness the power of GSH to promote skin health and resilience.

Disclaimer

**LAB USE ONLY**
*This information is for educational purposes only and does not constitute medical advice. THE PRODUCTS DESCRIBED HEREIN ARE FOR RESEARCH USE ONLY. All clinical research must be conducted with oversight from the appropriate Institutional Review Board (IRB). All preclinical research must be conducted with oversight from the appropriate Institutional Animal Care and Use Committee (IACUC) following the guidelines of the Animal Welfare Act (AWA).

Citations

[1] Richie JP Jr, Nichenametla S, Neidig W, et al. Randomized controlled trial of oral glutathione supplementation on body stores of glutathione. Eur J Nutr. 2015;54(2):251-263. doi:10.1007/s00394-014-0706-z

[2] Cui X, Mi T, Xiao X, et al. Topical glutathione amino acid precursors protect skin against environmental and oxidative stress. J Eur Acad Dermatol Venereol. 2024;38 Suppl 3:3-11. doi:10.1111/jdv.19717

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