A Randomized Controlled Clinical Trial in Healthy Older Adults to Determine Efficacy of Glycine and N-Acetylcysteine Supplementation on Glutathione Redox Status and Oxidative Damage

Abstract

Glycine and cysteine are non-essential amino acids that are required to generate glutathione, an intracellular tripeptide that neutralizes reactive oxygen species and prevents tissue damage. During aging glutathione demand is thought to increase, but whether additional dietary intake of glycine and cysteine contributes towards the generation of glutathione in healthy older adults is not well understood. We investigated supplementation with glycine and n-acetylcysteine (GlyNAC) at three different daily doses for 2 weeks (low dose: 2.4 g, medium dose: 4.8 g, or high dose: 7.2 g/day, 1:1 ratio) in a randomized, controlled clinical trial in 114 healthy volunteers. Despite representing a cohort of healthy older adults (age mean = 65 years), we found significantly higher baseline levels of markers of oxidative stress, including that of malondialdehyde (MDA, 0.158 vs. 0.136 µmol/L, p < 0.0001), total cysteine (Cysteine-T, 314.8 vs. 276 µM, p < 0.0001), oxidized glutathione (GSSG, 174.5 vs. 132.3 µmol/L, p < 0.0001), and a lower ratio of reduced to oxidized glutathione (GSH-F:GSSG) (11.78 vs. 15.26, p = 0.0018) compared to a young reference group (age mean = 31.7 years, n = 20). GlyNAC supplementation was safe and well tolerated by the subjects, but did not increase levels of GSH-F:GSSG (end of study, placebo = 12.49 vs. 7.2 g = 12.65, p-value = 0.739) or that of total glutathione (GSH-T) (end of study, placebo = 903.5 vs. 7.2 g = 959.6 mg/L, p-value = 0.278), the primary endpoint of the study. Post-hoc analyses revealed that a subset of subjects characterized by high oxidative stress (above the median for MDA) and low baseline GSH-T status (below the median), who received the medium and high doses of GlyNAC, presented increased glutathione generation (end of study, placebo = 819.7 vs. 4.8g/7.2 g = 905.4 mg/L, p-value = 0.016). In summary GlyNAC supplementation is safe, well tolerated, and may increase glutathione levels in older adults with high glutathione demand. Clinical Trial Registration: https://clinicaltrials.gov/ct2/show/NCT05041179, NCT05041179.

Keywords: cardiometabolic diseases; glutathione; glycine; healthy aging; n-acetylcysteine; nutrition; oxidative stress; total cysteine.

FIGURE 1

Flowchart of study design. Summary of recruitment process of healthy young subjects and healthy older adults and their randomization to the placebo arm and the three treatment groups. Follow-up indicates the interim time point at 3 days after first dosing (V3). V= Visit; N= Numbers.

FIGURE 2

Healthy older adults show increased levels of markers of oxidative stress. (A) Levels of MDA, (B) total cysteine and (C) glycine measured in plasma samples of the study volunteers. (D) Levels of GSH-T, (E) GSSG and (F) free to oxidized glutathione (GSH-F:GSSG) in whole blood samples. Boxplot shows the median, the first to third quartile, the 1.5x interquartile ranges, and outliers. p = p-values, parametric t-statistics of LS-means.

FIGURE 3

Dosing efficacy of GlyNAC. (A) Dose responses to acute oral intake of GlyNAC on glycine and (B) oxidized cysteine in plasma of older adults at Visit 2. Values were obtained at −60 min prior and 60 min after consumption of the active doses or placebo. Red lines represent the means. p = p-values, parametric paired t-tests between pre and post dose.

FIGURE 4

Effects of GlyNAC on circulating levels of GSH-T. (A) Effects of daily intake of placebo or three different doses of GlyNAC on GSH-T at Visit 4. Values were measured in whole blood samples in fasted subjects. The last GlyNAC dose was consumed the evening before the measurement. (B,C) Post-hoc subset analysis of response to (B) placebo or (C) GlyNAC treatment (4.8 and 7.2 g groups) in subjects characterized by elevated levels of oxidative stress (MDA above the median) and low baseline glutathione values (GSH-T below the median) in fasted state at Visit 2 and Visit 4. (D) Post-hoc subset analysis of response to high doses of GlyNAC treatment (4.8 and 7.2 g) in subjects characterized by low oxidative stress in fasted state at Visit 2 and Visit 4. Boxplots show the median, the first to third quartile, the 1.5x interquartile ranges, and outliers. p = p-value, nonparametric paired Wilcoxon/Mann-Whitney tests.

FIGURE 5

Dose dependent effects of GlyNAC supplementation on glycine status. Effects of daily intake of placebo or different doses of GlyNAC on whole blood glycine levels in a subpopulation of older adults (excluding the group above the third quartile). Glycine levels have been compared before (baseline, Visit 2) and after the 2-weeks of treatment (end of study, Visit 4) in samples taken prior to the dosing with GlyNAC (−60 min) The red dashed lines represent the median of the young adults. p = p-value, linear mixed model testing intra-individual changes from baseline to end of study within each dose group and from placebo group.