Nicotinamide mononucleotide (NMN) is a precursor of nicotinamide adenine dinucleotide (NAD), which declines with age. Supplementation of NMN has been shown to improve blood NAD concentration. However, the optimal NMN dose remains unclear. This is a post-hoc analysis of a double-blinded clinical trial involving 80 generally healthy adults aged 40 to 65 years. The participants received a placebo or daily 300 mg, 600 mg, or 900 mg NMN for 60 days. Blood NAD concentration, blood biological age, homeostatic model assessment for insulin resistance, 6-minute walk test, and 36-item short-form survey (SF-36) were measured at baseline and after supplement. A significant dose-dependent increase in NAD concentration change (NAD Δ) was observed following NMN supplementation, with a large coefficient of variation (29.2-113.3%) within group. The increase in NAD Δ was associated with an improvement in the walking distance of 6-minute walk test and the SF-36 score. The median effect dose of NAD Δ for the 6-minute walk test and SF-36 score was 15.7 nmol/L (95% CI: 10.9-20.5 nmol/L) and 13.5 nmol/L (95% CI; 10.5-16.5 nmol/L), respectively. Because of the high interindividual variability of the NAD Δ after NMN supplementation, monitoring NAD concentration can provide valuable insights for tailoring personalized dosage regimens and optimizing NMN utilization.
Nicotinamide mononucleotide (NMN) is a bioactive nucleotide formed by the reaction between a nucleoside containing ribose, nicotinamide (NAM), and a phosphate group. It is a precursor of nicotinamide adenine dinucleotide (NAD), a crucial cofactor for enzymes involved in major biological processes such as cellular redox regulation and metabolism, and DNA repair. Blood NAD concentration declines with chronological age in animals and humans due to the declined de novo synthesis of NAD and the hyperactivity of NAD-consuming enzymes. Preventing the depletion of NAD concentration in in-vitro studies had neuroprotective, cardioprotective effects, and promoted DNA repair.
Dietary supplementation with NMN and other NAD precursors has been shown to increase the level of NAD concentration in the blood of middle-aged and older individuals. While inter-individual variability in the increase of blood NAD after NMN supplementation has been observed in previous studies, there are no studies reporting the relationship between change in blood NAD concentration (NAD Δ) following NMN supplementation and aging-related clinical outcomes.
This study reports the determinants of blood NAD basal level and NAD Δ, the relationship between the blood NAD concentration and the change in blood biological age (blood biological age Δ), change in homeostatic model assessment for insulin resistance (HOMA-IR Δ), change in 6-minute walk test (6-minute walk test Δ), change in 36-item short form survey scores (SF-36 Δ), and the variance and median effective dose (ED 50) of NAD Δ after 30 or 60 days of NMN supplementation in healthy middle-aged individuals.
The study design of this double-blinded, randomized clinical trial was reported elsewhere, The trial was conducted in Lotus Healthcare and Aesthetics Clinic and Sunad Ayurved in Pune, India, and monitored by a clinical research organization (CRO) in Pune, ProRelix Services LLP. Briefly, generally healthy 40 to 65 years old individuals with a body mass index between (BMI) 18.5 to 35 kg/m2 were included in this study. Participants were randomized into four groups receiving placebo or daily NMN supplements of 300 mg, 600 mg, and 900 mg, respectively, for 60 days. Age, sex, BMI, blood biological age (calculated by Aging.AI 3.0) and HOMA-IR were recorded at baseline and day 60. In addition, blood NAD concentration, 6-minute walk test, and 36-item short-form survey (SF- 36) scores were measured at baseline, 30 days, and 60 days. All participants provided written informed consent. The ethical approval was obtained from the Royal Ethics Committee, Pune, India.
The normality of continuous variables was tested by the Shapiro-Wilk test. Data were presented as means and standard deviations (SD) or median and inter-quantile range (IQR) according to normality for continuous variables or numbers and percentages (%) for categorical variables. NAD Δ was defined as the difference in blood NAD concentration at day 30 or day 60 from the NAD concentration at baseline. The inter-group variance of NAD Δ was assessed by the coefficient of variance (CV). The difference in blood NAD concentration after supplementation was investigated by the Kruskal-Wallis test and the group-to-group difference was investigated by Dunn’s post-hoc test. Normalization for skewed data was conducted using square root transformation. The association between chronological age, blood biological age, sex, body mass index (BMI), and homeostatic model assessment for insulin resistance (HOMA-IR) and blood NAD baseline concentration or NAD Δ at day 30 or day 60 were investigated by generalized linear regression using Gaussian method.
Generalized linear regression was also used to investigate the association between NAD Δ and changes in blood biological age, HOMA-IR, and changes in the 6-minute walk test and SF-36 scores. The outlier for the generalized linear regression was defined as a data point where studentized residual over ±2. Outliers were excluded from the generalized linear regression analysis. Outcomes from generalized linear regression are shown as regression coefficient and 95% confidence interval (95% CI).
The median effective dose (ED 50) is a dose required to achieve a targeted effect in 50% of the population receiving the dose. In this study, ED 50 of NAD Δ after a 60-day NMN supplementation was calculated to determine a target NAD concentration improvement needed to achieve significant clinical improvement in a significant proportion of the population. Significant clinical improvement for the 6-minute walk test was defined as an increase of at least 30 meters from the baseline outcome and for the SF-36 score as an increase of at least ten points from the baseline score. To ensure a wide range of NAD Δ and a sufficient sample size in each group, the participants were divided into 20 groups (four individuals in each group) based on their NAD Δ, to simulate having 20 dose groups in pharmacological studies. The NAD Δ of each group was re-calculated as the mean value of the NAD Δ of the four individuals in the group. A four-parameter log-logistic model was used to assess the dose-response effect, where dose referred to NAD Δ, and response referred to achieving significant clinical improvement or not. The 95% confidence interval of ED 50 was obtained using the delta method, a method to calculate the approximated standard error for results generated from the function.
Data analysis was performed on R (version 4.2.1). ED 50 was calculated using the “drc” package. A p-value of less than 0.05 was considered statistically significant.
The demographic characteristics and responses are shown in Table 1. The number of male participants was eight in the placebo group, ten in the 300 mg group, six in the 600 mg group, and nine in the 900 mg group. All participants showed good compliance, ranging from 75% to 100%.
The NAD Δ from baseline to day 30 and day 60 is shown in Figure 1. The NAD Δ at both day 30 and day 60 increased dose-dependently (p<0.001), while the SD within the group was large, the CV of blood NAD concentration after supplementation ranging from 220% to 614% in the placebo group, and from 29.2% to 113.3% in intervention groups. The NAD Δ was dose-dependent with no significant difference between the 600 mg group and the 900 mg NMN group.
Figure 1.Blood NAD concentration change in each dose group NAD: nicotinamide adenine dinucleotide; Δ30: change at day 30; Δ60: change at day 60 Data are shown in mean (SD). *colour should be used
No significant association was found between blood NAD baseline concentration or NAD Δ at day 30 or day 60 and chronological age or blood biological age, sex, and BMI. A higher baseline HOMA-IR ratio was associated with higher baseline NAD concentration but not with NAD Δ (Table 2).
No significant association was found between blood baseline NAD concentration and 6-minute walk NAD Δ, SF-36 score Δ, blood biological age Δ, and HOMA-IR Δ (Table 3). Higher NAD Δ was significantly associated with better improvement in 6-minute walk Δ and SF-36 score Δ at both day 30 and day 60. Each 1 nmol/L increase in blood NAD concentration at day 30 was associated with 1.37 meters more in a 6-minute walk test. Each 1 nmol/L increase in blood NAD concentration was also associated with 0.02 more in the square root of SF-36 scores. For example, 5 nmol/L blood NAD concentration increase was associated with 5.76 (5*0.02+2.30 intercept)^2 higher SF-36 score at day 30. The improvements associated with higher NAD Δ were larger in measurements taken after 60 days of supplementation.
Each 1 unit increase of the NAD level on day 60 was associated with 2.42 meters more in the 6-minute walk test and 0.02 more in the square root of SF-36 scores. Thus, 5 nmol/L NAD concentration increase is associated with 8.82 (5*0.02+2.87 intercept)^2 higher SF-36 score on day 60. No significant association was found between NAD Δ and blood biological age Δ or HOMA-IR Δ.
After the 60-day supplementation, a total of 49 and 40 participants had a significant improvement in the 6-minute walk test and SF-36 score, respectively. The ED 50 of NAD Δ at day 60 for the clinically significant improvement of the 6-minute walk test and SF-36 score was 15.65 nmol/L (95% CI: 10.87-20.45 nmol/L) and 13.51 nmol/L (95% CI; 10.54-16.50 nmol/L) respectively. The dose-response curves of the NAD Δ and the percentage of respondents having a 6-minute walk test Δ at day 60 ≥30 meters or SF-36 Δ at day 60 ≥10 scores are shown in Figure 2.
