Abstract
To assess associations between blood nicotinamide adenine dinucleotide (NAD) levels and laboratory parameters at baseline and after 60 days of nicotinamide mononucleotide (NMN) supplementation. Post hoc analysis of a randomized, double-blind, clinical trial of daily NMN (300, 600, or 900 mg) or placebo in healthy middle-aged participants. Among the 80 participants (49.4 ± 6.8 year), the baseline NAD was 7.21 [5.5, 10.6] nM. Every 1 nM higher baseline NAD level was associated with 0.24% (95%CI: 0.05–0.44) higher lymphocytes, − 0.36% (95% CI: − 0.57 to − 0.14) lower neutrophils, 0.023% (95% CI: 0.011–0.036) higher triglycerides, − 0.009% (95% CI: − 0.015 to − 0.003) lower high-density lipoprotein, 0.02% (95% CI: 0.004–0.035) higher alanine transaminase, 0.01% (95% CI: 0.0003–0.021) aspartate transaminase. Every 1 nM increase in the NAD level was associated with an increase in hemoglobin of 0.027% (95% CI: 0.01–0.045), red blood cells (RBC) of 0.025% (95% CI: 0.009–0.042), mean corpuscular hemoglobin concentration of 0.016 g/dL (95% CI: 0.008–0.025), and uric acid of 0.02% (95%CI: 0.003–0.038). Higher baseline NAD levels are associated with inflammatory, lipid, and liver profiles. NMN-induced increases in blood NAD are associated with an increase in RBC parameters, potentially indicating enhanced oxygen-carrying capacity.
Supplementary information
The online version contains supplementary material available at 10.1007/s11357-025-01968-0.
Keywords: Blood nicotinamide adenine dinucleotide levels, Blood laboratory parameters, Nicotinamide mononucleotide supplementation, Aging
Introduction
Nicotinamide adenine dinucleotide (NAD) is a key molecule that functions as a substrate in oxidoreduction reactions and also plays essential roles in supporting metabolic pathways, repairing DNA damage, and inhibiting inflammation [1, 2]. In humans, NAD levels in capillary whole blood are significantly lower in older (50–80 years) compared to younger (20–50 years) individuals [3]. A lower baseline venous whole-blood NAD level is associated with a higher risk of age-related conditions, such as microcytic and normocytic anemia in healthy females [4], inflammation in patients with heart failure [5], and a lower baseline muscle tissue NAD level with Parkinson’s disease [6].
NAD precursors such as nicotinamide mononucleotide (NMN), nicotinamide riboside (NR), nicotinic acid (NA), and trigonelline enhance NAD biosynthesis, skeletal muscle mitochondrial function and insulin sensitivity, reduce inflammation, and improve clinical outcomes such as gait speed, grip strength, five-times sit-to-stand performance, subjective drowsiness, aerobic capacity, and quality of life in healthy middle-aged and older adults [7–12]. However, supplementation with niacin, another NAD precursor, has been reported to increase levels of the NAD metabolites N1-methyl-2-pyridone-5-carboxamide and N1-methyl-4-pyridone-3-carboxamide, which were associated with a significantly increased three-year risk of major adverse cardiac events in independent cohorts [13]. However, the response to NMN supplement varies largely across individuals. The coefficient of variation of NAD concentration changes of 29.2–113.3% within intervention group, thus a personalized regimen is needed for an optimal NAD concentration improvement [14].
It is unknown whether blood NAD levels are associated with changes in organ system function in healthy individuals, which may be detectable via standard laboratory tests.
This study assesses the association between blood NAD levels and blood laboratory parameters at baseline and their change after 60 days of intervention with either NMN supplementation or placebo.
Methods
Study population
Eighty healthy individuals (females and males) of Indian descent aged 40–65 years were included in a randomized, double-blinded, placebo-controlled clinical trial testing 300 mg, 600 mg, or 900 mg of NMN a day or placebo for 60 days. Inclusion criteria were body mass index between 18.5 and 35 kg/m2, no niacin-containing supplements for at least 7 days before baseline and during the trial, stable diet and lifestyle, use of effective contraception, ability to provide informed consent, and willingness to follow trial procedures and consume assigned supplements (NMN or placebo) for 60 days. Exclusion criteria included current or recent use of niacin supplements or statins, tobacco use within the past 6 months, history of substance or alcohol abuse, unstable mental illness within the past 6 months, pregnancy or breastfeeding, COVID-19 symptoms, planned weight loss during the trial, participation in another clinical trial within 30 days, hypersensitivity to trial ingredients, abnormal screening blood test indicating poor health, or inability to provide informed consent or a blood sample [11]. The trial was conducted in Lotus Healthcare and Aesthetics Clinic and Sunad Ayurved in Pune, India, and monitored by a clinical research organization in Pune. All participants provided written informed consent. Ethical approval was obtained from the Royal Ethics Committee in Pune, India (ECIV45/Indt/MII/2013/RR-19). Further information about the study design can be found in the previous publication [11].
Clinical trial supplement
In this study, the supplement, AbinoNutra™ NMN, was supplied by Aba Chemicals Co., Ltd. (Shanghai, China) and Abinopharm, Inc. (Connecticut, USA) and encapsulated by Polifarma (Nanjing, China) at a concentration of 150 mg NMN per capsule. Placebo capsules containing rice flour were also manufactured and provided by Polifarma to ensure consistency in appearance and administration. Participants received oral doses according to the study protocol, with dosages consisting of either two capsules (300 mg NMN or 300 mg placebo), four capsules (600 mg NMN or 600 mg placebo), or six capsules (900 mg NMN or 900 mg placebo), depending on the assigned treatment group [11].
Blood NAD levels and blood laboratory parameters
Fasting venous blood samples were collected at baseline and after 60-day NMN or placebo supplements. The colorimetric NAD test kit from MyBioSource, Inc. (catalog #MBS841786; California, USA) was utilized to measure NAD concentrations. This test quantifies the total levels of NAD and NADH in serum. Laboratory parameters included complete blood count (absolute neutrophils, eosinophils, hemoglobin, lymphocytes, mean corpuscular hemoglobin concentration [MCHC], mean corpuscular volume [MCV], monocytes, neutrophils, platelet count, red blood cell count [RBC], and total white blood cell count [WBC]), lipid profile (high-density lipoprotein [HDL], low-density lipoprotein [LDL], triglycerides, and total cholesterol), liver function (alkaline phosphatase, alanine aminotransferase [ALT], aspartate aminotransferase [AST], and total bilirubin), kidney function (blood urea nitrogen [BUN], glomerular filtration rate [GFR], creatinine, and urea), electrolytes and minerals (calcium, chloride, sodium, and potassium), proteins (albumin and total protein), coagulation markers (activated partial thromboplastin time [aPTT], patient prothrombin time [PT], and uric acid). Packed cell volume (PVC) was only measured at baseline.
Data analysis
The normality of each variable was assessed visually by histogram analysis. Normally distributed continuous numeric parameters data were presented as mean and standard deviation (SD). Skewed distributed continuous numeric parameters were presented as median and interquartile range [IQR]. Categorical parameters data were presented as numbers and percentage. The change in blood NAD and laboratory parameters is the difference in their levels after supplementation of NMN or placebo for 60 days compared to baseline.
Linear regression was used to assess the association between baseline blood NAD levels and blood laboratory parameters, as well as between changes in blood NAD levels and changes in blood laboratory parameters. For variables that are not normally distributed, log-transformation was performed before conducting linear regression. The 95% confidence intervals were calculated, and statistical analyses were performed using R. To demonstrate the associations in a clearer manner, Pearson’s correlation was performed between baseline blood laboratory parameters and blood NAD levels, as well as the changes of blood NAD levels and blood laboratory parameters after intervention, the correlation coefficients and their 95% CIs were presented in forest plots. A p-value of less than or equal to 0.05 was considered statistically significant.
Results
The demographic characteristics, NAD concentration, and blood laboratory biomarkers at baseline and post-intervention of the 60-day NMN supplementation are shown in Table 1. Among 80 participants, the mean chronological age was 49.4 (6.8) years, 47 participants were females, and the median [IQR] of the baseline blood NAD concentration was 7.21 [5.5, 10.6] nM. The change of NAD concentration after 60 days of supplement was 25 ± 21.2 nM, while the change of NAD concentration for placebo, 300 mg, 600 mg, and 900 mg groups were 3.66 ± 8.06, 20.8 ± 22.16, 37.39 ± 10.92, 38.07 ± 18.78 nM, respectively (Supplementary Table 1).
Table 1.
Characteristics of participants at baseline and post-intervention of a 60-day nicotinamide mononucleotide supplementation (N = 80)
| Baseline | Post-intervention | |
|---|---|---|
| Age, year | 49.4 ± 6.8 | - |
| Female, n | 47 | - |
| Weight, kg | 67.1 ± 11.7 | - |
| Height, cm | 157.4 ± 9.1 | - |
| BMI, kg/m2 | 27.1 ± 4.1 | - |
| NAD, nmol/L | 7.21 [5.5, 10.6] | 34.6 [18.3, 46.1] |
| Complete blood count | ||
| WBC, 109/L | 7.07 ± 1.70 | 7.69 ± 2.26 |
| Neutrophils, % | 57.7 [51.9, 62.8] | 53.7 [47.6, 59.42] |
| Lymphocytes, % | 35.6 ± 6.83 | 37.25 ± 7.09 |
| Monocytes, % | 3.6 [2.9, 4.4] | 5.25 [4.4, 6.23] |
| Eosinophils, % | 2.7 [1.78, 4.58] | 2.85 [1.98, 5.22] |
| Absolute neutrophils, 109/L | 4.06 ± 1.28 | 4.12 ± 1.43 |
| RBC, 1012/L | 4.68 ± 0.62 | 4.79 ± 0.68 |
| Hemoglobin, g/dL | 13.3 ± 1.84 | 13.57 ± 2.01 |
| MCHC, g/dL | 33.3 [32.5, 33.9] | 32.5 [31.7, 33.2] |
| MCV, fL | 86.3 ± 9.60 | 88.3 ± 10.2 |
| Platelet count, 109/L | 286 ± 74.9 | 314 ± 81.2 |
| Liver function | ||
| ALP, U/L | 85.4 [67.2, 103] | 88.8 [73.6, 111] |
| ALT, U/L | 19.4 [13.7, 26.6] | 17.4 [13.4, 24.2] |
| AST, U/L | 22.2 [17.6, 25.7] | 20.9 [17.5, 24.8] |
| Total bilirubin, mg/dL | 0.44 [0.36, 0.65] | 0.52 [0.37, 0.71] |
| Kidney function | ||
| BUN, mg/dL | 8.7 ± 2.75 | 8.99 ± 2.6 |
| GFR, mL/min/1.73 sqm | 103 ± 19.8 | 96.08 ± 20.61 |
| Creatinine, mg/dL | 0.7 [0.65, 0.85] | 0.76 [0.66, 0.95] |
| Urea, mg/dL | 18.6 ± 5.89 | 19.28 ± 5.52 |
| Uric acid, mg/dL | 5.1 [4.4, 5.9] | 5.1 [4, 5.82] |
| Lipid profile | ||
| HDL, mg/dL | 41.6 [36.7, 47.4] | 43.5 [39.0, 50.9] |
| LDL, mg/dL | 120 [102, 138] | 127 [109, 146] |
| Triglycerides, mg/dL | 148 [110, 201] | 130 [100, 175] |
| Total cholesterol, mg/dL | 182 [153.9, 198.7] | 180.4 [159.5, 204.9] |
| Electrolyte and minerals | ||
| Calcium, mg/dL | 9.3 [9.0, 9.5] | 9.4 [9.2, 9.8] |
| Chloride, mmol/L | 101.1 [99.6, 102.9] | 101.4 [100.1, 103.1] |
| Sodium, mmol/L | 139 [138, 141] | 140 [139, 142] |
| Potassium, mmol/L | 4.3 [4.07, 4.6] | 4.6 [4.3, 4.8] |
| Protein | ||
| Albumin, g/dL | 4.38 ± 0.28 | 4.41 ± 0.34 |
| Total protein, g/dL | 7.1 [6.9, 7.4] | 7.6 [7.2, 7.9] |
| Coagulation | ||
| aPTT, second | 32.35 [30.37, 35.52] | 32.2 [29.3, 34.8] |
| PT, second | 11.7 [11.47, 12.01] | 11.9 [11.4, 12.3] |
Data are presented as mean ± SD for normally distributed variables or median [IQR] for skewed distributed variables unless otherwise indicated
ALP, alkaline phosphatase; ALT, alanine transaminase; aPTT, activated partial thromboplastin time; AST, aspartate aminotransferase; BUN, blood urea nitrogen; GFR, glomerular filtration rate; HDL, high-density lipoprotein; LDL, low-density lipoprotein; MCHC, mean corpuscular hemoglobin concentration; MVC, mean corpuscular volume; NAD, nicotinamide adenine dinucleotide; PT, patient prothrombin time; PVC, packed cell volume; RBC, red blood cell; WBC, white blood cell
The association between baseline blood NAD levels and baseline blood parameters is reported in Table 2. Every 1 nM higher baseline blood NAD was associated with baseline complete blood count parameters (0.24% [95% CI: 0.05–0.44] higher lymphocyte levels, − 0.36% [95% CI: − 0.57–− 0.14] lower neutrophil levels), baseline lipid profile parameters (0.023% [95% CI: 0.011–0.036] higher triglyceride levels, and − 0.009% [95% CI: − 0.015 to − 0.003] lower high-density lipoprotein), baseline liver profile parameters (0.02% [95% CI: 0.004–0.035] higher ALT and 0.01% [95% CI: 0.0003–0.021] AST). There was no statistically significant association between baseline blood NAD levels and other baseline complete blood count parameters, lipid profile, liver and kidney function, electrolytes and minerals, proteins, and coagulation markers. The associations between baseline blood parameters and blood NAD levels were shown in Supplementary Fig. 1, and the correlation coefficient was shown in Supplementary Fig. 2.
Table 2.
The association of baseline blood nicotinamide adenine dinucleotide baseline level expressed as 1 nM higher level and baseline blood laboratory parameters
| B-value | 95% CIs | p-value | |
|---|---|---|---|
| Complete blood count | |||
| Absolute neutrophils, count/cmm* | − 0.008 | − 0.017–0.001 | 0.099 |
| Eosinophils, %* | 0.012 | − 0.008–0.032 | 0.223 |
| Hemoglobin, g/dL | 0.012 | − 0.043–0.067 | 0.665 |
| Lymphocytes, % | 0.24 | 0.047–0.439 | 0.017 |
| Monocytes, %* | 0.008 | − 0.003–0.018 | 0.140 |
| Neutrophils, % | − 0.36 | − 0.57 to − 0.14 | 0.002 |
| Platelet count, 109/L | − 4.42 | − 2233 to 2224 | 0.997 |
| RBC, 1012/L | − 0.002 | − 0.021 to 0.016 | 0.830 |
| MCHC, g/dL | 0.000 | − 0.001 to 0.001 | 0.926 |
| MCV, fL | 0.089 | − 0.20 to 0.37 | 0.541 |
| WBC, 109/L | − 8.54 | − 59.1 to 42.0 | 0.741 |
| Liver function | |||
| ALP, U/L | − 0.003 | − 0.01–0.006 | 0.496 |
| ALT, U/L* | 0.020 | 0.004–0.035 | 0.014 |
| AST, U/L* | 0.010 | 0.0003–0.021 | 0.044 |
| Total bilirubin, mg/dL* | 0.007 | − 0.009–0.023 | 0.382 |
| Kidney function | |||
| BUN, mg/dL | 0.014 | − 0.07–0.09 | 0.747 |
| GFR, mL/min/1.73 sqm | − 0.095 | − 0.683–0.493 | 0.751 |
| Creatinine, mg/dL* | 0.002 | − 0.005–0.008 | 0.621 |
| Urea, mg/dL* | 0.002 | − 0.008–0.012 | 0.681 |
| Lipid profile | |||
| HDL, mg/dL* | − 0.009 | − 0.015 to − 0.003 | 0.005 |
| LDL, mg/dL* | 0.001 | − 0.007–0.009 | 0.863 |
| Triglycerides, mg/dL* | 0.023 | 0.011–0.036 | < 0.001 |
| Total cholesterol, mg/dL* | 0 | − 0.006–0.006 | 0.874 |
| Electrolyte and minerals | |||
| Calcium, mg/dL | 0.009 | − 0.002–0.020 | 0.098 |
| Chloride, mmol/L | 0.082 | − 0.017–0.181 | 0.110 |
| Sodium, mmol/L* | 0 | − 0.0003–0.001 | 0.242 |
| Potassium, mmol/L | − 0.012 | − 0.024–0.000 | 0.058 |
| Protein | |||
| Albumin, g/dL | 0 | − 0.008–0.009 | 0.918 |
| Total protein, g/dL | 0.003 | − 0.009–0.015 | 0.622 |
| Coagulation | |||
| aPTT, seconds | 0.12 | − 0.006–0.245 | 0.065 |
| PT, seconds* | 0 | − 0.002–0.003 | 0.681 |
| Uric acid, mg/dL | 0.012 | − 0.03–0.054 | 0.587 |
*Indicates skewed data, data were log-transformed before conducting linear regression. Bolded values indicate statistically significant differences (p ≤ 0.05)
Data are presented as regression coefficient and 95% confident interval
ALP, alkaline phosphatase; ALT, alanine transaminase; aPTT, activated partial thromboplastin time; AST, aspartate aminotransferase; BUN, blood urea nitrogen; CIs, confidence intervals; GFR, glomerular filtration rate; HDL, high-density lipoprotein; LDL, low-density lipoprotein; MCHC, mean corpuscular hemoglobin concentration; MVC, mean corpuscular volume; NAD, nicotinamide adenine dinucleotide; PT, patient prothrombin time; RBC, red blood cell; WBC, white blood cell
The mean blood NAD change after 60-day NMN supplementation of placebo, 300 mg, 600 mg, and 900 mg NMN per day was 3.66 (8.06) nM, 20.8 (22.2) nM, 37.4 (10.9) nM, and 38.1 (18.8) nM, respectively. The association between blood NAD level change and blood laboratory parameters change after 60 days of NMN supplementation is reported in Table 3. Every 1 nM increase in the blood NAD level was associated with a change in complete blood count parameters (hemoglobin increase of 0.027% [95% CI: 0.01–0.045], RBC increase of 0.025% [95% CI: 0.009–0.042], and MCHC increase of 0.016 g/dL [95% CI: 0.008–0.025]), change in uric acid increase of 0.02% [95% CI: 0.003–0.038]). There was no statistically significant association between blood NAD level change and the change in other complete blood count parameters, lipid profile, liver and kidney function, electrolytes and minerals, proteins, and coagulation after 60 days of NMN supplementation. The associations between the changes of blood NAD levels and blood parameters were shown in Supplementary Fig. 3 according to their dose, and the correlation coefficient was shown in Supplementary Fig. 4.
Table 3.
Association of nicotinamide adenine dinucleotide change per 1 nM increase and blood parameters change (after 60 days)
| B-value | 95% CIs | p-value | |
|---|---|---|---|
| Complete blood count | |||
| Absolute neutrophils, count/cmm | 0.71 | − 10.92 to − 12.34 | 0.904 |
| Eosinophils, %* | 0.010 | − 0.010–0.030 | 0.313 |
| Hemoglobin, g/dL* | 0.027 | 0.01–0.045 | 0.003 |
| Lymphocytes, % | 0.030 | − 0.033 to − 0.003 | 0.353 |
| Monocytes, % | − 0.003 | − 0.017–0.006 | 0.676 |
| Neutrophils, % | − 0.02 | − 0.1–0.4 | 0.480 |
| Platelet count, 109/L | − 160 | − 771–451 | 0.603 |
| RBC, 1012/L* | 0.025 | 0.009–0.042 | 0.003 |
| MCHC, g/dL | 0.016 | 0.008–0.025 | < 0.001 |
| MCV, fL | − 0.057 | − 0.125–0.012 | 0.108 |
| WBC, 109/L | 4.17 | − 13.8–22.1 | 0.650 |
| Liver function | |||
| ALP, U/L | − 0.08 | − 0.24–0.08 | 0.302 |
| ALT, U/L* | − 0.002 | − 0.024–0.02 | 0.861 |
| AST, U/L* | − 0.003 | − 0.025–0.019 | 0.771 |
| Total bilirubin, mg/dL* | − 0.005 | − 0.022–0.011 | 0.520 |
| Kidney function | |||
| BUN, mg/dL | 0.018 | − 0.005–0.040 | 0.124 |
| GFR, mL/min/1.73 sqm | 0.016 | − 0.112–0.144 | 0.807 |
| Creatinine, mg/dL | 0.001 | − 0.001–0.001 | 0.393 |
| Urea, mg/dL | 0.04 | − 0.008–0.09 | 0.103 |
| Lipid profile | |||
| HDL, mg/dL* | 0.001 | − 0.019–0.022 | 0.908 |
| LDL, mg/dL | 0.18 | − 0.11–0.47 | 0.232 |
| Triglycerides, mg/dL | 0.48 | − 0.19–0.15 | 0.164 |
| Total cholesterol, mg/dL | 0.15 | − 0.15–0.44 | 0.324 |
| Electrolyte and minerals | |||
| Calcium, mg/dL | 0.0001 | − 0.004–0.004 | 0.875 |
| Chloride, mmol/L | − 0.20 | − 0.4–0.03 | 0.084 |
| Sodium, mmol/L | − 0.19 | − 0.051–0.013 | 0.258 |
| Potassium, mmol/L | − 0.004 | − 0.009–0.001 | 0.062 |
| Protein | |||
| Albumin, g/dL | 0.001 | − 0.003–0.005 | 0.654 |
| Total protein, g/dL | − 0.001 | − 0.006–0.004 | 0.735 |
| Coagulation | |||
| aPTT, seconds | − 0.004 | − 0.056–0.049 | 0.893 |
| PT, seconds | 0.000 | − 0.012–0.011 | 0.989 |
| Uric acid, mg/dL* | 0.020 | 0.003–0.038 | 0.026 |
*Indicates skewed data, data were log-transformed before conducting linear regression. Bolded values indicate statistically significant differences (p ≤ 0.05)
Data are presented as regression coefficient and 95% confident interval
abs. neutr., absolute neutrophils; ALP, alkaline phosphatase; ALT, alanine transaminase; aPTT, activated partial thromboplastin time; AST, aspartate aminotransferase; BUN, blood urea nitrogen; CIs, confidence intervals; GFR, glomerular filtration rate; HDL, high-density lipoprotein; LDL, low-density lipoprotein; MCHC, mean corpuscular hemoglobin concentration; MVC, mean corpuscular volume; NAD, nicotinamide adenine dinucleotide; PT, patient prothrombin time; RBC, red blood cell; WBC, white blood cell
Discussion
Higher baseline blood NAD levels were associated with higher lymphocyte and lower neutrophil levels, higher triglycerides, lower HDL levels, and higher ALT and AST levels, which were all within the reference range. After 60 days of NMN supplementation, an increase in blood NAD levels was associated with increases in hemoglobin, RBC, MCHC, and uric acid, which remained within the reference range. No statistically significant associations were found between baseline or NAD change and the baseline or change in other blood laboratory parameters, including blood count, liver and kidney function, lipid profile, electrolytes, proteins, and coagulation.
Lymphocytes and neutrophils are crucial components of the immune response. Higher baseline blood NAD levels were associated with higher lymphocyte and lower neutrophil counts, however, within the normal range. The change in blood NAD levels after 60 days of supplementation with NMN was not significantly associated with immune cell levels. These associations likely reflect individual physiological or metabolic setpoints such as differences in immune cell turnover, mitochondrial activity, or redox state and were not sustained following NMN supplementation, as increases in NAD levels over 60 days were not associated with changes in immune cell composition.
Higher baseline blood NAD levels were statistically significantly associated with higher triglyceride and lower HDL cholesterol. After 60 days of NMN administration, no statistically significant association was found between the change in blood NAD and the change in blood lipid levels. A study performed in healthy Japanese participants after a single administration of 300 mg/100 mL intravenous NMN reported that higher NAD levels were associated with a reduction in triglyceride levels (up to 60 mg/dL) and no difference in LDL, HDL, and total cholesterol levels from 0.5 to 5 h after administration [15]. Higher circulating NAD in individuals with less favorable lipid profiles may indicate a compensatory upregulation of NAD biosynthetic pathways in response to metabolic stress, inflammation, or oxidative load [16]. Although the participants in this study were of a stable health condition, this study had a wide range of participant BMI, with an upper threshold of 35 kg/m2. This value falls into the category of Class II obesity, which is a significant metabolic state with well-documented impacts on inflammation, lipid metabolism, and overall cellular function. Studies with a higher number of participants and a longer duration of NMN supplementation should be conducted to confirm the effect of blood NAD levels on triglycerides and HDL cholesterol, given their role in the onset of cardiovascular diseases [17].
Hemoglobin, RBC, and MCHC are key hematological indices that reflect the blood oxygen-carrying capacity [18]. An increase in blood NAD levels after 60 days of supplementation NMN was statistically significantly associated with an increase in hemoglobin, RBC, and MCHC, in line with another study where higher blood NAD levels were significantly associated with a lower prevalence of microcytic and normocytic anemia in females [4]. In clinical settings such as iron-deficiency anemia, chronic kidney disease, and transfusion practice, a hemoglobin increase of ≥ 1 g/dL (approximately 8–10%) is considered the minimal clinically significant improvement [19–21]. In contrast, study in trained athletes reported that each 1% increase in hemoglobin mass was associated with a 0.6–0.7% increase in VO₂max. Although we observed a statistically significant association between the increase in hemoglobin and the increase in NAD blood levels, the magnitude was well below this threshold and is unlikely to be clinically meaningful in healthy middle-aged individuals [22]. The association of NAD change and MCHC change could be explained by the activity of sirtuin-1 (SIRT1), a protein that depends on blood NAD for its function [23]. SIRT1 activity has been shown to enhance hemoglobin synthesis (for instance, by inducing fetal γ-globin gene expression) and to improve hematopoiesis in anemia models [24].
ALT and AST are important indicators of liver function [25, 26]. Higher baseline blood NAD levels were associated with higher ALT and AST levels, indicating less optimal liver function. There was no statistically significant association between blood NAD change after 60-day supplementation of NMN and ALT and AST levels. This appears contradictory to preclinical evidence, which reports that higher NAD levels can protect against ethanol-induced liver injuries by reducing oxidative stress through activating the SirT1-PGC-1α-mitochondrial biosynthesis pathway [27] and acetaminophen-induced hepatotoxicity by enhancing the antioxidant defense through promoting translocational activation of Nrf2 [28] in mice. Creatinine and uric acid are common markers of kidney function, with elevated levels suggesting potential impairment. Neither baseline NAD levels nor changes after 60 days of NMN supplementation were significantly associated with creatinine levels. However, an increase in NAD was associated with an increase in uric acid levels. While preclinical studies suggest NAD may protect liver and kidney function, these post hoc clinical findings, although remaining within reference ranges, raise the possibility of mild adverse effects in humans and underscore the need for further clinical observation.
The study’s internal validity is strengthened by its robust design as a double-blind randomized controlled trial, which minimizes potential biases and enhances reliability. Data collection over a 60-day period provides insights into both immediate and sustained intervention outcomes. However, limitations include exclusive focus on an Indian population and a small sample size. While this is valuable data, the findings may not generalize to other ethnicities, which may have different genetic predispositions, dietary habits, or baseline metabolic profiles. Further research with a larger sample and across diverse ethnic groups is recommended for broader applicability. Further research with a larger sample and across diverse ethnic groups is recommended for broader applicability. Furthermore, although some laboratory parameters showed statistically significant changes, these values remained within normal reference ranges, and their clinical relevance remains uncertain. It is also noted that given the exploratory nature of the analyses and the number of statistical tests performed, the risk of false positive associations cannot be excluded. The findings should be interpreted as exploratory, larger studies would be needed to confirm the findings in this study. Additionally, NAD levels in this study were measured in serum using an enzyme-linked immunosorbent assay method. While other studies [29, 30] also used the same sample and method. The NAD levels in other studies were measured in whole blood [4], plasma [31], and erythrocytes [32] with other analytic methods (e.g., liquid chromatography coupled to tandem mass spectrometry or high-performance liquid chromatography), which limits the comparability of results across studies.
Conclusion
Higher baseline NAD levels are associated with inflammatory, lipid, and liver profiles. Increasing blood NAD levels after NMN supplementation increases hemoglobin concentration, RBC count, MCHC, and uric acid levels. Although these values remained within reference ranges, the findings suggest potential improvements in blood oxygen-carrying capacity.
Supplementary information
Below are the links to the electronic supplementary materials.
Funding
The clinical trial is fully funded by Aba Chemicals Co. (Shanghai, China) and Abinopharm, Inc. (Connecticut, USA). The post hoc analysis was not based on funding.
Data availability
Data will be made available on request.
Declarations
Conflict of interest
Lin Yi is an employee of Abinopharm, Inc.; R.T. and Z.L. are employees of Aba Chemicals, Co. The other authors declare no conflict of interest.
Footnotes
Ajla H. Kuerec and Weilan Wang shared first author.
Publisher’s Note
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
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Data Availability Statement
Data will be made available on request.