Ethical approval, informed consent, and study location

All procedures were approved by the University of Colorado Boulder Institutional Review Board. The nature, benefits, and risks of the study were explained to all subjects, and their written informed consent was obtained prior to participation. All measurements were performed at the University of Colorado Boulder Clinical & Translational Research Center (CTRC) and in the Integrative Physiology of Aging Laboratory. The study was registered on ClinicalTrials.gov under the identifier NCT02921659.

Study participants

Middle-aged and older men and postmenopausal women aged 55−79 years were recruited from Boulder Colorado and surrounding communities. All subjects were free of clinical diseases, including peripheral artery disease (ankle-brachial index >0.90) and overt CVD as assessed by a graded exercise test, baseline blood panel, medical history, and physical examination by a physician. All subjects demonstrated age-related impairments in vascular endothelial function (defined as a flow-mediated dilation value <6%) and were excluded if they exhibited abnormal blood chemistries for renal or liver function (defined as 1 standard deviation outside of the normal range), had alcohol dependence, uncontrolled thyroid disease, severe obesity (body mass index >40 kg m−2), or were not weight stable for at least 3 months prior to enrolling in the study (defined as >2 kg change in body mass). Body mass, BMI, and waist and hip circumferences were measured by anthropometry, and total body fat percentage was measured using dual-energy x-ray absorptiometry (Lunar/Prodigy, GE). Fasting glucose and total, LDL, and HDL cholesterol levels were measured using standardized assays at the University of Colorado Boulder CTRC Core Laboratory at baseline and after each intervention phase of the study.

Study design, randomization, and intervention

The study design consisted of a 2 × 6-week randomized, double-blind, placebo-controlled crossover clinical trial. Subjects ingested nicotinamide riboside chloride (NIAGEN®; 500 mg, twice per day; ChromaDex, Inc.) and placebo capsules for 6 weeks each in a randomly determined order. Subjects were randomized after providing informed consent and meeting all inclusion criteria. Randomization was performed by a member of the study team not involved in the assessment of outcomes. The study participants and members of the study team involved in the collection and analysis of outcomes were blinded to the treatment condition. Capsules were consumed with meals in the morning and evening. Subjects refrained from taking any over-the-counter medications for 48 h and prescription medications for 24 h prior to all experimental testing. All assessments were performed after a 12 h overnight fast with the exception of motor function tests, which were performed 2 h after a light meal or snack in order to ensure that subjects had enough energy to complete the testing battery. Subjects refrained from consuming alcohol or engaging in vigorous exercise for 24 h and refrained from taking study pills for at least 12 h prior to all testing sessions.

Evaluation of safety and tolerability

Adherence to the intervention was assessed by pill count. Subjects reported to the laboratory every 2 weeks to receive a new bottle of capsules and to discuss any issues with tolerability or treatment-emergent AEs with a member of the research team who was not involved in data collection or analysis in order to ensure blinding of the investigators. Standard clinical markers of hematology, liver and kidney function and blood lipids were analyzed using standardized clinical assays at Boulder Community Hospital and any abnormal blood results were reviewed by the study physician.

Isolation of peripheral blood mononuclear cells

PBMCs were isolated from 35 ml of whole blood collected into EDTA-coated Vacutainer™ tubes. The blood was then centrifuged at 400 × g for 20 min and the majority of the plasma layer (~60%) was removed to increase the efficiency of the downstream PBMC isolation. The remaining sample was slowly added to a new 50 ml conical tube containing 10 ml of Histopaque 1.077 (Greiner Bio-One) and the mononuclear cell layer was isolated by density-dependent centrifugation at 400 × g for 20 min, washed and then frozen in 2 ml of PBS at −80°C.

Materials

NAAD, NAD+, NADP+, NaM, NmN, mono-, di- and triphosphate nucleotides and nucleosides were obtained from Sigma Aldrich (St. Louis, MO). Adenosine (ribose-13C5) and adenosine triphosphate (ribose-d4) were obtained from Cambridge Isotope Laboratories (Tewksbury, MA). NaM (13C6) was obtained from Cerilliant (Round Rock, TX). Nicotinamide riboside and doubly labeled nicotinamide riboside (13C1, H2-1) were obtained from ChromaDex Inc. (Irvine, CA). All HPLC solvents and extraction solvents were HPLC grade or better.

Preparation of calibration standards

Individual stock standards were prepared by dissolving 10 mg ml−1 in 1:1 methanol:water and then combining to obtain a stock mixture. The NAD+ metabolite combined stock was prepared at 100 μg ml−1 of each compound and the nucleoside/nucleotide combined stock was prepared at 400 μg ml−1 of each compound; both were frozen at −20 °C until use. The internal standard solution was prepared at 250 μg/ml adenosine-13C5, adenosine triphosphate-d4, doubly labeled nicotinamide riboside and 2.5 μg ml−1 of NaM-13C6 in 1:1 methanol:water. Immediately before analysis of each sample batch, the individual calibration curve standards were prepared by combining the NAD+ and nucleoside/nucleotide stocks at a ratio of 1:1 and then diluting them in 1:1 methanol:water to the required concentrations. Concentrations ranged from 0.025 to 25 μg ml−1 for the NAD+ metabolites and from 0.1 to 100 μg ml−1 for nucleosides/nucleotides. Internal standard concentrations in all calibration levels and samples were 50 μg ml−1 for adenosine-13C5, adenosine triphosphate-d4 and doubly labeled nicotinamide riboside, and 0.5 μg ml−1 for NaM-13C6.

Extraction of NAD+ metabolites and nucleosides/nucleotides

Frozen PBMCs (5×10E6 cells total) were thawed on ice. 500 μl of ice cold 70:30 methanol:water was added along with 20 μl of internal standard and samples vortexed for 10 s. The resulting extract was centrifuged at 8000 × g for 5 min at 4 °C. The resulting supernatant was transferred to a new centrifuge tube and stored on ice. To the remaining pellet, 500 μl of ice cold methanol was added and the sample vortexed for 10 s to resuspend the pellet. The sample was then centrifuged at 8000 × g for 5 min at 4 °C. The entire supernatant was removed and combined with the 70% methanol supernatant. The resulting pellet was reserved and frozen at −70 °C for protein concentration analysis using the Bradford assay. The combined supernatants were centrifuged at 18,000 × g for 15 min and the resulting supernatant was transferred to a new tube and dried in a vacuum centrifuge at 55 °C. The dried samples were reconstituted in 100 μl of 1:1 methanol:water and centrifuged at 18,000 × g for 10 min at 4 °C. The supernatant was then transferred to a reduced surface activity autosampler vial for analysis.

LC-MS

HPLC separation of NAD+ metabolites and nucleosides/nucleotides was performed using a method described by Evans et al.59 with minor modifications. Separation of NAD+ metabolites and nucleosides/nucleotides was performed on a 1200 series HPLC from Agilent (Santa Clara, CA) using a 100 × 2 mm 5 μm Luna NH2 column from Phenomenex (Torrance, CA) operated in HILIC mode. Buffer A consisted of 100% acetonitrile and buffer B consisted of 95:5 water with 20 mm ammonium acetate adjusted to pH 9.6 with 20 mm ammonium hydroxide. Ten microliters of the extracted sample was analyzed using the following gradient at a flow rate of 0.6 ml per min: linear gradient from 5 to 100% B over 6 min, hold at 100% B from 6 to 9.5 min, then 100−5% B from 9.5 to 10.5 min, followed by re-equilibration at 5% B from 10.5 to 14 min. The column temperature was held at 15 °C for the entire gradient. Mass spectrometric analysis was performed on an Agilent 6410 triple quadrupole mass spectrometer in positive ionization mode. The drying gas was 300 °C at a flow rate of 12 ml per min. The nebulizer pressure was 30 psi. The capillary voltage was 4000 V. Data for NAD+ metabolites and nucleosides/nucleotides were acquired in MRM mode using experimentally optimized conditions obtained by flow injection analysis of authentic standards (Supplementary Table 5). Calibration standards were analyzed over a range of concentrations from 0.25 to 250 ng on column for the NAD+ metabolites and from 1 to 1000 ng on column for nucleosides/nucleotides. Calibration curves for each NAD+ metabolite and nucleoside/nucleotide were constructed using Agilent Masshunter Quantitative Analysis software. Results for PBMCs were quantitated using the calibration curves to obtain the on-column concentration, followed by normalization of the results using the protein concentration of the pellet reserved from the PBMC extraction.

Assessments of cardiovascular function

Resting blood pressure was measured in the seated position after at least 10 min of quiet rest using a semi-automated blood pressure device (Dynamap™ XL, Johnson & Johnson, Arlington, TX, USA). Measurements were made multiple times from the non-dominant arm, with 2 min of quiet rest between recordings. Repeat measurements were made until three blood pressure values were obtained that were within 5 mmHg of one another. These values were then averaged to determine resting systolic and diastolic blood pressure and pulse pressure. Baseline blood pressure values were obtained using the above-described protocol on two separate testing days prior to the initiation of the first intervention arm and were averaged to determine baseline blood pressure status (i.e., normal vs. above normal) for subsequent analyses.

Aortic stiffness was measured using carotid-to-femoral PWV, the gold-standard assessment of elastic artery stiffness in humans38. Pressure waveforms were recorded simultaneously from the carotid and femoral arteries using applanation tonometry (Millar Inc., Houston, Texas) as previously described by our laboratory60,61,62. The transit time of the aortic pulse wave was determined by measuring the time-delay between the foot of the carotid and femoral pressure waves using LabChart analysis software. PWV was calculated by dividing the distance between the two measurement sites by the aortic transit time.

Carotid artery compliance was determined by the change in diameter of the right common carotid artery (assessed using high resolution ultrasonography, PowerVision 6000, Toshiba) relative to the change in carotid blood pressure (assessed using applanation tonometry, Millar Inc., Houston, TX) across the cardiac cycle. Carotid pressure was normalized to brachial artery pressure obtained using an automated blood pressure cuff (Dynamap™ XL, Johnson & Johnson, Arlington, TX, USA). Compliance was calculated as CC = π × DD2 × (ΔD DD−1)/(2 × PP), where DD is diastolic diameter, ΔD is the change in diameter and PP is the arterial pulse pressure, as has been described previously62,63,64,65.

Endothelium dependent dilation was measured as brachial artery flow-mediated dilation (FMD) to reactive hyperemia, using high-resolution ultrasonography (PowerVision 6000, Toshiba) as previously described66,67,68. FMD was expressed as the percentage change (%Δ) from baseline diameter.

Assessments of metabolic function

Three-day dietary records were collected at baseline and during the last week of each intervention phase to ensure stability of caloric intake. Results were analyzed by a registered dietician using the Nutrition Data System for Research (University of Minnesota) as previously described by our laboratory67,69.

Resting metabolic rate was measured by indirect calorimetry (ParvoMedics TrueOne 2400) as described previously by our laboratory70,71. Subjects rested in a supine position for 45–60 min with a ventilated hood placed over their head to collect concentrations of expired oxygen (O 2 ) and carbon dioxide (CO 2 ). Metabolic rate and respiratory exchange ratio (RER) were calculated in 1-min segments and averaged from at least 30 min of steady data.

Insulin sensitivity was assessed by measuring insulin-stimulated whole-body glucose uptake using a modified frequently sampled intravenous glucose tolerance test and the Minimal Model Method of analysis as described in detail elsewhere72. Insulin resistance and beta cell sensitivity were assessed using the homeostasis model assessment (HOMA) method as previously described73.

Assessments of exercise capacity and physical function

Cardiorespiratory fitness was determined from a graded treadmill exercise test to volitional exhaustion using a modified Balke protocol as previously described74. Oxygen consumption (VO 2 ) and RER were measured using open-circuit spirometry with an online, computer-assisted analysis system. Heart rate and ratings of perceived exertion (RPE) were also measured throughout the test.

Walking endurance was assessed by measuring the distance covered during a 6-min walking task on a 50-foot (out-and-back) indoor course as previously described75.

Muscle strength and rate of torque development were quantified by measuring the peak force produced during a maximal voluntary contraction, and rate of torque development was measured using the maximal rate of developing torque during a rapid, forceful contraction of the knee flexor and extensor muscles as previously described by our laboratory76. Handgrip strength was measured using a standard handgrip dynamometer.

Leg fatigability was assessed using performance until failure during a single-leg heel-rise task. Subjects were asked to perform one complete plantar flexion contraction every 2 s until failure, and the test was terminated when the subject voluntarily stopped due to discomfort or inability to achieve at least 50% of maximal plantar flexion without using upper extremities for more than balance77. The chronic attribute of fatigue was also assessed using a Fatigue Questionnaire and Fatigue Severity Scale78.

Dynamic balance was assessed using a rapid step test. Maximal step length was measured in each direction (forward, backward, left, right), and targets were placed on the ground at 80% of the subject’s maximum with lines of colored tape, as described previously79. Performance was quantified as the time taken and number of errors committed on average during three rounds of the rapid step balance test. Each round consisted of 18 commands instructing subjects to step with a random foot to a random direction (i.e. left front). An error was defined as failure to completely step beyond the target, loss of balance, failure to return to the initial starting position, taking multiple steps to completely reach a target, or stepping with the incorrect leg or to the wrong target.

Mobility was assessed as the time to complete a 4-m walk task (performed in duplicate at the subjects' preferred walking speed) and the five-repeated sit-to-stand test (performed in triplicate), as previously described75,78,80. The test involves rising from a seated position in a standard-height chair five times, as quickly as possible, without using their arms for momentum or support.

Manual dexterity was assessed as the time to complete a 9-hole pegboard test as previously described76. Subjects collected smooth, rounded pegs from a dish and placed them into a pegboard, then returned them to the dish as quickly as possible. Two trials were completed with each hand.

Statistical analyses

The sample size for this study was sufficient to detect at least a 50% increase in NAD+ concentration following NR supplementation vs. placebo (effect size = 0.7; mean of difference = 7; 1−β = 0.8; α = 0.05) as well as a clinically relevant improvement in the cardiovascular parameter with the lowest effect size (FMD; mean difference = >1%; effect size = 0.86). Estimate of effect size for NAD+ was determined from preliminary data of NAD+ metabolite concentrations in PBMCs collected from human subjects. Effect size for FMD was determined from our laboratory’s previous crossover interventions demonstrating improvements in vascular function67. The required sample size was determined to be 19 subjects. Assuming a 20% dropout (4 subjects) and 40% exclusion due to screen failures (consistent with other intervention studies in our laboratory using dietary supplements81,82), a total of 60 participants were consented for this study. Significance was set at α = 0.006 for all secondary outcomes to adjust for multiple testing of NR vs. placebo (paired t-tests) on each of the following nine pre-specified hypotheses: (1) NAD+ metabolites, (2) cardiovascular parameters, (3) hematology, (4) metabolic panel, (5) lipid profiles, (6) energy balance, (7) glycemic control, (8) motor function and (9) exercise performance. Because many of the measures within each hypothesis are correlated with one another (e.g., cardiovascular measures, Supplemental Table 9), each group of measures listed above was treated as one outcome when adjusting for multiple comparisons. With the exception of our primary outcome variables (NAD+ metabolites: NAD+, NAAD, NADP, NaM, NMN), in which inferences were based on an unadjusted alpha level set at 0.05, all inferences of significance are based on the Bonferroni-adjusted alpha level (α = 0.006).

The intent of this study was to translate promising preclinical evidence for the efficacy of chronic supplementation with NAD+ boosting compounds to humans. Therefore, each outcome was tested under a directional hypothesis that was determined a priori, based upon previous studies reported in the literature. Accordingly, one-tailed hypothesis tests were used to compare the proposed unidirectional effects of NR supplementation vs. placebo on these outcomes. This method has been recommended elsewhere for Phase I and II placebo-controlled clinical trials in which the goal is to gain early insight into the potential efficacy of a compound83.

Prior to analysis, all continuous outcome variables were assessed for normality using the Shapiro−Wilk test and by examining individual frequency histograms for each outcome. If a variable was non-normally distributed, it was log-transformed prior to analysis. If log-transformation did not normalize the data, treatment condition was analyzed using the non-parametric Wilcoxon signed rank test. For each variable, any subject with a missing value during either phase was excluded from that analysis. Based on the interpretation of the primary data, post-hoc analyses were performed to compare the change in blood pressure and aortic stiffness between subjects who exhibited normal vs. above normal baseline blood pressure using an un-paired two-tailed t-test. We also explored the relation between baseline NAD+ concentrations and the overall increase in NAD+ using a Pearson correlation. A formal washout period was not included in the study design; however, given the crossover design, we tested for presence of a carryover effect for each of the outcomes under study using linear regression modeled with an indicator for treatment order (no carryover effects were observed between conditions). All statistical analyses were performed using the R statistical computing platform (version 3.2.2) and GraphPad Prism 7 software.

Data availability

The data that support the findings of this study are available from the corresponding author upon reasonable request. This trial is registered on ClinicalTrials.gov under the identifier NCT02921659.