Nicotinic acid supplementation at a supraphysiological dose increases the bioavailability of NAD⁺ precursors in mares

Abstract

NAD+ deficiency has recently been linked with increased occurrences of congenital abnormalities and embryonic death in human and animal subjects. Early embryonic death is a major component of pregnancy loss in mares and very little is known regarding the requirement for NAD+ in horses. The aim of this study was to quantify NAD+ and its metabolites in the plasma and urine of mares after orally administering an acute dose of nicotinic acid and determine the absorption, metabolism and excretion of this essential precursor for NAD+ biosynthesis. Nicotinic acid (5 g per os) was administered to four mares via a dosing syringe. Blood samples were collected at 0, 0.25, 0.5, 1, 2, 4, 6 and 22 h, and urine samples were collected at 0, 3, 6 and 22 h. The samples were processed and analysed by mass spectrometry. A general additive model was applied to all metabolite concentration values followed by a post‐hoc multiple comparisons test. Nicotinic acid was rapidly absorbed into peripheral blood within 15 min of administration and the concentrations of nicotinic acid, nicotinamide (NAM), nicotinuric acid, nicotinic acid mononucleotide and nicotinic acid adenine dinucleotide (NaAD) increased significantly in plasma at 30 min. The concentrations of NAM, nicotinic acid riboside and NaAD increased significantly in urine at 3 h. The levels of NAM and NaAD remained significantly elevated in plasma at 22 h, sixfold and ninefold greater, respectively, than the basal levels at 0 h. While the extracellular levels of NAD+ in the samples remained undetected, the large, sustained elevation of NaAD levels in plasma indicates that the NAD+ levels were boosted within the cellular compartments. The results show that nicotinic acid supplementation increases the bioavailability of NAD+ precursors in mares, which is proposed to be beneficial during periods of peak NAD+ demand, such as during early embryo development.

Supplemental Nicotinic Acid Elevates NAD+ Precursors in the Follicular Fluid of Mares

“…The concentration of NA did not differ among the initial plasma samples and the plasma samples collected at the time of FF aspiration in both the vehicle-fed and NA-supplemented mares. In our previous study, we found that the concentration of NA in plasma peaked between 15 min and 1 h following the supplementation of NA [ 25 ]. Therefore, it is not surprising that the concentration of NA did not differ between the initial plasma and the plasma collected at the time of FF aspiration approximately 12–16 h after the final feed.…”

“…However, the absence of NaMN is surprising, considering that we previously detected it in the plasma of mares at low concentrations [ 25 ] that returned to baseline levels at 22 h following the supplementation of NA. In the present study, the NA dose was lower than that used in the previous study, and samples were collected 12–16 h following the final feed as opposed to the time points of 0, 0.25, 0.5, 1, 2, 4, 6 and 22 h [ 25 ] so it is likely that the concentration was below the limits of detection in this study. Additionally, the finding that NaAD was not elevated in the plasma or FF following the dosing period was unexpected, given our previous findings.…”

“…Additionally, the finding that NaAD was not elevated in the plasma or FF following the dosing period was unexpected, given our previous findings. NaAD is considered the most sensitive biomarker for NAD + metabolism [ 41 ] and was found to remain elevated in the plasma of mares for at least 22 h following the oral administration of a single supraphysiological dose of 5 g of NA [ 25 ]. In the present study, a NA dose of 3 g was used because the 5 g dose resulted in approximately 1.5 g of NA and associated metabolites being excreted in the urine, indicating it was excessive.…”

“…For the NA treatment, following a 12-day washout period, all mares were orally administered 3 g of NA mixed thoroughly in 10 mL of fresh apple sauce for a minimum of 3 consecutive days. This dose was chosen based on the findings of our previous study in which approximately 3.5 g of NA was absorbed, metabolised and distributed to cells via the circulation [ 25 ] and is about 30 times greater than the recommended daily requirement [ 15 ]. As before, the day of dominant follicle identification determined the number of consecutive days the mares received the NA treatment.…”

“…Recently, a mass spectrometric analysis of mare serum and urine samples following nicotinic acid supplementation at a supraphysiological dose (5 g) showed that the bioavailability of NAD+ precursors was significantly increased, with a sustained elevation of nicotinic acid adenine dinucleotide (NaAD) levels, indicating that the NAD + levels were boosted within the cellular compartments [ 25 ]. Furthermore, studies investigating dietary niacin requirements in humans have revealed that women require more niacin than men [ 26 , 27 , 28 , 29 ], particularly during pregnancy [ 30 ].…”