Purpose: The use of nutritional supplementation or the use of nutraceuticals has been a staple of athletes for decades. In this competitive market, there is an ongoing interest of increasing the stability and bioavailability of these substances. However, the effect of the salts, buffers and other components in nutraceutical formulations on their bioavailability is not well studied. One such common additive is trisodium citrate, a stable tribasic salt of citric acid. Trisodium citrate is commonly used as a flavoring agent, buffer, and/or preservative in food and drink and as an emulsifier in cheese. Previously it has been shown that the use of trisodium citrate may improve running performance by Ööpik et al. so there is interest in it not just as an excipient but as a nutraceutical as well. It has also been previously shown to modify cell membrane permeability (Anghileri, 1988).
In this study we examine the effect of trisodium citrate on the in vitro permeability through a CaCo 2 cell monolayer model for the intestinal lumen for leucine, isoleucine, glutathione, arginine, valine, citrulline, sarcosine, melatonin, and creatine monohydrate. To ensure that the effect was seen from trisodium citrate we additionally examined the effect of sodium chloride and calcium citrate on the permeability of leucine. Trisodium citrate concentration was optimized as well using leucine as a model amino acid.
Methods: Analytical separation was performed by HPLC on samples using a 250mm x 4.6mm 5um Thermo Scientific BDS Hypersil base deactivated C18 column with a mobile phase consisting of 0.05M ammonium sulphate at a flow rate of 1.5ml/min. Detection of standard samples was performed at 206nm using a photodiode array.
Briefly a confluent Caco2 Monolayer between (TEER ~900 Ohm*cm2) was equilibrated with DPBS. After 15 minutes apical solution was removed and replaced with experimental solution. Donor solution was immediately sampled and sealed for the duration of the experiment. Samples were taken from the basolateral chamber at 15 minute intervals for 90 minutes and replaced with fresh isotonic DPBS. At 90 minutes the apical chamber was also sampled. All samples were filtered and examined by HPLC.
Results: Optimal trisodium citrate concentration was found to be 10mM for the permeability of L-leucine. Calcium citrate and sodium chloride (at 10mM and 30mM respectively) had no effect on the permeability of L-leucine. L-leucine, isoleucine, creatine, glutathione, sarcosine, arginine, and valine showed a significant (p<0.05 by Students T-test) increase in permeability in the presence of 10mM trisodium citrate (Figure 1). Citrulline did show an increase in permeability in the presence of trisodium citrate, however it was not significant. Melatonin did not show a significant increase in permeability in the presence of trisodium citrate alone, however in combination with L-leucine there was a significant increase in melatonin permeability in the presence of trisodium citrate. Neither sodium chloride nor calcium citrate produced a significant increase in the permeability of L-leucine (Figure 2).
Conclusion: The common additive trisodium citrate resulted in a significant increase in the in vitro permeability of most of the nutraceuticals examined. This and other nutraceutical additives should be further studied for their effect both in vitro and in vivo.
Oopik V, Saaremets I, Medijainen L, Karelson K, Janson T, Timpmann S. Effects of sodium citrate ingestion before exercise on endurance performance in well trained college runners. Br J Sports Med. 2003;37(6):485–489.
Anghileri LJ, Crone-Escanye MC, Thouvenot P, Brunotte F, Robert J. Mechanisms of gallium-67 accumulation by tumors: role of cell membrane permeability. J Nucl Med. 1988; 29:663–668.