Salicylic Acid sans Aspirin Figure 2. Urinary SA + SU excreted throughout time course of 13C experiment: total SA + SU excreted during the 8 h urine collection periods. The columns represent the median value and the error bars the maximum and minimum values. The dotted line at 24 h shows the point at which the ﬁrst dose of benzoic acid was introduced. One subject was omitted because of incomplete data. Table 3. Isotopic Enrichment of Salicylic Acid (SA) and Salicyluric Acid (SU) in Individual Urinesa sample SA % 13C6 m/z 273/267 SU % 13C6 m/z 330/324 urine 1 urine 2 urine 3 urine 4 urine 5 urine 6 pooled urine (no 13C6) standard soln SA standard soln SU 0.06 3.31 0.41 10.91 3.50 5.45 0.00 0.00 0.00 31.29 43.07 6.82 36.53 23.96 36.98 0.00 0.00 0.00 a Urines 1-6 show the relevant m/z ratio values, for each individual, of 13C6- labeled urinary salicylic acid (SA) and salicyluric acid (SU) SA, unlabeled 267, labeled 273 SU, unlabeled 324, labeled 330. Also shown are results for pooled, unlabeled (day 1) urine and for standard solutions of SA and SU. able amounts of the expected 13C6-labeled hippuric acid were found (data not shown). DISCUSSION There is now no doubt that salicylic acid and its salts are components of the human diet (11, 19-22). Although the bioavailability of dietary salicylates was estimated to be low (23), it is clear that serum SA levels in some aspirin-free vegetarian subjects and populations (9, 11) overlap with those of patients on low-dose aspirin regimens. One particularly revealing recent observation, in a study which conﬁrmed that circulating SA levels related to fruit and vegetable consumption, was that <20% of the variability in serum SA derived from that source (12). Results of SA levels in a large variety of animals showed that those species regarded as primarily carnivorous had levels comparable to those measured in herbivores. Some bacteria, notably mycobacterial, yersinia, and pseudomonas species, synthesize SA to enhance iron chelation, so there was a possibility that gut, particularly colonic, bacteria might be the source of SA not readily explicable by lack of dietary exposure. However, preliminary study of two germ-free animal models reported here showed the net effect of minimizing or eliminating a contribution from colonic bacteria was, in fact, an increase in serum SA levels. It was these animal observations which led us to postulate that serum SA levels in aspirin-free individuals may be, at least partially, endogenous. That salicylate levels in serum and urine J. Agric. Food Chem., Vol. 56, No. 24, 2008 11651 plateaued in a subject on a water/milk diet for 3 days should be considered in light of a SA half-life of 4 h, which would cause the serum SA level to fall to ∼0.004% of its initial level at the end of this time interval. The levels of serum SA in the fasting surgical patients study supported these milk diet observa- tions. Persistence of low levels of serum SA and combined urinary SA + SU excretion in the two patients who had panproctocolectomy, and therefore no colonic or dietary source of SA, was considered to be particularly strong evidence for an endogenous source of this simple organic molecule. The recourse to benzoic acid as a possible SA precursor in man was directed by the relevant plant biochemistry (14) and its prevalence in fruits and berries (15, 16). Benzoic acid may also considered to be formed in vivo (17, 18) and is well-known to be an acceptable diet additive. Given the relatively high doses (1-2 g/day) used in the preliminary benzoic acid study, the increase in SA levels observed on the last day of that protocol might be thought to be a little disappointing. The ﬁndings led us to use a slightly higher benzoic acid dose for the labeled study. Even with that higher benzoic acid dose, the increased total SA and SU urinary excretion was not signiﬁcant, although clearly very marked in some individuals. Whereas the renal excretion of SA depends strongly on pH and the presence of organic acids as well as urinary ﬂow rate (3), these possible confounding factors should reduce SA plus SU excretion under the conditions of this small study. Moreover, in a study of SA metabolism (to SU) using the isolated perfused rat kidney, the addition of the competitive substrate benzoic acid produced a rapid formation and excretion of hippuric acid with corresponding inhibition of SU formation and excretion (24). It would not, therefore, be surprising if use of a larger dose of benzoic acid in a greater number of subjects should lead to signiﬁcantly increased total salicylate excretion. However, it is likely that naturally occurring benzoic acid was contributing to the modest 20% variability in serum SA from fruits and vegetables in the diet (12), although the added sodium benzoate content of diets with considerable fruit and vegetable content would probably be low. Given the widespread use of sodium benzoate as an additive food preservative, further work on its contribution to serum SA levels may be justiﬁed. However, set against the nonsigniﬁcant increase of total SA and SU levels in the day 2 8-16 h urines, the extent of SU 13C6 labeling we determined in these samples might be considered to be surprising and may point to bioregulation of endogenous serum SA. For more than a century the focus has predominantly been on the properties of aspirin, the pro-drug of SA. It is only recently that the in vivo efﬁcacy of SA as an anti-inﬂammatory has been adequately explainedsin terms of inhibition of Cox-2 gene transcription. Given the lability of its acetyl ester (aspirin) within the narrow pH range of 2-3, other in vitro Cox- independent effects, such as mediation of apoptosis (25) and inhibition of angiogenesis (26), may also be attributable to SA rather than its pro-drug. Other diverse effects of SA continue to be reported, such as the surprising protective effects of lower dose SA in a mouse heart model of myocardial ischemia (27). The mechanism by which aspirin (and SA in vitro) has antitumoral actions is unclear, but a recent observation that cell proliferation is dependent on mitochondrial Ca2+ uptake and inhibited by low-level SA may be relevant (28). SA is widely found in the animal kingdom. We have adduced evidence for its biosynthesis from benzoic acid in man. These observations are, we suggest, appropriately assessed in light of the many effects of aspirin, its pro-drug. It is, we suspect,