11648 J. Agric. Food Chem. 2008, 56, 11648–11652 Salicylic Acid sans Aspirin in Animals and Man: Persistence in Fasting and Biosynthesis from Benzoic Acid JOHN R. PATERSON,†,§ GWENDOLINE BAXTER,*,§ JACOB S. DREYER,§ JOHN M. HALKET,# ROBERT FLYNN,§ AND JAMES R. LAWRENCE§ Dumfries and Galloway Royal Inﬁrmary, Bankend Road, Dumfries DG1 4AP, United Kingdom, and Centre for Chemical Sciences, Royal Holloway, Egham TW20 0EX, United Kingdom Salicylic acid (SA), which is central to defense mechanisms in plants and the principal metabolite of aspirin, occurs naturally in man with higher levels of SA and its urinary metabolite salicyluric acid (SU) in vegetarians overlapping with levels in patients on low-dose aspirin regimens. SA is widely distributed in animal blood. Fasting for major colorectal surgery did not cause disappearance of SA from plasma, even in patients following total proctocolectomy. A 13C6 benzoic acid load ingested by six volunteers led, between 8 and 16 h, to a median 33.9% labeling of urinary salicyluric acid. The overall contribution of benzoic acid (and its salts) to the turnover of circulating SA thus requires further assessment. However, that SA appears to be, at least partially, an endogenous compound should lead to reassessment of its role in human (and animal) pathophysiology. KEYWORDS: Salicylic acid; aspirin; benzoic acid; synthesis; salicyluric acid INTRODUCTION In plants, salicylic acid (SA) is a ubiquitous secondary metabolite pivotally concerned in initiation of the response to a variety of physical, chemical, and biological insults (1). The potent analgesic and antipyretic properties of plant extracts, notably dried myrtle leaves and willow bark, had been known for many centuries before the isolation of salicylic acid, as the likely active principle, by Meyer in 1870 (2). Since synthesis of aspirin (acetylsalicylic acid) by Hoffman in 1897, investiga- tion has focused on the properties of that compoundsdesigned as a pro-drug. Aspirin is very rapidly hydrolyzed, having a plasma t1/2 of 20 min, to the deacetylated metabolite SA (2- hydroxybenzoic acid), which has a half-life of between 2 and 4 h (3). However, the demonstration that aspirin acts by serine side-chain acetylation of Cox-1 and Cox-2 isoforms, restricting substrate access to the active sites of these enzymes, has deﬂected attention from salicylic acid itself, although in vivo SA is, despite weak, reversible Cox-1 and absent Cox-2 inhibition, as effective as aspirin in suppressing inﬂammation (4). The inhibition of transcription of the Cox-2 gene by micromolar concentrations of SA (5, 6) is the likely explanation. Previous work by our group has demonstrated and conﬁrmed the presence of SA in serum (7) and urine (8), where its metabolite salicyluric acid (SU) predominates, from individuals who had not recently consumed aspirin. The measured levels * Corresponding author (e-mail Gwen.baxter@nhs.net). † Deceased. § Dumfries and Galloway Royal Inﬁrmary. # Centre for Chemical Sciences. were signiﬁcantly higher in vegetarians (9, 10), overlapping with levels in patients on low-dose aspirin regimens, and our past publications have focused on a dietary origin (11) of this naturally occurring SA in man. However, conﬁrmation of the contribution from fruit and vegetable consumption in the diet to circulating SA levels in man (12) has demonstrated that <20% of the variability in serum SA, measured by a sensitive speciﬁc method, may derive from that source. Some circulating SA in aspirin-na¨ıve or -free individuals could possibly derive from another dietary source or be of nondietetic origin. In plants, SA is synthesized through the shikimic acid pathway via isochorismate (13) or, probably predominantly, by the phenyl-propanoid route via cinnamic and benzoic acids (14). Benzoic acid is a natural constituent of plants, with high amounts being found in fruits and berries (15, 16). That hippuric acid, the main metabolite of benzoic acid, may be formed endog- enously in man was demonstrated by formula diet feeding (17, 18). In the Sprague-Dawley rat a radiolabel experiment showed phenylalanine to be the likely precursor (17). Use of the generally regarded as safe (GRAS) sodium benzoate as a food preservative also contributes to human intake. With regard to levels of regular intake, the FDA estimate is of 0.9-34 mg/ day as benzoic acid and 34-328 mg/day as sodium benzoate, although much higher levels of sodium benzoatesup to 5 g twice dailyshave been used therapeutically in the treatment of acute hepatic encephalopathy. Thus, in this paper we have determined whether the addition of benzoic acid to a standard- ized diet produced any change in serum or urinary salicylates. 10.1021/jf800974z CCC: $40.75  2008 American Chemical Society Published on Web 11/20/2008