Salicylic Acid sans Aspirin J. Agric. Food Chem., Vol. 56, No. 24, 2008 11649 Figure 1. Salicyluric acid in pooled urine sample: (a) partial mass spectrum obtained by gas chromatogrphaphy-mass spectrometry of a trimethylsilylated extract of pooled urine (isotopically labeled peaks are indicated by asterisks at m/z values 199, 212, and 330); (b) partial library mass spectrum of the di-trimethylsilyl derivative of standard salicyluric acid (o-hydroxyhippuric acid). Data were analyzed using the Automated Mass Spectral Deconvolution and Identiﬁcation System (AMDIS; NIST, Gaithersburg, MD; http://chemdata.nist.gov/mass-sps/amdis). MATERIALS AND METHODS Blood samples, serum or plasma, were obtained from animals at the London Zoo or at the Department of Biological Services, University of Glasgow. Blood was collected according to the approved codes of practice of these institutions. Six mice were treated with neomycin, 100 mg/kg/day, for 4 days prior to collection of blood to reduce as much as possible the bacterial content of their gastrointestinal tracts. Samples from germ-free Sprague-Dawley rats were obtained from Charles River Ltd., Margate, Kent, U.K. All human studies were approved by the Dumfries and Galloway Health Board Ethics Committee. Serum salicylic acid (SA) and urinary SA and salicyluric acid (SU) levels were assayed according to the HPLC methods described previously (7, 8). The absence of any SA from the milk diet used in the preliminary controlled diet study was conﬁrmed by total SA assay as we have previously described (11). A pilot study, to assess whether ingestion of benzoic acid might raise endogenous SA levels, utilized weighed preprepared meals (requiring only reheating) replicating exactly the same menu and ﬂuid intake over each 4 days. Throughout, urine samples were collected every 12 h. Fasting blood samples were obtained daily and on the morning of day 5. Two individuals were studied over 4 days of diet standardiza- tion after being aspirin-free for 2 weeks; the benzoic acid doses used were 1 g/day on days 3 and 4 in one subject and 2 g/day on these days in the other. Subsequently, a labeled study was undertaken over 3 days in six individuals (four males, two females, ages 30-62 years) using a standard protocol. On day 2 all six individuals received 1 g of uniformly ring-labeled 13C benzoic acid with each of their three main meals. Very minor gastric upset was the only side effect in an individual who preferred to take the benzoic acid unencapsulated. For that experiment uniformly ring-labeled 13C benzoic acid (ring- 13C 99% pure) was supplied by Cambridge Isotope Laboratories Inc. USA. Food and ﬂuid intake over the 3 days of the study were standardized. Each individual completed a detailed diary of all oral intake on day 1 and replicated that diet as closely as possible over the next two days. Cumulative urine samples were collected every 8 h over the whole 72 h of the study. The blood sampling protocol followed as closely as possible was day 1, 5 × 2 h, and days 2 and 3, 8 × 2 h, the ﬁrst sample on each day being taken fasting. The serum SA levels were analyzed as areas under the SA level time curves over 0-6 h only as missing values precluded 0-8 h analysis. Statistical analysis of the total urinary SA and SU results was by nonparametric Friedman two- way analysis of variance, using SPSS version 12. Gas chromatography-mass spectrometry (GC-MS) of the day 2, 8-16 h, urine samples was then performed. Preliminary fractionation was as described previously (8); samples prepared without internal standard were chromatographed under the usual conditions (with the electrochemical detector inactive), and the fraction between 1 min prior to the retention time (tR) of SU and 1 min after the tR of SA was collected. Trimethylsilyl derivatives were prepared by treating the dried extracts with a mixture of acetonitrile and N,O-bis(trimethylsilyl)triﬂuoroac- etamide (50:50, v/v, 30 min, 60 °C). GC-MS analysis was carried out on an Agilent 6890/5973 MSD system (30 m × 0.25 mm i.d. DB5MS column) using both scanning and selected ion monitoring methods. The main ion fragments of interest were for SA m/z 267 (unlabeled) and m/z 273 (labeled) and for SU m/z 324 (unlabeled) and m/z 330 (labeled). To obtain the mass spectra shown in part in Figure 1, aliquots of all of the second-day 8-16 h urine samples were initially pooled prior to derivatization. Data ﬁles were analyzed by the Automated Mass Spectral Deconvolution and Identiﬁcation System (AMDIS; NIST, Gaithersburg, MD; http://chemdata.nist.gov/mass-spc/amdis/). Further aliquots from individual samples were then fractionated, derivatized, and analyzed by selected ion monitoring to give the individual results. The ratios quoted were calculated from the m/z ratios of 273/267 for 13C6 SA and 330/324 for 13C6 SU. RESULTS As Table 1 shows, those species regarded as primarily carnivorous had blood SA levels comparable to those measured in herbivores. The highest levels we detected were in the range associated with aspirin use in man, as comparison with our appended published results (9, 11) shows, and only the crustacean samples examined did not contain SA. To examine the possibility of a gastrointestinal bacterial source for SA, two small groups of germ-free mice were assayed for SA. The pooled serum from six mice treated with neomycin, 100 mg/kg/day, for 4 days before sacriﬁce had a slightly higher concentration of SA in serum (0.309 µ mol/L) than the pooled serum sample from six untreated animals (0.268 µ mol/L). Another germ-free animal model studied was the Sprague- Dawley rat delivered by caesarean section, reared in a sterile environment, and fed sterilized food. A group of eight such germ-free animals had a pooled serum SA level of 0.166 µ mol/ L, approximately 2.5 times greater than the pooled serum salicylic acid level of 0.069 µ mol/L from a group of control animals. The net effect of minimizing or eliminating a contribu- tion from colonic bacteria in these animal models was therefore enhancement of serum SA levels in the host animal. Throughout the preliminary controlled diet study, in a subject who had taken no aspirin during the previous 2 weeks, there was persistence of measurable serum SA throughout 3 days on a milk/water diet (which was conﬁrmed by analysis to be free