11650 J. Agric. Food Chem., Vol. 56, No. 24, 2008 Table 1. Concentration of Salicylic Acid (SA) in the Blood of a Variety of Animals animal burrowing owl ne-ne Indian rhinoceros pygmy hippopotamus agouti Asian elephant Burmese python rabbit piglet Arabian oryx sheep phylogenetic class Aves Aves Mammalian Mammalian Mammalian Mammalian Reptilia Mammalian Mammalian Mammalian Mammalian [SA] (µ mol/L) 9.854 5.609 4.700 2.384 2.116 1.635 1.362 1.129 1.010 0.777 0.715 animal tiger brown trout giraffe donkey sacred ibis goat giant anteater collared peccary African lion cow Gelada baboon Results in Man (9, 11) Results in Animals phylogenetic class Mammalian Pisces Mammalian Mammalian Aves Mammalian Mammalian Reptilia Mammalian Mammalian Mammalian [SA] (µ mol/L) 0.661a 0.538 0.507 0.473 0.353 0.310 0.293 0.237 0.226a 0.216 0.210 range (µ mol/L) 0.04-2.47 0.02-2.00 0.05-0.64 0.23-25.40 median (µ mol/L) 0.110 0.070 0.763 10.03 vegetarians, n ) 37 nonvegetarians, n ) 39 southern Indian villagers, n ) 21 75 mg aspirin takers, n ) 14 limit of detection of the method ) 0.005 µ mol/L a Mean concentration in ﬁve animals. Table 2. Patient Proﬁle no. of obs 15 6 12 11 4 3 patient gender 1 2 3 4 5 6 male female female male female male age (years) at time of diagnosis 39 59 68 47 64 66 Paterson et al. animal Chinese alligator domestic cat pond heron gorilla red faced spider monkey mouse rat domestic cat (fed only meat) chimpanzee European shore crab prawn phylogenetic class Reptilia Mammalian Aves Mammalian Mammalian Mammalian Mammalian Mammalian Mammalian Crustacea Crustacea [SA] (µ mol/L) 0.156 0.144 0.136 0.125 0.080 0.078 0.069 0.058 0.033 <0.005 <0.005 diagnosis ulcerative colitis with toxic megacolon second colon cancer with multiple polyps carcinoma of rectum carcinoma of colon carcinoma of rectum carcinoma of rectum + second cancer in cecum procedure/operation total colectomy with ileostomy panproctocolectomy abdomino-perineal resection extended r. hemicolectomy low ant. resection of rectum panproctocolectomy with ileostomy days prior to eatinga 2 3 5 2 2 2 serum SA (µ mol/L) median (range) 0.0624 (0.054-0.082) 0.037 (0.013-0.074) 0.045 (0.012-0.132) 0.074 (0.061-0.088) 0.34 (0.079-0.847) 0.103 (0.063-0.174) lowest urine (SA + SU, µ mol/24 h, median) 0.368 0.470 7.707 0.288 1.01 1.84 a Patients were either fasting or receiving parenteral nutrition with no salicylic acid content (conﬁrmed by assay). from SA). In that experiment urine excretion of SA + SU continued at a rate of around 2.1 µ mol/24 h throughout the period of dietary restriction. Blood samples were assayed for SA at 12 h intervals, and the serum SA levels did not change signiﬁcantly, not falling below 0.1 µ mol/L (20 times the limit of detection of the assay) over the 72 h of the study. These data suggested that another source of SA was responsible for the persistence of serum SA and the steady rate of excretion of SA + SU throughout the time course of this experiment. In six patients (Table 2) who had total colectomy or rectal excision following standard preoperative bowel preparation low- level serum SA (range ) 0.012-0.0847 µ mol/L) was detected and urinary SA + SU excretion persisted (median of individual lowest levels ) 0.613 µ mol/24 h, range ) 0.184-7.607) in all subjects, for up to 5 days postoperatively, rising only on refeeding. During the pilot benzoic acid study in two subjects there was little variation in serum SA levels, which were in the ranges of 0.44-0.53 and 0.023-0.047, respectively, with no downward trend over the course of the experiment. The rate of combined SA and SU excretion in urine, however, increased from median levels of 3.87 (range ) 3.47-4.32) and 4.50 (range ) 4.39-4.62) µ mol/24 h over the ﬁrst 3 days of the study to 5.10 and 7.04 µ mol/24 h, respectively, on day 4. That increase was greater in the subject who had consumed 2 g of benzoic acid per day on days 3 and 4. Further examination of the possible biosynthesis of SA in man required a labeled precursor study. That showed variability in total serum SA during the study, but in every individual the highest serum SA measured during the 3 days occurred during day 2sat a median time of 4 h (range ) 2-8 h) after the ﬁrst dose of benzoic acid. The areas under the SA level time curves conﬁrmed the highest serum levels were reached on day 2, but that was not signiﬁcant. The highest level (Figure 2, p ) 0.052) of combined SA + SU urinary excretion was observed in the 8-16 h sample after the initial dose of benzoic acid. To ascertain whether any of the 13C6 label in the benzoic acid administered could be detected in salicylates, we therefore focused on the day 2, 8-16 h urine. No 13C6 was detected in the samples obtained prior to ingestion of labeled benzoic acid. In Figure 1 the marked peaks (/) indicate fragments from the presence of the (six ringed) 13C- labeled SU derivative (m/z 330), 6 mass units higher than the unlabeled SU derivative (m/z 324). The 13C isotope was also conﬁrmed in all six individual urine, day 2, 8-16 h samples and accounted, by selective ion monitoring, for 0.4-10.9% (median ) 3.4%) in the SA derivative and 6.8-43.1% (median ) 33.9%) in the SU derivative (Table 3). In addition, consider-