Salicylic Acid Sans Aspirin in Animals and Man

2.1 SA in blood

Examination of the chromatograms of blank serum or plasma from published methods of SA analysis revealed the presence of an unknown substance with a retention time (R_t) similar to SA [5, 6]. While Ruffin et al. [7] reported SA in the plasma from 17 of 53 subjects at baseline there was no information as to how they confirmed identification of the compound.

We examined samples from drug free volunteers: extracts of acidified serum were analysed by high performance liquid chromatography (HPLC) with electrochemical detection. Chromatographic conditions were altered and the R_ts of the unknown compounds compared against authentic SA, 2,3 DHBA and 2,5 DHBA. Serum samples (some spiked with SA) were also incubated with a bacterial salicylate hydroxylase and the substance which had a R_t identical to SA disappeared. Finally the trimethylsilyl (TMS) derivative of the unknown and SA had, using gas chromatography–mass spectrometry (GC–MS), a similar retention time and total ion chromatogram [8].

2.2 SA and salicyluric acid (SU) in urine

Armstrong et al. [9] had detected, by paper chromatography, a compound with characteristics similar to those of SU in the urine of 400 people who had not taken salicylate drugs. That was in an admixture of 49 compounds of predominantly, it was suggested, dietary origin. Von Studnitz and colleagues, who also used a paper system, suggested SA might be one of the phenolic acids in the urine of subjects on a diet restricted to glucose and citric acid [10]. Young reported a compound with thin layer chromatographic properties of SU [11] in a single individual on a synthetic diet, while Finnie and co-workers found a similar compound on their paper system in children not taking salicylate drugs [12]. At the time of all these earlier investigations methods for adequately characterising and quantifying the purported SA and SU were not readily available.

Other work [13] designed to assess the dietary importance of salicylates had examined acid-treated urine using HPLC with fluorescence detection but salicylates other than SA, salicylate precursors and structurally related compounds may have been included in the values reported.

We examined 24 h urine samples from 10 volunteers who had not taken any salicylate drugs during the previous 2 weeks. The acid hydrophobic compounds (=organic acids) were separated using HPLC and quantified electrochemically. The R_ts of the extracted substances and those of SA and SU were compared under two sets of chromatographic conditions and found very similar to those of authentic substances. The unknown substances, isolated by HPLC, and treated with acetyl chloride in methanol were compared with the methyl esters of SA and SU using GC–MS. After esterification the unknown compounds had mass spectra and R_ts comparable to those of methyl-SA and methyl-SU [14].

These findings in serum and urine led us on to explore, in some detail, the likely dietary sources for the salicylate compounds we found and confirmed. At the time interest was re-awakening in the potential benefits of salicylates commonly found in the diet [13]—as opposed to what were considered traditional plant medicine sources.

3.1 Previously

The occurrence of “natural salicylates”, such as SA, in strawberries and other fruits was raised in the Lancet in 1903 [15] and the matter of whether these natural salicylates were superior to synthetic salicylates was the subject of a JAMA editorial in 1913 [16]—no superiority was concluded! Interest then appeared to wane until re-invigorated by the popularity, from ∼1970, in therapeutic trials—arising from the apparent cross-reactivity of tartrazine and aspirin—of exclusion diets.

3.2 Background

Many plant derived non-nutritive compounds exert, in mammalian systems, biological activities that may have an impact on health and disease risk [17] and we proposed [18] that SA might provide a link between aspirin, diet and the prevention of colorectal cancer (CRC). There is good evidence that the regular intake of aspirin decreases the risk of developing cancer [19]. A mechanism for platelet mediated CRC tumorigenesis has been proposed [20]; that would, of course not be attributable to SA itself but a more balanced view is that both constituent groups of aspirin (acetyl and SA moieties) contribute to the anti-cancer effects [21].

Assessment of the extent of the contribution of diet to SA in blood and urine cannot be easily inferred from direct analysis of its concentration therein. There is considerable variability in peak serum levels of SA in subjects receiving a standard dose [7] while urinary salicylate is influenced by urine flow, pH, the presence of other organic acids and the saturability of SU formation and/or excretion [2].

3.3 Salicylates from food

It was unclear whether sufficient salicylic acid could be obtained from dietary sources to influence health and disease with estimated daily intakes ranging from 0.4 to 200 mg/day [13, 22, 23]. That range reflected the disparate information available on the salicylate content of foods.

Comparison, in the serum of subjects not taking aspirin, of SA levels in 37 vegetarians and 39 non-vegetarians found higher concentrations in the former [24]. That study revealed median concentrations of 0.11 (range 0.04–2.47) μmol/L and 0.07 (range 0.02–0.20) μmol/L respectively: the median of the difference was 0.05 μmol/L (95% confidence interval for difference 0.03–0.08; p < 0.0001). The median SA level measured in serum from 14 patients on aspirin 75 mg/day was, at 10.03 (range 0.23–25.40) μmol/L, significantly higher. However there was overlap in serum SA concentrations between the vegetarians (8 higher than lowest low dose aspirin) and patients taking aspirin (6 below the highest vegetarian value). These findings should be considered in light of the inhibition of COX2 transcription that has been shown to occur at SA levels as low as 0.1 μmol/L [4].

In a further study the urinary excretion of SA and SU was assessed in 24 h samples from 27 non-vegetarians, 21 vegetarians and 40 patients taking 75 or 150 mg aspirin/day [25]. For SU, the principle urinary salicylate, vegetarians excreted significantly more than the non-vegetarians (median 11.01; range 4.98–26.60 μmol/24 h compared with 3.91; range 0.87–12.23 μmol/24 h) but these amounts were significantly lower than those excreted by patients on aspirin. Significantly more SA was excreted by the vegetarians (median 1.19; range 0.02–3.55 μmol/24 h) than by the non-vegetarians (median 0.31; range 0.01–2.01 μmol/24 h). The median amounts of SA excreted by the vegetarians and the patients taking aspirin were not significantly different. These values were comparable to those found earlier, using less specific methodology, in a group of drug free volunteers on a variety of diets [13].

3.4 SA in food

Given that SA is a stress hormone in plants we can anticipate that locality, varietal and growing conditions could affect total salicylate content at harvesting before any variability in processing conditions and storage effects. Wide reported ranges for different brands assayed using standard conditions are therefore not particularly surprising. For example the SA content of five brands of orange juice obtained from Scottish retailers ranged from 0.47 to 3.01 mg/kg [29].

3.4.2 A Scottish overview

Clearly sample selection and methodology will affect estimates of SA content in the diet. Using the assay methodology our group developed [27, 30] to supplement published results, Wood et al. [29] prepared a comprehensive dietary database. They filtered published results of dietary constituent total salicylate content by specific criteria. Food items had to be randomly selected and purchased from various commercial outlets at different times of the year; food samples to be prepared using standard domestic practices; optimised sample extraction and hydrolysis conditions were to be clearly described or cited, and salicylate determination to be based on modern techniques of HPLC and mass spectrometry with validation and quality assurance methods summarised. Combination of such published data, which met these criteria, together with their in-house analyses resulted in a database of 27 types of fruit, 21 vegetables, 28 herbs, spices and condiments, 2 soups and 11 beverages—expressed as median values to reflect the non-normal distribution of the results.

Subsequently dietary intake was assessed by applying this salicylate database, using a validated questionnaire, to 237 healthy individuals age range (17–72) from Aberdeen [29]. Estimated median total salicylate intakes for men and women were 4.42 and 3.16 mg/day respectively, a gender difference not sustained when corrected for energy. Primary food sources of salicylates as shown in Figure 2 were alcoholic beverages (22%), herbs and spices (17%), fruits (16%), non-alcoholic beverages including fruit juice (13%), tomato-based sauces (12%) and vegetables (9%). Salicylate intake was significantly (p < 0.001) and positively associated with intakes of fibre, potassium, vitamin C and alcohol!

4.1 Background

There were clear suggestions in the literature that SA and SU in urine might not all derive from diet, given their presence in the urine of subjects on restricted diets [9, 11]. In addition quantification of the contribution from fruit and vegetable consumption to circulating SA levels in man had demonstrated, using a sensitive and specific method that <20% of the variability derived from these sources [31]. So some circulating SA in aspirin-naïve or -free individuals may derive from other dietary sources or be of non-dietetic origin.

4.2 SA in animal blood

Blood samples, serum or plasma, were obtained from animals at London Zoo or the Department of Biological Services, University of Glasgow in accordance with their approved codes of practice. The results (Table 2) showed that species regarded as primarily carnivorous had blood SA levels comparable to those measured in herbivores [28]. The highest levels detected were in the range associated with aspirin use in man and only the crustacean body fluid samples examined did not contain SA.

As some bacteria, notably Mycobacterial, Yersinia and Pseudomonas species, synthesise SA to enhance iron chelation (see Section 5.1) the possibility of a gastrointestinal particularly colonic, bacterial source for SA was assessed in two animal models. Pooled serum from six mice treated with neomycin 100 mg/kg/day for 4 days had a serum SA concentration which was, at 0.309 μmol/L, slightly higher than the level of 0.268 μmol/L measured in six untreated animals. Other measurements were done on serum samples from Sprague-Dawley rats delivered by caesarean section, raised in a sterile environment and fed sterilised food. A group of 8 such germ free animals had a pooled serum SA level which was, at 0.166 μmol/L, ∼2.5× greater than the level of 0.069 μmol/L in serum from a group of control animals [28].

4.3 SA in diet-restricted and fasting human subjects

A preliminary study followed serum SA and urinary SA + SU over 3 days on a water/milk diet (confirmed salicylate free on analysis) in a subject free of aspirin for at least 2 weeks. Excretion of SA + SU continued at a rate of ∼2.1 μmol/24 h throughout and serum SA did not fall—over the 72 h of the study—below 0.1 μmol/L (20× the limit of detection of the assay) [28].

In six patients who had total colectomy or rectal excision following standard pre-operative bowel preparation low level serum SA (range 0.012–0.085 μmol/L) was detected and urinary SA + SU excretion persisted (median lowest level 0.613 μmol/24 h; range 0.184–7.607) in all subjects for up to 5 days postoperatively, rising only on refeeding [28]. These results also, of course, have some relevance to Section 4.2.

4.4 SA formation from benzoic acid

Benzoic acid (BA) is a natural constituent of plants, with high levels found in fruits and vegetables. In plants synthesis of SA derives—at least partially—from phenylalanine via cinnamic and benzoic acids. Prior work, using formula diet feeding, also demonstrated that hippuric acid, the main metabolite of BA, may be formed endogenously in man, while a Sprague-Dawley rat radiolabeled experiment showed phenylalanine as the likely precursor [32]. Sodium benzoate as a food preservative also contributes to human intake and very high doses have been used in hepatic encephalopathy. These considerations led us to determine whether addition of BA to a very carefully standardised diet produced any change in serum or urinary salicylates—see Figure 1.

A preliminary study, over 4 days in two subjects, suggested that BA, 1 or 2 g/day on days 3 and 4 might be associated with a modest increase in urinary SA + SU excretion. Subsequently a labelled study was undertaken over 3 days in six individuals (4 M, 2F) who received 1 g of uniformly ring-labelled ¹³C BA with each of their main meals on day 2. They replicated their carefully recorded day 1 diet throughout and had regular blood sampling with complete urine collections. The total SA + SU urinary excretion increased, but not significantly (p = 0.052) and only in the 8–16 h sample after the first dose of BA. While no ¹³C was detected in samples prior to ingestion of the BA, the ¹³C isotope was confirmed in the 8–16 h urine sample from all six subjects. Its presence was determined by preliminary GC fractionation before subjecting the relevant fractions to derivatization and GC–MS. The ¹³C isotope 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. In addition considerable amounts of the expected ¹³C₆-labelled hippuric acid were found [28]. Set against the total SA + SU levels the extent of the SU ¹³C₆ labelling found might be considered surprising but could, as well as confirming the in vivo synthesis of SA, point to possible bioregulation of the levels of endogenous serum SA.

5.1 Bacteria

This complex area is here only briefly overviewed in relation to its potential for pointing to possible effects of SA preserved into animals.

Para-aminosalicylic acid (PAS), the earliest truly effective anti-tuberculous agent, was long thought an analogue for para-aminobenzoic acid and so an inhibitor of folic acid biosynthesis. That was before the discovery of the mycobacterial siderophore (iron binding molecule) mycobactin, and that SA (also formed, as an extracellular metabolite, by mycobacteria in iron deficient conditions) is its direct precursor. It appears PAS primarily inhibits the conversion of SA into myobactin. Possible secondary roles for SA are the transfer of Fe²⁺ across the cell membrane, either for direct incorporation into various porphyrins and apoproteins, or for storage of iron within the cytoplasm in bacterioferritin (both roles also potential targets for PAS) [33].

There are many kinds of bacterial siderophores but SA or one of its hydroxylated metabolites (2,3DHBA) are at the core of the aryl- capped molecules found in E. coli (Enterobactin); Yersinia sp. and Klebsiella pneumoniae (Yersinibactin); Pseudomonas sp.(Pyochelin); Vibrio sp. (Vibriobactin/Vulnibactin) and Acinetobacter baumanii (Acinetobactin) [34]. These authors described a probe for the initial aryl acid activation enzymatic step in the synthetic pathways of these “bactins” (via a non-ribosomal peptide synthetase pathway initiated by adenylation) and suggested lack of human homologues makes this a potential drug target—but see Section 5.3.4.

Intriguingly investigation into the bioinorganic chemistry of bacterial siderophores has revealed that many have functional capacities other than mere iron homeostasis. Examples include interactions with other metals such as zinc, copper and boron; signalling agents (referred to as “ferrimones”) in the regulation of genes related to iron metabolism; protection—by those with catecholate structures—from oxidative stress and an antibiotic function in sideromycins [35].

Finally bacterial growth in the presence of salicylate can be both beneficial and detrimental. On the one hand an intrinsic multiple antibiotic resistance phenotype can be induced and on the other reduced resistance to some antibiotics might result and bacterial virulence factors may be affected [36]. While the in vivo consequences of these observations is speculative the findings highlight, the authors suggest, the ability of salicylate to alter gene expression; they claim that the only life form not yet (then) shown to be affected by salicylate is the Archaea!

5.2 Plants

While salicylic acid (initially from plant sources) has been used in therapeutics for millennia detailed knowledge of its role in plants is relatively recent. Although plant phenolics are diverse and ubiquitous they were traditionally assumed to be unimportant secondary metabolites but SA in plants is a critical hormone playing a direct role in the regulation of many aspects of growth and development as well as in thermogenesis and disease resistance [37]. The first clear evidence came, intriguingly (in relation to its antipyretic qualities in animals), from its role in voodoo lily thermogenesis; that appears to be mediated by stimulation of the mitochondrial alternative respiratory pathway [38]. Soon thereafter its role as a defence signalling hormone was documented—though the ability of plants to develop acquired immunity after pathogen infection was first proposed many years earlier. In the acquired immunity—called “systemic acquired resistance” (SAR)—of plants to biotrophic (i.e. threatening living cells) pathogens, the role of SA is pivotal. Careful study has identified two pathways for its synthesis, numerous proteins that regulate its synthesis and metabolism and some signalling components, including a large number of potential targets/receptors, which operate downstream of SA [39]. This is a perplexing field; for example while the non-specialist can readily appreciate methylation of SA to a volatile ester for transport through the phloem (before demethylation at a site where SA levels are low) subsequent steps are complex. As these authors point out it is increasingly evident that SA does not signal immune response by itself but as part of an intricate network of other plant hormones. We would highlight, from the viewpoint of the present review, their suggestion that it is important to differentiate SA “targets” from the subset (whose criteria, they concede, will be difficult to specify) that meet additional conditions to be designated “receptors” [40]. That idea is particularly relevant when later considering the propensity of aspirin to acetylate many animal protein “receptors”—see Section 5.3.4.

The wide range of basal SA levels between and within plant species, and potential for a biphasic/concentration dependent response may explain some conflicting reports on the spectrum of plant processes it influences. Despite these caveats the long list affected by exogenous SA includes resistance to biotic (pathogen-associated) stress and tolerance to many abiotic stresses (drought, chilling, heat, metal, UV radiation, and salinity/osmotic stress) as well as multiple aspects of plant growth and development. These include photosynthesis, senescence, thermogenesis, respiration, glycolysis, the Krebs cycle and the alternative respiratory pathway [40].

5.3 Salicylic acid and aspirin (ASA) in animals and man

Although the use of willow extracts had been known for centuries the report of its first well documented use—as a cheaper remedy for “the agues” than expensive cinchona bark—focused on its antipyretic properties. Then, particularly in the decades following the isolation of SA as the active principle, evidence steadily accrued for its efficacy as an analgesic and anti-inflammatory in e.g. acute rheumatic fever.