**4. Endogenous salicylic acid**

#### **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

*Concentration of salicylic acid (SA) in the blood or body fluid of a variety of animals.*

*a*

**Table 2.**

**183**

*Mean concentration in five animals.*

*\*Red faced spider monkey. \*\*Fed only meat. \*\*\*European shore crab.*

**Animal Phylogenetic class SA (μmol/L)** Burrowing Owl Aves 9.854 Ne-Ne Aves 5.609 Indian Rhinoceros Mammalia 4.700 Pigmy Hippopotamus Mammalia 2.384 Agouti Mammalia 2.116 Asian Elephant Mammalia 1.635 Burmese Python Reptilia 1.362 Rabbit Mammalia 1.129 Piglet Mammalia 1.010 Arabian Onyx Mammalia 0.777 Sheep Mammalia 0.715 Tiger<sup>a</sup> Mammalia 0.661 Brown Trout Pisces 0.538 Giraffe Mammalia 0.507 Donkey Mammalia 0.473 Sacred Ibis Aves 0.353 Goat Mammalia 0.310 Giant Anteater Mammalia 0.293 Collared Peccary Reptilia 0.237 African Lion<sup>a</sup> Mammalia 0.226 Cow Mammalia 0.216 Gelada Baboon Mammalia 0.210 Chinese Alligator Mammalia 0.156 Domestic Cat Mammalia 0.144 Pond Heron Aves 0.136 Gorilla Mammalia 0.125 Monkey\* Mammalia 0.080 Mouse Mammalia 0.078 Rat Mammalia 0.069 Domestic Cat\*\* Mammalia 0.058 Chimpanzee Mammalia 0.033 Crab\*\*\* Crustacea <0.005 Prawn Crustacea <0.005

*Salicylic Acid Sans Aspirin in Animals and Man DOI: http://dx.doi.org/10.5772/intechopen.91706*

## *Salicylic Acid Sans Aspirin in Animals and Man DOI: http://dx.doi.org/10.5772/intechopen.91706*


*a Mean concentration in five animals.*

*\*Red faced spider monkey.*

*\*\*Fed only meat.*

*\*\*\*European shore crab.*

#### **Table 2.**

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

*Relative contributions of different food groups to total salicylate intake of a Scottish population. Source: [29].*

*Drug Repurposing - Hypothesis, Molecular Aspects and Therapeutic Applications*

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

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

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

Blood samples, serum or plasma, were obtained from animals at London Zoo or the Department of Biological Services, University of Glasgow in accordance with

4.42 and 3.16 mg/day respectively, a gender difference not sustained when

intakes of fibre, potassium, vitamin C and alcohol!

dietary sources or be of non-dietetic origin.

**4. Endogenous salicylic acid**

**4.1 Background**

**4.2 SA in animal blood**

**182**

the results.

**Figure 2.**

*Concentration of salicylic acid (SA) in the blood or body fluid of a variety of animals.*

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.

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

The protean actions of aspirin in animals and man are here, in a short review by JRL an GJB, set against what is known of the role of its SA precursor in earlier life forms.

This complex area is here only briefly overviewed in relation to its potential for

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 nonribosomal 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

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

form not yet (then) shown to be affected by salicylate is the Archaea!

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

SA, point to possible bioregulation of the levels of endogenous serum SA.

pointing to possible effects of SA preserved into animals.

**5. Salicylic acid in the biosphere**

*Salicylic Acid Sans Aspirin in Animals and Man DOI: http://dx.doi.org/10.5772/intechopen.91706*

**5.1 Bacteria**

targets for PAS) [33].

**5.2 Plants**

**185**

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 preoperative 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.
