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J Clin Pathol 2001;54:553–555
Salicylic acid in the serum of subjects not takingaspirin. Comparison of salicylic acidconcentrations in the serum of vegetarians,non-vegetarians, and patients taking low doseaspirin C J Blacklock, J R Lawrence, D Wiles, E A Malcolm, I H Gibson, C J Kelly, J R Paterson Keywords: salicylic acid; salicylates; diet; low dose Aims—To determine serum salicylic acid
concentrations in non-vegetarians and
vegetarians not taking salicylate drugs,
Over the past few decades, evidence has and to compare these concentrations with
emerged to suggest that regular aspirin use those found in patients taking aspirin,
reduces the risk of cardiovascular disease1 and 75 mg daily.
of developing colorectal cancer,2 and that it Methods—Serum samples were obtained
might also reduce the risk of developing lung and breast cancer3 4 and Alzheimer's disease.5 vegetarians (n = 39) not taking salicylate
In vivo, aspirin probably acts as an anti- drugs. Non-vegetarians and vegetarians
inflammatory prodrug, with the active compo- were recruited from the community and
nent being salicylic acid (SA).6 7 The risks of from a Buddhist monastery, respectively,
developing colorectal cancer and cardiovas- cular disease are also lower in those who Patients (n = 14) taking aspirin (75 mg
consume diets rich in fruits and vegetables, daily) were recruited from the Dumfries
although the chemical components responsible diabetic clinic. Serum salicylic acid con-
have not been identified. Nutritional research centrations were determined using a high
has centred on identifying the fruit and vegeta- ble components that are responsible for those method with electrochemical detection.
health benefits but these remain, as yet, Results—Salicylic acid was detected in
elusive.8 9 Recently, we identified SA and twoother salicylates—2,3-dihydroxybenzoic acid every serum sample analysed. Higher
and 2,5-dihydroxybenzoic acid—as normal serum concentrations of salicylic acid
constituents of serum in subjects not consum- were found in vegetarians than non-
ing salicylate drugs; because these compounds are components of fruits and vegetables, we 0.11 (range, 0.04–2.47) µmol/litre and 0.07
speculated that they originate from dietary (range, 0.02–0.20) µmol/litre, respectively;
plant sources.10 Because aspirin is a prodrug of the median of the diVerence was 0.05
SA,6 7 and a diet rich in fruits and vegetables µmol/litre (95% confidence interval for
and Galloway Royal
appears to help prevent similar conditions to diVerence, 0.03 to 0.08; p < 0.0001). The
aspirin, we sought quantitative evidence that median serum concentration of salicylic
might support SA as the common factor. Only Scotland, DG1 4AP,
acid in patients taking aspirin (75 mg
one other study has investigated salicylates in daily) was 10.03 (range, 0.23–25.40) µmol/
humans not taking salicylate drugs, measuring litre, which was significantly higher than
urinary "total salicylates" in 17 volunteers.11 D WilesJ R Paterson that found in non-vegetarians and veg-
Hydrolysis of urine with subsequent analysis, etarians. There was overlap in serum sali-
however, provides no information on the cylic acid concentrations between the
concentrations of SA found in blood or in vegetarians and patients taking aspirin.
tissue, especially because SA is metabolised and Galloway Royal
Infirmary
and salicylates may be concentrated in urine.
We have developed a method to measure SA present in fruits and vegetables and is
in sera from volunteers not consuming sali- found in higher concentrations in vegetar-
cylate drugs and have determined serum ians than non-vegetarians. This suggests
concentrations in non-vegetarians, vegetarians, Department of
Pathology, Dumfries
that a diet rich in fruits and vegetables
and patients taking aspirin, 75 mg daily.
and Galloway Royal
contributes to the presence of salicylic
acid in vivo. There is overlap between the
serum concentrations of salicylic acid in
VOLUNTEERS AND PATIENTS vegetarians and patients taking aspirin,
Non-vegetarians (n = 39; mean age, 40.5 Correspondence to:Dr Paterson 75 mg daily. These findings may explain,
years; SD, 10.9; 21 men) were recruited from in part, the health promoting eVects of
the community in Dumfries and Galloway, Accepted for publication dietary fruits and vegetables.
southwest Scotland. Vegetarians (n = 37; mean (J Clin Pathol 2001;54:553–555)
age, 41.7 years; SD, 10.5; 24 men) were Blacklock, Lawrence, Wiles, et al Serum concentrations of salicylic acid (µmol/litre) in non-vegetarians, vegetarians, and patients taking aspirin(75 mg daily) Patients taking low dose aspirin Aqueous standard solution Values are median (range).
*Significantly greater than non-vegetarians, median of the diVerence 0.05 µmol/litre (95% confidence interval for oltammetric response (mV) diVerence, 0.03 to 0.08; p <0.0001).
B). The prodigy ODS 3 HPLC column was Retention time (minutes) maintained at 30°C. Before use, all glassware High performance liquid chromatography chromatograms of an extract of serum was rinsed in double distilled water followed by and of an aqueous standard solution of salicylic acid (SA) and 4-methylsalicylic acid single distilled acetone. Each sample was analysed in duplicate. The intra-assay coef- recruited from a group of Buddhist monks, of ficient of variation (CV) was 7% at a serum SA European origin, who were in retreat (being concentration of 0.059 µmol/litre (10 determi- catered for from a communal kitchen) at the nations) and the interassay CV was 6% at a Samye Ling Monastery in Upper Eskdale, serum SA concentration of 0.095 µmol/litre southwest Scotland. Patients taking aspirin, (six assays carried out on six diVerent days). SA 75 mg daily (n = 14; mean age, 58.9 years; SD, was not detected when water samples were 12; seven men) were recruited from the Dum- analysed using the same needles, syringes, and fries diabetic clinic. In this last group the blood collection tubes as used for serum sam- median time between aspirin ingestion and venesection was 215 minutes (range, 105–1200). A drug history was obtained in the non- STATISTICAL ANALYSIS vegetarians and vegetarians to ensure no Serum SA concentrations were not normally volunteer was taking salicylate drugs. Blood distributed and median values (range) are samples (non-fasting) were taken in the morn- reported. Non-parametric analysis was carried ing from the vegetarians and patients, and from out using the Mann Whitney U test.
most of the non-vegetarians, although in thislast group some samples were taken in theafternoon. Venous blood samples were allowed to coagulate before separating the serum by SA was completely resolved from other com- (2000 ×g for 10 minutes).
pounds, having a retention time of approxi- Serum samples were then stored at −28°C.
mately 32–35 minutes (fig 1), and was found to These investigations were approved by the local be present in every serum sample analysed.
research ethics committee and informed con- Higher serum concentrations of SA were found sent was obtained.
vegetarians (table 1); the median of the diVer-ence was 0.05 µmol/litre (95% confidence The analysis of serum samples for SA was car- p < 0.0001). There was overlap in the serum ried out by high performance liquid chroma- concentrations between the vegetarian group tography (HPLC) with electrochemical detec- and the aspirin group. Eight of the 37 vegetar- tion, as described previously,10 but with the ians had serum SA concentrations above the following modifications. Briefly, we added lowest serum SA concentration found in the EDTA (final concentration of 100 µmol/litre), aspirin group and six of the 14 patients in the 4-methyl SA (internal standard, final concen- aspirin group had serum SA concentrations tration of 2.0 µmol/litre), and HCl (1.0 µmol/ below the highest vegetarian value. However, litre) to 0.5 ml aliquots of serum to produce a there was a large variation in the SA concentra- pH of 2.0; 2.0 ml of ethyl acetate was then tions within each of these groups, with the added. The extraction tubes were shaken for 15 aspirin group having a median SA concentra- minutes, centrifuged at 2000 ×g and the tion approximately 100 times greater than that organic phase removed. The extraction was of the vegetarian group.
repeated and the combined organic phasesevaporated to dryness at 70°C under oxygenfree nitrogen. The combined extracts were reconstituted in HPLC mobile phase B con- This is the first study to measure serum SA concentrations in subjects not taking salicylate 1.0 µmol/litre). The proportions of two mobile drugs. Our main findings were that SA was phases (A: 50% methanol (vol/vol) in citrate present in every serum sample analysed, and buVer, 30 mmol/litre, pH 4.0; B: 1% methanol that sera from vegetarians had higher SA con- buVer, 30 mmol/litre, centrations than those obtained from non- pH 3.8) in the elution mixture were modified vegetarians. This suggests that fruits and in a four step programme, namely: (1) 0–5 vegetables contribute to the presence of SA in minutes (25% A, 75% B); (2) 5–28 minutes vivo. Many fruits and vegetables contain (0% A, 100% B); (3) 28–43 minutes (100% A, salicylates but, in particular, herbs and spices 0% B); and (4) 43–58 minutes (25% A, 75% contain the greatest concentrations.12 We found Salicylic acid in the serum of subjects not taking aspirin a large variation in the serum SA concentra- The results of our study highlight the tions within both the vegetarian and aspirin presence of SA in blood, probably originating groups. In the last group, the blood sampling from fruits and vegetables. The presence of SA, time post-dose probably explains some of the especially in vegetarians, was at concentrations variation. It is more diYcult to explain the large that are known to inhibit the transcription of variation seen in the vegetarian group who COX 2, a key inflammatory enzyme in various were fed the same meals and all of whom were pathologies. SA concentrations also overlapped bled within a single two hour session. The with those measured in patients taking aspirin, samples, however, were non-fasting and the 75 mg daily. If the anti-inflammatory actions of serum SA concentrations may have been influ- SA at these concentrations can be confirmed in enced by the timing of the specimens relative to vivo, it is possible that dietary salicylates will breakfast. In this respect the diVerence be- prove to be one of the reasons why diets rich in tween the non-vegetarians and vegetarians may fruits and vegetables protect against colorectal be an underestimate because some of the sam- cancer and cardiovascular disease.
ples from the first group were taken in theafternoon.
We wish to thank the doctors and patients of the general prac- Studies are needed to establish the biological tices in Dumfries (33 Castle Street), Sanquhar, and Wigtown, eVects of the concentrations of SA found in the and the Buddhist monks at the Samye Ling centre, the diabeticpatients attending the diabetic clinic at Dumfries and Galloway vegetarian and non-vegetarian volunteers not Royal Infirmary, Dr M Murphy, and Dr M McMahon for help- taking aspirin. Other investigators have consid- ful comments on the manuscript, and P Moore for secretarialassistance. The authors gratefully acknowledge the financial ered dietary salicylate intake as unimportant assistance of Newton Stewart and District Branch of the BDA, because dietary salicylates are not in the form Dumfries and Galloway Acute and Maternity Hospitals NHSTrust, and the Chief Scientist's OYce, Edinburgh, UK.
of aspirin.11 The antiplatelet action of aspirin isnot shared by the other salicylates. However,most cardiovascular disease is the result of 1 Antiplatelet Trialists' Collaboration. Collaborative overview of randomised trials of antiplatelet therapy. I: Prevention of atherosclerosis, a chronic inflammatory disease death, myocardial infarction, and stroke by prolonged process.13 SA is an anti-inflammatory drug that antiplatelet therapy in various categories of patients. BMJ
1994;308:81–106.
was used clinically before aspirin, with recent 2 Marcus AJ. Aspirin as the prophylaxis against colorectal cancer. N Engl J Med 1995;333:656–8.
3 Schreinmachers DM, Everson RB. Aspirin use and lung, action of aspirin is the result of its predominant colon, and breast cancer incidence in a prospective study.
metabolite, SA.6 7 SA inhibits the transcription of cyclooxygenase 2 (COX 2), a key enzyme 4 Vainio H, Morgan G. Cyclo-oxygenase 2 and breast cancer prevention. BMJ 1998;317:328.
involved in inflammation and certain cancers.
5 Rich JB, Rasmusson DX, Folstein MF, et al. Nonsteroidal The inhibition of COX 2 transcription has anti-inflammatory drugs in Alzheimer's disease. Neurology
1995;45:51–5.
been shown to occur at SA concentrations as 6 Higgs GA, Salmon JA, Henderson B, et al. Pharmacokinet- low as 0.1 µmol/litre, concentrations that we ics of aspirin and salicylate in relation to inhibition of ara-chidonate cyclooxygenase and anti-inflammatory activity.
have found in volunteers not taking salicylate Proc Natl Acad Sci U S A 1987;84:1417–20.
drugs, especially vegetarians.
7 Xu X-M, Sansores-Garcia L, Chen X-M, et al. Suppression of inducible cyclooxygenase 2 gene transcription by aspirin Further evidence to support the view that and sodium salicylate. Proc Natl Acad Sci U S A the serum concentrations of SA found in 8 Stavric B. Role of chemopreventers in human diet. Clin Bio- vegetarians may have biological eVects is found in the overlap between vegetarians and patients 9 Greenberg ER, Sporn MB. Antioxidant vitamins, cancer, and cardiovascular disease. N Engl J Med 1996;334:1189–
taking aspirin. RuYn et al have shown that low dose aspirin (equivalent to 31 mg SA) reduced 10 Paterson JR, Blacklock CJ, Campbell G, et al. The identifi- cation of salicylates as normal constituents of serum: a link prostaglandin F2á concentrations in colorectal between diet and health? J Clin Pathol 1998;51:502–5.
mucosa at 24–30 and 72–78 hours after the last 11 Janssen PLTMK, Hollman PCH, Reichman E, et al.
Urinary salicylate excretion in subjects eating a variety of dose of aspirin.14 The mean peak plasma SA diets shows that amounts of bioavailable salicylates in foods concentration measured was 11.8 (SD, 8.18) are low. Am J Clin Nutr 1996;64:743–7.
µmol/litre in this group and the large standard 12 Venema DP, Hollman PCH, Janssen PLTMK, et al. Deter- mination of acetylsalicylic and salicylic acid in foods, using deviation indicates a wide range of plasma SA HPLC with fluorescence detection. J Agric Food Chem concentrations in subjects taking the same dose 13 Ross R. Atherosclerosis—an inflammatory disease. N Engl J of aspirin. At an aspirin dose of 81 mg daily, mucosal prostaglandin concentrations of both 14 RuYn MT, Krishman K, Rock CL, et al. Suppression of human colorectal mucosal prostaglandins: the lowest eVec- E2 and F2á were decreased.14 tive aspirin dose. J Natl Cancer Inst 1997;8:1152–60.

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