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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;
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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;
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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;
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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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Contents lists available at journal homepage: Detection of pyrrolizidine alkaloids in German licensed herbalmedicinal teas M. Schulz J. Meins S. Diemert P. ZR. Goebel D. SM. Schubert-Zsilavecz M. A a Drug Commission of German Pharmacists (AMK), Jaegerstrasse 49/50, 10117 Berlin, Germanyb Central Laboratory of German Pharmacists, Carl-Mannich-Strasse 20, 65760 Eschborn, Germanyc Department of Food Chemistry and Toxicology, Technische Universität Kaiserslautern, Erwin-Schrödinger-Strasse, 67663 Kaiserslautern, Germanyd Department of Pharmaceutical Chemistry, Goethe-University Frankfurt, Max-von-Laue Strasse 9, 60438 Frankfurt am Main, Germany