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in Smoking and Nonsmoking Men and Women1
School of Biomolecular Sciences, Liverpool John Moores University, Byrom Street, Liverpool, L3 3AF, England, UK
2To whom correspondence should be addressed. E-mail: k.rahman{at}livjm.ac.uk
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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(8-iso-PGF2). Because smokers are exposed to increased oxidative stress, this study was performed in both smoking and nonsmoking subjects. Plasma and urine concentrations of 8-iso-PGF2 in nonsmoking individuals were 1.25 ± 0.19 nmol/L and 272 ± 53 pmol/mmol of creatinine, respectively. In age- and sex-matched smokers, plasma and urine concentrations of 8-iso-PGF2 were 58% and 85% higher, respectively. Dietary supplementation with AGE for 14 d reduced plasma and urine concentrations of 8-iso-PGF2 by 29% and 37% in nonsmokers and by 35% and 48% in smokers. Fourteen days after cessation of dietary supplementation, plasma and urine concentrations of 8-iso-PGF2 returned to values not different from those before ingestion of AGE in both groups. Thus, dietary supplementation with AGE may be useful in reducing oxidative stress in humans.
KEY WORDS: • F2-isoprostanes • garlic • smoking • humans
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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). As a result, increases in antioxidative capacity are believed to be protective against such diseases. Garlic (Allium sativum) has attracted considerable interest as a potential cardioprotective agent (2
–4
), and the antioxidant properties of one such preparation, aged garlic extract (AGE)3
or Kyolic (Wakunaga of America, Mission Viejo, CA), have been demonstrated. Thus, AGE inhibits lipid peroxidation in vascular endothelial cells induced by both hydrogen peroxide (5
) and oxidized LDL (6
). Recently, it has been reported that AGE supplementation reduces the propensity of human LDL to undergo Cu2+-induced lipid peroxidation in vitro (7
).
Most of the traditional methods used to assess oxidative stress in clinical settings are nonspecific, unreliable or inaccurate. Recently, a family of novel prostaglandin F2 isomers called F2-isoprostanes has been described (8
). F2-isoprostanes are produced in vivo by cyclooxygenase-independent free radical peroxidation of arachidonic acid and are released from membrane phospholipids in response to cellular activation, presumably through a phospholipase-mediated mechanism. They circulate in plasma as the free form or as esters in phospholipids while the free form is excreted in urine. Quantification of F2-isoprostanes in plasma and urine is a sensitive and specific indicator of lipid peroxidation in vivo (9
). Indeed, F2-isoprostanes are increased in human diseases thought to be associated with increased oxidative stress, such as smoking, hypercholesterolemia, diabetes and alcoholic liver disease (10
–15
). Recent studies have shown that plasma concentrations of F2-isoprostanes are not influenced by the fat content of the diet (16
).
Due to its vasoconstrictive platelet activation and mitogenic properties, the F2-isoprostane 8-iso-prostaglandin F2 (8-iso-PGF2, also referred to as 8-epi-PGF2 or iPF2-III) has received much attention (17
–20
). Plasma and urinary concentrations of total F2-isoprostanes (and specifically 8-iso-PGF2) are elevated in smokers, while concentrations return to control values upon cessation of smoking or after supplementation with vitamin C and/or vitamin E (11
,21
). In this article we report the results of a small clinical trial that had two aims. The first aim was to confirm the differences in plasma and urinary 8-iso-PGF2 concentrations between smokers and nonsmokers. The second aim was to investigate whether AGE taken as a dietary supplement had any antioxidant effect on plasma and urinary concentrations of 8-iso-PGF2 in both smoking and nonsmoking individuals. AGE was used in this study because it is widely available as a dietary supplement and, unlike other garlic preparations, is standardized to its major organosulfur constituent, S-allylcysteine.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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AGE (Kyolic), kindly provided by Wakunaga of America, is formulated by soaking sliced raw garlic (A. sativum) in 15–20% aqueous ethanol for up to 20 mo at room temperature. The extract is then filtered and concentrated under reduced pressure at low temperature. The content of water-soluble compounds is relatively high, whereas that of oil-soluble compounds is low. The AGE used in this trial contained 305 g/L extracted solids; S-allyl cysteine, the most abundant water-soluble organosulfur compound in AGE, was present at 1.47 g/L.
Subjects.
Apparently healthy subjects who were not taking any medication for any known disease participated in the study, which had the approval of the Ethics Committee of Liverpool John Moores University. They were divided into two groups, nonsmokers (n = 10; five men and five women; age, 41.6 ± 4.0 y) and smokers (n = 10; five men and five women; age, 41.0 ± 4.1 y). All subjects answered a simple questionnaire about their age, height, weight, average weekly alcohol consumption and number of cigarettes smoked per day. Body mass index (BMI) was determined as weight/height2 (kg/m2). On d 0 all subjects provided 10-mL samples of mid-stream urine and venous blood between 1000 and 1200 h. Subjects then consumed 5 mL of AGE (taken in a small volume of fruit juice) daily for 14 d between 0700 and 0900 h; otherwise, subjects followed their usual diet and lifestyle, including alcohol intake. This dose of AGE is that recommended as a dietary supplement by the manufacturers and was used in our previous study, which showed inhibition of ADP-induced platelet aggregation (3
). On d 14, blood and urine samples were collected in exactly the same way. All subjects then discontinued taking AGE and a final set of blood and urine samples were collected on d 28 of the trial (i.e., 14 d after cessation of AGE supplementation). All blood samples were collected into 100 U heparin and plasma was obtained by centrifugation at 2000 x g for 20 min. Plasma and urine was stored at -70°C as 0.5-mL aliquots. Plasma and urine samples specifically used for 8-iso-PGF2 analysis also included 10 mg/L meclofenamic acid to prevent any in vitro formation of 8-iso-PGF2.
Biochemical analyses.
Plasma and urinary creatinine were determined using diagnostic kits supplied by Sigma-Aldrich Company (Dorset, UK). The method was a colorimetric endpoint assay and relied upon creatinine reacting with alkaline picrate to form an orange/yellow-colored complex. Plasma creatinine concentrations were used to estimate creatinine clearance (mL/min), which is a measure of glomerular filtration rate. Plasma protein concentrations were determined by the Bradford assay using human serum albumin as the standard. Total plasma cholesterol concentrations were determined enzymatically using Infinity Cholesterol Reagent kits supplied by Sigma-Aldrich Company. Total plasma triglyceride concentrations were determined enzymatically using Peridochrom GPO-PAP kits supplied by Boehringer Manheim (East Sussex, UK).
Enzyme immunoassay of 8-iso-PGF2.
Plasma total (i.e., esterified plus free) and urine free 8-iso-PGF2 concentrations were assayed using competitive enzyme immunoassay kits purchased from Assay Designs Inc. (Ann Arbor, MI).
Essentially plasma samples were thawed and incubated at 42°C for 10 min to melt any lipid or salt crystals. To release 8-iso-PGF2 esterified to plasma lipids, four volumes of plasma were subjected to alkaline hydrolysis with one volume of 10 mol/L NaOH for 2 h at 45 oC. The hydrolysis mixture was allowed to cool and was neutralized by adding an equal volume of 2 mol/L HCl. The samples were then centrifuged at 500 x g for 5 min to remove any particulate matter. All analyses were carried out in duplicate as described in the kit manufacturers instructions; 25 µL of either sample or standards (range, 0–140 nmol/L) were incubated in 96-well plates coated with a goat antibody specific to rabbit IgG followed by addition of solutions of alkaline phosphatase conjugated 8-iso-PGF2 and a polyclonal rabbit antibody to 8-iso-PGF2. Wells were then washed thoroughly and incubated with a p-nitrophenyl phosphate substrate solution for 45 min at room temperature. A stop solution was added to all wells and the absorbance was read immediately at 405 nm using a microtiter plate reader. Results are expressed as nmol/L plasma.
Urine samples were thawed and incubated at 42°C for 10 min to dissolve any precipitated materials, centrifuged at 500 x g for 5 min to remove residual particulate matter and diluted 1:10 with Tris-buffered saline, pH 7.4. All samples and standards (range, 0–280 nmol/L) were assayed in duplicate as described previously. Because 24-h urine collections were not possible, urinary creatinine concentrations (mmol/L) were used to standardize urinary 8-iso-PGF2 output. Results are expressed as pmol/mmol creatinine.
Statistical analysis.
The significance of differences between the smoking or nonsmoking groups was assessed by Student’s unpaired t test, while differences before and after AGE supplementation were assessed by Student’s paired t test. Analyses were performed using the Minitab (State College, PA) statistical package and P values < 0.05 were considered significant. Values are means ± SEM.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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. All subjects were normolipidemic and had normal kidney function. The groups consumed similar amounts of alcohol and did not differ in age, weight and height. The only observed difference between the two groups was in smoking habits. Dietary supplementation for 14 d with AGE had no significant effect on plasma lipid profiles or plasma and urinary creatinine concentrations in either the smoking or nonsmoking groups (results not shown).
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concentrations 
58% greater than nonsmokers (Table 2
). After 14 d of supplementation with AGE, there was a 29% reduction in the plasma total concentration of 8-iso-PGF2 in nonsmokers and a 35% reduction in smokers (Table 2)
. Interestingly, the difference in the concentration of plasma total 8-iso-PGF2 between nonsmokers and smokers before the ingestion of AGE was not significant after 2 wk of supplementation (Table 2)
. However, after the 2-wk washout period, plasma concentrations of total 8-iso-PGF2 increased by 26% in nonsmokers (P = 0.14) and 34% in smokers (P = 0.07) resulting in plasma total 8-iso-PGF2 concentrations not different from those before ingestion of AGE (Table 2)
.
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(expressed as pmol/mmol of creatinine) in the urine of smokers was approximately twice that of nonsmokers (Table 2)
. The changes observed in the urinary concentrations of 8-iso-PGF2 after supplementation with AGE followed a similar pattern to those observed with plasma total 8-iso-PGF2 concentrations. Thus, after 14 d of supplementation with AGE, the urinary concentration of free 8-iso-PGF2 was decreased by 37% in nonsmokers and 48% in smokers (Table 2)
. The urinary concentration of 8-iso-PGF2 increased by 21% in nonsmokers (P = 0.23) and 60% in smokers (P < 0.05) after the 2-wk washout period. Indeed, no significant differences were observed in the concentration of urinary free 8-iso-PGF2 in either the smoking or nonsmoking group before supplementation with AGE and after the 2-wk washout period. |
DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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). Indeed, smoking increases oxidative stress and is a causative factor in coronary artery disease (23
,24
). Various in vitro studies have demonstrated that certain antioxidants can inhibit LDL oxidation (25
), and these studies have stimulated an impressive number of epidemiological studies that have investigated whether dietary supplementation with antioxidants is protective against atherosclerosis and coronary heart disease (26
–28
).
Most of the traditional methods used to assess oxidative stress have short comings. For example, the traditional method of assessing lipid peroxidation in vivo includes the measurement of thiobarbituric acid-reactive substances or lipid peroxides, both of which suffer from ex vivo artifactual generation, instability and nonspecificity of analytes (29
,30
). Another common method of monitoring lipid peroxidation and assessing the efficacy of antioxidants in vivo is to estimate the susceptibility of isolated LDL to Cu2+-induced oxidation in vitro. The obvious problem with this method is that it does not directly relate to oxidant stress in vivo. Recently, a novel family of prostaglandin F2 isomers called F2-isoprostanes has been reported. These are produced in vivo by a free radical peroxidation of arachidonic acid and can produce physiological or pathological effects due to their ability to alter smooth muscle and platelet functions (8
). Urinary excretion of free F2-isoprostanes also takes place and is positively correlated with age, indicating increased oxidative stress during the normal aging process. High plasma F2-isoprostane concentrations have been described in diseases such as ischemic heart disease, diabetes mellitus, Alzheimer’s disease and hepatic cirrhosis (8
). Indeed, the correlation of F2-isoprostane concentrations and the pathological severity of hepatic cirrhosis, cardiac failure and diabetes suggests that these compounds may be useful as predictive markers. The other advantage of measuring F2-isoprostanes is that they offer an intermediate endpoint for clinical studies of antioxidant therapies.
Thus, this study investigated plasma and urine concentrations of F2-isoprostanes in smokers (who have increased oxidative stress) and nonsmokers. Dietary intervention with AGE was then carried out to see whether its known antioxidant properties result in reduced plasma and urinary F2-isoprostane concentrations.
This study confirmed that smokers are subject to increased oxidative stress as evident by increases of 58% and 85% in the concentrations of plasma total and urinary free 8-iso-PGF2 compared with age- and sex-matched nonsmokers. These data are in agreement with other studies that report that the urinary excretion of 8-iso-PGF2 is increased by 42% in moderate smokers and 69% in heavy smokers (11
), while plasma free and esterified F2-isoprostanes are increased by 57% and 39%, respectively, in smokers (21
). Both studies also observed reductions in F2-isoprostane/8-iso-PGF2 concentrations after 2 wk of smoking cessation.
After 14 d of dietary supplementation with AGE, plasma and urine concentrations of 8-iso-PGF2 were significantly decreased by 29% and 37%, respectively, in nonsmokers. Perhaps more importantly, a greater reduction was seen in smokers such that their plasma concentration of 8-iso-PGF2 was reduced by 35%, while its urine concentration was reduced by 48%. The plasma and urine concentrations of 8-iso-PGF2 increased in both groups after the 2-wk washout period, such that values were no longer different from the initial baseline values. A similar 30% reduction in the urinary excretion of 8-iso-PGF2 has been observed in smokers after dietary supplementation with the antioxidant vitamin C (11
). Vitamin C or vitamin E supplementation has also been shown to reduce 8-iso-PGF2 concentrations in other syndromes associated with increased oxidative stress, such as diabetes, hypercholestrolemia and alcoholic liver disease (10
,12
–15
). In addition, the amount of 8-iso-PGF2 esterified to LDL has been reported to be 63% higher in hypercholesterolemic patients compared with age- and sex-matched controls (12
).
F2-isoprostanes have several biological and pathological effects. Thus, 8-iso-PGF2 has been shown to enhance platelet activation, promote vasoconstriction and smooth cell proliferation and has been shown to be present in increased amounts in human atherosclerotic vascular tissue compared with healthy tissue (17
,18
,20
,31
,32
). These pro-atherogenic properties of 8-iso-PGF2 together with the finding that elevated plasma concentrations of 8-iso-PGF2 are associated with traditional risk factors confirm its association with the development of cardiovascular disease. In addition, because 8-iso-PGF2 is pro-aggregatory to platelets, the decrease in circulating concentrations after supplementation with AGE may explain, at least in part, the inhibition of platelet aggregation in a previous trial involving dietary supplementation with AGE (3
). Thus, the data presented in this article suggest that dietary supplementation with AGE may protect against diseases, such as atherosclerosis, which are associated with increased oxidative stress.
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FOOTNOTES |
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3 Abbreviations used: AGE, aged garlic extract; BMI, body mass index; PG, prostaglandin.
Manuscript received 10 September 2001. Revision accepted 16 November 2001.
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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