Dietary supplementation with 11trans- and 12trans-18:1 and oxidative stress in humans

¹ From the Institute of Nutrition, Friedrich Schiller University, Jena, Germany (KK, JK, and GJ), and the Division of Clinical Nutrition and Metabolism, Department of Public Health and Caring Sciences, Faculty of Medicine, Uppsala University, Uppsala, Sweden (SB)

² Supported by grant no. JA 893 from the Deutsche Forschungsgemeinschaft.

³ Reprints not available. Address correspondence to G Jahreis, Institute of Nutrition, Friedrich Schiller University, Dornburger Strasse 24, D-07743 Jena, Germany. E-mail: b6jage{at}uni-jena.de.

ABSTRACT  
Background: High consumption of trans fat has been associated with high oxidative stress in humans, which could increase the risk of the development or acceleration of several diseases, such as atherosclerosis, cancer, and type 2 diabetes.

Objective: Several urinary and blood biomarkers of oxidative stress [8-iso-prostaglandin-F₂ (PGF₂), 15-keto-dihydro-PGF₂, and 7,8-dihydro-8-oxo-2'-deoxy-guanosine in urine and -,ß-,-,-tocopherol, and retinol in plasma] were monitored to evaluate the oxidative stress induced by dietary supplementation of 11trans- and 12trans-18:1 isomers in humans during a 6-wk intervention.

Design: After a 14-d adaptation period free of trans fatty acid supplementation (baseline), the test group (n = 12) received 3.0 g 11trans-18:1/d and 3.0 g 12trans-18:1/d ( 6.0 g/d), and the control group (n = 12) consumed a control oil free of trans fatty acids and conjugated linoleic acids for 6 wk.

Results: The postintervention concentration of urinary 8-iso-PGF₂ (free radical–induced lipid peroxidation) in the test group was significantly higher than baseline and significantly higher than that observed in the control group. The concentrations of 15-keto-dihydro-PGF₂ (cyclooxygenase-mediated inflammatory response indicator) and 7,8-dihydro-8-oxo-2'-deoxy-guanosine (oxidative DNA damage) were not affected by the 11trans- and 12trans-18:1 supplementation.

Conclusions: Although an increase in urinary 8-iso-PGF₂ was observed and the effects of prolonged high (ie, >5.0 g/d) consumption of trans fat could be relevant to the development of disease, the mean intakes of 11trans- and 12trans-18:1 in Europeans are estimated to be significantly below the amounts administered in this study (ie, 6.0 g/d); such low intakes could minimize the possible risk of detrimental effects on human health.

Key Words: Oxidative stress • trans fatty acids • conjugated linoleic acids • isoprostanes • prostaglandins • 7,8-dihydro-8-oxo-2'-deoxyguanosine

INTRODUCTION  
Oxidative stress is a term commonly used to describe the steady state level of oxidative damage in a cell, tissue, or organ that is caused by the reactive oxygen species, such as free radicals and peroxides, within a biological organism. Oxidative stress is the result of an imbalance between prooxidant and antioxidant processes within that organism in favor of the former. In fact, a greater consumption of trans fat has been associated with higher levels of oxidative stress in humans (1-5), and prolonged exposure to high levels of oxidative stress has been implicated in the development or acceleration of several dysfunctions and diseases, such as cardiovascular disease (6-11), inflammation (12, 13), type 2 diabetes (14), and breast, colon, and prostate cancers (15, 16).

The trans fats are a class of unsaturated fatty acids that possess at least one double bond in trans configuration. Generally, these trans fatty acids occur naturally in ruminant fats formed by the enzymatic hydrogenation of several polyunsaturated fatty acids (eg, linoleic acid) in the rumen. They are also formed during industrial processes such as the hydrogenation of vegetable oils. Although ruminant fats and partially hydrogenated vegetable oils (PHVOs) contain the same trans fatty acid isomers, their isomeric profiles are clearly different; that is, ruminant fats have the 11trans-18:1 (11t-18:1; vaccenic acid), which is 60–80% of total 18:1 (17), and PHVOs mainly have the 9t-18:1 (elaidic acid) (18). During hydrogenation by rumen bacteria, numerous geometric (t,t, c,t, t,c, and, c,c) and positional (2,4 to 14,16) isomers of conjugated linoleic acids (CLAs) are created, but 9cis,11trans-18:2 (9c,11t-CLA) is the predominant isomer formed (19). In addition, the major source of 9c,11t-CLA in milk fat is the created via endogenous synthesis of t11-18:1 by 9-desaturase (20). The endogenous synthesis of 9c,11t-CLA has also been found in humans (21, 22). The intakes of trans fatty acids and their effects on human health are still under review (23). Estimates for dietary intake of trans fatty acids differ from country to country. In European countries, the intake of trans fatty acids varies from 2.0 to 2.7 g/d (60% from ruminant fats and 40% from PHVOs; 24), whereas the intakes of 11t-18:1 range from 0.7 to 1.0 g/d (25) and those of 9c,11t-CLA range from 0.3 to 0.5 g/d (26). For comparison, in US and Canadian populations, the mean trans fatty acid intake was estimated to be higher, ie, 5.8 g/d (27, 28), and, in Canadian women, it was exceedingly high, ie, 10.6 g/d (29). In the US and Canadian populations, 80% of total trans fatty acids is currently derived from PHVOs (28). However, to date, very few human intervention studies have been carried out to evaluate the specific effects of the individual trans isomers of 18:1.

At present, the measurement of F₂-isoprostanes is regarded as the gold standard by which to evaluate the level of oxidative stress in vivo; 8-iso-prostaglandin F₂ (8-iso-PGF₂) is the major F₂-isoprostane successfully evaluated in many experimental and clinical studies (30). It is derived from arachidonic acid by using nonenzymatic free radical–induced peroxidation (30-32). The 15-keto-13,14-dihydro-PGF₂ (15-ketodihydro-PGF₂) can be used as an indicator for lipid peroxidation through the cyclooxygenase pathway (33). The 7,8-dihydro-8-oxo-2'-deoxy-guanosine (8-oxodG), a sensitive biomarker monitoring in vivo DNA damage, is eliminated via DNA repair mechanisms and excreted in urine (1, 34). Tocopherols and retinol are measured to assess the status of individual antioxidants (35).

Several studies have shown the association of the effects of the consumption of trans fat with higher levels of oxidative stress. In a study with mice fed a trans diet (13.6% of energy as fat), an increased plasma concentration of F₂-isoprostanes was found (36). Increases in the concentrations of urinary 8-iso-PGF₂ were observed in several studies on the effects of supplementation with 11t-18:1, linoleic acid, or CLAs in humans (5, 21, 37-39). In another study, the oxidative DNA damage in women was higher after supplementation with a diet rich in linoleic acid (40). To evaluate oxidative stress during the dietary intake of the 2 trans 18:1 isomers (11t- and 12t-18:1; a total of 6 g/d) for a 6-wk intervention period, several biomarkers of oxidative stress were measured—8-iso-PGF₂, 15-ketodihydro-PGF₂, and 8-oxodG in urine and -, ß-, -, -tocopherol, and retinol in plasma.

SUBJECTS AND METHODS  
Subjects
Twenty-four healthy subjects (12 women and 12 men) were recruited. The volunteers were informed of the purpose, course, and possible risks of the study. Subjects had no diagnosed diseases, were not taking any medications (eg, aspirin), were not abusers of alcohol, and were not taking any dietary supplements. The volunteers had a mean ± SD age of 24 ± 3 y (range: 20–28 y) and a normal weight with a mean body mass index (in kg/m²) of 21 ± 2 (range: 19–26). The treatment groups did not differ significantly in anthropometric data (22). Each study group consisted of 6 men and 6 women.

All subjects gave informed written consent. The study was approved by the ethics committee of the Friedrich Schiller University (Jena, Germany).

Diets
Throughout the entire study (8 wk), the diet consumed contained only traces of trans fatty acids and CLA, because the food supplied in this study was so chosen to minimize the extraneous amounts of these fatty acids. All volunteers were subjected to a 14-d adaptation period without supplementation to establish baseline conditions for the trans fatty acids and CLA concentrations. During the 42-d intervention period, the diet of the test group (n = 12) contained no CLA and was supplemented with 3.0 g 11t-18:1/d and 3.0 g 12t-18:1/d (a total of 6 g/d; trans-isomer mixture; Natural ASA, Hovdebygda, Norway; 22). The diet of the control group (n = 12) was supplemented with a control oil (palm kernel oil and rapeseed oil, 1:1). The control oil was free of trans fatty acids and CLA.

During the adaptation period, all volunteers consumed 20 g of a pure (ie, without supplements), commercially available chocolate spread (Nutella; Ferrero, Frankfurt am Main, Germany) to make the diets isocaloric. For the intervention period, control oil and the trans-isomer mixture were added to the chocolate spread to achieve good acceptability. During the intervention period, each subject consumed daily a total of 20 g of the corresponding combined supplement and chocolate spread mixture.

In the last 7 d of each study period, the volunteers were given a standardized diet (Table 1). During this time, the food residues were returned and weighed each day to allow for more accurate measurements of food consumption. Duplicate portions of the dietary supplies were collected, freeze-dried, homogenized, and sampled for the nutritional analysis of the study diet. The chemical analyses of the study diet and volunteer's blood samples were conducted by using previously described procedures (22).

View this table:
TABLE 1. Nutritional evaluation of daily dietary intakes in the control and test groups during the adaptation and intervention periods of the dietary supplementation study¹

 
Blood and urine sampling
Blood samples were collected on the last day of the standardized diet during the adaptation period (0 d–baseline) and the 42-d intervention period. Blood samples were drawn between 0730 and 0830 by venipuncture into evacuated tubes (BD Vacutainer Systems, Heidelberg, Germany) containing EDTA as an anticoagulant for plasma preparation after overnight fasting.

Urine samples were collected during the last 5 d of the standardized diet of each period. The morning urine on the first collection day was not kept, but urinations during the rest of the day up to the morning urine on the next day (ie, 24-h urine collection) were collected in a special 24-h urine-collection tank (Sarstedt, Nümbrecht, Germany). One percent (by vol) of each 24-h urine was taken with urine-monovettes (Sarstedt) and stored in a frozen state at –80 °C until analysis. In a previous study, Helmerrson and Basu (41) found no significant difference between 8-iso-PGF₂ concentrations in urine taken from 24-h samples from different days. However, to minimize the variation in each subject, in the current study, 1% (by vol) of each 24 h-urine sample from 5 consecutive collection days of each subject was pooled before analysis.

Biomarkers of lipid peroxidation (nonenzymatic and enzymatic)
The concentrations of free 8-iso-PGF₂ in urine samples were analyzed without extraction by using a highly specific and sensitive radioimmunoassay as previously described (42). Urinary samples were analyzed for 15-ketodihydro-PGF₂ without any extraction by using a radioimmunoassay as described previously (33).

The urinary concentrations of 15-ketodihydro-PGF₂ and 8-iso-PGF₂ were adjusted by creatinine values to correct for variations in the glomerular filtration rate. Urinary creatinine concentrations were determined by using a commercial kit (IL Test; Monarch Instruments, Amherst, MA).

Measurement of urinary 7,8-dihydro-8-oxo-2'-deoxyguanosine
Urine samples were acidified by using acetic acid (pH 6.5). After centrifugation (4000 U/min; 1800 x g at 21 °C for 15 min), urine samples were purified by using solid-phase extraction with C18 EC columns (Macherey-Nagel, Dueren, Germany). The analysis of 8-oxodG was conducted with the use of HPLC (column: Hypersil C18 ODS II, 5 µm, 250 x 4 mm; Agilent, Waldbronn, Germany) by using a gradient elution of sodium citrate (pH 5) as solvent A and acetonitrile as solvent B at a flow rate of 1 mL/min. The detailed gradient profile was as follows: isocratic elution with 2% solvent B for 10 min, a gradient of 10% solvent B for 5 min, further elution with 10% solvent B for 3 min, and reequilibration with 2% solvent B for 5 min. The detection of 8-oxodG was followed by electrochemical detection (0.550 V) and diode-array detection at different wave lengths (254, 260, and 280 nm). Standards (8-bromoguanosin, isocytosin, and 8-oxodG) were purchased from Sigma-Aldrich (Munich, Germany).

Measurement of plasma tocopherols and retinol
Retinol and tocopherols behave as antioxidants in lipid peroxidation in biological systems (43). Plasma concentrations of -, ß-, -, and -tocopherol and retinol were analyzed by using HPLC. Plasma (250 µL) was extracted with 1 mL n-hexane containing 0.045% of 2.6-Di-tert-butyl-p-kresol and 250 µL ethanol. After centrifugation (2500 U/min, at 700 x g at 21 °C for 5 min), a 20-µL volume of the supernatant solution was used for injection. Tocopherols and retinol were separated by using a Shimadzu 10A series HPLC with a 250 x 4-mm, 5-µm Nucleosil–100 NH₂ column (Macherey-Nagel) by isocratic elution with a ratio of n-hexane to 2-propanol (96:4 by vol) at a flow rate of 0.8 mL/min. The tocopherols and retinol were detected by using an RF 10AXL fluorescence detector (excitation wave length 295 nm, emission wave length 335 nm) and diode-array detector (325 nm), respectively. The tocopherol (, ß, , ) and all-trans-retinol standards were purchased from Calbiochem (Merck Biosciences, Nottingham, United Kingdom).

Measurement of tocopherols and retinol in supplements and food
Retinol and - and - tocopherol were measured in food and supplements (trans-isomer mixture and control oil). The ß- and -tocopherol concentrations were below the limit of quantification (0.0001 ng/µL). Lyophilized and homogenized food samples from duplicate portions of the standardized diet (1 g) were mixed with 1 g ascorbic acid, saponified by using a ratio of potassium hydroxide to distilled water to ethanol (12:20:100 wt by vol), heated for 40 min at 80 °C, extracted with the use of n-hexane in the presence of 0.045% 2.6-Di-tert-butyl-p-kresol, and washed with 2 mL distilled water. The analyses were based on conditions similar to those described above. The tocopherol and retinol content in food was calculated by using the consumed amount of each food item (wet wt) during the standardized diet to ascertain the total intakes of tocopherols and retinol.

Statistical analysis
All statistical analyses were performed by using SAS software (version 9.1; SAS Institute Inc, Cary, NC). P 0.05 was regarded as significant. The data values are stated as means ± SDs. The Kolmogorov-Smirnov test was used to test the distribution of the data, and all measures were normally distributed. The 2-factor analysis of variance was used to compare the data of the 2 treatments. The covariate value was the measurement from day 0 (baseline) of the study. The treatment x sex interaction was not significant. Correlations were calculated by using the Pearson correlation analysis. Correlation factors were compared by using Fisher's z-transformation (z test).

RESULTS  
All subjects successfully completed the study, and all measured variables were within their normal physiologic range.

Dietary intake
Male subjects tended to have higher daily intakes of food than did the female subjects (baseline: total men 11.0 ± 1.2 MJ/d; total women 8.9 ± 1.7 MJ/d). Nevertheless, the distribution (by % of energy) of carbohydrates, protein, and total fat did not differ between the treatment groups (Table 1). The intakes of monounsaturated fatty acids (trans 18:1 was not included) and polyunsaturated fatty acids in the test group were significantly lower than those in the control group, which corresponded to the increase in trans fatty acids (11t- and 12t-18:1) in the test group diet (Table 1).

The intakes of tocopherol equivalents and retinol did not differ significantly between the treatment groups (Table 1). The -tocopherol intake during the intervention period was greater in the control group than in the test group. The portions of retinol, -, and -tocopherol in the daily consumed dose of chocolate spread during the adaptation period and the portions received with control oil or the trans-isomer mixture during the intervention period did not differ significantly (Table 1).

Plasma tocopherol and retinol concentrations
The mean concentration of plasma -tocopherol was 0.001 µmol/L (data not shown). The - and -tocopherol concentrations in plasma remained unchanged after the intervention. The ß-tocopherol concentrations in the control group were significantly higher than those in the test group (Table 2). Unfortunately, the ß-tocopherol concentration of the diet was not measured. The retinol concentration in the control group was significantly lower than that in the test group, although the groups' retinol intakes did not differ significantly (Tables 1 and 2).

View this table:
TABLE 2. Blood and urinary biomarker concentrations in the control and test groups during the adaptation and intervention periods of dietary supplementation study¹

 
9cis,11trans–conjugated linoleic acid of serum and red blood cell (RBC) membranes
The combined serum and RBC membrane 9c,11t-CLA concentrations in the test group during the intervention period were significantly higher than those in the test group during baseline or in the control group during the intervention period. In contrast, the combined serum and RBC membrane 9c,11t-CLA concentrations in the control group decreased from the adaptation to the intervention (Table 2). The 9c,11t-CLA content of the RBC membranes in the intervention period differed between male and female subjects (control group: 0.06 ± 0.02 in males, 0.09 ± 0.02 in females; P = 0.022; test group: 0.16 ± 0.03 in males, 0.21 ± 0.05 in females; P = 0.084).

Urinary concentrations
The urinary 8-iso-PGF₂ excretion of the test group was significantly greater than the baseline concentration. In contrast, the control group's 8-iso-PGF₂ excretion remained constant throughout the study. The test groups 8-iso-PGF₂ concentrations after the intervention were significantly higher than those of the control group (Table 2).

The concentrations of urinary 15-ketodihydro-PGF₂ detected in both treatment groups' samples were constant throughout the study. No significant differences between the treatment groups or sexes were observed (Table 2).

The comparison between the control and test groups' baseline urinary 8-oxodG concentrations found no significant differences. Overall, the intervention period urinary concentration of 8-oxodG remained constant for both treatment groups throughout the study (Table 2).

Correlation analysis
During the adaptation period, urinary 8-iso-PGF₂ concentrations correlated significantly with urinary 15-ketodihydro-PGF₂ concentrations in the control and test groups (Table 3). It is interesting that the comparison of postintervention and baseline urinary 8-iso-PGF₂ concentrations found a significantly lower correlation coefficient (r = 0.011) in the test group, whereas the correlation coefficient of the control group was unchanged. The urinary 8-oxodG concentration did not correlate with the urinary 8-iso-PGF₂ or 15-ketodihydro-PGF₂ concentration (Table 3).

View this table:
TABLE 3. Correlation coefficients of the urinary biomarker concentrations of control and test groups during a dietary supplementation study¹

 
In both groups and both study periods, no correlations were found between urinary 8-iso-PGF₂ and 15-ketodihydro-PGF₂, respectively, and plasma tocopherols and retinol, respectively (data not shown). Moreover, no correlation between the intakes of tocopherols and retinol, their plasma concentrations, and urinary 8-iso-PGF₂ and 15-ketodihydro-PGF₂ concentration was observed.

DISCUSSION  
Healthy rats fed a diet containing 0.5% and 1% 11t- and 12t-18:1 at a ratio of 1:1 over 9 d had increased urinary concentrations of 8-iso-PGF_{2 In the current study, the test group's urinary 8-iso-PGF2 concentration after supplementation with 11t- and 12t-18:1 over 42 d was significantly higher then its baseline concentration. This group's postintervention urinary 8-iso-PGF2 concentration also was significantly higher than that of the control group (Table 2). A more distinctive increase in the urinary 8-iso-PGF2 concentrations was detected in the female subjects than in the male subjects.}

In contrast to the current study, Tholstrup et al (45) reported no significant difference in urinary 8-iso-PGF₂ concentrations between subjects who consumed a diet containing a naturally 11t-18:1–enriched butter (3.6 g 11t-18:1/d) supplement and those subjects who consumed a diet low in 11t-18:1. Unfortunately, the 12t-18:1 content of the butter supplement used in that study was not stated.

Although the trans fatty acid intake is associated with inflammatory processes (12) in both the study by Turpeinen et al (21) and the current study, the urinary excretion of 15-ketodihydro-PGF₂, which reflects the proinflammatory response (33), was unaffected by supplementation with the trans fatty acids. In addition, the biomarkers 8-iso-PGF₂ and 15-ketodihydro-PGF₂ correlated with each other after the adaptation period in both treatment groups. After the intervention with the trans isomers, the coefficient of correlation between 8-iso-PGF₂ and 15-ketodihydro-PGF₂ had decreased significantly in the test group, whereas the correlation coefficient of these biomarkers in the control group was unchanged (Table 3). This trend would suggest that the supplemented trans isomers increased the radical-induced lipid peroxidation without influencing the cyclooxygenase-dependent inflammatory response.

Obviously, in studies with 11t-18:1 supplementation (21, 44) and in the current study, the portion of 9c,11t-CLA in serum and RBC membranes was greater (Table 2). Thus, the endogenous conversion of 11t-18:1 to 9c,11t-CLA in the body was proven. However, there was no significant correlation between the greater urinary 8-iso-PGF₂ concentration and the greater amount of 9c,11t-CLA in serum and RBC membranes (r = –0.245, P = 0.468 and r = –0.085, P = 0.804, respectively) in any of the studies. Moreover, in the above described studies and in the current study, 11t- and 12t-18:1 were supplemented together because no highly pure 11t 18:1 preparations (ie, those with only a single isomer) in adequate amounts (5 kg) were commercially available.

At present, it is possible that the two trans isomers, 11t- and 12t-18:1, in combination or alone can induce an increase in the 8-iso-PGF₂ response, the biomarker of nonenzymatic lipid peroxidation type of oxidative stress. In addition, it is possible that 9c,11t-CLA, endogenously synthesized from 9-desaturation of 11t-18:1, was responsible for the observed increase in this biomarker. The induction of lipid peroxidation during CLA supplementation is supported by previous studies in humans (37-39). However, in those studies, the increases in 8-iso-PGF₂ (eg, from 0.5 to 1.7 nmol/mmol Cr) and in 15-ketodihydro-PGF₂ (eg, from 0.7 to 1.3 nmol/mmol Cr) were more distinct. The comparison of the 9c,11t-CLA dose supplied via endogenous synthesis in this study (20–25% of dietary 11t-18:1; 22) with the doses in CLA supplementation studies previously mentioned (isomer mixture containing 10t,12c-CLA) showed a significantly higher supplementation dose in the CLA supplementation studies (2.2–4.2 g/d). Moreover, the prooxidative effect of 10t,12c CLA is more pronounced than that of 9c,11t CLA (46-48). Apparently, the higher the 10t,12c portion of the CLA mixture, the more pronounced the lipid peroxidation.

A meta-analysis of 60 controlled trials in humans found that the ratio of total to HDL cholesterol was higher during consumption of trans fatty acids than during consumption of cis-unsaturated fatty acids (49). However, in the current study, after consumption of the diet enriched with 6 g 11t and 12t 18:1 over 42 d, the atherogenic risk ratio compared with that in the control group was unchanged (total:HDL cholesterol, 3.35 ± 0.66 in the test group and 3.21 ± 0.53 in the control group; P = 0.863).

In general, concentrations of 8-iso-PGF₂ are elevated in conditions thought to be associated with free radical–induced oxidative injury in humans, such as smoking, hypercholesterolemia, diabetes mellitus, overweight, and obesity (3, 32). At present, the clinical relevance of higher 8-iso-PGF₂ concentrations in urine and in plasma, in particular after CLA and 11t,12t-18:1 supplementation in healthy subjects, is unclear. Kumar et al (50) proposed that greater lipid peroxidation could stimulate endogenous defense systems and indicated a potential antiinflammatory effect of 8-iso-PGF₂ in the microvasculature. In general, the role of CLA in oxidative stress is controversial (51). Some authors state that CLA has prooxidative properties, which are responsible for the CLA-induced anti-cancer activity (52, 53). In contrast, CLA reduced lipid peroxidation in animal studies and may have antioxidative properties associated with scavenging radicals (43, 54).

Dietary fats can induce oxidative DNA damage in different matrixes. A diet high in fat increased the urinary excretion of 8-oxodG in rats (55), whereas the extent of unsaturation was related to the 8-oxodG concentration in mammary gland DNA in the same animals (56). In contrast, de Kok et al (57) observed no significant increase, after supplementation with linoleic acid (7.5 or 15 g/d over 6 wk), in 8-oxoG in DNA from human peripheral lymphocytes. Under physiologic conditions in humans, the urinary 8-oxodG concentration ranges from 0.5 to 1.7 nmol/mmol Cr (58; A Wagner, unpublished observations, 2004), whereas, in cancer patients, smokers, and obese subjects, increased urinary 8-oxodG has been found (1, 59).

The effect of the supplementation with 11t- and 12t-18:1 on urinary 8-oxodG concentrations has not been previously reported. In the current study, the values of urinary 8-oxodG were within the physiologic range and showed no differences between the sexes or the treatment groups (Table 2). Park and Floyd (4) postulated that lipid peroxidation products mediate the formation of 8-oxodG. In the current study, the greater 8-iso-PGF₂ excretion of the test group was not associated with elevated 8-oxodG excretion (Table 3). This lack of association indicates no oxidative effect on DNA during the intervention with 6 g 11t/12t 18:1.

In addition, modifications in dietary antioxidants—in particular, -tocopherol and retinol—can induce changes in the levels of biomarkers of oxidative stress (35, 57, 60). Supplementation with high doses of -tocopherol in rats decreased the basal urine concentration of both 8-iso-PGF₂ and 15-ketodihydro-PGF₂ (61). In contrast, the CLA-induced increase in urinary 8-iso-PGF₂ with additional supplementation could not reduce moderate concentrations of -tocopherol in humans (47). Mice fed a trans diet developed plasma tocopherol depletion accompanied by a higher concentration of plasma F₂-isoprostanes (36). In the current study, despite an increase in 8-iso-PGF₂ excretion, no change was found in the plasma concentrations of -tocopherol (Table 2). The plasma concentrations of retinol were significantly higher in the test group than in the control group, but, in the case of plasma ß-tocopherol, the reverse was true. In addition, the comparison of the total tocopherol equivalents and retinol between the treatment groups found no significant differences throughout the study.

After supplementation with 11t- and 12t-18:1, the biomarker of the free radical–induced lipid peroxidation increased from the baseline concentrations (diet without these trans isomers). However, no major effects could be observed on cyclooxygenase-induced lipid peroxidation, DNA damage, or antioxidant status.

In conclusion, with respect to the high intake of trans fatty acids in the US and Canadian populations (>5.0 g/d, especially by PHVOs), our findings indicate that trans fatty acids could be relevant to the development of the previously mentioned diseases. Although an increase in urinary 8-iso-PGF₂ was observed in this study, and the effects of prolongd high consumption of trans fat (>5.0 g/d) could be relevant to the development of disease, the intakes of 11t- and 12t-18:1 in Europeans tend to be estimated as significantly below those in this study (6.0 g/d), which could minimize the possible risk of detrimental effects on human health. Further research is merited to investigate and more clearly define the effects of individual trans fatty acid isomers on oxidative stress and their relation to disease.

ACKNOWLEDGMENTS  
KK and JK were responsible for the conception and design of the study; KK was responsible for the conduct of the study; KK and AW were responsible for data acquisition; KK was responsible for data analysis; KK, AQ, SB, and GJ were responsible for data interpretation; KK was responsible for the statistical analysis; KK was responsible for drafting the manuscript; KK, AQ, JK, SB, and GJ were responsible for critical revision of the manuscript; and JK was responsible for obtaining funding. None of the authors had any personal or financial conflict of interest.

REFERENCES  

1. Kasai H, Iwamoto-Tanaka N, Miyamoto T, et al. Life style and urinary 8-hydroxydeoxy-guanosine, a marker of oxidative DNA damage: effects of exercise, working conditions, meat intakes, body mass index, and smoking. Jpn J Cancer Res2001; 92 :9 –15.
2. Leinonen J, Lehtimaki T, Toyokuni S, et al. New biomarker evidence of oxidative DNA damage in patients with non-insulin dependent diabetes mellitus. FEBS Lett1997; 417 :150 –2.
3. Morrow JD. Quantification of isoprostanes as indices of oxidant stress and the risk of atherosclerosis in humans. Arterioscler Thromb Vasc Biol2005; 25 :279 –86.
4. Park JW, Floyd RA. Lipid peroxidation products mediate the formation of 8-hydroxydeoxy-guanosine in DNA. Free Radic Biol Med1992; 12 :245 –50.
5. Turpeinen AM, Basu S, Mutanen M. A high linoleic acid diet increases oxidative stress in vivo and affects nitric oxide metabolism in humans. Prostaglandins Leukot Essent Fatty Acids1999; 59 :229 –33.
6. Troisi R, Willet WC, Weiss ST. Trans fatty acid intake in relation to serum lipid concentrations in adult men. Am J Clin Nutr1992; 56 :1019 –24.
7. Zock PL, Katan MB. Hydrogenation alternatives: effects of trans fatty acids and stearic acid versus linoleic acid on serum lipids and lipoproteins in humans. J Lipid Res1992; 33 :399 –410.
8. Willett WC, Stampfer MJ, Manson JE, et al. Intake of trans fatty acids and risk of coronary heart diseases among women. Lancet1993; 341 :581 –5.
9. Katan MB, Zock PL, Mensink RP. Trans fatty acids and their effects on lipoproteins in humans. Annu Rev Nutr1995; 15 :473 –93.
10. Hu FB, Manson JE, Willett WC. Types of dietary fat and risk of coronary heart disease: a critical review. J Am Coll Nutr2001; 20 :5 –19.
11. Oomen CM, Ocke MC, Feskens EJ, van Erp-Baart MA, Kok FJ, Kromhout D. Association between trans fatty acid intake and 10-year risk of coronary heart disease in the Zutphen Elderly Study: a prospective population based study. Lancet2001; 357 :746 –51.
12. Mozaffarian D, Pischon T, Hankinson SE, et al. Dietary intake of trans fatty acids and systemic inflammation in women. Am J Clin Nutr2004; 79 :606 –12.
13. Lopez-Garcia E, Schulze MB, Meigs JB, et al. Consumption of trans fatty acids is related to plasma biomarkers of inflammation and endothelial dysfunction. J Nutr2005; 135 :562 –6.
14. Salmeron J, Hu FB, Manson JE, et al. Dietary fat intake and risk of type 2 diabetes in women. Am J Clin Nutr2001; 73 :1019 –26.
15. Rissanen H, Knekt P, Jarvinen R, Salminen I, Hakulinen T. Serum fatty acids and breast cancer incidence. Nutr Cancer2003; 45 :168 –75.
16. King IB, Kristal AR, Schaffer S, Thornquist M, Goodman GE. Serum trans-fatty acids are associated with risk of prostate cancer in beta-carotene and retinol efficacy trial. Cancer Epidemiol Biomarkers Prev2005; 14 :988 –92.
17. Kraft J, Collomb M, Moeckel P, Sieber R, Jahreis G. Differences in CLA isomer distribution of cow's milk lipids. Lipids2003; 38 :657 –64.
18. Aro A, Kosmeijer-Schuil T, van de Bovenkamp P, Hulshof P, Zock P, Katan MB. Analysis of C18:1 cis and trans fatty acid isomers by the combination of gas-liquid chromatography of 4,4-dimethyloxazoline derivatives and methyl esters. J Am Oil Chem Soc1998; 75 :977 –85.
19. Delmonte P, Roach JAG, Mossoba MM, Losi G, Yurawecz MP. Synthesis, isolation, and GC analysis of all the 6,8- to 13,15-cis/trans conjugated linoleic acid isomers. Lipids2004; 39 :185 –191.
20. Piperova LS, Sampugna J, Teter BB, Kalscheur KF, Yurawecz MP, Ku Y, Morehouse KM, Erdman RA. Doudenal and milk trans octadecenoic acid and conjugated linoleic acid (CLA) isomers indicate that postabsorptive synthesis is the predominant source of cis-9-containing CLA in lactating dairy cows. J Nutr2002; 132 :1235 –41.
21. Turpeinen AM, Mutanen MAA, Salminen I, Basu S, Palmquist DL, Griinari JM. Bioconversion of vaccenic acid to conjugated linoleic acid in humans. Am J Clin Nutr2002; 76 :504 –10.
22. Kuhnt K, Kraft J, Moeckel P, Jahreis G. Trans-11–18:1 is effectively 9-desaturated compared with trans-12–18:1 in humans. Br J Nutr2006; 95 :752 –61.
23. Weggemans RM, Rudrum M, Trautwein EA. Intake of ruminant versus industrial trans fatty acids and risk of coronary heart disease—what is the evidence? Eur J Lipd Sci Technol2004; 106 :390 –7.
24. van de Vijver LP, Kardinaal AF, Couet C, et al. Association between trans fatty acid intake and cardiovascular risk factors in Europe: the TRANSFAIR study. Eur J Clin Nutr2000; 54 :126 –35.
25. Voorrips LE, Brants HAM, Kardinaal AFM, Hiddink GJ, van den Brandt PA, Goldbohm RA. Intake of conjugated linoleic acid, fat, and other fatty acids in relation to postmenopausal breast cancer: the Netherlands Cohort Study on Diet and Cancer. Am J Clin Nutr2002; 76 :873 –82.
26. Jahreis G, Kraft J. Sources of conjugated linoleic acid in the human diet. Lipid Tech2002; 14 :29 –32.
27. Ascherio A, Katan MB, Zock PL, Stampfer MJ, Willett WC. Trans fatty acids and coronary heart disease. N Engl J Med1999; 340 :1994 –8.
28. Food and Drug Administration. Food labeling: trans fatty acids in nutrition labeling, nutrient content claims, and health claims. Fed Regist2003; 68 :41434 –506.
29. Chen ZY, Pelletier G, Hollywood R, Ratnayake WMN. Trans fatty acid isomers in Canadian human milk. Lipids1995; 30 :15 –21.
30. Basu S. Isoprostanes: novel bioactive compounds of lipid peroxidation. Free Radic Res2004; 38 :105 –22.
31. Montuschi P, Barnes PJ, Roberts LJ. Isoprostanes: markers and mediators of oxidative stress. FASEB J2004; 84 :1791 –800.
32. Basu S, Helmersson J. Factors regulating isoprostane formation in vivo. Antioxid Redox Signal2005; 7 :221 –35.
33. Basu S. Radioimmunoassay of 15-keto-13,14-dihydro-prostaglandin F₂: an index for inflammation via cyclooxygenase catalysed lipid peroxidation. Prostaglandins Leukot Essent Fatty Acids1998; 58 :347 –52.
34. Shigenaga MK, Ames BN. Assays for 8-hydroxy-2-deoxyguanosine: a biomarker of in vivo oxidative DNA damage. Free Radic Biol Med1991; 10 :211 –6.
35. Urso ML, Clarkson PM. Oxidative stress, exercise, and antioxidant supplementation. Toxicology2003; 189 :41 –54.
36. Cassagno N, Palos-Pinto A, Costet P, Breilh D, Darmon M, Berard AM. Low amounts of trans 18:1 fatty acids elevate plasma triacylglycerols but not cholesterol and alter the cellular defence to oxidative stress in mice. Br J Nutr2005; 94 :346 –52.
37. Basu S, Risérus U, Turpeinen A, Vessby B. Conjugated linoleic acid induces lipid peroxidation in men with abdominal obesity. Clin Sci2000; 99 :511 –6.
38. Basu S, Smedman A, Vessby B. Conjugated linoleic acid induces lipid peroxidation in humans. FEBS Lett2000; 468 :33 –6.
39. Risérus U, Vessby B, Arnlov J, Basu S. Effects of cis-9,trans-11 conjugated linoleic acid supplementation on insulin sensitivity, lipid peroxidation, and proinflammatory markers in obese men. Am J Clin Nutr2004; 80 :279 –83.
40. Nair J, Vaca CE, Velic I, Mutanen M, Valsta LM, Bartsch H. High dietary omega-6 polyunsaturated fatty acids drastically increase the formation of etheno-DNA base adducts in white blood cells of female subjects. Cancer Epidemiol Biomarkers Prev1997; 6 :597 –601.
41. Helmersson J, Basu S. F₂-isoprostane excretion rate and diurnal variation in human urine. Prostaglandins Leukot Essent Fatty Acids1999; 61 :203 –5.
42. Basu S. Radioimmunoassay of 8-iso-prostaglandin F₂: an index for oxidative injury via free radical catalysed lipid peroxidation. Prostaglandins Leukot Essent Fatty Acids1998; 58 :319 –25.
43. Palacios A, Piergiacomi V, Catalá A. Antioxidant effect of conjugated linoleic acid and vitamin A during nonenzymatic lipid peroxidation of rat liver microsomes and mitochondria. Mol Cell Biochem2003; 250 :107 –13.
44. Kraft J, Hanske L, Moeckel P, Zimmermann S, Härtl A, Kramer JKG, Jahreis G. The conversion efficiency of trans-11 and trans-12 18:1 by delta9-desaturation differs in rats. J Nutr2006; 136 :1209 –14.
45. Tholstrup T, Raff M, Basu S, Nonboe P, Sejrsen K, Straarup EM. Effects of butter high in ruminant trans and monounsaturated fatty acids on lipoproteins, incorporation of fatty acids into lipid classes, plasma C-reactive protein, oxidative stress, hemostatic variables, and insulin in healthy young men. Am J Clin Nutr2006; 83 :237 –43.
46. Risérus U, Smedman A, Basu S, Vessby B. Metabolic effects of conjugated linoleic acid in humans: the Swedish experience. Am J Clin Nutr2004; 79 (suppl):1146S –8S.
47. Smedman A, Vessby B, Basu S. Isomer-specific effects of conjugated linoleic acid on lipid peroxidation in humans: regulation by alpha-tocopherol and cyclo-oxygenase-2 inhibitor. Clin Sci2004; 106 :67 –73.
48. Risérus U, Basu S, Jovinge S, Fredrikson GN, Arnlov J, Vessby B. Supplementation with conjugated linoleic acid causes isomer-dependent oxidative stress and elevated C-reactive protein—a potential link to fatty acid-induced insulin resistance. Circulation2002; 106 :1925 –9.
49. Mensink RP, Zock PL, Kester AD, Katan MB. Effects of dietary fatty acids and carbohydrates on the ratio of serum total to HDL cholesterol and on serum lipids and apolipoproteins: a meta-analysis of 60 controlled trials. Am J Clin Nutr2003; 77 :1146 –55.
50. Kumar A, Kingdon E, Norman J. The isoprostane 8-iso-PGF(2 alpha) suppresses monocyte adhesion to human microvascular endothelial cells via two independent mechanisms. FASEB J2005; 19 :443 –5.
51. Pardos SC, Torre PD, Sanchez-Muniz FJ. [lsqb[CLA: antioxidant or prooxidant? ] Grasa Aceites2000; 51 :268 –74 (in Spanish).
52. Schonberg S, Krokan HE. The inhibitory effect of conjugated dienoic derivatives (CLA) of linoleic acid on the growth of human tumor cell lines is in part due to increased lipid peroxidation. Anticancer Res1995; 15 :1241 –6.
53. Bergamo P, Luongo D, Rossi M. Conjugated linoleic acid-mediated apoptosis in Jurkat T cells involves the production of reactive oxygen species. Cell Physiol Biochem2004; 14 :57 –64.
54. Kim HK, Kim SR, Ahn JY, Cho IJ, Yoon CS, Ha TY. Dietary conjugated linoleic acid reduces lipid peroxidation by increasing oxidative stability in rats. J Nutr Sci Vitaminol (Tokyo)2005; 51 :8 –15.
55. Loft S, Thorling EB, Poulsen HE. High fat diet induced oxidative DNA damage estimated by 8-oxo-7,8-dihydro-2-deoxyguanosine excretion in rats. Free Radic Res1998; 29 :595 –600.
56. Haegele AD, Briggs SP, Thompson HJ. Antioxidant status and dietary lipid unsaturation modulated oxidative DNA damage. Free Radic Biol Med1994; 16 :111 –5.
57. de Kok TM, Zwingman I, Moonen EJ, et al. Analysis of oxidative DNA damage after human dietary supplementation with linoleic acid. Food Chem Toxicol2003; 41 :351 –8.
58. Nakano M, Kawanishi Y, Kamohara S, et al. Oxidative DNA damage (8-hydroxy-deoxyguanosine) and body iron status: a study of 2507 healthy people. Free Radic Biol Med2003; 35 :826 –32.
59. Wagner A, Jahreis G. Nachweis von DNA-Schaeden mittels Analyse von oxidierten Nucleosiden und deren Anwendung als Biomarker. (Determination of DNA damage by analysis of oxidized nucleosides and their use as biomarkers. ) Ernaehrungs-Umschau2004; 51 :178 –84 (in German).
60. Landi L, Cipollone M, Cabrini L, Fiorentini D, Farruggia G, Galli MC. Injury of rat thymocytes caused by exogenous peroxyl radicals in vitro. Biochim Biophys Acta1995; 1239 :207 –12.
61. Soedergren E, Cederberg J, Basu S, Vessby B. Vitamin E supplementation decreases basal levels of F-2-isoprostanes and prostaglandin F-2 alpha in rats. J Nutr2000; 130 :10 –4.