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-Linolenic Acids in HepG2 Cells1
Institut für Ernährungswissenschaften, Martin-Luther-Universität Halle-Wittenberg, D-06108 Halle/Saale, Germany
2To whom correspondence should be addressed. E-mail: eder{at}landw.uni-halle.de
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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-linoleic acid to linoleic acid and of arachidonic acid to linoleic acid in phospholipids than control cells; with 
-linolenic acid as substrate, they had a lower ratio of eicosapentaenoic acid to -linolenic acid in phospholipids than control cells. Cells treated with cis-9,trans-11 CLA did not differ in these ratios from control cells. Cells treated with trans-10,cis-12 CLA had also a markedly lower ratio of mononunsaturated fatty acids (MUFA) to saturated fatty acids (SFA) in lipids than control cells; cells treated with cis-9,trans-11 CLA had a slightly lower MUFA:SFA ratio than control cells. These findings suggest that trans-10,cis-12 CLA suppresses 
9-, 6- and 5-desaturation in HepG2 cells; cis-9,trans-11 CLA slightly reduces 9-desaturation but does not inhibit 6- and 5-desaturation. Moreover, HepG2 cells treated with 100 µmol/L of trans-10,cis-12 CLA released larger amounts of 6-keto-prostaglandin F1 and prostaglandin F2 than control cells. Treatment of cells with cis-9,trans-11 CLA did not alter the release of these eicosanoids compared with control cells. In conclusion, this study suggests that trans-10,cis-12 CLA has significant effects on the metabolism of essential fatty acids in HepG2 cells, whereas cis-9, trans-11 CLA does not have any effect in this respect.
KEY WORDS: • human hepatoma cells • conjugated linoleic acid • fatty acids • desaturation • eicosanoids
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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,2
). In platelets, treatment with CLA inhibited the formation of thromboxane (TX) A2, followed by a strong inhibition of their aggregation (3
). In contrast, in human breast cancer cells, treatment with pure cis-9,trans-11 CLA did not reduce the secretion of PGE2 (4
); in splenocytes of mice fed a diet supplemented with CLA, the release of PGE2 was slightly increased relative to splenocytes of control mice fed diets without CLA (5
). Although there is experimental evidence that CLA modulates the synthesis of eicosanoids, the results are conflicting and little information is available on the effects of individual CLA isomers.
Highly unsaturated fatty acids with 20 carbon atoms such as arachidonic acid, EPA or di-homo--linoleic acid are substrates for the synthesis of eicosanoids by cyclooxygenase or lipoxygenase. These fatty acids are formed from their precursors, linoleic acid or -linolenic acid, by the action of 6- and 5-desaturases. There is some evidence that CLA inhibits the desaturation of linoleic acid and the formation of arachidonic acid. However, results in this regard are also contradictory. A study by Bretillon et al. (6
) found that incubation of liver microsomes with either cis-9,trans-11 CLA or trans-10,cis-12 CLA lowered the activity of 6-desaturase. In agreement with that finding, feeding diets with a mixture of various CLA isomers to chickens reduced the concentrations of arachidonic acid in some tissues such as liver, heart and muscle (7
). However, there are also studies that do not confirm that CLA impair the desaturation of linoleic acid. In rats, dietary CLA treatment did not reduce the concentration of arachidonic acid in the liver (5
,8
). Treating HepG2 cells with a mixture of CLA isomers did also not reduce the concentration of arachidonic acid in cell lipids (9
,10
).
There is strong evidence that trans-10,cis-12 CLA inhibits the desaturation of saturated fatty acids by 9-desaturase. This has been shown by measuring the activity of this enzyme in the liver of mice fed diets with CLA (11
) as well as in hepatocytes (12
) and adipocytes (13
) treated with CLA. Whether the cis-9,trans-11 isomer also affects the activity of that enzyme is less clear. Treating hepatic microsomes of mice and rats or HepG2 cells with cis-9,trans-11 CLA did not suppress the activity of 9-desaturase (6
,12
), whereas treating adipocytes with cis-9,trans-11 CLA slightly suppressed the activity of that enzyme (13
).
The aim of this study was to investigate the effects of two different isomers of CLA, cis-9,trans-11 CLA and trans-10,cis-12 CLA, on fatty acid desaturation and the formation of some eicosanoids. The cis-9,trans-11 isomer was considered because it is the predominant CLA isomer of milk and dairy products. More than 80% of CLA present in milk, corresponding to 0.1 to 0.7 g/L, exists as cis-9,trans-11 isomer (14
). Trans-10,cis-12 CLA exists in milk and most other foods only in traces but is of particular interest because it has been shown to be more active than the cis-9,trans-11 isomer in several respects (12
,15
). We decided to conduct studies on hepatocytes because they exhibit the highest activities of 9, 6, and 5 desaturase of all the body cells. As a model of hepatocytes, a human hepatoma-derived cell line (HepG2) was chosen because these cells have been reported to retain many normal hepatic metabolic functions, including those of lipid metabolism (16
). Fatty acid desaturation was assessed indirectly, by measuring the fatty acid composition of cellular lipids. To assess 5- and 6-desaturation, cells were treated with linoleic acid or -linolenic acid as exogenous substrates of these reactions and their conversion to higher unsaturated fatty acids such as arachidonic acid and EPA was determined.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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Trans-10,cis-12 CLA (>98% pure), cis-9,trans-11 CLA isomers (>96% pure) and -linolenic acid (>98% pure) were purchased from Cayman Chemical (Ann Arbor, MI). Linoleic acid (
99% pure), indomethacin and bovine serum albumin (BSA) were from Sigma-Aldrich Chemicals (Deisenhofen, Germany). Fetal bovine serum (FBS) was purchased from Biochrom AG (Berlin, Germany). RPMI 1640 medium (containing 0.3 g/L L-glutamine and 2.0 g/L sodium bicarbonate), trypsin-ethylene diamine tetra acetic acid (0.5 g/L trypsin 0.2 g/L EDTA) in Hank’s balanced salt solution (HBSS), HBSS without phenol red and gentamicin were obtained from Gibco/Invitrogen Corp. (Karlsruhe, Germany).
Cell culture.
The human hepatoma HepG2 cell line was obtained from the American Type Culture Collection (WAK-Chemie Medical GmbH, Bad Soden, Germany). The cells were grown in RPMI 1640 medium supplemented with 10% FBS and 0.5% gentamicin (growth medium). Stock cultures were maintained in T-75-cm2 flasks (Greiner, Frickenhausen, Germany) at 37°C in a humidified atmosphere of 95% air and 5% CO2. The medium was changed every 3rd d. When cells had grown to 80–90% confluence, fresh cultures were started. The growth medium was removed and the cells were harvested by trypsinization, added immediately to RPMI 1640 containing 10% FBS to inhibit tryptic activity and centrifuged at 100 x g for 5 min to sediment cells. Cells were washed twice with PBS. Aliquots of cell suspensions in the growth medium (8 or 16 x 106 cells) were seeded and grown in T-75-cm2 flasks. Incubations were carried out over periods of either 30 min or 24 h with medium alone (control) or with medium supplemented with either cis-9,trans-11 CLA or trans-10,cis-12 CLA at final concentrations of 1 to 100 µmol/L. To study the desaturation of linoleic acid or -linolenic acid, the medium was supplemented with either linoleic acid or -linolenic acid at final concentrations of 25 µmol/L.
Cell count and viability.
At the end of the incubation periods, cells were harvested with HBSS containing 0.5 g/L trypsin and 0.2 g/L EDTA, pelleted by centrifugation at 100 x g for 5 min, washed twice with PBS and counted in a Neubauer chamber. Cell viability was checked by the trypan blue dye exclusion method. Cell pellets were lysed with 0.2 mol/L sodium hydroxide solution and the protein concentration was measured by the method of Bradford with BSA as standard (17
).
Fatty acid solutions.
Stock solutions of fatty acids (cis-9,trans-12 CLA, trans-10,cis-12 CLA, linoleic acid, -linolenic acid) were prepared in ethanol at a concentration of 100 mmol/L. For the preparation of the test media, aliquots of these stock solutions were used. The solvent was evaporated under nitrogen, and the fatty acids were converted into their sodium salts by adding equimolar amounts of sodium hydroxide solution. The solution containing the fatty acid salts was added to the growth medium to achieve the final concentrations of fatty acids.
Lipid analysis.
Total cellular lipids were extracted with 1.5 mL of a mixture of hexane and isopropanol (3:2 v/v) (18
). Aliquots of the lipid extracts were dried under nitrogen. Lipids were separated by a solid-phase extraction method described by Suzuki et al. (19
) with modifications. The unpolar fraction of the dried lipids was extracted with acetone. The acetone was evaporated under nitrogen and the neutral lipid fraction resolved in hexane/isopropanol (8:2 v/v); this solution was applied to a SI-phase cartridge (Supelclean, LC-Si; Supelco, Bellefonte, PA) and neutral lipids were eluted with a mixture of hexane and isopropanol (8:2 v/v). The polar fraction of dried lipids was extracted with a mixture of chloroform and methanol (2:1 v/v) and applied to the SI-phase cartridge. Phosphatidyl ethanolamine (PE) was eluted with chloroform/methanol (2:1 v/v); phosphatidyl choline (PC) was eluted with methanol. The individual phospholipids in the eluates were identified by thin layer chromatography with a mixture of chloroform, methanol and ammonia (26:10:1.6 v/v/v) as developing system. Total lipids of the extracts and individual phospholipids were transmethylated with trimethylsulfonium hydroxide (20
). Fatty acid methyl esters (FAME) were separated by gas chromatography using a gas chromatographic system (Hp 5890; Hewlett Packard, Waldbronn, Germany) fitted with an automatic split injection system, a flame ionization detector and a FFAP fused silica capillary column (30 m x 0.53 mm i.d.; Macherey and Nagel, Düren, Germany). FAME were identified by comparing their retention times with those of individual purified standards (21
).
Release of eicosanoids from HepG2 cells.
The effect of CLA isomers on the release of eicosanoids was tested in two different ways.
In the first test, the cells were incubated with growth medium alone (control) or with growth medium supplemented with either 100 µmol/L of cis-9,trans-11 CLA or 100 µmol/L trans-10,cis-12 CLA for 24 h. Cells were then harvested with HBSS containing 0.5 g/L trypsin and 0.2 g/L EDTA, washed twice with PBS, centrifuged (100 x g, 5 min) and counted. Cells (2 x 106) were incubated at 37°C for 30 min in HBSS. The incubation was terminated by addition of 50 µL of ice-cold 4.5 mmol/L indomethacin solution. After centrifugation (1800 x g for 10 min, 4°C) the supernatants were assayed for 6-keto-PGF1, PGF2 and TXB2 using ELISA-kits purchased from Cayman Chemical. 6-keto-PGF1 was determined as a measure of the unstable PGI2, and TXB2 was determined as a measure of the unstable TXA2 (22
).
In the second test, a pool of untreated HepG2 cells was used and the effect of adding the CLA isomers at a final concentration of 100 µmol/L to the assay medium consisting of HBSS was investigated. Incubations were carried out at 37°C for 30 min. Eicosanoids were determined as described above.
Statistical analysis.
Treatment effects were analyzed using one-way ANOVA. In the case of large differences in the variances of means, data were transformed into their logarithms before ANOVA. For statistically significant F-values, means were compared by Fisher’s multiple range test. Differences with P < 0.05 were considered significant. Values are means ± SD.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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The growth and viability of the cells were not affected by incubation with either CLA isomer. Taking the average of all experiments conducted, the cell counts after incubation with 100 µmol/L of cis-9,trans-11 CLA and 100 µmol/L of trans-10,cis-12 CLA were 94 ± 11% and 91 ± 18% (means ± SD, n = 16), respectively, compared with cells treated with growth medium alone (control = 100%). Cell viability was 91 ± 4% for control cells, 89 ± 5% for cells treated with 100 µmol/L of cis-9,trans-11 CLA and 88 ± 7% for cells treated with 100 µmol/L of trans-10,cis-12 CLA (n = 15). The protein levels were also similar in control cells (1.28 ± 0.34 mg per 8 x 106 cells), cells treated with 100 µmol/L of cis-9,trans-11 CLA (1.30 ± 0.34 mg per 8 x 106 cells) and cells treated with 100 µmol/L of trans-10,cis-12 CLA (1.32 ± 0.36 mg per 8 x 106 cells) (n = 15). When the monolayer of the cells was examined under the light microscope, the cells appeared normal.
Incorporation of CLA isomers into HepG2 lipids.
The incorporation of both CLA isomers in total HepG2 lipids was dependent on their concentrations in the medium and the period of incubation (Fig. 1
). The highest concentrations of both CLA isomers were found in the fraction of neutral lipids (Fig. 2
). After a 0.5-h incubation with either cis-9,trans-11 CLA or trans-10,cis-12-CLA, the concentration of cis-9,trans-11 CLA in neutral lipids was much higher than that of trans-10,cis-12-CLA. In contrast, after incubation for 24 h, the concentrations of both CLA isomers in neutral lipids were similar. The concentrations of both CLA isomers in phospholipids were only one-half those of neutral lipids. Cis-9,trans-11 CLA was predominantly incorporated into PE; after an incubation period of 24 h, the concentration of cis-9,trans-11 CLA in PE was approximately two times higher than that in PC. In contrast, the concentration of trans-10,cis-12 CLA after a 24-h incubation was similar in PC and PE.
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9-desaturation in HepG2 cells.
As an index of 9-desaturation, the ratios of MUFA to SFA in HepG2 total lipids, neutral lipids and phospholipids were determined (Figs. 3
and 4
). Both CLA isomers reduced the ratio of MUFA to SFA in a concentration-dependent manner, but the effect was clearly stronger for trans-10,cis-12 CLA than for cis-9,trans-11 CLA. Treating HepG2 cells with low concentrations (1 µmol/L) of both CLA isomers for 24 h did not alter the concentrations of SFA and MUFA in their total lipids compared with untreated control cells. Trans-10,cis-12 CLA reduced the ratio of MUFA to SFA in HepG2 total lipids at a concentration of 10 µmol/L in the medium (corresponding to a concentration of 1.7 g trans-10,cis-12 CLA per 100 g of total fatty acids in HepG2 cells) compared with untreated controls (Fig. 3)
. The reduction of this ratio by trans-10,cis-12 CLA was due to increased concentrations of myristic, palmitic, stearic and eicosanoic acid and reduced concentrations of palmitoleic and oleic acid (data not shown). Treating HepG2 cells with 100 µmol/L trans-10,cis-12 CLA reduced the ratio of MUFA to SFA in total lipids to one-half of that in untreated control cells. Cis-9,trans-11 CLA reduced the ratio of MUFA to SFA in HepG2 total lipids only at a concentration of 100 µmol/L, corresponding to a concentration of 23 g cis-9,trans-11 CLA per 100 g total fatty acids in HepG2 cells. The magnitude of the reduction in the ratio of MUFA to SFA caused by 10 µmol/L trans-10,cis-12 CLA was similar to that caused by 100 µmol/L cis-9,trans-11 CLA.
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9-desaturation was reflected mostly in neutral lipids and PE (Fig. 4)
. HepG2 cells treated with 25 µmol/L trans-10,cis-12 CLA for 24 h had higher concentrations of myristic, palmitic, stearic and eicosanoic acids and lower concentrations of palmitoleic and oleic acids in these lipids than untreated control cells (data not shown); in contrast, the concentrations of SFA and MUFA in PC were similar in HepG2 cells treated with 25 µmol/L cis-9,trans-11 CLA and in untreated controls. HepG2 cells treated with 25 µmol/L trans-10,cis-12 CLA tended to have lower concentrations (P = 0.12) of MUFA in their lipid fractions than untreated control cells.
Effect of cis-9,trans-11 CLA and trans-10,cis-12 CLA on the desaturation of linoleic acid and -linolenic acid in HepG2 cells.
To assess the effects of the CLA isomers on desaturation of linoleic acid and -linolenic acid, the concentrations of individual (n-6) PUFA (Table 1
) and (n-3) PUFA (Table 2
) in phospholipid fractions of HepG2 cells were determined. The ratio 20:3 (n-6)/18:2 (n-6) was calculated as an indicator of 6-desaturation; ratios 20:4 (n-6)/18:2 (n-6) and 20:5 (n-3)/18:3 (n-3) were calculated as indicators of 5- and 6-desaturation; the ratio 22:6 (n-3)/22:5 (n-3) was calculated as an indicator of 4-desaturation.
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. The concentrations of 22:4 (n-6) and 22:5 (n-6) were generally below the detection limit of 0.1 g/100 g fatty acids and, therefore, were not recorded. HepG2 cells treated with 25 µmol/L trans-10,cis-12 CLA for 24 h had lower concentrations of 20:3 (n-6) and 20:4 (n-6) and lower ratios 20:3 (n-6)/18:2 (n-6) in PC and PE and 20:4 (n-6)/18:2 (n-6) in PC than untreated control cells. In contrast, the ratios in HepG2 cells treated with cis-9,trans-11 CLA did not differ from those in untreated HepG2 cells.
Incubation of HepG2 cells with linoleic acid markedly increased the concentrations of 18:2 (n-6) and 20:3 (n-6) in PC and PE and that of 20:4 (n-6) in PE compared with HepG2 cells that were not treated with linoleic acid. Incubation of HepG2 cells with linoleic acid and trans-10,cis-12 CLA significantly raised the concentration of 18:2 (n-6) and reduced the concentrations of 20:3 (n-6) and 20:4 (n-6) in PC and PE compared with cells treated with linoleic acid alone. The ratios 20:3 (n-6)/18:2 (n-6) and 20:4 (n-6)/18:2 (n-6) in PC and PE were also lower in HepG2 cells treated with linoleic acid and trans-10,cis-12 CLA than in HepG2 cells treated with linoleic acid alone. In contrast, cells treated with linoleic acid and cis-9,trans-11 CLA did not differ in their concentrations of individual (n-6) PUFA from cells treated with linoleic acid alone.
The concentrations of (n-3) PUFA in HepG2 cells were low in both phospholipids (Table 2)
. HepG2 cells treated with cis-9,trans-11 CLA and trans-10,cis-12 CLA did not significantly differ in their concentrations of individual (n-3) PUFA in cell lipids from control cells.
Incubation of HepG2 cells with -linolenic acid increased the concentrations of 18:3 (n-3) and 20:5 (n-3) in both phospholipids fractions compared with cells that were not treated with -linolenic acid. Cells treated with -linolenic acid and trans-10,cis-12 CLA had higher concentrations of 18:3 (n-3) and lower concentrations of 20:5 (n-3) and 22:6 (n-3) in PC and PE than in HepG2 cells treated with -linolenic acid alone. The ratio 20:5 (n-3)/18:3 (n-3) in PC and PE was also lower in HepG2 cells treated with -linolenic acid and trans-10,cis-12 CLA than in HepG2 cells treated with -linolenic acid alone; the ratio 22:6 (n-3)/22:5 (n-3) in PC and PE tended to be lower in HepG2 cells treated with -linolenic acid (P = 0.11) and trans-10,cis-12 CLA (P = 0.08) than in HepG2 cells treated with -linolenic acid alone. The concentrations of individual (n-3) PUFA and the ratios between different (n-3) PUFA in HepG2 cells treated with -linolenic acid and cis-9,trans-11 CLA did not differ from those treated with -linolenic acid alone.
The effect of cis-9,trans-11 CLA and trans-10,cis-12 CLA on the release of eicosanoids from HepG2 cells.
HepG2 cells which were treated for 24 h with 100 µmol/L trans-10,cis-12 CLA released more 6-keto-PGF1 and PGF2 during a 30-min incubation in HBSS than control cells (Fig. 5
). The release of TXB2 did not differ between control cells and cells treated with trans-10,cis-12 CLA. Treating HepG2 cells with 100 µmol/L cis-9,trans-11 CLA over a 24-h period did not alter the release of any of the eicosanoids assayed.
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from HepG2 cells during a 30-min incubation (Fig. 6
). The addition of 100 µmol/L cis-9,trans-11 CLA to the assay medium did not alter the release of PGF2. The release of 6-keto- PGF1 and TXB2, in contrast, was not altered by adding either isomer to the assay medium.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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,23
). This study, thus, must be considered as a model study that cannot be directly extrapolated to animals. The finding that HepG2 cells incorporate most of the CLA into their neutral lipid fraction agrees with another study (9
). In mice fed diets containing a mixture of CLA isomers, the concentration of CLA in the liver was also highest in the triglyceride fraction (5
).
Our study clearly showed that treating HepG2 cells with trans-10,cis-12 CLA increases the concentrations of SFA in various cell lipid fractions at the expense of MUFA. This finding confirms several investigations with model animals or cell systems that clearly demonstrated that trans-10,cis-12 CLA strongly suppresses 9-desaturase (4
,11
–13
). We also found a reduction of the ratio of MUFA to SFA in HepG2 cell lipids after incubating these cells with cis-9,trans-11 CLA. This finding suggests that cis-9,trans-11 CLA is also able to suppress 9-desaturation. However, this effect was seen only at very high concentrations of cis-9,trans-11 CLA, which are not physiologically relevant. Thus, we assume that at physiologic concentrations in tissue lipids such as obtained by dietary treatment, cis-9,trans-11 CLA does not significantly affect 9-desaturation.
Moreover, our study showed elevated concentrations of substrates and reduced concentrations of products of 5- and 6-desaturase in lipids of HepG2 cells treated with trans-10,cis-12 CLA. This finding suggests that trans-10,cis-12 CLA suppresses the desaturation of linoleic acid and -linolenic acid to more unsaturated fatty acids. Other investigators (6
,24
) found that CLA inhibited the activity of 6-desaturase in liver microsomes and torula yeast. However, contrary to our study, those studies found an inhibiting effect of cis-9,trans-11 CLA as well as of trans-10,cis-12 CLA. An inhibition of the desaturation of linoleic acid and -linolenic acid is physiologically relevant because the products of this process are important constituents of membrane lipids which determine important membrane properties (25
); some of them, the C20-PUFA, are substrates for the formation of eicosanoids (26
). A reduction of the arachidonic acid concentration in membrane lipids leads to reduced formation of eicosanoids. Accordingly, some studies have found that a reduction of the concentration of arachidonic acid in cell lipids caused by CLA treatment leads to a reduced formation of some eicosanoids such as PGE2 (1
,2
). In our study, the release of PGF2 and 6-keto-PGF1 from cells into the medium was enhanced by treating cells with trans-10,cis-12 CLA, although the concentrations of arachidonic acid in cell lipids were lowered. Treating cells with high concentrations of trans-10,cis-12 CLA could have enhanced the deacylation of PUFA from phospholipids and, thus, enlarged the pool of free arachidonic acid as a substrate for eicosanoid formation. An increased rate of deacylation also could have contributed to reduced concentrations of arachidonic acid observed in lipids of HepG2 cells treated with trans-10,cis-12 CLA. Because the concentrations of CLA used in this study were rather high, it is questionable whether the effect of trans-10,cis-12 CLA observed on the release of eicosanoids is physiologically relevant. In cells treated with cis-9,trans-11 CLA neither the concentration of arachidonic acid nor the amounts of eicosanoids released into the medium were different from untreated cells.
In conclusion, this study suggests that trans-10,cis-12 CLA at high concentrations affects the desaturation of fatty acids and the formation of eicosanoids in HepG2 cells, whereas cis-9,trans-11 CLA has little, if any, effect in this respect.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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3 Abbreviations used: BSA, bovine serum albumin; CLA, conjugated linoleic acid; EDTA, ethylenediamine tetra-acetic acid; EPA, eicosapentaenoic acid; FBS, fetal bovine serum; HBSS, Hank’s balanced salt solution; MUFA, monounsaturated fatty acids; PBS, phosphate buffered saline; PC, phosphatidyl choline; PE, phosphatidyl ethanolamine; PG, prostaglandin; PUFA, polyunsaturated fatty acids; SFA, saturated fatty acids; TX, thromboxane.
Manuscript received 26 November 2001. Initial review completed 2 February 2002. Revision accepted 4 March 2002.
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LITERATURE CITED |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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