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Nutrient Interactions and Toxicity

Dietary (+)-Catechin and BHT Markedly Increase -Tocopherol Concentrations in Rats by a Tocopherol--Hydroxylase–Independent Mechanism^1,2

Jan Frank³, Torbjörn Lundh^*, Robert S. Parker, Joy E. Swanson, Bengt Vessby^** and Afaf Kamal-Eldin

Department of Food Science, Swedish University of Agricultural Sciences, S-750 07 Uppsala, Sweden; ^* Department of Animal Nutrition and Management, Swedish University of Agricultural Sciences, S-750 07 Uppsala, Sweden; Division of Nutritional Sciences, Cornell University, Ithaca, NY; and ^** Department of Public Health and Caring Sciences/Geriatrics, University of Uppsala, S-751 25 Uppsala, Sweden

³To whom correspondence should be addressed. E-mail: Jan.Frank{at}lmv.slu.se.

	ABSTRACT

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

 
The effects of dietary (+)-catechin (CAT) and BHT on plasma and tissue concentrations of -tocopherol (-T), -tocopherol (-T) and cholesterol (C) were studied in male Sprague-Dawley rats. The rats were fed the compounds during a 4-wk period at concentrations of 2 g/kg in standardized diets, low but adequate in vitamin E, with 2 g/kg cholesterol. The CAT-regimen did not affect weight gain, feed intake or organ weights. BHT did not affect feed intake but lowered the body weight and the amount of liver lipids and increased the weights of livers and lungs relative to the body weight. Rats consuming CAT had 2.5–3.5-fold increased plasma, liver and lung -T concentrations, but C concentrations remained unchanged. BHT-feeding resulted in 2.4- and 1.7-fold elevation in -T but 50% decrease in -T concentrations in blood plasma and liver, respectively. BHT also lowered total C in the liver without affecting the concentration of C in the liver lipids. To investigate whether the -T–sparing action of the studied compounds was due to the inhibition of tocopherol--hydroxylase, HepG2 cells were incubated with CAT or BHT in the presence of -tocopherol (-T) and the 3'- and 5'--carboxychromanol metabolites in the media were analyzed by GC/MS. Neither CAT nor BHT inhibited tocopherol--hydroxylase activity in hepatocyte cultures; CAT was also inactive in a rat microsomal assay. In conclusion, both dietary CAT and BHT markedly increased -T concentrations in plasma and organs of Sprague-Dawley rats by a mechanism that apparently does not involve inhibition of tocopherol--hydroxylase, a key enzyme in tocopherol catabolism.

KEY WORDS: • BHT • catechin • cytochrome P₄₅₀ • tocopherols • tocopherol--hydroxylase.

Vitamin E was recognized as a factor essential for reproduction in rats by Evans and Bishop in 1922 and subsequently acknowledged as a vitamin in 1925 (1). Vitamin E is a generic name for all natural and synthetic substances exerting the biological activity of -tocopherol (-T).³ The eight natural vitamin E compounds are derivatives of 6-chromanol and classified into tocopherols, substituted with a saturated phytyl side chain, and tocotrienols, substituted with an unsaturated isoprenoid side chain. The prefixes -, ß-, - and - are added according to the number and position of methyl groups substituted at the chromanol ring (2).

-T is the major lipid-soluble antioxidant (3) and the predominant E-vitamer in human and animal tissues despite the fact that -tocopherol (-T) is the major dietary form of vitamin E (4,5). Among other things, the body specifically retains -T due to the action of a hepatic -tocopherol transfer protein, which has a higher affinity for -T than for the other vitamers. Also, tocopherol--hydroxylase, the enzyme catalyzing the initial step in the catabolism of tocopherols to their water-soluble metabolites, has a much higher catalytic activity toward -T than -T (6). Because the body makes use of specific mechanisms to retain -T, it has been regarded as the biologically most important form of the vitamin. Natural -T also has the highest biological activity of all forms of vitamin E, which traditionally has been assessed in animal model systems, such as the rat fetal resorption-gestation test, the curative myopathy test or the dialuric acid-induced test for RBC hemolysis (5). One major concern with the values obtained with these tests is that the animals are given experimental feeds that do not reflect a natural diet but contain the studied vitamer as the only antioxidant (7–9). The concentration of a certain E-vitamer in the body (10–12), its availability for physiological tasks, and, as a result, the values for its biological activity would probably be different if assessed with diets including coantioxidants.

Because of its potent function as an antioxidant, vitamin E is believed to be helpful in the prevention of diseases associated with oxidative stress, such as cancer, cardiovascular disease, Alzheimer’s disease and chronic inflammation. Furthermore, -T has specific (nonantioxidant) functions in cellular signaling, gene activity, immune function and apoptosis that may contribute to its role in disease prevention [reviewed in (13)]. Reduced serum concentrations of vitamin E were reported for patients suffering from cardiovascular disease (14). Hence, increasing vitamin E concentrations by dietary means may be beneficial in the prevention of such diseases.

Previously, we demonstrated that certain dietary antioxidants can modify the availability of vitamin E in vivo. Although BHT and sesamin markedly increased vitamin E concentrations in our rat model, cyanidin-3-O-glucoside, and caffeic and chlorogenic acids exerted only a minor elevating effect, curcumin and ferulic acid did not affect tocopherols at all, and secoisolariciresinol diglucoside and its oligomer even reduced vitamin E concentrations [(10–12,15) and unpublished data]. The underlying mechanisms are not known at present, except for sesamin, which inhibits the catabolism of -T to its water-soluble carboxychroman-metabolites (6,16).

In the course of our investigations on the interactions of minor dietary compounds with vitamin E, we chose to study the effects of (+)-catechin (CAT) and BHT in a rat model, because both compounds are strong antioxidants (17–25) and are commonly found in the human diet (26–30). CAT has been shown to reduce the oxidative consumption of vitamin E in vitro (20,22) and BHT is commonly used to protect vitamin E during analytical procedures. Both compounds are absorbed and metabolized in humans and rats (31–39) and, accordingly, have the potential to interact with vitamin E in vivo.

	MATERIALS AND METHODS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

 
Experimental animals and diets.

Male, 21-d-old Sprague-Dawley rats (n = 24) with a mean body weight of 58 g (B&K Universal AB, Sollentuna, Sweden) were used for this study. The rats were housed individually in Macrolon IV cages (Ehret GmbH, Emmendingen, Germany) with aspen wood bedding (Beekay bedding; B&K Universal AB) under controlled conditions of 25°C and 60% relative humidity with a 12-h light:dark cycle (light 0700 to 1900h). Each cage was equipped with a water bottle with a metal lid, a feed container attached to a stainless steel plate to avoid overthrowing and spilling, two black plastic tubes, which the rats used for resting and hiding, and a table tennis ball for playing. The rats had free access to feed and water throughout the experiment, which was carried out in accordance with the guidelines of and approved by the Ethical Committee for Animal Experiments in the Uppsala region.

The composition of the basal diet is shown in Table 1. Rapeseed oil was a gift from Karlshamns AB (Karlshamn, Sweden). Cholesterol (C) and the phenolic compounds (+)-catechin (CAT; CAS # 154–23-4) and BHT (CAS # 128–37-0), added to the basal diet at concentrations of 2 g/kg, were purchased from Sigma Chemical (St. Louis, MO).

Rats (n = 24) were randomly divided into groups of eight with similar mean body weights of (means ± SEM) 59.8 ± 2.0, 58.0 ± 1.1 and 60.2 ± 1.4 g for the control, CAT and BHT groups, respectively; they were fed the control diet for an acclimation period of 5 d, and then fed their respective diets without added antioxidants (control) or with 2 mg/kg CAT or BHT for 28 d. Body weights were measured weekly. At the end of the experiment, the rats were food deprived for 12 h before intraperitoneal injection of an overdose of sodium pentobarbital and killed by exsanguination. Blood samples were withdrawn from the heart and collected in tubes containing EDTA as anticoagulant, centrifuged (1000 x g; 10 min); the blood plasma was transferred to test tubes with screw caps and stored at -20°C until analyzed. Liver and lung tissues were excised, weighed and stored in 2-propanol at -80°C until analyzed.

Extraction and analyses of tissue lipids.

For the extraction of plasma T, blood plasma (500 µL) was mixed with ethanol containing 0.005% BHT (500 µL) and extracted with hexane (2 mL) after manual shaking for 3 min. The lipids from livers and lungs were extracted according to the method developed by Hara and Radin (40) as described previously (10–12). Briefly, the liver tissue was homogenized in hexane/2-propanol (3:2, v/v), centrifuged (4000 x g) and the lipid extract collected in a separatory funnel. The extract was washed with aqueous sodium sulfate and the supernatant evaporated; the lipids were weighed and dissolved in 10 mL hexane. The extract was stored at -20°C until analyzed. Plasma and tissue concentrations of tocopherols, cholesterol and triacylglycerols were determined by standard methods as previously described (10–12).

Tocopherol--hydroxylase activity.

The effect of CAT and BHT on tocopherol--hydroxylase activity was evaluated in a hepatocyte cell culture assay and sesamin (Cayman Chemical, Ann Arbor, MI) was used as a positive control. HepG2 cells (subclone C3A; American Type Culture Collection, Manassas, VA) were grown in DMEM containing 10% fetal bovine serum (FBS) under conditions recommended by the supplier and used 3–5 d postconfluence. Test compounds (BHT or CAT), in ethanol stock solutions, were first added drop-wise to FBS, which was then diluted 10-fold with DMEM for a final concentration of 20 µmol/L BHT or CAT. Cells were preincubated with medium containing BHT or CAT for 4 h, after which the medium was changed to one containing 20 µmol/L -tocopherol and 20 µmol/L BHT or CAT. After 48 h of incubation, the concentration of 3'- and 5'--carboxychromanol metabolites in the medium was determined by GC/MS of their trimethylsilyl ethers, using d₉--3'-carboxychromanol as an internal standard, as previously described (16). Total cell protein was determined by dye binding (Bio-Rad Protein Assay; Bio-Rad Laboratories, Hercules, CA). Due to a somewhat larger variation of the results obtained for CAT in the hepatocyte assay (see below), the effect of 5 µmol/L CAT on rat liver microsomal tocopherol--hydroxylase activity was evaluated as previously described, using 25 µmol/L -T as substrate (6). Experiments were replicated three times and representative results are shown. -T and -T were used as substrates for enzyme activity because both are substantially better substrates than -T, as mentioned above, and therefore offer more sensitivity in assays of enzyme activity and inhibition.

Statistical analyses.

Statistical analysis of the registered variables was performed by ANOVA and the general linear model (GLM) of SAS (41). Least significant differences from the t test function of the SAS GLM procedure were used to make statistical comparisons. Values are means ± SEM. Differences were considered significant at P < 0.05.

	RESULTS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

 
In this experiment, the rats consumed all of the diet provided; consequently, feed intake (380 g/28 d) did not differ between groups and amounted to a mean intake of 27.1 mg/d of CAT or BHT. Although CAT-feeding did not affect rat performance assessed by measuring feed intake, total body weight, weight gain and organ weights, BHT-treatment impaired growth and increased liver weight relative to body weight (Table 2).

	DISCUSSION

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

 
To study the interactions of dietary antioxidants with vitamin E in vivo, we chose the natural plant phenolic compound (+)-catechin (CAT) and the synthetic food additive BHT because of their frequent occurrence in the human diet and their reported interactions with vitamin E in vitro (20,22) or strong antioxidative capacity (25), suggesting a potential for vitamin E sparing in vivo. Especially high concentrations of CAT are found in black chocolate and infusions of black tea (26,27), whereas BHT is used industrially as a preservative in lipid-containing foodstuffs and chewing gum (29). The daily intake of tea catechins by humans has been estimated to be 100–500 mg (29,30) or 1–7 mg/kg for a human weighing 70 kg. For BHT, Leclercq et al. (28) calculated a theoretical maximum daily intake in Italy equivalent to 0.27 mg/kg for a human of 70 kg. We treated the rats throughout this experiment with high doses of the compounds to provoke potential physiologic effects. The daily intake of the phenolic compounds ranged from 350 mg/kg in the beginning of the experiment to 165 mg/kg before killing because of the larger gain in body weight compared with the increase in feed consumption.

To study the effects of the phenolic antioxidants, we established them as the only variables in the experimental diets and compared their effects in rats to those in controls fed diets void of these compounds (Table 1). We chose rats as model animals because their physiology and metabolism are studied and documented extensively, organ samples are readily available and rat experiments are easy to reproduce. To detect changes in tocopherol concentrations, we chose blood plasma, the liver, the central organ for vitamin E metabolism, and the lung as an example of a tissue under permanent oxidative challenge. To investigate whether the effects on tocopherols are accompanied by changes in cholesterol concentrations, the diets were prepared with 2% cholesterol.

Dietary CAT is absorbed and metabolized by rats (31–33) and humans (34–36). In this study, CAT feeding elevated -T levels in blood plasma, livers and lungs (Table 3) without any adverse effects in rats (Table 2) in line with results from other studies (42,43). Feeding male Wistar rats diets containing 8 g/kg flavonoids (quercetin and CAT, 2:1 wt/wt) for 4 wk, increased -T in blood plasma and liver microsomes (42). In another study, feeding a mixture of tea catechins (10 g/kg diet, amounting to 140 mg CAT/kg diet) to rats resulted in a significant increase in -T concentrations in plasma and erythrocytes (43). High doses of CAT (3 g/d for 1 mo or 1.5 g/d for 2 mo) increased blood levels of vitamin E and the activities of the antioxidant enzymes catalase and glutathione peroxidase in patients with chronic hepatitis (44). A pronounced -T-sparing action of CAT was reported in different in vitro models (17–20). Liao and Yin found that CAT and -T synergistically delayed lipid oxidation in human erythrocyte membrane ghosts (21) and Pedrielli and Skibsted demonstrated a direct regeneration of the -tocopheroxyl radical by CAT in homogenous solutions of peroxidating methyl linoleate (22).

The lipid-soluble antioxidant BHT has been studied extensively for its metabolism and its physiologic and toxicologic actions because of its use as a preservative in the food industry, and metabolites as well as metabolizing enzymes have been described (37–39). BHT feeding resulted in lower body weights and an increase in liver weight relative to the body weight in our rats (Table 2), which are well known effects of BHT consumption (10,37,45,46). In plasma and liver from rats fed BHT, there was a pronounced elevation of -T and a significant reduction of -T concentrations (Table 3). The effects of BHT on -T confirm our previous results (10) when a higher dose of BHT (4 g/kg feed) was fed. The -T–lowering effect, on the other hand, was not observed in the previous experiment (10) and cannot be explained at present. Contrary to our findings, Simán and Eriksson (46) observed decreased -T concentrations in the liver and elevated concentrations in adipose tissue of BHT-fed female Sprague-Dawley rats. These authors used older and heavier female rats, and BHT affects growing and adult rats differently (37). Moreover, female rats have been shown to be less responsive to growth impairment caused by BHT (45). Our male rats administered the BHT-regimen were clearly growing more slowly than control rats, whereas the female rats of Simán and Eriksson were not. The early work of Daniel and Gage (47) indicated a faster excretion of BHT in female rats, which may contribute to the observed differences in rats of opposing sexes. BHT did not affect the C and TAG levels in plasma, but decreased the total C and lipid concentrations in the liver (Tables 2, and 4), in agreement with our previous results (10) and another publication on the effects of BHT administered to rats in normo- and high cholesterolemic diets (48). The percentage of total C in liver lipids was not affected in the present investigation, but was lowered in our previous experiment (10). Consequently, the lowering of total C in the liver tissue in the present study appears to be caused by the reduction in total lipids.

In earlier studies, dietary sesamin, a sesame seed lignan, increased concentrations of tocopherols in rat tissues and plasma (10,15). The mechanism of this effect was attributed to the inhibition of tocopherol--hydroxylase, a key enzyme in the catabolism of tocopherols to water-soluble urinary metabolites (6,16). To determine whether the -T–enhancing effect of CAT or BHT might involve a similar mechanism, we assessed their effects on tocopherol--hydroxylase activity in HepG2 cells and rat liver microsomes. Neither CAT nor BHT inhibited tocopherol--hydroxylase activity in HepG2 cultures. The lack of effect on tocopherol--hydroxylase activity of CAT was confirmed in the rat liver microsomes. The fact that both CAT and BHT resulted in substantially elevated plasma and tissue levels of -T, but not -T, additionally supports the lack of involvement of inhibition of tocopherol--hydroxylase. -T, present in the diet at a concentration 1.8-fold that of -T, is a better substrate for -oxidation, and would therefore be expected to respond even more strongly than -T to its inhibition (6), as was the case with sesamin-treated rats (10,15). The data presented here suggest involvement of a mechanism selective to the trafficking or metabolism of -T, whereas the tocopherol--hydroxylase pathway appears to be involved mainly with the elimination of those vitamin E forms other than -T.

Although CAT and BHT can be found in their free forms in vivo, the major part of these compounds is rapidly metabolized and subsequently excreted in the form of conjugated metabolites, mainly glucuronides (31,49). In rats, a large quantity of the ingested CAT seems to be glucuronidated in the enterocytes (33). Therefore, it is possible that these metabolites are also involved in the observed tocopherol-sparing effect. The major metabolites of CAT in biological fluids of rats were found to be equally or even more efficient superoxide anion radical scavengers than the parent compound (31). In view of the vast in vitro data supporting a synergistic action of -T and CAT (17–20), coantioxidant mechanisms represent the most likely explanation for the -T–enhancing effects of CAT in the present study. However, the additional or alternative involvement of one or more of the following mechanisms facilitating the effects of the dietary antioxidants cannot be excluded: 1) reaction with reactive oxygen species that consume T in vivo, such as peroxyl radicals (2); 2) effects on enzyme systems involved in the production of such radicals (e.g., lipoxygenase and cyclooxygenase); 3) inhibitory or stimulatory effects on antioxidant enzyme systems such as catalase and glutathione peroxidase (44); and 4) enhancement of vitamin E absorption. The latter could result in an increase in -T but not -T because of the high capacity of rodents to eliminate -T. Because free radical species are involved in the development of certain degenerative diseases (13), the addition of vitamin E–sparing antioxidants may be helpful in their prevention.

In conclusion, our data show that dietary CAT and BHT markedly increase -T concentrations in plasma and organs of Sprague-Dawley rats. Our in vitro data suggest that the underlying mechanism does not involve inhibition of tocopherol--hydroxylase, a key enzyme in tocopherol catabolism. These findings warrant further investigations of the importance of this effect in humans.

	ACKNOWLEDGMENTS

 
We acknowledge Timothy Sontag (Division of Nutritional Sciences, Cornell University) for his skilful help with the microsomal tocopherol--hydroxylase assay.

	FOOTNOTES

 
¹ Presented in part at the Oxygen Club Of California 2002 World Congress, March 2002, Santa Barbara, CA [Frank, J., Kamal-Eldin, A., Lundh, T. & Vessby, B. (2002) Sesamin, (+)-catechin, and butylated hydroxytoluene elevate vitamin E levels in male Sprague-Dawley rats].

² Supported by the Swedish Council for Forestry and Agricultural Research (SJFR, Grant 50.0496/98).

⁴ Abbreviations used: C, cholesterol; CAT, (+)-catechin; FBS, fetal bovine serum; T, tocopherol; TAG, triacylglycerols.

Manuscript received 4 June 2003. Initial review completed 3 July 2003. Revision accepted 24 July 2003.

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