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Gamma-Tocotrienol Is Required for Normal Vitamin D Metabolism in Female Rats

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Gamma-Tocotrienol Is Required for Normal Vitamin D Metabolism in Female Rats Research Paper Gamma-tocotrienol is required for normal vitamin D metabolism in female rats M. Norazlina, F.W. Ng*, S. Ima-Nirwana ABSTRACT Objective: To study the effects of vitamin E deficiency and supplementation of vitamin Dept of Pharmacology, D and bone metabolism in female Sprague-Dawley rats. Faculty of Medicine & *Dept of Biomedical Sciences, Materials and Methods: Rats weighing between 200 and 250 g were divided into six Faculty of Allied Health Sciences, groups, that is, rats fed on (a) normal rat chow diet (RC), (b) vitamin E–deficient diet Universiti Kebangsaan Malaysia, (VED) (c) VED diet supplemented with 60 mg/kg α-tocopherol acetate (ATF), (d) VED Kuala Lumpur, Malaysia. diet supplemented with 60 mg/kg γ-tocotrienol (GTT), (e) VED diet supplemented with 60 mg/kg Tocomin® (TOC) and (f) baseline control group which was killed without Received: 24.2.2005 treatment (BC). Treatment was given for 2 months. Serum 1,25-dihydroxyvitamin D3, Revised: 24.8.2005 serum total calcium, urine calcium, left femur, and fourth lumbar vertebra calcium Accepted: 31.8.2005 content and left femur length were measured. Results: In the VED and ATF groups, activation of vitamin D to 1,25-dihydroxyvitamin Correspondence to: D was inhibited and calcium reabsorption in the kidneys were increased. Both the Ima Nirwana Soelaiman 3 Department of Pharmacology, effects seen in ATF and GTT groups were observed in the TOC group. The GTT group Faculty of Medicine, was protected from the effects of the vitamin E–deficient diet. Calcium content of the Universiti Kebangsaan Malaysia, fourth lumbar vertebra was also decreased by vitamin E– deficiency, which was not Jalan Raja Muda Abdul Aziz reversed by vitamin E supplementation. 50300 Kuala Lumpur, Malaysia. Conclusion: γ-tocotrienol and not α-tocopherol protects vitamin D metabolism and E-mail: [email protected] calcium homeostasis from the effects of vitamin E–deficiency. KEY WORDS: Calcium homeostasis, α-tocopherol, vitamin E–deficiency. Introduction postmenopausal women.[5] Tocotrienol was shown to reduce Vitamin D and its metabolites are among the factors that carcinogenesis,[6] and to inhibit the production of HMG-CoA reductase – the enzyme responsible for cholesterol synthesis.[7] are important in regulating bone metabolism. Vitamin D3 is a prohormone, which is hydroxylated in the liver and in the kidney Palm oil is the major source of vitamin E, mainly the tocotrienol type. Vitamin E–rich extract from palm oil protected to the active metabolite 1,25-dihydroxyvitamin D3 and 24,25- the bone against the toxic effects of the oxidizing agent ferric dihydroxyvitamin D3. In a previous study, supplementation with nitrilotriacetate,[8] reduced bone resorption, and increased the α 1, 25-dihydroxyvitamin D3 analogue was able to increase bone mineral density in osteoporotic women.[1] The most potent survival rate of thyrotoxic rats[9] and prevented the decline in bone mineral density, and bone calcium loss seen in vitamin D metabolite is 1,25-dihydroxyvitamin D3 (calcitriol), which stimulates calcium and phosphate transport from the orchidectomized rats.[10] intestine and calcium reabsorption from bone.[2] 1,25- Palm vitamin E (60 mg/kg) was comparable to α-tocopherol dihydroxyvitamin D3 has also been reported to act directly on in maintaining bone mineral density and bone calcium content circulating osteoclast precursors to influence osteoclast in ovariectomized rats.[11] Tocotrienols and δ-tocopherol were differentiation.[3] able to induce apoptosis in human mammary gland tumor Vitamin E, a lipid-soluble vitamin with antioxidant cells.[12] However, a few studies found that tocotrienol is properties has an important role in protecting biological superior compared to tocopherol. Alpha-tocotrienol was able systems. There are two types of vitamin E; tocopherol and to protect against glutamate-induced neuron cell death tocotrienol, and there are 4 isomers for each type: alpha (α), compared to α-tocopherol.[13] Palm vitamin E improved bone beta (β), gamma (γ) and delta (δ). Tocopherol has been shown calcium content in vitamin E– deficient rats compared to α- to prevent lipid peroxidation[4] and heart disease in tocopherol.[14] Indian J Pharmacol | October 2005 | Vol 37 | Issue 5 | 309-314 309 Norazlina et al. Vitamin E deficiency impaired calcification[15] and retarded Table 1 bone growth[16] in normal and ovariectomized rats. Its mechanism is unclear. One hypothesis is that vitamin E Rat Chow diet composition (Gold Coin, Malaysia) deficiency may influence the hormones regulating bone Composition Amount/Percentage metabolism (e.g., vitamin D metabolism). Other studies have Crude protein 21-23% shown that the presence of vitamin E is crucial in vitamin D Soybean meal metabolism and calcium absorption from the intestines in Skimmed milk powder which they found that vitamin E deficiency reduced calcium absorption from the intestines[17] and inhibited the conversion Crude fibre (max.) 5.0% of vitamin D to its active metabolites.[18] To further examine Crude fat (min.) 3.0% this hypothesis, this study was carried out, in which the effects Crude palm oil of a vitamin E–deficient diet on vitamin D metabolism and Moisture (max.) 3.0% bone calcium homeostasis were determined. The effects of Calcium 0.8-1.2% supplementation with three different types of vitamin E on Phosphorus 0.6-1.0% vitamin D metabolism were also studied. Nitrogen free extract (approx.) 49.0% Materials and Methods Additives per metric tonne of feed (approx.) Animals and treatment Vitamin A 10 M.I.U. Three-month old female Sprague-Dawley rats weighing 200 Vitamin D3 2.5 M.I.U. to 250 g, obtained from the University Breeding Centre, were Vitamin E 15 g used. This study was a continuation of our previous study which Vitamin K trace Vitamin B trace used female rats, and for this study the gender of the animals 12 was maintained. The rats were divided into 6 groups of 10 Thiamine trace each: (1) control group fed normal rat chow diet (RC), and Riboflavin trace treatment groups fed with (2) vitamin E–deficient diet (VED), Pantothenic acid trace (3) vitamin E–deficient diet supplemented with 60 mg/kg pure Niacin trace Pyridoxine trace α-tocopherol acetate (ATF), (4) vitamin E–deficient diet sup- Choline trace plemented with 60 mg/kg γ-tocotrienol (GTT) and (5) vitamin E–deficient diet supplemented with 60 mg/kg Tocomin® (TOC). Antioxidants per metric tonne of feed The respective diets were given ad libitum. Treatment was Santoquin 125 g carried out for 2 months. The sixth group, which acts as base- Microminerals trace line control (BC), was untreated and was killed at the begin- Iron ning of the study (day zero). This group acts as the control for Cobalt bone calcium content and femur length. The other parameters Manganese were measured in either the serum or urine. For control val- Iodine ues of these parameters, blood and urine were collected from Selenium each treatment group at day zero. Five rats were kept per cage under 12 h natural light/dark cycles and given distilled Serum was stored at –700C for the measurement of 1,25- water ad libitum. dihydroxyvitamin D3 and calcium. Diets At the end of the treatment period, the rats were Normal rat chow was obtained from Gold Coin, Port Klang, anesthetized and then sacrificed. Left femoral and 4th lumbar Selangor, Malaysia. Vitamin E–deficient diet and α-tocopherol bones were dissected out and cleansed of all soft tissues. acetate were purchased from ICN Biomedicals, Costa Mesa, Femur length was measured and the bones were then stored CA, USA. The composition of rat chow diet and vitamin E– at –700C for the measurement of bone calcium content. deficient diet are shown in Table 1 and 2, respectively. Gamma- Urine collection tocotrienol, which contains 82% γ-tocotrienol, and Tocomin® Urine samples were collected before the start of treatment were supplied by Carotech Sdn Bhd, Ipoh Perak, Malaysia. and at the end of the treatment period. For this purpose, rats Tocomin® contains 22.1% γ-tocotrienol, 6.8% δ-tocotrienol, were placed in individual metabolic cages beginning at 4 o’clock 10.7% α-tocotrienol, and 11.5% α-tocopherol. Vitamin E was in the afternoon until 8 o’clock the next morning. Urine excreted dissolved in olive oil, produced by Bertolli Classico, Italy. during that period was collected and centrifuged at 3000 rpm Alpha-tocopherol (60 mg/kg, body weight) was prepared for 10 min at 40C. Hydrochloric acid 6M was added to the by mixing 3 g of α-tocopherol in 47 g olive oil. The same supernatant obtained until the final pH of the urine was less procedure was applied in preparing γ-tocotrienol and Tocomin® than 2.0 to dissolve the calcium ions in the urine. (60 mg/kg, b.w.). 0.1 mL/100 g rat weight of the respective Serum 1,25-dihydroxyvitamin D3 level solutions was given by oral gavage 6 days a week for 2 months. Measurement was carried out using the ELISA kit from Bone and blood samples collection ImmunDiagnostik, Germany (catalog no. K2112). Color Blood samples were taken before the start of treatment intensity, which reflects 1,25-dihydroxyvitamin D3 and at the end of treatment period. The rats were anesthetized concentration, was determined by using an ELISA reader with diethyl ether and blood was taken via the orbital sinus. (Tecan Spectra Classic, Austria). 310 Indian J Pharmacol | October 2005 | Vol 37 | Issue 5 | 309-314 Vitamin E deficiency on vitamin D and calcium homeostasis Table 2 Serum and urine calcium levels Total serum and urine calcium levels were measured on Vitamin E–deficient diet composition (Draper et al) the basis of the complex produced because of the reaction Composition Fraction/Percentage between calcium and complexion o-cresol phthalein.
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