利用同位素示踪技术对β-胡萝卜素在大鼠体内分布的研究
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摘要
首先，利用螺旋藻能够合成β-胡萝卜素的特性建立了~3H-β-胡萝卜素(~3H-β-Carotene，~3H-βc)的合成方法。实验分为两步，首先在普通Zarrouk培养基中培养极大螺旋藻，获得一定数量的藻体；第二步，将收集的藻体转入用氚水配制的放射性活度为6mCi／L的Zarrouk培养基中，培养2周后收获螺旋藻，并从中提取~3H标记βc。用高效液相色谱、可见光分光光度计和液体闪烁计数器测定了所得~3H-βc的纯度大于95％、放射性活度为2.4×10~(-4)Ci／mol。本方法为利用同位素技术研究类胡萝卜素在动物体内吸收、分布提供了前提条件。
采用高效液相色谱分析技术，用摇瓶法测定了β-胡萝卜素的正辛醇／水分配(n-octanol-water partition coefficient，K_(ow))系数。β-胡萝卜素的LgK_(ow)为3.98±0.27。在定量描述β-胡萝卜素亲脂性的基础上，根据文献报道的回归方程对β胡萝卜素的水中溶解度(Solubility，S_w)进行了估算，β-胡萝卜素的LgS_w为-4.406±0.39。表明βc是一种脂溶性强的营养素，易于透过生物膜的亲脂屏障。
通过对大鼠腹腔注射~3H-β-胡萝卜素，研究βc在大鼠各组织中的沉积情况。结果表明：放射性46.54％沉积于肝脏，21.90％沉积于脾脏；此外，心脏、肾脏、肺组织、脑组织、胸腺和肾上腺中也能检测到少量放射性。肌肉和脂肪组织中未检测到放射性沉积。同时发现，大鼠各组织中沉积放射性总量的比例与各组织中粗脂肪含量之间存在着较强的负相关，相关系数为-0.9170。由此可以推测：动物脂肪对βc沉积能力强弱的不同造成了不同动物间脂肪组织中βc含量的不同。结合βc的物理化学性质分析，βc在动物细胞中可能是以与遗传物质相结合的形式存在的。
This article aims to study distribution of beta-carotene in rats' tissues by isotopic trace technique.
In the first experiment, the biological synthetic method of 3H-β -carotene (3H- βc) had been established. The Spirulina maxima was grown in Zarrouk's medium using initially H2O and subsequently 3H2O enriched water (specific activity 6mCi/L). After suspension cultured in normal Zarrouk culture medium for producing maximum biomass and P -carotene ( P c), Spirulina was kept in radioactive culture medium 2 weeks in which some H atoms of the Spirulina was replaced by 3H atoms of the culture. Then, tritium labeling P c could be extracted from the Spirulina by chromatogram. The radioactive culture medium could be used in several cycles for metabolic labeling of carotenoids whereby the cost of production was reduced. At last, purity of 3H-β c was about to 95% and specific activity was 2.4 10 4Ci/mol.
In the second experiment, n-octanol/water partition coefficient (Kow)of β c was determined for describing lipophilicity quantitatively with shake-flask method and HPLC. In addition, water solubility of 0 c was estimated by regression equation. The result showed the 1gKow was 3.98 0.27 and the lgSw was -4.406 0.39.
In the third experiment, distribution of 3H-βc in rats was determined. After injecting 3H- βc into their abdominal cavity for 84h, the liver and the spleen accounted for 46.54% and 21.90%, respectively, of the total recovered 3H radioactivity in rats. In addition, a little radioactivity also can be detected in heart, kidney, brain, chest gland and adrenal. No radioactivity was found in muscle and fat. At the same time, negative correlation was discovered between distribution of radioactivity and fat concentration in rats' tissues. The correlation coefficient is -
0.9170. The results suggest that, in different animals, the amount of P c lies on the deposit ability of β c in the fat and P c is likely to bound with germ plasm in animal cell according to nature of physical chemistry.
采用高效液相色谱分析技术，用摇瓶法测定了β-胡萝卜素的正辛醇／水分配(n-octanol-water partition coefficient，K_(ow))系数。β-胡萝卜素的LgK_(ow)为3.98±0.27。在定量描述β-胡萝卜素亲脂性的基础上，根据文献报道的回归方程对β胡萝卜素的水中溶解度(Solubility，S_w)进行了估算，β-胡萝卜素的LgS_w为-4.406±0.39。表明βc是一种脂溶性强的营养素，易于透过生物膜的亲脂屏障。
通过对大鼠腹腔注射~3H-β-胡萝卜素，研究βc在大鼠各组织中的沉积情况。结果表明：放射性46.54％沉积于肝脏，21.90％沉积于脾脏；此外，心脏、肾脏、肺组织、脑组织、胸腺和肾上腺中也能检测到少量放射性。肌肉和脂肪组织中未检测到放射性沉积。同时发现，大鼠各组织中沉积放射性总量的比例与各组织中粗脂肪含量之间存在着较强的负相关，相关系数为-0.9170。由此可以推测：动物脂肪对βc沉积能力强弱的不同造成了不同动物间脂肪组织中βc含量的不同。结合βc的物理化学性质分析，βc在动物细胞中可能是以与遗传物质相结合的形式存在的。
This article aims to study distribution of beta-carotene in rats' tissues by isotopic trace technique.
In the first experiment, the biological synthetic method of 3H-β -carotene (3H- βc) had been established. The Spirulina maxima was grown in Zarrouk's medium using initially H2O and subsequently 3H2O enriched water (specific activity 6mCi/L). After suspension cultured in normal Zarrouk culture medium for producing maximum biomass and P -carotene ( P c), Spirulina was kept in radioactive culture medium 2 weeks in which some H atoms of the Spirulina was replaced by 3H atoms of the culture. Then, tritium labeling P c could be extracted from the Spirulina by chromatogram. The radioactive culture medium could be used in several cycles for metabolic labeling of carotenoids whereby the cost of production was reduced. At last, purity of 3H-β c was about to 95% and specific activity was 2.4 10 4Ci/mol.
In the second experiment, n-octanol/water partition coefficient (Kow)of β c was determined for describing lipophilicity quantitatively with shake-flask method and HPLC. In addition, water solubility of 0 c was estimated by regression equation. The result showed the 1gKow was 3.98 0.27 and the lgSw was -4.406 0.39.
In the third experiment, distribution of 3H-βc in rats was determined. After injecting 3H- βc into their abdominal cavity for 84h, the liver and the spleen accounted for 46.54% and 21.90%, respectively, of the total recovered 3H radioactivity in rats. In addition, a little radioactivity also can be detected in heart, kidney, brain, chest gland and adrenal. No radioactivity was found in muscle and fat. At the same time, negative correlation was discovered between distribution of radioactivity and fat concentration in rats' tissues. The correlation coefficient is -
0.9170. The results suggest that, in different animals, the amount of P c lies on the deposit ability of β c in the fat and P c is likely to bound with germ plasm in animal cell according to nature of physical chemistry.
引文
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