Abstract
A high performance liquid chromatography with spectrofluorometer assay was applied for the determination of α-, β-, γ- and δ-tocopherols in serum, bile and intravenous fat emulsions.
The tocopherols were extracted from 0.5ml of serum or bile with 5ml of n-hexane after precipitation of proteins with ethanol containing tocol as an internal standard. Concerning fat emulsions, extractions were repeated three times from 0.2ml of emulsions. After centrifugation (3,000 r. p.m., 5min.) 4ml of the supernatant fluid was evaporated at 40° under nitrogen stream. The residue was dissolved in 50μl of n-hexane and 20μl of this was injected into the HPLC column (Nucleosil-5NH2, 4mmφ×250mm). Isopropylether-hexane mixture was applied as mobile phase and the tocopherols were determined separately by using a spectrofluorometer (Ex. 298nm, Em. 325nm). The procedure was standardized by chromatographing standard mixtures containing known mass ratios of tocopherols and tocol. Linear relationships between peak-height ratios and mass ratios were recognized. Within-run precisions of 1.9 to 21% were obtained analyzing 10 standard samples. Analytical recoveries of tocopherols for fortified serum and bile were from 94 to 100% and for fortified fat emulsion were from 103 to 117%.
The levels of total tocopherols in normal human sera ranged from 0.78 to 1.22mg/dl; a-toco-pherol from 0.67 to 1.19mg/dl; β-tocopherol from 0.01 to 0.02mg/dl; γ-tocopherol from 0.02 to 0.13mg/dl, and δ-tocopherol was less than 0.01mg/dl. α-Tocopherol occupied more than 87% of the total tocopherol.
Serum levels of tocopherols in various diseases were examined. Pancreatic diseases and congenital choledochal cyst showed lower levels of a-tocopherol.
One to four days after operation, all the patients who didn't get blood or plasma transfusions showed lower serum levels of total and a-tocopherol than the preoperative levels (mean level was 80% of the preoperative value). They recovered within several days by sufficient food intake.
The levels of α- or total tocopherol had positive correlation with cholesterol and β-lipoprotein. Negative correlation with lipid peroxide was not recognized in this series.
Fat emulsions for intravenous injection contained from 20 to 30mg/500ml of total tocopherols and about 60% was γ-tocopherol and 30 % was δ-tocopherol. They may be derived from soybean oil which consists main compartment of the fat emulsion.
When fat emulsions were injected intravenously in the patient with T-tube drainage, the serum and biliary levels of γ- and δ-tocopherol markedly increased.
