The outline of the special lecture presented by the author at the 20th Annual Meeting of the Japan Vitamin Society, which consists of the studies on the natural occurence, physicochemical properties, metabolic functions and action mechanism of vitamin B_<12> coenzyme and its related compounds carried out by the author and his coworkers.
When weanling rats were fed for 7 days on a mild choline-deficient diet, their kidney increased in size and weight and congested. The concentration of both cephalin and lecithin has decreased in kidney of deficient animals while in liver, in contrast to lecithin which has decreased in deficient animals, cephalin has increased significantly in comparison with that in control animals. When the feeding was continued for additional one month they were recovered from deficient symptoms and the difference of phospholipids pattern between the deficient and normal rats have disappeared. On a more severe deficient diet, rats were suffered from a severe hemorrhagic degeneration in kidney even only after 5 days of feeding. Many of them lost their weight and were moribund. The renal changes were characterized grossly by an increase in size, weight and hemorrhagic discoloration. In this case cephalin and lecithin have a lower concentration in both kidney and liver than normal animals. When the red cells were tested for osmotic fragility using hypotonic saline solution, it was found that those of deficient animals were more fragile and were hemolyzed more easily than those of control animals.
The effects of vitamin B_6 on blood coagulation and fibrinolytic activity were investigated. In vivo, PAL-P prolonged whole blood clotting time and plasma recalcification time and activated fibrinolysis, while PAM-P or PIN showed no such effects, except that PAM-P slightly prolonged whole blood clotting time. Also in vitro, PAL-P prolonged plasma recalcification time and had the antithrombin activity, inhibiting the thrombin fibrinogen reaction, while PAL, PAM-P, PAM, PIN-P and PIN did not show such effects. Cocarboxylase and ATP did not prolong plasma recalcification time in vitro. PAL-P inhibited the hydrolysis of p-toluene sulfonylarginine methyl ester by thrombin and increased plasma antithrombin activity. Using thromboelastogram of citrated plasma, it was observed that PAL-P in low concentration increased the effect of ε-aminocaproic acid on the fibrinolytic system produced by streptokinase. These effects of PAL-P do not seem physiological ones of vitamin B_6,but pharmacological.
It was found that the percentage recovery of O, S-bis (ethoxycarbonyl) thiamine (DCET) was decreased by the presence of ascorbic acid (AsA) when DCET was determined by the thiochrome method after preliminary treatment with alkali. This alkali-method was also interfered by hydroquinone and other compounds susceptible to oxidation. An explanation is proposed for the interference by AsA that DCET is converted by the alkali-treatment into thiochrome reaction-positive compounds, which are successively oxidized to thiamine disulfide (TDS) by the presence of AsA. This was supported by the fact that TDS was detected in the reaction mixture when DCET was treated with alkali in the presence of AsA. DCET can be determined without any interference when cysteine is preliminarily added before the alkali treatment to the sample solution containing DCET and AsA. Other methods for the determination of DCET in the presence of AsA are discussed.
Experiments were made on the stabilities of thiamine (B_1) and six B_1-derivatives in ascorbic acid (AsA) solution (50 mg/ml) at pH 4.0,45 and 100℃ in comparison with those in acetic acid solution of the equimolar concentration to the AsA solution at the same pH. Stabilities of B_1 and O-ethoxycarbonylthiamine (OCET) in the AsA solution were similar to those in the acetate buffer. S-Ethoxycarbonylthiamine (CET) was converted into OCET and B_1 both in the acetate buffer and in the AsA solution, and the formation of B_1 was accelerated by AsA. Main product from O, S-bis (ethoxycarbonyl) thiamine (DCET) in the AsA solution was OCET. These findings suggest that CET and DCET are decomposed by AsA mainly through the cleavage of S-ethoxycarbonyl ester linkage. This was supported by the easy formation of O-butoxycarbonylthiamine from S-ethoxycarbonyl-O-butoxycarbonyl-thiamine in the presence of AsA. Thiamine disulfide (TDS) and thiamine propyldisulfide (TPD) were converted to B_1 by AsA, where TDS was less stable than TPD.
Several derivatives of 2-(3-hydroxy-5-hydroxymethyl-2-methylpyridyl-4)-tetrahydro-1,3-thiazine-4-carboxylic acid (PAL-HCySH) and 2-(3-hydroxy-2-methyl-5-phosphoryloxymethyl-pyridyl-4)-tetrahydro-1,3-thiazine-4-carboxylic acid (PAL-P-HCySH), which have potential vitamin B_6 activity, were synthesized. Alkylesters of PAL-HCySH, and PAL-P-HCySH were prepared in 12-70% yield by the reactions of pyridoxal or pyridoxal phosphate with alkyl DL-homocysteinates which were obtained from the ring-cleavage of DL-homocysteine thiolactone-HCl with sodium alcoxides in the corresponding alcohols. The acetylations of methyl 2-(3-hydroxy-5-hydroxymethyl-2-methylpyridyl-4)-tetrahydro-1,3-thiazine-4-carboxylate (PAL-HCySOMe) with acetic anhydride were also investigated. The alcoholic O-acylates (acetate, benzoate, butyrate, and nicotinate) of PAL-HCySH were prepared by the condensation of pyridoxal-5-acylates with DL-homocysteine in high yield. Further, the spectrophotometric studies of the stabilities and dissociations of PAL-HCySOMe, 2-(5-acetoxymethyl-3-hydroxy-2-methylpyridyl-4)-tetrahydro-1,3-thiazine-4-carboxylic acid (PAL-5-Ac-HCySH) and methyl 2-(5-acetoxymethyl-3-hydroxy-2-methylpyridyl-4)-tetrahydro-1,3-thiazine-4-carboxylate (PAL-5-Ac-HCySOMe) were carried out to compare with those of PAL-HCySH.
A microautoradiographic detection of the injected CoQ_7 in animals has been performed. Two to five mμc/g body weight of ^<14>C-CoQ_7 was administered intraperitoneally in a rat and six mice. The animals were sacrificed at the 24th hour after injection and all organs and tissues were excised and embedded in paraffin as usual. Sliced tissue sections were covered with Sakura NR-M2 nuclear track emulsion and after 4 weeks' exposure they were developed in D-19. Autoradiographic grains were observed mostly in the intestinal contents, and much in the liver parenchymal cells, intestinal epithelium, various exocrine organs (salivary gland and pancreas) and fat tissues. After considering those results, it may be given as a conclusion that the possible route of the absorbed CoQ_7 from the peritoneal cavity will be as follows : from circulation it first goes into the cytoplasma of the liver parenchymal cells, is secreted out with bile and most of them will be discharged with feces. However, some of them will be reabsorbed through intestinal walls into the portal system. A part of the CoQ_7 in the systemic circulations will also be secreted from many exocrine glands such as pancreas and salivary glands. It also has an affinity to bind with fat substances. There is no preferential localization in the endocrine systems such as pancreatic islets.