In vivo formation of 25-hydroxycholesterol from endogenous cholesterol after a single meal, dietary cholesterol challenge.

The role of oxysterols as regulatory molecules in the suppression of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase activity was investigated in the intact rat in response to an acute dietary cholesterol challenge. When rats were fed highly purified cholesterol as a single meal at a level of 5% of the diet, maximal inhibition of enzyme activity (66%) occurred 120 min after the completion of the meal. Furthermore, when nonsaponifiable liver extracts were chromatographically resolved and analyzed by high performance liquid chromatography (HPLC) and capillary gas chromatography-mass spectrometry (GC-MS), 25-hydroxycholesterol was identified in the livers of rats 120 min after the completion of the single cholesterol meal. Significantly, only barely detectable amounts of 25-hydroxycholesterol were observed in the livers from control rats fed a sterol-free diet. The biosynthetic origin of 25-hydroxycholesterol was investigated with the use of deuterated water. Rats were fed deuterium oxide (33%) ad libitum for 3 days and then killed 120 min after the completion of a single cholesterol meal. As before, 25-hydroxycholesterol was detected in the livers from cholesterol-fed rats, but not to a significant extent in livers from control-fed rats receiving a sterol-free diet. Isotope ratio mass spectrometry revealed that the fractional incorporation of deuterium into 25-hydroxycholesterol (21%) was less than that observed for cholesterol (24%) isolated from the same livers, indicating that 25-hydroxycholesterol was produced endogenously from exogenous cholesterol and not from autoxidation of cholesterol. In a separate experiment it was also shown that [3H]mevalonate was incorporated into 25-hydroxycholesterol after a single meal cholesterol challenge, but was barely detected in the livers of control rats. The evidence obtained in the present article supports the hypothesis that 25-hydroxycholesterol is endogenously produced from cholesterol at early time intervals after an acute dietary cholesterol challenge. In addition, rat liver HMG-CoA reductase was inhibited by the administration of a single intragastric dose (1 microgram/kg) of an aqueous solution of 25-hydroxycholesterol. Thus, the results provide strong support for the conclusion that 25-hydroxycholesterol plays a significant role in the in vivo regulation of rat liver cholesterol biosynthesis after an acute dietary cholesterol challenge.

Vitalethine modulates erythropoiesis and neoplasia.

Novel compounds based upon the thiol N-(carboxy)-beta-alanyl-cysteamine (vitaletheine) have strikingly potent and seemingly diverse biological activities. Concentrations of vitaletheine modulators from 1 femtograms/ml to 100 picograms/ml medium regulate RBC production from progenitors initially deprived of erythropoietin. Similarly, as little as attograms/ml concentrations of the disulfide vitalethine stimulate immunological responses of murine splenocytes toward sheep RBC in a hemolytic plaque assay. Because dosages of vitalethine as low as femtograms/kg substantially diminish tumor size and incidence and increase survival to 80% in mice inoculated with a uniformly fatal melanoma (Cloudman S-91), activities of these compounds have in vivo significance. A preliminary probe of the benzyl derivative of vitalethine in a myeloma model (NS-1) suggests efficacy (100% survival) as well. The high potencies of the vitaletheine modulators, both in cell culture and in vivo, indicate that these or similar regulatory components, if constitutively present, probably occur endogenously at vanishingly small concentrations and may be prone to deficiency resulting from metabolic imbalances, irradiation, aging, diet, pathogenic or parasitic infections, or exposure to environmental pollutants. Pathways for the biosynthesis of vitaletheine are proposed and chemical syntheses of the vitaletheine modulators are described. Possible molecular mechanisms of action, including interactions with peptidyl hormones, other endogenous effectors, and xenobiotic and pharmaceutical compounds, are explored. Indications for the treatment of other diseases are identified.

Seemingly diverse activities of beta-alethine.

beta-Alethine (beta-alanyl-cysteamine disulfide) exhibits striking biological activities in diverse systems. At an optimum of about 10 ng/ml, beta-alethine (a) adapts murine liver cells to culture (53 colonies/10(6) cells versus none in controls), (b) delays aging of human IMR-90 fetal lung fibroblasts (102 population doubling levels versus 47 in controls, producing 3 x 10(16) greater biomass), and (c) markedly stimulates antibody-producing plaque-forming cells from murine splenocytes (16,875/10(6) cells versus 55/10(6) cells in controls) or human peripheral blood leukocytes (1826/10(6) cells versus 0/10(6) cells in controls). Early interventions with beta-alethine (1 ng/kg to 100 micrograms/kg) successfully treat NS-1 myeloma in a syngeneic murine tumor model (NS-1 myeloma). Although there are indications in this model that beta-alethine is also effective when intervention is late, beta-alethine is ineffective in an allogeneic murine melanoma model (Cloudman S-91 melanoma). It is inferred that beta-alethine enhances cellular phenotypic expression, function, and vitality in diverse biological systems and may treat certain types of neoplasia. Because atomic spacings between the amide moieties in beta-alethine are the same as in the differentiating agent hexamethylene-bis-acetamide and because the radioprotectors WR 2721 and WR 1065 lack only the carbonyl oxygen of the thiol form (beta-aletheine), biological activities already reported for these compounds are compared with those presented herein for beta-alethine. Although these comparisons have not been made in the same systems, the tentative conclusion is that the amide moieties of beta-alethine may be critical to its potency and lack of obvious toxicity in cell culture and animal models.