Endogenous formation of morphine in human cells

Morphine is a plant (opium poppy)-derived alkaloid and one of the strongest known analgesic compounds. Studies from several laboratories have suggested that animal and human tissue or fluids contain trace amounts of morphine. Its origin in mammals has been believed to be of dietary origin. Here, we address the question of whether morphine is of endogenous origin or derived from exogenous sources. Benzylisoquinoline alkaloids present in human neuroblastoma cells (SH-SY5Y) and human pancreas carcinoma cells (DAN-G) were identified by GC/tandem MS (MS/MS) as norlaudanosoline (DAN-G), reticuline (DAN-G and SH-SY5Y), and morphine (10 nM, SH-SY5Y). The stereochemistry of reticuline was determined to be 1-(S). Growth of the SH-SY5Y cell line in the presence of (18)O(2) led to the [(18)O]-labeled morphine that had the molecular weight 4 mass units higher than if grown in (16)O(2), indicating the presence of two atoms of (18)O per molecule of morphine. Growth of DAN-G cells in an (18)O(2) atmosphere yielded norlaudanosoline and (S)-reticuline, both labeled at only two of the four oxygen atoms. This result clearly demonstrates that all three alkaloids are of biosynthetic origin and suggests that norlaudanosoline and (S)-reticuline are endogenous precursors of morphine. Feeding of [ring-(13)C(6)]-tyramine, [1-(13)C, N-(13)CH(3)]-(S)-reticuline and [N-CD(3)]-thebaine to the neuroblastoma cells led each to the position-specific labeling of morphine, as established by GC/MS/MS. Without doubt, human cells can produce the alkaloid morphine. The studies presented here serve as a platform for the exploration of the function of "endogenous morphine" in the neurosciences and immunosciences.     

Substrate Specificity and Mechanism of the Intestinal Clonidine Uptake by Caco-2 Cells

Purpose This study was performed to characterize the substrate specificity and mechanism of the intestinal clonidine transport. Methods Uptake of [³H]clonidine into Caco-2 cells was investigated. Interaction with drugs was studied in competition assays. Results Uptake of [³H]clonidine was linear for up to 2 min, Na⁺-independent, and insensitive to changes in membrane potential, but strongly H⁺-dependent. The uptake rate of clonidine was saturable with kinetic parameters of 0.5 ± 0.1 mM (K_t) and 16.6 ± 1.8 nmol/2 min per mg of protein (V_max) at an outside pH of 7.5. Many drugs such as clonidine, guanabenz, methamphetamine, imipramine, clomipramine, nortriptyline, quinine, xylazine, ephedrine, and diphenhydramine strongly inhibited the [³H]clonidine uptake with K_i values between 0.15 and 1 mM. Conclusions Clonidine is transported by a carrier-mediated process. Substrate specificity and mechanism are very similar to the transport described in blood–brain barrier endothelial cells. The transport characteristics do not correspond to carriers for organic cations of the SLC22 family or the choline transporters CHT1 and CLT1. The system might be identical to the H⁺/tertiary amine antiporter. It interacts with a large number of both hydrophilic and lipophilic cationic drugs, and also, interestingly, with opiates.     

Lupin protein compared to casein lowers the LDL cholesterol:HDL cholesterol-ratio of hypercholesterolemic adults

Background  

Lupin protein had hypocholesterolemic effects in laboratory animals. However, the effect in humans has not been elucidated till now.     

The intestinal H+/peptide symporter PEPT1: structure-affinity relationships.

Peptide transporter 1, PEPT1, of the mammalian enterocyte is presently under intense investigation in many laboratories because of its nutritional importance in the absorption of protein hydrolysis products and because more recent studies have shown that many drugs and prodrugs gain entry into the systemic circulation via PEPT1. Until the exact structural features of the substrate binding site of PEPT1 become available, for example by X-ray crystallography, determination of affinities followed by proof of actual membrane translocation will have to suffice when testing for possible new substrates for PEPT1. Affinity constants reflect the strength of their interaction with the binding site of the transporter. A review of the literature shows a wide range of affinity constants between 2 microM and 30 mM. We consider affinity constants for substrates or inhibitors of PEPT1 lower than 0.5 mM as high affinity, between 0.5 and 5.0 mM as medium affinity and above 5 mM as low affinity. Values above 15 mM we consider with great caution. In this mini-review we discuss affinities and structural determinants which affect affinities of a variety of substrates for PEPT1.     

Supplementation of vitamins C and E increases the vitamin E status but does not prevent the formation of oxysterols in the liver of guinea pigs fed an oxidised fat

Summary Background Dietary oxidised fats are a source of oxidative stress. They cause deleterious effects in animal organism by lowering the antioxidant status of tissues and enhancement of the formation of lipid oxidation products. The vitamins E and C might be useful to prevent the formation of oxidation products by dietary oxidised fats. Aim of the study The purpose of this study was to investigate whether or not supplementation of diets with vitamins E and C is able to prevent oxidative stress and the formation of lipid oxidation products caused by dietary oxidised fats. Among lipid oxidation products, oxysterols should be particularly considered because of their high pathophysiological effects. Methods Male guinea pigs were divided into five groups. Four groups were fed diets with an oxidised fat supplemented with 35 or 175 mg -tocopherol equivalents/kg and 300 or 1000 mg of vitamin C/kg for 29 days. One group, used as a control, was fed the same basal diet with fresh fat with 35 mg -tocopherol equivalents/kg and 300 mg of vitamin C/kg. Results The guinea pigs fed the oxidised fat diet with 35 mg -tocopherol equivalents/kg and 300 mg vitamin C/kg had significantly lower concentrations of tocopherols in various tissues, higher concentrations of various oxysterols and thiobarbituric acid-reactive substances in the liver, higher concentrations of glutathione in the liver and lower concentrations of glutathione in erythrocytes than the control animals fed the fresh fat. Increasing the dietary vitamin E concentration from 35 to 175 mg -tocopherol equivalents/kg and/or the dietary vitamin C concentration from 300 to 1000 mg/kg increased tissue tocopherol concentrations in guinea pigs fed the oxidised fat but did not influence concentrations of oxidation products in the liver and glutathione concentrations in liver and erythrocytes. Conclusion The results demonstrated that supplementation of vitamins E and C improves the vitamin E status but does not prevent the formation of lipid oxidation products in the liver of guinea pigs fed oxidised fats.     

Functional characterization of a high-affinity choline transport system in human keratinocytes

This study was performed to characterize the mechanism of choline transport into human keratinocytes. Uptake of [3H]choline was measured both in the HaCaT cell line and in native keratinocytes. Uptake in HaCaT cells was linear with time at least up to 10 min. There was little dependence of choline transport on sodium. Choline uptake was slightly stimulated by extracellular H+ with the pH optimum being 7.5. The uptake rate was saturable and indicated participation of a single transport system (Kt = 14.8 +/- 1.0 micro M, Vmax = 1.0 +/- 0.01 nmol per 10 min per mg protein). The choline uptake into HaCaT cells was inhibited by unlabeled choline, hemicholinium-3, and acetylcholine. The prototypical organic cation tetraethylammonium showed very little affinity for the choline uptake system in these cells. Several cationic drugs such as diphenhydramine, clonidine, and atropine also interacted with the transport system. Choline uptake in normal keratinocytes was very similar to that in HaCaT cells with respect to substrate specificity and affinity. We conclude that keratinocytes express a Na+ independent, high-affinity choline transport system. This system accepts many pharmacologically important organic cations as substrates. It is similar or identical to the choline carrier described in intestinal epithelial cells and in endothelial cells of the blood-brain barrier. The choline carrier seems to have relevance not only for the uptake of cationic drugs into the keratinocytes but also for the biosynthesis of skin lipids.     

Excess dietary vitamin E lowers the activities of antioxidative enzymes in erythrocytes of rats fed salmon oil

In vitro studies suggest that high vitamin E supplementation has prooxidative activity, but very few studies have investigated this effect in vivo. We investigated the effect of excess vitamin E on the antioxidative status of rat erythrocytes and indicators of hemolysis. Six groups of growing male Sprague-Dawley rats were fed purified diets with three different vitamin E doses [100, 1000 and 10,000 mg all-rac-alpha-tocopheryl acetate (TA)/kg diet] and two different dietary fats (salmon oil and lard) for 8 wk. The rats whose diet contained salmon oil and 10,000 mg TA/kg had lower activities of superoxide dismutase (P < 0.05), glutathione peroxidase (P < 0.05), catalase (P < 0.05) and glucose-6-phosphate dehydrogenase (P < 0.05) and a lower concentration of glutathione (P < 0.05) in the erythrocyte cytosol than rats whose diet contained 100 mg TA/kg. The concentration of free hemoglobin and the binding capacity of haptoglobin in plasma, both indicators of in vivo hemolysis, did not differ between rats fed the salmon oil diet with 100 or 10,000 mg TA/kg. In the rats whose diet contained lard, the activities of antioxidant enzymes in erythrocytes and indicators of in vivo hemolysis were independent of the dietary vitamin E concentration. The results of the study suggest that an excessive vitamin E intake, when combined with salmon oil in the diet, lowers the activities of antioxidant enzymes in erythrocytes without affecting in vivo hemolysis.