Possible origin of extremely high contents of vitamin D3 in some kinds of fish liver

• 1.1. Comparative studies on the possible origin of extremely high contents of vitamin D₃ in some kinds of fish liver were performed.
• 2.2. Neither photochemical formation of vitamin D₃ in fish skin by solar radiation of 7-dehydrocholesterol (7-DHC) nor nonphotochemical enzymatic formation of vitamin D₃ from 7-DHC in fish liver was demonstrated as the origin of vitamin D₃.
• 3.3. On the other hand, when bastard halibuts and carps were farmed from fingerlings to adults with feedstuff's containing vitamin D₂ or D₃, significant amounts of the vitamins were accumulated in the fish liver.
• 4.4. The contents of vitamins D₂ and D₃ in bastard halibut liver increased according to the duration of farming and dose responses of the vitamins in carp liver were observed.
• 5.5. Significant amounts of vitamins D₂ and D₃ in phytoplankton and vitamin D₃ in Zooplankton and small fish were detected.
• 6.6. Therefore, we have concluded that the most probable origin of vitamin D₃ in fish liver is a result of food chains from plankton.

     

Dose-dependent vitamin D3 and 1,25-dihydroxyvitamin D3-induced hypercalcemia and hyperphosphatemia in male cyprinoid Cyprinus carpio

• 1.1. Vitamin D₃ (10IU and 100IU/100 g body weight) and 1,25(OH)₂D₃ (0.5, 5 and 50 U) were administered daily to unfed male carp Cyprinus carpio for 10 days. The serum calcium and inorganic phosphate levels were measured colorimetrically.
• 2.2. The serum calcium level increased significantly in all treated groups; this increase is dose-dependent.
• 3.3. The serum inorganic phosphate was elevated in the treated groups on days 3 and 5.

     

Dietary supplementation of vitamin E fails to prevent the development of hyperoxic lung injury in the premature guinea pig

• 1.1. The benefit of dietary vitamin E supplementation in preventing oxidative-induced lung injury was investigated. Three day preterm guinea pig pups were exposed to hyperoxic (85% O₂) or normoxic (21% O₂) conditions. The animals were fed either a standard low birthweight human infant formula milk (6.4 mg/l vitamin E), or a vitamin E supplemented milk (100 mg/l) for up to 7 days.
• 2.2. After 3 days vitamin E supplementation, plasma but not erythrocyte vitamin E concentrations were elevated, while following 7 days both plasma and erythrocyte vitamin E concentrations were significantly increased.
• 3.3. Lung and liver vitamin E concentrations were elevated at both 3 and 7 days. At 3 days the increase in lung vitamin E was oxygen-dependent, suggesting that the lung increases uptake of vitamin E in response to oxidative stress.
• 4.4. Despite an increase in the vitamin E concentration of the lungs of preterm guinea pigs, no amelioration of the lung injury was observed. These results suggest that although vitamin E is a potent antioxidant, it is unable to protect adequately the lungs from reactive oxygen species in the absence of sufficient primary enzymatic antioxidant defences.

     

In vitro metabolism of vitamin D3 by isolated liver cells

Summary Liver cells were prepared from rats fed a rachitogenic diet to investigate the hepatic metabolism of [ — 1,2 —³H₂] vitamin D₃. Rat hepatocytes suspended in Hanks medium rapidly took up labeled vitamin D₃ from the incubation medium and converted this sterol to various metabolites, including 25-hydroxy vitamin D₃ (25-OH-D₃). There was a steady increment in the cellular production of 25-OH-D₃ and of the more polar metabolites of vitamin D₃ over 3 hr of incubation as determined by thin layer chromatography. Neither the addition of cyclic nucleotides or dexamethasone to, nor the removal of calcium or phosphate from the medium resulted in changes in the rate of conversion of vitamin D₃ to its products. Rats pretreated with sodium diphenylhydantoin converted labeled vitamin D₃ to its metabolites at the same rate as control rats. These data indicate that isolated liver cells retain the capacity for vitamin D₃ hydroxylation, but suggest that the rate of this process does not undergo rapid changes in response to metabolic stimulation.Recipient of Research Career Development Award 1 K04 HL-00089.     

Action of 1,25-dihydroxyvitamin d3 on calcium metabolism in the crustacean Orchestia cavimana during the molt cycle

• 1.1. The possible involvement of vitamin D₃ in the calcium metabolism of the terrestrial crustacean Orchestia during the molt cycle was further investigated by measuring the effects of administration of exogenous 1,25 (OH)₂D₃ on three parameters of the calcium balance in two different compartments of the body.
• 2.2. At the hemolymph level, a strong hypocalcémie effect was observed in intermolt and early premolt. Within the posterior caeca of the midgut, stimulation of calcium storage and calcium release were noticed during a short period surrounding the time of exuviation, with concomitant variations of the epithelial carbonic anhydrase activity.
• 3.3. These results, together with other data, are discussed to determine the possible functions of vitamin D₃, or related molecules, in the calcium turnover within the different compartments of the body, according to the successive stages of the molt cycle.

     

Bioconversion pathway of astaxanthin into retinol2 in mature rainbow trout (Salmo Gairdneri Rich.)

• 1.1. Carotenoid and retinoid forms were analysed by HPLC in various tissues of mature rainbow trout fed for 11 months (7–9°C) with astaxanthin (50 and 100 mg/kg diet) or canthaxanthin (100 mg/kg diet).
• 2.2. Decreasing concentrations of canthaxanthin, echinenone and β-carotene, but no retinol₁, were found in the liver and skin of canthaxanthin-fed fish.
• 3.3. Higher retinol₂ concentrations were found in ovaries and testes of astaxanthin-fed fish compared to canthaxanthin and control groups.
• 4.4. A new metabolic pathway for direct conversion of astaxanthin into retinol₂ in gonads is proposed.

     

Regulation of fish glycolysis—gluconeogenesis: role of fructose 2,6 P2 and PFK-2

• 1.1. Fructose 2,6 P₂ and PFK-2 have a key role in the regulation of glycolysis-gluconeogenesis in fish
• 2.2. PFK-1 and FBPase-1, as in mammals, are the target enzymes for fructose 2,6 P₂, this in turn may be controlled by glucagon and insulin.
• 3.3. PFK-2 from fish liver seems to be a bifunctional enzyme regulated by phosphorylation/dephosphorylation.
• 4.4. Starvation, refeeding, diet composition and anoxia studies provide a general view of the fructose 2,6 P₂ fish system from which the differences between fish and mammal glycolysis-gluconeogenesis may be ascertained.

     

Changes of the fatty acid composition in smolts of masu salmon (Oncorhynchus masou), associated with desmoltification and sea-water transfer

• 1.1. Tissue lipid compositions of desmoltified yearlings of masu salmon (Oncorhynchus masou) obtained by keeping smoltified fish in fresh water, were examined and compared to those of smoltified fish before and after transfer to sea-water (SW).
• 2.2. Lipid contents of muscle, liver, gut and gills of desmolts tended to increase compared to those of initial smolts.
• 3.3. The increased proportion of triacylglycerol (TG) and decreased proportion of phospholipids (PL) characterized the tissue lipids of desmolts.
• 4.4. Liver and muscle lipids showed no distinct differences both in content and proportion between initial and SW smolts, but gut and gill lipids of SW smolts decreased in content accompanied by a decrease of TG and an increase of PL in proportion.
• 5.5. Excepting muscle non-polar lipids, tissue lipids of desmolts contained more mono-unsaturated fatty acids and saturated fatty acids and less polyunsaturated fatty acids (PUFA), especially (n-3) PUFA such as 22:6(n-3), than those of initial and SW smolts.
• 6.6. No large differences in fatty acid composition were seen between initial and SW smolts except for the gut.
• 7.7. The proportion of (n-3) PUFA in the gut of SW smolts was higher than that of initial smolts.
• 8.8. The results indicated that masu salmon smolts can modify their lipid metabolism to adapt to ambient salinity changes. The proportion of (n-3) PUFA particularly in polar lipids, or in osmoregulatory organs such as gut and gills, was seen to be critical in lipid types of freshwater- or sea-water-adapted fish.

     

The effect of d2o on glycolysis by rat hepatocytes

• 1.1. The effect of incorporating D₂O into the incubation medium on glycolysis and gluconeogenesis by hepatocytes from fasted rats was examined.
• 2.2. The substitution by heavy water, D₂O, at concentrations from 10 to 40%, stimulated glucose uptake, lactate production and CO₂ yields from glucose. At 10 mM glucose, 40% D₂O doubled glucose uptake, increased CO₂ production by 40%, and increased lactate production by 350%.
• 3.3. The stimulation of lactate production decreased at higher glucose concentrations, but was still substantial even at 80 mM glucose.
• 4.4. There was no effect on CO₂ production above glucose concentrations of 30 mM.
• 5.5. Ten percent D₂O showed little inhibition of lactate uptake, its oxidation and gluconeogenesis. At 40% D₂O the inhibition ranged from 10 to 20%.
• 6.6. No effect of D₂O on the rate of glucokinase or glucose-6-phosphatase was observed.
• 7.7. The concentration of fructose, 2,6-P was not affected by D₂O

     

Identification and characterization of a third form (D3) of cyclic 3'5'-nucleotide phosphodiesterase from rhizobwm fredii

• 1.1. A third form (D3) of cyclic nucleotide phosphodiesterase from Rhizobiumfrediiv/as detected and characterized for the first time.
• 2.2. The enzyme could hydrolyse both cyclic AMP and cyclic GMP with apparent K_m for cyclic AMP of approx. 0.2 μM.
• 3.3. D3 cyclic nucleotide phosphodiesterase had a pH optimum of about 6.0 when hydrolysing cyclic AMP.
• 4.4. The enzyme lost almost all its activity when heated to 60°C for 20 min.
• 5.5. Gel filtration with Sephadex G-100 gave a mol. wt of approx. 42.5 kD for the native enzyme.

     

Ostrich fructose-1,6-bisphosphatase: Kinetic characterization of the liver isoenzyme

• 1.1. Purified ostrich (Struthio camelus) liver fructose-1,6-bisphosphatase exhibited an absolute requirement for Mg²⁺.
• 2.2. The enzyme catalyzed the hydrolysis of fructose-1,6-bisphosphate, sedoheptulose-l,7-bisphosphate and ribulose-l,5-bisphosphate.
• 3.3. S_0.5 for substrate was 1.4 μM.
• 4.4. AMP was a potent non-competitive inhibitor with respect to substrate (K_i of 25 μM).
• 5.5. Fructose-2,6-bisphosphate was a potent competitive inhibitor of the enzyme (K_i of 4.8 μM).

     

Effect of triton X-100 treatment on the lipid composition,biochemical and functional properties of hake (Merluccius hubbsi) myofibrils purified from pre- and post-spawned fish

• 1.1. The effects of Triton X-100 treatment on the lipid contents and functional properties of hake myofibrils from pre- and post-spawned fish were investigated.
• 2.2. Differences in lipids, biochemical and functional properties of hake myofibrils related to the gonadal condition of fish were observed.
• 3.3. Triton X-100 treatment removed 65% of polar lipids in myofibrils from pre-spawned fish and only 10% in myofibrils from post-spawned fish.
• 4.4. Triton X-100 increased the Hill coefficient to 1.5 in an allosteric type of reaction for the myofibrillar Mg²⁺-ATPase from pre-spawned hake.
• 5.5. The detergent effect observed on the contraction response was greater in myofibrils from prespawned fish than in post-spawned fish.

     

Mixed-function oxygenase in juvenile rainbow trout exposed to hexachlorobenzene or 3, 3′, 4, 4′-tetrachlorobiphenyl

• 1.1. 3,3',4,4'-tetrachlorobiphenyl (PCB 77), but not hexachlorobenzene, induced liver micro-somal cytochrome P-450 (Cyt P-450), ethoxycoumarin-O-deethylase (ECOD) and ethoxyresorufin-O-deethylase (EROD) in rainbow trout. Maximum induction was observed in a PCB 77 injected group of fish (1.0mg/kg, i.p. injection) 13 days after the injections being 2, 10 and 50 times the values of non-induced fish, respectively.
• 2.2. The apparent K_m value of ethoxyresorufin of this induced group of fish differed only slightly from that of non-induced fish. The apparent V_max value (EROD) was 50 times higher.
• 3.3. Freezing small pieces of liver in liquid nitrogen did not produce cytochrome P-420.
• 4.4. Fluorimetric and spectrophotometric measurements of EROD correlated.

     

Proteolytic cleavage of band 3 protein in relation to anion transport in fish (Oncorhynchus mykiss) red blood cells

• 1.1. The effects of trypsin and chymotrypsin on HCO₃⁻/Cl⁻ exchange through red blood cell membranes of humans and trout were studied.
• 2.2. To measure the anion exchange we used a right-angle light-scattering technique by applying the Jacobs-Stewart cycle in ammonium solution and the osmotiration method at constant cell volume.
• 3.3. The Cl⁻ flux in human red blood cells remained unaltered after treatment with external trypsin and chymotrypsin while in trout red blood cells the flux decreased.
• 4.4. This partial inhibition of anion transport in fish, ranging from 30 to 40%,suggest that one or several of the cleavage sites in band 3 protein, essential for anion transport function, are exposed in fish red blood cells.
• 5.5. In human red blood cells the fragments of band 3 which are affected by proteolytic digestion, retain their tertiary structure because there is no influence on anion transport.

     

Generation of C3- and C2-deuterated l-lactic acid by human erythrocytes exposed to d-[1-13C]glucose,d-[2-13c]glucose and d-[6-13C]glucose in the presence of D2O

• 1.1. The generation of C₂- and C₃-deuterated l-lactate was monitored by ¹³C NMR in human erythrocytes exposed to d-[1-¹³glucose, d-[2-¹³C]glucose or d-te-¹³C]glucose and incubated in a medium prepared in D₂O.
• 2.2. The results suggested that the deuteration of the C₁ of d-fructose 6-phosphate in the phosphoglucoisomerase reaction, the deuteration of the C₁ of d-glyceraldehyde-3-phosphate in the sequence of reactions catalyzed by triose phosphate isomerase and aldolase and the deuteration of the C₃ of pyruvate in the reaction catalyzed by pyruvate kinase were all lower than expected from equilibration with D₂O.
• 3.3. Moreover, about 40% of the molecules of pyruvate generated by glycolysis apparently underwent deuteration on their C₃ during interconversion of the 2-keto acid and l-alanine in the reaction catalyzed by glutamate-pyruvate transaminase.
• 4.4. The occurrence of the latter process was also documented in cells exposed to exogenous [3-¹³C]pyruvate.
• 5.5. This methodological approach is proposed to provide a new tool to assess in intact cells the extent of back-and-forth interconversion of selected metabolic intermediates.

     

Effects of feeding,starvation, and refeeding on the fatty acid composition of channel catfish,ICtalurus punctatus,tissues

• 1.1. The effects of feeding, food deprivation (14 and 28 days) and refeeding (starved 14 then fed 14 days) on the fatty acid composition of white muscle, liver and brain of pond-raised channel catfish (Ictalurus punctatus) were investigated.
• 2.2. Levels of n-3 fatty acids were significantly higher (P < 0.05) in white muscle of fish starved 28 days (10.7%) than in fish fed throughout the study (8.0%), due primarily to an increase in 22:6(n-3) docosahexaenoic acid or DHA.
• 3.3. Significantly higher levels of 20:5(n-3) (eicosapentaenoic acid or EPA) were found in livers offish starved 28 days (P < 0.05) compared to fish fed throughout the study.
• 4.4. Results suggest that the fatty acid compositions of channel catfish white muscle and liver are subject to only limited perturbation during periods of starvation and refeeding and that the brain is extremely well protected.

     

Lysosomal perturbations in fish liver as indicators for toxic effects of environmental pollution

• 1.1. Lysomones play a key role in liver injury in fish caused by organic and inorganic xenobiotics. The lysosomal stability test was transferred to fish liver with the aim of testing responsive and practicable methods for biological-effects monitoring.
• 2.2. A two-step response of lysosomes in fish liver could be discerned, reflected by the activity (number and size of lysosomes) and the injury (membrane destabilisation) of the lysosomal detoxifying system.
• 3.3. Significant differences, with respect to lysosomal enlargement, membrane stability and pathological lipid accumulation, were found along a pollution gradient throughout the year.
• 4.4. The lysosomal tests clearly reflect the breakdown of the adaptive capacity of the fish liver to toxic injury. Therefore, a test battery measuring lysosomal perturbations should be recommended for the biological-effects monitoring.

     

Effects of inhibition of nadph: cytochrome p-450 reductase on benzo(a)pyrene metabolism in mouse liver microsomes

• 1.1. Effects of antioxidants (butylated hydroxytoluene and nor-dihydroguaiaretic acid), vitamin K-related quinones (vitamin K₁ and coenzyme Q₁₀) and inorganic copper (CuSO₄), in concentrations inhibiting NADPH: cytochrome P -450 reductase, were re-examined on benzo(a)pyrene metabolism in mouse liver uninduced microsomes.
• 2.2. It was found that all these compounds decrease production of the two-electron oxygenation products of benzo(a)pyrene (monophenoles, diols) and the amounts of glucuronides in a manner parallel to their inhibitory potency against NADPH: cytochrome P-450 reductase.
• 3.3. No correlation was found between amounts of one-electron oxidation products of benzo(a)pyrene and inhibition of NADPH: cytochrome P-450 reductase.
• 4.4. Without added UDPGA the compounds studied decreased protein associated benzo(a)pyrene metabolites in parallel to the decreased overall metabolism of this polyaromatic hydrocarbon.
• 5.5. The mode of action of the studied compounds is discussed.

     

IDentification of a microsomal retinoic acid synthase as a microsomal cytochrome P-450-linked monooxygenase system

• 1.1. To characterize an enzyme which metabolizes retinal in liver microsomes, several properties of the enzymatic reaction from retinal to retinoic acid were investigated using rabbit liver microsomes.
• 2.2. The maximum pH of the reaction in the liver microsomes was 7.6.
• 3.3. The K_m and V_max values for all-trans, 9-cis and 13-cis-retinals were determined.
• 4.4. The reaction proceeded in the presence of NADPH and molecular oxygen.
• 5.5. The incorporation of one atom of molecular oxygen into retinal was confirmed by using oxygen-18, showing that the reaction comprised monooxygenation, not dehydrogenation.
• 6.6. The monooxygenase activity was inhibited by carbon monoxide, phenylisocyanide and antiNADPH-cytochrome P-450 reductase IgG, but not by anti-cytochrome b₅ IgG.
• 7.7. The enzymatic activity inhibited by carbon monoxide was photoreversibly restored by light of a wavelength of around 450 nm.
• 8.8. The retinal-induced spectra of liver microsomes with three isomeric retinals were type I spectra.
• 9.9. The microsomal monooxygenase activity induced by phenobarbital or ethanol were more effective than that by 3-methylcholanthrene, clotrimazole or β-naphthoflavone.
• 10.10. These results showed that the monooxygenase reaction from retinal to retinoic acid in liver microsomes is catalyzed by a cytochrome P-450-linked monooxygenase system.