Synthesis and metabolism of all-trans-[11-3H]retinyl beta-glucuronide in rats in vivo.

All-trans-[11-3H]retinyl beta-glucuronide (all-trans-[11-3H]ROG) was synthesized from [3H]retinol by an improved synthetic procedure. After its intraperitoneal injection into rats, ROG is initially found as the predominant labelled component in the serum, but then is distributed to the liver, intestine, kidney and other organs of the body. Esters of vitamin A, which constituted the major metabolite of ROG, were detected in the liver as well as in other tissues. Of the labelled vitamin A esters derived from tritiated ROG in the liver and intestine, about 50% contained 5,6-epoxyretinol, which was characterized by its chromatographic behaviour, formation of an acetyl ester and lack of reactivity with diazomethane. Thus ROG, although converted to retinol in vivo, might also act physiologically in an intact form.     

Uptake and cellular transport of [11-3H] all-trans-retinoic acid in the liver of vitamin A-deficient hamsters

In this study, we examined, by ultrastructural autoradiography, the uptake and intracellular transport of [3H]all-trans-retinoic acid ([3H]RA) in the livers of vitamin A-deficient hamsters. Four-week-old animals were administered 25 microCi of [3H]RA by gavage, and, at different intervals thereafter, one animal was sacrificed. Their livers were excised and processed for autoradiography. Radioactive grains were observed to pass randomly through the plasma membrane by diffusion. No evidence of retinoid internalization by endocytosis was observed. Between 1 and 30 min after gavage, the radioactivity in parenchymal cells was associated mainly with rough endoplasmic reticulum (RER) and mitochondria. The labeling over nuclei was apparent at 1 min, remained relatively high up to 30 min, and subsequently decreased. At 2 and 5 hr, only a few grains were observed over nuclei, RER and mitochondria. At 24 hr, most of the labeling was associated with endothelial cells and sinusoidal spaces, indicating mobilization of [3H]RA from the liver. The results indicate that [3H]RA is transported through the plasma membrane by transmembrane diffusion without endocytosis and, after entering the cells, the ligand is rapidly translocated into nuclei.     

Metabolism of [6,7-3H, 35S] estradiol 17-sulfate in rats

To confirm whether or not the sulfo group of estradiol 17-sulfate (ES) is removed during in vivo metabolism in rats, the doubly labeled conjugate [6,7-3H, 35S] ES was injected into rats, and its biliary and urinary metabolites were determined by reverse isotope dilution method (RIDM). In male rats, the major radioactivity was detected in biliary disulfate fraction, which was composed of mainly ES and its two minor metabolites, 2-hydroxyestradiol 17-sulfate (2-OH-ES) and 2-methoxyestradiol 17-sulfate (2-MeO-ES). In female rats, in contrast, the radioactivity was dispersed into three fractions:biliary monosulfate, biliary disulfate, and urinary monosulfate fractions (Frs.) In both monosulfate Frs., 7beta-hydroxyestradiol 17-sulfate was detected as the major metabolite followed by 6alpha-, 6beta-, and 15beta-hydroxyestradiol 17-sulfates. Like male rats, 2-OH-ES and 2-Meo-ES as the minor products were detected in biliary disulfate fraction. The isotope ratios of ES and its metabolites in both sexes were essentially the same as that of the dose except that of 6alpha-hydroxylated metabolite, which may be derived from the loss of the tritium labeled at C6. These results confirm the occurrence of the direct metabolism of ES in rats.     

Metabolism of pyridoxine in the liver of vitamin B-6-deficient rats

The metabolism of [6-³H]pyridoxine · HCl was investigated in the liver of vitamin B-6-deficient rats. Rats were made vitamin B-6 deficient by feeding adlititum for 42 days a diet lacking pyridoxine but otherwise optimal. Animals were each injected intraperitoneally with 33 μCi of [6-³H]pyridoxine · HCl and killed at different time intervals afterwards up to 7 days. Radioactively labeled hepatic B-6 compounds were extracted with acid and chromatographically separated on Dowex-X8 (H⁺) columns and the percent radioactivity for each vitamin compound was then calculated. Maximal uptake in control and deficient animals was observed 30 and 60 min, respectively, after administration of label. Radioactivity was not retained by the control animals but decreased steadily in a linear fashion after 30 min, reaching a low level after 3 h. On the other hand, vitamin deficient animals accumulated almost twice as much radioactivity in their liver as the controls and retained it through 7 days.In vitamin B-6-deficient animals 93% of the injected radioactivity was metabolized within 2 min at which time pyridoxine 5′-P and pyridoxal 5′-P reached 36 and 44% levels, respectively. Pyridoxine 5′-P dropped to minimal values (3%) within 15 min and remained unchanged for 7 days while pyridoxal 5′-P reached a peak (79%) level at 15 min and then began to drop linearly reaching a plateau (29%) at 5 days. Further, as the level of pyridoxal 5′-P was falling, pyridoxamine 5′-P was linearly synthesized reaching a plateau level (62%) in 5 days which also remained unchaged through 7 days. Some pyridoxal was also formed (7% at 1 h) which by 12 h had dropped to a plateau low level (3%). The specific activity level of pyridoxal kinase decreased 3.2 times and that of pyridoxine 5′-phosphate oxidase increased 1.5 times in the state of deficiency. The results presented show that metabolism of [³H]pyridoxine in deficiency is characterized by (a) a delayed, two-fold increase in label uptake as well as an extended label retention period, (b) a rapid pyridoxal 5′-P synthesis, and (c) a continuouus synthesis (and accumulation) of pyridoxamine 5′-P which is not utilized or further metabolized.     

Metabolism of [3H]leupeptin by rat liver

Tritium-labeled leupeptin was used to study how this tripeptide proteinase inhibitor interacts with the liver, including the mechanism of its transport into the cell, its subcellular distribution after uptake, and its metabolism once in the tissue. Experiments were done in situ and in a perfused liver. At low concentrations (1 to 10 μm) the uptake of radioactive inhibitor was competed by chemically reduced leupeptin. At high concentrations at least up to 400 μm the uptake was directly proportional to the external concentration of tripeptide. Entry into the tissue essentially stopped at low temperature (<21 °C). [³H]Leupeptin initially was located in the soluble fraction of the liver homogenate and by 15 to 30 min became concentrated in the lysosome-rich fraction. During 2 h of perfusion almost 50% of [³H]leupeptin that had entered the liver was secreted intact into the bile. In addition, a portion of the leupeptin that remained in the liver was degraded during this time period.     

Metabolism of pyridoxine in the liver of vitamin B-6-deficient rats.

The metabolism of [6-3H]pyridoxine - HCl was investigated in the liver of vitamin B-6-deficient rats. Rats were made vitamin B-6 deficient by feeding ad libitum for 42 days a diet lacking pyridoxine but otherwise optimal. Animals were each injected intraperitoneally with 33 muCi of [6-3H] pyridoxine - HCl and killed at different time intervals afterwards up to 7 days. Radioactively labeled hepatic B-6 compounds were extracted with acid and chromatographically separated on Dowex-X8 (H+) columns and the percent radioactivity for each vitamin compound was then calculated. Maximal uptake in control and deficient animals was observed 30 and 60 min, respectively, after administration of label. Radioactivity was not retained by the control animals but decreased steadily in a linear fashion after 30 min, reaching a low level after 3 h. On the other hand, vitamin deficient animals accumulated almost twice as much radioactivity in their liver as the controls and retained it through 7 days. In vitamin B-6 deficient animals 93% of the injected radioactivity was metabolized within 2 min at which time pyridoxine 5'-P and pyridoxal 5'-P reached 36 and 44% levels, respectively. Pyridoxine 5'-P dropped to minimal values (3%) within 15 min and remained unchanged for 7 days while pyridoxal 5'-P reached a peak (79%) level at 15 min and then began to drop linearly reaching a plateau (29%) at 5 days. Further, as the level of pyridoxal 5-P was falling, pyridoxamine 5'-P was linearly synthesized reaching a platuau low level (3%). The specific activity level of pyridoxal kinase decreased 3.2 times and that of pyridoxine 5'-phosphate oxidase increased 1.5 times in the state of deficiency. The results presented show that metabolism of [3H]pyridoxine in deficiency is characterized by (a) a delayed, two-fold increase in label uptake as well as an extended label retention period, (b) a rapid pyridoxal 5'-P synthesis, and (c) a continuous synthesis (and accumulation) of pyridoxamine 5'-P which is not utilized or further metabolized.     

Syntheses of 24R,25-dihydroxy-[6,19,19-3H]vitamin D3 and 24R,25-dihydroxy-[6,19,19-2H]vitamin D3

24R,25-Dihydroxy-[6,19,19-3H]vitamin D3 with a specific activity of 54 Ci/mmol and 24R,25-dihydroxy-[6,19,19-2H]vitamin D3 with 2.6 deuterium atoms/mol were synthesized in four steps starting from 24R,25-Dihydroxyvitamin D3 via its sulfur dioxide adduct.     

Spontaneous transfer of retinoic acid, retinyl acetate, and retinyl palmitate between single unilamellar vesicles

The transfer of retinoic acid, retinyl acetate, and retinyl palmitate between single unilamellar vesicles was studied by resonance energy transfer. The retinoic acid transfers spontaneously between single unilamellar vesicles with a first order rate constant of 9.6 s-1 at 15 degrees C and pH 7.4. At 30 degrees C, the transfer rate was 3.5 times faster than that at 10 degrees C. At pH 7.4, the transfer rate was almost 2 orders of magnitude faster than that observed at pH 1.6. Increasing the concentration of NaCl decreased the retinoic acid transfer rate significantly. Retinyl acetate transfers with a rate constant of 0.15 s-1, but no spontaneous transfer of retinyl palmitate was observed over 60 min. The evidence supports the proposal that retinoic acid and retinyl acetate transfer between single unilamellar vesicles occur via the aqueous phase. In contrast, no spontaneous transfer of retinyl palmitate was observed. However, linear free energy relationships and the thermodynamic parameters for retinyl acetate transfer permit the calculation of rate constant for retinyl palmitate transfer.     

Effect of depletion of vitamin A, followed by supplementation with retinyl acetate or retinoic acid, on regeneration of rat liver.

After partial hepatectomy the net increase in tissue weight and in RNA, DNA and proteins in the regenerating liver was markedly less in vitamin A-deleted or retinoic acid-supplemented male rats, compared with the corresponding normal control or retinyl acetate-supplemented ones.     

Synthesis of 1alpha-hydroxy [6-3H]vitamin D3 and its metabolism to 1alpha, 25-dihydroxy [6-3H]vitamin D3 in the rat.

1alpha-Hydroxy [6-3H]vitamin D3 has been synthesized with a specific activity of 4 Ci/mmol, and its metabolism in rats has been studied. It is rapidly converted to 1alpha,25-dihydroxy [6-3H]vitamin D3 in vivo. Following an intravenous or oral dose, a maximal concentration of 1alpha,25-dihydroxy [6-3H]vitamin D3 is found 2 and 4 hours, respectively, before the maximal intestinal calcium transport response is observed. Similarly, 1alpha,25-dihydroxy[6-3H]vitamin D3 accumulation in bone precedes the bone calcium mobilization response. It appears, therefore, that the biological activity of 1alpha-hydroxyvitamin D3 is largely, if not exclusively, due to its conversion to 1alpha,25-dihydroxy[6-3H]vitamin D3 1alpha-Hydroxy[6-3H]vitamin D3 and 1alpha,25-dihydroxy[6-3H]vitamin D3 appear in intestine equally well after an oral or an intravenous dose of 1alpha-hydroxy[6-3H]vitamin D3. However, much less of both 1alpha-hydroxy[6-3H]vitamin D3 and 1alpha,25-dihydroxy[6-3H]vitamin D3 appears in bone and blood after an oral than after an intravenous dose. A much reduced bone calcium mobilization response is also noted following an oral dose as compared to an intravenous dose of 1alpha-hydroxyvitamin D3, suggesting that oral 1alpha-hydroxyvitamin D3 is not utilized as well as intravenously administered material.     

Synthesis of [1,2-3H2]cholecalciferol and metabolism of [4-14C,1,2-3H2]- and [4-14C,1-3H]-cholecalciferol in rachitic rats and chicks

[1,2-(3)H(2)]Cholecalciferol has been synthesized with a specific radioactivity of 508mCi/mmol by using tristriphenylphosphinerhodium chloride, the homogeneous hydrogen catalyst. With doses of 125ng (5i.u.) of [4-(14)C,1-(3)H(2)]cholecalciferol the tissue distribution in rachitic rats of cholecalciferol and its metabolites (25-hydroxycholecalciferol and peak P material) was similar to that found in chicken with 500ng doses of the double-labelled vitamin. The only exceptions were rat kidney, with a very high concentration of vitamin D, and rat blood, with a higher proportion of peak P material, containing a substance formed from vitamin D with the loss of hydrogen from C-1. Substance P formed from [4-(14)C,1,2-(3)H(2)]cholecalciferol retained 36% of (3)H, the amount expected from its distribution between C-1 and C-2, the (3)H at C-1 being lost. 25-Hydroxycholecalciferol does not seem to have any specific intracellular localization within the intestine of rachitic chicks. The (3)H-deficient substance P was present in the intestine and bone 1h after a dose of vitamin D and 30min after 25-hydroxycholecalciferol. There was very little 25-hydroxycholecalciferol in intestine at any time-interval, but bone and blood continued to take it up over the 8h experimental period. It is suggested that the intestinal (3)H-deficient substance P originates from outside this tissue. The polar metabolite found in blood and which has retained its (3)H at C-1 is not a precursor of the intestinal (3)H-deficient substance P.     

Synthesis of 25-hydroxy[23,24-3H]vitamin D3

A synthesis of 25-hydroxy[23,24-³H]vitamin D₃ leading to a radiochemically pure product with a specific acitivity of 78 Ci/mmol is described. The structure of the product was confirmed by comparison with unlabeled material and its biological activity was established by in vitro conversion to 1α,25-dihydroxy[23,24-³H]vitamin D₃ using the chick kidney 1α-hydroxylase system.     

Synthesis of 25-hydroxy-[26,27-3H]vitamin D2, 1,25-dihydroxy-[26,27-3H]vitamin D2 and their (24R)-epimers

Synthesis of a C-24-epimeric mixture of 25-hydroxy-[26,27-3H]vitamin D2 and a C-24-epimeric mixture of 1,25-dihydroxy-[26,27-3H]vitamin D2 by the Grignard reaction of the corresponding 25-keto-27-nor-vitamin D2 and 1 alpha-acetoxy-25-keto-27-nor-vitamin D3 with tritiated methyl magnesium bromide is described. Separation of epimers by high-performance liquid chromatography afforded pure radiolabeled vitamins of high specific activity (80 Ci/mmol). The identities and radiochemical purities of 25-hydroxy-[26,27-3H[vitamin D2 and 1,25-dihydroxy-[26,27-3H]vitamin D2 D2 were established by cochromatography with synthetic 25-hydroxyvitamin D2 or 1,25-dihydroxyvitamin D2. Biological activity of 25-hydroxy-[26,27-3H]vitamin D2 was demonstrated by its binding to the rat plasma binding protein for vitamin D compounds, and by its in vitro conversion to 1,25-dihydroxy-[26,27-3H]vitamin D2 by kidney homogenate prepared from vitamin D-deficient chickens. The biological activity of 1,25-dihydroxy-[26,27-3H]vitamin D2 was demonstrated by its binding to the chick intestinal receptor for 1,25-dihydroxyvitamin D3.     

Tetrapyrrole biosynthesis in Anacystis nidulans; incorporation of [1-13C]-, [2-13C]-, [1,2-13C]- and [2-13C, 2-2H3]acetate

Labelling experiments with [2-¹³C]- and [1,2-¹³C]acetate showed that both photopigments of Anacystis nidulans, chlorophyll a and phycocyanobilin, share a common biosynthetic pathway from glutamate. The fate of deuterium during these biosynthetic events was studied using [2-¹³C, 2-²H₃]acetate as a precursor and determining the labelling pattern by ¹³C NMR spectroscopy with simultaneous [¹H, ²H]-broadband decoupling. The loss of ²H (ca 20%) from the precursor occurred at an early stage during the tricarboxylic acid cycle. After formation of glutamate there was no further loss of ²H in the assembly of the cyclic tetrapyrrole intermediates or during decarboxylation and modification of the side-chains. Thus the labelling data support a divergence in the pathway to cyclic and linear tetrapyrroles after protoporphyrin IX.     

Liquid-gel partition chromatography of vitamin A compounds; formation of retinoic acid from retinyl acetate in vivo

A clear separation of retinol, retinal, and retinoic acid has been achieved by liquid-gel partition chromatography on Sephadex LH-20 with solvent mixtures of chloroform, Skellysolve B, and methanol. A mixture of retinyl esters, retinol, retinal, and retinoic acid has been resolved on hydroxyalkoxypropyl Sephadex using Skellysolve B and acetone. There is no decomposition of any of the vitamin A compounds during chromatography, and recovery is complete. The combination of mildness and potential for resolution makes liquid-gel partition chromatography a superior tool for the separation of vitamin A compounds. This method has been applied to the study of vitamin A metabolism at physiological levels in the vitamin A-deficient rat. Retinyl palmitate, an ester of retinoic acid, retinal, retinol, retinoic acid, and a polar metabolite have been demonstrated in various tissues of the rat 12 hr after a dose of 2 micro g of [11-(14)C]retinyl acetate.     

The intracellular distribution of [1-3H]-cholecalciferol in the intestine of vitamin D-deficient and supplemented rats

1. [1-(3)H]Cholecalciferol was administered orally at two dosages to vitamin D-deficient and -supplemented rats, and the intracellular distribution of the vitamin in the intestinal mucosa studied. 2. The concentration of cholecalciferol was highest in a fraction consisting of brush borders and nuclei. The microsomal fraction contained a higher concentration of the vitamin than the mitochondrial fraction in deficient rats, irrespective of the dose, whereas in the vitamin D-supplemented rats the concentration was the same in the two fractions. 3. Appreciable metabolism of the cholecalciferol occurred only in the supplemented rats and the metabolites were found predominantly in the mitochondrial fraction. 4. The cholecalciferol is more tightly bound to the microsomal fraction than to the mitochondrial fraction. 5. Experiments conducted in vitro have shown that all the intracellular particles combine with the vitamin either when dissolved in ethanol or bound to albumin. However, such an uptake does not account for the high concentration of radioactivity found in vivo in the fraction containing nuclei and brush border, nor for the tightly bound vitamin in the microsomal fraction.