Cellular distribution and clearance of aerosolized dipalmitoyl lecithin.

Wistar-Lewis rats were anesthetized anc connected to a 3-MHz nebulizer which aerosolized 250 muCi l-alpha-1-palmitoyl-2palmitoyl-[9-10-3H]phosphatidylcholine ([3H]DPL) for 3 min. Appleton frozen-section autoradiographs showed greater than 4 times background radioactivity in approximately 30% of alveoli at 1 min and 2 h after aerosol. As tritium content in the lung decreased, it increased in liver, spleen, kidney, blood, and urine. Percentage of radioactivity from [3H]phosphatidylcholine in the liver declined with time, while [3H]phosphatidylethanolamine doubled between 2 and 12 h. One minute postaerosol 2,500 +/- 500 (SE) type I cells/mm3 lung and 2,500 +/- 750 type II cells/mm3 lung had greater than 20 times background radioactivity; 2 h later only 950 +/- 250 type I cells/-m3 lung still had levels of radioactivity greater than 20 times background while 3,150 +/- 600 type II cells/mm3 lumg now had this level of 3HIDPL. Corresponding numbers of alveolar macrophages were 450 +/- 250 1 min postaerosol and 1,100 +/- 200 after 2 h. Aerosolized DPL as a synthetic surfactant is hampered by significantly faster clearance from the alveolar surface as compared with normal in vivo DPL.     

Metabolic dehydration of prostaglandin E2 and cellular uptake of the dehydration product: correlation with prostaglandin E2-induced growth inhibition

When L-1210 murine leukemia cells were incubated with 60 microM PGE2 in culture medium containing fetal calf serum for various time, cell proliferation was inhibited dependent on incubation time. However, when the medium containing PGE2 was changed every 6 h during 24 h exposure to cells, growth inhibition became much weaker. Moreover, when the medium containing PGE2 was aged by preincubating without cells at 37 degrees C, growth inhibitory effect of the medium was enhanced with preincubation time, suggesting that active growth inhibitory compound(s) accumulated during preincubation. In culture medium containing fetal calf serum, PGE2 degraded time-dependently and the major product was identified as PGA2 by HPLC. Furthermore, when cells were incubated with the medium containing 60 microM[3H]PGE2 or the same medium aged by preincubation, we observed that the radioactivity was taken up by the cells time-dependently, and identified the incorporated radioactivity as PGA2. This uptake was closely correlated with decrease in viable cell number during incubation. These results suggested that growth inhibitory effect of PGE2 was due to the metabolic dehydration of PGE2 to PGA2, and PGA2, after taken up by cells, exerted cell growth inhibition.     

Solubilization and Characterization of Prostaglandin E2 Binding Protein from Porcine Cerebral Cortex

The specific binding protein for prostaglandin (PG) E2 was solubilized in an active form from the crude mitochondrial (P2) fraction of porcine cerebral cortex. After incubation with 3-[(3-cholamidopropyl)dimethylammonio]-1-propane sulfonate (CHAPS) at 4 degree C for 30 min, the PGE2 binding to the supernatant fraction (103,000 g, 60 min) was determined by the polyethylene glycol method. The maximum yield (approximately 30% of the binding activity to the P2 fraction) was obtained with 10 mM CHAPS. The specific [3H]PGE2 binding to the solubilized fraction was time-dependent and the equilibrium was reached at around 60 min at 37 degrees C. By dilution of the reaction mixture, the binding site-[3H]PGE2 complex formed after 5-min incubation slowly dissociated, whereas that formed after 60-min incubation did not dissociate to a significant extent. The binding was highly specific for PGE2 and inhibited by unlabeled PGs in the following order: PGE2 greater than PGE1 much greater than PGF2 alpha greater than PGE2 methyl ester greater than PGA2 greater than 13,14-dihydro-15-keto-PGE2 greater than PGD2. Scatchard analyses of the solubilized fraction suggested the presence of high- and low-affinity sites. Heat treatment and preincubation with trypsin or proteinase K markedly reduced the binding. The binding activity was eluted in a single peak both from gel filtration and from ion-exchange columns using HPLC. These results suggest that a specific protein solubilized may be responsible for the binding site.     

Incorporation of orotic acid into nucleotides and RNA in mouse organs during 60 minutes.

Mouse kidney and liver were found to increase their levels of radioactivity above that of serum from 2 to 60 min after administration of [6-14C]orotic acid. In spleen, thymus and brain, the radioactivity level reached a maximum soon after the injection and then decreased, as did that in serum. Sixty minutes after the injection, 44% of the administered isotope dose was found in the kidneys, 22% in the liver and 0.75% in the spleen. The 14C activity in liver UTP increased rapidly and then remained constant for 60 min. The ratio between the activities in uridine phosphates and UDP-sugars was 3:4 from 10- 60 min after injection. In the liver and kidneys, the RNA 14C activities at 60 min after injection were 15% of the activity in their acid-soluble fractions. Intraperitoneal administration was found to be preferable to intravenous administration for studies on nucleotides and RNA in mouse liver, due to the delayed incorporation of the [14C]orotic acid activity into the nucleotide pool.     

Extremely rapid degradation of [3H] methionine-enkephalin by various rat tissues in vivo and in vitro

Thirty sec after the intrajugular injection of [³H] methionine-enkephalin (met-enkephalin) in the rat, the radioactivity was already distributed in an apparent volume of 53 ml and the metabolic clearance rate calculated from the characteristics of the plasma disappearance curve was 10 ml/min. As shown by partition chromatography plasma extracts obtained 15 sec after injection of [³H] met-enkephalin, only 5% of the total radioactivity migrated as the intact pentapeptide, while no detectable intact pentapeptide remained 2 min after injection, thus indicating a half-life of [³H] met-enkephalin of the order of 2 to 4 sec. Incubation of rat cerebral tissue with [³H] met-enkephalin indicates that the first step in the breakdown of met-enkephalin in both plasma and brain tissue is cleavage of the Tyr-Gly amide bond. These data offer an explanation for the low activity of met-enkephalin after intraventricular or intravenous administration.     

The incorporation and distribution of H oleic acid in the isolated perfused guinea-pig heart: A biochemical and EM autoradiographic study

The incorporation of 3H oleic acid into tissue lipids of guinea pig heart was studied after 15, 30, 60 or 120 sec perfusion using EM autoradiography with 'hypothetical grain' analysis and lipid analysis by thin-layer chromatography. Radioactivity in triacylglycerol and phospholipid increased and in free fatty acid decreased with time. This corresponded to an increase in radioactivity associated with lipid droplets in the autoradiographs. High levels of radioactivity were found associated with the mitochondria after only 15 sec. The movement of fatty acids is interpreted in terms of transport mechanisms, concentration gradients and bound and unbound molecules.     

Enzymatic synthesis of (15s)-[15-3h]prostaglandins and their use in the development of a simple and sensitive assay for 15-hydroxyprostaglandin dehydrogenase.

The stereospecificity of swine renal NAD+-dependent 15-hydroxyprostaglandin dehydrogenase has been determined. It was found that the enzyme is a B-side specific dehydrogenase. (15S)-[15-3H]Prostaglandins were synthesized by stereospecific transfer of the tritium label of D-[1-3H]galactose to prostaglandins by coupling 15-hydroxyprostaglandin dehydrogenase with beta-D-galactose dehydrogenase, an enzyme of the same stereospecificity. A simple and sensitive assay for 15-hydroxyprostaglandin dehydrogenase was developed based on the stereospecific transfer of the tritium label of tritiated prostaglandins to glutamate by coupling 15-hydroxyprostaglandin dehydrogenase with glutamate dehydrogenase. The amount of prostaglandin oxidized is determined by the radioactivity of labeled glutamate present in the supernatant after charcoal precipitation of labeled prostaglandin. Concurrent assays with the present tritium release method and the thin-layer chromatography method indicated excellent correlation. The assay was employed to study some of the properties of swine renal 15-hydroxyprostaglandin dehydrogenase in crude extract and the distribution of enzyme activity in various tissues of rat. Enzyme activity was linear for the first 10 min studied and was nonlinear with increasing amounts of crude enzyme, indicating the possible presence of endogenous inhibitor(s). Apparent Km's for PGE2, PGF2alpha, and PGA2 were found to be 2.5, 12.5, and 3.9 muM, respectively. The distribution pattern indicated high levels of enzyme activity in gastrointestinal tract, lung, kidney, and spleen. The assay method may prove to be valuable for studying enzyme turnover and enzyme regulation by hormonal and pharmacological agents.     

Effects of pleural pressure and abdominal pressure on diaphragmatic blood flow

Alveolar transfer of prostaglandin E2 (PGE2) was characterized in isolated perfused guinea pig lungs (n = 19) by measuring radioactivity appearing in the venous effluent during 30 min after intratracheal instillation of [3H]PGE2, [14C]-mannitol, and [125I]iodoantipyrine. Recovery of lipid-soluble [125I]iodoantipyrine [91 +/- 3% (SE)] after 30 min was used to estimate total 3H and 14C delivered to the exchanging region of lung at time 0. In seven control lungs, 58 +/- 4% of [14C]mannitol and 16 +/- 4% of [3H]PGE2 was retained 10 min after instillation. Neither perfusion with diphloretin phosphate (10 micrograms/ml; n = 4) nor hypothermia (5 degrees C; n = 5) significantly affected the amount of [14C]mannitol retained; however, [3H]PGE2 remaining in these lungs increased significantly to 36 +/- 4 and 53 +/- 2%, respectively. Addition of unlabeled PGE2 (200 micrograms) to the instilled solution (n = 3) increased retention of both [14C]mannitol (80 +/- 3%) and [3H]PGE2 (65 +/- 4%). Alveolar transfer of [3H]PGE2 was calculated as the difference in percent retention of [14C]mannitol and [3H]PGE2 and normalized to that of [14C]mannitol. After 10 min, alveolar transfer of [3H]PGE2 was 71 +/- 8% in control lungs but was decreased to 26 +/- 7, 10 +/- 5, and 19 +/- 6% by diphloretin phosphate, hypothermia, or unlabeled PGE2, respectively. These data suggest that alveolar clearance of PGE2 involves a saturable drug- and temperature-sensitive process.     

Pharmacokinetics,Tissue Distribution and Metabolism of Intravenously Administered Digalactosyldiacylglycerol and Monogalactosyldiacylglycerol in the Rat

Abstract

A bilayer forming galactolipid, digalactosyldiacylglycerol (DGalDG) has been identified as a tool with suitable physicochemichal properties for pharmaceutical formulation work. One possible application is as a carrier for liposome entrapped drugs for intravenous administration. The fate of intravenously administered galactolipids is not known. In this study liposomal dispersions of galactolipids, containing [³H]fatty acid labelled DGalDG or monogalactosyldiacylglycerol (MGalDG) were injected intravenously in the rat and the disappearance from blood and uptake by tissues were examined. The T1/2 of [³H]DGalDG in plasma was 3 to 5 minutes. Of the tissues examined (liver, spleen, kidneys, lung, heart, stomach, upper and lower small intestine and colon), the liver contained the highest radioactivity per g tissue after both 15 min. and 4 h. Autoradiographic examinations after 15 min, 1 h and 4 h showed that the uptake of radiolabeled DGalDG and MGalDG occurred mainly to the hepatocytes. Less than 6 % of the injected [³H]DGalDG remained in liver and plasma as [³H]DGalDG after 4 h. [³H]MGalDG exhibited a similar pattern of metabolism although the initial disappearance rate was faster than for [³H]DGalDG. The study thus shows that the hepatocytes take up and hydrolyse galactolipids after intravenous administration.     

Prostaglandin E1 binds to Z protein of rat liver

Z protein or fatty-acid-binding protein is abundant in the cytosol of many cell types including liver cells. It is considered to play an important role in intracellular transport and metabolism of long-chain fatty acids and other organic anions. We studied the role of Z protein in the metabolism of prostaglandin E1 (PGE1). Binding of tritiated prostaglandin E1 to this fatty-acid-binding protein (Z protein) purified from rat liver was determined. The binding of [3H]prostaglandin E1 to Z protein is rapid, saturable and reversible. Scatchard analysis of [3H]PGE1 binding to Z protein showed a single class of binding sites with a dissociation constant (Kd) of 37 nM. The binding capacity is 110 nmol/mg Z protein. Optimal [3H]PGE1 binding occurred at pH 7.4. The presence of 3 mM MgCl2 stimulated the prostaglandin E1 binding to Z protein. Competition experiments show that the binding of this autacoid to Z protein is highly specific. It could not be displaced by other prostaglandins (PGA1, PGA2, PGE2, PGB1, PGI2, PGD2, PGF2 alpha, and 6-keto PGF1 alpha). Z protein might be involved in the metabolism of prostaglandins in the cytosol.     

Serum half-life, distribution, hepatic uptake and biliary excretion of alpha-tocopherol in rats

The serum clearance of alpha-[3H]tocopherol has been studied after intravenous injection of intestinal lymph labeled in vivo with radioactive alpha-tocopherol. The half-life of the injected alpha-[3H]tocopherol was approx. 12 min. Fractionation of plasma by ultracentrifugation 10 min after injection of lymph showed that 91% of the radioactive alpha-tocopherol remaining in plasma was located in chylomicrons (d less than 1.006 g/ml) and 7.8% in high-density lipoproteins (HDL, 1.05 less than d less than 1.21 g/ml). 2 h after administration of alpha-tocopherol, about 35% of the radioactivity recovered in plasma was associated with chylomicrons and approx. 51% with HDLs. alpha-[3H]Tocopherol was initially taken up by the liver, which contained more than 50% of the injected radioactivity after 45-60 min. Separation of parenchymal and nonparenchymal cells demonstrated a preferential uptake of alpha-[3H]tocopherol by the parenchymal liver cells. After 24 h about 11% of the injected dose was recovered in the liver. Considering whole organs the liver, adipose tissue and skeletal muscle had the highest content of radioactivity after 24 h. Furthermore, about 14% of the administered dose was recovered in bile during 24 h draining.     

Comparison of in vivo and in vitro binding of polycyclic hydrocarbons to DNA

The in vivo binding of [³H]benzo(a)pyrene (BP) and 3-[³H]methylcholanthrene (3MC) to liver and lung DNA was studied in A/J mice. Only in liver was there any reduction in total DNA-bound radioactivity between 4 h and 24 h after administration of the hydrocarbon. DNA was fractionated on Sephadex LH-20 after enzymatic digestion. A single deoxyribonucleoside-BP adduct was detected whereas two major 3MC-adducts were observed. With both BP and 3MC, three additional peaks of radioactivity eluted rapidly in the lung DNA experiments while a fourth was noted with liver DNA. The nucleoside-bound adducts from lung represented a much larger proportion of the total radioactivity than with liver. In vitro analysis of 3MC binding to DNA showed the nucleoside-bound adducts to be predominantly deoxyguanosine-dependent but that the early peaks were independent of base suggesting binding to another part of the DNA molecule, perhaps phosphate, i.e., phosphotriesters.     

Inosine metabolism in the rat

1. Uptake and subsequent metabolism of purine and ribose moieties was monitored after intravenous administration of doubly labelled inosine. 2. More than 95% was cleared from the plasma within 5 min, and 99% within 20 min. 3. Approx. 50% of the 160 mumol total was rapidly incorporated into liver and kidney. Kidney removed the greatest amount (21 mumol/g wet wt.), about 10-fold more than heart, lung or liver. Lung and heart accounted for only 3%. These tissues then lost radioactivity during the remainder of the experiment. Radioactivity in the skeletal muscle, in contrast, increased from 8% of the injected dose at 5 min to 40% at 60 min. 4. In liver, kidney, heart and lung there was a significant difference in the fate of inosine. After initial incorporation of inosine, kidney predominantly lost inosine; heart preferentially lost purines; lung preferentially lost ribose radioactivity; and in liver the ribose radioactivity was rapidly lost, whereas purine was retained. Some of the ribose moiety was metabolized to glucose, presumably in the liver, and then released into the blood. Ribose radioactivity (probably as glucose) and radioactive hypoxanthine accumulated in skeletal muscle throughout the experiment. 5. Inosine caused a rapid and prolonged increase in the blood glucose content, from 6 to 15 mM in 60 min. This was accompanied by a small increase in plasma insulin. 6. It is concluded that the purine and ribose radioactivity lost from the kidney, liver and other tissues becomes incorporated into skeletal muscle.     

Prostaglandin catabolism in fetal and maternal tissues--a study of 15-hydroxyprostaglandin dehydrogenase and delta 13 reductase with specific assay methods

Recent studies have demonstrated that extraductal tissues such as lung are important sources of prostaglandin E2 which maintains the patency of ductus arteriosus in fetuses and prematurely-born infants. Also, organs such as lung are known to be active in the catabolism of PGE2. Earlier studies of enzymes involved in the catabolism of PGE2 such as 15-hydroxyprostaglandin dehydrogenase (15-PGDH) and delta 13 reductase all used non-specific methods. In the present report, we studied 15-PGDH in fetal and maternal rat lung, kidney, and fetal lamb lung, kidney and ductus arteriosus with the use of a specific substrate (15-S)-[15(3)H-PGE2]. In addition, we measured the activity of delta 13 reductase in these tissues by measuring the conversion of [1-14C]-15-keto PGE2 to [1-14C]-15-keto-13,14-dihydro PGE2. The results from these studies demonstrated that in fetal rat lung and kidney, 15-PGDH activities increased rapidly while delta 13 reductase remained unchanged during late gestation. Ductus arteriosus possessed little 15-PGDH activities. These results strongly suggest that extraductal regulation of PGE2 metabolism is important in determining ductal caliber in fetuses and prematurely delivered neonates.     

In vivo studies on the metabolism of pyrimidine nucleosides (author's transl)]

3H-labelled metabolites were determined in the perchloric acid-soluble fraction of blood plasma and liver of adult male Wistar rats, following the application of [5 - 3H]uridine. Ten minutes after the injection of uridine, only 20% of the total 3H activity of the plasma could be attributed to [3H]uridine. The remaining radioactivity was found chiefly in [3H]uracil (40%) and 3H2O (20%). In the liver, at 10 min, [3H]-uridine and [3H]uracil together accounted for less than 0.5% of the total radioactivity; about 70% of the radioactivity was due to [3H]beta-alanine, and 15% to 3H2O. 45 min after the injection, 70% of the radioactivity in the plasma was due to 3H2O, whereas uridine and uracil represented about 4% and 6%, respectively. At this time, about 55% of the radioactivity in the liver was due to [3H]beta-alanine, about 40% to 3H2O, and about 5% to unidentified metabolites; [3H]uridine and [3H]uracil were not observed. A comparison of the rate of catabolism of [5-3H]-uridine, [5-3H]cytidine and [6-3H]thymidine showed that cytidine is degraded in the organism 25 times more slowly than uridine or thymidine. The biological half lives for the total degradation of the [3H]nucleosides to 3H2O, based on the values in the plasma, were: uridine 1.1 h; thymidine 1.3 h; cytidine 25 h. Furthermore, the turnover time of exogenous uridine in the plasma was found to be 9 min, which gives a half life of 6 min for the metabolism of exogenous uridine to uracil.     

Disposition of 125I-labeled recombinant human tumor necrosis factor in the tumor-bearing mouse

Disposition of [125I]rHu-TNF was elucidated in BALB/c mice bearing Meth A fibrosarcoma 7 days after transplantation. After i.v. administration, [125I]rHu-TNF measured by radioactivity and immunoreactivity biphasically decreased in plasma. Tumor level of [125I]rHu-TNF was the maximum at 1 h, then decreased and finally remained essentially constant. After i.t. administration, plasma level reached the maximum at 1 h. Tumor level decreased quickly and then became essentially constant. [125I]rHu-TNF was suggested to be degraded to small fragments in the tumor. Significant distribution of [125I]rHu-TNF was found in the kidney, lung, liver and tumor. Most tissue levels decreased with time in parallel with plasma levels. [125I]rHu-TNF radioactivity was found in proximal convoluted tubules of kidney and in those areas of tumor consisting of degenerating cells with pyknotic nuclei. Urine contained most of administered radioactivity, which being neither immunoreactive nor protein-bound.     

Fate of bradykinin-potentiating peptide 9a after intravenous injection.

The fat of less than Glu1-3H-labelled bradykinin-potentiating peptide 9a [BPP9a; less than Glu-Trp-Pro-Arg-Pro-Gln-Ile-Pro-Pro, an inhibitor of angiotensin-converting enzyme (peptidyl dipeptidase)] was studied in the rabbit. After intravenous injection, BPP9a was rapidly removed from blood and much of the associated radioactivity was excreted in urine. Approx. 8% of the radioactivity in urine collected 2h after drug administration occurred in the form of BPP9a itself, the remainder occurring in three lower homologues: less than Glu-Trp (60%), less Glu-Trp-Pro-Arg-Pro-Gln (20%) and less than Glu-Trp-Pro-Arg-Pro-Gln-Ile (12%). Hydrolysis was not accounted for by enzymes in blood or urine. Apparently hydrolysis occurred within the kidney, as less than Gl-Trp was obtained in 60% yield in urine of isolated rat kidney perfused with [less than Glu1-3H]BPP9a.     

Problems associated with the labelling of RNA with radioactive precursors in vivo (author's transl)]

The radioactivity of RNA, DNA and proteins in the liver, muscles and cerebrum of 30-day-old rats after labelling with [3H]uridine, [14C]uridine, [3H]cytidine or [3H]orotic acid was measured. It was found that after administration of [3H]uridine, the proteins were 5 - 10 times more radioactive than the RNA. After administration of [14C]uridine, the proteins were 1 - 2 times more heavily labelled than the RNA. Hydrolysis of the proteins followed by chromatography of the amino acids revealed that the protein labelling was mostly due to [3H]glutamate. In the liver, [3H]orotic acid produced very specific labelling of the RNA. The radioactivity of the proteins is very slight. However, the specific labelling of the RNA in the muscles and cerebrum is not so pronounced with this precursor. [3H]Cytidine is an ideal precursor for RNA. The labelling of protein in all three organs examined is very slight, and furthermore, the specific activity of the RNA is 10 - 20 times higher than after labelling with uridine. We were also able to show that after labelling with radioactive uridine, the method of isolation of RNA by alkaline hydrolysis gives incorrect results, because [3H]amino acids interfere with the measurement of the specific activity of the RNA. The heavy labelling of proteins by [3H]-uridine must also be taken into account in histoautoradiography, because our experiments showed that in liver, the proteins in the cell nucleus are 3 times as radioactive as the nucleic acids. The particulate components of the cytoplasm are even 20 times more radioactive than the nucleic acids.     

The dynamics of [3H]-cholesterol incorporation into the liver and aortic tissue of guinea pigs on long-term ethanol administration]

The effect of ethanol administration to guinea pigs (4 g/kg, per os) on the dynamics of [3H]-cholesterol incorporation into the liver and aorta tissues was studied for 3 months. It has been discovered that specific radioactivity of the control animals linearly increased during 24 hours in the blood serum. Ethanol reduced it as compared with the control only 0.5 h after a label has been introduced. Cholesterol renovation in the liver remained unchanged under the prolonged effect of ethanol. In the aorta the ethanol effect was characterized by a decrease of [3H]-cholesterol specific radioactivity 0.5 h after its administration. However, in this case the ratio of aorta/blood serum radioactivity increased. A day after the labelled cholesterol administration to alcoholized animals the radioactivity calculated per 1 mg of cholesterol and per unit of tissue weight and referred to the blood serum radioactivity was lower as compared to the control level.     

Metabolic fate of [3H]deoxycorticosterone and [14C]progesterone--I. Early tissue metabolites and analysis of 21-hydroxysteroids by high pressure liquid chromatography

Rabbits injected with mixtures of [3H]deoxycorticosterone and [14C]progesterone had significant levels of both 3H and 14C in several tissues and fluids extracted 10-45 min later. The distribution of radioactivity between 21-deoxysteroid, 21-hydroxysteroid, steroid acid and steroid glucuronide fractions was determined by alumina adsorption chromatography. Steroid acids derived from both steroids accumulated in the liver, kidney and urine, but were quantitatively less significant in the bile, duodenum, uterus, spleen and lung and were detected in the blood for the first time. Different 21-hydroxysteroid profiles were detected in the tissue and fluid extracts by reverse and straight phase high pressure liquid chromatography. [3H]Deoxycorticosterone accumulated in the kidney, lung, spleen and uterus, whereas tetra and hexahydro reduced metabolites predominated in the liver, bile and duodenum. By contrast, [14C]progesterone was metabolised to more polar 21-hydroxylated metabolites which were detected in the liver, kidney and urine. These results show the influence of a steroid 21-hydroxyl function, when administered, as opposed to being formed in in vivo, on the metabolic fate and excretory pathways of 21-hydroxysteroids by the rabbit.