Inhibition of sterologenesis but not glycolysis in 2,5-hexanedione-induced distal axonopathy in the rat

If glycolysis is inhibited in distal axonopathy, there should be a concomitant inhibition of lipogenesis from glucose. To investigate this possibility, lipogenesis from [¹⁴C]glucose and [³H]acetate was studied in sciatic nerves incubated with iodoacetate, a known inhibitor of glycolysis, in sciatic nerves incubated with 2,5-hexanedione, a putative inhibitor of glycolysis, and in sciatic nerves from rats exhibiting clinical signs of peripheral neuropathy induced by 2,5-hexanedione. Nerves incubated with 1.0 mm iodoacetate, in comparison with untreated nerves, exhibited decreased incorporation of [¹⁴C]glucose into sterols + diacylglycerols (33-fold), free fatty acids (14-fold), triacylglycerols (27-fold), and phospholipids (21-fold). In addition, these nerves exhibited decreased incorporation of [³H]acetate into sterols + diacylglycerols (30-fold), free fatty acids (2-fold), triacylglycerols (23-fold), and phospholipids (12-fold). In contrast, the incorporation of [¹⁴C]glucose into sterols + diacylglycerols, free fatty acids, and triacylglycerols was not affected by 1.0 mm 2,5-hexanedione. Compared to untreated nerves, nerves incubated with 1.0 mm 2,5-hexanedione exhibited a small decrease (15%) in the incorporation of [¹⁴C]glucose into phospholipids. Nerves from rats given 1% 2,5-hexanedione in the drinking water for 6 weeks, in comparison with those from pair-fed control rats, exhibited decreased (45%) incorporation of [¹⁴C]glucose and [³H]acetate into digitonin-precipitable sterols. Nerves from 2,5-hexanedione-treated and pair-fed control rats exhibited similar incorporation of [¹⁴C]glucose and [³C]acetate into free fatty acids, triacylglycerols, and phospholipids. The data indicate that while sterologenesis is inhibited in distal axonopathy, glycolysis is not.     

Effect of 2,5-Hexanedione on lipid biosynthesis in sciatic nerve and brain of the rat

Distal axonopathy induced by n-hexane and methyl n-butyl ketone has been attributed to a common metabolite, 2,5-hexanedione. Since altered lipid metabolism is frequently associated with neuropathy, the effects of 2,5-hexanedione on lipid biosynthesis from [1-¹⁴C]acetate in sciatic nerve and brain of rats given 1% 2,5-hexanedione in drinking water have been studied, in vitro. Clinical signs of neuropathy appeared after 6 weeks. Loss of body weight induced by 2,5-hexanedione was similar to that observed in pair-fed control rats. Compared to nerves from pair-fed controls, nerves from rats fed 2,5-hexanedione exhibited decreased incorporation of [1-¹⁴C]acetate into triacylglycerols (32%), total sterols + diacylglycerols (54%), digitonin-precipitable sterols (55%), squalene (55%), and ubiquinone (43%). Incorporation of [1-¹⁴C]acetate into phospholipids, fatty acids, and cholesteryl esters was similar in nerves of 2,5-hexanedione-treated rats and pair-fed controls. In brain, incorporation of [1-¹⁴C]acetate into lipids was similar in 2,5-hexanedione-treated and pair-fed control rats, except into the fatty acid fraction which was significantly decreased by 11%. The data support the hypothesis that lipid metabolism, in particular sterol metabolism, is altered in hexacarbon-induced distal axonopathy.     

Inhibition of sciatic nerve sterologenesis in hexacarbon-induced distal axonopathy in the rat

Sterologenesis from [¹⁴C]acetate and [³H]mevalonolactone has been studied, in vitro, in sciatic nerves of rats fed 1% 2,5-hexanedione in the drinking water for 6 weeks. Nerves from rats given 2,5-hexanedione, in comparison with those from pair-fed control rats, exhibited reduced incorporation of [¹⁴C]acetate, but not of [³H]mevalonolactone, into the sterol precursor squalene (38%), C₃₀ sterols (53%), C₂₇ sterols (46%), and digitonin-precipitable sterols (41%). Sterologenesis was not inhibited in nerves obtained from untreated rats and incubated in vitro with 1 mm 2,5-hexanedione, nor in nerves of rats that had inhaled 1000 ppm n-hexane for 6 hr. Incorporation of [¹⁴C]acetate into triacylglycerols of nerves from rats exposed to n-hexane was increased by 50%; [¹⁴C]acetate incorporation into other lipid fractions was similar in n-hexane-exposed and control rats. The concentration of 2,5-hexanedione in nerves of rats receiving 1% 2,5-hexanedione in drinking water for 6 weeks or exposed to 1000 ppm n-hexane for 6 hr was determined by gas chromatography-mass spectrometry and found to be 0.8 ± 0.2 and 5.2 ± 0.6 μg/g tissue wet weight, respectively.     

Liver lipid metabolism in T-2 toxicosis

The acute effects of oral administration of a single dose of T-2 toxin (2.0 mg/kg body wt) to rats on whole liver lipid metabolism were studied at 8, 16 and 24 h post-treatment. Administration of T-2 toxin significantly increased liver and microsomal total lipids, free cholesterol, esterified cholesterol and triglycerides initially at 8 h, which subsequently returned to control values at 24 h. However, no significant alterations were observed in the contents of whole liver and liver microsomal total phospholipids and phosphatidyl choline, except that phosphatidyl ethanolamine and sphingomyelin + lysophosphatidyl ethanolamine contents in liver at 16 and 24 h and sphingomyelin + lysophosphatidyl ethanolamine content in liver microsomes at all three periods were significantly lower. The incorporation of 1-¹⁴C-acetate into whole liver and liver microsomal total lipids was reduced at 16 and 24 h post feeding. However, the incorporation of 1-¹⁴C-acetate into liver and microsomal free cholesterol, esterified cholesterol and triglycerides was significantly higher at 8 h, subsequently returning to the control value at 24 h; incorporation was significantly lower even into microsomal triglycerides. The incorporation of 1-¹⁴C-acetate into liver and its microsomal total phospholipids, phosphatidyl choline, phosphatidyl ehtanolamine and sphingomyelin + lysophosphatidyl ethanolamine, was significantly decreased at all three periods post toxin treatment. The results suggested that T-2 toxin inhibited the incorporation of ¹⁴C-acetate mainly into liver and its microsomal phospholipids and their subfractions in rats.     

Effects of chloroquine on lipid metabolism of mouse pancreatic islets

Pancreatic islets accumulate the amphiphilic drug, chloroquine, which leads to a marked impairment of the insulin production of the B-cells in vitro. In this study the effects of the drug on islet lipid metabolism in vitro were investigated. It was found that exposure of islets to chloroquine (10^?5 M) for one week induced an increase of d-[U-¹⁴C]glucose incorporation into phospholipids and triacylglycerols of about two-fold and into diacylglycerols of about six-fold. Furthermore, two-dimensional thin-layer chromatography showed an increased rate of d-[U-¹⁴C]glucose incorporation into all major phospholipid classes. A 17% increase of the total islet phospholipid content indicated that an accumulation of islet cell phospholipids occurred. Pulse-chase experiments with d[U-¹⁴C]glucose showed that the rate of degradation of islet phospholipids was decreased after prolonged exposure to chloroquine. No short-term effects of chloroquine upon either the de novo biosynthesis of islet lipids or upon phospholipid degradation could be demonstrated. The present data therefore suggest that chloroquine impairs the rate of islet lipid degradation in long-term experiments, which may be a primary lesion in the sequence of events leading to functional impairment of the B-cell.     

Resistance of HepG2 cells against the adverse effects of ethanol related to neutral lipid and phospholipid metabolism

The influence of both short- and long-term ethanol exposure on the lipid metabolism was determined in the human hepatoma cell line HepG2. Ethanol did not cause any cytotoxicity or lipid peroxidation even after 7 days of 100 mM ethanol treatment of HepG2 cells. Incubation of cells in the presence of [1-(14)C]ethanol demonstrated that these cells actively metabolize ethanol to acetyl CoA, incorporating the radioactive label into neutral lipids and phospholipids. [1,2,3-(3)H]glycerol was efficiently used in phospholipid and neutral lipid biosynthesis, showing higher radioactivity in phosphatidylcholine, phosphatidylethanolamine and triacylglycerols. Exposure of HepG2 cells to 100 mM ethanol for 24 hr did not significantly modify the incorporation of glycerol into newly synthesized phospholipids and neutral lipids, nor was lipid degradation affected by the presence of ethanol. When the alcohol treatment was prolonged for 7 days, incorporation of [1,2,3-(3)H]glycerol into triacylglycerols and diacylglycerols showed a slight increase concomitantly with decreased radioactivity in the major phospholipids, phosphatidylcholine and phosphatidylethanolamine. In addition, these changes were associated with a greater release of radiolabeled triacylglycerols into the culture medium. These results indicate that ethanol does not cause in HepG2 cells the marked lipogenic stimulation widely shown in hepatocytes, and demonstrate that HepG2 cells strongly resist the adverse effects of ethanol. Since these cells lack the isoenzymatic form of cytochrome P(450) mainly involved in the ethanol metabolism (namely cytochrome P(450)2E1) and also are devoid of alcohol dehydrogenase activity, we propose that the toxic actions of ethanol on liver must be linked to the activity of one or both of these systems.     

Effect of typical inducers of microsomal drug-metabolizing enzymes on phospholipid metabolism in rat liver.

The effect of administration of phenobarbital (PB), polychlorinated biphenyls(PCBs) or 3-methyl-cholanthrene (3-MC) on the metabolism of phospholipids in rat liver was studied by the i.p. injection of [³²P]orthophosphate or [Me-¹⁴C]choline chloride. The inducers were given to animals for 2 successive days PB had no significant effect on the incorporation rate of ³²Pi into liver microsomal phospholipid classes 48 hr after the first administration. The rate of incorporation of [¹⁴C]choline into phosphatidylcholine (PC) in both subcellular components of the liver and blood plasma decreased slightly at this experimental period. In addition, the ratio of [¹⁴C] sp. act. of phosphorylcholinc to that of microsomal phosphulipid was considerably higher on PB-trcated rats as compared with the ratio in control rats. These and previous findings strongly suggest that proliferation of liver endoplasmic reticulum (ER) membranes induced by PB would be accompanied by the stimulation of phospholipid synthesis at the early process of induction and subsequently followed by the decrease in turnover rate of microsomal phospholipids. The administration of PCBs, on the other hand, caused strong inhibition of both ³²Pi and ¹⁴C incorporation into PC in liver subcellular fractions. The secretion of PC from liver cells to blood plasma was also strongly depressed. In addition, phospholipid catabolizing activity was found to be depressed in the liver. These results indicate that hypertrophy of ER membranes in the liver after PCBs administration could be due to a depression of both secretion of lipoprotein from liver cells to plasma and catabolic activity toward membranous phospholipids in liver cells. The decrease in ³²Pi incorporation into liver microsomal PC was also observed in rats treated with 3-MC. There occurred a considerable accumulation of ¹⁴C activity in phosphorylcholine in the liver of rats treated with either PCBs or 3-MC, suggesting strongly that these drugs caused an inhibition of PC synthesis at the site of CDP-choline formation, namely the inhibition of the reaction catalyzed by cholinephosphate cytidylyltransferase.     

The effect of ethanol on phospholipid metabolism in rat pancreas

The phospholipid effect involves agonist-induced breakdown of phosphatidyl inositol (or polyinositides) generating second messengers followed by increased incorporation of 32P during the resynthetic phase of the cycle. Ethanol, an aetiological factor in pancreatitis, has been shown to have various effects on pancreatic secretion. In this study ethanol decreased the incorporation of 32P into phosphatidyl inositol but had no effect on the stimulated breakdown of prelabelled phosphatidyl inositol. However, in addition to recycling of phosphatidyl inositol stimulation of pancreatic tissue results in increased incorporation of precursors into other phospholipids. Cholecystokinin increased the incorporation of both [U-14C] glucose and 32P into phosphatidyl ethanolamine 3-fold but had no effect on 32P incorporation into phosphatidyl choline. As well as increased incorporation of 32P into phosphatidyl inositol (8-fold) cholecystokinin also increased the incorporation of [U-14C] glucose into phosphatidyl inositol (4-5-fold) implying significant de novo synthesis of 1,2 diacyl glycerol in addition to the currently accepted recycling of the 1,2 diacyl glycerol back to phosphatidyl inositol. Ethanol caused an inhibition of 32P incorporation into total phospholipid of rat pancreas during basal and stimulated conditions. When individual phospholipids were separated ethanol was found to decrease the incorporation of 32P into phosphatidyl choline under basal conditions and into all phospholipids during cholecystokinin stimulation. With [U-14C] glucose as the precursor, ethanol inhibited its incorporation into phosphatidyl choline only. Ethanol did not alter the total 32P radioactivity in the aqueous phase of the pancreatic extract nor the percent incorporated into nucleotides. This excluded decreased uptake of 32P and incorporation into nucleotides as a mechanism for the differential inhibition of 32P versus [U-14C] glucose incorporation into phospholipids other than phosphatidyl choline under stimulated conditions.     

Effect of polychlorinated biphenyls (PCBs) administration on phospholipid biosynthesis in rat liver

The effect of PCBs or phenobarbital on the biosynthesis of phospholipids in hepatic endoplasmic reticulum of rats was studied by the intraperitoneal injection of [³²P]orthophosphate, [Me?¹⁴ C]choline or [2?³H]glycerol. Significant increases in liver microsomal phospholipid content after the administration of either PCBs or phenobarbital indicated the actual proliferation of endoplasmic reticulum membranes. The rate of both [³²P] and [¹⁴C] incorporations into microsomal choline-containing phospholipids, such as phosphatidylcholine, sphingomyelin and lysophosphatidylcholine, was reduced to one fifth by PCBs administration compared with control animals. The incorporation of [³²P]orthophosphate into phosphatidylethanolamine or other phospholipid classes was less or not affected, respectively, by PCBs administration. The specific inhibitory effect of PCBs on the incorporation into cholinecontaining phospholipids was not observed when [2?³-H]glycerol was used as a precursor. Phenobarbital administration, however, increased significantly the rate of [³²P] incorporation into liver phospholipids, especially phosphatidylcholine. It is suggested that the increase in microsomal phospholipid content by PCBs administration is not due to the stimulation of synthesis but to the inhibition of the catabolism of membrane phospholipids and that the increase in content caused by phenobarbital is due at least in part, to the stimulation of synthesis. The possible site(s) of PCBs-induced inhibition of phospholipid biosynthesis in rat liver is discussed.     

AMIODARONE-INDUCED PHOSPHOLIPIDOSIS: AN IN VIVO [14C]-ACETATE UPTAKE STUDY IN RAT

ABSTRACT

Amiodarone (AD), a potent antiarrhythmic drug, is often associated with several adverse effects. It is shown to accumulate phospholipids in various tissues, and the impaired catabolism of phospholipids has been implicated in AD-induced phospholipidosis. The synthesis of phospholipids in tissues has not been dealt with. Hence, the incorporation of [¹⁴C]-acetate into phospholipids has been studied to understand the AD-induced phospholipidosis in lung and liver. A significant increase in lung and liver phospholipids was observed after 21 and 28 days of AD (175 mg/kg body weight/day) treatment. In the lung and liver, the incorporation of [¹⁴C]-acetate into all phospholipid fractions was elevated, while in the lung mitochondria phosphatidylcholine, phosphatidyl ethanolamine and the cardiolipin levels were significantly increased. The results indicate that, in addition to the impaired catabolism of phospholipid, AD treatment resulted in increased phospholipid synthesis.     

Effect of phenobarbital on methyl transfer between methylated drugs and hepatic microsomal phospholipids.

The effect of phenobarbital on the incorporation of the label from N-[¹⁴C-Me]nicotine and [¹⁴C]formaldehyde into hepatic phospholipids of the rat has been studied. ¹⁴C was utilized for the formation of methylated phospholipids from both precursors. Phenobarbital elicited no significant action either on the synthesis of total hepatic phospholipids or on the incorporation of radioactivity into the total or individual liver phospholipid fractions. However, this treatment increased phospholipid content and the uptake of the label from nicotine into microsomal phospholipids. Phenobarbital raised microsomal phosphatidylethanolamine, -choline (PC), -serine (PS), and lysophosphatidylcholine contents and the incorporation of ¹⁴C-labeled methyl groups from nicotine into PC and PS fractions. Radioactivity from [¹⁴C]formaldehyde was also incorporated into hepatic phospholipids. Phenobarbital however, had no significant effect on the incorporation either into total or microsomal phospholipids. Comparing the utilization of ¹⁴C for synthesis of liver microsomal phospholipids from N-[¹⁴C-Me]nicotine or [¹⁴C]formaldehyde with the natural methyl donor, l-[¹⁴C]-Me]methionine, greater amounts were taken up from methionine than nicotine or formaldehyde. The methyl group of nicotine was probably incorporated into phospholipids via the metabolic pool; the enhancing effect of phenobarbital on this process was associated with increased metabolism and with increased methyl transfer into methyl group containing microsomal phospholipids.     

Effects of hexafluoroacetone on testicular morphology and lipid metabolism in the rat

Rats dosed dermally with 39 or 130 mg/kg/day hexafluoroacetone sesquihydrate (HFA) for 14 days developed moderate or severe testicular atrophy, respectively; rats dosed with 13 mg/kg/day HFA for 14 days did not. Histologic evaluation of the testes revealed that spermatids, followed by spermatocytes, were the germ cells most affected by HFA; spermatogonia and Sertoli cells appeared to be less vulnerable. Lipogenesis from [³H]acetate and [¹⁴C]glucose was investigated in vitro in testes from HFA-treated and pair-fed control rats. Triacylglycerol and phospholipid synthesis was increased whereas sterol synthesis was decreased in testes from HFA-treated rats. Vitamin A and zinc were measured in the testes of control and HFA-treated rats; no differences in the levels of these nutrients were observed between the two groups. The data support the hypothesis that altered lipid metabolism, in particular sterol metabolism, is associated with the development of HFA-induced testicular atrophy.     

A study of ozone-induced edema in the isolated rat lung in relation to arachidonic acid metabolism,mixed-function oxidases and angiotensin converting enzyme activities

In order to elucidate the role of arachidonic acid in the pathogenesis of ozone-induced pulmonary edema, isolated rat lungs were exposed to ¹⁴C-arachidonic acid in the presence or absence of ozone and the incorporation of radiolabelled arachidonate into pulmonary cell lipids was studied. The perfusates from these studies were also subjected to differential extraction and thin layer chromatography (t.l.c.) to determine synthesis of both cyclo-oxygenase and lipoxygenase products. In the presence of an edemagenic concentration of ozone, isolated lungs incorporated significantly less exogenous arachidonic acid into phosphatidyl choline and phosphatidyl ethanolamine, whereas incorporation into phosphatidyl inositol or serine was not affected. The edemagenic concentration of ozone also increased production of a variety of arachidonic acid metabolites via cyclo-oxygenase and lipoxygenase pathways. In separate studies, a similar ozone exposure did not affect ¹⁴CO₂ production, resulting from the metabolism of ¹⁴C-antipyrine by mixed function oxidases (MFO). Similarly, an edemagenic concentration of ozone did not affect pulmonary angiotensin converting enzyme activity (ACE) as determined by the rate of formation of ¹⁴C-hippuric acid from ¹⁴C-hippuryl-histidyl-leucine (¹⁴C-HHL). Thus, acute ozone exposure is specifically associated with a reduced incorporation of arachidonate into phospholipids and with an increased conversion of arachidonate into bio-active metabolites.     

Association of inhaled [14C]methyl chloride with macromolecules from various rat tissues

Inhalation of ¹⁴C-labeled methyl chloride (CH₃Cl) by rats resulted in accumulation of radioactivity in lipid, RNA, DNA, and protein isolated from lung, liver, kidney, testes, brain, muscle, and intestine. At the end of a 6-hr exposure to 500 or 1500 ppm ¹⁴CH₃Cl, 8 to 20% of the radioactivity present in whole tissue homogenates was associated with acid-insoluble material. Most of this activity represented labeling of protein and lipid, although the concentration of ¹⁴C was usually over 10-fold higher in nucleic acids, per mole of nucleotide, compared with protein, per amino acid residue. Sephasorb HP chromatography of DNA revealed that radioactivity derived from ¹⁴CH₃Cl was associated with normal purine bases and no methylated bases were detected. Similarly, 82% of the radioactivity present in the lipid fraction copurified with the major species of phospholipids upon thin-layer chromatography. Pretreatment of rats with cycloheximide reduced the amount of CH₃Cl-derived radioactivity associated with tissue protein by 42 to 58%, whereas [³H]leucine incorporation was inhibited by 75 to 85%, indicating that most, but not necessarily all, of the uptake of ¹⁴CH₃Cl into protein was dependent on normal protein synthesis. Pretreatment with methotrexate inhibited the uptake of ¹⁴CH₃Cl into lipid, acid-insoluble material, RNA, and DNA by 47, 64, 65, and 93%, respectively. Pretreatment with methanol inhibited ¹⁴CH₃Cl uptake into acid-insoluble material by 66%. These findings indicate that most of the ¹⁴C appearing in macromolecules following inhalation of ¹⁴CH₃Cl arose through metabolic incorporation, stemming from metabolism of CH₃Cl via the one-carbon pool. Methanol apparently competed with CH₃Cl for entry into this pool, although the failure of ethanol, 4-methylpyrazole, or 3-amino-1,2,4-triazole to inhibit ¹⁴CH₃Cl incorporation indicated that CH₃Cl was not metabolized to methanol per se. Methanol pretreatment also inhibited ¹⁴CO₂ evolution from ¹⁴CH₃Cl, indicating that metabolism of CH₃Cl via single-carbon pathways may be of major quantitative significance.     

Effect of T-2 toxin administration to rats on lipid metabolism in liver

The effect of oral dosing of rats with 1.5 mg T-2 toxin/kg body weight daily for 4 days on metabolism of liver lipids was studied. T-2 toxin significantly elevated total liver lipids, triglycerides, free cholesterol, total phospholipids and phosphatidyl choline, whereas the level of sphingomyelin + lysophosphatidyl ethanolamine was reduced. No change in the esterified cholesterol and phosphatidyl ethanolamine contents was observed. Incorporation of [1-14C]acetate into liver lipids, esterified cholesterol, triglycerides, free cholesterol and phosphatidyl ethanolamine was reduced in T-2 toxin-treated animals, implying reduced lipogenesis. Increased lipids in liver in T-2 toxin-treated rats are possibly due to an impaired secretion of lipids from the liver.     

Stimulation of fatty acid utilization by sodium clofibrate in rat and monkey hepatocytes

The acute effects of sodium clofibrate (NaCPIB) on the metabolism of [1-14C]palmitate, [1-14C]octanoate, [1-14C]butyrate, and [2-3H]glycerol by freshly isolated hepatocytes were tested to explore its mechanism of action. Labeled long-, medium-, and short-chain fatty acids were incorporated into all the major lipid classes and were oxidized to 14CO2 by the liver cells. The partitioning of labeled fatty acids from lipogenic towards oxidative pathways was inversely related to fatty acid chain length. [1-14C]Palmitate was incorporated mainly into cellular triglycerides and phospholipids; [1-14C]octanoate, mainly into triglycerides and free cholesterol; and [1-14C]butyrate, mainly into free cholesterol and phospholipids of the cells. NaCPIB (1-3 mM) rapidly stimulated the esterification of labeled palmitate or glycerol to triglycerides, but drug levels greater than 5 mM were inhibitory to esterification. NaCPIB (1 mM) increased the oxidation of [1-14C]palmitate to 14CO2 by either rat or monkey hepatocytes and enhanced the release of labeled lipids from [2-3H]glycerol-prelabeled cells into the extracellular medium. Accelerated [1-14C]octanoate incorporation into glycerolipids and sterols and increased [1-14C]octanoate conversion to 14CO2 were observed in rat liver cells incubated with 1 mM NaCPIB. In contrast, the same drug level stimulated the oxidation of [1-14C]butyrate to 14CO2 but greatly diminished its incorporation into hepatocellular sterols or glycerolipids. These results indicate that (a) NaCPIB acutely alters hepatic ultilization of fatty acids by actions at diverse loci; (b) these metabolic alterations vary with fatty acid chain length; and (c) these effects are probably due to rapid changes in biochemical regulatory mechanism and/or in substrate channelling within the cells. These data further suggest that the early hypolipidemic effect of the drug in rats and primates may be related to an enhanced hepatic oxidation of long-chain fatty acids, but cannot be attributed simply to a reduction in their esterification to complex lipids.     

The Effect of Low-, Medium- and High-Tar Cigarette Smoke on the Fate of Arachidonic Acid in Isolated Hamster Lungs

Abstract: Isolated hamster lungs were ventilated with smoke from low-, medium- and high-tar cigarettes when 45 nmol of ¹⁴C-arachidonic acid was infused into the pulmonary circulation. Most of the infused radioactivity was found in different phospholipid and neutral lipid fractions of the perfused lungs and a smaller amount was found in the nonrecirculating perfusion effluent mainly as metabolites. Cigarette smoke ventilation increased the amount of ¹⁴C-arachidonic acid in the triacylglycerols of the perfused lungs but had usually no effect on the amount of radioactivity in diacylglycerols or in different phospholipids. The increased amount of radiolabel in triacylglycerols was significantly greater in the lungs ventilated with smoke from medium- or high-tar cigarettes than in those ventilated with low-tar cigarette smoke. Cigarette smoke ventilation increased the amount of unmetabolized arachidonate in the perfusion effluent and decreased the amount of many metabolites. The present study indicates that low-, medium- and high-tar cigarettes have effects of the fate and metabolism of arachidonic acid in isolated hamster lungs and that the effects of medium- and high-tar cigarettes are more clear that those of low-tar cigarettes when the cigarettes are burned with a constant puff volume and rate.     

Cardenolide and Neutral Lipid Biosynthesis from Malonate in Digitalis lanata

DIGITALIS LANATA plants were grown on water culture in a controlled environment and in the young, growing leaves free sterols (0.335 micromol/g FW), triacylglycerols (0.97 micromol/g FW) and cardenolides (1.82 micromol/g FW) were the major apolar and polar lipids. The cardenolide-containing fraction from these tissues was separated into 26 cardenolides by HPLC. The 5 major components (accounting for 60 % of the occurring glycosides) lanatosides A and C, acetyldigoxin, acetyldigitoxin, and glucoevatromonoside were identified by FABMS. Incorporation experiments with [2- (14)C]-acetate, [2- (14)C]-malonate, [2- (14)C]-mevalonate, and [U- (14)C]-sucrose (absorbed by excised, young, growing leaves) showed the labelling of all the occurring cardenolides after a 3 day incorporation period (as judged by HPLC). Comparing the simultaneous synthesis of labelled sterols and triacylglycerols, malonate could be considered as the most effective precursor in cardenolide synthesis, reaching an incorporation value of 4.1 % in a 4 day incorporation period. A time-course experiment revealed a temporary accumulation of (14)C in glucoevatromonoside, which may play a role as an intermediate in cardenolide production of DIGITALIS LANATA.     

Drug protein conjugates—I. A study of the covalent binding of [14C]captopril to plasma proteins in the rat

The metabolism of [¹⁴C]captopril has been investigated in vitro and in vivo in male Wistar rats. The formation of conjugates of [¹⁴C]captopril with plasma proteins was observed both in vitro and in vivo: 180 min after intravenous infusion of [¹⁴C]captopril 35 ± 5% of total radioactivity was covalently bound to plasma proteins. The fate of [¹⁴C]captopril-plasma protein conjugates was investigated in vivo. [¹⁴C]Captopril was incubated in vitro with rat and human plasma and the resulting captopril-protein conjugates were infused into male rats. The plasma concentration of [¹⁴C]captopril-rat plasma protein conjugates declined monoexponentially with a half-life of 71.1 ± 2.2 min. After 180 min 28 ± 3% of the radioactivity was excreted in urine, largely as [¹⁴C]captopril-cysteine mixed disulphide (67%). Thus although captopril readily forms covalent bonds with plasma proteins the resulting conjugates dissociate in vivo. The toxicological implications of these findings are discussed.     

Effects of dl-propranolol on the synthesis of glycerolipids by rabbit iris muscle

Propranolol (0.03?0.3 mM), an amphiphilic cationic drug which is used therapeutically as a β-blocker, was found to alter significantly the incorporation of [¹⁴C]glucose, [¹⁴C]glycerol, [¹⁴C]acetate, ³²Pi, [³H]cytidine, [³H]inositol, [¹⁴C]choline, [¹⁴C]ethanolamine and [¹⁴C]serine into phospholipids of the iris muscle. Furthermore, it was found to exert a stimulatory effect on the [¹⁴C]serine incorporation into phosphatidylserine of the muscle and microsomes. In contrast, sotalol, another β-blocker-but lacking the hydrophobicity of propranolol-exerted no effect on lipid metabolism. Whereas norepinephrine stimulated only the turnover of the phosphate moiety of phosphatidic acid and phosphatidylinositol, in general propranolol caused the following changes: (a) it stimulated by 2- to 6-fold the labelling of phosphatidic acid and phosphatidylinositol from [¹⁴C]glucose, [¹⁴C]glycerol, [¹⁴C]acetate, ³²Pi and [³H]inositol, (b) it increased by 5- and 38-fold the incorporation of ³²Pi and [³H]cytidine, respectively into CDP-diglyceride, (c) it inhibited appreciably the incorporation of [¹⁴C]glucose, [¹⁴C]glycerol, [¹⁴C]acetate and ³²Pi into phosphatidylcholine and phosphatidylethanoalmine. However, while it inhibited significantly the [¹⁴C]choline incorporation into the former, it stimulated by 60 per cent the ethanolamine incorporation into the latter phospholipid. These results indicate that propranolol probably redirects phospholipid synthesis de novo, by inhibiting phosphatidate phosphohydrolase, such that the increase obtained in the biosynthesis of phosphatidylinositol is accompanied by a corresponding decrease in the synthesis of phosphatidylcholine and phosphatidylethanolamine.Propranolol also caused a 250 per cent increase in the [¹⁴C]serine incorporation into phosphatidylserine of the iris muscle and 28 per cent increase in that of microsomes. The drug appears to stimulate the Ca²⁺ -uptake by muscle and microsomes, which in turn could act to stimulate the Ca²⁺-catalyzed base-exchange reaction.In addition the metabolic pathways involved in the biosynthesis of the major phospholipids of the iris, a smooth muscle, are reported for the first time. These pathways were found to be essentially similar to those reported for other tissues.