Alkylthioacetic acids (3-thia fatty acids) are metabolized and excreted as shortened dicarboxylic acids in vivo.

The metabolism of 1-14C-labeled long-chain alkylthioacetic acids (3-thia fatty acids) which are blocked for normal beta-oxidation by a sulfur atom in the beta-position has been investigated in vivo. Most of the injected radioactivity (greater than 50%) was excreted in the urine within the first 48 h. The recovered and identified metabolites were all short sulfoxydicarboxylic acids. The main metabolite from dodecylthioacetic acid was carboxypropylsulfoxy acetic acid. Some bis(carboxymethyl)sulfoxide (dithioglycolic acid sulfoxide) was also found. The main metabolite from nonylthioacetic acid was carboxyethylsulfoxyacetic acid. No sulfones were found. Less than 1% of the 1-14C from the dodecylthioacetic acid was recovered in respiratory CO2 and about 3% of the 1-14C from nonylthioacetic acid. [1-14C]Dodecyl-sulfonylacetic acid was recovered almost quantitatively as carboxypropylsulfonylacetic acid in the urine after 3 h. A significant fraction (10% of the dodecylthioacetic acid was recovered in the phospholipids and triacylglycerols from liver and epidymal fat pad 4 h after injection. These experiments show that the alkylthioacetic acids undergo an initial omega-oxidation followed by beta-oxidation to short dicarboxylic acids.

Metabolism of dicarboxylic acids in vivo and in the perfused kidney of the rat.

After intraperitoneal injection of (1-14C)-labelled suberic or dodecanedioic acid, the acids themselves and their metabolites were excreted in urine and as 14CO2. There was a striking difference in the capacity to oxidize the two dicarboxylic acids. Most of the suberic acid was excreted unchanged in the urine, and less was recovered as 14CO2. A trace was excreted as adipic acid. Dodecanedioic acid was more efficiently oxidized; 2-3-times more was expired as 14CO2, and the urine contained only a trace of the unchanged acid. Adipic acid was the main metabolite. Kidney perfusion experiments confirmed these results by showing that unmetabolized suberic acid was actively excreted by the kidneys. Dodecanedioic acid was oxidized and shorter dicarboxylic acids were excreted. The perfused hindquarter did not metabolize the dicarboxylic acids. Our results show that dodecanedioic acid can be completely oxidized both in the whole animal and in the kidneys. Dicarboxylic acids in the urine may to a significant extent be formed in the kidneys themselves.

Metabolism of dicarboxylic acids in rat hepatocytes.

[carboxyl-14C]Dodecanedioic acid (DC12) is metabolized in hepatocytes at a rate about two thirds that of [1-14C]palmitate. Shorter dicarboxylates (sebacic (DC10), suberic (DC8), and adipic (DC6) acid) are formed, mainly DC6, less DC8 and only a little DC10. In hepatocytes from clofibrate-treated rats, more polar products account for most of the breakdown products, presumably because the beta-oxidation proceeds all the way to succinate and acetyl-CoA. [carboxyl-14C]Suberic acid (DC8) is oxidized at a rate only one fifth that of dodecanedioic acid. (+)-Decanoylcarnitine inhibits palmitate oxidation but not the oxidation of dodecanedioic acid. At low concentrations of [carboxyl-14C]dodecanedioic acid or of [1-14C]palmitate, acetylsulfanilamide is more efficiently labeled by the former. High concentrations of dodecanedioic acid inhibit palmitate oxidation and the acetylation of sulfanilamide, presumably because their CoA-esters accumulate in the cytosol. These results indicate that medium-chain dicarboxylic acids are beta-oxidized mainly in the peroxisomes.

The effects of alkylthioacetic acids (3-thia fatty acids) on fatty acid metabolism in isolated hepatocytes.

Long-chain alkylthioacetic acids (3-thia fatty acids) inhibit fatty acid synthesis from [1-14C]acetate in isolated hepatocytes, while fatty acid oxidation is nearly unaffected or even stimulated. Desaturation of [1-14C]stearate (delta 9-desaturase) is also unaffected. [1-14C]Dodecylthioacetic acid (a 3-thia fatty acid) is incorporated in triacylglycerol and in phospholipids more efficiently than [1-14C]palmitate in isolated hepatocytes. The metabolism of [1-14C]dodecylthioacetic acid to acid-soluble products (by omega-oxidation) is slow compared to the oxidation of [1-14C]palmitate. In hepatocytes from adapted rats (rats fed tetradecylthioacetic acid for 4 days) the rate of [1-14C]palmitate oxidation is increased and its rate of esterification is decreased. Stearate desaturation is also decreased. The rate of cyanide-insensitive peroxisomal fatty acid beta-oxidation is several-fold increased. The metabolic effects of long-chain 3-thia fatty acids are discussed and it is concluded that they behave essentially like normal fatty acids except for their slow breakdown due to the sulfur atom in the 3 position, which blocks normal beta-oxidation.

The effect of adaptation on the metabolism of dodecylthioacetic acid (a 3-thia fatty acid) in rat tissues.

Dodecylthioacetic acid (DTA) was both omega-hydroxylated and sulfur-oxygenated at about equal rates by the microsomal fraction from liver and kidney. Feeding tetradecylthioacetic acid (TTA) for 4 days increased omega-hydroxylation 4-fold only in the liver. The sulfur oxygenation rate was similar in liver, kidney and lung, barely detectable in heart and absent in intestinal mucosa. In isolated hepatocytes from normal rats the major metabolite from dodecylthioacetic acid was carboxypropylsulfoxyacetic acid. In hepatocytes from adapted rats, the main product was identified as bis(carboxymethyl)sulfide. In kidney perfusion experiments dodecylthioacetic acid was metabolized to carboxypropyl-sulfoxyacetic acid and preferentially excreted in the urine. In hindquarter perfusion experiments no oxidative metabolites were detected. These experiments show that only liver and kidney can metabolize dodecylthioacetic acid completely and that omega-hydroxylation in the liver is the only inducible activity, in addition to the beta-oxidation.

Carnitine palmitoyltransferase: activation and inactivation in liver mitochondria from fed, fasted, hypo- and hyperthyroid rats.

The sensitivity of carnitine palmitoyltransferase to malonyl-CoA is lost when liver mitochondria are preincubated in a KCl-containing medium. This loss of sensitivity is slowed down in mitochondria from hypothyroid rats and accelerated in mitochondria from fasted and hyperthyroid rats. Glucagon seems to enhance the effect of fasting. The loss of sensitivity is significantly slowed down by 50-500 nM malonyl-CoA and accelerated by small amounts of palmitoyl-CoA in the preincubation medium.

Microsomal oxidation of dodecylthioacetic acid (a 3-thia fatty acid) in rat liver.

[1-14C]Dodecylthioacetic acid (DTA), a 3-thia fatty acid, is omega (omega-1)-hydroxylated and sulfur oxygenated at about equal rates in rat liver microsomes. In prolonged incubations DTA is converted to omega-hydroxydodecylsulfoxyacetic acid. omega-Hydroxylation of DTA is catalysed by cytochrome P450IVA1 (or a very closely related isoenzyme in the same gene family), the fatty acid omega-hydroxylating enzyme. It is absolutely dependent on NADPH and inhibited by CO, and lauric acid is a competing substrate. omega-Hydroxylation of DTA is increased by feeding tetradecylthioacetic acid (TTA), a 3-thia fatty acid, for 4 days to rats. omega-Hydroxylation of [1-14C]lauric acid is also induced by TTA and other 3-thia carboxylic acids. A close relationship was observed between induction of microsomal omega-hydroxylation of fatty acid and palmitoyl-CoA hydrolase activity. DTA is omega-hydroxylated at about the same rate as the physiological substrate lauric acid. The sulfur oxygenation of DTA is catalysed by liver microsomal flavin-containing monooxygenase (FMO) (EC 1.14.13.8). It is dependent on either NADH or NADPH. The Km value for NADH was approx. five times larger than the Km value for NADPH. It is inhibited by methimazole and not affected by CO. It is not induced by TTA.

Expression of the second epidermal growth factor-like domain of human factor VII in Escherichia coli.

The second epidermal growth factor (EGF)-like domain of human coagulation factor VII is a potent inhibitor of the FVIIa/tissue factor complex, the predominant initiator of coagulation in vivo. This domain has now for the first time been cloned and expressed in Escherichia coli as an affinity fusion protein. The fusion protein was secreted into the periplasm of E. coli and purified by affinity chromatography. The purified protein consisted of a fusion protein with the expected molecular weight, and in addition, a significant fraction of oligomers cross-linked by intermolecular disulfide bonds. Despite the presence of oligomers, the purified protein was a potent inhibitor of the extrinsic blood coagulation pathway with an IC50 value of about 20 microM. The biological activity was retained after liberation of the EGF domain by proteolytic cleavage.

The effect of feeding fish oils, vegetable oils and clofibrate on the ketogenesis from long chain fatty acids in hepatocytes.

Groups of rats were fed diets containing 25% fish oil (FO), 25% soybean oil, 25% partially hydrogenated fish oil (PHFO), 25% partially hydrogenated soybean oil (PHSO), 25% partially hydrogenated coconut oil or 0.3% clofibrate for 3 wk. After the animals were fasted for 24 hr, hepatocytes were isolated and ketogenesis from added palmitate, linoleate cis and trans, arachidonate and docosahexaenoate was measured. Ketogenesis after oil feeding was significantly stimulated (two- to threefold) only in cells from the PHFO- and PHSO-fed rats. The stimulation was most apparent with the long chain unsaturated fatty acids as substrates. These fatty acids were relatively poor ketone body precursors in control hepatocytes. Essential fatty acid deficiency did not seem to be the reason for this stimulation. Clofibrate also stimulated ketogenesis significantly (1.5- to 3-fold). The degree of stimulation increased with chain length and degree of unsaturation of the substrate. The activity of the enzyme 2,4-dienoyl-CoA reductase was also studied in the same groups. Its activity was stimulated about fourfold in the clofibrate-treated rats and to a lesser extent by the PHFO, PHSO and FO diets. The activity showed no correlation with the content of unsaturated fatty acids in the diet or their oxidation in isolated hepatocytes. The 2,4-dienoyl-CoA reductase, therefore, does not seem to be a regulatory enzyme in the metabolism of dietary polyunsaturated fatty acids. It is concluded that an induction of the peroxisomal beta-oxidation system most likely is involved in the reported increases in ketogenesis from very long chain polyunsaturated fatty acids.

Subcellular localization of vitamin D3 25-hydroxylase in human liver.

Vitamin D3 25-hydroxylase activity was measured in subcellular and submitochondrial fractions of human liver. Quantitation of 25-hydroxyvitamin D3 was based on high performance liquid chromatography. Vitamin D3 25-hydroxylase activity was detected in the mitochondrial fraction only. The mitochondrial 25-hydroxylase activity was linear with time up to 60 min and with mitochondrial protein up to 1 mg/ml. An apparent Km value of about 10(-5) M was found. Substrate satuation level was not reached. In the presence of 2.4 X 10(-4) M vitamin D3, the rate of 25-hydroxyvitamin D3 formation was 0.19 nmol X mg of protein-1 X h-1 After fractionation of the mitochondria, 86% of the 25-hydroxylase activity was recovered in the mitoplast fraction. The outer membrane fraction was devoid of activity. It is concluded that human liver contains only one detectable vitamin D3 25-hydroxylase enzyme localized to the mitochondrial inner membrane.

Effects of thia-substituted fatty acids on mitochondrial and peroxisomal beta-oxidation. Studies in vivo and in vitro.

1. The effects of 3-, 4- and 5-thia-substituted fatty acids on mitochondrial and peroxisomal beta-oxidation have been investigated. When the sulphur atom is in the 4-position, the resulting thia-substituted fatty acid becomes a powerful inhibitor of beta-oxidation. 2. This inhibition cannot be explained in terms of simple competitive inhibition, a phenomenon which characterizes the inhibitory effects of 3- and 5-thia-substituted fatty acids. The inhibitory sites for 4-thia-substituted fatty acids are most likely to be the acyl-CoA dehydrogenase in mitochondria and the acyl-CoA oxidase in peroxisomes. 3. The inhibitory effect of 4-thia-substituted fatty acids is expressed both in vitro and in vivo. The effect in vitro is instantaneous, with up to 95% inhibition of palmitoylcarnitine oxidation. The effect in vivo, in contrast, is dose-dependent and increases with duration of treatment. 4. Pretreatment of rats with a 3-thia-substituted fatty acid rendered mitochondrial beta-oxidation less sensitive to inhibition by 4-thia-substituted fatty acids.

Free acetate production by rat hepatocytes during peroxisomal fatty acid and dicarboxylic acid oxidation.

The fate of the acetyl-CoA units released during peroxisomal fatty acid oxidation was studied in isolated hepatocytes from normal and peroxisome-proliferated rats. Ketogenesis and hydrogen peroxide generation were employed as indicators of mitochondrial and peroxisomal fatty acid oxidation, respectively. Butyric and hexanoic acids were employed as mitochondrial substrates, 1, omega-dicarboxylic acids as predominantly peroxisomal substrates, and lauric acid as a substrate for both mitochondria and peroxisomes. Ketogenesis from dicarboxylic acids was either absent or very low in normal and peroxisome-proliferated hepatocytes, but free acetate release was detected at rates that could account for all the acetyl-CoA produced in peroxisomes by dicarboxylic and also by monocarboxylic acids. Mitochondrial fatty acid oxidation also led to free acetate generation but at low rates relative to ketogenesis. The origin of the acetate released was confirmed employing [1-14C]dodecanedioic acid. Thus, the activity of peroxisomes might contribute significantly to the free acetate generation known to occur during fatty acid oxidation in rats and possibly also in humans.

25-Hydroxyvitamin D3-24-hydroxylase in rat kidney mitochondria.

Assay conditions for the measurement of 25-hydroxyvitamin D3-24-hydroxylase activity in rat kidney mitochondria have been worked out. The product, 24,25-dihydroxyvitamin D3 was quantitated either by high pressure liquid chromatography or by isotope dilution-mass spectrometry. By these procedures, the enzyme activity could be measured with saturating concentration (greater than 2.5 X 10(-6) M) of substrate. Pretreatment of the animals by aminophylline (Kulkowski, J. A., Chow, T., Martinez, J., and Ghazarian, J. G. (1979) Biochem. Biophys. Res. Commun. 90, 50-57) stimulated the 24-hydroxylase activity in vitro at least 2 to 3-fold. The identity of the product was verified by gas chromatography-mass spectrometry. The rates of the reaction varied between 1.5 and 5 pmol/mg of mitochondrial protein.min (at 25 degrees C), and the K'm was determined to be 4.2 X 10(-7) M. Malate, succinate, and isocitrate were all able to support the reaction. Low O2 tension, CO, KCN, and the uncoupler carbonyl cyanide m-chlorophenylhydrazone inhibited the reaction, while the respiratory inhibitor rotenone had no effect. Metyrapone inhibited the reaction with 50% inhibition at a concentration of 2.5 mumol/ml. The enzyme was found to be localized inside the inner mitochondrial membrane. The results indicate that in the rat the renal mitochondrial 25-hydroxyvitamin D3-24-hydroxylase is a cytochrome P-450 and that the reducing equivalents are primarily supplied by NADPH via the energy-dependent transhydrogenase.