Jon Norseth

Clinical and biochemical heterogeneity in conditions with phytanic acid accumulation

Phytanic acid accumulation has for more than 20 years been used as a diagnostic criterion of Refsums disease. Recently, however, phytanic acid has also been found in peroxisomal disorders (Zellwegers syndrome, neonatal adrenoleukodystrophy, infantile Refsums syndrome, rhizomelic chondrodysplasia punctata). The 17 patients with Refsums disease in the present study had serum phytanic acid values differing from 73 to less than 0.5 mg/dl (normal). alpha-Oxidation of phytanic acid in skin fibroblast cultures showed a defective capacity in all, with only small differences in residual activity. Phytanic acid determinations in serum from 3 of the 7 patients with peroxisomal disorders showed slightly elevated levels in 2. The alpha-oxidation capacity in the fibroblasts was defective in all, with a residual activity similar to that of Refsums disease. An assay of the alpha-oxidation capacity may be useful in the diagnosis of both Refsums disease and the peroxisomal disorders. The distinction between Refsums disease and the peroxisomal disorders can easily be done on a clinical basis.

Stimulation of microperoxisomal β-oxidation in rat heart by high-fat diets

Abstract 1. 1. Heart microperoxisomal β-oxidation activity, measured as cyanide-insensitive palmitoyl-CoA-dependent NAD + -reduction, was detected in a microperoxisome-enriched fraction from rat myocardium. The effect on this microperoxisomal β-oxidation of the fatty acid composition of the dietary oils was investigated. 2. 2. Feeding 15% (w/w) high erucic acid rapeseed oil or partially hydrogenated marine oil for 3 weeks increased the microperoxisomal β-oxidation in the heart 4–5-fold, compared to a soybean oil diet. Increasing amounts (5–30%, w/w) of partially hydrogenated marine oil in the diet led to a 3-fold increase in the microperoxisomal β-oxidation capacity at 20% or more of this oil in the diet. 3. 3. The activity of the microperoxisomal marker enzyme catalase followed closely the cyanide-insensitive palmitoyl-CoA-dependent NAD + -reduction, except when feeding more than 20% (w/w) partially hydrogenated marine oil where a significant decrease in the catalase activity was observed. 4. 4. In rapeseed oil-fed animals the extent of increase of microperoxisomal β-oxidation was directly correlated to the amount of erucic acid (22:1, n−9 cis ) in the diet. 5. 5. Feeding partially hydrogenated rapeseed oil or partially hydrogenated soybean oil resulted in activities of microperoxisomal β-oxidation significantly lower than in the corresponding unhydrogenated oils. No significant difference could be detected between diets containing hydrogenated or unhydrogenated marine oil. 6. 6. Addition of 5% soybean oil to the essential fatty acid-deficient, partially hydrogenated marine oil diet did not change the effect on the microperoxisomal β-oxidation activity. 7. 7. Clofibrate feeding increased the heart microperoxisomal β-oxidation capacity 2.5-fold, as compared to a standard pelleted diet. 8. 8. These findings are discussed in relation to the transient nature of the cardiac lipidosis observed with animals fed on diets rich in C 22:1 fatty acids. It is concluded that the heart plays an important part in the adaptation process.

The effect of feeding rats with partially hydrogenated marine oil or rapeseed oil on the chain shortening of erucic acid in perfused heart.

1. The metabolism of [14(-14)C]erucic acid and [U-14C]palmitic acid was studied in perfused hearts from rats fed diets containing hydrogenated marine oil, rapeseed oil or peanut oil for three weeks. 2. [14C]Erucic acid was shortened to [14C]eicosenoic acid (20 : 1, n -- 9) and [14C]oleic acid (18 : 1, n -- 9) in perfused rat hearts from all diet groups. The rapeseed oil diet caused a three-fold increase and the marine oil diet a four-fold increase in the amount of chain-shortened products recovered in heart lipids at the end of perfusion, compared to peanut oil diet. 3. The content of C16:1, C18:1 and C20:1 fatty acids was increased in heart lipids of rats fed hydrogenated marine oil or rapseed oil diet, compared to peanut oil diet. 4. Feeding hydrogenated marine oil or rapeseed oil to the rats induced a 85% increase in catalase activity, a 20% increase in the activity of cytochrome oxidase and a 30--40% increase in the content of total CoA in the heart compared to rats fed peanut oil diet. 5. It is suggested that [14(-14)C]erucic acid is shortened by the beta-oxidation system of peroxisomes in the heart. The increased chain shortening in the hearts from animals fed rapeseed oil or partially hydrogenated marine oil for three weeks may be an important part of an adaptation process.

Chain shortening of erucic acid in isolated liver cells

C22 monounsaturated fatty acids are abundant in some types of rapeseed oil and some marine oils used for human consumption in many countries. In feeding experiments rapeseed oil, rich in erucic acid (22:1, n-9cis) causes a fatty infiltration in the heart of rats and several other species [1 ]. Previous studies with isolated heart and liver mitochondria have shown that erucic acid is oxidized at a distinctly slower rate than palmitic acid. It has also been shown that erucic acid inhibits the oxidation of other fatty acids in mitochondria, probably by an inhibitory effect on acyl CoA dehydrogenase [2]. In vivo experiments with 14C-labelled erucic acid given to rats have shown that part of the injected radioactivity is recovered in shorter monounsaturated fatty acids, particularly in oleic acid in liver [3]. A chain shortening of erucic acid in cultured myocytes has also been reported [4]. In the present work the metabolism of erucic acid in isolated liver cells has been studied.

Studies on the regulation of arachidonic acid synthesis in isolated rat liver cells

Isolated liver cells from rats fed a diet deficient in essential fatty acids were used to study the oxidation, esterification and, especially, the desaturation and chain elongation of [1-14C]linoleic acid. 14C-labelled arachidonic acid (20:4) and smaller amounts of eicosatrienoic acid (20:3) were recovered mainly in the phospholipids, while gamma-linolenic acid (18:3) was found in both the phospholipids and the triacylglycerol fraction. Lactate strongly increased the formation of arachidonic acid, which was found mainly in the phosphatidylcholine and the phosphatidylinositol fractions. Lactate reduced the amounts of gamma-linolenic acid. Glucagon and (+)-decanoylcarnitine reduced the formation of arachidonic acid, and (+)-decanoylcarnitine increased the incorporation of gamma-linolenic acid especially, in the triacylglycerol fraction. Increasing concentrations of the [1-14C]linoleic acid substrate increased the formation of arachidonic acid and of the other chain-elongated or desaturated fatty acids. Lactate also stimulated the formation of arachidonic acid in liver cells from animals fed adequate amounts of essential fatty acids. It is suggested that dietary and hormonal factors which can change the intracellular levels of malonyl-CoA may influence both the ratio of arachidonic acid/gamma-linolenic acid formed and the total amounts of desaturated and chain-elongated fatty acids formed from linoleic acid.

Increased β-oxidation of erucic acid in perfused hearts from rats fed clofibrate

1. The metabolism of [14-14C]erucate and [U-14C]palmitate has been investigated in perfused heart from rats fed 0.3% clofibrate for 10 days and from control rats. 2. The total uptake of fatty acids in the heart increased in the clofibrate fed group. Clofibrate increased the oxidation of [14-14C]erucic acid by 100% and the oxidation of [U-14C]palmitic acid by 30% compared to controls. 3. The chain-shortening of erucate to C20:1 and C18:1 fatty acids in the perfused heart was stimulated at least two-fold by clofibrate feeding. 4. The activity of the peroxisomal marker enzyme catalase increased 60%, the activity of cytochrome oxidase increased approx. 16% and the content of total coenzyme A increased 30% in heart homogenates from rats fed clofibrate compared to controls. 5. The isolated mitochondrial fraction from clofibrate fed rats showed an increased capacity for oxidation of palmitoylcarnitine and decanoylcarnitine, while the oxidation of erucoylcarnitine showed little change. 6. It is suggested that clofibrate increases the oxidation of [14-14C]erucic acid in the perfused heart by increasing the capacity for chain-shortening of [14-14C]erucate in the peroxisomal beta-oxidation system.

Long-term effects of high-fat diets on peroxisomal β-oxidation in male and female rats

In weanling male rats a 4-fold increase of heart triacylglycerols was observed after three days on a high-fat diet containing partially hydrogenated fish oil (PHFO). In female rats this increase was only about 50%. No significant differences were observed between female and male rats in the fatty acid composition of the accumulated lipids.The initial level of peroxisomal β-oxidation activity was similar in male and female rats in both liver and heart. After three weeks of receiving high-fat diets, the rats showed a marked increase in peroxisomal β-oxidation activity with PHFO in the diet and less with soybean oil (SO), confirming previous studies with male rats. Catalase activity was similarly affected in hearts of both sexes.In male rats the levels of peroxisomal β-oxidation observed after three weeks of feeding on the high-fat diets were found to be maintained, both in liver and heart, during a feeding period of three months. The response to high-fat diets in females, however, seems to be further accentuated after three months of feeding, resulting in a capacity of peroxisomal β-oxidation in liver of about three times that of the male rats when calculated on a total body-weight basis.

The effect of clofibrate on heart and plasma lipids in rats fed a diet containing rapeseed oil

The effect of clofibrate on heart and plasma lipids in rats fed a diet containing 30% of the calories as peanut oil (PO) or rapeseed oil (RSO) (42.7% erucic acid and 0.5% eicosenoic acid) was studied. A decrease of erucic acid content to one-third and concomitant increase in the content of 18∶1, 16∶1 and 16∶0 fatty acids in plasma triacylglycerols were observed after administration of clofibrate to rats fed the RSO-diet. It is suggested that these changes reflect the increased capacity of the liver to chainshorten very long chain length fatty acids. The extent of lipidosis in the heart of rats fed the RSO-diet was decreased by 50% by clofibrate. However, the concentration of erucic acid in heart triacylglycerols decreased much less (30%) than the concentration of all other fatty acids (50–65%). It is concluded that the clofibrate administration increased the oxidative capacity of the heart mitochondria and that the heart cell does not have an efficient system to handle very long chain length monounsaturated fatty acids as does the liver.

Hydrodynamic parameters and isolation of mitochondria, microperoxisomes and microsomes of rat heart.

1. Analytical differential centrifugation of rat heart homogenates revealed a single population of mitochondria and microperoxisomes. Using cytochrome c oxidase, malate dehydrogenase and amine oxidase as mitochondrial marker enzymes, the s-value of mitochondria was estimated to s = 10326 +/- 406 S (average for the three marker enzymes). The s-value of microperoxisomes was found to be s = 1381 +/- 40 S using catalase as the marker enzyme. The s-value for the two organelles did not change significantly when the isoosmotic sucrose medium was substituted by an isoosmotic mannitol medium. 2. Analytical differential centrifugation revealed a polydispercity of the microsomal fraction using glucose-6-phosphatase and NADPH-cytochrome c reductase as the marker enzymes. The s-values were found to be sH1 = 1569 +/- 412 S (NADPH-cytochrome c reductase), sH2 = 1195 +/- 400 S (glucose-6-phosphatase) and sL = 153 +/- 28 S (NADPH-cytochrome c reductase and glucose-6-phosphatase). The recovery of marker enzymes in the isolated subcellular fractions was in the range of 84-94%. 3. When the mitochondrial and microperoxisomal fractions were subjected to isopycnic gradient centrifugation, using a self-generating gradient of polyvinylpyrrolidone-coated colloidal silica particles (Percoll) in 0.25 M sucrose medium, buoyant densities of 1.10 g/cm3 (main fraction of mitochondria) and 1.06 g/cm3 (main fraction of microperoxisomes) were obtained. The density gradient centrifugation separated microperoxisomes from contaminating lysosomes of high specific activity in acid phosphatase. A value 1.04 g/cm3 was found for the density of the microsomal fraction. 4. Based on the estimated s-values, an optimal procedure is described for the isolation of mitochondrial and microperoxisomal fractions from rat heart muscle.

Arachidonic acid synthesis studied in isolated liver cells: Effects of (−)-carnitine and of (+)-decanoylcarnitine

The biosynthesis of arachidonic acid from linoleic acid has been extensively studied in isolated liver microsomes and in vivo [ 11. The mechanisms regulating the synthesis of polyunsaturated fatty acids have been little studied in isolated cells and perfused organs and are to a great extent still unknown. The aim of this work was to study whether isolated hepatocytes can be used as a suitable in vitro system to study the regulation of arachidonic acid synthesis. In most experiments the incubation medium was as in [5]. Linoleic acid with spec. act. 7 mCi/mmol was used. Cell suspension (1 ml) was mixed with 1 ml incubation medium containing the additions. The final linoleic acid level was 0.07 mM, and 5 mM glucose was present. Incubation was for 45 min. When indicated the hepatocytes were preincubated for 20 min with 1 mM (-)-carnitine or 1 mM (t)-decanoylcarnitine before the addition of linoleic acid.