Joel Avigan

A defect in pyruvate decarboxylase in a child with an intermittent movement disorder

A patient with an intermittent movement disorder has been found to have an inherited defect in pyruvate decarboxylase ((2-oxo-acid carboxy-lyase, E.C. 4.1.1.1.). The patient is a 9 yr old boy who since infancy has had repeated episodes of a combined cerebellar and choreoathetoid movement disorder. He has an elevated level of pyruvic acid in his blood, an elevated urinary alanine content, and less marked elevations in blood alanine and lactate. Methods were developed to study his metabolic abnormality in dilute suspensions of white blood cells and cultured skin fibroblasts, as well as in cell-free sonicates of fibroblasts. Oxidation of pyruvic acid-1-(14)C and pyruvic acid-2-(14)C by his cells and pyruvate decarboxylase activity in sonicates of his cells were less than 20% of those in cells from control subjects. Oxidation of glutamic acid-U-(14)C, acetate-1-(14)C, and palmitate-1-(14)C was normal, as was incorporation of alanine-U-(14)C into protein. The rate of oxidation of pyruvic acid by the fathers cells and the activity of pyruvate decarboxylase in the fathers sonicated fibroblasts were intermediate between those of the patient and those of controls. Values for the mother were at or just below the lower limits of the ranges in controls. Kinetic data suggested the posibility of several forms of pyruvate decarboxylase in this family. Possible mechanisms relating the chemical abnormality and the clinical symptoms in this patient are discussed.

Adrenoleukodystrophy Oleic acid lowers fibroblast saturated C22‐26 fatty acids

Monounsaturated fatty acids, especially oleic acid (C18:1), decreased the content of saturated very-long-chain fatty acids (VLFA) in cultured skin fibroblasts from patients with adrenoleukodystrophy (ALD) and controls. When confluent ALD fibroblasts were incubated with oleic acid for 5 days in lipid-free medium, which eliminates uptake of exogenous VLFA, the mean cell content of C26:0 was decreased by 33.7 ± 10.1%. Oleic acid inhibited C26:0 synthesis in ALD fibroblasts by 58% and total fatty acid synthesis by 68 to 78%. Therefore, the elevated C26:0 levels in ALD cells may be lowered by inhibiting fatty acid biosynthesis, and inhibition of saturated VLFA synthesis by oleate may be useful in treating ALD.

Alpha-decarboxylation, an important pathway for degradation of phytanic acid in animals

Abstract Patients suffering from Refsums disease, an inherited disorder of the nervous system ( Refsum, 1946 ), accumulate large stores of phytanic acid (3,7,11,15-tetramethylhexadecanoic acid) in their blood and tissues ( Klenk and Kahlke, 1963 ). There appears to be little or no endogenous synthesis of phytanic acid either in normal experimental animals ( Avigan, Steinberg and Cammermeyer, 1966 ; Mize et al, 1966 ) or in patients with Refsums disease ( Steinberg et al, 1965 , 1966a ) and the metabolic error is presumed to lie in a relative inability to degrade phytanic acid. It has been suggested on the basis of indirect evidence that an error in omega-oxidation might be involved ( Eldjarn, 1965 ), but there is as yet no direct information on the normal pathway for degradation of phytanic acid. The results reported below show that the normal rat rapidly converts phytanic acid (3,7,11,15-tetramethylhexadecanoic acid) to its α-decarboxylation product, pristanic acid (2,6,10,14-tetramethylpentadecanoic acid).

EFFECTS OF TRIPARANOL (MER-29) ON CHOLESTEROL BIOSYNTHESIS AND ON BLOOD STEROL LEVELS IN MAN*

The possibility of reducing elevated blood cholesterol levels with agents that inhibit endogenous biosynthesis of cholesterol is an attractive one, since dietary treatment is sometimes difficult to achieve and almost always difficult to maintain. Some of the theoretical aspects of such an approach have been outlined (3, 4), and the feasibility of the approach has been established in experimental animals. For example, it has been shown that A4-cholestenone, a potent inhibitor of cholesterol biosynthesis (5), can indeed lower levels of blood cholesterol (3, 6). Unfortunately, the feeding of A4-cholestenone in man and animals leads to accumulation of dihydrocholesterol in the serum (7) and this latter compound is known to be itself atherogenic (8). Certain other inhibitory substances have been explored but either have proved to be relatively ineffective (9, 10) or have not yet been adequately tested clinically (11, 12). In 1959 Blohm, MacKenzie, Kariya and Laughlin at the William S. Merrell Company reported their finding of a new inhibitor of cholesterol synthesis, MER-29 (1[p-,8-diethylaminoethoxy) phenyl] -1(p-tolyl) -2(p-chlorophenyl) ethanol). Their studies indicated that the compound blocked synthesis at some point subsequent to the formation of lanosterol (13) and that serum and tissue levels of cholesterol were reduced in experimental animals (14). Animal studies in this laboratory demonstrated the accumulation of desmosterol (24-dehydrocholesterol) in the tissues of MER29-treated animals, and a series of isotopic studies established that the major site of action of the drug was at the last step in cholesterol biosynthesis-namely, in the reduction of desmosterol to

Studies on the Metabolic Error in Refsum's Disease*

Studies utilizing mevalonic acid-2-(14)C and D(2)O as precursors failed to provide evidence for an appreciable rate of endogenous biosynthesis of phytanic acid in a patient with Refsums disease. Orally administered tracer doses of phytol-U-(14)C were well absorbed both by seven normal control subjects (61 to 94%) and by two patients with Refsums disease (74 and 80%). The fraction of the absorbed dose converted to (14)CO(2) in 12 hours was 3.5 and 5.8% in Refsums disease patients and averaged 20.9% in seven control subjects. Labeled phytanic acid was demonstrated in the plasma of both control subjects and patients given phytol-U-(14)C, establishing phytol in the diet as a potential precursor of phytanic acid. This labeled phytanic acid had disappeared almost completely from the plasma of the seven control subjects by 24 to 48 hours, whereas it persisted at high concentrations in the plasma of the two patients for many days. We conclude that the phytanic acid accumulating in Refsums disease is primarily of exogenous origin and that patients with Refsums disease have a relative block in the degradation of phytanic acid and possibly other similar branched-chain compounds. This may relate to a deficiency in mechanisms for release of phytanic acid from stored ester forms or, more probably, to reactions essential to oxidative degradation of the carbon skeleton.

Effects of Saturated and Unsaturated Fat on Cholesterol Metabolism in the Rat

Summary 1. Rats fed a diet containing 20% corn oil have significantly lower concentrations of serum cholesterol than those fed equal amounts of coconut oil but both high-fat diets lead to elevation of serum cholesterol above that seen on control diets. 2. There is a very marked increase in esterified cholesterol of livers of rats fed corn oil whereas coconut oil-fed rats show no significant changes in liver cholesterol compared to controls. 3. Rate of incorporation of 1-C14-acetate or of T2O is higher in corn oil-fed rats than in others. 4. Some implications with respect to the mechanism by which unsaturated fats alter cholesterol metabolism are discussed.

In vitro effects of serum proteins and lipids on lipid synthesis in human skin fibroblasts and leukocytes grown in culture.

Abstract Cultures of human skin fibroblasts incubated overnight in medium containing solvent-extracted serum showed a large increase in incorporation of labeled acetate into nonsaponifiable lipids. Freshly prepared monolayers of blood leukocytes were similarly stimulated following an incubation with solvent-extracted serum. No stimulation occurred with native serum. Incorporation of 3 H 2 O into nonsaponifiable lipids also increased substantially in both systems after the solvent-extracted serum treatment. The effect of solvent-extracted serum on nonsaponifiable lipids synthesis was paralleled in some cells by a stimulation of incorporation of acetate into fatty acids and of alanine into proteins. The stimulation of acetate incorporation into nonsaponifiable lipids was rapidly reversed in fibroblast cultures by whole serum, by low- and high-density lipoproteins, or by suspensions of cholesterol. These findings indicate that a feedback regulation of cholesterol synthesis may be operating in vitro both in cells grown in culture and in cells directly derived from the organism.

Adrenoleukodystrophy Very long‐chain fatty acid metabolism in fibroblasts

We studied very long-chain fatty acid (VLFA) metabolism in cultured fibroblasts from patients with adrenoleukodystrophy (ALD). Total hexacosanoate (C26:0) content of ALD fibroblasts was sixfold higher than normal and did not return to normal when cells were grown in lipid-free medium. When normal or ALD fibroblasts were grown in medium containing 10% ALD serum (which is enriched in C26:0), there was no further increase in C26:0 content compared with cells grown in 10% normal human serum. Uptake and loss of 1-14C-palmitate (C16:0) and 1-14C-lignocerate (C24:0) by ALD fibroblasts were similar to normal fibroblasts. Catabolism of exogenous H-C26:0 to H2O was about 307 of normal. Oxidation of exogenous 1-14C-hexacosanoate, 1-14C-lignocerate, and 1-14C-palmitate in intact ALD fibroblasts was 42%, 27 ± 13% (SD), and 73 ± 47%, respectively, of normal. These results are consistent with, but do not conclusively prove, a VLFA oxidation defect in ALD fibroblasts.

A major pathway for the mammalian oxidative degradation of phytanic acid.

Abstract A series of branched-chain saturated fatty acids accumulated in tissues of weanling mice fed regular chow supplemented with 2% phytanic acid (3,7,11,15 tetramethylhexadecanoic acid). They were identified by combined gas-liquid chromatography-mass spectrometry as phytanic acid, α-hydroxyphytanic acid, and the 19-, 16- and 14-carbon homologs of phytanic acid. Trace quantities of the 9-carbon homolog have also been detected by gas-liquid chromatography. When the regular chow is supplemented with 2% phytol (3,7,11,15-tetramethylhexadec-2-en-1-ol), Δ 2 -phytenic acid accumulated in liver in addition to the compounds listed above. No evidence was found for the presence of α-ketophytanic acid, or of 15- and 17-carbon intermediates. The latter two would be expected if carbon dioxide fixation to the branch-methyl group were required in phytol or phytanate metabolism, as in the bacterial metabolism of farnesoic acid. Dietary supplementation with 3,7,11-trime thyldodecanoic acid produces accumulation in liver of the fed compound only. Intravenous injection of uniformly 14 C-labeled phytanic acid in mice led to significant incorporation of radioactivity associated on gas-liquid chromatography with 19-16-, 14- and 11-carbon isoprenoid acids. Rat liver in vivo also rapidly converted in high yield uniformly 14 C-labeled phytanic acid or 2,3-dideuterophytanic acid to uniformly 14 C-labeled pristanic acid and 2-deuteropristanic acid, respectively. The oxidation of uniformly 14 C-labeled phytanic acid to 14 CO 2 was not impaired in biotindeficient rats. The proposed main metabolic pathway of phytanic acid in mammals consists of an α-oxidative process leading through α-hydroxyphytanic acid to pristanic acid, followed by a series of β-oxidative steps. The chemical syntheses of a number of isoprenoid acids are described.

Absorption and metabolism of uniformly 14C-labeled phytol and phytanic acid by the intestine of the rat studied with thoracic duct cannulation

Abstract 1. 1. Tracer doses of uniformly 14C-labeled phytol were absorbed from the intestine of the rat to the extent of approx. 50% in 24 h. When the doses were increased to 300 mg, a smaller fraction was absorbed but the total quantity absorbed reached values as high as 65 mg. Over 70% of the absorbed phytol entered through the intestinal lymphatics. Phytol and cetyl alcohol were absorbed more slowly and less completely than were phytanic acid and palmitic acid, and less exclusively by way of the lymphatic route. 2. 2. Over half of the [14C] phytol radioactivity absorbed into the thoracic-duct lymph was still in phytol, some of which was free but a larger part of which was in the form of phytyl esters of fatty acids. The remainder of the label was in acidic derivatives of phytol, principally phytanic acid and phytenic acid (probably at least 3 isomers), which were present almost entirely in triglycerides and other complex lipids. The same derivatives were found in germ-free rats. No dihydrophytol was found in the lymph after phytol had been fed, and no phytenic acid was found after phytanic acid had been fed. Administered phytanic acid was absorbed into the lymph largely without change, except for its incorporation into triglycerides and other complex lipids. In contrast to the results with phytol, cetyl alcohol was largely converted to fatty acid during absorption. 3. 3. Significant quantities of radioactivity from [14C]phytol (administered to rats with intact thoracic ducts) were excreted in the bile, principally in the form of derivatives much more polar than phytanic acid.