T. Sanjeeva Reddy

Docosahexaenoic acid (22:6, n-3) is metabolized to lipoxygenase reaction products in the retina

Docosahexaenoic acid (22:6, n-3), a major component of retinal phospholipids, is a substrate for active lipoxygenation in intact canine retinas incubated in vitro with [U-14C]docosahexaenoic acid. The major lipoxygenase reaction product was identified by high performance liquid chromatography and gas chromatography-mass spectrometry as 11-hydroxy-4,7,9-(trans)13,16,19 docosahexaenoic acid. Other mono- and di-hydroxy derivatives also were detected. The synthesis of these compounds was inhibited by the antioxidant and lipoxygenase inhibitor, nordihydroguaiaretic acid, but was not inhibited by indomethacin or esculetin.

Kinetic properties of arachidonoyl-coenzyme A synthetase in rat brain microsomes

Free arachidonic acid is released rapidly in the brain at the onset of ischemia and during convulsions. The transient nature of this phenomenon indicates the existence of an active reacylation system for this fatty acid, likely an arachidonoyl-CoA synthetase-arachidonoyl transferase. The first of these enzymatic activities in brain microsomes was studied and it was found that [1-14C]arachidonic acid is rapidly activated and shows an absolute requirement for ATP and CoA. MgCl2 enhances this activity 10-fold. The optimum pH is 8.5, and the apparent Km values for the radiolabeled substrate, ATP, CoA, and MgCl2 are 36, 154, 8, and 182 microM, respectively. The apparent Vmax is 32.4 nmol/min/mg protein for arachidonic acid. The presence of Triton X-100 (0.1%) in the assay medium caused a significant reduction in apparent Km (9.4 microM) and Vmax (25.7 nmol/min/mg protein) values. The enzymatic activity is thermolabile with a T1/2 of less than 1 min at 45 degrees C and a maximal activity at 40 degrees C. The breaking point or transition temperature is 25 degrees C in an Arrhenius plot. The activation energies were 95 kJ/mol from 0 to 25 degrees C and 30 kJ/mol from 25 to 40 degrees C. Fatty acid competition studies showed inhibition by unlabeled docosahexaenoic and arachidonic acids with a Ki of 31 and 37 microM, respectively, in the absence and 18 and 7.7 microM in the presence of Triton X-100. Palmitic acid and oleic acid slightly inhibited the reaction whereas linoleic acid inhibited it to a moderate extent. It is concluded that this very active enzyme can activate arachidonic acid as well as docosahexaenoic acid in brain microsomes. In addition, this reaction may be involved in regulating the pool size of these free fatty acids in brain by rapid removal through activation, thus limiting eicosanoid formation. Moreover, the rapid formation of polyenoic acyl-coenzyme A may participate in the retention of essential fatty acids in the central nervous system.

Synthesis of arachidonoyl coenzyme A and docosahexaenoyl coenzyme A in retina

The synthesis of 14C-labeled arachidonoyl coenzyme A (CoA) and docosahexaenoyl CoA was studied in the human, bovine, rat and frog retina. The synthesis of arachidonoyl CoA was two- to fourfold higher than that of docosahexaenoyl CoA in the retinal membranes examined. The enzyme involved in the synthesis of these polyenoyl CoAs, long-chain acyl-CoA synthetase, had a species variation and was most active in microsomal membranes from frog retina. In the retina, 60% of the enzymes total activity was in the microsomal fraction, whereas only 4-7% of the long-chain acyl-CoA synthetase activity was in the rod outer segments, which contain large amounts of docosahexaenoate and arachidonate esterified to phospholipids. This fact implies that despite the high concentration of docosahexaenoate and arachidonate in rod outer segments, there is little turnover of these acyl chains, at least through the formation of thioesters. The apparent Km (uM) and Vmax (nmol/min/mg protein) values for the labeled docosahexaenoate were 9.84 +/- 0.86 and 5.26 +/- 0.46, respectively. The presence of Triton X-100- lowered the Km and Vmax values to 7.64 +/- 0.11 and 3.03 +/- 0.12, respectively. The Km and Vmax values for arachidonate were 40 and 13.3, respectively. The apparent Km value for ATP was 270 +/- 33 uM and for CoA, 3.70 +/- 0.23 uM. The transition temperatures obtained from Arrhenius plots for docosahexaenoate and arachidonate were 24 degrees C and 28 degrees C, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)

Change in content, incorporation and lipoxygenation of docosahexaenoic acid in retina and retinal pigment epithelium in canine ceroid lipofuscinosis

We have examined the metabolism of docosahexaenoic acid (22:6, n-3) in retina and retinal pigment epithelium of normal dogs and those affected with canine ceroid lipofuscinosis (CCL), a hereditary degenerative neurological disorder. In the CCL retina, there was a decrease in 22:6 content in phospholipids, particularly phosphatidylethanolamine. This decrease in 22:6 was compensated by an increase in arachidonic acid (20:4, n-6). In contrast, CCL retinal pigment epithelium had higher levels of 22:6 and lower levels of 20:4 in phosphatidylethanolamine. The in vitro incorporation of radiolabeled 22:6 into glycerolipids of CCL retina and retinal pigment epithelium was increased as compared to control. The major lipoxygenase reaction product of 22:6, (11-hydroxy-4,7,9(trans)13,16,19)-22:6, increased 31% in CCL retina, but not in the retinal pigment epithelium. This is the first report describing alterations in content, incorporation and lipoxygenation of 22:6 in an animal model of a human disease (Battens disease).

Synthesis of docosahexaenoyl-, arachidonoyl- and palmitoyl-coenzyme a in ocular tissues

The synthesis of long-chain acyl coenzyme A (CoA) was studied in the cornea, lens, vitreous, retina and pigment epithelium (PE) in the rat using [14C]-labeled palmitic, arachidonic and docosahexaenoic acids as substrates. Except for retina and PE, the ocular tissues studied showed relatively little enzyme activity with the fatty acid substrates. In addition, the enzyme activities were studied in homogenates and microsomal fractions from retina, pigment epithelial cells and choroid of frog, bovine and human eyes. Long-chain acyl CoA synthetase from the microsomal fraction exhibited three- to fivefold greater activity than homogenates in retina and PE. The enzyme activity was highest with palmitic acid, followed by arachidonic acid and docosahexaenoic acid. There were significant differences in enzyme activity between the species. The apparent Km (microM) and Vmax [nmol min-1 (mg protein)-1] values for the enzyme in bovine retinal microsomes were 7.91 +/- 0.39 (S.E.) and 21.6 +/- 1.04, respectively, for palmitic acid substrate and 5.88 +/- 0.25 and 4.58 +/- 0.21, respectively, for docosahexaenoic acid substrate. These values for bovine pigment epithelial microsomes were 13.0 +/- 0.27 and 36.9 +/- 1.18, respectively, for palmitic acid and 15.8 +/- 0.40 and 13.2 +/- 0.56, respectively, for docosahexaenoic acid. The synthesis of acyl CoA may play a central role in controlling the availability of free arachidonic acid for eicosanoid formation and in the retention of polyunsaturated fatty acid families (18:2, n-6 and 18:3, n-3) within cells of ocular tissues, particularly retina and retinal PE.

Docosahexaenoate metabolism and fatty-acid composition in developing retinas of normal and rd mutant mice.

The fatty-acid composition of retinal lipids in developing control and rd mice (C57BL/6J) was determined. In addition, fatty-acid composition in brain and retina of normal and rd adult animals was compared. At 11 days of postnatal age, rd retinas contained proportionally less docosahexaenoate than controls, whereas the reverse relationship held for oleate at 20 days of age. In contrast, no differences in the fatty-acid composition of brain lipids were observed between rd and control animals. We also examined docosahexaenoate metabolism in rd and control retinas of different postnatal ages in vitro. These studies of [1-14C]docosahexaenoic-acid incorporation into retinal phospholipids and neutral lipids demonstrated significantly higher incorporation into triacylglycerols of the rd retina, beginning at 14-15 days of postnatal age. Incorporation into diacylglycerols and phospholipids was also higher in rd retinas than in controls at 14 days of age and older. Moreover, the concentration of ganglioside (a glycolipid class probably enriched within inner retinal layers) was higher in adult rd retinas. Degeneration of the rd retina can be detected histologically as early as 8-9 days. Therefore, the alterations of fatty-acid composition and docosahexaenoate metabolism described here are probably an effect, rather than a cause, of retinal degeneration in the rd mouse.

Activation of polyunsaturated fatty acids by rat tissues in vitro.

The conversion of labeled palmitic, linoleic, arachidonic and docosahexaenoic acids to their respective acyl CoA’s was studied in homogenates and microsomes of rat tissues. The highest activity, both in homogenates and microsomes, was seen in liver and heart. There was moderate activity in retina, brain, lung, kidney and testes and the lowest activity was found in spleen. Docosahexaenoic acid was activated much less actively in heart tissue than the other fatty acids. In all tissues examined, the highest activation was observed with arachidonic acid and the lowest with docosahexaenoic acid. Except for liver, those tissues that contained high levels of docosahexaenoic acid also had the highest activation capacity for this fatty acid.

Fatty acid composition and arachidonic acid metabolism in vitreous lipids from canine and human eyes

About 55% of the acyl groups of dog and human vitreous are unsaturated fatty acids. The major components are oleate (18:1, n-9) and arachidonate (20:4, n-6) with moderate amounts of linoleate (18:2, n-6) and docosahexaenoate (22:6, n-3). Palmitate (16:0) and stearate (18:0) are the major saturated fatty acids. There are no significant changes between ages 37-82 years in the fatty acyl group content and composition of human vitreous. In vitreous from Irish setters with hereditary rod-cone dysplasia (RCD) the levels of oleate are decreased with a concomitant increase in arachidonate. [1-14C]Arachidonic acid was actively incorporated into canine vitreous glycerolipids both in vitro and in vivo. The incorporation was mainly into phosphatidylinositol, triacylglycerol, phosphatidylcholine and phosphatidylethanolamine. There were some differences in the pattern of incorporation between human and dog and between in vivo and in vitro incubations of canine vitreous. Glycerolipid acylation was significantly increased in phosphatidylinositol and phosphatidylcholine in RCD canine vitreous. The pattern of incorporation of [U-14C]docosahexaenoic acid into vitreous glycerolipids was different from arachidonic acid incorporation. Although vitreous did not produce any measurable enzymatic synthesis of cyclooxygenase and lipoxygenase products from [1-14C]-arachidonic acid in vitro, there was significant generation of autooxidation products. These results suggest an active lipid metabolism in vitreous.

Long-chain acyl coenzyme A synthetase activity during the postnatal development of the mouse brain.

Palmitic, linoleic, arachidonic, and docosahexaenoic acids were used as substrates to study fatty acid activation in the mouse brain and liver during postnatal development. Long‐chain acyl coenzyme A synthetase showed peak activity during the period of rapid oligodendroglial proliferation and myelination. In brain, activation of linoleic and arachidonic acids was highest, followed by palmitic and docosahexaenoic acids. In liver, no appreciable change in enzyme activity was seen during the period of development studied. Palmitic and arachidonic acids showed the highest rate of activation, followed by docosahexaenoic acid. These ontogenic data suggest the presence of a single long‐chain acyl coenzyme A synthetase in brain.

Long-chain acyl CoA synthetase in microsomes from rat brain gray matter and white matter

Long-chain acyl coenzyme A (CoA) synthetase in homogenates and microsomes from rat brain gray and white matter was studied. The formation of the thioesters of CoA was studied upon addition of [1-14C]-labeled fatty acids. The maximal activities were seen with linoleic acid, followed by arachidonic, palmitic, and docosahexaenoic acids in both gray and white matter homogenates and microsomes. The specific activities in microsomes were 3–5 times higher than in homogenates. The presence of Triton X-100 in the assay system enhanced the activity of long-chain acyl CoA synthetase in homogenates. The effect was more pronounced in palmitic and docosahexaenoic acid activation. The apparentKm values andVmax values for palmitic and docosahexaenoic acids were much lower than for linoleic and arachidonic acids. The presence of Triton X-100 in the medium caused a definite decrease in the apparentKm and Vmax values for all the fatty acid except palmitic acid in which case the reverse was true. There were no significant differences observed in the kinetic measurements between gray and white matter microsomes. These findings are similar to those resulting from the known interference of Triton X-100 in the measurement of kinetic variables of long-chain acyl CoA synthetase of liver microsomes. In this work, no correlation was observed between the fatty acid composition of gray and white matter and the capacity of these tissues for the activation of different fatty acids.