Neurochemical Research, Vol. 22, No. 7, 1997, pp. 759-765
Relation Between Free Fatty Acid and Acyl-CoA Concentrations in Rat Brain Following Decapitation Joseph Deutsch,2 S. I. Rapoport,1 and A. David Pardon1,3 (Accepted February 10, 1997)
To ascertain effects of total ischemia on brain phospholipid metabolism, anesthetized rats were decapitated and unesterified fatty acids and long chain acyl-CoA concentrations were analyzed in brain after 3 or 15 min. Control brain was taken from rats that were microwaved. Fatty acids were quantitated by extraction, thin layer chromatography and gas chromatography. Long-chain acylCoAs were quantitated by solubilization, solid phase extraction with an oligonucleotide purification cartridge and HPLC. Unesterified fatty acid concentrations increased significantly after decapitation, most dramatically for arachidonic acid (76 fold at 15 min) followed by docosahexaenoic acid. Of the acyl-CoA molecular species only the concentration of arachidonoyl-CoA was increased at 3 min and 15 min after decapitation, by 3-4 fold compared with microwaved brain. The concentration of docosahexaenoyl-CoA fell whereas concentrations of the other acyl-CoAs were unchanged. The increase in arachidonoyl-CoA after decapitation indicates that reincorporation of arachidonic acid into membrane phospholipids is possible during ischemia, likely at the expense of docosahexaenoic acid.
KEY WORDS: Ischemia; stroke; brain; phospholipid; fatty acids; acyl-CoA; rats; arachidonic acid; docosahexaenoic acid.
13), due to activation of phospholipases (2,3,5). Comparable levels of arachidonic acid and stearic acid at the onset of ischemia suggest combined actions of phospholipase C and diglyceride lipase on phosphatidylinositol (10). However, activation of phospholipase A2 during ischemia has also been reported (14). Long chain acyl-CoAs are major intermediates for reacylation of lysolipids through the action of acyltransferases (15,16), but brain acyl-CoA concentrations and regulation of reacylation following ischemia are not understood. However, during reperfusion following ischemia, high arachidonic acid levels decrease due to reincorporation into lysophospholipids (17-19). Therefore, it is of interest to investigate the effects of ischemia on acyl-CoA which is the crucial metabolic intermediate in reacylation. We recently reported a rapid method to quantitate long-chain acyl-CoAs in brain, using phosphate buffer:
INTRODUCTION Energy depletion, loss of ion homeostasis, and altered calcium metabolism contribute to cell damage in brain ischemia (1-3). Release of excitatory amino acids may augment these processes and further increase cytosolic free calcium (4,5). An early biochemical change is release of lipid mediators such as arachidonic acid (68), diglyceride (9-11) and platelet activating factor (12Laboratory of Neurosciences, National Institute on Aging, National Institutes of Health, Bethesda, Maryland 20892. 2 The Department of Pharmaceutical Chemistry, School of Pharmacy, The Hebrew University of Jerusalem, Jerusalem, Israel. 3 Address reprint requests to: Dr. A. David Purdon, Laboratory of Neurosciences, National Institute on Aging, National Institutes of Health, Bldg 10, Room 6C103, 9000 Rockville Pike, Bethesda, Maryland 20892-1582. Phone: (301) 594-5080; fax: (301) 402-0074; e-mail
[email protected] 1
759 0364-3190/97/0700-0759$12.50/0 C 1997 Plenum Publishing Corporation
760 organic solvent solubilization at pH 4.9, solid phase extraction with an oligonucleotide purification cartridge and HPLC separation (20). We now use a refinement of this method to resolve the polyunsaturated molecular species of acyl-CoA which could not be distinguished in the earlier procedure, and thus are able to determine time-dependent changes in acyl-CoAs compared with free fatty acids following decapitation. Unesterified fatty acids and other lipid mediators increase in brain immediately following decapitation (6-8). Although decapitation cannot be considered a complete model for studying ischemia, the dramatic increase in lipid mediators is comparable to the modification of complex lipids and fatty acids seen in more refined models. Therefore decapitated brain is a good model to observe the effects of ischemia on acyl-CoA metabolism and ascertain the suitability of our analytical approach. Our results indicate that brain phospholipid metabolism is a robust recycling system that responds to an ischemic insult with very selective differences related to each fatty acid species.
EXPERIMENTAL PROCEDURE Brain Isolation. Experiments followed Guidelines for the Care and Use of Laboratory Animals (NIH Publication NI. 80-23) and were approved by the NICHD Animal Care and Use Committee. Male Sprague-Dawley rats (weighing 180 g to 210 g) were purchased from Charles River Laboratories (Wilmington, MA) and were maintained in a 12 h/12 h light/dark cycle with free access to food and water. An animal was anesthetized with sodium pentobarbital (40 mg/kg, i.p.) and was killed by focused-beam microwave irradiation if a control animal (5.5 kW, 3.0 s) (Cober Electronics, Stanford, CT), or by decapitation if an ischemic animal. The brain was removed within 3 min while the rat was kept under a heat lamp to maintain body temperature. Decapitated heads also were put in a plastic bag at 37°C for up to 15 min. Brains were excised by 3 min or 15 min from these heads and were immediately frozen in dry ice pellets. In control experiments heads were also microwaved twice at half intensity after incubation of the decapitated head for the same time interval. No major difference in acyl-CoA concentrations compared with nonmicrowaved decapitated heads was observed. Acyl-CoA Analysis. Acyl-CoA esters were purchased from Sigma Chemical Co. (St. Louis, MO) except for docosahexaenoyl-CoA which was synthesized from coenzyme A and the acid chloride (NuChek Prep, Elysian, MN) by a published method (21) and purified by solid phase extraction using an ABI oligonucleotide cartridge (20). Other chemicals and solvents of analytical and HPLC grade were obtained from Baxter Health-Care Corporation (Muskegon, MI). Acyl-CoA was isolated as follows (20). Microwaved brain tissue (0.5 to 0.8 g) was broken into small fragments and together with internal standard was suspended in 25 mM KH2PO4, pH 4.5 (2.0 ml), kept on ice at 4°C. The sample was homogenized for 20 s by a probe sonicator (Heat Systems-Ultrasonics, Inc., Farmingdel, NY) still while on ice at 4°C. After adding 2-propanol (2.0 ml) (kept cold at 4°C), the sample was sonicated for another 20 s. Saturated (NH4)2SO4 (0.25 ml) and then acetonitrile (4.0 ml) were added and the emulsion was vortexed for 5
Deutsch, Rapoport, and Purdon min, then centrifuged at 1,900 g for 5 min. The supernatant was diluted with 25 mM KH2PO4, pH 4.5 (10.0 ml). Solid phase extraction was carried out with an oligonucleotide purification cartridge (Applied Biosystems, Foster City, CA). The dry cartridge was washed with 5 ml acetonitrile and solvent completely removed with air. The sample was applied and completely pushed through the support. Next the bound sample was washed with 5 ml of 25 mM KH2PO4 and again dried with air. Acyl-CoA was eluted using 70% CH3CN in 25 mM KH2PO4 (0.15 ml) and was collected from the last 0.10 ml. Material from the first 0.05 ml of the eluate appeared in the flow-through fraction in the subsequent HPLC step. Fifty microliters were injected into an HPLC system for quantitation. High pressure liquid chromatography (HPLC) was performed with a Waters HPLC system (Millipore Corp., Milford, MA) comprised of a Waters HPLC pump (Model 510), Wisp 710B autosampler, and Waters 484 tunable absorbance detector set to 260 nm. The system was controlled by a Waters Millennium 2010 software chromatographic manager. Reversed-phase HPLC separation of acyl-CoA molecular species was performed using a Symmetry C-18, 5-um column (250 X 4.6 mm) (Waters; Millipore Corp., Milford, MA) with a stainless steel filter (20). Chromatography was performed using a gradient system of two mobile phases, (A) 75 mM KH2PO4 and (B) 100% CH3CN, with a flow rate of 1.0 ml/min. Starting conditions were 56% buffer A and 44% B. B was increased to 49% over 25 min and then to 70% during the next 5 min, kept at 70% for 9 min and decreased to 44% over 4 min, and held at 44% for an additional 4 min before returning to the starting conditions. The 484 variable wave length detector was set to monitor absorbance at 260 nm the Lmax of the chromophoric portion of the acyl-CoA. Acyl-CoA standards were solubilized in a minimum volume of 70% acetonitrile:KH2PO4. To ensure complete solubilization, a few drops of concentrated phosphoric acid (Fluka, Tonkonkoma, N.Y) were added. The standard acyl-CoAs were purified using an oligonucleotide purification cartridge (20). Concentration was determined from Absorbance260 using an molar absorptivity of 15.4. When extracting brain, 20 M-g each of hexadecenoyl-CoA and heptadecanoyl-CoA were routinely added to each sample as internal standards. Acyl-CoA extraction was performed on one half of a brain (~0.5-0.7 g) using hexadecenoyl-CoA as internal standard. No evidence of either internal standard was seen in the endogenous acyl-CoA pool. The identity of the polyunsaturate acyl-CoAs was confirmed by coelution with standards. The arachidonoyl-CoA significantly increased in decapitated rat brain but co-eluted with the lower amount present in microwaved rat brain. The identity of the arachidonoyl-CoA peak was confirmed by gas chromatography after conversion to the methylester (22). This same peak was found to contain [3H]arachidonic acid radioactivity during [3H]arachidonic acid infusion (22). In preliminary experiments the docosahexaenoyl-CoA peak is also radiolabeled during infusion with [3H]docosahexaenoic acid. The limit of sensitivity of the method was 0.03 nmol on-line. Total acyl-CoA was determined by comparing peak areas of endogenous compounds with those of the internal standards. Palmitoleoyl-CoA was used as a standard for all acyl-CoAs except stearoyl-CoA, which was quantified by using heptadecanoyl-CoA as internal standard. The recovery of stearoyl-CoA and heptadecanoyl-CoA was 85% of the other molecular species. To determine percent recovery a calibration curve of peak area vs nmol injected was constructed for palmitoyl-CoA and arachidonoyl-CoA and gave coincident slopes. The total mass eluted from the column calculated from this standard curve was compared on a per gm basis with the value calculated from internal standards included in the extraction. Recovery of acyl-CoA using this procedure was 41 ±5% (N = 3, ±SEM)%.
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dropwise to 1 g KOH dispersed in a 1.6 ml H2O/5 ml 95% EtOH solution while the latter solution was stirred and held at 65°C. Diazomethane/diethyl ether distillate was collected as a yellow solution at 4°C. FAME were analyzed by gas chromatography (GC) using an HP 5890 Series II GC. Separation of FAME was done on a Supelco SP 2330 column (30m, 0.25 mm ID, 0.2 urn film thickness). Injection and detection ports were held at 250°C. The column temperature started at 80°C and was held at that temperature for 0.5 min following injection of sample. The oven temperature then increased at a rate of 10°C/min to 160°C and then at a rate of 3°C/min to 220°C where it was held for 5 min. A run lasts 37 min. The sample was suspended in 40 ul of isooctane which has a boiling point of 98°C so the sample was concentrated by cold trapping at the top of the column at the start of the run (i.e. at 80°C). This ensured narrow peak widths for each FAME during elution from the column. Quantitation was performed by comparing peak areas of known fatty acids with the area of the internal standard. The limit of detection was 2 ng while the linear range of detection was at least 2 to 50 ng FAME. The amount of each molecular species of acyl-CoA was compared among the three groups of animals (microwaved, 3 min decapitation, 15 min decapitation) and tested by the Student-Newman-Keuls multiple comparison test to determine significance. Means ± SEM are given (24).
RESULTS
Fig, 1. (A) Comparison of the unesterified fatty acids in rat brain 3 and 15 min after decapitation with corresponding concentrations found in microwaved brain. Mean ± SEM for each group of fatty acids (N = 3) are shown, p value is given at 3 min and 15 min after decapitation respectively, compared with microwaved brain, 16:0, p < 0.005, p < 0.005; 16:1, p < 0.01, n.s.; 18:0, p < 0.005, p < 0.05; 18:1, p < 0.05, p < 0.005; 18:2, n.s., n.s.; 20:4, p < 0.001, p < 0.001; 22:6, p < 0.001, p < 0.001. (B) Ratio of concentrations of unesterified fatty acids after 3 and 15 min decapitation to the concentrations in microwaved rat brain.
Analysis of Unesterified Fatty Acids in Rat Brain. An anesthetized rat was killed by microwave irradiation or decapitated as described above. The brain was excised and its lipids were extracted by the folch procedure (23). During extraction, 20 p.g of heptadecanoic acid was added as internal standard. Fatty acids were isolated by thin layer chromatography (TLC) (6,7) and eluted from the TLC plate with chloroform:methanol (5:1; v/v). The unesterified fatty acids were converted to fatty acid methyl esters (FAME) by addition of diazomethane in diethyl ether to the dried eluate in a glass tube for 15 min. Diazomethane was generated using a diazald kit from Aldrich Chem. Co (Milwaukee, WI), 5.0 g diazald in 50 ml diethyl ether was added
Unesterified Fatty Acids in Brain from Microwaved and Decapitated Rats. Concentrations of unesterified fatty acids in brain from microwaved and decapitated rats are shown in Fig. la. Following decapitation, there was a substantial increase in total unesterified fatty acid compared with values for microwaved brain. The total concentration of unesterified fatty acids in microwaved brain was 98.0 ± 10.1 (mean ± SEM) nmol/g brain; at 3 and 15 min after decapitation it was 475 ± 30 nmol/g and 703 ± 52 nmol/g, respectively. Very large increases occurred in concentrations of arachidonic acid and docosahexaenoic acids following decapitation. Arachidonic acid increased from 2.52 ± 0.17 nmol/g to 135.0 ± 8.7 nmol/g at 3 min and to 192.5 ± 20.6 nmol/g at 15 min, respectively. Docosahexaenoic acid increased from 2.92 ± 0.3 to 14.7 ± 0.9 nmol/g at 3 min and 22.13 ± 0.5 nmol/g at 15 min. Concentrations of palmitic acid, stearic acid and oleic acids also increased substantially during the same intervals. Ratios of unesterified fatty acid concentration in ischemic/ microwaved brain are shown in Fig. 1b. At 3 min after decapitation, ratios of unesterified fatty acids in ischemic brains compared with microwaved brains were 7.8 ± 0.7, 53.6 ± 3.7, and 5.03 ± 0.6 for 18:1, 20:4 and 22: 6, respectively. Ratios for the other fatty acids were less than 5.0. At 15 min, the three higher values had increased to 16.0 ± 1.7, 76.4 ± 8.5, and 7.7 ± 0.8, respectively, whereas for the other fatty acids values
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Fig. 2. (A) The HPLC ran is the elution profile of acyl-CoA molecular species purified from microwaved rat brain (0.5 g). 20 ug of each internal standard, hexadecenoyl-CoA (16:1) and heptadecanoyl-CoA (17:0), are added during extraction and are indicated on the figure. (B) The HPLC pattern is acyl-CoA molecular species isolated from rat brain after 3 min decapitation, and spiked with 20 ng internal standards hexadecenoyl-CoA and heptadecanoyl-CoA. There is a considerable increase in the arachidonoyl-CoA peak in the decapitated brain sample.
Table I. Concentrations of Acyl-CoA Molecular Species in Microwaved Rat Brain Molecular Species
16:0 18:0 18:1 18:2 20:4 22:6 Total
nmol/g brain
8.45 5.08 10.85 0.98 1.66 1.71 28.73
± 0.37 ± 0.27 ± 0.91 ± 0.14 ± 0.65 ± 0.25 ±1.10
Results are expressed as mean ± SEM, for N = 6.
were still low, 6.4 or less. The relative increase in arachidonic acid was considerably greater than for any other fatty acid. Acyl-CoA Composition in Microwaved and Ischemic Brain. Microwaved rat brain spiked with 20 ug of hexadecenoyl-CoA and 20 ug of heptadecanoyl-CoA as internal standards was extracted and analyzed by HPLC. The results are shown in Fig. 2A. Polyunsaturated molecular species were resolved and separated from the more saturated acyl-CoAs, which eluted at later times.
Fig. 3. Concentrations of acyl-CoA from microwaved brain and from brain of rats 3 and 15 min after decapitation. Mean ± SEM; N = 6 for microwaved rats, N = 6 for 3 min decapitated rats and N = 4 for 15 min decapitated rats. Mean ± SEM for each of the molecular species in decapitated brain were compared with values obtained for microwaved brain and significant differences are indicated *** p < 0.005, * p < 0.05.
Docosahexaenoyl-CoA and arachidonoyl-CoA are indicated and linoleoyl-CoA is a smaller peak eluting slightly later. Average concentrations for each of the molecular species of acyl-CoAs in microwaved brain are shown in Table I. Docosahexaenoyl-CoA, arachidonoylCoA and linoleoyl-CoA, the three polyunsaturate molecular species, represented 15% of the total molecular species of acyl-CoA. Of the three non-polyunsaturated components, oleoyl-CoA was present in larger amounts than palmitoyl-CoA or stearoyl-CoA. Rats were decapitated and the heads maintained at 37°C until the brains were excised by 3 min or 15 min and then rapidly frozen in dry ice. The HPLC elution profile of the acyl-CoA molecular species of a rat brain at 3 min after decapitation is shown in Fig. 2B, the lower part of the figure. Compared with the microwave results in Fig. 2A, there was a marked increase in arachidonoylCoA, a decrease in docosahexaenoyl-CoA and little apparent change in the other acyl-CoA species. The selective increase in arachidonoyl-CoA compared with the other molecular species is shown in Fig. 3 for groups of 6 and 4 rats at 3 and 15 min after decapitation, respectively. Arachidonoyl-CoA rose from 1.66 ± 0.65 nmol/g in microwaved brain to 6.29 ± 0.64 nmol/g at 3 min and 5.16 ± 0.22 nmol/g at 15 min after decapitation. However, docosahexaenoyl-CoA decreased significantly by 3 min and even more by 15 min
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comparable to those observed in more refined global and focal ischemic models and hence our observations can give insight into fatty acid metabolism in those systems.
Fig. 4. Ratio of nmol acyl-CoA/g brain for acyl-CoA species to nmol unesterified fatty acid/g brain in microwaved and decapitated rat brain. Values were calculated from Figs 1 and 3. In the microwaved rat brain, ratios are as follows; 16:0, 0.19 ± 0.03; 18:0, 0.16 ± 0.02; 18:1, 2.0 ± .17; 18:2, 0.28, 0.12; 20:4, 0.67 ± 0.075; and 22:6, 0.59 ± 0.06.
after decapitation. Of the predominant molecular species (16:0, 18:1, and 18:0), only oleoyl-CoA was decreased significantly at 15 min compared with microwaved brain. There was no significant change in linoleoyl-CoA, palmitoyl-CoA and stearoyl-CoA following decapitation. The mol % of arachidonoyl-CoA of the total acyl-CoA increased from 6 to 23 mol % during the experiment (by 15 min), whereas the mol % for docosahexaenoyl-CoA decreased from 6 to 0.6 mole %. The ratio of acyl-CoA to fatty acid concentrations for each molecular species and for brains from microwaved rats, 3 and 15 min after decapitation are shown in Fig. 4. In general the ratio was lower than unity except for oleic acid and decreased after decapitation. The average ratio in microwaved brain was 0.32; it decreased to 0.06 and to 0.02 at 3 and 15 min, respectively, after decapitation.
DISCUSSION In this work we have evaluated the effect of global ischemia on the precursor pools (unesterified fatty acid and acyl-CoA) required for reincorporation of fatty acid into brain phospholipid. Previously, we developed a fatty acid model which emphasized the importance of acyl-CoA turnover to the analysis of fatty acid metabolism in brain (25). We submit that the dramatic changes in phospholipid intermediates in decapitated rat brain are
Concentrations of brain unesterified fatty acids were significantly elevated at 3 min and 15 min after decapitation, compared with control values in microwaved brain. The relative increase was highest for arachidonic acid. This selectivity is in agreement with other reports (7,8,10). We report resolution of the polyunsaturated molecular species docosahexaenoyl-CoA, arachidonoyl-CoA, and linoleoyl-CoA for the first time. The total acyl-CoA concentration we found for brain was 28.7 nmol/g, considerably lower than reported for liver (e.g. 43.1 nmol/g) (26). Liver has a much higher acyl-CoA synthetase activity than brain, whereas in brain acyl-CoA hydrolase activity is much higher than liver (16). Thus total acyl-CoA levels appear to reflect a balance between acyl-CoA synthetase and hydrolase activities, as postulated by Waku (16). The concentrations of palmitoyl-, oleoyl-, and stearoyl-CoA appear to have been buffered from the increase in the brain unesterified fatty acid after decapitation, whereas concentrations of arachidonoyl-CoA and docosahexaenoyl-CoA were markedly affected indicating a difference in processing of elevated levels of unesterified fatty acids. The acyl-CoA/fatty acid concentration ratio (Fig. 4) was < 1 for each molecular species except oleic acid (ratio ~2.0), and decreased considerably after decapitation for all molecular species. The largest decrease after decapitation was for docosahexaenoic acid. The combined increase in the concentration of this fatty acid and decrease in its acyl-CoA after decapitation explain the dramatic change. The decrease in the ratio for arachidonic acid was attenuated by the dramatic increase in arachidonoyl-CoA. The acyl-CoA/unesterified fatty acid ratio can give some insight into acyl-CoA metabolism in the brain of the control animal. At steady state, the concentration ratio of acyl-CoA/fatty acid would equal 1.0 if the transfer coefficients into the acyl-CoA pool and out of the acylCoA pool (efflux coefficient) were equal. Values
