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Supplement: Waltham International Symposium

Conversion of Essential Fatty Acids by Delta 6-Desaturase in Dog Liver Microsomes

Comparative Nutrition Research Laboratory, Texas A&M University, College Station, TX

³To whom correspondence should be addressed. E-mail: jbauer{at}cvm.tamu.edu.

KEY WORDS: • 6-desaturase • liver • dog • microsomes • linoleic acid • kinetics

EXPANDED ABSTRACT

Dietary essential fatty acids (EFAs), linoleic acid [18:2(n-6), LA] and -linolenic acid [18:3(n-3), ALA] are converted to long-chain polyunsaturated fatty acids (LCPUFAs) by desaturase and chain-elongation enzyme systems (1). The LCPUFAs are important because they serve as eicosanoid precursors. In addition, several LCPUFAs have specific structural and functional roles in development or maintenance of neural tissues such as brain, retina and other tissues (2).

The rate-limiting step for desaturation and elongation is controlled by 6-desaturase, which adds a double bond at the sixth carbonyl carbon. Hence, LA is converted to 18:3(n-6) and ALA is converted to 18:4(n-3) and competition between these substrates for this enzymatic step exists among the fatty acid families. Some reports indicate a higher specificity for (n-3) fatty acid desaturation compared to that for (n-6) fatty acids (3).

Dogs are important to humans not only as companion animals but also serve as a model for human metabolism (4,5). This study addresses EFA metabolism in dogs using a classical enzyme kinetic approach.

	MATERIALS AND METHODS

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Animals

Fresh liver tissues were removed from 11 normal, healthy Coonhound dogs and three Sprague–Dawley rats at the termination of animal use protocols approved by Texas A&M University Laboratory Animal Use Committee. The animals had been fed once daily and were not denied food before euthanasia. Liver microsomes were prepared with rat serving as a positive control group for enzyme activity determinations.

Microsomal preparation

Liver tissue was immediately transferred to ice-cold saline after collection, blotted dry, minced and rinsed twice with saline. A phosphate buffer (40 mM, Buffer A) containing 0.1 M sucrose (pH 7.4) with tissue-to-buffer ratio of 1:6 was used for homogenization. Homogenates were centrifuged for 20 min at 4°C and 10,000 x g with fixed-angle rotor. Supernates were then centrifuged for 1 h at 4°C and 105,000 x g to pellet the microsomes. Microsomes were resuspended in fresh Buffer A and protein concentrations determined before freezing at -80°C.

Microsomal lipid composition determination

Microsomes were extracted by the method of Folch et al. (6) with internal standards for phospholipids (PL), nonesterified fatty acids (FFA) and triacylglyerol (TG) containing 22:1(n-9). Lipid subclasses were separated by thin-layer chromatography (TLC) on silica gel plates with 80:20:1 hexane:ether:glacial acetic acid (v/v/v). The PL, FFA and TG were scraped and fatty acid methyl esters (FAME) prepared. Samples were quantified by capillary gas chromatography (GC) on a Restek Stabilwax column (0.32 mm ID x 30 m x 0.25 mm film) with He gas carrier. A temperature program was begun at 170°C, held for 10 min, ramped to 228°C at 2°C/min then held for 20 min.

Incubations

Incubation conditions for ALA as substrate in the absence of malonyl-CoA were determined by independently varying C¹⁴-ALA or C¹⁴-LA substrate content, protein concentrations and incubation times. Protein (4 mg), 15-min incubation and 50 µM ALA substrate concentration were found to be suitable conditions for 6-desaturase assay. Incubations were performed at 37°C and each 2-mL incubation mixture contained ATP (6.67 µmol), coenzyme A (0.13 µmol), GSH (3.0 µmol), NADH (1.2 µmol), NADPH (2.5 µmol) and MgCl₂ (10 µmol). Reactions were terminated by adding 9 mL of 2:1 (v/v) chloroform/methanol containing 0.1% glacial acetic acid. Water (2 mL) was added and tubes were shaken for 10 min and centrifuged at low speed. A 5-mL aliquot of 3:48:47 (v/v/v) chloroform/methanol/water was used to wash the lipid-containing phase. Tubes were again shaken (10 min) and centrifuged, and chloroform layers were combined then dried under N₂ gas.

Saponification and free fatty acid phenacylation

Lipid residues were resuspended in 2 mL of 2.8% KOH in methanol. Samples were saponified under an atmosphere of N₂ for 30 min at 85–90°C. After cooling the mixture was acidified and extracted with hexane. To each saponified extract 100 µL 2-bromoacetophenone (10 mg/mL in acetone) was added and mixed, followed by the addition of 100 µL triethylamine solution (10 mg/mL in acetone) to prepare fatty acid phenacyl esters (FAPE) (7). The FAPE were dried under N₂ gas and resuspended in methanol.

High-performance liquid chromatography (HPLC) and liquid scintillation counting (LSC)

The radiolabeled FAPE were fractionated using HPLC on two Novapak C18 (4.6 x 150 mm) columns at 2 mL/min flow. Elution was conducted with a gradient of 70:5:25 acetonitrile:methanol:water (v/v/v) to 90:5:5 acetonitrile:methanol:water (v/v/v) both with 0.1 mL/L glacial acetic acid controlled for the first 25 min using a nonlinear gradient profile (Waters, no. 9; Waters Associates, Milford, MA) then held constant for 33 min. Detection of peaks at 242 nm was performed using retention times of authentic standards. Sample peaks were collected and scintillation cocktail (3 mL) was added to each. The tubes were mixed and radioactivity was counted twice by LSC for 2 min and values averaged.

Statistical analyses

Statistical comparisons between rat and dog lipid analyses were performed using Student’s t-test, with P < 0.05 considered significantly different.

	RESULTS

TOP MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

 
The amounts of conversion of LA to 18:3(n-6) and ALA to 18:4(n-3) were used to calculate maximal velocities (V_max) and Michaelis–Menten constants (K_m). In dogs, 6-desaturase exhibited a higher V_max for ALA (50.9 pmol mg protein^-1 min^-1) compared to that for LA substrate (5.4 pmol mg protein^-1 min^-1) (Fig. 1). By comparison, rat microsomes showed a 6-desaturase V_max of 36.6 pmol mg protein^-1 min^-1 with ALA substrate and 12.7 pmol mg protein^-1 min^-1 with LA substrate (data not shown).

View larger version (25K): [in this window] [in a new window]   FIGURE 1 Michaelis–Menten and Lineweaver–Burk plots of dog 6-desaturase with LA and ALA as substrates. (A) Activity vs. ALA concentration with endogenous free LA concentration < 50 µmol/L, open squares; and >50 µmol/L, open circles. (B) Lineweaver–Burk plot of 1/activity (1/V) vs. ALA-1 (1/S). Regression lines represent <50 µmol/L; open squares, >50 µmol/L; open circles, r2 = 0.95, P < 0.0001. (C) Activity vs. LA concentration. (D) Lineweaver–Burk plot of 1/activity vs. 1/LA (1/S). Each data point is the average ± SEM of at least three determinations for ALA and LA. Km and Vmax are as described in the text.

Endogenous microsomal fatty acid concentrations in the reaction mixtures were determined with internal standards and GC of the microsomal lipids. Of the three fractions studied (PL, FFA and TG), the PL fraction contributed a majority of endogenous lipid in dog and rat. Total lipids were not substantially different in the dog vs. rat, although PL and FFA were higher in dog than in rat, and TG concentrations were higher in rat. Dog liver microsomes contained approximately 6 times more total FFA than that in rat liver (Table 1) and its LA content was 10 times higher than rat (64.4 ± 61.8 vs. 6.5 ± 0.2 µmol/L ± SD). The contribution to the incubation mixtures from endogenous ALA substrate was 2.4 ± 2.5 µmol/L ± SD in dogs compared to none found in rat (Table 2). The concentrations of dog endogenous microsomal LA in the FFA fraction were considerable compared to rat, which resulted in inhibition of ALA conversion. To correct for this inhibition, a graphical method to correct ALA substrate kinetic values in the presence of high endogenous LA was devised. Given the short incubation times used, the contributions of the PL- and TG-LA were not considered in this correction.

View this table: [in this window] [in a new window]   TABLE 1 Concentration of endogenous microsomal total lipid fatty acids and EFAs in final assay mixture used for 6-desaturase assays1

View larger version (17K): [in this window] [in a new window]   FIGURE 2 Graphically corrected dog 6-desaturase activity vs. ALA concentration at extrapolated LA = 0 µmol/L. Insert graph: Lineweaver–Burk plot of corrected activity data. Km and Vmax are as described in the text.

	DISCUSSION

TOP MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

Using human fetal liver microsomes, Rodriguez et al. (8) found a V_max for ALA 6-desaturation with ALA substrate higher than that with LA substrate (24.5 vs. 7.5 pmol mg protein^-1 min^-1, respectively). Also, the K_m for ALA (24.5 µmol/L) was higher than that for LA (6.5 µmol/L). However, the endogenous LA contribution was low in the human fetal liver microsomes compared to that in adult rat liver microsomes. Ivanetich et al. (9) also found FFA to significantly contribute to the substrate pool of rat liver microsomes. They reported an endogenous FFA-LA concentration of 2.9 µmol/L (compared to 64.4 µmol/L in dog and 6.5 µmol/L in rat in the present study) and noted that membrane phospholipids make an insignificant contribution to the endogenous fatty acid substrate pool in microsomes. It was concluded that endogenous LA would affect total substrate concentration, specific activity of substrate and product, and rate of product formation as in the present study. Purvis et al. (10) reported that the 2.85–30 µg of endogenous LA substrate/mg protein in pig liver microsomes should also be considered as available substrate. This range of LA is consistent with that seen here for dogs (9.0 µg/mg microsomal protein).

In conclusion, dog liver microsomes are capable of desaturating EFAs. Also, the maximal velocity for 6-desaturation of ALA is considerably higher than that for LA in vitro, yet the K_m constant for LA was at least twice as high as that of ALA. When corrected for endogenous LA concentrations of microsomal enzyme preparations, the differences in V_max and K_m between these two substrates become even more pronounced. Physiologically, ALA concentration may never exceed the K_m for desaturation in the absence of high dietary amounts. By contrast, LA amounts are readily converted because most diets for dogs are replete in LA and because microsomal concentrations (64.4 µmol/L) appear to widely exceed the K_m. These phenomena help explain low in vivo conversion of ALA in dogs and other species. The findings also suggest that high levels of ALA supplementation may be necessary to exceed the 6-desaturase K_m for this substrate and to significantly affect physiological levels of (n-3) LCPUFA in dogs.

Despite reports of endogenous fatty acids contributing to the total substrate pool for the 6-desaturase reaction, many authors do not quantitatively report microsomal lipid and fatty acid concentrations in liver microsomes. The elevated endogenous fatty acids present in dog liver microsomes not only act as inhibitors of desaturation in vitro, but also serve as competitive substrate for the reaction. Either highly purified preparations or a correction as applied in the present study is therefore needed. Finally, it should be noted that studies designed to simply measure mRNA abundance of 6-desaturase mass cannot evaluate the nature of the competitive interactions between LA and ALA. The graphic correction technique used here allows characterization of such enzyme–substrate competition.

	ACKNOWLEDGMENTS

 
Special thanks to Karen Bigley for assistance with assays.

	FOOTNOTES

 
¹ Presented as part of the Waltham International Symposium: Pet Nutrition Coming of Age held in Vancouver, Canada, August 6–7, 2001. This symposium and the publication of symposium proceedings were sponsored by the Waltham Centre for Pet Nutrition. Guest editors for this supplement were James G. Morris, University of California, Davis, Ivan H. Burger, consultant to Mars UK Limited, Carl L. Keen, University of California, Davis, and D’Ann Finley, University of California, Davis.

² Supported, in part, by the Mark L. Morris Professorship in Clinical Nutrition at Texas A&M University.

⁴ Abbreviations used: EFA, essential fatty acid; ALA, -linolenic acid; LA, linoleic acid, LCPUFA, long-chain polyunsaturated fatty acid; GC, gas chromatography; TLC, thin-layer chromatography; FAPE, fatty acid phenacyl esters; TG, triacylglycerol; PL, phospholipid; FFA, nonesterifed fatty acid.

	LITERATURE CITED

TOP MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

 

1. Holman, R. T. (1998) The slow recovery of the importance of omega 3 essential fatty acids in human health. J. Nutr. 128:427S-433S.

2. Birch, E. E., Hoffman, D. R., Uauy, R., Birch, D. G. & Prestidge, C. (1998) Visual acuity and the essentiality of docosahexaenoic acid and arachidonic acid in the diet of term infants. Pediatr. Res. 44:201-209.[Medline]

3. Maniongui, C., Blond, J. P., Ulmann, L., Durand, G., Poisson, J. P. & Bezard, J. (1993) Age-related changes in 6 and 5 desaturase activities in rat liver microsomes. Lipids 28:291-297.[Medline]

4. Lock, E. A., Mitchell, A. M. & Elcombe, C. R. (1989) Biochemical mechanisms of induction of hepatic peroxisome proliferation. Ann. Rev. Pharmacol. Toxicol. 29:145-163.[Medline]

5. Hulman, S., Kleigman, R., Heng, J. & Crouser, E. (1988) Relationship of substrate level to turnover rate in fasted adult and newborn dogs. Am. J. Physiol. Endocrinol. Metab. 254:E137-E143.[Abstract/Free Full Text]

6. Folch, J., Lees, M. & Sloane-Stanley, G. H. (1957) A simple method for the isolation and purification of total lipids from animal tissues. J. Biol. Chem. 226:497-509.[Free Full Text]

7. Chen, H. & Anderson, R. E. (1992) Quantitation of phenacyl esters of retinal fatty acids by high-performance liquid chromatography. J. Chromatogr. 578:124-129.[Medline]

8. Rodriquez, A., Sarda, P., Nessmann, C., Boulot, P., Leger, C. L. & Descomps, B. (1998) 6- and 5-Desaturase activities in the human fetal liver: kinetic aspects. J. Lipid Res. 39:1825-1832.[Abstract/Free Full Text]

9. Ivanetich, K. M., Bradshaw, J. J. & Ziman, M. R. (1996) 6-Desaturase: improved methodology and analysis of the kinetics in a multi-enzyme system. Biochim. Biophys. Acta 1292:120-132.[Medline]

10. Purvis, J. M., Clandinin, M. T. & Hacker, R. R. (1983) Chain elongation-desaturation of linoleic acid during the development of the pig. Implication for the supply of polyenoic fatty acids to the developing brain. Comp. Biochem. Physiol. B 75:199-204.[Medline]

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