Journal of Orthopaedic Research 9341-347 Raven Press, Ltd., New York 0 1991 Orthopaedic Research Society
Effect of Extracellular Fatty Acids on Lipid Metabolism in Cultured Rabbit Articular Chondrocytes Masato Nagao, Seiichi Ishii, "Yoshihisa Murata, and "Toyoaki Akino Departments of Orthopedic Surgery and *Biochemistry, Sapporo Medical College, Sapporo, Japan
Summary: Rabbit articular chondrocytes were cultured for 8 h in the presence of various concentrations (5-500 pA4) of [I4C] oleic, [I4C] linoleic, and E3H] arachidonic acids. The radioactive unsaturated fatty acids were incorporated into triacylglycerol (TG) and phosphatidylcholine (PC) in a concentrationdependent manner; more fatty acids were incorporated into TG than into PC, at higher concentrations of extracellular fatty acids. Among these fatty acids, arachidonic acid was incorporated into TG much more than into PC, in spite of a very low concentration of arachidonic acid in TG. After transfer of the labeled cells to maintenance medium, the radioactivity in TG declined rapidly and [3H]arachidonic acid radioactivity in PC increased continuously during the chase time periods. Palmitoyl-unsaturated species were mainly formed in PC when cultured at a concentration of 5 pM of each fatty acid. However, when cultured at 500 FM, unsaturated-unsaturated species, specific for each unsaturated fatty acid were actively formed. These findings indicate that (1) fatty acid composition of TG and PC in articular chondrocytes is influenced by the degree of fatty acid supply, (2) formation and turnover of TG plays a role in fatty acid metabolism of cells, and (3) fatty acid pairing in PC is modulated by extracellular fatty acid concentrations. Key Words: Articular chondrocytesLipid metabolism-Arachidonic acid-Phosphatidylcholine molecular species.
were shown to accumulate in intra- and extracellular regions of the cartilage. On the other hand, an important role of the synovial membrane in the pathogenesis of osteoarthritis has been reported recently by Stockwell et al. (24) and Lukoschek et al. (14). As with early osteoarthritic changes in experimental animal models, synovial membrane changes and lipid accumulation in superficial chondrocytes have been noted. Since the synovial fluid is a source of nutrition to the cartilage, lipids might be released into the synovial fluid from damaged synovial membranes and be taken up by chondrocytes, which would affect the lipid metabolism in the cells. However, there has been no detailed study on lipid metabolism of articular chondrocytes, particularly with respect to lipid storage in the cells. The present study was done to gain insight into the mechanism by which lipid metabolism is regu-
It has been reported that perturbation in lipid metabolism, particularly lipid storage, in articular chondrocytes might be associated with the degradation process, of articular cartilage, that occurs with aging (6,11,23). Stockwell (23) was the first to find increased amounts of triacylglycerol (TG) and phospholipid in the superficial matrix of articular cartilage, isolated from aged humans. Subsequently, Bonner et al. (6) performed histochemical and chemical studies on human articular cartilage and described an increase in lipid content, with increasing age; TG, cholesterol, and phospholipids
Received February 2, 1990; accepted November 14, 1990. Address correspondence and reprint requests to Dr. Toyoaki Akino, at Department of Biochemistry, Sapporo Medical College, South-1, West-17, Chuo-Ku, Sapporo 060, Japan.
341
M . NAGAO ET AL.
342
lated in articular chondrocytes. There have been several reports regarding the regulation of lipid metabolism by extracellular fatty acids in cultured cells: endothelial cells (7), fibroblasts (21,26), smooth muscle cells (8) and hepatocytes (15). Here, effects of extracellular fatty acids on lipid metabolism in cultured articular chondrocytes were studied. Results show that extracellular fatty acids can regulate the TG and phosphatidylcholine (PC) metabolism of articular chondrocytes in a concentration-dependent manner. Among the fatty acids studied, arachidonic acid was found to be the most potent regulator of lipid metabolism. To our knowledge, this is the first study on the regulation of fatty acid turnover in lipids of articular chondrocytes. MATERIALS AND METHODS Materials
[1-14C] oleic acid (56 mCi/mmol), [1-14C] linoleic acid (59 mCi/mmol) and [5,6,8,9,11,12,14,15-3H] arachidonic acid (163 Ci/mmol) were purchased from Amersham (Buckinghamshire, U.K.). Collagenase (Clostridiopeptidase A, type I), phospholipase C (Clostridium perfringens, type I) and phospholipase A, (Nuja nuju) were obtained from Sigma Chemical Co. (St. Louis, MO, U.S.A.). Fetal bovine serum (FBS, Lot. 3C045) was purchased from M.A. Bioproducts (Walkersville, MD, U.S.A.). Ham’s F12 medium was from Nissui Pharmaceutical Co. (Tokyo, Japan). Authentic oleic, linoleic, and arachidonic acids were obtained from Nakarai Chemical Co. (Kyoto, Japan). Dioleoyl (18: 1/18:1) and dilinoleoyl (18:2/18:2) phosphatid ylcholines were obtained from Funakoshi Co. (Tokyo, Japan). Neutral lipids and phospholipids, which were used as carrier substances in chromatographic analysis, were prepared in our laboratory (19). Analytical grades of all other chemicals were commercially obtained. CeIl Culture
Articular chondrocytes were obtained from pooled articular cartilage of 4-week-old Japanese white rabbits (800-900 g) as previously described (22). Freshly isolated chondrocytes (2 X lo5 cells) were inoculated into 35 mm culture dishes (Nunc, Denmark) in a maintenance medium of Ham’s F12 supplemented with 10% FBS and 0.01% streptomy-
J Orthop Res, Vol. 9,No. 3, 1991
cin, and cultured at 37°C for 72 h in 5% C02/air to obtain about 80% confluency. Preparation of Fatty Acid-Supplemented Media
Fatty acids were dissolved in ethanol. After adding 1 drop of 1N NaOH, the material was dried under a N, stream and redissolved in a small amount of warm distilled water. To the sodium salt of the fatty acids, FBS was added with mechanical stirring and the pH adjusted immediately to 7.4. Before supplementation, the serum contained 27.8 pA4 oleic, 5.4 p M linoleic and 14.7 p M arachidonic acids, as determined by the measurement of free fatty acid concentration and analysis of fatty acid composition by gas chromatography (GC), as reported previously (25). Experimental media, which contained various concentrations of radioactive fatty acids, were prepared by adding the fatty acidsupplemented FBS to Ham’s F12 medium containing 0.01% streptomycin. Fatty Acid Incorporation
After culturing for 72 h, experimental media, containing [14C]oleic, [14C]linoleic, or [3H]arachidonic acid, were added to culture media to adjust the final concentration of each fatty acid to 5,50,250, or 500 pM; the chondrocytes were further incubated with the radioactive fatty acids for 8 h at 37°C in 5% C02/air. After incubation, cells were washed three times with ice-cold phosphate buffered saline (PBS), containing 1% bovine serum albumin (BSA) (fatty acid free; Sigma, St. Louis, MO, U.S.A.); then methanol (1.5 ml) was added to stop the biological reactions. Cells were then scraped, transferred into tubes with methanol, and lipids were extracted with a mixture of chlorofordmethanol. To measure the turnover of the incorporated radioactive fatty acids, chase experiments were carried out. Confluent monolayers were exposed initially for 8 h at 37°C to 500 p M radioactive fatty acid. Cells were then washed with warm PBS, containing 1% BSA, and incubated for various times at 37°C in a maintenance medium, containing low concentrations of free fatty acids. After incubation, cells were washed and harvested to determine the amount of the remaining radioactivity. Lipid Analysis
Distribution of radioactivity among lipid classes was determined as follows: After incubation with
LIPID METABOLISM IN ARTICULAR CHONDROCYTES various concentrations of radioactive fatty acids, cells were washed three times with PBS, containing 1% BSA, to remove as much of the unincorporated free fatty acids as possible. After addition of an aliquot of methanol to the cells to stop the biological reactions, cells were scraped into tubes and lipids were extracted by the method of Bligh and Dyer (4). Distribution of radioactivity in the cellular lipid fractions was determined by spotting aliquots of the lipid extracts on silica gel G plates (Merck, Darmstadt, Germany), along with the standard lipid mixture. For isolation of the main lipid classes, such as cholesterol ester, TG, free fatty acid, cholesterol, and phospholipid, the plates were developed with a solvent system of n-hexanelethyl ethedacetic acid (80:20:0.5, v/v). Individual phospholipids were separated by two-dimensional, thin-layer chromatography, as described previously (1). After development, plates were stained with I, and spots, corresponding to the different lipid fractions, were scraped into scintillation vials containing 10 ml ACS-I1 scintillationfluid (Amersham, Buckinghamshire, U.K.). All radioactivity measurements were made with a Beckman LS9000 liquid scintillation spectrometer. For the preparative separation of labeled PC, the PC spot on the plates was visualized using a fluorescein reagent and then was recovered using Arvidson’s method (2). Purified PC was hydrolyzed by phospholipase C, according to the method described by Okano et al. (19). The diacylglycerol (DG) thus formed was acetylated and purified (12). The DG acetates, derived from PC, were resolved into molecular species using high-performance liquid chromatography (HPLC), as described by Itoh et al. (10) in our laboratory. The effluent was collected in counting vials with a constant time interval, and radioactivity was determined. The molecular species contained in the radioactive peaks were identified by comparison to the retention time of authentic molecular species of PC. The mass composition of lipid classes was determined as follows: Lipids of freshly isolated rabbit articular chondrocytes were extracted by the method of Bligh and Dyer (4). Amount of TG, cholesterol, and free fatty acids were determined using assay kits (Kyowa Medex, Tokyo), based on enzymatic methods; phospholipid content was determined by measuring phospholipid P (3). For analysis of phospholipid composition, individual phospholipids were separated and detected as described above, the phospholipid P content of each spot be-
343
ing determined. TG and PC were isolated from rabbit articular chondrocytes and from serum and liver of the same animals, as described above. Fatty acid methyl esters, prepared with BF,/methanol (17), were analyzed by GC, as reported previously (25). Purified PC was also treated with phospholipase A, and the degradation products isolated by thin-layer chromatography (18). Fatty acids and lysophosphatidylcholine were eluted from the gels, and each fatty acid methyl ester was prepared for analysis by GC . RESULTS The lipids of freshly isolated articular chondrocytes were composed mainly of phospholipids (63.7% of total lipid) and TG (22.4%). The prominent phospholipid was PC. As shown in Table 1, the PC of the chondrocytes contained much more oleic than linoleic and arachidonic acids, as unsaturated fatty acids. The main fatty acids of TG were oleic, palmitic, and linoleic acids, while the amount of arachidonic acid was less than 1%. Fatty acid composition of PC and TG in chondrocytes was distinctly different from that of liver and serum. It is, therefore, likely that high oleic and very low arachidonic acid contents are characteristics of articular chondrocytes. The effect of concentration on incorporation of unsaturated fatty acids, i.e., oleic, linoleic, and arachidonic acids, into chondrocyte TG and PC, after an 8 h-incubation, is shown in Fig. 1. Since fatty acid incorporations into cellular lipids have been shown to reach a plateau within 8 h, an 8 hincubation, which appears to be an approximation of the steady state of fatty acid uptake, was used in this study. As shown in Fig. 1, these radioactive unsaturated fatty acids were incorporated into TG and PC in a concentration-dependent manner. At concentrations higher than 5 p M of fatty acids in the medium, more radioactive fatty acids were incorporated into TG than into PC, although labelings of PC were also increased, with an increase of fatty acid concentration. At a concentration of arachidonic acid of 500 p M , incorporation of the [,HIlabeled fatty acid into TG was about 4-fold higher than that into PC, while at 500 p M oleic and linoleic acids, incorporation into TG was only about 2-fold higher than into PC. Thus, among the unsaturated fatty acids examined, arachidonic acid was incorporated much more into TG than into PC, in spite of the very low content of arachidonic acid in TG. The
J Orthop Rrs. Vol. 9, No. 3, 1991
M . NAGAO ET AL.
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TABLE 1. Fatty acid composition of phosphatidylcholine and triacylglycerol in rabbit articular chondrocytes Fatty acid composition (%) Phosphatidylcholine Articular chondrocytes
Triacylglycerol Liver ~
Total" 14:O 14:O DMA 16:O 16:O DMA 16: 1 18:O 18:O DMA 18: 1 18:2 20:3 20:4 22:4 225 22:6
1-position
2-position
Serum
Articular chondrocytes
~
Total
Total
Total"
Liver
Serum
__
__
Total
Total
1.3
1.1
0.1
0.1
tr
tr
ND
ND
ND
31.1 2 0 . 5
54.7
9.0
20.0
21.0
tr
tr tr
ND
ND tr
14.7
5.3 2.4
ND tr 25.2
21.5
8.0
3.2
tr
ND
ND
ND
ND
ND
ND
1.7 1.2
22.1 1.o
tr 0.1
10.2 30.6 1.7 11.8
36.3 f 1.2 19.3 f 0.1 0.2 f 0.3 0.8 2 0.1 0.5 f 0.1 0.2 f 0.1 0.2 2 0.2 4.0 2 0.6
28.8 26.6
tr
4.4 25.3 3 .O 18.5 0.9 0.7 0.4 1.5
29.6 20.8
3.6 f 1.2 1.1 f 0.2 2.9 2 2.5 0.2 f 0.4 3.3 f 0.8
54.5 7.8 3.3 6.6 2.7 5.1 0.4 1.8
2.0
f 0.2
4.7 10.1
f 0.6 f 1.1
tr 37.3 3.7
Others
f f
tr tr tr 6.1
0.6 0.1 0.3 1.5
2.2
f 0.1
ND 31.0
f 0.6
ND tr 5.3
f
1.3
1.4
1.3
ND
ND
29.6
28.9
ND tr
ND tr
tr
tr
5.9 0.5 0.2
4.9 0.3 0.2 0.6 5.2
tr 4.0
" Values are mean
2 SD from four separate analyses. Other values are mean from two separate analyses. DMA, dimethylacetal; ND, not detected; tr, trace (
Effect of Extracellular Fatty Acids on Lipid Metabolism in Cultured Rabbit Articular Chondrocytes Masato Nagao, Seiichi Ishii, "Yoshihisa Murata, and "Toyoaki Akino Departments of Orthopedic Surgery and *Biochemistry, Sapporo Medical College, Sapporo, Japan
Summary: Rabbit articular chondrocytes were cultured for 8 h in the presence of various concentrations (5-500 pA4) of [I4C] oleic, [I4C] linoleic, and E3H] arachidonic acids. The radioactive unsaturated fatty acids were incorporated into triacylglycerol (TG) and phosphatidylcholine (PC) in a concentrationdependent manner; more fatty acids were incorporated into TG than into PC, at higher concentrations of extracellular fatty acids. Among these fatty acids, arachidonic acid was incorporated into TG much more than into PC, in spite of a very low concentration of arachidonic acid in TG. After transfer of the labeled cells to maintenance medium, the radioactivity in TG declined rapidly and [3H]arachidonic acid radioactivity in PC increased continuously during the chase time periods. Palmitoyl-unsaturated species were mainly formed in PC when cultured at a concentration of 5 pM of each fatty acid. However, when cultured at 500 FM, unsaturated-unsaturated species, specific for each unsaturated fatty acid were actively formed. These findings indicate that (1) fatty acid composition of TG and PC in articular chondrocytes is influenced by the degree of fatty acid supply, (2) formation and turnover of TG plays a role in fatty acid metabolism of cells, and (3) fatty acid pairing in PC is modulated by extracellular fatty acid concentrations. Key Words: Articular chondrocytesLipid metabolism-Arachidonic acid-Phosphatidylcholine molecular species.
were shown to accumulate in intra- and extracellular regions of the cartilage. On the other hand, an important role of the synovial membrane in the pathogenesis of osteoarthritis has been reported recently by Stockwell et al. (24) and Lukoschek et al. (14). As with early osteoarthritic changes in experimental animal models, synovial membrane changes and lipid accumulation in superficial chondrocytes have been noted. Since the synovial fluid is a source of nutrition to the cartilage, lipids might be released into the synovial fluid from damaged synovial membranes and be taken up by chondrocytes, which would affect the lipid metabolism in the cells. However, there has been no detailed study on lipid metabolism of articular chondrocytes, particularly with respect to lipid storage in the cells. The present study was done to gain insight into the mechanism by which lipid metabolism is regu-
It has been reported that perturbation in lipid metabolism, particularly lipid storage, in articular chondrocytes might be associated with the degradation process, of articular cartilage, that occurs with aging (6,11,23). Stockwell (23) was the first to find increased amounts of triacylglycerol (TG) and phospholipid in the superficial matrix of articular cartilage, isolated from aged humans. Subsequently, Bonner et al. (6) performed histochemical and chemical studies on human articular cartilage and described an increase in lipid content, with increasing age; TG, cholesterol, and phospholipids
Received February 2, 1990; accepted November 14, 1990. Address correspondence and reprint requests to Dr. Toyoaki Akino, at Department of Biochemistry, Sapporo Medical College, South-1, West-17, Chuo-Ku, Sapporo 060, Japan.
341
M . NAGAO ET AL.
342
lated in articular chondrocytes. There have been several reports regarding the regulation of lipid metabolism by extracellular fatty acids in cultured cells: endothelial cells (7), fibroblasts (21,26), smooth muscle cells (8) and hepatocytes (15). Here, effects of extracellular fatty acids on lipid metabolism in cultured articular chondrocytes were studied. Results show that extracellular fatty acids can regulate the TG and phosphatidylcholine (PC) metabolism of articular chondrocytes in a concentration-dependent manner. Among the fatty acids studied, arachidonic acid was found to be the most potent regulator of lipid metabolism. To our knowledge, this is the first study on the regulation of fatty acid turnover in lipids of articular chondrocytes. MATERIALS AND METHODS Materials
[1-14C] oleic acid (56 mCi/mmol), [1-14C] linoleic acid (59 mCi/mmol) and [5,6,8,9,11,12,14,15-3H] arachidonic acid (163 Ci/mmol) were purchased from Amersham (Buckinghamshire, U.K.). Collagenase (Clostridiopeptidase A, type I), phospholipase C (Clostridium perfringens, type I) and phospholipase A, (Nuja nuju) were obtained from Sigma Chemical Co. (St. Louis, MO, U.S.A.). Fetal bovine serum (FBS, Lot. 3C045) was purchased from M.A. Bioproducts (Walkersville, MD, U.S.A.). Ham’s F12 medium was from Nissui Pharmaceutical Co. (Tokyo, Japan). Authentic oleic, linoleic, and arachidonic acids were obtained from Nakarai Chemical Co. (Kyoto, Japan). Dioleoyl (18: 1/18:1) and dilinoleoyl (18:2/18:2) phosphatid ylcholines were obtained from Funakoshi Co. (Tokyo, Japan). Neutral lipids and phospholipids, which were used as carrier substances in chromatographic analysis, were prepared in our laboratory (19). Analytical grades of all other chemicals were commercially obtained. CeIl Culture
Articular chondrocytes were obtained from pooled articular cartilage of 4-week-old Japanese white rabbits (800-900 g) as previously described (22). Freshly isolated chondrocytes (2 X lo5 cells) were inoculated into 35 mm culture dishes (Nunc, Denmark) in a maintenance medium of Ham’s F12 supplemented with 10% FBS and 0.01% streptomy-
J Orthop Res, Vol. 9,No. 3, 1991
cin, and cultured at 37°C for 72 h in 5% C02/air to obtain about 80% confluency. Preparation of Fatty Acid-Supplemented Media
Fatty acids were dissolved in ethanol. After adding 1 drop of 1N NaOH, the material was dried under a N, stream and redissolved in a small amount of warm distilled water. To the sodium salt of the fatty acids, FBS was added with mechanical stirring and the pH adjusted immediately to 7.4. Before supplementation, the serum contained 27.8 pA4 oleic, 5.4 p M linoleic and 14.7 p M arachidonic acids, as determined by the measurement of free fatty acid concentration and analysis of fatty acid composition by gas chromatography (GC), as reported previously (25). Experimental media, which contained various concentrations of radioactive fatty acids, were prepared by adding the fatty acidsupplemented FBS to Ham’s F12 medium containing 0.01% streptomycin. Fatty Acid Incorporation
After culturing for 72 h, experimental media, containing [14C]oleic, [14C]linoleic, or [3H]arachidonic acid, were added to culture media to adjust the final concentration of each fatty acid to 5,50,250, or 500 pM; the chondrocytes were further incubated with the radioactive fatty acids for 8 h at 37°C in 5% C02/air. After incubation, cells were washed three times with ice-cold phosphate buffered saline (PBS), containing 1% bovine serum albumin (BSA) (fatty acid free; Sigma, St. Louis, MO, U.S.A.); then methanol (1.5 ml) was added to stop the biological reactions. Cells were then scraped, transferred into tubes with methanol, and lipids were extracted with a mixture of chlorofordmethanol. To measure the turnover of the incorporated radioactive fatty acids, chase experiments were carried out. Confluent monolayers were exposed initially for 8 h at 37°C to 500 p M radioactive fatty acid. Cells were then washed with warm PBS, containing 1% BSA, and incubated for various times at 37°C in a maintenance medium, containing low concentrations of free fatty acids. After incubation, cells were washed and harvested to determine the amount of the remaining radioactivity. Lipid Analysis
Distribution of radioactivity among lipid classes was determined as follows: After incubation with
LIPID METABOLISM IN ARTICULAR CHONDROCYTES various concentrations of radioactive fatty acids, cells were washed three times with PBS, containing 1% BSA, to remove as much of the unincorporated free fatty acids as possible. After addition of an aliquot of methanol to the cells to stop the biological reactions, cells were scraped into tubes and lipids were extracted by the method of Bligh and Dyer (4). Distribution of radioactivity in the cellular lipid fractions was determined by spotting aliquots of the lipid extracts on silica gel G plates (Merck, Darmstadt, Germany), along with the standard lipid mixture. For isolation of the main lipid classes, such as cholesterol ester, TG, free fatty acid, cholesterol, and phospholipid, the plates were developed with a solvent system of n-hexanelethyl ethedacetic acid (80:20:0.5, v/v). Individual phospholipids were separated by two-dimensional, thin-layer chromatography, as described previously (1). After development, plates were stained with I, and spots, corresponding to the different lipid fractions, were scraped into scintillation vials containing 10 ml ACS-I1 scintillationfluid (Amersham, Buckinghamshire, U.K.). All radioactivity measurements were made with a Beckman LS9000 liquid scintillation spectrometer. For the preparative separation of labeled PC, the PC spot on the plates was visualized using a fluorescein reagent and then was recovered using Arvidson’s method (2). Purified PC was hydrolyzed by phospholipase C, according to the method described by Okano et al. (19). The diacylglycerol (DG) thus formed was acetylated and purified (12). The DG acetates, derived from PC, were resolved into molecular species using high-performance liquid chromatography (HPLC), as described by Itoh et al. (10) in our laboratory. The effluent was collected in counting vials with a constant time interval, and radioactivity was determined. The molecular species contained in the radioactive peaks were identified by comparison to the retention time of authentic molecular species of PC. The mass composition of lipid classes was determined as follows: Lipids of freshly isolated rabbit articular chondrocytes were extracted by the method of Bligh and Dyer (4). Amount of TG, cholesterol, and free fatty acids were determined using assay kits (Kyowa Medex, Tokyo), based on enzymatic methods; phospholipid content was determined by measuring phospholipid P (3). For analysis of phospholipid composition, individual phospholipids were separated and detected as described above, the phospholipid P content of each spot be-
343
ing determined. TG and PC were isolated from rabbit articular chondrocytes and from serum and liver of the same animals, as described above. Fatty acid methyl esters, prepared with BF,/methanol (17), were analyzed by GC, as reported previously (25). Purified PC was also treated with phospholipase A, and the degradation products isolated by thin-layer chromatography (18). Fatty acids and lysophosphatidylcholine were eluted from the gels, and each fatty acid methyl ester was prepared for analysis by GC . RESULTS The lipids of freshly isolated articular chondrocytes were composed mainly of phospholipids (63.7% of total lipid) and TG (22.4%). The prominent phospholipid was PC. As shown in Table 1, the PC of the chondrocytes contained much more oleic than linoleic and arachidonic acids, as unsaturated fatty acids. The main fatty acids of TG were oleic, palmitic, and linoleic acids, while the amount of arachidonic acid was less than 1%. Fatty acid composition of PC and TG in chondrocytes was distinctly different from that of liver and serum. It is, therefore, likely that high oleic and very low arachidonic acid contents are characteristics of articular chondrocytes. The effect of concentration on incorporation of unsaturated fatty acids, i.e., oleic, linoleic, and arachidonic acids, into chondrocyte TG and PC, after an 8 h-incubation, is shown in Fig. 1. Since fatty acid incorporations into cellular lipids have been shown to reach a plateau within 8 h, an 8 hincubation, which appears to be an approximation of the steady state of fatty acid uptake, was used in this study. As shown in Fig. 1, these radioactive unsaturated fatty acids were incorporated into TG and PC in a concentration-dependent manner. At concentrations higher than 5 p M of fatty acids in the medium, more radioactive fatty acids were incorporated into TG than into PC, although labelings of PC were also increased, with an increase of fatty acid concentration. At a concentration of arachidonic acid of 500 p M , incorporation of the [,HIlabeled fatty acid into TG was about 4-fold higher than that into PC, while at 500 p M oleic and linoleic acids, incorporation into TG was only about 2-fold higher than into PC. Thus, among the unsaturated fatty acids examined, arachidonic acid was incorporated much more into TG than into PC, in spite of the very low content of arachidonic acid in TG. The
J Orthop Rrs. Vol. 9, No. 3, 1991
M . NAGAO ET AL.
344
TABLE 1. Fatty acid composition of phosphatidylcholine and triacylglycerol in rabbit articular chondrocytes Fatty acid composition (%) Phosphatidylcholine Articular chondrocytes
Triacylglycerol Liver ~
Total" 14:O 14:O DMA 16:O 16:O DMA 16: 1 18:O 18:O DMA 18: 1 18:2 20:3 20:4 22:4 225 22:6
1-position
2-position
Serum
Articular chondrocytes
~
Total
Total
Total"
Liver
Serum
__
__
Total
Total
1.3
1.1
0.1
0.1
tr
tr
ND
ND
ND
31.1 2 0 . 5
54.7
9.0
20.0
21.0
tr
tr tr
ND
ND tr
14.7
5.3 2.4
ND tr 25.2
21.5
8.0
3.2
tr
ND
ND
ND
ND
ND
ND
1.7 1.2
22.1 1.o
tr 0.1
10.2 30.6 1.7 11.8
36.3 f 1.2 19.3 f 0.1 0.2 f 0.3 0.8 2 0.1 0.5 f 0.1 0.2 f 0.1 0.2 2 0.2 4.0 2 0.6
28.8 26.6
tr
4.4 25.3 3 .O 18.5 0.9 0.7 0.4 1.5
29.6 20.8
3.6 f 1.2 1.1 f 0.2 2.9 2 2.5 0.2 f 0.4 3.3 f 0.8
54.5 7.8 3.3 6.6 2.7 5.1 0.4 1.8
2.0
f 0.2
4.7 10.1
f 0.6 f 1.1
tr 37.3 3.7
Others
f f
tr tr tr 6.1
0.6 0.1 0.3 1.5
2.2
f 0.1
ND 31.0
f 0.6
ND tr 5.3
f
1.3
1.4
1.3
ND
ND
29.6
28.9
ND tr
ND tr
tr
tr
5.9 0.5 0.2
4.9 0.3 0.2 0.6 5.2
tr 4.0
" Values are mean
2 SD from four separate analyses. Other values are mean from two separate analyses. DMA, dimethylacetal; ND, not detected; tr, trace (
