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Phytanic acid α-oxidation in rat liver mitochondria
ELSEVIER
Biochimica et Biophysica Acta 1201 (1994) 491-497
Biochi~mic~a et BiophysicaA~ta
Phytanic acid a-oxidation in rat liver mitochondria Kalipada Pahan, Sukhvarsha Gulati, Inderjit Singh * Department of Pediatrics, Medical University of South Carolina, 171 Ashley Avenue, Charleston, SC 29425, USA Received 15 November 1993; revised 12 April 1994
Abstract
The a-oxidation of phytanic acid in rat liver is a mitochondrial function. The inhibition of phytanic acid oxidation activity by inhibitors of acyl-CoA ligases (Naproxen and Triacsin C) and that of carnitine acyltransferase I (2-(5-(4-chlorophenyl)pentyl)oxirane-2 carboxylic acid (POCA) and 2-bromopalmitate) and increase in phytanic acid oxidation activity by the addition of exogenous carnitine and CoA to purified mitochondria suggests that phytanoyl-CoA ligase and carnitine acyltransferase I are essential for the activation and transport of phytanic acid across the mitochondrial membrane. This was further supported by the fact that activation of phytanic acid to phytanoyl-CoA was required only in intact mitochondria but not in mitochondria permealized with digitonin. DesulfoCoA, Naproxen and POCA treatment resulted in a significant decrease in phytanic acid oxidation in intact mitochondria but not in digitonin permealized mitochondria. These results show that a-oxidation of phytanic acid to pristanic acid, in contrast to fl-oxidation of fatty acids, requires free fatty acid as substrate. The inhibition of a-oxidation (~ 90%) of phytanic acid by different cytochrome P-450 enzyme inhibitors indicated that a-oxidation of phytanic acid is mediated through cytochrome P-450 containing enzyme system. Similar to the to-hydroxylation system in endoplasmic reticulum, a-hydroxylation and the subsequent a-oxidation of phytanic acid in mitochondria is induced by ciprofibrate, a hypolipidemic drug. Keywords: Mitochondrion; Phytanic acid oxidation; Ciprofibrate; Cytochrome P-450; (Rat liver)
1. Introduction
Phytanic acid (3,7,11,15-tetramethylhexadecanoic acid), a highly branched chain fatty acid, originates from dietary sources, and little, if any, is synthesized in mammals [1-3]. Phytol, a constituent of chlorophyll, is a precursor of phytanic acid. Due to the presence of methyl group at the fl-carbon of phytanic acid it cannot be degraded by fl-oxidation, a major catabolic pathway for fatty acids. It first undergoes a-oxidation to yield pristanic acid and CO 2 which in turn is degraded by fl-oxidation pathway to yield 3 mol of acetate, 3 mol of propionate and a mole of butyrate [1,4,5]. Recent studies from our laboratory have clearly demonstrated that in rodents the phytanic acid is a-oxidized in mitochondria and in humans it is a peroxisomal function [6]. The oxidation of phytanic acid in intact peroxisomes requires its prior activation to phytanoyl-CoA [6,7]. We have demonstrated that phytanoyl-CoA ligase, an enzyme which activates phytanic acid to phytanoyl-CoA, in the
* Corresponding author. Fax: + 1 (803) 7922033. 0304-4165/94/$07.00 © 1994 Elsevier Science B.V. All rights reserved SSDI 0 3 0 4 - 4 1 6 5 ( 9 4 ) 0 0 1 0 3 - 0
peroxisomal membrane is an enzyme distinct from palmitoyl-CoA and lignoceroyl-CoA ligases, the other known peroxisomal acyl-CoA ligases [7]. A number of reports are available on the requirement of different cofactors for a-oxidation of phytanic acid in rat liver [4,8-10]; however, the results are still very controversial. The lack of effect of exogenous addition of CoASH and carnitine suggested that the enzyme system, carnitine acyltransferase, for translocation of regular chain saturated and unsaturated fatty acids may not be involved in the transport of branched chain fatty acids (phytanic acid) for its a-oxidation in mitochondria [8,10]. However, inhibition of a-oxidation of phytanic acid by POCA, an inhibitor of carnitine acyltransferase I, in rat tissue suggested that phytanoyl-CoA ligase as well as carnitine acyltransferase system are involved in mitochondrial a-oxidation [6]. The oxidation of phytanic acid to pristanic acid possibly involves a number of steps starting with hydroxylation at a-position. The formation of hydroxyphytanic acid, as an intermediate, in the catabolism of phytanic acid has been demonstrated previously [1,11]. The hydroxylation of fatty acids at the to-position is mediated by the cytochrome P-450 containing enzyme system present in the endoplas-
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K. Pahan et al. / Biochimica et Biophysica Acta 1201 (1994) 491-497
mic reticulum (ER). The activity of this enzyme system in ER is markedly enhanced by the administration of hypolipidemic drugs [12,13]. This raises an interesting question of whether a-oxidation of fatty acid in mitochondria is mediated by a P-450 containing enzyme system and also if the activity of this enzyme system is modulated by hypolipidemic drugs. The present study was undertaken to evaluate the involvement of various cofactors (CoASH, carnitine) and cytochrome P-450 in a-oxidation of phytanic acid to pristanic acid in rat liver mitochondria.
2. Materials and methods
2.1. Materials
Nycodenz was obtained from Accurate Chemical and Scientific, Westbury, NY. ATP, Carnitine and CoASH were obtained from P-L Biochemicals, Milwaukee, WI. DesulfoCoA, Naproxen, Triacsin C, Nicardipine, Clotrimazole, Miconazole, Bifonazole and Digitonin were obtained from Sigma, St. Louis, MO. Ketoconazole was obtained from Biomol, Plymouth Meeting, PA. [1-14C]Phytanic acid (55 mCi/mmol) was purchased from Amersham International, Arlington Heights, IL, and [1-14C]phytanoyl-CoA was synthesized as described [14]. 2.2. Preparation of subcellular fractions
Isolation of mitochondria: male Sprague Dawley rats were used to isolate rat liver mitochondria. Rats were decapitated after an overnight fasting. The livers were removed and homogenized with 10 volumes ( w / v ) of 0.25 M sucrose containing 1 mM NaeEDTA, 1 /zg/ml antipain, 0.7/xg/ml pepstatin, 1 /zg/ml leupeptin, 2 /zg/ml aprotinin, 0.2 mM phenylmethylsulfonyl fluoride, 0.1% ethanol and 3 mM imidazole buffer pH 7.4 at 4°C. Nuclei and cellular debris were removed by centrifugation at 700 × g for 10 min. Heavy mitochondria were obtained by centrifugation of the postnuclear fraction at 5000 × g for 10 min. They were washed thrice and suspended in known volume of homogenizing buffer and used for further studies. 2.3. Effect of ciprofibrate
To study the effect of ciprofibrate on phytanic acid oxidation in different subcellular fractions, the rats were fed either a standard pellet diet or diet supplemented with 0.025% ( w / v ) ciprofibrate for 2 weeks. The standard diet was supplemented with ciprofibrate by soaking the pellets (500 g) in 60 ml of ethanol containing 125 mg of ciprofibrate. The control pellet diet was soaked only in the ethanol. The food was air dried to remove ethanol. The rats were decapitated after an overnight fasting and livers
were removed and homogenized in sucrose buffer as described above. The homogenate was fractionated by differential centrifugation to prepare lambda fraction (light mitochondrial fraction) as described by Leighton et al. [15]. This lambda fraction was layered over nycodenz density gradient and centrifuged at 33700 X g for 1 h in JV-20 Beckman vertical rotor with low acceleration and deceleration as described earlier [16]. The gradient fractions were analyzed for the subcellular markers; cytochrome c oxidase for mitochondria [17], NADPH cytochrome c reductase for microsomes [18], catalase for peroxisomes [19], and N-acetylglucosaminidase for lysosomes [20]. The protein concentrations were determined by the procedure of Bradford [21]. 2.4. Enzyme assay for activation and oxidation of [114C]phytanic acid
Phytanoyl-CoA ligase activity was measured as mentioned earlier [7,16]. Enzyme activity for the oxidation of phytanic acid to pristanic acid was measured as [14C]O2 released from [1-14C]phytanic acid using medium of the following composition (modified from [22]): [114C]phytanic acid (12 /zM) suspended in a-cyclodextrin (1.6 m g / m l assay volume) was added to the enzyme assay medium (final volume 250 /~I) containing 20 mM MopsHC1, pH 7.8, 10 mM Na2ATP, 80 /zM CoASH, 0.25 mM NAD, 30 mM KCI, 5 mM MgC12, 0.17 mM FAD, 2.5 mM L-carnitine, 0.25 mM NADPH, and 0.43 mM L-malic acid. The reaction was started by the addition of 10-50 /zg of protein and was stopped with 100 /zl of 5 M H2SO 4 after 2 h of incubation at 37°C. [14C]O2 was collected in KOH wetted cotton by shaking overnight. The cotton was transferred into a scintillation vial, and the radioactivity was measured. For solubilization of phytanic acid with acyclodextrin, the fatty acid (20- 106 dpm) was first dried in a tube under nitrogen and then resuspended in 3.5 ml (20 m g / m l ) of a-cyclodextrin by sonication.
3. Results
3.1. Requirement of CoA and carnitine for phytanic acid a-oxidation
Consistent with the previous reports [8-10] the addition of different concentrations of CoA and carnitine in isolated mitochondria did not show any significant effect on phytanic acid oxidation (data not shown) indicating that these cofactors are not required for the a-oxidation of phytanic acid. However, when isolated mitochondria were preincubated with desulfo-CoA, an analog of CoASH which does not support fatty acid activation, this resulted in an inhibition of phytanic acid oxidation (Fig. 1) suggesting that activation of phytanic acid to phytanoyl-CoA is essential for its oxidation. In the next step we depleted endogenous
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Fig. 1. Effect of different concentrations of desulfo-CoA on phytanic acid oxidation in isolated rat liver mitochondria. Mitochondrial preparation was incubated with different concentrations of desulfo-CoA for 30 min and the phytanic acid oxidation was then measured as described in the methodology section. Control (100%) activity represents 178.6+_13.2 p m o l / h per mg protein. Data is expressed as mean + S.D. of six values.
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CoASH and carnitine by preincubation of mitochondria with 4-pentenoic acid and studied the effect of the subsequent addition of exogenous CoASH and carnitine on mitochondrial phytanic acid oxidation (Fig. 2). Addition of CoASH and carnitine increased the phytanic acid oxidation significantly, suggesting that the carnitine acyltransferase system is involved in the transport of phytanic acid into mitochondria. To further support our observations that CoASH and carnitine systems are necessary for the oxidation of phytanic acid we studied the effect of specific inhibitors of acyl-CoA ligases (Naproxen and Triacsin C) and carnitine acyltransferase I (POCA and 2-bromopalmitate) on phytanic acid oxidation. Fig. 3a and b shows the effect of different concentrations of Naproxen and Triacsin C on phytanoyl-CoA ligase activity. About 80% of the total phytanoyl-CoA ligase activity was inhibited at 60 /xM concentration of Naproxen, whereas Triacsin C resuited in the same degree of inhibition at 200/xM concentration. Similar to ligase activity, phytanic acid oxidation activity was also found to decrease with addition of both Naproxen and Triacsin C (Fig. 4a and b) demonstrating that phytanic acid needs to be converted to its CoA-derivative prior to its oxidation to pristanic acid further confirming our observations that CoA-dependent activation to phytanoyl-CoA is necessary for the oxidation of phytanic acid. Inhibitors of carnitine acyltransferase I (CPT-I) (POCA or 2-bromopalmitate) inhibited the oxidation of phytanic acid (Fig. 5a and b). The inhibition of phytanic acid oxidation by inhibitors of phytanoyl-CoA ligases and CPT-I suggests that the carnitine system is essential for the mitochondrial phytanic acid oxidation. While studying the enzyme system for the a-oxidation of phytanic acid in mitochondria we observed that for its
Fig. 2. Effect of different concentrations of CoASH (A) and carnitine (B) on phytanic acid oxidation after depleting endogenous CoASH and carnitine. To deplete endogenous CoASH and carnitine isolated mitochondria was preincubated for 1 h with 100 /.tM 4-penteonic acid and 50 /zM acetyl CoA and the effect of exogenous addition of CoASH and carnitine on the rate of oxidation of phytanic acid was studied as described in the text. Results are mean + S.D. of six values.
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Fig. 6. Effect of cofactors and inhibitors of acyl-CoA ]igases and carnitine acyltransferase on the rate of phytanie acid a-oxidation (A) and palmitic acid fl-oxidation (B) in rat liver mitochondria. Enzyme activities are expressed as mean ± S.D. of three experiments, a, complete medium containing ATP, CoASH and MgCI2; b, plus 50 /xM POCA; c, plus 50 /xM desulfo-CoA; d, plus 50 /zM naproxen; e, plus 20 /xg/ml digitonin; f, plus POCA and digitonin; g, plus desulfo-CoA and digitonin; h, plus naproxen and digitonin; i, minus cofactors; j, minus cofactors plus 20 / x g / m l digitonin.
oxidation, activation of phytanic acid to phytanoyl-CoA was required only in intact mitochondria (Fig. 6Aa) but not in mitochondria permealized with digitonin (Fig. 6Aj). Deletion of ATP, CoASH and MgC12 (fatty acid activating cofactors) from the assay medium decreased the phytanic acid oxidation activity to 25 percent (Fig. 6Ai), and permealization of mitochondria with digitonin increased the activity to 80 percent of the values observed in the presence of fatty acid activating co-factors (Fig. 6Ae). DesulfoCoASH, an analog of CoASH (Fig. 6Ac), and Naproxen, an inhibitor of acyl-CoA ligase (Fig. 6Ad) resulted in significant inhibition ( P < 0.001) of phytanic acid oxidation in intact mitochondria but not in the digitonin permealized mitochondria (Fig. 6Ag and Ah). Similarly, addition of POCA, an inhibitor of CPT-1, led to a significant inhibition of c~-oxidation only in intact mitochondria (Fig. 6Ab) and not in digitonin permealized mitochondria (Fig. 6Af). For comparison, we studied the oxidation of palmitic acid under similar conditions (Fig. 6B). Similar to the oxidation of phytanic acid, the oxidation of palmitic acid in intact mitochondria was inhibited by POCA, desulfoCoA and Naproxen (Fig. 6Bb, Bc and Bd). In contrast to phytanic acid oxidation, the oxidation of palmitic acid was inhibited by desulfo-CoA and Naproxen in digitonin permealized mitochondria as well (Fig. 6Bg and Bh). However, addition of POCA did not result in inhibition of
K. Pahan et aL / Biochimica et Biophysica Acta 1201 (1994) 491-497 Table 1 Effect of cytochrome P-450 inhibitors on phytanic acid oxidation by rat liver mitochondria Inhibitors
Phytanic acid oxidation (% of control)
Ketoconazole Clotrimazole Miconazole Nicardipine Bifonazole
4.97 ± 1.25 7.33 + 2.12 7.89 + 2.36 9.45 +_2.75 10.76 ± 3.45
Ketoconazole, Clotrimazole and Bifonazole were solubilized in ethanol, whereas nicardipine and miconazole were solubilized in water. Purified rat liver mitochondria were preincubated for 30 min with 50 ~M of these compounds in 5 tzl of alcohol or water, and phytanic acid oxidation was assayed as described in the text. If inhibitors were solubilized in alcohol, the control had the same amount of alcohol. Control activity represents 178.6 ± 13.2 p m o l / h per mg protein.
palm±tic acid oxidation in digitonin permealized mitochondria (Fig. 6Bf). Similar to phytanic acid oxidation, the deletion of fatty acid activating cofactors from assay system decreased the palm±tic acid oxidation to 20 percent (Fig. 6Bi); however, permealization of mitochondria in these conditions increased the activity of phytanic acid a-oxidation (Fig. 6Aj) but not of palm±tic acid /3-oxidation (Fig. 6Bj).
3.2. Involvement of cytochrome P-450 in a-oxidation of phytanic acid In the present study we wanted to elucidate if cytochrome P-450 dependent hydroxylase system is involved in a-oxidation of phytanic acid. For this we studied the effect of Ketoconazole, Clotrimazole, Nicardipine, Miconazole and Bifonazole, potent inhibitors of cytochrome P-450, on phytanic acid oxidation. All of the these drugs used to inhibit cytochrome P-450 resulted in more than 90% inhibition of phytanic acid oxidation at 50 /zM concentration suggesting that a-hydroxylation of phytanic acid is mediated through cytochrome P-450 hydroxylase system (Table 1). It has been reported that administration of clofibrate, and various other hypolipidemic agents result in induction of microsomal cytochrome P-450 IVA-1 de-
495
pendent fatty acid omega hydroxylase activity [12,13]. Hence, it was interesting to study if these hypolipidemic drugs have any effect on the mitochondrial a-hydroxylat±on system. For this the rate of oxidation of phytanic acid was studied in mitochondria, microsomes, and peroxisomes isolated from ciprofibrate treated rat liver. The relative specific activities of marker enzymes and the percent of contamination of peak fractions of peroxisomes, mitochondria, and microsomes are shown in Table 2. These results show that mitochondria, microsomes, and peroxisomes isolated by the Nycodenz gradient were relatively pure, and their composition did not change significantly after ciprofibrate treatment. In agreement with our previous observations [6], the mitochondrial fraction showed the highest rate of oxidation of phytanic acid (198 + 42 pmol/h per mg protein) as compared to peroxisomes (11 ___5 pmol/h per mg protein) and microsomes ( 7 _ 3.0 pmol/h per mg protein) indicating that in rat phytanic acid oxidation is a mitochondrial function. Ciprofibrate treatment led to a significant increase (2-fold) in phytanic acid oxidation in mitochondrial fraction (Table 2).
4. Discussion:
Phytanic acid accumulates in excessive amounts in tissues and body fluids of patients with classical Refsum disease and diseases with abnormality in the biogenesis of peroxisomes. The catabolism of phytanic to pristanic acid via a-hydroxyphytanic acid was proposed many years ago; however, the actual identification of the organelle responsible and the various cofactors involved in the a-oxidation of phytanic acid to pristanic acid has been a matter of debate. The phytanic acid oxidation in isolated organelles provided evidence that phytanic acid is oxidized in mitochondria in rat and human liver [4,8,23,24] and also in rat liver endoplasmic reticulum in the presence of nonphysiological amounts of Fe 2÷ [25]. However, this conclusion is not consistent with the excessive accumulation and lack of oxidation of phytanic acid in disorders of biogenesis of peroxisomes and rhizomelic chondrodysplasia punctata
Table 2 Effect of ciprofibrate treatment on phytanic acid oxidation in different subcellular fractions isolated by Nycodenz gradient Activity:
Control (RSA) mitochondria
Catalase Cytochrome c oxidase NADPH cytochrome c reductase N-Acetyl fl-glucosaminidase Phytanic acid oxidation ( p m o l / h per mg protein)
0.19 4.45 0.31 0.28
_ 0.4 + 1.2 ± 0.11 ± 0.12
198.0 ± 42.0
Ciprofibrate (RSA) peroxisomes
microsomes
41.3 0.04 0.10 0.10
0.23 0.06 4.4 0.73
_ + ± ±
7.2 0.01 0.03 0.03
11.0 ± 5.0
The results are expressed as an average of six different gradients + S.D. RSA, relative specific activity.
+ + ± +
0.11 0.03 1.52 0.18
7.0 ± 3.0
mitochondria 0.22 3.8 0.39 0.39 415.0
+ + ± ±
0.09 0.9 0.13 0.16
± 91.0
peroxisomes 23.2 0.07 0.51 0.51
+ + ± +
4.8 0.01 0.01 0.1
21.0 ± 5.0
microsomes 0.35 0.09 3.62 1.53
+ + ± ±
0.14 0.04 1.41 0.66
15.0 ± 5.0
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K. Pahan et aL/ Biochimica et BiophysicaActa 1201 (1994) 491-497
[26-29]. A detailed study of oxidation of phytanic acid in liver and cultured skin fibroblasts demonstrated that in humans phytanic acid is predominantly oxidized in peroxisomes, and in rodents it is a mitochondrial function [6,30]. We also observed that Nycodenz, a gradient material used for isolation of subcellular organelles, has an inhibitory effect on the oxidation of phytanic acid to pristanic acid [6] and this inhibition may have contributed to misleading previous results [9,10]. The studies reported in this manuscript demonstrate that for a-oxidation of phytanic acid in mitochondria, it needs to be converted to phytanoyl-CoA for its transport into the mitochondria by the carnitine acyltransferase system. However, unlike the oxidation of acyl-CoA derivatives of saturated and unsaturated fatty acids (unbranched) by /3-oxidation in mitochondria, the phytanic acid is a-oxidized as free fatty acid to pristanic acid. Also, the a-oxidation of phytanic acid in rat mitochondria is mediated by a cytochrome P-450 containing enzyme system which is inducible by ciprofibrate, a hypolipidemic drug. 4.1. Requirement of CoASH and carnitine
For /3-oxidation of fatty acids in mitochondria the fatty acids are activated to acyl-CoA derivatives by acyl-CoA ligases on the cytoplasmic surface of the outer mitochondrial membrane and then are converted to acyl-carnitine derivatives by carnitine acyltransferase I localized on the inner mitochondrial membrane, and the fatty acids are transported into mitochondria as acyl-carnitine derivatives. In the matrix of mitochondria the acyl-carnitine derivatives are converted to acyl-CoA derivatives by carnitine acyltransferase II, and the acyl-CoAs are further oxidized by the /3-oxidation enzyme system. However, consistent with previous reports [8-10], the addition of different concentrations of CoASH and carnitine to isolated mitochondria did not show any effect on the a-oxidation of phytanic acid (data not shown here) indicating that these cofactors and the associated enzyme system (e.g., acyl-CoA ligase and carnitine acyltransferase for transport of fatty acids into mitochondria) are not involved in the a-oxidation of phytanic acid. However, these results were contradictory to the observed inhibitory effect of POCA [6], an inhibitor of carnitine palmitoyltransferase I on phytanic acid oxidation. We examined the possibility that mitochondrial preparation had significant amount of endogenous carnitine and CoASH; therefore, the exogenous addition of these cofactors may not have any effect. The observed increase in phytanic acid oxidation on addition of CoASH and carnitine in mitochondria depleted of their endogenous CoASH and carnitine content (Fig. 2) suggested that the carnitine acyl transferase system is involved in the transport of phytanic acid into mitochondria. The inhibition of phytanoyl-CoA ligase and phytanic acid oxidation by Naproxen and Triacsin C (Figs. 3 and 4), the inhibitors of acyl-CoA ligases, and inhibition of phytanic acid oxidation by POCA
and 2-bromopalmitate, inhibitors of CPT-I (Fig. 5) support the conclusion that for phytanic acid oxidation in mitochondria the phytanoyl-CoA ligase, for synthesis of phytanoyl-CoA, and CPT-I for its transport into mitochondria, are essential for its a-oxidation to pristanic acid. Substitution of CoA by desulfo-CoA inhibited a-oxidation of phytanic acid (Fig. 1). The observed activity (20-25%) of phytanic acid a-oxidation in intact mitochondrial preparation in the presence of various concentrations of desulfoCoA may only reflect the percentage of disrupted mitochondria during the experimental conditions. The strict requirements for activation of phytanic acid to phytanoyl-CoA for a-oxidation in intact mitochondria, but not in digitonin permealized mitochondria (Fig. 6) suggests that the function of phytanoyl-CoA ligase may be to synthesize phytanoyl-CoA for its transport into mitochondria. The inhibition of phytanic acid oxidation by desulfo-CoA, inhibitors of acyl-CoA ligases and CPT-I only in intact mitochondria as compared to near normal activity in digitonin permealized mitochondria clearly demonstrates that free phytanic acid may be the substrate for its oxidation to pristanic acid in mitochondria. The lack of inhibition by POCA in digitonin permealized mitochondria suggests that phytanic acid was able to enter in these permealized mitochondria independent of the CPT-I system. Once inside the mitochondria the phytanoyl-CoA is hydrolyzed to free phytanic acid and then a-oxidized to pristanic acid. In contrast to phytanic acid oxidation, the oxidation of palmitic acid was inhibited by desulfo-CoA and Naproxen, an inhibitor of acyl-CoA ligases, in digitonin-permealized mitochondria suggesting that for its oxidation palmitic acid needs to be converted to its acyl-CoA derivatives even in permealized mitochondria (Fig. 6B). 4.2. Involvement of cytochrome P-450 in a-oxidation of phytanic acid
The observed inhibition of phytanic acid oxidation in the presence of various cytochrome P450 inhibitors (Table 1) suggested that a-hydroxylation of phytanic acid is mediated through cytochrome P-450 hydroxylase system. These results are supported by previous findings [8,11] which showed partial inhibition of phytanic acid hydroxylation and oxidation by carbon monoxide. It has been very well documented that cytochrome P-450 IVA1 is involved in the w-hydroxylation of fatty acids [31]. This cytochrome P-450 is induced by hypolipidemic drugs [13,32] and is very specific for fatty acid hydroxylation and has no detectable activity towards other substrates [33,34]. Ciprofibrate treatment led to a significant increase (2fold) in phytanic acid oxidation in mitochondrial fraction (Table 2). Relatively this is a smaller increase than the increase in oJ-fatty acid oxidation in endoplasmic reticulum observed previously [13,31-34]. Although peroxisomal and microsomal activities show an increase on ciprofibrate treatment, their activities are negligible when com-
K. Pahan et al. /Biochimica et Biophysica Acta 1201 (1994) 491-497
pared with mitochondrial activity after ciprofibrate treatment. From the relative specific activities in Table 2 and the fact that mitochondria, microsomes and peroxisomes each constitute ~ 20, 20 and 2%, respectively, of total liver protein [15,35] we calculated that mitochondria contributed 0.8% in control and 1.4% in ciprofibrate treated peroxisomal peak as protein, whereas in microsomes, the contamination was 1.2 and 1.8% in control and ciprofibrate treated, respectively. From these calculations one can deduce that phytanic acid oxidation activity coming from the peroxisomal peak was actually contributed by its mitochondrial contamination. The residual activity observed in microsomes could be contributed by its mitochondrial contamination and by other non specific hydroxylase systems. Administration of ciprofibrate causes not only peroxisome proliferation, but also alteration in mitochondrial number and structure with concomitant increase in certain enzyme levels [36]. These results demonstrate that cytochrome P-450 is associated with mitochondrial a-hydroxylase enzyme system and its activity is modulated by hypolipidemic drugs. In summary, our results show that phytanic acid is transported in mitochondria by carnitine acyltransferase enzyme system; however, free fatty acid is a-oxidized to pristanic acid by cytochrome P-450 associated a-oxidation and treatment with ciprofibrate resulted in 2.17 fold increase in this activity in rat liver mitochondria.
Acknowledgements This work was supported by a grant from the National Institutes of Health (NS22576). The authors would like to thank Mrs. Carol Frazier for typing the manuscript.
References [1] Steinberg, D. (1989) The Metabolic Basis of Inherited Diseases (Scriver, C.R., Beaudet, A.L., Sly, W.S. and Valle, D., eds.), 6th Edn., pp. 1533-1550, McGraw-Hill Books, New York. [2] Lazarow, P.B. and Moser H.W. (1989) The Metabolic Basis of Inherited Disease (Scriver, C.R., Beaudet, A.L., Sly, W.S. and Valle, D., eds.), 6th Edn., pp. 1479-1509, McGraw-Hill Books, New York. [3] Stoffel, W. and Kahlke, W. (1965) Biochem. Biophys. Res. Commun. 19, 33-36. [4] Tsai, S.C., Avigan, J. and Steinberg, D. (1969) J. Biol. Chem. 244, 2682-2692. [5] Herndon, J.H. Jr., Steinberg, D., Uhlendorf, B.W., and Fales, H.M. (1969) J. Clin. Invest. 48, 1017-1032. [6] Singh, I., Pahan, K., Dhaunsi, G.S., Lazo, O. and Ozand, P. (1993) J. Biol. Chem. 268, 9972-9979. [7] Pahan, K., Cofer, J., Baliga, P. and Singh, I. (1993) FEBS Lett. 322, 101-104.
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[8] Muralidharan, V.B. and Kishimoto, Y. (1984) J. Biol. Chem. 259, 13021-13026. [9] Mihalik, S.J., Soliman, T.M., Day, R.F. and Watkins, P.A. (1992) Progr. Clin. Biol. Res. 375, 239-244. [10] Wanders, R.A. and VanRoermund, C.W.T. (1993) Biochim. Biophys. Acta 1167, 345-350. [11] Muralidharan, F.N. and Muralidharan, V.B. (1987) Int. J. Biochem. 19, 663-670. [12] Sharma, R., Lake, B.G., Foster, J. and Gibson, G.G. (1988) Biochem. Pharmacol. 37, 1193-1201. [13] Gibson, G.G. (1989) Xenobiotica 19, 1123-1148. [14] Akanuma, H. and Kishimoto, Y. (1979) J. Biol. Chem. 254, 10501056. [15] Leighton, F., Poole, B., Beaufay, H. and Bauduin, P.B. (1968) J. Cell Biol. 37, 482-572. [16] Lazo, O., Contreras, M., Bhushan, A., Stanley, W. and Singh, I. (1989) Arch. Biochem. Biophys. 270, 722-728. [17] Cooperstein, S.J. and Lazarow, P. (1971) J. Biol. Chem. 189, 655-670. [18] Beaufay, H., Amar-Cortesec, A., Feytmans, E., Thines-Sempoux, D., Wibo, M. Robbi, M.N. and Berthet, T. (1974) J. Cell Biol. 61, 188-200. [19] Baudhuin, P., Beaufay, Y.L., Rehman, O., Sellinger, Z., Wattiaux, R., Jacques, P. and deDuve, C. (1964) Biochem. J. 92, 179-184. [20] Sellinger, O.Z., Beaufay, H., Jacques, P., Doyer, A. and deDuve, C. (1960) Biochem. J. 74, 450-456. [21] Bradford, M. (1976) Anal. Biochem. 72, 248-254. [22] Singh, I., Moser, A.E., Goldfischer, S. and Moser H.W. (1984) Proc. Natl. Acad. Sci. USA 81, 4203-4207. [23] Skjeldal, O.H. and Stokke, O. (1987) Biochim. Biophys. Acta 921, 38-42. [24] Skjeldal, O.H. and Stokke, O. (1988) Scand. J. Clin. Lab Invest. 48, 97-102. [25] Huang, S., Van Veldhoven, P.P., Vanhoutte, F., Parrnentier, G., Eyssen, H.F. and Mannaerts, G.P. (1992) Arch. Biochem. Biophys. 296, 214-223. [26] Poulos, A., Sharp, P. and Whiting, M. (1984) Clin. Genet. 26, 579-586. [27] Stokke, O., Skrede, S., Ek, J. and Bjorkhem, I. (1984) Scand. J. Clin. Lab. Invest. 44, 463-464. [28] Singh, I., Johnson, G.H. and Brown, F.R. (1988) Am. J. Dis. Child 142, 1297-1301. [29] Lazarow, P.B. and Moser, H.W. (1989) in The Metabolic Basis of Inherited Diseases (Scriver, C.A., Beaudet, A.L., Sly, W.S. and Valle, D., eds.), Vol. 2, pp. 1501-1532, McGraw-Hill Books, New York. [30] Singh, I., Lazo, O., Pahan, K. and Singh, A.K. (1992) Biochim. Biophys. Acta 1180, 221-224. [31] Ortiz de Montellano, P.R. and Reich, N.O. (1984) J. Biol. Chem. 259, 4136-4141. [32] Hardwick, J.P., Song, B.J. Huberman, E. and Gonzalez, F.J. (1987) J. Biol. Chem. 262, 801-810. [33] Parker, G.L. and Orton, T.C. (1980) In Biochemistry, Biophysics and Regulation of Cytochrome P-450 (Gustafsson, J.A., Duke, J.C., Mode, A. and Rafter, J., eds.), pp. 373-377, Elsevier/North-Holland Press, Amsterdam. [34] Gibson, G.G., Orton, T.C. and Tamburini, P.P. (1982) Biochem. J. 203, 161-168. [35] Amar-Costesec, A.H., Beaufay, M., Wibo, D., Thines-Sempoux, E., Leytmans, M., Robbi and Berth, J. (1974) J. Cell Biol. 61, 201-212. [36] Hawkins, J.M., Jones, W.E., Bonner, F.W. and Gibson, G.G. (1987) Drug. Metab. Rev. 18, 441-515.
Biochimica et Biophysica Acta 1201 (1994) 491-497
Biochi~mic~a et BiophysicaA~ta
Phytanic acid a-oxidation in rat liver mitochondria Kalipada Pahan, Sukhvarsha Gulati, Inderjit Singh * Department of Pediatrics, Medical University of South Carolina, 171 Ashley Avenue, Charleston, SC 29425, USA Received 15 November 1993; revised 12 April 1994
Abstract
The a-oxidation of phytanic acid in rat liver is a mitochondrial function. The inhibition of phytanic acid oxidation activity by inhibitors of acyl-CoA ligases (Naproxen and Triacsin C) and that of carnitine acyltransferase I (2-(5-(4-chlorophenyl)pentyl)oxirane-2 carboxylic acid (POCA) and 2-bromopalmitate) and increase in phytanic acid oxidation activity by the addition of exogenous carnitine and CoA to purified mitochondria suggests that phytanoyl-CoA ligase and carnitine acyltransferase I are essential for the activation and transport of phytanic acid across the mitochondrial membrane. This was further supported by the fact that activation of phytanic acid to phytanoyl-CoA was required only in intact mitochondria but not in mitochondria permealized with digitonin. DesulfoCoA, Naproxen and POCA treatment resulted in a significant decrease in phytanic acid oxidation in intact mitochondria but not in digitonin permealized mitochondria. These results show that a-oxidation of phytanic acid to pristanic acid, in contrast to fl-oxidation of fatty acids, requires free fatty acid as substrate. The inhibition of a-oxidation (~ 90%) of phytanic acid by different cytochrome P-450 enzyme inhibitors indicated that a-oxidation of phytanic acid is mediated through cytochrome P-450 containing enzyme system. Similar to the to-hydroxylation system in endoplasmic reticulum, a-hydroxylation and the subsequent a-oxidation of phytanic acid in mitochondria is induced by ciprofibrate, a hypolipidemic drug. Keywords: Mitochondrion; Phytanic acid oxidation; Ciprofibrate; Cytochrome P-450; (Rat liver)
1. Introduction
Phytanic acid (3,7,11,15-tetramethylhexadecanoic acid), a highly branched chain fatty acid, originates from dietary sources, and little, if any, is synthesized in mammals [1-3]. Phytol, a constituent of chlorophyll, is a precursor of phytanic acid. Due to the presence of methyl group at the fl-carbon of phytanic acid it cannot be degraded by fl-oxidation, a major catabolic pathway for fatty acids. It first undergoes a-oxidation to yield pristanic acid and CO 2 which in turn is degraded by fl-oxidation pathway to yield 3 mol of acetate, 3 mol of propionate and a mole of butyrate [1,4,5]. Recent studies from our laboratory have clearly demonstrated that in rodents the phytanic acid is a-oxidized in mitochondria and in humans it is a peroxisomal function [6]. The oxidation of phytanic acid in intact peroxisomes requires its prior activation to phytanoyl-CoA [6,7]. We have demonstrated that phytanoyl-CoA ligase, an enzyme which activates phytanic acid to phytanoyl-CoA, in the
* Corresponding author. Fax: + 1 (803) 7922033. 0304-4165/94/$07.00 © 1994 Elsevier Science B.V. All rights reserved SSDI 0 3 0 4 - 4 1 6 5 ( 9 4 ) 0 0 1 0 3 - 0
peroxisomal membrane is an enzyme distinct from palmitoyl-CoA and lignoceroyl-CoA ligases, the other known peroxisomal acyl-CoA ligases [7]. A number of reports are available on the requirement of different cofactors for a-oxidation of phytanic acid in rat liver [4,8-10]; however, the results are still very controversial. The lack of effect of exogenous addition of CoASH and carnitine suggested that the enzyme system, carnitine acyltransferase, for translocation of regular chain saturated and unsaturated fatty acids may not be involved in the transport of branched chain fatty acids (phytanic acid) for its a-oxidation in mitochondria [8,10]. However, inhibition of a-oxidation of phytanic acid by POCA, an inhibitor of carnitine acyltransferase I, in rat tissue suggested that phytanoyl-CoA ligase as well as carnitine acyltransferase system are involved in mitochondrial a-oxidation [6]. The oxidation of phytanic acid to pristanic acid possibly involves a number of steps starting with hydroxylation at a-position. The formation of hydroxyphytanic acid, as an intermediate, in the catabolism of phytanic acid has been demonstrated previously [1,11]. The hydroxylation of fatty acids at the to-position is mediated by the cytochrome P-450 containing enzyme system present in the endoplas-
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mic reticulum (ER). The activity of this enzyme system in ER is markedly enhanced by the administration of hypolipidemic drugs [12,13]. This raises an interesting question of whether a-oxidation of fatty acid in mitochondria is mediated by a P-450 containing enzyme system and also if the activity of this enzyme system is modulated by hypolipidemic drugs. The present study was undertaken to evaluate the involvement of various cofactors (CoASH, carnitine) and cytochrome P-450 in a-oxidation of phytanic acid to pristanic acid in rat liver mitochondria.
2. Materials and methods
2.1. Materials
Nycodenz was obtained from Accurate Chemical and Scientific, Westbury, NY. ATP, Carnitine and CoASH were obtained from P-L Biochemicals, Milwaukee, WI. DesulfoCoA, Naproxen, Triacsin C, Nicardipine, Clotrimazole, Miconazole, Bifonazole and Digitonin were obtained from Sigma, St. Louis, MO. Ketoconazole was obtained from Biomol, Plymouth Meeting, PA. [1-14C]Phytanic acid (55 mCi/mmol) was purchased from Amersham International, Arlington Heights, IL, and [1-14C]phytanoyl-CoA was synthesized as described [14]. 2.2. Preparation of subcellular fractions
Isolation of mitochondria: male Sprague Dawley rats were used to isolate rat liver mitochondria. Rats were decapitated after an overnight fasting. The livers were removed and homogenized with 10 volumes ( w / v ) of 0.25 M sucrose containing 1 mM NaeEDTA, 1 /zg/ml antipain, 0.7/xg/ml pepstatin, 1 /zg/ml leupeptin, 2 /zg/ml aprotinin, 0.2 mM phenylmethylsulfonyl fluoride, 0.1% ethanol and 3 mM imidazole buffer pH 7.4 at 4°C. Nuclei and cellular debris were removed by centrifugation at 700 × g for 10 min. Heavy mitochondria were obtained by centrifugation of the postnuclear fraction at 5000 × g for 10 min. They were washed thrice and suspended in known volume of homogenizing buffer and used for further studies. 2.3. Effect of ciprofibrate
To study the effect of ciprofibrate on phytanic acid oxidation in different subcellular fractions, the rats were fed either a standard pellet diet or diet supplemented with 0.025% ( w / v ) ciprofibrate for 2 weeks. The standard diet was supplemented with ciprofibrate by soaking the pellets (500 g) in 60 ml of ethanol containing 125 mg of ciprofibrate. The control pellet diet was soaked only in the ethanol. The food was air dried to remove ethanol. The rats were decapitated after an overnight fasting and livers
were removed and homogenized in sucrose buffer as described above. The homogenate was fractionated by differential centrifugation to prepare lambda fraction (light mitochondrial fraction) as described by Leighton et al. [15]. This lambda fraction was layered over nycodenz density gradient and centrifuged at 33700 X g for 1 h in JV-20 Beckman vertical rotor with low acceleration and deceleration as described earlier [16]. The gradient fractions were analyzed for the subcellular markers; cytochrome c oxidase for mitochondria [17], NADPH cytochrome c reductase for microsomes [18], catalase for peroxisomes [19], and N-acetylglucosaminidase for lysosomes [20]. The protein concentrations were determined by the procedure of Bradford [21]. 2.4. Enzyme assay for activation and oxidation of [114C]phytanic acid
Phytanoyl-CoA ligase activity was measured as mentioned earlier [7,16]. Enzyme activity for the oxidation of phytanic acid to pristanic acid was measured as [14C]O2 released from [1-14C]phytanic acid using medium of the following composition (modified from [22]): [114C]phytanic acid (12 /zM) suspended in a-cyclodextrin (1.6 m g / m l assay volume) was added to the enzyme assay medium (final volume 250 /~I) containing 20 mM MopsHC1, pH 7.8, 10 mM Na2ATP, 80 /zM CoASH, 0.25 mM NAD, 30 mM KCI, 5 mM MgC12, 0.17 mM FAD, 2.5 mM L-carnitine, 0.25 mM NADPH, and 0.43 mM L-malic acid. The reaction was started by the addition of 10-50 /zg of protein and was stopped with 100 /zl of 5 M H2SO 4 after 2 h of incubation at 37°C. [14C]O2 was collected in KOH wetted cotton by shaking overnight. The cotton was transferred into a scintillation vial, and the radioactivity was measured. For solubilization of phytanic acid with acyclodextrin, the fatty acid (20- 106 dpm) was first dried in a tube under nitrogen and then resuspended in 3.5 ml (20 m g / m l ) of a-cyclodextrin by sonication.
3. Results
3.1. Requirement of CoA and carnitine for phytanic acid a-oxidation
Consistent with the previous reports [8-10] the addition of different concentrations of CoA and carnitine in isolated mitochondria did not show any significant effect on phytanic acid oxidation (data not shown) indicating that these cofactors are not required for the a-oxidation of phytanic acid. However, when isolated mitochondria were preincubated with desulfo-CoA, an analog of CoASH which does not support fatty acid activation, this resulted in an inhibition of phytanic acid oxidation (Fig. 1) suggesting that activation of phytanic acid to phytanoyl-CoA is essential for its oxidation. In the next step we depleted endogenous
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CoASH and carnitine by preincubation of mitochondria with 4-pentenoic acid and studied the effect of the subsequent addition of exogenous CoASH and carnitine on mitochondrial phytanic acid oxidation (Fig. 2). Addition of CoASH and carnitine increased the phytanic acid oxidation significantly, suggesting that the carnitine acyltransferase system is involved in the transport of phytanic acid into mitochondria. To further support our observations that CoASH and carnitine systems are necessary for the oxidation of phytanic acid we studied the effect of specific inhibitors of acyl-CoA ligases (Naproxen and Triacsin C) and carnitine acyltransferase I (POCA and 2-bromopalmitate) on phytanic acid oxidation. Fig. 3a and b shows the effect of different concentrations of Naproxen and Triacsin C on phytanoyl-CoA ligase activity. About 80% of the total phytanoyl-CoA ligase activity was inhibited at 60 /xM concentration of Naproxen, whereas Triacsin C resuited in the same degree of inhibition at 200/xM concentration. Similar to ligase activity, phytanic acid oxidation activity was also found to decrease with addition of both Naproxen and Triacsin C (Fig. 4a and b) demonstrating that phytanic acid needs to be converted to its CoA-derivative prior to its oxidation to pristanic acid further confirming our observations that CoA-dependent activation to phytanoyl-CoA is necessary for the oxidation of phytanic acid. Inhibitors of carnitine acyltransferase I (CPT-I) (POCA or 2-bromopalmitate) inhibited the oxidation of phytanic acid (Fig. 5a and b). The inhibition of phytanic acid oxidation by inhibitors of phytanoyl-CoA ligases and CPT-I suggests that the carnitine system is essential for the mitochondrial phytanic acid oxidation. While studying the enzyme system for the a-oxidation of phytanic acid in mitochondria we observed that for its
Fig. 2. Effect of different concentrations of CoASH (A) and carnitine (B) on phytanic acid oxidation after depleting endogenous CoASH and carnitine. To deplete endogenous CoASH and carnitine isolated mitochondria was preincubated for 1 h with 100 /.tM 4-penteonic acid and 50 /zM acetyl CoA and the effect of exogenous addition of CoASH and carnitine on the rate of oxidation of phytanic acid was studied as described in the text. Results are mean + S.D. of six values.
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494
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Fig. 6. Effect of cofactors and inhibitors of acyl-CoA ]igases and carnitine acyltransferase on the rate of phytanie acid a-oxidation (A) and palmitic acid fl-oxidation (B) in rat liver mitochondria. Enzyme activities are expressed as mean ± S.D. of three experiments, a, complete medium containing ATP, CoASH and MgCI2; b, plus 50 /xM POCA; c, plus 50 /xM desulfo-CoA; d, plus 50 /zM naproxen; e, plus 20 /xg/ml digitonin; f, plus POCA and digitonin; g, plus desulfo-CoA and digitonin; h, plus naproxen and digitonin; i, minus cofactors; j, minus cofactors plus 20 / x g / m l digitonin.
oxidation, activation of phytanic acid to phytanoyl-CoA was required only in intact mitochondria (Fig. 6Aa) but not in mitochondria permealized with digitonin (Fig. 6Aj). Deletion of ATP, CoASH and MgC12 (fatty acid activating cofactors) from the assay medium decreased the phytanic acid oxidation activity to 25 percent (Fig. 6Ai), and permealization of mitochondria with digitonin increased the activity to 80 percent of the values observed in the presence of fatty acid activating co-factors (Fig. 6Ae). DesulfoCoASH, an analog of CoASH (Fig. 6Ac), and Naproxen, an inhibitor of acyl-CoA ligase (Fig. 6Ad) resulted in significant inhibition ( P < 0.001) of phytanic acid oxidation in intact mitochondria but not in the digitonin permealized mitochondria (Fig. 6Ag and Ah). Similarly, addition of POCA, an inhibitor of CPT-1, led to a significant inhibition of c~-oxidation only in intact mitochondria (Fig. 6Ab) and not in digitonin permealized mitochondria (Fig. 6Af). For comparison, we studied the oxidation of palmitic acid under similar conditions (Fig. 6B). Similar to the oxidation of phytanic acid, the oxidation of palmitic acid in intact mitochondria was inhibited by POCA, desulfoCoA and Naproxen (Fig. 6Bb, Bc and Bd). In contrast to phytanic acid oxidation, the oxidation of palmitic acid was inhibited by desulfo-CoA and Naproxen in digitonin permealized mitochondria as well (Fig. 6Bg and Bh). However, addition of POCA did not result in inhibition of
K. Pahan et aL / Biochimica et Biophysica Acta 1201 (1994) 491-497 Table 1 Effect of cytochrome P-450 inhibitors on phytanic acid oxidation by rat liver mitochondria Inhibitors
Phytanic acid oxidation (% of control)
Ketoconazole Clotrimazole Miconazole Nicardipine Bifonazole
4.97 ± 1.25 7.33 + 2.12 7.89 + 2.36 9.45 +_2.75 10.76 ± 3.45
Ketoconazole, Clotrimazole and Bifonazole were solubilized in ethanol, whereas nicardipine and miconazole were solubilized in water. Purified rat liver mitochondria were preincubated for 30 min with 50 ~M of these compounds in 5 tzl of alcohol or water, and phytanic acid oxidation was assayed as described in the text. If inhibitors were solubilized in alcohol, the control had the same amount of alcohol. Control activity represents 178.6 ± 13.2 p m o l / h per mg protein.
palm±tic acid oxidation in digitonin permealized mitochondria (Fig. 6Bf). Similar to phytanic acid oxidation, the deletion of fatty acid activating cofactors from assay system decreased the palm±tic acid oxidation to 20 percent (Fig. 6Bi); however, permealization of mitochondria in these conditions increased the activity of phytanic acid a-oxidation (Fig. 6Aj) but not of palm±tic acid /3-oxidation (Fig. 6Bj).
3.2. Involvement of cytochrome P-450 in a-oxidation of phytanic acid In the present study we wanted to elucidate if cytochrome P-450 dependent hydroxylase system is involved in a-oxidation of phytanic acid. For this we studied the effect of Ketoconazole, Clotrimazole, Nicardipine, Miconazole and Bifonazole, potent inhibitors of cytochrome P-450, on phytanic acid oxidation. All of the these drugs used to inhibit cytochrome P-450 resulted in more than 90% inhibition of phytanic acid oxidation at 50 /zM concentration suggesting that a-hydroxylation of phytanic acid is mediated through cytochrome P-450 hydroxylase system (Table 1). It has been reported that administration of clofibrate, and various other hypolipidemic agents result in induction of microsomal cytochrome P-450 IVA-1 de-
495
pendent fatty acid omega hydroxylase activity [12,13]. Hence, it was interesting to study if these hypolipidemic drugs have any effect on the mitochondrial a-hydroxylat±on system. For this the rate of oxidation of phytanic acid was studied in mitochondria, microsomes, and peroxisomes isolated from ciprofibrate treated rat liver. The relative specific activities of marker enzymes and the percent of contamination of peak fractions of peroxisomes, mitochondria, and microsomes are shown in Table 2. These results show that mitochondria, microsomes, and peroxisomes isolated by the Nycodenz gradient were relatively pure, and their composition did not change significantly after ciprofibrate treatment. In agreement with our previous observations [6], the mitochondrial fraction showed the highest rate of oxidation of phytanic acid (198 + 42 pmol/h per mg protein) as compared to peroxisomes (11 ___5 pmol/h per mg protein) and microsomes ( 7 _ 3.0 pmol/h per mg protein) indicating that in rat phytanic acid oxidation is a mitochondrial function. Ciprofibrate treatment led to a significant increase (2-fold) in phytanic acid oxidation in mitochondrial fraction (Table 2).
4. Discussion:
Phytanic acid accumulates in excessive amounts in tissues and body fluids of patients with classical Refsum disease and diseases with abnormality in the biogenesis of peroxisomes. The catabolism of phytanic to pristanic acid via a-hydroxyphytanic acid was proposed many years ago; however, the actual identification of the organelle responsible and the various cofactors involved in the a-oxidation of phytanic acid to pristanic acid has been a matter of debate. The phytanic acid oxidation in isolated organelles provided evidence that phytanic acid is oxidized in mitochondria in rat and human liver [4,8,23,24] and also in rat liver endoplasmic reticulum in the presence of nonphysiological amounts of Fe 2÷ [25]. However, this conclusion is not consistent with the excessive accumulation and lack of oxidation of phytanic acid in disorders of biogenesis of peroxisomes and rhizomelic chondrodysplasia punctata
Table 2 Effect of ciprofibrate treatment on phytanic acid oxidation in different subcellular fractions isolated by Nycodenz gradient Activity:
Control (RSA) mitochondria
Catalase Cytochrome c oxidase NADPH cytochrome c reductase N-Acetyl fl-glucosaminidase Phytanic acid oxidation ( p m o l / h per mg protein)
0.19 4.45 0.31 0.28
_ 0.4 + 1.2 ± 0.11 ± 0.12
198.0 ± 42.0
Ciprofibrate (RSA) peroxisomes
microsomes
41.3 0.04 0.10 0.10
0.23 0.06 4.4 0.73
_ + ± ±
7.2 0.01 0.03 0.03
11.0 ± 5.0
The results are expressed as an average of six different gradients + S.D. RSA, relative specific activity.
+ + ± +
0.11 0.03 1.52 0.18
7.0 ± 3.0
mitochondria 0.22 3.8 0.39 0.39 415.0
+ + ± ±
0.09 0.9 0.13 0.16
± 91.0
peroxisomes 23.2 0.07 0.51 0.51
+ + ± +
4.8 0.01 0.01 0.1
21.0 ± 5.0
microsomes 0.35 0.09 3.62 1.53
+ + ± ±
0.14 0.04 1.41 0.66
15.0 ± 5.0
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K. Pahan et aL/ Biochimica et BiophysicaActa 1201 (1994) 491-497
[26-29]. A detailed study of oxidation of phytanic acid in liver and cultured skin fibroblasts demonstrated that in humans phytanic acid is predominantly oxidized in peroxisomes, and in rodents it is a mitochondrial function [6,30]. We also observed that Nycodenz, a gradient material used for isolation of subcellular organelles, has an inhibitory effect on the oxidation of phytanic acid to pristanic acid [6] and this inhibition may have contributed to misleading previous results [9,10]. The studies reported in this manuscript demonstrate that for a-oxidation of phytanic acid in mitochondria, it needs to be converted to phytanoyl-CoA for its transport into the mitochondria by the carnitine acyltransferase system. However, unlike the oxidation of acyl-CoA derivatives of saturated and unsaturated fatty acids (unbranched) by /3-oxidation in mitochondria, the phytanic acid is a-oxidized as free fatty acid to pristanic acid. Also, the a-oxidation of phytanic acid in rat mitochondria is mediated by a cytochrome P-450 containing enzyme system which is inducible by ciprofibrate, a hypolipidemic drug. 4.1. Requirement of CoASH and carnitine
For /3-oxidation of fatty acids in mitochondria the fatty acids are activated to acyl-CoA derivatives by acyl-CoA ligases on the cytoplasmic surface of the outer mitochondrial membrane and then are converted to acyl-carnitine derivatives by carnitine acyltransferase I localized on the inner mitochondrial membrane, and the fatty acids are transported into mitochondria as acyl-carnitine derivatives. In the matrix of mitochondria the acyl-carnitine derivatives are converted to acyl-CoA derivatives by carnitine acyltransferase II, and the acyl-CoAs are further oxidized by the /3-oxidation enzyme system. However, consistent with previous reports [8-10], the addition of different concentrations of CoASH and carnitine to isolated mitochondria did not show any effect on the a-oxidation of phytanic acid (data not shown here) indicating that these cofactors and the associated enzyme system (e.g., acyl-CoA ligase and carnitine acyltransferase for transport of fatty acids into mitochondria) are not involved in the a-oxidation of phytanic acid. However, these results were contradictory to the observed inhibitory effect of POCA [6], an inhibitor of carnitine palmitoyltransferase I on phytanic acid oxidation. We examined the possibility that mitochondrial preparation had significant amount of endogenous carnitine and CoASH; therefore, the exogenous addition of these cofactors may not have any effect. The observed increase in phytanic acid oxidation on addition of CoASH and carnitine in mitochondria depleted of their endogenous CoASH and carnitine content (Fig. 2) suggested that the carnitine acyl transferase system is involved in the transport of phytanic acid into mitochondria. The inhibition of phytanoyl-CoA ligase and phytanic acid oxidation by Naproxen and Triacsin C (Figs. 3 and 4), the inhibitors of acyl-CoA ligases, and inhibition of phytanic acid oxidation by POCA
and 2-bromopalmitate, inhibitors of CPT-I (Fig. 5) support the conclusion that for phytanic acid oxidation in mitochondria the phytanoyl-CoA ligase, for synthesis of phytanoyl-CoA, and CPT-I for its transport into mitochondria, are essential for its a-oxidation to pristanic acid. Substitution of CoA by desulfo-CoA inhibited a-oxidation of phytanic acid (Fig. 1). The observed activity (20-25%) of phytanic acid a-oxidation in intact mitochondrial preparation in the presence of various concentrations of desulfoCoA may only reflect the percentage of disrupted mitochondria during the experimental conditions. The strict requirements for activation of phytanic acid to phytanoyl-CoA for a-oxidation in intact mitochondria, but not in digitonin permealized mitochondria (Fig. 6) suggests that the function of phytanoyl-CoA ligase may be to synthesize phytanoyl-CoA for its transport into mitochondria. The inhibition of phytanic acid oxidation by desulfo-CoA, inhibitors of acyl-CoA ligases and CPT-I only in intact mitochondria as compared to near normal activity in digitonin permealized mitochondria clearly demonstrates that free phytanic acid may be the substrate for its oxidation to pristanic acid in mitochondria. The lack of inhibition by POCA in digitonin permealized mitochondria suggests that phytanic acid was able to enter in these permealized mitochondria independent of the CPT-I system. Once inside the mitochondria the phytanoyl-CoA is hydrolyzed to free phytanic acid and then a-oxidized to pristanic acid. In contrast to phytanic acid oxidation, the oxidation of palmitic acid was inhibited by desulfo-CoA and Naproxen, an inhibitor of acyl-CoA ligases, in digitonin-permealized mitochondria suggesting that for its oxidation palmitic acid needs to be converted to its acyl-CoA derivatives even in permealized mitochondria (Fig. 6B). 4.2. Involvement of cytochrome P-450 in a-oxidation of phytanic acid
The observed inhibition of phytanic acid oxidation in the presence of various cytochrome P450 inhibitors (Table 1) suggested that a-hydroxylation of phytanic acid is mediated through cytochrome P-450 hydroxylase system. These results are supported by previous findings [8,11] which showed partial inhibition of phytanic acid hydroxylation and oxidation by carbon monoxide. It has been very well documented that cytochrome P-450 IVA1 is involved in the w-hydroxylation of fatty acids [31]. This cytochrome P-450 is induced by hypolipidemic drugs [13,32] and is very specific for fatty acid hydroxylation and has no detectable activity towards other substrates [33,34]. Ciprofibrate treatment led to a significant increase (2fold) in phytanic acid oxidation in mitochondrial fraction (Table 2). Relatively this is a smaller increase than the increase in oJ-fatty acid oxidation in endoplasmic reticulum observed previously [13,31-34]. Although peroxisomal and microsomal activities show an increase on ciprofibrate treatment, their activities are negligible when com-
K. Pahan et al. /Biochimica et Biophysica Acta 1201 (1994) 491-497
pared with mitochondrial activity after ciprofibrate treatment. From the relative specific activities in Table 2 and the fact that mitochondria, microsomes and peroxisomes each constitute ~ 20, 20 and 2%, respectively, of total liver protein [15,35] we calculated that mitochondria contributed 0.8% in control and 1.4% in ciprofibrate treated peroxisomal peak as protein, whereas in microsomes, the contamination was 1.2 and 1.8% in control and ciprofibrate treated, respectively. From these calculations one can deduce that phytanic acid oxidation activity coming from the peroxisomal peak was actually contributed by its mitochondrial contamination. The residual activity observed in microsomes could be contributed by its mitochondrial contamination and by other non specific hydroxylase systems. Administration of ciprofibrate causes not only peroxisome proliferation, but also alteration in mitochondrial number and structure with concomitant increase in certain enzyme levels [36]. These results demonstrate that cytochrome P-450 is associated with mitochondrial a-hydroxylase enzyme system and its activity is modulated by hypolipidemic drugs. In summary, our results show that phytanic acid is transported in mitochondria by carnitine acyltransferase enzyme system; however, free fatty acid is a-oxidized to pristanic acid by cytochrome P-450 associated a-oxidation and treatment with ciprofibrate resulted in 2.17 fold increase in this activity in rat liver mitochondria.
Acknowledgements This work was supported by a grant from the National Institutes of Health (NS22576). The authors would like to thank Mrs. Carol Frazier for typing the manuscript.
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