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Department of Pediatrics, University of Arkansas for Medical Sciences and the Arkansas Children's Hospital Research Institute, Little Rock, AR 72202
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
ACKNOWLEDGMENT
FOOTNOTES
LITERATURE CITED
ABSTRACT 
In the past, lipoic acid has been administered to patients and test animals as therapy for diabetic neuropathy and various intoxications. Lipoic acid and the vitamin biotin have structural similarities. We sought to determine whether the chronic administration of lipoic acid affects the activities of biotin-dependent carboxylases. For 28 d, rats received daily intraperitoneal injections of one of the following: 1 ) a small dose of lipoic acid [4.3 µmol/( kg·d)]; 2 ) a large dose of lipoic acid [15.6 µmol/(kg·d)]; or 3 ) a large dose of lipoic acid plus biotin [15.6 and 2.0 µmol/(kg·d), respectively]. Another group received n-hexanoic acid [14.5 µmol/(kg·d)], which has structural similarities to lipoic acid and biotin and thus served as a control for the specificity of lipoic acid. A fifth group received phosphatidylcholine in saline injections and served as the vehicle control. The rat livers were assayed for the activities of acetyl-CoA carboxylase, pyruvate carboxylase, propionyl-CoA carboxylase, and 
-methylcrotonyl-CoA carboxylase. Urine was analyzed for lipoic acid; serum was analyzed for indicators of liver damage and metabolic aberrations. The mean activities of pyruvate carboxylase and -methylcrotonyl-CoA carboxylase were 28-36% lower in the lipoic acid-treated rats compared with vehicle controls (P < 0.05). Rats treated with lipoic acid plus biotin had normal carboxylase activities. Carboxylase activities in livers of n-hexanoic acid-treated rats were normal despite some evidence of liver injury. Propionyl-CoA carboxylase and acetyl-CoA carboxylase were not significantly affected by administration of lipoic acid. This study provides evidence consistent with the hypothesis that chronic administration of lipoic acid lowers the activities of pyruvate carboxylase and -methylcrotonyl-CoA carboxylase in vivo by competing with biotin.
INTRODUCTION
Lipoic acid (thioctic acid) is a cofactor in the transacylation reactions catalyzed by the various 
-keto acid dehydrogenase complexes; these multienzyme complexes play a central role in carbohydrate metabolism and the Krebs cycle (Garrett and Grisham 1995
). Dihydrolipoic acid, the reduced form of lipoic acid, is also capable of scavenging peroxyl radicals and of reducing semidehydroascorbate and dehydroascorbate (Kagan et al. 1992, Tsuchiya et al. 1992). The ascorbate-regenerating properties of dihydrolipoic acid apparently account for the -tocopherol sparing effect of lipoic acid (Kagan et al. 1992, Podda et al. 1994, Stoyanovsky et al. 1995).
Lipoic acid has been administered successfully to patients or test animals to reduce the signs of diabetic neuropathy (Jörg et al. 1988, Ziegler et al. 1995), to enhance glucose disposal in patients with noninsulin-dependent diabetes mellitus (Jacob et al. 1995), and to treat heavy-metal intoxication (Grunert 1960, Muller 1989). Lipoic acid treatment has been credited for reduced mortalities among patients with amanita poisoning. However, the efficacy of lipoic acid to treat amanita poisoning and the mechanism of action remain uncertain (Mitchel 1980, Piering and Bratanow 1990, Plotzker et al. 1982). On the basis of successful animal studies or in vitro studies, the administration of lipoic acid has been proposed to reduce neuronal injury and to improve cardiac recovery after ischemia (Haramaki et al. 1993, Prehn et al. 1992), to reduce HIV transcription via blocking the signal transduction in cells (Packer and Suzuki 1993), to prevent formation of cataracts (Packer 1994) and to treat hexacarbon-induced neuropathies (Altenkirch and Stoltenburg-Didinger 1990). To our knowledge, no clinical studies in humans are available to provide evidence for or against the efficacy of lipoic acid therapy in these latter clinical circumstances.
The chemical structure of lipoic acid is similar to that of the vitamin biotin (Fig. 1). Consequently, both lipoic acid and biotin bind to the protein avidin (Green 1963 and 1975, Hale et al. 1992) and are degraded in similar metabolic pathways (Chang et al. 1975, Furr et al. 1978, Lee et al. 1972, McCormick 1975, McCormick and Wright 1971, Nilsson and Ronge 1992, Shih et al. 1972, Spence et al. 1976, Wang et al. 1995). On the basis of the structural similarities between lipoic acid and biotin, we hypothesized that the chronic administration of lipoic acid at doses used in the therapeutic regimens discussed above might affect the activities of biotin-dependent carboxylases, namely, the mitochondrial -methylcrotonyl-CoA carboxylase (EC 6.4.1.4), propionyl-CoA carboxylase (EC 6.4.1.3) and pyruvate carboxylase (EC 6.4.1.1), and the cytosolic acetyl-CoA carboxylase (EC 6.4.1.2). Theoretically, lipoic acid could affect the carboxylase activities by competing with biotin at two steps in biotin transport and catalysis as follows: 1 ) by displacing biotin from holocarboxylase synthetase, the enzyme that catalyzes the incorporation of biotin into the apocarboxylases to form the holoenzymes; or 2 ) by competing with biotin for binding to biotin transporters responsible for the transport of biotin across cell membranes, thereby reducing the intracellular concentrations of biotin. Both mechanisms would reduce the activities of biotin-dependent carboxylases.
MATERIALS AND METHODS
= 150·(mol/L)
1·cm1] (Shih et al. 1974). A fourth group of rats received injections of n-hexanoic acid in phosphatidylcholine/saline (denoted as hexanoate group). n-Hexanoic acid served as a specificity control, because, like lipoic acid and biotin, n-hexanoic acid is degraded by -oxidation and binds to the protein avidin (Green 1975). A fifth group of rats received phosphatidylcholine in saline, denoted as vehicle control (Table 1). All solutions for ip injection were taken from the same batches. The solutions were kept frozen in small aliquots at 
70°C until they were used.
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Table 1. Composition of the solutions and intraperitoneal doses |
, Mitchel 1980, Piering and Bratanow 1990, Plotzker et al. 1982, Ziegler et al. 1995). The volume of fluid injected was adjusted daily to maintain a constant dose per gram body weight.
, Witters et al. 1979). Briefly, an aliquot of liver was homogenized on ice in a Potter-Elvehjem homogenizer in 5 volumes of buffer containing 50 mmol/L Tris chloride (pH 7.5), 1 mmol/L EDTA, 10 mmol/L 2-mercaptoethanol and 0.25 mol/L sucrose. The lysate was centrifuged at 14,000 × g for 30 min at 7°C. An aliquot of the liver supernatant (1 mL) was transferred onto a DG 10 column (exclusion molecular weight 6000; Bio-Rad, Hercules, CA) that had been equilibrated with the homogenizing buffer before and was eluted using the homogenizing buffer; 1-mL fractions were collected. Fractions 4 and 5 contained the acetyl-CoA carboxylase and were pooled. To activate the enzyme, 9 µL of the combined column fractions was mixed with 57 µL of a solution containing 96 mmol/L Tris buffer (pH 7.5), 8 mmol/L magnesium chloride, 1.2 mmol/L -mercaptoethanol, 1.6 g/L fatty acid-free bovine serum albumin and 8 mmol/L sodium citrate. The mixture was incubated for 30 min at 37°C. Then, 34 µL of a solution containing 5.6 mmol/L ATP, 0.4 mmol/L acetyl-CoA and 48 mmol/L NaH[14C]O3 (specific radioactivity 39.2 MBq/mmol) was added. For blanks, acetyl-CoA was omitted. After 8 min of incubation at 37°C, 17 µL of 1 mol/L perchloric acid was added to terminate the reaction. A 100-µL aliquot was transferred into a scintillation vial and dried at 55°C to remove unbound [14C]carbon dioxide. 14C was quantitated in 4 mL of Ultima Gold XR scintillation fluid (Packard Instrument, Meriden, CT) in a liquid scintillation analyzer Tri-Carb 1900-TR (Packard Instrument). Acetyl-CoA carboxylase activity is expressed as units per milligram of protein in purified liver fractions, where 1 unit equals 1 pmol of H[14C]O3 incorporated into malonyl-CoA per minute at 37°C.
-methylcrotonyl-CoA carboxylase (6.4.1.4). For each, liver was homogenized on ice in five volumes of buffer using a Potter-Elvejhem homogenizer. The buffer contained 0.25 mol/L sucrose, 50 mmol/L Tris (pH 7.9), 5 mmol/L reduced glutathione and 1 mmol/L disodium EDTA. The homogenate was sonicated on ice four times for 30 s each and centrifuged at 105,000 × g for 45 min at 4°C. Aliquots of supernatant were used for the various carboxylase assays.
). Instead of measuring the disappearance of NADH, we measured the incorporation of [14C]bicarbonate into oxalacetate. Therefore 18 mmol/L NaH[14C]O3 (specific radioactivity 39.2 MBq/mmol) was used in the incubation buffer instead of unlabeled bicarbonate. In blanks, pyruvate was omitted. The sample was incubated at 30°C for 5 min; 17 µL of 1 mol/L perchloric acid was added to terminate the reaction. A 100-µL aliquot was dried and 14C determined as described above. Pyruvate carboxylase activity is expressed as units per milligram of protein in purified liver fractions, where 1 unit equals 1 nmol of H[14C]O3 incorporated into oxaloacetate per minute at 30°C.
, as modified by Suormala et al. (1985). The sample was incubated at 30°C for 8 min. Then 17 µL of 1 mol/L perchloric acid was added to terminate the reaction. A 100-µL aliquot was dried and 14C determined as described above. Propionyl-CoA carboxylase activity is expressed as units per milligram of protein in purified liver fractions, where 1 unit equals 1 nmol of H[14C]O3 incorporated into methylmalonyl-CoA per minute at 30°C.
-methylcrotonyl-CoA carboxylase, 25 µL of liver supernatant was incubated with 100 µL of prewarmed buffer as described previously (Suormala et al. 1985). The sample was incubated at 30°C for 15 min. Then 20 µL of 1 mol/L perchloric acid was added to terminate the reaction. A 120-µL aliquot was dried and 14C determined as described above. -Methylcrotonyl-CoA carboxylase activity is expressed as units per milligram of protein in purified liver fractions, where 1 unit equals 1 nmol of H[14C]O3 incorporated into -methylglutaconyl-CoA per minute at 30°C.
, Furr et al. 1978). The acidified phases were extracted four times with one-fifth volumes of benzene. The benzene phases were combined and were evaporated to dryness. The lipoic acid analogs were quantitated by spectroscopic absorbance at 440 nm after derivatization with 2,6-dibromoquinone-4-chloroimide (Saccani and Neri 1970). For calibration, d,l-lipoic acid was used.
RESULTS
-methylcrotonyl-CoA carboxylase. The small and the large dose of lipoic acid reduced pyruvate carboxylase activity by ~28 and 35%, respectively, compared with vehicle controls (P < 0.05, Fig. 2, upper panel). The small and the large dose of lipoic acid reduced -methylcrotonyl-CoA carboxylase activity by ~36 and 29%, respectively, compared with vehicle controls (P < 0.05, Fig. 2, lower panel). When lipoic acid was administered with biotin, neither pyruvate carboxylase activity nor -methylcrotonyl-CoA carboxylase activity was significantly different from vehicle controls. When hexanoate was administered, neither pyruvate carboxylase activity nor -methylcrotonyl-CoA carboxylase activity was significantly different from vehicle controls.
-methylcrotonyl-CoA carboxylase (lower panel) in control rats and four treatment groups after 28 d (treatment as per Table 1). Values are means ± SD, n = 4-5. *P < 0.05 vs. vehicle controls.
-methylcrotonyl-CoA carboxylase for one rat in the group that received the large dose of lipoic acid was more than 4 SD greater than the mean of the other rats in the same group. This artifact likely arose from inadequate outgassing of [14C]O2 . This value was excluded from statistical analysis.
Table 2.
Urinary excretion of lipoic acid plus metabolites in the control rats and four treatment groups after 28 days1
Table 3.
Serum indicators of liver injury and metabolic dysfunction in control rats and four treatment groups after 28 days1,2
DISCUSSION
-methylcrotonyl-CoA carboxylase in livers from lipoic acid-treated rats were significantly less than in controls. We offer the following two mechanisms that could account for the small enzyme activities: 1 ) Lipoic acid at sufficiently great concentrations might displace biotin from its binding site at holocarboxylase synthetase. Consequently, lipoic acid would prevent biotin from being incorporated into one or more of the holocarboxylases. To our knowledge, inhibitory effects on holocarboxylase synthetase by compounds that are structurally related to biotin have not yet been reported. 2 ) Lipoic acid at great serum concentrations might compete with biotin for transport into cells by binding to the biotin transporter in the cell membrane. Biotin transport across membranes of rat liver cells and human hepatoma cells is an active process that is inhibited by biotin analogs with ring modifications (e.g., desthiobiotin) and by lipoic acid (Bowers-Komro and McCormick 1985, Said et al. 1994). In analogy, studies of intestinal biotin absorption are consistent with the presence of both saturable and nonsaturable components of biotin uptake (Bowman et al. 1986). Lipoic acid might interfere with biotin uptake at either level, i.e., intestinal absorption or uptake by hepatocytes. One might expect that intracellular biotin deficiency would reduce all of the carboxylase activities. To explain the selective effects we observed, a differential effect of intracellular biotin deficiency would be required, for example, a differential effect on protein turnover or differential affinities of the apocarboxylases as substrates for the holocarboxylase synthetase.
, Witters et al. 1979 and 1988). In contrast, pyruvate carboxylase and -methylcrotonyl-CoA carboxylase might be less strictly regulated by hormones. However, the present study did not address the hormonal regulation of carboxylases, and the reasons for the greater susceptibility of pyruvate carboxylase and -methylcrotonyl-CoA carboxylase to lipoate treatment remain uncertain.
and 1980). In a mouse preadipocyte cell line (3T3-L1), the half-life of holo-pyruvate carboxylase (28-35 h) is similar to the half-life of biotin associated with pyruvate carboxylase (31-32 h) (Freytag and Utter 1983). This is consistent with the hypothesis that biotin is released by enzyme degradation. The half-life of rat liver acetyl-CoA carboxylase is 48-59 h (Majerus and Kilburn 1969, Nakanishi and Numa 1970). We are not aware of studies on the half-lives of -methylcrotonyl-CoA carboxylase and propionyl-CoA carboxylase. However, the half-lives of other mitochondrial enzymes vary between 70 min and 8.1 d (Weinberg and Utter 1979). We conclude that lipoic acid must be administered chronically (~1-2 half-lives of enzymes) to exert an effect on carboxylases.
). We attribute the apparent discrepancy between that study and our studies to the short-term incubations (4-24 h) of Weiner and Wolf. These short times may not have allowed time for lipoic acid to displace biotin that is already covalently bound in holocarboxylases or to inhibit cellular biotin uptake producing intracellular biotin depletion and low carboxylase activities.
, Spence and McCormick 1976). In rat urine, 81% of an ip dose of lipoic acid (~24 µmol/kg body weight) was recovered within 24 h (Spence and McCormick 1976). In the present study, ANOVA with Dunnett's post-hoc procedure was chosen for statistical comparisons; this procedure does not permit post-hoc testing among the treatment groups but only comparisons between treatment groups and controls. Notwithstanding, the urinary excretion of lipoic acid was similar after the small and the large dose of lipoic acid. Hypothetically, the absorption of lipoic acid from the injection site might be limited, or large amounts of lipoic acid could have been converted to metabolites that were not detected by our assay procedure. Our assay for lipoic acid is semiquantitative and allows detection of only about one third of the lipoate analogs present in urine (Spence and McCormick 1976).
). Hence, the d,l-racemate was used in the pioneering rat studies by McCormick and co-workers (Spence and McCormick 1976). The data from the study presented here do not indicate whether the reductions in carboxylase activities are caused by d-isomer, l-isomer or both.
). The absorption is very fast and most of the compound is absorbed within 2 h, causing an immediate systemic availability similar to parenteral administration. Absorption of lipoic acid from the stomach contributes to this fast availability. The elimination half-life from plasma is similar for oral (t1/2 = 51.9 h) and intravenous (t1/2 = 60.6 h) administration, indicating that the elimination kinetics do not depend on the route of administration (Peter and Borbe 1995). Orally administered lipoic acid might affect carboxylase activities even more than parenterally administered lipoic acid. Lipoate inhibits the intestinal absorption of biotin (Said and Redha 1987).
-methylcrotonyl-CoA carboxylase; enzyme activities remain normal if biotin at pharmacological doses is administered together with lipoic acid. Even without supplemental biotin, the decreases in enzyme activities are not dramatic and would presumably not cause pathology in patients. For example, in individuals who are heterozygous for carboxylase deficiencies, carboxylase activities may be 50% of the activities of controls; these individuals are characteristically asymptomatic (Wolf 1995, Wolf and Feldman 1982). However, lipoic acid administered to such individuals could theoretically cause deleterious effects.
ACKNOWLEDGMENT
FOOTNOTES
Manuscript received 7 March 1997. Initial reviews completed 15 April 1997. Revision accepted 2 June 1997.
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