Relationship between acid-soluble carnitine and coenzyme A pools in vivo

The relationship between the acid-soluble carnitine and coenzyme A pools was studied in fed and 24-h-starved rats after carnitine administration. Carnitine given by intravenous injection at a dose of 60μmol/100g body wt. was integrated into the animal's endogenous carnitine pool. Large amounts of acylcarnitines appeared in the plasma and liver within 5min of carnitine injection. Differences in acid-soluble acylcarnitine concentrations were observed between fed and starved rats after injection and reflected the acylcarnitine/carnitine relationship seen in the endogenous carnitine pool of the two metabolic states. Thus, a larger acylcarnitine production was seen in starved animals and indicated a greater source of accessible acyl-CoA molecules. In addition to changes in the amount of acylcarnitines present, the specific acyl groups present also varied between groups of animals. Acetylcarnitine made up 37 and 53% of liver acid-soluble acylcarnitines in uninjected fed and starved animals respectively. At 5min after carnitine injection hepatic acid-soluble acylcarnitines were 41 and 73% in the form of acetylcarnitine in fed and starved rats respectively. Despite these large changes in carnitine and acylcarnitines, no changes were observed in plasma non-esterified fatty acid or β-hydroxybutyrate concentrations in either fed or starved rats. Additionally, measurement of acetyl-CoA, coenzyme A, total acid-soluble CoA and acid-insoluble CoA demonstrated that the hepatic CoA pool was resistant to carnitine-induced changes. This lack of change in the hepatic CoA pool or ketone-body production while acyl groups are shunted from acyl-CoA molecules to acylcarnitines suggests a low flux through the carnitine pool compared with the CoA pool. These results support the concept that the carnitine/acid-soluble acylcarnitine pool reflects changes in, rather than inducing changes in, the hepatic CoA/acyl-CoA pool.     

Interorgan cooperativity in carnitine metabolism in the trained state

This study was designed to evaluate the effects of chronic exercise training on carnitine acetyl- and palmitoyltransferase activity and the distribution of carnitine forms and concentrations in various organs and tissues of female rats. Sprague-Dawley rats were swim trained 6 days/wk and progressed to 75-min swims twice daily (with 3% of their total body weight attached to the medial portion of the tail) at the end of 5 wk of training. Sedentary (S, n = 12) and trained (T, n = 13) animals were killed by decapitation, and the livers, kidneys, hearts, and several skeletal muscle types were removed and immediately frozen in liquid N2 and/or extracted for enzyme activity assays. Blood was collected and plasma was stored frozen. Samples were assayed for free, acid-soluble, and acid-insoluble carnitine. Free carnitine increased significantly (P less than 0.03) in T hearts. Free carnitine remained unchanged in liver, but short-chain acylcarnitines increased significantly (P less than 0.001). There was a significant (P less than 0.001) reduction in long-chain acylcarnitines in kidney in the trained rats, and plasma short-chain acylcarnitine levels also decreased (P less than 0.001). Several significant changes in carnitine distribution also occurred in the superficial and deep portions of the vastus lateralis and in the mixed gastrocnemius muscles. There was a significant reduction in carnitine acetyltransferase activity with training in both the soleus (P less than 0.02) and superficial gastrocnemius (P less than 0.002) muscles. The deep portion of the gastrocnemius muscle contained significantly higher activity than either the superficial portion or the soleus.(ABSTRACT TRUNCATED AT 250 WORDS)     

Uptake, metabolism and release of carnitine and acylcarnitines in the perfused rat liver

The uptake and release of carnitine and isovalerylcarnitine have been studied in the perfused rat liver. Labelled carnitine accumulates in rat livers perfused with 50 or 500 microM [3H]carnitine. When alpha-ketoisocaproate (5 mM) is added to the perfusate after 30 min of perfusion, the net uptake of carnitine in the liver stops, and there is even a decrease in liver radioactivity. The decrease in liver carnitine can be attributed to an enhanced formation and efflux to the perfusate of short-chain acylcarnitines. Thin-layer chromatography of liver and perfusate extracts showed that efflux rates for branched-chain acylcarnitines (isovalerylcarnitine) formed are at least 2.5-fold the efflux rate for carnitine. Acetylcarnitine is released about twice as fast as carnitine from the liver. Perfusion with 50 microM [3H]isovalerylcarnitine showed that the influx rate of isovalerylcarnitine exceeds that of carnitine 1.5-fold. Since the efflux rate is still higher, a net loss of carnitine from the liver to the perfusate will result when branched-chain acylcarnitines are formed in the perfused liver. The addition of 500 microM unlabelled carnitine to the perfusate does not influence the release of labelled carnitine or acylcarnitines from the liver, showing that uptake and release are independent processes. Isovalerylcarnitine accumulates faster than carnitine does, also in the perfused rat heart. A mechanism for the development of secondary carnitine deficiencies associated with organic acidemia is proposed.     

Surplus acylcarnitines in the plasma of starved rats derive from the liver

The method used here to assess the contribution of liver to plasma acylcarnitine is based on the idea that in rat, shortly after administration of [3H]butyrobetaine the [3H]carnitine appearing in the plasma derives from the liver and so does the acyl moiety of [acyl-3H] carnitine. In the perchloric acid extracts of plasma and liver, the ester fraction of total carnitine was determined by enzymatic analysis and that of [3H]carnitines was determined by high performance liquid chromatography. The ester fraction of total carnitine in the plasma of fed rats was 32.6% while that of [3H]carnitines was 67.9%, 1 h following injection of [3H]butyrobetaine. For 48 h starved rats the equivalent values were 54.2 and 84.0%, respectively. 24 h after the administration of [3H]butyrobetaine, the ester content became the same in the total and [3H]carnitines. That the newly synthesized carnitine was more acylated (67.9 versus 32.6%, fed) indicates that liver exports acyl groups with carnitine as carrier. The observation that the ester fraction in the newly synthesized plasma carnitine increased with fasting (84.0 versus 67.9%) indicates that the surplus plasma acylcarnitine in fasting ketosis derives from the liver. Perfused livers, however, released carnitine with the same ester content (60-61%) whether they were from fed or fasted animals. Probably, the increased plasma [acylcarnitine] in fasting develops not by an increased ester output from the liver but by an altered handling in extrahepatic tissues.     

Carnitine metabolism in the vitamin B-12-deficient rat.

In vitamin B-12 (cobalamin) deficiency the metabolism of propionyl-CoA and methylmalonyl-CoA are inhibited secondarily to decreased L-methylmalonyl-CoA mutase activity. Production of acylcarnitines provides a mechanism for removing acyl groups and liberating CoA under conditions of impaired acyl-CoA utilization. Carnitine metabolism was studied in the vitamin B-12-deficient rat to define the relationship between alterations in acylcarnitine generation and the development of methylmalonic aciduria. Urinary excretion of methylmalonic acid was increased 200-fold in vitamin B-12-deficient rats as compared with controls. Urinary acylcarnitine excretion was increased in the vitamin B-12-deficient animals by 70%. This increase in urinary acylcarnitine excretion correlated with the degree of metabolic impairment as measured by the urinary methylmalonic acid elimination. Urinary propionylcarnitine excretion averaged 11 nmol/day in control rats and 120 nmol/day in the vitamin B-12-deficient group. The fraction of total carnitine present as short-chain acylcarnitines in the plasma and liver of vitamin B-12-deficient rats was increased as compared with controls. When the rats were fasted for 48 h, relative or absolute increases were seen in the urine, plasma, liver and skeletal-muscle acylcarnitine content of the vitamin B-12-deficient rats as compared with controls. Thus vitamin B-12 deficiency was associated with a redistribution of carnitine towards acylcarnitines. Propionylcarnitine was a significant constituent of the acylcarnitine pool in the vitamin B-12-deficient animals. The changes in carnitine metabolism were consistent with the changes in CoA metabolism known to occur with vitamin B-12 deficiency. The vitamin B-12-deficient rat provides a model system for studying carnitine metabolism in the methylmalonic acidurias.     

Butyrobetaine availability in liver is a regulatory factor for carnitine biosynthesis in rat. Flux through butyrobetaine hydroxylase in fasting state

Urinary excretion of total carnitine in 48-h fasted rats dropped to 0.30 +/- 0.01 mumol/day from 2.23 +/- 0.4 mumol/day found in fed, control animals (mean +/- SEM). Despite this marked retention, the total carnitine content of the whole body remained constant, about 83 mumol, predicting a slow-down in biosynthesis. The conversion of butyrobetaine into carnitine takes place only in the liver in rats. 48 h of starvation caused a decrease in the liver butyrobetaine level from 11.6 +/- 1.19 nmol/g to 9.30 +/- 1.19 nmol/g, which in whole livers corresponds to a decrease from 138 nmol to 61.3 nmol. The conversion rate of butyrobetaine into carnitine was studied with radiolabelled butyrobetaine. 30 min after injection of [3H]butyrobetaine the carnitine pool in the liver of fasted rats was labelled to about the same extent as that in fed rats, but from a butyrobetaine pool with higher specific radioactivity. Therefore, the conversion rate of butyrobetaine into carnitine was reduced. The newly formed carnitine found in the whole body of fasted rats was estimated to be 59% of controls. We conclude that the biosynthesis of carnitine in fasted rats slows down, for which a decreased availability of butyrobetaine in the liver is responsible. Urinary excretion of butyrobetaine in the fasted group decreased to 74.1 nmol/day from the 222-nmol/day control value while the butyrobetaine content of whole body did not significantly decrease (2.85 mumol vs. 3.04 mumol). Urinary excretion of trimethyllysine was also depressed.     

Liver carnitine metabolism after partial hepatectomy in the rat: Effects of nutritional status and inhibition of carnitine palmitoyltransferase

This study examined the effects of partial hepatectony on hepatic carnitine and acylcarnitine concentrations in fed or 24 h-starved partially hepatectonized (PH) or sham-operated (SO) rats at 1 or 4 days after surgery. The ratio of free to esterified carnitine was low in fed PH rats at day 1 : the low ratio was increased to the SO value when mitochondrial fat oxidation was inhibited by 2-tetradecylglycidate. Starvation (24 h) increased plasma [non-esterified fatty acid] in PH or SO rats, the increases being greater at day 1 than at day 4. Hepatic [long-chain acylcarnitine] were also increased. These latter increases were a consequence of increased mitochondrial fat oxidation since they were not observed in PH or SO rats treated with 2-tetradecylglycidate. Whereas the starvation-induced increase in long-chain acylcarnitine was associated with increased [ketone body] in livers of SO rats at both day 1 and day 4 after surgery, [ketone body] was inappropriately low for the steady-state long-chain [acylcarnitine] in livers of PH rats at the first post-operative day. This was not a consequence of a decrease in [total carnitine] in the liver. The results are discussed with reference to the role of the liver in determining the relative proportions of the fat fuels available for extrahepatic tissues and the effects of liver cell proliferation on hepatic triacylclycerol metabolism.     

Release of carnitine from the perfused rat liver

Perfused rat liver was shown to be the proper model for studies on hepatic cellular transport of carnitine. During recirculating perfusion the livers kept equilibrium with 45 nmol/ml total carnitine in perfusate, exhibited concentrative uptake and there was no sign of artificial leakage. The release side of the carnitine transport was characterized by utilizing outflow perfusions. The livers from fed rats exported daily 9.93 mumol per 100 g body weight total carnitine. This release rate is 4- or 10-fold higher than the estimated daily turnover in vivo or the measured urinary excretion. Therefore, the major part of the released carnitine has to re-enter the liver. The outward carnitine transport does not depend on energy or the Na+-K+ pump, since it did not respond to metabolic poisons and ouabain. However, the release rate was strongly inhibited by mersalyl and showed saturability in function of tissue carnitine levels. The Vmax of the saturable outward transport system was 2.47 nmol . min-1 . g-1 liver, the apparent Km was 0.27 mM tissue level (both as compared to total carnitine). These data showed the outward transport of carnitine from the liver to be protein mediated. The contribution of a diffusion (nonsaturable) component was estimated to be 20-25% in the range of tissue levels occurring in vivo. The rate of carnitine release from the liver decreased as an effect of 24 h starvation from the daily 9.92 mumol release to 6.55 mumol on 100 g body weight basis. This decrease is more pronounced when the release rates are expressed on the basis of tissue carnitine levels. The resulting value can be called rate constant (at the linear part of the saturation curve, Fig. 5) and it decreased to 5.00 min-1 from 8.41 min-1 as an effect of starvation. We have concluded that the altered parameters of carnitine transport across the liver cell is decisive in developing the higher hepatic carnitine concentration in the fasted state.     

Effect of insulin and glucagon on the uptake of carnitine by perfused rat liver

The possible direct effects of insulin and glucagon on carnitine uptake by perfused rat liver were studied with L-[3H]carnitine of an initial concentration of 50 microM in the perfusate. Insulin (10 nM) did not significantly affect the uptake by livers from fed animals. However, insulin could reverse the stimulated transport by livers from 24-h fasted animals, reducing the uptake rate from 852 +/- 54.1 to 480 +/- 39.9 (mean +/- S.E.), P less than 0.01 (rates are expressed as nmol per h per 100 g body wt). Glucagon (50 nM) stimulated the uptake rate when livers were either from fed (551 +/- 40.1 vs. 915 +/- 55.3, P less than 0.01) or from fasted animals (852 +/- 54.1 vs. 1142 +/- 88.1, P less than 0.02). Based on these and earlier observations, we propose that the carnitine concentration in rat liver is controlled by insulin and glucagon via cellular transport processes.     

Carnitine metabolism during exercise in patients with peripheral vascular disease

The distribution between carnitine and the acyl derivatives of carnitine reflects changes in the metabolic state of a variety of tissues. Patients with peripheral vascular disease (PVD) develop skeletal muscle ischemia with exertion. This impairment in oxidative metabolism during exercise may result in the generation of acylcarnitines. To test this hypothesis, 11 patients with PVD and 7 age-matched control subjects were evaluated with graded treadmill exercise. Subjects with PVD walked to maximal claudication pain at a peak O2 consumption (VO2) of 19.9 +/- 1.3 ml X kg-1 X min-1 (mean +/- SE). Control subjects were taken to a near-maximal work load at a VO2 of 31.3 +/- 1.0 ml X kg-1 X min-1. In patients with PVD, the plasma concentration of total acid-soluble, long-chain acylcarnitine and total carnitine was increased at peak exercise compared with resting values. Four minutes postexercise, the plasma short-chain acylcarnitine concentration was also increased. In control subjects taken to the higher work load, only the long-chain acylcarnitine concentration was increased at peak exercise. In patients with PVD, plasma short-chain acylcarnitine concentration at rest was negatively correlated with subsequent maximal walking time (r = -0.51, P less than 0.05). In conclusion, acylcarnitines increased in patients with PVD who walked to maximal claudication pain, whereas control subjects did not show equivalent changes even when taken to a higher work load. The relationship between short-chain acylcarnitine concentration at rest and subsequent exercise performance suggests that repeated episodes of ischemia may cause chronic accumulation of short-chain acylcarnitine in plasma in proportion to the severity of disease.(ABSTRACT TRUNCATED AT 250 WORDS)     

Perfused liver carnitine palmitoyl-transferase activity and ketogenesis in streptozotocin treated and genetic hyperinsulinemic rats. Effect of glucagon.

Perfused liver carnitine palmitoyl transferase (CPT) activity and ketone body output were determined in streptozotocin -- treated and untreated Sprague-Dawley and Zucker rats. Streptozotocin enhanced liver ketogenic capacity and CPT activity in both these strains. No difference was observed in CPT activity or in ketone body production between the fatty and lean Zucker strains. Glucagon, added directly to the perfusate, had no influence on ketone body output and only in the livers of obese Zücker rats increased CPT activity.     

Effects of oleic acid on the biosynthesis of lipoprotein apoproteins and distribution into the very-low-density lipoprotein by the isolated perfused rat liver.

The effects of oleic acid on the biosynthesis and secretion of VLDL (very-low-density-lipoprotein) apoproteins and lipids were investigated in isolated perfused rat liver. Protein synthesis was measured by the incorporation of L-[4,5-3H]leucine into the VLDL apoproteins (d less than 1.006) and into apolipoproteins of the whole perfusate (d less than 1.21). Oleate did not affect incorporation of [3H]leucine into total-perfusate or hepatic protein. The infusion of oleate, however, increased the mass and radioactivity of the VLDL apoprotein in proportion to the concentration of oleate infused. Uptake of oleate was similar with livers from fed or fasted animals. Fasting itself (24 h) decreased the net secretion and incorporation of [3H]leucine into total VLDL apoprotein and decreased the output of VLDL protein by the liver. A linear relationship existed between the output of VLDL triacylglycerol (mumol/h per g of liver) and secretion and/or synthesis of VLDL protein. Net output of VLDL cholesterol and phospholipid also increased linearly with VLDL-triacylglycerol output. Oleate stimulated incorporation of [3H]leucine into VLDL apo (apolipoprotein) E and apo C by livers from fed animals, and into VLDL apo Bh, B1, E and C by livers from fasted rats. The incorporation of [3H]leucine into individual apolipoproteins of the total perfusate lipoprotein (d less than 1.210 ultracentrifugal fraction) was not changed significantly by oleate during perfusion of livers from fed rats, suggesting that the synthesis de novo of each apolipoprotein was not stimulated by oleate. This is in contrast with that observed with livers from fasted rats, in which the synthesis of the total-perfusate lipoprotein (d less than 1.210 fraction) apo B, E and C was apparently stimulated by oleate. The observations with livers from fed rats suggest redistribution of radioactive apolipoproteins to the VLDL during or after the process of secretion, rather than an increase of apoprotein synthesis de novo. It appears, however, that the biosynthesis of apo B1, Bh, E and C was stimulated by oleic acid in livers from fasted rats. Since the incorporations of [3H]leucine into the VLDL and total-perfusate apolipoproteins were increased in fasted-rat liver when the fatty acid was infused, part of the apparent stimulated synthesis of the VLDL apoprotein may be in response to the increased formation and secretion of VLDL lipid.     

Secretion and uptake of nascent hepatic very low density lipoprotein by perfused livers from fed and fasted rats

Livers from fed or 24-hr fasted male rats were perfused in a recycling system. VLDL labeled with [1-14C]oleate (95% in triglyceride), produced in separate perfusions of livers from fed rats, was added to the medium as a pulse. Uptake of VLDL 14C-labeled triglyceride by livers from fasted rats was less than that from fed rats regardless of addition of oleate. During the interval in which radioactive triglyceride was taken up, the mass of triglyceride in the medium increased, indicative of the synthesis and net secretion of triglycerides. The rates of secretion of VLDL and uptake of VLDL were both more rapid in livers from fed rats in comparison to those from fasted animals. It was calculated that about 50% of the triglyceride synthesized and secreted by the liver was taken back by livers from fed rats. The VLDL from livers of fasted rats did not contain any apoE detectable by SDS gel electrophoresis or by radioimmunoassay when no fatty acid or 166 mumol of oleic acid was infused. In contrast, apoE comprised 6% of the VLDL apoprotein derived from perfusion of livers from fed animals in the absence of added fatty acid, and 20% when the fed livers were infused with 166 mumol of oleic acid. However, the net output (accumulation) of apoE by fasted liver was only two-thirds that from fed livers. When lipoprotein-free rat plasma containing apoE (4 mg/dl) was used in place of bovine serum albumin, the VLDL secreted by livers from either fed or fasted rats contained apoE and was taken up to a similar extent by such livers. These data suggested that the apoE of the d greater than 1.21 g/ml fraction was transferred to newly secreted VLDL which then stimulated uptake of the VLDL by livers from fasted rats. With further stimulation of secretion of VLDL triglyceride by infusion of 332 mumol of oleic acid/hr, the percent of apoE in the VLDL secreted by livers from fasted rats increased to 20%, which was similar to that of the VLDL produced by livers from fed rats when either 166 or 332 mumol/hr was infused. These data suggest a relationship between rates of hepatic secretion of VLDL (TG) and apoE, and the association of apoE with the secreted VLDL. During fasting, reduced secretion of both VLDL and apoE resulted in a VLDL particle that was considerably diminished in content of apoE and, therefore, that would be taken up by the liver at a reduced rate, in comparison to that observed in the fed animal.     

Tissue carnitine fluxes in vitamin C depleted-repleted guinea pigs

The biosynthesis of carnitine requires vitamin C as a cofactor for two separate hydroxylation steps. The majority of body carnitine (approximately 98%) is located in muscle and less than 0.5% is present in plasma. We examined the physiologic dynamics of plasma free carnitine and muscle total acid-soluble carnitine in vitamin C-depleted guinea pigs repleted with increasing amounts of vitamin C. Animals were fed a vitamin C-deficient diet for 3 weeks at which time symptoms of scurvy were evident. Animals were repleted with increasing doses of vitamin C, from 0.5 to 10.0 mg vitamin C/100 g body weight daily. Muscle total acid-soluble carnitine concentrations tended to correlate directly with plasma vitamin C (r = 0.41, P = 0.087) during the repletion phase of the study. Conversely, plasma free carnitine was inversely related to liver vitamin C (r = −0.54, P = 0.020) and to muscle total acid-soluble carnitine (r = −0.56, P = 0.015). Mean plasma free carnitine values fell 30% over the course of vitamin C repletion (P > 0.05) and mean muscle total acid-soluble carnitine rose by 30% (P > 0.05). These data suggest that elevated plasma free carnitine may indicate a low to marginal vitamin C status.     

Rapid stimulation of calcium uptake into rat liver by L-tri-iodothyronine.

The short-term effect of L-tri-iodothyronine (T3) on hepatic Ca2+ uptake from perfusate was compared with changes induced by T3 on cellular respiration and glucose output in isolated perfused livers from fasted and fed rats. The same parameters were also studied after the addition of glucagon or vasopressin. T3 (1 microM) induced Ca2+ uptake from the perfusate into the liver within minutes, and the time course was similar to that for stimulation of respiration and gluconeogenesis in livers from fasted rats, and for the stimulation of respiration and glucose output in livers from fed rats. The effects were dose-dependent in the range 1 microM-0.1 nM. Similar changes in the same parameters could be observed with glucagon and vasopressin, but with a completely different time course. Also, the influence of the T3 analogues L-thyroxine (L-T4), 3,5-di-iodo-L-thyronine (L-T2) and 3,3',5-tri-iodo-D-thyronine (D-T3) on hepatic energy metabolism was examined. Whereas D-T3 had practically no effect, L-T4 and L-T2 caused changes in Ca2+ uptake, O2 consumption and gluconeogenesis in livers from fasted rats similar to those with T3. It is concluded that changes in mitochondrial and cytosolic Ca2+ concentrations are involved in the stimulation of respiration and glucose metabolism observed with T3, glucagon and vasopressin.     

Effect of the oral hypoglycemic agent 2-tetradecylglycidic acid on fatty acid oxidation in suckling rats in vivo and in perfused liver

The hypoglycemic agent, 2-tetradecylglycidic acid (TDGA), administered in vivo lowered the concentration of plasma glucose and ketone bodies but raised the concentration of liver and plasma triglycerides in 10-day-old suckling rats. Phospholipid and cholesterol content of the plasma and liver were unaffected by drug treatment. TDGA inhibited the in vivo oxidation of [1-¹⁴C]palmitate but not that of [1-¹⁴C]decanoate. In suckling rat liver perfusion, TDGA totally inhibited ketone body formation from palmitate and depressed ketone body production from decanoate by 20%. Liver ATP and ADP content in the presence of TDGA decreased although this was probably a reflection of the increased triglyceride content of the liver since the $\text{ATP}\text{ADP}$ was the same as control livers. The results are discussed in relation to the diet and to the inhibition of carnitine acyl transferase in suckling rats.     

Effect of short- and long-term diabetes on carnitine and myo-inositol in rats.

1. The effect of short- (2 wk) and long-term (20 wk) streptozotocin diabetes was studied on urine, blood, liver, heart, brain, skeletal muscle, pancreas and kidney concentrations of acid-soluble carnitine and free myo-inositol. 2. Short-term diabetic rats excreted significantly higher concentrations of carnitine as well as myoinositol than normal rats. Blood carnitine and myo-inositol were not different between normal and diabetic rats. Diabetes caused a decrease in liver, brain and pancreatic carnitine, but not in heart, skeletal muscle and kidney. Myo-inositol concentration was decreased in liver, heart and kidney but not in brain, pancreas and skeletal muscle. 3. Long-term diabetic rats had higher urinary excretions of both carnitine and myo-inositol. Blood carnitine did not change; however, myo-inositol was higher in diabetic than in normal rats. Diabetes caused a significant increase in liver and a decrease in heart, brain, skeletal muscle and pancreatic content of carnitine; no difference in kidney carnitine was noted. Myo-inositol content was elevated only in liver of diabetic rats. 4. We suggest that carnitine and myo-inositol concentrations are influenced both by short- and long-term diabetes through changes in tissue metabolism.     

Separation of acylcarnitines from biological samples using high-performance liquid chromatography

A reverse-phase high-performance liquid chromatography technique to separate carnitine and acylcarnitines from a biological matrix is described. The method utilizes a step gradient to provide baseline resolution of acylcarnitines (individually or by class) for subsequent quantification using a sensitive radioenzymatic assay. The method requires minimal sample preparation and prevents any contamination among groups of acylcarnitines. This technique has been applied to liver tissues of rats obtained under a variety of conditions. These studies demonstrate the validity and utility of the HPLC method while confirming the applicability of the perchloric acid fractionation of acylcarnitines by functional class. The present HPLC method permits resolution of long-chain acylcarnitines in the presence of large excess concentrations of carnitine and short-chain acylcarnitines (coelution of unesterified carnitine with long-chain acylcarnitines less than or equal to 0.05%). Thus, the method will be of use in the study of acylcarnitines in biological systems over a broad spectrum of metabolic conditions.     

Carnitine metabolism in livers and hearts of 48h-starved rats: the contribution of fat and carbohydrate to acylcarnitine formation

The work investigated the effects of administration of 2-tetradecylglycidate (TDG), an inhibitor of mitochondrial long-chain fatty acid oxidation, alone or in combination with glucose, on concentrations of free and acylated carnitine in livers and hearts of 48 h-starved rats. The only significant effect of TDG in the heart was to decrease [short-chain acylcarnitine]. This demonstrates that in heart, fat oxidation is linked to the formation of short-chain acylcarnitine. Cardiac [short-chain acylcarnitine] was not significantly decreased by TDG if the rats were also administered glucose, suggesting that acyl CoA derived from glucose may be used for short-chain acylcarnitine formation in TDG-treated rats. TDG significantly decreased in [free carnitine]. No changes in [short-chain acylcarnitine] were observed. This indicates that formation of short-chain acylcarnitine in liver is not determined by the rates of fat oxidation. It was calculated that at least 63% of the acyl-groups esterified to carnitine were generated by intramitochondrial beta-oxidation. The effects of glucose and TDG on hepatic concentrations of free and long-chain acylcarnitine were additive, suggesting that extramitochondrial fat oxidation can contribute to acylcarnitine formation in liver.     

Disassociation between acidinsoluble acylcarnitines and ketogenesis following carnitine administration in vivo

1-Carnitine was administered to fed rats and the changes in plasma beta-hydroxybutrate concentration and liver acid-insoluble acylcarnitine content were assessed. One hour following injection of carnitine in doses greater than 1 mumol/100 g of body weight there was a dose-dependent increase in liver acid-insoluble acylcarnitine content to levels comparable to those seen in fasting. These increased levels were maintained for a least 2 h following injection. During the period following carnitine administration there was no increase in ketogenesis as evidenced by plasma beta-hydroxybutyrate concentrations. Since acid-insoluble acylcarnitines represent the product of carnitine palmitoyltransferase A, the results are interpreted as contradictory to the theory that this enzyme is rate-limiting and regulatory for ketogenesis.