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.

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 [acyL3H] 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] 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.
In ketotic states, whether in starvation or diabetes, the contribution of short-chain acylcarnitines to the total plasma carnitine markedly increases at the expense of free carnitine. This fact has been established in animal (l-3) as well as in human (4-7) studies. The mechanism of the development of increased plasma [acylcarnitine], however, has not quite been revealed, and the tissue of origin is also obscure. The candidate with the most potential as the source of extra acylcarnitines was the liver since it responds uniquely by enhancing its capacity to oxidize fatty acids in ketotic states. Really, the participation of acylcarnitines in the total carnitine released by the liver cells (8) or perfused liver (3,9,10) is relatively high, about 50-60%.
However, we also observed that the percentage of acylcarnitines in the perfusate of liver did not increase when the liver was from fasted animals (3,9). These latter observations seem to challenge the view that liver supplies the surplus plasma acylcarnitines in ketosis. Alternatively, muscle or kidney may be the source. To approach the problem, a method was required which is able to detect the tissue origin of plasma carnitine and acylcarnitines. We analyzed in this work the composition of ["HI carnitine in the plasma shortly after injection of ["Hlbutyrobetaine.
The fact that the liver is the only organ in rat which is able to convert butyrobetaine into carnitine ( were killed by decapitation between 8:00 and 10:00 a.m., blood was collected into heparinized tubes, and livers were quickly removed and frozen in liquid N,. Neutralized perchloric acid extracts were prepared on the day of experiment, and the extracts were stored at -20 "C until analysis. Liver Perfusion-The perfusions were performed in situ in outflow fashion as previously described (9). The perfusion medium was composed of human erythrocytes suspended to a hematocrit of 15% in Krebs bicarbonate buffer, pH 7.4, containing 4% bovine albumin. Animals were injected with ["Hlbutyrobetaine 1 h prior to perfusion. Sample Preparation and Analyses-Neutralized perchloric acid extract of 0.5 g of liver was purified on 0.5 X 8-cm columns of Dowex 5OW-X8, NH:, and Dowex l-X8, F-resins as previously described (14). The final methanol extract was evaporated, reconstituted in 1.0 ml of water, neutralized to pH 7.2, and subjected to a second purification on resins. In the latter procedure, one-half amounts of resins (0.5 x 4-cm columns) were used. The extract of 1.0 ml of plasma was purified in the same way, but in the second purification on resins, Dowex 1 was employed in acetate-form (instead of F-form at 1000 x g for 20 min (less than 5% of radioactivity was found in the pellet). The resulting supernatant was treated as plasma, but 10 ml was processed.
The extract of 10 ml of supernatant was purified first on 1.0 x 8-cm columns of resins, and in the second step of purification the size of resins was reduced to 0.5 x 4 cm. The final sample was reconstituted in 400 ~1 of water, of which 200 ~1 was used for HPLC and the other half was made up to 1.0 ml with water and used for carnitine assay. A portion of perfusion fluid, which was not ' The abbreviation used is: HPLC, high performance liquid chromatography.  in the acid-soluble fractions of liver (Table I) and plasma (Table II) of fed and fasted rats. In liver (Table   I), the total carnitine level increased with fasting in accord with earlier observations (16), while the percent contribution of esters did not change (1,2,16). It is also seen that the ester content of [3H]carnitines (obtained by HPLC separation) was very close to that of total carnitine (obtained by enzymatic analysis). (In the l-h experiment, however, the ester [3H] carnitine fraction tended to be higher, indicating that the newly formed carnitine did not quite equilibrate with the total pool in this time.) The contribution of acid-insoluble carnitine to total increased from 6.45 to 12.0% with fasting, and the same percentages of [3H]carnitine were found in this fraction (not shown).

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Data obtained in the plasma are shown in Table II. As appears from the enzymatic analysis, the percentage of carnitine esters markedly increased with fasting. An essential question for this work is the organ origin of this surplus acylcarnitine.  Rats were injected with 15 x lo6 cpm [3H]butyrobetaine 1 h before the experiment. Livers from fed and 48-h starved rats were perfused in outflowing fashion at the rate of 10 ml/min as previously described (9). The perchloric acid extract of liver tissue and perfusate were analyzed for acid-soluble total and ester carnitine as specified under "Experimental Procedures." Values are means + S.E. for three animals.
Liver  to extrahepatic tissues via carnitine as carrier, in accord with recent observations by Snoswell and co-workers (17) on sheep.
The second question addressed is how the increment in plasma acylcarnitines develops with fasting. As shown in Table II, the acylation of the newly synthesized [3H]carnitine also markedly increased with fasting (84.0 uersus 67.9% in the l-h experiment and 77.9 uer.w.s 61.5% in the Z-h experiment). This observation, together with the fact that [3H] carnitine was acylated in the liver, proves' that the surplus plasma acylcarnitines derive from the liver. The contribution of newly synthesized carnitine from the liver to circulating carnitine can be calculated by dividing the plasma carnitine counts/min by the apparent specific activity in the corresponding liver. Calculating with data from the 2h experiment (Tables I and II), we found 23.7 nmol/ml "new" carnitine in the plasma of fed rats and 36.6 nmol/ml in that of fasted ones. The value for the fasted animals seems to be higher on a per ml basis; however, the smaller total plasma volume likely compensates for it. To estimate the whole carnitine turnover between liver and extrahepatic tissues requires special studies involving all tissues. (The data calculated above for plasma may be overestimated, because we divided plasma counts/min by the specific activity of liver tissue, presuming that the organ released carnitine with the same specific activity. However, in the early period after injection of [3H]butyrobetaine, the liver releases carnitine with higher specific activity, as shown in Table III.) A further question posed in this work is what mechanism leads to the increased plasma [acylcarnitine] in fasting? Since the surplus plasma acylcarnitine derives from the liver, the most plausible mechanism would be that liver in fasting released carnitine with a higher ester content. To answer this we perfused livers from fed and fasted rats and measured the ester content in total carnitine and [3H]carnitines.
The perfusions in this case were performed in outflowing fashion to prevent reuptake, unlike former recirculating perfusions (3,9). From the results of Table III, it can be seen that in the absence of added substrates the ester content of released carnitine did not change when the livers were either from fed or fasted rats. In another experiment (Table IV, Experiment I), the ester content did not change either when the perfusates were supplemented with substrates at physiological concentrations (18). An increased release of carnitine esters could be forced only with 3.5 mM oleate (Table IV, Experiment II) which was 5 times physiological (and caused hemolysis of red blood cells). The results show that livers even from fed rats release carnitine and ["Hlcarnitine (Table III) with ester content as high as (or a little higher) that found in the plasma of fasted rats (Table II). Not surprisingly, this value did not increase further in the perfusate when livers were from fasted animals, suggesting that liver does not supply extra acylcarnitines into the bloodstream in the fasting state. This result could also be predicted from the fact that the ester fraction did not increase in the liver tissue either with fasting (Tables  I, III, and IV).
To account for these observations, we propose that the increase of [acylcarnitines] in the plasma of fasted rats is caused by an altered handling in extrahepatic tissues. This may be either a decreased uptake and utilization of acylcarnitines or an increased uptake of free carnitine by extrahepatic tissues in the fasted state. The lowered total plasma carnitine in fasting (Table II) suggests an increased uptake of free carnitine. To investigate this possibility, we would also need to know how carnitine turnover changes in fasting, which requires further studies. An altered renal handling cannot play a significant role because carnitine excretion is markedly