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C14O2 excretion after the intravenous administration of albumin-bound palmitate-1-C14 to intact rats
ARCHIVES
OF
BIOCHEMISTRY
AND
BIOPHYSICS
71,
346-351 (1957)
CY402Excretion after the Intravenous Administration Albumin-Bound Pahnitate- 1-C’* to Intact Rats. Charles McCalla,’
Herbert
S. Gates, Jr. and Robert
S. Gordon, Jr.
From the Laboratory of Cellular Physiology and Metabolism, National Institute, National Institutes of Health, Bethesda, Maryland Received January
of
Heart
17, 1957
Recent work in this laboratory (1,2) has implicated the small quantity of unesterified fatty acid (UFA) found in human plasma as an albuminbound complex as a major transport form of fatty acid, accounting for the bulk of the movement of fatty acids from adipose tissue depots to metabolically active viscera during fasting. The transport of fatty acid in this form has been observed to cease, coincident with a marked decrease in the arterial UFA concentration, in human subjects to whom intravenous glucose (together with insulin) is administered. The simultaneous occurrence of a cessation of tissue extraction of UFA and a decrease in the arterial level has been interpreted to mean that the transport ceasesprimarily because of a reduction in the output of UFA from the depot fat of the subject. If the conclusions noted above are correct, and if the metabolism of UFA in rats is comparable to that in human subjects, it should be possible to demonstrate in fasting rats conversion of isotopically labeled unesterified fatty acid to carbon dioxide. It is to be anticipated that such conversion will be rapid, inasmuch as the amount of UFA in circulation is small compared to the quantity that must be oxidized to account for a major portion of the metabolic needsof the organism. The fate of isotopitally labeled UFA administered to a carbohydrate-fed rat should also be of interest; if the secretion of UFA from the adipose tissue into the blood is the only step in fat metabolism to be inhibited by carbohydrate feeding, a rapid conversion of UFA to carbon dioxide is to be anticipated in the fed rat. If, on the other hand, the belief of Lossow and Chaikoff * Fellow,
National
Science Foundation. 346
CONVERSION
OF
ALBUMIN-BOUND
PALMITATE
347
(3) that carbohydrate feeding inhibits the enzymic degradation of fatty acids is correct, the rate of conversion ought to be markedly decreased. In the work cited, such was found to be the case for higher fatty acids, whereas the lower fatty acids were converted to carbon dioxide even in the presence of carbohydrate. However, they did not administer the higher acids, myristic and pahnitic, in a form corresponding to the naturally occurring UFA, but rather as complex emulsions of free acid dissolved in olive oil. It is possible, therefore, that the effects they observed were related to an action of carbohydrate on the rate of removal of this emulsion from the blood and its conversion into a metabolically active form. The lower fatty acids, on the other hand, were administered as water solutions of sodium salts. Such soap solutions, on mixing with plasma, give rise to the albumin-fatty acid complex rapidly and spontaneously, so that in the case of the lower acids, the material administered did in fact correspond exactly with the naturally occurring transport material (except as regards chain length; there is no evidence for the occurrence of significant amounts of these lower fatty acids in the plasma of most higher mammals). The experiments here reported were designed to investigate these possibilities in albino rats, the labeled UFA being administered intravenously as a preformed complex of sodium palmitate-l-Cl4 with the albumin of normal rat serum. The administration of the tracer substance in a form chemically identical with the naturally occurring substrate should serve to demonstrate the effects of oxidative processes as opposed to processes related to the liberation of metabolically active UFA from either depot fat or from other forms of test substrate. MATERIALS
AND
METHODS
Palmitic acid-l-Cl4 of specific activity 0.98 mc./mmole was obtained from the Nuclear Instrument and Chemical Company, Chicago, Illinois. This material was dissolved in warm water as the sodium salt, and added to fresh normal rat serum. After standing overnight in the refrigerator, the resulting mixture was freed of insoluble particles by centrifugation, and stored frozen until used. For each experiment, 1.0 ml. of the serum-palmitate mixture was injected into the tail vein of an albino rat of the Sprague-Dawley strain, weighing 200-250 g. The rats designated as fasting (four rats) had been without food for a 16-hr. period but had had free access to water. Those designated as carbohydrate-fed had had no pellets for a corresponding period, but had received 10% glucose in water ad Zibitum, and, in addition, were force-fed 2 ml. of 5Ooj, glucose 1 hr. before and again immediately before the administration of the isotopic material. After receiving the injection, the rat was placed with all possible speed into a metabolism cham-
348
MCCALLA,
GATES, JR. AND GORDON, JR.
ber through which a stream of carbon dioxide-free air was drawn, the expired carbon dioxide being collected in bubbling towers containing sodium hydroxide solution. The dead space within the collecting system was kept to a minimum, being of such size that the air within the chamber was completely changed within each 30 sec. during the experiments. The bubblers were changed at 2, 4,8, 15, 30, 60, and 90 min. after the time of the injection. Suitable aliquots of the resultant sodium carbonate solutions were precipitated with barium chloride, the precipitates were washed, and the barium carbonate was plated on stainless steel planchets of area 1.43 sq. cm. Aliquots of the serum-palmitate mixture were converted to carbon dioxide by a wet oxidation technique, and the carbon dioxide similarly collected and planchetted. Radioactivity of all samples was determined in the Robinson gas flow counter (4), and self-absorption corrections applied to reduce all counting rates to those for 5 mg. barium carbonate samples. OBSERVATIONS
The cumulative
radiochemical yield of isotopic carbon, expressed as per cent of the dose administered, is shown graphically in Fig. 1. The reproducibility of the results was such that it was deemed appropriate to
present only two curves, the mean curve for the four fasted animals, and the mean for the tpro given carbohydrate. At the 90min. point, the bracket indicates the extreme high and low values. It will be observed that the point of maximum slope of the output curve for the fasted rats, representing the collection of carbon dioxide of maximal specific
T I ME (minutes)
FIG. 1. UFA + CO2 conversion in rats. Palmitate-1-C” administered intravenously.
CONVERSION
OF ALBUMIN-BOUND
PALMITATE
349
activity, occurs at approximately the fifth minute after the administration of isotopic fatty acid. DISCUSSION
The rapidity of conversion of intravenously administered sodium palmitate to carbon dioxide, as judged by the rate of excretion of the tracer isotope, appears to justify the belief that the unesterified fatty acids circulating in plasma constitute a lipide available for oxidation in the tissues as a calorigenic substrate. The conversion occurs at a rate more rapid than that observed by Bloom and associates (5) for the oxidation of labeled glucose in the rat; injected acetate is converted to carbon dioxide at a rate exceeding the rate of pahnitate conversion by only a factor of two (6). The rate of excretion of the isotope in these experiments is roughly fivefold more rapid than that observed by Lossow (7) following the injection of isotopic palmitic acid in the form of a complex oily emulsion, and is comparable with the excretion rates observed by Lossow and Chaikoff after the administration of isotopic sodium salts of the shorter-chain fatty acids. The injection of a sodium salt of a fatty acid would be expected to give rise immediately to an albumin-fatty acid complex ion in the plasma, so that the data reported by Lossow and Chaikoff for the fatty acids of chain length 12 and below should be directly comparable with the figures reported here. Data presented by Weinman et al. (8) indicate that the release of the carboxyl carbon from palmitic acid into expired carbon dioxide may be taken as evidence of the complete oxidation of the palmitic acid molecule. By considering the plasma volume of a rat to approximate 4 % of the body weight, and the average plasma unesterified fatty acid concentration of fasting rats to be 0.5 meq./l.,2 it is possibleto estimate the specific activity of the carbon of the circulating unesterified fatty acids of the rats at the moment of injection of the isotopic material, The error introduced by assuming that all the unesterified fatty acids in circulation contain 16 carbon atoms is in all probability negligible (2). The specific activity so derived is approximately 100 times greater than the maximal specific activity of the expired carbon dioxide, observed at about the fifth minute in these rats. If it be assumed that all the expired carbon dioxide of a fasted rat is derived from the oxidation of fat, it follows that the “miscible pool” of fatty acids connected by reversible steps to the plasma unesterified fatty acids is on the order of 2 This value is taken from unpublished
data obtained
in this laboratory.
350
McCALLA,
GATES,
JR.
AND
GORDON,
JR.
100 times as great in quantity as the amount of UFA in circulation at any moment. For a 250-g. rat, this pool would contain approximately 140 mg. of fatty acid, an amount far less than the average fatty acid content of depot fat. The process, therefore, whereby adipose tissue secretes unesterified fatty acid into the plasma during fasting is, under these conditions, essentially irreversible. It also appears unlikely that the lOO-fold dilution of the isotope resulted from equilibration of the circulating UFA with the fatty acids of any one organ or tissue, since, in man at least, it is possible to show that the circulating unesterified fatty acid is extracted by a variety of tissues, and that the amount extracted in one transit through the capillaries comprises a large fraction of the total material present in the arterial plasma. The isotopic fatty acid would therefore be expected to have been extracted from the blood plasma into the tissues where oxidation is to occur before great dilution from a localized extravascular fatty acid store could occur, and the fatty acid pool responsible for the dilution effect would consequently be expected to be located within a wide variety of tissue cells. In fact, this supposition is strongly supported by preliminary observations in this laboratory, in which fasting rats killed 10 min. after being given intravenous albumin-palmitate-l-C14 complex showed radioactivity widely distributed, there being Cl4 compounds present in every major tissue except brain. The marked inhibition of the conversion of isotopic palmitic acid to carbon dioxide in the carbohydrate-fed rat indicates the occurrence of a block in fatty acid oxidation at the cellular level, as postulated by Lossow and Chaikoff (3). It is nevertheless necessary to assume that the adipose tissue has ceased to secrete UFA into the plasma in order to explain the decrease in circulating UFA levels observed in the human subject given glucose (1, 2). The mechanism for the control of UFA secretion has as yet not been elucidated, but it clearly is not a response to the accumulation of unoxidized UFA in the plasma. No attempt was made in the present series of experiments to discover the fate of the isotopic carbon which failed to appear in the expired air of the carbohydrate-fed rats. These experiments illustrate the need for careful consideration of the form and route of administration of an isotopic compound in biochemical research. Inasmuch as plasma unesterified fatty acids, which appear to be the most active of the plasma lipides in contributing substrate to oxidative processes, are present in low concentration,
CONVERSION
OF ALBUMIN-BOUND
PALMITATE
351
and are in addition strongly bound by serum albumin, it appears likely that the chemical potential at which the free fatty acid anions enter cellular metabolic processes must be very low indeed. It is therefore suggested that the anions of the higher fatty acids, when their presence in a biochemical experiment is desired, be added wherever possible as the albumin-fatty acid complex in order to preserve the low chemical potential at which these substances enter into natural life processes. SUMMARY
Sodium palmitate-l-Cl4 was administered intravenously to the form of the serum albumin-bound complex ion. Expired air tions indicated a rapid and efficient conversion of this substrate to dioxide in fasting rats, whereas rats fed carbohydrate before the was administered excreted only minimal amounts of the label same period.
rats in colleccarbon isotope in the
REFERENCES 1. GORDON, 2. GORDON, 3. Lossow, 4.
5. 6. 7. 8.
R. S., JR., AND CHERKES A., J. Clin. Invest. 36,206 (19%). R. S., JR., J. Clin. Invest. 36, 810 (1957). W. J., AND CHAIKOFF, I. L., Arch. Biochem. Biophys. 67, 23 (1955). ROBINSON, C. V., Science 112, 198 (1950). BLOOM, B., STETTEN, M. R., AND STETTEN, DEW., JR., J. Biol. Chem. 204, 681 (1953). GOULD, R. G., SINEX, F. M., ROSENBERG, I. N., SOLOMON, A. K., AND HASTINGS, A. B., J. Biol. Chem. 177, 295 (1949.). Lossow, W. J., “Importance of Fatty Acid Chain Length in Carbohydrate Sparing of Fat Oxidation.” Ph. D. Thesis, University of California, 19%. WEINMAN, E. O., CHAIKOFF, I. L., DAUBEN, W. G., GEE, M., AND ENTENMAN, C., J. Biol. Chem. 184, 735 (1950).
OF
BIOCHEMISTRY
AND
BIOPHYSICS
71,
346-351 (1957)
CY402Excretion after the Intravenous Administration Albumin-Bound Pahnitate- 1-C’* to Intact Rats. Charles McCalla,’
Herbert
S. Gates, Jr. and Robert
S. Gordon, Jr.
From the Laboratory of Cellular Physiology and Metabolism, National Institute, National Institutes of Health, Bethesda, Maryland Received January
of
Heart
17, 1957
Recent work in this laboratory (1,2) has implicated the small quantity of unesterified fatty acid (UFA) found in human plasma as an albuminbound complex as a major transport form of fatty acid, accounting for the bulk of the movement of fatty acids from adipose tissue depots to metabolically active viscera during fasting. The transport of fatty acid in this form has been observed to cease, coincident with a marked decrease in the arterial UFA concentration, in human subjects to whom intravenous glucose (together with insulin) is administered. The simultaneous occurrence of a cessation of tissue extraction of UFA and a decrease in the arterial level has been interpreted to mean that the transport ceasesprimarily because of a reduction in the output of UFA from the depot fat of the subject. If the conclusions noted above are correct, and if the metabolism of UFA in rats is comparable to that in human subjects, it should be possible to demonstrate in fasting rats conversion of isotopically labeled unesterified fatty acid to carbon dioxide. It is to be anticipated that such conversion will be rapid, inasmuch as the amount of UFA in circulation is small compared to the quantity that must be oxidized to account for a major portion of the metabolic needsof the organism. The fate of isotopitally labeled UFA administered to a carbohydrate-fed rat should also be of interest; if the secretion of UFA from the adipose tissue into the blood is the only step in fat metabolism to be inhibited by carbohydrate feeding, a rapid conversion of UFA to carbon dioxide is to be anticipated in the fed rat. If, on the other hand, the belief of Lossow and Chaikoff * Fellow,
National
Science Foundation. 346
CONVERSION
OF
ALBUMIN-BOUND
PALMITATE
347
(3) that carbohydrate feeding inhibits the enzymic degradation of fatty acids is correct, the rate of conversion ought to be markedly decreased. In the work cited, such was found to be the case for higher fatty acids, whereas the lower fatty acids were converted to carbon dioxide even in the presence of carbohydrate. However, they did not administer the higher acids, myristic and pahnitic, in a form corresponding to the naturally occurring UFA, but rather as complex emulsions of free acid dissolved in olive oil. It is possible, therefore, that the effects they observed were related to an action of carbohydrate on the rate of removal of this emulsion from the blood and its conversion into a metabolically active form. The lower fatty acids, on the other hand, were administered as water solutions of sodium salts. Such soap solutions, on mixing with plasma, give rise to the albumin-fatty acid complex rapidly and spontaneously, so that in the case of the lower acids, the material administered did in fact correspond exactly with the naturally occurring transport material (except as regards chain length; there is no evidence for the occurrence of significant amounts of these lower fatty acids in the plasma of most higher mammals). The experiments here reported were designed to investigate these possibilities in albino rats, the labeled UFA being administered intravenously as a preformed complex of sodium palmitate-l-Cl4 with the albumin of normal rat serum. The administration of the tracer substance in a form chemically identical with the naturally occurring substrate should serve to demonstrate the effects of oxidative processes as opposed to processes related to the liberation of metabolically active UFA from either depot fat or from other forms of test substrate. MATERIALS
AND
METHODS
Palmitic acid-l-Cl4 of specific activity 0.98 mc./mmole was obtained from the Nuclear Instrument and Chemical Company, Chicago, Illinois. This material was dissolved in warm water as the sodium salt, and added to fresh normal rat serum. After standing overnight in the refrigerator, the resulting mixture was freed of insoluble particles by centrifugation, and stored frozen until used. For each experiment, 1.0 ml. of the serum-palmitate mixture was injected into the tail vein of an albino rat of the Sprague-Dawley strain, weighing 200-250 g. The rats designated as fasting (four rats) had been without food for a 16-hr. period but had had free access to water. Those designated as carbohydrate-fed had had no pellets for a corresponding period, but had received 10% glucose in water ad Zibitum, and, in addition, were force-fed 2 ml. of 5Ooj, glucose 1 hr. before and again immediately before the administration of the isotopic material. After receiving the injection, the rat was placed with all possible speed into a metabolism cham-
348
MCCALLA,
GATES, JR. AND GORDON, JR.
ber through which a stream of carbon dioxide-free air was drawn, the expired carbon dioxide being collected in bubbling towers containing sodium hydroxide solution. The dead space within the collecting system was kept to a minimum, being of such size that the air within the chamber was completely changed within each 30 sec. during the experiments. The bubblers were changed at 2, 4,8, 15, 30, 60, and 90 min. after the time of the injection. Suitable aliquots of the resultant sodium carbonate solutions were precipitated with barium chloride, the precipitates were washed, and the barium carbonate was plated on stainless steel planchets of area 1.43 sq. cm. Aliquots of the serum-palmitate mixture were converted to carbon dioxide by a wet oxidation technique, and the carbon dioxide similarly collected and planchetted. Radioactivity of all samples was determined in the Robinson gas flow counter (4), and self-absorption corrections applied to reduce all counting rates to those for 5 mg. barium carbonate samples. OBSERVATIONS
The cumulative
radiochemical yield of isotopic carbon, expressed as per cent of the dose administered, is shown graphically in Fig. 1. The reproducibility of the results was such that it was deemed appropriate to
present only two curves, the mean curve for the four fasted animals, and the mean for the tpro given carbohydrate. At the 90min. point, the bracket indicates the extreme high and low values. It will be observed that the point of maximum slope of the output curve for the fasted rats, representing the collection of carbon dioxide of maximal specific
T I ME (minutes)
FIG. 1. UFA + CO2 conversion in rats. Palmitate-1-C” administered intravenously.
CONVERSION
OF ALBUMIN-BOUND
PALMITATE
349
activity, occurs at approximately the fifth minute after the administration of isotopic fatty acid. DISCUSSION
The rapidity of conversion of intravenously administered sodium palmitate to carbon dioxide, as judged by the rate of excretion of the tracer isotope, appears to justify the belief that the unesterified fatty acids circulating in plasma constitute a lipide available for oxidation in the tissues as a calorigenic substrate. The conversion occurs at a rate more rapid than that observed by Bloom and associates (5) for the oxidation of labeled glucose in the rat; injected acetate is converted to carbon dioxide at a rate exceeding the rate of pahnitate conversion by only a factor of two (6). The rate of excretion of the isotope in these experiments is roughly fivefold more rapid than that observed by Lossow (7) following the injection of isotopic palmitic acid in the form of a complex oily emulsion, and is comparable with the excretion rates observed by Lossow and Chaikoff after the administration of isotopic sodium salts of the shorter-chain fatty acids. The injection of a sodium salt of a fatty acid would be expected to give rise immediately to an albumin-fatty acid complex ion in the plasma, so that the data reported by Lossow and Chaikoff for the fatty acids of chain length 12 and below should be directly comparable with the figures reported here. Data presented by Weinman et al. (8) indicate that the release of the carboxyl carbon from palmitic acid into expired carbon dioxide may be taken as evidence of the complete oxidation of the palmitic acid molecule. By considering the plasma volume of a rat to approximate 4 % of the body weight, and the average plasma unesterified fatty acid concentration of fasting rats to be 0.5 meq./l.,2 it is possibleto estimate the specific activity of the carbon of the circulating unesterified fatty acids of the rats at the moment of injection of the isotopic material, The error introduced by assuming that all the unesterified fatty acids in circulation contain 16 carbon atoms is in all probability negligible (2). The specific activity so derived is approximately 100 times greater than the maximal specific activity of the expired carbon dioxide, observed at about the fifth minute in these rats. If it be assumed that all the expired carbon dioxide of a fasted rat is derived from the oxidation of fat, it follows that the “miscible pool” of fatty acids connected by reversible steps to the plasma unesterified fatty acids is on the order of 2 This value is taken from unpublished
data obtained
in this laboratory.
350
McCALLA,
GATES,
JR.
AND
GORDON,
JR.
100 times as great in quantity as the amount of UFA in circulation at any moment. For a 250-g. rat, this pool would contain approximately 140 mg. of fatty acid, an amount far less than the average fatty acid content of depot fat. The process, therefore, whereby adipose tissue secretes unesterified fatty acid into the plasma during fasting is, under these conditions, essentially irreversible. It also appears unlikely that the lOO-fold dilution of the isotope resulted from equilibration of the circulating UFA with the fatty acids of any one organ or tissue, since, in man at least, it is possible to show that the circulating unesterified fatty acid is extracted by a variety of tissues, and that the amount extracted in one transit through the capillaries comprises a large fraction of the total material present in the arterial plasma. The isotopic fatty acid would therefore be expected to have been extracted from the blood plasma into the tissues where oxidation is to occur before great dilution from a localized extravascular fatty acid store could occur, and the fatty acid pool responsible for the dilution effect would consequently be expected to be located within a wide variety of tissue cells. In fact, this supposition is strongly supported by preliminary observations in this laboratory, in which fasting rats killed 10 min. after being given intravenous albumin-palmitate-l-C14 complex showed radioactivity widely distributed, there being Cl4 compounds present in every major tissue except brain. The marked inhibition of the conversion of isotopic palmitic acid to carbon dioxide in the carbohydrate-fed rat indicates the occurrence of a block in fatty acid oxidation at the cellular level, as postulated by Lossow and Chaikoff (3). It is nevertheless necessary to assume that the adipose tissue has ceased to secrete UFA into the plasma in order to explain the decrease in circulating UFA levels observed in the human subject given glucose (1, 2). The mechanism for the control of UFA secretion has as yet not been elucidated, but it clearly is not a response to the accumulation of unoxidized UFA in the plasma. No attempt was made in the present series of experiments to discover the fate of the isotopic carbon which failed to appear in the expired air of the carbohydrate-fed rats. These experiments illustrate the need for careful consideration of the form and route of administration of an isotopic compound in biochemical research. Inasmuch as plasma unesterified fatty acids, which appear to be the most active of the plasma lipides in contributing substrate to oxidative processes, are present in low concentration,
CONVERSION
OF ALBUMIN-BOUND
PALMITATE
351
and are in addition strongly bound by serum albumin, it appears likely that the chemical potential at which the free fatty acid anions enter cellular metabolic processes must be very low indeed. It is therefore suggested that the anions of the higher fatty acids, when their presence in a biochemical experiment is desired, be added wherever possible as the albumin-fatty acid complex in order to preserve the low chemical potential at which these substances enter into natural life processes. SUMMARY
Sodium palmitate-l-Cl4 was administered intravenously to the form of the serum albumin-bound complex ion. Expired air tions indicated a rapid and efficient conversion of this substrate to dioxide in fasting rats, whereas rats fed carbohydrate before the was administered excreted only minimal amounts of the label same period.
rats in colleccarbon isotope in the
REFERENCES 1. GORDON, 2. GORDON, 3. Lossow, 4.
5. 6. 7. 8.
R. S., JR., AND CHERKES A., J. Clin. Invest. 36,206 (19%). R. S., JR., J. Clin. Invest. 36, 810 (1957). W. J., AND CHAIKOFF, I. L., Arch. Biochem. Biophys. 67, 23 (1955). ROBINSON, C. V., Science 112, 198 (1950). BLOOM, B., STETTEN, M. R., AND STETTEN, DEW., JR., J. Biol. Chem. 204, 681 (1953). GOULD, R. G., SINEX, F. M., ROSENBERG, I. N., SOLOMON, A. K., AND HASTINGS, A. B., J. Biol. Chem. 177, 295 (1949.). Lossow, W. J., “Importance of Fatty Acid Chain Length in Carbohydrate Sparing of Fat Oxidation.” Ph. D. Thesis, University of California, 19%. WEINMAN, E. O., CHAIKOFF, I. L., DAUBEN, W. G., GEE, M., AND ENTENMAN, C., J. Biol. Chem. 184, 735 (1950).