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The distribution of tritium in fatty acids synthesized from tritiated glucose and tritiated water by rat adipose tissue
422 BBA
BIOCHIMICA ET BIOPHYSICA
ACTA
55264
THE DISTRIBUTION TRITIATED
DANIEL
OF TRITIUM
IN FATTY
GLUCOSE AND TRITIATE~
W. FOSTER
AND
JOSEPH
ACIDS SYNTHESIZED
WATER
BY RAT ADIPOSE
FROM TISSUE
KATZ
The University of Texas Southwestern Medical School, Dallas, Texas and the Cedars-Sinai Medical Center, Los Angeles, Calif. (U.S.A.) (Received
May =$h, 1966)
SUMMARY
Fatty acids synthesized by rat epididymal fat-pad slices in the presence of [r-3H]glucose, [3-3H]glucose and tritiated water were degraded for the localization of tritium in the molecule. 75% of the tritium from [x-3H]glucose and 85% from [3-3Hjglucose was present on the odd carbons. The incorporation of 15% of the tritium from [3-3H]glucose in the even positions was unexpected. Tritium from 3H,O was incorporated to the same extent in the odd and even positions of the carbon chain. The presence of tritium from water in the odd positions confirms the participation of DPNH hydrogen in reductive fatty acid synthesis in the intact adipose tissue cell. The results with 3H,0 and [r-3H]glucose resemble those previously reported for liver and suggest that the mechanisms of fatty acid synthesis are essentially similar in the two tissues.
INTRODUCTION
When fatty acids are synthesized in the cell the hydrogens of the aliphatic chain are derived from three sources: substrate hydrogen (glucose, acetate, etc.), hydride ions from TPNH, and protons from H,O. According to theoretical considerations the hydrogens on the even carbons of the fatty acid (LX,y, etc.) would be derived from substrate and H,O while the reductive hydrogen of TPNH should be found on the odd carbons of the chain (8, 6, etc.). These predictions were previously tested for fatty acids synthesized by rat-liver slices in the presence of [r-3H]glucose and $H,O (ref. I). With [r-3H]glucose as substrate for the generation of radioactive TPNH in the cell, it was shown that over 05% of the tritium was present on the odd-numbered carbons of the fatty acid mole&le in accord with the theoretical predictions. Such was not the case in experiments with 3H,O. Assuming TPNH to be the sole donor of reductive hydrogen in fatty acid synthesis, little tritium was expected on the odd carbons of fatty acids synthesized in 3H,O. In actuality 5560% was present in the /J-position and only 40-45% on the C-U. These results suggested the possibility of an exchange Biochim. Biophys. Acta, 125 (1966) qzz-.p7
HYDROGEN
METABOLISM
IN FATTY ACID SYNTHESIS
423
reaction between TPNH and H,O prior to reduction of the intermediates of fatty acid synthesis. KATZ and co-workers8-4 have recently studied the incorporation of 3H into fatty acids synthesized by rat adipose tissue utilizing F120, [r-8H]glucose and [3-sH]glucose as substrates. It seemed of interest to determine the location of tritium in such fatty acids for two reasons: to compare the findings with [r-3H]glucose and 3H,O in adipose tissue with those in liver, and to compare the distribution of tritium arising in TPNH generated from jr-3H]glucose with that generated from [3-3H]glucose. EXPERIMENTAL
PROCEDURES
The experimental procedures used in these experiments have been previously described1T4. Briefly, epididymal fat-pad slices, 300 mg, from fasted-refed rats (2 days fast followed by 3 days high carbohydrate diet) were incubated for 3 h at 38” in 2.2 ml of Krebs-Henseleit bicarbonate buffer containing glucose specifically labeled with tritium or 3H,0. At the end of the incubation period the reaction was stopped and long-chain fatty acids were isolated and counted. The total fatty acids were then separated into saturated and unsaturated fractions by lead salt precipitation, and the saturated fraction used for the degradation procedure. After preparation of the silver salts of the acids decarboxylation was carried out with bromine. The alcohol containing one less carbon was next made and oxidation with CrO, was carried out to give the fatty acid of n-r chain length. In this oxidation the hydrogens present on the n-x carbon are released into the water of the reaction mixture. The water was then carried through two lyophilizations and assayed for tritium content. Completeness of the oxidative step was monitored by gas-liquid chromato~aphy which was also used for quantification and estimation of the radioactive content of the individual fatty acids of the adipose tissue. RESULTS AND DISCUSSION
Theoreticd considerations A summary of the pathways of hydrogen incorporation into fatty acids is helpful in the discussion of the results. The incorporation into the interior carbons (excluding the carboxyl and terminal methyl positions) is considered. Even carboss. According to accepted reaction mechanisms one of the hydrogens on the even carbons should arise from the methylene hydrogens of malonyl-CoA and the other from the proton pool during the condensation-decarboxylation reaction of malonyl-CoA* with acetyl-CoA (ref. 6). When fatty acids are synthesized from glucose an appreciable part of the malonyl-CoA hydrogen theoretically arising in substrate hydrogen is also derived from protons pJ. This incorporation occurs during conversion of glucose to pyruvate and is evidenced by a decrease of the 3H : I% ratio in lactate synthesized from [6-l*C, 6-3Hlglucose as compared to the ratio in gIucose4. Possible mechanisms for this dilution have been discussed elsewherea. The extent * The authors have used conventional terminology for the intermediates of this reaction, but realize that in actuality it is not the CoA derivatives but acetyl-acyl carrier protein and malonylacyl carrier protein that are involveds.
D. w. FORSTER, J. KATZ
424
of the exchange of substrate hydrogens in the conversion of glucose to fatty acids can be estimated from the 3H : 14C ratio in fatty acids synthesized from [6-3H,6X]glucose. If no loss of substrate hydrogen occurred the theoretical ratio in palmitic acid (relative to glucose) should be 0.42 (ref. 8). The observed ratio in adipose tissue was 0.25 (ref. 4) indicating an exchange of about half of the substrate hydrogen with H,O. Thus, of the two hydrogens of the even positions in adipose tissue, about 1.5 may be derived from protons. The ratio in liver slices is somewhat lower, 0.1, indicating that about 1.7 of the two hydrogens arise in water in this tissue7>@. Odd carbons. The hydrogens on the odd carbons should be derived from TPNH. However, it was found by FLATT ANDBALL lo and KATZ ANDLANDAU~~that TPNH generated by the glucose-6-phosphate dehydrogenase or 6-phosphogluconatede hydrogenase reactions accounted for only about half the hydrogen equivalents needed for fatty acid synthesis in the intact adipose-tissue cell. It has been proposed that this hydrogen deficit is filled through transh~rdrogenation from DPNH to TPNH, and KATZ ANDROGNSTAD~have presented evidence for the participation of DPNH in this reduction. As noted earlier, degradation of fatty acids synthesized by liver slices in sH,O showed extensive incorporation of tritium into the odd positions of the chain where theoretically only hydrogen from TPNH should be found. KATZ AND ROGNSTAD~showed that the reductive hydrogen of TPNH generated in the pentose phosphate pathway did not exchange with H,@ but found that the reductive hydrogen of DPNH formed in the oxidation of gly~eraldehyde 3-phosphate contained an extensive contribution from the proton pool. {The bulk of the exchange with H,O actually appeared to occur with the hydrogen on position I of giyceraldehyde 3-phosphate prior to its oxidation and the generation of DPNH (ref. 4)). The evidence that DPNH does participate in the reductive reactions of fatty acid synthesis together with the evidence of an exchange reaction between DPNH and H,O suggests that labeling of the odd positions of the fatty acid chain synthesized in 3H,0 should occur in adipose tissue as well as in liver, and that this labeling reflects the contribution of hydrogen equivalents from DPNH. Intermolecular distrib&iort of tritium The recovery of tritium in the various fatty acids of epididymal fat-pad slices following incubation with [I-3Hlglucose is shown in Table I. The major portion of the activity was found in the palmitic acid fraction with lesser amounts in stearic acid. In this respect adipose tissue closely resembles liver. Similar findings were obtained with [3-?H]glucose and 3H,O as substrates. The unusually high percentage of palmitic acid by weight probably reflects the fact that the animals were fasted and then refed a high carbohydrate diet. Ilztramolecular distribution of tritium The intramolecular distribution of tritium in the newly synthesized fatty acids is shown in Table II. When [I-3Hlglucose was the substrate approx. 5% of the total radioactivity in the molecule was present on the C-/I while about 2% was recovered in the c+ and y-positions. Since the /?-position is the site of incorporation of the reductive hydrogen of TPNH, it can be seen that about three quarters ~----5.1 x 100 = 73% 5.J+r.o Biochim.
Biophys.
Acta,
1’25 (1966)
422-427
D. W. FORSTER, J* EiATZ
426
The reason for this finding is not clear, A possible explanation would involve the reductive carboxylation of pyruvate to [GH]malate by TPNaH generated from glucose. Randomization through the fumarase reaction would result in the formation of [~,3-~R~]malate. Reversal of the malic enzyme reaction would yield [3JH]pyruvate which could then appear on the even carbons of the fatty acid. Similar results would be expected from the ~ecarboxylation of oxalacetate to pyrnvate or phosphoeuolpyruvate. Such a sequence was proposed by HBBERM~N~~to account for the labeling pattern found in glucose from [z-2H]glycerol and [@HIlactate. When fatty acids synthesized in the presence of 3H,0 were degraded (Table II), 6,874 of the isotope was found in the a-position and 6.3% in the ,&position. This approximately equal distribution closely resembles the pattern in liver*. In an attempt to give quantitative meaning to this distribution, the experiments shown in Table III were performed, It can be seen that about two atoms of TABLE
III
THE RELATIVE INCORPORATIQN BY
ADIPOSE
OF ACETYL
UNITS
AND
PROTONS
INTO
FATTY
ACIDS
SYNTHESfZED
TISSUE
Adipose-tissue slices, 350-500 mg, were incubated in 2.2 ml of Krebs bicarbonate buffer containing a5 pmoles of [r%,Jglucose (r-z ,uC). roe mC of VI,0 was also present in each flask. One unit of insulin was added as indicated. Gas phase was g5u/ 0,5 O/ 10 CO, and incubation time was 3 h. At the end of the incubation period, fatty acids were isolated and assayed for r*C and % content. Results are given as averages and ranges and expressed as percentage of added radioactivity recovered in the fatty acids as well as ,~moles of a&$ units or protons of hydrogen incorporated, ._...__--_ Nulnbev of f%s~li~ Incor#mration into fatty acids ftwnz fiatwas ~~~~~1~ ncety1 units experiments [‘“C,Jgzucose “H,O
4
0
7
I unit ._--
f%)
(vote)
(&243.1)
ifL6.0)
z-*3)
G-33)
--“_
-.
(% x IO=)
(patGm.s)
Q.34 (O.ZI-0.42)
8.9 (5.Sr1.a)
r.5 (0.88-2.6)
$73-67)
2.0 (1.6~-2.9)
tritium were incorporated into the fatty acid chain for each z carbon acetyl unit. Essentially similar results were obtained in the presence or absence of insulin. Correcting for the fact that no tritium would be present on the omega acetyl unit, it follows from the data of Table II that approx. 1.1 tritium atoms is bound on the average to each carbon in the interior of the fatty acid molecule. According to the theoretical considerations given above it was expected that about 2.5 atoms would be found on each interior acetyl unit, one on the odd carbon and 1.5 on the even. The discrepancy may be due to isotope discrimination effects in the exchange with %I+. In view of this possibility the agreement between theory and experiment seems reasonable. The present findings support the view that in both liver and adipose tissue * It should be noted that a molecule of palmitic acid synthesized de nozw would be expected to contain about 132/o of the tot& fatty acid tritium in each two carbon fragment of the molecule. In the experiments with *I&O, where the isotope pool was large, this expectation was met. In the glucose experiments only S-o% of the isotope was present on the combined L-a and L-B. The probable explanation for this discrepancy is that exogenous substrate was expended in the latter experiments and that utilization of endogenous substrate accounted for dilution of the label on the terminal carbons of the fatty acid molecule. B&him.
Biophys.
Acta, 125 (~966) 422-427
HYDROGEN
427
METABOLISM IN FATTY ACID SYNTHESIS
about half the reductive hydrogens for fatty acid synthesis are ultimately derived from DPNH which has equilibrated with the proton pool. They further suggest that the mechanisms of fatty acid synthesis in adipose tissue and liver are fundamentally the same. ACKNOWLEDGEMENTS
D. W. FOSTER is a Career Development Service.
J. KATZ
is an Established
Investigator
Awardee
of the U.S.
of the American
Heart
Public
Health
Association.
These studies were supported by U.S. Public Health Service grant No. CA 08269 (DWF) and grant No. AM 03682 and American Heart Association Grant-in-aid 61-F-17
(J.K.).
REFERENCES I D. W. FOSTER AND B. BLOOM, J. Biol. Chem., 238 (1963) 888. 2 B. R. LANDAU AND J.KATZ~~ A. E. RENOLD AND G. CAHILL,Handbook
3 4 5
6 7 8 g IO II
12
ofPhysiology, Vol.5, AmericanPhysiological Society, Washington,1965,p. 253. J. KATZ, R. ROGNSTAD AND R. KEMP,J. Biol. Chem.,240(1965)PC 1484. J. KATZ AND R. ROGNSTAD,~. BioZ.Chem., 241 (1966) 3600. P. W. MAJERUS, A. W. ALBERTS AND P. ROYVAGELOS, Proc. Natl. Acad. Sci. U.S., 51 (1964) 1231. R. BRESSLER AND S. J.WAKIL, J. BioZ.Chem., 236(1961) 1643. D. W. FOSTER AND B. BLOOM, Biochim. Biophys. Acta, 60 (1962) 189. S. ABRAHAM,J. KATZ,J.BENTLEYAND I.L.CHAIKOFF, Biochim.Biophys.Acta, 70(1963)6go. D. W. FOSTER AND B. BLOOM,J.B~~E.Chem.,236(Ig61) 2548. J. P. FLATT AND E. G. BALL, J. Biol. Chem., 239 (1964) 675. J. KATZ, B. R. LANDAU AND G. E. BARTSCH,J.BioZ.Chem., 241 (1966)727. H. D. HOBERMAN,J. BioZ.Chem., 233 (1958) 1045. Biochim.
Biophys.
Acta, 125 (1966) 422-427
BIOCHIMICA ET BIOPHYSICA
ACTA
55264
THE DISTRIBUTION TRITIATED
DANIEL
OF TRITIUM
IN FATTY
GLUCOSE AND TRITIATE~
W. FOSTER
AND
JOSEPH
ACIDS SYNTHESIZED
WATER
BY RAT ADIPOSE
FROM TISSUE
KATZ
The University of Texas Southwestern Medical School, Dallas, Texas and the Cedars-Sinai Medical Center, Los Angeles, Calif. (U.S.A.) (Received
May =$h, 1966)
SUMMARY
Fatty acids synthesized by rat epididymal fat-pad slices in the presence of [r-3H]glucose, [3-3H]glucose and tritiated water were degraded for the localization of tritium in the molecule. 75% of the tritium from [x-3H]glucose and 85% from [3-3Hjglucose was present on the odd carbons. The incorporation of 15% of the tritium from [3-3H]glucose in the even positions was unexpected. Tritium from 3H,O was incorporated to the same extent in the odd and even positions of the carbon chain. The presence of tritium from water in the odd positions confirms the participation of DPNH hydrogen in reductive fatty acid synthesis in the intact adipose tissue cell. The results with 3H,0 and [r-3H]glucose resemble those previously reported for liver and suggest that the mechanisms of fatty acid synthesis are essentially similar in the two tissues.
INTRODUCTION
When fatty acids are synthesized in the cell the hydrogens of the aliphatic chain are derived from three sources: substrate hydrogen (glucose, acetate, etc.), hydride ions from TPNH, and protons from H,O. According to theoretical considerations the hydrogens on the even carbons of the fatty acid (LX,y, etc.) would be derived from substrate and H,O while the reductive hydrogen of TPNH should be found on the odd carbons of the chain (8, 6, etc.). These predictions were previously tested for fatty acids synthesized by rat-liver slices in the presence of [r-3H]glucose and $H,O (ref. I). With [r-3H]glucose as substrate for the generation of radioactive TPNH in the cell, it was shown that over 05% of the tritium was present on the odd-numbered carbons of the fatty acid mole&le in accord with the theoretical predictions. Such was not the case in experiments with 3H,O. Assuming TPNH to be the sole donor of reductive hydrogen in fatty acid synthesis, little tritium was expected on the odd carbons of fatty acids synthesized in 3H,O. In actuality 5560% was present in the /J-position and only 40-45% on the C-U. These results suggested the possibility of an exchange Biochim. Biophys. Acta, 125 (1966) qzz-.p7
HYDROGEN
METABOLISM
IN FATTY ACID SYNTHESIS
423
reaction between TPNH and H,O prior to reduction of the intermediates of fatty acid synthesis. KATZ and co-workers8-4 have recently studied the incorporation of 3H into fatty acids synthesized by rat adipose tissue utilizing F120, [r-8H]glucose and [3-sH]glucose as substrates. It seemed of interest to determine the location of tritium in such fatty acids for two reasons: to compare the findings with [r-3H]glucose and 3H,O in adipose tissue with those in liver, and to compare the distribution of tritium arising in TPNH generated from jr-3H]glucose with that generated from [3-3H]glucose. EXPERIMENTAL
PROCEDURES
The experimental procedures used in these experiments have been previously described1T4. Briefly, epididymal fat-pad slices, 300 mg, from fasted-refed rats (2 days fast followed by 3 days high carbohydrate diet) were incubated for 3 h at 38” in 2.2 ml of Krebs-Henseleit bicarbonate buffer containing glucose specifically labeled with tritium or 3H,0. At the end of the incubation period the reaction was stopped and long-chain fatty acids were isolated and counted. The total fatty acids were then separated into saturated and unsaturated fractions by lead salt precipitation, and the saturated fraction used for the degradation procedure. After preparation of the silver salts of the acids decarboxylation was carried out with bromine. The alcohol containing one less carbon was next made and oxidation with CrO, was carried out to give the fatty acid of n-r chain length. In this oxidation the hydrogens present on the n-x carbon are released into the water of the reaction mixture. The water was then carried through two lyophilizations and assayed for tritium content. Completeness of the oxidative step was monitored by gas-liquid chromato~aphy which was also used for quantification and estimation of the radioactive content of the individual fatty acids of the adipose tissue. RESULTS AND DISCUSSION
Theoreticd considerations A summary of the pathways of hydrogen incorporation into fatty acids is helpful in the discussion of the results. The incorporation into the interior carbons (excluding the carboxyl and terminal methyl positions) is considered. Even carboss. According to accepted reaction mechanisms one of the hydrogens on the even carbons should arise from the methylene hydrogens of malonyl-CoA and the other from the proton pool during the condensation-decarboxylation reaction of malonyl-CoA* with acetyl-CoA (ref. 6). When fatty acids are synthesized from glucose an appreciable part of the malonyl-CoA hydrogen theoretically arising in substrate hydrogen is also derived from protons pJ. This incorporation occurs during conversion of glucose to pyruvate and is evidenced by a decrease of the 3H : I% ratio in lactate synthesized from [6-l*C, 6-3Hlglucose as compared to the ratio in gIucose4. Possible mechanisms for this dilution have been discussed elsewherea. The extent * The authors have used conventional terminology for the intermediates of this reaction, but realize that in actuality it is not the CoA derivatives but acetyl-acyl carrier protein and malonylacyl carrier protein that are involveds.
D. w. FORSTER, J. KATZ
424
of the exchange of substrate hydrogens in the conversion of glucose to fatty acids can be estimated from the 3H : 14C ratio in fatty acids synthesized from [6-3H,6X]glucose. If no loss of substrate hydrogen occurred the theoretical ratio in palmitic acid (relative to glucose) should be 0.42 (ref. 8). The observed ratio in adipose tissue was 0.25 (ref. 4) indicating an exchange of about half of the substrate hydrogen with H,O. Thus, of the two hydrogens of the even positions in adipose tissue, about 1.5 may be derived from protons. The ratio in liver slices is somewhat lower, 0.1, indicating that about 1.7 of the two hydrogens arise in water in this tissue7>@. Odd carbons. The hydrogens on the odd carbons should be derived from TPNH. However, it was found by FLATT ANDBALL lo and KATZ ANDLANDAU~~that TPNH generated by the glucose-6-phosphate dehydrogenase or 6-phosphogluconatede hydrogenase reactions accounted for only about half the hydrogen equivalents needed for fatty acid synthesis in the intact adipose-tissue cell. It has been proposed that this hydrogen deficit is filled through transh~rdrogenation from DPNH to TPNH, and KATZ ANDROGNSTAD~have presented evidence for the participation of DPNH in this reduction. As noted earlier, degradation of fatty acids synthesized by liver slices in sH,O showed extensive incorporation of tritium into the odd positions of the chain where theoretically only hydrogen from TPNH should be found. KATZ AND ROGNSTAD~showed that the reductive hydrogen of TPNH generated in the pentose phosphate pathway did not exchange with H,@ but found that the reductive hydrogen of DPNH formed in the oxidation of gly~eraldehyde 3-phosphate contained an extensive contribution from the proton pool. {The bulk of the exchange with H,O actually appeared to occur with the hydrogen on position I of giyceraldehyde 3-phosphate prior to its oxidation and the generation of DPNH (ref. 4)). The evidence that DPNH does participate in the reductive reactions of fatty acid synthesis together with the evidence of an exchange reaction between DPNH and H,O suggests that labeling of the odd positions of the fatty acid chain synthesized in 3H,0 should occur in adipose tissue as well as in liver, and that this labeling reflects the contribution of hydrogen equivalents from DPNH. Intermolecular distrib&iort of tritium The recovery of tritium in the various fatty acids of epididymal fat-pad slices following incubation with [I-3Hlglucose is shown in Table I. The major portion of the activity was found in the palmitic acid fraction with lesser amounts in stearic acid. In this respect adipose tissue closely resembles liver. Similar findings were obtained with [3-?H]glucose and 3H,O as substrates. The unusually high percentage of palmitic acid by weight probably reflects the fact that the animals were fasted and then refed a high carbohydrate diet. Ilztramolecular distribution of tritium The intramolecular distribution of tritium in the newly synthesized fatty acids is shown in Table II. When [I-3Hlglucose was the substrate approx. 5% of the total radioactivity in the molecule was present on the C-/I while about 2% was recovered in the c+ and y-positions. Since the /?-position is the site of incorporation of the reductive hydrogen of TPNH, it can be seen that about three quarters ~----5.1 x 100 = 73% 5.J+r.o Biochim.
Biophys.
Acta,
1’25 (1966)
422-427
D. W. FORSTER, J* EiATZ
426
The reason for this finding is not clear, A possible explanation would involve the reductive carboxylation of pyruvate to [GH]malate by TPNaH generated from glucose. Randomization through the fumarase reaction would result in the formation of [~,3-~R~]malate. Reversal of the malic enzyme reaction would yield [3JH]pyruvate which could then appear on the even carbons of the fatty acid. Similar results would be expected from the ~ecarboxylation of oxalacetate to pyrnvate or phosphoeuolpyruvate. Such a sequence was proposed by HBBERM~N~~to account for the labeling pattern found in glucose from [z-2H]glycerol and [@HIlactate. When fatty acids synthesized in the presence of 3H,0 were degraded (Table II), 6,874 of the isotope was found in the a-position and 6.3% in the ,&position. This approximately equal distribution closely resembles the pattern in liver*. In an attempt to give quantitative meaning to this distribution, the experiments shown in Table III were performed, It can be seen that about two atoms of TABLE
III
THE RELATIVE INCORPORATIQN BY
ADIPOSE
OF ACETYL
UNITS
AND
PROTONS
INTO
FATTY
ACIDS
SYNTHESfZED
TISSUE
Adipose-tissue slices, 350-500 mg, were incubated in 2.2 ml of Krebs bicarbonate buffer containing a5 pmoles of [r%,Jglucose (r-z ,uC). roe mC of VI,0 was also present in each flask. One unit of insulin was added as indicated. Gas phase was g5u/ 0,5 O/ 10 CO, and incubation time was 3 h. At the end of the incubation period, fatty acids were isolated and assayed for r*C and % content. Results are given as averages and ranges and expressed as percentage of added radioactivity recovered in the fatty acids as well as ,~moles of a&$ units or protons of hydrogen incorporated, ._...__--_ Nulnbev of f%s~li~ Incor#mration into fatty acids ftwnz fiatwas ~~~~~1~ ncety1 units experiments [‘“C,Jgzucose “H,O
4
0
7
I unit ._--
f%)
(vote)
(&243.1)
ifL6.0)
z-*3)
G-33)
--“_
-.
(% x IO=)
(patGm.s)
Q.34 (O.ZI-0.42)
8.9 (5.Sr1.a)
r.5 (0.88-2.6)
$73-67)
2.0 (1.6~-2.9)
tritium were incorporated into the fatty acid chain for each z carbon acetyl unit. Essentially similar results were obtained in the presence or absence of insulin. Correcting for the fact that no tritium would be present on the omega acetyl unit, it follows from the data of Table II that approx. 1.1 tritium atoms is bound on the average to each carbon in the interior of the fatty acid molecule. According to the theoretical considerations given above it was expected that about 2.5 atoms would be found on each interior acetyl unit, one on the odd carbon and 1.5 on the even. The discrepancy may be due to isotope discrimination effects in the exchange with %I+. In view of this possibility the agreement between theory and experiment seems reasonable. The present findings support the view that in both liver and adipose tissue * It should be noted that a molecule of palmitic acid synthesized de nozw would be expected to contain about 132/o of the tot& fatty acid tritium in each two carbon fragment of the molecule. In the experiments with *I&O, where the isotope pool was large, this expectation was met. In the glucose experiments only S-o% of the isotope was present on the combined L-a and L-B. The probable explanation for this discrepancy is that exogenous substrate was expended in the latter experiments and that utilization of endogenous substrate accounted for dilution of the label on the terminal carbons of the fatty acid molecule. B&him.
Biophys.
Acta, 125 (~966) 422-427
HYDROGEN
427
METABOLISM IN FATTY ACID SYNTHESIS
about half the reductive hydrogens for fatty acid synthesis are ultimately derived from DPNH which has equilibrated with the proton pool. They further suggest that the mechanisms of fatty acid synthesis in adipose tissue and liver are fundamentally the same. ACKNOWLEDGEMENTS
D. W. FOSTER is a Career Development Service.
J. KATZ
is an Established
Investigator
Awardee
of the U.S.
of the American
Heart
Public
Health
Association.
These studies were supported by U.S. Public Health Service grant No. CA 08269 (DWF) and grant No. AM 03682 and American Heart Association Grant-in-aid 61-F-17
(J.K.).
REFERENCES I D. W. FOSTER AND B. BLOOM, J. Biol. Chem., 238 (1963) 888. 2 B. R. LANDAU AND J.KATZ~~ A. E. RENOLD AND G. CAHILL,Handbook
3 4 5
6 7 8 g IO II
12
ofPhysiology, Vol.5, AmericanPhysiological Society, Washington,1965,p. 253. J. KATZ, R. ROGNSTAD AND R. KEMP,J. Biol. Chem.,240(1965)PC 1484. J. KATZ AND R. ROGNSTAD,~. BioZ.Chem., 241 (1966) 3600. P. W. MAJERUS, A. W. ALBERTS AND P. ROYVAGELOS, Proc. Natl. Acad. Sci. U.S., 51 (1964) 1231. R. BRESSLER AND S. J.WAKIL, J. BioZ.Chem., 236(1961) 1643. D. W. FOSTER AND B. BLOOM, Biochim. Biophys. Acta, 60 (1962) 189. S. ABRAHAM,J. KATZ,J.BENTLEYAND I.L.CHAIKOFF, Biochim.Biophys.Acta, 70(1963)6go. D. W. FOSTER AND B. BLOOM,J.B~~E.Chem.,236(Ig61) 2548. J. P. FLATT AND E. G. BALL, J. Biol. Chem., 239 (1964) 675. J. KATZ, B. R. LANDAU AND G. E. BARTSCH,J.BioZ.Chem., 241 (1966)727. H. D. HOBERMAN,J. BioZ.Chem., 233 (1958) 1045. Biochim.
Biophys.
Acta, 125 (1966) 422-427