- Email: [email protected]
Plasma free oleic and palmitic acid levels during vigorous exercise
Plasma Free Oleic and Palmitic Acid Levels during Vigorous Exercise ByPETER WOOD, &ENTER
SCHLIERF ANDLAURANCE KINSELL
The behavior of plasma free palmitic and oleic acids was studied without the use of radioactive tracers in fasting human subjects during vigorous exercise and following rest. The initial level of total plasma free fatty acids (FFA) fell during a period of vigorous exercise, and the proportions of palmitic and oleic acids in the total FFA rose and fell, respectively. Further exercise followed by rest resulted in a considerable increase of total FFA level; the pro-
portion of oleic acid rose towards the proportion present in the adipose tissue lipids of the particular subject, while the palmitic acid proportion fell. These changes are interpreted to mean that during muscular activity, uptake of FFA from plasma proceeds in such a manner that oleic acid has a measurably greater fractional turnover rate than has palmitic acid. (Metabolism 14: No. 10, October, 1095-1100, 1965)
P
LASMA FREE FATTY ACIDS (FFA) provide m important source of energy for working muscle, particularly in the fasting state.’ Previous studies on the turnover characteristics of the various constituent acids of the total plasma FFA have usually invohred either intravenous injection of radioactively-l.abeled FFA, bound to albumin in vitro, and measurement of the rate of disappearance of the label from the plasma FFA”-’ or study of the arteriovenous differences across single organs.5-i The present study is an attempt to obtain independent data on the behavior of plasma free oleic and palmitic acids in fasting human subjects during conditions of vigorous exercise, by simple procedures that avoid uncertainties associated with the isolation of a single organ or the use of injected tracers. MATERIALS AND METHODS Healthy undertook minutes
subjects (2 male and 2 female) aged 28-39. fasted for 15 hours and then the following program: very vigorous exercise was performed for about 5 in such a manner that the subject was extended ahnost to the limit of his ability;
less severe exercise was then continued for 20-40 minutes. followed by complete rest, sitting in a chair, for 30 minutes. Two male subjects exercised by running. the first exercise period consisting of a mile run in approximately 5 minutes. Two female subjects exercised on a stationary bicycle. Blood samples were taken from a forearm vein as follows: (a) resting. before exercise; (b) during the latter stages of the severe exercise in the case of the cyclists. and in the runners within 30 sconds of completing the mile run; and (c) at the encl of the final rest period _. -____.
in all subjects.
-
Radial
pulse rates were measured
in some
~__
From the Institute for Metaholir~ Research, Highland-Alameda County Ho.ypital, Oakland, California Receioed for pul&ation June 23, 1965. PETER WOOD, PH.D.: Research Riochemist, Institute for Mctaholic Research, HighlandAhmeda Corm@ Hospifaf, Oakland, Calif. CUENTER SCHLIERF, M.D.: Reseurch Fellow, I&it&e for Metabolic Research, Highland-Alumeda County Eloupital, Oakland, Calif. LAURANCE KINSELL, M.D.: Director, In&it& for Metabolic Research, Ifighland-Alameda County Hospital, Oakland, Calif. 1095
1096
WOOD,
Table I.-Pulse
Subject NO.
Rate and Plasma Lipid Levels during Exercise and Rest
Time of Determination*
Pulse Rate (beats/min.)
Plasma Triglyceride (mg./lOO ml.)
SCHLIERF
AND
KIKSELL
in 2 Male Subjects _ Pl83IXX3 Phospholipid (mg./lOO ml.)
Plasma Total Cholesterol (mg./lOO ml.)
1
i;; Cc)
50 180 64
50 52 32
244 269 261
232 256 250
68 2
i;‘, (c)
192 72
31 30 24
173 184 182
152 158 152
“(a)
resting,
before
exercise;
(1,)
immediately
after
vigorous
exercise
for $5 minutes;
(c) after further exercise followed by rest for 30 minutes. subjects. Adipose tissue samples needle biopsy from the buttock.* Blood was taken into EDTA separated within one hour. Lipids
were extracted
were obtained tubes
from plasma
which aliquots
(in 3 subjects were
chilled
without
delay
only)
on the same day, by
immediately
and
by the procedure
the
plasma
of Folch
et al.;!’ water containing tion. FFA were isolated
0.04 per cent (a/v) of calcium chloride was used as wash solufrom the extracts by thin-layer chromatography on plates coated Merck A.G.. Darmstadt, Germany) 250 p in thickness. and contain-
with Silica Gel G (E. ing a small quantity of 2’.7’-dichlorfluorescein. The solvent system was petroleum ether ( b.p. 30-60” )-diethyl ether-acetic acid ( 70: 30: 1 v/v/v/ ) ; separated lipids were visualized
under ultraviolet light, and the FFA area was accurately delineated. scraped off and eluted with diethyl ether from a small column. Recovery of FFA by this procedure was checked by addition to the plasma of tracer amounts of C 14-oleic or tritiated pahnitic acid. and was always better than 97 per cent. The absence of lipids other than FFA was confirmed by thin-layer chromatography. In this procedure. plasma lactic acid does not interfere. The total FFA content of the extracts was measured by 3 procedures: ester-linkage determination by the hlichaels method;rrr spot-size measurement on thin-layer chromatograms;il and treatment with Cr4- c1’. . omethane.rz The weight percentages of oleic and palmitic LIL acids in the total FFA were determined in duplicate. after methylation with diazomethane. by a gas-liquid chromatographic procedure previously described.rs Total adipose tissue lipids were extracted by the Folch procedure>rr transmethylated by heating with 2 per cent (v/v) sulfuric acid in methanol. and the methyl esters analyzed by gas-liquid chromatography.r:r Plasma triglycerides.10 phospholipidsrJ and total cholesterolra were determined for the male subjects. RESULTS
Radial pulse rates and plasma triglyceride, phospholipid and total cholesterol levels are shown in table 1 for the 2 male subjects who exercised by running. Plasma triglyceride levels, initially low after fasting, fell to still lower levels after exercise followed by rest. Plasma phospholipid and total cholesterol levels changed little, though there was a small rise in each after vigorous exercise for 5 minutes. Levels of plasma total FFA and proportions of free oleic and palmitic acids are shown for all subjects in figure 1, together with oleic and palmitic acid proportions found in adipose tissue lipids in 3 subjects. The 3 procedures used for determination of total FFA 1evels gave closely similar results. Agreement
OLEIC
AND
PALMITIC
ACID
LEVELS
DURING
VIGOROUS
1097
EXERCISE
+x@q
16:O
16:O l6:O
12 R
E
R
R’
4
E
R’
R
E
R’
R
E
R’
Fig. L-Changes in plasma FFA in subjects while exercising by running (1 and 2) or by pedalling a stationary bicycle (3 and 4), and after subsequent rest. Open circles: proportion of plasma free oleic acid (18:l) and palmitic acid (16:0:1 in the total FFA. Closed circles: proportions of oleic and palmitic acid in the adipose lipids of the subject. R, resting before exercise; E, immediately after vigorous exercise for 5 minutes; R’, after flIrther exercise followed lnr rest for 30 minutes. between uent
duplicate
fatty
determinations
acids
in FFA
and
by gas-liquid
in adipose
chromatography
tissue
was
good;
of constit-
mean
results
are
shown in the figure. In all subjects
acute
exercise
produced
a fall in total
FFA
by a rise in level after rest. In each case the concentration total
FFA
palmitic
had fallen
by the end of the acute
acid had risen.
The
cise and rest for 30 minutes and in the 3 subjects the 2 acids adipose
found
elevated
total
exercise
FFA
was accompanied
where
adipose
at this stage
tissue
period,
by a reversal had
followed
while
level seen after
was examined
in plasma
level,
of oleic acid in thr that
further
of these
of
exer-
changes,
the proportions
approached
those
present
asscciated
with
the
of in
tissue fatty acids. DISCUSSION
Design
Procedure.
of
labeled
fatty
radiochemical complex tion,
purity
avoided,
serial
sampling
of blood
muscular
In these
circumstances
poses represents
demand
the possibility
entirely
required
to reduce
use
regarding that
the
physiologically.li
in tracer
plasma
temporarily fate of FFA
that during
total
exceed
it is very probable
the major
,the evidence
tissue is minimal.
and
uncertainty
studies
of the
albumin In addi-
is difficult
to
exercise.
was used
making
instance,
does not beha\-e
during very vigorous exercise
difficulties
for
of the tracers,“.“’
in vitro
Vigorous
reviewed
In this study
were
formed
the
achieve
acids
supply
levels
from
that use by muscle
leaving
severe
FFA
the plasma.
esercise
uptake
rapidly
adipose for energy
by
tissue. pur-
Have1 et al.’ have
by liver and adipose
1098
WOOD, SCHLIERF AND XINSELL
During the final rest period, total plasma FFA rose to a high level, due almost certainly to rapid influx of FFA from adipose tissue at a time when muscular demand had suddenly decreased. At this stage some indication should be obtained of the nature of the fatty acid mixture being made available from adipose tissue. Special care was taken to ensure the accuracy of measurements of total FFA levels, and to check the purity and completeness of recovery of the total FFA fraction isolated for examination by gas-liquid chromatography. Site of Blood Sampling. Although sampling of arterial blood is ideal, it is difficult to achieve during very vigorous exercise. We feel that the changes of total FFA level seen in our venous blood samples reflect changes in arterial blood, since the changes we have found are similar to those seen in exercise studies where arterial blood has been examined.lsl’ Any addition of FFA from forearm adipose tissue would tend to elevate the venous FFA level above that in the arterial plasma, and as will be discussed below, would probably tend to raise the proportion of free oleic acid in the total FFA above that in the arterial plasma. Thus the changes seen in venous plasma immediately following exercise (sampling point E; fig. 1) would in all probability have been of somewhat greater degree had arterial plasma been examined. Change of 0leic:Palmitic Acid Ra.tio in FFA &ring Exercise. Our finding that the oleic:palmitic acid proportion in plasma FFA falls when the total level falls during acute exercise, and rises when the total FFA level rises during subsequent rest, is consistent with observations of Rothlin et al.‘!’ The latter workers used human subjects and anesthetized dogs; elevations of plasma FFA level were induced by norepinephrine infusions, and reductions of FFA level by infusion of glucose and insulin. It was noted that at high arterial FFA levels the composition of the FFA mixture in man approached that reported for depot fat by Hirsch et aLZn It seems probable to us that the changes seen when the total FFA level falls during vigorous exercise are the result of a preferential uptake of oleic rather than palmitic acid by the muscles, resulting in a greater fractional turnover rate for oleic acid compared with palmitic acid. This mechanism is in accord with several other observations. Rothlin and Ring” have found that the human heart extracts a consistently higher percentage of free oleic acid than of any other FFA present in arterial plasma. Imaichi et al.“l noted that during prolonged fasting in man the proportion of oleic acid in plasma FFA and triglycerides remained lower, and the proportion of palmitic acid rcmained higher, than was the case in the adipose tissue, for a given subject; this might suggest that oleic acid has a shorter turnover time than palmitic acid during fasting. Ono and Fredrickson 4 have found in anesthetized dogs that the rate of disappearance from the plasma of (2”‘-oleic acid bound to albumin was greater than that of simultaneously injected tritiated palmitic acid. Have1 et a1.l6 infused tritiated palmitic acid and C?j-oleic acid simultaneously into human subjects during rest and moderate exercise. While no large differences were found in rates of fractional turnover of these acids during exercise, small differences were probably not easily detectable by their pro-
OLEIC AND PALMITIC
ACID LEVELS
DURING
VIGOROUS
1099
EXERCISE
cedure. From measurement of espired C1”O, in subjects infused with either Cl”-oleic or C14-palmitic acid during exercise, these authors obtained some indication that oleate may be oxidized preferentially. Rothlin et al.” have considered other possible explanations for the variation of FFA composition as a function of total plasma FFA concentration. The existence of a small fraction of total FFA which is tightly bound to albumin, and thus not readily exchangeable, might explain our results, (a) if this fraction is extracted from albumin by the Folch procedure, and (b) if the fraction is relatively rich in palmitic acid and poor in oleic acid. Evidence in favor of these possibilities is apparently lacking. A further possibility is that the FFA supplied from adipose tissue during acute exercise is similar in composition to the FFA mixture seen at the low l)lasma FFA levels at the end of the exercise period; but again there is apparently no evidence for this. Oleic acid is released at a higher rate than palmitic acid by dog adipose tissue;F’ elevation of FFA level by release from adipose tissue following norepinephrine infusion in man increases the relative contribution of oleic acid to the total plasma FFA;‘” and the FFA composition in the final resting stage of the present work, when adipose tissue FFA were stiI1 being reIeased at a high rate, strongly suggests that the FFA mixture released into the plasma resembles that of adipose tissue, i.e., is rich in oleic acid. Level of Other Plasmu Lipids Followin, 0 Exercise. The small increase ir. plasma total cholesterol and phospholipid values found after vigorous exercise for 5 minutes is consistent with a report that some hemoconcentration occurs in such conditions.‘? In addition, increased production of norepinephrine during exercise may have contributed to the increased concentration of these lipids.‘3 In contrast, reduction of initial fasting plasma triglyceride levels was observed in 2 subjects following exercise and rest. This suggests some direct use of plasma triglycerides for energy purposes, as discussed by Have1 et al.’ and by Nikkilg and Konttinen.“’ Holloszy et al.?” have recently shown a substantial reduction of moderately elevated plasma triglyceride levels in middleaged men during a long-term program of vigorous exercise. Con&.sion.s. In summary, we interpret the changes in plasma FFA composition during vigorous exercise to indicate that oleic acid has a greater fractional turnover rate than has palmitic acid. Under our experimental conditions, removal by working muscle is probably the major factor responsible for this behavior. The results also suggest some direct use of plasma triglyceride for energy purposes during exercise. ACKNOWLEDGMENT This Products
study
was
supported
in part
by
PHS
Research
Grant
HE
00955
and
The
Corn
Company.
REFERENCES 1. Havel, R. J., Naimark. A., and Borchgrevink, C. F.: Turnover rate and oxidation of free fatty acids of blood plasma in man during exercise. J. Clin.
Invest.
42:1054,
1963.
2. Fredrickson, II. S., and Gordon, R. S., Jr.: The metabolism of albuminbound Cl+-labeled unesterified fatty acids in normal human subjects. J. Clin, Invest. 37; 1504, 1958.
1100
WOOD,
3. Nestel, P. J., Bezman, A., and Havel, R. J.: Metabolism of palmitate and linoleate in intact dogs. Amer. J. Physiol. 203:914, 1962. 4. Ono, K., and Fredrickson, D. S.: The metabolism of Cl*-labeled ci.s and tram isomers of octadecenoic and octadecadienoic acids. J. Biol. Chem. 239:2482, 1964. 5. Rothlin, M. E., and Bing, R. J.: Extraction and release of individual free fatty acicls by the heart and fat depots. J. Clin. Invest. 40:1380, 1961. 6. Ballarcl, F. B., Danforth, W. H., Naegle, S., and Bing, R. J.: Myocardial metabolism of fatty acids. J. Clin. Invest. 7. Harris,
39:717, 1960. P.. Chlouverakis.
C.,
Gloster,
J.> and
Jones, J. H.: Arterio-venous in the composition of differences
plasma regions
free fatty acids in various of the body. Clin. Sci. 22:
113. 1962. 8. Hirsch, J.: Adipose Tissue as an Organ. Springfield, Ill., Charles C Thomas, p. 81. 9. Folch, J., Lees, M., and Sloane-Stanley, C. H.: A simple method for the isolation and purification of total lipids Chem. 10. hlichaels.
from animal tissues. J. Biol. 226:497, 1957. C. D.: A method for the de-
termination of glycerides and free fatty acids. Metabolism 11: 833, 1962. 11. Schlirrf, G., and Wood, P.: Quantitative determination of plasma free fatty acids and triglycerides by thinlayer chromatography. J. Lipid Res. 6:317, 1965. 12. Wood, P. D. S., and Sodhi, H. S.: A method for determination of plasma free fatty acids. Proc. Sot. Exp. Biol. (N. Y.) 118:590, 1965. 13. Wood, P., Imaichi, K., Knowles, J., h/lichaels, G., and Kinsell, L.: The lipid composition of human plasma chylomicrons. J. Lipid Res. 5:225, 1964. 14. Youngburg, G. E., and Youngburg, M. V.: Phosphorus metabolism. I. A system ot blood phosphorus analysis.
J. Lab.
Clin.
SCHLIERF
Med.
AND KINSELL
16:158,
1930.
15. Technicon Autoanalyzer Methodology for Determination of Total Cholesterol. Technicon Laboratory File 24a. 16. Havel,
R. J., Carlson, L. A., Ekelund, and Holmgren, A.: Turnover L.. rate and oxidation of different free fatty acids in man during exercise. J. Appl. Physiol. 19:613, 1964.
17. Bates, M. W., and Olson, R. E.: Turnover rates of nonesterified fatty acids in the postabsorptive 19:230, 1960.
dog.
Fed.
Proc.
18. Friedberg, S. J., Sher, P. B., Bogdonoff, hl. D., and Estes, E. H., Jr.: The dynamics of plasma free fatty acid metabolism during exercise. J. Lipid Res. 4:34, 1963. 19. Rothlin, M. E., Rothlin, C. B., and Wendt, V. E.: Free fatty acid concentration and composition in arterial blood. Amer. J. Physiol. 203:306. 1962. 20.
Hirsch, J., F:Trqubar, J. H., Peterson, M. L., Studies of adipose Amer. J. Clin. Nutr.
21.
Imaichi, K., Michaels, G. D., Holton, L. W.: Plasma lipid S., and Kinsell, fatty acids during fasting. Amer. J. Clin. Nutr. 13:226, 1963.
22.
Bert, R. V., and Stead, E. A.: Demonstration that in normal man no reserves of blood are mobilized by exercise, epinephrine, and hemorrhage. 1941.
Amer.
J.
Med.
W., Ahrens, E. and Stoffel. W.: tissue in man. 8:499, 1960.
Sci.
201:655,
23.
Grasso, S., Michaels, G. D., and Kinsell, L. W.: Effects of norepinephrine upon lipid metabolism in a patient with biliary cirrhosis. Metabolism 13: 1333, 1964.
24.
NikkilL, E. A., and Konttinen, A.: Effect of physical activity on postprandial levels of fats in serum. Lancet 1: 1154, 1962.
25.
Holloszy, J. O., Skinner, J. S., Toro, G., and Cureton, T. K.: Effects of a six month program of endurance exercise on the serum lipids of middle-aged men.
Amer.
J. Cardiol.
14:753,
1964.
SCHLIERF ANDLAURANCE KINSELL
The behavior of plasma free palmitic and oleic acids was studied without the use of radioactive tracers in fasting human subjects during vigorous exercise and following rest. The initial level of total plasma free fatty acids (FFA) fell during a period of vigorous exercise, and the proportions of palmitic and oleic acids in the total FFA rose and fell, respectively. Further exercise followed by rest resulted in a considerable increase of total FFA level; the pro-
portion of oleic acid rose towards the proportion present in the adipose tissue lipids of the particular subject, while the palmitic acid proportion fell. These changes are interpreted to mean that during muscular activity, uptake of FFA from plasma proceeds in such a manner that oleic acid has a measurably greater fractional turnover rate than has palmitic acid. (Metabolism 14: No. 10, October, 1095-1100, 1965)
P
LASMA FREE FATTY ACIDS (FFA) provide m important source of energy for working muscle, particularly in the fasting state.’ Previous studies on the turnover characteristics of the various constituent acids of the total plasma FFA have usually invohred either intravenous injection of radioactively-l.abeled FFA, bound to albumin in vitro, and measurement of the rate of disappearance of the label from the plasma FFA”-’ or study of the arteriovenous differences across single organs.5-i The present study is an attempt to obtain independent data on the behavior of plasma free oleic and palmitic acids in fasting human subjects during conditions of vigorous exercise, by simple procedures that avoid uncertainties associated with the isolation of a single organ or the use of injected tracers. MATERIALS AND METHODS Healthy undertook minutes
subjects (2 male and 2 female) aged 28-39. fasted for 15 hours and then the following program: very vigorous exercise was performed for about 5 in such a manner that the subject was extended ahnost to the limit of his ability;
less severe exercise was then continued for 20-40 minutes. followed by complete rest, sitting in a chair, for 30 minutes. Two male subjects exercised by running. the first exercise period consisting of a mile run in approximately 5 minutes. Two female subjects exercised on a stationary bicycle. Blood samples were taken from a forearm vein as follows: (a) resting. before exercise; (b) during the latter stages of the severe exercise in the case of the cyclists. and in the runners within 30 sconds of completing the mile run; and (c) at the encl of the final rest period _. -____.
in all subjects.
-
Radial
pulse rates were measured
in some
~__
From the Institute for Metaholir~ Research, Highland-Alameda County Ho.ypital, Oakland, California Receioed for pul&ation June 23, 1965. PETER WOOD, PH.D.: Research Riochemist, Institute for Mctaholic Research, HighlandAhmeda Corm@ Hospifaf, Oakland, Calif. CUENTER SCHLIERF, M.D.: Reseurch Fellow, I&it&e for Metabolic Research, Highland-Alumeda County Eloupital, Oakland, Calif. LAURANCE KINSELL, M.D.: Director, In&it& for Metabolic Research, Ifighland-Alameda County Hospital, Oakland, Calif. 1095
1096
WOOD,
Table I.-Pulse
Subject NO.
Rate and Plasma Lipid Levels during Exercise and Rest
Time of Determination*
Pulse Rate (beats/min.)
Plasma Triglyceride (mg./lOO ml.)
SCHLIERF
AND
KIKSELL
in 2 Male Subjects _ Pl83IXX3 Phospholipid (mg./lOO ml.)
Plasma Total Cholesterol (mg./lOO ml.)
1
i;; Cc)
50 180 64
50 52 32
244 269 261
232 256 250
68 2
i;‘, (c)
192 72
31 30 24
173 184 182
152 158 152
“(a)
resting,
before
exercise;
(1,)
immediately
after
vigorous
exercise
for $5 minutes;
(c) after further exercise followed by rest for 30 minutes. subjects. Adipose tissue samples needle biopsy from the buttock.* Blood was taken into EDTA separated within one hour. Lipids
were extracted
were obtained tubes
from plasma
which aliquots
(in 3 subjects were
chilled
without
delay
only)
on the same day, by
immediately
and
by the procedure
the
plasma
of Folch
et al.;!’ water containing tion. FFA were isolated
0.04 per cent (a/v) of calcium chloride was used as wash solufrom the extracts by thin-layer chromatography on plates coated Merck A.G.. Darmstadt, Germany) 250 p in thickness. and contain-
with Silica Gel G (E. ing a small quantity of 2’.7’-dichlorfluorescein. The solvent system was petroleum ether ( b.p. 30-60” )-diethyl ether-acetic acid ( 70: 30: 1 v/v/v/ ) ; separated lipids were visualized
under ultraviolet light, and the FFA area was accurately delineated. scraped off and eluted with diethyl ether from a small column. Recovery of FFA by this procedure was checked by addition to the plasma of tracer amounts of C 14-oleic or tritiated pahnitic acid. and was always better than 97 per cent. The absence of lipids other than FFA was confirmed by thin-layer chromatography. In this procedure. plasma lactic acid does not interfere. The total FFA content of the extracts was measured by 3 procedures: ester-linkage determination by the hlichaels method;rrr spot-size measurement on thin-layer chromatograms;il and treatment with Cr4- c1’. . omethane.rz The weight percentages of oleic and palmitic LIL acids in the total FFA were determined in duplicate. after methylation with diazomethane. by a gas-liquid chromatographic procedure previously described.rs Total adipose tissue lipids were extracted by the Folch procedure>rr transmethylated by heating with 2 per cent (v/v) sulfuric acid in methanol. and the methyl esters analyzed by gas-liquid chromatography.r:r Plasma triglycerides.10 phospholipidsrJ and total cholesterolra were determined for the male subjects. RESULTS
Radial pulse rates and plasma triglyceride, phospholipid and total cholesterol levels are shown in table 1 for the 2 male subjects who exercised by running. Plasma triglyceride levels, initially low after fasting, fell to still lower levels after exercise followed by rest. Plasma phospholipid and total cholesterol levels changed little, though there was a small rise in each after vigorous exercise for 5 minutes. Levels of plasma total FFA and proportions of free oleic and palmitic acids are shown for all subjects in figure 1, together with oleic and palmitic acid proportions found in adipose tissue lipids in 3 subjects. The 3 procedures used for determination of total FFA 1evels gave closely similar results. Agreement
OLEIC
AND
PALMITIC
ACID
LEVELS
DURING
VIGOROUS
1097
EXERCISE
+x@q
16:O
16:O l6:O
12 R
E
R
R’
4
E
R’
R
E
R’
R
E
R’
Fig. L-Changes in plasma FFA in subjects while exercising by running (1 and 2) or by pedalling a stationary bicycle (3 and 4), and after subsequent rest. Open circles: proportion of plasma free oleic acid (18:l) and palmitic acid (16:0:1 in the total FFA. Closed circles: proportions of oleic and palmitic acid in the adipose lipids of the subject. R, resting before exercise; E, immediately after vigorous exercise for 5 minutes; R’, after flIrther exercise followed lnr rest for 30 minutes. between uent
duplicate
fatty
determinations
acids
in FFA
and
by gas-liquid
in adipose
chromatography
tissue
was
good;
of constit-
mean
results
are
shown in the figure. In all subjects
acute
exercise
produced
a fall in total
FFA
by a rise in level after rest. In each case the concentration total
FFA
palmitic
had fallen
by the end of the acute
acid had risen.
The
cise and rest for 30 minutes and in the 3 subjects the 2 acids adipose
found
elevated
total
exercise
FFA
was accompanied
where
adipose
at this stage
tissue
period,
by a reversal had
followed
while
level seen after
was examined
in plasma
level,
of oleic acid in thr that
further
of these
of
exer-
changes,
the proportions
approached
those
present
asscciated
with
the
of in
tissue fatty acids. DISCUSSION
Design
Procedure.
of
labeled
fatty
radiochemical complex tion,
purity
avoided,
serial
sampling
of blood
muscular
In these
circumstances
poses represents
demand
the possibility
entirely
required
to reduce
use
regarding that
the
physiologically.li
in tracer
plasma
temporarily fate of FFA
that during
total
exceed
it is very probable
the major
,the evidence
tissue is minimal.
and
uncertainty
studies
of the
albumin In addi-
is difficult
to
exercise.
was used
making
instance,
does not beha\-e
during very vigorous exercise
difficulties
for
of the tracers,“.“’
in vitro
Vigorous
reviewed
In this study
were
formed
the
achieve
acids
supply
levels
from
that use by muscle
leaving
severe
FFA
the plasma.
esercise
uptake
rapidly
adipose for energy
by
tissue. pur-
Have1 et al.’ have
by liver and adipose
1098
WOOD, SCHLIERF AND XINSELL
During the final rest period, total plasma FFA rose to a high level, due almost certainly to rapid influx of FFA from adipose tissue at a time when muscular demand had suddenly decreased. At this stage some indication should be obtained of the nature of the fatty acid mixture being made available from adipose tissue. Special care was taken to ensure the accuracy of measurements of total FFA levels, and to check the purity and completeness of recovery of the total FFA fraction isolated for examination by gas-liquid chromatography. Site of Blood Sampling. Although sampling of arterial blood is ideal, it is difficult to achieve during very vigorous exercise. We feel that the changes of total FFA level seen in our venous blood samples reflect changes in arterial blood, since the changes we have found are similar to those seen in exercise studies where arterial blood has been examined.lsl’ Any addition of FFA from forearm adipose tissue would tend to elevate the venous FFA level above that in the arterial plasma, and as will be discussed below, would probably tend to raise the proportion of free oleic acid in the total FFA above that in the arterial plasma. Thus the changes seen in venous plasma immediately following exercise (sampling point E; fig. 1) would in all probability have been of somewhat greater degree had arterial plasma been examined. Change of 0leic:Palmitic Acid Ra.tio in FFA &ring Exercise. Our finding that the oleic:palmitic acid proportion in plasma FFA falls when the total level falls during acute exercise, and rises when the total FFA level rises during subsequent rest, is consistent with observations of Rothlin et al.‘!’ The latter workers used human subjects and anesthetized dogs; elevations of plasma FFA level were induced by norepinephrine infusions, and reductions of FFA level by infusion of glucose and insulin. It was noted that at high arterial FFA levels the composition of the FFA mixture in man approached that reported for depot fat by Hirsch et aLZn It seems probable to us that the changes seen when the total FFA level falls during vigorous exercise are the result of a preferential uptake of oleic rather than palmitic acid by the muscles, resulting in a greater fractional turnover rate for oleic acid compared with palmitic acid. This mechanism is in accord with several other observations. Rothlin and Ring” have found that the human heart extracts a consistently higher percentage of free oleic acid than of any other FFA present in arterial plasma. Imaichi et al.“l noted that during prolonged fasting in man the proportion of oleic acid in plasma FFA and triglycerides remained lower, and the proportion of palmitic acid rcmained higher, than was the case in the adipose tissue, for a given subject; this might suggest that oleic acid has a shorter turnover time than palmitic acid during fasting. Ono and Fredrickson 4 have found in anesthetized dogs that the rate of disappearance from the plasma of (2”‘-oleic acid bound to albumin was greater than that of simultaneously injected tritiated palmitic acid. Have1 et a1.l6 infused tritiated palmitic acid and C?j-oleic acid simultaneously into human subjects during rest and moderate exercise. While no large differences were found in rates of fractional turnover of these acids during exercise, small differences were probably not easily detectable by their pro-
OLEIC AND PALMITIC
ACID LEVELS
DURING
VIGOROUS
1099
EXERCISE
cedure. From measurement of espired C1”O, in subjects infused with either Cl”-oleic or C14-palmitic acid during exercise, these authors obtained some indication that oleate may be oxidized preferentially. Rothlin et al.” have considered other possible explanations for the variation of FFA composition as a function of total plasma FFA concentration. The existence of a small fraction of total FFA which is tightly bound to albumin, and thus not readily exchangeable, might explain our results, (a) if this fraction is extracted from albumin by the Folch procedure, and (b) if the fraction is relatively rich in palmitic acid and poor in oleic acid. Evidence in favor of these possibilities is apparently lacking. A further possibility is that the FFA supplied from adipose tissue during acute exercise is similar in composition to the FFA mixture seen at the low l)lasma FFA levels at the end of the exercise period; but again there is apparently no evidence for this. Oleic acid is released at a higher rate than palmitic acid by dog adipose tissue;F’ elevation of FFA level by release from adipose tissue following norepinephrine infusion in man increases the relative contribution of oleic acid to the total plasma FFA;‘” and the FFA composition in the final resting stage of the present work, when adipose tissue FFA were stiI1 being reIeased at a high rate, strongly suggests that the FFA mixture released into the plasma resembles that of adipose tissue, i.e., is rich in oleic acid. Level of Other Plasmu Lipids Followin, 0 Exercise. The small increase ir. plasma total cholesterol and phospholipid values found after vigorous exercise for 5 minutes is consistent with a report that some hemoconcentration occurs in such conditions.‘? In addition, increased production of norepinephrine during exercise may have contributed to the increased concentration of these lipids.‘3 In contrast, reduction of initial fasting plasma triglyceride levels was observed in 2 subjects following exercise and rest. This suggests some direct use of plasma triglycerides for energy purposes, as discussed by Have1 et al.’ and by Nikkilg and Konttinen.“’ Holloszy et al.?” have recently shown a substantial reduction of moderately elevated plasma triglyceride levels in middleaged men during a long-term program of vigorous exercise. Con&.sion.s. In summary, we interpret the changes in plasma FFA composition during vigorous exercise to indicate that oleic acid has a greater fractional turnover rate than has palmitic acid. Under our experimental conditions, removal by working muscle is probably the major factor responsible for this behavior. The results also suggest some direct use of plasma triglyceride for energy purposes during exercise. ACKNOWLEDGMENT This Products
study
was
supported
in part
by
PHS
Research
Grant
HE
00955
and
The
Corn
Company.
REFERENCES 1. Havel, R. J., Naimark. A., and Borchgrevink, C. F.: Turnover rate and oxidation of free fatty acids of blood plasma in man during exercise. J. Clin.
Invest.
42:1054,
1963.
2. Fredrickson, II. S., and Gordon, R. S., Jr.: The metabolism of albuminbound Cl+-labeled unesterified fatty acids in normal human subjects. J. Clin, Invest. 37; 1504, 1958.
1100
WOOD,
3. Nestel, P. J., Bezman, A., and Havel, R. J.: Metabolism of palmitate and linoleate in intact dogs. Amer. J. Physiol. 203:914, 1962. 4. Ono, K., and Fredrickson, D. S.: The metabolism of Cl*-labeled ci.s and tram isomers of octadecenoic and octadecadienoic acids. J. Biol. Chem. 239:2482, 1964. 5. Rothlin, M. E., and Bing, R. J.: Extraction and release of individual free fatty acicls by the heart and fat depots. J. Clin. Invest. 40:1380, 1961. 6. Ballarcl, F. B., Danforth, W. H., Naegle, S., and Bing, R. J.: Myocardial metabolism of fatty acids. J. Clin. Invest. 7. Harris,
39:717, 1960. P.. Chlouverakis.
C.,
Gloster,
J.> and
Jones, J. H.: Arterio-venous in the composition of differences
plasma regions
free fatty acids in various of the body. Clin. Sci. 22:
113. 1962. 8. Hirsch, J.: Adipose Tissue as an Organ. Springfield, Ill., Charles C Thomas, p. 81. 9. Folch, J., Lees, M., and Sloane-Stanley, C. H.: A simple method for the isolation and purification of total lipids Chem. 10. hlichaels.
from animal tissues. J. Biol. 226:497, 1957. C. D.: A method for the de-
termination of glycerides and free fatty acids. Metabolism 11: 833, 1962. 11. Schlirrf, G., and Wood, P.: Quantitative determination of plasma free fatty acids and triglycerides by thinlayer chromatography. J. Lipid Res. 6:317, 1965. 12. Wood, P. D. S., and Sodhi, H. S.: A method for determination of plasma free fatty acids. Proc. Sot. Exp. Biol. (N. Y.) 118:590, 1965. 13. Wood, P., Imaichi, K., Knowles, J., h/lichaels, G., and Kinsell, L.: The lipid composition of human plasma chylomicrons. J. Lipid Res. 5:225, 1964. 14. Youngburg, G. E., and Youngburg, M. V.: Phosphorus metabolism. I. A system ot blood phosphorus analysis.
J. Lab.
Clin.
SCHLIERF
Med.
AND KINSELL
16:158,
1930.
15. Technicon Autoanalyzer Methodology for Determination of Total Cholesterol. Technicon Laboratory File 24a. 16. Havel,
R. J., Carlson, L. A., Ekelund, and Holmgren, A.: Turnover L.. rate and oxidation of different free fatty acids in man during exercise. J. Appl. Physiol. 19:613, 1964.
17. Bates, M. W., and Olson, R. E.: Turnover rates of nonesterified fatty acids in the postabsorptive 19:230, 1960.
dog.
Fed.
Proc.
18. Friedberg, S. J., Sher, P. B., Bogdonoff, hl. D., and Estes, E. H., Jr.: The dynamics of plasma free fatty acid metabolism during exercise. J. Lipid Res. 4:34, 1963. 19. Rothlin, M. E., Rothlin, C. B., and Wendt, V. E.: Free fatty acid concentration and composition in arterial blood. Amer. J. Physiol. 203:306. 1962. 20.
Hirsch, J., F:Trqubar, J. H., Peterson, M. L., Studies of adipose Amer. J. Clin. Nutr.
21.
Imaichi, K., Michaels, G. D., Holton, L. W.: Plasma lipid S., and Kinsell, fatty acids during fasting. Amer. J. Clin. Nutr. 13:226, 1963.
22.
Bert, R. V., and Stead, E. A.: Demonstration that in normal man no reserves of blood are mobilized by exercise, epinephrine, and hemorrhage. 1941.
Amer.
J.
Med.
W., Ahrens, E. and Stoffel. W.: tissue in man. 8:499, 1960.
Sci.
201:655,
23.
Grasso, S., Michaels, G. D., and Kinsell, L. W.: Effects of norepinephrine upon lipid metabolism in a patient with biliary cirrhosis. Metabolism 13: 1333, 1964.
24.
NikkilL, E. A., and Konttinen, A.: Effect of physical activity on postprandial levels of fats in serum. Lancet 1: 1154, 1962.
25.
Holloszy, J. O., Skinner, J. S., Toro, G., and Cureton, T. K.: Effects of a six month program of endurance exercise on the serum lipids of middle-aged men.
Amer.
J. Cardiol.
14:753,
1964.