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Some Kinetic Characteristics of Rumen Short-Chain Fatty Acids as Measured by the Isotope Dilution Method
1716
JOURNAL
OF DIIRY
nitrogen, such as those present in the free amino acids of 20-ml blood samples. A. A. L. J. C. J.
KOSHAROV ~ BENSADOUN ~ H. BREUER, JR. 3 K. LOOSLI J. M O R R I S T. REID
Animal Science Department and U. S. Plant, Soil and Nutrition Laboratory USDA Cornell University, Ithaca, New York
(2) (3) (4)
(5)
and J. O. LEGG
Soil and Water Conservation Research Division, USDA Beltsville, Maryland
(6)
(7) Present address: Physiology and Biochemistry, Moscow University, USSR. 2 Present address: Poultry Science Department, Cornell University, Ithaca, New York. Present address: Animal Science Department, Texas A & M University, College Station, Texas. References
(1) Bensadoun, A., and Reid, J. T. 1962. Estimation of Rate of Portal Blood Flow in Ru-
(8) (9)
SCIENCE
minants: Effect of Feeding, Fasting, and Anesthesia. J. Dairy Sci., 45: 540. Dude, G. D., and Handler, P. 1958. Kinetics of Ammonia Metabolism in Vivo J. Biol. Chem., 232: 303. Hoshino, S., Sarumaru, I(., and Morimoto, K. 1966. Ammonia Anabo]ism ix Ruminants. J. Dairy Sci., 49:1523. Land, H., and Virtanen, A. I. 1959. Ammonium Salts as Nitrogen Source in the Synthesis of Protein by the Ruminant. Acta Chem. Scand., 13:489. Liebster, J., Kopoldova, K., and Dobiasova, M. 1961. A Simplified Method for Separating Amino Acid Mixtures from Protein Hydrolysates on One Ion Exchange Column During Preparation. Nature, 191: 1198. Loosli, J. K., Williams, H. H., Thomas, W. F., Harris, F. It., and Maynard, L. A. 1949. Synthesis of Amino Acids in the Rumen. Science, 110: 144. Morris, C. J., and Thompson, J. F. 1962. The Isolation and Characterization of -LGlutamyl-L-tyrosine and -L-Glutamyl-Lphenylalanine from Soybeans. Biochem., 1 : 706. Stein, W. H., and Moore, S. 1954. The Free Amino Acids of Human Blood Plasma. J. Biol. Chem., 211:915. Thompson, J. F., Morris, C. J., and Gering, R. K. 1959. Purification of Plant Amino Acids for Paper Chromatography. Anal. Biochem., 31: 1028.
Some Kinetic Characteristics of Rumen Short-Chain Fatty Acids as Measured by the Isotope Dilution Method Quantitation of rumen fermentation products has interested investigators, because this would make available important data to evaluate ruminant diets. Volatile short-chain organic acids may provide ruminants with 40-70% of their energy needs (11, 14). To accurately assess this contribution, one must be able to determine the amounts of each acid produced. Barcroft and his coinvestigators (2) demonstrated that short-chain organic acids produced in the rumen were absorbed. Since the original discovery, numerous investigators have attempted with some success to quantitate the absorbed acids (1, 6, 13, 14). Most of the techniques used to study absorption have been disappearance measurements or changes in coecentration of acids in the blood draining the rumen. Some workers have measured production rates of rumen acids by in vivo (7) am! by in vitro (8) methods. Gray et el. (7) have used isotope dilution to study production rates of rumen organic acids and to overcome some difficulties of other techniques used. This technique does have the advantage that it allows in vivo measurement. I t does not, however, measure production alone, but the total flux of the acid in the tureen pool. J. DAII~Y SCIENCE VOL. 50, NO. 10
This includes production, metabolism, and interconversion to other acids. I f the total flux of rumen fatty acids through the respective pools can be determined, their contribution to the energy needs of the animal can be further assessed. The use of isotope dilution technique may be valuable for quantAtaring the flux through rumen fatty acid pools. To measure the fatty acid pool size and turnover rate by isotope dilution, certain assumptions must be made. The assumptions are: that the pool size remains constant, and that the processes influencing changes in the specific activity are simple dilution. Pool size can be determined if the rumen volume and the fatty acid concentration remain constant. Rumen volume may fluctuate among a given average, at least under normal water intake and salivation situations (7). I t is well documented that immediately after consumption of feed there is a rise in the concentration of short-chain fatty acids in the tureen, and then a decrease for 2-4 hr (10). The concentration of acids present reflects the intensity of production and absorption rates. I f production and absorption rates are constant, the concentration will be relatively stable; thus, the pool size should remain constant if the total
TECHNICAL
Z
~>
31. '
1717
NOTES
•
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2
3
4
5
6
7
8
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I0
TIME
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FIG. 1. Logarithmic (base e) regression of rumen acetate specific activity ( c / g a C ) introduction of acetate 2H.
>I--l.-L) <1:
7.0
m
after intraruminal
.~o PROPIONATE o
5.3
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-~
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,0
(HRS)
FIG. 2. Logarithmic (base e) regression of rumen propionate specific activity ruminal introduction of propionate-2-1~C. J.
(/zc/gaC) a f t e r intra-
DAIRY SCIENCE VOL. 50, NO. l 0
1718
J O U R N A L OF D A I R Y S C I E N C E
G . 9 ~
¥
BUTYRATE VVVV
•
< 5.7~ m_
V
0 t.~ G.
~
4.G
(1) 0
0
Y--820e-O'OO39X
...J
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TIME (HRS) FIG. 3. Logarithmic (base e) regression of rumen butyrate specific activity (l~c/gaC) after intraruminal introduction of butyrate-2-1~C. rumen volume does not change drastically. I£ a straight line results from a plot of the log of isotope concentration vs. time, then the data can be treated as a first-order process. Under these conditions one can derive certain constants that characterize the pool according to the following relations : dc/dt = --kc or c = coe - - k t k = rate constant of disappearance expressed as % per rain (turnover rate) c = concentration of isotope expressed as ~c/gaC at any time t co = concentration of isotope expressed as ,(¢c/gaC at zero time. Experimental
Procedure
I n three separate experiments acetate-SH, propionate-2-1~C, and butyrate-2-1'C were introduced into the tureen of a lactating Holstein cow. A t various time intervals after isotope introduction, tureen fluid samples were taken by aspiration through a sealed tureen cannula (9). Acetic, propionic, and butyric acids were separated by gas-liquid chromatography (9). A f t e r determining the quantity of each fatty acid by chromatography, each was assayed for radioactivity by liquid scintillation (9). Concentration of each fatty acid times estimated tureen volume. J, DAIRY SCIENCE ~OL. 50, No. 10
Results and Discussion
Semilog plots of acetate, propionate, and butyrate specific activities are shown in Figures 1-3. I n general, the specific activities of the injected f a t t y acid appeared to follow first order kinetics. There was considerable variation in the early samples. This was probably due to incomplete mixing of the radioactive material with rumen contents. Turnover rates, turnover times, and pool sizes calculated from the semilog plots are shown in Table 1. Independent estimations 1 of pool size indicated that these were 7.2 moles acetate, 2.3 moles propionate, and 1.3 moles of butyrate in the rumen at the time of these determinations. Pool sizes determined by isotope dilution were 7.58, 2.71, and 1.34 moles for acetate, propionate, and butyrate, respectively. Acetate and propionate leave the pool at comparable relative rates, while the relative butyrate flux is slightly faster (0.27, 0.30, and 0.39% per minute, respectively). The magnitude of these rates was not in agreement with the absorption or production rates reported by Pfander and Phillipson (13). They concluded that acetate, butyrate, and propionate were produced and absorbed in that order, with the absorption rate of acetate being 1.5 times greater than that of butyrate and 2.0 times greater than that of propionate. These corn-
TECtINICAL NOTES
1719
TABLE 1 Kinetic parameters of rumen fatty acid pools Acetate Turnover rate ~ ( %/min) Exchange rate b (mM/min) Pool size ¢ (moles)
0.30± 0.057 d 22.7 7.58
Propionate 0.27___ 0.039 7.3 2.71
Butyrate 0.39-----0.046 5.2 1.34
Turnover rate = exponential regression of specific activity of rumen fatty acid as time progressed. Regression line was calculated by method of least squares. b Exchange rate = pool size times turnover rate. Pool size = amount of isotope injected divided by specific activity at zero time, where specific activity = ttc/gaC and zero time activity is an extrapolated value. d Standard error of mean. parisons are not strictly valid, because the turnover rates reported here reflect total flux of the pool, i.e., production, absorption, removal to omasum, and metabolism. The results r e p o r t e d here are in agreement with those of B r o w n and Davis (4), using the same technique. These authors indicated that propionate exhibits a two-phase t u r n o v e r curve. Our data do not show such a curve (Fig. 2). The data herein also agree with results obtained by Cook and Ross (5) on a hay-fed calf. . H u n g a t e et al. (8), using an in vitro technique, reported f o r dairy cows 40, 12.8, and 10.5 moles p e r day, respectively, f o r acetate, propionate, and b u t y r a t e production. F r o m our exchange rate values it can be calculated that comparable rates would be 32.7, 10.5, and 7.5 moles per day. Brown and Davis (4), as well as others (7), r e p o r t their values as production rates. H o w ever, they are exchange rates or total flux of the rmnen fatty acid pool. The flux of this pool includes interconversion of f a t t y acids. Table 2 TABLE 2 Interconversion of volatile fatty acids Activity ratio a Isotope introduced Acetate-3K Propionate-14C Butyrate-2-14C
Acetate Proplonate Butyrate ...... 0.03 0.10
0.37 ...... 0.05
0.49 0.18 ......
e
f xdt a Activity ratio =
0
f ~r dt 0
where X = specific activity of product and ~r = specific activity of precursor. indicates there is considerable interconversion, especially of acetate to butyrate, while the reverse is similar but lower than that r e p o r t e d by B e r g m a n et al. (3), using the constant infusion technique in sheep. The data also agree in this respect with those of Cook and Ross (5). The t r a n s f e r o f tureen acetate to p r o p i o n a t e a p p e a r e d to be rather high in comparison to results of B e r g m a n et al. (3). This m a y have
been due to contamination from acetate in the isolation process. I n our procedure acetate is eluted from the chromatography column prior to p r o p i o n a t e (9). I f there had been any tailing of acetate from the column, it would have been collected in the propionate peak and, thus, contributed to the a p p a r e n t activity in propionate. This possibility would not seem to be important, since recoveries of standard radioactive f a t t y acids were 95% or greater (9). A more plausible explanation may be in use of acetate2H. Due to metabolism of tritiated acetate and exchange of -3H, it is likely there was ~H~O in the rumen. I n the isolation procedure a water peak is eluted p r i o r to the f a t t y acids. W a t e r elution was not at base line when the f a t t y acids were eluted. This may have contributed radioactivity to all acids collected. This source of contamination was not checked and, therefore, error f r o m ~I-I~O, if any, is not known. The data r e p o r t e d suggested that isotope dilution techniques may be useful in f u r t h e r evalu a t i n g the contribution of microbial metabolic end products to the energy needs of ruminants. The determination of total flux of rumen f a t t y acids has several advantages over measurement of absorption rates f r o m the rumen, which necessitate determination of portal vein blood flow which, in turn, involves extensive surgical manipulation (:14). Because of rumen epithelium metabolic activities (12), absorption rates do not yield an accurate picture of tureen fermentation products. F l u x determinations are relatively easy to do; sample acquisition and analysis procedures are well established (9). Steele et al. (15) have criticized single isotope injection in the isotope dilution procedure in f a v o r of continuous infusion of isotope. Kinetic parameters can be corrected f o r interconversion of f a t t y acids by continuous isotope infusion (3). However, i f single injections are used they should be accompanied by a marker substance such as ethylene glycol, to correct for tureen volume changes. K. L. KNOX 2, A. L. BLACK and M A X KLEIBER Department of Physiological Sciences University of California, Davis 2 Department of Animal Science, Colorado State University, Ft. Collins, Colorado. •]'.
DAIRY SOIEI~CE TC~OL. 50, No. 10
1720
J O U R N A L OF DAII~Y S C I E N C E
References
(1) Annison, E. F., and Lindsay~, D. B. 1962. The Measurement of Entry Rates of Propionate and of Butyrate in Sheep. J. Biochem., 85 : 474. (2) Barcroft, J., McAnally, R. A., and Phillipson, R. T. 1944. Absorption of Volatile Acids from the Alimentary Tract of the Sheep and Other Animals. J. Exptl. Biol., 20: 120. (3) Bergman, E. N., Reid, R. S., Murray, M. G., and Brockway, J. M. 1965. Interconversions and Production of Volatile F a t t y Acids in the Sheep Applying Infusion and Injection Methods. Nord. Vet. Med., 7:1001. (4) Brown, R. E., and Davis, C. L. 1962. Volatile F a t t y Acid Production in the Bovine Rumen. Federation Proc., 21: 396. (5) Cook, R. M., and Ross, R. H. 1964. The Turnover Rate of Rumen Acetate. J. Anim. Sci., 23: 601. (6) Gray, F. V. 1947. The Absorption of Volatile F a t t y Acids from the Rumen. J. Exptl. Biol., 24: 1. (7) Gra$, F. V., Jones, G. B., and Pilgrim, A. F. 1960. The Rates of Production of Volatile F a t t y Acids in Rumen. Australian J. Agr. Res., 11: 383. (8) Hungate, R. E., Mah, R. A., and Simesen, M. 1961. Rates of Production of Individual
(9)
(10)
(11) (12) (13) (14)
(15)
ASSOCIATION
Volatile F a t t y Acids in the Rumen of Lactating Cows. Appl. Microbiol., 9: 554. Knox, Kirvin Lee. 1964. The Metabolism of Isotopieally Labeled Volatile Fatty Acids and Barley Straw Introduced into the Rumen of Lactating Dairy Cows. Ph.D. thesis, University of California, Davis. Knox, K. L., and Ward, G. M. 1961. Rumen Concentration of Volatile F a t t y Acids as Affected by Feeding Frequency. J. Dairy Sci., 44: 1550. Marston, J. R. 1948. The Fermentation of Cellulose in Vitro by Organisms from the Rumen of Sheep. J. Biochem., 42: 564. Pennington, R. J. 1952. The Metabolism of Short-Chain Fatty Acids in the Sheep. J. Biochem., 51: 251. Pfander, W. H., and Phillipson, A. T. 1953. The Rate of Absorption of Acetic, Propionic, and N-Butyric Acids. J. Physiol., 122: 102. Sehambye, P. 1955. Experimental Estimation of the Portal Vein Blood Flow in Sheep. 2. Chronic Experiments in Cannulated Sheep Applying Infusion and Injection Methods. Nord. Vet. Meal., 7: 1001. Steele, R., Wall, J. S., DeBodo, R. C., and Altszuler, N. 1956. Measurement of Size and Turnover Rate of Body Glucose Pool by the Isotope Dilution Method. Am. J. Physiol., 187 : 15.
AFFAIRS
MEMORIAL Charles R. Gearhart 1893-1967 Charles R. Gearhart, P r o f e s s o r Emeritus of D a i r y Science at the Pennsylvania State University, died J u n e 3, 1967, in Manatee Memorial Hospital, Bradenton, Florida, at the age of 74. F u n e r a l services were held J u n e 7 at the Salem Church of Christ, Gilbert, Pennsylvania. A native of Effort, Pennsylvania, he graduated in 1920 f r o m the University of Missouri. A f t e r three years as extension dairy specialist at Kansas State College, he took a similar .position at the Pennsylvania State University in 1923. I n this capacity, he led the D a i r y H e r d I m p r o v e m e n t Association p r o g r a m until retirement in April, 1955. One of his many outstanding contributions to this p r o g r a m in Pennsylvania and the nation was his efforts to secure supervisors through the 1-W p r o g r a m during W o r l d W a r I I . Over 2,300 students
J . DAIRY SCIENCE VOL. 50, No. 10
attended short courses conducted by him to train supervisors. Following retirement f r o m the University, he served f o u r years as dairy advisor to the MinistlT of Ag~riculture in Turkey, working under the United States F o r e i g n Operations Administration. Since his r e t u r n from this assignment, Charles and Mrs. Gearhart have lived in Santa Maria, Florida. Besides h~s wife, Mable, he is survived by two sons: Gayle of Chevy Chase, MaiTland , and Gerald of t t i n g h a m , Massachusetts. Charles was an active member of the Association and in addition to work on various committees, served as Chairman of the Extension Section and two terms on the Board of Directors. I n 1954, he received the DeLaval Extension D a i r y m a n Award.
JOURNAL
OF DIIRY
nitrogen, such as those present in the free amino acids of 20-ml blood samples. A. A. L. J. C. J.
KOSHAROV ~ BENSADOUN ~ H. BREUER, JR. 3 K. LOOSLI J. M O R R I S T. REID
Animal Science Department and U. S. Plant, Soil and Nutrition Laboratory USDA Cornell University, Ithaca, New York
(2) (3) (4)
(5)
and J. O. LEGG
Soil and Water Conservation Research Division, USDA Beltsville, Maryland
(6)
(7) Present address: Physiology and Biochemistry, Moscow University, USSR. 2 Present address: Poultry Science Department, Cornell University, Ithaca, New York. Present address: Animal Science Department, Texas A & M University, College Station, Texas. References
(1) Bensadoun, A., and Reid, J. T. 1962. Estimation of Rate of Portal Blood Flow in Ru-
(8) (9)
SCIENCE
minants: Effect of Feeding, Fasting, and Anesthesia. J. Dairy Sci., 45: 540. Dude, G. D., and Handler, P. 1958. Kinetics of Ammonia Metabolism in Vivo J. Biol. Chem., 232: 303. Hoshino, S., Sarumaru, I(., and Morimoto, K. 1966. Ammonia Anabo]ism ix Ruminants. J. Dairy Sci., 49:1523. Land, H., and Virtanen, A. I. 1959. Ammonium Salts as Nitrogen Source in the Synthesis of Protein by the Ruminant. Acta Chem. Scand., 13:489. Liebster, J., Kopoldova, K., and Dobiasova, M. 1961. A Simplified Method for Separating Amino Acid Mixtures from Protein Hydrolysates on One Ion Exchange Column During Preparation. Nature, 191: 1198. Loosli, J. K., Williams, H. H., Thomas, W. F., Harris, F. It., and Maynard, L. A. 1949. Synthesis of Amino Acids in the Rumen. Science, 110: 144. Morris, C. J., and Thompson, J. F. 1962. The Isolation and Characterization of -LGlutamyl-L-tyrosine and -L-Glutamyl-Lphenylalanine from Soybeans. Biochem., 1 : 706. Stein, W. H., and Moore, S. 1954. The Free Amino Acids of Human Blood Plasma. J. Biol. Chem., 211:915. Thompson, J. F., Morris, C. J., and Gering, R. K. 1959. Purification of Plant Amino Acids for Paper Chromatography. Anal. Biochem., 31: 1028.
Some Kinetic Characteristics of Rumen Short-Chain Fatty Acids as Measured by the Isotope Dilution Method Quantitation of rumen fermentation products has interested investigators, because this would make available important data to evaluate ruminant diets. Volatile short-chain organic acids may provide ruminants with 40-70% of their energy needs (11, 14). To accurately assess this contribution, one must be able to determine the amounts of each acid produced. Barcroft and his coinvestigators (2) demonstrated that short-chain organic acids produced in the rumen were absorbed. Since the original discovery, numerous investigators have attempted with some success to quantitate the absorbed acids (1, 6, 13, 14). Most of the techniques used to study absorption have been disappearance measurements or changes in coecentration of acids in the blood draining the rumen. Some workers have measured production rates of rumen acids by in vivo (7) am! by in vitro (8) methods. Gray et el. (7) have used isotope dilution to study production rates of rumen organic acids and to overcome some difficulties of other techniques used. This technique does have the advantage that it allows in vivo measurement. I t does not, however, measure production alone, but the total flux of the acid in the tureen pool. J. DAII~Y SCIENCE VOL. 50, NO. 10
This includes production, metabolism, and interconversion to other acids. I f the total flux of rumen fatty acids through the respective pools can be determined, their contribution to the energy needs of the animal can be further assessed. The use of isotope dilution technique may be valuable for quantAtaring the flux through rumen fatty acid pools. To measure the fatty acid pool size and turnover rate by isotope dilution, certain assumptions must be made. The assumptions are: that the pool size remains constant, and that the processes influencing changes in the specific activity are simple dilution. Pool size can be determined if the rumen volume and the fatty acid concentration remain constant. Rumen volume may fluctuate among a given average, at least under normal water intake and salivation situations (7). I t is well documented that immediately after consumption of feed there is a rise in the concentration of short-chain fatty acids in the tureen, and then a decrease for 2-4 hr (10). The concentration of acids present reflects the intensity of production and absorption rates. I f production and absorption rates are constant, the concentration will be relatively stable; thus, the pool size should remain constant if the total
TECHNICAL
Z
~>
31. '
1717
NOTES
•
6.9
H-
o<~ u_
•.
o~" o %
ACETATE
•
"''"
6.2
o u_l n q)
~D J
Y=lO40e_O.O030 x ,5.3
O
I
I
I
I
I
I
I
I
I
I
I
2
3
4
5
6
7
8
9
I0
TIME
CHR$)
FIG. 1. Logarithmic (base e) regression of rumen acetate specific activity ( c / g a C ) introduction of acetate 2H.
>I--l.-L) <1:
7.0
m
after intraruminal
.~o PROPIONATE o
5.3
oooo
o
(D b_ (P LIJ 1:1_ U]
o
o
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o
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o
i
~
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~
L
-~
~
I
9
I
,0
(HRS)
FIG. 2. Logarithmic (base e) regression of rumen propionate specific activity ruminal introduction of propionate-2-1~C. J.
(/zc/gaC) a f t e r intra-
DAIRY SCIENCE VOL. 50, NO. l 0
1718
J O U R N A L OF D A I R Y S C I E N C E
G . 9 ~
¥
BUTYRATE VVVV
•
< 5.7~ m_
V
0 t.~ G.
~
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0
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TIME (HRS) FIG. 3. Logarithmic (base e) regression of rumen butyrate specific activity (l~c/gaC) after intraruminal introduction of butyrate-2-1~C. rumen volume does not change drastically. I£ a straight line results from a plot of the log of isotope concentration vs. time, then the data can be treated as a first-order process. Under these conditions one can derive certain constants that characterize the pool according to the following relations : dc/dt = --kc or c = coe - - k t k = rate constant of disappearance expressed as % per rain (turnover rate) c = concentration of isotope expressed as ~c/gaC at any time t co = concentration of isotope expressed as ,(¢c/gaC at zero time. Experimental
Procedure
I n three separate experiments acetate-SH, propionate-2-1~C, and butyrate-2-1'C were introduced into the tureen of a lactating Holstein cow. A t various time intervals after isotope introduction, tureen fluid samples were taken by aspiration through a sealed tureen cannula (9). Acetic, propionic, and butyric acids were separated by gas-liquid chromatography (9). A f t e r determining the quantity of each fatty acid by chromatography, each was assayed for radioactivity by liquid scintillation (9). Concentration of each fatty acid times estimated tureen volume. J, DAIRY SCIENCE ~OL. 50, No. 10
Results and Discussion
Semilog plots of acetate, propionate, and butyrate specific activities are shown in Figures 1-3. I n general, the specific activities of the injected f a t t y acid appeared to follow first order kinetics. There was considerable variation in the early samples. This was probably due to incomplete mixing of the radioactive material with rumen contents. Turnover rates, turnover times, and pool sizes calculated from the semilog plots are shown in Table 1. Independent estimations 1 of pool size indicated that these were 7.2 moles acetate, 2.3 moles propionate, and 1.3 moles of butyrate in the rumen at the time of these determinations. Pool sizes determined by isotope dilution were 7.58, 2.71, and 1.34 moles for acetate, propionate, and butyrate, respectively. Acetate and propionate leave the pool at comparable relative rates, while the relative butyrate flux is slightly faster (0.27, 0.30, and 0.39% per minute, respectively). The magnitude of these rates was not in agreement with the absorption or production rates reported by Pfander and Phillipson (13). They concluded that acetate, butyrate, and propionate were produced and absorbed in that order, with the absorption rate of acetate being 1.5 times greater than that of butyrate and 2.0 times greater than that of propionate. These corn-
TECtINICAL NOTES
1719
TABLE 1 Kinetic parameters of rumen fatty acid pools Acetate Turnover rate ~ ( %/min) Exchange rate b (mM/min) Pool size ¢ (moles)
0.30± 0.057 d 22.7 7.58
Propionate 0.27___ 0.039 7.3 2.71
Butyrate 0.39-----0.046 5.2 1.34
Turnover rate = exponential regression of specific activity of rumen fatty acid as time progressed. Regression line was calculated by method of least squares. b Exchange rate = pool size times turnover rate. Pool size = amount of isotope injected divided by specific activity at zero time, where specific activity = ttc/gaC and zero time activity is an extrapolated value. d Standard error of mean. parisons are not strictly valid, because the turnover rates reported here reflect total flux of the pool, i.e., production, absorption, removal to omasum, and metabolism. The results r e p o r t e d here are in agreement with those of B r o w n and Davis (4), using the same technique. These authors indicated that propionate exhibits a two-phase t u r n o v e r curve. Our data do not show such a curve (Fig. 2). The data herein also agree with results obtained by Cook and Ross (5) on a hay-fed calf. . H u n g a t e et al. (8), using an in vitro technique, reported f o r dairy cows 40, 12.8, and 10.5 moles p e r day, respectively, f o r acetate, propionate, and b u t y r a t e production. F r o m our exchange rate values it can be calculated that comparable rates would be 32.7, 10.5, and 7.5 moles per day. Brown and Davis (4), as well as others (7), r e p o r t their values as production rates. H o w ever, they are exchange rates or total flux of the rmnen fatty acid pool. The flux of this pool includes interconversion of f a t t y acids. Table 2 TABLE 2 Interconversion of volatile fatty acids Activity ratio a Isotope introduced Acetate-3K Propionate-14C Butyrate-2-14C
Acetate Proplonate Butyrate ...... 0.03 0.10
0.37 ...... 0.05
0.49 0.18 ......
e
f xdt a Activity ratio =
0
f ~r dt 0
where X = specific activity of product and ~r = specific activity of precursor. indicates there is considerable interconversion, especially of acetate to butyrate, while the reverse is similar but lower than that r e p o r t e d by B e r g m a n et al. (3), using the constant infusion technique in sheep. The data also agree in this respect with those of Cook and Ross (5). The t r a n s f e r o f tureen acetate to p r o p i o n a t e a p p e a r e d to be rather high in comparison to results of B e r g m a n et al. (3). This m a y have
been due to contamination from acetate in the isolation process. I n our procedure acetate is eluted from the chromatography column prior to p r o p i o n a t e (9). I f there had been any tailing of acetate from the column, it would have been collected in the propionate peak and, thus, contributed to the a p p a r e n t activity in propionate. This possibility would not seem to be important, since recoveries of standard radioactive f a t t y acids were 95% or greater (9). A more plausible explanation may be in use of acetate2H. Due to metabolism of tritiated acetate and exchange of -3H, it is likely there was ~H~O in the rumen. I n the isolation procedure a water peak is eluted p r i o r to the f a t t y acids. W a t e r elution was not at base line when the f a t t y acids were eluted. This may have contributed radioactivity to all acids collected. This source of contamination was not checked and, therefore, error f r o m ~I-I~O, if any, is not known. The data r e p o r t e d suggested that isotope dilution techniques may be useful in f u r t h e r evalu a t i n g the contribution of microbial metabolic end products to the energy needs of ruminants. The determination of total flux of rumen f a t t y acids has several advantages over measurement of absorption rates f r o m the rumen, which necessitate determination of portal vein blood flow which, in turn, involves extensive surgical manipulation (:14). Because of rumen epithelium metabolic activities (12), absorption rates do not yield an accurate picture of tureen fermentation products. F l u x determinations are relatively easy to do; sample acquisition and analysis procedures are well established (9). Steele et al. (15) have criticized single isotope injection in the isotope dilution procedure in f a v o r of continuous infusion of isotope. Kinetic parameters can be corrected f o r interconversion of f a t t y acids by continuous isotope infusion (3). However, i f single injections are used they should be accompanied by a marker substance such as ethylene glycol, to correct for tureen volume changes. K. L. KNOX 2, A. L. BLACK and M A X KLEIBER Department of Physiological Sciences University of California, Davis 2 Department of Animal Science, Colorado State University, Ft. Collins, Colorado. •]'.
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J O U R N A L OF DAII~Y S C I E N C E
References
(1) Annison, E. F., and Lindsay~, D. B. 1962. The Measurement of Entry Rates of Propionate and of Butyrate in Sheep. J. Biochem., 85 : 474. (2) Barcroft, J., McAnally, R. A., and Phillipson, R. T. 1944. Absorption of Volatile Acids from the Alimentary Tract of the Sheep and Other Animals. J. Exptl. Biol., 20: 120. (3) Bergman, E. N., Reid, R. S., Murray, M. G., and Brockway, J. M. 1965. Interconversions and Production of Volatile F a t t y Acids in the Sheep Applying Infusion and Injection Methods. Nord. Vet. Med., 7:1001. (4) Brown, R. E., and Davis, C. L. 1962. Volatile F a t t y Acid Production in the Bovine Rumen. Federation Proc., 21: 396. (5) Cook, R. M., and Ross, R. H. 1964. The Turnover Rate of Rumen Acetate. J. Anim. Sci., 23: 601. (6) Gray, F. V. 1947. The Absorption of Volatile F a t t y Acids from the Rumen. J. Exptl. Biol., 24: 1. (7) Gra$, F. V., Jones, G. B., and Pilgrim, A. F. 1960. The Rates of Production of Volatile F a t t y Acids in Rumen. Australian J. Agr. Res., 11: 383. (8) Hungate, R. E., Mah, R. A., and Simesen, M. 1961. Rates of Production of Individual
(9)
(10)
(11) (12) (13) (14)
(15)
ASSOCIATION
Volatile F a t t y Acids in the Rumen of Lactating Cows. Appl. Microbiol., 9: 554. Knox, Kirvin Lee. 1964. The Metabolism of Isotopieally Labeled Volatile Fatty Acids and Barley Straw Introduced into the Rumen of Lactating Dairy Cows. Ph.D. thesis, University of California, Davis. Knox, K. L., and Ward, G. M. 1961. Rumen Concentration of Volatile F a t t y Acids as Affected by Feeding Frequency. J. Dairy Sci., 44: 1550. Marston, J. R. 1948. The Fermentation of Cellulose in Vitro by Organisms from the Rumen of Sheep. J. Biochem., 42: 564. Pennington, R. J. 1952. The Metabolism of Short-Chain Fatty Acids in the Sheep. J. Biochem., 51: 251. Pfander, W. H., and Phillipson, A. T. 1953. The Rate of Absorption of Acetic, Propionic, and N-Butyric Acids. J. Physiol., 122: 102. Sehambye, P. 1955. Experimental Estimation of the Portal Vein Blood Flow in Sheep. 2. Chronic Experiments in Cannulated Sheep Applying Infusion and Injection Methods. Nord. Vet. Meal., 7: 1001. Steele, R., Wall, J. S., DeBodo, R. C., and Altszuler, N. 1956. Measurement of Size and Turnover Rate of Body Glucose Pool by the Isotope Dilution Method. Am. J. Physiol., 187 : 15.
AFFAIRS
MEMORIAL Charles R. Gearhart 1893-1967 Charles R. Gearhart, P r o f e s s o r Emeritus of D a i r y Science at the Pennsylvania State University, died J u n e 3, 1967, in Manatee Memorial Hospital, Bradenton, Florida, at the age of 74. F u n e r a l services were held J u n e 7 at the Salem Church of Christ, Gilbert, Pennsylvania. A native of Effort, Pennsylvania, he graduated in 1920 f r o m the University of Missouri. A f t e r three years as extension dairy specialist at Kansas State College, he took a similar .position at the Pennsylvania State University in 1923. I n this capacity, he led the D a i r y H e r d I m p r o v e m e n t Association p r o g r a m until retirement in April, 1955. One of his many outstanding contributions to this p r o g r a m in Pennsylvania and the nation was his efforts to secure supervisors through the 1-W p r o g r a m during W o r l d W a r I I . Over 2,300 students
J . DAIRY SCIENCE VOL. 50, No. 10
attended short courses conducted by him to train supervisors. Following retirement f r o m the University, he served f o u r years as dairy advisor to the MinistlT of Ag~riculture in Turkey, working under the United States F o r e i g n Operations Administration. Since his r e t u r n from this assignment, Charles and Mrs. Gearhart have lived in Santa Maria, Florida. Besides h~s wife, Mable, he is survived by two sons: Gayle of Chevy Chase, MaiTland , and Gerald of t t i n g h a m , Massachusetts. Charles was an active member of the Association and in addition to work on various committees, served as Chairman of the Extension Section and two terms on the Board of Directors. I n 1954, he received the DeLaval Extension D a i r y m a n Award.