BUTYRATE AS A PRECURSOR OF MILK CONSTITUENTS IN THE INTACT DAIRY COW*

BY MAX KLEIBER, ARTHUR L. BLACK, MARY A. BROWN, JACK LUICK,t CLAUDE F. BAXTER,1

AND BERT M. TOLBERT

(From the College of Agriculture and the Radiation Laboratory, University of California, Davis and Berkeley, California)

(Received for publication, February 1, 1954)

Butyric acid is a major product of feed fermentation in the intestinal tract of various animals, especially the rumen of cattle and sheep. This has been known for 70 years (1).

Elsden, Hitchcock, Marshall, and Phillipson (2) noted that the rumen and reticulum of an ox contained an average of 329 gm. of volatile acids (calculated as acetic acid), and that the mixture of volatile fatty acids in the intestines of cattle, sheep, horses, pigs, and rabbits had a similar com- position, namely an average of 67 per cent acetic, 19 per cent propionic, and 14 per cent butyric acid. (Th ese figures presumably represent molar percentages.)

The relative rate of absorption of fatty acids from the rumen depends on the acidity of the rumen content (3).

Kiddle, Marshall, and Phillipson (4) suggest that a greater part of the butyrate than of the other fatty acids is catabolized in the rumen wall, and Pennington (5) demonstrated that tissue slices of rumen epithelium convert butyrate to acetoacetate. Butyrate is thus a most interesting substance in the nutrition of cattle and sheep, its importance in biochemistry, how- ever, not being limited to ruminants. It may play a role as a link between fat and carbohydrate metabolism. Its function as such in rats may, at least in part, be independent of acetate (6), and a considerable part of its contribution to the synthesis of animal carbohydrate by-passes the car- bonate pool (7).

We injected C14-labeled butyrate as a single dose into the jugular vein of lactating cows. We then measured the Cl4 concentration in the expired COz and in lactose, casein, milk albumin, and butter fat as a function of time after injection of the tracer. These measurements permitted esti- mates of the relative rates at which butyrate provides carbon for respira- tory COZ and for the synthesis of the organic milk constituents.

* This investigation was supported by the United States Atomic Energy Commis- sion.

t Predoctoral Research Fellow, United States Atomic Energy Commission.

$ Research Fellow of the National Institutes of Health, United States Public Health Service.

239

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240 BUTYRATE AS PRECURSOR OF MILK

Method

Cows-Table I summarizes the characteristics of the three Jersey cows and one Holstein cow used in the experiments described here.

Tracer-The sodium butyrate-l-Cl4 was prepared by carbonation of propylmagnesium bromide with Cl402 at -20” (8). Yields were approxi- mately 95 per cent, based on the barium carbonate-C4 used.

The sodium butyrate-2-Cl4 was synthesized by condensation of potassium cyanide with propyl-l-Cl4 iodide, followed by hydrolysis of the nitrile and steam distillation of the butyric acid. Propyl-l-Cl4 iodide was prepared by high pressure hydrogenation of cadmium-nickel propionate and iodination of the resulting alcohol with phosphorus and iodine (9).

TABLE I

Data on Experiments with Cows

Trial No.

I II

III

IV

Body weight

kg.

905 414

963 547 1100 554

941 450

Food intake per day

H&Y ChCtXl-

trates

kg. kg.

2.2 2.3 7.0 3.0

6.3 2.9

Hilk yield ,,er day

kg.

6.4 6.8

22.4

16.3

Plasma volume

lilers

20.2 31.9 20.3

-

-

Relative injected

dose of C’4

PC. per “;eG&;

12.1 7.3

9.0 11.1

I

-

-

Position

“‘iG? xltyrate

1-W 1dp

2-w

2-P

I 1

_

-

hltyrate Injected

711M

9.7 7.2 4.4

4.4

Injection and Dose-The use of plastic tubes for the intravenous injec- tions performed in these trials has been described before (10).

The doses in microcuries per kilo of body weight and the amount of butyr- ate injected in millimoles are reported in Table I. From the data given by McClymont ((11) p. 95, Table I), we may estimate that 1 liter of ar- terial blood of a cow contains on the average 1.46 mM of acetate, 0.04 mM of propionate, 0.04 mM of butyrate, and 0.03 mM of higher fatty acids.

For an estimated average of 32 liters of blood, a cow thus contains in its blood about 1.3 mM of butyrate. According to this estimate, which ne- glects the difference between peripheral and intestinal blood, we injected about 3 to 8 times as much butyrate as is normally present in the blood of the cow. Considerable variation in the rate of production of fatty acids in the rumen and their appearance in the blood stream is to be expected. Our injection of butyrate, therefore, presumably did not produce abnormal conditions.

Respiration Trials and Cl4 Measurements-The methods of measuring

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KLEIBER, BLACK, BROWN, LUICK, BAXTER, TOLBERT 241

COZ production and radioactivity in the respiratory COZ have been de- scribed in two earlier papers (12, 13).

Cl4 in Respiratory CO2 of Cow after intravenous inject ion of Ci4 labeled Carbonate and Butyrate

A Butyrate Trial I, I-Cl4 l n ll II, ” 0 ” tl m, 2-c’4

x ” II ‘1p, II

- Carbonate Trial Ip

P = spec. Cl4 activity pc / mole CO2

in /.LC inject./Kg.Wt.

Time after injection, hours FIQ. 1

Results

In Fig. 1 the standardized specific activity of Cl4 in the expired CO2 ((microcuries of Cl4 per mole of COz)/( microcuries of Cl4 injected per kilo of body weight)) is plotted against time after the injection of the labeled butyrate. The curve for the specific activity after injection of carbonate- Cl4 is shown for comparison. As observed earlier with labeled acetate (14)

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242 BUTYRATE AS PRECURSOR OF MILK

and propionate (15), the butyrate-l-Cl4 leads to a higher specific activity in respiratory COz than does butyrate-2-C14. This result indicates that, from butyrate as well as from the other fatty acids, the carboxyl carbon is transferred to CO2 faster than the carbon in the 2 position of the molecule.

The maximal specific activity in COz from butyrate-l-Cl4 is reached in 10 minutes after injection, that from butyrate-2-Cl4 in 30 minutes. The curves for specific activity in COz from butyrate follow those from acetate more closely than those from propionate.

TABLE II

Influence of Position of Label in Butyrate on Mean Cl4 Level in Respiratory COZ As Function of Time

Time after injection (1)

hrs.

1

2 3

6 12 24

Mean 04 level in respiratory COz’

Position 1-C” Position 2-C”

Trial I Trial II Trial III Trial IV

27.6 28.1 10.8 12.8 2.36

22.0 22.5 9.8 11.4 2.10 18.1 18.6 8.6 9.7 2.00 12.0 11.4 6.3 6.2 1.87

6.7 6.2 3.6 3.5 1.82 3.5 3.2 2.0 1.9 1.72

Ratio of mean levels

(l-cq (2-W)

1 L * Cl4 levels are calculated as -

s t 0 pdt, where p is expressed as

pc. per mole COZ

pc. injected per kilo body weight

Table II shows that the mean level of Cl4 in the respiratory CO% for the 1st hour is over twice as high after injection of butyrate-l-Cl4 as it is after injection of butyrate-2-C14. With increasing time after injection, the two levels tend to become equal. The mean CL4 levels in CO2 after injection of butyrate-l-Cl4 are between the levels after acetate-l-Cl4 and those after propionate-1-C4.

The mean levels of Cl4 in COz after injection of butyrate-2-Cl4 are strik- ingly similar to those after acetate-2-Cl4 (14) and propionate-2-Cl4 (15).

The relative rate of loss of the carboxyl carbon of butyrate appears to be between that of acetate and of propionate, while the relative rate of oxida- tion at the Ct position, however, seems to be nearly the same for acetate, propionate, and butyrate. The injected acetate amounted to only 16 per cent of the normal acetate in the blood; the injected propionate and butyr-

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KLEIBER, BLACK, BROWN, LUICK, BAXTER, TOLBERT 243

ate, however, were from 3 to 8 times as great as the amount normally present in the blood. We do not have enough information to decide

TABLE III

Relative Cl4 Retention in Cow’s Body after Intravenous Injection of 04-Labeled

Butyrate and Carbonate

Tiie after injection

hrs.

0

1

2 3

Butyrate-1-C” Butyrate-Z-C’” Carbonate-C’4

Trial I Trial II Trial III Trial IV Trial IV

)er cent per cent )er cent per cent per cent

100 100 100 100 100 70 68 86 86 42

52 52 75 76 28 41 40 66 70 22

TABLE IV

Specijic Cl4 Activity in Milk Constituents

The results refer to microcuries per gm. atom of C in milk constituents per micro-

curies injected per kilo of body weight.

Milk constituents Period No.

Lactose

Casein

Fat

1 2

3 4 5

1 2 3

4 5 1

2 3 4

5

-

-

Time after injection

hrs. A

3 1.77 10 3.07

22 0.32

34 0.07 46 0.06

3 2.02

10 1.07 22 0.65

34 0.24 46 0.04

3 0.32

10 0.60 22 0.23 34 0.06

46 0.03

-

Cl4 in milk constituents from

Butyrate-1-C’”

Trial I Trial II

A

1.33

0.31 0.09 0.04

0.52 0.97 0.40

0.14 0.09 0.05

0.51 0.31 0.09

0.05

-

-

Butyrate-2-C”

Trial III Trial IV

A

3.76

0.20 0.09 0.06 2.64

1.36 0.82

0.29 0.10 0.29

0.49 0.13 0.04 0.02

A

2.55 2.24 0.12

0.09 0.05 2.14

1.63 0.60 0.19

0.18 0.07 0.31

0.29 0.09 0.04

whether or not this difference in the ratio between the injected dose and the average normal pool content affects the biokinetic behavior of the fatty acids.

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244 RUTYRATE AS PRECURSOR OF MILK

Our cows produced 102, 137, 157, and 108 liters of CO2 (O’, 1 atmos- phere, dry) per hour in Trials I to IV respectively. On the basis of these metabolic rates, the Cl4 retention during the 3 hours after injection has been calculated. The results are given in Table III. The relative reten- tion of carbon atom 2 of the butyrate is similar to that of carbon atom 2 of acetate (14) and propionate (15). The carboxyl of butyrate is retained for a longer time than that of propionate but not for as long a time as that of acetate.

TABLE V

Transfer of Cl4 from Injected Acetate (A) and Butyrate (B) to Organic Milk

Constituents

Position of 04 in injected acetate or butyrate

Per cent injected 04 in organic Per cent distribution of total 04 in organic milk constituents in 46 hrs. milk constituents among

Total Per kilo milk Lactose Casein Milk fat

A B A B A B A B A B

1-W 16 6 0.7 0.4 10 37 8 31 79 18 2-w 19 22 1.1 0.8 21 38 16 38 , 60 19

TABLE VI

Partition in Transfer of Cl4 from Butyrate to Milk Constituents

Transfer via COz in per cent of total transfer

Trial No. Position Comparison after from butyrate to injection

Lactose Casein Fat

hrs. I l-C’4 46 27 14 8

II l-04 46 * 21 7 III 2-C'4 48 * 6 6

IV 2.04 48 15 6 5

* Lactose sample lost.

Table IV shows the specific Cl4 activities in lactose, casein, and milk fat obtained during the 2 days following the injection of the labeled butyrate. As in previous trials (13-15), there is a definite delay in the labeling of milk fat in comparison to the labeling of lactose and casein. The high specific activity in lactose and casein in comparison to that of milk fat is surprising.

Table V summarizes the distribution of the Cl4 from butyrate in lactose, casein, and milk fat for a period of 46 hours following the injection. Table VI, calculated as described in our paper on acetate ((14) p. 377), indicates that only a fraction of the transfer of carbon from butyrate to organic milk constituents involves the carbonate pool.

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KLEIBER, BLBCK, BROWN, LUICK, BAXTER, TOLBERT 245

DISCUSSION

Our data indicate that, in the lactating cow, butyrate in contrast to acetate is more closely related to the synthesis of lactose than to that of milk fat.

This result is in line with that of Buchanan, Hastings, and Nesbett (6), who fed I-CF-labeled acetate, propionate, and butyrate mixed with glucose to fasted rats, and concluded that propionic and butyric acids are converted to liver glycogen but that acetate is not. The latter statement is derived from the observation that the labeling of glycogen after acetate feeding could be entirely accounted for by transfer of the carboxyl carbon to sugar via carbonate.

Lifson et al. (7) fed CF-labeled acetate and butyrate to rats, and then determined the Cl3 excess in the liver glycogen and its degradation prod- ucts. In contrast to Buchanan et al. (6), Lifson and his coworkers con- cluded that, at least, the a-carbon of acetate can enter glycogen by a path- way other than COz fixation. The labeling pattern of glycogen after feeding butyrate-Cl3 was consistent with /3 oxidation of butyrate and trans- fer of carbon from butyrate to glycogen via acetate.

Our trials indicate that a considerable part of the transfer of C from butyrate as well as from acetate (14) in either position 1 or 2 by-passes the carbonate pool.

The comparison of the Cl4 distribution among organic milk constituents after injection of labeled acetate and butyrate (Table V) reveals a radical difference between the metabolic behavior of butyrate and that of acetate, which supports the idea that a considerable part of the carbon transfer from butyrate to lactose by-passes the acetate pool.

These observations, combined with earlier results of other workers, favor the hypothesis that the transfer of carbon from butyrate to lactose is part of a net synthesis of lactose rather than the result of a redistribution process of C atoms in a closed Krebs cycle. Blixenkrone-Moller (16) observed an increase of glycogen in cat liver perfused with butyrate. That was un- doubtedly a net synthesis. Since he did not use a tracer, he had no direct evidence that the carbon in the glycogen formed came from the butyrate. But the combination of Blixenkrone-MMler’s results with those of Bu- chanan et al. (6) and Lifson et al. (7) on rats, of those of Schambye, Wood, and Popjak (17) on lactating rabbits, and of our own on lactating cows with labeled carbon forms a rather strong case for the hypothesis that buty- rate participates in a net carbohydrate synthesis.

Blixenkrone-Moller’s scheme of a transfer of butyrate to carbohydrate via oxidation to succinate does not fit the labeling pattern obtained by J,ifson et nl. (7); namely, an excess labeling of the 3 and 4 carbons of glu- cose after feeding of butyrate-3-C4. Also, the more recent scheme of

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246 BUTYRATE AS PRECURSOR OF MILK

acetone formation from butyrate by oxidation at the 3 position and de- carboxylation (Plaut and Lardy (18)) and the subsequent direct oxidation of the acetone to pyruvate (Sakami and Lafaye (19)) would lead to the prediction that injected or fed butyrate-3-Cl4 labels glucose in the 2 and 5 rather than in the 3 and 4 positions.

Our observation, that the labeling pattern of milk constituents after the injection of C-l- and C-2-labeled butyrate differs markedly from that observed after the injection of labeled acetate, does not preclude the ,B oxidation of butyrate if interpreted on the basis of the “two species theory” of Cz intermediate metabolites of Crandall et al. (20). Such an explana- tion, however, would indicate that the carboxyl type C2 unit is more di- rectly involved in carbohydrate than in fat synthesis.

A trial is under way in our laboratory to measure directly the involve- ment of the acetate and the blood glucose pools in the transfer of carbon from butyrate to organic milk constituents.

SUMMARY

1. Intact normal dairy cows were injected with sodium butyrate-l-Cl4 and -2-C’* in single doses ranging from 7 to 12 PC. of Cl4 per kilo of body weight.

2. The specific activity of Cl4 in the expired COP reached a maximum 10 minutes after injection of butyrate-l-U4 and 30 minutes after injection of butyrate-2-CY4.

3. The relative rate of oxidation of the carboxyl carbon of butyrate ap- pears to be between that of acetate and that of propionate, and the rela- tive oxidation rate of the carbon in the 2 position of the molecule nearly the same for butyrate as for acetate and propionate.

4. About one-third of the transfer of carboxyl carbon of butyrate to lactose apparently passes through the carbonate pool, and about one-fifth of the corresponding transfer to casein and less than one-tenth of that to milk fat pass via carbonate. The carbonate pool is by-passed by over 80 per cent of the transfer of carbon atom 2 in butyrate to lactose and by over 90 per cent of that to casein and milk fat.

5. About 6 per cent of the l-Cl4 and 22 per cent of the 2-C’* of the in- jected butyrate appeared in organic milk constituents.

6. More of the l-C14, as well as of the 2-CY4, of the injected butyrate appeared in lactose and in casein than appeared in milk fat.

7. This pattern of labeling milk constituents differs from the correspond- ing one after injection of C14-labeled acetate.

8. In contrast to acetate, butyrate behaved in the lactating cow in a more glyconeogenic than lipogenic manner.

9. Our results are consistent with the participation of but#yrate in net synthesis of carbohydrate.

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KLEIBER, BLACK, BROWN, LUICK, BAXTER, TOLBERT 247

We are pleased to acknowledge the valuable help of Patricia Adams of the Radiation Laboratory at Berkeley, who synthesized the C14-labeled butyrate for us, and the faithful assistance of Mr. T. Chernikoff of the Department of Animal Husbandry, who analyzed all the gas samples for our Cl4 trials.

BIBLIOGRAPHY

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M. TolbertBrown, Jack Luick, Claude F. Baxter and Bert

Max Kleiber, Arthur L. Black, Mary A.DAIRY COW

MILK CONSTITUENTS IN THE INTACT BUTYRATE AS A PRECURSOR OF

1954, 210:239-247.J. Biol. Chem. 

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