The Absorption of Volatile Fatty Acids from the Rumen: II. The Influence of pH on Absorption

Danielli and his colleagues (Danielli et al. 1945), however, concluded that the relative rates of absorption were reversed when the medium was slightly alkaline, and experimental data were presented which, it was claimed, proved that the relative velocities of absorption from a mixture of fatty acids at pH 7·5.fell in the series acetic > propionic > butyric. These latter experimental data were unconvincing. The experiments conducted in this laboratory which are described here lend no support to the claim, or to the general theory in which Danielli and his colleagues suggested that diffusion from acid solutions may proceed through both the intercellular substance and the limiting membranes of the cells themselves, and that only the former pathway is traversed when the solution is slightly alkaline. There is QO satisfactory evidence to support the contention that fatty acids diffuse through the rumen wall from a slightly alkaline medium.

As in the earlier studies (Gray, 1947 a), pectin was used as a marker to indicate changes in composition of the aqueous solutions of fatty acids introduced into the rumen. In the course of these experiments it has been proved that pectin does not pass through the rumen wall and that it is not modified when it is introduced in solution into the empty washed rumen.

The concentrations of the fatty acids were estimated by methods (Gray, 19476) which, for the amounts used in these experiments, seem to be more precise than those based on partition chromatography.

For the most part the marker-ratio technique was employed with sheep in which permanent rumen fistulae had been established previously. This allowed observations to be made on the essentially intact rumen in its physiological state. The procedure first used by Barcroft, McAnally & Phillipson (1944) and adopted by Danielli et al. (1945) was, however, repeated in some experiments. In these the rumen and its associated organs were isolated from the rest of the alimentary tract by tying off the abomasum just below the omasum, taking care to exclude the epiploic vessels. The oesophagus was occluded in the cervical region. The rumen in this state will be referred to as the ‘isolated’ rumen.

Four experiments were conducted to observe changes in the composition of solutions containing a mixture of sodium acetate and sodium propionate at pH 6-5 and at 8-5 when introduced into an empty, ‘intact’ rumen. Phosphate was introduced in one solution at each reaction, for there was some suggestion from earlier experiments that the rates of absorption might be influenced by its presence.

The procedure for emptying and washing out the rumen and the methods for the estimation of the fatty acids were those employed previously (Gray, 1947a). Between 3 and 41. of solution were introduced into the empty, washed rumen. Samples were withdrawn immediately and after 6 hr.

From the results set out in Table 1 it is clear that there were significant changes in the composition of solutions introduced at acid reactions (pH 6·5), indicating, as in previous experiments, the preferential absorption of propionic acid. There was, however, no evidence of preferential absorption from the alkaline solutions (pH 8.5), since there were no changes in the proportions of the two acids. Nevertheless, these findings do not preclude the absorption of fatty acids from the alkaline medium, for there would be no such changes if the rates of absorption of the two acids were proportional to their concentrations.

Experiments to test this point were undertaken. In these, the absolute amounts of the acids absorbed under acid and alkaline conditions were determined.

If fatty acids are absorbed from an alkaline medium, the fact could be demonstrated by changes in the concentrations of the acids in relation to the concentration of an unabsorbed marker (Gray, 1947 a). But to measure the actual amounts of each acid which pass through the rumen wall, further information is required. The method used previously to determine the relative rates of absorption of two acids involved the addition of pectin to the solution to act as marker, the assumption being made that pectin could not be absorbed through the rumen wall. It was pointed out then that the absolute amounts of the acids absorbed could be determined only if estimations were made of the quantities of each acid which passed into the abomasum during the experimental period, and of the amounts of each which remained in the rumert at the end of it.

In consequence, experiments were carried out (a) to determine whether pectin is or is not absorbed through the rumen wall, and (b) to measure the absorption of acetic and propionic acids from solutions of different reactions when introduced into the rumen of an unanaesthetized sheep in a normal physiological state.

A sheep which had been starved for 3 days was anaesthetized with nembutal and the rumen was isolated according to the procedure described by Barcroft et al. (1944). A canula was tied into the rumen, the contents siphoned out and the organs thoroughly washed with several lots of warm water. A solution of pectin was introduced into the empty, washed rumen and the animal was maintained under light nembutal anaesthesia for a further period of $412$ hr. The whole contents were then very carefully collected and the organs washed clean, particular attention being paid to the reticulum and omasum.

Pectin was determined in the original and residual solutions by the method of Nanji & Norman (1928). From the data shown in Table 2, which indicate a recovery of 97 % of the pectin introduced into the rumen, it is evident that little or no pectin was absorbed. It may be claimed therefore that pectin is a suitable marker for reference when measuring the absorption of other compounds from the rumen.

Five trials were carried out to determine the absolute amounts of acetic and prppionic acids absorbed at different pH levels, both in the presence and in the absence of inorganic phosphate.

Trials 5–8. Mixtures of acetic acid, propionic acid, and pectin, with or without added phosphate, were introduced into the ‘intact’, empty rumen in distinctly alkaline and acid solutions (pH 10·6 and pH 4·9). In each trial a 400 ml. sample was withdrawn after 2–3 hr. and a 500 ml. sample after 6 hr. The remaining contents of the rumen, together with a small amount of water used to complete the removal, were then collected. Throughout each of these trials the pH of the rumen contents was determined with a glass electrode in samples withdrawn by syringe at half-hourly intervals.

Trial 9. A mixture of acetic acid, propionic acid and pectin at pH 7·5 was introduced into the rumen. A single sample was withdrawn after 5 hr. and the residual contents collected immediately afterwards. The pH of the contents was measured at the end of the experimental period.

In all five trials determinations of the acids and of pectin were made on the original solutions used, and on each sample. The pectin concentrations in the residues were also measured. The analytical data are set out in Table 3 and from these the amounts of each acid absorbéd from the rumen and the amounts that passed on to the abomasum may be assessed. The calculations are shown below in full for trial 5 ; those for the other trials follow the same procedure.

In these calculations the assumption is made that the amount of each acid passing into the abomasum during the experimental period can be estimated from a knowledge of the average concentrations of the constituents in the rumen, and of the amount of pectin lost’from the rumen. This is equivalent to assuming that the passage of material from the rumen to the abomasum takes place at regular intervals. The calculations for trial 5 are as follows :

Thus the partition of the acids was as follows:

Therefore

Acetic acid absorbed = 74 ml. N acid = 15 % of the original acetic acid.

Propionic acid absorbed = 246 ml. N acid = 47% of the original propionic acid.

An extra significant figure has been retained in the data and throughout the calculations in order to minimize the accumulation of arithmetical errors. Results for all five trials are summarized in Table 4.

Changes in the reaction of the rumen contents during the first four of these trials are plotted in Fig. 1. In trial 9 the reaction was not significantly altered (pH 7.4) at the end of the experimental period, and may be considered to have remained constant throughout the trial.

It may be concluded from trials 5 and 7 that while the reaction of the solution in the rumen was changing from about pH 5 to 7, absorption of both acids took place rapidly. The presence of inorganic phosphate had no significant effect on the total quantities of the acids absorbed, and it may therefore be concluded that the apparent effect of phosphate noticed in the earlier experiments (Gray, 1947 a) was, in fact, an effect due to the different pH levels of the solutions used in those trials where phosphate was included.

In trials 6 and 8 little or no absorption of acid took place while the reaction of the solution in the rumen was changing from about pH 10-5 to 7, and in trial 9 no significant absorption took place while the reaction of the solution in the rumen remained practically constant at pH 7-5.

As the claim that the fatty acids are absorbed through the rumen wall at pH 7.5 was not supported by the evidence in the trials just described, it was decided to check the results obtained when using the indirect method of Danielli et al. by a direct measurement of the recovery of acetic acid introduced into an ‘isolated’ rumen. Both methods were employed at the same time in each of two trials so that the experimental conditions were identical for each procedure. Acetic acid was used alone since, according to Danielli et al., it is absorbed more rapidly than its higher homologues from an alkaline solution.

The operative procedure for isolating the rumen from the rest of the alimentary tract has already been referred to. In these trials solutions of sodium acetate at reactions between pH 7.5 and 8.0 were introduced into the rumen for periods of 3 hr., after which samples were withdrawn for analysis. In one trial sodium acetate solution was then added to the rumen contents ; in the other acetic acid was used instead. In each case 5 min. were allowed for mixing, and this was assisted by massaging the abdomen of the animal. Then another sample was withdrawn so that the change in acid concentration could be measured. Immediately afterwards, the whole contents of the isolated organs, together with washings used to complete their removal, were collected.

The original solutions, the residues, and the two intermediate samples were steam-distilled according to the usual procedure and the distillate titrated with standard alkali to determine the concentrations of acid present. Data from these trials are given in Table 5.

The loss of acetic acid from the rumen is calculated directly from these measurements and also indirectly by the procedure used by Danielli et al.

(1) By direct measurement

(2) By indirect measurement (procedure of Danielli et al.)

Three significant figures have been quoted throughout the data from these experiments, and used in the calculations from them, but no claim is made for the order of accuracy that this implies. The amount of acetic acid computed from the direct measurement probably involves an error of the order of 1 % ; but the result from the indirect method is undoubtedly less accurate. Further reference is made to this point in the discussion.

The experiments that have been described here prove that, while there is rapid absorption of volatile acids from solutions introduced into the rumen at acid reactions, there is little or no absorption when the reaction of the rumen contents is alkaline.

This latter finding is in conflict with the conclusion of Danielli et al. (1945). The direct measurement of the absorption of acetic acid from an ‘isolated’ rumen, carried out at the same time as the indirect measurement employed by these workers, has shown that their procedure may give rise to misleading results. One reason for this inaccuracy may be inherent, not in the method itself, but in certain limitations which were arbitrarily imposed in its application. The accuracy of the calculation of the volume of the rumen contents at the end of the experimental period depends upon the accuracy with which the difference in concentrations of acid can be measured before and after the addition of a known amount of acid. A small error in the estimation of either of these concentrations will lead to a very much larger error in the computed difference between them so long as that difference is small in relation to the original and final concentrations. This larger error will then be reflected in the volume calculated, and therefore in the estimation of the acid present at the end of the experimental period. In the three experiments described by Danielli et al., the proportions of acid absorbed in 2 hr. were claimed to be approximately 30, 7 and 16%. The change in acid concentration at the end of the first of the experiments, in which the most significant amount of absorption was calculated, was from 19.7 to 21.8^* ml. N acid/100 ml. If these measurements were each subject to an error of only ±0·5%, then the amount of acid calculated to remain in the rumen at the end of the experimental period would have been subject to an error of approximately ±10%. The smaller absorption (7%) claimed in their second experiment, in which a much greater change was brought about in the acid concentration, lends support to the view that this source of error may have complicated the interpretation of their first experiment. The acid concentrations were determined in 2 ml. samples by the method of McAnally (1944), the accuracy of which was not stated.

A second possible source of error lies in the fact that the acid added at the end of the experimental period must be completely mixed with the whole contents of the reticulum and omasum, as well as with that of the rumen; the former two organs are not always in free communication with the rumen, and while massage of the abdomen will promote a more rapid mixing of the contents, some uncertainty of its completeness remains. If mixing between these organs were incomplete, the estimation of the volume by this means would tend to give low results and the amount of absorption calculated from this would therefore be too great.

The exact separation of the abomasum from the omasum in the isolation of the rumen is not easily achieved. In some cases during the present work it was found that a small pocket of the abomasum had been left above the ligature. If such a pocket were extensive enough, it might offer an avenue for the absorption of significant amounts of acid which would therefore be wrongly attributed to passage of the acid through the rumen wall.

It was claimed by Danielli et al. that not only were the fatty acids absorbed in the alkaline range, but also that the order of the rates of absorption was the reverse of that obtaining under acid conditions. Their data, however, show only very small changes in the composition of three-acid mixtures placed in the rumen and these do not all indicate the order of absorption claimed. The partition chromatographic method of Elsden (1946) was used for these analyses, and for this Elsden quotes figures showing a standard deviation of between 2 and 3 % from the mean recovery in each of the three acids determined in such a mixture. The changes in the proportions of the acids which were present in the alkaline mixtures used by Danielli et al. do not therefore appear to be significant.

Two minor points of interest arise incidentally out of the present trials. In the first place a rapid change of reaction took place in the rumen when a solution was introduced at pH 5 or at 10. Both solutions were brought approximately to neutrality within 3 hr. when no inorganic phosphate had been included, and within about 5 hr. when phosphate was present. Danielli et al. (1945) have pointed out that the change from acid to neutral reaction must, in part, be brought about by the passage of free acid through the rumen wall. In the older view the change would have been ascribed entirely to the advent of alkaline saliva into the rumen. The second point to be noted is that, while it has been claimed that only small quantities of fatty acids are to be found in the abomasum (Phillipson & McAnally, 1942), and that significant quantities of such acids do not appear in the blood draining the abomasum (Barcroft et al. 1944), yet the trials described in part (2) (b) demonstrate the passage of considerable amounts of acid into the abomasum under the conditions in which the experiments were made.

1. Mixtures of acetic and propionic acids introduced into the rumen at pH 8·5 showed no significant changes in the relative proportions of the acids in a period of 6 hr.

2. Pectin was shown to remain unchanged and unabsorbed in the ‘isolated’ rumen. By using it as a marker the course taken by fatty acids introduced into the empty rumen was followed. The acids found their way from the rumen, in part by absorption through the rumen wall and in part by passing on to the abomasum.

3. Absorption of both acetic and propionic acids took place readily at acid reactions. Between 10 and 14% of the acetic acid and about 47% of the propionic acid introduced was absorbed within 6 hr. The amounts absorbed were not altered by the inclusion of inorganic phosphate in the mixture.

   Absorption did not occur to any significant extent from similar solutions introduced at reactions ⩾ pH 7.5.

4. In two experiments acetic acid was not absorbed from an ‘isolated’ rumen when introduced as a slightly alkaline (about pH 7.5) solution of sodium acetate. Recovery after 3 hr. was measured directly and found to be 96%. In the same experiments the indirect method of Danielli et al. (1945) yielded varying results (89 and 109% recovery).

5. It is concluded that while rapid absorption of fatty acids from the rumen takes place at acid reactions, there is no absorption at all from alkaline solutions.

The author is greatly indebted to Mr I. G. Jarrett who carried out all the necessary operative procedures, and to Mr H. R. Marston, Chief of the Division, for his continued interest and advice.