JOURNAL OF THE SCIENTIFIC AGRICULTURAL SOCIETY OF FINLAND
Maataloustieteellinen A ikakauskirja

69

Vol. 52: 69- 74, 1980

Incorporation of radioactive carbon from [ l4C] alanine in the
rumen of a cow given feed containing urea
as the sole source of nitrogen

Eeva-Liisa Syväoja and Matti Kreula
Biochemical Research Institute, Kalevankatu 56 B, 00180 Helsinki 18, Finland

Abstract. The utilisation of exogenic amino acid in a cow given feed containing urea
and ammonium salts as the sole sources of nitrogen was studied by means of intraruminal
administration of [l4C]alanine. The labelling of the trichloracetic acid-precipitated
bacterial cell mass, the main volatile fatty acids (acetic, propionic and butyric acid)
and a number of isolated amino acids after 1,3, 8 and 26 h was determined. The rumen
micro-organisms rapidly incorporated the [l4 C]alanine into their cellular constituents.
After the above-mentioned times the microbial cell mass was found to contain 47.4,
49.7, 70.0 and 80.0 % of the total activity of the rumen contents. Although the carbon
skeleton of alanine can be used for the formation of many amino acids the rumen bacteria
studied were shown to utilise only small amounts of this amino acid in their synthesis
of Asp, Glu, Tyr and Phe. The combined label present in these 4 amino acids was 1.5,
1.9, 2.9 and 5.5 % of the total activity of the rumen fluid at the stated times. A con-
siderable proportion of the [ l4C]alanine was degraded to volatile fatty acids: label
present in acetic, propionic and butyric acid totalled 40.8, 32.3, 23.0 and 5.0 % of that
in the rumen fluid. Of these, acetic acid had the strongest labelling after 1 and 3 h, and
propionic acid the weakest at all stages.

Introduction

The concentration of extracellular amino acids in the rumen is usually
relatively low (Wright and Hungate 1967). Feed protein is degraded very
rapidly in the rumen to peptides and free amino acids, which undergo exten-
sive metabolism before passing further down the digestive tract (McDonald
1948, 1952; El-Shazly 1952, Lewis 1955 and Annison 1956). Lewis and
Emery (1962) studied the degradation of amino acids in rumen fluid in vitro
and found that they form three groups according to their rate of deamination.
Ser, Cys, Asp, Thr and Arg are deaminated almost completely, followed by
Phe, Glu, Lys and Cys-Cys. Those with the lowest deamination rate are Try,
Met, Ala, Vai, He, Orn, His, Gly, Pro, Pro(OH) and <r-aminovaleric acid.
Mangan (1972) found, in in vivo studies, that the half-life of casein in the
cow’s rumen fell in the range 5.6—21.5 minutes, in degradation to peptides,
free amino acids and ammonia. He further observed that there was a complete
loss of amino acids from casein hydrolysate, Cys and Arg in the rumen in 15

https://www.c-info.fi/en/info/?token=AinnGaPCWjSlUrRt.0A9DQMIL_ZU69IBQ2WFEjQ.s7Zcj0n3Ty-4EPQBpOODy6Dl3E_RvKCOaTocKQJtHMn5_5HoRZYS6hO9GOaw9FexS7FeuwiiSALZpJIs7BbKx288E8mqhaJAuoKGMXioegXZPqyDIWDBBFYFGlJz0GHUu3ygcSiK85PEDISMoepB--62lDX9M50ku1fw47Tnh-PhmZ_yRmnwsJe3hVNUutR4oNADP6ax


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minutes, and of Asp, Ser, Thr, Gly, Ala, Met, Tyr, Phe and His in 30 minutes.
Also Val, Leu. He and Lys disappeared quickly, but their reappearance was
regarded as evidence of the synthesis of these four amino acids in the rumen.

Ammonia is the principal source of nitrogen in microbial protein synthesis
(Bryant and Robinson 1962). Not all rumen bacteria can use exogenic amino
acids, and those which can will grow better when ammonia is available in
addition. The most probable explanation for the apparent failure of amino
acids to compete with or inhibit the utilisation of ammonia is a low activity,
or even absence, of mechanisms for the transport of amino acids through the
membrane of the bacterial cell (Allison 1969).

The significance of these biosynthetic reactions can readily be demonstrated
in cows which produce milk and meat from protein-free feed, in which urea and
ammonium salts form the sole source of nitrogen and purified carbohydrates
the sole energy source (Virtanen 1963, 1966, 1971). The level of free amino
acids in the rumina of these cows is very low (Mäkinen 1972), but the rate of
synthesis of microbial protein is greater than that in cows given normal,
proteinaceous feed (Virtanen 1966). In the work described below the utilisa-
tion of carbon from a single exogenous amino acid, namely [l4C]alanine, by
the rumen bacteria of a cow on protein-free feed was examined. The work is
one of a series of studies on the metabolism by such cows of many 14 C-labelled
compounds (Kreula and Rauramaa 1979).

Materials and Methods

A single dose of 237 /iCi L-[U- 14C]alanine (Radiochemical Centre, Amersham)
was given in 360 g of 0.1% unlabelled alanine to the cow, via a rumen fistula
and mixing thoroughly with the rumen contents. The feed of the cow was
composed mainly of purified carbohydrates, urea and ammonium salts being
the only source of nitrogen (Virtanen 1963, 1966, 1971).

Rumen samples were taken just before the administration of the labelled
alanine and 1,3, 8 and 26 h afterwards. No drinking water was given for 3 h
after the alanine dose. The rumen liquor was strained immediately through
glass-fibre cloth. The bacteria were isolated by centrifugation, washed several
times with phosphate buffer containing inactive alanine and finally twice
with distilled water; they were then suspended homogenously in water and
lyophilised. Protein was precipitated from rumen liquor and lyophilised
bacteria with 10 % trichloracetic acid (TCA). The precipitates were washed
4 times with the same reagent and the TCA was then removed with ether.
Asp, Glu, Tyr and Phe were isolated by means of Dowex IXB anion exchange
resin from the bacterial protein, following our published method (Syväoja
and Kreula 1979).

Volatile fatty acid contents in rumen liquor were determined as described
by Cottyn and Boucque (1968). The main components, namely acetic,
propionic and butyric acid, were isolated by the method of Harper (1953)
and the purity of the isolates checked by gas chromatography.

14C was determined either witha liquid scintillation counter or in an ionisation
chamber after oxidation of the sample to 14C0 2 using CuO catalyst.



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Results and Discussion

The distribution of radioactivity in the rumen liquor is shown in Figure 1.
1,3, 8 and 26 h after the alanine dose the proportion of the activity present in
one litre of rumen fluid was 1.05, 0.85, 0.52 and 0.25 % of the total given.
Most of the activity was found in the fraction precipitated with 10 % TCA,
which comprised mainly rumen bacteria. The protein content of this fraction
was 40, 45, 35 and 50 % after the above-mentioned intervals. The proportion
of total rumen liquor activity precipitated with TCA increased throughout
the 1-day period: 47.4 % after 1 h, 49.7 after 3, 70.0 after 8 and 80.0 %

after 26 h. Though the incorporation of [l4 C]alanine into TCA-precipited
material and into microbial cells was very vigorous most of this activity was
not present in the microbial protein. Thus while the carbon skeleton of alanine
can be used for the biosynthesis of many amino acids it is evident that the
rumen bacteria utilised only small amounts of it in the formation of cellular
protein. The labelling of the isolated amino acids, namely Tyr, Phe, Asp
and Glu was slight: 3.2, 3.9, 4.1 and 6.6 % respectively of the activity of the
TCA precipitate and 1.5, 1.9, 2.9 and 5.5 % of the total activity of the rumen
liquor. These amino acids together accounted for about 36 % of the total
amino acid.

Figure 1. Labelling of volatile
fatty acids and trichloracetic
acid precipitate from rumen
fluid 1—26 h after the admi-
nistration of [l4C]alanine.



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Figure 2 compares the labelling of the 4 isolated amino acids in the present
work with that found when [U- 14C]sucrose was fed to a cow on urea-rich
low-protein feed (Syväoja and Kreula 1979). It is evident that the bacteria
synthesised these amino acids from sucrose at about double the rate from
alanine. The retention time of sucrose in the rumen was shorter than that of
alanine.

In studies with sheep fed a diet of pelleted, dried grass meal, Portugal

and Sutherland (1966) found very little incorporation of labelled aspartic
and glutamic acid into rumen microbial protein; both amino acids were meta-
bolised rapidly, with incorporation of much of the 14C into short chain fatty
acids and C0 2. Similar results were obtained by Borchers (1967): he used
[U- 14C]glucose and 14C-labelled amino acids from an acid hydrolysate of
Chlordla and found that rumen microorganisms utilised glucose more readily
than exogenic amino acid carbons for the synthesis of cellular protein.

Measurements of the labelling of the main volatile fatty acids of rumen
fluid 1,3, 8 and 26 h after feeding [l4 C]alanine showed that acetic, propionic
and butyric acid, which together accounted for 92—94 % of the total rumen
volatile fatty acids, contained 40.8, 32.3, 23.0 and 5.0 % of the total activity
of the rumen fluid. Acetic acid carried the strongest labelling in the first two
samples (29.9 and 19.6 % of the total activity of the rumen fluid), butyric
acid in the last two samples (11.1 and 2.6 %), and propionic acid had the
weakest labelling in all samples (2.1 —O.B %), as shown in Figure 1.

Lactose is synthesised in the udder from blood glucose. Propionate is the
most important source of glucose in fed ruminants not absorbing glucose from
the small intestine. Lindsay (1979) estimated that 40—60 % of the glucose
entry rate derives from propionate. That the lactose was labelled 7 and 21 h
after feeding the [ l4C]alanine (11 and 15 nCi/g respectively, Kreula and
Kauramaa, unpublished results) showed that the blood glucose had carried
label. The above-mentioned weak labelling of propionic acid after feeding
[l4C]alanine does not by itself account for the incorporation of 14C into the
lactose glucose must have been formed via pyruvate and oxaloacetate
directly from alanine. Further study is needed before the contribution of amino

Figure 2. Specific activity
of amino acids isolated from
rumen bacterial protein after
intraruminal administration of
237 [U- 14C] alanine to
a cow on protein-free feed
( ) and 220 [U-14C]
sucrose (Syväoja and Kreula
1979) to a cow on urea-rich
low-protein feed (—). In the
alanine study the rumen fluid
samples were taken 1, 3 and
26 h after giving the label,
in the sucrose study 1, 5 and
24 h.



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acids to gluconeogenesis can be assessed. Bergman and Heitmann (1978)
estimated that 15—25 % of the glucose is derived from amino acids in amply-
fed sheep, glutamic acid and alanine being the most important substrates. In
starved sheep 35 % of the glucose entry rate derives from amino acids (Lindsay
1979). Black (1968) and Black et al. (1968) reported that amino acids can
provide 33—SO % of the carbon required for glucose synthesis in the lactating
cow.

Acknowledgement: This work was supported by a grant from the Academy of Finland.
The authors wish to thank Miss Terttu Ettala, M.Sc., for assistance in obtaining the samples.

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SELOSTUS

Radioaktiivisen hiilen inkorporoituminen ureaa ainoana typenlähteenään
saaneen lehmän pötsissä 14 C-alaniinista

Eeva-Liisa Syväoja ja Matti Kreula
Biokemiallinen Tutkimuslaitos, Kalevankatu 56 B, 00180 Helsinki 18

Eksogeenisen aminohapon hyväksikäyttöä tutkittiin ureaa ja ammoniumsuoloja ainoana
typen lähteenään saaneella O-lehmällä antamalla pötsiin 14C-alaniinia. Leimaantuminen määri-
tettiin 1,3, 8 ja 26 h kuluttua trikloretikkahapolla saostuvasta bakteerimassasta, haihtuvien
rasvahappojen pääkomponenteista (etikka-, propioni- ja voihaposta) sekä muutamista eriste-
tyistä aminohapoista. Pötsin mikro-organismit inkorporoivat 14C-alaniinia nopeasti soluma-
teriaaliinsa. Yhden pötsinestelitran sisältämästä kokonaisaktiiviisuudesta todettiin mikro-
bimassan sisältävän yllä mainittujen aikojen kuluttua 47.4, 49.7, 70.0 ja 80.0 %. Vaikka
alaniinin hiilirankoa voidaan käyttää monien aminohappojen muodostumisessa bakteerit
käyttivät tätä aminohappoa vain vähän Asp, Glu, Tyr ja Phe muodostumiseen. Näiden eris-
tettyjen aminohappojen leimaantuminen oli 1.5, 1.9, 2.9 ja 5.5 % pötsinestelitran totaali-
aktiivisuudesta mainittuina näytteenottoaikoina. Huomattava osa 14C-alaniinista hajosi haih-
tuviksi rasvahapoiksi. Etikka-, propioni- ja voihapossa todettiin 40.8, 32.3, 23.0 ja 5.0 %

annetusta aktiivisuudesta samoina näytteenottoaikoina. Haihtuvista rasvahapoista etikka-
hapon leimaantuminen oli voimakkain ja propionihapon heikoin.