JOURNAL OF THE SCIENTIFIC AGRICULTURAL SOCIETY OF FINLAND

177

Maataloustieteellinen A ikakauskirja
Vol. 50: 177-181, 1978

On the metabolism of benzoic acid by cows on purified
protein-free and low-protein feed

M. Kreula, A. Kauramaa and M. Tegengren

Biochemical Research Institute, Kalevankatu 56 B, 00180 Helsinki 18,Finland

Abstract. Two test cows, one adapted to purified, protein-free feed (0-feed, 0-cow)
and the other to a low-protein feed (ULP-feed, ULP-cow) in which 60 % of the nitrogen
was derived from urea, were fed single doses of 212 and 193 /<Ci [ring U- 14C] benzoic
acid respectively in 0.01 % benzoic acid solution. For five days the milk, faeces and
urine were collected quantitatively. Fat, protein and lactose contents of the milk
samples were determined and these components were isolated in pure form. 23.5 and
98.8 % respectively of the 14C-activity given to the 0- and ULP-cows was found in the
urine, and 2.5 % and 3.0 % in the faeces. Totals of 0.5 % and 0.8 % were recovered
in the milk, as found from analyses of whole milk. The sum of the activities in isolated
fat, protein and lactose components was 0.2 % for both cows, and only the protein
being labelled.

Introduction

In long-term feeding experiments with purified protein-free feed, urea being
the sole source of nitrogen (0-feed), and with feeds containing small amounts
of true protein and large supplements of urea (ULP-feed), considerable dif-
ferences were found in the milk production of dairy cows. The annual milk
yields of the ULP-cows were in general 1 000—2 000 kg higher than those of
0-cows. Reasons for this were sought ever since the start of the experiments,
and metabolic differences were also studied (Virtanen 1963, 1966, Virtanen
et ai. 1972, Mäkinen 1972, Ettala and Kreula 1976).

One of the most characteristic feed differences is the lack of aromatic
precursors in 0-feed. As regards ruminants, very little information is available
about the role of the different components of the feed in the formation and
metabolism of benzoic acid and its precursors.

In the present study the ability of 0-cows and ULP-cows to metabolise
benzoic acid was investigated by feeding two cows I4C-labelled benzoic acid.
The excretion of the carbon derived from the benzoic acid in the urine and
faeces, as well as its utilisation in the biosynthesis of milk components, was
followed.

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Materials and methods

A single dose containing 212 /uCi [ring U- l4 C] benzoic acid (Radiochemical
Centre, Amersham, England) in 700 g 0.01 % benzoic acid\ solution was fed
orally to 0-cow Oona. Oona, a 8.5 year-old Ayrshire, had been on the protein-
free feed ever since it was a heifer. It had last calved in December 1973. During
the test period (Nov. 22—27, 1974) the cow consumed an average of 1.4 kg
cellulose strips, 2.8 kg starch, 1.2 kg sucrose, 0.1 kg glucose, 0.1 kg straw, and
0.26 kg urea daily. Also the cow was given daily 0.13 kg vegetable oil (maize/
soyabean 1: 3), 0.54 kg mineral mixture, vitamin A (100 000 1.U.) and vitamin D
(D 2 -f- D 3, 20 000 1.U.). Once a week the cow received 400 mg DL-a-tocopherol.
The digestibility of the organic matter was 84 % and the cow drank an average
of 45 1 water per day.

A single dose of 193 /id [ring U- 14C] benzoic acid (Radiochemical Centre,
Amersham, England) in 650 g 0.01 % benzoic acid solution was fed orally to
ULP-cow Euru. Euru was a 17-year old Ayrshire in its second month of lactation.
The cow had been on ULP-feed since 1966. During the test period (Feb. 18
22, 1975) Euru consumed daily 9.5 kg organic matter, of which 1.7 kg was
maize, 6.0 kg sugar beet pulp, 1.4 kg hemicellulose powder and 1.5 kg straw.
The cow was also given daily 0.1 kg vegetable oil, 0.35 kg urea, 0.37 kg mineral
mixture, vitamin A (100 000 1.U.) and vitamin D (D 2 + D 3, 5 000 1.U.), and
further 330 mg DL-a-tocopherol per week. The proportion of urea was about
60 % of the total nitrogen intake; the digestibility of the organic matter was
72 %.

The cows were milked in the morning and evening. The milk, faeces and
urine were collected quantitatively. The methods of storage and analysis of
the samples were those reported earlier (Kauramaa and Kreula, 1975).

Results

During the test period Oona produced a total of 26.2 kg milk, of average
compostion: 12.1 % dry matter, 3.1 % fat, 3.5 % protein and 4.7 % lactose.

The corresponding figures for ULP-cow Euru were as follows: 55.3 kg milk,
average composition 12.1 % dry matter, 4.0 % fat, 3.1 % protein and 4.3 %

lactose.
The 14C-activities of the milk and its components were measured 8.1 118 h

after the administration of the [ l4 C] benzoic acid. Of the total activity fed,
0.5 % was transferred to the whole milk with the 0-cow and 0.8 % with the
ULP-cow (Table 1).

Table 1. Distribution of 14C in milk and its components obtained after administration of 212
»Ci [ring U-14C] benzoic acid to 0-cow Oona and of 193 iiCi [ring U-14C] benzoic acid to ULP-
cow Euru.

14C-activity, % of 14C fed
Whole milk Fat Protein Lactose

0-cow Oona 0.5 0
0

0.2 O
OULP-cow Euru 0.8 0.2

178



179

Table 2. Excretion of 14C in the faeces and urine of 0-cow Oona after administration of 212
/<Ci {ring U-14C) benzoic acid.

Hours Faeces Urine
after 14C-activity, 14C-activity,

feeding kg %of 14C fed kg %of 14C fed

9.1 2.6 0.0 1.6 7.9
22.8 5.5 1.2 12.4 13.6
33.1 3.5 0.0 11.4 1.1
46.7 3.7 0.3 8.5 0.4
57.0 3.3 0.2 4.7 0.1
70.5 3.3 0.2 4.8 0.1
81.3 3.9 0.2 6.6 0.1
94.5 4.4 0.2 10.5 0.1

105 0 3.1 0.1 7.1 0.1
118.8 4.9 0.1 14.8 0.0

Total 2.5 23.5

Table 3. Excretion of 14C in the faeces and urine of ULP-cow Euru after administration of
193 ,uCi [ring U-14C] benzoic acid.

Hours Faeces Urine
after 14C-activity, 14C-activity,

feeding kg % of 14C fed kg % of 14C fed

8.1 5.9 0.0 4.6 92.8
22.0 9.7 0.5 3.8 5.4
32.2 7.7 0.6 3.8 0.3
45.5 7.5 0.4 6.0 0.1
56.2 12.7 0.0 4.6 0.1
69.8 9.9 0.3 4.7 0.0
80.3 7.0 0.3 5.6 0.0
93.4 10.6 0.7 6.5 0.0

104.1 7.0 0.1 5.4 0.0
117.8 12.0 0.1 7.3 0.1

Total 3.0 98.8

In the faeces and urine of the 0-cow totals of 2.5 % and 23.5 %, respectively,
of the 14C fed were excreted (Table 2). The corresponding figures for the
ULP-cow were 3.0 % and 98.8 % (Table 3).

Discussion
The feed of ruminants generally contains large amounts of benzoic acid

precursors, for example aromatic amino acids, cyclohexane compounds, and
they are also formed in rumen fermentation. The detoxication of these com-
pounds in the organism usually takes place via benzoic acid, which is conjugated
in the liver with glycine and removed, as hippuric acid, from the organism via
the kidney and urine. The conjugation of feed benzoic acid with glycine is
usually quantitative, and it has been observed with rabbits that the reaction



180

rate is independent of the amount of benzoic acid fed (Bray et al. 1951).
According to studies performed with non-ruminants, the reaction rate may be
governed by the amount of glycine available, the activity of the two enzymes
required and the presence of inhibitory factors (Irjala 1972).

The use of labelled benzoic acid in studies on ruminant metabolism has not
apparently been reported previously in the literature. In the present study,
98.2 % of the 14C-activity fed was recovered in the urine of the ULP-cow
during the first day. It seems that [I4 CJ benzoic acid was metabolised during
this time almost quantitatively in the normal way. In contrast, with the 0-cow
the 14C-activity in the urine was considerably lower; during the first 24 h only
21.5 % of the activity fed was excreted. During the following day a further 1.5 %

was recovered, after which the rate of excretion dropped to level of about 0.1%
per day. The capacity of 0-cow Oona to metabolise feed benzoic acid was less
complete and slower than that of ULP-cow Euru. The high amounts of
glycine found in the blood plasma of the 0-cow exclude the possibility of a
deficiency of glycine (Virtanen et ai. 1972). As the 14C activity of the urine,
faeces and milk fell (in two days) to a constant level of about 0.1 %, it can be
deduced that the later metabolism of benzoic acid remained very slow.

The 14C-activity recovered in the faeces (2.5 % with the 0-cow and 3.0 %

with the ULP-cow) results either from alimentary canal secretions, or from a
slight utilisation of benzoic acid by the rumen microbes for the synthesis of
structural cell components, which remain undigested.

In the amounts of 14C excreted in the milk a slight difference (0.5 % and
0.8 %) was found between the 0-cow and the ULP-cow. The total of the 14C
contents in the major components of the milk from both cows (0.2 %) was less
than that in the whole milk (Table 1). Svensen (1974) has shown that cow’s
milk contains small amounts of hippuric acid. It is transferred from the blood
plasma by passive diffusion in the mammary gland to the milk plasma (Miller
et al. 1967).

As to the biosynthesis of milk components, benzoic acid is used only for
protein synthesis, and even then in very small amounts.

On the basis of the feeding experiments it can be stated that cows adapted
to low-protein, urea-rich feed are able to metabolise benzoic acid normally,
whereas the detoxication of this compound by cows adapted to protein-free,
purified feed is slower and less complete. However, this may be an indication
of the small amount of aromatic compounds in a metabolic system in which
all the necessary aromatic compounds have to be synthesised solely from hexose-
based carbon sources.

REFERENCES

Bray, H. G., Thorpe, W. V. & White, K. 1951. Kinetic studies of the metabolism of foreign
organic compounds. Biochem. J. 48: 88 96.

Ettala, X. & Kreula, M. 1976. Urinary nitrogen compounds in dairy cows fed urea as the
sole or partial source ot nitrogen. J. Sciest. Agric. Soc. Finl. 48: 323—335.

Irjala, K. 1972. Synthesis of p-aminohippuric, hippuric, and salicyuric acids in experimental
animals and man. Ann. Acad. Sei. Fenn. A. V Med. 154: 40 pp.



181

Miller, G. E., Banerjee, N. C. & Stowe, C. M. Jr. 1967. Diffusion of certain weak organic
acids and bases across the bovine mammary gland membrane after systemic admist-
ration. J. Pharm. Exp. Ther. 157: 245 253.

Mäkinen, S. 1972. Aspects of the nitrogen metabolism and nutritional status of urea-fed
dairy cattle. Ann. Acad. Sei. Fenn. A. II Chem. 165: 67 pp.

Kauramaa, A. & Kreula, M. 1975. Utilization of acetate in the biosynthesis of milkcomponents
by a cow on purified protein-free feed. Finn. Chem. Lett. 140 154.

Svensen, A. 1974. Hippursyre og benzoesyre i melk og forskjellige meieriprodukter. Meieri-
posten 35 36: 731—734.

Virtanen, A. I. 1963. Production der Kuhmilch ohne Protein mit Harnstoff und Ammoni-
umsalzcn als Stickstoffquelle und gereinigten Kohlenhydrate als Energiequelle. Biochem.
Z. 338:443-453.

» 1966. Milk production of cows on protein-free feed. Science 153: 1603 1614.
»

, Ettala, T. & Mäkinen, S. 1972. Milk production of cows on purified protein-free
feed with urea and ammonium salts as the only nitrogen source and on non-purified
feed with rising amounts of true protein. Festskrift til Prof. Dr. Agr. h.c. Knut Breirem
til hans 70-ärs dag. pp. 249—275.

Ms received March 15, 1978

SELOSTUS

Bentsoehapon metaboliasta proteiinittomilla puhdistetuilla rehuilla ja vähän
proteiinia sisältävillä rehuilla ruokituilla koelehmillä

M. Kreula, A. Kauramaa ja M. Tegengren

Biokemiallinen Tutkimuslaitos, Kalevankatu 56 B, 00180 Helsinki 18

Proteiinittomalla, puhdasrehuruokinnalla olleen lehmän (0-lehmä) ja vähän proteiinia,
runsaasti ureaa sisältävällä ruokinnalla olleen lehmän (ULP-lehmä) bentsoehappometaboliaa
on seurattu käyttämällä [l4C]:llä leimattua bentsoehappoa. 0- ja ULP-lehmälle on
annettu kerta-annoksena suun kautta vastaavasti 212 j«Ci:tä ja 193 /iCi:tä [rengas- 14C] bent-
soehappoa 0.01 % bentsoehappoliuoksessa. Viiden vuorokauden ajan maito, sonta ja virtsa on
kerätty kvantitatiivisesti. Maitonäytteistä on määritetty rasva-, proteiini- ja laktoosipitoi-
suudet, nämä aineosat on eristetty myös puhtaina. 23,5 % ja 98,8 % vastaavasti 0- ja ULP-
lehmälle annetusta 14C-aktiivisuudesta on löydetty virtsasta ja 2,5 % ja 3,0 % sonnasta. Kaik-
kiaan 0,5 % ja 0,8 % on löydetty kokomaidosta. Eristettyjen rasva-, proteiini- ja laktoosi-
aineosien 14C-aktiivisuuksien summa on ollut 0,2 % molemmilla lehmillä vain proteiinin lei-
maantuessa.

Näyttää siltä, että ULP-lehmä pystyy metaboloimaan bentsoehappoa normaaliin tapaan,
mutta 0-lehmän bentsoehappo-detoksikaatio on normaalia hitaampaa ja epätäydellisempää.