Download Liver Proteolytic Activity in Tannic Acid-Fed Birds

Research Notes
Liver Proteolytic Activity in Tannic Acid-Fed Birds
F. Marzo,*,1 E. Urdaneta,* and S. Santidrián†
*Laboratory of Animal Physiology and Nutrition, School of Agromomy, Universidad Pública de Navarra,
31006 Pamplona (Navarra), Spain; and †Physiology Department, School of Medicine,
Universidad de Navarra 31008, Pamplona (Navarra), Spain
ABSTRACT The influence of tannic acid in the rate of
growth (BWG), feed intake, protein efficiency ratio, and
liver proteolytic activities (cathepsin A and D) were measured in growing male chickens. These birds were fed ad
libitum over a 15-d experiment on 20% protein standard
diets containing heated soybean (control, C) as the main
source of protein. Tannic acid (TA; 25 g/kg diet) was
added to all diets, except the control. It has been found
that in comparison to control-fed birds, TA-fed birds
showed a significant reduction (P < 0.01) in BWG, protein
efficiency ratio, and relative weight of liver, together with
a significant increase (P < 0.01) in the activities of cathepsin A and D in liver. Addition of TA to the control diet had
no significant effect on feed intake. The possible nature of
these results is discussed.
(Key words: chicken, tannic acid, liver, cathepsin A, cathepsin D)
2002 Poultry Science 81:92–94
growing animals diets containing these compounds
brings about several undesirable physiological and biochemical effects. These effects are reflected by growth
inhibition, negative nitrogen balances, reduced intestinal
absorption of sugars and amino acids, reduced immune
response, and increased liver and protein catabolism
(Santidrian, 1981; Santidrian and Marzo, 1989). The liver
is one of the body’s most sensitive organs to toxic factors
or protein deficiency. Administration of diets to animals
containing tannins should result in a greater decrease in
liver protein than in other organs. Proteins seem to be
influenced by protein catabolic processes (Cenarruzabeitia et al., 1979; Lasheras et al., 1980), which have an obvious relevance in farm animal nutrition. Lysosomal proteolytic activity plays a decisive role in protein metabolism, with cathepsin A and D being the main enzymes
involved in the catabolism process in the liver. For this
reason, we have undertaken the present investigation to
further elucidate catabolic action of tannins on chicken
protein metabolism by studying the effect of the TA on
the activities of cathepsin A and D in liver.
INTRODUCTION
Tannins are present in many products of vegetable origin that are used as human foods or animal feeds. Although tannins are chemically not well defined, they are
usually divided into hydrolyzable and condensed tannins. Hydrolyzable tannins have a central carbohydrate
core whose hydroxyl groups are esterified to phenolic
carboxylic acids, such as gallic acid, ellagic acid, and hexahydroxydiphenic acid. Esters of the first two acids are
referred to as gallotannins. Tannic acid (TA) is a wellknown gallotannin and contains 8 to 10 mol of gallic
acids/mol of glucose. These types of tannins are readily
hydrolyzed by acids, alkali, or some enzymes. Upon hydrolysis, they yield glucose or some other polyhydroxy
alcohol and gallic acid or some phenolic acids related to
it (Freudenberg and Weinges, 1962; Haslam, 1966; Salunkhe et al., 1990; Jansman, 1993; Hagerman et al., 1997).
A number of adverse nutritional effects have been attributed to tannins. Studies have been conducted on the
effects of tannins in feedstuffs on animal performance.
Some of them have been carried out with tannins isolated
from feedstuffs or with standard commercial tannins,
such as TA, that were thought to be representative of
tannins in many feedstuffs and with raw legumes containing different levels of tannins (Mitjavila et al., 1977;
Wareham, 1993). It has been demonstrated that feeding
MATERIALS AND METHODS
One-day-old White Leghorn male chickens, weighing
40 to 50 g, were randomly assigned to two dietary groups
of 10 birds each and were housed in battery brooders
with raised floors. Groups were arranged as follows: one
was fed a standard control diet, and the other was fed
the same diet to which 25 g TA/kg diet was added. Both
2002 Poultry Science Association, Inc.
Received for publication April 5, 2001.
Accepted for publication September 27, 2001.
1
To whom correspondence should be addressed: [email protected].
Abbreviation Key: BWG = rate of growth; TA = tannic acid.
92
93
RESEARCH NOTE
TABLE 1. Feed intake (FI), body weight gain (BWG), protein efficiency ratio (PER), liver weight (LW), and cathepsin A and D
in chickens fed 20% protein diets of heated soybean (control) with or without tannic acid1
Diet
FI
(g 100/g BW)
BWG
(g/d)
163 ± 4
172 ± 5a
12.1 ± 0.2
4.4 ± 0.1b
Control
Tannic acid (25 g/kg diet)
b
a
PER
(g BW/g protein)
LW
(g 100/g BW)
2.5 ± 0.1
2.0 ± 0.2b
4.3 ± 0.2
2.2 ± 0.3b
a
Liver cathepsin A
Liver cathepsin D
Total
Total
Free
41 ± 1
60 ± 3a
a
b
17 ± 1
28 ± 2a
b
Free
32 ± 2
64 ± 3a
b
19 ± 2b
39 ± 2a
Means not sharing a common superscript within a column are significantly different (P < 0.01).
Values are means of enzymatic units (×102)/mg of protein from 10 chickens.
a,b
1
water and feed were supplied ad libitum. The total protein content of each diet was 20%; the source was heated
soybeans. Diets were prepared according to criteria previously reported (Marzo et al., 1989, 1991). Total caloric
content was about 3,000 kcal/kg. Body weight changes
were individually recorded every 3 d.
Within each experimental group, 10 chickens were
killed by decapitation when they were 15 d of age. Immediately after bleeding, livers were removed and weighed.
Liver protein content was assayed by the method of
Lowry et al. (1951). Free liver cathepsin D activities were
measured in control and TA-treated birds according to
the method of Gianetto and DeDuve (1955). As in previous work (Santidrian et al., 1987), activities of cathepsin
D are given in units per milligram of liver. One unit
is defined as the amount of enzyme that catalyzes the
liberation of 1 µmol of tyrosine (measured by ninhydrin
reaction) from hemoglobin during incubation (30 min at
37 C).
Cathepsin A was assayed by the method of Iodice and
Weinstock (1965); the steps involved were very similar
to those mentioned for the cathepsin D, with the difference that N-CBZ-glutamyl-L-tyrosine was used as the
substrate. TA (from Phaseolus helvetica extracts), hemoglobin, and N-CBZ-glutamyl-L-tyrosine were purchased.2
Statistical Analysis
For each parameter investigated, the data were statistically evaluated by one-way ANOVA. Comparisons between TA- and control-fed birds were made. Least-significant differences for P < 0.01 were calculated (Miller
and Miller, 1993).
RESULTS AND DISCUSSION
Results of the experiment are summarized in Table 1.
As shown, the rate of growth (BWG) and relative weight
of liver were significantly reduced (P < 0.01) in TA-fed
chickens as compared with control-fed birds. Nevertheless, no significant changes were observed in feed intake,
measured in grams of food consumed per bird over time.
Protein efficiency ratios were diminished 20% (P < 0.01)
in TA-fed birds. The reduced BWG and relative weight
of other organs displayed by TA-fed chickens or those
2
Sigma Chemical Co., St. Louis, MO 63178.
fed raw legumes correlate with previously reported data
from this laboratory (Marzo et al., 1989, 1990, 1991) and
others (Catañon and Marquardt, 1991). The toxicity of
raw legume beans has been described in relation to tannin
content. Although there is general agreement that feeding
a diet containing these legume as the sole source of protein exerts a toxic effect, it has been found that the toxic
action of tannins may account, at least in part, for this
growth impairment (Elkin et al., 1990).
Some studies have determined the effects of commercially available hydrolyzable tannins on chicks and rats.
TA, when fed to different animal species, has been shown
to affect the digestibility and absorption of nutrients but
also to affect different internal organs (Chang and Fuller,
1964; Singleton, 1981). However, biochemical changes in
the cathepsin activities in response to tannic acid content
in diets have not been studied previously. Table 1 shows
that, as compared with control birds, those fed the diet
with TA exhibited a marked increase in the activities of
cathepsin A and D in liver. This finding relates to the
fact that feeding growing animals a diet containing raw
legumes brings about an increase in protein catabolism.
Published evidence has shown increases in the activity
of a number of hepatic amino acid-degrading enzymes
in rats and chickens fed a raw faba bean diet (Cenarruzabeitia et al., 1979; Santidrian et al., 1981). Reports have
also shown that TA or degradation products are absorbed
from small intestine and cause toxic effects (Vohra et al.,
1966; Kardivel et al., 1969; Karin et al., 1978). To explain,
at least in part, the catabolic effect caused by tannins from
raw legumes or by TA, the results of this experiment have
shown relationship between TA and liver cathepsin A
and D activities. Moreover, a relatoinship between the
marked increase in liver poteolytic activity and the reduction in BWG may suggest that use of TA affects protein
catabolism and the nutritive value of diets.
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