Herbivore Growth Reduction by Tannins: Use of Waldbauer Ratio Techniques and Manipulation of Salivary Protein Production to Elucidate Mechanisms of Action
SIMON MOLE,*1" JOHN C. ROGLER,~ CARLOS J. MORELL* and LARRY G. BUTLER* *Department of Biochemistry, Purdue University, West Lafayette, IN 47907, U.S.A.;
~:Department of Animal Science, Purdue University, West Lafayette, IN 47907, U.S.A.
Abstract--The consumption and utilization of food by rats fed diets both high and low in condensed tannin was analysed on a dry weight basis and on a Kjeldahl nitrogen basis. Growth analyses followed the protocol devised for insects by Waldbauer. The hypothesis that tannin-induced proline-rich salivary proteins (PRPs) diminish the antinutritional effects of dietary poly- phenolics in mammals was investigated by supplementing diets with 0.05% propranolol to suppress PRP production. Rats fed diets high in tannin gained less weight than those fed low tannin diets. This effect was too pronounced to be explained by differences in feed consumption and could only be attributed to depressed efficiency in converting injested food to body matter. The reduction in approximate digestibility (AD) with high tannin sorghum diets was low relative to the severe reduction in the conversion of digested food into body matter (ECD) for high tannin diets. The main growth reducing effect of tannin thus appears to have been on the post-absorptive utilization of nutrients rather than on the digestibility of the diet. On a nitrogen basis, AD(N) is reduced in the presence of tannins so tannins did act preferentially on the digestion and absorption of nitrogen. Nevertheless, on a nitrogen basis ECD(N) was even more substantially depressed by tannins and so it could not be concluded that tannins act only or even mainly as digestibility-reducing agents. Propranolol only produced adverse effects in combination with tannin but these were much more severe than those with tannin alone. Further evidence suggested that the cause was the suppression of PRP production. PRPs are known to bind to tannins in competition with other proteins and their absence denies the animal the means to neutralize dietary tannin. This study also examines the effect of sodium chloride (0.5%) in the diet following a previous report that salt alleviates the effect of dietary tannins in mice. In our experiments this had negligible effects. We conclude by comparing our data for a mammal to previous studies on insects using these same parameters to assess consumption and utilization of foods.
Introduction Tannins and related polyphenolics have been widely reported to reduce the growth rates of animals consuming them in their diet. The mechanism for this effect has usually been con- sidered to be the inhibition of digestion [1]. Abundant evidence from in vitro models of digestion shows that tannins can indeed reduce protein and carbohydrate digestion [2-4]. How- ever, in vivo evidence for digestibility reduction is far from unequivocal [1, 5-7]. For instance, mammals such as the wood rat naturally thrive on diets rich in tannins [8]. In contrast, there are other cases in which tannins have negative effects on animal performance but where these negative effects are indicative of metabolic
1"Present address: School of Biological Sciences, Univer- sity of Nebraska at Lincoln, Lincoln, NE 68588, U.S.A.
(Received 15 November 1989)
poisoning by substances absorbed into the body [9, 10]. Recent in vitro models of digestion in the presence of tannins reveal situations in which tannins promote rather than inhibit enzymic digestion [11] or in which intestinal surfactants and specificity effects in tannin-protein inter- actions force us to reconsider the potential for tannins to impair digestive processes [1, 11-13].
The broad object of these experiments was to test the hypothesis that the major effect of tannins is manifested by digestibility reduction, the alternative hypothesis being that tannins act as toxins within the body. To make this test we have quantified the extent to which tannins reduce digestion, and specifically nitrogen diges- tion, relative to subsequent processes involved in growth and development. In doing this, we have borrowed a methodology from entomological studies in order to provide truly comparative insect-mammal data for the first time.
183
184 SIMON MOLE E f A L
Proline-rich salivary proteins and salt One other major goal in this work, was to examine the in vivo interaction of proline-rich salivary proteins with tannins. Several species in each of three major groups of mammals (rodents, ruminants and primates) are known to produce large quantities of a group of proline- rich proteins (PRPs) in their saliva [14-16]. The reasons for the production of these proteins remain to be resolved. Although they can repre- sent 60-70% of the proteins in parotid saliva [16], no physiological role has yet been confidently and generally attributed to them. In some animals (e.g. humans), PRPs appear to be con- stitutively produced while in others (e.g. rats) they are normally present only in trace amounts. In such animals, production of PRPs can reach high levels once induced, and any general explanation of the function of these proteins should help explain their regulation. In this context, it is of note that dietary tannins induce PRP production in rats [16].
Tannins and other polyphenolic chemicals of plant origin are known to bind to PRPs with an affinity which is as much as four or more orders of magnitude greater than that for other proteins [12, 17]. Animals which produce salivary PRPs may gain some protection against the activity of dietary polyphenolics further down the gastro- intestinal tract, as the polyphenolics are specifi- cally bound to salivary PRPs, and are thus removed from the digestive process [16]. While we do not wish to discount any other proposed functions for these proteins [14], we nevertheless believe that there is a strong case to be made that PRPs do have an adaptive role in animals naturally consuming plant material containing tannins. Here we test the prediction that the growth-reducing effects of tannins are poten- tiated in animals prevented from producing PRPs. A final and minor objective of this work was to investigate the finding of Freeland et al. [18] that supplementation of the diet with 0.5% salt (NaCI) may reduce the growth depressing effect of tannins in mammals.
Methodological approach Waldbauer [17] examined techniques for dividing the efficiency with which animals convert ingested food into new body substance (ECI) into separate components. These are the approxi-
mate digestibility of the food (AD) and the effi- ciency with which digested and absorbed food is then converted to new body substance (ECD). Equations which define these terms are given in Table 1. It can be seen that if tannins only reduce digestibility then if an animal is fed a high tannin diet, AD will be diminished while the ECD term should not be affected as it is a measure of the post-absorptive fate of the nutrients. This scheme of analysis provides a powerful way to quantify the relative contribution of reduced AD and ECD to lower ECI and thus growth rate (GR) for animals consuming tannins. Although the molecular mechanism by which tannins exert their effects cannot be revealed by this method,
TABLE 1. EQUATIONS DEFINING TERMS USED TO ANALYSE THE
CONSUMPTION AND UTILIZATION OF FOOD, ADAPTED FROM
WALDBAUER [1]
Growth rate (GR) o= animal weight gain/tirne
Consumption rate (CR) = feed ingested/time
Efficiency of conversion of ingested food to body matter (ECII is
calculated as:
ECI = (animal weight gain/feed ingested) 100%
Where all weights are made on dry weight basis GR can be
expressed in terms of CR and ECI, i.e. GR = CR v ECI
Approximate digestibility (ADI is calculated as
AD weight of feed ingested weight of feces . 100t, weight of feed ingested
Efficiency of conversion of digested food to body matter (ECD) is
calculated as:
animal weight gain ECD ." 100%
weight of feed ingested weight of feces
Where all weights are made on a dry weight basis ECI can be
expressed in terms of AD and ECD, i.e. ECt AD .- ECD.
Equations defining terms used to analyse the consumption and
utilization of nitrogen are similar, thus .
Nitrogen accumulation rate (NAR) .~ body nitrogen gain/time
Nitrogen consumption rate (NCR) = N in feed ingested/time
AD(N)* nitrogen ingested nitrogen in feces z 100% nitrogen ingested
Efficiency of conversion of digested food to body matter (ECD} is
calculated as:
AD body nitrogen gain , 100% nitrogen ingested nitrogen in feces
As with ECI = AD × ECD, ECI(N) = AD(N) ~ ECD(N)
~Termed nitrogen utilization efficiency (NUE) by Slansky and Feeny
[38]
MECHANISM OF ACTION OF TANNINS 185
if tannins depress ECD an examination of pro- cesses other than the inhibition of digestion is indicated.
Practical determination of AD and ECD in a feeding trial requires that all the component measurements are made in the same units, otherwise the relationship ECI=ADXECD will not hold. Measurements are most easily made on a dry weight basis but they can also be made in terms of specific nutrients (e.g. grams of nitrogen). We have done both of these.
In order to test the hypothesis that PRPs reduce the effects of tannins on rats, we repli- cated the experiments using animals which were and were not able to produce PRPs, using the drug propranolol, a j]-antagonist, to suppress PRP production. Without propranolol, PRPs are induced during the first three days that animals are fed high tannin diets [19]. To eliminate the brief depression of growth rate when proprano- Iol is first introduced into the diet, all rats given the propranolol treatment were pre-fed a low tannin, propranolol-containing diet for one week. Although the animals exhibited normal growth at the end of this period, they were lighter in body weight than animals of the same age fed the same diet differing only in the omission of pro- pranolol. To eliminate low initial weight as a confounding factor in the experiment, the pro- pranolol treated rats were put on the high- and low-tannin diets one week later than the untreated animals. The intention was that at the beginning of the experiments, the animals acclimatized to propranolol would weigh as much or more as their younger counterparts not fed propranolol. This leaves age, but not initial body weight, as a confounding factor in the design. We believe this helps strengthen con- clusions about the negative effect of tannins on growth (i.e. weight gain).
In these experiments eight groups of five experimental animals were each fed one of eight diets based on ground sorghum. Table 2 sum- marizes the construction of the diets, which are numbered 1 to 8 for reference.
Results Tannins and other polyphenolics in the feeds
Assays of the experimental diets revealed that those based on the high tannin (Savanna) sorghum (3,4,7,8) contained over 10 times more
TABLE 2. DIETS FED TO EACH GROUP OF FIVE ANIMALS*
Diets without propranolol
1 RS610 basal diet plus 0,55% cornstarch 2 RS610 basal diet plus 0.5% salt, 0.05% cornstarch 3 Savanna basal diet plus 0.5% cornstarch 4 Savanna basal diet plus 0.5% salt, 0.05% cornstarch
Diets with 0.05% propranolol
5 RS610 basal diet plus 0.5% cornstarch 6 RS610 basal diet plus 0.5% salt 7 Savanna basal diet plus 0.5 cornstarch 8 Savanna basal diet plus 0.5% salt
*Numbers are those used for reference in the text.
condensed tannin than those based on the low tannin hybrid RS610 (1,2,5,6). On a weight basis, and using quebracho tannin as a standard, these groups of diets contained 7 and 0.4% condensed tannin, respectively. Because quebracho is a crude plant extract, the actual quantity of tannin present may be half these figures. Condensed tannins were not the only flavonoids present in the diets. Both diets contained flavan-4-ols which were present in the low tannin diet at a level approximately half that in the high tannin diet. Other types of phenolics were undoubtedly present too, but the major phenolic component of high tannin sorghum is condensed tannin [20].
Growth analysis on a weight basis Weight gains. The mean starting weights for
the groups of animals not fed propranolol (diets 1-4) were within the range 70.6-71.0 g. After one week's pre-feeding on a low tannin propranolol containing diet, animals fed propranolol during the experiment (diets 5-8) had slightly higher mean starting weights (range 72.2-75.6 g). Table 3 gives data for the final group mean weights and weight gains. By inspection, it is clear that weight gain on the low tannin diets was far in excess of that on the high tannin diets. Indeed, there was very little weight gain seen with the groups of animals on the high tannin diets 3 and 4. For some individual animals, slight losses in weight were observed. In the presence of pro- pranolol (compare diets 7 and 8 with 3 and 4) weight losses were clearly evident indicating the interaction of tannins and propranolol to produce more severe effects than seen with tannin alone. Salt did not alleviate the effect of tannin on weight gain (compare diets 3 with 4, and 7 with 8).
186 SIMON MOLE ETAL
TABLE 3. WEIGHTS, WI=IGHT GAINS AND FEED CONSUMPTION BY RATS DURING THE TWO-WEEK FEEDING TRIAL (ALL WEIGHTS IN GRAMS)
Diet*
I t 2 3 4
Measurements Mean S.E.M. Mean S.E.M, Mean SE.M. Mean S.E.M
*Diets numbered as in Table 2, which gives details of their composition.
1-Means and their associated standard errors are given for the groups of five animals fed each diet.
Food consumption. Table 3 also gives the amounts of feed consumed during the two week period. This may be taken as a measure of con- sumption rate (CR, Table 1) while the parameter known as relative consumption rate (RCR) can be estimated as RCR=CR/dry wt (final dry weight of animal). Comparing the data for high and low tannin diets, CR for high tannin diets 3,4,7,8 was between 60 and 85% of that on diets 1,2,5 and 6. It seems unlikely that this would cause the observed differences in dry weight gain, which approximates to an order of magnitude difference between high and low tannin diets. Mean values of RCR are also given in Table 3. Although RCR is more properly calculated as CR relative to a weighted average of body dry weight during the experiment [17], the data for RCR shows that when the size of the animal is considered, differences in CR are small. Animals on high tannin diets (3,4,7,8) may actually consume somewhat more than animals on low tannin diets (1,2,5,6).
Analysis of ECl. Given that low CR does not appear to be the primary cause of low growth rate on high tannin diets, then from the GR = CR × ECI model (see Table 1) an analysis of ECI is warranted. This is presented in Table 4 and shows that ECI was much lower for the high tannin diets relative to the low tannin diets; i.e. low ECI was the main component of low GR,
where GR was estimated from the differences in animal dry weight gain during the experiment. Inspection of the data for AD and ECD shows that the values of both these parameters were lower for the high tannin diets relative to the low tannin diets. However, comparing AD and ECD, the depression of ECD was much more profound than that for AD; thus digestibility reduction does not appear to be the main cause of reduced ECI.
An examination of the data for AD using t-tests indicates that differences in AD between diets 1 v 3 and 2 v 4 were significant (P>0.999 in both cases); however, these differences are only 8.2 and 12.2% between AD values in the range 88.2-76%, and so differences in AD seem to be significant but a relatively minor component of the differences seen for ECI. For the equivalent comparison of animals fed propranolol, t-tests indicate that differences in AD between diets 5 v 7 and 6 v 8 were also significant (P>0.999). Again these differences were small (12.7 and 11.2%) relative to AD values in the range 88.7-- 76%. AD was positive and substantial for diets 1-8 and the effect of tannins on AD cannot account for the negative values of ECI.
Salt supplementation had no effect on AD for animals consuming low tannin diets. No major effects were seen in the presence of tannin either, although salt supplemented diet 4 was less digestible than diet 3 (t-test, P>0.999). The
MECHANISM OF ACTION OF TANNINS
TABLE 4. GROWTH ANALYSIS ON A DRY WEIGHT BASIS, FOLLOWING CALCULATIONS DETAILED IN TABLE 1 AND BELOW
187
Diet* I t 2 3
Measurements Mean S.EM. Mean S.EM. Mean S.EM, 4 Mean S.E.M
*Diets numbered as in Table 2, which gives details of their composition.
tMeans and their associated standard errors are given for the groups of five animals fed each diet. ~:URL = dry weight of feed -- dry weight of feces -- dry weight gain of body.
difference between the mean digestibilities was small (4%). Differences betweeen salt and salt- free diets will not be further considered as salt does not appear to be a significant factor influencing the outcome of these experiments.
Examination of the data for ECD shows approximately order of magnitude differences between data for the high and low tannin diets, for which more refined statistical analysis is superfluous; ECD was therefore the major factor responsible for low ECI on high tannin diets, not AD. This held true for diets with and without pro- pranolol.
By the application of conservation laws, a combined estimate of urinary and respiratory weight losses (URL) can be made. This requires the assumption that these are the only routes for material loss from ingested feed, besides that loss to body weight gain and feces. Data is given in Table 4 which shows that animals on high tannin diets lost less material by this route, but these differences are insignificant if differences in consumption are corrected by dividing URL by the dry weight of feed consumed. Differences in URL between diets are similary diminished if the weights of these losses are divided by final body dry weight. This exercise yields relative urinary and respiratory weight loses corrected for differ- ences in body size. Mean values for diets are also given in Table 4. The point of interest in this second set of data is that their similarity shows basal tissue respiration to be relatively un- affected by tannins.
Growth analysis on a nitrogen basis Nitrogen gains. Table 5 presents data for the
mean percentage of protein in the rats at the end of the experiment, t-Tests between the means indicate that after the two-week feeding trial, animals on the high tannin diet without salt had a higher proportion of protein in their bodies than those on the low tannin diet without salt (P>0.995). For the equivalent salt containing diets (2 v 4) the difference between these means is not significant (P>0.90). If it is assumed that measurements for the pairs of high and low tannin diets are independent measures of these effects, then by combining these probabilities [21 ] the hypothesis of equal mean values for high and low tannin diets can be rejected (P>0.995). For the propranolol containing diets the situation was more clear cut. t-Tests between the means indicate that after the two week feeding trial, animals on both of the high tannin diets had a higher proportion of protein in their bodies than those on the comparable low tannin diet (P>0.999).
It may appear counter-intuitive that the per- centages of body proteins for animals on high tannin diets exceed those for animals fed low tannin diets. However, the percentage of protein present at the beginning of the experiment (52%), as estimated from the animals killed at this time, was very close to the final values for animals on diets 3,4,7 and 8. Considering the very low dry weight gain of these animals and their near zero gain in body protein (Table 5),
188 SIMON MOLE ETAL
TABLE 5. PROTEIN CONTENTS OF RAT BODY AND FECAL MATERIALS, GAINS IN 8ODY PROTEIN BY RATS DURING THE TWO WEEK FEEDING TRIAL
AND RAT BODY LIPID CONTENTS. Proteins estimated as N × 6.25, weights in g, lipid contents as a percentage of total body weight
Diet ~ I t 2 3 4
Measurements Mean S.E.M Mean S .EM Mean SE M Mean SE .M
Protein in rats (%) 46.8 077 477 0.64 52.4 1 68 505 189
Protein in feces (%) 18.3 0.40 16.9 0.58 223 0.52 21,3 0 51
BNG1- g protein 313 0.26 2 27 0.37 0.33 0~11 0 16 0.55
*Diets numbered as in Table 2, which gives details of their consumption
/Means and their associated standard errors are given for the groups of five animals fed each diet. :I:BNG =: body nitrogen gain.
tannin appears to preserve the juvenile condition of the animals and prevent growth. In contrast, animals fed low tannin diets showed high net gains of body protein, estimated in terms of new nitrogen gained (Table 5). It was clear by inspec- tion that increased protein biomass on diets 1 and 2 was far in excess of that for diets 3 and 4. As well as this gain of nitrogenous material, other substances must have been synthesized to a relatively higher degree due to the lower percentage of protein in these animals' bodies. Estimates of body lipid content suggest that acquisition of fat reserves on low tannin diets may account, at least in part, for the above data. The data for this is presented in Table 5. t-Tests for the equality of mean values using combined data for high and low tannin diets allow for the rejection of the hypothesis of equal fat contents (P>0.95) in animals subjected to these two treatments. As with body nitrogen, animals on high tannin diets appeared to remain closer to the juvenile condition as the animals had an average of 11.8% lipid content at the beginning of the experiment.
Nitrogen consumption. In the analysis of growth on a dry weight basis (GR), it was shown that decreased consumption (CR) did not account for the depression of GR in rats on high tannin diets. The two sorghum cultivars used were chosen so as to have similar nitrogen con- tents so that a given quantity of food uptake entails approximately the same protein intake
from either feed. Analyses of the actual experimental diets showed that the protein con- tents of the high and low tannin diets were 8.54 and 8.35%, respectively, and differences in nitrogen consumption rate cannot explain dif- ferences in GR. The mean percentage of protein in fecal material (Table 5) shows that fecal protein levels were relatively high for animals on the high tannin diets. Comparing diets, t-tests indicate that fecal protein levels were higher on the high tannin diet (P3>0.99) irrespective of salt or propranolol treatment. This analysis suggests that low GR for high tannin diets should be examined through an analysis of ECI(N), the nitrogen based equivalent of ECI.
Analysis of ECI(N). An analysis of ECI(N) is presented in Table 6. First comparisons were made of diets without propranolol (1-4). As with ECI, ECI(N) was considerably lower for animals on high tannin diets. Like the data for AD, AD(N) was lower for animals consuming high tannin diets but unlike the data for AD, AD(N) was rather more severely affected so that the values for animals on high tannin diets were approx. 60% of those on low tannin diets. From this data, tannins do appear to have a disproportionately larger effect on protein digestibility than on the digestibility of the diet as a whole. This is con- sistent with the long held view that tannins act primarily by reducing the availability of nitrogen entering the body via the products of protein digestion. Evidence that this is not the whole
MECHANISM OF ACTION OF TANNINS
TABLE 6. GROWTH ANALYSIS ON A WEIGHT OF NITROGEN BASIS, FOLLOWING CALCULATIONS DETAILED IN TABLE 1 AND BELOW
189
Diet* I t 2 3 4
Measurements Mean S.EM. Mean S.E.M Mean S.E.M. Mean S.EM.
*Diets numbered as in Table 2, which gives details of their composition.
tMeans and their asociated standard errors are given for the groups of five animals fed each
~UNO-urinary nitrogen output=nitrogen ingested -- nitrogen in feces -- body nitrogen gain. diet.
story lies in the fact that AD(N) was substantially above zero and positive for each diet. To explain the near zero body nitrogen gain data for tannin containing diets (see Table 6), low ECD(N) must also be a component of the low ECI(N) values.
Now we consider the added effect of pro- pranolol (diets 5-8). Again AD(N) was lower for animals consuming high tannin diets and more severely affected than AD, so that the values for animals on high tannin diets were approx. 30- 34% of those on low tannin diets. Importantly, this was much more considerable reduction than seen previously for diets without propranolol. This confirms that tannins have a disproportion- ately large effect on protein digestibility and, more importantly, it shows that propranolol exacerbates this effect. Values of AD(N) for tannin-containing diets were approximately halved again in the presence of propranolol.
Generally, these results support the view that tannins can reduce protein digestion in a signifi- cant way. The effect of propranolol can be explained by a reduced proportion of proline-rich proteins, leading to tannins being bound to a larger proportion of dietary and other proteins in the gut. Evidence that low AD(N) is not the whole story behind the negative ECI values seen in Table 6 is demonstrated again by the fact that AD(N) was positive for each diet. To explain the loss of nitrogen, ECD(N) must be negative for negative ECI(N) values.
Examination of the data for ECD(N) shows that animals on high tannin diets containing pro-
pranolol had substantial negative ECD(N) values compared to those on low tannin diets. ECD(N) was also very low for high tannin diets without propranolol. Using t-tests the probability that ECD(N) for low tannin diets exceeded that on high tannin diets is highly significant (P>0.995).
Analysis of urine The main focus of these experiments at the time of their conception was to concentrate on measurements of animal growth and of feed and feces, but it quickly became apparent that the diets profoundly affected urine volume. The method for urine collection was unsatisfactory in that urine was simply allowed to collect into a test-tube via funnel. Evaporation losses will have been disproportionately large from animals pro- ducing urine in small amounts such that it wetted and then dried onto the funnel before reaching the collection tube. As this will have magnified the between treatment differences, the absolute values for urinary volume output need to be examined with caution. From data presented in Table 7, however, it is clear that animals on high tannin diets produce less urine than those on low tannin diets. Even if the mean values are divided by the final dry weights of the animals in the experiment to estimate the relative urinary volume output, the volume produced by animals on the high tannin diets still seems anomalously low (see Table 7). From these data it can be seen that both absolute and relative urine volumes were higher for the animals on salt sup-
190 SIMON MOLE ETAL.
TABLE 7. URINE VOLUME OUTPUT (UVO, mt) AND OUTPUTS OF UREA (Urea, g nitrogen), PROTEIN (UPO, mg), SOLIDS (USO, g) AND ELECTRO
LYTES (UEO, g NaCI)
Diet"
I t 2 3 4
Measurements Mean S.E.M. Mean S.E.M Mean S.EM Mean S.E.M
UVO 32.6 7.49 46.0 4.34 6.8 205 167 5.58
Urea 0.18 0.02 0.11 0.01 0.08 0.01 0 12 0.04
UPO 1.64 0.36 2.08 0.22 0.33 0.21 0.87 0.38
USO 1.78 2.35 0.72 0.69
UEO 1.52 0.61 111 0.41 0.38 0.68 0.69 0.33
5 6 7 8
Mean S.EM. Mean S.E.M. Mean SE.M. Mean S.E.M.
UVO 14.2 5.22 38.6 8.73 1.8 0.84 27 1 35
Urea 0.21 0.05 0.30 0.03 0.03 0001 0.08 0.02
UPO 0.98 0.41 2.04 0.86 0.86 034 0.12 005
USO 1.25 2.38 0.42 1.30
UEO 1.21 0.31 1.83 0.10 0.20 012 0.31 0.14
*Diets numbered as in Table 2, which gives details of their composition.
l-Means and their associated standard errors are given for the groups of five animals fed each diet.
plemented diets, but that salt did not eliminate the apparently antidiuretic effect of the tannin- containing diets.
Analyses on the urine were made to deter- mine the output of urea and protein in the urine. The data are presented in Table 7 in units com- parable with the urinary nitrogen output data in Table 6. The discrepancy between the directly measured urea output is large and probably due to bacterial contamination and ammonia losses. Considering all treatments together, the two sets of data show less urinary loss of nitrogen for the animals on high tannin diets (but see urea data for diet 4). When a correction was made for dif- ferences in N intake and AD(N), the percentage difference in loss of absorbed N is not greatly different between rats fed low and high tannin sorghum diets. Dividing these data by those for total urine volume showed that the urine was more concentrated in animals fed the high tannin diets. Thus urinary protein output can be seen to be a trivial source of nitrogen loss (see Table 7). Data on the output of solid material in the urine as well as the output of electrolytes estimated as salt are given in Table 7. This data reinforces the picture given above that the animals fed high tannin diets excreted less material in the urine but did so in a more concen- trated solution. The small weights of material recovered from urine samples also justifies ignoring urine as a factor in calculations of ECI, ECD and AD.
Intestinal enzyme activity Results for rats fed the basal diet un- supplemented with salt are given in Table 8. For each assay, the mean (and S.E.M.) for each enzyme activity is given for tissues sampled from four animals. For protease and amylase activities the data are somewhat variable due to the small sample sizes; however, no differences are apparent between the values obtained for high and low tannin diets in either of the assays. By contrast, the range of values for alkaline phos- phatase activities do not overlap and levels of this enzyme would appear to be low in the animals fed the high tannin diet. Alkaline phos- phatase is produced by the gut wall, not the pancreas (unlike trypsin, chymotrypsin and amylase). Using this as a marker, these results may indicate direct damage to the gut wall by tannins rather than inhibition of digestive enzymes, because the activities of predomi- nantly pancreatic enzyme activities (protease and amylase) are unaffected by tannins.
Fate of dietary phenolics Recoveries of (i) condensed tannins, (ii) flavan-4- ols and (iii) total phenolic material in the rats' feces were measured and calculated as the per- centages of these materials consumed in the feed which were obtained in the feces. On high tannin diets without propranolol these three recoveries were 1.6, 18 and 22%, respectively, while on low tannin diets they were 26, 55 and
MECHANISM OF ACTION OF TANNINS
TABLE 8. INTESTINAL ENZYME ACTIVITIES
191
Enzyme* Amylase Protease Phosphatase Mean S.E.M. Mean S.EM. Mean S.E.M
Diet Low tannin with no propranolol 1.21 0.28 0,13 0.01 0.90 0.07 Low tannin with propranolol 0.94 0,22 0,11 0,02 0.94 0.05 High tannin with no propranolol 0.79 0.29 0.14 0.02 0.36 0.01 High tannin with propranolol 0.68 0.08 0.26 0.02 0.62 0.07
*Activities expressed in units per g fresh tissue using Sigma Chemical Co. 0(-amylase, trypsin and alkaline phosphatase to prepare calibration curves.
20%, respectively. For diets with propranolol, recoveries of condensed tannins, flavan-4-ols and total phenolic material were 3.5, 33 and 21% while on low tannin diets recoveries were 30, 33 and 57%, respectively. The data for low tannin diets need cautious interpretation given the low initial level of phenolics. Insignificant amounts of phenolic material were excreted in the urine. From this it follows that the unrecovered phenolic material was either metabolized to non- phenolic materials or it became bound to fecal material in an inextractable form. We consider both these fates to be likely.
Comparing the recovery data for animals on diets with and without propranolol, recoveries were higher for animals fed propranolol for every comparison of the means for high tannin diets and in most cases for the low tannin diets. This may indicate either metabolic differences or differences in extractability. To help resolve this question, extracts of feed and fecal materials were compared by HPLC techniques optimized to separate low molecular weight sorghum phenolics (D. Netzley, unpublished data) or optimized to separate condensed tannins by polymer length [22]. Chromatograms of feed and fecal samples were quite similar. No evidence could be found by way of altered retention times or diminished peak heights that suggested any significant metabolism of the phenolics during their passage through the gut. For the high tannin diets, we performed the simple experi- ment of wetting samples of the diets (3,4,7,8) and incubating them at 37°C overnight. This process reduced the extractability of tannins and other phenolics by 90% relative to samples of diet which were extracted (in methanol) without first being wetted. This and our HPLC data suggests that low recoveries of dietary phenolics
in fecal material does not imply any metabolism during their passage through the gut. Rather, the simple process of wetting the diet during masti- cation, which allows combination of dietary phenols and proteins, may explain the apparent "digestion" of the phenolics in terms of altered extractability [23].
Levels of fecal proline The critical result required to support the hypoth- esis that tannins bind to proline-rich proteins (PRPs) in vivo is that PRPs should be found in the feces of animals consuming tannins and which are able to produce PRPs. The proline richness of the feces is given in Table 9. Animals with the most proline-rich feces were those fed high tannin diets in the absence of propranolol. These animals produced PRPs which were rendered relatively indigestible by combination with tannin so that they remained in the gut and exited in the feces. The lowest fecal proline richness was found for diets 1 and 2 for animals not fed tannin or propranolol. These animals produced rela- tively low levels of PRPs [23] which are digested in their passage through the gut. Sorghum grain itself contains a moderately proline-rich kafirin protein which strongly binds tannins and may account for the proline richness of the feces on diets containing propranolol [14]. The lower proline richness of the feces for diets 7 and 8 (tannin plus propranolol treatment) and the low AD(N) for these diets does support a mechanism of action for tannins that involves reduced levels of PRP leading to reduced protein digestion.
Discussion Effect of tannins on a mammalian system These results show that in the monogastric digestive system studied, the major effect of
192
TABLE 9. PROLINE CONTENT (PRO) AND PROLINE RICHNESS (PR) OF FECAL PROTEIN
SIMON MOLE ETAL
Diets*
1 2 3
PR 0.076 0.017 0071 0.005 0.214
PROt 754 0.36 555 0.46 19.6
5 6 7
Mean S.E.M. Mean S.E.M. Mean
PR O. 11E 0.022 0110 0.007 0.147
PRO 7.45 0.38 7.52 0.45 17.6
4
0016 0178 0010
046 188 0 69
6
SE.M. Mean S EM
0.037 0127 0006
0.91 187 ] ! 3
*See Table 2.
tUnits of mg g sample '.
tannins is not to reduce the consumption or the gross digestibility of the diet. Instead, the major effect is on the efficiency with which digested and absorbed material is converted to new body substance (ECD). When the impact of tannins on nitrogen nutrition is considered, more of an effect on AD(N) is seen although tannins still depress ECD(N) to a great extent. A possible interpretation is that tannins cause nitrogen starvation by reduced consumption and protein digestion and that low AD(N) leads to the in- efficient utilization of non-nitrogenous nutrients and thus to low ECD values. This assumes that carbohydrates are digested and absorbed but they are then respired due to the lack of nitrogen available for biosyntheses and further anabol- ism. The low ECD(N) values might be explained by arguing that they reflect the proportionally greater contribution of basal nitrogen excretion. Nitrogen starvation caused by a reduction in protein digestibility could thus be seen as the main effect of tannin while lowered ECD(N) is the necessary consequence of a fixed and obligate turnover of body nitrogen.
The data are also consistent with another and essentially opposite interpretation. It is conceiv- able that tannins, associated phenolics or tannin degradation products require detoxification to such an extent that carbohydrates are utilized and energy is no longer available for growth. Here reduced ECD, not nitrogen starvation, is seen as the primary effect of tannins. This view is consistent with the greater body fat content of animals on low tannin diets and the lack of any effect on protease and amylase activity by tannins.
The last point concerning digestive enzymes raises a problem when considering the result
that tannins reduced AD(N) more than AD. Because the processes of digestion and absorp- tion require active enzymes and other proteins for their correct function, then if protein digestion is specifically reduced, tannins must be shown to inhibit proteases more than other enzymes, e.g. amylases. We did not find this. The result indica- ting that only alkaline phosphatase activity was depressed suggests that pancreatic enzyme production is unaffected, although enzymes produced by the gut wall may be affected. This could be interpreted as evidence that tannins damage the gut wall and inhibit absorptive or other processes located there. Thus, digestion in the gut lumen could be relatively unhindered, but absorptive processes might be impaired. Even though gross morphological changes in the gut wall due to condensed tannins have not been found [24], this does not rule out such effects at the biochemical level.
The specificity with which tannins and pro- teins interact [12] also leads to the possibility that tannins bind selectively to particular proteins. This could explain the differences in enzyme activity seen for protease and amylase activity vs that for alkaline phosphatase activity. The very reason that enzyme activity can be measured at all may be because tannins are bound to other proteins such as those from the diet or the mucous and the mucosal lining of the gut (i.e. endogenous, non-dietary protein). An interesting question arises as to whether the tannins in the diet have a higher affinity for binding to protein or dietary or endogenous origin. It is entirely possible that tannins bind to non-dietary proteins or specific classes of dietary proteins, and prevent their digestion and re-absorption or absorption into the body. Most dietary proteins
MECHANISM OF ACTION OF TANNINS 193
could then be digested and absorbed in the presence of tannins. In terms of the present fecal nitrogen data, it is not possible to distinguish endogenous vs dietary protein losses. Only future experiments, for instance using 15N- labeled dietary proteins, will determine the extent to which tannins reduce the digestibility of dietary protein relative to those originating from the animal's body.
Because both ECD and ECD(N) were de- pressed by high tannin diets, the diets may have contained substances altering metabolism after absorption from the gut. It has been an article of faith that condensed tannins, such as those present in sorghum grain, are of too large a molecular weight and too polar in nature to be absorbed into the body. In contrast, it is import- ant to realize that reports of leg abnormalities [9], acute toxicity [25], unusual urinary metabolites [25], increased liver UDP-glucuronyl transferase [26], altered thyroid hormones [27] and the induction of proline-rich protein synthesis [19] all indicate effects of condensed tannin containing diets that are not readily explicable in terms of this viewpoint. Resolution of this situation is a goal for future research. A possible explanation is that tannins perse do not act as causal agents for these effects, but that they co-occur with other phenolics (e.g. flavonoids) which are readily absorbed and are pharmacologically active. It is also possible that microbes present in the intesti- nal microflora produce toxins from either the breakdown of tannins or by the modification of co-occurring phenolics. Low molecular weight phenolics present in, or produced from, the high tannin diet may have been the antidiuretic agents in the present experiment. The results indicating low recoveries of tannins and other phenolics in fecal material leave the problem of toxic metabolic product(s) of tannins open for investigation.
Tannins and nutritional significance of PRPs The absence of PRPs, produced as the effect of dietary propranolol, is clearly to exacerbate the growth reducing action of tannins. This result is all the more convincing because the animals fed low tannin RS610 diets with propranolol (diets 5 and 6) performed as well or better than those fed the same diets without propranolol (diets 1 and 2). No formal statistical interaction between
tannin and propranolol has been attempted using analysis of variance techniques because of the confounding factor of time, introduced by starting animals on high tannin diets with pro- pranolol a week later than those without pro- pranolol. The interaction can readily be seen by inspection [e.g. consider results for AD(N)]. We now have results (unpublished) from the alterna- tive experimental design where animals are all put on experimental diets at the same age but where exposure to propranolol also starts at the same time: this data confirms the indication of an interaction suggested here.
Apparently, the absence of PRPs causes an animal on a high tannin diet to digest nitro- genous nutrients less well [low AD(N)]. We may hypothesize that the PRPs in animals on diets 3 and 4 bind some of the tannin allowing for more complete protein digestion than on diets 7 and 8 where more dietary protein is lost in tannin- bound complexes. This hypothesis is supported by the results for the proline richness of the fecal material, notably by the low PR values for diets 7 and 8 vs 3 and 4.
The mechanism of action of tannins The present results provide clear evidence that the effects of propranolol and tannins interact in the statistical sense. Their limitation is that they do not provide direct evidence as to how propranolol and tannins interact in the chemical sense. If the effects observed above are not due to PRPs and tannins interacting in protein diges- tion, then the possibility has to be considered that tannins or related phenolics enter the body tissues and interact with the antagonist adrener- gic activity of propranolol. If tannins were to act this way, it would be an effect not previously considered. Our lack of evidence for any metab- olism of dietary phenolics (based on recovery experiments), suggests that the current prejudice against the possibility of toxic polyphenolics entering the body may be correct. However, we cannot rule out a highly potent component acting at low concentrations, masked by the other dietary phenolics present. The fact that tannins have little effect on AD as opposed to AD(N) suggests that carbohydrate digestion may be little affected. The digestion of protein and the subsequent absorption of amino acids seem to be the two main possible sites for the action of
194 SIMON MOLE ETAL.
dietary tannin. The absence of any effect of tannins on levels of amylase or protease activity suggests that tannins may in some way be prevented from direct inhibition of enzymes. Experiments to study the effect of tannins on absorptive processes might be revealing, for instance it would be interesting to repeat the present work using amino acids or protein hydrolysates in place of the dietary protein. Previous studies demonstrated that the toxic effects of tannin could be overcome with intact protein but not with crystalline amino acids in the same concentrations supplied by the intact protein [10]. Little consideration has been given to possible effects of tannins on non-digestive aspects of physiology. The antidiuretic effects reported here may indicate a pharmacological activity within the body although it could be a secondary effect of tannin disordered metabol- ism. The absence of any effect of salt supple- mentation contradicts the reported mineral- tannin interaction seen in mice [17] and it is not helpful in interpreting urinary volume output.
Insect-mammal comparison One important goal of the present work has been to provide data comparable with that available for insects. Understanding the comparative effects of tannins on both insects and mammals will, at the ecological level, help us understand the allelochemical role of tannins in plants which must defend themselves against both types of herbivore. In broad terms, rats fed tannins give results comparable to insects in that AD is only slightly affected relative to ECD. However, the reduced growth of insects fed tannins can also be attributed to lower CR (and RCR). For instance larvae of the southern armyworm (Spodoptera eridania) show a 14% reduction in RCR when fed a diet supplemented by 2.5% quebracho tannin [28]. These larvae showed AD and ECD values of 41.9 and 80.7%, respectively, for a control diet, but 41.9 and 49.4% for the quebracho supplemented diet [28]. The main effect of tannin was on ECD, but low consumption also contributed to diminished growth. A further example of an insect that is affected by tannins is provided by the desert locust (Locusta migra- toria). Bernays et al. [7] showed that by supple- menting a diet with tannic acid, ECD was reduced from 42 to 18% while AD remained at
34%; nevertheless lowered feed consumption also contributed to poor growth. Manuwoto and Scriber [28] also fed a lepidopteran tree feeding specialist (promethea silkmoth, Callasamia prom- ethea) the same diets as the armyworm and in this species growth was not affected by tannins. More impressive evidence that tannins do not always reduce GR in insects is that of Bernays and Woodhead [29] who showed that the acridid Anacridium melanorhodon thrives on tannic acid to the extent that AD and ECD are increased by supplementing a lettuce diet with this tannin. It remains to be seen whether any of the tannin specialist mammals can perform in this way or whether the variability in effects seen in insects is absent in mammals.
An interesting comparison on which further information is needed concerns AD(N). Manu- woto and Scriber [28] showed that AD(N) (NUE in their paper) was not affected by tannins in the diet of the armyworm. This is in contrast to the present results where we show that AD(N) is depressed by tannins. Further evidence with other species is needed to resolve the question of whether these different results are typical for mammalian and insect systems. If they are, then this will point to different biochemistries in these digestive systems. Exploration of this point will yield insights both into comparative animal physiology and into the way plants utilize tannins as a defense against insect and mammalian herbivores.
Use of Waldbauer and conventional growth analyses This work has utilized the type of growth analysis commonly seen in entomological studies but not usually applied to mammals. Some consider- ation of the merits of this kind of analysis is worthwhile as we see advantages beyond the simple fact that comparisons with insect studies can be made by using it with mammals.
At the outset it is worth stating that we do not see conventional growth analysis techniques used with mammals as either wrong or generally inferior to entomological methods; indeed, both methods have much in common. For instance AD in this study is identical to the "apparent digestibility" normally calculated in feeding trials, assuming that both are calculated on a dry weight basis. ECl is also the reciprocal of the feed
~r
MECHANISM OF ACTION OF TANNINS 195
to gain ratio commonly calculated in feeding trials with vertebrates. As such, AD and ECI yield no additional information as a single index of performance. To be of any additional value, as in the context of this paper, they must both be calculated in terms of dry weights and it is diffi- cult and uncommon to do this, especially with feed/gain. The term ECD, new to vertebrate growth analysis, is entirely dependent on dry weight measurements. It would make little sense to express body weight gain in terms of net food and water intake. It is the ability to construct the expression ECI~AD×ECD on a dry weight or nitrogen basis that makes the present analysis interesting in the context of digestive physiology. The main benefit is in being able to separate digestive (AD) and postidigestive (ECD) contribu- tions to ECI. Additionally, the analysis of growth in terms of consumption and ECI (GR =CR × ECI) is also useful in feeding studies.
There may be little to be gained in using these Waldbauer ratios in extensive studies with a single species. For instance, optimizing poultry feeds, feed/gain measurements may yield all the required information so long as animal water balance is not affected. Even in this event, the fresh weight yield of meat may be the ultimate interest of the feeding trial. However, when making estimates of the comparative feed efficiencies of radically different species, e.g. bird, mammal, reptile on similar diets, relative water contents and changes in water content become a problem. Consider the initial weights of animals in the present experiments where the older propranolol-treated animals began the trial with heavier live weights but lower dry weights than their younger counterparts not fed pro- pranolol. Including the water in an animal's tissue weight may lead to overestimation of its metabolic capacity to which water per se does not contribute.
For the above situations where comparative measurements between distantly related taxa are required, the use of experiments based on the ECI=ADXECD model seems to be the best way to compare these ecological and physio- logical efficiencies. Such comparisons may be interesting with both nutritious and challenging diets, e.g. those containing allelochemicals such as tannins. The fact that the effect on AD and the digestibility of a specific nutrient differed [AD(N)]
shows that allelochemical effects on specific components of the diet can be traced using this methodology. The demonstration that the drug propranolol interacts with tannin to alter AC)(N) shows that the interaction between drugs or toxic allelochemicals can be examined in synergism with materials such as polyphenolics.
In further work with tannins and their effects on different mammals, some acknowledgement of the limits to this type of work must be made. The need to dry weigh the animals would seem to preclude the use of large herbivores. Further- more, the need to kill animals necessitates the use of specimens from laboratory breeding colonies and/or large natural populations able to sustain such losses.
Further studies with insects would be desir- able for comparative purposes. With regard to mammals, a number of species of rodent have been the subject of recent field studies [30, 31] and these would be of suitable size and com- parability with the rats of the present work. Squirrels, which can be found in abundance and which consume tannin-rich acorns, would also make for an interesting study by this kind of growth analysis. If tannin-induced synthesis of PRPs occurs in squirrels, a further experiment would be to follow PRP production through seasonal- or damage-induced fluctuations in the dietary intake of tannins. Small marsupial species would be an interesting comparison as many are hind-gut fermentors and consume tannin-rich eucalyptus foliage. The examination of a coprophagous herbivore eating normally and restrained from eating fecal pellets would be amenable to an ECI=AD×ECD analysis, especially to show the stage at which nitrogen became digestible and whether tannins affected this.
Experimental Animals. Weanling rats of the Sprague-Dawley strain (Harlan Laboratories, Indianapolis) fed diets containing ground sorghum grain were used.
Formulat/on of d/ets. The diets were of ground sorghum supplemented with vitamins, minerals and lipids to satisfy National Research Council (U.S.A.) requirements. The sorghum grain was from the 1984 crop grown at the Purdue University Agronomy Farm. Grain was from sorghum hybrids RS610 and Savanna (DeKalb), the latter is a high tannin bird resistant type. Experimental diets were constructed from the basal diet supplemented with either 0.5% salt (NaCI) and/or 0.05% propranolol with the balance (to 0.55% weight) being made up
196 SIMON MOLE ETAL
with corn starch. The construction of diets 1-8 are summarized in Table 2. In a second set of experiments where the animals were grown to provide samples of intestinal tissues, the diets were as above except for the omission of the 0.5% salt or corn- starch.
Feeding and sample collection. For the main experiment, 52 male rats were fed Rodent Block (Wayne Feeds) for one day after delivery. Forty animals were then weighed and allocated to eight treatment groups of five animals based on their body weight so that the mean weights of each group at the start of the experiment were approximately equal. The remaining twelve animals also had a similar group mean weight, and six of these were killed with CO 2 gas and deep frozen for future use. The 20 experimental animals to be fed diets without propranolol were individually housed in metabolism cages and placed on experimental diets for a two week period. All other animals were individually housed and fed diet 5 for one week, after which a group of six animals was killed and frozen. The remaining four groups of five animals were then placed on the four propranolol containing diets for two weeks.
During the two-week period of the experiment in which the experimental diets were fed to the eight groups of animals, the animals were fed and watered ad libitum and the weight of feed consumed was recorded, Fecal pellets were collected every two days from a wire retaining grid under the cages. Urine passed through this grid and was collected via a funnel into a test tube. A small plug of glass wool prevented solid material from entering the urine collection tube. Urine was collected every two days and frozen. Collections of fecal material were also stored frozen. At the end of the two-week period on the experimental diets, all of the animals were killed with CO 2 and frozen intact.
In a subsequent experiment animals were grown to provide samples of intestinal tissues. Twenty male weanling rats (same strain and source) were fed Rodent Block to the same age as the animals in the main experiment. They were then divided into two groups of equal mean weight placed in individual cages and fed diets similar to those of experiment one (but no salt treatment) for a period of 10 days. They were then killed and immediately dissected; approximately 45 cm of intestine proximal to the stomach was removed and frozen by immer- sion in liquid nitrogen. These samples were stored frozen for future use.
Laboratory procedures. After the fresh and frozen weights of the 40 rats from the main experiment were recorded, the bodies were rapidly crushed into small pieces and each quickly transferred to a pre-weighed paper bag. It was found to be unnecessary to correct for weight losses due to the crushing procedure. The bag containing the crushed material was then lyophilized to dryness and the dry weight of the animal was recorded. The initial live weights of the 20 animals which underwent the two week experiment were recorded directly, but their initial dry weights had to be estimated from the dry weights of the six animals killed at the beginning of the experi- ment. The estimated values of initial dry weight were calcu- lated as the product of their initial live weight and the mean fresh weight/dry weight ratio of the six animals killed at the time the feeding trial began. Dry and fresh weight gains were calculated as the differences between final and initial weights.
After weighing, the lyophilized material was ground to a fine powder in a Wiley mill. All the fecal material produced by each rat over the two-week period was lyophilized, weighed, ground to a powder, and bulked for each animal. Samples of
the feeds were lyophilized to measure their fresh weight/dry weight ratio and the weight of feed consumed by the animals during the experiment was converted to a dry weight equivalent using these ratios. Urine samples from each rat were bulked to give one sample per animal, and total urine volume output was measured. A sample of urine from each rat was lyophylized to estimate the concentration of urinary solids. The product of this and the total urine volume yielded the weight of dry matter output in the urine.
Samples (1 g) of pulverized rat were extracted with CHCI:~, filtered, dried and re-weighed to give an estimate of lipid content in the material. Samples of feed, powdered rat and feces were analysed for their total nitrogen content by the Kjedahl method. The protein content in these materials was estimated as N% x 6.25. The total quantities of protein in feed, fecal and animal body matter were determined from these and their dry weight data. Body nitrogen gain was calculated as the difference between final and initial weights of protein in the body.
Samples (0.5 g) of feed and powdered feces were extracted with 80% v/v aqueous MeOH for 2 h and the filtered extracts were assayed for total phenolics by the Prussian Blue method [32] and for condensed tannins and flavan-4-ols by the butanol/HCI method [33] in order to estimate the recoveries of these substances in the feces. Dry weighed samples of feed were also moistened with water to form a stiff paste and then incubated for 24 h at 37°C. This material was then dried and assayed for tannins and phenolics to measure the quantity of these substances rendered inextractable simply by wetting.
Analyses were also made to determine the quantity of proline residues in feed and fecal protein on (i) an absolute basis and (ii) relative to the quantity of other amino acids present. To accomplish this, samples (500 mg) of dry ground material were hydrolysed in 8 ml of 6 N HCI maintained at 100°C for 8 h in a screw-capped test tube. After this time, 4 ml of 11 N NaOH was added to partially neutralize the acid. Unhydrolysed controls were made by mixing the same quantity of fecal material with 4 M NaCI solution (controls were not heated). Both the hydrolysates and the unhydrolysed controls were assayed for proline and amino acids. For amino acids, a colorimetric ninhydrin based procedure was used following Mole and Waterman [11]. Proline was assayed following Trol and Lindsley [34], which again employs ninhydrin but under different reaction conditions.
The absorbances measured in these two assays were used to calculate the proline content and proline richness of protein present in the sample using measurements on unhydrolysed samples to control for free amino acids and proline. Where Ap is the absorbance in the assay for proline and Aa that for the assay of amino acids, proline richness of the protein (PR) was calculated as a unitless ratio of two absorbances.
Ap (hydrotysed)-- Ap (unhydrolysed) PR=
As(hydrolysed)- Aa(unhydrolysed)
In calculating PR, dilution factors have been employed to account for the differing volumes used in the two assays such that PR is the ratio of A ; values from the two measurements. In addition, results are also presented for the absorbance due to the proline hydrolytically released from protein (PRO). Thus PRO is equal to "Ap(hydrolysed)--Ap(unhydryolysed)" in the terms used above, with the values for absorbance expressed in mg of proline per gram of dry sample,
MECHANISM OF ACTION OF TANNINS 197
Samples of urine were assayed for total phenolics as well as for urea, protein and electrical conductivity. Urea was measured using the methodology and reagents of the Sigma Chemical Co. Kit (No. 640). Urea output is given as mg urea m1-1, the total output being the product of this and total urine volume. Protein was measured by the dye binding method of Watanabe etal. [35], using a solution of bovine serum albumin (BSA) to provide a calibration curve so as to express results in terms of mg BSA. Conductivity was measured potentio- metrically using a conductivity bridge (Yellow Springs Instru- ment Co.) and results were expressed in terms of sodium chloride by reference to a calibration curve.
Assay of enzyme activities. Frozen intestines were carefully cleaned of extraneous material such as mesentery or adipose tissue. The intestine was then pulverized into small pieces and 0.5 g of this was thawed in a culture tube containing buffer (100 mM potassium phosphate, pH 6.8). The contents of the tube was then homogenized with a polytron homogenizer and kept cool during the process by immersion in ice. The homogenate was then centrifuged at 5000 g and the super- natant was kept on ice and assayed as follows. Protease activity was determined by the release of TCA-soluble, ninhydrin-reactive products from the degradation of BSA at pH 6.8 using the method of Mole and Waterman [11]. Amylase activity was measured using Bernfield's reagent [36], while alkaline phosphatase activity was measured using the method of Landt and Butler [37]. All assays were calibrated relative to commercial standards of known purity obtained from Sigma Chemical Co., and results are expressed as quantities of these standards per g of fresh intestinal tissues.
Data analysis and terminology. The elegance of Waldbauer's method lies in the systemic analysis of the variables measured above. Defining formulae for the indices presented by Wald- bauer [18] and parallel indices which are appropriate to nitrogen nutrition are given in Table 1. To ease comparison with other published work, the terminology of Slansky and Feeny [38] is also given. Formulae for additional indices which exploit the fact that excreted and egested products exit the mammalian body separately as urine and feces are given in Tables 4 and 6 (these products are combined as frass in insects). Means, standard errors, confidence limits, t-tests and combined probability procedures have all been calculated following Sokal and Rohlf [21].
Acknowledgement--This work was supported by USAID/ INTSORMIL, Journal paper number 11,849 from the Purdue University Agricultural Experiment Station.
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