Drug Metabolism Reviews
ISSN: 0360-2532 (Print) 1097-9883 (Online) Journal homepage: http://www.tandfonline.com/loi/idmr20
The Influence of Dietary Factors on Drug Metabolism in Animals T. Colin Campbell, Johnnie R. Hayes, Alfred H. Merrill, Martha Maso & Mabel Goetchius To cite this article: T. Colin Campbell, Johnnie R. Hayes, Alfred H. Merrill, Martha Maso & Mabel Goetchius (1979) The Influence of Dietary Factors on Drug Metabolism in Animals, Drug Metabolism Reviews, 9:2, 173-184, DOI: 10.3109/03602537908993889 To link to this article: http://dx.doi.org/10.3109/03602537908993889
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DRUG METABOLISM REVIEWS, 9(2), 173-184 (1979)
The Influence of Dietary Factors on Drug Metabolism in Animals* T. COLIN CAMPBELL, JOHNNIE H. HAYES, ALFRED H. MERRILL, JR., hlARTHA MASO, and MABEL GOETCHIUS Division of Nutritional Sciences Cornell University Ithaca, New York 14853
I. 11.
111.
IV.
.. .-.... . ... .. -. ..-. DIETARY PROTEIN E F F E C T .- - ... -- - -A. In Vitro Metabolism .......... .......... .......... B. In Vivo Metabolism.. ................ ....... . ..... EXTRAPOLATION O F IN VITRO ENZYME ACTIVITIES ........................................ A. In Vitro Substrate Concentration ....... ............ U. In Vitro Weight Basis ... .......................... C. Relative Rate Limitations ......................... .... . .-..... .... CONCLUDING REMARKS ..... Acknowledgments ................... ........... ...... References ......................................... INTRODUCTION
m
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*. *
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174 175 175 176 179 180
181 181 182 182 183
*Presented a t Symposium on Drug Disposition in Man held in Sarasota, Florida, November 6-11, 1977 under the a u s p i c e s of the American Society for Pharmacology and Experimental Therapeutics. 173 Copyright 0 1979 hy Marcel Dekker. I n c . All Rlghts Reserved. Neither this work nor any part may he reproduced or transmitted in any form or by any means, electronic or mechanicd. including photocopying. microfilming, and recordinp.. or by any information storage and retrieval system. without permission in writing from the pub sher.
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I. INTRODUCTION One hardly needs to belabor the point that nutrient intake, when not in balance, can markedly alter the activities of so-called drug metabolism reactions. This general area has been recently reviewed elsewhere [l, 23 and has become a topic for discussion at numerous symposia and workshops in the last 2 to 3 years. Various suggestions have been made that both pharmacological response and risk to toxic chemicals may be influenced by nutritional status when metabolism of such chemicals determines the duration and intensity of response. This type of interaction should not be confused with the effect of nonnutrient components of food on drug metabolism reactions, These latter components, which may include synthetic and adventitious chemicals, intentional food additives, and natural compounds, a r e known to be effectors of drug metabolism, primarily a s inducers of enzyme activity [3-51. A rather large literature which has recently evolved shows that virtually every nutrient, when not ingested at optimum levels, can influence drug metabolism reactions in the experimental animal. With the exception of the guinea pig, which has been employed for research on vitamin C, most of these studies have been conducted with the laboratory rat. In general, a suboptimal intake of the nutrient tends to depress enzyme activities, although there a r e important exceptions. For example, intakes of iron [6] and thiamine [7] a r e said to vary inversely with the rate of metabolism for certain mixed function oxidation (MFO) catalyzed reactions, Furthermore, the activities of certain transferases [8, 9 1 may be increased with a low dietary intake of protein, Because consistency of nutrient effects on enzyme activities has not yet been found, it is somewhat premature in most cases to relate in vitro activity measurements with toxio o r pharmacologic response in the intact animal. Nevertheless, the ultimate clinical significance of this type of information would be greatly enhanced if in vitro enzyme measurement could be related with drug response for the intact animal. Thus the purpose of this paper will be to present some basic data on the intake of one of the major nutrients, i. e., protein, as it relates to drug metabolism reactions and then to discuss how such experimental data may o r may not relate to whole animal response, Dietary protein intake is selected for this discussion because 1) the effect on drug metabolism reactions i s striking, 2) its intake i s known to vary severalfold between and within human population groups, 3) relationships between metabolism and whole body response are somewhat better known, and 4) the research data a r e best known to the authors,
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11. DIETARY PROTEIN EFFECT
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A.
In Vitro Metabolism
When weanling rats were fed a 5% casein semipurified diet for 2 weeks, MFO enzyme activities a r e depressed by 60 to 75% [lo, 111. About one-fourth of this depression can be accounted for by a decreased quantity of microsomal protein; the remaining three-fourths of the decrease results from a specific effect on the existing enzyme system. Other than certain minor differences, animal response is quite similar throughout the growth period between 3 and 9 weeks of age. Feeding the 5% casein diet at either 22, 37, o r 52 days of age immediately and completely interrupted body growth and increased the liver size relative to body weight (unpublished data). The livers of the low protein animals a r e characterized by an approximately 50% increase in cell size for 50 to 75% of the cells and 220% more lipid after 2 weeks of feeding. Liver microsomal protein, which inoludes the MFO enzyme, is quickly depressed by the low protein diet, primarily because of losses per cell, even though there is a compensatory increase in tissue size in these animals. Tissue hyperpktsia, as indicated by total tissue DNA content, immediately ceases when the low protein diet is fed. These responses a r e somewhat more significant in animals 22 days of age than in animals 37 days of age. When the 22-day old animals a r e fed the 5% protein diets for 1 to 2 weeks and then refed the 20'& diets, complete restoration of cellular microsonial protein and tissue growth requires about 2 to 4 weeks, These studies therefore indicate that that portion (25%) of the enzyme activity which is depressed because of the depressed rate of cell proliferation and companion intracellular microsomal enzyme content can be readily recovered if protein deprivation is not too severe. The more significant effect of protein deprivation on IVIFO activity, a s mentioned above, is that which depressed the existing enzyme activity. We originally found that both cytochrome P-450 content and cytochrome P-450 reductase activity were reduced t o about the same extent a s the depression of total MFO activity [lo, 113. The most obvious conclusion drawn from such data was that either one of these components could have accounted for the depression, depending on which one was rate-limiting. However, in more recent studies [12) we fractionated the microsomes into their three major components (cytochrome P-450, cytochrome P-450 reductase, phospholipid) and then reconstituted the components from each diet treatment group s o that an independent examination of the activity
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of each component from each group could be made. There was no significant effect of diet on the phosphatidylcholine activity even though it is known that fatty acid substitution may be influenced by dietary protein and even though such chemical substitution may influence in vitro MFO activities, The MFO components most affected by diet treatment were the reductase and cytochrome P-450 fractions. Both of these protein components obtained from the 5y0 protein animals, when recombined on an activity basis equivalent to the ZOo/o protein animals, were not able to reconstitute the expected activities, This suggested that the interaction between these components could be the primary mechanism responsible for the dietary protein effect a s opposed to the effect on the specific activity of either component alone, Further physiochemical measurements of the membrane-bound enzyme system (unpublished data) has suggested to u s that the arrangement of components within the membrane is more rigid in the 5% protein animals and perhaps is less able to permit translational mobility of one component relative to the other. In more recent studies we have found that the depression of MFO activity associated with low dietary protein intake occurs extremely rapidly (Fig, 1). When weanling rats were pair-fed either the 5 or 20% protein diets, a greater than twofold difference in MFO activity was established within 24 hr. This difference became progressively greater up to 4 days a t which time the activity for ethylmorphine Ndemethylation in the 5% protein animals was almost too low to measure, The decline observed after 4 days is due to the method of expressing activity on a whole body basis; activity per gram of tissue o r milligram of microsomal protein begins to plateau at this age. In other studies (Fig. 2), animals were fed the high and low protein diets for 2 weeks and then reversed to the opposite diet. If animals were first fed the 5% protein diet, then re-fed the 20% protein diet, the enzyme activity was recovered in about 2 to 4 weeks. Thus the MFO activity seems to be quickly depressed upon feeding a low protein diet but is less responsive to recovery upon feeding the high protein diet. B. In Vivo Metabolism Extrapolation in vitro of MFO enzyme activities to the whole animal response may be possible where drug metabolism remains straight forward, simple, and rate limiting, That is, i f the drug is cleared primarily by an MFO-catalyzed reaction to a product(s) with lesser activity, then low protein intake should prolong clearance and
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f
DAYS ON DIET FIG. 1. Rat liver ethylmorphine N-demethylase activity at; related to the consumption of semipurified diets containing either 5% casein o r 207" casein. Weanling rats, 22 days of age, were pair fed the two diets to equalize total feed and caloric intake.
duration of action. Thus Kato et al. [13] were able to show that the level of dietary protein intake was directly correlated with delayed serum clearance and prolonged anesthesia when phenobarbital was administered to rats, Numerous other examples were discussed by Burns [14]. On the other hand, for those compounds where toxic activities depend on the production of a chemically reactive intermediate (.RI), interpretation of dietary effects on MFO activity is less clear. There a r e at least two major reasons for this. First, it is not the rate at which the RI is produced by the MFO system but the quantity which is produced and which reacts covalently with the target site [15, IS]. Second, the quantity of this MFO product which is available for covalent interaction may be affected by the activity of ancillary clegradative reactions rather than the MFO system which forms the R1.
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8
15
30
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DAYS ON DIET FIG. 2. Effect of dietary protein replenishment on rat liver ethylmorphine N-demethylation and aniline hydroxylation. Weanling rats, 22 days of age, with 7 to 1 0 groups and 3 to 6 animals per group, were fed semipurified diets ad libitum a t different times in same animal facilities. Diets: 20% casein (solid line), 5% casein (dashed line).
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The absence of a clear relationship between MFO activity anti toxic response is probably best illustrated by the data which show that low protein intake and phenobarbital administration produce contrasting effects on the metabolism and carcinogenicity of aflatoxin. MFO activity is depressed by protein intake but increased by phenobarbital administration. Yet both treatments apparently decrease the availability of the MFO-catalyzed aflatoxin epoxide, considered to be the chemically reactive intermediate which covalently binds DNA [ 117, 183. Moreover, both treatments depress hepatoma formation [ 193. Hypothetically, MFO activity may depress tumorigenicity with the protein-deficient animals but this activity cannot explain the phenobarbital effect. However, it is somewhat less tenable to assume that MFO metabolism is rate limiting for the controls in one case and not the other. Therefore, it would be useful to know the relative rake limitations before the effects of these various treatments can be extrapolated to the whole animal.
111.
EXTRAPOLATION O F IN VITRO ENZYME ACTIVITIEiS
This last section will briefly consider, then, a few questions which prevent simple interpretation and extrapolation of in vitro measurements of MFO activity to whole animal response. Our own research, a s well a s that of most other groups, has either explicitly or implicitly depended on a couple of important assumptions; namely, that for whole animal response, 1) metabolism is rate limiting and 2) in vitro enzyme activity i s an acceptable index for in vivo metabolism. Because reports on nutritional effects often reach contrasting conclusions, it becomes worthwhile to list a few questions concerned with the extrapolation of such in vitro data. For the purpose of this discussion, I will accept the first assumption that metabolism is rate limiting when compared to gastrointestinal absorption, protein binding, cellular uptake, etc. There is considerable evidence that this is true in many instances since the rates of plasma clearance of compounds such a s phenobarbital [ZO], aminopyrine [21], and theophylline [21] appear to be primarily a function of metabolism by the MFO system. This type of research in humans is, in fact, largely limited to in vivo measurements of pilasma clearance times and is a subject to be discussed elsewhere in this symposium (Kappus). Nevertheless, a comparison of in vitro data with in vivo response is oftentimes in conflict and is the focus of the next sect ion.
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TABLE 1
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Hypothetical Kinetic Parameters for Enzyme Reaction under Influence of Two Treatments
Treatment
'max (nmoles/mg/min)
Km
Vn
A
100
1.0
0.99
B
50
0.1
4.55
a
Assuming substrate concentration of 0.01 mM and v A, with A equal to substrate concentration. A.
-
(V)(A)/k,
+
In Vitro Substrate Concentration
What can be said about rates of metabolism with these preparations.? Conventionally, the enzyme activities contained therein a r e measured with excess substrate. When determined kinetically with varying substrate concentrations, enzyme activity is expressed a s maximal velocity, o r V m a . In these latter experiments a Michaelis constant, o r Km, can also be estimated. Both of these kinetic parameters must be considered "apparent" only, in that the enzyme system is multicomponent. That is, neither Vmax is considered to be a classical turnover number nor is the Km thought to be an affinity constant a s is often represented for simpler solubilized enzymes. We generally assume that Vmax i s an index of enzyme quantity and that the Km reflects a rate limitation for one of the reaction components within the enzyme complex. In most studies, simultaneous measurement of both parameters is advised, regardless of their mechanistic interpretation, because it then becomes possible to estimate physiologically meaningful enzyme reaction velocities. Measuring the reaction velocity a t a single excess substrate concentration implies the existence of saturating substrate concentrations in vivo, which is a gross oversimplification. This is illustrated in Table 1 with a set of hypothetical data. Treatment A appears to be causing greater rates of metabolism if only maximal velocities a r e measured where the rates a r e zero order with respect to substrate concentration. This i s what most investigators report, However, this is clearly irrelevant since the enzyme in vivo is not presented with saturating substrate concentrations. On
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the other hand, consider the more physiological reaction velocities calculated from the equation, v = (Vmax)(A)/km + A, where A can be given a value for a substrate concentration at a more physiological level. In this case the relative velocities a r e reversed and the reaction rate is first order with respect to substrate concentraticm. B.
In Vitro Weight Basis
Another practice that i s quite inconsistent in the literature is the weight unit chosen to express the in vitro reaction velocity. Most often, either liver weight or microsomal protein is chosen. If these velocities a r e to be more meaningful for the intact animal or human organism, then one should also consider using a reaction rate p e r total organ weight o r body weight since ingestion of the chemical carcinogen is more related to body weight than to milligrams of microsoma1 protein. If mechanistic o r molecular information on M I 7 0 hemoprotein activity is desired, one could choose to express the r a t e per unit of hemoprotein o r some other MFO component. Probably the best practice to follow would be to simultaneously provide all weight bases for the expression of relative reaction rates. These concepts a r e illustrated in previous studies from this laboratory [ 10, 11, 22, 231. C. Relative Rate Limitations Compounds whose toxicity o r pharmacological activity a r e regulated by their metabolism may be conveniently considered in one of two categories. Either they a r e cleared primarily a s a function of MFO metabolism and exhibit their activity through reversible interaction with the target site or they can yield MFO-catalyzed products which a r e chemically reactive and produce their activity through irreversible covalent interaction. Extrapolation of in vitro enzyme activities in the first case may be fairly straightforward and is discussed above. However, for those compounds which a r e metabolized by the MFO enzyme system to a chemically reactive intermediate (RI), a different set of kinetic relationships applies. Gillette [ 15, 161 has developed a pharmacokinetic model to show that it is the total amount of RI produced and not the rate at which it is produced. In turn, the amount of RI which reacts covalently with the target site to form an adduct will be a function of RI concentration. Actually, this latter concentration may be determined by any of several reaction rates. Either its rate of formation by the MFO system o r its rate of tlegradation might be involved. If any of these reactions a r e saturated,
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CAMPBELL E T AL.
i. e., rate limited, then nutritional modification of that reaction becomes the key interaction that should receive the highest priority for study. If none of these reactions i s rate limited and all a r e first order, then the rate of formation of FU, perhaps a s a consequence of earlier rate-limiting events, will determine RI concentration. Therefore, in many cases a study of nutrient interaction with MFO activity, after allowance for the correct substrate concentration and weight basis, will be satisfactory.
IV.
CONCLUDING REMARKS
What can be said about the value of using in vitro MFO assays a s indicators of nutrient effects? A t the very least, such data need to be confirmed with in vivo studies. If in vivo MFO activity is positively correlated with whole animal response under various experimental conditions, one may conclude that MFO metabolism is a primary determinant of response. If other reactions o r co-substrate availabilities can be influenced by nutrient interaction and these correlate with whole animal response, then they a r e likely to be the rate-limiting reactions. That this is the case has been demonstrated with acetaminophen-induced liver necrosis [ 241 and acetylaminofluorene alkylation of DNA [25]. When liver glutathione, which is required for the formation of a mercapturate of acetaminophen, is depleted, i . e., becomes rate limiting, the tissue concentration of a reactive metabolite Hypothetically, if and liver necrosis a r e sharply increased [24]. nutrient factors could limit glutathione availability, liver necrosis would be affected. The alkylation of DNA by the ultimate carcinogen of acetylaminofluorene, which is presumably the N-hydroxy sulfate ester, can be influenced by dietary sulfate [ 251. Thus the availability of sulfate for the sulfotransferase enzyme reaction is critical. The most significant conclusion that can be presently drawn about dietary protein effects, a s an exhibitor of nutrient interactions, is that the alteration of MFO activities is striking both in terms of time required after protein ingestion and in terms of magnitude of response. What this means for pharmacological response remains to be determined, but if one generalization is permitted, then one should anticipate that duration and intensity of response for most drugs would be enhanced. Acknowledgments The authors gratefully acknowledge the financial assistance provided by the Hoffmann LaRoche Research Foundation and NIH Grants
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R01 ES00336 and R01 CA20079, and the typing of the manuscript by Eleanor Parker.
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REFERENCES
c21
[ 31 c41
T. C. Campbell and J. R. Hayes, Pharmacol. Rev,, 26, 171 (1974). T. C. Campbell, "Influence of Nutrition on Metabolism of Carcinogens, ' I Adv. Nutr. Res., 2 (1977). A. H. Conney, Pharmacol. Rev., E , 317 (1967). A. H. Conney, in Fundamentals of Drug Metabolism and Drug Disposition (B. N. LaDu, H. G. Mandel, and E. L. Way, eds.), Williams and Wilkins, Baltimore, 1972, pp. 253-278. L. W. Wattenberg, Toxicol. Appl. Pharmacol., 2,711 (1972). E. D. Wills, Biochem. Pharmacol., 21, 239 (1972). A. E. Wade, F. E. Greene, R. H. Ciordia, J. S. Meadows, and W. 0. Caster, I-8, 2288 (1969). B. G. Woodcock and G. C. Wood, E d . , 2713 (1971). M. Eriksson, C. Catz, and S. J. Yaffe, Biol. Neonate, ?'7, 339 (1975). M. U. K. Mgbodile and T. C. Campbell, J. Nutr., E 2 , 5 3 (1972). M. U. K. Mgbodile, J. R. Hayes, and T. C. Campbell, &B chem. Pharmacol., 22, 1125 (1973). L. S. Nerurkar, J. R. Hayes, and T. C. Campbell, J. blutr., In Press, R. Kato, T. Oshima, and S. Tomizawa, Jpn. J. Pharmaool., 18, 356 (1968). J. Burns, in Fundamentals of Drug Metabolism and Drug Disposition (B. N. LaDu, H. G. Mandel, and E. L. Way, eds. ), Williams and Wilkins, Baltimore, 1971, pp. 340-366. J. R. Gillette, Biochem. Pharmacol., 2, 2785 (1974a). J. R. Gillette, 3,2927 (1947b). R. C. Garner, g., 24, 1553 (1975). R. S. Preston, J. R. Hayes, and T. C. Campbell, Life Sci., 19, 1191 (1976). V. Madhavan and C. Gopalan, Arch. Pathol., 133 (1968). R. Kato, E. Chiesara, and P. Vassanelli, Biochem. Pharmacol., 11, 211 (1962). A. P. Alvares, K. E. Anderson, A. H. Conney, and A. Kappas, Proc. Natl. Acad. Sci. USA, 73, 2501 (1976).
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[ 251
CAMPBELL E T AL. J. R. Hayes, M. U. K. Mgbodile, and T. C. Campbell, Kchem. Pharmacol., 22, 1005 (1973). J. R. Hayes and T. C. Campbell, g d . , 23, 1721 (1974). J. R. Gillette, J. R. Mitchell, and B. B. Brodie, Ann. Rev. Pharmacol., 2, 271 (1974). T. A. Miller, Cancer Res., 2, 559 (1970).
ISSN: 0360-2532 (Print) 1097-9883 (Online) Journal homepage: http://www.tandfonline.com/loi/idmr20
The Influence of Dietary Factors on Drug Metabolism in Animals T. Colin Campbell, Johnnie R. Hayes, Alfred H. Merrill, Martha Maso & Mabel Goetchius To cite this article: T. Colin Campbell, Johnnie R. Hayes, Alfred H. Merrill, Martha Maso & Mabel Goetchius (1979) The Influence of Dietary Factors on Drug Metabolism in Animals, Drug Metabolism Reviews, 9:2, 173-184, DOI: 10.3109/03602537908993889 To link to this article: http://dx.doi.org/10.3109/03602537908993889
Published online: 22 Sep 2008.
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Article views: 12
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Date: 12 November 2015, At: 12:14
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DRUG METABOLISM REVIEWS, 9(2), 173-184 (1979)
The Influence of Dietary Factors on Drug Metabolism in Animals* T. COLIN CAMPBELL, JOHNNIE H. HAYES, ALFRED H. MERRILL, JR., hlARTHA MASO, and MABEL GOETCHIUS Division of Nutritional Sciences Cornell University Ithaca, New York 14853
I. 11.
111.
IV.
.. .-.... . ... .. -. ..-. DIETARY PROTEIN E F F E C T .- - ... -- - -A. In Vitro Metabolism .......... .......... .......... B. In Vivo Metabolism.. ................ ....... . ..... EXTRAPOLATION O F IN VITRO ENZYME ACTIVITIES ........................................ A. In Vitro Substrate Concentration ....... ............ U. In Vitro Weight Basis ... .......................... C. Relative Rate Limitations ......................... .... . .-..... .... CONCLUDING REMARKS ..... Acknowledgments ................... ........... ...... References ......................................... INTRODUCTION
m
*
s..
*
*. *
*
174 175 175 176 179 180
181 181 182 182 183
*Presented a t Symposium on Drug Disposition in Man held in Sarasota, Florida, November 6-11, 1977 under the a u s p i c e s of the American Society for Pharmacology and Experimental Therapeutics. 173 Copyright 0 1979 hy Marcel Dekker. I n c . All Rlghts Reserved. Neither this work nor any part may he reproduced or transmitted in any form or by any means, electronic or mechanicd. including photocopying. microfilming, and recordinp.. or by any information storage and retrieval system. without permission in writing from the pub sher.
C A M P B E L L E T AL.
174
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I. INTRODUCTION One hardly needs to belabor the point that nutrient intake, when not in balance, can markedly alter the activities of so-called drug metabolism reactions. This general area has been recently reviewed elsewhere [l, 23 and has become a topic for discussion at numerous symposia and workshops in the last 2 to 3 years. Various suggestions have been made that both pharmacological response and risk to toxic chemicals may be influenced by nutritional status when metabolism of such chemicals determines the duration and intensity of response. This type of interaction should not be confused with the effect of nonnutrient components of food on drug metabolism reactions, These latter components, which may include synthetic and adventitious chemicals, intentional food additives, and natural compounds, a r e known to be effectors of drug metabolism, primarily a s inducers of enzyme activity [3-51. A rather large literature which has recently evolved shows that virtually every nutrient, when not ingested at optimum levels, can influence drug metabolism reactions in the experimental animal. With the exception of the guinea pig, which has been employed for research on vitamin C, most of these studies have been conducted with the laboratory rat. In general, a suboptimal intake of the nutrient tends to depress enzyme activities, although there a r e important exceptions. For example, intakes of iron [6] and thiamine [7] a r e said to vary inversely with the rate of metabolism for certain mixed function oxidation (MFO) catalyzed reactions, Furthermore, the activities of certain transferases [8, 9 1 may be increased with a low dietary intake of protein, Because consistency of nutrient effects on enzyme activities has not yet been found, it is somewhat premature in most cases to relate in vitro activity measurements with toxio o r pharmacologic response in the intact animal. Nevertheless, the ultimate clinical significance of this type of information would be greatly enhanced if in vitro enzyme measurement could be related with drug response for the intact animal. Thus the purpose of this paper will be to present some basic data on the intake of one of the major nutrients, i. e., protein, as it relates to drug metabolism reactions and then to discuss how such experimental data may o r may not relate to whole animal response, Dietary protein intake is selected for this discussion because 1) the effect on drug metabolism reactions i s striking, 2) its intake i s known to vary severalfold between and within human population groups, 3) relationships between metabolism and whole body response are somewhat better known, and 4) the research data a r e best known to the authors,
INFLUENCE OF DIETARY FACTORS
175
11. DIETARY PROTEIN EFFECT
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A.
In Vitro Metabolism
When weanling rats were fed a 5% casein semipurified diet for 2 weeks, MFO enzyme activities a r e depressed by 60 to 75% [lo, 111. About one-fourth of this depression can be accounted for by a decreased quantity of microsomal protein; the remaining three-fourths of the decrease results from a specific effect on the existing enzyme system. Other than certain minor differences, animal response is quite similar throughout the growth period between 3 and 9 weeks of age. Feeding the 5% casein diet at either 22, 37, o r 52 days of age immediately and completely interrupted body growth and increased the liver size relative to body weight (unpublished data). The livers of the low protein animals a r e characterized by an approximately 50% increase in cell size for 50 to 75% of the cells and 220% more lipid after 2 weeks of feeding. Liver microsomal protein, which inoludes the MFO enzyme, is quickly depressed by the low protein diet, primarily because of losses per cell, even though there is a compensatory increase in tissue size in these animals. Tissue hyperpktsia, as indicated by total tissue DNA content, immediately ceases when the low protein diet is fed. These responses a r e somewhat more significant in animals 22 days of age than in animals 37 days of age. When the 22-day old animals a r e fed the 5% protein diets for 1 to 2 weeks and then refed the 20'& diets, complete restoration of cellular microsonial protein and tissue growth requires about 2 to 4 weeks, These studies therefore indicate that that portion (25%) of the enzyme activity which is depressed because of the depressed rate of cell proliferation and companion intracellular microsomal enzyme content can be readily recovered if protein deprivation is not too severe. The more significant effect of protein deprivation on IVIFO activity, a s mentioned above, is that which depressed the existing enzyme activity. We originally found that both cytochrome P-450 content and cytochrome P-450 reductase activity were reduced t o about the same extent a s the depression of total MFO activity [lo, 113. The most obvious conclusion drawn from such data was that either one of these components could have accounted for the depression, depending on which one was rate-limiting. However, in more recent studies [12) we fractionated the microsomes into their three major components (cytochrome P-450, cytochrome P-450 reductase, phospholipid) and then reconstituted the components from each diet treatment group s o that an independent examination of the activity
CAMPBELL ET AL.
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of each component from each group could be made. There was no significant effect of diet on the phosphatidylcholine activity even though it is known that fatty acid substitution may be influenced by dietary protein and even though such chemical substitution may influence in vitro MFO activities, The MFO components most affected by diet treatment were the reductase and cytochrome P-450 fractions. Both of these protein components obtained from the 5y0 protein animals, when recombined on an activity basis equivalent to the ZOo/o protein animals, were not able to reconstitute the expected activities, This suggested that the interaction between these components could be the primary mechanism responsible for the dietary protein effect a s opposed to the effect on the specific activity of either component alone, Further physiochemical measurements of the membrane-bound enzyme system (unpublished data) has suggested to u s that the arrangement of components within the membrane is more rigid in the 5% protein animals and perhaps is less able to permit translational mobility of one component relative to the other. In more recent studies we have found that the depression of MFO activity associated with low dietary protein intake occurs extremely rapidly (Fig, 1). When weanling rats were pair-fed either the 5 or 20% protein diets, a greater than twofold difference in MFO activity was established within 24 hr. This difference became progressively greater up to 4 days a t which time the activity for ethylmorphine Ndemethylation in the 5% protein animals was almost too low to measure, The decline observed after 4 days is due to the method of expressing activity on a whole body basis; activity per gram of tissue o r milligram of microsomal protein begins to plateau at this age. In other studies (Fig. 2), animals were fed the high and low protein diets for 2 weeks and then reversed to the opposite diet. If animals were first fed the 5% protein diet, then re-fed the 20% protein diet, the enzyme activity was recovered in about 2 to 4 weeks. Thus the MFO activity seems to be quickly depressed upon feeding a low protein diet but is less responsive to recovery upon feeding the high protein diet. B. In Vivo Metabolism Extrapolation in vitro of MFO enzyme activities to the whole animal response may be possible where drug metabolism remains straight forward, simple, and rate limiting, That is, i f the drug is cleared primarily by an MFO-catalyzed reaction to a product(s) with lesser activity, then low protein intake should prolong clearance and
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X
f
DAYS ON DIET FIG. 1. Rat liver ethylmorphine N-demethylase activity at; related to the consumption of semipurified diets containing either 5% casein o r 207" casein. Weanling rats, 22 days of age, were pair fed the two diets to equalize total feed and caloric intake.
duration of action. Thus Kato et al. [13] were able to show that the level of dietary protein intake was directly correlated with delayed serum clearance and prolonged anesthesia when phenobarbital was administered to rats, Numerous other examples were discussed by Burns [14]. On the other hand, for those compounds where toxic activities depend on the production of a chemically reactive intermediate (.RI), interpretation of dietary effects on MFO activity is less clear. There a r e at least two major reasons for this. First, it is not the rate at which the RI is produced by the MFO system but the quantity which is produced and which reacts covalently with the target site [15, IS]. Second, the quantity of this MFO product which is available for covalent interaction may be affected by the activity of ancillary clegradative reactions rather than the MFO system which forms the R1.
CAMPBELL E T AL.
178 h
n
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I
I
I
1
8
15
30
I 45
DAYS ON DIET FIG. 2. Effect of dietary protein replenishment on rat liver ethylmorphine N-demethylation and aniline hydroxylation. Weanling rats, 22 days of age, with 7 to 1 0 groups and 3 to 6 animals per group, were fed semipurified diets ad libitum a t different times in same animal facilities. Diets: 20% casein (solid line), 5% casein (dashed line).
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The absence of a clear relationship between MFO activity anti toxic response is probably best illustrated by the data which show that low protein intake and phenobarbital administration produce contrasting effects on the metabolism and carcinogenicity of aflatoxin. MFO activity is depressed by protein intake but increased by phenobarbital administration. Yet both treatments apparently decrease the availability of the MFO-catalyzed aflatoxin epoxide, considered to be the chemically reactive intermediate which covalently binds DNA [ 117, 183. Moreover, both treatments depress hepatoma formation [ 193. Hypothetically, MFO activity may depress tumorigenicity with the protein-deficient animals but this activity cannot explain the phenobarbital effect. However, it is somewhat less tenable to assume that MFO metabolism is rate limiting for the controls in one case and not the other. Therefore, it would be useful to know the relative rake limitations before the effects of these various treatments can be extrapolated to the whole animal.
111.
EXTRAPOLATION O F IN VITRO ENZYME ACTIVITIEiS
This last section will briefly consider, then, a few questions which prevent simple interpretation and extrapolation of in vitro measurements of MFO activity to whole animal response. Our own research, a s well a s that of most other groups, has either explicitly or implicitly depended on a couple of important assumptions; namely, that for whole animal response, 1) metabolism is rate limiting and 2) in vitro enzyme activity i s an acceptable index for in vivo metabolism. Because reports on nutritional effects often reach contrasting conclusions, it becomes worthwhile to list a few questions concerned with the extrapolation of such in vitro data. For the purpose of this discussion, I will accept the first assumption that metabolism is rate limiting when compared to gastrointestinal absorption, protein binding, cellular uptake, etc. There is considerable evidence that this is true in many instances since the rates of plasma clearance of compounds such a s phenobarbital [ZO], aminopyrine [21], and theophylline [21] appear to be primarily a function of metabolism by the MFO system. This type of research in humans is, in fact, largely limited to in vivo measurements of pilasma clearance times and is a subject to be discussed elsewhere in this symposium (Kappus). Nevertheless, a comparison of in vitro data with in vivo response is oftentimes in conflict and is the focus of the next sect ion.
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TABLE 1
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Hypothetical Kinetic Parameters for Enzyme Reaction under Influence of Two Treatments
Treatment
'max (nmoles/mg/min)
Km
Vn
A
100
1.0
0.99
B
50
0.1
4.55
a
Assuming substrate concentration of 0.01 mM and v A, with A equal to substrate concentration. A.
-
(V)(A)/k,
+
In Vitro Substrate Concentration
What can be said about rates of metabolism with these preparations.? Conventionally, the enzyme activities contained therein a r e measured with excess substrate. When determined kinetically with varying substrate concentrations, enzyme activity is expressed a s maximal velocity, o r V m a . In these latter experiments a Michaelis constant, o r Km, can also be estimated. Both of these kinetic parameters must be considered "apparent" only, in that the enzyme system is multicomponent. That is, neither Vmax is considered to be a classical turnover number nor is the Km thought to be an affinity constant a s is often represented for simpler solubilized enzymes. We generally assume that Vmax i s an index of enzyme quantity and that the Km reflects a rate limitation for one of the reaction components within the enzyme complex. In most studies, simultaneous measurement of both parameters is advised, regardless of their mechanistic interpretation, because it then becomes possible to estimate physiologically meaningful enzyme reaction velocities. Measuring the reaction velocity a t a single excess substrate concentration implies the existence of saturating substrate concentrations in vivo, which is a gross oversimplification. This is illustrated in Table 1 with a set of hypothetical data. Treatment A appears to be causing greater rates of metabolism if only maximal velocities a r e measured where the rates a r e zero order with respect to substrate concentration. This i s what most investigators report, However, this is clearly irrelevant since the enzyme in vivo is not presented with saturating substrate concentrations. On
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the other hand, consider the more physiological reaction velocities calculated from the equation, v = (Vmax)(A)/km + A, where A can be given a value for a substrate concentration at a more physiological level. In this case the relative velocities a r e reversed and the reaction rate is first order with respect to substrate concentraticm. B.
In Vitro Weight Basis
Another practice that i s quite inconsistent in the literature is the weight unit chosen to express the in vitro reaction velocity. Most often, either liver weight or microsomal protein is chosen. If these velocities a r e to be more meaningful for the intact animal or human organism, then one should also consider using a reaction rate p e r total organ weight o r body weight since ingestion of the chemical carcinogen is more related to body weight than to milligrams of microsoma1 protein. If mechanistic o r molecular information on M I 7 0 hemoprotein activity is desired, one could choose to express the r a t e per unit of hemoprotein o r some other MFO component. Probably the best practice to follow would be to simultaneously provide all weight bases for the expression of relative reaction rates. These concepts a r e illustrated in previous studies from this laboratory [ 10, 11, 22, 231. C. Relative Rate Limitations Compounds whose toxicity o r pharmacological activity a r e regulated by their metabolism may be conveniently considered in one of two categories. Either they a r e cleared primarily a s a function of MFO metabolism and exhibit their activity through reversible interaction with the target site or they can yield MFO-catalyzed products which a r e chemically reactive and produce their activity through irreversible covalent interaction. Extrapolation of in vitro enzyme activities in the first case may be fairly straightforward and is discussed above. However, for those compounds which a r e metabolized by the MFO enzyme system to a chemically reactive intermediate (RI), a different set of kinetic relationships applies. Gillette [ 15, 161 has developed a pharmacokinetic model to show that it is the total amount of RI produced and not the rate at which it is produced. In turn, the amount of RI which reacts covalently with the target site to form an adduct will be a function of RI concentration. Actually, this latter concentration may be determined by any of several reaction rates. Either its rate of formation by the MFO system o r its rate of tlegradation might be involved. If any of these reactions a r e saturated,
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i. e., rate limited, then nutritional modification of that reaction becomes the key interaction that should receive the highest priority for study. If none of these reactions i s rate limited and all a r e first order, then the rate of formation of FU, perhaps a s a consequence of earlier rate-limiting events, will determine RI concentration. Therefore, in many cases a study of nutrient interaction with MFO activity, after allowance for the correct substrate concentration and weight basis, will be satisfactory.
IV.
CONCLUDING REMARKS
What can be said about the value of using in vitro MFO assays a s indicators of nutrient effects? A t the very least, such data need to be confirmed with in vivo studies. If in vivo MFO activity is positively correlated with whole animal response under various experimental conditions, one may conclude that MFO metabolism is a primary determinant of response. If other reactions o r co-substrate availabilities can be influenced by nutrient interaction and these correlate with whole animal response, then they a r e likely to be the rate-limiting reactions. That this is the case has been demonstrated with acetaminophen-induced liver necrosis [ 241 and acetylaminofluorene alkylation of DNA [25]. When liver glutathione, which is required for the formation of a mercapturate of acetaminophen, is depleted, i . e., becomes rate limiting, the tissue concentration of a reactive metabolite Hypothetically, if and liver necrosis a r e sharply increased [24]. nutrient factors could limit glutathione availability, liver necrosis would be affected. The alkylation of DNA by the ultimate carcinogen of acetylaminofluorene, which is presumably the N-hydroxy sulfate ester, can be influenced by dietary sulfate [ 251. Thus the availability of sulfate for the sulfotransferase enzyme reaction is critical. The most significant conclusion that can be presently drawn about dietary protein effects, a s an exhibitor of nutrient interactions, is that the alteration of MFO activities is striking both in terms of time required after protein ingestion and in terms of magnitude of response. What this means for pharmacological response remains to be determined, but if one generalization is permitted, then one should anticipate that duration and intensity of response for most drugs would be enhanced. Acknowledgments The authors gratefully acknowledge the financial assistance provided by the Hoffmann LaRoche Research Foundation and NIH Grants
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R01 ES00336 and R01 CA20079, and the typing of the manuscript by Eleanor Parker.
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[ 31 c41
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