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The vitamin B6 requirement of the rat as estimated with weight gain and general activity measurements
Physiology and Behavior. Vol. 3, pp, 301-303. Pergamon Press, 1968 Printed in Great Britain
The Vitamin B6 Requirement of the Rat as Estimated with Weight Gain and General Activity Measurements L. A. H O F F A N D L. J. P E A C O C K
Psychology Department AND J. S. M E A D O W S
A N D W . O. C A S T E R
Home Economics Nutrition and Institute of Comparative Medicine, University of Georgia, Athens, Georgia 30601 (Received 28 A p r i l 1967) PEACOCK, J. S. MEADOWSAND W. O. CASTER. The vitamin B6 requirement of the rat as estimated with weight gain andgeneral activity measurements. PHYSIOL.BEHAV.3 (2) 301-303, 1968.--Tbe minimal nutritional require-
HOFF, L. A., L. J.
ment for a particular substance has been defined traditionally in terms of weight gain and the prevention of deficiency signs. Many nutrients are known to be involved in several specific but different points in metabolic processes, which suggests that nutritional requirements are multiple rather than singular for a particular substance. In the present study, the use of different physiological measurements led to very different estimates of nutritional requirement. The present study employs a "half-change intake" value to estimate the minimal nutritional requirement for vitamin Be (pyridoxine) as measured by body weight gain and by the general activity of rats. The minimal nutritional requirement for pyridoxine for the rat determined in this manner is 5 ~tg per day for the weight gain data and 0.8 ttg per day for the general activity data. These values compare with the standard NRC figures of 8-15 ~tg per rat per day, providing evidence for the multiplicity of requirements. Vitamin Be, pyridoxine activity
Minimal nutritional requirement
ONE OF THE persistent problems of nutritional science has been the relative lack of demonstrable relationships between various functional evidences of nutritional deficiency and what is known by the biochemist concerning the metabolic function of a given nutrient. Present definitions of nutritional requirement are concerned largely with descriptive statements of the presence or absence of certain "deficiency signs". Such an approach is not amenable to the use of biochemical terms, or to quantitative procedures designed to relate nutritional requirements, weight gain, physiological performance changes, or deficiency signs to tissue biochemistry. Classically, the minimal nutrient requirement (MNR) is defined as that amount of a given nutrient which will prevent development of deficiency signs and produce a maximal rate of weight gain. Conversely, an intake of nutrient below minimal requirement is defined as one which does not allow a maximum growth rate, or at which metabolic changes indicative of the deficiency condition may be demonstrated. Examination of biochemical data suggests one further problem with current methodology. Many nutrients, vitamin Be (pyridoxine) in particular, are known to be involved at many specific but different points in the metabolic processes.
General activity
Ultrasonic measurement of
The nutritional requirement, thus, is not singular but is multiple. Differences in the different enzyme systems involved, in regard to affinity for the vitamin B8 coenzyme, and in regard to certain rate-controlling factors could easily lead to a series of distinctly different nutritional requirements for such a nutrient. Recently [3], a different approach to the problem of measuring the quantitative relationship between nutrient intake and certain functional and metabolic factors has been taken that allows each of these M N R values to be estimated individually. In the method described by Caster, et al. [3], exponential equations were fitted to experimental data points. The most characteristic feature of such an exponential function is the "half-life period". In this instance the half-life period represents nutrient intake rather than time and therefore is spoken of as a "half-change intake" value (I1/~), and is defined as the nutrient intake which provides a biochemical or physiological response lying midway between that found at a zero intake level and that found at an adequate intake level of the critical nutrient. Since a characteristic of a simple exponential curve is that the abscissa of any point on it may be defined in terms of the Ii/~ units, it follows that whatever point is chosen
XThis work was supported in part by U.S. Public Health Service Grant AM08595 from the National Institute of Arthritis and Metabolic Diseases and in part by Grant HD00946 from the National Institute of Child Health and Human Development. This is publication No. 667 of the Institute of Comparative Medicine. 301
302
HOFF, PEACOCK, MEADOWS AND CASTER
to represent the MNR is equal to some constant times the half-change intake value: MNR = nI~/~ where n is a constant of proportionality assumed by Caster et aL [3] to be approximately 1.7. This value is based on both human and animal data [4, 6, 7]. The purpose of the present report is to use this relationship for the determination of the pyridoxine requirements of the rat on the basis of weight gain data and on the basis of activity measurements. That measurements of general activity level of organisms reflect physiological changes in body metabolism has been well demonstrated and accepted [8].
METHOD
Animals were 13 weanling male white rats of the SpragueDawley strain. The animals were fed purified diets containing known and different amounts of pyridoxine [0, 1, 3, 10, or 30 ~g per 10 g of diet) for a period of 60 days before activity measurements were made. Classical deficiency signs of dermatitis and weight loss were well developed in those rats whose daily pyridoxine intake was lowest. Activity measures were obtained with the ultrasonic technique devised by Peacock and Williams [ll]. A detailed description of the equipment used in portions of this particular experiment has been presented elsewhere [10]. Animals were housed in individual cages, each o f which was equipped with an ultrasonic measuring device. As an animal moves through the sound field generated by the ultrasonic unit, a signal is produced which is led to an integrator circuit that stores the signal as a charge on a capacitor. When the charge reaches a specified value, it is discharged through a relay that pulses a counter. The number of discharges is proportional to the extent of the animal's movement. Data from 8 of the rats were individually collected automatically every 5 min, 24 hr/day by means of an I B M 024 key punch. Animals were kept in a sound shielded, light controlled room with an ambient temperature of 74 4- 2°F. Illumination of the room was provided by fluorescent light programmed to follow a 12 hr light-dark cycle with the dark period beginning at 1800 hr. Each day the diet and water supplies were replenished from 1650-1700 hr. The diet included 16 per cent casein, up to 76 per cent sucrose, up to 3 per cent corn oil, 4 per cent Jones-Foster salt mix, 4 per cent cellulose (Alphacel), and vitamins (except for pyridoxine which was varied systematically) in amounts suggested by Cuthbertson [5]. Activity data, excepting the required 10 minute daily maintenance period which were discarded, were collected continuously for 2 days on all animals. RESULTS
Each animal's activity measure during each of the two day periods was expressed as a percentage of daily activity with respect to a group of 22 control animals given access ad libitum to the same laboratory diet containing greater amounts of pyridoxine. The results of these measurements are shown in Fig. 1, where the daily activity is plotted as a function of B, intake level. An exponential curve has been drawn which has an Ii/2-value of 0.8 ~.g of pyridoxine per day. Figure 2 provides the weight-gain data for these same animals. The exponential curve, in this case, has an I,/2-value of 5 Fg of pyridoxine per day.
0
120 rr ~z~O0 '
0 0
60-
~40. ~-2C
UG
PYRIDOXINE
/ DAY
FIG. 1. General activity of individual rats, expressed as a percentage of control group activity, for several dietary levels of vitamin Be. Circles: measurements obtained in the Animal Behavior Laboratory. Crosses: Measurements independently obtained in the Nutrition Laboratory. Open circles: animals on fat-free diet. Solid circles and crosses: diet contained 3 % corn oil.
Although small numbers of animals were used, the significance of the obtained activity measurements is enhanced by the fact that data from 8 of the rats (circles in Fig. 1) were obtained by two of us (LJP and LAH) in the Animal Behavior Laboratory while the other measurements from the other 5 rats (crosses in Fig. 1) were obtained independently by the other two investigators (WOC and JSM) in the Nutrition Laboratory with different but functionally similar equipment. The two sets of data represented by circles relate to the fact that the diet of 4 animals was devoid of fatty acid, while the diet of the other 4 contained 3 per cent corn oil substituted isocalorically for part of the sucrose.
180
~ 160
,i/
/
~140 120
/
/
'~100
~6o uJ
2O
o ,o 20 30 X6 ~o OG PYR~DOX,NE/ DAY
FIG. 2. Weight gain for groups of animals represented in Fig. 1. Data represent increase in total body weight expressed in grams and measured over a period of 40 days on diet starting from weaning at 22 days of age. Circles and crosses represent the same groups described in Fig. I.
DISCUSSION
Weight-gain data, such as those of Fig. 2, have been interpreted in various ways in the past. The MNR has been suggested by the NRC [9] as being in the range of 8-15 ~g of pyridoxine per day for the rat. In terms of the above equation, this corresponds with values of n ranging from 1.6-5.0. Even higher requirements have been seriously proposed [1] on the basis of weight gain data. Because of this uncertainty, Caster
VITAMIN B6 REQUIREMENT OF THE RAT
303
et aL [3] suggested that the I1/2-value was the more funda-
mental and reliable statistic. There is a 6-fold difference between the I1/~-value associated with the activity data (Fig. 1) and the weight-gain data (Fig. 2) obtained simultaneously on the same animals. This is evidence of the multiplicity of the vitamin Bs requirement.
An interdependence between the requirements for essential fatty acid and vitamin B8 has been suggested [2, 12, 13, 14, 15]. The agreement between the data represented by open and solid circles in Figs. 1 and 2 provide no evidence for an effect of essential fatty acid upon the two different vitamin B~ requirements measured in this experiment.
REFERENCES
1. Beaton, G. H. and M. C. Cheney. Vitamin Be Requirement of the Rat. Fed. Proc. 24: 624, 1965. 2. Birch, T. W. and P. Gyorgy. A Study of the Chemical Nature of Vitamin B0 and Methods for Its Preparation in a Concentrated State. Biochem. J. 30: 304-315, 1936. 3. Caster, W. O., P. Alan, E. G. Hill, H. Mohrhauer, and R. T. Holman. Determination of Linoleate Requirement of Swine by a New Method of Estimating Nutritional Requirement. J. Nutr. 78: 147-154, 1962. 4. Caster, W. O. and R. T. Holman. Statistical Study of the Relationship Between Dietary Linoleate and The Fatty Acids of Heart and Blood Lipids. J. Nutr. 73: 337-346, 1961. 5. Cuthbertson, W. F. J. Nutrient Requirements of Rats and Mice. Proc. Nutr. Soc. 16: 70-76, 1957. 6. Hill, E. G., E. L. Warmanen, C. L. Silbernick, and R. T. Holman. Essential Fatty Acid Nutrition in Swine. I. Linoleate Requirement Estimated from Triene: Tetraene Ratio of Tissue Lipids. J. Nutr. 74: 335-341, 1961. 7. Mickelsen, O., W. O. Caster, and A. Keys. A Statistical Evaluation of the Tfiiamine and Pyramin Excretions of Normal Young Men on Controlled Intakes of Thiamine. J. Biol. Chem. 168: 415~,31, 1947.
8. Morgan, C. T. Physiological Psychology, New York: McGrawHill, 1965. 9. NRC Publication 990. Nutrient Requirements o f Laboratory Animals, 1962. 10. Peacock, L. J., M. H. Hodge, and R. K. Thomas. Ultrasonic Measurement and Automatic Analysis of General Activity in the Rat. J. comp. physiol. Psychol. 62: 284-288, 1966. 11. Peacock, L. J. and M. Williams. An Ultrasonic Device for Recording Activity. Am. J. Psychol. 75: 648-652, 1962. 12. Salmon, W. D. The Supplementary Relationship of vitamin Be and Unsaturated Fatty Acids. J. Biol. Chem. 133: 33, 1940. 13. Sewell, L., M. D. Law, P. E. Schools and C. R. Treadwell, Tissue Lipid Fatty Acid Composition in Pyridoxine-Deficient Rats. J. Nutr. 74: 148-156, 1961. 14. Sherman, H. C. Pyridoxine and Fat Metabolism. Vitam. Horm. 8: 55-68, 1950. 15. Witten, P. W. and R. T. Holman. Polyethenoid Fatty Acid Metabolism. VI. Effect of Pyridoxine on Essential Fatty Acid Conversions. Archs. Biochem. Biophys. 41: 266-273, 1952.
The Vitamin B6 Requirement of the Rat as Estimated with Weight Gain and General Activity Measurements L. A. H O F F A N D L. J. P E A C O C K
Psychology Department AND J. S. M E A D O W S
A N D W . O. C A S T E R
Home Economics Nutrition and Institute of Comparative Medicine, University of Georgia, Athens, Georgia 30601 (Received 28 A p r i l 1967) PEACOCK, J. S. MEADOWSAND W. O. CASTER. The vitamin B6 requirement of the rat as estimated with weight gain andgeneral activity measurements. PHYSIOL.BEHAV.3 (2) 301-303, 1968.--Tbe minimal nutritional require-
HOFF, L. A., L. J.
ment for a particular substance has been defined traditionally in terms of weight gain and the prevention of deficiency signs. Many nutrients are known to be involved in several specific but different points in metabolic processes, which suggests that nutritional requirements are multiple rather than singular for a particular substance. In the present study, the use of different physiological measurements led to very different estimates of nutritional requirement. The present study employs a "half-change intake" value to estimate the minimal nutritional requirement for vitamin Be (pyridoxine) as measured by body weight gain and by the general activity of rats. The minimal nutritional requirement for pyridoxine for the rat determined in this manner is 5 ~tg per day for the weight gain data and 0.8 ttg per day for the general activity data. These values compare with the standard NRC figures of 8-15 ~tg per rat per day, providing evidence for the multiplicity of requirements. Vitamin Be, pyridoxine activity
Minimal nutritional requirement
ONE OF THE persistent problems of nutritional science has been the relative lack of demonstrable relationships between various functional evidences of nutritional deficiency and what is known by the biochemist concerning the metabolic function of a given nutrient. Present definitions of nutritional requirement are concerned largely with descriptive statements of the presence or absence of certain "deficiency signs". Such an approach is not amenable to the use of biochemical terms, or to quantitative procedures designed to relate nutritional requirements, weight gain, physiological performance changes, or deficiency signs to tissue biochemistry. Classically, the minimal nutrient requirement (MNR) is defined as that amount of a given nutrient which will prevent development of deficiency signs and produce a maximal rate of weight gain. Conversely, an intake of nutrient below minimal requirement is defined as one which does not allow a maximum growth rate, or at which metabolic changes indicative of the deficiency condition may be demonstrated. Examination of biochemical data suggests one further problem with current methodology. Many nutrients, vitamin Be (pyridoxine) in particular, are known to be involved at many specific but different points in the metabolic processes.
General activity
Ultrasonic measurement of
The nutritional requirement, thus, is not singular but is multiple. Differences in the different enzyme systems involved, in regard to affinity for the vitamin B8 coenzyme, and in regard to certain rate-controlling factors could easily lead to a series of distinctly different nutritional requirements for such a nutrient. Recently [3], a different approach to the problem of measuring the quantitative relationship between nutrient intake and certain functional and metabolic factors has been taken that allows each of these M N R values to be estimated individually. In the method described by Caster, et al. [3], exponential equations were fitted to experimental data points. The most characteristic feature of such an exponential function is the "half-life period". In this instance the half-life period represents nutrient intake rather than time and therefore is spoken of as a "half-change intake" value (I1/~), and is defined as the nutrient intake which provides a biochemical or physiological response lying midway between that found at a zero intake level and that found at an adequate intake level of the critical nutrient. Since a characteristic of a simple exponential curve is that the abscissa of any point on it may be defined in terms of the Ii/~ units, it follows that whatever point is chosen
XThis work was supported in part by U.S. Public Health Service Grant AM08595 from the National Institute of Arthritis and Metabolic Diseases and in part by Grant HD00946 from the National Institute of Child Health and Human Development. This is publication No. 667 of the Institute of Comparative Medicine. 301
302
HOFF, PEACOCK, MEADOWS AND CASTER
to represent the MNR is equal to some constant times the half-change intake value: MNR = nI~/~ where n is a constant of proportionality assumed by Caster et aL [3] to be approximately 1.7. This value is based on both human and animal data [4, 6, 7]. The purpose of the present report is to use this relationship for the determination of the pyridoxine requirements of the rat on the basis of weight gain data and on the basis of activity measurements. That measurements of general activity level of organisms reflect physiological changes in body metabolism has been well demonstrated and accepted [8].
METHOD
Animals were 13 weanling male white rats of the SpragueDawley strain. The animals were fed purified diets containing known and different amounts of pyridoxine [0, 1, 3, 10, or 30 ~g per 10 g of diet) for a period of 60 days before activity measurements were made. Classical deficiency signs of dermatitis and weight loss were well developed in those rats whose daily pyridoxine intake was lowest. Activity measures were obtained with the ultrasonic technique devised by Peacock and Williams [ll]. A detailed description of the equipment used in portions of this particular experiment has been presented elsewhere [10]. Animals were housed in individual cages, each o f which was equipped with an ultrasonic measuring device. As an animal moves through the sound field generated by the ultrasonic unit, a signal is produced which is led to an integrator circuit that stores the signal as a charge on a capacitor. When the charge reaches a specified value, it is discharged through a relay that pulses a counter. The number of discharges is proportional to the extent of the animal's movement. Data from 8 of the rats were individually collected automatically every 5 min, 24 hr/day by means of an I B M 024 key punch. Animals were kept in a sound shielded, light controlled room with an ambient temperature of 74 4- 2°F. Illumination of the room was provided by fluorescent light programmed to follow a 12 hr light-dark cycle with the dark period beginning at 1800 hr. Each day the diet and water supplies were replenished from 1650-1700 hr. The diet included 16 per cent casein, up to 76 per cent sucrose, up to 3 per cent corn oil, 4 per cent Jones-Foster salt mix, 4 per cent cellulose (Alphacel), and vitamins (except for pyridoxine which was varied systematically) in amounts suggested by Cuthbertson [5]. Activity data, excepting the required 10 minute daily maintenance period which were discarded, were collected continuously for 2 days on all animals. RESULTS
Each animal's activity measure during each of the two day periods was expressed as a percentage of daily activity with respect to a group of 22 control animals given access ad libitum to the same laboratory diet containing greater amounts of pyridoxine. The results of these measurements are shown in Fig. 1, where the daily activity is plotted as a function of B, intake level. An exponential curve has been drawn which has an Ii/2-value of 0.8 ~.g of pyridoxine per day. Figure 2 provides the weight-gain data for these same animals. The exponential curve, in this case, has an I,/2-value of 5 Fg of pyridoxine per day.
0
120 rr ~z~O0 '
0 0
60-
~40. ~-2C
UG
PYRIDOXINE
/ DAY
FIG. 1. General activity of individual rats, expressed as a percentage of control group activity, for several dietary levels of vitamin Be. Circles: measurements obtained in the Animal Behavior Laboratory. Crosses: Measurements independently obtained in the Nutrition Laboratory. Open circles: animals on fat-free diet. Solid circles and crosses: diet contained 3 % corn oil.
Although small numbers of animals were used, the significance of the obtained activity measurements is enhanced by the fact that data from 8 of the rats (circles in Fig. 1) were obtained by two of us (LJP and LAH) in the Animal Behavior Laboratory while the other measurements from the other 5 rats (crosses in Fig. 1) were obtained independently by the other two investigators (WOC and JSM) in the Nutrition Laboratory with different but functionally similar equipment. The two sets of data represented by circles relate to the fact that the diet of 4 animals was devoid of fatty acid, while the diet of the other 4 contained 3 per cent corn oil substituted isocalorically for part of the sucrose.
180
~ 160
,i/
/
~140 120
/
/
'~100
~6o uJ
2O
o ,o 20 30 X6 ~o OG PYR~DOX,NE/ DAY
FIG. 2. Weight gain for groups of animals represented in Fig. 1. Data represent increase in total body weight expressed in grams and measured over a period of 40 days on diet starting from weaning at 22 days of age. Circles and crosses represent the same groups described in Fig. I.
DISCUSSION
Weight-gain data, such as those of Fig. 2, have been interpreted in various ways in the past. The MNR has been suggested by the NRC [9] as being in the range of 8-15 ~g of pyridoxine per day for the rat. In terms of the above equation, this corresponds with values of n ranging from 1.6-5.0. Even higher requirements have been seriously proposed [1] on the basis of weight gain data. Because of this uncertainty, Caster
VITAMIN B6 REQUIREMENT OF THE RAT
303
et aL [3] suggested that the I1/2-value was the more funda-
mental and reliable statistic. There is a 6-fold difference between the I1/~-value associated with the activity data (Fig. 1) and the weight-gain data (Fig. 2) obtained simultaneously on the same animals. This is evidence of the multiplicity of the vitamin Bs requirement.
An interdependence between the requirements for essential fatty acid and vitamin B8 has been suggested [2, 12, 13, 14, 15]. The agreement between the data represented by open and solid circles in Figs. 1 and 2 provide no evidence for an effect of essential fatty acid upon the two different vitamin B~ requirements measured in this experiment.
REFERENCES
1. Beaton, G. H. and M. C. Cheney. Vitamin Be Requirement of the Rat. Fed. Proc. 24: 624, 1965. 2. Birch, T. W. and P. Gyorgy. A Study of the Chemical Nature of Vitamin B0 and Methods for Its Preparation in a Concentrated State. Biochem. J. 30: 304-315, 1936. 3. Caster, W. O., P. Alan, E. G. Hill, H. Mohrhauer, and R. T. Holman. Determination of Linoleate Requirement of Swine by a New Method of Estimating Nutritional Requirement. J. Nutr. 78: 147-154, 1962. 4. Caster, W. O. and R. T. Holman. Statistical Study of the Relationship Between Dietary Linoleate and The Fatty Acids of Heart and Blood Lipids. J. Nutr. 73: 337-346, 1961. 5. Cuthbertson, W. F. J. Nutrient Requirements of Rats and Mice. Proc. Nutr. Soc. 16: 70-76, 1957. 6. Hill, E. G., E. L. Warmanen, C. L. Silbernick, and R. T. Holman. Essential Fatty Acid Nutrition in Swine. I. Linoleate Requirement Estimated from Triene: Tetraene Ratio of Tissue Lipids. J. Nutr. 74: 335-341, 1961. 7. Mickelsen, O., W. O. Caster, and A. Keys. A Statistical Evaluation of the Tfiiamine and Pyramin Excretions of Normal Young Men on Controlled Intakes of Thiamine. J. Biol. Chem. 168: 415~,31, 1947.
8. Morgan, C. T. Physiological Psychology, New York: McGrawHill, 1965. 9. NRC Publication 990. Nutrient Requirements o f Laboratory Animals, 1962. 10. Peacock, L. J., M. H. Hodge, and R. K. Thomas. Ultrasonic Measurement and Automatic Analysis of General Activity in the Rat. J. comp. physiol. Psychol. 62: 284-288, 1966. 11. Peacock, L. J. and M. Williams. An Ultrasonic Device for Recording Activity. Am. J. Psychol. 75: 648-652, 1962. 12. Salmon, W. D. The Supplementary Relationship of vitamin Be and Unsaturated Fatty Acids. J. Biol. Chem. 133: 33, 1940. 13. Sewell, L., M. D. Law, P. E. Schools and C. R. Treadwell, Tissue Lipid Fatty Acid Composition in Pyridoxine-Deficient Rats. J. Nutr. 74: 148-156, 1961. 14. Sherman, H. C. Pyridoxine and Fat Metabolism. Vitam. Horm. 8: 55-68, 1950. 15. Witten, P. W. and R. T. Holman. Polyethenoid Fatty Acid Metabolism. VI. Effect of Pyridoxine on Essential Fatty Acid Conversions. Archs. Biochem. Biophys. 41: 266-273, 1952.