Biochemical SocietyTransactions ( 1 992) 20
S t u d i e s on t h e r o l e of ascorbic acid i n olfactory tissue.
199s
Table 1. Ascorbic acid levels in animal tissues. Results represent the mean f standard deviation of 3 independent experiments (triplicate readings from single animals).
ROSEMARY BLAND, STAN LOVETT and GEORGE H. DODD ua A N a wet weiaht. Mean f SD
Tissue
Olfaction Research Group, University of Warwick, Coventry, CV4 7AL, U.K. Ascorbic acid (AA)’ accumulates in the olfactory bulb [l] and it has been suggested that AA may be a cofactor in olfactory transduction [2].The levels of free AA (approximately 20% of AA may be bound, [3]) were determined in the brain, hepatic, olfactory and respiratory tissue of rats, rabbits and guinea-pigs (G-P’s). Olfactory tissue was examined for the presence of gulonolactone oxidase (GLO), the final enzyme in the biosynthetic pathway of AA. To investigate whether or not AA was essential for olfactory tissue to respond to odours, G-P’s were fed a diet deficient i n AA to reduce the levels of AA in the olfactory tissue. Electro-olfactograms (EOG’s) were performed on the olfactory tissue of the G-P’s. Ascorbic acid was determined using the 2,4dinitrophenylhydrazine (DNPH) colorimetric method [4]. Tissue samples were prepared by homogenisation in 5% TCA and the precipitated protein was removed by centrifugation (1 O,OOOg, 20 minutes). The supernatant was incubated with the DNPH solution for 3 hours, and the reaction was stopped and the colour developed with 65% H2S04. GLO was measured in terms of AA produced from gulonolactone. Tissue was prepared in phosphate buffer (O.lM, pH 7.5) and incubated with gulonolactone (2.5mM) for 15 minutes at 37°C. The reaction was stopped with 50% TCA [5]. Ascorbic acid production was measured by the DNPH method. For the deficiency study, experimental and control diets were supplied by Special Diet Services. G-P’s were maintained on the deficient diet for between 15-18 days. EOG’s were performed as previously described [6], except that AA was omitted from the Ringer solution. Eugenol, cineole and iso-amylacetate were the odours tested. The quantity of AA present in the tissues is shown i n Table 1 . No significant activity of GLO was found in rat olfactory or brain tissue, although it was detected in hepatic tissue of rats. In the deficiency study, 5 control and 6 scorbutic G-P’s were examined. The G-P’s were not pair-fed, but their weights were monitored and the weight change of the experimental groups closely matched that of the control group. ’Abbreviations used: AA, ascorbic acid; DNPH, 2,4-dinitrophenylhydrazine; EOG, electro-olfactograrn; GLO, gulonolactone oxidase; G-P’s, guinea-pigs; TCA, trichloroacetic acid.
Rat
+ 178
Rabbit
Guinea-pig
253 f 33
5 5 0 f 24
Olfactory
722
Respiratory
31 6 f 86
157 f 16
457 f 40
Brain
156 f 1
155 f 7.0
143 f 22
Hepatic
347 f 45
170 f 39
169 f 25
Therefore any differences in the response of the groups would not be due to differing nutritional states. After 15-18 days on the scorbutic diet the levels of AA in the G-P tissues had fallen. Hepatic, olfactory and respiratory tissue were 5.2%, 8.1% and 26.1% of the control G-P’s respectively. The brain, which is known to retain AA [7], had only fallen to 60% of the control value. The levels of AA in scorbutic G-P’s returned to control levels, in all tissues, after 7 days on a normal diet. As table 1 shows the levels of AA in the olfactory tissue are higher than the respiratory, hepatic and brain tissue in the animals examined. Interestingly there is also a high concentration of AA in another sensory tissue, retina [8]. However, GLO was not detected in the olfactory tissue so the high levels are not caused by AA production. When the G-P’s were maintained on an AA deficient diet AA was not preferentially stored i n the olfactory tissue. The response to odours of olfactory tissue from deficient G-P’s, as measured by EOG amplitude, was not significantly altered. However the number of G-P’s used was small and the range of odours was very limited. These results indicate that whilst the high levels of AA suggest it has an important role in olfactory tissue, it may not be essential for transduction. We would like to thank Michael Skinner lor performing the Ems. 1. Hornig, D. (1975) Anals. N.Y. Acad. Sci. 2 5 8 , 1 0 3 - 1 1 8 2. Ash, K.O. (1969) Science 1 6 5 , 9 0 1 - 9 0 2 3. Surnmerwell, W.N. 8 Sealock,R.R. (1952) J. Biol. Chern. 1 9 6 , 753-759 4. Roe, J.H. 8 Kuether. C.A. (1’943) J. Biol. Chem. 1 4 7 , 3 9 9 407 5. Nishikirni, M. (1979) Methods in Enzymology 6 2 , 2 4 - 3 0 6. Shirley, S.,Polak, E. 8 Dodd, G.H. (1983) Eur. J. Biochern. 1 3 2 , 485-494 7. Hughes, R.E., Hurley, R.J. 8 Jones, P.R. (1971) Br. J. Nutr. 2 6 , 433-438 8. Varrna, S.D. (1987) Anals. N.Y. Acad. Sci. 4 9 8 , 2 8 0 - 3 0 6
S t u d i e s on t h e r o l e of ascorbic acid i n olfactory tissue.
199s
Table 1. Ascorbic acid levels in animal tissues. Results represent the mean f standard deviation of 3 independent experiments (triplicate readings from single animals).
ROSEMARY BLAND, STAN LOVETT and GEORGE H. DODD ua A N a wet weiaht. Mean f SD
Tissue
Olfaction Research Group, University of Warwick, Coventry, CV4 7AL, U.K. Ascorbic acid (AA)’ accumulates in the olfactory bulb [l] and it has been suggested that AA may be a cofactor in olfactory transduction [2].The levels of free AA (approximately 20% of AA may be bound, [3]) were determined in the brain, hepatic, olfactory and respiratory tissue of rats, rabbits and guinea-pigs (G-P’s). Olfactory tissue was examined for the presence of gulonolactone oxidase (GLO), the final enzyme in the biosynthetic pathway of AA. To investigate whether or not AA was essential for olfactory tissue to respond to odours, G-P’s were fed a diet deficient i n AA to reduce the levels of AA in the olfactory tissue. Electro-olfactograms (EOG’s) were performed on the olfactory tissue of the G-P’s. Ascorbic acid was determined using the 2,4dinitrophenylhydrazine (DNPH) colorimetric method [4]. Tissue samples were prepared by homogenisation in 5% TCA and the precipitated protein was removed by centrifugation (1 O,OOOg, 20 minutes). The supernatant was incubated with the DNPH solution for 3 hours, and the reaction was stopped and the colour developed with 65% H2S04. GLO was measured in terms of AA produced from gulonolactone. Tissue was prepared in phosphate buffer (O.lM, pH 7.5) and incubated with gulonolactone (2.5mM) for 15 minutes at 37°C. The reaction was stopped with 50% TCA [5]. Ascorbic acid production was measured by the DNPH method. For the deficiency study, experimental and control diets were supplied by Special Diet Services. G-P’s were maintained on the deficient diet for between 15-18 days. EOG’s were performed as previously described [6], except that AA was omitted from the Ringer solution. Eugenol, cineole and iso-amylacetate were the odours tested. The quantity of AA present in the tissues is shown i n Table 1 . No significant activity of GLO was found in rat olfactory or brain tissue, although it was detected in hepatic tissue of rats. In the deficiency study, 5 control and 6 scorbutic G-P’s were examined. The G-P’s were not pair-fed, but their weights were monitored and the weight change of the experimental groups closely matched that of the control group. ’Abbreviations used: AA, ascorbic acid; DNPH, 2,4-dinitrophenylhydrazine; EOG, electro-olfactograrn; GLO, gulonolactone oxidase; G-P’s, guinea-pigs; TCA, trichloroacetic acid.
Rat
+ 178
Rabbit
Guinea-pig
253 f 33
5 5 0 f 24
Olfactory
722
Respiratory
31 6 f 86
157 f 16
457 f 40
Brain
156 f 1
155 f 7.0
143 f 22
Hepatic
347 f 45
170 f 39
169 f 25
Therefore any differences in the response of the groups would not be due to differing nutritional states. After 15-18 days on the scorbutic diet the levels of AA in the G-P tissues had fallen. Hepatic, olfactory and respiratory tissue were 5.2%, 8.1% and 26.1% of the control G-P’s respectively. The brain, which is known to retain AA [7], had only fallen to 60% of the control value. The levels of AA in scorbutic G-P’s returned to control levels, in all tissues, after 7 days on a normal diet. As table 1 shows the levels of AA in the olfactory tissue are higher than the respiratory, hepatic and brain tissue in the animals examined. Interestingly there is also a high concentration of AA in another sensory tissue, retina [8]. However, GLO was not detected in the olfactory tissue so the high levels are not caused by AA production. When the G-P’s were maintained on an AA deficient diet AA was not preferentially stored i n the olfactory tissue. The response to odours of olfactory tissue from deficient G-P’s, as measured by EOG amplitude, was not significantly altered. However the number of G-P’s used was small and the range of odours was very limited. These results indicate that whilst the high levels of AA suggest it has an important role in olfactory tissue, it may not be essential for transduction. We would like to thank Michael Skinner lor performing the Ems. 1. Hornig, D. (1975) Anals. N.Y. Acad. Sci. 2 5 8 , 1 0 3 - 1 1 8 2. Ash, K.O. (1969) Science 1 6 5 , 9 0 1 - 9 0 2 3. Surnmerwell, W.N. 8 Sealock,R.R. (1952) J. Biol. Chern. 1 9 6 , 753-759 4. Roe, J.H. 8 Kuether. C.A. (1’943) J. Biol. Chem. 1 4 7 , 3 9 9 407 5. Nishikirni, M. (1979) Methods in Enzymology 6 2 , 2 4 - 3 0 6. Shirley, S.,Polak, E. 8 Dodd, G.H. (1983) Eur. J. Biochern. 1 3 2 , 485-494 7. Hughes, R.E., Hurley, R.J. 8 Jones, P.R. (1971) Br. J. Nutr. 2 6 , 433-438 8. Varrna, S.D. (1987) Anals. N.Y. Acad. Sci. 4 9 8 , 2 8 0 - 3 0 6
