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A relationship between ascorbic acid and threonic acid in guinea-pigs
Fd Chem. Toxic. Vol. 21, no. 4, pp. 449M.52, 1983
0015-6264/83503.00+0.00 Copyright ~, 1983 PergamonPress Ltd
Printed in Great Britain. All rights reserved
A RELATIONSHIP BETWEEN ASCORBIC ACID A N D THREONIC ACID IN GUINEA-PIGS M. THOMASand R. E. HUGHES University of Wales Institute of Science and Technology, Cardiff, Wales (Received 16 July 1982; revision received 18 November 1982)
Abstract--Threonic acid is a major breakdown product of ascorbic acid used as a food additive. When administered orally to guinea-pigs (100 mg/kg body weight) for periods of 4 or 28 days, it produced a significant fall in the ascorbic acid concentration of certain organs but was without effect on other physiological and biochemical characteristics. The lifespan of scorbutic guinea-pigs was significantly reduced by dietary threonic acid (100 mg/kg body weight). The results indicate that threonic acid may modify the metabolism of ascorbic acid in guinea-pigs.
INTRODUCTION Ascorbic acid (L-xyloascorbic acid; 3-keto-L-gulofuranolactone; vitamin C) is widely used as a technological aid in the food industry. It has been calculated that the amount used as a bread improver alone amounts to some 70 tonnes annually in the UK (Chamberlain, 1981). Substantial amounts are used in the preparation of other foodstuffs such as sausages and other meat products and canned mushrooms. Because of its extreme lability during processing and storage, little of this added ascorbic acid is present in the commodities purchased by the consumer. Of the ascorbic acid added during the Chorleywood Bread Process (75 ppm) virtually none was present in the marketed product (Chamberlain, Collins, Elton et al. 1966), and samples of canned mushrooms were found on analysis to have lost over 98% of their added ascorbic acid (M. Thomas, unpublished results 1981). In contrast, ascorbic acid in fruit-juice preparations is stable over comparatively long periods, presumably because of the protective influence of the bioflavonoids present in many fruit juices (Hughes & Wilson, 1977). The average consumer may, therefore, ingest annually from additive sources some 2-10 g ascorbic acid breakdown products. Few of these breakdown products have been identified with certainty and their nature and formation are influenced by the prevailing conditions of processing and storage. In one case, the in vitro formation of some 20 different breakdown products was reported (Tatum, Shaw & Berry, 1969), and 15 of these were identified. Among the products it would appear that threonic acid (threo-2,3,4trihydroxybutyric acid) is of major significance. Thewliss (1971) reported that 52% of the ascorbic acid used in the Chorleywood Bread Process was left in the bread as threonic acid. Threonic acid has also been detected by gas-liquid chromatography in various processed meat products to which ascorbic acid had been added as a technological aid, although in no case did the threonic acid concentration exceed 10% of the ascorbic acid added (M. Thomas, unpublished results 1982). Significant amounts of threonic acid may, therefore, be ingested by the average consumer. Yet nothing is known of its metabolic fate in, or its 449
influence on, the body. Threonic acid has recently acquired an added significance because of reports that ascorbic acid or its breakdown products may have mutagenic activity (Hughes, 1981; Omura, Shinohara, Maeda et al. 1978; Stich, Wei & Whiting, 1979). The experiments described in this report were designed to investigate the influence of ingested threonic acid on the metabolism of guinea-pigs and also to study the relationship between ingested threonic acid and ascorbic acid in hypovitaminotic C guinea-pigs.
EXPERIMENTAL
Threonic acid. Calcium threonate was prepared by oxidation of ascorbic acid with hydrogen peroxide (Isbell & Frush, 1979). Analysis of the final product gave: C, 29.26; H, 4.87; Ca, 12.04 (calculated: C, 29.27; H, 4.91; Ca, 12.20). The specific rotation was [~]~2 + 13.62: (c = 1.0 in H20, in close agreement with the value of [~]~ + 13.80 (c = 1.0 in H20 ) reported by Isbell & Frush (1979). Threonic acid was prepared by passing a solution of calcium threonate (12.15 g in 800ml warm glassdistilled water) through an Amberlite Resin column (IR-120 H + form). The column was washed with 1000 ml distilled water to ensure complete removal of the threonate ion. The eluate was concentrated in a rotary evaporator under high vacuum at 4ff'C. Lactonization of the threonic acid occurred during the concentrating process. Thin-layer chromatography of the product on Merck cellulose plastic sheets (without fluorescent indicator) and detection with hydroxamate (Dawson, 1969) gave Rv values of 0.55 (lactone) and 0.37 (threonic acid) with the solvent mixture butanol-ethanol-water (4:1 : 1, by vol.) and 0.66 (lactone) and 0.44 (threonic acid) with propanol formic acid-water (6:3: 1, by vol.). Contaminants (e.g. ascorbic acid and oxalic acid) were not detected on spraying the sheets with 2,6-dichlorophenolindophenol and ammoniacal silver nitrate (Dawson, 1969). The melting point of the recrystallized threonolactone was 65 67°C. Gatzi & Reichstein (1937) reported a value of 65-68"C.
450
M. THOMASand R. E. HUGHES Table 1. Organ weights and ascorbic acid status of guinea-pigs dosed orally with threonic acid for 4 days Values for group:
Parameter
Supplements (mg/kg body weight/day): (a) Ascorbic acid... (b) Threonic acid...
Body weight initial (g) final (g) Food intake (g/100 g body weight) Liver weight (g) (g/100 g body weight) --ascorbic acid (rag/100 g tissue) Adrenals weight (g) (g/100 g body weight) --ascorbic acid (mg/100 g tissue) Spleen weight (g) (g/100 g body weight) --ascorbic acid (mg/100 g tissue) Testes--weight (g) (g/100 g body weight) ascorbic acid (rag/100 g tissue) Kidneys--weight (g) (g/100 g body weight)
A (control)
B (test)
5 0
5 100
428 _+ 13 436 _+ 14 10.2 + 1.2 14.93 _+0.67 3.42 +_0.07 2.70 _+0.14 0.203 + 0.016 0.047 _+ 0.005 14.22 + 1.26 0.561 + 0.037 0.129 _+0.007 5.59 _+0.65 2.64 _+0.18 0.61 _+ 0,03 5.33 ,+ 0.16 3.40 ,+ 0~09 0.78 _+0.02
427 _+9 437 _+ 10 10.8 _+0.9 14.97 _+0.48 3.43 +_0.07 1.52 + 0.22* 0.229 _+0.010 0.052 _+0.003 8.24 _+ 1.18" 0.589 _+0.030 0.135 + 0.008 4.54 _+0.68 2.46 _+0.19 0.56 ,+ 0.04 4.27 ,+ 0.30* 3.51 +_0.10 0.80 _+0.02
Values are means _+ SEM for groups of ten guinea-pigs. Those marked with an asterisk differ significantly (P < 0.01 by Student's t test) from the control.
Animals and diet. Young male albino guinea-pigs, Dunkin Hartley strain (initial body weight c. 300 g) were used. They received the semisynthetic scorbutogenic diet prepared as previously described (Williams & Hughes, 1972). Conditions of feeding, dosing and husbandry were also as previously described (Williams & Hughes, 1972). Animals were killed by stunning followed by decapitation. Analytical procedures. Organs were removed, freed from adventitious tissue, dried rapidly with filter paper and weighed within 5 min of their removal from the body. Ascorbic acid was determined in 10~/~ metaphosphoric acid extracts using the 2,6-dichlorophenolindophenol-dye technique (Williams & Hughes, 1972). Packed cell volume and haemoglobin content of terminal blood samples were determined using standard procedures (Archer, 1965). Cholesterol (von Roschlau, Bernt & Gruber, 1974), alkaline phosphatase (Bessey, Lowry & Brock, 1946), alanine aminotransferase (Wr6blewski & LaDue, 1956) and aspartate aminotransferase (Karmen, 1955) were determined on plasma from heparinized blood using Boehringer-Mannheim Test Combinations.
weight at 15.00hr. For the control group (A) the threonic acid dose was omitted. Experiment 2. Two groups (C and D) each of ten guinea-pigs received exactly the same treatment as in Experiment 1 but the test period was extended from 4 to 28 days. Experiment 3. This was designed to study the influence of dietary threonic acid on the lifespan of scorbutic guinea-pigs and to assess the nutritional significance of the threonic acid-induced depletion of ascorbic acid recorded in Experiments I and 2 (Results, Tables 1 & 2). Two groups of guinea-pigs (E and F) were used. After the initial 3-day saturation period no further ascorbic acid was administered. G r o u p F received a daily oral supplement of 100 mg threonic acid/kg body weight; group E (control) received no threonic acid. Food and water were available ad lib. The survival periods of the guineapigs were recorded.
Statistical analysis Differences between test and control groups were investigated using Student's t test. The level of significance chosen was P < 0.05.
Dosing schedules Experiment I. Two groups (A and B) each of ten
RESULTS
guinea-pigs, received a l"J~ solution of ascorbic acid as drinking-water for 3 days to produce tissue saturation with ascorbic acid. This was followed by a 10-day period of ascorbic acid depletion. This procedure was adopted to eliminate differences between the initial ascorbic acid status of the guinea-pigs and to produce a uniform basal concentration in all animals (Hughes, Hurley & Jones, 1971; Hurley, Jones & Hughes, 1972). For a further 4 days, group B received a daily oral dose of 100rag threonic acid/kg body weight at 09.00 hr and a daily oral 'maintenance' dose of 5 mg ascorbic acid/kg body
Results are summarized in Tables l and 2. There was no significant difference between the food intakes of treated and control animals (Tables I & 2). There was no evidence that threonic acid influenced organ weights (relative or absolute), haematological characteristics or the plasma concentrations of cholesterol and certain enzymes frequently used in toxicity assessments. However, in both the 4-day and the 28-day experiments there was evidence of a threonic acidinduced fall in the ascorbic acid concentration in certain organs. In the 4-day experiment this fall was apparent in the adrenals, testes and liver but in the
Ascorbic acid and threonic acid
451
Table 2. Organ weights, ascorbic acid status, haematological values and plasma determinations for guinea-pigs dosed orally with threonic acid for 28 days Values for group:
Parameter
Supplements (mg/kg body weight/day): Ascorbic acid... Threonic acid...
Body weight --initial (g) --final (g) Food intake (g/100 g body weight) Liver--weight (g) (g/100 g body weight) --ascorbic acid (mg/100 g tissue) Adrenals--weight (g) (g/100 g body weight) --ascorbic acid (mg/100 g tissue) Spleen--weight (g) (g/100 g body weight) --ascorbic acid (mg/100 g tissue) Teste~weight (g) (g/100 g body weight) --ascorbic acid (mg/100 g tissue) Kidneys--weight (g) (g/100 g body weight) Blood --PCV (0%) --haemoglobin (g/100 ml) Plasma---cholesterol (mmol/litre) alk. phosphatase (U/litre, 37C) - - G P T (U/litre, 25'C) - - G O T (U/litre, 25~'C)
C (control)
D (test)
5 0
5 100
457 ± 10 606 ± 12 9.7 ± 0.3 20.18 ± 0.57 3.33 ± 0.06 5.11 ± 0.40 0.282 ± 0.021 0.047 ± 0.003 31.87 _+ 2.09 0.760 + 0.060 0.125 ± 0.009 11.50 ± 0.57 3.70 ± 0.15 0.61 ± 0.02 6.04 ± 0.20 3.95 ± 0.09 0.65 ± 0.01 42.6 ± 0.4 18.9 ± 1.0 2.15 ± 0.16 130.7 ± 6.2 100.7 ± 18.7 147.2 ± 20.3
476 ± 11 644 ± 12 10.4 _+0.6 19.06 ± 0.40 2.96 ± 0.07 2.63 + 0.47* 0.288 ± 0.014 0.045 ± 0.002 32.74 _+2.63 0.773 ± 0.028 0.120 ± 0.005 8.81 ± 0.33* 4.07 ± 0.14 0.63 ± 0.02 5.62 _+0.20 4.27 ± 0.14 0.66 ± 0.02 42.6 ± 0.3 17.3 ± 0.5 2.32 ± 0.18 153.8 + 12.5 102.8 ± 19.7 148.3 ± 19. I
GPT = Glutamic-pyruvic transaminase (alanine aminotransferase) GOT = Glutamic-oxalacetic transaminase (aspartate aminotransferase) Values are means ± SEM for groups of ten guinea-pigs. Those marked with an asterisk differ significantly (P < 0.01 by Student's t test) from the control.
28-day experiment it was confined to the liver and spleen (Tables 1 & 2). The ascorbic acid concentrations in experiment 1 were in general lower than the corresponding ones in experiment 2, presumably because after 4 days the tissue levels had not attained the equilibrium concentration found after 28 days. The survival experiment (experiment 3) indicated that orally administered threonic acid shortened significantly ( P < 0 . 0 1 ) the lifespan of scorbutic guinea-pigs. In group F, fed 100 mg threonic acid/kg body weight/day, the mean survival time ( + SEM) alter the start of ascorbic acid deprivation was 30.0 _+ 0.72 days, compared with 32.4 _+ 0.70 days in the control group given no threonic acid supplement. DISCUSSION Threonic acid is formed in insignificant amounts from ascorbic acid used as a technological aid in the food industry. The amount of threonic acid administered in the experiments described in this paper (100 mg/kg body weight) was some hundreds of times greater, on a body-weight basis, than the probable maximum intake by man from the breakdown of ascorbic acid used as a food additive. However, such ascorbic acid is not the only source of threonic acid. Some may be formed in the gastro-intestinal tract by the breakdown of ascorbic acid ingested p e r se. This could be of especial significance in 'vitamin C megatherapy' the ingestion for supposedly therapeutic
reasons of amounts of ascorbic acid far in excess of the Recommended Daily A m o u n t a circumstance in which up to 50% of a 1 g dose is unabsorbed and may undergo chemical and/or microbiological degradation (Hughes, 1981). The two short-term experiments described in this paper indicated that, in guinea-pigs, threonic acid is without effect on a number of physiological and biochemical characteristics customarily regarded as being of significance in toxicity trials. The only clearly defined effect of the threonic acid was a depression of the ascorbic acid content of certain organs. This occurred in the adrenals, testes and liver after 4 days but after 28 days was limited to the liver and spleen, possibly suggesting an adaptation on the part of the guinea-pig. As the threonic acid was administered separately from the ascorbic acid, it is unlikely that the fall in tissue ascorbic acid reflects any interference with its absorption from the gastro-intestinal tract. It would appear to be more likely that the threonic acid modifies the uptake/retention of ascorbic acid by the tissues--or, possibly, that it influences its subsequent availability. Experiment 3 indicated that the threonic acidinduced fall in ascorbic acid is sufficient to influence significantly the survival time of ascorbic aciddepleted guinea-pigs. The absence of any concomitant changes in organ weights and blood biochemical patterns would suggest that this is a specific threonic acid-ascorbic acid relationship, rather than a pointer
452
M. THOMAS and R. E. HUGHES
to a more general toxicity on the part o f threonic acid. These findings with guinea-pigs, if equally valid in h u m a n nutrition, could be of some significance for certain categories currently d i s a d v a n t a g e d with respect to ascorbic acid intake. G r o u p s such as the institutionalized elderly could have their already ina d e q u a t e body stores of ascorbic acid (Hughes, 1981) still further reduced by the intake of threonic acid. In more general terms, the study underlines the imp o r t a n c e in the food industry of assessing the significance n o t only of p a r e n t additives, but also of any k n o w n or suspected b r e a k d o w n products. The practice of some m a n u f a c t u r e r s of including "ascorbic acid" in the p r o d u c t description, when the original additive, ascorbic acid, has u n d e r g o n e m o r e or less complete b r e a k d o w n is b o t h scientifically incorrect and nutritionally misleading. REFERENCES
Archer R. K. (1965). Haematological Techniques for Use on Animals'. Blackwell Scientific Publications, Oxford. Bessey O. A., Lowry O. H. & Brock M. J. (1946). Method for the determination of alkaline phosphatase with five cubic millimeters of serum. J. biol. Chem. 164, 321. Chamberlain N. (1981). The use of ascorbic acid in breadmaking. In Vitamin C (Ascorbic Acid). Edited by J. N. Counsell & D. H. Hornig. p. 87. Applied Science Publishers, London. Chamberlain N., CoNns T. H., E|ton G. A., Hollingsworth D. F., Lisle D. B. & Payne P, R. (1966). Studies on the composition of food. 2. Comparison of the nutrient content of bread made conventionally and by the Chorleywood Bread Process. Br. J. Nutr. 20, 747. Dawson R. M. C. (Editor) 0969). Data for Biochemical Research. pp. 571 & 590. Oxford University Press, Oxford.
Gatzi K. & Reichstein T. (1937). Crystallized L-threonic acid lactone and synthesis of L-threonic acid 2-methyl ether. Helv. chim. Acta 20, 1298. Hughes R. E. (1981). Vitamin C: Some Current Problems. British Nutrition Foundation, London. Hughes R. E., Hurley R. J. & Jones P. R. (1971). The retention of ascorbic acid by guinea-pig tissues. Br. J. Nutr. 26, 433. Hughes R. E. & Wilson H. K. (1977). Flavonoids: some physiological and nutritional considerations. Prog. mednl Chem. 14, 285. Hurley R. J., Jones P. R. & Hughes R. E. (1972). The uptake of ascorbic acid by the tissues of ascorbic acid-deficient guinea-pigs. Nutr. Metab. 14, 136. Isbell H. S. & Frush H. L. (1979). Oxidation of L-ascorbic acid by hydrogen peroxide: preparation of e-threonic acid. Carb. Res. 72, 301. Karmen A. (1955). A note on the spectrophotometric assay of glutamic-oxaloacetic transaminase in human blood serum. J. clin. Invest. 34, 131. Omura H., Shinohara K., Maeda H., Nonaka M. & Murakami H. (1978). Mutagenic action of triose reductone and ascorbic acid on Salmonella typhimurium TA 100 strain. J. Nutr. Sci. Vitaminol. 24, 185. Stich H. F., Wei L. & Whiting R. F. (1979). Enhancement of the chromosome-damaging action of ascorbate by transition metals. Cancer Res. 39, 4145. Tatum J. H., Shaw P. E. & Berry R. E. (1969). Degradation products from ascorbic acid. d. agric. Fd Chem. 17, 38. Thewliss B. H. (1971). Fate of ascorbic acid in the Chorleywood Bread Process. J. Sci. Fd Agric. 22, 16. von Roschlau P., Bernt E. & Gruber W. (1974). Enzymatische Bestimmung des Gesamt-Cholesterins in Serum. Z. klin. chem. klin. Biochem. 12, 403. Williams R. S. & Hughes R. E. (1972). Dietary protein, growth and retention of aseorbic acid in guinea-pigs. Br. J. Nutr. 28, 167. Wr6blewski F. & LaDue J. S. (1956). Serum glutamic pyruvic transaminase in cardiac and hepatic disease. Proc. Soc. expl Biol. Med. 91, 569.
0015-6264/83503.00+0.00 Copyright ~, 1983 PergamonPress Ltd
Printed in Great Britain. All rights reserved
A RELATIONSHIP BETWEEN ASCORBIC ACID A N D THREONIC ACID IN GUINEA-PIGS M. THOMASand R. E. HUGHES University of Wales Institute of Science and Technology, Cardiff, Wales (Received 16 July 1982; revision received 18 November 1982)
Abstract--Threonic acid is a major breakdown product of ascorbic acid used as a food additive. When administered orally to guinea-pigs (100 mg/kg body weight) for periods of 4 or 28 days, it produced a significant fall in the ascorbic acid concentration of certain organs but was without effect on other physiological and biochemical characteristics. The lifespan of scorbutic guinea-pigs was significantly reduced by dietary threonic acid (100 mg/kg body weight). The results indicate that threonic acid may modify the metabolism of ascorbic acid in guinea-pigs.
INTRODUCTION Ascorbic acid (L-xyloascorbic acid; 3-keto-L-gulofuranolactone; vitamin C) is widely used as a technological aid in the food industry. It has been calculated that the amount used as a bread improver alone amounts to some 70 tonnes annually in the UK (Chamberlain, 1981). Substantial amounts are used in the preparation of other foodstuffs such as sausages and other meat products and canned mushrooms. Because of its extreme lability during processing and storage, little of this added ascorbic acid is present in the commodities purchased by the consumer. Of the ascorbic acid added during the Chorleywood Bread Process (75 ppm) virtually none was present in the marketed product (Chamberlain, Collins, Elton et al. 1966), and samples of canned mushrooms were found on analysis to have lost over 98% of their added ascorbic acid (M. Thomas, unpublished results 1981). In contrast, ascorbic acid in fruit-juice preparations is stable over comparatively long periods, presumably because of the protective influence of the bioflavonoids present in many fruit juices (Hughes & Wilson, 1977). The average consumer may, therefore, ingest annually from additive sources some 2-10 g ascorbic acid breakdown products. Few of these breakdown products have been identified with certainty and their nature and formation are influenced by the prevailing conditions of processing and storage. In one case, the in vitro formation of some 20 different breakdown products was reported (Tatum, Shaw & Berry, 1969), and 15 of these were identified. Among the products it would appear that threonic acid (threo-2,3,4trihydroxybutyric acid) is of major significance. Thewliss (1971) reported that 52% of the ascorbic acid used in the Chorleywood Bread Process was left in the bread as threonic acid. Threonic acid has also been detected by gas-liquid chromatography in various processed meat products to which ascorbic acid had been added as a technological aid, although in no case did the threonic acid concentration exceed 10% of the ascorbic acid added (M. Thomas, unpublished results 1982). Significant amounts of threonic acid may, therefore, be ingested by the average consumer. Yet nothing is known of its metabolic fate in, or its 449
influence on, the body. Threonic acid has recently acquired an added significance because of reports that ascorbic acid or its breakdown products may have mutagenic activity (Hughes, 1981; Omura, Shinohara, Maeda et al. 1978; Stich, Wei & Whiting, 1979). The experiments described in this report were designed to investigate the influence of ingested threonic acid on the metabolism of guinea-pigs and also to study the relationship between ingested threonic acid and ascorbic acid in hypovitaminotic C guinea-pigs.
EXPERIMENTAL
Threonic acid. Calcium threonate was prepared by oxidation of ascorbic acid with hydrogen peroxide (Isbell & Frush, 1979). Analysis of the final product gave: C, 29.26; H, 4.87; Ca, 12.04 (calculated: C, 29.27; H, 4.91; Ca, 12.20). The specific rotation was [~]~2 + 13.62: (c = 1.0 in H20, in close agreement with the value of [~]~ + 13.80 (c = 1.0 in H20 ) reported by Isbell & Frush (1979). Threonic acid was prepared by passing a solution of calcium threonate (12.15 g in 800ml warm glassdistilled water) through an Amberlite Resin column (IR-120 H + form). The column was washed with 1000 ml distilled water to ensure complete removal of the threonate ion. The eluate was concentrated in a rotary evaporator under high vacuum at 4ff'C. Lactonization of the threonic acid occurred during the concentrating process. Thin-layer chromatography of the product on Merck cellulose plastic sheets (without fluorescent indicator) and detection with hydroxamate (Dawson, 1969) gave Rv values of 0.55 (lactone) and 0.37 (threonic acid) with the solvent mixture butanol-ethanol-water (4:1 : 1, by vol.) and 0.66 (lactone) and 0.44 (threonic acid) with propanol formic acid-water (6:3: 1, by vol.). Contaminants (e.g. ascorbic acid and oxalic acid) were not detected on spraying the sheets with 2,6-dichlorophenolindophenol and ammoniacal silver nitrate (Dawson, 1969). The melting point of the recrystallized threonolactone was 65 67°C. Gatzi & Reichstein (1937) reported a value of 65-68"C.
450
M. THOMASand R. E. HUGHES Table 1. Organ weights and ascorbic acid status of guinea-pigs dosed orally with threonic acid for 4 days Values for group:
Parameter
Supplements (mg/kg body weight/day): (a) Ascorbic acid... (b) Threonic acid...
Body weight initial (g) final (g) Food intake (g/100 g body weight) Liver weight (g) (g/100 g body weight) --ascorbic acid (rag/100 g tissue) Adrenals weight (g) (g/100 g body weight) --ascorbic acid (mg/100 g tissue) Spleen weight (g) (g/100 g body weight) --ascorbic acid (mg/100 g tissue) Testes--weight (g) (g/100 g body weight) ascorbic acid (rag/100 g tissue) Kidneys--weight (g) (g/100 g body weight)
A (control)
B (test)
5 0
5 100
428 _+ 13 436 _+ 14 10.2 + 1.2 14.93 _+0.67 3.42 +_0.07 2.70 _+0.14 0.203 + 0.016 0.047 _+ 0.005 14.22 + 1.26 0.561 + 0.037 0.129 _+0.007 5.59 _+0.65 2.64 _+0.18 0.61 _+ 0,03 5.33 ,+ 0.16 3.40 ,+ 0~09 0.78 _+0.02
427 _+9 437 _+ 10 10.8 _+0.9 14.97 _+0.48 3.43 +_0.07 1.52 + 0.22* 0.229 _+0.010 0.052 _+0.003 8.24 _+ 1.18" 0.589 _+0.030 0.135 + 0.008 4.54 _+0.68 2.46 _+0.19 0.56 ,+ 0.04 4.27 ,+ 0.30* 3.51 +_0.10 0.80 _+0.02
Values are means _+ SEM for groups of ten guinea-pigs. Those marked with an asterisk differ significantly (P < 0.01 by Student's t test) from the control.
Animals and diet. Young male albino guinea-pigs, Dunkin Hartley strain (initial body weight c. 300 g) were used. They received the semisynthetic scorbutogenic diet prepared as previously described (Williams & Hughes, 1972). Conditions of feeding, dosing and husbandry were also as previously described (Williams & Hughes, 1972). Animals were killed by stunning followed by decapitation. Analytical procedures. Organs were removed, freed from adventitious tissue, dried rapidly with filter paper and weighed within 5 min of their removal from the body. Ascorbic acid was determined in 10~/~ metaphosphoric acid extracts using the 2,6-dichlorophenolindophenol-dye technique (Williams & Hughes, 1972). Packed cell volume and haemoglobin content of terminal blood samples were determined using standard procedures (Archer, 1965). Cholesterol (von Roschlau, Bernt & Gruber, 1974), alkaline phosphatase (Bessey, Lowry & Brock, 1946), alanine aminotransferase (Wr6blewski & LaDue, 1956) and aspartate aminotransferase (Karmen, 1955) were determined on plasma from heparinized blood using Boehringer-Mannheim Test Combinations.
weight at 15.00hr. For the control group (A) the threonic acid dose was omitted. Experiment 2. Two groups (C and D) each of ten guinea-pigs received exactly the same treatment as in Experiment 1 but the test period was extended from 4 to 28 days. Experiment 3. This was designed to study the influence of dietary threonic acid on the lifespan of scorbutic guinea-pigs and to assess the nutritional significance of the threonic acid-induced depletion of ascorbic acid recorded in Experiments I and 2 (Results, Tables 1 & 2). Two groups of guinea-pigs (E and F) were used. After the initial 3-day saturation period no further ascorbic acid was administered. G r o u p F received a daily oral supplement of 100 mg threonic acid/kg body weight; group E (control) received no threonic acid. Food and water were available ad lib. The survival periods of the guineapigs were recorded.
Statistical analysis Differences between test and control groups were investigated using Student's t test. The level of significance chosen was P < 0.05.
Dosing schedules Experiment I. Two groups (A and B) each of ten
RESULTS
guinea-pigs, received a l"J~ solution of ascorbic acid as drinking-water for 3 days to produce tissue saturation with ascorbic acid. This was followed by a 10-day period of ascorbic acid depletion. This procedure was adopted to eliminate differences between the initial ascorbic acid status of the guinea-pigs and to produce a uniform basal concentration in all animals (Hughes, Hurley & Jones, 1971; Hurley, Jones & Hughes, 1972). For a further 4 days, group B received a daily oral dose of 100rag threonic acid/kg body weight at 09.00 hr and a daily oral 'maintenance' dose of 5 mg ascorbic acid/kg body
Results are summarized in Tables l and 2. There was no significant difference between the food intakes of treated and control animals (Tables I & 2). There was no evidence that threonic acid influenced organ weights (relative or absolute), haematological characteristics or the plasma concentrations of cholesterol and certain enzymes frequently used in toxicity assessments. However, in both the 4-day and the 28-day experiments there was evidence of a threonic acidinduced fall in the ascorbic acid concentration in certain organs. In the 4-day experiment this fall was apparent in the adrenals, testes and liver but in the
Ascorbic acid and threonic acid
451
Table 2. Organ weights, ascorbic acid status, haematological values and plasma determinations for guinea-pigs dosed orally with threonic acid for 28 days Values for group:
Parameter
Supplements (mg/kg body weight/day): Ascorbic acid... Threonic acid...
Body weight --initial (g) --final (g) Food intake (g/100 g body weight) Liver--weight (g) (g/100 g body weight) --ascorbic acid (mg/100 g tissue) Adrenals--weight (g) (g/100 g body weight) --ascorbic acid (mg/100 g tissue) Spleen--weight (g) (g/100 g body weight) --ascorbic acid (mg/100 g tissue) Teste~weight (g) (g/100 g body weight) --ascorbic acid (mg/100 g tissue) Kidneys--weight (g) (g/100 g body weight) Blood --PCV (0%) --haemoglobin (g/100 ml) Plasma---cholesterol (mmol/litre) alk. phosphatase (U/litre, 37C) - - G P T (U/litre, 25'C) - - G O T (U/litre, 25~'C)
C (control)
D (test)
5 0
5 100
457 ± 10 606 ± 12 9.7 ± 0.3 20.18 ± 0.57 3.33 ± 0.06 5.11 ± 0.40 0.282 ± 0.021 0.047 ± 0.003 31.87 _+ 2.09 0.760 + 0.060 0.125 ± 0.009 11.50 ± 0.57 3.70 ± 0.15 0.61 ± 0.02 6.04 ± 0.20 3.95 ± 0.09 0.65 ± 0.01 42.6 ± 0.4 18.9 ± 1.0 2.15 ± 0.16 130.7 ± 6.2 100.7 ± 18.7 147.2 ± 20.3
476 ± 11 644 ± 12 10.4 _+0.6 19.06 ± 0.40 2.96 ± 0.07 2.63 + 0.47* 0.288 ± 0.014 0.045 ± 0.002 32.74 _+2.63 0.773 ± 0.028 0.120 ± 0.005 8.81 ± 0.33* 4.07 ± 0.14 0.63 ± 0.02 5.62 _+0.20 4.27 ± 0.14 0.66 ± 0.02 42.6 ± 0.3 17.3 ± 0.5 2.32 ± 0.18 153.8 + 12.5 102.8 ± 19.7 148.3 ± 19. I
GPT = Glutamic-pyruvic transaminase (alanine aminotransferase) GOT = Glutamic-oxalacetic transaminase (aspartate aminotransferase) Values are means ± SEM for groups of ten guinea-pigs. Those marked with an asterisk differ significantly (P < 0.01 by Student's t test) from the control.
28-day experiment it was confined to the liver and spleen (Tables 1 & 2). The ascorbic acid concentrations in experiment 1 were in general lower than the corresponding ones in experiment 2, presumably because after 4 days the tissue levels had not attained the equilibrium concentration found after 28 days. The survival experiment (experiment 3) indicated that orally administered threonic acid shortened significantly ( P < 0 . 0 1 ) the lifespan of scorbutic guinea-pigs. In group F, fed 100 mg threonic acid/kg body weight/day, the mean survival time ( + SEM) alter the start of ascorbic acid deprivation was 30.0 _+ 0.72 days, compared with 32.4 _+ 0.70 days in the control group given no threonic acid supplement. DISCUSSION Threonic acid is formed in insignificant amounts from ascorbic acid used as a technological aid in the food industry. The amount of threonic acid administered in the experiments described in this paper (100 mg/kg body weight) was some hundreds of times greater, on a body-weight basis, than the probable maximum intake by man from the breakdown of ascorbic acid used as a food additive. However, such ascorbic acid is not the only source of threonic acid. Some may be formed in the gastro-intestinal tract by the breakdown of ascorbic acid ingested p e r se. This could be of especial significance in 'vitamin C megatherapy' the ingestion for supposedly therapeutic
reasons of amounts of ascorbic acid far in excess of the Recommended Daily A m o u n t a circumstance in which up to 50% of a 1 g dose is unabsorbed and may undergo chemical and/or microbiological degradation (Hughes, 1981). The two short-term experiments described in this paper indicated that, in guinea-pigs, threonic acid is without effect on a number of physiological and biochemical characteristics customarily regarded as being of significance in toxicity trials. The only clearly defined effect of the threonic acid was a depression of the ascorbic acid content of certain organs. This occurred in the adrenals, testes and liver after 4 days but after 28 days was limited to the liver and spleen, possibly suggesting an adaptation on the part of the guinea-pig. As the threonic acid was administered separately from the ascorbic acid, it is unlikely that the fall in tissue ascorbic acid reflects any interference with its absorption from the gastro-intestinal tract. It would appear to be more likely that the threonic acid modifies the uptake/retention of ascorbic acid by the tissues--or, possibly, that it influences its subsequent availability. Experiment 3 indicated that the threonic acidinduced fall in ascorbic acid is sufficient to influence significantly the survival time of ascorbic aciddepleted guinea-pigs. The absence of any concomitant changes in organ weights and blood biochemical patterns would suggest that this is a specific threonic acid-ascorbic acid relationship, rather than a pointer
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M. THOMAS and R. E. HUGHES
to a more general toxicity on the part o f threonic acid. These findings with guinea-pigs, if equally valid in h u m a n nutrition, could be of some significance for certain categories currently d i s a d v a n t a g e d with respect to ascorbic acid intake. G r o u p s such as the institutionalized elderly could have their already ina d e q u a t e body stores of ascorbic acid (Hughes, 1981) still further reduced by the intake of threonic acid. In more general terms, the study underlines the imp o r t a n c e in the food industry of assessing the significance n o t only of p a r e n t additives, but also of any k n o w n or suspected b r e a k d o w n products. The practice of some m a n u f a c t u r e r s of including "ascorbic acid" in the p r o d u c t description, when the original additive, ascorbic acid, has u n d e r g o n e m o r e or less complete b r e a k d o w n is b o t h scientifically incorrect and nutritionally misleading. REFERENCES
Archer R. K. (1965). Haematological Techniques for Use on Animals'. Blackwell Scientific Publications, Oxford. Bessey O. A., Lowry O. H. & Brock M. J. (1946). Method for the determination of alkaline phosphatase with five cubic millimeters of serum. J. biol. Chem. 164, 321. Chamberlain N. (1981). The use of ascorbic acid in breadmaking. In Vitamin C (Ascorbic Acid). Edited by J. N. Counsell & D. H. Hornig. p. 87. Applied Science Publishers, London. Chamberlain N., CoNns T. H., E|ton G. A., Hollingsworth D. F., Lisle D. B. & Payne P, R. (1966). Studies on the composition of food. 2. Comparison of the nutrient content of bread made conventionally and by the Chorleywood Bread Process. Br. J. Nutr. 20, 747. Dawson R. M. C. (Editor) 0969). Data for Biochemical Research. pp. 571 & 590. Oxford University Press, Oxford.
Gatzi K. & Reichstein T. (1937). Crystallized L-threonic acid lactone and synthesis of L-threonic acid 2-methyl ether. Helv. chim. Acta 20, 1298. Hughes R. E. (1981). Vitamin C: Some Current Problems. British Nutrition Foundation, London. Hughes R. E., Hurley R. J. & Jones P. R. (1971). The retention of ascorbic acid by guinea-pig tissues. Br. J. Nutr. 26, 433. Hughes R. E. & Wilson H. K. (1977). Flavonoids: some physiological and nutritional considerations. Prog. mednl Chem. 14, 285. Hurley R. J., Jones P. R. & Hughes R. E. (1972). The uptake of ascorbic acid by the tissues of ascorbic acid-deficient guinea-pigs. Nutr. Metab. 14, 136. Isbell H. S. & Frush H. L. (1979). Oxidation of L-ascorbic acid by hydrogen peroxide: preparation of e-threonic acid. Carb. Res. 72, 301. Karmen A. (1955). A note on the spectrophotometric assay of glutamic-oxaloacetic transaminase in human blood serum. J. clin. Invest. 34, 131. Omura H., Shinohara K., Maeda H., Nonaka M. & Murakami H. (1978). Mutagenic action of triose reductone and ascorbic acid on Salmonella typhimurium TA 100 strain. J. Nutr. Sci. Vitaminol. 24, 185. Stich H. F., Wei L. & Whiting R. F. (1979). Enhancement of the chromosome-damaging action of ascorbate by transition metals. Cancer Res. 39, 4145. Tatum J. H., Shaw P. E. & Berry R. E. (1969). Degradation products from ascorbic acid. d. agric. Fd Chem. 17, 38. Thewliss B. H. (1971). Fate of ascorbic acid in the Chorleywood Bread Process. J. Sci. Fd Agric. 22, 16. von Roschlau P., Bernt E. & Gruber W. (1974). Enzymatische Bestimmung des Gesamt-Cholesterins in Serum. Z. klin. chem. klin. Biochem. 12, 403. Williams R. S. & Hughes R. E. (1972). Dietary protein, growth and retention of aseorbic acid in guinea-pigs. Br. J. Nutr. 28, 167. Wr6blewski F. & LaDue J. S. (1956). Serum glutamic pyruvic transaminase in cardiac and hepatic disease. Proc. Soc. expl Biol. Med. 91, 569.