THE ASCORBIC ACID SYSTEM IN PLANT TISSUES

Transcription

1 THE ASCORBIC ACID SYSTEM IN PLANT TISSUES I. INFLUENCE OF VARIOUS METHODS OF EXTRACTION IN THE ESTIMATION OF DEHYDROASCORBIC ACID BY J. BARKER Botany School, University of Cambridge AND L. W. MAP80N Low Temperature Station for Research in Biochemistry and Biophysics, University of Cambridge and Department of Scientific and fndustrial Research {Received ii January 1958) (With I figure in the text) SUMMARY Evidence is presented which shows that when plant tissues are extracted with metaphosphoric acid at room temperature, some oxidation of ascorbic acid to dehydroascorbic acid occurs. This oxidation was diminished, though no conclusive proof was obtained that it was eliminated, by extracting the tissue with either metaphosphoric acid at 3 C or with methanol-metaphosphoric acid at 70"" C, in the absence of oxygen. An effect attributed to disorganization occurred if the tissue was held in the frozen state and as a result ascorbic acid was oxidized during disintegration in an acid extractant. The effect of disorganization was greater when the period in the frozen state was extended. The lowest values for dehydroascorbic acid in plant tissues have been obtained, either by extraction in metaphosphoric acid at 3" C or by rapid freezing and immediate extraction with methanol-metaphosphoric acid solution at 70 C, both extractions being carried out in the absence of oxygen. These procedures gave similar results within the limits of experimental error. INTRODUCTION Ascorbic acid (AA) and dehydroascorbic acid () are usually estimated after extraction of the tissue with acids such as metaphosphoric or oxalic acid at room temperature. In most plant tissues ascorbic acid is rapidly oxidized when the structure is disorganized as by homogenizing. Since both AA and are relatively stable in metaphosphoric acid (Mapson and Mawson, 1943), a common extraction procedure is to homogenize the tissue in this acid, the intention being to inactivate oxidizing enzymes and to complex copper so rapidly that none of the AA is oxidized to during extraction. The decrease in the content of ascorbic acid, usually observed when potato tubers or detached leaves arc held under aerobic conditions, may be prevented by excluding oxygen (Barker and Mapson, 1932). A simple interpretation of these observations is that 58

2 Estimation of dehydroascorbic acid 59 AA is no longer oxidized to under anaerobic conditions, the irreversible breakdown of to 2-3 dioxo-l-gulonic acid therefore does not occur and the level of AA is maintained. Yet this explanation was not supported by the results of earlier work, for even in tissue maintained under anaerobic conditions was found (Wood, Mercer and Pedlow, 1944; Barker and Mapson, 1952). This prompted us to re-examine the methods of extraction used for estimating this substance in plant tissue. The critical study of Isherwood and Niavis (1956) has emphasized the importance of maintaining a low temperature during extraction procedures designed to stabilize the content of certain labile cell components. Using techniques recommended by these authors the amounts of as determined in our earlier work (Barker and Mapson, 1952, 1955) have been shown to be largely artefacts of the extraction procedure. METHODS Sampling. Either radial longitudinal segments or median longitudinal slices were cut from potato fubers (variety King Edward VII) for extraction as described below. In comparing two different extraction techniques alternate segments or slices were segregated fo give two samples (termed below initially comparable samples) each weighing about 10 g. Each strawberry leaflet and cabbage leaf was cut into two sections to exclude the midrib (with cabbage the two sections were then subdivided further), and a number of such pairs of segments were separated to give two initially comparable samples. Extraction with metaphosphoric acid. Each segment of potato or leaf as cut was dropped directly into metaphosphoric acid 4'','3 w/v, and the sample was homogenized in a blender (Measuring and Scientific Equipment Ltd.), with top drive stainless steel knives. Before and during extraction with metaphosphoric acid at 3 C, the bowl of the blender was cooled by immersion in a methanol-ice freezing mixture. Disintegration of the samples was complete under these conditions within 2 min. Oxygen was excluded during extraction by passing a rapid stream of oxygen-free nitrogen through the metaphosphoric acid before and during bbnding. Extraction with methanol-mefaplwsphoric acid at 70' C. Extraction at 70 ' C was carried out in a solution of methanol 2.^ vol. metaphosphoric acid 16",, w/v i vol. to which solid COs was added to reduce the temperature to 70' C. During blending the mixture was kept at 70 C by immersion in a bath of methanol-solid CO^. This solution was then diluted with an equal volume of metaphosphoric acid 4 ",3 ^^'/v before estimation of ascorbic acid. Oxygen was excluded in most experiments in the manner described above. Extraction ivith boiling methanol-metaphosphoric acid. The plant tissue was dropped into a solution of boiling methanol-metaphosphoric acid of the same composition as that described above, the solution boiled for i min, after which it-w as rapidly cooled to room temperature and blended. Estimation of ascorbic and dehydroascorhic acids. Ascorbic acid was determined by titration against 2-6 dichlorophenolindophenol, and dehydroascorbic acid was estimated by determining the increase in titre after reduction by Eschcrichia coli (Mapson and Ingram, 1950) or by the use of homocysteine (Hughes, 1956). The former method gave consistently very slightly higher results than the latter method, which, bt-ing more convenient, has been generally used. Impurities present in some samples of KH.POj used for the neutralization of the

3 6o J. BARKER AND L. W. MAPSON metaphosphoric acid extracts were found to promote the oxidation of ascorbic acid; these impurities were removed on recrystallization. All analytical results are expressed as mg per ioo g fresh wt. RESULTS The effect of temperature of extraction. Potato slices were extracted with metaphosphoric acid (2 (, w/v) at temperatures between 3 and +37 C. The content of as found in the extract decreased with decreasing temperature of extraction (Fig. i). In a series of comparisons, including some with cabbage leaves, the values of I E X a / 02 1 I 1 1 \ Temp, of Extraction Fig. I. Relation between temperature of extraction and estimated content of potato tubers. found after extraction in metaphosphoric acid at 3" C (subsequently termed cold metaphosphoric acid), were always lower than those obtained by extraction at room temperature (17-20" C) (Table i). Many determinations have now been carried out on potatoes using cold metaphosphoric acid; of ten stocks tested two stocks (E and F, Table i) bave given high values for ; in the other stocks the content of was below 0.4 F.W. These low values compare with a range of F.W. obtained with various stocks in our earlier work, using metaphosphoric acid at room temperature. Extraction in metaphosphoric acid at room temperature icith and without removal of oxygen. A simple explanation of the above results is that the natural content of is low but that during blending some oxidation of ascorbic acid occurs either chemically or enzymically, in the interval between disorganization of the tissue and penetration of the acid. The rate of this oxidation would be expected to be more rapid the higher the

4 Tissue Potato Stock Cabbage leaves , A B c C D E EF G II.2 i Estimation of dehydroascorbic acid Table i. Effect of temperature of extraction Acid extraction at room temperature Acid extraction at -3'C AA AA II Q Q temperature. If this interpretation is correct the postulated oxidation might be retarded by excluding oxygen. The results given in Table 2 show that the values of as estimated in extracts prepared at room temperature in the absence of oxygen were lower than in corresponding extracts prepared without excluding oxygen. The values recorded for potatoes in the former case were, however, still almost double those found when comparable samples were extracted with cold metaphosphoric acid (cf. Table i), possibly because the procedure used does not remove the oxygen from the tissue itself. Table 2. The effect of excluding oxygen dtiring extraction at room temperature AA Acid extraction at room temperature O2 present O2 excluded AA Potato 4-I II.2 II.I 0.2 II.3 S-i-SS II.7 II II.8 I8-4-5S I9-4-S Strawberry leaves Cabbage Extraction in cold metaphosphoric acid zvith and without exclusion of oxygen or the addition of inhibitors. The observations recorded above are in accord with the view that the natural content of is low and that an appreciable amount of additional acid is produced during extraction unless measures are taken to retard the oxidation of ascorbic acid. On this view the low values for obtained by blending the tissue in cold metaphosphoric acid may also be in part artefact. We have already shown that at room temperature exclusion of oxygen reduces tbe to a low level; if, therefore, the level of found after extraction with cold metaphosphoric acid is still artificially high, the exclusion of oxygen during this procedure might be expected to give even lower values. The results (Table 3) show that the further decrease as a result of excluding oxygen at 3 C was not significant with potatoes, and only small with either of the leafy tissues. Since, however, in these experiments a further decrease was observed with strawberry and cabbage leaves, there remains the possibility that even with these precautions, the values were in part artefact. Two inhibitors, known to he inhibitors of the oxidases oxidizing ascorbic acid, were therefore tested to determine if, by their use, diiterent 6i

5 62 Tissue Potato 22,6, Strawberry leaves ,3, Cabbage J. Table 3. AA , ,2 309, BARKER AND L. W. Extraction zvith metaphosphoric acid at - Oxvgen presenit With exclusion of AA mg/100 g ,2 1,9 2,2 2, S ,1 312, MAPSON ,0 265, yc mg/100 g ,13 1, ,8 1.6 oxygen values for were obtained. Extraction of potatoes with cold metaphosphoric acid to which cyanide or azide were added in a concentration of 0.0iM did not with the exception of cyanide reduce the values further. The results with cyanide (Table 4) Tissue Potato 16, ,55 AA 8,41 8.II Table 4. Effect of cytinide."^eid extraction Without inhibitor nig/100 0, ,14 g , at 3" C A A ,35 8,40 7,17 With KCN (IO-^^ mg/100 g showed a small decrease, but this was only slightly greater than the effect of excluding oxygen, and may have been due to the greater instability of in the presence of cyanide (cf. Mapson and Ingram, 1950). The effect of increasing the concentration of metaphosphoric acid was also tested with potatoes, the values obtained for being slightly lower with cold ^% w/v metaphosphoric acid than with a 2% solution. Extraction with cold 6% acid did not further decrease the values obtained so that no further advantage would be gained by using acid of a higher concentration than 4'Jo w/v. Effect of holding slices of potato in cold metaphosphoric acid. When initially comparable samples of slices were used to compare the eflfect of different extraction techniques, the slices blended first (i.e.firstsample) were in cold metaphosphoric acid for a shorter time than those of the sample which \\ as blended second (second sample). A series of comparisons showed that the content of dehydroascorbic acid was lower in the first than in the second sample (Table s). In using initially comparable samples the procedure was, therefore, either to employ two blenders with simultaneous blending or to use one Tissue Potato , I 0,12 0,13 I Table 5. Effect of holding slices in cold metaphosphoric acid at 3 C Sample extracted in acid immediately A A ,69 6, nig/i00 g ,03 0,04 0,09 0, , Time sample left in acid before extraction (min) 5 5 S Tota ,28 Sample extracted after immersion acid for different times AA Tota , H

6 Estimation of dehydroascorbic acid 63 blender, the first and second samples being alternatively the control and the treated sample. In the lower part of Table 5 are recorded the results of holding slices in metaphosphoric acid for an extra 10 and 30 min respectively prior to blending. Freezing at 70 C or below followed by blending in metaphosphoric acid at 3 C. In theory rapid freezing to 70 C or below should arrest enzymic or chemical activity more quickly than the procedures used above. Isherwood and Niavis (1956) found that the contents of a-keto acids were closely similar when comparable samples of strawberry leaves were extracted by {a) disintegration in cold metaphosphoric acid and {b) freezing at 70 C followed by blending in cold metaphosphoric acid. Table 6 shows the results obtained when these and other procedures were used to determine in potato and strawberry leaves; similar results were obtained with cabbage. When the tissues were held frozen in either methanol-solid CO2 at 70" C or in liquid nitrogen at 179 C for only 3 min before disintegration, the value obtained for was either the same as or slightly higher than the amount determined simply by blending in cold metaphosphoric acid. If, however, the samples were held in the frozen state for min before blending, markedly higher values of were recorded. Indeed, with strawberry leaves (Table 6) and with a different stock of potatoes (stock B see below, page 65, Table 7) even the short period of 3 min at 179 C gave a large increase in the value for. While, then, short periods in the frozen state may or may not cause disorganization, longer periods appear to cause serious disorganization. These results are interpreted not as indicating that ascorbic acid is oxidized at the low temperatures, but that holding the tissue in the frozen state initiates cellular disorganization, the Table 6. Effect of freezing before disintegration Tissue Treatment AA Disintegrated directly in HPO3 at 3 C Frozen and held 3 min at 70" C, disintegrated in HPO3 at -3" C but held 15 min at 70" to 50' C Frozen and held 3 min at 179 C in liquid N2 before disintegration in HPO3 at 3 C but held for 30 min at iig" C but held for 30 min at 179 C but held for 30 min at 179" C, and disintegrated with exclusion of oxygen is I II.8 II.8 IO but held frozen for 2 niin before disintegration but held frozen for 2 min before disintegration but held frozen for 2 min before disintegration but held for 30 min before disintegration held 30 min before disintegration and disintegrated in absence of oxygen but held for 30 min at 70'^ C held 30 min before disintegration and disintegrated in absence of oxygen i-s , I-S

7 64 J. BARKER AND L. W. MAPSON effect of which becomes apparent only when the frozen tissue is disintegrated in cold metaphosphoric acid. If this view is correct the postulated formation of might be prevented by excluding oxygen during disintegration. This procedure, though only partly successful with potatoes was fully effective with strawberry leaves in eliminating the formation of 'extra' (Table 6). Thus the of strawberry leaves, held at 70" C for only 2 min prior to extraction with cold metaphosphoric acid, was about the same as that obtained by simple extraction with cold metaphosphoric acid (Table 6), but, as with potatoes, prolonging the duration of freezing at 70' C markedly increased the value obtained for. When, however, the longer freezing treatment was followed by disintegration in cold metaphosphoric acid, with oxygen excluded, values for were obtained which were similar to those obtained by direct extraction with cold metaphosphoric acid. This is evidence that, whose formation is attributed to the disorganization caused by freezing, was produced during disintegration of the tissue in cold metaphosphoric acid. As mentioned above, with another stock of potatoes (stock B) the effect of the disorganization, attributed to the freezing procedure, was more marked than with the potatoes of stock A used for the tests given in Table 6. After freezing and holding in liquid nitrogen for only 3 min, the amount of 'extra' was much greater with stock B (tests of , D, Table 7) than with stock A (tests of C, Table 6). The reason for the difference was not ascertained but all samples of stock B disintegrated in cold metaphosphoric acid gave higher values for dehydroascorbic acid (e.g. see (A) of test of , Table 7) than the samples of stock A extracted in this manner (Table i). Stock B may thus have been potentially more liable than stock A to disorganization whether this was produced by freezing or merely by disintegration in cold metaphosphoric acid. Extraction with methatwl-metaphosphoric acid after freezing. On the assumption that the rate of chemical or enzymic change during disintegration would be the more retarded the lower the temperature of disruption, a solution of metaphosphoric acid in methanol at 70^ C, previously used by Isherwood and Niavis (1956), was tested. Disintegration in methanol-metaphosphoric acid solution at 3 C gave values for of the same order as those obtained by the standard procedure, i.e. disintegration in cold metaphosphoric acid (Table 7, stock B). Disintegration in methanol-metaphosphoric acid at 70" C after freezing the tissues for I min in this solution also gave similar values for to those obtained by disintegration in metaphosphoric acid at 3 C (Table 7, stock F). Disintegration in methanol-metaphosphoric acid at 70 C was also tested after a period of freezing, i.e. 3 min in liquid No, which produced severe disorganization. With potato tissue, the values for were lower than those of initially comparable samples disintegrated immediately in metaphosphoric acid at 3 C following freezing in liquid nitrogen, though higher than those obtained from samples disintegrated directly into metaphosphoric acid at 3" C without prior freezing (Table 7). Several variations of this procedure were tested on frozen tissues, including holding at 70" C following disintegration (see test of , Table 7) with the exclusion of oxygen throughout the operations, but no method gave a value for as low as that obtained by direct disintegration in cold metaphosphoric acid. The inference from this and other similar tests is that after a freezing treatment in liquid nitrogen 'killing' was too slow even when the tissue was disintegrated in methanol-metaphosphoric acid solution at 70" C.

8 Tissue Potatoes (Stock F) (Stock B) J. -^ D Strawberry leaves Cabbage Estimation, of dehydroascorbic acid Table 7. Extractions with methanol-inetaphosphoric acid solution Treatment Disintegrated in HPO3 at 3 C oxygen excluded Frozen i min methanol-hpoj at 70 C, disintegrated at 70^ C with oxygen excluded Disintegrated in methanol-hpo3 at 3" C Frozen 3 min liquid N, before disintegration in HPO3 at -3 C Frozen 3 min liquid N^ before disintegration for 5 min in methanol-hpo, at 70'' C, further disintegration with HPO3 at 3 C Following disintegration in methanol-hpo3 at 70 C, held for 90 min at 70' C before disintegration at 3" C AA II Extraction in hotmethanol-metaphosphoric acid. For the extraction of various phosphate compounds from plant tissues boiling alcohol has, so far, proved preferable to other methods of extraction (Benson, 1950; Bean and Hassid, 1951). Markedly higher values for were, however, obtained following extraction in boiling methanol-metaphosphoric acid than with the standard extraction in cold metaphosphoric acid DISCUSSION Extraction in metaphosphoric acid at room temperatitre and at 3 C. Using one of the 'cold' methods, i.e. extraction in metaphosphoric acid at 3" C, recommended by Isherwood and Niavis (1956) for the stabilization of a-keto acids, we have obtained lower values for the content of of potato tubers, strawberry and cabbage leaves, than by extraction in metaphosphoric acid at room temperature (Table i). Our interpretation of these results is that the natural content of of these plant tissues is low and that the higher value obtained by extraction in metaphosphoric acid at room temperature is due to oxidation of ascorbic acid during the disruption. This suggestion was supported by excluding oxygen during the extraction. Thus, with potatoes, extraction in the absence of air in metaphosphoric acid at room temperature gave values for (Table 2) which were nearly as low as those obtained by blending in cold metaphosphoric acid. The suggestion that artefact is formed during extraction in metaphosphoric acid at room temperature also provides a simple explanation of the anomaly mentioned above (see Introduction). Thus although the natural decrease in the content of AA of potatoes was prevented by holding under anaerobic conditions, yet extracts prepared from these potatoes by blending in metaphosphoric acid at room temperature contained E N.P.

9 66 J. BARKER AND L. W. MAPSON (Barker and Mapson, I9s2)- When, however, such potatoes were extracted in cold metaphosphoric acid, with precautions to exclude oxygen, the value for was virtually zero. Frecsinv and disoigani:::ation. In their study of extraction procedures tor determining a-keto acids, Isherwood and Niavis (1956) obtained values of the same order either when the leaves were disintegrated in cold metaphosphoric acid or when rapid freezing at 70 C was followed by blending in cold metaphosphoric acid. In contrast our investigation of the extraction of another labile cell-component, namely, showed for each tissue studied that freezing, followed by disintegration in cold metaphosphoric acid, might, under certain conditions, give a higher value for the content of than when the material was extracted in cold metaphosphoric acid without preliniinary freezing (Table 6). P^ranke (1957) has also reported higher values for in potato tissue after freezing. We have used the term disorganization to describe the change which is induced by freezing and which viltimately results in a higher value for dehydroascorbic acid. Our hypothesis is that freezing, when it causes disorganization, does so by eliminating barriers either betweeti substrates and etizymes or between certain cell components; the eifect of the disorganization is to produce dehydroascorbic acid, presumably by oxidation of ascorbic acid. The postulated oxidation would not be expected to proceed at a perceptible rate at -70 C or below. The disorganization, which is initiated by the freezing treatment, is, ho\\ever, made manifest during the blending in cold acid at 3 C by oxidation of ascorbic to dehydroascorbic acid. This suggestion is supported by results which show that the extra dehydroascorbic acid resulting from freezing can be partly or completely eliminated by excluding oxygen during the blending in cold metaphosphoric acid at 3 C (Table 6). That freezing can produce or initiate disorganization, presumably by destroying barriers within the cell, has long been recognized. For example, with many plant tissues thawing after freezing causes a rapid oxidation of AA and the development of discolorations; these changes also proceed, though more slowly, in the frozen state at 20 C. The data presented above show that the degree of disorganization produced by freezing was, in general, greater when the tissue \\as frozen at 179 C instead of at 70 C and when the period in the frozen state was extended. The extent of disorganization also varied in different samples of the same plant material. Thus with one stock of potatoes a period of only 3 min in liquid nitrogen produced a large degree of disorganization (Table 7); in contrast another stock of potatoes showed only slight disorganization following this freezing treatment (Table 6). The eliniination of artefact in extracting from tissues. We have shown in this paper that by extracting plant tissues in the cold ( 3 C) with metaphosphoric acid, and by taking precautions to exclude oxygen, the oxidation of ascorbic acid to dehydroascorbic acid may be greatly reduced, if not completely eliminated. But so far we have no proof that this method will entirely prevent oxidation of ascorbic acid to during extraction either in the ttssues of the present study or in other types of plant tissue or in plant tissues in diflerent metabolic states. In fact the content of strawberry leaves analysed during the summer months has amounted to y'];-, of the total ascorbic acid content of the leaf, which compares with a value of 1,, or less for similar leaves analysed during the colder months These very high values we suspect may be due to some oxidation occurring duritig the extraction. \\ ith the tissues stiidted a procedure involving preliminary freezing has given a value

10 Estimation of dehydroascorbic acid 67 for of the same order as that obtained by extraction in cold metaphosphoric acid without freezing. Since freezing has not given markedly lower values than those obtained by direct disintegration in cold metaphosphoric acid, it might be argued (cf, Isherwood and Niavis, 1956) that cold acid effectively eliminates any oxidation of ascorbic acid during extraction. But this does not take into account our observations that freezing may cause cellular disorganization which accelerates the rate of oxidation of ascorbic acid during disintegration procedures. We cannot, therefore, be certain that any method so far used will prevent completely the oxidation of ascorbic acid during extraction. Using the standard procedure of extraction in cold metaphosphoric acid iti the absence of oxygen, we have found zero values for in potatoes after these tissues had been held in nitrogen, which shows that in these circumstances no oxidation of ascorbic acid occurred during the extraction process. However, it does not follow that no oxidation would occur during extraction in a tissue previously held in air. ACKNOWLEDGMENTS We wish to thank Drs. Isherwood and Niavis for their interest and advice. Mr. F. Lenton gave technical assistance. One of us (L. W. M) participated in this work as part of the programme of the Food Investigation Organizatioti of the Department of Scientific and Industrial Research. REFERENCES BARKER, J. & MAPSON, L, W, (1952). The ascorbic acid content of potato tubers. III, The influence of storage in nitrogen, air and pure oxygen. New Phytol., 51, 90, BARKER, J, & MAPSON, L, W, (1955), Studies in the respiratory and carbohydrate metabolism of plant tissues. VII, Experimental studies with potato tubers of an inhibition of the respiration and of a 'block' in the tricarboxylic acid cycle induced by 'oxygen poisoning', Proc. Roy. Soc. B,, 143, 523. BEAN, R, C, & HASSID, W, Z. (1951), In Phosphorus Metabolism, i, 11, BENSON, A, A, (1950). In 'Assimilation in Biological Systems', Brookhaven Conference Rep. (Assoc, Univ. Inc, Upton, N,Y,), p, 119, FRANKE, V, W, (1957), Uber das vorkommen der dehydroascorbinsaure in pflanzen und ihre Beziehungen zum Vitamin-C-Problen:i, Biolog. Zentralblatt, Id, 148, HUGHES, R, E. (1956), The use of homocysteine in the estimation of dehydroascorbic acid, Biochem. J-, 64, 203, ISHERWOOD, F, A, & NIAVIS, C, A. (1956). Estimation of a-keto acids in plant tissue: a critical study of various methods of extraction as applied to strawberry leaves, washed potato slices and peas. Biochem. J., 64, 549, MAFSON, L, W, & MAWSON, C, A, (1943), The stability of ascorbic acid in metaphosphoric acid extracts. Nature, 151, 222, MAPSON, L, W, & INGRAM, M, (1950), Observations on the use of Escherichia coli for the reduction and estimation of dehydroascorbic acid, Biochem. jf., 48,551, WOOD, J, G,, MERCER, F, V, & PEDLOW, C, (19.4.4). Metabolism of starving leaves, 4, Respiration rate and metabolism of leaves of Kikuya grass during air-nitrogen transfers, Aust. J. Exp. Biol. Med. Sci.,

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