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The Effect of Cortisone and Adrenocorticotropic Hormone on the Dehydroascorbic Acid of Human Plasma

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The Effect of Cortisone and Adrenocorticotropic Hormone on the Dehydroascorbic Acid of Human Plasma 254 I953 The Effect of Cortisone and Adrenocorticotropic Hormone on the Dehydroascorbic Acid of Human Plasma By C. P. STEWART, D. B. HORN AND J. S. ROBSON Department of Clinical Chemistry, University of Edinburgh (Received 30 May 1952) It is well known that ascorbic acid in aqueous solu- added to 2-0 ml. of plasma, and the precipitated proteins tion is easily and reversibly oxidized to dehydro- separated by centrifugation and filtration. A known excess ascorbic acid, and it is believed that above pH 4 the of standard solution of the indophenol was added to a sample of the filtrate, and the residual colour was measured latter can undergo irreversible change, possibly to in a spectrophotometer (Unicam SP 500) at a wavelength of diketogulonic acid, as a preliminary to further 480m,u., 30sec. from the time of addition of the indophenol. oxidation. The possibility of the existence of de- The method is similar in principle to that of Mindlin & hydroascorbic acid or diketogulonic acid in blood Butler (1937-8), except that it was found possible to dis- plasma, however, has been generally ignored since pense with the buffer, and greater accuracy was obtained by the time of Borsook, Davenport, Jeffreys & constructing a standard curve instead of using a single Warner (1937) who, with van Eekelen (1935), standard solution. Kellie & Zilva (1936), Farmer & Abt (1936) and The validity of the procedure was first tested by Ralli & Sherry (1941), claimed that vitamin C measuring the rate of fading of the indophenol solution under and the results are shown in exists in plasma in the reduced state as ascorbic acid. varying conditions, Fig. 1. The indophenol was decolorized rather slowly in the The brief statement that dehydroascorbic acid presence of aqueous metaphosphoric acid (curveA) and more exists in human blood has recently been made by rapidly in the presence of trichloroacetic acid (curve B) at Chen & Schuck (1950) but no supporting evidence the same pH. Ascorbic acid added to the metaphosphoric was given. Apart from its academic interest, the acid solution (curve C) completely stabilized the indophenol relation between dehydroascorbic acid and experi- colour after the initial reduction due to oxidation of the mental diabetes (Patterson, 1950) and the close ascorbic acid. Unfortunately, it was impossible to obtain association of ascorbic acid with the adrenal cortex readings earlier than 20 sec. after the addition of the indo- and stress (Selye, 1946) suggest that the form in phenol, but the reduction of the dye by ascorbic acid is which vitamin C exists in blood is of in- certainly completed within 30 sec. When the indophenol worthy was added to a metaphosphoric acid solution containing a vestigation. The experiments described in this paper trace ofH2S (as an example ofa substance oxidized slowly by establish the facts that a significant part of the total the indophenol) fading continued to occur after 30 sec. and vitamin C present in normal human plasma exists for as long as determinations were made (curve D). A similar as dehydroascorbic acid, that diketogulonic acid slow reduction of the dye occurred in the presence of meta- does not exist in plasma in significant quantities, phosphoric acid and Na2S203 (curve E) in a concentration and that the ratio between dehydroascorbic acid comparable with that in plasma; the curve, however, can be and ascorbic acid can be consistently influenced by superimposed on that for metaphosphoric acid alone, and adrenocortical steroids. it can be concluded that thiosulphate had no appreciable effect. It is of interest to note also that stabilization of the indophenol colour did not occur when ascorbic acid was added to trichloroacetic acid (curve F). Similar results were EXPERIMENTAL AND RESULTS obtained when the indophenol was added to metaphosphoric acid filtrates of plasma. With normal plasma the reaction The existence ofdehydroascorbic acid in humanp1asma with the dye was complete in 30 sec. (curve G) and this also Chemical method8. Plasma from normal human subjects occurred when the plasma was treated with H2S which was was submitted to the technique of Roe& Keuther(1943) and subsequently removed by N. (curve H). This curve shows to a procedure employing 2:6-dichlorophenolindophenol. that the H2S, used in this way, does not interfere with the The former involves preliminary oxidation of ascorbic acid determination of ascorbic acid, although if the removal of with activated charcoal and the colorimetric estimation of H2S is incomplete (curve J) stabilization of colour is delayed the reaction product with 2:4-dinitrophenylhydrazine and estimatiQn of ascorbic acid is impossible. The general under defined conditions. It is claimed to estimate the conclusion is that with spectrophotometer readings taken dehydroascorbic acid produced by oxidation ofascorbic acid 30-60 sec. after addition of the dye, with metaphosphoric plus any preformed dehydroascorbic acid and diketogulonic acid but not trichloroacetic acid as the protein precipitant acid (i.e. 'total ascorbic acid'). The procedure involving 2:6- and with no residual H2S, the readings represent ascorbic dichlorophenolindophenol was applied to a sample of the acid concentrations, and that the presence of interfering same plasma in order to estimate ascorbic acid itself. For this substances is indicated by a steady change of spectro- purpose 3-0 ml. of 3% (w/v) metaphosphoric acid were photometer readings during the chosen period. VoI. 53 EFFECT OF HORMONES ON DEHYDROASCORBIC ACID 255 To test the reproducibility of results, twenty determina- became 0-82 + 0-06 (Table 3, column 6), the absolute tions were made on a solution of pure ascorbic acid; the differences remaining about the same. mean result was 1 81 ±0-025 mg./100 ml., the range being There are several possible explanations of this 1-77-1-83; similarly, a sample ofplasma with added ascorbic acid gave twenty results between 2-09 and 2-13 mg./100 ml., phenomenon other than the obvious one that the with a mean of 2-11 and S.D. of 0-019. At a lower ascorbic difference represents dehydroascorbic acid and/or acid concentration the figures for twenty replicate deter- diketogulonic acidexisting in the circulating plasma. minations were: mean, 0.43 mg./100 ml.; S.D., 0-023; range, Oxidation of ascorbic acid to dehydroascorbic acid 0-40-0-48. and subsequent change to diketogulonic acid may 780- 90 _ H I I I I I l l I I 20 40 60 80 100 20 40 60 80 100 Time after addition of indophenol (sec.) Fig. 1. The effect of time on the transmission at 480 mp. of 1-0 ml. of the indophenol solution (4 mg./100 ml.) in the presence of 2-0 ml. of various reagents. A, metaphosphoric acid 3% (w/v); B, trichloroacetic acid 5% (w/v); C, HPO3 3% (w/v) plus ascorbic acid, 1 mg./100 ml.; D, HPO3 3% (w/v) +H2S; E, HPO3 3% (w/v) +Na2S203, 8 mg./100 ml.; F, trichloroacetic acid 5% (w/v) + ascorbic acid, 1 mg./100 ml.; G, 2 ml. plasma +3 ml. HPO2, 3% (w/v); H, as G, after treatmentwith H2S and removal of excess H2S by N2; J, as H, butwith H2S incompletelyremoved. The results of recovery experiments when pure ascorbic occur spontaneously after withdrawal of blood or acid was added to plasma are shown in Table 1. Recoveries during the manipulation involved in the indophenol were always within 0-05 mg. ascorbic acid/100 ml. method prior to the estimation. Alternatively, interfering substances may be reacting with 2:4- Table 1. Recovery of a8corbic acid added to human dinitrophenylhydrazine in the method of Roe & plasma using the indophenol method Keuther (1943), giving a falsely high figure for the Ascorbic acid (mg./100 ml. plasma) concentration of total ascorbic acid. Indeed, both Added Found Calculated Differenc possibilities may be involved. Added Found C:alculated Difference In regard to the first of these suggestions, the 0-15 stability of ascorbic acid was investigated in blood, 0-25 0-37 0-4 -0-03 plasma and plasma filtrate. For this purpose, 0-5 0-64 0-65 -0-01 1-0 1-17 1-15 +0-02 venous blood was withdrawn into an oiled syringe; 1-5 1-70 1-65 +0-05 part of the sample was allowed to stand at room 2-0 2-20 2-15 +0-05 temperature with occasional mixing, the remainder being used for the immediate determination of Invariably the results obtained by the method of plasma total ascorbic acid and of ascorbic acid by Roe & Keuther (1943) were higher than those by the the method of Roe & Keuther (1943), and by the method using the indophenol, although both indophenol method respectively. After about 60 methods were standardized against the same original and 120 min., further determinations were made by solution of pure ascorbic acid. A similar difference both methods. Similar experiments were carried was found in the plasma from subjects who had been out on plasma separated immediately after the given a diet rich in vitamin C for a few weeks. These withdrawal of blood. A third series was done on results are shown in columns 4 and 5 of Tables 2 plasma filtrate prepared as quickly as possible after and 3. In the normal fasting subjects on an ordinary the withdrawal ofthe blood. The earliest determina- diet, the mean ratio of plasma ascorbic acid tion possible in any of these experiments was about determined by the indophenol method to the 'total 30 min. after withdrawal. Typical results of these ascorbic acid' determined by the method of Roe & experiments are shown in Fig. 2, where it is seen Keuther (1943) was-0-68 + 0-15 (Table 2, column 6).
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