Corbet Page Stewart

The reduction of dehydroascorbic acid by human erythrocytes.

Abstract 1. 1. Dehydroascorbic acid (DHA) easily penetrates human erythrocytes. It is rapidly reduced to ascorbic acid by the intact cells or by preparations of erythrocytes haemolysed with water and centrifuged to remove cell debris. The ascorbic acid (AA) produced diffuses slowly from intact cells to the surrounding medium. The reaction continues until practically no DHA remains, the main product apart from AA being dioxogulonic acid (DGA). 2. 2. Ascorbic acid penetrates the erythrocytes very slowly. 3. 3. Neither glutathione (GSH) nor total SH disappears during the reduction of DHA. The reaction is, however, prevented by SH inhibitors. If, as seem probable, SH compounds are involved they must be regenerated very efficiently, possibly through the agency of glutathione reductase. 4. 4. Heated haemolysed erythrocytes reduce DHA at a rate comparable to that occurring in simple solution with similar DHA and GSH concentrations. 5. 5. The findings suggest that an enzyme other than glutathione reductase is involved in the reduction of DHA by human erythrocytes.

The simultaneous fluorimetric microdetermination of cortisol and corticosterone in plasma

Abstract 1. (1) A simplified fluorimetric procedure for the estimation of free cortisol and corticosterone in 1-ml samples of human plasma has been described. 2. (2) A correction procedure for non-specific fluorescence has been described which is applicable to both human and rat plasma. It depends on the differing rates of development of fluorescence of plasma and of solutions of cortisol and corticosterone. 3. (3) The mean value for free plasma cortisol in 27 normal adults was 9.6 ± 2.7, and for free plasma corticosterone 0.4 ± 0.2 μg/100 ml. Elevated values were foundin Cushings syndrome, and normal levels in hirsutism and in Addisons disease. Low values occurred in hypopituitarism.

Studies of hydrocortisone production as measured by isotope dilution techniques in adrenocortical diseases and in idiopathic hirsutism

Abstract Two methods are described for the measurement of the endogenous hydrocortisone production rate using the isotope dilution technique. The blood method largely follows that proposed by Peterson and Wyngaarden 1 and the urine method is based upon that proposed by Cope and Black 2. Measurement of cortisol production was made in 7 patients in hospital free from endocrine abnormality (blood method), in 7 healthy ambulant individuals (urine method), in 8 patients suffering from Cushings syndrome, in 3 patients with adrenocortical insufficiency and in 7 women with idiopathic hirsutism. Cortisol production rate was increased in all 8 patients with Cushings syndrome. Urinary 17-ketosteroids and 17-ketogenic steroids were found to be elevated in only 2 and 6 of these patients, respectively. The determination of cortisol production rates appears to be the most sensitive method to detect adrenocortical overfunction. The results obtained in patients with idiopathic hirsutism lend support to the view that cortisol synthesis is not depressed in this condition.

The determination of vitamin C in normal human plasma and erythrocytes.

Abstract 1. 1. Various methods based on (a) reduction of indophenol, (b) formation of a derivative with 2: 4-dinitrophenylhydrazine, and designed to measure ascorbic acid, dehydroascorbic acid and diketogulonic acid have been applied to the analysis of normal human plasma and erythrocytes. 2. 2. It is concluded that measurement of indophenol reduction at pH ca. 4 in protein-free filtrates of plasma, or of carbon monoxide saturated erythrocytes, treated with p-chloromercuribenzoic acid provides the most accurate means of determining ascorbic acid in these materials. 3. 3. Comparison of values for total vitamin C and ascorbic acid of human plasma indicates that the amount of dehydroascorbic acid and/or diketogulonic acid in normal plasma is smaller than can be reliably determined by this procedure. The similar, and contrary, findings of others have been discussed in relation to the methods used. 4. 4. In erythrocytes the results obtained were consistent with the presence of a small amount of dehydroascorbic acid and/or diketogulonic acid. 5. 5. Measurement of indophenol reduction of plasma or erythrocyte filtrates treated with hydrogen sulphide gives results which are believed to include some non-ascorbic acid material and thus to overestimate any dehydroascorbic acid present. 6. 6. The concentration of total vitamin C in plasma is higher than that in erythrocytes, freed as far as possible from leucocytes, when the concentration in plasma is high. When the concentration in plasma is low the reverse relationship holds. In contrast, the concentration of ascorbic acid in plasma tends to be higher than that in erythrocytes at all levels.

The metabolic response to adrenalectomy and hypophysectomy

Abstract 1. 1. Observations have been made upon the balance of sodium, potassium, chloride and nitrogen and the excretion of corticosteroids in the urine in three patients subjected to bilateral adrenalectomy and given constant doses of cortisone acetate, and in two patients undergoing hypophysectomy, only one of whom received cortisone acetate. 2. 2. The usual post-operative metabolic changes of sodium and chloride retention with loss of potassium and nitrogen in the urine occurred in all patients. In the three patients subjected to second stage adrenalectomy, the metabolic response was not associated with a rise in the urinary excretion of adrenocorticosteroids (acid-stable formaldehydogenic or 17-hydroxy-steroids). Increase in urinary corticosteroids occurred in both patients after hypophysectomy and in the one patient studied on whom a first stage adrenalectomy was performed. 3. 3. It is concluded that the metabolic responses observed after second stage adrenalectomy occurred in the absence of significant alterations in the rate of adrenocortical secretion. The observations we have made do not eliminate the possibility that trauma and surgical operation in patients with intact adrenal glands increase the secretory activity of the adrenal cortex. However the occurrence of alterations in electrolyte and nitrogen balance following surgical trauma may not be used as evidence that such an increase in secretion occurs. 4. 4. The role of the cortisone acetate given to the patients before and after removal of the adrenal glands in the metabolic response is discussed. Although the results are not inconsistent with the “permissive” action of the hormone as suggested by Ingle , the possibility that alterations in metabolism and the rate of excretion of the hormone result in an increase in the blood concentration of biologically effective hormone with consequent alterations in electrolyte and nitrogen balance cannot be excluded. Nevertheless, if such an alteration in the rate of disposal of biologically active hormone occurs for some days after the operation, it must do so without causing a rise in the urinary content of the measured steroid metabolites such as is normally found after operations not involving the adrenal glands. 5. 5. Four possible interpretations of the increase in corticosteroids in the urine after hypophysectomy are suggested but it is not possible to decide which of these explanations is correct.

The relationship of aldosterone excretion to the metabolic response to adrenalectomy

Abstract 1. 1. Observations have been made on the balance of sodium, potassium, chloride and nitrogen, and the urinary excretion of corticosteroids and aldosterone in three patients undergoing, respectively, 1st stage adrenalectomy, 2nd stage adrenalectomy and ovariectomy after previous bilateral adrenalectomy. 2. 2. Post-operative metabolic changes of sodium and chloride retention with urinary loss of potassium and nitrogen occurred. In the patient subjected to 1st stage adrenalectomy, the metabolic response was accompanied by rises in the urinary excretion of aldosterone and total corticosteroids. In the patient undergoing 2nd-stage adrenalectomy and in the patient undergoing ovariectomy, the metabolic response was not associated with any rise in urinary corticosteroids, and urinary aldosterone was not detectable post-operatively. 3. 3. It is concluded that the metabolic responses ordinarily observed affecting sodium, potassium and nitrogen excretion after surgery can occur without concomitant rises in excretion of aldosterone.

A note on the determination of serum proteins by paper electrophoresis.

Abstract The serum protein fractions separated by electrophoresis on a paper strip and dyed have been determined by densitometry with three alternative planimetric methods. For various reasons, the method of choice is to cut out and weigh the paper in the scanner diagram corresponding to the various protein fractions.

Isolation of ascorbic acid from urine.

HINSBERG and Ammon1 have been unsuccessful in an attempt to isolate ascorbic acid from urine. Recently E. C. Noyons2 has described a method for the isolation of ascorbic acid from tomato juice involving chromatographic adsorption, and has announced his intention of applying a similar procedure to the isolation of ascorbic acid from urine.