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CHRONIC ULCERATION OF THE LEG TREATED WITH AMNION AND CHLORAMPHENICOL IN PROPYLENE GLYCOL
225 observed when rheumatic
activity
is
continuing.
In
rheumatic fever many factors affect the patient’s weight, among which may be the following : (1) In the early stages the increase in body-fluids found
to
take place in rheumatic fever by Bradley (1938), Reid et al. (.1950), and Cochran (1951), may mask the wasting, with the result that, when the patient recovers, reduction in both processes may lead to a lack of change in weight. (2) In spite of continuing rheumatic activity the placing of a rheumatic child at rest in bed may be sufficient to stop a loss of weight which would as in fig. 2. Note four- have Fig. 3-Symbols occurred had the month period of no growth though child child remained was well. ambulant. (3) A child may be ill fed at home but, when placed on a good diet in hospital, may gain weight in spite of rheumatic
activity. SUMMARY
of
The problems assessing weight changes in disease discussed. The underlying principle and advantages of Wetzel’s "grid" as a solution are set out and illustrated by reference to rheumatic fever. are
caked in the the
penetrate was
displaced.
chloramphenicol in propylene glycol was Dr. J. D. Gray, who had been using a suggested by solution containing 15 g. in 100 ml. in chronic otorrhoea (Lewis and Gray 1951). Propylene glycol has proved a satisfactory vehicle ; it is non-toxic, miscible with water, and hygroscopic, and it can penetrate denuded surfaces. The
REFERENCES
-
CHRONIC ULCERATION OF THE LEG TREATED WITH AMNION AND CHLORAMPHENICOL IN PROPYLENE GLYCOL
SIDNEY SHAW
E. TROENSEGAARD-HANSEN
M.D. Lond.
F.R.C.S.
CLINICAL PATHOLOGIST
Using the cup-plate technique ten organisms that were found resistant to 0’25% chloramphenicol in water were tested against 2’5% chloramphenicol in propylene glycol; there was a large zone of inhibition in each case. The organisms included Ps. mruginosa, two strains of Staphylococcus aureus, Bacteriu-m coli, Proteus vulgaris, and two strains of streptococcus, (non-haemolytic and faecal). Propylene glycol alone gave no inhibition. RESULTS OF TREATMENT
The treatment previously described (TroensegaardHansen 1950) was used up to and including the scraping of the ulcer under general anaesthesia. A solution of 2-5% chloramphenicol in propylene glycol was then sprayed on the wound once daily. Most wounds were free from infection within 7 days. Amnion was then applied and covered with tulle gras, cotton-wool, and The patient stayed in bed, and dressa light bandage. ings were left in place for six weeks if possible. who had had ulceration of the legs for at least treated in this way ; 5 had associated varicose veins, and 4 had been unsuccessfully treated with phenoxetol and amnion. 7 of these ulcers healed in six weeks and 1 in three weeks ; they have now remained healed for from six to eight months. The 9th patient was a lady of 80 ; her ulcer healed in six weeks but recurred five months later. The 10th patient had a chronic ulcer with eczema and swelling of the leg and foot. Local penicillin had previously produced a rash on the leg, but there was no other history of allergy or sensitivity, or of fungus infection. After two days’ spraying with chloramphenicol in propylene glycol an urticarial rash appeared on the arms, the backs of hands, and the cheeks, but there was no local reaction. Treatment was stopped and saline dressings applied. On the eighth day she had an asthmatic attack ; the acute phase subsided in twenty-four hours but her chest was not completely clear for a month. The ulcer remained clean, and amnion was applied after two weeks’ treatment. The ulcer healed in six weeks, and the swelling and eczema of the leg also subsided, but a mild scaly eczema of the hands and forearms 10
patients
leg were treated by coverlayer of amnion (Troensegaard-Hansen 1950, 1951), healing was sometimes delayed by bacterial infection. It is well known that Pseudomonas curuginosa may become established in chronic leg ulcers and is particularly difficult to eradicate. them with
One of these patients had a similar ulcer on the other which was treated with chloramphenicol in propylene glycol, but no amnion. At six weeks this ulcer was clean but unhealed ; amnion was applied and the ulcer healed
leg
HOSPITAL
WHEN chronic ulcers of the
ing
were
persisted.
SURGICAL REGISTRAR
CHARING CROSS
of
use
two years
Bradley, W. H. (1938) Proc. int. Congr. Rheum. p. 86. Bransby, E. R., Gelling, J. W. (1946) Med. Offr, 75, 213. Cochran, J. B. (1951) Brit. med. J. ii, 637. Forbes, H. A. W. (1952) D.M. thesis, Oxford. Friend, G. E., Bransby, E. R. (1947) Lancet, ii, 677. Ministry of Food (1943) Body Weight Survey. Nelson, W. E. (1945) Mitchell-Nelson Textbook of Pediatrics. 4th ed., London. Reid, J., Watson, R. D., Sproull, D. H. (1950) Quart. J. Med. 19, 1. Wetzel, N. C. (1941) J. Amer. med. Ass. 116, 1187. (1944) In Glasser, O. Medical Physics. Chicago ; p. 513.
wound, and the antibiotic did not seem to tissues ; much pus formed and the membrane
a
after
a
further six weeks.
INHIBITION ACETIC
OF
ACID,
Pseudomonas AND
mm.
in diameter
were
PHENOXETOL, IN
DIFFERENT
Base
Ung. alcoh.
Ian.
Water Ung Ung. water
emulsif.
cut from nutrient agar
Three pseudomonas strains from chronic ulcers
plates. spread placed in
were
the plates, and the various preparations were the cups. The results, using three different bases for the medium, are shown in the table. Inhibition depends largely on the ability of the drug to diffuse into the medium. Trials of terramycin were not satisfactory. A powder containing 1 part of terramycin in 200 parts of prepared starch (Boots K285) was applied to 6 chronic ulcers before covering them with amnion. The powder on
BY
PROPIONATE
Reagent
A 2%’Phenoxetol’ cream was used in the original treatment of ulcers with amnion, and acetic acid and sodium propionate have also been used in the past against pseudomonas infections. The inhibitory action of these compounds was tested as follows : 8
coruginosa
BASES
PRELIMINARY TESTS
Cups
SODIUM
shown on three strains of Ps. œruginosa. inhibition. + == slight zone of-inhibition at 8 hours, when growth first obvious, but no inhibition at 20 hours. + + = slight zone persisting at 20 hours. + -!- + = moderate zone persisting at 20 hours.
Equal effects -
=
were
no
was
226 A patient who was not treated with amnion had a band of ulceration, 6 in. wide, round the leg. Spraying with chloramphenicol in propylene glycol every other day has reduced it to 21/2 X 1 in. DISCUSSION
All the
sensitive
organisms isolated from these ulcers were chloramphenicol in propylene glycol, as
to
shown by a definite zone of inhibition in the cup test. The possibility of local or generalised allergy can best be assessed by further trial of the method. Intradermal tests which we performed on our patient who had urticaria and asthma did not suggest that chloramphenicol was the sensitising agent. Pure propylene glycol produced a marked weal, but no surrounding erythema. A healthy adult control reacted similarly to the same amount intradermally. The weal was probably the result of absorption by the hygroscopic propylene glycol. In the 10th case it may be that the infection was overcome so rapidly that there was a sudden release of bacterial products. The patient may have been sensitive to the bacterial protein or to something else produced by the action of the antibiotic. If cultures were kept of organisms isolated from an ulcer, it would be possible to check this hypothesis by injecting a killed bacterial suspension, should an allergic reaction occur. Despite the one reaction in this small series, chloramphenicol in propylene glycol seems to be a very useful adjunct to treatment with amnion. We are grateful to Dr. J. Stanley White and Dr. Robert of the department of clinical investigation of Messrs. Parke, Davis for supplies of chloramphenicol in
Hodgkinson
propylene glycol. REFERENCES
Lewis, R. S., Gray, J. D. (1951) Brit. med. J. ii, 939. Troensegaard-Hansen, E. (1950) Lancet, i. 859. (1951) Charing Cr. Hosp. Gaz. 49, 79. -
SECRETION OF A SALT-RETAINING HORMONE BY THE MAMMALIAN ADRENAL CORTEX S. A. SIMPSON J. F. TAIT B.Sc. Lond.
B.Sc., Ph.D. Leeds, A.Inst.P.
ASSISTANT IN BIOLOGICAL
RESEARCH, COURTAULD INSTITUTE OF BIOCHEMISTRY
ASSISTANT IN
RESEARCH,
BARNATO-JOEL RESEARCH LABORATORIES
MIDDLESEX HOSPITAL, LONDON
I. E. BUSH B.A. Camb. RESEARCH
ASST.,
NATIONAL INSTITUTE FOR MEDICAL RESEARCH
IN recent years the division of adrenal cortical steroid hormones into glucocorticoids and " niineralocorticoids (Selye 1950) has been seriously questioned by some authors (Verzar 1952), and Conn, Louis, and Fajans (1951) have gone so far as to suggest that all the actions of adrenocorticotropic hormone (A.C.T.H.) in humans could be accounted for if the human adrenal be supposed "
to secrete 17-hydroxycorticosterone (Compound F) alone. Thus the three most active known glucocorticoids, cortisone, Compound F, and Compound B, all produce sodium retention in man (Sprague et al. 1950, Conn, Fajans et al. 1951, Conn, Louis, and Fajans 1951), and cortisone does so in dogs (Roberts and Pitts 1952). Similarlv all the active cortical hormones have been found to have some effect in depressing the urinary Na24/K42 ratio of adrenalectomised rats (Simpson and Tait 1952). Since chemical estimations of the corticoids in adrenal venous blood have shown Compound F and Compound B (corticosterone) to be by far the major secretory products of the adrenal cortex of many mammalian species (Nelson et al. 1950, Hechter et al. 1951, Bush 1951) it has appeared to some (see Sayers 1950) unnecessary to postulate" the secretion of a specific " mineralocorticoid " or salt-retaining hormone " by
the mammalian adrenal cortex.
However Selye’s comprehensive theory of the.existence of "diseases of adaptation" (Selye 1950) depends at least in part on the’postulate that the adrenal cortex secretes a mineralocorticoid distinct from and antagonistic in action to the glucocorticoids. Furthermore the glucocorticoid "cortisone" is not fully effective in controlling Addison’s disease (Conn, Fajans, et al. 1951, Forsham 1951), nor is it as effective as whole adrenal extract in correcting various deficiencies of adrenalectomised animals (Ingle et al. 1952). Many objections to the theory of mineralocorticoid secretion by the adrenal- were made on the grounds that the actual secretion of desoxycorticosterone (deoxycortone) had not been demonstrated and that this substance was possibly only an artefact in adrenal gland extracts (Dobriner 1952). At the time no other potent mineralocorticoid was known. The whole question became more complex when it was discovered (Grundy, Simpson, and Tait 1952) that " by far the greater part of the, " salt-retaining activity" of whole-beef adrenal extract was contained in a hitherto unknown substance of high potency, which could only be separated from " cortisone " by prolonged chromatography. We wish to present evidence in this communication that a similar or identical substance is actually secreted into the blood-stream by the adrenal of the dog and the monkey. It thus appears to be an active hormone of physiological significance and not only a component of gland extracts or simply an artefact. METHODS
Monkcey.—The isolated left adrenal of a 6-35 kg. rhesus monkey was perfused with blood by a technique similar to that of Vogt (1951) and the effluent blood collected in an ice-cooled flask. The blood was extracted at the end of the experiment with ethyl acetate and the extract purified and concentrated by washing quickly with cold N/5 Na2CO3, water, distillation, and partition between petrol and 70% ethanol. The final extract was divided into several parts and fractionated by paper chromatography (Bush 1952). The content of &agr;,&bgr;unsaturated ketonic steroids was determined by their fluorescence reaction with NaOH in two fractions of the extract and the position of " cortisone " on the chromat-ogram was marked with a pencil. The remaining fractions were run on the same chromatogram but not treated with NaOH. The region of the chromatogram containing the cortisone from these fractions was eluted with ethanol, and the ethanol eluate was evaporated and assayed (table i) by the method of Simpson and Tait (1952). Dog.-Blood from the left adrenal vein of a 21-5 kg. bitch was collected in vivo under pentobarbitone antsthesia and after evisceration (Vogt 1943). The splanchic nerves were intact. The blood was extracted and was fractionated by chromatography. The whole of a chromatogram run in the system toluene : methanol : water (100 : 75 : 25 by volume) was divided into regions and assayed in parallel by the sodium/potassium ratio assay and the fluorescence reaction with NaOH. A measured portion of the cortisone fraction of the blood extract was acetylated and chromatographed with the benzene : formanide system of Burton et al. (1951). RESULTS
The results of the two types of assay when applied to these extracts of adrenal blood are given in table I. It is seen that the " cortisone spot " on the chromatogram of the monkey’s adrenal blood extract contained 1-2 g. of cortisone, which would be equivalent to 0.2 .g. deoxycortone in the mineral ratio bioassay used. However, the biological activity of this spot when assayed in the adrenalectomised mouse was equivalent to that of 15 ug. deoxycortone and could not be accounted for by the small amount of cortisone present in the extract. A more complete study was made of the extract of dog In this experiment the whole adrenal venous blood.
activity
is
continuing.
In
rheumatic fever many factors affect the patient’s weight, among which may be the following : (1) In the early stages the increase in body-fluids found
to
take place in rheumatic fever by Bradley (1938), Reid et al. (.1950), and Cochran (1951), may mask the wasting, with the result that, when the patient recovers, reduction in both processes may lead to a lack of change in weight. (2) In spite of continuing rheumatic activity the placing of a rheumatic child at rest in bed may be sufficient to stop a loss of weight which would as in fig. 2. Note four- have Fig. 3-Symbols occurred had the month period of no growth though child child remained was well. ambulant. (3) A child may be ill fed at home but, when placed on a good diet in hospital, may gain weight in spite of rheumatic
activity. SUMMARY
of
The problems assessing weight changes in disease discussed. The underlying principle and advantages of Wetzel’s "grid" as a solution are set out and illustrated by reference to rheumatic fever. are
caked in the the
penetrate was
displaced.
chloramphenicol in propylene glycol was Dr. J. D. Gray, who had been using a suggested by solution containing 15 g. in 100 ml. in chronic otorrhoea (Lewis and Gray 1951). Propylene glycol has proved a satisfactory vehicle ; it is non-toxic, miscible with water, and hygroscopic, and it can penetrate denuded surfaces. The
REFERENCES
-
CHRONIC ULCERATION OF THE LEG TREATED WITH AMNION AND CHLORAMPHENICOL IN PROPYLENE GLYCOL
SIDNEY SHAW
E. TROENSEGAARD-HANSEN
M.D. Lond.
F.R.C.S.
CLINICAL PATHOLOGIST
Using the cup-plate technique ten organisms that were found resistant to 0’25% chloramphenicol in water were tested against 2’5% chloramphenicol in propylene glycol; there was a large zone of inhibition in each case. The organisms included Ps. mruginosa, two strains of Staphylococcus aureus, Bacteriu-m coli, Proteus vulgaris, and two strains of streptococcus, (non-haemolytic and faecal). Propylene glycol alone gave no inhibition. RESULTS OF TREATMENT
The treatment previously described (TroensegaardHansen 1950) was used up to and including the scraping of the ulcer under general anaesthesia. A solution of 2-5% chloramphenicol in propylene glycol was then sprayed on the wound once daily. Most wounds were free from infection within 7 days. Amnion was then applied and covered with tulle gras, cotton-wool, and The patient stayed in bed, and dressa light bandage. ings were left in place for six weeks if possible. who had had ulceration of the legs for at least treated in this way ; 5 had associated varicose veins, and 4 had been unsuccessfully treated with phenoxetol and amnion. 7 of these ulcers healed in six weeks and 1 in three weeks ; they have now remained healed for from six to eight months. The 9th patient was a lady of 80 ; her ulcer healed in six weeks but recurred five months later. The 10th patient had a chronic ulcer with eczema and swelling of the leg and foot. Local penicillin had previously produced a rash on the leg, but there was no other history of allergy or sensitivity, or of fungus infection. After two days’ spraying with chloramphenicol in propylene glycol an urticarial rash appeared on the arms, the backs of hands, and the cheeks, but there was no local reaction. Treatment was stopped and saline dressings applied. On the eighth day she had an asthmatic attack ; the acute phase subsided in twenty-four hours but her chest was not completely clear for a month. The ulcer remained clean, and amnion was applied after two weeks’ treatment. The ulcer healed in six weeks, and the swelling and eczema of the leg also subsided, but a mild scaly eczema of the hands and forearms 10
patients
leg were treated by coverlayer of amnion (Troensegaard-Hansen 1950, 1951), healing was sometimes delayed by bacterial infection. It is well known that Pseudomonas curuginosa may become established in chronic leg ulcers and is particularly difficult to eradicate. them with
One of these patients had a similar ulcer on the other which was treated with chloramphenicol in propylene glycol, but no amnion. At six weeks this ulcer was clean but unhealed ; amnion was applied and the ulcer healed
leg
HOSPITAL
WHEN chronic ulcers of the
ing
were
persisted.
SURGICAL REGISTRAR
CHARING CROSS
of
use
two years
Bradley, W. H. (1938) Proc. int. Congr. Rheum. p. 86. Bransby, E. R., Gelling, J. W. (1946) Med. Offr, 75, 213. Cochran, J. B. (1951) Brit. med. J. ii, 637. Forbes, H. A. W. (1952) D.M. thesis, Oxford. Friend, G. E., Bransby, E. R. (1947) Lancet, ii, 677. Ministry of Food (1943) Body Weight Survey. Nelson, W. E. (1945) Mitchell-Nelson Textbook of Pediatrics. 4th ed., London. Reid, J., Watson, R. D., Sproull, D. H. (1950) Quart. J. Med. 19, 1. Wetzel, N. C. (1941) J. Amer. med. Ass. 116, 1187. (1944) In Glasser, O. Medical Physics. Chicago ; p. 513.
wound, and the antibiotic did not seem to tissues ; much pus formed and the membrane
a
after
a
further six weeks.
INHIBITION ACETIC
OF
ACID,
Pseudomonas AND
mm.
in diameter
were
PHENOXETOL, IN
DIFFERENT
Base
Ung. alcoh.
Ian.
Water Ung Ung. water
emulsif.
cut from nutrient agar
Three pseudomonas strains from chronic ulcers
plates. spread placed in
were
the plates, and the various preparations were the cups. The results, using three different bases for the medium, are shown in the table. Inhibition depends largely on the ability of the drug to diffuse into the medium. Trials of terramycin were not satisfactory. A powder containing 1 part of terramycin in 200 parts of prepared starch (Boots K285) was applied to 6 chronic ulcers before covering them with amnion. The powder on
BY
PROPIONATE
Reagent
A 2%’Phenoxetol’ cream was used in the original treatment of ulcers with amnion, and acetic acid and sodium propionate have also been used in the past against pseudomonas infections. The inhibitory action of these compounds was tested as follows : 8
coruginosa
BASES
PRELIMINARY TESTS
Cups
SODIUM
shown on three strains of Ps. œruginosa. inhibition. + == slight zone of-inhibition at 8 hours, when growth first obvious, but no inhibition at 20 hours. + + = slight zone persisting at 20 hours. + -!- + = moderate zone persisting at 20 hours.
Equal effects -
=
were
no
was
226 A patient who was not treated with amnion had a band of ulceration, 6 in. wide, round the leg. Spraying with chloramphenicol in propylene glycol every other day has reduced it to 21/2 X 1 in. DISCUSSION
All the
sensitive
organisms isolated from these ulcers were chloramphenicol in propylene glycol, as
to
shown by a definite zone of inhibition in the cup test. The possibility of local or generalised allergy can best be assessed by further trial of the method. Intradermal tests which we performed on our patient who had urticaria and asthma did not suggest that chloramphenicol was the sensitising agent. Pure propylene glycol produced a marked weal, but no surrounding erythema. A healthy adult control reacted similarly to the same amount intradermally. The weal was probably the result of absorption by the hygroscopic propylene glycol. In the 10th case it may be that the infection was overcome so rapidly that there was a sudden release of bacterial products. The patient may have been sensitive to the bacterial protein or to something else produced by the action of the antibiotic. If cultures were kept of organisms isolated from an ulcer, it would be possible to check this hypothesis by injecting a killed bacterial suspension, should an allergic reaction occur. Despite the one reaction in this small series, chloramphenicol in propylene glycol seems to be a very useful adjunct to treatment with amnion. We are grateful to Dr. J. Stanley White and Dr. Robert of the department of clinical investigation of Messrs. Parke, Davis for supplies of chloramphenicol in
Hodgkinson
propylene glycol. REFERENCES
Lewis, R. S., Gray, J. D. (1951) Brit. med. J. ii, 939. Troensegaard-Hansen, E. (1950) Lancet, i. 859. (1951) Charing Cr. Hosp. Gaz. 49, 79. -
SECRETION OF A SALT-RETAINING HORMONE BY THE MAMMALIAN ADRENAL CORTEX S. A. SIMPSON J. F. TAIT B.Sc. Lond.
B.Sc., Ph.D. Leeds, A.Inst.P.
ASSISTANT IN BIOLOGICAL
RESEARCH, COURTAULD INSTITUTE OF BIOCHEMISTRY
ASSISTANT IN
RESEARCH,
BARNATO-JOEL RESEARCH LABORATORIES
MIDDLESEX HOSPITAL, LONDON
I. E. BUSH B.A. Camb. RESEARCH
ASST.,
NATIONAL INSTITUTE FOR MEDICAL RESEARCH
IN recent years the division of adrenal cortical steroid hormones into glucocorticoids and " niineralocorticoids (Selye 1950) has been seriously questioned by some authors (Verzar 1952), and Conn, Louis, and Fajans (1951) have gone so far as to suggest that all the actions of adrenocorticotropic hormone (A.C.T.H.) in humans could be accounted for if the human adrenal be supposed "
to secrete 17-hydroxycorticosterone (Compound F) alone. Thus the three most active known glucocorticoids, cortisone, Compound F, and Compound B, all produce sodium retention in man (Sprague et al. 1950, Conn, Fajans et al. 1951, Conn, Louis, and Fajans 1951), and cortisone does so in dogs (Roberts and Pitts 1952). Similarlv all the active cortical hormones have been found to have some effect in depressing the urinary Na24/K42 ratio of adrenalectomised rats (Simpson and Tait 1952). Since chemical estimations of the corticoids in adrenal venous blood have shown Compound F and Compound B (corticosterone) to be by far the major secretory products of the adrenal cortex of many mammalian species (Nelson et al. 1950, Hechter et al. 1951, Bush 1951) it has appeared to some (see Sayers 1950) unnecessary to postulate" the secretion of a specific " mineralocorticoid " or salt-retaining hormone " by
the mammalian adrenal cortex.
However Selye’s comprehensive theory of the.existence of "diseases of adaptation" (Selye 1950) depends at least in part on the’postulate that the adrenal cortex secretes a mineralocorticoid distinct from and antagonistic in action to the glucocorticoids. Furthermore the glucocorticoid "cortisone" is not fully effective in controlling Addison’s disease (Conn, Fajans, et al. 1951, Forsham 1951), nor is it as effective as whole adrenal extract in correcting various deficiencies of adrenalectomised animals (Ingle et al. 1952). Many objections to the theory of mineralocorticoid secretion by the adrenal- were made on the grounds that the actual secretion of desoxycorticosterone (deoxycortone) had not been demonstrated and that this substance was possibly only an artefact in adrenal gland extracts (Dobriner 1952). At the time no other potent mineralocorticoid was known. The whole question became more complex when it was discovered (Grundy, Simpson, and Tait 1952) that " by far the greater part of the, " salt-retaining activity" of whole-beef adrenal extract was contained in a hitherto unknown substance of high potency, which could only be separated from " cortisone " by prolonged chromatography. We wish to present evidence in this communication that a similar or identical substance is actually secreted into the blood-stream by the adrenal of the dog and the monkey. It thus appears to be an active hormone of physiological significance and not only a component of gland extracts or simply an artefact. METHODS
Monkcey.—The isolated left adrenal of a 6-35 kg. rhesus monkey was perfused with blood by a technique similar to that of Vogt (1951) and the effluent blood collected in an ice-cooled flask. The blood was extracted at the end of the experiment with ethyl acetate and the extract purified and concentrated by washing quickly with cold N/5 Na2CO3, water, distillation, and partition between petrol and 70% ethanol. The final extract was divided into several parts and fractionated by paper chromatography (Bush 1952). The content of &agr;,&bgr;unsaturated ketonic steroids was determined by their fluorescence reaction with NaOH in two fractions of the extract and the position of " cortisone " on the chromat-ogram was marked with a pencil. The remaining fractions were run on the same chromatogram but not treated with NaOH. The region of the chromatogram containing the cortisone from these fractions was eluted with ethanol, and the ethanol eluate was evaporated and assayed (table i) by the method of Simpson and Tait (1952). Dog.-Blood from the left adrenal vein of a 21-5 kg. bitch was collected in vivo under pentobarbitone antsthesia and after evisceration (Vogt 1943). The splanchic nerves were intact. The blood was extracted and was fractionated by chromatography. The whole of a chromatogram run in the system toluene : methanol : water (100 : 75 : 25 by volume) was divided into regions and assayed in parallel by the sodium/potassium ratio assay and the fluorescence reaction with NaOH. A measured portion of the cortisone fraction of the blood extract was acetylated and chromatographed with the benzene : formanide system of Burton et al. (1951). RESULTS
The results of the two types of assay when applied to these extracts of adrenal blood are given in table I. It is seen that the " cortisone spot " on the chromatogram of the monkey’s adrenal blood extract contained 1-2 g. of cortisone, which would be equivalent to 0.2 .g. deoxycortone in the mineral ratio bioassay used. However, the biological activity of this spot when assayed in the adrenalectomised mouse was equivalent to that of 15 ug. deoxycortone and could not be accounted for by the small amount of cortisone present in the extract. A more complete study was made of the extract of dog In this experiment the whole adrenal venous blood.