Document VJN2ro5dyRY8NKLrYyYJ77NKg

I -iEPT. 29, 1951 mates of patients 'Ik, leave out of 1 1 liese severely misery that so Jousive scarring ""mic handicap '"attires may bo " of the inarch a of the severely I ho first critical f slow healing, and its wider icli to alleviate to givo a child ' equivalent of \. ilional attempt Wo have stood is to tho Burns pital has been auses of those idonts, a third 'd a third rvero > children under : I cen. ring fire caused i act with a fire, responsible for lis due to this >ared with only period of six of 60,998 days ml and Wales) I that aboutI ed to hospital hospital beds approximately arning injuries a is needed to icstic fires, by 'fared for salo ms (and coal can be over dal to mako lax from all "operation of I ho safer fires I in this paper "I the views of a c.s., and Dr. 1 allowing us to lical Research also to thank Is. lission of lira. 1 lined by her ; coal, gas, and y Council on Stephen, has m a fire-guard 'U'aUh, Scotland, THE LANCET] SPECIAL ARTICLES [sect, 29, 1951 58;) PROPOSED STANDARD METHOD OF 17-KETOSTEROID DETERMINATION M.R.C. COMMITTEE ON CLINICAL EN DOC KINO I.OCT Ziiumcrmann (1935) first suggested that steroid sex hormones could be determined quantitatively by the use of m-dinitrobcnZcno, which gives a red colour in presence of alkali with compounds containing an aclivo methylene group, lie described (1936) a method applicable to pure substances and to urine exlraefs. Wu and Chou (1937) described a modification of this method using androstcrono as reference substance. Oesling and Webster (1938) used the Zimmermann technique and roughly correlated the colorimetric assays with biological determinations on capons. In a very careful and complete investigation of tho Zimmermann reaction as applied both to puro substances and to urine oxtracts Callow et al. (1938) described a modified and improv'd! method for determining urinary 17-ketostcroids. .Many variations and modifications of the method described by Callow et al. have subsequently been published ; but it probably still remains t-lio most satisfactory and reproducible procedure. This method, called for convenience tho Callow-Zimmennann method, is, therefore, tho basis of tbo technique described below. Laboratories now use various techniques to estimate 17-ketosteroids so that there is need for some standard by which all results can be made comparable. Tho committee therefore suggest that the following method be used as such a reference standard. The Method COLLECTION OF URINE SPECIMEN Since there is considerable diurnnl variation in excretion of 17-ketostoroids (cf. Pincus 1943), complete 24-hour collections of urine are necessary. Subjects should bo instructed to reject the first, morning urine on tbo first day of collection and then to collect all urine up to and including tho first morning urine on tho second day. Addition of preservatives is usually unnecessary. Formation of inconvenient amounts of ammo nium carbonate can be prevented by addition of a salt of a heavy metal (to inhibit urease activity)--e.g., copper sulphate 1 mg. Pr m'\ HYDROLYSIS AND EXTRACTION OF URINE Published procedures for extraction and hydrolysis of urine are legion and have given rise to much controversy. Tho following method, based on the work of Robbie and Gibson (1943), has been found to be convenient and rapid. Direct comparison with other recommended procedures lias shown close agreement in amounts of ketosteroid extracted. A somplo of urine (100 ml.) is brought to boiling under rollux over a bunsen flame. Concentrated 1IC1 (10 ml.) is added down the condenser and boiling maintained for 10 minutes. The urine is then allowed to cool somewhat and 30 ml. of carbon tetrachloride added down the condenser. Tho contents of the flask are again muintained at the boil for 10 minutes. The flask is then cooled, the CCI, layer removed and replaced by tho same volume of fresh solvent, and tho mixture, again refluxed for 10 minutes. The CCI, layer is then removed and added to tho first extract. Tlie total CCI, extract (about 60 ml.) is washed successively with: (1) 20 ml. water, (2) 20 ml. 2A' NaOK, (3) 20 ml. water. (4) 20 ml. water containing a pinch of sodium dithionite (N'ajSjO,). Tho washed CCI, extract is then evaporated to dryness on a wator-bath using water-pump vacuum to remove last traces. Tho dry residue is dissolved in aldehyde-1 roe absolute ethanol. Tho volume of ethanol may be varied according to the expected ketosteroid content. With normal urines 4 ml. of ethanol is convenient, but 2 ml. or oven 1 ml. may he more appropriate for urines of low ketosteroid content. Bthanolic extracts thus prepared appear to bo quite stable and need protection only from evaporation. COLORIMETRIC ESTIMATION Reagents . (1) Eth/inol.--The suitability of tho absoluto alcohol is the most important factor in achieving satisfactory results. Some grades of commorcial " absoluto " alcohol can bo used without preliminary purification but usually some treatment is necos- sary. Tho following mothod described by Callow ofc al. (1939) may bo employed. Commorcial " absoluto " alcohol is treated with 4 g. per litre of m-phcnj'lonodininino hydrochloride, allowed to stand in tho dark for a week, with occasional shaking, and then distilled, tho bond and tail fractions being rejected. This purified alcohol should bo used for all purposes in connection with tho estimation of 17-kctostcroids. (2) in-Dinitrobenzene,--A well-crystal!iscd and fairly pure specimen is further purified thus: 20 g. is dissolved in 750 ml. of 95% otlianol, warmed "to 40 C, and 100 ml. of 2*V NaOH is added. After five minutes, tho solution is cooled and 2500 ml. of water is added. The precipitated m-dinitrobenzono is collected on a Buchner funnel and washed very* thoroughly with water, sucked dry, and reorystallisod twico in succession from 120 ml. and SO ml. of absolute ethanol. Tho material should bo well crystallised in almost colourloss needles m.pt. 90*5-91"C. Tho reagent is a 2% wjv solution of this material in absoluto ethanol. It is stored in a brown bottle with a glass stopper and kept- in tbo dark. Under thoso conditions it is stable for 10-14 days. (3) Potassium hydroxide..--The reagent solution is 2*5aV KOH in absolute ethanoL KOH (9 g.) is dissolved with shaking or mechanical stirring in 50 ml. of absoluto ethanol and the solution filtered through a hardened filter paper. The con centration is chockod by titration with acid (mothyl orange indicator) and adjusted between the limits 2*48-2,52Ar. The solution is stored in a refrigerator and must be discarded as soon as tho faintest colour is perceptible. Mode of Operation The following tubes should always bo set up : (1) Rcaucnt Rlnnk.--0-2 ml. ethanol, 0-2 ml. m-clinitrobctr/.cno rciurcnt (u.N.n.), 0-2 ml. KOH. (2) Urine Extract.--0-2 ml. urine extract, 0-2 ml. d.n.b., 0*2 ml. KOH. (3) Standard.--0-2 ml. standard, 9*2 ml. i.n.b., 0-2 ml. K01I. A convenient standard is one containing 0*1 mg. androsterono or dehydroepi-androsterone in 0*2 ml. absolute ethanol. Tho .tubes are stoppered and placed in a thermostat at 25d: 1C for (50 minutes. During incubation tho tubes should bo protected from bright light. It is most convenient to keep them in complete darkness. At tho end of CO minutes 10 ml. of absolute ethanol is addod to each tube and tbo contents mixed. The urine extract and tho standard are then read in a photo-electric colorimeter against the reagent blank tube. Colorimeter readings should always bo made with two different filters, one green (approximate wave-length maximum ' 5200 A), and one blue or violet (approximate wave-length maximum 4300 A). Thoso correspond to ` Ilford Spectrum ' filters nos. 604 and 601 respectively. Readings with both filters are necessary for correcting for interfering chroraogcns by the method given below. There is a straight-lino relation between extinction and amount of ketosteroid up to about 0T mg. of androsterono. With larger quantities of sterono tho calibration deviates from a straight line. If very high readings aro obtained it is necessary to dilute the original urino extract and repeat colour development. Dilution of tho final coloured solution should not bo employed as it gives erroneous results. CORRECTION FOR INTERFERING CHROMOGENS Substances other than 17-ketosteroids develop colour with tho reagents. Talbot et al. (1942) suggested tho use of tho following formula for correcting the observed green extinction values for interfering chromogens : Observed (1 -- 0-6 V Corrected Green =-------------- <>73------------ That is, from tho obsorved green extinction subtract 6/10 of the observed violet extinction and divide by 0-73. Tho corrected green extinction is converted into mg. of standard by comparison with tho extinction of tho latter with tho green filter. Tho validity of this correction has been chocked by comparison with values obtained on urinary neutral ketone fractions obtained by tho use of Girard's reagent (Talbot et al. 1942) and by simultaneous determinations by colorimetric and polarographic methods (Butt ct al. 1951). Ar.7>--If the original paper of Talbot ct al. is consulted it should he noted that there is a confusing misprint in discussing the ratios of the extinctions in green and blue light. They are reversed In the text.. Tho preface to vol. 143 of J. biol. Chem. carries a' corrigendum on this point. DSW 255056 STLCOPCB4060747 586 TUB LANCET] SPECIAL ARTICLES [SEPT. 29, 1951 THE L.' I ;i . !:s NORMAL EXCRETION OF 17-KETOSTEROIOS periods. In the first ten yoars Reichstcin and his col- j Bamett ct al. (1940) lmvo tabulated most of the data for leagues at Zurich, his own team at tho Mayo Clinic, and * Some steroids _j I excretions by normal subjects published from 1939 to 1940. Wint.orsteintsr and lTilTncr of Columbia University, g 'i in disoa- Younger and middle-aged subjects showed an average excretion made fundamental contributions to tho detection, ' support i per 24 hours of about 13 mg. for men and 9 mg. for women. A later report by Forbes ct al. (1947) gave an average of 12-5 mg. for 73 young men and 8-2 mg. for 05 young women. Variations in oxcrelion of kctnstcroids by normal subjects with special reference to age. and sex are discussed in publica isolation, and identification of the adrenal steroids. By 1940 it was known that'4 groups of sleroids could be. isolated from adrenal glands. The first group were sex hormones, not peculiar to the adrenal ; the producii a so-cal first injt might n i *k .3 = > tions by Robinson (1948a) and by Hamburger (1948). Evaluation of Colorimetric Determination In a review of ketosteroitl excretion in health and disease, Robinson (1948b) concludes : " With the advent of a colorimetric method a wido survey of normal and pathological urines became possible and during the, past ten years many thousands of specimens have been examined in laboratories in all parts of the world. At first much painstaking work was done in simultaneous comparisons of androgenic potencies as determined by capon assay with the results of chemical estimations. Correlation between tbe two methods was fairly good in general but not invariably so. But it soon became apparent that, at least in somo clinical conditions, tliero were important correlations between tho chemically determined values and the disease. Tho valuo of the chemical determination today rests upon these empiri second group were inactive steroids found only in the adrenals ; (lie third group were steroids with adrenal hormonal activity ; while tho fourth group was an unidentified " amorphous residue," probably of steroids, which also possessed considerable hormonal activity. But the world supply of adrenal glands was limited. To apply these, hormones to clinical investigation it was necessary to develop methods for their synthesis from non-adrenal sources. From 1940 onwards tho various workers concentrated on this aspect of the problem, which was full of difficulty. On paper the transition from the steroid acids of bile to cortisone might appear easy, hut in fact ove.r 30 difficult chemical steps are required. However, each year brought improvements and alternatives, and there wore now easier and more fruitful methods : indeed, so much knowledge had accrued that K. B. Woodward had recently reported ( product proved I)r. 1 seems t from tli the pr< theorcli This toi I)esc: toid ar; the Ma; only 1 This p parentc 1. To sympto: 2. To from tb cally established relations rather than upon their moro the total synthesis of cortisone from simplo aromatic 3. Tc 'i doubtful value as measures of biologically potent compounds. Tribute must bo paid to the workers in the require! 1 androgens." references laboratories of Messrs. Merck, who had made many 4. Tc Barnett, J., Hcnlv, A. A., Morris, D. J. O. R., Warren, F. L. (1940) important advances. Chemical manipulation of the Biochcm. J. 40, 778. steroid nucleus could now bo controlled in many ways, 5. T< The tri Butt, \Y\ K., Morris, C. J. (>. R., Robinson, A. M., Warren, F. L. (1951) J. Endocrinol, 7, xii. Callow, N. H., Callow, U. K., Emmons, C. W. (1938) Biochem. J. and ring C was now in reach of the chemists, thus opening the way for the production of active adrenal hormones were a was as 32 1312 -- -- --- Stroud, S. W. (1939) J. Endocrinol. 1, 70. Forbes, A. I\, Donaldson. K. O., Reifcnsfem, K. C., Albright, F. (1947), J. clin. Endocrinol. 7. 204. Hamburger, C. (1948) Acta cndnrrinol. 1, 19. Oestinf?, R. R,, Webster, B. (1938) Endocrinology, 22, 307. Pincus, G. (1943) J. clin. Endocrinol. 3. 195. Robbie, W. A., Gibson, R. B. (19 13) Ibid, p. 200. Robinson, A. M. (1948a) Brit. J. Cancer, 2, 13. -- (1948b) SI. Bart's llosp. J. 52, 40. Talbot, N. B., Berman, R. A., MacLaolilan, E. A. (1942) J. biol. Ckem. 143, 211. from plant steroids. In addition, organic chemists had produced 40 or more steroids closely related to cortisone. Other chemists had invented better methods for the detection and assay of steroids in blood and urine. These chemical methods had also enabled physiologists to study accurately tho hormones released from tho adrenal gland under stimulation with adrenocorticotropic hormone (a.c.t.h.). The discovery of Sept. 21, 1948, had proved 1 Initial cortiso Pose (n. Wu, H., Chou, C. Y. (1937) Chin. J. Physiol. 11, 413. Zimmermann, W. (1935) Iloppc-Scyl. Z. 233, 257. to be a great stimulus to further work in this field. 4 -- (1936) Ibid, 245, 47. Was cortisone specific ? Apparently it was. No simple substitute (analogous to stilbocstrol as a substitute for At thi THE ADRENAL CORTEX AND oestradiol) was known. Other steroids had been claimed bring RHEUMATOID ARTHRITIS as having antirbeumatic activity, but their evaluation, which must he in patients and not in animals, had initial doses -1 I* THE IIEBERDEN ORATION sometimes failed to take into account tho psychic On Sept. 19, in London, I'rof. E. C. Kendall, d.sc., factors in patients. In this way dcsoxycorticosterone patiei were delivered the annual Ileberden oration before tho (deoxycortone) and ascorbic acid had enjoyed a brief reduc Heberden Society. lie recalled that it was three years spell of popularity. In fact, so specific was the structure (in 72 since the first injection of cortisone (then known as of corlisono that two otherwiso identical isomers--one Compound E) was given with successful results to a with no doublo bond in tho 4-G position, and one patient with rheumatoid arthritis at the Mayo Clinic. with an extra double bond fi-7--were both devoid of It had been given, in fact, seven days before, his colleague, physiological activity. Furthermore, the effects were Dr. Philip Hench, left for England to deliver a previous not simply those of toxic overdosage, since the dose Ileberden oration. Dr. Henck's script, which had been of cortisono required to produce remissions of rheumatoid ii I prepared some weeks previously, had contained no reference to this trial. Instead, Dr. Hench had set out his reasons for believing that rheumatoid arthritis was a reversible disease--that there was, in fact, some sub stance x, produced in the body during pregnancy and in jaundice, which could cause, complete, remission of tho activity of the rheumatoid process. One could imagine the conflict in Dr. Bench's mind when ho was slating his arthritis was within the physiological range--i.e., the normal human gland could product) enough hormones to keep the symptoms of rheumatoid arthritis from . appearing. Under normal conditions human urine might contain 30-00 mg. of steroids of adrenal origin every day, as determined by the methods of Venning, Corcoran, Mason, and others. Furthermore, under conditions of stress the output of steroids increased So patiei lint o and ; of trr 40 di dose omitt reasons for believing in tho existence, of a certain sub stance but was unable to disclose its discovery because this bad not been confirmed. The startling efficiency of cortisone in causing a remission in rheumatoid arthritis and considerably. When cortisone, was given therapeutically, doses of less than 100 mg. a day were effective, and as it was probable that the endogenous contribution of adrenal steroids is suppressed the total availablo cortisone was as ro were sidc-t many other diseases was now known, and it seemed possible thus in the normal range. That the adrenals could, but At that cortisone may bo tho substance x of pregnancy, do not, produce enough hormones to ensure clinical although the substance x of jaundice remains obscure. remission, pointed to a failure in the control of adrenal moda tho I hv Si Tracing the history of investigations of the adrenal output. Dr. Kendall thought it might be profitable to r.R.C. hormones, Dr. Kendall divided it into two ten-year investigate pituitary factors in rheumatic diseases. , Lord DSW 255057 I STLCOPCB4060748