ISSN:0003-2654    

DOI:10.1039/AN95176FX029

出版商:RSC    

年代:1951

数据来源: RSC

ISSN:0003-2654    

DOI:10.1039/AN95176BX031

出版商:RSC    

年代:1951

数据来源: RSC

ISSN:0003-2654    

DOI:10.1039/AN95176FP069

出版商:RSC    

年代:1951

数据来源: RSC

摘要:

THE ANALYST viiSOUTHERS RHODESIA GOVERNMENTVACANCY: FOOD TECHNOLOGIST: DEPARTMENTOF HEALTHPPLICATIONS for the above post are invited from malesAunder the age of 40 years and holders of the B.Sc. or M.Sc.degree or their equivalent, w)ith experience in food technology.Knowledge and experience of biochemistry, including micro-biological assay and of tropical food is also desirable.Duties will include the investigation of native foods, theirstorage, preservation and processing, and research with a viewto introducing improvements; also the biochemical investiga-tion of clinical material required in connection with nutritionsurveys.Salary Scale: L468 X L66 to L600 x A34 to L668 x L33to €800 x €100 to €900 x 640 to El140 per mum, but thecommencing salary may be advanced up to four steps in thescale for approved experience subsequent to qualification.The minimum salary applicable to the holder of an M.Sc.degree is €600 per annum.The successful applicant will bestationed in Salisbury and will be required to provide his ownaccommoda t ion.Application forms and details of leave conditions, pensionscheme, cost of living, mamage and children’s allowances,income tax, medical examination, refund of sea and rail fareson appointment etc may be obtained from the Secretaryto the High Co&&oner for Southern Rhodesia, RhodesiaHouse 429 Strand London W.C.2 to whom completedforms khoul6 be retdned not liter than‘ the 31st August, 1961.Canvassing will disqualify applicants.SOUTHERN RHODESIA GOVERNMENTVACANCY : ANALYTICAL CHEMIST : DEPARTMENTOF GEOLOGICAL SURVEYPPLICATIONS are invited for the post of Assistant Ac hemkt in the Department of Geological Survey.Applicants should possess a Specialised Honours Degreeof a recognised University, or the A.R.I.C.qualification.Experience in inorganic analysis will be a strong recommenda-tion.Salary Scale: L468 x E66 to €600 x €34 to €668 x E33 toQ300 x flO0 to €900 x €40 to €1140 perannum.In addition to salary a cost of living allowance, at presentapproximately 20 per cent. of salary, is payable. A successfulapplicant with an Honours Degree or A.R.I.C. qualificationwill be appointed to the L600 per annum step in the scale.Additional increments, not exceeding four, may be grantedin recognition of previous experience.The appointment will be subject to the rules and regula-tions of the Southern Rhodesia Civil Service.Application forms may be obtained from the Secretary,Office of the High Commissioner for Southern Rhodesia,Rhodesia House, 429, Strand, London, W.C.2, to whomcompleted forms should be returned not later than the 31stAugust, 1951.Details of leave mnditions, pension scheme, cost of living,marriage and children’s allowances. income tax, medicalexamin-ation, refund of sea and rail.fares on appointment,etc., may be obtained from the High Commissioner’s Office.Canvassing will disqualify applicants.ROCHE PRODUCTS LIMITED have an opening for assist-ants in their Analytical Department, age about twenty-five years, of B.Sc. or A.R.I.C.standard, who are trained orwish to do chemical analysis. Write stating qualifications,experience and salary required to the Secretary, RocheProducts Limited, Welwyn Garden City, Herts.T H E Research and Development Dept. of the DistillersCo. Ltd. have a vacancy for a chemist to work on bio-chemical analysis connected with antibiotic fermentationresearch. Age under 28. Applicants must possess anHonours Degree in Chemistry or equivalent, and experiencein biochemical analysis is essential. The vacancy affordsgood opportunities for any chemist wishing to enter theantibiotic research field, with good prospects of advancement.Salary will depend on qualifications and experience. Non-contributory pension scheme.Apply: Staff Manager, 21,St. James’s Square, London, S.W.1.HEMIST qualified required for control of anal icalcsection df Laborat& at our Shaftmoor Lane grks,Birmingham. F‘revious experience in this class of work isessential. The position is permanent and pensionable andoffers excellent scope for initiative. State age qualificationsand experience to Personnel Manager, Joseph Lucas Ltd.,Great King Street, Birmingham.OSEPH LUCAS LIMITED require Engineers to fill theJfollowing vacancies in the factory which they propose toopen in Liverpool for the manufacture of Fuel Injectionequipment.METALLURGICAL ASSISTANTS and experience in at least oneof the following:-1. Production Heat Treatment Checking.2. Physical Testing of Raw Materials.3.Pyrometry and Radiography.1.2. Analysis of Raw Materials.3. Analysis of Plating Solutions.CHEMICAL LABORATORY ASSISTANTS and experience in oneProcess Control (Plating, Blacking, etc.).Selected applicants will be expected to spend a short periodat the Company’s Laboratories in Birmingham prior totaking up their positions in Liverpool. The Company willpay the expenses in connection with such visits.Application, giving full details of previous experience,should be made in writing to the Personnel Manager, JosephLucas (Gas Turbine Equipment) Limited, Shaftmoor Lane,Hall Green, Birmingham, 28.or more of the following:-UNLOP have two vacancies for assistant chemists atDthe Research Centre, Birmingham. Applicants shouldbe aged 20-25 with a degree in chemistry.One post isconcerned with ;he development of new analytical methods inthe field of rubber and plastics, the other with microchemicalanalysis. Previous analytical experience, though desirable,is not-essential. Salary according to age, qualifications andexpenence. Applications in writing, quoting ref. A.S 100,To: Personnel Manager, Dunlop Rubber Co. Ltd., FortDunlop, Erdhgton, Birmingham. 24.ICRO ANALYST desires appointment with an organisa- M tion where 18 years’ experience and a knowledge ofmodern methods would be appreciated. Write Box 3780,THE ANALYST, 47, Gresham Street, London, E.C.2.CHELSEAPOLYTECHNICMANRESA ROAD, S.W.3A Course of Lectures and Practical Work onTHE CHEMISTRY ANDMICROSCOPY OF FOOD,DRUGS AND WATERwill be givenSEPTEMBER, 1951 to JULY, 1952Lecturer in Charge :R. G. MINOR, Ph.C., F.R.I.C.The course is based on the Syllabus of theExamination for the Fellowship of the RoyalInstitute of Chemistry (Branch E), andincludes Analysis and Microscopy of Food,Drugs and Water, Toxicological Analysis,Acts and Regulations relating to Food,Drugs and Poisons.Mr. Minor will attend on Wednesday,19th September, 1951, 6-8 p.m., to advisestudents.Full particulars can be obtained on appli-cation to the Secretary

ISSN:0003-2654    

DOI:10.1039/AN95176BP073

出版商:RSC    

年代:1951

数据来源: RSC

摘要:

AUGUST, 1951 Vol. 76, No. 905 The Precision of the 3-Point Correction Method of Spectrophotometric Assay of Vitamin A BY D. C . M. ADAMSON, W. F. ELVIDGE, N. T. GRIDGEMAN, E. H. HOPKINS, R. E. STUCKEY AND R. J. TAYLOR A seven-laboratory determination of Ei:& a t 328 mp, geometrically corrected for absorption irrelevant to vitamin A, was carried out on each of five oils containing vitamin A. Readings were made in duplicate on photo-electric instruments. From a statistical analysis of the results it is concluded that the limits of error of a determination in duplicate from any one laboratory are about f15 per cent. for P = 0.05. The corresponding figure for gross, i.e., uncorrected, E values is about f2 per cent. THE practice of taking the vitamin-A content of any oil to be directly proportional to its absorption of ultra-violet light at 328mp is moribund, despite the fact that within certain classes of oils and concentrates an empirical relation between the two can be assumed with some degree of confidence and roughly checked by colorimetric reactions; for an analytical method, the interpretation of which depends on extraneous information, does not lend itself to standardisation and so must eventually be laid aside in favour of some more nearly absolute method-even if the new method is technically more complex.Most new methods now being developed, and some already in use, for t.he estimation of vitamin A depend on the elimination of, or allowance for, the ultra-violet absorption due to the presence in the sample of com- pounds other than the vitamin.Knowing the spectrophotometric characteristics of the 445446 ADAMSON, ELVIDGE, GRIDGEMAN, HOPKINS, STUCKEY AND TAYLOR : LVol. 76 pure vitamin ia the particular solvent used, we can then calculate the vitamin-A content of the sample by simple proportion. In practice it is customary to express the result not in percentages (parts per 100) but in International Units per gram (parts per 107/3), the I.U. being 0.3 pg of vitamin A as alcohol.’ The ingenuity and simplicity of one method of allowing for irrelevant absorption have earned it considerable attention. This is the Morton - Stubbs 3-point geometric correction meth0d.~s3s~s6 Its basic assumption is that the irrelevant absorption of most ‘oils and con- centrates is linear at three points in the regions 310 to 313 mp, 325 to 328 mp and 336 to 339 mp.Now it conveniently happens that there are points within the same regions on the absorption curve of vitamin A itself whose ordinakes bear the relation 6:7:6 and this makes for a simple geometry. The exact points chosen and the constants of the correction formula depend upon whether the whole oil or the unsaponifiable fraction is being examined and upon the solvent used. For the whole oil in cyclohexane the formula is- E at 328 mp (corr.) = 7 x E at 328 mp - 2.882 x E at 313 mp - 4.118 x E at 3386 mp. Hence the substitution of the Morton-Stubbs method for the old method involves only the taking of two extra spectrophotometric readings on the same solution and a consequent calculation. The theory of the correction procedure is discussed elsewhere6; the present paper is concerned with its reproducibility, a matter that does not yet seem to have been investigated.EXPERIMENTAL Seven independent laboratories undertook to determine, by photo-electric spectro- photometry, E:& at 313 mp, 328 mp and 338.5 mp, each in duplicate (separate weighings), on five oils, the solvent to be cyclohexane. As a subsidiary investigation, they were asked for complete ultra-violet absorption curves between about 250 mp and 350 mp. This secondary information was mainly for another purpose,6 but in the present context it served to show that none of the five curves conformed to the requirement’ that it should “agree closely with that of the international standard measured under the same conditions and compensated with a solution of the diluent oil,” and in particular that intensities of absorption “in the region 310 to 350mp expressed as decimal factions of the maximum should not differ between sample and standard by more than 0.02.” As these requirements were not met, the curves were presumed to contain irrelevant absorption and to be in need of rectifica- tion.Not all the absorption curves rose to a maximum at exactly 328 mp, but none of the maxima fell outside the critical range 325 to 328 mp. For the sake of simplicity the figure 328 will be used in the remainder of this paper for all the maxima. Four of the samples were- A refined fish-liver oil concentrate, E at 328 mp (gross) . . 16-61 A hake-liver oil, E at 328mp (gross) .... . . - 6.31 = 154.4 - A halibut-liver oil, E at 328mp (gross) . . . . . . - A cod-liver oil, E at 328mp (gross) . . . . .. .. - - 0.628 - The fifth sample was a blend of these oils. Its composition was afterwards revealed as 20 parts of concentrate, 31 parts of halibut-liver oil, 20 parts of hake-liver oil and 30 parts of cod-liver oil. Its gross E at 328 mp could therefore be expected to be 37.11; the average value found was reasonably close, viz., 36.82. REswLTs Before application of the correction formula, the submitted estimates of the gross EiZL at 328mp for the five samples were themselves statistically analysed, and a coefficient of variation of 1.48 for any one estimate in one laboratory emerged. Additionally, the corre- sponding coefficient for estimates at any of the three wavelengths was extracted and found to be 1.26.These are normal values and compare favourably, for instance, with the figure of 1-55 found in a recent investigation of the basic limits of error of photo-electric instruments.7 The mean results in the form of percentage variation per :;ample are shown in Table I. As expected, the individual estimates (not tabulated) show a wider range. Not all the single estimates were available; three of the seven laboratories submitted averages of duplicate estimates only, and this necessitated a The correction formula was then applied to each triad of E estimates. The values in Table I range from 92.3 to 117.2.August, 19511 SPECTROPHOTOMETRIC ASSAY OF VITAMIN A 447 correspondingly restricted estimate of the residual coefficient of variation.of the analysis of variance of the corrected E values are set out in Table 11. The essentials TABLE I ESTIMATES OF CORRECTED Ei& AT 328 mp ARRANGED AS PERCENTAGES OF OVER-ALL AVERAGES, EACH ENTRY BEING THE MEAN OF TWO ESTIMATES Laboratory A . . .. B . . .. c .. . . D . . . . E .. . . F . . . . G .. . . Means .. Concentrate, % . . 93.8 . . 104.8 . . 100.5 . . 100.9 . . 95.7 . . 102.6 . . 101-6 .. 100 Absolute mean E values 138.7 Halibut-liver Hake-liver oil, Oil, % % 117.2 94-9 92.3 104.6 99.0 100.4 98.5 100.5 96.7 97.1 95-9 97.6 100.4 104.9 100 100 15.21 5-62 Cod-liver oil, 97.5 104-9 105.9 104.8 97.1 92.6 97.2 % 100 0.541 Blend, 107.2 111.7 93.5 103.8 93.7 95.4 94.7 34.80 % 100 TABLE I1 ANALYSIS OF VARIANCE OF THE ESTIMATES OF CORRECTED Ei& AT 328 mp Source of Variance D.F.Mean square Between laboratories . . . . .. 6 77-49 Laboratories x oils . . .. .. 24 68.61 Residual error . . .. . . .. 20 161.54 Although the interaction term “laboratories x oils” in Table I1 has a mean square lower than that for the residual error, it will be wise to take it into account inasmuch as it covers more of the experimental data than does the residual term. We may therefore combine the two estimates and hence take the square root of the weighted mean square of 68.61 and 161.54, i.e., 10.5, as the fairest estimate of the residual coefficient of variation. esented in another form as the percentage of irrelevant absorption at 328 mp estimated by correction. Table I11 shows these means. The expected values for the blend are calculated from each laboratory’s findings on the four main oils and the known composition of the blend. The seven laboratories are entered in descending order of agreement between the values found and expected for the blend.The results of the trial may be TABLE I11 ESTIMATES OF PERCENTAGE OF IRRELEVANT ABSORPTION AT 328mp BY THE CORRECTION METHOD Each figure represents the mean of at Laboratory G . . .. c .. .. F . . . . D .. . . E .. .. A . . .. B . . .. Means . . Concentrate, % . . 6.9 . . 10.3 . . 3.6 . . 10.9 . . 11.8 . . 17.0 . . 10.7 . . 10.2 Halibut-liver Hake-liver oil, oil, % 7. 9.6 5.4 13.8 12.1 13.3 10.9 9.7 7.7 13.2 12.6 4.8 17.0 20.3 10.3 10.7 10.9 least two estimates Blend Cod-liver +- 7 oil, Found, Expected, % % % 16.0 11.6 16.7 11.8 13.9 16.3 13.1 14.2 7.1 12.2 6.8 7.5 8.1 7.0 4 4 7.5 7-0 10.9 5-2 10.6 12.0 14.0 12.0 10.2 The distribution of results in Table I11 and, in particular, the results for the blend give further credence to the derived errors.It is interesting to note that analysis of variance of the contents of Table I11 shows no significant difference between samples; if all the oils had in truth contained the same amount of irrelevant absorption (on the “correction” criteria) a distribution such as that now found would occur in 1 in 5 trials of this size. It might strictly be claimed that, as far as these oils are concerned, the gross E value provides448 ADAMSON, ELVIDGE, GRIDGEMAN, HOPKINS, STUCKEY AND TAYLOR [Vol. 76 a measure of the presumptive relative potency that is as good as the corrected value.In fact, however, the tendency for the cod-liver oil figure to be higher, and perhaps to be more consistent, cannot be ignored, especially as there is some evidences to show a fair reliability of the method when applied to this type of oil. DISCUSSION OF RESULTS That this estimate of 106 for the residual coefficient of variation of one result in one laboratory is not unreasonable can be inferred from a theoretical consideration of the influence of observational errors on corrected E values.6 The magnification predicted is of the order of 9.5, so that from the coefficient of variation found for the gross estimates (see above), 1.25, we should expect the coefficient for the corrected values to be nearly 12. I t concerns the variations in the relative readings of the three optical densities from one test solution to another.Inter- laboratory discrepancies in the calibration of instruments and experimental errors in the preparation of test solutions are not directly concerned ; these are factors that operate equally at all three wavelengths, and the calculation of the corrected result involves no magnification of this type of error. In this particular set of results no significant inter-laboratory differences arose (see Tables I and 11). This is unusual; work by the Photo-electric Spectrometry Group, published in part ,’ indicates that , in general, inter-laboratory (more strictly, perhaps, inter-instrument) differences can be represented by a coefficient of variation of about 1.6. If we take the published value of 1.62 and the companion value of 1.55 for the residual coefficient of variation of one result on one test solution, it follows that the standard deviation of any one duplicated assay in one laboratory is- It is important to make clear the meaning of this coefficient., i.e., *Z.O per cent., which means (a) that two-thirds of all such duplicates, each from a different laboratory, will fall within the range 98 to 104 per cent. of the over-all means, and (b) that there is a probability of 2 in 3 that the true result, defined as the mean assay from an infinite number of laboratories, lies within the range 98 to 104 per cent. about any one (duplicate) result. On the same basis, the corresponding deviation for corrected E values can be expected to amroximate to- J( 1.622 + (1.55 : g‘5r), i.e., 1106‘per cent. The value found for this deviation of duplicates in the present investigation (with no significant inter-laboratory differences) is + 7 4 per cent., and this is probably a fair guide (erring, if at all, on the low side) to what can normally be expected. The equivalent P = 0.05 limits of error are +15 per cent. (again for the mean of two estimates from one laboratory). REFER:ENCES 1. 2. 3. 4. -,- , Ibid., 1948, 42, 195. 5. Morton, R. A., J . Pharm. Pharmacol., 1950 2, 129. 6. Gridgeman, N. T., Analyst, 1951, 76, 440. 7. “Report of the Expert Committee on Biological Standardization,” World Health Organisation Morton, R. A., and Stubbs, A. L., Analyst, 1946, 71, 348. -,- , Biochem. J., 1947, 41, 525. Technical Report Series, No. 3, 1950. Edisbury, J . R., Phot. Spect. Group Bull. No. 1, April, 1940, 10. GLAXO LABORATORIES LIMITED, GREENFORD, MIDDIXSEX BOOTS PURE DRUG COMPANY LIMITED, NOTTINGHAM LEVER BROTHERS & UNILEVER LIMITED, SHARNBROOK, BEDFORD, and PORT SUNLIGHT, CHESHIRE THE CROOKES LABORATORIES LIMITED, PARK ROYAL, LONDOS, S.W. 10 THE BRITISH DRUG HOUSES LIMITED, LONDON, N.l, and POOLE, DORSET January, 195 1

ISSN:0003-2654    

DOI:10.1039/AN951760445b

出版商:RSC    

年代:1951

数据来源: RSC

摘要:

August, 19511 GRIDGEMAN 449 Observations on the Spectrophotometric Assay of Vitamin A by Geometric Correction of Absorption Curves BY N. T. GRIDGEMAN The accuracy of the spectrophotometric assay of vitamin A by geometric correction of absorption curves depends on the valid scope of two assumptions, viz., that the absorption curves of natural forms of the vitamin are indistin- guishable from that of pure all-trans vitamin A, and that the ultra-violet absorption curves of materials other than vitamin A in natural oils have three linear points a t certain wavelengths. There is evidence that these assumptions are not always correct, and it is shown that comparatively small departures from these conditions may be associated with appreciable loss of accuracy. An attempt is made to quantify degrees of departure in terms of the accuracy of the result.The influence of the normal observational errors of spectrophotometry on the precision of the result is also considered. The application of the method to cod-liver oil, as originally proposed, appears to be more firmly based than its application to richer oils. IN 1946, Morton and Stubbsl described a geometric method of “breaking down” compound spectrophotometric absorption curves into two parts, the major part being the established and characteristic curve of the compound in the original mixture whose quantitative analysis r E” i S S r Fig. 1. The geometry of correction S is sought and the minor part being the remaining “irrelevant” absorption about whose shape certain simple assumptions must be made.The method was applied in particular to the estimation of vitamin A in cod-liver oil. Later, the same authors extended their work to the analysis of other liver ~ i l ~ . ~ ~ ~ OseI“‘ and McGillivray6 have also published papers on the application of the method to oils containing vitamin A. The’present paper records some observations on the theory and on the reliability of the method as applied to the analysis of vitamin A in fish-liver oils and concentrates; in this context, reliability includes both precision (reproducibility of results) and accuracy (approximation to truth).460 GRIDGEMAN : OBSERVATIONS ON THE SPECTROPHOTOMETRIC ASSAY OF [VOl. 76 DERIVATION OF METHOD Consider three vertical parallel lines, as shown in Fig. 1, of length EL, EM and EH, rising from a horizontal base at distances apart r and s.Suppose these verticals to be the sum of the two lower sets, about which we know, first, the values a and b in set I, and secondly, that the tops of the three verticals in set I1 fall on a straight line. Given EL, EM and EH, Y , s, a and b, we are required to find X. I t can easily be shown that- and the expressions for ax, b X , P and Q and 12 readily follow. Applying this to spectrophotometry we can see that, given (2) the optical densities (verticals) at any three wavelengths (horizontals) of a solution of a mixture, (ii) the relative optical densities at those wavelengths of one component (a) of the mixture and (iii) the fact that the optical densities at the same wavelengths of the other component, or the sum of the other components (p), lie on a straight line, WE: can calculate for each wavelength the exact and unique partition of the original optical density between the contributions of cc and p.Further, knowing the cell thickness, the streng1:h of the solution and the light absorption by pure cc at the three wavelengths, we can transpose our results into quantitative analysis. In practice this means working in terms of EiZa. It is important to note that there is no assumption of linearity of the optical densities of the irrelevant component /3 at wavelengths other than the specified triad; in between them the optical densities may take any value. In practice we can simplify things by so choosing r and s in equation (1) that a = b = (say) k. Then- To apply this equation to the assay of vitamin A, Morton and Stubbs determined the relative optical densities in various solvents over a wide wavelength range for vitamin-A, acetate (assumed to have precisely the same curve as the fatty-acid esters of vitamin A existing in fish-liver oils) and for vitamin-A, al.coho1 (assumed to have the same curve as the free vitamin A that is split off into the unsaponiiiable matter of fish-liver oils).They then assumed that, in most fish-liver oils, “the irrelevant absorption ‘curve’ is linear over the approximate range 310 to 340mp, i.e., that no impurity or artefact shows a maximum very close to that of vitamin A,” adding that “a good deal is now known concerning the spectra of vitamin A, and oxidation products of vitamin A, and it can be said that over the range 310 to 340 mp their absorption curves are at least approximately linear.”S With this in mind they chose, for vitamin-A acetate in cyclohexane, Y = 15 and s = 10.5 on either side of A,,,.= 328 mp. So in the present notation, EM = E at 328 mp, EL = E at 313 mp, and EH = E at 338.5 mp, where E is an abreviation for E:& These fixation points give k = 6/7, and the equation becomes- = 7 [E at 328 mp - 0.4118 :E at 313 mp - 0.5882 E at 338-5 mp], . . (3) X, of course, being E at 328 mp (.net), or (cow.), i.e., “corrected” to eliminate all absorption other than that due to vitamin A, in contradistinction to the E at 328 mb in the brackets, which is gross, i.e., as recorded on the original sample. cyclohexane, Morton and Stubbs find- Other, very similar, equations hold for other conditions.For vitamin-A X = 7 alcohol in = 7@ at 326 mp - 0.4 E at 311 mp - 0-6 E at 336 mpj.August, 19511 451 Oser-4 finds that the above Morton - Stubbs equation for vitamin-A acetate in alcohol applies equally to the same compound in isopropanol. For vitamin-A alcohol in isopropanol he uses the equation- VITAMIN A BY GEOMETRIC CORRECTION OF ABSORPTION CURVES 5 EH] X = 7 E M - - E L - iI l2 = 7[E at 326 mp - 0.4167 E at 312 mp - 0,5833 E at 336 mp], but the U.S.P. Assaye of Vitamin A specifies, for vitamin-A alcohol in isopropanol, the slightly different equation- = 7[E at 325 mp - 0.375 E at 310 mp - 0.625 E at 334 rnpu], the constants being based on a 23-laboratory co-operative examination of the U.S.P.Standard Vitamin-A acetate via the unsaponifiable matter. The recently published 1951 Addendum (Appendix XV, p. 92) to the B.P. 1948 specifies “6/7” correction formulae based on A,,,. = 327.5 mp, with r = 15 and s = 10-2, for the esterified vitamin, and on Amax. = 326.5 mp, with r = 14 and s = 10.2, for the alcohol form. No worker appears to have considered any value for k other than 6/7. From the Morton - Stubbs curve for vitamin-A acetate we can derive numerous equations geometrically no less valid than the “6/7” ones, although, of course, there may be dispositional objections to them. Some examples are- Both refer to cydohexane solutions. k 1/(1 - k) L, mp HJ mp s I ( y + 4 y / ( y + s) 9/10 10 316 336 0.4 0.6 5 310 341 0.4194 0-5806 4 308 343 0-4286 0.5714 4/5 3/4 Morton and Stubbs remark that the “only significance of the ratio 6/7 is that it is empirically appropriate in relation to the wavelength range covered, and to the performance of the spectrophotometer.JJ3 They had principally in mind high-potency oils, to which the formula originally devised for cod-liver oil was then being applied.There was in practice a special reason for the choice of the 313-mp fixation point on the absorption curves of cod- liver oil: it seemed least likely to disturb the mathematical assumption of %point linearity on the absorption curve of the irrelevant material. Some of the curves obtained by sub- traction of the vitamin-A curve from the gross curves of cod-liver oil exhibited small but distinctive peaks at about 305 mp and 320 mp associable with conjugated tetra-ene acids, and the trough in between usually reached a minimum at 313 mp.A comparison of the results from the standard “6/7” equation with those from the equations corresponding to the sets of constants tabulated above is made later in this paper. McGillivray6 uses the correction procedure in principle, but adopts a slightly different geometry that “lends itself to a simpler calculation.” Instead of taking fixation points at which the two “shoulder” absorptions are equal (ie., a = b = k), he prefers two points equidistant in wavelength from the point of maximum absorption (k, Y = s). He chooses 310mp and 340mp for vitamin-A alcohol and the equation is- X = EM - 2.611 (EL + EH) = 2Eat325mp -2.611 (Eat310mp+Eat340mp). SOURCES OF ERROR OBSERVATIONAL ERRORS- A result that is a function of three E values will clearly have wider limits of error than it result depending on one.To solve the complex problem of how much wider the limits of error are, it must be recalled that what is normally described as “error” in spectrophotometry can be ascribed to several variables, such as the effect of temperature and solvent, and the accuracies of the optical density scale, of the make-up of the test solution, and of the cell dimensions; all these will have in common an exactly parallel influence on the three E values, so that the corrected E value, being a function of the three, will be, as far as these and simiIa.r452 GRIDGEMAN : OBSERVATIONS ON THE SPECTROPHOTOMETRIC ASSAY OF [VOl. 76 sources of variation are concerned, in error to exactly the same extent as the uncorrected value.On the contrary, the reproducibility of the d a t i v e optical densities at the three wavelengths-covered by the term “residual error” in the analysis of variance-has necessarily an inflated influence on the error of the corrected E value. This residual error, therefore, independently affects the three readings, and its inflationary influence can be calculated. Let us assume that EM, EL and EH have a common coefficient of variation e; in other words, that the standard deviations of the three values are, respectively, eE@00, eEL/100 and eEH/100. Now, if we write the correction formula as- it follows from the theory of errors that the standard deviation of X will be- X = 7 [EM - CIEL - CZEH], - If we take the constants C, and C, appropriate for vitamin-A ester in cyclohexane (equation 3, above) and if, for simplicity, we suppose that the irrelevant absorption has zero slope over the three wavelengths (b., EM - X = EL - 6X/7 = EH - 6X/7), then the coefficient of variation, which is the percentage standard deviation of X, can readily be derived.It is- This means that the coefficient of variation of a corrected E value is greater than that of the original gross E value by a factor that depends on the amount of irrelevant absorption (EM - X) present at the central wavelength, 328 mp. If, for example, the amount is 10 per cent., then EM/X is 1.1 and the factor is 9.2. Some representative evaluations are as follows- Irrelevant absorption at 328 mp, yo .. .. 6 10 16 20 This special case of equality of irrelevant absorption at the three wavelengths is in practice very representative geometrically; it is obvious that within the range of slopes of the irrelevant contribution likely to be encountered the difference between (EL - 6X/7) and (EM - X) will be very close to that between (EM - X) and (EH - 6X/7), and the corresponding differences introduced into the error equation will almost compensate. So that, as far as this point is concerned, we can assume that t.he factors given above hold for all normal circumstances. The assumption that EL, EM arid EH have equal coefficients of variation is not wholly justified; in practice EL and EH can be expected to have slightly higher coefficients; this means that the given factors may be very slightly low.It appears, then, that for every 1 per cent. of observational error in a gross reading, an error of the order of 8 to 10 per cent. is to be expected in a corrected reading. Let us now reconsider the total error of variation of an E value, made up of the residual coefficient of variation e, and the summated variation due to the factors discussed above and whose effect is not magnified by correction: let us call its coefficient d. A duplicate E value from one laboratory will have an effective coefficient of variation of 2/(# + e 2 / 2 ) . If three E values are determined and two of them are used to “correct” the third, the resultant corrected E value will have an effective coefficient of variation of d ( d 2 + e2f2/2), where f is the error factor derived as shown above and whose value is here, say, 9.Typical values for e and d are, respectively, 1-5 and 2; hence, on evaluation it is found that the expected coefficients of variation of results of the type described are 2.3 for normal, uncorrected, E values and 9-8 for corrected E values. A recent co-operative trial designed t o ascertain the error of corrected E vaIues has yielded evidence not inconsistent with this theoretical derivation.’ Error factor .. .. .. .. . . 8.7 9.2 9.7 10.4 ASSUMPTIVE ERRORS- Assumptive errors are those introduced by departures from the basic assumptions of the shapes and positions of the tWQ absorption curves, that of vitamin A and that of the irrelevant material. Let us first consider departures from the assumed characteristics of the curve for vitamin A.Suppose, to begin with, that the true position of the vitamin A is displaced, without distortion, along the wavelength scale. (Such a shift might be artificial, caused by unknown solvent effects or maladjustnient of the wavelength scale or, alternatively, natural; in thatAugust, 19511 VITAMIN A BY GEOMETRIC CORRECTION OF ABSORPTION CURVES 463 the particular esterified vitamin A in the oil might be slightly different chemically or stereo- isomerically, and therefore spectrophotometrically, from vitamin-A, acetate.) It can be shown that the resulting displacement errors are as follows- Displacement of vitamin-A curve from assumed position Corrected E a t 328 mp as percentage of true value -2 mp 98-3 -1Illp 98.7 zero 100 104.1 2mCL 108.6 It appears that the errors so introduced are small if the vitamin-A curve reaches a maximum at a wavelength one or two millimicrons less than that of the assumed maximum, but appreciable if the displacement is in the other direction. Robeson and BaxterS have evidence that suggests that the vitamin A of the common fish-liver oils is two- thirds vitamin A, and one-third neovitamin A,, the two forms being equally potent.The The presence of neovitamin A may appropriately be considered here. I 1 I12 I I I I I I I I I I I 1 I I I L w \ 1 300 328 340 Wavelength, m l (4 I I 90.5 I I I : 21.5 1 , I ‘ 1 I I 1 * & I 1 300 328 340 Wavelength, mp (c) Fig. 2. correction of its absorption curve. as shown a t (c) How a hypothetical mixture of vitamin A and “irrelevant” material responds to 3-point The 3-point correction of the curve shown a t (b) partitions it absorption curve of the neovitamin seems to be similar to that of the ordinary (all-trans) material, but shifted 3 mp further up the wavelength scale, while its absorption at A,,,.is 10 per cent. lower than that of the ordinary vitamin A at its Amax.. Suppose that a 2 to 1 mixture of the two forms, uncontaminated and saponified, is assayed according to U.S.P. XIV.6 At the fixation points the true breakdown would be- E at 310 m p E at 325 mp E at 334 mp Vitamin A . . . . . . .. 59.5 69.4 59.5 Neovitamin A . . . . .. 25-5 30-6 28.1 Totalobserved . . . . . . 85.0 100.0 87-6454 GRIDGEMAN : OBSERVATIONS ON THE SPECTROPHOTOMETRIC ASSAY OF [VOl.76 and on applying the correction formula to the observed values the assay would be 93.6 per cent. of vitamin A, instead of the true (biological) value of 100 per cent. Let us now consider “distortion,” i e . , the possibility that the fixation points of the particular sample of vitamin A under test are not quite those assumed in the fonnula. To set out all the possible kinds and degrees of distortion would be impracticable, but a repre- sentative selection is shown in Table I. TABLE I EFFECT OF FIXATION-POINT DISPLACEMENT ON E AT 328mp (CORR.) OF VITAMIN-A ACETATE IN CYCZOHEXANE “Corrected” observed values when the true value is 100 If the higher 6/7 point, assumed to be a t 338-5 mp, is actually at 336.6 337.5 338.5 339.5 340.6 106.0 11 11.2 117.0 123.3 128.8 98.2 1LO3-4 109.2 116.5 121.0 to be at 313 mp, 314 80.9 86.1 91.9 98.2 103.7 is actually a t II; 315 73.5 78.7 84.5 90.8 96.3 f A \ point, assumed 313 89.1 94.3 100 106-4 111.9 Finally, there are possible faults to be reckoned with in the assumption of effective linearity of the curve of the irrelevant material.The term “effective” is used because, as already pointed out, the assumption concerns the E values a t the fixation points only, the contour of the curve between and beyond these points being immaterial. Curvilinearity of the three E values, as of any three points in a plane, can, of course, theoretically cover great distributional variety, ‘but fortunately for the present purpose all distributions can be considered as vertical displacements of the centre point, Le., values of E a t 328 mp greater or smaller than those interpolated from E at 313 mp and E a t 338.5 mp of the irrelevant material.It can readily be shown from the fundamental formula that one unit of departure from linearity in this measure (IZ at 328 mp) will result in an error of 7 units in the estimation of E at 328 mp (net) by correction. A hypothetical example is shown in Fig. 2 and in the following- If the lower E a t ,313 mp E a t 328 mp E a t 338.6 m p Vitamin A .. .. .. . . 85.7 100.0 85.7 Irrelevant . . .. .. .. I. 8.5 12.0 9.8 Sum .. .. . . 104.2 112.0 95.6 The three E values for the irrelevant material fall on a curve, which may be considered Correction as a 1.4-unit middle-point displacement from the straight line 18.5, 13.4, 9.8.of the summated E values yields the following analysis- E at 313 m p E a t 328 mp E at 338.5 m p Vitamin A .. .. .. :I 7.2 90.1 77-2 Irrelevant . . .. .. .. 27-0 21.9 18.3 Sum. .. .. . . 104.2 112.0 95.5 in which E at 328 mp for the vitamin-A fraction is 9-9 units (k., approximately 7 x 1-4) too low. This is not, it may be mentioned, an exaggerated example: if, as in Fig. 2, the curve through the three points, E at 313 mp = 18.5, E at 328 mp = 12-0 and E at 338.5 mp = 9-8, is plotted, its shape will be found not implausible for the irrelevant material likely to occur in fish-liver oils. Yet there would be a 10 per cent. error if such a mixture were analysed by 3-point correction. ALTERNATIVE FIXATION POINTS I t is useful at this stage to consider the effect of using correction formulae based on fixation points other than the customary ones, i.e., based on values of k (in formula 2) other than 6/7.Constants for the equations corresponding to k = 9/10, 4/5 and 3/4 have already been given; to compare their corrective values to typical curves for oils containing vitamin A the equations have been applied to the absorption data of five samples described and discussed by Adamson et aZ.’ Of the seven sets of data (one from each of seven independent laboratories) three had for this purpose to be excluded, because one did not include detailed curves andAugust, 19511 456 two included curves insufficiently detailed to permit interpolation of the new fixation points. The mean results from the other four laboratories are set out in Table 11, the normal k = 6/7 values being included for comparison.VITAMIN A BY GEOMETRIC CORRECTION OF ABSORPTION CURVES TABLE 11 PERCENTAGE OF IRRELEVANT ABSORPTION AT 328 mp CALCULATED FOR (Mean values for four laboratories) VARIOUS FIXATION POINTS OF FIVE SAMPLES Sample A r \ Halibut-liver Hake-liver Cod-liver k Concentrate* oil oil oil Blend t Mean 9/10 16.2 21.9 16-7 17.8 15-3 (17-7) 17.6 10.9 14.2 10.7 12.6 8.0 (11.5) 11.3 10.5 13.3 9-3 18.9 7.2 (1 1.0) 11-8 9.5 12.4 11.8 16.6 5-9 (10.1) 10.2 617 4/5 314 Mean 11-8 16.6 16.5 12.1 8.1 * A processed high-potency fish-liver oil concentrate. t A mixture of the first four samples. The bracketed figures are the expected values calculated from The 80 values condensed into Table I1 have been analysed for variance with the results the known composition of the blend.shown in Table 111. TABLE I11 ANALYSIS OF VARIANCE OF DATA SUMMARISED IN TABLE 11 Source of variation Mean Variance Significance D.F. square ratio (at P = 0.05) Between laboratories . . .. .. 3 80.3 1-29 not significant Between samples . . .. .. 4 142.4 2.29 not significant Between formulae . . .. .. 3 188.6 19.36 significant Laboratories x samples . . .. 12 6 2 4 6.40 significant Laboratories x formulae . . .. 9 3.0 < 1 not significant Samples x formulae . . .. .. 12 16.7 1.71 not significant Interactions- Laboratories x samples x formulae 36 9.74 1 From Tables I1 and I11 the following inferences can be drawn- (i) There is no significant over-all difference between the four laboratories. (ii) No significant differences are shown between the quantities of irrelevant absorption in the five oils.Nevertheless, there is a tendency for the halibut- and cod-liver oils to give higher, and for the blend to give lower, values than the average. (iii) Laboratories differ in their relative placing of the oils: it is only when the results of all laboratories are pooled that these differences are submerged. (iv) The quantities of irrelevant absorption indicated by the four formulae differ. This is almost wholly accounted for by the high “9/10JJ corrections. But the figures for cod-liver oil can be specially treated: with k = 6/7 the yield of irrelevant absorption is markedly less than that given with the other values of k . This difference does not quite reach significance at P = 0.05, but is still noteworthy.(v) The values calculated from the absomtion curves of the blend, and those calculated from the curves of the component oils, appropriately weighted, show discrepancies. Those for k = 9/10 are least discrepant. (vi) Except perhaps for cod-liver oil, where there is some chemical evidence for the suitability of the k = 6/7 points, there is nothing on which to base a decision as to which of the four sets of fixation points is best. MECHANISM OF CORRECTION Analytically, attention can be confined to the E values at the three specified wavelengths. But, having calculated the partition, we may plot the gross absorption curve in extertso, subtract from it the vitamin-A curve and so obtain the presumptive curve of the irrelevant material.456 GRIDGEMAN OBSERVATIONS ON THE SPECTROPHOTOMETRIC ASSAY OF [VOl.76 Morton and Stubbs have published a number of such curve partitions in illustration of the results of %point correction. The irrelevant components usually slope down gradually and unevenly, froq the region of low to that of high wavelength. Sometimes, however, the slope is in the other direction and accompanied by slight peaks on the higher wavelength side: the presence of vitamin A, (Amax. = 350 ‘rip) or of anhydrovitamin A (Amax. = 350, 370 and 390mp) can often be inferred in irrelevant-component curves of this type. The Wavelength, mp (0) CONCENTRATE I O( >c w 0 L I I I l r I 250 300 350 Wavelength, rnp (c) COD i $ 1 1 1 00 - I oc s W 0 Wavelength, mp (6) HALIBUT 250 300 350 wavelength, my (d) HAKE Fig.3. Absorption curves for the four oils. Upper curves, whole oils; lower curves, “irrelevant” absorptions assumed in %point correction at fixation points shown by vertical broken lines a t wavelengths of 313, 328 and 338-5mp. partitioned curves of cod-liver oil come into a special category because of the low vitamin-A potency of the oil: this means that the ultra-violet absorption of the fatty acids is large in relation to that of the vitamin and plays a big part in shaping the irrelevantwomponent curve. In Fig. 3 the absorption curves of the four main oils analysed in the co-operative study reported by Adamson et a1.’ have been partitioned on the basis of ordinaxy (K = 6/7) 3-point correction (the fifth sample, a blend, was not included as it was too heavily weighted withAUWSt, 19611 VITAMIN A BY GEOMETRIC CORRECTION OF ABSORPTION CURVES 467 the high-potency concentrate to be illustratively useful in this context).The irrelevant components clearly resemble those described elsewhere. To understand the mechanism of the correction procedure, it is necessary first to realise that any given gross curve can be partitioned into an infinite number of pairs ranging from 20 ,\“ UJ 10 ol ’ I I 250 300 350 Wavelength, rnp. (0) CONCENTRATE 250 300 350 Wavelength, (b) HALIBUT Fig. 4. Theoretical breakdown of whole-oil curves into vitamin-A ester portions (not The curves labelled M-S are those assumed in the 3-point correction because the marked E:L& a t 328 m p of whole-oil curve = 100 The figures a t the right of the curves indicate the possible percentage of “irrelevant” shown) and “irrelevant” portions points &it 313, 328 and 338.5mp) fall on a straight line absorptions at 328 mp a certain upper limit (particular to each curve) downward.For example, a curve exhibiting E at 313 mp = 98.0, E at 328 mp = 100.0 and E at 338.5 mp = 79.7 cannot (on the basis of the standard vitamin-A acetate curve) “contain” more vitamin A than that represented by458 GRIDGEMAN : OBSERVATIONS ON THE SPECTROPHOTOMETRIC ASSAY OF [Vol. 76 E at 328 mp = 93 and E at 313 mp = E at 338.5 mp = 79.7 If this maximum is expressed as 93 per cent. of vitamin A and 7 per cent. irrelevant material, this is but the first of an infinite series of theoretical partitions of the original curve ranging down to no vitamin A and 100 per cent.of irrelevant material. Obviously, the h;t is highly improbable and perhaps the first is unlikely, but somewhere in the range is the tnie partition, and adjacent to it, and on either side, are numerous theoretical partitions that on graph paper would look equally plausible. Among them is that selected by the correcticln procedure, viz.- E at 313 m p E a t 328 mp E a t 338.5 m p Vitamin A . . . . . . .. 76.6 89.3 76.6 Irrelevant . . . . . . . . 21-4 10.7 3.1 Sum . . . . . . 98-0 100.0 79.7 whose only distinctive feature is the linearity of the three points on the lesser curve. Let us now return to the partitioned curves in Fig. 3. Any one of these lesser curves selected by the correction procedure can be set in its appropriate place in a representative selection of the companion curves whose theoretical existence is discussed above.This has been done for the concentrate and the halibut-liver oil in Fig. 4, where the arbitrariness of the selection made by the correction procedure is reflected in the similarity of the other curves in the vicinity-and even although the extremes in these sets can be rejected as constitu- tionally improbable, there remains a wide range of possibilities. Another feature emphasised in Fig. 4 is the not inconsiderable discrepancy between the spectroscopic assumption of linearity and the mathematical requirement of linearity of the fixation points, for in between these points the curve is far from smooth. If these curves are examined further, the similarity in general shape of the two sets is particularly noticeable. Roughly the same absorption peaks and troughs occur, apparently, in the curves of the irrelevant material in two oils of very different natures-one a natural oil and the other a processed concentrate.What the two oils do contain in common is vitamin A and, as the most marked irregularities of the curves of the irrelevant material lie at about the same wavelength as the irregularity near the peak of the vitamin-A curve, the possibility must be considered of some degree of error in the assumption, in the correction method, that the absorption curves of the vitamin-A fatty-acid esters in these oils are identical with the published vitamin-A acetate curve. Graphically it can be predicted that a slight shift of the vitamin-A curves along the wavelength scale would tend to smooth the comple- mentary curves of irrelevant absorption.In Fig. 5, this is done for three representative members of the family of curves of irrelevant absorption from the halibut-liver oil in Fig. 4. Not only does this trial amendment of the assumption smooth the curves, but it materially alters the general slopes. An obvious and simple explanation of the peculiar shapes of the curves as left by the original subtractions woilld be that the wavelength scale was instru- mentally in error. This is unlikely, as the curves are averages obtained from several instruments in different laboratories, all agreeing on the position of Amax.. Moreover, there is evidence that the absorption curve of the vitamin A chromatographically separated from the concentrate was positioned 2 mp further up the scale than that of ordinary vitamin-A alcohol.What could cause the postdated differences between the accepted curves and those encountered in certain-for it is not possible, o:E course, to say all-oils? First, it is at least conceivable that the acid radicals of vitamin-A esters should slightly influence the contour of the absorption curve, i.e., that the curve for vitamin-A acetate, for instance, was not the same, spectrophotometrically, as that for naturally-occurring esters. Secondly, there may be differences between the absorption curves of the four geometric isomers of vitamin A whose occurrence and characteristics have not yet been fully established.Robeson and Baxters have distinguished between two isomers that occur together in fish-liver oil, and it is significant that they appear to have absorption curves similar in shape but 3 mp out of step (vide supra). VALIDITY OF RESULTS The previous section bears on the question of the validity of results in that it deals with the assumptions fundamental to 3-point correction of vitamin-A absorption curves. What is further needed is some direct evidence, i.e., comparisons between correction estimates and estimates by physico-chemical methods. The literature is not rich in information of thisAugust, 19511 VITAMIN A BY GEOMETRIC CORRECTION OF ABSORPTION CURVES 469 kind. Morton and Stubbs have discussed three samples of tunny-liver oil with this in mind, but there is not enough range among the samples to give the evidence great strength: it can be concluded that the corrected results are indeed of the same order as the physico- chemical results, but as they are also similar among themselves we cannot be sure that other 20 s u1 10 0 s w 1 I I I I 300 3 10 320 330 340 (4 Wavelength, mp 20 I 15% I I I I 0 300 3 10 320 330 340 Wavelength, mp (4 Fig.5. The curves labelled M-S are those selected by 3-point correction Ei& at 328 mp of whole-oil curve = 100 (a) Typical trio of the family of Halibut “irrelevant” curves, re-scaled from Fig. 4 (b) Comparable curves derived after assumption of a 3-mp upward shift of the complementary vitamin-A ester curve Effect of assumed wavelength shift on shape of postulated “irrelevant” absorption curves samples containing greater or less irrelevant material would respond in proportion.No information on sensitivity therefore emerges. Data on the 3-point corrected E at 328 mp values of several fish-liver oils, a distilled ester and vitamin-A acetate have been given by Coet~ee,~ but they contribute little to the460 GRIDGEM AN [Vol. 76 present problem. They turn on biological assays against /3-carotene, so that the precision and accuraey are insufficient to show up critical differences. The apparent percentages of irrelevant absorption in the samples are 0.6 per cent. for the acetate, 3.4 per cent. for the distilled ester, and 6.6 to 12.3 per cent. for the fish-liver oils, but the bio-assays correlate almost as well with the gross as with the corrected values (if., the coefficients of variation for the gross and corrected “conversion” factors are, respectively, 11 and 9).More interesting is Morton and Stubbs’s collection of assays on cod-liver oil given in their original paper.l Each sample was assayed by correction of the whole-oil absorption curve and by determination of the E value via the unsaponifiable matter (it has long been observed that most of the irrelevant material irz cod-liver oils can be removed by saponifi- cation). Morton and Stubb’s own tabulation of their results does not make it clear whether the correlation between the two methods is good or bad. In fact it is good, as can be seen from the summary in Table IV. TABLE I’V CONDENSATION AND REARRANGEMENT OF E AT 328mp DATA ON COD-LIVER OILS FROM MORTON AND STUBBS’S TABLE 111 (A?Za&d, 1946, 71, 355) The 20 samples are grouped in five equal sets and arranged in order of percentages of E at 328 mp due to vitamin A “Irrelevant” E at 328 mp Mean E at 328 m.p (gross) 1st set of 4 samples . . . . 1.086 2nd 9 ) .. . . 0.899 3rd 19 .. . . 0.832 4th 99 .. . . 0.836 6th 99 .. . . 0.609 , by 3-point correction, Yo 9.1 14.4 20-8 23-6 42-5 1 by determination of unsaponifiable matter, 10.4 14.5 19-5 21.6 34.6 % These figures suggest that the ultra-violet absorption of cod-liver oil fatty-acids is linear at the fixation points, and therefore, that the method has some validity for this oil. Evidence of a similar kind is needed before the application of the method to other oils can be established. At present its reliability is sub judace and its use requires caution. I . 2. 3. 4. 5. 6. 7. 8. 9. REFERENCES Morton, R. A., and Stubbs, A. L., Analyst, 1946, 71, 348. -- , Ibid., 1948, 42, 195. Oser: B. L., Anal. Ckem., 1949, 21, 529, McGillivray, W. A., Ibid., 1950, 22, 494. “United States Pharmacopoeia XIV,” Mack Publishing Co., New I’ork, 1950, p. 785. Adamson, D. C. M., Elvidge, W. F., Gridgeman, IX. T., Hopkins, E. H., Stuckey, R. E., and Taylor, Robeson, C. D., and Baxter, J. G., J . Anzer. CIzen2. SOC., 1947, 69, 136. Coetzee, W. H. K., Biochem. J., 1949, 45, 628. I , Biochem. J., 1947, 41, 525. -- R. J., Autalyst, 1951, 76, 445. LEVER BROTHERS & UNILEVER LIMITED FOOD RESEARCH DEPT., COLWORTH HOUSE SHARNBROOK, BEDFORD Janzcary, 195 1

ISSN:0003-2654    

DOI:10.1039/AN9517600449

出版商:RSC    

年代:1951

数据来源: RSC

摘要:

August, 19511 REISS, HALKERSTON, BA4DRICK AND HALKERSTON 461 Assessment of Adrenocorticotrophic Hormone Activity BY M. REISS, I. D. K. HALKERSTON, F. E. BADRICK AND JEAN M. M. HALKERSTON' (Presented at the meeting oj YJze Biologicnl Methods Group on Tuesday, December 19th, 1950) -4 review of attempts to assess adrenocorticotrophic hormone (ACTH) activity biologically is given. The methods discussed include those based on change of adrenal weight and those u tilising histological changes within the adrenal cortex. Experiences with the Sayers 'adrenal ascorbic acid depletion assay are described, together with an account of pituitary blockade by desoxycorticosterone acetate (DOCA) in an attempt to improve the precision of the method. The use of phosphorus-32 as an index of adrenal function is described.Following a review of indirect assay methods based on changes due to adrenal hormones mobilised by ACTH, attempts to utilise the modification of insulin sensitivity brought about by ACTH are discussed. Some results of clinical adrenal cortex response tests are reported. The possibility of the assay of ACTH by using surviving tissue in place of the assay animal is envisaged. SINCE it was first recognised that extracts made from the pituitary anterior lobe had an adrenocorticotrophic action, there have been numerous attempts a t its qualitative and quantitative assay. The earliest investigators used the weight increase of the adrenal as a basis for the assay of adrenocorticotrophic hormone (ACTH) . Jores and Beck,l for example, used the adrenal weight of normal male mice, which are particularly sensitive ; Moon2 used the adrenal weight of 3-day old rats and later of 21-day old rats3; Bates, Riddle and Miller-4 used the adrenal weight of 2-day old chicks.Results of assays of this type, employing intact as opposed to hypophysectomised animals, should be regarded with the greatest reserve, for many factors, such as changes in environ- mental temperatures or the presence of toxic substances in the extracts used, can influence the weight of the adrenal by mobilising endogenously produced ACTH. CollipJ5 in his first attempts at standardisation, made use of hypophysectomised animals for assays based on adrenal weight change, but experiments published later from his laboratory by Neufelds showed a complete lack of quantitative correlation between dose and response.Investigations were carried out a few years ago in our laboratory on hypophysectomised male Wistar rats of 100-g body weight, using ACTH extracts prepared from cattle pituitaries. The results were disappointing, as can be seen from Table I, in which the individual weights of the right adrenals, removed 7 days after hypophysectomy, are recorded below the weights of the left adrenals removed at autopsy after a 7-day injection period. The very large scatter of the results is indicated by the standard deviation of each response; the dose- response curve in Fig. 1 constructed from the data of Table I only emphasises the unsatisfactory nature of the assay procedure. The causes of the large variation in response are at present being investigated in our laboratory and some are discussed below.Recently, Cortis- Jones, Crooke, Henly, Morris and Morris7 reported a satisfactory linear relationship between the logarithms of the doses injected and the increase in adrenal weights of hypophysectomised rats. They define one ACTH unit, arbitrarily, as an activity causing a 50 per cent. increase in the mean adrenal weight over that of the hypophysectomised control animals. Weight maintenance methods developed by Astwood and Tyslowitzs and by Sayers, White and Longg seemed to yield better results. For a number of years we used as a method of assay the restitution, produced by ACTH injection, of the sudanophobic zone of the adrenal cortex of hypophysectomised rats, onghdly described by Reiss, B a h t , Oestreicher and AronsonlO and later by Simpson, Evans and Li.I1 This assay gave a satisfactory linear relationship between the percentage of animals giving positive response (or better still, the probit of the percentage) and the logarithm462 REISS, HALKERSTON, BADRICK AND HALKERSTON : TA~LE I [Vol.76 INVESTIGATION INTO THE ASSAY OF ADRENOCORTICOTROPHIC HORMONE (ACTH) EXTRACTS WITH 100-g HYPOPHYSECTOMISED MALE WISTAR RATS Mean of individual Number Weight of adrenals, mg percentage weight Dose of of Left adren,al after treatment difference between right ACTH, animals Right adrenal before treatment and left adrenals mg f S.E. of mean 40 8 9 8 8 5 6 8 1 0 8 -z 3 14 -2 3 3-4 3 + 76 & 16.6 + 42 11.3 1 0 1 2 8 6 6 6 1 2 9 -- 8 7 z z 6 8 - 3 7 20 8 + 76 & 15.1 11 10 9 8 7 11 12 7 -3.3 s 3 z 3 - i b 10 8 6 3 7 6 6 6 6 1 0 8 8 S ; ; i 5 6 5 i i + 22 & 8.8 $- 33 & 16.0 Controls .... €4 - 22 6.9 of the dose. If one compares the original adrenal weight method illustrated in Fig. 1 with Fig. 2, showing response curves for standard and unknown ACTH preparations by the histological method, the superiority of the sudanophobic zone restitution method is evident without further calculation. With the histological method 10 to 35 rats per dose and 3 to 4 doses per preparation were required to characterise the response curves, which were found to be very reproducible 420- . # * 22 * 10- * - v) ; +loo- - / - 0 - 0 -10- 0 -30- ’ 0 -20- ,/- I I I -0 310 $0 16.0 20.0 40.0 Total dose, mg Fig.1. Relation between log-d.ose of adrenocorticotrophic hormone and change in weight of adreuals in hypophysectomised rats The dotted lines are one standard deviation from the line of best -fitAugust, 19511 ASSESSMENT .OF ACTH ACTIVITY 463 and in this respect much superior to those we have since obtained with the adrenal ascorbic acid depletion assay of Sayers and Sayers,12 to be discussed later. It is a more time-consuming method, however, requiring the maintenance of hypophysectomised animals for 7 to 11 days before assay and for a 3 to 4-day injection period, so that a considerably higher standard of hypophysectomy technique is involved. Though more consistent, it is less sensitive, requiring 50 to 100 times as much material as the adrenal ascorbic acid depletion method.Dose, mg Fig. 2. Comparison between adrenocorticotrophic hormone of unknown strength and laboratory standard by the histological method Standard - a- Unknown - o ___ Because of the changes observed in the sudanophobic zone, it might have been worth while to follow up more intensively the changes in cholesterol and steroid contents of the adrenals after ACTH treatment, described by Sayers, Sayers, White and Long13 and later by Carryett, Golla and Reiss.14 However, interest in this, and for that matter in any other form of assay, was overshadowed by the introduction of the adrenal ascorbic acid depletion assay by Sayers and Sayers.12 This method makes it possible to obtain results more quickly, with much less material and with comparatively less labour than does, for instance, the sudanophobic zone restitution assay.It has therefore been of great help in the development of methods for preparing ACTH concentrates. It is carried out as a routine method in our laboratory essentially as described by Sayers, Sayers and Woodbury,15 with a few alterations. The use of ether anaesthesia throughout, the choice of the femoral vein as the injection site, a personal preference for the right adrenal as the control and the use of an electrotitrimetric method for ascorbic acid determinations are the only important differences between our technique and that of the originators. Table I1 summarises the results of some of the assays carried out in our department by this method. I t shows the number of rats used, the slopes of the response curves for standard and unknown, the value of 2 (the test for heterogeneity between the two lines), the standard deviation of the assay and the standard error of M (the logarithm of the ratio of potencies) for P = 0.95 and the approximate limits of error calculated from the standard error of M.The first three entries refer to separate one-day (3 + 3) assays comparing the same preparations. The fourth entry gives the evaluation of the combined results of the three assays, which does not show a significant improvement in precision over that of the individual assays. On the other hand the assay carried out on September 27th with only 30 animals gave a result of far greater precision. The irregular nature of the response curves indicated by Table I1 caused much misgiving when we first began to use this method of assay. It appeared that several hundreds of ’ animals would be required to obtain satisfactory limits of error.464 REISS, HALKERSTON, BADRICK AND HALKERSTON (Vol.76 The occurrence every now and then of an assay completely in agreement with results published by Sayers, Sayers and Woodbury,lS by Morris and his co-w~rkers,~ by Prunty16 and by other authors, in that reasonable limits of error were obtained with approximately 30 animals, led us to investigate as fully as possible the causes for the apparently chance variation of our results. Over the question of the suitability or otherwise of the strain of Wistar rats bred in our colony we received considerable help, obtaining, through the courtesy of Dr.Morris, Wistar rats of his own strain. We hope to breed from these animals and compare the results with those obtained on our own rats, which are partly derived from the original Glaxo strain and partly from the strain bred by Dr. Paterson in Porton. That the solution lies solely in the use of a different strain of rats seems unlikely, for it does not explain why our own animals sometimes respond as satisfactorily as those of other workers. Date of assay 2 1.4.50 26.4.50 5.5.50 21.4.50 26.4.50 6.5.50 I- ; ;: : ::} 17.5.50 27.9.50 9.11.50 14.11.60 8.12.60 7.6.50 TABLE I1 SUMMARY OF RESULTS OF SOME ASSAYS OF ACTH BY THE ADRENAL ASCORBIC ACID DEPIXTION METHOD Kumber of rats 44 40 47 131 78 16 30 23 25 26 9 Slupe of log-dose - response curves Standard deviation Standard Approximate limits of 7- of the Error of.M* Standard Unknown X2 assay (P = 0.95) 58.2 63.1 0.059 2 26.7 0.2634 91.3 95.6 0.037 2 33-1 0.2300 144.3 117.4 3.610 f 31.0 0.1355 104.5 85.2 3.210 5 32-3 0.1331 101.0 79.1 2.0 & 24.9 0.1701 149.0 139.0 0.103 & 22.0 0.1510 116.3 114.3 0.0'77 13.7 0,085 1 125.5 142.8 0.049 k 22.6 0.1448 84.25 86.25 0.008 & 25.5 0.2455 116.4 78.1 0.100 * 22.2 0.2028 Log-dose - response curve for 60-g hypophysectomised rats & 34.9 s / b = 0.280 124.6 - -- * M is the logarithm of the ratio of potencies. error, % 2 64 + 56 z 32 - + 31 & 40 & 36.5 19.5 * 34 & 48.5 & 54-45 It would appear more likely that the variation is due to the fact that no other anterior pituitary hormone can be so readily mobilised endogenously as ACTH.The ascorbic acid depletion index shows how even small manipulations on an intact animal, e.g., anaesthesia, injection of test material or even changes in environmental temperature, can bring about a mobilisation of ACTH with a consequent fall in adrenal ascorbic acid concentration. It is obvious that such stresses as fights with other rats and draughts in the animal house can occur in the life of a growing rat, mobilising temporarily increased amounts of endogenous ACTH with its prevailing effect upon the adrenal cortex. This might be the sole explanation for the variation in adrenal weight per unit body weight found among the members of a large rat colony. The point is important, because the dose injected is calculated per 100 g of body weight and may therefore be called upon to influence adrenals of very different weights.Obviously the younger the rat the fewer the occasions for irreversible changes in the adrenal cortex. This consideration, together with the observation that it is easier to induce weight changes in the adrenals of younger than of older rats, led us to use 60-g hypophysectomised rats for assay purposes ; the result of one rather promising experiment is included in Table 11. The precision obtained is comparable with that of most of the assays on older animals, although the scatter of the responses is still unsatisfactory. It would be preferable to use even younger rats, but in a 40-g animal the stress of hypophysectomy is very much greater than in older ones. For this reason we studied the known effect of desoxycorticosterone acetate (DOCA) in blocking the pituitary function of ACTH mobilisa- tion, with a view to using animals pre-treated in this way for assay purposes.August, 19511 ASSESSMENT OF ACTH ACTIVITY 465 In Table I11 is shown the effect of injecting DOCA into 40-g rats on the endogenous production of ACTH by the pituitary, following stress.The stress applied consisted in anaesthesia with ether and the rapid removal of one adrenal. One hour later the animals were killed, the second adrenal was removed and the concentrations of ascorbic acid in the adrenals were compared. TABLE I11 EFFECT OF DESOXYCORTICOSTERONE ACETATE (DOCA) ON THE ENDOGENOUS PRODUCTION OF ACTH BY THE PITUITARY, FOLLOWING STRESS Treatment A r \ Material injected Time before and dose stress, hours 18 18 18 18 No injection - 1 ml of arachis oil 1 mg of DOCA in 1 ml of oil 10 mg of DOCA in 1 ml of oil 10 mg of DOCA in 1 ml of oil Concentration of Number ascorbic acid in of rats right adrenal, 5 343 & 38-1 4 385 & 954 4 337 & 18.4 6 386 & 50.5 4 385 2 50.2 mg per 100 g, & S.D.Mean difference in ascorbic acid concentration between right and left adrenals, mgper lOOg, f S.D. - 73 rf: 23.4 - 63 & 26.2 - 94 f 4.2 - 6 f 10.9 + 62 & 39.9 The response to the stress, in the un-pre-treated control group, is indicated by the considerable drop in concentration of ascorbic acid in the second adrenal. One millilitre of arachis oil or 1 mg of DOCA in 1 ml of arachis oil injected 18 hours before stress did not change this reaction.However, 10mg of DOCA given 18 hours before stress completely prevented depletion of ascorbic acid. It may be pointed out that it is necessary to treat with DOCA at least 18 hours before assay, as the injection is itself a stress: 4 hours after such an injection the concentration of ascorbic acid in the adrenal is still considerably reduced, so that no further decrease can be achieved by a further stress. Only after 18 hours at the earliest is the ascorbic acid concentration sufficiently high to allow the effect of another stress to be recorded efficiently. Table IV shows the reduction of the adrenal ascorbic acid by different doses of ACTH in 40-g male Wistar rats pre-treated with 10 mg of DOCA 18 hours before assay. The scatter of results is still considerable, and we are investigating the possi- bility of improving the technique of pituitary block. TABLE IV REDUCTION OF ASCORBIC ACID IN THE ADRENALS BY DIFFERENT DOSES OF ACTH INJECTED INTO 4O-g RATS PRE-TREATED BY INJECTION OF 10 mg OF DOCA 18 HOURS BEFORE ASSAY Difference between amount and left adrenal, mg per 100 g, f S.D.Dose of ACTH, Number of ascorbic acid in right Log-dose - response curve pg per 100 g of body weight of rats ch axac teris tics 1.0 3 41 & 29.4 Slope ( b ) = 60.3 2.0 4 51 1- 30.7 Standard deviation 4.0 6 76 & 20-0 Index of precision The difficulties encountered in the adrenal ascorbic acid depletion assay will be found in any assay method with rats, owing to the extreme ease in mobilising endogenous ACTH during the animal’s lifetime.It is in our experience particularly true of adrenal repair tests or those based on hypertrophy of the adrenals, when a further complication occurs because the glands are continuously atrophying during the three weeks following hypophysectomy and the weight increases achieved after treatment are at the best a result of the action of the several factors concerned. It might therefore be advantageous instead to investigate the biochemical changes that are the primary basis of the growth phenomenon. Previous investigations by Reissl7 and by Reiss, Druckery and FischP8 on the ovary showed up to 100 per cent. increases in oxygen consumption and glycolysis 1 hour after injection of gonadotrophic hormone, long before any growth could be seen.The adrenal cortexlg also shows similar increases in oxygen consumption and glycolytic processes soon after injection of ACTH. The methods used in such investigations are too cumbersome for application to routine assay procedure, and the (s) = 26.16 (s/b) = 0.433466 REISS, HALKERSTON, BADRICK AND HALKERSTON : [Vol. 76 dose - response relationships are not satisfactory. It is therefore simpler to investigate phosphorylation processes in the adrenal cortex, particularly as Gemzel120 has shown that ACTH increases phosphorylation rate in the adrenal. Changes in the uptake of phosphorus-32, soluble in trichloro-acetic acid, by the adrenal, as an arbitrary index of phosphorylation rate, were investigated in numerous experiments by Reiss and Halkerston.21 The effect of endogenously mobilised ACTH was studied in animals exposed to cold, and increases of several hundred per cent.in uptake of phosphorus-32 by the adrenals were recorded after exposure for 1 hour. This change was not shown by hypophysectomised animals. It was also interesting to note that new-born animals at the age of three and eight days are already able to mclbilise considerable amounts of endogenous ACTH. Table V shows that even the removal of litter mates from the warm environment of the mother and leaving them at room temperature resulted in a considerable increase in the uptake of phosphorous-32 by the adrenals. Removal to a cool room still further enhanced this reaction. These changes in the uptake of phosphorus-32 by the adrenal parallel the ascorbic acid depletion known to occur under similar conditions.TABLE 1' EFFECT OF ENVIRONMENT ON ADRENAL UPTAKE OF PHOSPHORUS New-born rats, litter mates, injected with 6 pc of phosphorus-32 intraperitoneally and killed 60 to 80 minutes later. Trichloro-acetic acid extracts of adrenals measured in a liquid counter. Impulses per Litter number Rat number Treatment 100 mg of adrenal 1 (3 days old) 1 Left with mother 550 2 Kept 1 hour at room temperature 850 3 Kept 1 hoiir in cold room 1165 2 (8 days old) 1 Left with mother 465 2 Left with mother 455 3 Kept 1 hour at room temperature 660 4 Kept 1 hour in cold room 830 6 Kept 1 hoiir in cold room 1940 Recently we have taken advantage of the fact that during routine use of the ascorbic acid depletion assay it is possible to introduce by intravenous injection, after the removal of the control adrenal, a dose of phosphorus-32 together with the ACTH test solution.An aliquot of the metaphosphoric acid filtrate from the left adrenal contains a greater amount of phosphorus-32 than that of control animals, and Table VI shows that promising dose - response relationships are obtained between the percentage of the phosphorus-32 taken up by the adrenal and the dose of ACTH injected. TABLE VI DOSE - RESPONSE RELATIONSHIP BETWEEN PHOSPHORUS-32 TAKEN U P BY . ADRENALS AND DOSE OF ACTH INJECTED Standard preparation of ACTH Unknown preparation of ACTH A A f \ f Percentage of dose Percentage of dose Dose in pg per Number of taken up by Dose in pg per Number of 32P taken up by 100 g of body weight of rats left adrenal 100 g of body weight of rats left adrenal 0-5 5 0.049 0.75 4 0.061 0-25 6 0.03 7 0.375 5 0.052 1.0 5 0.054 0-5 6 0.04 1 0-25 2 0.03 1 1.0 0.5 0.25 4 5 7 0.058 0.0475 0.0435 Such changes in oxygen consumption and uptake of phosphorus-32 are by no means confined to the adrenal cortex.The turnover rate for phosphorus-32 is increased in most growing tissue, particularly tumours. Similarly, changes in the ascorbic acid concentration of the adrenal need not necessarily be connected with the endocrine function of the adrenal cortex; one should therefore be somewhat reluctant to use this index as a basis for the biologicalAugust, 19511 ASSESSMENT O F ACTH ACTIVITY 467 assay of preparations having adrenocorticotrophic action. The use of the adrenal ascorbic acid depletion assay before the mechanism of the depletion is known reminds one of previous attempts to standardise the hormonal activity of ‘pituitary posterior lobe extracts, prepared for their clinical pressor, oxytocic and antidiuretic activities, by their action on the melano- phores of frogs.Indirect tests, based on the quantitative action of adrenocortical hormones mobilised by exogenous doses of ACTH, might therefore be more satisfactory from the point of view of the physiologist and the clinician. Several workers have shown that animals may become more sensitive to certain injurious factors after hypophysectomy and have indicated the possibility of increasing their resistance by treatment with ACTH or adrenocortical hormones. Tyslowitz and Astwood,22 for instance, reported the effect of ACTH on the resistance of hypophysectomised rats to low environmental temperatures and later reportedB the increased resistance of these animals to low environmental pressures after treatment with ACTH.Li, Simpson and EvansM described the action of ACTH in increasing resistance to cold, starvation and anoxia, while Reiss, MacLeod and Golla? showed that rats treated with ACTH exhibit increased resistance to secondary shock symptoms (rapid fall in oxygen con- sumption and body temperature, rise of blood haematocrit value and fall in blood sodium) induced by intraperitoneal injections of hypertonic glucose solution. Recently, Buttle26 reported on the increased resistance of guinea-pigs to histamine after ACTH administration, and Pruntyl6 has based an assay on the antagonistic effect of ACTH to wound-healing in mice.All of these actions can, of course, be used as a basis for quantitative bio-assay as soon as standardised conditions can be worked out. Recently we have investigated the effect of ACTH in increasing the resistance of mice to insulin. Table VII shows the results of two such experiments where groups of about TABLE VII EFFECT OF ACTH ON SENSITIVITY OF MICE TO INSULIN Dose of ACTH, given 1 hour before injection mg per animal Percentage of mice reacting 2 hours after injection of of insulin, Number of mice insulin (a) 0-2 units of insulin injected subcutaneously- nil (controls) 20 0.1 19 0.4 20 1.0 20 (b) 0.045 units of insulin injected subcutaneousIy- nil (controls) 30 0.1 18 1.0 22 65 32 25 20 93 39 4.5 20 mice were injected with ACTH 1 hour before insulin administration.In the first experi- ment the occurrence of insulin coma or death 2 hours after 0.2 units of insulin was taken as a criterion of insulin action, while in the second experiment the criterion was the percentage of animals showing at least paralysis of the hind limbs 2 hours after a lower insulin dose. In both experiments the degree of protection increased with dose. The results appear encouraging, particularly since it should be possible for all laboratories with well-established facilities for mouse insulin tests to apply them to the bio-assay for ACTH. The method could doubtless be considerably improved in precision and sensitivity by pre-treatment of the animals with DOCA, which increases the insulin sensitivity considerably by blockage of the pituitary anterior lobe.A discussion of the assessment of ACTH activity would not be complete without reference to the response of human subjects to ACTH administration. Specific response tests for adrenocortical and adrenocorticotrophic efficiency worked out by Forsham, Thorn , Prunty and Hills,27 Pincus28 and other authors are becoming more and more a routine procedure in pathological laboratories. It is at once apparent when reviewing the different indirect methods of assay that different adrenocortical products are concerned in such effects as resistance to cold, low atmospheric pressure, starvation, insulin and secondary shock .symptoms. This becomes still more obvious with the responses of different human subjects to a single injection of ACTH ;468 REISS, HALKERSTON, BADRICK.AND HALKERSTON [Vol. 76 even if the response tests are restricted to three secondary actions, vix., decrease in circulating eosinophils, increase in uric acid - creatinine ratio and increase in 17-ketosteroid excretion after ACTH administration, very different reaction types can be found among human subjects. In a large proportion of the tests the results show an eosinophil count decreased by at least TABLE VIII RESPONSE OF OVER 400 PATIENTS TO INJECTION OF DOSES OF ACTH AND TO STIMULATIOK OF ENDOGENOUS ACTH BY INJECTION OF GLUCOSE AND BY ELECTRIC SHOCK TREATMENT Response measured by A 7- 7 Circulating Uric acid - 17-Ketosteroid Treatment eosinophils, creatinine ratio, excretion, 0 ’ % % /O Glucose injection .. .. .. 41 34 9 Low ACTH dose, 15 to 26 mg . . 76 58 18 High ACTH dose, 50 mg . . .. 95 62 86 Electric shock . . .. .. .. 100 85 35 30 per cent., an increase in uric acid - creatinine ratio by at least 25 per cent. and an hourly 17-ketosteroid excretion increased by at least 20 per cent. In other tests, however, only the eosinophil count falls, while the other two indices do not alter. Other cases show an un- changed eosinophil count, one or both of the other components showing significant increases. The fact that an increase in the ACTH dose produces a full response, when the low dose failed to do so, suggests that different independent mechanisms are concerned, some of which are TABLE IX INDIVIDUAL RESPONSES OF PATIENTS TO INJECTIOle OF DOSES OF ACTH -4ND TO STIMULATION OF ENDOGENOUS ACTH BY ADMINISTRATION OF GLUCOSE AND BY ELECTRIC SHOCK TREATMENT Patient 1 2 3 4 5 6 7 8 9 10 11 Patient 1 2 3 4 5 6 7 8 9 10 11 Age 25 42 26 28 30 32 42 40 26 40 46 Age 25 42 26 28 30 32 42 40 26 40 46 Response to small dose of ACTH (15 to 26 mg) A I 7 Uric acid - 17-Keto- creatinine steroid Eosinophils ratio excretion + 0 0 0 + 0 0 0 + + -t + + + + + -t + 0 + + + + + 0 + + 0 0 + 0 0 Response to glucose (150 g administered orally) I A 7 Uric acid - 17-Keto- creatinine steroid Eosinophils ratio excretion + 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 + 0 0 + 0 + 0 + + 0 + Response to higher dose of ACTH (50 mg) r > Uric acid - 17-Keto- creatinine steroid Eosinophils ratio excretion + 0 0 + + 0 + -+ + I - + 0 + 4- 0 Response to electric shock treatment Eosinophils 1- + + + + + + + + + + Uric acid - creatinine ratio + + + 0 + + + 0 + + 0 3 17-Keto- steroid excretion 4- + 0 0 0 0 0 0 0 0 +August, 19511 ASSESSMENT OF ACTH ACTIVITY 469 disturbed under pathological conditions.The threshold becomes increased and, therefore, the particular response is absent at low dose levels. In Table VIII are shown the results of response tests performed on over 400 patients29 each of whom received in turn 25 mg of ACTH, 50 to 100 mg of ACTH, then, as mobilisers of endogenous ACTH, 150 g of glucose by injection, and finally electric shock treatment. From this table the general pattern of the response would appear to be an enhanced reaction with increase in the strength of stimulus.That this is only true for the low and high ACTH dose is shown by the individual responses set out in Table IX. Patients 1 to 3, for example, react according to the over-all picture in Table VIII, while patients 4, 5, 6 and 8 show a complete response to even the low ACTH dose, though not to the greater stimulus of electric shock. In other patients it would appear that different adrenal cortex responses are evoked by the different stimuli. Many of the results are difficult to explain and the whole picture remains obscure, but it seems difficult to avoid the conclusion that there are different adrenocorticotrophic components. A review of all the bio-assays and possibilities of bio-assay for ACTH emphasises the shortcomings of purely biological methods. It would seem that it will not be possible to overcome all the inherent difficulties of assays based on living animals with their individual demands for adaptation.This demand is a prominent feature of the whole life of the animal before being taken into the assay experiment and is concomitant with the mobilisation of adrenal cortex hormones. In other words, it would be a great advantage if one could dispense with the assay animal altogether and investigate the influence of ACTH and different ACTH fractions on adrenal cortex tissue in vitro. As a step in this direction we are trying to determine the amount of “cortin-like” substances (i.e., neutral lipoid-soluble reducing substances) produced by adrenal cortex tissue in vitro and to find how this production can be influenced by ACTH added to the medium.The results obtained so far have proved encouraging and we shall report more fully when the experimental conditions have been stabilised. 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26. 27. 28. 29. REFERENCES Jores, A., and Beck, H., 2. ges. ex$. Med., 1936, 97, 622. Moon, H. D.. Proc. SOC. Exp. Biol. Med., 1937, 35, 649. Bates, R. W., Riddle, O., and Miller, R. A., Endocvinology, 1940, 27, 781. Collip, J. B., J . Mount Sinai Hosp., 1934, 1, 28. Neufeld, A. H., Proc. Soc. Exp. Biol. Med., 1943, 54, 91. Cortis-Jones, B., Crooke, A. C., Henly, A. A., Morris, P., and Morris, C. J. 0. R., Biochem. J., Astwood, E. B., and Tyslowitz, R., Fed. Proc., 1942, 1, 4. Sayers, G., White, A., and Long, C.N. H., J . Biol. Chem., 1943, 149, 426. Reiss, M., Balint, J., Oestreicher, F., and Aronson, V., Endokrinologie, 1936, 18, 1. Simpson, M. E., Evans, H. M., and Li, C. H., Endocrinology, 1943, 33, 261. Sayers, G., and Sayers, M. A., Fed. Pvoc., 1946, 5, 200. Sayers, G., Sayers, M. A., White, A., and Long, C. N. H., PYOC. SOG. Ex?. Biol. Med., 1943, 52, Carreyett, R., Golla, Y. M. L., and Reiss, M., J . Physiol., 1945, 104, 106. Sayers, G., Sayers, M. A., and Woodbury, L. A., Endocrinology, 1948, 42, 379. Prunty, F. T. G., “Proceedings of the Meeting of the Society of Endocrinology,” Oct. 25th, 1960, Reiss, M., Med. Klin., 1932, 28, 992. Reiss, M., Druckney, H., and Fischl, F., Endocrinology, 1932, 10, 241. Carpenter, R. K., MacLeod, L. D., and Reiss, M., J . Physiol., 1946, 105, 231. Gemzell, C. A., Acta Endocrinol., 1948, 1, Supplementum 1. Reiss, M., and Halkerston, J. M. M., J . Endocrinol., 1950, 6, 369. Tyslowitz, R., and Astwood, E. B., Amer. J . Physiol., 1941, 133, 472. -- , Ibid., 1942, 136, 22. Li, C. H., Simpson, M. E., and Evans, H. M., Science, 1942, 96, 450. Reiss, M., MacLeod, L. D., and Golla, Y . M. L., J . EndocrinoZ., 1943, 3, 292. Buttle, G. A. H., “Proceedings of the Meeting of the Society of Endocrinology,” Oct. 25th. 1960. Forsham, P. H., Thorn, G. W., Prunty, F. T. G., and Hills, A. G., J . Clin. Endocrinol., 1948,8, 16. Pincus, G., Psychosom. Med., 1949, 2, 74. Hemphill, R. E., and Reiss, M., “Proceedings of International Congress of Psychiatry,” Pans, -, Ibid., 1940, 43, 42. 1950, 46, 173. 200. in the Press. 1950, in the Press. BIOCHEMICAL AND ENDOCRINOLOGICAL RESEARCH DEPARTMENT BRISTOL MENTAL HOSPITALS FISHPONDS, BRISTOL470 MORRIS : ADRENAL ASCURBK: ACID DEPLETION AND ADRENAL DISCUSSION [Vol. 76 DR. W. PERRY said that variations introduced by stress could be reduced by animal training. He thought i t might be possible to use non-hypophysectomised animals for assay purposes on a basis of the modification of their immunity responses. DR. L. J. HARRIS enquired whether the electrometric; method used by the authors for the estimation of ascorbic acid was a photo-electric or a potentiometric method and, if the latter, the nature of the electrode used. DRS. HALKERSTON and h r s s said, in reply, that they had not tried any of the possibilities suggested by Dr. Perry, but they did not think that any advantage could be gained by their use. The method used or ascorbic acid estimation was potentiometric, with clean platinum electrodes and a small potential applied across them for a dead-stop end-point. The oxidising agent was 2 :6-dichlorophenolindophenol

ISSN:0003-2654    

DOI:10.1039/AN9517600461

出版商:RSC    

年代:1951

数据来源: RSC

摘要:

470 MORRIS : ADRENAL ASCURBK: ACID DEPLETION AND ADRENAL [Vol. 76 The Adrenal Ascorbic Acid Depletion and Adrenal Repair Methods for the Bio-Assay of Adrenocorticotrophic Hormone BY C. J. 0. R. MORRIS (Presented at the meeting of the Biological Methods Group on Tuesday, October 24th., 1950) The methods for the bio-assay of adrenocorticotrophic hormone (ACTH) are discussed. An example of the adrenal repair methods is included in the form of a detailed description of the technique used in the author’s laboratory on male Wistar rats, 35 to 42 days old. Methods that use histological evidence of repair as the criterion of ACTH activity are discussed; these are much more sensitive than those involving adrenal weight. The adrenal ascorbic acid depletion m.ethod is described and discussed, and modifications are mentioned.THE first methods to be used for the bio-assay of the pituitary adrenocorticotrophic hormone (ACTH) were based on increase in size of the adrenals after subcutaneous injection of the hormone. As the technique of hypophysectomy in the rat became more generally practised, it was soon realised that removal of the experimental animal’s endogenous supply of ACTH was essential for reliable assay. This point cannot be too strongly stressed. The use of immature normal animals for ACTH assay by maay workers has led to the publication of much erroneous information. Investigation in our own laboratory has indicated that, even under the most carefully controlled conditions, reliable results cannot be obtained on the intact animal.The point also arises in the assessment of the effect of ACTH on the human subject and must always be borne in mind in such experiments. ,IDRENAL REPAIR METHODS- The technique of the adrenal repair methods for the assay of ACTH on the hypo- physectomised rat is broadly as follows. The animals are hypophysectomised at 5 to 6 weeks old and a period of 7 to 14 days is allowed for the adrenals to atrophy. The test material is then injected, usually twice daily for 3 days, and the animals are killed on the day following the last injection. Adrenals and testes are dissected free of extraneous tissue and weighed. The weight of the testes provides a criterion of completeness of hypophysectomy, although it must be remembered that, in the assay of crude preparations, the presence of gonadotrophic hormones may lead to an increase in weight of the testes.As an example the technique as practised in our laboratory will be described in detail. Nale Wistar rats, 35 to 42 days old, are used. The animals are bred in our own colony, a s it appears that strain and uniform maintenance conditions from birth are essential for repro- ducible results. In particular, adequate nutritional status is most important. Animals of this age and strain should weigh 90 to 110 g, and rats outside this range should not be used. The animals are hypophysectomised by the parapharyngeal approach. They are kept forAugust, 19511 REPAIR METHODS FOR BIO-ASSAY OF ACTH 47 I 10 to 14 days in a ventilated enclosure thermostatically maintained at 25" C. The test material is injected subcutaneously in a volume of 0.5 to 2.0 ml twice dairy for 3 days.Groups of 4 or 5 animals per dose level are used. A similar group may be injected with saline as a control. The animals are killed on the fourth day, and the adrenals are carefully dissected free of extraneous tissue, quickly dried with filter-paper and weighed to 0.1 mg. The testes are also dissected, dried and weighed to 6mg. Under our conditions a combined testis weight of less than 600mg is taken as the criterion of completeness of hypophysectomy. The adrenal weight is expressed as milligrams per 1oOg of body weight a t the time of the * - 1 0 0.2 0.4 0.6 0 8 1-0 Log 10 xdore. p~ Fig. 2. Assay of adrenocorticotrophic hormone by adrenal ascorbic acid depletion method first injection.An arbitrary unit has been taken as equivalent to a 50 per cent. increase in mean adrenal weight over that of the controls. Results for a typicz.1 assay are shown in Fig. 1. It will be seen that there is a satisfactory linear relation between log-dose and biological response. The preparation used was slightly more active than Armour Standard La-1-a ( x 1-18), so that 1 mg of such a preparation gives a response of 0-4 unit. The useful range of the method is between 1 and 4 units (P < 0.05). It will be seen that under carefully controlled conditions the method is at least as accurate as any other and has the advantage of simplicity. It is, however, rather insensitive and requires 4 days, compared with 1 day for the adrenal ascorbic acid depletion method.Various modifications of the adrenal repair method have been suggested. Collipl used repair of the remaining adrenal in the hypophysectomised unilaterally adrenalectomised rat, the animal serving as its own control. In view of the marked difference in size between the adrenals of a rat, this procedure has little to recommend it. Sayers, White and Long2 used three daily intraperitoneal injections of test material. Comparison of this method with our own of two subcutaneous injections revealed little difference. METHODS MAKING USE OF VISTOLOGICAL EVIDENCE OF REPAIR- In a somewhat different category are the methods that use histological evidence of repair as the criterion of activity. Reiss, Balint, Oestreicher and AronsonS first described a dis- appearance of lipids from the greater part of the adrenal cortex following hypophysectomy.The remaining lipids in the zona glomerulosa become irregular in distribution. Simpson,MORRIS : ADRENAL ASCORBIC ACID DEPLETION AND ADRENAL [Vol. 76 472 Evans and Li4 based an assay method on the ability of ACTH preparations to reverse these changes. A total of 25 pg of a preparation roughly equivalent to Armour Standard injected intraperitoneally, 6.25 pg once daily for 4 days, was sufficient to produce the first detectable signs of repair. The assay is thus much more sensitive than the methods making use of adrenal weight. In our own experience the method is somewhat difficult to carry out, owing to lack of sharpness at the end-point, and in any event, in common with most “all or none” bio-assays, it requires a large number of animals for precision.Here the test material is injected for a period of 14 days, beginning the day after hypophysectomy. The unit is defined as the least quantity of the preparation sufficient to maintain the adrenals at a weight equal to those of normal controls. A total dose of 350pg of a preparation about equal to Armour Standard is adequate. The method is thus roughly ten times as sensitive as the repair test, but requires a large number of animals. It has been described in detail by Sayers, White and Long2 and by Simpson, Evans and Li4; we have ourselves had no experience with it. Another method of this type is the adrenal maintenance method. ADRENAL ASCORBIC ACID DEPLETION METHOD- The method of ACTH assay most generally used at present is the adrenal ascorbic acid depletion method.This is based on the observation of Sayers, Sayers, Liang and Long5 that a single injection of ACTH causes a rapid fall in the ascorbic acid and cholesterol contents of the adrenals. This process is now interpreted as being the first stage in the synthesis of adrenocortical hormones, for it is soon followed by the increase in liver glycogen typical of treatment with certain of these steroids. As this mechanism is set in action by any stress stimulus, it is essential to hypophysectomise the test animal. Long et aL6 showed, however, that the response was diminished and finally disappeared a few days after hypophysectomy, and it is now customary to use the animals the clay after operation.Sayers and Sayers6 and later Sayers, Sayers and Woodbury’ developed the method into an extremely sensitive, rapid and specific bio-assay method for ACTH. We use rats of 90 to 110 g body weight. It appears that in this method the strain is extremely important, and many strains are entirely unsuitable owing to insensitivity and irregularity of response. The literature shows that some of the standard curves quoted have a very low slope, and the common strains of rat appear to fall into two groups in this respect, We have used our own albino Wistar strain and are very fortunate in having a type of rat of extremely high sensitivity. The animals are hypophysectomised by the usual method; as evidence of completeness of hypo- physectomy is difficult to obtain, care should be taken to use only those animals in which the operator is satisfied that removal is complete.The method thus requires a high standard of operative technique. The following day the animal’s left adrenal is removed under anaesthesia, dissected free of extraneous tissue, dried, weighed to 0.1 mg and immediately homogenised in a modified Potter - Elvehjem homcgeniser with the solvent used for extrac- tion. The test material is then injected intravenously in the proportion of 0-2 ml per 100 g of body weight. Sayers, Sayers and Woodbury7 used the tail vein, but we have preferred the jugular vein for this purpose. Exactly one hour after injection, the second adrenal is removed under anaesthesia, dissected, dried, weighed and homogenised as before. The animal is killed and the sella turcica examined under a low power microscope for remnants of the pituitary.Sayers, Sayers and Woodbury7 used pentobarbital, but we have had most unsatisfactory experience with this anaesthetic and now use ether exclusively. Some American workers have had a similar experience. This effect also appears to be related to the strain of animal. Analysis of the adrenals for ascorbic acid can be carried out by any of the common methods. Sayers, Sayers and Woodbury7 used the Roe-Kuether methods but we have used a modified indophenol decolorisation m e t h ~ d . ~ In this method the decolorisation of a buffered 2 :6-dichlorophenol- indophenol solution is measured photo-electrically exactly 30 seconds after addition of the test solution. It is at least as precise as the Roe - Kuether method and is probably more reliable.It is in routine use by Armour Laboratories, who have probably carried out more assays by the Sayers method than any other group. The adrenal ascorbic acid value is expressed in pg per 100mg of adrenal tissue. Typical results for an assay of a sample of unknown potency against Armour Standard La-1-a are shown in Fig. 2. These data give a log-potency ratio of 0.07, a standard error of the mean of 0.066 and a combined standard deviation of 19.7. At the usual significance level of 1 in 20 this The choice of anaesthetic is of importance. It is also much quicker.August, 19511 REPAIR METHODS FOR BIO-ASSAY OF ACTH 473 corresponds to an accuracy of 3.16 per cent. for the use of 28 animals. This is well within the limits of precision quoted by Sayers, Sayers and W~odbury.~ By suitable choice of strain of animal and meticulous experimental technique this precision is always possible, despite reports to the contrary that have appeared in the literature.However, 2 to 3 per cent. of the animals give completely anomalous values, which can easily be discerned. This effect can also be found in the adrenal repair assay. The adrenal ascorbic acid depletion assay is thus about 2000 times as sensitive as the repair assay, while precision and specificity are probably comparable. The form?r is also very much quicker, but requires more labour and the most meticulous attention to detail. The high sensitivity may in some ways be a drawback, as the extremely dilute test solutions may show the instability characteristic of ACTH in neutral solutions.In routine work we make up our solutions for injection in 0.02 N acetic acid, which is tolerated well on intravenous injection and prevents adsorption and other inactivation effects. Some modifications of the Sayers, Sayers and Woodbury7 technique have been suggested. Munson, Barry and KochlO do not perform the unilateral adrenalectomy before injection but compare the mean total adrenal ascorbic acid in an injected group with the mean of a control group. Sayers, Sayers and Woodbury7 made a statistical comparison of both methods by using the same experimental data and right-adrenal ascorbic acid values as Munson responses so that identical statistical treatment could be applied to both. They concluded that, to attain the same precision, twice as many animals were required for the Munson method as for the difference method.The Munson method decreases the number of analyses to be performed. It is used in routine work by Amour Laboratories. Stoerk, Porter and Silberll have developed a modified Sayers assay in which normal rats are used, the pituitary being blocked by prior administration of adrenal cortical extract. No details are available of this method, but it is understood that this group is now using the usual Sayers assay. This paper is an attempt to give an account of the chief methods for the assay of ACTH. Only two seem to be of practical value, the adrenal ascorbic acid depletion method and adrenal repair method. The choice between these must depend on the user’s special requirements. REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. Collip, J. B., J . Mount S i n a i Hosp., 1934, 1, 28. Sayers, G., White, A., and Long, C. N. H., J . Biol. Chem., 1943, 149, 425. Reiss, M., B a h t , J., Oestreicher, F., and Aronson, V., Endokrinologie, 1936, 18, 1. Simpson, M. E., Evans, H. M., and Li, C. H., Endocrinology, 1943, 33, 261. Sayers, G., Sayers, M., Liang, T. V., and Long, C. N. H., Ibid., 1945, 37, 96. Sayers, M., and Sayers, G., Fed. Proc., 1946, 5, 200. Sayers, G., Sayers, M., and Woodbury, L. A., Endocrinology, 1948, 42, 379. Roe, J. H., and Kuether, C. A., J . Biol. Chem., 1943, 147, 399. Mindlin, R. L., and Butler, A. M., Ibid., 1938, 122, 673. Munson, P. L., Barry, A. G., and Koch, F. C., quoted in Endocrinology, 1948, 42, 379. Stoerk, H. C., Porter, C. C., and Silber, R. H., quoted in J . Amer. Chem. SOC., 1950, 72, 1040. ENDOCRINE UNIT THE LONDON HOSPITAL WHITECHAPEL ROAD LONDON, E.l

ISSN:0003-2654    

DOI:10.1039/AN9517600470

出版商:RSC    

年代:1951

数据来源: RSC

摘要:

474 CLAYTON AND PRUNTY: ASCORBIC ACID DEPLETION METHOD [Vol. 76 The Ascorbic Acid Depletion Method for the Bio-Assay of Adrenocorticotrophic Hormone, and Preliminary Observations on the Use of Inhibition of Tissue Repair BY BARBARA E. CLAYTON AND F. T. G. PRUNTY (Presented at the meeting of the Biological Methods Group 0s Tuesday, October 24th, 1950) The adrenal ascorbic acid depletion method for the assay of adreno- corticotrophic hormone has been examined. Certain modifications -are given and some resuIts obtained by the authors. For the colony of rats used, A, the index of precision, had the value 0.2911 -4 0.044. ACTH inhibits the formation of granu'lation tissue in response to trauma. This fact can be used as the basis of its assay on mice, in which a quanta1 response is measured.SAYERS, Sayers and Woodburyl in 1948 published a method for the assay of adrenocortico- trophic hormone (ACTH) that depended on depletion of ascorbic acid in the adrenals of hypophysectomised rats. During the last six months this method has been used in our laboratory with certain modifications ; some results thereby obtained are described below. ADREN-4L ASCORBIC ACID DEPLETION TECHNIQUE- Wistar male rats weighing 75 to 130g were brought into the laboratory at least three days before use; they were fed ad lib. on Rowett diet 46* before operation and afterwards on bread and milk. Attempts to maintain their environment a t 70" to 75" C were not always successful owing to difficulties with the heating system. When sudden marked fluctuations occurred the technique of hypophysectomy and adrenalectomy were made more difficult, as blood clotting was sometimes impaired.Hypophysectomy was performed under ether anaesthesia. The freshness of the ether' was found to be most important if respiratory difficulties were to be avoided. The parapharyngeal approach was used, and, largely by feel, the initial hole in the skull was made with a fine pair of forceps and enlarged with a thick blunt probe, the gland being then removed by suction. A tracheotomy tube was not necessary after some experience had been obtained and is now no longer used. At autopsy the sella was examined for completeness of hypophysectomy. The solution being assayed was injected into the exposed left external iliac vein and one hour later the right adrenal was removed. Excised adrenals were cleaned, weighed to the nearest 0.1 mg on an analytical balance and transferred to trichloro-acetic acid.Ascorbic acid was determined by the dinitrophenylhydrazine method of Roe and Kuethner.2 New bottles of trichloro-acetic acid should be tested before use, as some sample:; give a precipitate during the final colour reaction. Soluble preparations of ACTH were injected in normal saline, less soluble ones were taken up in glacial acetic acid and diluted to 0.02 to 0.01 N , as suggested by Morris? The solutions were allowed to stand during the tests in a beaker of water over a tray of ice. The left adrenal was removed under ether anaesthesia 18 to 21 hours later. RESULTS The Sprague - Dawley rats in our colony showed great variation in the sizes of adrenal glands, the difference in an individual rat occasionally amounting to as much as 20 mg.In our Wistar rats the adrenal glands usually weighed 7 to 12 mg. In 90 per cent. of the rats the difference in weight between the two adrenals was less than 2 mg and in 23 per cent, the right gland was larger The strain of rat used for assay appears to be most important. * Obtained from Heygate and Sons.August, 19511 FOR THE BIO-ASSAY OF ACTH 4754 than the left. The ascorbic acid concentrations in the adrenals of hypophysectomised but otherwise untreated rats are given in Table I. Some of the percentage differences are much larger than those observed by Sayers et aZ.l Where this occurs it is due to the considerably higher concentration of ascorbic acid in the second gland (right) ; one example of this is given in Table I.TABLE I A RANDOM SELECTION OF THE ASCORBIC ACID CONCENTRATION OF THE ADRENALS OF UNTREATED HYPOPHYSECTOMISED WISTAR RATS Adrenal ascorbic acid, mg per 100 g Left Right A I 3 438.2 404.7 366.8 494.8 367.0 440.0 666.0 600.0 680.8 644.2 715-0 650-0 420.4 402.9 426.6 500.0 346.0 454.6 568.4 583.0 642.6 615.2 721-8 623.0 Difference in mg (left minus right) + 17.8 + 1.8 - 60.8 - 5.2 + 21.0 - 14.6 - 3.4 + 17.0 + 38-2 + 29.0 + 27.0 - 6.8 Difference per cent. of left adrenal 4-0 0.4 16-7 1-0 5.7 3.3 0.6 2-8 6.6 4.6 0.9 4.1 Average: 4-1 Standard Error: & 1-62 An analysis of variance of body weight and initial concentration of ascorbic acid showed no significant effect on the potency of ACTH determined.The precision of the method is given in Table 11. The standard error of the slope is approximately 60, and the standard deviation of 10 slopes is approximately 56, so no evidence of heterogeneity of slopes has yet occurred. The index of precision, A, is 0.291 2 0.044, compared with 0.176 & 0.016 obtained by Sayers et aZ.l The method is thus less precise with this strain of rats, and about 24 to 3 times as many animals are needed by us to give the same degree of precision as these workers got. TABLE I1 PRECISION OF THE ASCORBIC ACID DEPLETION METHOD Month June June June September September October July Preparation 23 200 : 365 203 : 935 J 10602 British Organon La-1-A J20507 Number of rats used per preparation 11 13 11 12 10 12 12 s 62.7 38.0 40.7 71.9 61.3 57.2 83.6 b 197-8 160-3 128-7 263.2 272.2 161.4 262.3 h 0.317 0.237 0.316 0.273 0.225 0.354 0.318 Mean = 0.291 Standard error = +0.044 Very occasionally it has been found in an otherwise satisfactory assay that one animal failed to respond.Several times, however, when assays have been carried out, it has been found that the responses of the individual animals have shown great variations independent of the dosage of ACTH given; indeed, nearly half may have failed to respond at all. This has occurred both with the old.er, poorly soluble, preparations and with the newer soluble ones. We have had correspondence with Dr. Hayes and his colleagues of the Armour Company about the results obtained by us on one of these poorly soluble preparations. They noted that the discrepancies we have found were within the limits of variation obtained by them, by both the Sayers methodl and the Munson m0dification.l They occasionally appear to have similar occurrences in large groups (40 to 50) of animals on any one day, and they ascribe this to biological variation.Nevertheless, for most determinations they reported h = 0.25. We believe that such aberrant results occur when we periodically use the offspring of certain parents in our colony, and this is being further investigated.476 CLAYTON AND PRUNTY: ASCORBIC ACID DEPLETION METHOD [Vol. 76 INHIBITION OF TISSUE REPAIR During the treatment of patients with ACTH: and cortisone it was noticed by one of us that healing of biopsy wounds was inhibited. This happening independently prompted Howes, Plotz, Meyer and Blunt3 to study the effect of cortisone on granulation tissue formation in the ears of rabbits.They found that 12.5mg of cortisone acetate per day per rabbit for 8 to 11 days produced inhibition. It seemed as though the inhibition of healing might be used for the assay of ACTH if smaller laboratory animals, preferably mice, proved suitable. At first some difficulty was experienced in consistently obtaining good granulation tissue in mice. Among methods tried were burning and the injection of turpentine and formalin, but the following method was finally devised. They failed to obtain good responses in rats. TECHNIQUE- Male albino mice weighing 12 to 18 g were maintained on a diet of Thomson cubes and water. Operation was performed under ether, and an aseptic technique was adopted as far as possible.Fur was removed from the anterior abdominal wall with scissors, a piece of skin about 2 to 3 mm in diameter in the midline of the anterior part of the ventral surface of the abdomen was lifted with fine forceps and clipped off with sharp scissors. The wound was covered with three thicknesses of sterile Vaseline gauze, over which was placed the cap of a bottle about 15mm in diameter. The dressing was held in place with adhesive tape, and the cap prevented any pressure on the wound and any contamination with urine and faeces. Care and attention to detail are essential if consistent controls are to be obtained. Twenty-eight and a half hours later the mouse was killed, and the dressing was carefully removed.The ACTH was given in normal saline as 25 subcutaneous injections, each of 0.1 ml. Injections were given one hour and half an hour before the ulcer was made in the morning, immediately after making it and then hourly, except between 11.30 p.m. and 7.30 a.m. when only two injections were given. With a sufficient dose of ACTH complete inhibition of healing is obtained. By this technique well-defined granulation tissue was consistently obtained. APPEARANCES OF THE TISSUES- two feet beneath and one foot to the side of an electric light. ances of the ulcers. Two to three minutes after removal of the dressing, the ulcers were examined about Figs. 1 and 2 show the appear- The points to be noted are summarised as follows- Control 1. 2. 3. 4. healing ulcer Inhibited ulcer Marked hyperaemia.I.. No hyperaemia. Sloping margins. 2. Sharp, clear-cut margins; this appear- ance is emphasised by the shadow in the third lesion (Fig. 2). Rough, thick floor. 3. Smooth, thin floor. The original vessels in the floor are 4. The original vessels in the floor are obscured by new tissue. still visible. Histological examination of most ulcers was at first carried out. The ulcer and a large piece of the surrounding skin were removed and placed flat on a thick piece of blotting paper. After 1 minute the skin and paper were immersed in formalin. The paper was removed twenty-four hours later, and the skin was trimmed. The ulcers were sectioned in the trans- verse diameter and stained with haemotoxylin and eosin. The histological appearances are shown in Figs.3, 4 and 5. The same differences occur as are seen macroscopically, and it is also found that the thick floor of the control ulcer consists of granulation tissue with marked polymorph infiltration, proliferating fibroblasts and numerous capillaries, while the floor of the inhibited ulcer is very thin and contains only a few polymorphs, some flattened fat cells and no capillaries. ASSAY METHOD In the assays, a preparation of ACTH, Armour 7911, was used as a laboratory standard. Doses of this, ranging between 33 and 62 pg, were given to groups of 10 mice. The percentageFig. 1 . Left, control healing nlccr; rifiht, inhibited ulcer Fig. 2.- I .eft, control healing ulcer; centre, partly inhibited ulcer; right, completely inliiliited ulcerFig. 3 .Section cut immediately after the ulcer was made; stained with H. a i d 17. 1 x 30 Fig. 4, Section of a healing ulcer; stained 14. and 1:. 1 x 30August, 19511 FOR THE BIO-ASSAY OF ACTH 477 healed in a group was plotted against the logarithm of the dose and gave a sigmoid curve, as shown in Fig. 6. A probit line was fitted, and it was calculated that the approximate 95 per cent. limits of error would be 78 to 128 for 10 animals on each of two preparations, 84 to 119 for 20 animals, and 90 to 112 for 50 animals. The results of assays on two preparations of ACTH, one from Organon Ltd. and one made by Dr. M. Reiss, are shown in Table 111. Thus the potency ratio of Organon ACTH in terms of standard 7911 is estimated at 0-033 with approximate limits of error, 0.071 and 20 - 10’ 1 I I LOG DOSE 1.6 I .I 1.8 I.! I I DOSE 33.7pg 61.3pg 7.0 6-5 6.0 7 5.5 g a 5.0 4.5 10 4.0 C 2 CID U 3.5 3.0 Fig.6. Dosage - healing curve with adrenocorticotrophic hormone (7911) : 7 to 10 mice per group 0.097. The potency ratio of Reiss ACTH in terms of standard 7911 is estimated at 0.265 with approximate 95 per cent. limits a t 0.228 and 0.309. This may be compared with estimates by the method of Sayers et aZ.l Owing to shortage of mice these figures were not all obtained on the same day; they are based on the assumption that the dose - response curve did not change in any respect over the period of assay. The test for differences between the three slopes shows no evidence that the slope of the probit line has changed during the period of assay. These preliminary observations appear to be encouraging.The method is simple and accurate for assaying preparations known to consist of ACTH, and may therefore be of value in controlling its production. The method lacks the specificity of that of Sayers et aL,l but, in view of its simplicity, may be useful for certain purposes. TABLE I11 RESULTS OF A MOUSE HEALING INHIBITION ASSAY Organon Reiss Dose in pg animals healed Dose in p g animals healed 418.0 4 75 135.0 4 100 469.0 4 75 151-5 4 50 526.0 4 50 169.4 4 50 590.5 4 50 190.6 4 25 Standard 79ll/Organon = 0.083 (Sayer’s method 0.070; Limits = 75 to 132 per cent.). Limits* (P = 0.95) = 0-071 to 0-097. Standard 7911/Reiss = 0.265 (Sayer’s method 0.234; Limits = 46 to 212 per cent.). Limits* (P = 0.95) = 0.228 to 0.309. L A 7 \ r \ Kumber of Per cent.Number of Per cent. * These limits are approximate 95 per cent. fiducial limits.478 VOGT : ASSAYS OF ADRENOCORTICAL HORMONES [Vol. 76 ACKNOWLEDGMENTS We are grateful for statistical assistance given us by Mr. P. Armitage of the M.R.C. Statistics Unit; for technical assistance from Miss J. Hammant in carrying out the assays; from Mr. A. E. Clark in histological work and from Miss J. Dewe in the preparation of the colour drawing. We acknowledge with thanks gifts of ACTH from Dr. J. R. Mote (Armour Company), Dr. W. J. Tindall (Organon) and Dr. Max Reiss (Bristol). REFERENCES 1. 2. 3. 4. Sayers, M. A., Sayers, G. S., and Woodbury, L..A., Endocrinology, 1948, 42, 379. Roe, J. H., and Kuethner, C. A., J . Biol. Chem., 1943, 147, 399. Moms, C. J. 0. R., personal communication. Howes, E. L., Plotz, C. M., Meyer, K., and Blunt, J. W., Proc. SOC. Exp. B i d . Med., 1949, DEPARTMENT OF CHEMICAL -PATHOLOGY 72, 718. ST. THOMAS’S HOSPITAL LONDON, S.E.1 DISCUSSION DR. PRUNTY commented that it was important to stress the remarks made by Dr. Overbeck in the discussion on tlie preceding paper, about the need for repeated injections of ACTH to produce any given response. They had noted in their laboratory that a patient being treated with 40mg of ACTH daily, divided into four doses of 10mg intramuscularly at 6-hourly intervals, gave about l/lOth of the bio- chemical response elicited by the continuous intravenous infusion of 40 mg of ACTH over a 24-hour period. In his paper, Dr. Morris stressed the need for hypophysectomy in any ACTH assay. We, too, held the same opinion until we obtained the results we have reported. We do not know of assays based on multiple injections of ACTH at short intervals. One wonders if this technique does not, in fact, produce a “biological hypophysectomy” in the animal, perhaps by direct action on the pituitary. If this were so, it would explain the constant results obtained in their experiments. This stresses the importance of continuous administration.

ISSN:0003-2654    

DOI:10.1039/AN9517600474

出版商:RSC    

年代:1951

数据来源: RSC

摘要:

478 VOGT : ASSAYS OF ADRENOCORTICAL HORMONES [Vol. 76 Assays of Adrenocortical Hormones on Small Laboratory Animals BY MARTHE VOGT (Presented at the meeting of the Biological Methods Group on Tuesday, October 24h, 1950) All biological assays of cortical hormojnes are camed out on adrenalec- Mice and young rats are the animals of choice because Practical tomised animals. of the ease and speed with which their adrenals can be removed. details are given for the performance of three such assays. ALL attempts at assaying cortical hormones either on normal animals or on isolated organs have so far failed; only methods using adrenalectomised animals have met with some measure of success. In most tests the potency of the assayed material is measured by partial restoration of a functional deficiency resulting from the removal of the adrenals.One test, however, which uses as a measure of activity the fall in circulating eosinophils produced by cortical hormone, is carried out on adrenalectomised animals for the sole purpose of excluding effects due to stimulation of the animal’s own adrenals. Since, then, adrenalectomy has to precede any further assay procedure, it is obvious that those species that are easiest to adrenal- ectomise are also most suitable for these hormone assays. Since the rat is the animal on which mass-adrenalectomy is most conveniently performed, all the tests I shall discuss were originally tried on rats. Soon, however, it was found that, in all but one of these tests, mice show a greater sensitivity to the hormone, not only because of their smaller body weight, but also because of an inherent greater susceptibility.In spite, therefore, of their being somewhat less pleasant to handle and to anaesthetise than rats, all except one of the tests are nowadays done on mice.August, 19611 ON SMALL LABORATORY ANIMALS 479 There are three biological assays of cortical hormone on rodents of which enough is (1) The survival test in low environmental temperature, originated in 1938 by Selye and Schenker-l (“Cold test.”) (2) The test using deposition of liver glycogen in fasting mice given glucose parenterally, described by Venning, Kazmin and Bell2 (3) The test using the fall of circulating eosinophils in the mouse, devised by Speirs and Meyer.3 A method very similar to that of Venning, Kazmin and Bell2 was published simul- taneously by Eggleston, Johnston and D~briner.~ Fewer people seem to have used this second method, so that I shall omit its description.There is no reason, however, to suppose that it is less good. Before describing the different methods, it will be useful to compare their merits or disadvantages- (a) Of the three tests, the “cold test” is easiest to perform, since it requires observation of survival times for a period of an average of 12 to 14 hours, but no further chemical or biological measurements on the adrenalectomised animals. (b) The amount of material required is a little less for the “cold test” than for the liver glycogen test and much less for the eosinophil test. (c) The “cold test” is the only test applicable both t o desoxycorticosterone-like compounds and to compounds oxygenated at Cll.The former compounds, however, have to be administered in a different way owing to their slow action and do not give a graded response at different dose levels. The other two methods, as far as we know, assay exclusively compounds carrying an oxygen atom in the 11-position. (d) The precision is highest for the glycogen deposition test, lowest for the eosinophil test and appears to be intermediate for the “cold test.” The desirable number of animals on each dose or compound is not less than 10 for all tests. (e) Standard and unknown have to be assayed simultaneously in every experiment that uses the “cold test,” whereas a standard curve can be constructed for the glycogen deposition test.This means that experiments can be done with unknown solutions alone, and the results read off on a previously prepared standard curve. It is hoped, though it is not certain, that the same will be true for the eosinophil test. (f) The “cold test” gives significant differences between different doses only if the group of rats is made up of litter mates of very nearly the same weight. No such restriction applies to the other two tests. It may be asked whether there is no method of testing for the influence of cortical substances on electrolyte metabolism. Many such tests have been tried, but none has been found satisfactory. Recently, however, Spencer5 has developed a method that, though laborious, appears to be very promising. It permits the assay of small quantities of deoxy- corticosterone acetate (DOCA) by measuring the rate of sodium excretion after a given load of sodium chloride injected subcutaneously.There has not been enough time since the publication of the method for experience to be gained with it in other laboratories. The method is lengthy and requires precision in all manipulations; it appears to require 0-5 to 4 pg of DOCA per mouse and 12 “selected” adrenalectomised mice per test. The 12 animals are selected from 18 in a preliminary sodium load test carried out without injecting any steroids. On the next day, test, standard and control solutions are given intraperitoneally to 4 mice each and the sodium excretions measured; similar tests are performed on the sub- sequent two days, changing over the mice used for a particular solution until all mice have in turn received test, standard and control.A brief description of each test follows, though there is little to add to the published accounts except for the third test. known to justify their use as routine or screening tests. (1) “COLD TEST”- The principle of the test is to accelerate the death of adrenalectomised rats by keeping them at a low environmental temperature. Whereas death at room temperature would occur in 4 to 20 days, the cold environment (in the absence of food) precipitates it, so that it occurs within as many hours. It can be delayed by administration of cortical hormone, and the length of this delay is a measure of the potency of the hormone preparation. The480 VOGT : ASSAYS OF ADRENOCORTICAL HORMONES wol.76 exposure to low temperature not only shortens the duration of the assay, but also increases its sensitivity and, to a lesser degree, its precision. Age and size of the rat and the litter from which the animal is taken considerably influence its survival time and make careful matching of the groups imperative for obtaining significant differences in survival times with varying doses of hormone (Vogt6). The optimal age and size of the animals depends on the particular strain, successful assays requiring vigorous, fast-growing animals. Mice can be used instead of rats, but offer no advantage. Suitable ages and sizes for a particular colony of Wistar rats are, for instance, 21 to 25 days and 37 to 50 g. The variation of weight within a litter should not exceed a few grams, the precision of the results being highest when this variation is smallest.If X samples are tested on X groups of rats in a particular experiment, it is desirable to have 10 litters of X rats each, and to allot one rat of each litter to every group. The effect of variations in weight within a litter is minimised by making the mean weight of all groups equal. It is also advisable to make the sex ratio the same in all groups. The rats are adrenalectomised on one morning, well fed overnight and used for the assay on the following day. The material to be tested is injected subcutaneously in four doses spaced at intervals of one hour and a half. After the first injection, all animals are simul- taneously placed in a cold-room or large refrigerator and only removed for brief periods for further injections or rapid inspection in order to determine the times of death. If doubt is felt about whether a rat is dead or not, it is counted as dead when its corneal reflex has disappeared.The mean survival time of each group measures the potency of each sample. There is a straight line relationship between mean survival time and log-dose of hormone, provided the samples are free from toxic substances. A control group injected with the solvent and two groups treated with different doses of a standard have to be used in every assay in which a quantitative result is desired. If the samples differ in potency by less than 250 per cent. they will rarely produce significant differences in mean survival time.A suitable total dose of extract is 0.1 ml per rat. This may produce an increase of 18 to 4 hours in mean survival time over the controls, depending among other things on the temperature in the refrigerator. The temperature should be somewhere between +2” and +7” C, according to the vitality of the rats, which will vary with their size and with the strain, (2) THE GLYCOGEN-DEPOSITION TEST- The principle of the test is the observation that fasted, adrenalectomised mice only deposit injected glucose as liver glycogen when supplied with cortical steroids carrying an oxygen atom at C1,. Mice weighing from 20 to 25 g are fed on a specified diet, adrenalectomised and kept a t a constant temperature. Food is withdrawn on the third post-operative day, and the test is begun on the following morning.Seven hypodermic injections of a mixture of glucose, alcohol and the test substance are given at inte:rvals of 45 to 60 minutes. The mouse is anaesthetised one hour after the last injection. The liver is excised and hydrolysed and its glycogen content is determined. A glycogen deposition of about 30 mg per 100 g of liver is significant. Since there are seasonal variations in the response, frequent checking with a standard preparation is desirable, but is not required on every occasion as it is in the “cold test.” The most accurate results are obtained when between 10 and 40pg of cortisone are given to each mouse. The test involves liver glycogen estimations on every animal. Not less than 6 mice are required for each sample. (3) THE MOUSE EOSINOPHIL TEST- The test is based on the fact that injection of‘ cortical steroids oxygenated at CI1 causes a fall in the circulating eosinophils in the blood of any mammal.The assay is still in the experimental stage, but its sensitivity is such that this fact alone warrants its discussion in this survey. The counting of eosinophils in the blood of the mouse requires some practice, but by using the solution recommended by Speirs and Meier,3 which dissolves all blood cells except the eosinophils, counting is greatly facilitated. In the description of the details of the test, I shall quote the procedure developed by Dr. Bibile at the Pharmacological Laboratory, Edinburgh (unpublished), but I wish to emphasise that it is still in the process of development.From 6 to 10 large mice are adrenalectomised, special care being taken to remove all fat surrounding the adrenal along with the gland, in order to avoid regeneration of glandular tissue.August, 19611 ON SMALL LABORATORY ANIMALS 481 The animals are kept at a steady temperature and supplied with 0.9 per cent. sodium chloride solution instead of drinking water. On the fifth post-operative day, the mouse is warmed until its tail vessels are well dilated, and a drop of blood is obtained from a tail vein for a first eosinophil count. At the same time an injection of 5 pg of adrenaline is given subcutaneously, and a second eosinophil count is made 3 hours later. Those mice responding with a fall in the count are suspect of glandular remnants and are discarded.Immediately after obtaining the second sample of blood, the test substance is injected and its effect on the eosinophil count is observed 4 hours later. A fall indicates adrenocortical activity in a sensitive strain. The test is applicable to doses of 2 pg of cortisone, and possibly to less. The dose-response curve, however, appears to be even flatter than that for the “cold test.” I t is possible to improve the practicability of the test by using adrenalectomised mice maintained with implants of desoxycort icost erone, If we assay a mixture like a cortical extract, it may or may not be justified to use a simple steroid, say cortisone, as a standard. In the “cold test” it is not justified because the dose - response curves are not parallel. In the glycogen deposition test it has been the practice to use Kendall’s compound E as standard, and this probably introduces little, if any, error, since in that test the potency of an extract is likely to be determined by its content of compounds E and the very similar F.The same may hold for the eosinophil test, but there is as yet no information on this point. It is the ardent wish of every worker in the field of biological assays to see the biological test disappear and the assay replaced by a chemical estimation. Until recently, this hope was faint indeed for adrenocortical hormones. New chemical micro-methods, particularly chromatography, have also opened new avenues in steroid chemistry. I hope that the task of biological assays will, in the not too distant future, be relegated to its proper field, namely, to help the chemist in the isolation and identification of compounds manufactured by the adrenal gland, and to test synthetic substitutes. The question of standards has not yet been raised. To-day, the picture is changing. REFERENCES 1. 2. 3. 4. 5. 6. Selye, H., and Schenker, V., Proc. Soc. Exp. Biol., 1938, 39, 618. Venning, E. H., Kazmin, V. E., and Bell, J. C., Endocrinology, 1946, 38, 79. Speirs, R. S., and Meyer, R. K., Ibid., 1949, 45, 403. Eggleston, N. M., Johnston, B. J., and Dobriner, K., Ibid., 1946, 38, 197. Spencer, A. G., Nature, 1950, 166, 32. Vogt, M., J . Physiol., 1943, 102, 341. THE DEPARTMENT OF PHARMACOLOGY UNIVERSITY OF EDINBURGH

ISSN:0003-2654    

DOI:10.1039/AN9517600478

出版商:RSC    

年代:1951

数据来源: RSC