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A colorimetric method for the estimation of testosterone
451
A COLORIMEBRIC MEIXOD BQR THE ESTIMATIOFOF TESTOSTKRONE bY D.GUPTA%nd EILEENHoCAFFKRTT Departmentof Growthand Development, Inatituteof Child Health,Universityof London.
ReceivedMay 16, 1966
A new colorimetrio method for the estimationof pure testosterone with oeric %ulph%tein sulphuric acid is reported. The method is Of %ll speoific,sensitive,reliableand cronvenient for routine u8e. the steroidsexaminedwith this reagentonly a few gave positive reactionwhich may aid in identification and yet requirelittle material. Work is now in progressto apply this colourmethod to eetimateurinarytestosterone in developingchildren.
Testoeteroneis properlyconsideredthe meet potentof the known androgens(Dorfmanet al, 1962) and severalmethodshave been proposed for estimatingit in the pure form.
Finkelsteinet al (1961)
describeda method which involvedfluorimetric assag of oestradiol-17$ formedenzymatically from testosterone. Rosneret al (1965)estimated testosterone by oxidizingit to %ndrostenedione, %nd then carryingout a modifiedmicro-Zimmerman reaction, The fluorescence given by testosterone and a few other adrenocortical steroidswhen treatedwith certainconcentratedacidshas been utilizedby other workers(WinterSteinerand Pfiffner,1936; Beichsteinand Shoppee,1943).
Sulphuric
acid reagentsfor the apectrophotometric assay of testosterone have been describedby Koeniget al (1941),Allen et al (1950),Mastin (1956) Kalant (1958).Oertel (1961),Wilson (1960). Sachs (1964)used a sulphuricacid mixturecontainingNK4Fe(SO4~+ 12 X20 and WyaO4,
452
8:4
STEROIDS
We have investigatedthe complexchromogenicity developedby authentictestosteronein combinationwith metallicions.
7!hi.S
paper describeda relativelysimplebut quite sensitiveand specific s~ctrophotometri~method for the measurementof pure testosterone with ceric sulphate. This method seems to be accurateaud at the ssme time convenientfor day to day routineuse.
We are now
lookinginto the possibilities of adaptingthis method for the study of the urinaryexcretionpatternsof testosterone in childrenduring growth and development.
AnaJ.sr sulphuricacid suppliedby BritishDrug HousesLtd. (BDH) is specified; reagentspreparedwith this acid keep the reagentblank values down. These shouldnot be higher than 1% of the readinggiven by the lowestreferencestandardused. Cerio sulphate(low in other rare earths) ......BBH Ammoniumferric sulphate(Analar) .....BBH Acetic acid glacial (Analar) BBH ...**. Ceric sulphate-H2S04 reagent ....... Preparea 2.y~ (w/v) solution of ammoniumferric sulphate with 1 ml of l@& (v/v) sulphuric acid solutioncontainingO.OOl$ (w/v)of eerie sulphate. Heat mixturein a boilingwater bath for two minutes. Cool and bring to 100 ml with cont. sulphuric acid. Store at 4OC. l
a) Use of ceric suluhateas an oxidizingaaent: Ceric sulphateas an oxidizingagent is well known and has msny advantages(Vogel,1953) especiallyits stabilityin strongI5$504 solution. A detailedcomparativestudy of the developmentof the colour given by testosteronewith -04 (Sachs,1964) and ceric aulphatewas carriedout. Ssmplesof differentconcentrations of testosterone, evaporatedto drynessunder nitrogen,were dissolved in 0.01 ml of ethanol. To these were added 0.5 ml eitherof KMn04 reagent (as per Sachs)or of eerie sulphatereagent,as
Oct. 1966
STEROIDS
453
deearibedabove, The mixtureswere heatedin a boilingwater bath for 5 min, cooledin ice water for 3 min, aat3 then 3 ml of 3% =etfc on @kiCsm acid was added. The blue colourdevelopedwas msEt,stired SP 800 between400 and 700 mu, An increaseof over 2091in ohromogsnic intensity(at 620 pq~)was obtc&nedby the use of oeric fmlphate w contrastedwith IWLLO~, the moleoulsrabsorptivity being 15,300with the formerand 12,600with the latter. b) Influenceof reaRentconcentration durti incubation: To determinethe optimalreagentaoncentration during incubation a seriesof testosterone samplesof differentconcentrations was treatedwith 0.5 ml of ceric sulphate-H2SQreagent mixture. Another seriesof similartestosterone concentrations was treatedwith 1 ml of After incubationthe colourWE@ developedin the the ssme reagent, wsy describedabove and measuredon the Unicam SP 800 between400 to in increaseof over 3 times (at 620 mu) in chromogenicity 700 W. was observedwith the more concentrated reagent, c} w>mnce of initial, concentration of sulphuricacid in the reaRent: Ceric sulphate-H2SO4 reagentmixturewas preparedwith different strengthsof sulphuricacid (lO$,2& 39% and 4%). 1 ml of the reagentmixturewas added to each tube containing testosterone and incubatedfor 5 min. in a boilingwater bath. The colourwas developedand measuredas above. Table I shows that the resultsare slightlybetterwith the reagentmixturewith lO$ sulphuricacid, higher concentrations being a littleless effective. d) Influenceof differentmetallicions used in combinationwith ceric suluhate: An investigation was carriedout on the influenceof various metallicions on the chromogenicity developedby testosterone with ceric sulphateand sulphuricacid. For this purposewe prepared reagentsin the similarway to the usual ammoniumferric sulphate, viz ammoniumcupricsulphate,smmoniumcobaltsulphate,smmonium manganesesulphateand ammoniumchromicsulphate. '&th most of the met&3 testosterone gave two peaks. Table II gives the wavelengths where peaks were observedfor individualmetals. Only the ferric ion gave a singlepeak, which was aIs0 comparatively much more sensitive, e) Influenceof dilutionafter incubation: TO investigate the influenceof the final dilutionmixtureon the developmentof chromogenicit , we tried,in additionto the nOI?IUdly used 3$ aceticacid (v v), l$ aceticacid (v/v),51: acetic acid (v/v>,lC$ aceticacid (v/v), water and 3$ propionicacid (V/V), The COlour developedwas messuredas before, The results(TableIII) show that the loG/o aceticacid solutionproducedslightlybettervalues. This concentration was used in later experiments, f> Influenceof incubationtime: A seriesOf equal quantitiesof testosterone snd a numberof reagentblankswere treatedwith ceric sulphate-H@O4rsegentmixture8s describedabove in a boilingwater bath at 100°C, At intervalsof 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20 and 25 minutesafter innaersion in
454
STEROIDS
8:4
the bath a set of tubes (containing10 t.lg of testosterone) were removed, cooled and treatedwith lO$ acetic acid (v/v). The chromogenicity developedwas measuredas above. We found no significantdifference in the developmentof colourwith time. The colourseemedto get destroyed(8$ in two hours)when left at room temperatureand exposed to light. In later experiments2 minutesincubationtime was adopted. g) Extractionof the chromogenicsubstancesinto a solventlaver: To see how far the chromogenicsubstancesdevelopedby testosterone with ceric sulfate-~~~ reagentmixturewere extractablein solventlayersmethylenechloridesnd ethyl acetate were used. The colour complexdevelopedwas found to be much more stablein an aqueousphase than in a solventphase. In the latter cases peaks were at differentwavelength8and vanishedin less than Ii.5 minutes.
A sampleof solutionknown to containfrom 1 trgto 15 tig steroidis evaporatedto drynessin a C-10 Exe10 tube (10 x 100 mm) under nitrogen. Absoluteethanol(0.01ml) is added and the tube is rotatedso that the steroidresidueis thoroughlywettedby akohol snd 0.3 ml of ceric sulphate-E2SOq reagentis added. After thoroughmixing,the stopperedtube is placedwithoutdelay in a water bath at 100°C for 2 minutes. The size of the tube is not critical but the bath shouldbe such that the liquidin the tube is fully immersed. The mouth of the tube is protectedfrom condensing steam by atoppering. After heating,the tubes ere cooledin icewater and 1 ml of lCY$aceticacid (v/v)is added. The copperblue colourdevelopedis measuredspectrophotometrically in a semi-micro cell (1.3 ml capacity)on a Iiecording Spectrophotometer (Unicsm SP 800) between500 and 700 w. The colourdevelopedis stable for at least 30 minutesand absorbslightmaximallyat 620 mu, Measuringthe opticaldensitiesat differentwavelengths (530, 620 and 670 mrr)and correctingfor non-specificabsorption by the method of Allen (1950)is necessaryto correctfor colours due to other contaminating substancesfrom the chemicals. Optical densityreadings(D) are correctedby the followingformula: Correctedi?eading = 2 D620 - (D570+ D670) RESULTS
To demonstratethe reproducibility of the procedure,equal ssmplesof testosterone were measuredin 10 successiveasssys, In Table IV the averageresults (Allencorrectedless reagentblank) are shown with standarddeviationfor each .group. The last column
Oct. 1966
STEROIDS
455
demonstratesthe reproducibility of the Allen correctedvalues at differentconcentration levelsof testosterone when calculatedper trgper ml. Out of the steroidssubjectedto this colourreactiona positive epi-testosterone, ILhydroxytestoeterone test was given by testosterone, audrostenedione and 6$-hydroxyprogeaterone.Table IV shows the differentcoloursgiven by differentsteroidswhen subjectedto this colourreaction. It ala0 gives the percentageof chromogenicity given by positive-reacting steroids,takingtestosterone as 100. DISCUSSIOIV The value of this apectrophotometric method of testosteroue lies in its simplicityaud sensitivity. The lattercau be further increasedby using smallervolumesof reagentsin micro-cells (capacity0.5 ml>.
0.3 pg of testosterone cau be satisfactorily
assayed. The validityof resultsobtainedby a method depends,to a considerable extent,on the effectiveness of the densitycorrection. Correctedopticaldensitiesby the presentprocedureobeyed Deer's Law closelybetween1 ug and 15 ug.
Pure testosterone added to
paper ohromatogra~~ and elutedwith ethanolafter the run gave similarpeaks and obeyedDeer*&Law like the authenticstandard. The method cau claim a relativelyhigh degreeof speoificity towardsa restrictednumberof steroids. As most steroids ocmxring iu urine gave no similarcolourat the concentration of lo-20 ug in 1.5 ml reactionmixture,it seems that no appreoiable interference can be expeotedin oolourdevelopmentby known
STEROIDS
456
8:4
urinarysteroidsof polarityaimilarto that of testosterone (eyeterns:Gupta and Tanner,1965).
At the moment work is in
progressto adapt this calorimetric method for the study of the urinaryexcretionpatternsof testosterone in developing prepubertalchildren.
This work was eupportedby a grant from the MedicalResearch Councilto Dr. 3, M. Tannerwhom we wish to thank for his criticism and encouragement. REFERENCES Allen,
W.M., Hayward, and Pinto,A.
S.J.
Dorfman,R.I., Soroini,G. M. Brust,N., Nightingale, and Forohielli,E.
J. CLIN. ENDOCR.2, 54 0950) PROC. 44THMEE?pING ENDOCR.SOC. P 15 (1962)
Finkeletein, M., Forchielli,E. J, CLIN. ENDOCR.& 98 (1961) and Dorfmsn,R.I. HATUBE203, 187 (1964) Gupta, D. snd Tanner,J.M. Gupta,D. snd Tanner,J.M.
BIOCHEM.3. &,, 25 P
Kalant,H.
BIOCHEM.J. a, 79
VA., Mslraer, F., Srtego, C,MI.and Ssmuels,L.T.
(1965)
(1958)
Koenig,
J. BIOL. CHEM.141, 487 (1941)
REV. CANAD.BIOL.I;e,399 (1956) Martin,E.A. ACTA ENDOCRINOL.x, 237 (1961) Oertel,G.W. Reichstein,T, and Sboppee,C.W. VITAM. AND HORM. A, 345 (1943) Rosner,J.M., Conte,N.F., Briggs,J.H., Chao, P.Y., Sudman,E.M. snd Forsham,P.H. J. CLIN. ENDOCH.25, 95 (1965) NATURE201, 296 (1964) Sachs,L, QUANTITATIVEINORGANICANALYSIS. Vogel, 8.1. 2nd ed. Longmane,Green:London (1953) Wilson,H. Wintersteiner, 0. and Pfiffner,J,J,
ANALYT,BIOCHEM.1, 402 (1960) J. BIOL, CHEM,116, 291 (1936)
STEROIDS
Oct. 1966
457
TABLE1 INF'LUENCEOFSULPHDRI CACID CONCENTRATION ONTHEDEVELOPMJZNTOFCHROMOGEX?ICITY
Concentration of H2SO4 in the ceric sulphate-H2S04 reagent
Maximumabsorbancegiven by 20 ug of testosterone
lWj5
0.78 0.76 0.72 0.74
2% 3% 4%
TABLE II INFLUJZNCE OFDIFFEZfENT~TALS ON THF, DEVEWPMENT OFcHFKNOGmCITP Metals used
F@lTiC
Manganese
Cobalt chromic
Cupric
Maxmum abaorbaucegiven by 20 ug of testosterone Peak at 620
Peak at 595
Peak at 470
mu
Mu
mu
0.66 None None None 0.24
None 0.29 0.30 0.26 None
None 0.32 0.35 0.38 0.24
458
8:4
STEROIDS
TABLE III IRFLUENCE 09 ACETIC ACID CONCEXJTRATIOX AS A DILUTIKG PLZCIDON THE DEXELOPPIEKTOF CIIRO~~OGEZICITY
Diluting fluid used
Maximum absorbance given by 20 ug of testosterone
0.64
l$ Acetic acid 3$ Acetic acid 3: Acetic acid lC& Acetic acid Water 2; Propionic acid
0.68
0.66 0.71
0.58 0.56
TABLE IV
AIJ&N CORE0Xl'EDFUZSULTSOF TKE CXROMOGEKICITY GIVEN BY TESTOSTEXOI'JB AT DIFJ?EZREXl' CONCEl4Pl'RATICNS
Testosterone (w)
1.0 ::; 7.5
10.0 12.5 15.0
Allen corrected results (average of 10)
S.D.
Allen corrected results (per w per ~1)
0.038 0.105 0.212
.009 .012 .028
0.026 0.028 0.028
0.312 0.403 0.500 0.594
-039 .028 0034 .023
0.027 0.027 0.027 0.027
STEROIDS
Oct. 1966
459
EFFECT OFTHECIBIC SULPEAT%H2SO4RlUGENTOrJVARIOUSSTEROIDS Steroid
Colour
copperblue Teetoaterone 11 n Epi-testosterone " n Androatenedione " n i-Androstenolone II n Ii-OH-Testosterone n n 6p-OH-Progesterone green Z&OH-Te8tosterone ll+OH-bndrostenedione pale yellow n n 7-oxo-DEA n (1 +bndrost-16-ene-3-01 Androsta-5-en-3 ,17 -dial peach/yellow Androsta-l,+diene-3,17-dione peach 5&Androstan-3,17-dione colourless 5B-dndrostan-3,11,17-~ione palebuff Androetenetrione colourless Androeterone yellow Aetiocholanolone pale yellow Dehydroepiandrosterone buff Epi-androsterone pale yellow ll&OH-=Audrosterone peachjiplnk 38-21-~~~regn-5-en-20-one colourlese Ecgosterol-Dacetate colourless Cholicacid oolourless B-Ionone PM X-Substance(Guptasnd Tanner,1964)peaoh Corticosterone colourless Cortisone colourless Tetrahydrocortisone colourless ll- Deoxycortisol(Reichstein's S) colorless Tetrshydro-ll-dehydrocorticosterone colourless (TW
Peak at Chr0lRO620 mu genicity (T - 100) 100 3-0 66 3-s 99 3-e 3-e Yes
yes NO
NO NO
No No No No No No No No NO
No NO NO
NO NO
No No No No No No No
71 63
A COLORIMEBRIC MEIXOD BQR THE ESTIMATIOFOF TESTOSTKRONE bY D.GUPTA%nd EILEENHoCAFFKRTT Departmentof Growthand Development, Inatituteof Child Health,Universityof London.
ReceivedMay 16, 1966
A new colorimetrio method for the estimationof pure testosterone with oeric %ulph%tein sulphuric acid is reported. The method is Of %ll speoific,sensitive,reliableand cronvenient for routine u8e. the steroidsexaminedwith this reagentonly a few gave positive reactionwhich may aid in identification and yet requirelittle material. Work is now in progressto apply this colourmethod to eetimateurinarytestosterone in developingchildren.
Testoeteroneis properlyconsideredthe meet potentof the known androgens(Dorfmanet al, 1962) and severalmethodshave been proposed for estimatingit in the pure form.
Finkelsteinet al (1961)
describeda method which involvedfluorimetric assag of oestradiol-17$ formedenzymatically from testosterone. Rosneret al (1965)estimated testosterone by oxidizingit to %ndrostenedione, %nd then carryingout a modifiedmicro-Zimmerman reaction, The fluorescence given by testosterone and a few other adrenocortical steroidswhen treatedwith certainconcentratedacidshas been utilizedby other workers(WinterSteinerand Pfiffner,1936; Beichsteinand Shoppee,1943).
Sulphuric
acid reagentsfor the apectrophotometric assay of testosterone have been describedby Koeniget al (1941),Allen et al (1950),Mastin (1956) Kalant (1958).Oertel (1961),Wilson (1960). Sachs (1964)used a sulphuricacid mixturecontainingNK4Fe(SO4~+ 12 X20 and WyaO4,
452
8:4
STEROIDS
We have investigatedthe complexchromogenicity developedby authentictestosteronein combinationwith metallicions.
7!hi.S
paper describeda relativelysimplebut quite sensitiveand specific s~ctrophotometri~method for the measurementof pure testosterone with ceric sulphate. This method seems to be accurateaud at the ssme time convenientfor day to day routineuse.
We are now
lookinginto the possibilities of adaptingthis method for the study of the urinaryexcretionpatternsof testosterone in childrenduring growth and development.
AnaJ.sr sulphuricacid suppliedby BritishDrug HousesLtd. (BDH) is specified; reagentspreparedwith this acid keep the reagentblank values down. These shouldnot be higher than 1% of the readinggiven by the lowestreferencestandardused. Cerio sulphate(low in other rare earths) ......BBH Ammoniumferric sulphate(Analar) .....BBH Acetic acid glacial (Analar) BBH ...**. Ceric sulphate-H2S04 reagent ....... Preparea 2.y~ (w/v) solution of ammoniumferric sulphate with 1 ml of l@& (v/v) sulphuric acid solutioncontainingO.OOl$ (w/v)of eerie sulphate. Heat mixturein a boilingwater bath for two minutes. Cool and bring to 100 ml with cont. sulphuric acid. Store at 4OC. l
a) Use of ceric suluhateas an oxidizingaaent: Ceric sulphateas an oxidizingagent is well known and has msny advantages(Vogel,1953) especiallyits stabilityin strongI5$504 solution. A detailedcomparativestudy of the developmentof the colour given by testosteronewith -04 (Sachs,1964) and ceric aulphatewas carriedout. Ssmplesof differentconcentrations of testosterone, evaporatedto drynessunder nitrogen,were dissolved in 0.01 ml of ethanol. To these were added 0.5 ml eitherof KMn04 reagent (as per Sachs)or of eerie sulphatereagent,as
Oct. 1966
STEROIDS
453
deearibedabove, The mixtureswere heatedin a boilingwater bath for 5 min, cooledin ice water for 3 min, aat3 then 3 ml of 3% =etfc on @kiCsm acid was added. The blue colourdevelopedwas msEt,stired SP 800 between400 and 700 mu, An increaseof over 2091in ohromogsnic intensity(at 620 pq~)was obtc&nedby the use of oeric fmlphate w contrastedwith IWLLO~, the moleoulsrabsorptivity being 15,300with the formerand 12,600with the latter. b) Influenceof reaRentconcentration durti incubation: To determinethe optimalreagentaoncentration during incubation a seriesof testosterone samplesof differentconcentrations was treatedwith 0.5 ml of ceric sulphate-H2SQreagent mixture. Another seriesof similartestosterone concentrations was treatedwith 1 ml of After incubationthe colourWE@ developedin the the ssme reagent, wsy describedabove and measuredon the Unicam SP 800 between400 to in increaseof over 3 times (at 620 mu) in chromogenicity 700 W. was observedwith the more concentrated reagent, c} w>mnce of initial, concentration of sulphuricacid in the reaRent: Ceric sulphate-H2SO4 reagentmixturewas preparedwith different strengthsof sulphuricacid (lO$,2& 39% and 4%). 1 ml of the reagentmixturewas added to each tube containing testosterone and incubatedfor 5 min. in a boilingwater bath. The colourwas developedand measuredas above. Table I shows that the resultsare slightlybetterwith the reagentmixturewith lO$ sulphuricacid, higher concentrations being a littleless effective. d) Influenceof differentmetallicions used in combinationwith ceric suluhate: An investigation was carriedout on the influenceof various metallicions on the chromogenicity developedby testosterone with ceric sulphateand sulphuricacid. For this purposewe prepared reagentsin the similarway to the usual ammoniumferric sulphate, viz ammoniumcupricsulphate,smmoniumcobaltsulphate,smmonium manganesesulphateand ammoniumchromicsulphate. '&th most of the met&3 testosterone gave two peaks. Table II gives the wavelengths where peaks were observedfor individualmetals. Only the ferric ion gave a singlepeak, which was aIs0 comparatively much more sensitive, e) Influenceof dilutionafter incubation: TO investigate the influenceof the final dilutionmixtureon the developmentof chromogenicit , we tried,in additionto the nOI?IUdly used 3$ aceticacid (v v), l$ aceticacid (v/v),51: acetic acid (v/v>,lC$ aceticacid (v/v), water and 3$ propionicacid (V/V), The COlour developedwas messuredas before, The results(TableIII) show that the loG/o aceticacid solutionproducedslightlybettervalues. This concentration was used in later experiments, f> Influenceof incubationtime: A seriesOf equal quantitiesof testosterone snd a numberof reagentblankswere treatedwith ceric sulphate-H@O4rsegentmixture8s describedabove in a boilingwater bath at 100°C, At intervalsof 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20 and 25 minutesafter innaersion in
454
STEROIDS
8:4
the bath a set of tubes (containing10 t.lg of testosterone) were removed, cooled and treatedwith lO$ acetic acid (v/v). The chromogenicity developedwas measuredas above. We found no significantdifference in the developmentof colourwith time. The colourseemedto get destroyed(8$ in two hours)when left at room temperatureand exposed to light. In later experiments2 minutesincubationtime was adopted. g) Extractionof the chromogenicsubstancesinto a solventlaver: To see how far the chromogenicsubstancesdevelopedby testosterone with ceric sulfate-~~~ reagentmixturewere extractablein solventlayersmethylenechloridesnd ethyl acetate were used. The colour complexdevelopedwas found to be much more stablein an aqueousphase than in a solventphase. In the latter cases peaks were at differentwavelength8and vanishedin less than Ii.5 minutes.
A sampleof solutionknown to containfrom 1 trgto 15 tig steroidis evaporatedto drynessin a C-10 Exe10 tube (10 x 100 mm) under nitrogen. Absoluteethanol(0.01ml) is added and the tube is rotatedso that the steroidresidueis thoroughlywettedby akohol snd 0.3 ml of ceric sulphate-E2SOq reagentis added. After thoroughmixing,the stopperedtube is placedwithoutdelay in a water bath at 100°C for 2 minutes. The size of the tube is not critical but the bath shouldbe such that the liquidin the tube is fully immersed. The mouth of the tube is protectedfrom condensing steam by atoppering. After heating,the tubes ere cooledin icewater and 1 ml of lCY$aceticacid (v/v)is added. The copperblue colourdevelopedis measuredspectrophotometrically in a semi-micro cell (1.3 ml capacity)on a Iiecording Spectrophotometer (Unicsm SP 800) between500 and 700 w. The colourdevelopedis stable for at least 30 minutesand absorbslightmaximallyat 620 mu, Measuringthe opticaldensitiesat differentwavelengths (530, 620 and 670 mrr)and correctingfor non-specificabsorption by the method of Allen (1950)is necessaryto correctfor colours due to other contaminating substancesfrom the chemicals. Optical densityreadings(D) are correctedby the followingformula: Correctedi?eading = 2 D620 - (D570+ D670) RESULTS
To demonstratethe reproducibility of the procedure,equal ssmplesof testosterone were measuredin 10 successiveasssys, In Table IV the averageresults (Allencorrectedless reagentblank) are shown with standarddeviationfor each .group. The last column
Oct. 1966
STEROIDS
455
demonstratesthe reproducibility of the Allen correctedvalues at differentconcentration levelsof testosterone when calculatedper trgper ml. Out of the steroidssubjectedto this colourreactiona positive epi-testosterone, ILhydroxytestoeterone test was given by testosterone, audrostenedione and 6$-hydroxyprogeaterone.Table IV shows the differentcoloursgiven by differentsteroidswhen subjectedto this colourreaction. It ala0 gives the percentageof chromogenicity given by positive-reacting steroids,takingtestosterone as 100. DISCUSSIOIV The value of this apectrophotometric method of testosteroue lies in its simplicityaud sensitivity. The lattercau be further increasedby using smallervolumesof reagentsin micro-cells (capacity0.5 ml>.
0.3 pg of testosterone cau be satisfactorily
assayed. The validityof resultsobtainedby a method depends,to a considerable extent,on the effectiveness of the densitycorrection. Correctedopticaldensitiesby the presentprocedureobeyed Deer's Law closelybetween1 ug and 15 ug.
Pure testosterone added to
paper ohromatogra~~ and elutedwith ethanolafter the run gave similarpeaks and obeyedDeer*&Law like the authenticstandard. The method cau claim a relativelyhigh degreeof speoificity towardsa restrictednumberof steroids. As most steroids ocmxring iu urine gave no similarcolourat the concentration of lo-20 ug in 1.5 ml reactionmixture,it seems that no appreoiable interference can be expeotedin oolourdevelopmentby known
STEROIDS
456
8:4
urinarysteroidsof polarityaimilarto that of testosterone (eyeterns:Gupta and Tanner,1965).
At the moment work is in
progressto adapt this calorimetric method for the study of the urinaryexcretionpatternsof testosterone in developing prepubertalchildren.
This work was eupportedby a grant from the MedicalResearch Councilto Dr. 3, M. Tannerwhom we wish to thank for his criticism and encouragement. REFERENCES Allen,
W.M., Hayward, and Pinto,A.
S.J.
Dorfman,R.I., Soroini,G. M. Brust,N., Nightingale, and Forohielli,E.
J. CLIN. ENDOCR.2, 54 0950) PROC. 44THMEE?pING ENDOCR.SOC. P 15 (1962)
Finkeletein, M., Forchielli,E. J, CLIN. ENDOCR.& 98 (1961) and Dorfmsn,R.I. HATUBE203, 187 (1964) Gupta, D. snd Tanner,J.M. Gupta,D. snd Tanner,J.M.
BIOCHEM.3. &,, 25 P
Kalant,H.
BIOCHEM.J. a, 79
VA., Mslraer, F., Srtego, C,MI.and Ssmuels,L.T.
(1965)
(1958)
Koenig,
J. BIOL. CHEM.141, 487 (1941)
REV. CANAD.BIOL.I;e,399 (1956) Martin,E.A. ACTA ENDOCRINOL.x, 237 (1961) Oertel,G.W. Reichstein,T, and Sboppee,C.W. VITAM. AND HORM. A, 345 (1943) Rosner,J.M., Conte,N.F., Briggs,J.H., Chao, P.Y., Sudman,E.M. snd Forsham,P.H. J. CLIN. ENDOCH.25, 95 (1965) NATURE201, 296 (1964) Sachs,L, QUANTITATIVEINORGANICANALYSIS. Vogel, 8.1. 2nd ed. Longmane,Green:London (1953) Wilson,H. Wintersteiner, 0. and Pfiffner,J,J,
ANALYT,BIOCHEM.1, 402 (1960) J. BIOL, CHEM,116, 291 (1936)
STEROIDS
Oct. 1966
457
TABLE1 INF'LUENCEOFSULPHDRI CACID CONCENTRATION ONTHEDEVELOPMJZNTOFCHROMOGEX?ICITY
Concentration of H2SO4 in the ceric sulphate-H2S04 reagent
Maximumabsorbancegiven by 20 ug of testosterone
lWj5
0.78 0.76 0.72 0.74
2% 3% 4%
TABLE II INFLUJZNCE OFDIFFEZfENT~TALS ON THF, DEVEWPMENT OFcHFKNOGmCITP Metals used
F@lTiC
Manganese
Cobalt chromic
Cupric
Maxmum abaorbaucegiven by 20 ug of testosterone Peak at 620
Peak at 595
Peak at 470
mu
Mu
mu
0.66 None None None 0.24
None 0.29 0.30 0.26 None
None 0.32 0.35 0.38 0.24
458
8:4
STEROIDS
TABLE III IRFLUENCE 09 ACETIC ACID CONCEXJTRATIOX AS A DILUTIKG PLZCIDON THE DEXELOPPIEKTOF CIIRO~~OGEZICITY
Diluting fluid used
Maximum absorbance given by 20 ug of testosterone
0.64
l$ Acetic acid 3$ Acetic acid 3: Acetic acid lC& Acetic acid Water 2; Propionic acid
0.68
0.66 0.71
0.58 0.56
TABLE IV
AIJ&N CORE0Xl'EDFUZSULTSOF TKE CXROMOGEKICITY GIVEN BY TESTOSTEXOI'JB AT DIFJ?EZREXl' CONCEl4Pl'RATICNS
Testosterone (w)
1.0 ::; 7.5
10.0 12.5 15.0
Allen corrected results (average of 10)
S.D.
Allen corrected results (per w per ~1)
0.038 0.105 0.212
.009 .012 .028
0.026 0.028 0.028
0.312 0.403 0.500 0.594
-039 .028 0034 .023
0.027 0.027 0.027 0.027
STEROIDS
Oct. 1966
459
EFFECT OFTHECIBIC SULPEAT%H2SO4RlUGENTOrJVARIOUSSTEROIDS Steroid
Colour
copperblue Teetoaterone 11 n Epi-testosterone " n Androatenedione " n i-Androstenolone II n Ii-OH-Testosterone n n 6p-OH-Progesterone green Z&OH-Te8tosterone ll+OH-bndrostenedione pale yellow n n 7-oxo-DEA n (1 +bndrost-16-ene-3-01 Androsta-5-en-3 ,17 -dial peach/yellow Androsta-l,+diene-3,17-dione peach 5&Androstan-3,17-dione colourless 5B-dndrostan-3,11,17-~ione palebuff Androetenetrione colourless Androeterone yellow Aetiocholanolone pale yellow Dehydroepiandrosterone buff Epi-androsterone pale yellow ll&OH-=Audrosterone peachjiplnk 38-21-~~~regn-5-en-20-one colourlese Ecgosterol-Dacetate colourless Cholicacid oolourless B-Ionone PM X-Substance(Guptasnd Tanner,1964)peaoh Corticosterone colourless Cortisone colourless Tetrahydrocortisone colourless ll- Deoxycortisol(Reichstein's S) colorless Tetrshydro-ll-dehydrocorticosterone colourless (TW
Peak at Chr0lRO620 mu genicity (T - 100) 100 3-0 66 3-s 99 3-e 3-e Yes
yes NO
NO NO
No No No No No No No No NO
No NO NO
NO NO
No No No No No No No
71 63