A sephadex extraction of urinary steroid conjugates

195

D. GUPTA and JAIUT GOODWIN Departmentof Growthand Development, Instituteof ChildHealth,Universityof London.

ReceivedMay 16, 1966.

ABSTRACT

A simplemethod for the isolationof the C 9 and $1 steroid conjugatesfrom urine with SephadexG-50 (coarse ? is described. Followingcontractionof the urine volume by Sephadex,the Sephadex cake was washedwith ethanol:methanol(1:lv/v) mixtureand this was added to the contractedurine sampleand evaporatedto dryness. Two additionsof Sephadexreducedthe urine volumeby about 65$. The yield of 17 oxosteroidsand 17 hydroxycorticosteroids averaged106s and 97% respectivelyof that obtainedby a standardsolventextraction method and the recovery of individualsteroidconjugatesadded to water varied from Y& to 103$. Comparisonof individualeteroids by fractionation after hydrolysiswith the resultsby the normalmethod showed that they were unalteredby Sephadextreatment. Severaltechniquesfor the isolationof steroidconjugates from urine are currentlyemployed. Norymberski's(1960)method in which the pyridiniumsalts of steroidsulphatesare extractedinto chloroform,is only useful for steroidsulphates. Liquidion-exchangetechniques (Kushinskyand Tang, 1963) producetroublesomeemulsions. The extractionof steroid. conjugates by charcoalabsorption(Grant and Beall, 1950; Arcos and Lieberman,1965) is still in the stage of development. RecentlyZumoffand Bradlow(1963)have describeda method of precipitation of steroidconjugateswith egg albumin. The most widelyused methodsof extractionare with organicsolvents(Talbotet al.,1943; Edwards

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et al, 1953; Jayle, 1961; Kernel,1963). In our laboratorythe method of Edwardset al, modifiedby using iso-propenolinsteadof ethanol &q$a

8~‘hmer,

19651, hae been successful,but when large

volumesof urine are consideredit becomeslaboriousand unsattifactory. There is also the dangerof fire becauseof the considerablevolumesof iuflammablesolventsinvolved. Large amountsof ammoniumsulphateare carriedinto the organicextractsand there are possibilities of structural. changesdue to the drastictreatmentof urine with hydrochloricacid. This paper describesa method which eliminatesthese disadvantages by wing Sephadex.

Sephadex was first used by Plodin et al (1960)to

isolatecelluloseby conceutratiug solutesof high molecularsubstances, such as dye-proteinconjugates(George & Walton,19611,haemoglobin (Deutschet al, 19631, fibrinolysiusolutionscontainingglycerol (Painter& NcVicar,1963) and erythropoietin from plasma and kidney extracts(Hansen,1963). Materials and Hethode

All solventswere of analyticalgrade and doubledistilledin the laboratory. Sephadex(G-25Fine, G-25 Coarse,G-w Fdne, G50 Medium,G-50 Coarse,G-75 Coarse and GlOO Coarse)was obtatiedfrm! Pharmacia(GreatBritain)Ltd. Solventextraction,hydrolysisand fractionation of individualsteroidswere done using procedures previouslydescribed(Gupta,1965; Gupta & Tamer, 1965). The extractsand filtrate8from the Sephadexexperimentswere evaporated to dryuessunder reducedpressureand tetnperature by using a rotary eva rator. Spectrophotometric measurementswere made on the Unicem SP EO . All the referencesteroidswere obtainedfrom the Steroid ReferenceCollection(MedicalResearchCouncil). DEA-sulphatewas generouslysuppliedby OrganonLaboratoriesLtd., and the glwuronide salts of androsteroneand aetiocholanolone were Preparedin the laboratory(Gupta,1965).

Aug. 1966

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197

Reeults Prelim~ lh

Ebsperiments: a series of preliminary experiments the parameter8investigated

were: the ratio of Sephadexto the volumeof urine, the volume and nature of solvents used to wash the Sephadexffltercake, the nature of the Sephadex and the effectof time, temperatureand PH.

Experienceshowedthat occ~eionalshakingof urine samplesfor 120 minuteswith the appropriateamountof Sephadexwas sufficientfor optimal swellingof the Sephadexgrains. It was also found that pH

and temperaturedid not have any significanteffecton the swellingof Sephadex. Variationfrom 4OC to 3’7’Cia the temperatureat which the concentration was carriedout and variationin the pH of the urine from 2 to 10 did not alter the percentagerecovery* All subsequentexperiments were done at room temperaturewithoutadjueiting the pR of the urine samples. The followinggradesof Sephadexwere used: 625 @ins), 6-25 (Coarse),G-50 (J?ine)J G-50 (Medium),6-50 (Coarse),G75, G-100. To each 100 ml sampleof urine 5g of Sephadexof a particulargrade wae added. The samplewas then left at room temperaturefor 120 minutes and filtered. The Sephadexfiltercake was waehedwith 50 ml of ethanol. The re8ult.s on the wash were comparedwith the resultson the unconcentrated urine. The most satisfactoryresultwas obtainedwith the G50 coarsegrade where the concentrated volumewae low and the recovery quantitative. To determinethe optimalweight of SephadexG50 coarse100 ml urine sampleswere stirredwith variousamountsof Sephadexfor 120

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TABLE I

E~TOFV~Y~GT~~O~OFS~H~~~50

(COARSE)UPONTHE

RECOVERYOF STEROIDCONJUGATESFROM URINE

Mean and individualrecoveriesa6 % of that extractedby the standardprocedure of ether: i6o-propanol(3:l v/v).

174xosteroid6

2

x06 (115,96,lo8,1o5)

3

105 W+,log,llo,g8,lo7,

17-hydroxycorticosteroids 101

98 (95,m;,;vjw,

1121 5

lo6

(116,97,1o:;s2,95,

(1o7,9t,lo5,98)

s

97 @w;y;~6, 9

8

98 (94,96,m5,9?~

92

10

89 (@,85,99

85 (88,86,81)

12

84 (87,83,82)

80 (74,78,88)

(97,8$%94)

minutesat room temperature. The sampleswere filteredand the Sephadexfiltercake was washed with ethanol (6&/g

of Sephadex).

The resultsobtainedwere comparedwith those obtainedby the standard extractionprocedureused in the laboratory. Table I shows that from 2 to 5g of Sephadexper 100 ml of urine producedgood recoveries,and as 5 g contractsthe urine volume more than lower quantitiesthis wa8 adoptedas the standardamount.

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199

8olvents

6.mlh of senhadex 17-oxosteroids

106(116,97,103,112 95,110s 24sthanol

92(98,87,91)

E-01: methsnol(lrl v/v) 105(93,110,110,99 106,112j Ethanol: propyleneglycol (3d

17=hydroxycorticosteroids 97(92,llo,93,96,9o,lol) 107(101,110,111) 107(109,115,107,111,98,102)

93(91,95)

S7~81,93)

Ethanol: ethyl acetate(2tl) 87(83,91)

69(61,77)

BWhanol: ethyl acetate(2:X)

73(67,79)

78(81,75)

Ether; iso-propsnol(1:s)

72(75,69)

71(65,77)

Next the efficiencyof variousorganicsolventsas elutingagents was studied. In all cases 5 g of SephadexG_To coarseper 100 ml of urine

and 6 ml of solventper gram of Sephadexwere used. It was

found (TableII) that the best overallresultswere obtainedwith methanol: ethanolmixture (l:l),

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TAELE III liS!FEZT OF SUCCESSIVE CONC~TION

S!EROIDCONJUGATXSFRONURINE,

STEPS ON TEE BECOVERY 03' SZEB.&D~G-5O.CO~SE

(5GPHL

100 ML OF URINE) AND 6 MLOF ETHANOL: MEEHANOL (It1 V/V) PER G OF~~~t~U~.

Mean and individual recoveries exmeesed

as % of that extrsctedb_vstandard stem,

urine vol.

170oxosteroids

1

35

105~93,110,110,99 106,112j

17&ydroxycorticosteroids 107(10g,l15,107,111 98,1023

2

50

105(110,101,98)

107(103,106,112)

3

64

106(107,100,111}

105(lO7,109,99)

4

77

102(lO4,lO5,97)

lol(lO5,96,lO3)

Table III shows the recoveries when successive concentration steps were carried out. Two concentration steps gave a 5%

reduction

in urinary volume without any loss OC steroidconjugates. With three concentration steps the volume reduction averaged 64%. The recoveries,

expressedas the percentages of the results obtained by the standard extraction procedure by solvent8 do not differ essentially as the number of successive concentrations increases. Table IV shows the recoveries of the reference steroid conjugates when added to water. In each case 5g of Sephadex G-50 coarse and 6 ml of methanol: ethanol mixture (1:l) per gram of Sephadex were used. With free steroids the recoveries were low.

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STEROIDS

Steroidsadded to water Conziurrates Nhydroepiandrosterone eulphate Androsteronesulphate

92 ~i02,80,9i,84,i05,~) 103 (96,108,107,101)

Androsteroneglucuronitie

97 (99,92,103,94)

Aetiocholanolone glucuronide

93 (96,901

Free steroids Androsterone

71 (80,62)

Dehydroepisndrosterone

75 (72,78)

Cortisol

83 @7,79)

Cortisone

81 {77,86)

Corticosterone

77 @5,69)

Tetrahydro-ll-deoxy-cortisol

71 (68,74)

After finalisation of the method,in two cases (not selected) the extractedconjugateswere subsequentlyexaminedby completefractionation and individualqua&it&ion, and comparedwith the results obtainedby the standard method of extraction. The urine specimen was dividedin two: one portionW&B extractedwith ether: isopropanol mixture,and the other was treatedwith Sephadex, The results (Table 5) show the comparisonsare as good as duplicatesby the standard method (Gupta,1965).

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TABLEV COMPdRISONOF AMOUNTSOF VARIOUS Clp AND C71 STEROIDSISOLATEDBY SEFflADEX ANDTHE STANDARDSOLVENTEKTRACTPBOCEDURES. AJ?TEEt EXTRACTIONOF THZ CONJUGATES.HYDHOLYSISAND INDIVIDUALFRBCTIONATION WERE CARRIEDOUT BY THE PROCEDlJMX OF GUPTA AND TANNER (1965). (G - GLUCURONIDES; S = SULPHATES)

t-----m--STEROIDS ISOLATED __-.... 11-OH-Aetio

Fmmurs = w/24 hr. SUBJECT A

SEPHADEX G IS

’ 0.43 11-OH-Andre 0.39 11-oxoaetio 0.72 ll-oxoandro 0.27 Dehydroepiandro 0.66 0.20 Aetiocholenolone1.95 0.23 Androsterone 3.12 0.32

Cortol Cortolone

SUBJPTB

__OLVENT EXT. S

or46 1 0.40 ’ 0.59 0.22 0.53 0.18 1.78 0.27 2.94 0.30

y=P$-+

y-y+

0.41 0.49 0.78

0.36

0.10 0.17 0.13

0.46 0.73 0.22 0.28 0.25 0.71 0.19 0.57 1.98 0.30 2.05 0.25 2.95 0.48 2.71 0.42

0.34 0.55 0.63 1.57 2.40

0.11 0.23 0.35 1.23 1.32

0.32 0.52 0.55 1.54 2.33

0.12

0.21

0.19 0.40 1.05 1.15

0.72 0.25 0.75 0.68 0.43 0.74 0.95 0.77 0.84 3.0 0.71 2.56 - ,-

0.33 0.30 0.40 0.49 0.35

0.35

0.35 0.32 0.33 0.54 0.38

0.38 0.39 0.22 0.32 0.23 0.40 0.20 0.43 0.32 1.17 0.32 1.03 0.28 0.44 0.40 0.41 0.20 0.10 0.13 0.12

0.28

0.27 0.51 0.21

-'

0.24 0.38 0.71 0.69

0.27 0.17 0.24 0.31 0.17

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1966

203

STEROIDS

Procedure8 for the extraction of urinary steroid conjugate8 using Seph8dex G-50 (ooarse) a8 an ab8orbent of the aqueou8 uha80: The technique finally adopted wa8 as followe:

5g of Sephadex G-50 (coaree) were added to 100 ml of urine without any prior adjustment of pR. The suspension wa8 left at room temperature with occasional stirring. After 120 minute8 it was filtered on a Buchner funnel. The urine filtrate wae collected. The filter cake was then washed with 6 ml of methanol: ethanol (1:l v/v) per gram of Sephadex used and the Wash wa8 collected separately. The urine filtrate vas then subjected to another concentrationstep with Sephadex a8before. The two urine filtrates and the two washes were pooled and dried on a rotary evaporator under reduced pressure and temperature. Ii at this stage the VOhmIO of the solution i8 not small enough for handling, further concentrationsteps can be carried out. The concentration in two steps can be accomplished in less than 5 hours with enough spare time to carry on with other job8 in between. The Sephadex cake after washing was regenerated by suepending it twice in water followed by once in ethanol. It wae filtered on a Buchner funnel and washed with ethanol several times until the Sephadex grains turned completely white. It was then broken, spread on a shallow glass tray, and dried at 8O'C. The dried substance can be used for new concentration steps without any detectable difference in it8 properties at least for 6 operations. Discussion An ideal technique for the recovery of conjugated steroid8 from urine would be one which was quantitative,which permitted the easy manipulation of large volumes, provided an extract relatively free of extraneous material6 and which caused no undeeirable structural alterations in the steroids involved. The results obtained demonstrate that concentrationof urine volumes by Sephadex meets their criteria. Specimens derived from Sephadex treatment were much less pigmented than those from ether: isopropanol extract. Moreover, when water

WaS

taken up by the swollen grains, salts and other 8olutes of low moleculer weight were removed from the urine to an extent that ionic strength and pH remained approximately constant during the procedure. We found this method particularly useful for the isolation

204

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of a steroid-likesubstance(Gupta& Tanner,1964) from a large pool (30 litres)of urine, as the substanceis excretedin minute quantities. ACKNOWLEDGEMENTS This work was supportedbY a grant from the MedicalResearch Councilto Dr. J.M. Tannerwhom we wish to thank for criticismand encouragement. REFERENCES Amos, PI,& Lieberman,S,

J. CLIN. ENDCCR.3, 808 (1965)

Deutsch,B., Levere,R.D. & Levin, J.

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Edwards,R.W.H.,Kellie,A.E. & Wade, A.P. MEW SOC. ENDOCR.& 53 (1953) Flodiu,P., Gelotte,3. & Porath,Y.

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Gupta, D.

THE DEVELCPMENTOF INDIVIDUALDIFFERENCES IN STERCIDEKCR-ION PATTERNSDURING HUMAN GROWTH. THESIS,LOND. UNIV. (1965)

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Gupta, D. & Tamer, J.M.

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Kornel,L.

J. CLIN. ENDCCR.2, 1192, (1963)

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ACTA. ENDOCR. (KBH)2, 345, (1963)

McKema, J. & Norymberski,J.K.

BIOCHEM.J. 2, 60, &XXI>

1188 (1961)

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Painter,R.H. & McVicar,G.A.CANAD. J. BIOCHEM.PHYSIOL. 4l, 2269 (1965) !&rnoff, B. & Bradlow,H.L.

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