A sensitive spectrophotometric method for adrenaline and noradrenaline

CLINICACHII\uCA ACTA

VOL.4 (1959)

307

SHORT COMMUNICATIONS

A sensitive spectrophotometric

method for

adrenaline and noradrenaline” Calorimetric methods for adrenaline which are based on oxidation to the red compound, adrenochrome, are relatively insensitive because the molecular extinction coefficient of this compound at its maximum in the visible spectrum is relatively low. These methods require between 20 and 200 pg of adrenalineI. Fluorometric methods are much more sensitive, requiring less than I pg of the medullary hormones. However, these methods are attended by various technical difficulties and require rigid control to maintain reasonable accuracy. The present method is intermediate in sensitivity between the fluorometric methods and the calorimetric methods mentioned above, being useful with the amounts usually available following paper chromatographic separation, i.e. between I and 20 pg. It retains the simplicity of colorimetric methods, although it cannot be classed as a calorimetric method since it measures the absorption of light of a wave length slightly below the visible range. In this region the light source may be either a tungsten or a hydrogen lamp. The cuvettes may be either glass or quartz. The increased sensitivity over the methods based on determination of adrenaline as adrenochrome (or iodoadrenochrome) is made possible by use of a derivative or transformation product (I) of adrenochrome which has a much higher absorption peak than the adrenochrome peak in the visible range. Adrenaline is determined by oxidation to adrenochrome (or iodoadrenochrome), then converted to a compound (I) which is remarkably stable at pH 5 and has a strong absorption at 347 rnp. It is apparently identical with intermediate (V) of HARLEYMASONSfor which he has proposed a zwitterionic structure. Both compounds absorb maximally at 347 rnp and both are readily converted to 3,5,6-trihydroxy-r-methylindole (adrenolutin) on addition of alkali. Similarly, noradrenaline is determined by oxidation to noradrenochrome (or iodonoradrenochrome) followed by conversion to the analogous compound (II) absorbing at 350 m,u. Using the procedure outlined below, adrenaline or noradrenaline, when separated chromatographically, may be estimated within an error of less than 30/ when 20 ,ug or more are available for analysis. With IO pug the error is within 5%. Although perceptible readings can be obtained with as little as I ,ug, the y0 error is much higher. The reagents are the same as those of the modified VON EULER AND HAMBERG method described earlier3, except that I N acetate is used rather than 0.1 1L’.The increased concentration of acetate facilitates formation of (I) from adrenochrome or iodoadrenochrome. The latter are formed by oxidizing adrenaline with iodine followed by removal of excess iodine with thiosulfate. The reaction products of iodine and thiosulfate apparently include a small amount of the reducing compound, hydrosulfite (dithionite), which is capable of producing the HARLEY-MASONreaction under these * This investigation was supported in part by research grant H-3231 from the Xational Heart Institute, U.S. Public Health Service. References fi. 309

conditions. The reducing activity of thiosulfate alone does not produce this rcactiorl, as shown by the failure of sodium thiosulfatc to yield (I) w IWJI added to ;I solution of adrenochrome” in acetate buffer. The reaction products, potassium iodide and sodium tctrathionate**, when added to adrcnochrome or iodoadrenocllrom~,” * * alto g:i\.c, negative results. However, addition of the premised iodine a~lcl thiosulfatc~ solution!does give rise to (I) OJI standing.

Test spots containing 2 to 25 ,ug of adrenaline or noradrcnalinc on tiltcr paper are cut out and placed in ro-ml flasks. To each is added 2 ml of I X acctatc buffer, pH 5.0. The flasks arc placed in a water bath at 30” and 0.4 ml of 0.1 A’ iodine solu-

0.7.

0.6-

0

I

2 MicVogroms

3

4 of

the

5

6

free

base

7 per

8 ml

9 of

final

IO

I

solution

tion 3 is added. After mixing and standing I min for adrenaline and 5 min for noradrenaline, 0.9 ml of 0.05 N sodium thiosulfate is added. The mixture is shaken until the iodine color disappears. It is allowed to stand in stoppered flask at room temperature for 18 to 20 h, transferred to cuvctte, and read against a reagent blank prepared simultaneously. \I:ave length 347 rnp is used for adrenaline and 350 for noradrenalinc. A sensitive instrument such as the Beckman IIT2 Spectrophotometcr is used, with the standard r-cm cuvettc. The calibration curves shown here were prepared by analyzing z-ml portions of standard solutions of adrenaline bitartrate or noradrenaline hydrochloride freshly prepared in I N acetate lxrffer, pH 5. Essentially the same results are * TVe are grateful to b. NORMAX I&%RSEL of [nternational Hormones, Inc. for kindly supplying crystalline adrenochrume freshly prepared and to Dr. J. D. CHANLEY, Mt. Sinai Hospital, for his suggestion with regard to obtaining the freshly prepared compound. * * I’repared by a method recommended by Dr. JOHN H. SPEER of G. 1). Searlc & Co. *** l’rq~%red b\- the method of %BOTK.k ASI) AKST1.u’.

VOL. 4

SHORT C‘OMMUNICATIONS

(1959)

309

obtained when the standards are introduced on filter paper and eluted above. Further studies concerning the structure of (I) are in progress. Department

IiOSE L. ‘l-SE*

of Physiological Chemistry, Woman’s Jledical Philadelfihia, Pa. (CT.S.A .)

College of Pennsylvania,

31. JASE

in D. GLICK, Methods of Biochemical .1?lnlyses, Vol. II, Interscience Sew York, 1955, p. 57. 2 J. HARLEY-MASON, J. Chem. Sot., (1950) 1276. 3 hf. J. OESTERLING, Biochim. Biophys. .1cta, ‘4 (1957) 175. a H. SOBOTKA AND J. AUSTIN,,/. <~VJZ. Chew. .%c., 73 (1951) 3078. 1 H. PERSKY

Received * Medical

Student

Adenosine

Research

Fellow,

and inosine

I‘.S.

Public

Health

nucleotides

as described

Service,

content

()ESTERI.IS(;

l’ul~lishers,

Sovember

Summer

Inc.,

rst, 195s

19.5s

of human

placenta

Recently we described methods1 for the separation and determination of adenosine and inosine nucleotides and nucleosides in microgram quantities in tissues. This has allowed us to study the content of these substances in human placenta. Small pieces were excised from the placenta of a normal pregnancy and delivery immediately after the expulsion, rapidly washed from blood under running tap water and frozen in acetone-dry ice mixture. The samples were then homogenized at o” in z vol. (wet wt.) ice-cold zoo/, trichloroacetic acid. The homogenate was centrifuged at -2’ and an aliquot of the supernatant analyzed by paper chromatography as previously described’. The quantitative determination of adenosine and inosine nucleotides was done by the orcine reaction after elution of the spots. Results are given in Table I. TABLE ADENIKE

jtg/ 100 g wet weight * AMP, phosphate.

ADI’

AND

INOSINE KUCLEOTIDE

1 CONTENT

OFHUMAN

PLACENTA*

Na,A TP

Na,.-I LIP

NaJ

MP

46.5 + 6.4 ,I = 9

_I’..+ 2 6.S P, = Y

37.7 i n=7

2.4

and

ATI’

= adenosine

mono-,

di- nnd

triphosphate;

.Va.JMP ‘i

ir 4.i ?l = q

IMl’ = inosine

mono-

It can be seen that for the content in ATP and ADP, variability is much greater than for AMP. This can partly be due to the presence in placenta of a strong ATPase, ADPasc and adenylate kinase activity, which will be described in detail elsewhere.

Institute of Biological Chemistry and I&it&e qf Ohsfefrics and Gyrcaecology of the 7’niversity of Rome (Italy)

I’AOLO ~ERLETTI CLARA

liROXTICEIL1

EMANUELE ’ 1’. CERLETTI,

r'.1,.IPAT. AND

9. SILIPRANDI, .‘f1zd. Chim.

LAVRICELLA

.-l&Z,16 (1957) 54%

Received

August

rxth,

195s