Pergamon Prat
Lils Soisnces Vol. 16, pp " 375-384 Printed in the II .8 .11.
A SBZISITIVS BADIO~YMATIC ASSèY POB NOBEPINSPHNIIiS IN TISSIISS AND PLASMA David P. Hoary, Barbra J . Sterna, David G. Jobneon, and Bobort H. Williass Departseat of Medicine, ünivereity of Waehington,School of Medicine, Seattle, Washington 98195 (Received in final form January 1, 1975) Swoary A procedure for assuring picogras quantities of norapinephrine has been developed utilising partially purified bovine adrenal phenylethaaolasine-N-aethyl-tranafnrase and 3[H]-S-adenósyl snthioninn . The sensitivity of the assay is 25 pg, and the procedure is applicable to many body tissues and fluids, including brain and plash . Several radioeasy~atic assay procedures for assaying catecholaaines have been developed in recent yare (1,2,3) .
This report describes a new sethod
utilising the co~nrsion of norepinephriae (NB) to tritiated npiaaphrine (8) by partially purified bovine adrenal phenylethaaolasine-N-methyl-transferaae (P1~lf) and tritiated S-adenoeyl-sethionine (SAMS) .
The athod is staple, affi-
cleat, and applicable to all tissue and fluids tested, including brain and plasma .
The sensitivity of the assay ie approsamataly 25 pg, and largo numbers
of samples can be processed in a relatively short tile . ?laterials and Methods Batortals:
Diathylhe:ylphosphoric acid (DHBP) vas obtained fron Basten
Rodak; [~ SAID (8 .5 Ci/mmol) a~
[~ epinephrine (6 .5 Ct/snol) ware obtained
fron New Bugland Nuclear Corp ; dithiothreitol, norepinaphrine bitartrate, epi nephrine-bitartrate and SAME-chloride were obtained fron Cal-Bloche:.
Alusina
vas obtains fron Merck and Co . and acid-washed by the sathod of Anton and Sayer (4) .
All other reagents were of analytical grade .
PNMT Preparation PNKf va~ partially purified Eros fresh bovine adrenals .
Fros 15 adrenals
33 g of medullary tissue vea dissected a~ homogenised in 280 sl of cold 1.19x 375
Radioenzymatic Norepinephrine Assay
376
KC1 for 45 sec in a blaring blender .
Vol . 16, No . 3
The homogenate was centrifuged at 40,000
The supernatant was filtered through cheese cloth and recen
x g for 60 min.
trifuged at 100,000 x g for 60 min . 20,000 x g for 10 min.
All subsequent centrifugatione were
5 ml of 0 .5 K sodium phosphate buffer, pH 7 .5, was
added to 250 ml of the supernatant at 5°C, and, over a 45 min period, 44 g of solid (NH4)2504 was added .
The precipitate was removed by centrifugation, and
19 .8 g of (NH4) 2504 per 100 ml was slowly added .
After centrifugation the
supernatant was discarded and the precipitate resuspendeü in 100 ml of 55Z saturated (NH4)250~0.0125 M sodium phosphate buffer, pH 7 .5, and slowly stirred for 30 min.
After centrifugation the procedure was repeated with 35X
saturated (NH4) 2504-0 .0125 M sodium phosphate buffer, pH 7.5 .
Any insoluble
materíal that remained after 30 min of stirring was removed by centrifugation and discarded .
For each 100 ml of supernatant 17 g of
(NH4) 250 4 was added and,
after 30 min of stirring, the precipitate removed by centrifugation .
The pre-
capitate was resuspe~ded in 100 ml of 0 .005 M Tris-HC1, pH 7 .4, containing 0 .1 mK dithiothreitol and dialyzed in 1 L of the same buffer with four buffer changes in a 12 h period .
The dialyzed enzyme was then centrifuged at 40,000
z g for 10 min and frozen in small aliquots . so that 10
ul
The PNMT was subsequently diluted
of the enzyme contained twice the activity necessary to produce
the maximum amount of E from 10 ng NE under the conditions of assay.
The Vmaz
of the diluted enzyme was 1 .39 pmole E/min~yl enzyme, and the protein concen tration was 7 .5 ug/ul.
The enzyme preparation from 33 g of adrenal is adequate
for greater than 20,000 assays and is stable when stored at -20°C for more than 12 months . Sample Preparation When samples contained NE in conceatrationa greater than 10 pg/mg, they were homogenized in 10 volumes (weight :vol) of cold 0 .1 N perchloric acid and centrifuged at 4°C for 10 min at 10,000 x g . so that 50 to 100
ul
The supernatant was then diluted
aliquots contained between 50 and 5000 pg NE .
For plasma
or tissue perfusates containing less than 10 pg NE/mg as initial alumina chro-
Rndioenzymatic Norepinaphrine Away
Vol. 16, No . 3
matographic step was necaaeary.
377
IIP to 10 sl of perfusate vas placed in a 15 nl
Hunan blood vas anticoagulated with either SDTA or
conical centrifuge tube .
heparin and the plasma vas rapidly separated from the cells by low speed centrifugation .
The plasma was then stored at -20°C.
Immediately before assay
2 ml of plasma was diluted 1:1 with distilled water and placed in a centrifuge
ul
of freshly prepared sodium~tabiaulfite solution (50 mg/
100 ml) was added.
The pH of the samples was adjusted to at least pH 8 .0 by
tuba .
Nezt 100
the addition of 0.5 ml of 1 .0 M Tris-HC1, pH 8 .6, containing 2 g/100 ml SDTA . Alumina vas added by a scoop prepared from a small polyethylene tube which reproducably dispensed 50 mg of alumiaa .
The samples were vortezed 3 base for
1-3 sec and centrifuged briefly in a clinical centrifuge .
The supernatant was
The ahmina was washed with 3-5 ml of water by vor-
aspirated and~diecarded .
teeing 2-5 sec, centrifuging, and decanting the supernatant . cedure was repeated three times . teeing three tiles for 1-3 sec.
N8 was eluted with 250
a
The washing pro-
of 0.2 N HC1 by vor-
The recovery of this step was detersined with
each assay by adding 5 ng NE to a duplicate sample before alumna adsorption . Assay Procedure:
Aliquots of 50-100
ul
of the tissue hosogenates, or
100 J1 of the alumina slants were placed in 15 ml conical centrifuge tubes . Internal standards consisting of 1000 pg NS in 50 with each assay.
ul
of 0.01 N HC1 were run
Blanks consisted of perchloric acid or HC1 only .
were adjusted to the sema volume . 35
ul
All samples
Freshly prepared reaction siz consisting of
of 1 M Trie-HC1, pH 8 .6, in 5 g/100 sl SDTA, 0.1 mM dithiothraitol, 5
[3H]SAMg (2 .5 uCi) and 10
ul
PNMT was added .
ul
ühea samples were preadsorbed on
alumina, 2 M Tris-HC1, pS 9 .2, in 5 g/100 ml BDTA wan used as buffer . samples were then incubated for 30 min at 37° C.
The
The reaction was stopped by
the addition of 2 .0 ml of 0 .5 M sodium phosphate, pH 10 .0, containing 5 g/100 ml SDTA and 0.1 mM dithiothreitol . penned 150 mg of alumina.
Alumina vas added by a scoop which die-
The samples ware vortezed 3 times each for 1-3 sec.
After settling for 30 sec, the liquid was aspirated.
The alwina was washed
3 base by vortezing 1-3 sec after the addition of 3-5 ml H20 .
After each
378
Radioenaymatic Norepinephrine Assay
Vol . 16, No .
3
wash, the alumina was allowed to settle for 30 sec and the water removed by aspiration .
After the third wash and aspiration, the [~i]-epinephrine was
extracted with 1.0 ml of cold 0.1 N perchloric acid by vortezing 3 times for 2-5 sec.
Neat 50 yl of freshly prepared 0.2 N acetic acid containing 25 ug
epinephrine and 100 ug SAME-chloride was added . addition of 100 H20) .
ul
This was followed by the
of a saturated solution of phoaphotungetic acid (25 g/100 ml
The tubes were allowed to sit in ice for 5 min and then centrifuged for
5 min at a low speed in a clinical centrifuge .
A 1.0 ml aliquot of the super-
natant was transferred to a second 15 ml conical centrifuge tube containing 1 .0 ml of 1 .0 M potassium phosphate, pH 7 .15, and 10 ml of 1X (v :v) DEHPtoluene .
The tubes were shaken vigorously on a reciprocating shaker for 3 min.
After a 10 min low speed centrifugation to separate phases, 9 ml of the clear organic phase was transferred to a plastic ecintillatioa vial containing 400 yl of Liquiflor (New England Nuclear) .
The samples were then quantitated by am-
Meat temperature liquid scintillation epectrophotonetry .
Phase separation
occurred if the samples ware counted at low temperature . Results The recovery of NE in the initial alumna concentration step averaged 50X.
Recoveries of E throughout the product isolation procedure were followed
using commercially prepared [3H]E pre-purified immediately before use by alu mina adsorption and elution.
The recovery after the alumina step was 52X .
After the phosphotuagstic acid precipitation step there was a 30X loss due to aliquotting .
The DEHP extraction was 95X efficient, but again there was a lOX
aliquotting lose, since only 9 nl of the DEHP was counted . of epinephrine was 33X.
Blank recovery was only 0.005X .
Overall recovery The absolute counting
efficiency for tritium depends oa the liquid scintillation counter used .
The
counting efficiency of a 1X DSHP-toluene liquiflor solution ie, however, equal to that of a toluene-liquiflor solution, which is considered to have the highest counting efficiency generally available for liquid scintillation counting . The assay can accurately measure amounts of norepinephrine from 25 pg to
vol . 16, No . 3 at least 15 ng (Fig . 1) .
Radioaasysatic Norepinephria~ Aasay
The assay is specific for NB .
37 9
None of the naturally-
occurring catecholasines or their setabolic products eith~ar interfere with or are detected by the assay (Table 1) . TABLE 1 NS-P1~ Substrate Specificity cps-blank Alone
Plus 1 ng NB
7,248
-
Epinephrine
142
7,580
Dopamine
-26
7,449
Norsetanephrine
10
7,279
Metanephrine
48
7,135
Methozytyramine
30
7,287
Octopasine
72
7,464
Tyramine
16
7,355
Norepinephrine
Average S.D . S.S .N .
7,364 147 55
Blank consisted of 100 ul of 0 .01 N HC1 . Each of the quines vas tested alone and in the presence of an equal amount of NS in a final vohme of 100 ul . Results are ezpressed as cpm/ng amies . Blank vas 195 cpu. The procedure vas applicable to all tissues tested, including brain and the plassa of a variety of animals (Table 2) .
The assay vas linear over a
vide range of tissue earact concentrations when NE internal staadards were included to correct for [~]SAC dilution by endogenous SAMB (Fig . 2) .
When
alusina preadsorption is not needed, 50 saaplee caa be processed in a day. Discussion In this assay NB is N-sethylated by PNI~ a~ [3H]SAT~ to form [38]B, which is selectively isolated and seaeured by liquid scintillation apectrophotosetry.
The specificity of the assay ie the result of prafarcntial siethyla
tion of NS by PNlif and selection adsorption of the B product by alud.na .
380
Radioenzymatic Norepinephrine Assay
DV
Vol . 16, No . 3
NE
FIG . 1 Norepinephrine standard curve . Samples were diluted in 100 U1 of 0 .01 N HC1 . Blanks consisting of 100 ul of 0 .01 N HC1 gave 155 cpm . TABLE 2 NE Content in Tísauee and Plasma
Superior cervical ganglia (rat)
25 .715 .3 ng/ganglia n - 8
Iris (rat)
6 .211 .2 ng/iris n - 8
Hypothalamus (rat)
1 .10 ng/mg n - 4
Plasma (human)
0 .25410 .077 ng/ml n - 7
Plasma (dog)
0 .28510 .084 ng/ml n - 7
Plasma (baboon)
1 .51±0 .59 ng/ml n - 7
Serum (rat)
1 .9511 .52 ng/ml n - 11 Tissues were homogenized in 0 .1 N perchloric acid . Human venous plasma was obtained from recümbeät fSeYed subyects . Dog plasma was from file femoral artery under barbiturate anesthesiâ . Baboon plasma was from the inferior vena cava of chair-trained animale . Rat serum was from the tail vein . Ttie results are expressed as ng NE ± S .D .
Vol. 16, No . 3
Radiosaaymatic Norapinephriaa Assay
381
Highly efficient reaoval of contaainating radioactivity is acco~liehed by perforaing the alumina adsorption at high pH in the presence of phosphate and by selective precipitation of SA?~ with phosphotungetic acid .
Ion pair solvent
eztraction with DSPH-toluene ie also used to increase the sensitivity and specificity of the assay. PNMT aethylatea a variety of phenylethanola~ine derivatives but is epacific for the phenylethanolamine moiety .
Aulrod (5) found that the naturally-
occurring seines, NS, 8, normetaaephrine, aetanaphrine, and octopamina, ae well ae a variety of phenylethanolaaine-derived droga, such as amphetamine, were substrates for PNMT .
Fuller et sl . (6) also found that ring-halogenated and
alpha-methylated pheaylethanolamines were PNMT substrates or inhibitors .
How-
ever, these investigators demonstrated that NS was the preferred substrate at low eubetrate concentrations, having the lowest Ra and highest Va~:.
Saelene
et al . (1) also demonstrated that NS was prefareatially methylated at low concentrations of asines .
In the picograa range of this sassy, NB was the only
generally-encountered pheaylethanolaaine derivative detected (Table 1) .
Other
potential substrates either were not N-methylated or were not eztracted. Fuller et al . demonstrated that NS produced eubetrate inhibition at high concentrations
(6) .
This has been confirmed by Saelens et al . (1) .
In our
assay, inhibition has not bees observed when sale NS content was kept below 15 .0 ng .
Other substrates for PN!!P are coapetitive inhibitors of PNMT when
the Nraethylation of N8 ie followed .
None of the naturally-occurring catacho-
laaines, however, inhibited the comiareion of N8 to B (Table 1) .
It should be
noted that drugs that contain both the phenylethanolaaine and catechol moiety are potential substrates or inhibitors of the PNKf reaction and their influence upon this assay should be evaluated before they are included in ezperimental samples . Alumina adsorption at alkaline pH is a standard isolation procedure for catechol compounds (7) .
Non-catechol substrates for PNMT are not adsorbed .
Ae noted by Drell (8), adsorption at pH 10 in the presence of phosphate Baits
382
Radioenzymatic Norepinephrine Assay
Vol . 16, No . 3
FIG . 2 Whole rat brain was hoaogenized in 10 volmnes (w :v) cold 0 .1 N perchloric acid and centrifuged at 10,000 x g for 10 min . The supernatant was diluted with perchloric acid so that a 50 ul eagle contained the indicated amount of tissue . Internal standards (1 ng NE in 50 ul~of 0 .01 N HC1) were included with each dilution and were used to calculate the content of NB . Aliquots containing 20 ng of tissue reduced internal standard counts by 52X . the non-specific binding to alumina of many compounds, including SAME, without significantly reducing E recovery .
We have found that the inclusion of phos-
phate considerably reduces blanks in our procedure .
Additionally, phospho-
tungatic acid is an effective precipitating agent for sulfonium compounds and has been used in a preparative procedure for SAME (9) .
The combination of
batch alumine adsorption chromatography and phosphotungatic acid precipitation efficiently eliminates the SAME blank and specifically isolates catecholamines . DSBP, a well-characterized ion-pair solvent extraction reagent, has been used previously to isolate catecholamines with high efficiency (10) .
Thin solvent
extraction step reduces the blank and increases specificity . Perchloric acid does not adversely affect the PNMT reaction ae long as pR is maintained at 8 .4, and, therefore, tissue extracts can be used directly without neutralization and the associated oxidative lose which has been aeso dated with this procedure (11) .
Thin assay can also be used with tissues or
Vol . 16, No . 3
383
Radiosnzymatic Norepinsphrina 1lseap
body fluids which contain NS in low concentrations, since NS can be concentrated efficiently by en initial alumina chromatographic procedure . As noted by Iversen and Jarrott (11), previous PN1+ffP-based NB assays have not been applicable to brain tissue because of variable results .
Our proce-
dure is easily applied to brain tissue and can measure N8 in as little as 20 ug of rat hypothalamus .
Large brain samples reduce the radioactivity pro-
duced from NE and [3H]SAML, but samples of any size can be analysed if internal standards of NS are used (Fig . 2) . SA1~ present in brain tissue .
tiost of the reduction of coasts is due to
Haldasearini and Ropin (12) have found that rat
brain contains 0.011 ug SA1~/mg.
The assay system described contains only
0 .12 ug [3H]SAl~ in each incubation tube .
Therefore, 10 mg of brain entract
would reduce the specific activity of 3Al~ to approzimately SOx of control values and reduce the specific activity of the 8 formed to an equivalent degree . Previous ensymatic assays for catecholamines have had serious deficiencies .
The procedures of 3selens and ~mracsica (1), as modified by Iversm sad
Jarrott (11), also utilise PlalT.
However, this assay ie limited to a 10
sample sad requires a 12 h chromatographic step . ie approzimately 100 pg .
ul
The sensitivity of the assay
Sagelmsn et al . (2) have developed a procedure uti-
using COIRr, cation exchange chromatography, periodate azidation and solvent eztraction .
The procedure ie laborious and has a sensitivity in our laboratory
of approzímately 1000 pg .
The sway procedure does not distinguish between
norepinephrine and epinephrine .
Sogelman has modified the procedure by includ-
ing a thin layer chromatographic separation of the ormethylated catecholamines . Thus, N8 and B can be assayed individually, but only with a significant increase in camplezity and reduced sensitivity .
Thn aasymatic method of Coyle
and Henry (3) uses CQQ, solvent eatractíon, and periodate oxidation.
This
assay has a sensitivity of 100 pg and can be performed in a short time period, but does not distinguish between NS and 8 .
Furthermore, it ie not possible to
assay catecholamines with this method after en initial preadsorption on alumine
Vol . 16, No . 3
Radioenzymatic Norepinephrine Assay
384
The PNMT-based assay for NE described in this paper is highly specific and more sensitive than existing radioenzymatic procedures .
It employs a com-
bination of well-characterized procedures and requires little special equip ment .
It is anticipated that this method will be particularly useful in
measuring NE in small amounts of tissue, such ae discrete areas of brain or is fluids, such se plasma, where it is present in minute concentrations . Acknowl~ta This research was supported by NIH research grants AM 05020-19 and AM 02456 .
D.P . Henry was a NIH Special Research Fellow (F03 AM 53427) and D .G .
Johnson is tha recipient of a NIH Career Development Award (K04 AM 70727) . Sincere thanks are eztended to Dr . I .J . Ropin for encouragement and support during the early portions of this work . References 1.
J .K . SAELSNS, M.S . SCHOEN, and G.B . ROVACSICS, Biochem. Pharm . 16, 1043 (1967) .
2.
K. ENGELMAN, B . PORTNOY, and W. LOVENBERG, Am . J. Med. Sci . 255, (1968) .
3.
J .T . COYLE, and D.P . HENRY, J. Neurochem. 21, 61
4.
A.H . ANTON, and D.F . SAYRE, J. Pharmacol . Exp. Therap . 138, 360 (1962) .
5.
J. AZELROD, J. Biol . Chew . 237, 1657
6.
R.W . FULLER, B .J . WARREN, and B.B . MALLOY, Biochem. Biophys. Acta 222, 212 (1970) .
7.
T. NAGATSU, Biocheaietry of Catecholamine : University Park Prese, Baltimore (1973) .
8.
W. DRELL, Anal . Bioche~. 34, 142 (1970) .
9.
J .A . STEKOL, Methode in Enzymology , Vol. 6, p . 566, Academic Press, New York (1963) .
259
(1973) .
(1962) .
the Biochemical Method , p . 212,
10 .
R. MODIN, and M. JOHANSSON, Acta Pharm . Suec . S, 501 (1971) .
11 .
L .L . IVERSSN, and B. JABR0IT, Biochea. Pharm . 19, 1841 (1970) .
12 .
R.J . BALDASSARINI, and I .J . KOPIN, J . Neurochem . 13, 769 (1966) .
Lils Soisnces Vol. 16, pp " 375-384 Printed in the II .8 .11.
A SBZISITIVS BADIO~YMATIC ASSèY POB NOBEPINSPHNIIiS IN TISSIISS AND PLASMA David P. Hoary, Barbra J . Sterna, David G. Jobneon, and Bobort H. Williass Departseat of Medicine, ünivereity of Waehington,School of Medicine, Seattle, Washington 98195 (Received in final form January 1, 1975) Swoary A procedure for assuring picogras quantities of norapinephrine has been developed utilising partially purified bovine adrenal phenylethaaolasine-N-aethyl-tranafnrase and 3[H]-S-adenósyl snthioninn . The sensitivity of the assay is 25 pg, and the procedure is applicable to many body tissues and fluids, including brain and plash . Several radioeasy~atic assay procedures for assaying catecholaaines have been developed in recent yare (1,2,3) .
This report describes a new sethod
utilising the co~nrsion of norepinephriae (NB) to tritiated npiaaphrine (8) by partially purified bovine adrenal phenylethaaolasine-N-methyl-transferaae (P1~lf) and tritiated S-adenoeyl-sethionine (SAMS) .
The athod is staple, affi-
cleat, and applicable to all tissue and fluids tested, including brain and plasma .
The sensitivity of the assay ie approsamataly 25 pg, and largo numbers
of samples can be processed in a relatively short tile . ?laterials and Methods Batortals:
Diathylhe:ylphosphoric acid (DHBP) vas obtained fron Basten
Rodak; [~ SAID (8 .5 Ci/mmol) a~
[~ epinephrine (6 .5 Ct/snol) ware obtained
fron New Bugland Nuclear Corp ; dithiothreitol, norepinaphrine bitartrate, epi nephrine-bitartrate and SAME-chloride were obtained fron Cal-Bloche:.
Alusina
vas obtains fron Merck and Co . and acid-washed by the sathod of Anton and Sayer (4) .
All other reagents were of analytical grade .
PNMT Preparation PNKf va~ partially purified Eros fresh bovine adrenals .
Fros 15 adrenals
33 g of medullary tissue vea dissected a~ homogenised in 280 sl of cold 1.19x 375
Radioenzymatic Norepinephrine Assay
376
KC1 for 45 sec in a blaring blender .
Vol . 16, No . 3
The homogenate was centrifuged at 40,000
The supernatant was filtered through cheese cloth and recen
x g for 60 min.
trifuged at 100,000 x g for 60 min . 20,000 x g for 10 min.
All subsequent centrifugatione were
5 ml of 0 .5 K sodium phosphate buffer, pH 7 .5, was
added to 250 ml of the supernatant at 5°C, and, over a 45 min period, 44 g of solid (NH4)2504 was added .
The precipitate was removed by centrifugation, and
19 .8 g of (NH4) 2504 per 100 ml was slowly added .
After centrifugation the
supernatant was discarded and the precipitate resuspendeü in 100 ml of 55Z saturated (NH4)250~0.0125 M sodium phosphate buffer, pH 7 .5, and slowly stirred for 30 min.
After centrifugation the procedure was repeated with 35X
saturated (NH4) 2504-0 .0125 M sodium phosphate buffer, pH 7.5 .
Any insoluble
materíal that remained after 30 min of stirring was removed by centrifugation and discarded .
For each 100 ml of supernatant 17 g of
(NH4) 250 4 was added and,
after 30 min of stirring, the precipitate removed by centrifugation .
The pre-
capitate was resuspe~ded in 100 ml of 0 .005 M Tris-HC1, pH 7 .4, containing 0 .1 mK dithiothreitol and dialyzed in 1 L of the same buffer with four buffer changes in a 12 h period .
The dialyzed enzyme was then centrifuged at 40,000
z g for 10 min and frozen in small aliquots . so that 10
ul
The PNMT was subsequently diluted
of the enzyme contained twice the activity necessary to produce
the maximum amount of E from 10 ng NE under the conditions of assay.
The Vmaz
of the diluted enzyme was 1 .39 pmole E/min~yl enzyme, and the protein concen tration was 7 .5 ug/ul.
The enzyme preparation from 33 g of adrenal is adequate
for greater than 20,000 assays and is stable when stored at -20°C for more than 12 months . Sample Preparation When samples contained NE in conceatrationa greater than 10 pg/mg, they were homogenized in 10 volumes (weight :vol) of cold 0 .1 N perchloric acid and centrifuged at 4°C for 10 min at 10,000 x g . so that 50 to 100
ul
The supernatant was then diluted
aliquots contained between 50 and 5000 pg NE .
For plasma
or tissue perfusates containing less than 10 pg NE/mg as initial alumina chro-
Rndioenzymatic Norepinaphrine Away
Vol. 16, No . 3
matographic step was necaaeary.
377
IIP to 10 sl of perfusate vas placed in a 15 nl
Hunan blood vas anticoagulated with either SDTA or
conical centrifuge tube .
heparin and the plasma vas rapidly separated from the cells by low speed centrifugation .
The plasma was then stored at -20°C.
Immediately before assay
2 ml of plasma was diluted 1:1 with distilled water and placed in a centrifuge
ul
of freshly prepared sodium~tabiaulfite solution (50 mg/
100 ml) was added.
The pH of the samples was adjusted to at least pH 8 .0 by
tuba .
Nezt 100
the addition of 0.5 ml of 1 .0 M Tris-HC1, pH 8 .6, containing 2 g/100 ml SDTA . Alumina vas added by a scoop prepared from a small polyethylene tube which reproducably dispensed 50 mg of alumiaa .
The samples were vortezed 3 base for
1-3 sec and centrifuged briefly in a clinical centrifuge .
The supernatant was
The ahmina was washed with 3-5 ml of water by vor-
aspirated and~diecarded .
teeing 2-5 sec, centrifuging, and decanting the supernatant . cedure was repeated three times . teeing three tiles for 1-3 sec.
N8 was eluted with 250
a
The washing pro-
of 0.2 N HC1 by vor-
The recovery of this step was detersined with
each assay by adding 5 ng NE to a duplicate sample before alumna adsorption . Assay Procedure:
Aliquots of 50-100
ul
of the tissue hosogenates, or
100 J1 of the alumina slants were placed in 15 ml conical centrifuge tubes . Internal standards consisting of 1000 pg NS in 50 with each assay.
ul
of 0.01 N HC1 were run
Blanks consisted of perchloric acid or HC1 only .
were adjusted to the sema volume . 35
ul
All samples
Freshly prepared reaction siz consisting of
of 1 M Trie-HC1, pH 8 .6, in 5 g/100 sl SDTA, 0.1 mM dithiothraitol, 5
[3H]SAMg (2 .5 uCi) and 10
ul
PNMT was added .
ul
ühea samples were preadsorbed on
alumina, 2 M Tris-HC1, pS 9 .2, in 5 g/100 ml BDTA wan used as buffer . samples were then incubated for 30 min at 37° C.
The
The reaction was stopped by
the addition of 2 .0 ml of 0 .5 M sodium phosphate, pH 10 .0, containing 5 g/100 ml SDTA and 0.1 mM dithiothreitol . penned 150 mg of alumina.
Alumina vas added by a scoop which die-
The samples ware vortezed 3 times each for 1-3 sec.
After settling for 30 sec, the liquid was aspirated.
The alwina was washed
3 base by vortezing 1-3 sec after the addition of 3-5 ml H20 .
After each
378
Radioenaymatic Norepinephrine Assay
Vol . 16, No .
3
wash, the alumina was allowed to settle for 30 sec and the water removed by aspiration .
After the third wash and aspiration, the [~i]-epinephrine was
extracted with 1.0 ml of cold 0.1 N perchloric acid by vortezing 3 times for 2-5 sec.
Neat 50 yl of freshly prepared 0.2 N acetic acid containing 25 ug
epinephrine and 100 ug SAME-chloride was added . addition of 100 H20) .
ul
This was followed by the
of a saturated solution of phoaphotungetic acid (25 g/100 ml
The tubes were allowed to sit in ice for 5 min and then centrifuged for
5 min at a low speed in a clinical centrifuge .
A 1.0 ml aliquot of the super-
natant was transferred to a second 15 ml conical centrifuge tube containing 1 .0 ml of 1 .0 M potassium phosphate, pH 7 .15, and 10 ml of 1X (v :v) DEHPtoluene .
The tubes were shaken vigorously on a reciprocating shaker for 3 min.
After a 10 min low speed centrifugation to separate phases, 9 ml of the clear organic phase was transferred to a plastic ecintillatioa vial containing 400 yl of Liquiflor (New England Nuclear) .
The samples were then quantitated by am-
Meat temperature liquid scintillation epectrophotonetry .
Phase separation
occurred if the samples ware counted at low temperature . Results The recovery of NE in the initial alumna concentration step averaged 50X.
Recoveries of E throughout the product isolation procedure were followed
using commercially prepared [3H]E pre-purified immediately before use by alu mina adsorption and elution.
The recovery after the alumina step was 52X .
After the phosphotuagstic acid precipitation step there was a 30X loss due to aliquotting .
The DEHP extraction was 95X efficient, but again there was a lOX
aliquotting lose, since only 9 nl of the DEHP was counted . of epinephrine was 33X.
Blank recovery was only 0.005X .
Overall recovery The absolute counting
efficiency for tritium depends oa the liquid scintillation counter used .
The
counting efficiency of a 1X DSHP-toluene liquiflor solution ie, however, equal to that of a toluene-liquiflor solution, which is considered to have the highest counting efficiency generally available for liquid scintillation counting . The assay can accurately measure amounts of norepinephrine from 25 pg to
vol . 16, No . 3 at least 15 ng (Fig . 1) .
Radioaasysatic Norepinephria~ Aasay
The assay is specific for NB .
37 9
None of the naturally-
occurring catecholasines or their setabolic products eith~ar interfere with or are detected by the assay (Table 1) . TABLE 1 NS-P1~ Substrate Specificity cps-blank Alone
Plus 1 ng NB
7,248
-
Epinephrine
142
7,580
Dopamine
-26
7,449
Norsetanephrine
10
7,279
Metanephrine
48
7,135
Methozytyramine
30
7,287
Octopasine
72
7,464
Tyramine
16
7,355
Norepinephrine
Average S.D . S.S .N .
7,364 147 55
Blank consisted of 100 ul of 0 .01 N HC1 . Each of the quines vas tested alone and in the presence of an equal amount of NS in a final vohme of 100 ul . Results are ezpressed as cpm/ng amies . Blank vas 195 cpu. The procedure vas applicable to all tissues tested, including brain and the plassa of a variety of animals (Table 2) .
The assay vas linear over a
vide range of tissue earact concentrations when NE internal staadards were included to correct for [~]SAC dilution by endogenous SAMB (Fig . 2) .
When
alusina preadsorption is not needed, 50 saaplee caa be processed in a day. Discussion In this assay NB is N-sethylated by PNI~ a~ [3H]SAT~ to form [38]B, which is selectively isolated and seaeured by liquid scintillation apectrophotosetry.
The specificity of the assay ie the result of prafarcntial siethyla
tion of NS by PNlif and selection adsorption of the B product by alud.na .
380
Radioenzymatic Norepinephrine Assay
DV
Vol . 16, No . 3
NE
FIG . 1 Norepinephrine standard curve . Samples were diluted in 100 U1 of 0 .01 N HC1 . Blanks consisting of 100 ul of 0 .01 N HC1 gave 155 cpm . TABLE 2 NE Content in Tísauee and Plasma
Superior cervical ganglia (rat)
25 .715 .3 ng/ganglia n - 8
Iris (rat)
6 .211 .2 ng/iris n - 8
Hypothalamus (rat)
1 .10 ng/mg n - 4
Plasma (human)
0 .25410 .077 ng/ml n - 7
Plasma (dog)
0 .28510 .084 ng/ml n - 7
Plasma (baboon)
1 .51±0 .59 ng/ml n - 7
Serum (rat)
1 .9511 .52 ng/ml n - 11 Tissues were homogenized in 0 .1 N perchloric acid . Human venous plasma was obtained from recümbeät fSeYed subyects . Dog plasma was from file femoral artery under barbiturate anesthesiâ . Baboon plasma was from the inferior vena cava of chair-trained animale . Rat serum was from the tail vein . Ttie results are expressed as ng NE ± S .D .
Vol. 16, No . 3
Radiosaaymatic Norapinephriaa Assay
381
Highly efficient reaoval of contaainating radioactivity is acco~liehed by perforaing the alumina adsorption at high pH in the presence of phosphate and by selective precipitation of SA?~ with phosphotungetic acid .
Ion pair solvent
eztraction with DSPH-toluene ie also used to increase the sensitivity and specificity of the assay. PNMT aethylatea a variety of phenylethanola~ine derivatives but is epacific for the phenylethanolamine moiety .
Aulrod (5) found that the naturally-
occurring seines, NS, 8, normetaaephrine, aetanaphrine, and octopamina, ae well ae a variety of phenylethanolaaine-derived droga, such as amphetamine, were substrates for PNMT .
Fuller et sl . (6) also found that ring-halogenated and
alpha-methylated pheaylethanolamines were PNMT substrates or inhibitors .
How-
ever, these investigators demonstrated that NS was the preferred substrate at low eubetrate concentrations, having the lowest Ra and highest Va~:.
Saelene
et al . (1) also demonstrated that NS was prefareatially methylated at low concentrations of asines .
In the picograa range of this sassy, NB was the only
generally-encountered pheaylethanolaaine derivative detected (Table 1) .
Other
potential substrates either were not N-methylated or were not eztracted. Fuller et al . demonstrated that NS produced eubetrate inhibition at high concentrations
(6) .
This has been confirmed by Saelens et al . (1) .
In our
assay, inhibition has not bees observed when sale NS content was kept below 15 .0 ng .
Other substrates for PN!!P are coapetitive inhibitors of PNMT when
the Nraethylation of N8 ie followed .
None of the naturally-occurring catacho-
laaines, however, inhibited the comiareion of N8 to B (Table 1) .
It should be
noted that drugs that contain both the phenylethanolaaine and catechol moiety are potential substrates or inhibitors of the PNKf reaction and their influence upon this assay should be evaluated before they are included in ezperimental samples . Alumina adsorption at alkaline pH is a standard isolation procedure for catechol compounds (7) .
Non-catechol substrates for PNMT are not adsorbed .
Ae noted by Drell (8), adsorption at pH 10 in the presence of phosphate Baits
382
Radioenzymatic Norepinephrine Assay
Vol . 16, No . 3
FIG . 2 Whole rat brain was hoaogenized in 10 volmnes (w :v) cold 0 .1 N perchloric acid and centrifuged at 10,000 x g for 10 min . The supernatant was diluted with perchloric acid so that a 50 ul eagle contained the indicated amount of tissue . Internal standards (1 ng NE in 50 ul~of 0 .01 N HC1) were included with each dilution and were used to calculate the content of NB . Aliquots containing 20 ng of tissue reduced internal standard counts by 52X . the non-specific binding to alumina of many compounds, including SAME, without significantly reducing E recovery .
We have found that the inclusion of phos-
phate considerably reduces blanks in our procedure .
Additionally, phospho-
tungatic acid is an effective precipitating agent for sulfonium compounds and has been used in a preparative procedure for SAME (9) .
The combination of
batch alumine adsorption chromatography and phosphotungatic acid precipitation efficiently eliminates the SAME blank and specifically isolates catecholamines . DSBP, a well-characterized ion-pair solvent extraction reagent, has been used previously to isolate catecholamines with high efficiency (10) .
Thin solvent
extraction step reduces the blank and increases specificity . Perchloric acid does not adversely affect the PNMT reaction ae long as pR is maintained at 8 .4, and, therefore, tissue extracts can be used directly without neutralization and the associated oxidative lose which has been aeso dated with this procedure (11) .
Thin assay can also be used with tissues or
Vol . 16, No . 3
383
Radiosnzymatic Norepinsphrina 1lseap
body fluids which contain NS in low concentrations, since NS can be concentrated efficiently by en initial alumina chromatographic procedure . As noted by Iversen and Jarrott (11), previous PN1+ffP-based NB assays have not been applicable to brain tissue because of variable results .
Our proce-
dure is easily applied to brain tissue and can measure N8 in as little as 20 ug of rat hypothalamus .
Large brain samples reduce the radioactivity pro-
duced from NE and [3H]SAML, but samples of any size can be analysed if internal standards of NS are used (Fig . 2) . SA1~ present in brain tissue .
tiost of the reduction of coasts is due to
Haldasearini and Ropin (12) have found that rat
brain contains 0.011 ug SA1~/mg.
The assay system described contains only
0 .12 ug [3H]SAl~ in each incubation tube .
Therefore, 10 mg of brain entract
would reduce the specific activity of 3Al~ to approzimately SOx of control values and reduce the specific activity of the 8 formed to an equivalent degree . Previous ensymatic assays for catecholamines have had serious deficiencies .
The procedures of 3selens and ~mracsica (1), as modified by Iversm sad
Jarrott (11), also utilise PlalT.
However, this assay ie limited to a 10
sample sad requires a 12 h chromatographic step . ie approzimately 100 pg .
ul
The sensitivity of the assay
Sagelmsn et al . (2) have developed a procedure uti-
using COIRr, cation exchange chromatography, periodate azidation and solvent eztraction .
The procedure ie laborious and has a sensitivity in our laboratory
of approzímately 1000 pg .
The sway procedure does not distinguish between
norepinephrine and epinephrine .
Sogelman has modified the procedure by includ-
ing a thin layer chromatographic separation of the ormethylated catecholamines . Thus, N8 and B can be assayed individually, but only with a significant increase in camplezity and reduced sensitivity .
Thn aasymatic method of Coyle
and Henry (3) uses CQQ, solvent eatractíon, and periodate oxidation.
This
assay has a sensitivity of 100 pg and can be performed in a short time period, but does not distinguish between NS and 8 .
Furthermore, it ie not possible to
assay catecholamines with this method after en initial preadsorption on alumine
Vol . 16, No . 3
Radioenzymatic Norepinephrine Assay
384
The PNMT-based assay for NE described in this paper is highly specific and more sensitive than existing radioenzymatic procedures .
It employs a com-
bination of well-characterized procedures and requires little special equip ment .
It is anticipated that this method will be particularly useful in
measuring NE in small amounts of tissue, such ae discrete areas of brain or is fluids, such se plasma, where it is present in minute concentrations . Acknowl~ta This research was supported by NIH research grants AM 05020-19 and AM 02456 .
D.P . Henry was a NIH Special Research Fellow (F03 AM 53427) and D .G .
Johnson is tha recipient of a NIH Career Development Award (K04 AM 70727) . Sincere thanks are eztended to Dr . I .J . Ropin for encouragement and support during the early portions of this work . References 1.
J .K . SAELSNS, M.S . SCHOEN, and G.B . ROVACSICS, Biochem. Pharm . 16, 1043 (1967) .
2.
K. ENGELMAN, B . PORTNOY, and W. LOVENBERG, Am . J. Med. Sci . 255, (1968) .
3.
J .T . COYLE, and D.P . HENRY, J. Neurochem. 21, 61
4.
A.H . ANTON, and D.F . SAYRE, J. Pharmacol . Exp. Therap . 138, 360 (1962) .
5.
J. AZELROD, J. Biol . Chew . 237, 1657
6.
R.W . FULLER, B .J . WARREN, and B.B . MALLOY, Biochem. Biophys. Acta 222, 212 (1970) .
7.
T. NAGATSU, Biocheaietry of Catecholamine : University Park Prese, Baltimore (1973) .
8.
W. DRELL, Anal . Bioche~. 34, 142 (1970) .
9.
J .A . STEKOL, Methode in Enzymology , Vol. 6, p . 566, Academic Press, New York (1963) .
259
(1973) .
(1962) .
the Biochemical Method , p . 212,
10 .
R. MODIN, and M. JOHANSSON, Acta Pharm . Suec . S, 501 (1971) .
11 .
L .L . IVERSSN, and B. JABR0IT, Biochea. Pharm . 19, 1841 (1970) .
12 .
R.J . BALDASSARINI, and I .J . KOPIN, J . Neurochem . 13, 769 (1966) .
