Breaking the lipid barrier in partition chromatography a steroid memoir

Ian E. Bush BREAKING

June

1985

THE LIPID

BARRIER IN PARTITION A STEROID MEMOIR

Steroids

CHROMATOGRAPHY

Volume

45, Number

6

SC

480

BREAKING

WBEOTDI

THE LIPID BARRIER IN PARTITION A STEROID MEMOIR

CHROMATOGRAPHY

Ian E. Bush Research Service Veterans Administration Medical and Regional Off%zeCenter White River Junction, VT 05001

It was an honor to be asked to contribute Steroids.

Contemplation

the most important, is to illuminate in scientific

experience

But this cannot

to catch

father

paper,

a longer

experiment a bottle

and made a number

the effect

purpose of scientific

that

in Great

of improvements

he wrapped

at various

of the coin on the paper on the other

And an autobiography

side.

I

to say.

will find

tale.

Britain

out with a sheet

bit, and a flashlamp. to adopt

in the technique.

it around

distances

described

one has very little

milk, a threepenny

band, and then hung the coin on a thread

and held the flashlamp

that are not ordinarily

I took part in was carried

radiologists

that

through a memoir of one young man’s

but blander

of diluted

in

autobiography

period, in the hope that readers

of beam size on the image produced

a small hole in the paper

unless

glimpses of this process

was one of the first

practice,

elastic

on a more limited

of it, than to digest

wrapping

summary

series

me to the conclusion

be done by summary alone.

must be mostly

concentrated

The first serious green

soon forced

those parts of the process of discovery

of some fifteen pages

it more valuable

however,

and perhaps the only respectable,

papers.

have therefore

of the task,

to the new autobiographical

by a scattering

tomography He wanted

medium.

the milk bottle, inside the bottle.

fastened

of My

in his to study

After cutting it with an

I was his assistant

from the hole while he observed

the shadow

“The coin,” he said, “is like a small tumor.

The bottle

I want to see how we might sharpen

could be, say, the liver.

up that shadow

a bit.” We spent I was twelve, passing

the bbby

of Britain

that

was raging

my father

of intuition

to be confined

epitomizes

had on it.

combined

Burch.

I wanted

introduction. miles away bicycles snoring

later to

make

A remarkable in the valley

an

below

one warm afternoon loudly

in a deckchair.

the lawn and declared

After gesture

that

moon before Leaping

that

had fallen

personality

six weeks before,

over a hundred

to the

east.

life and the considerable he did, it taught

me the

was far too important

hours; and that

one could do a and a clear idea.

in the school vacations

telescope,

in Physics at Bristol

our house.

and

my father

University,

with CR. secured

an

Burch lived a few

and I arrived

When my brother

in early September,

that it was tea-time.

miles

and the

began to be drawn forth more sinfully

to study optics

We helped

to hear the news on

as long as one had ingenuity

on our

Burch was taking a nap in his workshop,

his

wife pick apples

until

he appeared

on

He was tall, thin to the point of emaciation,

with short grey hair and a small, sharp chin. the notion

as they hurried

working

astronomical

lecturer

The few vacationers

also that science

and normal

when 1 began

on Exmoor.

Like most of the things

with almost any apparatus

years

of milk, and sizes of holes.

one side of my scientific

with practicality:

The other side of my scientific some three

France

in the skies a Iittie

to the workplace

useful experiment

dilutions

in a hotel

It was 1940.

This early experience

virtues

distances,

gave us hardly a glance

in the lounge.

influence

various

and we were on vacation

through

the radio Battle

an hour trying

My younger

brother

seriously

entertained

he came from Mars.

tea on the lawn, Burch bent his tiny jaw to his chest made him look like a quizzical you go home to supper,

from his deekchair.

we could hardly keep up with.

jackdaw:

“If we’re

young man, we’d better

he lead the way to his workshop I was in a daze.

start

in a characteristic going

to look

on that

with springy

at

the

telescope!” steps

that

1 had read a lot about the painstaking

grinding or

and

months;

cleaning

But his

Burch

bench,

cast

and

iron

lap,

Foucault

test

mirror

of and

found

about

f.12.

blinded

Once

polish

pinhole

namely, have

Tycho

in

and

a rewarmed

went

lesson

I believe

perfectly

tidy.

But

slowly

over

valuable:

the

primacy

of

telescope

he made I should

expect

Cassegrain

reflector

2dull?1’

followed

and

Schwarzchild’s scribbled

altitude

I was more

to was

--

interested

branches

of

chemistry

Geoffrey

Harris

had

with

immediately! All

I started

War Two

Much

greeted

in my notebook,

time

and then

obtain.

reconnaissance

the

World

him why,

alternative.

it down

By

me tell

conditions

the

given

the

next

my

he was putting

dictated finishing Air

the

Royal

research

in

the

Physiology

I had switched

the

stimulating

me a strong

than

from

anything teaching

bent

towards

the

telescope

of

and

were

experimentation: do not taught

to make

Why?

Isn’t

making that

the

horrendous

a larger

in resolving of

of

me

a

rather

theory

algebra

to a large

of and

lens

I

for

Force. Laboratory physics

else, of

fix

increase

touches

the

fifty-fold.

intention

--

the

the

to

Burch

I wanted the

of

experiments

years

on a

and ingenuity,

good

development

for

and

few

on

diameter

lawn of

first

“Oh?

basics

two-inch

darkening

announced

a four-hour Burch

the

Galilean

of

When

clutter

sealing-wax

made me calculate

by

in neurophysiology and

achieved,

camera

was over,

the

are

later,

while

of

feature

an incredulous -

lap,

uses of improvisation

theory.

weeks

Carborundum

a magnification

is a crucial

be

more

essential

to

at

in the

as the

of

serviceable out

took

incredible

grades

touch

entirely

we

in the

the resulting

Another but

mirror

a day!

pitch

fixed

Eratosthenes

a dazzling

what

four

and had

o’clock

of

me through on

hewn

seven

part lying

as long

even

1949,

rouge

a roughly

I received

something

a high

with

taken

telescope

metal

that to

power

he had

a small

better

of speculum

sealing-wax.

of

instruction

the

with

after

glories

disc

Even

and razorblade,

lath had

again

early

needed

a small

fair

we

the

silvering

a

A little

by

surfaces.

hours

with

and

optical

in two

on a wooden

eyepiece,

and

polishing

but

Cambridge

to premedical extra

W. Feldberg, general

at

and

courses Basil humoral

in

sciences. in various Verney,

and

physiology.

Two specific

problems

fascinated

of unknown

function

in the primate

secretion

of the adrenal

Lord Adriani adrenal

cortex

me: the huge number of smatl, poorly myelinated pyramidal

steroids

but suggested

intelligent

that

have time to switch

was mandatory

He was helping

supervisor. nerve

membranes

cheerful. he

to my first

fortunate

and was that

Keynes

rarest

the

chromatography

theoretical

“It would be nice to have some

project

and take

to Peter

steroids

measure

ion movements

of birds, an analytical for theory.

foundations

which was by then barely

I pointed out that Reichstein

using

alumina

promising separations

of

the

five years

had isolated

chromatographyf2);

of carotenoids

and

that

tried

since

Tswett’s

to find a simple partition

RF value on plain

filter

either

phenol,

various

And from the very beginning immense

was

well

success

had achieved

DattafSf filter

of

paper

But what had

his then amazing S.P.

cuttlefish

who wes always

into its stride(I).

on alumina-impregnated

little

success

range of adrenal

had just

paper.

obtained

But adsorption

had been stuck in a rut

work?

Dutifully

I put empirical

system

that would move testosterone

with a reasonable

However I juggled the proportions

of light petroleum,

US.

alcohols,

and water,

the steroid

stuck at the origin or ran with the solvent

testosterone

course

temptations

aside and

paper.

But theory seemed to fail benzene,

across

chemist

was a dirty word to Lewis: why did I think that chromatography for forty years

the etectronics

Lewis as my semi-official

worked so well for amino acids, sugars, and other hydrophilics with lipids.

He gave two reasons.

at Cambridge.

Like C.R. Burch, he was a stickler

emphasized

-

in being assigned

Richard

the first

And, if my ideas hadn’t worked out by the Fall

for neurophysiologists

I was immensely

(later

of the

that I try out my ideas on separating

at Cambridge

people around in other fields.”

I would stiil

nature

E.D. Adrian

in the Long Vacation.

by paper chromatography

We had too many neurophysiologists

and the chemical

My chairman,

in mammals.

liked the first project

tracts;

fibers

known

to

be

eluted

front. from

(detected

by iodine

vapor)

This was doubly odd, because highly

active

alumina

by

low

concentrations

of benzene

in hexane:

on plain

run with pure benzene!

We theorized

that

activity

for steroids

because

Lewis

but low capacity gracefully

gave

his results

and was obtaining

testosterone,

was nothing

to have to admit

less than

drunk after

congratulations

a snare

small

but gently

smudge

reiterated

of

steroid

that

plans and asking the great

weeks later

about

impeccable,

almost

I wrote,

as an adsorbent

extended,

when

of high

fused ring structure.

work on Datta’s

separations

procedure

for

of progesterone,

reservations.

of adrenal

interest

script

precious

of

the

with the pure steroids

looked

to Reichstein,

by return

arrived

explaining

To my amazement

reference

my and

of post, and a few

from Basel, labelled

of von Euw, his long-time

1 to 5 mg of the then uniquely

origin

on the

steroids.

almost

packets

the

comet-shaped,

when I started

support,

for help with reference

small cellophane

at

of

blood.

the results

with C.W. Shoppee’s

microscopic

By the standards

“have trouble”

cheerfully,

keen

stuck

can resist

Lewis offered

But the spots were slightly stubbornly

of empiricism

student

in a new technique?

his theoretical

in extracts

man expressed

this small triumph

But what graduate

But by midsummer

tentatively

sixteen

that

success

remained

steroids

How right he was! so promising

and a delusion.

were spectacular.

real work of separating

contained

to budge

Within a few weeks I had reproduced

in hindsight

I might, Lewis warned

chromatogram.

delight,

of their

and clearcut

the first wholly personal

the time the separations a

behaved

and I started

reproducible

it refused

and deoxycorticosterone.

It is awful

and

paper

cellulose

filter paper with aluminum hydroxide.

impregnating

getting

in to my pleas

filter

collaborator.

steroids

in the

Each

from Reichstein’s

collection. In the Fall, Adrian decreed large

laboratory

on the second

famous study with Geoffrey stalk,

and John Wolstencroft

that I was well launched floor,

where

Dora Jacobsen

Harris on the regeneration was studying

respiratory

and gave me a bench was installed

of blood vessels gases.

in a

doing her

in the pituitary

About one quarter

of the

floor space

was occupied

had been

used by Sir Joseph

respiratory

problems

and fuel was short. a reasonably swings

the war.

Later,

that

thermal

of temperature

chemicals

of plate

for experiments

winter,

I used Bareroft’s

in East

tanks and no official

that

and military

of postwar

Britain

grisly room-within-a-room tanks

against

But when I first

Anglia.

budget;

gases

period”

for my chromatography

efficient

was administered

saw

only some dusty glassware

and charming

by an austere

largely

man of vague

but totipotent

With his thin lips and lantern

Attwood.

to write.

him with polite

fear;

contempt

summer that my research I had known fear! I had managed

by Adrian’s

But supplies

woman.

that Dickens had never quite dared or abject

as

the wild

it I had no

and bottles

It was only through

to assemble

each of testosterone Attwood

consumables

for physioIogists

of Barcroft’s inverted

made wonderful

According

of

that

sample

chemicals

- “YOU -had benzene

of solvents

and gave me access

that

covered

Upside-down

with obsessional

to seniority,

the staff

with

treated in the

light petroleum,

in other departments

respectable

that

in oil, and a few to give up trying

class of laboratory

last month, Mr. Bush!” - and embarked Friends

fitted

the glass plates

in Organic

Chemistry

donated

lamp; and I discovered

perfectly

between

on which they

the

normally

two

legs of sat,

they

tanks.

were rapidly

would separate

aspect

to Attwood

Now, I decided

to an ultraviolet they

a

came from some novel

of progesterone

were a perfectly

Miss Elton,

and forbidding

when I had first suggested

and deoxycorticosterone.

chromatography

Lewis’ warnings

functions

the kindness of faculty

a gynecological

old bell-jars.

lab stools;

managed

jaw, Attwood

on a major campaign of begging and improvisation. and solvents

were

secretary,

would need at least one liter of good quality

to persuade

reagents

on poison

iron frames

left behind by Hugh Blaschko when he had left for Oxford eight years before.

parsimony

crystals

glass and black

It was the tlausterity

buffer

common

The laboratory

eaHed

Barcroft

during

effective

chromatography

quietly

by a huge chamber

borne out.

it was easy enough to find combinations

most of the pure adrenal

steroids

OR “alumina-paper,”

But

the

were

tailing

even

Robert

was

less

blood

on

severe,

satisfactory.

Comline,

adrenolumbar

more

and

By the

alumina-paper,

I

therefore

on the quantity

the

specific

to By

enough

iodine

by

thin-layer

achievable

with

carburettor

fun,

learnt

made

zero

progress

but would

the

in

of

they

shout

in detail,

steroids

buying

about my

was of

in the

ultraviolet

light

scope

of

and the

glowing

in

alumina, -

I was

with

the

dog’s

of adrenal

venous

obviously

getting

the

spots

depended

extracts. was

on

my

he had been

Further, sufficiently

faults

image my

of

mind, For

and

discovering

the

I knew in general.

later

realized

steroids

beautiful

I unwisely

nearly

cellulose,

-

with

the

own.

right

alumina-paper its

automobile.

goal

surgery

cannulate

of material

Lewis,

and

a new

I

positions

and that

immense;

youth,

to

extracts

trouble.

with

potential

of

how

extract

chromatograms!

chats

was

amount

of

were

of

a case

American of

normal.

supposed

disciplines instructions

readers

utterly

Cambridge

It was quite

most

-

towards

benefit

seem,

University

courses;

early

of

the

gland

seemed

separations decided

a year physical

I had

to

immense

chemistry,

chemical

fix

nature

but of

the

secretion.

might

expense.

The

polar

a tremendous

For

the

less

in

adrenal

experimental

acetate

wavelength

wrong

rashness

instead

adrenocortical

as it

the the

short

was

strong.

was

composition

occasional

what

learning

chromatograms:

nor

chromatography With

I

and the

from

too

I started

that

mess on those

to know was

explicable.

the

apart

theory

intuition

the

a commercial

I had run two ethyl

“elution”

reaction

unravel now,

But

than

with

me a demonstration

knew

rather

neither

gave time

“displacement” both

results

Nevertheless

Feldberg

vein.

and

you from

to

I should

irresponsible a reckless

British

were the

waste

bank

of

in for

time

students

the the

river first

and

was and

few

that

this

of a graduate

generally

Mine

educated.

thrown

explain

abandonment

graduate

to be fully

probably

resources received

possibly told

miles.

to But

was not,

student at

the

public

no “required”

an extreme swim. if

by

The you

were

case, coach still

afloat

in Minneapolis,

Baton

Rouge.

memories

But not just memories. Both the colleges

heaven. snobberies. the

diffraction

Site

were

structures

had just been

structure metabolism

with Dorothy

was worth

a dozen

controversy

designed

between -

less robust

Eccles

that

woefully

I doubt

four years in Cambridge. in the New Year that sent

first

his honorarium

somebody,

I suppose!”

on growth

hormone.

gleanings and

lacking

-- “Too

He passed

Feldberg

expertise

in the one field that

heartache

I decided

to take

of obtaining

with

over

the

Gregorio

discuss

role

of acetylcholine

policy

in structure,

of the British

and wasteful

sunk

wouldn’t have swum very far. still

regarded

Ph.D. system

the full three

supervisor

But you have

more than encouragement

I needed:

adrenal

friendly

hints

Cambridge

steroids

-- x

that

Feldberg

is but or

Adrian discovered

him as my official

for all its richness,

to me by

of much genuine

if I had stayed

in

bomb.

me to F.G. Young who had an active

up the

Weber,

the violent

for the views expressed

much for my conscience.

that

a definite

Discussing carbohydrate

with the problem of the atomic

“F.G.” could offer little realized

action-potential

had got far enough with

in radio-astronomy,

1 would have

the University

low-resolution

And in the Bun Shop or the Eagle one could

But I certainly

back

need -- and I suddenly

in the nerve

spectroscopy

the sink-or-swim

that

and their

on and around

the first

the very possibility

or ultraviolet

or simply relax

academics

talent.

denied

back on it, I have some sympathy

for an elite,

their politics,

had obtained

to

time was sheer

of the laboratories

the role of sodium

courses.

of Ryle’s

transmission

many American

Perutz

you through

at that

were going to be proved wrong.

Needham,

required

raging

But looking

who still

molecule

drink beer and hear

student

by Hodgkin and Huxley; and Sanger

those

for a protein

to carry

had their cliques,

and friendliness

overwhelming.

discovered

were supposed

for a graduate

and the University

of hemoglobin;

to know that

synaptic

Cambridge

But the freedom of spirit

Downing

insulin

of Mark Twain

and

to have

group working -

which I didn’t

could not provide

steroids!

With much

had dropped

before

leaving

in the Fall to head up the Physiology

intitute

for Medical

the Chemistry

Division,

Mill Hill under I left worshipped

Cambridge Adrian;

in Mill Hill,

supervision

invitation

of Callow and Feldberg

in mid-summer

Division at the National

He put me in touch with R.K. Callow

and in a few weeks an official

the joint

with the people course,

Research

and Pharmacology

feeling

terribly

for me to transfer

was sent

and I had to make a final break with a girl.

at Mill Hill but surprisingly

we don’t really

and

Adrian had been angry

kind to me about

it -

here. I* His generosity

have any chemistry

to

to Adrian.

I loved the place

guilty.

of

You’re

of spirit

right

of

only made

me feel worse. The atmosphere the chief technician of chirpy,

at Mill Hill was also splendid, of the Chemistry

intelligent

had just unloaded

Cockney.

When his eyes fell on Barcroft’s I had obtained

permission

Division was white-coated

He surveyed

from my ancient

least.

overrated

the lab stools

But you won’t be needing

the best sort

equipment

eyebrows

that

them here,

with me.

I

and open mouth. that

He shook his head slowly

of one who had always known that Cambridge

“Well, they’re

place.

and neat:

Mr. Clark,

he flinched, and I assured him ingenuously

bell-jars

to bring

way.

the pile of homemade

Woisey Hornet with raised

and then smiled with the sublime confidence was a greatly

but in a different

quite

good stools -

of the old sort,

Mr. Bush, I can assure you.

at

We’ll soon get

you some real apparatus!” And he kept his promise. time spent

on innovation,

no research

over

begging,

Lewis’

and learnt strictures

on adsorption

by the publication

separation

of adrenal

The

steroids

theft

I improved

chemistry

were

well-known

on paper impregnated was less than

the proportion

of my

mil~imum without

which

my methods of making My nagging

from Callow.

chromatography

of Zaffaroni’s

shock of being scooped

fell to that

six months

a lot of steroid

Christmas

(4).

or sheer

For another

can proceed.

alumina-paper,

I soon had all that I needed,and

brought

paper in Science

with propylene that

doubts

to a head

that

describing

the

glycol or formamide

of remembering

that

these

two

sofvents

had been Lewis’ favorite

half a liter

of formamide

By then Prudence

pride

in ~ambridga

I was a year

and empiricism

blood using Zaffaroni’s were

into

solvent

before

were in all probabifity

earlier

idea that had failed

work with

genuine

after

redoubled

partition

in Cambridge

on paper

by adding except

volatile

to control

dared

ethanol,

but

in the

even

fluctuating

for it.

work on that adrenat

venous

But for once ctose

my respect

inspection

and

of the

Which meant

under the superb

that

an

conditions

adsorption

pairs.

was distributed

temperatures

theory

for Lewis and said that

the postulated

hydrocarbon/water

ethanol

little

of steroids

None had shown any

unfavorably

and

was too

I had never

in Cambridge.

even

to try metbanoI. I obtained

strange

Callow’s support

adsorption

the Institute’s capable

af steroids

and decided

to celfutose

shop produced

to increase

a thermo~tatic%lly

controlled

a concentration

of 30% methanoi

of eortisol

absorbing positions

zones

and cortisone in the adrenal

were still

The next

but I had achieved day I raised

disappeared. methanol/water

Over ratio

the

the

gland extract

up to 6O*C,

methanol

concentration

month

with increasing

I recovered

confidence

but

imperfect,

as I tested

was On

of 3VC. the

The

ultraviolet-

I had not e~imjnated

partition

to 40% and from

later

for the first time, and their

of the extract,

if somewhat

that

the

as mobile phase with

comet-shaded,

were distinct

concentrations

a genuine,

next

at temperatures tried benzene

slightly

A month

box to my design

in the aqueous phase and a temperature

were the same at different

adsorption,

tanks

methods I gingerly

classic

my chances of defeating

by using raised temperatures.

of ho~d~~y~four large chromatography

the basis of E.C. Kendall’s

spots

to various

to show

a very

WBS worth trying

at Mill Will. f had once tried to eliminate

promise

very

cb~omatograms~

available

alcohols

to use it.

it’s too late!

theory,

chromatograms~

these

tried

your pride and start

For

alliance.

of Zaffaroni’s

but had never

systems

I had even ~‘bo~rowed”

for ~onsidefat~on~

my thesis

said swallow

in rational

photographs

candidates

the

chromatogram.

atl signs

shock

and

of taifing raised

less polar hydrocarbons

the as

mobile phases.

Fly the time 1 reached

set of solvent

systems

3,20-dione:

of handling

i.e., all major classes

The harvest pointed

capable

on alternative one year

with about

student,

David Taylor,

species

with harvestable

six months

was attempting amounts

of Taylor’s

with a passion.

specimen

forms.”

The chromatogram

revealed

chemistry:

dinitrobenzoate!

With a hundred

his scales

than

of Strophanthus

from Africa

plants.

Another

methods,

looking for

with oxygen functions

from which

By the end of 1951 I had endured

over

the questionable

value

for some reason organic

of colored

chemists

Early in 1952 he approached

of S. sarmentosus quite

clearly

at least

and ninety-three

s

and unravelled that

that

loathed

me more politely

“appeared

the weakness

four spots

Paul on the road to Tarsus

next few months we collaborated(9) biochemistry

had returned

asked if I could possibly help him “sew up a difficulty”

from his seventh

faster

purposes -

of the

two teams working

them by classical

glycosides

ribbing

As Carl Djerassi

of Strophanthus

could be generated.

a genin

in steroid

melabolites.

was enormous.

lie himself

to analyse

good-humored

and with much tergiversation

as evidence

and their

specimens

of cardiac

spots on paper for serious scientific

more polar than 5 a-pregnane-

and Callow was coordinating

two-hundred

I had a graded

at the time over the discovery

and syntheses.

group of cortisone

chromatography

excitement

of cortisone,

starting-materials

the 11-ketone

hormones

from this month of work, of course,

actions

before

and light petroleum

any steroid

of steroid

outf5), th ere was tremendous

anti-inflammatory

paper

96% methanol

with

to exist

in two

of mixed-melting

points

were detected

more specimens

with alkaline to go, Taylor

and was a changed the fascinating

man.

confusion

Callowt71 (and independently,

mlost

Over the in botanical

Reichstein

(81) had

set out to solve. From my own point of view, I was at last able to start venous blood with a tool that I could rely on of the ultraviolet

absorption

-- on the basis of Marthe

except

method for detecting Vogt’s workf9) -

on the steroids

for one last step.

a,@-unsaturated

going to be adequate;

ketones

of adrenal

The sensitivity was probably

but its specificity

S for

biological

to

see

whether

alkaline had

extracts the

in

ultraviolet

steroids,

and,

cortisol

and

not

came the

fluorescence;

maximaily have

sublimed reaction

one

of

most

interesting solvents

to to

the

or

given

the

steroid

is both

reduce

the

After

proceed

quenching;

after

sensitive

the

and Thus

And

specific it

it

and

ethanol for

cyclohex-2-enone,

by

the

damp

been

steroid

is completely

nor

the

clinical

of

the

the

British

driven

360

of

alkali

discover

what

quenched

and

of

adrenal

positions

that

to

reaction;

enough

-

I was 370

using

nm

and

m-dinitrobenzene atmosphere

must

had

allowed

off.

The

reaction

A 4-3-ketones

and

has

a number

but

not

by

by

water

presence

R-unsaturated

in

who

of whatever

showed

optimum

abolished on the

for

m-dinitrobenzene

the

24 hours,

had

is dependent

The

curtailing from

spots

with

Callow,

method

I was lucky

reaction.

far

from

chromatogram

I soon

Indeed,

rapidly,

by m-dinitrobenzene.

t-butanol. by

responsible.

first.

J-ketones

chromatograms,

a day-old

at

steroid

reliable

all

I decided

reasons

advice

fluorescent

there

fluorescence

which

a

to examine

orange

been

evaporates

peculiarities.

not

of

ketone

the

fused

groups

A6

remains of

non-polar rings:

in other

it is

parts

of

nucleustll).

A number the

soda

nm ,

254

not

by

expert

ketosteroids

I reexamined faint

was

as the

quenched

the

had

ethanol

of

day

saw

m-dinitrobenzene

be known

ultraviolet

it a habit

that

l’i’-

theoretical

given

I had

improved.

ago made One

purely

reaction

for

surprise,

cortisone

the

to

my

be

color

reaction

light.

to

weak

could

1 had long

For

to be desired.

relatively

Zimmerman’s

chemistsflO).

and

much

m-dinitrobenzene

made

sort,

left

TIIEOXD6

discovery

of

of

controversies

the

therapeutic

crisis

in

endocrinology

“Back

to

Blood”

Ingle,

and

G.

SaYerst13)

some

sort

of

glucocorticoid.

a mineralocorticoid

about

at

actions

the

time,

in his address

was

had

adrenocortical

to

well

the

secreted

summarized Society

most

there at

cortisone.

Royal

convinced But

of

all.

secretion

people

was serious These

There by

A.S.

had

been

doubt

“unitarians”

the

up by

was also

a more

general

Parkes’

vigorous

slogan

of Medicinef12). that

heated

C.N.R.

adrenal

Long,

cortex

D.J.

secreted

in some

minds

as to

whether

were

made

more

militant

by

Selye%(14)

imbalance came

grandiloquent

of the secretion

from

striking

Marthe

discoveries

of -

more

a few

minutes’

than gamble.

Vogt’s

work

feasible been

If

showed

volumes

due

to

be nothing after

all

what

known

worth

new

my

known The

ultraviolet

absorption

nor

in reasonable

volumes

cats

experiments

pattern

very

revealed

had

to

examine

emerged

glucocorticoids,

only

but

in

proportions

that

evolutionary

position

of

the

or

no evidence

of a known

Dunne of

(of

F.G.

“adrenal

flogging

Young’s

exhaustion” with

lipid-free corticosterone

cells

I disproved

long-acting hanging like

ACTH, in

the

a sponge

gangbusters!

rat’s of

There

steroids, in had

there (the

might oil

was so great detect

the

venous

blood

engorged could

species

left that

active

and

rats,

secreted

and

major

with

one

indeed capillaries;

have

but

like it

been

or to

the was

Stack-

that

a Stage

six

a string

was still a

both

Mickey

After

looked

time

There

-

syndrome’(lg).

Vogt’s

a strange

either

tenets

from

By the

relationship

secreted;

hardly

from

of life or diet(l@.

Selye’s

“alarm adrenal

rabbits,

obvious

habits

of

expected

studies(16p17)).

All

being

one

initial

amounts

ferrets,

no

their

team)

a

detected

fraction”

of adrenal

and Hechter’s

bore

mineralocorticoid

his

in

feature.

“pure”

followed

extracts

monkeys,

to

had

would

not

compounds

fraction”(15)

test

was

contained

the

extracts)

most

blood.

corticosterone

common

species,

adrenal

the

active

she

an

contained

my project

activity

amorphous

of

never

detect

to

inspiration

cortex

biologically to

due

one

and

the “amorphous

my first

rhesus

one

the

it, But

fluorescence

by Samuels’

sheep,

with

of

venous

when and

if

made adrenal

secreted

be able

from

soda

the

known

as “the

crystallized

happy

anticipated

the

activity

adrenal

cortisol

(partially

I had managed

of

were My own

she had

products.

But

days

the

adaptation”

that

it

should

blood.

biological

as

of

methods

in those

been

and

steroids

one

venous

assay

namely

secretory

that adrenal

a biological

its

of

of

and glucocorticoids.

steroid

secreted

therefore

“diseases

new

adrenal

was

the

endocrinology,

synthesized

to detect!

I was dogs

of

using

had

neither agent

it

the

work:

modern

organ

that

of mineralocorticoids

Vogt’s

a storage

big

theory

better

weeks’ bag

of

secreting example

vindicating criteria,

For, on the basis of then conventional

Parkes’ slogan.

endocrinological

Selye’s claim of adrenal “exhaustion” had been perfectly sound. I had almost convinced myself to join the “unitarians” when Grundy, Simpson and

Tait(20) published their epoch-making letter to Nature describing a new mineralocorticoid in adrenal extract that they called “electrocortin4” what we now call aldosterone

In one of Zaffaroni’s solvent systems,

ran with cortisone,

but the mineralocorticoid

was far too large to be explained by the amount of cortisone in the zone.

activity

It was clear

to me immediately thet they had at Iast cracked the problem of “the amorphous fraction” and that it was immensely important to demonstrate this substance

was actually

secreted

as soon as possible whether or not

into the blood.

whetted, and we agreed to collaborate

on two critical

Jim and Sylvia’s appetite experiments.

was

By doing parallel

biological and chemical assays on carefully sectioned

paper chromatograms of extracts

of adrenal venous blood, we were able to demonstrate

in clearcut

substance

was secreted

fashion that the new

by both the dog and the rhesus monkeyt21f.

allowed to publish on the basis on one animal of each species today?

I doubt it.

one that I believe we were justified,

the time, the result was such a critical

Editor of the Lancet agreed after briefly challenging us on the point. so pleased that he sent us all complimentary Stress!

And simultaneously

Would we be But at and the

Hans Selye was

copies of that year’s Annual Review of

T. Reichstein and A. Wettstein in Base], and H. Mason and

V. Mattox in Rochester, MN, started

their race to isolate and identify the new compound

from adrenal extracts. Younger apparatus excitement

readers,

of the theoretical

chromatography

debt

for his tenacious

almost

all

their

chromatograph~e

houses may find it hard to appreciate

and technical

at least those that are any good.

great

to purchase

and supplies from commercial

theory of partition -

accustomed

twists I have described

here.

the

But the

is built into every piece of equipment sold today And it was hard-won.

insistence

on theoretical

We owe A.J.P. Martin a

rigor at every step

in its

development

-- from the first silica

gel columns,

through

filter

paper,

to the gas-liquid

chromatogram(22s1*23*24). In later who used these

methods

The most poignant Schneider, vocally,

I found

years

to solve

applications

were

and convenience by champions a Laurentian

systems

the ordinary

it: in Marrian’s

for handling for

was broken

was going

steroid the

systems

collectors,

to devise something

very

case, to open up a totally

new

to develop

neater

guy to get into the great

for years,

and John often

with Marvin

conjugates(26).

isolation

of

Lewbart

Some of the most

aldosteronet27)

but slowly lost popularity

it was a pleasure

and

the

After

as the speed

much early dispute

to meet Alex Zaffaroni

at

that we had shared at least one purpose.

for paper chromatography

and infra-red

forwards.

like Guy Marrian

became recognized.

and discover

to be impossible

field

reactionst2*).

were very effective

Hormone Conference

chemists

of paper chromatography

of the two sorts of systems,

fraction

determined

organic

for biosynthetic

of the volatile

Until the lipid barrier biochemistry

-

number of workers

and move the steroid

in John Schneider%,

undoubtedly

of microorganisms

Zaffaroni’s

columns,

systems

in the amazing

problems

“discovered”

metabolites(25);

a new family of solvent

screening

their

the very validity

and then suddenly

useful

satisfaction

were the converts

who rejected

field of estrogen

great

without

expensive

spectrometers(12). and less costly

it looked as though steroid batteries

Both Alex and 1 had been

-- something

game of steroids.

of huge alumina

that

would enable

S

TEIEOSDl

495

REFERENCES (1)

R. Consden,

A.H. Gordon and A.J.P.

(2)

T. Reichstein

(3)

S.P. Datta,

(4)

A. Zaffaroni,

(5)

C. Djerassi,

(6)

I.E. Bush and D.A.H. Taylor,

(7)

R.K. Callow,

(8)

See references

(9)

M. Vogt, 2. Physiol.,

(10)

N.H. Callow,

(11)

I.E. Bush, “The Chromatography

(12)

A.S. Parkes,

(13)

G. Sayers,

(14)

H. Sefye, “Stress, I* Aeta

(15)

L.F. Fieser

(16)

D.H. Nelson, H. Reich and L.T. Samuels,

(17)

0.

and C.W. Shoppee, B.G. Overefl

Martin,

Biochem. H., 2,

Disc. Faraday

Sot., _I, 305 (1949.

and M. Stack-Dunne,

Nature, 164, 673 (1949).

,R.B. Burton and E.H. Keutmann, Steroids,

3,

224 (1944).

Science,

111, 6 (1950).

351 (1984). Biochem.,

jz,

643 (1952).

R-D. Meikle end D.A.H. Tayfor, to Table

Chem. and Ind., p. 336 (1981).

2 in Ref. 6 above.

102, 341 (1943).

R.K. Callow and C.W. Emmens, Biochem. g., 32, 1312 (1938).

Proc. Physiol.

Rev., 2,

UK, 1961

Recent

241 (1950).

Inc.,

A. Zaffaroni,

and G. Pincus,

Press, Oxford,

Roy. Sot. Med., 43_, 361 (1950).

and M. Fieser,

Hechter,

of Steroids, It Pergamon

Montreal;

1950.

“Steroids ,I’ Reinhold, Science,

R.P. Jacobson,

Prog.

New York, 1953, p. 701. 111, 578 (1950).

M. Levy,

R.W. Jeanloz,

V. Sch>

Hormone ~Res. _’ 6 215 (1951).

(18)

I.E. Bush, 2. Endocrinol.,

2, 95 (1953).

(19)

I.E. Bush, --Ph.D. Thesis,

(20)

H. Grundy,

(21)

S.A. Simpson,

J.F. Tait and I.E. Bush, m.

(22)

A.J.P.

Martin

and R.L.M Synge,

(23)

A.J.P.

Martin,

Symp. Biochem. s.,

(24)

A.J.P.

Martin

and A.T. James,

(25)

K.H. Loke, G.F. Marrian

Cambridge

University,

S.A. Simpson and J.F. Tait,

United

Nature,

Kingdom,

1953.

169, 795 (1952). 2, 226 (1951).

Biochem. 2.. 2,

1358 (1941).

3, 4 (1949).

Biochem. J., E,

and E.J.D. Watson,

138 (1956).

Biochem. A., 7l_, 43 (1959).

WDEOXDI

S

496

(26)

S.J.

Schneider

and

M. Lewbart,

(27)

S.A.

Simpson,

J.F.

Tait,

(28)

T. Reichstein,

Helv. --

L.M.

Analyt.

Reineke,

Recent

A. Wettstein,

Chim.

Acta,

Chem.,

2,

Progress

Hormone

I&.,

R. Neher,

J. von

Euw,

37, 1163 (1954). 1853 (1956).

5, 0.

201 (1959). Schindler

and