Fat-Mobilizing Urine

T. M. ChALMERS, M.D., F.R.C.P.,

Activity Extract
L. S.
PAWAN,

of Human

G.

B.Sc.,

AND

A.

KEKWICK,

MA.,

M.B.

I
urine ing.

1947 Weil
alkaline
was

and extract recently23

activity

Stetten reported of urine from

in increasing liver

that an fasting
fat in

ficient ing for

function thirty-six

of

the hours

anterior or on

pituitary, a diet of

fast1 ,)()

rabbits

effective

calories
any

and
activity

91) per
in the

cent
urine

fat,

failed
III).

to

produce

mice.
fat-mobilizing

More
under

we
is conditions

have
present

shown
in including

that
human fast-

(Table
EFFECTS

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certain

BIOLOGICAL

The
CONDITIONS IN WHICH ACTIVITY IS FOUND

effects material
II.

of into

subcutaneous nice
blood the effects

injection are summarized

lipids being amid blood maximimal

of in

active found Table

ketones
i.

The
activity Carbohydrate least ficiency. appears as

conditions
in the urine

in
deprivation

which
are

we
shown appears as calories,

have
in to calorie

Liver all increase,

fat,

Table be activity de

about
III).

six
Values

hours
in

after
this our

injectiomm
and error most

(Fig.

1 , Table
tables

at

subsequent of the active nmean. extracts

important With when diets the

a of

stimulus 1,000

are
is

means

standard of

The
for

minimal

dose

carbohydrate

content

reduced
sively

below
with further

100

gm. with
found diet

and

increases diffuse

progreslipoatrophy,

obtaining about (>

rises in

the 10 zg. are and

liver for
lipids

fat each
(total

and mouse.

ketone Larger

effects doses unequivocal

is

carbohydrate in was the

restriction. urine although consumed.

In
activity a

two
has

patients
been mixed

2OOg.)
blood

needed

to produce
lipids,

phospholipid,

cholesterol well in

non-esterified

fatty well

acids). fall as in blood niobilihas

normal

being

One of them was controlled diabetes

addition was As found cinoma few all days these not in Table with after patients low normally to be and to diabetic.
i

an

adolescent and accelerated

The shows, activity in and surgical

girl
other

with growth

There is an early and transient sugar (Fig. 1). Utilization as

zation of body fat is increased.

This

lipoatrophy. also wasting, major the there

patient has the been carfirst In was about pituitary

been demonstrated using labeled ri However, it is denmonstrated more simply the

single (Figs.
2

by after
ones appe-

diabetic

ketosis,

widespread during operations. of calories doubt

loss

of body
large doses

weight
or iv).

and
repeated
No

carcass
change

fat
small in

to 4, Table food intake

intake is some

tite

and

accompanies of body therefore, increase. utilization of

this

in-

relatively

the
A appears

interpretation

of the
functioning necessary

finding.
anterior for the production of

creased catabolism simplest assumption, iture of energy must the efficiency

are in

fat. On the total expendAlternatively, of energy the uptake may of

of the Measurements
progress.

the

active
Fromn the

material.
Department W. at 1, England. the Brook Balance, 29,

Six
of Medicine, Lodge sponsored 1959,

patients
Middlesex Invitational by at Brook

with

deHospi-

diminish.
oxygen

tal, sium

London,

PREPARATION

OF

EXTRACTS

Presented
on Company, gusta,
American

SymnpoThe Lodge, Upjohn Au-

Energy September

Our trated alkaline

present
in

method 3. may The be

Vol.

of

extraction preparation conveniently

is of

illusthe

Figure extract

Michigan.
Journal of

carried
1960

Clinical

Nutrition

728

8, September-October

Fat-Mobilizing

Activity

of Human

Urine

Extract
TABLE
I

729

90 -

Fat-Mobihizimig

and

Ketogenic

Activity

ism Urinme

-.

70

BLOOD SODAR

(sg./loO

ml.)

Present (Fastimmg)

Absent (Normimal

I)iet)

50

1 ,000
cent

Calories,
Fat

91) per

.3
3
BLOOD

1 , 000 Calories, cemmt Proteism 2 000 Calories, cent I)iffuse

ET0N (wg.ilOO

90
83

per per

1 ,000 Calories, CHO Lipodystroplmy Late Brief Pregnmammcy Exercise

90

per

cemit

imosm-fasting

Fat hipoatropimy

ml.)

(mmomi-fastimmg) Diabetic ktosis* Carcinomatosis Postoperative Calorie ismtake low.

#{149}=.::#{149}:::-::-=:::- ---.-.

.--.-

-a---

* -#{149}

TABLE

II

Effects
LIVER (g./lOO PA? g.)

of Active

Extracts

in Mice

Increased

Dimiminished

3
HOSJRB

Liver fat Blood lipids Blood ketones Fat utilization

Blood Body Carcass

sugar weight fat

FIG.

1. and hourly

Tim1me-course (FMS) liver

on blood

of in

effects
sugar,

of
ketone

fat-mmmobilizing
bodies (as

subaceTABLE III

stance tone), at vithm Brokemi

fat

mice. after results

Groups mm c(Ismtrol

of animals animals

killed ismjection. injected

intervals lines indicate

subcutasmeous

Effects Deficient

of

Urine Subjects

Extracts fronm omi Liver Fat Mice

Nornmal amid Pituitaryand Blood Ketones

in

saline.
30

Material Injected Six Hours Previously

Liver Fat (gnm./100 gnm.)

Blood Ketones (nmg./100 miii. as acetone)

28

t-..
26

.4

AVERAGE
BODY WEIGHT (gm.)
2L

-.---.

-...

Salimme NFU FU FU(Hypopituitarisnm)

4.3 0. 14 4.80.24 7.20.33 3.9 0. 17

1.68 0.37 2.100.46 4.080.45 1.35 0.33

NOTE:
extract
(If

NFU
urine

Non-fasting

collected

22

20

MEAN Control Experimental

C.

FOOD

INTAKE
6.87 6.36 gm./day gm/day
15 20

fat, 1,000 calorie observations on six three weeks previously, mmecrosis amid three All patiemmts were cortisone and thyroid.

FU = per cent diet. Hypopituitarissmm = eight subjects, one hypophysectomized tilV(I with postpartunm pituitary with chromophobe adenomas. receiving nmaintenmance doses of
during fasting or

urine

extract.

90

1-. DAYS

out
(broken
weekly for in

in
of

an
twenty

M.S.E.
to thirty

basket
(Eastman) times

centrifuge.
increases (compared

The
the with

FIG. active

2. on

Effect
extract

of
(solid

injectiomm
line) three

of (ten

saline
timnes

limse)
twenty

or

use potency

oxycellulose

days The six

hiodv

weight imi food For

of mnice intake carcass

mice

each

group). in iv. Table

difference experimmments.

is time largest asmalysis see

observed

the 25

ultrafiltrate). to 50 per cent

Our of

extract oxycellulose

is shaken

with

overnight

730

Chalmers,

Pawan

and

Kekwick
TABLE IV

I.

Carcass

Analysis for

After Treatment with Active Three Weeks (gm./100 gin.)

Extract

-.9,

p.,AVERAGE

#{149}

Substances Analyzed Control

BODY

WEIGIIT

Experimental

Average

N et

Change

(gm. mouse)
/

(gm.)

(gm. /100 gm.)

Fat
Water Protein

14:3 0.5 64.1 0.7 11.80.4

91J

0.4
0.4

-125
-0.80 +0.07

-38
-5.5 +2.5

07.3 53.50.3

MEAN FOOD Control Experimental

INTAKE

(gm 6.1 6.2

/day) 6.8 6.9

.

NOTE: The same experiment amount of water lost is somewhat for by depletion of fat depots. cant gain or loss (If protein after extracts.

illustrated in Figure 2. The greater than can be accounted We have not observed any signitlprolonged treatment with active

as

DAYS

twenty-four
in days. No Figure Note difference 2 but recovery in food groups in time

hour

period

has

rammged

fronm

1 to

FIG.

3.

Time samime experimsmemmt

were weight between part of the stopped in treated commtrol study. after grotsp. amid ten

as

mg.
CHEMICAL PROPERTIES

inmjectionms of body intake eitimer

experimnental

The about
About 10 extracted or more per cent during extractions of the this yields

oxycel S per
the

extract cent.
following

has After
amino

a miitrogemm content acid

acids

of it

hydrolysis,
: histidine,

(sixteen original

hours). activity is

not

single therefore The

exposure . two will be needed for the

phenylalanine, leucine, sen ne, acid and a trace of alanine.

weakly positive Molisch test

cysti ne, aspartic It gives only

for carbohydrate

for

quantitative of urine
FOOD

work. over
INTAKE
7.0

yield

excretion

a
(gm.) 8.1

material.

c
0.

7.9
8.0

10

_.
-

CONTROL

.-

BODY (per

WEIGHT cent)

o.I

R.

1.

2
DAYS as per graded Control, 10.3 per cent single 14.9 cent. of initial doses per cent of value oxycel mug.
,

FIG. Single

4.

Body mice
05i day

weight receiving 7 :

plotted 12.4

against Fat cent per

time

in days. content of
,

mnaterial.

carcasses per cent;

; 0.4

; 4 Big.

1 1.7

40

nmg.,

Fat-Mobilizing
URDIE

Activity

of Human

Urine

Extract
TABLE V

731

II,
RENZOIC ACID

Alcoholic
PRECIPITATE Wsehd with

Ransom

oid

Release of Adipose

Noim-esterified Tissue Incubated

Fatty with

Acid Oxycel

( NEFA
Extract

) frosmi

Ethanol

Concentration Extract (,g./ml.)

0 (18)t

of

NEFA Reieased (iM./10() mg.)*

RESImiE

M..olv.d
Sodius

in
Crbonat

0.5%

0. 18

0.08

0.06 0.6
1.6 6 with
Ethanol

(4)
(4) (5) (4) (3) NEFA are mean

0.23

0.16

CRUDE AI.LALDIE

EkCT Pr.oipitated atpff5.3 120

,1..
ALCOHOLIC

0.440.10 1.030.39 2.200.31 5.030.45 standard error of

PRECIPITATE

a Values
the

for

F
RESIW!

t
Washed with Ethanol

mean. Figures

in paremmtheses

show

time smumsmber of observa-

tions.

I
ALXALThE

TABLE Msaolved
0.1 UTRLCT N.

VI

in

Comimparison

of Effects

of Urinary

Material

and

Corticotropin

on Blood
Eosinophil

Ketones,
Count

Liver

Fat

and

in Mice*

Ultrafiltered

Adsorbed on to ol after Iautralization

0XT3L

Material injected
Six Hours Previously

ACTIVE

ULTRAPILTRLIE

Blood Ketones (mg./100 ml.

Liver
(gm./100 gm.)

Fat

Eosinophils

I muted 0.1 l.
ACTIVE FIG.
EWATE

as acetone)

(cu./

mm.)

with

NaOfl Saline eluate is 2.45 2.60 4.00

5.

Extraction

proce(lure.

The

final

3.75
7.40 7.85

165 149 145

141

neutralized

and

freeze-dried.

The stable
destroyed

biologically active up to 80#{176}c.in

by boiling for less

niaterial 0. 1 N
two than

is thermoalkali. It is
It After is

Oxycel Extract
(50ig.)

4.60
5. 15

minutes.

ACTH
(0.5

ultrafiltrable
molecular

through
weight

Visking activity
destroyed

membrane
l8,00).

(i.e. but
trypsin

U.)

3.75 4. 15

5.95
6.50

75 60
urinary large

peptic
activity

digestion
is

some

remains,
by

NOTE

completely

extract

: in

Absence a close

of effect sufficient

on to

eosinophils of produce relatively

effects
*

and

by chymotrypsin.
EFFECT ON ADIPOSE TISSUE iN VITRO

Two

on blood ketones mice per group.

and

liver

fat.

sponse

is

linearly

related

to

the

logarithm

have used a modification of the method of White and Engel.4 Pieces of epididymal fat weighing about 50 mg. from rats weighing 9() to 1 10 gmu. have been incubated in a bicarbonate Inedium containing 4 per cent
We

concentration. Similar tamed using the cruder tion at concentrations greater.

RELATION TO CORTICOTROPIN HORMONE

data have been obultrafiltrate preparaabout twenty times

bovine
(NEFA)

albunmin.
release

Non-esterified
into the medium

fatty
during

acid
a

ANDGROWTH

three Doles6

hour

incubation

has Addition

been of

deterlnined oxycel
,

by

The properties namely, effect

active in affinity in vitro

urinary common for and

substance with oxycellulose, capacity to

has

certain lipolytic

method.

material the threshThe re-

corticotropin, lower blood

increases N EPA old concentration

release being

(Table v) 1 g./ml.

732
URINARY FitS. POSITIVE NEGATIVE

Chalmers,
NEGATIVE

Pawan

and

Kekwick and exanmined

derived

ASSAYS
1.5

filtrability We In have two

fragment

its

affinity the
fronm the

for
growth

oxycellulose. that urine of case

or

possibility persons,

it is a colthe for could

in
IWO

hormone.

PLASMA NEFA
mM/L

pituitary-deficient after growth an intranmuscular hormone* activity.

be (Fig. 6).
SUMMARY

1.0

lected human
0.5

injection has In
by

been neither
in vitro

assayed

fat-mobilizing
any activity methods
CONTROL FASTING HYPOPITUITARY FASTING
}IYPOPITUITARY

detected

FASTING GROWTH HORMONE

5

mg./I.M.

In
at two with

people fat, urine

who a

are

actively can cause

in

mnobilizing

and

FIG.
IR)Ofl

6.

Plasma subjects deficiency, and the effect subjects.

NEFA fasted ; other of second

(InC

commcentrations overnight column, was growth (I.M.) collected for present the urine fat-mnobilizing in the from recently hornmone in a chromophobe : two

at 8 AM. First columnn, subjects

and

imi subjects

utilizing from the

substance which will

be extracted increased fat

mice, with de-

normal pituitary mized columnn, mninistered deficient and (FMS). urines noon

hypopimysectoadenoma (5 the between normal the mg.) pituitary8 substance fasting pituitaryAM.

had human Urine

; third ad-

mobilization and catabolism pletion of the body fat stores. active tissue
in vitro in releasing

The
from

material
adipose

is

NEFA

intrammiuscularlv was Activity but absent assayed was in

at a concentration

of less

than

I j.tg./ml.

The pituitary is necessary for its production. It is not corticotropin or the growth hormone.
Its cussed.
REFERENCES 1. WElL, R. of amid
STETTEN, I)EW.,
JR.

relation

to

these

hormones

is briefly

dis-

deficiemmt
administered. admiministration

subjects
(If

even
Urine growth

after
collected

the

growth
for several was

hormone
days also negative.

was
after

hormone

Urimmary J. Biol.

excreCheat.,

sugar tones. is in

and The the

to effect

increase most on The

liver obvious body urinary

fat point weight

and of and

blood difference carcass also

ke2.

tion
CHALMERS,

fat-mimobilizimmg T. M. I. M.
, KEKVICK,

agesmt. A. I : 866, G. amid L. to 2: 6, L.

,

168: 129, 1947.

PA\VAN,

G. L.
activity

S. of

and

SMITII, urine. T. extracts.

On Lancet,

the

fat-snobilizismg 1958.

composition.

material

ap-

hunman

pears
not

to
depress

be

more
the

stable

in
count

alkali.
in the

It

does
mouse

3.

CHALMERS,

, PAWAN,

S. amid KECKWICK, activity of aimd actiomi in vitro. fatty J. of J. corticotropimm 1960. Lipolytic tissue

eosinophil

A. urine growth 4.
WHITE,

Fat-nmobilizing hormnone.

ketogemmic

(Table This
mone and olism. more in differs growth of

vi). See substance

the With active in properties increasing

also Chalmers shares with

of fat to and lowering NEFA mobilization

et al.3 the growth

blood and release less

Relatiomm Lancet,
ENGEL,

horsugar catabit active is

J. E. and
Invest., V

F. adipose

corticotropimm Clin.

omi rat 37 : 1556, Relation plasnma amid

1958. between non-esterified

(It

respect vitro

5.

DOLE,

P. iii

possibly

acids

nmetabolismim

glucose.

vivo.
from

It
nor

does
the the

not
deposition growth

increase
of hormone

the
protein. also

rate
in

of
It its
by search
*

Clin.

Invest.,

35: 150, 1956.

gr(lwth hormmmosme Voummg tlmroi.mgh

The
Professor

human
F. Council.

was
the

kindly
Medical

supplied
Re-

G.

chemical

properties,

especially

in

its

ultra-