American Journal of Clinical Nutrition 728 Vol. 8, September-October 1960

Fat-Mobilizing Activity of Human

Urine Extract

T. M. ChALMERS, M.D., F.R.C.P., G. L. S. PAWAN, B.Sc., AND A. KEKWICK, MA., M.B.

I N 1947 Weil and Stetten’ reported that an

alkaline extract of urine from fasting

rabbits was effective in increasing liver fat in

mice. More recently2’3 we have shown that

fat-mobilizing activity is present in human

urine under certain conditions including fast-

ing.

CONDITIONS IN WHICH ACTIVITY IS FOUND

The conditions in which we have found

activity in the urine are shown in Table i.

Carbohydrate deprivation appears to be at

least as important a stimulus as calorie de

ficiency. With diets of 1,000 calories, activity

appears when the carbohydrate content is

reduced below 100 gm. and increases progres-

sively with further carbohydrate restriction.

In two patients with diffuse lipoatrophy,

activity has been found in the urine although

a normal mixed diet was being consumed.

One of them was an adolescent girl with well

controlled diabetes and accelerated growth in

addition to lipoatrophy. The other patient

was not diabetic.

As Table i also shows, activity has been

found in diabetic ketosis, in widespread car-

cinoma with wasting, and during the first

few days after major surgical operations. In

all these patients the intake of calories was

relatively low and there is some doubt about

the interpretation of the finding.

A normally functioning anterior pituitary

appears to be necessary for the production of

the active material. Six patients with de-

Fromn the Department of Medicine, Middlesex Hospi-

tal, London, W. 1, England.

Presented at the Brook Lodge Invitational Symnpo-

sium on Energy Balance, sponsored by The Upjohn

Company, September 29, 1959, at Brook Lodge, Au-

gusta, Michigan.

ficient function of the anterior pituitary, fast-

ing for thirty-six hours or on a diet of 1 ,�)()

calories and 91) per cent fat, failed to produce

any activity in the urine (Table III).

BIOLOGICAL EFFECTS

The effects of subcutaneous injection of

active material into nice are summarized in

Table II. Liver fat, blood lipids amid blood

ketones all increase, the effects being maximimal

about six hours after injectiomm (Fig. 1 , Table

III). Values in this and subsequent tables

are means ± standard error of the nmean. The

minimal dose of our most active extracts for

obtaining the liver fat and ketone effects is

about 10 �zg. for each mouse. Larger doses

(> 2OO�g.) are needed to produce unequivocal

rises in blood lipids (total lipids, phospholipid,

cholesterol and non-esterified fatty acids).

There is an early and transient fall in blood

sugar (Fig. 1). Utilization as well as niobili-

zation of body fat is increased. This has

been demonstrated using labeled ri�

However, it is denmonstrated more simply by

the loss of body weight and carcass fat after

single large doses or repeated small ones

(Figs. 2 to 4, Table iv). No change in appe-

tite and food intake accompanies this in-

creased catabolism of body fat. On the

simplest assumption, therefore, total expend-

iture of energy must increase. Alternatively,

the efficiency of the utilization of energy may

diminish. Measurements of the uptake of

oxygen are in progress.

PREPARATION OF EXTRACTS

Our present method of extraction is illus-

trated in Figure 3. The preparation of the

alkaline extract may be conveniently carried

by guest on October 31, 2013

ajcn.nutrition.orgD

ownloaded from

by guest on O

ctober 31, 2013ajcn.nutrition.org

Dow

nloaded from

by guest on October 31, 2013

ajcn.nutrition.orgD

ownloaded from

by guest on O

ctober 31, 2013ajcn.nutrition.org

Dow

nloaded from

by guest on October 31, 2013

ajcn.nutrition.orgD

ownloaded from

90

70

- -.� � �-

TABLE I

Fat-Mobihizimig and Ketogenic Activity ism Urinme

50

.3�

3

BLOOD�ET0N�

(wg.i’lOO ml.)2

1 #{149}=.::#{149}:�::-::-=:�::- ---.-. - .--.- - _ -a--- -‘#{149}

30

28

t-.. ‘.4’

AVERAGEBODY WEIGHT

(gm.) 2L�

22

20

Fat-Mobilizing Activity of Human Urine Extract 729

BLOODSODAR

(sg./loO ml.)

LIVER PA?(g./lOO g.)

0 1 2 3 Z� 5 6

HOSJRB

FIG. 1. Tim1me-course of effects of fat-mmmobilizing sub-

stance (FMS) on blood sugar, ketone bodies (as ace-

tone), and liver fat in mice. Groups of animals killed

at hourly intervals after subcutasmeous ismjection.

Brokemi lines indicate results mm c(Ismtrol animals injected

�vithm saline.

26

-.---. -...

MEAN FOOD INTAKEControl 6.87 gm./day

Experimental 6.36 gm/day

C. 5 1-. 15 20

DAYS

FIG. 2. Effect of injectiomm of saline (broken limse) or

active extract (solid line) three timnes weekly for twenty

days on hiodv weight of mnice (ten mice in each group).

The difference imi food intake is time largest observed in

six experimmments. For carcass asmalysis see Table iv.

Present(Fastimmg)

Absent

(Normimal I)iet)

1 ,000 Calories, 91) per

cent Fat

1 , 000 Calories, 90 per

cemmt Proteism2 000 Calories, 83 per

cent FatI)iffuse hipoatropimy

(mmomi-fastimmg)Diabetic ktosis*

Carcinomatosis �

Postoperative

1 ,000 Calories, 90 per cemit

CHO

Lipodystroplmy

Late Pregnmammcy imosm-fasting

Brief Exercise

* Calorie ismtake low.

TABLE II

Effects of Active Extracts in Mice

Increased Dimiminished

Liver fat

Blood lipids

Blood ketones

Fat utilization

Blood sugar

Body weight

Carcass fat

TABLE III

Effects of Urine Extracts fronm Nornmal amid Pituitary-

Deficient Subjects omi Liver Fat and Blood Ketones inMice

Material Injected

Six Hours

Previously

Liver Fat

(gnm./100

gnm.)

Blood Ketones

(nmg./100 miii.

as acetone)

Salimme

NFU

FU

FU(Hypopituitarisnm)

4.3 ± 0. 14

4.8±0.24

7.2±0.33

3.9 ± 0. 17

1.68 ± 0.37

2.10±0.46

4.08±0.45

1.35 ± 0.33

NOTE: NFU = Non-fasting urine extract. FU =

extract (If urine collected during fasting or 90 per cent

fat, 1,000 calorie diet. Hypopituitarissmm = eight

observations on six subjects, one hypophysectomized

three weeks previously, tilV(I with postpartunm pituitary

mmecrosis amid three with chromophobe adenomas.

All patiemmts were receiving nmaintenmance doses of

cortisone and thyroid.

out in an M.S.E. basket centrifuge. The

use of oxycellulose (Eastman) increases the

potency twenty to thirty times (compared with

the ultrafiltrate). Our extract is shaken with

25 to 50 per cent of oxycellulose overnight

730

AVERAGE

BODY WEIGIIT(gm.)

Chalmers, Pawan and Kekwick

‘I.

-.9,

p.,-#{149}‘

p

MEAN FOOD INTAKE (gm . /day)Control 6.1 6.8

Experimental 6.2 6.9

NOTE: The same experiment as illustrated in Figure 2. The

amount of water lost is somewhat greater than can be accountedfor by depletion of fat depots. We have not observed any signitl-

cant gain or loss (If protein after prolonged treatment with active

extracts.

c� 7.00.� 8.1‘� 7.9

� 8.0+ 10

- .-- -

_. C CONTROL

o.I� R�.

‘ �1.

0 2

DAYS

8

DAYS

FIG. 3. Time samime experimsmemmt as in Figure 2 but time

inmjectionms were stopped after ten days. Note recovery

of body weight in treated grotsp. No difference in food

intake between commtrol amid experimnental groups in

eitimer part of the study.

(sixteen hours). About 10 per cent of the

original activity is not extracted during this

single exposure . two or more extractions

therefore will be needed for quantitative work.

The yield for the excretion of urine over a

TABLE IV

Carcass Analysis After Treatment with Active Extract

for Three Weeks (gm./100 gin.)

Average N et Change

Sub-stances

AnalyzedControl

.Experi-mental

-(gm. /

mouse)

(gm. /100

� gm.)

Fat 14:3 ±0.5 91J ±0.4 -125 -38Water 64.1 ± 0.7 07.3 ± 0.4 -0.80 -5.5

Protein 11.8±0.4 53.5±0.3 +0.07 +2.5

twenty-four hour period has rammged fronm 1 to 7

mg.

CHEMICAL PROPERTIES

The oxycel extract has a miitrogemm content of

about S per cent. After acid hydrolysis, it

yields the following amino acids : histidine,

phenylalanine, leucine, sen ne, cysti ne, aspartic

acid and a trace of alanine. It gives only a

weakly positive Molisch test for carbohydrate

material.

0

BODY WEIGHT

(per cent)

FOOD INTAKE (gm.)

FIG. 4. Body weight as per cent of initial value plotted against time in days.

Single mice receiving graded single doses of oxycel mnaterial. Fat content of

carcasses 05i day 7 : Control, 14.9 per cent ; 0.4 mug. , 12.4 per cent ; 4 Big. , 1 1.7

per cent; 40 nmg., 10.3 per cent.

URDIE

II, Alcoholic Ransom �oid

RENZOIC ACID PRECIPITATE

TABLE V

Release of Noim-esterified Fatty Acid ( NEFA ) frosmiAdipose Tissue Incubated with Oxycel Extract

TABLE VI

Comimparison of Effects of Urinary Material and

Corticotropin on Blood Ketones, Liver Fat andEosinophil Count in Mice*

Materialinjected

Six Hours

Previously

Saline

OxycelExtract(50ig.)

ACTH

(0.5 U.)

Blood Ketones

(mg./100 ml.as acetone)

2.45

2.60

4.605. 15

3.75

4. 15

Liver Fat(gm./100

gm.)

4.00

3.75

7.40

7.85

5.956.50

Eosinophils

(cu./ mm.)

165149

145141

7560

Fat-Mobilizing Activity of Human Urine Extract

Wsehd with Ethanol

RESImiE1 M..olv.d in 0.5%

Sodius Crbonat

CRUDE AI.LALDIE �EkCT

I Pr.oipitated with Ethanol

,�1.. atpff5.3

ALCOHOLIC PRECIPITATE

F Washed with Ethanol

RESIW!

731

Concentration ofExtract

(,�g./ml.)

NEFA Reieased(�iM./10() mg.)*

0. 18 ± 0.080 (18)t0.06 (4) 0.23 ± 0.16

0.6 (4) 0.44±0.10

1.6 (5) 1.03±0.39

6 (4) 2.20±0.31

120 (3) 5.03±0.45

a Values for NEFA are mean ± standard error of

the mean.

t Figures in paremmtheses show time smumsmber of observa-tions.

I Msaolved in0.1 N.

ALXALThE UTRLCT

Ultrafiltered Adsorbed on to �‘olafter Iautralization

ACTIVE ULTRAPILTRLI’E 0XT�3L

I muted with0.1 l�. NaOfl

ACTIVE EWATE

FIG. 5. Extraction proce(lure. The final eluate is

neutralized and freeze-dried.

The biologically active niaterial is thermo-

stable up to 80#{176}c.in 0. 1 N alkali. It is

destroyed by boiling for two minutes. It is

ultrafiltrable through Visking membrane (i.e.

molecular weight less than l8,00�). After

peptic digestion some activity remains, but

activity is completely destroyed by trypsin

and by chymotrypsin.

EFFECT ON ADIPOSE TISSUE iN VITRO

We 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 bi-

carbonate Inedium containing 4 per cent

bovine albunmin. Non-esterified fatty acid

(NEFA) release into the medium during a

three hour incubation has been deterlnined by

Dole’s6 method. Addition of oxycel material

increases N EPA release (Table v) , the thresh-

old concentration being 1 �g./ml. The re-

NOTE : Absence of effect on eosinophils of urinary

extract in a close sufficient to produce relatively large

effects on blood ketones and liver fat.* Two mice per group.

sponse is linearly related to the logarithm

concentration. Similar data have been ob-

tamed using the cruder ultrafiltrate prepara-

tion at concentrations about twenty times

greater.

RELATION TO CORTICOTROPIN AND�GROWTH

HORMONE

The active urinary substance has certain

properties in common with corticotropin,

namely, affinity for oxycellulose, lipolytic

effect in vitro and capacity to lower blood

URINARY FitS. POSITIVE NEGATIVE NEGATIVE

ASSAYS

PLASMANEFAmM/L

1.5

1.0

0.5

0�

filtrability and its affinity for oxycellulose.

We have exanmined the possibility that it is a

fragment derived fronm the growth hormone.

In two pituitary-deficient persons, urine col-

lected after an intranmuscular injection of the

human growth hormone* has been assayed for

fat-mobilizing activity. In neither case could

any activity be detected by in vitro or in IWO

methods (Fig. 6).

SUMMARY

In people who are actively mnobilizing and

utilizing fat, a substance can be extracted

from the urine which will cause increased fat

mobilization and catabolism in mice, with de-

pletion of the body fat stores. The material is

active in vitro in releasing NEFA from adipose

tissue 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 relation to these hormones is briefly dis-

cussed.

sugar and to increase liver fat and blood ke-

tones. The most obvious point of difference

is in the effect on body weight and carcass

composition. The urinary material also ap-

pears to be more stable in alkali. It does

not depress the eosinophil count in the mouse

(Table vi). See also Chalmers et al.3

This substance shares with the growth hor-

mone the properties of lowering blood sugar

and of increasing fat mobilization and catab-

olism. With respect to NEFA release it is

more active in vitro and possibly less active

in vivo. It does not increase the rate of

growth nor the deposition of protein. It

differs from the growth hormone also in its

chemical properties, especially in its ultra-

732 Chalmers, Pawan and Kekwick

CONTROL HYPOPITUITARY }IYPOPITUITARYFASTING FASTING FASTING

GROWTH HORMONE

5 mg./I.M.

FIG. 6. Plasma NEFA commcentrations at 8 AM. and at

IR)Ofl imi subjects fasted overnight : First columnn, two

normal subjects ; second column, two subjects with

pituitary deficiency, (InC was recently hypopimysecto-

mized and the other had a chromophobe adenoma ; third

columnn, effect of human growth hornmone (5 mg.) ad-

mninistered intrammiuscularlv (I.M.) in the pituitary-

deficient subjects. Urine collected between 8 AM.

and noon was assayed for fat-mnobilizing substance

(FMS). Activity was present in the normal fasting

urines but absent in the urine from the pituitary-

deficiemmt subjects even after the growth hormone wasadministered. Urine collected for several days after

admiministration (If growth hormone was also negative. REFERENCES

1. WElL, R. amid STETTEN, I)EW., JR. Urimmary excre-

tion of fat-mimobilizimmg agesmt. J. Biol. Cheat.,

168: 129, 1947.

2. CHALMERS, T. M. , KEK�VICK, A. , PA\VAN, G. L. S.

and SMITII, I. On the fat-snobilizismg activity of

hunman urine. Lancet, I : 866, 1958.

3. CHALMERS, T. M. , PAWAN, G. L. S. amid KECKWICK,

A. Fat-nmobilizing amid ketogemmic activity of

urine extracts. Relatiomm to corticotropimm aimd

growth hormnone. Lancet, 2: 6, 1960.

4. WHITE, J. E. and ENGEL, F. L. Lipolytic actiomi of

corticotropimm omi rat adipose tissue in vitro. J.

Clin. Invest., 37 : 1556, 1958.

5. DOLE, V . P. Relation between non-esterified fatty

acids iii plasnma amid nmetabolismim (It glucose. J.

Clin. Invest., 35: 150, 1956.

* The human gr(lwth hormmmosme was kindly supplied

by Professor F. G. Voummg tlmroi.mgh the Medical Re-

search Council.