Vol. 88, No. 3, 1979

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

June 13, 1979

Pages 1030-1036

FATTY Miguel

ACID

Bronfman, Department

OXIDATION Nibaldo

C. Inestrosa

of Cell

Biology,

de Chile, Received

April

BY HUMAN LIVER

Casilla

PEROXISOMES

and

Federico

Universidad

114-D,

Leighton

Catblica

Santiago,

Chile

30, 1979

SUMMARY A cyanide insensitive fatty acid oxidation system is detected in human liver and is shown to be localized in peroxisomes by subcellular fractionation in Metrizamide continuous density gradients. Fatty acylCoA oxidase, its characteristic enzyme, acts maximally on C12-C18 saturated fatty acids and on oleoyl-CoA and requires FAD. These results, together with the already estab1ishe.d properties of the system in rat liver, support its potential contribution to lipid metabolism and to the hypolipidemic effect of Clofibrate and related drugs in humans. INTRODUCTION The has

not

acids

role been

also The

known

been

rats

also

they

In lism

from

rat fact, and

some drugs, eventually

and

proliferative

these

to evaluate their

of

this

is

have the

their presence

properties,

not

role

with

in must

acid

be studied.

1030

in the

liver

exert

in

so,

humans acid

since

detected

in

(6,7) in

Furthermore,

human

lipid

system,

liver human

(9).

composition

hypolipidemic

oxidizing

(3,5).

fatty

been

(8).

of peroxisomes

a fatty

rat

p-oxidation

enzymatic

to

(3,4)

also

not

Nafenopin

contribution

compounds

necessarily

has

a different

by Clo-

in

acid

fatty

inducible

peroxisomal

fatty

0006-291X/79/111030-07$01.00/0 Copyright @ 1979 by Academic Press. Inc. All rights of reproduction in any form reserved.

the

of lipids

oxidize

is

drugs

response

treated

to understand

its

proliferation

mitochondrial

peroxisomes

which

(I)

peroxisome which

metabolism

peroxisomes

hypolipidemic

However

stimulate

the

other

effect

patients

liver

system

(2)

(1).

in

In rats,

to an enhancement

and a peroxisomal biopsies

humans.

to induce

attributed system

peroxisomes

insensitive

hypolipidemic

oxidizing

and

in

Nafenopin

(I),

drugs

by liver

studied

by a cyanide

fibrate

has

played

metaboaction and

of

Vol. 88, No. 3, 1979

The zes

BIOCHEMICAL

peroxisomal

fatty

acid

actions

which,

sembles

that

acyl-CoA

fatty

p-oxidation with

mediated to the

peroxisomes

by a fatty

fatty

(2,121.

acids

protein

of

system has

in

been

146,000

the

a diet

The present peroxisomal of its MATERIAL

fatty

report acid

characteristic

(IO).

The

in

mitochondria

oxidase,

oxidase,

control

in

saturated

establishes oxidizing enzyme

desaturation

of

is

(II),

mainly

rate

and

of rats

acids

the

in

on C,2-C18 as a flavoenzyme

in

which (2)

the

of

the

system

or 2.4

fold

liver

of

(14).

existence

acyl-CoA

re-

catalyzed

limiting

administration

system , and fatty

of re-

reaction,

characterized

fatty

cataly-

by a dehydrogenase

active

the

liver

sequence

LX,/? -

chain

(2)

by Nafenopin

rat

oxidative

as the

rats

from

first

partially

appears

RESEARCH COMMUNICATIONS

insensitive

the

acyl-CoA

T-fold rich

of

transport

mw (131, of

system

electron

This

liver

enhanced

by feeding

exception

of mitochondria

coupled

oxidizing

by a cyanide

the

derivative,

tightly

acid

AND BIOPHYSICAL

in

describes

human

a

some properties

oxidase.

AND METHODS

Liver biopsies were obtained from four patients with normal liver function tests undergoing surgery for uncomplicated gall-stone or gastroduodenal ulcer disease. Informed consent was obtained following the procedures established by the Ethics Committee from the Medical School of the Universidad Catblica de Chile. The biopsies, obtained at the beginning of surgery, were placed in ice cold 0.25 M-sucrose. Homogenization was performed in 0.25 M-sucrose containing 3 mM-imidazole HCl, pH 7.4, and 1 mM-dithiothreitol at 20-2596 w/v tissue concentration. The homogenates were fractionated in continuous Metrizamide density gradients by a modification of the procedure developed by Wattiaux et al. for the isolation of lysosomes from an enriched subcellular fraction (15). 0.5 ml aliquots of the homogenate were layered on top of gradients extending from 1.10 to 1.28 g/ml in tubes from an SW-65 rotor (Beckman Instruments Inc.) and were centrifuged 90 min at 39,000 r-pm, 4OC. The activity of the peroxisomal fatty acid oxidizing system was determined from palmitoyl-CoA dependent NAD+ reduction, as described by Lazarow & de Duve (3) with some modifications (2). The assay medium contained, at pH 8.3, 60 mM-Tris HCl, 24 pM-palmitoyl-CoA, 120 ,uM-FAD, 370 PM-NAD+, 94 mM-nicotinamide, 2.8 mM-dithio50 PM-CoA, threitol, 2 mM-potassium cyanide, and 0.15 mg/ml of bovine serum albumin in a final volume of 0.8 ml. The enzyme containing samples were diluted to 1/2 with 1% (w/v) Triton X-100 and 5-20 ~1 was added to each test. Units correspond to pmol NAD+ reduced . min-1. g wet wt. liver-l. The activity of the fatty acyl-CoA oxidase was determined polarographically from palmitoyl-CoA dependent oxygen consumption. The assay medium contained, at pH 8.3, 100 mM-Tris HCl,

1031

Vol. 88, No. 3, 1979

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

1 mM-potassium cyanide, 200 PM-NAD+, 31 PM-palmitoyl-CoA, 33 mM-nicotinamide, 170 PM-CoA, 50 PM-FAD, 0.6 mg/ml of bovine serum albumin, and 0.01% (w/v) Triton X-100 in a total volume of 1.6 ml. After addition of IO-50 ~"1 of the enzyme containing sample, the basal 02 uptake was recorder for 5 min, and the reaction started by addition of palmitoyl-CoA (2). Units correspond to pmol 02 consumed . min-1. g wet wt. liver-l. Established procedures were employed for the determination of marker enzymes: catalase (16) for peroxisomes , glutamate dehydrogenase (16) for mitochondria, NADPH cytochrome c reductase (17) for endoplasmic reticulum microsomes, and acid pFosphatase (18) for lysosomes. Protein was measured as described by Wattiaux et al. (15) using bovine serum albumin as standard. Crude fatty acyl-CoA oxidase preparations were made diluting the homogenates with 4 volumes of 100 mM-Tris HCl pH 8.3 and rehomogenizing in a tissue desintegrator (Virtis 45, The Virtis Co., Gardiner, New York). The supernatant obtained after centrifugation, 30 min at 100,000 g, was made 50% w/v with (NH412 SO4. The precipitate, collected by centrifugation, was dissolved and dialyzed overnight in the same buffer. This preparation frees the enzyme of some components responsible for high blank readings in the assay. The final specific activity was twice that of the homogenate. Metrizamide (2-(3-acetamido-5-N-methylacetamido-2-4-6 triiodobenzamido)-2-deoxy-D-glucose) was obtained from Nyegaard and CO., Oslo. Substrates and coenzymes were from Sigma Chemical Co., St. Louis, Missouri. RESULTS In

AND DISCUSSION four

oxidase,

liver

with liver;

system,

measured

2 0.06

the

+ 0.05 The

activity

(S.D.)

(S-D.)

the

liver,

palmitoyl-CoA

I).

The

gave

good

resolution

of peroxisomes

microsomes

and

reticulum activity system, top

of fatty follows

fractions

subcellular

together

rat

were

0.81

2 0.07

the

bound with

the

catalase. rest

of

1032

oxidizing was

liver,

applying

(S.D.)

in

was clearly

developed

mitochondria,

endoplasmic one half

of

the

fatty

acid

oxidizing

The

other

half,

stays

catalase

and

the

liver

peroxisomal

procedure

of

the

and

detected

Approximately and

acid

activities

from

oxidase

(S.D.)

(2).

fractionation

lysosomes.

acyl-CoA particle

normal

as substrate,

(Fig

acyl-CoA

NAD+ reduction,

In

of

fatty

+ 0.06

fatty

respectively

distribution

novel

of

was 0.16

dependent

found

units/g

using

activity

peroxisomal

liver.

values

subcellular

homogenates,

of

units/g the

the

as substrate,

as palmitoyl-CoA

same procedures, 0.81

studied,

palmitoyl-Cob

units/g

0.24

biopsies

of

the

the

in

the

other

Vol. 88, No. 3, 1979

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

20

10 DPH

cyt. c 20

10

20

,lO

-20

-10 110 FIGURE

115

1.20 1.25 110 115 Density (g/ml)

1.20

1.25

1

Subcellular distribution of fatty acyl-CoA oxidase (FAO) and of the peroxisomal fatty acid oxidizing system (PFAOS). Complete homogenate, from human liver,fractionated in a continuous density gradient of Metrizamide. The distribution of peroxisomes,mitochondria, endoplasmic reticulum microsomes and lysosomes was determined from marker enzymes, catalase, glutamate dehydrogenase, NADPH cytochrome c reductase and acid phosphatase, respectively. Ordinates represent average frequency of the components for each fraction, Q/EQAp where Q represents the activity found in the fraction, CQ the total activity recovered and Ap the increment in density from top to bottom of the Frequency is plotted against gradient in the centrifuge tube (20). density in a histogram form. The hatched area represents the proportion of the total activity of each component recovered in the top 0.5 ml of the tube contents which correspond to the volume of the initial homogenate layer.

marker

enzymes.

better

resolution

fractions, lase, soluble fraction attributed

it fatty

In

separate

of

organelles

was established acyl-CoA

components of

the

Metrizamide

of

peroxisomal to

enzyme

the

and

release

the the

low

In

from

has

also

the

1033

in

density

oxidizing

homogenate.

enzymes

designed

equilibrating

that

oxidase,

gradients,

rat been

organelles

the

low

fraction system,

liver,

during

density of

cata-

follows

a large

observed

for

the soluble

and has

been

fractionation

Vol. 88, No. 3, 1979

Whatever

(5,191. release

might

oxidase. acid

BIOCHEMICAL

the

be,

It

Human fatty preparation

section.

This in

together

with is

little

our

liver

more

fatty

was

at

observation

of

acyl-CoA

in

the

acyl-CoA and

the

FAD (Fig

2).

that bound,

the

mitochondria. employing and

Methods

and a 5.5-fold The

effect

oxidase

suggests

similar

fatty

subcellular

Materials

kinetics

are

enzymes

characterized

assay

FAD weakly oxidases

of

all

in

establishing

with

fatty oxidase

further

linear

addition

than

pattern

as described gave

peroxisomal

acyl-CoA

no activity

oxidase

upon

fatty

that

of FAD, from

rat

human

flavoproteins

(13).

I

25

02

or

obtained

activity

for

a peroxisomal

preparation

I

FIGURE

have

a flavoprotein

and rat

that

acyl-CoA

a crude

responsible catalase

concluded

with

increase

affects

system

distribution,

liver

it

was

oxidizing

mechanism

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

50

75

i

16

16

Chain Lengthof substrate

Enzyme ( pl) 2

Effect of 50 PM-FAD on the rate of palmitoyl-CoA dependent oxygen consumption by a crude preparation of peroxisomal fatty acyl-CoA oxidase from human liver. The assays were performed as described in the Materials and Methods section, omitting NAT)+ and nicotinamide. FIGURE

3

Substrate specificity of human liver oxidase with saturated fatty acids, The rate of acyl-CoA dependent oxygen consumption was measured as described in the Materials and Methods section, omitting NAD+ and nicotinamide. A crude preparation of the enzyme was employed. Bars represent mean + S.D. of three independent determinations expressed as percent of the activity observed with palmitoyl-CoA.

1034

Vol. 88, No. 3, 1979

The with

substrate

three

fatty

described

for enzyme

90-100% in

of

rat

liver

crude

gave,

in

with

it

liver

has

fatty

system,

in

could

humans

by changes

with

(2).

The

the

that

is

the

present

work

oxidase

and

in

activities

findings

the

remains total that

the

human

saturated,

as well

mitochondria

to be established. of peroxiso-

of mitochondria

establishes of

that

that

of

were

contrast,

contribution

the

that

of peroxisomes

in

fatty lipid

and

(21).

presence

peroxisomal

possibility

capacity

In

of

the

that

suggest

pharmacologically

the

oleoyl-CoA

contribution

to

as already

to 40% of

metabolism

similar

supports

be controlled

induced

specific

amounts

the

oxidation

and

With

relative

for

31,

(2,12).

metabolism

suggested

(Fig

palmitoyl-CoA.

These in

acid

acyl-CoA

oxidizing

enzyme

was checked

activities

C12-C18

oleoyl-CoA

involved

been

conclusion,

of

with

oxidase

Maximal with

observed

fatty

acyl-CoA

preparation,

acids.

to

mes to palmitate In

each

are fatty

fatty

liver

palmitoyl-CoA

peroxisomes rats,

rat

activity

peroxisomes

and

of

observed

activities the

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

preparations.

were

the

as unsaturated

In

crude

acids

the

liver

observed

specificity

different

saturated

human

BIOCHEMICAL

human

acid metabolism

physiologically

to oxidize

fatty

acids. ACKNOWLEDGEMENTS This work was supported by the Fondo de Investigaciones de la Universidad Cat6lica de Chile, proyecto 212/77, and by funds from the American States Organization. We thank Drs. F. Nervi for the clinical monitoring of the human volunteers, and R. Wattiaux for Metrizamide and a pre-publication copy of his work. The skilful participation of Cecilia Necochea and Maria Nelly Morales is gratefully acknowledged. REFERENCES I.

Lazarow, U.S.A.

73,

P.B., and de Duve, 2043-2046.

2.

Inestrosa, (accepted

N-C., Bronfman, for publication).

3.

Lazarow,

4.

Osumi,

5.

Leighton, F., 67, 281-309.

P.B. T.,

and

(1977)

M.,

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Hashimoto, Coloma,

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T. L.,

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6.

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Mannaerts, G.P., Thomas, J., D.W. (1978) Biochim. Biophys.

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507-524. pp 1-26, Tolbert,

Fatty acid oxidation by human liver peroxisomes.

Vol. 88, No. 3, 1979 BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS June 13, 1979 Pages 1030-1036 FATTY Miguel ACID Bronfman, Department...
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