isolation of peroxisomes from frozen human liver samples

ANALYTICAL BIOCHEMISTRY 2 0 6 , 147-154 (1992)

Isolation of Peroxisomes from Frozen Human Liver Samples

Alejandra Alvarez,* Ulises Hidalgo,* Maria E. Kawada,* Alejandro Munizaga,* Alvaro Zflfiiga,t Luis Ibfinez,t Cecilia S. Koenig,* and Manuel J. Santos* *Department of Cell & Molecular Biology, Faculty of Biological Sciences, and ~fDepartment of Surgery, Faculty of Medicine, Catholic University of Chile, Santiago, Chile

Received April 13, 1992

T h i s p a p e r s h o w s the s u c c e s s f u l i s o l a t i o n o f p e r o x i - s o m e s f r o m h u m a n l i v e r s a m p l e s t h a t w e r e k e p t f r o z e n at - 7 0 ° C . P u r i f i c a t i o n o f t h e s e p e r o x i s o m e s w a s ob- t a i n e d b y a c o m b i n a t i o n o f t w o s u b c e l l u l a r f r a c t i o n - a t i o n t e c h n i q u e s : d i f f e r e n t i a l c e n t r i f u g a t i o n an d iso- p y c n i c f r a c t i o n a t i o n in N y c o d e n z d e n s i t y g r a d i e n t s . P e r o x i s o m e i n t e g r i t y w a s e v a l u a t e d by l a t e n c y m e a - s u r e m e n t s and b y u l t r a s t r u c t u r a l o b s e r v a t i o n . T h e pro- c e d u r e d e s c r i b e d h e r e m a y be u s e f u l for the i s o l a t i o n o f o t h e r s u b c e l l u l a r o r g a n e l l e s f r o m f r o z e n h u m a n s a m p l e s . © 1992 Academic Press, Inc.

Peroxisomes are ubiquitous subcellular organelles. In animals they carry out several metabolic functions: #- oxidation of fatty acids, synthesis of plasmalogens, cellular respiration (with formation of H202), synthesis of bile acids, and others (1-4). Peroxisomal proteins are synthesized on free ribosomes and imported post-trans- lationally into pre-existing organelles. Therefore, peroxisomes seem to form by growth and division of pre- existing organelles (5,6). Recently, a group of human genetic disorders involving peroxisomal functions has been described (7,8). Some of these disorders affect peroxisome biogenesis, such as the Zellweger Syndrome (7-10).

The existence of these peroxisomal disorders has mo- tivated extensive research on the biology of the organelle (8). However, very little is known about the function and biogenesis of human peroxisomes, in contrast to the vast information accumulated on rat peroxisomes over the past years (2,3). There are important differences between rat and human liver peroxisomes. Therefore, information obtained on rat liver peroxisomes may be inapplicable to human peroxisomes. Hence, it is neces- sary to obtain direct information from human peroxi-

0003-2697/92 $5.00 Copyright © 1992 by Academic Press, Inc. All rights of reproduction in any form reserved.

somes. The main reason for the rather limited information on human peroxisomes is the difficult access to the main source of peroxisomes, the human liver. Since in some medical centers, biopsy or autopsy liver samples, obtained from control subjects or patients affected by peroxisomal disorders, can be maintained frozen at -70°C, these samples could represent an alternative source of peroxisomes.

Here we report the successful isolation of peroxisomes from frozen human liver samples, by using subcellular fractionation techniques. The integrity of the isolated organelles was evaluated biochemically by organelle latency measurements and morphologically by electron microscopy and cytochemistry.

MATERIALS AND METHODS

Human Liver Samples

Liver biopsies (200-500 mg) were obtained from patients undergoing surgery for uncomplicated gallstone disease. Informed consent from the patients was obtained by following procedures approved by the Ethics Committee of the Medical School of the Catholic Uni- versity of Chile. Liver-function tests were normal in all cases. In one case, a large sample of 200 g, histologically normal and tumor free, was surgically obtained, due to the presence of an encapsulated liver metastatic tumor. A portion of this sample was used immediately for frac- t ionation and the rest was quickly frozen and kept at -70°C. When needed, a portion of this frozen sample was sectioned (using a meat knife) on dry ice, avoiding any thawing of the unused sample.

Homogenization

Fresh and frozen liver samples were received in homogenization solution (0.25 M sucrose, 3 mM imidazole,

147

148 ALVAREZ ET AL.

H 6 0 0 g x 10 min.

I E

3 0 0 0 g x lOmin.

I I LPS M

J 25000 g x 10 min. x 2

I PS

I O0000 g x 35 min.

FIG. 1.

I I N ~

12000 g

I ~ 0 0 0 0 0 g x 35min.

I ! I L S P

H

r 7oog x lO min. x2

I 1,1

x 20min. x2

I A

I P

Centrifugation conditions used for fractionation by differential centrifugation.

pH 7.4, 1 mM EDTA, 0.1% (v/v) ethanol). The capsule was removed and the sample was cut in small pieces, weighed, diluted in 2 vol of homogenization solution, and was homogenized in a Pot ter -Elvehjem homoge- nizer at 1000 rpm three times at 0°C, similar to the procedure described by Leighton et al. (11). This homogenate was diluted 1:10 and divided in two aliquots, each of which was fractionated by differential centrifugation and, subsequently, by isopycnic centrifugation procedures, as shown below.

Fractionation by Differential Centrifugation

Two different fractionation protocols were used:

a. Fractionation NMLPS. This method proceeds according to de Duve et al. (12). This procedure fraction- ates the homogenate into five fractions: N (nuclear), M (heavy mitochondria), L (light mitochondria), P (microsomes), and S (supernatant). The centrifugation conditions are shown in Fig. 1.

b. Fractionation v-h-¢ . This method proceeds according to Leighton et al. (11). This protocol is a modifi- cation of the previous method. Briefly, the homogenate is fractionated into three fractions: v (equivalent to the N fraction), h (equivalent to the L fraction, but having more contamination with mitochondria), and ¢ (corre- sponding to the supernatant of the h fraction and equivalent to the P and S fractions altogether). We further fractionated the ¢ fraction into P and S following the same conditions indicated above. The centrifugation conditions for this procedure are also shown in Fig. 1.

Fractionation by Isopycnic Centrifugation in Density Gradients

Nycodenz density gradients were made according to Santos et al. (13), with the exception that we used a

25-ml Nycodenz gradient (instead of metrizamide) with density limits ranging from 1.0000 to 1.3000 and a cush- ion of 4 ml of 47% Nycodenz in 0.25 M sucrose/3 mM imidazole, pH 7.4, 1 mM EDTA, 0.1% (v/v) ethanol); 4.5 ml of an L fraction was layered on top of the gradient and centrifugation was performed at 18,000 rpm for 60 min at 8°C in the SV 288 vertical rotor, with slow accel- eration and deceleration, in a Sorvall RC-5B rate-con- trolled centrifuge (DuPont Instruments, Sorvall Divi- sion). One-milliliter fractions were collected from the bottom of the gradient and density measurement was done by determining the refractive index of each fraction (14).

Enzyme and Protein Assays

Established procedures for the determination of marker enzymes were as follows: catalase (11) and fatty acyl-CoA oxidase (lauroyl-CoA as a substrate) (15) for peroxisomes, glutamate dehydrogenase (11) for mitochondria; N-acetyl-3-glucosaminidase (16) for lysosomes, NADPH-cytochrome-c reductase (17) for microsomes, and lactate dehydrogenase (18) for cytosol. Latency measurements of catalase and N-acetyl-3- glucosaminidase were performed in the presence and absence of detergent (19). Proteins were measured by the Bradford method (20).

Electron Microscopy and Cytochemistry

Fractions enriched in peroxisomal enzymes from fractionation of fresh and frozen liver samples, by differential centrifugation (L fractions) and density gradients were diluted, centrifuged, and fixed for 3 h, as a pellet, with 4% paraformaldehyde, 0.5% glutaraldehyde in 0.1 M Pipes buffer, containing 0.2 M sucrose. Then, the samples were postfixed in 1% osmium tetroxide, dehy-

I S O L A T I O N O F H U M A N L I V E R P E R O X I S O M E S 149

T A B L E 1

Specific Activities of Marker Enzymes in Human Fresh and Frozen Liver Homogenates

Specific activities ( m U / m g protein) of h u m a n liver homogena tes

Frozen Frozen Enzyme Fresh Li tera ture a 1 m o n t h 6 months

Cata lase 220.00 173.30 144.21 118.33 N-Acetyl-~-glucosaminidase 23.35 ND 38.55 16.62 Glu tama te dehydrogenase 26.09 39.45 23.51 12.32 NADPH-cy toch rome-c

reductase 20.90 20.93 10.22 4.11 Lac ta te dehydrogenase 204.74 ND 133.58 69.98

a D a t a o b t a i n e d f r o m B r o n f m a n e t al. (24) . Note . V a l u e s s h o w n c o r r e s p o n d t o t h e a v e r a g e o f t w o i n d e p e n d e n t

e x p e r i m e n t s . N D , n o t d e t e r m i n e d .

drated, and embedded in Epon. Ultrathin sections were further stained with uranyl acetate and lead citrate and examined in a Phillips electron microscope.

Cytochemistry for catalase was performed by a modi- fication of the alkaline diaminobenzidine method {21). Incubations were performed at 37°C for 15 h. Incuba- tions in the absence of H202 served as controls.

Calculation and Presentation of Results

The distribution of enzyme markers in fractionation experiments by differential centrifugation and in density gradients was calculated and represented according to de Duve (22) and Bowers and de Duve (23).

RESULTS

Enzyme Activities in Fresh and Frozen Human Liver Homogenates

The specific activities of marker enzymes in fresh and frozen liver homogenates are shown in Table 1. The values obtained for fresh samples are in good agreement with the literature. The activities of these enzymes re- main detectable up to 6 months in frozen samples. Ca- talase, the peroxisomal marker enzyme, decreases only slightly after the liver sample is kept frozen for 6 months. The same pat tern was found for the mitochondrial marker enzyme, glutamate dehydrogenase. The longer exposure to the freezing procedure seems to more dramatically affect the lysosomal and microsomal marker enzymes, N-acetyl-/~-glucosaminidase and NADPH-cytochrome-c reductase, respectively.

Cell Fractionation Studies

The subcellular distribution of different marker enzymes obtained after fractionation of frozen human liver homogenates by two different protocols of differential centrifugation is shown in Fig. 2. Catalase, as ex-

pected, has a higher specific activity in the L (Fig. 2A) and ~ fractions (Fig. 2B) of the two fractionation protocols, with approximately 45% of the activity displaying a soluble pattern. This finding indicates tha t a significant portion of catalase (55%) might be present in a sedi- mentable particle. The distribution of the other marker enzymes is the usual: the marker for cytosol (lactate dehydrogenase), for lysosomes (N-acetyl-~-glucosaminidase), for microsomes, (NADPH-cytochrome-c reductase), and for mitochondria (glutamate dehydrogenase) exhibited maximal relative specific activities in fractions S, L-h, P, and NM-~, respectively. It is worth mentioning that all markers have a considerable pro- portion of their activities recovered in the N fraction, which may reflect unbroken cells. The frozen liver sample is particularly difficult to homogenize; therefore, to avoid organelle breakage, homogenization was stopped before completion. In general, the distribution pat tern of the different marker enzymes is roughly similar to the pat terns reported for fresh human liver samples (24). However, the specific activities for these markers in frozen liver are generally lower than those in fresh samples.

The integrity of the organelles of the frozen liver sample during the fractionation experiments was evaluated by measuring the latency of catalase and N-acetyl-~- glucosaminidase in different subcellular fractions. These results are shown in Table 2. About 40 to 80% of the peroxisomal and lysosomal markers are latent. This means that in frozen samples, these enzymes are com- partmentalized in organelles surrounded by a mem- brane.

To further characterize the distribution of the peroxisomal marker catalase, isopycnic fractionation of human liver sample was performed in Nycodenz density gradients. L fractions from the same liver sample under both conditions, fresh and frozen, were utilized for this type of fractionation experiments. In fresh samples, catalase is largely recovered in the high-density region of the gradient, as expected for peroxisomal equilibrium density (Fig. 3A). Two peaks are seen in this region at densities of 1.22 and 1.17 g/ml. The higher density peak is well separated from the mitochondrial (glutamate dehydrogenase), lysosomal (N-acetyl-~-glucosaminidase), and microsomal (NADPH-cytochrome-c reductase) markers. Catalase also displays a third peak at the low- density area of the gradient, where cytosolic markers are recovered (data not shown). This is also an expected finding since some portion of catalase might leak out of the organelles, during the fractionation procedure. Ta- ble 3 shows some of the properties of peroxisomes purified from this density gradient. Human peroxisomes were purified about 15 times. Rat liver peroxisomes, using the classical purification protocol of Leighton et al. (11), yielded about 30 times. Human liver peroxisomes are slightly contaminated by mitochondria and, to a

150 A L V A R E Z E T AL.

4_

2_

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o 4 U . I

a . m

w

p-

W

2_

4

2_

A

Cata lase

L N

I N- Acet y I-.R-Glucosa m in idase

G l u t a m a t e Dhse.

I I NADPH Cyt.c

I I L a c t a t e Dhse.

h r e d u c t a s e

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- C a t a l a s e

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,1 NADPH

I

0

PROT EIN I%)

A u S

, [7 1 I N-Acetyl-j3- Glucosaminidase

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0 210 4'0 8'0 100 2'0 '~40 60 8'0 100

F I G . 2 . Subce l lu la r f r ac t iona t ion of f rozen h u m a n liver by different ial cent r i fuga t ion . (A) T h e h o m o g e n a t e was f rac t iona ted into N, M, L, P, a n d S f rac t ions . (B) T h e h o m o g e n a t e was f r ac t iona ted into ~, ~, ~, P, a n d S fract ions. For each d is t r ibu t ion p a t t e r n t he absc issa r ep r e sen t s t h e cumula t ive p ro t e in c o n t e n t for each f rac t ion as a pe rcen tage of t he to ta l p ro te in of t he homogena t e . T h e o rd ina te r ep r e sen t s relat ive specific act ivi ty, i.e., pe rcen t age in t he f rac t ion of t he h o m o g e n a t e c o n t e n t of t h e m a r k e r e n z y m e over the pe rcen tage of h o m o g e n a t e p ro te in in t h e f ract ion. T h e d i s t r ibu t ion p a t t e r n co r r e sponds to a r ep resen ta t ive f rac t iona t ion expe r imen t .

lesser extent, by microsomes. These results are in good agreement with the data obtained by Bronfman et al. (24) using fractionation of L human liver fractions in metrizamide gradients.

The human liver sample not used for the previous experiment was frozen quickly, kept frozen at -70°C, thawed, and fractionated. The distribution of the marker enzymes in the same type of Nycodenz density gradient is shown in Fig. 2B. Remarkably, the peroxisomal marker is largely recovered in the high-density region of the gradient, in the "peroxisomal area," which is consistent with the idea of particles containing catalase. The peroxisomal fat ty acid oxidase was also determined across the gradient and found to commigrate with catalase (data not shown). In comparison to the fresh condi-

tion, only one peak of catalase of density 1.22 g/ml was found in this region. The catalase distribution in this region is more contaminated with the mitochondrial and microsomal markers than that in the case of the fresh liver condition. This might be due to the presence of damaged mitochondria and microsomes. Human peroxisomes from this density gradient were purified about 15 times, which is similar to the fresh liver condition (Table 3).

Electron Microscopy (EM) 1 Studies

Subcellular fractions containing peroxisomes derived from a frozen human liver fractionated by differential

1 Abbrev ia t ion used: EM, e lec t ron microscopy.

ISOLATION OF HUMAN LIVER PEROXISOMES 151

TABLE 2

Latency Measurements of Peroxisome and Lysosome En- zymes during Subcellular Fractionation of Frozen Human Liver Samples

Total activity Free activity % Latency

Fraction Catalase NaBgase Catalase Na/~gase Catalase Na/~gase

H 20.8 7.7 16.4 5.6 20.9 27.6 L 0.8 0.6 0.5 0.4 65.3 81.9 Lambda 3.0 1.2 0.6 0.7 81.2 37.8

Note. Enzyme activity is expressed in U/g of liver. Free activity corresponds to enzyme activity determination under nonlytic conditions. Values shown are the averages of two independent experiments. Na/3gase, N-acetyl-/~-glucosaminidase.

a n d isopycnic cen t r i fuga t ions were e x a m i n e d by elec- t r o n microscopy. As shown in Fig. 4A, the L f rac t ion con ta in s severa l types of m e m b r a n o u s organel les , such as pe rox i somes (arrows) , mi tochondr i a , lysosomes , a n d microsomes . T o conf i rm the p resence of pe rox i somes , c y t o c h e m i s t r y for ca t a l a se was pe r fo rmed . F igure 4B shows the e l ec t ron -dense reac t ion p r o d u c t in peroxi -

somes. Some of these organe l les are well p rese rved , as i nd i ca t ed by the r eac t ion p r o d u c t t h a t fills the o rgan- elle. O the r s seemed more ex t rac ted .

Th i s L f rac t ion was s u b f r a c t i o n a t e d in a N y c o d e n z dens i ty g r ad i en t (Fig. 2B) a n d the dense r ca t a l a se p e a k was fixed and e x a m i n e d by EM. C o n v e n t i o n a l E M shows the p re sence of a b u n d a n t we l l -p r e se rved perox i - somes in these g rad i en t f rac t ions . Very few mi tochondr ia and mic rosomes a n d some o the r un i de n t i f i ed s t ruc- t u r e s a re also found (Fig. 4C). Ca t a l a se c y t o c h e m i s t r y shows the r eac t ion c o n c e n t r a t e d in p e r o x i s o m e s (Fig. 4D). T h e s e p e r o x i s o m e s a p p e a r more e x t r a c t e d t h a n the o rgane l l e s found in the L f rac t ion (Figs. 4A a n d 4B). M o s t p e r o x i s o m e s i so l a t ed u n d e r t hese cond i t i ons con- t a i n e d a nuc leo id- l ike s t ruc ture . T h e s e s t r uc tu r e s were ra re ly seen in f resh h u m a n l iver p e r o x i s o m e s (da ta no t shown).

DISCUSSION

P e r o x i s o m e s in h u m a n l iver were iden t i f i ed by Biem- p ica et al. (25,26) as microbodies . Some yea r s la ter , No- vikoff et al. (27), us ing his cy tochemica l m e t h o d devel- oped for the in s i tu d e m o n s t r a t i o n of ca ta lase , t he

A

>- ¢J Z

50-' Catalase

0 50 N - AcetyI-B-GIL_ _m~_ _minkl~

LU

0 LU

b .

o 5o NADPH Cyt,c reductase

Protein

Glutamate Dhse. ?

Catalase

B

50 ] Catalase Protein

a~uJ° ~'''z° >" 50 N-Acetyl-J3-Gluc0saminidase Glutamate .~ o

50 NADPH Cyt.c reductose Catalase

105 1.15 1.25 1.05 1.15 1.25 1.05 1.15 1.25 1.05 1,15 1.25 DENSITY (g/ml) DENSITY (g/ml)

FIG. 3. Nycodenz gradient isopycnic fractionation of L fractions obtained from fresh (A) and frozen (B) human liver samples. The distribution pattern of marker enzymes corresponds to a representative fractionation experiment. For each distribution pattern, the ordinate represents the average frequency of the components for each fraction, Q/~QAp, where Q represents the activity found in the fraction, F~Q is the total activity recovered from the gradient, and Ap is the increment in density of the gradient for each fraction. Frequency is plotted against density in a histogram form. Dashed lines represent the distribution of catalase.

152 ALVAREZ ET AL.

TABLE 3

Properties of Purified Peroxisomes a

Fresh sample

Glutamate NADPH-cytochrome-c Catalase dehydrogenase reductase Catalase

Frozen sample

Glutamate NADPH-cytochrome-c dehydrogenase reductase

Peroxisome SA b 3.51 0.036 0.015 2.12 Homogenate SA 0.22 0.023 0.002 0.144 Relative specific activity

(peroxisome SA/ homogenate SA) 15.9 0.64 0.13 14.7

0.030 0.011 0.050 0.004

1.67 0.36

a Peroxisomes were taken from the high-density peroxisomal peak from a Nycodenz gradient. b SA, specific activity in U/mg protein; U are defined as by Leighton et al. (11).

p ro to type of peroxisomal enzyme, formally demon- s t ra ted the presence of peroxisomes in human hepatic t issues. T h e y were recognized as abundan t single-mem- b rane organelles, of about 0.5 ~m in d iameter and with a granular matr ix. In con t r a s t to ra t liver peroxisomes, which have a ura te oxidase-conta ining nucleoid, h u m an liver peroxisomes lack nucleoid s t ruc tures (28). Conse- quent ly , the identif icat ion of h u m a n liver peroxisomes by convent iona l e lec t ron microscopy can be difficult. Fo r this reason, cytochemical (28-30) and immunocyto- chemical (31) techniques are commonly used for the in s i t u detec t ion of peroxisomes.

Only a few repor ts concern ing the isolat ion and character izat ion of liver peroxisomes f rom human samples are found in the l i te ra ture (24,32-34). Th is s i tuat ion is s tr ikingly different when consider ing rat liver peroxisomes (2,3). Al though ra t liver peroxisomes have been extensively studied, the available in format ion canno t be appl ied to h u m a n peroxisomes. Several impor t an t differences between h u m a n and ra t peroxisomes have been demons t ra ted . For example, h u m a n peroxisomes lack ura te oxidase (and nucleoids) (1), conta in a different p ropor t ion of peroxisomal enzymes (such as the ~3-oxi- dat ion enzymes, catalase, (24,32), and do not exhibit the peroxisomal induct ion p h e n o m e n o n upon adminis t ra- t ion of hypolipidemic drugs (35). There fore , informa- t ion ob ta ined direct ly f rom human peroxisomes is required. T h e main reason for the l imited character iza- t ion of the h u m a n peroxisome is the l imited availabili ty of h u m a n samples. Perox isomes are most f requent ly found in liver cells (28). T h e sources of h u m a n liver can be needle and surgical biopsies, surgical resections, and au topsy material . It is very difficult to access these sources. However , h u m a n liver samples f rom surgical resect ions or f rom fresh au topsy mater ia l could be used as a po ten t ia l source of peroxisomes, if the subcellular organelles are not damaged dur ing the freezing, storage, and thawing procedures . Such samples are cur ren t ly available in some medical inst i tut ions. Therefore , it seems impor t a n t to devise a procedure to isolate organ-

elles f rom frozen samples. W h e n we s ta r ted this work, no in format ion on the isolat ion of any membranous organelles f rom any frozen t issue sample was available.

As men t ioned above, several h u m an genetic disorders involving peroxisome biogenesis have been identif ied (7,8). Th e Zellweger Syndrome is the pro to type of the peroxisomal assembly defect. This is a rare disorder charac ter ized by craniofacial dysmorphia , neurological impai rment , severe metabol ic disturbances, and neona- tal dea th (36). Peroxisomes seemed to be absent in this syndrome, as first repor ted in liver biopsies by Gold- fischer et al. (37). However , the presence of peroxisomal m em b ran e ghosts was found in Zellweger fibroblasts, which suggested a defect in the impor t mach inery for peroxisomal prote ins (9,10). Most of the in format ion cur ren t ly available on this syndrome and o ther peroxisomal disorders has been obta ined f rom peroxisomal studies on skin fibroblast samples (7,8). Since some human liver samples f rom pa t ien ts affected by peroxisomal disorders are cur ren t ly kep t frozen, we decided to devise a protocol for the isolat ion of peroxisomes f rom frozen liver samples. This protocol might be useful in the future for peroxisomal studies related to the character izat ion of the basic defects producing h u man peroxisomal disorders. We were able to isolate peroxisomes f rom the same control liver sample, under bo th fresh and frozen (up to 6 months) conditions, in Nycodenz densi ty gradients. Similar resul ts were obta ined using metr izamide densi ty gradients (data not shown). A significant por t ion of peroxisomes isolated from the frozen sample was well p reserved (by biochemical and morpho- logical cri teria). These peroxisomes were also reasonably pure. At the electron microscopy level the major i ty of these peroxisomes showed a nucleoid-like s t ructure . In this respect , it is wor th ment ion ing tha t similar mor- phological findings have been repor ted in liver biopsies f rom control subjects and some pat ients affected by several diseases (28,29). In the liver samples tha t we ana- lyzed for the presence of nucleoid-like s t ructures , we could not rule out the possibil i ty of a freezing art ifact .

ISOLATION OF HUMAN LIVER PEROXISOMES 153

"h

-% ,: ~ ¢

FIG. 4. Electron microscopy (EM) and catalase cytochemistry in subcellular fractions obtained from frozen liver samples. Fractions enriched, in peroxisomal enzymes were fixed and subjected to conventional EM (A and C) and cytochemistry for catalase (B and D), as indicated under Materials and Methods. (A) and (B) represent L fractions and (C) and (D) represent "peroxisomal fractions" from density gradients. Peroxisomes are abundant and reasonably well preserved and contain a nucleoid-type structure.

154 ALVAREZ ET AL.

T h e r e s u l t s s h o w n in t h i s p a p e r i n d i c a t e t h a t f r o z e n l ive r s a m p l e s m a y be u s e d as an a l t e r n a t i v e s o u r c e for t h e i s o l a t i o n o f p e r o x i s o m e s . T h e s e i s o l a t e d pe rox i - s o m e s c a n be u s e d fo r bas ic b io logy a n d m e d i c a l - r e l a t e d s tud ies . In gene ra l , t h e p r o t o c o l p r e s e n t e d h e r e c o u l d p o t e n t i a l l y be u t i l i z e d for t h e i s o l a t i o n o f o t h e r subce l - l u l a r o r g a n e l l e s f r o m f r o z e n l ive r s amp le s .

ACKNOWLEDGMENTS

This work was supported by Grant 718/90 from FONDECYT, Grant INT90-02001 from NSF, and Grant AAAS from the John D. and Catherine T. MacArthur Foundation. We thank Dr. Miguel Bronfman for helpful suggestions and critical reading of this manu- script.

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isolation of peroxisomes from frozen human liver samples

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