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
This paper shows the successful isolation of peroxisomes from human liver samples that were kept frozen 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 obtained by a combination of two subcellular fractiona 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 a n d isopycnic fractionation in Nycodenz density gradients. Peroxisome integrity was evaluated by latency meas u r e m e n t s a n d 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 procedure described here may be useful for the isolation of other subcellular organelles from frozen human 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-translationally into pre-existing organelles. Therefore, peroxisomes seem to form by growth and division of preexisting organelles (5,6). Recently, a group of hum an genetic disorders involving peroxisomal functions has been described (7,8). Some of these disorders affect peroxisome biogenesis, such as the Zellweger Syndrome (7-10). T h e existence of these peroxisomal disorders has motivated extensive research on the biology of the organelle (8). However, very little is known about the function and biogenesis of hum a n peroxisomes, in contrast to the vast information accumulated on rat peroxisomes over the past years (2,3). T h e r e are important differences between rat and hum an liver peroxisomes. Therefore, information obtained on rat liver peroxisomes may be inapplicable to h u m a n peroxisomes. Hence, it is necessary to obtain direct information from hum an peroxi0003-2697/92 $5.00 Copyright © 1992 by Academic Press, Inc. All rights of reproduction in any form reserved.
somes. T h e main reason for the rat her limited information on hum an peroxisomes is the difficult access to the main source of peroxisomes, the h u m a n 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 hum an liver samples, by using subcellular fractionation techniques. T he 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 University of Chile. Liver-function tests were normal in all cases. In one case, a large sample of 200 g, histologically normal and t u m o r free, was surgically obtained, due to the presence of an encapsulated liver metastatic tumor. A portion of this sample was used immediately for fractionation 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
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p H 7.4, 1 mM EDTA, 0.1% (v/v) ethanol). T h e capsule was removed and the sample was cut in small pieces, weighed, diluted in 2 vol of homogenization solution, and was homogenized in a P o t t e r - E l v e h j e m homogenizer 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 N M L P S . This method proceeds according to de Duve et al. (12). This procedure fractionates the homogenate into five fractions: N (nuclear), M (heavy mitochondria), L (light mitochondria), P (microsomes), and S (supernatant). T h e centrifugation conditions are shown in Fig. 1. b. Fractionation v - h - ¢ . This method proceeds according to Leighton et al. (11). This protocol is a modification 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 ¢ (corresponding to the s uper na t a nt 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 t ha t we used a
25-ml Nycodenz gradient (instead of metrizamide) with density limits ranging from 1.0000 to 1.3000 and a cushion of 4 ml of 47% Nycodenz in 0.25 M sucrose/3 mM imidazole, p H 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 acceleration and deceleration, in a Sorvall RC-5B rate-controlled centrifuge (D uPont Instruments, Sorvall Division). 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-3glucosaminidase 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-
ISOLATION
TABLE
OF HUMAN
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 h o m o g e n a t e s
Enzyme
Fresh
Literature a
Frozen 1 month
Frozen 6 months
Catalase N-Acetyl-~-glucosaminidase Glutamate dehydrogenase NADPH-cytochrome-c reductase L a c t a t e dehydrogenase
220.00 23.35 26.09
173.30 ND 39.45
144.21 38.55 23.51
118.33 16.62 12.32
20.90 204.74
20.93 ND
10.22 133.58
4.11 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. Values shown correspond to the average of two independent experiments. ND, not determined.
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 modification of the alkaline diaminobenzidine method {21). Incubations were performed at 37°C for 15 h. Incubations in the absence of H202 served as controls. Calculation and Presentation of Results T h e 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 H u m a n Liver Homogenates T h e specific activities of marker enzymes in fresh and frozen liver homogenates are shown in Table 1. T h e values obtained for fresh samples are in good agreement with the literature. T he activities of these enzymes remain detectable up to 6 months in frozen samples. Catalase, the peroxisomal marker enzyme, decreases only slightly after the liver sample is kept frozen for 6 months. Th e same pa t t e r n was found for the mitochondrial mar k er enzyme, glutamate dehydrogenase. T h e 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 T h e subcellular distribution of different marker enzymes obtained after fractionation of frozen hum an liver homogenates by two different protocols of differential centrifugation is shown in Fig. 2. Catalase, as ex-
LIVER
PEROXISOMES
149
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 t h a t a significant portion of catalase (55%) might be present in a sedimentable particle. T h e distribution of the other m a rk e r 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 t h a t all markers have a considerable proportion of their activities recovered in the N fraction, which may reflect unbroken cells. T h e frozen liver sample is particularly difficult to homogenize; therefore, to avoid organelle breakage, homogenization was stopped before completion. In general, the distribution p a t t e r n of the different marker enzymes is roughly similar to the patterns reported for fresh hum an liver samples (24). However, the specific activities for these markers in frozen liver are generally lower t han those in fresh samples. T he 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 t hat in frozen samples, these enzymes are compartmentalized in organelles surrounded by a membrane. T o 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 u n d e r 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. T h e 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 lowdensity 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. Table 3 shows some of the properties of peroxisomes purified from this density gradient. H u m a n peroxisomes were purified about 15 times. Rat liver peroxisomes, using the classical purification protocol of Leighton et al. (11), yielded about 30 times. H u m a n liver peroxisomes are slightly contaminated by mitochondria and, to a
150
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P R O T EIN I%) F I G . 2. S u b c e l l u l a r f r a c t i o n a t i o n of frozen h u m a n liver by differential c e n t r i f u g a t i o n . (A) T h e h o m o g e n a t e w a s f r a c t i o n a t e d into N, M, L, P, a n d S fractions. (B) T h e h o m o g e n a t e w a s f r a c t i o n a t e d into ~, ~, ~, P, a n d S fractions. F o r e a c h d i s t r i b u t i o n p a t t e r n t h e abscissa r e p r e s e n t s t h e c u m u l a t i v e p r o t e i n c o n t e n t for e a c h f r a c t i o n as a p e r c e n t a g e of t h e t o t a l p r o t e i n of t h e h o m o g e n a t e . T h e o r d i n a t e r e p r e s e n t s relative specific activity, i.e., p e r c e n t a g e in t h e fraction of t h e 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 t h e p e r c e n t a g e of h o m o g e n a t e p r o t e i n in t h e fraction. T h e d i s t r i b u t i o n p a t t e r n c o r r e s p o n d s to a r e p r e s e n t a t i v e f r a c t i o n a t i o n e x p e r i m e n t .
lesser extent, by microsomes. These results are in good agreement with the data obtained by B r onf m an et al. (24) using fractionation of L hum a n liver fractions in metrizamide gradients. T h e h u m a n liver sample not used for the previous experiment was frozen quickly, kept frozen at -70° C, thawed, and fractionated. T h e 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. T h e peroxisomal fatty 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. T h e catalase distribution in this region is more contaminated with the mitochondrial and microsomal markers t han t h a t in the case of the fresh liver condition. This might be due to the presence of damaged mitochondria and microsomes. H u m a n peroxisomes from this density gradient were purified about 15 times, which is similar to the fresh liver condition (Table 3). Electron Microscopy ( E M ) 1 Studies
Subcellular fractions containing peroxisomes derived from a frozen human liver fractionated by differential 1 A b b r e v i a t i o n used: E M , electron microscopy.
ISOLATION OF HUMAN LIVER PEROXISOMES TABLE 2 Latency Measurements of Peroxisome and Lysosome Enzymes during Subcellular Fractionation of Frozen Human Liver Samples Total activity
Free activity
% Latency
Fraction
Catalase
NaBgase
Catalase
Na/~gase
Catalase
Na/~gase
H L Lambda
20.8 0.8 3.0
7.7 0.6 1.2
16.4 0.5 0.6
5.6 0.4 0.7
20.9 65.3 81.2
27.6 81.9 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 i s o p y c n i c c e n t r i f u g a t i o n s were e x a m i n e d b y elect r o n m i c r o s c o p y . As s h o w n in Fig. 4A, t h e L f r a c t i o n c o n t a i n s s e v e r a l t y p e s of m e m b r a n o u s o r g a n e l l e s , such as p e r o x i s o m e s ( a r r o w s ) , m i t o c h o n d r i a , l y s o s o m e s , a n d m i c r o s o m e s . T o c o n f i r m t h e p r e s e n c e of p e r o x i s o m e s , c y t o c h e m i s t r y for c a t a l a s e was p e r f o r m e d . F i g u r e 4B s h o w s t h e e l e c t r o n - d e n s e r e a c t i o n p r o d u c t in p e r o x i -
151
somes. S o m e of t h e s e o r g a n e l l e s a r e well p r e s e r v e d , as i n d i c a t e d by t h e r e a c t i o n p r o d u c t t h a t fills t h e o r g a n elle. O t h e r s s e e m e d m o r e e x t r a c t e d . T h i s L f r a c t i o n w a s 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 d e n s i t y g r a d i e n t (Fig. 2B) a n d t h e d e n s e r c a t a l a s e p e a k w a s fixed a n d e x a m i n e d b y E M . C o n v e n t i o n a l E M shows t h e p r e s e n c e of a b u n d a n t w e l l - p r e s e r v e d p e r o x i s o m e s in t h e s e g r a d i e n t f r a c t i o n s . V e r y few m i t o c h o n dria and microsomes and some other unidentified struct u r e s a r e also f o u n d (Fig. 4C). C a t a l a s e c y t o c h e m i s t r y shows t h e r e a c t i o n 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 m o r e e x t r a c t e d t h a n t h e o r g a n e l l e s f o u n d in t h e L f r a c t i o n (Figs. 4A a n d 4B). M o s t p e r o x i s o m e s i s o l a t e d u n d e r t h e s e c o n d i t i o n s contained a nucleoid-like structure. These structures were r a r e l y s e e n in f r e s h h u m a n liver p e r o x i s o m e s ( d a t a n o t shown). DISCUSSION P e r o x i s o m e s in h u m a n liver were i d e n t i f i e d b y B i e m p i c a et al. (25,26) as m i c r o b o d i e s . S o m e y e a r s l a t e r , N o v i k o f f et al. (27), u s i n g his c y t o c h e m i c a l m e t h o d develo p e d for t h e in s i t u d e m o n s t r a t i o n of c a t a l a s e , t h e
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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~Qis 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 Catalase
Peroxisome SAb Homogenate SA Relative specific activity (peroxisome SA/ homogenate SA)
3.51 0.22 15.9
Frozen sample
Glutamate dehydrogenase
NADPH-cytochrome-c reductase
0.036 0.023
0.015 0.002
0.64
0.13
Catalase 2.12 0.144 14.7
Glutamate dehydrogenase
NADPH-cytochrome-c reductase
0.030 0.050
0.011 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 r o t o t y p e of p e r o x i s o m a l enzyme, formally d e m o n s t r a t e d the p r e s e n c e of p e r o x i s o m e s in h u m a n hepatic tissues. T h e y were recognized as a b u n d a n t single-memb r a n e organelles, of a b o u t 0.5 ~m in d i a m e t e r and with a g r a n u l a r matrix. In c o n t r a s t to rat liver peroxisomes, which have a u r a t e oxidase-containing nucleoid, h u m a n liver p e r o x i s o m e s lack nucleoid s t r u c t u r e s (28). Consequently, the identification of h u m a n liver peroxisomes by c o n v e n t i o n a l e l e c t r o n microscopy can be difficult. F o r this reason, c y t o c h e m i c a l (28-30) a n d i m m u n o c y t o chemical (31) t e c h n i q u e s are c o m m o n l y used for the in s i t u d e t e c t i o n of peroxisomes. Only a few reports c o n c e r n i n g the isolation and characterization of liver p e r o x i s o m e s from h u m a n samples are f o u n d in the l i t e r a t u r e (24,32-34). T h i s situation is strikingly different w h e n considering rat liver peroxisomes (2,3). Although rat liver peroxisomes have been extensively studied, the available i n f o r m a t i o n c a n n o t be applied to h u m a n peroxisomes. Several i m p o r t a n t differences between h u m a n a n d rat p e r o x i s o m e s have been d e m o n s t r a t e d . F o r example, h u m a n peroxisomes lack u r a t e oxidase (and nucleoids) (1), contain a different p r o p o r t i o n of p e r o x i s o m a l e n z y m e s (such as the ~3-oxidation enzymes, catalase, (24,32), and do not exhibit the p e r o x i s o m a l induction p h e n o m e n o n u p o n administration of hypolipidemic drugs (35). T h e r e f o r e , information o b t a i n e d directly from h u m a n peroxisomes is required. T h e main r e a s o n for the limited characterization of the h u m a n p e r o x i s o m e is the limited availability of h u m a n samples. P e r o x i s o m e s are most f r e q u e n t l y f o u n d in liver cells (28). T h e sources of h u m a n liver can be needle a n d surgical biopsies, surgical resections, a n d a u t o p s y material. It is very difficult to access these sources. However, h u m a n liver samples from surgical resections or f r o m fresh a u t o p s y material could be u s e d as a p o t e n t i a l source of peroxisomes, if the subcellular organelles are not d a m a g e d during the freezing, storage, a n d thawing procedures. Such samples are c u r r e n t l y available in some medical institutions. T h e r e f o r e , it seems i m p o r t a n t to devise a p r o c e d u r e to isolate organ-
elles from frozen samples. W h e n we s t a r t e d this work, no i n f o r m a t i o n on the isolation of any m e m b r a n o u s organelles from any frozen tissue sample was available. As m e n t i o n e d above, several h u m a n genetic disorders involving peroxisome biogenesis have been identified (7,8). T h e Zellweger S y n d r o m e is the p r o t o t y p e of the peroxisomal assembly defect. T h i s is a rare disorder c h a r a c t e r i z e d by craniofacial dysmorphia, neurological i m p a i r m e n t , severe metabolic disturbances, and n e o n a tal d e a t h (36). P e r o x i s o m e s s e e m e d to be a b s e n t in this syndrome, as first r e p o r t e d in liver biopsies by Goldfischer et al. (37). However, the presence of peroxisomal m e m b r a n e ghosts was f o u n d in Zellweger fibroblasts, which suggested a defect in the i m p o r t m a c h i n e r y for peroxisomal proteins (9,10). Most of the i n f o r m a t i o n c u r r e n t l y available on this s y n d r o m e and o t h e r peroxisomal disorders has been o b t a i n e d from p e r o x i s o m a l studies on skin fibroblast samples (7,8). Since some hum a n liver samples from p a t i e n t s affected by peroxisomal disorders are c u r r e n t l y k e p t frozen, we decided to devise a protocol for the isolation of peroxisomes f r o m frozen liver samples. T h i s protocol might be useful in the future for peroxisomal studies related to the characterization of the basic defects producing h u m a n peroxisomal disorders. We were able to isolate peroxisomes from the same control liver sample, u n d e r b o t h fresh and frozen (up to 6 m o n t h s ) conditions, in N y c o d e n z density gradients. Similar results were obtained using metrizamide density gradients (data not shown). A significant p o r t i o n of peroxisomes isolated from the frozen sample was well p r e s e r v e d (by biochemical and m o r p h o logical criteria). T h e s e peroxisomes were also reasonably pure. At the electron microscopy level the majority of these peroxisomes showed a nucleoid-like structure. In this respect, it is w o r t h m e n t i o n i n g t h a t similar morphological findings have b e e n r e p o r t e d in liver biopsies f r o m control subjects a n d some patients affected by several diseases (28,29). In the liver samples t h a t we analyzed for the presence of nucleoid-like structures, we could not rule out the possibility of a freezing artifact.
153
ISOLATION OF HUMAN LIVER P E R O X I S O M E S
"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 results s h o w n in this p a p e r indicate t h a t frozen l i v e r s a m p l e s m a y b e u s e d as a n a l t e r n a t i v e s o u r c e f o r the isolation of peroxisomes. These isolated peroxisomes can be used for basic biology and medical-related studies. In general, the protocol presented here could p o t e n t i a l l y be utilized for the isolation of other subcellular organelles from frozen liver samples. 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 manuscript. REFERENCES 1. 2. 3. 4.
5. 6. 7.
8.
9. 10. 11. 12. 13. 14.
de Duve, C., and Baudhuin, P. (1966) Physiol. Rev. 46, 323-357. Tolbert, N. E. (1981) Annu. Rev. Biochem. 5 0 , 133-157. de Duve, C. (1983) Sci. Am. 248, 74-81. Lazarow, P. B. (1988) in The Liver: Biology and Pathobiology (Arias, I. M., Jakoby, W. P., Popper, H., Schachter, D., and Shafritz, D. A., Eds.), pp. 241-254, Raven Press, New York. Lazarow, P. B., and Fujiki, Y. (1985) Annu. Rev. CellBiol. 1,489530. Borst, P. (1989) Biochem. Biophys. Acta 1006, 1-13. Schutgens, R. B. H., Heymans, H. S. A., Wanders, R. J. A., van den Bosch, H., and Tager, J. M. (1986) Eur. J. Pediatr. 144, 430-440. Lazarow, P. B., and Moser, H. (1989) in The Metabolic Basis of Inherited Diseases (Scriver, C. R., Beaudet, A. L., Sly, W. S., and Valle, D., Eds.), pp. 1470-1509, McGraw-Hill, New York. Santos, M. J., Imanaka, T., Shio, H., Small, G. M., and Lazarow, P. B. (1988) Science 239, 1536-1538. Santos, M. J., Imanaka, T., Shio, H., and Lazarow, P. B. (1988) J. Biol. Chem. 263, 10502-10509. Leighton, F., Poole, B., Beaufay, H., Baudhuin, P., Coffey, J. W., Fowler, S., and de Duve, C. (1968) J. Cell Biol. 37, 482-513. de Duve, C., Pressman, B. C., Gianetto, R., Wattiaux, R., and Appelmans, F. (1955) Biochem. J. 60, 604-617. Santos, M. J., Ojeda, J. M., Garrido, J., and Leighton, F. (1985) Proc. Natl. Acad. Sci. USA 82, 6556-6560. Rome, L., Garvin, A., Allietta, M. A., and Neufeld, E. (1979) Cell 17, 143-153.
15. Small, G., Brolly, D., and Connock, M. J. (1985) Biochem. J. 227, 205-210. 16. Sellinger, O. Z., Beaufay, H., Jacques, P., Doyen, A., and de Duve, C. (1960) Biochem. J. 74, 450-456. 17. Beaufay, H., Amar-Costesec, A., Feytmans, E., Thines-Sempoux, D., Wibo, Robbi, M., and Berthet, J. (1974) J. CeUBiol. 61,188200. 18. Bergmeyer, H. U., Bernt, E., and Hess, R. (1963) in Methods of Enzymatic Analysis (Bergmeyer, H. U., Ed.), pp. 452-552, Academic Press, London/New York. 19. Berthet, J., and de Duve, C. (1951) Biochem. J. 50, 174-181. 20. Bradford, M. (1976) Anal. Biochem. 72,248-254. 21. Roels, F., and Goldfischer, S. (1979) J. Histochem. Cytochem. 27, 1471-1477. 22. de Duve, C. (1967) in Enzyme Cytology (Roodyn, D. B., Ed.), pp. 1-26, Academic Press, London. 23. Bowers, W. E., and de Duve, C. {1967) J. Cell Biol. 32, 339-348. 24. Bronfman, M., Inestrosa, N., Nervi, F., and Leighton, F. (1984) Biochem. J. 224,709-720. 25. Biempica, L. (1966) J. Cell Biol. 29, 383-386. 26. Ma, M. H., and Biempica, L. (1971) Am. J. Pathol. 62, 353-375. 27. Novikoff, P. M., Novikoff, A. B., Quintana, N., and Davis, C. (1973) J. Histochem. Cytochem. 21,540-558. 28. Sternlieb, I., and Quintana, N. (1977) Lab. Invest. 36, 140-149. 29. Roels, F., Pauwels, M., Cornelis, A., Kerckaert, I., Van Der Spek, P., Goovaerts, G., Versieck, J., and Goldfischer, S. (1983) J. Histochem. Cytochem. 31,235-237.
30. De Craemer, D., Espeel, M., Langendries, M., Schutgens, R. B. H., Hashimoto, T., and Roels, F. (1990) Histochem. J. 22, 36-44. 31. Litwin, J. A., VSlkl, A., Miiller-HScker, J., Hashimoto, T., and Fahimi, D. (1987) Am. J. Pathol. 126, 141-150. 32. Bronfman, M., and Leighton, F. (1984) Biochem. J. 224, 721730. 33. Chandonga, J., Krizco, J., Moravec, R., Vician, M., and Juraskova, E. (1986) BiolUgia (Bratislava) 41,383-392. 34. Wilson, G. N., and King, T. E. (1991) Biochem. Med. Metab. Biol. 46, 235-245. 35. Hanefeld, M., Kemmer, C., and Kadner, E. (1983) Atherosclerosis 46, 239-246. 36. Kelley, R. I. {1983) Am. J. Med. Genet. 16, 503-517. 37. Goldfischer, S., Moore, C. L., Johnson, A. B., Spiro, A. J., Valsamis, M. P., Wisniewski, H. K., Ritch, R. H., Norton, W. T., Raspin, I., and Gartner, L. M. (1973) Science 182, 62-64.
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
This paper shows the successful isolation of peroxisomes from human liver samples that were kept frozen 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 obtained by a combination of two subcellular fractiona 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 a n d isopycnic fractionation in Nycodenz density gradients. Peroxisome integrity was evaluated by latency meas u r e m e n t s a n d 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 procedure described here may be useful for the isolation of other subcellular organelles from frozen human 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-translationally into pre-existing organelles. Therefore, peroxisomes seem to form by growth and division of preexisting organelles (5,6). Recently, a group of hum an genetic disorders involving peroxisomal functions has been described (7,8). Some of these disorders affect peroxisome biogenesis, such as the Zellweger Syndrome (7-10). T h e existence of these peroxisomal disorders has motivated extensive research on the biology of the organelle (8). However, very little is known about the function and biogenesis of hum a n peroxisomes, in contrast to the vast information accumulated on rat peroxisomes over the past years (2,3). T h e r e are important differences between rat and hum an liver peroxisomes. Therefore, information obtained on rat liver peroxisomes may be inapplicable to h u m a n peroxisomes. Hence, it is necessary to obtain direct information from hum an peroxi0003-2697/92 $5.00 Copyright © 1992 by Academic Press, Inc. All rights of reproduction in any form reserved.
somes. T h e main reason for the rat her limited information on hum an peroxisomes is the difficult access to the main source of peroxisomes, the h u m a n 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 hum an liver samples, by using subcellular fractionation techniques. T he 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 University of Chile. Liver-function tests were normal in all cases. In one case, a large sample of 200 g, histologically normal and t u m o r free, was surgically obtained, due to the presence of an encapsulated liver metastatic tumor. A portion of this sample was used immediately for fractionation 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
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p H 7.4, 1 mM EDTA, 0.1% (v/v) ethanol). T h e capsule was removed and the sample was cut in small pieces, weighed, diluted in 2 vol of homogenization solution, and was homogenized in a P o t t e r - E l v e h j e m homogenizer 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 N M L P S . This method proceeds according to de Duve et al. (12). This procedure fractionates the homogenate into five fractions: N (nuclear), M (heavy mitochondria), L (light mitochondria), P (microsomes), and S (supernatant). T h e centrifugation conditions are shown in Fig. 1. b. Fractionation v - h - ¢ . This method proceeds according to Leighton et al. (11). This protocol is a modification 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 ¢ (corresponding to the s uper na t a nt 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 t ha t we used a
25-ml Nycodenz gradient (instead of metrizamide) with density limits ranging from 1.0000 to 1.3000 and a cushion of 4 ml of 47% Nycodenz in 0.25 M sucrose/3 mM imidazole, p H 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 acceleration and deceleration, in a Sorvall RC-5B rate-controlled centrifuge (D uPont Instruments, Sorvall Division). 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-3glucosaminidase 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-
ISOLATION
TABLE
OF HUMAN
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 h o m o g e n a t e s
Enzyme
Fresh
Literature a
Frozen 1 month
Frozen 6 months
Catalase N-Acetyl-~-glucosaminidase Glutamate dehydrogenase NADPH-cytochrome-c reductase L a c t a t e dehydrogenase
220.00 23.35 26.09
173.30 ND 39.45
144.21 38.55 23.51
118.33 16.62 12.32
20.90 204.74
20.93 ND
10.22 133.58
4.11 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. Values shown correspond to the average of two independent experiments. ND, not determined.
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 modification of the alkaline diaminobenzidine method {21). Incubations were performed at 37°C for 15 h. Incubations in the absence of H202 served as controls. Calculation and Presentation of Results T h e 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 H u m a n Liver Homogenates T h e specific activities of marker enzymes in fresh and frozen liver homogenates are shown in Table 1. T h e values obtained for fresh samples are in good agreement with the literature. T he activities of these enzymes remain detectable up to 6 months in frozen samples. Catalase, the peroxisomal marker enzyme, decreases only slightly after the liver sample is kept frozen for 6 months. Th e same pa t t e r n was found for the mitochondrial mar k er enzyme, glutamate dehydrogenase. T h e 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 T h e subcellular distribution of different marker enzymes obtained after fractionation of frozen hum an liver homogenates by two different protocols of differential centrifugation is shown in Fig. 2. Catalase, as ex-
LIVER
PEROXISOMES
149
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 t h a t a significant portion of catalase (55%) might be present in a sedimentable particle. T h e distribution of the other m a rk e r 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 t h a t all markers have a considerable proportion of their activities recovered in the N fraction, which may reflect unbroken cells. T h e frozen liver sample is particularly difficult to homogenize; therefore, to avoid organelle breakage, homogenization was stopped before completion. In general, the distribution p a t t e r n of the different marker enzymes is roughly similar to the patterns reported for fresh hum an liver samples (24). However, the specific activities for these markers in frozen liver are generally lower t han those in fresh samples. T he 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 t hat in frozen samples, these enzymes are compartmentalized in organelles surrounded by a membrane. T o 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 u n d e r 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. T h e 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 lowdensity 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. Table 3 shows some of the properties of peroxisomes purified from this density gradient. H u m a n peroxisomes were purified about 15 times. Rat liver peroxisomes, using the classical purification protocol of Leighton et al. (11), yielded about 30 times. H u m a n liver peroxisomes are slightly contaminated by mitochondria and, to a
150
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P R O T EIN I%) F I G . 2. S u b c e l l u l a r f r a c t i o n a t i o n of frozen h u m a n liver by differential c e n t r i f u g a t i o n . (A) T h e h o m o g e n a t e w a s f r a c t i o n a t e d into N, M, L, P, a n d S fractions. (B) T h e h o m o g e n a t e w a s f r a c t i o n a t e d into ~, ~, ~, P, a n d S fractions. F o r e a c h d i s t r i b u t i o n p a t t e r n t h e abscissa r e p r e s e n t s t h e c u m u l a t i v e p r o t e i n c o n t e n t for e a c h f r a c t i o n as a p e r c e n t a g e of t h e t o t a l p r o t e i n of t h e h o m o g e n a t e . T h e o r d i n a t e r e p r e s e n t s relative specific activity, i.e., p e r c e n t a g e in t h e fraction of t h e 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 t h e p e r c e n t a g e of h o m o g e n a t e p r o t e i n in t h e fraction. T h e d i s t r i b u t i o n p a t t e r n c o r r e s p o n d s to a r e p r e s e n t a t i v e f r a c t i o n a t i o n e x p e r i m e n t .
lesser extent, by microsomes. These results are in good agreement with the data obtained by B r onf m an et al. (24) using fractionation of L hum a n liver fractions in metrizamide gradients. T h e h u m a n liver sample not used for the previous experiment was frozen quickly, kept frozen at -70° C, thawed, and fractionated. T h e 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. T h e peroxisomal fatty 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. T h e catalase distribution in this region is more contaminated with the mitochondrial and microsomal markers t han t h a t in the case of the fresh liver condition. This might be due to the presence of damaged mitochondria and microsomes. H u m a n peroxisomes from this density gradient were purified about 15 times, which is similar to the fresh liver condition (Table 3). Electron Microscopy ( E M ) 1 Studies
Subcellular fractions containing peroxisomes derived from a frozen human liver fractionated by differential 1 A b b r e v i a t i o n used: E M , electron microscopy.
ISOLATION OF HUMAN LIVER PEROXISOMES TABLE 2 Latency Measurements of Peroxisome and Lysosome Enzymes during Subcellular Fractionation of Frozen Human Liver Samples Total activity
Free activity
% Latency
Fraction
Catalase
NaBgase
Catalase
Na/~gase
Catalase
Na/~gase
H L Lambda
20.8 0.8 3.0
7.7 0.6 1.2
16.4 0.5 0.6
5.6 0.4 0.7
20.9 65.3 81.2
27.6 81.9 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 i s o p y c n i c c e n t r i f u g a t i o n s were e x a m i n e d b y elect r o n m i c r o s c o p y . As s h o w n in Fig. 4A, t h e L f r a c t i o n c o n t a i n s s e v e r a l t y p e s of m e m b r a n o u s o r g a n e l l e s , such as p e r o x i s o m e s ( a r r o w s ) , m i t o c h o n d r i a , l y s o s o m e s , a n d m i c r o s o m e s . T o c o n f i r m t h e p r e s e n c e of p e r o x i s o m e s , c y t o c h e m i s t r y for c a t a l a s e was p e r f o r m e d . F i g u r e 4B s h o w s t h e e l e c t r o n - d e n s e r e a c t i o n p r o d u c t in p e r o x i -
151
somes. S o m e of t h e s e o r g a n e l l e s a r e well p r e s e r v e d , as i n d i c a t e d by t h e r e a c t i o n p r o d u c t t h a t fills t h e o r g a n elle. O t h e r s s e e m e d m o r e e x t r a c t e d . T h i s L f r a c t i o n w a s 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 d e n s i t y g r a d i e n t (Fig. 2B) a n d t h e d e n s e r c a t a l a s e p e a k w a s fixed a n d e x a m i n e d b y E M . C o n v e n t i o n a l E M shows t h e p r e s e n c e of a b u n d a n t w e l l - p r e s e r v e d p e r o x i s o m e s in t h e s e g r a d i e n t f r a c t i o n s . V e r y few m i t o c h o n dria and microsomes and some other unidentified struct u r e s a r e also f o u n d (Fig. 4C). C a t a l a s e c y t o c h e m i s t r y shows t h e r e a c t i o n 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 m o r e e x t r a c t e d t h a n t h e o r g a n e l l e s f o u n d in t h e L f r a c t i o n (Figs. 4A a n d 4B). M o s t p e r o x i s o m e s i s o l a t e d u n d e r t h e s e c o n d i t i o n s contained a nucleoid-like structure. These structures were r a r e l y s e e n in f r e s h h u m a n liver p e r o x i s o m e s ( d a t a n o t shown). DISCUSSION P e r o x i s o m e s in h u m a n liver were i d e n t i f i e d b y B i e m p i c a et al. (25,26) as m i c r o b o d i e s . S o m e y e a r s l a t e r , N o v i k o f f et al. (27), u s i n g his c y t o c h e m i c a l m e t h o d develo p e d for t h e in s i t u d e m o n s t r a t i o n of c a t a l a s e , t h e
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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~Qis 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 Catalase
Peroxisome SAb Homogenate SA Relative specific activity (peroxisome SA/ homogenate SA)
3.51 0.22 15.9
Frozen sample
Glutamate dehydrogenase
NADPH-cytochrome-c reductase
0.036 0.023
0.015 0.002
0.64
0.13
Catalase 2.12 0.144 14.7
Glutamate dehydrogenase
NADPH-cytochrome-c reductase
0.030 0.050
0.011 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 r o t o t y p e of p e r o x i s o m a l enzyme, formally d e m o n s t r a t e d the p r e s e n c e of p e r o x i s o m e s in h u m a n hepatic tissues. T h e y were recognized as a b u n d a n t single-memb r a n e organelles, of a b o u t 0.5 ~m in d i a m e t e r and with a g r a n u l a r matrix. In c o n t r a s t to rat liver peroxisomes, which have a u r a t e oxidase-containing nucleoid, h u m a n liver p e r o x i s o m e s lack nucleoid s t r u c t u r e s (28). Consequently, the identification of h u m a n liver peroxisomes by c o n v e n t i o n a l e l e c t r o n microscopy can be difficult. F o r this reason, c y t o c h e m i c a l (28-30) a n d i m m u n o c y t o chemical (31) t e c h n i q u e s are c o m m o n l y used for the in s i t u d e t e c t i o n of peroxisomes. Only a few reports c o n c e r n i n g the isolation and characterization of liver p e r o x i s o m e s from h u m a n samples are f o u n d in the l i t e r a t u r e (24,32-34). T h i s situation is strikingly different w h e n considering rat liver peroxisomes (2,3). Although rat liver peroxisomes have been extensively studied, the available i n f o r m a t i o n c a n n o t be applied to h u m a n peroxisomes. Several i m p o r t a n t differences between h u m a n a n d rat p e r o x i s o m e s have been d e m o n s t r a t e d . F o r example, h u m a n peroxisomes lack u r a t e oxidase (and nucleoids) (1), contain a different p r o p o r t i o n of p e r o x i s o m a l e n z y m e s (such as the ~3-oxidation enzymes, catalase, (24,32), and do not exhibit the p e r o x i s o m a l induction p h e n o m e n o n u p o n administration of hypolipidemic drugs (35). T h e r e f o r e , information o b t a i n e d directly from h u m a n peroxisomes is required. T h e main r e a s o n for the limited characterization of the h u m a n p e r o x i s o m e is the limited availability of h u m a n samples. P e r o x i s o m e s are most f r e q u e n t l y f o u n d in liver cells (28). T h e sources of h u m a n liver can be needle a n d surgical biopsies, surgical resections, a n d a u t o p s y material. It is very difficult to access these sources. However, h u m a n liver samples from surgical resections or f r o m fresh a u t o p s y material could be u s e d as a p o t e n t i a l source of peroxisomes, if the subcellular organelles are not d a m a g e d during the freezing, storage, a n d thawing procedures. Such samples are c u r r e n t l y available in some medical institutions. T h e r e f o r e , it seems i m p o r t a n t to devise a p r o c e d u r e to isolate organ-
elles from frozen samples. W h e n we s t a r t e d this work, no i n f o r m a t i o n on the isolation of any m e m b r a n o u s organelles from any frozen tissue sample was available. As m e n t i o n e d above, several h u m a n genetic disorders involving peroxisome biogenesis have been identified (7,8). T h e Zellweger S y n d r o m e is the p r o t o t y p e of the peroxisomal assembly defect. T h i s is a rare disorder c h a r a c t e r i z e d by craniofacial dysmorphia, neurological i m p a i r m e n t , severe metabolic disturbances, and n e o n a tal d e a t h (36). P e r o x i s o m e s s e e m e d to be a b s e n t in this syndrome, as first r e p o r t e d in liver biopsies by Goldfischer et al. (37). However, the presence of peroxisomal m e m b r a n e ghosts was f o u n d in Zellweger fibroblasts, which suggested a defect in the i m p o r t m a c h i n e r y for peroxisomal proteins (9,10). Most of the i n f o r m a t i o n c u r r e n t l y available on this s y n d r o m e and o t h e r peroxisomal disorders has been o b t a i n e d from p e r o x i s o m a l studies on skin fibroblast samples (7,8). Since some hum a n liver samples from p a t i e n t s affected by peroxisomal disorders are c u r r e n t l y k e p t frozen, we decided to devise a protocol for the isolation of peroxisomes f r o m frozen liver samples. T h i s protocol might be useful in the future for peroxisomal studies related to the characterization of the basic defects producing h u m a n peroxisomal disorders. We were able to isolate peroxisomes from the same control liver sample, u n d e r b o t h fresh and frozen (up to 6 m o n t h s ) conditions, in N y c o d e n z density gradients. Similar results were obtained using metrizamide density gradients (data not shown). A significant p o r t i o n of peroxisomes isolated from the frozen sample was well p r e s e r v e d (by biochemical and m o r p h o logical criteria). T h e s e peroxisomes were also reasonably pure. At the electron microscopy level the majority of these peroxisomes showed a nucleoid-like structure. In this respect, it is w o r t h m e n t i o n i n g t h a t similar morphological findings have b e e n r e p o r t e d in liver biopsies f r o m control subjects a n d some patients affected by several diseases (28,29). In the liver samples t h a t we analyzed for the presence of nucleoid-like structures, we could not rule out the possibility of a freezing artifact.
153
ISOLATION OF HUMAN LIVER P E R O X I S O M E S
"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.
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T h e results s h o w n in this p a p e r indicate t h a t frozen l i v e r s a m p l e s m a y b e u s e d as a n a l t e r n a t i v e s o u r c e f o r the isolation of peroxisomes. These isolated peroxisomes can be used for basic biology and medical-related studies. In general, the protocol presented here could p o t e n t i a l l y be utilized for the isolation of other subcellular organelles from frozen liver samples. 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 manuscript. REFERENCES 1. 2. 3. 4.
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