Peroxisomes isolation

B. Gerhardt, Localization of B-oxidation enzymes in peroxisomes isolated from nonfatty plant tissues. Planta 159 238 (1983). 

Thiolester hydrolases are present in most tissues and cell compartments. High concentrations are found in liver microsomes and in brown adipose tissue mitochondria and peroxisomes. Several acyl-CoA hydrolases have shown a close relationship to the nonspecific carboxylesterases EC 3.1.1.1. Thus, palmitoyl-CoA hydrolase purified from rat liver microsomes was found to be identical to esterase pI 6.2I6A (ES4 type). An acyl-CoA hydrolase was isolated that showed high similarity to esterase pI 6.1 [74a] [129] [130]. These few examples are further illustrations of the unsatisfying situation of the traditional classification of esterases. 

Mannaerts G.P. et al., (1979). Mitochondrial and peroxisomal fatty acid oxidation in hver homogenates and isolated hepatocytes from control and clofibrate-treated rats. Journal of Biological Chemistry 254 4585-4595. 

Hepatocytes isolated from male Wistar rats (180-250 g) were treated with 0.2 mM mono(2-ethylhexyl) phthalate or 1 mM 2-ethylhexanol for 48 h (Gray et al., 1982). Both di(2-ethylhexyl) phthalate metabolites increased carnitine acetyltransferase activity about nine-fold. In studies with hepatocytes from male Sprague-Dawley rats (180-220 g), treatment with 0.2 mM mono(2-ethylhexyl) phthalate and 1.0 mM 2-ethylhexanol for 48 h resulted in induction of carnitine acetyltransferase activity about 15-fold and six-fold, respectively (Gray et al., 1983). Mono(2-ethylhexyl) phthalate was also shown to induce cyanide-insensitive palmitoyl-CoA oxidation and, by ultra-structural examination, to increase numbers of peroxisomes. Hepatocytes were isolated from Wistar-derived rats (180-220 g) and treated for 72 h with 0-0.5 mM mono(2-ethylhexyl) phthalate and some mono(2-ethylhexyl) phthalate metabolites (Mitchell etal., 1985). Treatment with mono(2-ethylhexyl) phthalate and metabolites VI and IX (see Figure 1) resulted in a concentration-dependent induction of cyanide-insensitive palmitoyl-CoA oxidation. In addition, 0-0.5 mM mono(2-ethylhexyl) phthalate and 0-1.0 mM metabolite VI produced concentration-dependent increases in lauric acid hydroxylation. Treatment with metabolites I and V resulted in only small effects on the enzymatic markers of peroxisome proliferation. In another study with hepatocytes from Wistar-derived rats (180-220 g), metabolite VI was shown by subjective ultrastructural examination to cause proliferation of peroxisomes (Elcombe Mitchell, 1986). 

Primary hepatocyte cultures may also be employed to study species differences in hepatic peroxisome proliferation (lARC, 1995 Doull et al, 1999). Hepatocytes were isolated from male Sprague-Dawley rats (180-220 g), male Syrian hamsters (70-80 g) and male Dunkin-Hartley guinea-pigs (400-450 g). Treatment with 20-200 0,M mono(2-ethylhexyl) phthalate for 70 h caused strong induction of cyanide-insensitive palmitoyl-CoA oxidation activity in rat hepatocytes (up to 600% of control levels), while no marked effect was observed in Syrian hamster (up to 120% of control) or guinea-pig (down to 80% of control) hepatocytes (Lake et al., 1986). 

Species comparisons of hepatic peroxisomal proliferation have also included studies of human and non-human primate primary hepatocyte cultures. Hepatocytes isolated from Wistar-derived rats (180-220 g), male Alderley Park guinea-pigs (400-500 g), male marmosets (350-500 g) and three human liver samples (renal transplant donors) were treated with 0-0.5 mM mono(2-ethylhexyl) phthalate for 72 h (Elcombe Mitchell, 1986). While there was a concentration-dependent induction of cyanide-insensitive palmitoyl-CoA oxidation in rat hepatocytes, no induction was observed in guinea-pig or human hepatocytes and only small non-concentration-dependent effects were observed in marmoset hepatocytes. Metabolite VI induced cyanide-insensitive palmitoyl-CoA oxidation and lauric acid hydroxylation in cultured... 

Peroxisome proliferators have also been shown to induce replicative DNA synthesis in cultured rodent hepatocytes (lARC, 1995). In contrast, several peroxisome proliferators have failed to induce replicative DNA s mthesis in human hepatocyte cultures (Doull et al., 1999). Hepatocytes were isolated from male Wistar-derived rats and from three human liver samples (liver transplantation donors) and treated with 2-ethylhexanoic acid and some other peroxisome proliferators for 72 h (Elcombe et al, 1996). While 2-ethylhexanoic acid induced replicative DNA s5mthesis in cultured rat hepatocytes, no effect was observed in human hepatocytes. Hepatocytes were isolated from male Fischer 344 rats and three humans and treated in culture with 250-2000 pM mono(2-ethylhexyl) phthalate (Hasmall et al, 1999). Increased peroxisomal (O-oxi-dation (at 250-750 pM), replicative DNA s mthesis (at 500-1000 pM), and inhibition of apoptosis (at 250-1000 pM) were observed in rat hepatocytes. None of these parameters was affected by mono(2-ethylhexyl) phthalate in human hepatocytes. 

Zeiger, E. Haworth, S. (1985) Tests with a preincubation modification of the Salmonellal microsome assay. Prog. Mutat. Res., 5, 187-199 Zeiger, E., Haworth, S., Mortelmans, K. Speck, W. (1985) Mutagenicity testing of di(2-ethylhexyl)phthalate and related chemicals in Salmonella. Environ. Mutag., 7, 213-232 Zhu,Y, Qi, Ch., Jain, S., Rao, M.S. Reddy, J.K. (1997) Isolation and characterization of PBP, a protein that interacts with peroxisome proliferator-activated receptor. J. biol. Chem., 272, 25500-25506... 

In hepatocytes isolated from rats and mice, treatment of primary cultures with metabolites of di(2-ethylhexyl) adipate increased peroxisomal palmitoyl-coenzyme A oxidation activity. The same treatment of primary cultures of hepatocytes from guinea-pigs and marmosets failed to cause any similar increase in activity. 

Tugwood, JD., Holden, P.R., James, N.H., Prince, R.A. Roberts, R.A. (1998) Isolation and characterization of a functional peroxisome proliferator-activated receptor-alpha from guinea pig. Toxicol., 72, 169-177... 

The presence of PA in patients with defective peroxisomal metabohsm may be a diagnostic tool in distinguishing the peroxisome biogenesis defects from isolated enzyme... 

Kerckaert I, Poll-The , Espeel M, Duran M, Roeleveld AB, Wanders RJA, Roels F (2000) Hepatic peroxisomes in isolated hyperpipecolic acidaemia evidence supporting its classifications as a single peroxisomal enzyme deficiency. Virchows Arch 436 459-465... 

The most logical material for the analysis of PUFA and plasmalogens are the erythrocytes. Fatty acids can also be quantitated in plasma. The plasmalogens are also easily detectable in homogenates of cultured fibroblasts and may add to the definitive diagnosis of patients with a generalised or isolated peroxisomal dysfunction. 

Patients with an inherited defect of peroxisomal function usually have both an increased C26 0 level and an increased C24 C22 ratio. An isolated moderate increase of C26 0 has only rarely led to a true diagnosis. Nevertheless, every persistent abnormality of VLCFA levels and/or ratios should be checked by studies in fibroblasts in order to arrive at an accurate diagnosis so that genetic counselling of the family can be arranged. Erroneously increased VLCFA levels are rare a ketogenic diet is the only well-known example. 

Density gradients are widely used in separating and purifying biological samples. In addition to this preparative application, measurements of s can be made. Gradient techniques have been used to isolate and purify the sub-cellular components, microsomes, ribosomes, lysosomes, mitochondria, peroxisomes, chloroplasts, and others. After isolation, they have been biochemically characterized as to their protein, lipid, nucleic acid, and enzyme contents. 

Rather, the isolated subcellular fraction is a mitochondrial fraction, which indicates that its major components are mitochondria. Other cellular components that may be present are lysosomes, cell fragments, nuclear fragments, and microbodies (peroxisomes). The purity of the fraction depends on the source of the extract and the method chosen for isolation. 

Main intracellular compartments of an animal cell. Cytosol, endoplasmic reticulum, Golgi complex, nucleus, mitochondria, endosome, lysosome, and peroxisome are distinct compartments that are isolated from the rest of the cell by at least one selective membrane. 

For many years, it was assumed that the inability of patients with X-ALD to degrade VLCFA was due to a primary deficiency of the first enzyme in this pathway, very long chain fatty acyl-CoA (VLCF-CoA) synthase. Indeed, studies in cultured skin fibroblasts from patients with X-ALD have revealed that this enzyme activity is decreased when compared to cells from normal controls. However, when the X-ALD gene was isolated in 1993, it was discovered that the underlying defect is the deficiency of a peroxisomal membrane protein, called ALD... 

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