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Keywords:

  • hepatic stellate cells;
  • vitamin A-storing cells;
  • polar bear;
  • arctic fox;
  • hepatic lobule;
  • morphometry;
  • heterogeneity

Abstract

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. Acknowledgements
  7. LITERATURE CITED

To investigate the storage mechanisms of vitamin A, we examined the liver of adult polar bears and arctic foxes, which physiologically store a large amount of vitamin A, by high-performance liquid chromatography (HPLC), transmission electron microscopy (TEM) morphometry, gold chloride staining, fluorescence microscopy for the detection of autofluorescence of vitamin A, staining with hematoxylin-eosin (H&E), Masson's trichrome, and Ishii and Ishii's silver impregnation. HPLC revealed that the polar bears and arctic foxes contained 1.8–1.9 × 104 nmol total retinol (retinol plus retinyl esters) per gram liver. In the arctic foxes, the composition of the retinyl esters was found to be 51.1% palmitate, 26.6% oleate, 15.4% stearate, and 7% linoleate. The hepatic stellate cells of the arctic animals were demonstrated by TEM to contain the bulk of the vitamin A-lipid droplets in their cytoplasm. The liver lobules of the arctic animals showed a zonal gradient in the storage of vitamin A. The gradient was expressed as a symmetric crescendo-decrescendo profile starting at the periportal zone, peaking at the middle zone, and sloping down toward the central zone in the liver lobule. The density (i.e., cell number per area) of hepatic stellate cells was essentially the same among the zones. The gradient and the composition of the retinyl esters in storing vitamin A were not changed by differences in the vitamin A amount in the livers. These results indicate that the heterogeneity of vitamin A-storage capacity in hepatic stellate cells of arctic foxes and polar bears is genetically determined. Anat Rec Part A 271A:240–248, 2003. © 2003 Wiley-Liss, Inc.

Vitamin A is essential for the growth, reproduction, and maintenance of differentiated epithelia (Blomhoff, 1994; Sporn et al., 1994; Chambon, 1996). Hepatic stellate cells (vitamin A-storing cells, fat-storing cells, lipocytes, and interstitial cells) are located in the perisinusoidal space of Disse, and extend their thin fibrillar processes into this space (Wake, 1971, 1980; Imai and Senoo, 1998, 2000; Imai et al., 2000a). Under physiological conditions, the hepatic stellate cells can store 80% of the total vitamin A in the whole body as retinyl esters in lipid droplets in the cytoplasm, and play pivotal roles in regulation of vitamin A homeostasis (Senoo and Wake, 1985; Blomhoff et al., 1990; Senoo et al., 1990, 1993a,b, 1997; Blomhoff and Wake, 1991; Imai et al., 2000b).

To elucidate the cellular and molecular mechanisms of vitamin A storage, we studied the liver of arctic animals, such as polar bears, arctic foxes, and glaucous gulls, because it is well known that these animals physiologically store a large amount of vitamin A in their liver (Rodahl and Davies, 1943; Rodahl and Moore, 1943; Rodahl, 1949). In previous studies we reported that the hepatic stellate cells are responsible for the storage of a large amount of vitamin A in these animals (Senoo et al., 1999).

The value of total retinol (retinol plus retinyl esters) in the liver (Nagy et al., 1997), the composition of retinyl esters in storing vitamin A (Kato et al., 1984), and an intralobular gradient of vitamin A storage (Wake and Sato, 1993; Zou et al., 1995, 1998) have been assessed under physiological conditions of vitamin A homeostasis in experimental animals. For example, it was found that vitamin A storage in the stellate cells is well developed in the middle zone, but gradually decreases toward the central region in the porcine liver lobule.

However, these parameters in arctic animals, which physiologically store a large amount of vitamin A in their liver, are still unknown. To elucidate the storage mechanisms of vitamin A, we conducted the present study on polar bears and arctic foxes.

MATERIALS AND METHODS

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. Acknowledgements
  7. LITERATURE CITED

After receiving permission from the District Governor of Svalbard, Norway, to hunt animals, we obtained 11 arctic foxes (Alopex lagopus) near Longyearbyen (78° N, 15° E) in the Svalbard archipelago during the period of August 1996 to September 2001. We then analyzed liver tissue samples from these animals. Three polar bears (Ursus maritimus) were shot in self-defense at Ny Ålesund and Hornsund in Svalbard in February and August 1998. Three male Wistar strain rats weighing 200–230 g were used as controls. The rats had been maintained on a standard cake diet (Clea Japan, Tokyo, Japan). The protocols used in this study for specimen preparation were previously approved by the Animal Research Committee, Akita University School of Medicine; all subsequent specimen preparation adhered to the university's guidelines for animal experimentation.

Analysis of Retinol and Retinyl Esters by High-Performance Liquid Chromatography (HPLC)

The content of vitamin A in the livers was analyzed by HPLC. The amount of retinyl esters (palmitate, oleate, stearate, and linoleate) and retinol in the livers was estimated. The retinyl esters were extracted according to the method of Barua et al. (1993), with some minor modifications. First, 5–10 mg of tissue were homogenized in 90 μl ice-cold phosphate-buffered saline (PBS), and then 400 μl ice-cold 2-propanol/aceton (50:50 v/v) were added. This mixture was vigorously shaken for 5 min and centrifuged for 15 min at 3,500 g, at 10°C. An aliquot of 80 μl was injected on to the HPLC system with a 250 × 4.6 mm C30 column from YMC (YMC, Inc., Milford, MA) and an acetonitril/dichloromethane (70:30 v/v) mobile phase delivered at 2 ml/min (Furr et al., 1986).

For the retinol analyses, 50 mg of tissue were homogenized in 450 μl ice-cold PBS before adding 2,000 μl ice-cold 2-propanol containing TMMP-retinol as an internal standard and BHT (10 mg/L) as an antioxidant. After the mixture was shaken and centrifuged as described above, an aliquot of 1,000 μl was injected onto the HPLC system combining on-line solid-phase extraction and column switching (Gundersen and Blomhoff, 1998). Mobile phases M1 (2.2 ml/min), M2 (1 ml/min), and M3 (0.5 ml/min) from pumps 1, 2, and 3 were 100% water, 100% methanol, and acetonitrile/water (85:15 v/v), respectively. Both retinyl esters and retinol were detected at 325 nm. All analyses were performed in duplicate from each sample.

Light Microscopy

For gold chloride staining, the excised liver of these animals was quickly cut into blocks (30 mm × 30 mm × 20 mm) and subjected to a specific vitamin A-staining technique, the gold chloride-staining method as modified by Wake et al. (1986). To detect the autofluorescence of vitamin A, parts of the excised livers were quickly cut into slices (30 mm × 30 mm × 5 mm) and immersed in 3.7% formaldehyde for 24 hr at 4°C in total darkness, and 20-μm-thick sections were made with a freezing microtome. The sections were examined with a Zeiss Axioskop 20 FL (excitation filter BP365/12, barrier filter BP495/40) for the detection of the rapidly fading green autofluorescence that is characteristic of vitamin A (Popper and Greenberg, 1941). For hematoxylin-eosin (H&E) staining, Masson's trichrome method, and Ishii and Ishii's silver impregnation (Ishii and Ishii, 1965), parts of the excised livers were fixed in 3.7% formaldehyde for 24 hr at room temperature. The specimens were then quickly cut into slices (20 mm × 10 mm × 5 mm), and the slices were stored in the same fixative for 2 days. After dehydration by passage through a graded ethanol series, the pieces of the liver were embedded in paraffin and sectioned at a 5-μm thickness. These sections were stained with H&E in order to observe the structure of the liver lobule, and were also stained by Masson's trichrome method and Ishii and Ishii's silver impregnation method to visualize the connective tissue of the liver. Photomicrographs were taken with an Olympus OHD-2000 microscope (Olympus, Tokyo, Japan).

Electron Microscopy

Part of the excised livers was divided into blocks (30 mm × 30 mm × 20 mm) and perfused (with 1.5% glutaraldehyde in 0.062 M cacodylate buffer, pH 7.4, containing 1% sucrose) for 1 or 2 min by injection through blood vessels whose lumens appeared on the cut surface of the blocks. After perfusion, the blocks (2 mm × 2 mm × 2 mm) were prepared as described previously (Senoo et al., 1999).

Morphometry

To create zonal maps of the liver lobule, we examined semithin sections of the specimens embedded in Epon-812 under a photomicroscope, after staining them with 1% toluindine blue. The lobular mass was divided into five zones (zones I–V) of equal width, extending from the portal area (Glisson's sheath) to the central vein (Fig. 1). To measure the areas of lipid droplets in each stellate cell, and to calculate hepatic stellate cell density (i.e., cell number per area), we scanned printed electron micrographs of the cells with an image scanner (Epson GT-7000S) connected to a personal computer system (Power Macintosh G3400, Apple Computer). Each area was recognized with Adobe Photoshop 5.0J software, measured, and calculated with NIH Image 1.61. The hepatic stellate cells, which were recognized by their cell body (including the nucleus), were counted in the electron micrographs.

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Figure 1. Zonal division of the liver lobule. To make a zonal morphometric analysis, we divided the liver lobule histologically into five zonal areas (zones I–V) of equal widths from the portal vein (pv) to the central vein (cv).

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Statistical analyses were performed with Scheffe's multiple comparison at P < 0.05, using the Statistical Package for the Biosciences (Winesteem Institute of Community Medicine, Toda, Saitama, Japan) run on a computer using Windows© (Microsoft Corp.). Data were expressed as the means ± standard deviation (SD). As a control, we examined rat livers by the same procedures as described for the arctic animals.

RESULTS

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. Acknowledgements
  7. LITERATURE CITED

Hepatic Storage of Total Retinol in Arctic Animals

The HPLC analysis of vitamin A stored in livers (Table 1) showed that the polar bears and arctic foxes contained 1.8–1.9 × 104 nmol total retinol (retinol plus retinyl esters) per gram liver. In arctic fox, the composition of the retinyl esters was found to be 51.1% palmitate, 26.5% oleate, 15.4% stearate, and 7% linoleate (Table 2).

Table 1. Concentration of vitamin A in liver
SpeciesTotal retinol concentration
Concentration (nmole/g wet weight)Median (min-max)
  • The total retinol (retinol plus retinyl esters) was measured as described under “Materials and Methods.” The results presented are mean in rats and median in polar bears and arctic foxes.

  • a

    This result has been reported by Nagy et al. (1997).

Polar bear (n = 3)18297(16566–23689)
Arctic fox (n = 8)18641(14507–24394)
Rat (n = 3)a1106 
Table 2. Retinyl esters in liver of arctic fox and rat
SpeciesRetinyl ester (% of total retinyl esters)
PalmitateOleateStearateLinolate
  • The composition of retinyl esters was identified and quantitated as described under “Materials and Methods.” Percentages in this table may not add up to exactly 100% because of rounding.

  • a

    The result has been reported by Blomhoff et al. (1985).

Arctic fox51.126.615.47
Rata87.83.19.2 

Electron Microscopy

In polar bears, most stellate cells contained only a single, large lipid droplet that compressed the nucleus (Fig. 2a–c). In the arctic foxes, a number of medium-sized lipid droplets occupied almost all of the cytoplasm of the hepatic stellate cells (Fig. 2d–f). The stellate cells of the normal rat liver contained few lipid droplets (Fig. 2g–i). The area occupied by lipid droplets in the hepatic stellate cells was significantly larger in the liver of polar bears and arctic foxes than in the rat liver. The hepatic stellate cells of zone III (Fig. 2b, e, and h) had a larger area of lipid droplets than the cells in zones I and V (Fig. 2a, c, d, f, g, and i). The average diameter of the lipid droplets was 15 μm in polar bears, 5 μm in arctic foxes, and 1 μm in rats.

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Figure 2. Transmission electron micrographs of liver sections. Electron micrographs of the liver of polar bears (a–c), arctic foxes (d–f), and rats (g–i) were taken in (a, d, and g) zone I, (b, e, and h) zone III, and (c, f, and i) zone V, as described in the Materials and Methods section. The hepatic stellate cells (SC) in zone III (b, e, and h) contain more vitamin A-storing lipid droplets (L) than those in zones I and V (a, c, d, f, g, and i). cv, central vein; K, Kupffer cell; PC, parenchymal cell; E, endothelial cell; s, sinusoid. Scale bar = 5 μm.

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Morphometry

In the polar bears, there was a definite zonal gradient in the five zones (Fig. 3). The mean area of lipid droplets was smallest in zone V (0.021 ± 0.022 μm2) and largest in zone III (0.058 ± 0.028 μm2). The mean area of lipid droplets in zone III was approximately three times as large as the areas in zones I and V. In addition, it was significantly larger than that in zone V. The lipid area increased from zone I to zone III, and then decreased gradually from zone III to zone V.

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Figure 3. Intralobular zonal gradient of vitamin A storage in hepatic stellate cells. The zonal gradient of vitamin A storage is expressed as an asymmetric crescendo-decrescendo profile with a peak at zone III and a downward slope toward zone V. Characteristically, the area of lipid droplets in zone V is slightly smaller than that in zone I. Graphs were plotted with the mean values, depicting three animal species: polar bear (•), arctic fox (▴), and rat (⧫).

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In arctic foxes, the area of these lipid droplets differed among the five zones along the sinusoidal axes (Fig. 3). The mean area of lipid droplets was smallest in zone V (0.007 ± 0.001 μm2) and largest in zone III (0.019 ± 0.005 μm2). The mean area of lipid droplets in zone III was approximately three times as large as that in zone I or V. In addition, it was significantly larger than that in zone V. The lipid area increased from zone I to zone III, and then decreased gradually from zone III to zone V.

The same tendency toward intralobular zonation of lipid droplets was also found in the rats (Fig. 3). The mean area of lipid droplets was smallest in zone V (0.00038 ± 0.00033 μm2) and largest in zone III (0.0013 ± 0.00063 μm2). The mean area of lipid droplets in zone III was approximately three times as large as that in zone I or V. The lipid area increased from zone I to zone III, and then decreased gradually from zone III to zone V.

The liver lobules in all of the animals examined in this study thus showed a zonal gradient in vitamin A storage. The zonal gradient of vitamin A storage capacity was expressed as a symmetric crescendo-decrescendo profile starting at zone I, peaking at zone III, and sloping down toward zone V. Characteristically, the area of lipid droplets in zone V was slightly smaller than that in zone I. The area of these lipid droplets in zone III of polar bears was the largest in all zones evaluated. It was about three times as large as that of zone III in arctic foxes, and about 43 times as large as that in the corresponding zone in rats. We also compared the cell density of stellate cells in each zone, but no significant differences were found (Table 3).

Table 3. Comparison of stellate cell density in zones
SpeciesCell density (cell/10,000 μm2)
Zone IZone IIZone IIIZone IVZone V
  1. Cell density (cell/10,000 μm2) was determined as described under “Materials and Methods.” No significant difference was found among zones. Data are expressed as mean ± SD.

Polar bear1.1 ± 0.71.5 ± 0.91.5 ± 1.01.2 ± 0.81.2 ± 0.4
Arctic fox2.2 ± 1.11.9 ± 1.11.9 ± 0.31.5 ± 0.92.7 ± 0.2
Rat0.9 ± 0.31.1 ± 0.50.9 ± 0.31.1 ± 0.11.2 ± 0.1

Light Microscopy

Gold chloride staining specifically demonstrated black-stained hepatic stellate cells in polar bears (Fig. 4a and b), arctic foxes (Fig. 4c and d), and rats (Fig. 4e and f). Most stellate cells of polar bears contained only a single lipid droplet (Fig. 4b), but most stellate cells of arctic foxes and rats contained a number of the droplets (Fig. 4d and f). The zonal difference in gold precipitation in the hepatic lobules in control rats (Fig. 4e) was essentially the same as that in normal rats as reported by Wake (1971). Thus, the distribution of stellate cells stained by the gold chloride method was consistent with the electron microscopic morphometry.

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Figure 4. Gold chloride staining specifically demonstrating black-stained hepatic stellate cells of (a and b) polar bears, (c and d) arctic foxes, and (e and f) rats. Gold chloride staining was performed as described in the Materials and Methods section. The zonal difference in gold precipitation in the hepatic lobule of the control rats was essentially the same as that previously reported for normal rats (Wake, 1971). Scale bars = 100 μm.

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Fluorescence micrographs showed vitamin A autofluorescence in the hepatic stellate cells of polar bears (Fig. 5a), arctic foxes (Fig. 5b), and rats (Fig. 5c and d). The fluorescing objects in the hepatic stellate cells of the arctic foxes and rats often appeared like stars in the night sky (Fig. 5). The intralobular distribution of stored vitamin A in the arctic foxes and rats (Fig. 5b and c) was similar to that previously described for normal rats (Wake, 1971). Thus, the distribution of fluorescing cells was not inconsistent with the stellate cells observed by electron microscopic morphometry.

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Figure 5. Fluorescence micrographs demonstrating vitamin A autofluorescence in hepatic stellate cells of (a) polar bears, (b) arctic foxes, and (c and d) rats. Fluorescence micrographs were taken as described in the Materials and Methods section. No specific autofluorescence was seen in any of the other cell types in the liver (e.g., parenchymal cells, endothelial cells, and Kupffer cells). Scale bars = 100 μm.

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To examine the arctic animals pathologically, we analyzed the liver with H&E staining, Masson's trichrome method, and Ishii and Ishii's silver impregnation method (Fig. 6). The liver lobules were approximately 1.0 mm in diameter in all of the animals (Fig. 6a, d, and g). The liver lobules had a greater abundance of connective tissue in the polar bears (Fig. 6b and c) than in the arctic foxes (Fig. 6e and f). No pathological signs (such as liver cirrhosis or hepatic fibrosis) were observed in these animals.

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Figure 6. Light photomicrographs of the liver. The livers of (a–c) polar bears, (d–f) arctic foxes, and (g–i) rats were stained (a, d, and g) with H&E, (b, e, and h) by Masson's trichrome method, and (c, f, and i) with Ishii and Ishii's silver impregnation, as described in the Materials and Methods section. Arrows indicate collagen fibers. cv, central vein; pv, portal vein. Scale bars = 250 μm.

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DISCUSSION

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. Acknowledgements
  7. LITERATURE CITED

This study revealed three major findings. First, the arctic animals demonstrated intralobular heterogeneity of vitamin A-storage capacity in the hepatic stellate cells. Second, the large amount of vitamin A stored in the arctic-animal livers, as determined by morphometry, agreed with the amount of retinol measured by HPLC. Third, the composition of retinyl esters in stored vitamin A in the liver of arctic foxes was quite similar to that previously reported for rats (Blomhoff et al., 1985).

In earlier studies, a specific zonality for intralobular vitamin A storage was found; i.e., the zone of highest storage was not located outermost but slightly inward, and the zone of lowest storage was located centrilobularly (Wake and Sato, 1993; Zou et al., 1995, 1998). Our results agree with those from other studies as regards the heterogeneity of the vitamin A-storing lipid droplets in the hepatic stellate cells of the liver lobule. However, the location of the highest-storage zone as determined in the present work differs from that found in previous studies. This discrepancy may be explained by the observation methods (Wake and Sato, 1993; Zou et al., 1995, 1998) used in the earlier studies. Whereas those authors used only the light microscopy method, in the present study we examined all tissues by electron microscopy. Moreover, we certified that the pattern of vitamin A storage observed in liver lobules by light microscopy strongly supported that of intralobular heterogeneity as revealed by electron microscopical morphometry.

The intralobular heterogeneity of vitamin A storage may be related to the number of hepatic stellate cells within each zone, or to the maturation of the hepatic stellate cells themselves (Wake et al., 1991). At the molecular level, this heterogeneity may involve various regulatory factors, e.g., the retinol metabolism rate of hepatocytes (Shintaku et al., 1997), and the mechanism for mobilization of retinol from stellate cells to the plasma retinol pool (Blomhoff et al., 1990). However, regarding the quantification of cell density in each zone, the average density calculated in all the animal species examined in our study did not differ among the zones. In previous works we reported that the morphology, proliferation, and function of hepatic stellate cells can be regulated by the three-dimensional structure of the extracellular matrix (ECM) (Kojima et al., 1998; Li et al., 1999; Miura et al., 1999; Sato et al., 1999). The hepatic stellate cells displayed rounded shapes when they were cultured on Matrigel containing basement membrane components, but they exhibited elongated cellular processes when cultured on interstitial type I collagen gel. The heterogeneity of ECM in the liver lobule (Reid et al., 1992; Rojkind and Greenwel, 1994), and the modulation of the cellular retinol-binding protein (CRBP) level by the extracellular collagen matrix in hepatic stellate cells (Davis et al., 1987) were previously described. CRBP plays an important role in retinol metabolism, and has also been reported to be indispensable for efficient retinyl ester synthesis and storage (Ghyselinck et al., 1999). Furthermore, Kato et al. (1984) found intralobular heterogeneity of CRBP in the liver of rats. Therefore, we speculate that the intralobular distribution of CRBP in the liver of polar bears and arctic foxes may also show similar heterogeneity to that in rats, and have effects on the intralobular heterogeneity of vitamin A storage in the liver. As to the agreement between the vitamin A measurements revealed by morphometry and HPLC in the present work, we consider that the study by Nagy et al. (1997) in rats fully supports our findings. Regarding the composition of retinyl esters, the largest amount of retinyl ester found in the arctic foxes in our study agrees with that previously reported in rats (Blomhoff et al., 1985). Therefore, our interpretation is that the capacity for vitamin A storage does not correlate with the composition of vitamin A.

In the light of accruing evidence, including the present results, we conclude that the heterogeneity of vitamin A storage capacity in the hepatic stellate cells of arctic foxes and polar bears is genetically determined. The precise mechanisms by which the hepatic stellate cells have different capacities for vitamin A storage in the liver lobule, as well as the effect of heterogeneity on vitamin A homeostasis, are still unknown and are under investigation in this laboratory.

Acknowledgements

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. Acknowledgements
  7. LITERATURE CITED

The authors thank Heidi L. Wold; Drs. Rune Blomhoff, Jan Øivind Moskaug, and Kaare R. Norum (Institute for Nutrition Research, Faculty of Medicine, University of Oslo); Norbert Roos (Electronmicroscopical Unit for Biological Science, Faculty of Mathematics and Natural Sciences, University of Oslo); Trond Berg (Division of Molecular Cell Biology, Institute of Biology, University of Oslo); Kenjiro Wake (Liver Research Unit, Minophagen Pharmaceutical Co, Ltd.); and Mitsuru Sato, Katsuyuki Imai, and Takeya Sato (Department of Anatomy, Akita University School of Medicine) for valuable discussions. The expert technical support provided by Mitsutaka Miura and Naosuke Kojima was also greatly appreciated. The authors are grateful to Jørn Eldar Fortun, Anders Friberg, and Trond Østaas for their excellent hunting skills. We also thank the Norwegian Polar Institute, the District Governor at Svalbard, and the University Courses at Svalbard for generously allowing us to use their laboratory facilities in Longyearbyen.

LITERATURE CITED

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. Acknowledgements
  7. LITERATURE CITED
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