Stereological analysis of hepatic fine structure in the Fischer 344 rat. Influence of sublobular location and animal age

Stereological analysis of hepatic fine structure in Fischer 344 male rats at 1, 6, 10, 16, 20, 25, and 30 mo of age revealed differences in the amounts and distributions of hepatocellular organelles as a function of sublobular location or animal age. Between 1 and 16 mo of age, both the centrolobular and periportal hepatocytes increased in volume by 65 and 35%, respectively. Subsequently, the cell volumes declined until the hepatocytes of 30-mo-old rats approached the size of those found in the youngest animals. Regardless of animal age, the centrolobular cells were consistently larger than the corresponding periportal hepatocytes. The cytoplasmic and ground substance compartments reflected similar changes in their volumes, although there was no significant alteration in the nuclear volume. The volumes of the mitochondrial and microbody compartments increased and decreased concomitant with the changes in average hepatocyte size. Both lobular zones in the 30-mo-old rats contained significantly smaller relative volumes of mitochondria than similar parenchyma in 16-mo-old animals. The volume density of the dense bodies (lysosomes) increased markedly in both lobular zones between 1 and 30 mo of age, confirming reports of an age-dependent increase in this organelle. The surface area of the endoplasmic reticulum in the centrolobular and periportal hepatocytes reached its maximum level in the 10-mo-old rats and subsequently declined to amounts which approximated those measured in the 1-mo-old animals. This age-related loss of intracellular membrane is attributable to a significant reduction in the surface area of the smooth-surfaced endoplasmic reticulum (SER) in animals beyond 16 mo of age. The amount of rough-surfaced endoplasmic reticulum (RER) in the periportal parenchymal cells was unaffected by aging, but the centrolobular hepatocytes of 30-mo-old animals contained 90% more RER than similar cells in the youngest rats. The centrolobular parenchyma contained more SER and the portal zones more RER throughout the age span studied. These quantitative data suggest that (a) certain hepatic fine structural parameters undergo marked changes as a function of animal age, (b) there exists a gradient in hepatocellular fine structure across the entire liver lobule, and (c) there are remarkable similarities in hepatocyte ultrastructure between very young and senescent animals, including cell size and the amount of SER.     

Variations in the volumes of parenchyma and stroma of the liver and in the cytology of hepatocytes are associated with gonadal stages in female Ohrid trout (Salmo letnica)

Quantitative microscopic studies of season- and breeding-related changes in the liver of farm-raised fish are very scarce, and those of wild fish populations are virtually nonexistent. Moreover, none of the available studies investigated breeding-related changes in hepatic stroma, although it is already known that changes might exist in the parenchyma. To the best of our knowledge, ours is the first study using wild adult Ohrid trout females to evaluate quantitative influences of the breeding cycle on the volumes of the two liver compartments—parenchyma and stroma. Quantitative microscopy (stereology) using light microscopy was supplemented with semi-quantitative and quantitative analyses of hepatocyte cytology to express in numbers the seasonal changes in the extent of vacuolated versus basophilic cytoplasm. The liver volume and that of each compartment changed from that at the time of pre-vitellogenesis to the end of the spawning season. The changes in total volumes of parenchyma and stroma—increasing from pre-vitellogenesis to late vitellogenesis—followed the changes in total liver volume. Despite all the changes in total volumes, no differences existed in the relative proportions between the two major compartments. After spawning, the stromal volume continued to increase while that of the parenchyma reduced despite no detectable statistically significant impacts on the liver volume. Changes in both the liver and parenchyma volumes were correlated with the gonadosomatic index and plasma oestradiol levels. With this study, we first establish that fish liver remodelling might also occur in the stroma. When comparing Ohrid trout with other fish species, we noted interspecies similarities and differences with regard to the hepatocyte cytology during the breeding cycle. In our study, the semi-quantitative and stereological studies revealed that, from pre-vitellogenesis to post-spawning, although cytoplasmic vacuolation of the hepatocytes decreased, the basophilia increased. Overall, these cytological changes were only partially in agreement with the data from other trout studies. We believe that this is because of intrinsic interspecies differences in association with natural conditions. Thus, establishing normal patterns is crucial, at least for flagship species, namely to support interpretation of histopathological changes in biomonitoring.     

Cellular composition of the cyclic corpus luteum of the cow

The cellular composition of CL from 6 cows on approximately Day 12 of the oestrous cycle, after synchronization with cloprostenol, was studied by ultrastructural morphometry. Point-count measurements of volume density (mean +/- s.d.) showed that large luteal cells occupied 40.2 +/- 7.0% of the luteal tissue, and small luteal cells 27.7 +/- 6.3%. Of the total of 393.4 +/- 52.0 x 10(3) cells per mm3 of luteal tissue, large luteal cells made up only 3.5% and small luteal cells 26.7%, a ratio of 1:7.6. Endothelial cells/pericytes, at 52.3%, were the most numerous cell type. The mean volume per large luteal cell was 29.6 +/- 6.3 x 10(3) microns 3, while that of small luteal cells was 2.7 +/- 0.4 x 10(3) microns 3. In spherical form, these volumes would represent mean diameters of 38.4 microns and 17.2 microns respectively, and are consistent with published measurements on dispersed luteal cells. However, the values for cell numbers are much higher than published values based on luteal tissue dispersion, suggesting that dispersion may result in substantial and possibly selective losses of luteal cells.     

A QUANTITATIVE STEREOLOGICAL DESCRIPTION OF THE ULTRASTRUCTURE OF NORMAL RAT LIVER PARENCHYMAL CELLS

The principles of stereology have been applied to a morphometric analysis of parenchymal cells from the peripheral, midzonal, and central regions of normal rat liver lobules. The fractional volumes of cytoplasm occupied by mitochondria, peroxisomes, lysosomes, lipid, and glycogen have been determined. The surface densities of smooth- and rough-surfaced endoplasmic reticulum and of mitochondrial envelope and cristae have also been measured. The average number and dimensions of mitochondria and peroxisomes have been evaluated. By the use of an independent measurement of the average cytoplasmic volume, these data have been expressed as the actual volumes, areas, and numbers per cell in the different parts of the hepatic lobule. Similarly, the volumes of the envelope, cristae, and matrix compartments and the area of cristae membranes have been calculated for the average-sized mitochondrion in each lobular zone. Structural homogeneity is found in over 80% of normal rat liver parenchymal cells, with most of the significant differences being confined to those cells immediately surrounding the central veins.     

Temperature and gender influences on the hepatic stroma (and associated pancreatic acini) of Nile tilapia, Oreochromis niloticus (Teleostei, Cichlidae): a stereological analysis by light microscopy

The normal organ morphology and function in fishes varies according to several natural factors, and such variability is found in liver. Knowledge about the normal liver microanatomy is fundamental to pathological evaluation. Even though gender and temperature are important factors for modulating morphophysiological processes in fishes, their influences on liver stroma are virtually unknown. Because temperature- and gender-related changes exist in liver parenchyma, we predict both factors should also influence the normal stromal structure. Using Nile tilapia as a model, we undertook a study to: 1) establish baseline quantitative structural data on the hepatic stroma (and intimately associated pancreatic acini); 2) compare data with those available from other species, namely, salmonids that do not have a liver with pancreatic acini; and 3) test our hypothesis that, within normal healthy limits, the stroma and its structural components may vary significantly with temperature and gender. We used 1-year-old male and female specimens acclimated to 17 degrees C (breeding noncompatible) and 27 degrees C (breeding compatible) for 45 days. Basic morphometric fish parameters were recorded. After estimation of liver volume, the organ was sliced and pieces systematically sampled for light microscopy. Stereology allowed estimation of the relative volumes of organ components. The total volumes were computed by combining the relative volumes with the total liver volumes. Nile tilapia of both genders, held at 17 vs. 27 degrees C, showed structural quantitative differences in the relative volumes of stroma and most of its components, and in the total volumes of certain stromal elements. The total volume of the stroma and of associated pancreatic acini did not differ. We first established that, in fishes, the total amount (volume) of liver biliary ducts and of eosinophilic granule cells might significantly change (increase and decrease, respectively) with a higher acclimation temperature. Indeed, virtually all the stereological changes were, essentially, temperature- and not gender-related. At 27 degrees C, parallel changes in the parenchyma caused a decreased liver volume and hepatic-somatic index (HSI). The relative volumetric proportion of stroma vs. parenchyma in tilapia is higher than in salmonids. The differences found in this study could not be detected with a qualitative approach, thus stressing the importance of using stereology for analyzing histological patterns and for establishing reliable baseline values in healthy conditions. It was also anticipated that in experimental settings with fish the baseline liver stromal architecture may be different according to temperature and breeding status; in consequence, the effects of the tested variable may also diverge. Our data do not fully explain the lower liver volume and HSI at 27 degrees C, thus justifying studies on the parenchyma, particularly on cell size and number.     

Variability of parenchymal expansion measured by computed tomography

Computed tomography scans of isolated dog lung lobes at different lobe volumes were used to determine the variability of parenchymal tissue density and the variability of parenchymal volume changes on the scale of a voxel, a cube 1.5 mm on a side. The variability of tissue density increased with decreasing lobe volume. The variability of tissue density of neighboring voxels was positively correlated; the spatial correlation decreased exponentially with distance with an exponential scale of 0.3 cm. The ratio of the volume of the parenchyma within a voxel to its volume at total lobe capacity was calculated from the tissue density data at two lobe volumes. At a lobe volume of 40% total lobe capacity, the local fractional volumes were 0.42 +/- 0.12. The variability of ventilation that corresponds to this variability of fractional volume is large enough to explain the inefficiency of mixing in the isolated lobe and the slope of the alveolar plateau of nitrogen concentration in the expirate after a breath of oxygen. These results are consistent with data reported earlier on the variability of parenchymal volumes at a scale of 1-10 cm3.     

Microbiochemical approach to liver cell heterogeneity around terminal hepatic venules

Using lyophilized cryostat sections of liver the activities of alanine aminotransferase, lactate dehydrogenase, and pyruvate kinase were measured using a Lowry technique in the first layer of hepatocytes adjacent to terminal hepatic venules and in the residual parenchymal of the perivenous zone of the acinus in normally fed adult male Wistar rats. Alanine aminotransferase was homogeneously distributed in the two areas measured (ratio hepatocytes adjacent to terminal hepatic venules/residual parenchyma of the perivenous zone: 1.05). Enzyme activities of the lactate dehydrogenase were significantly lower in the hepatocytes adjacent to terminal hepatic venules (ratio: 0.65) and those of the pyruvate kinase significantly higher (ratio: 1.12) than in the residual parenchyma of the perivenous zone indicating liver cell heterogeneity in this zone of the liver acinus.     

Differences in rat kidney morphology between males, females and testosterone-treated females.

Kidneys of normal female and male Wistar-Kyoto rats were studied by standard morphological techniques and morphometry in order to evaluate possible differences in the overall kidney morphology between both sexes. Furthermore, we investigated the role of testosterone (DHT) on kidney morphology by treating females with daily DHT injections. Kidney weight and volume in relation to body weight were not significantly different between males and females and were not affected by DHT. Differences were found in the volume distribution among the kidney zones. The cortex was larger in males than in females, whereas the medulla was conspicuously larger in females than in males. The greater volume of the cortex in males was mainly due to a more extensive development of proximal tubules. DHT treatment in females increased the volume of their proximal tubules. Glomerular volume was similar among the three groups. Within the medulla, the difference was most prominent in the inner stripe (14.9% of the total kidney volume in females vs. 8.9% in males) and was also important in the inner medulla (7.0 vs. 4.8%). The absolute epithelial volume of thick ascending limbs in this zone was larger in females than in males. This difference was more pronounced in short loops (approximately 20%) than in long loops (approximately 10%). The values of the DHT-treated females ranged in between. In spite of the greater development of medulla and thick ascending limbs in females, urine concentration was higher in males than in females and maximum urinary concentrating ability after 48 h dehydration was not different between both sexes.     

Hepatocyte transplantation in a model of toxin-induced liver disease: variable therapeutic effect during replacement of damaged parenchyma by donor cells

To provide long-term therapy in patients with severe toxin-induced hepatic parenchymal damage, donor hepatocytes would need to replicate and replace a large portion of the damaged parenchyma. Using a mouse model developed to reproduce this type of hepatic injury, we found that hepatocyte transplantation only slightly improved survival after transplantation despite the fact that many non-survivors showed moderate liver repopulation by donor cells. Perhaps accounting for this outcome, donor parenchyma in non-survivors did not have typical lobular organization. These results indicate that the re-creation of functional parenchyma by transplanted hepatocytes requires time, during which donor cells proliferate and then establish normal parenchymal architecture.     

ELECTRON-OPAQUE, LIPID-CONTAINING BODIES IN MOUSE LIVER AT EARLY INTERVALS AFTER PARTIAL HEPATECTOMY AND SHAM OPERATION

The appearance and distribution of electron-opaque, lipid-containing bodies have been studied in liver of adult male mice of the C3H strain. The mice were either partially hepatectomized or sham-operated, and the liver was fixed in Veronal acetate-buffered 2 per cent osmium tetroxide at various postoperative intervals (10, 20, 40, 60, and 120 minutes). Normal, non-operated mice served as controls. As early as 10 minutes after both sham operation and partial hepatectomy, lipid-containing bodies have been observed, not only in the cytoplasm of hepatic parenchymal cells, but also in the space of Disse. At the very early postoperative intervals studied, minute lipid bodies are repeatedly found to be more numerous in the space of Disse than at later intervals. It is suggested that the lipid-containing bodies enter the parenchymal cell from the circulation. At the cell membrane, numerous invaginations, each containing a lipid body, have been observed; this suggests that the lipid bodies enter the hepatic parenchymal cells by the process of pinocytosis.The fact that only hepatic parenchymal cells contain the lipid bodies, whereas von Kupffer, endothelial lining, and Ito's fat-storing cells do not, may indicate a specific lipid mobilization response on the part of the cells of the hepatic parenchyma.     

The involvement of parenchymal,Kupffer and endothelial liver cells in the hepatic uptake of intravenously injected liposomes effects of lanthanum and gadolinium salts

¹²⁵I-labeled albumin or poly(vinyl pyrrolidone) encapsulated in intermediate size multilamellar or unilamellar liposomes with 30–40% of cholesterol were injected intravenously into rats. In other experiments liposomes containing phosphatidyl[Me-¹⁴C]choline were injected. 1 h after injection parenchymal or non-parenchymal cells were isolated. Non-parenchymal cells were separated by elutriation centrifugation into a Kupffer cell fraction and an endothelial cell fraction. From the measurements of radioactivities in the various cell fractions it was concluded that the liposomes are almost exclusively taken up by the Kupffer cells. Endothelial cells did not contribute at all and hepatocytes only to a very low extent to total hepatic uptake of the ¹²⁵I-labels. Of the ¹⁴C-label, which orginates from the phosphatidylcholine moiety of the liposomes, much larger proportions were recovered in the hepatocytes. A time-dependence study suggested that besides the involvement of phosphatidylcholine exchange between liposomes and high density lipoprotein, a process of intercellular transfer of lipid label from Kupffer cells to the hepatocytes may be involved in this phenomenon. Lanthanum or gadolinium salts, which effectively block Kupffer cell activity, failed to accomplish an increase in the fraction of liposomal material recovered in the parenchymal cells. This is compatible with the notion that liposomes of the type used in these experiments have no, or at most very limited, access to the liver parenchyma following their intravenous administration to rats.     

Morphology of the green livers in upstream migrants of Petromyzon marinus L.

A morphological comparison was made of the green livers of male and female lampreys (Petromyzon marinus L.) collected during the upstream (prespawning) migration. Light and electron microscope histochemistry for iron, and both thin sections and freeze-fracture replicas in the electron microscope, revealed some sexual dimorphism in these livers. Ferric iron is much more abundant in the liver of females and is present in the cytoplasmic matrix, in dense bodies, and in vacuoles of hepatocytes. The numerous vacuoles of females may be the deposition site of biliverdin and other bile components that would account for the darker green coloration of the liver compared to males. Hepatocytes in females are also characterized by prominent rough endoplasmic reticulum and Golgi apparatus that reflect the involvement of the cells in vitellogenesis. The presence of numerous lipid droplets in the hepatocytes of males indicates that the liver is an important storage site for fat. The lipid droplets are associated with electron-dense deposits of unknown nature. Large gap junctions typify the parenchymal cells of both male and female livers. Perisinusoidal and sinusoidal cells are similar to those in the nonparenchymal region in other vertebrate livers, namely, endothelial and Kupffer cells, lipocytes (Ito), and some granulated cells. The relationship of lipocytes to fibrous tissue and fibrogenesis is discussed.     

Characterization and cell cycle kinetics of hepatocyte populations isolated from adult liver tissue by a nonenzymatic procedure

Suspensions of liver cells enriched in lobular parenchymal hepatocytes were isolated from adult mouse hepatic tissue by nonenzymatic dispersion in chelating buffer and sedimentation of the released cells at unit g. Single cell suspensions so obtained were suitable for flow cytometric measurements of hepatic ploidy class distributions. The more quickly sedimenting cell population consisted of 88% albumin/transferrin-positive epithelial hepatocytes, the nuclei of which were bimodally distributed with respect to RNA content. This dual G1 population was observed in 2C DNA content liver nuclei prepared by several methods and appears to be a general cytochemical characteristic of adult liver parenchymal cells.     

The hepatocytes of the brown trout (Salmo trutta f. fario): a quantitative study using design-based stereology

A stereological study was performed on brown trout hepatocytes aiming to disclose whether there are basic gender differences when minimal levels of sex hormones exist, and also to establish a platform for both interspecific comparisons and physiological correlations. We used the so-called "design-based stereology" (with no shape, size or orientation assumptions) and also some new related statistics. Two-year-old brown trout were collected in April, and the livers were fixed by perfusion. From liver slicing to microscopical field selection, systematic sampling was used. Stereology was applied at light and electron microscopy. Target parameters were the relative and total hepatocyte number, the mean individual hepatocyte volume and surface, and also both relative and total volumes, and surfaces, either of organelles or of cell compartments. Observed variability was usually high, but the precision of estimates was proved to be globally adequate facing the true biological variation amongst specimens. Females had more hepatocytes per liver (1.79x10(9) vs. 1.12x10(9)). Considering the individual hepatocytes, whereas no gender differences were detected in the cell volume, males had higher values of nuclear volume (199 vs. 151 microm3) and surface (170 vs. 131 microm2), endoplasmic reticulum volume (1,300 vs. 824 microm3), and microvilli volume (82 vs. 54 microm3) and surface (1,445 vs. 975 microm2). However, when dealing with quantities per liver, gender differences were found only in the volumes of dense bodies (56 vs. 97 mm3) and of residual cytoplasm (169 vs. 341 mm3)--both volumes were higher in females. Functional implications of data are discussed, namely that females seem to have basic structural traits for coping with the later demands of breeding. Data also support that structural remodelling of hepatocytes occurs after breeding, urging to pursue seasonal studies (namely on lysosomes). We advanced the hypothesis that genders differ in microvilli surface just to maintain an optimal physiological surface-to-volume ratio. Interspecific similarities and differences were disclosed. For example, the number of hepatocytes/cm3 of parenchyma of brown trout was much lower than those reported in rainbow trout, but in both trouts females seem to have an higher cell number. In addition, when comparing the size of hepatocytes of brown trout with that from other fish and mammals it was suggested that major interspecific differences exist.     

Lung growth of the turkey, Meleagris gallopavo: I. Morphologic and morphometric description

To describe lung growth qualitatively and quantitatively from prehatch to adulthood of an unselected line of turkey, a precocial avian species, 36 male turkeys, three in each age group, were killed at 22 and 25 days of incubation, on hatch day, and at 1, 4, 7, 10, 14, 21, 28, 112, and 420 days of age. Body weight and lung volume were measured. A three-level cascade sampling system was used to prepare lung tissue for morphologic and morphometric observation by light microscopy. Point and intersection counting were used to estimate volume and surface densities of lung compartments relative to lung volume. Absolute volumes and surfaces of lung compartments were calculated. Bilogarithmic regressions provided allometric equations to describe growth of the lung in three phases: Tissue proliferation--explosive growth of lung volume relative to body weight and of the gas-exchange compartment within the lung. At 22 days of incubation there were few air and blood capillaries and a great deal of tissue that looked like mesenchyme between the parabronchi. Within the 6 days prior to hatch, the surface area of air capillaries increased 11-fold and of blood capillaries 27-fold, whereas the volume of interparabronchial tissue decreased 58%. Equilibrated growth--from hatch day to 28 days of age, most lung compartments grew evenly with lung volume. Regulated growth--from 28 days of age to adult, all lung compartments, except large vessels and exchange compartment, grew more slowly than the entire lung. Interatrial septa lengthened and their epithelial covering thinned, infundibula became more apparent, and interparabronchial connective tissue reached a minimal volume density in the adult lung.     

Peroxisomes in guinea pig liver: their peculiar morphological features may reflect certain aspects of lipoprotein metabolism in this species

Summary We have studied the ultrastructural characteristics and the distribution of peroxisomes in guinea pig liver using electron-microscopic cytochemistry for catalase and morphometry. By light microscopy, peroxisomes appear as dark 0.2–0.5 m granules in the cytoplasm of liver parenchymal cells, often forming large clusters that measure up to 5 m across. Rows of single peroxisomes or their aggregates line the sinusoidal surface of hepatocytes. Electron microscopy reveals that clusters of up to 25 individual peroxisomes are usually located in the subsinusoidal region of parenchymal cells. The mean diameter and the volume density of peroxisomes are larger in pericentral than in periportal regions of the liver lobule. Whereas large amounts of lipoprotein particles with a mean diameter of 160 nm (chylomicrons) are present in the Disse space, the cytoplasm of parenchymal cells contains multivesicular bodies and abundant lipid droplets. In addition, the Golgi complexes show distended lipoprotein-filled vesicles suggesting active biosynthesis of lipoproteins. We propose that the unique features of peroxisomes in guinea pig liver, such as cluster formation and alignment along the sinusoidal surface, may be related to the high levels of lipoproteins in the portal circulation and their hepatic catabolism in this species.     

Lesions in mink (Mustela vison) infected with giant kidney worm (Dioctophyma renale).

Adult Dioctophyma renale occupied the enlarged renal pelvis of the right kidney of naturally infected mink. Lesions in the kidney parenchyma consisted of connective tissue proliferation in the interstitial tissue, tubular atrophy and fibrosis, and periglomerular fibrosis. The luminal surface of the renal pelvis wall was formed of numerous papillae covered with transitional epithelium. The nematodes in the lumen were bathed in an albuminous fluid containing red blood cells, epithelial cells and D. renale eggs. The left (uninfected) kidney was 60% larger than the left kidney of normal mink.     

New observations on the fine structure of the liver in goldfish (Carassius auratus)

Summary A re-examination of goldfish liver was made through the use of SEM of fractured samples and TEM of ultrathin-sections and freeze-etch replicas. Several new hepatic fine structures described in the present study are morphologically similar to those reported previously in many higher vertebrates including mammals. Hepatic sinusoids of goldfish contain fenestrations which are arranged into sieve plates. Although the hepatic plates are made up of two layers of hepatocytes, the parenchymal cells of goldfish liver are morphologically similar to mammalian hepatocytes, particularly with respect to the sinusoidal surfaces which are studded with numerous microvilli. The intercellular surfaces of hepatocytes have both nexus and desmosomal junctions, similar to those found in various epithelial cells of higher vertebrates, as cell attachments and communication foci. Tight junctions are found mainly between the openings of the intracellular bile canaliculi and the intralobular bile ductules which are situated in the center of the bicellular hepatic plate.Supported in part by Grants # GM92 and ES07017     

Subcellular volumes and metabolite concentrations in barley leaves

Metabolite concentrations in subcellular compartments from mature barley (Hordeum vulgare L. cv. Apex) leaves after 9 h of illumination and 5 h of darkness were determined by nonaqueous fractionation and by the stereological evaluation of cellular and subcellular volumes from light and electron micrographs. Twenty one-day-old primary leaves of barley with a total leaf volume of 902 μL per mg chlorophyll were found to be composed of 27% epidermis, 42% mesophyll cells, 6% veins, 4.5% apoplast and 23% gas space. While in epidermal cells 99% of the volume was occupied by the vacuole, mesophyll cells with an average volume of 31.3 pL consisted of 23 pL (73%) vacuole, 4.6 pL (19%) chloroplasts, 2.06 pL (6,7%) cytosol (including smaller organelles and vesicles), 0.34 pL (1%) mitochondria and 107 fL (0.34%) nucleus. The differences between leaves harvested after 9 h of illumination and after 5 h of darkness were in the size of the stromal compartment and the starch grains therein. Subcellular metabolite concentrations were calculated from the compartmental volumes and metabolite contents of the compartments as determined by nonaqueous fractionation. The amino-acid concentrations in stroma and cytosol were rather similar after 9 h of illumination and 5 h of darkness. In contrast, the vacuolar amino-acid concentrations were about one order of magnitude lower than the stroma and cytosol values, and there was a slight increase in concentration after 5 h of darkness.