Three-dimensional organization of the hepatic microcirculation in the rodent as observed by scanning electron microscopy of corrosion casts

GASTROENTEROLOGY

LIVER

PHYSIOLOGY

AND

79:72-81, 1980

DISEASE

Three-Dimensional Organization of the Hepatk Microcirculation in the Rodent as Observed by Scanning Electron Microscopy of Corrosion Casts RANDY

H. KARDON

Departments

of Pharmacology

and RICHARD

G. KESSEL

and Zoology, University

The hepatic microcirculation was studied in three dimensions by scanning electron microscopy of corrosion casts. A methyl methacrylate casting medium was used which was formulated into a low viscosity mixture so that arterial, venous, and capillary divisions were filled and, hence, casted. Under injection conditions of constant flow and physiologic pressure, the casting medium appeared to distribute according to the intrahepatic vascular resistances. The vascular connections within the hepatic microcirculation and their extent could be easily assessed using this technique. The sinusoids were observed to be organized into dense, anastomosing capillary masses which were supplied by short inlet venules and terminal divisions of the distributing portal vein as well as by arteriolar capillaries. In some c&es, the arteriolar capillaries connected with sinusoids at a point midway between the central and portal veins. The density of capillaries that was casted immediately adjacent to the portal areas varied extensively. Portal areas containing a paucity of capillaries appeared to define perivenular capillary thickets, the vessels of which radiated toward a centrally located collecting venule. The thickets followed the distribution of the hepatic venous divisions. These capillary masses produced patterns corresponding to the organization of the hepatic parenchyma. Increased permeability of the sinusoids in some areas Received July 3, 1979. Accepted January 27, 1980. Address requests for reprints to: Randy H. Kardon, M.D., Department of Pharmacology, University of Iowa College of Medicine, Iowa City, Iowa 52242. Support for use of the scanning electron microscope was provided by an allocation from the Graduate College, University of Iowa. Randy Kardon was supported by the Insurance Medical Scientist Scholarship Fund (The Prudential Co., sponsor) during the period of this investigation. The authors would like to thank Cynthia Stiffin for printing and labeling the photomicrographs. 0 1980 by the American Gastroenterological Association 0018-5085/80/070072-10W2.25

of Iowa, Iowa City, Iowa

was evidenced by leakage of casting medium from sinusoids into the spaces of Disse and Mall. Scanning electron microscopy of corrosion casts are useful in evaluating the three-dimensional organization of the normal hepatic microcirculation and could be applied to the study of perturbations that occur in the vasculature in pathologic conditions. The morphology and distribution of the liver microvasculature have been studied over a period of many years by the injection of dyes and po1ymers.‘-4 AIthough much useful information has been revealed with respect to vascular connections, such studies were limited by the resolution, depth of focus, and plane of section of the light microscope. More recently, the technique of microvascular corrosion casting has been coupled with scanning electron microscopy to permit the three-dimensional distribution of the microcirculation and the interconnections between small vascular subdivisions to be revealed in a variety of organs.5-8 The technique has recently been applied to the investigation of the liver vasculature; specifically, the distribution of vessels supplying and draining the peribiliary plexus has been investigated in the monkeys and rat, and the terminal distribution of hepatic arterioles has also received attention.‘O However, the distribution of the sinusoidal capillaries within the boundaries of the defined classic liver lobule and within the liver acinus have not been investigated with this technique. This may be due to the inherent difficulty of exposing the interior of large, complex, and brittle microvascular casts. The goal of this study was to expose the internal organization of the liver microsvasculature so that sinusoidal patterns of organization could be assessed. In addition, the nature of connections between branches of the portal vein and sinusoids as well as between the hepatic arterial terminations

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and sinusoids were studied. The extent of sinusoidal anastomoses was also investigated.

Materials and Methods Vascular Perfusion Male and female albino rats weighing between 200 and 306 g were anesthetized with ether. The vascular system was first perfused free of blood with Tyrode-Ringer solution containing 1000U of heparin. The Tyrode-Ringer solution was perfused through the heart after the inferior vena cava was severed for a drainage route. The solution was introduced by way of a constant pressure infusion apparatus in which the infusion pressure was maintained at 100 mmHg. Pre-perfusion was terminated when complete branching of the kidneys, liver, and intestine had occurred. During the pre-perfusion, Batson’s No. 17 anatomic corrosion compound (Polysciences, Inc., Warrington, Pa.) was formulated for use as the casting medium. The proportion of constituents was modified so that the resulting mixture was of low viscosity (0.1 poise as measured by a capillary tube kinematic viscosimeter). The modified mixture consisted of 40.0 ml of monomer base, 10.0 ml of catalyst, and 6 drops of promoter. The promoter was not added until the mixture was to be introduced. Initially, the identification of hepatic arterial and portal venous divisions was accomplished by arterial injection of casting medium containing red dye, and concomitant injection of casting medium containing blue dye into the portal vein. After maceration, specific vascular divisions were identified with the dissecting microscope on the basis of color. These same divisions were then viewed with the scanning electron microscope and could be traced along their course. The terminal portal venules were identified by tracing their continuity with larger portal divisions in the portal tract. The portal venules also possessed a much larger and more irregular luminal diameter compared with the arteriolar divisions. In subsequent experiments, casting medium was infused only through the aorta, which served to replicate the hepatic microcirculation by way of both hepatic arterial and portal venous routes. The descending aorta was cannulated with a short segment of PE160 polyethylene tubing at a point just proximal to its bifurcation into the iliac arteries. The tubing was connected to a larger diameter tubing which was connected to a disposable 20-ml syringe by way of a three-way stop-cock. At the start of the retrograde infusion of the casting medium into the aorta, the aorta was ligated above the celiac branch, serving to restrict the casting medium to the abdominal organs. In more recent infusions, the casting medium was introduced

intraarterially by way of a cannula introduced retrograde into one iliac artery. The other iliac artery was cannulated with polyethylene tubing connected to a pressure transducer and recorder. Using a Harvard constant-flow syringe pump, the rate of infusion was adjusted to produce an intraarterial pressure of 100 mmHg. In experiments in which the volume of casting medium was limited, the infusion was terminated after a venous effluent appeared from the liver, but before the capsular circulation of the liver was observed to fill with polymer.

Processing

of Casts

Polymerization is complete in approximately 15 min at room temperature, as evidenced by hardening of the uninfused mixture. During this period the liver was bathed in Ringer’s solution while still in the abdominal cavity to prevent displacement or distortion of the casting medium. After polymerization, some of the livers were cut was a sharp razor ilade before maceration. The organs were then excised and placed in tap water. After 3-4 hr, the tissue was macerated in a solution of potassium hydroxide (340 g/liter) at 60°C. The solution was changed at least once a day with a distilled water rince between changes. A total maceration time of l-2 days was usually required. When no tissue debris was present in the solution, the maceration was complete. The casts were then dehydrated in ethanol, critical point dried, and mounted by affixing them to alluminum specimen holders and adhesive cooper tape. Specimens were sputter coated with gold (approximately 300 A thick) and subsequently viewed in a JEOL 35C scanning electron microscope at an accelerating voltage of 16 kv.

Results General During the infusion of the casting medium, the areas of the microvasculature with the lowest resistance were the first to be filled. An hepatic venous effluent of casting medium appeared long before the more peripheral capsular surface of the liver was filled. If the casting medium was infused long enough, all of the potential vascular space could be filled as was apparent even before tissue digestion by the color imparted to the capsular surface of the lobes by the dye in the injected casting medium. After tissue digestion, casted areas at the surface appeared very flat (Figure 1). In scanning electron micrographs of the capsular surface, the radiating pattern of sinusoids was apparent, but other vascu-

Figure 1. Survey of casted hepatic microcirculation in both capsular (CAPS) and subcapsular regions. The capsular surface is shown in upper right, of figure and the adjacent regions of the cast are from areas underlying this plane. Zones one (l), two (2), and three (3) of the liver acinus are denoted at the bottom of the figure. Adjacent areas also contain: sinusoids (Si), the arrangement of which corresponds to a lobular (Lob) parenchymal organization, conducting portal vein (CPV), distributing portal vein (DPV), collecting venule (CV), hepatic arteriole (HA), and noncasted areas in portal tracts (*) (X 65).

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Figures 2-3. Figure 2 is an enlargement of the lower portion of Figure 1, while Figure 3 represents an enlargement of the area enclosed by the rectangle in Figure 2. The approximate boundary of zone 1 of the liver acinus is denoted by broken lines in Figure 3. Conducting portal vein (CPV), terminal divisions of the distributing portal vein (DPV), hepatic arteriole (HA), collecting venule (CV), blind-ends of portal vein (*), and anastomoses of sinusoids (arrows). Note that the hepatic arteriole in Figure 2 extends into acinar zones 2 and 3 (Figure 2, x 75; Figure 3 x ZOO).

lar divisions such as central collecting veins, branches of the portal vein and hepatic arterioles were obscured from view. From the cut, planar surfaces of these casts, the vessels in the inferior of the lobe could be distinguished, but a three-dimensional picture of the vasculature was not afforded (Figure 9) making it difficult to study the distribution of the vascular divisions. In preparations where the infusion of casting medium was terminated before the capsular surface was completely filled, more of the three-dimensionality of the microvasculature was revealed. This was true because vascular structures underlying the capsular surface were not obscured from view (Figure 1). Using this method, preferential areas of the microcirculation were casted, and adjacent areas often exhibited a paucity of casted capillaries which contained narrow, blind-ended divisions of the portal vein and hepatic artery. Areas of the cast exhibiting a paucity of casted capillaries were usually directly adjacent to the vessels traversing the portal tracts (Figures 2,4, and 5). These areas surrounded, and visually subdivided, thickets of casted capillaries (Fig-

ure 1) which radiated toward central collecting venules. The capillary thickets correspond to the lobular arrangement of the hepatic parenchyma about the collecting venule, and this lobular vascular appearance is most likely due to the heterogeneous filling of sinusoids surrounding the portal tract. The perivenular capillary thickets followed the distribution of the collecting venules and divisions of the hepatic vein. A limited number of sinusoids at the periphery of such capillary thickets were usually supplied by short inlet venules originating from the distributing portal veins. The paucity of casted capillaries surrounding the portal areas allowed the afferent vascular divisions to be unobscured from view so that they could be studied along their length for patterns of branching and distribution, vessel diameter, and connections with sinusoids. Since the hepatic artery was injected with casting medium containing a red dye, and the portal vein was injected with medium containing a blue dye, the identities of casted divisions of the two afferent blood supplies were easily made under the dissecting microscope. Identical vessels were also viewed by scanning electron micros-

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copy. Thus, the identity of the venous and arterial divisions viewed by scanning electron microscopy could be substantiated by this correlative method. In portal areas where sinusoids were casted, they were primarily supplied by terminal arborizations of the distributing portal veins, and these sinusoids were part of a dense interconnecting capillary network which eventually drained toward the central venules. In these areas one could arbitrarily define the three zones (Figures 1 and 3) of the liver acinus as defined by Rappaport and Schneiderman.” Hepatic Arterioles After the initial correlative studies with dyes, the arterial divisions were easily identified in subsequent specimens on the basis of diameter, nature of branching, continuity with larger arteries in the portal tract, and characteristic oval surface depressions which presumably are made by endothelial cells projecting into the arterial 1umen.6.‘2.‘3The arterioles often accompanied the conducting and distributing branches of the portal vein within the portal tract (Figures 1-5).By comparison, the diameter of the arteriolar divisions was much less than those of the portal vein, and often appeared as thin strands. After passing outside the portal tract, the arteriolar capillaries were easily distinguished from adjacent sinusoids (Figures 2 and 5), because they exhibited no irregular luminal diameter or extensive branching and anastomoses, as was the case for sinusoids.

Figures 4-5.

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The terminal divisions of the hepatic arterioles were observed to consist of two main types. In the first type, which was least frequently encountered, a short branch from a long slender arteriole accompanying divisions of the portal vein formed a longitudinal plexus of capillaries in close association with the portal venous division (Figure 6). The capillary plexus represents the peribiliary portal system, previously demonstrated using a similar technique.“” In the second type, the terminal arteriolar divisions often diverged from the venous divisions associated with the portal tract and arborized to supply sinusoids (Figure 7). The terminal arteriolar divisions were continuous with sinusoids adjacent to the portal areas at sites (Figure 5) corresponding to zone 1 of the liver acinus. In a few cases (Figure 2, lower left) arteriolar capillaries penetrated deeper into the acinus, without branching, to a location approximately equidistant from the central venule and portal tract. In areas where casted sinusoids were particularly sparse (Figure ll), the distribution of the central venules and their relationship to the portal vessels could be more easily discerned. In such cases, the course of the arterioles and their proximity to the central venules could be assessed, as exemplified by the small arrows in Figure 11,where the distal termination of some arterioles appears to occur in close proximity to the central venules, corresponding to acinar zones 2 and 3. These arterioles possessed blind ends that almost touched the collecting ven-

Shown are examples of sinusoidal arrangements which distribute according to the parenchymal organization of the liver lobule. Conducting portal vein (CPV), distributing portal vein (DPV), hepatic arteriole (HA), collecting venule (0, inlet venules (IV), and anastomoses of sinusoids (arrows) (Figure 4, X 110; Figure 5, x 95).

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*e 6. Peribiliary capillary next to distributing vein (DPV) (X 330).

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plexus portal

re 7. Arborization of hepat ic arterioles (HA) supplying ! sinusoids (Si) (X 220).

ules, however, no direct connection served (Figure 13).

has yet been ob-

Divisions of the Portal Vein The conducting divisions of the portal vein could be followed as they branched to form the distributing divisions (Figure 1).It was apparent that dense networks of anastomosing sinusoids were supplied at specific sites, located at various distances from the portal tract, by different distributing portal veins. Short inlet venules supplying adjacent sinusoids were also observed to arise from the distributing portal venules along their length (Figures 4 and 8). Inlet venules were sometimes observed to narrow at their point of origin from distributing branches of the portal vein (Figure 8), and such regions may correspond to locations where sphincters have been previously reported to exist.14 At its distal end, the distributing portal vein repeatedly branched into smaller terminal divisions that became continuous with sinusoids (Figure 3). In limited cases, small divisions ended without making contact with sinusoids (Figures 1,2, and 5).

Collecting Veins In cases where the injected volume of casting medium was limited, large areas of the hepatic microcirculation were untasted, revealing an existing pattern of casted sinusoids which could then be followed along the distribution of the collecting and sublobular divisions of the venous system (Figures 11-13). In some areas of these specimens, the central venules appeared to end blindly and sinusoids originating from such divisions appeared to “back-fill” and also end blindly. The casted sinusoids formed right angles where they became continuous with the collecting venule.

Sinusoids The low viscosity of the casting medium allowed large areas of the vast network of sinusoids to be filled. The high resolution and depth of field of the scanning electron microscope revealed the extensive anastomoses within the capillary network and the enormous density of the hepatic capillary system. The organizational picture emphasized that

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only specific sites of the dense capillary mass were directly continuous with an afferent blood supply. Specimens cut with a razor blade before tissue digestion revealed an unusual pattern of density on the cut surface of the cast after prolonged tissue digestion (Figure 9). At higher magnification, the areas of the increased density of casting medium appeared as thin strands between the sinusoids (Figure lo), and may represent areas where the casting medium passed into the space of Disse through areas of increased permeability in the porous sinusoidal lining. Another feature of the cut surface was the presence of a “shell” of casting medium which ‘surrounded divisions of the portal vein and hepatic artery in the portal tract (Figure 9). These areas may be continuous with the casting medium that passed into the space of Disse and likely represent the space of Mall which had filled with casting medium.

Discussion

Figure 8. Distributing portal vein (DPV), inlet venules (IV), areas of constriction (arrows), rounded, blind ends of sinusoids (*) (X 330).

The organization of the hepatic microcirculation and the nature of its afferent blood supply has been revealed by scanning electron microscopy using the corrosion casting technique. By partially injecting the liver with casting medium under conditions of constant flow and at physiologic pressure, the low resistance vascular pathways and those closest to the liver hilus were first to be filled. The lack of casted vessels in adjacent areas (presum-

Figures 9-10. Cut surface of cast interior. Figure 10 is an enlargement of the area enclosed by the rectangle in Figure 9. Divisions of the hepatic artery (AR) and portal vein (PV), areas of casted density (‘) between sinusoids (Si), shell of casting medium (arrows) around portal tract is delineated. Figure 9, X 65; Figure 10, X 365.

Figures 11-13. Brushlike arrangement of sinusoids (Si) about collecting (CV) and sublobular venous (SV) divisions resulting from partial casting of the hepatic microcirculation. Areas where hepatic arterioles (HA) terminate in close proximity to collecting venules are denoted by thin arrows in Figures 11 and 13. Figure 12 is an enlargement of the region enclosed by the rectangle in Figure 11.Note areas where sinusoids end blindly (thick arrows). Figure 13 is an enlargement of an area directly below the rectangle in Figure 11, and illustrates the distribution of hepatic arteriolar divisions and their relationship to surrounding collecting venules (Figure 11, X 40; Figure 12, X 160; Figure 13, x 100).

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ably areas of higher resistance) permits those areas that are casted to be more clearly studied with regard to their three-dimensional organization and vascular connections. This situation is analogous to the difficulty in discerning the complex branching of a leaf-ladened tree in the summer compared with the ease of this task in the winter. The extensive anastomoses of sinusoids that was observed could permit blood that is supplied directly to some sinusoids by arterioles or portal venules to reach adjacent, connecting sinusoids. This condition would obviate the necessity for a direct afferent blood supply to each sinusoid. The anastomoses, therefore, probably play an important functional role in the regional distribution of blood borne substances through the liver acinus. In addition, the branches of the hepatic arterioles were observed to terminate at sinusoids located adjacent to the portal areas and at sinusoids close to the collecting venule as has been previously reported.’ This result would indicate that the point of mixing of portal, venous, and arterial blood may occur at a variety of locations within the hepatic microcirculation. The different locations where arterioles supply sinusoids could also produce pressure gradients influencing the direction and magnitude of regional blood flow. The observation that the terminal ends of arteriolar divisions in some cases distributed very close to collecting venules was unexpected. It has been widely accepted that hepatic arterioles do not penetrate further than zone 1 of the hepatic acinus. Functional studies have established the existence of a PO, gradient from acinar zone 1 to zone 3 by determinations of oxidation reduction potentials in hepatic tissue.15 It is worthwhile to point out that some measurements are made at the capsular surface of the liver and not deep in the liver lobe where we have observed hepatic arterioles to become closely associated with collecting venules. Measurement of oxidation-reduction potentials may not necessarily correlate with anatomic distribution of some branches of the arterial tree. Plasma skimming could occur in many hepatic arteriolar divisions due to the angle of branching and the small diameter (5 pm) that the terminal arteriolar capillaries can attain. The existence of plasma skimming would not allow a direct correlation to be made between PO, and arteriolar distribution. Because the terminal ends of the deeply penetrating arterioles were usually blind ended, we have not yet been able to establish their points of connection to other vessels. The blind ends may represent areas of high resistance, and further studies to clarify the significance of this finding may be warranted. To account for the presence of blind-ended sinusoids observed in the casts, the possibility should be

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considered that such vessels might have originated embryologically as an outgrowth from divisions of the collecting and sublobular hepatic veins, whereas, those sinusoids directly connecting with an afferent blood supply might have their embryologic origin from portal divisions. The formation of sinusoidal anastomoses would then establish an interconnected system. Some of the ends of the vessels may not connect, leaving them blind-ended. The application of the casting technique to vascular morphogenesis in the liver during embryologic development or regeneration might provide evidence for this possibility. An alternative explanation is that casts of blind-ended vessels may result from areas of high vascular resistance or from vessels with an occluded lumen. However, the blind ends of the casted sinusoids are neither constricted nor irregular; rather, the ends appear smooth, rounded, and often expanded (Figure 8).A further possibility which could account for the blind-ended vessels is that the volume of casting medium injected was not enough to fill the entire capillary. It would be difficult to explain, however, why some adjacent sinusoids do not appear blind-ended. In some areas the sinusoids appeared to radiate only short distances from the collecting venules and exhibited no evidence of an afferent vascular supply. It is conceivable that this condition was due to backfilling from the collecting venule. Although the infusion of casting medium was within a physiologic range of pressure and in the direction of normal blood flow, we cannot discount that back-filling may be an artifact of preparation. However, it is possible that retrograde venous blood flow may normally exist in selected regions depending upon pressure gradients established by patterns of vascular resistance or vascular shunts. Backfilling of casting medium has also been observed in venous divisions of the renal medulla (unpublished observations). In summary, the corrosion casting technique reveals the organizational pattern of sinusoids, the nature of their afferent vascular connections, and their efferent route of drainage. The technique could be applied to the investigation of pathologic changes in the hepatic microcirculation.

References 1. Lozano DR, Andrews WH: A study by means of vascular casts of small vessels related to the mammalian portal vein. J Anat 160665-673, 1966 2. Elias H, Petty D: Terminal distribution of the hepatic artery. Anat Ret 116:9-l& 1953 3. Hase T, Brim J: Observation on the microcirculatory architecture of the rat liver. Anat Ret X6:157-174, 1966 4. Mitra SK: The terminal distribution of the hepatic artery with

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5.

6.

7.

8.

9.

10.

special reference to arterio-portal anastomoses. J Anat 166:651-663, 1966 Kessel RG, Kardon RH: Tissues and Organs: A Text-Atlas of Scanning Electron Microscopy. First Edition. San Francisco, California, W. H. Greeman & Company, 1979 Kardon RH, Kessel RG: SEM studied of vascular casts of the rat ovary. In: Scanning Electron Microscopy/l979/11. Edited by RP Becker, 0 Johari. AMF O’Hare, Chicago, Illinois, Scanning Electron Microscopy, Inc. 1979, p 743-756 Murakami T: Methyl methacrylate injection replica method. In: Principles and Techniques of Scanning Electron Microscopy. Edited by MA Hayat. New York, Van Nostrand Reinhold Company, 1978, 6:159-169 Gannon B: Vascular casting. In: Principles and Techniques of Scanning Electron Microscopy. Edited by MA Hayat. New York, Van Nostrand Reinhold Company, 1978, 6:170-193 Murakami T, Itoshima T, Shimada Y: Peribiliary portal system in the monkey liver as evidenced by the injection replica scanning electron microscope method. Arch Histol Jap 37:245-266, 1974 Nopanitaya W, Grisham JW, Aghajanian JG, et al: Intrahepatic microcirculation: SEM study of the terminal distribution of the hepatic artery. In: Scanning Electron Microscopy/

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11.

12.

13.

14.

15.

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1978/B. Edited by RP Becker, 0 Johari, AMF O’Hare, Chicago, Illinois, Scanning Electron Microscopy, Inc., 1978, p 837-842 Rappaport AM, Schneiderman JH: The function of the hepatic artery. Rev Physiol Biochem Pharmacol 76:129-175, 1976 Hodde K, Miodonski A, Bakker C, et al: Scanning electron microscopy of microcorrosion casts with special attention on arterio-venous differences. In: Scanning Electron Microscopy/ 1977/B. Edited by RP Becker, 0 Johari. Chicago, Illinois, Illinois Institute of Technology Research Institute, 1977, p 477484 Hodde K, et al: The vascularization of the pineal gland of the rat. In: Scanning Electron Microscopy/l979/11. Edited by RP Becker, 0 Johari. AMF O’Hare, Chicago, Illinois, Scanning Electron Microscopy, Inc., 1979 Popper H, Schaffner F: Structure of hepatic blood vessels. In: Liver: Structure and Function. New York, McGraw-Hill Book Company, 1957, p 130 Quistorff B, Chance B, Takeda H: Two and three dimensional redox hetrogeneity of rat liver. Effects of anoxia and alcohol on the lobular redox pattern. In: Frontiers of Biological Energetics. New York, Academic Press Inc., 2:1487, 1978