1Medical Hypotheses i
1 03069877MVOO33
Me&u1 Hypofherrs (19%) 33, 27s -281 0 Longman Group UK Ltd 1990
Multiple Roles for Surface-Active Hypertension
- 0275/$10.00
Phospholipid in
B.A. HILLS Department
of Physiology,
University
of New
England,
Armidale,
NSW,
235 7, Australia
Abstract - The elusiveness of the agent responsible for primary hypertension and the diversity of its impact upon the body, as reflected by the widely differing and almost independent avenues of research in this field, indicate that the answer could lie with a particularly common substance in the body acting at various levels of fundamental physiological function. This hypothesis pursues some basic physics of phospholipid whereby a change in quantity or quality can affect the capability to generate extreme surface activity manifest as the numerous properties which this agent can impart to blood and to adjacent surfaces by adsorption. Nine possible roles are traced by which surface-active phospholipid could impinge upon neurogenic control of blood pressure, the effects of circulating relaxing factors, blood rheology, atherosclerosis, and the major renal aspects of control by diuresis and association with the antihypertensive neutral renomedullary lipid. This multi-faceted approach offers mechanisms by which diet can affect blood pressure in addition to the traditional emphasis upon the deposition of atheroma.
Introduction Despite the high mortality associated with the disorder and the bewildering amount of published data, the mechanism(s) underlying essential hypertension remain obscure. For many decades there have been several schools of thought, each searching for the ‘one-and-only’ humoral pressor agent, neurogenic or renal mechanism, renal antihypertensive hormone or a physical factor operating by a single mode such as atheroma or hyperviscosity. Each such unitary hypothesis has eventually ‘ended in a blind alley’, as it has been deDate received 28 October 1989 Date accepted 21 February 1990
scribed so aptly by Folkow (l), who describes the present concensus of settling for a ‘multi-faceted approach’. However, this does not rule out a single agent if that agent operates through all of the modes mentioned above. Moreover the net effect would be even more perplexing to investigators if, in some modes, there were multiple roles of the same agent with some detrimental in maintaining normal blood pressure and others beneficial. One circulating agent which might act in such diverse ways, and in as many as nine possible roles is surface-active phospholipid @APL). Plasma contains amounts of non-membranous phospho-
276 lipid considered very high by standards for surface studies (2), the levels in serum being quoted as 211 mg/lOO ml (3). When this very insoluble material is extracted from the protein carriers to which it is reversibly bound as lipoprotein, many of the components are highly surface active, especially the phosphatidylcholines (alias lecithins) and, in particular, the disaturated derivatives. Until recently (4), SAPL was known only for its vital role as ‘surfactant’ in the lung. Gershfeld (5) has shown a strong correlation between ischaemic heart disease and the plasma concentration of disaturated lecithins (DSL). Moreover, the incidence of atherosclerosis confirmed by coronary angiography not only displayed a highly significant association with DSL statistically, but correlated more strongly with DSL than with cholesterol or triglycerides. Mean plasma concentrations were 2.92 +0.25 for atherosclerotics compared to 1.74 +O. 13 mg/lOO ml for the average population. Highly surface-active DSL has been shown to absorb strongly to inorganic surfaces such as glass (4, 5) and platinum (6), and to epithelium (7 - 9), attaching by the polar ends of the surfactant molecules which are then orientated with their fatty-acid chains facing outwards to present a hydrocarbon exterior (4). Such a surface would be most compatible with triglycerides and cholesterol and, if occurring as a lining to the vascular lumen, could be most conducive to the deposition of these substances in the formation of atheroma (5). Surfactants could also be involved in the reverse process of elutriating and mobilising neutral lipids from atheroma since they are very potent emulsifying agents (4) and, in the form of unsaturated lecithins, have long been prescribed as a ‘health food’ in the unproven belief (10) that they amelioriate atherosclerosis. However the hypothesis (5) that this disease is potentiated by the di-saturated DSL is entirely dependent upon demonstrating adsorption of the vascular lining. Hence it has hitherto received little attention largely because the hydrocarbon surface resulting from surfactant adsorption should be hydrophobic whereas vessel walls have been generally regarded as hydrophilic (11). Moreover, methods employed specifically to measure hydrophobicity as the contact angle (0) produced when a droplet of saline is applied to the surface have produced values of no more than 23 ‘(12). The contact angle is the angle between the solid surface and liquid-air interface at the triple point where all three phases meet and ranges from 0 for a perfectly wettable surface to 108x
MEDICAL
HYPOTHESES
for materials such as Teflon (2). This finding is consistent with the hydrophilic endothelial membrane envisaged as the standard lipid bilayer with fatty-acid chains orientated inwards while the polar (hydrophilic) groups form the outer surface (13, 14) which should therefore be readily wettable. Endothelial lining In view of the preceding argument it was therefore most surprising to find that ‘lawns’ of endothelial cells cultured by standard methods (15) from human umbilical cords were particularly hydrophobic, displaying contact angles of 50” - 70’(4). Recent repetition of these studies in this laboratory has largely confirmed these findings with 8 = 76.3”+4.2”(N = 10) on fresh human umbilical endothelium after rigorous rinsing with saline and 8 = 78.4”+4.1 “(N = 20) on rat aorta, histological examination showing that the rinsing had not displaced endothelial cells. Hence a major objection to the adsorption theory (5) for the role of DSL in promoting the deposition of neutral lipids in atheroma formation has now been removed. Vascular collapse An equally insidious consequence of an adsorbed surfactant layer is the high interfacial energy which must result from the direct contact between such a hydrophobic surface and blood. The surface area will tend to reduce to a minimum, thereby applying a collapsing pressure (AP) to the contents just as the pressure inside a soap bubble exceeds that outside. When the contact angle recorded above (78”) is substituted in standard equations (16), the interfacial energy for the blood-vessel interface (y) is 55 dynes/cm (mN/m). Substitution of this value in the Laplace equation for a vessel (AP = y/r) for the arterioles (r = 15 pm) as the calibre of resistance vessel at which flow resistance is known to be increased in hypertension (17), the collapsing pressure is derived as 35 mmHg. This simple physical force could thus explain the difference between hypertensives and normotensives (4). Barostat setting The vital question which is difficult to resolve for so many theories of hypertension (1) is why the
MULTIPLE
ROLES FOR SURFACE-ACTIVE
PHOSPHOLIPID
IN HYPERTENSION
central mechanism controlling arterial blood pressure via peripheral autonomic and/or renal effectors does not sense the change and reset the barostat accordingly. If the hydrophobic surface were responsible, the baroreceptors would not be exposed to any elevation in pressure because these are external to the lumen, i.e. on the convex side, where they would therefore detect a normal pressure. There might appear to be a fallacy in this explanation in that many of the baroreceptors monitoring arterial pressure are located in the wall of the aorta where the collapsing pressure (v/r) would be much lower due to the larger diameter. However, the walls of such large vessels have their own circulation in the form of the vasa vasorum consisting of a plexus of arterioles (18), i.e. vessels of diameter comparable to those of the relevant resistance vessels with which the baroreceptors are controlling the peripheral circulation (17). These stretch receptors are located in the layer of adventitia adjacent to the border with the tunica media (19) and would therefore be closer to the vasa vasorum than to the lumen of the aorta. Baroreceptors in other arterial locations such as carotid bodies (19) are, yet again, on the convex side of a plexus of arterioles. If these arterioles are as hydrophobic as the aorta, then the original argument would still be valid, and the central mechanism would be unable to sense primary hypertension due to vascular collapse and, hence, would not reset the barostat. Humoral wetting agent The situation on the luminal surfaces could be more complex in so far as the net hydrophobicity of the vessel wall would be determined not only by adsorption of disaturated phospholipid but also by the subsequent deposition of a wetting agent derived from plasma to reduce the high interfacial energy which could otherwise result. A comparable situation is the manner in which gastric mucus - as a superb wetting agent (9) - reduces what is otherwise a very high surface energy imparted by the monolayer of adsorbed surfactant, thus stabilising this lining claimed to provide the ‘gastric mucosal barrier’. This concept is compatible with the much lower contact angle recorded on vessel walls prior to rinsing (4, 12) and offers an explanation for the wettable nature of indwelling Teflon catheters removed after long periods in vivo. Thus both the collapsing and lipid-deposition aspects of a
277
hydrophobic vascular lining could reflect a deficiency of wetting agent as much as the adsorption of surfactant, i.e. displacement of a balance between the opposing effects of water repellents and wetting agents. Endothelial barrier The same monolayer of phospholipid believed to be deposited on the luminal lining of blood vessels could act as a barrier to diffusion (20). Thus, it could exclude various solutes and circulating humoral agents which might include neurotransmitters and/or releasing factors for vascular smooth muscle. It is well established in the physical sciences that transmission properties are highly dependent upon the surface energy (21) and, hence, upon surfactants (22) which can modify it. This concept has been expounded in more detail as a means of differentiating membrane function (20) and providing barriers by adsorption of phospholipid, two of particular interest being the gastric mucosal barrier (8,9) and blood-brain barrier (4, 20). As a barrier derived by adsorption from adjacent fluid, SAPL has a particular theoretical advantage (20) over barriers based upon cells per se, however tight their junctions. This is the ability to maintain the barrier during cell shedding and regeneration when direct adsorption to basement membrane would immediately seal any breach before adjacent cells were able to divide or otherwise fill the gap left by the sloughed cell. Morphological evidence for direct adsorption to basement membrane is discussed later. Acting as both a permeability barrier and the interfacial agent inducing vascular collapse, a monolayer of SAPL could provide the ‘mechanical tension’ which Bradbury (23) claims to characterise the blood-brain barrier (BBB) after his most comprehensive review of the literature. This conclusion is based largely upon the coincidence of two major features - breakdown of the BBB with breakdown of cerebral autoregulation, especially upon elevating blood pressure. Returning to the arterial wall, SAPL could provide the barrier which Griffith et al (24) needed to disrupt by localised damage before an otherwise unresponsive artery would elicit a conventional constrictor response to a variety of vasoconstrictor agents. The same barrier could be the one localising the effects of endothelium-derived relaxant factor (25, 26) to vascular smooth muscle by preventing its escape into the vascular space where its half-life is likely to be much less
278 than the 6 s recorded in well oxygenated aqueous buffer at 37°C (24). Surface-active phospholipid would also satisfy the criterion for the heat-labile plasma component providing the endothelial barrier blocking EDFR activity in aortic strips.
MEDICAL
HYPOTHESES
tion correlates so well with antihypertensive activity shows a remarkable resemblance to the lamellar bodies found in cells adjacent to proximal and distal renal tubules as described above. When first discovered in the alveolar Type II cells, lamellar bodies were described as ‘granules’ (35).
Renal implications The capability of plasma to deposit a surfactant layer which can cause permeability changes to adjacent membrane surfaces has particular implications to the kidney. At the glomerular level there is the parallel with reverse osmosis in synthetic membranes (21,22) where more hydrophobic materials tend to retain more sodium ions than hydrophilic membranes of the same pore size, sodium retention being a major feature of hypertension (1). At the level of the renal tubule, recent studies of one model - the toad bladder - have shown an appreciable decrease in water permeability and elimination of the response to ADH by adsorbing surface-active phospholipid (27). Moreover, decreased permeability corresponded to increased hydrophobicity as monitored by the contact angle described above - just as the ability to absorb water and electrolytes along the GI tract is exactly reciprocated by surface hydrophobicity (4, 19). Returning to ADH, this is one of the few hormones found consistently at elevated levels in hypertensives (28). The stores of SAPL in the kidney are probably the ‘whorls’ which are enlarged by administration of suramin (29) - a phospholipase inhibitor and which have been identified as lamellar bodies (4). In another model of the renal tubule - frog skin - sodium permeability is also increased by phospholipase ‘C’ (30) which breaks down SAPL. Thus reabsorption by the renal tubule could be inhibited by adsorption of surfactant with a resulting fall in blood pressure. By comparison, the adverse effects of a surfactant monolayer in the rest of the body, e.g. in promoting vascular collapse, could be ameliorated by its partial removal by an enzyme or a solvent. One particularly effective group of solvents for SAPL is neutral lipids which are known for their effectiveness in opening the blood-brain barrier (4,31) for reasons outlined above. Hence it could be most pertinent that the antihypertensive agent released by the kidney and so effective in renoaspillary transplants in the absence of both kidneys (32) is a neutral lipid ANRL (33). On the other hand there is some question that the lipid really is neutral (34), while the densely osmiophilic ‘granules’ whose degranula-
Haemodynamic
aspects
Many aspects of primary hypertension have been attributed directly or indirectly to hyperviscosity (36) although blood pressure can be permanently elevated when blood rheology is normal (1). One aspect apparently overlooked is the possible role of ‘viscosity modifiers’, especially since phospholipid in its commercial from (lecithin) is very widely used for this purpose in food processing. In confectionery (37) it requires the addition of only 0.1% lecithin to lower by 60% the viscosity of molten chocolate - a two-phase fluid not unlike blood rheologically. The concentration in serum is 0.2% (3). Another aspect of surface-active phospholipid discovered in connection with sliding of the pleura (38) and articular surface (39) is its remarkable lubricating properties. Measurements using the standard apparatus and method used for studying joints have revealed coefficients of kinetic friction for oligolamellar layers of DPL in the range of 0.002-0.004 and occasionally as low as 0.0007 (40). If such a layer existed at the vascular wall, as already demonstrated (41), then viscous forces would exceed shear forces and slippage would occur at the wall. This has been tested recently in a very simple experiment in which sheep blood has been pumped through a capillary tube of 2mm dia. at a fixed rate of 100 ml/min. The pressure gradient (AP) of 200 cm.H20 was reduced by 32 cm.HzO, i.e. by 16%, when the inside was coated with a layer of L -aDPL deposited as a layer of 20 pg/cm from solution in chloroform followed by rotation of a close-fitting glass rod to promote formation of a lamellated coating. A coating would have reduced the tube diameter to which BP is particularly sensitive (AP ar4) by the Poiseuille equation so that the decrease of 16% is a conservative estimate of the effect of slippage. A reduction of 16% for a subject with a systolic pressure of 150 mmHg amounts to 24mmHg which is significant. This finding is compatible with a long-known phenomenon - the LindquistFahraeus effect (42) - whereby the apparent viscosity of blood is decreased as the radius of the capillary tube used for measuring is reduced. The
MULTIPLE ROLES FOR SURFACE-ACTIVE
PHOSPHOLIPID
IN HYPERTENSION
effect has been attributed to a lubricating layer at the wall (43) and comparison with other explanations largely supports this view (44) although the identitiy of the lubricating layer has never been established. Hence this study could provide that identitiy as the surface-active phospholipid normally present in plasma. Lubricating layers also protect against erosion (4) and, hence, it would be expected that replenishment of the phospholipid lining should reduce injury to endothelium. Hence it is interesting to find that post-infusion injury to venous endothelium is reduced by i.v. infusions of soyabean oil known (45) to be ‘loaded’ with unsaturated phospholipid (4). Morphology Before discussing the implications of so many roles for surface-active phospholipid in affecting blood pressure, it is necessary to address the vital question common to them all - the morphological evidence for a luminal lining. Apart from indirect evidence provided by studies of endothelial hydrophobicity, why do we not see multiple layers by electron microscopy? This question has been addressed in much detail elsewhere (4) but the answer could lie with the fixatives. Since Sabatini et al (46) introduced glutaraldehyde in 1963, this has been used almost universally and produces e.m.s of membranes as simple ‘tramlines’ in agreement with popular lipid bilayer theories (11, 12). However, aldehydes are well known for their ability to remove hydrophobic surfaces (40), and the studies of contact angle leave no doubt that there is a hydrophobic lining to the aorta and some other blood vessels. The only tissue surface on which the preservation of surfactant has been studied extensively is the alveolar surface and here the principle of vascular fixation or ‘fixing from behind’ is almost universal practice (48). Presumably the fixative reaches the polar ends of the molecules before the hydrophobic surface. Vascular fixation would thus be unlikely to succeed for a vascular lining. The only organ in which a vascular lining could be ‘fixed from behind’ is the lung where the airways offer a unique route for non-vascular fixation. Hence it could be particularly significant that, when Ueda et al (49) ventilated the airways with a fixative in which much of the glutaraldehyde was replaced by tannic acid, they found clear evidence of surfactant on the endothelial surface and on the basement membrane of junctions in multi-lamellated form. These findings have now
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been confirmed in this laboratory and extended to the luminal surface of the aorta where 3 -5 layers have been demonstrated and 8 - 10 layers on cerebral endothelium (41). It must also be emphasized that many membranes were seen as trilaminar structures in early electron microscopy before glutaraldehyde was introduced and simple fixatives such as potassium permanganate were in vogue (50). A trilaminar model is also used by those studying membranes by X-ray diffraction (51). Discussion If substantially correct, the foregoing hypothesis would tend to emphasize the well accepted need (52) to reduce the intake of saturated fats not only for the triglycerides themselves but for the accompanying phospholipids which tend to reflect the overall fatty-acid distribution in plassma (53). IJnsaturated PL should be beneficial in reducing blood viscosity, lubricating blood flow and emulsifying neutral lipids in reversing atheroma formation, while most of the undesirable aspects, such as collapse due to hydrophobic surfaces, are associated with the very high surface activity resulting (4) from saturation of the component fatty acids. This aspect could be relevant to the other schools of thought besides atherosclerosis and, in that context, may explain such current topics as the beneficial effects of fish oil, especially since this is known (54) to reduce the saturation of plasma phospholipids. However, the ubiquitous nature of phospholipid and the complex surface physics which are attractive theoretically in enabling SAPL to be considered for nine roles impinging upon all of the five or so schools of thought upon hypertension also make this hypothesis difficult to prove. Keferences 1. Folkow B. Physiological aspects of primary hypertension. Phvsiol Rev 62: 347 - 504. 1982. 2. Adamson AW. Physical Chemistry of Surfaces. 2nd ed: pp 352 - 367. New York: Wiley 1967. 3. Spector WS. Handbook of Biological Data. p. 52. Philadelphia: Saunders, 1956. 4. Hills BA. The Biology of Surfactant. Cambridge: Cambridge University Press, 1988. 5. Gershfeld NL. Selective phospholipid adsorption and atherosclerosis. Science, 204: 5Of- 8, 1979. 6. Barrow RE, Hills BA. A critical assessment of the Wilhelmy method in studying lung surfactants. .I Physiol, 1295: 217-227, 1979. 7. Hills BA, Barrow RE. The contact angle induced by DPL at pulmonary epithelial surfaces. Respirat Physiol 38:
280 173 - 183, 1979. 8. Hills BA, Butler BD, Lichtenberger LM. Gastric mucosal barrier: hydrophobic lining to the lumen of the stomach. Am J. Physiol: Gastrointest and Liver Physiol. 244: G561-68, 1983. 9. Hills BA. Gastric mucosal barrier: stabilization of hydrophobic lining to the stomach by mucus. Amer J Physiol: Gastrointest and Liver Physiol. 249: G342 - 49, 1985. 10. Mustard JF. Composition and method for correcting foods and blood conditions having clot promoting characteristics. Canadian patent 774, 004, 1958. 11. Harvey EN. Physical factors in bubble formation. In: Fulton JF, ed. Decompression Sickness. pp90 - 114, Philadelphia: Saunders, 1951. 12. Sherman IA. Interfacial tension effects in the microvasculature. Microvasc. Res. 22: 296- 307, 1981. 13. Singer SJ, Nicolson GL. The fluid mosaic model of the structure of cell membranes. Science 175: 720- 31, 1972. 14. Danielli JF, Davson H. A contribution to the theory of permeability of thin films. J Cell Comp Physiol. 5: 495 - 508, 1935. 15. Jaffe EA. Culture and identification of the large vessel endothelial cells. In: Jaffe EA ed, The Biology of Endothelial Cells pp 1 - 13, The Hague: Nijhoff. 16. Neufeld RJ, Zajic JE, Gerson DF. Cell surface measurements in hydorcarbon and carbohydrate fermentations. Appl Environ Microbial 39: 511- 17, 1980. 17. Folkow B. Cardiovascular structural adaptation: its role in the initiation and maintenance of primary hypertension. Clin Sci Molec Med, 55: 3 - 22, 1978. 18. Bloom W, Fawcett DW. A Textbook of Histology. 10th ed. p 402. Philadelphia: Saunders, 1975. 19. Abraham A. The structure of baroreceptors in pathological conditions in man. Proc Symp Baroreceptors and Hypertension. Oxford: Pergamon. 20. Hills BA. Possible role of adsorbed phospholipid in controlling membrane permeability and function. Med Hypoth 28: 85 -92, 1989. 21. Sourirajan S, Matsuura T. Science of reverse osmosis an essential tool for the chemical engineer. Chem Engr, 385: 359-68, 1982. 22. Fane AG, Fell CJD, Kim KJ. The effect of surfactant pretreatment on the ultrafiltration of proteins. Desalination 53: 37-55, 1985. 23. Bradbury M. The Concept of a Blood-Brain Barrier. New York: Wiley. 24. Griffith TM, Edwards DH, Lewis MJ et al. The nature of endothelium-derived relaxing factor. Nature 308: 645 -47, 1984. 25. Furchgott RF, Zawadzki JV. The obligatory role of endothelial cells in the relaxation of arterial smooth muscle by acetylcholine. Nature 288: 373 - 76, 1980. 26. Peach MJ, Loeb AL, Singer HA, Saye J. Endotheliumderived relaxing factor. Hypertension 7: 194- 100, 1985. 27. Dial EJ, Huang J, O’Neill RG, et al. Surface hydrophobicity and water transport of the toad urinary bladder: effect of vasopressin. J Membrane Biol 106: 119- 122, 1988. 28. Bowman WC, Rand MJ. Textbook of Pharmacology. 2nd ed. p23 - 32. Oxford: Blackwell 1980. 29. Rees S. Membranous neuronal and neuroglia inclusions produced by intracerebral injection of suramin. J Neural Sci 36: 97- 109, 1978. 30. Tarapoom N, Royce R, Yorio T. Inhibition of the antidiuretic hormone hydroosmotic response of phospholipids
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31. 32.
33. 34.
35. 36. 37.
38.
39.
40. 41. 42. 43. 44.
45.
46.
47.
48.
49.
50. 51.
52. 53.
HYPOTHESES
and phospholipids metabolites. Lipids 21: 596- 602, 1986. Broman T. quoted by Bradbury (23). Muirhead EE, Kosinski M. Renal medulla and renoprival hypertension: relationship between corticorenal (renin) and medellorenal extracts. Circ Res 11: 647 - 80, 1962. Muirhead EE, Pitcock JA. The renal antihypertensive hormone. J Hypertension, 3: 1- 8, 1984. Pitcock JA, Brown PS, Byers LW et al. Degranulation of renomedullary interstitial cells during reversal of hypertension. Hypertension, 3: (suppl II): 75 - 80, 1981. Macklin CC. The pulmonary alveolar mucoid film and pneumocytes. Lancet i: 1099- 104, 1954. Dintenfass L. Hyperviscosity in Hypertension. Sydney: Pergamon, 1981. Schoen M. Confectionery and cacao products. In: Jacobs MB ed. The Chemistry of Technology of Food and Food Products. Vol III, pp 2138-2140. New York: Interscience, 1951. Hills BA, Butler BD, Barrow RE. Boundary lubrication imparted by pleural surfactants and their identification. J Appl Physiol: Respirat. Environ Exercise Physiol. 53: 463 - 69, 1982. Hills BA, Butler BD. Surfactants identified in synovial fluid and their ability to act as boundary lubricants. Ann Rheum Dis 48: 51- 57, 1984. Hills BA. Oligolamellar lubrication of joints by surfaceactive phospholipid. J Rheumatol 16: 82 - 91, 1989. Hills BA. A physical identity for the blood-brain barrier. Proc Roy Sot NSW (in press). Fahraeus R, Lindquist T. The viscosity of blood in narrow capillary tubes. Amer J Physiol 96: 562, 1931. Schofield RK, Scott-Blair GW. (1930) quoted by Haynes (44). Haynes RH. Physical basis of the dependence of blood viscosity on tube radius. Amer J Physiol 198: 1193- 200, 1960. Fujiwara T, Karawasaki H, Fonkalsrud EW. Reduction of postinfusion venous endothelial injury with intralipid. Surgery, Gynecology and Obstetrics 158: 57 - 65, 1984. Sabatini DD, Bensch K, Barnett RJ. Cytochemistry and electron microscopy: preservation of cellular ultrastructure and enzymatic activity by aldehyde fixation. J Cell Biol 17: 19-26, 1963. Untersee P, Gil J, Weibel ER. Visualization of extracellular lining layer of lung alveoli by freezing-etching. Respirat Physiol 13: 171-85, 1971. Gil J, Weibel ER. Morphological study of pressure-volume hysteresis in rat lungs fixed by vascular perfusion. Respir Physiol 15: 190-213, 1972. Ueda S, Kawamura K, Ishii N et al. Morphological studies on surface lining layer of the lungs. Part VI. Surfactantlike substance in other organs (pleural cavity, vascular lumen and gastric lumen) than lungs. J Jap Med Sot Biol Interface 17:132 - 56, 1986. Robertson JD. The ultrastructure of cell membranes and their derivatives. Symp Biochem Sot 16: 3 - 5, 1959. Oimatov M, L’Vov YM, Aripov TF, et al. Location of cytotoxin in model membranes and its influence on the packing of hydrocarbon lipid chains. X-ray small-angle analysis. Soviet Physics and Crystallography, 31: 942- 50, 1986. Puskas P et al. Controlled, randomised trial of the effect of dietary fat on blood pressure. Lancet l/83: 1 - 5, 1983. Dougherty RM, Galli C, Ferro-Luzzi A et al. Lipid and
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phospholipid fatty acid composition of plasma, red blood cells, and platelets and how they are affected by dietary lipids: A study of normal subjects from Italy, Finland and the U.S.A. Amer J Clin Nutr 45: 443 - 55, 1987.
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54. Barcelli UO, Beach DC, Pollak VE. The influence of n-6 and n-3 fatty acids on kidney phospholipid composition and on eicosanoid production in aging rats. Lipids, 23: 309-312, 1988.
1 03069877MVOO33
Me&u1 Hypofherrs (19%) 33, 27s -281 0 Longman Group UK Ltd 1990
Multiple Roles for Surface-Active Hypertension
- 0275/$10.00
Phospholipid in
B.A. HILLS Department
of Physiology,
University
of New
England,
Armidale,
NSW,
235 7, Australia
Abstract - The elusiveness of the agent responsible for primary hypertension and the diversity of its impact upon the body, as reflected by the widely differing and almost independent avenues of research in this field, indicate that the answer could lie with a particularly common substance in the body acting at various levels of fundamental physiological function. This hypothesis pursues some basic physics of phospholipid whereby a change in quantity or quality can affect the capability to generate extreme surface activity manifest as the numerous properties which this agent can impart to blood and to adjacent surfaces by adsorption. Nine possible roles are traced by which surface-active phospholipid could impinge upon neurogenic control of blood pressure, the effects of circulating relaxing factors, blood rheology, atherosclerosis, and the major renal aspects of control by diuresis and association with the antihypertensive neutral renomedullary lipid. This multi-faceted approach offers mechanisms by which diet can affect blood pressure in addition to the traditional emphasis upon the deposition of atheroma.
Introduction Despite the high mortality associated with the disorder and the bewildering amount of published data, the mechanism(s) underlying essential hypertension remain obscure. For many decades there have been several schools of thought, each searching for the ‘one-and-only’ humoral pressor agent, neurogenic or renal mechanism, renal antihypertensive hormone or a physical factor operating by a single mode such as atheroma or hyperviscosity. Each such unitary hypothesis has eventually ‘ended in a blind alley’, as it has been deDate received 28 October 1989 Date accepted 21 February 1990
scribed so aptly by Folkow (l), who describes the present concensus of settling for a ‘multi-faceted approach’. However, this does not rule out a single agent if that agent operates through all of the modes mentioned above. Moreover the net effect would be even more perplexing to investigators if, in some modes, there were multiple roles of the same agent with some detrimental in maintaining normal blood pressure and others beneficial. One circulating agent which might act in such diverse ways, and in as many as nine possible roles is surface-active phospholipid @APL). Plasma contains amounts of non-membranous phospho-
276 lipid considered very high by standards for surface studies (2), the levels in serum being quoted as 211 mg/lOO ml (3). When this very insoluble material is extracted from the protein carriers to which it is reversibly bound as lipoprotein, many of the components are highly surface active, especially the phosphatidylcholines (alias lecithins) and, in particular, the disaturated derivatives. Until recently (4), SAPL was known only for its vital role as ‘surfactant’ in the lung. Gershfeld (5) has shown a strong correlation between ischaemic heart disease and the plasma concentration of disaturated lecithins (DSL). Moreover, the incidence of atherosclerosis confirmed by coronary angiography not only displayed a highly significant association with DSL statistically, but correlated more strongly with DSL than with cholesterol or triglycerides. Mean plasma concentrations were 2.92 +0.25 for atherosclerotics compared to 1.74 +O. 13 mg/lOO ml for the average population. Highly surface-active DSL has been shown to absorb strongly to inorganic surfaces such as glass (4, 5) and platinum (6), and to epithelium (7 - 9), attaching by the polar ends of the surfactant molecules which are then orientated with their fatty-acid chains facing outwards to present a hydrocarbon exterior (4). Such a surface would be most compatible with triglycerides and cholesterol and, if occurring as a lining to the vascular lumen, could be most conducive to the deposition of these substances in the formation of atheroma (5). Surfactants could also be involved in the reverse process of elutriating and mobilising neutral lipids from atheroma since they are very potent emulsifying agents (4) and, in the form of unsaturated lecithins, have long been prescribed as a ‘health food’ in the unproven belief (10) that they amelioriate atherosclerosis. However the hypothesis (5) that this disease is potentiated by the di-saturated DSL is entirely dependent upon demonstrating adsorption of the vascular lining. Hence it has hitherto received little attention largely because the hydrocarbon surface resulting from surfactant adsorption should be hydrophobic whereas vessel walls have been generally regarded as hydrophilic (11). Moreover, methods employed specifically to measure hydrophobicity as the contact angle (0) produced when a droplet of saline is applied to the surface have produced values of no more than 23 ‘(12). The contact angle is the angle between the solid surface and liquid-air interface at the triple point where all three phases meet and ranges from 0 for a perfectly wettable surface to 108x
MEDICAL
HYPOTHESES
for materials such as Teflon (2). This finding is consistent with the hydrophilic endothelial membrane envisaged as the standard lipid bilayer with fatty-acid chains orientated inwards while the polar (hydrophilic) groups form the outer surface (13, 14) which should therefore be readily wettable. Endothelial lining In view of the preceding argument it was therefore most surprising to find that ‘lawns’ of endothelial cells cultured by standard methods (15) from human umbilical cords were particularly hydrophobic, displaying contact angles of 50” - 70’(4). Recent repetition of these studies in this laboratory has largely confirmed these findings with 8 = 76.3”+4.2”(N = 10) on fresh human umbilical endothelium after rigorous rinsing with saline and 8 = 78.4”+4.1 “(N = 20) on rat aorta, histological examination showing that the rinsing had not displaced endothelial cells. Hence a major objection to the adsorption theory (5) for the role of DSL in promoting the deposition of neutral lipids in atheroma formation has now been removed. Vascular collapse An equally insidious consequence of an adsorbed surfactant layer is the high interfacial energy which must result from the direct contact between such a hydrophobic surface and blood. The surface area will tend to reduce to a minimum, thereby applying a collapsing pressure (AP) to the contents just as the pressure inside a soap bubble exceeds that outside. When the contact angle recorded above (78”) is substituted in standard equations (16), the interfacial energy for the blood-vessel interface (y) is 55 dynes/cm (mN/m). Substitution of this value in the Laplace equation for a vessel (AP = y/r) for the arterioles (r = 15 pm) as the calibre of resistance vessel at which flow resistance is known to be increased in hypertension (17), the collapsing pressure is derived as 35 mmHg. This simple physical force could thus explain the difference between hypertensives and normotensives (4). Barostat setting The vital question which is difficult to resolve for so many theories of hypertension (1) is why the
MULTIPLE
ROLES FOR SURFACE-ACTIVE
PHOSPHOLIPID
IN HYPERTENSION
central mechanism controlling arterial blood pressure via peripheral autonomic and/or renal effectors does not sense the change and reset the barostat accordingly. If the hydrophobic surface were responsible, the baroreceptors would not be exposed to any elevation in pressure because these are external to the lumen, i.e. on the convex side, where they would therefore detect a normal pressure. There might appear to be a fallacy in this explanation in that many of the baroreceptors monitoring arterial pressure are located in the wall of the aorta where the collapsing pressure (v/r) would be much lower due to the larger diameter. However, the walls of such large vessels have their own circulation in the form of the vasa vasorum consisting of a plexus of arterioles (18), i.e. vessels of diameter comparable to those of the relevant resistance vessels with which the baroreceptors are controlling the peripheral circulation (17). These stretch receptors are located in the layer of adventitia adjacent to the border with the tunica media (19) and would therefore be closer to the vasa vasorum than to the lumen of the aorta. Baroreceptors in other arterial locations such as carotid bodies (19) are, yet again, on the convex side of a plexus of arterioles. If these arterioles are as hydrophobic as the aorta, then the original argument would still be valid, and the central mechanism would be unable to sense primary hypertension due to vascular collapse and, hence, would not reset the barostat. Humoral wetting agent The situation on the luminal surfaces could be more complex in so far as the net hydrophobicity of the vessel wall would be determined not only by adsorption of disaturated phospholipid but also by the subsequent deposition of a wetting agent derived from plasma to reduce the high interfacial energy which could otherwise result. A comparable situation is the manner in which gastric mucus - as a superb wetting agent (9) - reduces what is otherwise a very high surface energy imparted by the monolayer of adsorbed surfactant, thus stabilising this lining claimed to provide the ‘gastric mucosal barrier’. This concept is compatible with the much lower contact angle recorded on vessel walls prior to rinsing (4, 12) and offers an explanation for the wettable nature of indwelling Teflon catheters removed after long periods in vivo. Thus both the collapsing and lipid-deposition aspects of a
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hydrophobic vascular lining could reflect a deficiency of wetting agent as much as the adsorption of surfactant, i.e. displacement of a balance between the opposing effects of water repellents and wetting agents. Endothelial barrier The same monolayer of phospholipid believed to be deposited on the luminal lining of blood vessels could act as a barrier to diffusion (20). Thus, it could exclude various solutes and circulating humoral agents which might include neurotransmitters and/or releasing factors for vascular smooth muscle. It is well established in the physical sciences that transmission properties are highly dependent upon the surface energy (21) and, hence, upon surfactants (22) which can modify it. This concept has been expounded in more detail as a means of differentiating membrane function (20) and providing barriers by adsorption of phospholipid, two of particular interest being the gastric mucosal barrier (8,9) and blood-brain barrier (4, 20). As a barrier derived by adsorption from adjacent fluid, SAPL has a particular theoretical advantage (20) over barriers based upon cells per se, however tight their junctions. This is the ability to maintain the barrier during cell shedding and regeneration when direct adsorption to basement membrane would immediately seal any breach before adjacent cells were able to divide or otherwise fill the gap left by the sloughed cell. Morphological evidence for direct adsorption to basement membrane is discussed later. Acting as both a permeability barrier and the interfacial agent inducing vascular collapse, a monolayer of SAPL could provide the ‘mechanical tension’ which Bradbury (23) claims to characterise the blood-brain barrier (BBB) after his most comprehensive review of the literature. This conclusion is based largely upon the coincidence of two major features - breakdown of the BBB with breakdown of cerebral autoregulation, especially upon elevating blood pressure. Returning to the arterial wall, SAPL could provide the barrier which Griffith et al (24) needed to disrupt by localised damage before an otherwise unresponsive artery would elicit a conventional constrictor response to a variety of vasoconstrictor agents. The same barrier could be the one localising the effects of endothelium-derived relaxant factor (25, 26) to vascular smooth muscle by preventing its escape into the vascular space where its half-life is likely to be much less
278 than the 6 s recorded in well oxygenated aqueous buffer at 37°C (24). Surface-active phospholipid would also satisfy the criterion for the heat-labile plasma component providing the endothelial barrier blocking EDFR activity in aortic strips.
MEDICAL
HYPOTHESES
tion correlates so well with antihypertensive activity shows a remarkable resemblance to the lamellar bodies found in cells adjacent to proximal and distal renal tubules as described above. When first discovered in the alveolar Type II cells, lamellar bodies were described as ‘granules’ (35).
Renal implications The capability of plasma to deposit a surfactant layer which can cause permeability changes to adjacent membrane surfaces has particular implications to the kidney. At the glomerular level there is the parallel with reverse osmosis in synthetic membranes (21,22) where more hydrophobic materials tend to retain more sodium ions than hydrophilic membranes of the same pore size, sodium retention being a major feature of hypertension (1). At the level of the renal tubule, recent studies of one model - the toad bladder - have shown an appreciable decrease in water permeability and elimination of the response to ADH by adsorbing surface-active phospholipid (27). Moreover, decreased permeability corresponded to increased hydrophobicity as monitored by the contact angle described above - just as the ability to absorb water and electrolytes along the GI tract is exactly reciprocated by surface hydrophobicity (4, 19). Returning to ADH, this is one of the few hormones found consistently at elevated levels in hypertensives (28). The stores of SAPL in the kidney are probably the ‘whorls’ which are enlarged by administration of suramin (29) - a phospholipase inhibitor and which have been identified as lamellar bodies (4). In another model of the renal tubule - frog skin - sodium permeability is also increased by phospholipase ‘C’ (30) which breaks down SAPL. Thus reabsorption by the renal tubule could be inhibited by adsorption of surfactant with a resulting fall in blood pressure. By comparison, the adverse effects of a surfactant monolayer in the rest of the body, e.g. in promoting vascular collapse, could be ameliorated by its partial removal by an enzyme or a solvent. One particularly effective group of solvents for SAPL is neutral lipids which are known for their effectiveness in opening the blood-brain barrier (4,31) for reasons outlined above. Hence it could be most pertinent that the antihypertensive agent released by the kidney and so effective in renoaspillary transplants in the absence of both kidneys (32) is a neutral lipid ANRL (33). On the other hand there is some question that the lipid really is neutral (34), while the densely osmiophilic ‘granules’ whose degranula-
Haemodynamic
aspects
Many aspects of primary hypertension have been attributed directly or indirectly to hyperviscosity (36) although blood pressure can be permanently elevated when blood rheology is normal (1). One aspect apparently overlooked is the possible role of ‘viscosity modifiers’, especially since phospholipid in its commercial from (lecithin) is very widely used for this purpose in food processing. In confectionery (37) it requires the addition of only 0.1% lecithin to lower by 60% the viscosity of molten chocolate - a two-phase fluid not unlike blood rheologically. The concentration in serum is 0.2% (3). Another aspect of surface-active phospholipid discovered in connection with sliding of the pleura (38) and articular surface (39) is its remarkable lubricating properties. Measurements using the standard apparatus and method used for studying joints have revealed coefficients of kinetic friction for oligolamellar layers of DPL in the range of 0.002-0.004 and occasionally as low as 0.0007 (40). If such a layer existed at the vascular wall, as already demonstrated (41), then viscous forces would exceed shear forces and slippage would occur at the wall. This has been tested recently in a very simple experiment in which sheep blood has been pumped through a capillary tube of 2mm dia. at a fixed rate of 100 ml/min. The pressure gradient (AP) of 200 cm.H20 was reduced by 32 cm.HzO, i.e. by 16%, when the inside was coated with a layer of L -aDPL deposited as a layer of 20 pg/cm from solution in chloroform followed by rotation of a close-fitting glass rod to promote formation of a lamellated coating. A coating would have reduced the tube diameter to which BP is particularly sensitive (AP ar4) by the Poiseuille equation so that the decrease of 16% is a conservative estimate of the effect of slippage. A reduction of 16% for a subject with a systolic pressure of 150 mmHg amounts to 24mmHg which is significant. This finding is compatible with a long-known phenomenon - the LindquistFahraeus effect (42) - whereby the apparent viscosity of blood is decreased as the radius of the capillary tube used for measuring is reduced. The
MULTIPLE ROLES FOR SURFACE-ACTIVE
PHOSPHOLIPID
IN HYPERTENSION
effect has been attributed to a lubricating layer at the wall (43) and comparison with other explanations largely supports this view (44) although the identitiy of the lubricating layer has never been established. Hence this study could provide that identitiy as the surface-active phospholipid normally present in plasma. Lubricating layers also protect against erosion (4) and, hence, it would be expected that replenishment of the phospholipid lining should reduce injury to endothelium. Hence it is interesting to find that post-infusion injury to venous endothelium is reduced by i.v. infusions of soyabean oil known (45) to be ‘loaded’ with unsaturated phospholipid (4). Morphology Before discussing the implications of so many roles for surface-active phospholipid in affecting blood pressure, it is necessary to address the vital question common to them all - the morphological evidence for a luminal lining. Apart from indirect evidence provided by studies of endothelial hydrophobicity, why do we not see multiple layers by electron microscopy? This question has been addressed in much detail elsewhere (4) but the answer could lie with the fixatives. Since Sabatini et al (46) introduced glutaraldehyde in 1963, this has been used almost universally and produces e.m.s of membranes as simple ‘tramlines’ in agreement with popular lipid bilayer theories (11, 12). However, aldehydes are well known for their ability to remove hydrophobic surfaces (40), and the studies of contact angle leave no doubt that there is a hydrophobic lining to the aorta and some other blood vessels. The only tissue surface on which the preservation of surfactant has been studied extensively is the alveolar surface and here the principle of vascular fixation or ‘fixing from behind’ is almost universal practice (48). Presumably the fixative reaches the polar ends of the molecules before the hydrophobic surface. Vascular fixation would thus be unlikely to succeed for a vascular lining. The only organ in which a vascular lining could be ‘fixed from behind’ is the lung where the airways offer a unique route for non-vascular fixation. Hence it could be particularly significant that, when Ueda et al (49) ventilated the airways with a fixative in which much of the glutaraldehyde was replaced by tannic acid, they found clear evidence of surfactant on the endothelial surface and on the basement membrane of junctions in multi-lamellated form. These findings have now
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been confirmed in this laboratory and extended to the luminal surface of the aorta where 3 -5 layers have been demonstrated and 8 - 10 layers on cerebral endothelium (41). It must also be emphasized that many membranes were seen as trilaminar structures in early electron microscopy before glutaraldehyde was introduced and simple fixatives such as potassium permanganate were in vogue (50). A trilaminar model is also used by those studying membranes by X-ray diffraction (51). Discussion If substantially correct, the foregoing hypothesis would tend to emphasize the well accepted need (52) to reduce the intake of saturated fats not only for the triglycerides themselves but for the accompanying phospholipids which tend to reflect the overall fatty-acid distribution in plassma (53). IJnsaturated PL should be beneficial in reducing blood viscosity, lubricating blood flow and emulsifying neutral lipids in reversing atheroma formation, while most of the undesirable aspects, such as collapse due to hydrophobic surfaces, are associated with the very high surface activity resulting (4) from saturation of the component fatty acids. This aspect could be relevant to the other schools of thought besides atherosclerosis and, in that context, may explain such current topics as the beneficial effects of fish oil, especially since this is known (54) to reduce the saturation of plasma phospholipids. However, the ubiquitous nature of phospholipid and the complex surface physics which are attractive theoretically in enabling SAPL to be considered for nine roles impinging upon all of the five or so schools of thought upon hypertension also make this hypothesis difficult to prove. Keferences 1. Folkow B. Physiological aspects of primary hypertension. Phvsiol Rev 62: 347 - 504. 1982. 2. Adamson AW. Physical Chemistry of Surfaces. 2nd ed: pp 352 - 367. New York: Wiley 1967. 3. Spector WS. Handbook of Biological Data. p. 52. Philadelphia: Saunders, 1956. 4. Hills BA. The Biology of Surfactant. Cambridge: Cambridge University Press, 1988. 5. Gershfeld NL. Selective phospholipid adsorption and atherosclerosis. Science, 204: 5Of- 8, 1979. 6. Barrow RE, Hills BA. A critical assessment of the Wilhelmy method in studying lung surfactants. .I Physiol, 1295: 217-227, 1979. 7. Hills BA, Barrow RE. The contact angle induced by DPL at pulmonary epithelial surfaces. Respirat Physiol 38:
280 173 - 183, 1979. 8. Hills BA, Butler BD, Lichtenberger LM. Gastric mucosal barrier: hydrophobic lining to the lumen of the stomach. Am J. Physiol: Gastrointest and Liver Physiol. 244: G561-68, 1983. 9. Hills BA. Gastric mucosal barrier: stabilization of hydrophobic lining to the stomach by mucus. Amer J Physiol: Gastrointest and Liver Physiol. 249: G342 - 49, 1985. 10. Mustard JF. Composition and method for correcting foods and blood conditions having clot promoting characteristics. Canadian patent 774, 004, 1958. 11. Harvey EN. Physical factors in bubble formation. In: Fulton JF, ed. Decompression Sickness. pp90 - 114, Philadelphia: Saunders, 1951. 12. Sherman IA. Interfacial tension effects in the microvasculature. Microvasc. Res. 22: 296- 307, 1981. 13. Singer SJ, Nicolson GL. The fluid mosaic model of the structure of cell membranes. Science 175: 720- 31, 1972. 14. Danielli JF, Davson H. A contribution to the theory of permeability of thin films. J Cell Comp Physiol. 5: 495 - 508, 1935. 15. Jaffe EA. Culture and identification of the large vessel endothelial cells. In: Jaffe EA ed, The Biology of Endothelial Cells pp 1 - 13, The Hague: Nijhoff. 16. Neufeld RJ, Zajic JE, Gerson DF. Cell surface measurements in hydorcarbon and carbohydrate fermentations. Appl Environ Microbial 39: 511- 17, 1980. 17. Folkow B. Cardiovascular structural adaptation: its role in the initiation and maintenance of primary hypertension. Clin Sci Molec Med, 55: 3 - 22, 1978. 18. Bloom W, Fawcett DW. A Textbook of Histology. 10th ed. p 402. Philadelphia: Saunders, 1975. 19. Abraham A. The structure of baroreceptors in pathological conditions in man. Proc Symp Baroreceptors and Hypertension. Oxford: Pergamon. 20. Hills BA. Possible role of adsorbed phospholipid in controlling membrane permeability and function. Med Hypoth 28: 85 -92, 1989. 21. Sourirajan S, Matsuura T. Science of reverse osmosis an essential tool for the chemical engineer. Chem Engr, 385: 359-68, 1982. 22. Fane AG, Fell CJD, Kim KJ. The effect of surfactant pretreatment on the ultrafiltration of proteins. Desalination 53: 37-55, 1985. 23. Bradbury M. The Concept of a Blood-Brain Barrier. New York: Wiley. 24. Griffith TM, Edwards DH, Lewis MJ et al. The nature of endothelium-derived relaxing factor. Nature 308: 645 -47, 1984. 25. Furchgott RF, Zawadzki JV. The obligatory role of endothelial cells in the relaxation of arterial smooth muscle by acetylcholine. Nature 288: 373 - 76, 1980. 26. Peach MJ, Loeb AL, Singer HA, Saye J. Endotheliumderived relaxing factor. Hypertension 7: 194- 100, 1985. 27. Dial EJ, Huang J, O’Neill RG, et al. Surface hydrophobicity and water transport of the toad urinary bladder: effect of vasopressin. J Membrane Biol 106: 119- 122, 1988. 28. Bowman WC, Rand MJ. Textbook of Pharmacology. 2nd ed. p23 - 32. Oxford: Blackwell 1980. 29. Rees S. Membranous neuronal and neuroglia inclusions produced by intracerebral injection of suramin. J Neural Sci 36: 97- 109, 1978. 30. Tarapoom N, Royce R, Yorio T. Inhibition of the antidiuretic hormone hydroosmotic response of phospholipids
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31. 32.
33. 34.
35. 36. 37.
38.
39.
40. 41. 42. 43. 44.
45.
46.
47.
48.
49.
50. 51.
52. 53.
HYPOTHESES
and phospholipids metabolites. Lipids 21: 596- 602, 1986. Broman T. quoted by Bradbury (23). Muirhead EE, Kosinski M. Renal medulla and renoprival hypertension: relationship between corticorenal (renin) and medellorenal extracts. Circ Res 11: 647 - 80, 1962. Muirhead EE, Pitcock JA. The renal antihypertensive hormone. J Hypertension, 3: 1- 8, 1984. Pitcock JA, Brown PS, Byers LW et al. Degranulation of renomedullary interstitial cells during reversal of hypertension. Hypertension, 3: (suppl II): 75 - 80, 1981. Macklin CC. The pulmonary alveolar mucoid film and pneumocytes. Lancet i: 1099- 104, 1954. Dintenfass L. Hyperviscosity in Hypertension. Sydney: Pergamon, 1981. Schoen M. Confectionery and cacao products. In: Jacobs MB ed. The Chemistry of Technology of Food and Food Products. Vol III, pp 2138-2140. New York: Interscience, 1951. Hills BA, Butler BD, Barrow RE. Boundary lubrication imparted by pleural surfactants and their identification. J Appl Physiol: Respirat. Environ Exercise Physiol. 53: 463 - 69, 1982. Hills BA, Butler BD. Surfactants identified in synovial fluid and their ability to act as boundary lubricants. Ann Rheum Dis 48: 51- 57, 1984. Hills BA. Oligolamellar lubrication of joints by surfaceactive phospholipid. J Rheumatol 16: 82 - 91, 1989. Hills BA. A physical identity for the blood-brain barrier. Proc Roy Sot NSW (in press). Fahraeus R, Lindquist T. The viscosity of blood in narrow capillary tubes. Amer J Physiol 96: 562, 1931. Schofield RK, Scott-Blair GW. (1930) quoted by Haynes (44). Haynes RH. Physical basis of the dependence of blood viscosity on tube radius. Amer J Physiol 198: 1193- 200, 1960. Fujiwara T, Karawasaki H, Fonkalsrud EW. Reduction of postinfusion venous endothelial injury with intralipid. Surgery, Gynecology and Obstetrics 158: 57 - 65, 1984. Sabatini DD, Bensch K, Barnett RJ. Cytochemistry and electron microscopy: preservation of cellular ultrastructure and enzymatic activity by aldehyde fixation. J Cell Biol 17: 19-26, 1963. Untersee P, Gil J, Weibel ER. Visualization of extracellular lining layer of lung alveoli by freezing-etching. Respirat Physiol 13: 171-85, 1971. Gil J, Weibel ER. Morphological study of pressure-volume hysteresis in rat lungs fixed by vascular perfusion. Respir Physiol 15: 190-213, 1972. Ueda S, Kawamura K, Ishii N et al. Morphological studies on surface lining layer of the lungs. Part VI. Surfactantlike substance in other organs (pleural cavity, vascular lumen and gastric lumen) than lungs. J Jap Med Sot Biol Interface 17:132 - 56, 1986. Robertson JD. The ultrastructure of cell membranes and their derivatives. Symp Biochem Sot 16: 3 - 5, 1959. Oimatov M, L’Vov YM, Aripov TF, et al. Location of cytotoxin in model membranes and its influence on the packing of hydrocarbon lipid chains. X-ray small-angle analysis. Soviet Physics and Crystallography, 31: 942- 50, 1986. Puskas P et al. Controlled, randomised trial of the effect of dietary fat on blood pressure. Lancet l/83: 1 - 5, 1983. Dougherty RM, Galli C, Ferro-Luzzi A et al. Lipid and
MULTIPLE
ROLES FOR SURFACE-ACTIVE
PHOSPHOLIPID
IN HYPERTENSION
phospholipid fatty acid composition of plasma, red blood cells, and platelets and how they are affected by dietary lipids: A study of normal subjects from Italy, Finland and the U.S.A. Amer J Clin Nutr 45: 443 - 55, 1987.
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54. Barcelli UO, Beach DC, Pollak VE. The influence of n-6 and n-3 fatty acids on kidney phospholipid composition and on eicosanoid production in aging rats. Lipids, 23: 309-312, 1988.
