Lipoids

LIPOIDS, a group of substances, differing in their chemical structure, including fats and substances resembling fats in certain physical and chemical properties, especially solubility, namely phosphatides, sterols, cerebrosides, lecithin, and cholesterol (see). According to the nomenclature adopted by the International Commission for the Reform of Biological-Chemical Nomenclature, fats and other complex esters close to fats, sterols, and phosphatides form the class of lipids. Simple lipids include: 1) glycerides (fats belong here); 2) cerides, containing higher monohydric alcohols instead of glycerol (wax, spermaceti, etc.); 3) sterides (cholesterol and other sterols). Complex lipids include phosphoaminolipids (phosphatides), subdivided into: 1) glycerophosphoaminolipids (lecithins, cephalin) and 2) sphingophosphoaminolipids, containing the unsaturated dihydric amino alcohol sphingosine (sphingomyelin). Cerebrosides, according to this nomenclature, belong not to lipids, but to heteroglycosides.

Physiological role of L. is extremely diverse. Representing an obligatory, vital component of cellular protoplasm, they have fundamental significance for the function of the latter, with cholesterol, in contrast to lecithins, not exhibiting species specificity. Lipoids, as surface-active substances, accumulate in the boundary layer of cells and exert a significant influence on the metabolism in cells. In this, the significance of hydrophobic cholesterol and hydrophilic lecithin are directly opposite. The presence of lecithin, capable of swelling and increasing the bioelectric potential difference on cell surfaces, facilitates the penetration of substances into cells. The presence of cholesterol, possessing opposite properties and acting as a kind of "insulator" of cells, lowers their permeability. The views of Overton, that cell permeability depends on the solubility of substances in the lipoids of the boundary cell layer, are currently abandoned. The significance of the boundary layer, rich in lipoids, for cell permeability is explained by the processes of swelling and bioelectric phenomena occurring within it.

The particularly important significance of lipoids for the entry of substances into the cell is conditioned by adsorption phenomena occurring at the water-lipoid boundary layer (Traube). Many substances that do not possess surface activity at the water-air interface exhibit such at the water-lipoid interface and therefore, according to the theory of Gibbs, accumulate at this interface (Okunev). The indicated antagonistic influence of cholesterol and lecithin manifests itself in very many processes. Therefore, physiologically very important are the constants (so-called lipocytic coefficients) (Mayer and Schaeffer, Terroine and Weill), as well as the cholesterol/cholesterol-ester ratio. It has long been known that lecithins and cholesterol have an antagonistic effect during hemolysis: lecithin possesses an activating role in this, while cholesterol possesses an inhibitory role (see Hemolysis). These lipoids act in the same direction regarding certain enzymes, in particular lipase (Jagic, Remezov), as well as regarding blood coagulation (Dorle, Szenes). An important role belongs to lipoids, and especially cholesterol, in the process of muscle contraction (Embden). For example, the presence of cholesterol significantly enhances the action of adrenaline on smooth musculature (Westphal and Herrmann). The process of gas exchange in muscles depends to a significant degree on the presence of lipoids in their boundary layer (Embden and Lange). Lecithin, easily undergoing swelling, contributes to the enhancement of gas exchange; cholesterol apparently exerts the opposite effect. The presence of a lipoid boundary layer is attributed important significance in explaining the phenomenon of narcosis.

The presence of lipoids in plasma influences the erythrocyte sedimentation rate, whereby cholesterol, by lowering the surface charge of erythrocytes, accelerates this process, while lecithin retards it. The phagocytic ability of leukocytes is weakened in the presence of cholesterol and strengthened under the influence of lecithin and cerebrosides (Stuber, Rothschild). The ability of lipoids, especially cholesterol, to bind certain poisons (e.g., saponin, cobra venom) likely explains in part their favorable influence on the course of certain infections, although this is far from constant. One should also note the important significance of lipoids in certain immune reactions, which is connected with the production of immune bodies. By regulating the permeability of animal membranes, lipoids take part in the exchange of water in tissues. A significant content of cholesterol in membranes, by causing electrical insulation of tissue elements, retards the movement of ions and thus causes the accumulation of water in tissues. The accumulation of lecithin causes the opposite effect (Brinkmann and van Damm). Connected with the influence of lipoids on tissue metabolism is their significance for the process of growth (e.g., for the growth of tumors; Robertson and Burnet, Borst). Especially important is the significance of lipoids as carriers of certain vitamins (vitasterols) (see Vitamins, Avitaminoses). The rapid death of animals when fed food devoid of lipoids (Stepp) depends on the lack of vitamins. During starvation, the ratio of cholesterol to phosphatides does not change, but a decrease in higher fatty acids occurs in tissues, and the ratio of cholesterol to fatty acids falls, especially in muscles. Finally, the action of certain hormones, especially the sex hormone, is apparently also closely connected with the presence of lipoids. Furthermore, cholesterol retards the action of insulin and enhances the action of adrenaline. By lowering surface tension, lipoids facilitate the emulsification of neutral fat, whereby lecithins favor the formation of an emulsion of fat in water, and cholesterol favors the dispersion of water in fat. One or another type of emulsification therefore depends on the ratio of cholesterol and lecithins in the body's fluids, which in turn influences the physical-chemical state of fat depots.

Not having significance as energy material in the body, lipoids are a necessary component of intermediary fat metabolism. According to Walker, Hueck, and Leathes, the breakdown of neutral fat occurs in such a way that the fatty acid radical from the triglyceride combines with cholesterol to form cholesterol ester; the remaining distearylglyceride forms lecithin with choline-phosphoric acid, which easily undergoes oxidation; cholesterol ester, reacting with glycerol, can again form neutral fat and free cholesterol. The question of the synthesis of lipoids in the animal organism can apparently be considered clarified at the present time in the positive sense (Wessner and Lehmann, Gardner, et al.). The source of cholesterol formation is still unclear; apparently, derivatives of bile acids (Thannhauser) and possibly higher fatty acids (Reicher, Leites) have significance in this regard. Likewise, it is not clarified whether the breakdown of cholesterol occurs in the body; the opinion that bile acids are products of cholesterol cleavage must still be considered unproven. As for phosphatides, the latter can be cleaved in the body into their components. The cycle of lipoids in the body and the role of individual organs in it still present much that is unclear. A central role apparently belongs to the liver; in it occurs the accumulation and resynthesis of lecithins (Franchini, Eichholtz); their breakdown also occurs there (Leathes). In the liver, processes of cholesterol accumulation (Anichkov, Khalatov), its esterification (Thannhauser), partial excretion by bile, and possibly synthesis (Artom, Lombroso, Reicher) also take place. Among other organs having significance in the processes of accumulation and apparently synthesis of cholesterol and lecithins, one should note the spleen, the cortical layer of the adrenal gland, the lungs, as well as cells of the so-called reticulo-endothelial system; adipose tissue plays an important role as a depot for lipoids. It is necessary to emphasize that the cycle of lipoids in the body is closely connected with the cycle of fats, starting from the processes of absorption in the intestine (cholesterol is absorbed only in the presence of fat). During passage through organs, quantitative changes in lipoids are always accompanied by certain quantitative changes in fats (Reicher, Leites). The regulation of lipoid metabolism and the role of the endocrine and vegetative nervous systems in it are an almost uninvestigated area. Individual works concern mainly blood lipoids and do not allow for definite conclusions, especially since it is not possible to establish whether changes in lipoids are primary or (which is more likely) appear secondary, depending on changes in fat metabolism. The scheme put forward by Dresel and Sternheimer: sympathicus - cholesterol, vagus - lecithin, is disputed (Weiss and Paul) and is far from proven. Under pathological conditions, the deposition of lipoids (lipoidosis) in tissues (both in cells and in the interstitial substance) is observed frequently, and usually mixtures of various lipoids are observed. In the presence of degenerative phenomena in the corresponding tissues, one sometimes speaks of lipoid degeneration (see Fatty degeneration).

S. Leites. Methodology of histological examination of lipoids. The study of lipoids in tissues can be performed either by osmium treatment of small pieces of tissue before they are sectioned, or by various staining methods of the latter.* Osmium treatment is performed by placing freshly cut and, if possible, small pieces of tissue into fixatives containing osmium. The most commonly used mixtures are: a) Flemming's fluid: 1% chromic acid - 15 cm³, 2% osmic acid - 2 or 4 cm³, glacial acetic acid - 6-10 * Below, under the name "lipoids," according to the terminology of Ivar Bang, all fats in general are implied.

drops; b) Altmann's fluid: 5% solution of potassium dichromate, mixed before use with an equal volume of 2% osmic acid. Fixation continues for 1-3 days, after which follows a daily washing in running water, embedding in celloidin or paraffin, and sectioning. Most lipoids are stained black during this process. Not only the pieces themselves, but also the sections obtained from them (e.g., with preliminary fixation of the pieces in formalin) can be subjected to the action of osmic acid. At the present time, osmium is rarely used for studying lipoids contained in tissues, mainly because, on the one hand, not all types of lipoids are revealed by this method, and on the other hand, a number of structural formations that have nothing in common with lipoids take on a black stain. The most commonly used method at the present time is the staining of sections obtained by freezing. Pieces of tissue up to 1/2-1 cm thick are fixed for 1-2 days in a 20% formalin solution (1 part commercial formalin to 4 parts tap water), after which they are sectioned on a freezing microtome into sections 10-15 microns thick. To detect the total amount of lipoids, the sections are stained with a solution of Scharlachrot or Sudan III (a saturated solution of the dye in a mixture of equal volumes of 70% alcohol and acetone). Staining lasts 10-15 minutes, after which the sections are rinsed in water and counterstained with hematoxylin. Lipoids are stained in various shades of red and reddish-yellow (see table). For preservation, the sections are mounted in Apathy's gum syrup (50 cm³ of water, 50 g of gum arabic, 20 g of powdered sugar, and a crystal of thymol) or in glycerin-gelatin. Gum syrup makes it possible to obtain more durable and permanent preparations (the section is picked up from the water onto a microscope slide, slightly dried, a drop of gum syrup is placed on it, and it is covered with a coverslip). To differentiate various groups of lipoids, there are a number of methods, of which the following are of the greatest importance. Staining with Nile blue sulfate (saturated aqueous solution of the dye); sections are stained for 10-15 minutes, rinsed in water, and differentiated (preferably under microscopic control) in a weak (1-5%) solution of acetic acid. Neutral fats are stained pinkish-red, cerebrosides and phosphatides - bluish-blue, soaps and fatty acids - dark blue. The stained sections are mounted in gum syrup and examined immediately after the preparation is made; after some time, the staining may change quite significantly. Staining with Nile blue sulfate is important for preliminary orientation in the qualitative composition of the lipoids present in the tissue. Fischler's method is used to detect fatty acids and soaps. Sections fixed in a 20% formalin solution are placed for a day in a mordant (saturated aqueous solution of copper acetate), then washed in water and stained with hematoxylin, prepared as follows: hematoxylin - 1.0, absolute alcohol - 10 cm³, saturated solution of lithium carbonate - 1 cm³, water - 9 cm³. After washing in water, the sections are differentiated with Weigert's fluid (borax - 2.0, potassium ferricyanide - 2.5, water - 100 cm³), washed in water, and mounted in gum syrup. Fatty acids are stained blue-black. Another piece of the same tissue is fixed in 20% formalin saturated with calcium salicylate. Sections from such pieces are stained by the method just described, in which case, in addition to fatty acids, soaps are also stained blue-black. By comparing the preparations obtained using the various fixation methods described, one can form an idea of the ratio of fatty acids and soaps. The remaining lipoids can be counterstained on the same sections with Scharlachrot. Ciaccio's method is based on preliminary chroming of tissue pieces, after which part of the lipoids is not extracted when the pieces are passed through alcohols and xylene. After fixing the piece for 2 days in a mixture of 80 parts of 5% potassium dichromate, 20 parts of formalin, and 5 parts of acetic acid, it is transferred for several (5-6) days into a 3% solution of potassium dichromate, washed with water for a day, and embedded in paraffin via xylene. The sections, freed from paraffin, are stained with Sudan. Phosphatides and some of their mixtures, fatty acids, soaps, and some other lipoids take up the stain (see table). Smith-Dietrich method. Sections obtained by freezing are chromed for 48 hours in a thermostat at 37° (in a saturated solution of potassium dichromate), after which, having been previously washed, they are stained at 37° for several hours in Kulchitsky's hematoxylin and differentiated in Weigert's fluid (see Fischler's method above). Mixtures of phosphatides, soaps, and fatty acids with cholesterol esters are mainly stained blue-black (see table). Staining with Neutral red (saturated solution) can be used to detect soaps, fatty acids, and phosphatides, which are stained red (see table). Benda's method can be used to detect soaps and fatty acids (and some of their mixtures). The method was proposed for staining necrosis of adipose tissue. Pieces fixed in formalin are cut on a freezing microtome and placed for 2 days in a thermostat at 37° in Weigert's mordant for neuroglia (fluorochrome and copper acetate). Soaps and fatty acids formed at the site of fat necrosis are stained green. Examination using a polarizing microscope makes it possible to identify, first of all, cholesterol and cholesterol esters. They possess birefringence, which disappears upon heating and reappears after cooling. Some phosphatides possess birefringence that does not disappear upon heating. Neutral fats, soaps, and fatty acids do not possess birefringence (see table). Examination with the help of a polarizing microscope can be performed both on unstained sections placed in a drop of water under a coverslip and on permanent preparations stained with Nile blue sulfate. Although the above-mentioned methods do not allow for the determination of the exact chemical composition of lipoids under a microscope, they do, in the aggregate, make it possible to orient oneself as to which main groups of lipoids one is dealing with (see table). If it is necessary to examine lipoids on smears, the latter are fixed with formalin vapors and stained with Scharlachrot or Nile blue sulfate. Some lipoids can be detected in the form of shiny globules and grains in a drop of liquid placed under a coverslip. They can be stained by adding a few drops of Scharlachrot.

S. Weil.