Milos Chvapil

New aspects in the biological role of zinc: a stabilizer of macromolecules and biological membranes.

Abstract It has long been known that zinc is essential to life as an integral part of a number of enzymes (1,2). Increasing evidence also suggests that zinc is important in the stability of macromolecules, particularly the components of various biological membranes. This brief review presents a personal and admittedly incomplete view of the latter aspect of the role of zinc. In this review, I shall combine experimental data with speculations in the hopes of stimulating research along new lines.

Perivascular myofibroblasts and microvascular occlusion in hypertrophic scars and keloids

Microvessels in normal skin, granulation tissue, hypertrophic scar, keloid, and mature scar from human subjects were studied by transmission electron microscopy. Comparative observations suggested that most microvessels in hypertrophic scar and keloid are occluded or partially occluded, apparently owing to an excess of endothelial cells. Endothelial cell contraction was also supported by the observations, and perivascular satellite cells (pericytes), some of which were identified as myofibroblasts, were observed in hypertrophic scars and keloids. Among findings from statistical analyses were that 1) the patency of microvessels in hypertrophic scar and granulation tissue is similar, as is that of microvessels in keloid and mature scar, but the patency of all these microvessels is significantly less than that of microvessels in normal skin, and 2) endothelial cell density is greater in nonpatent vessels than in patent vessels. The observed extent of microvascular occlusion supports a previously published theory that hypoxia is involved in the generation of hypertrophic scar.

Influence of shock waves on fracture healing

During the last decades the influence of physical factors on fracture healing has been widely described. With the use of shock waves for the treatment of urolithiasis, a new mechanical medium has been introduced into medicine. For the first time the influence of shock waves on fracture healing was studied in rats. With fractioned shock-wave treatment (5 times 100 shock waves at 14 or 18 kV) an enhancement in healing could be achieved.

Identification of the depth of burn injury by collagen stainability.

Heat-denatured collagen in burned skin stains red instead of blue in Massons trichrome stain. This change in stainability corresponds to the loss of birefringence in slides examined in polarized light. The depth of the abnormal staining of the skin slices was proportional to the time and temperature of the heat exposure. It is concluded that the change in collagen stainability from blue to red relates to the loss of crystallinity or parallel alignment of the collagen fibers. It is further proposed that change in the stainability of collagen in the burns could be used to delineate the depth of the thermal skin injury or the effectiveness of the surgical excision or debridement of the wound by dressing materials.

Protective effect of zinc on carbon tetrachloride-induced liver injury in rats

In rats treated for 20 or 34 days with CCl4 (0.2 ml/100 g, ip, twice weekly) a significant increase in the content of malonaldehyde product in microsomal and mitochondrial fractions of the liver occurred. The raise in this indirect index of lipid peroxidation correlated with labilization of lysosomes, as evidenced by the increased portion of free β-glucuronidase present in the 15,000g supernatant of the liver homogenate. At the same time, the amount and the synthesis of collagen was significantly elevated. After zinc acetate administration (5 mg/100 g/day, intragastrically) to rats receiving CCl4, the content of malonaldehyde in the microsomal, as well as the mitochondrial fraction of the liver, was significantly lower. This correlated with a decreased portion of free β-glucuronidase and a decrease in the amount and in the rate of biosynthesis of collagen. Administration of zinc to normal rats had no effect on the amount and synthesis of collagen in the liver. The free and total activity of lysosomal β-glucuronidase in the liver was also unaltered by zinc in normal animals. The level of malonaldehyde was inhibited only in the microsomal fraction. In rats kept on a diet high in zinc (1000 ppm), the content of endogenously-, as well as exogenously-induced malonaldehyde in the liver was lower. Some factors influencing the content of malonaldehyde in intact rat liver are described. We believe that zinc protects the liver against the noxious effect of CCl4 primarily by interference with lipid-peroxidation-related tissue damage. Other possibilities of the protective effect of zinc administration in tissue injury have not been ruled out.

The Effect of Zinc and Other Metals on the Stability of Lysosomes

Summary The effects of zinc, certain other metals (Cu, Hg, Pb, Cd, Ni and Au), 8-hydroxyquinoline (8-HQ), and various stoichiometric complexes of Zn:8-HQ on the release of β-glucuronidase from lysosomes isolated from rat liver were tested. Zinc and Zn:8-HQ (1:1 and 1:2 complexes) markedly stabilized lysosomes in a concentration-related fashion. The saturated 1:3 complex and 8-HQ alone showed a labilizing effect, while the nonchelating substitute of 8-HQ (8-methoxyquinoline) did not affect lysosomal stability. Cd and Pb also stabilized lysosomes, although they were both less effective than zinc. The distribution of zinc between the membrane and the interior of isolated liver lysosomes is 2:1. After treatment with zinc, the distribution is unchanged but the total content of zinc increased 10-fold. In samples treated with unsaturated complexes of Zn:8-HQ (1:1 or 1:2) the total amount of zinc present is the same as in zinc-treated particles, but the metal is bound exclusively to the membrane fraction. The effect of pH on lysosomal fragility in the presence of zinc was almost opposite to that observed when calcium was tested. The results of this study favor the concept that the stabilizing effect of zinc occurs at the surface of the membrane. The effect does not seem to be related to the function of known phospholipases.

Effect of shock waves on the healing of partial-thickness wounds in piglets

During the last 20 years, the role of various physical factors in wound healing has been widely studied and recognized. With the use of shock waves for the treatment of urolithiasis, a new mechanical medium has been introduced into medicine. The influence of shock waves on the reepithelialization of partial-thickness wounds was studied in four Yorkshire piglets by a quantitative morphometric method. Wounds were inflicted either in intact skin (three pigs) or in skin irradiated with 1500 rads to achieve delayed healing. A significant enhancement in normal or delayed healing was found with low-dose treatment (10 SW at 14 kV). High-dose application of shock waves (100 SW at 18 kV) resulted in inhibition of the rate of reepithelialization of the wounds. Shock waves of intermediate energies were without effect. The stimulating effect of low-energy shock waves coincides with significantly increased vascularization of the upper dermis and thicker layer of the newly formed epithelial cells covering the wound.

Effect of zinc on lipid peroxidation in liver microsomes and mitochondria.

While studying the role of certain metals and chelating agents in controlling tissue injury, we found that zinc salts stabilize lysosomal membranes in vitro (1, 2): the rupture of these particles with the resulting release of their enzymes was decreased roughly 50% by low (millimolar) concentrations of zinc. The lysis of cells or subcellular structures is frequently attributed to peroxidation of the lipid components of their membranes (3-5). Thus, one possible explanation of this effect of zinc is that it interferes in some way with peroxidation of unsaturated fatty acids in the lysosomal membrane. This hypothesis was evaluated in in vivo and in vitro experiments using carbon tetrachloride to induce lipid peroxidation. The toxic effects of CCl4 in vivo are due to the metabolism of this agent by the liver microsomal drug oxidizing system to the trichloromethyl radical CCl3 (6). This free radical attacks unsaturated lipids in intracellular membranes, oxidizing them and causing membrane distortion. This process terminates in cell necrosis (7). Lipid peroxidation can also be induced in vitro by incubating isolated liver microsomes with CCl4 in the presence of NADPH or a NADPH-generating system (8). We have found that zinc prevents or significantly reduces the CCl4-induced formation of lipid peroxides both in vivo and in vitro. Methods. Male rats (Simonson, 200 ± 15 g) fed standard Purina lab chow diet were injected sc twice weekly with CCl4 at the dose 0.1 ml/100 g. Zinc acetate (5 mg/100 g) was administered daily by intragastric gavage. Control rats were gavaged accordingly with saline. Rats were sacrificed by decapitation after 20 and 34 days. The livers were perfused in situ with 20 ml of ice cold saline and homogenized by hand with all-glass homogenizers in three volumes of Tris-KCl buffer (0.05 M, pH 7.4).

Effects of selected chelating agents and metals on the stability of liver lysosomes

Abstract Diethyldithiocarbamate (DDC), penicillamine and sodium ethyldiaminetetraacetate (Na-EDTA) in 1 mM concentrations significantly stabilized the membrane of isolated liver lysosomes, as shown by a decreased release of β-glucuronidase upon incubation at 37° and pH 5·0. Thiol compounds which are readily auto-oxidized (cysteine, glutathione and dithiothreitol) did not affect lysosomal fragility when subjected to 1 mM concentration; at a higher concentration (5 mM), these substances increased the release of β-glucuronidase. Stabilization by DDC and penicillamine, both of which contain sulphydryl-like groups, may be related to their nonauto-oxidizability, although their chelation effect cannot be excluded. Various chelating agents relatively specific for Fe 2+ or Fe 3+ , such as l,10-phenanthroline, 5,6-dimethyl-l,10-phenanthroline, 2-2′-dipyridyl, Desferal, and pyrocatechol-3,5-disulfonic acid (Tiron), had no effect on lysosomal stability. Zinc ions (0·05–2·5 mM) significantly stabilized lysosomal membranes; this stabilization was concentration dependent, was not affected by thiol compounds, and was potentiated by equimolar complex with 8-hydroxyquinoline (8-HQ) ; 8-HQ alone (1 mM) labilized lysosomes. Calcium ions had no effect, whereas Cu 2+ and Hg 2+ labilized the lysosomes. Labilization by Cu 2+ was reversed by chelating agents and decreased by thiol compounds. Incubation of lysosomes in nitrogen and dark prevented labilization by copper, and did not affect stabilization by zinc. The effects of certain chelating agents (8-HQ, DDC, penicillamine, EDTA) or metals (Zn 2+ , Cu 2+ , Hg 2+ ) on the stability of the lysosomal membranes are discussed in terms of the reactivity of these agents with components of the membrane, or interference with metal-catalyzed lipid peroxidation.

Effect of electrocautery on wound healing in midline laparotomy incisions

The effect of electrocautery on midline fascial wound healing was studied in 108 Sprague-Dawley rats. Midline wound tensile strength was significantly reduced in fascia incised with the coagulation current compared with the cutting current or scalpel. In addition, tissue necrosis and inflammation as well as adhesion formation between the incision and abdominal viscera were more extensive in animals with incisions made using coagulation current. The results of the study indicate that the use of electrocautery coagulation current is associated with increased tissue damage and a significant reduction in the tensile strength of healing wounds. The contribution of electrocautery to wound complications in patients needs further evaluation.