Faculty Bibliography

The alveolar surfactant is a prime target of reactive oxygen species present in air. Alveolar surfactant is supplemented with vitamin E during its assembly in type II pneumocytes. However, it is unknown which of the lipoproteins supply type II pneumocytes with vitamin E. The measurement of the uptake kinetics indicates that HDL might be the primary source of the vitamin E uptake by type II pneumocytes. Vitamin E depletion of rats caused an increase of vitamin E uptake by isolated type II pneumocytes from HDL but not from LDL or VLDL. We demonstrated that type II pneumocytes express the scavenger receptor class B type 1 (SR-B1), an HDL-specific receptor. Vitamin E depletion caused an increased expression of SR-B1 by a post-transcriptional mechanism. The increased vitamin E uptake from HDL and the increased expression of the SR-B1 were reversed by refeeding the vitamin. We propose that HDL is the primary source of vitamin E for type II pneumocytes. The rate of uptake of vitamin E by this cell type might be regulated by the expression of SR-B1

Cardiolipin is a specific mitochondrial phospholipid that is present in mammalian tissues in low concentration. To measure cardiolipin in small biopsies from patients with mitochondrial disease, we developed a new technique that can detect subnanomolar levels of well-resolved molecular species, the most abundant of which are tetralinoleoyl-cardiolipin (L(4)) and trilinoleoyl-oleoyl-cardiolipin (L(3)O). To this end, a fluorescence-labeled derivative of cardiolipin (2-[naphthyl-1'-acetyl]-cardiolipin dimethyl ester) was formed and analyzed by high performance liquid chromatography. Cardiolipin was measured in skeletal muscle biopsies from 8 patients with mitochondrial disease and in 17 control subjects. In 5 patients with mitochondrial disease, cardiolipin content was higher than normal (2. 4;-7.0 vs. 0.4;-2.2 nmol/mg protein). In 3 patients with mitochondrial disease, the L(4)/L(3)O ratio was lower than normal (2;-4 vs. 4;-6). Cardiolipin was also measured in various rat and dog muscle tissues. The L(4)/L(3)O ratio was higher in condensed 'muscle' type mitochondria (heart ventricle, skeletal muscle, ratios 4;-7) than in orthodox 'liver' type mitochondria (liver, smooth muscle, heart auricular appendage, H9c2 myoblasts, ratios 0.4;-3), suggesting that the L(4)/L(3)O proportion is important for cristae membrane structure. We concluded that the L(4)/L(3)O ratio is a tissue-specific variable that may change in the presence of mitochondrial disease. The new method is suitable to measure cardiolipin in muscle biopsies in order to estimate concentration of mitochondria

Oxidation of unsaturated phosphatidylcholine (PC) produces fragmented phospholipids which have similar bioactivities as the platelet-activating factor (PAF, 1-O-alkyl-2-acetyl-PC). Since a large number of molecular species are produced upon PC oxidation, the active ingredients have not been identified. We synthesized several short-chain PCs which are known to be characteristic PC oxidation products to test their PAF-like activity. The synthetic PCs contained palmitoyl or hexadecyl residues (both C16) in sn-1 position, and propionyl (C3), valeroyl (C5), succinyl (C4 with omega-carboxyl), glutaroyl (C5 with omega-carboxyl), or suberoyl (C8 with omega-carboxyl) residues in sn-2 position. Biological activity was measured by: (1) increase of intracellular calcium in human monocytes; (2) [3H]serotonin release from rabbit platelets; and (3) aggregation of human platelets. Specificity of the cellular response was tested by inhibition with the PAF-receptor antagonists BN 52021 and WEB 2086. Synthetic PC oxidation products activated both monocytes and platelets in a PAF-specific manner. The effective concentration varied with respect to assay system and chemical structure. In general, 1-hexadecyl-PCs were more effective than 1-palmitoyl-PCs, while increasing chain length in sn-2 position lowered biological activity. However, several 1-palmitoyl-PCs activated monocytes in concentrations between 10-8 and 10-6 M. In contrast, platelets were less susceptible to 1-palmitoyl-PCs. No significant difference was found between 2-valeroyl-PC (C5 with omega-methyl) and 2-glutaroyl-PC (C5 with omega-carboxyl). The data suggest that typical products of PC oxidation, containing propionyl, succinyl, or glutaroyl residues in sn-2 position, display PAF-like activity at micromolar concentrations

After operations with cardiopulmonary bypass, patients often show early symptoms of the systemic inflammatory response syndrome (SIRS). Potential mediators of SIRS include the platelet-activating factor (PAF), which has been linked to septic shock and multiple organ dysfunction. We studied the effect of cardiac surgery on PAF acetylhydrolase, the PAF-degrading plasma enzyme, as well as the relationship between the enzyme and the postoperative state of the patients. PAF acetylhydrolase activity decreased by 38+/-8% after instituting cardiopulmonary bypass because of plasma dilution and returned to near-preoperative levels within 6 h postsurgery. After that, enzyme levels decreased again, resulting in a 24+/-12% reduction until at least 3 days postsurgery. Patients in poor postoperative condition (Acute Physiology Score >9) had a lower preoperative PAF acetylhydrolase activity than did normal patients (12+/-4 vs. 17+/-4 nmol min(-1) mL(-1); p < .05). Likewise, patients who developed postoperative SIRS had a lower preoperative PAF acetylhydrolase activity than did patients without SIRS (12+/-3 vs. 17+/-4 nmol min(-1) mL(-1); p < .05). The data suggest that PAF acetylhydrolase deficiency is among the factors associated with postoperative distress after cardiac surgery

alpha-Tocopherol transfer protein (alpha-TTP) supplements nascent very-low-density lipoprotein (VLDL) preferentially with alpha-tocopherol by selecting the alpha-isomers against other stereoisomers of tocopherol. It is exclusively expressed in liver. We investigated whether the expression of the hepatic alpha-TTP can be induced by dietary tocopherols. Vitamin E-depleted rats were fed with a diet containing alpha- and delta-tocopherol (ratio 1:3). The expression of alpha-TTP mRNA was measured in liver tissue. The ratio of tocopherol stereoisomers was determined in plasma, plasma lipoproteins and tissues to measure the metabolic action of alpha-TTP. Refeeding a diet containing either alpha- or delta-tocopherol, or both, caused a steady increase of the expression of alpha-TTP mRNA. In parallel the alpha/delta-tocopherol ratio increased in plasma, VLDL, high-density lipoprotein and low-density lipoprotein as well as in liver tissue, when the diet was fed containing both isomers. The alpha-tocopherol/delta-tocopherol ratio of heart, kidney, lung, lamellar bodies of lung and in lung lavage showed no or a comparatively low increase. The data show that both tocopherol isomers were able to induce alpha-TTP mRNA in rat liver and, thus, the ability of liver to select for the alpha-isomer. On the other hand, tocopherol depletion did not change the expression of hepatic alpha-TTP mRNA in the rat

The phospholipid cardiolipin (CL) is ubiquitous in eucaryotes and is unique in structure, subcellular localization, and potential function. Previous studies have shown that CL is associated with major respiratory complexes in the mitochondrial membrane. To determine whether CL biosynthesis requires the presence of intact respiratory complexes, we measured activity of CL synthase, which catalyzes the synthesis of CL from cytidine diphosphate diacylglycerol and phosphatidylglycerol, in Saccharomyces cerevisiae strains with genetic defects in the oxidative phosphorylation system. Assembly mutants of cytochrome oxidase had significantly reduced CL synthase activity, while assembly mutants of respiratory complex III and the F0F1-ATPase were less inhibited. To obtain further information on the activity of CL synthase, we purified the enzyme and compared the size of the catalytic protein with the functional molecular mass. The enzyme was solubilized by Triton X-100 from KSCN-extracted mitochondrial membranes of S. cerevisiae. The functional molecular mass of Triton-solubilized CL synthase, determined by radiation inactivation, was 150-240 kDa, indicating that the functional enzyme was a large complex. After partial purification, the enzyme eluted from a Superose 12 gel filtration column with an apparent molecular mass of 70 kDa. CL synthase was further purified by hydroxylapatite and cytidine diphosphate diacylglycerol affinity chromatographies, Mono Q anion exchange FPLC, and preparative gel electrophoresis. These steps led to identification of a 28-kDa protein, which had catalytic activity when eluted from an SDS-polyacrylamide gel. This 28-kDa protein also reacted with an antiserum that inactivated the enzyme. We conclude that yeast CL synthase is a 28-kDa protein, which forms an oligomeric complex whose biogenesis and/or activity is influenced by the assembly of cyto-chrome oxidase

Cardiolipin was first isolated from beef heart and was shown to contain an unusually high content of linoleic acid ester residues. Cardiolipin is found throughout the eukaryotes including animals, plants and fungi. In mammalian tissue and in yeast, cardiolipin is found exclusively in mitochondria. Mitochondrial synthesis of cardiolipin utilizes phosphatidylglycerol and CDP-diacylglycerol as substrates in a reaction which requires a divalent cation (Mg2+, Mn2+ or Co2+). Cardiolipin synthase has been purified to near-homogeneity from rat liver by solubilization with Zwittergent 3-14 followed by FPLC anion exchange, gel permeation and chromatofocusing steps. Cardiolipin synthase has a molecular mass of 50 kDa, a pH optimum of 8.0, and requires added phospholipids (phosphatidylethanolamine and cardiolipin) and 4 mM Co2+ for optimal activity. Except for the effects of divalent cations and the requirement for phospholipids, little is known about the regulation of cardiolipin synthase. Cardiolipin deficiency in aging mitochondria has been linked to decreased metabolite transport across the inner membrane. Both cardiolipin levels and cardiolipin synthase activity are increased in hyperthyroidism and decreased in hypothyroidism suggesting regulation by thyroid hormone. Mammalian cardiolipin synthase has not been sequenced or cloned and its biological role in mitochondria is not yet fully understood

Cardiolipin synthase catalyzes the synthesis of the mitochondrial phospholipid cardiolipin. Cardiolipin synthase is a unique membrane-bound enzyme in that it utilizes two phospholipids, both insoluble in water, as substrates. Kinetic analysis suggests that the enzyme forms a ternary complex with the two lipid substrates, and that a divalent metal ion directly associates with cardiolipin synthase to form the active enzyme. While little is known about the regulation of cardiolipin synthase in yeast, activity is reduced in mutants in which the mitochondrial genome is deleted, and in mutants with defective respiratory complexes. In p0 mutants, which contain no mitochondrial DNA and are defective in the assembly of many mitochondrial membrane protein complexes, cardiolipin synthase activity is reduced by 50%. Mutants defective in respiratory complexes, particularly those incapable of cytochrome oxidase assembly, also have reduced cardiolipin synthase activity. Thus it is likely that respiration and cardiolipin formation are interdependent. The enzyme was recently purified from the budding yeast Saccharomyces cerevisiae. Enzyme activity was associated with a 25-30-kDa protein. The amino acid sequence of this protein, combined with the availability of the complete yeast genome sequence, will hopefully lead to the identification of the structural gene for this enzyme in the near future

Experimental and clinical studies have provided evidence for the involvement of oxygen free radicals in development of acute and chronic lung diseases. Hyperoxia is very often an indispensable therapeutic intervention which seems to impose oxidative stress on lung tissue. We measured the effect of hyperoxia (80% O2 for 20 h) (1) on the lipid composition of pulmonary surfactant treated in vitro, (2) on surfactant lipid synthesis and secretion of type II pneumocytes in primary culture, (3) on the lipid composition and on the SP-A content of rat lung lavages and (4) on the turnover of phospholipids, cholesterol, plasmalogens and vitamin E in type II pneumocytes, lamellar bodies and lavages of adult rat lungs. (1) Hyperoxia of lung lavages in vitro reduces the vitamin E content significantly but does not change the relative proportion of PUFA or the content of plasmalogens. (2) Hyperoxia does not affect the biosynthesis or secretion of surfactant lipids and plasmalogens by type pneumocytes in primary culture. (3) Hyperoxic treatment of rats increases the SP-A content and reduces the vitamin E content significantly but does not change the concentration of other lipid components of lung lavage. (4) The vitamin E turnover, measured in type II pneumocytes, lamellar bodies and lung lavages, is increased 2-fold in these fractions. In contrast, the turnover of surfactant cholesterol and surfactant lipids does not change. (5) Hyperoxia caused an increase of the vitamin E uptake by type II pneumocytes resulting in a vitamin E enrichment of lamellar bodies. From these results we conclude that type II pneumocytes are able to regulate the turnover of lipophilic constituents of the alveolar surfactant independently of each other. Hyperoxia caused type II pneumocytes to increase the vitamin E content of lamellar bodies. The lipid and SP-A content of alveolar fluid can be regulated independently each other