Thomas P. Krick

Furanoid fatty acids from fish lipids

Fatty acids, recently reported as constitutents of certain fish lipids, were identified to be derivatives of furan (furanoid fish fatty acids). 12,15-Epoxy-13,14-dimethyleicosa-12,14-dienoic acid is predominant among the furan acids and is associated withbis-homologs in regard to chain length. Monomethyl acids, such as 12,15-epoxy-13-methyleicosa-12,14-dienoic, are present in appreciable amounts. The structures were concluded from oxidative degradations, from mass spectrometry of methyl esters of the novel acids and fatty acids derived from them by opening the ring, and from nuclear magnetic resonance, infrared, and Raman spectra. The results from chemical procedures and from spectrometric methods were in aggreement with those obtained with authentic methyl 9,12-epoxyoctadeca-9,11-dienoate. The number of substituents at the furan ring greatly influences hydrogenation, hydrogenolysis, and hydrolysis reactions of the ring.

New series of fatty acids in Northern Pike (Esox lucius)

The presence in Northern Pike (Esox locius) liver and testes lipids of a group of eight homologous fatty acids of as yet unknown structure is reported. They occur esterified to cholesterol and to glycerol as triglycerides but are absent from the phospholipids. They contain three oxygens and are characterized further by being more resistant to hydrogenation than normal unsaturated fatty acids and by an inability to form urea inclusion compounds. They also have been found to be major constituents of the liver fatty acids of four additional species of fish.

The occurrence and distribution of furan fatty acids in spawning male freshwater fish.

Furan fatty acids (F acids) have been found in the livers and/or testes of 20 species, representing 9 families of male freshwater fish. In 9 species they are major components of the lipids while in the remaining 11 species they occur to a much lesser extent. The F acids in some species reach a maximum concentration in the testes lipids, and minimum liver lipid concentration, at spawning. In all species in the testes, the F acids are confined almost exclusively to the triglyceride fraction while, in the liver lipids, they are found, in order of decreasing concentration, in the cholesteryl esters, the triglycerides, and the phospholipids. In the lipids of many individuals F6 12,15-epoxy-13,14-dimethyleicosa-12,14-dienoic acid, is the major fatty acid present. It is presumed that these acids perform some as yet unidentified metabolic function. Isolation technology and identification of F acids by a specific thin layer chromatographic spray reagent are discussed.

Posttranslational modifications in the CP43 subunit of photosystem II

Photosystem II (PSII) catalyzes the light-driven oxidation of water and the reduction of plastoquinone; the oxidation of water occurs at a cluster of four manganese. The PSII CP43 subunit functions in light harvesting, and mutations in the fifth luminal loop (E) of CP43 have established its importance in PSII structure and/or assembly [Kuhn, M. G. & Vermaas, V. F. J. (1993) Plant Mol. Biol. 23, 123–133]. The sequence A350PWLEPLR357 in luminal loop E is conserved in CP43 genes from 50 organisms. To map important posttranslational modifications in this sequence, tandem mass spectrometry (MS/MS) was used. These data show that the indole side chain of Trp-352 is posttranslationally modified to give mass shifts of +4, +16, and +18 daltons. The masses of the modifications suggest that the tryptophan is modified to kynurenine (+4), a keto-/amino-/hydroxy- (+16) derivative, and a dihydro-hydroxy- (+18) derivative of the indole side chain. Peptide synthesis and MS/MS confirmed the kynurenine assignment. The +16 and +18 tryptophan modifications may be intermediates formed during the oxidative cleavage of the indole ring to give kynurenine. The site-directed mutations, W352C, W352L, and W352A, exhibit an increased rate of photoinhibition relative to wild type. We hypothesize that Trp-352 oxidative modifications are a byproduct of PSII water-splitting or electron transfer reactions and that these modifications target PSII for turnover. As a step toward understanding the tertiary structure of this CP43 peptide, structural modeling was performed by using molecular dynamics.

Ferrous iron mediated oxidation of arachidonic acid: studies employing nitroblue tetrazolium (NBT).

The oxidation of arachidonic acid by ferrous sulfate provides a useful model to study the role of iron in lipid oxidation reactions. We have employed nitroblue tetrazolium (NBT) in the present investigation to evaluate the mechanism of this reaction. In the presence of arachidonic acid, Fe +++, and O2, the yellow dye NBT was rapidly reduced to the blue form, NBTH2. The molar ratio of arachidonic acid to Fe++ in this rapid reaction was 1:1, showing an interaction of one fatty acid molecule per iron molecule. Approximately one molecule of NBT was reduced per four molecules of arachidonic acid and Fe++. Reduction of NBT was accompanied by oxidation of Fe++ to Fe+++, suggesting the transfer of four electrons from the Fe++ to NBT to reduce the dye. Arachidonic acid was found to be unchanged when extracted at the end of the reaction, indicating formation of a complex that could dissociate leaving intact arachidonic acid. Evidence for the presence of such a complex which slowly dissociates during the reaction was obtained after longer incubations with small amounts of arachidonic acid. NBT reduction was not inhibited by agents which hydrolyze superoxide, by catalase or by agents which trap hydroxy radicals. We, therefore, propose a model in which NBT traps a radical generated on the arachidonic acid molecule. The proposed model suggests mechanisms for other fatty acid oxidation reactions such as prostaglandin and hydroperoxy fatty acid synthesis.

Hexose monophosphate shunt measurement in cultured cells with [1-13C]glucose: correction for endogenous carbon sources using [6-13C]glucose

Hexose monophosphate shunt (HMPS) activity can be measured with 1H nuclear magnetic resonance spectroscopy or gas chromatography--mass spectrometry by monitoring the differential production of [3-13C]lactate and [3-12C]lactate from the degradation of [1-13C]-glucose. Errors in measurement of HMPS activity can arise from unlabeled lactate precursors, by recycling of HMPS products, and by incomplete fractional enrichment of labeled glucose. A method utilizing cultured cells incubated with [1-13C]glucose in parallel with incubations using [6-13C]glucose to correct for all these problems is presented. In cultured rat C6 glioma and 9L gliosarcoma cells, failure to apply this correction results in an approximately twofold overestimation of HMPS activity.

(3R*,5S*,6R*)-3,5-dimethyl-6-(methylethyl)-3,4,5,6-tetrahydropyran-2-one, a third sex pheromone component forMacrocentrus grandii (goidanich) (Hymenoptera: Braconidae) and evidence for its utility at eclosion

The compound (3R*,5S*,6R*)-3,5-dimethyl-6-(methylethyl)-3, 4,5,6-tetrahydropyran-2-one was identified as a sex pheromone component ofM. grandii. Laboratory and field bioassays demonstrated that it elicits flight initiation, upwind anemotaxis, and casting in male wasps. The compound acts synergistically with (Z)-4-tridecenal, a previously identified sex pheromone component of femaleM. grandii, to increase male response to the aldehyde component. The source of the lactone was determined to be the mandibular glands of male and female wasps. At eclosion a majority of male-female and female-only cocoon masses released the lactone and attracted male wasps. Male-only cocoon masses were not attractive at eclosion and the lactone component was either not released or released at below-threshold concentration. Mating was observed to occur following eclosion in laboratory and field studies.

Syntheses of radioactive furan fatty acids

A novel route for the synthesis of naturally occurring furan fatty acids with particular emphasis on labeling with14C is described. Methyl [2-14C] 9-(5-pentyl-3,4-dimethyl-2-furyl)nonanoate was synthesized from 3,4-dimethyl-2-pentylfuran by a new route. [3-14C]11-(5-Pentyl-3-methyl-2-furyl)undecanoate and [2-14C] 9-(5-pentyl-2-furyl)nonanoate were prepared from their lower homologs. The label was introduced in all cases by means of the Arndt-Eistert method for chain elongation, using14CH2N2. Comparisons of yields show that, with increasing number of substituents on the ring, the furan compounds are increasingly subject to uncontrollable side reactions.

Cuticular hydrocarbons of the yellowheaded spruce sawfly, Pikonema alaskensis∗☆

Cuticular hydrocarbons of adult yellowheaded spruce sawflies, Pikonema alaskensis (Rohwer) (Hymenoptera: Tenthredinidae) were analyzed. Saturated hydrocarbons in males and females included n-alkanes and 2-methyl-, 3-methyl- and internally branched methylalkanes. The most abundant hydrocarbons were unsaturated: (Z)-9-pentacosene in males and (Z)-9-heptacosene in females. Additional (Z)-9-alkenes, traces of 6-, 7-, 8- and 10-alkenes, (Z,Z)-6,9-alkadienes and traces of alkatrienes were also found in both sexes. Pheromonally active (Z,Z)-9,19-alkadienes amounted to approx. 10% of the hydrocarbons in females but only to a maximum of approx. 0.1% in males. The amount of these dienes per female increased from 10.2 μg at 0–0.5 days old to 25.0 μg at 5–5.5 days old. These dienes were located on wings, legs, heads, thoraces and abdomens. They were also identified in field-collected mated females. The amount of (Z,Z)-9,19-dienes as a percentage of total hydrocarbons remained relatively constant with respect to age, location on body or occurrence of mating.

Analysis of hydroxy and keto cholesterols in oxidized brain synaptosomes

A rapid method for the simultaneous determination of cholesterol and its oxidation products as well as α-tocopherol and tocopherolquinone in brain subcellular fractions is described. The samples are saponified and extracted with hexane. It is not necessary to remove cholesterol in the sample before analyzing for oxysterols. The hexane extract can be used for the assay of cholesterol compounds by capillary gas chromatography and tocopherol compounds by liquid chromatography using a procedure reported previously. Oxidation of synaptosomes by a mixture of Fe2+ plus ascorbate resulted in the production of 7-keto-, 7α-hydroxy-, 7β-hydroxy-, and 5α, 6α-epoxycholesterols. The identities of these products were confirmed with gas chromatography/mass spectrometry. Cholesterol oxidase treatment did not result in the formation of any of the above compounds. Thus the types and amounts of the products of oxidation of cholesterol were dependent upon the oxidizing agent. Extraction of the oxysterols under milder conditions without saponification using sodium dodecyl sulfate cannot be used since such treatment results in low recovery of oxysterols. Oxidation of synaptosomes by low concentrations of ferrous iron and ascorbate resulted in (i) low levels of oxidation of cholesterol which could be followed by estimating the production of oxysterols and (ii) oxidation of a substantial percentage of α-tocopherol. The proposed procedure will be useful in monitoring the oxidation of small quantities of membrane cholesterol in vitro.