Books and Software: A complete lesson on chromatography.

A review of Advances in Chromatography, Volume 39.

Photochemical reactions and phototoxicity of sterols: novel self-perpetuating mechanisms for lipid photooxidation.

Sterols are important lipid components that may contribute to phototoxicity. We have found that phototoxic response in earthworms is related to sterols extractable with lipophilic solvents. The photochemically active compounds in worm lipids are 5,7,9(11),22-ergostatetraen-3 beta-ol (9-DHE) and 5,7,9(11)-cholestartien-3 beta-ol (9-DDHC), respectively. Human skin lipids are known to contain 9-DHE. We have also found 9-DDHC in human skin, which is reported here for the first time. In the presence of an excess of the corresponding 5,7-dienes (ergosterol of 7-dehydrocholesterol), these photoactive sterols constitute a self-regenerating source of singlet molecular oxygen (1O2) during irradiation in vivo or in vitro with UVA (315-400 nm). The quantum yield for photosensitization of 1O2 by 9-DHE was estimated to be 0.09. The 1O2 is scavenged by the dienes and the rate constant for 1O2 quenching by ergosterol was found to be 1.2 x 10(7) M-1 s-1 in methyl t-butyl ether (MTBE). This scavenging ultimately leads to the production of 5,8-endoperoxide and hydrogen peroxide. Photochemically induced superoxide radical was also produced on irradiation of sterol 5,7,9-trienes and trapped with the spin trap 5,5-dimethyl-1-pyrroline N-oxide (DMPO). The production of singlet oxygen, peroxides and radicals by the sterols may be significant in the cell damaging and tumor promoting action of UVA light on skin.

Doubly allylic hydroperoxide formed in the reaction between sterol 5,7-dienes and singlet oxygen.

Ergosterol and 7-dehydrocholesterol, common 5,7-conjugated diene sterols, react with photochemically produced singlet oxygen very efficiently to yield, in parallel pathways, the corresponding 5,8-endoperoxides and the 7 beta-hydroperoxy-5,8(9),22-trienol or -5,8(9)-dienol, respectively. The hydroperoxides decompose in an acid-catalyzed reaction to generate hydrogen peroxide and the 5,7,9(11),22-tetraenol or 5,7,9(11) trienol, respectively, with 1:1 stochiometry. The molar ratio of endoperoxide to hydroperoxide was constant (16:5) with two different reaction solvents, two different photosensitizers, and at all time points between 5 min and 3 h from the start of irradiation. Ergosterol did not react with either hydrogen peroxide or superoxide ion under our reaction conditions. Inhibition studies with nitrogen, 2,5-dimethylfuran, beta-carotene, and tert-butanol confirmed the involvement of singlet oxygen in these reactions. The unstable hydroperoxide would be expected to have undesirable biological consequences if formed in vivo.

Endogenous lipids of the earthworm Lumbricus terrestris.

Earthworms (Lumbricus terrestris) were given [1-14C]-labeled palmitic acid by gavage on days 0 and 3, and sacrificed on day 7. The distribution of label among lipid classes indicated that glycerides, sterol esters, cerebrosides, sulfatides, phosphatidylethanolamine, phosphatidylserine and (or) phosphatidylinositol, phosphatidylcholine, and sphingomyelin turn over in, or are synthesized by, the earthworm. Free fatty acids still had the highest specific radioactivity of any lipid class at the end of the experiment. Incorporation of label into sterol and hydrocarbon fractions was insignificant and there was no detectable label incorporated into gangliosides. Phosphatidylethanolamine apparently turned over quite slowly compared with other lipid classes, while the cerebroside fraction became highly labeled. Elongation of palmitic acid to stearate and oxidation to CO2 occurred extensively, but there was no evidence for desaturation.

The metabolism of di(2-ethylhexyl)phthalate in the earthworm Lumbricus terrestris.

1. Earthworms can hydrolyze di-(2-ethylhexyl) phthalate (DEHP) to mono-2-ethylhexyl phthalate (MEHP) and phthalic acid (PA). 2. They apparently cannot produce the side-chain-oxidized derivatives of MEHP that constitute the major DEHP metabolites in higher animals. 3. With the assistance of intestinal bacterial Pseudomonas, the worm-derived PA is degraded through protocatechuic and beta-carboxymuconic acids to CO2. 4. There is an indication of a second pathway for degradation of PA leading through benzoic acid.

Isolation and characterization of the initial radical adduct formed from linoleic acid and alpha-(4-pyridyl 1-oxide)-N-tert-butylnitrone in the presence of soybean lipoxygenase.

The spin trapping agent alpha-(4-pyridyl-1-oxide)-N-tert-butylnitrone (POBN) was used to trap the initial radical formed from [U-14C]linoleic acid in the reaction with soybean lipoxygenase. By using low levels of enzyme and relatively short incubation times it was possible to avoid the formation of secondary oxidation products and polymers. The adduct was extracted after methyl esterification, and isolated by a combination of open column chromatography on silicic acid and high pressure liquid chromatography on Spherisorb S5 CN with non-aqueous solvents. The 1:1 POBN-linoleate adduct was characterized by UV, IR and ESR spectra of the appropriate HPLC column fraction, by the ratio of the UV absorption to 14C content, and by mass spectrometry of the reduced (hydroxylamine) form. The results indicated that POBN trapped a linoleic acid carbon-centered radical such that POBN was attached to the fatty acid chain at C-13 or C-9 (two isomers), the linoleate double bonds having become conjugated in the process. The exact locations of the bridges in the two isomers were only tentatively determined. There was no evidence for the presence of oxygen-bridged adducts. The trapped linoleoyl radical adduct provides evidence for the production of a free radical as part of the enzymatic mechanism of soybean lipoxygenase.

Chamber and gavage technique for metabolic studies of earthworms.

Earthworms make very suitable laboratory animals for metabolic studies in vivo using radiolabeled test chemicals. We describe the construction and operation of a metabolic chamber to enable the collection of labeled CO2, volatile organics, material excreted into the bedding, and labeled material remaining in the worms. A gavage technique has been developed that permits the administration of water-soluble and lipid-soluble test chemicals in spite of the extremely low level of triglyceride lipase activity in the earthworm gut. This technique is less likely to puncture the worm tissue than previous methods. Radiolabeled DDT and diethylhexyl adipate were used to provide examples of the use of these techniques and the metabolic chamber. Results were qualitatively similar to those that have been noted in vertebrates.

Lipids of the earthworm Lumbricus terrestris.

The lipid composition of the earthworm Lumbricus terrestris has been reexamined under conditions intended to avoid enzymatic and chemical alterations during storage, extraction, and fractionation procedures. The simple lipids included aliphatic hydrocarbons, steryl esters, glycerides, and at least nine different sterols, all thought to be derived from the diet. Free fatty acids, previously considered to be major components of worm lipids, comprised only 0.3% of the total lipid weight. Phospholipids included (in order of relative abundance) phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, and phosphatidylinositol, as well as sphingomyelin. Glycolipids included cerebrosides and sulfatides containing both glucose and galactose, and gangliosides containing glucosamine and sialic acid. The fatty acid compositions of these lipid classes appeared to be a mixture of what are considered typical plant, bacterial, and animal acids. Several fatty acids found in the worms, including cis-vaccenic and eicosapentaenoic acids, were essentially absent from the dietary components, and it is concluded that these acids were synthesized in the worms. The earthworm derives much of its lipid adventitiously, but exerts at least some control over its tissue lipid composition.

Isolation and identification of alpha-(4-pyridyl-1-oxide)-N-tert-butylnitrone radical adducts formed by the decomposition of the hydroperoxides of linoleic acid, linolenic acid, and arachidonic acid by soybean lipoxygenase.

alpha-(4-Pyridyl-1-oxide)-N-tert-butylnitrone (4-POBN) radical adducts, which are formed in the reactions of soybean lipoxygenase with linoleic acid, arachidonic acid, and linolenic acid, were isolated using HPLC-ESR spectroscopy. Both linoleic acid and arachidonic acid gave one radical adduct, whereas in the case of linolenic acid, two radical adducts were isolated. These radical adducts all showed virtually identical uv spectra with lambda max at 292 and 220 nm in hexane. The absence of absorbance with lambda max at 234 nm indicates that a conjugated diene structure is not contained in these radical adducts. The mass spectra of the radical adducts formed from linoleic and arachidonic acids were identical and contained a molecular ion of m/z 264, consistent with the trapping of the pentyl radical by 4-POBN. Indeed, authentic 4-POBN pentyl radical adduct obtained from the reaction between pentylhydrazine and 4-POBN gave the same mass spectrum as the product obtained from the reaction of linoleic acid and arachidonic acid with 4-POBN. The two 4-POBN radical adducts formed in the linolenic acid reaction were shown by mass spectrometry to be isomers of pentenyl radicals. The 4-POBN-pentyl radical adduct was also detected in the reaction mixture of 13-hydroperoxy-linoleic acid, soybean lipoxygenase, and 4-POBN, indicating that the pentyl radical and pentenyl radical are formed by the decomposition of the hydroperoxides.

Mono-2-ethylhexyl phthalate, a metabolite of di-(2-ethylhexyl) phthalate, causally linked to testicular atrophy in rats.

Acute testicular atrophy results when appropriate dosages of di-(2-ethylhexyl) phthalate (DEHP) or its hydrolysis product mono-2-ethylhexyl phthalate (MEHP) are given to male rats. Events thought to be involved in this pathological effect also occur in cultures of testicular cells in vitro, but require MEHP rather than DEHP. Primary cultures of hepatocytes, Sertoli cells, and Leydig cells were incubated with 14C-labeled MEHP [8 microM] for up to 24 hr. No significant reduction in viability was produced under these conditions. In contrast to the hepatocytes, which extensively metabolized MEHP to a variety of products in 1 hr, the testicular cell cultures were apparently unable to metabolize MEHP (beyond a slight hydrolysis to phthalic acid by Sertoli cells) in 18-24 hr. MEHP was efficiently taken up by hepatocytes, but much less so by testicular cells. These results, combined with related observations from the literature, support the hypothesis that MEHP itself is the metabolite of DEHP responsible for testicular atrophy in rats.

In vitro studies of the inhibition of protein kinase C from rat brain by di-(2-ethylhexyl)phthalate.

The environmental contaminant di(2-ethylhexyl)phthalate (DEHP) has been shown to inhibit the phosphorylation of histone by purified protein kinase C (PK-C) from rat brain in a concentration-dependent manner. The inhibition does not involve making the substrate unavailable, although DEHP does bind to some extent to histone. DEHP displaces phorbol dibutyrate from PK-C, indicating that DEHP binds to the regulatory domain of the enzyme. Since DEHP does not affect the PK-C dependent phosphorylation of protamine, DEHP probably does not bind at the catalytic site. DEHP non-competitively blocked activation of PK-C by either phosphatidyl serine or calcium ion. Inhibition of histone phosphorylation by DEHP was enhanced if diglyceride was present, and the enhancement was stereoselective for the isomeric form of the diglyceride. The mechanism of the inhibition is thought to involve interference with the interaction between calcium ion and the regulatory domain of PK-C, and would have significance only for those PK-C substrates that require calcium activation of the enzyme. Thus the presence of DEHP in the high nanomolar concentration range alters the effective substrate specificity of PK-C.

Comparison of the effects of carbon tetrachloride and of 2,3,7,8-tetrachlorodibenzo-p-dioxin on the disposition of linoleic acid in rat liver in vitro.

Both 2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) and carbon tetrachloride (CCl4) have conspicuous effects on lipid metabolism in rat liver. Although it is generally accepted that CCl4 administration leads to hepatic lipid peroxidation in vivo, conflicting reports from different laboratories make it unclear whether or not lipid peroxidation is involved in the mechanism of toxicity of TCDD. The present study involved pretreating F344 rats with CCl4 or TCDD, then at predetermined times thereafter, giving [U-14C]linoleic acid. A variety of compound classes were monitored in extracts of liver taken 30 min after the label was given. A previously unreported effect of CCl4 was a conspicuous increase in turnover of 1,2-diglycerides. That CCl4 did cause lipid peroxidation was evident from the presence of allylic hydroxyacids not seen in vehicle-treated controls, greatly increased radioactivity in protein-bound material, and decreased levels of arachidonate without decreased synthesis from linolate. Where effects of TCDD pretreatment could be seen, they were much less than the corresponding effects of CCl4. No allylic hydroxyacids were detected in livers of TCDD-treated rats. The concentration of arachidonate was not reduced, and elongation of linolate was not stimulated, indicating that TCDD did not cause extensive-but-repaired peroxidation. It is concluded that while TCDD may slightly increase hepatic lipid peroxidation in rats in vivo, the extent of such stimulation appears to be too slight to account for the toxicity of TCDD.

Rapid isolation of microsomes for studies of lipid peroxidation.

Conventional isolation of microsomes by high-speed centrifugation from isotonic sucrose requires exposure to air for several hours, leading to the formation of low levels of lipid peroxidation products. Sucrose interferes in protein and malondialdehyde assays and provides no protection against lipid peroxidation during workup. A new procedure for the purification of microsomes from rat liver substitutes mannitol (a hydroxyl radical scavenger) for sucrose and takes advantage of the properties of morpholinopropane sulfonic acid (MOPS) buffer and triethylenetetramine to provide protection against lipid peroxidation during the rapid (less than one hour) workup and subsequent low-temperature storage. The microsomal fractions prepared by the proposed method are free of detectable mitochondrial contamination and at least as pure overall as those prepared by the conventional method, but they have higher glucose-6-phosphatase and laurate hydroxylase activities and significantly less malondialdehyde than conventional microsomes at the time isolation is complete. Laurate hydroxylase activity is more stable during frozen storage in mannitol medium. The kinetics of lipid peroxidation in vitro are quite different for microsomes prepared by the two methods.

Increase in cholesterol sulfotransferase activity during in vitro squamous differentiation of rabbit tracheal epithelial cells and its inhibition by retinoic acid.

It has previously been demonstrated that rabbit tracheal epithelial cells in primary culture undergo terminal differentiation at confluence to yield cornified cells much in analogy to epidermal keratinocytes and that one biochemical marker of this process seems to be the accumulation of cholesterol sulfate by the cells. The current work addresses the possible causes of this accumulation. Our studies show that the stimulation of cholesterol sulfate is paralleled by an increased activity of the biosynthetic enzyme cholesterol sulfotransferase. Squamous differentiated cells exhibited 20- to 30- fold higher levels of this enzyme activity than that in undifferentiated cells. As with other markers of squamous cell differentiation, the increase in cholesterol sulfotransferase can be prevented by the inclusion of retinoids in the cell culture medium. Inhibition of sulfotransferase levels can be observed at concentration of retinoic acid as low as 10(-11) M. The enzyme activity is optimal at pH 7 in buffers containing 0.2 M NaCl and 0.01% Triton X-100. Apparent Michaelis constants for the substrates 3'-phosphoadenosine-5'-phosphosulfate and cholesterol are 1 microM and 0.6 mM, respectively. Our results indicate that the increase in cholesterol sulfotransferase is the proximate cause for the accumulation of cholesterol sulfate in rabbit tracheal epithelial cells during squamous cell differentiation.

Beta-oxidation of 2-ethyl-5-carboxypentyl phthalate in rodent liver.

[7-14C]-2-Ethyl-5-carboxypentyl phthalate was isolated and purified from urine of rats given [7-14C]-di-(2-ethylhexyl) phthalate. This metabolite was shown to serve as a precursor for 2-ethyl-3-carboxypropyl phthalate in vivo. 2-Ethyl-5-carboxypentyl phthalate was oxidized to 2-ethyl-3-carboxypropyl phthalate in liver slices from control or, much more rapidly, from clofibrate-pretreated rats. Inhibition by KCN in liver slices from untreated rats, and strong inhibition by acrylate, suggested that formation of 2-ethyl-3-carboxypropyl phthalate involved mitochondrial beta-oxidation. The strong enhancement of the production of this compound by clofibrate (a very weak inducer for mitochondrial dehydrogenases), and strong inhibition by chlorpromazine suggested that peroxisomes may also be able to oxidize 2-ethyl-5-carboxypentyl phthalate. We were able to detect beta-oxidation of 2-ethyl-5-carboxypentyl phthalate to 2-ethyl-3-carboxypropyl phthalate using purified mitochondria, but strong phthalate monoester hydrolase activity observed during incubation of the former compound with purified peroxisomes made it impossible to determine whether 2-ethyl-3-carboxypropyl phthalate could be produced in the latter organelle or not. 2-Ethyl-5-carboxypentyl phthalate was such an inefficient substrate for beta-oxidation compared to palmitic acid that it is unlikely that it contributes significantly to the production of H2O2 in rats chronically exposed to di-(2-ethylhexyl) phthalate. Normal fatty acids are most likely to serve as the dominant substrates for peroxisomal beta-oxidase.

In-vitro modulation of protein kinase C activity by environmental chemical pollutants.

A number of environmental chemical pollutants have been reported to cause tumors or help in the propagation of tumors in experimental animals. The in-vitro effects of a few chemical contaminants were studied on the histone phosphorylation and 3H Phorbol dibutyrate (PdBu) binding of partially purified Ca2+/phospholipid dependent protein kinase c (PKC) from the brains of Fischer F344 and B6C3F1 mice. The enzyme was prepared by a modified method which gave approximately 75-fold purification. A differential effect of various compounds was observed on the phosphorylation activity and PdBu binding of PKC from rats and mice. The reported tumor promoting ability and effect on protein kinase C activity appeared to be related in the case of the rat enzyme, although causality cannot be inferred.

Application of the thiobarbiturate assay to the measurement of lipid peroxidation products in microsomes.

By applying two different thiobarbiturate assay procedures in parallel to aliquots of a microsomal incubation mixture one can simultaneously monitor free malondialdehyde and malondialdehyde plus labile lipid peroxidation products. The levels of malondialdehyde increase continuously during the incubation of microsomes, NADPH and ferrous-ADP complex, while the lipid precursors of MDA stop forming when the system becomes depleted in NADPH. In contrast to systems in which lipids are undergoing autooxidation, NADPH-dependent lipid peroxidation does not appear to generate significant amounts of water-soluble malondialdehyde precursors. As a result, quantitative interpretation of results is straightforward in the microsomal system. In spite of the lack of specificity of the thiobarbiturate coupling reaction, interferences can be easily compensated for by using zero time controls.

Generation of hydrogen peroxide by incidental metal ion-catalyzed autooxidation of glutathione.

Autooxidation of reduced glutathione in 50 mM buffer at pH 7.9 is indetectably slow in the presence of 1 mM DETAPAC, EDTA, TET, or tripyridine, but passing buffer through Chelex resin was insufficient to remove traces of catalytically active metals. Production of hydrogen peroxide during glutathione autooxidation was catalyzed by traces of Fe+2 or Cu+2, and to a much lesser extent by Cu+1 and Ni+2, but not to a detectable extent by Na+1, K+1, Fe+3, Al+3, Cd+2, Zn+2, Ca+2, Mg+2, Mn+2, or Hg+2. Cysteine was a much better precursor for hydrogen peroxide production than were cysteine sulfinic or sulfonic acids. The chelators EGTA, NTA, bipyridine, dimethyl glyoxime, salicylate, and Desferal were ineffective at preventing autooxidation. EDDA and 8-hydroxyquinoline were partially effective. Catalase could completely prevent the accumulation of detectable H2O2, but superoxide dismutase was only slightly inhibitory. Hydroxyl radical and singlet oxygen quenching agents (mannitol and histidine) stimulated. A mechanism for the production of H2O2 during trace metal catalyzed oxidation of glutathione is proposed, involving glutathione-complexed metal and dissolved oxygen. Although a radical intermediate can not be ruled out, no radical initiated chain reaction is necessary.

Absorption, metabolism, and excretion of di(2-ethylhexyl) phthalate by rats and mice.

There is convincing evidence in the literature that most of the adverse biological effects of phthalate diesters are actually effects of metabolites rather than of the parent compounds. If so, the dramatic species differences in endpoint metabolic profiles make it essential that metabolism of phthalates be understood in detail, including the factors that may alter the metabolism. A metabolic pathway for phthalates having saturated alkyl groups has been postulated based on identification of metabolites produced in vivo and excreted in urine. The first few steps in the postulated pathway have been confirmed in vitro using enzymatically active preparations from rats and mice; some details of the nature of these early steps have been learned. Although some information concerning later steps is available, much remains to be learned in this area. Species differences are postulated to involve kinetics of several biochemical and physiological events acting in concert or competition. Among these interacting factors are competition of at least three enzymes for phthalate monoesters as substrate, relative kidney clearance rates for different metabolites, relative Km values of oxidative enzymes for the same precursors in different species, and relative equilibria between glucuronide formation and hydrolysis. Essential information that must be obtained in the future includes which metabolites play a causal role in which biological effects, and what factors (age, diet, state of health, etc.) can modify the metabolism of phthalate esters and in what way.