Phillip W. Albro

Metabolism of diethylhexyl phthalate by rats. Isolation and characterization of the urinary metabolites.

Abstract The metabolites appearing in the urine of rats fed di(2-ethylhexyl)phthalate have been isolated by two procedures, thin-layer chromatography of the free metabolites, or thin-layer and gas-liquid chromatography after treatment with diazomethane. The metabolites were characterized by infrared spectroscopy, nuclear magnetic resonance spectroscopy, and mass spectrometry. The only metabolites found were those to be expected from ω- and (ω—1)-oxidation of mono(2-ethylhexyl) phthalate without attack on the aromatic ring. Conjugates were apparently not formed, and free phthalic acid amounted to less than 3% of the urinary metabolites.

Enzymatic hydrolysis of Di-(2-ethylhexyl) phthalate by lipases

Abstract Di-(2-ethylhexyl) phthalate was hydrolyzed by lipases from a variety of rat tissues. Of the preparations studied, only glycerol-ester hydrolase (pancreatic lipase, EC 3.1.1.3) and sterol-ester -hydrolase (cholesteryl ester hydrolase, EC 3.1.1.13) were unable to hydrolyze the phthalate diester. However, out of 15 different tissue preparations, only the liver alkaline lipase preparation could hydrolyze mono-(2-ethylhexyl) phthalate at a significant rate. The quantitative data on rates of phthalate ester hydrolysis by intestinal enzymes of rat, mouse, hamster, and guinea pig suggested that orally ingested di-(2-ethylhexyl) phthalate would have little opportunity to be absorbed intact. Injected di-(2-ethylhexyl) phthalate, however, would probably survive long enough for all of the tested tissue lipases and lipoprotein lipases to play some role in the metabolism of this compound.

Identification of the metabolites of simple phthalate diesters in rat urine

Abstract Rat urinary metabolites of orally-administered dimethyl, di- n -butyl and di- n -octyl phthalates have been isolated by high-pressure liquid chromatography on Porasil A. A linear gradient of tetrahydrofuran into n -heptane resolved the following classes of diazomethane-treated metabolites: diesters, triesters, ketodiesters, and two classes of hydroxydiesters. Phthalic acid was a very minor metabolite except in the case of dimethyl phthalate precursor. The corresponding monoesters become more significant as they become more polar (methyl > butyl > n -octyl ≈ ethylhexyl). The remaining metabolites are those that woudl be expected from ω - 1 and ω- followed by α-and β-oxidation of the monoesters. Intact diester was excreted as a trace component when dibutyl phthalate was fed, and as a significant component when dimethyl phthalate was fed.

Comparison of the compositions of aroclor 1242 and aroclor 1016

The complete polychlorinated biphenyl compositions of two American products, Aroclor 1242 and its more modern replacement, Aroclor 1016, have been determined by gas-liquid chromatography (GLC) on twelve liquid phases of differing selectivities. Attempts were made to determine the degree of contamination of these Aroclors with chlorinated naphthalenes, using GLC with multiple ion-monitoring mass spectrometry. Chlorinated dibenzofurans, indetectable in Aroclor 1016, were tentatively identified by negative chemical-ionization mass spectrometry and their retention times relative to dieldrin on two GLC liquid phases. Quantitation of the dibenzofurans was initially accomplished using an electron-capture detector, and confirmed by negative chemical-ionization mass spectrometry. Aroclor 1242 contained less than 0.05 mol.% chloronaphthalenes, while Aroclor 1016 contained less than 0.06 mol.% of these compounds. Aroclor 1242 had approximately 150 ppb of chlorinated dibenzofurans, of which 43% was the toxic 2,3,7,8-tetrachloro isomer.

The Metabolism of 2-Ethylhexanol in Rats

1. 2-Ethylhexanol was efficiently absorbed following oral administration to rats. 14C associated with 2-ethyl[1-14C]hexanol was rapidly excreted in respiratory CO2 (6-7%), faeces (8-9%) and urine (80-82%), with essentially complete elimination by 28 h after administration. 2. The amount of label recovered in 14CO2 matched the amount of unlabelled 2-heptanone plus 4-heptanone recovered from urine, suggesting that both types of metabolite may have been derived form the major urinary metabolite, 2-ethylhexanoic acid, by decarboxylation following partial beta-oxidation. The 14CO2 appeared not to be derived from acetate (urinary acetic acid and liver and brain cholesterol were not labelled) or by reductive decarboxylation (heptane was not present.) 3. Other identified metabolites were 2-ethyl-5-hydroxyhexanoic acid, 2-ethyl-5-ketohexanoic acid, and 2-ethyl-1,6-hexanedioic acid. Only about 3% of the ethylhexanol was excreted unchanged. 4. Ethylhexanol was a competitive inhibitor of yeast alcohol dehydrogenase, but a good substrate for horse alcohol dehydrogenase. 5. Other relationships between metabolism and toxicity of 2-ethylhexanol are discussed.

Determination of protein in preparations of microsomes

Abstract Nine protein assay procedures based on the phenol, biuret, and picryl sulfonate reactions were compared as to their suitability for the determination of protein in microsomal preparations. All of the methods gave comparable results except the “macrobiuret,” which showed light-scattering interference, and the routine assays of Lowry et al. (1) and Schacterle and Pollack (14) which gave low values with calcium-precipitated microsomes. The advantages and disadvantages of the various assays relative to simplicity, interferences, and sensitivity are discussed. Three of the assay methods, the “insoluble biuret” (17) “nonaqueous biruet” (18), and a modification of the assay described by Schacterle and Pollack (14) (which is a modification of the assay of Lowry et al. (1), are particularly recommended.

Bacterial hydrocarbons: occurrence, structure and metabolism.

The chemical structures, general pathways of catabolism and the biosynthesis of bacterial, nonisoprenoid hydrocarbons are reviewed with emphasis on recent work on the chemistry and biosynthesis of the hydrocarbons ofSarcina lutea.

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.

Isolation and identification of α-(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.

Absorption of aliphatic hydrocarbons by rats

The percentage retention of aliphatic hydrocarbons from relatively simple mixtures administered intragastrically to rats is shown to be an inverse linear function of carbon number, at least for major components of the mixtures. Retention (intake minus excretion) is reduced by treatment with certain antibiotics or by feeding amounts of hydrocarbon in excess of 320 mg/kg body weight as a single dose. Bacterial degradation of paraffin in the feces, and recirculation of intact hydrocarbon in the bile apparently do not occur to a significant extent under the conditions tested. The major, if not the only significant, site of absorption of ingested hydrocarbons in rats is the small intestine. No conspicuous difference in ability to take up hydrocarbons in everted sac experiments was seen between duodenum, jejunum and ileum, although duodenal sacs released hydrocarbon or its metabolic products into the serosal medium more rapidly than did sacs of ileum. The primary recipient of absorbed paraffin in vivo was the lymph, but there was some evidence for absorption of hexadecane or, perhaps more likely, its metabolic products, directly into the portal blood.