Hepatitis E Virus Antibodies in Finnish Veterinarians.

We investigated hepatitis E virus (HEV) infections in Finnish veterinarians engaged in different practice specialties and evaluated the effect of different background factors on HEV exposure by examining total HEV antibodies in samples collected from the participants of the 2009 National Veterinary Congress in Helsinki, Finland. Finnish veterinarians commonly have total HEV antibodies with seroprevalence of 10.2%. Of the non-veterinarians, 5.8% were seropositive. Increasing age was associated with HEV seropositivity, and, surprisingly, the highest HEV seroprevalence (17.8%) among veterinarians was detected among small animal practitioners. Although no positive correlation between swine contacts and HEV seropositivity was found, 22.7% of veterinarians who had had needle stick by a needle that had previously been injected into a pig versus 9.0% of those who had not were seropositive, even though the finding was statistically non-significant (P = 0.07). Our results suggest that, although contact with swine is a known risk factor for HEV infection, the sources of HEV infections are probably numerous, including travelling abroad and possibly also other reservoirs of HEV than pigs.

12-[(5-iodo-4-azido-2-hydroxybenzoyl)amino]dodecanoic acid: biological recognition by cholesterol esterase and acyl-CoA:cholesterol O-acyltransferase.

Potential probes of protein cholesterol and fatty acid binding sites, namely, 12-[(5-iodo-4-azido-2-hydroxybenzoyl)amino]dodecanoate (IFA) and its coenzyme A (IFA:CoA) and cholesteryl (IFA:CEA) esters, were synthesized. These radioactive, photoreactive lipid analogues were recognized as substrates and inhibitors of acyl-CoA:cholesterol O-acyltransferase (ACAT) and cholesterol esterase, neutral lipid binding enzymes which are key elements in the regulation of cellular cholesterol metabolism. In the dark, IFA reversibly inhibited cholesteryl [14C]oleate hydrolysis by purified bovine pancreatic cholesterol esterase with an apparent Ki of 150 microM. Cholesterol esterase inhibition by IFA became irreversible after photolysis with UV light and oleic acid (1 mM) provided 50% protection against inactivation. Incubation of homogeneous bovine pancreatic cholesterol esterase with IFA:CEA resulted in its hydrolysis to IFA and cholesterol, indicating recognition of IFA:CEA as a substrate by cholesterol esterase. The coenzyme A ester, IFA:CoA, was a reversible inhibitor of microsomal ACAT activity under dark conditions (apparent Ki = 20 microM), and photolysis resulted in irreversible inhibition of enzyme activity with 87% efficiency. IFA:CoA was also recognized as a substrate by both liver and aortic microsomal ACATs, with resultant synthesis of 125IFA:CEA. IFA and its derivatives, IFA:CEA and IFA:CoA, are thus inhibitors and substrates for cholesterol esterase and ACAT. Biological recognition of these photoaffinity lipid analogues will facilitate the identification and structural analysis of hitherto uncharacterized protein lipid binding sites.

Chemical modification of acyl-CoA:cholesterol O-acyltransferase. 1. Identification of acyl-CoA:cholesterol O-acyltransferase subtypes by differential diethyl pyrocarbonate sensitivity.

Acyl-CoA:cholesterol O-acyltransferase (EC 2.3.1.26) (ACAT) catalyzes the intracellular synthesis of cholesteryl esters from cholesterol and fatty acyl-CoA at neutral pH. Despite the probable pathophysiologic role of ACAT in vascular cholesteryl ester accumulation during atherogenesis, its mechanism of action and its regulation remain to be elucidated because the enzyme polypeptide has never been identified or purified. Present chemical modification results identify two distinct tissue types of ACAT, based on marked differences in reactivity of an active-site histidine residue toward diethyl pyrocarbonate (DEP) and acetic anhydride. The apparent Ki of the DEP-sensitive ACAT subtype, typified by aortic ACAT, was 40 microM, but the apparent Ki of the DEP-resistant ACAT subtype, typified by liver ACAT, was 1500 microM, indicating a 38-fold difference in sensitivity to DEP. Apparent Ki's of aortic and liver ACAT for inhibition by acetic anhydride were also discordant (less than 500 microM and greater than 5 mM, respectively). On the basis of the reversibility of inhibition by hydroxylamine, a neutral pKa for maximal modification, and acetic anhydride protection against DEP inactivation, DEP and acetic anhydride appear to modify a common histidine residue. Oleoyl-CoA provided partial protection against inactivation by DEP and acetic anhydride, suggesting that the modified histidine is at or near the active site of ACAT. Systematic investigation of ACAT activity from 14 different organs confirmed the existence of 2 subtypes of ACAT on the basis of their different reactivities toward DEP and acetic anhydride.(ABSTRACT TRUNCATED AT 250 WORDS)

Chemical modification of acyl-CoA:cholesterol O-acyltransferase. 2. Identification of a coenzyme A regulatory site by p-mercuribenzoate modification.

Acyl-CoA:cholesterol O-acyltransferase (EC 2.3.1.26, ACAT) is the major intracellular cholesterol-esterifying activity in vascular tissue and is potentially a key regulator of intracellular cholesterol homeostasis during atherogenesis. We have previously reported inhibition of microsomal ACAT by histidine and sulfhydryl-selective chemical modification reagents and present here a more detailed analysis of the effect of sulfhydryl modification on ACAT activity. This analysis indicated two effects of sulfhydryl modification on ACAT activity. Modification of aortic microsomes with relatively low concentrations of p-mercuribenzoate (PMB) (100-200 microM) identified an inhibitory coenzyme A binding site on ACAT which contains a modifiable sulfhydryl group. This site binds CoA tightly (Ki = 20 microM), and PMB modification prevented subsequent ACAT inhibition by CoA without itself inhibiting enzyme activity. At higher concentrations (1-2 mM), PMB inhibited ACAT activity, indicating the presence of a modifiable sulfhydryl group necessary for cholesterol esterification by ACAT. Modification of both sites by PMB was reversible by thiols, and protection against modification was afforded in both cases by oleoyl-CoA, indicating that these sites may also bind oleoyl-CoA. Thus, at least two sulfhydryl groups influence ACAT activity: one is necessary for cholesterol esterification by ACAT, and one is at or near an inhibitory CoA binding site, which may be occupied at intracellular concentrations of CoA.

Nonoxidative ethanol metabolism in human leukocytes: detection of fatty acid ethyl ester synthase activity.

Oxidative pathways of alcohol metabolism such as alcohol dehydrogenase usually are not present in human blood and therefore clinical studies correlating ethanol metabolism with alcohol abuse syndromes have not been performed. To assess the activity of nonoxidative ethanol metabolism in blood, we assayed for the activity of fatty acid ethyl ester synthase, a pathway recently described as abundant in the human organs most commonly damaged by alcohol. Indeed, peripheral human leukocytes contain detectable fatty acid ethyl ester synthase activity: 1.2 X 10(6) leukocytes from 10 ml blood catalyze the synthesis of ethyl oleate at 1.4 nmol/4 hr. The reaction is linear with respect to cell number and expended time; Km oleate = 600 microM, Km ethanol = 600 mM. DEAE cellulose chromatography partially purifies synthase activity into a minor and major form (activity ratio = 10/1). Thus, gene products exist in human blood that recognize ethanol and whose biological activity is conveniently assayable for clinical investigations of alcohol metabolism and abuse.

Identification and quantitation of fatty acid ethyl esters in biological specimens.

Fatty acid ethyl esters, recently described as enzymatic products of nonoxidative ethanol metabolism in the heart, may represent a mediator or marker of ethanol-induced organ pathology such as alcoholic cardiomyopathy. This study was designed to develop a method for the extraction, quantitation, and definitive identification of fatty acid ethyl esters formed both in biological specimens and during enzymatic incubations. First, several potential sources of error were identified and characterized. Tissue extraction with alcohols led to the time, temperature, and concentration-dependent nonenzymatic formation of fatty acid alcohol esters. Contamination of both substrates, [14C]ethanol and 14C-fatty acid, used to measure enzymatically mediated fatty acid ethyl ester synthesis, could be removed by purification. Accurate quantitation of fatty acid ethyl esters in tissue was achieved using acetone as an extraction solvent, after which isolated lipids were thin-layer chromatographed on silica gel developed with an apolar solvent system (petroleum ether:diethyl ether:acetic acid, 75:5:1). Gas chromatography and mass spectroscopy identified individual fatty acid ethyl esters. The reproducibility of this assay was high, as assessed by quintuplicate determinations of fatty acid ethyl esters formed in liver and heart homogenates, a method with standard deviations 4 to 11% of the mean.

Partial purification and product characterization of fatty acid ethyl ester synthases in rabbit myocardium.

Homogenates of rabbit ventricular myocardium synthesize fatty acid ethyl esters using as substrates nonesterified fatty acid and ethanol in the absence of coenzyme A and ATP. This catalytic activity resides in two soluble cytosolic enzymes accounting for 19 and 81% of total fatty acid ethyl ester synthetic capability. These enzymes have been separated and partially purified by anion exchange chromatography. Gas chromatographic/mass spectrometric analyses of the catalytic products formed by these enzymes from nonesterified fatty acid and ethanol confirm their identity as ethyl esters of fatty acids. Kinetic studies indicate apparent Km values for ethanol of 0.65 M and 0.75 M for the minor and major activities, respectively. These data confirm the presence of a myocardial pathway for nonoxidative ethanol metabolism and for a metabolism of fatty acids independent of coenzyme A.