Effects of dietary oil related to the toxic oil syndrome on the lipids of guinea pig liver microsomes.

The potential effects of oil specimens related to cases of toxic oil syndrome (TOS) on the liver microsomal lipid composition from guinea pigs were investigated. For four weeks, animals were fed diets supplemented with either "case oil" (oil related to cases of TOS) or "control oil" (oil unrelated to cases of TOS), either previously heated or not. Results were compared with those from guinea pigs fed the same diet with no oil. The administration of case oil produced changes in liver microsomal lipid composition. Statistically significant differences were also found between heated case and heated control oils. The cholesterol/phospholipid molar ratios and the major phospholipid class distribution were unaffected under these diet conditions. However, increases in the relative contents of linoleic and arachidonic acids and, simultaneously, a reduction in palmitic and palmitoleic acid levels were observed by diet effects. Heated oil administration decreased the saturated/unsaturated ratios in all cases. Our data suggest that changes observed in the fatty acid composition are attributable to the free fatty acid contents of administered oils. The toxic constituents of case oil seem to be able to alter the liver microsomal lipid composition.

Lysosomal enzyme activities in liver and sera from guinea pigs fed oil related to the toxic oil syndrome.

beta-N-Acetylglucosaminidase and beta-galactosidase activities were determined in serum and liver from guinea pigs fed "toxic oil" (related to cases of TOS) under different experimental conditions. The results obtained were compared with those of guinea pigs fed non "toxic oil" (case-unrelated oil; controls 1) and animals fed no oil (controls 2). In serum, both activities were significantly increased after all treatments with case-related oil as compared with controls 1 and 2. In the liver, beta-galactosidase activity did not show significant differences in any of the treatments when compared with controls 2. However, NAG activity decreased significantly after 7 days of treatment with non-heated oil--either case-related or not--when compared with controls 2; it also decreased significantly after 28 days of treatment with heated case-unrelated oil, both with respect to controls 2 and the animals fed case-related oil. Liver weights tended to increase in the animals fed oil--toxic or not--with respect to those of the livers from untreated animals. Morphologically, a slight vacuolization of the hepatocytes was observed in some of the samples from the treated animals.

Toxic oil syndrome: a study of renal function in guinea pigs fed toxic oil.

1. Comparative studies of beta-N-acetylglucosaminidase (NAG, EC 3.2.1. 52) and beta-galactosidase (EC 3.2.1. 23) activities, protein concentration and creatinine clearance, were carried out in urine and kidney from guinea pigs treated with "toxic oil", orally administered under different conditions, related to controls. 2. Enzyme activities did not vary significantly in urine with any of the studied conditions of oil administration. By contrast, in kidney, beta-D-galactosidase disclosed a significant increase in all the treatments studied when compared to controls without treatment. 3. Urinary protein excretion, creatinine concentration and creatinine clearance were significantly greater in treated animals than in controls after 4 weeks of treatment. 4. Relative kidney weight (g/100 g body wt), was significantly lower in animals treated for 28 days with previously heated oil.

Age-related excretion of six glycosidases in rat urine.

The activity of beta-N-acetylglucosaminidase (NAG), beta-galactosidase, alpha-L-fucosidase, beta-glucuronidase, beta-glucosidase and alpha-mannosidase was determined in the urine of rats at progressive ages from newborn to old animals. The age-dependence of urinary creatinine, protein and pH values was also studied. Enzyme activity, related to urinary creatinine, was significantly higher in the newborn group than other ages. The excretion of NAG increased significantly in adult rats (3-6 months old) compared to young rats (1 month old). Most of the enzyme activities were diminished in old rats (25 months old). Increased proteinuria and creatinine excretion were observed in rats since 3 months of age. Age-related differences among enzyme activities therefore should be considered when these urinary glycosidases are to be studied in rats.

Increased affinity to concanavalin A and enhanced secretion of alpha 1-acid glycoprotein by hepatocytes isolated from turpentine-treated rats.

Hepatocytes were isolated from adult rats at various times after subcutaneous injection of turpentine (1 ml). The affinity to concanavalin A (Con A) of alpha 1-acid glycoprotein (AGP) and the intracellular content and rate of secretion of AGP and albumin were evaluated over a period of 19 days. Inflamed hepatocytes secreted mainly the Con A-reactive form of AGP whereas control hepatocytes secreted a higher amount of the Con A-non-reactive form. The intracellular content and rate of secretion of AGP by inflamed hepatocytes increased markedly whereas those of albumin decreased. However, when the residence time (ratio of intracellular content to rate of secretion) was evaluated, it appeared that the efficiency of secretion of both proteins was higher than in control hepatocytes. The changes in the affinity of AGP to Con A and in the secretion of AGP and albumin were reversible. These findings indicate that acute inflammation leads to posttranslational alterations during the intracellular transit of these secretory proteins.

One-step purification of rat plasma alpha 1-acid glycoprotein by antibody affinity chromatography: application to normal and inflamed rat sera.

Most purification procedures used previously to isolate alpha 1-acid glycoprotein (AGP) from plasma can lead to some alterations in its carbohydrate moiety. An immunoaffinity chromatographic method is proposed for purifying in one step rat plasma AGP without any detectable modification of its glycan moiety. Crossed immunoaffinoelectrophoresis with concanavalin A before and after purification showed identical patterns, suggesting no glycan selection during the purification. In the same way no desialylation occurred during the purification step. This immunoaffinity chromatographic procedure provided evidence of a decreased level of fucosyl residues in turpentine oil rat plasma AGP compared with normal rat plasma AGP.

Purification and characterization of acid beta-D-galactosidase from rabbit spleen.

beta-D-Galactosidase has been purified to apparent homogeneity from rabbit spleen. The purification steps involved ammonium sulphate precipitation, DEAE-cellulose, concanavalin A-Sepharose, Sephadex G-200, and Sepharose 4B-(epsilon-aminocaproyl)-2-deoxy-beta-D-glucosylamine affinity chromatographies. In the DEAE-cellulose step, the beta-D-galactosidase was separated into two molecular forms, designated I and II, with similar pH optimum, Km, substrate specificity, and sensitivity to substrate analogues and other substances. Form I was purified 1,800-fold with a yield of about 2% of the total activity. This form is heat-labile, it has an acid optimal pH (4.0), an isoelectric point of 6.7 and a molecular weight of 75,000 daltons. Form II has an optimal pH of 3.6 and three different pI values (5.3, 5.7, and 6.7) whose relative proportions can be modified by treatment with neuraminidase. Form II appeared to be a multimeric form (IIA) of about 600,000 daltons at pH 4.0, which was reversibly dissociated to an oligomeric form (IIB) with an apparent molecular weight of 120,000 at neutral pH values. Both IIA and IIB were purified separately and showed an acid pH optimum and an heterogeneous pI (from 4.6 to 7.2). The dissociation of IIA into IIB can be generated spontaneously, but is increased by the presence of urea in the elution buffer, suggesting that both are aggregates of a common subunit.

Properties and kinetics of a neutral beta-galactosidase from rabbit kidney.

A neutral beta-galactosidase has been purified by concanavalin A-Sepharose affinity chromatography, DEAE-cellulose chromatography, Sephadex G-200 gel filtration and hydroxylapatite chromatography. The enzyme was purified 126-fold with a yield of about 21%. This form has a neutral optimal pH (7.5) and it is located in the cytosolic fraction. It shows a wide pH stability from pH 4.5 to 8.0, but it is very unstable at low pH values. Its isoelectric point is 4.9 and this value does not change on neuraminidase treatment. The estimated molecular weight was 47 000. The neutral form shows beta-D-galactosidase, beta-D-fucosidase and beta-D-glucosidase activities, all of them associated in a single peak in all the purification steps. p-Nitrophenyl beta-D-galactosides, p-nitrophenyl beta-D-fucosides and p-nitrophenyl beta-D-glucosides competed fully for a common active site in mixed-substrate experiments. Using gamma-D-galactonolactone as competitive inhibitor the Ki values were always coincident for the three activities. The effect of NaCl, methyl mannoside and some sugars (fucose, galactose and glucose) was studied.

Inhibition of A and B N-acetyl-beta-D-glucosaminidase urinary isoenzymes by urea.

A urinary fraction which inhibits the activity of N-acetyl-beta-D-glucosaminidase (NAG) has been isolated and identified as being urea. Usually present in high concentration, urea appears to be the only urinary component responsible for the frequently observed urinary NAG inhibition. The inhibition of the two urinary NAG isoenzymes A and B is competitive with respective Ki values of about 70 mmol/l and 60 mmol/l. With routine assay conditions, it seems that a dilution of urine prior to enzyme assay is sufficient to abolish the inhibition of the two isoenzymes A and B by endogenous urea.