Matti Laitinen

Changes in the protein-lipid interaction in rat liver microsomes after pretreatment of the rat with barbiturates and polycyclic hydrocarbons

Abstract 1. 1. Liver microsomes were assayed fluorometrically after pretreatment of rats with 3-methylcholanthrene, chrysene and phenobarbital. A difference in the fluorescence was obtained after pretreatment with phenobarbital when ANS fluorescence was combined with variable temperature. 2. 2. UDP Glucuronosyltransferase was solubilized after different pretreatments of the rats using trypsin digestion and digitonin solubilization. It was possible to get more enzyme into solution using 3-methylcholanthrene and chrysene as the inducer. Digitonin did not release as much enzyme after phenobarbital treatment of rats. 3. 3. Differently-acting inducers make changes in the protein-lipid interaction in the membranes. These changes are reflected also in the enzyme activities under in vitro conditions.

Inducibility of Mucosal Drug-Metabolizing Enzymes of Rats Fed on a Cholesterol-Rich Diet by Polychlorinated Biphenyl, 3-Methylcholanthrene and Phenobarbitone

The results show that a cholesterol-rich diet changes the composition of mucosal membranes. A high cholesterol diet increases mucosal cholesterol and phospholipid contents. Cholesterol enhanced mucosal NADPH cytochrome c reductase and aryl hydrocarbon hydroxylase activities as well as mucosal UDP glucuronosyltransferase activity. When phenobarbitone or Clophen A 50 or 60 were administered intraperitoneally to cholesterol-fed rats, the hydroxylation and glucuronidation activities decreased to a lower level. 3-Methylcholanthrene was, however, able to maintain or increase mucosal hydroxylative enzymes and UDP glucuronosyltransferase. These results indicate that the drug-metabolizing enzymes of the intestinal mucosa behave very differently from those in the liver. Diet apparently has a regulatory effect on the induction of drug-metabolizing enzymes because only a very potent inducer, 3-methylcholanthrene, was able to maintain and even induce mucosal drug-metabolizing enzymes in rats fed on a high cholesterol diet, possibly through changes in the microenvironment of enzymes caused by cholesterol.

Hepatic drug metabolism during ethanol ingestion in riboflavin deficient rats

Riboflavin deficiency was induced by feeding rats a riboflavin-deficient diet for 1 month. In order to find out if there are any combined effects of ethanol and riboflavin deficiency on drug metabolism, a group of riboflavin-deficient rats were also given ethanol in their drinking water. At the end of the feeding period, hepatic drug-metabolizing enzyme activities were determined. The hepatic phospholipid and protein contents were the same in rats receiving a standard diet and in those on a riboflavin-deficient diet. However, ethanol ingestion in both groups enhanced significantly the phospholipid content. Ethanol ingestion also markedly enhanced the hepatic cytochrome P-450 concentration in rats fed either a standard or riboflavin-deficient diet. Ethoxycoumarin O-deethylase activity was significantly lower in riboflavin-deficient rat livers than in those of the controls. In both groups ethanol ingestion nearly doubled the activities. Aryl hydrocarbon hydroxylase activity was also significantly decreased during riboflavin deficiency. However, ethanol administration did not change the activities of this enzyme. UDP glucuronosyltransferase activity was slightly lower in riboflavin-deficient rat livers than in those fed a standard diet. No significant decrease was found in the epoxide hydrase activity in the riboflavin-deficient rats. However, the riboflavin-deficient rats had enhanced activity after the ethanol ingestion.

Effects of anaesthesia with tricaine (MS 222) on the blood composition of the splake (salvelinus fontinalis × salvelinus namaycush)

1. The effects of anaesthesia with tricaine (MS 222) and tagging on the blood composition were studied in 123 splakes in Northern Finland. 2. Anaesthetization with tricaine clearly influenced all blood values, except plasma enzyme activities (ASAT, ALAT and SAP). Blood haemoglobin and packed cell volume increased during anaesthesia, and the peak of PCV was reached within 15 min of anaesthesia. 3. the calculated E-MCHC values decreased during anaesthesia and tagging, suggesting swelling of the erythrocytes. The initial haematological values were regained with 3-4 hr of recovery. 4. Anaesthetization elevated the blood lactate concentration and the peak values were measured for splakes anaesthetised and tagged at 1 hr after recovery. 5. The blood glucose was rather stable until the peak was reached within 1 hr of recovery. The initial blood lactate and glucose were regained within 3 and 48 hr, respectively. 6. Repeated anaesthetization had only a slight effect on blood composition, but blood glucose elevated significantly (P less than 0.001) after 4 anaesthetizations.

Dose-Response Relationships in Blood Lipids during Moderate Freshwater Fish Diet

Fish and fish oils are known to counteract coronary heart disease risk factors. We have previously showed that eating moderate amounts of freshwater fish modifies lipid and prostanoid metabolism in healthy students. In this study, the dose response relationship was clarified. One hundred male students took part and were randomly divided into control and four fish diet groups with different fish diets. Hematological, serum lipid and vitamin E and A analyses were performed. Already 1.5 fish containing meals per week increased n-3 to n-6 ratio of fatty acids in erythrocyte ghost phosphatidylethanolamine. The serum triglyceride and apolipoprotein B concentration fell significantly in the group eating 3.8 fish containing meals a week for 12 weeks. At two lower doses (1.5 and 2.3 meals/week) the tendency to lower values was already seen. No significant changes were observed in the serum cholesterol, apolipoprotein A1 and vitamin E and A concentrations. The hematological variables also remained unchanged. The results show that moderate amounts of fish as a constituent of normal diet have beneficial effects on lipid metabolism.

Effect of administration route of 3-methylcholanthrene on the inducibility of intestinal drug-metabolizing enzymes.

The inducibility of the mucosal drug-metabolizing enzymes of rat small intestine was studied by administering 3-methylcholanthrene either intragastrically or intraperitoneally. The aryl hydrocarbon hydroxylase activity was 4.1 times higher after intragastric than after intraperitoneal administration of methylcholanthrene. The ethoxycoumarin-O-diethylase activity was 11 times and UDP-glucuronosyltransferase (with p-nitrophenol as substrate) activity was 3 times higher after intragastric administration than after intraperitoneal administration. The epoxide hydratase activity was, on the other hand, 38% lower after intragastric administration of 3-methylcholanthrene than after intraperitoneal administration. The results suggest that compounds entering the body intragastrically, e.e. in the diet, might have profound enzyme-specific effects on the intestinal metabolic rates of drugs.

Separation of the apoprotein components of human serum high density lipoprotein: chromatofocusing, a new simple technique

The high-density lipoproteins (HDL) are a heterogeneous group of particles operationally defined as those lipoproteins isolated between the densities of 1.063 and 1.210 g/ml. About 50% of HDL mass is protein, 30% is phospholipid, and 20% is cholesterol [l]. Of the several apolipoproteins present, apo A-I and apo A-II are major components; minor polypeptides include apo C-I, apo C-II, apo C-III, apo D and apo E [2-41. Apoprotein A-I and apoprotein A-II constitute about 90% of total HDL protein, with a ratio of apo A-I to APO-II of 3 : 1 in both HDL, and HDL, [5]. Apoprotein C is present in the HDL density range of humans in small amounts, and comprises 5-10% of HDL, protein, and l-2% of HDL, protein [6]. The most used methods to separate different apoproteins from HDL are gel filtration combined with DEAE cellulose ion-exchange chromatography [7,8]. In this study, chromatofocusing of human serum apoHDL with and without urea has been employed to fractionate different apoproteins, and slab gel electrophoresis has been performed to study the purity of fractions.

Dietary cholesterol-induced enhancement of hepatic biotransformation rate in male rats

Male rats were fed a cholesterol-free diet for 5 weeks, followed by a 2% cholesterol diet for 4 weeks. Another group of rats was continuously fed a cholesterol-free diet. A third group was fed standard pelllets during the whole experiment. Hepatic microsomal protein and cholesterol contents and drug-metabolizing enzyme activities were measured. The cholesterol-rich diet increased microsomal protein content and this increase disappeared after trypsin digestion of microsomal membranes. Microsomal cholesterol content was enhanced three-fold by cholesterol feeding. Cytochrome P-450 concentration, NADPH cytochrome c reductase and aryl hydrocarbon hydroxylase activities showed only minor changes following cholesterol feeding. The p-nitroanisole O-demethylase and ethoxycoumarin deethylase activities were doubled by cholesterol in comparison to cholesterol-free diet. Trypsin digestion activated the UDP-glucuronosyltransferase enzyme eight- to ten-fold on a protein basis. Trypsin treatment increased the cholesterol activation of UDP-glucuronosyltransferase when compared to the activity in native microsomes. The data suggest that dietary cholesterol regulates the cholesterol content of microsomal membranes. The activities of drug-metabolizing enzymes are also altered, possibly due to the compositional changes of the membranes.

The relationships between human serum pseudocholinesterase, lipoproteins, and apolipoproteins (APOHDL)

During recent years there has been an increasing number of studies concerning metabolizing enzymes during lipoprotein catabolism. It is well documented that lecithin-cholesterol acyltransferase (LCAT, ED 2.3.1.43) and lipoprotein lipase (EC 3.1.1.34) take part with these reactions (1). In addition to these enzymes there has been evidence that pseudocholinesterase (EC 3.1.1.8.) is also associated with lipid metabolism (2). A relationship between serum pseudocholinesterase and low density lipoprotein (LDL) has been shown on the basis of ultrasonication studies (3) and after artifical induction of hyperlipidemia in vitro and in vivo (4). Because pseudocholinesterase activity has been previously measured only in whole serum and in DLD, our purpose has been to find out if there is any activity in other lipoprotein fractions. In addition to whole enzyme activity we investigated the distribution of pseudocholinesterase (PCE) isoenzymes in different fractions. As previously reported, LCAT and lipoprotein lipase need as cofactor, a specific apolipoprotein (5). We also studied a possible connection between HDL (high density lipoprotein) apolipoproteins (apoA-I, A-II, E, and total apoC) and PCE activity.

Enhancement of hepatic drug biotransformation rate by polychlorinated biphenyls in rats fed cholesterol-rich diet.

A combined effect of cholesterol and polychlorinated biphenyls (PCBs) on the microsomal drug hydroxylation and glucuronidation in the liver of the rat was studied. PCBs, Clophen A-50 and A-60, having an average chlorination degree of 50 and 60% affect the structure of microsomal membranes. It was found that Clophen A-60 increased the binding of trypsin- and digitonin-sensitive proteins to the membranes. Also it was found that PCBs enhanced the phsopholipid content of microsomes. PCBs increased the activity of hepatic NADPH cytochrome c reductase about 1.5-fold. Aryl hydrocarbon hydroxylase activity doubled with Clophen A-50 and quadrupled with Clophen A-60. Hepatic UDPglucuronosyltransferase activity was doubled with both PCBs. The enhancement in hepatic aryl hydrocarbon hydroxylase and in UDPglucuronosyltransferase was found to be lower in the presence of high cholesterol level in the diet when compared to earlier results. This is supposed to be due to the membraneous effects of cholesterol.