To obtain further information on lipid metabolism in the histidine-excess and copper-deficiency, rats were fed basal, histidine-excess (the addition of 50 g L-histidine/kg diet) or copper-deficient diets for 0, 7, 21 and 42d ad libitum. Liver triacylglycerol accumulated and the serum triacylglycerol level decreased after feeding of the histidine-excess diet for 21 or 42d, but not after feeding of the copper-deficient diet. Serum cholesterol level increased in rats fed the histidine-excess diet for 7, 21 and 42d, but not in rats fed the copper-deficient diet. Copper content in the liver and serum significantly decreased in rats fed the histidine-excess diet. Copper content in the liver and serum was markedly decreased in rats fed the copper-deficient diet. Liver zinc content was constant, but the serum zinc level decreased in rats fed the histidine-excess diet. Feeding of the copper-deficient diet hardly affected zinc content in the liver and serum. Urinary copper and zinc increased in rats fed the histidine-excess diet, and decreased or showed a decreasing tendency in rats fed the copper-deficient diet. Overall results indicated that feeding the histidine-excess diet caused copper deficiency, whereas hypercholesterolemia was not shown in rats fed the copper-deficient diet although the livers of rats fed the copper-deficient diet contained less copper than those of rats fed the histidine-excess diet. Thus, the responses on liver triacylglycerol and serum cholesterol to copper deficiency induced by the feeding of a histidine-excess diet are different from those to copper deficiency induced by feeding of a copper-deficient diet.

Administration of xenobiotics to rats results in hypercholesterolemia and in the induction of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase and malic enzyme. To investigate the mechanism of the induction of the enzymes by xenobiotics, the effects of xenobiotics on gene expressions for HMG-CoA reductase, malic enzyme, and cytochrome P-450 in rat liver and in cultured hepatocyte were investigated. The treatment of rats with polychlorinated biphenyls (PCB) as a xenobiotic induced mRNAs for HMG-CoA reductase and malic enzyme as well as CYP2B1/2 (cytochrome P-450b/e). Other xenobiotics, 1,1,1-trichloro-2,2-bis(p-chlorophenyl)ethane (DDT), and chloretone, also increased HMG-CoA reductase mRNA. In an investigation of diurnal rhythm of mRNA for HMG-CoA reductase, the induction by PCB was observed in a dark period. Induced expressions of HMG-CoA reductase gene and malic enzyme gene by PCB were observed in primary cultured rat hepatocytes and showed that the action of PCB on the gene expression relating to lipid metabolism was directed on hepatocytes. The induction was observed only in hepatocytes cultured on Engelbreth-Holm-Swarm sarcoma basement membrane gel (EHS-gel), not on type I collagen, which is usually used for monolayer culture of hepatocytes. The induction of CYP2B1/2 gene expression also was observed only in the cells cultured on EHS-gel. The induction of HMG-CoA reductase and malic enzyme by PCB required dexamethasone. However, the addition of dexamethasone per se to medium containing insulin did not show an inductive effect on levels of mRNA for HMG-CoA reductase and malic enzyme. From the data of diurnal variation and hepatocyte culture experiment, HMG-CoA reductase and malic enzyme are considered to be induced by PCB through the so-called “permissive effect” of glucocorticoid.

The oxidative break down of the membrane polyunsaturated fatty acids is known to be accompanied by the formation of a complex mixture of lipidhydroperoxides and secondary products. These compounds are highly reactive and are capable of rapid reaction with cellular nucleophiles such as phospholipids and proteins, and it was found that these reaction products are candidates as impotant biomarkers to evaluate antioxidative activity of dietary antioxidants. The author has been involved in developing immunochemical detection methods for oxidative stress by application of polyclonal and monoclonal antibodies. From the hypothesis that endogenous antioxidants in plants must play an important role for antioxidative defense systems from oxidative stress, an intensive search for novel type of natural antioxidants has been carried out from numerous plant materials, including those used as foods, and we have isolated and identified a number of lipid-soluble and water-soluble dietary antioxidants from crop seeds, sesame seeds and some spices. In this paper, the recent progress of research on functions of dietary antioxidants is reviewed.

Addition of excess cystine to a wheat gluten diet did not alter rat liver triacylglycerols or serum cholesterol. However, if the cystine-enriched diet was supplemented with lysine and threonine, rats accumulate triacylglycerols and show increased serum cholesterol. Increases in hepatic triacylglycerols can be prevented by the further addition of methionine. This diet further increases serum cholesterol. We conclude that accumulation of triacylglycerols in the liver might be due to an increased methionine requirement, induced by the addition of excess cystine, and therefore to choline deficiency.

We recently demonstrated that pentosidine, an advanced glycation end product, accumulates markedly as albumin-linked form (Palb) and in free-form (Pfree) in the plasma of patients with end-stage renal failure. The present study was undertaken to examine the clearance of Palb and Pfree by different modalities of renal replacement therapy, that is, hemodialysis (HD), continuous ambulatory peritoneal dialysis (CAPD), and renal transplantation. HD cleared Pfree (9.4 +/- 4.3 nmol/kg/HD) but not Palb, by diffusion but not by membrane adsorption, whereas CAPD cleared both Palb (4.03 +/- 2.01 nmol/kg/day) and Pfree (2.43 +/- 1.24 nmol/kg/day). Plasma total pentosidine levels were significantly (P < 0.05) lower in CAPD (0.97 +/- 0.41 nmol/ml) than in HD (1.19 +/- 0.41 nmol/ml), as the result of a lower serum albumin level in the former patients. Indeed, Palb expressed per mg albumin was virtually identical in HD and CAPD. By contrast, Pfree was significantly lower in CAPD than in HD. Palb levels were significantly correlated with plasma Pfree levels in both HD and CAPD patients, but not in the CAPD dialysate. Pentosidine transport across the peritoneum occurs mainly by diffusion, both as Palb and Pfree. Interestingly, peritoneal Palb clearance (0.17 +/- 0.07 ml/min) significantly (P < 0.00001) exceeded albumin clearance (0.11 +/- 0.05 ml/min). Palb levels being significantly higher (P < 0.0005) in the peritoneal fluid (36.28 +/- 18.55 pmol/mg) than in the serum (27.12 +/- 11.71 pmol/mg), thus raises the possibility of a facilitated diffusion of Palb or an active transport mechanism for protein-linked pentosidine into the peritoneal cavity. After renal transplantation, plasma Pfree fell rapidly, remained barely detectable after one month, and returned to normal at six months. By contrast, Palb fell more slowly and remained significantly above normal at six months, but returned eventually to normal levels. These findings demonstrate that: (1) both HD and CAPD remove Pfree; (2) the peritoneal clearance of Palb, might contribute to the lower level of plasma pentosidine in CAPD than in HD patients; and (3) renal transplantation is the best therapeutic modality to normalize both Palb and Pfree levels.

Using the ODS rat (genotype od/od) as a model, we investigated the effect of ascorbic acid deficiency on the expression of the apolipoprotein A-I gene. Male ODS rats (7 wk old, body weight approximately 140 g) were fed a basal diet containing ascorbic acid (300 mg/kg) or a diet without ascorbic acid for 14 d. Ascorbic acid deficiency lowered the serum apolipoprotein A-I concentration. The apolipoprotein A-I mRNA level in the liver of ascorbic acid-deficient rats was lowered to about 40% (P < 0.05) of that of control rats fed sufficient ascorbic acid. The mRNA level in jejunum was not affected by ascorbic acid deficiency. Ascorbic acid deficiency did not change the transcriptional rate of the hepatic apolipoprotein A-I gene, suggesting that post-transcriptional regulation was involved in lowering the mRNA level. The low level of hepatic apolipoprotein A-I mRNA was restored to the control level within 3 d after the administration of sufficient ascorbic acid. These data indicate that ascorbic acid deficiency lowers serum apolipoprotein A-I concentration through lowering its mRNA level and subsequent depression of its synthesis in liver.

In the present study, immunohistochemical and immuno-electron microscopic techniques were used to differentiate Langerhans cells (LC) from interdigitating cells (IDC) in the lymph nodes (LN) of dermatopathic lymphadenopathy. The majority of the dendritic cells that existed in the LN of dermatopathic lymphadenopathy were positive for OKT-6 (CD 1a) antibody. It was concluded that these dendritic cells were not IDC, but LC. Electron microscopically, LC in these LN contained a few Birbeck granules (BG). In order to prove the fact that these dendritic cells were LC, the existence of BG was investigated ultrastructurally by examining serial sections, and immunoelectron microscopically for CD 1a positive cells. Most of the LC in the lymph nodes we examined were negative for the anti-proliferating nuclear antigen (PCNA) antibody. This finding may mean that LC in the LN are fully developed cells and do not divide in the LN. Langerhans cells may migrate from the skin lesions to the paracortical areas in the LN, which then may become enlarged.

We have examined the effects of dietary level of protein (5% or 30% in casein) on enzyme activities and mRNA levels of two xenobiotic-inducible UDPglucuronosyltransferase (UDPGT) enzymes, such as chloramphenicol-UDPGT (CP-UDPGT) and 4-nitrophenol-UDPGT (4NP-UDPGT), in the livers of rats treated or not treated with polychlorinated biphenyls (PCB). In animals fed 5% casein and not treated with PCB, CP-UDPGT activity tended to be lower than in rats fed 30% casein, In contrast, 4NP-UDPGT activity was higher in rats fed 5% casein, In rats treated with PCB, the activities of both enzymes were increased, CP-UDPGT mRNA level was higher (1.4-fold) in rats fed 30% dietary casein than in those fed 5% casein, In contrast, 4NP-UDPGT mRNA level was higher (2.8-fold) in rats fed 5% casein than in those fed 30% casein, These changes in mRNA levels paralleled those in the activities of the two enzymes, The data indicate that the dietary protein is an important factor controlling the expression of hepatic xenobiotic-inducible UDPGT genes and modulates the capacity of the liver to metabolize foreign compounds.

The present study shows that Langerhans cells can be differentiated from interdigitating cells at the light microscopic level. Superficial lymph nodes and skin taken from necropsies and the lymph nodes of dermatopathic lymphadenopathy (DPL) were used for this experiment. Sections of lymph node and skin were embedded using the acetone, methyl benzoate and xylene (AMeX) method and dendritic cells were immunostained with anti S-100 protein antibody (S-100, and OKT-6 (CD1a) using the restaining method. Langerhans cells in the skin were positive for both CD1a and S-100. Dendritic cells positive for both CD1a and S-100, and dendritic cells positive for S-100, but not for CD1a were observed in superficial lymph nodes. In normal superficial lymph nodes, there were more interdigitating cells than Langerhans cells. The majority of the dendritic cells in the DPL were Langerhans cells. We conclude that the S-100 and CD1a positive cells are Langerhans cells, and the S-100 positive-CD1a negative cells are interdigitating cells.

The effects on serum cholesterol level were examined in rats fed on various xenobiotics. The hypercholesterolemia induced by polychlorinated biphenyls (PCB) was characterized in rats, from which lipoproteins were isolated by ultracentrifugation. A dietary addition of 0.03% PCB, 0.3% chloretone, 0.1% aminopyrine, or 0.2% 2,6-di-tert-butyl-p-cresol (BHT) resulted in a significant increase in serum cholesterol, although the chemical structure of each of these xenobiotics was different. The serum cholesterol level was markedly increased by one month of PCB feeding, the effect of PCB on the serum phospholipid level being similar. The serum triglyceride level transiently increased within 7 days of feeding with PCB diet. PCB feeding resulted in the elevation of all lipoproteins, including VLDL, LDL, HDL(1), and HDL(2), a marked increase being observed in HDL(1). Both HDL(1) and HDL(2) isolated from PCB-treated rats contained more apolipoprotein A-I (apo A-I) and less apo E than normal. VLDL isolated from PCB-treated rats had more cholesterol and apo E, but less apo C than that of the control animals. These data demonstrate that PCB feeding resulted in increased VLDL rich in cholesterol and apo E, and increased HDL rich in apo A-I. This experimentally induced hypercholesterolemia resulting in apo A-I-rich HDL would be a useful model for investigating the metabolism of apo-A-I and HDL.

Feeding xenobiotics such as polychlorinated biphenyls (PCB) causes hypercholesterolemia and fatty liver in rats. The hypercholesterolemia was characterized by high levels of high density lipoproteins (HDL) and apolipoprotein A-I (ape A-I), and by very low density lipoproteins (VLDL) rich in cholesterol and ape E (designated ''PCB-VLDL''). The mechanisms for the generation of ''PCB-VLDL'' and fatty liver, and for hyper-alpha-lipoproteinemia in rats fed PCB were investigated. The secretion rate of VLDL-lipids was increased by PCB on day 3, while the secretion rate of only VLDL-cholesterol and phospholipid were increased by PCB on days 8 and 57. Although all liver lipids were accumulated by PCB, the accumulation of esterified cholesterol was the most drastic. These results suggested that PCB stimulated the secretion of VLDL at the early period of PCB feeding (on day 3), and that cholesterol-rich VLDL, ''PCB-VLDL'', was not generated in the circulation, but was originally secreted from the liver. In spite of the stimulation of VLDL secretion, liver lipids accumulated within 8 days on the PCB diet. On days 3 and 8, serum levels of free fatty acids were not changed by PCB feeding. These data and our previous findings that PCB induced hepatic lipogenic enzymes lead us to speculate that fatty liver induced by PCB may be attributed to a stimulation of de novo synthesis of liver lipids. Even when hepatic secretion of VLDL was blocked by erotic acid, HDL-cholesterol was increased by PCB feeding, suggesting that the increase in serum level of HDL by PCB was not due to stimulation of cholesterol transport into HDL from VLDL.

Excess dietary lysine fed to previously starved rats caused lipid accumulation in the liver after refeeding for 7 days and increased excretion of potassium into urine for 2 days after the initiation of refeeding. We investigated whether dietary supplement of potassium compounds to the "lysine-excess" diet could prevent the lipid accumulation in the liver. Potassium acetate tended to improve the growth rate and significantly reduced lipid accumulation in the liver. Neither potassium bicarbonate nor potassium citrate had any effect on growth rate and liver lipid level. Non-starved rats, rate fed a lysine-excess diet for 20 days did not demonstrate liver lipid accumulation. The addition of potassium acetate to the lysine-excess diet reduced growth retardation and had no effect on liver lipid content. It is suggested that the effect of potassium acetate might be due to acetate reduction of lysine-arginine antagonism.

Wistar-Shi (genotype +/+), heterozygous Gunn (j/+) and homozygous Gunn (j/j) rats was injected intraperitoneally with 3-methylcholanthrene (3MC) dissolved in corn oil. In rats of all genotypes the hepatic concentration of UDP glucuronosyltransferase (UDPGT) mRNA was increased at 48 and 96 h after the treatment with 3MC. Hepatic activity of 4-nitrophenol UDPGT was increased by 3MC in Wistar-Shi rats and heterozygous Gunn rats but not in homozygous Gunn rats. Urinary ascorbic acid excretion increased 72 and 96 h after the injection with 3MC in Wistar-Shi and heterozygous Gunn rats but not in homozygous Gunn rats. Ninety-six hours after the injection with 3MC, the hepatic concentration of ascorbic acid in Wistar-Shi rats was 90% higher than that in the corresponding control group, whereas in heterozygous and homozygous Gunn rats the increases were 70 and 30%, respectively. Wistar-Shi rats and homozygous Gunn rats were also injected daily for 3 d with sodium phenobarbital. In rats of both genotypes, the activity and hepatic concentration of chloramphenicol-UDPGT mRNA and liver and urine ascorbic acid concentration were increased by sodium phenobarbital. The data indicate that the stimulation of the expression of both the 4-nitrophenol and chloramphenicol UDPGT genes plays a key role in the ascorbic acid biosynthesis induced by 3MC and sodium phenobarbital.

L-Ascorbic acid (AsA) is synthesized from D-glucose in rats; the terminal step of this synthetic pathway is catalyzed by L-gulono-gamma-lactone oxidase (GLO). In this study, we examined the effects of phenobarbital (PB) and 3-methylcholanthrene (MC), both of which are known to stimulate AsA biosynthesis in rats, on the hepatic levels of GLO activity, GLO mRNA, and AsA. Firstly, the existence of GLO mRNA was examined in the liver, kidney, lung, small intestine, spleen, testis, and prostate from a male rat; and GLO mRNA was found to be present only in the liver, in which GLO activity was also detected. The intraperitoneal injection with PB (100 mg/day/kg body weight, once a day for 2 days) or MC (20 mg/day/kg body weight, once) significantly elevated the hepatic level of AsA and the urinary excretion of AsA in rats (5-week-old males). The hepatic levels of cytochrome P-450IIB1 mRNA and cytochrome P-450IIB2 mRNA and those of cytochrome P-450IA1 mRNA and cytochrome P-450IA2 mRNA were also elevated in the rats treated with PB and MC, respectively, indicating a normal response of these animals to these compounds. However, the level of GLO mRNA and the activity of GLO in the liver tended to be slightly decreased by the administration of PB or MC, though the differences were not significant. Thus it is clear that the treatment with PB or MC stimulates the biosynthesis of AsA by increasing the activity of some enzyme(s) participating in the synthesis prior to GLO.

A nitrogen-sparing action of sulfur-containing amino acids and threonine in rats fed a non-protein diet has been reported from our laboratory. The nutritional significance of dietary sulfur-containing amino acids and threonine on the expression of the albumin gene was investigated. An elevated level of serum albumin and its mRNA in liver was observed in rats fed a non-protein diet supplemented with sulfur-containing amino acids and threonine, but the level of albumin mRNA of the group supplemented with cystine and threonine was much higher than that of the methionine and threonine supplemented group. Nuclear run-on assay showed an enhanced rate of albumin gene transcription in liver nuclei of rats fed the cystine and threonine supplemented diet. We showed the evidence that cystine is a more efficient amino acid than methionine to maintain the expression of the albumin gene at a high level in rats fed a non-protein diet.

Ascorbic acid (AsA) deficiency causes a decrease in hepatic concentration of cytochrome P-450 and a decrease in hepatic activity of drug-metabolizing enzymes in rats unable to synthesize AsA (ODS rats). To study the mechanism of the decrease in hepatic concentration of cytochrome P-450 isozymes by AsA deficiency, we chose the xenobiotics-inducible cytochrome P-450 and performed the experiments indicated below. AsA-deficient rats were fed polychlorinated biphenyls (PCB) which markedly induce both CYP1A subfamily and several isozymes in CYP2B subfamily. First, we assayed the activities of two drug-metabolizing enzymes so that one could be functionally distinguished from another. AsA deficiency significantly reduced the hepatic activity of aminopyrine-N-demethylase in ODS rats with and without dietary PCB, but had no effect on benzo(a)pyrene hydroxylase activity. Secondly, quantitative immunoblot analyses demonstrated that the levels of CYP2B1/2B2 and CYP1A1 in the AsA-deficiency rats fed PCB were approximately 60 and 80% lower than those found in rats fed AsA-supplemented diet. The degree of reduction in CYP2B1/2B2 was greater than CYP1A1. Thirdly, AsA deficiency caused a decrease in hepatic abundance of CYP2B1/2B2 mRNA, whereas it had no effect on the levels of CYP1A1 and 1A2 mRNA. These results indicated that dietary AsA selectively affects the levels of CYP2B1/2B2 mRNA among cytochrome P-450 induced by PCB and plays important roles for optimum induction of drug-inducible cytochrome P-450. We concluded that AsA deficiency decreases specific froms of drug-inducible cytochrome P-450, especially CYP2B1/2B2 and that the reduction of CYP2B1/2B2 mRNA level in AsA-deficient rats caused a decrease in cytochrome P-450 concentration and hepatic activity of drug-metabolizing enzymes.

The induction of NADPH-generating enzymes by polychlorinated biphenyls (PCB) in rats was investigated. The administration of PCB to rats for 3 and 14 days increased the activities of malic enzyme (ME, EC 1.1.1.40), glucose-6-phosphate dehydrogenase (G6PD, EC 1.1.1.49), and 6-phosphogluconate dehydrogenase (6PGD, EC 1.1.1.44) about 2-fold above the control level in the liver. Hepatic mRNA levels of ME, G6PD, and 6PGD, except for G6PD mRNA of the 14-day group, were also elevated to the same degree as the enzyme activities in PCB-treated rats. In rats fed a PCB-containing diet for 1 day, the hepatic mRNA levels of ME and G6PD were elevated prior to the induction of enzyme activity. In the kidney, lung, spleen, heart, and testis, the mRNA levels of ME, G6PD, and 6PGD were not affected by PCB. The induction of hepatic NADPH-generating enzymes would imply an increased demand of NADPH in the liver of rats fed with a PCB-containing diet.

The purpose of the present study was to determine whether the regulation of urea synthesis was mediated through the activation of N-acetylglutamate synthesis by arginine and whether the concentration of glutamate or activity of N-acetylglutamate synthetase might control N-acetylglutamate concentration when the thyroid status was manipulated. Three groups of rats were given 6-propyl-2-thiouracil (PTU, thyroid inhibitor) without triiodothyronine (T3) treatment, treated with PTU + T3 or treated with neither PTU nor T3 (control). Urinary excretion of urea, liver concentration of N-acetylglutamate and plasma concentration of arginine in rats given PTU + T3 were significantly lower than in rats given PTU alone. Liver concentration of N-acetylglutamate was correlated with plasma concentration of arginine (r = 0.842, P < 0.001). The activity of N-acetylglutamate synthetase and glutamate concentration in liver of the PTU + T3-treated group were significantly higher than in the group treated with PTU alone. Arginine administration (0.5 mmol/100 g body wt) elevated the liver concentration of N-acetylglutamate in all three groups. The results suggest that the greater concentration of arginine in the hypothyroid (PTU alone) rats is likely to increase the N-acetylglutamate concentration and stimulate urea synthesis.

The purpose of the present study was to determine whether the regulation of urea synthesis was mediated through the supply of nitrogen by amino acid-catabolizing enzymes and whether the concentration of acetylCoA would control the N-acetylglutamate concentration when the thyroid status was manipulated. Experiments were conducted on three groups of rats, each being given 6-propyl-2-thiouracil (PTU, thyroid inhibitor) without a triiodothyronine (T3) treatment, or PTU + T3, or neither PTU nor T3 (control), respectively. The urinary excretion of urea, the liver concentration of N-acetylglutamate, and the hepatic activities of serine dehydratase, threonine dehydratase, alanine transaminase (GPT) and aspartate transaminase (GOT) in rats given PTU + T3 were significantly lower than those in rats given PTU alone. The activity of glutamate dehydrogenase, and the concentrations of free amino acids and acetylCoA in the liver of the PTU + T3-treated group were significantly higher than those in the group treated with PTU alone. These results suggest that the higher activity of amino acid-catabolizing enzymes in the hypothyroid (with PTU alone) rats is likely to stimulate urea synthesis.

1. Glycogen is accumulated in the liver of rats given a histidine-excess diet. 2. In order to know the regulatory point, the activities of eight hepatic key enzymes in the carbohydrate metabolism were measured after feeding either a basal or a histidine-excess diet for 0, 3 and 7 days. 3. The active glycogen synthase level was about 2.6- and 3.0-fold higher after feeding for 3 and 7 days, respectively, but the total activity of this enzyme was not affected after feeding for 3 days and was increased after feeding for 7 days. 4. The active glycogen phosphorylase, glucose 6-phosphatase and pyruvate kinase activities were significantly decreased, and fructose 1,6-bisphosphatase activity was increased on a histidine-excess diet for 3 days. 5. Thus, of these enzymatic changes, mainly the enhancement of active glycogen synthase might lead to the substantial accumulation of hepatic glycogen. 6. The in vivo incorporation of radioactive glucose into liver glycogen was stimulated in rats given a histidine-excess diet.