Investigation on Acute Biochemical Effects of Ce（NO3 ）3 on Liver and Kidney Tissues by MAS 1H NMR Spectroscopic-Based Metabonomic Approach

High resolution1Hmagicanglespinning(MAS)NMRspectroscopyisincreasinglybeingusedasause fultechniqueforinvestigatingthemetabolicstateof variousintacttissuesintheareasofdrugtoxicity[1,2]anddiseasediagnosis[3,4].Biologicaltissuesaresemi solidheterogeneouscomplexcontainingspecieswitha widerangeofmolecularweightssuchasproteins,lip ids,andlow molecularweightmetabolites[5].1HNMR spectraoftissuesufferfrommajorline broadeningcon tributionswhenacquiredbyconventionalsolution state NMRspectroscopybecau…     

Metabolic alterations in organs of Meriones unguiculatus infected with Echinococcus multilocularis

1. 1H-NMR spectra of liver, spleen and kidney extracts from Meriones unguiculatus infected with Echinococcus multilocularis showed decreased levels of glucose. In addition, the liver extracts were severely glycogen-depleted. 2. Both livers and spleens contained less glycine, taurine and acetate. Spleens also had fewer cholines and less lactate but more betaine and alanine. 3. In the kidneys, elevated concentrations of succinate, acetate and lactate were found.     

Time course study of the influence of acute iron overload on Kupffer cell functioning and hepatotoxicity assessed in the isolated perfused rat liver

This study tested the hypothesis that acute iron overload (500 mg/kg) alters Kupffer cell functioning by promoting free radical reactions associated with the respiratory burst of liver macrophages, assessed in the isolated perfused rat liver under conditions of Kupffer cell stimulation by carbon infusion and inactivation by gadolinium chloride pretreatment. Total serum and hepatic iron levels were markedly enhanced compared with control values 2 to 24 hours after iron treatment. Total liver O2 uptake progressively increased by iron overload reaching a maximum at 6 hours after treatment, an effect that was completely blocked by GdCl3. Concomitantly, carbon-induced GdCl3-sensitive liver O2 uptake was either enhanced by 119% at 2 hours after iron overload, diminished compared with control values at 4 hours, or abolished at 6 hours. Iron-overloaded rats showed a marked increase in liver sinusoidal lactate dehydrogenase efflux at 4 and 6 hours after treatment, an effect that is exacerbated by carbon infusion and reduced (69%-89%) by GdCl3 pretreatment. Both basal and carbon-induced lactate dehydrogenase effluxes returned to control values at 24 hours after iron overload concomitantly with depression of the basal O2 uptake, without development of iron-induced GdCl3-sensitive respiration or Kupffer cell activation by carbon infusion. It is concluded that iron overload induces a derangement in the Kupffer cell functional status represented by early increases in macrophage-dependent respiratory activity, which may contribute to the concomitant liver injury that developed and to the impairment of both hepatic respiration and the macrophage response to particle stimulation observed at later times after treatment.     

Inactivation of Kupffer cells prevents early alcohol-induced liver injury

It is well recognized that consumption of alcohol leads to liver disease in a dose-dependent manner; however, the exact mechanisms remain unclear. Hypoxia subsequent to a hypermetabolic state may be involved; therefore, when it was observed recently that inactivation of Kupffer cells prevented stimulation of hepatic oxygen uptake by alcohol, the idea that Kupffer cells participate in early events that ultimately lead to alcohol-induced liver disease became a real possibility. The purpose of this study was to test that hypothesis. Male Wistar rats were exposed to ethanol continuously by means of intragastric feeding for up to 4 weeks using the model developed by Tsukamoto and French. In this model, ethanol causes fatty liver, necrosis and inflammation--changes characteristic of alcohol-induced liver disease in human beings. Kupffer cells were inactivated by twice weekly treatment with gadolinium chloride (GdCl3), a selective Kupffer cell toxicant. AST levels were elevated to 192 +/- 13 and 244 +/- 56 IU/L in rats exposed to ethanol for 2 and 4 wk, respectively (control value, 88 +/- 7). This injury was prevented almost completely by GdCl3 treatment. Fatty changes, inflammation and necrosis were also all reduced dramatically by GdCl3 treatment. The average hepatic pathological score of rats treated with ethanol for 4 wk was 4.3 +/- 0.6, which was reduced significantly in ethanol- and GdCl3-treated rats to 1.8 +/- 0.5 (p < 0.05). Rates of ethanol elimination were elevated 2- to 3-fold in rats exposed to ethanol for 2 to 4 wk. This elevation was blocked by GdCl3 treatment.(ABSTRACT TRUNCATED AT 250 WORDS)     

The role of splenic macrophages in plasma tumor necrosis factor levels in endotoxemia

We investigated the function of splenic macrophages (M phi s) with respect to changes in plasma tumor necrosis factor (TNF) in lethally endotoxemic rats treated with gadolinium chloride (GdCl3), which blocks the phagocytosis of large liver M phi s. We also carried out an immunohistochemical study to investigate the change of populations of liver and splenic M phi s under the condition of dysfunction of liver M phi s with or without splenectomy. Twenty-four-hour survival rates were 100% in the GdCl3-treated group (n = 6) and 0% in the nontreated group (n = 6). These rates did not change with splenectomy. Immunohistochemical examination with the primary monoclonal antibodies ED1, ED2 and ED3 revealed that large liver M phi s were eliminated after GdCl3 injection, and that this was not related to splenectomy. The splenic M phi s were not affected by GdCl3 treatment. Plasma TNF levels did not differ between the GdCl3-treated and the nontreated groups, irrespective of whether splenectomy was performed. It was suggested that plasma TNF levels are not affected by the splenic M phi s and that they do not compensate for dysfunction of liver M phi s after GdCl3 treatment.     

1H NMR study of metabolic alterations in the small intestine of rats infected with Hymenolepis diminuta

1H NMR spectra of the duodenum, jejunum and ileum tissues of the small intestine of a rat showed metabolic gradients. 2. The concentrations of metabolites in these gut regions were altered by the presence of the tapeworm Hymenolepis diminuta. 3. In the infected duodenum there was significantly less glycogen, glucose and phosphocreatine/creatine, but significantly more lactate than in the corresponding controls. 4. Infected jejunum contained significantly less betaine but significantly more succinate, alanine and lactate. 5. Infected ileum had significantly less glycogen and taurine but significantly more alanine and lactate.     

Gadolinium chloride inhibits Kupffer cell nitric oxide synthase (iNOS) induction

Kupffer cells (KC) are the phagocytic macrophages of the liver. The rare earth metal, gadolinium (GdCl3), is a lanthanide, which, after phagocytosis by the KC, has been found to alter various aspects of KC physiology. In this study, we describe for the first time that the in vivo administration of GdCl3 to rats decreases the release of NO by isolated rat KC in response to lipopolysaccharide. Western blot analysis shows decreased expression of both inducible nitric oxide synthase as well as total cellular calmodulin after GdCl3 treatment. Possible mechanisms for this phenomenon are suggested.     

Daunomycin unfolds compactly packed DNA

Metabolic degradation of a soluble highly branched (-->3)-beta-D-glucan, SSG, was examined in mice using a macrophage blocker, gadolinium chloride (GdCl3). Intraperitoneally administered SSG distributed in the liver was slowly degraded, and after 5 weeks about 30% of the SSG became anionic. In addition, it is suggested that the metabolites would contain fewer branching points as assessed by the reactivity to limulus factor G. On the other hand, in the spleen, the molecular weight and the degree of branching of SSG were not changed for at least 5 weeks. Blockade of Kupffer cells by GdCl3 did not significantly change the distribution ratio of SSG in the liver. However, the treatment significantly delayed the degradation of SSG. These results suggested that Kupffer cells play important roles, not in the distribution, but in the oxidative degradation of SSG in the liver. In addition, splenic macrophages did not significantly contribute to the metabolic degradation of SSG.     

Kupffer cell inactivation prevents lipopolysaccharide-induced structural changes in the rat liver sinusoid: an electron-microscopic study

Scanning and transmission electron-microscopic examination of the rat liver sinusoid was performed in this study after in vivo treatment of rats with gram-negative bacterial lipopolysaccharide (LPS, 1 mg/Kg(-1) body weight), with or without pretreatment with gadolinium chloride (GdCl3 10 mg(Kg(-1) body weight). Twenty-seven and 48 hours after GdCl3 administration, to inactivate/eliminate part of the Kupffer cell population, a decrease in the number of visualized Kupffer cells was observed, without evident effects on the sinusoidal endothelial cell or on the hepatocyte. Three and 24 hours after its administration, LPS produced ultrastructural changes in the sinusoid characterized by morphological evidence of Kupffer cell activation (i.e., swelling and expanded philopodia anchoring the Kupffer cell to the luminal surface of the sinusoidal wall), and a marked decrease in the population of endothelial cell fenestration. The reduction in the number of fenestrae was associated with a change in the diameter of fenestrae and can be interpreted as a component of the "capillarization" process of the hepatic sinusoid. Such ultrastructural changes were prevented by the administration of GdCl3 24 hours before LPS injection. Hence, these findings suggest that LPS-induced structural changes in the liver sinusoid are mediated by an LPS-induced Kupffer cell activation. Coupled with previous experimental data, showing similar effects of GdCl3 on one of the hepatic sinusoidal endothelial cell (SEC) functions, i.e., hyaluronan scavenging, the data presented in this study strongly support the view that Kupffer cells modulate both the hepatic SEC's functional as well as ultrastructural properties.     

Proton nuclear magnetic resonance spectral profiles of urine in type II diabetic patients

Serial urine samples of 33 type II diabetic patients and 20 control subjects were examined by 1H nuclear magnetic resonance (NMR). Metabolites including lactate, citrate, glycine, alanine, hippurate, trimethylamine-N-oxide, and dimethylamine were identified in all subjects although in higher concentrations in diabetic patients. Other analytes, such as creatine, acetate, betaine, and ketone bodies, were found more frequently and in greater concentrations in diabetics than in controls. In addition, although lactate, citrate, alanine, and hippurate concentrations increased with increasing glycosuria and glycohemoglobin, trimethylamine-N-oxide and dimethylamine were present at high concentrations even in diabetics with good metabolic control. 1H NMR spectroscopy permitted us to explore the relationships among the metabolites present in the urine samples and to obtain information about the disease status in type II diabetic patients.     

The effect of dietary flaxseed supplementation on organic anion and osmolyte content and excretion in rat polycystic kidney disease

Progression of chronic renal failure in the Han:SPRD-cy rat polycystic kidney disease is associated with renal depletion of citric acid cycle metabolites and betaine. Amelioration of this disease by a soy protein diet is associated with retention of citric acid cycle anions, despite increased excretion, and preservation of tissue levels of betaine. As we have recently found that modest dietary supplementation with flaxseed preserves renal function and reduces histologic injury in the Han:SPRD-cy rat, we undertook a high-resolution 1H NMR spectroscopic study of urine and renal tissue extracts from Han:SPRD-cy rats to explore the renal biochemical consequences of a flaxseed diet. There was no significant dietary effect upon organic anion, methylamine, or osmolyte excretion in healthy animals. There was increased citrate excretion in Han:SPRD-cy rats fed flaxseed. Urinary ammonium excretion did not differ, suggesting that the observed increase in citrate excretion was not due to an alkaline effect of diet. Tissue extract studies revealed that disease amelioration was associated with tissue retention of succinate and betaine. Amelioration of Han:SPRD-cy rat polycystic kidney disease by diet is associated with alteration in the handling of citric acid cycle metabolites. Betaine may have a metabolic role in the reduction of chronic renal injury.     

Experimental allergic encephalomyelitis raises betaine levels in the spinal cord of strain 13 guinea-pigs

Chronic relapsing experimental allergic encephalomyelitis, an animal model of multiple sclerosis, was induced in Strain 13 guinea-pigs by subcutaneous injection of spinal cord homogenate and Freund's incomplete adjuvant supplemented with Mycobacterium tuberculosis. High resolution 1H NMR spectra of CNS tissue extracts indicated that the levels of choline metabolites, particularly betaine, were elevated in the spinal cord tissue, the principal site of lesion formation in this guinea-pig strain. The spectra also show that N-acetylated compounds are slightly depleted in the disease. The results are discussed in relation to the biochemical interpretation of NMR spectra obtained in vivo from patients with multiple sclerosis.     

Two cases of pulmonary disease due to Mycobacterium szulgai

1. Groups of five male and five female CD-1 mice received a single intravenous injection of gadolinium chloride at dosages of 0 (saline control), 0.05, 0.1 and 0.2 mmol/ kg. All mice were necropsied 48 h post dose. 2. Plasma analysis showed increases in concentrations of lactate dehydrogenase (both sexes), aspartate aminotransferase and alanine aminotransferase (females only) in the 0.2 mmol/kg group. Cholesterol was elevated at all dosages in both sexes whilst globulin was raised in both sexes at 0.1 and 0.2 mmol/kg. 3. Histological lesions were present at all dosages and increased in severity in a dose-related fashion. The most common lesions were: mineral emboli in capillaries, accumulation of mineral in the mononuclear phagocytic system, hepatocellular necrosis, and lymphoid depletion, necrosis and mineralisation in the spleen. 4. Such observations are similar to those in rats given gadolinium chloride and should be assessed when evaluating the toxicological profile of gadolinium containing compounds being developed for nuclear magnetic resonance imaging.     

Effects of singly administered betaine on hepatotoxicity of chloroform in mice

Effects of a single dose of betaine on the chloroform-induced hepatotoxicity were examined in adult male ICR mice. Administration of betaine (1000 mg/kg, ip) 1 to 7 hr prior to a chloroform challenge (0.25 ml/kg, ip) resulted in remarkable enhancement of hepatotoxicity as indicated by increases in serum sorbitol dehydrogenase (SDH), alanine aminotransferase (ALT) and aspartate aminotransferase (AST) activities. The potentiation of hepatotoxicity was most significant when mice were treated with betaine 4 hr earlier than chloroform. However, a 24 hr prior administration of betaine protected the animals from induction of the chloroform hepatotoxicity. Thus, its effect appeared to be highly dependent on the time lapse from the betaine pretreatment to the challenge of mice with chloroform. Betaine treated either 4 or 24 hr prior to sacrifice did not alter the hepatic contents of cytochrome P-450, cytochrome b5, or NADPH cytochrome P-450 reductase activity. Accordingly the hepatic microsomal p-nitroanisole O-demethylase, aminopyrine N-demethylase, or p-nitrophenol hydroxylase activities were not influenced by the betaine pretreatment. Betaine was shown not to affect any of the enzyme activities associated with glutathione (GSH) conjugation reaction, such as glutathione S-transferases (GSTs), glutathione disulfide (GSSG) reductase and GSH peroxidase irrespective of the time of its administration. When betaine was administered to mice 2-6 hr prior to sacrifice, hepatic GSH level, but not plasma GSH, was decreased significantly. Enhancement of the chloroform hepatotoxicity by betaine correlated well with the reduction in hepatic GSH levels. Both hepatic and plasma GSH levels were elevated in mice 24 hr following the betaine treatment. The results suggest that betaine affects induction of the chloroform hepatotoxicity by modulating the availability of hepatic GSH, which appears to be associated with its role in the transsulfuration pathway in the liver.     

Lactic acid and protein interactions: implications for the NMR visibility of lactate in biological systems

The addition of bovine serum albumin (BSA) to a solution of lactate and alanine resulted in the disappearance of the 1H-NMR resonances from lactate but not alanine. As temperature is increased lactate becomes increasingly NMR visible and after heating above 65 degreesC and cooling to 25 degreesC lactate binding is reduced. With a concentration of 0.2 mM BSA, there was a linear relationship between NMR visible lactate versus total lactate over a range of lactate concentrations of 0.2-35 mM (slope 0.384+/-0.003) indicating that approx. 60% of the added lactate is not visible in the 1H-NMR spectrum. With a 0.1 mM BSA solution, however, the slope was markedly higher indicating that under these conditions only 25-30% of the lactate was NMR invisible. The results from this study indicate that decreased NMR visibility of lactate in proteinaceous solutions is due to non-specific binding which is dependent on the tertiary structure of the protein. This has important implications not only for the interpretation of in vivo 1H-NMR experiments but also for 13C, and 14C studies of metabolism.     

Time course of stomach mineralization, plasma, and urinary changes after a single intravenous administration of gadolinium(III) chloride in the male rat

In a previous experiment it was reported that the intravenous administration of gadolinium chloride (GdCl3) to rats results in a discrete band of interstitial mineralization in the fundic glandular mucosa of the stomach. To investigate the time course for the development of this lesion and its relationship to plasma calcium and phosphate concentrations, 2 experiments were carried out in male Sprague-Dawley rats given a single intravenous dose of 0.07 mmol/kg GdCl3. Plasma calcium and phosphate concentrations approximately doubled between 30 min and 12 hr postdose but had regressed back to near normal values by 24 hr. However, there were no observable clinical signs in treated animals. Histologically, there was progressive mineralization of the lamina propria of the neck region of the fundic glands from 6 hr postdose, forming a distinctive mineral band by 12 hr postdose. At 7 and 14 days postdose the mineral deposits were accompanied by mucous cell hyperplasia, interstitial fibrosis, and a very sparse infiltration of inflammatory cells. By 56 days postdose only occasional mineral deposits remained. Transmission electron microscopy showed mineral first nucleated on collagen in the interstitium, but there was no evidence of cell necrosis. X-ray microanalysis showed that the interstitial mineral was composed of calcium and phosphate in the form of hydroxyapatite; gadolinium (Gd) was only very rarely identified. These findings are consistent with metastatic mineralization. The source, cause, and the exact nature of the excess plasma calcium and phosphate are unknown, and the possible significance of this effect for clinical use of Gd-containing chelates in nuclear magnetic resonance imaging requires further investigation.     

Study of the metabolism of choline and phosphatidylcholine in tumors in vivo using phosphonium-choline

The results of an initial study on the feasibility of using the phosphonium analog of choline to follow the metabolism of phosphatidylcholine in tumors in vivo using 31P NMR are reported. C3H/He mice bearing a mammary carcinoma tumor on the foot pad were fed a choline-free diet supplemented with the phosphonium analog of choline. Metabolites of this compound, including the phosphonium analogs of phosphatidylcholine, phosphocholine, glycerophosphocholine, and betaine were observed noninvasively in vivo in tumors by 31P NMR after 2-3 weeks of feeding. Clearance of these phosphonium-labeled metabolites from tumors was measured after a change to a choline-containing diet. Significant decreases were seen in the levels of the analogs of betaine (P < 0.003) and phosphatidylcholine (P < 0.004) by Day 4. A significant increase in the level of authentic phosphocholine (P < 0.003) occurred over the same time period.     

Primary immunodeficiency diseases at Red Cross War Memorial Children''s Hospital

BACKGROUND: Normally, a Buffalo (BUF) recipient (RT1b) rejects a heterotopically transplanted Lewis (LEW) heart (RT1l) drained into the portal vein (PV) within 14 days. However, the addition of PV administration of 25x10(6) ultraviolet B (UVB)-treated LEW spleen cells (SC) to BUF recipients 7 days before cardiac transplantation results in 70% long-term allograft survival. METHODS: In this study, we used gadolinium chloride (GdCl3) (7 mg/kg/day) to selectively block the phagocytosis of recipient hepatic Kupffer cells before PV injection of UVB-treated donor SC to examine the mechanism of tolerance induction, as measured by in vitro analysis of mixed lymphocyte culture (MLC), T cell cytotoxicity, T helper cell precursors (pTH), and cytotoxic T cell precursors (pCTL) by limiting dilution analysis. RESULTS: A BUF recipient that received untreated or gamma-irradiated LEW SC intraportally reacted to in vitro stimulation by LEW alloantigen with increased MLC proliferation, T cell cytotoxicity, pTH and pCTL frequencies, and interleukin-2 production. In contrast, SC from BUF that received UVB-treated LEW SC were hyporesponsive on MLC stimulation by donor LEW alloantigen and exhibited markedly reduced cytotoxicity, pTH and pCTL frequency, and interleukin-2 production. However, normal in vitro responsiveness resulted with stimulation by third-party Brown-Norway (RT1n) SC, thus indicating that the systemic hyporesponsiveness was specific for the UVB donor alloantigen given PV. On the other hand, GdCl3 given by intravenous injection daily for 3 days before PV alloantigen blocked the induction of in vitro hyporesponsiveness. CONCLUSION: Therefore, prevention of alloantigen sequestration by GdCl3 inhibition of hepatic Kupffer cell phagocytosis was pivotal in preventing the development of portal venous tolerance.     

Hepatic protein synthesis rate of liver specimens as a predictor of viability in rat cold ischemia liver transplantation model

BACKGROUND/AIMS: We have previously reported that the hepatic protein synthesis rate, calculated as the uptake rate of L-[4.5 3H] leucine by the protein fraction during a 10-min incubation of a 16-G needle biopsy specimen of liver tissue, represents a high level of liver function and is therefore useful for evaluating liver function. We investigated the hepatic protein synthesis rate level in a pre-transplant liver to learn if it might predict the outcome in a rat orthotopic liver transplantation model. METHODS: Grafts were stored, liver specimens were obtained using a 21-G Chiba type II skinny needle, and the hepatic protein synthesis rate was calculated. Subsequently, liver transplantation was performed, and the hepatic protein synthesis rate level of revascularized liver, tissue blood flow rate, serum alanine aminotransferase, lactate dehydrogenase, hyaluronic acid, ketone body rate, and 2-week survival were examined. RESULTS: The hepatic protein synthesis rate of pretransplant liver was correlated with parameters of post-transplant liver function: hepatic protein synthesis rate of the revascularized liver (r=0.92, p<0.0001), tissue blood flow rate (r=0.77, p<0.004), serum alanine aminotransferase (r=-0.69, p<0.003), lactate dehydrogenase (r=-0.54, p<0.03), hyaluronic acid (r=-0.86, p<0.0002), and ketone body rate (r=0.57, p<0.02). Pretransplant hepatic protein synthesis rate in survivors was 263.6+/-54.2 nmol/mg protein/10 min, while that in nonsurvivors was significantly lower at 162.0+/-39.0 (p<0.0001). When evaluation was made using a logistic regression model, the accuracy predicted using the value of hepatic protein synthesis rate was 95% (19/20). CONCLUSIONS: These results suggest that measuring the hepatic protein synthesis rate of the grafts with a 21-G Chiba type II skinny needle may be a predictive criterion in the assessment of graft viability.     

Sinusoidal endothelial cell damage by activated macrophages in rat liver necrosis

BACKGROUND: Massive hepatic necrosis caused by fibrin deposition in the hepatic sinusoids develops with hepatic macrophage activation in rats given endotoxin after administration of heat-killed Corynebacterium parvum. Targeted cells of such macrophages were investigated. METHODS: In C. parvum-treated rats, the pathological appearance of liver cells was serially measured in serum following endotoxin administration and compared with the appearance in the perfusate during closed liver perfusion with endotoxin. RESULTS: Serum activities of tumor necrosis factor, purine nucleoside phosphorylase present in both hepatocytes and sinusoidal endothelial cells, and levels of alanine aminotransferase were higher after 30 minutes, 1 hour, and 3 hours, respectively. Pretreatment of rats with gadolinium chloride, an inhibitor of macrophage function, reduced this liver injury. Although alanine aminotransferase activity remained almost unchanged in the liver perfusate, purine nucleoside phosphorylase activity increased. This increase was reduced when rats were pretreated with gadolinium chloride. There was sinusoidal endothelial cell damage around hepatic macrophages in the liver perfused with endotoxin. CONCLUSIONS: Activated hepatic macrophages may cause sinusoidal endothelial cell damage leading to hepatocyte necrosis in rats given C. parvum and endotoxin.