Enzymatic determination of 1-14C-glucose in pig plasma

Summary A simple and cheap one-step enzymatic method has been developed for the determination of 1-¹⁴C-glucose in plasma. C-1 of glucose is cleaved off as CO₂ by treatment with hexokinase, glucose-6-phosphate dehydrogenase, and 6-phosphogluconic dehydrogenase. True 1-¹⁴C-glucose activity is then calculated as the difference between total radioactivity and radioactivity remaining after enzyme treatment and evaporation. The reaction is shown to be quantitative and specific, thus eliminating both labelled metabolites and label recycled to other positions in glucose. Two different types of pig experiments show that 1-¹⁴C-glucose, when determined by this method, is as irreversible a tracer as the commonly used 3-³H-glucose.     

The role of hepatocyte-specific staining in liver pathology

The advantages of MRI in the investigation of liver disease are well documented. Recent developments, including fast scanning technique and new MRI contrast agents, enable improved detection and characterization of focal liver lesions. Therefore, a definitive diagnosis can be made avoiding invasive procedures, such as liver biopsy. In this article, a special emphasis is placed on the clinical use of combined perfusional and hepatocyte-selective MRI contrast agents, which allow us to obtain morphologic and vascular information, owing to the dynamic study, as well as functional information, owing to the hepatocyte-selective phase of enhancement. Different clinical scenarios are considered in order to highlight the proper use of the hepatocyte phase to noninvasively characterize and detect different focal liver lesions.     

Evaluation of a novel non-invasive 13C-glucose breath test for the identification of diabetes mellitus in cirrhotic patients

Aim: Diabetes mellitus (DM) has been reported to worsen the long-term prognosis of cirrhotic patients, and many studies have reported that DM is an independent risk factor for hepatocellular carcinoma. However, an accurate diagnosis of DM is sometimes difficult in cirrhotic patients. Recently, a novel non-invasive ¹³C-glucose breath test has been reported to be useful for diagnosing insulin resistance in non-cirrhotic patients. The aim of this study was to evaluate the efficacy of this tool for the identification of DM in cirrhotic patients. Methods: Thirty eight cirrhotic patients with normal fasting serum glucose and hemoglobin A1c levels underwent the ¹³C-glucose breath test and the oral glucose tolerance test. Blood and breath samples were collected at baseline and at 30, 60 and 120 min after ingestion of 100 mg ¹³C-labeled glucose and 75 g glucose. Results: There was a strong correlation between the change in the concentrations at 2 h for the measured ¹³C-glucose breath test (2h-BT) and the 2 h plasma glucose level (r = −0.60, P < 0.0001). In a receiver–operator curve analysis using the 2h-BT, the area under the curve was determined to be 0.88, with a sensitivity and specificity (cut-off value of 3.5‰) of 82% and 85%, respectively, for the detection of DM. Multivariate analysis showed the 2h-BT to be an independent parameter to identify DM. Conclusion: The ¹³C-glucose breath test is a useful tool and has the potential to become a routine outpatient examination for the screening of DM in cirrhotic patients.     

Estimation of 14C-Triolein Assimilation as a Test of Lipid Assimilation

Two tests of lipid assimilation based on estimation of ¹⁴C-triolein assimilation from expiratory ¹⁴CO₂ (breath test) and from serum radioactivity of ¹⁴C, respectively, were investigated in 48 consecutive patients suspected of having malassimilation. Patients with proven malassimilation had significantly lower expiration of ¹⁴CO₂ and lower serum radioactivity of ¹⁴C than patients with normal lipid assimilation. The se-¹⁴C test correctly diagnosed significantly more patients with malassimilation than the breath test; the diagnostic efficiencies were 0.87 and 0.74, respectively. The results of both tests correlated with measurement of faecal fat. However, within the group of patients with proven malassimilation the results of the breath test correlated poorly with faecal fat, whereas a significant correlation was found between the se-¹⁴C test and faecal fat within this group. Correspondingly, the correlation between the results of the breath test and the se-¹⁴C test was poor, indicating that intermediate metabolism influences the results.     

Evaluation of [14C] aminopyrine breath test,peripheral clearance of [99mTc]EHIDA,and serum bile acid levels in liver function and disease

The purpose of this study is to evaluate the diagnostic value of the following tests in the assessment of patients with chronic liver disease (CLD) and cholestatic syndrome (CS):(1) aminopyrine breath test, measuring¹⁴CO₂ excretion in the expired air, (2) peripheral clearance of [^99mTc]EHIDA, and (3) postprandial levels of glycocholic acid (GCA) and glycochenodeoxycholic acid (GCDCA). The results indicate that: (1)¹⁴CO₂ 2-hr excretion rate is a specific and sensitive marker of liver function, with good correlation with postprandial bile acid levels, [^99mTc]EHIDA retention, and the conventional tests of serum albumin and prothrombin time. (2) Peripheral clearance and retention of [^99mTc]EHIDA increased in both groups of CLD and CS vs controls, but it does not discriminate between the two. (3) Postprandial bile acids were elevated in CLD, particularly those of GCDCA, whereas GCA levels were significantly elevated in CS compared with CLD. This may be due to increased synthesis and entry into the blood. (4) The combination of [¹⁴C]aminopyrine breath test and postprandial levels of GCDCA enhance the diagnostic value, specificity, and sensitivity in the assessment of patients with CLD.     

Study of the metabolism in vitro of glucose in the rat epididymal fat tissue

Summary The rat epididymal fat tissue was incubated with glucose differently labelled with ¹⁴C, and the effect of palmitate bound to albumin was studied in the absence or presence of insulin. No inhibition by palmitate of the glucose metabolism was observed. On the contrary, it was demonstrated that palmitate stimulates the different pathways of the glucose metabolism, particularly the fatty acid neosynthesis. The results are discussed in relation to the glucose and palmitate metabolism in the adipose cell, and in relation to the role in vivo of the adipose tissue in the decrease of the glucose assimilation observed in conditions which abnormally increase the serum level of free fatty acids.We should like to thank Miss M. Plattenburg for her technical assistance.     

Insulin responsiveness of adipose tissue from normal weight subjects with early diabetes

Summary In normal weight subjects, classified by a 2-h glucose infusion test as having normal (11), borderline (3) or pathological (9) carbohydrate tolerance (CHT), subcutaneous adipose tissue was removed under intracutaneous anesthesia by surgical biopsy. The biological responsiveness of isolated adipocytes as well as adipose tissue fragments measured as incorporation of (1-¹⁴C) glucose into CO₂ or triglycerides was studied in the absence or presence of different insulin concentrations. In persons with normal CHT the insulin-stimulated (62.5 μU/ml) glucose conversion to CO₂ by adipocytes as well as fat pads increased significantly up to 156 ± 14% and 285 ± 30%, respectively. Insulin enhanced the glucose incorporation into triglycerides up to 154 ± 20% (fat cells) and 258 ± 30% (fat pads) in adipose tissue from subjects displaying a normal CHT. Rates of glucose oxidation and triglyceride synthesis were markedly reduced in adipose tissue obtained from patients with borderline or pathological CHT. A significant positive relationship was found between glucose oxidation to CO₂ and triglyceride production of fat cells and fat pads (r=0.964 and 0.783, respectively). There was no correlation with responsiveness of adipose tissue to insulin and insulin secretion during glucose infusion test. The results indicate that sensitivity to insulin of target cells might be important for the development of carbohydrate intolerance also in normal weight subjects. Early diabetes: all stages of carbohydrate intolerance before overt diabetes. Investigations carried out within the medical research project ‘Diabetes mellitus and disturbances of lipid metabolism’, Ministry of Health, GDR.     

Role of the 13C-methacetin breath test in the assessment of acute liver injury in a rat model

AIM: To evaluate the role of the ¹³C-methacetin breath test (¹³C-MBT) in the assessment of acute liver injury in a rat model.METHODS: Acute liver injury in rats was induced by a single intraperitoneal injection of D-galactosamine (D-GalN). Forty-eight male Sprague-Dawley rats were randomly assigned to a control group (n = 8) and five model groups (each n = 8), and acute liver injury was assessed at different time points (6, 12, 24, 48 and 72 h) after D-GalN injection. The ¹³C-MBT, biochemical tests, 15-min retention rate of indocyanine green (ICG_R15), and liver biopsy were performed and compared between the control and model groups. Correlations between parameters of the ¹³C-MBT (T_max, MV_max, CUM₁₂₀ and DOB_max), biochemical tests, ICG_R15 and liver necrosis score were also analyzed using Spearman’s correlation analysis.RESULTS: T_max, MV_max, CUM₁₂₀ and DOB_max, as well as most of the traditional methods, correlated with the liver necrosis score (r = 0.493, P < 0.05; r = -0.731, P < 0.01; r = -0.618, P < 0.01; r = -0.592, P < 0.01, respectively). MV_max, CUM₁₂₀ and DOB_max rapidly decreased and were lower than those in the controls as early as 6 h after D-GalN injection (3.84 ± 0.84 vs 5.06 ± 0.78, P < 0.01; 3.35 ± 0.72 vs 4.21 ± 1.44, P < 0.05; 52.3 ± 20.58 vs 75.1 ± 9.57, P < 0.05, respectively) and reached the lowest point 24 h after D-GalN injection. MV_max, CUM₁₂₀ and DOB_max returned to normal levels 72 h after D-GalN injection and preceded most of the traditional methods, including liver biopsy.CONCLUSION: The ¹³C-MBT is a sensitive tool for the timely detection of acute liver injury and early prediction of recovery in a rat model. Further clinical studies are warranted to validate its role in patients with acute liver injury.     

Hyperglycaemia induced by glucose infusion in the unrestrained pregnant rat during the last three days of gestation: Metabolic and hormonal changes in the mother and the fetuses

Summary Continuous glucose infusion was used to induce mild hyperglycaemia in unrestrained pregnant rats during the last three days of pregnancy. Control pregnant rats were infused with distilled water. Fetuses were studied after normal or prolonged pregnancy. Fetuses from glucose-infused rats, compared with controls, showed higher plasma glucose levels, increased plasma insulin and lower plasma glucagon concentrations. Pregnancy prolonged until day 23.5 resulted in a rise in the glucagon/insulin ratio from 6.5 to 67 in fetuses from control rats and from 1.3 to 13 in fetuses from glucose-infused rats. Concurrently in fetuses from control rats, liver phosphoenolpyruvate carboxykinase activity increased markedly and liver glycogen stores decreased sharply. In fetuses from glucose-infused rats, liver phosphoenolpyruvate carboxykinase activity rose and glycogen content decreased, but to a lesser extent. These results show that both the A and B cells of the rat fetal pancreas are sensitive to chronic glucose stimulation.     

NO-mediated vasodilation in the rat liver: Role of hepatocytes and liver endothelial cells

Background/Aims: Nitric oxide (NO) is a potent vasodilator. We investigated the mechanisms responsible for this effect in the liver.Methods: Isolated perfused rat liver and cultures of endothelial sinusoidal cells and hepatocytes were used.Results: L-arginine (10⁻³ M) and NO donor Sin-1 (10⁻⁵ M) respectively increased the liver flow by 52% (p<0.01) and 93% (p<0.01) vs controls. The NO synthase inhibitor N^w-nitro-L-arginine (10⁻³ M) and the guanylate cyclase inhibitor methylene blue (10⁻⁵ M) respectively decreased the basal liver flow by 26% and 16% (p<0.05) and inhibited the vasodilating effects of L-arginine. L-arginine (10⁻³ M) increased nitrite concentration in hepatocyte culture (77.25±7.40 μmol · 1⁻¹ vs 14.70±3.55 μmol · 1⁻¹ in controls; p<0.01) and in liver endothelial cell culture (0.36±0.09 μmol · 1⁻¹ vs 0.12±0.05 μmol · 1⁻¹ in controls; p<0.05). N^w-nitro-L-arginine inhibited the basal production and abolished the L-arginine-induced production of nitrites both in hepatocyte and in liver endothelial cell cultures. The concentration of nitrites in the hepatocyte supernatant rose from 14.70±3.55 μmol⁻¹ · 1⁻ to 150.50±45.55 μmol · 1⁻¹ in the presence of a combination of interleukin-1β, TNF a and interferon γ.Conclusions: Under basal conditions, NO regulates the vascular tone of liver circulation. Both liver endothelial cells and hepatocytes can be implicated. NO production by hepatocytes may increase during inflammation.     

Extrapancreatic action of the sulphonylurea gliquidone: post-receptor effect on insulin-stimulated glycogen synthesis in rat hepatocytes in primary culture

Summary The effects of a sulphonylurea, gliquidone, on insulin binding and the insulin induced rate of glycogen synthesis, were studied in rat hepatocytes in primary culture. Hepatocytes were cultured for 48 h. During the second 24 h of this period, the hepatocytes were incubated with or without gliquidone (5 mg/l). The binding of ¹²⁵I-insulin and the insulin stimulation of glycogen synthesis from ¹⁴C-glucose were measured. Gliquidone influenced neither insulin binding nor the basal rate of glycogen synthesis, but it did enhance the effect of insulin on glycogen synthesis. Responsiveness was increased by gliquidone at all insulin concentrations used (10–10,000 mU/l); at 1000 mil/l the drug increased glycogen synthesis from 310 to 430% above the basal rate. Half-maximal stimulation was reached in control cells at an insulin concentration of 95 mU/l and in gliquidone-treated cells at 94 mU/l, which indicates unchanged insulin sensitivity. Based on these experiments with cultured rat hepatocytes it appears that the extrapancreatic action of gliquidone is not mediated by an effect on insulin binding.     

The metabolism of recombinant erythropoietin in the isolated perfused rat liver

ABSTRACT— Indirect evidence points to extrarenal organs, presumably the liver, as the site of degradation of erythropoietin (EPO). The metabolism of both fully glycosylated and desialated intrinsically labelled ³⁵S-Cysteine recombinant human erythropoietin (rhEPO) was therefore studied in isolated Wistar rat livers perfused in a recirculating mode for 180 min with a hemoglobin-free medium containing rhEPO. Perfusate and bile levels of rhEPO were measured by RIA. Total ³⁵S-radioactivity in liver, bile and perfusate as well as non-acid precipitable radioactivity in perfusate were determined. In addition, detection of ³⁵S-radioactivity was performed after subcellular fractionation of rat livers perfused with desialo-³⁵S-Cysteine rhEPO. While concentrations of fully glycosylated ³⁵S-Cysteine rhEPO did not exhibit any detectable decrease during perfusion, desialo-³⁵S-Cysteine rhEPO was rapidly cleared from the perfusate. After 60 min of perfusion, only 32% of the initial levels of both immunoreactive rhEPO and total radioactivity remained in the perfusate. Quantitative hepatic accumulation of desialated tracer was demonstrated. Subcellular fractionation showed extensive hepatic degradation of the desialated tracer. Furthermore, during perfusion progressively larger amounts of small molecular weight degradation products of the tracer were found in the perfusate. Bile excretion of both fully glycosylated and desialated tracer was negligible. The significance of hepatic metabolism of desialo-³⁵S-Cysteine rhEPO was supported by reduced removal of desialo-³⁵S-Cysteine rhEPO from plasma in hepatectomized rats. It is hypothesized that continuous in vivo desialation is a crucial rate-limiting step in the degradation of circulating EPO.     

Proliferating cell nuclear antigen (PCNA) expression in regenerating rat liver after partial hepatectomy

Proliferating cell nuclear antigen (PCNA) is a nuclear protein maximally elevated in the S phase of proliferating and transformed cells and is recognized by the monoclonal antibody PC-10 in paraffin tissue sections. The liver regenerative process after partial hepatectomy in rats was estimated with thein vivo incorporation of [³H]thymidine into liver DNA and the liver thymidine kinase activity. The expression of PCNA in rat liver after partial hepatectomy was performed by immunohistochemical staining with PC-10 in paraffin embedded tissues, at different time intervals up to 240 hr. Proliferating cell nuclear antigen expression, [³H]thymidine incorporation into DNA, and liver thymidine kinase activity exhibited marked oscillations during the liver regenerative process. A close relationship was demonstrated among DNA synthesis, thymidine kinase activity, and PC-10 score. Our results suggest that PC-10 monoclonal antibody may be used as a worthwhile proliferation index in the evaluation of the rate of liver regeneration in rats.     

Interactions of glucagon and fructose in the control of glycogenolysis in perfused rat liver.

By measuring the specific radioactivity of glucose released from isolated perfused livers of normal, fed rats in the presence of [U-14C]fructose, the gluconeogenetic and glycogenolytic contributions to glucose production were estimated. After 20 min of perfusion with 4 mM fructose, glycogenolysis was inhibited by 40% in the absence and by 70% in the presence of glucagon (3 nM). Glucagon decreased the release of lactate plus pyruvate and enhanced glucose formation from fructose without affecting its uptake. Glycerol (4 mM) and xylitol (3 mM) had qualitatively similar, but smaller effects on glucagon-stimulated glycogenolysis. The glucagon-mediated phosphorylase b to a conversion was not altered by fructose, indicating that glycogenolysis was decreased as a consequence of an inhibition of phosphorylase a. During the first minutes after the addition of fructose, decreased ATP/AMP ratios and tissue Pi levels correlated with a transient increase of phosphorylase a activity. It was concluded that the effects of fructose on the control of hepatic glycogenolysis and glucose production were the result of a complex interplay between a transient b to a conversion of phosphorylase and an inhibition of the a-form of the enzyme, possibly by fructose 1-phosphate and other phosphorylated metabolites.     

Expression of lipopolysaccharide binding protein and its receptor CD14 in experimental alcoholic liver disease

AIM:To evaluate the relationship between the expression oflipopolysaccharides(LPS)binding protein(LBP)and CD14mRNA and the severity of liver injury in alcohol-fed rats.METHODS:Twenty Wistar rats were divided into twogroups:ethanol-fed group(group E)and control group(group C).Group E was fed with ethanol(5-12g.kg~(-1).d~(-1))and group C received dextrose instead of ethanol.Rats ofthe two groups were sacrificed at 4 weeks and 8 weeks.Levels of endotoxin and alanine transaminase(ALT)inblood were measured,and liver pathology was observedunder light and electronic microscopy.Expressions of LBPand CD14 mRNA In liver tlssues were determined by RT-PCRanalysis.RESULTS:Plasma endotoxln levels were increased moresignificantly in group E(129±21 )ng.L~(-1) and(187±35)ng·L~(-1) at 4 and 8 wk than in control rats(48±9) ng·L~(-1) and(53±11)ng·L~(-1),respectively(P<0.05).Mean values ofplasma ALT levels were(1867±250)nkat·L~(-1) and(2450±367) nkat.L~(-1) in Group E.The values were increased moredramatically In ethanol-fed rats than in Group C after 4 and 8weeds.In liver section from ethanol-fed rats,there weremarked pathological changes(steatosis,cell infiltration andnecrosis).In ethanol-fed rats,ethanol administration led toa significant increase in LBP and CD14 mRNA levelscompared with the control group(P<0.05).CONCLUSION:Ethanol administration led to a significantIncrease in endotoxin levels in serum and LBP and CD14mRNA expressions in liver tissues.The increase of LBP andCD14 mRNA expression might wake the liver more sensitiveto endotoxin and liver injury.     

O6-Methylguanine repair of methylated DNA in vitro: Cell cycle-dependence of rat liver methyltransferase activity

Summary O⁶-Methylguanine DNA transferase activity was investigated in liver proteins obtained at various intervals after partial hepatectomy and/or after hydroxyurea-induced synchronization of the liver cell cycle. Liver proteins were incubated with ³H-methylated calf thymus DNA as previously described by Pegg et al. (1981). The loss of O⁶-methylguanine was measured by radiochromatography of DNA hydrolysates. The extent of O⁶-methylguanine repair differed during the cell cycle: the activity increased in late G₁, reached a maximum in early S phase and declined in late S phase and G₂M. These results indicate that hepatocytes are endowed with an increased DNA repair capacity for this promutagenic lesion during the period of highest transformation sensitivity in the cell cycle. Though increased, however, this repair potential does not, because of its exhaustibility, appear to be sufficient to prevent initiation of transformation after high doses of alkylating carcinogens.Supported by grants from Deutsche Forschungsgemeinschaft     

Different concentrations of various radiopharmaceuticals in the two main liver lobes: a preliminary study in clinical patients

Background At clinical scintigraphic examinations of the abdomen using single photon emission computed tomography (SPECT), we have observed a different distribution between the left and right main liver lobes of various radiopharmaceuticals. This was studied retrospectively in clinical patients.Methods Examinations with [¹²³I]-metaiodobenzylguanidine MIBG; (n = 19), a ^99mTc-labelled monoclonal antibody against granulocytes (n = 18), and ¹¹¹In-pentetreotide (n = 26) were assessed. There was no known history of, or risk factor for liver disease, and all lobes showed a uniform activity distribution. Twenty healthy volunteers underwent consecutive examinations with ^99mTc-dimethyliminodiacetic acid (HIDA). The activity ratios between the left and right main liver lobes were calculated from the transverse tomographic (SPECT) sections.Results The left : right lobar activity ratio for [¹²³I]-MIBG was (mean ± SD) 1.25 ± 0.21 (null hypothesis = 1.00; P < 0.001); for the antibody, acquisition after 3–5 h was 0.98 ± 0.06 (NS) and after 20–24 h, 0.99 ± 0.11 (NS); for ¹¹¹In-pentetreotide, 0.90 ± 0.09 (P < 0.001); for ^99mTc-HIDA, immediate acquisition, 0.68 ± 0.12 (P < 0.001) and acquisition at 7 min, 0.66 ± 0.12 (P < 0.001).Conclusions The differences in tracer uptake between the liver lobes cannot be caused only by differences in blood flow. One explanation of the higher uptake of [¹²³I]-MIBG by the left lobe may be a greater presence of catecholamines and a higher sympathetic nerve density in this liver portion. Consequently, there may be a functional difference between the two main liver lobes.     

C-Phenylalanine and C-Methacetin breath test to evaluate functional capacity of hepatocyte in chronic liver disease

BACKGROUND: To grade liver damage, Child-Pugh classification is used but these tests do not reflect the quantitative functional hepatic reserve. AIMS: 13C-Phenylalanine Breath Test and 13C-Methacetin Breath Test are evaluated as possible tools, being both safe and easy to perform, to quantify functional hepatic reserve in chronic liver disease patients. PATIENTS: Both tests were performed in 48 healthy volunteers and 48 chronic liver disease patients. METHODS: Breath samples were collected after taking 13C-Phenylalanine (100 mg) and 13C-Methacetin (75 mg). 13CO2 enrichment was measured using mass spectrometry RESULTS: Both tests discriminated the hepatic function, decreasing results of the 13CO2 enrichment agreeing with the increasing severity of the hepatic patient (13C-Phenylalanine Breath Test multiple correlation coefficient: 0.72, global p<0.001; Methacetin Breath Test: 0.73, p<0.001). Correlation between 13C-Phenylalanine Breath Test and Methacetin Breath Test was 0.63, p<0.001. If both tests were pathological, the sensitivity for the diagnosis of hepatic dysfunction was high (98%), although the specificity decreased to 60%. Best results were obtained at 30 minutes with 13C-Phenylalanine Breath Test and at 10 minutes with Methacetin Breath Test. CONCLUSIONS: Both 13C-Phenylalanine Breath Test and Methacetin Breath Test are safe and easy tests to perform and both are able to discriminate the hepatic functional capacity between the different groups studied.     

Glomerular hyperfiltration during the onset of diabetes mellitus in two strains of diabetic mice (C57BL/6Jdb/db and C57BL/KsJdb/db)

Summary GFR estimated by the total clearence of⁵¹Cr-EDTA increases from 41 to 60 ml/min·m² at the onset and during the development of marked hyperglycaemia and obesity in female diabetic mice (db/db) of the C57BL/6J and the C57BL/KsJ strains (blood sugar rises from 160 to 400 mg/100 ml). GFR decreases slowly in older diabetic mice (>120 days old) approaching the control values (42±7 ml/ min·m²) and then decreases still further. The total clearance of¹⁴C-hippuric acid is unaltered indb/db mice and controls between 45 and 150 days of age (96±20 ml/min·m²). This suggests no alteration of RPF during the development of the diabetic syndrome. The glomerular hyperfiltration of diabetic mice lasts until they are 120 days old and shows no correlation with the different blood glucose levels typical for diabetic mice (db/db) of the two strains.     

Biomarkers of liver fibrosis: prospective comparison of multimodal magnetic resonance,serum algorithms and transient elastography

Abstract

Background and aims

Accurate biomarkers for quantifying liver fibrosis are important for clinical practice and trial end-points. We compared the diagnostic performance of magnetic resonance imaging (MRI), including gadoxetate-enhanced MRI and ³¹P-MR spectroscopy, with fibrosis stage and serum fibrosis algorithms in a clinical setting. Also, in a subset of patients, MR- and transient elastography (MRE and TE) was evaluated when available.