Sirius red histophotometry and spectrophotometry of sections in the assessment of the collagen content of liver tissue and its application in growing rat liver.

By means of staining with Sirius Red F3BA in a saturated picric acid solution, the collagen contents of rat livers with varying degrees of fibrosis have been measured quantitatively in fixed and sectioned material, using both histophotometry in situ and extraction of bound dye with colorimetric analysis. These findings have been correlated with chemical assays of the hydroxyproline content in homogenates from the same livers. It appears that a highly significant correlation exists between both section-based analysis methods and the hydroxyproline content, the Spearman-Rank correlation coefficients being virtually identical. For analysis of collagen accumulation in rat liver, both section-based methods seem to be useful and reliable, the extraction method giving the quickest results for large-scale screening, and the histophotometric method being more appropriate to take readings in selected areas. With human liver material, indications have been obtained for the existence of large sampling errors due to inhomogeneous distribution of collagen deposits. Using the extraction method, no significant changes could be observed in the volume density of collagen during postnatal growth from 1 week to 21 months in rat liver: only on the third day after birth was a higher value of collagen/total protein obtained, possibly due to a higher water content of the hepatocytes. Partial hepatectomy was found to have no influence at all on the collagen content of rat liver during the period of restorative growth or after it.

Assessment of tissue iron overload by nuclear magnetic resonance imaging.

PURPOSE: The ability of stored intracellular iron to enhance magnetic susceptibility forms the basis by which tissue iron can be detected by nuclear magnetic resonance (NMR) imaging. We used this technique to assess myocardial, spleen, and liver iron content in patients with known or suspected iron overload disorders. PATIENTS AND METHODS: Spin echo NMR images were obtained in 30 patients; 20 had chronic anemias treated by multiple blood transfusions, five had idiopathic hemochromatosis, and five had non-hemochromatotic liver disease with elevated serum ferritin levels and no stainable iron on liver biopsy. The acquisition of oblique images through the short axis of the left ventricle permitted assessment of left ventricular function, while demonstrating the liver and spleen on the same image. Iron content was assessed using a signal intensity ratio of organ (spleen, liver, or myocardium) to skeletal muscle. RESULTS: In patients with multiple blood transfusions, iron content was highest in liver, followed by the spleen. Significant iron overload was detected in the myocardium of only one patient. Left ventricular systolic wall thickening was normal in patients receiving multiple blood transfusions. Two patients with treated idiopathic hemochromatosis had normal signal intensity ratios, and three untreated patients had evidence of significant deposits of iron in the liver and spleen as indicated by a reduction in signal intensity ratios (0.2 +/- 0.01 and 0.9 +/- 0.01, respectively). Five patients with non-hemochromatotic liver disease and high serum ferritin levels had normal signal intensity ratios by NMR imaging. CONCLUSION: NMR imaging is a useful method of detecting tissue iron and distinguishing disease due to iron overload. Myocardial iron deposition is a late event, occurring after accumulation of iron in the spleen and liver.

Criteria for reference value assessment of elements in human tissues.

The availability of updated and reliable reference values for elements in biological tissues and fluids plays a key role in the correct planning and performance of clinical, toxicological and environmental studies. The basic principles for a proper analytical approach in order to obtain such data are outlined. This in turn should significantly reduce the wide range of figures reported for most elements as "normal" intervals in the scientific literature. Some applications of these guidelines are illustrated in detail in the case of human lungs, liver and kidneys for which tentative reference values are assessed for a number of minor and trace elements, namely, Al, Ba, Cd, Cr, Cu, Li, Mg, Mn, Pb, Sr, V and Zn.

Studies of marginal zinc deprivation in rhesus monkeys. III. Use of liver biopsy in the assessment of zinc status.

Studies of marginal zinc deficiency in rhesus monkeys have demonstrated that plasma Zn levels are often a poor indication of Zn status. To better assess the Zn status of these animals, we examined their liver concentration of Zn as well as of other minerals, metallothionein (MT), and superoxide dismutase (SOD). Liver-wedge biopsies were obtained from adult rhesus monkeys fed for 15 mo, either a control (100 micrograms Zn/g) or a marginally Zn deficient diet (4 micrograms/g; ZD). Liver Zn and MT concentrations were lower in ZD monkeys than in controls whereas iron concentration was higher in ZD monkeys than in controls. Liver copper, manganese, and magnesium concentrations and activities of CuZnSOD and MnSOD were similar in the two groups. Data from the groups were pooled for regression analysis. Measurement of liver Zn and MT concentrations are useful in the assessment of the effects of long-term Zn deprivation in primates.

Presence of diguanosine tri, tetra-, and pentaphosphates in commercial samples of GTP and guanosine 5'-tetraphosphate.

Commercial samples of GTP and guanosine 5'-tetraphosphate were analyzed, with or without previous treatment with alkaline phosphatase, by high-pressure liquid chromatography on a Hypersil ODS column. They showed the presence of diguanosine 5',5"'-Pl,Pn-tri, tetra-, and pentaphosphates in varying amounts depending on the sample, but usually in proportions of around 0.3%.

Assessment of taurine deficiency in cystic fibrosis.

Bile acid taurine deficiency is common in cystic fibrosis (CF) and is thought to be associated with impaired fat absorption. The relationship between the glycine: taurine bile acid conjugation ratio (bile acid G/T ratio) and taurine concentrations in plasma, urine and leucocytes was examined in 27 CF children aged 4-15 yr. The bile acid G/T ratio was elevated in serum in 14 of the 27 and in duodenal juice in 5 of 6 children. Subgroups of CF children with elevated and normal bile acid G/T ratio and controls (n = 8) all had similar plasma, urine and leucocyte taurine concentrations. The results suggest that either taurine measurement in plasma, urine and leucocytes does not accurately reflect stores elsewhere in CF, or that taurine deficiency is confined to bile acids.

Nuclear magnetic resonance assessment of adenosine triphosphate (ATP) dynamics in ischemic mouse livers perfused with adenine and ribose.

Hepatic energy stores are essential to liver viability. We used a mouse liver perfusion model and MR spectroscopy to study the effect of adding two precursors of ATP (adenine and ribose) on ATP dynamics during ischemia and reperfusion. Using Krebs-Henseleit buffer with or without added adenine and ribose made little difference in the ATP decay rate during ischemia, but the recovery of ATP during reperfusion was more complete when adenine and ribose were added to the buffer. These findings suggest that the addition of the precursors of ATP, adenine and ribose, to perfusate after ischemia can accelerate and enhance ATP recovery.

The manganese(II) economy of rat hepatocytes.

The liver is known to play a central role in regulating the content of manganese in the rat. Experiments with isolated hepatocytes have demonstrated a specific high-affinity transport system for Mn(II). Manganese that is transported into hepatocytes is partitioned between the cytosol and noncytosolic compartments. The cytosolic ion is tightly bound by macromolecules. After uptake of Mn(II), incubation of hepatocytes in the absence of Mn(II) or in the presence of chelating agents fails to release the metal unless the plasma membrane is disrupted.

Assessment of lymphocyte function during vitamin A deficiency.

Two variables of immune status were measured in vitamin A-deficient (A-), vitamin A-sufficient pair-fed (A + PF), and healthy control (A+) rats to examine the mechanisms of immune regulation by vitamin A. Parallel samples were run in bovine fetal serum (BFS)-containing medium and serum-free medium to control BFS-origin vitamin A in the cultures. Splenocytes derived from A- rats showed significantly depressed blastogenic response to all lectins tested in both mediums when compared with responses of A + PF and A+ rats. The splenocyte blastogenic response of A + PF rats was significantly lower than that of A+ (control) rats only when cultured in BFS medium and stimulated by lentil lectin, lipopolysaccharide, or concanavalin A mitogens. Thymic lymphocyte blastogenic transformation assays were equivocal. Splenic immunosuppression could not be linked to significant reductions in surface glycoprotein receptor availability, since splenocytes of A- rats, as well as thymocytes, were capable of binding fluorescein isothiocyanate-conjugated lectins in the same capacity as splenocytes of A+ and A + PF rats. The combination of depressed cellular function and adequate lectin binding to viable cells implies that the regulation of immunohomeostasis by vitamin A was achieved through intracellular mechanisms, possibly selective genomic expression.

Characteristics and specificity of the glucocorticoid "carrier" of rat liver plasma membrane.

The uptake of corticosterone by highly purified plasma membrane vesicles of rat liver was studied by a rapid-centrifugation technique which allows uptake measurements within 5 s. The vesicles are free of soluble cytoplasmic constituents. Therefore, association of hormone with the vesicle is attributed entirely to components of the vesicle-membrane. Half maximal uptake is reached at 8 s at 21 degrees C. At 15 degrees C transition of the lipid state in the membrane leads to a decrease of uptake, a characteristic property common to membrane mediated processes. The uptake of corticosterone is saturable and reversible but does not follow normal saturation kinetics. The apparent dissociation constants of three uptake systems bear direct relation to the concentration of free corticosterone in rat plasma (4-16 nM) supporting a physiological role for the system. Uptake of corticosterone decreases with decreases in vesicular volume; about 50% of the hormone is bound specifically and 50% is transported to the lumen of the vesicle. Since outflow of intravesicular hormone also occurs readily, the uptake and transport is proposed to be mediated by putative "carriers". The "carrier" preferentially transports glucocorticoids; dexamethasone is not taken up by this putative molecule. Steroids with 5 alpha conformation are more potent inhibitors of the "carrier" for corticosterone than 5 beta-steroids. Androgens and estrogens are weak competitors of corticosterone. The affinity of the "carrier" for several hormones differs considerably from that of the cytoplasmic receptor. Morris hepatoma cells (MH 3924) do not take up corticosterone. Our results prompt us to propose the hypothesis that the transport function of the "carrier" and the binding of the hormone by the cytoplasmic receptor are two different entities; perturbation of the "carrier" may lead to steroid unresponsiveness. Normal expression of steroid hormone activity is manifested in the concerted action of the functionally sound cell membrane "carrier" and the intracellular receptor.

[Biochemical quality assessment of broiler production using a hydrogen bacteria biomass in the diet]. / Biokhimicheskaia otsenka kachestva produktsii broilerov pri ispol'zovanii v ratsione biomassy vodorodnykh bakterii.

The authors studied the biochemical composition of the meat of broilers, eggs, liver and muscles of laying hens of 3 generations on a 5, 10, 25, 50 and 100% (broilers), 10 and 20% (laying hens) replacement of the animal protein quota in the diet by protein obtained from the hydrogen bacteria Alcaligenes eutrophus L-1. No deterioration of the quality of the produce was found from the standpoint of the main biochemical parameters.

Biochemical and histological assessment of hepatic lipid in sudden infant death syndrome.

A biochemical and histological study of hepatic lipid in children dying from the sudden infant death syndrome (SIDS) and children of a similar age dying explicably are reported. Contrary to a previous report based on histological assessment of hepatic lipid, no significant increase of total lipid content in livers of children dying from SIDS was found. Analysis of hepatic phospholipid fatty acid esters, however, revealed a significant difference between SIDS and children of similar age dying acutely and explicably. The phospholipid abnormality found in SIDS was similar to that found in children dying subacutely with hypoxia and would be consistent with increased cell membrane fluidity. The implications of these findings in the pathogenesis of SIDS are discussed.

Value of copper-associated protein in diagnostic assessment of liver biopsy.

Of 1361 consecutive liver biopsy specimens, 24% contained orcein-positive granules. The highest incidence of positivity was found in biliary disease (90.9%), long before cirrhosis had developed, whereas in chronic non-primarily biliary disease, positive results were almost exclusively in patients with well established cirrhosis. Orcein-positive granules were never found in acute liver disease. These granules were also demonstrated in tumour cells of primary hepatocellular tumours (benign 4 of 4 cases; malignant 9 of 37 cases), while all the secondary tumour deposits were negative. In our view the additional information obtained by this technique warrants its adoption as a routine procedure.

A simplified isotope dilution procedure for the assessment of vitamin A status in rats.

In four separate experiments, rats with liver reserves of vitamin A ranging from deficient to high were administered physiological doses (5, 10 or 20 micrograms) of [11,12-3H] retinol, either by gastric intubation or intraperitoneal injection, and sacrificed after either 7 days or 5 hours. Measurements were then made of radioactivity and vitamin A in plasma and liver. In all four experiments there was a highly significant negative correlation between the logarithm of the specific activity of plasma vitamin A and the logarithm of total liver vitamin A.

An assessment of the immunofluorescence technique as a method for demonstrating the histological localization of tetrahydrocannabinol in mammalian tissues.

The use of the indirect immunofluorescence technique as a method for demonstrating the histological localization of tetrahydrocannabinol (delta-THC) has been examined. The experimental protocol was designed in order that optimal staining conditions with respect to temperature, the length of time of incubations and washes, and the dilution of the antisera should be defined. No marked differences were detected between frozen sections of liver from normal and delta-THC-injected mice. Results from radiotracer experiments using human liver suggest that the success of the method is dependent upon the solubility characteristics of the antigen-antibody complex.

Assessment of liver iron content in 271 patients: a reevaluation of direct and indirect methods.

Two direct methods for hepatic iron assessment (liver iron concentration and stainable liver iron--judged by a new histologic grading) and three indirect methods (serum iron and transferrin saturation, deferoxamine-chelation test, and ferritinemia) were reevaluated in 271 patients. These patients consisted of: 103 with idiopathic hemochromatosis, 39 with alcoholic cirrhosis, 54 with noncirrhotic alcoholic liver diseases, 13 with nonalcoholic liver diseases, and 62 with miscellaneous disorders. The results indicate that: (a) liver iron concentration, well correlated with mobilized excess iron (r = 0.88; p < 0.01), is the method of reference and validates the proposed histologic grading; (b) serum ferritin, which is in good correlation with liver iron concentration (r = 0.80; p < 0.01), is a valuable indirect method for hepatic iron evaluation; (c) regarding the other indirect methods a "boundary zone" may be delimited, thus corresponding to liver iron concentration values of 10.7 mumol/100 mg dry liver weight, beyond which values of serum iron less than 28.6 micrometer or transferrin saturation less than 45% or chelatable iron less than 45 mumol/24 h are rare; and (d) using the various indirect methods, there is a marked risk in idiopathic hemochromatosis to underestimate and in alcoholic liver diseases to overestimate hepatic iron content.