Blood Plasma Metabolome Profiling at Different Stages of Renal Cell Carcinoma.

Early diagnostics significantly improves the survival of patients with renal cell carcinoma (RCC), which is the prevailing type of adult kidney cancer. However, the absence of clinically obvious symptoms and effective screening strategies at the early stages result to disease progression and survival rate reducing. The study was focused on revealing of potential low molecular biomarkers for early-stage RCC. The untargeted direct injection mass spectrometry-based metabolite profiling of blood plasma samples from 51 non-cancer volunteers (control) and 78 patients with different RCC subtypes and stages (early stages of clear cell RCC (ccRCC), papillary RCC (pRCC), chromophobe RCC (chrRCC) and advanced stages of ccRCC) was performed. Comparative analysis of the blood plasma metabolites between the control and cancer groups provided the detection of metabolites associated with different tumor stages. The designed model based on the revealed metabolites demonstrated high diagnostic power and accuracy. Overall, using the metabolomics approach the study revealed the metabolites demonstrating a high value for design of plasma-based test to improve early ccRCC diagnosis.

Non-enzymic glycosylation of horse spleen and rat liver ferritins.

Incubation of horse spleen ferritin and rat liver ferritin with [14C]glucose, [14C]mannose or [14C]fucose resulted in the covalent incorporation of the sugar into ferritin. The rate of reaction depended on the concentrations of ferritin and sugar and time of incubation. The order of this nonenzymic incorporation was glucose greater than mannose greater than or equal to fucose. Glucosylated or mannosylated ferritin was not retained by concanavalin A. The plasma half-life of rat liver ferritin and apoferritin was found to be 20 min. This value remained unaffected by in vitro glucosylation or mannosylation of ferritin. It is suggested that varying degrees of glycosylation might account for the occurrence of isoferritins.

Influence of zinc on iron uptake by monolayer cultures of rat hepatocytes and the hepatocellular ferritin.

Monolayer cultures of isolated rat hepatocytes on collagen gels were used to study the effect of zinc on (a) 59Fe uptake by the hepatocytes; (b) 59Fe uptake by the hepatocellular ferritin and (c) biosynthesis of ferritin and total proteins. While deposition of iron into the cellular ferritin was inhibited by zinc in a dose-dependent manner, iron uptake by the hepatocytes was inhibited significantly only at high concentrations of zinc. Biosynthesis of total proteins or ferritin was not affected by zinc. Since only a small fraction of the cellular 65Zn was found to be associated with ferritin, it was concluded that zinc was not deposited in ferritin. Intracellular distribution of 59Fe revealed that newly accumulated iron went into an iron pool the size of which could be increased by the presence of zinc.

Studies on the mechanism of transferrin iron uptake by rat reticulocytes.

Mechanism of transferrin iron uptake by rat reticulocytes was studied using 59Fe- and 125I-labelled rat transferrin. Whereas more than 80% of the reticulocyte-bound 59Fe was located in the cytoplasmic fraction, only 25-30% of 125I-labelled transferrin was found inside the cells. As shown by the presence of acetylcholine esterase, 10-15% of the cytoplasmic 125I-labelled transferrin might have been derived from the contamination of this fraction by the plasma membrane fragments. Electron microscopic autoradiography indicated 26% of the cell-bound 125I-labelled transferrin to be inside the reticulocytes. Both the electron microscopic and biochemical studies showed that the rat reticulocytes endocytosed their plasma membrane independently of transferrin. Sepharose-linked transferrin was found to be capable of delivering 59Fe to the reticulocytes. Our results suggest that penetration of the cell membrane by transferrin is not necessary for the delivery of iron and that, although it might make a contribution to the cellular iron uptake, internalization of transferrin reflects endocytotic activity of the reticulocyte cell membrane.

Bimodal role of endogenous interleukin-6 in concanavalin A-induced hepatitis in mice.

Acute concanavalin A (Con A)-induced hepatitis in mice is an animal model for hepatic injury induced by activated T cells. The evolution of hepatic involvement can be followed from hour to hour by measuring serum transaminase levels. We investigated the possible role of endogenous interleukin-6 (IL-6) in this model. We found serum IL-6 levels and splenic IL-6 mRNA during Con A-induced hepatitis to be significantly lower in interferon-gamma (IFN-gamma)-deficient mice, which are resistant against the Con A-induced syndrome, than in wild-type ones, suggesting that systemic IL-6 production favors development of hepatic injury. However, IL-6-deficient mice proved to be more susceptible to the disease than wild-type mice, indicating that endogenous IL-6 plays a predominantly hepatoprotective role. Experiments in which wild-type mice were treated with anti-IL-6 antibodies, before or after Con A challenge, allowed us to reconcile these contrasting observations. The antibody injections resulted in a biphasic alteration of serum IL-6 levels, initial neutralization being followed by rebound increased levels due to accumulation of IL-6 in the form of antigen-antibody complexes. The effect of antibody on disease severity differed depending on the time of injection. Antibody injection at 2.5 h post Con A resulted in delayed disease manifestation, whereas treatment initiated before Con A resulted in accelerated disease. We conclude that endogenous IL-6 plays a bimodal role. IL-6 present before Con A challenge as well as that induced in the very early phase after Con A injection triggers hepatoprotective pathways. Continuation of IL-6 production beyond this early phase, by some other pathway, seems to be harmful to hepatocytes.

Calciotrophic hormones during experimental hypocalcaemia and hypercalcaemia in spontaneously diabetic rats.

1,25-Dihydroxyvitamin D(3) (1,25(OH)(2)D(3)) concentrations have been found to be decreased in diabetic humans and rats. To investigate further the regulation of plasma Ca in diabetes, first we measured Ca(2+), P, Mg, parathyroid hormone(1-34) (PTH), and total and free 1,25(OH)(2)D(3) in male spontaneously diabetic rats 7 and 28 days after the onset of glycosuria. Secondly, we studied changes in the levels of PTH and 1,25(OH)(2)D(3) in response to hypocalcaemia induced by an i.v. infusion of EGTA (2.5%, wt/vol.) for 24 h, and changes in the levels of 1,25(OH)(2)D(3) in response to an i.v. infusion of rat PTH (10 microgram over 24 h) without or with concomitant EGTA infusion (producing hypercalcaemia or normo/hypocalcaemia respectively), in diabetic and control rats. Ca(2+), P, Mg and PTH concentrations remained within the control ranges after 7 and 28 days of glycosuria; 1,25(OH)(2)D(3) concentrations were decreased after 7, but not after 28, days of glycosuria. PTH concentrations showed a similar rise during EGTA-induced hypocalcaemia in control and diabetic rats compared with saline-infused rats, whereas 1,25(OH)(2)D(3) concentrations were unchanged in both groups. Total and free 1,25(OH)(2)D(3) levels were comparably (about 3-fold) increased during PTH, but not during combined PTH and EGTA infusion in control and diabetic rats. Total 1, 25(OH)(2)D(3) concentrations were lower in the diabetic groups infused with saline or PTH than in their respective controls, and there was a similar trend in the PTH+EGTA-infused group; free 1, 25(OH)(2)D(3) levels, however, were normal or increased in the diabetic groups, confirming our previous data. The novel finding of this study is that, despite severe insulin deficiency and altered 1, 25(OH)(2)D(3) levels, the in vivo response of PTH levels to hypocalcaemia and the in vivo response of 1,25(OH)(2)D(3) levels to PTH in diabetic rats are comparable with those found in nondiabetic rats.

Blood antioxidant levels in patients with alcoholic liver disease correlate with the degree of liver impairment and are not specific to alcoholic liver injury itself.

BACKGROUND: Enhanced production of reactive oxygen species may play a pathogenic role in alcoholic liver injury. AIMS: To investigate whether various antioxidant parameters in blood are affected in different stages of alcoholic liver disease and how specific the changes are relative to non-alcoholic cirrhosis. METHODS: Patients with alcohol abuse without cirrhosis (n=14), with alcoholic cirrhosis [Child-Pugh scores A (n=9), B (n=5) and C (n=18)] and with non-alcoholic cirrhosis [Child-Pugh score C (n=6)] and healthy controls (n=13) were studied. Levels of reduced glutathione and glutathione peroxidase activity in blood, erythrocytic superoxide dismutase activity and carotenoids, alpha-tocopherol and malondialdehyde in plasma were measured. RESULTS: Levels of reduced glutathione were significantly decreased in Child-Pugh score C cirrhotics, alcoholic or not in origin, whereas oxidized glutathione and glutathione peroxidase activity were not affected. Superoxide dismutase activity and alpha-tocopherol levels were not significantly different in the various groups. Carotenoid levels were significantly lower in alcoholic cirrhotics (Child-Pugh score C) vs. controls. Malondialdehyde levels were elevated only in cirrhotics Child-Pugh score C, alcoholic or non-alcoholic. CONCLUSIONS: Levels of reduced glutathione and malondialdehyde reflect the degree of liver impairment, more than the relation with alcohol intake. Decreases in several antioxidant levels are not specific to alcoholic liver injury.

Safely reducing manual urine microscopy analyses by combining urine flow cytometer and strip results.

We aimed to reduce the number of manual urine microscopy examinations safely by cross-interpretation of the Sysmex UF-100 (TOA Medical Electronics, Kobe, Japan) and urine strip results such that microscopy would be performed if there was discordance between the UF-100 and urine strip results. We also evaluated the usefulness of the optional UF-100 expert software. We performed 2 studies: study 1 to establish review rules for eventual microscopic examination; study 2, a validation study. Our review rates were 40% and 48% and those of UF-100 software were 16% and 32% for the 2 studies. Our false-positive and false-negative results, among the samples not flagged for microscopic review, were acceptably low. We did not find a good correlation between the microscopic classification of RBC morphologic features and the classification given by the UF-100. Since incorporation of the automated urine strip reader and the UF-100 in routine use, our manual microscopy has been reduced to less than 40%.

Disappointing dipstick screening for urinary tract infection in hospital inpatients.

AIM: To compare the performance of leucocyte esterase and nitrite dipstick tests with microscopic examination and culture of first morning urines (n = 420) of hospital inpatients. RESULTS: The sensitivity, specificity, and negative predictive value of the leucocyte esterase test for the cutoff of > 10 WBC/microliter were 57%, 94%, and 68%, respectively. For > 5 WBC per high power field (HPF) these variables were 84%, 90%, and 93%. For > 10(5) colony counts/ml, the sensitivity of the nitrite test was 27%, specificity 94%, and negative predictive value 87%. When either leucocyte esterase or nitrite positivity was accepted as a marker of urinary tract infection, the sensitivity was 78%, specificity 75%, and negative predictive value 94%, and there were 22% false negative results. Semiquantitative microscopic estimation of bacteria per HPF yielded 40% false positives. CONCLUSIONS: Leucocyte esterase and nitrite dipstick tests are not suitable for screening for urinary tract infections.

Blood gas analysis: POCT versus central laboratory on samples sent by a pneumatic tube system.

BACKGROUND: We aimed to compare the results of blood gas analyses, performed at point-of-care and in the central laboratory (CL), on samples sent via a pneumatic tube system (PTS). METHOD: Specimens from two locations (lung function laboratory (LFL) and pneumology wards) were first analysed locally and then sent to the CL via PTS. RESULTS: While none of the blood gas samples from the LFL had air bubbles, 21 samples from the wards (n=31) had air bubbles in them. The mean time difference between the first (POCT) and the second (CL) determinations from LFL was 13.3+/-5.4 min (n=27) and from the wards 20.2+/-11 min. For pO2 the differences between LFL and CL results, for patients undergoing a 100% O2 test, were unacceptably large. For pO2 range 41-407 mm Hg, the difference was -2.4+/-3.2 (n=25). For the samples from the wards, the difference in pO2 between ward (range 37-183 mm Hg) and CL was -13+/-18 mm Hg. CONCLUSION: Irrespective of air bubbles, the transport by PTS has very little or no effect on pH and pCO2. If air bubbles cannot be excluded with certainty, PTS is not an appropriate transport medium for measurement of pO2 on blood gas samples.

Multiphasic modelling of ligand/acceptor interactions. The hydrophobicity-dependent binding of relatively small amphiphilic substances to acceptor proteins and the nature and facedness of acceptor sites.

The modelling of multiphasic ligand/acceptor equilibrium binding systems proceeds at three logically distinct levels: (1) A suitable response quantity, e.g. the amount of acceptor-bound ligand nEL, is expressed as a function of the ligand concentrations [Li] (L = A,B,...) in the compartment i that contains the acceptor sites. One thus obtains a response function nEL = f1([Li]). In general, the equilibrium constants KL contained in such mathematical models are physically ill-defined. (2) Each local concentration [Li] is further expressed as a function of [Laq], the corresponding concentration in the aqueous phase, leading to nEL = f2([Laq]). In this way, the constants KL are transformed into effective constants K'L which (i) can be assessed experimentally and (ii) depend on ligand hydrophobicity in a way that is characteristic of the binding site. Formulation of the functions f1 and F2 only requires knowledge of the reactions in which the acceptor sites participate directly. (3) For each ligand, the experimentally accessible total ligand concentration Lt is expressed as a function of [Laq], leading to concentration balance equations Lt = Lt([Laq]). The latter transformation takes account of any reactions, distinct from ligand/acceptor interaction, in which the ligands are involved, e.g. binding to additional protein sites. As a result of steps 2 and 3, each binding system is described by a set of simultaneous equations dependent on the auxiliary variable [Laq]: (i) the response function f2([Laq]) and (ii) a concentration balance for each ligand Lt = Lt([Laq]). The formulae are rendered more conscise and their discussion and application to data fitting are simplified by introducing, for each ligand L, a function FL characterising the distribution of unbound monomeric ligand over the various partition compartments. When the acceptor acts on unbound ligand, the formulae are further expressed in terms of a new auxiliary variable i.e. the total concentration of unbound monomeric ligand microL. In contrast to data analysis as a function of local concentrations, analysis in terms of total ligand concentrations avoids losing sight of alternate hypotheses about the nature of the binding sites. The present formulation has also permitted clarification of several consequences of the multiphasic nature of the binding systems that, as yet, have been poorly recognised.(ABSTRACT TRUNCATED AT 400 WORDS)

Microscopic haematuria in giant cell arteritis.

Giant cell arteritis (GCA) is traditionally considered to spare the kidney, although an uncontrolled study reported microscopic haematuria in 10 out of 30 patients with GCA. To study the frequency and the characteristics of microscopic haematuria in GCA, we retrospectively studied 42 patients with biopsy-proven GCA, 39 patients with polymyalgia rheumatica (PMR) and 62 control patients >or=60 years of age, admitted to the general internal medicine unit. Patients with pyuria, significant bacteriuria or a known haematuric disorder were excluded. Microscopic haematuria was defined as the presence of >5 red blood cells (RBC) per high-power field (sediment counts) or of >8 RBC/microl (direct counting). Microscopic haematuria was present at presentation in 47.6% of the GCA patients, versus 17.9% of the PMR patients (P = 0.005) and 21.0% of the control patients (P = 0.008). Urinary RBC were predominantly dysmorphic in all GCA patients in whom RBC morphology was assessed (n = 7). Presenting symptoms, renal function, arterial blood pressure and degree of leukocyturia did not differ significantly between GCA patients with or without haematuria. After the initiation of corticosteroid therapy, microscopic haematuria was no longer detectable in 25 of 35 GCA patients (71.4%). Microscopic haematuria of renal origin is frequent but generally benign in patients with GCA. Its presence, if unassociated with blood pressure elevation or renal function deterioration, helps to rule in rather than to rule out the diagnosis of GCA. In the typical setting invasive urologic and nephrologic work-up may not be warranted.

Comparison of planned and measured rectal dose in-vivo during high dose rate Cobalt-60 brachytherapy of cervical cancer.

Cobalt-60 (Co-60) is a relatively new source for the application of high-dose rate (HDR) brachytherapy. Radiation dose to the rectum is often a limiting factor in achieving the full prescribed dose to the target during brachytherapy of cervical cancer. The aim of this study was to measure radiation doses to the rectum in-vivo during HDR Co-60 brachytherapy. A total of eleven HDR brachytherapy treatments of cervical cancer were recruited in this study. A series of diodes incorporated in a rectal probe was inserted into the patient's rectum during each brachytherapy procedure. Real-time measured rectal doses were compared to calculated doses by the treatment planning system (TPS). The differences between calculated and measured dose ranged from 8.5% to 41.2%. This corresponds to absolute dose differences ranging from 0.3 Gy to 1.5 Gy. A linear relationship was observed between calculated and measured doses with linear regression R(2) value of 0.88, indicating close association between the measured and calculated doses. In general, absorbed doses for the rectum as calculated by TPS were observed to be higher than the doses measured using the diode probe. In-vivo dosimetry is an important quality assurance method for HDR brachytherapy of cervical cancer. It provides information that can contribute to the reduction of errors and discrepancies in dose delivery. Our study has shown that in-vivo dosimetry is feasible and can be performed to estimate the dose to the rectum during HDR brachytherapy using Co-60.

Dysmorphic erythrocytes and G1 cells as markers of glomerular hematuria.

We aimed to assess the utility of the percentage of G cells and dysmorphic erythrocytes in the diagnosis of glomerular hematuria in pediatric patients. We determined the percentage of dysmorphic erythrocytes and G1 cells in urine samples from patients with glomerulonephritis and other renal diseases of non-glomerular origin. There was excellent correlation and agreement between results obtained by counting the cells in counting chambers and in urine sediments. With cut-off values of > or =1%, > or =2%, > or =5%, and 10% for G1 cells, sensitivities for the detection of glomerulonephritis were 62%, 40%, 28%, and 10% and specificities were 89%, 95%, 95%, and 98%. For the dysmorphic erythrocytes cut-off values of > or =10%, > or =20%, 50%, and > or =90% gave respective sensitivities of 95%, 95%, 93%, and 62% and specificities of 24%, 34%, 43%, and 85%. In 38% of cases of biopsy-proven glomerulonephritis no G1 cells were found. For cut-off values of > or =50% dysmorphic erythrocytes and > or =1% G1 cells, the sensitivity and specificity were 60% and 91%. For cut-off values of > or =50% dysmorphic or > or =1% G1 cells, sensitivity and specificity were 93% and 44%. Our results show that neither the percentage of dysmorphic erythrocytes nor the G1 cell count is of adequate sensitivity and specificity to enable reliable differentiation of glomerular and non-glomerular hematurias. Both tests are needed to achieve >90% sensitivity and specificity.

Mechanism and stereochemistry of vinyl-group formation in haem biosynthesis.

5-Aminolaevulinate containing tritium at C-3 and C-5 was converted into haem using a preparation of anaemic chicken blood. The biosynthetic haem was degraded to ethylmethyl maleimide and haematinic acid which had relative tritium radioactivity of 0.58 and 1.0 respectively. These results indicated that in the formation of the vinyl group of haem only one of the hydrogen atoms from the beta-positions of two propionate side chains of coproporphyrinogne III was removed. Haem was also biosynthesised from [(3R)-3H1]2-oxoglutarate. The determination of relative radioactivity in ethylmethyl maleimide and haematinic acid endorsed the above conclusion and further indicated that the pro-R hydrogen atoms located at the beta-positions of the propionate side chains are retained in haem biosynthesis. In order to explore the status of hydrogen atoms located at the alpha-positions of propionate side chains haem was biosynthesised using [2RS)-3H2]succinate, [(2R)-3H1]succinate and [(2S)-3H1]succinate. Degradation of the three samples of haem into ethylmethyl maleimide and haematinic acid showed that both the vinyl groups of haem are formed through the loss of pro-S hydrogen atoms located at the beta-positions of the propionic acid side chains of coproporphyrinogen III. The results further showed that the hydrogen atoms located at the alpha-positions of the side chains are not involved in the biosynthesis of haem. Various mechanisms for the formation of vinyl groups in the biosynthesis are discussed.

Unsatisfactory performance of flow cytometer UF-100 and urine strips in predicting outcome of urine cultures.

UF-100 flow cytometer and urine strip results were cross-interpreted to predict culture outcomes. The best negative predictive value was obtained with bacteria at > or =1,000/microl, white blood cells at > or =20/microl, or leukocyte esterase positivity. Nine of 24 false negatives were clinically significant. Thus, UF-100 and urine strip results do not accurately predict the outcome of cultures.

Effect of acute lead intoxication on the ultrastructure of rat erythroblasts and reticulocytes. Morphometric analysis and röntgen micro-analysis.

Effect of 17 hr intoxication of lead on the different maturation stages of erythroid cells were studied in rat. Morphometric methods were used to analyse the lead-induced ultrastructural changes in the early, intermediate, late erythroblasts and reticulocytes. It was found that the toxic effect of lead increases with maturation. Energy-dispersive Röntgen micro-analysis showed that fibrillar structures within vesicles and endoplasmic reticulum contained lead.

Simultaneous determination of vitamins A and E and carotenoids in plasma by reversed-phase HPLC in elderly and younger subjects.

A reversed-phase high-performance liquid-chromatographic method for the simultaneous determination of retinol, alpha-tocopherol, alpha-carotene, beta-carotene, cryptoxanthin, lutein/zeaxanthin, and lycopene is described. This method was applied to plasma measurements in healthy young and elderly subjects. The plasma, deproteinized with ethanol, is extracted twice with n-hexane. After evaporation, the residue is dissolved in 50 microL of tetrahydrofuran and made up to 200 microL with ethanol. Samples (50 microL) are injected onto a 250 x 4.6 mm column of 5-microns-particle Spherisorb ODS1 (Phase Separations) that had been equilibrated with solvent mixture A:B (90:10 by vol) [A = 100 mmol/L ammonium acetate in methanol: acetonitrile (80:20 by vol) and B = 100 mmol/L ammonium acetate in water] at 2 mL/min. The analytes are eluted by running a 12-min linear gradient to 100% A; solvent A is then maintained for 10 min. Intrabatch CVs were 2.3%, 3.3%, 2.8%, 3.6%, 3.6%, and 3.0% for retinol, alpha-tocopherol, lutein/zeaxanthin, cryptoxanthin, lycopene, and beta-carotene, respectively. The corresponding interbatch CVs were 4.9%, 5.8%, 12.3%, 6.5%, 8.0%, and 3.4%.

Mechanism and stereochemistry of the 5-aminolaevulinate synthetase reaction.

1. Two mechanisms for the biosynthesis of 5-aminolaevulinate from glycine and succinyl-CoA (3-carboxypropionyl-CoA) are considered. One of the mechanisms involves the retention of both the C-2 H atoms of glycine during the synthesis of 5-aminolaevulinate, whereas the other predicts the retention of only one of the C-2 H atoms of glycine. 2. Highly purified 5-aminolaevulinate synthetase from Rhodopseudomonas spheroides was used to show that the C-2 H atom of glycine with R configuration is specifically removed during the biosynthesis of 5-aminolaevulinate. 3. The mechanism of the condensation therefore differs from the analogous reaction of the biosynthesis of sphinganine from palmitoyl-CoA and serine, in which the C-2 H of serine is retained (Wiess, 1963).