Diuresis-dependent excretions of low-molecular mass proteins in urine: beta 2-microglobulin, lysozyme, and ribonuclease

An investigation was carried out into how the low-molecular mass proteins beta 2-microglobulin, lysozyme, and ribonuclease were excreted over 8 h after high fluid intake (22 ml/kg of body weight in 15 min). With increasing urine flow rate the amount of lysozyme excreted per hour or per millimole creatinine increased more markedly than that of beta 2-microglobulin while at the same time the excretion rate of ribonuclease decreased. The effect of urinary flow upon the excretion rates of the various low-molecular mass proteins has to be considered as a preanalytical factor when these proteins are used as indicators of tubular dysfunction.     

Circulating histocompatibility antigen (HLA) gene products may help differentiate benign from malignant indeterminate pulmonary lesions

Background

This study explores the potential diagnostic utility of soluble Human Leukocyte Antigen (sHLA) molecules differentially released by lung adenocarcinoma and benign lung lesions.

Urinary enzymes and low-molecular-mass proteins as indicators of diabetic nephropathy

We measured the excretion rates of six urinary enzymes that either originate from the proximal renal tubule, like alanine aminopeptidase (EC 3.4.11.2), alkaline phosphatase (EC 3.1.3.1), gamma-glutamyltransferase (EC 2.3.2.2), and N-acetyl-beta-D-glucosaminidase (EC 3.2.1.30), or that are typical low-molecular-mass proteins, like lysozyme (EC 3.2.1.17) and pancreatic ribonuclease (EC 3.1.27.5). These rates were compared with those of total protein and albumin in urine of 36 insulin-dependent diabetic men and 30 healthy men. Seventeen of the diabetics had "clinical proteinuria," defined as excretion of more than 7.5 g of protein per mole of urinary creatinine (group B). Group A comprised the 19 diabetics without proteinuria. Except for gamma-glutamyltransferase, the excretions of enzymes and proteins were significantly higher in diabetics than in controls and were greater in group B than in group A. N-Acetyl-beta-D-glucosaminidase was the analyte most often increased in group A (89%), followed by albumin and alkaline phosphatase (each 32%). All patients in group B showed increased excretion of N-acetyl-beta-D-glucosaminidase. We conclude from the comparative data that this enzyme may be useful as an early predictor of diabetic nephropathy.     

Stability of enzymes in urine at 37°C

The activity of alanine aminopeptidase, alkaline phosphatase, γ-gluta-myltransferase, lactate dehydrogenase and β-$\text{N-}\text{acetyl}\text{-}\text{d}\text{-}\text{glucosaminidase}$ in urine at 37°C was investigated by a model simulating in vivo conditions.The stability of these urinary enzymes is influenced particularly by pH. At low pH values in urine (about pH 5.0) the four first-mentioned enzymes rapidly lose a considerable part of their activity, whereas β-$\text{N-}\text{acetyl}\text{-}\text{d}\text{-}\text{glucosaminidase}$ is inactivated at higher pH values in urine (about at pH 8.0).This inactivation effect is also time-dependent and can be modified by urinary substances such as creatinine, urea and electrolytes. To avoid misinterpretation of enzyme activity determinations in urine, the simultaneous measurement of urinary pH should be performed.     

Immunohistochemical evidence for flupirtine acting as an antagonist on theN-methyl-d-aspartate and homocysteic acid-induced release of GABA in the rabbit retina

When rabbit retinas are exposed in vitro to specific excitatory amino acid receptor agonists certain GABAergic amacrine cells are activated to cause a release of GABA. The GABA that is not released can be detected by immunohistochemistry. Exposure of tissues to kainate or NMDA each caused a characteristic change in the GABA immunoreactivity. CNQX antagonised the kainate effect specifically while MK-801 counteracted the influence of NMDA The effect produced by kainate was mimicked by domoic acid while the influence of homocysteic acid was identical with NMDA. Flupirtine alone did not influence the nature of the GABA immunoreactivity and so did not act as a kainate or NMDA agonist. However, flupirtine counteracted the influence produced by NMDA and homocysteic acid but had no effect on the kainate and domoic acid responses. Thus in this system flupirtine acts as an NMDA antagonist.     

Flupirtine reduces functional deficits and neuronal damage after global ischemia in rats

Global cerebral ischemia leads to selective neuronal damage in the CA1 sector of the hippocampus and in the dorsolateral striatum. In addition, it results in deficits in spatial learning and memory as shown by an increase in escape latency and swim distance during the escape trials and a reduction of time spent in the quadrant of the former platform position during the probe trial of the water maze. Flupirtine is a non-opioid, centrally acting analgesic which has been shown to be neuroprotective against N-methyl-

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-aspartate (NMDA)-mediated toxicity in vitro. The purpose of the present study was to investigate the potential protective effect of flupirtine in vivo with both behavioural and histological measures of global cerebral ischemia. Global ischemia was induced by four-vessel-occlusion (4VO) for 20 min in rats. Flupirtine was administered at a dose of 5 mg/kg i.p. either 20 min before and 50 min after occlusion (pre-treatment) or directly and 70 min after occlusion (post-treatment). 1 week after surgery, spatial learning and memory was tested in the Morris water maze. Pre-treatment with flupirtine reduced the increase in escape latency and in swim distance induced by 4VO. It also diminished the deficit in spatial memory as revealed by an increase in time spent in the quadrant of the former platform position during the probe trial which was reduced by 4VO. Post-treatment with flupirtine had no effect on the deficits in spatial learning and memory induced by 4VO. Neuronal damage in the CA1 sector of the hippocampus and in the striatum produced by 4VO was significantly attenuated with pre-treatment of flupirtine whereas post-treatment did not affect this neuronal damage. The present data demonstrate that pre-treatment with flupirtine exerts a protective effect on hippocampal and striatal neuronal damage and on deficits in spatial learning induced by 4VO. © 1997 Elsevier Science B.V. All rights reserved.     

Prevention of glutathione depletion-induced apoptosis in cultured human RPE cells by flupirtine

We have recently reported that the non-opiate analgesic, flupirtine, counteracts apoptosis in cultures of human retinal pigmented epithelial (RPE) cells induced by deprivation of serum, oxygen and glucose (experimental ischaemia). In the present study, human RPE cells grown on coverslips were treated with buthionine sulphoxamine (BSO), a compound that inhibits glutathione biosynthesis. BSO caused a dose-dependent reduction in culture density and an increase in the number of cell nuclei that were positively labelled by the terminal deoxynucleotidyl transferase-mediated dUTP nick end labelling (TUNEL) procedure. These data show that reduction of glutathione levels causes apoptosis in the RPE cultures. When flupirtine gluconate was co-incubated with BSO, it dose-dependently prevented the induction of apoptosis. The most effective concentration of flupitine found to inhibit cell death caused by BSO (1 micro M - 1 mM) was 100 micro M. The presence of serum (2% or 10%) in the culture medium did not have any effect on the outcome of apoptosis and overall cell death caused by BSO. Futhermore, melatonin, also known to reduce experimental ischaemia-induced overall cell death and apoptosis of cultured RPE cells had only a mild protective effect at 1 mM. The combined data suggest that flupirtine prevents apoptosis by increasing the cellular levels of reduced glutathione and/or protects the cells against the damaging effects of reactive oxygen species (ROS) that are produced subsequent to inhibition of glutathione production.