Improved determination of urinary oxalate with alkylamine glass bound barley oxalate oxidase

The measurement of oxalate in urine was improved by employing barley oxalate oxidase immobilized on alkylamine glass beads affixed in a glass beaker. The minimum detection limit was 3.6 mg l(-1) urine. The recovery of added oxalate was 88.9+/-9.2%. Within- and between-assay coefficients of variation (CV) were <4.0 and <9.4%, respectively. The urinary oxalate values were obtained by a commercial kit method and the present method showed a good correlation (0.999). The method is free from tedious handling of glass beads and Cl- interference.     

Determination of urinary oxalate with alkylamine glass-bound sorghum oxalate oxidase and horseradish peroxidase

Oxalate in urine was analyzed using sorghum oxalate oxidase and horseradish peroxidase immobilized on alkylamine glass through glutaraldehyde. The minimum detection limit was 0.46 g/0.1 ml urine. The recovery of added oxalate was 97.5%. Within and between assay coefficients of variation were <3.5% and <6.5% respectively. A good correlation (r=0.9234) was found between oxalate values obtained by a commercial kit method and the present method. The method is unaffected by Cl– and NO3– found in urine.     

Spectrophotometric determination of oxalate in urine and plasma with oxalate oxidase

In order to establish a standard procedure for the spectrophotometric determination of urinary and plasma oxalate with oxalate oxidase (Laker, M.F., et al. (1980) Clin. Chem. 26, 827-830; Sugiura, M., et al. (1980) Clin. Chim. Acta 105, 393-399) and to define the limitations of the method, the procedures and reactions involved in the assay have been examined. Among the chromogenic hydrogen donors for peroxidase tested, a combination of 3-methyl-2-benzothiazolinone hydrazone (MBTH) and sodium N-sulfopropylaniline (HALPS) was found to be best for the oxalate determination under the conditions used. Urine contained substance(s) which were inhibitory to the measurement of hydrogen peroxide by the peroxidase-catalyzed oxidative condensation of MBTH and HALPS, but they were largely removed by charcoal treatment at pH 5.6 without significant loss of oxalate. Deproteinization of plasma was carried out by ultrafiltration through a membrane cone (Centriflo CF-25) at neutral pH. The plasma oxalate ultrafiltrability under the conditions employed was calculated to be approximately 95%. A standard assay system for oxalate in these urine and plasma samples was then set up based on a series of studies on the reactions involved in the assay. In the case of normal plasma, however, the absorbance change was very small due to the low concentration of oxalate, and in addition, pretreatment of plasma with excess oxalate decarboxylase followed by the ultrafiltration and oxalate determination did not abolish completely the oxalate oxidase-dependent absorbance increase. It was concluded that the enzymic method was useful for the assay of urinary oxalate and in detecting elevated levels of plasma oxalate such as those in hemodialysis patients but was not sensitive enough to determine accurately the normal or decreased level of oxalate in plasma. The apparent concentration of oxalate in normal human plasma was measured in this work as 3.5 +/- 0.8 microM (mean +/- S.D., n = 8), and this result was interpreted to mean that the concentration of plasma oxalate was less than approximately 3.5 microM, as estimated by the present method.     

Ileal oxalate absorption and urinary oxalate excretion are enhanced in Slc26a6 null mice

Intestinal oxalate transport, mediated by anion exchange proteins, is important to oxalate homeostasis and consequently to calcium oxalate stone diseases. To assess the contribution of the putative anion transporter (PAT)1 (Slc26a6) to transepithelial oxalate transport, we compared the unidirectional and net fluxes of oxalate across isolated, short-circuited segments of the distal ileum of wild-type (WT) mice and Slc26a6 null mice [knockout (KO)]. Additionally, urinary oxalate excretion was measured in both groups. In WT mouse ileum, there was a small net secretion of oxalate (J(net)(Ox) = -5.0 +/-5.0 pmol.cm(-2).h(-1)), whereas in KO mice J(net)(Ox) was significantly absorptive (75 +/- 10 pmol.cm(-2)h.h(-1)), which was the result of a smaller serosal-to-mucosal oxalate flux (J(sm)(Ox)) and a larger mucosal-to-serosal oxalate flux (J(ms)(Ox)). Mucosal DIDS (200 microM) reduced J(sm)(Ox) in WT mice, leading to reversal of the direction of net oxalate transport from secretion to absorption (J(net)(Ox) = 15.0 +/- 5.0 pmol.cm(-2).h(-1)) , but DIDS had no significant effect on KO ileum. In WT mice in the absence of mucosal Cl(-), there were small increases in J(ms)(Ox) and decreases in J(sm)(Ox) that led to a small net oxalate absorption. In KO mice, J(net)(Ox) was 1.5-fold greater in the absence of mucosal Cl(-), due solely to an increase in J(ms)(Ox). Urinary oxalate excretion was about fourfold greater in KO mice compared with WT littermates. We conclude that PAT1 is DIDS sensitive and mediates a significant fraction of oxalate efflux across the apical membrane in exchange for Cl(-); as such, PAT1 represents a major apical membrane pathway mediating J(sm)(Ox).     

Entrapment of Sorghum root oxalate oxidase into polyvinyl alcohol membrane

A Cl- and NO3- insensitive oxalate oxidase, purified from the roots of 10-day old seedlings of grain Sorghum has been immobilized on polyvinyl alcohol (PVA) membrane through entrapment with 96.07% retention of initial activity. The membrane bound enzyme showed an increase in optimum pH (from 5.0 to 6.5), time of incubation (from 5 to 10 min) and Km for oxalate (from 0.38 to 6.23 mM), but decrease in incubation temperature for maximum activity (from 37 to 30 degrees C) and Vmax (from 70 nmol/min/ml to 9.7 nmol H2O2/min) and was unaffected by Cl- and NO3. The membrane bound enzyme lost 50% of its initial activity after 30 days of storage at room temperature. The use of membrane bound oxalate oxidase in determination of serum oxalate of urinary stone patients is demonstrated.     

Degradation of oxalate in rats implanted with immobilized oxalate oxidase

Accumulation of oxalate leads to hyperoxaluria and calcium oxalate nephrolithiasis in man. Since oxalate is a metabolic end product in mammals, the feasibility of its enzymic degradation has been tested in vivo in rats by administering exogenous oxalate oxidase. Oxalate oxidase, isolated from banana fruit peels, in its native form was found to be non-active at the physiological pH of the recipient animal. However, its functional viability in the recipient animal was ensured by its prior binding with ethylenemaleic anhydride, thus shifting its pH activity curve towards the alkaline range. Rats implanted with dialysis membrane capsules containing such immobilized oxalate oxidase in their peritoneal cavities effectively metabolized intraperitoneally injected [14C]oxalate as well as its precursor [14C]glyoxalate. The implantation of capsules containing coentrapped multienzyme preparations of oxalate oxidase, catalase and peroxidase led to a further degradation of administered [14C]oxalate in rats.     

Immobilization of Amaranthus leaf oxalate oxidase on alkylamine glass

A membrane bound oxalate oxidase from leaves of Amaranthus spionsus has been partially purified and immobilized on alkylamine glass with a yield of 9.2 mg protein/g support. The enzyme retained 99.4% of initial activity of free enzyme after immobilization. There was no change in the optimum pH (3.5) and Vmax but the temperature for maximum activity was slightly decreased (35 degrees C) and energy of activation (Ea) and Km for oxalate were increased after immobilization. The immobilized enzyme preparation was stable for 6 months, when stored in distilled water at 4 degrees C. Presence of Cl- did not affect the activity of immobilized enzyme.     

Activation of polyvinyl chloride sheet surface for covalent immobilization of oxalate oxidase and its evaluation as inert support in urinary oxalate determination

Polyvinyl chloride (PVC) sheets are a promising material for enzyme immobilization owing to the PVC’s properties such as being chemically inert, corrosion free, weather resistant, tough, lightweight, and maintenance free and having a high strength-to-weight ratio. In this study, this attractive material surface was chemically modified and exploited for covalent immobilization of oxalate oxidase using glutaraldehyde as a coupling agent. The enzyme was immobilized on activated PVC surface with a conjugation yield of 360 μg/cm². The scanning electron micrographs showed the microstructures on the PVC sheet surface revealing the successful immobilization of oxalate oxidase. A colorimetric method was adopted in evaluating enzymatic activity of immobilized and native oxalate oxidase. The immobilized enzyme retained 65% of specific activity of free enzyme. Slight changes were observed in the optimal pH, incubation temperature, and time for maximum activity of immobilized oxalate oxidase. PVC support showed no interference when immobilized oxalate oxidase was used for estimation of oxalic acid concentration in urine samples and showed a correlation of 0.998 with the values estimated with a commercially available Sigma kit. The overall results strengthen our view that PVC sheet can be used as a solid support for immobilization of enzymes and in the field of clinical diagnostics, environmental monitoring and remediation.     

Measurement of oxalate in human plasma ultrafiltrate by ion chromatography

An improved ion chromatographic method for the measurement of oxalate in human plasma ultrafiltrate is described. Ultrafiltration was carried out using an appropriate device and procedure. Centrifugation of 0.5 ml heparin plasma at 4°C for 50 min yielded water-clear ultrafiltrate in amounts allowing replicate measurements of oxalate. The specificity of the method was confirmed. The recovery of oxalate added to plasma was approximately 80%, whereas dilution of plasma, and of an oxalate-containing salt solution, resulted in falsely high values; the mechanism(s) underlying this phenomenon are insufficiently understood at present. The intra-assay of the method was assessed and from replicates of a pool plasma, the inter-assay precision from ten measurements of the same plasma on different days; the observed ranges of oxalate were 1.32-1.56 (mean 1.42) and 1.42-1.64 (mean 1.53) μmol/l, respectively. In plasma ultrafiltrate of a limited number of healthy volunteers the range of oxalate was 1.81-2.50 μmol/l, thus permitting renal oxalate handling to be studied.     

Beta-adrenergic receptors couple to CFTR chloride channels of intercalated mitochondria-rich cells in the heterocellular toad skin epithelium

In the heterocellular toad skin epithelium the beta-adrenergic receptor agonist isoproterenol activates cyclic AMP-dependent Cl(-) channels that are not located in the principal cells. With four experimental approaches, in the present study, we tested the hypothesis that the signalling pathway targets cystic fibrosis transmembrane conductance regulator (CFTR)-chloride channels of mitochondria-rich cells. (i) Serosal application of isoproterenol (log(10)EC(50)=-7.1+/-0.2; Hill coefficient=1.1+/-0.2), as well as noradrenaline, activated an anion pathway with an apical selectivity sequence, G(Cl)>G(Br)> or =G(NO(3))>G(I), comparable to the published selectivity sequence of cloned human CFTR expressed in Xenopus oocytes. (ii) Known modulators of human CFTR, glibenclamide (200 micromol/l) and genistein (50 micromol/l), depressed and activated, respectively, the receptor-stimulated G(Cl). Genistein did not modify the anion selectivity. (iii) Transcellular voltage clamp studies of single isolated mitochondria-rich cells revealed functional beta-adrenergic receptors on the basolateral membrane. With approximately 60,000 mitochondria-rich cells per cm(2), the saturating activation of 11.9+/-1.6 nS/cell accounted for the measured isoproterenol-activated transepithelial conductance of 600-900 microS/cm(2). In forskolin-stimulated cells, glibenclamide (200 micromol/l) reversibly inhibited the transcellular conductance by 9.6+/-1.6 nS/cell. (iv) With primers constructed from cloned Xenopus CFTR and PCR amplification of reverse-transcribed mRNA from toad skin, full-length Bufo CFTR cDNA was generated. The derived protein of 1466 residues shows 86% homology with xCFTR and 89% homology with hCFTR. All major functional sequences, that is, the R- and the NBF1- and NBF2-domains are well-conserved as are the predicted transmembrane segments proposed to form the pore of the channel protein. These new results taken together with our previously identified small-conductance CFTR-like Cl(-) channel in the apical membrane of the mitochondria-rich cells lead to the conclusion that the toad's CFTR gene codes for a functional Cl(-) channel in the apical plasma membrane of this minority cell type.     

Role of nitric oxide in the regulation of glucose kinetics in response to endotoxin in dogs.

The purpose of the present in vivo study was to determine the role of nitric oxide (NO) in the regulation of glucose metabolism in response to endotoxin by blocking NO synthesis with N(G)-monomethyl-L-arginine (L-NMMA). In five dogs, the appearance and disappearance rates of glucose (by infusion of [6,6-(2)H(2)]glucose), plasma glucose concentration, and plasma hormone concentrations were measured on five different occasions: saline infusion, endotoxin alone (E coli, 1.0 microg/kg i.v.), and endotoxin administration plus three different doses of primed, continuous infusion of L-NMMA. Endotoxin increased rate of appearance of glucose from 13.7 +/- 1.6 to 23.6 +/- 3.3 micromol x kg(-1) x min(-1) (P < 0.05), rate of disappearance of glucose from 13.9 +/- 1.1 to 24.8 +/- 3.1 micromol x kg(-1) x min(-1) (P < 0.001), plasma lactate from 0.5 +/- 0.1 to 1.7 +/- 0.1 mmol/l (P < 0.01), and counterregulatory hormone concentrations. L-NMMA did not affect the rise in rate of appearance and disappearance of glucose, plasma lactate, or the counterregulatory hormone response to endoxin. Plasma glucose levels were not affected by endotoxin with or without L-NMMA. In conclusion, in vivo inhibition of NO synthesis by high doses of L-NMMA does not affect glucose metabolism in response to endotoxin, indicating that NO is not a major mediator of glucose metabolism during endotoxemia in dogs.     

Discrete analysis of serum uric acid with immobilized uricase and peroxidase.

Commercially available uricase and peroxidase have been immobilized onto alkylamine glass and arylamine glass beads respectively. A discrete method has been developed to determine uric acid in serum using immobilized uricase and peroxidase. The method is based on generation of H2O2 from serum uric acid by immobilized uricase and its measurement by a colour reaction catalyzed by immobilized peroxidase. The minimum detection limit of the method was 8 microg/0.1 ml sample. The mean analytical recovery of added uric acid in serum was 87.5%. The within and between assay coefficient of variation (C.V.) were <6.58% and <10.77% respectively. The serum uric acid in apparently healthy adults and persons suffering from different disease was found to be 25-55 microg/ml, 32+/-2.25 (range, mean+/-S.D.) and 55-200 microg/ml; 52+/-6.4 (range, mean+/-S.D.) respectively by our method. A good correlation (r = 0.8170) was obtained between the serum urate values by this method and with those obtained by commercial Enzo-kit method.     

Shift in metabolic substrate uptake by the heart during development of alloxan-induced diabetes

Inhibition of endothelial nitric oxide (NO) synthase (eNOS) is associated with an increase in glucose uptake by the heart. We have already shown that Type I diabetes also causes a decrease in eNOS protein expression and altered NO control of both coronary vascular resistance and oxygen consumption. Therefore, we predict that the increase in plasma glucose and the reduction in eNOS during diabetes together would result in a large increase in cardiac glucose uptake. Arterial (A) and coronary sinus (C) plasma levels of glucose, free fatty acid (FFA), beta-hydroxybutyric acid (beta-HBA), and lactate were measured, and myocardial uptake was calculated before and at week 1, 2, 3, and 4 of alloxan-induced diabetes. The heart of healthy dogs consumed FFA (19.2 +/- 2.6 microeq/min) and lactate (19.7 +/- 3.4 micromol/min). Dogs in the late stage of diabetes (at week 4) had elevated arterial beta-HBA concentrations (1.6 +/- 0.7 micromol/l) that were accompanied by an increased beta-HBA uptake (0.3 +/- 0.2 micromol/min). In contrast, myocardial lactate (-4.8 +/- 3.0 micromol/min) and FFA uptake (2.5 +/- 1.9 microeq/min) were significantly reduced in diabetic animals. Despite a marked hyperglycemia (449 +/- 25 mg/dl), the heart did not take up glucose (-7.9 +/- 4.1 mg/dl). Our results indicate significant changes in the myocardial substrate utilization in dogs only in the late stage of diabetes, at a time when myocardial NO production is already decreased.     

Altered physicochemical characteristics of polyethylene glycol linked beet stem oxalate oxidase

Oxalate oxidase (EC 1.2.3.4), obtained from the beet stem, was covalently linked to polyethylene glycol (PEG). Compared with native enzyme, the modified oxalate oxidase exhibited decreased electrophoretic mobility, increased storage stability, higher thermal stability, and resistance to heavy metal inactivation and proteolytic digestion. The chemical modification of oxalate oxidase with PEG also brought about a marked shift in its optimal pH, from pH 4.5 to 6.5, without altering its Michaelis constant (K(m)) significantly. These acquired properties of the immobilized oxalate oxidase render it suitable for possible applications in clinical, nutritional, and medical fields. (c) 1995 John Wiley & Sons, Inc.     

Eosinophils are a major source of nitric oxide-derived oxidants in severe asthma: characterization of pathways available to eosinophils for generating reactive nitrogen species

Eosinophil recruitment and enhanced production of NO are characteristic features of asthma. However, neither the ability of eosinophils to generate NO-derived oxidants nor their role in nitration of targets during asthma is established. Using gas chromatography-mass spectrometry we demonstrate a 10-fold increase in 3-nitrotyrosine (NO(2)Y) content, a global marker of protein modification by reactive nitrogen species, in proteins recovered from bronchoalveolar lavage of severe asthmatic patients (480 +/- 198 micromol/mol tyrosine; n = 11) compared with nonasthmatic subjects (52.5 +/- 40.7 micromol/mol tyrosine; n = 12). Parallel gas chromatography-mass spectrometry analyses of bronchoalveolar lavage proteins for 3-bromotyrosine (BrY) and 3-chlorotyrosine (ClY), selective markers of eosinophil peroxidase (EPO)- and myeloperoxidase-catalyzed oxidation, respectively, demonstrated a dramatic preferential formation of BrY in asthmatic (1093 +/- 457 micromol BrY/mol tyrosine; 161 +/- 88 micromol ClY/mol tyrosine; n = 11 each) compared with nonasthmatic subjects (13 +/- 14.5 micromol BrY/mol tyrosine; 65 +/- 69 micromol ClY/mol tyrosine; n = 12 each). Bronchial tissue from individuals who died of asthma demonstrated the most intense anti-NO(2)Y immunostaining in epitopes that colocalized with eosinophils. Although eosinophils from normal subjects failed to generate detectable levels of NO, NO(2-), NO(3-), or NO(2)Y, tyrosine nitration was promoted by eosinophils activated either in the presence of physiological levels of NO(2-) or an exogenous NO source. At low, but not high (e.g., >2 microM/min), rates of NO flux, EPO inhibitors and catalase markedly attenuated aromatic nitration. These results identify eosinophils as a major source of oxidants during asthma. They also demonstrate that eosinophils use distinct mechanisms for generating NO-derived oxidants and identify EPO as an enzymatic source of nitrating intermediates in eosinophils.     

The relation between selenium, zinc and copper concentration and the trace element dependent antioxidative status.

An imbalance in the antioxidative system was connected with the development of a number of pathological processes. In order to receive values of a healthy group and to evaluate pathological changes of the trace element dependent antioxidative status in future, we investigated 99 healthy volunteers (45 male and 54 female, mean age 37.4 +/- 11.7 years). We determined the concentrations of Se, Cu and Zn, the concentrations of malondialdehyde (MDA) and the activities of the Se dependent glutathione peroxidase (GSH-Px) and the Zn/Cu dependent superoxide dismutase (SOD). The plasma concentrations (mean +/- SD) for Se, Cu and Zn were 0.84 +/- 0.10 micromol/l, 15.6 +/- 2.78 micromol/l and 12.6 +/- 1.80 micromol/l, resp., and for non protein-bound and protein bound MDA 0.27 +/- 0.07 micromol/l and 1.11 +/- 0.25 micromol/l, resp. The activity of GSH-Px in plasma and erythrocytes was 130 +/- 20.8 U/l and 19.8 +/- 4.18 U/mg Hb, resp. and of SOD in erythrocytes 3,159 +/- 847.2 U/g Hb. In plasma positive correlations have been found between Se concentrations and GSH-Px activities (p < 0.002, r = 0.31) and between GSH-Px activities and concentrations of non protein-bound MDA (p = 0.004, r = 0.28). A negative correlation has been observed between GSH-Px activities in plasma and in erythrocytes. The higher the concentrations of Cu in erythrocytes, the higher were the activities of SOD (p = 0.03, r = 0.22) and GSH-Px in erythrocytes (r = 0.26, p = 0.01), while an increasing activity of GSH-Px in these cells correlated with a decreasing concentration of non protein-bound MDA (r = -0,31, p = 0.002). An increase in BMI was connected with an increase in protein-bound MDA and a decrease in GSH-Px activities in pLasma (p = 0.002 and r = 0.23). As the results demonstrate, Se and Cu concentrations in erythrocytes can improve the trace element dependent antioxidative status.     

Characterization of oxalate transport by the human erythrocyte band 3 protein

This paper describes characteristics of the transport of oxalate across the human erythrocyte membrane. Treatment of cells with low concentrations of H2DIDS (4,4'-diisothiocyanatostilbene-2,2'- disulfonate) inhibits Cl(-)-Cl- and oxalate-oxalate exchange to the same extent, suggesting that band 3 is the major transport pathway for oxalate. The kinetics of oxalate and Cl- self-exchange fluxes indicate that the two ions compete for a common transport site; the apparent Cl- affinity is two to three times higher than that of oxalate. The net exchange of oxalate for Cl-, in either direction, is accompanied by a flux of H+ with oxalate, as is also true of net Cl(-)-SO4(2-) exchange. The transport of oxalate, however, is much faster than that of SO4(2-) or other divalent anions. Oxalate influx into Cl(-)-containing cells has an extracellular pH optimum of approximately 5.5 at 0 degrees C. At extracellular pH below 5.5 (neutral intracellular pH), net Cl(-)- oxalate exchange is nearly as fast as Cl(-)-Cl- exchange. The rapid Cl(- )-oxalate exchange at acid extracellular pH is not likely to be a consequence of Cl- exchange for monovalent oxalate (HOOC-COO-; pKa = 4.2) because monocarboxylates of similar structure exchange for Cl- much more slowly than does oxalate. The activation energy of Cl(-)- oxalate exchange is about 35 kCal/mol at temperatures between 0 and 15 degrees C; the rapid oxalate influx is therefore not a consequence of a low activation energy. The protein phosphatase inhibitor okadaic acid has no detectable effect on oxalate self-exchange, in contrast to a recent finding in another laboratory (Baggio, B., L. Bordin, G. Clari, G. Gambaro, and V. Moret. 1993. Biochim. Biophys. Acta. 1148:157-160.); our data provide no evidence for physiological regulation of anion exchange in red cells.     

Use of caffeic acid phenethyl ester and cortisone may prevent proliferative vitreoretinopathy

PURPOSE: To investigate whether caffeic acid phenethyl ester (CAPE) and cortisone prevent proliferative vitreoretinopathy (PVR). METHODS: Twenty pigmented rabbits were used in this study. All rabbits except controls received an intravitreal injection of 0.15 ml (75,000 U) of platelet-rich plasma into their left eye. The animals were divided into four groups: group I was treated with intraperitoneal injection of 0.5 ml (15 micromol/kg) of CAPE for 3 days, group II received 0.15 ml (4 mg/kg) of intravitreal cortisone, group III received nothing (blank group), and group IV (control group) received only 1 ml of 1% ethanol intraperitoneally daily for 3 days. Proliferative changes were graded in a masked fashion by indirect ophthalmoscopy for a 15-day follow-up period. The malondialdehyde (MDA), reduced glutathione (GSH) and total nitrite (NO) levels were measured in the vitreous humor. RESULTS: The grades of PVR were B-C in group I, and C-D in group II. The PVR grade in the control group was C-D. The mean MDA level in group I (4.0+/-0.8 micromol/l) was significantly lower than in the blank group (6.0 micromol/l) (p < 0.05). The mean GSH level in group I (71.0+/-11.2 micromol/l) was significantly different than in the blank group (p < 0.05). The MDA and GSH levels in group II were 4.7+/-0.6 micromol/l and 53.8+/-7.8 micromol/l, respectively. Both these levels were not significantly different from the blank group (p > 0.05). The NO levels in both treatment groups were significantly lower than in the blank group (p < 0.001). CONCLUSION: These findings suggest an inhibitory effect of CAPE on PVR. The inhibitory effect was supported by lower MDA and NO with higher GSH levels in treatment groups than in the blank group. There was no detected significant effect of cortisone for preventing PVR experimentally.     

Effects of cadmium and copper on peroxidase, NADH oxidase and IAA oxidase activities in cell wall, soluble and microsomal membrane fractions of pea roots

Twelve-day-old seedlings of pea (Pisum sativum L.) that were treated for 4 days by 20 and 100 micromol/l Cd(NO3)2 or CuSO4 showed a growth reduction in all organs. From root protein extracts, the activities of guaiacol peroxidase (GPX; EC 1.11.1.7), ascorbate peroxidase (APX; EC 1.11.1.11), coniferyl alcohol peroxidase (CAPX), NADH oxidase, and indole-3-acetic acid (IAA) oxidase were measured in covalently--and ionically--[symbol: see text] bound cell wall, soluble, and microsomal membrane fractions. With the exception of 20 micromol/l Cu, metal treatments enhanced GPX activity in all fractions. Only IAA oxidase activity was metal-elevated in the covalently bound cell wall fraction, while the ionic one showed Cd stimulation for all assayed enzymic activities. These effects were not entirely observed in Cu-treated plants, since APX and IAA oxidase activities were only enhanced in this fraction. However, soluble extract showed stimulation of APX activity, while in the microsomal fraction metal exposure also increased the activities of CAPX and NADH oxidase. Differential responses of root cell fractions to the presence of cadmium and copper ions are discussed in regard to the contribution of their enzymic capacities in antioxidant, lignification, and auxin degradation pathways. Comparisons between metals and dose effects are also underlined.     

Exercise-induced stimulation of K(+) transport in human erythrocytes.

The hypothesis was tested that exercise-induced changes in plasma composition stimulate unidirectional K(+) transport (J(in)K) in human red blood cells (RBCs). Ten men performed two 30-s high-intensity leg-cycling tests separated by 4 min of rest. Antecubital venous blood was sampled before exercise and at the end of the second exercise bout. RBCs were separated from true exercise plasma, (42)K was added to plasma, and RBC K(+) transport was studied in vitro at 37 degrees C. In the second part of the study, blood from nine healthy men studied in vitro at 37 degrees C was used to test the hypothesis that exercise-simulated (ES) plasma stimulates net K(+) transport and J(in)K (measured using (86)Rb) in human RBCs. The J(in)K of resting RBCs added to true exercise plasma was 1,574 +/- 200 (SE) micromol. h(-1). l(-1) vs. 1,236 +/- 256 micromol. h(-1). l(-1) in true resting plasma at 2 min (controls). In true exercise and ES plasma, J(in)K was increased through activation of the ouabain-sensitive Na(+)-K(+) pump and the bumetanide-sensitive Na(+)-K(+)-2Cl(-) cotransporter. Increases in plasma osmolality and K(+), H(+), and epinephrine concentrations independently and in combination stimulated K(+) transport into human RBCs. In a third series of experiments, in which ES plasma K(+) concentration was continuously measured during the first 5 min of incubation of RBCs, a 1.6 +/- 0.3 mmol/l decrease in plasma K(+) concentration occurred during the first 2 min. It is concluded that RBCs transport K(+) at elevated rates in response to exercise-induced changes in plasma composition.