[The clinic Heilanstalt Weidenplan in Halle (Saale), origin of the German urology. Otto Kneise's founding of the first independent urology department in Germany]. / Die Klinik "Heilanstalt Weidenplan" in Halle (Saale), eine Pionierstätte der Deutschen Urologie. Otto Kneises Weg zur ersten selbständigen Urologie in Deutschland.

The routine use of cystoscope initiated the development of the modern urology. Otto Kneise (1875-1953) extended the targets of cystoscopy by including examinations of the male bladder and prostate. He achieved the goal that "cystoscopy is part of general work in urology and not a pure gynecological act". He, thus, founded the specialty gynecological urology in the field urology, which prevented it from becoming an independent field. Under the leadership of Otto Kneise, the first independent urology department in Germany was created in the hospital Heilanstalt Weidenplan.

Implementation of evidence-based therapy in patients with systolic heart failure from 1998-2000.

BACKGROUND: In recent years, the incidence of systolic heart failure has increased. Besides a complete revascularization, guideline-based medication represents the most effective therapeutic approach. AIM: Analysis of adherence of guideline-recommended and actual medication during inpatient cardiac rehabilitation as well as under subsequent outpatient conditions. METHODS: From 01/1998 to 12/ 2000, 1346 consecutive patients (64 +/- 10 years, 73% male, LVEF 36.3 +/- 8%, 88% ischemic, 6.7% valvular cardiomyopathy, 5.3% other causes, 11.8% atrial fibrillation) were included in a singlecenter prospective register. Medication was recorded at discharge and after the follow-up period of 731 +/- 215 days. Trends in prescription rates were analyzed based on nonparametric correlations (Spearman's-Rho). Changes in medication from in- to outpatient settings were analyzed using exact McNemar test. RESULTS: At discharge 75.3% (67.9%/68.9%/ 86.6% in 1998/1999/2000, p <0.001) of the patients were treated as recommended. This rate dropped to 68.3% at followup (p <0.0001). Mortality within the follow-up period was low (12.6%). CONCLUSION: It could be shown that from 1998 to 2000 inpatient guideline conformity was implementable adequately. Outpatient conformity was significantly lower. Although a high proportion of correctly prescribed CHF medication could be demonstrated, a further effort to improve guideline adherence in the management of heart failure patients is desirable.

Novel spermine-amino acid conjugates and basic tripeptides enhance cleavage of the hairpin ribozyme at low magnesium ion concentration.

Combinations of the polyamine spermine and magnesium ions synergize to dramatically enhance cleavage of the hairpin ribozyme. Certain synthetic basic tripeptides stimulate hairpin cleavage significantly at limiting magnesium ion concentration, notably the tripeptide of L-diaminobutyric acid (Dab). Of a range of novel synthetic spermine-amino acid conjugates, L-Dab-spermine (but not D-Dab nor other amino acid conjugates) was more effective than spermine itself.

Increased kynurenic acid in the brain after neonatal asphyxia.

In the brain, L-kynurenine is an intermediate for the formation of kynurenic acid, a metabolite with neuroprotective activities, and a substrate for the synthesis of 3-hydroxy-kynurenine, a metabolite with neurotoxic properties. In the present study, alterations of L-kynurenine, 3-hydroxy-kynurenine and kynurenic acid levels were examined in the brain of neonatal (10 minutes old) rats after 5, 10, 15 or 20 minutes of asphyxia, and in the brain of the corresponding caesarean-delivered controls, using sensitive high-performance liquid chromatographic methods. Among kynurenines we found a marked time-dependent increase of kynurenic acid levels, a moderately delayed increase of 3-hydroxy-kynurenine, and a trend for a decrease of L-kynurenine content. Thus, the brain reacted rapidly to the oxygen deficit by increasing kynurenic acid levels by 44% already after 5 minutes of asphyxia, and the most prominent elevation of kynurenic acid (302% of control) was found after 20 minutes of asphyxia--the critical time limit of survival.

Kynurenic acid influences the respiratory parameters of rat heart mitochondria.

In the present study the effect of L-kynurenine, kynurenic acid and quinolinic acid on the heart mitochondrial function were investigated. Mitochondria were incubated with saturating concentrations of respiratory substrates glutamate/malate (5 mmol/l), succinate (10 mmol/l) or NADH (1 mmol/l), with and without kynurenines. The concentration of kynurenines varied between 1.25 micromol/l and 10 mmol/l. From all investigated kynurenines, only kynurenic acid affected dose-dependently the respiratory parameters of heart mitochondria. Respiratory control and P/O values were reduced significantly with glutamate/malate and moderately with succinate as substrates in the presence of 125 micromol/l to 10 mmol/l kynurenic acid. A known elevation of L-kynurenine in the blood of patients with ischemic heart disease or essential hypertension may suggest the involvement of L-kynurenine metabolites in the impairment of heart mitochondrial function, for example in cardiomyopathy.

Spin trapping of lipid radicals with DEPMPO-derived spin traps: detection of superoxide, alkyl and alkoxyl radicals in aqueous and lipid phase.

The spin trap 5-(diethoxyphosphoryl)-5-methyl-1-pyrroline N-oxide (DEPMPO) forms a superoxide adduct with a half-life of almost 15 min. DEPMPO is very hydrophilic and its use for the detection of radicals in the lipid phase (lipid-derived radicals and superoxide generated in the lipid phase) is therefore limited due to its very low concentration in the lipid phase. For the detection of lipid-derived radicals, three derivatives of DEPMPO with increasing degree of lipid solubility have been investigated: 5-(di-n-propoxyphosphoryl)-5-methyl-1-pyrroline N-oxide (DPPMPO), 5-(di-n-butoxyphosphoryl)-5-methyl-1-pyrroline N-oxide (DBPMPO), and 5-(bis-(2-ethylhexyloxy)phosphoryl)-5-methyl-1-pyrroline N-oxide (DEHPMPO). As compared with the spin trap DMPO, the half-lives of the respective superoxide adducts were clearly higher in aqueous solutions of the spin traps, which facilitates qualitative ESR measurements. The stability of the superoxide spin adducts formed with the various lipophilic spin traps in aqueous buffer were similar to those observed with DEPMPO (half-life: 7-11 min.). In model experiments using Fe(3+)-catalyzed nucleophilic addition of methanol or tert-butanol to the respective spin trap the respective alkoxyl radical adducts were formed in aqueous solution as transient species in the presence of high concentrations of the alcohol. Upon dilution with water the alkoxyl group was substituted by water, giving the respective hydroxyl adduct of the spin trap. Care must therefore be taken when Fenton-type reactions are used for the generation of radicals such as the use of Fe(2+) complexes with phosphate or DTPA or inactivation of iron by addition of "Desferal" (Novarti's Pharma GmbH, Vienna, Austria) after a short incubation time. Addition of Fe(2+) under anaerobic conditions to an aqueous suspension of linoleic acid hydroperoxide and the spin trap resulted in the detection of three different species: a carbon-centered radical adduct, an acyl radical adduct, and the hydroxyl adduct. In the presence of oxygen a different species was observed with DEPMPO, DPPMPO, and DBPMPO, which was only slightly suppressed upon the addition of SOD, possibly the respective spin adduct of either the alkylperoxyl radical or, in analogy to DMPO, a secondary alkoxyl radical.

Lipid radicals: properties and detection by spin trapping.

Unsaturated lipids are rapidly oxidized to toxic products such as lipid hydroperoxides, especially when transition metals such as iron or copper are present. In a Fenton-type reaction Fe2+ converts lipid hydroperoxides to the very short-lived lipid alkoxyl radicals. The reaction was started upon the addition of Fe2+ to an aqueous linoleic acid hydroperoxide (LOOH) emulsion and the spin trap in the absence of oxygen. Even when high concentrations of spin traps were added to the incubation mixture, only secondary radical adducts were detected, probably due to the rapid re-arrangement of the primary alkoxyl radicals. With the commercially available nitroso spin trap MNP we observed a slightly immobilized ESR spectrum with only one hydrogen splitting, indicating the trapping of a methinyl fragment of a lipid radical. With DMPO or 5-diethoxyphosphoryl-5-methyl-1-pyrroline N-oxide (DEPMPO) adducts were detected with carbon-centered lipid radical, with acyl radical, and with the hydroxyl radical. We also synthesized lipophilic derivatives of the spin trap DEPMPO in order to detect lipid radical species generated in the lipid phase. With all spin traps studied a lipid-derived carbon-centered radical was obtained in the anaerobic incubation system Fe2+/LOOH indicating the trapping of a lipid radical, possibly generated as a secondary reaction product of the primary lipid alkoxyl radical formed. Under aerobic conditions an SOD-insensitive oxygen-centered radical adduct was formed with DEPMPO and its lipophilic derivatives. The observed ESR parameters were similar to those of alkoxyl radical adducts, which were independently synthesized in model experiments using Fe3+-catalyzed nucleophilic addition of methanol or t-butanol to the respective spin trap.

Free radical formation and erythrocyte membrane alterations during MetHb formation induced by the BHA metabolite, tert-butylhydroquinone.

Erythrocyte membranes are altered as a consequence of oxidative stress following the incubation of intact erythrocytes with one of the major metabolites of the antioxidant butylated hydroxyanisole (BHA), tertbutylhydroquinone(tBHQ). Arather persistent semiquinone radical was observed by electron spin resonance (ESR) spectroscopy when tBHQ was incubated with either homogeneous oxyhemoglobin solutions or suspensions of intact erythrocytes. Erythrocyte ghosts prepared from fresh control erythrocytes and ghosts from erythrocytes preincubated with BHA and its metabolite, tBHQ, were subjected to polyacrylamide gel electrophoresis (SDS-PAGE). Only minor changes of the electrophoresis pattern relative to the control was observed in the BHA incubations whereas tBHQ significantly increased the amount of high molecular weight degradation products of erythrocyte membrane constituents. These changes were only observed when incubations were performed in the presence of oxygen. In control experiments where heme oxygen was replaced by carbon monoxide, no membrane degradation products appeared. These observations can be interpreted in terms of metabolic activation of the antioxidant BHAvia tBHQ to the tert-butylsemiquinone free radical and finally to the corresponding quinone, thereby leading to harmful effects on erythrocyte membrane structures. Moreover, deleterious effects on other biological membranes are also likely to occur.

The effects of xenobiotics on erythrocytes.

1. Methemoglobin formation was observed when erythrocytes were incubated with xenobiotics such as hydroxylamines or phenols, other metabolites resulting from the interaction of these compounds with erythrocytes being reactive free radicals derived from the respective xenobiotic, and a ferryl-heme oxo-complex. 2. Steady-state levels of these reaction products depended on the permeability of the erythrocyte membrane for the various methemoglobin (MetHb) generators and the presence of antioxidants that downregulate the radicals formed. 3. Electron spin resonance (ESR) spectra of xenobiotic-derived free radicals could be obtained only from the readily water soluble hydroxylamines, whereas the poorly water soluble phenolic compounds did not allow the use of concentrations required for the generation of detectable amounts of ESR-sensitive metabolites in erythrocytes. 4. Previous investigations with oxyhemoglobin solutions and with the MetHb/H2O2 model systems have shown that, apart from ESR-sensitive radical species, excited reaction intermediates such as compound 1 ferryl hemoglobin can be detected as well by using chemiluminescence measurements. 5. A strong correlation was found between the intensity of the emitted light and the MetHb formation rate, indicating that the production of compound 1 ferryl hemoglobin is closely related to the MetHb formation step. 6. The sensitivity of the photon-counting method allowed measurements of excited species in intact erythrocytes not only with the readily soluble hydroxylamines, but also with the less soluble phenolic compounds. 7. In addition, parameters indicative of xenobiotic-induced oxidative alterations were found: a significant decrease in intraerythrocytic thiol levels was a result of all compounds that initiate MetHb formation, as also described for slowly reacting xenobiotics. 8. With the most reactive compound investigated, unsubstituted hydroxylamine, a significant release of iron from the oxidatively modified hemoglobin was detected, facilitated by binding of this transition metal to hydroxylamine and its final oxidation product, nitric oxide. 9. The use of the ESR spin-labeling technique revealed membrane alterations of erythrocytes exposed to the reducing MetHb generators presented in this study. 10. A direct action of BHA and BHT on the integrity of the erythrocyte membrane was observed, leading to hemolysis independent of the formation of prooxidant species. 11. The presence of strong prooxidants (radicals) was indicated both by fluidity changes in the membrane and by an oxidative decrease in cytosolic thiol levels.

Light emission resulting from hydroxylamine-induced singlet oxygen formation of oxidizing LDL particles.

Oxidation of low-density lipoprotein (LDL) by low amounts of cupric ions resulted in the formation of singlet oxygen (1O2, 1 delta g) when hydroxylamine (NH2OH) was added. Direct evidence on this excited species came from partial spectral resolution of the emitted light in the red spectral region (634 nm and 703 nm), which can be attributed to the dimol decay of singlet oxygen. Additional evidence for the existence of singlet oxygen came from the enhancing effect of deuterium oxide buffer (D2O) on chemiluminescence intensity and the quenching effect of sodium azide. A linear correlation between NH2OH-dependent chemiluminescence intensity and the amount of diene conjugates (DC) formed in this reaction was observed. Removal of adventitious transition metals by adequate chelators prevented chemiluminescence in this system; NH2OH was also found to efficiently decrease metabolites of lipid peroxidation (LPO). Our findings are consistent with a sequence of reactions in which NH2OH first converts transition metals to their reduced state, thereby stimulating the formation of alkoxy- and peroxyradicals. Peroxyradicals decompose in a bimolecular Russel reaction to hydroxyl compounds and singlet oxygen while the majority of alkoxy radicals are eliminated by a secondary reaction with NH2OH. Identical effects were observed when reducing antioxidants such as ascorbic acid or trolox C were used instead of hydroxylamine.

Hydroxylamine and phenol-induced formation of methemoglobin and free radical intermediates in erythrocytes.

As previously shown with isolated oxyhemoglobin, methemoglobin formation can also be induced in intact erythrocytes by hydroxylamine compounds and substituted phenols such as butylated hydroxyanisole (BHA). Electron spin resonance investigations revealed that, accordingly, free radical intermediates were formed in erythrocytes from hydroxylamine, N,N-dimethylhydroxylamine, and N-hydroxyurea. Due to the low stability of the dihydronitroxyl radicals, their detection required the use of a continuous flow system and relatively high amounts of the reactants. As has already been demonstrated with the solubilized hemoglobin system, hemoglobin of intact erythrocytes also reacts with the more hydrophilic xenobiotics such as hydroxylamine. However, the reaction rate was slightly reduced, indicating the existence of an incomplete permeability barrier for these compounds. The limited solubility of phenolic compounds in the aqueous buffer of suspended erythrocytes (in combination with the strict requirement of osmolarity in order to prevent hemolysis) impeded the direct detection of the respective phenoxyl radicals previously reported in hemoglobin solutions. However, in accordance with earlier findings in homogeneous reaction systems, chemiluminescence was observed as well, indicating the existence of a further reaction intermediate, which was also obtained in pure hemoglobin solutions when mixed with the respective reactants. As has recently been demonstrated, this light emission is indicative of the existence of highly prooxidative compound I intermediates during methemoglobin formation. Prooxidant formation in erythrocytes is reflected by a significant decrease in thiol levels even with those compounds where free radical formation was not directly detectable by ESR spectroscopy. The use of the spin-labeling technique revealed membrane effects as a result of oxidative stress. Oxidative metabolism of hemoglobin with hydroxylamine caused a release of low molecular weight iron. The marked hemolysis observed in the presence of BHA results from a direct membrane effect of this compound rather than a consequence of free radical-induced oxidative stress. A correlation of the different results is discussed in terms of possible toxicological consequences.

Reactions of reducing xenobiotics with oxymyoglobin. Formation of metmyoglobin, ferryl myoglobin and free radicals: an electron spin resonance and chemiluminescence study.

The oxygen-haem centre of oxymyoglobin reacts with reducing xenobiotics such as hydroxylamines and phenols with the concomitant formation of metmyoglobin and oxidation of the respective xenobiotic. Metmyoglobin formation rates were measured by visible spectroscopy with xenobiotic concentrations ranging from 100 microM to 30 mM. Analogous to previous results obtained with oxyhaemoglobin, the first step in the reaction of hydroxylamines with oxymyoglobin leads to the formation of the one-electron oxidation product of hydroxylamine, a nitroxyl radical detectable by electron spin resonance. A variety of paramagnetic secondary products were also found. The terminal oxidation product of hydroxylamine and hydroxyurea was the myoglobin-nitric oxide complex, one showing similar spectral characteristics to the analogous haemoglobin-nitric oxide adduct found in our previous experiments. On the other hand, the amount of low-spin ferric complexes obtained from metmyoglobin and an excess of the respective hydroxylamine was considerably lower than the corresponding results with methaemoglobin. A second important reaction intermediate was the compound I-type ferryl haem-species detected by a recently-published chemiluminescence assay. Partial spectral resolution of the emitted light using a set of cut-off filters indicated that maximum light emission occurred above 600 nm, most probably involving excited porphyrin states. The intensity of oxymyoglobin-related light emission was considerably higher than that reported earlier with oxyhaemoglobin. This indicates a difference in the excitation mechanism which leads to the formation of the compound I-type ferry haem species.

Chemiluminescence from activated heme compounds detected in the reaction of various xenobiotics with oxyhemoglobin: comparison with several heme/hydrogen peroxide systems.

Chemiluminescence was detected in the reaction of oxyhemoglobin with various hydroxylamines and phenols, which have previously been shown to produce free radicals. The emitted light intensity correlated roughly with the methemoglobin formation rate, indicating the involvement of a photoemissive species as a reaction intermediate. In our previous work, we postulated the involvement of a catalase-insensitive, heme-bound hydrogen peroxide species in the methemoglobin formation reaction. In a series of experiments, we showed that intensive chemiluminescence occurred when hydrogen peroxide was mixed with either methemoglobin or metmyoglobin but not with hematin, which lacks the globin moiety. This suggests the involvement of the globin moiety in the light-emitting reaction sequence. The detection of paramagnetic globin species exhibiting similar kinetics as the corresponding light-emitting compound demonstrated that the assumed H2O2-heme compound has strong oxidizing properties. Accordingly, addition of bovine serum albumin to the hematin-hydrogen peroxide system also resulted in a strong chemiluminescence due to the formation of a paramagnetic transient species which could be detected by electron spin resonance (ESR). Several other heme compounds, such as cytochrome c or cytochrome c oxidase which have no vacant ligand site, did not show any light emission under similar conditions. This means that hydrogen peroxide must have access to a free-binding position on the heme. Chemiluminescence most probably stems from the transition of the initially formed heme-H2O2 adduct to the compound II type species. Due to their oxidizing nature, these species might be responsible for deleterious toxic effects such as lipid peroxidation and protein degradation.

Ischemia/reperfusion impairs mitochondrial energy conservation and triggers O2.- release as a byproduct of respiration.

The aim of the present study was to elucidate the role of mitochondria in the development of heart failure following ischemia/reperfusion. Although mitochondria were increasingly assumed to be responsible for the establishment of an oxidative stress situation the lack of suitable methods to prove it required new concepts for an evaluation of the validity of this hypothesis. The principal idea was to expose isolated mitochondria to metabolic conditions which are developed during ischemia/reperfusion in the cell (anoxia, lactogenesis) and study how they respond. Heart mitochondria treated in that way responded with an incomplete collapse of the transmembraneous proton gradient, thereby impairing respiration-linked ATP generation. The membrane effect affected also the proper control of e- transfer through redox-cycling ubisemiquinone. Electrons were found to leak at this site from its normal pathway to O2 suggesting that ubisemiquinone becomes an active O2.- generator. It was concluded from these observations that mitochondria are likely to play a pathogenetic role in the reperfusion injury of the heart both, by an impairment of energy conservation and their transition to a potent O2.(-)-radical generator. Furthermore, there is considerable evidence that the exogenous NADH-dehydrogenase of heart mitochondria is mainly responsible for functional changes of these organelles during ischemia/reperfusion.

Ubisemiquinones of the mitochondrial respiratory chain do not interact with molecular oxygen.

The present investigation deals with the suggested role of redox-cycling ubisemiquinones in mitochondrial O2-. generation. Due to the functional complexity of electron-transferring ubiquinones in the respiratory chain, model experiments were designed to study whether ubisemiquinones will directly react with oxygen, thereby generating O2-. radicals. Based on the fact that mitochondrial ubiquinone was reported to operate in an aprotic surrounding of the inner mitochondrial membrane, the reactivity of ubisemiquinones with oxygen was tested in water-free acetonitrile. Our results prove that autoxidation of ubisemiquinones requires the addition of protons to the non-polar reaction system. An experimental evaluation of the validity of this finding with respect to mitochondrial ubiquinones is impeded by the biochemical role that oxygen plays in the establishment of ubisemiquinone populations. To differentiate between a possible direct interaction of oxygen on redox-cycling ubisemiquinones and this indirect biochemical O2 effect, we have successfully introduced ferricyanide instead of oxygen to establish mitochondrial ubisemiquinone pools. Ubisemiquinones in this reaction system were not susceptible to oxygen and no O2-. radicals were released unless the inner mitochondrial membrane was protonated by toluene pretreatment. Since the inner mitochondrial membrane is normally not permeable to protons (which is a prerequisite of the chemiosmotic theory of energy conservation) based on our experiments we can exclude the involvement of redox-cycling ubisemiquinones in mitochondrial O2-. generation.

Methemoglobin formation from butylated hydroxyanisole and oxyhemoglobin. Comparison with butylated hydroxytoluene and p-hydroxyanisole.

The widely used food additives butylated hydroxyanisole (BHA) and butylated hydroxytoluene (BHT) react with oxyhemoglobin, thereby forming methemoglobin. The reaction rates were measured using visible spectroscopy, and second order rate constants were established for BHA and compared with p-hydroxyanisole. Using ESR we investigated the involvement of free radical reaction intermediates. The expected one-electron oxidation product of BHA and BHT, the phenoxyl radical, could only be detected with pure 3-t-butyl-4-hydroxyanisole and oxyhemoglobin. With the commercial mixture of 2- and 3-t-butyl-4-hydroxyanisole a very strong ESR signal of a secondary free radical species was observed, similar to the one observed earlier with p-hydroxyanisole and dependent on the presence of free thiol groups, so that we assumed the intermediate existence of a perferryl species, the MetHb-H2O2 adduct. In a second series of experiments we investigated the reactivity of this postulated intermediate with BHA and BHT, starting with a pure MetHb/H2O2-phenol mixture in a stopped-flow apparatus linked to the ESR spectrometer, detecting the expected phenoxyl radicals from BHA and p-hydroxyanisole. Due to the low solubility and decreased reactivity of BHT only traces of phenoxyl type radical were found together with a high concentration of unreacted perferryl species. The reactivity of BHA, BHT and p-hydroxyanisole with free thiol groups is demonstrated by an increased reaction rate in the presence of the thiol group blocking substance NEM.

ESR spectroscopy of flow-oriented cation radicals of phenothiazine derivatives and phenoxathiin intercalated in DNA.

Several derivatives of the phenothiazine cation radicals intercalated into DNA have been investigated using a new flow orientation technique. The anisotropic hyperfine coupling constants of both the parallel and the perpendicular orientation relative to the magnetic field were measured and compared to previous results, which used different techniques. The phenoxathiin cation radical could also be stabilized by intercalation into DNA at pH 4, but the orientation technique revealed no further information due to the poor resolution of the experimental spectra.

Is oxidative stress primarily involved in reperfusion injury of the ischemic heart?

Reperfusion injury of ischemic organs is suggested to result from metabolic derangements initiating an imbalanced formation of free oxygen radicals. Most investigators in this field have used the spin-trap 5,5'-dimethyl-N-pyrroline-N-oxide (DMPO) to stabilize these short-lived radicals and make them visible by means of the electron spin resonance (ESR) technique. ESR signals obtained from intravascular DMPO were reported to indicate the formation of free OH. radicals and, in some cases, also carbon-centered radicals. We were unable to confirm these findings. Carbon-centered radicals were not obtained irrespectively of conditions studied, while oxygen-centered DMPO-adducts could only be detected in minor amounts. Instead, we observed an ascorbyl-related ESR signal. The addition of ethylenediaminetetraacetic acid (EDTA), which was used by many investigators in this field, was found to greatly influence ESR-spectra of the reperfusion fluid. The ascorbyl radical concentration was clearly reduced and the DMPO-OH. adduct became more prominent. The addition of iron further stimulated this change eliciting a Fenton-type reaction responsible for DMPO-OH.-related ESR spectra in the perfusate after ischemia. Accordingly, we observed the release of iron and ascorbic acid into the perfusate as a consequence of ischemia. We could demonstrate that iron in the presence of ascorbate and EDTA causes both types of radicals detected in the perfusate. DMPO-OH. generation in the presence of EDTA was found to result from free OH. radicals that were not generated in the absence of EDTA.(ABSTRACT TRUNCATED AT 250 WORDS)