High-performance liquid chromatographic determination of isoniazid, acetylisoniazid and hydrazine in biological fluids

The basic principle of derivatization of a hydrazide moiety with an aldehyde as applied in the method developed by Lacroix et al. [J. Chromatogr., 307 (1984) 137–144] for the quantitation of isoniazid and acetylisoniazid was imppoved by modification, standardization and extension to allow quantitation of hydrazine in patient samples. It could be shown that 40 μl of 1% methanonic cinnamaldehyde per 200 μl of deproteinized analysate gave maximal chromophoric isoniazid-cinnamaldehyde conjugate, read at 340 nm. The hydrolytic loss of isoniazid, crucial to the quantitation of acetylisoniazid, could be compensated for by introduction of an appropriate set of calibration curves. Although the method described here allows quantitation of monoacetylhydrazie and diacetylhydrazine, in addition to hydrazine, in mono-spiked samples, the method cannot be used for the quantitation of the acetylated metabolites of hydrazine in patient samples because of a lack of specificity. Linear calibration curves in the range 1–25 μg/ml for isoniazid and acetylisoniazid, 10–400 ng/ml for hydrazine and 50–1000 ng/ml for mono-acetylhydrazine and diacetylhydrazine, could be constructed; analyte recoveries approaching 100% could be achieved in all instances.     

Nitrogenase IX. Effect of the MgATP generator on the catalytic and EPR properties of the enzyme in vitro.

Nitrogenase(nitrogen:(acceptor) oxidoreduction, EC 1.7.99.2) of Clostridium pasteuranium is very sensitive to the ratio of MgADP/MgATP in dithionite oxidation assays. Variation of concentration of creatine kinase, an ATP-regenerating enzyme, can be used to control the ratio of ADP/ATP and thereby the dithionite oxidation activity of nitrogenase. The in vitro properties of nitrogenase support the suggestion of Haaker (Haaker, H., deKok, A. and Veeger, C. (1974) Biochim. Biophys. Acta 357, 344-357) that in vivo the nucleotide ratio and not the electron supply normally regulates nitrogenase activity. In EPR experiments it has been shown that the "steady state" varies as a function of the concentration of creatine kinase. The spectral differences are interpreted as being a function of the ratio of MgADP/MgATP obtained in the pseudo steady-state condition, which occurs as a result of variation in relative rates of ATP-utilizing and ATP-generating reactions, that is, the relative nitrogenase and creatine kinase activities. Implications of these finding for interpretation of previously reported kinetic and EPR studies are discussed.     

Liquid Chromatographic-Fluorescence Determination of Ammonia from Nitrogenase Reactions: A 2-Min Assay

The analytical potential of the reaction of ammonia with o-phthalaldehyde mercaptoethanol reagent at pH 7 (an atypical fluorescence) has already been demonstrated. This, coupled with additional findings reported here, has led to an ammonia determination well suited to nitrogenase studies. As a result, large numbers of samples can be rapidly analyzed by high-pressure liquid chromatrography methods under mild conditions and without prior microdiffusion. Neither sodium dithionite (or other components of the usual nitrogenase assay), nor alternative substrates (cyanide, azide, methyl isonitrile), nor their products (methylamine, dimethylamine, hydrazine) interfere. High-pressure liquid chromatography showed that the fluorescent “product” of the o-phthalaldehyde mercaptoethanol reagent-ammonia reaction was, in fact, more than just a single compound. Despite this, once the proper solvent composition was found, high-pressure liquid chromatography with a small inexpensive C₁₈ “guard” column proved quite fast and reproducible for this measurement. Fluorescence response to ammonia was linear to at least 40 nmol/ml. A previous problem, long-term stability of the fluorescence, was solved by running the reactions in the dark. Background ammonia in the buffer could be substantially reduced by an analogous o-phthalaldehyde mercaptoethanol reagent reaction, using t-butyl mercaptan, and solvent extraction.     

Effect of intravenous infusion of 15-methyl-PGF2α on plasma hormone levels during early pregnancy

Ten pregnant women (7th–8th week of pregnancy) obtained an intravenous infusion of 15-methyl-prostaglandin-F_2α (2.5 μg/min) until clinical signs of abortion occurred or up to 7 hours. Surgical removal of the products of conception was performed 4–7 hours after the start of the infusion. Blood samples were taken prior to and during the infusion and then during the post-abortion period. The plasma levels of both progesterone and estradiol exhibited a significant decrease (p<0.001 and p<0.05, respectively) one hour after the beginning of infusion and remained reduced by approximately 35 and 45 per cent, respectively, during the entire infusion period. A drop in the levels of both steroids was seen after surgical interruption. This was followed by a gradual decrease to non-pregnancy levels. The levels of cortisol increased significantly (p<0.01) by approximately 60 per cent, starting with the second hour of infusion. Following surgical interruption, the levels dropped to pre-infusion values. 17-Hydroxyprogesterone levels increased (p<0.05) above the pretreatment levels by approximately 25 per cent, starting with the third hour of infusion. These levels were not correlated with those of cortisol during the infusion period. Following surgical interruption the plasma levels of 17-hydroxyprogesterone returned to non-pregnancy levels.     

Luminometric assays of ATP, phosphocreatine, and creatine for estimation of free ADP and free AMP.

We present methods to measure ATP, phosphocreatine, and total creatine (the sum of creatine and phosphocreatine) in alkaline cell extracts. Knowledge of these parameters, together with the known equilibrium constants for the creatine kinase and adenylate kinase-catalyzed reactions, allows one to estimate the levels of free ADP and free AMP inside cells. The enzymatic assays for the above-mentioned metabolites all lead up to the production of ATP, which is measured luminometrically with the ATP-dependent oxidation of luciferin catalyzed by firefly luciferase. To determine phosphocreatine, endogenous ATP is first destroyed, and phosphocreatine is then quantitatively reacted with exogenous ADP to form ATP. Total creatine is measured after quantitative conversion of creatine to phosphocreatine with a large excess of exogenous ATP, conversion of all ATP to ADP, and final reaction of phosphocreatine with ADP to form ATP. We used 5-microl samples in 0.5-ml microcentrifuge tubes and subsequent 5-microl additions of analytical reagents. We expect that the volumes can be changed easily. We tested the methods with glucagon- and insulin-secreting cells. Estimates of free ADP and AMP are expected to be useful in many different areas of research, such as cellular energy metabolism, purine nucleotide metabolism, adenine nucleotide gating of ion channels, and release of vasoactive or angiogenic factors.     

The nitrogen fixation system of photosynthetic bacteria. I. Preparation and properties of a cell-free extract from Chromatium

The preparation and properties of cell-free extracts of the photosynthetic bacterium Chromatium which give consistent and appreciable nitrogen fixation are described. Nitrogen fixation required ATP and reducing power. Reducing power was supplied by reduced ferredoxin or, in its absence, by sodium dithionite or H₂, in the presence of catalytic amounts of a viologen dye. ATP was supplied by phosphocreatine and creatine phosphokinase, with a catalytic amount of free ATP.

The Chromatium nitrogenase complex was found to have the typical properties of other nitrogenases, i.e., reduction of N₂ to ammonia, and of acetylene to ethylene, ATP-dependent H₂ evolution and reductant-dependent ATP hydrolysis.     

A P-NMR saturation transfer study of the regulation of creatine kinase in the rat heart

(1) ³¹P nuclear magnetic resonance was used to measure the creatine kinase-catalysed fluxes in Langendorff-perfused rat hearts consuming oxygen at different rates and using either of two exogenous substrates (11 mM glucose or 5 mM acetate). (2) Fluxes in the direction of ATP synthesis were between 3.5–12-times the steady-state rates of ATP utilization (estimated from rates of O₂-consumption), demonstrating that the reaction is sufficiently rapid to maintain the cytosolic reactants near their equilibrium concentrations. (3) Under all conditions studied, the cytosolic free [ADP] was primarily responsible for regulating the creatine kinase fluxes. The enzyme displayed a K_m for cytosolic ADP of 35 μM and an apparent V_max of 5.5 mM/s in the intact tissue. (4) Although the reaction is maintained in an overall steady-state, the measured ratio of the forward flux (ATP synthesis) to the reverse flux (phosphocreatine synthesis) was significantly greater than unity under some conditions. It is proposed that this discrepancy may be a consequence of participation of ATP in reactions other than the PCr /ag ATP or ATP /ag ADP + P_i interconversions specifically considered in the analysis. (5) The results support the view that creatine kinase functions primarily to maintain low cytosolic concentrations of ADP during transient periods in which energy utilization exceeds production.     

Glutamine Synthetase/Glutamine: alpha-Ketoglutarate Aminotransferase in Chloroplasts from the Marine Alga Caulerpa simpliciuscula

The enzymic capacities for ammonia assimilation into amino acids have been investigated in chloroplasts from the siphonous green alga Caulerpa simpliciuscula (Turner) C. Ag. The results show that these chloroplasts differ from those of higher plants in having present simultaneously the enzymic capacities to permit assimilation of ammonia by two pathways. Glutamine synthetase (EC 6.3.1.2) activity at levels up to 4 μmoles per mg chlorophyll per hour were found in soluble extracts of the chloroplasts. Glutamine(amide):α-ketoglutarate aminotransferase (oxidoreductase ferredoxin) (EC 1.4.7.1) activity at levels up to 1.4 μmoles per mg chlorophyll per hour was detected by incubation of photosynthetically active chloroplasts either in light or with reduced ferredoxin. Together these enzymes provide the capacity for the conventional pathway of ammonium assimilation in chloroplasts via glutamine. A similar level of a glutamate dehydrogenase with an unusually low K_m for ammonia which has been described previously in these chloroplasts provides the second potential pathway.     

ATP concentration dependency of the tubule-extrusion velocity from the axonemes

ATP-driven tubule-extrusion from trypsintreated axonemes of sea urchin sperm flagella was induced by adding ATP. Using a CTC-9000 Night Vision Camera system including a video tape recorder, velocity of the tubule-extrusion process was measured and found to increase with increasing ATP concentration. Minimum concentration of ATP inducing the tubule-extrusion was around 4 μM. The velocity at 18–22 °C was around 1.8 μm/sec at 10 μM ATP and 6.6 μm/ sec at 1 mM ATP.     

Diazotrophy and Nitrogenase Activity in the Archaebacterium Methanosarcina barkeri 227

Nitrogen fixation (diazotrophy) has recently been demonstrated in several methanogenic archaebacteria. To compare the process in an archaebacterium with that in eubacteria, we examined the properties of diazotrophic growth and nitrogenase activity in Methanosarcina barkeri 227. Growth yields with methanol or acetate as a growth substrate were significantly lower in N₂-grown cultures than in NH₄⁺-grown cultures, and the culture doubling times were increased, indicating that diazotrophy was energetically costly, as it is in eubacteria. Growth of nitrogen-fixing cells was inhibited when molybdenum was omitted from the medium; addition of 10 nM molybdate stimulated growth, while 1 μM molybdate restored maximum diazotrophic growth. Omission of molybdenum did not inhibit growth of ammonia-grown cells. Tungstate (100 μM) strongly inhibited growth of molybdenum-deficient diazotrophic cells, while ammonia-grown cells were unaffected. The addition of 100 nM vanadate or chromate did not stimulate diazotrophic growth of molybdenum-starved cells. These results are consistent with the presence of a molybdenum-containing nitrogenase in M. barkeri. Acetylene, the usual substrate for assaying nitrogenase activity, inhibited methanogenesis by M. barkeri and consequently needed to be used at a low partial pressure (0.3% of the headspace) when acetylene reduction by whole cells was assayed. Whole cells reduced 0.3% acetylene to ethylene at a very low rate (1 to 2 nmol h⁻¹ mg of protein⁻¹), and they “switched off” acetylene reduction in response to added ammonia or glutamine. Crude extracts from diazotrophic cells reduced 10% acetylene at a rate of 4 to 5 nmol of C₂H₄ formed h⁻¹ mg of protein⁻¹ when supplied with ATP and reducing power, while extracts of Klebsiella pneumoniae prepared by the same procedures had rates 100-fold higher. Acetylene reduction by extracts required ATP and was completely inhibited by 1 mM ADP in the presence of 5 mM ATP. The low rates of C₂H₂ reduction could be due to improper assay conditions, to switched-off enzyme, or to the nitrogenase's having lower activity towards acetylene than towards dinitrogen.     

Regulation of nitrogenase activity by light in the azolla-anabaena symbiosis

Nitrogenase activity and the rate of photosynthesis were measured simultaneously in Azolla by a continuous gas flow system. The mode of interaction between light, photosynthesis and nitrogenase activity was analysed.Nitrogenase activity dropped off when either Azolla plants or the cyanobiont Anabaena were transferred from light to dark. This decline was immediate and was independent of length or intensity of the prior light phase. Reillumination restored nitrogenase activity.Nitrogenase activity did not depend on the rate of photosynthesis at light intensities below 10 μE m⁻² s⁻¹. Its activity was saturated at 200 μE m⁻² s⁻¹ while CO₂ fixation was saturated at a light intensity of 850 μE m⁻² s⁻¹. Azolla photosynthetic activity followed the absorption spectrum of chlorophyll a, while nitrogenase activity markedly increased between 690 and 710 nm. Inhibition of photosynthesis by DCMU was accompanied by an increase in nitrogenase activity. These results suggest direct light regulation of nitrogenase activity in Azolla independent of CO₂ fixation, and a possible inhibition of nitrogenase activity by the oxygen produced in photosynthesis.     

A method for low volume and low Se concentration samples and application to paired cerebrospinal fluid and serum samples

The well-known beneficial health effects of Se have demanded the development of rapid and accurate methods for its analysis. A flow injection (FI) method with inductively coupled plasma mass spectrometry (ICP-MS) as a selenium-selective detector was optimized. Flow injection was carried out using a Knauer 1100 smartline inert series liquid chromatograph coupled with a Perkin Elmer DRC II ICP-mass spectrometer. For sample injection a Perkin Elmer electronic valve equipped with a 25 μL sample loop was employed. Before measurement, standards or samples were administered with 1 μg/L rhodium as internal standard for correction of changes in detector response according to changes in sample electrolyte concentration. The method characterization parameters are: LOD (3σ criterion): 26 ng/L, LOQ (10σ criterion): 86 ng/L, linearity: 0.05–>10 μg/L, r²=0.9999, serial or day-to-day precision at 2 μg/L: 4.48% or 5.6%. Accuracy was determined by (a) recovery experiments (CSF spiked with 2 μg/L Se); (b) comparison of FI-ICP-MS measurement with graphite furnace atomic absorption (GFAAS) measurements of 1:10 diluted serum samples; (c) Se determination in urine and serum control materials. Recovery (a) was 101.4%, measurement comparison with GFAAS (b) showed 98.8% (5 serum samples, 1:10 diluted in the range of 0.5–1.3 μg/L, compared to GFAAS determination, which was set to 100%), and accuracy was 96.8% or 105.6% for the serum or urine control material. Analysis time per sample was short and typically below 2 min for the complete measurement, including sample introduction, sample-line purge and quadruplicate Se determination.This method was used to determine Se in cerebrospinal fluid (CSF) and plasma (here parallel to GFAAS) in 35 paired serum and CSF samples. Se determination gave values in the range of 42–130 μg/L for serum and 1.63–6.66 μg/L for CSF. The median for Se in 35 individual CSF samples was 3.28 μg/L, the mean (±SD) was 3.67 (1.35) μg/L, whilst for individual serum samples the median was 81 μg/L and the mean (±SD) was 85 (26) μg/L. When relating the paired Se concentrations of CSF samples to respective serum samples it turned out that Se-CSF (behind blood brain barrier (BBB)) is independent on Se-serum concentration (before BBB).     

The Metabolism of the Volatile Amines: VII. The Clinical Significance of Two Components of the Blood Ammonia Level

Blood ammonia levels consist of two components: ammonia present in blood at the time of shedding, termed “free” ammonia, and ammonia produced by the deamidating action of the alkali reagents. Blood of healthy people contained little or no “free” ammonia while blood of patients with chronic liver disease occasionally showed levels up to 1.2 μg./ml. Patients with hepatic encephalopathy had significantly elevated levels which usually fell to zero following therapy. Levels of “free” ammonia above 0.6 μg./ml. were diagnostic of hepatic encephalopathy in patients suffering from unexplained neurological disorders.The rate of formation of ammonia by the alkali reagents was increased in patients with hepatic necrosis and was depressed in those with chronic hepatitis. The ammonia appeared to arise from the deamidation of glutamine and asparagine, present in blood in both the free and peptide forms.     

Catecholamines and dopamine-β-hydroxylase activity during therapeutic abortion induced by sulprostone

To evaluate the changes in circulating norepinephrine (NE), epinephrine (E) and dopamine-β-hydroxylase (DBH) caused by an intravenous infusion of a derivate of PGE₂, sulprostone, in connection with legal termination of pregnancy, serial plasma samples were analyzed for six gravidae. Plasma catecholamines were measured by a sensitive radioenzymatic method (9,10) and DBH activities by a photometric assay (11). Intravenous infusion of sulprostone, in abortifacient doses as an intravenous infusion of 3–4 μg per minute for six to eight hours produced a decrease in circulating norepinephrine. No significant alteration was found in plasma epinephrine or dopamine-β-hydroxylase activity. The finding suggests an inhibitory effect of sulprostone on the release of norepinephrine from the adrenergic terminals without inhibition of the adrenal medulla.     

Comparative study of the pulmonary effects of intravenous leukotrienes and other bronchoconstrictors in anaesthetized guinea pig

Pulmonary responses to intravenous leukotrienes C₄, D₄ and E₄ administered as a bolus injection and by continuous infusion were studied in anesthetized guinea pigs. LTD₄, LTC₄ and LTE₄ (respective ED₅₀ of 0.21 ± .1, 0.64 ± .2 and 2.0 ± .1 μg kg⁻¹) produced dose-dependent increases in insufflation pressure when given as a bolus injection to anesthetized guinea pigs (Konzett-Rössler). Bronchoconstriction was antagonized by FPL-55712 (50–200 μg kg⁻¹), and indomethacin (50–200 μg kg⁻¹) but was not significantly altered by mepyramine (1.0 mg kg⁻¹), methysergide (0.1 mg kg⁻¹), intal (10 mg kg⁻¹) mepacrine (5 mg kg⁻¹) or dexamethasone (10 mg kg⁻¹). The beta adrenoceptor blocker, timolol (5 μg kg⁻¹) produced a significantly greater potentiation of the responses to the leukotrienes than to arachidonic acid, histamine and acetylcholine. Responses to bolus injection of LTE₄ but not LTD₄ or LTC₄ were partially antagonized by atropine (100 μg kg⁻¹) and bilateral vagotomy. In experiments of a different design, continuous infusion of LTD₄ and LTE₄ (2.8–3.2 μg kg⁻¹ min⁻¹) into indomethacin-treated animals produced slowly developing increases in pulmonary resistance and decreases in compliance. The increase in resistance produced by LTE₄ and LTD₄ was partly reversed by intravenous FPL-55712 (1.0 mg kg⁻¹) and atropine (100 μg kg⁻¹) but was almost completely reversed by FPL-55712 (3 – 10 mg kg⁻¹). These findings indicate that leukotrienes can produce bronchoconstriction in guinea pigs through cyclooxygenase-dependent and cyclooxygenase independent mechanisms both of which are blocked by FPL-55712. Cholinergic mechanisms are involved in the mediation of part of the response to bolus injection of LTE₄ as well as a small part of the initial response to continuous infusion of LTD₄ and LTE₄. Intrinsic beta adrenoceptor activation serves to down modulate responses to the leukotrienes to a greater extent than responses to arachidonic acid, histamine and acetylcholine.     

Simultaneous determination of amiodarone and its major metabolite desethylamiodarone in plasma, urine and tissues by high-performance liquid chromatography

A simple and sensitive high-performance liquid chromatographic method for the simultaneous assay of amiodarone and desethylarniodarone in plasma, urine and tissues has been developed. The method for plasma samples and tissue samples after homogenizing with 50% ethanol, involves deproteinization with acetonitrile containing the internal standard followed by centrifugation and direct injection of the supernatant into the liquid chromatograph. The method for urine specimens includes extraction with a diisopropyl ether—acetonitrile (95:5, v/v) mixture at pH 7.0 using disposable Clin-Elut 1003 columns, followed by evaporation of the eluate, reconstitution of the residue in methanol—acetonitrile (1:2, v/v) mixture and injection into the chromatograph. Separation was obtained using a Radial-Pak C₁₈ column operating in combination with a radial compression separation unit and a methanol–25% ammonia (99.3:0.7, v/v) mobile phase. A wavelength of 242 nm was used to monitor amiodarone, desethylamiodarone and the internal standard. The influence of the ammonia concentration in the mobile phase on the capacity factors of amiodarone, desethylamiodarone and two other potential metabolites, monoiodoamiodarone (L6355) and desiodoamiodarone (L3937) were investigated. Endogenous substances or a variety of drugs concomitantly used in amiodarone therapy did not interfere with the assay.The limit of sensitivity of the assay was 0.025 μg/ml with a precision of ± 17%. The inter- and intra-day coefficient of variation for replicate analyses of spiked plasma samples was less than 6%. This method has been demonstrated to be suitable for pharmacokinetic and metabolism studies of amiodarone in man.     

Physiological ecology of acetylene reduction (nitrogen fixation) in a Delaware salt marsh

The effects of several fixed nitrogen compounds on acetylene reduction activity (nitrogen fixation) of surface sediments from a Delaware salt marsh were studied. Ammonia addition caused little decrease in activity early in the summer but resulted in a considerable decrease (85–95%) in activity late in the summer and early in the fall. Nitrate caused a near complete suppression of activity at all times. Other compounds such as glutamate, urea, and yeast extract caused a slight increase in activity in tallSpartina sediments and caused more than a 2.5-fold increase in shortSpartina sediments. There was a lag period (1–2 days) before the commencement of in vitro acetylene reduction activity during the spring and early summer, but this lag period was not present in the late summer. The addition of chloramphenicol to samples from a shortSpartina zone caused decreases in activity similar to those obtained with ammonia, whereas chlorate amendments yielded results which, when compared on an electron basis, were comparable to those obtained with nitrate. These results indicated that the observed lag period may be the result of a physiological response to the in situ levels of ammonia and/or nitrate. It is suggested here that in situ nitrogenase activity may be controlled by two processes: (a) repression and derepression of nitrogenase synthesis mediated by the levels of ammonia, and (b) competition for reducing power (electrons) and energy (ATP) between the processes of nitrate reduction and nitrogen fixation.     

On-line monitoring of lipolytic activity by sequential injection analysis

An automated sequential injection analysis using stop-flow technique for the on-line determination of lipolytic activity has been developed. It is based on a colorimetric method using a chromogenic substrate, 1,2-O-dilauryl-rac-glycero-3-glutaric acid-(6-methylresorufin)-ester. The system permits a linear range analysis between 5–100 lipolytic activity units ml^–1, without external dilution of the sample, a sampling frequency of 5 samples per hour and a relative standard deviation (RSD) of 5%. The analyser has been used for the on-line monitoring of Candida rugosa fed-batch fermentation with excellent performance, regarding its reliability and reproducibility.     

Oxygen Uptake and Hydrogen-Stimulated Nitrogenase Activity from Azorhizobium caulinodans ORS571 Grown in a Succinate-Limited Chemostat

Succinate-limited continuous cultures of an Azorhizobium caulinodans strain were grown on ammonia or nitrogen gas as a nitrogen source. Ammonia-grown cells became oxygen limited at 1.7 μM dissolved oxygen, whereas nitrogen-fixing cells remained succinate limited even at dissolved oxygen concentrations as low as 0.9 μM. Nitrogen-fixing cells tolerated dissolved oxygen concentrations as high as 41 μM. Succinate-dependent oxygen uptake rates of cells from the different steady states ranged from 178 to 236 nmol min⁻¹ mg of protein⁻¹ and were not affected by varying chemostat-dissolved oxygen concentration or nitrogen source. When equimolar concentrations of succinate and β-hydroxybutyrate were combined, oxygen uptake rates were greater than when either substrate was used alone. Azide could also used alone as a respiratory substrate regardless of nitrogen source; however, when azide was added following succinate additions, oxygen uptake was inhibited in ammonia-grown cells and stimulated in nitrogen-fixing cells. Use of 25 mM succinate in the chemostat resevoir at a dilution rate of 0.1 h⁻¹ resulted in high levels of background respiration and nitrogenase activity, indicating that the cells were not energy limited. Lowering the reservoir succinate to 5 mM imposed energy limitation. Maximum succinate-dependent nitrogenase activity was 1,741 nmol of C₂H₄h⁻¹ mg (dry weight)⁻¹, and maximum hydrogen-dependent nitrogenase activity was 949 nmol of C₂H₄ h⁻¹ mg (dry weight)⁻¹. However, when concentration of 5% (vol/vol) hydrogen or greater were combined with succinate, nitrogenase activity decreased by 35% in comparison to when succinate was used alone. Substitution of argon for nitrogen in the chemostat inflow gas resulted in “washout,” proving that ORS571 can grow on N₂ and that there was not a nitrogen source in the medium that could substitute.     

Estimation of nitric oxide level in vivo by microdialysis with water-soluble iron-N-methyl-d-dithiocarbamate complexes as NO traps: A novel approach to nitric oxide spin trapping in animal tissues

It was found that microdialysis, i.e., passage of aqueous solutions of iron-N-methyl-d-glucamine dithiocarbamate complexes through dialysis fibers implanted into heart, kidney and liver tissues of narcotized rats, was accompanied by effective binding of the complexes to nitric oxide from interstitial fluid. The walls of dialysis fibers used in this study were permeable for compounds with molecular weight not exceeding 5 kDa. The dialyzate samples collected every 20 min and containing diamagnetic nitrosyl Fe³⁺-MGD adducts were reduced to the paramagnetic state with sodium dithionite; their concentration was measured by the EPR method. The basic level of the adducts, which represented mononitrosyl iron complexes with MGD (MNIC–MGD), in the dialyzate samples of all tested organs were similar (1 μМ). Treatment of animals with the water-soluble nitroglycerine analog Isoket or a low-molecular dinitrosyl iron thiosulfate complex as a NO donor increased the concentration of MNIC–MGD with going out into a plateau. The novel approach allows determination of nitric oxide levels in tissue interstitial fluid from concentration of MNIC–MGD formed during microdialysis.