Presence Of Oxyhaemoglobin Research Articles

New method for the determination of the net bilirubin absorbance in cerebrospinal fluid that minimizes the interference of oxyhaemoglobin and biliverdin

The presence of oxyhaemoglobin and biliverdin interferes with the method recommended by the UK NEQAS Specialist Advisory group for EQA of CSF Proteins and Biochemistry for estimating of the net bilirubin absorbance in CSF. This is easily demonstrated by using solutions with different concentrations of these three substances.The two secondary peaks of the oxyhaemoglobin spectrum at 540 nm and 577 nm are used as reference to minimize these interferences. Those peaks have the same absorbance as at 456 nm in the oxyhaemoglobin spectrum, independent of its concentration. This wavelength is very close to the maximum absorption of bilirubin and, therefore, is suitable for estimating the net bilirubin absorbance.A preliminary study with 48 spectrophotometric analyses of CSF from patients who were suspected of having subarachnoid haemorrhage were used to compare both net bilirubin absorbance estimation methods.The new method is practically free of oxyhaemoglobin and biliverdin interference. This allows for higher sensitivity and a more realistic estimation of the bilirubin concentration in a sample.A better estimation of the bilirubin concentration can have special relevance for diminishing the amount of equivocal or inconclusive cases and also to improve the prematurity of the diagnosis.

Specificity of elevated cerebrospinal fluid bilirubin in the investigation of subarachnoid haemorrhage.

The spectrophotometric examination of cerebrospinal fluid for bilirubin is an established investigation in patients with suspected subarachnoid haemorrhage. This study assesses the diagnostic specificity of an elevated cerebrospinal fluid bilirubin and how this may be influenced by the presence of oxyhaemoglobin and the concentration of cerebrospinal fluid total protein. One thousand cerebrospinal fluid spectroscopy reports were reviewed. Electronic patient records were examined to determine the clinical outcome in patients with an elevated cerebrospinal fluid bilirubin. Forty-four out of 1000 cerebrospinal fluid scans showed an increase in cerebrospinal fluid bilirubin unrelated to elevated serum bilirubin concentrations. This was associated with subarachnoid haemorrhage in 16 (36%) cases. Subarachnoid haemorrhage was confirmed in 5/17 (29%) patients positive for cerebrospinal fluid bilirubin alone and in 11/27 (41%) patients positive for both cerebrospinal fluid bilirubin and oxyhaemoglobin. At cerebrospinal fluid total protein concentrations <1 g/L, the specificity for subarachnoid haemorrhage improved: 4/9 (44%) vs. 1/8 (13%) with an increase in cerebrospinal fluid bilirubin alone; 6/10 (60%) vs. 5/17 (29%) in patients with increases in both bilirubin and oxyhaemoglobin. While an increase in cerebrospinal fluid bilirubin may be considered consistent with subarachnoid haemorrhage, the likelihood that a subarachnoid haemorrhage has occurred is influenced by the presence of oxyhaemoglobin and the concentration of total protein in the cerebrospinal fluid.

Studies of the potential genotoxic effects of furoxans: The case of CAS 1609 and of the water-soluble analogue of CHF 2363

CAS 1609 (compound 1) and CHF 2363 (compound 2) are two furoxan derivatives able to release nitric oxide (NO) under physiological conditions, and display typical NO-dependent vasodilator activity. The potential genotoxic effects of compound 1 and of the water-soluble analogue of CHF 2363 (compound 2a) were investigated. The results show that the two compounds induce genotoxic effects only at concentrations that significantly reduce cell viability. However, in the case of compound 1 this range of concentrations is one order of magnitude higher than the one leading to the beneficial effects, while in the case of compound 2a these ranges partially overlap. In both cases the release of NO plays a key role in the induction of the cytotoxic and genotoxic effects, since the non-NO-donating furazan analogues display a different toxicological profile, and since the effects were reduced in the presence of oxyhaemoglobin, a well-known NO-scavenger.

Synthesis and Vasodilating Properties of N‐Alkylamide Derivatives of 4‐Amino‐3‐furoxancarboxylic Acid and Related Azo Derivatives.

AbstractFor Abstract see ChemInform Abstract in Full Text.

Synthesis and vasodilating properties of N-alkylamide derivatives of 4-amino-3-furoxancarboxylic acid and related azo derivatives

A series of N-alkylamide derivatives of 4-amino-3-furoxancarboxylic acids 5a– 11a and their oxidation products, the azo derivatives 5b– 11b, were synthesised and studied for their vasodilating properties. All the products were able to release rat aorta strips precontracted with (−)noradrenaline. Azo derivatives proved to be 20–200 times more potent than the parent amines. The large variation of lipophilicity within the two series does not seem to influence significantly the activity. Experiments carried out in the presence of oxyhaemoglobin (HbO 2) suggest the involvement of nitric oxide (NO ) in the vasodilation.

IN VITRO AND IN VIVO VASODILATING ACTIVITY OF NITROSO DERIVATIVES GEM -SUBSTITUTED WITH ELECTRON-WITHDRAWING GROUPS

A series of nitroso compounds gem -substituted with electron-withdrawing groups (R2C(X)NO, R=alkyl, X=NO2, CN, Cl), were studied for their in vitro and in vivo vasodilating properties as well as for their ability to activate soluble guanylate cyclase (sGC) in RFL-6 cells. All the compounds, with the sole exception of chloro derivative, display good in vitro vasodilating action and are able to increase the basal level of cGMP. Their potencies as vasodilators decrease in the presence of oxyhaemoglobin, a scavenger of nitric oxide (NO). The haemodynamic profile of the most interesting compounds, assessed in anaesthetized pigs, is also in line with a release of NO from these compounds.

Evidence that nitric oxide causes calcium-independent release of [3H] dopamine from rat striatum in vitro.

Nitric oxide (NO), liberated from the photoactive donor Roussin's black salt (RBS), was investigated for its ability to release tritium from [3H]dopamine-loaded rat striatal slices. Our results show that illumination of RBS-pretreated striatal slices caused an increase in basal dopamine release, which was reduced by approximately 73% in the presence of oxyhaemoglobin (10 microM), indicating that it was mediated by liberation of NO. The release was insensitive to removal of extracellular calcium yet was not due to gross cellular damage of the tissue, as there was no detectable increase in lactate dehydrogenase release. Chelation of intracellular calcium with 1,2-bis(o-amino-phenoxy)ethane-N,N,N',N'-tetraacetic acid tetra(acetoxymethyl) ester (BAPTA-AM; 10 microM) had no effect on the dopamine release stimulated by illumination of RBS-pretreated slices. The concentration of BAPTA-AM was adequate to chelate intracellular calcium because it inhibited release evoked by the calcium ionophore ionomycin (10 microM). Superfusion with zaprinast (10 microM) had no effect on RBS-induced dopamine release, suggesting that a mechanism independent of cyclic GMP is involved. This study indicates that NO has a stimulatory effect on striatal dopamine release in vitro that is independent of calcium.

The Antiplatelet Effects of a New Nitroderivative of Acetylsalicylic Acid - An In Vitro Study of Inhibition on the Early Phase of Platelet Activation and on TXA2 Production

We studied in vitro the antiplatelet activity of a new nitroderivative chemically related to acetylsalicylic acid: 2 acetoxybenzoate 2-[1-nitroxy-methyl]-phenyl ester (NCX 4016), in order to identify any effects due to the release of nitric oxide and the blockade of cyclo-oxygenase. The effects of scalar doses of NCX 4016 on the early phase of platelet activation, platelet aggregation and thromboxane A2 production were investigated. We observed inhibitory effects of NCX 4016 on platelet adhesion (IC50 = 7.3 x 10(-5) M), platelet cytosolic calcium concentration, assayed by fluorescent probe Fura 2, and the expression of glycoprotein IIb/IIIa (CD41/alpha IIb beta 3) (IC50 = 3.4 x 10(-5) M) and P-selectin (CD62/GMP-140) (IC50 = 4.9 x 10(-5) M) measured by flow cytometry. NCX 4016 also prevented thrombin-induced platelet aggregation (IC50 = 3.9 x 10(-5) M). None of these parameters were affected by acetylsalicylic acid. These inhibitory activities of NCX 4016 were abolished by oxyhaemoglobin and methylene blue. Intracellular cyclic GMP observed during thrombin-induced aggregation was increased by incubation with NCX 4016. These results appear to be attributable to the release of nitric oxide, which activates soluble platelet guanylylcyclase and promotes intracellular cyclic GMP increase. NCX 4016 almost completely inhibited platelet thromboxane A2 production and arachidonic acid-induced platelet aggregation. This also occurred in the presence of oxyhaemoglobin and methylene blue, indicating that its antiplatelet activity can be attributed not only to nitric oxide release but also to cyclo-oxygenase inhibition.

Characterization of furoxans as a new class of tolerance-resistant nitrovasodilators

The vasodilator effects of C92-4609 (4-hydroxymethyl-furoxan-3-carboxamide, CAS 1609), C92-4678(4-phenyl-furoxan-3-carboxylic acid (pyridyl-3-yl-methyl)-amide), C92-4679 (3-phenyl-furoxan-4-carboxylic acid (pyridyl-3-yl-methyl)-amide) and C93-4759 (3-hydroxymethyl-furoxan-4-carboxamide) were studied in the isolated rabbit femoral artery and jugular vein. All furoxans were potent vasodilators in the femoral artery (EC50 0.1-50 microM), while they were less potent in the jugular vein by at least one order of magnitude. Apart from C92-4679, the vasodilatory potency of the furoxans correlated well with their nitric oxide (NO)-releasing capacity which was estimated both by stimulation of purified soluble guanylyl cyclase activity and electron spin resonance spectroscopy with a trapping agent for NO. The hypothesis that furoxans stimulate soluble guanylyl cyclase in the smooth muscle by spontaneously releasing NO was supported by the marked attenuation of their vasodilator effect in the presence of oxyhaemoglobin (10 microM) or following treatment with methylene blue (30 microM). In contrast to earlier findings, NO release from these furoxans was not thiol-dependent, as demonstrated for C92-4609, the relaxant effect of which in the femoral artery was not altered in the presence of N-acetyl-L-cysteine (1 mM). Moreover the KCa+ channel inhibitor, tetrabutylammonium (3 mM), but not the KATP+ channel inhibitor, glibenclamide (3 microM), significantly attenuated the dilator response to C92-4679 in the femoral artery. Pretreatment of these segments with the cytochrome P450 inhibitor, SKF525a (30 microM), also reduced the C92-4679-induced relaxation in this vascular bed.(ABSTRACT TRUNCATED AT 250 WORDS)

Effects of endothelial cell removal on constrictor and dilator responses of aortae of pregnant rats.

1. The possibility has been examined that changes in sensitivity of the aorta occurring during pregnancy in the rat are due to changes in output of endothelium-derived relaxing factor (EDRF). 2. Concentration-isometric response curves, obtained in vitro, from thoracic aortic rings of non-pregnant rats in oestrous and 20-day pregnant rats to noradrenaline (NA) and phenylephrine (PE) were shallower with lower maxima when compared to those obtained to the thromboxane A2-mimetic U46619. 3. Removal of endothelium from aortae of non-pregnant animals caused significant shifts to the left of the curves obtained using all three agonists. In the case of NA or PE, EC50 values were reduced approximately fourfold, with maximum responses also increasing significantly. Endothelial cell removal caused an approximately eightfold increase in sensitivity to U46619 but no change in maximum response. The presence of oxyhaemoglobin (Hb) had a similar effect to endothelial removal on responses to NA and PE. 4. Rings obtained from 20-day pregnant rats showed significantly increased maximum responses to NA and PE when compared with those from non-pregnant animals. Removal of the endothelium or the presence of Hb caused significant shifts of the dose-response curves to the left and further increases in maxima. 5. In contrast, no differences in maximum responses to U46619 could be detected between the aortic rings of pregnant and non-pregnant animals. Removal of the endothelium from either caused increases in sensitivity which did not differ significantly.(ABSTRACT TRUNCATED AT 250 WORDS)

The oxidation of oxyhaemoglobin by glyceraldehyde and other simple monosaccharides.

Glyceraldehyde and other simple monosaccharides oxidize oxyhaemoglobin to methaemoglobin in phosphate buffer at pH 7.4 and 37 degrees C, with the concomitant production of H2O2 and an alpha-oxo aldehyde derivative of the monosaccharide. Simple monosaccharides also reduce methaemoglobin to ferrohaemichromes (non-intact haemoglobin) at pH 7.4 and 37 degrees C. Carbonmonoxyhaemoglobin is unreactive towards oxidation by autoxidizing glyceraldehyde. Free-radical production from autoxidizing monosaccharides with haemoglobins was observed by the e.s.r. technique of spin trapping with the spin trap 5,5-dimethyl-l-pyrroline N-oxide. Hydroxyl and l-hydroxyalkyl radical production observed from monosaccharide autoxidation was quenched in the presence of oxyhaemoglobin and methaemoglobin. The haemoglobins appear to quench the free radicals by reaction with the free radicals and/or the ene-diol precursor of the free radical.

In vitroandin vivoevaluation of the toxicity of 1,4-naphthoquinone and 1,2-naphthoquinone derivatives againstTrypanosoma cruzi

The toxic effect of lapachol, beta-lapachone and several 1,2-naphthoquinone derivatives on the growth, viability and infectivity of Trypanosoma cruzi were compared. beta-lapachone was the most active compound in vitro. No inhibition was observed in suspensions which contained inactivated foetal calf serum or rabbit haemoglobin solution. The infectivity of trypomastigotes in mice was not affected when cells were previously incubated with beta-lapachone or one of several other naphthoquinone derivatives in vitro in the presence of blood. It is suggested that beta-lapachone and the other compounds can be inactivated either by reduction in the presence of oxyhaemoglobin or by interaction with serum proteins. A beta-lapachone derivative, allyl-beta-lapachone, was not inactivated in the presence of blood and remained effective in suppressing trypomastigote infectivity.

Oxidation of phenylhydrazines in the presence of oxyhaemoglobin and the origin of Heinz bodies in erythrocytes.

Oxidation of Phenyl-Hydrazines in the Presence of Oxyhaemoglobin and the Origin of Heinz Bodies in Erythrocytes