Intestinal current measurement and nasal potential difference to make a diagnosis of cases with inconclusive CFTR genetics and sweat test.

BACKGROUND: Nasal potential difference (NPD) and intestinal current measurements (ICM) are cystic fibrosis transmembrane conductance regulator (CFTR) biomarkers recommended to make a diagnosis in individuals with inconclusive sweat test and CFTR genetics and a clinical suspicion for cystic fibrosis (CF) or CFTR-related disorder (CFTR-RD). METHODS: NPD and ICM were measured according to standard operating procedures of the European Cystic Fibrosis Society Diagnostic Network Working Group. RESULTS: We assessed 219 individuals by NPD or ICM who had been referred to our laboratory due to clinical symptoms suggestive of CF, but inconclusive sweat test and CFTR genetics (median age: 16.3 years, range 0.4 to 76 years). CF or CFTR-related disorder was diagnosed in 22 of 29 patients (76%) with a CFTR genotype of unknown or variable clinical significance and in 51 of 190 carriers (27%) of one (35/42) or no (16/148) identified CFTR mutation. If two CFTR sequence variants had been identified, the outcome of NPD and ICM was consistent with the classification of the CFTR2 database. Moreover, a suspected false-positive diagnosis of CF was confirmed in seven and withdrawn in eight patients. Of 26 individuals assessed by both NPD and ICM, eleven individuals exhibited discordant tracings of ICM and NPD, with one measurement being in the CF range and the other in the normal range. CONCLUSION: The majority of patients whom we diagnosed with CF or CFTR-RD by extended electrophysiology are carriers of the wild-type CFTR coding sequence on at least one of their CF alleles. The disease-causing genetic lesions should reside in the non-coding region of CFTR or elsewhere in the genome, affecting the regulation of CFTR expression in a tissue-depending fashion which may explain the large within-group variability of CFTR activity in the respiratory and intestinal epithelium seen in this group.

Reduction of myocardial nitrosyl complex formation by a nitric oxide scavenger prolongs cardiac allograft survival.

Nitric oxide synthase (NOS) inhibitors have been shown to reduce NO but yield conflicting results on cardiac allograft survival. In this study, we provide an alternative approach specifically to examine the efficacy of a NO scavenger on nitrosyl complex formation and graft survival in a model of heterotopic cardiac transplantation. Efficacy was examined under both acute and chronic conditions (i.e., without or with immunosuppression, respectively). Electron paramagnetic resonance (EPR) spectroscopy of frozen myocardial tissue from untreated allografts showed progressive increases in nitrosylheme and nitrosomyoglobin before graft failure. These signals were not seen in either isografts or native hearts of allograft recipients. Both plasma nitrate plus nitrite and myocardial nitrosyl complex formation in cardiac allografts were significantly decreased in recipient animals treated with the NO scavenger, NOX-100, or by low-dose cyclosporine (CsA). Both interventions were nearly equivalent in significantly prolonging graft survival. The short-term combination treatment of both NOX-100 plus CsA completely eliminated myocardial nitrosyl complex formation and synergistically prolonged graft survival. Long-term combination drug treatment (days 0-100) followed by cessation of therapy resulted in permanent graft acceptance with no evidence for nitrosyl complex formation. These studies support a role of NO in cardiac allograft rejection. Furthermore, these studies indicate a potential therapeutic value of NO scavengers in preventing organ rejection.

Detection of thiyl radical adducts formed during hydroxyl radical- and peroxynitrite-mediated oxidation of thiols--a high resolution ESR spin-trapping study at Q-band (35 GHz).

Thiyl radicals (RS.) formed during peroxynitrite- or hydroxyl radical-dependent oxidation of thiols, i.e., glutathione (GSH) and L-cysteine (CySH) were trapped with 5,5'-dimethyl-1-pyrroline N-oxide (DMPO) and analyzed by X-band and Q-band electron spin resonance (ESR) spectroscopy. At X-band, the ESR parameters of DMPO-glutathionyl radical adduct (DMPO/.SG) and DMPO-hydroxyl radical adduct (DMPO/.OH) are nearly similar in aqueous solutions and as a result, except for the lowfield spectral line, the remaining spectral lines of DMPO/ .SG virtually over-lap with those of the DMPO/.OH adduct. In contrast, at Q-band, most of the spectral lines due to the DMPO/.SG were separated from the DMPO/ .OH. Inclusion of a superoxide dismutase (SOD) mimic completely abolished the formation of the DMPO/.OH adduct and not the DMPO/.SG adduct during ONOO(-)-mediated oxidation of GSH and DMPO. In the presence of formate, the DMPO/.SG spectrum was replaced by the DMPO/.CO2- spectrum which was monitored by Q-band ESR spectroscopy. Thus, spin-trapping at Q-band provides unambiguous proof for the glutathionyl radical-dependent oxidation of formate by peroxynitrite. High resolution Q-band ESR spectra of DMPO/.Scys were also obtained. Biological applications of the Q-band spin-trapping technique to detect thiyl radicals in cellular systems are discussed.

Detection by ESR of DMPO hydroxyl adduct formation from islets of Langerhans.

Electron spin resonance (ESR) spectroscopy together with spin trapping techniques and the application of state-of-the-art loop gap resonators was used to provide a direct measure of spontaneous oxygen radical production by homogenates of freshly isolated and cultured rat pancreatic islets. Using the spin trap agent, 5,5-dimethyl-1-pyrroline-N-oxide (DMPO), we were able to detect production by islets of an ESR-sensitive radical signal consisting of a quartet with intensity ratio of 1:2:2:1 and hyperfine splitting of aN = aH = 14.9 Gauss, which is consistent with the DMPO-OH adduct. The amplitude of the signal was decreased by decreasing amount of islets and not detected in the absence of islets. Formation of the DMPO-OH adduct was diminished by the hydroxyl radical scavengers (e.g., ethanol, dimethylsulfoxide, and dimethylthiourea). Only partial attenuation of signal was produced by incubation with an iron chelator or using chelex-treated buffers. The ESR signal was insensitive to the xanthine oxidase inhibitor, oxypurinol, or to superoxide dismutase, but was eliminated in a concentration-dependent manner by either potassium cyanide or catalase (but not heat-inactivated catalase). These observations suggest that the origin of the DMPO-OH arose not from free hydroxyl radicals but primarily from endogenous hydrogen peroxide production perhaps of mitochondrial origin. The development of this technology has implications for the potential measure of oxygen radical production in islet homogenates under pathologic conditions as well as to the application of other cell culture systems.

Multiquantum EPR of the mixed valence copper site in nitrous oxide reductase.

This work demonstrates the use of multiquantum EPR to study the magnetic properties of copper complexes and copper proteins. Pure absorption spectra are obtained because of the absence of field modulation. The signal intensity of 3-quantum spectra is proportional to the spin lattice relaxation time T1, while its linewidth in a frequency difference sweep is T1(-1). A change in lineshape for the EPR detectable mixed value [Cu(1.5) . . . Cu(1.5)] site in nitrous oxide reductase is attributed to suppression of the forbidden transitions. The data confirm the unusually fast relaxation time for this site, which requires temperatures of less than 100 K to resolve hyperfine structure. The T1's for the mixed valence [Cu(1.5) . . . Cu(1.5)] site in nitrous oxide reductase are very similar to T1's for the Cua site in cytochrome c oxidase. The similar relaxation properties, together with previous multifrequency EPR results, support the hypothesis that the EPR detectable sites in cytochrome c oxidase and nitrous oxide reductase are mixed valence [Cu(1.5) . . . Cu(1.5)] configurations.

Characterization of the pigment from homogentisic acid and urine and tissue from an alkaptonuria patient.

When urine samples from alkaptonuria patients are allowed to stand, they turn black, presumably owing to the oxidation of homogentisic acid to a melanin-like substance. We report the characterization of the pigments formed by polymerization of (a) the components in the urine from a patient with alkaptonuria and (b) homogentisic acid. The absorption spectra and electron spin resonance signals of these pigments are similar to those of eumelanins. Irradiation of the pigments with nitroblue tetrazolium caused reduction of the tetrazolium; this was partially inhibited by superoxide dismutase. Irradiation of Ehrlich ascites carcinoma cells with the pigments from homogentisic acid or urine caused cell lysis. Since this lysis was inhibited by catalase, we have concluded that it was mediated by H2O2. A similar pigment was also extracted from the tissue from an alkaptonuria patient. It is suggested that the degeneration of tissue in vivo may be due to the deposition of melanin-like pigments in the tissues, probably in combination with metal ions.

Oxygen activation during the interaction between MPTP metabolites and synthetic neuromelanin--an ESR-spin trapping, optical, and oxidase electrode study.

Oxidase electrode measurements as well as optical and electron spin resonance spectroscopic data have shown that synthetic neuromelanin oxidizes the neurotoxin metabolite 1-methyl-4-phenyl-2,3-dihydropyridinium in a dose-dependent manner forming 1-methyl-4-phenylpyridinium and hydrogen peroxide. Hydroxyl radicals are formed in this reaction which is promoted by iron chelates. In contrast, neither 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine nor 1-methyl-4-phenylpyridinium reacts with synthetic neuromelanin in a similar fashion. The mechanism of selective toxicity of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine in pigmented neuronal cells is discussed in the light of these findings.

Myocardial ischemia and reperfusion: direct evidence for free radical generation by electron spin resonance spectroscopy.

Electron spin resonance spectroscopy has recently been used by others to detect directly radical species in isolated perfused hearts. Sample processing prior to spectroscopy in this study involved pulverization of tissue, which can artifactually generate radical species. We assessed in isolated perfused hearts the influence of tissue pulverization on the identity of radical species detected by spectroscopy and then, using a processing technique less likely to induce artifacts, whether myocardial ischemia and reperfusion generate radical species. Rat and rabbit hearts (n = 8) were perfused aerobically for 10 min and freeze-clamped to -196 degrees C. Frozen tissue was processed at -196 degrees C for spectroscopic analysis by pulverization vs. chopping. Spectra of pulverized tissue consisted of three components: a semiquinone (g = 2.004), a lipid peroxy radical (g [ = 2.04 and g = 2.006), and a carbon-centered radical that is possibly a lipid radical (giso = 2.002 and AHzz approximately equal to 50 G). Chopped tissue consisted of a single component, a semiquinone (g = 2.004). Rat hearts (n = 8 per group) also underwent 10-min global no-flow normothermic ischemia followed by 5-60 sec of either aerobic or anaerobic reperfusion, with frozen tissue chopped prior to spectroscopy. Spectra of ischemic tissue consisted of an iron-sulfur center and a semiquinone. Aerobic reperfusion resulted in a spectrum similar to the control but with increased amplitude that peaked after 10-15 sec of reflow. Anaerobic reperfusion yielded a spectrum identical to that of ischemic tissue. We conclude that pulverization of frozen myocardial tissue arti-factually generates radical species. Using a nonpulverization technique for tissue processing, we found that myocardial ischemia and reperfusion produce radical species but that molecular oxygen is necessary for the burst of radical production during reflow.

Evidence for the one-electron oxidation of 1-methyl-4-phenyl-2,3-dihydropyridinium (MPDP+).

Optical data have shown that the neurotoxin metabolite 1-methyl-4-phenyl-2,3-dihydropyridinium undergoes one-electron oxidation/reduction in the presence of iron chelates. The activation energy for one-electron oxidation of 1-methyl-4-phenyl-2,3-dihydropyridinium is less than that for two-electron oxidation. Horseradish peroxidase catalyzes the oxidation of 1-methyl-4-phenyl-2,3-dihydropyridinium. Reactivity of 1-methyl-4-phenylpyridinyl radical is discussed in relation to the well-known pyridinyl radicals.

Is there any difference in the photobiological properties of melanins isolated from human blue and brown eyes?

Investigations were carried out to determine whether the melanin present in the blue and brown eyes were eumelanin, the melanin present in black hair and dark skin, or pheomelanin, the melanin present in red hair and the skin of people with red hair. Our results showed that UV-visible irradiation of blue or brown eye melanin did not produce any superoxide. Irradiation of 51Cr-labelled Ehrlich ascites carcinoma cells in the presence of blue or brown eye melanin did not produce significant cell lysis. The electron spin resonance (ESR) signals of blue and brown eye melanins were very similar to those of eumelanin. Comparison of these findings with our previous results indicated that the blue and brown eye melanins are essentially eumelanin. The ESR signals further suggested that in the case of both blue and brown eye melanins the iris, ciliary body, choroid, and retinal pigment epithelium did not differ.

Mechanism of oxidation of 1-methyl-4-phenyl-2,3-dihydropyridinium (MPDP+).

Oxidase electrode measurements have shown that the neurotoxin metabolite 1-methyl-4-phenyl-2,3-dihydropyridinium autoxidizes to hydrogen peroxide and 1-methyl-4-phenylpyridinium in a reaction promoted by iron chelates. The mechanism of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine toxicity is discussed in the light of these findings.

Semiquinone anion radicals of catechol(amine)s, catechol estrogens, and their metal ion complexes.

The characterization and identification of semiquinone radicals from catechol(amine)s and catechol estrogens by electron spin resonance spectroscopy is addressed. The use of diamagnetic metal ions, especially Mg2+ and Zn2+ ions, to detect transient semiquinone radicals in biological systems and to monitor their reactions, is discussed. A brief account of the identification and reactions of quinones is also presented.

Identification by electron spin resonance spectroscopy of free radicals produced during autoxidative melanogenesis.

Free radicals produced during the autoxidation of 3,4-dihydroxyphenylalanine (DOPA) and other catechol(amine)s to melanins have been studied using electron spin resonance spectroscopy. Magnetic parameters for the radical intermediates have been determined, allowing the radicals to be unambiguously identified. Three types of radical are formed: the primary radical from one-electron oxidation of the parent catechol(amine); and two secondary radicals, one formed via OH- substitution, the other via cyclization. The formation of these radical species can be linked to molecular products formed during catecholamine oxidation and melanin formation.

Peroxidatic oxidation of catecholamines. A kinetic electron spin resonance investigation using the spin stabilization approach.

Using spin stabilization, ESR measurements have been made of o-semiquinone production from the horseradish peroxidase-H2O2 oxidation of catecholamine substrates. The termination rate constant for semiquinones stabilized with Zn2+ at pH 5 is about 10(4) times smaller than for uncomplexed semiquinones at neutral pH. Stabilization allows steady state concentrations of semiquinones to be obtained. The duration of the steady state is dependent upon the concentrations of enzyme, hydrogen peroxide, and catecholamine substrate. The relative reactivity of the substrates 3,4-dihydroxyphenylalanine, norepinephrine, and dopamine at pH 5 is 1:8:40. The effects of phenol and ascorbate were studied and shown to be consistent with scavenging of phenoxyl radicals by catecholamine and semiquinone radicals by ascorbate, respectively.

Electron spin resonance-spin stabilization of semiquinones produced during oxidation of epinephrine and its analogues.

The ESR-spin stabilization approach has been employed to detect and characterize o-semiquinone radicals from the oxidation of epinephrine and related materials (norepinephrine, 3,4-dihydroxynorephedrine, isoproterenol, and adrenalone) in aqueous solutions. Semiquinones were generated by various oxidative procedures--enzymatic oxidation (with horseradish peroxidase/H2O2), chemical oxidation (with Ag2O) and photooxidation--and subsequently kinetically stabilized through complexation with Zn2+ ions. This "spin stabilization" affords high radical concentrations, which has allowed unambiguous identification of the radical intermediates. Where appropriate, spectral assignments have been supported by deuterium substitution experiments and computer simulations of spectra. Two types of free radical have been identified: primary "open chain" semiquinones, formed by one-electron oxidation of the parent catecholamines, and secondary semiquinones, formed subsequent to cyclization reactions. The mechanism of formation of the secondary radicals is discussed, and it is concluded that they are derived from product aminochromes. Thus, oxidation of adrenochrome gives a radical identical to the secondary species observed from oxidation of epinephrine.

Melanin endocytosis by cultured mammalian cells. A model for melanin in a cellular environment.

The incorporation of natural eumelanin from bovine eyes and synthetic 3,4-dihydroxy-phenylalanine (dopa) melanin into Chinese hamster ovary (CHO) cells is reported. The process is linear for at least 8 h. Electron microscopy showed phagocytosis of melanin, either as a single granule or in groups of granules, into cell lysosomes with subsequent degradation of the granule. The general features of the ingestion and degradation processes mimic those of the incorporation of melanosomes into keratinocytes. CHO cells with ingested melanin in general revealed properties very similar to those of the pigment-free CHO cell: cell division, oxygen consumption and plating efficiency were not greatly altered by moderate concentrations of pigment. This suggests that the CHO cell system may be useful for the study of pigment in a cellular environment; pigment-free CHO cells are well characterized and can serve as a good control. Preliminary applications are reported: demonstrations of (1) incorporation of metal ions (Al3+) into CHO cells using melanin as a carrier; (2) the ability of melanin to enhance the rate of oxygen consumption during photo-irradiation of the cells.