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Background: P21, a cyclin kinase inhibitor, is upregulated by renal 'ischemic preconditioning' (IPC), and induces a 'cytoresistant' state. However, P21-induced cell cycle inhibition can also contribute to cellular senescence, a potential adverse renal event. Hence, this study assessed whether: (i) IPC-induced P21 upregulation is associated with subsequent renal senescence; and (ii) preconditioning can be established 'independent' of P21 induction and avoid a post-ischemic senescent state?, Methods: CD-1 mice were subjected to either IPC (5-15 min) or to a recently proposed 'oxidant-induced preconditioning' (OIP) strategy (tin protoporphyrin-induced heme oxygenase inhibition +/- parental iron administration). P21 induction [messenger RNA (mRNA)/protein], cell proliferation (KI-67, phosphohistone H3 nuclear staining), kidney senescence (P16ink4a; P19Arf mRNAs; senescence-associated beta-galactosidase levels) and resistance to ischemic acute kidney injury were assessed., Results: IPC induced dramatic (10-25×) and persistent P21 activation and 'downstream' tubular senescence. Conversely, OIP did not upregulate P21, it increased, rather than decreased, cell proliferation markers, and it avoided a senescence state. OIP markedly suppressed ischemia-induced P21 up-regulation, it inhibited the development of post-ischemic senescence and it conferred near-complete protection against ischemic acute renal failure (ARF). To assess OIP's impact on a non-P21-dependent cytoprotective pathway, its ability to activate Nrf2, the so-called 'master regulator' of endogenous cell defenses, was assessed. Within 4 h, OIP activated each of three canonical Nrf2-regulated genes (NQO1, SRXN1, GCLC; 3- to 5-fold mRNA increases). Conversely, this gene activation pathway was absent in Nrf2-/- mice, confirming Nrf2 specificity. Nrf2-/- mice also did not develop significant OIP-mediated protection against ischemic ARF., Conclusions: OIP (i) activates the cytoprotective Nrf2, but not the P21, pathway; (ii) suppresses post-ischemic P21 induction and renal senescence; and (iii) confers marked protection against ischemic ARF. In sum, these findings suggest that OIP may be a clinically feasible approach for safely activating the Nrf2 pathway, and thereby confer protection against clinical renal injury.

Background: Although heme-oxygenase (HO) is cytoprotective, its effects on podocyte regulators like podocalyxin, podocin, CD2-associated protein (CD2AP) in renal dysfunction in N (ω)-nitro-l-arginine-methyl ester (l-NAME) hypertension are largely unclear., Methods: Hypertension was induced in normotensive Sprague Dawley rats by administering l-NAME for 4 weeks. Enzyme immunoassay, enzyme-linked immunosorbent, histology/morphology, spectrophotometry, and western immunoblotting were used. HO was enhanced with heme-arginate (HA) or inhibited with chromium mesoporphyrin (CrMP)., Results: Treatment with heme-arginate reduced several renal histo-pathological lesions including renal arteriolar thickening, glomerular abnormalities, tubular cast, tubular atrophy/fibrosis, and mononuclear cell infiltration in l-NAME-hypertensive rats. Similarly, HA abated the elevated levels of renal extracellular matrix/profibrotic proteins like collagen and fibronectin that deplete nephrin, a fundamental transmembrane protein that forms the scaffoldings of the podocyte slit diaphragm permitting small ions to filter, but not massive excretion of proteins, hence proteinuria. Correspondingly, HA enhanced the aberrant expression of nephrin alongside other important regulators of podocyte like podocalyxin, podocin, and CD2AP, and improved renal function by reducing albuminuria/proteinuria, while increasing creatinine clearance. The renoprotection by HA were accompanied by significant reduction of inflammatory/oxidative mediators including nuclear factor-kappaB, macrophage inflammatory protein-1-alpha, macrophage chemoattractant protein-1, tumor necrosis factor-alpha, interleukin (IL)-6, IL1β, 8-isoprostane, endothelin-1, and aldosterone. These were associated with increased levels of adiponectin, HO-1, HO activity, cyclic guanosine monophosphate, and atrial natriuretic peptide (ANP), whereas the HO inhibitor, CrMP annulled the renoprotection and exacerbated renal dysfunction., Conclusions: HA improves renal function by attenuating histopathological lesions, suppressing inflammatory/oxidative mediators, abating profibrotic/extracellular matrix proteins, and reducing albuminuria/proteinuria, while concomitantly potentiating the HO-adiponectin-ANP axis, enhancing nephrin, podocin, podocalyxin, CD2AP and increasing creatinine clearance. Our study underscores the benefit of potentiating the HO-adiponectin-ANP against nephropathy., (© American Journal of Hypertension, Ltd 2014. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

Background: The mechanisms by which heme oxygenase (HO) improves glucose metabolism in essential hypertension are not completely understood. Because dysfunctional insulin signaling is associated with elevated inflammation and high cholesterol and triglycerides, we investigated the effects of HO on the proinflammatory macrophage M1 phenotype and the anti-inflammatory macrophage M2 phenotype in spontaneously hypertensive rats (SHRs). SHRs are a model of human essential hypertension with features of metabolic syndrome, including impaired glucose metabolism., Methods: Spectrophotometric analysis, enzyme immunoassay, enzyme-linked immunosorbent assay, and Western immunoblotting were used. HO was enhanced with hemin or inhibited with chromium-mesoporphyrin (CrMP)., Results: Hemin suppressed inflammation by abating proinflammatory macro phage M1 phenotype (ED1) and chemokines such as macrophage chemoattractant protein 1 (MCP-1) and macrophage inflammatory protein 1 alpha (MIP-1α) while enhancing anti-inflammatory macrophage M2 phenotype by potentiating ED2, CD206, and CD14. Similarly, hemin improved insulin signaling by enhancing insulin receptor substrate 1 (IRS-1), IRS-2, phosphatidylinositol 3 kinase (PI3K), and glucose transporter 4 (GLUT4) but reduced total cholesterol and triglycerides. These effects were accompanied by increased HO-1, HO activity, and cyclic guanosine monophosphate (cGMP), whereas the HO inhibitor CrMP nullified the hemin effects. Importantly, the effects of the HO system on ED1, ED2, CD206, and CD14 in SHRs are novel., Conclusions: Hemin abated inflammation in SHRs by selectively enhancing the anti-inflammatory macrophage M2 phenotype that dampens inflammation while suppressing the pronflammatory macrophage M1 phenotype and related chemokines such as MCP-1 and MIP-1α. Importantly, the reduction of inflammation, total cholesterol, and triglycerides was accompanied by the enhancement of important proteins implicated in insulin signaling, including IRS-1, IRS-2, PI3K, and GLUT4. Thus, the concomitant reduction of inflammation, total cholesterol and triglycerides and the corresponding potentiation of insulin signaling are among the multifaceted mechanisms by which the HO system improves glucose metabolism in essential hypertension.

Exogenous δ-aminolevulinic acid (ALA)-induced photodynamic therapy (PDT) has been used in the treatment of cancer. To obtain a high efficacy of ALA-PDT, we have screened various chemicals affecting ALA-induced accumulation of protoporphyrin in cancerous cells. When HeLa cells were treated with quinolone chemicals including enoxacin, ciprofloxacin or norfloxacin, the ALA-induced photodamage accompanied by the accumulation of protoporphyrin was stronger than that with ALA alone. Thus, quinolone compounds such as enoxacin, ciprofloxacin and norfloxacin enhanced ALA-induced photodamage. The increased ALA-induced photodamage in enoxacin-treated HeLa cells was decreased by haemin or ferric-nitrilotriacetate (Fe-NTA), suggesting that an increase in iron supply cancels the accumulation of protoporphyrin. On the other hand, the treatment of the cells with ALA plus an inhibitor of haem oxygenase, Sn-protoporphyrin, led to an increase in the photodamage and the accumulation of protoporphyrin compared with those upon treatment with ALA alone, indicating that the cessation of recycling of iron from haem augments the accumulation. The use of quinolones plus Sn-protoporphyrin strongly enhances ALA-induced photodamage. To examine the mechanisms involved in the increased accumulation of protoporphyrin, we incubated ferric chloride with an equivalent amount of quinolones. Iron-quinolone complexes with visible colours with a maximum at 450 nm were formed. The levels of iron-metabolizing proteins in enoxacin- or ciprofloxacin-treated cells changed, indicating that quinolones decrease iron utilization for haem biosynthesis. Hence, we now propose that the use of quinolones in combination with ALA may be an extremely effective approach for the treatment modalities for PDT of various tumour tissues in clinical practice.

Heme oxygenase (HMOX) regulates vascular tone and blood pressure through the production of carbon monoxide (CO), a vasodilator derived from the heme degradation pathway. During pregnancy, the maternal circulation undergoes significant adaptations to accommodate the hemodynamic demands of the developing fetus. Our objective was to investigate the role of HMOX on maternal and fetal hemodynamics during pregnancy in a mouse model. We measured and compared maternal tissue and placental HMOX activity and endogenous CO production, represented by excreted CO and carboxyhemoglobin levels, during pregnancy (Embryonic Days 12.5-15.5) to nonpregnant controls. Micro-ultrasound was used to monitor maternal abdominal aorta diameters as well as blood flow velocities and diameters of fetal umbilical arteries. Tin mesoporphyrin, a potent HMOX inhibitor, was used to inhibit HMOX activity. Changes in maternal vascular tone were monitored by tail cuff blood pressure measurements. Effects of HMOX inhibition on placental structures were assessed by histology. We showed that maternal tissue and placental HMOX activity and CO production were significantly elevated during pregnancy. When HMOX in the placenta was inhibited, maternal and fetal hemodynamics underwent significant changes, with maternal blood pressures increasing. We concluded that increases in maternal tissue and placental HMOX activity contribute to the regulation of peripheral vascular resistance and therefore are important for the maintenance of normal maternal vascular tone and fetal hemodynamic functions during pregnancy.

With potentially neuroprotective properties, heme oxygenase-1 (HO-1) has been suggested to be the main mediator of cholesterol-independent anti-inflammatory and antioxidant actions of statins. However, we had demonstrated that simvastatin-induced HO-1 increased apoptosis of Neuro 2A cells in glucose deprivation, and iron production from HO-1 activity may be responsible for the toxicity. This study was designed to explore the effect of simvastatin-induced HO-1 on cultured Neuro 2A and C6 cells exposed to lipopolysaccharide (LPS). We found that the HO-1 upregulation was significantly associated with increased nuclear factor kappa B (NF-kappaB) activation, manifested as IkappaBalpha phosphorylation and p65 nuclear translocation, as well as increased production of superoxides. Inhibition of the induced HO-1 by zinc protoporphyrin reduced the increased NF-kappaB activation and superoxides production. RNA interference with HO-1 siRNA reduced the expression of HO-1 transcripts and protein as well as oxygen radical production. Addition of the iron chelator desferrioxamine to reduce the accumulation of ferric iron from heme by HO-1 resulted in blockade of the aggravated oxygen radical production. There was no significant effect on production of oxygen radicals under these conditions in the presence of a CO donor (RuCO) or a CO scavenger (hemoglobin). In addition, the viable cells were significantly decreased in 48 h in those cells receiving simvastatin pretreatment plus LPS compared to those in control or exposed to simvastatin or LPS alone. This study revealed that simvastatin-induced HO-1 led to increased NF-kappaB activation and superoxides production in the neuronal cells when exposed to LPS, and iron production may play a role in such a response.

Background: Heme oxygenase-1 (HO-1) seems to have an important protective role in acute and chronic inflammation. The products of heme catalysis, biliverdin/bilirubin, carbon monoxide (CO), and iron (that induces apoferritin) mediate the beneficial effects of HO-1. Blockade of HO-1 activity results in exacerbation of experimental colitis. We tested whether HO-1 has protective effects in the development of colitis and determined that specific enzymatic products of HO-1 are responsible for these effects., Methods: Colitis was induced by oral administration of dextran sodium sulfate (5%) to C57BL/6 mice for 7 days. HO-1 was up-regulated by cobalt-protoporphyrin (5 mg/kg, intraperitoneally). Biliverdin, exogenous CO, or the iron chelator desferrioxamine was administered to other groups., Results: Cobalt-protoporphyrin treatment resulted in significant up-regulation of HO-1 protein in mucosal and submucosal cells. Induction of HO-1 was associated with significantly less loss of body weight in mice with induced colitis (-12% versus -22% in the control animals, P < 0.001). Development of diarrhea and gastrointestinal hemorrhage was substantially delayed in animals in which HO-1 was induced, and mucosal injury was significantly attenuated. Administration of CO or desferrioxamine alone had no significant effects, whereas enhanced protection with lesser evidence of bowel inflammation was observed with systemic biliverdin administration (50 micromol/kg, 3 times per day, intraperitoneally)., Conclusions: We conclude that heightened HO-1 expression or administration of biliverdin ameliorates dextran sodium sulfate-induced experimental colitis. Novel therapeutic strategies based on HO-1 and/or biliverdin administration may have use in inflammatory bowel disease.

The prognosis of intracerebral hemorrhage (ICH) is unfavorable. Beyond immediate mass effect and tissue destruction, ICHs cause additional neuronal loss in a "perifocal reactive zone." Heme in ICH induces heme oxygenase-1 (HO-1), and the action of this enzyme on heme yields ferrous iron, biliverdin, and carbon monoxide. Iron is ultimately converted to ferritin and hemosiderin. Free iron is tissue-toxic, and inhibition of HO-1 should provide protection against additional damage. Experimental ICHs were made in adult rabbits by the stereotaxic injection of autologous blood, and the induction of HO-1 and increase in ferritin were followed by confocal immunofluorescence microscopy. Heme diffused rapidly through perivascular spaces, and HO-1 reaction product first occurred in perivascular cells and microglia. At this stage, HO-1 and ferritin showed extensive colocalization. As ICH resolution progressed, HO-1 immunoreactivity faded while ferritin and hemosiderin continued to accumulate. This process was accompanied by a gradient of destruction of neuronal cell bodies and dendrites in the perifocal reactive zone. In an effort to inhibit HO-1, repeated intravenous injections of tin-mesoporphyrin IX (SnMP) were given to ICH-bearing rabbits. The ICH disrupted the blood-brain barrier sufficiently to allow SnMP to enter the brain in pharmacological amounts, and the metalloporphyrin provided significant protection against neuronal loss.

The human lung cell line A549 is frequently used as an in vitro model for studying lung toxicity and genotoxicity of environmental mutagens and carcinogens. Hyperbaric oxygen (HBO) treatment has been shown to be an excellent model for investigating the genetic consequences of oxidative stress in vitro and in vivo. We have now studied the genotoxic effects of and adaptive protection by HBO in A549 cells. Using the alkaline Comet assay, our results show that HBO exposure directly induces DNA damage but reduces the DNA-damaging effect of a second HBO treatment of the cells 24 h later. HBO pretreatment also reduces potassium chromate-induced genotoxicity in A549 cells. Heme oxygenase-1 (HO-1) protein level is increased 24 h after HBO exposure and inhibition of HO-1 activity by tin-mesoporphyrin increased HBO genotoxicity. These results are in accordance with previous findings suggesting an involvement of HO-1 in the adaptive protection. Our study indicates that A549 cells are an appropriate cell culture system for the evaluation of genetic effects induced by HBO in lung cells.

The article highlighted in this issue is "2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD)-Induced Accumulation of Biliverdin and Hepatic Peliosis in Rats," by Marjo Nittynen, Jouni T. Tuomisto, Seppo Auriola, Raimo Pohjanvirta, Paula Syrjälä, Ulla Simanainen, Matti Viluksela, and Jouko Tuomisto (pp. 112-123).

Background: Vascular endothelium and smooth muscle express heme oxygenase (HO) that metabolizes heme to biliverdin, iron and carbon monoxide (CO). Carbon monoxide promotes endothelium-independent vasodilation, but also inhibits nitric oxide formation. This study examines the hypothesis that an inhibitor of HO promotes endothelium-independent vasoconstriction, which is attenuated in the presence of unabated nitric oxide formation., Methods: In vivo studies were conducted in anesthetized male Sprague-Dawley (SD) rats instrumented with flow probes and arterial catheters. In vitro experiments were performed on pressurized first-order gracilis muscle arterioles isolated from male SD rats superfused with oxygenated modified Krebs buffer., Results: Vascular smooth muscle and endothelium showed positive HO-1 and HO-2 immunostaining. In anesthetized rats the HO inhibitor chromium mesoporphyrin (CrMP; 45 micromol/kg intraperitoneally) had minimal effect on hindlimb resistance. However, in animals pretreated with N(omega)-nitro-L-arginine methyl ester (L-NAME; 300 mg/kg intraperitoneally), CrMP substantially increased hindlimb resistance. In contrast, in rats infused with phenylephrine to increase blood pressure and vascular tone, CrMP had no effect on hindlimb resistance. In isolated arterioles denuded of endothelium, CrMP (15 micromol/L) caused a powerful vasoconstriction, which was abolished in the presence of a functional endothelium. In arterioles with intact endothelium pretreated with L-NAME (1 mmol/L), or with L-NAME and sodium nitroprusside (10 to 30 nmol/L), CrMP promoted a similarly powerful vasoconstriction as in vessels denuded of endothelium., Conclusions: These results suggest that smooth muscle-derived CO may contribute to endothelium-independent regulation of vascular tone by providing a vasodilatory influence. Furthermore, the dilatory effects of endogenous CO are offset by a unique interaction between the CO and nitric oxide systems.

Background: Stress conditioning is thought to improve microvascular free flap survival but the mechanisms of protection are not clear. The aim of this study was to determine whether local induction of heat-shock protein (HSP) 32 improves microvascular perfusion in transferred osteomyocutaneous flaps., Methods: The hindlimb harvest region of osteomyocutaneous flaps in Wistar rats was subjected to stress conditioning by local heating (30 min, 42.5 degrees C) 24 h before microvascular flap transfer. In a second group of animals, after heat-shock priming, the action of HSP-32 was inhibited by tin protoporphyrin IX. Animals with unconditioned flaps served as controls. After transfer, the microcirculation of the muscle, cutaneous, subcutaneous and periosteal tissue of the flap was analysed quantitatively for 6 h using intravital fluorescence microscopy., Results: Immunohistochemistry revealed that HSP-32 was detectable only after priming and not in unconditioned flaps. Priming did not alter functional capillary density or capillary red blood cell velocity compared with that in unconditioned flaps. However, heat-shock priming induced significant capillary dilatation (P < 0.05) and thus a substantial increase in capillary blood flow volume (P < 0.05) in all tissues of the transferred flaps. Inhibition of HSP-32 by tin protoporphyrin IX completely abolished the priming-induced improvement in capillary perfusion, as indicated by the lack of increased capillary diameters and volumetric blood flow., Conclusion: The present study demonstrated that stress conditioning by local heat-shock priming improves nutritive perfusion in osteomyocutaneous flaps by capillary dilatation, probably mediated through the vasoactive action of HSP-32.

We tested the hypothesis that nitric oxide and carbon monoxide, which are produced in the brain by nitric oxide synthase (NOS) and heme oxygenase (HO), modulate the hypothalamic-pituitary-adrenal response to physico-emotional stressors by acting at the hypothalamus. Accordingly, we determined 1) whether the intracerebroventricular (icv) injection of NOS or HO inhibitors at doses that were confined to the brain attenuated electroshock-induced ACTH release; and 2) whether the decreases in this ACTH response were concurrent with decreases in NOS or HO activity levels at the hypothalamus. Icv injection of the NOS inhibitor Nomega-nitro-L-arginine-methylester (L-NAME; 50 microg) or the HO inhibitor tin protoporphyrin (SnPP; 20-25 microg) significantly blunted the plasma ACTH response to a 45-min session of intermittent electroshocks. Importantly, in these same animals there were concurrent decreases in hypothalamic NOS or HO activities, respectively. There were little or no effects of these inhibitors on anterior pituitary NOS or HO activities, indicating that there was only minimal leakage of the drug from the brain after icv administration. The specificity of action of these inhibitors was confirmed by the fact that SnPP did not affect NOS activity, and L-NAME did not affect HO activity. Finally, L-NAME produced no effect, whereas SnPP produced only transient increases in blood pressure, suggesting that these inhibitors do not affect activity indirectly through alterations in blood pressure. These data support the hypothesis that in the whole animal, both NO and CO exert a stimulatory influence on the acute ACTH response to physico-emotional stressors, and that the hypothalamus is the critical site of their actions.

Zinc protoporphyrin (ZnPP) is a normal metabolite that is formed in trace amounts during heme biosynthesis. The final reaction in the biosynthetic pathway of heme is the chelation of iron with protoporphyrin. During periods of iron insufficiency or impaired iron utilization, zinc becomes an alternative metal substrate for ferrochelatase, leading to increased ZnPP formation. Evidence suggests that this metal substitution is one of the first biochemical responses to iron depletion, causing increased ZnPP to appear in circulating erythrocytes. Because this zinc-for-iron substitution occurs predominantly within the bone marrow, the ZnPP/heme ratio in erythrocytes reflects iron status in the bone marrow. In addition, ZnPP may regulate heme catabolism through competitive inhibition of heme oxygenase, the rate-limiting enzyme in the heme degradation pathway that produces bilirubin and carbon monoxide. Physiological roles, especially relating to carbon monoxide and possibly nitric oxide production, have been suggested for ZnPP. Clinically, ZnPP quantification is valuable as a sensitive and specific tool for evaluating iron nutrition and metabolism. Diagnostic determinations are applicable in a variety of clinical settings, including pediatrics, obstetrics, and blood banking. ZnPP analytical methodologies for clinical studies are discussed. In addition to diagnostic tests and metabolic studies, ZnPP has a potential therapeutic application in controlling bilirubin formation in neonates as a preventive measure for hyperbilirubinemia. Biochemical research techniques, both in vivo and in vitro, are described for further studies into the role of ZnPP in metabolism and physiology.

In this report, we found that arsenite-resistant human lung adenocarcinoma cells, CL3R15, were more susceptible to CuCl2 than the parental CL3 cells. With the aid of atomic absorption spectrophotometry, we observed that CL3R15 cells accumulated more copper than CL3 cells. We further demonstrated that sodium arsenite treatment resulted in a dose-dependent increase of copper accumulation in the parental CL3 cells. In contrast, copper did not alter the levels of intracellular arsenite in CL3 cells treated in combination with sodium arsenite and CuCl2. Pretreatment of CL3 cells with sodium arsenite resulted in a significant increase of copper accumulation and cytotoxicity. These results indicate that intracellular copper accumulation is enhanced by arsenite. However, arsenite-enhanced copper accumulation was not observed in two fibroblastic cells, GM00220 and GM03700, derived from Menkes patients. The Menkes gene encodes a membrane pump responsible for copper exportation. Our results suggest that Menkes protein is a potential target of arsenite.

Background: Ferritin and the percentage of transferrin saturation (TS) are established parameters with which to evaluate endogenous iron availability during treatment of renal anaemia with recombinant human erythropoietin (rHuEpo). Zinc protoporphyrin (ZPP) has been proposed as another valid marker in this setting., Methods: We determined the following parameters in 127 patients, including 117 haemodialysis patients: haemoglobin, erythrocytes, haematocrit, mean corpuscular volume (MCV), iron, ferritin, transferrin saturation and ZPP. Of the patients treated in a cross-sectional study, 38.5% were treated with rHuEpo; 30.7% with intravenous iron; and 13.6% with intravenous iron and rHuEpo simultaneously. Median ferritin was 304 ng/ml and median transferrin saturation was 21.2%., Results: Including cases with manifest storage iron deficiency, a concordant elevated ZPP ( > 40 mumol/mol haem) and a decreased transferrin saturation ( < 20%) were found in 23 of our dialysis patients (19.6%) while 55 cases (47%) were classified as concordantly negative. However, as many as 39 cases (33.3%) showed discrepant results: in 16 cases (13.6%) ZPP was elevated but transferrin saturation was in the normal range, while in 23 cases (19.6%) the opposite results were observed., Conclusions: We conclude that beyond the border of manifest storage iron deficiency, defined as a ferritin < 30 ng/ml in male and < 15 ng/ml in female patients, ZPP and TS cover different ranges of functional iron deficiency which is reflected in the lack of a correlation of ZPP to any other of the above-mentioned parameters. Our data suggest that a TS < 20% as a diagnostic, and thus intervention, criterion in the evaluation of functional iron deficiency and iron substitution beyond manifest storage iron deficiency might result in overestimation of iron requirements. It remains to be shown in a longitudinal study, also reflecting the course of haemoglobin and the mean rHuEpo dose, whether ZPP or TS is the more appropriate parameter in the evaluation of functional iron availability.

Background: Zinc protoporphyrin (ZPP), a metabolic intermediate generated in the red blood cell by incorporation of zinc instead of iron, has been suggested to be a sensitive and specific parameter of absolute iron deficiency in haemodialysis (HD) patients., Methods: We studied 62 HD patients, 29-86 years old, with ZPP levels > 50 mumol/mol haeme (normal value of ZPP < 40 mumol/mol haeme) assessing the value of ZPP as a marker of functional iron deficiency at different cut-off points of ZPP. None of the patients had apparent inflammatory disease, infectious disease, or malignancy. ZPP, haemoglobin, iron and ferritin levels were determined before, and after a 24-week period of once-weekly i.v. administration of 40 mg iron, to determine whether ZPP levels return to normal during adequate iron supplementation (960 mg iron/ patient)., Results: There was no significant change in ZPP levels after iron supplementation in patients with a ZPP > 50 mumol/mol haeme (96.7 +/- 49.8 versus 88.4 +/- 43.5 mumol/mol haeme before and after iron administration respectively, P = n.s.). However, in patients with a ZPP > 90 mumol/mol haeme, there was a significant reduction in ZPP levels (141.2 +/- 54.5 versus 108.0 +/- 48.8 mumol/mol haeme, P < 0.001). Serum ferritin increased significantly in both groups. There was no correlation between ZPP and serum ferritin at any time during the study. There was also no correlation between serum aluminium levels and ZPP and no significant difference in changes in ZPP in patients receiving desferrioxamine therapy compared to those not receiving desferrioxamine therapy. We did find a significant correlation between moderately elevated total blood lead concentrations and ZPP levels at the end of the study. The ZPP levels were not significantly different in the range from 50-110 mumol/mol haeme before and after i.v. iron supplementation in the responders (10% increase of haemoglobin or 20% decrease of the recombinant human erythropoietin dose) compared with the non-responders., Conclusions: Our data indicate that ZPP cannot be used to predict the erythropoietic response to iron supplementation. However, ZPP levels may be an indicator of functional iron deficiency due to blockade of the reticuloendothelial iron release in haemodialysis patients.

Although recent evidence suggests that the gas nitric oxide (NO) can modulate the secretion of corticotropin-releasing hormone (CRH) from acute rat hypothalamic explants, another gas, carbon monoxide (CO), has been suggested to play a role in neural signaling in the brain; CO may complement the activity of NO in long term potentiation. In this study, we have investigated whether CO shares with NO the ability to modify the release of CRH from the rat hypothalamus. Hemin, a specific CO precursor through the enzyme heme oxygenase (the enzymatic pathway synthesizing endogenous CO), was found to inhibit in a dose-dependent manner KCl-stimulated CRH release, with a maximal effect at 1 microM, while showing no effect on basal CRH secretion. The stimulation of CRH by interleukin-1 beta (100 ng/ml) was also significantly antagonized by hemin (1 microM). An inhibitor of heme oxygenase, zinc-protoporphyrin-9, had no effect on basal or stimulated CRH release up to a maximal dose of 10 microM. When hemin and zinc-protoporphyrin-9 were given together, the hemin-induced inhibition of CRH release was completely antagonized by the enzyme inhibitor. These findings provide evidence that endogenous CO may play a role in the control of CRH release; by analogy with NO, CO may represent a major new neuroendocrine modulator.

Photo-reversal of the carbon monoxide inhibition of heme oxygenase reaction by monochromatic light was investigated. Heme degradation in either the microsomal or the reconstituted heme oxygenase system was inhibited by CO. In both systems the extents of Co inhibition were dependent on the CO/O2 ratio and were nearly equal at a given CO/O2 ratio. In the reconstituted heme oxygenase reaction using a highly purified heme oxygenase preparation the relationship between the intensity of light and the degree of reversal of the CO inhibition of heme degradation as expressed in terms of delta K/Kd was not linear, but the tentatively obtained photochemical action spectrum exhibited the peaks of reversal at about 420, 540, 570, and 640 nm and suggested the occurrence of at least two steps of CO inhibition in the overall sequence of heme degradation. One could be ascribed to protoheme and the other was supposed to be the 688 nm compound which is an intermediate locating between hydroxyheme and the biliverdin-iron complex in the sequence of heme degradation.