Novel Aminomethyl Derivatives of 4-Methyl-2-prenylphenol: Synthesis and Antioxidant Properties.

4-Methyl-2-prenylphenol (1) was synthesized from para-cresol and prenol, natural alcohol under the conditions of heterogeneous catalysis. A series of nine new aminomethyl derivatives with secondary and tertiary amino groups were obtained on the basis of compound 1. A comparative evaluation of their antioxidant properties was carried out using in vitro models. It was established that Mannich base with octylaminomethyl group has radical-scavenging activity, high Fe2+ -chelation ability as well as the ability to inhibit oxidative hemolysis of red blood cells.

Effects of hypnotics on prefrontal cortex activity during a verbal fluency task in healthy male subjects: A near-infrared spectroscopy study.

OBJECTIVE: To assess the effects of hypnotics on prefrontal cortex activity in healthy subjects using near-infrared spectroscopy (NIRS) in a double-blind, placebo-controlled crossover trial. METHODS: Eighteen healthy males received acute doses of ramelteon (8 mg), triazolam (0.125 mg), or placebo in a predetermined randomization schedule, with a washout period of more than 1 week. All subjects performed a verbal fluency task during NIRS assessments at baseline and at 1 and 4 hr post-dose. The number of words correctly generated during the task (behavioral performance) and scores on the Stanford Sleepiness Scale (SSS) were also recorded at each test time. RESULTS: Compared with the placebo, triazolam (0.125 mg) significantly decreased oxyhemoglobin (oxy-Hb) concentration change in NIRS during the posttask period and significantly increased behavioral performance, whereas triazolam (0.125 mg) and ramelteon (8 mg) significantly increased SSS scores. CONCLUSIONS: The differential effects of two types of hypnotics on oxy-Hb change measured by NIRS were observed in acute dosing, suggesting that when assessing brain activity of patients with psychiatric disorders, researchers should consider how certain types of hypnotics can influence brain function. This would also provide useful information to clinicians when prescribing hypnotics suitable for their patients' conditions.

Frontal activity measured by near-infrared spectroscopy in patients taking different atypical antipsychotic drugs: A cross-sectional study.

Using near-infrared spectroscopy, we examined changes in the concentration of oxygenated hemoglobin ([oxy-Hb]) in the frontal lobe during a verbal fluency task in 20 patients with schizophrenia (10 patients each receiving olanzapine [OLZ] and risperidone [RIS]) and 10 healthy controls. We found that [oxy-Hb] levels in the prefrontal region were higher in the patients receiving OLZ than in those receiving RIS. These results suggest that antipsychotic drugs have different effects on cerebral hemodynamic patterns, which may reflect frontal lobe function. Further studies with a larger sample size are needed to verify our preliminary findings.

Hemodynamic Changes Associated with Interictal Spikes Induced by Acute Models of Focal Epilepsy in Rats: A Simultaneous Electrocorticography and Near-Infrared Spectroscopy Study.

Interictal spikes can be generated by blocking GABAA receptor-mediated inhibition. The nature of the hemodynamic activities associated with interictal spikes in acute models of focal epilepsy based on GABA deactivation has not been determined. We analyzed systemic changes in hemodynamic signals associated with interictal spikes generated by acute models of focal epilepsy. Simultaneous ElectroCorticoGraphy (ECoG) and Near-InfraRed Spectroscopy (NIRS) recordings were obtained in vivo from adult Sprague-Dawley rat brain during semi-periodic focal interictal spikes induced by local cortical application of low doses of Penicillin G (PG) and Bicuculline Methiodide (BM) as GABA deactivation agents. The Finite Impulse Response deconvolution technique was used to estimate the profile of hemodynamic changes in oxyhemoglobin (HbO) and deoxyhemoglobin (HbR) concentrations associated with interictal ECoG spikes in each rat. Our results show that, in both acute models of focal epilepsy, the hemodynamic changes associated with interictal spikes were characterized by pre-spike and post-spike primary NIRS responses, and recovery periods with slight differences in amplitude and latency. The pre-spike period starting at least 2 s prior to the onset of ECoG spikes was characterized by a significant decrease in HbO concomitant with an increase in HbR with respect to baseline. The post-spike primary NIRS response exhibited the expected changes described according to the classical view of neurovascular coupling, i.e., a significant increase in HbO and a significant decrease in HbR in response to interictal spikes. The recovery period was characterized by a decreased HbO signal and an increased HbR signal, followed by a return to baseline. Compared to the BM epilepsy model, the PG model was more stable and showed lower variability in the shape, amplitude and latency of the components of spike-related hemodynamic changes. Our findings support a prominent role for pre-spike hemodynamic changes in the initiation of interictal spikes. The mechanism of interactions between neuronal and vascular networks during the pre-spike period constitutes a complex process, resulting in increased sensitivity of the epileptogenic focus to induce neuronal spiking.

Increased hemoglobin-oxygen affinity ameliorates bleomycin-induced hypoxemia and pulmonary fibrosis.

Although exertional dyspnea and worsening hypoxia are hallmark clinical features of idiopathic pulmonary fibrosis (IPF), no drug currently available could treat them. GBT1118 is a novel orally bioavailable small molecule that binds to hemoglobin and produces a concentration-dependent left shift of the oxygen-hemoglobin dissociation curve with subsequent increase in hemoglobin-oxygen affinity and arterial oxygen loading. To assess whether pharmacological modification of hemoglobin-oxygen affinity could ameliorate hypoxemia associated with lung fibrosis, we evaluated GBT1118 in a bleomycin-induced mouse model of hypoxemia and fibrosis. After pulmonary fibrosis and hypoxemia were induced, GBT1118 was administered for eight consecutive days. Hypoxemia was determined by monitoring arterial oxygen saturation, while the severity of pulmonary fibrosis was assessed by histopathological evaluation and determination of collagen and leukocyte levels in bronchoalveolar lavage fluid. We found that hemoglobin modification by GBT1118 had strong antihypoxemic therapeutic effects with improved arterial oxygen saturation to near normal level. Moreover, GBT1118 treatment significantly attenuated bleomycin-induced lung fibrosis, collagen accumulation, body weight loss, and leukocyte infiltration. This study is the first to suggest the beneficial effects of hemoglobin modification in fibrotic lungs and offers a promising and novel therapeutic strategy for the treatment of hypoxemia associated with chronic fibrotic lung disorders in human, including IPF.

Sildenafil Increases the p50 and Shifts the Oxygen-Hemoglobin Dissociation Curve to the Right.

INTRODUCTION: Sildenafil (Viagra®) is a selective phosphodiesterase type 5 (PDE5) inhibitor that block the breakdown of cyclic guanyl monophosphate (cGMP) leading to relaxation of the smooth muscles of the corpus cavernous and an increase in blood flow resulting in penile erection. It is hypothesized that sildenafil will increase the release of oxygen from erythrocytes and shift the oxygen-hemoglobin curve to the right. AIM: The aim of this study was to investigate the effect of varying doses of sildenafil on the p50 of the oxygen-hemoglobin dissociation curve in blood samples from eight (8) healthy adult male volunteers with normal hemoglobin HbAA. METHOD: The hemox-analyzer was used to generate the p50 and the oxygen-hemoglobin dissociation curves. MAIN OUTCOME MEASURES: The effect of different doses of sildenafil on the p50 values and shift of the oxygen-hemoglobin curve were the main outcome measures. RESULT: Sildenafil caused a statistically significant increase in the p50 values and rightward shift of the oxygen-hemoglobin dissociation curve. CONCLUSION: Sildenafil caused a dose-dependent increase in the release of oxygen from the erythrocytes as shown by the increased p50 values and rightward shift of the oxygen-hemoglobin dissociation curve. Ellis SS and Pepple DJ. Sildenafil increases the p50 and shifts the oxygen-hemoglobin dissociation curve to the right.

Effects of bicarbonate on oxyhaemoglobin desaturation and exercise performance in athletes.

AIM: Decrease in peripheral oxygen delivery may impact exercise performance in athletes with exercise-related arterial oxygen desaturation (ERD). We evaluated whether sodium bicarbonate ingestion would be effective to reduce ERD and what is the consequences upon exercise performance. METHODS: Seventy highly trained athletes performed an incremental treadmill cardiopulmonary exercise test (incCPX) and a high intensity constant speed test (ctCPX) on separate days. Subjects who developed ERD by pulse oximetry were randomly allocated to oral sodium bicarbonate or placebo during 5 days. At the end of treatment subjects repeated both tests. RESULTS: ERD prevalence was 33% during the incCPX (17 % severe, 48% moderate and 35% mild) and 34% (5 % severe, 37% moderate and 58% mild) in the ctCPX. Athletes who developed ERD have greater aerobic capacity (incCPX) and endurance time (ctCPX). Active treatment, but not placebo, reduced ERD during ctCPX (P<0.05). However, there were no significant positive effects on main parameters of aerobic function and endurance exercise capacity (P>0.05). CONCLUSION: Sodium bicarbonate was effective in lessening ERD during ctCPX in athletes. However, this intervention failed to improve maximal and submaximal exercise capacity in these subjects.

Nocturnal transcutaneous carbon dioxide tension in postmenopausal estrogen users and non-users.

OBJECTIVE: The effect of menopause on breathing is not fully understood. We have previously shown that postmenopausal women have a higher sleep-induced increase in transcutaneously measured carbon dioxide tension (TcCO(2)) than premenopausal women. Therefore, we hypothesized that estrogen therapy (ET) would normalize this sleep-induced TcCO(2) increase. METHODS: Nine postmenopausal ET users and nine non-users went through an overnight polygraphic sleep study including continuous monitoring of TcCO(2). RESULTS: TcCO(2) levels were higher during sleep than evening wakefulness (awake median 6.55 kPa versus sleep median 6.90 kPa, P = 0.001). ET users had a greater sleep-induced increase in TcCO(2) than non-users when comparing the difference between wakefulness and slow-wave sleep (0.85 kPa versus 0.28 kPa, P = 0.004). Lower sleep efficiency was associated with higher sleep-induced increase in TcCO(2). CONCLUSIONS: In contrast to our initial hypothesis, postmenopausal ET users have a higher sleep-induced increase in TcCO(2) than women without ET. Thus, TcCO(2) may be sensitive in measuring the peripheral estrogen effect. These findings warrant placebo-controlled intervention studies to confirm the effects of ET on TcCO(2) measurements.

Reactive oxidant species and oxidation of protein and haemoglobin as biomarkers of susceptibility to stress caused by chloramphenicol.

Chemiluminescence assay was suitable for rapid determination of alterations caused by chloramphenicol (Ch) in blood. The new aspects observed indicated oxidative stress with: (a) biphasic response of reactive oxidant species (ROS) with different concentration of Ch; (b) increase of carbonyl residues and advanced oxidation protein products in blood as a consequence of oxidative stress; (c) oxidation of haemoglobin with rise of oxyhaemoglobin; protein and haemoglobin oxidation was more pronounced in the samples that exhibited high stimuli of ROS; and (d) the ferrous reduction antioxidant potential (FRAP) was the lowest in the maximum ROS-producer sample, which exhibited the lowest consumption of FRAP in response to Ch treatment. The present investigation could contribute to a rapid detection of persons with elevated susceptibility to Ch previously to its application in chemotherapy.

Caffeine stimulates ventilation in athletes with exercise-induced hypoxemia.

INTRODUCTION/PURPOSE: Many athletes with exercise-induced hypoxemia (EIH) show an insufficient ventilatory response to exercise and low resting ventilatory responsiveness. The purpose of this project was to determine whether a moderate dosage of caffeine, a common ventilatory stimulant, could augment resting ventilatory responsiveness, exercise ventilation (V E), end-tidal O2 partial pressure (PetO2), and arterial oxyhemoglobin saturation (HbSaO2) in athletes with EIH. METHODS: Eight highly trained males (V[spacing dot above]O2max, 69.2 +/- 4.0 mL.[kg.min]) who demonstrated EIH at V[spacing dot above]O2max (HbSaO2, 88.0 +/- 1.7%), ingested in a randomized design a placebo or caffeine (CAF, 8 mg.kg body wt) 1 h before testing. Ventilatory responsiveness at rest was assessed via the isocapnic hypoxic and hyperoxic hypercapnic ventilatory responses (HVR and HCVR, respectively). Dependent measures of metabolic variables, ventilation, and saturation were determined during progressive treadmill exercise to exhaustion. RESULTS: V E was higher at 75%, 80%, and 100% of V[spacing dot above]O2max with CAF (P < 0.05). V E/V O2, PetO2, and HbSaO2 were increased at 75%, 80%, and 90% of [formula: see text] with CAF but were not different at V[spacing dot above]O2max despite an increase in V e. No change in V[spacing dot above]O2max was observed between treatments. HVR and HCVR were not different between the two conditions, indicating that the increased V E likely came from central stimulation or secondary effects of CAF. CONCLUSION: The failure of HbSaO2 to increase at [formula: see text] despite an increase in V E suggests that mechanisms influencing HbSaO2 other than an inadequate hyperventilatory response may operate to different degrees across individuals as V[spacing dot above]O2max is approached.

Mild induced hypertension improves blood flow and oxygen metabolism in transient focal cerebral ischemia.

BACKGROUND AND PURPOSE: In focal ischemic cortex, cerebral blood flow autoregulation is impaired, and perfusion passively follows blood pressure variations. Although it is generally agreed that profound hypotension is harmful in acute stroke, the hemodynamic and metabolic impact of increased blood pressure on the ischemic core and penumbra are less well understood. We, therefore, tested whether pharmacologically induced hypertension improves cerebral blood flow and metabolism and tissue outcome in acute stroke using optical imaging with high spatiotemporal resolution. METHODS: Cerebral blood flow, oxyhemoglobin, and cerebral metabolic rate of oxygen were measured noninvasively using simultaneous multispectral reflectance imaging and laser speckle flowmetry during distal middle cerebral artery occlusion in mice. Hypertension was induced by phenylephrine infusion starting 10 or 60 minutes after ischemia to raise blood pressure by 30% for the duration of ischemia; control groups received saline infusion. RESULTS: Mild induced hypertension rapidly increased cerebral blood flow, oxyhemoglobin, and cerebral metabolic rate of oxygen in both the core and penumbra and prevented the expansion of cerebral blood flow deficit during 1 hour distal middle cerebral artery occlusion. Induced hypertension also diminished the deleterious effects of periinfarct depolarizations on cerebral blood flow, oxyhemoglobin, and cerebral metabolic rate of oxygen without altering their frequency. Consistent with this, mild induced hypertension reduced infarct volume by 48% without exacerbating tissue swelling when measured 2 days after 1 hour transient distal middle cerebral artery occlusion. CONCLUSIONS: Our data suggest that mild induced hypertension increases collateral cerebral blood flow and oxygenation and improves cerebral metabolic rate of oxygen in the core and penumbra, supporting its use as bridging therapy in acute ischemic stroke until arterial recanalization is achieved.

Noninvasive monitoring of estrogen effects against ischemic stroke in rats by near-infrared spectroscopy.

The aim of this study was to assess hemodynamic changes by near-infrared spectroscopy (NIRS) during acute focal cerebral ischemia and reperfusion. The study also has evaluated the therapeutic effects of estrogen against vascular dysfunction. Focal cerebral ischemia was induced in nine bilaterally ovariectomized rats, using an endovascular occlusion technique of the middle cerebral artery (MCA). Four out of nine rats had estrogen pretreatment before MCA occlusion (MCAO). The other five rats had MCAO with no pretreatment. The occlusion time was 60 min, followed by 40-60 min of reperfusion. Real-time monitoring of changes in hemoglobin concentrations was performed by a steady-state, two-channel, NIRS system through the period of occlusion and reperfusion. Both changes in total and oxygenated hemoglobin concentrations (D[HbT] and D[HbO(2)]) display apparent periodic fluctuations during occlusion for the rats without estrogen pretreatment, while no rhythmic fluctuation was observed in the rats with the pretreatment. This rhythmic fluctuation is a microvascular dysfunction. Fourier power spectral analysis was performed on the D[HbO(2)] profiles in both rat groups. The results show that the cumulative frequency power of D[HbO(2)] in the range of 0.0025-0.01 Hz for the rats without pretreatment is significantly higher than that with pretreatment. The study implies that the dysfunctional fluctuations disappear in the rats with estrogen pretreatment, demonstrating a new application of NIRS, i.e., to detect focal cerebral ischemia and to monitor cerebral responses to therapy against vascular dysfunction in animal models.

Menopausal estrogen therapy predicts better nocturnal oxyhemoglobin saturation.

OBJECTIVES: The respiratory responses in the few previous studies evaluating the effects of short-term unopposed estrogen therapy on breathing in postmenopausal women have been inconsistent. We performed a study to investigate whether long-term estrogen therapy would prevent age-related decline in nocturnal arterial oxyhemoglobin saturation and whether higher serum estradiol concentration is associated with better arterial oxyhemoglobin saturation. METHODS: Sixty-four healthy postmenopausal women were followed-up for 5 years in a 5-year prospective open follow-up study. The women were users or non-users of estrogen therapy according to their personal preference. RESULTS: Mean overnight arterial oxyhemoglobin saturation was similar at baseline (94.3 +/- 1.1%) and after follow-up (94.5 +/- 1.6%). Present estrogen users had higher mean arterial oxyhemoglobin saturation (95.2 +/- 1.4%) than present non-users (94.0 +/- 1.5%), when adjusted for age and body mass index (p = 0.042). The change in mean arterial oxyhemoglobin saturation during follow-up was not associated with serum estradiol concentration at baseline but associated with estradiol at follow-up (p = 0.042), when adjusted for age and body mass index. At follow-up, women with higher serum estradiol concentration had also higher mean nocturnal arterial oxyhemoglobin saturation (Pearson r = 0.29, p = 0.019) and lower apnea-hypopnea index (Spearman r = -0.28, p = 0.031). The pooled current estrogen users spent proportionally less time with SaO(2) below 90% than non-users (ANCOVA adjusted for age and BMI, p = 0.017). CONCLUSIONS: Estrogen use and especially high serum estradiol concentration predict higher mean overnight arterial oxyhemoglobin saturation. The present data suggest that estrogen therapy has favorable respiratory effects.

Interaction of arsine with hemoglobin in arsine-induced hemolysis.

The mechanism of arsine (AsH3) toxicity is not completely understood, but hemoglobin (Hb) has long been recognized as a necessary component of the overall mechanism of AsH3-induced hemolysis. In this study, the role of Hb in AsH3-induced hemolysis was investigated. The purpose was to determine whether exposure to AsH3 altered the structure of the heme or globin constituents of Hb. Arsine was incubated with isolated, human oxyhemoglobin (oxyHb) and carboxyhemoglobin (carboxyHb), and the release of heme and formation of AsH3-induced hemoglobin modifications were examined. Arsine increased the amount of heme released from oxyHb by 18%. When carboxyHb was incubated with AsH3, there was no change in heme release, suggesting that the sixth ligand position on the heme iron may be critical in the interaction with AsH3. Arsine-Hb interactions were studied by mass spectral analysis of heme, alpha-chain globin, and beta-chain globin. Arsine had no significant effect on the alpha- or beta-chain LCMS spectra in oxyHb and carboxyHb, but in oxyHb, arsine consistently increased the frequency of methyl acetate ion fragment (.CH2OOH, m/z = 59) loss from heme in the matrix-assisted laser desorption/ionization-mass spectrometry (MALDI-MS) spectra. The formation of Hb-protein crosslinks was investigated by Western blotting using an anti-Hb antibody in isolated membranes from AsH3-treated erythrocytes, but no Hb-membrane adducts were found. These results suggest that the interaction between AsH3 and hemoglobin result in an increase in heme release which may contribute to the hemolytic mechanism of AsH3.

Acetazolamide improves central sleep apnea in heart failure: a double-blind, prospective study.

RATIONALE: Acetazolamide is a mild diuretic and a respiratory stimulant. It is used to treat periodic breathing at high altitude. OBJECTIVES: To determine the therapeutic efficacy of acetazolamide on central sleep apnea associated with heart failure. METHODS: Twelve male patients with stable systolic heart failure whose initial polysomnograms showed more than 15 episodes per hour of apnea and hypopnea participated in the study. The patients were randomized to a double-blind cross-over protocol with acetazolamide or placebo, taken 1 h before bedtime for six nights with 2 wk of washout. MEASUREMENTS: Polysomnography, pulmonary function tests, arterial blood gases, and left ventricular ejection fraction were obtained initially along with a sleep questionnaire, history, and physical examination. Baseline measurements were repeated at the end of each arm. MAIN RESULTS: There were no significant differences between parameters at baseline and placebo. In comparing placebo with acetazolamide, the hourly number of episodes of central apnea (49 +/- 28 vs. 23 +/- 21 [mean +/- SD]; p = 0.004) and the percentage of total sleep time spent below an arterial oxyhemoglobin saturation of 90% (19 +/- 32 vs. 6 +/- 13%; p = 0.01) decreased significantly. Acetazolamide improved subjective perception of overall sleep quality (p = 0.003), feeling rested on awakening (p = 0.007), daytime fatigue (p = 0.02), and falling asleep unintentionally during daytime (p = 0.002). CONCLUSIONS: In patients with heart failure, administration of a single dose of acetazolamide before sleep improves central sleep apnea and related daytime symptoms.

A new sensitive assay reveals that hemoglobin is oxidatively modified in vivo.

Free radical formation in heme proteins is recognised as a factor in mediating the toxicity of peroxides in oxidative stress. As well as initiating free radical damage, heme proteins damage themselves. Under extreme conditions, where oxidative stress and low pH coincide (e.g., myoglobin in the kidney following rhabdomyolysis and hemoglobin in the CSF subsequent to subarachnoid hemorrhage), peroxide can induce covalent heme to protein cross-linking. In this paper we show that, even at neutral pH, the heme in hemoglobin is covalently modified by oxidation. The product, which we term OxHm, is a "green heme" iron chlorin with a distinct optical spectrum. OxHm formation can be quantitatively prevented by reductants of ferryl iron, e.g., ascorbate. We have developed a simple, robust, and reproducible HPLC assay to study the extent of OxHm formation in the red cell in vivo. We show that hemoglobin is oxidatively damaged even in normal blood; approximately 1 in 2,000 heme groups exist as OxHm in the steady state. We used a simple model (physical exercise) to demonstrate that OxHm increases significantly during acute oxidative stress. The exercise-induced increase is short-lived, suggesting the existence of an active mechanism for repairing or removing the damaged heme proteins.

Effects of a pharmacologically-induced shift of hemoglobin-oxygen dissociation on myocardial energetics during ischemia in patients with coronary artery disease.

BACKGROUND: Conventional strategies to treat myocardial ischemia include interventions that reduce oxygen demand and/or increase myocardial blood flow. Animal experiments suggest that right-shifting the hemoglobin-oxygen dissociation curve may also attenuate the metabolic consequences of myocardial ischemia. We evaluated whether exercise-induced myocardial ischemia can be alleviated in subjects with coronary artery disease (CAD) by enhancing oxygen release with an allosteric modifier of hemoglobin's affinity for oxygen (RSR13). METHODS AND RESULTS: Seven subjects with CAD underwent a randomized, double-blind, cross-over study of the metabolic consequences of RSR13 administration on myocardial ischemia. Myocardial high-energy phosphates were quantified with 31P nuclear magnetic resonance (NMR) spectroscopy before, during, and after isometric handgrip-exercise. Subjects underwent NMR studies at baseline and on two separate occasions following the infusion of RSR13 (100 mg/kg) or placebo. RSR13 infusion significantly increased mean p50 by 8.1 +/- 2.7 mmHg at the end of the infusion, and it was still elevated by 4.9 +/- 3.3 mmHg after the completion of the treadmill tests while placebo had no effect. The myocardial creatine-phosphate (PCr) to adenosine-triphosphate (ATP) ratio decreased during handgrip-exercise in the baseline studies (from 1.39 +/- 0.23 before exercise to 0.95 +/- 0.21 during handgrip-exercise, p = 0.0001) and in the placebo studies (from 1.29 +/- 0.16 to 0.98 +/- 0.37, p = 0.06) but not during administration of RSR13 (from 1.28 +/- 0.18 to 1.02 +/- 0.24, p = 0.12). However, the mean values of cardiac PCr/ATP during handgrip-exercise did not differ significantly among the three measurements (baseline, placebo, RSR13). CONCLUSIONS: A single infusion of RSR13 to subjects with CAD increased mean p50 by 4.9-8.1 mmHg but did not significantly alter myocardial PCr/ATP during exercise. This is the largest right-shift in hemoglobin-oxygen binding affinity achieved in CAD subjects, and it did not provide clear evidence of protection from cardiac ischemia.

Formation of meso, N-diphenylprotoporphyrin IX by an aerobic reaction of phenylhydrazine with oxyhemoglobins.

Administration of phenylhydrazine to rabbits resulted in the denaturation of hemoglobins in erythrocytes, causing the formation of intracellular precipitates known as Heinz bodies, severe hemolytic anemia, and reticulocytosis. To elucidate the molecular mechanism of the destabilization, we allowed human oxyhemoglobins to react aerobically with phenylhydrazine. After treatment with acetic acid/HCl and H2SO4/methanol, the chloroform extract contained blue-green pigments of major products accompanied by different minor products. Each product was isolated by column chromatography. By fast-atom-bombardment mass spectrometry (FAB-MS) and proton nuclear magnetic resonance (1H-NMR) spectrometry, dimethyl esters of N-phenylprotoporphyrin IX and meso, N-diphenylprotoporphyrin IX were determined. Other major products also were determined to be dimethyl esters of triphenyl-and tetraphenyl-substituted protoporphyrins by FAB-MS. The formation of meso, N-diphenylprotoporphyrin indicated that the addition of a phenyl radical to the meso-carbon atom of the protoporphyrin ring occurred. Triphenyl and tetraphenyl adducts also indicated the formation of phenyl radicals in the aerobic reaction of phenylhydrazine with oxyhemoglobins. From these results, we suggest that the formation of phenyl radicals and the replacement of heme with phenyl-substituted protoporphyrins cause the destabilization of hemoglobins to induce Heinz bodies and hemolytic anemia with phenylhydrazine.