Quantitative analysis of heme and hemoglobin for the detection of intravascular hemolysis.

BACKGROUND: Intravascular hemolysis is associated with massive release of hemoglobin and consequently labile heme into the blood, resulting in prothrombotic and proinflammatory events in patients. Though heme is well-known to participate in these adverse effects, it is not monitored. Instead, haptoglobin and hemoglobin serve as clinical biomarkers. The quantification of labile heme together with hemoglobin, however, should be considered in clinical diagnosis as well, to obtain a complete picture of the hemolytic state in patients. So far, quantification techniques for labile heme were not yet systematically analyzed and compared for their clinical application potential, especially in the presence of hemoglobin. RESULTS: Two commercial assays (Heme Assay Kit®, Hemin Assay Kit®) and five common approaches (pyridine hemochromogen assay, apo-horseradish peroxidase-based assay, UV/Vis spectroscopy, HPLC, mass spectrometry) were analyzed concerning their linearity, accuracy, and precision, as well as their ability to distinguish between hemoglobin-bound heme and labile heme. Further, techniques for the quantification of hemoglobin (Harboe method, SLS method, Hemastix®) were included to study their selectivity for hemoglobin and potential interference by the presence of labile heme. Both, indirect and direct approaches were suitable for the determination of a wide concentration of heme (∼0.02-45 µM) and hemoglobin (∼0.002-17 µM). A clear distinction between hemoglobin-bound heme and labile heme with one method was not possible. Thus, a novel combined approach is presented and applied to human and porcine plasma samples for the determination of hemoglobin and labile heme. SIGNIFICANCE: Our results demonstrate the need to develop improved techniques to differentiate labile and protein-bound heme for early detection of intravascular hemolysis. Here, we present a novel strategy by combining two spectroscopic methods, which is most reliable as an easy-to-use tool for the determination of hemoglobin and heme levels in plasma samples for the diagnosis of intravascular hemolysis and in basic biomedical research.

Detecting labile heme and ferroptosis through 'turn-on' fluorescence and lipid droplet localization post Fe2+ sensing.

Iron, a crucial biologically active ion essential for metabolic processes in living organisms, plays a vital role in biological functions, and imbalances in iron levels can lead to various diseases. In this study, we have developed two simple "turn-on" fluorescent probes, NOPy and NOCN, for the quick and selective detection of Fe2+ at nanomolar levels (LOD of 35 nM), accompanied by significant absorption and emission shifts, along with colorimetric demarcation. Both fluorophores exhibit an excellent "turn-on" emission response upon encountering Fe2+ in the cells. Flow cytometry and confocal fluorescence imaging studies demonstrate enhanced fluorescence signals in response to labile iron, efficiently detecting heme during erastin-induced ferroptosis. Interestingly, we also observed that the product formed after Fe2+ sensing localizes within the lipid droplets. These water-soluble and highly sensitive reactive probes, NOPy and NOCN, enable investigations of iron-dependent physiological and pathological conditions. The development of these probes represents an advancement in the field, offering a rapid and selective means for detecting Fe2+ with minimal cytotoxicity.

Zinc-protoporphyrin formation in nitrite-free Parma Ham and its relationship with intrinsic parameters and red color profile of processed hams.

A total of 134 fresh hams, assayed for Ferrochelatase (FeCH) activity and ultimate pH (pH48), were processed in compliance with the procedures established for PDO Parma ham and finally, analyzed for salt, moisture, Zinc Protoporphyrin IX (ZnPP), heme, iron and zinc contents, and proteolysis index (PI). The variation in ZnPP content was related to the intrinsic parameters of fresh and matured hams by a Partial Least Square Regression model. The most favorable factors on the formation of ZnPP were total iron content (representative of the initial hemoprotein content), and FeCH activity, demonstrating the main role played by these raw matter-specific predictors in the long matured dry-cured hams. To a lesser extent, zinc content and pH48 were involved with a positive and negative role, respectively. Salt content and PI of matured hams showed an inhibitory and a favorable influence, respectively, toward the ZnPP formation. Principal Component Analysis showed the associations between the sensory red color profile and the physicochemical traits of matured hams. The red color intensity increased in agreement with the red-violet and red-pink hues scores. The formation of a high amount of ZnPP was associated with the increased perception of the red-violet shade, with a lower lightness (L*) and Hue angle (h°). Moisture increase contributed to the shift in color perception to red-pink, while marked progress in PI strengthened the perception of the red-brown shade. ZnPP and final heme favored the red color of matured hams, although a high concentration of these pigments increased in particular the red-violet perception.

Improved methods for the detection of heme and protoporphyrin IX adducts and quantification of heme B from cytochrome P450 containing systems.

Heme B is a critical prosthetic group for the function of numerous proteins including the cytochrome P450 (CYP) family of enzymes. CYP enzymes are involved in the metabolism of endogenous and xenobiotic molecules that are of central interest in drug development. Formation of reactive metabolites by CYPs can lead to heme modification and destruction of the enzyme. The structure of the adducted heme can provide key information on the mechanism of inactivation, which is of great interest during preclinical drug discovery. Historically, techniques to extract the modified heme or protoporphyrin IX species involved harsh extraction conditions and esterification of propionate groups to aid chromatography. We have developed a simplified extraction method and LC/MS chromatography system that does not require derivatization to quantify heme B and identify modified heme B species from multiple CYP-containing matrices. The method uses mass defect filter triggered data dependent MS2 scans to rapidly identify heme and protoporphyrin IX adducts. These methods may also be useful for the analysis of other heme variants and hemoproteins.

Meconium-stained amniotic fluid.

Green-stained amniotic fluid, often referred to as meconium-stained amniotic fluid, is present in 5% to 20% of patients in labor and is considered an obstetric hazard. The condition has been attributed to the passage of fetal colonic content (meconium), intraamniotic bleeding with the presence of heme catabolic products, or both. The frequency of green-stained amniotic fluid increases as a function of gestational age, reaching approximately 27% in post-term gestation. Green-stained amniotic fluid during labor has been associated with fetal acidemia (umbilical artery pH <7.00), neonatal respiratory distress, and seizures as well as cerebral palsy. Hypoxia is widely considered a mechanism responsible for fetal defecation and meconium-stained amniotic fluid; however, most fetuses with meconium-stained amniotic fluid do not have fetal acidemia. Intraamniotic infection/inflammation has emerged as an important factor in meconium-stained amniotic fluid in term and preterm gestations, as patients with these conditions have a higher rate of clinical chorioamnionitis and neonatal sepsis. The precise mechanisms linking intraamniotic inflammation to green-stained amniotic fluid have not been determined, but the effects of oxidative stress in heme catabolism have been implicated. Two randomized clinical trials suggest that antibiotic administration decreases the rate of clinical chorioamnionitis in patients with meconium-stained amniotic fluid. A serious complication of meconium-stained amniotic fluid is meconium aspiration syndrome. This condition develops in 5% of cases presenting with meconium-stained amniotic fluid and is a severe complication typical of term newborns. Meconium aspiration syndrome is attributed to the mechanical and chemical effects of aspirated meconium coupled with local and systemic fetal inflammation. Routine naso/oropharyngeal suctioning and tracheal intubation in cases of meconium-stained amniotic fluid have not been shown to be beneficial and are no longer recommended in obstetrical practice. A systematic review of randomized controlled trials suggested that amnioinfusion may decrease the rate of meconium aspiration syndrome. Histologic examination of the fetal membranes for meconium has been invoked in medical legal litigation to time the occurrence of fetal injury. However, inferences have been largely based on the results of in vitro experiments, and extrapolation of such findings to the clinical setting warrants caution. Fetal defecation throughout gestation appears to be a physiologic phenomenon based on ultrasound as well as in observations in animals.

Timing of iron deficiency and recognition memory in infancy.

Objective: To determine the relationship between iron deficiency (or iron-deficient, ID) and neural correlates of recognition memory depending on ID timing (gestation vs. infancy) and infant age at testing (9 vs. 18 months).Study design: Event-related potentials (ERP) were used in a visual recognition memory task (mother vs. stranger face) to compare healthy term infants according to iron status at birth and 9 months. Fetal-neonatal ID was defined as cord serum ferritin < 75 µg/l or zinc protoporphrin/heme ratio > 118 µmol/mol, postnatal ID as ≥ 2 abnormal iron measures at 9 months with normal cord-blood iron status, and iron-sufficient as not ID at birth or 9 months. Recognition of mother faces was measured by negative component (Nc) and late slow wave (LSW). These ERP components reflect attention and memory updating processes, respectively.Results: All groups showed differences in Nc amplitude elicited by mother and stranger faces at 9 months. At 18 months, only postnatal ID and iron-sufficient groups showed condition differences in Nc amplitude. However, the 2 groups were different in the involved brain regions. For LSW, only the 2 ID groups showed condition differences in amplitude at 9 months. At 18 months, condition differences were not observed in any group.Conclusions: This study indicates that the timing of ID in early life (fetal-neonatal vs. postnatal) modulates the impact of ID on recognition memory. Such impact also varies depending on the age of infants at testing (9 vs. 18 months).

Targeting elevated heme levels to treat a mouse model for Diamond-Blackfan Anemia.

Diamond-Blackfan anemia (DBA) is a rare genetic disorder in which patients present a scarcity of erythroid precursors in an otherwise normocellular bone marrow. Most, but not all, patients carry mutations in ribosomal proteins such as RPS19, suggesting that compromised mRNA translation and ribosomal stress are pathogenic mechanisms causing depletion of erythroid precursors. To gain further insight to disease mechanisms in DBA, we performed a custom short hairpin RNA (shRNA) based screen against 750 genes hypothesized to affect DBA pathophysiology. Among the hits were two shRNAs against the erythroid specific heme-regulated eIF2α kinase (HRI), which is a negative regulator of mRNA translation. This study shows that shRNA-mediated HRI silencing or loss of one HRI allele improves expansion of Rps19-deficient erythroid precursors, as well as improves the anemic phenotype in Rps19-deficient animals. We found that Rps19-deficient erythroblasts have elevated levels of unbound intracellular heme, which is normalized by HRI heterozygosity. Additionally, targeting elevated heme levels by treating cells with the heme scavenger alpha-1-microglobulin (A1M), increased proliferation of Rps19-deficient erythroid precursors and decreased heme levels in a disease-specific manner. HRI heterozygosity, but not A1M treatment, also decreased the elevated p53 activity observed in Rps19-deficient cells, indicating that p53 activation is caused by ribosomal stress and aberrant mRNA translation and not heme overload in Rps19-deficiency. Together, these findings suggest that targeting elevated heme levels is a promising new treatment strategy for DBA.

Sedimentary Cobalt Protoporphyrin as a Potential Precursor of Prosthetic Heme Group for Bacteria Inhabiting Fossil Organic Matter-Rich Shale Rock.

This study hypothesizes that bacteria inhabiting shale rock affect the content of the sedimentary cobalt protoporphyrin present in it and can use it as a precursor for heme synthesis. To verify this hypothesis, we conducted qualitative and quantitative comparative analyses of cobalt protoporphyrin as well as heme, and heme iron in shale rock that were (i) inhabited by bacteria in the field, (ii) treated with bacteria in the laboratory, and with (iii) bacterial culture on synthetic cobalt protoporphyrin. Additionally, we examined the above-mentioned samples for the presence of enzymes involved in the heme biosynthesis and uptake as well as hemoproteins. We found depletion of cobalt protoporphyrin and a much higher heme concentration in the shale rock inhabited by bacteria in the field as well as the shale rock treated with bacteria in the laboratory. Similarly, we observed the accumulation of protoporphyrin in bacterial cells grown on synthetic cobalt protoporphyrin. We detected numerous hemoproteins in metaproteome of bacteria inhabited shale rock in the field and in proteomes of bacteria inhabited shale rock and synthetic cobalt protoporhyrin in the laboratory, but none of them had all the enzymes involved in the heme biosynthesis. However, proteins responsible for heme uptake, ferrochelatase and sirohydrochlorin cobaltochelatase/sirohydrochlorin cobalt-lyase were detected in all studied samples.

Effects of Iron Supplements on Heme Scavengers in Pregnancy.

In malaria endemic countries, anemia in pregnant women occurs as a result of erythrocyte destruction by Plasmodium infections and other causes including malnutrition. Iron supplementation is recommended as treatment of iron-deficiency anemia. Erythrocyte destruction results in increased release of cytotoxic free heme that is scavenged by haptoglobin (Hp), hemopexin (Hx) and heme oxygenase-1 (HO-1). Paradoxically, iron supplementation in pregnant women has been reported to enhance parasitemia and increase levels of free heme. The relationship between free heme, heme scavengers, and birth outcomes has not been investigated, especially in women who are on iron supplementation. We hypothesized that parasite-infected pregnant women on routine iron supplementation have elevated heme and altered expression of heme scavengers. A cross-sectional study was conducted to determine the association between plasma levels of free heme, HO-1, Hp, Hx, and malaria status in pregnant women who received routine iron supplementation and their birth outcomes. Heme was quantified by colorimetric assay and scavenger protein concentration by ELISA. We demonstrated that iron-supplemented women with asymptomatic parasitemia had increased free heme (mean 75.6 µM; interquartile range [IQR] 38.8-96.5) compared with nonmalaria iron-supplemented women (mean 34.9 µM; IQR 17.4-43.8, P < 0.0001). Women with preterm delivery had lower levels of Hx (mean 656.0 µg/mL; IQR 410.9-861.3) compared with women with full-term delivery (mean: 860.9 µg/mL; IQR 715.2-1055.8, P = 0.0388). Our results indicate that iron supplementation without assessment of circulating levels of free heme and heme scavengers may increase the risk for adverse pregnancy outcomes.

UPLC-MS/MS Determination of Linezolid and Heme in Plasma of Infected Patients and Correlation Analysis.

Linezolid can cause serious haematological toxicity, such as thrombocytopenia and aneamia. Heme, composed of iron and porphyrin, is an important component of hemoglobin. In order to investigate the relationship between the concentration of linezolid and heme in the plasma of infected patients, a UPLC-MS/MS method that can determine the concentrations of linezolid and heme simultaneously was developed and validated. A total of 96 healthy subjects and 81 infected patients, who received blood routine blood tests, were included and determined by the UPLC-MS/MS method. The results showed that the concentration of linezolid was 5.08 ± 3.46 µg/mL in infected patients who were treated with linezolid. The heme in healthy subjects was 7.05 ± 8.68 µg/mL, and it was significantly decreased to 0.88 ± 0.79 µg/mL in infected patients (P < 0.01). Spearman correlation analysis showed that linezolid had a high negative correlation with platelet (PLT) (R = -0.309). Heme had a high positive correlation with hemoglobin (Hb) (R = 0.249) in healthy subjects and infected patients. The ROC analysis showed that heme had diagnostic value to distinguish low Hb (110 g/L). In conclusion, there was a positive correlation between heme and Hb, and this correlation was also observed in infected patients. A high concentration of linezolid was inclined to decrease PLT. Monitoring of heme and linezolid helps in the early diagnose of low Hb and PLT.

Effect of hydroxyurea therapy on intravascular hemolysis and endothelial dysfunction markers in sickle cell anemia patients.

Intravascular hemolysis (IH) contributes to the development of endothelial dysfunction (ED) in sickle cell anemia (SCA), and the effects of hydroxyurea (HU, the only approved drug that decreases the frequency and severity of vaso-oclussive crises) on IH and ED in SCA remain unclear. We evaluated and compared the markers of IH among steady-state adult Brazilians with SCA and HbAA individuals. Overall, this cross-sectional study enrolled 30 SCA patients not receiving HU therapy (HbSS), 25 SCA patients receiving HU therapy (HbSS_HU), and 32 HbAA volunteers (HbAA). The IH markers evaluated were serum Lactate Dehydrogenase (LDH), total heme, plasma hemoglobin (pHb), and soluble CD163 (sCD163). The ED markers analyzed were plasma von Willebrand factor (VWF:Ag), VWF ristocetin cofactor activity (VWF:RCo) levels, antigen of VWF-cleaving protease (ADAMTS13:Ag), thrombospondin-1, endothelin-1 levels, and ADAMTS13 Activity (ADAMTS13:Act). The levels of VWF:Ag, VWF:RCo, total heme, thrombospondin-1, and endothelin-1 were significantly higher in SCA patients (HbSS and HbSS_HU) compared to HbAA individuals. Also, pHb, LDH, and thrombospondin-1 levels were significantly higher in the HbSS group than in the HbSS_HU group. Contrarily, the levels of sCD163, ADAMTS13:Ag, and ADAMTS13:Act were significantly lower in both groups of SCA patients than HbAA controls, and ADAMTS13:Act levels were significantly lower in HbSS compared to HbSS_HU patients. The higher ADAMTS13 activity levels in those on HU therapy may be attributed to lower pHb and thrombospondin-1 levels as previously shown by in vitro studies that thrombospondin-1 and pHb are bound to VWF. Thus, VWF is restrained from ADAMTS13 activity and cleavage.

The roles of free iron, heme, haemoglobin, and the scavenger proteins haemopexin and alpha-1-microglobulin in preeclampsia and fetal growth restriction.

BACKGROUND: Preeclampsia (PE) is a complex pregnancy syndrome characterised by maternal hypertension and organ damage after 20 weeks of gestation and is associated with an increased risk of cardiovascular disease later in life. Extracellular haemoglobin (Hb) and its metabolites heme and iron are highly toxic molecules and several defence mechanisms have evolved to protect the tissue. OBJECTIVES: We will discuss the roles of free iron, heme, Hb, and the scavenger proteins haemopexin and alpha-1-microglobulin in pregnancies complicated by PE and fetal growth restriction (FGR). CONCLUSION: In PE, oxidative stress causes syncytiotrophoblast (STB) stress and increased shedding of placental STB-derived extracellular vesicles (STBEV). The level in maternal circulation correlates with the severity of hypertension and supports the involvement of STBEVs in causing maternal symptoms in PE. In PE and FGR, iron homeostasis is changed, and iron levels significantly correlate with the severity of the disease. The normal increase in plasma volume taking place during pregnancy is less for PE and FGR and therefore have a different impact on, for example, iron concentration, compared to normal pregnancy. Excess iron promotes ferroptosis is suggested to play a role in trophoblast stress and lipotoxicity. Non-erythroid α-globin regulates vasodilation through the endothelial nitric oxide synthase pathway, and hypoxia-induced α-globin expression in STBs in PE placentas is suggested to contribute to hypertension in PE. Underlying placental pathology in PE with and without FGR might be amplified by iron and heme overload causing oxidative stress and ferroptosis. As the placenta becomes stressed, the release of STBEVs increases and affects the maternal vasculature.

Unravelling the mechanisms controlling heme supply and demand.

In addition to heme's role as the prosthetic group buried inside many different proteins that are ubiquitous in biology, there is new evidence that heme has substantive roles in cellular signaling and regulation. This means that heme must be available in locations distant from its place of synthesis (mitochondria) in response to transient cellular demands. A longstanding question has been to establish the mechanisms that control the supply and demand for cellular heme. By fusing a monomeric heme-binding peroxidase (ascorbate peroxidase, mAPX) to a monomeric form of green-fluorescent protein (mEGFP), we have developed a heme sensor (mAPXmEGFP) that can respond to heme availability. By means of fluorescence lifetime imaging, this heme sensor can be used to quantify heme concentrations; values of the mean fluorescence lifetime (τMean) for mAPX-mEGFP are shown to be responsive to changes in free (unbound) heme concentration in cells. The results demonstrate that concentrations are typically limited to one molecule or less within cellular compartments. These miniscule amounts of free heme are consistent with a system that sequesters the heme and is able to buffer changes in heme availability while retaining the capability to mobilize heme when and where it is needed. We propose that this exchangeable supply of heme can operate using mechanisms for heme transfer that are analogous to classical ligand-exchange mechanisms. This exquisite control, in which heme is made available for transfer one molecule at a time, protects the cell against the toxic effect of excess heme and offers a simple mechanism for heme-dependent regulation in single-molecule steps.

Severe homocysteinemia in two givosiran-treated porphyria patients: is free heme deficiency the culprit?

Givosiran is a novel approach to treat patients with acute intermittent porphyrias (AIP) by silencing of ∂-ALA-synthase 1, the first enzyme of heme biosynthesis in the liver. We included two patients in the Envision study who responded clinically well to this treatment. However, in both patients, therapy had to be discontinued because of severe adverse effects: One patient (A) developed local injection reactions which continued to spread all over her body with increasing number of injections and eventually caused a severe systemic allergic reaction. Patient B was hospitalized because of a fulminant pancreatitis. Searching for possible causes, we also measured the patients plasma homocysteine (Hcy) levels in fluoride-containing collection tubes: by LC-MS/MS unexpectedly, plasma Hcy levels were 100 and 200 in patient A and between 100 and 400 µmol/l in patient B. Searching for germline mutations in 10 genes that are relevant for homocysteine metabolism only revealed hetero- and homozygous polymorphisms in the MTHFR gene. Alternatively, an acquired inhibition of cystathionine-beta-synthase which is important for homocysteine metabolism could explain the plasma homocysteine increase. This enzyme is heme-dependent: when we gave heme arginate to our patients, Hcy levels rapidly dropped. Hence, we conclude that inhibition of ∂-ALA-synthase 1 by givosiran causes a drop of free heme in the hepatocyte and therefore the excessive increase of plasma homocysteine. Hyperhomocysteinemia may contribute to the adverse effects seen in givosiran-treated patients which may be due to protein-N-homocysteinylation.

Measurement of labile and protein-bound heme in fixed prostate cancer cells and in cellular fractions.

Labile heme is present in the cells at very low concentrations, either unbound or loosely bound to molecules, and accessible for signaling as alarmin. Our recent work suggests that extracellular heme can be taken up and detected in the nuclei of cancer cells. Here, we describe the detailed protocol for detection of labile and total heme in prostate cancer cells and its measurement in subcellular compartments in vitro. The protocol can be adapted to be used for other cell types. For complete details on the use and execution of this protocol, please refer to Canesin et al. (2020).

In vivo evaluation of heme and non-heme iron content and neuronal density in human basal ganglia.

Non-heme iron is an important element supporting the structure and functioning of biological tissues. Imbalance in non-heme iron can lead to different neurological disorders. Several MRI approaches have been developed for iron quantification relying either on the relaxation properties of MRI signal or measuring tissue magnetic susceptibility. Specific quantification of the non-heme iron can, however, be constrained by the presence of the heme iron in the deoxygenated blood and contribution of cellular composition. The goal of this paper is to introduce theoretical background and experimental MRI method allowing disentangling contributions of heme and non-heme irons simultaneously with evaluation of tissue neuronal density in the iron-rich basal ganglia. Our approach is based on the quantitative Gradient Recalled Echo (qGRE) MRI technique that allows separation of the total R2* metric characterizing decay of GRE signal into tissue-specific (R2t*) and the baseline blood oxygen level-dependent (BOLD) contributions. A combination with the QSM data (also available from the qGRE signal phase) allowed further separation of the tissue-specific R2t* metric in a cell-specific and non-heme-iron-specific contributions. It is shown that the non-heme iron contribution to R2t* relaxation can be described with the previously developed Gaussian Phase Approximation (GPA) approach. qGRE data were obtained from 22 healthy control participants (ages 26-63 years). Results suggest that the ferritin complexes are aggregated in clusters with an average radius about 100nm comprising approximately 2600 individual ferritin units. It is also demonstrated that the concentrations of heme and non-heme iron tend to increase with age. The strongest age effect was seen in the pallidum region, where the highest age-related non-heme iron accumulation was observed.

Relationship of hemoglobin level and plasma coproporphyrin-I concentrations as an endogenous probe for phenotyping OATP1B.

Plasma coproporphyrin-I (CP-I) concentration is used as a sensitive and selective endogenous probe for phenotyping organic anion transporting polypeptides 1B (OATP1B) activity in many studies. CP-I is produced in the process of heme synthesis, but the relationship between plasma CP-I concentrations and heme synthesis activity is unknown. In this study, we evaluated the relationship between plasma CP-I concentration and hemoglobin level as a biomarker of heme synthesis activity. The data of 391 subjects selected from the Japanese general population were analyzed. One hundred twenty-six participants had OATP1B1*15 allele, 11 of whom were homozygous (OATP1B1*15/*15). Multiple regression analysis identified hemoglobin level as an independent variable associated with plasma CP-I concentration (p < 0.0001). A significant positive correlation was observed between hemoglobin level and plasma CP-I concentration in participants without OATP1B1*15 allele (n = 265; rs  = 0.35, p < 0.0001) and with OATP1B1*15 allele (n = 126; rs  =0.27, p = 0.0022). However, Kruskal-Wallis test showed no large difference in Kruskal-Wallis statistics between the distribution of plasma CP-I concentrations and that of ratio of plasma CP-I to hemoglobin among six OATP1B1 polymorphism groups. These findings suggest that the hemoglobin level seems to reflect biosynthesis of CP-I. However, correction by hemoglobin level is not required when using basal plasma CP-I concentration for phenotyping OATP1B activity.

Nanocluster-assisted protein-film voltammetry for direct electrochemical signal acquisition.

Acquisition of the direct electrochemical response of protein is the cornerstone for the development of the third generation of electrochemical biosensors. In this work, we developed a nanocluster-assisted protein-film voltammetry technique (NCA-PFV) which can achieve the acquisition of the electrochemical signal and maintain the activity without affecting of the protein's structure. With this strategy, a lipid bilayer membrane is used to immobilize the membrane protein so as to maintain its natural state. Copper nanoclusters with a size smaller than most proteins are then used to function at sub-protein scale and to mediate the electron hopping from the electroactive center of the electrode. As a model, the direct electrochemical signal of cyclooxygenase (COX) is successfully obtained, with a pair of well-defined redox peaks located at -0.39 mV and -0.31 mV, which characterize the heme center of the enzyme. Its catalytic activity towards the substrate arachidonic acid (AA) is also retained. The detection range for AA is 10-1000 µM and the detection limit is 2.4 µM. Electrochemical monitoring of the regulation of the catalytic activity by an inhibitor DuP-697 is also achieved. This work provides a powerful tool for the fabrication of enzyme-based electrochemical biosensors, and is also of great significance for promoting the development and application of next-generation electrochemical biosensors.

Quantification and Carbon and Nitrogen Isotopic Measurements of Heme B in Environmental Samples.

Heme B is an iron-coordinated tetrapyrrole molecule that acts as a cofactor in hemoproteins. It is expected to be ubiquitous in the environment, as b-type hemoproteins catalyze a variety of essential biochemical reactions. In this study, we developed an analytical method to quantify heme B in biological and environmental samples using high-performance liquid chromatography (HPLC) coupled to a photodiode array detector. The applicability of our method was further extended by the use of liquid chromatography/mass spectrometry (LC/MS; detection limit: ∼1 fmol), which enabled the quantification of a trace amount of dissolved heme B in filtered seawater and sedimentary heme B coexisting with an abundant interfering organic matrix. For compound-specific carbon and nitrogen isotopic measurements, heme B was successfully isolated and purified from biological and environmental samples by a combination of anion-exchange column chromatography, methyl esterification, and dual-step HPLC. While carbon and nitrogen isotopic compositions of heme B in phototrophs were mostly comparable to those of chlorophyll a, heme B in suspended particulate materials in coastal water and an intertidal sediment was 13C-depleted and 15N-enriched relative to chlorophyll a, suggesting that nonphototrophic microorganisms are also a significant source of heme B in natural environments.