Is carbonic anhydrase inhibition useful as a complementary therapy of Covid-19 infection?

The ongoing Covid-19 is a contagious disease, and it is characterised by different symptoms such as fever, cough, and shortness of breath. Rising concerns about Covid-19 have severely affected the healthcare system in all countries as the Covid-19 outbreak has developed at a rapid rate all around the globe. Intriguing, a clinically used drug, acetazolamide (a specific inhibitor of carbonic anhydrase, CA, EC 4.2.1.1), is used to treat high-altitude pulmonary oedema (HAPE), showing a high degree of clinical similarities with the pulmonary disease caused by Covid-19. In this context, this preliminary study aims to provide insights into some factors affecting the Covid-19 patients, such as hypoxaemia, hypoxia as well as the blood CA activity. We hypothesise that patients with Covid-19 problems could show a dysregulated acid-base status influenced by CA activity. These preliminary results suggest that the use of CA inhibitors as a pharmacological treatment for Covid-19 may be beneficial.

Carbonic anhydrase, obstructive sleep apnea and hypertension: Effects of intervention.

Whole blood carbonic anhydrase activity (CAa) is increased in patients with obstructive sleep apnea (OSA). Our study investigated the influence of positive airway pressure (PAP) or CA inhibitor acetazolamide (ACT) therapy on CAa, OSA and blood pressure. Thirty-three OSA patients (21 hypertensive, body mass index (BMI) 37 ± 7 kg/m2 and apnea-hypopnea index (AHI) of 47 ± 31 events/hr) were followed-up after PAP treatment (compliance, 4.7 ± 1.5 hr/day; duration, median 6 [IQR 6,6] months) (Cohort A). A second OSA Cohort (B) contained nine hypertensive patients (BMI, 29 ± 4 kg/m2 ; AHI, 39 ± 20 events/hr) with 2-week treatment of ACT, PAP or ACT + PAP in an open crossover study. CAa was assessed at baseline and at the end of each treatment period. In Cohort A, baseline CAa was higher in hypertensive, compared with normotensive, patients (1,033 ± 204 versus 861 ± 201 units, p = .028). PAP treatment reduced systolic/diastolic blood pressure but not CAa (-9 ± 11/-5 ± 7 mmHg and -20 ± 289 units, p < .001, <.001 and .70). In Cohort B, blood pressure was reduced in both ACT-treated groups (-10 ± 10/-5 ± 7 mmHg, p = .043 and .019; and -5 ± 5/-13 ± 13 mmHg, p < .001 and .009). AHI was reduced in both groups: ACT only, -17 ± 9 events/hr p = .001; and ACT + PAP, -39 ± 19 events/hr, p < .001. PAP did not change CAa (p = .98) but activity tended to decrease after ACT with or without PAP (p = .081 and .056). CAa is elevated in hypertensive OSA patients. Long-term PAP reduced blood pressure without affecting CAa. ACT reduced blood pressure and CAa. Increased CAa may constitute a physiological characteristic in OSA, contributing to comorbid hypertension.

Comparison of blood carbonic anhydrase activity of athletes performing interval and continuous running exercise at high altitude.

The effects of high-intensity interval and continuous exercise on erythrocytes carbonic anhydrase (CA, EC 4.2.1.1) activity levels were scarcely investigated up until now. Here we present a study focused on the CA activity from erythrocytes of athletes experiencing interval and continuous training for 6 weeks, during cold weather and at high altitude (> 1600 m). We observed a 50% increase in the blood CA activity at the second week after initiation of the training in both interval and continuos running groups, whereas the control group did not experience any variation in the enzyme activity levels. In the trained individuals a mild decrease in their body mass, BMI and an increased [Formula: see text] were also observed. The CA activity returned at the basal values after 4-6 weeks after the training started, probably proving that a metabolic compensation occurred without the need of an enhanced enzyme activity. The unexpected 50% rise of activity for an enzyme which acts as a very efficient catalyst for CO2 hydration/bicarbonate dehydration, such as the blood CA, deserves further investigations for better understanding the physiologic basis of this phenomenon.

Do CO2 and oxidative stress induce cancer?: a brief study about the evaluation of PON 1, CAT, CA and XO enzyme levels on head and neck cancer patients.

Head and neck cancer (HNC) is one of the most common malignancies in the world. HNC is a group of cancers that starts in the mouth, nose, throat, larynx, sinuses, or salivary glands. According to this section of the body parts; induction of cancer can be associated with CO2 and oxidative stress. The aim of this study is to assess the activities of carbonic anhydrase (CA), catalase (CAT), paraoxonase1 (PON1), and xanthine oxidase (XO) activities in 89 HNC patients and 115 healthy volunteers. Paraoxonase1 activity was found lower in HNC cancer patients. There is no statistically significant difference between patients and controls for catalase, carbonic anhydrase, and xanthine oxidase enzyme levels. According to this results, paraoxonase1 levels could be a candidate as an oxidative marker in HNC patients, but further studies are needed to investigate the other type of cancer related PON1 and the other enzyme levels.

Time course of red blood cell intracellular pH recovery following short-circuiting in relation to venous transit times in rainbow trout, Oncorhynchus mykiss.

Accumulating evidence is highlighting the importance of a system of enhanced hemoglobin-oxygen (Hb-O2) unloading for cardiovascular O2 transport in teleosts. Adrenergically stimulated sodium-proton exchangers (ß-NHE) create H+ gradients across the red blood cell (RBC) membrane that are short-circuited in the presence of plasma-accessible carbonic anhydrase (paCA) at the tissues; the result is a large arterial-venous pH shift that greatly enhances O2 unloading from pH-sensitive Hb. However, RBC intracellular pH (pHi) must recover during venous transit (31-90 s) to enable O2 loading at the gills. The halftimes ( t1/2) and magnitudes of RBC ß-adrenergic stimulation, short-circuiting with paCA and recovery of RBC pHi, were assessed in vitro, on rainbow trout whole blood, and using changes in closed-system partial pressure of O2 as a sensitive indicator for changes in RBC pHi. In addition, the recovery rate of RBC pHi was assessed in a continuous-flow apparatus that more closely mimics RBC transit through the circulation. Results indicate that: 1) the t1/2 of ß-NHE short-circuiting is likely within the residence time of blood in the capillaries, 2) the t1/2 of RBC pHi recovery is 17 s and within the time of RBC venous transit, and 3) after short-circuiting, RBCs reestablish the initial H+ gradient across the membrane and can potentially undergo repeated cycles of short-circuiting and recovery. Thus, teleosts have evolved a system that greatly enhances O2 unloading from pH-sensitive Hb at the tissues, while protecting O2 loading at the gills; the resulting increase in O2 transport per unit of blood flow may enable the tremendous athletic ability of salmonids.

Effects of zinc oxide nanoparticles on the egg quality, immune response, zinc retention, and blood parameters of laying hens in the late phase of production.

The objective of this study was to evaluate the effects of dietary supplementation with zinc oxide nanoparticles (ZnO-NPs) on the performance, egg quality, Zn retention, immunity responses, superoxide dismutase activity (SOD), egg malondialdehyde (MDA) content, and serum parameters in laying hens in the late phase of production. A total of 288 laying hens at 64 weeks of age were randomly assigned to 4 treatments with 6 replicates, and 12 birds within each group. Experimental diets included a corn-soybean meal-based diet (without Zn supplementation) and a basal diet supplemented with 80 mg/kg of Zn-oxide, ZnO-NPs, and Zn-methionine. The results indicated that egg production and egg mass were significantly higher in the Zn-methionine and ZnO-NPs groups (p < .05). Also, eggshell thickness and shell strength increased in the ZnO-NPs group as compared with the other groups (p < .05). Moreover, Zn supplementation decreased egg loss (p < .05). There were significant differences among treatments in Zn deposition in tibiotarsus, liver, pancreas, eggs, and excreta (p < .01). Antibody titre, heterophil (%(, and phytohemagglutinin (PHA) were significantly higher in birds fed with Zn-supplemented diets (p < .05). In treatments supplemented with ZnO-NPs and Zn-methionine, the SOD activity in the liver, pancreas, and plasma was greater as compared with the other treatments (p < .05). The MDA content in eggs was significantly reduced in groups supplemented with Zn (p < .01). Moreover, dietary Zn supplementation significantly affected serum total protein, albumin, glucose, alkaline phosphatase activity, carbonic anhydrase activity, and Zn level (p < .05). In conclusion, this study demonstrated that dietary supplementation with ZnO-NPs can improve the performance of laying hens. Therefore, ZnO-NPs can enhance zinc absorption in the intestine of aged layers and can be a more suitable source of zinc than regular Zn-oxide in diets.

Carbonic anhydrase inhibitory properties of some uracil derivatives.

Inhibitors of carbonic anhydrase (CA) have been carried out in many therapeutic applications, especially antiglaucoma activity. In this study, we investigated some uracil derivatives (4-12) to inhibit human CA I (hCA I) and II (hCA II) isoenzymes. The KI values of the compounds 4-12 are in the range of 0.085-428 µM for hCA I and of 0.1715-645 µM against hCA II, respectively. It is concluded from the kinetic investigations, all compounds used in the study act as competitive inhibitors with substrate, 4-NPA. Uracil derivatives are emerging agents for the inhibiton of carbonic anhydrase which could be used in biomedicine.

Carbonic anhydrase 1 is a promising biomarker for early detection of non-small cell lung cancer.

This study aimed to identify candidate biomarkers associated with stage I non-small cell lung cancer (NSCLC). Sera from three groups, a lung cancer group (n = 11), benign control group (n = 12), and normal control group (n = 10), were collected and pooled. Protein expression profiles were analyzed by a combination of two-dimensional electrophoresis (2DE) and matrix-assisted laser desorption ionization mass spectrometry (MALDI-MS). These methods were used to separate, screen, and identify proteins that were differentially expressed between stage I NSCLC and controls. Differentially expressed proteins were validated by both Western blot and ELISA in an expanded sample size (22, 18, and 18 in three groups, respectively). MALDI-MS identified 12 differentially expressed proteins in the lung cancer group compared to the two control groups. Expression of carbonic anhydrase 1 (CA1) was validated by Western blot. CA1 was significantly elevated in the lung cancer group compared to controls. ELISA results confirmed that CA1 in the lung cancer group (3.18 ± 1.27 ng/mL, n = 22) was highly expressed in stage I NSCLC patients compared to those in the benign control group (2.21 ± 0.71 ng/mL, n = 18) and the normal control group (2.04 ± 0.63 ng/mL, n = 18) (P = 0.001). In conclusion, we provide evidence that CA1 is highly expressed in the sera of stage I NSCLC patients. Additionally, CA1 might serve as a novel biomarker for early detection of NSCLC.

High serum carbonic anhydrase IX predicts shorter survival in head and neck cancer.

OBJECTIVES: The objective of the study was to investigate prognostic and predictive value of pretreatment soluble carbonic anhydrase IX (CA IX) blood serum concentration in patients with locally advanced head and neck cancer. BACKGROUND: Increased expression of CA IX in tumor tissues has been associated with treatment resistance and worth prognosis. Soluble form of CA IX, released from tumor cells, is detectable in blood serum and could be a convenient predictive factor of treatment effectiveness that would enable treatment individualization. METHODS: The prospective study evaluated 48 patients with locally advanced squamous cell carcinomas of head and neck, treated by radiotherapy or chemo-radiotherapy. Pretreatment soluble CA IX serum concentrations were examined using enzyme-linked immunosorbent assay. RESULTS: Soluble CA IX serum concentration failed to predict radiotherapy effectiveness in the studied patient population (p = 0.26). However, high CA IX serum concentrations have been associated with shorter overall survival (p = 0.035) CONCLUSION: High pretreatment CA IX serum concentration is a negative prognostic factor in locally advanced head and neck cancer patients (Tab. 1, Fig. 2, Ref. 23).

Early increase in circulating carbonic anhydrase IX during neoadjuvant treatment predicts favourable outcome in locally advanced rectal cancer.

BACKGROUND: Locally advanced rectal cancer (LARC) comprises heterogeneous tumours with predominant hypoxic components. The hypoxia-inducible metabolic shift causes microenvironmental acidification generated by carbonic anhydrase IX (CAIX) and facilitates metastatic progression, the dominant cause of failure in LARC. METHODS: Using a commercially available immunoassay, circulating CAIX was assessed in prospectively archived serial serum samples collected during combined-modality neoadjuvant treatment of LARC patients and correlated to histologic tumour response and progression-free survival (PFS). RESULTS: Patients who from their individual baseline level displayed serum CAIX increase above a threshold of 224 pg/ml (with 96 % specificity and 39 % sensitivity) after completion of short-course neoadjuvant chemotherapy (NACT) prior to long-course chemoradiotherapy and definitive surgery had significantly better 5-year PFS (94 %) than patients with below-threshold post-NACT versus baseline alteration (PFS rate of 56 %; p < 0.01). This particular CAIX parameter, ΔNACT, was significantly correlated with histologic ypT0-2 and ypN0 outcome (p < 0.01) and remained an independent PFS predictor in multivariate analysis wherein it was entered as continuous variable (p = 0.04). CONCLUSIONS: Our results indicate that low ΔNACT, i.e., a weak increase in serum CAIX level following initial neoadjuvant treatment (in this case two cycles of the Nordic FLOX regimen), might be used as risk-adapted stratification to postoperative therapy or other modes of intensification of the combined-modality protocol in LARC. TRIAL REGISTRATION: ClinicalTrials.gov NCT00278694.

Antibody-free, targeted mass-spectrometric approach for quantification of proteins at low picogram per milliliter levels in human plasma/serum.

Sensitive detection of low-abundance proteins in complex biological samples has typically been achieved by immunoassays that use antibodies specific to target proteins; however, de novo development of antibodies is associated with high costs, long development lead times, and high failure rates. To address these challenges, we developed an antibody-free strategy that involves PRISM (high-pressure, high-resolution separations coupled with intelligent selection and multiplexing) for sensitive selected reaction monitoring (SRM)-based targeted protein quantification. The strategy capitalizes on high-resolution reversed-phase liquid chromatographic separations for analyte enrichment, intelligent selection of target fractions via on-line SRM monitoring of internal standards, and fraction multiplexing before nano-liquid chromatography-SRM quantification. Application of this strategy to human plasma/serum demonstrated accurate and reproducible quantification of proteins at concentrations in the 50-100 pg/mL range, which represents a major advance in the sensitivity of targeted protein quantification without the need for specific-affinity reagents. Application to a set of clinical serum samples illustrated an excellent correlation between the results obtained from the PRISM-SRM assay and those from clinical immunoassay for the prostate-specific antigen level.

A pilot study on potential plasma hypoxia markers in the radiotherapy of non-small cell lung cancer. Osteopontin, carbonic anhydrase IX and vascular endothelial growth factor.

BACKGROUND: Hypoxic radioresistance plays a critical role in the radiotherapy of cancer and adversely impacts prognosis and treatment response. This prospective study investigated the interrelationship and the prognostic significance of several hypoxia-related proteins in non-small cell lung cancer (NSCLC) patients treated by radiotherapy ± chemotherapy. MATERIAL AND METHODS: Pretreatment osteopontin (OPN), vascular endothelial growth factor (VEGF) and carbonic anhydrase IX (CA IX) plasma levels were determined by ELISA in 55 NSCLC (M0) patients receiving 66 Gy curative-intent radiotherapy or chemoradiation. Marker correlation, association with clinicopathological parameters and the prognostic value of a biomarker combination was evaluated. RESULTS: All biomarkers were linearly correlated and linked to different clinical parameters including lung function, weight loss (OPN), gross tumor volume (VEGF) and T stage (CA IX). High OPN (p = 0.03), VEGF (p = 0.02) and CA IX (p = 0.04) values were significantly associated with poor survival. Double marker combination additively increased the risk of death by a factor of 2 and high plasma levels of the triple combination OPN/VEGF/CA IX yielded a 5.9-fold risk of death (p = 0.009). The combined assessment of OPN/VEGF/CA IX correlated independently with prognosis (p = 0.03) in a multivariate Cox regression model including N stage, T stage and GTV. CONCLUSION: This pilot study suggests that a co-detection augments the prognostic value of single markers and that the integration of OPN, VEGF and CA IX into a hypoxic biomarker profile for the identification of patients with largely hypoxic and radioresistant tumors should be further evaluated.

Increased oxidation-related glutathionylation and carbonic anhydrase activity in endometriosis.

This study examined the possible involvement of carbonic anhydrase activation in response to an endometriosis-related increase in oxidative stress. Peripheral blood samples obtained from 27 healthy controls and 30 endometriosis patients, classified as having endometriosis by histological examination of surgical specimens, were analysed by multiple immunoassay and carbonic anhydrase activity assay. Red blood cells (RBC) were analysed for glutathionylated protein (GSSP) content in the membrane, total glutathione (GSH) in the cytosol and carbonic anhydrase concentration and activity. In association with a membrane increase of GSSP and a cytosolic decrease of GSH content in endometriosis patients, carbonic anhydrase significantly increased (P < 0.0001) both monomerization and activity compared with controls. This oxidation-induced activation of carbonic anhydrase was positively and significantly correlated with the GSH content of RBC (r = 0.9735, P < 0.001) and with the amount of the 30-kDa monomer of carbonic anhydrase (r = 0.9750, P < 0.001). Because carbonic anhydrase activation is implied in many physiological and biochemical processes linked to pathologies such as glaucoma, hypertension, obesity and infections, carbonic anhydrase activity should be closely monitored in endometriosis. These data open promising working perspectives for diagnosis and treatment of endometriosis and hopefully of other oxidative stress-related diseases. Endometriosis is a chronic disease associated with infertility and local inflammatory response, which is thought to spread rapidly throughout the body as a systemic subclinical inflammation. One of the causes in the pathogenesis/evolution of endometriosis is oxidative stress, which occurs when reactive oxygen species are produced faster than the endogenous antioxidant defence systems can neutralize them. Once produced, reactive oxygen species can alter the morphological and functional properties of endothelial cells, including permeability and adhesion molecule expression, thus contributing to ongoing inflammation. Due to their main cellular functions--delivery of O2 from lung to tissue and removal of CO2 from tissue to lung--red blood cells (RBC) are exposed to oxidative stress. Carbon dioxide in tissue capillaries diffuses into red cells, where it is rapidly hydrated by the action of cytosolic carbonic anhydrase. Analysis of the oxidation status of endometriotic RBC membranes showed a high content of glutathionylated proteins, indicating pre-existing oxidation-related alterations. The increase in glutathionylated proteins was correlated to increased carbonic anhydrase activity in endometriotic RBC compared with healthy controls. Carbonic anhydrase is a family of metalloenzymes involved in many physiological processes such as acid-base homeostasis, respiration, carbon dioxide and ion transport, and bone resorption, and in the regulation of ureagenesis, gluconeogenesis, lipogenesis and tumourigenesis. Due to the potential implication of carbonic anhydrase activation in many pathologies, such as glaucoma, hypertension, obesity and infections, carbonic anhydrase activity should be closely monitored in endometriosis to prevent possible complications and/or worsening of related conditions.

Increased expression of carbonic anhydrase IX in oral submucous fibrosis and oral squamous cell carcinoma.

BACKGROUND: Cumulative evidence has demonstrated that carbonic anhydrase IX (CAIX) is upregulated in many types of human cancers. We attempted to evaluate plasma levels of CAIX in patients with oral cancer and investigated whether plasma CAIX is correlated with the progression of this disease. METHOD: In total, 191 patients with oral cancer, 30 patients with oral submucous fibrosis and 100 controls were recruited in this study. The plasma samples were collected and the levels of soluble CAIX in plasma were determined by the enzyme-linked immunosorbent assay (ELISA). Furthermore, the normal buccal mucosa fibroblast was challenged by arecoline, the major areca nut alkaloid, to assess the relationship between the levels of CAIX and areca nut chewing in oral cancer patients. RESULTS: Results showed that patients with oral cancer exhibited significantly higher levels of soluble CAIX compared to controls (p<0.001). Plasma levels of CAIX in oral cancer patients were associated with clinical stages after adjusting for age and areca nut chewing (p<0.05). In addition, patients with areca nuts chewing had higher CAIX levels than those who have not chewed areca nuts. Total carbonic anhydrase activity and CAIX mRNA levels were significantly higher in oral submucous fibrosis fibroblasts than in normal buccal mucosa fibroblasts. Moreover, arecoline elevated CAIX expression in a dose-dependent manner in normal buccal mucosa fibroblasts. CONCLUSIONS: Our results suggest that determining plasma levels of CAIX may be used as a non-invasive method for monitoring oral cancer progression and the involvement of areca quid chewing in oral carcinogenesis may be related to a higher expression of CAIX.

Synthesis and carbonic anhydrase inhibitory properties of novel 1,4-dihydropyrimidinone substituted diarylureas.

Abstract A new series of 1,4-dihydropyrimidinone (DHPM) substituted diaryl urea and thiourea derivatives were synthesized and their inhibitory effects on the activity of purified human carbonic anhydrase (hCA) I and II were evaluated. 4-Nitrophenyl-1,4-DHPM was prepared with dimedone, nitrobenzaldehyde and urea or thiourea and nitro group was reduced to amine derivative. The compound was reacted with isocyanates and isothiocyanates to get the final products. The results showed that all the synthesized compounds inhibited the carbonic anhydrase isoenzyme activity; 4c (IC50=66.23 µM for hCA I) and 4f (IC50=63.09 µM for hCA II) have the most inhibitory effect. The synthesized compounds are very bulky to be able to bind near the zinc ion and they much more probably bind as the coumarins and activators.

[Relation between carbonic anhydrase IX serum level, hypoxia and radiation resistance of head and neck cancers]. / Vztah medzi sérovou hladinou karboanhydrázy IX, hypoxiou a rádiorezistenciou nádorov hlavy a krku.

BACKGROUND: Hypoxia of locally advanced head and neck cancers is one of the main causes of their radiation resistance that presents clinically as a persistence of residual tumor disease after radiation therapy. Therefore, detection of tumor hypoxia could be an important predictor of treatment efficacy. Carbonic anhydrase IX (CA IX) is a protein, coded by a homonymous gene, the expression of which increases in tumor tissues at hypoxic conditions. Hence, CA IX represents an endogenic marker of tumor hypoxia, identifiable in tumor tissues, and its soluble extracellular domain can also be detected in body fluids of the patient. The primary endpoint of this study was to explore whether a correlation exists between CA IX serum level and the residual tumor disease after therapy. The secondary endpoint was to find out how the serum concentration of CA IX changes during the course of fractionated radiation therapy. MATERIALS AND METHODS: The presented prospective monocentric clinical study evaluated a population of 30 patients with locally advanced squamous cell head and neck cancers, treated by radiation therapy or concurrent chemo radiation therapy with a curative intent. The serum concentration of the soluble form of CA IX was examined from a venous blood sample, using sandwich enzyme linked immunosorbent assay (ELISA). The blood samples were obtained before the treatment initiation, in the middle of radiation therapy, at the time of finishing radiation therapy and six weeks after the treatment completion. RESULTS: We found a substantial variability in the CA IX levels measured in the examined population, ranging 0- 1,696 pg/ ml. We found no significant changes in the mean value of CA IX concentration during the course of radiation therapy and after the treatment completion. In 11 patients (36.7%), the treatment resulted in complete remission of the disease. In these patients, lower average pretreatment levels of CA IX were noted when compared to patients with persistence of residual tumor disease (37.57 vs 77.47; p = 0.154). CONCLUSION: The results indicate that serum level of CA IX in patients with locally advanced head and neck cancers does not change significantly during the course of fractionated radiation therapy. The relation between CA IX serum level and residual tumor disease after radiation therapy requires verification on a larger population of patients.

Heterogeneity of F cells in ß-thalassemia.

BACKGROUND: Fetal hemoglobin (HbF), which is largely replaced after birth by the adult Hb, is concentrated in a few "F cells." Their number significantly increases in certain physiologic and clinical situations, including in ß-thalassemia (ß-thal). Their quantification is used to detect fetal-maternal hemorrhage (FMH), where fetal cells enter the maternal circulation. We were confronted with a pregnant woman with ß-thal who was suspected to have FMH. To establish the usefulness of a flow cytometric procedure to differentiate between fetal cells and the maternal F cells, we screened adult ß-thal patients. STUDY DESIGN AND METHODS: Blood samples were simultaneously stained with fluorescent antibodies to HbF and to carbonic anhydrase (CA) isotype II, which is specific to adult red blood cells (RBCs). RESULTS: A heterogeneous distribution of RBCs with respect to HbF and CA expression was observed: adult non-F cells (CA+HbF-) and F cells (CA+HbF+/HbF++) as well as F cells with characteristics of fetal cells (CA-HbF++). CONCLUSIONS: The presence of CA-HbF++ RBCs in nonpregnant women, and even men, with thal indicates that the CA/HbF method is inappropriate for detection of FMH. The coexistence of F cells carrying fetal or adult markers suggests that they originate from two types of stem cell, adult and fetal, lineages. Normally, the fetal lineage is insignificant, but in ß-thal, as HbF-containing RBCs have a selective advantage, the "fetal" lineage gains significance.

Measuring binding of protein to gel-bound ligands using magnetic levitation.

This paper describes the use of magnetic levitation (MagLev) to measure the association of proteins and ligands. The method starts with diamagnetic gel beads that are functionalized covalently with small molecules (putative ligands). Binding of protein to the ligands within the bead causes a change in the density of the bead. When these beads are suspended in a paramagnetic aqueous buffer and placed between the poles of two NbFeB magnets with like poles facing, the changes in the density of the bead on binding of protein result in changes in the levitation height of the bead that can be used to quantify the amount of protein bound. This paper uses a reaction-diffusion model to examine the physical principles that determine the values of rate and equilibrium constants measured by this system, using the well-defined model system of carbonic anhydrase and aryl sulfonamides. By tuning the experimental protocol, the method is capable of quantifying either the concentration of protein in a solution, or the binding affinities of a protein to several resin-bound small molecules simultaneously. Since this method requires no electricity and only a single piece of inexpensive equipment, it may find use in situations where portability and low cost are important, such as in bioanalysis in resource-limited settings, point-of-care diagnosis, veterinary medicine, and plant pathology. It still has several practical disadvantages. Most notably, the method requires relatively long assay times and cannot be applied to large proteins (>70 kDa), including antibodies. The design and synthesis of beads with improved characteristics (e.g., larger pore size) has the potential to resolve these problems.

Comparison of haemoglobin F detection by the acid elution test, flow cytometry and high-performance liquid chromatography in maternal blood samples analysed for fetomaternal haemorrhage.

BACKGROUND AND OBJECTIVE: All pregnant women undergoing blood grouping at Southmead Hospital are offered haemoglobinopathy screening by high-performance liquid chromatography (HPLC). RhD-negative women who deliver RhD-positive infants are tested for fetomaternal haemorrhage (FMH) by acid elution (AE). The effectiveness of these two assays for quantitation of FMH was compared with flow cytometry (FC). MATERIALS AND METHODS: The relationship between expression of haemoglobin F (HbF) in individual cells by AE and FC and quantitation of HbF in haemolysates by HPLC was investigated, using maternal samples with unusually high levels of HbF-positive maternal cells (F cells) or with large FMH (fetal cells). Standard anti-D FC was performed to quantitate fetal D-positive cells in D-negative women and compared with FMH estimated by AE and HbF FC. RESULTS: AE overestimated FMH when maternal F cells were increased. HbF FC distinguished F cells from fetal cells. Values of HbF determined by HPLC were less than the level of 5% used for investigation of raised fetal haemoglobin, even in the maternal samples with elevated F cells or massive FMH. CONCLUSIONS: To quantitate FMH, measurement of HbF using FC was more sensitive and accurate than AE or HPLC. HbF FC is the method of choice when results from routine investigation using AE or standard anti-D FC are discrepant or when there is maternal and fetal RhD compatibility.

Effects of dopaminergic compounds on carbonic anhydrase isozymes I, II, and VI.

Studies on carbonic anhydrase (CA, EC 4.2.1.1) inhibitors have increased due to several therapeutic applications while there are few investigations on activators. Here we investigated CA inhibitory and activatory capacities of a series of dopaminergic compounds on human carbonic anhydrase (hCA) isozymes I, II, and VI. 2-Amino-1,2,3,4-tetrahydronaphthalene-6,7-diol hydrobromide and 2-amino-1,2,3,4-tetrahydronaphthalene-5,6-diol hydrobromide were found to show effective inhibitory action on hCA I and II whereas 2-amino-5,6-dibromoindan hydrobromide and 2-amino-5-bromoindan hydrobromide exhibited only moderate inhibition against both isoforms, being more effective inhibitors of hCA VI. K(i) values of the molecules 3-6 were in the range of 41.12-363 µM against hCA I, of 0.381-470 µM against hCA II and of 0.578-1.152 µM against hCA VI, respectively. Compound 7 behaved as a CA activator with K(A) values of 27.3 µM against hCA I, of 18.4 µM against hCA II and of 8.73 µM against hCA VI, respectively.