Range Of Hemoglobin Concentrations Research Articles

#1533 The relationship between the severity of anemia, the level of inflammation markers and the dose of epoetin in patients in pre-dialysis stages of CKD

Abstract Background and Aims Inflammation is known to be an important factor associated with hemoglobin variability in patients with anemia of chronic disease. However, the question remains whether high levels of C-reactive protein, a widely used marker of inflammatory activity, and other inflammatory markers predict less stable anemia control in patients with CKD. The purpose of this study was to establish the relationship between the severity of anemia with the levels of inflammatory markers (C-reactive protein, IL-6) and the dose of epoetin in patients with pre-dialysis stages of CKD. The purpose of our study was to study the age-related characteristics of the nutritional status in patients with CKD of the dialysis stages who are on parenteral nutrition. Method 180 patients (96 men and 84 women) with CKD as a result of nephropathies of diabetic and non-diabetic etiology were examined. Chronic pyelonephritis was diagnosed in 96 patients, chronic glomerulonephritis - in 73, polycystic kidney disease - in 11 patients. Glomerular filtration rate was calculated based on serum creatinine concentration using the CKD-EPI formula (2011). The patients were divided into 2 groups, depending on the achievement of the target hemoglobin value. Thus, group 1 included patients with CKD C3, C4, whose hemoglobin concentration was above 110 g/l (n = 101). Group 2 included patients whose hemoglobin concentration was less than 110 g/l in at least one analysis (n = 79). Results In the examined patients, a significant correlation was found between the range of hemoglobin concentration and the range of CRP concentration (p < 0.05), as well as between the range of hemoglobin concentration and the variation in the percentage of hypochromic erythrocytes (p < 0.05), between the range of hemoglobin concentration and the variation of ferritin ( p < 0.05). The change in the percentage of hypochromic erythrocytes also correlated with the change in IL-6 (p < 0.05) and the change in CRP ( p < 0.05). We found an association between the average weekly dose of epoetin and the levels of CRP, IL-6 and TNF-α. Patients with TNF-α levels ≥2 ng/mL and IL-6 ≥40 ng/mL required a significantly higher dose of epoetin than patients with lower levels of these markers (128 U/kg per week versus 57 U/kg at week). Conclusion The establishment of a relationship between inflammatory markers and hemogram and iron parameters suggests that inflammation had an indirect effect on the increase in hemoglobin through its effect on iron metabolism. The results of the study indicate the presence of diabetic and non-diabetic etiology of systemic inflammation in CKD, more so in the group of patients with type 2 diabetes mellitus.

Study on biosensor and its whole blood detection under the background of integrated traditional Chinese and western medicine

The quantitative detection of biomolecules related to various diseases in blood is the basis of clinical diagnosis, prognosis monitoring, early disease prevention and controllability detection. However, so far, it is difficult to detect whole blood by clinical detection methods. Biosensors can be widely used in detection and analysis in the fields of medicine, environmental protection and industrial and agricultural production. Under the background of integrated traditional Chinese and western medicine, countries all over the world have invested heavily in technical research and product development of biosensors. The starting point of this paper is that it is difficult to realize direct detection of whole blood by fluorescence immunoassay technology at present. Based on the research of biosensors with solid-phase optical fiber and glass slide as biosensors, innovative research has been carried out in new probes and sensing methods of biomarkers. The research shows that combining the advantages of nano-probe with optical fiber evanescent wave biosensor can effectively reduce the influence of spectral scattering, light absorption and autofluorescence background in whole blood on detection signals, improve the accuracy of whole blood detection, realize the rapid detection of hemoglobin by biosensor, and meet the needs of clinical detection of hemoglobin concentration range.

A dynamic spectrum optimized single-trail extraction method for noninvasive analysis of human blood components

Dynamic spectroscopy (DS) is a new noninvasive detection method of human blood components. Theoretically, it can eliminate the individual differences of static tissues such as skin and muscle and the influence of measurement conditions on measurement accuracy, and realize high-precision noninvasive blood component analysis. Obtaining the dynamic spectrum with a high signal-to-noise ratio is the key to modeling and analysis. To further improve the quality of the dynamic spectrum and ensure the robustness of the model, this paper proposes an optimized single-trail extraction method, which is improved for the main single-trail extraction method at present. In this method, the sampling points deviating from the fitting line in the rising edge signal of photoplethysmography (PPG) are eliminated by statistical method many times and using the superposition average of the slope between each sampling point on the rising edge to obtain the high-quality DS. To verify the effectiveness of the method proposed in this paper, we collected the spectral PPG data of the near-infrared transmission of the fingers of 231 volunteers, the wavelength range was 591.8 nm-1100 nm, and the hemoglobin concentration range of the total sample was 90–176 g / L, then used the optimized single-trail extraction method and the single-trail extraction method to extract DS from the spectral PPG data of all samples and divided them into an experimental group and a control group according to the different extraction methods. Two methods of spectral smoothness and modeling analysis were selected to evaluate the effectiveness of the spectrum. The experimental data are as follows: when the DS is evaluated with spectral smoothness as an index, the mean square error of the experimental group's spectrum is reduced from 0.011 to 0.0059, which is 53.6 % of the control group; and when using the least partial squares method to establish a hemoglobin model for prediction, compared with the control group, the related coefficient of the correction set (Rc) in the experimental group reached 0.91, an increase of 7.96 %, and the root mean square error of the correction set (RMSEC) of the calibration set decreased to 5.63g/L, decreased by 22.17 %, the related coefficient of the prediction set (Rp) reached 0.86, increased by 10.90 %, and the root mean square error of the prediction set (RMSEP) decreased to 6.27g/L, decreased by 19.76 %. The experimental results show that the optimized single-trail extraction method proposed in this paper improves the spectral smoothness and modeling effect compared with the original single-trail extraction method effectively improving the quality of DS. This method provides a new way for further improving the accuracy of noninvasive blood component quantitative analysis based on dynamic spectrum and helps to promote the application process of noninvasive detection of blood components.

Highly sensitive fiber optic biosensors with graphene-MoS2 heterostructure for hemoglobin detection

Two-dimensional materials, which can be used to modify sensor surfaces to increase sensor sensitivity, have important research in the field of sensors. In this paper, we design a highly sensitive D-shaped photonic crystal fiber sensor with graphene-MoS2 heterostructure for hemoglobin detection. The research utilized the finite element method and involved addition of different layers of graphene and MoS2 to the optical fiber sensing area, and it was determined that the hybrid nano-heterostructures made of monolayer graphene and bilayer MoS2 provided the greatest improvement in sensor performance. The sensor shows excellent detection performance in 1.33∼1.38 refractive index units. Using incident light in the wavelength ranges of 650 nm and 850 nm, the proposed sensor has a maximum wavelength sensitivity of 4700 nm/RIU, a maximum amplitude sensitivity of 327.5 RIU-1, and a resolution of 2.17×10−5 RIU. The range of hemoglobin concentrations detected with this sensor was 0 g/L∼241 g/L, with an average sensitivity of 0.7 nm/(g/L). A fiber biosensor was enhanced with graphene-MoS2 hybrid nanostructures, which exhibit excellent photoelectric properties and detection performance, enabling highly sensitive, highly accurate, and real-time hemoglobin detection. The result shows the significant research value and application prospects in the field of biomedical detection.

Nonlinear optical and photoacoustic properties of aqueous crystalline hemoglobin. Towards facile detection of hemoglobin concentration in blood

In this work, nonlinear optical properties of hemoglobin are studied under the irradiation of a blue LED laser beam. The z-scan setup is applied in order to determine nonlinear optical responses. Photoacoustic waves are created in a hearable sound region. Sounds are recorded by a microphone and analyzed by using Fourier transform to evaluate the sonic frequencies. The suspensions of hemoglobin in water are prepared in the range of 120–160 mg/mL that is in the range of hemoglobin concentration in blood. It is found that the amplitude of photoacoustic (PA) signals and nonlinear absorption coefficients depend directly to the hemoglobin concentration. On the other hand, blood of rabbit is studied using this method and results are compared with the crystalline hemoglobin findings. It is concluded that the frequency domain of photoacoustic waves created by hemoglobin is similar to that observed for the blood sample. It means that the main portion of photoacoustic response in blood is due to its hemoglobin content and amplitude of the sonic signals is directly related to the concentration of hemoglobin. FT-IR studies confirmed that the vibrational levels of hemoglobin had no a significant effect on the recorded PA signals and the main PA effect has been originated from explosion of the micro- and nano-bubbles. Moreover, from the UV–Vis spectrum of hemoglobin, it can be conferred that absorption of the blue photons by the porphyrin ring leads to the evaporation of water molecules and generation of the mentioned bubbles responsible for the created sonic waves after explosion. This study would be considered as a fast and simple method to determine hemoglobin concentration in the real blood samples using nonlinear optical and photoacoustic characterizations.

Optimization of surface plasmon resonance-based biosensors for monitoring hemoglobin levels in human blood

Biological sensors based on surface plasmon resonance (SPR) are used in a wide range of fields such as pharmacy, medicine, food and drink safety to measure biological and chemical parameters. These sensors function by detecting changes in the refractive index of the sample environment. In this paper a biosensor design based on surface Plasmon resonance is introduced for the purpose of monitoring hemoglobin concentrations in human blood. Our design is based on a Kretschmann structure which contains an SF10 prism, with metal, graphene and metal dichalcogenide layers. The optical source considered here is a He–Ne laser with the wavelength of 632.8 nm. The effect of adding graphene and metal dichalcogenide layers to the structure of these sensors has been investigated. Our investigation of this structure shows that depending on the specific structure of metal dichalcogenide, the addition of this layer increases the sensitivity of the sensor. Using this optimized structure, our main goal of a blood health monitoring device can be achieved. We use this structure to determine the reflection coefficients that correspond with the healthy range of hemoglobin concentration in human blood based on the refractive index of human blood. This sensor is first optimized for sensitivity using water as a sample, this optimized sensor is then used to investigate the range of human blood in a healthy state of blood iron for men and women between the ages of 20 until 80 years. Results show that the reflection coefficients of the optimized structure for healthy range of hemoglobin concentrations for an adult man is from 0.6805(a.u) to 0.7067(a.u) and for an adult female is from 0.6547(a.u) to 0.6583(a.u).

Interference of hemolysis, hyperlipidemia, and icterus on plasma infrared spectral profile.

Recently, pre-analytical, analytical, and post-analytical issues have been addressed to implement biofluid FTIR spectroscopy as a novel diagnostic tool in the clinical setting. Although hemolysis, icterus, and hyperlipidemia are known to interfere with colorimetric and turbidimetric biochemical methods, there are no data on their impact on serum/plasma FTIR spectra. This study aimed at investigating the impact of hemoglobin, bilirubin, and triglycerides concentrations on plasma spectral analysis. Plasma samples with high concentrations of hemoglobin, conjugated bilirubin, or triglycerides were studied. To mimic the various concentrations observed in clinical setting, samples were diluted using normal plasma and analyzed using high-throughput FTIR spectroscopy. Hemolytic, icteric, and hyperlipidemic plasma spectra were compared with control plasma spectra. Unsupervised analysis of all spectra was performed using principal component analysis. The comparison between control and hemolytic plasmas did not show spectral differences in the range of hemoglobin concentrations observed in spurious or pathological hemolysis. By contrast, spectra from lipidemic plasmas had different spectral profiles compared with control plasma, exhibiting increased absorbance in lipid bands. Differences in the same spectral regions were observed in spectra from icteric plasma, which may be explained by the hyperlipidemia associated with cholestasis. PCA did not discriminate between control and hemolytic plasmas up to 1g/L hemoglobin but confirmed the interference of bilirubin and triglycerides concentrations on spectral classification. Our results show that hemolysis does not have an impact on the plasma spectral profile except for high concentrations of hemoglobin rarely observed in clinical practice, whereas icterus and hyperlipidemia constitute significant confounding factors. Graphical abstract.

An Accurate and Rapid Color-Based Point-of-Care Assay for the Detection of Severe Anemia in Low Resource Settings

▪Background: Severe anemia is a leading cause of morbidity and mortality among children in low-resource countries, particulalrly in sub-Saharan Africa, where malaria is endemic and sickle cell disease is prevalent. In many low-resource regions, particularly more remote areas, laboratory diagnostics are not always readily available. The utility of available equipment can be limited by lack of technical expertise, maintenance, and trained personnel, as well as lack of affordable reagents and reliable power. In a setting where severe, life-threatening anemia is common, it is critical for providers to have access to a rapid and accurate diagnostic tool to determine which patients need acute evaluation and treatment. A simple, rapid, accurate, and disposable point-of-care assay (AnemoCheck¨) has recently been tested and published(Tyburski et al. JCI 2014, in press), Hemoglobin concentration is measured by assessing the color of a chemical solution containing hydrogen peroxide and 3,3′,5,5′-tetramethylbenzidine, after mixing with 10μL blood. The AnemoCheck assay is self-contained and does not require electricity, complicated sensors, or additional equipment. The color scale of the original assay correlated well with mild anemia (Hb 9-13 g/dL) but was not designed to discriminate lower hemoglobin concentrations. Accordingly, the test was modified to allow the color scale to detect more severe anemia (Hb 2.5-9.1 g/dL), but needs to be tested in a real-world setting where severe anemia is prevalent.Methods: The primary objective of this study was to determine whether AnemoCheck tests could measure hemoglobin concentrations at least as accurately as currently used standard laboratory techniques in low resource settings where severe anemia is common. The study was performed in the sickle cell clinic at Hospital Pediatrico David Bernardino, a large, public pediatric hospital in Luanda, Angola. After receiving informed consent from a parent or guardian, capillary blood was collected by fingerstick as per routine to measure hemoglobin using a BioSystems BTS-350 Hemoglobin Analyzer. A small sample of capillary blood was also collected for the AnemoCheck assay using a 10μL end-to-end capillary tube via capillary action (Sanguis Counting, Germany). Venous blood was also collected to measure hemoglobin using a calibrated hematology analyzer (Sysmex XT-2000i), which was considered the true hemoglobin concentration for comparison purposes. Hemoglobin was determined first by AnemoCheck by placing the 10μL capillary into a 2mL screw cap polypropylene tube containing the chemical reagents. The tube was then vigorously shaken and after 60 seconds, was compared to a standardized color scale and the hemoglobin concentration was determined. The AnemoCheck results were obtained and recorded before any additional machine-determined results were available, to avoid potential bias.Results: For this pilot study, samples were collected from 40 children for hemoglobin determination by all three methods. The range of hemoglobin concentrations, based on results from the Sysmex hematology analyzer, was 4.8 – 9.2 g/dL (median 7.0). As illustrated in the Figure, the hemoglobin values obtained from the AnemoCheck assay correlated well with the Sysmex hematology analyzer results, r=0.74, p<0.0001. The AnemoCheck results were more accurate than the hemoglobin values obtained by the BioSystems Hemoglobin Analyzer (r=0.47, p=0.020), which is the primary mode of hemoglobin determination in the clinic. On average, the hemoglobin obtained by AnemoCheck was within 0.5 g/dL of the Sysmex value (range 0-1.9 g/dL), compared to the Biosystems value (absolute mean difference=0.7 g/dL, range 0-2.2 g/dL). [Display omitted] Conclusions: Laboratory diagnosis of anemia is expensive and difficult in low resource settings such as Angola, where severe anemia is common and life-threatening. Our pilot data demonstrate that a novel, point-of-care, color-based assay that does not require electricity or expensive reagents is able to accurately estimate low hemoglobin concentrations. Further refinements of the AnemoCheck assay will include photographic color assessment and automated hemoglobin estimation, which will be helpful in resource-poor settings. This test has the potential to become extremely useful diagnostic tool in low resource hospitals and health centers, where sophisticated equipment and reagents may not be available. DisclosuresNo relevant conflicts of interest to declare.

Resolved

Resolved

Relation of Hemoglobin to Level of Cognitive Function in Older Persons

Background: While decreased hemoglobin concentration is common in the elderly, the relationship of the entire range of hemoglobin concentrations with cognitive function is not well understood. Methods: Cross-sectional analyses were conducted utilizing data from community-dwelling, older persons participating in the Rush Memory and Aging Project. Proximate to first available hemoglobin measurement, 21 cognitive tests were administered to measure global cognitive function along with semantic memory, episodic memory, working memory, perceptual speed and visuospatial abilities. Results: For 793 participants without clinical dementia, stroke or Parkinson’s disease, the mean age was 81.0 years (SD = 7.2); 595 (75%) were women, and 94% were white. The mean hemoglobin concentration was 13.3 g/dl (SD = 1.3). 17% of the cohort had anemia. Using linear regression models adjusted for age, education, gender, body mass index, mean corpuscular volume and glomerular filtration rate, both low and high hemoglobin levels were associated with lower global cognitive function (parameter estimate = –0.015, SE = 0.007, p = 0.019). Low and high hemoglobin levels were associated with worse performance on semantic memory (parameter estimate = –0.201, SE = 0.008, p = 0.010) and perceptual speed (parameter estimate = –0.030, SE = 0.010, p = 0.004), but not the other specific cognitive functions. Conclusions: Low and high hemoglobin concentrations in older persons are associated with a lower level of cognitive function in old age, particularly in semantic memory and perceptual speed.

Commentary on Point-Counterpoint

This letter is in response to the Point:Counterpoint series “Positive effects of intermittent hypoxia (live high:train low) on exercise performance are/are not mediated primarily by augmented red cell volume that appeared in the November issue (vol. 99: 2053–2058, 2005; doi: 10.1152/japplphysiol

The HemoCue® for point‐of‐care hemoglobin measurement and packed cell volume estimation in cats

AbstractObjective: To determine conversion formulas so that values from the HemoCue® hemoglobinometer (HbHQ) could be equated to hemoglobin concentrations measured by the gold‐standard cyanomethemoglobin (HbCY) method and used for estimation of packed cell volume (PCV) in cats.Study design: Prospective, in vitro.Animals: Twelve healthy, adult, client‐owned cats.Interventions: The PCV of 12 parent blood samples was manipulated between ∼3 and ∼80% by removing or adding autologous plasma. Hemoglobin was measured by the HbCY method at a university clinical pathology laboratory and by the azidemethemoglobin method in a HbHQ. PCV was determined by micro‐centrifugation.Measurements and main results: Hemoglobin varied from 1–26 g/dL. Repeated‐measures regression of HbCY on HbHQ demonstrated that the Y‐intercept was not different from zero. When the regression was repeated, forcing the line through the origin the coefficient for converting HbHQ to HbCY was not significantly different from 1.0 (adjusted R2=99.7%). The conversion formula was HbCY=HbHQ. Using similar methods, the conversion formula for estimating PCV was PCV=3.1 HbHQ (adjusted R2=99.8%).Conclusion: Hemoglobin concentration measured by the HemoCue® was indistinguishable from the (HbCY) method in feline blood over a wide range of hemoglobin concentrations. This study demonstrates that in cats PCV=3.1HbHQ.Clinical relevance: The HemoCue® is useful for point‐of‐care hemoglobinometry and for estimating PCV in cats.

Determination of glucose in whole blood samples by mid-infrared spectroscopy.

We have determined the glucose concentration of whole blood from mid-infrared spectra without sample preparation or use of chemical reagents. We selected 1119-1022 cm(-1) as the optimal wavelength range for our measurement by making a first-loading vector analysis based on partial least-squares regression. We examined the influence of hemoglobin on samples by using different calibration and prediction sets. The accuracy of glucose prediction depended on the hemoglobin level in the calibration model; the sample set should represent the entire range of hemoglobin concentration. We obtained an accuracy of 5.9% in glucose prediction, and this value is well within a clinically acceptable range.

Determination of traces of hemoglobin by square wave stripping voltammetry at a silver microelectrode

An electrochemical method was developed for the determination of traces of hemoglobin (Hb) by adsorption square wave voltammetric stripping at a bare silver microelectrode. Under optimum conditions the proposed method provided a linear response over the hemoglobin concentration range 5 to 100 nmol/L. The detection limit was 3 nmol/L. The relative standard deviation was 4.5% for 6 successive determinations at 50 nmol/L Hb. Other chemicals present in the sample did not interfere.

The influence of time of storage, temperature of storage, platelet number in platelet-rich plasma, packed cell, mean platelet volume, hemoglobin concentration, age, and sex on platelet aggregation test

The influences of time of storage of platelet-rich plasma (PRP), temperature of storage of PRP, platelet number in PRP, mean platelet volume in whole blood, sex, age, hemoglobin concentration, and different forms of PRP storage on platelet aggregation (PAG) tests, performed with epinephrine, collagen, arachidonate, and ristocetin by a four-channel aggregation profiler (Platelet Aggregation Profiler, Model PAP-4, Bio/Data Corporation, Hatboro, PA 19040, U.S.A.), were evaluated in four groups of subjects (52 men, 22 women, age range 20-85 years, hemoglobin concentration range 8.4-16.8 g/dl). The PRP was stored with or without packed cells, at room temperature or at 4 degrees C, for 0-6 h. The ideal platelet number of PRP for performing the PAG test fell between 150 and 500 x 10(9)/l. If the number was less than 150 x 10(9)/l, the result of PAG should be meaningless. No significant change was noted for up to 6 h when the PRP was stored either at room temperature or at 4 degrees C. Hemoglobin concentration and mean platelet volume did not affect the PAG. However, there was significant but weak correlation (p = 0.0125, r = 0.3696) between age and PAG when using arachidonic acid as the agonist. Men had significantly increased PAG when collagen and ristocetin were used as the agonists. The PRP was stored best at room temperature, without packed cells. In conclusion, to obtain the best result from a PAG test, the PRP should be kept without packed cells at room temperature for no longer than 6 h, and the platelet number should fall between 150 and 500 x 10(9)/l.

Pulse Oximetry Is Accurate in Acute Anemia From Hemorrhage

Hypothesis: Pulse oximetry is an accurate, noninvasive assay of oxygen saturation percentage (SaO 2) in acutely severe anemia (less than 5 g/dL). Design: A paired comparison of SaO 2 by pulse oximetry (SpO 2) and arterial blood gas analysis (SaO 2) in patients with either acute gastrointestinal bleeding or blunt trauma with hematocrit of less than 20%. Setting: An academic emergency department in a tertiary referral hospital with a Level I trauma center. Participants: Seventeen nonhypoxic patients were identified with initial hematocrit of less than 20% over a 9-month period. Measurements and main results: Determination of SaO 2 by pulse oximetry and arterial blood analysis was not statistically significantly different ( P <.05) over a range of hemoglobin levels from 2.3 to 8.7 g/dL. The observed mean difference between SpO 2 and SaO 2 was 0.53±0.23% (mean±SEM; n=17). The 95% confidence interval associated with this difference score ranged from 0.044 to 1.014. Conclusion: Despite reports that pulse oximetry is inaccurate below a hemoglobin concentration of 5 g/dL, our study suggested that this technology is accurate and reliable at 2.3 g/dL for nonhypoxic SaO 2 values (SaO 2 of more than 93%). The anemic endpoint where pulse oximetry either becomes inaccurate or simply fails to work has not been determined. Pulse oximetry overestimates SaO 2 on average by 0.53% over a range of hemoglobin concentrations from 2.3 to 8.7 g/dL. [Jay GD, Hughes L, Renzi FP: Pulse oximetry is accurate in acute anemia from hemorrhage. Ann Emerg Med July 1994;24:32-35.]

Determination of Rebreathing Oxygen Consumption and Derivation of Oxygen Delivery in Intubated Mongrel Dogs after Phlebotomy and Isoproterenol Infusion

This study attempted to determine the correlation between oxygen consumption (VO2), pulmonary-capillary blood flow (QEPC), and oxygen delivery (DO2) by rebreathing and invasive techniques obtained over a range of hemoglobin concentration and cardiac output. Twenty mongrel dogs were instrumented with central arterial and venous catheters to determine DO2 by thermodilution cardiac output and standard formulas. The animals were administered isoproterenol in doses that increased DO2 and subsequently were serially phlebotomized by 30%, 40%, and 50% to decrease DO2. All animals were studied using a rebreathing technique to determine noninvasively VO2, QEPC, and DO2. Sixteen dogs completed the experimental protocol. A correlation analysis was carried out for VO2, QEPC, and DO2 obtained by the rebreathing and invasive methods. Thermodilution cardiac output increased from 3.91 +/- 1.77 L/min at baseline to 8.19 +/- 2.50 L/min during isoproterenol infusion. Hemoglobin varied from 12.21 +/- 1.26 gm% at baseline to 5.21 +/- 1.36 g% at 50% phlebotomy. Over this range of conditions, significant correlations were obtained between rebreathing VO2 and invasive VO2 (r = 0.80, p < 0.0001), rebreathing QEPC and invasive QEPC (r = 0.79, p < 0.0001), and rebreathing DO2 and invasive DO2 (r = 0.82 p < 0.0001). These data demonstrate that the rebreathing technique can be used to monitor oxygen metabolism over wide ranges of DO2.

Linkage between cooperative oxygenation and subunit assembly of cobaltous human hemoglobin

The thermodynamic linkage between cooperative oxygenation and dimer-tetramer subunit assembly has been determined for cobaltous human hemoglobin in which iron(II) protoporphyrin IX is replaced by cobalt(II) protoporphyrin IX. The equilibrium parameters of the linkage system were determined by global nonlinear least-squares regression of oxygenation isotherms measured over a range of hemoglobin concentrations together with the deoxygenated dimer-tetramer assembly free energy determined independently from forward and reverse reaction rates. The total cooperative free energy of tetrameric cobalt hemoglobin (over all four binding steps) is found to be 1.84 (+/- 0.13) kcal, compared with the native ferrous hemoglobin value of 6.30 (+/- 0.14) kcal. Detailed investigation of stepwise cooperativity effects shows the following: (1) The largest change occurs at the first ligation step and is determined on model-independent grounds by knowledge of the intermediate subunit assembly free energies. (2) Cooperativity in the shape of the tetrameric isotherm occurs mainly during the middle two steps and is concomitant with the release of quaternary constraints. (3) Although evaluation of the pure tetrameric isotherm portrays identical binding affinity between the last two steps, this apparent noncooperativity is the result of a "hidden" oxygen affinity enhancement at the last step of 0.48 (+/- 0.12) kcal. This quaternary enhancement energy is revealed by the difference in subunit assembly free energies of the triply and fully ligated species and is manifested visually by the oxygenation isotherms at high versus low hemoglobin concentration. (4) Cobaltous hemoglobin dimers exhibit apparent anticooperativity of 0.49 (+/- 0.16) kcal (presumed to arise from heterogeneity of subunit affinities).(ABSTRACT TRUNCATED AT 250 WORDS)

Binding of diphosphoglycerate and ATP to oxyhemoglobin dimers

The relative affinity of diphosphoglycerate and ATP for hemoglobin dimers and tetramers can be measured under conditions where the protein is in large molar excess over the polyphosphate. Binding of both compounds to dimers was about 25 times stronger than to tetramers in the case of the three low-spin hemoglobins, oxyhemoglobin, carboxyhemoglobin and cyanomethemoglobin. The mutation in hemoglobin Kansas leads to an increased dissociation into αβ dimers. The increase in diphosphoglycerate binding by this hemoglobin was in good agreement with that expected from the dimer-tetramer dissociation constant over a wide range of hemoglobin concentrations. In contrast to the liganded hemoglobins, both deoxyhemoglobin and aquomethemoglobin bind the two polyanions as tetramers.

A pteroylpolyglutamate binds to tetramers in deoxyhemoglobin but to dimers in oxyhemoglobin.

The binding of a physiological concentration of pteroylhepta(glutamate) to oxy- and deoxyhemoglobin in large excess was measured by ultrafiltration. The variation of free to bound folate with hemoglobin concentration showed that a single molecule of the pteroylpolyglutamate is bound by deoxyhemoglobin tetramers and by alpha beta dimers in oxyhemoglobin. Although the binding sites are different, the affinity constants are the same and very similar to the 2,3-bisphosphoglycerate binding energy. Nevertheless, in view of the small proportion of dimers in oxyhemoglobin much more pteroylhepta(glutamate) is bound by deoxyhemoglobin over a wide range of hemoglobin concentrations. Because even 2% deoxyhemoglobin is enough to bind all of the erythrocyte folate as polyglutamate, the bulk of it will be bound at physiological oxygen pressures. Free folate could only be expected in fully oxygenated erythrocytes. Therefore, the reaction of pteroylpolyglutamates with hemoglobin represents an oxygenation-dependent storage mechanism that can account for the 40-fold excess of the vitamin in the erythrocyte over the amounts in the serum. Because methotrexate is also converted to polyglutamate derivatives in the erythrocyte, this drug is likely to be concentrated and stored there by the same mechanism.