Quantitation of drug content in a low dosage formulation by transmission near infrared spectroscopy

A transmission near infrared (NIR) spectroscopic method has been developed for the nondestructive determination of drug content in tablets with less than 1% weight of active ingredient per weight of formulation (m/m) drug content. Tablets were manufactured with drug concentrations of ∼0.5%, 0.7%, and 1.0% (m/m) and ranging in drug content from 0.71 to 2.51 mg per tablet. Transmission NIR spectra were obtained for 110 tablets that constituted the training set for the calibration model developed with partial least squares regression. The reference method for the calibration model was a validated UV spectrophotometric method. Several data preprocessing methods were used to reduce the effect of scattering on the NIR spectra and base the calibration model on spectral changes related to the drug concentration changes. The final calibration model included the spectral range from 11 216 to 8662 cm⁻¹ the standard normal variate (SNV), and first derivative spectral pretreatments. This model was used to predict an independent set of 48 tablets with a root mean standard error of prediction (RMSEP) of 0.14 mg, and a bias of only −0.05 mg per tablet. The study showed that transmission NIR spectroscopy is a viable alternative for nondestructive testing of low drug content tablets, available for the analysis of large numbers of tablets during process development and as a tool to detect drug agglomeration and evaluate process improvement efforts. Published: March 24, 2006     

On-line multi-analyzer monitoring of biomass, glucose and acetate for growth rate control of a Vibrio cholerae fed-batch cultivation

In situ near-infrared (NIR) spectroscopy and in-line electronic nose (EN) mapping were used to monitor and control a cholera-toxin producing Vibrio cholerae fed-batch cultivation carried out with a laboratory method as well as with a production method. Prediction models for biomass, glucose and acetate using NIR spectroscopy were developed based on spectral identification and partial-least squares (PLS) regression resulting in high correlation to reference data (standard errors of prediction for biomass, glucose and acetate were 0.20 gl(-1), 0.26 gl(-1) and 0.28 gl(-1)). A compensation algorithm for aerated bioreactor disturbances was integrated in the model computation, which in particular improved the prediction by the biomass model. First, the NIR data were applied together with EN in-line data selected by principal component analysis (PCA) for generating a trajectory representation of the fed-batch cultivation. A correlation between the culture progression and EN signals was demonstrated, which proved to be beneficial in monitoring the culture quality. It was shown that a deviation from a normal cultivation behavior could easily be recognized and that the trajectory was able to alarm a bacterial contamination. Second, the NIR data indicated the potential of predicting the concentration of formed cholera toxin with a model prediction error of 0.020 gl(-1). Third, the on-line biomass prediction based on the NIR model was used to control the overflow metabolism acetate formation of the V. cholerae culture. The controller compared actual specific growth rate as estimated from the prediction with the critical acetate formation growth rate, and from that difference adjusted the glucose feed rate.     

Quantitation of cerebral blood volume in human infants by near-infrared spectroscopy

Current methods for measuring cerebral blood volume (CBV) in newborn infants are unsatisfactory. A new method is described in which the effect of a small change (5-10%) in arterial oxygen saturation (SaO2) on cerebral oxyhemoglobin [HbO2] and deoxyhemoglobin [Hb] concentration is observed by near-infrared (NIR) spectroscopy. Previous experiments in which the NIR absorption characteristics of HbO2 and Hb and the pathlength of NIR light through the brain were defined allowed changes in [HbO2] and [Hb] to be quantified from the Beer-Lambert law. It is shown here that CBV can then be derived from the expression CBV = (delta[HbO2] - delta[Hb])/(2. delta SaO2.H.R.), where H is the large vessel total hemoglobin concentration and R to the cerebral-to-large vessel hematocrit ratio. Observations on 12 newborn infants with normal brains, born at 25-40 wk of gestation and aged 10-240 h, gave a mean value for CBV of 2.22 +/- 0.40 (SD) ml/100 g, whereas mean CBV was significantly higher 3.00 +/- 1.04 ml/100 g in 10 infants with brain injury born at 24 to 42 wk of gestation and aged 4-168 h (P less than 0.05).     

Determination of total protein in hyperimmune serum samples by near-infrared spectrometry and multivariate calibration

In the present work, the determination of the total protein concentration in hyperimmune serum samples was performed through a partial least-squares near-infrared (NIR-PLS) method. The method was based on the chemometric treatment of the NIR spectra of samples. The influences of spectra preprocessing and spectral window utilized in the construction of PLS model were studied. Models were built using reference data of 19 samples selected through the use of hierarchical cluster analysis (HCA) of NIR spectra of samples and another 24 samples were employed for external validation of the method. A model with better prediction capacity was obtained after whole spectra preprocessing by multiplicative scattering correction (MSC) algorithm and using data in the spectral range of 2158-2209 nm. Under optimized conditions a RMSEP of 0.21 g dl⁻¹ and a quality coefficient value (QC) of only 5.8% were obtained for the prediction of total protein content in the samples used for external validation. Also, a determination coefficient, r², of 0.97 was obtained in the correlation of predicted and reference data of samples situated in the validation set.     

Hemoglobin plus myoglobin concentrations and near infrared light pathlength in phantom and pig hearts determined by diffuse reflectance spectroscopy

To noninvasively determine absolute concentrations of hemoglobin (Hb) plus myoglobin (Mb) in cardiac tissue by means of regular near infrared (NIR) light diffuse reflectance measurements, a first derivative approach was applied. The method was developed to separately calculate oxygenated and deoxygenated [Hb + Mb] as well as an effective pathlength, which NIR light passes through in the tissue between optodes. Applying a cotton wool-based phantom, which mimics muscle tissue, it was shown that the intensity of the pseudo-optical density first derivative depends linearly on both oxygenated and deoxygenated Hb concentration, thereby validating the Lambert-Beer law in the range of 0 to 0.25 mM tetrameric Hb. A high correlation (R = 0.995) was found between concentrations of Hb loaded onto the phantom and those determined spectrophotometrically, thereby verifying the first derivative method validity. The efficiency of the method was tested using in vivo pig hearts prior to and after ischemia initiated experimentally by left anterior descending artery branches occlusion. The results showed that the total [Hb + Mb] was 0.9-1.2 mM heme, the average tissue oxygen saturation was approximately 70% (which reduced to nearly 0% after occlusion), and the NIR (700-965 nm) light pathlength was 2.3 mm (differential pathlength factor [DPF] = 2.7-2.8) in a living heart tissue.     

A label-free biosensor based on gold nanoshell monolayers for monitoring biomolecular interactions in diluted whole blood

Gold nanoshells (GNSs) were self-assembled on the surface of transparent glasses modified with 3-aminopropyltrimethoxysilane (APTES) to form GNS self-assembled monolayers (SAMs). Because the localized surface plasmon resonance (LSPR) of GNSs can be controlled in the near-infrared (NIR) region of the spectrum, where the optical transmission through tissue and whole blood is optimal, GNSs would be used as an effective signal transduction in whole blood. Accordingly, after modified with cystamine and biotin-NHS (N-hydroxy succinimide), GNS SAMs were used as a novel optical biosensor for real-time detection of streptavidin-biotin interactions in diluted human whole blood within short assay time, without any sample purification/separation. An UV-vis-NIR spectrophotometer was used to monitor the absorbance changes at 730 nm as a function of time for different concentrations of streptavidin in 20% whole blood, and the results showed that the biosensor displayed low detection limit of approximately 3 microg/mL and wide dynamic range of approximately 3-50 microg/mL. This approach provides an opportunity to construct LSPR biosensor for protein sensing and cellular analysis in diluted whole blood.     

Nondestructive near-infrared spectroscopic measurement of multiple analytes in undiluted samples of serum-based cell culture media.

An adaptive calibration procedure is used to build selective multivariate calibration models for the measurement of glucose, lactate, glutamine, and ammonia in undiluted serum-based cell culture media. This adaptive procedure removes metabolism-induced covariance between these analytes in a series of calibration samples collected during the cultivation of PC-3 human prostate cancer cells. Partial least-squares calibration models are generated from single-beam near-infrared (NIR) spectra collected over the 4800- to 4200-cm(-1) combination spectral range. Calibration models were generated with both the full spectral range and optimized spectral ranges. In both cases, the number of model factors was optimized and model validity was determined by comparing analyte concentrations predicted from a series of independent and unaltered samples that were obtained during a subsequent cultivation of the PC-3 cells. Similar analytical performance was achieved with fewer model factors when the optimized spectral range was used. The lowest standard errors of prediction were 0.82, 0.94, 0.55, and 0.76 mM for glucose, lactate, glutamine, and ammonia, respectively. Different spectral ranges were optimal for each analyte and the optimized spectral range coincided with the distinguishing spectral features of the analyte. The results of this study demonstrate that NIR spectroscopy can be used effectively in the off-line measurement of important nutrients (glucose and glutamine) and byproducts (lactate and ammonia) in a serum-based animal cell culture medium.     

基于小波理论的干旱区内陆湖泊叶绿素a的TM影像遥感反演

叶绿素a(Chl-a)是衡量湖泊富营养化的重要指标,利用遥感技术动态监测面积较大的湖区水体中Chl-a浓度对了解湖区水质具有重要意义。以内蒙古乌梁素海为例,提出利用TM影像中的水体实测光谱进行小波去噪和光谱信号重构,并结合水质采样实测数据进行神经网络拟合,建立光谱反射率比值与Chl-a浓度的反演模型的方法。结果显示:小波理论和神经网络相结合的模型可以适用于估算乌梁素海Chl-a浓度,去噪后Chl-a浓度与光谱信号的相关系数(-0.575)较去噪前(-0.417)明显增强,去噪后的采样点光谱信号与Chl-a浓度之间表现出比原始信号更强的负相关性,证明了去噪后的观测值可进一步减弱随机误差的干扰和去除噪声,使观测数据更加逼近Chl-a浓度的真实情况,图像去噪重构结果显示重构后的光谱范围较之前有所缩窄,部分信号点得到了增强,但基本剖面结构并没有产生较大变化,反演模型的平均相对误差为0.142,与其他研究相比差别不大。反演得出的乌梁素海Chl-a浓度分布反映了污染源的分布,同时说明了乌梁素海Chl-a浓度在时空分布上呈现一定的差异,表现为丰水期呈现浅水区Chl-a浓度值高于湖心区,来水区高于其他湖区的分布趋势,枯水期乌梁素海中部呈现由西向东Chl-a浓度逐步降低的分布规律,西部呈均一化分布。反演模型基本可以满足实际预测的需要。但模型在具体应用中在影像数据采集、数据量及算法方面还有很大的改进空间,该方法的提出为干旱区大型内陆水体富营养化的实时定量遥感监测提供了新的解决方案。     

Formation spectra of the EPR split signals from the S0, S1, and S3 states in photosystem II induced by monochromatic light at 5 K

The interaction EPR split signals from photosystem II (PSII) have been reported from the S0, S1, and S3 states. The signals are induced by illumination at cryogenic temperatures and are proposed to reflect the magnetic interaction between YZ* and the Mn4Ca cluster. We have investigated the formation spectra of these split EPR signals induced in PSII enriched membranes at 5 K using monochromatic laser light from 400 to 900 nm. We found that the formation spectra of the split S0, split S1, and split S3 EPR signals were quite similar, but not identical, between 400 and 690 nm, with maximum formation at 550 nm. The major deviations were found between 440 and 480 nm and between 580 and 680 nm. In the regions around 460 and 680 nm the amplitudes of the formation spectra were 25-50% of that at 550 nm. A similar formation spectrum was found for the S2-state multiline EPR signal induced at 0 degrees C. In general, the formation spectra of these signals in the visible region resemble the reciprocal of the absorption spectra of our PSII membranes. This reflects the high chlorophyll concentration necessary for the EPR measurements which mask the spectral properties of other absorbing species. No split signal formation was found by the application of infrared laser illumination between 730 and 900 nm from PSII in the S0 and S1 states. However, when such illumination was applied to PSII membranes poised in the S3 state, formation of the split S3 EPR signal was observed with maximum formation at 740 nm. The quantum yield was much less than in the visible region, but the application of intensive illumination at 830 nm resulted in accumulation of the signal to an amplitude comparable to that obtained with illumination with visible light. The split S3 EPR signal induced by NIR light was much more stable at 5 K (no observable decay within 60 min) than the split S3 signal induced by visible light (50% of the signal decayed within 30 min). The split S3 signals induced by each of these light regimes showed the same EPR spectral features and microwave power saturation properties, indicating that illumination of PSII in the S3 state by visible light or by NIR light produces a similar configuration of YZ* and the Mn4Ca cluster.     

Hemoglobin oxygen saturation measurements using resonance Raman intravital microscopy

A system is described for in vivo noninvasive measurements of hemoglobin oxygen saturation (HbO2Sat) at the microscopic level. The spectroscopic basis for the application is resonant Raman enhancement of Hb in the violet/ultraviolet region, allowing simultaneous identification of oxy- and deoxyhemoglobin with the same excitation wavelength. The heme vibrational bands are well known, but the technique has never been used to determine microvascular HbO2Sat in vivo. A diode laser light (power: 0.3 mW) was focused onto sample areas 15-30 microm in diameter. Raman spectra were obtained in backscattering geometry by using a microscope coupled to a spectrometer and a cooled detector. Calibration was performed in vitro by using glass capillaries containing blood at several Hb concentrations, equilibrated at various oxygen tensions. HbO2Sat was estimated using the Raman band intensities at 1,360 and 1,375 cm(-1). Glass capillary path length and Hb concentration had no effect on HbO2Sat estimated from Raman spectra. In vivo observations were made in blood flowing in microvessels of the rat mesentery. The Hb Raman peaks observed in oxygenated and deoxygenated blood were consistent with earlier Raman studies that used Hb solutions and isolated cells. The method allowed HbO2Sat determinations in the whole range of arterioles, venules, and capillaries. Tissue transillumination allowed diameter and erythrocyte velocity measurements in the same vessels. Raman microspectroscopy offers distinct advantages over other currently used techniques by providing noninvasive and reliable in vivo determinations of HbO2Sat in thin tissues as well as in solid organs and tissues, which are unsuitable for techniques requiring transillumination.     

Miniaturized multichannel near infrared endoscope for mouse imaging

We describe the design and construction of a miniaturized multichannel near infrared (NIR) endoscopic imaging system developed for high-resolution imaging of mice. The device allows for simultaneous real-time video images in white light and two independent NIR channels. Testing demonstrated independent acquisition of nanomolar concentrations of fluorochromes Cy5.5 and Cy7. Cross-talk between the NIR channels, partially a result of broad tails in the spectra of commonly used organic fluorochromes, was assessed, modeled for the linear range of the concentration/signal intensity function, and compensated. The calculated compensation was 5.5% and 22% of the total signal intensity in the two channels NIR700 and NIR780, respectively, at equal concentrations of the two fluorochromes. Using a mouse model of colonic adenomatosis, we show that both perfusion and protease activity can be detected simultaneously, independently, and repeatedly in live mice. The developed device should be useful for in vivo imaging of diverse molecular targets.     

A new modified Edman procedure for analysis of N-terminal valine adducts in hemoglobin by LC–MS/MS

A rapid and sensitive method using liquid chromatography–tandem mass spectrometry (LC–MS/MS) for simultaneous determination of adducts from acrylamide, glycidamide and ethylene oxide to N-terminal valines in hemoglobin (Hb) was developed. This new procedure is based on the same principles as the N-alkyl Edman procedure for analysis of adducts from electrophilic agents to N-terminal valines in Hb. The N-substituted valines can be detached, enriched and measured selectively as thiohydantoins by the use of an Edman reagent, in this case fluorescein isothiocyanate (FITC). This procedure is denoted as the “adduct FIRE procedure” as the FITC reagent is used for measurement of adducts (R) formed from electrophilic compounds with a modified Edman procedure. In this study, fluorescein thiohydantoin (FTH) analytes of N-substituted valines from acrylamide, glycidamide and ethylene oxide, as well as their corresponding hepta- and tri-deuterium-substituted analogues, were synthesized. These analytes (n = 8) were then characterized by LC–MS/MS (ESI, positive ion mode) and obtained product ions were interpreted. A considerable work with optimization of the FIRE procedure™, resulted in a procedure in which low background levels of the studied adducts could be measured from 250 μL lyzed whole blood samples (human non-smokers). The analytes were enriched and purified with solid phase extraction columns and analyzed by LC–MS/MS with LOQ down to 1 pmol adduct/g Hb. Compared to other procedures for determination of N-terminal Hb adducts, the introduction of FITC has led to a simplified procedure, where whole blood also can be used, giving new opportunities and reduced hand on time with increased sample throughput.     

Vibrational communication along plants by the stink bugs Nezara viridula and Murgantia histrionica

The velocity and spectral characteristics of vibrational signals of Nezara viridula (L.) and Murgantia histrionica (Hahn) (Heteroptera: Pentatomidae) were analyzed as the signals were transmitted through different plants. The velocity parameter of the body vibrations ranges from 0.1 to 1 mm/s. According to the mechanical properties of different substrates, the signal is attenuated or amplified during transmission from the insect's body to the substrate. Attenuation of up to 20 dB occurs during transmission of signals from leaves to stalks or stems. The velocity decrease with distance is below 0.5 dB/cm during transmission through less dense green stems, whereas it ranges between 0.6 and 1.6 dB/cm during transmission through more dense, woody stems. Signal velocity decreases non-linearly with increasing distance from the signal source. Regularly repeated velocity minima (nodes) and maxima (internodes) spaced 10-15 cm apart are characteristic of signal transmission through green plants but not woody stems. The signal velocity at some internodes exceeds the input value for N. viridula but not M. histrionica signals. The relative amplitude of the dominant frequency spectral peak varies with distance, along with overall signal velocity. Variable ratios of spectral peak amplitudes are characteristic for signals recorded at different distances from the source.     

Measurement of hemoglobin oxygen saturation using Raman microspectroscopy and 532-nm excitation.

The resonant Raman enhancement of hemoglobin (Hb) in the Q band region allows simultaneous identification of oxy- and deoxy-Hb. The heme vibrational bands are well known at 532 nm, but the technique has never been used to determine microvascular Hb oxygen saturation (So(2)) in vivo. We implemented a system for in vivo noninvasive measurements of So(2). A laser light was focused onto areas of 15-30 microm in diameter. Using a microscope coupled to a spectrometer and a cooled detector, Raman spectra were obtained in backscattering geometry. Calibration was performed in vitro using blood at several Hb concentrations, equilibrated at various oxygen tensions. So(2) was estimated by measuring the intensity of Raman signals (peaks) in the 1,355- to 1,380-cm(-1) range (oxidation state marker band nu(4)), as well as from the nu(19) and nu(10) bands (1,500- to 1,650-cm(-1) range). In vivo observations were made in microvessels of anesthetized rats. Glass capillary path length and Hb concentration did not affect So(2) estimations from Raman spectra. The Hb Raman peaks observed in blood were consistent with earlier Raman studies using Hb solutions and isolated cells. The correlation between Raman-based So(2) estimations and So(2) measured by CO-oximetry was highly significant for nu(4), nu(10), and nu(19) bands. The method allowed So(2) determinations in all microvessel types, while diameter and erythrocyte velocity could be measured in the same vessels. Raman microspectroscopy has advantages over other techniques by providing noninvasive and reliable in vivo So(2) determinations in thin tissues, as well as in solid organs and tissues in which transillumination is not possible.     

Monitoring for occupational exposure to 4,4′-methylenebis(2-chloroaniline) by gas chromatographic-mass spectrometric analysis of haemoglobin adducts, blood, plasma and urine

The feasibility of using plasma, blood and haemoglobin adducts for monitoring occupational exposure to the suspected human carcinogen 4,4′-methylenebis(2-chloroaniline) (MOCA) was investigated. A method utilising capillary gas chromatography-negative-ion chemical-ionisation mass spectrometry (GC-MS) for the determination of pentafluoropropionyl (PFP) derivatives of MOCA, released by alkaline hydrolysis from protein adducts and conjugates, was both sensitive and selective. When selected ion monitoring was used, sub-femtomole amounts of PFP-MOCA could be measured. The detection limit for haemoglobin adducts of MOCA was below 10 fmol/g Hb, well below the levels found for occupationally exposed individuals. Capillary GC with electron-capture detection also had the required sensitivity for the determination of MOCA in blood and urine of five individuals who were exposed to MOCA during the manufacture of polyurethane elastomers were determined by the GC-MS method. The MOCA concentrations for the various blood fractions and urine were within the following ranges: haemoglobin adducts, 0.73–43.3 pmol MOCA/g Hb; plasma alkaline hydrolysate, 0.05–22.0 nmol/l; whole blood, 0.13–17.4nmol/l; urine, 4.5–2390 nmol/l. Because the products of MOCA in the blood reflect metabolic activation of MOCA and integrate exposure over a period of weeks, the use of blood samples for monitoring exposure to MOCA offers advantages over the currently used urinary MOCA measurements.     

New ultra-micro high-performance liquid chromatographic method for determining the γ chain composition of hemoglobin F in normal adults

The difficulty in isolating the minute quantity of Hb F (<1%) present in the red blood cells of normal adults greatly complicates the determination of its γ chain composition. We have developed a rapid ultra-micro high-performance liquid chromatographic (HPLC) method and used it to analyze the γ chain composition of Hb F in 47 adults with Hb F levels between 0.1–3.4%. The method involves the isolation of Hb F from as little as 50 μl of whole blood on an analytical size cation-exchange HPLC column, followed by concentration in a Centricon micro concentrator unit and by reversed-phase HPLC analysis. The entire procedure can be completed in one day and 3–4 analyses can be made simultaneously. We reanalyzed the blood samples from 22 subjects with known β-globin gene cluster haplotypes, and confirmed the association of high, low, and very low ^Gγ levels with haplotypes A, B, and C, respectively. Also included are the results of DNA sequence analyses of the ^Gγ and β promoters, and of the locus-control-region hypersensitive site-2 (LCR-HS-2) of the β-globin gene cluster in five subjects homozygous for haplotypes A, B or C; the data obtained failed to provide a satisfactory explanation for all the variations in the ^Gγ levels that have been observed.     

Purification of Concentrated Hemoglobin Using Organic Solvent and Heat Treatment

A simple method for obtaining a purified and concentrated hemoglobin (Hb) solution (25 g/100 ml) from human red blood cells has been established. To prevent MetHb formation during the purification procedure, Hb in red blood cells was carbonylated in advance, and then washed red blood cells were mixed with organic solvents such as diethyl ether or dichloromethane for hemolysis and removal of stroma. The Hb solution was isolated by centrifugation (1 900g) with the high removal efficiency of phospholipid (>99.8%). After the solution was heated (60°C, 1 h), the precipitates were removed by centrifugation. The purity of Hb was confirmed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Isoelectric focusing and oxygen-binding properties of the obtained Hb solution demonstrated its purity and showed no denaturation of globin. This purification procedure is applicable to large-scale production of the purified Hb.     

Skin blood flow influences near-infrared spectroscopy-derived measurements of tissue oxygenation during heat stress.

Near-infrared (NIR) spectroscopy is a noninvasive optical technique that is increasingly used to assess muscle oxygenation during exercise with the assumption that the contribution of skin blood flow to the NIR signal is minor or nonexistent. We tested this assumption in humans by monitoring forearm tissue oxygenation during selective cutaneous vasodilation induced by locally applied heat (n = 6) or indirect whole body heating (i.e., heating subject but not area surrounding NIR probes; n = 8). Neither perturbation has been shown to cause a measurable change in muscle blood flow or metabolism. Local heating (approximately 41 degrees C) caused large increases in the NIR-derived tissue oxygenation signal [before heating = 0.82 +/- 0.89 optical density (OD), after heating = 18.21 +/- 2.44 OD; P < 0.001]. Similarly, whole body heating (increase internal temperature 0.9 degrees C) also caused large increases in the tissue oxygenation signal (before heating = -0.31 +/- 1.47 OD, after heating = 12.48 +/- 1.82 OD; P < 0.001). These increases in the tissue oxygenation signal were closely correlated with increases in skin blood flow during both local heating (mean r = 0.95 +/- 0.02) and whole body heating (mean r = 0.89 +/- 0.04). These data suggest that the contribution of skin blood flow to NIR measurements of tissue oxygenation can be significant, potentially confounding interpretation of the NIR-derived signal during conditions where both skin and muscle blood flows are elevated concomitantly (e.g., high-intensity and/or prolonged exercise).     

Tetramer-dimer equilibrium of oxyhemoglobin mutants determined from auto-oxidation rates.

One of the main difficulties with blood substitutes based on hemoglobin (Hb) solutions is the auto-oxidation of the hemes, a problem aggravated by the dimerization of Hb tetramers. We have employed a method to study the oxyHb tetramer-dimer equilibrium based on the rate of auto-oxidation as a function of protein concentration. The 16-fold difference in dimer and tetramer auto-oxidation rates (in 20 mM phosphate buffer at pH 7.0, 37 degrees C) was exploited to determine the fraction dimer. The results show a transition of the auto-oxidation rate from low to high protein concentrations, allowing the determination of the tetramer-dimer dissociation coefficient K4,2 = [Dimer] 2/[Tetramer]. A 14-fold increase in K4,2 was observed for addition of 10 mM of the allosteric effector inositol hexaphosphate (IHP). Recombinant hemoglobins (rHb) were genetically engineered to obtain Hb with a lower oxygen affinity than native Hb (Hb A). The rHb alpha2beta2 [(C7) F41Y/(G4) N102Y] shows a fivefold increase in K4,2 at pH 7.0, 37 degrees C. An atmosphere of pure oxygen is necessary in this case to insure fully oxygenated Hb. When this condition is satisfied, this method provides an efficient technique to characterize both the tetramer-dimer equilibrium and the auto-oxidation rates of various oxyHb. For low oxygen affinity Hb equilibrated under air, the presence of deoxy subunits accelerates the auto-oxidation. Although a full analysis is complicated, the auto-oxidation studies for air equilibrated samples are more relevant to the development of a blood substitute based on Hb solutions. The double mutants, rHb alpha2beta2 [(C7) F41Y/(G4) N102A] and rHb alpha2beta2 [(C7) F41Y/(E10) K66T], show a lower oxygen affinity and a higher rate of oxidation than Hb A. Simulations of the auto-oxidation rate versus Hb concentration indicate that very high protein concentrations are required to observe the tetramer auto-oxidation rate. Because the dimers oxidize much more rapidly, even a small fraction dimer will influence the observed oxidation rate.     

Glycosylated haemoglobin concentrations in newly diagnosed diabetics before and during treatment.

Concentrations of total glycosylated haemoglobins (Hb A1) were measured in 40 diabetics at diagnosis and at monthly intervals after treatment with chlorpropamide, insulin, or diet alone was begun. The mean Hb A1 concentration at presentation in 16 patients treated with chlorpropamide was significantly higher than that in 12 patients treated with insulin, and the duration of glycaemic symptoms was much longer in the chlorpropamide-treated group. In contrast, the mean plasma glucose concentration was similar in both groups. The mean concentrations of Hb A1 and plasma glucose at diagnosis in the 12 patients treated by diet alone were lower than those in the other two groups, and most of these patients were free of symptoms. Treatment quickly relieved symptoms and lowered plasma glucose in all patients. The Hb A1 concentration fell significantly with treatment such that after two months there was no significant difference between the three groups, although results remained above the normal range. These findings support the theory that the Hb A1 concentration reflects the blood glucose control over the previous one to two months and suggest that the duration of hyperglycaemia may be important in determining the Hb A1 concentration as well as the absolute blood glucose concentration.