STUDY OF THE ELECTRODE PROCESS OF HEMOGLOBIN AT A POLYMERIZED AZURE A FILM ELECTRODE

The electrochemically polymerized azure A film electrode is reported. The resulting film on a platinum electrode surface was analyzed with electron spectroscopy for chemical analysis (ESCA). The heterogeneous electron transfer processes of hemoglobin at the polymerized azure A film electrode have been investigated using in situ UV-visible spectroelectrochemistry. The formal potential (E-degrees') and electron transfer number (n) of hemoglobin were calculated as E = 0.088 V versus NHE (standard deviation +/- 0.5, N = 4) and n = 1.8 (standard deviation +/- 0.5, N = 4). Exhaustive reduction and oxidation electrolysis are achieved in 80 and 380 seconds, respectively, during a potential step between -0.3 and +0.3 V. A formal heterogeneous electron-transfer rate constant (k(sh)) of 3.54(+/- 0.12) X 10(-6) cm/s and a transfer coefficient (alpha) of 0.28(+/- 0.01) were obtained by cyclic voltabsorptometry, which indicated that the poly-azure A film electrode is able to catalyze the direct reduction and oxidation of hemoglobin.

CATALYTIC REDUCTION OF HEMOGLOBIN AT THIONINE CHEMICALLY MODIFIED ELECTRODE AND FIA APPLICATIONS

Thionine-containing chemically modified electrode (cme) was constructed with glassy carbon substrate by potential sweep oxidation, electrodeposition and adsorption procedures, and electrocatalytic reduction of hemoglobin was carried out and characterized at the cme under batch and flow conditions. Comparison of the catalytic response toward hemoglobir obtained at the cme was made mainly in terms of the potential dependence, the detectability and long-term stability. When used in flow injection analysis (FIA) experiments with the detector monitored at a constant potential applied at -0.35 V vs sce, detection limit of 0.15-1.5 pmol level of hemoglobin injected was achieved at the cme, with linear response range over 2 orders of magnitude. All the cme s retained more than 70% of their initial hemoglobin response level over 8 h of continuous service in the flow-through system.

FLOW-INJECTION ANALYSIS OF MYOGLOBIN AND HEMOGLOBIN AT TOLUIDINE BLUE CHEMICALLY MODIFIED ELECTRODE

Electrodeposition of the phenothiazine mediator titrant toluidine blue onto a glassy carbon substrate at an appropriate potential was used to construct a toluidine blue chemically modified electrode (CME) exhibiting electrocatalytic reduction for myoglobin and hemoglobin. The CME catalyzed the hemoprotein electroreduction at the reduction potential of the mediator molecule. When the CME as used as a detector for flow injection analysis at a constant applied potential of -0.30 V vs. a saturated calomel electrode, it gave detection limits of 20 and 50 ng (1.2 and 0.78 pmol) injected myoglobin and hemoglobin, respectively, with a dynamic linear concentration range over 2 orders of magnitude. After a brief equilibration period, the CME retained nearly 90% of its initial myoglobin response over 8 hours of continuous exposure to the flow-through system.

Allylation of intraerythrocytic hemoglobin by raw garlic extracts.

Recent studies have shown that deoxygenated human red blood cells (RBCs) converted garlic-derived polysulfides into hydrogen sulfide, which in turn produced vasorelaxation in aortic ring preparations. The vasoactivity was proposed to occur via glucose- and thiol-dependent acellular reactions. In the present study, we investigated the interaction of garlic extracts with human deoxygenated RBCs and its effect on intracellular hemoglobin molecules. The results showed that garlic extract covalently modified intraerythrocytic deoxygenated hemoglobin. The modification identified consisted of an addition of 71 atomic mass units, suggesting allylation of the cysteine residues. Consistently, purified human deoxyhemoglobin reacted with chemically pure diallyl disulfide, showing the same modification as garlic extracts. Tandem mass spectrometry analysis demonstrated that garlic extract and diallyl disulfide modified hemoglobin's beta-chain at cysteine-93 (beta-93C) or cysteine-112 (beta-112C). These results indicate that garlic-derived organic disulfides as well as pure diallyl disulfide must permeate the RBC membrane and modified deoxyhemoglobin at beta-93C or beta-112C. Although the physiological role of the reported garlic extract-induced allyl modification on human hemoglobin warrants further study, the results indicate that constituents of natural products, such as those from garlic extract, modify intracellular proteins.

Noninvasive monitoring of tissue hemoglobin using UV-VIS diffuse reflectance spectroscopy: a pilot study.

We conducted a pilot study on 10 patients undergoing general surgery to test the feasibility of diffuse reflectance spectroscopy in the visible wavelength range as a noninvasive monitoring tool for blood loss during surgery. Ratios of raw diffuse reflectance at wavelength pairs were tested as a first-pass for estimating hemoglobin concentration. Ratios can be calculated easily and rapidly with limited post-processing, and so this can be considered a near real-time monitoring device. We found the best hemoglobin correlations were when ratios at isosbestic points of oxy- and deoxyhemoglobin were used, specifically 529/500 nm. Baseline subtraction improved correlations, specifically at 520/509 nm. These results demonstrate proof-of-concept for the ability of this noninvasive device to monitor hemoglobin concentration changes due to surgical blood loss. The 529/500 nm ratio also appears to account for variations in probe pressure, as determined from measurements on two volunteers.

Rapid ratiometric determination of hemoglobin concentration using UV-VIS diffuse reflectance at isosbestic wavelengths.

We developed a ratiometric method capable of estimating total hemoglobin concentration from optically measured diffuse reflectance spectra. The three isosbestic wavelength ratio pairs that best correlated to total hemoglobin concentration independent of saturation and scattering were 545/390, 452/390, and 529/390 nm. These wavelength pairs were selected using forward Monte Carlo simulations which were used to extract hemoglobin concentration from experimental phantom measurements. Linear regression coefficients from the simulated data were directly applied to the phantom data, by calibrating for instrument throughput using a single phantom. Phantoms with variable scattering and hemoglobin saturation were tested with two different instruments, and the average percent errors between the expected and ratiometrically-extracted hemoglobin concentration were as low as 6.3%. A correlation of r = 0.88 between hemoglobin concentration extracted using the 529/390 nm isosbestic ratio and a scalable inverse Monte Carlo model was achieved for in vivo dysplastic cervical measurements (hemoglobin concentrations have been shown to be diagnostic for the detection of cervical pre-cancer by our group). These results indicate that use of such a simple ratiometric method has the potential to be used in clinical applications where tissue hemoglobin concentrations need to be rapidly quantified in vivo.

Demonstration of increased concentrations of circulating glycated insulin in human Type 2 diabetes using a novel and specific radioimmunoassay.

Aims/hypothesis: Glycation of insulin, resulting in impaired bioactivity, has been shown within pancreatic beta cells. We have used a novel and specific radioimmunoassay to detect glycated insulin in plasma of Type 2 diabetic subjects.

Methods: Blood samples were collected from 102 Type 2 diabetic patients in three main categories: those with good glycaemic control with a HbA1c less than 7%, moderate glycaemic control (HbA1c 7–9%) and poor glycaemic control (HBA1c greater than 9%). We used 75 age- and sex-matched non-diabetic subjects as controls. Samples were analysed for HbA1c, glucose and plasma concentrations of glycated insulin and insulin.

Results: Glycated insulin was readily detected in control and Type 2 diabetic subjects. The mean circulating concentration of glycated insulin in control subjects was 12.6±0.9 pmol/l (n=75). Glycated insulin in the good, moderate and poorly controlled diabetic groups was increased 2.4-fold (p<0.001, n=44), 2.2- fold (p<0.001, n=41) and 1.1-fold (n=17) corresponding to 29.8±5.4, 27.3±5.7 and 13.5±2.9 pmol/l, respectively.

Conclusion/interpretation: Glycated insulin circulates at noticeably increased concentrations in Type 2 diabetic subjects. [Diabetologia (2003) 46:475–478]

Demonstration of glycated insulin in human diabetic plasma and decreased biological activity assessed by euglycemic-hyperinsulinemic clamp technique in humans

The presence and biological significance of circulating glycated insulin has been evaluated by high-pressure liquid chromatography (HPLC), electrospray ionization mass spectrometry (ESI-MS), radioimmunoassay (RIA), receptor binding, and hyperinsulinemic-euglycemic clamp techniques. ESI-MS analysis of an HPLC-purified plasma pool from four male type 2 diabetic subjects (HbA(1e) 8.1 +/- 0.2%, plasma glucose 8.7 +/- 1.3 mmol/l [means +/- SE]) revealed two major insulin-like peaks with retention times of 14-16 min. After spectral averaging, the peak with retention time of 14.32 min exhibited a prominent triply charged (M+3H)(3+) species at 1,991.1 m/z, representing monoglycated insulin with an intact M-r of 5,970.3 Da. The second peak (retention time 15.70 min) corresponded to native insulin (M-r 5,807.6 Da), with the difference between the two peptides (162.7 Da) representing a single glucitol adduct (theoretical 164 Da). Measurement of glycated insulin in plasma of type 2 diabetic subjects by specific RIA gave circulating levels of 10.1 +/- 2.3 pmol/l, corresponding to -9% total insulin. Biological activity of pure synthetic monoglycated insulin (insulin B-chain Phe(1)-glucitol adduct) was evaluated in seven overnight-fasted healthy nonobese male volunteers using two-step euglycemic-hyperinsulinemic clamps (2 h at 16.6 mug (.) kg(-1) (.) min(-1), followed by 2 h at 83.0 mug (.) kg(-1) (.) min(-1); corresponding to 0.4 and 2.0 mU (.) kg(-1) (.) min(-1)). At the lower dose, the exogenons glucose infusion rates required to maintain euglycemia during steady state were significantly lower with glycated insulin (P