Volume and enthalpy changes associated with O2 dissociation from Mb in the temperature range 6 - 10C.
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Ligand-induced conformational transitions play an eminent role in the biological activity of proteins including recognition, signal transduction, and membrane trafficking. Conformational transitions occur over a broad time range starting from picosecond transitions that reflect reorientation of amino acid side chains to slower dynamics on the millisecond time-scale that correspond to larger domain reorganization (Henzler-Wildman et al., 2007). Direct characterization of the dynamics and energetics associated with conformational changes over such a broad time range remains challenging due to limitations in experimental protocols and often due to the absence of a suitable molecular probe through which to detect structural reorganization. Photothermal methods such as photoacoustic calorimetry (PAC) and photothermal beam deflection provide a unique approach to characterize conformational transitions in terms of time resolved volume and enthalpy changes (Gensch&Viappiani, 2003; Miksovska&Larsen, 2003). Unlike traditional spectroscopic techniques that are sensitive to structural changes confined to the vicinity of a chromophore, photothermal methods monitor overall changes in volume and enthalpy allowing for the detection of structural transitions that are spectroscopically silent (i.e. do not lead to optical perturbations of either intrinsic or extrinsic chromophores).
Myoglobin (Mb) and hemoglobin (Hb) play a crucial role in the storage and transport of oxygen molecules in vertebrates and have served as model systems for understanding the mechanism through which protein dynamics regulate ligand access to the active site, ligand affinity and specificity, and, in the case of hemoglobin, oxygen binding cooperativity. Mb and individual α- and β- subunits of Hb exhibit significant structural similarities, i.e. the presence of a five coordinate heme iron with a His residue coordinated to the central iron (proximal ligand) and a characteristic “3-on-3” globin fold (Fig. 1)(Park et al., 2006; Yang&Phillips Jr, 1996). Both proteins reversibly bind small gaseous ligands such as O2, CO, and NO. The photo-cleavable Fe-ligand bond allows for the monitoring of transient deoxy intermediates using time-resolved absorption spectroscopy (Carver et al., 1990; Esquerra et al., 2010; Gibson et al., 1986) and time resolved X-ray crystallography (Milani et al., 2008; Šrajer et al., 2001). Based on spectroscopic data and molecular dynamics approaches (Bossa et al., 2004; Mouawad et al., 2005), a comprehensive molecular mechanism for ligand migration in Mb was proposed including an initial diffusion of the photo-dissociated COmoleculeinto the internal network of hydrophobic cavities, followed by a return
Left: Ribbon representation of the tetrameric human Hb structure (PBD entry 1FDH). Right: horse heart Mb structure (PDB entry 1WLA). The heme prosthetic groups are shown as sticks. In the case of Mb, the distal and proximal histidine are visualized.
into the distal pocket and subsequent rebinding to heme iron or escape from the protein through a distal histidine gate. The ligand migration into internal cavities induces a structural deformation, which promotes a transient opening of a gate in the CO migration channel. Such transitional reorganization of the internal cavities is ultimately associated with a change in volume and/or enthalpy and thus can be probed using photothermal techniques. Indeed, CO photo-dissociation from Mb has been intensively investigated using PAC by our group and others (Belogortseva et al., 2007; Peters et al., 1992; Vetromile, et al., 2011; Westrick&Peters, 1990; Westrick et al., 1990) and these results lead to a thermodynamic description of the transient “deoxy intermediate” that is populated upon CO photo-dissociation.
The mechanism of ligand migration in Hb is more complex, since it is determined by the tertiary structure of individual subunits as well as by the tetramer quaternary structure. Crystallographic data have shown that the structure of the fully unliganded tense (T) state of Hb and the fully ligated relaxed (R) states differ at both the tertiary and quaternary level (Park et al., 2006). Crystallographic and NMR studies suggest that the fully ligated relaxed state corresponds to the ensemble of conformations with distinct structures (Mueser et al., 2000; Silva et al., 1992). Moreover, Hb interactions with diatomic ligands is modulated by physiological effectors such as protons, chloride, and phosphate ions, and non-physiological ligands including inositol hexakisphosphate (IHP) and bezafibrate (BZF) (Yonetani et al., 2002). Despite a structural homology between Hb and Mb, the network of internal hydrophobic cavities identified in Mb is not conserved in Hb suggesting distinct ligand migration pathways in this protein (Mouawad et al., 2005; Savino et al., 2009). Here we present thermodynamic profiles of CO photo-dissociation from human Hb in the presence of heterotropic allosteric effectors IHP and BZF. In addition, we include an acoustic study of oxygen photo-dissociation from Mb that has not been investigated previously using photothermal methods, despite the fact that oxygen is the physiological ligand for Mb.
Mb, Hb, inositol hexakisphosphate (IHP), and bezafibrate (BZF) were purchased from Sigma-Aldrich and used as received. Fe(III) tetrakis(4-sulfonatophenyl)porphine (Fe(III)4SP) was obtained from Frontier-Scientific Inc. Oxymyoglobin (O2-Mb) samples were prepared by dissolving the protein in 50 mM HEPES buffer pH 7.0. The sample was then purged with Ar for 10 min and reduced by addition of a freshly prepared solution of sodium dithionite. The quality of the deoxymyoglobin (deoxyMb) was verified by UV-visible spectroscopy. (O2-Mb) was obtained by bubbling air through deoxyMb sample. The CO bound hemoglobin sample was prepared by desolving Hb in 100 mM HEPES buffer pH 7.0 in a 0.5 x 1cm quartz cuvette. The concentration of allosteric effectors was 5 mM for BZF and 1 mM for IHP. The sample was then sealed with a septum cap and purged with Ar for 10 min, reduced with a small amount of sodium dithionite to prepare deoxyhemoglobin (deoxyHb), and subsequently bubbled with CO for approximately 1 min. Preparation of O2-Mb and CO-Hb aducts was checked by UV-vis spectroscopy (Cary50, Varian).
The quantum yield () was determined as described previously (Belogortseva et al., 2007). All transient absorption measurements were carried out on 50 µM samples in 50 or 100 mM HEPES buffer, pH 7.0, placed in a 2mm path quartz cell. The cell was placed into a temperature controlled holder (Quantum Northwest) and the ligand photo-dissociation was triggered using a 532 nm output from a Nd:YAG laser (Minilite II, Continuum). The probe beam, an output from the Xe arc lamp (200 W, Newport) was propagated through the center of the cell and then focused on the input of a monochromator (Yvon-Jovin ). The intensity of the probe beam was detected using an amplified photodiode (PDA 10A, Thornlabs) and subsequently digitized (Wave Surfer 42Xs, 400 MHz). The power of the pump beam was kept below 50 µJ to match the laser power used in photoacoustic measurements. The quantum yield was determined by comparing the change in the sample absorbance at 440 nm with that of the reference, CO bound myoglobin of known quantum yield (ref= 0.96 (Henry et al., 1983)) according to Eq 1:
where ΔAsam and ΔAref are the absorbance change of the sample and reference at 440 nm, respectively, and Δsam and Δref are the change in the extinction coefficient between the CO bound and reduced form of the sample and the reference, respectively.
The photo-acoustic set-up used in our lab was described previously (Miksovska&Larsen, 2003). Briefly, the sample in a quartz cell was placed in a temperature controlled holder (Quantum Northwest). The 532 nm output from a Nd:YAG laser (7 ns pulse width, < 50 µJ power) was shaped using a narrow slit (100 µm) and focused on the center of the quartz cell. An acoustic detector (1 MHz, RV103, Panametrix) was coupled to the side of a quartz cell using a thin layer of honey and the detector output was amplified using an ultrasonic preamplifier (Panametrics 5662). The signal was then stored in a digitizer (Wave Surfer 42Xs, 400 MHz). The data were analyzed using Sound Analysis software (Quantum Northwest).
The excitation of the photocleavable iron-ligand bond in heme proteins generates at least two processes that contribute to the photoacoustic wave: the volume change due to the heat released during the reaction (Q), and the volume change (ΔV’) due to the photo-triggered structural changes (including bond cleavage / formation, electrostriction, solvation, etc.). The amplitude of the sample acoustic wave (Asam) can be expressed as:
where K is the instrument response constant, Ea is number of Einsteins absorbed, β is the expansion coefficient, ρ is the density, and Cp is the heat capacity. For water, the (β/Cpρ) term strongly varies with temperature mainly due to the temperature dependence of the β term. To evaluate the instrument response constant, the photo-acoustic traces are measured for a reference compound under experimental conditions (laser power, temperature, etc.) identical to those for the sample measurements. We have used Fe(III)4SP as a reference since it is non-fluorescent and photo-chemically stable. The amplitude of the reference acoustic trace can be described as:
where Ehν is the energy of a photon at 532 nm (Ehν= 53.7 kcal mol-1). The amount of heat deposited to the solvent and the non-thermal volume changes can then be determined by measuring the acoustic wave for the sample and the reference for several temperatures and plotting the ratio of the sample and reference acoustic wave () as a function of (Cpρ/β) according to Eq. 4:
For a multi-step process that exhibits volume and enthalpy changes on the time-scale between ~ 20 ns to 5 µs, the thermodynamic parameters for each individual step and corresponding lifetimes can be determined due to the sensitivity of the acoustic detector to the temporal profile of the pressure change. The time dependent sample acoustic signal E(t)obs can be expressed as a convolution of the time dependent function describing the volume change H(t) with the instrument response T(t) function (the reference acoustic wave):
where 1 and 2 correspond to the
For processes that occur with a quantum yield that is temperature dependent in the temperature range used in PAC measurements, the thermodynamic parameters for the fast phase (<20ns) are determined by plotting [Ehν(1-)]/] versus (Cpρ/β) according to Eq.7 and the volume and enthalpy changes for the subsequent steps are obtained by plotting (Ehν/) versus (Cpρ/β) according to Eq. 8 (Peters et al., 1992).
where ΔH and ΔV correspond to the reaction enthalpy and volume change, respectively.
Ligand migration in heme proteins is often described using the sequential three-state model (Henry et al., 1983) shown in Scheme 2. Upon cleavage of the coordination bond between the ligand and heme iron, the ligand is temporarily trapped within the protein matrix and then it either directly rebinds back to the heme iron in the so called “geminate rebinding” or diffuses from the protein matrix into the surrounding solvent. The subsequent bimolecular ligand binding to heme iron occurs on significantly longer time scales, hundreds of microseconds to milliseconds. The quantum yield for the geminate rebinding and for bimolecular association is strongly dependent on the character of the ligand and the protein. For example, CO rebinds to Mb predominantly through a bimolecular reaction with quantum yield close to unity (bim = 0.96 )(Henry et al., 1983), whereas the quantum yield for bimolecular O2 rebinding to heme proteins is significantly lower (Carver et al., 1990; Walda et al., 1994), and NO rebinds predominantly through geminate rebinding (Ye et al., 2002). To determine the thermodynamic parameters from acoustic data, the quantum yields for CO and O2 bimolecular rebinding to Hb and Mb, respectively, have to be known. The quantum yield for O2 binding to Mb was measured in the temperature range from 5 C to 35 C (Fig. 2) and the values show a weak temperature dependence with the quantum yield decreasing with increasing temperature. At 20 C the quantum yield is 0.09 ± 0.01 that is within the range of values reported previously ( = 0.057 (Walda et al., 1994) and ( = 0.12 (Carver et al., 1990)). We have also measured the quantum yield for CO bimolecular rebinding to Hb, and to Hb in the presence of effector molecules (Fig. 2). The quantum yield increases linearly with temperature and at 20 C, CO binds to Hb with quantum yield of 0.68 and in the presence of IHP and BZF 0.62 and 0.46, respectively. A similar quantum yield for CO bimolecular rebinding to Hb was reported previously by Unno et al. (bim =0.7 at 20 C) (Unno et al., 1990) and by Saffran and Gibson (=0.7 for CO binding to Hb and = 0.73 for CO association to Hb in the presence of IHP at 40 C) (Saffran&Gibson, 1977).
The photo-acoustic traces for O2 dissociation from Fe(II)Mb at pH 7.0 are shown in Fig. 3. At low temperatures (6 C to 15 C), the sample photoacoustic traces show a phase shift with respect to the reference trace indicating the presence of thermodynamic process(es) that occurs between 50 ns and ~ 5 µs. The sample traces were deconvoluted as described in the Materials and Methods section and the i values were plotted as a function of the temperature dependent factor (Cpρ/β) (Fig. 4). The extrapolated volume and enthalpy changes are listed in Table 1. The photo-cleavage of the Fe-O2 bond is associated with a fast structural relaxation (< 20 ns) forming a transient “deoxy-Mb intermediate”. This initial transition is endothermic (ΔH = 21 ± 9 kcal mol-1) and leads to a small volume contraction of – 3.0 ± 0.5 mL mol-1. This initial relaxation is followed by ~ 250 ns kinetics that exhibit a volume increase of 5.5 ± 0.4 mL mol-1 and a very small enthalpy change of -8.9 ± 8.0 kcal mol-1. We associate the initial process with the photo-cleavage of the Fe-O2 bond. A similar volume decrease of approximately -3 mL mol-1 has been observed previously for the photo-dissociation of Fe-CO bond in Mb (Westrick&Peters, 1990; Westrick et al., 1990). The observed volume contraction reflects a fast relaxation of the heme binding pocket including: i) cleavage of the hydrogen bond between the distal histidine and oxygen molecule (Phillips&Schoenborn, 1981) ii) reorientation of distal residues within the heme binding pocket (Olson et al., 2007), and iii) fast migration of the photo-released ligand into the primary docking site and then into the internal cavities (Xe4 or Xe1) (Hummer et al., 2004). Also, the positive enthalpy change is consistent with the photo-cleavage of Fe-O2 bond.
The subsequent 250 ns kinetics may reflect either the nanosecond geminate rebinding of the O2 molecule or the ligand diffusion from the protein matrix into the surrounding solvent. The kinetics for the geminate O2 rebinding were studied on femtosecond timescale by Petrich et al. (Petrich et al., 1988), and on picosecond and nanosecond timescales (Carver et al., 1990; Miller et al., 1996). These studies identified two distinct sub-states of the “deoxyMb” intermediate: a “barrier-less” and a “photolyzable” sub-state. In the “barrier-less” sub-state, oxygen rebinds to heme iron on sub-picosecond timescale whereas the oxygen association to the “photolyzable” substate occurs on nanosecond and microsecond timescales. Carver et al. (Carver et al., 1990) have reported the time constant for O2 nanosecond geminate rebinding to be 52 ± 14 ns at room temperature. This kinetic step has a lifetime that is comparable to the time resolution of our PAC instrument ( ~ 50 ns) and therefore it was not resolved in this study. The 250 ns step thus corresponds to the O2 escape from the transient “deoxy-Mb” intermediate into the surrounding solvent and is approximately 3 times faster than the rate of the CO escape (Westrick et al., 1990), which suggests that O2 diffuses from the protein matrix through a transient channel with a lower activation barrier than CO. This result is consistent with the transient absorption studies that estimated the rate for O2 release to be approximately two times faster than that for CO (Carver et al., 1990). Interestingly, a similar time-constant of 200 ns to 300 ns was determined for CO escape from Mb at pH 3.5 (Angeloni&Feis, 2003). At acidic pH Mb adopts an open conformation with His 64 displaced toward the solvent giving a direct access to the distal cavity. These data suggest that the reorientation of His 64 may not be a rate limiting step for the O2 escape.
Quantum yield for bimolecular photo-dissociation of O2 from the O2-Mb complex (bottom) and CO from the CO- Hb complex (top) as a function of temperature. The error of quantum yield is ± 0.05. The solid line demonstrates the trend.
PAC traces for O2 photo-dissociation from O2-Mb complex at 6 ºC. Conditions: 40 µM Mb dissolved in 50 mM Hepes buffer pH 7.0. The absorbance of the reference compound, Fe(III)4SP, at excitation wavelength of 532 nm was identical as that of O2-Mb.
Plot of the ratio of the acoustic amplitude for the photo-dissociation of the O2-Mb complex and the reference compound as a function of (Cpρ/β) term. 1 values that correspond to the prompt phase are shown as solid circles and the 2 values corresponding to the slow phase are shown as open squares. The data were fit with a linear curve and the corresponding volume and enthalpy changes were determined using Eq. 6 and Eq. 7.
The reaction volume change observed for the slow phase includes several factors: i) volume change due to the O2 escape into the surrounding solvent, ii) volume change associated with the heme hydration in deoxyMb and iii) volume change due to the structural changes. The reaction volume can be expressed as the difference between the partial molar volume of products and reactants according to: ΔVslow= VO2 + VdeoxyMb- VO2-Mb - VH2O, where VO2 is the partial molar volume of oxygen, VH2O is the partial molar volume of water, VdeoxyMb is the partial molar volume of transient “deoxyMb” intermediate and VO2-Mb is the partial molar volume of oxy-Mb. Using VO2 = 28 mL mol-1(Projahn et al., 1990) and VH2O = 15 mL mol-1 (the partial molar volume of water scaled to the occupancy of water molecule hydrogen bound to distal histidine) (Belogortseva et al., 2007), we estimate that the O2 release from Mb results in a structural volume change (VdoxyMb- VO2-Mb) of - 7.5 mL mol-1. This value is very similar to that reported previously for CO escape from Mb (ΔVstructural= VdoxyMb- VCO-Mb = - 6 mL mol-1) (Vetromile, et al., 2011) demonstrating that the overall structural changes accompanying the ligand bound to ligand free transition in Mb are very similar for both ligands. This is in agreement with the close resemblance of the X-ray structure of both the CO-bound and O2-bound Mb(Yang&Phillips Jr, 1996). The small enthalpy change measured for the 250 ns relaxation (ΔH = -8.9 ± 8.0 kcal mol-1) includes the enthalpy change for O2 solvation (ΔHsolv = -2.9 kcal mol-1(Mills et al., 1979)) and the enthalpy change associated with H2O binding to the heme binding pocket (ΔHsolv = -7 kcal mol-1(Vetromile, et al., 2011) indicating that the structural relaxation coupled to the ligand escape from the protein is entropy driven.
The overall enthalpy change for O2 dissociation from Mb was determined to be 11.6 ± 8.5 kcal mol-1 and this value is in agreement with the value of 10 kcal mol-1 reported previously (Projahn et al., 1990). The overall reaction volume change determined here (ΔVoverall = +2.5 mL mol-1) is somewhat larger than the reaction volume change determined from the measurement of the equilibrium constant as a function of pressure (ΔV= - 2.9 mL mol-1) (Hasinoff, 1974) and significantly smaller than the reaction volume change determined as a difference between the activation volume for oxygen binding and dissociation from Mb that was reported to be 18 mL mol-1(Projahn et al., 1990). Unlike photoacoustic studies that allow for reaction volume determination at ambient pressure, the high pressure measurements of equilibrium constant and/or rate constants (to determine activation volumes) may cause a pressure induced protein denaturation and/or structural changes, which may influence the magnitude of reaction volume changes in high pressure studies.
ΔV (mL mol-1) | ΔH (kcal mol-1) | |
Fast phase | -3.0 ± 0.5 | 20.5 ± 8.5 |
Slow phase | 5.5 ± 0.4 | -8.9 ± 8.0 |
Volume and enthalpy changes associated with O2 dissociation from Mb in the temperature range 6 - 10C.
We have also probed the thermodynamic parameters associated with the CO photo-dissociation from Hb and the impact of the binding of BZF and IHP on the thermodynamics associated with the ligand migration between the heme binding pocket and surrounding solvent. The photo-acoustic traces for CO photo-dissociation from Hb are shown in Fig. 5. The sample and the reference acoustic wave overlay in phase indicating that the observed thermodynamic processes take place within 50 ns upon photo-dissociation, which is consistent with the fast CO diffusion from the heme matrix into the surrounding solvent. The fast ligand escape from the heme binding pocket was observed in the presence of effectors (data not shown). Previous transient absorption studies showed that the CO photo-release from the fully ligated R-state Hb is followed by three relaxations with lifetimes of 50 ns, 1 µs, and 20 µs that were assigned to the unimolecular geminate rebinding, the tertiary structural relaxation, and the RT quaternary change, respectively (Goldbeck et al., 1996). The geminate rebinding occurs too fast to be resolved by our PAC detector, whereas the 20 µs RT transition, which strongly depends on the extent of heme ligation, is too slow to be resolved in PAC measurements. The 1 µs relaxation is within the time-window accessible by our detection system, however we were unable to resolve this step. Since this relaxation was observed as a small perturbation of the deoxy-Soret band (Goldbeck et al., 1996), it may reflect the structural relaxation localized within the vicinity of the heme binding pocket, which does not lead to measurable volume and enthalpy changes.
The volume and enthalpy changes associated with the diffusion of the photo-dissociated ligand to the surrounding solvent can be determined from the plot of the ratio of the amplitude of the acoustic trace for CO photo-dissociation from Hb and the reference as a function of temperature according to Eq. 7 (Fig. 6). The extrapolated thermodynamic values
PAC traces for the CO photo-dissociation from the CO-Hb complex and the reference compound Fe(III)4SP. Conditions: 40 µM Hb in 100 mM HEPES buffer pH 7.0 and 20 C. The absorbance of the reference compound matched the absorbance of the sample at 532 nm.
are shown in Table 2. The CO photo-release from Hb is associated with a positive volume change of 21.5 ± 0.9 mL mol-1 and enthalpy change of 19.4 ± 1.2 kcal mol-1. These results are in agreement with the thermodynamic parameters reported previously by Peters et al: ΔV = 23.4 ± 0.5 mL mol-1 and ΔH = 18.0 ± 2.9 kcal mol-1(Peters et al., 1992). Since the laser power used in this study was kept below 50 µJ, the low level of photo-dissociation was achieved that corresponds to 1 CO molecule per hemoglobin photo-released. Thus the observed thermodynamic parameters reflect the transition between fully ligated (CO)4Hb and triple ligated (CO)3Hb. Consequently, the observed reaction enthalpy corresponds to the enthalpy change due to the cleavage of the Fe-CO bond (ΔHFe-CO=17.5 kcal mol-1 (Leung et al., 1987; Miksovska et al., 2005)), the enthalpy change due to the solvation of a CO molecule (ΔHsolv = 2.6 kcal mol-1(Leung et al., 1987)), the enthalpy change of structural relaxation associated with the ligand release from the protein matrix, and enthalpy of the distal pocket hydration. The occupancy of water molecules in the distal pocket of deoxyHb was determined to be significantly lower than that in Mb (~0.64 for the Hb - chain and ~ 0.33 for the Hb β-chain (Esquerra et al., 2010)). Using an average occupancy of 0.48, we estimate that the distal pocket hydration contributes to the overall enthalpy change by ~ - 3 kcal mol-1(Vetromile, et al., 2011). Therefore, the structural relaxation coupled to the CO dissociation and diffusion into the surrounding solvent is accompanied by a small enthalpy change of 2 kcal mol-1.
The plot of the ratio of the acoustic amplitude for the CO photo-dissociation from the CO-Hb complex and the reference compound as a function of the temperature dependent factor (Cpρ/β) term. The reaction volume and enthalpy changes were extrapolated according to Eq. 5
Analogous to O2 photo-release from Mb, the observed reaction volume change for CO photorelease from Hb, ΔV=21.5 mL mol-1, can be expressed as: ΔV= VCO + V(CO)3Hb - V(CO)4 Hb - VH2O,where VCO is the partial molar volume of CO and V(CO)3Hb and V(CO)4 Hb are the partial molar volume of (CO)3Hb and (CO)4Hb, respectively. Using VCO = 35 mL mol-1 (Projahn et al., 1990) and VH2O = 9 mL mol-1 (partial molar volume of water scaled by the average occupancy of the Hb chain), we estimate that upon release of one CO molecule per Hb, the protein undergoes a small contraction of -7 mL mol-1. The small volume change observed here is consistent with the minor structural changes due to deligation of Hb in the R-state as observed in the X-ray structure that are predominantly localized in the the -chain and include reposition of the F-helix and shift of the EF and CD corner (Wilson et al., 1996).
ΔHprompt (kcal mol-1) | ΔVprompt (mL mol-1) | |
CO-Hb | 19.4 ± 1.2 | 21. 5 ± 0.9 |
CO-Hb + BZF | 21.7 ± 7.9 | 22.3 ± 1.7 |
CO-Hb + IHP | -9.9 ± 6.1 | 11.4 ± 1.3 |
Volume and enthalpy changes associated with CO photo-dissociation from Hb.
The thermodynamic profile for CO photo-dissociation from Hb in the absence of effector and in the presence of BZF and IHP.
We have also determined volume and enthalpy changes associated with the CO photo-dissociation from Hb in the presence of heterogenous effectors BZT and IHP (Fig. 6) and the thermodynamic profiles for CO photo-dissociation from CO-Hb complex in the presence and absence of effectors are presented in Fig.7. Both effectors bind to Hb in the T-state and R-state and modulate the interaction of Hb with diatomic ligands (Coletta et al., 1999b; Marden et al., 1990). For example, the binding of BZF or IHP to CO-Hb complex decreases the CO association rate approximately four or eight times, respectively (Marden et al., 1990), and decreases the affinity of R state deoxy-Hb for oxygen (Tsuneshige et al., 2002). Coletta et al (Coletta et al., 1999a) have reported that simultaneous binding or IHP and BZF effectors to Hb at ambient pressure leads to the Hb intermediate with tertiary T-like structure in the quaternary R- conformation. Recently, using NMR spectroscopy Song et al. have shown that binding of IHP to the fully ligated Hb increase the conformational fluctuation of the R-state in both the - and β-chain (Song et al., 2008).
The photoacoustic data presented here show that BZF binding to CO-Hb complex does not impact the reaction volume and enthalpy changes associated with CO photo-release. The crystal structure of horse CO-Hb in complex with BZF indicates that the structural changes due to BZF association to fully ligated Hb are localized in the -subunits (Shibayama et al., 2002). BZF binds to the surface of each -chain E-helix and decreases the distance between the heme iron and distal His and its binding site is surrounded by hydrophobic residues such as Ala 65, Leu 68, Leu 80 and Leu 83 (Shibayama et al., 2002). Such minor structural changes caused by BZF association are unlikely to alter the overall structural volume and enthalpy changes associated with the CO photo-release. However, due to the lower solubility of BZF, the effector concentration used is this study was 5 mM that results in a Hb fractional saturation of 0.25 (using KD of 15 mM (Ascenzi et al., 1993)). Such lower fractional saturation may prevent detection of BZF induced changes in Hb conformational dynamics.
On the other hand, the binding of IHP has a significant impact on the observed volume and enthalpy changes (Table 2). The reaction volume decreases by 10 mL mol-1 and the enthalpy change is more exothermic by nearly 30 kcal mol-1 compared to the thermodynamic parameters determined in the absence of effector molecules. Such negative reaction volume and exothermic enthalpy change indicates that electrostriction of solvent molecules caused by reorganization of salt bridges or redistribution of charges on protein surface contributes to the overall reaction volume and enthalpy change associated with the CO photo-release. Indeed, IHP interacts with charged residues along the Hb central cavity. At the Hb T-state, the IHP binding site is located at the interface of the β-chains involving Val 1, His2, Lys 82 and His 141 from each chain (Riccio et al., 2001); whereas at the R-state Hb, the IHP molecule interacts with the charged residues Lys 99 and Arg 141 from each -chain (Laberge et al., 2005). In the absence of the X-ray structure of IHP bound fully ligated and partially photolyzed CO-Hb, it is difficult to point out the factors that contribute to the observed volume and enthalpy changes on the molecular level. Arg 141 forms a salt bridge with Asp 126 in the T-state deoxy Hb that is absent in the fully ligated R- state (Park et al., 2006). We speculate that the transition between the fully ligated (CO)4Hb and partially ligated (CO)3Hb may be associated with a repositioning of the Arg 141 side chain leading to a partial exposure of either the IHP molecule and/or the Arg 141 side chain to the surrounding solvent molecules. Also, the ligand photo-release may be associated with the repositioning of the IHP molecule within the Hb central cavity. Based on a molecular dynamics simulation of IHP binding sides in south polar skua deoxyHb, an IHP migration pathway connecting the binding site at the interface between the -chains and the second binding site located between the β-chains was proposed suggesting that IHP interactions with Hb are dynamic and involve numerous positively charged residues situated along the central cavity (Riccio et al., 2001). Therefore, CO photo-release may trigger relocation of IHP within the central cavity resulting in larger exposure of IHP phosphate groups and/or charged amino acid residues and concomitant electrostriction of solvent molecules.
The photoacoustic data for the ligand photo-dissociation from Mb shows that the structural volume changes associated with the O2 diffusion from the Mb active site are similar to those determined previously for CO in agreement with the crystallographic data. On the other hand, the time constant for O2 escape from the distal pocket to the surrounding solvent is two to three time faster than that for CO suggesting a distinct migration pathway for diatomic ligands in Mb. Our PAC study also indicates that IHP binding to Hb-CO complex alters the volume and enthalpy changes associated with the CO photo-dissociation from the heme iron indicating that the transition between the fully ligated (CO)4Hb and partially ligated (CO)3Hb complex is associated with the reorientation of IHP molecule within the central cavity and/ or charged amino acid residues interacting with IHP.
This work was supported by J. & E. Biomedical Research Program (Florida Department of Health) and National Science Foundation (MCB 1021831).
Four hundred years back, Paracelsus stated that, “All substances are poisons; there is none which is not a poison.” If the right dose is taken, it could become a remedy, otherwise poisonous [1, 2]. The therapeutic index or ratio, i.e., LD50/ED50, tells whether the chemical is safe or not.
Poisons are generally found in cases of homicides, suicides, or accidents. They have a significant role to play as the silent weapon to destroy life mysteriously and secretively.
Every poison has almost similar action on the victim’s body. In many cases, they either stop the transfer of O2 to the tissues or create an obstacle in the respiratory system by inhibition of enzymes which are associated with the process. In this, the myoneural junction and the ganglions and synapses are the sites of action. In some cases of insecticidal poisoning, hyperexcitement of voluntary and involuntary muscles can cause death. There are four categories of action of poisons—(i) local action, (ii) remote action, (iii) local and remote actions, and (iv) general action.
Local action: Local action means direct action on the affected site of the body. Examples include irritation and inflammation in strong mineral acids and alkalis, congestion and inflammation by irritants, the effect on motor and sensory nerves, etc.
Remote action: Remote action affects the person due to absorption of that poison into the system of that person. For example, alcohol is absorbed in the system and then it affects the person.
Local and remote actions: Some poisons can affect both local and remote organs. Thus, they not only affect the area with contact to the poison but also cause toxic effect after absorption into the system, for example, oxalic acid.
General action: General action means the absorbed poison affects more than one system of the body, for example, mercury, arsenic, etc.
Toxicity of a poison depends upon its inherent properties such as physiochemical as well as pharmacological properties.
The action of poisons mainly depends upon the following factors discussed below:
Forms of poison: There are three forms of poison:
Physical form: Gaseous/volatile/vaporous forms of poisons act faster than liquid poisons as they are quickly absorbed. Similarly, liquid poisons act faster than solid poisons.
Gaseous or volatile > liquid > solid.
For solid poisons, powdered poisons act quickly than the lumps. For example, there are certain seeds that escape the gastrointestinal tract as they are solid, but when crushed, they can be fatal.
For solids: powdered > lumps
Chemical form: Few substances like mercury or arsenic are not poisonous as they are insoluble and cannot be absorbed when they are in combination with other substances like mercuric chloride, arsenic oxide, etc.
In other cases, the action is vice versa. For example, there are some substances that become inert in combination with silver nitrate and hydrochloric acid and are deadly and poisonous when present in pure forms.
Mechanical combination: The effect of poisons is significantly altered when they are combined with inert substances.
Quantity: Large doses of toxin cause much lethal effect. But this statement is not always true. For example, sometimes when a toxin is taken in very large amount, the body produces a mechanism against it such as vomiting, and thus the intensity of the toxin is reduced.
Concentration: The absorption speed of poison is dependent on concentration; thus poison of higher concentration is fatal. However, there are still some exceptions. For example, a dilute oxalic acid is less corrosive, but the absorption rate is high and so it is more dangerous.
Methods of administration: It has a unique role in the process of absorption. It is fastest through inhalation and then through injection as compared to the oral mode.
Condition of the body: Different persons react differently when exposed to a poison. It is because the condition of our body is also responsible for the increase or decrease of the effect of a poison on the body:
Age: Children and older people are more affected than an adult by the same quantity of toxin.
Sleep: The body functions are slower during sleep; thus toxin circulation in the body is also slower.
Health: Healthy persons can tolerate a toxin better than a weak or ill person.
Dosage: The effect of the poison depends upon its dosage. It is said that the dose determines whether a substance is a poison or remedy. A substance is usually considered a poison after a certain fixed quantity. Although this quantity is not fixed for all people, it is considered according to the average effect on the population. There are two considerable effects of poison on the body of a person; these are the subtle long-term chronic toxicity and immediate fatality.
Some poisons are lethal in microquantities, while others can affect in large doses. The significance of a dose can be understood by taking an example of a metal essential in the food, for example, iron, copper, manganese, zinc, etc.; if its dose is higher than the body requires, it can be lethal.
Effective dose (ED): The effective dose is the quantity of a substance at which it shows its effect in the population. In most cases, ED50 is measured as a dose which induces a response in half of the targeted population.
Lethal dose: The lethal dose (LD) 50 is the amount of drug which is expected to cause death of 50% population.
Hypersensitivity: It is basically the type of reaction initiated by the body against any other substances. Sometimes, it could be related to allergy. There is an assumption that hypersensitivity does not depend on wrong doses. Every person who is hypersensitive to a particular substance has a dose related that defines the quantity required to cause hypersensitivity to that person. The allergic response is actually a toxic response and can be sometimes fatal.
Idiosyncrasy: It is defined as a reaction produced by the body to a chemical genetically. It is a type of person that affects only those people who are genetically sensitized to that particular chemical or substance but will show no effect on others. In such cases, the person experiences discomfort for several hours or if the dose is high can be fatal also. For example- peanut allergy in some people.
Tolerance: It is the capability of a person to not produce any effect against a chemical that usually causes reaction to normal persons. It is a state of reduced or no reaction to a chemical. There are basically two types of mechanism that induces tolerance. First is when the toxin reaches the effective site, its quantity is very less. This is called dispositional tolerance. The second is because the tissues show reduced response to the toxin.
Tolerance can also be achieved if a drug is taken in a small quantity on a regular basis. This can be explained by taking the example of alcohol. When any human consume alcohol for the first time, he/she will show an effect even when the quantity is small, but eventually the effect will decrease and the person can tolerate a large amount also.
Individual susceptibility: It is defined as the different kinds of responses produced by different individuals to a particular harmful compound. It can be due to occupational or environmental factors and exposures. It is determined by complex genetic factors. Its effect depends upon the intensity of exposure. There is a gene uniqueness that varies from person to person; thus the same amount of exposure can show no effect in one individual, cause illness to other individual, and also could be fatal to someone as well.
The route of administration is the path through which a drug, toxin, or poison is taken or administered into the body of a person which is distinguished by the location where any drug is applied. It is mostly classified on the basis of its target:
Topical—which has a local effect
Enteral—which has a wide effect, i.e., affect the whole system
Parental—which follows a systemic action
Poisons are given or taken so that death can occur at once by shock due to stoppage of body’s vital systems. Drug addicts take drugs through inhalation or injection.
Route of administration plays a very important role in determination of death by poison as time in which death occurs are fastest in inhaled poisons, relatively slow in injected and lastly when ingested orally.
Some important features that are considered during the administration of poisons and can make a poison fatal are:
Rate of dissolution of the poison that depends upon the physical form of the poison, i.e., gaseous, vapors, liquid, solid, etc.
The surface area affected at the site of administration of the poison
The circulation rate of blood in that route
The solubility of the poison, i.e., lipid soluble or water soluble
The concentration of the poison
The time required by the poison to be absorbed completely from the site of administration
Routes of administration can be classified into two categories:
Enteral routes/gastrointestinal routes.
Parenteral routes.
Enteral routes: When the drug is administered through the gastrointestinal tract, it is defined as an enteral route. It has both oral and rectal routes. It also includes sublingual and sublabial routes. It is comparatively a slower mode of action for absorption of drugs:
Oral route: Generally absorption takes place in the tongue and the gums of the oral passage. The pH of the buccal cavity and mouth ranges from 4 to 5. Sublingual and supralingual routes have a significant role in absorption. The sublingual absorption is faster as the toxin is transformed directly to the heart, but it takes more time.
Rectal route: Administration of drugs can be done through anus which directly absorbed in bloodstream through membrane of mucous. This administration can cause the burning of tissues or bleeding in rectum as the area is very sensitive.
Parental route: It includes all the other routes that does not involve the gastrointestinal tract. It has a systemic effect on the body. It has the following categories of administration:
Intradermal: Here, the administration of drugs takes place from surface of skin. This type of poisoning is mostly found in chronic poisoning cases.
Intravenous: It is one of the fastest modes of drug administration as the injection is directly taken and the drug is transferred directly into the veins and thus is directly circulated into the blood quickly. Immediate death might be caused by this type of drug.
Intraosseous: It involves an administration of a drug directly into the bone marrow. This mode is actually used for administration of drugs for medical purposes.
Intra-arterial: It involves an administration of a drug into the artery directly through injection. It is a fast mode of administration.
Intramuscular: In this mode, the drug or poison is administered into the muscle of the thigh, upper arm, or buttock. The time required in this mode is greater than other parental modes.
Subcutaneous: In this mode, the drug is injected into the layer beneath the skin, i.e., the subcutaneous layer. The drug then goes to the small blood vessels and then to the bloodstream. This mode is used for mostly those protein drugs that would be destroyed if administered through the gastrointestinal tract.
Inhalation: In this mode, the nose is the primary path. Because of the presence of mucous membrane, the nasal aperture is very absorptive. The microparticles of poisons are easily absorbed and transported quickly to the lungs. From the lungs, they are circulated into the blood.
Poisons are classified into two ways:
Based on their action on the body.
Based on their physical and chemical properties [1].
Classification based upon the effect of poison on the body:
Corrosive: The poisons burn the tissues or organs when they come in contact with them, e.g.:
Strong acids such as H2SO4, HNO3, HCL, etc.
Strong alkalis such as hydroxides of Na, K, NH4, etc.
Irritants: The poisons irritate the tissues or organs when they come in contact with them [3]:
Inorganic:
Nonmetallic phosphorous, chlorine, bromine, iodine, etc.
Metallic salts of arsenic, antimony, mercury, copper, lead, zinc, etc.
Organic:
Vegetable—castor oil, madar, croton oil, etc.
Animals—snake venom, cantharides, insect bites, etc.
Mechanical—glass powder, needles, diamond dust, hair, etc.
Neurotics: Poisons affect the nervous system and the brain [3]:
Cerebral:
Narcotic—opium and its alkaloids
Inebriant (depressant)—alcohol, ether, chloroform, and chloral hydrate
Spinal:
Excitant (stimulants)—nux vomica and strychnine
Depressant—gelsemium
Cardiorespiratory:
Cardiac—aconite, digitalis, oleander, and hydrocyanic acid (HCN)
Asphyxiants—carbon monoxide, carbon dioxide, and hydrogen sulfide
Miscellaneous: A number of chemicals having diverse actions on their body are included in this group [4]:
Animal poisons
Curare (an arrow poison)
Poisonous food articles
Industrial poisons—methyl isocyanate (MIC)
Fuels—petroleum and kerosene
Insecticides—endrin, dichlorodiphenyltrichloroethane (DDT), and
naphthalene
Radioactive substances
Classification of poisons based upon their properties:
Inorganic poisons
Metallic poisons:
Arsenic: It has been the most known and exclusively used throughout
the ages to poison men and animals [1].
It is a white tasteless powder and a pinch of the poisons can kill two adult persons.
Arsenic for homicidal purposes is mixed with various food articles, e.g., cooked food, milk, tea, liquors, or medicines.
Arsenic in a metal form is not poisonous; its oxides are highly poisonous. It is extensively used in insecticides, etc. [5].
Mercury: Chloride and nitrites of mercury are highly poisonous. They
are used in chemical industry and as fungicides.
Lead: Most of its compounds are poisonous. This is a slow poison,
e.g., Sindoor adulterated with red lead oxide.
Copper: Its salts are used in electroplating; copper sulfate is a poison.
Thallium: Thallium salt is used as rat poison [6].
Antimony: Its effect is like that of arsenic.
Nonmetallic poisons:
Cyanides: Cyanides of potassium and sodium are extremely
poisonous, even in small quantities. They react with the acid of
gastric juices in the stomach to form hydrocyanic acid, which
paralyzes the respiratory center in the brain resulting in death due to
respiratory failure [4].
Yellow phosphorus: In olden days it was used in match industry and
several times proved highly poisonous.
Iodine: Only elemental iodine in high quantity is poisonous.
Strong acids and alkalis: These are highly poisonous with corrosive
effects, e.g., sulfuric acid, nitric acid, sodium, potassium
hydroxides, etc.
Gases: Phosphine gas kills rats when used on the rat holes and is
poisonous for infants. MIC killed over 2000 persons and invalidated
several others in a gas leak tragedy in Bhopal in 1984. Some other
poisonous gases are HCN, carbon monoxide, hydrogen sulfide,
arsine, etc. [3].
Organic poisons
Volatile poisons:
Ethyl alcohol: It is poisonous if taken in excess.
Other alcohols: Methyl alcohol and isopropyl alcohol are poisonous.
Methanol, used in polish and chemical industries, is used in illicit
liquor, and its intake causes paralysis, blindness, and death [3].
Phenol: Phenol or carbolic acid could be poisonous. It is mostly used
as a disinfectant [6].
Miscellaneous substances: Various industrial chemicals like
chlorinated hydrocarbons, benzene, chloral hydrate, etc. are
poisonous. In several cases of poisoning, chloral hydrate could be
used in illicit liquors.
Nonvolatile substances:
Alkaloids: Several narcotics and vegetable poisons contain alkaloids,
e.g., strychnine, morphine, cocaine, nicotine, etc.
Barbiturates: These drugs are synthetic and induce sleep [1].
Glycosides: These drugs can cause cardiac arrest and could be fatal
such as aconite, oleander digitalis, etc.
Insecticides and pesticides
Poisoning: It is known as the injurious effect caused by the action of a poison or a detrimental chemical substance. It leads to the development of adverse reaction toward the harmful chemicals or drugs. It is basically differentiated in three categories: suicidal, homicidal, and accidental. Cattle poisoning is the poisoning related to animals. Accidental poisoning is caused by negligence and carelessness. Homicidal poisoning includes the killing of a person due to the poison. Suicidal poisoning refers to the use of toxic chemicals in order to kill oneself.
Corrosive poisoning: It is caused by poisons such as acids and alkalis. They produce a corrosive action on the human body by causing ulcers and acute inflammation.
Metallic poisoning: Metals such as arsenic, mercury, lead, etc., when ingested, cause a deleterious effect. This is known as metallic poisoning.
Plant poison: The study of plant poisons is known as phytotoxicology. Plant poisons, or phytotoxins, comprise a vast range of biologically active chemical substances, such as alkaloids, polypeptides, amines, glycosides, oxalates, resins, toxalbumins, etc.
An alcohol is a drink that contains ethanol. Ethanol is made by fermentation of grains, fruits, and some resources of sugar. Chemically, it is a group of compounds whose saturated carbon chain has a “-OH” group. Alcohol is also a depressant, and in low dose, it can reduce tension, cause euphoria, and improve sociability, but in high dose it can cause stupor, drunkenness, and even death. Regular alcohol intake can cause cancer, alcoholism, dependency, etc. 33% of the total people in the world consumes alcohol. Drinks containing alcohol are broadly classified into three classes, i.e., beer, spirit, and wine, whose alcohol content varies between 3% and 50%. When diluted, alcohol has nearly sweet taste, but when concentrated it gives a burning sensation. 90% of the absorbed alcohol is metabolized by the liver and broken down into less toxic metabolites. Alcohol acts on the central nervous system (CNS) as a depressant on the cells of the cerebral cortex. Its adverse effects like a decrease in cognitive and psychomotive skills are well documented. Alcohol percentage (ABV) differs from one brand to another, for example, beers contain 5%, wines contain typically 13.5%, fortified wines contain 15–22%, spirits contain 30–40%, fruit juice contains less than 0.1%, and cider/wine coolers contain 4–8% ABV [1].
The goal of blood alcohol test is to check the concentration of alcohol in the body. This test result is known as blood alcohol concentration (BAC) which indicates alcohol % in the blood. It is directly proportional to the alcohol in the body, and alcohol hinders with people’s decision, control on them and other characteristics [3]. This test can tell the presence of alcohol in blood for 12 hours [4]. Blood quickly absorbs alcohol and is measured within minutes of consuming alcoholic drink. The highest level of BAC result can be reached within an hour of consuming alcohol. Intake of food can vary the result. Liver breaks down almost 90% of alcohol and rest are given out from exhalation and urine [5].
In case of deaths due to alcoholic intoxication, the viscera is collected and preserved in saturated saline. Preservation of sample is very important as if wrongly preserved it can ruin the examination. Generally, urine and blood are taken as samples.
A sterile needle must be cleaned up by the swab of a nonalcoholic disinfectant like aqueous mercuric chloride and aqueous benzalkonium chloride (Zephiran) before the suspect’s skin is punctured with it. The use of an alcoholic disinfectant either may give false-positive results or may contribute to falsely high alcohol contents of blood. About 5–10 ml of the sample (blood) is taken in a test tube; an anticoagulant such as potassium oxide and EDTA and a preservative such as NaF are added and stored in the refrigerator at 40°C. The anticoagulant will prevent blood from clotting, and the preservative will inhibit the presence of microorganisms. The urine sample is also collected in the usual manner and preserved with 30 mg of phenyl mercuric nitrate for every 10 ml of urine [6].
Ethyl alcohol is isolated from biological materials by acid distillation. Viscera, vomit, stomach contents, and other materials should be analyzed separately. About 50–100 g of the viscera is taken and is finally minced by thin gruel and adding water (3–5 times) and sulfuric acid. It is passed to steam distillation which is generally heating it on the water bath. The condenser and the receiving flask should be well cooled with ice especially in the hot season, the outlet of the condenser being dipped in little water or NaOH solution. Some pieces of pumice stone are stored in the flask to avoid bumping. It is better to collect the distillate in 4–5 fractions, out of which the first one should not exceed 20 ml and the remaining fractions should be 50 ml each. The distillate contains alcohol and other volatile acids, etc. [6].
There are some tests which show the presence of ethyl alcohol in the exhibits.
Also known as triiodomethane reaction, it is used in the detection of CH3CH (OH) which is present in alcohol. There are mainly two types of different mixtures used in this reaction which are mainly chemically equivalent. A pale yellow precipitate occurs if the result is positive [6].
In the above structure, “R” can be hydrogen or alkyl group or any other hydrocarbon group. In case when R denotes hydrogen, then the compound we have the possibility to find is primary alcohol ethanol. Ethanol is the only alcohol that gives an iodoform reaction. In case R is any hydrocarbon group, then it gives secondary alcohol groups. Tertiary alcohol is not able to contain R group because of the absence of hydrogen atom [7].
In 1 ml of distillate, a few drops of 10% NaOH are added dropwise till the solution becomes brown and warmed for a few minutes. A few drops of iodoform solution are added to change the color to yellow. The mixture has to be again heated on low flame/water bath; a yellow-colored precipitate is formed on standing. The precipitate has to be observed under a microscope. Characteristic hexagonal crystals of iodoform are seen which usually shows the presence of ethanol, acetaldehyde, isopropanol which on standing for long time breaks into flower like structure. This test initially involves oxidation followed by substitution and hydrolysis [6].
Add 1 gm of molybdic acid in 25 ml of a concentrated sulfuric acid which has the reagent. Mix 2 ml of this reagent when hot and with 2 ml of distillate. At the junction of both liquids, a ring will be formed which is deep blue in color. On shaking, the whole mixture will become deep blue which is due to ethyl alcohol. This test is very sensitive and it gives a negative result with acetone, acetaldehyde, and dilute solution of methyl alcohol. Only the strong solution of methyl alcohol gives a light blue color after several minutes [6].
Mix two drops of benzoyl chloride with 2 ml of the distillate. Add 10% of sodium hydroxide drop by drop till the solution becomes alkaline. By providing heat the irritating smell of benzoyl chloride will be replaced by sweet fruity odor of ethyl benzoate. Methyl alcohol gives this test also but not the iodoform test [6].
In case of drunkenness, alcohol detection in the body is very important. Observing behavioral abnormalities of the suspect is the best method, but analyzing the breath, blood, and urine is the only way of confirming it. The analysis of breath alcohol can be performed on the spot with the help of breath-analyzer instruments like Alco-Sensor, Breathalyzer, etc. However, the alcohol content of the blood could be determined by using the modified version of the Kozelka and Hine/Cavett method [6].
In recent years, several methods in determining the alcohol in body fluids are described. Kent-Jones and Taylor reported the results of an investigation into the merits of two methods—the micro Cavett and that of Kozelka and Hine. The micro Cavett method is more accurate, but it suffered from serious inconsistencies in reproducibility, but the Kozelka and Hine method is less accurate and more time-consuming but gives good reproducibility.
Nickolls modified the micro Cavett method which appears to give a more accurate result in comparison with the unmodified method. The simplicity of this procedure increases its use for routine work in laboratory [8].
The principle behind this method is the oxidation of alcohol, which is easy with acetic acid in the presence of oxidizing agents such as sulfuric acid and potassium dichromate. Reduction of each mL of N/20 potassium dichromate solution takes place that is equivalent to 0.575 mg of alcohol [6].
This formula is used to estimate the amount in which alcohol is present in the body.
a. For blood analysis
Here, a = Total amount of alcohol absorbed in the body; p = Weight of the person; c = Concentration of alcohol in the blood; r = Constant which is 0.5 in women and 0.68 in men
b. In urine analysis.
Here, a = Total alcohol content present in the body; p = Total weight of the person; q = Alcohol concentration in the urine; r = Constant, namely, 0.68 for men and 0.5 in women [6].
There are several methods in determining ethanol in the blood, urine, and serum. One of the most important methods is gas chromatography (GC). The sample is injected in a heating chamber, and due to its high temperature, alcohol converts in vapors which are carried by inert carrier gas such as nitrogen through the column which is packed by an adsorbent material. Separation of different types of components depends on their different affinity, i.e., partition coefficient toward adsorbent phase which is stationary and later detected as shown in the figure below. A chromatogram so obtained helps in qualitative as well as quantitative analysis [6].
Various components of gas chromatography are [9]:
Carrier gas
Flow regulator
Injector
Column
Stationary phase
Oven
Detectors
Display device
The area covered by the peak represents the amount and position of a particular type of compound [6].
Operating conditions [10]:
Column: Porapak polymer bead 80–100 mesh or its equivalent, which can separate or resolve the ethanol.
Column temperature: 1600°C.
Carrier gas: Nitrogen.
Rate of gas flow: 50 ml/minute.
Detector: Flame ionization detector.
Alternative operating conditions:
Column: 0.3% Carbowax 20 M on 80–100 mesh Carbopak C, 2 m × 2 mm ID or its equivalent.
Column temperature: 350°C for 2 minutes and then programmed at 50°C per minute to 1750°C and hold for at least 8 minutes.
Carrier gas: Nitrogen at 30 ml/minute [6].
The purpose of this chapter is to discuss the mode of action and function of poisons once they reached in the human body. The impacts of poisons are severe and even cause death if not treated properly.
Edited by Jan Oxholm Gordeladze, ISBN 978-953-51-3020-8, Print ISBN 978-953-51-3019-2, 336 pages,
\nPublisher: IntechOpen
\nChapters published March 22, 2017 under CC BY 3.0 license
\nDOI: 10.5772/61430
\nEdited Volume
This book serves as a comprehensive survey of the impact of vitamin K2 on cellular functions and organ systems, indicating that vitamin K2 plays an important role in the differentiation/preservation of various cell phenotypes and as a stimulator and/or mediator of interorgan cross talk. Vitamin K2 binds to the transcription factor SXR/PXR, thus acting like a hormone (very much in the same manner as vitamin A and vitamin D). Therefore, vitamin K2 affects a multitude of organ systems, and it is reckoned to be one positive factor in bringing about "longevity" to the human body, e.g., supporting the functions/health of different organ systems, as well as correcting the functioning or even "curing" ailments striking several organs in our body.
\\n\\nChapter 1 Introductory Chapter: Vitamin K2 by Jan Oxholm Gordeladze
\\n\\nChapter 2 Vitamin K, SXR, and GGCX by Kotaro Azuma and Satoshi Inoue
\\n\\nChapter 3 Vitamin K2 Rich Food Products by Muhammad Yasin, Masood Sadiq Butt and Aurang Zeb
\\n\\nChapter 4 Menaquinones, Bacteria, and Foods: Vitamin K2 in the Diet by Barbara Walther and Magali Chollet
\\n\\nChapter 5 The Impact of Vitamin K2 on Energy Metabolism by Mona Møller, Serena Tonstad, Tone Bathen and Jan Oxholm Gordeladze
\\n\\nChapter 6 Vitamin K2 and Bone Health by Niels Erik Frandsen and Jan Oxholm Gordeladze
\\n\\nChapter 7 Vitamin K2 and its Impact on Tooth Epigenetics by Jan Oxholm Gordeladze, Maria A. Landin, Gaute Floer Johnsen, Håvard Jostein Haugen and Harald Osmundsen
\\n\\nChapter 8 Anti-Inflammatory Actions of Vitamin K by Stephen J. Hodges, Andrew A. Pitsillides, Lars M. Ytrebø and Robin Soper
\\n\\nChapter 9 Vitamin K2: Implications for Cardiovascular Health in the Context of Plant-Based Diets, with Applications for Prostate Health by Michael S. Donaldson
\\n\\nChapter 11 Vitamin K2 Facilitating Inter-Organ Cross-Talk by Jan O. Gordeladze, Håvard J. Haugen, Gaute Floer Johnsen and Mona Møller
\\n\\nChapter 13 Medicinal Chemistry of Vitamin K Derivatives and Metabolites by Shinya Fujii and Hiroyuki Kagechika
\\n"}]'},components:[{type:"htmlEditorComponent",content:'This book serves as a comprehensive survey of the impact of vitamin K2 on cellular functions and organ systems, indicating that vitamin K2 plays an important role in the differentiation/preservation of various cell phenotypes and as a stimulator and/or mediator of interorgan cross talk. Vitamin K2 binds to the transcription factor SXR/PXR, thus acting like a hormone (very much in the same manner as vitamin A and vitamin D). Therefore, vitamin K2 affects a multitude of organ systems, and it is reckoned to be one positive factor in bringing about "longevity" to the human body, e.g., supporting the functions/health of different organ systems, as well as correcting the functioning or even "curing" ailments striking several organs in our body.
\n\nChapter 1 Introductory Chapter: Vitamin K2 by Jan Oxholm Gordeladze
\n\nChapter 2 Vitamin K, SXR, and GGCX by Kotaro Azuma and Satoshi Inoue
\n\nChapter 3 Vitamin K2 Rich Food Products by Muhammad Yasin, Masood Sadiq Butt and Aurang Zeb
\n\nChapter 4 Menaquinones, Bacteria, and Foods: Vitamin K2 in the Diet by Barbara Walther and Magali Chollet
\n\nChapter 5 The Impact of Vitamin K2 on Energy Metabolism by Mona Møller, Serena Tonstad, Tone Bathen and Jan Oxholm Gordeladze
\n\nChapter 6 Vitamin K2 and Bone Health by Niels Erik Frandsen and Jan Oxholm Gordeladze
\n\nChapter 7 Vitamin K2 and its Impact on Tooth Epigenetics by Jan Oxholm Gordeladze, Maria A. Landin, Gaute Floer Johnsen, Håvard Jostein Haugen and Harald Osmundsen
\n\nChapter 8 Anti-Inflammatory Actions of Vitamin K by Stephen J. Hodges, Andrew A. Pitsillides, Lars M. Ytrebø and Robin Soper
\n\nChapter 9 Vitamin K2: Implications for Cardiovascular Health in the Context of Plant-Based Diets, with Applications for Prostate Health by Michael S. Donaldson
\n\nChapter 11 Vitamin K2 Facilitating Inter-Organ Cross-Talk by Jan O. Gordeladze, Håvard J. Haugen, Gaute Floer Johnsen and Mona Møller
\n\nChapter 13 Medicinal Chemistry of Vitamin K Derivatives and Metabolites by Shinya Fujii and Hiroyuki Kagechika
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I am also a member of the team in charge for the supervision of Ph.D. students in the fields of development of silicon based planar waveguide sensor devices, study of inelastic electron tunnelling in planar tunnelling nanostructures for sensing applications and development of organotellurium(IV) compounds for semiconductor applications. I am a specialist in data analysis techniques and nanosurface structure. 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