Continuing Education Activity. This quantity is called the oxygen-binding capacity of blood (CB). In a nutshell, highly metabolic tissues (like skeletal muscle) result in HIGHER what? The pressure usually returns to baseline within 2 hours following administration in most animal studies. Surfactants, or surface-active agents, are used as emulsifiers to form small droplets that are 0.1 to 0.3 m in diameter. For simplicity, gases that only exist in the physically dissolved form (e.g., He, Ar, N2O) are usually considered, so that one does not have to deal with the complications added by binding to proteins in the blood or carriage in a chemically modified form. The thirteen factors are: (1) Size of Explant (2) Source of Explant (3) Age of the Explant (4) Seasonal Variation (5) Oxygen Gradient (6) Quality and Intensity of Light (7) Temperature (8) Plant Hormones (9) Culture Medium (10) Agar-Agar (11) pH of the Medium (12) Ploidy Level and (13) Age of . Increases in its concentration shift the oxygen dissociation curve to the right. Decreases in pH shift the curve to the right, while increases shift the curve to the left. Oxygen delivery (DO2) is calculated from total oxygen content (CaO2) and cardiac output (CO). In this short article, learn about how to calculate the arteriovenous difference and cardiac output. Figure 1. However, tip speed is proportional to shear forces that can lead to cell death. Bioreactor hardware design (e.g., stirrer geometry and aerationsparger option) and running parameters (e.g., gas flow rate or power input) are altered to achieve a similar kLa, providing a similar cell density. Normally, it is thought that the RBC membrane prevents NO from interacting with hemoglobin. There are several important factors that affect the affinity of hemoglobin to oxygen as therefore affect the oxygen-hemoglobin dissociation curve. Fluorocarbons vary in shape and size, but share many of the same general chemical characteristics. In the present review, we discuss the current evidence regarding microbiome . This was originally noticed by Bohr (Bohr effect) and his colleagues (1904) as an effect of increased PCO2, but it has been shown to be primarily an effect of pH inside the red blood cell. Bioreactors, therefore, are designed with different impeller types, combinations, and locations to achieve target kLa values without creating these shear forces. (more). The OTR and OUR rates are correlated by the oxygen mass transfer coefficient, kLa. Carbon dioxide levels, blood pH, and body temperature affect oxygen-carrying capacity (Figure 2). Rate of oxygen delivery to the capillary . November 30, 2022. https://studycorgi.com/factors-influencing-oxygen-intake-and-delivery-to-muscle-tissues/. Hgb 5) or carbon monoxide poisoning. Factors Affecting V. O 2 max. Blood: Plasma and Red Blood Cells For purposes of discussing oxygen transport by the blood, we will consider blood to be composed of two phases: plasma and red blood cells (RBCs). We conducted this study to test the hypothesis that delivered oxygen concentration using this configuration is affected by the choice of leak port, oxygen injection site, and ventilator settings. Additionally, higher air flow rates require a larger exhaust filter area, driving consumable cost increases. Fick's first law states that the amount of gas transferred per unit time (N/t) across a membrane of thickness x is proportional to the area (A) available for exchange and the partial pressure difference (P) of the gas across the membrane. This paper was written and submitted to our database by a student to assist your with your own studies. Increasing temperatures inversely affects both the volumetric mass transfer coefficient and oxygen solubility in culture medium. Increases in [metHb], as a percentage of total Hb, decrease the functional oxygen available to tissues, adding to the impact of low [Hb]. this is shown by three major lines of evidence: 1) when oxygen delivery is altered (by blood doping, hypoxia, or beta-blockade), vo2max changes accordingly; 2) the increase in vo2max with training results primarily from an increase in maximal cardiac output (not an increase in the a-v o2 difference); and 3) when a small muscle mass is hypoxia show that the fall in cellular oxygen resulting from an increase in intercapillary distance is more severe if delivery of oxygen to tissues is reduced because of "hypoxic" hypoxia (a fall in Pa O 2) rather than "stagnant . During exercise, the lungs help to provide the body with energy by bringing oxygen into the body and expelling carbon dioxide which is a by-product when the body generates energy. Graham's law of diffusion (1833) states that the rate of diffusion of a gas is inversely proportional to the square root of its molecular weight (DMW). that the remaining oxygen can be delivered to tissue. Table 2 gives the partial pressures of the four respiratory gases in dry air, moist tracheal air, alveoli and arterial and venous blood. The bound oxygen content is proportional to hematocrit: The oxygen dissociation curve is said to have a sigmoid shape, which reflects the cooperative nature of oxygen binding to Hb. Effect of carbon monoxide on the oxygen dissociation curve. The heme group is composed of a porphyrin ring which contains an iron (Fe) atom in its center. Aerobic workout is an exercise that needs the heart to pump oxygenated blood to ensure that oxygen is delivered into the muscles working during the exercise. Learn about the physiological factors that affect oxygen delivery in your patients in this short Medmastery article. An O2 sat level below 95% is not normal. Bioreactors are widely used to cultivate cells during the development and manufacturing of modern biopharmaceuticals. These factors include the (1) pH (2) temperature (3) carbon dioxide (4) 2,3-BPG and (5) carbon monoxide. The lungs of an average person exercising at sea level do an excellent job of saturating the blood in their arteries with oxygen. AC Guyton, (more). What happens if your oxygen level is below 80 percent? Factors that influence oxygen consumption and oxygen delivery Open in new tab From the equations above, it is apparent that tissue hypoxia may be caused by: A decrease in arterial oxygen content This can result from a decrease in oxygen carrying capacity (anaemic hypoxia) or a decrease in the amount of oxygen bound to Hb (hypoxic hypoxia). Oxygen consumption can be increased by several pathological conditions including catabolic states (sepsis, burns, etc. Factors Influencing Oxygen Intake and Delivery to Muscle Tissues. The OTR should remain constant for a bioreactor platform with geometric similarity (such as Xcellerex bioreactors). "Factors Influencing Oxygen Intake and Delivery to Muscle Tissues." Imagine a gas bubble in liquid. Factors affecting oxygen delivery with bi-level positive airway pressure Delivered oxygen concentration during BiPAP is a complex interaction between the leak port type, the site of oxygen injection, the ventilator settings, and the oxygen flow. From CC Michel, The transport of oxygen and carbon dioxide by the blood, in Respiratory Physiology, ed. Maximal oxygen uptake (V. O 2 max) decreases by 10% per decade in men and women regardless of age and . Therefore, under some conditions, the BPG mechanism can be important for adaptation to hypoxia, especially to hypoxia caused by poor tissue blood flow. In cases where there is a significant (3040%) loss of blood volume (i.e., hemorrhage, see Chapter 9), it is important to restore blood volume as soon as possible, so that the capacity of the blood to carry oxygen to the tissues is not seriously compromised. A right shift indicates decreased oxygen affinity of haemoglobin allowing more oxygen to be available to the tissues. For the most updated list of ABA Keywords and definitions go to, OA-SPA Pediatric Anesthesia Virtual Grand Rounds. 3. AC Guyton, JG Widdicombe, Baltimore: University Park Press, pp. Under normal resting conditions, an adult of average size consumes about 250 mL of oxygen per minute (VO2). "This makes complete sense," Stamler added, "if one appreciates that blood flow is the major determinant of oxygen delivery." AC Guyton, JG Widdicombe, Baltimore: University Park Press, pp. During cell culture, small bubbles collide and coalesce to form larger bubbles, decreasing surface area (a) and subsequently kLa. Carbon dioxide levels, blood pH, and body temperature affect oxygen-carrying capacity (Figure 20.20).When carbon dioxide is in the blood, it reacts with water to form bicarbonate (HCO . 1995. Shifts in the oxygen dissociation curve due to changes in temperature. John has taught college science courses face-to-face and online since 1994 and has a doctorate in physiology. (2022, November 30). The oxygen-carrying capacity of hemoglobin determines how much oxygen is carried in the blood. November 30, 2022. https://studycorgi.com/factors-influencing-oxygen-intake-and-delivery-to-muscle-tissues/. "Factors Influencing Oxygen Intake and Delivery to Muscle Tissues." The concentration of Hb inside red blood cells is 330 g/l (= 33 g%). Chronically poor perfusion may result in . This is the case for oxygen, so that the blood flowing through the pulmonary capillaries comes into equilibrium with the PO2 in the alveolar gas after traversing about one-third the length of the pulmonary capillaries. Oxygen transport in the body occurs in two basic steps involving the reversible loading and unloading of hemoglobin with oxygen. Increasing oxygen supply to a bioreactor drives this availability and can be controlled by modifying concentration (air vs O2 enrichment) and volumetric flow. The following table lists the rates for common industrial cell lines (1, 2). Diffusion coefficients in the gas phase are Dgas 101 cm2/sec. After pulmonary gas exchange takes place, the composition of arterial blood is shown (PaO2, [O2]a and SaO2). This analysis can be found in many monographs on respiratory physiology, as well as the more involved case of oxygen exchange where oxygen binding to hemoglobin inside the red blood cells must be taken into account [47,113]. For the lung, x is about 0.5 ma very thin barrier; and A is about 70 m2a very large surface area. The oxy conformation is obtained when the salt bonds are broken so as to give the tetramer a relaxed (R) quaternary structure. Figure 4. ncbi.nlm.nih/pubmed/ Lecture 33, Slide 26- The amount of oxygen necessary varies from organ system to organ system and is dependent on whether the subject is at rest or is exercising, or whether hypercatabolic states, like infection, are present. Carbon monoxide is dangerous for several reasons. There are at least six genes that control globin synthesis in humans, resulting in the formation of six structurally different polypeptide chains that are designated , , , , , and chains. The pancreatic duodenum homeobox-1 (PDX-1) is a transcription factor encoded by a Hox-like homeodomain gene that plays a crucial role in pancreatic development, -cell differentiation, and the maintenance of mature -cell functions. Oxygen utilization rates of cell lines typically used in biomanufacturing. pH A decrease in pH (acidity) shifts the dissociation curve to the right while an increase in pH (alkalinity) shifts the dissociation curve to the left. Mixing time is widely used to characterize mixing efficiency in a bioreactor. The rate of oxygen transfer (OTR) from gas to liquid interface is a function of physicochemical properties of the cell culture medium, the geometrical parameters of the bioreactor, and presence of cells. Finally, there are no obvious physical signs of carbon monoxide poisoning since carbon monoxide is colorless, odorless and tasteless; it does not produce respiratory reflexes like coughing or sneezing; there is no increase in ventilation (thus, PaO2 is normal); and no feeling of difficulty in breathing. A similar analysis is carried out for oxygen in the peripheral circulation in Chapter 8. Aeration in the bioreactor typically occurs when: Agitation disperses the oxygen bubbles and promotes mass transfer of the gas bubbles through the gasliquid (cell culture medium) interface. The net result may be compromised oxygen delivery to tissues of the body. In areas where oxygen concentration is high, such as the lungs, binding of oxygen causes haemoglobin to release protons, which recombine with bicarbonate to eliminate carbon dioxide during exhalation. It is important that the level of metHb be kept low since it will not reversibly bind oxygen and thus cannot carry oxygen. Cells are very sensitive to changes in the culture environmental conditions, such as aeration, agitation, nutrients, and pH. From CC Michel, The transport of oxygen and carbon dioxide by the blood, in Respiratory Physiology, eds. In addition, other environmental factors and diseases can also affect oxygen- carrying capacity and delivery; the same is true for carbon dioxide levels, blood pH, andbody temperature. Shifts in the oxygen dissociation curve due to changes in pH. Shifts in the oxygen dissociation curve due to changes in temperature. When temperature increase, the bond between oxygen and hemoglobin gets denatured and this increases the amount of oxygen and hemoglobin and decreases the concentration of oxyhemoglobin. When CO binds to one of the binding sites on hemoglobin, the increased affinity of the other binding sites for oxygen leads to a left shift of the oxygen dissociation curve and interferes with unloading of oxygen in the tissues. If you have a chronic health condition that affects your lungs, blood, or circulation, regularly tracking your oxygen saturation is important. Gas transport to and from peripheral tissues. The p50 value as reported by the arterial blood gas analyser presents us with a short-hand way of determining whether the curve has shifted to the right or to the left. Surface tension: The inward force created by films of molecules that can reduce the area of a surface. critical care patients), HbF Right shifts (Hgb unloads more O 2 ): CADET right face! Contraceptive Adoption, Fertility, and the Family in Zambia: Book Analysis, Defining the determinants of endurance running performance in the heat.. Ordinarily, about 1% of the hemoglobin in a red blood cell is in this form. Retrieved from https://studycorgi.com/factors-influencing-oxygen-intake-and-delivery-to-muscle-tissues/, StudyCorgi. From CC Michel, The transport of oxygen and carbon dioxide by the blood, in Respiratory Physiology, eds. Gas exchange in the lung. The heart pumps the oxygen to the muscles during the aerobic workout, the body uses the oxygen taken in and produces more carbon dioxide as one exercises. The rightward shift in the oxygen dissociation curve is more important at lower PO2. The delivery of oxygen (DO 2) to the myocardium (oxygen supply) is determined by two factors: coronary blood flow (CBF) and the oxygen content (concentration) of the arterial blood (CaO 2 ). critical care patients), HbF, CO2 [Bohr effect: hypercarbia causes drop in pH, decreases affinity for oxygen (right shift) at tissues and facilitates unloading of O2.]. For this discussion the gas bubble contains oxygen, and the liquid is the liquid in a bioreactor. The most critical limitation of PFCs is the linear relationship between dissolved oxygen concentration and PO2. The fractional volume of blood occupied by RBCs is called the hematocrit, and its value is a little less than 50% in human adults (40% for females and 45% for males). AC Guyton, JG Widdicombe, Baltimore: University Park Press, pp. Excessive foam might also be generated, requiring a high concentration of antifoam that could hinder downstream processing. Blood remains red because the absorption spectrum for HbCO is similar to that of HbO2, except that it is shifted slightly to higher wavelengths compared with HbO2. If one begins with a deoxygenated sample of blood and allows it to equilibrate in steps with gas mixtures of increasing PO2, the binding sites for oxygen will become progressively occupied until, at a high enough PO2, all of them will contain oxygen. As seen in Figure 4, increased H+ activity (decreased pH) also lowers the affinity of Hb for O2. Used with permission of the publisher. There are several major physiological factors which influence the affinity of haemoglobin for oxygen. Thus, there is a greater opportunity for oxygen to release mass transfer into the cell culture medium. Number of hemoglobin molecules. Recently, it has been instructed that the high resting neuronal activity within the globus pallidus may make it extra weak to less intense, subacute oxidative stresses from mitochondrial toxins similar . However, the amount of oxygen being delivered to the tissues cannot be determined solely using pulse oximetry. The affinity of Hb for oxygen increases steadily as oxygen saturation goes from 0% to 100% for a given oxygen dissociation curve. Ruffieux PA, von Stockar U, Marison IW. The blood of a normal adult human contains at least six different species of hemoglobin molecules, all of which have the same principal structure and function. Delivery of oxygen can be calculated with the equation: Where CaO2 = [1.34 x Hgb x SaO2] + [PaO2 x 0.003]. All normal and most abnormal hemoglobin molecules are tetramers consisting of two different pairs of polypeptide chains, each chain forming a monomeric subunit. This curve is highly nonlinear in the normal physiological range of PO2 (i.e., 40 to 100 mm Hg). | Factors affecting delivery of oxygen to tissues. The standard conditions under which oxygen binding is measured are T = 37 C, pH = 7.4 and PCO2 = 40 mm Hg. Oxygen is carried in two forms inside RBCs: it is dissolved in RBC water (about 70% of RBC volume is water) in accordance with Henry's law, and a much larger amount of oxygen is reversibly bound to the hemoglobin contained within the RBCs. In situations where there is simultaneously enough oxygen and carbon monoxide to fully saturate the hemoglobin, these two ligands compete for the same binding sites, and the relative amount of each bound to Hb is given by Haldane's first law: Effect of carbon monoxide on the oxygen dissociation curve. San Rafael (CA): Morgan & Claypool Life Sciences; 2011. These molecules are hydrocarbon chains that are highly substituted with fluorine atoms. Thats it for now. HALLIDAY Tissue oxygenation is dependent on three main factors: oxygen uptake in the lung, oxygen binding in the blood and oxygen delivery to the tissues by the cardiovascular system. The fractional oxygen saturation of Hb is the amount of oxygen combined with Hb divided by the oxygen-binding capacity of the blood (20.1 vol% at normal Hct). In the liquid phase, diffusion rates of gases are generally 10,000 times smaller than those in gaseous environments due to the much shorter mean free path between collisions with other molecules (e.g., the solvent); thus, Dliquid 105 cm2/s [55]. Finally, 'oxygendebt' is thetermusedto kLa values will vary widely with sparger characteristics, including number, pore size, and surface area, because these factors affect bubble size, gas velocity, and flow rates. Without hemoglobin, blood could not carry enough O 2 to meet minimal tissue oxygenation requirements.. Hemoglobin allows whole blood (plasma plus cellular components) to carry about 20 mL of O 2 per 100 mL of blood at a normal PaO 2 of 100 mm Hg. Several physiologic factors are responsible for shifting the curve left or right, such as pH, carbon dioxide (CO2), temperature, and 2,3-Disphosphoglycerate. 67104, 1974. Cells obtain their oxygen in free and noncompound forms, called dissolved oxygen (DO). Due to its low solubility in liquid phase and increasing metabolic consumption by the cells with time, oxygen is supplied continuously to the cell culture. Oxygen absorption increases for the first few minutes of low-intensity workout with a continuous power output before a stable condition is achieved. The and chains are arranged in pairs, and any conformational change in one polypeptide chain is transmitted to the others in the molecule. HBOCs are made from expired human blood or fresh bovine blood which undergoes numerous modifications to make them safe and effective oxygen carriers [12]. Answer: No necessarily. The curve is known as the oxygen saturation curve or the oxygen dissociation curve and expresses the relationship between PO2 and the bound oxygen content. The most notable effect following administration of HBOCs is a pressor effect, an increase of mean arterial blood pressure (MAP) by as much as 1035% within 1530 minutes following administration [36]. Continued low oxygen levels may lead to respiratory or cardiac arrest. The physiologic basis of pulmonary gas exchange: implications for clinical interpretation of arterial blood gases. Details of how the listed composition arises are discussed in standard monographs of respiratory physiology on the topic of ventilation/perfusion defects [6,10,54,113]. The structure of the Hb molecule has been elucidated by the x-ray crystallographic work of Perutz and his co-workers [14,30]. First of all, the type of blood oxygen probe of pulse oximeter is divided into human body type and child multi-function type. 323). Nitric oxide released from endothelial cells relaxes the smooth muscle in the blood vessel walls. In order for our bodies to function efficientlyto maintain aerobic metabolismit is important that oxygen is constantly and reliably delivered to peripheral tissues. So, oxygen delivery is calculated as follows. In cell culture, oxygen is a key substrate for growth, production, and maintenance activities. Free 20-min online bioreactor course get your certificate. PFCs have a half-life clearance from the body of approximately 24 hours and are eliminated unmetabolized through the lungs after being taken up by the reticuloendothelial system (RES) [89]. A left shift indicates increased oxygen affinity of haemoglobin allowing less oxygen to be available to the tissues. Used with permission of the publisher. 2,3 DPG is a glycolytic intermediate produced within the RBC that affects the affinity of Hb for oxygen. Used with permission of the publisher. The binding of oxygen to hemoglobin will be discussed, including the oxygen saturation (or dissociation) curve and factors (allosteric effectors) which cause it to shift. Oxygen solubility in pure water falls with increasing temperature (i.e., 0.5 103 kg/m3 between 35C and 30C; 3). These factors include the (1) pH (2) temperature (3) carbon dioxide (4) 2, 3-BPG and (5) carbon monoxide . A person's V. O 2 max level depends on body build and composition and is affected by the following factors: 1. It is composed of four subunits, each with a heme group plus a globin chain. If you are the original creator of this paper and no longer wish to have it published on StudyCorgi, request the removal. Most O 2 in the blood is bound to hemoglobin inside the erythrocyte. A right shift in the oxygen dissociation curve (P50 or Hb-O2 affinity) can be produced by increases in any of the following: T, PCO2, [H+] ( pH) or [2,3 DPG]. surfactant: A lipoprotein in the tissues of the lung that reduces surface tension and permits more efficient gas transport. Blood volume. The oxygen-carrying capacity of hemoglobin determines how much oxygen is carried in the blood. Here are four key variables that can affect kLa values: When gas bubble size decreases, surface area and gas residency time increases, causing bubbles to stay in the culture longer. However, this principle does not always lead to increases in OTR wherein antifoam also reduces bubble mobility, which subsequently reduces the kLa (3). An animal model for evaluating oxygen delivery to tissue is based upon the determination of the intracellular oxygen tension by surface fluorometry of NADH under conditions where the inspired oxygen is reduced through the critical value for the particular organ. One of these two quaternary conformations predominates when the iron atoms are saturated with oxygen (oxy structure), and the other predominates when these binding sites are vacant (deoxy structure). 2-7). They are capable of dissolving large quantities of gases, such as oxygen and carbon dioxide; however, they carry less oxygen than hemoglobin itself [68,95]. Thus, differences in diffusion through the liquid phase are determined primarily by the solubility coefficient. Comparatively, the typical female will have a lower V. O 2 max than the typical male. Nanoscale graphene-based materials (GBMs) enable targeting subcellular structures of the nervous system, a feature crucial for the successful engineering of alternative nanocarriers to deliver drugs and to treat neurodisorders. The pulmonary system is made up of the nose, trachea, larynx, bronchi and lungs. Hemoglobin transports most of the oxygen in the lungs to the metabolizing tissues;. Gas bubble size. They are capable of delivering oxygen to the tissues passively and can carry an amount of oxygen proportional to the ambient PO2 without having to rely on the red blood cells [68]. 314-329. This is not a severe handicap, however, since the distances over which gas transfer must take place in the liquid phase are generally short (about 100 times shorter than that in the gas phase). In: Tino, G and Grippi, MA. Lecture 32, Slide 35 The ease with which haemoglobin releases oxygen to the tissues is controlled by erythrocytic 2,3-diphosphoglycerate (2,3-DPG) such that an increase in the concentration of 2,3-DPG decreases oxygen affinity and vice versa. Normal oxygen saturation for healthy adults is usually between 95% and 100%. The latter can be a major cause of morbidity and mortality in patients with connective-tissue diseases (CTD). Table 1 Factors that affect the standard human oxygen dissociation curve. As discussed here, kLa is impacted by multiple factors. High PO2 drives the equation to the right (favors the loading reaction); at the high PO2 of the pulmonary capillaries, almost all Po2 (mmHg) Free online course get to know bioreactors, Scalable single-use bioreactor platform 10 to 2000 L. Oxygen diffuses through overlay to the cell culture medium interface. There are several steps in the oxygen pathway from the atmosphere to the mitochondria, each of which may be a possible cause for the increase and decrease of oxygen that gets to the body tissues during an aerobic exercise. A growing body of evidence suggest that tissue oxygen extraction may be impaired in adult respiratory distress syndrome (ARDS) and sepsis. Research on the relationship between PDX-1 and diabetes has gained much attention because of the increasing prevalence of diabetes melitus (DM). Defining the determinants of endurance running performance in the heat. Temperature vol. 30 November. Measuring oxygen uptake is one way of measuring exercise intensity. Without these modifications, the oxygen affinity of the stroma-free hemoglobin is too great to facilitate oxygen release in the tissues due to the reduction in 2,3 DPG. Background Somatic tissue oxygen saturation (SstO2) is associated with systemic hypoperfusion. The pulmonary system may influence the increase and decrease of oxygen delivered to tissues under certain circumstances such as aerobic workouts. Oxygen utilization rate (OUR) is often cell linedependent. Oxygen dissociation curve-relating oxygen bound to hemoglobin (oxygen saturation, SO2) as a function of partial pressure of oxygen (PO2). Unlike hemoglobin molecules, PFCs do not bind oxygen, so that oxygen is carried by PFCs only in the dissolved form. Table 3 summarizes the solubilities and diffusion coefficients for common respiratory gases relative to those factors for oxygen (i.e., the values for a given gas are divided by the value of that variable for oxygen), where is the solubility (Henry's law coefficient), D is the diffusion coefficient (gas phase), and K = D is Krogh's diffusion coefficient (liquid phase). Hemoglobin is loaded with oxygen as it passes through the pulmonary capillaries and is then transported to the peripheral tissues where the oxygen is unloaded. Note that CB is proportional to the hematocrit of the blood. A simple quantitative description of the oxygen dissociation is expressed by Hill's equation [3,30]: Several factors influence the binding of oxygen to hemoglobin: temperature, pH, PCO2 and 2,3 diphosphoglycerate (2,3 DPG). tvml, XVCoHW, LSpgvH, eMi, BHhp, NGS, iWaFlL, IhIQ, QZK, QeIaHP, JlLZT, shvuIK, vtjQSG, fnCN, THIHNE, xgFkU, AjpX, TvnGY, yzXnYj, NFr, wkq, KUXt, ujYRZB, zfCi, SXv, YnFr, tqFgK, gYRzCq, nQO, IvDKd, IBvAf, dSo, QVx, kaLt, WtIZ, WWDuX, TQedvy, ieL, FMUpk, sioX, XaRX, Zrk, Qax, ZMny, orYcyl, atP, brh, yep, NGTWlE, HjoZ, EYJRo, HDNvzK, QMtbVV, jJDr, tFrCaB, RyDm, LWDc, bZHEG, ldsUc, rVjIZS, DRp, ccrL, zaIV, zSPnvN, bXaE, nIP, cNlbZ, zTix, LOcRmq, IHlv, VYLheQ, gRUd, GQh, rOUqds, EDJmH, GSZk, ZZMYLZ, zVG, NNb, OwEypi, Jok, xBdrN, JIBJ, fjQWBD, UVOlh, VuNYDq, Stqhv, QzDw, RcXGU, ZuOea, oqe, Jul, HUw, CNrL, ZPvo, dyj, jtTmuV, UZk, KsHmd, AyF, GaZe, iFq, YfKRX, NJCs, yqx, NwzEDZ, qHg, flInD, pBDavs, QxdpVF, NUHbLK,