Hypoxia Acidosis Research Articles

Effect of peritoneal dialysis solution with different pyruvate concentrations on intestinal injury.

To investigate the effects of direct peritoneal resuscitation with pyruvate-peritoneal dialysis solution (Pyr-PDS) of different concentrations combined with intravenous resuscitation on acid–base imbalance and intestinal ischemia reperfusion injury in rats with hemorrhagic shock. Sixty rats were randomly assigned to group SHAM, group intravenous resuscitation, and four direct peritoneal resuscitation groups combined with intravenous resuscitation: group NS, LA, PY1, and PY2, that is, normal saline, lactate-PDS (Lac-PDS), lower concentration Pyr-PDS (Pyr-PDS1), and higher concentration Pyr-PDS (Pyr-PDS2), respectively. Two hours after hemorrhagic shock and resuscitation, the pH, oxygen partial pressure, carbon dioxide partial pressure (PCO2), base excess, and bicarbonate ion concentration (HCO3−) of the arterial blood were measured. The intestinal mucosal damage index and intercellular adhesion molecule 1 (ICAM-1), tumor necrosis factor-α, interleukin-6, zonula occludens-1, claudin-1, and occludin levels in intestinal issues were detected. Two hours after resuscitation, group PY2 had higher mean arterial pressure, pH, oxygen partial pressure, and base excess and lower PCO2of arterial blood than group PY1 ( P < 0.05). Tumor necrosis factor-α and interleukin-6 levels in group PY2 were significantly lower than those in group PY1 ( P < 0.05). Zonula occludens-1, claudin-1, and occludin expression levels were significantly higher in group PY2 than in group PY1 ( P < 0.05). Direct peritoneal resuscitation with Pyr-PDS2 combined with intravenous resuscitation enhanced the hemodynamics, improved the acid–base balance, and alleviated intestinal ischemia reperfusion injury from hemorrhagic shock and resuscitation in rats. The mechanisms might include correction of acidosis, inhibition of inflammatory response, enhancement of systemic immune status, regulation of intestinal epithelial permeability, and maintenance of intestinal mucosal barrier function.Impact statementHemorrhagic shock is a life-threatening condition after trauma or during surgery. Acid–base imbalance and intestinal ischemia reperfusion injury are two significant causes in the pathogenetic process and multiple organ dysfunction. As a result, it is urgent and necessary to find an effective method of resuscitation in order to reverse the acid–base imbalance and protect organ function. This current study confirmed the protection against hypoxic acidosis and intestinal ischemia reperfusion injury by peritoneal resuscitation with pyruvate combined with intravenous resuscitation in rats with hemorrhagic shock. And the peritoneal dialysis solution with pyruvate of high concentration plays a crucial role in the process. It provided a new idea and possible direction of fluid resuscitation for alleviating organ injuries, protecting organ functions, and improving clinical prognosis after hemorrhagic shock and resuscitation.

A Preliminary Computational Model for Hypoxic Acidosis in Cardiac Myocytes.

Intracellular acidosis induced by hypoxia resulted from myocardial ischemia damages myocardium. However, the detailed mechanisms of hypoxic acidosis are not quantitatively explained. The purpose of this study is to create a novel computational model which can reproduce intracellular acidosis caused by myocardial ischemia. We constructed a computational model of myocardium, by using a mathematical ventricular cell model which includes pH regulation and a computational model of myocardial microcirculation for calculating extracellular conditions. The present model reproduced cellular hypoxia in an ischemic condition simulated by a reduced blood flow, and intracellular pH reduction in response to the hypoxia.

The relationship between acidosis and hypercapnia with Cor pulmonale in patients with chronic obstructive pulmonary disease

Introduction: Cor pulmonale or right ventricular (RV) enlargement is cardiovascular complication of the chronic obstructive pulmonary disease (COPD). Hypoxic pulmonary vasoconstriction, hypercapnia, acidosis and pulmonary vascular remodeling are suggested as possible mechanisms of cor pulmonale. In this study, we aimed to evaluate the correlation between acidosis and hypercapnia with corpulmonale in patients with COPD. Methods: In this cross-sectional analytical study, 100 patients (56 men and 44 women with mean age of 66.53 ± 10.63 years) with moderate to severe COPD exacerbation were included. Complete history taking and physical examination as well as atrial blood gas, pulmonary function test (PFT) and echocardiography were performed. Disease severity was defined according to global initiative for obstructive lung disease (GOLD), Modified Medical Research Council (mMRC) and COPD Assessment Test (CAT) criteria. Patients with cor pulmonale were defined and findings were compared between patients with and without cor pulmonale. Results: Forty-two patients had cor pulmonale. There was no significant difference in hypercapnia between groups. Cor pulmonale patients, compared to non-cor pulmonale, had significantly lower forced expiratory volume in the first second (FEV1) (P = 0.020), higher tricuspid regurgitation (TR) (P = 0.001) and pulmonary hypertension (P = 0.020). There was significantly negative correlation between RV thickness with FEV1/forced vital capacity (FCV) (r = -0.239, P = 0.010) and RV size with FEV1/FVC (r = -0.312, P = 0.002) and positive correlation with partial pressure of carbon dioxide (PaCO2) (r = 0.312, P = 0.002) and bicarbonate (HCO3) (r = 0.258, P = 0.009). Conclusion: Cor pulmonale in the course of COPD accompanies with adverse outcome. These patients have worse spirometry and left ventricle echocardiographic findings, but have no difference in arterial blood gas (ABG) findings.

Sutureless anastomoses using magnetic rings in canine liver transplantation model

BackgroundIn the first posttransplant month, the most frequent complications are due to technical problems related to complex vascular and bile duct reconstructions during the operation. Moreover, despite great improvements in suturing technique and materials, severe organ ischemia-reperfusion caused by time-consuming hand suturing is still an important factor in graft survival. During the operation, severe hypotension, hypoxic acidosis, hyperkalemia, and renal dysfunction may occur during the anhepatic phase due to the prolonged venous clamping time required for hand suturing. Therefore, hand suturing is a handicap in the development of further advancements in liver transplantation. In this study, we aimed to test a new “mechanical installation method” for rapid vascular reconstruction. MethodsThe magnetic pinning-ring device was developed consisting of paired magnetic rings coated with titanium oxide and embedded in a polypropylene shell. The rings were equipped with alternately spaced holes and titanium pins. Forty adult mongrel dogs were randomly divided into groups: A (n = 16), all vascular and bile duct reconstruction by magnetic ring without venous bypass; B (n = 16), all vascular and bile duct reconstruction by hand suturing with venous bypass; C (n = 8), sham transplantation group, transection of all vessels and common bile duct followed by anastomosis with the magnetic rings without liver transplantation. From groups A and B, dogs were randomly selected as donors (n = 8) or recipients (n = 8) of liver transplantations. We recorded operation time, vascular and bile duct anastomosis time, anhepatic time, administration of supplemental fluids during operation, and survival; blood samples were collected for the detection of liver damage (alanine aminotransferase [ALT] and aspartate aminotransferase [AST]) and tumor necrosis factor α level. Patency was confirmed using ultrasound scans at various time points as late as 24 wk after surgery. Angiography was used to evaluate the anastomoses formed with magnetic rings. In group C, gross observation, histologic staining, and scanning electron microscopy were used to evaluate the vessels and bile ducts 12 wk postoperatively. ResultsIn group A, the total operation time, inferior vena cava, and portal vein anastomosis times were significantly shortened, and the anhepatic phase was reduced to about one-fifth that of group B, which was a significant difference between the two groups (P < 0.01). The mean total operative time was 2.54 ± 0.45 h. In order to maintain adequate blood pressure, the mean fluid volume infused was 800.56 ± 60.56 mL in the recipients of group A, which was lower than that in group B (2241.67 ± 390.78 mL, P < 0.01). Use of a pressor agent in group A was unnecessary. After operation, five of eight animals in group A survived more than 7 d after operation. The main cause of death was acute rejection. Only three of eight animals in group B survived more than 1 wk after operation due to chronic anastomotic bleeding, kidney failure, heart failure, and gastrointestinal bleeding. There was a statistically significant difference (P < 0.01) between the short-term survival rate in the two groups (75.0% versus 37.5%). The ALT (1544.46 ± 286.27) U/L and AST (1710.74 ± 252.27) U/L levels after operation in the animals with hand suturing were significantly higher than those in the sutureless group (ALT = 1116.41 ± 210.55 U/L; AST = 1176.95 ± 248.25) U/L after reperfusion (P < 0.01). The serum tumor necrosis factor α levels (45.56 ± 10.78) ng/L in group B were significantly higher than those of group A (26.64 ± 10.84) ng/L after reperfusion (P < 0.01). Re-endothelialization was confirmed in all vessels in group C, with neither formation of aneurysms nor thickening of the vascular wall noted after 12 wk. The bile duct anastomoses also healed well. ConclusionsThe magnetic pinning-ring device offers a simple, fast, reliable, and efficacious technique for nonsuturing vascular and bile duct anastomoses. Use of this device shortens operation time, maintains a high patency rate, and improves the healing of tissue. Application of the magnetic ring anastomosis technique can effectively reduce the complications caused by hand suturing, and can reduce the extent of ischemia-reperfusion injury, leading to smoother operations and improved prognosis.

Abstract A18: Genetic elements of cancer cell survival in tumor microenvironment stresses

Abstract As solid human cancer tumors form there are a number of physiological changes that occur within the tumor itself, including low oxygen levels (hypoxia) and an accumulation of lactic acid with concomitant lowered pH levels (lactic acidosis). In order to adapt to and survive in these tumor microenvironmental (TME) stresses, human cancer cells in solid tumors exhibit significant genetic and metabolic changes. While transcriptional responses and downstream signaling events are known under certain stresses, a systematic genome-wide investigation of which genes are critical or restrictive for cell survival in these TME stresses has not been examined. The central hypothesis of this research is that understanding the function of genes that modulate cell survival under TME stresses will allow for the development of treatments to specifically target cancer cells under these stress conditions. To investigate this hypothesis, the concept of synthetic lethality was applied to cultured cancer cells by combining individual gene expression knockdown with the application of hypoxia or lactic acidosis. Specifically, we transduced lung cancer cells with the genome-wide, pSM2C short-hairpin RNA (shRNA) retroviral library before subjecting them to a hypoxic or lactic acidosis stress in culture. Genomic DNA was recovered from surviving cells and the resulting shRNAs amplified by PCR before de-convolution by microarray. A number of criteria were used to select top candidate hits, including a cut off of multiple shRNAs targeting a single gene needing to pass stringent statistical criteria. We have successfully validated a number of candidate genes with multiple short-hairpin RNAs. For example, HIF2a was identified and validated as critical for these lung cancer cells to survive hypoxia. Other top candidate genes have a wide variety of unexpected biological functions, as could only be found in a genome-wide screen. A top gene candidate indicates that blocking lipid synthesis protects cells under hypoxia. This protection is HIF-independent and more specific to a low serum growth condition. Another top candidate gene reveals a new role for a transcription factor previously implicated in leukemias and lymphomas as important for cell survival under lactic acidosis. Detailed mechanistic studies of these findings are ongoing. The goal of these studies is to realize how these genes impact the survival of cancer cells under TME stresses and how they play a role in the selection of somatic mutations under long-term stresses. Accomplishing this goal will allow for a better understanding of the genetic changes selected by the TME and improve therapies to target solid human cancers in the future. Citation Format: Melissa M. Keenan, Beiyu Liu, Jianli Wu, Derek Cyr, Joseph Lucas, Joseph Nevins, So Young Kim, Jen-Tsan Chi. Genetic elements of cancer cell survival in tumor microenvironment stresses. [abstract]. In: Proceedings of the AACR Precision Medicine Series: Synthetic Lethal Approaches to Cancer Vulnerabilities; May 17-20, 2013; Bellevue, WA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(5 Suppl):Abstract nr A18.

Neonatal exposure to high concentration of carbon dioxide produces persistent learning deficits with impaired hippocampal synaptic plasticity

Although respiratory complications with blood gas abnormalities contribute significantly to neurodevelopment in the immature brain, little is known about the mechanisms via which blood gas abnormalities, such as hypoxic hypercapnia, impair neurocognitive outcomes. To investigate the possible long-term consequences of neonatal exposure to hypoxic hypercapnia regarding learning ability, we investigated the effect of neonatal hypoxic hypercapnia on later functions in the hippocampus, which is a structure that has been implicated in many learning and memory processes. Neonatal rat pups (postnatal day 7; P7) were exposed to a high concentration of carbon dioxide (CO2; 13%) for 2 or 4h. Exposure to CO2 in P7 rat pups caused blood gas abnormalities, including hypercapnia, hypoxia, and acidosis, and disrupted later learning acquisition, as assessed in 10-week-old adult rats subjected to a Morris water maze test. Induction of long-term potentiation (LTP) in the synapses of the hippocampal CA1 area was also impaired, whereas the paired-pulse responses of population spikes exhibited a significant increase, in CO2-exposed rats, suggesting decreased recurrent inhibition in the hippocampus. Such long-lasting modifications in hippocampal synaptic plasticity may contribute to the learning impairments associated with perinatal hypoxic hypercapnia and acidosis.

DNase activation by hypoxia–acidosis parallels but is independent of programmed cell death

AimsHypoxia, acidosis and programmed cell death are each hallmarks of acute myocardial infarction (AMI). We previously described a death pathway of cardiac myocytes mediated by hypoxia–acidosis that was characterized by activation of the Bcl2-family protein Bnip3 and programmed necrosis. The pathway included extensive DNA fragmentation that was sensitive to inhibition of the mitochondrial permeability transition pore (mPTP) and calpain inhibitors, but not caspase inhibitors. We did not identify the DNases responsible for DNA cleavage. Main methodsNeonatal rat cardiomyocytes were subjected to hypoxia with and without concurrent acidosis, and the cellular localization of apoptosis-inducing factor (AIF), DNase II and caspase-dependent DNase (CAD) were determined. Key findingsHere we report the occurrence of biphasic pH-dependent translocations of AIF and DNase II but no change in CAD or its inhibitor ICAD. AIF co-localized with the mitochondria under aerobic and hypoxia-neutral conditions but translocated to the nucleus at pH ~6.7 coincident with a decrease of the mitochondrial membrane potential. DNase II co-localized with lysosomes under normoxia and hypoxia-neutral conditions, and translocated to the nucleus at pH ~6.1 coincident with the appearance of single strand DNA cuts. Inhibition of the mPTP pore with BH4-TAT peptide, calpain inhibition with PD150606, or knockdown (KD) of Bnip3 failed to prevent nuclear translocation of these DNase although Bnip3 KD blocked mitochondrial fission. SignificanceThese results suggest that caspase-independent DNA fragmentation is precisely regulated and occurs in parallel but independently from programmed necrosis mediated by hypoxia–acidosis.

Mechanism of glutathione depletion during simulated ischemia-reperfusion of H9c2 cardiac myocytes

It has been reported that myocardial glutathione content is decreased during ischemia-reperfusion, but the mechanism of glutathione depletion has remained unclear. The present study tested whether osmotic stress is involved in the glutathione depletion during ischemia. Six hours of hypoxic acidosis with either high CO2 tension or low HCO3− concentration, which simulates the ischemic condition, resulted in a significant decrease of glutathione content and the glutathione depletion was prevented by hyperosmolarity. High-CO2 acidosis alone without hypoxia induced a similar degree of glutathione depletion. Intracellular pH was lowered by high-CO2 acidosis to 6.41 ± 0.03 in 15 min. Meanwhile, the cell size gradually increased and reached ∼110% in 10 min and the increased cell size was maintained for at least 30 min, which was also prevented by hyperosmolarity. Subsequent experiments observed the effects of simulated reperfusion on the glutathione content. Measured in 1 h after the hypoxic acidotic reperfusion, the glutathione content was further decreased compared to the level at the end of ischemia, which was not suppressed by increasing the osmolarity of reperfusion solution. The degree of glutathione depletion during hypoxic reperfusion with normal pH was similar to the hypoxic acidotic reperfusion group. On the other hand, normoxic reperfusion was not accompanied by further depletion of glutathione content. Based on these results, it was concluded that ischemia induces the glutathione depletion via osmotic stress, which results from intracellular acidification, and the glutathione content is further decreased during reperfusion through a mechanism other than oxygen toxicity.

Nitrites can be reduced in retinal vessels during hypoxia and protect the retina against ischemia and apoptosis

The possibility and the mechanism of the reduction of nitrites in retinal vessels under the acute hypoxia in vivo have been investigated. An experimental model of the rat retina ischemia was elaborated using the laser coagulation of retinal vessels. It was demonstrated that the vessel thrombosis does not occur if the nitrite concentration in the vessels is increased. It was proposed that, under acute hypoxia, nitrites are reduced to NO, which results in a drastic vasodilatation. Considering that the effect takes less than a minute, this reduction cannot be due to hypoxic acidosis but is more likely associated with NO reduction by heme proteins. It was found that the increased concentration of nitrites protects the retina from the development of ischemia progress and that the preliminary administration of nitrites prevents apoptosis in the retina and a decrease in its photoelectric activity.

Near-drowning and clinical laboratory changes

Opposite to clinical laboratory findings in experimental drowning of animals (erythrocytic lysis, hyperkalemia, and final cardial fibrillation) are the observations in drowned humans (increase of pCO 2, hypoxic encephalopathy), which leads to a different pathophysiological interpretation of the drowning process. This process, however, is recently discussed again, therefore an additional study seemed to be recommended. In a retrospective study, 31 cases of near-drowning (23 cases: fresh water; 8 cases: brackish water) clinical laboratory data were analysed. While 21 of the cases were fatal with a delay of up to 180 days, 10 individuals survived the accident, four cases with severe neurological deficits. Data of pH, potassium, sodium, chloride, hemoglobin and total protein were collected during the very early post-drowning period. Nearly all cases (96%) revealed a reduction of pH due to hypoxic acidosis, and only two cases (6.5%) exhibited a slight hyperkalemia. The hemoglobin level was normal in most of the cases (83%) and slightly reduced in the others (17%) while the protein level was slightly reduced in most of the fatalities (80%). As a result of our investigation we have to state the lack of hyperkalemia as well as of an increase of the hemoglobin level indicate that there is no distinct intravascular red cell lysis due to influx of water into the vascular compartment. Therefore the death by drowning in humans in most cases is the result of a hypoxic cerebral process. A comparison with animal experiments obviously is not helpful because the drowning process in humans leads to an aspiration of only 2–4 ml water/kg, while in animal experiments more than 10 ml water/kg will be artificially aspirated leading to red cell lysis as well as to electrolyte disturbances and cardial fibrillation.

Purinergic P2Y 2 receptors promote hepatocyte resistance to hypoxia

ATP stimulation of purinergic P2 receptors (P2YR and P2XR) regulates several hepatic functions. Here we report the involvement of ATP-mediated signals in enhancing hepatocyte tolerance to lethal stress. The protection given by purinergic agonists was investigated in rat hepatocytes exposed to hypoxia. ATP released after hypotonic stress (200 mOsm/L) as well as P2YR agonists prevented hepatocyte killing by hypoxia with efficiency ranking UTP > ATPgammaS > ADPbetaS, whereas the P2XR agonist, methylene-adenosine-5'-triphosphate, was ineffective. Adenosine-5'-O-3-thiotriphosphate (ATPgammaS; 100 micromol/L) also prevented Na+ -overload in hypoxic cells by inhibiting the Na+/H+ exchanger, without interfering with hypoxic acidosis. ATPgammaS activated Src and promoted a Src-dependent stimulation of both ERK1/2 and p38MAPK. Blocking p38MAPK with SB203580 reverted the protection given by ATPgammaS on both cell viability and Na+ accumulation, whereas ERK1/2 inhibition with PD98058 was ineffective. An increased phosphorylation of ERK1/2 was also evident in untreated hypoxic hepatocytes. PD98058 ameliorated Na+ accumulation and cell death caused by hypoxia. Hepatocyte pre-treatment with ATPgammaS reverted ERK1/2 activation in hypoxic cells. SB203580 blocked the effects of ATPgammaS on both ERK1/2 and Na+/H+ exchanger. The activation of p38MAPK by P2Y2R increases hepatocyte resistance to hypoxia by down-modulating ERK1/2-mediated signals that promote Na+ influx through the Na+/H+ exchanger.

A unique pathway of cardiac myocyte death caused by hypoxia-acidosis.

Chronic hypoxia in the presence of high glucose leads to progressive acidosis of cardiac myocytes in culture. The condition parallels myocardial ischemia in vivo, where ischemic tissue becomes rapidly hypoxic and acidotic. Cardiac myocytes are resistant to chronic hypoxia at neutral pH but undergo extensive death when the extracellular pH (pH[o]) drops below 6.5. A microarray analysis of 20 000 genes (cDNAs and expressed sequence tags) screened with cDNAs from aerobic and hypoxic cardiac myocytes identified >100 genes that were induced by >2-fold and approximately 20 genes that were induced by >5-fold. One of the most strongly induced transcripts was identified as the gene encoding the pro-apoptotic Bcl-2 family member BNIP3. Northern and western blot analyses confirmed that BNIP3 was induced by 12-fold (mRNA) and 6-fold (protein) during 24 h of hypoxia. BNIP3 protein, but not the mRNA, accumulated 3.5-fold more rapidly under hypoxia-acidosis. Cell fractionation experiments indicated that BNIP3 was loosely bound to mitochondria under conditions of neutral hypoxia but was translocated into the membrane when the myocytes were acidotic. Translocation of BNIP3 coincided with opening of the mitochondrial permeability pore (MPTP). Paradoxically, mitochondrial pore opening did not promote caspase activation, and broad-range caspase inhibitors do not block this cell death pathway. The pathway was blocked by antisense BNIP3 oligonucleotides and MPTP inhibitors. Therefore, cardiac myocyte death during hypoxia-acidosis involves two distinct steps: (1) hypoxia activates transcription of the death-promoting BNIP3 gene through a hypoxia-inducible factor-1 (HIF-1) site in the promoter and (2) acidosis activates BNIP3 by promoting membrane translocation. This is an atypical programmed death pathway involving a combination of the features of apoptosis and necrosis. In this article, we will review the evidence for this unique pathway of cell death and discuss its relevance to ischemic heart disease. The article also contains new evidence that chronic hypoxia at neutral pH does not promote apoptosis or activate caspases in neonatal cardiac myocytes.

Variability in hypoxia-induced corneal swelling is associated with variability in corneal metabolism and endothelial function.

To determine if the variability in contact lens-induced corneal swelling is associated with variability in corneal oxygen consumption (Q(C)) or corneal endothelial function. Corneal swelling was induced in 30 non-contact lens wearers by 2 hours of closed-eye contact lens wear, using thick (oxygen transmission [Dk/t] = 4.0 x 10-9) and thin (Dk/t = 12 x 10-9) hydrogel lenses of identical design. Following the induction of swelling, corneal thickness was continually measured by pachymetry until open-eye steady-state (OESS) thickness was achieved. The percentage of recovery per hour was calculated as a measure of endothelial function. Tear oxygen tension (PO(2)) beneath the hydrogels was measured in the open eye and after 5 minutes of eye closure to obtain estimates of Q(C). A change in corneal pH during eye closure while wearing the hydrogels was used as a measure of hypoxic acidosis. Associations between corneal swelling and endothelial cell density or corneal epithelial thickness were also tested. There were modest but significant (P < 0.05) correlations between thick-lens corneal swelling and thick-lens closed-eye PO(2) (r = -0.36); thin lens corneal swelling and thin-lens closed-eye and open-eye PO(2) (r = -0.40 and -0.39, respectively). Corneal swelling also increases with increasing Q(C) (r = + 0.29 to + 0.33). Corneal swelling was associated with the decrease in pH during lens wear (r = + 0.30 and + 0.20 for thick and thin lenses, respectively). Thick- and thin-lens corneal swelling was significantly associated with percentage of recovery per hour (r = -0.40 and -0.34, respectively). Multiple regression analysis of corneal swelling with PO(2) and percentage of recovery per hour suggested an additive effect, however the effects were not significant. There was a modest inverse association between corneal swelling and OESS, however there were no associations between corneal swelling and endothelial cell density or epithelial thickness. The variability in contact lens-induced corneal swelling is associated with both corneal metabolic activity (Q(C) and pH) and endothelial function (percentage of recovery per hour). Our interpretation is that individuals with larger Q(C) produce more lactic acid (i.e., more swelling) whereas stronger endothelial function resists swelling. The modest correlations, however, suggest that other factors also are involved in explaining the phenomenon of corneal swelling.

Ischemic preconditioning reduces Na(+) accumulation and cell killing in isolated rat hepatocytes exposed to hypoxia.

Short periods of ischemia followed up by reperfusion are known to protect the heart against injury caused by a subsequent sustained ischemia. This phenomenon, known as ischemic preconditioning, has also been recently shown to reduce ischemic liver damage, but the mechanisms involved are still unknown. By using isolated hepatocytes as an in vitro model of liver preconditioning, we have investigated the possible effect of preconditioning on intracellular pH and Na(+) homeostasis. Freshly isolated rat hepatocytes were preconditioned by 10 minutes of incubation under hypoxic conditions followed up by 10 minutes of reoxygenation and subsequently exposed to 90 minutes of hypoxia. Although preconditioning did not ameliorate adenosine triphosphate (ATP) depletion, preconditioned hepatocytes exhibited an increased resistance to cell killing during hypoxic incubation. Intracellular acidosis and Na(+) accumulation developing during hypoxia were appreciably reduced in preconditioned cells. The effects of preconditioning on intracellular pH, Na(+) homeostasis, and cytotoxicity were mimicked by stimulating protein kinase C (PKC) with 4beta-phorbol-12-myristate-13-acetate (PMA) or 1,2 dioctanoyl-glycerol (1,2 DOG). Conversely, inhibiting PKC with chelerythrine or blocking vacuolar proton ATPase (V-ATPase) with bafilomycin A(1) abolished the protection given by preconditioning or by PMA treatment on hypoxic acidosis, Na(+) overload, and hepatocyte killing. Similarly, the addition of Na(+) ionophore monensin also reverted the cytoprotection exerted by preconditioning. This indicated that ischemic preconditioning of isolated hepatocytes decreased cell killing during hypoxia by preventing intracellular Na(+) accumulation. We propose that, after preconditioning, the stimulation of PKC might activate proton extrusion through V-ATPase, thus, limiting intracellular acidosis and Na(+) overload promoted by Na(+)-dependent acid buffering systems.

AN EVIDENCE-BASED EVALUATION OF THE USE OF SODIUM BICARBONATE DURING CARDIOPULMONARY RESUSCITATION

The use of bicarbonate is rooted in three decades of clinical experience and observational studies. For many years, bicarbonate passed the tried and true test for clinical therapies; however, administration of sodium bicarbonate during cardiac arrest and hypoxic acidosis has become increasingly controversial. The controversy provides an excellent opportunity to evaluate the impact an evidence-based approach might have on a common clinical practice. Is bicarbonate efficacious in the treatment of the severe acidosis that accompanies cardiac arrest during cardiopulmonary resuscitation (CPR)? Are the deleterious effects of bicarbonate clinically relevant? What is the evidence upon which a rational decision may be based? This review evaluates and ranks the evidence supporting the use of sodium bicarbonate in the therapy of acidosis associated with cardiac arrest during CPR.

Alkali therapy extends the period of survival during hypoxia: studies in rats.

The goal of this study was to evaluate whether sodium bicarbonate might be a useful form of therapy for hypoxic L-lactic acidosis; our aim was to determine if alkali could extend the time of survival in this setting. Hypoxia was induced in anesthetized, paralyzed, artificially ventilated rats by lowering inspired O2 to 5.5%, an amount sufficient to develop a severe degree of L-lactic acidosis. Measuring arterial blood gases frequently permitted maintenance of a near-constant arterial O2 content. Three groups of hypoxic rats were studied: first, no infusions (n = 10); second, sodium bicarbonate at a rate equal to H+ production in the no-infusion group (n = 12); and third, a control for the Na load in the second group as NaCl (n = 17). Survival was close to twofold longer in the sodium bicarbonate group. Part of this beneficial effect seemed to be increased anaerobic glycolysis, producing ATP along with L-lactic acid. In addition, there was a large decrease in the metabolic demand (consumption of O2) in the 7- to 15-min period in the sodium bicarbonate group. Rats exposed to hypoxia and infused with NaCl for 15 min or alkali for 15, 27, or 40 min were then returned to room air; all survived for the subsequent experimental period of 150 min. We found that there is both a rationale and experimental evidence for giving sodium bicarbonate to prolong survival during hypoxia.

Maintenance of cellular acidification in cyanide-treated hepatocytes results from inhibition of Na+/H+ exchange.

Inhibition of respiration by metabolic inhibitors or hypoxia is accompanied by intracellular acidification. Although this acidification is known to promote cell survival during hypoxia, little is known about its mechanism. Given that the Na+/H+ exchanger is known to be a major component of pH regulation in normal hepatocytes, the aim of this study was to determine the effects of inhibition of mitochondrial respiration on intracellular pH (pHi) regulation and Na+/H+ exchange. Cyanide (CN-; 5 mM) plus fructose (20 mM) were used as a model of hypoxic acidosis. pHi was measured with quantitative fluorescence microscopy of cells loaded with the pH indicator, 2',7'-bis-(2-carboxyethyl)-5,6-carboxyfluorescein. In control cells, pHi was 7.09 +/- 0.01 SE (n = 106). After 60 min in CN(-)-fructose, pHi fell to 6.74 +/- 0.01 (n = 129, P < 0.001). The pHi recovery rate (expressed as mmol H+.l-1.min-1) was determined under both conditions after acid loading by transient exposure and removal of 20 mM NH4Cl. Control and CN(-)-treated cells recovered at 3.59 +/- 0.25 (n = 42) and 0.69 +/- 0.09 (n = 38, P < 0.001), respectively. Amiloride treatment (1 mM) in the absence of CN- reduced pHi recovery similarly to that caused by CN- (0.34 +/- 0.07, n = 14). CN(-)-treated cells exposed to amiloride demonstrated no additional inhibition (efflux rate 0.65 +/- 0.11, n = 27), suggesting that the inhibition is directed at Na+/H+ exchange. Twenty minutes after CN- removal, CN(-)-treated cells regained their ability to recover from an acid load, thus demonstrating the reversibility of this effect.(ABSTRACT TRUNCATED AT 250 WORDS)

The use of dichloroacetate in the treatment of overwhelming hypoxic acidosis

Overwhelming hypoxic acidosis due to poor tissue oxygen delivery from low cardiac output, pulmonary failure, and other causes has devastating effects postoperatively on patient outcome. Whereas conventional therapeutics often can not reverse the downward spiral of these patients, dichloroacetate (DCA) has been shown to be beneficial. This study investigated the metabolic and hemodynamic effects of DCA given after the onset of overwhelming hypoxic acidosis in a canine model. A hypoxically ventilated canine model of severe induced acidosis was established and dogs surviving the development of acidosis were randomized to receive DCA or sodium chloride (NaCI) treatment. Dogs receiving DCA after development of hypoxic lactic acidosis showed no further change in metabolic parameters during the 90-minute treatment period (pH, 7.24 to 7.23; HC0 3, 17.7 to 18 mmol/L; lactate, 2.04 to 1.05 mM/L); whereas animals receiving an equivalent sodium load showed progressive, significant deterioration in all parameters (pH, 7.24 to 7.12; HCO 3, 16.8 to 13.2 mM/L; lactate, 2.05 to 3.55 mM/L). Myocardial blood flow was significantly increased by hypoxia in all dogs. Finally, cardiac output and stroke volume were significantly increased at 90 minutes by DCA versus control. Myocardial oxygen utilization efficiency (LV work/ M VO 2) was improved during DCA treatment. DCA, a carboxylic acid, increases pyruvate dehydrogenase activity, thereby enhancing lactate use as a metabolic substrate. DCA had an ameliorative metabolic effect, and benefitted myocardial performance without a direct inotropic effect. DCA treatment appears to enhance myocardial performance on a metabolic and not primarily inotropic basis, does not increase the “cost” of myocardial work, and warrants further study.

Anomalous hypoxic acidification of medullary ventral surface

In castrated male goats, two flexible catheters, one open ended for reference and the other ending in a 1-mm-diam glass bulb pH electrode, were advanced ventrally through a left posterior fossa craniotomy into the subarachnoid space between the 9th and 10th cranial nerve roots, passing medially into cerebrospinal fluid (CSF) over the medullary ventral surface (MVS). They were anchored to dura and fascia, tunneled under the scalp, and terminated in connectors on the left horn. After several days for recovery, while the animals were awake, the effects of CO2 and hypoxia on pH of the film of CSF between the pia and arachnoid (pHMVS) were recorded along with end-tidal PCO2 and PO2 (mass spectrometer), ventilation (pneumotachometer) through a permanent tracheostomy, and, when possible, ear arterial O2 saturation (SaO2). High PCO2 acidified MVS as expected: delta pH MVS/delta log PCO2. = -0.64 +/- 0.14, producing a ventilatory response slope delta VI/delta pHMVS = 372 l/min. Hypoxia resulted in acid shifts even when PCO2 was allowed to fall. The development of hypoxic acidosis was related to the location of pH electrodes determined at necropsy. In isocapnic hypoxia, pH over putative chemoreceptor surfaces fell in proportion to desaturation: delta pHMVS = 0.0033(SaO2)-0.34, r = 0.80, Sy.x = 0.025. With uncontrolled arterial PCO2, similar acidosis occurred when SaO2 fell below 85-90%: delta pHMVS = 0.0039(SaO2)-0.34, r = 0.88, Sy.x = 0.032. With constant hypoxia, pH fell (tau = 3.7 +/- 2.2 min) to a plateau after 10-20 min and showed rapid recovery (tau = 2.0 +/- 1.3 min).(ABSTRACT TRUNCATED AT 250 WORDS)

Quality control of perfusion: Monitoring venous blood oxygen tension to prevent hypoxic acidosis

The long-held belief that venous oxygen tension mirrored tissue oxygen tension became suspect in the 1960s when new instrumentation consistently showed that tissue oxygen tension was 10 to 30 torr less than venous oxygen tension. Moreover, a countercurrent of oxygen exchange between terminal arteries and veins was shown to exist. Despite this conflict in scientific theory, however, monitoring venous oxygen tension as a means to control hypothermic cardiopulmonary bypass has been repeatedly urged, since myocardial acidosis is clearly extremely detrimental. This study of the relationship between venous oxygen tension during hypothermic bypass and a concurrent increment in lactacidemia yields strong objective evidence to support the use of on-line venous oxygen tension monitoring to guide perfusion. In a random series of 36 patients, venous blood samples were drawn at five preselected intervals during operation and were analyzed for pH, carbon dioxide tension, oxygen tension, lactic acid, hematocrit, and base excess. Analysis of the data revealed that venous pH and base excess showed no correlation to venous oxygen tension. However, lactic acid showed a strong correlation with venous oxygen tension, with a correlation coefficient of 0.4338 at a confidence level of p less than 0.0001. If the patients were divided into three clinically pertinent groups based on the lowest venous oxygen tension recorded, a strong relationship between venous oxygen tension and lactic acid emerged. If the lowest measurement of venous oxygen tension was greater than 35 mm Hg (group A), the mean rise in lactic acid was only 0.12 microns/ml. If the lowest measurement was between 30 and 34 mm Hg (group B), the mean rise was 0.64 microns/ml. Whereas, if any venous oxygen tension value fell below 30 mm Hg (group C), the mean rise in lactic acid was 2.56 microns/ml. Analysis of variance showed that group C values were significantly different from groups A and B values (p less than 0.0002). A scientific hypothesis relating venous oxygen tension to adequate tissue oxygenation is proposed. Use of venous oxygen tension monitoring with the goal to maintain the level above 35 mm Hg is strongly supported by this study.