G6PD Levels Research Articles

Met-Flow analyses of the metabolic heterogeneity associated with different stages of cord blood-derived hematopoietic cell differentiation.

The differentiation of hematopoietic cells is significantly affected by cell metabolic activity. However, despite increasing interest in this field, there has been no comprehensive investigation of the metabolic functions of human hematopoietic cells during specific phases of differentiation. Thus, this study was conducted to develop a method for comparing hematopoietic cell lineage differentiation based on the metabolic functions of the cell. The metabolic activity of human umbilical cord-derived hematopoietic cells was examined during various phases of differentiation, specifically, hematopoietic stem cells (HSCs), hematopoietic progenitor cells, and differentiated blood cells. This approach was used to develop comprehensive metabolic maps corresponding to the different stages. HSCs were found to have robust fatty acid (FA) synthesis, FA oxidation, pentose phosphate pathway (PPP) activity, and glucose uptake, shown by their significantly higher expression of ACAC, CPT1A, G6PD, and GLUT1 as compared to differentiated pluripotent progenitor cells, common myeloid progenitors, megakaryocyte erythroid progenitors, lympho-myeloid primed progenitors, and granulocyte-macrophage progenitor cell populations. In monocytic differentiation, higher levels of ACAC, ASS1, ATP5A, CPT1A, G6PD, GLUT1, IDH2, PRDX2, and HK1 protein expression were evident in classical and intermediate monocytes relative to non-classical monocytes, consistent with high anabolic and catabolic levels. Compared with myelocytes and mature cells, the meta-myelocyte and pro-myelocyte populations of granulocytes show significantly elevated levels of ACAC, ASS1, ATP5A, CPT1A, G6PD, IDH2, PRDX2, and HK. In contrast to naïve and regulatory B cells, pro-B cells had higher levels of oxidative phosphorylation, while regulatory B cells showed greater PPP activity, glucose uptake, and tricarboxylic acid cycle activity. The analyses of T cells also indicated significantly higher ACAC, ASS1, ATP5A, CPT1A, G6PD, GLUT1, IDH2, PRDX2, and HK1 expression levels in CD4+ populations compared with CD8+ populations. The results provide comprehensive analytical methods and reference values for future systematic studies into the metabolic functions of various cord blood-derived hematopoietic cell populations in different pathological or physiological conditions. These findings could also contribute to research on the connection between cellular metabolism and cancer or aging.

Abstract A103: Targeting mutant p53-R248W reactivates WT p53 function and alters the onco-metabolic profile

Abstract The tumor suppressor p53 is mutated (mt-p53) in over 50% of human cancers causing gain-of-function oncogenic effects, including metabolic changes that reduce tumor responsiveness to radio/chemotherapy. Common hot-spot mutations within the DNA-binding domain can be categorized as conformational (R175H) or DNA binding (R248W). NSC59984 has been characterized as a small molecule that targets mt-p53 for degradation and restores wt-p53 signaling. Using esophageal adenocarcinoma cells and CRISPR generated isogenic cell lines bearing matching hot-spot p53 mutations, we aim to understand how the molecular features of mt-p53 affect drug efficiency and enable the development of targeted therapies to limit cancer cell growth. We found that NSC59984 covalently modifies p53 by Michael addition at cysteine residues 124 and 229, which promote interactions that would stabilize the protein/DNA complex leading to increased p53 transcriptional activity. In cells, the effects of NSC59984 were substantially greater in cells harboring the R248W mutation compared with the R175H mutation. Treatment with NSC59984 reduced proliferation and increased apoptosis via the intrinsic mitochondrial pathway. It also induced changes in OXPHOS, ATP level, mitochondrial membrane potential, glycolysis, and lactate production. Furthermore, treatment of cells with NSC59984 increased reactive oxygen species production and decreased glutathione levels; effects were enhanced by the addition of buthionine sulfoximine and inhibited by N-acetyl cysteine. NSC59984 treatment increased G6PD activity, total NADPH levels, and expression of TIGAR. Knockout of TIGAR partially removed the antiproliferative effects of the drug and reduced G6PD levels in the p53-R248W cells. Incorporation of [13C6] into cellular metabolites suggests that p53-regulated transcription of TIGAR increased utilization of the pentose phosphate pathway and inhibited glycolysis at the fructose-6-P fructose-1,6-bisphosphate junction, supported by an increase in Hexokinase 2 and a decrease of phosphofructokinase-1. Thermal proteome profiling identified TIGAR as an additional reaction target of NSC59984, suggesting increased involvement in modulating these metabolic effects. Combining currently available therapeutic metabolic inhibitors with NSC59984 enhanced the antiproliferative effects in cells harboring p53-R248W creating a therapeutic window when compared to the wt-p53 expressing cells. This suggests these combinations could be used in a clinically relevant setting. Overall, this work has identified a distinctive mode of action for p53 reactivation resulting in not only transcriptional activity, but also a unique effect on cellular energetics. This study shows evidence of variation in responsiveness of different mt-p53 forms and allows the development of specific therapeutics directed to individuals for patient-centered precision medicine. Importantly, we have shown that targeting p53 signaling has significant effects on the metabolic profiles of cancer cells rendering them more vulnerable to neoadjuvant therapy. Citation Format: Kate Brown, Lisa Jenkins, Dan Crooks, Deborah Surman, Sharlyn Mazur, Yuan Xu, Bhargav Arimilli, Ye Yang, Andrew Lane, Stewart Durell, Teresa Fan, David Schrump, Marston Marston, Taylor Ripley, Ettore Appella, Gaelyn Lyons, Andrew Perciaccante, Jerry Dinan, Marco Robello, Herman Nikolayevskiy, Robert O’Connor, Daniel Appella. Targeting mutant p53-R248W reactivates WT p53 function and alters the onco-metabolic profile [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr A103.

Hemoglobin Monza: A New Variant of Unstable Hemoglobin

Introduction: Unstable hemoglobins are variants with structural abnormalities; while not hindering protein formation, they lead to protein destabilization and can cause hemolytic crises. Most unstable hemoglobins are caused by point mutations that result in the premature dissociation of heme from the globin chain and usually involve His residues (Weatherall et al. 2001 Nat.Rev.Genetics; Scheps et al. 2018 EJH). Here a new unstable hemoglobin, named Hb-Monza is presented, exhibiting a long duplication in the HBB gene without apparent alterations in α-β subunit interaction or involvement of specific His residues. The variant was identified in a family, with four out of six members involved and is associated with mild chronic hemolysis and acute severe hemolytic crises during febrile infections. Methods: The presence of erythrocyte membrane abnormalities was tested by glycerol lysis, Pink, osmotic resistance and by EMA binding test. G6PD and PK activity levels were tested both during the hemolytic crisis and at resolution. Abnormal variants were screened by HPLC and electrophoresis. Stability of the Hb variant was assessed by the Isopropanol Precipitation Test. DNA was extracted from peripheral blood leukocytes, followed by PCR amplification of HBB, HBA1 and HBA2 genes. Sequencing was performed using Genetic Analyzer 3500. A 3D model of the variants was produced by SWISS-MODEL using the crystal structure of the deoxy human hemoglobin (PDB 1A3N) as template and refined by Maestro Suite. Results: The index patient (pt) was a 13-year-old girl of Chinese origin; she was the firstborn of 4 children (2 sisters and 1 brother) and came to ER with fever and hyperchromic urine. Blood tests revealed severe anemia (Hb 5.2 g/dL) and signs of hemolysis. Direct Coombs test was negative both for warm and cold antibodies. She tested positive for Mycoplasma Pneumoniae, Epstein Barr Virus (EBV) and Parvovirus B19 (IgG and IgM) antibodies. The child's infection cleared during hospitalization, and hemoglobin levels increased without transfusion. Her hemoglobin further rose to 11 g/dL in outpatient follow-up although signs of low-level chronic hemolysis persisted. All affected family members exhibit mild chronic hemolytic anemia, with all, except the mother, experiencing at least one acute severe hemolytic state triggered by infection. HPLC showed the presence of an abnormal fraction of hemoglobin with slow migration speed in alkaline electrophoresis and extremely slow elution in HPLC (4.8 min). The abnormal Hb fraction in the proband, mother, brother and sister was 20.8%, 17.7%, 28.1%, and 25.6% respectively. The father and the youngest sibling did not show this band. Sequencing of HBB demonstrated an in-frame duplication of 69 bases corresponding to 23 amino acids located in exon 2 (c.176_244dup; p.Pro59_Asn81dup); this was present in all pts exhibiting the abnormal fraction at HPLC. A functional validation of Hb Monza utilizing the isopropanol test confirmed the unstable nature of the variant (Figure 1). The 3D-model analysis (Figure 2) showed that the duplication is situated amidst helix E and F of HBB resulting in a disordered conformation protruding from the Hb structure The binding of the heme group remains unaffected and the coordination of the heme group with His residues of both the E and F helix is preserved. The tertiary structure of the Hb beta chain is scarcely affected. Conclusions: This report introduces a novel HBB gene variant, Hb Monza (HBB: c.176_244dup), associated with subclinical chronic hemolysis and acute hemolytic crises during fever-inducing infections. Only a few HBB duplications exceeding 20 bp have been documented, all leading to Beta-Thalassemia clinical features through production of a truncated beta globin chain or failure of HBB to bind the alpha chain (e.g. Frischknecht et al. 2007 Haematologica). However, 3D-modeling of Hb Monza indicates minimal tertiary structure alteration. The inclusion of 23 additional amino acids results in a disordered conformation protruding from the Hb structure possibly facilitates direct bonding between the iron molecule in the heme group and internal amino acid side chains due to increased structural flexibility. This could lead during thermal or metabolic stress, to Hb precipitation. Further data from molecular dynamic experiments will be presented. Dr. Civettini and Dr. Corti contributed equally to this article

Real World Analysis of G6PD Testing Prior to the Use of Rasburicase in Hematologic Malignancies; A Single Center Experience

Introduction: The use of Rasburicase is contraindicated in patients with (Glucose -6-Phosphate Dehydrogenase) G6PD deficiency. The FDA recommends screening for G6PD deficiency prior to the use of Rasburicase, especially in those of African or Mediterranean ancestry. This recommendation, however, is not implemented uniformly. Methods: This was an IRB approved study. A retrospective chart analysis of adult patients with hematological malignancies treated in both the inpatient and outpatient setting between 2019 and 2022 was conducted. Data was reviewed and collected by investigators and compiled in a confidential platform. Results: A total of 437 patients were reviewed. The median age was 67 years. 54.9% were male and 45.1% were female. 65.6% of the patients were White, 23.5% were Black. Other ethnicities included Hispanics 3.8%, Asians 3.7%, Native American or Alaskan Natives 0.4% and unknown ethnicity in 4.3%. 37.3% of the patients had a diagnosis of Diffuse Large B Cell Lymphoma/High Grade B Cell Lymphoma, 34.1% had AML, 29.1% had CLL, 6.2 % had B or T-ALL, 2% had Burkitt lymphoma, and 1.6% had anaplastic T cell lymphoma. Rasburicase was utilized in 85/437 (19.5%) patients for the treatment of hyperuricemia of malignancy 50/85 (59%) and in 35/85 (41%) patients for the treatment of tumor lysis syndrome. G6PD was tested in 197 patients (45%) of which 39.5% were White, 21.8% were Black, 3.5% were Hispanic, 1% were Asian and 2.5% of unknown ethnicity. G6PD status was unknown in 55% patients. 7 cases of G6PD deficiency were identified, and 2 cases were highly suspicious for it due to the development of hemolysis after Rasburicase administration. 8/9 of these patients were African-American. In those patients who received Rasburicase, 70/85 (82.3%) received the medication prior to the G6PD results being available; 6 patients developed hemolysis and 1 patient developed methemoglobinemia. Of these 7 patients, 4 had unknown G6PD status, 2 had G6PD levels which were elevated but sent after signs of hemolysis were present and 1 was G6PD deficient. Of these patients, 3 were Black, 3 were Hispanic and 1 was White. Conclusions: G6PD testing was requested in 45% of patients being evaluated for hematologic malignancies. Of these, only 17.7% had results available before the administration of Rasburicase. 2% of this sample were found to either be G6PD deficient or had clinical suspicion for the same. 89% of them were Black. 8.2% of patients receiving Rasburicase developed complications related to oxidant injury caused by G6PD of which 43% were Black and 43% were Hispanic, though they constituted only 27.3% of the population. This study goes on to highlight the ethnic prevalence of G6PD deficiency, especially in the Black population. Strategies to increase testing for G6PD for all new diagnoses of hematological malignancies known to have a risk of TLS and reducing the time to resulting of G6PD levels can mitigate known adverse events from Rasburicase. The incorporation of G6PD testing as part of chemotherapy order set labs or use of hard stops prior to signing the treatment plan can be potential options to increase testing and screening.

Incidence of G6pd Deficiency in Young Asymptomatic Males in Pakistan

Background: NADPH is crucial for glutathione reduction. Deficiecy of G6PD leads to excessive oxidised glutathione that denatures hemoglobin and forms Heinz bodies. Heinz bodies damages RBC membrane and cause premature removal of cells by spleen. G6PD deficiency is the most common X-linked inherited red cell enzymopathy that leads to hemolytic anemia and neonatal jaundice in enzyme deficient patients under oxidative stress. G6PD is the rate limiting enzyme in pentose phosphate pathway, a dominant form of glycolysis in RBCs. G6PD converts glucose-6 phosphate into 6-phosphonogluconolactone and reduces NADP to NADPH. Objective: To determine incidence of G6PD deficiency in young healthy males in Pakistan. Study design: A descriptive cross sectional study Place and duration: Study was carried out at PAF Hospital Sargodha from Jan 2021 to Feb 2023 Methodology: G6PD levels were checked in young healthy male volunteers of 12 to 18 years of age, after taking informed consent from subjects and their parents. Results: Out of 1493 volunteers, only 61 were G6PD deficient and rest all had normal levels. This shows an incidence of 4.08% in our population. Conclusion: Comparing with previous studies suggests 2-4% incidence of G6PD deficiency in Pakistani population. A larger study including females and studying variants of disease is needed. Patients with lab confirmed P.Vivax should get screened for G6PD deficiency, so that they receive Primaquine. Keywords: Incidence, G6PD deficiency, Malaria, Primaquine.

A retrospective comparison of phototherapy need in O-B versus O-A incompatibility in a single Saudi institution.

ABO incompatibility is a major risk factor for neonatal indirect hyperbilirubinemia (NIH), requiring treatment. It has been shown that there are racial differences in direct antiglobulin test (DAT) positivity and phototherapy need in the O--B versus (vs) O--A incompatibility. The comparison between the O--B and O--A incompatibility is not well studied in Saudi Arabia. We aimed to compare DAT positivity and phototherapy need in O-B vs O-A incompatibility in Saudi Arabia. This retrospective cohort study was conducted in one Saudi hospital. We included a convenience sample of neonates born between 01 January 2013 and 31 December 2021. We included healthy neonates admitted to the nursery care unit only, born at≥38 weeks gestation, and had normal G6PD levels. Neonates that had no G6PD level measurement or lost follow-up post-discharge were excluded. The data span was the first 14 days of life. A total of 611 neonates met our inclusion criteria. Positive DAT was more prevalent in the O-B than the O-A incompatibility [43.5% vs 29.2%, p < 0.001). A greater odd of phototherapy need was observed in the O--B vs O-A incompatibility across various strata. Readmission for NIH, use of 360° exposure phototherapy, or intravenous immunoglobulin administration was more prevalent in the O-B than the O-A incompatibility (13.2% vs 5.0%, p < 0.001). A logistic regression analysis revealed that the O-B incompatibility modified the association between DAT positivity and phototherapy need. The O-B incompatibility had a mediator effect on the relationship between DAT positivity and the need for phototherapy in the study population, which emphasizes that the O-B and O-A are not the same from the NIH point of view.

The Effects of Sodium Tetraborate against Lead Toxicity in Rats: The Behavior of Some Metabolic Enzymes.

This study was planned to research the in vivo effects of lead (Pb) ions and sodium tetraborate (Na2B4O7) on G6PD and 6PGD, which are some of the enzymes of the pentose phosphate pathway, which carries vital importance for metabolism, and GR and GST, which are glutathione metabolism enzymes, and the in vitro effects of the same agents on the 6PGD enzyme. According to the in vivo analysis results, in comparison to the control group, the rat liver G6PD (p < 0.05), and 6PGD (p < 0.01) enzyme activities in the Na2B4O7 group were significantly lower. In addition, GR and GST enzyme activities were insignificantly lower in the Na2B4O7 group compared to the control group (p > 0.05). The Pb group had lower G6PD and 6PGD enzyme activity levels and higher GR and GST enzyme activity levels compared to the control group, while these changes did not reach statistical significance (p > 0.05). In the in vitro analyses of the effects of Pb ions on the 6PGD enzyme that was purified out of rat liver with the 2',5'-ADP-Sepharose 4B affinity chromatography method, it was determined that Pb ions (200-1200 μM) increased the rat liver 6PGD enzyme activity levels by 33%. On the other hand Na2B4O7 was not significantly effective on 6PGD activity. These results will also contribute to future studies in understanding the physiopathology of the states triggered by Pb ions and sodium tetraborate (Na2B4O7).

The promising role of hypoxia-resistant insulin-producing cells in ameliorating diabetes mellitus in vivo.

Aim:This study aimed to evaluate the efficacy of hypoxia-persistent insulin-producing cells (IPCs) against diabetes in vivo.Materials & methods:Mesenchymal stem cells (MSCs) differentiation into IPCs in the presence of Se/Ti (III) or CeO2 nanomaterials. IPCs were subjected to hypoxia and hypoxia genes were analyzed. PKH-26-labeled IPCs were infused in diabetic rats to evaluate their anti-diabetic potential.Results:MSCs were differentiated into functional IPCs. IPCs exhibited overexpression of anti-apoptotic genes and down-expression of hypoxia and apoptotic genes. IPCs implantation elicited glucose depletion and elevated insulin, HK and G6PD levels. They provoked VEGF and PDX-1 upregulation and HIF-1α and Caspase-3 down-regulation. IPCs transplantation ameliorated the destabilization of pancreatic tissue architecture.Conclusion:The chosen nanomaterials were impressive in generating hypoxia-resistant IPCs that could be an inspirational strategy for curing diabetes.

The Transcription Coregulator RIP140 Inhibits Cancer Cell Proliferation by Targeting the Pentose Phosphate Pathway.

Cancer cells switch their metabolism toward glucose metabolism to sustain their uncontrolled proliferation. Consequently, glycolytic intermediates are diverted into the pentose phosphate pathway (PPP) to produce macromolecules necessary for cell growth. The transcription regulator RIP140 controls glucose metabolism in tumor cells, but its role in cancer-associated reprogramming of cell metabolism remains poorly understood. Here, we show that, in human breast cancer cells and mouse embryonic fibroblasts, RIP140 inhibits the expression of the gene-encoding G6PD, the first enzyme of the PPP. RIP140 deficiency increases G6PD activity as well as the level of NADPH, a reducing cofactor essential for macromolecule synthesis. Moreover, G6PD knock-down inhibits the gain of proliferation observed when RIP140 expression is reduced. Importantly, RIP140-deficient cells are more sensitive to G6PD inhibition in cell proliferation assays and tumor growth experiments. Altogether, this study describes a novel role for RIP140 in regulating G6PD levels, which links its effect on breast cancer cell proliferation to metabolic rewiring.

G6PD Namoru in Heterozygous Twins—A Case Report

Glucose-6-phosphate dehydrogenase deficiency remains the most prevalent enzyme deficiency in the world. The global prevalence is 4.9%. There are around 92 mutations detected and both hemizygous and heterozygous can be symptomatic. Case Report: Twin babies were born at 32 weeks gestation. Initially they had mild respiratory distress and settled. On day 3 of life, they had neonatal jaundice and received phototherapy for 36 h. On day 8 of life, the boy became symptomatic and diagnosed as Escherichia coli sepsis. On 9th day, he developed jaundice and by day 10 of life he expired. G6PD was suspected but newborn screening showed normal G6PD reports. Girl was screened and had severe hemolysis requiring blood transfusion. Mutation analysis done revealed boy has homozygous mutation and girl had heterozygous mutation. Discussion: G6PD deficiency is a very common hemolytic genetic disorder, especially where malaria is endemic. It is more common in males and hence neonatal sepsis. They are several mutations detected. They can have severe hemolysis and severe sepsis with catalase positive organism. Boy had homozygous mutation and manifested as severe hemolysis and in turn sepsis. Girl had heterozygous mutation and manifested as hemolysis but not sepsis. During hemolysis period, G6PD levels can be falsely normal and so, to do Sanger sequencing analysis of G6PD levels. Conclusion: Newborn screening should be initiated by government in all areas The males who had sepsis or severe hemolysis during newborn period should undergo Sanger sequencing analysis of G6PD gene to avoid false negative normal levels.

Role of glucose phosphate deficiency in neonatal hyperbilirubinemia among local population of Pakistan

Due to the prevalence of new-born jaundice (65%) and neonatal hyperbilirubinemia (85%) in the first 24 hours of life, Objectives of the study: The study's main purpose is to investigate G6PD insufficiency in neonates. Material and methods: The Punjab Health Department conducted a descriptive study from July to December 2021. The hospital's ethics committee approved this probe. The research included 197 patients. The research included infants aged 0-20 days. A survey was used to collect data. Gender, gestational age, age of presentation, and length of hospitalisation were all noted. Results: The study included 197 patients, 61 of whom were female and 136 males. Participants in this research had bilirubin levels between 10 and 30 mg/dl. The maximum age in this study was 18 days, while the minimum was a new-born infant. G6DP insufficiency was seen in 21 of the 197 patients. 176 individuals had normal G6PD levels. Conclusion: The study found that G6PD deficiency is common in new-borns. Severe hyperbilirubinemia has several risk factors

Glucose-6-phosphate dehydrogenase gene Ala365Thr mutation in an Iraqi family with confusing clinical differences

Abstract Objectives Glucose-6-phosphate dehydrogenase (G6PD) has role in the Embden Meyerhof road. Any loss of its function causes NADPH to cease, leaving erythrocytes susceptible to oxidative damage resulting in acute hemolytic anemia attacks secondary to drugs or infection and favism. Because of X-linked recessive inheritance males are mainly affected. Being heterozygous, females have less severe clinical presentation. Case presentation G6PD deficiency was suspected in a six-year-old girl from an Iraqi family with a history of yellowing of skin and darkening of urine after eating broad beans. Besides the patient, G6PD levels were found low in the father and in two sisters who showed no symptoms. The father was found hemizygous and the three sisters were found heterozygous for NM_000402.4c.1093G&gt;A(p.A365T)(6.Ala365Thr) mutation while the mother was normal. Conclusions G6PD enzyme deficiency can be seen in both genders, and it may be presented with different clinical manifestations even within the people having the same mutation.

EYES DO NOT SEE WHAT MIND DOESN'T SEEK: A PUZZLING CASE OF METHEMOGLOBINEMIA

TOPIC: Critical Care TYPE: Medical Student/Resident Case Reports INTRODUCTION: Methemoglobinemia must be considered in the differential diagnosis of hypoxia not improving with supplemental oxygen. CASE PRESENTATION: An 84-year-old male patient with a past medical history of Atrial fibrillation, scleritis, hypertension presented with complaints of fever, chills, shortness of breath for 5 days. Medication history included triamterene/hydrochlorothiazide, Amiodarone, warfarin, ezetimibe, prednisone, and methotrexate. On presentation, the patient was saturating 79% on ambient air and was put on Hiflo nasal oxygen, and saturation improved to 95% on Fi02 100% at 35 l/min. The lab was significant for WBC count of 12.1, procalcitonin 1.06, a chest x-ray revealed bilateral opacities left greater than right with air bronchograms. Chest CT done showed bilateral ground-glass opacities with interposed consolidation. Bronchoscopy was deferred due to the clinical condition of the patient The patient was started on cefepime, doxycycline, methylprednisone, primaquine, and clindamycin for empiric treatment of PCP pneumonia with superimposed bacterial pneumonia and was transferred to ICU for hypotension secondary to sepsis. Pt initially improved clinically and Hiflo was weaned to Fi02 of 50% and maintained saturation above 95%. Over the course of 3-4 days after downgrading to the medical floor, the patient's oxygen requirement uptrend, though a repeat CT scan of the chest showed radiological improvement. An ABG with co-oximetry done showed Po2 of 302 mmHg, saturation of 91 % on Fi02 of 100%, and Methemoglobin content of 7.8%. IV methylene blue infusion was given and the patient's respiratory status improved with the Fio2 requirement coming down. Repeat ABG with co-oximetry showed a methemoglobin level of 2.4%. Repeat labs did not show evidence of hemolysis and G6PD levels were normal. The patient's increased susceptibility to methemoglobinemia would have been due to drug interaction as he is on thiazide diuretic and was started on primaquine increasing the oxidative stress coupled with oxidative stress secondary to sepsis. Primaquine was discontinued and started on atovaquone for PCP treatment. The patient continued to improve clinically. DISCUSSION: Methemoglobinemia can occur after exposure to a number of medications, including primaquine, sulfonamides, nitrates, topical anesthetics. Methemoglobin is the oxidized form of Hb, Oxygen does not bind as easily to the heme subunit with the ferric form of iron. The remaining heme sites have increased affinity for oxygen, resulting in decreased ability to release oxygen to tissues, causing left-shift of the oxygen-hemoglobin dissociation curve leading to tissue hypoxia. A clue to diagnosis is the cyanosis-saturation gap. CONCLUSIONS: An obvious mismatch between the PaO2 (which is >>100 mm) versus the pulse oximetry ( typically ~80-90%), known as the PaO2-saturation gap, is a clue to the diagnosis of methemoglobinemia REFERENCE #1: Gibson QH. The reduction of methemoglobin in red blood cells and studies on the cause of idiopathic methemoglobinemia. Biochem J 1948;42: 13-23. REFERENCE #2: Ohashi K, Yukioka H, Hayashi M, Asada A. Elevated methemoglobin in patients with sepsis. Acta Anaesthesiol Scand 1998;42: 713-716. REFERENCE #3: Beutler E. Methemoglobinemia and other causes of cyanosis. In: Beutler E, Lichtman MA, Coller BS, Kipps TJ, eds. Williams Hematology. 5th ed. New York: McGraw-Hill; 1994: 654-662. DISCLOSURES: No relevant relationships by Santhosh Bayambe, source=Web Response No relevant relationships by Nadish Garg, source=Web Response No relevant relationships by Killol Patel, source=Web Response No relevant relationships by ARCHANA SREEKANTAN NAIR, source=Web Response

Intravenous Methylene Blue as a Rescue Therapy in the Management of Refractory Hypoxia in COVID-19 ARDS Patients: A Case Series.

ABSTRACTObjectives: To describe the clinical outcomes of hypoxic coronavirus disease 2019 (COVID-19) patients treated with intravenous methylene blue (MB) in a tertiary care hospital.Materials and methods: We conducted a case series of 50 patients with hypoxic COVID-19 treated with intravenous MB admitted to our hospital between June 01 and September 10, 2020. Intravenous MB was administered as rescue therapy in dosage of 1 mg/kg body weight, with a maximum of five doses, to patients with high oxygen requirements (SpO2/FiO2 <200) apart from the standard of care after obtaining G6PD levels. Data were abstracted from multiple electronic data sources or patient charts to provide information on patient characteristics, clinical and laboratory variables and outcomes.Results: The median age of the patients was 53.3 (range 25–74 years) and most patients (74%) were men. About 68% of patients had pre-existing comorbidity. Median SpO2/FiO2 ratio progressively improved from 132.5 (predose) to 284 before the terminal event (death or discharge), ventilator-free days, and decrease in the proinflammatory biochemical parameter was significantly higher after the second dose of MB. A total of six patients out of 50 required invasive mechanical ventilation (IMV). Thirty patients were discharged with a recovery rate of 60%, while 20 patients succumbed to the illness. There was no major side effect or adverse event reported in any of the patients.Conclusion: MB due to its polypharmacological action against SARS‐CoV‐2, an inexpensive and widely available drug with minimal side effects, has a significant potential in the treatment of COVID-19.How to cite this article: Mahale N, Godavarthy P, Marreddy S, Gokhale SD, Funde P, Rajhans PA, et al. Intravenous Methylene Blue as a Rescue Therapy in the Management of Refractory Hypoxia in COVID-19 ARDS Patients: A Case Series. Indian J Crit Care Med 2021;25(8):934–938.

Lactate Modulates Cellular Metabolism Through Histone Lactylation-Mediated Gene Expression in Non-Small Cell Lung Cancer.

Lactate has been observed to fuel TCA cycle and is associated with cancer progression in human lung cancer, the leading cause of cancer deaths worldwide, but the effect of lactate on lung cancer metabolism is rarely reported. In this study, disordered metabolism in non-small cell lung cancer was demonstrated by increased G6PD and SDHA protein levels via immunofluorescence, and up-regulated lactate dehydrogenase was found to be associated with poor prognosis. Then flow cytometry and Seahorse XFe analyzer were utilized to detect the effect of lactate on glycolysis and mitochondrial function in non-small cell lung cancer cells. The results show that in non-small cell lung cancer cells lactate attenuates glucose uptake and glycolysis while maintaining mitochondrial homeostasis as indicated by improved mitochondrial membrane potential. Further exploration found that mRNA levels of glycolytic enzymes (HK-1, PKM) and TCA cycle enzymes (SDHA, IDH3G) are respectively down-regulated and up-regulated by lactate, and increased histone lactylation was observed in promoters of HK-1 and IDH3G via chromatin immunoprecipitation assay. Taken together, the above results indicate that lactate modulates cellular metabolism at least in part through histone lactylation-mediated gene expression in non-small cell lung cancer.

Screening of the defi ciency of glucose-6-phosphate dehydrogenase of newborn babies

Deficiency of glucose - 6 - phosphate dehydrogenase (G6PD) - X - linked enzyme defect, which can manifest itself as hemolysis after acute infections or ingestionoxidants (salicylates and sulfonamides). Diagnosis is based on G6PD levels, although the test often gives a false negative result in the presence of acute hemolysis. Treatment is supportive. Since this pathology is X-linked, the complete clinical picture is observed in men and homozygous women; in heterozygous women, clinical manifestations are variable.

Protective effects of nobiletin on cisplatin induced neurotoxicity in rats

Objectives This study was designed to investigate the possible antioxidant, antiapoptotic and neuroprotective effects of nobiletin on cisplatin-induced neurotoxicity rat model by evaluating neurotrophins, antioxidants and histopathology. Methods Forty male Wistar Albino rats were divided into four groups: control, cisplatin (CIS), cisplatin + nobiletin (CIS + NOB) and nobiletin + cisplatin (NOB + CIS). CIS + NOB was applied nobiletin (10 mg/kg, i.p.) during the last four days whereas NOB + CIS was applied nobiletin during the first four days of the study. Cisplatin (4 mg/kg, i.p. twice a day) was administered to the experimental groups on the 5th day of the study. All rats were sacrificed on the 10th day of the study. BDNF, NGF, G6PD, GPx, tGSH and MDA levels were determined in brain. In addition, routin histolopathological analysis and caspase-3 immunoreactivity assay were conducted. Results BDNF concentrations increased in nobiletin-administered groups, compared to Control and CIS and that the increase was statistically significant in NOB + CIS (p < 0.05). It was also found that G6PD activity increased (p < 0.05) in the nobiletin-administered groups, compared to control and CIS. Histopathologically, neuronal degeneration, oedema and gliosis increased in CIS compared to Control, and nobiletin administration decreased neuronal degeneration and oedema compared to CIS (p < 0.05). Cisplatin increased (p < 0.05) caspase-3 immunoreactivity in cerebrovascular endothelium and neurons compared to Control, while nobiletin administration decreased caspase-3 immunoreactivity in cerebrovascular endothelium. Caspase-3 immunoreactivity in neurons decreased only in NOB + CIS (p < 0.05). Conclusion Nobiletin increased BDNF concentration and G6PD activity in brain and when evaluated together with histopathological and immunohistochemical findings, it may have antioxidant, antiapoptotic and neuroprotective effects against cisplatin.

A Case of Concurrent Sickle Cell Trait, Alpha Thalassemia, and G6PD Deficiency in a Pediatric Patient

Objective: The aim of this study is to highlight the hospital course of a pediatric patient with concurrent sickle cell trait, alpha thalassemia, and G6PD deficiency. Methods: The patient’s direct bilirubin remained less than 0.2 mg/dl throughout his hospitalization but his total bilirubin peaked at 18.7 mg/dl at 84 hours of life. While the patient’s bilirubin levels decreased after this, the decline was not as rapid as anticipated so a peripheral smear was performed which showed spherocytosis. Hemoglobin electrophoresis was also conducted just prior to discharge after the patient’s hyperbilirubinemia had resolved. Results: The results of the patient’s hemoglobin electrophoresis revealed that the patient was a sickle cell trait carrier and also showed evidence of trace Hb Barts consistent with alpha thalassemia. In addition, given the patient’s peripheral smear showing spherocytosis, G6PD levels were also assessed and found to be low consistent with mild to moderate G6PD deficiency. The patient’s family was educated about precautions to take to reduce the risk of excessive oxidative stress that could precipitate acute hemolytic anemia episodes in the future. Conclusion: The concurrent presentation of sickle cell trait, alpha thalassemia, and G6PD deficiency is rare and it is theorized that each trait respectively confers an evolutionary advantage against malaria.

Case Report: Hemolysis in a G6PD-deficient patient, a dose-dependent effect of metformin

Glucose-6-phosphate deficiency is an X-linked genetic disorder, which predisposes erythrocytes to oxidative stress, resulting in hemolysis. It is the most common enzymatic deficiency, and typically affects African, Asian, Mediterranean, and Middle Eastern lineages. It can be induced by some medications, chemicals, and foods. Metformin is an uncommon drug to cause hemolysis in G6PD-deficient patients. We report a case of a 52-year old African American male with G6PD-related hemolysis secondary to toxic metformin accumulation with acute kidney injury (AKI). The patient was a type-2 diabetic and was taking metformin (500mg twice daily) for three years. He presented to the ER with nausea, vomiting, and diarrhea for last three days with severe hemodynamic instability. Labs revealed hemoglobin 15mg/dl, white blood count 28 mm3/L, creatinine 10 mg/dl, blood urea nitrogen 100 mg/dl, bicarbonate 7 mEq/L, lactic acid 17 mg/dl, pH 6.8, pCO2 21mmHg, metformin 41 mcg/ml, albumin-globulin 41. Severe sepsis protocol was activated; IV fluids 30ml/kg bolus and antibiotics were given. CT abdomen revealed colitis. The patient was started on continuous renal replacement therapy. The next day, the patient’s hemoglobin dropped to 12.6 mg/dl. A hemolytic panel was unswerving with hemolysis and G6PD levels reported low at 1.72. The patient improved with antibiotics, but the hemolysis continued. Metformin toxicity induced hemolysis was suspected. The patient’s hemoglobin dropped to 6g/dl and he received blood transfusions. His hemoglobin started to improve with hemodialysis sessions, as metformin levels started to normalize, emphasizing the fact that patient was clearing metformin. Unlike most cases reported, in which hemolysis occurs within days to months of starting metformin, in our patient it occurred due to the cumulative effect of metformin because of the patient’s underlying AKI. This led us to propose that the hemolytic effect of metformin may not only be time-dependent but also dose-dependent.

S2627 A Case of K2-Induced Hyperbilirubinemia

INTRODUCTION: Over the last decade, synthetic cannabinoids (SCB) have been marketed as a natural and safer version of marijuana, leading to an increase in its recreational use. We present a case of isolated hyperbilirubinemia caused by K2 and, to our knowledge, this is the first reported case of K2-induced indirect hyperbilirubinemia. CASE DESCRIPTION/METHODS: An 18-year-old male with no significant medical history presented with behavioral changes. He admitted to using K2 daily. His sibling and paternal uncle had Gilbert's syndrome. On physical exam he was withdrawn and agitated. He had Scleral icterus and jaundice. Slit Lamp exam was equivocal. On labs: Total Bilirubin 11.2 mg/dL, Indirect bilirubin 10.8 mg/dL, Urine toxicology positive for marijuana.Serum ceruloplasmin, serum copper, 24h urinary copper excretion, anti-mitochondrial Ab, anti-Nuclear Ab, anti-LKM, Soluble Liver antigen, ferritin, iron, TIBC, A1AT, A1AT phenotype, IgG, IgM, IgA, hepatitis A, B, C serologies, LDH, haptoglobin, uric acid, Coombs test, and G6PD levels were all within normal limits. His peripheral blood smear was normal. Imaging: MRI brain: unremarkable, Liver biopsy:normal hepatic architecture without severe pathological process (Figures 2a and 2b). He was treated with supportive care and indirect hyperbilirubinemia improved. DISCUSSION: • There is no current literature that correlates indirect hyperbilirubinemia and SCB use. In our otherwise healthy patient, this hyperbilirubinemia was likely a consequence of his K2 ingestion. We hypothesize that K2 may impair hepatic glucuronidation which is the process by which indirect bilirubin is converted to direct bilirubin. • During the time of exacerbation of Gilbert's disease, most often the indirect bilirubin levels remain < 5mg/dL. In the case of our patient, he presented with an indirect bilirubin level of 10.8 mg/dL. His work-up excluded other causes of indirect hyperbilirubinemia. • This leads us to hypothesize that K2 may induce significant isolated indirect hyperbilirubinemia in patients with a predisposition to impaired hepatic glucuronidation. • We believe that removal of the insulting agent is required for resolution of the hyperbilirubinemia. • With the use of SCB increasing recreationally, it is crucial we understand the effects these drugs can have. SCB do not appear on standard hospital drug screens. Therefore, a focused history and exam should be obtained from the patient. Our case serves to increase awareness of SCB use as a cause of indirect hyperbilirubinemia.Figure 1Figure (2a).: Liver biopsy stained with Hematoxylin & Eosin, showing normal hepatic architecture with no periductal or ductular inflammatory infiltration.Figure (2b).: Hepatic tissue stained with Periodid acid-Schiff Diastase and for copper; normal pathology & negative copper staining.