ISSN  2587-2362  |  E-ISSN  2618-642X
International Journal of Medical Biochemistry - Int J Med Biochem : 6 (2)
Volume: 6  Issue: 2 - 2023
1.Front Matter 2023-2

Pages I - X

2.Evaluation of coagulation parameters according to serum CRP levels in early stage COVID-19 patients
Sema Kardesler, Ahmet Erkin Bozdemir, Inanc Karakoyun, Fatma Demet Arslan, Hulya Parildar, Nisel Yilmaz, Banu Isbilen Basok, Ayfer Colak
doi: 10.14744/ijmb.2023.77698  Pages 63 - 68
INTRODUCTION: An increase in the levels of inflammatory biomarkers is observed in coronavirus disease (COVID-19). Coagulopathy occurring during the course of the disease has also been associated with inflammation. In our study, we aimed to evaluate the coagulation parameters according to the severity of inflammation in patients with early stage COVID-19 disease.
METHODS: The study was carried out retrospectively in a third-level hospital between April 8 and August 20, 2020. The patients were divided into two groups according to polymerase chain reaction (PCR) results. Non-COVID-19 group consisted of 72 patients with negative, and COVID-19 group consisted of 247 patients with positive PCR results. According to the serum C-reactive protein (CRP) levels the COVID-19 patients were divided into three groups as follows: Group1 (CRP<10 mg/L; n=105), Group 2 ( CRP 10-50 mg/L; n=72), and Group 3 (CRP >50 mg/L; n=70). Age, CRP, and coagulation parameters including fibrinogen, D-dimer, aPTT, and PT were compared between the groups.
RESULTS: There were significant differences between the non-COVID-19 and COVID-19 patients in terms of age, CRP and coagulation parameters. Likewise, there was a significant difference among 3 groups regarding coagulation parameters. In the multinomial logistic regression analysis, only level of D-dimer was an independent risk factor among all groups, while PT was an independent risk factor between Groups 1, and 3.
DISCUSSION AND CONCLUSION: Our findings suggest that coagulopathy occurs in the early stage in relation to the severity of inflammation. For the diagnosis of COVID-19 disease and the detection of thrombotic complications; it is important to monitor results of the coagulation tests along with markers of inflammation from the early stages of the disease.

3.Evaluation of Vitamin D binding protein and 25-hydroxy Vitamin D metabolites in COVID-19 patients
Cigdem Karakukcu, Ayca Elibol, Esma Eren, Hatice Saracoglu, Fatma Mutlu Sariguzel, Aysun Gorkem, Ozlem Gulbahar, Ilhami Celik
doi: 10.14744/ijmb.2023.54775  Pages 69 - 74
INTRODUCTION: The immunomodulatory roles of Vitamin D and Vitamin D binding protein (VDBP) are in interest with incidence or outcome of coronavirus disease-2019 (COVID-19). This study aimed to investigate the association between the severity of COVID-19 with VDBP, total 25-hydroxy Vitamin D (25(OH)D), and its metabolites free Vitamin D (VDfree) and bioavailable Vitamin D (VDbio).
METHODS: Study group consisted of 68 COVID-19 patients and 20 healthy subjects. Patients were subgrouped as asymptotic, mild/moderately pneumonia, or severe pneumonia. Plasma total 25(OH)D was quantitated by liquid chromatography with mass spectrometry and serum VDBP by a polyclonal sandwich enzyme immunoassay. In addition, routinely used laboratory parameters in follow-up were recorded. VDfree and VDbio were calculated using total 25(OH)D, VDBP, and albumin levels.
RESULTS: Plasma total 25(OH)D (13.3±5.7 vs. 30.3±13.3 ng/dL), VDfree (2.18 [1.52–3.44] vs. 4.34 [3.74–6.48] pg/mL), and VDbio (1.86 [1.09–2.81] vs. 4.28 [3.45–6.34] nmol/L) levels were lower in COVID-19 patients (p<0.001). Despite the in-significance of 25(OH)D and metabolites between COVID-19 severity subgroups, serum VDBP was highest in mild/moderately pneumonia (601.8±278.6 ng/mL) and lowest in severe pneumonia (427.9±147.2 ng/mL) (p<0.001). In addition, VDBP was positively correlated with lymphocyte counts (B: 87.9, r2=0.068, p=0.031) and negatively correlated with D-Dimer levels (B: −0.024, r2=0.081, p=0.032).
DISCUSSION AND CONCLUSION: COVID-19 patients have lower plasma 25(OH)D levels and lower 25(OH)D metabolites VDfree, VDbio which are physiologically active. In addition, serum VDBP concentrations significantly decrease in critically ill patients which needs further studies to be associated in the etiopathogenesis of the disease severity.

4.Evaluation of systemic inflammation markers in predicting cardiac risk in patients with acute chest pain
Levent Deniz, Yasin Yuksel, Hilmi Furkan Arslan, Hale Aral, Umit Bulut
doi: 10.14744/ijmb.2023.71473  Pages 75 - 83
INTRODUCTION: This study aimed to evaluate systemic inflammation markers in predicting cardiac risk in patients with acute chest pain (ACP), in this way identifying cases with acute coronary syndrome (ACS) in admission to the hospital. In addition, the relationship between these markers and the HEART score was investigated.
METHODS: By evaluating the laboratory/clinical data, patients with ACP (n=308) aged 18–70 were included in the study. As a result of clinical follow-up, patients were categorized into two groups: those diagnosed with ACS and those with non-ACS. Low-risk, moderate-risk, and high-risk patient groups were formed using the HEART score. From the routinely studied hemogram data, systemic immune inflammation index, systemic inflammation response index (SIRI), neutrophil-to-lymphocyte ratio (NLR), monocyte-to-lymphocyte ratio, and platelet-to-lymphocyte ratio (PLR) were calculated.
RESULTS: In determining the high-risk group, the highest area under the curve (AUC) was observed as 0.862 (95% confidence interval [CI]=0.818-0.898) at a cutoff value of 2.9 (69.3% sensitivity and 90.3% specificity) for NLR. For SIRI at a cutoff value of 2.0, the AUC value was found as 0.855 (95% CI=0.811 to 0.893), having 72.6% sensitivity and 85.2% specificity. The strongest association was between the HEART score and SIRI (r=0.612; p<0.001). Comparing patients without ACS and patients with ACS, there was no difference in lymphocyte counts, platelet counts, and PLR. In the ROC analysis for ACS, the SIRI performed that the highest AUC value was 0.858 (95% CI= 0.814 to 0.895), presenting 77.3% sensitivity and 79.5% specificity at a cutoff value of 1.19.
DISCUSSION AND CONCLUSION: When pre-pandemic data were evaluated, higher NLR or SIRI might help risk stratification for individuals with ACP, and it could be recommended for clinical benefit in the emergency department. SIRI, which includes the number of monocytes, may be helpful as a novel index in identifying individuals with ACS.

5.Evaluation of analytical quality of coagulation parameters by sigmametric methodology
Murat Aksit, Ayfer Colak, Banu Isbilen Basok, Merve Zeytinli Aksit, Mesut Fidan, Musab Kazar, Tuba Kansu Altan
doi: 10.14744/ijmb.2023.09609  Pages 84 - 89
INTRODUCTION: The total testing process in the laboratory is divided into pre-analytical, analytical, and post-analytical phases. Evaluating the quality of these phases and carrying out improvement studies will increase the quality of the healthcare system. In this study, we aimed to evaluate the analytical phase of the coagulation tests using the six-sigma methodology, which is a quality management tool that aims at zero error.
METHODS: The coefficient of variation (CV%) values were calculated from the two-level internal quality control (IQC) data between January and June 2022 of the coagulation tests (prothrombin time (PT), activated partial thromboplastin time (aPTT), fibrinogen, and D-dimer). External quality control (EQC) data were retrieved from the EQC program of external quality assurance services and used in the calculation of bias %. Total allowable error (TEa) data were obtained from the database of international organizations. The sigma value for each parameter was calculated according to the formula “(TEa%–Bias %)/ CV%.”
RESULTS: Level 1 and level 2 sigma values were calculated as 4.41 and 2.93 for PT; 5.27 and 4.31 for aPTT; 2.72 and 2.73 for fibrinogen, and 4.36 and 4.08 for D-dimer.
DISCUSSION AND CONCLUSION: Among coagulation tests in which we evaluated their analytical performances using the six sigma methodology, PT (level 2) and fibrinogen (level 1 and 2) showed poor performance (sigma value <3). We decided to follow the tests with 13s/22s/R4s/41s/8x Westgard control rules. Using these tests improvement can be achieved in the analytical process, and the overall testing process may improve by performing pre-analytical, post-analytical, and analytical assessments.

6.Conventional cigarette and electronic nicotine delivery systems exacerbate high-fat diet-induced inflammation and oxidative stress
Naufal Arif Ismail, Gavin Izza Muhammad, Ahmad Alvin Noor Muchtar, Arif Jamal Ar Rasyid, Zalfa Nihamuyassari Kanilla, Dwi Nur Ahsani
doi: 10.14744/ijmb.2023.99815  Pages 90 - 95
INTRODUCTION: There is a community habit that conventional cigarette or electronic nicotine delivery systems (ENDS) are used after meals. Also, they tend to consume a high-fat diet (HFD). The effects of this behavior on health remain unclear. Our study focuses on oxidative stress and inflammation in HFD-fed rats model exposed to conventional cigarettes and ENDS.
METHODS: Twenty-four male rats were equitably separated into the following four different groups: (1) NDC: normal diet and fresh air control, (2) HFDC: HFD and fresh air control, (3) HC: HFD+conventional cigarette, and (4) HE: HFD+ENDS. Conventional cigarettes and ENDS were exposed to the same nicotine dose of 12 mg/mL/group. Oxidative stress markers comprising malondialdehyde (MDA), superoxide dismutase (SOD), catalase (CAT), and glutathione (GSH), as well as inflammation markers comprising interleukin (IL)-1β, IL-6, tumor necrosis factor-α, and high sensitivity C-reactive pro-tein were assayed after 6 weeks of treatment. Statistical analysis of one-way ANOVA with post hoc test was performed.
RESULTS: HFD independently increased MDA and IL-6; simultaneously, it decreased SOD, CAT, and GSH. Both conventional cigarettes and ENDS exposure significantly increased entire inflammation markers and MDA, simultaneously decreasing SOD, CAT, and GSH compared to the normal and HFD control rats. Furthermore, a significant difference was observed between conventional cigarettes and ENDS-exposed groups.
DISCUSSION AND CONCLUSION: This study revealed that ENDS is less harmful than conventional cigarettes. Nevertheless, both exposures significantly exacerbated oxidative stress and inflammation in HFD-fed rats, potentially leading to related diseases.

7.Acacetin ameliorates acetylsalicylic acid-induced gastric ulcer in rats by interfering with oxidative stress, inflammation, and apoptosis
Hasan Simsek, Nurhan Akaras
doi: 10.14744/ijmb.2023.07830  Pages 96 - 103
INTRODUCTION: Gastric ulcer (GU) is a benign lesion in which excessive acid and pepsin activity affects the mucosal epithelium and is common worldwide. Gastrointestinal disturbances come to the fore among the side effects observed in the treatment with drugs such as aspirin. Acacetin is a plant-derived flavonoid with intriguing properties such as anti-inflammatory, antioxidant, and anticancer. The aim of the study is to investigate the effects of acacetin in GU model caused by aspirin active ingredient acetylsalicylic acid.
METHODS: Thirty-two Wistar albino rats were divided into four groups: Control, GU, acacetin, and GU +acacetin. Acetyl-salicylic acid (150 mg/kg) and acacetin (25 mg/kg) were administered intraperitoneally as a single dose. Gastric lesions were examined microscopically and macroscopically. TNF-a, cyclooxygenase-2 (COX-2), and nuclear factor kappa B (NF-kB) for inflammation; Caspase-3 and Bcl-2 for apoptosis, total antioxidant status (TAS), total oxidant status (TOS), and oxidative stress index (OSI) for oxidative stress were analyzed.
RESULTS: Bcl-2 and TAS values were decreased, while Tumor necrosis factor-alpha (TNF-α), COX-2, NF-kB, Caspase-3, TOS, and OSI values were increased in the GU group compared to the control group. Bcl-2 and TAS values were increased and TNF-α, COX-2, NF-kB, Caspase-3, TOS, and OSI values were decreased in the GU +acacetin group compared to the GU group. The GU index (GUI) detected in the GU group decreased significantly with the administration of acacetin.
DISCUSSION AND CONCLUSION: High doses of ASA contributed to the formation of GU in the stomach tissue by increasing the levels of inflammation, oxidative stress, and apoptosis, whereas ACA reduced the ulcer damage by reducing the increase in all these pathways.

8.Suberoylanilide hydroxamic acid and 3-deazaneplocin a decrease lncRNA hox transcript antisense RNA in liver fibrosis
Merve Ozel
doi: 10.14744/ijmb.2023.95914  Pages 104 - 108
INTRODUCTION: Liver fibrosis stimulates the abnormal wound repair response of chronic tissue damage, leading to the emergence of many chronic liver diseases such as cirrhosis. In addition, this process is characterized by excessive accumulation of extracellular matrix (ECM) components. The activation of silent hepatic stellate cells (HSCs) in liver fibrosis leads to the release of more ECM. HOX transcript antisense RNA (HOTAIR) is a long non-coding RNAs (lncRNAs) that are overexpressed in numerous types of cancer and are also associated with a number of various fibrosis processes. Recent studies have shown that lncRNAs are important in epigenetic modification. In our study, we aimed to evaluate the effect of the epigenetic drugs suberoylanilide hydroxamic acid (SAHA) and 3-deazaneplanocin A (DZNep) on HOTAIR expression in HSC line (LX2).
METHODS: LX2 cells were scraped with TRIzol using a scraper and total RNAs were taken into ependorphs. After cDNA synthesis from RNA was obtained with the appropriate kit, cDNAs were amplified with FAM-labeled primer probes specific to the mRNA sequence of the genes HOTAIR expression levels which were calculated 2(-ΔΔ)(CT) method and GAPDH was used for control gene.
RESULTS: Both SAHA and DZNep statistically decreased HOTAIR gene levels in LX2 cells (p<0.001; p<0.001, respectively).
DISCUSSION AND CONCLUSION: Due to the fact that both DZNep and SAHA reduced HOTAIR expression, it can be thought that the combined use of both drugs synergistically could be an important approach in preventing hepatic fibrosis. However, further mechanisms related to HOTAIR inhibition should be investigated in hepatic fibrosis.

9.Impact of preanalytical storage on the accuracy of CD3, CD4, CD8 testing results using the BD FACSLyric™ Clinical Flow Cytometry System
Claudio Ilardo, Delphine Tabone, Martine Becker, Laure Deloison, Nathalia Arias Rojas, Margaux Dirat
doi: 10.14744/ijmb.2023.18189  Pages 109 - 113
INTRODUCTION: The development of flow cytometry has facilitated the phenotypic characterization of different populations of lymphocytes. The number of technical platforms performing this analysis is limited and requires transportation of samples from the field site to the laboratory. The aim of our study was to evaluate the agreement of absolute CD3+, CD4+ and CD8+ T cells count measurements from whole blood specimens stored at room temperature (15–25°C) tested 24 h and 72 h post-collection.
METHODS: Forty-one EDTA-anticoagulated blood samples stored at room temperature (15–25°C) after sampling were assayed after 24 h and 72 h, respectively. The BD FACSLyric™ system was used to identify and enumerate cell subsets.
RESULTS: After 72 h, CD3+, CD4+, and CD8+ T lymphocytes, data showed non-significant differences with p-values of 0.766, 0.855, and 0.754, respectively. The Boxplot showed substantial convergence between the two measurement periods.
DISCUSSION AND CONCLUSION: Our data indicated that whole blood can be stored for up to 72 h at room temperature before analysis without affecting the result.

10.Effect of underfilling of tubes with EDTA on PTH assay measured by cobas analyzer
Berrak Guven, Ismail Benice
doi: 10.14744/ijmb.2023.50876  Pages 114 - 116
INTRODUCTION: The purpose of this study is to evaluate cobas parathyroid hormone (PTH) measurement effect of plasma samples obtained from underfilled tubes with ethylenediaminotetraacetic acid (EDTA).
METHODS: Two blood collection tubes with K3-EDTA from 67 patients at the same time were taken. One of them was for routine PTH measurement, which was filled to its capacity, while another tube was underfilled. All EDTA tubes were immediately centrifuged at 4°C. Plasma PTH concentrations were measured by electrochemiluminescence immunoassay method on the cobas e 601 (Roche Diagnostics GmbH, Mannheim, Germany) analyzer.
RESULTS: The underfilled sample tubes were grouped according to their being up to 25% (n=9), 25–50% (n=35), and 50–100% (n=23) of the appropriate amount. In all groups of underfilled tubes, the PTH values were found to decrease concerning those fulfilled (p<0.001). The agreement among underfilled and fulfilled for measuring PTH demonstrated a bias of −3.2 pg/mL (−5.9%) for the underfilled tubes.
DISCUSSION AND CONCLUSION: Insufficiently filled blood in tubes with EDTA has a significant effect on PTH levels. However, further studies are needed to show whether this effect is clinically significant.

11.Soluble FMS-like tyrosine kinase-1: An overview
Sathya Selvarajan, Jothimalar Ramalingam, Veena Venugopal
doi: 10.14744/ijmb.2023.66933  Pages 117 - 123
The soluble FMS-like tyrosine kinase-1 (sFLT-1) or soluble vascular endothelial growth factor receptor 1 (VEGF-R1) is a receptor tyrosine kinase which inhibits the mitogenic activity of VEGF by decreasing its availability for binding with transmembrane receptors. VEGFRs exist in various isoforms due to alternative splicing from the same gene. sFLT-1 along with another of its isoform sFLT1-14 is found in abundance in the cytotrophoblast of placenta, and also in the cornea, liver, brain, and kidney. Hypoxia is the key trigger in inducing production of sFLT-1. In normal pregnancy, the soluble receptor regulates the process of vascular transformation by modulating the balance between VEGF-R1 and sFLT-1 activity. Proteinuria and hypertension in pre-eclamptic women have been found to be associated with an elevated level of sFLT-1. sFLT-1 blocks the podocytederived VEGF and induces damage of the glomerular endothelium leading to proteinuria and blockage of endothelial nitric oxide signaling pathways resulting in hypertension. In ectopic pregnancy, the abnormal implantation of the embryo simulates a hypoxic environment inducing excess production of VEGF and its consequent binding to sFLT-1. The usage of sFLT-1 as an early biomarker of preeclampsia, ectopic pregnancy, and other failing pregnancies is being studied. Variation in sFLT-1 levels has also been identified in cardiovascular diseases, chronic kidney disease, non-healing ulcers, and liver cirrhosis to name a few. Therapeutic use of sFLT-1 to reduce angiogenesis in various conditions like cancer is currently being pursued.

12.Adapter protein – FADD bridges the apoptosis
Faiz Marikar, Chun Zi Hua
doi: 10.14744/ijmb.2023.85619  Pages 124 - 132
More than a century ago, the very first adverse human health effects of Fas-associated death domain (FADD) were re-ported with cell death. It is most well-known role in apoptosis, FADD has also been seen to play a role in other processes including proliferation, cell cycle regulation, and development. It contains two main domains: A C terminal death domain (DD) and an N terminal death effector domain. On stimulation by the Fas ligand, the Fas receptor trimerises. Many receptors, including Fas, contain a cytoplasmic DD and are therefore named death receptors. FADD binds to the DD of this trimeric structure through its DD leads to apoptosis. FADD also plays a role in regulating necroptosis, a process requiring the serine/threonine kinases. Activated caspase 8 cleaves these kinases, inhibiting necroptosis. Application of Taxol is a drug used in anticancer therapies due to its ability to interfere with microtubule assembly, which leads to cell cycle arrest. It has been suggested that inhibition of FADD might work as a potential targeted therapy for drug-resistant ovarian cancer.

13.Irreversible neurological effects of late diagnosis phenylketonuria: A case presentation
Khyati Gupta, Aviral Jain, Vishal Sawant, Sushma Save
doi: 10.14744/ijmb.2023.54376  Pages 133 - 136
Phenylketonuria (PKU) is an autosomal recessive hereditary disorder due to deficiency of enzyme phenylalanine hydroxylase or the cofactor tetrahydrobiopterin. Its late manifestation leads to irreversible neurological changes. The aim of our work is to emphasize the difference in presentation in late diagnosis of the disease verses classical presentation making it difficult to correctly diagnose and also point out the reasons for late diagnosis and missed cases in India. A 9-year-old patient presented with global developmental delay and severe behavioral problems. Hypertonia and spasticity with low intelligence quotient (IQ) were seen. Baseline investigations such as renal function, thyroid function, electrolytes, uric acid, folate, and blood adrenocorticotropic hormone level were within range; hence, magnetic resonance imaging (MRI) was advised which revealed areas of bilateral demyelination suggesting metabolic leukodystrophy. PKU was thereafter confirmed with metabolic profile and clinical exome study. Early routine screening of all new-borns for common inherited and metabolic disorders should be mandatory to later prevent irreversible damage. Cases of delayed diagnosis deviate considerably from classical clinical and radiological findings of the disease making correct and prompt diagnosis difficult. Education of parents and prenatal counseling should be encouraged.

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