175 EFFECTS OF GLUCURONIC ACID AND N-ACETYL-D-GLUCOSAMINE SUPPLEMENTATION ON THE PERIVITELLINE SPACE DURING THE IN VITRO MATURATION OF PORCINE OOCYTES

2017 ◽  
Vol 29 (1) ◽  
pp. 196
Author(s):  
J. Z. Current ◽  
B. D. Whitaker
Keyword(s):  
Hyaluronic Acid ◽  
Embryonic Development ◽  
Glucuronic Acid ◽  
Assay Method ◽  
Enzyme Linked Immunosorbent Assay ◽  
Cortical Granule ◽  
Perivitelline Space ◽  
Significant Difference ◽  
Pronuclear Formation

Pig oocytes fertilized in vitro experience high polyspermic penetration rates due to inadequate cortical granule exocytosis into reduced perivitelline space (PVS) thickness. The objective of this study was to minimize polyspermic penetration by increasing the PVS thickness through supplementation of its hyaluronic acid components, glucuronic acid (GA), and N-acetyl-d-glucosamine (GlcNAc) during maturation. Oocytes were supplemented during the first 24 h or second 24 h of maturation with 0.01 mM GA and 0.01 mM GlcNAc and then evaluated for nuclear maturation (n = 200), PVS thickness (n = 245), and the amount of hyaluronic acid (n = 245) present. The PVS thickness was determined at the equatorial plane of the oocyte using a micrometer. Hyaluronic acid concentrations were determined using an enzyme-linked immunosorbent assay method. Oocytes (n = 800) were fertilized using frozen-thawed semen and evaluated for fertilization characteristics and subsequent embryonic development at 48 and 144 h for cleavage and blastocyst formation, respectively. The PVS thickness was significantly thicker (P < 0.05) with oocytes supplemented with 0.01 mM GA and 0.01 mM GlcNAc during the first half of maturation (3.20 ± 0.29) and all of maturation (2.78 ± 0.21) compared with no supplementation (2.22 ± 0.13) and supplementation during only the second half of maturation (2.02 ± 0.16). The amount of hyaluronic acid present at 24 h of maturation was significantly greater (P < 0.05) in oocytes supplemented with the PVS components (2.03 ± 0.07 pg/oocyte) compared with no supplementation (0.21 ± 0.02 pg/oocyte). At the end of maturation, oocytes supplemented with 0.01 mM GA and 0.01 mM GlcNAc during the entire maturation had significantly greater (P < 0.05) amounts of hyaluronic acid present (4.16 ± 0.19 pg/oocyte) compared with all other groups. There was no significant difference in penetration rates between the groups. Polyspermic penetration was significantly less (P < 0.05) in oocytes supplemented with 0.01 mM GA and 0.01 mM GlcNAc during the first half of maturation compared with no supplementation or supplementation during only the second half of maturation. Oocytes supplemented with 0.01 mM GA and 0.01 mM GlcNAc during the first half of maturation (87.36 ± 4.01) compared with no supplementation (76.47 ± 5.67) or supplementation during only the second half of maturation (80.23 ± 3.21) had significantly higher percentages (P < 0.05) of male pronuclear formation by 12 h after IVF. Supplementing 0.01 mM GA and 0.01 mM GlcNAc during maturation significantly increased (P < 0.05) the percentage of cleaved embryos by 48 h after IVF and the percentage of those reaching the blastocyst stage by 144 h after IVF compared with those that were not supplemented during maturation (55.00 ± 6.43, 20.00 ± 6.16). There were no significant differences between the supplementation treatment groups at 48 or 144 h after IVF. These results indicate that supplementing GA and GlcNAc to the media during maturation, specifically during the first 24 h, decreases polyspermic penetration by increasing PVS thickness, hyaluronic acid amount, and male pronuclear formation, which improves subsequent embryonic development.

scholarly journals Effect of D-Glucuronic Acid and N-acetyl-D-Glucosamine Treatment during In Vitro Maturation on Embryonic Development after Parthenogenesis and Somatic Cell Nuclear Transfer in Pigs

Animals ◽  
2021 ◽  
Vol 11 (4) ◽  
pp. 1034
Author(s):  
Joohyeong Lee ◽  
Eunhye Kim ◽  
Seon-Ung Hwang ◽  
Lian Cai ◽  
Mirae Kim ◽  
...  
Keyword(s):  
Embryonic Development ◽  
Somatic Cell ◽  
Somatic Cell Nuclear Transfer ◽  
Nuclear Transfer ◽  
In Vitro Maturation ◽  
Glucuronic Acid ◽  
Cumulus Cell ◽  
Cortical Granule ◽  
Oocyte Diameter

This study aimed to examine the effects of treatment with glucuronic acid (GA) and N-acetyl-D-glucosamine (AG), which are components of hyaluronic acid (HA), during porcine oocyte in vitro maturation (IVM). We measured the diameter of the oocyte, the thickness of the perivitelline space (PVS), the reactive oxygen species (ROS) level, and the expression of cumulus cell expansion and ROS-related genes and examined the cortical granule (CG) reaction of oocytes. The addition of 0.05 mM GA and 0.05 mM AG during the first 22 h of oocyte IVM significantly increased oocyte diameter and PVS size compared with the control (non-treatment). The addition of GA and AG reduced the intra-oocyte ROS content and improved the CG of the oocyte. GA and AG treatment increased the expression of CD44 and CX43 in cumulus cells and PRDX1 and TXN2 in oocytes. In both the chemically defined and the complex medium (Medium-199 + porcine follicular fluid), oocytes derived from the GA and AG treatments presented significantly higher blastocyst rates than the control after parthenogenesis (PA) and somatic cell nuclear transfer (SCNT). In conclusion, the addition of GA and AG during IVM in pig oocytes has beneficial effects on oocyte IVM and early embryonic development after PA and SCNT.

The effects of glucuronic acid and N-acetyl-D-glucosamine on in vitro fertilisation of porcine oocytes

2016 ◽  
Vol 28 (8) ◽  
pp. 1223 ◽  
Author(s):  
K. Schmidt ◽  
A. Clark ◽  
A. Mello ◽  
C. Durfey ◽  
A. Buck ◽  
...  
Keyword(s):  
Oocyte Maturation ◽  
Zona Pellucida ◽  
Glucuronic Acid ◽  
In Vitro Fertilisation ◽  
Cortical Granule ◽  
Perivitelline Space ◽  
Blastocyst Formation ◽  
Space Thickness ◽  
Intracellular Glutathione

High incidences of polyspermic penetration continue to challenge researchers during porcine in vitro fertilisation (IVF). The aim of this study was to reduce the incidence of polyspermy by increasing the perivitelline space thickness with glucuronic acid and N-acetyl-D-glucosamine (GlcNAc) supplementation during oocyte maturation. After maturation, zona pellucida and perivitelline space thicknesses, intracellular glutathione concentrations and fertilisation kinetics were measured, in addition to embryonic cleavage and blastocyst formation at 48 h and 144 h after IVF, respectively. There were no significant differences between the treatments for zona pellucida thickness, penetration rates, male pronuclear formation or cortical granule exocytosis. Glucuronic acid supplementation significantly increased (P < 0.05) the perivitelline space thickness and significantly lowered the incidence (P < 0.05) of polyspermy. GlcNAc supplementation significantly increased (P < 0.05) intracellular glutathione concentrations. Supplementation with 0.005 mM glucuronic acid plus 0.005 mM GlcNAc during oocyte maturation produced significantly higher rates (P < 0.05) of cleavage and blastocyst formation by 48 and 144 h after IVF compared with all other groups. These results indicate that supplementing with 0.005 mM glucuronic acid and 0.005 mM GlcNAc during oocyte maturation decreases the incidence of polyspermic penetration by increasing perivitelline space thickness and improving embryo development in pigs.

Effects of glucuronic acid and N-acetyl-D-glucosamine supplementation on the perivitelline space during the IVM of pig oocytes

2020 ◽  
Vol 32 (10) ◽  
pp. 941
Author(s):  
J. Z. Current ◽  
B. D. Whitaker
Keyword(s):  
Hyaluronic Acid ◽  
Glutathione Peroxidase ◽  
Embryo Development ◽  
Glucuronic Acid ◽  
The Other ◽  
Perivitelline Space

The objective of this study was to minimise polyspermic penetration by increasing the perivitelline space (PVS) thickness through supplementation of the hyaluronic acid components glucuronic acid and N-acetyl-d-glucosamine (GlcNAc). Oocytes (n=4690) were supplemented during the first 24h and/or the remainder of maturation (final 16–18h) with 0.01mM glucuronic acid and 0.01mM GlcNAc and then evaluated for PVS thickness, hyaluronic acid, glutathione and glutathione peroxidase concentrations. Fertilised oocytes were evaluated for polyspermic penetration and embryo development. The PVS thickness and amount of hyaluronic acid was significantly (P&lt;0.05) greater in oocytes supplemented with 0.01mM glucuronic acid and 0.01mM GlcNAc during the second part or all of maturation compared with the other treatments. In addition, polyspermic penetration was significantly (P&lt;0.05) less in oocytes supplemented with 0.01mM glucuronic acid and 0.01mM GlcNAc during the second part or all of maturation compared with the other treatments. Supplementing 0.01mM glucuronic acid and GlcNAc during maturation significantly (P&lt;0.05) increased the percentage of cleaved embryos by 48h after IVF and blastocysts formed by 144h after IVF compared those not supplemented. These results indicate that supplementing PVS components during maturation decreases polyspermic penetration by increasing PVS thickness.

scholarly journals Comparative study of estrogen receptor α, β mRNA expressions of endometriosis and normal endometrium in women and analysis of potential synthetic anti-estrogens in silico

2018 ◽  
Vol 91 (2) ◽  
Author(s):  
Eldafira Eldafira ◽  
Abinawanto Abinawanto ◽  
Luthfiralda Sjahfirdi ◽  
Asmarinah Asmarinah ◽  
Purnomo Soeharso ◽  
...  
Keyword(s):  
Estrogen Receptor ◽  
Mrna Expression ◽  
Estrogen Receptor Α ◽  
Assay Method ◽  
Enzyme Linked Immunosorbent Assay ◽  
Normal Endometrium ◽  
Pcr Method ◽  
Genetic And Environmental Factors

Endometriosis is a multifactorial disease in which genetic and environmental factors interact causing its pathogenesis. The aim of this study was to investigate the expression pattern of estrogen receptor α (ERα) and β (ERβ) in endometriosis patients compared to normal endometrioum (n=18) as a control by using Quantitative Real Time PCR method. Moreover, we also measured serum estradiol levels of endometriosis patients in the proliferation phase of the menstrual cycle using the enzyme-linked immunosorbent assay method. The mRNA expression of ERβ was significantly higher in the endometriosis group compared to control, and the result of t-test showed that were significantly different (P<0.05). Overexpression of ERβ in endometriosis was likely to have other significant important impacts in the pathology of endometriosis that allowed ERβ to stimulate prostaglandin production in endometriosis tissue and cells. Estradiol content did not correlate with the ERα expression, and it is weakly correlated with ERβ mRNA expression. Molecular docking analysis showed that ERα and ERβ have different binding interactions with synthetic antiestrogens, whereas the best inhibitor was Ral2 to ERα and Aco1 to ERβ. Thus, both inhibitors could be used as leads in further investigation of ERα, ERβ inhibitory activities in vitro and in vivo.

In vitro synthesis of glycosaminoglycans in endocrine ophthalmopathy

1992 ◽  
Vol 127 (5) ◽  
pp. 397-402 ◽  
Author(s):  
G Kahaly ◽  
C Stover ◽  
J Beyer ◽  
E Otto
Keyword(s):  
Hyaluronic Acid ◽  
Acid Concentration ◽  
Cetylpyridinium Chloride ◽  
Acid Synthesis ◽  
Cell Mediated Immunity ◽  
Glycosaminoglycan Synthesis ◽  
In Vitro Synthesis ◽  
Significant Difference ◽  
Endocrine Ophthalmopathy

The effects of humoral and cell-mediated immunity on the glycosaminoglycan synthesis of retrobulbar fibroblasts was evaluated in patients with endocrine ophthalmopathy. After incubation with IgG and sera, secreted glycosaminoglycans, radiolabeled with D-6-3H-glucosamine and 35sulfate, were precipitated with cetylpyridinium chloride and ethanol. Hyaluronic acid synthesis of human retrobulbar fibroblasts after incubation with sera and IgG and after co-culture with lymphocytes was assessed by means of a radiometric test. Patients' IgG, compared to controls', accounted for a higher secretory stimulation of porcine retrobulbar fibroblasts (as measured by cetylpyridinium chloride precipitation) after 24 and 48 h. Contrasting with 24 h incubation time, glycosaminoglycan values after 48 h were increased two to threefold. Patients' and controls' sera caused earlier and stronger, yet indistinguishable glycosaminoglycan production. Non-sulfated hyaluronic acid was the preponderant glycosaminoglycan secreted into the media by retrobulbar fibroblasts. As assessed with the radiometric test, incubation with patients' and controls' sera and IgG did not reveal a significant difference in stimulating the hyaluronic synthesis of patients' and controls' retrobulbar fibroblasts. When measuring the hyaluronic acid synthesis of controls' and patients' retrobulbar fibroblasts after co-cultivation of lymphocytes, however, patients' lymphocytes had a marked ability to increase the hyaluronic acid concentration compared to controls' lymphocytes. The hyaluronic acid concentration after incubation of a patient's retrobulbar fibroblasts with autologous lymphocytes was markedly more elevated than the intrinsic hyaluronic acid production of retrobulbar fibroblasts. In conclusion, though a significant in vitro influence of patients' IgG and sera on the glycosaminoglycan release of both porcine and human (patients' as well as controls') retrobulbar fibroblasts could not be observed in this study, the indications of a marked stimulatory influence of lymphocytes on the hyaluronic acid secretion of retrobulbar fibroblasts demand further investigation.

4 SUPPRESSION OF ASH2L ALTERS DNA METHYLATION AND HISTONE PATTERNS DURING BOVINE EMBRYONIC DEVELOPMENT

2016 ◽  
Vol 28 (2) ◽  
pp. 131
Author(s):  
M. D. Snyder ◽  
J. H. Pryor ◽  
M. D. Peoples ◽  
G. L. Williamson ◽  
M. C. Golding ◽  
...  
Keyword(s):  
Dna Methylation ◽  
Embryonic Development ◽  
Blastocyst Stage ◽  
Early Embryonic Development ◽  
Standard Laboratory ◽  
Significant Difference ◽  
Commercial Supplier ◽  
Secondary Antibodies ◽  
Laboratory Protocol

Epigenetic patterns established during early bovine embryogenesis via DNA methylation and histone modification patterns are essential for proper gene expression and embryonic development. We have previously discovered that suppression of absent, small, or homeotic-like (ASH2L) with small interfering RNA (siRNA) had no significant effect during in vitro embryo development when compared with its respective control (31.3 ± 2.0% standard error of the mean, n = 466 v. 34.8 ± 1.9%, n = 418). Analysing DNA methylation and histone modifications via immunocytochemistry will further explain the role of ASH2L during embryonic development, specifically at the blastocyst stage. In this experiment, we obtained mature bovine oocytes from a commercial supplier (De Soto Biosciences, Seymour, TN) and preformed IVF following standard laboratory protocol. Eighteen hours after IVF, presumptive zygotes were divided into 3 treatments: noninjected controls, nontargeting siRNA injected controls (siNULL), and injection with siRNA targeting ASH2L (siASH2L). Each embryo was injected with ~100 pL of 20 nM siRNA previously verified to suppress expression of ASH2L by ~79%. Embryos were cultured in Bovine Evolve (Zenith Biotech, Guilford, CT) supplemented with 4 mg mL–1 of BSA (Probumin, Millipore) for 7 days. Blastocysts from each treatment (N = 601) were fixed and prepared for immunocytochemistry following standard laboratory protocol. The following primary antibodies were used to target specific DNA and histone methylation marks: 5mc mAb (Epigentek, Farmingdale, NY), 5hmc pAb, H3K4me3 pAb (Active Motif, Carlsbad, CA), H3K4me2 pAb, H3K9me2–3 mAb, and H3K27me3 mAb (Abcam, Cambridge, MA). Embryos were fluorescently labelled with the following secondary antibodies: Alexa Flour 488 Goat Anti-Rabbit, Alexa 488 Donkey Anti-Goat, and Alexa Flour 594 Goat Anti-Mouse (Invitrogen, Carlsbad, CA). The DNA was stained with Hoechst 33342 (Invitrogen). Fluorescent images were captured using the Zeiss Stallion digital imaging work station. Ratio averages (targeting mark/DNA) were calculated and statistical analysis performed using one-way ANOVA and Tukey’s honestly significant difference to assess treatment effects. The ratio of DNA methylation to total DNA increased in siASH2L as compared with control and siNULL embryos (0.35 ± 0.01, 0.26 ± 0.02, and 0.30 ± 0.01, respectively; P < 0.01). The 5hmC was inversely related to 5mC levels and decreased in siASH2L embryos (0.75 ± 0.01, 0.93 ± 0.02, 0.87 ± 0.02, respectively; P < 0.0001). The H3K4me3 and H3K27me3 are also inversely related with decreased H3K4me3 in siASH2L versus control and siNULL embryos (0.48 ± 0.02, 0.57 ± 0.02, 0.58 ± 0.02, respectively; P < 0.001) and increased H3K27me3 (0.62 ± 0.02, 0.053 ± 0.01, 0.54 ± 0.02, respectively; P < 0.001). No differences were observed in H3K9me2–3 or H3K4me2 labelling across treatments. These results indicate that ASH2L may play a role in DNA methylation by decreasing 5mc and 5hmc conversion, which is a key event during early embryonic development. Suppression of ASH2L also alters global levels of H3H4me3 and H3K27me3, which may lead to transcription aberrations. Further analysis of siASH2L embryos via RNA-seq will help define its role during early embryonic development.

scholarly journals Effect of Denture Base Reinforcement Using Light Cured E- Glass Fibers on the Level of Salivary Immunoglobulin A

2018 ◽  
Vol 6 (11) ◽  
pp. 2168-2172
Author(s):  
Shady M. El Naggar ◽  
Mohamed I. Seif El Nasr ◽  
Hassan M. Sakr ◽  
Sherihan M. Eissa ◽  
Asmaa N. Elboraey ◽  
...  
Keyword(s):  
Immunoglobulin A ◽  
Acrylic Resin ◽  
Enzyme Linked Immunosorbent Assay ◽  
Complete Dentures ◽  
Denture Base ◽  
Mean Values ◽  
Glass Fibres ◽  
Significant Difference ◽  
Salivary Immunoglobulin A

BACKGROUND: A gap still exists between in vitro and clinical studies concerning the biocompatibility of the material in the oral environment and their potential to cause immunological undesirable side effects. The uses of glass fibres to improve the mechanical properties of acrylic resin denture base polymers are well documented in vitro. AIM: The present study aimed to evaluate the effect of denture base reinforcement using light-cured E- glass fibres mesh on the level of salivary immunoglobulin A (S-IgA) in patients wearing complete dentures. MATERIAL AND METHODS: Fourteen completely edentulous patients, in need of complete dentures, participated in the study. The patients were divided into two groups (n = 7) according to the treatment protocol. In the first group, patients received conventional heat-cured acrylic resin dentures. In the second group, the mandibular dentures were reinforced using light cured resin impregnated E glass fibres mesh. In both groups, salivary samples were collected using passive drool technique. The level IgA was assessed by enzyme-linked immunosorbent assay (ELISA) technique at different time intervals. Statistical analysis was carried out using one-way ANOVA followed by Tukey`s post-hoc test and independent t-test. The significant level was set at P ≤ 0.05. RESULTS: Acrylic resin dentures and reinforced ones demonstrated an increase in the mean values of IgA level at the end of the follow-up intervals. And this increase was statistically significant (P ≤ 0.05). Although, the reinforced dentures revealed higher mean values, there was no statistically significant difference between the two groups (P > 0.05) CONCLUSIONS: Within the limitations of the present study, the following could be concluded: (1) the insertion of complete dentures induced changes in the level of IgA; and (2) denture base reinforcement using light cured resin impregnated E-glass fibres mesh had a similar effect to that of heat cured acrylic resin on the level of IgA.

scholarly journals Cumulus Cell Expansion, Its Role in Oocyte Biology and Perspectives of Measurement: A Review

2015 ◽  
Vol 45 (4) ◽  
pp. 212-225 ◽  
Author(s):  
J. Nevoral ◽  
M. Orsák ◽  
P. Klein ◽  
J. Petr ◽  
M. Dvořáková ◽  
...  
Keyword(s):  
Extracellular Matrix ◽  
Hyaluronic Acid ◽  
High Performance ◽  
Uv Light ◽  
Cumulus Cell ◽  
Developmental Competence ◽  
Enzyme Linked Immunosorbent Assay ◽  
Cumulus Expansion

Abstract Cumulus expansion of the cumulus-oocyte complex is necessary for meiotic maturation and acquiring developmental competence. Cumulus expansion is based on extracellular matrix synthesis by cumulus cells. Hyaluronic acid is the most abundant component of this extracellular matrix. Cumulus expansion takes place during meiotic oocyte maturation under in vivo and in vitro conditions. Quantification and measurement of cumulus expansion intensity is one possible method of determining oocyte quality and optimizing conditions for in vitro cultivation. Currently, subjective methods of expanded area and more exact cumulus expansion measurement by hyaluronic acid assessment are available. Among the methods of hyaluronic acid measurement is the use of radioactively labelled synthesis precursors. Alternatively, immunological and analytical methods, including enzyme-linked immunosorbent assay (ELISA), spectrophotometry, and high-performance liquid chromatography (HPLC) in UV light, could be utilized. The high sensitivity of these methods could provide a precise analysis of cumulus expansion without the use of radioisotopes. Therefore, the aim of this review is to summarize and compare available approaches of cumulus expansion measurement, respecting special biological features of expanded cumuli, and to suggest possible solutions for exact cumulus expansion analysis.

scholarly journals CYTOTOXICITY OF IONS RELEASED FROM 17-4 PRECIPITATION HARDENING STAINLESS STEEL ORTHODONTIC BRACKETS IN ARTIFICIAL SALIVA

2018 ◽  
Vol 9 ◽  
pp. 71
Author(s):  
Tjokro Prasetyadi ◽  
Bambang Irawan ◽  
Miesje Karmiati Purwanegara ◽  
Bambang Suharno ◽  
Sugeng Supriadi
Keyword(s):  
Stainless Steel ◽  
Cell Viability ◽  
Precipitation Hardening ◽  
Artificial Saliva ◽  
Nickel Content ◽  
Orthodontic Brackets ◽  
Assay Method ◽  
Cytotoxicity Test ◽  
Significant Difference

Objective: 17-4 precipitation hardening (PH) stainless steel has a low nickel content, which can reduce the risk of allergic reactions. It also has good mechanical properties against the stress caused by the archwire slot brackets in orthodontic treatments. The main focus of this study to evaluate the metal ions released into artificial saliva from different orthodontic brackets with the same 17-4 PH stainless steel and to examine the in vitro cytotoxicity of the metal.Methods: Material properties were analyzed by energy dispersive spectroscopy. The 3-(4,5-dimethylthiazol-2-y1)2,5-diphenyltetrazolium bromide (MTT) assay method was used to examine the cytotoxicity of Gemini and Synergy brackets.Results: The cytotoxicity test on all the orthodontic brackets showed a mean cell viability value above 80% in each immersion group, which means that this material is not cytotoxic to the human immortalized keratinocyte cell line.Conclusions: The results showed cell viability in the extracts of both groups of brackets, and there was no statistically significant difference between the groups (p>0.05).

scholarly journals N-TERMINAL PRO-BRAIN NATRIURETIC PEPTIDE (NT-proBNP) IN STAGE 1 AND STAGE 2 HYPERTENSION PATIENTS

2019 ◽  
Vol 1 (2) ◽  
pp. 65-67
Author(s):  
Supriati Wila Djami
Keyword(s):  
Left Ventricular ◽  
Assay Method ◽  
Enzyme Linked Immunosorbent Assay ◽  
Hypertensive Patients ◽  
Hypertensive Group ◽  
Undiagnosed Hypertension ◽  
Increased Risk ◽  
Study Results ◽  
Significant Difference ◽  
Stage 1

The increased levels of NT-proBNP in the blood occur when heart function, especially the left ventricular muscle chambers of the heart increases. Therefore, NT-proBNP is used as a biomarker to detect heart failure.The level of N Terminal – Pro Brain Natriuetic Peptide was independently associated with an increased risk of hypertension. This study aimed to determine the difference of NT-proBNP serum levels and the correlation between the levels of NT-proBNP in patients with stage 1 and stage 2 hypertension. This research was conducted at RSUP dr. Wahidin Sudirohusodo in August - September 2018. The study used a cross-sectional design with the total of 72 hypertensive patients, who had met the inclusive criteria. NT-proBNP levels were measured using the ELISA (Enzyme Linked Immunosorbent Assay) method.  The collected data was processed using Mann Whitney Different Test and Spearman's rho Correlation Test. The study results indicated that the level of NT-proBNP in the hypertensive patients with stage 2 was higher and significantly different (p = <0.001) compared to stage 1 hypertensive patients. NT-proBNP levels were higher in the hypertensive group of >6 years than in the hypertensive group <6 years. There were significant differences between the two groups statistically (p=0.010). It can be Conclude that there is a significant difference in the levels of NT-proBNP with a degree of hypertension where NT-proBNP levels were higher in patients with stage 2 hypertension compared to stage 1 hypertension, although  there was not statistically significant correlation between levels of NT-proBNP with Hypertension degree. Further research was needed to determine the relationship of NT-proBNP levels with the degree of hypertension, which can confirm the diagnosis, especially in patients with hypertension. Also, it is suggested to consider the accuracy of the data length of a patient suffering from undiagnosed hypertension.

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