Pathophysiology of Mild Hypercortisolism: From the Bench to the Bedside

Mild hypercortisolism is defined as biochemical evidence of abnormal cortisol secretion without the classical detectable manifestations of overt Cushing’s syndrome and, above all, lacking catabolic characteristics such as central muscle weakness, adipose tissue redistribution, skin fragility and unusual infections. Mild hypercortisolism is frequently discovered in patients with adrenal incidentalomas, with a prevalence ranging between 5 and 50%. This high variability is mainly due to the different criteria used for defining this condition. This subtle cortisol excess has also been described in patients with incidentally discovered pituitary tumors with an estimated prevalence of 5%. To date, the mechanisms responsible for the pathogenesis of mild hypercortisolism of pituitary origin are still not well clarified. At variance, recent advances have been made in understanding the genetic background of bilateral and unilateral adrenal adenomas causing mild hypercortisolism. Some recent data suggest that the clinical effects of glucocorticoid (GC) exposure on peripheral tissues are determined not only by the amount of the adrenal GC production but also by the peripheral GC metabolism and by the GC sensitivity. Indeed, in subjects with normal cortisol secretion, the combined estimate of cortisol secretion, cortisone-to-cortisol peripheral activation by the 11 beta-hydroxysteroid dehydrogenase enzyme and GC receptor sensitizing variants have been suggested to be associated with the presence of hypertension, diabetes and bone fragility, which are three well-known consequences of hypercortisolism. This review focuses on the pathophysiologic mechanism underlying both the different sources of mild hypercortisolism and their clinical consequences (bone fragility, arterial hypertension, subclinical atherosclerosis, cardiovascular remodeling, dyslipidemia, glucose metabolism impairment, visceral adiposity, infections, muscle damage, mood disorders and coagulation).

Comparison between LDH with Zap-70 and CD38 as prognostic Markers in Chronic Lymphocytic Leukemia Sudanese patients - A Pilot Study in Khartoum State

Background: Several prognostic markers are used in clinical stages of Chronic Lymphocytic Leukemia (CLL); including lactate dehydrogenase enzyme (LDH), CD38 and Zeta associated protein kinase (ZAP70). This study aimed to compare LDH with ZAP70 and CD38 as prognostic markers in Sudanese patients with CLL. Materials and Methods: Fifty newly diagnosed patients with CLL were enrolled in this cross-sectional study, the age of patients ranged between 36 - 85 years, with mean of 62 years. Of total patients; there were 34 males (68%) and 16 females (32%). Blood samples were obtained and LDH measurement was done by using Dirui CS-T240 automated analyzer while CD38 and ZAP-70 were measured by flowcytometry. Data were analyzed by statistical package for social science (SPSS) computer software program version 21. Results: The present study revealed a significant association between LDH and Rai staging (p. value= 0.002), when LDH compared with ZA-70 and CD38 as reference prognostic markers; poor correlation between CD38 and LDH was found (R2= 0.086, P value= 0.034) and no correlation between LDH level and ZAP-70 expression (R2= 000, P value= 0.960). In conclusion: According to the outcomes of this study; ZAP-70 and CD38 cannot be substituted by LDH as a prognostic marker.

Potential added value of combined DPYD/DPD genotyping and phenotyping to prevent severe toxicity in patients with a DPYD variant and decreased dihydropyrimidine dehydrogenase enzyme activity

Decreased dihydropyrimidine dehydrogenase enzyme activity is associated with severe fluoropyrimidine-associated toxicity. Four clinically relevant variants in the DPYD gene are associated with decreased dihydropyrimidine dehydrogenase activity. However, only ∼25% of DPYD variant carriers show a decreased dihydropyrimidine dehydrogenase activity in peripheral blood mononuclear cells. Objective To investigate if dihydropyrimidine dehydrogenase phenotyping has added value when combined with DPYD genotyping in predicting fluoropyrimidine-related toxicity. Methods Retrospective cohort study in which treatment and toxicity data were collected of 228 patients genotyped for four DPYD variants and phenotyped using an ex vivo peripheral blood mononuclear cell assay. Results Severe toxicity occurred in 25% of patients with a variant and normal dihydropyrimidine dehydrogenase activity, in 21% of patients without a variant and with decreased dihydropyrimidine dehydrogenase activity, and in 29% of patients without a variant and with normal dihydropyrimidine dehydrogenase activity (controls). The majority of patients with a variant or a decreased dihydropyrimidine dehydrogenase activity received an initial dose reduction (68% and 53% vs 19% in controls) and had a lower mean dose intensity (75% and 81% vs 91% in controls). Fifty percent of patients with a variant and decreased enzyme activity experienced severe toxicity, despite the lowest initial dose and whole treatment dose intensity. They also experienced more grade 4/5 toxicities. Conclusions Our results indicate that a combined genotype–phenotype approach could be useful to identify patients at increased risk for fluoropyrimidine-associated toxicity (e.g. patients with a variant and decreased dihydropyrimidine dehydrogenase activity). Because the group sizes are too small to demonstrate statistically significant differences, this warrants further research in a prospective study in a larger cohort.

Evaluation of the Antioxidant, Antidiabetic, and Antiplasmodial Activities of Xanthones Isolated from Garcinia forbesii and Their In Silico Studies

This study aimed to isolate xanthones from Garcinia forbesii and evaluated their activity in vitro and in silico. The isolated compounds were evaluated for their antioxidant activity by DPPH, ABTS and FRAP methods. The antidiabetic activity was performed against α-glucosidase and α-amylase enzymes. The antiplasmodial activity was evaluated using Plasmodium falciparum strain 3D7 sensitive to chloroquine. Molecular docking analysis on the human lysosomal acid-alpha-glucosidase enzyme (5NN8) and P. falciparum lactate dehydrogenase enzyme (1CET) and prediction of ADMET for the active compound, were also studied. For the first time, lichexanthone (1), subelliptenone H (2), 12b-hydroxy-des-D-garcigerrin A (3), garciniaxanthone B (4) and garcigerin A (5) were isolated from the CH2Cl2 extract of the stem bark of G. forbesii. Four xanthones (Compounds 2–5) showed strong antioxidant activity. In vitro α-glucosidase test showed that Compounds 2 and 5 were more active than the others, while Compound 4 was the strongest against α-amylase enzymes. In vitro antiplasmodial evaluation revealed that Compounds 2 and 3 showed inhibitory activity on P. falciparum. Molecular docking studies confirmed in vitro activity. ADMET predictions suggested that Compounds 1–5 were potential candidates for oral drugs. The isolated 2–5 can be used as promising phytotherapy in antidiabetic and antiplasmodial treatment.

Molecular cloning and characterization of NAD+ dependent isocitrate dehydrogenase enzyme from Shewanella putrefaciens

Isocitrate dehydrogenase (IDH) is a fundamental enzyme for carbon metabolism in the Krebs cycle. This enzyme is required for oxidation-reduction reactions in both eukaryotic and prokaryotic cells and plays a critical role in their growth and pathogenesis. In this study, we cloned the gene encoding NAD+ dependent isocitrate dehydrogenase from Shewanella putrefaciens. The expression of recombinant protein was induced with 0.5 mM of IPTG. His-tagged IDH overexpressed in E. coli was purified and characterized. The expressed IDH enzyme was purified in an active soluble form. The molecular weight of the enzyme was confirmed with Western blotting. High sequence homology was observed with IDH sequences of other Shewanella strains and remarkable sequence homology was found with other bacteria reported in the database. The open reading frame of the gene encoding IDH of S. putrefaciens was 1069 bp in length, which codes a polypeptide composed of 356 amino acids and with a 38 KDa molecular weight. The optimum enzyme activity was obtained at pH 8. We cloned, purified and characterized Shewanella putrefaciens isocitrate dehydrogenase enzyme (SpIDH). The recombinant enzyme demonstrated a specific activity of Vmax 4.6±0.3 μM/min and Km 334 μM using NAD+ as a cofactor.

In silico docking of Novel Phytoalkaloid Camalexin in the Management of Benomyl Induced Parkinson's disease and its In vivo Evaluation by Zebrafish Model

Background: Parkinson’s disease (PD) exhibits the extra pyramidal symptoms caused due to the dopaminergic neuronal degeneration in the substantia nigra of the brain and depletion of aldehyde dehydrogenase (ALDH) enzyme. Objective: This study was designed to enlighten the importance of Aldehyde dehydrogenase enzyme in protecting the dopamine levels in a living system. Camalexin, a potentially active compound has been evaluated for its dopamine enhancing and aldehyde dehydrogenase protecting role in pesticide induced Parkinson’s disease. Methods: AutoDock 4.2 software was employed to perform the docking simulations between the ligand camalexin and standard drugs Alda-1, Ropirinole with three proteins 4WJR, 3INL, 5AER. Consequently, the compound was evaluated for its in vivo neuroprotective role in zebrafish model by attaining Institutional Animal Ethical Committee permission. The behavioral assessments and catecholamine analysis in zebrafish were performed. Results: The Autodock result shows that the ligand camalexin has a lower binding energy (-3.84) that indicate higher affinity with the proteins when compared to the standard drug of proteins (-3.42). In zebrafish model, behavioral studies provided an evidence that camalexin helps in improvement of motor functions and cognition. The catecholamine assay has proved there is an enhancement in dopamine levels, as well as an improvement in aldehyde dehydrogenase enzyme also. Conclusion: The novel compound, camalexin, hence offers a protective role in Parkinson’s disease model by its interaction with neurochemical proteins and also in alternative in vivo model.

Dehydrogenation Mechanism of Three Stereoisomers of Butane-2,3-Diol in Pseudomonas putida KT2440

Pseudomonas putida KT2440 is a promising chassis of industrial biotechnology due to its metabolic versatility. Butane-2,3-diol (2,3-BDO) is a precursor of numerous value-added chemicals. It is also a microbial metabolite which widely exists in various habiting environments of P. putida KT2440. It was reported that P. putida KT2440 is able to use 2,3-BDO as a sole carbon source for growth. There are three stereoisomeric forms of 2,3-BDO: (2R,3R)-2,3-BDO, meso-2,3-BDO and (2S,3S)-2,3-BDO. However, whether P. putida KT2440 can utilize three stereoisomeric forms of 2,3-BDO has not been elucidated. Here, we revealed the genomic and enzymic basis of P. putida KT2440 for dehydrogenation of different stereoisomers of 2,3-BDO into acetoin, which will be channeled to central mechanism via acetoin dehydrogenase enzyme system. (2R,3R)-2,3-BDO dehydrogenase (PP0552) was detailedly characterized and identified to participate in (2R,3R)-2,3-BDO and meso-2,3-BDO dehydrogenation. Two quinoprotein alcohol dehydrogenases, PedE (PP2674) and PedH (PP2679), were confirmed to be responsible for (2S,3S)-2,3-BDO dehydrogenation. The function redundancy and inverse regulation of PedH and PedE by lanthanide availability provides a mechanism for the adaption of P. putida KT2440 to variable environmental conditions. Elucidation of the mechanism of 2,3-BDO catabolism in P. putida KT2440 would provide new insights for bioproduction of 2,3-BDO-derived chemicals based on this robust chassis.

Evaluation of Serum Levels of Lactate Dehydrogenase Isoenzymes in COVID- 19 Patients

Background: This study aimed to determine the serum levels of lactate dehydrogenase isoenzymes (LDH1, LDH2, LDH3, LDH4, and LDH5) and their contribution to the total lactate dehydrogenase enzyme elevation observed in COVID-19 patients. Design of study: This study was conducted in collaboration between Al-Nahrain University/College of Medicine/Chemistry and Biochemistry Department and Al- Yarmouk Teaching Hospital, Baghdad, Iraq. The study included 90 patients with confirmed COVID-19 infection: 45 with severe symptoms, and 45 with mild symptoms during the period from February 2021 to June 2021. The different LDH isoenzymes (LDH1, LDH2, LDH3, LDH4, and LDH5) were determined by enzyme-linked immunosorbent assay (ELISA) kits. Results: This study showed high correlation between total lactate dehydrogenase (LDH) enzyme and disease progression and severity in patients with COVID-19. The study also showed significantly higher levels of the lactate dehydrogenase isoenzymes (LDH1, LDH3, and LDH4) in patients with severe symptoms. Conclusion: This study suggests that elevation of serum lactate dehydrogenase (LDH) in patients with COVID-19 may be associated with the release of more than one of LDH isoenzymes into the bloodstream, therefore the use of total LDH as a specific biomarker for lung affectation in patients with COVID-19 is not specific, but the assay of all LDH isoenzymes could give a better understanding of the tissues most affected by the virus.