Competitive oxidation of key pentose phosphate pathway enzymes modulates the fate of intermediates and NAPDH production.

The oxidative phase of the pentose phosphate pathway (PPP) involving the enzymes glucose-6-phosphate dehydrogenase (G6PDH), 6-phosphogluconolactonase (6PGL), and 6-phosphogluconate dehydrogenase (6PGDH), is critical to NADPH generation within cells, with these enzymes catalyzing the conversion of glucose-6-phosphate (G6P) into ribulose-5-phosphate (Ribu5-P). We have previously studied peroxyl radical (ROO•) mediated oxidative inactivation of E. coli G6PDH, 6PGL, and 6PGDH. However, these data were obtained from experiments where each enzyme was independently exposed to ROO•, a condition not reflecting biological reality. In this work we investigated how NADPH production is modulated when these enzymes are jointly exposed to ROO•. Enzyme mixtures (1:1:1 ratio) were exposed to ROO• produced from thermolysis of 100 mM 2,2'-azobis(2-methylpropionamidine) dihydrochloride (AAPH). NADPH was quantified at 340 nm, and protein oxidation analyzed by liquid chromatography with mass spectrometric detection (LC-MS). The data obtained were rationalized using a mathematical model. The mixture of non-oxidized enzymes, G6P and NADP+ generated ∼175 µM NADPH. Computational simulations showed a constant decrease of G6P associated with NADPH formation, consistent with experimental data. When the enzyme mixture was exposed to AAPH (3 h, 37 ºC), lower levels of NADPH were detected (∼100 µM) which also fitted with computational simulations. LC-MS analyses indicated modifications at Tyr, Trp, and Met residues but at lower concentrations than detected for the isolated enzymes. Quantification of NADPH generation showed that the pathway activity was not altered during the initial stages of the oxidations, consistent with a buffering role of G6PDH towards inactivation of the oxidative phase of the pathway.

Understanding the dosing-time-dependent antihypertensive effect of valsartan and aspirin through mathematical modeling.

Chronopharmacology of arterial hypertension impacts the long-term cardiovascular risk of hypertensive subjects. Therefore, clinical and computational studies have proposed optimizing antihypertensive medications' dosing time (Ta). However, the causes and mechanisms underlying the Ta-dependency antihypertensive effect have not been elucidated. Here we propose using a Ta- dependent effect model to understand and predict the antihypertensive effect of valsartan and aspirin throughout the day in subjects with grade I or II essential hypertension. The model based on physiological regulation mechanisms includes a periodic function for each parameter that changes significantly after treatment. Circadian variations of parameters depending on the dosing time allowed the determination of regulation mechanisms dependent on the circadian rhythm that were most relevant for the action of each drug. In the case of valsartan, it is the regulation of vasodilation and systemic vascular resistance. In the case of aspirin, the antithrombotic effect generates changes in the sensitivity of systemic vascular resistance and heart rate to changes in physical activity. Dosing time-dependent models predict a more significant effect on systemic vascular resistance and blood pressure when administering valsartan or aspirin at bedtime. However, circadian dependence on the regulation mechanisms showed different sensitivity of their circadian parameters and shapes of functions, presenting different phase shifts and amplitude. Therefore, different mechanisms of action and pharmacokinetic properties of each drug can generate different profiles of Ta-dependence of antihypertensive effect and optimal dosing times.

Dosing time optimization of antihypertensive medications by including the circadian rhythm in pharmacokinetic-pharmacodynamic models.

Blood pressure (BP) follows a circadian variation, increasing during active hours, showing a small postprandial valley and a deeper decrease during sleep. Nighttime reduction of 10-20% relative to daytime BP is defined as a dipper pattern, and a reduction of less than 10%, as a non-dipper pattern. Despite this BP variability, hypertension's diagnostic criteria and therapeutic objectives are usually based on BP average values. Indeed, studies have shown that chrono-pharmacological optimization significantly reduces long-term cardiovascular risk if a BP dipper pattern is maintained. Changes in the effect of antihypertensive medications can be explained by circadian variations in their pharmacokinetics (PK) and pharmacodynamics (PD). Nevertheless, BP circadian variation has been scarcely included in PK-PD models of antihypertensive medications to date. In this work, we developed PK-PD models that include circadian rhythm to find the optimal dosing time (Ta) of first-line antihypertensive medications for dipper and non-dipper patterns. The parameters of the PK-PD models were estimated using global optimization, and models were selected according to the lowest corrected Akaike information criterion value. Simultaneously, sensitivity and identifiability analysis were performed to determine the relevance of the parameters and establish those that can be estimated. Subsequently, Ta parameters were optimized to maximize the effect on BP average, BP peaks, and sleep-time dip. As a result, all selected models included at least one circadian PK component, and circadian parameters had the highest sensitivity. Furthermore, Ta with which BP>130/80 mmHg and a dip of 10-20% are achieved were proposed when possible. We show that the optimal Ta depends on the therapeutic objective, the medication, and the BP profile. Therefore, our results suggest making chrono-pharmacological recommendations in a personalized way.

Protein quantification by bicinchoninic acid (BCA) assay follows complex kinetics and can be performed at short incubation times.

Amongst the available methodologies for protein determination, the bicinchoninic acid (BCA) assay highlights for its simplicity, sensitivity, repeatability and reproducibility. Nevertheless, in spite that the general principle behind this methodology is known, there are still unanswered questions regarding the chemistry behind the assay and the experimental conditions commonly employed. The present work explored the kinetics, and the analytical response of the assay to free amino acids, peptides (containing tryptophan and tyrosine), and proteins. Results revealed kinetic profiles characterized by the absence of plateaus, with behaviors depending on the type of the sample. The latter, along with contribution to the BCA index elicited by oxidation products generated at the side chain of tryptophan and tyrosine, as well as pre-oxidized ß-casein, evidenced the presence of complex reaction mechanisms. In spite of such complexity, our results showed that the BCA index is not modulated by the incubation time. This applies for responses producing absorbance intensities (at 562 nm) higher than 0.1. Therefore, we propose that the assay can be applied at shorter incubation times (15 min) than those indicated in manufactures specifications, and usually used by researches and industry (30 min at 37 °C).

Circadian Rhythm of Blood Pressure of Dipper and Non-dipper Patients With Essential Hypertension: A Mathematical Modeling Approach.

Blood pressure in humans presents a circadian variation profile with a morning increase, a small postprandial valley, and a deeper descent during night-time rest. Under certain conditions, the nocturnal decline in blood pressure can be reduced or even reversed (non-dipper), which is related to a significantly worse prognosis than a normal fall pattern (dipper). Despite several advances in recent years, our understanding of blood pressure's temporal structure, its sources and mechanisms is far from complete. In this work, we developed an ordinary differential equation-based mathematical model capable of capturing the circadian rhythm of blood pressure in dipper and non-dipper patients with arterial hypertension. The model was calibrated by means of global optimization, using 24-h data of systolic and diastolic blood pressure, physical activity, heart rate, blood glucose and norepinephrine, obtained from the literature. After fitting the model, the mean of the normalized error for each data point was <0.2%, and confidence intervals indicate that all parameters were identifiable. Sensitivity analysis allowed identifying the most relevant parameters and therefore inferring the most important blood pressure regulatory mechanisms involved in the non-dipper status, namely, increase in sympathetic over parasympathetic nervous tone, lower influence of physical activity on heart rate and greater influence of physical activity and glucose on the systemic vascular resistance. In summary, this model allows explaining the circadian rhythm of blood pressure and deepening the understanding of the underlying mechanisms and interactions integrating the results of previous works.

Quantification of carbonate radical formation by the bicarbonate-dependent peroxidase activity of superoxide dismutase 1 using pyrogallol red bleaching.

Carbonate radicals (CO3-) are generated by the bicarbonate-dependent peroxidase activity of cytosolic superoxide dismutase (Cu,Zn-SOD, SOD-1). The present work explored the use of bleaching of pyrogallol red (PGR) dye to quantify the rate of CO3- formation from bovine and human SOD-1 (bSOD-1 and hSOD-1, respectively). This approach was compared to previously reported methods using electron paramagnetic resonance spin trapping with DMPO, and the oxidation of ABTS (2,2-azino-bis(3-ethylbenzothiazoline)-6-sulfonic acid). The kinetics of PGR consumption elicited by CO3- was followed by visible spectrophotometry. Solutions containing PGR (5-200 µM), SOD-1 (0.3-3 µM), H2O2 (2 mM) in bicarbonate buffer (200 mM, pH 7.4) showed a rapid loss of the PGR absorption band centered at 540 nm. The initial consumption rate (Ri) gave values independent of the initial PGR concentration allowing an estimate to be made of the rate of CO3- release of 24.6 ±â€¯4.3 µM min-1 for 3 µM bSOD-1. Both bSOD-1 and hSOD-1 showed a similar peroxidase activity, with enzymatic inactivation occurring over a period of 20 min. The single Trp residue (Trp32) present in hSOD-1 was rapidly consumed (initial consumption rate 1.2 ±â€¯0.1 µM min-1) with this occurring more rapidly than hSOD-1 inactivation, suggesting that these processes are not directly related. Added free Trp was rapidly oxidized in competition with PGR. These data indicate that PGR reacts rapidly and efficiently with CO3- resulting from the peroxidase activity of SOD-1, and that PGR-bleaching is a simple, fast and cheap method to quantify CO3- release from bSOD-1 and hSOD-1 peroxidase activity.