Lactate dehydrogenase ldh

The amount of lactate the cancer cells secreted into the culture medium was measured using an enzymatic assay using L-lactate dehydrogenase (Sigma). In this assay, the lactate secreted into the culture medium sample is reduced to pyruvate and NADH in the presence of lactate dehydrogenase (LDH) (Sigma) and excess NAD. The amount of NADH formed in the reaction, measured by the change in absorbance at 340 nm, is proportional to the concentration of lactate present in the sample. To avoid interference with the LDH that may already be present in the serum used to supplement the culture medium, the samples were subjected to deproteinization with 8% trichloroacetic acid (TCA) to render them protein-free prior to the assay.

Lyophilized preparations of enzymes were produced in the Laboratory of Nanobiotechnology and Bioluminescence at the Institute of Biophysics, Siberian Branch, Russian Academy of Sciences (Krasnoyarsk, Russia). Luciferase (Luc) Photobacterium leiognathi (0.5 mg) was purified from the recombinant strain of Escherichia coli, while NAD(P)H:FMN-oxidoreductase (Red) (0.15 units) from luminous bacteria Vibrio fischeri. Lactate dehydrogenase (LDH) from rabbit muscles was procured from Sigma (Type XI, catalog No. L1254, 5000 units). NAD⁺ (AppliChem, Darmstadt, Germany), and DL-lactic acid (Sigma, Steinheim, Germany) were used as substrates of LDH; FMN (Serva, Heidelberg, Germany) and tetradecanal (Merck, Steinheim, Germany) were used as substrates for the Red + Luc enzyme system. To prepare the R + L enzyme solutions, 5 mL of potassium phosphate buffer (pH 7.2) was added to a vial containing the enzymes. For preparing the LDH enzyme solution, 0.5 mL of potassium phosphate buffer (pH 7.2) was added to a vial. The tetradecanal solution [0.0025% (v/v)] was prepared by mixing 50 μL of 0.25% (v/v) ethanol solution of aldehyde and 5 mL of 0.05 M potassium phosphate buffer (pH 7.2). The NAD⁺ solution was prepared in a 0.05 M potassium phosphate buffer (pH 7.2). The samples of FMN and lactic acid were dissolved in distilled water.

Lactate dehydrogenase (LDH; rabbit muscle, type IX, lyophilized powder, 900 units/mg protein), pyruvate and reduced NADH were purchased from Sigma Chemical Company (Mississauga, ON, Canada). Polymerized F-actin were produced as follows: G-actine (500 ug/mL) was allowed to polymerize overnight at 4 °C in the following media: 100 mM KCl, 0.2 mM ATP, 2 mM MgCl₂, 0.2 mM CaCl₂ in 10 mM Hepes-NaOH, pH 7.5. The formation of F-actin was determined by absorbance at 450 nm. The activity in LDH were determined at 1 mM NADH and pyruvate at 25 °C in the assay buffer composed of 50 mM NaCl, 10 mM Hepes-NaOH, pH 7.5 with 0.1 μg/mL of commercial LDH. The activity was also measured with varying concentration in pyruvate (0.1 to 1 mM) in the presence of 1 mM NADH to determine the fractal kinetic constants (K_M and V_Max) as described below. The reaction was allowed to proceed for 30 min where NADH fluorescence (350 nm excitation and 450 nm emission) were measured each 30 s under flash mode for quick readings in dark 96-well microplates (Synergy-4, Biotek Instruments, USA). The influence of space crowding (fractal dimension) was determined by adding 10 μg/mL of F-actin during the incubation reaction or with 10 ug/mL of polystyrene NPs of 50 nm diameter (Polyscience Inc., USA). The blanks consisted of freshly prepared F-actin buffer where ATP was replaced with ADP (no polymerization).

To check the effect of pre-treatment with activated carbon cloth, plasma collected from ALF rats 1–2 h post induction of injury, was first perfused through activated carbon cloth as described earlier and then perfused for 3 h at a flow rate of 0.2 mL/min through a 5-day cell-seeded cryogel bioreactor (3D) or incubated for 3 h with cells cultured in monolayer (2D) (density 5 × 10⁴ cells/well). Viability of the cells was measured using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) (Sigma Aldrich, USA) assay and Lactate dehydrogenase (LDH) (Sigma Aldrich, USA) activity before and after treatment with plasma. Absorbance values from the MTT assay were normalized to cell number using a standard curve. Functionality in terms of albumin synthesis was measured before and after treatment with ALF plasma using standard sandwich ELISA as per manufacturer’s protocol (Bethyl Laboratories, USA). For microscopic analysis on the effect of ALF plasma, cells were fixed using 4% paraformaldehyde followed by staining with the nuclear stain, 4′,6-diamidino-2-phenylindole (DAPI) (Sigma Aldrich, USA).