Krebs-Henseleit buffer

The cores are subsequently placed in the sheer, and the slicing procedure is performed by advancing the core over an oscillating knife in a controlled environment (Figure 12.1). Cold (4°C) Krebs-Henseleit buffer (pH = 7.4, saturated with 95% O2 and 5% CO2) supplemented with 25 mM glucose is commonly used in preparing the slices [35,38 0], but Williams medium E [41], Earle s balanced salt solutions [37], Sacks preservation medium [42] and V-7 preservation buffer [43,44] are also used. 

Typical experimental procedures are as follows The test drug candidate is incubated with pooled human liver microsomes (e.g., 1 mg protein/mL) that were previously preincubated with ABT (1 or 2 mM) for 30 minutes at (37 1)°C in the presence of an NADPH-generating system. Incubations of the drug candidate in the absence of ABT serve as controls. For hepatocytes, suspensions of freshly isolated or cryopreserved hepatocytes (lx 106 cells/ mL) are preincubated with 100-pM ABT for 30 minutes in 0.25 mL of Krebs-Henseleit buffer or Waymouth s medium (without phenol red) supplemented with FBS (4.5%), insulin (5.6 pg/mL), glutamine (3.6 mM), sodium pyruvate (4.5 mM), and dexamethasone (0.9 pM) at the final concentrations indicated. After the preincubation, the drug candidate is added to the incubation and the rate of metabolism of the drug candidate is compared in hepatocytes or microsomes with and without ABT treatment. A marked difference in metabolism caused by ABT is evidence that CYP plays a prominent role in the metabolism of the drug candidate. 

Krebs-Henseleit buffer without CaCl, pH 7.4 (see Subheading 2.2). 

In situ rat kidney perfusion experiments were carried out as described in Ref. [5.4]. Briefly, the perfusion medium used was Krebs-Henseleit buffer at pH7.4 containing glucose (5.6 mmol/L), 5.5% bovine serum albumin (fraction V, Sigma), 5-6% washed rat erythrocytes and different amino acids. After cannulation of the ureter and the renal vein and artery, the right kidney was perfused via the renal artery at a constant arterial pressure of 14.5 kPa at 37"C. After an equilibration period of 30-35 min, the agent was added and the perfusion continued for 60 min. Urine samples were collected every 10 min, and midpoint samples of the perfusate were also obtained. Inuhn was used as the standard for the measurement of the glomerular filtration rate. Elimination parameters of labelled peptides in the perfused rat kidney were characterized by the values of total renal clearance (CLr) and free fraction of the peptide in the perfusate (FJ. These values were compared with the glomerular filtration rate (GFR). 

Allis and co-workers54 used 87Rb NMR to measure the K+ flux into cardiac muscle. Hearts from Wistar rats were perfused in the Langendorff mode91,92 with modified Krebs-Henseleit buffer (KHB).93 The heart hung freely within the radiofrequency coil, thus eliminating the problems associated with Rb+ accumulation within any enclosing perfusion chamber. The temporal resolution between successive spectra was 250 s. Quantification of absolute amounts... 

Fig. 3. Electron micrograph of synaptic vesicles from rat brain cortex, incubated in Krebs-Henseleit buffer in the presence of phospholipase A for 30 sec. x 80,000,...

In the isolated rat heart perfused according to Langendorff s technique, the 8-hydroxyguanine content in the DNA significantly increased after an ischaemia of 30 or 60 min followed by reperfusion with oxygenated Krebs-Henseleit buffer (You et al. 2000). The levels of 8-hydroxyguanine did not increase either in the ischaemic hearts reperfused with a nitrogenated solution or in the ischaemic-reperfused hearts treated with superoxide dismut-ase, mannitol or allopurinol. 

After the equilibration period, clearance periods of 20 min are used. Urine samples are collected and perfusate is obtained at midpoint of the clearance period for the evaluation of overall kidney function. For determination of glomerular filtration rate (GFR) and fluid transport, 3H-labelled polyethylene glycol is added to a modified Krebs-Henseleit bicarbonate buffer. Electrolytes are determined in urine by standard flame photometry. Fractional excretions of water, electrolytes and test compounds are calculated. 

Hearts from CD-I male rats (250-300 g n=4 each group, 34 total) are excised and perfused within 25 s to 2 min with non-recirculating oxygenated Krebs-Henseleit bicarbonate buffer (120 mM NaCl, 25 mM NaHCOj, 1.2 mM MgSO -ZHp, 5 mM KCl, 1.7 mM CaCl-2H20, 10 mM glucose, pH 7.4, 37°C) at a constant coronary perfusion pressure (CPP) of 80 mm Hg (18, 19). 

Dissolve 220 mg of adenosine-5 -triphosphate in 5 mL of Kreb s-Henseleit buffer and adjust pH to 7.4 with 1 M NaOH. 

COS cells transfected with pcDNA3.1/V7-3 and the vector pCDNA3.1 served as positive and negative controls, respectively. Parallel incubations with 30 pM of unlabeled anandamide allowed estimation of nonspecific uptake and binding. Uptake assays were carried out for 5 min at 3TC, followed by two washes each with 2 mL of Krebs-Ringer-Henseleit buffer. 

Isolated rat liver hepatocytes were prepared as described by Seglen (9), except that the cells were suspended and incubated in Krebs-Henseleit bicarbonate buffer (K-H buffer), supplemented as specified. 

Figure 51. Representative traces of heat output (a) and O2 consumption (b) rates by isolated brown adipocytes from cafeteria-fed rats after the sequential addition of glucose, noradrenaline, propranolol and oleic acid. Isolated brown adipocytes (2.65x 10 cells/cm ) were prepared from two cafeteria-fed rats. Noradrenaline, propranolol and oleic acid were prepared in Krebs-Henseleit bicarbonate-buffered saline, equilibrated at 37 C for 3.0 min, and added in a concentrated form at the times indicated (Reproduced from Reference [146] with permission).

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