Modified Krebs-Ringer Bicarbonate Buffer Research Articles

Factors influencing theophylline serum protein binding.

We studied a number of influences on theophylline binding to serum proteins using equilibrium dialysis (37 degrees), a modified Krebs-Ringer bicarbonate buffer (pH 7.4), and 8-14C-theophylline with unlabeled theophylline (30 microgram/ml) added to sera from healthy subjects. Theophylline protein binding rose by 18.6% as pH rose from 7.0 to 7.8 (percent theophylline bound = 28.2 +/- 4.3 at pH 7.0 and 46.8 +/- 4.9 at pH 7.8, n = 5). Average theophylline binding to the proteins at 37 degrees in serum samples from 10 normal adults was 39.3 +/- 3.44%, which is 89.9% lower than the average of 48.2 +/- 3.74% for the same samples at 26 degrees. Theophylline binding was 6.1% higher with 0.1 mole/l phosphate buffer, pH 7.4, than with a modified Krebs-Ringer bicarbonate buffer, pH 7.4. Of the 19 drugs and metabolites tested for competition with theophylline for binding sites on serum proteins, 10 induced decreases in binding ranging from 6.8% in the case of furosemide to 18.3% for sodium salicylate. The latter was the only drug that induced a decrease in theophylline binding at concentrations that would be achieved in the therapy of same patients (i.e., patients on long-term salicylate therapy). All the other drugs that decreased theophylline binding did so at much greater concentrations than their usual therapeutic levels. The mean +/- SD of theophylline bound in 51 fresh serum samples from healthy adults was 48.6 +/- 10.2%; the pH of these specimens varied from 7.6 to 8.7. After adjusting pH to 7.4, theophylline binding was lowered to 37.6 +/- 4.5% and intersubject variability decreased. We recommend that the pH of serum specimens be adjusted to 7.4, or to the original pH of the blood specimen if it differs significantly from 7.4 (i.e., in acidotic or alkalotic patients). The wide range of reported values for theophylline binding to serum proteins in normal and asthmatic adults at least partly results from differences in the conditions used for the separation of free from bound drug.

Effect of Amino Acid Concentration on Orotic Acid Production by Bovine Mammary Tissue

Investigations were to determine if bovine mammary tissue could synthesize orotate and to assess the influence of amino acids on production of orotate. Mammary tissue from 6 Holstein cows was obtained immediately after slaughter. Tissue slices were incubated in either a modified Krebs-Ringer bicarbonate buffer, the same medium containing a physiological concentration of amino acids, or a third medium containing a fourfold concentration of the amino acid mixture. In all experiments, tissue slices were preincubated in the physiological amino acid medium for 5, 10, and 15min (30 total min) with fresh medium changes after each incubation interval. Tissue slices then were incubated with one of the three treatment media for three consecutive 20-min periods. Tissue slices incubated in the physiological amino acid medium for a total of 90min synthesized 28.5µg orotate/g wet tissue after correction for the initial tissue contribution. Orotate secreted by the tissue slices increased as concentration of amino acids increased. Lactose released into the medium was not affected by amino acid concentration. Bovine mammary tissue can synthesize substantial amounts of orotate, and tissue syndiesis of orotate but not lactose increased as amino acid concentration increased.

Amino acid uptake by isolated renal brush border membrane vesicles in various buffers

The uptake of amino acids by isolated rat renal brush border membrane vesicles in a modified Krebs-Ringer bicarbonate buffer and a phosphate buffer was compared to the uptake in the standard membrane vesicle buffer, Tris-Hepes-mannitol. The uptake in the modified Krebs-Ringer bicarbonate buffer was similar to that in the Tris-Hepes-mannitol buffer. Removal of the ionic constituents other than NaCl and NaHCO 3 in the modified Krebs-Ringer bicarbonate buffer (KCl, CaCl 2, KH 2PO 4 and MgSO 4) did not affect the amino acid uptake by the isolated membrane vesicles. The timed uptake of proline under sodium gradient conditions in a phosphate buffer had a markedly dampened overshoot. Kinetic analysis of the initial rate of proline uptake in a phosphate buffer compared to a Tris-Hepes-mannitol buffer showed two entry systems for proline in each buffer with similar K m values, but the maximal rate of transport ( V) for each system in the phosphate buffer was much lower than that in the Tris-Hepes-mannitol buffer. From these data, phosphate buffer does not appear to be a suitable medium for the study of amino acid uptake by isolated brush border membrane vesicles.

Role of pancreozymin-cholecystokinin and structurally related compounds as calcitonin secretogogues.

Surgically isolated pig thyroid glands were perfused in situ under controlled conditions with blood, or modified Krebs-Ringer bicarbonate buffer, to which individual gastrointestinal hormones or related compounds were added at a constant rate. The thyroid venous effluent was collected quantitatively and the calcitonin (CT) concentration measured by either bioassay or radioimmunoassay so that the CT secretion rate could be calculated. Porcine secretin (50 pM to 145 nM) did not stimulate CT secretion, in contrast to a significant effect with porcine pancreozymin (1.3–25 nM). Stimulation of CT secretion was also produced by the Cterminal octapeptide of pancreozymin (1.8–17 nM), whereas the N-terminal hexapeptide (70 nM) was inactive. This stimulatory action was reduced by mild oxidation. Cerulein (1–14 nM) and pentagastrin (0.5 μM), which share with pancreozymin the same C-terminal tetrapeptide, also stimulated CT release. (Endocrinology 89: 262,1971)

Serotonin inhibition of in vitro insulin release from golden hamster pancreas.

To assess the possible functional role of serotonin on insulin secretion, pieces of golden hamster pancreas were incubated in a modified Krebs-Ringer bicarbonate buffer. In 10-4M concentration, serotonin suppressed basal insulin secretion and also insulin secretion that was stimulated by high glucose, tolbutamide, or dibutyryl cyclic AMP. Inhibition of insulin secretion by serotonin occurred over the entire range examined (1.0×10-4 to 5.8 ×10-4M). In a similar in vitro system, concentrations of serotonin as high as 5.8 ×10-4M were without significant effects on basal or high glucose (3 mg/ml) stimulated insulin release from mouse pancreas. Thus, serotonin effect on the pancreatic β cell has some species difference. Serotonin may have a role as a physiological inhibitor of insulin secretion in some species. (Endocrinology 86: 66, 1970)

Lipid metabolism in normal and rachitic rat epiphyseal cartilage.

SummaryLipid synthesis in rat epiphyseal cartilage has been studied in relation to normal growth and the production of rickets. Cartilage was incubated in vitro in modified Krebs-Ringer bicarbonate buffer with added I-14C palmitic acid. In normal 28-day-old male weanling rats, the total uptake of 1-14C palmitic acid from the media showed a gradual increase with time. Fractionation of the lipids showed 64.6% of the label in free fatty acids; 19.2% in triglycerides; 12.8% in phospholipids; and 6.2% in cholesterol and cholesterol esters. In rachitic rats of comparable age, the total uptake of 1-14C palmitic acid was increased significantly, indicating an increased rate of lipid synthesis.

Insulin disposition in the isolated perfused rat heart. Effect of "bound" insulin.

Hearts from fed and fasted rats were perfused with modified Krebs-Ringer bicarbonate buffer containing 3 per cent human serum albumin, 2 mg./ml. glucose and 100 or 200 μU./ml. crystalline insulin. The concentration of immunoassayable insulin in the medium decreased, during two hours of perfusion, to approximately 50 per cent of the initial concentration. Perfusion of hearts with insulin-I-131 (100 μU./ml.) resulted in: (1) a decrease in immunoprecipitable radioactivity corresponding in rate and extent to that of crystalline insulin; (2) a significantly smaller decrease in trichloracetic acid (TCA)-precipitable radioactivity; (3) a corresponding accumulation of a fraction of radioactivity which was TCA-precipitable but not immunoprecipitable (TCA-nonimmunoprecipitable) as well as an increase in TCA-soluble radioactivity. Immunoprecipitable-I-131 and not TCA-precipitable I-131 more accurately reflects the fate of intact insulin. Analysis of the two-hour perfusate by gel nitration chromatography (Sephadex G-100) indicated that radioactivity was associated with both the insulin-I-131 peak (M.W. 12,000) and with substances exhibiting a lower molecular weight. Thus, the disappearance of insulin during heart perfusion may be largely accounted for by degradation of insulin. Both immunoassayable insulin and immunoprecipitable insulin-I-131 were demonstrated to decrease in concentration in cell-free medium which had been previously perfused through the heart for two hours, but not in unperfused medium. The extent of disappearance was less than that during a two-hour heart perfusion. Thus, a portion of the degradation of insulin occurring during heart perfusion is probably due to the action of enzymes which diffuse into the medium from the heart. “Bound” insulin inhibited the disappearance of both crystalline insulin and immunoprecipitable I-131 radioactivity from the medium during heart perfusion. “Bound” insulin was shown to inhibit the rate of disappearance of immunoprecipitable radioactivity from preperfused medium. During perfusions with “Bound” insulin alone, the level of bioassayable activity in the medium remained constant throughout the perfusion, indicating a lack of disappearance of “Bound” insulin from the medium and vascular space of the heart. Studies of glucose uptake during heart perfusion demonstrated that “Bound” insulin has minimal or no effect on basal and insulin-stimulated glucose uptake. “Bound” insulin may inhibit the disappearance of insulin by interfering with the transport of insulin across the capillary wall and/or by inhibiting the degradation of insulin in the medium.

ACCUMULATION OF VITAMIN A BY SMALL INTESTINE OF THE RAT IN VITRO.

The vitamin A accumulation by the small intestine of the rat was investigated, using the method of everted intestinal sacs as modified by the authors. For incubation, modified Krebs-Ringer bicarbonate buffer with vitamin A-acetate stabilized with human albumin was used. Vitamin A accumulates in the intestinal wall and the kinetics of this process are analogous to the Michaelis-Menten kinetics. Accumulation is inhibited by KCN and NaF. The transfer of vitamin A into the serosal contents of sacs was minimal. There were no significant differences in the accumulation of vitamin A in various parts of small intestine. It is concluded that the accumulation of vitamin A has a complex and active character.