An in vitro Coupled Assay for PEPC with Control of Bicarbonate Concentration

BIO-PROTOCOL ◽  
2021 ◽  
Vol 11 (24) ◽  
Author(s):  
Nicholas Moody ◽  
Chatawal Phansopal ◽  
James Reid
Parasitology ◽  
2002 ◽  
Vol 125 (1) ◽  
pp. 65-70 ◽  
Author(s):  
H. HERTZBERG ◽  
U. HUWYLER ◽  
L. KOHLER ◽  
St REHBEIN ◽  
M. WANNER

The aim of the study was to investigate the longitudinal changes of exsheathment of ovine and bovine 3rd-stage strongylid larvae in an artificial rumen (RUSITEC) and to compare the results with in vivo data obtained from rumen-fistulated sheep. Infective larvae were incubated in nylon mesh bags in the sheep rumen or the RUSITEC apparatus for periods of 1, 6 and 12 h, respectively. The 12 h exsheathment rates in the rumen and the RUSITEC apparatus (in parentheses) were as follows: Haemonchus contortus: 100% (100%), Ostertagia circumcincta: 100% (76%), O. leptospicularis: 100% (100%), O. ostertagi: 53% (59%), Trichostrongylus axei: 100% (100%), T. colubriformis: 37% (36%), Cooperia curticei: 94% (76%), C. oncophora: 95% (89%), Nematodirus filicollis: 0% (N.D.), N. spathiger: 11% (15%), N. battus: 7% (5%), Oesophagostomum venulosum: 17% (9%), Chabertia ovina: 7% (2%), Dictyocaulus filaria: 1% (N.D.). Larvae of Nematodirus spp. and T. colubriformis showed a quick rise of the exsheathment rate 2 h after transfer into the abomasum. These results confirm that exsheathment generally occurs in the part of the gastrointestinal tract immediately anterior to the habitat of the adult parasite. The overall similar course of exsheathment in both systems indicates that the essential stimuli for exsheathment were generated and maintained under in vitro conditions of the artificial rumen. In both systems, the bicarbonate concentration and the pH reflected a similar status of the H2CO3/HCO buffer system, which is known to provide the essential stimuli for larval exsheathment of the abomasal species. These results give evidence that the RUSITEC system represents a valid system for studying the kinetics of exsheathment of strongylid nematodes under in vitro conditions. For 7 of the species investigated the obtained results represent the first data on larval exsheathment in vivo and in vitro.


Development ◽  
1978 ◽  
Vol 48 (1) ◽  
pp. 1-21
Author(s):  
D. J. Pritchard ◽  
R. M. Clayton ◽  
D. I. De Pomerai

The in vitro transdifferentiation of chicken embryo neural retina into pigment epithelium and lens cells was investigated under a variety of experimental conditions. Our findings suggest that some aspects of the phenomena are a function of medium composition and volume, whereas others depend upon conditions which develop during culture growth. Before melanin is visible, potential pigment cells are recognized as foci within epithelialsheets which remain in contact with the dish. The final area occupied by colonies of potential pigment cells is directly proportional to bicarbonate concentration. Low total medium volume also favours formation of potential pigment cells. In contrast the extent of cells other than potential pigment cells is not related to bicarbonate and is favoured when the volume of medium is large. Accumulation of melanin within the potential pigment cell colonies is suppressed when cells are crowded together. Lentoid bodies are formed from cells which are distinct from potential pigment cells and arise in crowded situations, in association with multilayering. Another type of structure superficially resembling a lentoid is derived from cell aggregates formed during the initial establishment of cultures. The survival of these ‘aggregate bodies’ is inversely related to bicarbonate concentration. Crystallin content is unrelated to lentoid numbers. The results provide the basis for a new hypothesis concerning cytodifferentiation in this system.


Author(s):  
Cristina Soriano-Úbeda ◽  
Jon Romero-Aguirregomezcorta ◽  
Carmen Matás ◽  
Pablo E. Visconti ◽  
Francisco A. García-Vázquez

Perfusion ◽  
2019 ◽  
Vol 34 (7) ◽  
pp. 578-583 ◽  
Author(s):  
Lien H Vu ◽  
John A Kellum ◽  
William J Federspiel ◽  
Matthew E Cove

Background: Extracorporeal carbon dioxide removal may be used to manage hypercapnia, but compared to dialysis, it’s not widely available. A recent in vitro study showed that dialysis with low bicarbonate dialysates removes CO2. Objective: To show that bicarbonate dialysis removes CO2 in an animal model to validate in-vitro findings and quantify the effect on arterial pH. Methods: Male Sprague-Dawley hypercapnic rats were dialyzed with either a conventional dialysate (PrismasolTM) or a bicarbonate-free dialysate (Bicarb0). The effect of dialysis on standard blood gases and electrolytes was measured. Results: Partial pressure of CO2 and bicarbonate concentration in blood decreased significantly after exposure to Bicarb0 compared to PrismasolTM (filter outflow values 12.8 vs 81.1 mmHg; p < 0.01 for CO2 and 3.5 vs 22.0 mmol/L; p < 0.01 for bicarbonate). Total CO2 content of blood was reduced by 459 mL/L during dialysis with Bicarb0 (filter inflow 546 ± 91 vs filter outflow 87 ± 52 mL/L; p < 0.01), but was not significantly reduced with PrismasolTM. Conclusions: Bicarbonate dialysis removes CO2 at rates comparable to existing low-flow ECCO2R.


1996 ◽  
Vol 16 (1_suppl) ◽  
pp. 109-113 ◽  
Author(s):  
Jutta Passlick-Deetjen ◽  
Judith Kirchgessner

For a long time bicarbonate, the physiological buffer of the body, was suggested to be the best buffer for peritoneal dialysis. However, since the production of bicarbonate containing solutions is associated with technical problems, lactate was favored. To avoid the well-known disadvantages of lactate solution concerning biocompatibility and possible metabolic side effects, different attempts have been made to use bicarbonate as a buffer in peritoneal dialysis. One of the major approaches was the total replacement of lactate by bicarbonate combined with storage of the fluid in a specially designed double-chamber bag. Further solutions of the above-mentioned problem were the on-line preparation of bicarbonate fluids for intermittent peritoneal dialysis, the addition of bicarbonate just before use, the combination of bicarbonate with organic acids, or its combination with the dipeptide glycylglycine as a stabilizing agent. By now, the beneficial effect of the neutral bicarbonate fluid, for example, on cell viability and cell functions, has been demonstrated in many different in vitro and animal studies. However, only few reports on clinical experience have been published. These investigations demonstrated independently that bicarbonate fluids diminish inflow pain, are well tolerated by the patients, and may correct metabolic acidosis of uremic patients. A controlled randomized multicenter trial using 34 mmol/L bicarbonate for at least three months confirmed that bicarbonate is as efficacious as lactate in equimolar concentrations. Concomitant investigations on energy metabolism and redox state of red blood cells and phospholipid secretion of mesothelial cells additionally demonstrated the improvement of cell function with bicarbonate solutions. For some patients with severe metabolic acidosis the bicarbonate concentration used in the multicenter trial seemed to be too low. Thus, a fluid containing a higher bicarbonate concentration was tested in a pilot study resulting in the expected significant increase of arterial bicarbonate levels. In summary, bicarbonate-containing peritoneal dialysis solutions are a promising alternative to lactate, especially if bicarbonate concentrations are adjusted individually to the patient's need.


1985 ◽  
Vol 248 (6) ◽  
pp. F821-F829 ◽  
Author(s):  
D. W. Good

In vitro microperfusion experiments were performed to investigate the mechanism of bicarbonate absorption in the cortical thick ascending limb of the rat. Tubules were perfused at 1.0-1.5 nl X min-1 X mm-1 and bicarbonate concentration was 25 mM in the perfusate and bath. Bicarbonate absorption rates were determined by microcalorimetry. Control tubules absorbed bicarbonate at a mean rate of 9.5 +/- 0.6 pmol X min-1 X mm-1. The limiting luminal bicarbonate concentration was approximately 5 mM for tubules perfused at slow rates with 25 mM bicarbonate in the bath. Acetazolamide (10(-4)M) in the bath reduced bicarbonate absorption by 76% without significant effect on transepithelial voltage. Removing sodium from the perfusate and bath or removing potassium from the bath reduced bicarbonate absorption and transepithelial voltage to near zero. Adding amiloride (5 X 10(-4) or 10(-3) M) to the perfusate reduced bicarbonate absorption by 60-75% without detectable effect on transepithelial voltage. Adding furosemide (10(-4)M) to the perfusate increased bicarbonate absorption significantly by 40-50% while decreasing transepithelial voltage from 17 to 1.8 mV. Thus, bicarbonate absorption by cortical thick ascending limbs requires carbonic anhydrase activity and sodium transport but is not dependent on transepithelial voltage. When considered together, the results are consistent with mediation of the bicarbonate absorption by apical membrane sodium-hydrogen exchange.


1979 ◽  
Vol 236 (4) ◽  
pp. F387-F391 ◽  
Author(s):  
Y. Iino ◽  
M. B. Burg

The effect of parathyroid hormone on bicarbonate absorption was tested in rabbit proximal renal tubules perfused in vitro. In proximal straight tubules 0.05 U/ml of parathyroid hormone caused a large and reversible increase in the steady-state bicarbonate concentration in tubule fluid. Further, the rates of bicarbonate and fluid absorption (measured at faster flow rates) were inhibited approximately 50% by the hormone. We conclude that parathyroid hormone directly inhibits fluid and bicarbonate absorption by proximal straight tubules, causing an increase in the bicarbonate concentration in the tubule fluid, and we suggest that this action of the hormone contributes to the increase in renal bicarbonate excretion that is generally caused by the hormone. In proximal convoluted tubules, parathyroid hormone was previously demonstrated by other investigators to inhibit fluid and bicarbonate absorption approximately proportionally, so that there was little or no change in the bicarbonate concentration in tubule fluid. In agreement we found in the present studies that 0.05 U/ml of the hormone did not affect the steady-state bicarbonate concentration in proximal convoluted tubule fluid and that 5 U/ml caused only an equivocal increase in tubule fluid bicarbonate concentration.


1979 ◽  
Vol 56 (4) ◽  
pp. 353-364 ◽  
Author(s):  
R. L. Tannen ◽  
B. D. Ross

1. The effect of metabolic acidosis simulated in vitro on ammoniagenesis was investigated by using the isolated kidney of the rat perfused with an albumin Krebs—Henseleit medium containing glutamine and glucose. 2. Addition of HCl to a perfusate of normal bicarbonate concentration resulted in a prompt increase in urine flow rate, decrease in fractional sodium reabsorption and decrease in urine pH. 3. A minimum urine pH as low as 5·15 was achieved, with an average value of 5·92, indicating that this preparation has the capacity to acidify normally. 4. In contrast with studies in vitro with other preparations, with the functional perfused kidney a diminution in perfusate bicarbonate concentration resulted in a prompt increase in ammonia production, which was strikingly correlated with the decrease in urine pH. 5. The increase in ammonia production was diminished in studies carried out with a non-urinating kidney, in comparison with those that exhibited significant urine acidification. 6. These data suggest that a decrease in urine pH with trapping of ammonia in the urine may be a critical stimulus for increased ammonia production in acute metabolic acidosis.


1986 ◽  
Vol 250 (2) ◽  
pp. F267-F272 ◽  
Author(s):  
B. A. Biagi ◽  
M. Sohtell

Conventional microelectrodes were used to examine the electrogenic pathways for bicarbonate transport across the basolateral membranes of proximal convoluted (PCT) and straight (PST) tubule segments of the rabbit kidney perfused in vitro. When bath bicarbonate concentration was reduced from 22 to 6.6 mM at a constant pH, transient depolarizations lasting several seconds with a peak value of approximately 15 mV were seen in both tubule segments. Acetazolamide (0.1 mM) in the lumen and bath solutions reduced the magnitude and increased the duration fo this response. The final pH of the bathing solution influenced both the peak height and steady-state values of the intracellular potential when bicarbonate concentration was reduced either with constant CO2 or with an increase in CO2. Reducing bath sodium concentration by replacement with either tetramethylammonium or N-methyl-D-glucamine resulted in a sustained depolarization of both PCT and PST cells. This response was inhibited by the addition of 10(-4) M 4-acetamido-4'-isothiocyanostilbene-2,2'-disulfonate (SITS) in the bathing solution. By analogy with bicarbonate transport in rat and amphibian proximal tubules, these data suggest that bicarbonate exit across the basolateral membrane of the rabbit proximal tubule is electrogenic and coupled to sodium and that basolateral bicarbonate exit can be inhibited by both acetazolamide and SITS in the bathing solution.


1976 ◽  
Vol 50 (4) ◽  
pp. 293-299 ◽  
Author(s):  
G. E. Levin ◽  
P. Collinson ◽  
D. N. Baron

1. Viable human leucocytes were isolated from venous blood and suspended in artificial media. Intracellular pH measurements were made by the dimethyloxazolidinedione technique in conditions simulating ‘respiratory’ or ‘metabolic’ acid-base disturbances. 2. Normal intracellular pH was 7·11 ± 0·02 (mean ± 2 sd) at an extracellular Pco2 of 5·8 kPa and a bicarbonate concentration of 25 mmol/l. 3. ‘Respiratory’ and ‘metabolic’ acidosis caused little change in pH1 although increases in Pco2 led to relatively greater falls in pH1 than did reduction in external bicarbonate concentration. 4. ‘Respiratory’ and ‘metabolic’ alkalosis caused similar and relatively greater increases in the pH1 when compared with the response to an external acidosis.


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