Bicarbonate in the treatment of metabolic acidosis: Effects on hepatic intracellular pH, gluconeogenesis, and lactate disposal in rats

Bicarbonate in the Treatment of Metabolic Acidosis: Effects Intracellular pH, Gluconeogenesis, and Lactate Disposal J.S. Beech,

on Hepatic in Rats

R.A. Iles, and R.D. Cohen

The effects of agents used in the treatment of metabolic acidosis could depend on the induced changes in intracellular pH (pHi). To determine the effect of sodium bicarbonate on hepatic pHi and function, this agent was infused into anesthetized rats with acute metabolic acidosis due to either diabetic ketoacidosis (DKA) or HCI infusion. Hepatic pHi was measured by 31P-magnetic resonance spectroscopy (MRS). A substantial increase in pHi occurred (from 7.13 2 0.08 to 7.32 + 0.08, P < .05) despite an increase in mixed venous Pcoz. Isolated livers from normal rats or those with DKA were perfused at pH 6.8 and normal Pco~. With infusion of sodium bicarbonate, there was again an increase in pHi (ApHi, + 0.27 f 0.06, P < .02) despite increases in both portal and hepatic venous Pco~. Lactate uptake was increased twofold to threefold (P c .OOl) by bicarbonate infusion in perfusions from both types of animals. Glucose output was increased twofold (P c .OOl) only in livers from normal animals. Copyright 87 1993 by W.B. Saunders Company

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HE:RE IS CONSIDERABLE controversy over the role of sodium bicarbonate in the treatment of metabolic acidosis. Failure to resolve this argument has resulted from difficulty in mounting informative randomized controlled trials in seriously ill patients, whose physiological states may vary widely cm presentation. In the absence of such evidence, the argument has mainly been developed from the results of studies in animal models in which the initial conditions may be relatively tightly definedi. and from theoretical considerations. Thus Arieff et al’ and Graf et al’ have demonstrated adverse hemodynamic effects and elevations of arterial lactate concentrations in dogs with either hypoxic or phenformin-induced lactic acidosis treated with bicarbonate; these effects were accompanied by a decrease in hepatic intracellular pH (pH,). Hberti et al3 found no advantage in using bicarbonate instead of sodium chloride to treat dogs with lactic acidosis resulting from hemorrhagic shock, and Shapiro et ala found that bicarbonate caused a decrease in brain pH, in ammoniurn-chloride or respiratory acidosis in rats. The main theorel.ical objection to the use of bicarbonate is based on the observation that in isolated cell suspensions the highly lipophilic CO: molecule diffuses much more rapidly across cell membranes than does the charged HCOlanion. Intravenous (IV) infusion of bicarbonate in metabolic acidosis, leading to immediate generation of CO? by titration, could thus result in “paradoxical” intracellular acidification. There is in vitro evidence that decreased cardiac pH, has a negative inotropic effects,h and that hepatic intracellular acidosis inhibits removal of lactate and its conversion to glucose and bicarbonate.7-4 In the only attempt at a formal randomized comparison of the effects of rapidly administered sodium bicarbonate and sodium chloride in patients with lactic acidosis, no differences were found in the hemodynamic effects achieved, but bicarbonate was associatcd with a transient increase in arterial Pco~.~” In contrast, the arguments that withholding bicarbonate from patients with sserious metabolic acidosis is unjustified have been summarized by Narins and Cohen.” Because the control of pHi varies quantitatively and qualitatively between organs, 1?,13it seems improbable that any generalizations about the effects of bicarbonate on individual organ pH, and function can be made from the ensuing changes in arterial pH (pH,) and PCO: (P,C@). In

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the present study, WC have examined the elfcct of bicarbonate treatment of metabolic acidosis in the rat on hepatic cell pH (pH,) both in the whole animal and in perfused livers obtained from animals with acute metabolic acidosis, using “‘P-magnetic resonance spectroscopy (MRS). In the perfused-liver model, the effect of Row changes that might have occurred in vivo could be eliminated. The perfusedliver studies also permitted observations of the effect of bicarbonate administration on lactate uptake and glucose output, the rates of which themselves contribute to acidbase regulation.x We have concentrated on diabetic ketoacidosis (DKA), but also report results in acidosis induced by infusion of HCI. MATERIALS

AND METHODS

Inbred 4X-hour-starved male Wistar rats weighing

1hO to ‘40 g were used. All animals had an indwelling right atrial cannula placed under ether anesthesia 48 hours before the studies for blood sampling and infusion. In some animals. DKA was induced by IV injection of streptozotocin 100 mg kg-’ immediately after the cannulation. For induction of HCI acidosis. 4 to h mL 0.3 mol L ’ HCI was infused at 0.05 mL min I over the 7- to j-hour period before the start of the “P-MRS observations.

After right atrial samples had been ohtained from conscious animals resting in a restraining cage for pH (PHI(~). P(‘o: (PKA~ c.0~). lactate, and 3.hydroxybutyrate estimations. the animals were anesthetized with Inactin [5-ethyl-(methylpropyl)-Z-thiobarbiturate; Byk-Gulden. Konstanz, Germany] 100 mg kg ] and placed in a Bruker AM360 8.46T (Bruker Spectrospin, Conventry. UK) or Sisco 4.7T (Spectroscopic Imaging Systems, Sunny Vale, CA) wide-bore spectrometer. No appreciable differences have been observed between results obtained with the two spectrometers in either treatment group. The technique of obtaining phosphorus spectra from the liver using an externally placed surface coil has been previously described in detail. I4 In brief. spectra from liver

From the Celhdur Mechunisms Research Group. The Londorl Hospital Medical College, London. UK. Submitted December IO. 1991: acceptedAptil9. 1992. Supported by the Uttited Kingdom Medical Research C‘outlcil. Address reprint requests to R.A. Iles. PhD. The Medical Unit, The Royal London Hospital, Whitechapel Road, London. El 1BB UK. Copwight 0 1993 by W.B. Saunders Compan) 0026-0395193/4203-0012$03.00i0

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tissue were obtained by progressively increasing the pulse angle until the phosphocreatine signal from skeletal muscle was minimized; the ratio of phosphocreatine to inorganic phosphate (P,) in muscle is such that the small residual phosphocreatine signals indicated only minimal contamination of the liver P, signal by muscle P,. At the pulse interval used. the signal from extracellular P, is heavily saturated because of its long spin lattice relaxation time (Tl) compared with that of intracellular P,.” Extracellular P, therefore does not contribute appreciably to the P, signal; the pH values derived from the chemical shift of the P, signal therefore refer to pH,. This discrimination is further enhanced by the low relative volume ( - 25%) of the extracellular space, and the overall contribution of extracellular P, to the P, signal is less than 5%. This was verified by observations on a dummy system in which perfusate (see below) was circulated within the spectrometer through a capsule of volume equal to or less than that of rat liver. The other potential contributor to the P, signal is erythrocyte 2.3-bisphosphoglycerate (2,3-BPG), whose signal has a similar chemical shift. In vivo, erythrocyte 2,3-BPG concentration is 5 mmol t L-l, and with a packed cell volume of 50% and a liver blood volume fraction of 1.5%. the contribution to the apparent P, signal might be equivalent to 0.35 mmol L-’ P,. However, since the relaxation time of 2.3.BPG in blood is more than 3 seconds. it is almost completely saturated at a pulse repetition rate of 0.6 seconds, and its contribution to the P, signal is therefore negligible. After baseline spectra were obtained from summation of 64 scans at 06second intervals, either 1 mol. L-’ sodium bicarbonate or I mol L-’ sodium chloride was infused through the right atrial cannula over IO minutes. The volume infused was calculated in milliliters as (0.4 x body weight [kg] x base deficit [mmol L-l]). pH, observations were continued throughout the infusion and for IO more minutes, during which little further change in pH, was observed; pH, observations after infusion are therefore recorded as the average of observations made in the period IO to 20 minutes. The animal was then removed from the spectrometer, the cannula was carefully washed out with 154 mmol L- ’ sodium chloride. and another blood sample was obtained for measurement of ~HRA, PRACOL,and metabolites. For measurement of adenosine triphosphate (ATP)/P,. saturation factors were derived by varying the pulse repetition rate. The P-ATP resonance was found to be completely relaxed; for P,, a saturation factor of 1.23 was derived and applied to the calculation of peak areas.

Perfused Liver Studieb Isolated perfused liver preparations were established from either normal animals or animals pretreated with streptozocin as previously described. Livers were perfused at low pH ( - 6.8) but at normal PCOZ (- 5.3 kPa [40 mm Hg]) before infusion of either bicarbonate or saline. The technique of liver perfusion in situ was a minor modification of that previously described,‘5,‘0 using a medium containing 40 g/L bovine serum albumin and washed expired human erythrocytes to give a packed cell volume of 0.17. A pH of 6.8 was obtained as previously described by reducing the bicarbonate concentration and replacing it with chloride while maintaining a constant Pcoz. The lactate concentration was adjusted to 5 mmol L-’ by addition of sodium L(+)-lactate. Perfusions were performed at 37°C and at a flow rate of 7.5 mL min-’ 100 g rat weight-‘. After 20 minutes of perfusion in the recirculatory mode. the perfusion was switched to nonrecirculation. Two baseline samples of portal and hepatic venous perfusate were obtained at an interval of 5 minutes and then either 1 mol. L-’ NaCl or 1 mol L-’ NaHC03 was infused into the portal venous line at rates described

in the Results for 20 minutes. The entire study was pertormed in the Bruker AM360 spectrometer using the technique described by lies et al’” for observation of pH,. which excludes the rignal from extracellular P, by the same method described above for the in vivo studies. Portal and hepatic venous samples were again obtained in duplicate IO and 20 minutes after the start of infusion. To determine the amount of increase in Pco:. pH, and hicarhonate at the point of entry of the perfusate into the liver during bicarbonate infusion. a bench simulation using the same length of tubing and flow rates of the perfusion studies was performed. This procedure was adopted because of the inaccessibility of the preparation for sampling while in the magnet. ATPIP, ratios were calculated as described for the in vivo studies.

Analyticcd Methods Lactate. glucose, and 3-hydroxybutyrate levels were determined in neutralized perchloric acid extracts of blood or perfusate using standard enzymatic methods.” pH, Pco: and bicarbonate levels were determined with an Instrumentation Laboratories (Warrington Cheshire. UK) 413 or 1304 blood gas analyzer.

Statistical Methods arzd Other Calculations All results are means ? SEM unless otherwise stated. In the perfusion studies, rates of lactate uptake and glucose output were calculated by the Fick principle and are means of the average rates in each individual experiment before and after hicarbonate or saline treatment. PCO~ and extracellular pH values are the means of observations during the S-minute period before infusion and those values obtained 20 minutes after commencement of the infusion. Comparisons (two-tailed) before and after treatment were made using a paired I test or the Wilcoxon test in the case of differing variances. and between-group comparisons were made using an unpaired t test. RESULTS

Before infusion of bicarbonate. mean pHKA in animals with DKA was 6.90 -+ 0.05, mean PRAcol was 4.86 + 0.31 kPa (36.5 2 2.33 mm Hg), blood 3-hydroxybutyrate level was 6.50 t 0.24 mmol . L-l. and glucose level was 18.87 2 1.29 mmol L-l. Figure 1 shows serial observations of pH, in a single animal with DKA before and during bicarbonate infusion; figure 2 shows the effect of bicarbonate in six animals with DKA. A substantial increase in pHKA was achieved. Despite the significant increase in PRAco2 (P < .Ol), there was a highly significant increase in pH, (from 7.13 + 0.08 to 7.32 * 0.08, P < .05). Qualitatively similar effects were obtained in two animals with acidosis due to infusion of HCI (Fig 2). In five animals with DKA that were infused with sodium chloride instead of bicarbonate, there was no significant change in pHRA, pHi, or PCO~ (Fig 2). In DKA animals, the mean change in pHi with bicarbonate infusion (+0.19 ? 0.04) was significantly greater (P < .OOl) than that with sodium chloride infusion (-0.02 ? 0.03). Blood lactate level was measured in four of the six animals with DKA that were infused with bicarbonate and was not significantly changed during the infusion (mean, I .02 2 0. IS mmol L-l before; mean, 1.23 t 0.38 mmol . L-l after).

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pHi

PH

RA

= 7.12

PCO,= 11.85

10 Time

20

(min)

Fig 1. Serial observations of pH, in a DKA rat infused with sodium bicarbonate according to the protocol described, starting at the arrow. Corresponding values of pH,,* and Pco, are shown (1 kPa = 7.5 mm Hgl

As described in the Methods, isolated liver preparations were obtained either from DKA or normal animals and perfused at a pH of approximately 6.8, with normal Pco:. Five preparations from normal animals and five from DKA animals were infused with 1 mol . L-l NaHC03 at 0.57 mmol min’: another four preparations from DKA animals were infused at a lower rate (0.39 mmol . mint).

76

PH

74

yArP

L’ ?

pre

pHnv and Puvco z values are from hepatic venous effluent. In two bench perfusions simulating the tubing lengths, flow rates, and bicarbonate infusion rates (0.57 mmol . mini) used in the MRS studies, the pH and PCO~ measured at a point 0.7 m downstream from the point of infusion were 7.25 to 7.31 and 11.8 to 13.2 kPa (89 to 99 mm Hg), respectively; at a second point near the “entry” of perfusate into the liver, pH was 7.23 to 7.26 and Pco2 was Il.5 to 12.5 kPa (86 to 94 mm Hg). The mean pH of blood obtained from the inferior vena cava of 12 DKA rats immediately before establishing liver perfusion was 6.85 -+ 0.05, mean PCO? was 5.91 * 0.42 kPa (44.3 + 3.2 mm Hg). mean glucose level was 17.95 2 0.51 mmol L-l. and mean 3-hydroxybutyrate level was 7.16 ? 0.40 mmol . L-t. Figure 3 shows a spectrum obtained from a perfused liver, and Figure 4 shows pH, changes seen during bicarbonate infusion. Figure 5 demonstrates that, as during the in vivo situation, a highly significant increase occurred in pHi despite the increase in PCO~ in the portal influent and hepatic venous effluent. The mean changes are similar in preparations from normal and DKA animals given the same rate of bicarbonate infusion. Correspondingly smaller but still significant changes took place in perfused DKA livers having the lower rate of bicarbonate infusion (mean pH, in low-bicarbonate series +0.15 ‘_ 0.03 compared with +0.27 + 0.06 in high-bicarbonate series). It is noteworthy that the mean pH, before bicarbonate infusion was significantly higher in DKA livers (normal liver pH,, 7.12 + 0.02, n = 5; DKA liver pH,, 7.24 ? 0.02, n = 9; P = ,005). an observation referred to previously.*A In three studies on livers from DKA rats in which sodium chloride (1 mol L-I) was added to the perfusate at 0.57 mmol min-I. mean ApHnv was +0.04 ? 0.017, APnvco2 was -0.38 * 0.21 kPa (-2.9 ? 1.6 mm Hg), and ApH, was -0.007 t 0.018; none of these changes was significant. Figure 6 demonstrates the effects of bicarbonate on lactate uptake and glucose output in groups given the higher dose of bicarbonate. As previously noted,‘” livers

--...:

sXATP

--P post

pre

post BATP

we

post

Fig 2. Summary of effects of sodium bicarbonate infusion on pHa,, Pco2, and pH, in DKA rats (0, n = 6) or in two animals with acidosis induced by infusion of HCI (W). The amount of sodium bicarbonate infused was calculated as described in the Methods. (0) Mean results from five DKA rats infused with sodium chloride. P values referring to paired comparisons before and at the end of the infusion in bicarbonate-treated DKA animals are as follows: pHs,,, P < .05; pHi, P < .05; and Pco,, P < .Ol. Error bars are SEM (1 kPa = 7.5 mm Hg).

::_

Fig 3. )‘P-MRS spectrum recorded from an isolated perfused liver. The Pi peak and the o, (3, and y phosphorus peaks of ATP are shown. The spectrum is the sum of 30 scans using approximately 90” pulses (at center of coil) and a pulse interval of 0.6 seconds.

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,,s_

Venous pH= 6.19

LACTATE REMOVAL

T

GLUCOSE OUTPUT (X2)

M 8

T

.5 E

2

E

PHI

3.

Venous pH = 1.42. PCO, = 9.31 I 20

10

Time (ruin) Fig 4. Serial changes in pH, in a perfused liver obtained from an animal with DKA. The format is the same as in Fig 1 (1 kPa = 7.5 mm Hg)

from DKA rats show higher lactate uptake and glucose output than those from normal animals. Bicarbonate infusion produced marked increases in lactate uptake in both types of livers. However, although glucose output was approximately doubled by bicarbonate infusion in livers from normal animals. it was unchanged in those from DKA animals. If all of the lactate taken up is converted to glucose, the increment in glucose output on a molar basis should bc half that in lactate uptake. For this reason. the glucose scale in Fig 6 has been doubled so that a direct 76

1

74 i

72

70

1

Fig 6. Effect of bicarbonate infusion (higher rate protocol) on lactate removal and glucose output in perfusions at acidotic pH (-6.8) of livers from normal (n = 5) and DKA (n = 9) rats. Results are expressed as mmol min-’ . 100 g rat weight ‘. Glucose output is shown as twice the actual amount for reasons described in the text. P values are from paired comparisons between values before bicarbonate infusion (c) and at the end of infusion (n), and are indicated as follows: ns, not significant; l*P < .02; l**P < ,001.

comparison can be made with lactate uptake: equal column heights for lactate uptake and glucose output indicate stoichiometric equivalence of these two processes. In the three studies on livers from DKA rats in which sodium chloride was added to the perfusate instead of bicarbonate. the mean change in lactate uptake was -0.X1 t 0.47 mmol mini 100 g rat weight, which is not significant. Mean ATPIP, in normal livers (n = 5) was 2.65 f 0.41 before and 2.33 + 0.41 20 minutes after the start of infusion. The corresponding values in DKA livers (n = 4) were 2.16 ? 0.25 and 2.49 ? 0.39: neither of thcsc changes is significant. DISCUSSION

-1 6

Fig 5. Summary of effects of bicarbonate infusion on hepatic venous pH, Pco~, and pH; in livers from five DKA (0) and five normal (8) animals perfused at approximately pH 6.8. For both series, the higher bicarbonate infusion rate protocol (see the Methods) was used. Also shown are results from three perfusions of livers from DKA animals (0) in which sodium chloride (1 mol L-‘) was infused (0.57 mmol min-I) instead of bicarbonate. P values from paired comparisons before and after the infusion are indicated as follows: lP < .02, l*P < .Ol,‘**P < ,001 (1 kPa = 7.5 mm Hg).

Our results show that not only does bicarbonate rapidly increase intrahepatic pH in vivo when used to treat either DKA or HCI-induced acidosis, it also increases lactate uptake in livers perfused at low pH from both normal and DKA animals. In addition, glucose output is doubled in perfused livers from normal rats. It has previously been shown that gluconeogcnesis from lactate is inhibited by both metabolic and respiratory acidosis7-“.‘“~” This is the first demonstration of the reverse process. In the DKA series. there was no change in glucose output after bicarbonate infusion, suggesting an effect on the disposal of lactate separate from stimulation of gluconeogencsis. which may already have been maximal in these livers; the routes of extra nongluconeogenic lactate disposal were not dctermined, but possibilities include pyruvate and alanine formation, ketogencsis and lipogenesis, and complete oxidation. In livers from normal animals, bicarbonate also increases lactate removal in excess of that accountable for by increased gluconeogenesis. Increased lactate removal is of potential benefit in all types of metabolic acidosis, as this

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process contributes to the removal of protons.lY We found no increase in blood lactate concentration after bicarbonate administration, in contrast to observations in dogs.?” In both in vivo and in vitro studies, an increase in pHi was detecteid within 1 minute of initiating bicarbonate infusion. Measurements of portal and hepatic venous PCO~ in the perfusion studies suggest that the liver was exposed to an increase of 3 to 8 kPa. Using the relationship between Pco? and pH, at constant extracellular bicarbonate levels previously cstablished,7-y,Z1 this increase in PCO* would be expected to result in a decrease in pHi of 0.08 to 0.2 units, an effesct that should be detectable by “‘P-MRS. In one perfusion and in one in vivo study, at the first pHi point recorded after initiation of the infusion (after -30 seconds) a small decrease in pHi was in fact recorded: all subsequent points showed the main effect, ie, an increase. Although the initial transient decrease may not be significant, it could represent a genuine effect. In vivo, a substantial amount of CO? released by titration with bicarbonate during IV infusion is lost during passage reasons, it was not through the lungs.‘? For technical possibll: to determine arterial PCO~ (P,co~) and portal venous PCO~ (Ppvco2) in the animals studied, and we cannot therefore be certain that these were not within normal limits. However, this cannot be the case in the perfused-liver studies, where a substantial increase in PCO~ was demonstrated during bicarbonate infusion in the portal venous line. Notwithstanding this increase in portal influent Pro?, pHi increased and lactate disposal was stimulated. These results may be considered surprising in view of other observations in the literature. Shapiro et al4 reported that infusion of bicarbonate in acidotic rats resulted in a decrease in brain pHi. However, the amount of bicarbonate infused was considerably smaller than that in the present study, and calculations using data for brain CO2 buffering power:‘” indicate that the small increase in CO? could not have accounted for the decrease in pHi observed. Bersin and Ariefi? infused bicarbonate to dogs with hypoxic lactic acidosns; arterial pH decreased slightly and there was no change in arterial Pco?, but there was a slight increase in mixed venous Pco~. They observed a decrease in hepatic pHi, which was related to a decrease in portal vein pH and an increase in Pco?. Shapiro et alz4 infused bicarbonate 2.5 times more rapidly than we did (but at N half the total dose) to rats with acute ammonium-chloride acidosis and found a

small transient decrease in hepatic pHi and an increase in P,cor. It is therefore not immediately clear from these previous observations and our own to what extent intracellular acidification after exposure to bicarbonate can be directly due to CO2 entry. Despite suggestions to the contrary,?“JsJh COZ generated in a tissue specifically because of titration of protons in or derived from the intracellular compartment in that tissue cannot decrease pHi, although it can contribute to elevation of mixed venous Pco?.?~ In contrast, an increase in PCO~ in blood entering the tissue could lead to a decrease in pH,; however, elevation of P,co? can be prevented simply by administering IV bicarbonate sufficiently ~lowly.?~ We suggest that differences in behavior of pH, between tissues might be explained at least partially by different ratios of effective plasma membrane permeability to CO? and bicarbonate. We postulate that in the liver bicarbonate entry via Na+/HC03cotransportlx and Natdependent Cl-/HCO?exchange together with Na+ /H+ exchangeZy results in rapid alkalinization of the hepatocyte on application of bicarbonate, and only occasionally is the acidifying effect of CO1 entry transiently observed. A possible reason for the difference between our results and those of Bersin and Aries” could be the lower starting pH, and the larger dose of bicarbonate given in our in vivo studies; systemic alkalinization was achieved in our studies, but not in those of Bersin and Arieff. Finally, the stimulation of lactate disposal observed during bicarbonate infusion itself generates additional intracellular bicarbonate3” and augments alkalinization of the hepatocyte. In conclusion, in the models of severe acidosis that we have used, there appears to be no danger of inducing hepatic intracellular acidosis by bicarbonate infusion at the rates used; indeed the stimulatory effects on lactate removal and gluconeogenesis are likely to be beneficial. But our results certainly do not exclude the possibility that the effect of bicarbonate infusion on pHi in other critical organs such as the heart might be opposite to that observed in the liver. If this were the case, cardiac output might decrease, accounting for the findings of Arieff s group in dogs and the advantages in their models of alternative alkalinizing agents. ACKNOWLEDGMENT

We are grateful to Professor D.G. Gadian and Drs S. Williams and S.C.R. Williams for advice and use of MRS facilities.

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