MAGNETIC RESONANCE IN MEDICINE 16,403-410 ( 1990)

Hemodynamic and Hepatic pH Responses to Sodium Bicarbonate and Carbicarb during Systemic Acidosis

J. I. SHAPIRO, M. WHALEN, AND L. CHAN

The Giles Filley Laboratory, Department of Medicine and Webb- Waring Lung Institute, University of Colorado Health Sciences Center, Denver, Colorado 80262

Received July 10, 1989; revised November 15, 1989

Rats subjected to ammonium chloride-induced metabolic acidosis were given alkalin- ization therapy with either sodium bicarbonate or Carbicarb. Ammonium chloride-induced Severe metabolic acidosis had minimal effect on mean arterial blood pressure and cardiac output. This acidosis resulted in a small but statistically significant fall in intracellular liver pH (pHi) as measured with 3'P magnetic resonance spectroscopy (7.01 f 0.05 vs 7.08 f 0.04, p < 0.05 ). Sodium bicarbonate treatment resulted in systemic alkalinization and increases in arterial pC02 as well as transient but extreme decreases in cardiac output and mean arterial pressure. Alkalinization with sodium bicarbonate also resulted in a transient but significant decrease in intracellular liver pH (7.02 f 0.06 at 5 min vs 7.09 f 0.06 at baseline, p < 0.05). Carbicarb therapy resulted in systemic alkalinization without major changes in arterial pCOz, cardiac output, or mean arterial blood pressure. Moreover, Carbicarb effected a sustained intracellular alkalinization of the liver (pH, = 7.12 t 0.07 at 5 min, p 4 0.05, pH, = 7.19 -f 0.07 at 10 min, p < 0.01, pH, = 7.16 f 0.06 at 15 min, p .c 0.01, vs baseline pH, = 7.05 0.06). These data suggest that Carbicarb may be a more effective buffer than sodium bicarbonate during conditions where ventilation is limited and hemodynamic instability is present. 0 1990 Academic Press, Inc.

INTRODUCTION

The current clinical treatment of systemic acidosis is controversial ( 1 , 2). It is well established that severe acidosis compromises function in multiple organs, especially the heart (3-10). However, clinical treatment of patients with acute metabolic acidosis with sodium bicarbonate has become less popular, primarily because of its lack of efficacy in improving clinical outcome ( 2 ) . Although much of the lack of clinical efficacy of bicarbonate therapy may relate to the severity of most patients' underlying medical illness ( I ), it is likely that bicarbonate does indeed have deleterious effects on hemodynamics as well as on other organ functions ( 2 , 11-15). Many of these deleterious effects of sodium bicarbonate have been attributed to its tendency to gen- erate COz when added to acidotic blood, a phenomenon which has been demonstrated in both in vizro and in vivo animal models as well in selected clinical circumstances

Carbicarb is an experimental buffer made of disodium carbonate and sodium bi- carbonate which has been formulated to avoid generation of C02 when added to acidotic blood ( 19, 20). In previous studies, we have demonstrated that where bicar- bonate therapy was associated with both an increase in the arterial tension of C02

(11-14, 16-18).

403 0740-3194/90 $3.00 Copyright 0 1990 by Academic Press, Inc. All rights of reproduction in any form reserved.

404 SHAPIRO, WHALEN, AND CHAN

(P,CO,) and a decrease in intracellular brain pH as measured by 31P NMR, Carbicarb had less effect on the P,CO2 and effected intracellular brain alkalinization in these studies (21 , 22). We performed the following studies to measure the effects of am- monium chloride acidosis and alkalinization therapy on cardiac function as well as on the intracellular pH of the liver, an organ critical in total body acid-base homeostasis ( 1 7, 18, 23).

METHODS

Animals. Sprague-Dawley rats (approximately 300 g body weight) were used in these experiments. For cardiac output experiments, rats were anesthetized with Ket- amine (0.4 ml, 50 mg/dl) and Xylazine (0.2 ml, 20 mg/ml), after which the right jugular vein, right carotid artery, right femoral artery, and right femoral vein were cannulated with PE-50 tubing. One catheter in the right jugular vein was advanced into the right ventricle and was used to withdraw mixed venous blood gases (vide infra) while the other right jugular catheter was left in the distal superior vena cava. Rats were then placed on a warming pad which maintained core temperature (measured by rectal temperature probe) between 35 and 37°C. A tracheostomy was performed (PE- 160 tubing) and the rat was ventilated using a rodent ventilator ( Harvard Instru- ments) employing a tidal volume of 4.0 ml and 45 breaths per minute with approx- imately 12 ft of tubing ending in a “T” connection to the tracheostomy tube. Rats were then subjected to neuromuscular blockade with 0.7 ml of metubine ( 2 mg/ml) fixing their ventilation at a constant level. After priming the system with 7% albumin (bovine albumin, Fraction V), an arteriovenous shunt from right carotid artery to right jugular vein containing an extracorporeal volume of approximately 2 ml, which was driven by a peristaltic pump and traversed a densitometer (Waters Inc., Baltimore, MD) for on-line determination of the blood concentration of indocyanine green (Car- dio-Green, Hynson, Westcott and Dunning, Inc., Baltimore, MD) , was opened. As the pump speed driving the shunt was increased to 3 mlfmin, an additional 1-3 ml Of 7% albumin was infused to maintain hemodynamic stability. For magnetic resonance spectroscopy ( MRS ) experiments for monitoring liver pH, the surgical preparation was identical except for the omission of the femoral catheters, the right ventricular catheter, and the arteriovenous shunt.

Ammonium chloride acidosis was induced by administering 10 mmol/ kg ammo- nium chloride (2 M solution) by gavage. Saline (0.9%), sodium bicarbonate ( 1 M ) , and Carbicarb were placed in numbered vials and administered (4 ml/ kg or approx- imately 1.4 ml/animal) over 2 min intravenously. This dose corresponded to 4 mmol/ kg of sodium bicarbonate which was chosen on the basis of typical initial clinical dosing for, severe acidosis.

Hemodynamic measurements. Arterial blood pressure was monitored continuously from the femoral artery (in cardiac output experiments) or the carotid artery (in MRS experiments) with a pressure transducer and recorded on a Gould 4-channel chart recorder (Gould Instruments, Cleveland, OH). Cardiac outputs were measured serially in the same animal using the indicator dilution method. Basically, the establishment of an arteriovenous shunt allowed for continuous measurement of the blood concen- tration of indocyanine green using a densitometer. For each cardiac-output measure- ment, indocyanine green was injected into the femoral catheter and on-line measure-

HEMODYNAMIC AND HEPATIC pH RESPONSES 405

ment of the densitometer readout was established for 40 s. Using an A-D converter (DASH-8, Metrabyte Corp., Taunton, MA) and a personal computer (IBM PC, In- ternational Business Systems) and software written by us (J.S. and M.W.), the evolution and decay of the blood concentration of indocyanine green following injection was recorded. This allowed for integration of the monoexponential portion of the concen- tration decay ( A ) following establishment of a 0 baseline and solving of the equation CO = Q / A in ml/min, where Q is a densitometer reading on a standard concentration of indocyanine green ( 2 4 ) . Hard copy printouts of these indicator dilution curves were also made using the chart recorder.

Blood-gas determinations. Arterial blood obtained from the carotid catheter was analyzed for p 0 2 , pC02 and pH using a blood-gas instrument (Radiometer Instru- ments, Copenhagen, Denmark). Volumes of arterial blood removed during the course of experiments for blood-gas determinations were replaced by equal volumes of 7% albumin. During cardiac output experiments, blood drawn from the pulmonary artery was also analyzed for mixed venous tensions of 0 2 (Pm,Oz) and C 0 2 (PmvC02) as well as pH.

Magnetic resonance spectroscopy. The determination of intracellular liver pH was performed using a 3P MRS technique. A 1.89-T, 30-cm horizontal-bore cryomagnet with Biospec spectrometer (Bruker Medical Instruments, Billerica, MA was used for all MRS studies. A 2-cm-diameter three-turn surface coil made from 2-mm-thick copper wire, homebuilt by the authors (J.S. and L.C.), was surgically placed through a midline abdominal incision on the anterior surface of the liver and shielded from surrounding viscera and abdominal wall musculature with gauze. Using variable ca- pacitors, the probe was then tuned to the resonance frequency of 31P (32.6 MHz) at the field strength used. The Bo field was shimmed on a water proton signal from the liver after positioning the rate in the homogeneous volume of the magnet until the proton linewidth was 30-60 Hz (25 ) . 31P spectra were then continually acquired every 2.5 min using a sweep width of 3000 Hz, 1K data arrays zero-filled to 4K, and 1200 transients employing a pulse width of 8 ps (corresponding to a 45" tip at the center of the coil) and a repetition time of 0.125 s. This relatively short repetition time was chosen to decrease contributions from extracellular inorganic phosphate, which has a spin-lattice relaxation time ( TI ) much longer than that of intracellular inorganic phosphate ( T I of rat plasma inorganic phosphate = 8.5 -t 0.50 s ( N = 5) vs TI of liver inorganic phosphate peak = 0.9 k 0.07 s ( N = 5 ), p < 0.0 1, T I values measured with saturation recovery method), as well as to maximize the signal-to-noise ratio (which equaled or exceeded 40: 1 for all spectra analyzed). The free induction decay was then Fourier transformed following exponential multiplication with 10 Hz line- broadening. As little creatine phosphate was visible on the 31P MRS spectrum from liver, the gamma ATP resonance was used as an internal chemical shift reference for the resonance frequency of the inorganic phosphate peak as described by Gadian (26). The chemical shift of the inorganic phosphate peak relative to the water proton res- onance, using a method described by Ackerman and colleagues, agreed quite well with this on the first of serial 31P spectra ( 2 5 ) , but because of Bo drift during collection of serial spectra, was not generally applicable. The resolution of this measurement of liver pH was to about 0.04 ppm or approximately 0.09 pH units. The liver pH values obtained on the spectra before and after each time point were averaged to yield each

406 SHAPIRO, WHALEN, AND CHAN

experimental time point value (e.g., spectra obtained from 2.5 to 5 min and from 5 to 7.5 min following a drug would be used to calculate the 5-min data point). A representative 3'P MRS spectrum from rat liver is shown in Fig. 1.

Experimental design. Following induction of ammonium chloride acidosis for 30 min, normal saline, sodium bicarbonate and Carbicarb (all stored in coded vials) were administered in random order with the spectroscopist (J.S.) blinded as to the identity of the drug.

Statistics. One- and two-way analysis of variance were performed where it was appropriate to demonstrate differences among group means. Comparison of group means with the control was done using Student's t-test for unpaired or paired data with Bonferroni's correction (27).

RESULTS

Ammonium chloride administration resulted in severe systemic acidosis by 30 min (7.08 k 0.02 vs 7.40 f 0.01, p < 0.01 ), as we have described in previous work ( 2 1 ) , which was accompanied by minimal changes in mean arterial pressure (82 t- 4 vs 88 k 5 mm Hg, P = NS) and cardiac output (390 f 30 vs 397 t- 40 ml/min/kg, P = NS) compared to baseline values. Intracellular liver pH was decreased a small but significant amount by 30 min following ammonium chloride administration (7.01 f 0.05 vs 7.08 k 0.04, p < 0.05).

, ' " ' 1 ' ~ ' ~ I " " I " ' I ' ~ ' ~ I ~ ' " I ' ' ' ' 1 ' ' " 1 ' ~ ' 15 10 5 0 -5 -10 -15 -20 -25

PPM

FIG. 1. Representative 3'P MRS spectrum of rat liver. Spectrum acquired with 1200 transients, 0.125-s relaxation delays, and a pulse width of 8 ps (i.e., 45" at the center of the coil) with a sweep width of 3000 using a 1 K data array (zero-filled to 4 K ) . The FID was exponentially multiplied using 1 0-Hz line-broadening prior to Fourier transformation. Peak assignments were as follows: ( 1 ) phosphomonoesters, (2) inorganic phosphate, (3 ) phosphodiesters and mobile membrane phospholipids ( 3 4 ) , (4) gamma phosphate group of ATP, (5 ) alpha phosphate group of ATP, and (6 ) beta phosphate group of ATP.

HEMODYNAMIC AND HEPATIC pH RESPONSES 407

The hemodynamic responses of animals to the different treatments are summarized in Table 1. Administration of normal saline did not significantly affect cardiac output or mean arterial blood pressure. Administration of sodium bicarbonate resulted in a transient but marked drop in cardiac output (23 1 +. 35 vs 36 1 t 39, p < 0.05 at 2 min; Fig. 2) associated with a transient fall in mean arterial pressure (40 f 7 vs 78 f 3, P < 0.01 at 2 min). In contrast, Carbicarb infusion had less effect on the he- modynamic parameters, causing no significant change in cardiac output and a marked but transient fall in mean arterial pressure (46 f 5 vs 80 f 4, p < 0.01 at 2 min).

Examining the acid-base measurements (Table 2), we note that, again, saline in- fusion had no significant effect on any parameter. Sodium bicarbonate caused a sus- tained extracellular alkalinization which was associated with an increase in P,C02 and Pm.,C02. Intracellular liver pH was noted to fall transiently (7.02 f 0.06 vs 7.09 f 0.06, p < 0.05 at 5 min), then return to baseline after bicarbonate administration. Carbicarb also caused extracellular alkalinization with little effect on PaCO2 and PmvC02. Moreover, Carbicarb treatment induced a sustained increase in the intra- cellular liver pH (pHi = 7.12 f 0.07 at 5 min, p < 0.05, pHi = 7.19 f 0.07 at 10 min, p < 0.01, pHi = 7.16 k 0.06 at 15 min, p < 0.01, vs baseline pHi = 7.05 f 0.06; Fig. 3).

DISCUSSION

In these studies, we first demonstrated that our model of metabolic acidosis is a hemodynamically stable one, with little change in blood pressure or cardiac output observed despite relatively severe decreases in arterial pH. Moreover, the lack of effect

TABLE 1

Effect of Treatment on Hemodynamic Measurements

Time Cardiac output Mean arterial pressure Drug (min) (ml/min/kg) (mm Hg)

Saline 0 2 5

10 15

Bicarbonate 0 2 5

10 15 0 2 5

10 15

Car b i car b

318 -+ 17 325 f 14 346 -+ 27

304 t 15 361 f 39 231 f 35* 358 f 21 354 f 24 316 t 21 366 t 35 320 t 21 365 2 3 0 380 t 30 331 rt-21

343 f 26

82 f 3 80 f 3 78 t- 3 80 L 2 77 + 2 78 f 3 40 k I** 7 3 + 4 80 f 4 77 f 3 80 rt_ 4 46 f 5** 80 f 5 I7 f 4 15 rt- 5

Note. Results expressed as mean f sem. For cardiac output determinations, n = 9 for each drug and time point. For the blood pressure determinations, n = 20 for each drug and time point. * p < 0.05, **p c 0.01 vs baseline (0 min) by paired t-test.

408

7.20-

7.' 0

7.00 -

SHAPIRO, WHALEN, AND CHAN

0- [I

pHi

7.301

1 i * 6.90

0 5 10 15

TIME (min)

FIG. 2. Response of intracellular liver pH (pH,) to saline (0), sodium bicarbonate ( ~ ), and Carbicarb (0) administration. Data presented as mean f sem. Data derived from N = 1 I experiments for each drug treatment. * p < 0.05, * * p < 0.01 vs baseline (time, 0 min) by paired t-test.

of saline on any of the parameters which we measured is additional proof of the short- term stability of this preparation.

Bicarbonate therapy was found to transiently decrease the intracellular liver pH, a phenomenon which could be explained both on the basis of its effect of transiently decreasing the cardiac output which probably was associated with decreases in liver

TABLE 2

Effect of Treatment on Acid-Base Measurements

Time pacoz PIn"CO2 Drug (min) P H ~ (mm Hg) PH,, (mm Hg)

Saline 0 7.18 f 0.02 3 6 f 1 7.18 k 0.03 44 f 2 5 7. I5 f 0.02 38 f 1 7.15 f 0.03 45 f 1

10 7.16 f 0.02 36 k 1 7.15 f 0.03 45 f 2 15 7.15 k 0.02 3 8 k 2 7.15 k 0.03 44 f 2

Bicarbonate 0 7.11 k 0.02 3 6 k 1 7.04 f 0.04 46 f 2

10 7.26 f 0.02** 40 f 1** 7.22 f 0.03** 48 f 1 15 7.25 f 0.02** 38 f 1 7.21 ? 0.03** 46 f 1

Carbicarb 0 7.09 f 0.02 35 f 1 7.07 f 0.03 41 f 2 5 7.30 f 0.02** 38 f 1 7.29 f 0.03** 44 f 3

10 7.25 f 0.02** 37 * 1 7.24 f 0.03** 44 f 2 15 7.23 -t 0.02** 36 5 1 7.22 f 0.03 43 f 3

5 7.29 f 0.02** 46 f 1** 7.24 f 0.03** 51 f 2 *

Note. Results expressed as mean f sem. For each drug and time point, arterial pH (pH,) and P,C02 determinations came from 20 experiments and mixed venous pH (pH,,) and P,,,,C02 determinations came from 9 experiments (i.e., same as 9 cardiac output experiments, Table I). *p < 0.05, **p < 0.01 vs baseline (0 min) by paired t-test.

HEMODYNAMIC AND HEPATIC pH RESPONSES 409

blood flow and on the basis of the acute increase in PaCOz which resulted from its infusion. Which of these effects is dominant is difficult to discern from this study. However, the relatively rapid return of intracellular liver pH to baseline values in the face of a persistently elevated PaC02 suggests that hernodynamic effects may be im- portant, at least in minimizing the liver cells’ ability to buffer the effects of the increase in PaC02.

Carbicarb therapy, in contrast, was observed to dramatically increase the intracellular liver pH in a sustained manner. This effect could not be explained by observed changes in PaCOz or Pm,C02 or by any observed hemodynamic effects. Although Carbicarb could have effects on tissue tensions of COz not reflected by alterations in Pm,C02 or PaC02, this explanation is less than satisfying. Alternately, it is possible that admin- istration of Carbicarb induces an increase in the intracellular concentration of the carbonate ion which could buffer cytosolic calcium and accomplish alkalinization of the cell (28-31). What is more likely is that sustained increases in the extracellular pH mediated by increases in bicarbonate concentration which are unopposed by in- creases in PaC02 effect this intracellular alkalinization, a phenomenon which has been well documented (30, 32, 33). Certainly, this issue needs further investigation in simpler biological systems.

In summary, we have demonstrated marked differences in the effects of sodium bicarbonate and Carbicarb on hemodynamics and intracellular liver pH homeostasis in an experimental model of metabolic acidosis where ventilation is fixed. If these differences are confirmed in humans, Carbicarb may have some advantages over so- dium bicarbonate in the treatment of acute metabolic acidosis, especially under con- ditions where ventilation is fixed or limited, such as during cardiac arrest resuscitation.

ACKNOWLEDGMENTS

We thank Drs. Thomas Petty for his helpful criticism of these studies. The NMR instrument has been purchased through an NIH Biotechnology Instrumentation Grant ( 1870 RR02436-Ol ), Division of Research Resources. This work was supported by grants from the American Heart Association and the Colorado Heart Association. Dr. Shapiro is supported by an American Heart Association-Squibb Corporation Clinician Scientist Award.

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