Kinetics

of Lactate

Transport

Into Rat Liver In Vivo

Michael A. Lupo, William T. Cefalu, and William M. Pardridge Lactate clearance by liver plays an important role in lactate homeostasis and in the development of lactic acidosis. The role of lactate delivery to liver as a limiting factor in hepatic uptake of lactate is unclear. Lactate delivery mechanisms could be important if rates of lactate transport approximate rates of lactate metabolism by liver. The rates of lactate transport into liver have been determined in vitro with isolated liver cells and the results have been conflicting. Therefore, the present studies measure the rate of transport of [?I-L-lactate, and its poorly metabolizeable stereoisomer. [‘4C]-o-lactate, into rat liver in vivo using a portal vein injection technique. The transport of [3H]-water and of [‘*C]-sucrose, an extracellular reference compound, were also studied. Portal blood flow was determined from the kinetics of [3H]-water efflux in liver and was 1.93 2 0.22 mL/min/g. The volumes of distribution of [“Cl-L-lactate. [?,]-o-lactate, and [‘?Z]-sucrose were 1.31 + 0.22, 0.71 + 0.07, and 0.22 _t 0.07 mL/g, respectively. The extraction of unidirectional influx of [‘?I-L-lactate and [‘4C]-o-Iactate by rat liver was 93% * 10% and 91% * 9%. respectively. The rate of lactate transport into rat liver in vivo, 1.8 pm01 - mini’ . g-‘, .IS approximately twofold greater than the rate of lactate metabolism by rat liver reported in the literature. Therefore, lactate uptake by liver may not be limited by transport under normal conditions. However, conditions such as decreased portal blood flow, which slow lactate delivery to liver by 50% or more, could cause lactate uptake by liver to be limited by transport of circulating lactate. 0 1990 by W.B. Saunders Company.

T

HE LIVER CLEARANCE of lactic acid plays a central role in lactate homeostasis.’ Therefore, derangements

in hepatic lactate clearance mechanisms may play a role in lactic acidosis.‘,2 The role of lactate transport from blood into liver in regulating overall lactate uptake is unclear. If rates of lactate transport were comparable to rates of lactate metabolism, then transport could become rate-limiting under conditions that slowed transport.2 Estimates of liver transport rates of lactic acid have been made in isolated liver cells and

these studies give conflicting estimates. One study suggests lactate transport into liver cells is normally an order of magnitude greater than lactate metabolic rates3 whereas another recent study indicates lactate transport approximates rates of lactate metabolism.4 Therefore, the purpose of the present studies was to determine the rates of lactate transport into liver in vivo by measuring lactate extraction and hepatic blood flow. Since rapid metabolism of L-lactate could potentially confound interpretation of transport data, the present studies also examine the hepatic transport of D-lactate, which is relatively metabolically inert in 1iver.r” MATERIALS AND METHODS Materials

All isotopes were purchased from DuPont-New England Nuclear (Boston, MA). The specific activities of the isotopes were [i4C(U)]-

From the Department of Medicine, Division of Endocrinology, UCLA School of Medicine, Los Angeles, CA. Michael A. Lupo was recipient of an American Heart Association Summer Student Fellowship during the course of these studies. William T. Cefalu was sponsored by National Institutes of Health Training Grant No. AM-07094. This work was supported in part by the American Diabetes Association of Southern California and by NIH Grant No. DK-25744. Address reprint requests to William M. Pardridge, MD, Department of Medicine/Endocrinology. UCLA School of Medicine, Los Angeles, CA 90024. Q 1990 by W.B. Saunders Company. 0026-0495/90/3904-0007$3.00/O

374

L-lactate, 139 pCi/fimOl; [ ‘“c( U)]-D-hCtatC, 40 &i/rmol; [‘“c(u)]sucrose, 3.6 &i/rmol. All unlabeled compounds were obtained from Sigma Chemical (St Louis, MO). Portal

Vein Injection Technique

The transport of [“‘Cl-lactate into rat liver was measured in male, Sprague-Dawley rats (180 to 250 g) anesthetized with intramuscular ketamine hydrochloride (230 mg/kg) and intramuscular xylazine (2.3 mg/kg) using a portal vein injection technique described previously.6 The abdomen was opened and the portal vein was cannulated with a 25-gauge needle attached to a l-cc syringe. Immediately after ligating the hepatic artery, approximately 250 PL of solution was rapidly injected (~0.5 seconds) so that the solution displaced blood from the portal vein during injection. The injection solution contained 2 rCi/mL [WI-compound and 10 &i/mL [‘HI-water, used as a freely diffusible internal reference of liver uptake, mixed in Ringer’s solution buffered with 10 mM HEPES (pH 7.4). After injection, the needle was left in the portal vein to prevent bleeding. The right major lobe of liver was rapidly excised at various times after injection (18, 30, 45, 60, and 90 seconds). The right lobe was routinely analyzed to minimize possible interlobular differences in uptake due to bolus streaming. The liver tissue was homogenized and approximately lOO-mg samples of liver tissue were solubilized in duplicate in 2 mL Soluene 350 (Packard Instrument, Downer’s Grove, IL). Similarly, a sample of injection solution was solubilized for double isotope liquid scintillation counting. The liver uptake index (LUI) at various times after injection was calculated as follows: LUI (%) =

[‘4C]/[3H] - liver [‘4C]/[3H] - injection solution

x 100.

The LUI(t) = E,(t)/E,(t), where E,(t), E,(t) are the extractions of the [?Z]-test compound and the [3H]-water reference compound at the respective time period after portal injection. Therefore, E, = (LUI)E,. The E,(t) was determined in separate studies by measuring the weight of the entire liver, the [‘HI-dpm/g of liver tissue analyzed, and the [‘HI-dpm/mL portal vein injection solution. In these studies, exactly 0.2 mL of solution was injected into the portal vein. From these results, the extraction at any time point was determined for either [‘HI-water or [‘JC]-compounds. The unidirectional extraction, E(O), and rate constant, K, of

Metabolism, Vol39,

No

4

(Aprd), 1990: pp 374-377

LACTATE TRANSPORT IN LIVER

375

monoexponential washout of 13H]-water or [‘4C]-compound was determined with linear regression analysis by fitting the extraction at various times after pulse injection, ie, E(t), to the following: E(t) = E(0)e-K’.‘.8 Portal blood flow (F) was measured as reported previously,’ ie, F = KV,/[E(O)], where V, = 0.88 mL/g, ie, the distribution space of water in liver (mL/g) to blood (mL/mL).’ The volume of distribution (V,) of test compound was computed from V, = [E(O)]F/K,* and the rate of lactate influx (J) into liver is given by J = EFC,,’ where C, = the portal vein concentration of lactic acid and E, the unidirectional extraction of [‘4C]-lactate by hepatocytes, equals E(0)lac’ - E(O)‘“‘/[ 1 - E(O)‘“‘], where E(0) for lactate or sucrose is determined from the y-intercept of the monoexponential washout curve. RESULTS

The rate of [‘HI-water or [‘4C]-sucrose efflux from rat liver back to blood during the first 90 seconds after injection is shown in Fig 1. However, the extraction data at 90 seconds fall above the linear regression line owing to recirculation at this time. Therefore, only the data at 18 to 60 seconds after injection were used in the linear regression analysis. These FED

RATS

(3H) - WATER

EFFLUX

r = 0.99 slope= 2.19 +- 0.24 min-’ intercept = 1.14 + 0.20

.-

-4

(‘%)-SUCROSE

EFFLUX

r = 0.96 0.06 -

- 0.16 f 0.04 slope= 1.15 * 0,34min-

intercept

0.04 -

0.03 -

0.02

0.5

1.0 Minutes

1.5

Fig 1. Extraction of [3H]-water or [‘%]-sucrose at 18, 30, 80, and 90 seconds following rapid portal injection. Data are mean k SE (n = 4 to 8 rats per point). The intercept and slope were determined using a linear regression analysis and the method of least squares (Methods). Only the extraction data up to and including 1 .O minutes were used in the regression analysis, since recirculation of label (dashed line) was prominent after this time.

Table 1. Hapatic Transport K* (mini’)

Compound

Parameters

E(O)’

“,t (mL . g- ‘)

Water

2.19 + 0.25

1.14 * 0.20

L-lactate

1.15 + 0.19

0.94

D-lactate

2.07

+ 0.17

0.92

+ 0.09

0.71

r 0.07

Sucrose

1.15 k 0.34

0.16

+ 0.04

0.22

+ 0.07

k 0.10

1.31 + 0.22

*Determined from linear regression analyses of data in Figs 1 and 2 as described in Methods. tDetermined

from E(O) and K values and portal blood flow (F) =

1.93 + 0.22 mL/min . g as described in Methods.

extraction data were analyzed by linear regression analysis (Methods) to yield a slope and intercept of the curves between 0.3 and 1.0 minutes. These data were used to compute the K and E(0) values, which are listed in Table 1. Similarly, the rates of efflux of [‘4C]-L-lactate or [‘*cl-~lactate from rat liver back to blood were computed from the data in Fig 2, and the K and E(0) values calculated from these data are listed in Table 1. The data show that the extraction of unidirectional influx of L- and D-lactate by rat liver in vivo is 94% * 10% and 92% + 9%, respectively, indicating both compounds are nearly completely cleared by liver on a single passage. The rate constant of [‘4C]-D-kICtatC washout (2.07 + 0.17 min-‘) is not significantly different from the rate constant of [3H]-water washout, 2.19 k 0.25 min-’ (Table 1). Using E(0) = 1.0 for [3H]-water and V’ = 0.88 mL/g, the rate of portal blood flow under the present conditions is 1.93 + 0.22 ml/min . g (Methods). The V, values for L- and D-lactate were calculated (Methods) and are shown in Table 1. Studies were also performed to assess the saturability of L-lactate transport. The 18-second extraction of [14C]-~lactate (corrected for the corresponding extraction of [‘“Clsucrose) was 61% + 2%, 54% * 3%, 58% + 3%, 47% 2 2%, 42% * 3%, and 34% * 2%, respectively, at 0.01, 5, 10, 50, 100, and 200 mmol/L L-lactate added to the injection solution (mean + SE, n = 3 rats per point). DISCUSSION

The present studies describe the kinetics of lactate influx and efIIux across the liver cell membrane using a portal vein rats. The injection technique6q7 in ketamine-anesthetized volume of distribution of [“Cl-sucrose determined in the present studies (0.22 f 0.07 mL/g) is not significantly different from the sucrose distribution volume determined with the indicator dilution technique in liver of pentobarbital anesthetized rats (0.27 mL/g).’ Similarly, the rate of portal blood flow determined under the present conditions (1.93 * 0.22 mL/min/g) is comparable to rates of portal blood flow determined in liver of pentobarbital anesthetized rats using other techniques.” The volume of distribution of D-k3Ctate (0.71 + 0.07 mL/g) is not significantly different from the water volume determined in rat liver with the indicator dilution technique (0.69 mL/g): suggesting that this compound is relatively nonmetabolizeable in rat liver during short incubation times.3.5 The high apparent volume of distribution of L-lactate (1.3 1 k 0.22 mL/g) reflects the

LUPO. CEFALU, AND PARORIDGE

376

l.Or

n

(14C)-L- LACTATE EFFLUX

0.8

I = 0.99

slope

intercept

= 1.152 0.19min

= 0.94

+ 0.10

r = 0.99 slope = 2.07 ? 0.17 min-’ [ intercept = 0.92 4 0.09

. +$5Tk-

0.75

Minutes Fig 2. The extraction (on the y-axis) of [“Cl-L-lactate and [‘k]-o-lactate by rat liver is shown at 18.30. and 45 seconds after rapid portal injection. The slope and intercept of the monoexponential curves were determined by analyzing the data with a linear regression analysis (Methods). Data are mean + SE (n = 4 to 8 rats at each point).

lower rate of washout of L-lactate from rat liver back to blood, and this is attributed to sequestration of metabolites rapidly formed from L-lactate once it distributes into the liver intracellular compartment. It is unlikely that there is signifi-

cant washout of L-lactate metabolites during the 45 second period following portal injection, since the L-lactate apparent volume of distribution exceeds the D-kiCtate or water space (Table 1). The conversion of [‘“Cl-lactate to other [‘“Clmetabolites, and the differential washout of [‘4C]-lactate and [‘4C]-metabolites, is reflected in the high apparent volume of distribution for L-lactate. However, the metabolism of lactate subsequent to membrane transport does not influence the estimate of membrane permeability reflected in the E(0) parameter (Table 1). Both L- and D-h&de are rapidly cleared on a single passage by rat liver, since the E values for both compounds (94% 2 10% and 92% i 9%, respectively) are not significantly different from 100%. Therefore, the clearance of either L- or D-1Xtate by rat liver is essentially limited by portal blood flow. Stereospecific differences in D- and Llactate transport may exist in vitro,3.4 but because both are transported so fast, the differences cannot be detected in vivo. In this regard, it is of interest that marked stereospecific differences in D- and L-glucose are measurable in vivo using the present technique.6 The rates of lactate influx (J) into liver may be calculated (Methods) given E = 0.91 (Methods and data in Table 1), F = 1.93 (Results), and C, = 1 mmol/L, ie. the normal portal concentration of lactic acid.‘This calculation of transport rates assumes that lactate transport is nonsaturable in vivo in the 1 to 5 mmol/L range. The lack of transport saturability in this range has been shown previously in the perfused 1iver5,” and is confirmed by the present studies (Results). Given the measured parameters, the calculated influx = (0.91)(1.93)(1.0) = 1.8 pmol e min ’ . g-‘. Thus, the rate of lactate transport into liver in vivo correlates well with the transport rate in isolated liver cells, 1.6 pmol m&’ . g-‘,4 and approximates the rate of lactate metabolism by liver, 1.0 pmol . min-’ . g-‘.4 These considerations sug gest that, under normal conditions, lactate clearance by liver is not limited by membrane transport. However, if transport were decreased by 50%, then transport could be rate-limiting as postulated by Cohen and Woods.’ Since lactate extraction by liver is greater than 90%, the most common cause of decreased lactate delivery to liver would be a decrease in portal blood flow. Indeed, reduction in portal blood flow has been shown to limit lactate uptake in the perfused rat 1iver.12 A similar phenomenon may occur in humans and may explain why lactic acidosis is augmented by conditions of poor hepatic perfusion.‘.’

ACKNOWLEDGMENT

Dawn Brown skillfully prepared

the manuscript.

REFERENCES 1. Kreisberg

RA: Lactate homeostasis and lactic acidosis. Ann 1980 2. Cohen RD, Woods HF: Lactic acidosis revisited. Diabetes 32:181-191, 1983 3. Edlund CL, Halestrap AP: The kinetics of transport of lactate and pyruvate into rat hepatocytes. Biochem J 249: 117- 126, 1988 4. Fafournoux P, Demigne C, Remesy C: Carrier-mediated uptake of lactate in rat hepatocytes. J Biol Chem 260:292-299, 1985 Intern Med 92:227-237,

5. Schwab AJ, Bracht A, Scholz R: Transport of D-hAate in perfused rat liver. Eur J Biochem 102537-547, 1979 6. Pardridge WM, Jefferson LS: Liver uptake of amino acids and carbohydrates during a single circulatory passage. Am J Physiol 228:1155-1161, 1975 7. Pardridge WM: Unidirectional influx of glutamine and other neutral amino acids into the liver of the fed and fasted rat in vivo. Am J Physiol232:E492-E496, 1977

LACTATE TRANSPORT IN LIVER

8. Johnson JA, Wilson TA: A model for capillary exchange. Am J Physiol 210:1299-1303, 1966 9. Varin F, Huet P-M: Hepatic microcirculation in the perfused cirrhoticrat liver. J Clin Invest 76:1904-19i2, 1985 10. Ossenberg F-W, Denis P, Benhamou J-P: Hepatic blood flow in the rat: Effect of portacaval shunt. J Appl Physiol 37:806-808, 1974 Il. Schwab AJ, Zwiebel FM, Bracht A, et al: Transport and

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metabolism of L-lactate in perfused rat liver studied by multiple pulse labelling, in Yudilevich DL, Mann GE (eds): Carrier Mediated Transport of Solutes From Blood to Tissue. London, England, Longman, 1985, pp 339-344 12. Iles RA, Baron PG, Cohen RD: The effect of reduction of perfusion rate on lactate and oxygen uptake, glucose output and energy supply in the isolated perfused liver of starved rats. Biochem J 184:635-642, 1979