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AMERICAN JOURNAL OF PHYSIOLOGY Vol. 225, No. 3 September 1973. Printed in U.S.A.

Kinetics of unidirectional glucose transport into

the isolated dog brain

A. LORRIS BET& DAVID D. GILBOE, DAVID L. YUDILEVICH, AND LESTER R. DREWES (With the Technical Assistance of James H. Fitzpatrick, Jr.) Departmcn ts of Neurosurgery and Physiology, University of Wisconsin, Madison, Wisconsin 53706; and Department0 Biolugia, Facuhad de Ciencias, Universidad de Chile, Santiago, Chile

BETZ, A. LORRIS, DAVID D. GILBOE, DAVID L. YUDILEVICH, AND LESTER R. DREWES. Kinefics of unidz’rechzal glucose fransport into the isoluted dog brain. Am. J. Physiol. 225(3): 586592* 1973.- An indicator-dilution technique with 22Na as the intravascular marker was used to measure unidirectional transport of D-glucose- 6-3H from blood into the isolated dog brain. The rate of unidirectional glucose transport, v, was calculated from the equation v =

(E - 0.036) A Fp/W, where E is the fractional extraction of glucose from the blood, A is the arterial plasma glucose concentration, Fp/W is the plasma flow rate per unit weight of brain, and 0.036 is a correction for glucose diffusion. The diffusion correction was based on the fractional extraction of D-fructose-6-“H, a hexose that is not transported into brain. The results of experiments in which Fp/W was varied while A was held constant indicate that an increased flow rate results in an increased rate of glucose uptake. Because of the Aow effect, the kinetics of unidirectional glucose transport were studied in six brains in which Fp/W was held constant while A was varied between 4.3 and 60 mM. An apparent Kna for glucose transport of 8.26 =t 1.67 mM and a Vmax of 1.75 + .I 1 pmoles/g of brain per minute were calculated. The kinetics were not significantly altered in the presence of pentobarbital (25 mg/liter) or insulin (4 pg/lOO ml), This technique is generally applicable to any isolated, perfused tissue.

indicator-dilution technique; unidirectional extraction; brain fructose diffusion; blood flow rate; pentobarbital; insulin

THERE IS CONSIDERABLE EVIDENCE that the transfer of glucose from blood to brain occurs by means of a transport mechanism (4, 7, 8, 13, 14, 17, 18, 24, 28, 36, 45). A direct demonstration of this point was made by Crone ( 13), Yudilevich (45), and Cutler and Sipe (14) with the single- pass indicator-dilution technique. The rapid time course of the indicator-dilution experiment permits measurement of unidirectional uptake; thus it is theoretically possible to determine the kinetics of transport into the brain. The in- ability to measure and control the blood flow rate has not allowed this determination in in situ preparations (13, 14, 45).

Other techniques that have been used for the analysis of brain glucose transport in vivo are based on either i) determinations of flow rate and glucose arteriovenous differ- ence in the steady state (21, Setchell and Pappenheimer: personal communication), G) measurement of labeled

glucose uptake during a constant arterial infusion (4), or iii> uptake of nonmetabolizable sugars (8, 24).

We have used the indicator-dilution technique to determine the Km and Vmax for glucose transport into the isolated dog brain. Since the blood flow rate can readily be determined, the isolated brain preparation offers an excel- lent vehicle for the direct measurement of transport kinetics. Other advantages include the lack of recirculation of isotope and the ability to vary the composition of the blood perfusing the brain. The effects of sodium pentobarbital and insulin on cerebral glucose transport were also evalu- ated. Part of this work was presented in a preliminary communication (5).

MATERIALS AND METHODS

Brain isolation. Brains were isolated from 14 adult mon- grel dogs that were anesthetized with halothane (22). Arterial blood was supplied through the internal carotid arteries and the anastomotic branch of the internal maxillary arteries. Venous blood was collected through a threaded Luer connector cemented over a small hole drilled into the bone covering the confluence of sinuses. The perfusate consisted of compatible donor blood that had been diluted with dextran to a hematocrit of about 22 % and conditioned1

(20) In’dicator-dilution injections. The perfusion system consisted

of two separate pump-oxygenator combinations intercon- nected through a valve that permitted perfusion from only one oxygenator at a time (20). One system (containing 4.5- 7.0 mM glucose) was used to maintain normal brain glucose levels between indicator-dilution injections. The glucose concentration of the other system was increased stepwise to allow uptake determinations over a range of blood glucose concentrations. Blood with the lower glucose concentrations was obtained from dogs that were starved 24 hr prior to the experiment.

l Each donor was given sufficient 67, dextran to reduce the hematocrit to approximately X$& After the dog was anticoagulated with heparin, blood was pumped from the femoral artery through a 4 X 8 cm Dacron-wool filter and returned to the contralateral femoral vein. This conditioning process continues for 3-4 hr in order to re-equili- brate the diluted plasma with the low molecular weight compounds in the tissues and remove platelet aggregates, fibrin particles, and other debris.

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KINETICS OF GLUCOSE TRANSPORT INTO BRAIN 587

The 50-~1 injectate contained 2 PC of 22Na and 10 PC of D-glucose-6-3H (both obtained from New England Nu- clear Corp.) diluted with isotonic saline. Twenty seconds before each indicator-dilution injection, the valve was switched to start brain perfusion at the experimental glucose concentration. A 50-~1 syringe fitted with a 25-gauge needle was used for the rapid (less than 0.5 XC) injection of isotope. The injections were made directly into the blood stream through a rubber injection port located about 4 cm before the bifurcation of the common carotid artery into the internal and external carotid arteries. Sampling started 3 set after the injection. Thirty consecutive venous blood samples were collected at I-set intervals with a manually operated collection device (2). Immediately after the 30th sample, arterial and venous blood samples were taken for glucose analysis. The blood flow rate was determined by measuring the volume of a l-min collection of venous blood. This blood washed much of the remaining 3H from the brain; thus it was not returned to the oxygenator. The valve was then switched and the brain blood at a normal glucose concen tration

was for

perfused with 12 min before

the next injection was made. During this period of re- equilibration, the glucose concentration of the experimental oxygenator was increased. In this manner, 8-12 injections could be Although

made with a single the background in

isolated creased

brain preparation. slightly, the factor

limiting the number of injections was the supply of blood. Ex/m-imental groups. Control data were obtained from six

brains, In a second group of three brains, indicator-dilution injections were made 20 set after the start of perfusion with blood containing sodium pentobarbital at a concentration of 25 mg/liter (0.03 m$. Between pulses, however, these brains were perfused with normal blood. For the three brains in the third group, insulin (4 lug/100 ml, Iletin, Lilly) was added to both control and experimental blood at least 15 min before the first indicator-dilution injection was made.

In an analogous series of experiments performed on four brains, the indicator-dilution injectate contained 10 PC of D-fructose-6-3H (Amersham/Searle Corporation) and 2 PC of *?Na. The fructose concentration of the experimental blood was varied between 0.2 and 6.3 IIIM, while its glucose concentration was maintained at 5.7 mM 41 0.4 SE.

To evaluate the effect of plasma flow rate on unidirectional glucose uptake, 12 successive indicator-dilution injections were made in each of two dog brains during a period when the glucose concentration was held constant and the plasma flow rate was varied between 0.26 and 0.74 ml/g of brain per minute. Glucose was maintained between 5.7 Bnd 6.6 rn~ by the slow infusion of a glucose solution into the oxygenator. All injections were made 1 min after the flow rate was changed from the maintenance rate (0,50 ml/g per min) to the experimental rate.

Assay ;brocedures. Each sample was prepared for scintillation counting as follows (3 1). After adding 50 ~1 of whole blood to a scintillation vial containing 0.1 ml of 60 % per- chloric acid, the mixture was shaken vigorously with a Vortex mixer; 0.2 ml of 30 % hydrogen peroxide were added, and the vials were tightly capped, shaken, and dlaced in a 60-70 C oven for 1 hr to complete digestion and decolorization. Fifteen milliliters of scintillation fluid (5 g PPO, 520 ml toluene, 420 ml methyl Cellosolve, and 60

ml absolute ethanol) were added to each cooled vial. 3H and 22Na were counted simultaneously in a Packard Tri- Carb liquid scintillation counter by the exclusion (screening) method (35). The plasma arterial and venous glucose concentrations were determined in triplicate by a glucose oxi- dase method, with a Beckman glucose analyzer. In those experiments in which fructose was added to the blood, arterial and venous fructose concentrations were determined enzymatically with a spectrofluorometric method

(29) C&u&ions and statistical analysis. The data treatment was

similar to the method of Yudilevich et al. (45) The indicator-dilution calculations and plots were made with use of a program developed for the Wang 7OOB programable cal- culator with a printer-plotter attachment. C(t) and c(t) are the concentration of 22Na and 3H in each sample relative to their concentrations in the injectate (Fig. 1A). The fractional extraction of glucose for each sample, E(t), was calculated from the equation E(t) = 1 - c(t)/C(t). The plot of E(t) versus the percent recovery of the total injected 22Na (Fig. 1B) was used to determine the maximal fractional extraction of glucose, E. This point usually occurred near the peak of the indicator-dilution injection profile (Fig. IA).

The rate of glucose uptake, u, was calculated from the equation u = EAFp/W, where A is the arterial plasma glucose concentration, Fp is the plasma flow rate, and W is the brain weight. On the basis of experiments with fructose- 3H 3 a hexose that is thought to enter the brain only by simple diffusion (12, 13), the rate of glucose diffusion was estimated to be 3.6 % of uptake. The rate of unidirectional glucose transport into the brain, v, was calculated by in- corporating this diffusion correction into the glucose uptake equation, i.e., v = (E - 0.036) A Fp/W.

Values for glucose transport from each experimental group were fitted to the Michaelis-Menten equation

v A nzax

U=K,

where A, the average of the arterial and venous glucose concentrations, is used as an approximation of the average glucose concentration in the capillary. The computer program that was used (11) utilizes an iterative, least-squares method to fit this nonlinear equation directly and calcu- lates Km, Lax, and their respective standard errors (SE). This method was chosen because it is applicable to a variety of kinetic equations and permits the use of weighting factors during the fitting procedure. A weighted fit is necessary when the standard deviation (SD) of u is proportional to v itself (1 l), as was the case with our data. If it is assumed that ;) the SD of E is 0.014 (i.e., the SD of E for fructose), ii) the SD for glucose (A) is proportional to the arterial glucose concentration and is 0.013 rnM when A is 1.0 mM, and Z) the SD for the flow rate is 0.01 ml/g per min, then the weighting factor is inversely proportional to ~~(0.0006 + (0.014/E)2. Th us when v is high and/or E is low, the data are less important for the fit.

RESULTS

A typical indicator-dilution profile is shown in Fig* IA. The convergence of the two recoverv curves indicates that A

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BET& GILBOE, YUDILEVICH, AND DREWES 588

some of the 3H that entered the brain during this injection is returning to the blood. It is important to determine the unidirectional extraction of glucose before this backdiffusion of 3H is observed (46). Consequently, the maximal value of E(t) (Fig. 1B) was selected as the fractional extraction of glucose, E, for this injection.

The results of the experiments in which flow rate was varied while glucose concentration was held constant (A = 622 mM =t 24 SD) are seen in Fig. 2. Because of the increase in glucose uptake as the flow rate increased, all the experiments on glucose transport and fructose diffusion were performed at a constant plasma flow rate. The range 0.43- 0,53 ml/g of brain per minute was selected because it pro- vides proper oxygenation at a perfusion pressure of 80-120 mm Hg.

Figure 3 shows the results of experiments in which the fractional extraction of fructose was determined over a range of arterial fructose concentrations. E did not change as the fructose concentration increased. Therefore, we con- cluded that fructose enters the brain only by simple dif-

0 c (tHZ2No)

l c w(3H-Gl”cose)

"I 1 IlI~I~lIIII~~IIiIII11Il~I1~I~~ 246 8 IO 12 I4 16 18 20 22 24 26 28

the (S8C)

z 0.40-1 8 rJ 5 * * z o.zo- /

: - E s

o-------------------

2 5 -ozo- F z E -0.40 I 1 I 1 1 I 1 I 1 I

IO 20 30 40 50 60 70 80 90 IO0

PERCENT RECOVERY OF INJECTED 22Na

I I I 1 I I I I I I I I I I I 1 I I I I

5 6 7 8 9 IO il 12 14 I6 2030

TIME (set)

FIG. 1, A: typical dilution curves obtained after simultaneous arterial injection of 22Na and glucoseJH. C(t) and c(t) are isotope concentrations in venous blood relative to their concentrations in injectate. 23: plot of fractional extraction of glucose vs. total area under 22Na curve of A.

0.4

l l

*a a a l

: a

a -

I I I I I 0.2 0.4 0.6 0.8

plasma flow rate (ml /g / min)

FIG. 2. Plot of rate of unidirectional glucose uptake vs. plasma flow rate when A was held constant.

LX 1 I ’ ’ ’ ’ ’ ’ w IO 20 3.0 4.0 5.0 60 7.0

ARTERIAL FRUCTOSE CONCENTRATION. (mM)

FIG. 3. Plot of fractional extraction of fructose vs. arterial fructose concentration.

fusion. The average E for fructose was 0.036 & ,003 SJS

( 12 E 17) A plot bf the fractional extraction of glucose versus arterial

glucose concentration for the control experiments is shown in Fig. 4m The decrease in E as A increases implies that glucose is transported into the brain. Glucose also enters the brain by simple diffusion, probably at a rate very similar to the rate of fructose diffusion. The rate of glucose transport was therefore calculated after subtracting the average E for fructose uptake from the observed E for glucose uptake. The rate of glucose transport versus average capillary glucose concentration is shown in Fig. 5 for the control experiments. An apparent K, of 8.26 rnM and a V,,, of 1 l 75 pmoles/g of brain per minute (Table 1) were calculated.

Similar plots for the experiments in the presence of pentobarbital and insulin are shown in Figs. 6 and 7, respectively. I f it is assumed that neither pentobarbital nor insulin had an effect on the rate of simple diffusion of glucose, then the K m and Vrllax values for these two groups (Table 1) appear to be comparable to the corresponding control values. This observation was confirmed by a f test; however, the possibility of a slight effect is difficult to exclude on the basis of t tests alone (11).

DISCUSSION

The indicator-dilution technique used in these experiments is similar to the methods used by others (12-14, 26, 45, 46). As previously suggested (45), Wa was chosen as the reference material because most evidence indicates that it is confined to very nearly the same space as other intravascular indicators (26, 45). Its size eliminates the problem of separation of reference and test materials that may result from laminar blood flow (Taylor effect) when large molecules such as hemoglobin-Wr (45) or Evans blue-labeled albumin (26) are used as intravascular indicators. Furthermore, 2z-Ya emits beta particles of much

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KINETICS OF GLUCOSE TRANSPORT INTO BRAIN 589

1 I I I I I I I I

IO 20 A%1

40 500

FIG. 4. Plot of fraCtiOna! extraction of glucose vs. arterial glucose

concentration for control experiments. FIG. 6. Plot of rate of unidirectional glucose transport vs. average capillary glucose concentration after sodium pentobarbital (25 mg/

I PENTOBARBITAL

liter) was added to blood. Curve is rectangular hyperbola obtained CONTROL by a direct fit to experimental values of u and A.

l

K I I I I I Ii 1 I i I I I O 20 30 40 50 60

Z ImM) IO 20 3Q A IrnM40

50 60 7c

FIG. 5. Plot of rate of unidirectional glucose transport versus average capillary glucose concentration. Curve is rectangular hyperbola ob- FIG. 7. Plot of rate of unidirectional glucose transport vs. average

tained by a direct fit to experimental values of u and A. capillary glucose concentration in presence of insulin. Curve is rectangular hyperbola obtained by a direct fit to experimental values

TABLE 1. hhetic constants for unidirectional ghcose of v and A.

kansfmrt under various conditions results of indicator-dilution experiments in the stomach (1)

Vmax, WtIolesjg and the brain (14), other authors have suggested that this

?z Km, InM per min rise could represent ;) the presence of heterogeneous capillary beds, ~7) tissue shunting of the test material (i.e.,

Control 44 8.26 zt 1.12 1.75 =t -12 Pentobarbi tal 18 8.60 * .54 I.83 & l 07

rapid diffusion from artery to vein through the tissue), or

1 nsulin 18 8.06 AI 1.45 1.52 dz .12 ii;) the existence of arteriovenous shunts. Cutler and Sipe

(14) state that their use of an average of the E(t) values

K,, ad V,,, are shown with their standard errors. There are no determined on samples collected up to the peak isotope con- apparent differences in K,, or V,,, among the 3 groups. centration is valid as a measure of the fractional extraction

for the tissue as a whole only if the first alternative is cor- rect. Yudilevich et al. (46) h ave recently suggested that the

higher energy than “H, thus permitting accurate simul- maximal E(t) is the true measure of unidirectional flux. taneous counting of both isotopes. Since our pattern of extraction is reminiscent of the pattern

There are several important differences between our observed by Yudilevich et al. (45, 46) for the cortex, the technique and those used previously. Injection volumes of observed increase in extraction is not due to the presence of 0*4-2.0 ml (12-14, 26, 45, 46) may cause transitory in- both gray and white matter in our preparation. We have creases in cerebral blood flow and changes in solute con- noted the most variability at higher perfusion pressure, and centrations which could alter E. In our studies the rapid believe that the increase in E(t) is best explained by the injection of 0.05 ml has no detectable effect on blood pres- presence of A-V shunts. Thus the use of maximal E(t) de- sure or flow rate. Therefore, the E that we measure is valid emphasizes changes due to shunting, There may be several at the observed rate of perfusion. errors in using maximal E(t) as an estimate of the true E

Glucose moves into the erythrocyte as well as the brain for brain as a whole; however, two of the more obvious if the samples are not precipitated immediately after col- errors tend to offset each other. On the one hand, undetected lection; thus it is important that radioactivity be deter- backdiffusion of 3H causes our estimate of E to be too low, mined on whole blood rather than plasma in order to avoid while disregarding areas of the brain that have the lowest observing falsely high E values. The method used to de- extraction causes our estimate of E for whole brain to be colorize the blood for scintillation counting is a modification too high. From our experience with other procedures for of the technique described by Mahin and Lofberg (31). Be- calculating E, the maximal E(t) appears to be the most con- cause of its simplicity, this method allows rapid and accu- sistent and reliable measure of extraction. rate analysis of the many samples required for these studies. In general, kinetic analyses of enzyme-catalyzed reac-

The criteria for selection of an E value from an individual tions are carried out from initial reaction velocities with no injection have varied among investigators. In this study, as product present, Since it was not feasible to eliminate glu- well as in several earlier studies (12, 14, 45, 46), E(t) case from the brain in these experiments, we attempted to frequently increases from the time of isotope appearance to make velocity determinations at a constant brain glucose the peak of the indicator-dilution curve. In discussing the concentration. Changes in cerebral glucose levels were mini-

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590 BETZ, GILBOE, YUDILEVICH, AND DREWES

mized by the short period of time that the brain was in contact with the experimental blood and the 12-min recovery period on control blood. All extraction determinations were made 30-40 set after beginning perfusion with experimental blood.

A dependence of net uptake on cerebral blood flow rate was previously reported for glucose (2 1, 47) and valine (25). Zivin and Snarr (47) used a hyperbola to describe the re- lationship between net glucose uptake and blood flow rate in the rat brain. A definite tendency for unidirectional glucose uptake to increase as flow increases can be seen in Fig. 2; however, we were not able to distinguish between a linear or hyperbolic type of dependence. It is possible that the flow effect is an artifact of the method we use to estimate E, but similar results are obtained when alternate procedures are employed for this calculation. To minimize the effect of flow on our kinetic studies, all measurements were made at a constant plasma flow rate, Since Km and V rnax may also vary with flow, the values we have determined for these parameters are probably valid only over the limited range of flow rates used.

The observation that fructose is not transported into the brain was also made by Crone (12, 13) and Cutler and Sipe (14) who reported E values of 0.041 and 0.017, respectively. The possibility remains that fructose is transported into the brain via a transport mechanism with a very high Km for fructose. However, Oldendorf (36) reported that high concentrations (40 mM) of D-fructose or L-glucose had no effect on the extraction of D-glucose from a 0.42 mM D-glucose solution. In addition, these two hexoses had similar ex- tractions. We have performed experiments with mannitol- 3H and 22Na and calculated an E for mannitol of 0.050 =I= l OlO SD (n = 6). Because of the similarity between E values for fructose and mannitol, we conclude that transport of fructose is unlikely. Although this does not imply a uniform rate of diffusion of these substances into all areas of the brain, it does represent the average rate of diffusion of hexoses into the brain as a whole.

We calculated an apparent Km of 8.26 mM and a V,,, of 1.75 pmoles/g of brain per minute for unidirectional glucose transport under control conditions. Since we are collecting mixed venous blood from the entire organ, these values are the kinetic constants for the brain as a whole. The location of the transport system observed in indicator- dilution experiments has not been determined, but it is probably situated in the capillary endothelium (13, 45, 46). In deriving these kinetic constants, we applied a correction for glucose diffusion based on the diffusion of fructose-3H. The calculation not only corrects for those areas of the brain where diffusion of glucose is possible, but also eliminates errors due to use of 22Na as an intravascular reference. Since it is possible that a small fraction of the z2Na can enter some parts of the brain (26), the E values that we observe may be low because they are relative and not absolute extraction values. Therefore, subtraction of the fractional extraction due to simple diffusion, which was also determined relative to 22Na, eliminates this error in our calculation of the glucose transport rate.

The K, and V,,, for unidirectional glucose transport into the isolated dog brain are, in general, similar to values reported for the ra2: brain (7.12 mM and 1.24 pmoles/g per

min (4) and 7 mM and 1.5 pmoles/g per min (8)), the mouse brain (6 mM and 2.1 pmoles/g per min (24)), and the sheep brain (7 mM and 3.0 pmoles/g per min (Setchell and Pappenheimer : personal communication)). Atkinson and Weiss reported a Km of 10 mM for transport of glucose from blood to cerebrospinal fluid in dogs (3). The Km values reported for glucose transport into other tissues are also similar to the Km that we determined, varying from 6 to 10 mM for the erythrocyte (27, 40, 43), 8.7 mM for heart muscle (34), and 17 mM for liver (44).

Brain glucose levels reportedly increase during barbiturate anesthesia (16, 30, 33), and some investigators have attributed this increase to a facilitation of the glucose transport mechanism (16, 19, 33). The present study demon- strates that pentobarbital is not a direct activator of glucose transport from blood to brain, but it does not exclude a possible effect on transport from interstitial fluid to brain cells. There is also the possibility that pentobarbital has an in- direct effect on glucose transport as a result of its effects on brain metabolism. Such changes would not be detected in our experiments, since glucose uptake was measured about 1 min after the start of perfusion with the pentobarbital- containing blood. Preliminary experiments were conducted on brains during constant exposure to pentobarbital. Al- though the results were not significantly different from the data we have presented, the constant-exposure protocol was abandoned in order to avoid the effects of product inhibi- tion resulting from elevation of brain glucose levels during barbiturate anesthesia. We recently reported a decrease in net uptake of glucose after pentobarbital anesthesia (6). This observation is not inconsistent with the present study, since one would predict increased glucose efflux from the brain when cerebral glucose levels are elevated.

It is generally accepted that brain glucose uptake is un- affected by an elevation of blood insulin levels (8, 13, 15, 20, 37, 41); however, some investigators disagree (9, 23). In the present studv the plasma insulin concentration was about: 1,000 times greater than the normal value reported for the dog (32). N evertheless, there was no apparent increase of glucose flux from blood to brain * The existence of a verv rapid, short-term effect of insulin on gl ucose transport was examined by a series of experiments in which insulin was added to the injectate, but not the blood.2 The Km and VI,,, for this group were not different from the corresponding control values. The possibility remains that transport is maximally stimulated at normal blood insulin levels. Several studies have shown that insulin affects glucose transport when injected intracisternally (10, 42) or when placed in the media bathing brain or spinal cord slices (38, 39). I f insulin does cross the blood-brain barrier, as some studies indicate (32), it could very well have an effect on glucose transport from interstitial fluid into the brain cell. Uptake at this interface is not measured with our technique.

2 These experiments were conducted on four brains in a manner similar to the control experiments except that 50 pg of insulin were added to the radioisotope injectate. This would produce a peak blood insulin concentration that is 10,000 times normal if the dilution curves for 22Na and insulin are identical, The curve that best fit the 20 data points had a K, of 8.46 =t 1.67 HIM and a V,,, of 1.68 & .18 pmoles/g per min.

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KINETICS OF GLUCOSE TRANSPORT INTO BRAIN

Until now, it has been difficult to make direct kinetic measurements of transport processes on any tissue in viva. The techniques described in this study are applicable to

the analysis of transport kinetics of a solute into any tissue for which the circulation can be isolated and the blood flow

rate determined.

The authors are grateful to Miss Kathy Brown, Mr. Paul Conway, Mr. Wilbert Heiman, and Mr. Alton Mitmoen for their assistance.

Preliminary experiments for this study were made during a visit of one of the authors (DL>G) to Santiago, Chile, on a grant from the

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GILBOE, D. D., R. L. ANDREWS, AND G. DARDENNE. Factors affecting glucose uptake by the isolated dog brain. Am. J. Physiol. 219: 767-773, 1970, GILBOE, D. D., AND A. L. BETZ. Kinetics of glucose transport in the isolated dog brain. Am. J. Physiol. 219: 774-778, 1970.

GILBOE, D. D., A. L. BETZ, AND D. A. LANGEBARTEL. A guide for

591

Comision Fulbright. We are also grateful to Dr. Usvaldo Alvarez and Dr. Norina De Rose for their assistance during this stage of the

work. This investigation was supported by the following organizations:

Grants NS0596 1 and 6M01932 from the National Institutes of Health;

University Surgical Associates; Facultad de Medicina (C.I.C. no.

69/70); and Comisi6n Central de Xnvestigacidn Cientifica (nos. 83

and 197), Universidad de Chile.

A. L. Betz is the recipient of a Medical Scientist Training Program

Award.

Received for publication 24 October 19 72. I

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