The transport of ketone bodies into the brain of the rat (in vivo)

Journal of the Neurologwal Scwnces, 1977, 34 1-13 © Elsewer Scientific Pubhshmg Company, Amsterdam - Printed m The Netherlands

1

T H E T R A N S P O R T OF K E T O N E BODIES I N T O THE B R A I N OF T H E R A T (IN VIVO)

P M DANIEL*, E R LOVE, S R MOORHOUSE and O E PRATT Department of Neuropathologv, Institute of Psychiatry, De Cresptgny Park, London SE5 8AF (Great Brttam)

(Received 22 March, 1977)

SUMMARY The influx of ketone bodies (3-hydroxybutyrate and acetoacetate) into the brain was higher in rats fasted for 3 to 4 days than in normally fed animals This increase in influx was due only in part to the increase in level of the ketone bodies in the blood of the fasted animals, since, when in normally fed rats, the concentration of ketone bodies in the blood was raised to similar levels by injection, the influx into the brain was less than that found at the end of the fast Influx was high In suckling animals It decreased steadily through the first 16 weeks ofhfe This fall in influx with increasing age was greater than could be explained by the decrease in concentration of ketone bodies in the blood over this period There was a difference between the influx of the i~- and L-isomers of 3-hydroxybutyrate, 1 e the transport mechanism showed stereospeclfiClty The influx of each ketone body into the brain of the adult rat was far greater than it would have been if diffusion alone was responsible for entry The results show that prolonged ketonaemla, as in the new-born or during fastlng, Increases the activity of the transport process which carries ketone bodies into the brain The findings can only be explained if a carrier-mediated transport process is taking the ketone bodies into the brain, even though any appreciable degree of saturation of thts process cannot easily be achieved

The authors are grateful to the Research Fund of the Bethlem Royal and Maudsley Hospitals the Welicome Trust and the National Fund for Research into Cnpphng Diseases for support * Present address Department of Apphed Physiologyand Surgical Science, Institute of Basic Medical Sciences, Royal College of Surgeons, Lincoln's Inn Fields, London WC2A 3PN

INTRODUCTION There is now a substantial body of evidence showing that the ketone bodle~, D(-)3-hydroxybutyrate and acetoacetate, can be utilized by the brains of young or of fasted ammals, not only as sources of energy addlnonal to glucose (Owen, Morgan, Kemp, Sulhvan, Herrera and Cahlll 1967, Hawkins, Wflhamson and Krebs 1971, Van den Berg 1971, Ruderman, Ross, Berger and G o o d m a n 1974) but also, in young animals, as precursors of cerebral hplds (Edmond 1974, Patel and Owen 1976) Despite this evidence of the importance of ketone bodies m cerebral metabohsm, surprisingly little is known about the mechanisms by which these substances enter the brain It has been shown that the rate at which the brain uses ketone bodies l~ largely determined by the rate of their movement into the brain (Darnel, Love, Moorhouse, Pratt and Wilson 1971, 1972), whilst their rate of transport into the brain IS high in starvation (Gjedde and Crone 1975) and m early life (Cremer, Braun and Oldendorf 1976) N o quantltanve data are available, however, on the influx of ketone bodies into the brains of young or of fasted adult animals, although Hawkins et al (1971) have shown that m both cases the rate of ketone body unhzatlon is h~gh In the present paper, the reflux of ketone bodies into the brain of the rat has been measured m wvo, using a technique by means of which measurements have been made prewously of the influx either of glucose or of amino acids (Bachelard, Darnel, Love and Pratt 1973, Daniel, Love and Pratt 1975b, Bafios, Darnel, Moorhouse and Pratt 1973, 1975) Thus, the way m which the reflux of the ketone bodies into the brain changes during the first few months of hfe has been determined In addmon, the effect of raised concentrations of the ketone bod,es m the orculatmg blood upon the influx has been lnvesngated METHODS W~star rats of e~ther sex were used They were weaned at the end of the third week after birth and then were fed without restriction (unless otherwise specified) upon a commercial, pelleted diet (Oxold Breeding) which contained 20 4 ~ of protein and 48 8 ~o of carbohydrate Water was given ad hbltum In some experiments food was withheld for 3 to 4 days

lnjectton techntque The rats were anaesthenzed either with sodmm pentobarbltone B P C (35 mg kg -1 1 p ) or with ether A catheter was inserted into a femoral artery so that arterml blood samples could be taken and one or more catheters were put into a femoral vein, so that contmuous lnjecnons could be given Schedules for the continuous rejection of ketone bodies at a controlled variable rate were devised as follows The ketone body, radioactively labelled, was rejected rapidly by means of a synnge and needle, by hand, into a femoral veto Senal arterial blood samples were taken, so that the rate at which the radioactivity left the clrculanon could be measured (Fig 1A) From this curve, the rate at whmh the ketone body had to be rejected m order to replace its loss from the

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Fig 1 A a rapid i n t r a v e n o u s m j e c t m n o f [14C]acetoacetate was given at zero time a n d a series o f arterml blood samples were t a k e n over the next 30 m m to s h o w the rate at which radloactwlty left the o r c u l a t m g blood A curve h a s been fitted to the data by the m e t h o d o f least squares B to s h o w h o w a predetermined level o f [laC]acetoacetate could be obtained rapidly a n d m a i n t a i n e d steaddy m the o r c u l a t m g blood T h e c o n t i n u o u s i n t r a v e n o u s mject~on was started at zero t~me

circulation was calculated as described in Daniel, Donaldson and Pratt (1974a, 1975a) The programme which had been determined in this way was used to control the rate of injection, which was given by an electronically-controlled, mechanicallydriven syringe (Pratt 1974) In this way a steady level of one or more ketone bodies, either radloactwely labelled or unlabelled, could be maintained in the circulation (Fig 1B) In the present experiments, steady levels of either [14C]hydroxybutyrate or [14C]acetoacetate were maintained in the circulation, sometimes in combination wtth steady raised levels of the corresponding non-radioactive ketone body Whenever the term "inJection" is used hereafter, it means that a steady level of the injected substance is maintained in the circulating blood plasma by means of an 1 v injection at a controlled rate

Measurement of the mflux of ketone bodies mto the bram Preliminary experiments were carried out on the accumulation of each labelled ketone body in the brain, in order to determine how long it would be before the efflux of the radioactive tracer back from the brain into the circulating blood became appreciable In successive experiments, a steady level of radioactively labelled ketone body was maintained in the circulation for progressively longer periods and the radioactivity that accumulated in the brain was measured (Fig 2) It was clear from the results of these experiments that efflux from the brain back into the blood was not appreciable until about 5 or 6 min after the start of the tracer inJection Therefore, in further experiments, the radioactively labelled tracer was maintained in the circulation for periods which were not longer than 3 mln, so that influx could be measured during the period in which efflux was negligible During this injection 3 samples of arterial blood were taken so that the levels of radioactivity and of ketone bodies in the plasma could be estimated At the end of the injection, the vascular system was washed out for 20 sec with saline under pressure This procedure (Daniel, Love, Moorhouse, Pratt

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Fig 2 A the way m which radmactlvlty accumulates m the brain when a steady level of [t4C]hydroxybutyrate is maintained m the c~rculatmg blood The ratm, Rb/Rp, is the radmaet~vlty g-1 of bram at the end of the experiment, Rb, compared w~th the average radmaetlvlty m1-1 m the blood plasma, Rp, during the exl~nment The points with bars represent the means of 8 or more experiments, the bars showing the standard error of the mean Other points represent the results of single expenments B similar accumulatmn of [14C]acetoacetate and Wdson 1974b) removed more than 98 ~o of the blood from the brain (Darnel et al 1975b) The brain was then rapidly excised, frozen m hexane, cooled to - - 7 8 °C in sohd carbon dioxide and stored at - - 2 0 °C until assayed for ra&oactlvlty The reflux, v, was calculated from Rb, the ra&oaetlvlty found m the brain at the end of the experiment, Rp, the mean ra&oactlvlty in the blood plasma, during the period of the mjectmn, t, the duration of the mjecUon and s, the mean concentrataon of the ketone body m the blood plasma according to the following equation. v=--

Rh t

s ×-Ro

(I)

In this calculation we have taken the specific activity of the ketone body m the blood plasma to be Rp/s, and the possible conversion of some of the label to the other ketone body has been neglected I f any exchange of label &d take place during our expenments, the error introduce d would not be large, as the influx of 3-hydroxybutyrate was not apprecmbly different from that of aeetoacetate (F~g 4) It ~s not necessary to make any assumptmn about the blood flow through the b r a m The levels of the ketone bo&es m the blood were rinsed either by withholding food for 3--4 days or by a programmed i v mjectmn of the Na salt of the ketone body This mjectmn was started 1 m m before the lnjectmn of the radioactively labelled ketone body and continued throughout the experiment

Analytwal procedures Plasma was separated rapidly from cooled, hepannized blood The concentrations of D-3-hydroxybutyrate and of acetoacetate were measured m the plasma samples by the method of Wdliamson, Mellanby and Krebs (1962) Samples of plasma and of brain were assayed for ra&oactlvtty as follows To a welghed sample (40-60 rag) of brain was added 2 ml of a solution of an orgamc base (Soluene X-100, Packard In-

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Fig 3 The effect ot maintaining, by intravenous rejection, a steady raised level of 3-hydroxybutyrate m the clrculat~on upon the raho of acetoacetate to 3-hydroxybutyrate m the blood Raised levels of 3-hydroxybutyrate 0 6 mM (© ©), 30 mM (O O) The ratio before the rejection was started (&) The rejection was started at zero time struments) When the tissue had dissolved (usually 1-2 days) the mixture was neutrahsed with glacial acetic acid (0 3 ml) and 15 ml of a scintillation mixture, containing 5 g of 2,5-dlphenyloxazole and 0 3 g of 1,4-bis (2-(4-methyl-5-phenyloxazolyl)) benzene were added The radioactivity in the samples was measured in an automatic scintillation spectrophotometer (Model 2450, Packard Instruments) The results were corrected for quenching, if needed, by the channels ratio method

Matertals [3-14C]ethylacetoacetate, [3-14C]0(--) - and [3-14C]oL-3-hydroxybutyrate were obtamed from the Ra&ochemlcal Centre, Amersham, Bucks [3-14C]acetoacetate was prepared by hydrolysis of its ethyl ester with 2 N N a O H (Krebs, Hems, Weidemann and Speake 1966) D-3-Hydroxybutyrate dehydrogenase (EC 1 1 1 30) was purchased from the Boehringer Corporation L t d , Lewes, East Sussex and ethyl acetoacetate and DL-3-hydroxybutyrate (Na salt) were obtained from the Sigma Chemical Co L t d , Kingston upon Thames, Surrey RESULTS

The relattonshtp between the concentratton of a ketone body m the ctrculatmg blood and tts mflux mto the bram When a radioactively labelled ketone body (e g [14C]3-hydroxybutyrate) IS injected into the circulation, part of the label may be rapidly transferred to the other, unlabelled ketone body (acetoacetate) which is normally present in the blood (Barton 1973, Cremer and Heath 1974) In order to mitigate the effect of this transfer of label on the results of our experiments, we modified the experiments on influx in the followlng way When studying the influx of radioactively labelled 3-hydroxybutyrate we

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Ftg 4 The effect of raising the concentratzon of a ketone body Jn the c~rculatmn on its reflux into the brain A 3-hydroxybutyrate, B acetoacetate Each point represents either a single experiment or the mean of 3 experiments In the latter case the bar shows the standard error of the mean Curves have been fitted to each set of points by eye maintained a steady, raised level of this ketone body, unlabelled, m the clrculatlon for 1 min before, as well as throughout the period over which a tracer inJection of [14C]3-hydroxybutyrate was given in order to measure the influx By raising the concentration of one of the ketone bodies in this way, the ratio in the blood of the concentration of the other ketone body to the injected one, i e acetoacetate/3-hydroxybutyrate was kept low throughout the experiment (Fig 3) The concentration of the second ketone body, acetoacetate, rose slightly above the normal level, but not to anywhere near the level which had been attained by the Injected 3-hydroxybutyrate This disparity m the concentrations of the two ketone bodies made It possible to measure the influx of one of them separately When for example a tracer dose of [14C]3-hydroxybutyrate was injected 1 min after raising the blood level of this ketone body, only a small part of the label would be transferred to the acetoacetate, because of the disparity in the concentrations, which lasted throughout the period needed for the measurement of influx (Fig 3) In a similar way, when the influx of acetoacetate was being measured, a raised level of acetoacetate was maintained in the circulation immediately before and during the period when [14C]acetoacetate was injected When the results of the experiments m which the concentration of 3-hydroxybutyrate in the circulation was raised to successively higher levels, up to 25 mM (Fig 4A), were compared with those in which the concentration of acetoacetate was raised to successively higher levels, up to 40 mM (Fig 4B), there was no significant difference between the slopes of the hnes fitted to each series of points by linear regression or by eye We therefore concluded that the influx of acetoacetate into the brain was not significantly different from that of 3-hydroxybutyrate, when measured with similar concentrations of the ketone body in the blood Slightly curved lines were fitted by eye to each of the sets of points In Figs 4A and B, on the assumption that there may be some slight degree of saturation However, straight lines would give almost as good a fit The ratio of the influx of each ketone

2254-054 (13) 319±114 (8)

00485_0013 (13) 0035±0012 (8)

0021+0002 a (13) 00114-0001 (8)

ratio

1844-019 (5) 415±131 (3)

plasma concentration (raM)

reflux (#mol mm -1 g 1 of brain) plasma concentration (mM)

plasma concentration (mM)

reflux (~mol mm 1 g-1 of brain)

Acetoacetate

D-3-Hydroxybutyrate

0053d-0008 (5) 0052±0018 (3)

reflux (~mol mln -1 g 1 of brain)

a SJgmficantly different from the corresponding value for the fed animals given the ketone body (P < 0 001, t-test) Values d: SEM w~th number of experiments m parentheses

Fedtmjectlonof ketone body

Fasted 3-4 days

Animal

0028+0003 a (5) 0012~0001 (3)

ratio

reflux (/~mol mm -1 g-1 of brain) plasma concentration (mM)

KETONE BODY I N F L U X INTO THE BRAIN IN FASTED ANIMALS A N D IN N O R M A L L Y FED ANIMALS GIVEN A KETONE BODY BY AN INTRAVENOUS INJECTION

TABLE 1

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mM F~g 5 The relation between the influx of 3-hydroxybutyrate into the brain and ~ts concentration m the blood m fasted rats (©) Thirteen rats aged 8-10 weeks (young adults) were fasted for 3-4 days This raised the blood 3-hydroxybutyrate to levels which varied m mdlwdual ammals from 0 3 to 6 mM For comparison the relat|on between influx and blood concentration m normally fed ammals is shown (0) In these animals the concentration of 3-hydroxybutyrate was raJsed above normal by a controlled injection which was started only 1 mm before the measurement of the reflux (data taken from Fig 4A) The best straight hne has been fitted to each set of points by linear regression and the difference m slope between the two hnes is statistically significant (P < 0 005)

body to its concentration in the blood, v/s (as gwen by the slope of the curves m Fig 4), was approxamately 0 01 /~mol mm -1 g-1 of brain per mmole m the blood plasma Influx of ketone bodws into the bram when their level m the blood ts raised by fastmg W i t h h o l d i n g food from adult rats for 3-4 days p r o d u c e d a rise m the b l o o d levels of the ketone bodles which v a n e d in i n d i v i d u a l ammals, from 0 3 to 6 m M for 3-hyd r o x y b u t y r a t e a n d from I to 2 5 m M for acetoacetate The reflux of 3-hydroxybutyrate into the b r a i n of the fasted anamals increased m p r o p o r t i o n to the level of the substance in the b l o o d (Fig 5) F o r b o t h 3-hydroxybutyrate a n d acetoacetate, the ratao of the reflux into the b r a i n to the concentrataon an the b l o o d was sagmficantly higher m the fasted t h a n m the n o r m a l l y fed anamals whach had been rejected wtth the ketone body (Table 1, Fig 5)

TABLE 2 EFFECT OF STEREOISOMERISM UPON THE DISTRIBUTION RATIO OF [zac]3-HYDROXYBUTYRATE BETWEEN THE BRAIN AND THE BLOOD Radioactwe ~somer

Number of radioactivity g-Z of brain Dlstribut~on ratio experiments radioactivity ml-z of plasma

[3-14C]DL-3-hydroxybutyrate [3-x4C]D(-)-3-hydroxybutyrate

6 6

0 046 :[: 0 003 0 064 :k 0 006

The difference m the distribution ratio between the groups is statistically significant (P -< 0 025, t-test) Values :1: SEM

ratio

0 818±0 066 (14) a 0 260-t-0 060 (14)

0 086~-0 016 (13) a 0 005 ± 0 001 (14)

0 105£0 023 (13) a 0 020±0 001 (14)

plasma concentration (raM) 0 561+0001 (3) a 02514-0041 (3)

plasma concentration (raM)

influx (•mol mln -1 g-1 of brain)

plasma concentration (mM)

influx (~mol mln -1 g-1 of brain)

Acetoacetate

D-3-Hydroxybutyrate

Significantly different from the 8-16 week old group (P < 0 005, t-test) Values 2_ SEM with number of experiments in parentheses

8-16

1- 3

Age (weeks)

CHANGE WITH AGE IN THE I N F L U X OF KETONE BODIES INTO THE BRAIN

TABLE 3

0048±0004 (3) a 00047±00004 (3)

influx (,umol mln -1 g 1 of brain)

0 086±0008 (3) a 0018±0002 (3)

ratio

influx(,umolmin-1 g-1 of bram) plasma concentration (mM)

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Fig 6 To show the effect of age upon the reflux of ketone bodies into the bram A 3-hydroxybutyrate, B acetoacetate Each p o n t represents a separate expermaent or the mean of 3-6 experiments In the latter case the bars show the standard error of the mean The arrows ln&cate the age at which ammals were weaned Curves have been fitted to each set of points by eye

The effect of stereotsomertsm upon the dtstrtbutwn of [14C]3-hydroxybutyrate between the brain and the blood A racemic mixture ([lgC]oL-3-hydroxybutyrate) was injected into 6 fasted rats for 3 mln and at the end of this period the blood was washed out of the vascular system and the ratio of the radioactivity g-1 of brain to the radioactiwty m1-1 of plasma was determined In a further series of expenments, the [14C]D(-)isomer of 3-hydroxyhutyrate was injected Instead of the DE-mixture The ratio determined from the results of this series of experiments was significantly higher than that obtained in the prewous series in which the racemic mixture was injected (Table 2) Change m the mflux of ketone bodws mto the brain wlth increasing age The influx of ketone bo&es into the brain was measured in rats, aged from 1 to 16 weeks The influx of each ketone body fell rapidly at about the t~me of weaning (3 weeks) and the fall then continued at a slower rate (Fig. 6) The fall in the influx of each ketone body was significant when the results in suckhng rats were compared wlth those in young adults (Table 3) This reduced influx in the older animals is due, m part only, to a decrease in the concentration of each ketone body m their blood Thus, for each ketone body the ratio of influx into the brain to the concentratmn m the blood plasma was also significantly reduced in the mature compared with that in the suckhng animals (Table 3) DISCUSSION

The changes that we have found in the rates at which the ketone bodies move from the blood into the brain provide some in&cations about the mechamsm of this process It is tempting to assume that 3-hydroxybutyrate and acetoacetate must be transferred from the blood to the brain by a carrier-mediated transport system, comparable with the transport systems for glucose (Crone 1965, Bachelard et al 1973),

ll lactate and pyruvate (Daniel et al 1972, Oldendorf 1973, Gjedde, Andersson and Eklof 1975) and amino acids (Bafios et al 1973, 1975, Darnel, Moorhouse and Pratt 1976) Such carrier-mediated transport systems can normally be saturated by high blood levels of the substance being transported However, there is only hmlted evidence that this occurs for the ketone bodies Gjedde and Crone (1975) tried to find out whether saturation of a transport mechamsm occurred They used a semi-quantitative method in which they measured the uptake of 3-hydroxybutyrate and acetoacetate into the brain from a bolus injected into the carotid artery Both their results and our own (Fig 4) are not conclusive and suggest that, although complete saturation cannot be attained, there is evidence for a slight degree of saturation In addition there is a considerable body of indirect evidence that carrier-mediated processes must be responsible for at least part of the transport of ketone bodies into the brain The activity of a process by which a substance enters the brain from the blood can be determined by calculating the ratio v/s 1 e the influx/the concentration in the blood, when this concentratlon is low This is approximately equal to V/Kt for a process conforming to Michaelis kinetics In the case of a number of substances which enter the brain by diffusion this ratio is less than 2 nmol m m -1 g-1 of brain per mmole in the blood plasma (Bafios et al 1975) In the case of the ketone bodies this ratio is much larger, some l0 nmol min - i g-1 of brain per mmole In the plasma (Fig 4) This is comparable with the ratio for the essential amino acids, such as L-threonlne or L-vahne, which enter the brain by carrier-mediated transport (Bafios et al 1973) In addition the reduction in i n f l u x as the age of the animals Increases is greater than would be expected on the basis of the concomitant fall in the levels of the ketone bodies in the blood with increasing age This is shown in Table 3 by the fall in the ratio v/s (influx/concentration of ketone body In the circulation) There is other evidence for a decline in the activity of a transport system for ketone bodies with age Thus, the work of Moore, Lione, Sugden and Regen (1976) who studied the entry of [14C]3hydroxybutyrate into the brain by an redirect method and found that it was high during suckling and declined after weaning, also supports the view that a carrler-medmted transport system is operating The findings of Cremer et al (1976) show that during suckling there is a high rate of transport not only of ketone bodies, but also of other monocarboxyhc acids during this period The changes in influx seen in fasting, as well as those during early life, also support the view that a carrier-mediated system is operating, for the rise in the influx of ketone bodies into the brain after fasting is greater than would be expected on the basis of the concomitant rise in the ketone body levels in the blood (Table l) Thus, the significant difference in slope between the two lines in Fig 5 means that, for a given level of 3-hydroxybutyrate in the blood, the influx of this ketone body into the brain is significantly higher in fasted than it is m normally fed animals which have artificially raised levels of the ketone body in the blood The explanation of this finding must be that the prolonged high levels of ketone bodies in the blood, which are present during most of the 3 4 days of fasting, have induced an increase in the activity of the system which transports these substances into the brain, in a manner similar to that found by Gjedde and Crone 0975)

12 Our results show that in order to induce an increase m transport act~vity, ketonaemla must be prolonged, since an increase in actlwty was not induced by an intravenous injection of ketone bodies, given only a minute or two before the measurement of influx was made Therefore, it seems likely that it is the prolonged nature of the ketonaemla which lasts throughout the suckling period (Hawkins et al 1971) which causes such a large increase in the influx of the ketone bodies into the brain of these suckling animals (Table 3) It may well be that a prolonged ketonaemla from any cause, for example, that found in diabetic ketoacldOSlS, induces an increase in the activity of the mechanism transporting ketone bodtes into the brain Further evtdence for carrler-medlated transport is provided by the high influx of the D-Isomer of 3-hydroxybutyrate compared with the racemlc mixture At first sight our results (Table 2) appear to suggest that the L-isomer also enters the brain, albeit at less than half the rate of the D-form It is possible, however, that the t-3hydroxybutyrate does not enter the brain itself, but that some of the radioactive label has been transferred to acetoacetate (Barton 1973, Cremer and Heath 1974), which enters the brain readily It is also of interest that the influx of 3-hydroxybutyrate into the brain can be inhlbtted by rinsed levels of lactate in the circulation (Cremer et al 1976) for such an inhibition by a chemically related substance is characteristic of carrler-medmted transport The decline in the influx of ketone bodies into the brain as the age of the rat mcreases (Fig 6) suggests that the organ is beginning to use greater quantities of glucose and to rely less upon ketone bodies as a source of energy In the early postnatal period, fluctuations in the level of blood glucose occur readtly, since the regulatory mechanisms are poorly developed at this time The ability to use ketone bodies must be of considerable value under these circumstances The avoidance of damage to the brain from hypoglycaemla, which is a particular hazard to the premature baby (Anderson, Mllner and Stnch 1966, Strlch 1968) may depend to a large extent upon whether or not an increase in the transport of ketone bodies into the brain can be induced since an increase would provide a substantial alternative supply of energy-yielding substrate in place of glucose ACKNOWLEDGEMENT We are grateful to Mr Nigel Boonham for expert technical assistance

REFERENCES

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