THE Jonmu~ OF Bmmcxcar, CHEMISTRY

Vol. 245, No. 17, Issue of September 10, pp. 4382-4390, 1970

Printed in U.S.A.

Ketone Body Metabolisrn in the Ketosis of

Starvation and Alloxan Diabetes* (Received for publication, March 25, 1970)

J. DENIS MCGARRY, M. JOANNE GUEST, AND DANIEL W. FOSTERS

From the Departments of Internal Medicine and BiocheuGstry, The University of Texas (Southwestern) Medical School at Dallas, Dallas, Texas 75235

SUMMARY

Concentrations of acetoacetate and P-hydroxybutyrate and their specific radioactivities were shown to be assayable di- rectly in plasma of the rat without the need for prior depro- teinization. By utilizing this technique ketone body turn- over was estimated in the isotopic steady state and after the single injection of radioactive acetoacetate or P-hydroxy- butyrate. While difEculties exist in both types of experi- ments, the turnover rate of total ketones in the rat was shown to be in the range of 8.1 to 13.2 pmoles per min per 100 g of body weight during starvation ketosis with the true rate probably being closer to the lower figure. The turnover rate in acute diabetic ketosis approximated 15 Mmoles per min per 100 g of body weight. The liver was shown to play a central role in interconverting acetoacetate and P-hydroxy- butyrate in the intact rat. In functionally hepatectomized animals the capacity to bring radioactive acetoacetate or /3-hydroxybutyrate into equilibrium was virtually abolished while in diabetic animals this capability was enhanced.

Previous studies of ketone body synthesis by livers from fed, fasted, and diabetic rats have failed to show differences in the rates of acetoacetate formation despite the wide differences in blood levels of ketone bodies that are found ira viva under these three conditions (1, 2). Recently, Bates, Krebs, and William- son (3) reported estimates of ketone body turnover in the intact rat utilizing a single injection of fi-hydroxybutyrate-3-% which indicated turnover rates of 1.7, 4.2, and 10.9 pmoles per min per 100 g of body weight, respectively, in normal, starved, and dia- betic animals. We had previously observed that an isotopic steady state could be easily obtained in the rat by infusion of acetoacetate-3-14C at a constant rate (4). Therefore, it seemed advantageous to study ketone body synthesis by this technique in an attempt to gain further insight into the synthetic rates of ketone bodies in vio. We wish to report the results of these studies which, despite certain difficulties, give new insights into

* This investigation was supported by United States Public Health Service Grant CA 08269.

1 Research Career Development, Awardee 5-K3-AM 9968, United States Public Health Service.

acetoacetate and /?-hydroxybutyrate metabolism in the whole animal.

EXPERIMENTAL PROCEDURE

Animals-White male rats weighing approximately 150 g were used in all studies. The animals were maintained on a balanced diet containing 60% sucrose by weight. In the studies of fast- ing ketosis, food was withdrawn 48 hours prior to the experiment. Diabetes was produced by injection of alloxan as previously de- scribed (1). Diabetic rats were used only if acutely ketotic as de- termined by an immediate strongly positive urine test for ketones (Ketostix, Ames Company, Inc., Billerica, Massachusetts) at 48 hours.

Preparation of Animals and Xample Collection-Each rat was anesthetized with sodium pentobarbital given intraperitoneally. A femoral artery catheter (PE-10) was inserted on the right and a vena caval catheter was placed through the femoral vein on the left as previously described (4). The animals were then put in individual restraining cages and allowed to recover consciousness. Diabetic animals frequently failed to awaken completely, pre- sumably because of severe diabetic ketoacidosis, and were used after a l- to 2-hour postoperative period. Infusions were given via the vena caval catheter while blood samples were collected from the artery. The hydrostatic pressure of the blood is suffi- cient to allow rapid collection of samples directly into small plastic centrifuge tubes’ without requiring a syringe. Samples (0.2 ml) were collected and placed in ice until the entire esperi- ment was complete. Following centrifugation about 100 ~1 of plasma were available for analysis. Control experiments showed no loss of acetoacetate or fl-hydroxybutyrate during the storage period in ice.

For turnover studies a 0.9y0 NaCl solution containing 3.0 pmoles and 50 PCi of sodium D-( -)-P-hydroxybutyrate-3-14C per ml was infused at a rate of 1.2 ml per hour after the raIlid in- jection of 0.2 ml of the same solution as a primer. Preparation of the D-( -)-fl-hydroxybutyrate is described below. In other experiments acetoacetate-3-‘4C of the same concentration was utilized. In single injection experiments the concentration of isotope was doubled and 0.5 ml of solution containing 50 &I (3 pmoles) of either acetoacetate-3-*4C or D-( -)-@-hydroxy- butyrate-3-14C was given over a 30-set period.

Assay of Acetoacetate and fi-Hydroxybutyrate-The methods to be described, while modified slightly, are based on the pro- cedure of Williamson, Mellanby, and Krebs (5). All samples

1 Test tube 314326, Beckman-Spinco.

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were assayed spectrophotometrically at 340 rnp for NADH con- tent after the incubation periods were complete. Appropriate controls, lacking sample, enzyme, or both, were run with each set of determinations. The assay mixture for acetoacetate con- tained, in a final volume of 1 .O ml, 100 pmoles of potassium phos- phate buffer, pH 7.0, 0.2 pmole of NADH, and 0.06 unit of D-( -)-fi-hydroxybutyrate dehydrogenase. In the direct plasma assay 10 to 25 ~1 of sample were utilized. Incubation time was 45 min at room temperature. In the perchloric acid experiments 1.0 ml of plasma was added to I .O ml of 8% (w/v) HClO*. After standing for 10 min in ice the precipitated protein was re- moved by centrifugation and the supernatant was neutralized with 5 N KOH. The solution was again allowed to stand in ice for 10 min before the insoluble potassium perchlorate was re- moved. The supernatant was decanted, adjusted to a vohrme of 4.0 ml, and an aliquot, generally 0.2 ml, was assayed for aceto- acetate content. Similar ex periments were performed with samples deproteinized by the addition of 1 ml of plasma to 4 ml of 6.25% trichloracetic acid. The acid supernatant was neutralized by the addition of 2 M sodium acetate prior to assay under these circumstances.

TABLE I

Formation of LIenighs salt from P-hydroxybutyrate

nn-P-Hydroxybutyrate in the indicated amounts was added to weighed bacterial culture tubes containing H,SOd and HgSO4 as described in the text. After collection and washing of the precipitate the tubes were dried to constant weight in an oven at 100”. Percentage of conversion was calculated on the basis that each micromole of acetone derived from fi-hydroxybutyrate would yield 1.16 mg of Denigcs salt. (6).

m+Hydroxybutyrate added

Weight gfD~~r;igbs salt Conversion

0

/

0

I

200 41.4 17.8 200 42.5 18.3

400 77.8 16.8 400 77.7 16.7

Two systems were used for the determination of fi-hydroxy- butyrate. In the first the reaction mixture contained, in a final volume of 1.0 ml, 100 pmoles of Tris-HCl buffer, pH 8.5, 1.6 pmoles of NAD+, 300 pmoles of hydrazine-HCl, pH 8.5, and 0.06 unit of D-( -)-fl-hydroxybutyrate dehydrogenase. Un- treated and deproteinized plasma samples were processed as indicated for the acetoacetate experiments. Incubation time was 80 min at room temperature. When radioactivity was to be determined on the sample after conversion to acetoacetate, hy- drazine was omitted from the incubation mixture which con- tained, again in a volume of 1 ml, 500 pmoles of Tris-HCl, pH 9.3, 4 pmoles of NAD+, 0.06 unit of enzyme, and 10 to 25 ~1 of plasma.

Determination of Radioactivity Associated with Plasma Ketone Bodies-The principles of the methods outlined here were de- scribed by Bates et al. (3). Plasma (10 to 25 ~1) was taken for the preparation of the Deniges salt of acetoacetate according to the method of Van Slyke (6, 7). The distillation step described by Weichselbaum and Somogyi (8) and used previously in this laboratory (4) was omitted when parallel studies indicated no differences in the recovery of radioactivity in distilled and non- distilled samples from plasma. Accordingly, the sample was added to a 50-ml bacterial culture tube containing 20 ml of dis- tilled water. Nonradioactive acetoacetate (20 pmoles) was present as carrier. Eight milliliters of 18 N HzS04 and 7 ml of 10% HgS04 were added, and the tube was capped and placed in an autoclave for 30 min at 15 pounds of pressure. The acetone- mercury complex was collected by centrifugation, washed, dis- solved in 4 N HCl, and counted as previously described (9). Recovery of isotope in the DenigGs salt from authentic aceto- acetate-3-14C was about 80%.

An additional sample (10 to 25 ~1) was then assayed spectro- photometrically for /3-hydroxybutyrate content utilizing the second incubation mixture described above. When the reaction was complete and all the fl-hydroxybutyrate had been converted into acetoacetate, the entire mixture was washed into a culture tube and converted into the Deniges salt. The recovery of iso- tope in this sample represented the apparent total radioactivity in acetoacetate plus &hydroxybutyrate.

Deniges salt before and after conversion of fl-hydroxybutyrate into acetoacetate should represent the amount of isotope in the former compound. In control experiments, however, it was found that variable amounts of radioactivity from standard D-

(-)-fl-hydroxybutyrate-3-“C were recovered in the mercury- acetone complex prior to conversion into acetoacetate. This was not thought to be due to a radioactive contaminant since gas chromatographic analysis of the ethyl acetoacetate-3-r4C from which both acetoacetate-3-14C and P-hydroxybutyrate-3-14C were prepared showed no detectable radioactive contaminants and since D-( -)-P-hydroxybutyrate-3-r% purified by thin layer chromatography (10) also gave recovery of radioactivity in the Denig& salt. When nonradioactive P-hydroxybutyrate was sub- jected to the experimental conditions utilized in the routine as- say, the findings of the radioactive experiments were confirmed as shown in Table I. In this experiment approximately 17 to 18% of the P-hydroxybutyrate added was recovered in the precipitate, ruling out the possibility that a small radioactive contaminant accounted for the earlier results. As shown in Fig. 1, the extent of the conversion of P-hydroxybutyrate was related to the time of autoclaving, ranging from 4% at 15 min to 32a/, at 3 hours. In contrast, the reaction with acetoacetate was complete at 15 min. We assume that the formation of the Denigi% salt from @-hy- droxybutyrate represents low grade oxidation to acetoacetate in the presence of heat and strong acid, although an oxidizing agent such as potassium dichromate is required for complete conversion chemically (6, 7). Under any circumstance, accurate assessment of specific activities of both acetoacetate and /3-hydroxybutyrate requires correction of the crude data. The extent of conversion with 30 min of autoclaving, as routinely used here, averaged 147c, and this figure was utilized in the calculations, although standard /3-hydroxybutyrate samples gave values of 12 to 16% in various experiments. Actual radioactivity in acetoacetate and fi-hy- droxybutyrate was derived according to the following equa- tions2

2Results from a typical study will serve to underline the im- portance of this correction factor. In an experiment with a starved rat in which D-(-)-o-hydroxybutyrate-3J4C was given by constant infusion the following data were obt,ained for acetoace-

Theoretically the difference in the observed radioactivity in the tate and P-hydroxybutyrate, respectively, in the 60-min plasma sample: concentrations, 159 and 368 pmoles/lOO ml; specific radio-

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4384 Ketone Body Metabolism in Starvation and Diabetes Vol. 245, No. 17

-c

l @-hydroxybutyroie 0 Acetoacetate

iii 80- 3 0-~--~-------0-----0

2 5

I

n 60-f

0 E .- I

I 2 3 Hours

FIG. 1. The effect of the time of autoclaving on the recovery of radioactivity from acetoacetate-3-W or D-(I)-P-hydroxybutyr- ate-3-W in the Deni& salt. ADmoximatelv 200.000 dDm of ac- etoacetate-3-W or g-(-)-p-hydroxybutyrate-3-‘4i: we;e added to culture tubes containing the H&04-HgS04 solution described in the text. Carrier acetoacetate (20 pmoles) was present in each tube. Autoclaving was carried out for the indicated times.

If

2 = actual radioactivity in acetoacetate y = actual radioactivity in P-hydroxybutyrate

Z’ = apparent radioactivity in acetoacetate y’ = apparent radioactivity in P-hydroxybutyrate 2 = z + y = total radioactivity in acetoacet.ate plus p-

hydroxybutyrate 2’ = Z’ + y’ = apparent total radioactivity in acetoacetate

plus P-hydroxybutyrate P = percentage of recovery of fl-hydroxybutyrate in Deniges

salt prior to enzymatic conversion

Then

2’ = 0.8 2 (since 80% of standard acetoacetate-3-W is recovered in the Deniges salt)

And

x’ = 0.8 x + Py

Substituting

2’ = 0.8 x + P (1.25 2’ - x) = 0.8 x + 1.25 P 2’ - Px = x (0.8 - P) + 1.25 PZ’

Taking

P = 0.14, x = 1.52 x’ - 0.27 2’ y=z-x

Preparation of sodium D-( -)-/?-hydroxybutyrate-S-14C-Radio-

activities before correction, 1.78 X lo6 and 1.75 X 106 dpm per pmole. Thus, virtually complete equilibration between the two ketone bodies was indicated. However, after making the appro- priate correction, the true specific activities were found to be 0.90 X lo6 and 2.13 X lo5 dum Der umole. resaectivelv. As men- tioned in the text, the extent of*co&ersidn of P-hydrbxybutyrate into the Deniges salt (P) increases with the length of time of au- toclaving. Since the conditions of autoclaving will vary from one laboratory to another it is recommended that a standard sam- ple of p-hydroxybutyrate-3-W be included with each set of test samples in order to obtain an accurate value for P.

active /3-hydroxybutyrate was prepared enzymatically from acetoacetate-3-W. One millicurie of ethyl acetoacetate (specific activity 16.7 $Zi per pmole) was heated for 1 hour at 37” in 3 ml of 0.1 N NaOH. After neutralization with 4 N HCl the solution was lyophilizecl and the residue was dissolved in 3.0 ml of dis- tilled water. The yield of sodium acetoacetate-3J4C was 95% of the theoretical value.

The above solution (1.5 ml) was added to tl reaction mixture containing 150 pmoles of potassium phosphate buffer, pH 6.5, 46 pmoles of NADH, and 0.6 unit of D-( -)-P-hydroxpbutyrate clehydrogenase in a final volume of 5 ml. Incubation was car- ried out for 23 hours at room temperature. One drop of concen- trated H&O4 was added to bring the pH to approximately 2.0, and the mixture was continuously extracted for 12 hours with 100 ml of &ethyl ether. At the end of the extraction 5 ml of 1% NaHC03 were added to the ether which was then evaporated under a stream of nitrogen. The aqueous solution was brought to 10 ml in a volumetric flask with 0.9% NaCl, and the pH was adjusted to 7.4 with 4 N HCl. Analysis of the final solution showed no detectable acetoacetate while the concentration of &hydroxybutyrate was approximately 3.0 pmoles per ml. The 14C content was 50 &i per ml. When acetoacetate-3-14C was used as tracer, it was diluted to the same concentration with 0.9% NaCl solution.

Experiments with Aniline Citrate-In order to test the role of recycling in the turnover studies the amount of radioactivity in the carboxyl group of acetoacetate and /3-hyclroxybutyrate was determined utilizing a modification of the procedure described by Ontko (11). Plasma (50 ~1) was placed in the main compart- ment of a 25-ml center well flask in a volume of 1.0 ml of 0.1 M

potassium phosphate buffer, pH 7.0. One-ha,lf milliliter of 5 x KOH was in the center well. After standing at room tempera- ture for 80 min to remove 14C02 dissolved in the plasma, the flask was capped, and 1 ml of aniline citrate was added. After shak- ing at 37” for 1 hour, the KOH was removed and transferred to the main compartment of a second flask containing 0.5 ml of 1 RI methanolic Hyamine hydroxide solution in the center well. The flask was capped and shaken 45 min after the addition of 0.25 ml of 18 N HzSO4. The Hyamine was removed, the well was washed with methanol, and an aliquot was assayed for radioactivity in a liquid scintillation counter. The initial KOH step was added when it was found that about 12% of the labeled acetone clis- tilled into Hyamine after treatment of acetoacetate-3-*“C with aniline citrate. None was recovered in KOH under the same circumstances. The transfer of label to the Hyamine solutions was carried out to avoid the problem of chemiluminescence oc- curring with KOH in liquid scintillation systems. If conversion of P-hydroxybutyrate into acetoacetate is carried out, the incu- bation mixture, which is alkaline, must be acidified to remove dissolved 14C02 prior to addition of aniline citrate.

Experiments with Isolated Perfused Livers-Livers were iso- lated and perfused according to the technique of Exton and Park (12) except that washed human red blood cells prepared from bank blood at least 4 weeks old was used in place of rat erythro- cytes. The liver perfusion apparatus, which was of constant

flow and variable pressure type, was custom built in the appara- tus shop of Vanderbilt University. Initial volume of the per-

fusate was 72 ml and flow was maintained at 7 ml per min. A 0.1 M solution of sodium octanoate in Krebs bicarbonate buffer containing 29ib albumin was infused into the perfusate pool at a rate of 20 ~1 per min after a priming dose of 0.5 ml was given dur-

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TABLE II

Recovery of acetoacetate and p-hgdroxybutyrate from untreated and deproteinized plasma

Acetoacetate and o-hydroxybutyrate were added to 10 ml of plasma to give a final concentration of 4.75 and 3.80 pmoles per ml, respectively. Aliquots were added to additional plasma sufficient to give a total volume of 2 ml. Samples were then assayed di- rectly or after precipitation of protein with perchloric acid as described in the text. One hour postprandial plasma was used so that control ketone levels would be undetectable.

Treatment

None

Perchloric Acid

Acetoacetate

Added

&mzoles

0

0.95

1.90 2.85 3.80 4.75

0 0.95 1.90 2.85 3.80 4.75

0.85 1.83 2.69 3.52 4.31

0.59 1.15 1.76 2.31 2.70

c .-

-

90

96

95

93

91

62 61 62 61 57

p-Hydroxybutyrate

Added Found

pm&s

0

0.76 1.52 2.28 3.05 3.80

0.76 100 1.53 100 2.21 97 2.97 95 3.61 95

0 0.76 1.52 2.28 3.05 3.80

0.50 66 0.93 61 1.51 66 1.98 65 2.34 62

.ecovery

%

ing the 1st min. Isotopic octanoate and acetoacetate or ,&hy- drosybutyrate were added as indicated in the tables and figures.

Operative Procedures-Functional hepatectomies were carried out as described by Hotta and Chaikoff (13). In the portal vein infusion studies, the portal vein was catheterized by passing a PE-10 catheter through the splenic vein.

Materials-Ethyl acetoacetate-3-l% was obtained from New England Nuclear. D-( -)-P-Hydroxybutyrate dehydrogenase (EC 1.1.1.30) was purchased from Boehringer Mannheim. All other chemicals and reagents were commercial products of the highest available grades.

RESULTS

Recovery of Acetoacetate and /%Hydroxybutyrate from Plasma- The results of recovery studies for acetoacetate and P-hydroxy- butyrate added to plasma are shown in Table II. In this ex- periment recovery of acetoacetate in untreated plasma was greater than 90% in every instance and averaged 93%. Re- covery of P-hydroxybutyrate was slightly higher, in the 95 to 100% range. In contrast, when plasma was treated with per- chloric acid, recoveries of both acetoacetate and P-hydroxybuty- rate were about 60% of theoretical values. It should be noted that, these figures were obtained with the supernatant decanted from the two precipitates without washing. The bulk of the loss doubtless represents trapping of fluid in the precipitates and could be corrected either by several washes with perchloric acid or by including a tracer of tritiated water to evaluate the extent of trapping. All of these difficulties can be obviated, however, by using untreated plasma since recoveries are essentially com- plete. Losses similar to those found with perchloric acid were ob- served when trichloracetic acid was used for deproteinization. As expected, plasma ketone concentrations are about twice as high as concentrations in the same sample of whole blood.

Constant Infusion Studies in Starved Rats-Utilizing the direct

l p- hydroxybutyrote 0 Acetoacetate

I o--o-~--o--~----o

I I I I

20 40 60 80 Minutes

FIG. 2. The specific activities of acetoacetate and fl-hydroxy- butyrate after the constant infusion of D-(-)-P-hydroxybutyr- ate-3-W in starved rats. Isotopic p-hydroxybutyrate was infused into unanesthetized animals which had been starved for 48 hours as described in the text. Specific radioactivities were de- termined at the indicated time points. Acetoacetate and p-hy- droxybutyrate concentrations in the plasma were essentially con- stant throughout the experiment.

plasma technique, studies were undertaken to assess ketone body turnover in the isotopic steady state. D-( -)-/%Hydrosybuty- rate-3J4C was infused into four unanesthetized animals that had been fasted for 48 hours. As shown in Fig. 2 the specific activi- ties of both /3-hydroxybutyrate and acetoacetate remained con- stant after 30 min. Contrary to expectations, however, the

specific activity of acetoacetate was in each case lower than the specific activity of P-hydroxybutyrate, ranging from 44 to 77% and averaging 51a/, of the latter. As shown in Fig. 3, the dis- equilibrium was even more marked when acetoacetate-3J4C was infused. While an isotopic steady state was again obtained, the specific radioactivity of the P-hydroxybutyrate averaged only 3f ‘% of that of the acetoacetate. On theoretical grounds we had assumed that /3-hydroxybutyrate should be derived solely from acetoacetate and that specific activities would be identical be- cause of rapid equilibration between the two. It was conceiv- able, however, that alternate pathways of synthesis existed to account for the different specific activities. To test this possi- bility, labeled octanoic acid was infused into intact animals, and the specific activities of the newly synthesized ketone bodies were determined. Similar experiments were carried out in the isolated perfused liver system. The results are shown in Table III, where it can be seen that within the limits of experimental error identical specific activities were found in acetoacetate and P-hy- droxybutyrate. These findings clearly support the hypothesis of a common pathway for ketone body synthesis and rule out the possibility that the disequilibrium found in the constant infusion experiments was the result of alternate synthetic routes.

Another possibility was that the disequilibrium was an artifact of the experimental system. Since the isotopic ketone body was

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4386 Ketcme Body Metabolism in Starvation and Diabetes Vol. 245, No. 17

5 0 p-hydroxybutyrate o Acetoacetate

4 o----o._ 3

-z ‘0 -w---O

I I 1 I I

20 40 60 80

Minutes FIG. 3. The specific activities of acetoacetate and p-hydroxy-

butyrate after the constant infusion of acetoacetate-3-l% in starved rats. Conditions were as described for Fig. 2 except that acetoacetate-3-% was infused in place of D-(-)-p-hydroxybu- tyrate-3-l%.

TABLE III

Speci$c activities of acetoacetate and P-hydroxybutyrate synthesized from octanoate-f J4C in vivo and in

isolated perfused liver

Sodium octanoate-1-W (15 pmoles, 150 &i per ml) was infused into intact, unanesthetized rats fasted for 48 hours at a rate of 1.2 ml per hour after a priming dose of 0.2 ml had been given. In the isolated perfused liver system, 50 pmoles of octanoate-l-14C (8 PCi) were given as a primer, followed by a constant infusion at a rate of 2 rmoles (0.32 pCi) per min. Specific radioactivities of acetoacetate andfl-hydroxybutyrate were determined as described in t.he text.

Experiment and condition Time

Specific activity

1. In vivo

2. In vivo

3. Perfused liver

Acetoacetate ,9-Hydroxy-

butyrate

dpm/pmole x 10-s

0.52 0.45

0.72 0.77

0.41 0.47 0.38 0.38

infused into the inferior vena cava while samples were assayed on arterial blood, it was conceivable that the higher specific activity found in the ketone body being infused was due to sampling prior to passage through the liver which presumably effected equilibra- tion. This possibility was ruled out by the experiment shown in Table IV. In this experiment acetoacetate-3-‘4C was infused directly into the portal vein rather than into the vena cava. It can be seen that disequilibration between the two ketones was

TABLE IV

Specijic activities of acetoacetate and p-hydroxybutyrate after portal vein infusion of acetoacetate-W4C

Acetoacetate-3-l% was infused directly into the portal vein rather than into the vena cava. Specific radioactivities of aceto- acetate and P-hydroxybutyrate were determined as described in the text. Total blood ketones were 180 pmoles/lOO ml and re- mained essentially constant throughout the experimental period.

Specific activity

Time I

I Acetoacetate 1 p-Hydroxybutyrate

?nin a@?+& x 10-s 20 4.80 0.99 60 4.90 2.64 80 6.03 2.70

30 60 90 120

B

t

. /3-hydroxybutyrate oAcetoacetote

0 30 60 90 120 Minutes

FIG. 4. The specific activities of acetoacetate and P-hydroxy- butyrate after a single injection of acetoacetate-3-14C (A) or D- (-)-o-hydroxybutyrate-3J4C (B) in the isolated perfused liver. Sodium octanoate was infused as substrate throughout the exper- iment as described under “Experimental Procedure.” Radioac- tive ketone bodies were added in tracer amounts as a single in- jection at time zero.

again found, despite the fact that the rat was only mildly ketotic and t#hus did not have excessively large pools of acetoacetate and P-hydroxybutyrate to bring into equilibrium. This finding sug- gests that the capacity for interconversion of the two ketones in the liver is limited.

As shown in Fig. 4, a representative experiment utilizing the isolated perfused liver further supports this concept. Unlabeled octanoic acid was infused as substrate for ketone body formation from the beginning OF the experiment. A tracer quantity of either acetoacetate-3J4C or D-( -)-P-hydroxybutyrate-3-l% was given as a single injection at time zero. With the former, wide disequilibrium was still present at 15 min; 90 min were required to effect identical specific activities. In contrast, when P-hy- droxybutyrate was labeled, the initial specific activities were con- siderably closer, and isotopic equilibrium was obtained at 30 min. Thus, in the in vitro whole liver system a finite period of time was required to bring the two products into equilibrium, and the rate of equilibration was greater in the direction of fl-hydroxybutyrate to acetoacetic acid as was also the case in vivo. In experiments not shown, labeled acetoacetate and fl-hydroxybutyrate were introduced into the perfusion system by constant infusion to- gether with unlabeled octanoate to simulate the studies in vivo in the isotopic steady state. Disequilibrium in the specific ac-

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0 30 60 90 0 30 60 90 120

Minutes after hepatectomy

FIG. 5. The role of the liver in ketone body metabolism. Rats previously starved for 48 hours were functionally hepatectomixed at time zero. At 15 min, as indicated by the arrozus, a solution containing 150 pmoles of acetoacetate and 600 pmoles of nn-p-hy- droxybutyrate per ml was infused at a rate of 1.2 ml per hour. Acetoacetate-3-W (50 PCi per ml) was present in the infusate in the experiment shown in A, while a similar quantity of n-(-)-b- hydroxybutyrate-3-W was added in the experiment shown in B.

tivities of acetoacetate and /?-hydroxybutyrate was observed throughout the experiment with wider discrepancies noted when

acetoacetate contained the isotopic label, again conforming to the situation in the intact animal.

While the foregoing experiments unequivocally demonstrated that the liver is ultimately capable of bringing into equilibrium infused acetoacetate and &hydroxybutyrate, it was not certain that it played the major role in the process in Go. To answer

this question, the experiments shown in Fig. 5 were performed.

Rats were functionally hepatectomized at time zero. As ex- pected, blood ketones at 15 min dropped sharply. At this point a solution containing acetoacetate and P-hydroxybutyrate was given by constant infusion with one or the other of the com- pounds labeled with l4C. At 60 min after the infusion of aceto- acetate-3-K’), the specific activity of /3-hydroxybutyrate was only 6% of that found in acetoacetate in contrast to an average of 37% with the liver intact. At 90 min after the infusion of labeled fi- hydroxybutyrate, the specific activity of acetoacetate was 8.57,

of that in @-hydroxybutyrate compared with a mean figure of 51 y0 when the liver was present. Similar results were obtained in four other animals, strongly suggesting that the liver, rather than the peripheral tissues, is the primary site of equilibration between acetoacetate and /Lhydroxybutyrate.

It is of interest to note that the ketone bodies in these experi- ments were infused in a ratio of D-(-)-P-hydroxybutyrate to acetoacetate of 2:l but that the ratio became progressively greater than 2 in every case as the infusion progressed. It has

TABLE V

Distribution of radioactivity between acetone and carboxyl moieties of plasma ketones

Plasma samples taken at the indicated times during the con- stant infusion of n-(-)-&hydroxybutyrate-3-‘4C were treated with aniline citrate after enzymatic conversion of the entire sample into acetoacetate as described in the text. Values repre- sent the averages of duplicate determinations on 50.~1 samples.

Experiment

1 2 3

Time

min

50 90 90

Acetone Carboxyl

dPm

47 850 ) 325 33,060 160 58,980 370

TABLE VI

Calculation of turnover rate of plasma ketones in starved

animals during isotopic steady state

Acetoacetate -3 -1% or D - (-) -p - hydroxybutyrate -3 -1°C was infused as described in the text unt,il the isotopic steady state was obtained. Animals were fasted 48 hours prior to use. Mean concentrations and specific activities were obtained for samples taken between 40 and 80 min. Turnover rates were calculated on the basis of the specific activity in acetoacetate after aceto- acetate-3-W infusion, p-hydroxybutyrate after D-(-)-o-hydroxy- butyrate-3-‘4C infusion, or on the basis of the total ketone specific activity derived from the total radioactivity recovered in aceto- acetate plus P-hydroxybutyrate divided by the concentration of acetoacetate plus P-hydroxybutgrat,e.

Rat Weight

P-Hydroxybuty rate-3-W

1 2 3 4

Mean 119 100 393 1.10 2.16 8.1 9.G

Acetoacetate- 3-W

1 103 8G 279 3.20 1.29 2 100 123 484 4.37 1.34 3 110 71 85 4.54 1.31 4 110 73 92 5.95 2.G5

also been frequently noted that the ratio increases as blood ke- - I

Mean 106 88 237 4.51 1.65

--

P-BY- 4&o- droxy- icetate bu-

tyrate

WY- Aceto- droxy- 1cetate

tyrate

_

0

__

Aceto- lcetkte r@-Hy- Total drooxy- ketones

tyrate

p?mles/100 ml dPnt/ pmolrs/ )mde x 10-a &n/100 g

100 IG3 373 0.87 2.17 9.2 11.3 105 207 580 1.39 2.69 7.1 8.2 145 107 179 0.94 2.12 6.5 8.6 125 163 349 1.21 1.67 9.6 10.5

6. 1 4 G 4.0 3.1

4.5

11.2 10.2 6.5 4.4

8.1

MeaIl concentration

-

Mean specific activity Turnover rate

tones fall following hepatectomy. While other studies are needed to prove the point, the findings suggest the possibility introduced into the carboxyl carbon by oxidation of acetoacetate

that acetoacetate is preferentially used by peripheral tissues dur- to acetyl-CoA with subsequent resynthesis of acetoacetate would

ing ketosis. be missed. As shown in Table V, decarboxylation of plasma

One other experiment was required before calculation of turn- samples in which the total ketones were converted into acetoace- over rates could be undertaken. Since radioactivity in the two tate revealed essentially no radioactivity in the carboxyl carbon,

ketones was assayed by recovery of isotope in the acetone portion ruling out the possibility of significant recycling.

of the molecule trapped as the mercury salt, any radioactivity The blood concentrations and specific activities of acetoacetate

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4388 Ketone Body Metabolism in Starvation and Diabetes Vol. 245, No. 17

TABLE VII

Equilibrium between specijk activities of plasma acetoacetate and @-hydroxybutyrate after single injection of acetoacetate-S-14C

or P-hydroxybutyrate-S-W! in starved rats

Acetoacetate -3 -1% or D - (-) -p - hydroxybutyrate -3 - W (50 pCi (3 pmoles)) was injected into the inferior vena cava over a 30-set period. Specific activities of acetoacetate and p-hydroxy- butyrate were determined on plasma samples taken at the indi- cated times

min Acetoacetate-3-%

0 ..................... 10 ..................... 20 ..................... 30 ..................... 40 .....................

p-Hydroxybutyrate-3-1dC 0 .....................

10 ..................... 20 ..................... 30 ..................... 40 .....................

-

Concentration

/ Aceto- P-HY-

droxy- acetate butyrate

pm&5/100 ml

240 251 242 252 238 234 234 255 223 308

150 280 141 298 157 230 163 257 166 211

I-

Specific activity

Aceto- P-HY- acetate droxy-

butyrate

d@?z//.mole x 10-5

1.74 1.87 1.14 1.07 0.70 0.74 0.45 0.34

2.18 2.25 1.29 1.59 1.02 1.06 0.72 0.74

and /3-hydrosybutyrate during infusion with acetoacetate-3J4C or fl-hydroxybutyrate-3-14C are shown in Table VI. Turnover rates were calculated utilizing the specific radioactivities of the infused and the isolated plasma ketone bodies. In the studies with /3- hydroxybutyrate the calculated turnover rates ranged from 6.5 to 9.6 and averaged 8.1 pmoles per min per 100 g of body weight. This figure gives a minimum estimate of total ketone body syn- thesis and underestimates the true rate to the extent that equili- bration with acetoacetate did not occur (i.e. the specific activity observed in the fi-hydroxybutyrate fraction is higher than would have been the case had equilibration with acetoacetate occurred, resulting in an apparent lesser dilution of the isotope by the non- radioactive ketones produced by the liver.) By the same argu- ment, turnover rat.es calculated from the specific activity of acetoacetate following infusion with P-hydroxybutyrate-3J4C would overestimate total ketone body production. When aceto- acetate-3-‘4C was utilized as the tracer, turnover rates estimated from specific radioactivities of plasma acetoacetate ranged from 3.1 to 6.1 and averaged 4.5 pmolesper min per 100 g reflecting the lesser degree of equilibration found in these experiments and further underestimating the true turnover rate.

As indicated above, strong evidence has been obtained to indi- cate that the disequilibrium found in the isotopic steady state is due to a limited capacity of the liver to carry out the equilibra- tion of infused isotope between acetoacetate and P-hydroxybuty- rate. The best estimate of total ketone body turnover would appear, therefore, to be derived from total ketone body specific activity by ignoring the disequilibrium. Under these conditions the sum of the radioactivity recovered in acetoacetate and fi-hy- droxybutyrate divided by the total concentration of acetoacetate plus P-hydroxybutyrate is assumed to represent the specific ac- tivity that would have been attained if hepatic equilibration had not been limiting. Such calculations indicate turnover rates

averaging 9.6 pmoles per min per 100 g of body weight when p-

Minutes

FIG. 6. Specific activities of total plasma ketones after a single injection of acetoacetate-3-14C or n-(-)--p-hydroxybutyrate-3J4C in nine starved rats. Radioactive acetoacetate or p-hydroxybu- tyrate (50 &i (3 pmoles)) was given by rapid injection over a 30.set period. Specific radioactivities of plasma ketones were deter- mined at the indicated times. The zero time intercept equals a specific activity of 2.38 X lo6 dpm per rmole. Vertical lines repre- sent standard errors of the mean.

hydroxybutyrate-3-14C was infused and 8.1 pmoles per min per 100 g of body weight when acetoacetate-3-14C was the tracer. These values are very close, particularly when it is considered that two of the rats in the latter group were only moderately ketotic and had low turnover rates.

Single Injection Xtudies in Starved RatsIn order to check the turnover rate obtained in the isotopic steady state, additional

studies were done in which acetoacetate-3-14C or D-( -)-P-hy- droxybutyrate-3-i4C was given as a single rapid injection. In contrast to the report of Bates et al. (3), isotopic equilibrium was obtained whether radioactive acetoacetate or ,I-hydroxybutyrate was injected as shown by the two typical experiments recorded in Table VII. On the basis of these findings the specific activity of the total plasma ketones was used to calculate turnover rates. The results of such studies in nine rats (six utilizing D-( -)-p-hy- droxybutyrate-3-i4C and three utilizing acetoacetate-3-r4C as tracer) are shown in Fig. 6. It is clear that a straight line results when the data are plotted against time on a semilogarithmic scale and that the standard errors at each point are extremely small. The intercept corresponded to a specific activity at zero time of 2.38 x lo5 dpm per pmole. Turnover rate of total plasma ke- tones calculated from these data was 13.4 pmoles per min per 100 g of body weight.3 This figure represents a maximal estimate of

3 This rate was derived as follows (14) :

Total ketone pool = total dpm injected

specific activity of plasma ketones

11.5 X 1Or dpm =

2.38 X lo6

= 484 pmoles

total ketone pool 100 Turnover rate =

turnover time X

mean weight

484 100

= 28.2 x iii

= 13.4 fimoles per min per 100 g

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Issue of September 10, 1970 J. D. McGarry, M. J. Guest, and D. W. Foster 4389

3 l p- hydroxybutyrate 0 Acetoacetate

2

Minutes FIG. 7. The specific activities of acetoacetate and D-hydroxy-

butyrate after the constant infusion of D-(-)-~-hydroxybutyrate- 3-W in alloxan diabetic rats. Conditions were as described for Fig. 2.

the actual turnover rate since acetoacetate and P-hydroxybutyr- ate are rapidly oxidized in peripheral tissues. It is apparent, therefore, that a finite portion of the injected isotope would be utilized during the mixing phase and that the intercept specific activity is consequently too low resulting in an overestimation of the true turnover rate. In experiments not shown we have meas- ured ‘4C02 production from rats after the injection of radioac- tive acetoacetate and /?-hydroxybutyrate and found that ap- proximately 4’5” of the label was recovered in the first 10 min. Moreover, up to 30% of the radioactivity in the plasma at early time points was in CO*. While no accurate quantitative cor- rection can be made, it is conceivable that the calculated rate is too high by as much as 20%, an error which would bring the true rate close to that obtained in the isotopic steady state.

Studies in Alloxan Diabetes-Attempts were then made to measure the turnover rate of ketone bodies in alloxan diabetes. When D-( -)-/3-hydroxybutyrate-3-14C was infused the curves shown in Fig. 7 were obtained. Two points of interest are ap- parent. The first is that an isotopic steady state was not uni- formly attained over the 80-min infusion period. In other ex- periments, infusion of isotope was continued for up to 3 hours without attaining a steady state. While an increase in specific activity generally continued during this period, the change was minimal after 80 min. For this reason the turnover rate could not be accurately assessed in all the animals. The two animals that most closely approximated the steady state are shown in the two middle panels of Fig. 7. Turnover rates in these two animals (Table VIII) were 16.3 and 14.3 pmoles per min per 100 g of body weight assuming that the 60- and 80-min points were close to true steady state levels.

The second point of interest is that in the diabetic rats, which

TABLE VIII

Specijic activities of P-hydroxybutyrate and acetoacetate and calculation of turnover rates in alloxan diabetic animals

D-(-)-@Hydroxybutyrate-3-“C was infused as described in the text. Concentrations and specific activities are listed for the 80-min time point. Turnover rates were calculated as described in Table VI. Rats 2 and 3 were used for estimates of ketone body turnover in diabetes in the text since in these rats an iso- topic steady state was more closely approached than in Rats 1 and 4 (see Fig. 7).

Rat

Concentration Specific activity Turnover rate

pmoles/100 ml dpm/M?%ole x 10-6 p?noles/min/100 g

115 425 1398 1.19 0.89 23.0 21.4 125 425 788 1.09 0.98 17.0 16.3 150 519 1168 1.16 0.93 15.3 14.3 130 489 1111 0.86 1.11 14.0 15.0

Mean 130 465 1116 1.08 0.98 17.3 16.8

were severely ketotic (plasma ketones, 16 to 20 mM), equilibrium between the specific activities of acetoacetate and P-hydroxy- butyrate was present from the earliest time point when isotopic fl-hydroxybutyrate was infused, differing from the situation ob- served in starved animals. When acetoacetate-3J4C was infused, disequilibrium between the two groups of animals still existed, although differences were narrower than was the case in starva- tion.

DISCUSSION

The present studies have shown that concentrations of aceto- acetate and &hydroxybutyrate and their respective specific radioactivities can be accurately and easily measured in plasma without the need for deproteinization. Utilizing this technique total ketone body turnover rates were estimated by both constant infusion and single injection techniques in starvation ketosis. The minimal rate for rats fasted for 48 hours and infused with D-( -)-@-hydroxybutyrate-3J4C was calculated to be about 8.1 pmoles per min per 100 g of body weight. A maximal figure, calculated from single injection studies, was approximately 13 ymoles per min per 100 g of body weight. It is likely, therefore, that the average true production rate of ketones in rats starved for 48 hours is bracketed by these figures. Since it was shown that the liver produces ketone bodies from labeled fatty acids in isotopic equilibrium while exhibiting a limited capacity to equili- brate exogenously administered ketones, it appears reasonable to ignore the disequilibrium and calculate ketone body turnover from dilution of the administered label in the total ketone body pool. Under these circumstances the turnover rate was 9.6 pmoles per min per 100 g of body weight with labeled /3-hydroxy- butyrate and 8.1 pmoles per min per 100 g of body weight with la- beled acetoacetate. Since production rate varies linearly with total ketone body concentrations (3) and since two of the four rats given acetoacetate-3J4C were only moderately ketotic, it can be assumed that turnover rates calculated in this manner with either radioactive acetoacetate or P-hydroxybutyrate will give equivalent results.

Because of the difficulty in attaining the isotopic steady state

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4390 Ketone Body Metabolism in Starvation and Diabetes Vol. 245, No. 17

in the alloxan diabetic animals, estimation of turnover rates is less satisfactory than in starvation. Approximate rates of 14 and 16 pmoles per min per 100 g of body weight were obtained in two animals where the steady state was approached. Assuming a rate of about 15 pmoles per min per 100 g, the diabetic turnover rate is 1.6 times greater than that of the starved animals (15 pmoles per min per 100 g versus 9.6 pmoles per min per 100 g) while plasma total ketone concentrations were 3.4 times greater (1399 pmoles per 100 ml versus 417 pmoles per 100 ml). The differences in severity of ketosis cannot, therefore, be accounted for solely by rates of synthesis in the two conditions. It is likely that peripheral utilization approaches maximum at some given ketone body concentration beyond which small differences in production result in major differences in total plasma ketone con- centration (3, 15).

In a previous study (1) we have estimated the maximal rate of ketogenesis in vitro to be about 200 pmoles per hour per g of liver in normal, starved, and alloxan diabetic animals. Since a 100-g rat has roughly 4 g of liver tissue, the maximal rate of ketogenesis should be around 13 pmoles per min per 100 g of body weight. This would be very close to the estimated rate in diabetic ketosis according to the present figures and suggests that ketone body synthesis in this condition has reached the limit of hepatic ca- pacity, while ketogenesis in starvation is less than maximal. It is interesting that total ketone body turnover rates in the starved rat are proportionately much greater than those obtained in a large animal such as the sheep where values of 0.8 pmoles per min per 100 g of body weight are obtained during starvation (15).

Our studies differ from those of Bates et al. (3) in two respects. Turnover rates as determined here are more than double those reported by them. Secondly, we obtained equilibration between acetoacetate and P-hydroxybutyrate specific activities after single injections of either ketone labeled with I%. We are un- certain as to the reason for these differences. Our confidence in the results presented here is increased by the finding of turnover rates of the same order of magnitude using either the steady state or single injection techniques.

A final point deserving emphasis from the data obtained in these experiments concerns the central role played by the liver in over-all ketone body metabolism. Not only do ketone body concentrations drop to nondetectable levels after functional hepatectomy, but in the absence of the liver, interconversion of acetoacetate and &hydroxybutyrate appeared to be virtually abolished. On the other hand, when the liver was intact, equi-

librium was established within 10 min after a single injection of either radioactive compound, confirming the original report of Krebs (16) that the isolated perfused liver was able to adjust the ratio of the two ketone bodies after the addition of acetoacetate or fi-hydroxybutyrate to the system. It is of interest that D-( -)-

P-hydroxybutyrate-3-l4C given by constant infusion was also brought into isotopic equilibrium with acetoacetate in diabetic but not starved animals, despite the fact that the total ketone pool was several times larger in the former condition; this sug- gested an adaptive change in the diabetic state. The reason for such a change is not immediately apparent. One possibility, mentioned earlier, would be that acetoacetate is preferentially utilized by peripheral tissues at high plasma ketone concentra- tions requiring an accelerated hepatic capacity for interconver- sion in order to maintain a constant /3-hydroxybutyrate to aceto- acetate ratio in the plasma.

Ack~wledgmentsThe expert technical assistance of Miss Petra Contreras is gratefully acknowledged. We also wish to express appreciation to Dr. Philip W. Felts, Vanderbilt Univer- sity School of Medicine, for advice and assistance in the setting up of the isolated perfused liver system.

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J. Denis McGarry, M. Joanne Guest and Daniel W. FosterKetone Body Metabolism in the Ketosis of Starvation and Alloxan Diabetes

1970, 245:4382-4390.J. Biol. Chem. 

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