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Metabolism of labeled carnitine in the rat
ARCHIVES
OF BIOCHEMISTRY
AND
BIOPHYSICS
Metabolism GITTEN Department of Clinical
175, 173-180 (1976)
of Labeled CEDERBLAD
Carnitine AND
SVEN
in the Rat LINDSTEDT
Chemistry, University of Gothenburg, S-413 45 Gothenburg, Sweden
Sahlgren’s Hospital,
Received December 16, 1975 In two series of rats, the concentration of camitine in plasma was 39.9 and 37.8 pmol/ liter, in skeletal muscle tissue 2.97 and 3.26 pmol/g dry wt and the urinary excretion 3.2 and 2.4 pmo1/24 h. The renal clearance of camitine was calculated to 88 and 76 ml/24 h. L-[Me-14ClCarnitine and nr.-[Me-‘4Clcarnitine have been administered to rats. Only labeled Gcamitine has been found on chromatographic analysis of plasma, urine, and muscle tissue. The specific radioactivity of carnitine in plasma, urine, and muscle tissue has been followed for up to 16 days. A two-compartment metabolic model has been used to interpret the result of the experiment with labeled L-carnitine and the rate constants and compartment sizes have been calculated. The total body content of camitine was 57 pmol (about 35 pmol/lOO g body wt) and the daily turnover was about 7% of the body pool. The daily synthesis of camitine in the rat is estimated to about 2 pmol/lOO g body
wt. Carnitine (4-trimethylamino-3-hydroxybutyrate) plays a major role in the transport of activated long-chain fatty acyl groups from sites of activation to sites of poxidation in the mitochondria. Recent works (l-3) have established that there exists a pronounced species difference in carnitine concentration in skeletal muscle tissue. Less is known about the metabolism of carnitine itself. Apparently, nutritional and physiological conditions may influence carnitine metabolism (4). Khairallah and Wolf (4) identified a decarboxylation product of carnitine, P-methylcholine in rat urine. Starvation, alloxan diabetes, and feeding a high-fat diet were among the conditions that increased the decarboxylation reaction. The mechanisms behind the observed facts are not known. We considered it of interest to make a detailed study of carnitine metabolism in the intact normal rat involving both chemical determination of carnitine concentration in tissues and urine and an attempt to formulate a kinetic model for carnitine metabolism. MATERIALS AND METHODS Labeled carnitine. L-[MeJ4C]Carnitine was prepared according to Stokke and Bremer (5). Thin173 Copyright All rights
8 1976 by Academic Press, Inc. of reproduction in any form reserved.
layer chromatography on silica gel G in two different solvent systems of the prepared labeled L-carnitine gave one spot when the distribution of radioactivity was determined with a thin-layer scanner (Labor, Prof. Dr. Berthold Wildbad, West Germany). The R,-value was the same as for authentic camitine detected with iodine vapor. The two solvent systems used were methanol-concentrated ammonia (l:l, by volume) and chloroform-methanolwater-concentrated ammonia-formic acid (55:50: l&7.5:2.5, by volume). To establish that no racemization had occurred in the preparation of labeled ccamitine, we used the preparation in an enzymatic assay. After thin-layer separation the amount of Gacetylcarnitine formed was calculated. As seen in Table I, the amount formed was in good agreement with that expected if no racemization had occurred. nL-[Me-‘4C]Camitine had a specific radioactivity of 52 mCilmmo1 and was obtained from The Radiochemical Centre, Amersham, Bucks., England. Animals. Female rata of the Sprague-Dawley and Wistar strains, free of specific pathogen and weighing 150-220 g, were obtained from Miillegaards Avelslaboratoriet A/S, Skensved, Denmark. The rata had free access to water and a commercial pellet diet (EWOS, S&letilje, Sweden). Labeled nL-camitine, 6.66.106 cpm per rat, was injected intraperitoneally into 12 rats of the SpragueDawley strain. Labeled ticarnitine, 3.43.106 cpm per rat was injected intraperitoneally into 16 rata of the Wistar strain. The animals were kept in individ-
174
CEDERBLAD TABLE
FORMATION
1
OF L-ACETYLCARNITINE L- AND DL-CARNITINE"
Labeled carnitine
FROM
LABELED
Percentage recovered in acetylcarnitine Experimentally
L DL
AND LINDSTEDT
37 65
Calculated 39 59
o The labeled substrate was incubated with carnitine acetyltransferase and the reaction mixture was separated by thin-layer chromatography (chloroform-methanol-water-concentrated ammonia-formic acid 50:50:10:7.5:2.5 by volume, on silica gel G). The values were calculated by using 1.8 for the equilibrium constant of the reaction: carnitine + acetyl-CoA ti acetylcarnitine + CoASH (6). ual metabolic cages. Blood sampling was performed with a heparinized capillary tube in the medial angle of the eye during light ether anesthesia. After centrifugation, the plasma was kept frozen at - 20°C until analyzed. Urine was collected daily in bottles containing 0.5 ml of 6 M HCl as perservative and was kept frozen until analyzed. At different time intervals the animals were decapitated and preferentially red muscle tissue was removed from the gastrocnemius muscle (7). The muscle tissue was immediately frozen in liquid nitrogen and stored at -60°C until analyzed. Determination of carnitine. Camitine determination was performed with an enzymatic method USing acetyl-CoA:camitine-O-acetyltransferase (EC 2.3.1.7) and labeled [l-‘4Clacetyl-coensyme A as substrate (8). After precipitation with HClO, and neutralization with KOH, short-chain acylcarnitine was hydrolyzed in 0.1 M KOH according to Tubbs et al. (9). Radioactivity was measured in a Packard scintillation spectrometer (Model 3320, Packard Instrument Co., Downers Grove, Ill.). The amount of isotope in plasma was determined by adding 20 ~1 of plasma and 200 ~1 of water to 10 ml of In&a-Gel (Packard Instrument Co., Downers Grove, Ill. 1 in a plastic counting vial. The amount of isotope in urine was determined by adding 1 ml of urine to 10 ml of Insta-Gel and the amount of isotope in muscle by adding 300 ~1 of the muscle homogenate together with 800 ~1 of water to 10 ml of In&a-Gel. At least 5000 counts were recorded. RESULTS
Carnitine Concentration in Plasma and Muscle Tissue and Urinary Excretion The mean values of carnitine concentration in plasma were 39.9 and 37.8 pmol/ liter for the rats which had received la-
beled DL- and ccarnitine, respectively. The mean values of the concentration of perchloric acid soluble carnitine in muscle tissue were 2.97 and 3.26 pmol/g dry weight for the two groups of rata. No significant correlation was found between carnitine concentration in plasma and in muscle tissue. The average 24-h excretions of carnitine in the urine were 3.2 and 2.4 pmol for the rats injected with labeled DLcarnitine and L-carnitine, respectively. For rats given m-carnitine a computation of the components of intra- and interindividual variation (10) has been performed for carnitine concentration in plasma and for carnitine excretion in urine. In both cases, the intraindividual variance was the main contributory variance component. The mean values of renal carnitine clearance were 88 and 76 ml/24 h for the two groups (Table II). The carnitine concentration in plasma and the urinary excretion has been measured in individual rats for periods of 10 to 14 days and the renal clearance has been calculated (Table III). Characterization of Labeled Carnitine in Rat Tissue The extracts of plasma and muscle from rata injected with labeled Gcarnitine were chromatographed on an ion-exchange column, AG 50W X8 which was eluted with 1 M HCl. As shown in Fig. 1 only carnitine could be detected. The recoveries were 96108% of the applied radioactivity. The corresponding ion-exchange chromatograms of muscle homogenates are shown in Fig. 2. The homogenates were precipitated with HClO, and the supernatants were neutralized. In the chromatograms of both these supernatants and of alkali-hydrolyzed supernatants carnitine was the only radioactive peak. Labeled m-carnitine added to muscle homogenate which had been hydrolyzed coincided with the original peak, see Fig. 2. The recoveries were 96-100% of applied radioactivity. The chromatograms of two urine specimens from rats injected with labeled L-carnitine are shown in Fig. 3. The urine specimens had been collected during the first and the fourth days after administration of the iso-
METABOLISM
OF LABELED TABLE
CARNITINE
CONCENTRATION
IN PLASMA
Plasma concentration Muscle concentration Urinary
(~mol/liter) (pmol/g dry wt)
excretion (pmo1/24 h)
Renal clearance (ml/24 h)
II
AND MUSCLE
Injected isotope
TIWJE
AND URINARY
Mean value 2 SE 39.9 37.8 2.97 3.26 3.20
DL L DL L DL L DL L
175
CARNITINE
I$Lmdl;
+ f 2 k
2P
21.7 24.8 13.8 15.6 35.6 32.3 32.8 31.4
i-0.
suremerits
0.8 1.1 0.12 0.13 0.11
+
EXCRETIO#
Re3ez3ti&
112 69 12 16 111
41 79 31
2.41 + 0.12
88 k 3.2 76 2 4.3
n.s.
a Renal clearance has been calculated from urinary excretion per 24 h/plasma carnitine concentration. The values are given for rats injected with labeled m-camitine and L-carnitine, respectively. The two groups of rats have been compared with Student’s t test and the significance level is indicated. b (SD/mean) x 100. TABLE CARNITINE
Individual rata
IN PLASMA
Plasma concentration Mean + SE (~mol/liter)
III
AND URINE
excretion
Mean 2 SE @mob24 h)
19.2 3.73 2 35.1 I!?2.1 39.5 f 3.0 25.4 3.43 2 28.8 2.65 k 45.8 f 3.8 18.5 3.69 k 42.5 k 2.3 37.8 + 1.0 8.1 3.91 -+ 20.0 2.57 k 38.8 2 2.1 12.3 3.56 k 40.3 +: 1.4 L 1 32.2 2 1.1 13.8 2.59 iI 13.1 2.32 k 2 33.6 + 1.1 0 Camitine concentration in plasma, urinary excretion, rats injected with labeled DL- and L-carnitine. resnectively, . b (SD/mean) X 100. DL 1
2 3 4 5 6 7
tope. The main peak, at 5 column volumes, appeared at the position of carnitine. In these chromatograms there was also a few percent of radioactivity eluted after 1 column volume. No further analysis was done of this material. The recoveries of applied radioactivity were 92 and 83%, respectively. Compartmental
IN INDIVIDUAL
Urinary
Analysis
A semilogarithmic plot of the specific radioactivity of carnitine in plasma and in urine versus time for rats injected with labeled r,-carnitine is shown in Fig. 4. There was a good agreement between the specific radioactivities of carnitine over a period of 16 days. After an initial rapid fall
RAT@
Renal clearance yy2; m
flE -c 10 + 12 -t- 7 + 6
0.39 0.33 0.32 0.21
32.7 30.7 42.2 19.5
106 93 67 90
0.31
25.1
103 r 7
0.19 0.30 0.15
0.20 and and
Relative SD (%)b 31.0 41.5 31.5 22.5 22.1
26.9 71 ” 5 23.4 31.8 89 2 7 32.4 22.5 83 f 6 30.0 32.7 69 f 6 31.1 renal clearance of carnitine for individual followed for 10 to 14 days.
in specific radioactivity, the data fell on a straight line. Two-compartment open system. The two-compartment open system has been chosen as the simplest possible system to explain the distribution of labeled ccarnitine in the rat; see inset in Fig. 4. The assumption is made that the labeled substance is introduced into the first cornpartment, i.e., the absorption from the peritoneal cavity occurs at a greater rate than any subsequent transformation. Three different models can be distinguished in this system (ll), i.e., elimination occurs only from compartment 1 (model 11, elimination occurs only from compartment 2 (model 21, or elimination occurs from both compartments (model 3).
176
CEDERBLAD 300
300 2Lh
2h
;
AND LINDSTEDT
200
600
200
1 5," 9
100
100
-I--n
R
.5 E zl a B 5 u"
LOO
200
0
-0
30
60
90 fraction
rroct1on
no
FIG. 1. Ion-exchange chromatography of plasma extracts from rats injected with labeled n-carnitine. The animals had been killed at the time indicated in the figure. The samples were applied onto a column (0.5 x 4.5 cm) of AG 50W X8. The column was eluted with 1 M HCl in fractions of 0.12 ml.
-0
30
60
90
no
FIG. 3. Ion-exchange chromatography of urine samples or rats injected with labeled ccamitine. Collection periods are as indicated in the figure. Urine samples were applied onto a column (1.6 x 10 cm) of AG 50W X8. The column was eluted with 1 M HCl, fraction l-40 and with 4 M HCl, fraction 41-90. The fraction volume was 5 ml.
FIG. 4. Specific radioactivity of carnitine in plasma (+O) and urine (0-O) versus time for rate injected with labeled L-camitine. The curve represents the best-fitting function of all values of the specific radioactivity of camitine in plasma. The inset shows a two-compartment system with corresponding rate constants (k,-k,).
frachll
no
FIG. 2. Ion-exchange chromatography of skeletal muscle extracts from rats injected with labeled Lcamitine. The animals had been killed at the time indicated in the figure. Unhydrolyzed supematants (60) and the same supematants hydrolyzed in alkali (O0) were applied onto a column (0.5 X 4.5 cm) of AG 50W X8. The column was eluted with 1 M HCl in fractions of 0.12 ml. The dotted line represents the increase in radioactivity after addition of labeled nL-carnitine to the sample before application.
Selection of model and resulting pool sizes and rate constants. The specific radioactivities (SA and SB) in the two compartments in an open two-compartment model follow equations of the type S& = M1.e-h’t
+ M2-e-a’t
SBt = N-(e-h’t
- e-h’“),
where M, is SAt; (h, - k,MX, - A,), MZ is SA,; (k2 - X,)/oC, - A,) and N is SAt;W (A, - A,). The best-fitting curve of the measure-
METABOLISM
OF LABELED
ments of specific radioactivity of plasma was calculated using a computer program (12). This curve is shown in Fig. 4 for the specific radioactivity (SA,) of carnitine in plasma, i.e., in compartment 1. The values for A1 and hz are given in Table IV. Knowing the specific radioactivity of carnitine at t,,, the size of compartment 1 was calculated to be 14 pmol by the principle of isotope dilution. It is possible to determine all the rate constants for models 1 and 2 from measurements of compartment 1, whereas for model 3 additional information is necessary. As the specific radioactivities of carnitine in urine and plasma were the same, the urinary elimination occurred from compartment 1 and model 2 was thus rejected. The rate constant for the elimination in urine (hJ in model 1 was 0.011 h-l. The same rate constant also could be calculated from the known excretion of carnitine in urine by the relation k, = 2.4/(14 x 24) h-l. A value of 0.0072 h-l was then obtained. We used this value of It, to calculate the parameters in model 3, i.e., assuming an elimination also from compartment 2. As seen in Table V this resulted in a somewhat larger estimate of the size of compartment 2 than calculated according to model 1. Figure 5 shows the relative amounts of isotope present in compartment 1 and 2 as calculated with the rate constants for TABLE CURVES OF SPECIFIC
177
CARNITINE TABLE
KINETIC
PARAMETERS
OF CARNITINE
METABOLJSI@
Model 1
Model 3
0.060 0.020 0.011 -
0.064 0.019 0.007P 0.0014
14 39
14 47
Rate constant (h-9
k, k, k3 k, Size (pmol) Compartment Compartment
V
1 2
a Rate constants were estimated on the basis of two models: (a) model 1, elimination only from compartment 1; (b) model 3, elimination from both compartment 1 and 2, cf. inset in Fig. 4. b Estimated from rate of excretion of camitine in urine.
0.01111111111111111111 0
24 46 12
96
120 14L
166192 216 240264266 hours
3l2 33636038L
FIG. 5. Distribution of isotope expressed as fraction of administered amount. The curves have been calculated with the values of the rate constants in model 1 which are given in Table V. Experimental values are indicated by (0-O) and cumulative excretion in urine by (O-O).
IV
RADIOACTIVITY
OF CABNITINE?
A, (h-l)
X2 (h-l)
Specific radioactivity of car0.089 0.0026 nitine in plasma (n-carnitine? Specific radioactivity of car0.0038 nitine in plasma (m-camitine? Specific radioactivity of caro.oiw 0.0024 nitine in muscle (L-camitiney a Values of A, and A, in the exponential expression for specific radioactivities in plasma (SA,) and muscle (SB,) versus time for rats who had received labeled L-camitine and m-camitine, respectively. b SAt = M,.e-“,” + &.f,.e-~~‘f. c S& = N.(e-h,.f - e-b.t). d t = O-8 h.
model 1 given in Table V and the expected cumulative elimination of isotope as well as the measured cumulative excretion in urine. As mentioned, the calculated value (0.011 h-l) for the rate constant for elimination calculated from the specific radioactivity of carnitine in plasma was higher than the value calculated from the urinary excretion of carnitine (0.0072 h-l). Measurements of compartment 2. The specific radioactivity of carnitine has been determined in muscle tissue, i.e., a tissue belonging to compartment 2. Using the experimentally found values of the specific radioactivity of carnitine in muscle tissue, X1determined from values between 0 and 8 h was 0.078 h-l and AZwas 0.0024 h-l. The
178
CEDERBLAD
AND LINDSTEDT
specific radioactivity of carnitine measured in muscle tissue is lower than the calculated specific radioactivity of carnitine in compartment 2, (SB,) as seen in Fig. 6. This is consistent with the fact that heart and muscle tissue have the highest carnitine content in the rat (1). Distribution of Labeled m-carnitine. In the experiments with rats injected with labeled nL-carnitine the specific radioactivity of carnitine has been followed during the late phase only. Figure 7 shows the specific radioactivity of carnitine in plasma and urine as a function of time between the third and the fourteenth day after the administration of the isotope. As was the case with labeled L-carnitine, there was a good agreement between the specific radioactivities of carnitine in urine and plasma. The equation of the regression curve of the specific radioactivity of carnitine in plasma versus time after Day 3 was SA, = 67.6 f 2.7
(SJi~.~-0.0038
FIG. 7. Specific radioactivity of carnitine in plasma (+O) and urine (O-O) versus time of nine rats injected with labeled m-carnitine. Standard deviation is indicated by a vertical bar. The regression line of specific radioactivity of carnitine in plasma versus time for the late phase is logSA, = log 67.6 - 0.4343~0.0038~t. TABLE SPECIFIC
DISCUSSION
The carnitine concentration in muscle, blood, and urine has previously been determined in different species by chemical
FIG. 6. Comparison of the calculated specific radioactivity of carnitine in compartment 2 (-1 (SB,) and the experimental values of specific radioactivity of carnitine in muscle tissue (A-A) versus time for rats injected with labeled ccarnitine. The equation of the late part (72-384 hl of the experimental curve is log SBt = log 38.3 - 0.4343. 0.0024.t.
2 3 4
4 5 8 8
5
9
6 7
10 10 11 12 12 13
DL 1
k O.OOOSCSE).t.
Table VI shows the expression for SA, for nine rats, which had been followed between Days 4 and 12. The values of X2were 0.0030 to 0.0053 h-l, i.e., slightly higher than corresponding values for rats injected with labeled L-carnitine.
Days
Rat
8 L
9 1 2
VI
RADIOACTIWTY OF CARNITINE FOR INDIVIDUAL RAT@
IN PLASMA
Equation of regression curve y = 5g.g.e-0.0”2“ y = 70.3.c-0.0050.f y = fJo~o*~-0.0~o~t y = 64.fj.e-0.m0.f y = ~o~o.e~.0053”
y y y y y y
= = = = = =
56.g.e-0.0034.f
62.2.e-0.0034.t 67.6.ed.WO” 66.7.e”.N”f 57.O&l.W28’” 52.3.e+.00z7.t
r 0.86 0.96 0.98 0.95
0.85 0.88 0.92
0.89 0.94 0.80 0.94
a Equations of regression curve of specific radioactivity @A,) of carnitine in plasma afier Day 3 as a function of time for individual rats. The coefficient of correlation is indicated by r. The rats were injected with labeled m-camitine and tcarnitine, respectively. Blood was sampled during 4 to 13 days.
(131, enzymatic (14), or most recently, microbiological techniques (15). It is obvious that there is a considerable difference in the carnitine concentration between different species, e.g., with the same method (8) the carnitine concentration of skeletal muscle tissue in humans was 16.2 pmol/g dry wt (21, in rats 3.1 pmollg dry wt, and in mice 1.7 pmol/g dry wt (8). The carnitine concentration of skeletal muscle tissue in sheep is even higher, -50 pmol/g dry wt (3). For rats, most published figures fall in the range of 3 to 5 pmol/g dry wt, but there is a considerable variation in the
METABOLISM
OF
LABELED
published figures (1). The concentration of carnitine in rat plasma (38.3 ymollliter) is about the same as reported for humans, i.e., 51 pmol/liter (8) or 23-70 pmobliter (16). Both humans (17) and rats show a rather large variation in urinary excretion of carnitine, even for a single individual. The renal clearance of carnitine for the present rats was 82 ml/24 h, i.e., about 50 ml/24 h and 100 g body wt, whereas the glomerular filtration rate is about 1700 ml/24 h and 100 g body wt in the rat (18). Thus, carnitine is reabsorbed to about the same extent as amino acids. Based on observations of the rate of excretion of isotope in urine and respiratory CO, after administration of L-We14C]carnitine and it [cm-boxy14C]carnitine, Lindstedt and Lindstedt (19) concluded that carnitine has a “slow” fractional turnover rate. Mehlman et al. (20, 21) administered DL-[MeJ4C]carnitine and followed the change of specific radioactivity of carnitine in urine over an 8-day period beginning on the third day after administration of isotope in normal as well as in diabetic and in choline-deficient rats. The data were interpreted on the basis of a one-compartment metabolic model and yielded a turnover time of 14.2 days for the normal rats. In the present study we interpreted our data on the basis of a two-compartment model. The kinetic parameters in the model were calculated from measurements of specific radioactivity of carnitine in plasma. The model was then validated by comparing predicted and measured values for the specific radioactivity of carnitine in blood, muscle tissue and urine. For instance, the shape of the curve of measured specific radioactivity of carnitine in muscle vs time showed good agreement with that predicted from the model. The smaller compartment was estimated to contain 14 pm01 of carnitine indicating that it represents not only the extracellular water space but also tissues with a high fractional turnover rate, e.g., liver. The larger compartment was estimated to contain 40-50 ,umol of carnitine and would represent mainly muscle tissue. Assuming that a rat weighing 160 g contains about 75 g muscle tissue (22), the total muscle car-
CARNITINE
179
nitine would be about 55 pmol. An approximative figure for the body pool of carnitine arrived at by the compartmental analysis would be 35 kmol/lOO g body wt. Tsai et al. (23, 24) report a figure around 40 pmol/lOO g body wt based on direct chemical determination in carcass of rats. Mehlman et al. (20, 21) reported the body pool of carnitine in rats to be 159 pmol/lOO g body wt, which is considerably higher than our figure. However, their use of an one-compartment model would tend to overestimate the body pool. Their figures for carnitine concentration in muscle and for urinary carnitine excretion as determined by the bromphenolblue method (13) are also considerably higher than most published figures. Apart from the methodological differences, strain differences and differences in nutritional regime might also account for the discrepancy between the results. The slopes of the early and late component of the specific radioactivity curve of carnitine in plasma were 2.13 and 0.063124 hr for rats injected with labeled L-carnitine. For the rats injected with nL-carnitine, the slope of the late component was slightly steeper (0.091/24 h). The slopes reported by Mehlman et al. (20, 21) for four normal rats are in agreement with the values of the slope of the late component in the present study, as are also recent data by Brooks and McIntosh (25). In the present study, about 7%, i.e., 3.9 pmol (2.3 pmol/lOO g body wt) of the total body pool was eliminated per 24 h. Brooks and McIntosh (25) used data for specific radioactivity of plasma carnitine to calculate a daily turnover rate of 2.6 pmol/lOO g body wt. The dietary intake was estimated to be about 0.7 pmol (the daily intake of pellets determined for three rats was 15 g and the carnitine content in pellets was 0.047 pmol/g). Assuming that the rats were in steady state, the daily synthesis of carnitine would thus be about 3 pmol. The kinetic analysis indicated a higher elimination of carnitine than could be accounted for by chemical analysis in the urine. This could be caused by an error in determining the slope of the first part of
180
CEDERBLAD
AND
the curve of specific radioactivity vs time- which is less well defined than the later part of the curve-or by the presence of other routes of elimination. Brooks and McIntosh (25) could account for only 30% of the calculated turnover as urinary carnitine. Khairallah and Wolf (4) have reported the presence of a carnitine decarboxylase in rat tissues but neither we nor Brooks and McIntosh (25) have found evidence for the presence of labeled p-methylcholine or other metabolites in urine. In a previous study (19) only about 1% of an administered dose of L-[Me-14Clcarnitine was recovered as respiratory COz during a 24-h period. Thus, at present there exists an unexplained discrepancy between the elimination as calculated from kinetic data and the measured urinary excretion of carnitine. ACKNOWLEDGMENT This work was supported by Grant No. 13X-585 from The Swedish Medical Research Council and a grant from Geteborgs Lakareslillskap. REFERENCES 1. BROEKHYSEN, J., ROZENBLUM, C., GHISLAIN, M., AND DELTOUR, G. (1965) in Recent Research on Carnitine (Wolf, G., ed.), pp. 23-25, MIT Press, Cambridge, Mass. 2. CEDE-LAD, G., LINDSTEDT, S., AND LUNDHOLM, K. (1974) Clin. Chim. Actu 53, 311-321. 3. SNOSWELL, A. M., AND KOUNDAKJIAN, P. P. (1972) B&hem. J. 127, 133-141. 4. KHAIRALLAH, E. A., AND WOLF, G. (1967) J.
BioE. Chem. 242, 32-39. 5. ST~KKE,
O.,
AND
BREMER,
J.
(1970)
Biochim.
Biophys. Actu 218, 552-554. 6. FRITZ,
I. B., SCHULTZ,
S. K.,
AND SRERE, P. A.
LINDSTEDT
(1963) J. Bid. Chem. 238, 2509-2517. S. (1967) B&him. Biophys. Acta 144, 83-93. 8. CEDERBLAD, G., AND LINDSTEDT, S. (1972) Clin. Chim. Acta 37, 235-243. 9. TUBBS, P. K., PEARSON, D. J., AND CHASE, J. F. A. (1965) in Recent Research on Camitine 7. FROBERG,
10. 11. 12. 13. 14. 15.
(Wolf, G., ed.), pp. 117-125, MIT Press, Cambridge, Mass. SOKAL, R. R., AND ROHLF, F. J. (1969) in Biometry, p. 211, Freeman, San Francisco. RESCIGNO, A., AND SEGRE, G. (1966) in Drug and Tracer Kinetics, pp. 27-29, Blaisdell, London. CEDERBLAD, G., FLODERUS, A., AND KARLSSON, K.-E., Acta Physiol. Scund., in press. MEHLMAN, M. A., AND WOLF, G. (1962) Arch. B&hem. Biophys. 98, 146-153. MARQUIS, N. R.., AND FRITZ, I. B. (1964) J. Lipid Res. 5, 184-187. TRAVASXJS, L. R., AND SALES, C. 0. (1974)AnaZ.
Biochem. 58, 485-499. 16. DIMAURO, S., SCOTT, C., PENN, A. S., AND RowLAND, L. P. (1973) Arch. Neural. 28, 186-190. 17. CEDERBLAD, G., AND LINDSTEDT, S. (1971) Clin. Chim. Actu 33, 117-123. J. W., JR., AND SCHILLING PHILLIPS, 18. BAUMAN, E. (1970) Amer. J. Physiol. 218, 1605-1608. S., AND LINDSTEDT, G. (1961) Actu 19. LINDSTEDT, Chem. Scud. 15, 701-702. 20. MEHLMAN, M. A., ABDEL KADER, M. M., AND THERRIAULT, D. G. (1969) Life Sci. 8,465-472. 21. MEHLMAN, M. A., THERRIAULT, D. G., AND T+ BIN, R. B. (1971) Metabolism 20, 100-107. N., AND ASPIN, N. (1972) Amer. J. 22. MARCEAU, Physiol. 222, 106-110. G. A. 23. TSAI, A. C., ROMSOS, D. R., AND LEVEILLE, (1974) J. Nutr. 104, 782-792. 24. TSAI, A. C., R~MSOS, D. R., AND LEVEILLE, G. A. (1975) J. Nutr. 105, 301-307. J. E. A. (1975) 25. BROOKS, D. E., AND MCINTOSH, Biochem. J. 148, 439-445. T. (1974) Biochim. Biophys. Actn 343, 26. B~HMER, 551-557.
OF BIOCHEMISTRY
AND
BIOPHYSICS
Metabolism GITTEN Department of Clinical
175, 173-180 (1976)
of Labeled CEDERBLAD
Carnitine AND
SVEN
in the Rat LINDSTEDT
Chemistry, University of Gothenburg, S-413 45 Gothenburg, Sweden
Sahlgren’s Hospital,
Received December 16, 1975 In two series of rats, the concentration of camitine in plasma was 39.9 and 37.8 pmol/ liter, in skeletal muscle tissue 2.97 and 3.26 pmol/g dry wt and the urinary excretion 3.2 and 2.4 pmo1/24 h. The renal clearance of camitine was calculated to 88 and 76 ml/24 h. L-[Me-14ClCarnitine and nr.-[Me-‘4Clcarnitine have been administered to rats. Only labeled Gcamitine has been found on chromatographic analysis of plasma, urine, and muscle tissue. The specific radioactivity of carnitine in plasma, urine, and muscle tissue has been followed for up to 16 days. A two-compartment metabolic model has been used to interpret the result of the experiment with labeled L-carnitine and the rate constants and compartment sizes have been calculated. The total body content of camitine was 57 pmol (about 35 pmol/lOO g body wt) and the daily turnover was about 7% of the body pool. The daily synthesis of camitine in the rat is estimated to about 2 pmol/lOO g body
wt. Carnitine (4-trimethylamino-3-hydroxybutyrate) plays a major role in the transport of activated long-chain fatty acyl groups from sites of activation to sites of poxidation in the mitochondria. Recent works (l-3) have established that there exists a pronounced species difference in carnitine concentration in skeletal muscle tissue. Less is known about the metabolism of carnitine itself. Apparently, nutritional and physiological conditions may influence carnitine metabolism (4). Khairallah and Wolf (4) identified a decarboxylation product of carnitine, P-methylcholine in rat urine. Starvation, alloxan diabetes, and feeding a high-fat diet were among the conditions that increased the decarboxylation reaction. The mechanisms behind the observed facts are not known. We considered it of interest to make a detailed study of carnitine metabolism in the intact normal rat involving both chemical determination of carnitine concentration in tissues and urine and an attempt to formulate a kinetic model for carnitine metabolism. MATERIALS AND METHODS Labeled carnitine. L-[MeJ4C]Carnitine was prepared according to Stokke and Bremer (5). Thin173 Copyright All rights
8 1976 by Academic Press, Inc. of reproduction in any form reserved.
layer chromatography on silica gel G in two different solvent systems of the prepared labeled L-carnitine gave one spot when the distribution of radioactivity was determined with a thin-layer scanner (Labor, Prof. Dr. Berthold Wildbad, West Germany). The R,-value was the same as for authentic camitine detected with iodine vapor. The two solvent systems used were methanol-concentrated ammonia (l:l, by volume) and chloroform-methanolwater-concentrated ammonia-formic acid (55:50: l&7.5:2.5, by volume). To establish that no racemization had occurred in the preparation of labeled ccamitine, we used the preparation in an enzymatic assay. After thin-layer separation the amount of Gacetylcarnitine formed was calculated. As seen in Table I, the amount formed was in good agreement with that expected if no racemization had occurred. nL-[Me-‘4C]Camitine had a specific radioactivity of 52 mCilmmo1 and was obtained from The Radiochemical Centre, Amersham, Bucks., England. Animals. Female rata of the Sprague-Dawley and Wistar strains, free of specific pathogen and weighing 150-220 g, were obtained from Miillegaards Avelslaboratoriet A/S, Skensved, Denmark. The rata had free access to water and a commercial pellet diet (EWOS, S&letilje, Sweden). Labeled nL-camitine, 6.66.106 cpm per rat, was injected intraperitoneally into 12 rats of the SpragueDawley strain. Labeled ticarnitine, 3.43.106 cpm per rat was injected intraperitoneally into 16 rata of the Wistar strain. The animals were kept in individ-
174
CEDERBLAD TABLE
FORMATION
1
OF L-ACETYLCARNITINE L- AND DL-CARNITINE"
Labeled carnitine
FROM
LABELED
Percentage recovered in acetylcarnitine Experimentally
L DL
AND LINDSTEDT
37 65
Calculated 39 59
o The labeled substrate was incubated with carnitine acetyltransferase and the reaction mixture was separated by thin-layer chromatography (chloroform-methanol-water-concentrated ammonia-formic acid 50:50:10:7.5:2.5 by volume, on silica gel G). The values were calculated by using 1.8 for the equilibrium constant of the reaction: carnitine + acetyl-CoA ti acetylcarnitine + CoASH (6). ual metabolic cages. Blood sampling was performed with a heparinized capillary tube in the medial angle of the eye during light ether anesthesia. After centrifugation, the plasma was kept frozen at - 20°C until analyzed. Urine was collected daily in bottles containing 0.5 ml of 6 M HCl as perservative and was kept frozen until analyzed. At different time intervals the animals were decapitated and preferentially red muscle tissue was removed from the gastrocnemius muscle (7). The muscle tissue was immediately frozen in liquid nitrogen and stored at -60°C until analyzed. Determination of carnitine. Camitine determination was performed with an enzymatic method USing acetyl-CoA:camitine-O-acetyltransferase (EC 2.3.1.7) and labeled [l-‘4Clacetyl-coensyme A as substrate (8). After precipitation with HClO, and neutralization with KOH, short-chain acylcarnitine was hydrolyzed in 0.1 M KOH according to Tubbs et al. (9). Radioactivity was measured in a Packard scintillation spectrometer (Model 3320, Packard Instrument Co., Downers Grove, Ill.). The amount of isotope in plasma was determined by adding 20 ~1 of plasma and 200 ~1 of water to 10 ml of In&a-Gel (Packard Instrument Co., Downers Grove, Ill. 1 in a plastic counting vial. The amount of isotope in urine was determined by adding 1 ml of urine to 10 ml of Insta-Gel and the amount of isotope in muscle by adding 300 ~1 of the muscle homogenate together with 800 ~1 of water to 10 ml of In&a-Gel. At least 5000 counts were recorded. RESULTS
Carnitine Concentration in Plasma and Muscle Tissue and Urinary Excretion The mean values of carnitine concentration in plasma were 39.9 and 37.8 pmol/ liter for the rats which had received la-
beled DL- and ccarnitine, respectively. The mean values of the concentration of perchloric acid soluble carnitine in muscle tissue were 2.97 and 3.26 pmol/g dry weight for the two groups of rata. No significant correlation was found between carnitine concentration in plasma and in muscle tissue. The average 24-h excretions of carnitine in the urine were 3.2 and 2.4 pmol for the rats injected with labeled DLcarnitine and L-carnitine, respectively. For rats given m-carnitine a computation of the components of intra- and interindividual variation (10) has been performed for carnitine concentration in plasma and for carnitine excretion in urine. In both cases, the intraindividual variance was the main contributory variance component. The mean values of renal carnitine clearance were 88 and 76 ml/24 h for the two groups (Table II). The carnitine concentration in plasma and the urinary excretion has been measured in individual rats for periods of 10 to 14 days and the renal clearance has been calculated (Table III). Characterization of Labeled Carnitine in Rat Tissue The extracts of plasma and muscle from rata injected with labeled Gcarnitine were chromatographed on an ion-exchange column, AG 50W X8 which was eluted with 1 M HCl. As shown in Fig. 1 only carnitine could be detected. The recoveries were 96108% of the applied radioactivity. The corresponding ion-exchange chromatograms of muscle homogenates are shown in Fig. 2. The homogenates were precipitated with HClO, and the supernatants were neutralized. In the chromatograms of both these supernatants and of alkali-hydrolyzed supernatants carnitine was the only radioactive peak. Labeled m-carnitine added to muscle homogenate which had been hydrolyzed coincided with the original peak, see Fig. 2. The recoveries were 96-100% of applied radioactivity. The chromatograms of two urine specimens from rats injected with labeled L-carnitine are shown in Fig. 3. The urine specimens had been collected during the first and the fourth days after administration of the iso-
METABOLISM
OF LABELED TABLE
CARNITINE
CONCENTRATION
IN PLASMA
Plasma concentration Muscle concentration Urinary
(~mol/liter) (pmol/g dry wt)
excretion (pmo1/24 h)
Renal clearance (ml/24 h)
II
AND MUSCLE
Injected isotope
TIWJE
AND URINARY
Mean value 2 SE 39.9 37.8 2.97 3.26 3.20
DL L DL L DL L DL L
175
CARNITINE
I$Lmdl;
+ f 2 k
2P
21.7 24.8 13.8 15.6 35.6 32.3 32.8 31.4
i-0.
suremerits
0.8 1.1 0.12 0.13 0.11
+
EXCRETIO#
Re3ez3ti&
112 69 12 16 111
41 79 31
2.41 + 0.12
88 k 3.2 76 2 4.3
n.s.
a Renal clearance has been calculated from urinary excretion per 24 h/plasma carnitine concentration. The values are given for rats injected with labeled m-camitine and L-carnitine, respectively. The two groups of rats have been compared with Student’s t test and the significance level is indicated. b (SD/mean) x 100. TABLE CARNITINE
Individual rata
IN PLASMA
Plasma concentration Mean + SE (~mol/liter)
III
AND URINE
excretion
Mean 2 SE @mob24 h)
19.2 3.73 2 35.1 I!?2.1 39.5 f 3.0 25.4 3.43 2 28.8 2.65 k 45.8 f 3.8 18.5 3.69 k 42.5 k 2.3 37.8 + 1.0 8.1 3.91 -+ 20.0 2.57 k 38.8 2 2.1 12.3 3.56 k 40.3 +: 1.4 L 1 32.2 2 1.1 13.8 2.59 iI 13.1 2.32 k 2 33.6 + 1.1 0 Camitine concentration in plasma, urinary excretion, rats injected with labeled DL- and L-carnitine. resnectively, . b (SD/mean) X 100. DL 1
2 3 4 5 6 7
tope. The main peak, at 5 column volumes, appeared at the position of carnitine. In these chromatograms there was also a few percent of radioactivity eluted after 1 column volume. No further analysis was done of this material. The recoveries of applied radioactivity were 92 and 83%, respectively. Compartmental
IN INDIVIDUAL
Urinary
Analysis
A semilogarithmic plot of the specific radioactivity of carnitine in plasma and in urine versus time for rats injected with labeled r,-carnitine is shown in Fig. 4. There was a good agreement between the specific radioactivities of carnitine over a period of 16 days. After an initial rapid fall
RAT@
Renal clearance yy2; m
flE -c 10 + 12 -t- 7 + 6
0.39 0.33 0.32 0.21
32.7 30.7 42.2 19.5
106 93 67 90
0.31
25.1
103 r 7
0.19 0.30 0.15
0.20 and and
Relative SD (%)b 31.0 41.5 31.5 22.5 22.1
26.9 71 ” 5 23.4 31.8 89 2 7 32.4 22.5 83 f 6 30.0 32.7 69 f 6 31.1 renal clearance of carnitine for individual followed for 10 to 14 days.
in specific radioactivity, the data fell on a straight line. Two-compartment open system. The two-compartment open system has been chosen as the simplest possible system to explain the distribution of labeled ccarnitine in the rat; see inset in Fig. 4. The assumption is made that the labeled substance is introduced into the first cornpartment, i.e., the absorption from the peritoneal cavity occurs at a greater rate than any subsequent transformation. Three different models can be distinguished in this system (ll), i.e., elimination occurs only from compartment 1 (model 11, elimination occurs only from compartment 2 (model 21, or elimination occurs from both compartments (model 3).
176
CEDERBLAD 300
300 2Lh
2h
;
AND LINDSTEDT
200
600
200
1 5," 9
100
100
-I--n
R
.5 E zl a B 5 u"
LOO
200
0
-0
30
60
90 fraction
rroct1on
no
FIG. 1. Ion-exchange chromatography of plasma extracts from rats injected with labeled n-carnitine. The animals had been killed at the time indicated in the figure. The samples were applied onto a column (0.5 x 4.5 cm) of AG 50W X8. The column was eluted with 1 M HCl in fractions of 0.12 ml.
-0
30
60
90
no
FIG. 3. Ion-exchange chromatography of urine samples or rats injected with labeled ccamitine. Collection periods are as indicated in the figure. Urine samples were applied onto a column (1.6 x 10 cm) of AG 50W X8. The column was eluted with 1 M HCl, fraction l-40 and with 4 M HCl, fraction 41-90. The fraction volume was 5 ml.
FIG. 4. Specific radioactivity of carnitine in plasma (+O) and urine (0-O) versus time for rate injected with labeled L-camitine. The curve represents the best-fitting function of all values of the specific radioactivity of camitine in plasma. The inset shows a two-compartment system with corresponding rate constants (k,-k,).
frachll
no
FIG. 2. Ion-exchange chromatography of skeletal muscle extracts from rats injected with labeled Lcamitine. The animals had been killed at the time indicated in the figure. Unhydrolyzed supematants (60) and the same supematants hydrolyzed in alkali (O0) were applied onto a column (0.5 X 4.5 cm) of AG 50W X8. The column was eluted with 1 M HCl in fractions of 0.12 ml. The dotted line represents the increase in radioactivity after addition of labeled nL-carnitine to the sample before application.
Selection of model and resulting pool sizes and rate constants. The specific radioactivities (SA and SB) in the two compartments in an open two-compartment model follow equations of the type S& = M1.e-h’t
+ M2-e-a’t
SBt = N-(e-h’t
- e-h’“),
where M, is SAt; (h, - k,MX, - A,), MZ is SA,; (k2 - X,)/oC, - A,) and N is SAt;W (A, - A,). The best-fitting curve of the measure-
METABOLISM
OF LABELED
ments of specific radioactivity of plasma was calculated using a computer program (12). This curve is shown in Fig. 4 for the specific radioactivity (SA,) of carnitine in plasma, i.e., in compartment 1. The values for A1 and hz are given in Table IV. Knowing the specific radioactivity of carnitine at t,,, the size of compartment 1 was calculated to be 14 pmol by the principle of isotope dilution. It is possible to determine all the rate constants for models 1 and 2 from measurements of compartment 1, whereas for model 3 additional information is necessary. As the specific radioactivities of carnitine in urine and plasma were the same, the urinary elimination occurred from compartment 1 and model 2 was thus rejected. The rate constant for the elimination in urine (hJ in model 1 was 0.011 h-l. The same rate constant also could be calculated from the known excretion of carnitine in urine by the relation k, = 2.4/(14 x 24) h-l. A value of 0.0072 h-l was then obtained. We used this value of It, to calculate the parameters in model 3, i.e., assuming an elimination also from compartment 2. As seen in Table V this resulted in a somewhat larger estimate of the size of compartment 2 than calculated according to model 1. Figure 5 shows the relative amounts of isotope present in compartment 1 and 2 as calculated with the rate constants for TABLE CURVES OF SPECIFIC
177
CARNITINE TABLE
KINETIC
PARAMETERS
OF CARNITINE
METABOLJSI@
Model 1
Model 3
0.060 0.020 0.011 -
0.064 0.019 0.007P 0.0014
14 39
14 47
Rate constant (h-9
k, k, k3 k, Size (pmol) Compartment Compartment
V
1 2
a Rate constants were estimated on the basis of two models: (a) model 1, elimination only from compartment 1; (b) model 3, elimination from both compartment 1 and 2, cf. inset in Fig. 4. b Estimated from rate of excretion of camitine in urine.
0.01111111111111111111 0
24 46 12
96
120 14L
166192 216 240264266 hours
3l2 33636038L
FIG. 5. Distribution of isotope expressed as fraction of administered amount. The curves have been calculated with the values of the rate constants in model 1 which are given in Table V. Experimental values are indicated by (0-O) and cumulative excretion in urine by (O-O).
IV
RADIOACTIVITY
OF CABNITINE?
A, (h-l)
X2 (h-l)
Specific radioactivity of car0.089 0.0026 nitine in plasma (n-carnitine? Specific radioactivity of car0.0038 nitine in plasma (m-camitine? Specific radioactivity of caro.oiw 0.0024 nitine in muscle (L-camitiney a Values of A, and A, in the exponential expression for specific radioactivities in plasma (SA,) and muscle (SB,) versus time for rats who had received labeled L-camitine and m-camitine, respectively. b SAt = M,.e-“,” + &.f,.e-~~‘f. c S& = N.(e-h,.f - e-b.t). d t = O-8 h.
model 1 given in Table V and the expected cumulative elimination of isotope as well as the measured cumulative excretion in urine. As mentioned, the calculated value (0.011 h-l) for the rate constant for elimination calculated from the specific radioactivity of carnitine in plasma was higher than the value calculated from the urinary excretion of carnitine (0.0072 h-l). Measurements of compartment 2. The specific radioactivity of carnitine has been determined in muscle tissue, i.e., a tissue belonging to compartment 2. Using the experimentally found values of the specific radioactivity of carnitine in muscle tissue, X1determined from values between 0 and 8 h was 0.078 h-l and AZwas 0.0024 h-l. The
178
CEDERBLAD
AND LINDSTEDT
specific radioactivity of carnitine measured in muscle tissue is lower than the calculated specific radioactivity of carnitine in compartment 2, (SB,) as seen in Fig. 6. This is consistent with the fact that heart and muscle tissue have the highest carnitine content in the rat (1). Distribution of Labeled m-carnitine. In the experiments with rats injected with labeled nL-carnitine the specific radioactivity of carnitine has been followed during the late phase only. Figure 7 shows the specific radioactivity of carnitine in plasma and urine as a function of time between the third and the fourteenth day after the administration of the isotope. As was the case with labeled L-carnitine, there was a good agreement between the specific radioactivities of carnitine in urine and plasma. The equation of the regression curve of the specific radioactivity of carnitine in plasma versus time after Day 3 was SA, = 67.6 f 2.7
(SJi~.~-0.0038
FIG. 7. Specific radioactivity of carnitine in plasma (+O) and urine (O-O) versus time of nine rats injected with labeled m-carnitine. Standard deviation is indicated by a vertical bar. The regression line of specific radioactivity of carnitine in plasma versus time for the late phase is logSA, = log 67.6 - 0.4343~0.0038~t. TABLE SPECIFIC
DISCUSSION
The carnitine concentration in muscle, blood, and urine has previously been determined in different species by chemical
FIG. 6. Comparison of the calculated specific radioactivity of carnitine in compartment 2 (-1 (SB,) and the experimental values of specific radioactivity of carnitine in muscle tissue (A-A) versus time for rats injected with labeled ccarnitine. The equation of the late part (72-384 hl of the experimental curve is log SBt = log 38.3 - 0.4343. 0.0024.t.
2 3 4
4 5 8 8
5
9
6 7
10 10 11 12 12 13
DL 1
k O.OOOSCSE).t.
Table VI shows the expression for SA, for nine rats, which had been followed between Days 4 and 12. The values of X2were 0.0030 to 0.0053 h-l, i.e., slightly higher than corresponding values for rats injected with labeled L-carnitine.
Days
Rat
8 L
9 1 2
VI
RADIOACTIWTY OF CARNITINE FOR INDIVIDUAL RAT@
IN PLASMA
Equation of regression curve y = 5g.g.e-0.0”2“ y = 70.3.c-0.0050.f y = fJo~o*~-0.0~o~t y = 64.fj.e-0.m0.f y = ~o~o.e~.0053”
y y y y y y
= = = = = =
56.g.e-0.0034.f
62.2.e-0.0034.t 67.6.ed.WO” 66.7.e”.N”f 57.O&l.W28’” 52.3.e+.00z7.t
r 0.86 0.96 0.98 0.95
0.85 0.88 0.92
0.89 0.94 0.80 0.94
a Equations of regression curve of specific radioactivity @A,) of carnitine in plasma afier Day 3 as a function of time for individual rats. The coefficient of correlation is indicated by r. The rats were injected with labeled m-camitine and tcarnitine, respectively. Blood was sampled during 4 to 13 days.
(131, enzymatic (14), or most recently, microbiological techniques (15). It is obvious that there is a considerable difference in the carnitine concentration between different species, e.g., with the same method (8) the carnitine concentration of skeletal muscle tissue in humans was 16.2 pmol/g dry wt (21, in rats 3.1 pmollg dry wt, and in mice 1.7 pmol/g dry wt (8). The carnitine concentration of skeletal muscle tissue in sheep is even higher, -50 pmol/g dry wt (3). For rats, most published figures fall in the range of 3 to 5 pmol/g dry wt, but there is a considerable variation in the
METABOLISM
OF
LABELED
published figures (1). The concentration of carnitine in rat plasma (38.3 ymollliter) is about the same as reported for humans, i.e., 51 pmol/liter (8) or 23-70 pmobliter (16). Both humans (17) and rats show a rather large variation in urinary excretion of carnitine, even for a single individual. The renal clearance of carnitine for the present rats was 82 ml/24 h, i.e., about 50 ml/24 h and 100 g body wt, whereas the glomerular filtration rate is about 1700 ml/24 h and 100 g body wt in the rat (18). Thus, carnitine is reabsorbed to about the same extent as amino acids. Based on observations of the rate of excretion of isotope in urine and respiratory CO, after administration of L-We14C]carnitine and it [cm-boxy14C]carnitine, Lindstedt and Lindstedt (19) concluded that carnitine has a “slow” fractional turnover rate. Mehlman et al. (20, 21) administered DL-[MeJ4C]carnitine and followed the change of specific radioactivity of carnitine in urine over an 8-day period beginning on the third day after administration of isotope in normal as well as in diabetic and in choline-deficient rats. The data were interpreted on the basis of a one-compartment metabolic model and yielded a turnover time of 14.2 days for the normal rats. In the present study we interpreted our data on the basis of a two-compartment model. The kinetic parameters in the model were calculated from measurements of specific radioactivity of carnitine in plasma. The model was then validated by comparing predicted and measured values for the specific radioactivity of carnitine in blood, muscle tissue and urine. For instance, the shape of the curve of measured specific radioactivity of carnitine in muscle vs time showed good agreement with that predicted from the model. The smaller compartment was estimated to contain 14 pm01 of carnitine indicating that it represents not only the extracellular water space but also tissues with a high fractional turnover rate, e.g., liver. The larger compartment was estimated to contain 40-50 ,umol of carnitine and would represent mainly muscle tissue. Assuming that a rat weighing 160 g contains about 75 g muscle tissue (22), the total muscle car-
CARNITINE
179
nitine would be about 55 pmol. An approximative figure for the body pool of carnitine arrived at by the compartmental analysis would be 35 kmol/lOO g body wt. Tsai et al. (23, 24) report a figure around 40 pmol/lOO g body wt based on direct chemical determination in carcass of rats. Mehlman et al. (20, 21) reported the body pool of carnitine in rats to be 159 pmol/lOO g body wt, which is considerably higher than our figure. However, their use of an one-compartment model would tend to overestimate the body pool. Their figures for carnitine concentration in muscle and for urinary carnitine excretion as determined by the bromphenolblue method (13) are also considerably higher than most published figures. Apart from the methodological differences, strain differences and differences in nutritional regime might also account for the discrepancy between the results. The slopes of the early and late component of the specific radioactivity curve of carnitine in plasma were 2.13 and 0.063124 hr for rats injected with labeled L-carnitine. For the rats injected with nL-carnitine, the slope of the late component was slightly steeper (0.091/24 h). The slopes reported by Mehlman et al. (20, 21) for four normal rats are in agreement with the values of the slope of the late component in the present study, as are also recent data by Brooks and McIntosh (25). In the present study, about 7%, i.e., 3.9 pmol (2.3 pmol/lOO g body wt) of the total body pool was eliminated per 24 h. Brooks and McIntosh (25) used data for specific radioactivity of plasma carnitine to calculate a daily turnover rate of 2.6 pmol/lOO g body wt. The dietary intake was estimated to be about 0.7 pmol (the daily intake of pellets determined for three rats was 15 g and the carnitine content in pellets was 0.047 pmol/g). Assuming that the rats were in steady state, the daily synthesis of carnitine would thus be about 3 pmol. The kinetic analysis indicated a higher elimination of carnitine than could be accounted for by chemical analysis in the urine. This could be caused by an error in determining the slope of the first part of
180
CEDERBLAD
AND
the curve of specific radioactivity vs time- which is less well defined than the later part of the curve-or by the presence of other routes of elimination. Brooks and McIntosh (25) could account for only 30% of the calculated turnover as urinary carnitine. Khairallah and Wolf (4) have reported the presence of a carnitine decarboxylase in rat tissues but neither we nor Brooks and McIntosh (25) have found evidence for the presence of labeled p-methylcholine or other metabolites in urine. In a previous study (19) only about 1% of an administered dose of L-[Me-14Clcarnitine was recovered as respiratory COz during a 24-h period. Thus, at present there exists an unexplained discrepancy between the elimination as calculated from kinetic data and the measured urinary excretion of carnitine. ACKNOWLEDGMENT This work was supported by Grant No. 13X-585 from The Swedish Medical Research Council and a grant from Geteborgs Lakareslillskap. REFERENCES 1. BROEKHYSEN, J., ROZENBLUM, C., GHISLAIN, M., AND DELTOUR, G. (1965) in Recent Research on Carnitine (Wolf, G., ed.), pp. 23-25, MIT Press, Cambridge, Mass. 2. CEDE-LAD, G., LINDSTEDT, S., AND LUNDHOLM, K. (1974) Clin. Chim. Actu 53, 311-321. 3. SNOSWELL, A. M., AND KOUNDAKJIAN, P. P. (1972) B&hem. J. 127, 133-141. 4. KHAIRALLAH, E. A., AND WOLF, G. (1967) J.
BioE. Chem. 242, 32-39. 5. ST~KKE,
O.,
AND
BREMER,
J.
(1970)
Biochim.
Biophys. Actu 218, 552-554. 6. FRITZ,
I. B., SCHULTZ,
S. K.,
AND SRERE, P. A.
LINDSTEDT
(1963) J. Bid. Chem. 238, 2509-2517. S. (1967) B&him. Biophys. Acta 144, 83-93. 8. CEDERBLAD, G., AND LINDSTEDT, S. (1972) Clin. Chim. Acta 37, 235-243. 9. TUBBS, P. K., PEARSON, D. J., AND CHASE, J. F. A. (1965) in Recent Research on Camitine 7. FROBERG,
10. 11. 12. 13. 14. 15.
(Wolf, G., ed.), pp. 117-125, MIT Press, Cambridge, Mass. SOKAL, R. R., AND ROHLF, F. J. (1969) in Biometry, p. 211, Freeman, San Francisco. RESCIGNO, A., AND SEGRE, G. (1966) in Drug and Tracer Kinetics, pp. 27-29, Blaisdell, London. CEDERBLAD, G., FLODERUS, A., AND KARLSSON, K.-E., Acta Physiol. Scund., in press. MEHLMAN, M. A., AND WOLF, G. (1962) Arch. B&hem. Biophys. 98, 146-153. MARQUIS, N. R.., AND FRITZ, I. B. (1964) J. Lipid Res. 5, 184-187. TRAVASXJS, L. R., AND SALES, C. 0. (1974)AnaZ.
Biochem. 58, 485-499. 16. DIMAURO, S., SCOTT, C., PENN, A. S., AND RowLAND, L. P. (1973) Arch. Neural. 28, 186-190. 17. CEDERBLAD, G., AND LINDSTEDT, S. (1971) Clin. Chim. Actu 33, 117-123. J. W., JR., AND SCHILLING PHILLIPS, 18. BAUMAN, E. (1970) Amer. J. Physiol. 218, 1605-1608. S., AND LINDSTEDT, G. (1961) Actu 19. LINDSTEDT, Chem. Scud. 15, 701-702. 20. MEHLMAN, M. A., ABDEL KADER, M. M., AND THERRIAULT, D. G. (1969) Life Sci. 8,465-472. 21. MEHLMAN, M. A., THERRIAULT, D. G., AND T+ BIN, R. B. (1971) Metabolism 20, 100-107. N., AND ASPIN, N. (1972) Amer. J. 22. MARCEAU, Physiol. 222, 106-110. G. A. 23. TSAI, A. C., ROMSOS, D. R., AND LEVEILLE, (1974) J. Nutr. 104, 782-792. 24. TSAI, A. C., R~MSOS, D. R., AND LEVEILLE, G. A. (1975) J. Nutr. 105, 301-307. J. E. A. (1975) 25. BROOKS, D. E., AND MCINTOSH, Biochem. J. 148, 439-445. T. (1974) Biochim. Biophys. Actn 343, 26. B~HMER, 551-557.