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L-CARNITINE AND CARNITINE ESTER TRANSPORT IN THE RAT SMALL INTESTINE R MARCIANI*~f, C. LINDI*, A. MARZOt, E. ARRIGONI MARTELLIt, G. CARDACEt and the late G. ESPOSITO

*Istituto di Fisiologia Generale e Chimica Biologica, Facoltd di Farmacia, Via Saldini 50, 20133 Milan, Italy; tSigma Tau SpA, Via Pontina Kin. 30.400, 00040 Pomezia, Rome, Italy Received in final form 9 July 1990

SUMMARY L-Carnitine and its esters (acetyl-L-carnitine and propionyl-e-carnitine) at pharmacological doses (1, 5 and 10 raM) are absorbed by the rat jejunum by simple diffusion. Partition coefficients of carnitine esters determined in lipophilic media (diethyl ether/water and olive oil/water) are greater than that of L-carnitine. It would therefore seem that esters diffuse more easily through the lipid component of the intestinal barrier. The transport of acetyl- and propionyl-L-carnitine at pharmacological doses seems to be linearly and positively correlated with K + transport but not with Na + transport. KEYWORDS:L-carnitine,L-carnitineesters, intestinal absorption, Na + transport, K + transport. INTRODUCTION L-Carnitine is a substance of great significance since it is involved in fatty acid oxidation, i.e. in the translocation of long-chain fatty acids into mitochondria for/3oxidation. In addition, L-carnitine regulates the production of metabolic energy from proteins and carbohydrates [1-4]. Several pathological syndromes derive from systemic L-carnitine deficiency [5]. In spite of its physiological importance Lcarnitine is absorbed slowly by the intestine. Much effort has been devoted to seeking to understand the mechanisms involved in the intestinal absorption of exogenous L-carnitine. L-Carnitine is transported by the intestine via a Na+-dependent, carrier-mediated active transport system, which is easily demonstrated at intraluminal physiological doses (of the order of ~molar concentration), whereas at pharmacological doses (of the order of mmolar concentration) a diffusional component of the absorption is predominant; L-carnitine is preferentially and better absorbed than its biological inactive isomer, D-carnitine [6, 7]. ~To whom correspondence should be addressed.

1043-6618/91/020157-06/S03.00/0

© 1991 The Italian Pharmacological Society

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The aim of this investigation was to study the net transintestinal transport of Lcarnitine and its endogenous esters (acetyl- and propionyl-L-carnitine) at pharmacological doses, together with other properties, such as the relationship with the transport of electrolytes (Na + and K +), partition coefficients and intracellular concentration of solutes.

MATERIALS AND METHODS

Male albino rats (Wistar strain, Charles River Italiana) weighing 150-250 g and fasting overnight were used. Net transport of solutes was determined in the everted, cannulated and incubated sac of rat jejunum as described previously [8]. Incubation at 28°C in 50 ml of Krebs-Ringer bicarbonate buffer containing 1, 5 and 10 mM L-carnitine or acetyl-L-carnitine or propionyl-L-carnitine lasted 30 min. The solution was gassed with 95% 02 and 5% CO2. L-Carnitine and its esters were gifts from Sigma Tau, Rome, Italy. Trace amounts of L-(methyl-3H)carnitine'HC1 (specific activity 72 TBq/mmol, obtained from Amersham, Arlington Heights, I1) or acetyl-L-(methyl-3H)carnitine •HC1 or propionyl-k(methyl-BH)carnitine •HC1 were added to each incubating solution. Acetyl-c-(3H)carnitine and propionyl-L-(3H)carnitine were synthesized from L(methyl-3H)carnitine according to the following reactions catalysed by carnitine acetyltransferase (CAT) where the asterisk indicates labelled substances [9]: CAT

c-carnitine* + AcetylCoA ~ Acetyl-L-carnitine* + CoASH CAT

c-carnitine* + PropionylCoA ~ Propionyl-L-carnitine* + CoASH The cellular solute concentrations are expressed in mmol/1 of intracellular water; cell water is given in ml/g dry weight of scraped mucosa and net transport of solutes is expressed in ktmol or ml/g dry weight of scraped mucosa per hour. Intracellular water and, consequently, intracellular solute concentrations were obtained after determining extracellular spaces by means of polyethylene [14C]glycol (PEG 900) as previously reported [10]. Radioactivity was counted in the Liquid Scintillator Spectrometer Mod 1500 (Packard Instruments Company) using Instagel (Packard Instruments Company) as scintillator cocktail. Na + and K ÷ were measured by a Flame Fotometer Mod 9943 (Instrumentation Laboratory). In order to investigate partition coefficients, trace amounts of 3H-labelled compounds were added to cold substances at 10 mM concentration. Diethyl ether or n-butanol or olive oil were used as organic phase, whereas the buffer solution (120 mM NaC1, 20 mM Na2HPO4 and 50 m M KI-I2PO4) was at pH 6.7. Samples were thoroughly mixed, let to stand for a period lasting 0 (immediately after mixing), 5 h and 24 h, and radioactivity was then determined in the separated phases. Consistent results were found at the 24 h test.

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RESULTS Table I shows partition coefficients of c-carnitine, acetyl- and propionyl-c-carnitine between a water medium buffered at pH 6.7 and three organic solvents, namely nbutanol, diethyl ether and olive oil. With the three compounds investigated, partition coefficients are the highest with n-butanol and decreased with diethyl ether and olive oil in that order. Partition coefficients do not vary with the three substances in the case of n-butanol, whereas with diethyl ether and olive oil they increase in the two esterified L-carnitines, reaching the highest value with acetyl-ccarnitine.

Table I Partition coefficients (K) of c-carnitine and its acyl derivatives between three organic solvents and a buffer solution at pH 6.7 (120 mM NaCI, 20 mM NazHPO 4 and 50 mM KHzPO4) Organic phase

L-Carnitine

Acetyl-Lcamitine

Propionyl-Lcamitine

n-Butanol Diethyl ether Olive oil

4.7x 10 2 2.8 x 10 4 1.l x 10 ('

4.3 x 10 -2 8.3 X 10 -4 10.6 x 10 -~'

4.4x 10 -2 5.1~X 10 -~ 5.1 x 10 -('

Values are means of two determinations. Table II reports the solute transport values. It is interesting to note that there is a direct linear correlation between acetyl- and propionyl-L-carnitine transport and K + transport: y = 2 . 3 8 3 + 0 . 0 7 5 x (r 2=0.984) and y - = 3 4 . 5 3 8 + 0 . 4 1 x (r2=0.985) respectively. The slope of this correlation is more pronounced for propionyl- than for acetyl-L-carnitine. There is no correlation between L-carnitine and K + transport. In any case K + transport does not reach any statistically significant degree in the case of propionyl-L-carnitine. As shown in Fig. 1, there is a direct linear correlation between net transintestinal transport of c-carnitine, acetyl-c-carnitine, and propionyl-L-carnitine and their mucosal concentration, at least in the range of 1-10 mM. It should be noted that active transport is revealed only at ktmolar concentration and that physiological serum c-carnitine concentration is around 45-50 ,uM. Table III shows that cell electrolyt e concentrations are not affected by .the presence of the three substances and that, at the concentrations used, cell accumulation does not occur.

D I S C U S S I O N AND C O N C L U S I O N S Data obtained in this study allow the investigators to clarify better the transport of c-carnitine and its endogenous esters across the intestine epithelium in concentrations more related to pharmacological than physiological doses. All the

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Table II Solute transport in everted rat jejunum at three different L-carnitine, acetyl-Lcarnitine and propionyl-L-carnitine concentrations Incubating solution Krebs-Ringer bicarbonate buffer+ proper substance at the indicated concentration

Transport (~mol/g/h ) Na +

L-Carnitine 1 mM 5 mM 10 mM

1310 -+ 258 738-+225 679 + 151

Acetyl-L-carnitine 1 mM 5 mM 10 mM

679 + 124 916+--'279 898 -+ 183

Propionyl-L-carnitine 1 mM 5 mM 10 mM

1001 + 145 671 -+223 480 --+10

K+

Substance

59 -t- 15 83-+ 18 85 + 9

3.55 _+0.79 9.44--+2.81 23.32 -+ 5.93*

11 _+2 105 + 12"* 125 -+ 10"**

3.32 -+ 1.09 9.62+5.26 12.28 + 1.87"*

90+8 96--+25 11.4 -+ 4

2.03 --+0.45 5.75 --+0.97* 12.28 + 0.87***

Mean values of at least three findings _+SE. *P< 0.02; **P< 0.01; ***P< 0.001. The significance between either 5 or 10 versus 1 mM concentration was determined with the Student's/-test for independent findings.

L- C A R N I T I N E

AC E T Y L - L- CARNITINE

y ----__0 1 2 6 1 . 2 . 2 2 0 . x

PROPIONYL- L- CARN ITINE

y = 3.198 + 0.977. × r 2 = 0.916

T

25-

y = 0.576 + 1.146. x r== 0 . 9 9 1

ol 2 0 -

15-

10ca

5-

Z 0 0

1 L-carnitine

i

i

5

10

(mM)

0

1

5 AcetyI-L-carnitine

10 (raM)

|

i

f

1

5

10

P r o p i o n y l - L- car nitine (raM)

Fig. I. Relationship between net transintestinal transport of L-carnitine and its esters (ordinate) and their concentrations in the incubation media (abscissa). Each point is a mean of at least three experiments. Bars represent st.

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Table III Cellular solute concentration in everted rat jejunum af three different Lcarnitine, acetyl-L-carnitine and propionyl-L-carnitine concentrations Incubating solution Krebs- Ringer bicarbonate buffer+ proper substance at the indicated concentration

Cell concentration ( mmol/l of cell water) Na +

K+

Substance

L-Carnitine 1 mM 5 mM 10 mM

32_+7 40 _+ 1 35 --+3

123_+ 10 114 _+5 111 _+6

0.63_+0.11 1.50 _+0.06* 2.67 --+0.06**

Acetyl-L-carnitine 1 mM 5 m~ 10 mM

49 + 2 33+3 31 _+ 1

11 + 2 t13+5 101 + 3

0.26 + 0.03 0.66+0.06* 1.25 +0.03**

Propionyl-L-carnitine 1 mM 5 mr~ 10 mM

42 _+3 47 + 1 41 _+5

99 _+4 115 _+3 119 _+5

0.25 + 0.02 0.85 _+0.01"* 1.45 + 0.07**

Mean values of at least three findings _+sE. *P< 0.01; **P< 0.001. Significance of the differences found between either 5 or 10 mM versus 1 mM was checked with Student's t-test for independent findings.

three substances were in fact transferred across the intestine mainly through a passive process as the linear correlation with the concentration clearly demonstrates. A n apparent contrast arises from partition coefficients which are higher with Lcarnitine esters than with e-carnitine itself whereas the concentration/transfer shapes lead one to conclude that L-carnitine is transferred faster than its esters (Fig. 1 ). This contrast should be reconciled if one considers that this experiment shows a transport which is a result of an active saturated process and a passive diffusion. Evidence suggests that the active transport of L-carnitine should occur at a higher rate than that of its esters, as clearly demonstrated in renal epithelium which possesses a threshold for L-carnitine higher than its esters [11, 12]. A higher (even if saturated) active and a lower diffusional transport for L-carnitine combined with a higher diffusional and a lower active transport for its m o r e lipophilic esters could lead to a result of a certain balance in the concentration/transport relationship. These results are possibly affected by the metabolic processes occurring in all the tissues and thus also in the intestine cells aimed at buffering the L-carnitine components in the body, including enterocytes, through carnitine acetyltransferases which work with the aim of rebalancing the L-carnitine family components [12]. Marked acetylation of L-carnitine during its transport was in fact found when intraluminal physiological concentrations of L-carnitine were used

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(~M). In this study, however, high concentrations of these substances were used (up to 10 000 ~M). The esterification of L-carnitine could be therefore relatively and quantitatively less important. It was found in fact that, as the concentration of Lcarnitine was increased, the percentage of acetyl-c-carnitine decreased, indicating that carnitine acetyltransferase was becoming saturated with substrate [7]. Of great interest is the linear relationship between K + transport and acetyl- and propionyl-L-carnitine concentration, considering both K + and components of the Lcarnitine family are compartmented mainly into cells (K + ) and into the myocells (>carnitine family). This view needs to be confirmed considering in the case of K + that the differences encountered with the three concentrations used did not reach any statistically significant degree. In conclusion, it seems that at pharmacological doses, L-carnitine and its esters cross the intestine mainly by simple diffusion. Partition coefficients of carnitine esters determined in lipophilic media are higher than that of L-carnitine, indicating that the esters diffuse more easily through the lipid component of the intestinal epithelium. In addition, a positive linear correlation seems to exist between acetyland propionyl-l=carnitine transport and that of K + with a higher slope for the propionyl ester.

ACKNOW LEDGEMENTS The authors are greatly indebted to Dr M. Rimoldi (Neurological Institute C.Besta, Milan) for preparing labelled acetyl- and propionyl-L-carnitine.

REFERENCES 1. Borum PR. Role of carnitine in lipid metabolism. In: Horisberger M, Bracco U, ed. Lipids in modern nutrition. New York: Raven Press, 1987: 51-8. 2. Bremer J. Carnitine in intermediary metabolism. The metabolism of fatty acid esters of carnitine by mitochondria. JBiol Chem 1962; 237: 3628-32. 3. Bremer J. Carnitine and its role in fatty acid metabolism. Trends Biochem Sci 1977; 2: 207-9. 4. Sartorelli L, Ciman M, Rizzoli V, Siliprandi N. On the transport mechanisms of carnitine and its derivative in rat heart slices. Ital J Biochem 1982; 31:261-8. 5. Rebouche CJ, Engel AG. Carnitine metabolism and deficiency syndromes. Mayo Clin Proc 1983; 58: 533-40. 6. Hamilton JW, Li BUK, Shug AL, Olsen WA. Carnitine transport in human intestinal biopsy specimens. Gastroenterology 1986; 91: 10-16. 7. Shaw RD, Li BUK, Hamilton JW, Shug AL, Olsen WA. Carnitine transport in rat small intestine. A m J Physio11983; 245: G376-81. 8. Esposito G, Faelli A, Tosco M, Orsenigo MN, Battistessa R. Age related changes in rat intestinal transport of D-glucose, sodium and water. Am JPhysio11985; 249: G328-34. 9. Kerner J, Bieber LL. A radioisotopic-exchange method for quantitation of short-chain (acid-soluble) acylcarnitines. Anal Biochem 1983; 134: 459-66. 10. Esposito G, Faelli A, Tosco M, Burlini N, Capraro V. Extracellular space determination in rat small intestine by using markers of different molecular weight. Pfliigers Arch 1979; 382: 67-71. 11. Gross CJ, Henderson LM. Absorption of D- and L-carnitine by the intestine and kidney tubule in the rat. Biochim BiophysActa 1984; 772: 209-19. 12. Marzo A, Arrigoni Martelli E, Urso R, Rocchetti M, Rizza V, Kelly JG. Metabolism and disposition of intravenously administered acetyl-L-camitine in healthy volunteers. Eur J Clin Pharmaco11988; 37: 59-63.

L-carnitine and carnitine ester transport in the rat small intestine.

L-carnitine and its esters (acetyl-L-carnitine and propionyl-L-carnitine) at pharmacological doses (1, 5 and 10 mM) are absorbed by the rat jejunum by...
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