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l -carnitine and carnitine ester transport in the rat small intestine
Pharmacological Research, Vol. 23, No. 2, 1991
157
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
Pharmacological Research, Vol. 23, No. 2, 1991
158
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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Pharmacological Research, Vol. 23, No. 2, 1991
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
Pharmacological Research, VoL 23, No. 2, 1991
160
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.
161
Pharmacological Research, Vol.23, No. 2, 1991
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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Pharmacological Research, Vol. 23, No. 2, 1991
(~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.
157
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
Pharmacological Research, Vol. 23, No. 2, 1991
158
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.
159
Pharmacological Research, Vol. 23, No. 2, 1991
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
Pharmacological Research, VoL 23, No. 2, 1991
160
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.
161
Pharmacological Research, Vol.23, No. 2, 1991
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
162
Pharmacological Research, Vol. 23, No. 2, 1991
(~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.
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