L- L-carnitine a response - Hindawi Publishing Corporationdownloads.hindawi.com/journals/mi/1993/716950.pdf · L-carnitine improves immune response andlipid metabolism more than by

Research Paper

Mediators of Inflammation 2, S29-S32 (1993)

THE authors demonstrated that in vivo administered L-

carnitine strongly ameliorated the immune response inboth healthy individuals receiving Intralipid and ageingsubjects with cardiovascular diseases, as shown by theenhancement of mixed lymphocyte reaction. Notably, inthe latter group L-carnitine treatment also resultedin a significant reduction of serum levels of bothcholesterol and triglycerides. Therefore, the hypothesis isthat L-carnitine supplementation could ameliorate both thedysregulated immune response and the abnormal lipidmetabolism in several conditions.

Key words: Immune response, L-carnitine, Lipid metabolism

L-carnitine" a partner betweenimmune response and lipidmetabol ism ?

Giuseppe Famularo,z’cA Sonia Tzantzoglou,a

Gino Santini,a Vito Trinchieri,z SoniaMoretti,z Aleardo Koverech,4 and Claudio DeSimone

1Infectious Diseases and 2Department of InternalMedicine and Public Health, Via S. Sisto, 67100,University of L’Aquila; 3Infectious Diseases,University "’La Sapienza" Rome; 4SigmaTau, Pomezia, Italy.

ca Corresponding Author

Introduction

Carnitine (3-hydroxy-4-methyl-ammoniobutano-ate) is an essential intracellular constituent in higheranimals which is synthesized from peptide boundlysine. 1’2 In meat and dairy products, carnitine iseasily and almost completely absorbed, butendogenous biosynthesis can meet normal metabo-lic requirements of healthy adults. 3 Almost all thebody stores are in skeletal and cardiac muscles(98%), whereas liver and extracellular fluids containonly 1-6% of whole-body carnitine.4

L-carnitine, the physiological isomer, is a

non-toxic substance with an LDs0 similar to theLDs0 of amino acids. However, carnitine esters withlong-chain acyls are significantly more toxic, asshown for example by palmityl carnitine exhibitinga 23-fold toxicity with respect to free carnitine,

Carnitine and its derivatives have a key role inregulating substrate flux and energy balance acrosscell membranes.2’6’7 When the carnitine dependentmechanisms are impaired fatty acid oxidation isreduced and triglycerides accumulate, thereforeresulting into both microscopic fatty changes andimpaired hepatic ketogenesis.9 Carnitine is found inhigh concentrations in white blood cells.1 How-ever, the finding of most elevated carnitineconcentrations in circulating mononuclear cellsfirst suggested the possibility that carnitine and itsderivatives could modulate the function of immunecells and prompted us to assay the immuno-modulant properties of carnitine both in vitro andin vivo.

(C) 1993 Rapid Communications of Oxford Ltd

Materials and Methods

In vitro studies: L-carnitine was a gift fromSigma-Tau, Pomezia, Italy. Peripheral bloodmononuclear cells (PBMCs) were obtained fromhumans, as described previously.’2

For mitogen driven proliferative assays, PBMCswere resuspended at the concentration of 2 x 106cells/ml in RPM1 1640 (Gibco Bio Cult., Paisley,UK) containing 10% heat-inactivated foetal calfserum (FCS), 1% L-glutamine, and penicillin/streptomycin. PBMCs were cultured for 72 h in a

5% COg atmosphere at 37C. Then, the cells werepulse-labelled with 1/Ci of 3H-thymidine (Amer-sham, UK) in 20/1 medium 16 h before harvestingthe cultures. The incorporation of H-thymidinewas measured in a liquid scintillation beta counter.

In experiments carried out to evaluate the effectsof Intralipid(R) (Laboratorien Hausmann AG, StGallen, Switzerland) and Intralipid(R) plus L-

carnitine were added at various concentrations to

give a final volume of 60/1 at the beginning of thecultures.For the determination of lactate dehydrogenase

(LDH) isoenzyme patterns, PBMCs were incubatedin a 5% COg atmosphere at 37C for 48 h in RPMI1640 added with 10% FCS and antibiotics (controlsamples), with either Intralipid (100 #/ml) alone orIntralipid plus t-carnitine at various concentrations(1 and 10#g/ml). Then, PBMCs were freeze-thawed three times and spun at 3 000 rpm for 10min. The LDH isoenzymes of the supernatant fluidwere determined by cellulose acetate electrophoresis

Mediators of Inflammation. Vol 2 (Supplement) 1993 S29

G. Famularo et al.

of 5/1 samples for 30 min at 150 V. The areas ofenzyme activity were then shown by histochemicalprocedures employing phenazine methosulphateand tetranitro blue tetrazolium in a sandwichtechnique. The electrophoretic pattern was scannedin a densitometer equipped with an integrator.

Ex vivo studies: Six healthy volunteers were firsttreated with Intralipid (20%) for 6 h. Heparinized(10 U/ml) peripheral venous blood was obtained byvenipuncture before the infusion and then eachhour up to the end of the administration ofIntralipid. Peripheral venous blood was againobtained 24 h after the end of Intralipid infusion.The same subjects, after 2 days were treated withIntralipid plus L-carnitine (1 g/h) for the same time(6 h). Samples of heparinized venous blood wereobtained as described above.The ability of each plasma sample to inhibit

mixed lymphocyte reaction (MLR) was tested.Briefly, purified and washed PBMCs, obtained asdescribed above from healthy donors, were culturedin two-way mixed lymphocyte culture where foreach time point triplicate measurements wereperformed. Mixtures contained 1 x 106 cells fromeach of the two cell donors suspended in RPMI1640 supplemented with antibiotics and 50%normal pooled plasma or plasmas (50%) obtainedfrom the individuals treated with Intralipid andthen with Intralipid plus L-carnitine. After 72 h ofculture, PBMCs were pulse labelled with 3H-thymidine during the last 8 h of incubation. Then,the cells were collected into glass fibre filters andthe radioactivity was measured in a beta counter.The degree of inhibition of the MLR, incubatedwith plasma obtained from volunteers treated witheither Intralipid or with Intralipid plus -carnitine,was expressed as a percentage of MLR incubatedwith plasmas collected before the administration ofthe drugs.

Ex vivo and in vivo studies" Twenty-three hospitalizedpatients, 15 males and eight females (mean age75.5 8 years) with cardiovascular diseases(cardiomyopathy, atroventricular block, atrial fibril-lation, ventricular fibrillation, sick sinus syndrome)were enrolled into the study. The diagnosis was

supported by both clinical criteria and non-invasive procedures (electrocardiography, phono-cardiography, radiology and ultrasound).The patients were treated with I-carnitine (3 g

i.v. per day for 7 days). Peripheral blood sampleswere obtained at day 0 and at the end of the trial.Intradermal reactions to five recall antigens(candidine, parotitis, tricophytin, PPD and strepto-kinase-streptodornase) were tested by using theMultitest (Merieux Institute) immediately beforeand after treatment with L-carnitine.

S30 Mediators of Inflammation. Vol 2 (Supplement). 1993

Table 1. Effect of Intralipid (R) and Intralipid (R) plus L-carnitineon PHA-driven PBMC proliferation. The results are shown asmean cpm

___standard deviation of triplicate experiments

Intralipid (R) L-carnitine (#g/ml)(#llml)

0 10

0 57 062__+3441 57 518__+2883 58 623+__351225 28 209 __+ 2 994 33 542 __+ 2 098 34 778 -I- 2 74150 27 362+__2553 31 967+__2531 38 678+__2316"100 20164__+1537 24 250__+2274 26 250_____1883"250 4564+__ 625 8750+__ 933* 9893+__1132"

p < 0.01

Total lipaemia was measured by the manualturbidimetric method with a linearity up tO1 000mg/dl. Total cholesterol and triglycerideswere measured with an Abbott-UP bichromaticanalyser, by using a colorimetric enzymatic methodand an enzymatic UV method, respectively, bothwith a linearity up to 500 mg/dl.

Serum carnitine was measured by the radio-enzyme method described by McGarry and Foster,TM

with minor modifications as described previously, is

For the assays of MLR, either 50% normal pooledsera (from 60 healthy subjects) or sera from thepatients enrolled into the study were added to thecell suspensions, using microtitre plates withflat-bottomed wells. The radioactivity measurementwas performed as above described.

Results and Discussion

The proliferation of mitogen (PHA) drivenPBMCs from healthy individuals was stronglyreduced by lntralipid (Table 1). At Intralipidconcentrations up to 200 #l/ml, this inhibition wasnot due to cell death, as shown by PBMC samplestested for viability by trypan blue dye exclusion at

dierent time points. Above this concentration, thecell viability was slightly impaired resulting in celldeath of about 20% by the third day of culture (datanot shown). Notably, the immunotoxic eects ofIntralipid were dose-dependent. The addition of-carnitine at various doses (1 and 10#g/ml)exhibited a protective effect toward the immuno-toxic action of Intralipid. Furthermore, the moremarked the inhibition due to Intralipid themore marked was the restoring action ofL-carnitine.In fact, at the highest Intralipid doses tested(250/l/ml) I-carnitine increased thePBMCs incorp.oration of 3H-thymidine by about 20% (p < 0.001).The addition of L-carnitine at different dosesindicated that the chance of finding doseseective under a statistically significant standpointincreased as the concentration of Intralipid in themedium was augmented. Overall, these datasuggest that L-carnitine acts via a metabolic pathway

L-carnitine improves immune response and lipid metabolism

more than by exerting a direct immunoenhancingeffect and further evidence was found byperforming the isoenzymatic analysis of LDH inPBMCs cultured for 48 h. Both control PBMCs andPBMCs treated with Intralipid plus L-carnitinecontained more LDH1 isoenzyme and more heartsub-units compared to PBMCs treated withIntralipid alone (p < 0.01). This finding possiblyreflected a greater aerobic glycolysis, as the LDH1tissue content has been considered to be propor-tional to the degree of aerobic metabolism.Likewise, the LDH pattern of PBMCs treated withIntralipid alone contained more LDH5, likelypointing out the anaerobic metabolism of cellscultured in the presence of the fat emulsion.

In studying ex vivo the effects of Intralipid andL-carnitine in healthy individuals, a modification ofthe two-way MLR for the assessment of theimmunosuppressive activity was employed. Thechallenge of plasmas from subjects receivingIntralipid with lymphocytes from healthy unrelateddonors resulted in a mean 9% inhibition of thelymphocyte reaction after 1 h from the start of theinfusion. The administration of -carnitine inconjunction with Intralipid resulted, conversely, inan increase of the responses (mean 12% after 60min). Therefore, it resulted that after the first hourof infusion the total difference between plasmasfrom subjects treated with Intralipid and subjectstreated with Intralipid plus I-carnitine was about21% when tested for immunosuppressive activity ofMLR. This difference decreased to 7% after 24 h,probably due to host’s adaptative metabolicmechanisms.

Overall, the in vitro and in vivo studies pointed outthat the immunotoxic action of Intralipid could bereversed, at least to a certain degree, by adding-carnitine to PBMC cultures, even at minimalconcentrations. Since a direct immunoenhancingeffect of L-carnitine was ruled out, in the authors’opinion the most probable hypothesis is that-carnitine could restore the immune response byremoving some metabolic cofactor limitation ofPBMCs cultured in the presence of Intralipid.The treatment of elderly patients suffering from

cardiovascular diseases with L-carnitine (3 g per dayfor 7 days) resulted in a significant reduction oflipaemia, cholesterolaemia, and triglyceridaemia inthe majority of cases (Table 2). Furthermore, thisresult was paralleled by both an increase, in vivo, inthe delayed hypersensitivity response to commonrecall antigens (candidine, tricophytin, parotitis,PPD, streptokinase-streptodornase) and in theability of serum to stimulate MLR (Table 3).Notably, skin test reactivity increased in the samepatients in whom a reduction of serum lipid levelswas observed following -carnitine treatment.

Similarly, MLR was increased when the cells were

Table 2. Serum levels of cholesterol (normal range" 160-330mg/dl) and triglycerides (normal range: 40-160 mg/dl) before(A) and after (B) L-carnitine supplementation in ageing patientswith cardiovascular diseases

Patient Cholesterol Triglycerides

A B A B

231 212 138 1302 279 235 168 1453 288 231 171 1644 197 199 143 1445 281 243 177 1696 121 156 168 1127 200 203 136 1358 157 150 140 1149 181 251 184 28110 191 155 112 10911 231 196 117 11712 163 175 75 8313 199 164 114 10814 175 180 106 10615 269 198 134 13216 215 187 176 12217 152 136 163 12418 175 110 116 11319 230 242 114 12620 176 183 98 10621 157 136 87 8522 196 177 126 10923 226 185 130 130

Table 3. Delayed hypersensitivity skin test and mixedlymphocyte reactions (MLR) before (A) and after (B)L-carnitine supplementation in ageing patients with cardio-vascular diseases

Patient Skin tests M LR

A B % variation

+ +++ +142 + ++ +193 +++ ++++ +124 ++ ++ -35 + ++ +216 + +++ +357 +++ ++ 08 ++ +++ +169 -1210 + ++ +911 + +2312 013 ++ ++ +2314 + -1715 + -616 ++ +2217 + ++ +1618 + ++ +1419 020 021 + +1822 + +1223 + ++ +10

+ >5mm < 10mm" ++ >10mm < 15mm; +++>15 mm.

Mediators of Inflammation. Vol 2 (Supplement). 1993 S31

G. Famularo et al.

added to or preincubated with post-treatment seraobtained from the same patients, compared to theresults obtained by using the pre-treatment sera.Patients exhibiting no effect to I-carnitine admin-istration in lowering blood lipid levels also hadunmodified or even reduced skin test reactivity torecall antigens. Their sera were also unable toincrease MLR. The general trend in this study wastowards a lowering of lipaemia, cholesterolaemia,and triglyceridaemia and in enhanced host immuneresponsiveness following -carnitine supplementa-tion. The possibility that -carnitine had a directimmunoenhancing property was ruled out byfinding that high levels of serum -carnitine wereassociated with unchanged immunological para-meters in patients exhibiting no reduction of bloodlipids following -carnitine treatment.

In conclusion, the above experiments suggest a

direct relationship between L-carnitine, lipids andimmune responsiveness. The mechanisms account-

ing for the effects of L-carnitine on humanlymphocytes in the presence of a lipidic dysmeta-bolism have yet to be fully established.

References1. Rebouche CJ, Engel AG. Tissue distribution of carnitine biosynthetic

enzymes in Biochim Biophys Acta 1980; 630: 22-29.2. Siliprandi N, Di Lisa F, Menabo R, Ciman M, Sartorelli L. Transport and

functions of carnitine in muscles. J Clin Chem Clin Biochem 1990; 28: 303-306.3. Feller AG, Rudman D. Role of carnitine in human nutrition. J Nutr 1988;

118: 541-547.4. Engel AG, Rebouche CJ. Camitine metabolism and inborn errors. J Inherited

Metab Dis 1984; 7 (suppl. 1): 38-43.5. Haeckel R, Kaiser E, Oellerich M, Siliprandi N. Carnitine. Metabolism,

function and clinical application. J Clin Chem Clin Biocbem 1990; 28: 291-295.6. Bremer J. The role of carnitine in intracellular metabolism. J Clin Chem Clin

Biocbem 1990; 28: 297-301.7. Bremer J. Carnitine: metabolism and function. Physiol Rev 1983; 63:

1420-1480.8. Gilbert EF. Carnitine deficiency. Patholog 1985; 17: 161-169.9. Chapoy PR, Angelini C, Brown WJ, Stiff JE, Shug AL, Cederbaum SD.

Systemic camitine deficiency: treatable inherited lipid-storage disorderpresenting Reye’s syndrome. N Engl J Med 1980; 303: 1389-1394.

10. Deufel T. Determination of L-camitine in biological fluids and tissues. J ClinChem Clin Biochem 1990; 28: 307-311.

11. Famularo G, Giacomelli R, Valdarnini D, De Simone C, Tonietti G.Modulation by ST 789 of in vitro lymphocyte activation and cytokineproduction. Thymus 1992; 19 (suppl. 1): 571-578.

12. De Simone C, Arrigoni Martelli E, Foresta P, et al. Hypoxanthine derivativesin experimental infections. Cand J Infect Dis 1992; 3 (suppl. B): 106-110.

13. Famularo G, Giacomelli R, Di Giovanni S, Sacchetti S, Tonietti G. Cytokineproduction in patients with monoclonal gammapathies. J Clin Lab Immunol1991; 34: 63-70.

14. McGarry JD, Foster DW. Free and esterified carnitine: radio method. In:Bergmeyer J, Grassl MW, eds. Methods of EnwmaticAnaysis. Berlin: Verlag,1983; 474-481.

S32 Mediators of Inflammation. Vol 2 (Supplement) 1993

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L- L-carnitine a response - Hindawi Publishing Corporationdownloads.hindawi.com/journals/mi/1993/716950.pdf · L-carnitine improves immune response andlipid metabolism more than by