L-carnitine: a partner between immune response and lipid metabolism ?

The authors demonstrated that in vivo administered L-carnitine strongly ameliorated the immune response in both healthy individuals receiving Intralipid and ageing subjects with cardiovascular diseases, as shown by the enhancement of mixed lymphocyte reaction. Notably, in the latter group L-carnitine treatment also resulted in a significant reduction of serum levels of both cholesterol and triglycerides. Therefore, the hypothesis is that L-carnitine supplementation could ameliorate both the dysregulated immune response and the abnormal lipid metabolism in several conditions.


Introduction
Carnitine (3-hydroxy-4-methyl-ammoniobutanoate) is an essential intracellular constituent in higher animals which is synthesized from peptide bound lysine. 1 '2 In meat and dairy products, carnitine is easily and almost completely absorbed, but endogenous biosynthesis can meet normal metabolic requirements of healthy adults. 3 Almost all the body stores are in skeletal and cardiac muscles (98%), whereas liver and extracellular fluids contain only 1-6% of whole-body carnitine. 4 L-carnitine, the physiological isomer, is a non-toxic substance with an LDs0 similar to the LDs0 of amino acids. However, carnitine esters with long-chain acyls are significantly more toxic, as shown for example by palmityl carnitine exhibiting a 23-fold toxicity with respect to free carnitine, Carnitine and its derivatives have a key role in regulating substrate flux and energy balance across cell membranes. 2'6'7 When the carnitine dependent mechanisms are impaired fatty acid oxidation is reduced and triglycerides accumulate, therefore resulting into both microscopic fatty change s and impaired hepatic ketogenesis. 9 Carnitine is found in high concentrations in white blood cells. 1 However, the finding of most elevated carnitine concentrations in circulating mononuclear cells first suggested the possibility that carnitine and its derivatives could modulate the function of immune cells and prompted us to assay the immunomodulant properties of carnitine both in vitro and in vivo.
(C) 1993 Rapid Communications of Oxford Ltd

Materials and Methods
In vitro studies: L-carnitine was a gift from Sigma-Tau, Pomezia, Italy. Peripheral blood mononuclear cells (PBMCs) were obtained from humans, as described previously. '2 For mitogen driven proliferative assays, PBMCs For the determination of lactate dehydrogenase (LDH) isoenzyme patterns, PBMCs were incubated in a 5% COg atmosphere at 37C for 48 h in RPMI 1640 added with 10% FCS and antibiotics (control samples), with either Intralipid (100 #/ml) alone or Intralipid plus t-carnitine at various concentrations (1 and 10#g/ml). Then, PBMCs were freezethawed three times and spun at 3 000 rpm for 10 min. The LDH isoenzymes of the supernatant fluid were determined by cellulose acetate electrophoresis G. Famularo et al. of 5/1 samples for 30 min at 150 V. The areas of enzyme activity were then shown by histochemical procedures employing phenazine methosulphate and tetranitro blue tetrazolium in a sandwich technique. The electrophoretic pattern was scanned in a densitometer equipped with an integrator. Ex vivo studies: Six healthy volunteers were first treated with Intralipid (20%) for 6 h. Heparinized (10 U/ml) peripheral venous blood was obtained by venipuncture before the infusion and then each hour up to the end of the administration of Intralipid. Peripheral venous blood was again obtained 24 h after the end of Intralipid infusion. The same subjects, after 2 days were treated with Intralipid plus L-carnitine (1 g/h) for the same time (6 h). Samples of heparinized venous blood were obtained as described above.
The ability of each plasma sample to inhibit mixed lymphocyte reaction (MLR) was tested. Briefly, purified and washed PBMCs, obtained as described above from healthy donors, were cultured in two-way mixed lymphocyte culture where for each time point triplicate measurements were performed. Mixtures contained 1 x 106 cells from each of the two cell donors suspended in RPMI 1640 supplemented with antibiotics and 50% normal pooled plasma or plasmas (50%) obtained from the individuals treated with Intralipid and then with Intralipid plus L-carnitine. After 72 h of culture, PBMCs were pulse labelled with 3Hthymidine during the last 8 h of incubation. Then, the cells were collected into glass fibre filters and the radioactivity was measured in a beta counter. The degree of inhibition of the MLR, incubated with plasma obtained from volunteers treated with either Intralipid or with Intralipid plus -carnitine, was expressed as a percentage of MLR incubated with plasmas collected before the administration of the drugs. Ex vivo and in vivo studies" Twenty-three hospitalized patients, 15 males and eight females (mean age 75.5 8 years) with cardiovascular diseases (cardiomyopathy, atroventricular block, atrial fibrillation, ventricular fibrillation, sick sinus syndrome) were enrolled into the study. The diagnosis was supported by both clinical criteria and noninvasive procedures (electrocardiography, phonocardiography, radiology and ultrasound).
The patients were treated with I-carnitine (3 g i.v. per day for 7 days). Peripheral blood samples were obtained at day 0 and at the end of the trial. Intradermal reactions to five recall antigens (candidine, parotitis, tricophytin, PPD and streptokinase-streptodornase) were tested by using the Multitest (Merieux Institute) immediately before and after treatment with L-carnitine. analyser, by using a colorimetric enzymatic method and an enzymatic UV method, respectively, both with a linearity up to 500 mg/dl. Serum carnitine was measured by the radioenzyme method described by McGarry and Foster, TM with minor modifications as described previously, is For the assays of MLR, either 50% normal pooled sera (from 60 healthy subjects) or sera from the patients enrolled into the study were added to the cell suspensions, using microtitre plates with flat-bottomed wells. The radioactivity measurement was performed as above described.

Results and Discussion
The proliferation of mitogen (PHA) driven PBMCs from healthy individuals was strongly reduced by lntralipid (Table 1). At Intralipid concentrations up to 200 #l/ml, this inhibition was not due to cell death, as shown by PBMC samples tested for viability by trypan blue dye exclusion at dierent time points. Above this concentration, the cell viability was slightly impaired resulting in cell death of about 20% by the third day of culture (data not shown). Notably, the immunotoxic eects of Intralipid were dose-dependent. The addition of -carnitine at various doses (1 and 10#g/ml) exhibited a protective effect toward the immunotoxic action of Intralipid. Furthermore, the more marked the inhibition due to Intralipid the more marked was the restoring action ofL-carnitine. In fact, at the highest Intralipid doses tested (250/l/ml) I-carnitine increased the PBMCs incorp .oration of 3H-thymidine by about 20% (p < 0.001).
The addition of L-carnitine at different doses indicated that the chance of finding doses eective under a statistically significant standpoint increased as the concentration of Intralipid in the medium was augmented. Overall, these data suggest that L-carnitine acts via a metabolic pathway L-carnitine improves immune response and lipid metabolism more than by exerting a direct immunoenhancing effect and further evidence was found by performing the isoenzymatic analysis of LDH in PBMCs cultured for 48 h. Both control PBMCs and PBMCs treated with Intralipid plus L-carnitine contained more LDH1 isoenzyme and more heart sub-units compared to PBMCs treated with Intralipid alone (p < 0.01). This finding possibly reflected a greater aerobic glycolysis, as the LDH1 tissue content has been considered to be proportional to the degree of aerobic metabolism. Likewise, the LDH pattern of PBMCs treated with Intralipid alone contained more LDH5, likely pointing out the anaerobic metabolism of cells cultured in the presence of the fat emulsion. In studying ex vivo the effects of Intralipid and L-carnitine in healthy individuals, a modification of the two-way MLR for the assessment of the immunosuppressive activity was employed. The challenge of plasmas from subjects receiving Intralipid with lymphocytes from healthy unrelated donors resulted in a mean 9% inhibition of the lymphocyte reaction after 1 h from the start of the infusion. The administration of -carnitine in conjunction with Intralipid resulted, conversely, in an increase of the responses (mean 12% after 60 min). Therefore, it resulted that after the first hour of infusion the total difference between plasmas from subjects treated with Intralipid and subjects treated with Intralipid plus I-carnitine was about 21% when tested for immunosuppressive activity of MLR. This difference decreased to 7% after 24 h, probably due to host's adaptative metabolic mechanisms.
Overall, the in vitro and in vivo studies pointed out that the immunotoxic action of Intralipid could be reversed, at least to a certain degree, by adding -carnitine to PBMC cultures, even at minimal concentrations. Since a direct immunoenhancing effect of L-carnitine was ruled out, in the authors' opinion the most probable hypothesis is that -carnitine could restore the immune response by removing some metabolic cofactor limitation of PBMCs cultured in the presence of Intralipid.
The treatment of elderly patients suffering from cardiovascular diseases with L-carnitine (3 g per day for 7 days) resulted in a significant reduction of lipaemia, cholesterolaemia, and triglyceridaemia in the majority of cases (Table 2). Furthermore, this result was paralleled by both an increase, in vivo, in the delayed hypersensitivity response to common recall antigens (candidine, tricophytin, parotitis, PPD, streptokinase-streptodornase) and in the ability of serum to stimulate MLR (Table 3). Notably, skin test reactivity increased in the same patients in whom a reduction of serum lipid levels was observed following -carnitine treatment. Similarly, MLR was increased when the cells were  added to or preincubated with post-treatment sera obtained from the same patients, compared to the results obtained by using the pre-treatment sera. Patients exhibiting no effect to I-carnitine administration in lowering blood lipid levels also had unmodified or even reduced skin test reactivity to recall antigens. Their sera were also unable to increase MLR. The general trend in this study was towards a lowering of lipaemia, cholesterolaemia, and triglyceridaemia and in enhanced host immune responsiveness following -carnitine supplementation. The possibility that -carnitine had a direct immunoenhancing property was ruled out by finding that high levels of serum -carnitine were associated with unchanged immunological parameters in patients exhibiting no reduction of blood lipids following -carnitine treatment.
In conclusion, the above experiments suggest a direct relationship between L-carnitine, lipids and immune responsiveness. The mechanisms accounting for the effects of L-carnitine on human lymphocytes in the presence of a lipidic dysmetabolism have yet to be fully established.