Annals of Vascular Surgery
Volume 22, Issue 4 , Pages 552-558, July 2008

Propionyl-l-Carnitine in Leriche-Fontaine Stage II Peripheral Arterial Obstructive Disease

  • Claudio Allegra

      Affiliations

    • San Giovanni Hospital, Rome, Italy
    • Corresponding Author InformationReprint requests to: Claudio Allegra, MD, San Giovanni Hospital, Via del Colosseo 26, 00100 Rome, Italy
    • ,
  • Pier Luigi Antignani

      Affiliations

    • San Giovanni Hospital, Rome, Italy
    • ,
  • Ilana Schachter

      Affiliations

    • San Giovanni Hospital, Rome, Italy
    • ,
  • Aleardo Koverech

      Affiliations

    • Scientific and Clinical Department, Sigma Tau, Pomezia, Rome, Italy
    • ,
  • Masa Messano

      Affiliations

    • Scientific and Clinical Department, Sigma Tau, Pomezia, Rome, Italy
    • ,
  • Ashraf Virmani

      Affiliations

    • Scientific and Clinical Department, Sigma Tau, Pomezia, Rome, Italy
    • Corresponding Author InformationCorrespondence to: Ashraf Virmani, PhD, Medical and Scientific Affairs, R & D Sigma Tau HealthScience, Via Treviso 4, 00040 Pomezia (Rome), Italy

published online 27 May 2008.

Article Outline

Peripheral arterial obstructive disease (PAOD) of the lower limbs affects 5% of the adult population. Uncontrolled arteriopathy is established due to a microcirculatory deficit, which may be present despite a good Winsor index and which leads to exhaustion of the functional microcirculatory reserve. The target of this study was to examine possible improvements in microvascular and tissue homeostasis by the administration of propionyl-L-carnitine (PLC). A total of 26 patients were enrolled in this study, aged 65 ± 15 years; two males were diagnosed at stage IIA and 17 males and seven females at stage IIB PAOD. The main criterion of inclusion was the worsening of walking distance during the last month. In this study the duration of therapy was 33 days. PLC was administered in three flasks, each containing 300 mg in 250 cc saline by continuous infusion. The following parameters were measured before and after treatment: pain-free and maximum walking distance (measured on a treadmill at 3.2 km/hr with a gradient of 12%), recovery time from pain after maximum walking distance, ankle-brachial index by means of the Doppler apparatus, and evaluation of the microcirculation using capillaroscopy. The results showed that therapy with PLC was effective at restoring activity of skeletal muscle in ischemic conditions. In particular, capillaroscopy showed improvement in the angioarchitecture in the microcirculation fields, expressed as increased numbers of visible capillaries and diminution in the time of loss of sodium fluorescein marker. The clinical data showed increased walking distance and diminished time to recover from pain, and the clinical improvement correlated with improved microcirculatory function. From these preliminary data has emerged an indication of therapy with PLC for chronic obstructive arteriopathy of the lower limbs at stage II. Further studies with higher numbers of patients and more controlled variables are planned.

 

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Introduction 

Peripheral arterial obstructive disease (PAOD) of the lower limbs affects 5% of the adult population. It is also an index of cardiovascular risk since it is associated with coronary disease in 50% of cases and with carotid stenosis in 30% of cases.1

The classification of Leriche-Fontaine distinguishes four stages of disease progression2: an asymptomatic stage, a second stage characterized by claudication, a third by rest pain, and a fourth by trophic lesions.3, 4, 5, 6

Stage II of PAOD of the lower limbs is the expression of compensated ischemia. It is characterized by painful cramps in the lower limb due to the reduction in arterial flow, which creates a discrepancy in the quantity of oxygen available and the metabolic need.7

Intermittent claudication can be classified as initial or maximum (the former characterizes the interval that is free of pain while walking [pain-free walking distance] and the latter reflects the maximum walking distance traversed after the initiation of pain). In stage IIA the relative maximum walking distance (MWD) is more than 100 m, while in stage IIB the MWD is less than 100 m.

In studies carried out in patients affected by cardiovascular problems, an improvement in exercise capacity, possibly associated with restoration of the reduced levels of carnitine by administration of propionyl-l-carnitine (PLC), has been reported.8, 9

PLC is an analog of l-carnitine, which the human body produces naturally via an enzymatic reaction that is completely reversible.10, 11 Experimental findings suggest that PLC can enter mitochondria in a selective manner, and since mitochondria contain the enzyme carnitine acyltransferase, the final result is stimulation of the Krebs cycle and production of an excess of succinate.12 Some of the effects of PLC are also the result of its metabolism to l-carnitine, which in turn acts as a carrier for the transport of long chain fatty acids into the mitochondria, for ß-oxidation, and for the increase in level of free coenzyme A (CoA), essential for lipid and carbohydrate metabolism.10, 13 Thus, PLC stimulates energy production in ischemic muscles by increasing citric acid cycle flow and stimulating pyruvate dehydrogenase activity, and its free radical–scavenging activity may also be beneficial.10, 14

PLC has been suggested to be useful for patients affected by various cardiovascular pathologies.12, 15 The rationale for therapy with PLC is based on various synergistic mechanisms that act on the integrity of the architectural–biochemical–functional parameters of the microcirculation and muscle fibers.

The objective of this study was to quantify the clinical data of improved symptomatology in patients affected by stage II of POAD of the lower limbs in which the worsening of walking distance was found during the last month, measuring the increase in the initial and maximum walking distance and the time of recovery from pain. This was also correlated to the improved microcirculatory function by means of capillaroscopy.

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Patients and Methods 

A total of 26 patients attending the Angiology Unit at the San Giovanni Hospital (Rome, Italy) were enrolled in this study, aged 65 ± 15 years. Of these, two males were diagnosed at stage IIA and 17 males and seven females at stage IIB PAOD.

The main criterion of inclusion was worsening of walking distance during the last month.

PLC was administered as three flasks, each containing 300 mg in 250 cc saline by continuous infusion during the day with the following regimen (total duration of therapy 33 days): endovenous infusion for 12 consecutive days, followed by an infusion every alternate day (six times) and then every 3 days (three times).

We used endovenous infusion of the drug because of the particular nature of patients (quick worsening of claudication) and for acute evaluation of the instrumental results.

Parameters Evaluated 

The following parameters were measured before and after treatment:

•Initial and maximum walking distance (measured on a treadmill at 3.2 km/hr with a gradient of 12%)

•Recovery time from pain after absolute walking autonomy

•Ankle-brachial index by means of the Doppler apparatus

•The microcirculation, using capillariscopy

Capillaroscopy 

To explain possible clinical improvement resulting from the therapy, the modification at the level of the microcirculation was studied using a modified capillaroscopy (Capiflow, Kista, Sweden). The Capiflow system is able to evaluate the flow velocity and the relative hematocrit using a low velocity during the examination of images.16

The study on the arteriopathic patients was carried out with the microscope positioned on top of the nail of the big toe of the foot. It was possible to measure the capillary red blood cell velocity, the diameter (in micrometers), the number of capillaries per field (capillary density), and the number of perfused and open capillaries. Further, using a contrasting agent, sodium fluorescein (NaF) injected in the vein of the arm, with the dosage dependent on the age, sex, and weight of the patient, it was possible to measure and quantify the permeability of the capillaries and the time taken by NaF to arrive at the nail of the big toe. In this way, it was possible to evaluate the damage suffered by the capillaries as a result of ischemia. Due to the complexity of this invasive method and the time of examination, we studied only six patients.

Statistical Analysis 

All data are expressed in terms of means and standard deviation from the mean. Statistical comparison was done before and after treatment using the modified Student's t-test. p < 0.05 was considered statistically significant.

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Results 

All patients were treated with PLC, which was well tolerated. Out of the 26 patients enrolled in the study, 24 completed the treatment schedule as well as the walking tests; two abandoned the study due to failure of compliance. No adverse events were found in any of the patients.

The risk factors of patients were under control by means of specific drugs and observation of correct behavior. No drugs used influenced the metabolism of carnitine. No kidney or liver diseases were present.

Walking Autonomy and Recovery Time 

The initial walking distance increased significantly by an average of 157 m, from a mean of 140 m before treatment to 297 m after treatment (p < 0.001, n = 24) (Fig. 1). MWD went up by 135 m, from 224 to 359 m after treatment (p < 0.01, n = 21) (Fig. 2).

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  • Fig. 1 

    Pain-free walking capacity in 24 patients before and after intravenous PLC treatment. All measurements are in meters. Each point is the mean (±standard error) of 24 patients. ***Statistically significant difference at p < 0.001.

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  • Fig. 2 

    Maximum walking capacity in 21 patients before and after intravenous PLC treatment. All measurements are in meters. Each point is the mean (±standard error) of 21 patients. **Statistically significant difference at p < 0.01.

Although the study did not specifically set out to measure the recovery time, we found that this was decreased by about 25% in the PLC-treated group (results not shown).

Capillaroscopy 

The data from capillaroscopy for six patients, before and after therapy, were as follows. An increase in the velocity of red blood cells was noted from basal values of 0.15 ± 0.05 to 0.23 ± 0.07 mm/sec after treatment, an increase of 53.3% (p < 0.05) (Fig. 3). A slight, nonsignificant increase in the number of capillaries per field, from 15 ± 6 to 16 ± 7, was found. However, the actual number of open capillaries increased significantly from 4.0 ± 2.3 to 13 ± 7.1 (p < 0.05) (Fig. 4). The time of appearance of the contrast marker NaF was also significantly reduced, from 65 ± 18 to 35 ± 15 sec (p < 0.05) (Fig. 5, Fig. 6).

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  • Fig. 3 

    Red blood cell velocity measured by capillaroscopy before and after intravenous PLC treatment. All measurements are in millimeters per second, and each point is the mean (±standard error) of six patients. *Statistically significant difference at p < 0.05.

  • View full-size image.
  • Fig. 4 

    Number of open capillaries by capillaroscopy before and after intravenous PLC treatment. Each point is the mean (±standard error) of six patients. *Statistically significant difference at p < 0.05.

  • View full-size image.
  • Fig. 5 

    Time of appaearance of contrast marker NaF by capillaroscopy before and after intravenous PLC treatment. Each point is the mean (±standard error) of six patients. *Statistically significant difference at p < 0.05.

  • View full-size image.
  • Fig. 6 

    Representative images of videocapillaroscopy before (a) and after (b) PLC treatment. There is an increase of open capillaries with flow inside. The capillaries are the irregular dark tubes. The light area is the ground connective tissue.

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Discussion 

Chronic obstructive arteriopathy of the lower limbs at stage II can evolve in two ways: as compensated arteriopathy with improved symptomatology or as uncontrolled arteriopathy, with evolution toward stage III with pain at rest. The evolution depends on multidisciplinary interventions: surgical revascularization, medical therapy, and physical rehabilitation.

The amelioration of POAD by pharmacological treatment is related to the regression of clinical symptoms, mainly due to effects on the microcirculation; the adaptation to the workload in ischemia is compensated at the level of the preterminal and terminal beds. In the first phase the physiological defense mechanisms are activated, resulting in vasodilatation and use of the peripheral microcirculatory reserve. Uncontrolled arteriopathy, such as in our case report, was established due to a microcirculatory deficit which leads to exhaustion of the functional microcirculatory reserve. Therefore, the target of the medical therapy consists of improving the microvascular tissue homeostasis, and in the present study this mechanism was targeted by administration of PLC.

The microcirculation is composed of different structures which have different functions: nutritional capillaries (10-15% of hematic flow) and the paramicrovessel system (90% of microcirculatory flux), which form the functional reserve, consisting of the arterioles sensitive to variation in pressure, venules and postcapillary venules which have contractile pericytes and microvalves, meta-arterioles (with discontinuous lamina of myocells), arteriovenous anastomosis, and extracellular matrix. In POAD there is permanent hyperstomia, relative and absolute, of the intermediate segment with an increased flow.

It is the endothelial dysfunction which triggers pathology of the organ by priming the development of the inflammatory repair–proliferation process, which can manifest as atherosclerosis, arteriosclerosis, hypertension, or ischemia and consequent pathology of reperfusion.17 These factors can be further exacerbated by metabolic toxicities and other risk factors.18, 19, 20

Microvascular tissue homeostasis is regulated by systemic and local hemodynamic and biohistochemical mechanisms, correlated to various factors.21, 22, 23 The first is “vasomotion,” which regulates microcirculatory flow distribution on the basis of local metabolic necessity by means of the local myogenic reflex of the precapillary sphincter and the tone of the arteriole myocells. The myocells in the presence of adenosine triphosphate (ATP) and ionic Ca produce actinomyosin, which is indispensable to its activity. The second is the endothelial organ, which has metabolism related to its function, with specific properties according to its location, i.e., arterial, venous, and cerebral vessels.

Losing integrity initiates activation of prothrombotic substances and glycosaminoglycan, via the lipoprotein-associated coagulation inhibitor, with the formation of thrombin and microthrombus in the locality. In addition, the ischemic endothelial laboratory, to satisfy the vital functional requirements, converts its aerobic metabolism into mixed aerobic and anaerobic, which creates a further endothelial dysfunction, an increase in local acidosis, and worsening of symptomatology.

In POAD, the formation of l-carnitine and of acyl carnitine in the resting muscle shows a similar situation to submaximal force in the muscle.24, 25, 26 Repeated ischemic episodes provoke a depletion of l-carnitine, negative repercussions in the function of myocytes and endothelium, a vasogenic and interstitial edema, loss of vasomotion and of distribution of the microcirculatory flow, and local microthrombosis. Finally, the ischemia provokes further ischemia, establishing an adaptation in the skeletal muscle to the work under hypoxia.

The rationale for therapy with PLC is restoration of microcirculatory homeostasis, which it probably performs by at least five actions, of which the first two are correlated and act by potentiating each other:

1.Hemodynamic action25

2.Metabolic action26

3.Direct action on muscle fiber, probably at the nuclear level (the deficit of carnitine is associated with atrophy of the type 1 muscle fiber, which is characterized by high levels of oxidative enzymes)27

4.Hemorrheologic action for the restoration of normal stability and fluidity of the membrane of red blood cells28

5.Modulation of the glucose metabolism, in conditions of reduced carnitine, increases the acetyl-CoA in the mitochondria;29 therefore, the sugars cannot enter the cell, causing nonutilization of glucose. This represents a sort of insulin resistance in the cell.30

The mechanisms underlying the first two actions of PLC are related to its unique hemodynamic and metabolic properties. Firstly, its microhemodynamic action and the modulation of the vascular motility on the basis of functional need increase the energetic substrate (ATP) despite hypoxia. This anaplerosis mechanism was studied by Taylor et al. in 1996.31 They used 30P nuclear magnetic resonance spectroscopy to show changes consistent with improvement in muscle metabolism after PLC administration: a 30% decrease in reaction half-time of phosphocreatine (PCr), a 43% decrease in reaction half-time of adenosine diphosphate (ADP), and a 33% increase in maximal velocity (Vmax) for oxidative ATP synthesis. Changes in walking distance were correlated positively with changes in Vmax as well as with faster PCr and ADP recovery. In 1997, Thompson et al.32 showed that in patients treated with 2 g of PLC a day, normal levels of ATP and PCr were established. In the study by Di Marzo et al.,33 patients treated with PLC showed a faster recovery of PCr and a lesser decrease in tissue pH during exercise. The increased production of ATP has positive effects: it reequilibrates vasomotion, maintenance of arteriolar tone is facilitated with minor consumption of oxygen by the endothelium and the precapillary sphincter, maintenance of perfusion pressure, and restoration of equilibrium between the endothelin and nitric oxide produced by the endothelium. The resulting arteriolar vasodilatation constitutes the first response to ischemia.

Secondly, PLC probably remodels endothelial dynamic organs and the extracellular matrix. The tissue perfusion, intermittent for arteriolar vasomotion, in periods of nonperfusion allows the reabsorption of interstitial substances from interstitial area and the physiological prevention of edema. PLC is a scavenger of free radicals via a chelating mechanism on ferrous ions and reduction in intracellular calcium, with a protective effect on the endothelium due to the diminution of leukocyte adhesive molecules. This is demonstrated by the fall in the expression of endothelial l-selectin, a marker of adhesion molecules which are produced during ischemia and reperfusion following free radical formation.34 This restores the endothelial integrity with a reequilibrating effect on the permeability and the exchange of nutrients, catabolites, gases, and intra- and extracellular liquids (transmural pressure) and rebalance between the prothrombotic, profibronolytic, vasodilatory, and vasoconstrictive substances produced by the endothelium. PLC also has a vasoactive vasodilatory effect via a mechanism linked to prostaglandins.35

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Conclusion 

In conclusion, PLC therapy was demonstrated to be effective at restoring activity of skeletal muscle in ischemic conditions as demonstrated by capillaroscopy showing improvement in the angioarchitecture in the microcirculation fields, expressed as increased numbers of visible capillaries and diminution in the time of NaF loss. The clinical data reflect this microcirculatory function improvement by showing increased walking autonomy and diminished time to recover from pain. These preliminary data support therapy with PLC for “worsening” PAOD of the lower limbs at stage II. We suggest that further studies would be useful to elaborate the mechanisms underlying POAD and the therapeutic efficacy of PLC.

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PII: S0890-5096(08)00085-X

doi:10.1016/j.avsg.2008.02.010

Annals of Vascular Surgery
Volume 22, Issue 4 , Pages 552-558, July 2008

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