Water soluble palmitic acid-methylated cyclodextrin complex; a substrate oxidized by Mycobacterium leprae
To the Editor:
Mycobacterium leprae does not grow axenically on substrates which other mycobacteria use for growth, multiplication, and virulence. A search for decades for the oxidizable substrates for M. leprae resulted in the findings by Franzblau (2) and Franzblau and Harris (3) that palmitic acid is oxidized by M . leprae and that the reaction contributes to ATP formation and synthesis of PGL-I. Ishaque (4) confirmed these results and provided direct evidence that, indeed, M. leprae oxidizes palmitic acid through the tricarboxylic acid cycle and the electron-transport chain with oxygen as the terminal electron acceptor. These results can be considered as an important advance toward in vitro cultivation of M. leprae since palmitic acid, as oxidized, is a potent source of energy and a rich source of carbon. The key to in vitro cultivation of any microorganism is, of course, the incorporation into the media of an energy and carbon source. For M. leprae cultivation, palmitic acid could be such a substrate.
However, palmitic acid is insoluble in water and being hydrophobic in nature it remains in solid state in media, thus biologically not readily available. In biological systems, in culture media, and for metabolic studies, substrates insoluble in water-palmitic acid in particular-were used as finely dispersed crystals, suspensions, or liposomes. Bar (1) was the first to incorporate lipophilic substrates into media with hydrophobic substances as a fine suspension. Kato (5) used β -cyclodextrin to incorporate palmitates into media for the cultivation of noncultivable mycobacteria. In none of these systems was fatty acid directly available to the cells, since the insoluble substrate must be scavenged from a solid state.
Recognizing the importance of palmitic acid as a carbon and energy source for M. leprae, Kato initiated systematic studies to produce a water-soluble formulation of palmitic acid and palmitates as molecular dispersions soluble in water. Recently, Szejtli, et al. (personal communication, 1992) reported the preparation of a palmitate-heptakis-2,6-di-o-methyl- β -cyclodextrin complex practically soluble in water. This new formulation was incorporated into media for the cultivation of psychrophilic mycobacteria from M. leprae -infected tissues by Kato, et al. (personal communication, 1992).
The question thus arises, Can the watersoluble palmitate-methylated cyclodextrin complex serve as an energy source and be as potent as the water-insoluble mother substrate palmitic acid or palmitates? The bioavailabilities of the water-soluble and -insoluble palmitates were compared.
The heptakis-methylated cyclodextrin (DIMEB-CD) was synthesized by Cyclolab Research and Development, Budapest, Hungary. The water-soluble sodium palmitate complex was prepared by Kato (Catherine Booth Hospital, Montreal, Canada) according to Szejtli, et al. (personal communication, 1992). The complex was donated as a sterile solution containing 10 mg/ml sodium palmitate. The methylated cyclodextrin was used as a control.
Respiratory studies were conducted to determine whether or not soluble palmitate is oxidized by M. leprae bacilli. M. leprae bacilli were isolated from the foot pads of athymic nude mice previously infected with M. leprae, and purified suspensions were prepared by differential centrifugation in potassium phosphate buffer, pH 6.5. Our results indicated that although insoluble palmitate was oxidized by M . leprae suspensions after a lag period of 6-8 hr, soluble palmitate was actively oxidized after a lag period of 2 hr. Similar to insoluble palmitate, soluble palmitate was readily oxidized by M . phlei suspensions. Based on the effect of specific inhibitors, it is concluded that soluble palmitate oxidation is mediated through the electron-transport chain of both M. leprae and M. phlei. The methylated cyclodextrin, when used as a control, did not exhibit any oxidation.
The availability of heat-stable, water-soluble palmitate has made it possible to use palmitate as a substrate in both solid and liquid media. We have prepared solid media and soluble palmitate has been found to be uniformly dispersed in the media. Clear liquid media can also be prepared by using soluble palmitate. Soluble palmitate docs not interfere in spectrophotometric studies as well. It is of further advantage that the water-soluble palmitate complex assures a continuous release of the molecularly dispersed palmitate. This unique way of releasing the active substance results in an improved bioavailability, as is clear by the markedly reduced lag period of oxidation by M. leprae and M. phlei.
Based on preliminary results, the use of the soluble palmitate complex is highly encouraged for the in vitro cultivation trials of M. leprae as well as for other biological and metabolic studies.
- Mohammed Ishaque, Ph.D.
Professor
Applied Alicrobiology Research Centre
Institut Armand-Frappier
University of Québec
C.P. 100
Laval, Québec, Canada H7N 4Z3
Acknowledgment. This study was supported by grants from Le Secours aux Lepreux, Canada, Inc.
REFERENCES
1. BAR. R. A new cyclodextrin-agar medium for surface cultivation of microbes on lipophilic substrates. Appl. Microbiol. Biotechnol. 32(1980)470-472.
2. FRANZBLAU, S. G. Oxidation of palmitic acid by Mycobacterium leprae in an axenic medium. J.Clin. Microbiol. 26(1988)18-21.
3. FRANZBLAU, S. G. and HARRIS, E. Biophysical optima for metabolism of M. leprae. J. Clin. Microbiol. 16(1988)1124-1129.
4. ISHAQUL, M. Direct evidence for the oxidation of palmitic acid by host grown Mycobacterium leprae. Res. Microbiol. 140(1989)83-93.
5. KATO, L. Psychrophilie mycobacteria in M. leprae infected tissues. Int. J. Lepr. 56(1988)631-632.