Zinc Supplementation Combats Tuberculosis by Reverting Back to Normal Compartmentalized State of Iron and Hence Increasing Blood Hemoglobin Concentration

INTRODUCTION

Mycobacteria does require Fe as growth factor. In fact, as recorded in its genome, Fe is the cofactor of many enzymes meant for its metabolism like superoxide dismutase (SOD); enzymes involved with tricarboxylic acid (TCA) cycle, pyrimidine synthesis; 3-deoxy-D-arabino-heptulosonate-7-phosphate synthase, etc., numbering for at least forty.¹ To reduce Fe availability in mammalian tissue, almost all Fe is bound to proteins such as ferritin for storage, or to transferrin (present in circulating plasma) and lactoferrin (present in extracellular fluid) for transport or bound as a cofactor of haem in Hb or in Fe sulfur clusters.² To acquire Fe, Mtb expresses Fe⁺³-specific small iron-binding siderophores (mycobactin and carboxymycobactin) having extremely high affinity for Fe, which can scavenge Fe from host-insoluble and host protein-bound Fe-like transferrin, lactoferrin and ferritin.³⁻⁵ At the context of facing lower level of free Fe, Mtb through mycobacterial membrane proteins (Mmps) like MmpS4 and MmpS5 and along with associated MmpL4 and MmpL5 transport proteins releases desferricarboxymycobactin into the immediate environment and on release after chelating Fe⁺³ from insoluble or protein-bound Fe is converted to ferricarboxymycobactin.⁶ Mycobacterium tuberculosis then by using Irt A/Irt B transporter present on cytoplasmic membrane reductively removes Fe (from Fe⁺³ to Fe⁺²) by FAD-mediated reductase enzyme of Irt A protein and thus mediates internalization of Fe.⁷ Fe⁺² ion after being transported into the cytoplasm by an energy-dependent process can be utilized for various metabolic activities or excess Fe is stored as two Mtb-encoded storage proteins, Bfr A; a bacterioferritin; and Bfr B; a ferritin-like protein.⁸ In the experiment where Fe was made available as a supplement for macrophage culture or in experimental animals infected with Mtb, there was enhanced multiplication of the pathogen.⁹ Also, it was reported that with genetic disruption of siderophore expression, growth of Mtb could be impaired in mice and macrophages, demonstrating that virulence of mycobacteria is well dependent on its essential role of Fe acquisition by siderophores.¹⁰ For mycobacteria, intracellular Fe level is regulated through Hup B, a positive regulator on the expression of mycobactin, and also through IdeR, a negative regulator and Fe-dependent transcription factor, acting and controlling gene-transcription involved in Fe uptake, transport, and storage.^11,12 Macrophages have the capacity for high Fe flux through specific cell surface receptors for transferrin, lactoferrin, and hemoglobin−haptoglobin, and thus recycle Fe from senescent erythrocytes, and thus they are important niches for Mtb for acquiring Fe.¹³ Mature macrophages, the preferred abode of Mtb, have much less concentration of myeloperoxidase (MPO) in contrast to neutrophils and thus unable to catalyze production of hypochlorous acid (HOCl), highly toxic reactive oxygen species (ROS) from hydrogen peroxide in the presence of chloride ion (Cl^–), and thus mature macrophages are unable to kill intracellular Mtb by this microbicidal mechanism.¹⁴

This author in his previous published papers had demonstrated assay of three serum enzymes as early diagnostic for pulmonary tuberculosis (PTB) as well as extrapulmonary TB (EPTB) and also by serial assay of those enzymes during antituberculosis (A-TB) drug therapy; sensitivity toward drug treatment might be ascertained very early. The author had recorded the presence of L-methionine-S,R-sulfoximine (MSO)-sensitive serum glutamine synthetase (GS) would confirm diagnosis for TB.¹⁵ The same author had endorsed highly elevated and sodium cyanide (NaCN)-resistant serum SOD as a diagnostic marker for TB.¹⁶ The index author had also indoctrinated inhibited serum cholinesterase (ChE) activity as a reliable diagnostic aid for TB.¹⁷ The presence of Mtb origin GS in serum, elaboration of Mtb origin Fe-cofactored serum SOD in enhanced concentration and inhibited host origin serum ChE activity increases specificity to diagnose TB.¹⁷ Mycobacterium tuberculosis SOD having no leader peptide binds only with Fe and is exported extracellularly. Also, Fe⁺³ scavenged by siderophore secreted by Mtb might form acetylcholine-ferric hydroxamate complex binding more strongly with serum ChE resulting in inhibition of serum ChE activity in TB patients.¹⁷ The ratio of serum SOD and whole-blood glutathione peroxidise had been also reported by this author as a diagnostic for TB.¹⁸

Zinc (Zn), more electropositive than Fe, exists as divalent cation and is redox neutral unlike Fe and copper and so not redox active under physiological conditions. It (Zn) performs multiple physiological roles in different biological processes. Being more electro-positive, Zn might replace Fe from the binding site with critical thiol groups. By this process, Zn not only inhibits formation of superoxide (O₂-) 2but also brings back intracellular decompartmentalized state of Fe to normal compartmentalized state of Fe.

AIM AND OBJECTIVE

Keeping in mind the unique role engineered by Zn to revert back decompartmentalized state of Fe to normal compartmentalized state of Fe, the present author has pondered a study to construe the effect of Zn supplementation in TB patients as reflected by serum Fe level in those subjects. With Zn supplementation, when intracellular decompartmentalized state of Fe reverts back to normal compartmentalized state of Fe in host tissues and Fe is egressed out of cells, this author has progressed to assay Hb before and after Zn supplementation in TB patients to mull and interpret whether the egressed Fe can be used up by host for Hb synthesis.

MATERIALS AND METHODS

The cohort study was conducted at BS Medical College and Hospital, Bankura-722 102, West Bengal, India, from June 2004 to May 2009. Permission for research protocol along with a study on blood samples obtained from human subjects and also on zinc supplementation for TB patients was approved in writing by the Ethics Committee of BS Medical College and Hospital, Bankura (Vide Memo No. 3/BSMC/04 dt 14-05-2004).A total of 224 participants (aged 8−64 years), including normal control, lung disease control, PTB, and EPTB subjects, were enrolled for the project. The study was conducted in four phases with 56 participants being enrolled in each phase. The purpose of the study was explained to all participants, and before collection of blood, informed verbal consent was obtained from each of the subjects.

The following groups (Gp) of subjects were taken into consideration:

PROCEDURE METHODOLOGY

STATISTICAL ANALYSIS

Statistical analysis of the results was made using Statistical Software for Social Sciences (SPSS Version 21.0). The level of significance was assessed using Student’s t-test. p < 0.05 was considered to be significant statistically.

RESULT AND DISCUSSION

Baseline serum Fe in PTB and EPTB patients had shown a significant increase when compared with those of NC (p = 0.004) and LDC (p = 0.005) subjects. While there was no noteworthy change in serum Fe level after 30 days treatment in LDC subjects (p = 0.5), there was a noteworthy but statistically insignificant increase in serum Fe after 30 days’ additional A-TB drug treatment without Zn supplementation for both PTB and EPTB patients (p = 0.15). On other hand, for PTB and EPTB under Zn supplementation along with A-TB drugs for 1 month, there was a statistically significant increase in serum Fe level (p = 0.004) when compared with baseline serum Fe level in TB and EPTB patients (Table 1).

In the second part of this research study, it had been recorded that while both PTB and EPTB patients without Zn supplementation had shown increased but statistically insignificant increase in Hb concentration (p = 0.2), with Zn supplementation for 1 month, there was statistically significant increase in Hb percentage for PTB as well as EPTB patients (p = 0.02) (Table 2).

The increased serum Fe in TB patients is due to the presence of increased siderophore bound Fe⁺³ complexes in serum. In this study, it is evident that oral supplementation does increase significantly serum Fe level in TB patients, and also the increased serum Fe might be used up for Hb synthesis for them. So, the veritable question is what the mechanism behind it might be.

To begin with, it is worth mentioning that haem and Hb are well utilized by Mtb as Fe sources.²² As already mentioned in the “Introduction”, siderophores chelate Fe⁺³ from insoluble and protein-bound Fe. Again, several proteins like haem-binding protein, cell-surface protein, and RND efflux pumps had been implicated for Mtb to utilize haem and Hb as Fe sources.^23,24 Fe is readily accessible in aqueous medium to oxidative states ferrous (Fe⁺²) and ferric (Fe⁺³) and thus determines participation of a large variety of biochemical reactions concerned with electron transport, activation of molecular oxygen and nitrogen, and also binding of oxygen with Hb and myoglobin. In ferric state, Fe is liable to be hydrolyzed forming polynuclear hydroxide complexes, biologically unavailable molecules. So, redox reaction to release Fe as relatively water-soluble Fe⁺² might be essential combating the kinetic barrier. In view of the redox equilibrium of Fe⁺²−Fe−³ couple involving transfer of one electron in oxidation−reduction in biological system, it is very logical to infer that free radicals are likely to be involved in the process. Therefore, in Fe overload in biological systems, free radical intermediates may account for toxicity of Fe. Important factors for mobilization of Fe are ligand complexes of Fe⁺³, macromolecules for Fe storage and transport, and also the presence of lower-molecular-weight chelating molecules. Transferrin, the most dynamic Fe carrier in human, after binding to specific cell surface transferrin receptor (Tfr), e.g., erythroid precursors in bone marrow, leaves out Fe in endocytosed vesicles within the cell and comes back as apotransferrin. Nature has made provision that vital sites sensitive to Fe-catalyzed oxidation are buried in macromolecular structure preferably in hydrophobic milieu or by being bound to catabolically inert Zn ion (Zn⁺²) and thus well-protected from interaction with Fe. So, compartmentalization of Fe in the human system is very important as Fe-catalyzed oxidation of thiol groups results in formation of highly reactive and damaging free radicals.

With infection caused by Mtb, there is decompartmentalization of Fe in host tissue by binding of Fe⁺³ to siderophores secreted by Mtb, which are stronger binding sites as well as diffusible ones. Siderophores can chelate Fe⁺³ from insoluble and protein−bound Fe as already mentioned. It had been reported that two Mtb-encoded storage proteins Bfr A and Bfr B aggregate to form macromolecules having 24 subunits holding 600−2400 Fe atoms per molecule.¹² After Irt AB-mediated reduction of Fe⁺³ in internalized Fe⁺³- siderophore complex into Fe⁺² and release, Fe-free siderophore (desferricarboxymycobactin) is recycled through Mmps bound to transporter complex through inner membrane.⁶ Recycling of desferricarboxymycobactin, however, is an important mechanism for Mtb to acquire Fe at lower metabolic cost.²⁵ Jones et al. reported that Fe salts could not be utilized by Mtb in the absence of siderophores and genetic disruption of siderophore export, and recycling had reduced the capacity of Mtb to take up Fe and had caused siderophore-mediated self-poisoning.²⁵ Siderophore-mediated Fe uptake is essential for survival of Mtb in macrophages as knockout mutants, defective in siderophore synthesis and uptake, had inhibited growth in macrophages.¹⁰ It is interesting to note that three protons (H⁺) released per molecule of apo-transferrin being oxidized by molecular oxygen to Fe⁺³−transferrin−carbonate complex, are well-utilized by Mtb for dismutation reaction catalyzed by Fe-cofactored SOD secreted by it, thus helping to provide soluble oxygen for survival of the aerobe. It is also reported that within phagosomes, Mtb interacts early endosomes and does not acidify below pH 6.3−6.5.²⁶ But, reduction of pH of vesicles to 5.5 by hydrogen-proton pump (H⁺ATPase) is required for dissociation of Fe-bound transferrin to release its Fe.²⁷ Thus, Mtb by preventing phagosome acidification and lysosomal fusion is able to acquire Fe from host endosomal holotransferrin.

Therefore, in prevailing situation of infection by Mtb, there is a major decompartmentalized state of Fe in host tissues. In TB, it is then logical that siderophores having more affinity toward Fe⁺³ become major transporter of Fe in serum instead of transferrin. So, the major role of transferrin to play a pivotal role in innate immune system chelating free toxic Fe and thus acting as protective scavenger, gets a setback. Also, with increased degree of inflammation in TB, and hence decreased serum transferrin, major decompartmentalization of Fe prevails in host tissues. In that situation, there is generation of remarkable quantity of highly reactive superoxide (O₂-) by Fe-catalyzed oxidation of thiol groups. Interestingly, Mtb utilizes this O₂- for dismutation reaction to generate soluble oxygen for its survival. Also, Mtb utilizes O₂- produced by phagosome with the help of its membrane-bound NADPH oxidase system to generate soluble oxygen by dismutation reaction.²⁸

Notwithstanding, physical decompartmentalization of Fe in TB is by complexing of Fe with high-affinity siderophores that are diffusible ones, decreasing and damaging normal Fe-binding sites, and altering the barriers. These facts have really corroborated with the present study of this author. Definite increase in baseline serum Fe in TB patients is due to abundant extracellular release of high-affinity Fe⁺³ chelator siderophores that can scavenge Fe not only from insoluble and protein-bound Fe but also from mineral phases (Fe oxide and hydroxide) by formation of soluble Fe⁺³ complexes that can be taken up by active transport mechanism.²⁹ Also, nonsignificant increase of serum Fe (p = 0.15) has been reported in this study for TB patients after 30 days’ additional A-TB drug therapy (without Zn supplementation). This increase can be acclaimed that with A-TB drugs, there is decrease in mycobacterial load, resulting in decrease in siderophores and with decrease in the degree of inflammation, there is recovery of serum transferrin activity and thus; decompartmentalized state of Fe in host tissue begins to revert back to normal compartmentalized state of Fe. On other hand, highly significant increase in serum Fe (p = 0.004) with oral Zn supplementation along with A-TB drugs entails that Zn destabilizes decompartmentalized state of Fe in host tissues and hastens tremendously the process of reverting back to normal compartmentalized state of Fe and hence significant increase in serum Fe level.

Zn belonging to group 12 of periodic table is stable only as divalent cation (Zn⁺²) and due to its filled d-shell, it cannot directly take part in redox reactions, whereas two other bioactive metals Fe and copper (Cu) can stay in two different ionic states, e.g., Fe as ferrous (Fe⁺²) and ferric (Fe⁺³); and Cu as cuprous (Cu⁺) and cupric (Cu⁺²), and hence they are redox-active metals. Searle and Tomasi had demonstrated decrease in spin-trapped OH from Fe and cysteine in the presence of Zn. They inferred that competition between Fe (transition-active metal in biological system) and Zn for thiol amino acid had interfered with transfer of electron to O₂-.³⁰ So, Zn is able to antagonize Fe-mediated transport of electron and thus oxidation under normal and pathological conditions. Analogically, Zn might replace Fe bound to thiol groups. Hence, generation of O₂- is inhibited by Zn. With inhibition on generation of O₂-. Mycobacterium tuberculosis gets a jolt with remarkable decrease in formation of soluble oxygen by dismutation reaction, required badly for survival of this obligate aerobe. With hastening process of normal compartmentalization of Fe, supply of Fe for Mtb is curtailed, thus activity of Fe-cofactored SOD is diminished. In fact, as per co-ordination chemistry similarities, Zn can compete with Cu and Fe for certain types of binding, thus suppressing their ability in certain particular environment to transfer electron might suppress O₂- generation. Also, competition of Zn⁺² with Fe and Cu can lead to inhibition of NADPH-oxidase enzyme and thus can curtail O₂- production and thus can limit generation of soluble oxygen by dismutation reaction and thereby accelerate killing of Mtb in host tissue. It is recently reported that Zn⁺-limited Mtb is not only more resistant to oxidative stresses but also has increased replication in vivo and thus has increased survival as evidenced by formation of severe pulmonary granuloma in mice.³¹

In the second part of this work, TB patients with Hb% less than 9 gm/dL at baseline were spotted to observe the result on Hb% with Zn supplementation, which might be well-precipitating and well-marked keeping into view the veritable acceleration of decompartmentalized Fe to revert back to normal compartmentalized state of Fe in host tissues. It is interesting and unequivocal to note that TB patients with A-TB drugs for 1 month but without any Zn supplementation had shown nonsignificant increase in Hb%, however, TB patients with A-TB drugs and oral Zn supplementation for 1 month had shown significant increase in Hb% (p = 0.02). This entails that A-TB drugs can bring decompartmentalized state of Fe reverted back to normal compartmentalized state of Fe at low pace by decreasing mycobacterial load and hence Fe⁺³-chelator siderophores along with mycobacterial membrane protein and Irt A/Irt B proteins in host tissue. On other hand, with oral Zn supplementation and A-TB drugs, process of decompartmentalized state of Fe reversing to normal compartmentalized state of Fe gets accelerated to new height by curtailing superoxide formation and by other mechanisms as already elaborated and thus accelerating killing of Mtb in host tissue. With remarkable recovery from infection and hence inflammation serum transferrin level increases and with well-marked decrease in siderophore level caused by enhanced killing of Mtb; transferrin might get bound in greater number to specific cell surface transferrin receptors like erythroid precursors in bone marrow and leaving out Fe in endocytosed vesicles within the cells. This Fe can again be used up for Hb synthesis, which has been reflected by statistically significant increase in Hb% with oral Zn supplementation. Thus, oral Zn supplementation (25 mg elemental Zn daily) along with A-TB drug treatment might act as a great bactericidal against Mtb causing accelerated killing of Mtb, making an onslaught against virulence of Mtb, and thus also accelerating the general well-being of the patient as reflected in this study by significant increase in Hb%.

CONCLUSION

It is great solemnity to promulgate that oral Zn supplementation (25 mg elemental Zn daily) may be added to drug regime to treat TB for enhancing Mtb killing pertaining to general well-being of host as reflected by significant increase in Hb%.

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