Abstract
Background to our responses
It is worthy to mention that oxidative stress is a common pathogenic mechanism underlying the development of many diseases and conditions including malarial infection where both the host and the parasite are affected. Although oxidative stress, anaemia and malarial infection are closely linked, their complex relationships and the functional relevance of oxidative stress genes are poorly understood. 1,2
In Africa, the first line of therapy for the treatment of malaria is usually through the administration of antimalarial drugs. At present, the commonly used antimalarial drugs in Nigeria are artemisinin, artesunate, chloroquine, coartem and quinine with different mechanism of actions at the therapeutic doses. 3 In spite of this, a good number of these drugs have the potential to cause harm when they are misused or abused. 4 It is important to appreciate the facts that these drugs, when administered, are basically transported via blood stream and therefore can affect various blood components like erythrocytes, monocytes, neutrophils, among others. Therefore, it is safe to postulate that whole blood cells are the first line of contact and a major systemic reservoir of these xenobiotics.
Our published work ‘Alteration of redox status by commonly used antimalarial drugs in the north-western region of Nigeria’ is actually a preliminary work and a part of larger scope of our research intended to focus on the ‘effects of overdose/misuse of commonly used antimalarial drugs on specific blood cells components’ using in vitro and in vivo models. You will of course agree with us that it is more rational for a good scientist to initially commence such a research with the entire blood system in order to have a good picture and direction on the appropriate line of research, including cell types, to pursue with a view to answer our broad research question. Thus, the (high) concentration of the drugs was selected because the research was aimed at deciphering the biochemical implications of the overdose/abuse of antimalarials in relation to redox status. Hence, the selected concentrations may not necessarily reflect the final in vivo concentrations of the drugs when administered at the therapeutic doses.
Our responses
Query 1: Carefully looking at our experimental design, it is evident that for both blood of normal and malarial infected humans, we have negative control (untreated) samples which were used for result and statistical comparisons. However, we interchangeably used the words ‘phosphate buffer’ and ‘phosphate buffer saline (Sigma Aldrich, St Louis, Missouri, USA)’ and the latter was actually used in our experiments. Note that when publishing research findings, most journals discourage being explicit in materials and methods. In fact, some even give restrictions on the number of words/pages. In any case, we had untreated samples that serve as the basis of comparisons.
The issue of haemolysis and haemoglobin release has been critically addressed in our recent publication which is also the part of the larger project ‘Induction of haemolysis and dna fragmentation in a normal and malarial-infected blood by commonly-used antimalarial drugs in the north-western region of Nigeria. 4
For the query about the concentrations of drugs being reduced by half instead of threefold, we would like to emphasize that we are indeed correct for stating that it was reduced by half. This is because equal volumes (100 µL) of the blood and the buffer were mixed. However, in the reaction mixture, 100 µL of the diluted blood (above) was mixed with 100 µL of the drug and this unequivocably makes the concentration of the drug to be reduce by half as correctly captured in the published article.
Query 2: Again, we have untreated samples as the basis of comparisons; therefore, any effects that may be due to temperature variations would have been addressed, because all the experimental tubes were subjected to the same conditions. Furthermore, we were not working on platelets at that critical stage. Additionally, perhaps more importantly, the work was not conducted in a temperate climate country such as Poland where the temperatures are low. Rather, the work was conducted in northern Nigeria where room temperature can be as high as >30°C.
It is important to clarify at this stage that the incubation temperature for the administration of drugs was not targeted at culturing the parasite but for the drugs’ action which was adopted after our pilot studies on the same oxidative stress biomarkers which are of course part of the secondary effects of antimalarials. 5,6 Therefore, one can evidently mention that any changes in these biomarkers could be attributed to the presence of these drugs at varying concentrations.
Query 3: The parasitaemia level was not the part of the scope of the then published article. However, cognizance can be made to this for our future research. Likewise, we believe the number of participating individuals must not be stated because the published work was not an epidemiological/population-based study. Please note that in technical articles, such as those published by a reputable journal like Human and Experimental Toxicology, it is not uncommon for the methodology section to describe the key methods/information only and leave out the very basic and known scientific information.
Query 4: Again, the issue of haemolysis and haemoglobin release being the part of the total protein pool has been addressed in our recent publication which is also the part of the larger project ‘Induction of haemolysis and DNA fragmentation in a normal and malarial-infected blood by commonly-used antimalarial drugs in the north-western region of Nigeria’. 4 We are also not sure of the need for a corrective measurements because a number of previous authors have reported such a high protein concentration using the Biuret method. It is also important to note that assay protocols are prone to limitations which can vary across laboratories. 7
Second, we did not specify the isozyme of superoxide dismutase (SOD) or catalase investigated. This is because our interest is ‘alteration of redox status’ and therefore, whatever the forms of SOD or catalase perform the overall catalytic function may be inconsequential. Moreover, we used assays that detect all the isoenzymes (forms) of the SOD or catalase as the case may be. Perhaps our future assessment may specifically target such isoenzymes depending on the experimental question and design. Third, it is quite unfortunate that Karolina et al. decided to write a query letter to the editor on issues that were not clearly understood by them. This query alongside some previous ones make the enzymological and perhaps, the scientific backgrounds of the authors to be questionable. This reaction involves an enzyme (catalase from the blood samples) and a substrate (hydrogen peroxide) which was allowed to proceed before the addition of the chromogen and then incubation followed. Thus, the catalase reaction has been well completed before the incubation at 80°C and therefore the issue of catalase inhibition before enzymatic reaction is enzymologically very weak.
In fact, there was nothing in our article to indicate that dichromate/acetic acid solution was added at the beginning of the said procedure. Please read carefully the protocol in the published article: The mixture was allowed to stand for 4 h. Thereafter, 0.9 mL of distilled water was added to 0.1 mL of the treated sample. Then, 2.5 mL of phosphate buffer (pH 7.4) was added to 0.5 mL of the mixture followed by the addition of 2 mL of 0.2 M H2O2. It was mixed thoroughly and 1 mL of dichromate/acetic acid solution was added at the interval of 1 min for 3 min. 3 It is also important to note that same protocol has always been adopted elsewhere. 8
Query 5: It seems like Karolina et al. did not fully apprehend and appreciate our explanations under the ‘Discussions’ section. Our result was two-dimensional in nature, apparently healthy and malarial infected blood experiments. When we said normal blood samples, we do not refer to untreated samples in the malarial infected samples. This cuts across malondialdehyde (MDA), SOD, and so on. Meaning that, we have both results being compared within and across both samples after antimalarial administrations purposely for discussion’s sake.
Conclusions
While we appreciate the tireless effort made by Karolina et al. to pinpoint some things about our published article, we would like to draw their attention to the fact that every research starts and ends with questions. So also, in all scientific investigations, there is no single protocol without limitations vis-à-vis merits and demerits. Furthermore, there is a crucial need for sound scientific and conceptual backgrounds before writing a letter to an editor such as this which was apparently lacking. Therefore, we are maintaining our earlier stand that ‘commonly used antimalarials might alter the redox status in both healthy and non-healthy subjects from north-western region of Nigeria thereby inducing oxidative stress in vitro’. This is subject to further in-depth scientific investigations.