Parasitology International 64 (2015) 301–303
Contents lists available at ScienceDirect
Parasitology International journal homepage: www.elsevier.com/locate/parint
Short communication
Lactate retards the development of erythrocytic stages of the human malaria parasite Plasmodium falciparum Kenji Hikosaka a,b, Makoto Hirai c, Keisuke Komatsuya a, Yasuo Ono b, Kiyoshi Kita a,⁎ a b c
Department of Biomedical Chemistry, Graduate School of Medicine, The University of Tokyo, Bunkyo-ku, Tokyo, Japan Department of Microbiology and Immunology, Teikyo University School of Medicine, Itabashi, Tokyo, Japan Department of Molecular and Cellular Parasitology, Juntendo University School of Medicine, Bunkyo-ku, Tokyo, Japan
a r t i c l e
i n f o
Available online 28 August 2014 Keywords: Malaria Plasmodium Lactate In vitro culture Developmental retardation
a b s t r a c t The intraerythrocytic form of the human malaria parasite Plasmodium falciparum relies on glycolysis for its energy requirements. In glycolysis, lactate is an end product. It is therefore known that lactate accumulates in in vitro culture; however, its influence on parasite growth remains unknown. Here we investigated the effect of lactate on the development of P. falciparum during in vitro culture under lactate supplementation in detail. Results revealed that lactate retarded parasite development and reduced the number of merozoites in the schizont stage. These findings suggest that lactate has the potential to affect parasite development. © 2014 Elsevier Ireland Ltd. All rights reserved.
Malaria is one of the major global public health problems, annually truncating several hundred thousand lives, mostly in sub-Saharan African countries [1]. The disease is caused by parasitic protozoa, belonging to the Plasmodium genus. In this genus, Plasmodium falciparum, the human malaria parasite, causes cerebral malaria (often referred to as falciparum malaria), with a remarkably high mortality. P. falciparum has a complicated life cycle, in which there are two major cycles: asexual multiplication in humans and sexual multiplication in mosquitoes [2]. In humans, an erythrocytic cycle is composed of the ring form, trophozoite, and schizont stages. The erythrocytic stages can be cultured in vitro using human red blood cells [3]. In the early 1980s, many researchers tried to establish a culture method for high-efficiency production of the parasite. Some of these studies have reported that lactate accumulated in the supernatant of the culture medium and had a deleterious effect on parasite growth [4,5]. In the erythrocytic stages, ATP production by the parasite is highly dependent on glycolysis [6]. Lactate is an end product of the ATP production pathway by glycolysis; hence, it accumulates in the supernatant. Apart from the deleterious influence of lactate on parasite growth in vitro, hyperlactatemia has been used as an indicator of disease severity for malaria patients [7]. In most clinical studies on falciparum malaria, compared with the normal plasma lactate level (0.3 to 1.3 mM) [8], the levels of plasma lactate tend to be high and indicate significant variability, ranging from 2 to 26.7 mM [7, 9]. Consequently, the malaria parasite is often exposed to high concentrations of lactate in malaria patients and may be subjected to the ⁎ Corresponding author at: Department of Biomedical Chemistry, Graduate School of Medicine, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan. Tel.: +81 3 5841 3526; fax: +81 3 5841 3444. E-mail address: [email protected] (K. Kita).
http://dx.doi.org/10.1016/j.parint.2014.08.003 1383-5769/© 2014 Elsevier Ireland Ltd. All rights reserved.
adverse influence of lactate in the infected human body as well as in in vitro culture. Lactate is thus considered to be an intriguing molecule for parasite growth, although the mechanism by which lactate affects the parasite remains unclear. In this study, we conducted a cell biological study to assess the effect of lactate on the development of the human malaria parasite P. falciparum using an in vitro culture system under lactate supplementation. The human malaria parasite P. falciparum 3D7 strain was synchronized to the ring form stage using the single-step sorbitol method [10] and was cultured as described previously [3] with various concentrations (0, 1, 5, 10, 20, 30, and 60 mM) of sodium L-lactate (Sigma). As a result, the growth of synchronous parasites was suppressed by lactate in a dose-dependent manner (Fig. 1A). This is not caused by pH fluctuation because lactate supplementation did not change the pH of the culture medium (data not shown). Parasitemia of cultures supplemented with more than 10 mM lactate for 48 h was significantly lower than that of a culture without lactate (P b 0.01). When culturing parasites for 24 h, the percentage of each developmental stage was affected by lactate supplementation (Supplementary Fig. 1A). In these cultures, parasite development had a tendency to be retarded by each lactate concentration (e.g., the percentage of the ring form stage: 22% in 10 mM lactate; 13% in 20 mM lactate; 8% in 30 mM lactate; 3% in 60 mM lactate). In parasites cultured for 48 h, lactate supplementation at concentrations of 20, 30, and 60 mM affected the percentage of each developmental stage more remarkably (e.g., the percentage of the ring form stage: 65% in 0 mM lactate; 87% in 20 mM lactate; 93% in 30 mM lactate; 28% in 60 mM lactate) (Supplementary Fig. 1B). These results suggest that lactate would retard parasite development and consequently suppress parasite multiplication. To further investigate the parasite stages that are most affected by lactate, we prepared
302
K. Hikosaka et al. / Parasitology International 64 (2015) 301–303
A)
B)
100%
7
75%
Lactate (mM)
6
50% 25%
0
5
*
4
* *
0
1 5 10 20
3
0%
30
% of each stage
Parasitemia (%)
Control
60
2
16
24
32
40
48
24
32
40
48
24
32
40
48
Lactate 30 mM 100% 75% 50% 25% 0%
0
16
Lactate 60 mM
0
100%
*
1
75% 50%
0
24
25%
48
0%
Time (h)
0
16
Time (h)
Fig. 1. Growth suppression and developmental retardation of Plasmodium falciparum by lactate. (A) Parasite cultures synchronized with 5% (w/v) D-sorbitol were incubated at lactate concentrations of 0, 1, 5, 10, 20, 30, and 60 mM. Parasitemia at 0 h was 1.0% in each group. All experiments were performed in triplicate, and all data are represented as mean values and standard errors. *P b 0.01 compared with parasitemia of a culture not supplemented with lactate (0 mM). (B) Parasite cultures synchronized with sorbitol and Percoll density gradients were incubated at lactate concentrations of 0, 30, and 60 mM. White, gray, and black bars indicate percentages of ring form, trophozoite, and schizont, respectively. In a control culture, to which no lactate is added, it took 48 h for the parasite to develop from a ring form to the next ring form. Developmental retardation of the parasites was observed from 24 h to 48 h after supplementation with 30 mM lactate. When supplemented with 60 mM lactate, the retardation was even more marked.
25
Number of merozoite/segmenter
the parasites whose development was tightly synchronized to the ring form stage by sorbitol and Percoll (GE healthcare) density gradients [11]. As a result, when the parasites were cultured with lactate at 30 and 60 mM, a significant number of parasites remained in the schizont stage (Fig. 1B). This result implies that the schizont stage may be susceptible to the effect of lactate. In addition to the growth suppression, the effect of lactate on parasite morphology was observed. As shown in Supplementary Fig. 2, the ring form and trophozoite stages showed a normal shape even when these stages of parasites were cultured in the highest concentration of lactate (60 mM). In sharp contrast, when the schizont stage of the parasite was exposed to a high concentration of lactate at 60 mM but not at 0 and 30 mM, it showed an abnormal morphology: a smaller schizont in size and fewer purple dots in the schizont. Besides the morphological change in the schizont stage, we found a remarkable variation in the number of merozoites in a segmenter (the late stage of the schizont stage). To confirm this difference, we counted the number of merozoites using 50 representative segmenters in each group. In a culture not supplemented with lactate (control), approximately 19 merozoites were found in a segmenter (Fig. 2). When lactate was added to the culture at the concentrations of 30 and 60 mM, 16 and 10 merozoites were found in a segmenter, respectively. These numbers were significantly lower than those in the control culture, suggesting that lactate reduced the number of P. falciparum merozoites formed as well as retarded their development. In this study, we demonstrated that lactate, which is an end product of glycolysis, had an adverse impact on P. falciparum growth when it was present in excess in culture medium. In the erythrocytic stages, a trophozoite stage parasite has been reported to produce the largest amount of lactate [4]. It is thus reasonable to infer that the trophozoite may need to extrude lactate to the outside using a mechanism such as an efflux pump. In general, lactate is transported via the monocarboxylate transporter (MCT) family [12]. In P. falciparum, two genes encoding MCTs (PF3D7_0210300 and PF3D7_0926400) are identified in its
*
*
20
15
10
5
0
Control
Lactate 30 mM
Lactate 60 mM
Fig. 2. Comparison of the number of merozoites contained in one segmenter. In total, 50 segmenters were counted. *P b 0.01 compared with culture not supplemented with lactate (control). Bottom figures indicate representative segmenters, which were stained using Giemsa stain solution.
K. Hikosaka et al. / Parasitology International 64 (2015) 301–303
genome on the PlasmoDB (Version 11.0) website (http://plasmodb.org/ plasmo/). To elucidate the involvement of these MCTs in lactate influx/ efflux, we investigated the expression levels of the two genes by quantitative real-time PCR. The results exhibited that these MCT genes were differentially expressed at various developmental stages of the parasites (Supplementary Fig. 3). It is noteworthy that the expression levels of these genes are significantly enhanced at the trophozoite stage by lactate supplementation. This finding together with the fact that trophozoite-stage parasites exhibit the largest lactate production allows us to infer that these MCTs might be associated with lactate transport, probably functioning as an efflux pump even though further experiments are needed to address this point. Lactate dehydrogenase (LDH) is a final enzyme in glycolysis, converting pyruvate to lactate [13]. When lactate is present at a high concentration, LDH exhibits feedback inhibition and the conversion rate of pyruvate to lactate is decreased. Here we found that externally supplied lactate inhibited parasite growth in vitro. In this context, it is a logically acceptable notion that the excess amount of lactate may inhibit LDH activity, resulting in the retardation of parasite development. This assumption is supported by the fact that the expression levels of two MCT genes, putative lactate transporters, were enhanced by lactate supplementation at the trophozoite stage. In the present study, we demonstrated that lactate is a critical factor for parasite development. It is therefore likely that a study focusing on the lactate transporter would accelerate the development of antimalarial drugs. To apply the lactate transporter to research as a drug target for controlling malaria, further biochemical analysis of the transporter such as MCTs is required. Similarly, it is very important to reveal the molecular mechanism by which lactate retards the development of the parasite. Acknowledgments We would like to thank Y. Koyama (Pet Salon Flora) for the helpful discussion and E.O. Balogun (The University of Tokyo) for English corrections. This work was supported by Grant-in-Aids for Scientific Research from the Ministry of Education, Culture, Sports, Science, and
303
Technology of Japan (18073013 and 26253025) and Research Fellowships for Young Scientists of the Japan Society for the Promotion of Science (23•4161). Appendix A. Supplementary data Supplementary data to this article can be found online at http://dx. doi.org/10.1016/j.parint.2014.08.003. References [1] World Health Organization. World Malaria Report 2013. Available at: http://www. who.int/malaria/publications/world_malaria_report_2013/en/. (Accessed: march/ 2014). [2] Cowman AF, Crabb BS. Invasion of red blood cells by malaria parasites. Cell 2006; 124(4):755–66. [3] Trager W, Jensen JB. Human malaria parasites in continuous culture. Science 1976; 193(4254):673–5. [4] Pfaller MA, Krogstad DJ, Parquette AR, Nguyen-Dinh P. Plasmodium falciparum: stage-specific lactate production in synchronized cultures. Exp Parasitol 1982; 54(3):391–6. [5] Zolg JW, Macleod AJ, Scaife JG, Beaudoin RL. The accumulation of lactic acid and its influence on the growth of Plasmodium falciparum in synchronized cultures. In Vitro 1984;20(3 Pt 1):205–15. [6] Jensen MD, Conley M, Helstowski LD. Culture of Plasmodium falciparum: the role of pH, glucose, and lactate. J Parasitol 1983;69(6):1060–7. [7] Krishna SWD, ter Kuile F, Kwiatkowski D, Crawley J, Craddock CF, Nosten F, et al. Lactic acidosis and hypoglycaemia in children with severe malaria: pathophysiological and prognostic significance. Trans R Soc Trop Med Hyg 1994;88(1):67–73. [8] Phypers B, Pierce T. Lactate physiology in health and disease. Contin Educ Anaesth Crit Care Pain 2006;6(3):128–32. [9] Agbenyega T, Angus BJ, Bedu-Addo G, Baffoe-Bonnie B, Guyton T, Stacpoole PW, et al. Glucose and lactate kinetics in children with severe malaria. J Clin Endocrinol Metabol 2000;85(4):1569–76. [10] Lambros C, Vanderberg JP. Synchronization of Plasmodium falciparum erythrocytic stages in culture. J Parasitol 1979;65(3):418–20. [11] Nillni EA, Londner MV, Spira DT. A simple method for separation of uninfected erythrocytes from those infected with Plasmodium berghei and for isolation of artificially released parasites. Z Parasitenkd 1981;64:279–84. [12] Halestrap AP. The monocarboxylate transporter family — structure and functional characterization. IUBMB Life 2012;64(1):1–9. [13] Penna-Coutinho J, Cortopassi WA, Oliveira AA, França TC, Krettli AU. Antimalarial activity of potential inhibitors of Plasmodium falciparum lactate dehydrogenase enzyme selected by docking studies. PLoS One 2011;6(7):e21237.
Contents lists available at ScienceDirect
Parasitology International journal homepage: www.elsevier.com/locate/parint
Short communication
Lactate retards the development of erythrocytic stages of the human malaria parasite Plasmodium falciparum Kenji Hikosaka a,b, Makoto Hirai c, Keisuke Komatsuya a, Yasuo Ono b, Kiyoshi Kita a,⁎ a b c
Department of Biomedical Chemistry, Graduate School of Medicine, The University of Tokyo, Bunkyo-ku, Tokyo, Japan Department of Microbiology and Immunology, Teikyo University School of Medicine, Itabashi, Tokyo, Japan Department of Molecular and Cellular Parasitology, Juntendo University School of Medicine, Bunkyo-ku, Tokyo, Japan
a r t i c l e
i n f o
Available online 28 August 2014 Keywords: Malaria Plasmodium Lactate In vitro culture Developmental retardation
a b s t r a c t The intraerythrocytic form of the human malaria parasite Plasmodium falciparum relies on glycolysis for its energy requirements. In glycolysis, lactate is an end product. It is therefore known that lactate accumulates in in vitro culture; however, its influence on parasite growth remains unknown. Here we investigated the effect of lactate on the development of P. falciparum during in vitro culture under lactate supplementation in detail. Results revealed that lactate retarded parasite development and reduced the number of merozoites in the schizont stage. These findings suggest that lactate has the potential to affect parasite development. © 2014 Elsevier Ireland Ltd. All rights reserved.
Malaria is one of the major global public health problems, annually truncating several hundred thousand lives, mostly in sub-Saharan African countries [1]. The disease is caused by parasitic protozoa, belonging to the Plasmodium genus. In this genus, Plasmodium falciparum, the human malaria parasite, causes cerebral malaria (often referred to as falciparum malaria), with a remarkably high mortality. P. falciparum has a complicated life cycle, in which there are two major cycles: asexual multiplication in humans and sexual multiplication in mosquitoes [2]. In humans, an erythrocytic cycle is composed of the ring form, trophozoite, and schizont stages. The erythrocytic stages can be cultured in vitro using human red blood cells [3]. In the early 1980s, many researchers tried to establish a culture method for high-efficiency production of the parasite. Some of these studies have reported that lactate accumulated in the supernatant of the culture medium and had a deleterious effect on parasite growth [4,5]. In the erythrocytic stages, ATP production by the parasite is highly dependent on glycolysis [6]. Lactate is an end product of the ATP production pathway by glycolysis; hence, it accumulates in the supernatant. Apart from the deleterious influence of lactate on parasite growth in vitro, hyperlactatemia has been used as an indicator of disease severity for malaria patients [7]. In most clinical studies on falciparum malaria, compared with the normal plasma lactate level (0.3 to 1.3 mM) [8], the levels of plasma lactate tend to be high and indicate significant variability, ranging from 2 to 26.7 mM [7, 9]. Consequently, the malaria parasite is often exposed to high concentrations of lactate in malaria patients and may be subjected to the ⁎ Corresponding author at: Department of Biomedical Chemistry, Graduate School of Medicine, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan. Tel.: +81 3 5841 3526; fax: +81 3 5841 3444. E-mail address: [email protected] (K. Kita).
http://dx.doi.org/10.1016/j.parint.2014.08.003 1383-5769/© 2014 Elsevier Ireland Ltd. All rights reserved.
adverse influence of lactate in the infected human body as well as in in vitro culture. Lactate is thus considered to be an intriguing molecule for parasite growth, although the mechanism by which lactate affects the parasite remains unclear. In this study, we conducted a cell biological study to assess the effect of lactate on the development of the human malaria parasite P. falciparum using an in vitro culture system under lactate supplementation. The human malaria parasite P. falciparum 3D7 strain was synchronized to the ring form stage using the single-step sorbitol method [10] and was cultured as described previously [3] with various concentrations (0, 1, 5, 10, 20, 30, and 60 mM) of sodium L-lactate (Sigma). As a result, the growth of synchronous parasites was suppressed by lactate in a dose-dependent manner (Fig. 1A). This is not caused by pH fluctuation because lactate supplementation did not change the pH of the culture medium (data not shown). Parasitemia of cultures supplemented with more than 10 mM lactate for 48 h was significantly lower than that of a culture without lactate (P b 0.01). When culturing parasites for 24 h, the percentage of each developmental stage was affected by lactate supplementation (Supplementary Fig. 1A). In these cultures, parasite development had a tendency to be retarded by each lactate concentration (e.g., the percentage of the ring form stage: 22% in 10 mM lactate; 13% in 20 mM lactate; 8% in 30 mM lactate; 3% in 60 mM lactate). In parasites cultured for 48 h, lactate supplementation at concentrations of 20, 30, and 60 mM affected the percentage of each developmental stage more remarkably (e.g., the percentage of the ring form stage: 65% in 0 mM lactate; 87% in 20 mM lactate; 93% in 30 mM lactate; 28% in 60 mM lactate) (Supplementary Fig. 1B). These results suggest that lactate would retard parasite development and consequently suppress parasite multiplication. To further investigate the parasite stages that are most affected by lactate, we prepared
302
K. Hikosaka et al. / Parasitology International 64 (2015) 301–303
A)
B)
100%
7
75%
Lactate (mM)
6
50% 25%
0
5
*
4
* *
0
1 5 10 20
3
0%
30
% of each stage
Parasitemia (%)
Control
60
2
16
24
32
40
48
24
32
40
48
24
32
40
48
Lactate 30 mM 100% 75% 50% 25% 0%
0
16
Lactate 60 mM
0
100%
*
1
75% 50%
0
24
25%
48
0%
Time (h)
0
16
Time (h)
Fig. 1. Growth suppression and developmental retardation of Plasmodium falciparum by lactate. (A) Parasite cultures synchronized with 5% (w/v) D-sorbitol were incubated at lactate concentrations of 0, 1, 5, 10, 20, 30, and 60 mM. Parasitemia at 0 h was 1.0% in each group. All experiments were performed in triplicate, and all data are represented as mean values and standard errors. *P b 0.01 compared with parasitemia of a culture not supplemented with lactate (0 mM). (B) Parasite cultures synchronized with sorbitol and Percoll density gradients were incubated at lactate concentrations of 0, 30, and 60 mM. White, gray, and black bars indicate percentages of ring form, trophozoite, and schizont, respectively. In a control culture, to which no lactate is added, it took 48 h for the parasite to develop from a ring form to the next ring form. Developmental retardation of the parasites was observed from 24 h to 48 h after supplementation with 30 mM lactate. When supplemented with 60 mM lactate, the retardation was even more marked.
25
Number of merozoite/segmenter
the parasites whose development was tightly synchronized to the ring form stage by sorbitol and Percoll (GE healthcare) density gradients [11]. As a result, when the parasites were cultured with lactate at 30 and 60 mM, a significant number of parasites remained in the schizont stage (Fig. 1B). This result implies that the schizont stage may be susceptible to the effect of lactate. In addition to the growth suppression, the effect of lactate on parasite morphology was observed. As shown in Supplementary Fig. 2, the ring form and trophozoite stages showed a normal shape even when these stages of parasites were cultured in the highest concentration of lactate (60 mM). In sharp contrast, when the schizont stage of the parasite was exposed to a high concentration of lactate at 60 mM but not at 0 and 30 mM, it showed an abnormal morphology: a smaller schizont in size and fewer purple dots in the schizont. Besides the morphological change in the schizont stage, we found a remarkable variation in the number of merozoites in a segmenter (the late stage of the schizont stage). To confirm this difference, we counted the number of merozoites using 50 representative segmenters in each group. In a culture not supplemented with lactate (control), approximately 19 merozoites were found in a segmenter (Fig. 2). When lactate was added to the culture at the concentrations of 30 and 60 mM, 16 and 10 merozoites were found in a segmenter, respectively. These numbers were significantly lower than those in the control culture, suggesting that lactate reduced the number of P. falciparum merozoites formed as well as retarded their development. In this study, we demonstrated that lactate, which is an end product of glycolysis, had an adverse impact on P. falciparum growth when it was present in excess in culture medium. In the erythrocytic stages, a trophozoite stage parasite has been reported to produce the largest amount of lactate [4]. It is thus reasonable to infer that the trophozoite may need to extrude lactate to the outside using a mechanism such as an efflux pump. In general, lactate is transported via the monocarboxylate transporter (MCT) family [12]. In P. falciparum, two genes encoding MCTs (PF3D7_0210300 and PF3D7_0926400) are identified in its
*
*
20
15
10
5
0
Control
Lactate 30 mM
Lactate 60 mM
Fig. 2. Comparison of the number of merozoites contained in one segmenter. In total, 50 segmenters were counted. *P b 0.01 compared with culture not supplemented with lactate (control). Bottom figures indicate representative segmenters, which were stained using Giemsa stain solution.
K. Hikosaka et al. / Parasitology International 64 (2015) 301–303
genome on the PlasmoDB (Version 11.0) website (http://plasmodb.org/ plasmo/). To elucidate the involvement of these MCTs in lactate influx/ efflux, we investigated the expression levels of the two genes by quantitative real-time PCR. The results exhibited that these MCT genes were differentially expressed at various developmental stages of the parasites (Supplementary Fig. 3). It is noteworthy that the expression levels of these genes are significantly enhanced at the trophozoite stage by lactate supplementation. This finding together with the fact that trophozoite-stage parasites exhibit the largest lactate production allows us to infer that these MCTs might be associated with lactate transport, probably functioning as an efflux pump even though further experiments are needed to address this point. Lactate dehydrogenase (LDH) is a final enzyme in glycolysis, converting pyruvate to lactate [13]. When lactate is present at a high concentration, LDH exhibits feedback inhibition and the conversion rate of pyruvate to lactate is decreased. Here we found that externally supplied lactate inhibited parasite growth in vitro. In this context, it is a logically acceptable notion that the excess amount of lactate may inhibit LDH activity, resulting in the retardation of parasite development. This assumption is supported by the fact that the expression levels of two MCT genes, putative lactate transporters, were enhanced by lactate supplementation at the trophozoite stage. In the present study, we demonstrated that lactate is a critical factor for parasite development. It is therefore likely that a study focusing on the lactate transporter would accelerate the development of antimalarial drugs. To apply the lactate transporter to research as a drug target for controlling malaria, further biochemical analysis of the transporter such as MCTs is required. Similarly, it is very important to reveal the molecular mechanism by which lactate retards the development of the parasite. Acknowledgments We would like to thank Y. Koyama (Pet Salon Flora) for the helpful discussion and E.O. Balogun (The University of Tokyo) for English corrections. This work was supported by Grant-in-Aids for Scientific Research from the Ministry of Education, Culture, Sports, Science, and
303
Technology of Japan (18073013 and 26253025) and Research Fellowships for Young Scientists of the Japan Society for the Promotion of Science (23•4161). Appendix A. Supplementary data Supplementary data to this article can be found online at http://dx. doi.org/10.1016/j.parint.2014.08.003. References [1] World Health Organization. World Malaria Report 2013. Available at: http://www. who.int/malaria/publications/world_malaria_report_2013/en/. (Accessed: march/ 2014). [2] Cowman AF, Crabb BS. Invasion of red blood cells by malaria parasites. Cell 2006; 124(4):755–66. [3] Trager W, Jensen JB. Human malaria parasites in continuous culture. Science 1976; 193(4254):673–5. [4] Pfaller MA, Krogstad DJ, Parquette AR, Nguyen-Dinh P. Plasmodium falciparum: stage-specific lactate production in synchronized cultures. Exp Parasitol 1982; 54(3):391–6. [5] Zolg JW, Macleod AJ, Scaife JG, Beaudoin RL. The accumulation of lactic acid and its influence on the growth of Plasmodium falciparum in synchronized cultures. In Vitro 1984;20(3 Pt 1):205–15. [6] Jensen MD, Conley M, Helstowski LD. Culture of Plasmodium falciparum: the role of pH, glucose, and lactate. J Parasitol 1983;69(6):1060–7. [7] Krishna SWD, ter Kuile F, Kwiatkowski D, Crawley J, Craddock CF, Nosten F, et al. Lactic acidosis and hypoglycaemia in children with severe malaria: pathophysiological and prognostic significance. Trans R Soc Trop Med Hyg 1994;88(1):67–73. [8] Phypers B, Pierce T. Lactate physiology in health and disease. Contin Educ Anaesth Crit Care Pain 2006;6(3):128–32. [9] Agbenyega T, Angus BJ, Bedu-Addo G, Baffoe-Bonnie B, Guyton T, Stacpoole PW, et al. Glucose and lactate kinetics in children with severe malaria. J Clin Endocrinol Metabol 2000;85(4):1569–76. [10] Lambros C, Vanderberg JP. Synchronization of Plasmodium falciparum erythrocytic stages in culture. J Parasitol 1979;65(3):418–20. [11] Nillni EA, Londner MV, Spira DT. A simple method for separation of uninfected erythrocytes from those infected with Plasmodium berghei and for isolation of artificially released parasites. Z Parasitenkd 1981;64:279–84. [12] Halestrap AP. The monocarboxylate transporter family — structure and functional characterization. IUBMB Life 2012;64(1):1–9. [13] Penna-Coutinho J, Cortopassi WA, Oliveira AA, França TC, Krettli AU. Antimalarial activity of potential inhibitors of Plasmodium falciparum lactate dehydrogenase enzyme selected by docking studies. PLoS One 2011;6(7):e21237.
