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Improved methods for haemozoin quantification in tissues yield organ-and parasite-specific information in malaria-infected mice.

Deroost K, Lays N, Noppen S, Martens E, Opdenakker G, Van den Steen PE - Malar. J. (2012)

Bottom Line: Furthermore, total Hz contents correlated with peripheral parasitaemia and were significantly higher in mice with a lethal P. berghei ANKA or P. berghei NK65-infection than in mice with a self-resolving P. chabaudi AS-infection, despite similar peripheral parasitaemia levels.An organ-specific Hz deposition pattern was found and was independent of the parasite strain used.Highest Hz levels were identified in mice infected with lethal parasite strains suggesting that Hz accumulation in tissues is associated with malaria-related mortality.

View Article: PubMed Central - HTML - PubMed

Affiliation: Laboratory of Immunobiology, Rega Institute, University of Leuven, Leuven, Belgium.

ABSTRACT

Background: Despite intensive research, malaria remains a major health concern for non-immune residents and travelers in malaria-endemic regions. Efficient adjunctive therapies against life-threatening complications such as severe malarial anaemia, encephalopathy, placental malaria or respiratory problems are still lacking. Therefore, new insights into the pathogenesis of severe malaria are imperative. Haemozoin (Hz) or malaria pigment is produced during intra-erythrocytic parasite replication, released in the circulation after schizont rupture and accumulates inside multiple organs. Many in vitro and ex vivo immunomodulating effects are described for Hz but in vivo data are limited. This study aimed to improve methods for Hz quantification in tissues and to investigate the accumulation of Hz in different organs from mice infected with Plasmodium parasites with a varying degree of virulence.

Methods: An improved method for extraction of Hz from tissues was elaborated and coupled to an optimized, quantitative, microtiter plate-based luminescence assay with a high sensitivity. In addition, a technique for measuring Hz by semi-quantitative densitometry, applicable on transmitted light images, was developed. The methods were applied to measure Hz in various organs of C57BL/6 J mice infected with Plasmodium berghei ANKA, P. berghei NK65 or Plasmodium chabaudi AS. The used statistical methods were the Mann-Whitney U test and Pearsons correlation analysis.

Results: Most Hz was detected in livers and spleens, lower levels in lungs and kidneys, whereas sub-nanomolar amounts were observed in brains and hearts from infected mice, irrespectively of the parasite strain used. Furthermore, total Hz contents correlated with peripheral parasitaemia and were significantly higher in mice with a lethal P. berghei ANKA or P. berghei NK65-infection than in mice with a self-resolving P. chabaudi AS-infection, despite similar peripheral parasitaemia levels.

Conclusions: The developed techniques were useful to quantify Hz in different organs with a high reproducibility and sensitivity. An organ-specific Hz deposition pattern was found and was independent of the parasite strain used. Highest Hz levels were identified in mice infected with lethal parasite strains suggesting that Hz accumulation in tissues is associated with malaria-related mortality.

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Haem quantification by different techniques. Different concentrations of haematin (10 μM – 1.2 nM) were used to compare the sensitivity of previously described techniques to quantify haem in a 96-well based format. In panel A, haematin was measured with a spectrophotometer at 405 nm (n = 4 for each concentration). Panel B shows the measurements of haematin concentrations by haem-enhanced luminescence with the same reagent conditions as described by Schwarzer and colleagues (20) for a cuvette-based system. Background absorbance/luminescence from a blank sample was subtracted from all the measurements. The adjusted chemo-luminescence protocol (100-fold higher luminol and peroxide concentrations buffered around pH 10.4) was used to measure the same concentrations of haematin in panel C (n = 8 for each concentration in B and C). The horizontal dashed line denotes the accuracy limit of the assay. The time-dependence of the luminescence signal and the effect of 2% SDS are displayed in panel D and E, respectively. The arrow in panel D denotes the start of the kinetic reading after an initial delay of twelve seconds, and the vertical dashed line indicates the moment of luminescence detection during the experimental readings. In panel F, natural Hz was isolated from trophozoites (troph) and livers, its concentration was determined by absorption at 405 nm and the concentration-dependence of the haem-enhanced luminescence was measured. Inset of panel F is a picture demonstrating the birefringence of the isolated Hz. The amount of Hz/trophozoite was calculated and the corresponding number of trophozoites (# troph) is integrated in the figure as a second X-axis
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Figure 2: Haem quantification by different techniques. Different concentrations of haematin (10 μM – 1.2 nM) were used to compare the sensitivity of previously described techniques to quantify haem in a 96-well based format. In panel A, haematin was measured with a spectrophotometer at 405 nm (n = 4 for each concentration). Panel B shows the measurements of haematin concentrations by haem-enhanced luminescence with the same reagent conditions as described by Schwarzer and colleagues (20) for a cuvette-based system. Background absorbance/luminescence from a blank sample was subtracted from all the measurements. The adjusted chemo-luminescence protocol (100-fold higher luminol and peroxide concentrations buffered around pH 10.4) was used to measure the same concentrations of haematin in panel C (n = 8 for each concentration in B and C). The horizontal dashed line denotes the accuracy limit of the assay. The time-dependence of the luminescence signal and the effect of 2% SDS are displayed in panel D and E, respectively. The arrow in panel D denotes the start of the kinetic reading after an initial delay of twelve seconds, and the vertical dashed line indicates the moment of luminescence detection during the experimental readings. In panel F, natural Hz was isolated from trophozoites (troph) and livers, its concentration was determined by absorption at 405 nm and the concentration-dependence of the haem-enhanced luminescence was measured. Inset of panel F is a picture demonstrating the birefringence of the isolated Hz. The amount of Hz/trophozoite was calculated and the corresponding number of trophozoites (# troph) is integrated in the figure as a second X-axis

Mentions: Because the densitometric analysis is not suitable to quantify Hz in tissue sections from all organs, a more sensitive technique was designed to determine Hz in organ extracts. Several methods for quantifying Hz in blood are described in literature, including a colorimetric [23] and a chemo-luminescence assay [24]. These two techniques were adapted to a 96-well plate format and the sensitivity was compared by measuring different concentrations of haematin produced when haemin or Hz is dissolved in an alkaline environment. A sigmoidal relationship was obtained between the haematin concentration and the blank-subtracted absorbance at 405 nm (Figure 2A) or the blank-subtracted luminescence with the reagent concentrations used as described for a cuvette-based system by Schwarzer et al.[24] (Figure 2B). In the 96-well plate format adapted for measuring absorbance or luminescence with a plate reader, both techniques detected the presence of haematin starting from a minimal concentration of 1 μM. To improve the sensitivity, the chemo-luminescent assay was adjusted by stabilizing the pH around 10.4 and by raising the concentrations of luminol and peroxide 100-fold. In this way, a detection limit around 100 nM was obtained (Figure 2C). Furthermore, the time-dependence of the luminescence signal was examined by measuring the emitted light of a single concentration during a kinetic measurement. With the described protocol for haem-enhanced luminescence in a 96-well plate format, emitted light was measured when the luminescence signal was almost maximal (Figure 2D). The sensitivity of this 96-well plate-based assay is significantly lower than the sensitivity of the cuvette-based method described by Schwarzer et al.[24]. However, the current 96-well based method has the advantage of a higher throughput and the sensitivity appeared sufficient to measure Hz in organ extracts. To extract Hz from organs, the method described by Sullivan et al.[15] was optimized. The most important modification was the addition of an overnight proteinase K digestion step that eliminated high background signals e.g. in lung samples. After several wash steps to remove any free haemin, the Hz crystals were converted to free haematin by dissolving in a strong alkaline environment, so that the haematin concentration could be measured. Since the extraction procedure involved washing steps in the presence of SDS, possible quenching of the emitted light by SDS was investigated and had no effect on the luminescence catalyzed by haematin-Fe3+ (Figure 2E).


Improved methods for haemozoin quantification in tissues yield organ-and parasite-specific information in malaria-infected mice.

Deroost K, Lays N, Noppen S, Martens E, Opdenakker G, Van den Steen PE - Malar. J. (2012)

Haem quantification by different techniques. Different concentrations of haematin (10 μM – 1.2 nM) were used to compare the sensitivity of previously described techniques to quantify haem in a 96-well based format. In panel A, haematin was measured with a spectrophotometer at 405 nm (n = 4 for each concentration). Panel B shows the measurements of haematin concentrations by haem-enhanced luminescence with the same reagent conditions as described by Schwarzer and colleagues (20) for a cuvette-based system. Background absorbance/luminescence from a blank sample was subtracted from all the measurements. The adjusted chemo-luminescence protocol (100-fold higher luminol and peroxide concentrations buffered around pH 10.4) was used to measure the same concentrations of haematin in panel C (n = 8 for each concentration in B and C). The horizontal dashed line denotes the accuracy limit of the assay. The time-dependence of the luminescence signal and the effect of 2% SDS are displayed in panel D and E, respectively. The arrow in panel D denotes the start of the kinetic reading after an initial delay of twelve seconds, and the vertical dashed line indicates the moment of luminescence detection during the experimental readings. In panel F, natural Hz was isolated from trophozoites (troph) and livers, its concentration was determined by absorption at 405 nm and the concentration-dependence of the haem-enhanced luminescence was measured. Inset of panel F is a picture demonstrating the birefringence of the isolated Hz. The amount of Hz/trophozoite was calculated and the corresponding number of trophozoites (# troph) is integrated in the figure as a second X-axis
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3473299&req=5

Figure 2: Haem quantification by different techniques. Different concentrations of haematin (10 μM – 1.2 nM) were used to compare the sensitivity of previously described techniques to quantify haem in a 96-well based format. In panel A, haematin was measured with a spectrophotometer at 405 nm (n = 4 for each concentration). Panel B shows the measurements of haematin concentrations by haem-enhanced luminescence with the same reagent conditions as described by Schwarzer and colleagues (20) for a cuvette-based system. Background absorbance/luminescence from a blank sample was subtracted from all the measurements. The adjusted chemo-luminescence protocol (100-fold higher luminol and peroxide concentrations buffered around pH 10.4) was used to measure the same concentrations of haematin in panel C (n = 8 for each concentration in B and C). The horizontal dashed line denotes the accuracy limit of the assay. The time-dependence of the luminescence signal and the effect of 2% SDS are displayed in panel D and E, respectively. The arrow in panel D denotes the start of the kinetic reading after an initial delay of twelve seconds, and the vertical dashed line indicates the moment of luminescence detection during the experimental readings. In panel F, natural Hz was isolated from trophozoites (troph) and livers, its concentration was determined by absorption at 405 nm and the concentration-dependence of the haem-enhanced luminescence was measured. Inset of panel F is a picture demonstrating the birefringence of the isolated Hz. The amount of Hz/trophozoite was calculated and the corresponding number of trophozoites (# troph) is integrated in the figure as a second X-axis
Mentions: Because the densitometric analysis is not suitable to quantify Hz in tissue sections from all organs, a more sensitive technique was designed to determine Hz in organ extracts. Several methods for quantifying Hz in blood are described in literature, including a colorimetric [23] and a chemo-luminescence assay [24]. These two techniques were adapted to a 96-well plate format and the sensitivity was compared by measuring different concentrations of haematin produced when haemin or Hz is dissolved in an alkaline environment. A sigmoidal relationship was obtained between the haematin concentration and the blank-subtracted absorbance at 405 nm (Figure 2A) or the blank-subtracted luminescence with the reagent concentrations used as described for a cuvette-based system by Schwarzer et al.[24] (Figure 2B). In the 96-well plate format adapted for measuring absorbance or luminescence with a plate reader, both techniques detected the presence of haematin starting from a minimal concentration of 1 μM. To improve the sensitivity, the chemo-luminescent assay was adjusted by stabilizing the pH around 10.4 and by raising the concentrations of luminol and peroxide 100-fold. In this way, a detection limit around 100 nM was obtained (Figure 2C). Furthermore, the time-dependence of the luminescence signal was examined by measuring the emitted light of a single concentration during a kinetic measurement. With the described protocol for haem-enhanced luminescence in a 96-well plate format, emitted light was measured when the luminescence signal was almost maximal (Figure 2D). The sensitivity of this 96-well plate-based assay is significantly lower than the sensitivity of the cuvette-based method described by Schwarzer et al.[24]. However, the current 96-well based method has the advantage of a higher throughput and the sensitivity appeared sufficient to measure Hz in organ extracts. To extract Hz from organs, the method described by Sullivan et al.[15] was optimized. The most important modification was the addition of an overnight proteinase K digestion step that eliminated high background signals e.g. in lung samples. After several wash steps to remove any free haemin, the Hz crystals were converted to free haematin by dissolving in a strong alkaline environment, so that the haematin concentration could be measured. Since the extraction procedure involved washing steps in the presence of SDS, possible quenching of the emitted light by SDS was investigated and had no effect on the luminescence catalyzed by haematin-Fe3+ (Figure 2E).

Bottom Line: Furthermore, total Hz contents correlated with peripheral parasitaemia and were significantly higher in mice with a lethal P. berghei ANKA or P. berghei NK65-infection than in mice with a self-resolving P. chabaudi AS-infection, despite similar peripheral parasitaemia levels.An organ-specific Hz deposition pattern was found and was independent of the parasite strain used.Highest Hz levels were identified in mice infected with lethal parasite strains suggesting that Hz accumulation in tissues is associated with malaria-related mortality.

View Article: PubMed Central - HTML - PubMed

Affiliation: Laboratory of Immunobiology, Rega Institute, University of Leuven, Leuven, Belgium.

ABSTRACT

Background: Despite intensive research, malaria remains a major health concern for non-immune residents and travelers in malaria-endemic regions. Efficient adjunctive therapies against life-threatening complications such as severe malarial anaemia, encephalopathy, placental malaria or respiratory problems are still lacking. Therefore, new insights into the pathogenesis of severe malaria are imperative. Haemozoin (Hz) or malaria pigment is produced during intra-erythrocytic parasite replication, released in the circulation after schizont rupture and accumulates inside multiple organs. Many in vitro and ex vivo immunomodulating effects are described for Hz but in vivo data are limited. This study aimed to improve methods for Hz quantification in tissues and to investigate the accumulation of Hz in different organs from mice infected with Plasmodium parasites with a varying degree of virulence.

Methods: An improved method for extraction of Hz from tissues was elaborated and coupled to an optimized, quantitative, microtiter plate-based luminescence assay with a high sensitivity. In addition, a technique for measuring Hz by semi-quantitative densitometry, applicable on transmitted light images, was developed. The methods were applied to measure Hz in various organs of C57BL/6 J mice infected with Plasmodium berghei ANKA, P. berghei NK65 or Plasmodium chabaudi AS. The used statistical methods were the Mann-Whitney U test and Pearsons correlation analysis.

Results: Most Hz was detected in livers and spleens, lower levels in lungs and kidneys, whereas sub-nanomolar amounts were observed in brains and hearts from infected mice, irrespectively of the parasite strain used. Furthermore, total Hz contents correlated with peripheral parasitaemia and were significantly higher in mice with a lethal P. berghei ANKA or P. berghei NK65-infection than in mice with a self-resolving P. chabaudi AS-infection, despite similar peripheral parasitaemia levels.

Conclusions: The developed techniques were useful to quantify Hz in different organs with a high reproducibility and sensitivity. An organ-specific Hz deposition pattern was found and was independent of the parasite strain used. Highest Hz levels were identified in mice infected with lethal parasite strains suggesting that Hz accumulation in tissues is associated with malaria-related mortality.

Show MeSH
Related in: MedlinePlus