Lactic acid production from lime-treated wheat straw by Bacillus coagulans: neutralization of acid by fed-batch addition of alkaline substrate

Conventional processes for lignocellulose-to-organic acid conversion requires pretreatment, enzymatic hydrolysis, and microbial fermentation. In this study, lime-treated wheat straw was hydrolyzed and fermented simultaneously to lactic acid by an enzyme preparation and Bacillus coagulans DSM 2314. Decrease in pH because of lactic acid formation was partially adjusted by automatic addition of the alkaline substrate. After 55 h of incubation, the polymeric glucan, xylan, and arabinan present in the lime-treated straw were hydrolyzed for 55%, 75%, and 80%, respectively. Lactic acid (40.7 g/l) indicated a fermentation efficiency of 81% and a chiral l(+)-lactic acid purity of 97.2%. In total, 711 g lactic acid was produced out of 2,706 g lime-treated straw, representing 43% of the overall theoretical maximum yield. Approximately half of the lactic acid produced was neutralized by fed-batch feeding of lime-treated straw, whereas the remaining half was neutralized during the batch phase with a Ca(OH)2 suspension. Of the lime added during the pretreatment of straw, 61% was used for the neutralization of lactic acid. This is the first demonstration of a process having a combined alkaline pretreatment of lignocellulosic biomass and pH control in fermentation resulting in a significant saving of lime consumption and avoiding the necessity to recycle lime.
Introduction
Lactic acid is used throughout the world in manufacturing of food, chemicals, and pharmaceutical products. Recently, there is a lot of interest in biodegradable poly-lactic acid, which is an alternative to petrochemically derived plastic (Drumright et al. 2000). Chiral pure lactic acid is produced commercially by microbial fermentation of the carbohydrates glucose, sucrose, lactose, and starch/maltose derived from feedstocks such as beet sugar, molasses, whey, and barley malt (Narayanan et al. 2004). The choice of feedstock depends on its price, availability, and on the respective costs of lactic acid recovery and purification (Datta et al. 1995; Vaidya et al. 2005).
As an alternative to these traditional feedstocks, lignocellulosic biomass is an inexpensive and widely available renewable carbon source that has no competing food value. Lignocellulose consists primarily of cellulose and hemicellulose; polymers build up of mainly hexose sugars and pentose sugars, which are embedded in a matrix of the phenolic polymer lignin. The main pathway to derive fermentable sugars from lignocellulose is through enzymatic hydrolysis by cellulolytic and hemicellulolytic enzymes. A mechanical and chemical pretreatment of the lignocellulose is required to reduce particle size, to modify and/or to remove the lignin, and with that to enhance the accessibility of the polysaccharides for enzymatic hydrolysis (Claassen et al. 1999). Various chemical pretreatments of biomass have been studied in research and development of lignocellulose-to-ethanol production technology (Mosier et al. 2005). One is the use of lime (calcium hydroxide) at relatively mild temperature conditions (Chang et al. 1998). Lime as a pretreatment agent has promising potential because it is inexpensive, safe, and its use hardly results in sugar degradation products such as furfural and hydroxymethyl furfural. Nevertheless, this alkaline pretreatment features a relatively high pH value (>10) of the treated biomass, and at these pH levels, the activity of common cellulolytic and xylanolytic enzymes, necessary for the depolymerization of (hemi)-cellulose, is negligible low. Therefore, lowering the pH is essential to achieve an efficient enzymatic hydrolysis of the polysaccharides. One approach to remove calcium hydroxide is by washing the lime-treated biomass before enzymatic hydrolysis (Chang et al. 1998); however, this leads to the use of high amounts of water. Another way to lower the pH of the pretreated material is by neutralizing calcium hydroxide with sulfuric acid. Yet, this results in the formation of the low value byproduct gypsum.
As an alternative improvement to these approaches, we propose to use the calcium hydroxide present in lime-treated biomass as neutralizing agent for organic acids produced in microbial fermentation processes. To examine this proposed concept, lime-treated wheat straw (LTWS) was added fed-batch-wise during a simultaneous saccharification and fermentation (SSF) process in a 20-l controlled stirred fermenter containing hydrolytic enzymes and Bacillus coagulans DSM 2314, a thermophilic bacterium capable to convert both hexoses and pentoses homofermentative to l(+)-lactic acid (Otto 2004; Patel et al. 2006). The objective of this research was to evaluate whether high alkaline-treated lignocellulosic biomass (without neutralization) can be used directly in a SSF process by (1) providing a carbon source for enzymatic hydrolysis and fermentation and (2) providing a source of alkali to control the pH in the fermentation process.
Materials and methods
Feedstock and pretreatment
Wheat straw was selected as a lignocellulose model feedstock and was purchased from a farm in the Northeast of The Netherlands. The wheat straw was air dried (89.5% [w/w] dry matter [DM]) and ground through a 2-mm screen. The lime pretreatment was performed by filling two 15-l mixers (Terlet, The Netherlands), both with 1,650 g ground wheat straw, 13 kg tap water, and 165 g calcium hydroxide. This wheat straw suspension was heated and kept at 85°C for 16 h under continuously stirring at 30 rpm. The LTWS suspension was subsequently cooled to 30°C, dehydrated by placing the LTWS in a cotton bag, and pressing the suspension using a manual piston press at pressure up to 9.7 kg/m2. After dehydration, an amount of 11.45 kg LTWS with an average DM content of 27.0% (w/w) and pH 11.8 was obtained and served as substrate for further experiments. The chemical composition of LTWS was determined as described by van den Oever et al. (2003).
Enzyme preparation
The enzyme preparation GC 220 (Genencor-Danisco, Rochester, USA) containing cellulase, cellobiase, and xylanase activity of 116, 215, and 677 U/ml, respectively (Kabel et al. 2006), was used for this study. The preparation had a specific gravity of 1.2 g/ml and contained 4.5 mg/ml glucose, 2.9 mg/ml mannose, and 0.8 mg/ml galactose.
Microorganism and preculture
The bacterium B. coagulans strain DSM 2314 was used as the lactic acid-producing micro-organism. Bacterial cells were maintained in a 10% (w/w) glycerol stock solution and stored at −80°C. Chemicals, unless indicated otherwise, were purchased from Merck (Darmstadt, Germany). Gelrite plates were prepared with a medium containing (per liter): glucose, 10 g; Gelrite, 20 g (Duchefa, Haarlem, The Netherlands); yeast extract, 10 g (Duchefa); (NH4)2HPO4, 2 g; (NH4)2SO4, 3.5 g; Bis–Tris, 10 g (USB, Ohio, USA); MgCl2 6H2O, 0.02 g; and CaCl2.2H2O, 0.1 g. Glucose and Gelrite were dissolved in stock solution A (four times concentrated). The pH of this stock solution was adjusted to 6.4 with 2 M hydrochloric acid and autoclaved for 15 min at 125°C. The remaining nutrients were dissolved in stock solution B (1.33 times concentrated), which was also adjusted to pH 6.4 with 2 M hydrochloric acid but was filter sterilized (cellulose acetate filter with pore size of 0.2 μm, Minisart, Sartorius). After sterilization, the medium was prepared by combining stock solutions A and B and Gelrite plates were poured. The bacteria were cultivated on Gelrite plates for 48 h at 50°C.
An isolated colony was used to inoculate a 100-ml broth with similar composition and preparation as described above but without the addition of Gelrite. The culture was incubated statically for 24 h at 50°C and functioned as the inoculum for a 1,400-ml broth. This culture was incubated also statically for 12 h at 50°C and served as a 10% (v/v) preculture for the SSF experiments.
Simultaneous saccharification and fermentation
The SSF of LTWS was carried out in a 20-l fermenter (Applikon, Schiedam, The Netherlands) with pH and temperature control (biocontroller ADI 1020). At the start of SSF, the fermenter was filled with 6.0 kg tap water and 1,400 g dehydrated LTWS (DM content of 27.0% [w/w]). The following nutrients were then added to the LTWS suspension: yeast extract, 150 g (Duchefa); (NH4)2HPO4, 30 g; (NH4)2SO4, 52.5 g; MgCl2 6H2O, 0.3 g; and CaCl2 2H2O, 1.5 g. The LTWS suspension was then heated to 50°C, and the pH was adjusted to 6.0 with 101 g 3 M sulfuric acid (~30 g H2SO4).
The SSF process of LTWS to lactic acid consisted of three phases: (1) the prehydrolysis phase of preloaded LTWS, (2) the fed-batch phase with automatic feeding of LTWS from a screw feeder, and (3) the batch phase with pH control by a calcium hydroxide suspension and no LTWS feeding. A schematic representation of the experimental setup is shown in Fig. 1. The prehydrolysis was initiated by the addition of 40 ml enzyme preparation (88 mg enzyme/g DM substrate) to the LTWS suspension and was incubated for 2 h at 50°C under continuously stirring at 250 rpm. The fed-batch phase was initiated by the addition of 1,500-ml preculture of B. coagulans DSM 2314 to the fermenter. The lactic acid produced by the bacteria was neutralized by the automatic addition of 8,623 g dehydrated LTWS (DM of 27.0%) to the fermenter through a feeder (K-Tron Soder Feeders, Canada) and was regulated by the pH of the medium, which was set at 6.0. Throughout the fed-batch phase, an amount of 280 ml of enzyme preparation (total enzyme loading of 98 mg/g DM substrate) was added proportional to the LTWS addition rate into the fermenter. During the batch phase, the pH was controlled at 6.0 by the addition of 20.0% (w/v) calcium hydroxide suspension. Samples were withdrawn for DM, substrate, and (by)product analysis.
Fig. 1Schematic representation of the simultaneous saccharification and fermentation of lime-treated wheat straw to lactic acid
Analytical methods
For the analysis of monomeric sugars, the fermentation broth samples were centrifuged (3 min at 17,400 × g), and the pH of the supernatant was adjusted to 5.0 with barium carbonate using a pH indicator (Bromophenolblue) followed by filtration of the liquid. The analysis was performed by high-performance anion-exchange chromatography using a Carbopack PA1 column (column temperature of 30°C) and a pulsed amperometric detector (ED50; Dionex, Sunnyvale, CA, USA). Before injection, the system was equilibrated with 25.5 mM NaOH for 10 min at a flow rate of 1.0 ml/min. For the separation of monomeric sugars, at injection, the mobile phase was shifted to deionized water for 30 min. Postcolumn addition of sodium hydroxide was used for detection of the neutral monomeric sugars.
The determination of soluble oligomeric sugars was performed by centrifugation for 5 min at 3,000 rpm (Centaur 2, Beun de Ronde, The Netherlands) of preweighed samples and freeze drying the supernatant overnight. Pellets were weighed and hydrolyzed with sulfuric acid, and neutral monomeric sugars were determined according to the method as described by van den Oever et al. (2003). For the calculations, an average molecular weight of oligomers from glucan and xylan of 166 and 132 g/mol, respectively, were applied, resulting in a hydrolysis factor of 1.08 and 1.14, respectively.
For the analysis of insoluble polymeric sugars, samples of 25 g were centrifuged for 5 min at 3,000 rpm (Centaur 2, Beun de Ronde); the supernatant was removed, and the pellet was washed by resuspension in 25 ml fresh demineralized water followed by a centrifugation step of 5 min at 3,000 rpm (Centaur 2, Beun de Ronde). The sequence of resuspension and centrifugation was repeated three times. After the last removal of the supernatant, the pellets were freeze dried overnight. The pellets were weighed (values used for DM calculation), polymeric material was hydrolyzed with sulfuric acid, and neutral sugars were analyzed according to the method as described by van den Oever et al. (2003). For the calculations, a molecular weight of glucan and xylan of 162 and 132 g/mol, respectively, were applied, resulting in a hydrolysis factor of polymer to monomer of 1.11 and 1.14, respectively.
The analysis of organic acids was performed by high-pressure liquid chromatography according to the procedure described by Maas et al. (2006).
The chiral purity (%) of lactic acid was determined by derivatization of all lactates using methanol, after which both enantiomers of methyl lactate were separated on a chiral gas chromatography column and detected using a flame ionization detector. The chiral purity was expressed as the area of the main enantiomer divided by the sum of areas of both enantiomers.
Calculations
The theoretical maximum lactic acid (LAtheor. max. [g]) production was calculated according the following equation (Eq. 1):
where DMsubstrate = the total dry matter of substrate LTWS (g), Fpolysacch. = fraction polysaccharides per substrate (g/g), HFmonsacch./polysacch. = hydrolysis factor of polysaccharides, incorporation of water results in 1.11 g hexose from 1.00 g glucan and 1.14 g pentose from xylan and arabinan (g/g), and FF = fermentation factor of 1.00 g lactic acid per gram of monomeric sugar.
The efficiency of the enzymatic hydrolysis (%, w/w) was based on the amount of hydrolyzed polysaccharides (g; calculated by the difference between initial amounts and analyzed insoluble amounts) divided by the amount of polysaccharides (g) initially present in the substrate. The fermentation efficiency (%, w/w) is expressed as the amount of lactic acid produced (g) divided by the amount of monomeric sugars consumed (g) by the bacteria. The overall efficiency of the SSF (%, w/w) was calculated by the amount of lactic acid produced (g) divided by the theoretical maximum amount of lactic acid (g) determined as described in Eq. 1.
Results
Simultaneous saccharification and fermentation of LTWS to lactic acid
The polysaccharide composition of the LTWS consisted mainly of glucan, xylan, and arabinan of 33.0%, 19.0%, and 2.0% (w/w), respectively, whereas the remaining mass constituted of lignin, ash, extractives, and uronic acids. Some of the soluble components in wheat straw were partially removed by the solid/liquid separation (dehydration) of the LTWS. The focus of this study was on the conversion of glucan, xylan, and arabinan, which are the predominant polysaccharides present in LTWS and accounted for 99.8% (w/w) of the total polymeric sugars. Previous work showed that the cellulase preparation GC 220, used for the saccharification of polysaccharides, functioned optimally at 50°C and pH 5.0 (Maas et al., submitted for publication), whereas growth conditions for B. coagulans DSM 2314 were 54°C and pH 6.5 (Otto 2004). In this study, both the enzymatic hydrolysis and the fermentation occurred simultaneously in the same reactor at compromising conditions, which were set at 50°C and pH 6.0.
The SSF of LTWS to lactic acid was studied in a 20-l controlled stirred fermenter. Previous results showed that when this process was performed without a prehydrolysis of an initial amount of LTWS, the concentration of monomeric sugars was low and resulted, therefore, in relatively low lactic acid productivity. As a consequence, the fed-batch addition rate of the alkaline substrate to neutralize the produced lactic acid was low (results not shown). To start the fermentation with a substantial initial amount of fermentable sugars (>2 g/l), a prehydrolysis of 378 g LTWS and enzyme preparation (88 mg per g DM LTWS) in approximately 6 l volume at pH 6.0 for 2 h was introduced. This resulted in glucose, xylose, and arabinose concentrations of 2.0, 0.4, and 0.3 g/l, respectively (Fig. 3a).
The second phase (II) was initiated by introducing a 1,500-ml preculture of B. coagulans DSM 2314. A minor amount of lactic acid produced in the preculture caused a slight pH decrease and was automatically neutralized by the addition of LTWS (Fig. 2a,b). After a lag phase of 4 h, the dissolved oxygen concentration decreased rapidly within 1 h from 100% to oxygen-limiting conditions of below 1% (results not shown), and lactic acid production started. At that moment, concentrations of glucose, xylose, and arabinose of 3.3, 0.7, and 0.3 g/l, respectively, were present (Fig. 3a). These sugars were consumed simultaneously where glucose was utilized faster than xylose and arabinose. Simultaneous with the consumption of these monomeric sugars, lactic acid was produced, which was neutralized by the automatic addition of alkaline LTWS. By the addition of alkaline substrate throughout the fed-batch phase, the pH was maintained accurately at 6.0 ± 0.1 (Fig. 2a,b). At the end of phase II, a total amount of 10,023 g dehydrated LTWS (~2,706 g DM LTWS) and 320 ml of enzyme preparation was added to the fermenter. A lactic acid concentration of 20.5 g/l supernatant was detected (Fig. 3b), corresponding to a total of 342 g lactic acid. The chiral L(+) purity of lactic acid was determined at 99.4%, which is similar to that obtained with xylose as the sole carbon source (Otto 2004).
Fig. 2Control of pH (a) during simultaneous saccharification and fermentation of lime-treated wheat straw by commercial enzyme preparation GC 220 and B. coagulans DSM 2314 (b). The areas between the dotted lines represent the prehydrolysis phase (I), the fed-batch phase (II) with pH control by addition of alkaline LTWS and enzymes, and the batch phase (III) with pH control by addition of Ca(OH)2 suspension. Extra enzyme preparation GC220 was added at the times indicated by the arrowsFig. 3Profiles of glucose (empty square), xylose (empty diamonds), arabinose (empty triangles) (a) and lactic acid (filled diamonds) (b) in simultaneous saccharification and fermentation of lime-treated wheat straw by commercial enzyme preparation GC 220 and B. coagulans DSM 2314. The areas between the dotted lines represent the prehydrolysis phase (I), the fed-batch phase (II), and the batch phase (III). Extra enzyme preparation GC220 was added at the times indicated by the arrows
At the end of phase I, a low acetic acid concentration was detected in the medium, which increased to 1.5 g/l throughout phase II but remained constant during phase III (results not shown). This indicates that acetic acid was most likely not a fermentation product formed by B. coagulans. Acetic acid can be released upon solubilization and hydrolysis of hemicellulose during chemical pretreatment (Palmqvist et al. 1999). By the dehydration procedure of the LTWS, part of the acetic acid was easily separated from the substrate by removing the press water. Apparently, a remaining amount of acetic acid was fed together with the substrate to the fermenter. Furthermore, minor traces of other organic acids such as succinic acid and formic acid (<0.5 g/l) were detected in the fermentation broth.
Phase III was initiated by changing the pH control from the addition of alkaline LTWS to a 20% (w/v) calcium hydroxide suspension. To maintain the pH at 6.0, the addition of calcium hydroxide suspension occurred relatively fast but shifted, however, after a few hours to a lower addition rate indicating a decline of the volumetric lactic acid productivity (Figs. 2b, 3b). To exclude limitation (e.g., by inactivation) of enzymes, an extra dosage of enzyme preparation (80 ml) was added to the fermenter after 23.5 h of incubation. This resulted immediately in a slight acceleration of the calcium hydroxide addition rate indicating an increased lactic acid productivity and limitation of enzymatic activity (Fig. 3b). Nevertheless, after 29.7 h of incubation, a decline of the calcium hydroxide addition rate was observed again. Therefore, a second extra dosage of the enzyme preparation (240 ml) was added and resulted this time in a slight accumulation of glucose and xylose of 1.5 and 1.0 g/l (Fig. 3a), respectively, indicating that microbial conversion instead of enzymatic hydrolysis was rate limiting. After 32 h of incubation, a lactic acid concentration of 37.1 g/l was obtained, with a chiral l(+)-lactic acid purity of 99.4%. Continuation of the SSF process to a total incubation period of 55 h resulted in a slightly increased lactic acid concentration of 40.7 g/l supernatant (~37.8 g lactic acid/kg fermentation broth) with an overall volumetric lactic acid productivity of 0.74 g l−1 h−1. At this stage, a chiral l(+)-lactic acid purity of 97.2% was analyzed. This slight decline in lactic acid purity is possibly a result of infection with other undesired lactic acid-producing microorganisms. Because the substrate used was not sterile and also the chemical pretreatment and fermentation occurred in an open system under nonsterile conditions, microbial contamination throughout the SSF process is possible.
Conversion efficiency
The efficiency of the enzymatic hydrolysis of the polymeric material present in LTWS is shown in Fig. 4. The insoluble polymeric fraction was determined at various time points throughout the SSF experiment. At the end of the prehydrolysis (2 h) of 378 g LTWS, 36% of the insoluble glucan (Fig. 4a), 55% of xylan (Fig. 4b), and 62% of arabinan (Fig. 4c) were converted to soluble saccharides including monomeric sugars and oligomeric sugars. After the fed-batch phase (13 h), 2,706 g LTWS was added and resulted in a conversion of 42% of glucan, 57% of xylan, and 63% of arabinan to products including soluble saccharides and lactic acid. Between 13 and 32 h of incubation, further hydrolysis of the polymeric sugars was observed. However, during the last 23 h of the SSF, minor hydrolysis of the polysaccharides occurred, and this corresponded with the decline in lactic acid productivity during this phase. After 55 h, 398 g of glucan, 130 g of xylan, and 11 g of arabinan was still present as insoluble polymeric material. With these values, the hydrolysis efficiency of the initial glucan, xylan, and arabinan present in LTWS were calculated as 55%, 75%, and 80%, respectively.
Fig. 4Insoluble fraction (empty bars) and hydrolyzed soluble fraction (filled bars) (g; calculated by the difference between initial amounts and analyzed insoluble amounts) of the polysaccharide glucan (a), xylan (b), and arabinan (c) at various time points during the simultaneous saccharification and fermentation of lime-treated wheat straw. The figure represents also the percentage of polysaccharide hydrolyzed into soluble products (filled triangles). The error bars denote the deviation of duplicate analysis
The monomeric sugars, derived from the LTWS, were partly converted to lactic acid (711 g) by B. coagulans and accounted for 81% (w/w) of the theoretical maximum, indicating the formation of other products such as microbial biomass and carbon dioxide. An overall conversion yield of 43% (w/w) of the theoretical maximum was calculated according to Eq. 1. The fate of polysaccharides initially present in LTWS after 55 h of incubation is shown in Table 1. A part of the polysaccharides present in LTWS remained as insoluble polysaccharides (37% w/w), whereas a minor part was converted into soluble oligomeric (5% w/w) and monomeric (3% w/w) sugars. Another part of the initial polysaccharides present in the LTWS was not recovered in the form of saccharides or lactic acid and was therefore ascribed as ‘unaccounted.’
Table 1Fate of polysaccharidesa initially present in lime-treated wheat straw after 55 h of simultaneous saccharification and fermentationFractionPercentage (% w/w)Polysaccharides (insoluble)b37Oligosaccharides (soluble)5Monosaccharides (soluble)3Lactic acid (soluble)43Unaccounted (insoluble/soluble)c13Presented values are averages based on duplicate analytical measurements.aTotal of glucan, xylan, and arabinanbPart of the initial polysaccharides remained present as insoluble polysaccharides.cPart of the initial polysaccharides was not recovered and therefore denoted as ‘unaccounted.’
Neutralization of acid by alkaline substrate
The lactic acid produced (342 g) during the fed-batch phase (II) was neutralized with alkaline-pretreated wheat straw. During this phase, an amount of 2,328 g LTWS was added to the fermenter. Together with this substrate, an amount of 230 g calcium hydroxide was added to the fermenter and accounted for a ratio of 0.67 g calcium hydroxide per gram of lactic acid. The lactic acid (369 g) produced during the batch phase (III) was neutralized with 163 g calcium hydroxide resulting in a ratio of 0.44 g lactic acid per gram calcium hydroxide.
Discussion
Lignocellulosic feedstocks are considered as potential attractive substrates for the production of bulk chemicals. Pretreatment of biomass is required to break open the lignocellulosic matrix, and an enzymatic hydrolysis is necessary for the hydrolysis of polymeric carbohydrates. The lime pretreatment has proven to enhance enzymatic digestibility of the polysaccharides present in lignocelluloses (Chang et al. 1998; Kaar and Holtzapple 2000) and results, in comparison to other pretreatment routes, in minor inhibitor formation. However, before the enzymatic hydrolysis, it is essential to adjust the pH to a level optimal for enzymatic activity. In this study, the reduction in pH by washing or neutralization was omitted by using the alkaline character of LTWS to neutralize lactic acid produced by microbial fermentation during a SSF process.
The results showed that the largest part of the polysaccharides in LTWS was converted enzymatically and the resulting sugars were fermented simultaneously to mainly lactic acid by B. coagulans DSM 2314. Between 10 and 30 h of incubation, the bacteria utilized the monomeric sugars, as soon as they appeared in the medium, resulting in relatively low monomeric sugar concentrations (<2 g/l). This indicates that throughout this period, the enzymatic hydrolysis was the rate-controlling step. The highest lactic acid productivity was observed during the fed-batch phase and the initial hours of the batch phase and declined rapidly after approximately 20 h of incubation, as shown in Fig. 3b. An extra addition of enzyme preparation showed a slight improvement of the volumetric lactic acid productivity but shifted within a few hours again to a relatively low production rate. A second extra enzyme addition did not affect the lactic acid productivity significantly (Fig. 3b). This addition of new enzymes resulted in a modest liberation of hemicellulose sugars (xylose, arabinose), but no further hydrolysis of glucan occurred. This shows that the remaining glucan was too recalcitrant or not accessible for further hydrolysis, resulting in decreasing lactic acid productivity. Another possible explanation of the decreased lactic acid productivity is the inhibition of enzymes and/or bacteria by the increasing lactic acid concentration.
A lactic acid concentration of 40.7 g/l supernatant (~37.8 g lactic acid/kg fermentation broth) with a relatively high chiral purity was determined after 55 h of incubation, corresponding to an overall lactic acid yield of 43% of the theoretical maximum. Moreover, the efficiencies of the enzymatic saccharification and the fermentation were both determined. These calculations showed that, based on residue analysis, at the end of the SSF process (55 h), 55% of the glucan, 75% of the xylan, and 80% of the arabinan present in LTWS was enzymatically hydrolyzed, which agree well with previously obtained results from experiments aiming to convert LTWS to ethanol. To improve the yield, it is necessary to decrease the recalcitrance or improve the accessibility of polymeric sugars in the LTWS by optimization of the pretreatment procedure. The concentrations of soluble monosaccharides and oligosaccharides in the medium were relatively low, which can be expected in a SSF process. A fermentation yield of 81% was determined (related to the amount of monosaccharides released from the LTWS) and is slightly better than the results obtained by Otto (2004) who reported the production of 35 g/l lactic acid from 50 g/l xylose as the sole carbon source. Because no other soluble fermentation products were detected, the remaining 19% of the LTWS-derived monomeric sugars were most presumably converted to bacterial biomass and some carbon dioxide during the aerobic part of the fermentation. Several process parameters can be listed for enhancement of the overall lactic acid yield and productivity such as improving the accessibility of polysaccharides by a more severe lime pretreatment, enzyme dosage, type of enzymes, B. coagulans strain, size and growth phase of inoculum, pH gradient in SSF, and in situ product removal of lactic acid. These issues will be subject to further studies.
During the fed-batch phase (II), it was possible to counterbalance the pH decrease caused by lactic acid production by the addition of the alkaline feedstock. This suggests that it is possible to combine lime treatment with the production of other organic acids from lignocellulosic biomass. Throughout this phase, the ratio of calcium hydroxide in LTWS added per produced lactic acid was determined at 0.67 g/g. The theoretical stoichiometric neutralization of 1.00 g lactic acid requires 0.41 g calcium hydroxide. Therefore, only 61% of the calcium hydroxide initially added to the wheat straw was used for lactic acid neutralization. On the other hand, throughout the batch phase (III), an alkaline/acid ratio of 0.44 g/g was calculated corresponding to 93% of the added calcium hydroxide suspension used for lactic acid neutralization. The low efficiency of the calcium hydroxide added with the LTWS for lactic acid neutralization during phase II has three possible explanations. First, part of the calcium hydroxide could have been used during the chemical pretreatment of the wheat straw such as the neutralization of acetic acid or other organic acids and/or irreversible binding to the lignin. Second, the calcium hydroxide might be released slowly from the insoluble wheat straw fibers and could therefore partly have been used for lactic acid neutralization in the fed-batch phase. Finally, besides lactic acid production, other acidification reactions could have contributed to the decrease in pH and therefore the demand of alkaline substrate, for instance, the decrease in pH caused by the consumption of ammonium as the nitrogen source by microorganisms (Guebel et al. 1992).
The results in this paper show that it is possible to use lignocellulosic materials for the production of lactic acid. Lignocellulosic biomass is a relatively inexpensive substrate, and this affects feedstock costs for lactic acid production positively. Nevertheless, in comparison to the traditional relatively ‘clean’ feedstocks with a well-defined composition, using heterogenic lignocellulosic substrates will require a more intensified downstream processing (DSP) to recover and purify the lactic acid from the complex fermentation broth. The costs of feedstock materials and operational costs of the DSP contribute considerably to the overall production costs of lactic acid (Åkerberg and Zacchi 2000). Whether the cost decrease in using lignocellulosic feedstocks outweighs the potential increasing costs of DSP was not analyzed at the moment.
In summary, LTWS was converted into l(+)-lactic acid by B. coagulans throughout a SSF process at a 20-l bench scale. The pentose and hexose sugars derived from the polymeric material were utilized simultaneously by B. coagulans resulting in a final lactic acid concentration of 40.7 g/l supernatant, which accounted for 43% (w/w) of the theoretical yield. To our knowledge, this is the first paper demonstrating a process having a combined alkaline pretreatment of lignocellulosic biomass and pH control in organic acid fermentation resulting in a significant saving of lime consumption and avoiding the necessity to recycle lime.


Three-year survival of single- and two-surface ART restorations in a high-caries child population

The aim of this study was to evaluate the survival of single- and two-surface atraumatic restorative treatment (ART) restorations in the primary and permanent dentitions of children from a high-caries population, in a field setting. The study was conducted in the rainforest of Suriname, South America. ART restorations, made by four Dutch dentists, were evaluated after 6 months, 1, 2, and 3 years. Four hundred seventy-five ART restorations were placed in the primary dentition and 54 in first permanent molars of 194 children (mean age 6.09 ± 0.48 years). Three-year cumulative survivals of single- and two-surface ART restorations in the primary dentition were 43.4 and 12.2%, respectively. Main failure characteristics were gross marginal defects and total or partial losses. Three-year cumulative survival for single-surface ART restorations in the permanent dentition was 29.6%. Main failure characteristics were secondary caries and gross marginal defects. An operator effect was found only for two-surface restorations. The results show extremely low survival rates for single- and two-surface ART restorations in the primary and permanent dentitions. The variable success for ART may initiate further discussion about alternative treatment strategies, especially in those situations where choices have to be made with respect to a well-balanced, cost-effective package of basic oral health care.
Introduction
The concept of minimal invasive dentistry has evolved as a consequence of an increased understanding of caries and the development of adhesive restorative materials [27]. Within this concept, prevention and hard tissue preservation are the primary goals, and dentists are encouraged to prefer a more conservative and biological approach rather than a surgical approach, although the latter is sometimes unavoidable. The atraumatic restorative treatment (ART) technique is part of a minimal invasive approach and, as such, a technique that meets the specific goals mentioned above. In brief, with ART, soft demineralized carious tooth tissue is removed using hand instruments only, followed by restoration of the tooth with an adhesive restorative material, often glass ionomer cement [4, 7]. Because neither electricity nor running water is required for this treatment approach, ART can be applied in almost any setting. Although initially developed to provide restorative dental treatment in outreach or rural areas, ART or modified ART techniques are increasingly introduced into dental clinics in industrialized countries [1, 11, 14].
Since its introduction in the mid-1980s, ART has been evaluated in several community field trials. These studies served mainly to obtain information on technical aspects of the process, handling characteristics of the restorative material, and on the survival of the restorations. They led to improvement of the technique [20] and to the development of new, more appropriate glass ionomer restoration materials, especially for ART purposes.
Studies focussing on the survival of ART restorations have shown that the ART approach is very successful in restoring single-surface dentine lesions in the permanent dentition: 3-year survival rates of 71–92% have been reported [5, 6, 9, 10, 12, 20]. Regarding the survival rates of ART restorations in the primary dentition, only a few field studies were performed. They showed acceptable survival rates (65–96.7%) for single-surface ART restorations, but generally low success rates (31–76.1%) for multi-surface ART restorations, even with the newer glass ionomer materials [2, 3, 11–13, 19, 23, 24, 28]. Although its performance under multisurface conditions is disappointing, ART is considered a valuable approach towards the treatment of dental caries. The use of ART has resulted in the retention of many teeth that would otherwise have been extracted in a later stage. Nevertheless, there still remain some controversies towards the technique, presumably based on the inconsistency in survival results. Moreover, a recent study, investigating the influence of dental treatment on the oral health of a Surinamese child population, concluded that performing ART restorations only, did not contribute significantly to an improvement of the oral health, suggesting that ART alone is not a sufficient solution in the battle against dental decay (van Gemert-Schriks et al., submitted, 2007).
Frencken et al. [8] described comprehensively that ART should be part of a basic package of oral care in which prevention and urgent care are also represented. However, within this package, these three components should be geared to one another as much as possible and the individual effects of all three components must be sufficient and beneficial under different circumstances. When the success of either component, particularly ART, cannot be guaranteed, its contribution in the package should be reduced. Thus, the evaluation of ART in different countries or communities, among different kinds of caries-risk populations and under diverging conditions remains useful. Therefore, the aim of this study is to evaluate the survival of both single- and two-surface ART restorations in the primary and permanent dentitions of children from a high-caries population in a field setting on a longitudinal base.
Materials and methods
This cohort study was conducted in the rainforest of Suriname, South America. It was part of a large-scale project investigating the influence of dental treatment on the oral health of children (van Gemert-Schriks et al., submitted, 2007). Within the scope of that particular project, 380 6-year-old children were divided randomly among four different treatment groups. Material presented in the current article concerns only those children who received restorative treatment, according to the ART method, either in their primary or permanent dentitions.
The restorative treatments were performed in accordance with the ART guidelines [4, 7] and took place in empty classrooms where four children were treated at the same time. Ketac-Molar (3M-ESPE®) was used as the restorative material of choice. The treatments were carried out by four Dutch dentists who were trained in ART during a 1-week ART course and by using ART in children from their own practices, for a period of 3 months, before the start of the treatment phase of the study. They were assisted by six Surinamese health care assistants from the Medical Mission who completed an ART course supplemented with some basic dental knowledge. The dentists were asked to note any contamination with blood and/or saliva during the restoration of the cavity. Furthermore, the presence or absence of adjacent teeth was noted. During the treatment, one of the authors (MGS), who was not involved in the treatment phase, observed and classified the overall behavior of the child, based on a modified Venham scale [23, 29]. Before the study, this observer was trained in using the Venham behavior scale by scoring 42 videotapes of children in a dental situation. These observations were compared to the consensus score of two calibrated observers. This comparison resulted in a Cohen’s Kappa of 0.87, implying an excellent agreement.
Restorations were not assessed at the time of placement (T0). The children were revisited for evaluation of the ART restorations 6 months (T1), 1 year (T2), 2 years (T3), and 3 years (T4) after the initial treatment. The same author and dentist mentioned above (MGS), evaluated the restorations according to the ART criteria (Table 1) using a CPITN probe, a mouth mirror and a headlamp. Before the study, this person was calibrated against a “gold standard” (Kappa 0.94). This gold standard was achieved by the consensus of two experienced dentists during the assessment of 24 extracted molars with ART restorations. Restorations scored code 00 or 10 were considered successful, codes 11–40 were classified as failures, and codes 50–90 were assigned in case the tooth was unavailable for evaluation. If a tooth or restoration showed multiple defects, a marginal defect dominated an over- or underfilled restoration (10, 11 > 12, 13), secondary caries dominated a marginal defect (20, 21 > 10, 11), absence of a restoration dominated secondary caries (30 > 20, 21), and an overfilled cavity dominated an underfilled cavity (13 > 12).
Table 1Evaluation criteria for the ART restorationsCodeEvaluation characteristics00Restoration present, correct10Restoration present, slight marginal defect/wear of surface (<0.5 mm). No repair needed.11Restoration present, gross marginal defect/wear of surface (>0.5 mm). Repair needed.12Restoration present, underfilled (>0.5 mm). Repair needed.13Restoration present, overfilled (>0.5 mm). Repair needed.20Secondary caries, discoloration in depth, surface hard and intact, caries within dentin. Repair needed.21Secondary caries, surface defect, caries within dentin. Repair needed.30Restoration not present, bulk fracture, moving or partial lost. Repair needed.40Inflammation of the pulp; signs of dentogenic infection (abscesses, fistulae, pain complaints). Restoration might still be in situ. Extraction needed.50Tooth not present because of extraction60Tooth not present because of shedding70Tooth not present because of extraction or shedding90Patient not present
Statistical analysis
Statistical analyses were performed using SPSS for Windows, version 12.0.1 (SPSS, Chicago, USA). All significant differences were detected at a 95% confidence level.
Kaplan–Meier survival analyses were performed on the censored data of both single- and two-surface restorations. The significance of differences between survival curves was determined with log-rank tests. Possible confounding variables were taken into account using a Cox regression analysis.
Results
As stated in the “Materials and methods” section, the children in this study were derived from a larger study population of children participating in another project. The overall caries prevalence, expressed in terms of decayed, missing and filled surfaces (dmfs) among that group of children, was 11.51 (SD 10.5; range 0–53) in the primary dentition and 0.20 (SD 0.62; range 0–5) in the permanent dentition. According to the standards of the World Health Organisation [17], this denotes a high-caries child population based on the caries prevalence in the primary dentition. Within the larger group, 194 children (mean age 6.09 ± 0.48 years) received ART restorations in either their primary or permanent teeth, or both. Only these children were included in the current study. Their baseline caries prevalence was 12.75 (SD 9.88; range 0–53) in the primary dentition and 0.23 (SD 0.67; range 0–5) in the permanent dentition.
At baseline (T0), 475 ART restorations were placed in the primary dentition (mainly first and second molars) and 54 in the first permanent molars (predominantly mandibular). Table 2 presents data for the ART restorations, performed at baseline. A Mann–Whitney U test showed that children who received two-surface restorations scored higher on the Venham behavior scale (p = 0.005) than children that received single-surface restorations, in the primary dentition. Furthermore, dentists reported more contamination (chi-square = 25.02, df = 1, p < 0.001) when placing two-surface restorations than single-surface restorations.
Table 2Baseline data for the ART restorations Primary dentitionPermanent dentitionNumber of filled surfaces121Number of restorations13334254Number of children (N)6114734Mean number of restorations per child (SD; range)3.50 (1.61; 1–7)3.64 (1.73; 1–8)2.07 (0.97; 1–4)Dentist143 (32.3%)74 (21.6%)12 (22.2%)241 (30.8%)84 (24.6%)16 (29.6%)334 (25.6%)89 (26.0%)7 (13.0%)415 (11.3%)a95 (27.8%)19 (35.2%)Adjacent tooth presentYes117 (88.0%)303 (88.6%)45 (83.3%)No16 (12.0%)39 (11.4%)9 (16.7%)Contamination blood/salivaYes13 (9.8%)110 (32.2%)a5 (9.3%)No120 (90.2%)232 (67.8%)49 (90.7%)Venham behavior score050 (37.6%)78 (22.8%)8 (14.8%)144 (33.1%)137 (40.1%)27 (50.0%)226 (19.5%)82 (24.0%)13 (24.1%)313 (9.8%)33 (9.6%)6 (11.1%)4–12 (3.5%)a–5–––SD Standard deviationaStatistical significant difference at p = 0.005.
The lost-to-follow-up percentage of the restorations originally placed was 4.63%. After 3 years, the cumulative survival of the single-surface ART restorations in the primary dentition was 43.4% (standard error (SE) 10.9%). For the two-surface restorations, a cumulative survival of 12.2% (SE 2.99%) was observed. The survival curves, with censored data, are presented in Fig. 1a and b. The cumulative survival of the single-surface ART restorations in the permanent dentition was 29.6% (SE 8.2%) after 3 years (Fig. 2).
Fig. 1a Survival curve single surface ART restorations, primary dentition. b Survival curve multisurface ART restorations, primary dentitionFig. 2Survival curve single-surface ART restorations, permanent dentition
Table 3 represents the failure characteristics for the restorations in both primary and permanent dentitions at 3 years. The main failure characteristics of both single- and two-surface ART restorations in the primary dentition were gross marginal defects (score 11) and total or partial losses (score 30). For restorations in the permanent dentition, the main failure characteristics were secondary caries (score 21) and gross marginal defects (score 11).
Table 3Failure characteristics for the ART restorations at 3 years Primary dentitionPermanent dentition1 surface2 surface1 surfaceRestorations baseline (N)13334254Failures (N)4225132Failure scoreGross marginal defect (11)21 (15.8%)86 (25.1%)13 (24.1%)Restoration present, underfilled (12)1 (0.8%)9 (2.6%)–Restoration present, overfilled (13)2 (1.5%)11 (3.2%)2 (3.7%)Sec. caries, discoloration (20)–––Sec. caries, surface defect (21)5 (3.8%)1 (0.3%)14 (25.9%)Total or partial loss (30)13 (9.8%)120 (35.1%)3 (5.6%)Pulpal inflammation (40)–24 (7.0%)–Restoration missing, extracted (50)–––Scores 60–90 were not included (censored data)
A log-rank test indicated that there were no statistically significant differences in survival times between the four dentists regarding single-surface restorations in both primary and permanent teeth. However, regarding the two-surface restorations in the primary dentition, statistically significant differences between the four dentists appeared (log-rank statistic 11.7, df = 3, p = 0.009). The separate survival curves are presented in Fig. 3.
Fig. 3Survival curves per dentist, multi-surface ART restorations primary dentition
To detect any confounding variables on the survival of the ART restorations, a Cox regression analysis was performed. No significant relation could be found, indicating that neither the presence or absence of an adjacent tooth, nor contamination with blood and/or saliva, nor the behavior of the child during the restorative phase of the treatment had an influence on the 3-year survival of the restorations in the primary dentition. No effect also could be found regarding the number of restorations per child.
Neither of these variables had an effect on the survival rates in the permanent dentition, except for the presence of adjacent teeth. Restorations in teeth where no adjacent tooth was present were found to be more likely to fail (hazard ratio = 6.53, 95% CI 2.66–16.02, p < 0.001).
Discussion
In contrast with other studies, the results of this study show extremely low survival rates for both single- and two-surface ART restorations in the primary and permanent dentitions. An operator effect was observed for two-surface restorations only. Neither the behavior of the child during restoration, and the number of restorations per child, nor the contamination of preparations with blood or saliva had a significant influence on the survival of the restorations in this study.
This field study was performed correctly and the statistical power was sufficiently high to detect at least medium effects. However, because it was part of a large-scale randomized controlled clinical trial, no comprehensive criteria were formulated beforehand regarding, for example, the number of restorations per patient, and the location and the size of the cavities. This aspect is inherent to many cohort studies and it does not imply an inferior study quality, but it limits a meaningful comparison with other survival studies.
Although all possible efforts were exercised to trace the participating children over the evaluation period, 22 restorations (4.63%, eight children), all in primary molars, could not be evaluated at any of the recall visits. Either the children did not show up, or the teeth concerned had exfoliated before the first evaluation. These restorations were regarded as missing data and, therefore, excluded from further analysis. Twenty-six restorations (5.47%, 21 children) were “lost” for evaluation because the teeth either exfoliated or the child moved to another district during the course of the study, but after the first evaluation. These restorations (scores 60–90) were treated as censored data and not as true failures because they survived up to a certain moment.
Many causative factors could be suggested that might explain the failure of the ART restorations, such as secondary caries, cervical margin gaps, material properties, and field conditions (outside temperature, atmospheric humidity). However, many other ART studies face these or comparable problems and, therefore, these factors cannot sufficiently explain the extremely low survival rates found in this particular study. The operator difference for the survival rates of the two-surface restorations was not unique, and not a sufficient explanation for the disappointing survival results. Operator effects are often found in ART studies [4, 9, 15, 21, 26] and, as in every profession, there will always be individual differences in technical skills. The finding that the absence of an adjacent tooth was related to a lower 3-year survival of single-surface ART restorations in permanent molars could not be explained. One can only speculate about possible reasons for this relationship, such as that these freestanding molars experience larger occlusal forces.
A possible influence of the relatively high caries prevalence on the survival of the restorations could be hypothesized, but is very doubtful. A study in Indonesia, where the child population exhibited a much higher caries prevalence, also found disappointing survival rates for two-surface ART restorations [28], but these rates were not as extreme as those found in the current study. The survival rates for single-surface ART restorations, derived from other earlier cited studies, were all very promising regardless of the caries profile of the study populations. Furthermore, no effect on the survival rates of the restorations was found when the number of restorations per child was included in the analysis.
The ART protocol prescribes not to eat or drink within at least 1 h after the completion of the restorative treatment [7]. The children in the current study were not supervised after they received restorative treatment and consequently, their food intake could not be controlled. Future studies should take this aspect into account.
Other patient-related factors that may influence the survival of the restorations are the behavior and saliva flow of the child. The survival of the ART restorations in this study was analyzed at the restoration level. This method requires independency of the restoration data and, with respect to the mentioned patient-related possible bias, this assumption could not be guaranteed. To control for this lack of independency, the survival analyses also were performed at the patient level, including only one randomly selected restoration per child. These analyses did not render higher survival rates.
The predominant failure characteristics for both single- and two-surface ART restorations in the primary dentition were gross marginal defects and total or partial losses. This agrees with previous studies concerning the survival of ART restorations in the primary dentition [6, 13, 25, 26]. Gross marginal defects could be induced by occlusal forces or insufficient wear resistance of the restorative material. Ketac-Molar was specifically developed for ART purposes [13], and it has shown excellent results for posterior restorations in the primary dentition [16, 22]. Glass ionomer restorations can be dislodged for a number of reasons, such as insufficient cleaning and conditioning of the cavity, and improper mixing of the material. None of these conditions was recorded at the time the tooth was restored. However, all dentists and chair-side assistants followed the ART guidelines and the manufacturer’s instructions as much as possible under the given circumstances.
The main reasons for failure of the single-surface restorations in the permanent dentition were gross marginal defects and secondary caries. This latter finding is somewhat surprising and contrasts with earlier ART studies [5, 6, 10, 26]. Glass ionomer cement has been the restorative material of choice for the ART technique, based mainly on its fluoride releasing and, thus, caries-preventive properties [7]. Many studies underline these characteristics of glass ionomer [18, 26, 30, 31].
The extremely low survival of the ART restorations observed in this study remains unexplained. Circumstances that were not recognized as possible interfering factors at the start of the study might have played an important role, including cultural and seasonal dietary influences. People living in the rainforest of Suriname eat seasonal fruits such as mangos and fruits of the fiber palm (Awarra). In particular, the latter may influence the survival of the restorations, given the frequency and method in which they are consumed. The authors have seen unusual wear patterns, also in adult dentitions, which might have been caused by excessive consumption of Awarras. A possible causality between these dietary habits and the survival of the ART restorations can only be disclosed by future controlled studies.
Although previous studies have suggested that ART should not be considered as a routine procedure to restore multisurface cavities [13, 24], based on the results of this study, even the ART restoration of single-surface cavities might be reconsidered. This study underlines the inconsistency and variation in the success of the treatment. Apparently, certain conditions must be fulfilled to make ART successful. These conditions can be approached, but not always achieved, under all circumstances.
Conclusion
The uncertain predictability for the success of ART may introduce further discussion about alternative treatment strategies, especially in those situations where choices have to be made with respect to a well-balanced, cost-effective package of basic oral health care. To gain insight into factors determining the cumulative success rate of ART restorations, future studies should focus in more detail on variables that could possibly contribute to the failure of restorations.

art
primary dentition
cumulative survival
oral health


Luteal regression: a redefinition of the terms

The corpus luteum is a transient endocrine gland that is specialized for the production of progesterone and that plays a critical role in the establishment and maintenance of pregnancy. The life span of the corpus luteum varies between species and, within a species, can be dramatically altered by events such as mating or pregnancy. Regardless of the duration of its life span, the corpus luteum eventually enters a dynamic regression process during which it loses the capacity to produce progesterone and undergoes structural involution.
The overall process of luteal regression has been referred to by a variety of terms over the last several decades. In Table 1, we show the results of a cursory MEDLINE/PubMed search on luteal regression performed in November of 2002, which reveals that the most frequently utilized term for this process is "luteolysis," followed by "luteal regression," and then, to a lesser extent, by "functional luteolysis" and "structural luteolysis." Other terms, such as "luteal involution," "functional luteal regression," or "structural luteal regression" lag far behind in usage. The necessity of using several keywords to search for information on luteal regression is illustrated by the fact that in our sample MEDLINE/PubMed search, only 47% of the manuscripts retrieved using the keyword "luteal regression" were also retrieved when using the keyword "luteolysis." This inconsistency in terminology can result in serious under-retrieval and under-citation of papers using less popular keywords.
Table 1
Results of a cursory MEDLINE/PubMed search performed on November 2002 using the terms as listed.
Rank
Terms
Total Number of Articles Retrieved
Retrieval Period
Number of Articles Retrieved for Period 2001–2002
1
"luteolysis"
1251
1967–2002
96
2
"luteal regression"
403
1966–2002
35
3
"functional luteolysis"
82
1973–2002
5
4
"structural luteolysis"
72
1969–2002
14
5
"luteal involution"
41
1978–2002
8
6
"functional luteal regression"
10
1984–2002
0
7
"structural luteal regression"
5
1988–2002
1
To quote Rossdale and Cox in their communication entitled Terminology: a mark of scientific progress [1]: "Scientific terminology must be as exact as is possible within the state of knowledge available." As our knowledge of the molecular mechanisms participating in the process of luteal regression evolves, some of the commonly used terms cited above have been rendered obsolete, inappropriate, and even incorrect. The foremost of these is "luteolysis," still one of the most common terms used to define the process of luteal regression, most probably because it is listed among Medical Subject Headings (MeSH), and, therefore, used most frequently. MeSH is the U.S. National Library of Medicine's controlled vocabulary used for indexing articles in PubMed. MeSH terminology provides a consistent way to retrieve information that may use different terminology for the same concepts. In MeSH, "luteolysis" is defined as "degradation of corpus luteum" and further described as "...characterized by the involution and cessation of its endocrine function." From a cell biology point of view, "lysis" means "rupture of cell plasma membrane, leading to the release of cytoplasm and the death of the cell" [2]. If interpreted with strict accordance to the Latin, lysis refers to "disintegration," as recently discussed by Davis and Rueda [3]. Over the last decade, it has become clear that the involution of the corpus luteum is associated with a phenomenon of programmed cell death or apoptosis [4]. Because apoptosis is an organized process that does not involve a major inflammatory response, and because the majority of cells are removed prior to rupture, the term "lysis" when applied to luteal regression is largely inaccurate. Therefore, while the term luteolysis remains very popular – 1251 manuscripts were retrieved using the word "luteolysis" and only 403 were retrieved using the terms "luteal regression" in the MEDLINE/PubMed search (see Table 1) – this term no longer accurately reflects the mechanisms involved in luteal regression. In 1999, McCraken et al. [5] stated that "the term luteolysis may be something of a misnomer;" but also referred to the deep entrenchment of this term in the literature. However, the exponential increase in the number of scientific manuscripts published in recent years ensures that a prompt move to replace the older term with a more accurate descriptor will rapidly result in an overturn of the current ranking displayed in Table 1 and that only a minority of papers will continue to be retrieved using the older term.
Despite the fact that a strict definition of "luteolysis" appears to regard only the structural disintegration of the corpus luteum, broader interpretations are rife within the literature. Rothchild [6] defined luteolysis as "stopping the secretion of progesterone by the corpus luteum." Two other recent reviews take a middle road. Hoyer [7] refers to the fall in progesterone secretion as functional regression but considers structural regression, or cell death, the "true" regression. Niswender et al. [8], while referring to luteal regression as the structural demise of the corpus luteum, go on to describe the loss of progesterone synthesis and secretion – not a structural event – as part of the regression of the corpus luteum.
As the previous paragraph shows, the adjectives "structural" and "functional" cannot be overlooked when addressing terminology in the field of luteal regression. These words have had a consistent presence in literature concerning the regression of the corpus luteum since Malven and Sawyer [9] described the process of luteal regression in the rat as having two components: "1) an initial termination of luteal secretory function and 2) a subsequent morphological regression of the nonfunctioning corpora lutea." A few years later Malven et al. [10] used the term "structural luteolysis" to describe the morphological regression of persistent, nonfunctional corpora lutea in the hypophysectomized rat. The definition of "nonfunctional" provided in this manuscript expanded upon the 1966 description of the first component of luteal regression in the rat by explaining that the corpora lutea "did not secrete enough progesterone to induce deciduoma formation following endometrial traumatization." Thus, the corpora lutea were "nonfunctional" in terms of progesterone production and the potential for establishment of pregnancy.
Since the appearance of these early papers, the terms functional and structural regression of the corpus luteum (or the more popular phrasing "functional and structural luteolysis") have gained relative acceptance and usage. The cursory MEDLINE/PubMed search shown in Table 1 turned up 82 references containing the phrase "functional luteolysis," of which 5 were 2001 and 2002 publications, and 72 references containing the phrase "structural luteolysis," of which 14 were 2001 and 2002 publications. The prevalence of the term "structural luteolysis" over "functional luteolysis" in recently published articles may be due in part to a greater disparity in the perceived meaning of the latter term. This stems largely from studies in rats, in which it was revealed that corpora lutea which no longer secreted significant quantities of progesterone retained many functions, including active steroidogenesis [11-13].
It is our contention that these two frequently described components of the process of luteal regression (i.e. cessation of progesterone secretion and disappearance of the structure) merit equal attention. However, the timing and inter-relationship of these two types of regressive changes appears to vary by species. As new information on the molecular steps triggered during luteal regression is obtained, these mechanisms will need to be defined as they relate to these facets of the overall regressive process. Davis and Rueda [3] advised that the terms functional and structural used in the context of luteal regression should be adequately defined by the authors in the context of the particular species and reproductive stage being studied, with which we concur. It is not extreme to predict that with the advancement of knowledge in the process of luteal regression researchers will find increasing difficulties in establishing the limits as to what mechanisms belong to the "functional" facet of luteal regression, and which ones are part of the "structural" facet, as luteal regression is a process of synchronized events. For example, it now appears that progesterone itself is responsible for preventing the onset of apoptosis and structural disintegration [14-17].
Conclusions
These authors strongly recommend that the term luteal regression should be used to refer to the process of regression of the corpus luteum. Luteal regression represents a broad definition of the process of demise of the corpus luteum that is capable of accommodating all new knowledge evolved on the molecular mechanisms activated or inhibited during the process of regression of the corpus luteum regardless of species or reproductive stage. Further, according to the etymology of the word, luteolysis has a very limited meaning that no longer describes properly the complex sequence of synchronized molecular events associated with the demise of the corpus luteum. The modifying terms "functional" and "structural" can be used in conjunction with the term luteal regression to further identify aspects of the overall process but must be adequately defined within the context of the publication.

luteal regression
corpus luteum
luteolysis
functional luteolysis
structural luteolysis


Pharmacokinetics and tolerability of artesunate and amodiaquine alone and in combination in healthy volunteers

Objectives The WHO recommends artemisinin-based combination therapies for treatment of uncomplicated falciparum malaria. At least 15 African countries have adopted artesunate plus amodiaquine as treatment policy. As no pharmacokinetic data on this combination have been published to date, we investigated its pharmacokinetic interactions and tolerability in healthy volunteers in Africa.
Introduction
Since Plasmodium falciparum has developed resistance to almost all anti-malarial drugs, the use of combinations of effective antimalarials with different mechanisms of action is required to improve cure rates and delay drug resistance. The World Health Organisation (WHO) now recommends treatment of uncomplicated falciparum malaria with one of several artemisinin-based combination therapies (ACTs), including artesunate plus amodiaquine [1]. To date at least 15 African countries have adopted artesunate plus amodiaquine as first-line malaria treatment policy [2].
Although the limited data available suggest that the combination of artesunate and amodiaquine is well tolerated as short-course treatment, there are two safety concerns that have not yet been fully addressed. Neutropaenia and hepatitis curtailed the use of amodiaquine as prophylaxis in the 1980s [3–5]. A recent randomised, controlled trial comparing treatment with amodiaquine alone or in combination with artesunate found marked, asymptomatic neutropaenia on day 28 (NCI grade 3–4, absolute neutrophil counts 306–900 μL−1) in 6% of 153 children with uncomplicated falciparum malaria, all of whom had normal baseline absolute neutrophil counts [6]. We have previously reported a case of hepatitis in this group of volunteers that developed after the second dose of amodiaquine and was considered probably related to amodiaquine [7]. A similar report of delayed-onset asymptomatic hepatitis has been described in healthy American volunteers who had received artesunate plus amodiaquine alone followed by artesunate plus amodiaquine plus efavirenz, resulting in early study discontinuation [8]. In that study, efavirenz was thought to have caused an increase in amodiaquine concentrations by competitive inhibition of the P450 cytochrome enzymes that metabolise amodiaquine. With the widespread use of artesunate plus amodiaquine treatment in Africa, these studies support the need for laboratory monitoring of the treatment’s safety, particularly if antiretrovirals are used concomitantly [1].
Pharmacokinetic data are still sparse for amodiaquine. After oral administration, amodiaquine hydrochloride is rapidly absorbed and undergoes rapid and extensive metabolisation to desethylamodiaquine, the main active metabolite. Amodiaquine has a short elimination half-life (approximately 5 h), while that for desethylamodiaquine is much longer (6–18 days) [9, 10].
There are several pharmacokinetic studies on oral artesunate in healthy volunteers and malaria patients that give broadly consistent results [11, 12]. Artesunate is rapidly absorbed, rapidly converted to its main active metabolite dihydroartemisinin and rapidly cleared (elimination half-life generally <1 h). Dihydroartemisinin has similar pharmacokinetic characteristics but has a slightly longer elimination half-life. There is wide inter-individual variation in artesunate and dihydroartemisinin pharmacokinetic parameters.
No pharmacokinetic interaction data have yet been published on amodiaquine when used in combination with artesunate. In view of the forthcoming widespread use of this combination in Africa, our study aimed to investigate potential pharmacokinetic interactions between these antimalarials and assess their tolerability in healthy volunteers in Africa.
Methods
Study subjects and study design
Male or female healthy normal volunteers who provided written informed consent and met the following criteria were eligible to participate in the study: aged 18–45 years; no abnormalities on medical history, clinical examination, laboratory safety assessment (full blood count, differential white cell count, routine liver and renal function tests) or electrocardiogram; and a negative pregnancy test (for female volunteers). Volunteers were excluded if they were smokers (>5 cigarettes/day), had taken antimalarials or been in a malarial area in the preceding 8 weeks, had malaria parasites on a thick smear, used recreational drugs, or had ingested any alcohol or any medicines (including over-the-counter preparations) in the week preceding study commencement.
This was a randomised three-phase crossover study. All volunteers took artesunate (4 mg/kg as Arsumax, 50 mg tablets, Sanofi-Aventis), the drug with the shorter elimination half-life, in the first phase, prior to the administration of amodiaquine. In the second phase, 7 days later, the volunteers were randomly allocated to one of two treatment groups: group 1 received a single oral dose of artesunate (4 mg/kg) plus amodiaquine (10 mg base/kg as Camoquin, 200 mg tablets, Parke Davis), and group 2 received a single oral dose of amodiaquine (10 mg/kg) alone. In phase three, 21 days later, the groups received the alternative regimen. All trial drugs were given under direct supervision with 200 ml tap water on an empty stomach after an overnight fast. No caffeinated drinks were allowed during the study period. Standardised meals were offered, commencing with breakfast at 2 h post-dose. Volunteers in phase one had blood samples collected before taking artesunate and at 0.25, 0.50, 0.75, 1.0, 1.5, 2, 3, 4, 6, 8 and 12 h after artesunate administration. In phases two and three (i.e. volunteers receiving artesunate plus amodiaquine or amodiaquine alone) venous blood samples were collected pre-dose and at 0.25, 0.5, 0.75, 1, 1.5, 2, 3, 4, 6, 8, 12, 24, 48 and 72 h, and days 4, 5, 7, 10, 14 and 20 post-dose. At each time two 5-ml venous blood samples were collected in tubes containing sodium heparin anticoagulant, while three 5-ml samples were collected at each time point for the first 12 h from those volunteers receiving both drugs. Each sample was centrifuged within 5 min of collection and the plasma transferred to separate plastic cryotubes and frozen at −70°C until analysed for artesunate, dihydroartemisinin, amodiaquine and desethylamodiaquine. Volunteers remained in the study ward for the first 12 h of each phase; thereafter specimens were collected at outpatient visits.
A clinical examination and laboratory safety tests were performed during screening and 6 days after artesunate administration and 20 days after administration of both amodiaquine and amodiaquine plus artesunate. A 12-lead electrocardiogram (ECG) was recorded for each volunteer during screening and approximately 2 h after each drug dose. The corrected QT interval (QTc) was calculated by dividing the QT interval by the square root of the R-R interval. ECGs were reviewed by C.O. and by a consultant cardiologist. At follow-up visits, the volunteers were asked to report any new symptoms. A targeted physical examination was conducted if indicated. An adverse event together with its relationship to study drugs was defined according to the WHO guidelines [13] and the severity graded using the National Cancer Institute (NCI) Common Toxicity Criteria [14]. All adverse events and concomitant medications taken were recorded. Any significant clinical or laboratory abnormalities or the use of a concomitant medication with a previously described interaction resulted in the volunteer being withdrawn from the study.
Ethical approval
This clinical study was conducted in accordance with the principles laid down by the World Health Assembly of 1975 on Ethics in Human Experimentation and the Helsinki Declaration. The study adhered to the standards established for good clinical practice. The protocol was approved by the University of Cape Town Research Ethics Committee and the WHO Secretariat Committee for Research Involving Human Subjects (SCRIHS). Each volunteer was informed of the objectives, nature and possible risks of the trial. Written informed consent was obtained from every volunteer participating in the study. The volunteers were informed that they were free to withdraw consent at any time.
Drug assays
Artesunate and dihydroartemisinin concentrations in plasma were analysed using the method of Batty and colleagues [15] with minor modifications. Solid-phase extraction of samples was performed using Bond-Elut phenyl extraction cartridges (Varian, Harbour City, CA, USA). After washing the column with 2 ml of water, samples were eluted from the column with 1.5 ml of acetonitrile, which was then evaporated to dryness under nitrogen. Samples were reconstituted in 100 μl of mobile phase (45% acetonitrile, 0.05 M acetic acid, pH 6.0), and 50 μl was injected onto the column. HPLC was carried out on a Symmetry C 8, 5 μm, 15 cm × 4.6 mm HPLC column (Waters, Milford, MA, USA) using a Waters 600 pump and WISP 712 auto-sampler. The post-column reagent (1.2 M KOH in 90% methanol) was introduced using a Waters reagent delivery module and the mixture passed through a 1-ml reaction coil (Waters) kept at 69°C. Detection was at 290 nm using a Waters 484 Tunable Absorbance Detector, which produced a linear response over the range of the standard curve (20–1,000 ng/ml). Quality control samples of 15, 75 and 150 ng/ml for artesunate and 75, 450 and 750 ng/ml for DHA were included in each run. Within- and between-day coefficients of variation were below 14%. The lower limit of quantification was 24 ng/ml for both artesunate and dihydroartemisinin.
Amodiaquine and desethylamodiaquine were analysed by LC mass spectrometry using an Agilent 1100 Series LC/MS system. Protein was precipitated from plasma samples (200 ul) using three volumes of acetonitrile. Supernatant (5 μl) was injected onto the HPLC. Chromatography was carried out using a 50 × 4.6 mm C18 Xterra column (Waters). The mobile phase comprised 75% acetonitrile, 0.02 M ammonia, pH 10.2. The extracted ion for amodiaquine was m/z 356 and for desethylamodiaquine m/z 328. The calibration curve for amodiaquine was linear in the range 5–100 ng/ml and for desethylamodiaquine in the range 5–400 ng/ml. Quality control samples of 25, 75 and 250 ng/ml were used for desethylamodiaquine and 2.5, 7.5 and 25 ng/ml for amodiaquine. Within- and between-day coefficients of variation were below 12%. The lower limit of quantification was 5 ng/ml for both amodiaquine and desethylamodiaquine.
Statistical analysis
Pharmacokinetic parameters were calculated from the artesunate, dihydroartemisinin, amodiaquine and desethylamodiaquine concentration profiles using WinNonLin, version 3.3 (Pharsight), with concentrations outside the specified limits of quantification regarded as missing values. Clearance (Cl) was calculated as 0.693 × Vd/T1/2 where Vd is the apparent volume of distribution and T1/2 is the elimination half-life. Because there was no intravenous comparator arm in this study, equivalent bioavailability was assumed for comparisons of apparent volume of distribution (Vd/f) and clearance (Cl/f), where f is the oral bioavailability or fraction of the drug absorbed (unknown).
All four compounds were first analysed separately. Because both artesunate and dihydroartemisinin are highly parasiticidal in malaria patients, the pharmacokinetic parameters associated with therapeutic response [area under the plasma concentration time curve (AUC) and maximum clearance (Cmax)] are also reported together as dihydroartemisinin equivalents, defined as the sum of the measured dihydroartemisinin concentrations plus the artesunate concentration converted to dihydroartemisinin equivalents (using molecular weights of 384 and 284 for artesunate and dihydroartemisinin respectively) [16, 17]. As amodiaquine undergoes a rapid and extensive conversion to its active metabolite, desethylamodiaquine was considered the primary analyte [9, 10, 18].
The pharmacokinetic parameters following administration of artesunate and amodiaquine alone and in combination were compared using Stata, version 9.0 [Stata, College Station, TX, USA]. Data were log-transformed and then compared using the analysis of variance (ANOVA) for a cross-over design to take into account the repeated measures by study subject, treatment period and treatment groups and, for amodiaquine and desethylamodiaquine, a sequence effect. The treatment effects generated from the ANOVA were exponentiated in order to express comparisons between monotherapy and combination therapy as a ratio. Any apparent discrepancies between the difference in the group means and these ratios are due to the fact that the ratios are based on the within-patient differences in log-transformed values and not the group arithmetic means. Given the multiple testing, statistical significance of results should be interpreted with caution.
For the safety analysis, means and 95% confidence intervals (95% CI) for each haematological parameter (haemoglobin, haematocrit, platelets, white cell count, absolute lymphocyte and neutrophil count) were calculated at baseline and at the end of each treatment phase. Changes in these values from baseline were determined using a mixed effect regression model that took into account repeated measures within-patient and were adjusted for the period and period-treatment interaction effects.
Results
Subject demographics
Nineteen volunteers were screened; two were excluded because of neutropaenia and two withdrew consent. Fifteen healthy normal volunteers (10 male, 5 female) entered the study with a mean age, weight and height of 24.4 years, 67.3 kg and 171 cm, respectively. The mean dose of amodiaquine was 10.72 mg/kg and that of artesunate was 4.26 mg/kg. Safety analysis included all 15 volunteers; only data from the 13 volunteers who completed the study were included in the pharmacokinetic analyses. A female volunteer was withdrawn due to a new prescription of fluoxetine for depression, as this drug could potentially interact with artesunate and amodiaquine by inhibiting cytochrome P450 enzymes. One male volunteer was withdrawn due to a possible hypersensitivity reaction on his first exposure to amodiaquine.
Pharmacokinetic parameters
Effect of amodiaquine on artesunate pharmacokinetic parameters
Data were analysed using a non-compartmental model. Figure 1a shows the mean plasma concentration time curves of artesunate and dihydroartemisinin following administration of artesunate alone and artesunate plus amodiaquine. The AUC, Cmax and time to Cmax (Tmax) of artesunate were similar following monotherapy and combination therapy (Table 1). Figure 2a illustrates the impact of adding amodiaquine on the dihydroartemisinin AUC. Following the combination therapy, dihydroartemisinin AUC was lower [ratio 67% (95% CI 51–88%); P = 0.008], Cmax was lower [ratio 51% (95% CI 33–78%); P = 0.005], T1/2 was longer [ratio 157% (95% CI 115–213%); P = 0.008] and Vd was larger [ratio 192% (95% CI 133–275%); P = 0.003], when compared with artesunate monotherapy (Table 1). These ratios have been adjusted for repeated measures by subject and period effects. As a measure of the total exposure to artemisinin derivatives, the Cmax and AUC of the combined DHA-equivalents were also analysed and found to be lower following combination therapy [Cmax ratio 51% (95% CI 32–84%), P = 0.011; AUC ratio 72% (95% CI 54–95%), P = 0.023] when compared with monotherapy. Cl and Tmax of dihydroartemisinin were similar when monotherapy and combination therapy were administered (Table 1).
Fig. 1Profiles of mean drug concentrations over time following monotherapy (mono) and combination (combo) therapy for a artesunate (AS) and dihydroartemisinin (DHA) and b amodiaquine (AQ) and desethylamodiaquine (DEAQ)Table 1Pharmacokinetic parameters for artesunate and dihydroartemisinin when artesunate was administered alone and with amodiaquine (ACT) AUC (ng·h/ml)Cmax (ng/ml)Tmax (h)T1/2 (h)Vd/f (l)Cl/f (l/min)ArtesunateAS alone (mean ± SD)206.4 ± 135.5231.8 ± 155.00.62 ± 0.28NANANAACT (mean ± SD)183.3 ± 146.5141.6 ± 117.50.86 ± 0.67NANANAACT-to-monotherapy ratio (%) (mean, 95% CI)36 (8–173)25 (6–105)125 (76–202)NANANA  Significance (ANOVA)0.180.0570.33DihydroartemisininAS alone (mean ± SD)2,044.4 ± 564.2844.5 ± 309.41.10 ± 0.951.46 ± 0.484.89 ± 1.672.46 ± 0.86ACT (mean ± SD)1,410.5 ± 543.6446.2 ± 239.52.08 ± 1.722.20 ± 0.859.68 ± 4.163.08 ± 0.82ACT-to-monotherapy ratio (%) (mean, 95% CI)67 (51–88)51 (33–78)165 (82–334)157 (115–213)192 (133–275)122 (96–156)  Significance (ANOVA)0.0080.0050.150.0080.0030.097Treatment effects generated from the ANOVA were exponentiated to express within-subject comparisons between monotherapy and combination therapy as a ratio, adjusted for period effectsAUC Area under the plasma concentration time curve, Cmax maximum concentration, Tmax time to Cmax, T1/2 elimination half-life, Vd volume of distribution, f oral bioavailability or fraction of the drug absorbed, Cl clearance, AS artesunate, SD standard deviation, CI confidence interval, NA not available (too few detectable concentrations for calculation of T1/2, Cl, Vd)Fig. 2Stick plot comparing individual patient AUCs following monotherapy and combination therapy of a dihydroartemisinin (DHA) and b desethylamodiaquine (DEAQ). (Note: volunteer A was excluded from the statistical analyses)
Effect of artesunate on amodiaquine pharmacokinetic parameters
Amodiaquine samples for one volunteer were missing. Figure 1b shows the mean plasma concentrations of desethylamodiaquine over time following administration of amodiaquine alone and in combination. Figure 2b illustrates the impact of artesunate on the desethylamodiaquine AUC. The volunteer (subject A) who had the highest observed AUC for desethylamodiaquine following combination therapy [29,504 ng·h/ml compared with the mean of 8,437 (95% CI 5,744–11,131) ng·h/ml] was excluded from the pharmacokinetic analyses of desethylamodiaquine, as this was an extreme outlier and influential observation (Fig. 2b; Fig. 3).
Fig. 3Stick plot comparing individual patients. Day 7 concentrations of desethylamodiaquine when amodiaquine was administered as monotherapy and in combination with artesunate. (Note: volunteer A was excluded from the statistical analysis)
The impact of adding artesunate to amodiaquine on the pharmacokinetic parameters of amodiaquine and desethylamodiaquine is reported for the remaining 11 volunteers. The pharmacokinetic parameters of amodiaquine were similar when administered as monotherapy or in combination with artesunate (Table 2). The desethylamodiaquine AUC is lower [ratio 65% (95% CI 46–90%); P = 0.015], Tmax shorter [ratio 60% (95% CI 45–80%); P = 0.003] and clearance more rapid [ratio 164% (95% CI 112–243%); P = 0.016] when amodiaquine was administered in combination with artesunate rather than as monotherapy (Table 2). There was a trend towards a lower day 7 concentration of desethylamodiaquine [ratio 56% (95% CI 30–104%); P = 0.064] when amodiaquine was administered in combination (Fig. 3). These ratios have been adjusted for repeated measures by subject, period and sequence effects. The Vd and Cmax were not significantly different when monotherapy and combination therapy were administered.
Table 2Pharmacokinetic parameters for desethylamodiaquine (DEAQ) when amodiaquine was administered alone and with artesunate (ACT) AUC (ng·h/ml)Cmax (ng/ml)Tmax (h)[Day 7] (ng/ml)T1/2 (h)Vd/f (l)Cl/f (l/min)AmodiaquineAQ alone (mean ± SD)162.4 ± 101.429.2 ± 10.92.32 ± 1.16NA5.3 ± 4.1361.0 ± 128.369 ± 59ACT (mean ± SD)108.5 ± 56.022.7 ± 9.02.18 ± 1.61NA3.9 ± 1.2467.7 ± 180.586 ± 26ACT-to-monotherapy (%) (mean, 95% CI)77 (47–127)78 (58–103)92 (57–147)NA74 (29–189)128 (93–175)172 (84–357)  Significance (ANOVA)0.270.070.680.440.110.11DesethylamodiaquineAQ alone (mean ± SD)12,041 ± 3,480268.7 ± 70.83.68 ± 1.8519.4 ± 7.3240.8 ± 146.9234.1 ± 97.5768 ± 252ACT (mean ± SD)8,437 ± 4,009301.4 ± 166.12.18 ± 1.0313.3 ± 7.3136.9 ± 83.8210.8 ± 92.91,330 ± 735ACT-to-monotherapy ratio (%) (mean, 95% CI))65 (46–90)103 (73–147)60 (45–80)56 (30–104)53 (25–111)88 (51–149)164 (112–243)  Significance (ANOVA)0.0150.820.0030.0640.080.580.016Treatment effects generated from the ANOVA have been exponentiated to express within-subject comparisons between monotherapy and combination therapy as a ratio, adjusted for period and sequence effectsAUC Area under the plasma concentration time curve, Cmax maximum concentration, Tmax time to Cmax, [Day 7] day 7 concentration, T1/2 elimination half-life, Vd apparent volume of distribution, f oral bioavailability or fraction of the drug absorbed, Cl clearance, AQ amodiaquine, SD standard deviation, CI confidence interval, NA not available (as too few patients had quantifiable amodiaquine concentrations)
Safety
Twenty-nine adverse events were reported over the course of the study by 10 (67%) of the 15 volunteers. The frequency of adverse events was similar across all three treatment arms (P > 0.10): 4/15 (27%) following artesunate alone, 8/15 (53%) following amodiaquine alone, and 5/15 (33%) following the combination. All adverse events were consistent with the product information available, resolved spontaneously and, except for the transaminitis (see below) were mild or moderate in intensity.
A case of asymptomatic, prolonged, severe transaminitis (NCI grade 3, AST 5–20× upper limit of normal) that developed after phase 3 of the study has been previously published [7]. This was considered to be probably related to amodiaquine, rather than artesunate, before the pharmacokinetic results were available. The subsequent finding that this volunteer had the highest measured desethylamodiaquine AUC following the administration of artesunate plus amodiaquine [29,504 ng·h/ml compared with the combination therapy mean of 8,437 (95% CI 5,744–11,131) ng·h/ml] strengthened this assessment. There were no liver function abnormalities detected in any other volunteers. No renal or electrolyte abnormalities were detected in any volunteers.
Significant haematological changes were confined to the white cell counts. During phase 2 of the study, two volunteers (one following amodiaquine alone, one following the combination) developed asymptomatic, NCI grade 1 leucopaenia (3 to <4 × 109/L) and either a grade 1 (1.5 to <2 ×109/L) or grade 2 (1 to <1.5 × 109/L) neutropaenia. None of the other observed changes in haematological parameters were outside the normal range.
There were no significant changes following treatment seen on the ECGs; the mean (95% CI) QTc interval was 398 (390–406) ms at screening, 401 (393–409) ms following artesunate alone, 400 (385–414) ms following amodiaquine alone and 412 (400–424) ms following the combination. In two patients, the prolongation following treatment with amodiaquine or artesunate plus amodiaquine was considered of borderline clinical significance.
Discussion
The pharmacokinetic analyses in this randomised, cross-over study showed statistically significant pharmacokinetic interactions resulting in reductions in the AUC of both dihydroartemisinin and desethylamodiaquine when artesunate and amodiaquine were given in combination to healthy volunteers. Of further concern is that one healthy volunteer failed to reach quantifiable concentration of both artesunate and dihydroartemisinin throughout the initial 12 h. There is insufficient evidence currently available to explain the basis of these interactions.
The clinical significance of our findings is unclear because we studied healthy volunteers. In one study comparing pharmacokinetic parameters of dihydroartemisinin in patients and volunteers, patients had a significantly higher Cmax (1,948 vs. 1,192 nmol/l) and AUC0–12 (4,024 vs. 1,763 nmol·h/l), while their Vd and Cl were both lower. The Tmax and terminal elimination half-life of the patients were similar to those of the volunteers [19]. Desethylamodiaquine pharmacokinetic parameters in malaria-infected adults in Africa [20] and children with acute malaria in Papua New Guinea [21] were similar to those of normal volunteers.
In artemisinin-based combination therapy, the artemisinin is given to kill most of the parasites quickly, while the longer-acting partner drug (e.g. amodiaquine) is given to kill the residual parasites. For an antimalarial with a long elimination half-life like desethylamodiaquine, the key pharmacokinetic parameter for parasite killing is the AUC, which is a useful predictor of therapeutic response as it captures both the drug concentration and the duration of exposure [22, 23]. For such drugs the antimalarial concentration on day 7 is strongly correlated with AUC, and thus treatment response [23]. An association of desethylamodiaquine concentration on day 7 and amodiaquine treatment response has been found in children with uncomplicated falciparum malaria [18]. In our study, the AUC (and probably the day 7 desethylamodiaquine concentration) was reduced following the artemisinin-based combination; this may result in reduced parasite killing, particularly in areas where there is reduced amodiaquine sensitivity. Although there is a linear relationship between amodiaquine dose and the desethylamodiaquine AUC [24], the wide inter-individual variability in desethylamodiaquine levels (five-fold in our study), and the consequent potential for drug-induced toxicity, could preclude increasing the amodiaquine dose to compensate for the lower desethylamodiaquine AUC observed following co-administration with artesunate.
Extensive clinical trial data show that artesunate plus amodiaquine is more effective (at clearing parasitaemia and preventing recrudescence) than amodiaquine alone [1, 6]. Therefore, if reductions in the Cmax and AUC of desethylamodiaquine and dihydroartemisinin occur in patients, the negative effect on parasite killing may become clinically insignificant due to the additive (or possibly synergistic) parasiticidal effects of the combination. However, this may only apply to areas where the level of amodiaquine resistance is low. Although clinically significant resistance to the artemisinins has not yet been documented, amodiaquine resistance is well documented and this is probably the principal explanation for low artesunate plus amodiaquine cure rates observed in a number of African countries [1, 6].
Despite our small sample size, we detected the well-known amodiaquine-related adverse drug reactions. Clinically important adverse events were reported by a quarter of these volunteers, even though all were healthy and only two doses were administered 3 weeks apart (rather than the daily administration for 3 days recommended for malaria treatment). When the artesunate plus amodiaquine combination becomes widely used, monitoring of liver function tests and haematological parameters are warranted to define these risks in malaria patients, particularly in patients at special risk such as those at the extremes of age, with HIV co-infection or malnutrition [1]. However, the feasibility of this recommendation outside of the research setting is of concern.
To conclude, the total exposure to the main active metabolites of both artesunate and amodiaquine was significantly reduced when administered in combination to healthy African volunteers; this might be important clinically. However, because cure rates with this combination are generally higher than with amodiaquine monotherapy, artesunate and amodiaquine could remain in the armamentarium of drugs used to combat falciparum malaria, provided efficacy continues to be monitored and adequate safety precautions can be taken. Further pharmacokinetic research on artesunate plus amodiaquine, when administered concurrently or as a fixed dose combination, is urgently required in patients with malaria to establish the extent and clinical significance of these pharmacokinetic interactions.

pharmacokinetics
tolerability
artesunate
amodiaquine
healthy normal volunteers