**3.1 The ability of GFS components to sustain parasite growth**

We investigated the components of GFS and related substances that have shown an ability to sustain parasite growth (Asahi et al., 2005). A simple total lipid fraction of GFS obtained by lipid extraction has been shown to sustain complete parasite development. However, specific proteins such as bovine and human albumin, as well as the simple total lipid fraction of GFS, have also been shown to be important (Figure 1a). The simple total lipid fraction of GFS contained phospholipids, diacylglycerides (DAG), cholesterol, monoglycerides, nonesterified fatty acids (NEFA) and cholesteryl esters. The components of the NEFA fraction were mainly *cis*-9-octadecenoic acid (C18:1-*cis*-9, 43%), hexadecanoic acid

Fig. 1. Abilities of fractions derived from GFS (a) and a total simple lipid fraction of GFS (b) to sustain growth of *P. falciparum.* Growth rate was estimated 4 days after inoculation.

**I**ntraerythrocytic *Plasmodium falciparum* Growth in

influences their growth-promoting effects.

Serum-Free Medium with an Emphasis on Growth-Promoting Factors 77

Fig. 2. Effects of various classes of lipids and combinations of phospholipids on abilities of NEFA to sustain growth of *P. falciparum.* The combination of 30 μg/ml C18:1-*cis*-9 and 15 μg/ml C16:0 served as a control. Growth rate was estimated 4 days after inoculation.

C16:0, *cis*-13-octadecenoic acid (C18:1-*cis*-13) plus C16:0, and C18:2 plus C16:0 showed similar growth-promoting effects to that seen with C18:1-cis-9 plus C16:0 in the absence of phospholipids. Combinations of C18:1-*cis*-9 plus dodecanoic acid (C12:0), C18:1-*cis*-9 plus docosanoic acid (C22:0), *cis,cis,cis*-6,9,12-octadecatrienoic acid (C18:3) plus C16:0, C20:4 plus C16:0, C20:5 plus C16:0, and C22:6 plus C16:0 were detrimental to parasite growth. The combination of *trans*-9-octadecenoic acid (C18:1-*trans*-9) plus C16:0 also deterred parasite growth (Figure 4). The efficacies of NEFA in sustaining the growth of *P. falciparum* thus varied markedly, depending on the type, total amount, and combinations used; saturated or unsaturated NEFA with longer or shorter carbon-chain lengths than the optimal combination (C18:1-*cis*-9 plus C16:0) promoted growth to lesser extents, or were detrimental to the growth of *P. falciparum*. Higher degrees of unsaturation were also detrimental to parasite growth. The growth-promoting effects of NEFA with 18 carbons and one double bond are specific to the *cis*-form, and the position of the double bond in these NEFA

Various PC containing different fatty acid moieties, such as two of hexanoic acid, C12:0, C14:0, C16:0, C18:1, C18:1 in racemic form, C18:2, and C20:4, two different fatty acids of C18:1 and C16:0, and C20:4 and C16:0, and PC derived from soy beans and egg yolk, were tested at graded concentrations ranging from 20–320 μM, for their abilities to augment the effects of the NEFA mixture on parasite growth. Among the 12 tested PC, 1,2-dioleoyl-snglycero-3-phosphocholine (PC-di18:1) markedly amplified the growth-promoting ability of

(C16:0, 21%), octadecanoic acid (C18:0, 14%), *cis,cis*-9,12-octadecadienoic acid (C18:2), *cis*-9 hexadecenoic acid (C16:1), *cis*-5,8,11,14-eicosatetraenoic acid (C20:4), *cis*-5,8,11,14,17 eicosapentaenoic acid (C20:5), and *cis*-4,7,10,13,16,19-docosahexaenoic acid (C22:6). Each NEFA enriched with bovine albumin (fatty-acid free) was tested for its ability to promote parasite growth. Mixtures of NEFA, but not individual NEFA, sustained parasite growth to a low extent (Figure 1b), but parasite growth in the presence of various combinations of NEFA was still lower than that achieved with a simple total lipid fraction of GFS, or with GFS- or human serum-containing medium. These results implied that, although the NEFA components of the simple total lipid fraction are functional factors in promoting parasite growth, other factor (s) must also contribute to the high growth-promoting activity of GFS.

#### **3.2 CDM for intraerythrocytic growth of** *P. falciparum*

Initial experiments designed to determine the factor (s) responsible for the high growthpromoting activity of GFS involved culture of *P. falciparum* with the lipid classes found in the simple total lipid fraction of GFS, and different concentrations of a mixture of the two most abundant NEFA, C18:1-*cis*-9 (0–60 μg/ml [212.4 μM]) and C16:0 (0–30 µg/ml [117.0 μM]) at a ratio of 2:1. The growth rate was dependent on the concentrations of the NEFA in the mixture: the maximum effect was obtained with 30 μg/ml C18:1-*cis*-9 plus 15 μg/ml C16:0, with declines at lower and higher concentrations. However, the growth rates were much lower than that obtained with a simple total lipid fraction of GFS or with GFSRPMI.

A mixture of all the constituents detected in a simple total lipid fraction of GFS sustained complete parasite growth. In an attempt to identify the factor (s) responsible for this growthpromoting effect, each lipid was omitted from the medium in turn. Parasite growth in the absence of phosphatidylcholine (PC) decreased to a level similar to that seen with the NEFA mixture (Figure 2a). Phospholipids were also tested for their possible efficacies in augmenting the ability of NEFA to promote parasite growth, by adding each phospholipid to cultures with NEFA. Phospholipids, even PC alone, markedly amplified the growthpromoting ability of the NEFA mixture (Figure 2b). These results indicate the critical importance of PC for amplifying the parasite-growth-promoting ability of NEFA mixtures. Phospholipids other than PC, such as phosphatidylethanolamine (PE), phosphatidylserine (PS), and phosphatidic acid (PA), were also beneficial for parasite growth, while the growth rates in the absence of phosphatidylinositol (PI), cholesterol, and cholesterol ester were significantly higher (Figure 2a), indicating these phospholipids were detrimental. DAG had no effect on the growth rate of the parasite at the concentrations tested.

The effects of various types of NEFA mixtures enriched with phospholipids were tested for their abilities to promote parasite growth. The growth rate was dependent on the ratios of the two NEFA; the highest growth rate occurred at 2:1 (C18:1-*cis*-9 to C16:0) at a total concentration of 45 μg/ml. The growth rates with the best mixtures of NEFA in the presence of phospholipids were significantly higher than those with the same NEFA in the absence of phospholipids (Figure 3). The culture media were also reconstituted by mixing phospholipids with two types of NEFA (either C18:1-*cis*-9 plus a saturated NEFA or C16:0 plus an unsaturated NEFA). The best combination of NEFA was C18:1-*cis*-9 plus C16:0, followed by *cis*-11-octadecenoic acid (C18:1-*cis*-11) plus C16:0, C18:1-*cis*-9 plus pentadecanoic acid (C15:0), C18:1-*cis*-9 plus C18:0, and C18:1-*cis*-9 plus tetradecanoic acid (C14:0). The combinations of C16:1 plus C16:0, *cis*-6-octadecenoic acid (C18:1-*cis*-6) plus

(C16:0, 21%), octadecanoic acid (C18:0, 14%), *cis,cis*-9,12-octadecadienoic acid (C18:2), *cis*-9 hexadecenoic acid (C16:1), *cis*-5,8,11,14-eicosatetraenoic acid (C20:4), *cis*-5,8,11,14,17 eicosapentaenoic acid (C20:5), and *cis*-4,7,10,13,16,19-docosahexaenoic acid (C22:6). Each NEFA enriched with bovine albumin (fatty-acid free) was tested for its ability to promote parasite growth. Mixtures of NEFA, but not individual NEFA, sustained parasite growth to a low extent (Figure 1b), but parasite growth in the presence of various combinations of NEFA was still lower than that achieved with a simple total lipid fraction of GFS, or with GFS- or human serum-containing medium. These results implied that, although the NEFA components of the simple total lipid fraction are functional factors in promoting parasite growth, other factor (s) must also contribute to the high growth-promoting activity of GFS.

Initial experiments designed to determine the factor (s) responsible for the high growthpromoting activity of GFS involved culture of *P. falciparum* with the lipid classes found in the simple total lipid fraction of GFS, and different concentrations of a mixture of the two most abundant NEFA, C18:1-*cis*-9 (0–60 μg/ml [212.4 μM]) and C16:0 (0–30 µg/ml [117.0 μM]) at a ratio of 2:1. The growth rate was dependent on the concentrations of the NEFA in the mixture: the maximum effect was obtained with 30 μg/ml C18:1-*cis*-9 plus 15 μg/ml C16:0, with declines at lower and higher concentrations. However, the growth rates were much lower than that obtained with a simple total lipid fraction of GFS or with GFSRPMI. A mixture of all the constituents detected in a simple total lipid fraction of GFS sustained complete parasite growth. In an attempt to identify the factor (s) responsible for this growthpromoting effect, each lipid was omitted from the medium in turn. Parasite growth in the absence of phosphatidylcholine (PC) decreased to a level similar to that seen with the NEFA mixture (Figure 2a). Phospholipids were also tested for their possible efficacies in augmenting the ability of NEFA to promote parasite growth, by adding each phospholipid to cultures with NEFA. Phospholipids, even PC alone, markedly amplified the growthpromoting ability of the NEFA mixture (Figure 2b). These results indicate the critical importance of PC for amplifying the parasite-growth-promoting ability of NEFA mixtures. Phospholipids other than PC, such as phosphatidylethanolamine (PE), phosphatidylserine (PS), and phosphatidic acid (PA), were also beneficial for parasite growth, while the growth rates in the absence of phosphatidylinositol (PI), cholesterol, and cholesterol ester were significantly higher (Figure 2a), indicating these phospholipids were detrimental. DAG had

**3.2 CDM for intraerythrocytic growth of** *P. falciparum* 

no effect on the growth rate of the parasite at the concentrations tested.

The effects of various types of NEFA mixtures enriched with phospholipids were tested for their abilities to promote parasite growth. The growth rate was dependent on the ratios of the two NEFA; the highest growth rate occurred at 2:1 (C18:1-*cis*-9 to C16:0) at a total concentration of 45 μg/ml. The growth rates with the best mixtures of NEFA in the presence of phospholipids were significantly higher than those with the same NEFA in the absence of phospholipids (Figure 3). The culture media were also reconstituted by mixing phospholipids with two types of NEFA (either C18:1-*cis*-9 plus a saturated NEFA or C16:0 plus an unsaturated NEFA). The best combination of NEFA was C18:1-*cis*-9 plus C16:0, followed by *cis*-11-octadecenoic acid (C18:1-*cis*-11) plus C16:0, C18:1-*cis*-9 plus pentadecanoic acid (C15:0), C18:1-*cis*-9 plus C18:0, and C18:1-*cis*-9 plus tetradecanoic acid (C14:0). The combinations of C16:1 plus C16:0, *cis*-6-octadecenoic acid (C18:1-*cis*-6) plus

Fig. 2. Effects of various classes of lipids and combinations of phospholipids on abilities of NEFA to sustain growth of *P. falciparum.* The combination of 30 μg/ml C18:1-*cis*-9 and 15 μg/ml C16:0 served as a control. Growth rate was estimated 4 days after inoculation.

C16:0, *cis*-13-octadecenoic acid (C18:1-*cis*-13) plus C16:0, and C18:2 plus C16:0 showed similar growth-promoting effects to that seen with C18:1-cis-9 plus C16:0 in the absence of phospholipids. Combinations of C18:1-*cis*-9 plus dodecanoic acid (C12:0), C18:1-*cis*-9 plus docosanoic acid (C22:0), *cis,cis,cis*-6,9,12-octadecatrienoic acid (C18:3) plus C16:0, C20:4 plus C16:0, C20:5 plus C16:0, and C22:6 plus C16:0 were detrimental to parasite growth. The combination of *trans*-9-octadecenoic acid (C18:1-*trans*-9) plus C16:0 also deterred parasite growth (Figure 4). The efficacies of NEFA in sustaining the growth of *P. falciparum* thus varied markedly, depending on the type, total amount, and combinations used; saturated or unsaturated NEFA with longer or shorter carbon-chain lengths than the optimal combination (C18:1-*cis*-9 plus C16:0) promoted growth to lesser extents, or were detrimental to the growth of *P. falciparum*. Higher degrees of unsaturation were also detrimental to parasite growth. The growth-promoting effects of NEFA with 18 carbons and one double bond are specific to the *cis*-form, and the position of the double bond in these NEFA influences their growth-promoting effects.

Various PC containing different fatty acid moieties, such as two of hexanoic acid, C12:0, C14:0, C16:0, C18:1, C18:1 in racemic form, C18:2, and C20:4, two different fatty acids of C18:1 and C16:0, and C20:4 and C16:0, and PC derived from soy beans and egg yolk, were tested at graded concentrations ranging from 20–320 μM, for their abilities to augment the effects of the NEFA mixture on parasite growth. Among the 12 tested PC, 1,2-dioleoyl-snglycero-3-phosphocholine (PC-di18:1) markedly amplified the growth-promoting ability of

**I**ntraerythrocytic *Plasmodium falciparum* Growth in

Serum-Free Medium with an Emphasis on Growth-Promoting Factors 79

Fig. 4. Growth of *P. falciparum* in the presence of various combinations of paired NEFA. Each saturated NEFA was added at 15 μg/ml in the presence of 30 μg/ml C18:1-*cis*-9 (\*) and each unsaturated NEFA at 30 μg/ml in the presence of 15 μg/ml C16:0 (\*\*). These culture media contained phospholipids. #, NEFA (C18:1-*cis*-9 + C16:0) in the absence of phospholipids

**4. Development of a measure of intraerythrocytic growth of** *P. falciparum* 

Growth-promoting and antimalarial effects on plasmodia can be assessed both quantitatively and qualitatively by directly examining RBC smears from blood or cultures under a microscope; however, this method is tedious and subjective. Numerous novel *in vitro* assays have been introduced that are more objective, faster, more sensitive, and designed to be easier to handle. The most common of these include isotopic, enzymatic, and enzyme-linked immunosorbent assays (ELISA) (Noedl et al., 2003). Isotopic assays rely on the incorporation of radioactive 3H-hypoxanthine into the parasite DNA (Noedl et al., 2003; Webster et al., 1985; Yayon et al., 1983). These methods are relatively reliable and objective, but not sufficiently sensitive, and require the use of hazardous radioactive material. The assays are well suited for screening large numbers of compounds. Parasite lactate dehydrogenase levels have also been used to assess the growth of malarial parasites (Asahi, et al., 2005; Makler and Hinrichs, 1993; Noedl et al., 2003). ELISA-based assays can provide measures of parasite growth by quantifying biomolecules produced during parasite development, such as histidine-rich protein 2 or parasite lactate dehydrogenase, by double-

served as a control. Growth rate was estimated 4 days after inoculation.

**using flow cytometry and SYBR Green I** 

the NEFA mixture in a dose-dependent manner and over a wide range of concentrations, to a level similar to that seen with GFSRPMI (Figure 5). This was followed by 1,2-dipalmitoylsn-glycero-3-phosphocholine (PC-di16:0) and 2-oleoyl-1-palmitoyl-sn-glycero-3-phosphocholine (PC-18:1/16:0). The addition of PC other than PC-di18:1 at certain concentrations also augmented the growth-promoting ability of the NEFA mixture to various extents, ranging from 0–270%.

Fig. 3. Growth of *P. falciparum* with NEFA (C18:1-*cis*-9 plus C16:0) in the presence ( ) or absence ( ) of phospholipids. Growth rate was estimated 4 days after inoculation. #, A paired NEFA of C18:1-*cis*-9 and C16:0 served as a control.

Specific proteins such as bovine and human albumin were shown to be required for *P. falciparum* growth in serum-free culture with lipids, as stated above. Recombinant human albumin could replace serum albumin for sustaining parasite growth in the presence of lipids (Figure 6).

All stages of *P. falciparum* cultured in the formulated CDM containing the best combination of two NEFA, phospholipids, and human, bovine, or recombinant albumin were morphologically indistinguishable from growth in complete medium (Figure 7).

the NEFA mixture in a dose-dependent manner and over a wide range of concentrations, to a level similar to that seen with GFSRPMI (Figure 5). This was followed by 1,2-dipalmitoylsn-glycero-3-phosphocholine (PC-di16:0) and 2-oleoyl-1-palmitoyl-sn-glycero-3-phosphocholine (PC-18:1/16:0). The addition of PC other than PC-di18:1 at certain concentrations also augmented the growth-promoting ability of the NEFA mixture to various extents, ranging

Fig. 3. Growth of *P. falciparum* with NEFA (C18:1-*cis*-9 plus C16:0) in the presence ( ) or absence ( ) of phospholipids. Growth rate was estimated 4 days after inoculation. #, A

Specific proteins such as bovine and human albumin were shown to be required for *P. falciparum* growth in serum-free culture with lipids, as stated above. Recombinant human albumin could replace serum albumin for sustaining parasite growth in the presence of

All stages of *P. falciparum* cultured in the formulated CDM containing the best combination of two NEFA, phospholipids, and human, bovine, or recombinant albumin were

morphologically indistinguishable from growth in complete medium (Figure 7).

paired NEFA of C18:1-*cis*-9 and C16:0 served as a control.

from 0–270%.

lipids (Figure 6).

Fig. 4. Growth of *P. falciparum* in the presence of various combinations of paired NEFA. Each saturated NEFA was added at 15 μg/ml in the presence of 30 μg/ml C18:1-*cis*-9 (\*) and each unsaturated NEFA at 30 μg/ml in the presence of 15 μg/ml C16:0 (\*\*). These culture media contained phospholipids. #, NEFA (C18:1-*cis*-9 + C16:0) in the absence of phospholipids served as a control. Growth rate was estimated 4 days after inoculation.
