Abstract In addition to plasma membrane, Giardia lamblia contains numerous membrane-enveloped, primitive organelles, which house a variety of metabolic pro- cesses. It has been proposed earlier that this intes- tinal pathogen lacks the ability to synthesize the majority of its own lipids de novo and depends on supplies from outside sources. Therefore, the ques- tions as to how this ancient eukaryote utilizes ex- ogenous lipids and synthesizes membranes and organelles are extremely important. Does this para- site depend predominantly on remodeling path- ways, in which exogenous phospholipids undergo fatty acid and headgroup replacement reactions to generate new phospholipids? To answer this, and to better understand the overall pathway, we carried out a complete lipidomic analysis using electro- spray ionization quadrupole time-of-flight mass spectrometry (ESI-QTOF-MS). The results suggest that Giardia has the ability to generate new phos- pholipids de novo, most likely via the remodeling pathways. Among the newly synthesized lipids, phosphatidylglycerol (PG) is the major phospho- lipid followed by phosphatidylethanolamine. Gas chromatography-mass spectrometry (GC-MS) ana- lyses indicated that Giardia also has the ability to remodel fatty acids by chain elongation and desat- uration reactions. Thus, mass spectrometric analy- ses provided valuable information about lipid biosynthesis by Giardia and opened the possibility of investigating in greater detail the uptake and uti- lization of exogenous lipids for the synthesis of membranes and organelles. 7.1 Introduction Giardia trophozoites colonize the luminal surface of the human small intestine, below the bile duct and are exposed to dietary fatty acids and lipids, intestinal immunoglobulins, digestive enzymes, and newly syn- thesized bile acids (Gillin et al., 1986, 1987; Stevens et al., 1997; Subramanian et al., 2000). Because bile acid concentrations are particularly high in this site, it can be anticipated that bile and biliary lipids must regulate the growth and differentiation of G. lamblia (Das et al., 2002). Hegner and Eskridge (1938) pro- posed that bile might favor the growth of Giardia spe- cies in vivo. Furthermore, it was shown that bile from a number of mammals stimulated the in vitro growth of trophozoites (Farthing et al., 1983, 1985; Keister, 1983). Gillin et al. (1986) reported that the growth of G. lamblia on serum-free medium supplemented with phosphatidylcholine (PC), cholesterol, and a mixture of six bile salts can support the growth of the parasite in culture. Moreover, Das et al. (1988) found that con- jugated bile acids, when present above their critical micellar concentrations, protected trophozoites from lysis by free fatty acids that were produced during the digestion of the host’s dietary triglycerides. In addi- tion, the presence of bile salts in the growth medium has been proposed as being involved in transporting exogenous lipids to the trophozoite by forming mixed micelles (Das et al., 1997). Initial studies on the lipid biochemistry of Giardia concluded that this unicellular parasite lacked de novo lipid synthesis capacity and thus must obtain them from its microenvironment. This conclusion was built upon the observation that Giardia trophozoites failed 7 Mass Spectrometric Analysis of Phospholipids and Fatty Acids in Giardia lamblia Mayte Yichoy, Ernesto S. Nakayasu, Atasi Chatterjee, Stephen B. Aley, Igor C. Almeida and Siddhartha Das