Carbohydrate and Amino Acid Analyses of Giardia muris Cysts

ABSTRACT. Intact Giardia muris cysts were subjected to consecutive chloroform/methanol and 2% sodium dodecyl sulfate (SDS) extractions, and to amyloglucosidase treatment. The SDS-insoluble, amyloglucosidase-fast cyst walls (ACW) were further incubated with chymotrypsin, trypsin, papain, or pronase. Low voltage scanning electron microscopy revealed no discernible change in the ultrastructure of the filamentous layer of the cyst wall following any of these treatments. Affinity for cyst wall-specific monoclonal antibody (Meridian Diagnostics, Cincinnati. OH) was also retained after all treatments. Periodic acid-Schiff staining and gas chromatography/mass spectrometry (GC/MS) of intact and treated cyst hydrolysates showed a significant reduction in the amount of glucose associated with the cyst (72 nmoles/10⁶ intact cysts vs 1.9 nmoles/10⁶ ACW) as a result of amyloglucosidase treatment, indicating that glucose is stored with in Giardia as an SDS-insoluble polymer, Galactosamine was identified by GC/MS as the predominant sugar associated with both the ACW and the proteinase treated ACW (42 nmoles/10⁶ ACW). High performance liquid chromatographic analysis of amino acids from intact and treated cyst hydrolysates revealed a marked reduction, but not elimination, of detectable quantities of identifiable amino acid residues (255 nmoles/10⁶ intact cysts vs 6.8 nmoles/10⁶ proteinase treated ACW). These results suggest that the filamentous layer of the cyst wall is primarily a carbohydrate peptide complex.     

High-resolution immunogold localization of Giardia cyst wall antigens using field emission SEM with secondary and backscatter electron imaging

We describe here the ultrastructural localization of Giardia cyst antigens in the filaments associated with the outer portion of intact cysts and on developing cyst wall filaments in encysting trophozoites. Post-embedding immunogold labeling of thin sections of intact Giardia cysts with polyclonal and monoclonal antibodies specific for cyst wall antigens (major protein bands of approximately 29, 75, 88, and 102 KD on Western blots) showed strong labeling of the filamentous cyst wall, whereas no labeling was seen on the membranous portion. High-resolution field emission scanning electron microscopy (FESEM) of Giardia cysts revealed that the cyst wall-specific polyclonal rabbit antisera and monoclonal mouse antibody produced gold labeling of 20-nm filaments in the cyst wall as detected with secondary electron imaging (SEI) and backscatter electron imaging (BEI) at 10 kV, despite coating of the cells with platinum by ion sputtering. FESEM studies of encysting Giardia trophozoites demonstrated that immunostaining with antibodies to cyst wall antigens produced colloidal gold labeling of developing cyst wall filaments on the cell surface; however, the intervening membrane domains were unlabeled. Substitution of normal serum for cyst wall-specific antibodies, or preabsorption of specific antibodies with Giardia cysts, eliminated immunolabeling of the filaments.     

High-resolution electron microscopic evidence for the filamentous structure of the cyst wall in Giardia muris and Giardia duodenalis

High-resolution morphological studies of the cyst wall of Giardia spp. were performed using low-voltage scanning electron microscopy (LVSEM) and transmission electron microscopy (TEM). The cyst wall was composed of membranous and filamentous layers. The membranous layer consisted of an inner and an outer cyst membrane separated by a thin layer of cytoplasm. The filamentous layer contained individual filaments that ranged from 7 to 20 nm in diameter when measured by LVSEM, formed a dense meshwork with branches or interconnections, and were occasionally arranged on the surface in whorled patterns. Cysts of Giardia muris from mice, Giardia duodenalis from dogs, pigs, voles, beavers, muskrats, and humans, and Giardia psittaci from a bird (parakeet), possessed an essentially identical wall composed of filaments. Inducement of excystation in viable Giardia cysts produced a dramatic increase in the interfilament spacing over an entire cyst, but none was observed in heat-killed or chemically fixed control cysts. These results demonstrated that the cyst wall of Giardia spp. was composed of a complex arrangement of filaments, presumably formed during the process of encystment.     

Carbohydrate analysis of Acanthamoeba castellanii

We analyzed biochemically Acanthamoeba castellanii trophozoites, intact cysts and cyst walls belonging to the T4 genotype using gas chromatography combined with mass spectrometry. Cyst walls were prepared by removing intracellular material from cysts by pre-treating them with sodium dodecyl sulphate (SDS) containing dithiothreitol, and then subjecting these to a series of sequential enzymatic digestions using amyloglucosidase, papain, DNase, RNase and proteinase K. The resulting “cyst wall” material was subsequently lyophilized and subjected to glycosyl composition analysis. Transmission electron microscopy confirmed the removal of intracystic material following enzymatic treatment. Our results showed that treated A. castellanii trophozoites, intact cysts and cyst walls contained various sugar moieties, of which a high percentage was galactose and glucose, in addition to small amounts of mannose, and xylose. Linkage analysis revealed several types of glycosidic linkages including the 1,4-linked glucosyl conformation, indicative of cellulose. Inhibitor studies suggested that, beside sugar synthesis, cytoskeletal re-arrangement and mitogen-activated protein kinase-mediated pathways are involved in A. castellanii encystment.     

The activity of a developmentally regulated cysteine proteinase is required for cyst wall formation in the primitive eukaryote Giardia lamblia.

Giardia is an intestinal parasite that belongs to the earliest diverging branch of the eukaryotic lineage of descent. Giardia undergoes adaptation for survival outside the host's intestine by differentiating into infective cysts. Encystation involves the synthesis and transport of cyst wall constituents to the plasma membrane for release and extracellular organization. Nevertheless, little is known about the molecular events related to cyst wall biogenesis in Giardia. Among the components of the cyst wall there are two proteins that we have previously identified and characterized: CWP1 (26 kDa) and CWP2 (39 kDa). Expression of these proteins is coordinately induced, and both concentrated within encystation-specific secretory vesicles before their extracellular polymerization. Although highly similar to each other at the amino terminus, CWP2 includes a COOH-terminal 121-amino acid extension. Here, we show that this extension, rich in basic residues, is cleaved from CWP2 before cyst wall formation by an intracellular cysteine proteinase activity, which is induced during encystation like CWPs. Specific inhibitors prevent release of cyst wall materials, abolishing cyst wall formation. We also report the purification, cloning, and characterization of the encystation-specific cysteine proteinase responsible for the proteolytic processing of CWP2, which is homologue to lysosomal cathepsin C. Encystation-specific cysteine proteinase ESCP possesses unique characteristics compared with cathepsins from higher eukaryotes, such as a transmembrane domain and a short cytoplasmic tail. These features make this enzyme the most divergent cathepsin C identified to date and provide new insights regarding cyst wall formation in Giardia.     

A ten-year survey of Giardia cysts in drinking water supplies of Seoul, the Republic of Korea

To understand the distribution of Giardia cysts in drinking water supplies in Seoul, Korea, we collected water samples quarterly at 6 intakes in the Han River, its largest stream and 6 conventional water treatment plants (WTPs) serving drinking water, from 2000 to 2009. Giardia cysts in each of 10 L water were confirmed in 35.0% of intake water samples and the arithmetic mean was 1.65 cysts/10 L (range 0-35 cysts/10 L). The lowest cyst density was observed at Paldang and Kangbuk intakes, and the pollution level was higher at 4 intakes downstream. It seemed that these 4 intakes were under influence of Wangsuk stream at the end of which cysts were found in all samples with the mean of 140 cysts/10 L. The annual mean number of cysts was 0.21-4.21 cysts/10 L, and the cyst level at the second half of the 10 years was about 1/5 of that at first half on average. The cysts were more frequently found in winter, and their mean density was 3.74 cysts/10 L in winter and 0.80-1.08 cysts/10 L in other seasons. All finished water samples collected at 6 WTPs were negative for Giardia in each of 100 L sample for 10 years and cyst removal by physical process was average 2.9-log. It was concluded that conventional water treatment at 6 WTPs of Seoul appears to remove the cysts effectively under the present level of their source water. Domestic wastewater from the urban region could be an important source of Giardia pollution in the river.     

A comparison of Giardia microti and Spironucleus muris cysts in the vole: an immunocytochemical, light, and electron microscopic study

We have shown that cysts of the genus Spironucleus share many common morphological features with Giardia cysts including: 2-4 nuclei, flagellar axonemes, a distinct cyst wall, and they even display the same immunostaining as Giardia cysts when labeled with antibodies specific for Giardia cyst wall. A direct comparison of Spironucleus muris and Giardia microti cysts have revealed that cysts of S. muris are significantly smaller than cysts of G. miroti. At the ultrastructural level, the cyst walls are similar in fibrillar appearance, but the width of the S. muris cyst wall is significantly less than that of G. microti. The cysts of S. muris also differ from G. microti in that they contain a striated rootlet fiber, flagellar sheath, and numerous glycogen rosettes. Characteristic features of Giardia include the adhesive disc and median body. Although the cysts of Spironucleus and Giardia are similar in appearance, these unique morphological features can be used to distinguish between the 2 protozoa and should be employed in the detection of Giardia cysts in water samples.     

Formation of the Giardia Cyst Wall: Studies on Extracellular Assembly Using Immunogold Labeling and High Resolution Field Emission SEM

Encystment of the intestinal protozoan, Giardia, is a key step in the life cycle that enables this parasite to be transmitted from host to host via either fecal oral, waterborne, or foodborne transmission. The process of encystment was studied by localizing cyst wall specific antigens with immunofluorescence for light microscopy and immunogold staining for field emission scanning electron microscopy. Chronological sampling of Giardia cultures stimulated with endogenous bile permitted identification of an intracellular and extracellular phase in cyst wall formation, a process which required a total of 14-16 h. The intracellular phase lasted for 8-10 h, while the extracellular phase, involved the appearance of cyst wall antigen on the trophozoite membrane, and the assembly of the filamentous layer, a process requiring an additional 4-6 h for completion of mature cysts. The extracellular phase was initiated with the appearance of cyst wall antigen on small protrusions of the trophozoite membrane (-15 nm), which became enlarged with time to caplike structures ranging up to 100 nm in diameter. Caplike structures involved with filament growth were detected over the entire surface of the trophozoite including the adhesive disc and flagella. Encysting cells rounded up, lost attachment to the substratum, and became enclosed in a layer of filaments. Late stages in encystment included a “tailed” cyst in which flagella were not fully retracted into the cyst. Clusters of cysts were seen in which filaments at the surface of one cyst were connected with the surface of adjacent cysts or the “tailed” processes of adjacent cysts, suggesting that the growth of cyst wall filaments may be at the terminal end. In conclusion, the process of encystment has been shown to consist of two morphologically different stages (intracellular and extracellular) which requires 16 h for completion. Further investigation of the extracellular stage with regard to assembly of the filamentous layer of the cyst wall may lead to innovative methods for interfering with production of an intact functional cyst wall, and thereby, regulation of viable Giardia cyst release from the host.     

The ultrastructure of the cyst wall of Giardia lamblia

Giardiasis is the most common human protozoal infection. In their cystic phase, giardias are protected from the environment by a filamentous cyst wall made up of carbohydrates, proteins, and by two outer membranes separated from the plasma membrane of the parasite by a peripheral space. The present transmission electron microscope observations of G. lamblia cysts of human origin suggest that the extracellular peritrophic space originates from the growth, elongation, and fusion of large cytoplasmic vacuoles. As the large clear vacuoles grew in size, flattening against the inner face of the plasma membrane, they formed a single vacuole that surrounded the body of the parasite, eventually forming two outer membranes. In mature Giardia cysts, the original plasma membrane of the trophozoite becomes the outermost membrane of the cyst wall (CM1). The large vacuoles form a second membrane surrounding the cyst (CM2), and also form a third membrane (CM3), that becomes the new plasma membrane of the trophozoite. During excystation CM1 and CM2 attach to each other and fragment, leaving abundant membrane residues in the peritrophic space. Knowledge of the biochemical composition and functional properties of the complex outer membranous system of G. lamblia cysts here described will be of use to understand the survival of Giardia cysts in the environment, a major factor responsible for the high prevalence of giardiasis worldwide.     

Oligonucleotide probes for specific detection of Giardia lamblia cysts by fluorescent in situ hybridization

AIMS: Our study focused on the design of oligonucleotide probes and a suitable hybridization protocol that would allow rapid and specific identification of potentially viable cysts of the waterborne parasite Giardia lamblia. METHODS AND RESULTS: Comparative analysis of ribosomal RNA (rRNA) sequences of Giardia lamblia and a number of closely and more distantly related species identified six regions that appear to be specific for the G. lamblia 16S rRNA. Fluorescently labelled probes targeting these regions were produced and employed in fluorescent in situ hybridization (FISH) experiments. Two of the six probes tested successfully. CONCLUSION: Our study provides the first reported probes for specific FISH detection of G. lamblia. The method depends on sufficient amounts of intact rRNA in the target organism, which is unlikely to be present in nonviable cysts that have been exposed to the environment for a prolonged period. SIGNIFICANCE AND IMPACT OF THE STUDY: Currently, detection of G. lamblia cysts is largely based on immunofluorescence assays (IFA) targeting cyst wall surface antigens. These assays lack specificity and will detect species others than G. lamblia. Further, IFA will detect nonviable cysts and cyst wall fragments that do not pose a public health risk. In contrast, FISH probes allow specific detection and are likely to only detect viable, infectious cysts.     

Improved specificity for Giardia lamblia cyst quantification in wastewater by development of a real-time PCR method

The protozoan parasite Giardia lamblia is the most common cause of waterborne disease outbreaks associated with drinking water in the United States. The conventional method used for the enumeration of Giardia cysts in water is based on immunofluorescence with monoclonal antibodies. It is tedious and time-consuming and has the major drawback to be non-specific for the only species infecting humans, G. lamblia. We have developed a real-time polymerase chain reaction (PCR) method using fluorescent TaqMan technology, which improved the specificity of G. lamblia cyst quantification compared to the immunofluorescence assay (IFA). However, this PCR was not totally specific for G. lamblia species and amplified Giardia ardeae target as well. This method showed a sensitivity of 0.45 cysts per reaction and an efficiency of 95% in purified suspensions. We have then applied this quantification method to raw wastewater, a medium containing numerous debris, particles and PCR inhibitors. The adaptation to these environmental samples was realized by a screening of three cyst purification methods and six DNA extraction protocols. Real-time quantification was accomplished by the simultaneous amplification of unknown samples and a tenfold serial dilution of purified G. lamblia cysts. For all samples, the concentrations observed with TaqMan PCR method were compared to the IFA values. Giardia spp. cysts were detected in all non-spiked raw wastewater samples with IFA procedure and the concentrations of Giardia spp. cysts used for the comparison between the two methods ranged between 3.3x10(2)/l and 4.3x10(3)/l. The highest TaqMan PCR/IFA ratios were observed when Percoll/sucrose flotation was combined with DNA extraction protocol optimized for cyst wall lysis, impurities adsorption on a resin, and double step protein digestion and column purification. The concentrations observed with this TaqMan PCR method ranged from 2.5x10(2) to 2.4x10(3) G. lamblia cysts/l and only one sample resulted in a no amplification curve. Thus, we developed a TaqMan PCR method increasing the rapidity and specificity of G. lamblia cyst quantification. The combination of Percoll/sucrose flotation and DNA extraction optimized protocol before TaqMan assay has provided a good indication of the G. lamblia contamination level in raw sewage samples.     

UDP-N-acetylglucosamine 4'-epimerase from the intestinal protozoan Giardia intestinalis lacks UDP-glucose 4'-epimerase activity

The protozoan parasite Giardia intestinalis has a simple life cycle consisting of an intestinal trophozoite stage and an environmentally resistant cyst stage. The cyst is formed when a trophozoite encases itself within an external filamentous covering, the cyst wall, which is crucial to the cyst's survival outside of the host. The filaments in the cyst wall consist mainly of a beta (1-3) polymer of N-acetylgalactosamine. Its precursor, UDP-N-acetylgalactosamine, is synthesized from fructose 6-phosphate by a pathway of five inducible enzymes. The fifth, UDP-N-acetylglucosamine 4'-epimerase, epimerizes UDP-N-acetylglucosamine to UDP-N-acetylgalactosamine reversibly. The epimerase of G. intestinalis lacks UDP-glucose/UDP-galactose 4'-epimerase activity and shows characteristic amino acyl residues to allow binding of only the larger UDP-N-acetylhexosamines. While the Giardia epimerase catalyzes the reversible epimerization of UDP-N-acetylglucosamine to UDP-N-acetylgalactosamine, the reverse reaction apparently is favored. The enzyme has a higher Vmax and a smaller Km in this direction. Therefore, an excess of UDP-N-acetylglucosamine is required to drive the reaction towards the synthesis of UDP-N-acetylgalactosamine, when it is needed for cyst wall formation. This forms the ultimate regulatory step in cyst wall biosynthesis.     

The detection of Giardia muris and Giardia lamblia cysts by immunofluorescence in animal tissues and fecal samples subjected to cycles of freezing and thawing

The effects of freezing and thawing on the detection of selected Giardia spp. cysts were investigated using immunofluorescence, bright field microscopy, and low voltage scanning electron microscopy (SEM). Giardia muris cysts were obtained from either animal carcasses, fecal pellets, or isolated cyst preparations, whereas Giardia lamblia cysts were isolated from fecal samples. These samples were stained using an immunofluorescence technique after 1-3 freezing (-16 C) and thawing (20 C) cycles. Cysts were detected successfully by immunofluorescence in all samples. However, in those samples subjected to freeze-thawing, the cyst walls often became distorted and then were not detectable by bright field microscopy. Low voltage SEM demonstrated that the filaments in the distorted cyst wall underwent rearrangements of interfilament spacing. Quantitation of cyst recovery after freezing and thawing demonstrated that a substantial loss occurred after 1 cycle of alternating temperature when low concentrations of cysts were used, but not with high concentrations of cysts. Cyst recovery, after 3 freezing and thawing cycles, was dramatically lowered irrespective of the initial cyst concentration. These results demonstrated that immunofluorescence was an effective technique for the detection of Giardia spp. cysts in frozen samples and would suggest that freezing and thawing of fecal samples could prevent the detection of cysts when only bright field microscopy was employed.     

The Giardia intestinalis filamentous cyst wall contains a novel beta(1-3)-N-acetyl-D-galactosamine polymer: a structural and conformational study

Assembly of a protective cyst wall by Giardia is essential for the survival of the parasite outside the host intestine and for transmission among susceptible hosts. The structure of the G. intestinalis filamentous cyst wall was studied by chemical methods, mass spectrometry, and (1)H nuclear magnetic resonance spectroscopy. Isolated cyst wall material contains carbohydrate and protein in a ratio of 3:2 (w/w), and the carbohydrate moiety is composed of a beta(1-3)-N-acetyl-D-galactopyranosamine homopolymer. Conformational analysis by molecular dynamics and persistence length calculations of GalNAc oligomers in solution demonstrated a flexible structure consisting of left- and right-handed helical elements. It is most likely that in the solid state, the polysaccharide forms ordered helices or possibly multiple helical structures having strong interchain interactions. The highly insoluble nature of the Giardia cyst wall must be due to these strong interchain interactions and, probably, a strong association between the carbohydrate and the protein moiety.     

The chemical composition of the cyst wall of the potato cyst-nematode, Heterodera rostochiensis

1. Cyst walls of the potato cyst-nematode (Heterodera rostochiensis Woll.) were isolated by sieving a suspension of crushed cysts. About 12mg. of dried cyst walls was obtained from 1000 cysts. 2. The cyst walls contained mainly protein (72%, calculated from nitrogen content). On acid hydrolysis about 77% of the cyst wall went into solution. Of 19 amino acids present, proline, glycine, and alanine were the most abundant, and made up about 50% by weight of the total amino acids. The amino acid composition suggested that collagen-like proteins predominated in the cyst wall and larval cuticle. 3. A small amount of glucosamine (1.5%) was present in the hydrolysates, but chitin was not detected in the cyst walls. 4. Other components of the cyst walls were lipid (2%), carbohydrate (0.5%) and a small amount of inorganic matter (ash, 5%). Polyphenols (2% by wt. of the cyst walls) occurred in the acid hydrolysates. The dark pigments of the cyst wall were not indole-containing melanins.     

Detection of viable Giardia cysts by amplification of heat shock-induced mRNA.

Primers obtained from gene sequences coding for heat shock proteins (HSP) were used to specifically detect enteric protozoans of the genus Giardia. The HSP primers amplified Giardia DNA or the corresponding RNA sequences obtained from lysed cysts and gave a 163-bp product. Since the presence of the product did not indicate whether the cysts were viable, these amplifications are a presence/absence test only. In contrast, amplification of heat shock-induced mRNA utilizing the same HSP primers was indicative of viable Giardia cysts. The limit of sensitivity of the presence/absence test was 1 cyst, whereas for the viability test it was 10 cysts. Thus, viable Giardia cysts can be rapidly and specifically detected with great sensitivity through the use of PCR amplifications.     

Facts about the artifacts in the measurement of oxidative DNA base damage by gas chromatography-mass spectrometry

Recently, several papers reported an artifactual formation of a number of modified bases from intact DNA bases during derivatization of DNA hydrolysates to be analyzed by gas chromatography-mass spectrometry (GC/MS). These reports dealt with 8-hydroxyguanine (8-OH-Gua), 5-hydroxycytosine (5-OH-Cyt), 8-hydroxyadenine (8-OH-Ade), 5-hydroxymethyluracil (5-OHMeUra) and 5-formyluracil that represent only a small percentage of the 20 or so modified DNA bases that can be analyzed by GC/MS. Removal of intact DNA bases by prepurification of calf thymus DNA hydrolysates using HPLC was shown to prevent artifactual formation of these modified bases during derivatization. It needs to be emphasized that the procedures for hydrolysis of DNA and derivatization of DNA hydrolysates used in these papers substantially differed from the established procedures previously described. Furthermore, a large number of relevant papers reporting the levels of these modified bases in DNA of various sources have been ignored. Interestingly, the levels of modified bases reported in the literature were not as high as those reported prior to prepurification. Most values for the level of 5-OH-Cyt were even lower than the level measured after prepurification. Levels of 8-OH-Ade were quite close to, or even the same as, or smaller than the level reported after prepurification. The same holds true for 5-OHMeUra and 8-OH-Gua. All these facts raise the question of the validity of the claims about the measurement of these modified DNA bases by GC/MS.

A recent paper reported a complete destruction of 2, 6-diamino-4-hydroxy-5-formamidopyrimidine (FapyGua) and 4,6-diamino-5-formamidopyrimidine (FapyAde) by formic acid under the conditions of DNA hydrolysis prior to GC/MS. The complete destruction of FapyGua and FapyAde by formic acid is in disagreement with the data on these compounds in the literature. These two compounds were measured by GC/MS following formic acid hydrolysis for many years in our laboratory and by other researchers with no difficulties. These facts clearly raise the question of the validity of the claims made about the previous measurements of these compounds by GC/MS.     

Facts about the artifacts in the measurement of oxidative DNA base damage by gas chromatography-mass spectrometry

Recently, several papers reported an artifactual formation of a number of modified bases from intact DNA bases during derivatization of DNA hydrolysates to be analyzed by gas chromatography-mass spectrometry (GC/MS). These reports dealt with 8-hydroxyguanine (8-OH-Gua), 5-hydroxycytosine (5-OH-Cyt), 8-hydroxyadenine (8-OH-Ade), 5-hydroxymethyluracil (5-OHMeUra) and 5-formyluracil that represent only a small percentage of the 20 or so modified DNA bases that can be analyzed by GC/MS. Removal of intact DNA bases by prepurification of calf thymus DNA hydrolysates using HPLC was shown to prevent artifactual formation of these modified bases during derivatization. It needs to be emphasized that the procedures for hydrolysis of DNA and derivatization of DNA hydrolysates used in these papers substantially differed from the established procedures previously described. Furthermore, a large number of relevant papers reporting the levels of these modified bases in DNA of various sources have been ignored. Interestingly, the levels of modified bases reported in the literature were not as high as those reported prior to prepurification. Most values for the level of 5-OH-Cyt were even lower than the level measured after prepurification. Levels of 8-OH-Ade were quite close to, or even the same as, or smaller than the level reported after prepurification. The same holds true for 5-OHMeUra and 8-OH-Gua. All these facts raise the question of the validity of the claims about the measurement of these modified DNA bases by GC/MS.

A recent paper reported a complete destruction of 2, 6-diamino-4-hydroxy-5-formamidopyrimidine (FapyGua) and 4,6-diamino-5-formamidopyrimidine (FapyAde) by formic acid under the conditions of DNA hydrolysis prior to GC/MS. The complete destruction of FapyGua and FapyAde by formic acid is in disagreement with the data on these compounds in the literature. These two compounds were measured by GC/MS following formic acid hydrolysis for many years in our laboratory and by other researchers with no difficulties. These facts clearly raise the question of the validity of the claims made about the previous measurements of these compounds by GC/MS.