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FTIR microspectroscopy reveals fatty acid-induced biochemical changes in C. elegans
Accepted Manuscript Title: FTIR microspectroscopy reveals fatty acid-induced biochemical changes in C. elegans Authors: Amal Bouyanfif, Sumedha Liyanage, Eric Hequet, Naima Moustaid-Moussa, Noureddine Abidi PII: DOI: Reference:
S0924-2031(19)30014-1 https://doi.org/10.1016/j.vibspec.2019.03.002 VIBSPE 2907
To appear in:
VIBSPE
Received date: Revised date: Accepted date:
15 January 2019 4 March 2019 22 March 2019
Please cite this article as: Bouyanfif A, Liyanage S, Hequet E, MoustaidMoussa N, Abidi N, FTIR microspectroscopy reveals fatty acid-induced biochemical changes in C. elegans, Vibrational Spectroscopy (2019), https://doi.org/10.1016/j.vibspec.2019.03.002 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
FTIR microspectroscopy reveals fatty acid-induced biochemical changes in C. elegans Amal Bouyanfif a,b,c, Sumedha Liyanagea, Eric Hequeta, Naima Moustaid-Moussaa,b,c, Noureddine Abidia,c* Fiber and Biopolymer Research Institute, Texas Tech University Lubbock, TX, USA
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Department of Nutritional Sciences, Texas Tech University Lubbock, TX, USA
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Obesity Research Cluster, Texas Tech University, Lubbock, TX, USA
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* Corresponding author: Noureddine Abidi, Ph.D., 1001 East Loop 289, Lubbock, Texas 79403,
USA.
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Email: [email protected]
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Phone: 1-806-834-1221, Fax: 1-806-742-5343
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Abstract
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Fourier transform infrared microspectroscopy (FTIR) is a promising method for the
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analysis of biological samples. Recent studies reported that FTIR imaging allows determination of the distribution of several biomolecules in a sample with no staining or extraction. In this study,
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FTIR was used to monitor biochemical changes in C. elegans nematodes cultured in nematode maintenance media (CeMM) without supplementation and with supplementation with either a long
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chain polyunsatured omega 3 fatty acid, eicosapentaenoic acid (EPA) or a saturated fatty acid, palmitic acid (PA) at 100 M. EPA is an omega 3 fatty acid with documented health benefits while PA is generally consumed in diets. Worms were placed on BaF2 slides, and FTIR spectra were collected from single worms in transmission mode using a focal plane array detector. Principal
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component analysis grouped the FTIR spectra into three clusters corresponding to spectra of worms cultured with no supplementation, worms cultured with supplementation with EPA, and worms cultured with supplementation with PA. The major differences between the FTIR spectra
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reside in the vibrations corresponding to unsaturated fatty acids (3008 cm-1), lipids (2928, 2848, and 1744 cm-1), and proteins (1680, 1648, and 1515 cm-1). This indicates that supplementation with EPA or PA leads to biochemical alterations related to unsaturated fatty acids, lipids, and proteins. Furthermore, supplementing mutant strains (tub-1 and fat-3) CeMM with PA resulted in the appearance of the vibration 3008 cm-1, an increase in the intensity of the vibration 1744 cm-1,
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and a new vibration at 1632 cm-1, which is assigned to the amide I of -pleated sheet component
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of proteins, in the spectra of tub-1 and fat-3 mutant strains. The results illustrated the potential use
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of FTIR alongside other techniques such as gas chromatography and staining techniques to
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investigate lipid metabolism and fat accumulation as well as induced changes in protein structures.
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Abbreviations
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CeMM, C. elegans maintenance media; EPA, eicosapentaenoic acid; PA, palmitic acid; FPA, focal plane array; FTIR, Fourier transform infrared; NGM, nematode growth medium; PCA, principal
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component analysis; -3 PUFA, omega-3 polyunsaturated fatty acids; WT, wild-type; CGC,
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Caenorhabditis Genetics Center.
FTIR
microspectroscopy,
imaging,
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Keywords:
eicosapentaenoic acid
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olefinic,
palmitic
acid,
C.
elegans,
Introduction Caenorhabditis elegans (C. elegans) has attracted increasing attention and has become a major model organism in different research fields, such as biology (Witting and Schmitt-Kopplin
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2016), obesity, fat metabolism (Zheng and Greenway 2012; Ashrafi 2007; Ashrafi et al. 2003), drug discovery (O'Reilly et al. 2014; Artal-Sanz et al. 2006), biomedical toxicology, environmental toxicology (Leung et al. 2008), and nanoparticle toxicity (Gonzalez-Moragas et al. 2017; Gonzalez-Moragas et al. 2015; Meyer et al. 2010). The interest in using this nematode as a model organism instead of rodent models (e.g., mice) is attributed to several advantages, such as small
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size, short lifespan, quick turnover, reduced experimental timeline, complete genetic information,
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easy culture conditions, easy maintenance in the laboratory, low cost, possibility of long-term
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order animals (Bouyanfif et al. in press).
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storage as a frozen stock, no prior approval is required compared to studies on rodents and higher
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C. elegans has been used to investigate mechanisms that regulate lipid storage and
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metabolism (Watts 2009; Watts and Browse 2002; Hellerer et al. 2007; Folick et al. 2011; Ashrafi 2007; Witting and Schmitt-Kopplin 2016; Chen et al. 2016). In mammals, lipids are primarily
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stored as triglycerides in adipose tissue. In C. elegans, lipids are also stored primarily as
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triacylglycerols but in the gut granules and hypodermal cells (Ashrafi 2007; Ashrafi et al. 2003). The fat content in C. elegans has been determined by extracting total lipids from the whole worms, phospholipids
and
neutral
lipid
moieties,
and
then
performing
gas
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fractionating
chromatography/mass spectrometry analyses (Ashrafi 2007; Kniazeva et al. 2003; Satouchi et al. 1993; Watts and Browse 2002). The triacylglyceride content in the nematode is 40 to 55% of the total lipids, while phospholipids are composed of ~55% ethanolamine glycerophospholipid, ~32% choline
glycerophospholipid,
and
approximately 3
8%
sphingomyelin
(Ashrafi
2006).
Polyunsaturated fatty acids (PUFAs) play an important role, not only as structural components of membranes, but also as precursors to critical signaling molecules and lipid mediators (Kaja Reisner 2011; Watts and Browse 2002; Watts et al. 2003).
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Lipid metabolism pathways in C. elegans resemble those in mammals. However, a few exceptions exist. (1) unlike mammals, C. elegans possesses the fatty acid enzymes required to generate essential fatty acids, (2) C. elegans is not able to produce PUFAs longer than C20. Therefore, the -6 and -3 PUFAs biosynthesis pathways end with arachidonic acid (20:4n6) and eicosapentaenoic acid (20:5n3), respectively (Watts and Browse 2002). As indicated above, C.
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elegans stores fat primarily as droplets in their intestinal and skin-like epidermal cells (Ashrafi
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2007). Lipid staining techniques using Nile Red, Sudan Black, Oil Red O, and fluorescently
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labeled fatty acids are commonly used to assess changes in fat storage due to mutations or RNAi-
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mediated inactivation of genes encoding various lipid biosynthesis pathways (Ashrafi et al. 2003;
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Mukhopadhyay et al. 2005). This technique has some limitations, such as variability in dye
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labeling specificity and efficiency, which may lead to inconsistencies in lipid quantification (O'Rourke et al. 2009; Watts 2009; Soukas et al. 2009). Other tools used to visualize lipids include
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coherent anti-stokes Raman scattering, which allows visualization of lipids stored in both C. elegans epidermal and gut granules without the use of invasive methods (Hellerer et al. 2007).
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Tserevelakis et al. utilized label-free imaging using third-harmonic generation microscopy to study
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lipid deposition in C. elegans (Tserevelakis et al. 2014). Folick et al. combined anti-stokes Raman scattering and simulated Raman scattering microscopy to develop new methods to visualize the localization and regulation of lipids in C. elegans (Folick et al. 2011). We believe that metabolism of saturated and unsaturated fatty acids by C. elegans could result in biochemical changes that
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could be detected on intact worms (no chemical extraction no chemical staining) using Fourier transform infrared microspectroscopy (FTIR). FTIR is a powerful analytical technique to investigate the chemical composition of a
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sample. When combined with a microscope and focal plane array detectors (FPA), this technique allows imaging of a relatively large sample area, providing both spectroscopic information (chemical identification) and distribution of functional groups in the sample. Recently, we reviewed applications of FTIR to study the biochemical changes in C. elegans (Bouyanfif et al. 2018). FTIR imaging could be used to investigate intact nematodes. These applications include
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nematode identification (Ami et al. 2004; Ami et al. 2012), biochemical composition (Sheng et al.
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2016; Bouyanfif et al. 2017), and toxicity assessment of nanoparticles and drugs (Zanni et al. 2012;
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Diomede et al. 2010). We used FTIR imaging to illustrate changes in intact C. elegans (wild-type
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(N2) and mutant strains fat-3 and tub-1) when C. elegans maintenance media (CeMM) was
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supplemented with a polyunsaturated fatty acid, eicosapentaenoic acid (EPA) (Bouyanfif et al.
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2017). We used principal component analysis to discriminate WT from mutant strains (tub-1 and fat-3) cultured with or without EPA supplementation. This discrimination was mainly due to
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differences in unsaturated fatty acids, lipids, and protein profiles (Bouyanfif et al. 2017). These conclusions were further supported by performing digital subtraction of the FTIR spectra of mutant
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strains from those of WT. These studies validated the FTIR technique as an important tool to detect changes in biochemical compositions and lipid metabolism (Bouyanfif et al. 2017). This work has
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laid the foundation to explore further the use of FTIR imaging to dissect biochemical changes in C. elegans WT and mutant strains (tub-1 and fat-3) cultured in CeMM supplemented with EPA or palmitic acid (PA). We compared these two fatty acids as one of them, EPA is an omega 3 fatty acid with documented health benefits including metabolic diseases; while PA is a saturated fatty
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acid commonly consumed in the diet and also endogenously produced. In this study, FTIR spectra were acquired in transmission mode on a single intact worm with no special preparation. This stain-free analytical technique is able to provide information related to chemical functional groups
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that provide insight into biochemical changes in C. elegans fed saturated or unsaturated fatty acids. Materials and Methods
C. elegans strains, culture, and maintenance: Hermaphrodite adult WT and mutant strains tub-1 (nr2044) and fat-3 (wa22) were acquired from the Caenorhabditis Genetics Center (CGC, University of Minnesota, Minneapolis, MN, USA). WT (N2) is able to synthetize 20:4n6 and
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20:5n3 using saturated and monounsaturated fatty acids from bacteria as precursors (Watts et al.
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2003; Hutzell and Krusberg 1982). By contrast, the mutant strain fat-3 lacks 6 desaturase activity
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and fails to produce any of the common C20 PUFAs that are essential in regulating membrane
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structure, dynamics, and permeability (Watts et al. 2003). In the mutant strain tub-1, the functional
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loss of tubby ortholog called tub-1/F10B5.5 in C. elegans leads to the accumulation of
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triglycerides, the major form of stored fat (Ashrafi et al. 2003; Mukhopadhyay et al. 2005; Mak et al. 2006).
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Nematodes were first cultured in the nematode growth media (NGM) plates seeded with E. coli
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OP50 as the main food source (Stiernagle 2006b, a). Plates containing a large quantity of eggs and gravid adults were bleached, and eggs were left in sterile M9 Buffer to hatch overnight. The
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following day, starved L1 animals were transferred to CeMM (Cell Guidance Systems, Babraham, Cambridge, UK) containing 20 µg/mL of kanamycin sulfate (Fisher Scientific, Pittsburgh, PA, USA) and 200 µg/mL of streptomycin sulfate (Fisher Scientific, Pittsburgh, PA, USA) (Szewczyk et al. 2003). C. elegans strains were cultured in CeMM at 20°C for multiple generations to adapt to the media before being used in the experiments. 6
Fatty acids: Eicosapentaenoic acid (EPA) and palmitic acid (PA) were acquired from Nu-Check Prep (Elysian, MN, USA). EPA was supplied as a pure -3 ethyl esters. It was stored at -80°C, and exposure to heat, light, and oxygen was minimized to prevent oxidative degradation. PA was
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supplied as pure palmitic acid. EPA and PA solutions in ethanol were prepared and CeMM was supplemented at a final concentration of 100 µM; feeding was performed for 72 h on worm cultures of mixed ages.
FTIR microspectroscopy: Individual hermaphrodite adult nematodes were washed with distilled water and deposited on FTIR transparent BaF2 slides (PerkinElmer, MA, USA). Slides containing
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worms were then dried in a vacuum desiccator for 1 h. FTIR spectra were collected in the
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transmission mode between 4000 and 1000 cm-1 using a Spectrum 400 FTIR equipped with a
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Spotlight 400 microscope (PerkinElmer, MA, USA) accessory with a liquid nitrogen cooled
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128x128 Mercury-Cadmium-Telluride (MCT) focal plane array detector. To improve the signalto-noise ratio, 128 co-added spectra were collected from each pixel (6.25 x 6.25 µm) with a spectral
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resolution of 16 cm-1. Prior to sample measurements, a background spectrum from an empty area
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of the BaF2 slide was automatically subtracted from the spectra of the sample. The BaF2 slide does
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not show any absorption in the range of 4000 – 1000 cm-1. A visual image was first acquired from the sample (Figure 1-A). Figures 1-B and 1-C show
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FTIR image and the corresponding spectra extracted from the positions marked in the image. Point
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mode imaging was used in this study, which allowed us to acquire a large number of spectra in a short period of time (approximately 20 min). Spectra were acquired from a single worm along the length (4 to 6 worms for each treatment) with an aperture size of 15 x 15 µm. Four to eight data points were acquired from each worm (separated by approximately 50 m), and all the spectra were the result of 128 co-added scans. The acquired spectra were baseline corrected and 7
normalized with respect to the total absorbance over the entire range from 4000 to 1000 cm-1 using the Spectrum 10™ software (PerkinElmer, MA, USA). Principal component analysis was
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performed using the Unscrambler® X 10.3 software (CAMO Software AS, Norway).
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Figure 1: Image of the whole intact C. elegans illustrating the location from which spectra were
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recorded.
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Results and Discussion
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In previous work, we investigated the use of FTIR imaging to illustrate changes occurring in WT (N2), tub-1, and fat-3 C. elegans cultured with and without EPA supplementation at 25 or
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100 M (Bouyanfif et al. 2017). The results indicated that mutant strains have altered lipid
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compositions and that the major component of the middle part of the worms are fatty acyl groups. In this study, we investigated the chemical changes induced when the C. elegans diet was
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supplemented with and without EPA and PA at 100 M. Representative spectra acquired from WT (N2) cultured in CeMM alone and from WT (N2) cultured in CeMM either with EPA or PA supplementation at 100 M are exhibited in Figure 2. It is interesting to note that the only difference between these spectra resides in the intensities of the vibrations at 2928 cm-1 (asymmetric –CH2 stretch), at 2848 cm-1 (symmetric –CH2 stretch), and at 1744 cm-1 (C=O 9
stretch). These vibrations are associated with the presence of lipids, fatty acids, triglycerides, and phospholipids (Bouyanfif et al. 2017). Supplementing CeMM with PA resulted in a high intensity of these vibrations but did not affect other vibrations in the 1700 – 1000 cm-1 region. In this region,
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the vibration at 1648 cm-1 was assigned to the C=O stretch of amide I, mainly the -helix components of proteins (Bouyanfif et al. 2017; Hobro and Lendl 2011). The vibration at 1548 cm1
was attributed to amide II, which could originate from N-H bending and C-N stretching of the
protein amide group. The vibration at 1456 cm-1 was assigned to CH2 bending and deformation of methylene of lipids, proteins, or cholesterol esters. The vibration at 1392 cm-1 was assigned to the
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COO- stretch of carbohydrates, fatty acids, or amino acid side chains. The vibrations at 1232 and
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1084 cm-1 originated from PO2- antisymmetric and symmetric stretching of phosphodiesters,
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respectively. The vibration at approximately 1155 cm-1 was assigned to C-O stretching and
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possibly originated from glycogen (Vongsvivut et al. 2013). It is important to note the presence of the vibration at 3008 cm-1, which is attributed to -
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HC=CH- stretching (San-Blas et al. 2011; Holman et al. 2008). This vibration is due to unsaturated
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fatty acids and exists in the spectra acquired from WT (N2) with no supplementation. This
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vibration represents a very important feature in the FTIR spectra of WT (N2) C. elegans compared to the spectra acquired from tub-1 and fat-3 mutant strains. This result confirms previously
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reported results that indicated that WT (N2) C. elegans is capable of synthesizing a wide range of saturated, monounsaturated, and polyunsaturated fatty acids (Kniazeva et al. 2003; Kniazeva et al.
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2004; Satouchi et al. 1993; Watts and Browse 2002; Hutzell and Krusberg 1982). Previous fatty acid analyses of C. elegans and C. briggsae reported that lipids accounted for 19.1% of the dry weight of C. elegans (Hutzell and Krusberg 1982). The authors indicated that the major portion of the fatty acids contained 18 or 20 carbons, with unsaturated fatty acids making 70% of the total
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fatty acids (the principal fatty acid fraction was 18:1) (Hutzell and Krusberg 1982). Supplementation with EPA did not result in an increased intensity of the vibration at 3008 cm-1
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(slightly reduced intensity was noticed).
Figure 2: Representative FTIR spectra acquired from WT (N2) C. elegans cultured without and
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with either EPA or PA supplementation at 100 M. Several FTIR spectra were acquired, and principal component analysis (PCA) was
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performed to identify distinct groups of spectra that exhibit spectral similarities. PCA is a widely adopted multivariate statistical analysis used to reduce the dimensionality from a large number of
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interconnected variables (wavenumbers in the case of FTIR) to a few uncorrelated variables
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(Kemsley 1996; Chen et al. 1998). The reduction in dimensionality is achieved by means of a linear transformation to a new set of variables termed principal component scores. Thus, the sources of variability in the data are concentrated into the first 2 or 3 principal components. Plots of PC-1 against PC-2 or PC-3 revealed clustering in the FTIR spectra. We used PCA to analyze the FTIR spectra acquired from C. elegans WT and mutant strains tub-1 and fat-3 to detect diet 11
and genotype-dependent biochemical changes with and without EPA supplementation at 25 and 100 M (Bouyanfif et al. 2017). Figure 3 shows PC-1 and PC-2 scores of the FTIR spectra acquired from WT (N2) cultured with no supplementation and either with EPA or PA
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supplementation at 100 M. Three groups of spectra can be distinguished as follows: the first group corresponds to WT (N2) with no supplementation, the second group corresponds to WT (N2) with supplementation with EPA at 100 M, and the third group corresponds to WT (N2) with supplementation with PA at 100 M. It is worth pointing out the large variability in the spectra of WT (N2) cultured in CeMM with EPA or PA supplementation compared to the spectra acquired
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from WT (N2) cultured only in CeMM with no supplementation. The separation of the FTIR
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spectra into different groups indicates that biochemical changes are induced in worms when their
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diet is supplemented with saturated and unsaturated fatty acids.
Figure 3: PCA of the FTIR spectra acquired from WT (N2) cultured without and with either EPA or PA supplementation at 100 M. Each data point represents a spectrum obtained with 128 coadded scans. 12
The analysis of loading variables (or factors) as a function of wavenumbers can help identify the functional groups that are behind the grouping of the original spectra (Alonso-Simon et al. 2004). Plots of the first three component loadings as a function of wavenumbers are shown
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in Figure 4. PC-1, PC-2 and PC-3 account for 46%, 33% and 8% of the spectral variation, respectively. The PC-1 loading plot shows major peaks at 1744 cm-1 (C=O stretching of esters), 1632 cm-1 (amide I band of the -pleated sheet component of proteins, collagen proline residues (Hobro and Lendl 2011)). The PC-2 loading plot shows major peaks at the following vibrations: 2928 and 2848 cm-1 (assigned to asymmetric and symmetric stretching of the acyl –CH2 groups),
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1744 cm-1 and 1680 cm-1 (assigned to amide I band component originating from anti-parallel
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pleated sheets and -turns of proteins), and approximately 1550 cm-1 (assigned to amide II, N-H
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bending and C-N stretching of protein amide groups with some contribution from amino acid side
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chains such as arginine, aspartate, glutamate and tyrosine (Hobro and Lendl 2011)). The PC-3 loading plot shows major peaks at 2928 cm-1, 2848 cm-1, 1680 cm-1, 1632 cm-1, 1515 cm-1
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(assigned to tyrosine ring vibration), ~1232 cm-1 (PO2- antisymmetric stretching of
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phosphodiesters (Hobro and Lendl 2011)), and 1084 cm-1 (PO2- symmetric stretching of the
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phosphodiester backbone of nucleic acids and phospholipids).
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Figure 4: Loadings corresponding to PCA presented in Figure 3 as a function of wavenumbers for
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WT (N2) C. elegans cultured without and with either EPA or PA supplementation at 100 M.
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Representative spectra acquired from mutant strain tub-1 are shown in Figure 5. As
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reported in our previous work, the vibration at 3008 cm-1 (=CH– olefinic stretching), which
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generally arises from the presence of unsaturated fatty acids, is present only as a very small shoulder in the FTIR spectra of tub-1 cultured with no supplementation (Bouyanfif et al. 2017).
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The vibrations associated with triglycerides (2928, 2848, and 1744 cm-1) are present with high intensities. For this mutant strain, it was reported that the deletion of tubby ortholog (tub-1) leads
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to an accumulation of triglycerides, which are the major form of stored fat in C. elegans (Ashrafi et al. 2003; Mukhopadhyay et al. 2005). Supplementation of CeMM with 100 M EPA leads to the appearance of the vibration at 3008 cm-1 with only a minor increase in the intensity of the vibration at 1744 cm-1 (triglycerides, phospholipids, cholesterol esters, or fatty acids).
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Supplementing the growth media with palmitic acid (saturated fatty acid) not only resulted in the appearance of the vibration at 3008 cm-1 but also in a significant increase in the intensity of the vibration at 1744 cm-1. Palmitic acid can be integrated into triacylglycerides or phospholipids
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or can be modified by fatty acid elongases and desaturases to form a variety of long-chain polyunsaturated fatty acids (Watts 2009; Watts and Browse 2002). Furthermore, it is of particular interest to note the appearance of the vibration at 1632 cm-1, assigned to the carbonyl stretching in amide I of the -pleated sheet component of proteins (Hobro and Lendl 2011). Supplementing the worm diet with saturated palmitic acid resulted in the shift from the vibration at 1648 cm-1 assigned
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to amide I of the -helix component to 1632 cm-1 assigned to amide I of the -sheet components.
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However, this was not the case for the WT (N2) strain. Furthermore, supplementation with EPA
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and PA resulted in the appearance of a vibration at approximately 1520 cm-1, which has been
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assigned to the parallel mode of -helices (Vongsvivut et al. 2013).
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Figure 5: Representative FTIR spectra acquired from tub-1 C. elegans cultured without and with either EPA or PA supplementation at 100 M.
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Figure 6 shows the PCA of the FTIR spectra of the tub-1 C. elegans strain cultured without supplementation and with supplementation either with EPA or PA at 100 M. PC-1 and PC-2 account for 72% and 9% of the variance respectively and separate the FTIR spectra into 3 groups corresponding to tub-1 C. elegans cultured with no supplementation, tub-1 C. elegans cultured
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with EPA supplementation, and tub-1 C. elegans cultured with PA supplementation.
Figure 6: PCA of the FTIR spectra of tub-1 C. elegans cultured without and with either EPA or
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PA supplementation at 100 M. Each data point represents a spectrum obtained with 128 co-added scans. The PC score plots as a function of wavenumbers show that the major difference between the spectra come from lipids (2928, 2848, and 1744 cm-1) and proteins (1680, 1648, and 1515 cm16
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) (Figure 7). A small contribution from unsaturated fatty acids (vibration at 3008 cm-1) was also
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observed.
Figure 7: Loadings corresponding to PCA presented in Figure 6 as a function of wavenumbers for
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tub-1 C. elegans cultured without and with either EPA or PA supplementation at 100 M (PC-1:
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72%, PC-2: 9%, and PC-3: 8%).
Representative spectra acquired from the fat-3 C. elegans strain cultured without or with
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EPA or PA at 100 M are shown in Figure 8. The major differences between these spectra are the vibrations at 3008, 2928, 2848, and 1744 cm-1. The vibration at 3008 cm-1 is present only as a very small shoulder in the spectra of fat-3 C. elegans cultured with no supplementation. As mentioned previously, this vibration represents a fingerprint of –HC=CH-. The C. elegans mutant strain fat3 exhibits a deficiency in the synthesis of PUFAs due to the dysfunction of the desaturases, which 17
can cause defects in lipid regulation and reproduction (Chen et al. 2016). Watts et al. reported that fat-3 mutants lack 6 desaturase activity and fail to produce any of the common C20 PUFAs (Watts et al. 2003). Because of this deficiency, the growth and behavior of the worm are
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compromised along with neuromuscular defects, cuticle abnormalities, reduced brood size, and altered biological rhythms (Watts et al. 2003; Kaja Reisner 2011). Supplementation of the CeMM growth media with 100 M of EPA leads to the appearance of the vibration at 3008 cm-1 (Figure 8). Watts et al. indicated that although fat-3 mutants fail to produce any of the common C20 PUFAs, the resulting growth abnormalities could be biochemically complemented by dietary
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supplementation of various C20 PUFAs, such as eicosapentaenoic acid (Watts et al. 2003).
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Supplementing CeMM with EPA resulted in an increase in the intensity of the vibration at 1744
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cm-1 (assigned to C=O stretching).
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The supplementation of the CeMM growth media with 100 M of PA is accompanied by
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the appearance of the vibration at 3008 cm-1 and an increase of the intensity of the vibrations at
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2928, 2848, and 1744 cm-1. This result indicates that saturated palmitic acid is converted to unsaturated fatty acids. The results of gas chromatography analysis did not show EPA but showed
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a significant increase in the amount of linoleic acid (from approximately 6.9% in fat-3 C. elegans with no supplementation to approximately 15.7% with supplementation with PA) (result not
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shown). This result indicated that the vibration assigned to –CH=CH- observed in the spectra of
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fat-3 supplemented with PA likely originates from linoleic acid C18:2. It was reported that C. elegans synthesizes 7% of its palmitic acid (16:0) through acetyl Co-A carboxylase (ACC) and fatty acid synthase (FAS), and the rest is absorbed from bacterial diets (Zheng and Greenway 2012). Elongases and desaturases can then integrate palmitic acid into triglycerides or convert it to long-chain polyunsaturated fatty acids (Zheng and Greenway 2012). In contrast to nematode 18
growth media agar plates with E. Coli (NGM), CeMM does not contain palmitic acid. The FTIR results illustrated the ability of C. elegans to convert saturated fatty acids to unsaturated fatty acids. Because mutant strain fat-3 lacks -6-desaturase, it is likely that the conversion of palmitic acid
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results in the production of linoleic acid 18:2n6, which was detected by FTIR.
Figure 8: Representative FTIR spectra acquired from fat-3 C. elegans strain cultured without or
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with either EPA or PA supplementation at 100 M.
PCA of the FTIR spectra of mutant strain fat-3 is shown in Figure 9. Similar to the other
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strains, three groups of spectra could be identified, depending on the diet. The first three components explained 90% of the variance. The major differences between the spectra reside in lipids, proteins, and unsaturated fatty acids.
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Figure 9: PCA of the FTIR spectra: fat-3 strain cultured without or with either EPA or PA at 100
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M. Each data point represents spectra obtained with 128 co-added scans.
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Loadings of PC-1, PC-2, and PC-3 as a function of wavenumbers are shown in Figure 10. Similar to the results presented above, the plots show peaks corresponding to unsaturated fatty
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cm-1).
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acids (3008 cm-1), lipids (2928, 2848, 1744 cm-1), and proteins (1680, 1648, 1632, 1568, and 1515
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Figure 10: Loadings corresponding to PCA presented in Figure 9 as a function of wavenumbers
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for fat-3 C. elegans cultured without or with EPA or PA supplementation at 100 M (PC-1: 70%,
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PC-2: 12%, PC-3: 8%).
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FTIR analysis of the spectra acquired from single worms cultured with or without EPA or PA supplementation illustrates the occurrence of biochemical changes in the worms. These
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changes are essentially related to lipids, proteins, and unsaturated fatty acids. This study showed
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that FTIR could be used alongside gas chromatography and staining techniques to investigate lipid metabolism and fat accumulation as well as changes induced in the protein structure. When
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comparing the wild-type to mutant strains, relative quantification could be used by calculating the area under the peak or the peak intensity. Conclusions
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In C. elegans, lipid storage and dynamics have been studied using staining with Nile Red or Oil Red O followed by fluorescence microscopy imaging. Other studies monitored lipid storage using coherent anti-stokes Raman and stimulated Raman scattering microscopy. Here, we used
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FTIR microspectroscopy to investigate the effect of C. elegans diet supplementation with a saturated and an unsaturated fatty acid on the changes in the biochemical composition. The FTIR spectra were acquired on intact worms in transmission mode. The principal component analysis of the FTIR spectra grouped the spectra into three groups corresponding to the spectra acquired from worms cultured with no supplementation, worms cultured with supplementation with
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eicosapentaenoic acid, and worms cultured in CeMM supplemented with palmitic acid. The major
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differences between the spectra resided in the vibrations corresponding to unsaturated fatty acids,
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lipids, and proteins. Furthermore, relative quantification of the biochemical changes is possible by
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calculating the area under the peak of interest or its height. Comparison between two spectra could
Conflicts of interest
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biochemical compounds.
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allow us to conclude about the relative changes of the functional groups and, thus, the corresponding
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There are no conflicts to declare.
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Acknowledgments
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The authors would like to thank Dr. S.A. Vanapalli group for supplying C. elegans strains, he obtained from the Caenorhabditis Genetics Center (CGC), which is funded by the NIH Office of Research Infrastructure Programs (P40 OD010440). The research was supported by the Fiber and Biopolymer Research Institute and startup funds from the College of Human Sciences, Texas Tech University.
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S0924-2031(19)30014-1 https://doi.org/10.1016/j.vibspec.2019.03.002 VIBSPE 2907
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Please cite this article as: Bouyanfif A, Liyanage S, Hequet E, MoustaidMoussa N, Abidi N, FTIR microspectroscopy reveals fatty acid-induced biochemical changes in C. elegans, Vibrational Spectroscopy (2019), https://doi.org/10.1016/j.vibspec.2019.03.002 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
FTIR microspectroscopy reveals fatty acid-induced biochemical changes in C. elegans Amal Bouyanfif a,b,c, Sumedha Liyanagea, Eric Hequeta, Naima Moustaid-Moussaa,b,c, Noureddine Abidia,c* Fiber and Biopolymer Research Institute, Texas Tech University Lubbock, TX, USA
b
Department of Nutritional Sciences, Texas Tech University Lubbock, TX, USA
c
Obesity Research Cluster, Texas Tech University, Lubbock, TX, USA
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* Corresponding author: Noureddine Abidi, Ph.D., 1001 East Loop 289, Lubbock, Texas 79403,
USA.
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Email: [email protected]
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Phone: 1-806-834-1221, Fax: 1-806-742-5343
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Abstract
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Fourier transform infrared microspectroscopy (FTIR) is a promising method for the
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analysis of biological samples. Recent studies reported that FTIR imaging allows determination of the distribution of several biomolecules in a sample with no staining or extraction. In this study,
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FTIR was used to monitor biochemical changes in C. elegans nematodes cultured in nematode maintenance media (CeMM) without supplementation and with supplementation with either a long
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chain polyunsatured omega 3 fatty acid, eicosapentaenoic acid (EPA) or a saturated fatty acid, palmitic acid (PA) at 100 M. EPA is an omega 3 fatty acid with documented health benefits while PA is generally consumed in diets. Worms were placed on BaF2 slides, and FTIR spectra were collected from single worms in transmission mode using a focal plane array detector. Principal
1
component analysis grouped the FTIR spectra into three clusters corresponding to spectra of worms cultured with no supplementation, worms cultured with supplementation with EPA, and worms cultured with supplementation with PA. The major differences between the FTIR spectra
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reside in the vibrations corresponding to unsaturated fatty acids (3008 cm-1), lipids (2928, 2848, and 1744 cm-1), and proteins (1680, 1648, and 1515 cm-1). This indicates that supplementation with EPA or PA leads to biochemical alterations related to unsaturated fatty acids, lipids, and proteins. Furthermore, supplementing mutant strains (tub-1 and fat-3) CeMM with PA resulted in the appearance of the vibration 3008 cm-1, an increase in the intensity of the vibration 1744 cm-1,
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and a new vibration at 1632 cm-1, which is assigned to the amide I of -pleated sheet component
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of proteins, in the spectra of tub-1 and fat-3 mutant strains. The results illustrated the potential use
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of FTIR alongside other techniques such as gas chromatography and staining techniques to
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investigate lipid metabolism and fat accumulation as well as induced changes in protein structures.
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Abbreviations
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CeMM, C. elegans maintenance media; EPA, eicosapentaenoic acid; PA, palmitic acid; FPA, focal plane array; FTIR, Fourier transform infrared; NGM, nematode growth medium; PCA, principal
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component analysis; -3 PUFA, omega-3 polyunsaturated fatty acids; WT, wild-type; CGC,
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Caenorhabditis Genetics Center.
FTIR
microspectroscopy,
imaging,
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Keywords:
eicosapentaenoic acid
2
olefinic,
palmitic
acid,
C.
elegans,
Introduction Caenorhabditis elegans (C. elegans) has attracted increasing attention and has become a major model organism in different research fields, such as biology (Witting and Schmitt-Kopplin
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2016), obesity, fat metabolism (Zheng and Greenway 2012; Ashrafi 2007; Ashrafi et al. 2003), drug discovery (O'Reilly et al. 2014; Artal-Sanz et al. 2006), biomedical toxicology, environmental toxicology (Leung et al. 2008), and nanoparticle toxicity (Gonzalez-Moragas et al. 2017; Gonzalez-Moragas et al. 2015; Meyer et al. 2010). The interest in using this nematode as a model organism instead of rodent models (e.g., mice) is attributed to several advantages, such as small
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size, short lifespan, quick turnover, reduced experimental timeline, complete genetic information,
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easy culture conditions, easy maintenance in the laboratory, low cost, possibility of long-term
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order animals (Bouyanfif et al. in press).
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storage as a frozen stock, no prior approval is required compared to studies on rodents and higher
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C. elegans has been used to investigate mechanisms that regulate lipid storage and
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metabolism (Watts 2009; Watts and Browse 2002; Hellerer et al. 2007; Folick et al. 2011; Ashrafi 2007; Witting and Schmitt-Kopplin 2016; Chen et al. 2016). In mammals, lipids are primarily
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stored as triglycerides in adipose tissue. In C. elegans, lipids are also stored primarily as
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triacylglycerols but in the gut granules and hypodermal cells (Ashrafi 2007; Ashrafi et al. 2003). The fat content in C. elegans has been determined by extracting total lipids from the whole worms, phospholipids
and
neutral
lipid
moieties,
and
then
performing
gas
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fractionating
chromatography/mass spectrometry analyses (Ashrafi 2007; Kniazeva et al. 2003; Satouchi et al. 1993; Watts and Browse 2002). The triacylglyceride content in the nematode is 40 to 55% of the total lipids, while phospholipids are composed of ~55% ethanolamine glycerophospholipid, ~32% choline
glycerophospholipid,
and
approximately 3
8%
sphingomyelin
(Ashrafi
2006).
Polyunsaturated fatty acids (PUFAs) play an important role, not only as structural components of membranes, but also as precursors to critical signaling molecules and lipid mediators (Kaja Reisner 2011; Watts and Browse 2002; Watts et al. 2003).
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Lipid metabolism pathways in C. elegans resemble those in mammals. However, a few exceptions exist. (1) unlike mammals, C. elegans possesses the fatty acid enzymes required to generate essential fatty acids, (2) C. elegans is not able to produce PUFAs longer than C20. Therefore, the -6 and -3 PUFAs biosynthesis pathways end with arachidonic acid (20:4n6) and eicosapentaenoic acid (20:5n3), respectively (Watts and Browse 2002). As indicated above, C.
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elegans stores fat primarily as droplets in their intestinal and skin-like epidermal cells (Ashrafi
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2007). Lipid staining techniques using Nile Red, Sudan Black, Oil Red O, and fluorescently
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labeled fatty acids are commonly used to assess changes in fat storage due to mutations or RNAi-
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mediated inactivation of genes encoding various lipid biosynthesis pathways (Ashrafi et al. 2003;
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Mukhopadhyay et al. 2005). This technique has some limitations, such as variability in dye
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labeling specificity and efficiency, which may lead to inconsistencies in lipid quantification (O'Rourke et al. 2009; Watts 2009; Soukas et al. 2009). Other tools used to visualize lipids include
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coherent anti-stokes Raman scattering, which allows visualization of lipids stored in both C. elegans epidermal and gut granules without the use of invasive methods (Hellerer et al. 2007).
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Tserevelakis et al. utilized label-free imaging using third-harmonic generation microscopy to study
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lipid deposition in C. elegans (Tserevelakis et al. 2014). Folick et al. combined anti-stokes Raman scattering and simulated Raman scattering microscopy to develop new methods to visualize the localization and regulation of lipids in C. elegans (Folick et al. 2011). We believe that metabolism of saturated and unsaturated fatty acids by C. elegans could result in biochemical changes that
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could be detected on intact worms (no chemical extraction no chemical staining) using Fourier transform infrared microspectroscopy (FTIR). FTIR is a powerful analytical technique to investigate the chemical composition of a
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sample. When combined with a microscope and focal plane array detectors (FPA), this technique allows imaging of a relatively large sample area, providing both spectroscopic information (chemical identification) and distribution of functional groups in the sample. Recently, we reviewed applications of FTIR to study the biochemical changes in C. elegans (Bouyanfif et al. 2018). FTIR imaging could be used to investigate intact nematodes. These applications include
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nematode identification (Ami et al. 2004; Ami et al. 2012), biochemical composition (Sheng et al.
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2016; Bouyanfif et al. 2017), and toxicity assessment of nanoparticles and drugs (Zanni et al. 2012;
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Diomede et al. 2010). We used FTIR imaging to illustrate changes in intact C. elegans (wild-type
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(N2) and mutant strains fat-3 and tub-1) when C. elegans maintenance media (CeMM) was
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supplemented with a polyunsaturated fatty acid, eicosapentaenoic acid (EPA) (Bouyanfif et al.
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2017). We used principal component analysis to discriminate WT from mutant strains (tub-1 and fat-3) cultured with or without EPA supplementation. This discrimination was mainly due to
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differences in unsaturated fatty acids, lipids, and protein profiles (Bouyanfif et al. 2017). These conclusions were further supported by performing digital subtraction of the FTIR spectra of mutant
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strains from those of WT. These studies validated the FTIR technique as an important tool to detect changes in biochemical compositions and lipid metabolism (Bouyanfif et al. 2017). This work has
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laid the foundation to explore further the use of FTIR imaging to dissect biochemical changes in C. elegans WT and mutant strains (tub-1 and fat-3) cultured in CeMM supplemented with EPA or palmitic acid (PA). We compared these two fatty acids as one of them, EPA is an omega 3 fatty acid with documented health benefits including metabolic diseases; while PA is a saturated fatty
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acid commonly consumed in the diet and also endogenously produced. In this study, FTIR spectra were acquired in transmission mode on a single intact worm with no special preparation. This stain-free analytical technique is able to provide information related to chemical functional groups
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that provide insight into biochemical changes in C. elegans fed saturated or unsaturated fatty acids. Materials and Methods
C. elegans strains, culture, and maintenance: Hermaphrodite adult WT and mutant strains tub-1 (nr2044) and fat-3 (wa22) were acquired from the Caenorhabditis Genetics Center (CGC, University of Minnesota, Minneapolis, MN, USA). WT (N2) is able to synthetize 20:4n6 and
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20:5n3 using saturated and monounsaturated fatty acids from bacteria as precursors (Watts et al.
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2003; Hutzell and Krusberg 1982). By contrast, the mutant strain fat-3 lacks 6 desaturase activity
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and fails to produce any of the common C20 PUFAs that are essential in regulating membrane
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structure, dynamics, and permeability (Watts et al. 2003). In the mutant strain tub-1, the functional
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loss of tubby ortholog called tub-1/F10B5.5 in C. elegans leads to the accumulation of
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triglycerides, the major form of stored fat (Ashrafi et al. 2003; Mukhopadhyay et al. 2005; Mak et al. 2006).
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Nematodes were first cultured in the nematode growth media (NGM) plates seeded with E. coli
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OP50 as the main food source (Stiernagle 2006b, a). Plates containing a large quantity of eggs and gravid adults were bleached, and eggs were left in sterile M9 Buffer to hatch overnight. The
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following day, starved L1 animals were transferred to CeMM (Cell Guidance Systems, Babraham, Cambridge, UK) containing 20 µg/mL of kanamycin sulfate (Fisher Scientific, Pittsburgh, PA, USA) and 200 µg/mL of streptomycin sulfate (Fisher Scientific, Pittsburgh, PA, USA) (Szewczyk et al. 2003). C. elegans strains were cultured in CeMM at 20°C for multiple generations to adapt to the media before being used in the experiments. 6
Fatty acids: Eicosapentaenoic acid (EPA) and palmitic acid (PA) were acquired from Nu-Check Prep (Elysian, MN, USA). EPA was supplied as a pure -3 ethyl esters. It was stored at -80°C, and exposure to heat, light, and oxygen was minimized to prevent oxidative degradation. PA was
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supplied as pure palmitic acid. EPA and PA solutions in ethanol were prepared and CeMM was supplemented at a final concentration of 100 µM; feeding was performed for 72 h on worm cultures of mixed ages.
FTIR microspectroscopy: Individual hermaphrodite adult nematodes were washed with distilled water and deposited on FTIR transparent BaF2 slides (PerkinElmer, MA, USA). Slides containing
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worms were then dried in a vacuum desiccator for 1 h. FTIR spectra were collected in the
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transmission mode between 4000 and 1000 cm-1 using a Spectrum 400 FTIR equipped with a
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Spotlight 400 microscope (PerkinElmer, MA, USA) accessory with a liquid nitrogen cooled
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128x128 Mercury-Cadmium-Telluride (MCT) focal plane array detector. To improve the signalto-noise ratio, 128 co-added spectra were collected from each pixel (6.25 x 6.25 µm) with a spectral
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resolution of 16 cm-1. Prior to sample measurements, a background spectrum from an empty area
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of the BaF2 slide was automatically subtracted from the spectra of the sample. The BaF2 slide does
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not show any absorption in the range of 4000 – 1000 cm-1. A visual image was first acquired from the sample (Figure 1-A). Figures 1-B and 1-C show
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FTIR image and the corresponding spectra extracted from the positions marked in the image. Point
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mode imaging was used in this study, which allowed us to acquire a large number of spectra in a short period of time (approximately 20 min). Spectra were acquired from a single worm along the length (4 to 6 worms for each treatment) with an aperture size of 15 x 15 µm. Four to eight data points were acquired from each worm (separated by approximately 50 m), and all the spectra were the result of 128 co-added scans. The acquired spectra were baseline corrected and 7
normalized with respect to the total absorbance over the entire range from 4000 to 1000 cm-1 using the Spectrum 10™ software (PerkinElmer, MA, USA). Principal component analysis was
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performed using the Unscrambler® X 10.3 software (CAMO Software AS, Norway).
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Figure 1: Image of the whole intact C. elegans illustrating the location from which spectra were
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recorded.
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Results and Discussion
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In previous work, we investigated the use of FTIR imaging to illustrate changes occurring in WT (N2), tub-1, and fat-3 C. elegans cultured with and without EPA supplementation at 25 or
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100 M (Bouyanfif et al. 2017). The results indicated that mutant strains have altered lipid
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compositions and that the major component of the middle part of the worms are fatty acyl groups. In this study, we investigated the chemical changes induced when the C. elegans diet was
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supplemented with and without EPA and PA at 100 M. Representative spectra acquired from WT (N2) cultured in CeMM alone and from WT (N2) cultured in CeMM either with EPA or PA supplementation at 100 M are exhibited in Figure 2. It is interesting to note that the only difference between these spectra resides in the intensities of the vibrations at 2928 cm-1 (asymmetric –CH2 stretch), at 2848 cm-1 (symmetric –CH2 stretch), and at 1744 cm-1 (C=O 9
stretch). These vibrations are associated with the presence of lipids, fatty acids, triglycerides, and phospholipids (Bouyanfif et al. 2017). Supplementing CeMM with PA resulted in a high intensity of these vibrations but did not affect other vibrations in the 1700 – 1000 cm-1 region. In this region,
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the vibration at 1648 cm-1 was assigned to the C=O stretch of amide I, mainly the -helix components of proteins (Bouyanfif et al. 2017; Hobro and Lendl 2011). The vibration at 1548 cm1
was attributed to amide II, which could originate from N-H bending and C-N stretching of the
protein amide group. The vibration at 1456 cm-1 was assigned to CH2 bending and deformation of methylene of lipids, proteins, or cholesterol esters. The vibration at 1392 cm-1 was assigned to the
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COO- stretch of carbohydrates, fatty acids, or amino acid side chains. The vibrations at 1232 and
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1084 cm-1 originated from PO2- antisymmetric and symmetric stretching of phosphodiesters,
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respectively. The vibration at approximately 1155 cm-1 was assigned to C-O stretching and
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possibly originated from glycogen (Vongsvivut et al. 2013). It is important to note the presence of the vibration at 3008 cm-1, which is attributed to -
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HC=CH- stretching (San-Blas et al. 2011; Holman et al. 2008). This vibration is due to unsaturated
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fatty acids and exists in the spectra acquired from WT (N2) with no supplementation. This
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vibration represents a very important feature in the FTIR spectra of WT (N2) C. elegans compared to the spectra acquired from tub-1 and fat-3 mutant strains. This result confirms previously
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reported results that indicated that WT (N2) C. elegans is capable of synthesizing a wide range of saturated, monounsaturated, and polyunsaturated fatty acids (Kniazeva et al. 2003; Kniazeva et al.
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2004; Satouchi et al. 1993; Watts and Browse 2002; Hutzell and Krusberg 1982). Previous fatty acid analyses of C. elegans and C. briggsae reported that lipids accounted for 19.1% of the dry weight of C. elegans (Hutzell and Krusberg 1982). The authors indicated that the major portion of the fatty acids contained 18 or 20 carbons, with unsaturated fatty acids making 70% of the total
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fatty acids (the principal fatty acid fraction was 18:1) (Hutzell and Krusberg 1982). Supplementation with EPA did not result in an increased intensity of the vibration at 3008 cm-1
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(slightly reduced intensity was noticed).
Figure 2: Representative FTIR spectra acquired from WT (N2) C. elegans cultured without and
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with either EPA or PA supplementation at 100 M. Several FTIR spectra were acquired, and principal component analysis (PCA) was
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performed to identify distinct groups of spectra that exhibit spectral similarities. PCA is a widely adopted multivariate statistical analysis used to reduce the dimensionality from a large number of
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interconnected variables (wavenumbers in the case of FTIR) to a few uncorrelated variables
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(Kemsley 1996; Chen et al. 1998). The reduction in dimensionality is achieved by means of a linear transformation to a new set of variables termed principal component scores. Thus, the sources of variability in the data are concentrated into the first 2 or 3 principal components. Plots of PC-1 against PC-2 or PC-3 revealed clustering in the FTIR spectra. We used PCA to analyze the FTIR spectra acquired from C. elegans WT and mutant strains tub-1 and fat-3 to detect diet 11
and genotype-dependent biochemical changes with and without EPA supplementation at 25 and 100 M (Bouyanfif et al. 2017). Figure 3 shows PC-1 and PC-2 scores of the FTIR spectra acquired from WT (N2) cultured with no supplementation and either with EPA or PA
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supplementation at 100 M. Three groups of spectra can be distinguished as follows: the first group corresponds to WT (N2) with no supplementation, the second group corresponds to WT (N2) with supplementation with EPA at 100 M, and the third group corresponds to WT (N2) with supplementation with PA at 100 M. It is worth pointing out the large variability in the spectra of WT (N2) cultured in CeMM with EPA or PA supplementation compared to the spectra acquired
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from WT (N2) cultured only in CeMM with no supplementation. The separation of the FTIR
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spectra into different groups indicates that biochemical changes are induced in worms when their
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diet is supplemented with saturated and unsaturated fatty acids.
Figure 3: PCA of the FTIR spectra acquired from WT (N2) cultured without and with either EPA or PA supplementation at 100 M. Each data point represents a spectrum obtained with 128 coadded scans. 12
The analysis of loading variables (or factors) as a function of wavenumbers can help identify the functional groups that are behind the grouping of the original spectra (Alonso-Simon et al. 2004). Plots of the first three component loadings as a function of wavenumbers are shown
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in Figure 4. PC-1, PC-2 and PC-3 account for 46%, 33% and 8% of the spectral variation, respectively. The PC-1 loading plot shows major peaks at 1744 cm-1 (C=O stretching of esters), 1632 cm-1 (amide I band of the -pleated sheet component of proteins, collagen proline residues (Hobro and Lendl 2011)). The PC-2 loading plot shows major peaks at the following vibrations: 2928 and 2848 cm-1 (assigned to asymmetric and symmetric stretching of the acyl –CH2 groups),
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1744 cm-1 and 1680 cm-1 (assigned to amide I band component originating from anti-parallel
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pleated sheets and -turns of proteins), and approximately 1550 cm-1 (assigned to amide II, N-H
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bending and C-N stretching of protein amide groups with some contribution from amino acid side
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chains such as arginine, aspartate, glutamate and tyrosine (Hobro and Lendl 2011)). The PC-3 loading plot shows major peaks at 2928 cm-1, 2848 cm-1, 1680 cm-1, 1632 cm-1, 1515 cm-1
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(assigned to tyrosine ring vibration), ~1232 cm-1 (PO2- antisymmetric stretching of
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phosphodiesters (Hobro and Lendl 2011)), and 1084 cm-1 (PO2- symmetric stretching of the
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phosphodiester backbone of nucleic acids and phospholipids).
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Figure 4: Loadings corresponding to PCA presented in Figure 3 as a function of wavenumbers for
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WT (N2) C. elegans cultured without and with either EPA or PA supplementation at 100 M.
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Representative spectra acquired from mutant strain tub-1 are shown in Figure 5. As
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reported in our previous work, the vibration at 3008 cm-1 (=CH– olefinic stretching), which
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generally arises from the presence of unsaturated fatty acids, is present only as a very small shoulder in the FTIR spectra of tub-1 cultured with no supplementation (Bouyanfif et al. 2017).
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The vibrations associated with triglycerides (2928, 2848, and 1744 cm-1) are present with high intensities. For this mutant strain, it was reported that the deletion of tubby ortholog (tub-1) leads
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to an accumulation of triglycerides, which are the major form of stored fat in C. elegans (Ashrafi et al. 2003; Mukhopadhyay et al. 2005). Supplementation of CeMM with 100 M EPA leads to the appearance of the vibration at 3008 cm-1 with only a minor increase in the intensity of the vibration at 1744 cm-1 (triglycerides, phospholipids, cholesterol esters, or fatty acids).
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Supplementing the growth media with palmitic acid (saturated fatty acid) not only resulted in the appearance of the vibration at 3008 cm-1 but also in a significant increase in the intensity of the vibration at 1744 cm-1. Palmitic acid can be integrated into triacylglycerides or phospholipids
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or can be modified by fatty acid elongases and desaturases to form a variety of long-chain polyunsaturated fatty acids (Watts 2009; Watts and Browse 2002). Furthermore, it is of particular interest to note the appearance of the vibration at 1632 cm-1, assigned to the carbonyl stretching in amide I of the -pleated sheet component of proteins (Hobro and Lendl 2011). Supplementing the worm diet with saturated palmitic acid resulted in the shift from the vibration at 1648 cm-1 assigned
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to amide I of the -helix component to 1632 cm-1 assigned to amide I of the -sheet components.
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However, this was not the case for the WT (N2) strain. Furthermore, supplementation with EPA
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and PA resulted in the appearance of a vibration at approximately 1520 cm-1, which has been
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assigned to the parallel mode of -helices (Vongsvivut et al. 2013).
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Figure 5: Representative FTIR spectra acquired from tub-1 C. elegans cultured without and with either EPA or PA supplementation at 100 M.
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Figure 6 shows the PCA of the FTIR spectra of the tub-1 C. elegans strain cultured without supplementation and with supplementation either with EPA or PA at 100 M. PC-1 and PC-2 account for 72% and 9% of the variance respectively and separate the FTIR spectra into 3 groups corresponding to tub-1 C. elegans cultured with no supplementation, tub-1 C. elegans cultured
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with EPA supplementation, and tub-1 C. elegans cultured with PA supplementation.
Figure 6: PCA of the FTIR spectra of tub-1 C. elegans cultured without and with either EPA or
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PA supplementation at 100 M. Each data point represents a spectrum obtained with 128 co-added scans. The PC score plots as a function of wavenumbers show that the major difference between the spectra come from lipids (2928, 2848, and 1744 cm-1) and proteins (1680, 1648, and 1515 cm16
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) (Figure 7). A small contribution from unsaturated fatty acids (vibration at 3008 cm-1) was also
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observed.
Figure 7: Loadings corresponding to PCA presented in Figure 6 as a function of wavenumbers for
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tub-1 C. elegans cultured without and with either EPA or PA supplementation at 100 M (PC-1:
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72%, PC-2: 9%, and PC-3: 8%).
Representative spectra acquired from the fat-3 C. elegans strain cultured without or with
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EPA or PA at 100 M are shown in Figure 8. The major differences between these spectra are the vibrations at 3008, 2928, 2848, and 1744 cm-1. The vibration at 3008 cm-1 is present only as a very small shoulder in the spectra of fat-3 C. elegans cultured with no supplementation. As mentioned previously, this vibration represents a fingerprint of –HC=CH-. The C. elegans mutant strain fat3 exhibits a deficiency in the synthesis of PUFAs due to the dysfunction of the desaturases, which 17
can cause defects in lipid regulation and reproduction (Chen et al. 2016). Watts et al. reported that fat-3 mutants lack 6 desaturase activity and fail to produce any of the common C20 PUFAs (Watts et al. 2003). Because of this deficiency, the growth and behavior of the worm are
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compromised along with neuromuscular defects, cuticle abnormalities, reduced brood size, and altered biological rhythms (Watts et al. 2003; Kaja Reisner 2011). Supplementation of the CeMM growth media with 100 M of EPA leads to the appearance of the vibration at 3008 cm-1 (Figure 8). Watts et al. indicated that although fat-3 mutants fail to produce any of the common C20 PUFAs, the resulting growth abnormalities could be biochemically complemented by dietary
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supplementation of various C20 PUFAs, such as eicosapentaenoic acid (Watts et al. 2003).
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Supplementing CeMM with EPA resulted in an increase in the intensity of the vibration at 1744
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cm-1 (assigned to C=O stretching).
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The supplementation of the CeMM growth media with 100 M of PA is accompanied by
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the appearance of the vibration at 3008 cm-1 and an increase of the intensity of the vibrations at
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2928, 2848, and 1744 cm-1. This result indicates that saturated palmitic acid is converted to unsaturated fatty acids. The results of gas chromatography analysis did not show EPA but showed
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a significant increase in the amount of linoleic acid (from approximately 6.9% in fat-3 C. elegans with no supplementation to approximately 15.7% with supplementation with PA) (result not
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shown). This result indicated that the vibration assigned to –CH=CH- observed in the spectra of
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fat-3 supplemented with PA likely originates from linoleic acid C18:2. It was reported that C. elegans synthesizes 7% of its palmitic acid (16:0) through acetyl Co-A carboxylase (ACC) and fatty acid synthase (FAS), and the rest is absorbed from bacterial diets (Zheng and Greenway 2012). Elongases and desaturases can then integrate palmitic acid into triglycerides or convert it to long-chain polyunsaturated fatty acids (Zheng and Greenway 2012). In contrast to nematode 18
growth media agar plates with E. Coli (NGM), CeMM does not contain palmitic acid. The FTIR results illustrated the ability of C. elegans to convert saturated fatty acids to unsaturated fatty acids. Because mutant strain fat-3 lacks -6-desaturase, it is likely that the conversion of palmitic acid
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results in the production of linoleic acid 18:2n6, which was detected by FTIR.
Figure 8: Representative FTIR spectra acquired from fat-3 C. elegans strain cultured without or
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with either EPA or PA supplementation at 100 M.
PCA of the FTIR spectra of mutant strain fat-3 is shown in Figure 9. Similar to the other
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strains, three groups of spectra could be identified, depending on the diet. The first three components explained 90% of the variance. The major differences between the spectra reside in lipids, proteins, and unsaturated fatty acids.
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Figure 9: PCA of the FTIR spectra: fat-3 strain cultured without or with either EPA or PA at 100
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M. Each data point represents spectra obtained with 128 co-added scans.
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Loadings of PC-1, PC-2, and PC-3 as a function of wavenumbers are shown in Figure 10. Similar to the results presented above, the plots show peaks corresponding to unsaturated fatty
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cm-1).
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acids (3008 cm-1), lipids (2928, 2848, 1744 cm-1), and proteins (1680, 1648, 1632, 1568, and 1515
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Figure 10: Loadings corresponding to PCA presented in Figure 9 as a function of wavenumbers
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for fat-3 C. elegans cultured without or with EPA or PA supplementation at 100 M (PC-1: 70%,
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PC-2: 12%, PC-3: 8%).
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FTIR analysis of the spectra acquired from single worms cultured with or without EPA or PA supplementation illustrates the occurrence of biochemical changes in the worms. These
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changes are essentially related to lipids, proteins, and unsaturated fatty acids. This study showed
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that FTIR could be used alongside gas chromatography and staining techniques to investigate lipid metabolism and fat accumulation as well as changes induced in the protein structure. When
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comparing the wild-type to mutant strains, relative quantification could be used by calculating the area under the peak or the peak intensity. Conclusions
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In C. elegans, lipid storage and dynamics have been studied using staining with Nile Red or Oil Red O followed by fluorescence microscopy imaging. Other studies monitored lipid storage using coherent anti-stokes Raman and stimulated Raman scattering microscopy. Here, we used
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FTIR microspectroscopy to investigate the effect of C. elegans diet supplementation with a saturated and an unsaturated fatty acid on the changes in the biochemical composition. The FTIR spectra were acquired on intact worms in transmission mode. The principal component analysis of the FTIR spectra grouped the spectra into three groups corresponding to the spectra acquired from worms cultured with no supplementation, worms cultured with supplementation with
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eicosapentaenoic acid, and worms cultured in CeMM supplemented with palmitic acid. The major
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differences between the spectra resided in the vibrations corresponding to unsaturated fatty acids,
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lipids, and proteins. Furthermore, relative quantification of the biochemical changes is possible by
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calculating the area under the peak of interest or its height. Comparison between two spectra could
Conflicts of interest
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biochemical compounds.
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allow us to conclude about the relative changes of the functional groups and, thus, the corresponding
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There are no conflicts to declare.
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Acknowledgments
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The authors would like to thank Dr. S.A. Vanapalli group for supplying C. elegans strains, he obtained from the Caenorhabditis Genetics Center (CGC), which is funded by the NIH Office of Research Infrastructure Programs (P40 OD010440). The research was supported by the Fiber and Biopolymer Research Institute and startup funds from the College of Human Sciences, Texas Tech University.
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