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Analysis of short-chain fatty acids in human feces: A scoping review
Accepted Manuscript Analysis of short-chain fatty acids in human feces: A scoping review Maša Primec, Dušanka Mičetić-Turk, Tomaž Langerholc PII:
S0003-2697(17)30119-7
DOI:
10.1016/j.ab.2017.03.007
Reference:
YABIO 12648
To appear in:
Analytical Biochemistry
Received Date: 5 December 2016 Revised Date:
18 February 2017
Accepted Date: 7 March 2017
Please cite this article as: M. Primec, D. Mičetić-Turk, T. Langerholc, Analysis of short-chain fatty acids in human feces: A scoping review, Analytical Biochemistry (2017), doi: 10.1016/j.ab.2017.03.007. 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.
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Review
Analysis of short shortrt-chain fatty acids in human
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feces: a scoping review
a
Department
of
Microbiology,
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Maša Primeca*, Dušanka Mičetić-Turkb, Tomaž Langerholca Biochemistry,
Molecular
Biology
and
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Biotechnology, Faculty of Agriculture and Life Sciences, University of Maribor, Pivola 10, 2311 Hoče, Slovenia b
Department of Pediatrics, Faculty of medicine, University of Maribor,
Taborska ulica 8, 2000 Maribor, Slovenia
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Corresponding author*: E-mail: [email protected]; Phone: +386 2 320
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90 54, Fax: +386 2 616 11 58
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Abstract Short-chain fatty acids (SCFAs) play a crucial role in maintaining homeostasis in humans, therefore the importance of a good and reliable
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SCFAs analytical detection has raised a lot in the past few years. The aim of this scoping review is to show the trends in the development of different methods of SCFAs analysis in feces, based on the literature
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published in the last eleven years in all major indexing databases. The search criteria included analytical quantification techniques of SCFAs in
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different human clinical and in vivo studies. SCFAs analysis is still predominantly performed using gas chromatography (GC), followed by high
performance
resonance
liquid
chromatography
(HPLC),
nuclear
magnetic
(NMR) and capillary electrophoresis (CE). Performances,
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drawbacks and advantages of these methods are discussed, especially in the light of choosing a proper pretreatment, as feces is a complex
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biological material. Further optimization to develop a simple, cost effective
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and robust method for routine use is needed.
Keywords:
Short-chain fatty acids (SCFAs) Feces Human Detection methods
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Scoping review
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Abbreviations: bbreviations: SCFAs, short-chain fatty acids; FAs, fatty acids; GC, gas chromatography; liquid
chromatography;
chromatography;
RP-HPLC,
chromatography;
NMR,
HPLC,
reverse
nuclear
high
phase
magnetic
performance
high
liquid
performance
liquid
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LC,
resonance;
CE,
capillary
electrophoresis; IBD, inflammatory bowel disease; IBS, irritable bowel SPE,
solid
-
phase
extraction;
SPME,
solid
-
phase
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syndrome;
microextraction; FAME, fatty acid methyl esters; UV, ultra violet; PEG,
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polyethylene glycol; FFAP, free fatty acid phase column; FID, flame ionization detector; LOD, limit of detection; LOQ, limit of quantification; RSD, relative standard deviation; CV, coefficient of variation; RI, refractive index; PDA, photo diode array detector; VWD, variable
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ionization.
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wavelength detector ECD, electrochemical detector; ESI, electrospray
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1.
Introduction
The human intestinal microbiota plays an important role in food digestion, contributing to the catabolism of food and potential toxins, the synthesis
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of microelements and fermentation of human digestion resistant nutrients. Moreover, it participates in electrolyte and mineral absorption as well as in short chain fatty acid (SCFAs) production. SCFAs are carboxylic organic
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acids with an aliphatic tail and represent the largest group of metabolic nutrients obtained from the fermentation of resistant carbohydrates,
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which are otherwise undegradable via human digestion. The process predominantly takes place in the caecum and the colon (reviewed in Nicholson et al.) [1]. In fact, carbohydrate fermentation is the main origin of SCFAs, although SCFAs may be generated from protein and amino acid
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decompositions as well. 95% of all SCFAs are represented by the three main representatives, i.e. acetic acid (C2), propionic acid (C3) and butyric acid (C4). Their respective molecular ratio 60:20:20 is relatively constant
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in the colon as well as in the feces [2]. The type and the quantity of
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produced SCFAs depend on the quantity of available substrate for the microbiota digestion and the composition of the microbiota itself [1]. Beside the three main aliphatic SCFAs, lactic i.e. 2-hydroxy propanoic acid (C3), valeric (C5) and caproic acid (C6) are present in lower amounts. Moreover, lactic acid exists in two optical isomeric forms, L- and Dlactate, while the concentration of L-lactate exceeds D-lactate by 100-fold under physiological conditions [3].
ACCEPTED MANUSCRIPT There is an increasing evidence about the important role of SCFAs in health and homeostasis. SCFAs undergo an effective absorption from the colonic lumen and they represent 10% of the human daily energy intake. Unlike acetic and propionic acid, which are mainly absorbed, butyric acid
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serves as a principal energy source for colonocytes [4]. A direct link between SCFAs (qualitatively and quantitatively) and some human pathological conditions, such as inflammatory bowel disease (IBD) [5],
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irritable bowel syndrome (IBS) [6], diarrhea [7] and cancer [8] have been
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proposed. Pathological increase in D-lactate is attributed to an increase in lactic acid producing microbiota [9]. This points to the fact that there is a need for a good qualitative or quantitative detection of SCFAs in clinical laboratories on a daily basis, as well as understanding the complex
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relationship between food, microbiota and metabolic homeostasis [4]. SCFAs have been measured in various biological materials such as blood plasma [10–12] and serum [13], equine caecum liquor [14], brain [12]
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and complex fermentation media [15]. Moreover, SCFAs have been detected in different environmental samples [16], food [17], waste
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leachates [18] and even in asphaltene [19]. Nevertheless, only a few English language reviews have summarized the analysis of SCFAs in diverse
samples,
[16,20,21] pathological
or
mostly
potentially
conditions
focusing
on
applicable
[4,22].
a
specific
analytical
Comprehensive
analytical
methods reviews
in of
method specific available
methods for fecal SCFAs quantification are, to our knowledge, not available, although their determination in feces is non-invasive and most
ACCEPTED MANUSCRIPT popular. However, it should be noted that only 5% of all microbially produced
SCFAs
can
be
found
in
feces
[4].
Moreover,
actual
concentrations of SCFAs found in feces are, on average 80, 40, 15, 13, 2, 2.5, 2 and 0.5 (all mmol/kg feces) for total SCFAs, acetic, propionic, n-
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butyric, i-butyric, i-valeric, n-valeric and n-caproic acid, respectively [23]. The aim of this systematic scoping review is to summarize methods used
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for SCFAs determination in human feces published between 2004 and 2015. Trends in the development of different sample pretreatments,
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separation and detection methods are demonstrated and compared in terms of accuracy, time consumption, cost and reliability of the results. The conclusion contains gaps, problems, inconsistencies and future
Search strategy and selection of studies
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2.
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prospects of SCFAs analysis in feces.
A literature search was performed to identify scientific articles in English,
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published between January 2004 and December 2015. A comprehensive search was conducted using combinations of search terms ("short chain fatty acids; volatile acids; human; feces; faeces"). Preliminary hits were further screened for presence of inclusion criteria, that SCFAs were analyzed or detected in different human clinical and in vivo studies. In
vitro and cell models based studies were not taken into consideration. The literature search was conducted in databases that included PubMed
ACCEPTED MANUSCRIPT (http://www.ncbi.nlm.nih.gov/pubmed), (http://www.ncbi.nlm.nih.gov/pmc/),
PubMedCentral Science
Direct
(http://www.sciencedirect.com), Scopus (www.scopus.com) and Proquest Research
Library
(http://www.proquest.com).
The
initial
search
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(preliminary hits) returned 2458 matches in total. After the exclusion of duplicates and scientific studies not meeting the criteria previously mentioned, 134 full text articles were further screened for analytical
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techniques used for the detection of SCFAs in human feces. Due to
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insufficient data, abstracts were excluded from this review. Information obtained from the literature has been further divided into sections, according to the instrumentation and analytical procedures relevant for the analysis. Where applicable, information on method performance
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parameters in terms of specificity, LOD (limit of detection), LOQ (limit of quantification), recovery, reproducibility (inter assay) and repeatability
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(intra assay) has been provided.
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3.
General considerations about SCFAs SCFAs analysis in feces feces
SCFAs have become popular targets in scientific research that tries to link gut microbiota to pathological conditions and potential health-beneficial
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effects in humans. Feces or fecal water is a complex biological material, providing a challenge for a fast and reliable determination [24]. Although SCFAs analytical methods have improved a lot in the past 11 years, the search
showed
that
development
of
precise
and
fast
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literature
instrumental methods has not overcome gas chromatography (GC), which
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still appears to be the most commonly used quantification method of fecal SCFAs despite having some disadvantages [25]. Alternative methods include techniques related to liquid chromatography (LC), such as high performance liquid chromatography (HPLC), nuclear magnetic resonance
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(NMR) and capillary electrophoresis (CE).
Since SCFAs are volatile and feces contain high concentrations of
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microbes, it is important to keep the biological material in appropriate conditions after its collection, in order to prevent sample deterioration.
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Samples are usually kept at – 80 °C, although many researchers have successfully used -20 °C as well. Before the SCFAs analysis, two important pretreatment
steps
derivatization.
SCFAs
should are
be
considered,
partially
hydrophilic,
i.e.
extraction
which
makes
and their
quantitative extraction to hydrophobic organic solvents more difficult. However, sample acidification is generally applied to keep acids protonized and
less
hydrophilic
and
thus
facilitating
better
extraction
[26].
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SCFAs SCFAs analytical methods
4.1 4.1
Gas liquid chromatography (GC, GLC)
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4.. 4..
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readily detectable substances is therefore recommended.
Direct detection of fatty acids (FAs) with GC was first described in 1952
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[27]. The principle of GC relies on a carrier gas that serves as a mobile phase where sample compounds are separated by differential interaction with the column stationary phase. Depending on their chemical nature, retention time upon reaching a detector as well as its response are used
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for a qualitative and quantitative evaluation of compounds [27,28]. For a successful GC determination of SCFAs an appropriate pretreatment chromatographic column and detector are needed. It is important to note
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that some GC methods can cause a thermic degradation and structural
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modification of FAs during the methyl esterification process, or they can simply destroy the sample, disabling the possibility of its reanalysis [25].
4.1.1 Pretreatment in GC detection methods Feces pretreatment is crucial for the detection of SCFAs [29]. Studies that used GC for SCFAs detection along with potential pretreatment procedures are provided in Table 1.
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Table 1
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The physical pretreatment procedures, without any time-consuming extraction, such as filtration [31–39], ultrafiltration [30], centrifugation [40–42] or simple sample dilution [43,44] are likely to be the fastest and
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simplest. The disadvantage of these uncomplicated pretreatments are impurities, which may overload the system, resulting in an incomplete of
sample
substances
and
chromatographic
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separation
column
contamination with non-volatile particles. Moreover, this results in a shorter column life span and a frequent column change [133]. distillation
is
a
separation
technique,
used
to
separate
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Steam
temperature-sensitive material. The use of lower temperatures preserves the sample quality, but increases the likelihood of unspecific results [45].
to
more
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Moreover, steam distillation proved to be a less appropriate technique due variable
recovery
rates
found
especially
at
low
SCFA
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concentrations as 2-10 times higher coefficients of variance were determined, when compared to vacuum distillation [134]. In contrast, vacuum distillation is performed under low pressure and consequently at a lower temperature [46–51,53–65]. Most authors have implemented this pretreatment technique for SCFAs separation as done by Zijlstra et al. [134], with modifications introduced by Høverstad et al. [135]. Some authors used vacuum distillation after diluting samples with distilled water
ACCEPTED MANUSCRIPT [48–53,65] or acid, such as phosphoric [54–56] or sulfuric [57–65]. Acid driven protonation of SCFAs increases their volatility. The pretreatment procedure with vacuum distillation of volatile SCFAs is a considerably precise
method,
but
still
time
consuming
and
consequently
not
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appropriate in routine practice, where handling of large numbers of samples is required. Moreover, because the procedure is time consuming,
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the possibility of losing volatile acids can be expected [133].
Instead of the time-consuming purification by vacuum distillation, many
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authors applied simple sample acidification [67] before loading it into the GC column. The choice of acidification agent ranged from hydrochloric acid [26,69–73,76], phosphoric acid [79–83,86–92], formic acid [94–96], sulfuric acid [102–106,114] to oxalic acid [83,115,116]. Weir et al. [66]
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centrifuged and filtrated acidified samples before subjecting them to gas chromatography-mass
spectrometry
(GC-MS)
detection.
Simple
acidification may result in some disadvantages mentioned before, such as
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losing the column quality in a much shorter time, especially when no pre-
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column is applied [120].
Some authors used acetone as deproteinizing agent in addition to formic acid [6,100,101]. The use of formic acid as an acidification agent, among others, may better prevent the adsorption of SCFAs to the GC column than
phosphoric
acid,
resulting
consequently
in
less
analytical
disturbances such as peak tailing, peak broadening, and double peak formation [133].
ACCEPTED MANUSCRIPT Sample acidification has been frequently followed by organic solvent extraction, using chloroform [112,113] and more popularly, ether [74– 78,84,85,102,107–113,117,119]. For GC-MS analysis, García-Villalba et al. [120] tried two different acids before extraction with three different
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organic solvents, where ethyl acetate was found the most efficient.
Some authors used another step in the sample pretreatment procedure,
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i.e. derivatization, mostly silylation [75,78,121,122,132], which enhances sample purity. Derivatization not only provides chromophores, but in the
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case of GC derivatization, it should result in volatile and stable molecules, ready for the high temperatures used during GC separation. In fact, functional groups such as hydroxyl, carboxyl, amine, thiol, phosphate may be unstable at high temperatures applied in GC and therefore need to be
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derivatized by certain silylation reagents. The technique implicates replacement of the acidic hydrogen with an alkylsilyl group. Resulting silyl ethers,
such
as
trimethylsilyl
(TMS)
and
tert-butyldimethylsilyl
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(TBS/TBDMS) derivatives are highly volatile, less polar and thermally
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more stable. However, care should be paid to prevent evaporation of more volatile derivatives during the pretreatment procedure as potential loss of SCFAs
may
occur
[29].
To
avoid
that,
chloroformates
in
SCFAs
derivatization procedures have been introduced [24,123]. Besides the extraction with different solvents which leads to the separation of two reciprocally unmixed layers, a special extraction, where a solvent is not necessarily present, has also become popular. The so
ACCEPTED MANUSCRIPT called solid - phase extraction (SPE), especially the solid - phase microextraction (SPME) is a faster, more selective and more sensitive technique due to the lower presence of impurities [136]. However, an effective and successful GC-MS system demands purified samples without
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water, due to its negative effect on MS determination [120].
Recently, a SPME pretreatment technique in combination with GC-MS has
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shown promising results with water-soluble samples [124–129] as well. Couch et al. [128] performed the SPME with three different fibers.
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Moreover, in situ derivatization of FAs in combination with SPME and polymer coating (in-fibre derivatization) may generate very low detection limits in aqueous solutions (formic acid – 11 nmol/vial (0.011 mmol/kg dry feces), acetic acid 4 nmol/vial (0.004 mmol/kg dry feces), propionic
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acid 2 nmol/vial (0.002 mmol/kg dry feces) and C4 – C6 acids 1 -2 nmol/vial (0.001 – 0.002 mmol/kg dry feces)) [137]. The SPME pretreatment results in a higher sample purity and a longer life span of
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the chromatographic system. Still, fibres are expensive and the technique
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requires supplementary knowledge and instrumentation in order to provide an automated analysis [120]. However, a purge and trap sample pretreatment technique [130,131] has also been implemented in combination with GC-MS, with the capacity of achieving higher number of volatile compounds compared to SPME, but worse extraction capacities of volatiles having higher molecular mass.
ACCEPTED MANUSCRIPT Generally, two-step pretreatment methods are time-consuming, but they can be simplified by one-step transesterification of SCFAs in biological material through acid (i.e. hydrochloric acid or sulfuric acid in methanol) or basic (i.e. sodium methoxide or potassium hydroxide in methanol)
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catalysis. Fatty acid methyl esters (FAME) are stable derivatives, volatile and particularly suitable for SCFAs detection with GC [29]. In fact, some of the authors preferred the use of SCFAs transesterification with simple
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alcohols such as methanol [93,97–99], ethanol [54] or ethylene glycol
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[68]. Acid not only catalyzes transesterification but deproteinazes samples as well.
Nevertheless, the above described pretreatment procedures are timeconsuming and may have, due to the additional experimental steps, an
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effect on the decreased recovery rate, accuracy and reproducibility of the results [29]. Reagents and solvents needed during the pretreatment are hazardous because of their flammable characteristics. They may also lead
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to occupational exposure and thus health damage in terms of toxicity and
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allergies. Moreover, the use of large quantities of reagents increases the costs [26]. Advantages and disadvantages of different pretreatment techniques for the detection of SCFAs by GC are summarized in Table 2.
Table 2
4.1.2 GC columns
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a
). The
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stationary phase in capillary columns can be based on polysiloxanes and polyethylene glycol (PGE).
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The polysiloxanes are made of the most stable and robust material and characterized by the repeating siloxane backbone. Silicon atoms in bear
two
functional
groups
(methyl,
cyanopropyl,
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siloxanes
trifluoropropyl), where type and amount of substituted groups determine the different column properties [141]. Those used by different authors for SCFA analysis have been highly polar (designed to separate FAMEs) [30],
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non-polar [107,112,113,117,123,127]/polar [99], of low bleed and high temperature limit. The stationary phases that incorporate phenyl or phenyl type of functional groups are also known as arylenes. Their
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function results in very low column bleed and higher temperature limits.
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They are non-polar, with better sensitivity characteristics and mass spectral integrity and have also been successfully used for SCFA analysis [128].
PGE are also widely used as a stationary phase in capillary columns, but are less stable and robust and have lower temperature limits than most polysiloxanes. Therefore, a shorter lifespan and a higher susceptibility to damage caused by higher temperature are expected. Two general types of
ACCEPTED MANUSCRIPT PGE columns, considering temperature limits, are used, among which the one with the lower upper temperature limit and the lower low temperature limit
has
been
more
popular
for
separating
SCFAs
[31,80,81,85,92,97,100,103–106,108,124–126]. Moreover, this column
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exerts characteristics of high polarity, exhibits better reproducibility, inertness,
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and a higher resolution and temperature stability. The other type of PGE column has the characteristic of high polarity, but highest upper
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temperature limit and was used by one author only [115]. Furthermore, a faster and more accurate detection of SCFAs was first described by Zhao et al. [26]. The group described the usage of a free fatty acid phase (FFAP) column, which allows a direct SCFAs detection from water solutions
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without any derivatization. In fact, FFAP columns are of PGE type modified with terephthalic acid (pH modification), which are highly polar and widely used as stationary phases for the analysis of acidic compounds and
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therefore ideal for free fatty acid separation. Nevertheless, FFAP columns
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share certain characteristics of PGE columns and they are therefore also less stable and robust and have lower temperature limits than most polysiloxanes columns. The use of FFAP column has been subsequently performed by many authors (see Table 1 under
a(FFAP)
). Some acid
modified capillary columns with very similar or same characteristics as FFAP columns have also been widely used by many authors [37– 39,47,72,86–90].
ACCEPTED MANUSCRIPT 4.1.3 GC detectors The flame ionization detector (FID) (see Table 1 under b) is the most used conventional detector for SCFAs detection in GC. FID is sensitive to
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molecules that are ionized in a hydrogen–air flame, including most carbon-containing compounds, and produces a current that varies proportionally to the amount of organic species in a sample [142].
and
Nevertheless,
accuracy its
and
restriction
the
option
comes
with
of
a
temperature lack
of
regulation.
supplementary
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linearity
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Moreover, FID detectors are relatively quiet, have a wide spectrum of
information, besides the retention values, that could benefit in qualitative analysis of the analytes [29].
Instead of applying a conventional detector, GC can be bound to MS,
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resulting in a better sensitivity and selectivity of the analysis. MS ionizes molecules preferably into cations, which are separated on the basis of
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their mass to charge the ratio in a magnetic field. The ions are very reactive and unstable and the whole procedure is run under low pressure
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[143]. The scientific articles that used GC-MS methods for detecting SCFAs in feces are listed in Table 1 under c. If a tandem GC-MS-MS instrument is available, the use of such a system is also reasonable for the quantitation of analytes that are present in low concentrations in complex biological samples [129].
4.1.4 GC based methods performance
ACCEPTED MANUSCRIPT Some studies [35,36,69–72,118] using GC implemented their method according to Zhao et al [26], using the FFAP capillary column. The determined LOD for SCFAs was in a range lower than 10 µM (0.05 mmol/kg wet feces), while the LOQ was below 31 µM (0.155 mmol/kg wet
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feces). The recovery rates obtained varied from 87.7 - 114.5%, inter assay with relative standard deviation (RSD) values 1.5 – 4.9% with some exceptions and intra-assay with RSD values 1.0 – 6.0%. Schwiertz et al.,
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[115], who adapted the method after Schaefer et al. [144], reached LOD
mmol/kg
wet
acidification
feces
of
the
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of 50 – 100 µg/L in standard water solution (approximately 0.003 - 0.006 calculated sample
for
has
C3).
been
Vacuum
used
by
distillation many
after
researchers
[38,39,53,57–62,64,65,102] and was implemented from Høverstad et al.
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[135]. Despite being time consuming, LOD for SCFAs were found at 0.01 mmol/kg wet feces, recovery from 90-109%, intra assay with coefficient of variation (CV) of 2.6 – 8.9%, inter assay with CV of 6 – 19.7%, but
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Norin et al. [63] accomplished a higher LOD for SCFAs (0.2 mmol/kg wet feces). A big step towards more sensitive and faster GC method
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detections has been performed via the FID detector development. With limits of detection even in the low pg or fg range, FID has become the most widely and successfully used GC detector for volatile hydrocarbons and many carbon containing compounds. GC-FID detection methods are therefore mainly performed after a direct sample injection (or previously treated with acid). In fact, Schneider et al. [109] and Schneider et al. [110] performed a fast pretreatment and reported a sensitivity of 1 mmol.
ACCEPTED MANUSCRIPT Gråsten et al. [117] performed the SCFAs detection after Schooley et al., [145] who encountered some limiting factors for detection, due to the solvent
and
the
N-Methyl-N-t-butyldimethylsilyl
trifluoroacetamide
(MTBSTFA) peaks, which could present some problems in samples with
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trace levels of acids. In this case, derivatization was performed after the extraction and obtained an LOD and an LOQ which ranged from 0.3 – 3.0 ppm (0.3 ppm is 0.004 mmol/kg wet feces calculated for C3) and 2.0 –
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5.0 ppm (5.0 ppm is 0.067 mmol/kg wet feces calculated for C3),
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respectively. Furthermore, inter assay with CV of 3.49% for acetic acid and with CV of 8.70% for lactic acid and recovery rates between 92 109% were determined. An important role of pretreatment steps for the development of a more reliable SCFAs detection method in different
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biological materials has also been shown by Zheng et al. [123] and Gao et al. [24]. In fact, they performed chloroformate derivatization prior to GCMS and showed some advantages such as short reaction time at room
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temperature, acceptable efficiency (90.06 – 116.34% for SCFAs [123]), recovery (84.11 – 118.79% for analyte isotopes [123]) and a low LOD
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(from 20 to 1000 pg on-column of the test standards – approximately 0.058 mmol/kg wet feces calculated for C4 [123], from 10 to 500 pg oncolumn of the test standards – approximately 0.69 mmol/kg wet feces calculated for C5 [24]). However, LODs for acetic, propionic [24,123] and butyric [24] were not determined due to analytical interferences indicating a significant drawback as these SCFAs constitute the bulk of SCFAs in feces. Additionally, Zheng et al. [123] reported intra-day and inter-day
ACCEPTED MANUSCRIPT precision with an RSD below 5.99% and 8.43%, respectively. De Preter et al. [130] and Windey at al. [131] reached LODs from 0.25 mg/L to 25 mg/L in standard working solutions (i.e. approximately 3 mmol/kg wet feces calculated for C3) with a purge and trap pretreatment prior to GC-
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MS. Moreover, the intra assay variability was in the range of 3.3 – 9.7% RSD, the inter assay variability was demonstrated with an RSD range of 3.7 – 9.8% and the overall recovery of the compounds studied was 100%.
with
a
less
complicated
pretreatment
procedure
(without
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samples
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Nevertheless, García-Villalba et al. [120] analyzed the organic acid in fecal
derivatization) prior to GC-MS with LOD and LOQ ranging from 0.29 – 3.80 µg/g (0.006 mmol/kg wet feces calculated for C3) and 0.98 – 12.56 µg/g (0.02 mmol/kg wet feces calculated for C3), respectively. The inter-
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and intra assay variability values were below 2.6 and 5.6% respectively and the recovery values ranged from 82 - 105% for propionic, butyric,
High Performance Liquid Chromatography (HPLC)
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4.2
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isobutyric, valeric and isovaleric acid and around 65% for acetic acid.
HPLC is another method for SCFAs analysis and a good alternative to GC. The most commonly used technique is a reverse phase HPLC (RP-HPLC), where the stationary solid phase (column) is hydrophobic (non-polar) and the mobile liquid phase is hydrophilic (polar, watery). Due to the higher pressure, the mobile phase carrying analytes travels and the small stationary phase particles with a larger area allow for a better interaction
ACCEPTED MANUSCRIPT with the analytes. The greatest advantage of the HPLC over the GC technique is the use of lower running temperatures [146]. Similar to GC, specific combinations of pretreatments, columns, running conditions and
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detectors have to be optimized for a successful SCFAs analysis (Table 3).
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Table 3
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4.2.1 Pretreatment in HPLC detection methods
Like in GC, the sample pretreatment procedure in HPLC plays an important role. Derivatization of non-chromophoric SCFAs increases the and
Derivatization hydrazide
selectivity
techniques
of
the
derivatives
HPLC
that include
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sensitivity
have
been
the
detection
analysis
formation
performed
by
[25].
of fatty acid
using
a
nitro-
phenylhydrazine reagent alone [163], which is commonly used in
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derivatization of carboxylic acids, aldehydes and ketones, or by using a
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water-soluble carbodiimide reagent as the coupling agent that activates carboxyl groups for a spontaneous reaction with primary amines enabling a
peptide
immobilization
[165].
A
variety
of
purification
-
separation/extraction techniques have been applied before subjection of samples to HPLC, since samples must retain solubility in the liquid mobile phase. This may be argued by some authors, which used only simple centrifugation/filtration steps, before injecting the sample into HPLC [5,147,148].
ACCEPTED MANUSCRIPT The most widespread pretreatment method for fecal SCFAs analysis is simple acidification (deproteinization) with perchloric acid [149–158] or sulfuric acid [159–161]. Some authors pretreated the samples with steam [162] distillation before the acidification procedure. Especially with water-
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soluble samples, the extraction step may also be used. Some authors declared successful purification of samples by extraction with chloroform [167] or ether alone [165] or ether in combination with acetonitrile [164]
(acid
hydrazides)
before
the
extraction
step
with
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derivatization
SC
or hexane [163]. Tori et al. [165] and Underwood et al. [163] performed
disadvantages similar to those described at GC (see chapter 4.1.1).
4.2.2 HPLC columns
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RP columns, based on silica gel [29] bound to C4 (butyl), C8 (octyl, ODC), C18 (octadecyl), nitrile (cianopropyl) and phenyl (phenyl propyl) groups,
EP
are of common use in HPLC. Among RP-HPLC columns, the C18 phase column is most commonly used due to its high lipid resolution. However,
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in selected studies RP columns for SCFA analysis alone have been used in two publications [163,165]. . Nevertheless, some authors preferred using the ion-exclusion mode columns alone [149,157,160,161,164,166] or ion-exchange type of column
alone
[5,148,159,162].
The
basic
principle
of
the
ion
exclusion/exchange separation is in the interactions between ionic and polar analytes, ions in the eluent and ionic functional groups fixed to the
ACCEPTED MANUSCRIPT chromatographic support. The ion exchange mode is based on a competitive attraction between ions in the stationary and mobile phase. On the other hand, ion exclusion mode prevents ionized molecules from entering the pores of the support, leading to highly ionic molecules to
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elute first, followed by weakly ionized and non-ionic molecules last. When a mixture of weak acids, such as SCFAs in samples are expected, the use of the ion exclusion mode of column is more recommended as samples are
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not well separated, neither with ion exchange, nor with RP columns [168].
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In most cases the RP column has been used in combination with ionexclusion mode columns (in a dual-column mode, tandem mode) [150–
4.2.3 HPLC detectors
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156,158].
Several detectors have been successfully used with SCFAs detection upon
EP
HPLC separation. The most popular routine HPLC detector is based on UV absorption, which has the advantage of performing independently from
AC C
temperature changes, mobile phase flow speed and composition [29]. Literature revealed the use of UV detector by some authors [5,148,160– 165]. Nevertheless, the problem of SCFAs lacking chromophore [25,29] demands derivatization as a pretreatment when using an RP column [163,165], but not ion-exclusion or ion-exchange columns [5,147– 149,157,159–162,164,166,167]. Ferrario et al. [159] used a UV detector
ACCEPTED MANUSCRIPT concomitantly with a differential refractive index (RI) detector, which detects components in a solution based on the light refraction. A photo diode array (PDA) detector belongs to the group of variable
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wavelength detectors (VWD) where excited light is separated into different wavelengths. Absorption monitoring at different wavelengths allows for a simultaneous determination of more than one analyte. The advantage of
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such detectors are cost reductions on expensive solvents and time spent for performing an analysis [147].
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Apart from that, the literature review revealed a popularity in using conductivity detectors [150–156,158,167].. Conductivity detectors are mainly used to measure inorganic ions and small organic substances (organic acids and amines). It is a highly sensitive detector, but very to
temperature
variations.
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susceptible
Another
disadvantage
of
conductivity detectors, especially when dealing with a huge amount of
EP
samples, is its linkage to special and complicated instrumentation [169]. Kotani et al. [157] and Okazaki et al. [149] used an electrochemical
AC C
detector (ECD) which measures the oxidation – reduction potential of substances in the mobile phase. ECDs are very sensitive with some advantages [25], among which the most important may be a direct analysis of non-derivatized samples, skipping the pretreatment step and reducing analysis time [157]. The most advanced and sensitive detection is obtained by coupling HPLC to MS via electrospray ionization (ESI). Namely, Han et al. performed a
ACCEPTED MANUSCRIPT 13
combined chemical derivatization with LC/MS-MS.
C6 derivatives were
developed for the use as isotope-labelled internal standards, in order to compensate for ESI matrix effects and to obtain more specific and thorough quantitation of SCFAs in human fecal samples [169]. The
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combination enables a detection of analyte molecular mass, as well as
drawback is the instrumentation cost [165].
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4.2.4 HPLC-based methods performance
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type and local position of the substitutes in a carbon chain [29]. The major
Detection limits of fecal SCFAs by HPLC are apparently mostly influenced by the sensitivity of the applied detector (reviewed in Bielawska et al. [29]). The UV detector is the most commonly used detector in HPLC
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[170]. Moreover, Fritz et al. [148] performed a SCFAs detection after Ross et al. [171], who obtained sensitivities as low as 0.86 nmol (except for
EP
acetic acid) using a cation-exchange column and in most cases a UV detector. Recoveries ranged from 72 – 86% for most of the acids, except
AC C
for lactic acid, which was as low as 24%. The inter- and intra assay variability values were below 5.7 (8.4 for lactic acid) and 7.6%, respectively. Okazaki et al. [149] and Kotani et al. [157] used ECD and accomplished detection limits from 0.05 - 0.8 µmol/g (0.05 – 0.8 mmol/kg wet feces) and 40 pmol/single injection (0.04 mmol/kg wet feces; recovery in the range 92 - 98%, inter assay with RSD less than 2.7%) respectively. Valerio et al. [161] performed SCFAs detection after Valerio
ACCEPTED MANUSCRIPT et al. [172] who obtained an LOQ in the range between 0.09 - 1.72 µmol/g (0.09 - 1.72 mmol/kg wet feces).
Nuclear magnetic resonance (NMR)
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4.3
The NMR method is based on magnetic characteristics of particular atomic cores which absorb and emit electromagnetic radiations in a magnetic
SC
field. The produced energy appears at a specific resonance frequency which depends on the magnetic field and magnetic characteristics of
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specific isotopes. Isotope cores 1H [173–177] and
13
C [174] have been
used for SCFAs studies in fecal samples. NMR
is
a
quantitative
(absolute),
non-destructive,
repeatable
and
TE D
objective method. It has been mainly used for simultaneous tracing of different metabolites in a complex biological material in terms of chemical composition and concentrations. It is a comprehensive and enough
EP
sensitive method with the detection limits of metabolite concentration reaching the µM range and still decreasing [178,179]. The so called NMR
AC C
metabolic profiling enables the production of fast, stable and reproducible profiles [177]. It demands minimal sample pretreatment. Nevertheless, pretreatment methodology needs an optimization that enables the extraction of many different metabolites out of a complex biological material. Furthermore, it is recommended to use deuterated solvents in NMR experiments in order to correct the field strength and to avoid the huge solvent absorption that would otherwise spoil the 1H-NMR spectrum
ACCEPTED MANUSCRIPT [180]. This may result in an expensive, time-consuming procedure that may lead to significant analyte losses, especially when coping with big amounts of samples on a daily basis. At the same time, the method should be non-destructive and should cause only minor changes in
RI PT
material [177]. In fact, differences and contradictories in NMR's results show that the possible cause of low sensitivity (lower than that, obtained with GC-MS or LC-MS), may be indeed due to different pretreatment
SC
procedures and data acquisitions [173,177,179].
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The NMR application on feces [173] and fecal water samples [174–177] has been used to monitor human metabolic activity, especially the effect of microbiota on the regulation of host metabolism. NMR-based metabolic profiling could present the future disease diagnostic method because of
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the method’s advantages described above. However, instrumentation cost and sensitivity due to the poor pretreatment optimization methodology
Capillary Electrophoresis (CE)
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4.4
EP
still remain serious drawbacks.
CE is a technique that separates charged molecules in a capillary upon exposure to an electrical field. In the past 30 years, CE has been known as a good analytical technique for the detection of different organic molecules, including SCFAs. CE separation depends on the electrophoretic mobility of ions through the gel, being dependent on molecular mass, shape and charge of the molecule [181].
ACCEPTED MANUSCRIPT CE is especially used for separation of water-soluble compounds. The advantage of CE is its speed and minimal sample pretreatment procedure, which is very convenient in routine analysis. In fact, CE has been successfully implemented in analytical detection of SCFAs in different
RI PT
biological materials, including fecal samples [182]. Nevertheless, it has its disadvantages: low repeatability and reproducibility and the need for higher analyte concentration. Most of these limitations are due to the
SC
small amount of injected sample [183]. Garcia et al. [182] obtained the
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LOQ of standard solutions ranging from 10.4 to 49.6 µM (approximately 0.161 mmol/kg wet feces calculated for C3), accuracy (circa 68%, 18% and 68% for acetic, propionic and butyric acid, respectively), recovery (94.9 - 106.6%), 5.5 - 9.5% and from 8.1 - 14.5% for inter and intra
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assay respectively, by using a simple CE detection method. A combined detection technique with MS has solved many limitations in CE detection, raising the method’s reliability in detecting small molecules in the range
EP
from µM to nM. To our knowledge, combinations for the determination of
4.5
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SCFAs in fecal samples have not been applied yet.
Enzymatic detection of SCFAs SCFAs
One of the alternative SCFAs determination methods in feces is enzymatic detection, which relies on spectrophotometric measurement of enzymatic products obtained from SCFAs as substrates. Enzymes prefer stereospecific isomers in their metabolic reactions and they can therefore
ACCEPTED MANUSCRIPT differentiate between optical isomers. In this sense, lactate can be found in both D- and L-form and several research groups have enzymatically determined D- and L-lactate [6,85,96,97,99,102]. Although the assay is fast, disadvantages may be mainly related to cross-enzymatic reactions
RI PT
with other endogenous molecules as well as a potential presence of enzymatic inhibitors/accelerators in analytical samples. However, in terms of lactate determination, it is important to note that total lactate has been
AC C
EP
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successfully determined by GC [112,113] and HPLC [5,167] as well.
ACCEPTED MANUSCRIPT
5.
Summary and conclusion
Intestinal microbiota performs several important functions which maintain the human homeostasis in equilibrium. Changes in microbiota can lead to
production
of
bacterial
metabolites
like
RI PT
different pathological conditions in metabolism, such as disorders in the SCFAs.
A
comprehensive
understanding of SCFAs functional roles in the human body is essential
SC
due to several demonstrated connections between SCFAs, microbiota and metabolic diseases. Therefore, a need for a good and reliable analytical
pathological conditions.
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SCFA detection technique is crucial for the diagnosis of different
Due to the complicated composition of feces and the natural molecular characteristics of SCFAs, sample pretreatment procedures are almost
TE D
inevitable and the majority of research reports in the last 11 years have used it. However, additional steps prolong the time needed for analysis,
EP
they may lead to potential SCFAs losses and increase consumption of consumables and hence costs. Costs become a limiting factor when
AC C
dealing with large numbers of samples on a daily basis, in addition to the occupational exposure to chemicals needed during the pretreatment steps. Instead of traditional solvent extractions, researchers can opt for a modern SPME, solving many problems related to excessive solvent use and sample dilution. Few researchers applied simple acidification or even direct injection of samples into the chromatographic system. However,
ACCEPTED MANUSCRIPT negative effects of such pretreatment have merely caused a premature column failure and even decreased the detector lifespan. The choice of instrumentation for SCFAs analysis is usually limited to GC
most
analytical
and
biochemical
laboratories.
RI PT
and HPLC, as such equipment has become standard and thus accessible in Nonetheless,
several
detectors can be applied; from standard UV and PDA to more sensitive
SC
and expensive FID and MS detectors. GC-MS and HPLC-MS combinations have pushed the limits of detection well below the actual concentrations of
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SCFAs found in feces (see section 1 – Introduction). Therefore, for detection, MS could be a reasonable first choice in specific situations when low-concentrations of FAs are being followed, like branched chain fatty acids (BCFAs) as discussed below. In fact, GC methods, beside some
TE D
disadvantages, seem to be a justifiable choice for a reliable SCFAs detection. Emerging techniques like NMR based methods have satisfied the
need
for
a
simple
sample
pretreatment
procedure,
but
its
EP
contradictories in results related to sensitivity may be due to under-
AC C
optimized pretreatments. Moreover, NMR instrumentation availability and costs are beyond the reach of most laboratories. Likewise, CE needs minor and uncomplicated sample pretreatment, but lacks repeatability and reproducibility. Enzymatic detection is simple, but its application has been more or less applied for the distinction between optical isomers of lactate. Surprisingly, very few researchers have mentioned or showed method performance parameters such as LOD, LOQ, recovery and precision (in
ACCEPTED MANUSCRIPT terms of reproducibility and repeatability) of fecal SCFAs that would allow an
overall
estimation
concentrations
of
of
acetic,
method propionic
performance. and
butyric
In
some
acids
have
cases, been
determined, while less abundant lactic and BCFAs have not been
RI PT
measured. BCFAs are mainly saturated, bearing one or more methyl groups on the carbon chain [184]. They can be found in minor amounts in human feces [5], but their role in the gastrointestinal system is still not
SC
fully understood. On the other hand, lactate could be an important
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indicator in specific physiological or pathological issues, i.e. as a specific
Bifidobacterium metabolite [6,85,96] or an indicator of nonsteroidal antiinflammatory drug (NSAID) abuse [102].
In conclusion, most described SCFAs analytical methods have advantages
TE D
and drawbacks. In terms of expected SCFAs concentrations in fecal samples, the described method performance for SCFAs detection with GC/FID or MS, HPLC,NMR and even CE methods are all acceptable. It is recommended
that
EP
thus
researchers
consider
the
instrumentation
AC C
availability, time consumption and costs when choosing a proper detection method for fecal SCFAs in research, routine use in clinics and potential automation. This may however also depend on the future progress and knowledge about SCFAs in health and diseases.
Author contributions: contributions All the authors have accepted responsibility for the entire content of this submitted manuscript and approved submission.
ACCEPTED MANUSCRIPT Funding: unding This research was partially supported by the Slovenian research program P1-0164 (C) funded by the Slovenian Research Agency. Acknowledgements: Acknowledgements The authors would like to thank Katja Težak for
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language editing.
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Conflicts of interest: interest The authors declare no conflicts of interest.
ACCEPTED MANUSCRIPT
References [1]
J.K. Nicholson, E. Holmes, J. Kinross, R. Burcelin, G. Gibson, W. Jia, S. Pettersson, Host-gut microbiota metabolic interactions., Science.
[2]
RI PT
336 (2012) 1262–7. G. den Besten, K. van Eunen, A.K. Groen, K. Venema, D.-J.
Reijngoud, B.M. Bakker, The role of short-chain fatty acids in the
[3]
M AN U
J. Lipid Res. 54 (2013) 2325–40.
SC
interplay between diet, gut microbiota, and host energy metabolism.,
H. Hasegawa, T. Fukushima, J.-A. Lee, K. Tsukamoto, K. Moriya, Y. Ono, K. Imai, Determination of serum D-lactic and L-lactic acids in normal subjects and diabetic patients by column-switching HPLC with
(2003) 886–91. [4]
TE D
pre-column fluorescence derivatization., Anal. Bioanal. Chem. 377
E.P. Nyangale, D.S. Mottram, G.R. Gibson, Gut microbial activity,
EP
implications for health and disease: the potential role of metabolite
[5]
AC C
analysis., J. Proteome Res. 11 (2012) 5573–85. N. Huda-Faujan, A.S. Abdulamir, A.B. Fatimah, O.M. Anas, M.
Shuhaimi, A.M. Yazid, Y.Y. Loong, The impact of the level of the intestinal short chain Fatty acids in inflammatory bowel disease patients versus healthy subjects., Open Biochem. J. 4 (2010) 53–8.
[6]
R. Mohan, C. Koebnick, J. Schildt, M. Mueller, M. Radke, M. Blaut, Effects of Bifidobacterium lactis Bb12 supplementation on body
ACCEPTED MANUSCRIPT weight, fecal pH, acetate, lactate, calprotectin, and IgA in preterm infants., Pediatr. Res. 64 (2008) 418–22. [7]
B.S. Ramakrishna, V.I. Mathan, Colonic dysfunction in acute
RI PT
diarrhoea: the role of luminal short chain fatty acids., Gut. 34 (1993) 1215–8. [8]
W. Scheppach, H.P. Bartram, F. Richter, Role of short-chain fatty
SC
acids in the prevention of colorectal cancer., Eur. J. Cancer. 31A (1990) 1077–80.
L. Galland, The Gut Microbiome and the Brain, J. Med. Food. 17 (2014) 1261–1272.
M AN U
[9]
[10] N.M. Moreau, S.M. Goupry, J.P. Antignac, F.J. Monteau, B.J. Le Bizec,
TE D
M.M. Champ, L.J. Martin, H.J. Dumon, Simultaneous measurement of plasma concentrations and 13C-enrichment of short-chain fatty acids, lactic acid and ketone bodies by gas chromatography coupled
EP
to mass spectrometry., J. Chromatogr. B. Analyt. Technol. Biomed.
AC C
Life Sci. 784 (2003) 395–403. [11] I. Yanagisawa, M. Yamane, T. Urayama, Simultaneous separation and sensitive determination of free fatty acids in blood plasma by high-performance liquid chromatography, J. Chromatogr. B Biomed. Sci. Appl. 345 (1985) 229–240. [12] C. Bachmann, J.P. Colombo, J. Berüter, Short chain fatty acids in plasma and brain: quantitative determination by gas
ACCEPTED MANUSCRIPT chromatography., Clin. Chim. Acta. 92 (1979) 153–9. [13] G. Zhao, J. Liu, M. Nyman, J.Å. Jönsson, Determination of shortchain fatty acids in serum by hollow fiber supported liquid membrane
RI PT
extraction coupled with gas chromatography, J. Chromatogr. B. 846 (2007) 202–208.
[14] L.J. Horspool, Q.A. McKellar, Determination of short-chain fatty acids
SC
in equine caecal liquor by ion exchange high performance liquid
5 (1991) 202–6.
M AN U
chromatography after solid phase extraction., Biomed. Chromatogr.
[15] J. Schiffels, M.E.M. Baumann, T. Selmer, Facile analysis of shortchain fatty acids as 4-nitrophenyl esters in complex anaerobic fermentation samples by high performance liquid chromatography, J.
TE D
Chromatogr. A. 1218 (2011) 5848–5851. [16] K. Fischer, Environmental analysis of aliphatic carboxylic acids by
AC C
173.
EP
ion-exclusion chromatography, Anal. Chim. Acta. 465 (2002) 157–
[17] M.-H. Yang, Y.-M. Choong, A rapid gas chromatographic method for direct determination of short-chain (C2–C12) volatile organic acids in foods, Food Chem. 75 (2001) 101–108.
[18] G. Manni, F. Caron, Calibration and determination of volatile fatty acids in waste leachates by gas chromatography, J. Chromatogr. A. 690 (1995) 237–242.
ACCEPTED MANUSCRIPT [19] Y. Li, Y. Xiong, Q. Liang, C. Fang, C. Wang, Application of headspace single-drop microextraction coupled with gas chromatography for the determination of short-chain fatty acids in RuO4 oxidation products
RI PT
of asphaltenes, J. Chromatogr. A. 1217 (2010) 3561–3566. [20] B. Baena, A. Cifuentes, C. Barbas, Analysis of carboxylic acids in biological fluids by capillary electrophoresis., Electrophoresis. 26
SC
(2005) 2622–36.
[21] G.C. Cochran, A review of the analysis of free fatty acids [C2-C6]., J.
M AN U
Chromatogr. Sci. 13 (1975) 440–7.
[22] J.B. Ewaschuk, G.A. Zello, J.M. Naylor, D.R. Brocks, Metabolic acidosis: separation methods and biological relevance of organic acids and lactic acid enantiomers., J. Chromatogr. B. Analyt.
TE D
Technol. Biomed. Life Sci. 781 (2002) 39–56. [23] J. Fernandes, W. Su, S. Rahat-Rozenbloom, T.M.S. Wolever, E.M.
EP
Comelli, Adiposity, gut microbiota and faecal short chain fatty acids
AC C
are linked in adult humans, Nutr. Diabetes. 4 (2014) e121. [24] X. Gao, E. Pujos-Guillot, J.-F. Martin, P. Galan, C. Juste, W. Jia, J.-L. Sebedio, Metabolite analysis of human fecal water by gas chromatography/mass spectrometry with ethyl chloroformate derivatization., Anal. Biochem. 393 (2009) 163–75. [25] E.. Lima, D.S.. Abdalla, High-performance liquid chromatography of fatty acids in biological samples, Anal. Chim. Acta. 465 (2002) 8–91.
ACCEPTED MANUSCRIPT [26] G. Zhao, M. Nyman, J.A. Jönsson, Rapid determination of short-chain fatty acids in colonic contents and faeces of humans and rats by acidified water-extraction and direct-injection gas chromatography.,
RI PT
Biomed. Chromatogr. 20 (2006) 674–82. [27] J.A. T., M.A.J. P., Gas-liquid partition chromatography: the
separation and micro-estimation of volatile fatty acids from formic
SC
acid to dodecanoic acid, (1952).
[28] F.A. Guthrie, Introduction of instrumental analysis (Braun, Robert
M AN U
D.), J. Chem. Educ. 65 (1988) A336.
[29] K. Bielawska, I. Dziakowska, W. Roszkowska-Jakimiec, Chromatographic determination of fatty acids in biological material.,
TE D
Toxicol. Mech. Methods. 20 (2010) 526–37.
[30] J. Kopecný, J. Mrázek, K. Fliegerová, P. Frühauf, L. Tucková, The intestinal microflora of childhood patients with indicated celiac
EP
disease., Folia Microbiol. (Praha). 53 (2008) 214–6.
AC C
[31] H.-M. Chen, Y.-N. Yu, J.-L. Wang, Y.-W. Lin, X. Kong, C.-Q. Yang, L. Yang, Z.-J. Liu, Y.-Z. Yuan, F. Liu, J.-X. Wu, L. Zhong, D.-C. Fang, W. Zou, J.-Y. Fang, Decreased dietary fiber intake and structural alteration of gut microbiota in patients with advanced colorectal adenoma., Am. J. Clin. Nutr. 97 (2013) 1044–52. [32] A. Klein, U. Friedrich, H. Vogelsang, G. Jahreis, Lactobacillus acidophilus 74-2 and Bifidobacterium animalis subsp lactis DGCC 420
ACCEPTED MANUSCRIPT modulate unspecific cellular immune response in healthy adults., Eur. J. Clin. Nutr. 62 (2008) 584–93. [33] A. Fechner, K. Fenske, G. Jahreis, Effects of legume kernel fibres and
RI PT
citrus fibre on putative risk factors for colorectal cancer: a randomised, double-blind, crossover human intervention trial., Nutr. J. 12 (2013) 101.
SC
[34] A. Roessler, S.D. Forssten, M. Glei, A.C. Ouwehand, G. Jahreis, The effect of probiotics on faecal microbiota and genotoxic activity of
M AN U
faecal water in patients with atopic dermatitis: a randomized, placebo-controlled study., Clin. Nutr. 31 (2012) 22–9. [35] E. Nistal, A. Caminero, S. Vivas, J.M. Ruiz de Morales, L.E. Sáenz de Miera, L.B. Rodríguez-Aparicio, J. Casqueiro, Differences in faecal
TE D
bacteria populations and faecal bacteria metabolism in healthy adults and celiac disease patients., Biochimie. 94 (2012) 1724–9.
EP
[36] E. Beards, K. Tuohy, G. Gibson, A human volunteer study to assess the impact of confectionery sweeteners on the gut microbiota
AC C
composition., Br. J. Nutr. 104 (2010) 701–8.
[37] K. Turunen, E. Tsouvelakidou, T. Nomikos, K.C. Mountzouris, D. Karamanolis, J. Triantafillidis, A. Kyriacou, Impact of beta-glucan on the faecal microbiota of polypectomized patients: a pilot study., Anaerobe. 17 (2011) 403–6. [38] R.A. Kekkonen, R. Holma, K. Hatakka, T. Suomalainen, T. Poussa, H.
ACCEPTED MANUSCRIPT Adlercreutz, R. Korpela, A probiotic mixture including galactooligosaccharides decreases fecal β-glucosidase activity but does not affect serum enterolactone concentration in men during a
RI PT
two-week intervention., J. Nutr. 141 (2011) 870–6. [39] M. Finney, J. Smullen, H.A. Foster, S. Brokx, D.M. Storey, Effects of low doses of lactitol on faecal microflora, pH, short chain fatty acids
SC
and gastrointestinal symptomology., Eur. J. Nutr. 46 (2007) 307–14. [40] A. Cuervo, N. Salazar, P. Ruas-Madiedo, M. Gueimonde, S. González,
M AN U
Fiber from a regular diet is directly associated with fecal short-chain fatty acid concentrations in the elderly., Nutr. Res. 33 (2013) 811–6. [41] N. Salazar, E.M. Dewulf, A.M. Neyrinck, L.B. Bindels, P.D. Cani, J. Mahillon, W.M. de Vos, J.-P. Thissen, M. Gueimonde, C.G. de Los
TE D
Reyes-Gavilán, N.M. Delzenne, Inulin-type fructans modulate intestinal Bifidobacterium species populations and decrease fecal
EP
short-chain fatty acids in obese women., Clin. Nutr. (2014). [42] S. Arboleya, A. Binetti, N. Salazar, N. Fernández, G. Solís, A.
AC C
Hernández-Barranco, A. Margolles, C.G. de Los Reyes-Gavilán, M. Gueimonde, Establishment and development of intestinal microbiota in preterm neonates., FEMS Microbiol. Ecol. 79 (2012) 763–72.
[43] X.-M. Ben, J. Li, Z.-T. Feng, S.-Y. Shi, Y.-D. Lu, R. Chen, X.-Y. Zhou, Low level of galacto-oligosaccharide in infant formula stimulates growth of intestinal Bifidobacteria and Lactobacilli., World J. Gastroenterol. 14 (2008) 6564–8.
ACCEPTED MANUSCRIPT [44] J.G. Muir, E.G.W. Yeow, J. Keogh, C. Pizzey, A.R. Bird, K. Sharpe, K. O’Dea, F.A. Macrae, Combining wheat bran with resistant starch has more beneficial effects on fecal indexes than does wheat bran alone.,
RI PT
Am. J. Clin. Nutr. 79 (2004) 1020–8. [45] S. Wildt, I. Nordgaard-Lassen, F. Bendtsen, J.J. Rumessen, Metabolic and inflammatory faecal markers in collagenous colitis., Eur. J.
SC
Gastroenterol. Hepatol. 19 (2007) 567–74.
[46] J. Fernandes, A. Wang, W. Su, S.R. Rozenbloom, A. Taibi, E.M.
M AN U
Comelli, T.M.S. Wolever, Age, dietary fiber, breath methane, and fecal short chain fatty acids are interrelated in Archaea-positive humans., J. Nutr. 143 (2013) 1269–75.
[47] J. Ou, F. Carbonero, E.G. Zoetendal, J.P. DeLany, M. Wang, K.
TE D
Newton, H.R. Gaskins, S.J.D. O’Keefe, Diet, microbiota, and microbial metabolites in colon cancer risk in rural Africans and
EP
African Americans., Am. J. Clin. Nutr. 98 (2013) 111–20. [48] L. Wang, C.T. Christophersen, M.J. Sorich, J.P. Gerber, M.T. Angley,
AC C
M.A. Conlon, Elevated fecal short chain fatty acid and ammonia concentrations in children with autism spectrum disorder., Dig. Dis. Sci. 57 (2012) 2096–102.
[49] A.L. McOrist, G.C.J. Abell, C. Cooke, K. Nyland, Bacterial population dynamics and faecal short-chain fatty acid (SCFA) concentrations in healthy humans., Br. J. Nutr. 100 (2008) 138–46.
ACCEPTED MANUSCRIPT [50] A.L. McOrist, R.B. Miller, A.R. Bird, J.B. Keogh, M. Noakes, D.L. Topping, M.A. Conlon, Fecal butyrate levels vary widely among individuals but are usually increased by a diet high in resistant
RI PT
starch., J. Nutr. 141 (2011) 883–9. [51] D.L. Worthley, R.K. Le Leu, V.L. Whitehall, M. Conlon, C.
Christophersen, D. Belobrajdic, K.-A. Mallitt, Y. Hu, N. Irahara, S.
SC
Ogino, B.A. Leggett, G.P. Young, A human, double-blind, placebocontrolled, crossover trial of prebiotic, probiotic, and synbiotic
M AN U
supplementation: effects on luminal, inflammatory, epigenetic, and epithelial biomarkers of colorectal cancer., Am. J. Clin. Nutr. 90 (2009) 578–86.
[52] D.L. Worthley, V.L.J. Whitehall, R.K. Le Leu, N. Irahara, R.L.
TE D
Buttenshaw, K.-A. Mallitt, S.A. Greco, I. Ramsnes, J. Winter, Y. Hu, S. Ogino, G.P. Young, B.A. Leggett, DNA methylation in the rectal
EP
mucosa is associated with crypt proliferation and fecal short-chain fatty acids., Dig. Dis. Sci. 56 (2011) 387–96.
AC C
[53] K. Kajander, L. Krogius-Kurikka, T. Rinttilä, H. Karjalainen, A. Palva, R. Korpela, Effects of multispecies probiotic supplementation on intestinal microbiota in irritable bowel syndrome., Aliment. Pharmacol. Ther. 26 (2007) 463–73. [54] G.C.J. Abell, M.A. Conlon, A.L. Mcorist, Methanogenic archaea in adult human faecal samples are inversely related to butyrate concentration, Microb. Ecol. Heal. Dis. 18 (2006).
ACCEPTED MANUSCRIPT [55] G.D. Brinkworth, M. Noakes, P.M. Clifton, A.R. Bird, Comparative effects of very low-carbohydrate, high-fat and high-carbohydrate, low-fat weight-loss diets on bowel habit and faecal short-chain fatty
RI PT
acids and bacterial populations., Br. J. Nutr. 101 (2009) 1493–502. [56] A.R. Bird, M.S. Vuaran, R.A. King, M. Noakes, J. Keogh, M.K. Morell, D.L. Topping, Wholegrain foods made from a novel high-amylose
SC
barley variety (Himalaya 292) improve indices of bowel health in human subjects., Br. J. Nutr. 99 (2008) 1032–40.
M AN U
[57] J. Valeur, M.H. Morken, E. Norin, T. Midtvedt, A. Berstad, Intestinal fermentation in patients with self-reported food hypersensitivity: painful, but protective?, Clin. Exp. Gastroenterol. 3 (2010) 65–70. [58] J. Valeur, M.H. Morken, E. Norin, T. Midtvedt, A. Berstad,
TE D
Carbohydrate intolerance in patients with self-reported food hypersensitivity: comparison of lactulose and glucose., Scand. J.
EP
Gastroenterol. 44 (2009) 1416–23. [59] B. Tjellström, L. Högberg, L. Stenhammar, K.-E. Magnusson, T.
AC C
Midtvedt, E. Norin, T. Sundqvist, Effect of exclusive enteral nutrition on gut microflora function in children with Crohn’s disease., Scand. J. Gastroenterol. 47 (2012) 1454–9.
[60] B. Tjellström, L. Stenhammar, L. Högberg, K. Fälth-Magnusson, K.-E. Magnusson, T. Midtvedt, T. Sundqvist, E. Norin, Screening-detected and symptomatic untreated celiac children show similar gut microflora-associated characteristics., Scand. J. Gastroenterol. 45
ACCEPTED MANUSCRIPT (2010) 1059–62. [61] B. Tjellström, L. Stenhammar, T. Sundqvist, K. Fälth-Magnusson, E. Hollén, K.-E. Magnusson, E. Norin, T. Midtvedt, L. Högberg, The
RI PT
effects of oats on the function of gut microflora in children with coeliac disease., Aliment. Pharmacol. Ther. 39 (2014) 1156–60.
[62] B. Tjellström, L. Högberg, L. Stenhammar, K. Fälth-Magnusson, K.-E.
SC
Magnusson, E. Norin, T. Sundqvist, T. Midtvedt, Faecal short-chain fatty acid pattern in childhood coeliac disease is normalised after
M AN U
more than one year’s gluten-free diet., Microb. Ecol. Health Dis. 24 (2013).
[63] E. Norin, T. Midtvedt, B. Björkstén, Development of Faecal ShortChain Fatty Acid Pattern during the First Year of Life in Estonian and
TE D
Swedish Infants, Microb. Ecol. Heal. Dis. 16 (2004). [64] T. Midtvedt, E. Zabarovsky, E. Norin, J. Bark, R. Gizatullin, V.
EP
Kashuba, O. Ljungqvist, V. Zabarovska, R. Möllby, I. Ernberg, Increase of faecal tryptic activity relates to changes in the intestinal
AC C
microbiome: analysis of Crohn’s disease with a multidisciplinary platform., PLoS One. 8 (2013) e66074.
[65] A. Sandin, L. Bråbäck, E. Norin, B. Björkstén, Faecal short chain fatty acid pattern and allergy in early childhood., Acta Paediatr. 98 (2009) 823–7. [66] T.L. Weir, D.K. Manter, A.M. Sheflin, B.A. Barnett, A.L. Heuberger,
ACCEPTED MANUSCRIPT E.P. Ryan, Stool microbiome and metabolome differences between colorectal cancer patients and healthy adults., PLoS One. 8 (2013) e70803.
RI PT
[67] Y. Bouhnik, L. Raskine, K. Champion, C. Andrieux, S. Penven, H. Jacobs, G. Simoneau, Prolonged administration of low-dose inulin stimulates the growth of bifidobacteria in humans, Nutr. Res. 27
SC
(2007) 187–193.
[68] J.B. Adams, L.J. Johansen, L.D. Powell, D. Quig, R.A. Rubin,
M AN U
Gastrointestinal flora and gastrointestinal status in children with autism--comparisons to typical children and correlation with autism severity., BMC Gastroenterol. 11 (2011) 22.
[69] A. Caminero, E. Nistal, L. Arias, S. Vivas, I. Comino, A. Real, C.
TE D
Sousa, J.M.R. de Morales, M.A. Ferrero, L.B. Rodríguez-Aparicio, J. Casqueiro, A gluten metabolism study in healthy individuals shows
293–9.
EP
the presence of faecal glutenasic activity., Eur. J. Nutr. 51 (2012)
AC C
[70] A. Costabile, F. Fava, H. Röytiö, S.D. Forssten, K. Olli, J. Klievink, I.R. Rowland, A.C. Ouwehand, R.A. Rastall, G.R. Gibson, G.E. Walton, Impact of polydextrose on the faecal microbiota: a doubleblind, crossover, placebo-controlled feeding study in healthy human subjects., Br. J. Nutr. 108 (2012) 471–81. [71] F. Fava, R. Gitau, B.A. Griffin, G.R. Gibson, K.M. Tuohy, J.A. Lovegrove, The type and quantity of dietary fat and carbohydrate
ACCEPTED MANUSCRIPT alter faecal microbiome and short-chain fatty acid excretion in a metabolic syndrome “at-risk” population., Int. J. Obes. (Lond). 37 (2013) 216–23.
RI PT
[72] E.K. Mitsou, E. Kougia, T. Nomikos, M. Yannakoulia, K.C. Mountzouris, A. Kyriacou, Effect of banana consumption on faecal microbiota: a randomised, controlled trial., Anaerobe. 17 (2011)
SC
384–7. doi:10.1016/j.anaerobe.2011.03.018.
[73] G.E. Walton, R.A. Rastall, M. Martini, C. Williams, R. Jeffries, G.R.
M AN U
Gibson, A double-blind, placebo controlled human study investigating the effects of coffee derived manno-oligosaccharides on the faecal microbiota of a healthy adult population., Int. J. Probiotics Prebiotics. (2010).
TE D
[74] D. Goossens, D. Jonkers, M. Russel, A. Thijs, A. van den Bogaard, E. Stobberingh, R. Stockbrügger, Survival of the probiotic, L. plantarum
EP
299v and its effects on the faecal bacterial flora, with and without gastric acid inhibition, Dig. Liver Dis. 37 (2005) 44–50.
AC C
[75] C. Karlsson, S. Ahrné, G. Molin, A. Berggren, I. Palmquist, G.N. Fredrikson, B. Jeppsson, Probiotic therapy to men with incipient arteriosclerosis initiates increased bacterial diversity in colon: a randomized controlled trial., Atherosclerosis. 208 (2010) 228–33. [76] H. Zhang, J. Sun, X. Liu, C. Hong, Y. Zhu, A. Liu, S. Li, H. Guo, F. Ren, Lactobacillus paracasei subsp. paracasei LC01 positively modulates intestinal microflora in healthy young adults., J. Microbiol.
ACCEPTED MANUSCRIPT 51 (2013) 777–82. [77] A.J. Wallace, S.L. Eady, D.C. Hunter, M.A. Skinner, L. Huffman, J. Ansell, P. Blatchford, M. Wohlers, T.D. Herath, D. Hedderley, D.
RI PT
Rosendale, H. Stoklosinski, T. McGhie, D. Sun-Waterhouse, C. Redman, No difference in fecal levels of bacteria or short chain fatty acids in humans, when consuming fruit juice beverages containing
SC
fruit fiber, fruit polyphenols, and their combination., Nutr. Res. 35 (2015) 23–34.
M AN U
[78] P. Ramnani, A. Costabile, A.G.R. Bustillo, G.R. Gibson, A randomised, double- blind, cross-over study investigating the prebiotic effect of agave fructans in healthy human subjects., J. Nutr. Sci. 4 (2015) e10.
TE D
[79] L. Achour, S. Nancey, D. Moussata, I. Graber, B. Messing, B. Flourié, Patients with a short bowel syndrome.Faecal bacterial mass and
233–8.
EP
energetic losses in healthy humans, Eur. J. Clin. Nutr. 61 (2007)
AC C
[80] C.-H. Yen, Y.-H. Tseng, Y.-W. Kuo, M.-C. Lee, H.-L. Chen, Long-term supplementation of isomalto-oligosaccharides improved colonic microflora profile, bowel function, and blood cholesterol levels in constipated elderly people--a placebo-controlled, diet-controlled trial., Nutrition. 27 (2011) 445–50. [81] T.F.S. Teixeira, Ł. Grześkowiak, S.C.C. Franceschini, J. Bressan, C.L.L.F. Ferreira, M.C.G. Peluzio, Higher level of faecal SCFA in
ACCEPTED MANUSCRIPT women correlates with metabolic syndrome risk factors., Br. J. Nutr. 109 (2013) 914–9. [82] J.M. Clarke, D.L. Topping, C.T. Christophersen, A.R. Bird, K. Lange,
RI PT
I. Saunders, L. Cobiac, Butyrate esterified to starch is released in the human gastrointestinal tract., Am. J. Clin. Nutr. 94 (2011) 1276–83. [83] J. Slavin, J. Feirtag, Chicory inulin does not increase stool weight or
SC
speed up intestinal transit time in healthy male subjects., Food Funct. 2 (2011) 72–7.
M AN U
[84] G.A. Spiller, J.A. Story, E.J. Furumoto, J.C. Chezem, M. Spiller, Effect of tartaric acid and dietary fibre from sun-dried raisins on colonic function and on bile acid and volatile fatty acid excretion in healthy
TE D
adults., Br. J. Nutr. 90 (2003) 803–7.
[85] O.C. Thompson-Chagoyan, M. Fallani, J. Maldonado, J.M. Vieites, S. Khanna, C. Edwards, J. Doré, A. Gil, Faecal microbiota and short-
EP
chain fatty acid levels in faeces from infants with cow’s milk protein
AC C
allergy., Int. Arch. Allergy Immunol. 156 (2011) 325–32. [86] H.M. Staudacher, M.C.E. Lomer, J.L. Anderson, J.S. Barrett, J.G. Muir, P.M. Irving, K. Whelan, Fermentable carbohydrate restriction reduces luminal bifidobacteria and gastrointestinal symptoms in patients with irritable bowel syndrome., J. Nutr. 142 (2012) 1510–8. [87] K. Whelan, P.A. Judd, K.M. Tuohy, G.R. Gibson, V.R. Preedy, M.A. Taylor, Fecal microbiota in patients receiving enteral feeding are
ACCEPTED MANUSCRIPT highly variable and may be altered in those who develop diarrhea., Am. J. Clin. Nutr. 89 (2009) 240–7. [88] H.A. Majid, P.W. Emery, K. Whelan, Faecal microbiota and short-
RI PT
chain fatty acids in patients receiving enteral nutrition with standard or fructo-oligosaccharides and fibre-enriched formulas., J. Hum. Nutr. Diet. 24 (2011) 260–8.
SC
[89] K. Whelan, P.A. Judd, V.R. Preedy, R. Simmering, A. Jann, M.A. Taylor, Fructooligosaccharides and fiber partially prevent the
M AN U
alterations in fecal microbiota and short-chain fatty acid concentrations caused by standard enteral formula in healthy humans., J. Nutr. 135 (2005) 1896–902.
[90] H.A. Majid, J. Cole, P.W. Emery, K. Whelan, Additional
TE D
oligofructose/inulin does not increase faecal bifidobacteria in critically ill patients receiving enteral nutrition: a randomised controlled trial.,
EP
Clin. Nutr. 33 (2014) 966–72..
[91] C. Lefranc-Millot, L. Guérin-Deremaux, D. Wils, C. Neut, L.E. Miller,
AC C
M.H. Saniez-Degrave, Impact of a resistant dextrin on intestinal ecology: how altering the digestive ecosystem with NUTRIOSE®, a soluble fibre with prebiotic properties, may be beneficial for health., J. Int. Med. Res. 40 (2012) 211–24.
[92] R. Kumari, V. Ahuja, J. Paul, Fluctuations in butyrate-producing bacteria in ulcerative colitis patients of North India., World J. Gastroenterol. 19 (2013) 3404–14.
ACCEPTED MANUSCRIPT [93] A.L. Hovey, G.P. Jones, H.M. Devereux, K.Z. Walker, Whole cereal and legume seeds increase faecal short chain fatty acids compared to ground seeds., Asia Pac. J. Clin. Nutr. 12 (2003) 477–82.
RI PT
[94] P. Friederich, J. Verschuur, B.W.H. van Heumen, H.M.J. Roelofs, M. Berkhout, I.D. Nagtegaal, M.G.H. van Oijen, J.H.J.M. van Krieken, W.H.M. Peters, F.M. Nagengast, Effects of intervention with sulindac
SC
and inulin/VSL#3 on mucosal and luminal factors in the pouch of
patients with familial adenomatous polyposis., Int. J. Colorectal Dis.
M AN U
26 (2011) 575–82.
[95] H.D. Holscher, K.L. Faust, L.A. Czerkies, R. Litov, E.E. Ziegler, H. Lessin, T. Hatch, S. Sun, K.A. Tappenden, Effects of prebioticcontaining infant formula on gastrointestinal tolerance and fecal
TE D
microbiota in a randomized controlled trial., JPEN. J. Parenter. Enteral Nutr. 36 (2012) 95S–105S.
EP
[96] A.M. Bakker-Zierikzee, M.S. Alles, J. Knol, F.J. Kok, J.J.M. Tolboom, J.G. Bindels, Effects of infant formula containing a mixture of
AC C
galacto- and fructo-oligosaccharides or viable Bifidobacterium animalis on the intestinal microflora during the first 4 months of life., Br. J. Nutr. 94 (2005) 783–90.
[97] R.A. Reimer, X. Pelletier, I.G. Carabin, M.R. Lyon, R.J. Gahler, S. Wood, Faecal short chain fatty acids in healthy subjects participating in a randomised controlled trial examining a soluble highly viscous polysaccharide versus control., J. Hum. Nutr. Diet. 25 (2012) 373–7.
ACCEPTED MANUSCRIPT [98] S. Delgado, P. Ruas-Madiedo, A. Suárez, B. Mayo, Interindividual differences in microbial counts and biochemical-associated variables in the feces of healthy Spanish adults., Dig. Dis. Sci. 51 (2006) 737–
RI PT
43. [99] A. Gostner, M. Blaut, V. Schäffer, G. Kozianowski, S. Theis, M.
Klingeberg, Y. Dombrowski, D. Martin, S. Ehrhardt, D. Taras, A.
SC
Schwiertz, B. Kleessen, H. Lührs, J. Schauber, D. Dorbath, T. Menzel, W. Scheppach, Effect of isomalt consumption on faecal microflora
M AN U
and colonic metabolism in healthy volunteers., Br. J. Nutr. 95 (2006) 40–50.
[100] M.O. Weickert, A.M. Arafat, M. Blaut, C. Alpert, N. Becker, V. Leupelt, N. Rudovich, M. Möhlig, A.F. Pfeiffer, Changes in dominant
TE D
groups of the gut microbiota do not explain cereal-fiber induced improvement of whole-body insulin sensitivity., Nutr. Metab. (Lond).
EP
8 (2011) 90.
[101] B. Kleessen, S. Schwarz, A. Boehm, H. Fuhrmann, A. Richter, T.
AC C
Henle, M. Krueger, Jerusalem artichoke and chicory inulin in bakery products affect faecal microbiota of healthy volunteers., Br. J. Nutr. 98 (2007) 540–9.
[102] K. Tiihonen, S. Tynkkynen, A. Ouwehand, T. Ahlroos, N. Rautonen, The effect of ageing with and without non-steroidal antiinflammatory drugs on gastrointestinal microbiology and immunology., Br. J. Nutr. 100 (2008) 130–7.
ACCEPTED MANUSCRIPT [103] J. Maldonado, F. Lara-Villoslada, S. Sierra, L. Sempere, M. Gómez, J.M. Rodriguez, J. Boza, J. Xaus, M. Olivares, Safety and tolerance of the human milk probiotic strain Lactobacillus salivarius CECT5713 in
RI PT
6-month-old children., Nutrition. 26 (2010) 1082–7. [104] M. Olivares, M.A.P. Díaz-Ropero, N. Gómez, F. Lara-Villoslada, S. Sierra, J.A. Maldonado, R. Martín, E. López-Huertas, J.M. Rodríguez,
SC
J. Xaus, Oral administration of two probiotic strains, Lactobacillus gasseri CECT5714 and Lactobacillus coryniformis CECT5711,
M AN U
enhances the intestinal function of healthy adults., Int. J. Food Microbiol. 107 (2006) 104–11.
[105] Z. Liu, Z. Xu, M. Han, B.-H. Guo, Efficacy of pasteurised yoghurt in improving chronic constipation: A randomised, double-blind, placebo-
TE D
controlled trial, Int. Dairy J. 40 (2015) 1–5.
[106] S. Sierra, F. Lara-Villoslada, L. Sempere, M. Olivares, J. Boza, J.
EP
Xaus, Intestinal and immunological effects of daily oral administration of Lactobacillus salivarius CECT5713 to healthy
AC C
adults., Anaerobe. 16 (2010) 195–200.
[107] M.J. Bernal, M.J. Periago, R. Martínez, I. Ortuño, M. Sánchez-Solís, G. Ros, F. Romero, P. Abellán, Effects of infant cereals with different carbohydrate profiles on colonic function--randomised and doubleblind clinical trial in infants aged between 6 and 12 months--pilot study., Eur. J. Pediatr. 172 (2013) 1535–42. [108] A.S. Klosterbuer, M.A.J. Hullar, F. Li, E. Traylor, J.W. Lampe, W.
ACCEPTED MANUSCRIPT Thomas, J.L. Slavin, Gastrointestinal effects of resistant starch, soluble maize fibre and pullulan in healthy adults., Br. J. Nutr. 110 (2013) 1068–74.
RI PT
[109] S.-M. Schneider, F. Girard-Pipau, J. Filippi, X. Hebuterne, D. Moyse, G.-C. Hinojosa, A. Pompei, P. Rampal, Effects of Saccharomyces
boulardii on fecal short-chain fatty acids and microflora in patients on
SC
long-term total enteral nutrition., World J. Gastroenterol. 11 (2005) 6165–9.
M AN U
[110] S.M. Schneider, F. Girard-Pipau, R. Anty, E.G.M. van der Linde, B.J. Philipsen-Geerling, J. Knol, J. Filippi, K. Arab, X. Hébuterne, Effects of total enteral nutrition supplemented with a multi-fibre mix on
82–90.
TE D
faecal short-chain fatty acids and microbiota., Clin. Nutr. 25 (2006)
[111] C. Sierra, M.-J. Bernal, J. Blasco, R. Martínez, J. Dalmau, I. Ortuño,
EP
B. Espín, M.-I. Vasallo, D. Gil, M.-L. Vidal, D. Infante, R. Leis, J. Maldonado, J.-M. Moreno, E. Román, Prebiotic effect during the first
AC C
year of life in healthy infants fed formula containing GOS as the only prebiotic: a multicentre, randomised, double-blind and placebocontrolled trial., Eur. J. Nutr. 54 (2015) 89–99.
[112] E.J. Woodmansey, M.E.T. McMurdo, G.T. Macfarlane, S. Macfarlane, Comparison of compositions and metabolic activities of fecal microbiotas in young adults and in antibiotic-treated and nonantibiotic-treated elderly subjects., Appl. Environ. Microbiol. 70
ACCEPTED MANUSCRIPT (2004) 6113–22. [113] S. Macfarlane, S. Cleary, B. Bahrami, N. Reynolds, G.T. Macfarlane, Synbiotic consumption changes the metabolism and composition of
RI PT
the gut microbiota in older people and modifies inflammatory processes: a randomised, double-blind, placebo-controlled crossover study., Aliment. Pharmacol. Ther. 38 (2013) 804–16.
SC
[114] J.-M. Lecerf, F. Dépeint, E. Clerc, Y. Dugenet, C.N. Niamba, L. Rhazi, A. Cayzeele, G. Abdelnour, A. Jaruga, H. Younes, H. Jacobs, G.
M AN U
Lambrey, A.M. Abdelnour, P.R. Pouillart, Xylo-oligosaccharide (XOS) in combination with inulin modulates both the intestinal environment and immune status in healthy subjects, while XOS alone only shows prebiotic properties., Br. J. Nutr. 108 (2012) 1847–58.
TE D
[115] A. Schwiertz, D. Taras, K. Schäfer, S. Beijer, N.A. Bos, C. Donus, P.D. Hardt, Microbiota and SCFA in lean and overweight healthy
EP
subjects., Obesity (Silver Spring). 18 (2010) 190–5. [116] N. Larsen, F.K. Vogensen, R.J. Gøbel, K.F. Michaelsen, S.D.
AC C
Forssten, S.J. Lahtinen, M. Jakobsen, Effect of Lactobacillus salivarius Ls-33 on fecal microbiota in obese adolescents., Clin. Nutr. 32 (2013) 935–40.
[117] S.M. Gråsten, K.S. Juntunen, J. Mättö, O.T. Mykkänen, H. ElNezami, H. Adlercreutz, K.S. Poutanen, H.M. Mykkänen, High-fiber rye bread improves bowel function in postmenopausal women but does not cause other putatively positive changes in the metabolic
ACCEPTED MANUSCRIPT activity of intestinal microbiota, Nutr. Res. 27 (2007) 454–461. [118] A. Costabile, A. Klinder, F. Fava, A. Napolitano, V. Fogliano, C. Leonard, G.R. Gibson, K.M. Tuohy, Whole-grain wheat breakfast
RI PT
cereal has a prebiotic effect on the human gut microbiota: a doubleblind, placebo-controlled, crossover study., Br. J. Nutr. 99 (2008) 110–20.
SC
[119] I.E.J.A. François, O. Lescroart, W.S. Veraverbeke, M. Marzorati, S. Possemiers, H. Hamer, K. Windey, G.W. Welling, J.A. Delcour, C.M.
M AN U
Courtin, K. Verbeke, W.F. Broekaert, Effects of wheat bran extract containing arabinoxylan oligosaccharides on gastrointestinal parameters in healthy preadolescent children., J. Pediatr. Gastroenterol. Nutr. 58 (2014) 647–53.
TE D
[120] R. García-Villalba, J.A. Giménez-Bastida, M.T. García-Conesa, F.A. Tomás-Barberán, J. Carlos Espín, M. Larrosa, Alternative method for
EP
gas chromatography-mass spectrometry analysis of short-chain fatty acids in faecal samples., J. Sep. Sci. 35 (2012) 1906–13.
AC C
[121] M. Wullt, M.-L. Johansson Hagslätt, I. Odenholt, A. Berggren, Lactobacillus plantarum 299v enhances the concentrations of fecal short-chain fatty acids in patients with recurrent clostridium difficileassociated diarrhea., Dig. Dis. Sci. 52 (2007) 2082–6. [122] G.E. Walton, C. Lu, I. Trogh, F. Arnaut, G.R. Gibson, A randomised, double-blind, placebo controlled cross-over study to determine the gastrointestinal effects of consumption of arabinoxylan-
ACCEPTED MANUSCRIPT oligosaccharides enriched bread in healthy volunteers., Nutr. J. 11 (2012) 36. [123] X. Zheng, Y. Qiu, W. Zhong, S. Baxter, M. Su, Q. Li, G. Xie, B.M.
RI PT
Ore, S. Qiao, M.D. Spencer, S.H. Zeisel, Z. Zhou, A. Zhao, W. Jia, A targeted metabolomic protocol for short-chain fatty acids and
branched-chain amino acids., Metabolomics. 9 (2013) 818–827.
SC
[124] R. Di Cagno, M. De Angelis, I. De Pasquale, M. Ndagijimana, P. Vernocchi, P. Ricciuti, F. Gagliardi, L. Laghi, C. Crecchio, M.E.
M AN U
Guerzoni, M. Gobbetti, R. Francavilla, Duodenal and faecal microbiota of celiac children: molecular, phenotype and metabolome characterization., BMC Microbiol. 11 (2011) 219. [125] B. Vitali, M. Ndagijimana, F. Cruciani, P. Carnevali, M. Candela, M.E.
TE D
Guerzoni, P. Brigidi, Impact of a synbiotic food on the gut microbial ecology and metabolic profiles., BMC Microbiol. 10 (2010) 4.
EP
[126] I. Ahmed, R. Greenwood, B. de L. Costello, N.M. Ratcliffe, C.S. Probert, An investigation of fecal volatile organic metabolites in
AC C
irritable bowel syndrome., PLoS One. 8 (2013) e58204.
[127] D. Taneyo Saa, S. Turroni, D.I. Serrazanetti, S. Rampelli, S. Maccaferri, M. Candela, M. Severgnini, E. Simonetti, P. Brigidi, A. Gianotti, Impact of Kamut® Khorasan on gut microbiota and metabolome in healthy volunteers, Food Res. Int. 63 (2014) 227– 232.
ACCEPTED MANUSCRIPT [128] R.D. Couch, A. Dailey, F. Zaidi, K. Navarro, C.B. Forsyth, E. Mutlu, P.A. Engen, A. Keshavarzian, Alcohol induced alterations to the human fecal VOC metabolome., PLoS One. 10 (2015) e0119362.
RI PT
[129] M.-S. Kim, S.-S. Hwang, E.-J. Park, J.-W. Bae, Strict vegetarian diet improves the risk factors associated with metabolic diseases by
modulating gut microbiota and reducing intestinal inflammation.,
SC
Environ. Microbiol. Rep. 5 (2013) 765–75.
[130] V. De Preter, G. Van Staeyen, D. Esser, P. Rutgeerts, K. Verbeke,
M AN U
Development of a screening method to determine the pattern of fermentation metabolites in faecal samples using on-line purge-andtrap gas chromatographic-mass spectrometric analysis., J. Chromatogr. A. 1216 (2009) 1476–83.
TE D
[131] K. Windey, E. Houben, L. Deroover, K. Verbeke, Contribution of Colonic Fermentation and Fecal Water Toxicity to the
22.
EP
Pathophysiology of Lactose-Intolerance., Nutrients. 7 (2015) 7505–
AC C
[132] A.J. Wallace, S.L. Eady, D.C. Hunter, M.A. Skinner, L. Huffman, J. Ansell, P. Blatchford, M. Wohlers, T.D. Herath, D. Hedderley, D. Rosendale, H. Stoklosinski, T. McGhie, D. Sun-Waterhouse, C. Redman, No difference in fecal levels of bacteria or short chain fatty acids in humans, when consuming fruit juice beverages containing fruit fiber, fruit polyphenols, and their combination., Nutr. Res. 35 (2015) 23–34.
ACCEPTED MANUSCRIPT [133] A. Tangerman, F.M. Nagengast, A gas chromatographic analysis of fecal short-chain fatty acids, using the direct injection method., Anal. Biochem. 236 (1996) 1–8.
RI PT
[134] J.B. Zijlstra, J. Beukema, B.G. Wolthers, B.M. Byrne, A. Groen, J. Dankert, Pretreatment methods prior to gaschromatographic analysis of volatile fatty acids from faecal samples., Clin. Chim. Acta. 78
SC
(1977) 243–50.
[135] T. Høverstad, O. Fausa, A. Bjørneklett, T. Bøhmer, Short-chain fatty
M AN U
acids in the normal human feces., Scand. J. Gastroenterol. 19 (1984) 375–81.
[136] J. Pawliszyn, Solid Phase Microextraction: Theory and Practice,
TE D
1997.
[137] G.A. Mills, V. Walker, H. Mughal, Headspace solid-phase microextraction with 1-pyrenyldiazomethane in-fibre derivatisation
EP
for analysis of faecal short-chain fatty acids, J. Chromatogr. B
AC C
Biomed. Sci. Appl. 730 (1999) 113–122. [138] K. Grob, Gas chromatographic stationary phases analysed by capillary gas chromatography, 1980.
[139] Z. Ji, R.E. Majors, E.J. Guthrie, Porous layer open-tubular capillary columns: preparations, applications and future directions, J. Chromatogr. A. 842 (1999) 115–142. [140] W.J. Cheong, F. Ali, Y.S. Kim, J.W. Lee, Comprehensive overview of
ACCEPTED MANUSCRIPT recent preparation and application trends of various open tubular capillary columns in separation science, J. Chromatogr. A. 1308 (2013) 1–24.
RI PT
[141] J. Luong, R. Gras, W. Jennings, An advanced solventless column test for capillary GC columns, J. Sep. Sci. 30 (2007) 2480–2492.
[142] A.E. Thompson, A flame ionization detector for gas chromatography,
SC
J. Chromatogr. A. 2 (1959) 148–154.
[143] A. El-Aneed, A. Cohen, J. Banoub, Mass Spectrometry, Review of the
M AN U
Basics: Electrospray, MALDI, and Commonly Used Mass Analyzers, Appl. Spectrosc. Rev. 44 (2009) 210–230.
[144] K. Schäfer, Analysis of short chain fatty acids from different
TE D
intestinal samples by capillary gas chromatography, Chromatographia. 40 (1995) 550–556. [145] D.L. Schooley, F.M. Kubiak, J. V. Evans, Capillary Gas
EP
Chromatographic Analysis of Volatile and Non-Volatile Organic Acids
AC C
from Biological Samples as the t-Butyldimethylsilyl Derivatives, J. Chromatogr. Sci. 23 (1985) 385–390.
[146] G. Gutnikov, Fatty acid profiles of lipid samples., J. Chromatogr. B. Biomed. Appl. 671 (1995) 71–89.
[147] E.R. Farnworth, Y.P. Chouinard, H. Jacques, S. Venkatramanan, A.A. Maf, S. Defnoun, P.J.H. Jones, The effect of drinking milk containing conjugated linoleic acid on fecal microbiological profile, enzymatic
ACCEPTED MANUSCRIPT activity, and fecal characteristics in humans., Nutr. J. 6 (2007) 15. [148] E. Fritz, H.F. Hammer, R.W. Lipp, C. Högenauer, R. Stauber, J. Hammer, Effects of lactulose and polyethylene glycol on colonic
RI PT
transit., Aliment. Pharmacol. Ther. 21 (2005) 259–68. [149] M. Okazaki, S. Matsukuma, R. Suto, K. Miyazaki, M. Hidaka, M. Matsuo, S. Noshima, N. Zempo, T. Asahara, K. Nomoto,
SC
Perioperative synbiotic therapy in elderly patients undergoing
gastroenterological surgery: a prospective, randomized control trial.,
M AN U
Nutrition. 29 (2013) 1224–30.
[150] S. Nagata, T. Asahara, T. Ohta, T. Yamada, S. Kondo, L. Bian, C. Wang, Y. Yamashiro, K. Nomoto, Effect of the continuous intake of probiotic-fermented milk containing Lactobacillus casei strain Shirota
TE D
on fever in a mass outbreak of norovirus gastroenteritis and the faecal microflora in a health service facility for the aged., Br. J. Nutr.
EP
106 (2011) 549–56.
[151] K. Shimizu, H. Ogura, T. Asahara, K. Nomoto, M. Morotomi, Y.
AC C
Nakahori, A. Osuka, S. Yamano, M. Goto, A. Matsushima, O. Tasaki, Y. Kuwagata, H. Sugimoto, Gastrointestinal dysmotility is associated with altered gut flora and septic mortality in patients with severe systemic inflammatory response syndrome: a preliminary study., Neurogastroenterol. Motil. 23 (2011) 330–5, e157.
[152] K. Shimizu, H. Ogura, T. Asahara, K. Nomoto, A. Matsushima, K. Hayakawa, H. Ikegawa, O. Tasaki, Y. Kuwagata, T. Shimazu, Gut
ACCEPTED MANUSCRIPT microbiota and environment in patients with major burns – a preliminary report., Burns. 41 (2015) e28-33. [153] C. Tana, Y. Umesaki, A. Imaoka, T. Handa, M. Kanazawa, S.
RI PT
Fukudo, Altered profiles of intestinal microbiota and organic acids may be the origin of symptoms in irritable bowel syndrome., Neurogastroenterol. Motil. 22 (2010) 512–9, e114-5.
SC
[154] K. Kato, S. Mizuno, Y. Umesaki, Y. Ishii, M. Sugitani, A. Imaoka, M. Otsuka, O. Hasunuma, R. Kurihara, A. Iwasaki, Y. Arakawa,
M AN U
Randomized placebo-controlled trial assessing the effect of bifidobacteria-fermented milk on active ulcerative colitis., Aliment. Pharmacol. Ther. 20 (2004) 1133–41.
[155] H. Takaishi, T. Matsuki, A. Nakazawa, T. Takada, S. Kado, T.
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Asahara, N. Kamada, A. Sakuraba, T. Yajima, H. Higuchi, N. Inoue, H. Ogata, Y. Iwao, K. Nomoto, R. Tanaka, T. Hibi, Imbalance in
EP
intestinal microflora constitution could be involved in the pathogenesis of inflammatory bowel disease., Int. J. Med. Microbiol.
AC C
298 (2008) 463–72.
[156] Y. Kanamori, M. Sugiyama, K. Hashizume, N. Yuki, M. Morotomi, R. Tanaka, Experience of long-term synbiotic therapy in seven short bowel patients with refractory enterocolitis., J. Pediatr. Surg. 39 (2004) 1686–92. [157] A. Kotani, Y. Miyaguchi, M. Kohama, T. Ohtsuka, T. Shiratori, F. Kusu, Determination of short-chain fatty acids in rat and human
ACCEPTED MANUSCRIPT feces by high-performance liquid chromatography with electrochemical detection., Anal. Sci. 25 (2009) 1007–11. [158] S. Ohigashi, K. Sudo, D. Kobayashi, T. Takahashi, K. Nomoto, H.
RI PT
Onodera, Significant changes in the intestinal environment after surgery in patients with colorectal cancer., J. Gastrointest. Surg. 17 (2013) 1657–64.
SC
[159] C. Ferrario, V. Taverniti, C. Milani, W. Fiore, M. Laureati, I. De Noni, M. Stuknyte, B. Chouaia, P. Riso, S. Guglielmetti, Modulation of fecal
M AN U
Clostridiales bacteria and butyrate by probiotic intervention with Lactobacillus paracasei DG varies among healthy adults., J. Nutr. 144 (2014) 1787–96.
[160] G. Riezzo, A. Orlando, B. D’Attoma, V. Guerra, F. Valerio, P.
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Lavermicocca, S. De Candia, F. Russo, Randomised clinical trial: efficacy of Lactobacillus paracasei-enriched artichokes in the
EP
treatment of patients with functional constipation--a double-blind, controlled, crossover study., Aliment. Pharmacol. Ther. 35 (2012)
AC C
441–50.
[161] F. Valerio, S. de Candia, S.L. Lonigro, F. Russo, G. Riezzo, A. Orlando, P. De Bellis, A. Sisto, P. Lavermicocca, Role of the probiotic strain Lactobacillus paracasei LMGP22043 carried by artichokes in influencing faecal bacteria and biochemical parameters in human subjects., J. Appl. Microbiol. 111 (2011) 155–64. [162] Y. Bouhnik, C. Neut, L. Raskine, C. Michel, M. Riottot, C. Andrieux,
ACCEPTED MANUSCRIPT F. Guillemot, F. Dyard, B. Flourié, Prospective, randomized, parallelgroup trial to evaluate the effects of lactulose and polyethylene glycol-4000 on colonic flora in chronic idiopathic constipation.,
RI PT
Aliment. Pharmacol. Ther. 19 (2004) 889–99. [163] M.A. Underwood, N.H. Salzman, S.H. Bennett, M. Barman, D.A. Mills, A. Marcobal, D.J. Tancredi, C.L. Bevins, M.P. Sherman, A
SC
randomized placebo-controlled comparison of 2 prebiotic/probiotic combinations in preterm infants: impact on weight gain, intestinal
M AN U
microbiota, and fecal short-chain fatty acids., J. Pediatr. Gastroenterol. Nutr. 48 (2009) 216–25.
[164] S. Monira, M.M. Hoq, A.K.A. Chowdhury, A. Suau, F. Magne, H.P. Endtz, M. Alam, M. Rahman, P. Pochart, J.-F. Desjeux, N.H. Alam,
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Short-chain fatty acids and commensal microbiota in the faeces of severely malnourished children with cholera rehydrated with three
EP
different carbohydrates., Eur. J. Clin. Nutr. 64 (2010) 1116–24. [165] T. Torii, K. Kanemitsu, T. Wada, S. Itoh, K. Kinugawa, A. Hagiwara,
AC C
Measurement of short-chain fatty acids in human faeces using highperformance liquid chromatography: specimen stability., Ann. Clin. Biochem. 47 (2010) 447–52.
[166] A. Suzuki, K. Mitsuyama, H. Koga, N. Tomiyasu, J. Masuda, K. Takaki, O. Tsuruta, A. Toyonaga, M. Sata, Bifidogenic growth stimulator for the treatment of active ulcerative colitis: a pilot study., Nutrition. 22 (2006) 76–81.
ACCEPTED MANUSCRIPT [167] C. Wang, H. Shoji, H. Sato, S. Nagata, Y. Ohtsuka, T. Shimizu, Y. Yamashiro, Effects of oral administration of bifidobacterium breve on fecal lactic acid and short-chain fatty acids in low birth weight
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infants., J. Pediatr. Gastroenterol. Nutr. 44 (2007) 252–7. [168] K. Tanaka, K. Ohta, P.R. Haddad, J.S. Fritz, D.A. Miyanaga, W. Hu, K. Hasebe, Simultaneous ion-exclusion/cation-exchange
SC
chromatography of anions and cations in acid rain waters on a
weakly acidic cation-exchange resin by elution with sulfosalicylic
M AN U
acid., J. Chromatogr. A. 884 (2000) 167–74.
[169] J. Han, K. Lin, C. Sequeira, C.H. Borchers, An isotope-labeled chemical derivatization method for the quantitation of short-chain fatty acids in human feces by liquid chromatography-tandem mass
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spectrometry., Anal. Chim. Acta. 854 (2015) 86–94. [170] H. Miwa, M. Yamamoto, High-performance liquid chromatographic
EP
analysis of serum short-chain fatty acids by direct derivatization., J. Chromatogr. 421 (1987) 33–41.
AC C
[171] L. Fischer Ross, D.C. Chapital, Simultaneous Determination of Carbohydrates and Products of Carbohydrate Metabolism in Fermentation Mixtures by HPLC, J. Chromatogr. Sci. 25 (1987) 112– 117. [172] F. Valerio, F. Russo, S. de Candia, G. Riezzo, A. Orlando, S.L. Lonigro, P. Lavermicocca, Effects of probiotic Lactobacillus paracaseienriched artichokes on constipated patients: a pilot study., J. Clin.
ACCEPTED MANUSCRIPT Gastroenterol. 44 Suppl 1 (2010) S49-53. [173] J. Saric, Y. Wang, J. Li, M. Coen, J. Utzinger, J.R. Marchesi, J. Keiser, K. Veselkov, J.C. Lindon, J.K. Nicholson, E. Holmes, Species
RI PT
variation in the fecal metabolome gives insight into differential gastrointestinal function., J. Proteome Res. 7 (2008) 352–60.
[174] D. Monleón, J.M. Morales, A. Barrasa, J.A. López, C. Vázquez, B.
SC
Celda, Metabolite profiling of fecal water extracts from human colorectal cancer., NMR Biomed. 22 (2009) 342–8.
M AN U
[175] M. Ndagijimana, L. Laghi, B. Vitali, G. Placucci, P. Brigidi, M.E. Guerzoni, Effect of a synbiotic food consumption on human gut metabolic profiles evaluated by (1)H Nuclear Magnetic Resonance
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spectroscopy., Int. J. Food Microbiol. 134 (2009) 147–53. [176] G. Le Gall, S.O. Noor, K. Ridgway, L. Scovell, C. Jamieson, I.T. Johnson, I.J. Colquhoun, E.K. Kemsley, A. Narbad, Metabolomics of
EP
fecal extracts detects altered metabolic activity of gut microbiota in ulcerative colitis and irritable bowel syndrome., J. Proteome Res. 10
AC C
(2011) 4208–18.
[177] D.M. Jacobs, N. Deltimple, E. van Velzen, F.A. van Dorsten, M. Bingham, E.E. Vaughan, J. van Duynhoven, (1)H NMR metabolite profiling of feces as a tool to assess the impact of nutrition on the human microbiome., NMR Biomed. 21 (2008) 615–26. [178] J.C. Lindon, J.K. Nicholson, E. Holmes, J.R. Everett, Metabonomics:
ACCEPTED MANUSCRIPT Metabolic processes studied by NMR spectroscopy of biofluids, Concepts Magn. Reson. 12 (2000) 289–320. [179] A. Smolinska, L. Blanchet, L.M.C. Buydens, S.S. Wijmenga, NMR and
RI PT
pattern recognition methods in metabolomics: from data acquisition to biomarker discovery: a review., Anal. Chim. Acta. 750 (2012) 82– 97.
SC
[180] T.R.H. and, D.O. Koltun, An NMR Strategy for Determination of
Configuration of Remote Stereogenic Centers: 3-Methylcarboxylic
M AN U
Acids, (1998).
[181] H. Whatley, Basic principles and modes of capillary electrophoresis, Clin. Forensic Appl. Capill. Electrophor. (2001) 21–58.
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[182] A. Garcia, B. Olmo, A. Lopez-Gonzalvez, L. Cornejo, F.J. Rupérez, C. Barbas, Capillary electrophoresis for short chain organic acids in faeces Reference values in a Mediterranean elderly population., J.
EP
Pharm. Biomed. Anal. 46 (2008) 356–61.
AC C
[183] T. Toyo’oka, Use of derivatization to improve the chromatographic properties and detection selectivity of physiologically important carboxylic acids, J. Chromatogr. B Biomed. Sci. Appl. 671 (1995) 91– 112.
[184] R.R. Ran-Ressler, S. Devapatla, P. Lawrence, J.T. Brenna, Branched Chain Fatty Acids Are Constituents of the Normal Healthy Newborn Gastrointestinal Tract, Pediatr. Res. 64 (2008) 605–609.
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EP
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SC
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ACCEPTED MANUSCRIPT
ACCEPTED MANUSCRIPT
Table 2: Advantages and disadvantages of the main pre-treatment techniques in scientific reports using GC methods for SCFAs analysis.
centrifugation / filtration / ultrafiltration
Advantages
•
Disadvantages
RI PT
PrePre-treatment
speed
M AN U
•
separation with low temperature
TE D
steam distillation
column overload
•
unspecific results
SC
(direct injection)
•
•
frequent change of column
•
decomposition of acetyl-groups of other sample components
•
unspecific results
•
low recovery rate
•
separation with very low temperature
•
time consuming
acidified sample)
•
sensitive
•
not practical with a lot of samples
•
loss of SCFAs
EP
vacuum distillation (distilled water or
transesterification with different acids
•
direct method
•
column overload
•
fast
•
frequent change of column
•
direct method
•
column overload
•
relatively fast
AC C
simple acidification
ACCEPTED MANUSCRIPT
•
good purification
frequent change of column
•
time consuming (more steps)
•
loss of SCFAs
•
large quantities of reagents (costs)
•
occupational exposure (toxic, flammable,
RI PT
acidification / organic extraction
•
SC
solvent
•
very good purification
AC C
derivatization (chloroformates)
EP
TE D
derivatization (silylation)
M AN U
allergenic, organic solvents)
•
time consuming (more steps)
•
loss of SCFAs
•
large quantities of reagents (cost)
•
occupational exposure (toxic, flammable, allergenic organic solvents)
•
occupational exposure (toxic, flammable, allergenic organic solvents)
ACCEPTED MANUSCRIPT
•
very high purity of sample
•
longer life-span and usage of a
•
and knowledge of technique)
chromatographic system purge and trap technique
•
higher number of volatile compounds
•
extraction capacities of volatiles decrease with a higher molecular mass
AC C
EP
TE D
SCFAs, short-chain fatty acids; SPME, solid phase micro-extraction.
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SC
(compared to SPME)
high cost (fibres, supplementary instrumentation
RI PT
SPME
ACCEPTED MANUSCRIPT
Table 3
Column
filtration
ion-exchange
centrifugation; filtration
ion-exclusion
Detector
References
UV-VIS
Huda-Faujan 2010 [5]
SC
Pretreatment
RI PT
Scientific reports using HPLC methods for SCFAs analysis.
Farnworth 2007 [147]
ion-exchange
UV
Fritz 2005 [148]
ion-exclusion
ECD
Okazaki 2013 [149]
ion-exclusion; RP
conductivity Nagata 2011 [150]
ion-exclusion; RP
conductivity Shimizu 2011 [151], Shimizu 2015 [152]
ion-exclusion; RP
conductivity Tana 2010 [153]
ion-exclusion; RP
conductivity Kato 2004 [154]
ion-exclusion; RP
conductivity Takaishi 2008 [155]
ion-exclusion; RP
conductivity Kanamori 2004 [156]
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VWD
centrifugation; filtration
AC C
EP
TE D
perchloric acid; filtration
ACCEPTED MANUSCRIPT
ECD
ion-exclusion; RP
conductivity Ohigashi 2013 [158]
RI PT
ion-exclusion
Kotani 2009 [157]
cation-exchange
UV;RI
Ferrario 2014 [159]
sulfuric acid; filtration
ion-exclusion
UV
Riezzo 2012 [160], Valerio 2011 [161]
acid; steam distillation
ion-exchange
SC
sulfuric acid; centrifugation; filtration
sulfuric acid; sodium chloride; diethyl ether; acetonitrile; sodium
RP
UV
Underwood 2009 [163]
cation-exclusion
UV
Monira 2010 [164]
UV-VIS
Torii 2010 [165]
TE D
hydroxide; acetonitrile
Bouhnik 2004 [162]
M AN U
sulfuric acid; filtration; 2-NPH-HCl; HCl; hexane; diethyl-ether
UV
perchloric acid
NaOH; centrifugation; chloroform; filtration
AC C
ether; de-ionized water
EP
pyridine; 1-EDC-HCl; 2-NPH-HCl; potassium hydroxide; phosphoric acid; RP
ion-exclusion
absorbance Suzuki 2006 [166]
anion-exclusion
conductivity Wang 2007 [167]
NR, not reported; UV, ultraviolet detector; UV-VIS, ultraviolet visible detector; ECD, electrochemical detector; RP, Reverse Phase column; RI, refractive index detector; VWD, Variable Wavelength Detector.
ACCEPTED MANUSCRIPT
Table 1
RI PT
Scientific reports using GC methods for SCFAs analysis. References
ultrafiltration
Kopečný 2008 [30]a,b
filtration
Chen 2013 [31]a,b, Klein 2008 [32]a(FFAP),b, Fechner 2013 [33]a(FFAP),b,
SC
Pretreatment Pretreatment
M AN U
Roessler 2012 [34]a(FFAP),b, Nistal 2012 [35]a(FFAP),b, Beards 2010 [36]a,b, Turunen 2011 [37]a,b, Kekkonen 2011 [38]a, Finney 2007 [39]a,b Cuervo 2013 [40]c, Salazar 2014 [41]c, Arboleya 2012 [42]c
centrifugation; filtration sodium phosphate
vacuum distillation
phosphoric; vacuum distillation
TE D
copper sulphate, vacuum distillation
Wildt 2007 [45] Fernandes 2013 [46]a(FFAP),b
EP
steam distillation
Muir 2004 [44]
AC C
saline solution
Ben 2008 [43]
Ou 2013 [47]a,b, Wang 2012 [48]a(FFAP), McOrist 2008 [49]a(FFAP), McOrist 2011 [50]a(FFAP), Worthley 2009 [51]a(FFAP),b, Worthley, 2011 [52]a(FFAP),b, Kajander 2007 [53] Abell 2006 [54]
ACCEPTED MANUSCRIPT
Brinkworth 2009 [55]a(FFAP),b, Bird 2008 [56]a(FFAP),b
sulfuric acid; vacuum distillation
Valeur 2010 [57]b, Valeur 2009 [58]b, Tjellström 2012 [59]b, Tjellström
RI PT
orto-phosphoric acid; vacuum distillation
2010 [60]b, Tjellström 2014 [61]b, Tjellström 2013 [62]b, Norin 2004
SC
[63], Midtvedt 2013 [64], Sandin 2009 [65] Weir 2013 [66]c
water extraction; acid
Bouhnik 2007 [67]
ethylene glycol; homogenization; ultrafiltration; hydrochloric acid
M AN U
acidification; centrifugation; filtration
Adams 2011 [68]b Caminero 2012 [69]a(FFAP),b, Costabile 2012 [70]a(FFAP)b, Fava 2013
hydrochloric acid
TE D
[71]a(FFAP),b, Mitsou 2011 [72]a,b, Walton 2010 [73]a,b, Zhao 2006
[26]a(FFAP),b Goossens 2005 [74]a(FFAP),b
hydrochloric acid; diethyl ether
EP
hydrochloric acid; diethyl ether; silylation
AC C
diethyl ether; hydrochloric acid
Karlsson 2010 [75]a Zhang 2013 [76]a(FFAP)
hydrochloric acid; diethyl ether; MTBSTFA with TBDMSCI
Wallace 2015 [77]b
hydrochloric acid; diethyl ether; MTBSTFA
Ramnani 2015 [78]a,b
phosphoric acid; centrifugation phosphoric acid
Achour 2007 [79]b Yen 2011 [80]a,b, Teixeira 2013 [81]a,b
ACCEPTED MANUSCRIPT
Clarke 2011 [82]a(FFAP)
phosphoric acid; oxalic acid
Slavin 2011 [83]
phosphoric acid; diethyl ether
Spiller 2003 [84]
orto-phosphoric acid; diethyl ether
Thompson-Chagoyan 2011 [85]a,b
RI PT
phosphoric acid; sublimation;
SC
lactic acid: enzymatic test
Staudacher 2012 [86]a, Whelan 2009 [87]a, Majid 2011 [88]a, Whelan
phosphoric acid and mercury chloride
M AN U
2005 [89]a, Majid 2014 [90]a, Lefranc-Millot 2012 [91]a(FFAP),b Kumari 2013 [92]a,b
phosphoric acid in mercury chloride; filtration meta-phosphoric acid; methanol
Hovey 2003 [93] Friederich 2011 [94]b
TE D
ultracentrifugation; filtration; formic acid
Holscher 2012 [95]c
formic acid
Bakker-Zierikzee 2005 [96]b
formic acid
EP
lactic acid: heating, enzymatic test
L- and D-lactate: enzymatic test formic acid; methanol methyl esterification (methanol)
AC C
formic acid; methanol
lactic acid: lactate dehydrogenase enzymatic test
Reimer 2012 [97]a,b
Delgado 2006 [98]a(FFAP),b Gostner 2006 [99]a,b
ACCEPTED MANUSCRIPT
Weickert 2011 [100]a,b
perchloric acid in sodium hydroxide; lyophilisation; formic acid in acetone
Mohan 2008 [6]
RI PT
perchloric acid in sodium hydroxide; formic acid in acetone
lactic acid: enzymatic test
Kleessen 2007 [101]a,b
ultrasound; centrifugation; perchloric acid; sodium hydroxide;
sonification; NH2 (amino propyl-) in isopropanol–heptane (SPE); formic acid in diethyl ether lactic acid: perchloric acid in potassium hydroxide; enzymatic test
Tiihonen 2008 [102]a
M AN U
filtration; sonification; sulfuric acid; diethyl ether in sodium sulphate;
SC
liophylization; formic acid and acetone
Maldonado 2010 [103]a,b, Olivares 2006 [104]a,b, Liu 2015 [105]a,b, Sierra
acetate; sodium sulphate
2010 [106]a,b
sulfuric acid; ether lactic acid: methanol, chloroform sodium phosphate buffer; sulfuric acid; ether
EP AC C
sulfuric acid; diethyl ether; calcium chloride
TE D
sodium hydrogen carbonate in argon atmosphere; sulfuric acid, ethyl
Bernal 2013 [107]a,b, Klosterbuer 2013 [108]a, Schneider 2005 [109]b, Schneider 2006 [110]b, Sierra 2015 [111]a,b Woodmansey 2004 [112]a
Macfarlane 2013 [113]a
ACCEPTED MANUSCRIPT
methylation; chloroform (lactate and succinate) Lecerf 2012 [114]a(FFAP),b
oxalic acid; sodium azide; lyophilization
Schwiertz 2010 [115]a,b
oxalic acid
Larsen 2013 [116]a,b
sodium chloride in Tween solution; diethyl ether
Gråsten 2007 [117]a,b
M AN U
SC
RI PT
sulfuric acid
Costabile 2008 [118]a(FFAP),b
acetonitrile
Franc¸ois 2014 [119]a(FFAP),b
diethyl ether
formic acid or phosphoric acid; diethyl ether or dichloromethane or ethyl-
TE D
acetate
Wullt 2007 [121]a , Walton 2012 [122]a
propyl-chloroformate; propanol; pyridine; hexane
EP
silylation (TBDMS derivatives)
SPME (PDMS/CAR or PA, or DVB/CAR/PDMS)
AC C
ethyl chloroformate; ethanol; pyridine; sodium bicarbonate SPME (PDMS/CAR)
Garc´ıa-Villalba 2012 [120]c
Zheng 2013 [123]a,c Gao 2009 [24]c Di Cagno 2011a,c [124], Vitali 2010a,c [125], Ahmed 2013a,c [126], Taneyo Saa 2014 [127]a,c Couch 2015 [128]a,c
ACCEPTED MANUSCRIPT
Kim, 2013 [129]c
Purge and trap sample preparation system
De Preter 2009 [130]c, Windey 2015 [131]c
RI PT
SPME
SC
MTBSTFA, N-Methyl-N-t-butyldimethylsilyl trifluoroacetamide; TBDMSCI, tertbutyldimethylcholorosilane; TBDMS, tert-butyldimethylsilyl; SPE, solid phase extraction; PDMS/CAR, carboxen polydimethylsiloxane; SPME, Solid Phase Micro-Extraction; PA, polyacrylate; DVB/CAR/PDMS,
M AN U
divinylbenzene carboxen polydimethylsiloxane. a
, studies conducted with capillary column: [26,30–39,46–52,55,56,69–76,78,80–82,85–92,97–108,111–128].
a(FFAP)
, studies conducted with flame ionization detector (FID): [26,30–37,39,46,47,51,52,55–62,68–74,78–81,85,91,92,94,96–101,103–107,109–
111,114–119,132]. c
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b
, studies conducted with free fatty acid phase (FFAP) capillary column: [26,32–35,46,48–52,55,56,69–71,74,76,82,91,98,114,118,119].
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, studies conducted with mass spectrometry (MS) detector: [24,40–42,66,95,120,123–131].
S0003-2697(17)30119-7
DOI:
10.1016/j.ab.2017.03.007
Reference:
YABIO 12648
To appear in:
Analytical Biochemistry
Received Date: 5 December 2016 Revised Date:
18 February 2017
Accepted Date: 7 March 2017
Please cite this article as: M. Primec, D. Mičetić-Turk, T. Langerholc, Analysis of short-chain fatty acids in human feces: A scoping review, Analytical Biochemistry (2017), doi: 10.1016/j.ab.2017.03.007. 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.
ACCEPTED MANUSCRIPT
Review
Analysis of short shortrt-chain fatty acids in human
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feces: a scoping review
a
Department
of
Microbiology,
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Maša Primeca*, Dušanka Mičetić-Turkb, Tomaž Langerholca Biochemistry,
Molecular
Biology
and
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Biotechnology, Faculty of Agriculture and Life Sciences, University of Maribor, Pivola 10, 2311 Hoče, Slovenia b
Department of Pediatrics, Faculty of medicine, University of Maribor,
Taborska ulica 8, 2000 Maribor, Slovenia
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Corresponding author*: E-mail: [email protected]; Phone: +386 2 320
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90 54, Fax: +386 2 616 11 58
ACCEPTED MANUSCRIPT
Abstract Short-chain fatty acids (SCFAs) play a crucial role in maintaining homeostasis in humans, therefore the importance of a good and reliable
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SCFAs analytical detection has raised a lot in the past few years. The aim of this scoping review is to show the trends in the development of different methods of SCFAs analysis in feces, based on the literature
SC
published in the last eleven years in all major indexing databases. The search criteria included analytical quantification techniques of SCFAs in
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different human clinical and in vivo studies. SCFAs analysis is still predominantly performed using gas chromatography (GC), followed by high
performance
resonance
liquid
chromatography
(HPLC),
nuclear
magnetic
(NMR) and capillary electrophoresis (CE). Performances,
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drawbacks and advantages of these methods are discussed, especially in the light of choosing a proper pretreatment, as feces is a complex
EP
biological material. Further optimization to develop a simple, cost effective
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and robust method for routine use is needed.
Keywords:
Short-chain fatty acids (SCFAs) Feces Human Detection methods
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SC
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Scoping review
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Abbreviations: bbreviations: SCFAs, short-chain fatty acids; FAs, fatty acids; GC, gas chromatography; liquid
chromatography;
chromatography;
RP-HPLC,
chromatography;
NMR,
HPLC,
reverse
nuclear
high
phase
magnetic
performance
high
liquid
performance
liquid
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LC,
resonance;
CE,
capillary
electrophoresis; IBD, inflammatory bowel disease; IBS, irritable bowel SPE,
solid
-
phase
extraction;
SPME,
solid
-
phase
SC
syndrome;
microextraction; FAME, fatty acid methyl esters; UV, ultra violet; PEG,
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polyethylene glycol; FFAP, free fatty acid phase column; FID, flame ionization detector; LOD, limit of detection; LOQ, limit of quantification; RSD, relative standard deviation; CV, coefficient of variation; RI, refractive index; PDA, photo diode array detector; VWD, variable
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ionization.
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wavelength detector ECD, electrochemical detector; ESI, electrospray
ACCEPTED MANUSCRIPT
1.
Introduction
The human intestinal microbiota plays an important role in food digestion, contributing to the catabolism of food and potential toxins, the synthesis
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of microelements and fermentation of human digestion resistant nutrients. Moreover, it participates in electrolyte and mineral absorption as well as in short chain fatty acid (SCFAs) production. SCFAs are carboxylic organic
SC
acids with an aliphatic tail and represent the largest group of metabolic nutrients obtained from the fermentation of resistant carbohydrates,
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which are otherwise undegradable via human digestion. The process predominantly takes place in the caecum and the colon (reviewed in Nicholson et al.) [1]. In fact, carbohydrate fermentation is the main origin of SCFAs, although SCFAs may be generated from protein and amino acid
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decompositions as well. 95% of all SCFAs are represented by the three main representatives, i.e. acetic acid (C2), propionic acid (C3) and butyric acid (C4). Their respective molecular ratio 60:20:20 is relatively constant
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in the colon as well as in the feces [2]. The type and the quantity of
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produced SCFAs depend on the quantity of available substrate for the microbiota digestion and the composition of the microbiota itself [1]. Beside the three main aliphatic SCFAs, lactic i.e. 2-hydroxy propanoic acid (C3), valeric (C5) and caproic acid (C6) are present in lower amounts. Moreover, lactic acid exists in two optical isomeric forms, L- and Dlactate, while the concentration of L-lactate exceeds D-lactate by 100-fold under physiological conditions [3].
ACCEPTED MANUSCRIPT There is an increasing evidence about the important role of SCFAs in health and homeostasis. SCFAs undergo an effective absorption from the colonic lumen and they represent 10% of the human daily energy intake. Unlike acetic and propionic acid, which are mainly absorbed, butyric acid
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serves as a principal energy source for colonocytes [4]. A direct link between SCFAs (qualitatively and quantitatively) and some human pathological conditions, such as inflammatory bowel disease (IBD) [5],
SC
irritable bowel syndrome (IBS) [6], diarrhea [7] and cancer [8] have been
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proposed. Pathological increase in D-lactate is attributed to an increase in lactic acid producing microbiota [9]. This points to the fact that there is a need for a good qualitative or quantitative detection of SCFAs in clinical laboratories on a daily basis, as well as understanding the complex
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relationship between food, microbiota and metabolic homeostasis [4]. SCFAs have been measured in various biological materials such as blood plasma [10–12] and serum [13], equine caecum liquor [14], brain [12]
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and complex fermentation media [15]. Moreover, SCFAs have been detected in different environmental samples [16], food [17], waste
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leachates [18] and even in asphaltene [19]. Nevertheless, only a few English language reviews have summarized the analysis of SCFAs in diverse
samples,
[16,20,21] pathological
or
mostly
potentially
conditions
focusing
on
applicable
[4,22].
a
specific
analytical
Comprehensive
analytical
methods reviews
in of
method specific available
methods for fecal SCFAs quantification are, to our knowledge, not available, although their determination in feces is non-invasive and most
ACCEPTED MANUSCRIPT popular. However, it should be noted that only 5% of all microbially produced
SCFAs
can
be
found
in
feces
[4].
Moreover,
actual
concentrations of SCFAs found in feces are, on average 80, 40, 15, 13, 2, 2.5, 2 and 0.5 (all mmol/kg feces) for total SCFAs, acetic, propionic, n-
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butyric, i-butyric, i-valeric, n-valeric and n-caproic acid, respectively [23]. The aim of this systematic scoping review is to summarize methods used
SC
for SCFAs determination in human feces published between 2004 and 2015. Trends in the development of different sample pretreatments,
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separation and detection methods are demonstrated and compared in terms of accuracy, time consumption, cost and reliability of the results. The conclusion contains gaps, problems, inconsistencies and future
Search strategy and selection of studies
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2.
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prospects of SCFAs analysis in feces.
A literature search was performed to identify scientific articles in English,
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published between January 2004 and December 2015. A comprehensive search was conducted using combinations of search terms ("short chain fatty acids; volatile acids; human; feces; faeces"). Preliminary hits were further screened for presence of inclusion criteria, that SCFAs were analyzed or detected in different human clinical and in vivo studies. In
vitro and cell models based studies were not taken into consideration. The literature search was conducted in databases that included PubMed
ACCEPTED MANUSCRIPT (http://www.ncbi.nlm.nih.gov/pubmed), (http://www.ncbi.nlm.nih.gov/pmc/),
PubMedCentral Science
Direct
(http://www.sciencedirect.com), Scopus (www.scopus.com) and Proquest Research
Library
(http://www.proquest.com).
The
initial
search
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(preliminary hits) returned 2458 matches in total. After the exclusion of duplicates and scientific studies not meeting the criteria previously mentioned, 134 full text articles were further screened for analytical
SC
techniques used for the detection of SCFAs in human feces. Due to
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insufficient data, abstracts were excluded from this review. Information obtained from the literature has been further divided into sections, according to the instrumentation and analytical procedures relevant for the analysis. Where applicable, information on method performance
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parameters in terms of specificity, LOD (limit of detection), LOQ (limit of quantification), recovery, reproducibility (inter assay) and repeatability
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(intra assay) has been provided.
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3.
General considerations about SCFAs SCFAs analysis in feces feces
SCFAs have become popular targets in scientific research that tries to link gut microbiota to pathological conditions and potential health-beneficial
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effects in humans. Feces or fecal water is a complex biological material, providing a challenge for a fast and reliable determination [24]. Although SCFAs analytical methods have improved a lot in the past 11 years, the search
showed
that
development
of
precise
and
fast
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literature
instrumental methods has not overcome gas chromatography (GC), which
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still appears to be the most commonly used quantification method of fecal SCFAs despite having some disadvantages [25]. Alternative methods include techniques related to liquid chromatography (LC), such as high performance liquid chromatography (HPLC), nuclear magnetic resonance
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(NMR) and capillary electrophoresis (CE).
Since SCFAs are volatile and feces contain high concentrations of
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microbes, it is important to keep the biological material in appropriate conditions after its collection, in order to prevent sample deterioration.
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Samples are usually kept at – 80 °C, although many researchers have successfully used -20 °C as well. Before the SCFAs analysis, two important pretreatment
steps
derivatization.
SCFAs
should are
be
considered,
partially
hydrophilic,
i.e.
extraction
which
makes
and their
quantitative extraction to hydrophobic organic solvents more difficult. However, sample acidification is generally applied to keep acids protonized and
less
hydrophilic
and
thus
facilitating
better
extraction
[26].
ACCEPTED MANUSCRIPT Furthermore, SCFAs do not contain chromophores that would allow an easier detection in a UV or fluorescent spectrum. Derivatization into more
SCFAs SCFAs analytical methods
4.1 4.1
Gas liquid chromatography (GC, GLC)
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4.. 4..
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readily detectable substances is therefore recommended.
Direct detection of fatty acids (FAs) with GC was first described in 1952
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[27]. The principle of GC relies on a carrier gas that serves as a mobile phase where sample compounds are separated by differential interaction with the column stationary phase. Depending on their chemical nature, retention time upon reaching a detector as well as its response are used
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for a qualitative and quantitative evaluation of compounds [27,28]. For a successful GC determination of SCFAs an appropriate pretreatment chromatographic column and detector are needed. It is important to note
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that some GC methods can cause a thermic degradation and structural
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modification of FAs during the methyl esterification process, or they can simply destroy the sample, disabling the possibility of its reanalysis [25].
4.1.1 Pretreatment in GC detection methods Feces pretreatment is crucial for the detection of SCFAs [29]. Studies that used GC for SCFAs detection along with potential pretreatment procedures are provided in Table 1.
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Table 1
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The physical pretreatment procedures, without any time-consuming extraction, such as filtration [31–39], ultrafiltration [30], centrifugation [40–42] or simple sample dilution [43,44] are likely to be the fastest and
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simplest. The disadvantage of these uncomplicated pretreatments are impurities, which may overload the system, resulting in an incomplete of
sample
substances
and
chromatographic
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separation
column
contamination with non-volatile particles. Moreover, this results in a shorter column life span and a frequent column change [133]. distillation
is
a
separation
technique,
used
to
separate
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Steam
temperature-sensitive material. The use of lower temperatures preserves the sample quality, but increases the likelihood of unspecific results [45].
to
more
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Moreover, steam distillation proved to be a less appropriate technique due variable
recovery
rates
found
especially
at
low
SCFA
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concentrations as 2-10 times higher coefficients of variance were determined, when compared to vacuum distillation [134]. In contrast, vacuum distillation is performed under low pressure and consequently at a lower temperature [46–51,53–65]. Most authors have implemented this pretreatment technique for SCFAs separation as done by Zijlstra et al. [134], with modifications introduced by Høverstad et al. [135]. Some authors used vacuum distillation after diluting samples with distilled water
ACCEPTED MANUSCRIPT [48–53,65] or acid, such as phosphoric [54–56] or sulfuric [57–65]. Acid driven protonation of SCFAs increases their volatility. The pretreatment procedure with vacuum distillation of volatile SCFAs is a considerably precise
method,
but
still
time
consuming
and
consequently
not
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appropriate in routine practice, where handling of large numbers of samples is required. Moreover, because the procedure is time consuming,
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the possibility of losing volatile acids can be expected [133].
Instead of the time-consuming purification by vacuum distillation, many
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authors applied simple sample acidification [67] before loading it into the GC column. The choice of acidification agent ranged from hydrochloric acid [26,69–73,76], phosphoric acid [79–83,86–92], formic acid [94–96], sulfuric acid [102–106,114] to oxalic acid [83,115,116]. Weir et al. [66]
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centrifuged and filtrated acidified samples before subjecting them to gas chromatography-mass
spectrometry
(GC-MS)
detection.
Simple
acidification may result in some disadvantages mentioned before, such as
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losing the column quality in a much shorter time, especially when no pre-
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column is applied [120].
Some authors used acetone as deproteinizing agent in addition to formic acid [6,100,101]. The use of formic acid as an acidification agent, among others, may better prevent the adsorption of SCFAs to the GC column than
phosphoric
acid,
resulting
consequently
in
less
analytical
disturbances such as peak tailing, peak broadening, and double peak formation [133].
ACCEPTED MANUSCRIPT Sample acidification has been frequently followed by organic solvent extraction, using chloroform [112,113] and more popularly, ether [74– 78,84,85,102,107–113,117,119]. For GC-MS analysis, García-Villalba et al. [120] tried two different acids before extraction with three different
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organic solvents, where ethyl acetate was found the most efficient.
Some authors used another step in the sample pretreatment procedure,
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i.e. derivatization, mostly silylation [75,78,121,122,132], which enhances sample purity. Derivatization not only provides chromophores, but in the
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case of GC derivatization, it should result in volatile and stable molecules, ready for the high temperatures used during GC separation. In fact, functional groups such as hydroxyl, carboxyl, amine, thiol, phosphate may be unstable at high temperatures applied in GC and therefore need to be
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derivatized by certain silylation reagents. The technique implicates replacement of the acidic hydrogen with an alkylsilyl group. Resulting silyl ethers,
such
as
trimethylsilyl
(TMS)
and
tert-butyldimethylsilyl
EP
(TBS/TBDMS) derivatives are highly volatile, less polar and thermally
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more stable. However, care should be paid to prevent evaporation of more volatile derivatives during the pretreatment procedure as potential loss of SCFAs
may
occur
[29].
To
avoid
that,
chloroformates
in
SCFAs
derivatization procedures have been introduced [24,123]. Besides the extraction with different solvents which leads to the separation of two reciprocally unmixed layers, a special extraction, where a solvent is not necessarily present, has also become popular. The so
ACCEPTED MANUSCRIPT called solid - phase extraction (SPE), especially the solid - phase microextraction (SPME) is a faster, more selective and more sensitive technique due to the lower presence of impurities [136]. However, an effective and successful GC-MS system demands purified samples without
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water, due to its negative effect on MS determination [120].
Recently, a SPME pretreatment technique in combination with GC-MS has
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shown promising results with water-soluble samples [124–129] as well. Couch et al. [128] performed the SPME with three different fibers.
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Moreover, in situ derivatization of FAs in combination with SPME and polymer coating (in-fibre derivatization) may generate very low detection limits in aqueous solutions (formic acid – 11 nmol/vial (0.011 mmol/kg dry feces), acetic acid 4 nmol/vial (0.004 mmol/kg dry feces), propionic
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acid 2 nmol/vial (0.002 mmol/kg dry feces) and C4 – C6 acids 1 -2 nmol/vial (0.001 – 0.002 mmol/kg dry feces)) [137]. The SPME pretreatment results in a higher sample purity and a longer life span of
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the chromatographic system. Still, fibres are expensive and the technique
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requires supplementary knowledge and instrumentation in order to provide an automated analysis [120]. However, a purge and trap sample pretreatment technique [130,131] has also been implemented in combination with GC-MS, with the capacity of achieving higher number of volatile compounds compared to SPME, but worse extraction capacities of volatiles having higher molecular mass.
ACCEPTED MANUSCRIPT Generally, two-step pretreatment methods are time-consuming, but they can be simplified by one-step transesterification of SCFAs in biological material through acid (i.e. hydrochloric acid or sulfuric acid in methanol) or basic (i.e. sodium methoxide or potassium hydroxide in methanol)
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catalysis. Fatty acid methyl esters (FAME) are stable derivatives, volatile and particularly suitable for SCFAs detection with GC [29]. In fact, some of the authors preferred the use of SCFAs transesterification with simple
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alcohols such as methanol [93,97–99], ethanol [54] or ethylene glycol
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[68]. Acid not only catalyzes transesterification but deproteinazes samples as well.
Nevertheless, the above described pretreatment procedures are timeconsuming and may have, due to the additional experimental steps, an
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effect on the decreased recovery rate, accuracy and reproducibility of the results [29]. Reagents and solvents needed during the pretreatment are hazardous because of their flammable characteristics. They may also lead
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to occupational exposure and thus health damage in terms of toxicity and
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allergies. Moreover, the use of large quantities of reagents increases the costs [26]. Advantages and disadvantages of different pretreatment techniques for the detection of SCFAs by GC are summarized in Table 2.
Table 2
4.1.2 GC columns
ACCEPTED MANUSCRIPT Generally, in GC, two different types of columns are used: packed and capillary (or open tubular), the later being the more effective one [8]. Capillary columns share specific properties [138–140] and contain silica as the most common supporting material (see Table 1 under
a
). The
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stationary phase in capillary columns can be based on polysiloxanes and polyethylene glycol (PGE).
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The polysiloxanes are made of the most stable and robust material and characterized by the repeating siloxane backbone. Silicon atoms in bear
two
functional
groups
(methyl,
cyanopropyl,
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siloxanes
trifluoropropyl), where type and amount of substituted groups determine the different column properties [141]. Those used by different authors for SCFA analysis have been highly polar (designed to separate FAMEs) [30],
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non-polar [107,112,113,117,123,127]/polar [99], of low bleed and high temperature limit. The stationary phases that incorporate phenyl or phenyl type of functional groups are also known as arylenes. Their
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function results in very low column bleed and higher temperature limits.
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They are non-polar, with better sensitivity characteristics and mass spectral integrity and have also been successfully used for SCFA analysis [128].
PGE are also widely used as a stationary phase in capillary columns, but are less stable and robust and have lower temperature limits than most polysiloxanes. Therefore, a shorter lifespan and a higher susceptibility to damage caused by higher temperature are expected. Two general types of
ACCEPTED MANUSCRIPT PGE columns, considering temperature limits, are used, among which the one with the lower upper temperature limit and the lower low temperature limit
has
been
more
popular
for
separating
SCFAs
[31,80,81,85,92,97,100,103–106,108,124–126]. Moreover, this column
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exerts characteristics of high polarity, exhibits better reproducibility, inertness,
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and a higher resolution and temperature stability. The other type of PGE column has the characteristic of high polarity, but highest upper
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temperature limit and was used by one author only [115]. Furthermore, a faster and more accurate detection of SCFAs was first described by Zhao et al. [26]. The group described the usage of a free fatty acid phase (FFAP) column, which allows a direct SCFAs detection from water solutions
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without any derivatization. In fact, FFAP columns are of PGE type modified with terephthalic acid (pH modification), which are highly polar and widely used as stationary phases for the analysis of acidic compounds and
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therefore ideal for free fatty acid separation. Nevertheless, FFAP columns
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share certain characteristics of PGE columns and they are therefore also less stable and robust and have lower temperature limits than most polysiloxanes columns. The use of FFAP column has been subsequently performed by many authors (see Table 1 under
a(FFAP)
). Some acid
modified capillary columns with very similar or same characteristics as FFAP columns have also been widely used by many authors [37– 39,47,72,86–90].
ACCEPTED MANUSCRIPT 4.1.3 GC detectors The flame ionization detector (FID) (see Table 1 under b) is the most used conventional detector for SCFAs detection in GC. FID is sensitive to
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molecules that are ionized in a hydrogen–air flame, including most carbon-containing compounds, and produces a current that varies proportionally to the amount of organic species in a sample [142].
and
Nevertheless,
accuracy its
and
restriction
the
option
comes
with
of
a
temperature lack
of
regulation.
supplementary
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linearity
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Moreover, FID detectors are relatively quiet, have a wide spectrum of
information, besides the retention values, that could benefit in qualitative analysis of the analytes [29].
Instead of applying a conventional detector, GC can be bound to MS,
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resulting in a better sensitivity and selectivity of the analysis. MS ionizes molecules preferably into cations, which are separated on the basis of
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their mass to charge the ratio in a magnetic field. The ions are very reactive and unstable and the whole procedure is run under low pressure
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[143]. The scientific articles that used GC-MS methods for detecting SCFAs in feces are listed in Table 1 under c. If a tandem GC-MS-MS instrument is available, the use of such a system is also reasonable for the quantitation of analytes that are present in low concentrations in complex biological samples [129].
4.1.4 GC based methods performance
ACCEPTED MANUSCRIPT Some studies [35,36,69–72,118] using GC implemented their method according to Zhao et al [26], using the FFAP capillary column. The determined LOD for SCFAs was in a range lower than 10 µM (0.05 mmol/kg wet feces), while the LOQ was below 31 µM (0.155 mmol/kg wet
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feces). The recovery rates obtained varied from 87.7 - 114.5%, inter assay with relative standard deviation (RSD) values 1.5 – 4.9% with some exceptions and intra-assay with RSD values 1.0 – 6.0%. Schwiertz et al.,
SC
[115], who adapted the method after Schaefer et al. [144], reached LOD
mmol/kg
wet
acidification
feces
of
the
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of 50 – 100 µg/L in standard water solution (approximately 0.003 - 0.006 calculated sample
for
has
C3).
been
Vacuum
used
by
distillation many
after
researchers
[38,39,53,57–62,64,65,102] and was implemented from Høverstad et al.
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[135]. Despite being time consuming, LOD for SCFAs were found at 0.01 mmol/kg wet feces, recovery from 90-109%, intra assay with coefficient of variation (CV) of 2.6 – 8.9%, inter assay with CV of 6 – 19.7%, but
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Norin et al. [63] accomplished a higher LOD for SCFAs (0.2 mmol/kg wet feces). A big step towards more sensitive and faster GC method
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detections has been performed via the FID detector development. With limits of detection even in the low pg or fg range, FID has become the most widely and successfully used GC detector for volatile hydrocarbons and many carbon containing compounds. GC-FID detection methods are therefore mainly performed after a direct sample injection (or previously treated with acid). In fact, Schneider et al. [109] and Schneider et al. [110] performed a fast pretreatment and reported a sensitivity of 1 mmol.
ACCEPTED MANUSCRIPT Gråsten et al. [117] performed the SCFAs detection after Schooley et al., [145] who encountered some limiting factors for detection, due to the solvent
and
the
N-Methyl-N-t-butyldimethylsilyl
trifluoroacetamide
(MTBSTFA) peaks, which could present some problems in samples with
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trace levels of acids. In this case, derivatization was performed after the extraction and obtained an LOD and an LOQ which ranged from 0.3 – 3.0 ppm (0.3 ppm is 0.004 mmol/kg wet feces calculated for C3) and 2.0 –
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5.0 ppm (5.0 ppm is 0.067 mmol/kg wet feces calculated for C3),
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respectively. Furthermore, inter assay with CV of 3.49% for acetic acid and with CV of 8.70% for lactic acid and recovery rates between 92 109% were determined. An important role of pretreatment steps for the development of a more reliable SCFAs detection method in different
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biological materials has also been shown by Zheng et al. [123] and Gao et al. [24]. In fact, they performed chloroformate derivatization prior to GCMS and showed some advantages such as short reaction time at room
EP
temperature, acceptable efficiency (90.06 – 116.34% for SCFAs [123]), recovery (84.11 – 118.79% for analyte isotopes [123]) and a low LOD
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(from 20 to 1000 pg on-column of the test standards – approximately 0.058 mmol/kg wet feces calculated for C4 [123], from 10 to 500 pg oncolumn of the test standards – approximately 0.69 mmol/kg wet feces calculated for C5 [24]). However, LODs for acetic, propionic [24,123] and butyric [24] were not determined due to analytical interferences indicating a significant drawback as these SCFAs constitute the bulk of SCFAs in feces. Additionally, Zheng et al. [123] reported intra-day and inter-day
ACCEPTED MANUSCRIPT precision with an RSD below 5.99% and 8.43%, respectively. De Preter et al. [130] and Windey at al. [131] reached LODs from 0.25 mg/L to 25 mg/L in standard working solutions (i.e. approximately 3 mmol/kg wet feces calculated for C3) with a purge and trap pretreatment prior to GC-
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MS. Moreover, the intra assay variability was in the range of 3.3 – 9.7% RSD, the inter assay variability was demonstrated with an RSD range of 3.7 – 9.8% and the overall recovery of the compounds studied was 100%.
with
a
less
complicated
pretreatment
procedure
(without
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samples
SC
Nevertheless, García-Villalba et al. [120] analyzed the organic acid in fecal
derivatization) prior to GC-MS with LOD and LOQ ranging from 0.29 – 3.80 µg/g (0.006 mmol/kg wet feces calculated for C3) and 0.98 – 12.56 µg/g (0.02 mmol/kg wet feces calculated for C3), respectively. The inter-
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and intra assay variability values were below 2.6 and 5.6% respectively and the recovery values ranged from 82 - 105% for propionic, butyric,
High Performance Liquid Chromatography (HPLC)
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4.2
EP
isobutyric, valeric and isovaleric acid and around 65% for acetic acid.
HPLC is another method for SCFAs analysis and a good alternative to GC. The most commonly used technique is a reverse phase HPLC (RP-HPLC), where the stationary solid phase (column) is hydrophobic (non-polar) and the mobile liquid phase is hydrophilic (polar, watery). Due to the higher pressure, the mobile phase carrying analytes travels and the small stationary phase particles with a larger area allow for a better interaction
ACCEPTED MANUSCRIPT with the analytes. The greatest advantage of the HPLC over the GC technique is the use of lower running temperatures [146]. Similar to GC, specific combinations of pretreatments, columns, running conditions and
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detectors have to be optimized for a successful SCFAs analysis (Table 3).
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Table 3
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4.2.1 Pretreatment in HPLC detection methods
Like in GC, the sample pretreatment procedure in HPLC plays an important role. Derivatization of non-chromophoric SCFAs increases the and
Derivatization hydrazide
selectivity
techniques
of
the
derivatives
HPLC
that include
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sensitivity
have
been
the
detection
analysis
formation
performed
by
[25].
of fatty acid
using
a
nitro-
phenylhydrazine reagent alone [163], which is commonly used in
EP
derivatization of carboxylic acids, aldehydes and ketones, or by using a
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water-soluble carbodiimide reagent as the coupling agent that activates carboxyl groups for a spontaneous reaction with primary amines enabling a
peptide
immobilization
[165].
A
variety
of
purification
-
separation/extraction techniques have been applied before subjection of samples to HPLC, since samples must retain solubility in the liquid mobile phase. This may be argued by some authors, which used only simple centrifugation/filtration steps, before injecting the sample into HPLC [5,147,148].
ACCEPTED MANUSCRIPT The most widespread pretreatment method for fecal SCFAs analysis is simple acidification (deproteinization) with perchloric acid [149–158] or sulfuric acid [159–161]. Some authors pretreated the samples with steam [162] distillation before the acidification procedure. Especially with water-
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soluble samples, the extraction step may also be used. Some authors declared successful purification of samples by extraction with chloroform [167] or ether alone [165] or ether in combination with acetonitrile [164]
(acid
hydrazides)
before
the
extraction
step
with
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derivatization
SC
or hexane [163]. Tori et al. [165] and Underwood et al. [163] performed
disadvantages similar to those described at GC (see chapter 4.1.1).
4.2.2 HPLC columns
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RP columns, based on silica gel [29] bound to C4 (butyl), C8 (octyl, ODC), C18 (octadecyl), nitrile (cianopropyl) and phenyl (phenyl propyl) groups,
EP
are of common use in HPLC. Among RP-HPLC columns, the C18 phase column is most commonly used due to its high lipid resolution. However,
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in selected studies RP columns for SCFA analysis alone have been used in two publications [163,165]. . Nevertheless, some authors preferred using the ion-exclusion mode columns alone [149,157,160,161,164,166] or ion-exchange type of column
alone
[5,148,159,162].
The
basic
principle
of
the
ion
exclusion/exchange separation is in the interactions between ionic and polar analytes, ions in the eluent and ionic functional groups fixed to the
ACCEPTED MANUSCRIPT chromatographic support. The ion exchange mode is based on a competitive attraction between ions in the stationary and mobile phase. On the other hand, ion exclusion mode prevents ionized molecules from entering the pores of the support, leading to highly ionic molecules to
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elute first, followed by weakly ionized and non-ionic molecules last. When a mixture of weak acids, such as SCFAs in samples are expected, the use of the ion exclusion mode of column is more recommended as samples are
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not well separated, neither with ion exchange, nor with RP columns [168].
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In most cases the RP column has been used in combination with ionexclusion mode columns (in a dual-column mode, tandem mode) [150–
4.2.3 HPLC detectors
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156,158].
Several detectors have been successfully used with SCFAs detection upon
EP
HPLC separation. The most popular routine HPLC detector is based on UV absorption, which has the advantage of performing independently from
AC C
temperature changes, mobile phase flow speed and composition [29]. Literature revealed the use of UV detector by some authors [5,148,160– 165]. Nevertheless, the problem of SCFAs lacking chromophore [25,29] demands derivatization as a pretreatment when using an RP column [163,165], but not ion-exclusion or ion-exchange columns [5,147– 149,157,159–162,164,166,167]. Ferrario et al. [159] used a UV detector
ACCEPTED MANUSCRIPT concomitantly with a differential refractive index (RI) detector, which detects components in a solution based on the light refraction. A photo diode array (PDA) detector belongs to the group of variable
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wavelength detectors (VWD) where excited light is separated into different wavelengths. Absorption monitoring at different wavelengths allows for a simultaneous determination of more than one analyte. The advantage of
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such detectors are cost reductions on expensive solvents and time spent for performing an analysis [147].
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Apart from that, the literature review revealed a popularity in using conductivity detectors [150–156,158,167].. Conductivity detectors are mainly used to measure inorganic ions and small organic substances (organic acids and amines). It is a highly sensitive detector, but very to
temperature
variations.
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susceptible
Another
disadvantage
of
conductivity detectors, especially when dealing with a huge amount of
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samples, is its linkage to special and complicated instrumentation [169]. Kotani et al. [157] and Okazaki et al. [149] used an electrochemical
AC C
detector (ECD) which measures the oxidation – reduction potential of substances in the mobile phase. ECDs are very sensitive with some advantages [25], among which the most important may be a direct analysis of non-derivatized samples, skipping the pretreatment step and reducing analysis time [157]. The most advanced and sensitive detection is obtained by coupling HPLC to MS via electrospray ionization (ESI). Namely, Han et al. performed a
ACCEPTED MANUSCRIPT 13
combined chemical derivatization with LC/MS-MS.
C6 derivatives were
developed for the use as isotope-labelled internal standards, in order to compensate for ESI matrix effects and to obtain more specific and thorough quantitation of SCFAs in human fecal samples [169]. The
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combination enables a detection of analyte molecular mass, as well as
drawback is the instrumentation cost [165].
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4.2.4 HPLC-based methods performance
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type and local position of the substitutes in a carbon chain [29]. The major
Detection limits of fecal SCFAs by HPLC are apparently mostly influenced by the sensitivity of the applied detector (reviewed in Bielawska et al. [29]). The UV detector is the most commonly used detector in HPLC
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[170]. Moreover, Fritz et al. [148] performed a SCFAs detection after Ross et al. [171], who obtained sensitivities as low as 0.86 nmol (except for
EP
acetic acid) using a cation-exchange column and in most cases a UV detector. Recoveries ranged from 72 – 86% for most of the acids, except
AC C
for lactic acid, which was as low as 24%. The inter- and intra assay variability values were below 5.7 (8.4 for lactic acid) and 7.6%, respectively. Okazaki et al. [149] and Kotani et al. [157] used ECD and accomplished detection limits from 0.05 - 0.8 µmol/g (0.05 – 0.8 mmol/kg wet feces) and 40 pmol/single injection (0.04 mmol/kg wet feces; recovery in the range 92 - 98%, inter assay with RSD less than 2.7%) respectively. Valerio et al. [161] performed SCFAs detection after Valerio
ACCEPTED MANUSCRIPT et al. [172] who obtained an LOQ in the range between 0.09 - 1.72 µmol/g (0.09 - 1.72 mmol/kg wet feces).
Nuclear magnetic resonance (NMR)
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4.3
The NMR method is based on magnetic characteristics of particular atomic cores which absorb and emit electromagnetic radiations in a magnetic
SC
field. The produced energy appears at a specific resonance frequency which depends on the magnetic field and magnetic characteristics of
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specific isotopes. Isotope cores 1H [173–177] and
13
C [174] have been
used for SCFAs studies in fecal samples. NMR
is
a
quantitative
(absolute),
non-destructive,
repeatable
and
TE D
objective method. It has been mainly used for simultaneous tracing of different metabolites in a complex biological material in terms of chemical composition and concentrations. It is a comprehensive and enough
EP
sensitive method with the detection limits of metabolite concentration reaching the µM range and still decreasing [178,179]. The so called NMR
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metabolic profiling enables the production of fast, stable and reproducible profiles [177]. It demands minimal sample pretreatment. Nevertheless, pretreatment methodology needs an optimization that enables the extraction of many different metabolites out of a complex biological material. Furthermore, it is recommended to use deuterated solvents in NMR experiments in order to correct the field strength and to avoid the huge solvent absorption that would otherwise spoil the 1H-NMR spectrum
ACCEPTED MANUSCRIPT [180]. This may result in an expensive, time-consuming procedure that may lead to significant analyte losses, especially when coping with big amounts of samples on a daily basis. At the same time, the method should be non-destructive and should cause only minor changes in
RI PT
material [177]. In fact, differences and contradictories in NMR's results show that the possible cause of low sensitivity (lower than that, obtained with GC-MS or LC-MS), may be indeed due to different pretreatment
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procedures and data acquisitions [173,177,179].
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The NMR application on feces [173] and fecal water samples [174–177] has been used to monitor human metabolic activity, especially the effect of microbiota on the regulation of host metabolism. NMR-based metabolic profiling could present the future disease diagnostic method because of
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the method’s advantages described above. However, instrumentation cost and sensitivity due to the poor pretreatment optimization methodology
Capillary Electrophoresis (CE)
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4.4
EP
still remain serious drawbacks.
CE is a technique that separates charged molecules in a capillary upon exposure to an electrical field. In the past 30 years, CE has been known as a good analytical technique for the detection of different organic molecules, including SCFAs. CE separation depends on the electrophoretic mobility of ions through the gel, being dependent on molecular mass, shape and charge of the molecule [181].
ACCEPTED MANUSCRIPT CE is especially used for separation of water-soluble compounds. The advantage of CE is its speed and minimal sample pretreatment procedure, which is very convenient in routine analysis. In fact, CE has been successfully implemented in analytical detection of SCFAs in different
RI PT
biological materials, including fecal samples [182]. Nevertheless, it has its disadvantages: low repeatability and reproducibility and the need for higher analyte concentration. Most of these limitations are due to the
SC
small amount of injected sample [183]. Garcia et al. [182] obtained the
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LOQ of standard solutions ranging from 10.4 to 49.6 µM (approximately 0.161 mmol/kg wet feces calculated for C3), accuracy (circa 68%, 18% and 68% for acetic, propionic and butyric acid, respectively), recovery (94.9 - 106.6%), 5.5 - 9.5% and from 8.1 - 14.5% for inter and intra
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assay respectively, by using a simple CE detection method. A combined detection technique with MS has solved many limitations in CE detection, raising the method’s reliability in detecting small molecules in the range
EP
from µM to nM. To our knowledge, combinations for the determination of
4.5
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SCFAs in fecal samples have not been applied yet.
Enzymatic detection of SCFAs SCFAs
One of the alternative SCFAs determination methods in feces is enzymatic detection, which relies on spectrophotometric measurement of enzymatic products obtained from SCFAs as substrates. Enzymes prefer stereospecific isomers in their metabolic reactions and they can therefore
ACCEPTED MANUSCRIPT differentiate between optical isomers. In this sense, lactate can be found in both D- and L-form and several research groups have enzymatically determined D- and L-lactate [6,85,96,97,99,102]. Although the assay is fast, disadvantages may be mainly related to cross-enzymatic reactions
RI PT
with other endogenous molecules as well as a potential presence of enzymatic inhibitors/accelerators in analytical samples. However, in terms of lactate determination, it is important to note that total lactate has been
AC C
EP
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successfully determined by GC [112,113] and HPLC [5,167] as well.
ACCEPTED MANUSCRIPT
5.
Summary and conclusion
Intestinal microbiota performs several important functions which maintain the human homeostasis in equilibrium. Changes in microbiota can lead to
production
of
bacterial
metabolites
like
RI PT
different pathological conditions in metabolism, such as disorders in the SCFAs.
A
comprehensive
understanding of SCFAs functional roles in the human body is essential
SC
due to several demonstrated connections between SCFAs, microbiota and metabolic diseases. Therefore, a need for a good and reliable analytical
pathological conditions.
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SCFA detection technique is crucial for the diagnosis of different
Due to the complicated composition of feces and the natural molecular characteristics of SCFAs, sample pretreatment procedures are almost
TE D
inevitable and the majority of research reports in the last 11 years have used it. However, additional steps prolong the time needed for analysis,
EP
they may lead to potential SCFAs losses and increase consumption of consumables and hence costs. Costs become a limiting factor when
AC C
dealing with large numbers of samples on a daily basis, in addition to the occupational exposure to chemicals needed during the pretreatment steps. Instead of traditional solvent extractions, researchers can opt for a modern SPME, solving many problems related to excessive solvent use and sample dilution. Few researchers applied simple acidification or even direct injection of samples into the chromatographic system. However,
ACCEPTED MANUSCRIPT negative effects of such pretreatment have merely caused a premature column failure and even decreased the detector lifespan. The choice of instrumentation for SCFAs analysis is usually limited to GC
most
analytical
and
biochemical
laboratories.
RI PT
and HPLC, as such equipment has become standard and thus accessible in Nonetheless,
several
detectors can be applied; from standard UV and PDA to more sensitive
SC
and expensive FID and MS detectors. GC-MS and HPLC-MS combinations have pushed the limits of detection well below the actual concentrations of
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SCFAs found in feces (see section 1 – Introduction). Therefore, for detection, MS could be a reasonable first choice in specific situations when low-concentrations of FAs are being followed, like branched chain fatty acids (BCFAs) as discussed below. In fact, GC methods, beside some
TE D
disadvantages, seem to be a justifiable choice for a reliable SCFAs detection. Emerging techniques like NMR based methods have satisfied the
need
for
a
simple
sample
pretreatment
procedure,
but
its
EP
contradictories in results related to sensitivity may be due to under-
AC C
optimized pretreatments. Moreover, NMR instrumentation availability and costs are beyond the reach of most laboratories. Likewise, CE needs minor and uncomplicated sample pretreatment, but lacks repeatability and reproducibility. Enzymatic detection is simple, but its application has been more or less applied for the distinction between optical isomers of lactate. Surprisingly, very few researchers have mentioned or showed method performance parameters such as LOD, LOQ, recovery and precision (in
ACCEPTED MANUSCRIPT terms of reproducibility and repeatability) of fecal SCFAs that would allow an
overall
estimation
concentrations
of
of
acetic,
method propionic
performance. and
butyric
In
some
acids
have
cases, been
determined, while less abundant lactic and BCFAs have not been
RI PT
measured. BCFAs are mainly saturated, bearing one or more methyl groups on the carbon chain [184]. They can be found in minor amounts in human feces [5], but their role in the gastrointestinal system is still not
SC
fully understood. On the other hand, lactate could be an important
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indicator in specific physiological or pathological issues, i.e. as a specific
Bifidobacterium metabolite [6,85,96] or an indicator of nonsteroidal antiinflammatory drug (NSAID) abuse [102].
In conclusion, most described SCFAs analytical methods have advantages
TE D
and drawbacks. In terms of expected SCFAs concentrations in fecal samples, the described method performance for SCFAs detection with GC/FID or MS, HPLC,NMR and even CE methods are all acceptable. It is recommended
that
EP
thus
researchers
consider
the
instrumentation
AC C
availability, time consumption and costs when choosing a proper detection method for fecal SCFAs in research, routine use in clinics and potential automation. This may however also depend on the future progress and knowledge about SCFAs in health and diseases.
Author contributions: contributions All the authors have accepted responsibility for the entire content of this submitted manuscript and approved submission.
ACCEPTED MANUSCRIPT Funding: unding This research was partially supported by the Slovenian research program P1-0164 (C) funded by the Slovenian Research Agency. Acknowledgements: Acknowledgements The authors would like to thank Katja Težak for
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language editing.
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Conflicts of interest: interest The authors declare no conflicts of interest.
ACCEPTED MANUSCRIPT
References [1]
J.K. Nicholson, E. Holmes, J. Kinross, R. Burcelin, G. Gibson, W. Jia, S. Pettersson, Host-gut microbiota metabolic interactions., Science.
[2]
RI PT
336 (2012) 1262–7. G. den Besten, K. van Eunen, A.K. Groen, K. Venema, D.-J.
Reijngoud, B.M. Bakker, The role of short-chain fatty acids in the
[3]
M AN U
J. Lipid Res. 54 (2013) 2325–40.
SC
interplay between diet, gut microbiota, and host energy metabolism.,
H. Hasegawa, T. Fukushima, J.-A. Lee, K. Tsukamoto, K. Moriya, Y. Ono, K. Imai, Determination of serum D-lactic and L-lactic acids in normal subjects and diabetic patients by column-switching HPLC with
(2003) 886–91. [4]
TE D
pre-column fluorescence derivatization., Anal. Bioanal. Chem. 377
E.P. Nyangale, D.S. Mottram, G.R. Gibson, Gut microbial activity,
EP
implications for health and disease: the potential role of metabolite
[5]
AC C
analysis., J. Proteome Res. 11 (2012) 5573–85. N. Huda-Faujan, A.S. Abdulamir, A.B. Fatimah, O.M. Anas, M.
Shuhaimi, A.M. Yazid, Y.Y. Loong, The impact of the level of the intestinal short chain Fatty acids in inflammatory bowel disease patients versus healthy subjects., Open Biochem. J. 4 (2010) 53–8.
[6]
R. Mohan, C. Koebnick, J. Schildt, M. Mueller, M. Radke, M. Blaut, Effects of Bifidobacterium lactis Bb12 supplementation on body
ACCEPTED MANUSCRIPT weight, fecal pH, acetate, lactate, calprotectin, and IgA in preterm infants., Pediatr. Res. 64 (2008) 418–22. [7]
B.S. Ramakrishna, V.I. Mathan, Colonic dysfunction in acute
RI PT
diarrhoea: the role of luminal short chain fatty acids., Gut. 34 (1993) 1215–8. [8]
W. Scheppach, H.P. Bartram, F. Richter, Role of short-chain fatty
SC
acids in the prevention of colorectal cancer., Eur. J. Cancer. 31A (1990) 1077–80.
L. Galland, The Gut Microbiome and the Brain, J. Med. Food. 17 (2014) 1261–1272.
M AN U
[9]
[10] N.M. Moreau, S.M. Goupry, J.P. Antignac, F.J. Monteau, B.J. Le Bizec,
TE D
M.M. Champ, L.J. Martin, H.J. Dumon, Simultaneous measurement of plasma concentrations and 13C-enrichment of short-chain fatty acids, lactic acid and ketone bodies by gas chromatography coupled
EP
to mass spectrometry., J. Chromatogr. B. Analyt. Technol. Biomed.
AC C
Life Sci. 784 (2003) 395–403. [11] I. Yanagisawa, M. Yamane, T. Urayama, Simultaneous separation and sensitive determination of free fatty acids in blood plasma by high-performance liquid chromatography, J. Chromatogr. B Biomed. Sci. Appl. 345 (1985) 229–240. [12] C. Bachmann, J.P. Colombo, J. Berüter, Short chain fatty acids in plasma and brain: quantitative determination by gas
ACCEPTED MANUSCRIPT chromatography., Clin. Chim. Acta. 92 (1979) 153–9. [13] G. Zhao, J. Liu, M. Nyman, J.Å. Jönsson, Determination of shortchain fatty acids in serum by hollow fiber supported liquid membrane
RI PT
extraction coupled with gas chromatography, J. Chromatogr. B. 846 (2007) 202–208.
[14] L.J. Horspool, Q.A. McKellar, Determination of short-chain fatty acids
SC
in equine caecal liquor by ion exchange high performance liquid
5 (1991) 202–6.
M AN U
chromatography after solid phase extraction., Biomed. Chromatogr.
[15] J. Schiffels, M.E.M. Baumann, T. Selmer, Facile analysis of shortchain fatty acids as 4-nitrophenyl esters in complex anaerobic fermentation samples by high performance liquid chromatography, J.
TE D
Chromatogr. A. 1218 (2011) 5848–5851. [16] K. Fischer, Environmental analysis of aliphatic carboxylic acids by
AC C
173.
EP
ion-exclusion chromatography, Anal. Chim. Acta. 465 (2002) 157–
[17] M.-H. Yang, Y.-M. Choong, A rapid gas chromatographic method for direct determination of short-chain (C2–C12) volatile organic acids in foods, Food Chem. 75 (2001) 101–108.
[18] G. Manni, F. Caron, Calibration and determination of volatile fatty acids in waste leachates by gas chromatography, J. Chromatogr. A. 690 (1995) 237–242.
ACCEPTED MANUSCRIPT [19] Y. Li, Y. Xiong, Q. Liang, C. Fang, C. Wang, Application of headspace single-drop microextraction coupled with gas chromatography for the determination of short-chain fatty acids in RuO4 oxidation products
RI PT
of asphaltenes, J. Chromatogr. A. 1217 (2010) 3561–3566. [20] B. Baena, A. Cifuentes, C. Barbas, Analysis of carboxylic acids in biological fluids by capillary electrophoresis., Electrophoresis. 26
SC
(2005) 2622–36.
[21] G.C. Cochran, A review of the analysis of free fatty acids [C2-C6]., J.
M AN U
Chromatogr. Sci. 13 (1975) 440–7.
[22] J.B. Ewaschuk, G.A. Zello, J.M. Naylor, D.R. Brocks, Metabolic acidosis: separation methods and biological relevance of organic acids and lactic acid enantiomers., J. Chromatogr. B. Analyt.
TE D
Technol. Biomed. Life Sci. 781 (2002) 39–56. [23] J. Fernandes, W. Su, S. Rahat-Rozenbloom, T.M.S. Wolever, E.M.
EP
Comelli, Adiposity, gut microbiota and faecal short chain fatty acids
AC C
are linked in adult humans, Nutr. Diabetes. 4 (2014) e121. [24] X. Gao, E. Pujos-Guillot, J.-F. Martin, P. Galan, C. Juste, W. Jia, J.-L. Sebedio, Metabolite analysis of human fecal water by gas chromatography/mass spectrometry with ethyl chloroformate derivatization., Anal. Biochem. 393 (2009) 163–75. [25] E.. Lima, D.S.. Abdalla, High-performance liquid chromatography of fatty acids in biological samples, Anal. Chim. Acta. 465 (2002) 8–91.
ACCEPTED MANUSCRIPT [26] G. Zhao, M. Nyman, J.A. Jönsson, Rapid determination of short-chain fatty acids in colonic contents and faeces of humans and rats by acidified water-extraction and direct-injection gas chromatography.,
RI PT
Biomed. Chromatogr. 20 (2006) 674–82. [27] J.A. T., M.A.J. P., Gas-liquid partition chromatography: the
separation and micro-estimation of volatile fatty acids from formic
SC
acid to dodecanoic acid, (1952).
[28] F.A. Guthrie, Introduction of instrumental analysis (Braun, Robert
M AN U
D.), J. Chem. Educ. 65 (1988) A336.
[29] K. Bielawska, I. Dziakowska, W. Roszkowska-Jakimiec, Chromatographic determination of fatty acids in biological material.,
TE D
Toxicol. Mech. Methods. 20 (2010) 526–37.
[30] J. Kopecný, J. Mrázek, K. Fliegerová, P. Frühauf, L. Tucková, The intestinal microflora of childhood patients with indicated celiac
EP
disease., Folia Microbiol. (Praha). 53 (2008) 214–6.
AC C
[31] H.-M. Chen, Y.-N. Yu, J.-L. Wang, Y.-W. Lin, X. Kong, C.-Q. Yang, L. Yang, Z.-J. Liu, Y.-Z. Yuan, F. Liu, J.-X. Wu, L. Zhong, D.-C. Fang, W. Zou, J.-Y. Fang, Decreased dietary fiber intake and structural alteration of gut microbiota in patients with advanced colorectal adenoma., Am. J. Clin. Nutr. 97 (2013) 1044–52. [32] A. Klein, U. Friedrich, H. Vogelsang, G. Jahreis, Lactobacillus acidophilus 74-2 and Bifidobacterium animalis subsp lactis DGCC 420
ACCEPTED MANUSCRIPT modulate unspecific cellular immune response in healthy adults., Eur. J. Clin. Nutr. 62 (2008) 584–93. [33] A. Fechner, K. Fenske, G. Jahreis, Effects of legume kernel fibres and
RI PT
citrus fibre on putative risk factors for colorectal cancer: a randomised, double-blind, crossover human intervention trial., Nutr. J. 12 (2013) 101.
SC
[34] A. Roessler, S.D. Forssten, M. Glei, A.C. Ouwehand, G. Jahreis, The effect of probiotics on faecal microbiota and genotoxic activity of
M AN U
faecal water in patients with atopic dermatitis: a randomized, placebo-controlled study., Clin. Nutr. 31 (2012) 22–9. [35] E. Nistal, A. Caminero, S. Vivas, J.M. Ruiz de Morales, L.E. Sáenz de Miera, L.B. Rodríguez-Aparicio, J. Casqueiro, Differences in faecal
TE D
bacteria populations and faecal bacteria metabolism in healthy adults and celiac disease patients., Biochimie. 94 (2012) 1724–9.
EP
[36] E. Beards, K. Tuohy, G. Gibson, A human volunteer study to assess the impact of confectionery sweeteners on the gut microbiota
AC C
composition., Br. J. Nutr. 104 (2010) 701–8.
[37] K. Turunen, E. Tsouvelakidou, T. Nomikos, K.C. Mountzouris, D. Karamanolis, J. Triantafillidis, A. Kyriacou, Impact of beta-glucan on the faecal microbiota of polypectomized patients: a pilot study., Anaerobe. 17 (2011) 403–6. [38] R.A. Kekkonen, R. Holma, K. Hatakka, T. Suomalainen, T. Poussa, H.
ACCEPTED MANUSCRIPT Adlercreutz, R. Korpela, A probiotic mixture including galactooligosaccharides decreases fecal β-glucosidase activity but does not affect serum enterolactone concentration in men during a
RI PT
two-week intervention., J. Nutr. 141 (2011) 870–6. [39] M. Finney, J. Smullen, H.A. Foster, S. Brokx, D.M. Storey, Effects of low doses of lactitol on faecal microflora, pH, short chain fatty acids
SC
and gastrointestinal symptomology., Eur. J. Nutr. 46 (2007) 307–14. [40] A. Cuervo, N. Salazar, P. Ruas-Madiedo, M. Gueimonde, S. González,
M AN U
Fiber from a regular diet is directly associated with fecal short-chain fatty acid concentrations in the elderly., Nutr. Res. 33 (2013) 811–6. [41] N. Salazar, E.M. Dewulf, A.M. Neyrinck, L.B. Bindels, P.D. Cani, J. Mahillon, W.M. de Vos, J.-P. Thissen, M. Gueimonde, C.G. de Los
TE D
Reyes-Gavilán, N.M. Delzenne, Inulin-type fructans modulate intestinal Bifidobacterium species populations and decrease fecal
EP
short-chain fatty acids in obese women., Clin. Nutr. (2014). [42] S. Arboleya, A. Binetti, N. Salazar, N. Fernández, G. Solís, A.
AC C
Hernández-Barranco, A. Margolles, C.G. de Los Reyes-Gavilán, M. Gueimonde, Establishment and development of intestinal microbiota in preterm neonates., FEMS Microbiol. Ecol. 79 (2012) 763–72.
[43] X.-M. Ben, J. Li, Z.-T. Feng, S.-Y. Shi, Y.-D. Lu, R. Chen, X.-Y. Zhou, Low level of galacto-oligosaccharide in infant formula stimulates growth of intestinal Bifidobacteria and Lactobacilli., World J. Gastroenterol. 14 (2008) 6564–8.
ACCEPTED MANUSCRIPT [44] J.G. Muir, E.G.W. Yeow, J. Keogh, C. Pizzey, A.R. Bird, K. Sharpe, K. O’Dea, F.A. Macrae, Combining wheat bran with resistant starch has more beneficial effects on fecal indexes than does wheat bran alone.,
RI PT
Am. J. Clin. Nutr. 79 (2004) 1020–8. [45] S. Wildt, I. Nordgaard-Lassen, F. Bendtsen, J.J. Rumessen, Metabolic and inflammatory faecal markers in collagenous colitis., Eur. J.
SC
Gastroenterol. Hepatol. 19 (2007) 567–74.
[46] J. Fernandes, A. Wang, W. Su, S.R. Rozenbloom, A. Taibi, E.M.
M AN U
Comelli, T.M.S. Wolever, Age, dietary fiber, breath methane, and fecal short chain fatty acids are interrelated in Archaea-positive humans., J. Nutr. 143 (2013) 1269–75.
[47] J. Ou, F. Carbonero, E.G. Zoetendal, J.P. DeLany, M. Wang, K.
TE D
Newton, H.R. Gaskins, S.J.D. O’Keefe, Diet, microbiota, and microbial metabolites in colon cancer risk in rural Africans and
EP
African Americans., Am. J. Clin. Nutr. 98 (2013) 111–20. [48] L. Wang, C.T. Christophersen, M.J. Sorich, J.P. Gerber, M.T. Angley,
AC C
M.A. Conlon, Elevated fecal short chain fatty acid and ammonia concentrations in children with autism spectrum disorder., Dig. Dis. Sci. 57 (2012) 2096–102.
[49] A.L. McOrist, G.C.J. Abell, C. Cooke, K. Nyland, Bacterial population dynamics and faecal short-chain fatty acid (SCFA) concentrations in healthy humans., Br. J. Nutr. 100 (2008) 138–46.
ACCEPTED MANUSCRIPT [50] A.L. McOrist, R.B. Miller, A.R. Bird, J.B. Keogh, M. Noakes, D.L. Topping, M.A. Conlon, Fecal butyrate levels vary widely among individuals but are usually increased by a diet high in resistant
RI PT
starch., J. Nutr. 141 (2011) 883–9. [51] D.L. Worthley, R.K. Le Leu, V.L. Whitehall, M. Conlon, C.
Christophersen, D. Belobrajdic, K.-A. Mallitt, Y. Hu, N. Irahara, S.
SC
Ogino, B.A. Leggett, G.P. Young, A human, double-blind, placebocontrolled, crossover trial of prebiotic, probiotic, and synbiotic
M AN U
supplementation: effects on luminal, inflammatory, epigenetic, and epithelial biomarkers of colorectal cancer., Am. J. Clin. Nutr. 90 (2009) 578–86.
[52] D.L. Worthley, V.L.J. Whitehall, R.K. Le Leu, N. Irahara, R.L.
TE D
Buttenshaw, K.-A. Mallitt, S.A. Greco, I. Ramsnes, J. Winter, Y. Hu, S. Ogino, G.P. Young, B.A. Leggett, DNA methylation in the rectal
EP
mucosa is associated with crypt proliferation and fecal short-chain fatty acids., Dig. Dis. Sci. 56 (2011) 387–96.
AC C
[53] K. Kajander, L. Krogius-Kurikka, T. Rinttilä, H. Karjalainen, A. Palva, R. Korpela, Effects of multispecies probiotic supplementation on intestinal microbiota in irritable bowel syndrome., Aliment. Pharmacol. Ther. 26 (2007) 463–73. [54] G.C.J. Abell, M.A. Conlon, A.L. Mcorist, Methanogenic archaea in adult human faecal samples are inversely related to butyrate concentration, Microb. Ecol. Heal. Dis. 18 (2006).
ACCEPTED MANUSCRIPT [55] G.D. Brinkworth, M. Noakes, P.M. Clifton, A.R. Bird, Comparative effects of very low-carbohydrate, high-fat and high-carbohydrate, low-fat weight-loss diets on bowel habit and faecal short-chain fatty
RI PT
acids and bacterial populations., Br. J. Nutr. 101 (2009) 1493–502. [56] A.R. Bird, M.S. Vuaran, R.A. King, M. Noakes, J. Keogh, M.K. Morell, D.L. Topping, Wholegrain foods made from a novel high-amylose
SC
barley variety (Himalaya 292) improve indices of bowel health in human subjects., Br. J. Nutr. 99 (2008) 1032–40.
M AN U
[57] J. Valeur, M.H. Morken, E. Norin, T. Midtvedt, A. Berstad, Intestinal fermentation in patients with self-reported food hypersensitivity: painful, but protective?, Clin. Exp. Gastroenterol. 3 (2010) 65–70. [58] J. Valeur, M.H. Morken, E. Norin, T. Midtvedt, A. Berstad,
TE D
Carbohydrate intolerance in patients with self-reported food hypersensitivity: comparison of lactulose and glucose., Scand. J.
EP
Gastroenterol. 44 (2009) 1416–23. [59] B. Tjellström, L. Högberg, L. Stenhammar, K.-E. Magnusson, T.
AC C
Midtvedt, E. Norin, T. Sundqvist, Effect of exclusive enteral nutrition on gut microflora function in children with Crohn’s disease., Scand. J. Gastroenterol. 47 (2012) 1454–9.
[60] B. Tjellström, L. Stenhammar, L. Högberg, K. Fälth-Magnusson, K.-E. Magnusson, T. Midtvedt, T. Sundqvist, E. Norin, Screening-detected and symptomatic untreated celiac children show similar gut microflora-associated characteristics., Scand. J. Gastroenterol. 45
ACCEPTED MANUSCRIPT (2010) 1059–62. [61] B. Tjellström, L. Stenhammar, T. Sundqvist, K. Fälth-Magnusson, E. Hollén, K.-E. Magnusson, E. Norin, T. Midtvedt, L. Högberg, The
RI PT
effects of oats on the function of gut microflora in children with coeliac disease., Aliment. Pharmacol. Ther. 39 (2014) 1156–60.
[62] B. Tjellström, L. Högberg, L. Stenhammar, K. Fälth-Magnusson, K.-E.
SC
Magnusson, E. Norin, T. Sundqvist, T. Midtvedt, Faecal short-chain fatty acid pattern in childhood coeliac disease is normalised after
M AN U
more than one year’s gluten-free diet., Microb. Ecol. Health Dis. 24 (2013).
[63] E. Norin, T. Midtvedt, B. Björkstén, Development of Faecal ShortChain Fatty Acid Pattern during the First Year of Life in Estonian and
TE D
Swedish Infants, Microb. Ecol. Heal. Dis. 16 (2004). [64] T. Midtvedt, E. Zabarovsky, E. Norin, J. Bark, R. Gizatullin, V.
EP
Kashuba, O. Ljungqvist, V. Zabarovska, R. Möllby, I. Ernberg, Increase of faecal tryptic activity relates to changes in the intestinal
AC C
microbiome: analysis of Crohn’s disease with a multidisciplinary platform., PLoS One. 8 (2013) e66074.
[65] A. Sandin, L. Bråbäck, E. Norin, B. Björkstén, Faecal short chain fatty acid pattern and allergy in early childhood., Acta Paediatr. 98 (2009) 823–7. [66] T.L. Weir, D.K. Manter, A.M. Sheflin, B.A. Barnett, A.L. Heuberger,
ACCEPTED MANUSCRIPT E.P. Ryan, Stool microbiome and metabolome differences between colorectal cancer patients and healthy adults., PLoS One. 8 (2013) e70803.
RI PT
[67] Y. Bouhnik, L. Raskine, K. Champion, C. Andrieux, S. Penven, H. Jacobs, G. Simoneau, Prolonged administration of low-dose inulin stimulates the growth of bifidobacteria in humans, Nutr. Res. 27
SC
(2007) 187–193.
[68] J.B. Adams, L.J. Johansen, L.D. Powell, D. Quig, R.A. Rubin,
M AN U
Gastrointestinal flora and gastrointestinal status in children with autism--comparisons to typical children and correlation with autism severity., BMC Gastroenterol. 11 (2011) 22.
[69] A. Caminero, E. Nistal, L. Arias, S. Vivas, I. Comino, A. Real, C.
TE D
Sousa, J.M.R. de Morales, M.A. Ferrero, L.B. Rodríguez-Aparicio, J. Casqueiro, A gluten metabolism study in healthy individuals shows
293–9.
EP
the presence of faecal glutenasic activity., Eur. J. Nutr. 51 (2012)
AC C
[70] A. Costabile, F. Fava, H. Röytiö, S.D. Forssten, K. Olli, J. Klievink, I.R. Rowland, A.C. Ouwehand, R.A. Rastall, G.R. Gibson, G.E. Walton, Impact of polydextrose on the faecal microbiota: a doubleblind, crossover, placebo-controlled feeding study in healthy human subjects., Br. J. Nutr. 108 (2012) 471–81. [71] F. Fava, R. Gitau, B.A. Griffin, G.R. Gibson, K.M. Tuohy, J.A. Lovegrove, The type and quantity of dietary fat and carbohydrate
ACCEPTED MANUSCRIPT alter faecal microbiome and short-chain fatty acid excretion in a metabolic syndrome “at-risk” population., Int. J. Obes. (Lond). 37 (2013) 216–23.
RI PT
[72] E.K. Mitsou, E. Kougia, T. Nomikos, M. Yannakoulia, K.C. Mountzouris, A. Kyriacou, Effect of banana consumption on faecal microbiota: a randomised, controlled trial., Anaerobe. 17 (2011)
SC
384–7. doi:10.1016/j.anaerobe.2011.03.018.
[73] G.E. Walton, R.A. Rastall, M. Martini, C. Williams, R. Jeffries, G.R.
M AN U
Gibson, A double-blind, placebo controlled human study investigating the effects of coffee derived manno-oligosaccharides on the faecal microbiota of a healthy adult population., Int. J. Probiotics Prebiotics. (2010).
TE D
[74] D. Goossens, D. Jonkers, M. Russel, A. Thijs, A. van den Bogaard, E. Stobberingh, R. Stockbrügger, Survival of the probiotic, L. plantarum
EP
299v and its effects on the faecal bacterial flora, with and without gastric acid inhibition, Dig. Liver Dis. 37 (2005) 44–50.
AC C
[75] C. Karlsson, S. Ahrné, G. Molin, A. Berggren, I. Palmquist, G.N. Fredrikson, B. Jeppsson, Probiotic therapy to men with incipient arteriosclerosis initiates increased bacterial diversity in colon: a randomized controlled trial., Atherosclerosis. 208 (2010) 228–33. [76] H. Zhang, J. Sun, X. Liu, C. Hong, Y. Zhu, A. Liu, S. Li, H. Guo, F. Ren, Lactobacillus paracasei subsp. paracasei LC01 positively modulates intestinal microflora in healthy young adults., J. Microbiol.
ACCEPTED MANUSCRIPT 51 (2013) 777–82. [77] A.J. Wallace, S.L. Eady, D.C. Hunter, M.A. Skinner, L. Huffman, J. Ansell, P. Blatchford, M. Wohlers, T.D. Herath, D. Hedderley, D.
RI PT
Rosendale, H. Stoklosinski, T. McGhie, D. Sun-Waterhouse, C. Redman, No difference in fecal levels of bacteria or short chain fatty acids in humans, when consuming fruit juice beverages containing
SC
fruit fiber, fruit polyphenols, and their combination., Nutr. Res. 35 (2015) 23–34.
M AN U
[78] P. Ramnani, A. Costabile, A.G.R. Bustillo, G.R. Gibson, A randomised, double- blind, cross-over study investigating the prebiotic effect of agave fructans in healthy human subjects., J. Nutr. Sci. 4 (2015) e10.
TE D
[79] L. Achour, S. Nancey, D. Moussata, I. Graber, B. Messing, B. Flourié, Patients with a short bowel syndrome.Faecal bacterial mass and
233–8.
EP
energetic losses in healthy humans, Eur. J. Clin. Nutr. 61 (2007)
AC C
[80] C.-H. Yen, Y.-H. Tseng, Y.-W. Kuo, M.-C. Lee, H.-L. Chen, Long-term supplementation of isomalto-oligosaccharides improved colonic microflora profile, bowel function, and blood cholesterol levels in constipated elderly people--a placebo-controlled, diet-controlled trial., Nutrition. 27 (2011) 445–50. [81] T.F.S. Teixeira, Ł. Grześkowiak, S.C.C. Franceschini, J. Bressan, C.L.L.F. Ferreira, M.C.G. Peluzio, Higher level of faecal SCFA in
ACCEPTED MANUSCRIPT women correlates with metabolic syndrome risk factors., Br. J. Nutr. 109 (2013) 914–9. [82] J.M. Clarke, D.L. Topping, C.T. Christophersen, A.R. Bird, K. Lange,
RI PT
I. Saunders, L. Cobiac, Butyrate esterified to starch is released in the human gastrointestinal tract., Am. J. Clin. Nutr. 94 (2011) 1276–83. [83] J. Slavin, J. Feirtag, Chicory inulin does not increase stool weight or
SC
speed up intestinal transit time in healthy male subjects., Food Funct. 2 (2011) 72–7.
M AN U
[84] G.A. Spiller, J.A. Story, E.J. Furumoto, J.C. Chezem, M. Spiller, Effect of tartaric acid and dietary fibre from sun-dried raisins on colonic function and on bile acid and volatile fatty acid excretion in healthy
TE D
adults., Br. J. Nutr. 90 (2003) 803–7.
[85] O.C. Thompson-Chagoyan, M. Fallani, J. Maldonado, J.M. Vieites, S. Khanna, C. Edwards, J. Doré, A. Gil, Faecal microbiota and short-
EP
chain fatty acid levels in faeces from infants with cow’s milk protein
AC C
allergy., Int. Arch. Allergy Immunol. 156 (2011) 325–32. [86] H.M. Staudacher, M.C.E. Lomer, J.L. Anderson, J.S. Barrett, J.G. Muir, P.M. Irving, K. Whelan, Fermentable carbohydrate restriction reduces luminal bifidobacteria and gastrointestinal symptoms in patients with irritable bowel syndrome., J. Nutr. 142 (2012) 1510–8. [87] K. Whelan, P.A. Judd, K.M. Tuohy, G.R. Gibson, V.R. Preedy, M.A. Taylor, Fecal microbiota in patients receiving enteral feeding are
ACCEPTED MANUSCRIPT highly variable and may be altered in those who develop diarrhea., Am. J. Clin. Nutr. 89 (2009) 240–7. [88] H.A. Majid, P.W. Emery, K. Whelan, Faecal microbiota and short-
RI PT
chain fatty acids in patients receiving enteral nutrition with standard or fructo-oligosaccharides and fibre-enriched formulas., J. Hum. Nutr. Diet. 24 (2011) 260–8.
SC
[89] K. Whelan, P.A. Judd, V.R. Preedy, R. Simmering, A. Jann, M.A. Taylor, Fructooligosaccharides and fiber partially prevent the
M AN U
alterations in fecal microbiota and short-chain fatty acid concentrations caused by standard enteral formula in healthy humans., J. Nutr. 135 (2005) 1896–902.
[90] H.A. Majid, J. Cole, P.W. Emery, K. Whelan, Additional
TE D
oligofructose/inulin does not increase faecal bifidobacteria in critically ill patients receiving enteral nutrition: a randomised controlled trial.,
EP
Clin. Nutr. 33 (2014) 966–72..
[91] C. Lefranc-Millot, L. Guérin-Deremaux, D. Wils, C. Neut, L.E. Miller,
AC C
M.H. Saniez-Degrave, Impact of a resistant dextrin on intestinal ecology: how altering the digestive ecosystem with NUTRIOSE®, a soluble fibre with prebiotic properties, may be beneficial for health., J. Int. Med. Res. 40 (2012) 211–24.
[92] R. Kumari, V. Ahuja, J. Paul, Fluctuations in butyrate-producing bacteria in ulcerative colitis patients of North India., World J. Gastroenterol. 19 (2013) 3404–14.
ACCEPTED MANUSCRIPT [93] A.L. Hovey, G.P. Jones, H.M. Devereux, K.Z. Walker, Whole cereal and legume seeds increase faecal short chain fatty acids compared to ground seeds., Asia Pac. J. Clin. Nutr. 12 (2003) 477–82.
RI PT
[94] P. Friederich, J. Verschuur, B.W.H. van Heumen, H.M.J. Roelofs, M. Berkhout, I.D. Nagtegaal, M.G.H. van Oijen, J.H.J.M. van Krieken, W.H.M. Peters, F.M. Nagengast, Effects of intervention with sulindac
SC
and inulin/VSL#3 on mucosal and luminal factors in the pouch of
patients with familial adenomatous polyposis., Int. J. Colorectal Dis.
M AN U
26 (2011) 575–82.
[95] H.D. Holscher, K.L. Faust, L.A. Czerkies, R. Litov, E.E. Ziegler, H. Lessin, T. Hatch, S. Sun, K.A. Tappenden, Effects of prebioticcontaining infant formula on gastrointestinal tolerance and fecal
TE D
microbiota in a randomized controlled trial., JPEN. J. Parenter. Enteral Nutr. 36 (2012) 95S–105S.
EP
[96] A.M. Bakker-Zierikzee, M.S. Alles, J. Knol, F.J. Kok, J.J.M. Tolboom, J.G. Bindels, Effects of infant formula containing a mixture of
AC C
galacto- and fructo-oligosaccharides or viable Bifidobacterium animalis on the intestinal microflora during the first 4 months of life., Br. J. Nutr. 94 (2005) 783–90.
[97] R.A. Reimer, X. Pelletier, I.G. Carabin, M.R. Lyon, R.J. Gahler, S. Wood, Faecal short chain fatty acids in healthy subjects participating in a randomised controlled trial examining a soluble highly viscous polysaccharide versus control., J. Hum. Nutr. Diet. 25 (2012) 373–7.
ACCEPTED MANUSCRIPT [98] S. Delgado, P. Ruas-Madiedo, A. Suárez, B. Mayo, Interindividual differences in microbial counts and biochemical-associated variables in the feces of healthy Spanish adults., Dig. Dis. Sci. 51 (2006) 737–
RI PT
43. [99] A. Gostner, M. Blaut, V. Schäffer, G. Kozianowski, S. Theis, M.
Klingeberg, Y. Dombrowski, D. Martin, S. Ehrhardt, D. Taras, A.
SC
Schwiertz, B. Kleessen, H. Lührs, J. Schauber, D. Dorbath, T. Menzel, W. Scheppach, Effect of isomalt consumption on faecal microflora
M AN U
and colonic metabolism in healthy volunteers., Br. J. Nutr. 95 (2006) 40–50.
[100] M.O. Weickert, A.M. Arafat, M. Blaut, C. Alpert, N. Becker, V. Leupelt, N. Rudovich, M. Möhlig, A.F. Pfeiffer, Changes in dominant
TE D
groups of the gut microbiota do not explain cereal-fiber induced improvement of whole-body insulin sensitivity., Nutr. Metab. (Lond).
EP
8 (2011) 90.
[101] B. Kleessen, S. Schwarz, A. Boehm, H. Fuhrmann, A. Richter, T.
AC C
Henle, M. Krueger, Jerusalem artichoke and chicory inulin in bakery products affect faecal microbiota of healthy volunteers., Br. J. Nutr. 98 (2007) 540–9.
[102] K. Tiihonen, S. Tynkkynen, A. Ouwehand, T. Ahlroos, N. Rautonen, The effect of ageing with and without non-steroidal antiinflammatory drugs on gastrointestinal microbiology and immunology., Br. J. Nutr. 100 (2008) 130–7.
ACCEPTED MANUSCRIPT [103] J. Maldonado, F. Lara-Villoslada, S. Sierra, L. Sempere, M. Gómez, J.M. Rodriguez, J. Boza, J. Xaus, M. Olivares, Safety and tolerance of the human milk probiotic strain Lactobacillus salivarius CECT5713 in
RI PT
6-month-old children., Nutrition. 26 (2010) 1082–7. [104] M. Olivares, M.A.P. Díaz-Ropero, N. Gómez, F. Lara-Villoslada, S. Sierra, J.A. Maldonado, R. Martín, E. López-Huertas, J.M. Rodríguez,
SC
J. Xaus, Oral administration of two probiotic strains, Lactobacillus gasseri CECT5714 and Lactobacillus coryniformis CECT5711,
M AN U
enhances the intestinal function of healthy adults., Int. J. Food Microbiol. 107 (2006) 104–11.
[105] Z. Liu, Z. Xu, M. Han, B.-H. Guo, Efficacy of pasteurised yoghurt in improving chronic constipation: A randomised, double-blind, placebo-
TE D
controlled trial, Int. Dairy J. 40 (2015) 1–5.
[106] S. Sierra, F. Lara-Villoslada, L. Sempere, M. Olivares, J. Boza, J.
EP
Xaus, Intestinal and immunological effects of daily oral administration of Lactobacillus salivarius CECT5713 to healthy
AC C
adults., Anaerobe. 16 (2010) 195–200.
[107] M.J. Bernal, M.J. Periago, R. Martínez, I. Ortuño, M. Sánchez-Solís, G. Ros, F. Romero, P. Abellán, Effects of infant cereals with different carbohydrate profiles on colonic function--randomised and doubleblind clinical trial in infants aged between 6 and 12 months--pilot study., Eur. J. Pediatr. 172 (2013) 1535–42. [108] A.S. Klosterbuer, M.A.J. Hullar, F. Li, E. Traylor, J.W. Lampe, W.
ACCEPTED MANUSCRIPT Thomas, J.L. Slavin, Gastrointestinal effects of resistant starch, soluble maize fibre and pullulan in healthy adults., Br. J. Nutr. 110 (2013) 1068–74.
RI PT
[109] S.-M. Schneider, F. Girard-Pipau, J. Filippi, X. Hebuterne, D. Moyse, G.-C. Hinojosa, A. Pompei, P. Rampal, Effects of Saccharomyces
boulardii on fecal short-chain fatty acids and microflora in patients on
SC
long-term total enteral nutrition., World J. Gastroenterol. 11 (2005) 6165–9.
M AN U
[110] S.M. Schneider, F. Girard-Pipau, R. Anty, E.G.M. van der Linde, B.J. Philipsen-Geerling, J. Knol, J. Filippi, K. Arab, X. Hébuterne, Effects of total enteral nutrition supplemented with a multi-fibre mix on
82–90.
TE D
faecal short-chain fatty acids and microbiota., Clin. Nutr. 25 (2006)
[111] C. Sierra, M.-J. Bernal, J. Blasco, R. Martínez, J. Dalmau, I. Ortuño,
EP
B. Espín, M.-I. Vasallo, D. Gil, M.-L. Vidal, D. Infante, R. Leis, J. Maldonado, J.-M. Moreno, E. Román, Prebiotic effect during the first
AC C
year of life in healthy infants fed formula containing GOS as the only prebiotic: a multicentre, randomised, double-blind and placebocontrolled trial., Eur. J. Nutr. 54 (2015) 89–99.
[112] E.J. Woodmansey, M.E.T. McMurdo, G.T. Macfarlane, S. Macfarlane, Comparison of compositions and metabolic activities of fecal microbiotas in young adults and in antibiotic-treated and nonantibiotic-treated elderly subjects., Appl. Environ. Microbiol. 70
ACCEPTED MANUSCRIPT (2004) 6113–22. [113] S. Macfarlane, S. Cleary, B. Bahrami, N. Reynolds, G.T. Macfarlane, Synbiotic consumption changes the metabolism and composition of
RI PT
the gut microbiota in older people and modifies inflammatory processes: a randomised, double-blind, placebo-controlled crossover study., Aliment. Pharmacol. Ther. 38 (2013) 804–16.
SC
[114] J.-M. Lecerf, F. Dépeint, E. Clerc, Y. Dugenet, C.N. Niamba, L. Rhazi, A. Cayzeele, G. Abdelnour, A. Jaruga, H. Younes, H. Jacobs, G.
M AN U
Lambrey, A.M. Abdelnour, P.R. Pouillart, Xylo-oligosaccharide (XOS) in combination with inulin modulates both the intestinal environment and immune status in healthy subjects, while XOS alone only shows prebiotic properties., Br. J. Nutr. 108 (2012) 1847–58.
TE D
[115] A. Schwiertz, D. Taras, K. Schäfer, S. Beijer, N.A. Bos, C. Donus, P.D. Hardt, Microbiota and SCFA in lean and overweight healthy
EP
subjects., Obesity (Silver Spring). 18 (2010) 190–5. [116] N. Larsen, F.K. Vogensen, R.J. Gøbel, K.F. Michaelsen, S.D.
AC C
Forssten, S.J. Lahtinen, M. Jakobsen, Effect of Lactobacillus salivarius Ls-33 on fecal microbiota in obese adolescents., Clin. Nutr. 32 (2013) 935–40.
[117] S.M. Gråsten, K.S. Juntunen, J. Mättö, O.T. Mykkänen, H. ElNezami, H. Adlercreutz, K.S. Poutanen, H.M. Mykkänen, High-fiber rye bread improves bowel function in postmenopausal women but does not cause other putatively positive changes in the metabolic
ACCEPTED MANUSCRIPT activity of intestinal microbiota, Nutr. Res. 27 (2007) 454–461. [118] A. Costabile, A. Klinder, F. Fava, A. Napolitano, V. Fogliano, C. Leonard, G.R. Gibson, K.M. Tuohy, Whole-grain wheat breakfast
RI PT
cereal has a prebiotic effect on the human gut microbiota: a doubleblind, placebo-controlled, crossover study., Br. J. Nutr. 99 (2008) 110–20.
SC
[119] I.E.J.A. François, O. Lescroart, W.S. Veraverbeke, M. Marzorati, S. Possemiers, H. Hamer, K. Windey, G.W. Welling, J.A. Delcour, C.M.
M AN U
Courtin, K. Verbeke, W.F. Broekaert, Effects of wheat bran extract containing arabinoxylan oligosaccharides on gastrointestinal parameters in healthy preadolescent children., J. Pediatr. Gastroenterol. Nutr. 58 (2014) 647–53.
TE D
[120] R. García-Villalba, J.A. Giménez-Bastida, M.T. García-Conesa, F.A. Tomás-Barberán, J. Carlos Espín, M. Larrosa, Alternative method for
EP
gas chromatography-mass spectrometry analysis of short-chain fatty acids in faecal samples., J. Sep. Sci. 35 (2012) 1906–13.
AC C
[121] M. Wullt, M.-L. Johansson Hagslätt, I. Odenholt, A. Berggren, Lactobacillus plantarum 299v enhances the concentrations of fecal short-chain fatty acids in patients with recurrent clostridium difficileassociated diarrhea., Dig. Dis. Sci. 52 (2007) 2082–6. [122] G.E. Walton, C. Lu, I. Trogh, F. Arnaut, G.R. Gibson, A randomised, double-blind, placebo controlled cross-over study to determine the gastrointestinal effects of consumption of arabinoxylan-
ACCEPTED MANUSCRIPT oligosaccharides enriched bread in healthy volunteers., Nutr. J. 11 (2012) 36. [123] X. Zheng, Y. Qiu, W. Zhong, S. Baxter, M. Su, Q. Li, G. Xie, B.M.
RI PT
Ore, S. Qiao, M.D. Spencer, S.H. Zeisel, Z. Zhou, A. Zhao, W. Jia, A targeted metabolomic protocol for short-chain fatty acids and
branched-chain amino acids., Metabolomics. 9 (2013) 818–827.
SC
[124] R. Di Cagno, M. De Angelis, I. De Pasquale, M. Ndagijimana, P. Vernocchi, P. Ricciuti, F. Gagliardi, L. Laghi, C. Crecchio, M.E.
M AN U
Guerzoni, M. Gobbetti, R. Francavilla, Duodenal and faecal microbiota of celiac children: molecular, phenotype and metabolome characterization., BMC Microbiol. 11 (2011) 219. [125] B. Vitali, M. Ndagijimana, F. Cruciani, P. Carnevali, M. Candela, M.E.
TE D
Guerzoni, P. Brigidi, Impact of a synbiotic food on the gut microbial ecology and metabolic profiles., BMC Microbiol. 10 (2010) 4.
EP
[126] I. Ahmed, R. Greenwood, B. de L. Costello, N.M. Ratcliffe, C.S. Probert, An investigation of fecal volatile organic metabolites in
AC C
irritable bowel syndrome., PLoS One. 8 (2013) e58204.
[127] D. Taneyo Saa, S. Turroni, D.I. Serrazanetti, S. Rampelli, S. Maccaferri, M. Candela, M. Severgnini, E. Simonetti, P. Brigidi, A. Gianotti, Impact of Kamut® Khorasan on gut microbiota and metabolome in healthy volunteers, Food Res. Int. 63 (2014) 227– 232.
ACCEPTED MANUSCRIPT [128] R.D. Couch, A. Dailey, F. Zaidi, K. Navarro, C.B. Forsyth, E. Mutlu, P.A. Engen, A. Keshavarzian, Alcohol induced alterations to the human fecal VOC metabolome., PLoS One. 10 (2015) e0119362.
RI PT
[129] M.-S. Kim, S.-S. Hwang, E.-J. Park, J.-W. Bae, Strict vegetarian diet improves the risk factors associated with metabolic diseases by
modulating gut microbiota and reducing intestinal inflammation.,
SC
Environ. Microbiol. Rep. 5 (2013) 765–75.
[130] V. De Preter, G. Van Staeyen, D. Esser, P. Rutgeerts, K. Verbeke,
M AN U
Development of a screening method to determine the pattern of fermentation metabolites in faecal samples using on-line purge-andtrap gas chromatographic-mass spectrometric analysis., J. Chromatogr. A. 1216 (2009) 1476–83.
TE D
[131] K. Windey, E. Houben, L. Deroover, K. Verbeke, Contribution of Colonic Fermentation and Fecal Water Toxicity to the
22.
EP
Pathophysiology of Lactose-Intolerance., Nutrients. 7 (2015) 7505–
AC C
[132] A.J. Wallace, S.L. Eady, D.C. Hunter, M.A. Skinner, L. Huffman, J. Ansell, P. Blatchford, M. Wohlers, T.D. Herath, D. Hedderley, D. Rosendale, H. Stoklosinski, T. McGhie, D. Sun-Waterhouse, C. Redman, No difference in fecal levels of bacteria or short chain fatty acids in humans, when consuming fruit juice beverages containing fruit fiber, fruit polyphenols, and their combination., Nutr. Res. 35 (2015) 23–34.
ACCEPTED MANUSCRIPT [133] A. Tangerman, F.M. Nagengast, A gas chromatographic analysis of fecal short-chain fatty acids, using the direct injection method., Anal. Biochem. 236 (1996) 1–8.
RI PT
[134] J.B. Zijlstra, J. Beukema, B.G. Wolthers, B.M. Byrne, A. Groen, J. Dankert, Pretreatment methods prior to gaschromatographic analysis of volatile fatty acids from faecal samples., Clin. Chim. Acta. 78
SC
(1977) 243–50.
[135] T. Høverstad, O. Fausa, A. Bjørneklett, T. Bøhmer, Short-chain fatty
M AN U
acids in the normal human feces., Scand. J. Gastroenterol. 19 (1984) 375–81.
[136] J. Pawliszyn, Solid Phase Microextraction: Theory and Practice,
TE D
1997.
[137] G.A. Mills, V. Walker, H. Mughal, Headspace solid-phase microextraction with 1-pyrenyldiazomethane in-fibre derivatisation
EP
for analysis of faecal short-chain fatty acids, J. Chromatogr. B
AC C
Biomed. Sci. Appl. 730 (1999) 113–122. [138] K. Grob, Gas chromatographic stationary phases analysed by capillary gas chromatography, 1980.
[139] Z. Ji, R.E. Majors, E.J. Guthrie, Porous layer open-tubular capillary columns: preparations, applications and future directions, J. Chromatogr. A. 842 (1999) 115–142. [140] W.J. Cheong, F. Ali, Y.S. Kim, J.W. Lee, Comprehensive overview of
ACCEPTED MANUSCRIPT recent preparation and application trends of various open tubular capillary columns in separation science, J. Chromatogr. A. 1308 (2013) 1–24.
RI PT
[141] J. Luong, R. Gras, W. Jennings, An advanced solventless column test for capillary GC columns, J. Sep. Sci. 30 (2007) 2480–2492.
[142] A.E. Thompson, A flame ionization detector for gas chromatography,
SC
J. Chromatogr. A. 2 (1959) 148–154.
[143] A. El-Aneed, A. Cohen, J. Banoub, Mass Spectrometry, Review of the
M AN U
Basics: Electrospray, MALDI, and Commonly Used Mass Analyzers, Appl. Spectrosc. Rev. 44 (2009) 210–230.
[144] K. Schäfer, Analysis of short chain fatty acids from different
TE D
intestinal samples by capillary gas chromatography, Chromatographia. 40 (1995) 550–556. [145] D.L. Schooley, F.M. Kubiak, J. V. Evans, Capillary Gas
EP
Chromatographic Analysis of Volatile and Non-Volatile Organic Acids
AC C
from Biological Samples as the t-Butyldimethylsilyl Derivatives, J. Chromatogr. Sci. 23 (1985) 385–390.
[146] G. Gutnikov, Fatty acid profiles of lipid samples., J. Chromatogr. B. Biomed. Appl. 671 (1995) 71–89.
[147] E.R. Farnworth, Y.P. Chouinard, H. Jacques, S. Venkatramanan, A.A. Maf, S. Defnoun, P.J.H. Jones, The effect of drinking milk containing conjugated linoleic acid on fecal microbiological profile, enzymatic
ACCEPTED MANUSCRIPT activity, and fecal characteristics in humans., Nutr. J. 6 (2007) 15. [148] E. Fritz, H.F. Hammer, R.W. Lipp, C. Högenauer, R. Stauber, J. Hammer, Effects of lactulose and polyethylene glycol on colonic
RI PT
transit., Aliment. Pharmacol. Ther. 21 (2005) 259–68. [149] M. Okazaki, S. Matsukuma, R. Suto, K. Miyazaki, M. Hidaka, M. Matsuo, S. Noshima, N. Zempo, T. Asahara, K. Nomoto,
SC
Perioperative synbiotic therapy in elderly patients undergoing
gastroenterological surgery: a prospective, randomized control trial.,
M AN U
Nutrition. 29 (2013) 1224–30.
[150] S. Nagata, T. Asahara, T. Ohta, T. Yamada, S. Kondo, L. Bian, C. Wang, Y. Yamashiro, K. Nomoto, Effect of the continuous intake of probiotic-fermented milk containing Lactobacillus casei strain Shirota
TE D
on fever in a mass outbreak of norovirus gastroenteritis and the faecal microflora in a health service facility for the aged., Br. J. Nutr.
EP
106 (2011) 549–56.
[151] K. Shimizu, H. Ogura, T. Asahara, K. Nomoto, M. Morotomi, Y.
AC C
Nakahori, A. Osuka, S. Yamano, M. Goto, A. Matsushima, O. Tasaki, Y. Kuwagata, H. Sugimoto, Gastrointestinal dysmotility is associated with altered gut flora and septic mortality in patients with severe systemic inflammatory response syndrome: a preliminary study., Neurogastroenterol. Motil. 23 (2011) 330–5, e157.
[152] K. Shimizu, H. Ogura, T. Asahara, K. Nomoto, A. Matsushima, K. Hayakawa, H. Ikegawa, O. Tasaki, Y. Kuwagata, T. Shimazu, Gut
ACCEPTED MANUSCRIPT microbiota and environment in patients with major burns – a preliminary report., Burns. 41 (2015) e28-33. [153] C. Tana, Y. Umesaki, A. Imaoka, T. Handa, M. Kanazawa, S.
RI PT
Fukudo, Altered profiles of intestinal microbiota and organic acids may be the origin of symptoms in irritable bowel syndrome., Neurogastroenterol. Motil. 22 (2010) 512–9, e114-5.
SC
[154] K. Kato, S. Mizuno, Y. Umesaki, Y. Ishii, M. Sugitani, A. Imaoka, M. Otsuka, O. Hasunuma, R. Kurihara, A. Iwasaki, Y. Arakawa,
M AN U
Randomized placebo-controlled trial assessing the effect of bifidobacteria-fermented milk on active ulcerative colitis., Aliment. Pharmacol. Ther. 20 (2004) 1133–41.
[155] H. Takaishi, T. Matsuki, A. Nakazawa, T. Takada, S. Kado, T.
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Asahara, N. Kamada, A. Sakuraba, T. Yajima, H. Higuchi, N. Inoue, H. Ogata, Y. Iwao, K. Nomoto, R. Tanaka, T. Hibi, Imbalance in
EP
intestinal microflora constitution could be involved in the pathogenesis of inflammatory bowel disease., Int. J. Med. Microbiol.
AC C
298 (2008) 463–72.
[156] Y. Kanamori, M. Sugiyama, K. Hashizume, N. Yuki, M. Morotomi, R. Tanaka, Experience of long-term synbiotic therapy in seven short bowel patients with refractory enterocolitis., J. Pediatr. Surg. 39 (2004) 1686–92. [157] A. Kotani, Y. Miyaguchi, M. Kohama, T. Ohtsuka, T. Shiratori, F. Kusu, Determination of short-chain fatty acids in rat and human
ACCEPTED MANUSCRIPT feces by high-performance liquid chromatography with electrochemical detection., Anal. Sci. 25 (2009) 1007–11. [158] S. Ohigashi, K. Sudo, D. Kobayashi, T. Takahashi, K. Nomoto, H.
RI PT
Onodera, Significant changes in the intestinal environment after surgery in patients with colorectal cancer., J. Gastrointest. Surg. 17 (2013) 1657–64.
SC
[159] C. Ferrario, V. Taverniti, C. Milani, W. Fiore, M. Laureati, I. De Noni, M. Stuknyte, B. Chouaia, P. Riso, S. Guglielmetti, Modulation of fecal
M AN U
Clostridiales bacteria and butyrate by probiotic intervention with Lactobacillus paracasei DG varies among healthy adults., J. Nutr. 144 (2014) 1787–96.
[160] G. Riezzo, A. Orlando, B. D’Attoma, V. Guerra, F. Valerio, P.
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Lavermicocca, S. De Candia, F. Russo, Randomised clinical trial: efficacy of Lactobacillus paracasei-enriched artichokes in the
EP
treatment of patients with functional constipation--a double-blind, controlled, crossover study., Aliment. Pharmacol. Ther. 35 (2012)
AC C
441–50.
[161] F. Valerio, S. de Candia, S.L. Lonigro, F. Russo, G. Riezzo, A. Orlando, P. De Bellis, A. Sisto, P. Lavermicocca, Role of the probiotic strain Lactobacillus paracasei LMGP22043 carried by artichokes in influencing faecal bacteria and biochemical parameters in human subjects., J. Appl. Microbiol. 111 (2011) 155–64. [162] Y. Bouhnik, C. Neut, L. Raskine, C. Michel, M. Riottot, C. Andrieux,
ACCEPTED MANUSCRIPT F. Guillemot, F. Dyard, B. Flourié, Prospective, randomized, parallelgroup trial to evaluate the effects of lactulose and polyethylene glycol-4000 on colonic flora in chronic idiopathic constipation.,
RI PT
Aliment. Pharmacol. Ther. 19 (2004) 889–99. [163] M.A. Underwood, N.H. Salzman, S.H. Bennett, M. Barman, D.A. Mills, A. Marcobal, D.J. Tancredi, C.L. Bevins, M.P. Sherman, A
SC
randomized placebo-controlled comparison of 2 prebiotic/probiotic combinations in preterm infants: impact on weight gain, intestinal
M AN U
microbiota, and fecal short-chain fatty acids., J. Pediatr. Gastroenterol. Nutr. 48 (2009) 216–25.
[164] S. Monira, M.M. Hoq, A.K.A. Chowdhury, A. Suau, F. Magne, H.P. Endtz, M. Alam, M. Rahman, P. Pochart, J.-F. Desjeux, N.H. Alam,
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Short-chain fatty acids and commensal microbiota in the faeces of severely malnourished children with cholera rehydrated with three
EP
different carbohydrates., Eur. J. Clin. Nutr. 64 (2010) 1116–24. [165] T. Torii, K. Kanemitsu, T. Wada, S. Itoh, K. Kinugawa, A. Hagiwara,
AC C
Measurement of short-chain fatty acids in human faeces using highperformance liquid chromatography: specimen stability., Ann. Clin. Biochem. 47 (2010) 447–52.
[166] A. Suzuki, K. Mitsuyama, H. Koga, N. Tomiyasu, J. Masuda, K. Takaki, O. Tsuruta, A. Toyonaga, M. Sata, Bifidogenic growth stimulator for the treatment of active ulcerative colitis: a pilot study., Nutrition. 22 (2006) 76–81.
ACCEPTED MANUSCRIPT [167] C. Wang, H. Shoji, H. Sato, S. Nagata, Y. Ohtsuka, T. Shimizu, Y. Yamashiro, Effects of oral administration of bifidobacterium breve on fecal lactic acid and short-chain fatty acids in low birth weight
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infants., J. Pediatr. Gastroenterol. Nutr. 44 (2007) 252–7. [168] K. Tanaka, K. Ohta, P.R. Haddad, J.S. Fritz, D.A. Miyanaga, W. Hu, K. Hasebe, Simultaneous ion-exclusion/cation-exchange
SC
chromatography of anions and cations in acid rain waters on a
weakly acidic cation-exchange resin by elution with sulfosalicylic
M AN U
acid., J. Chromatogr. A. 884 (2000) 167–74.
[169] J. Han, K. Lin, C. Sequeira, C.H. Borchers, An isotope-labeled chemical derivatization method for the quantitation of short-chain fatty acids in human feces by liquid chromatography-tandem mass
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spectrometry., Anal. Chim. Acta. 854 (2015) 86–94. [170] H. Miwa, M. Yamamoto, High-performance liquid chromatographic
EP
analysis of serum short-chain fatty acids by direct derivatization., J. Chromatogr. 421 (1987) 33–41.
AC C
[171] L. Fischer Ross, D.C. Chapital, Simultaneous Determination of Carbohydrates and Products of Carbohydrate Metabolism in Fermentation Mixtures by HPLC, J. Chromatogr. Sci. 25 (1987) 112– 117. [172] F. Valerio, F. Russo, S. de Candia, G. Riezzo, A. Orlando, S.L. Lonigro, P. Lavermicocca, Effects of probiotic Lactobacillus paracaseienriched artichokes on constipated patients: a pilot study., J. Clin.
ACCEPTED MANUSCRIPT Gastroenterol. 44 Suppl 1 (2010) S49-53. [173] J. Saric, Y. Wang, J. Li, M. Coen, J. Utzinger, J.R. Marchesi, J. Keiser, K. Veselkov, J.C. Lindon, J.K. Nicholson, E. Holmes, Species
RI PT
variation in the fecal metabolome gives insight into differential gastrointestinal function., J. Proteome Res. 7 (2008) 352–60.
[174] D. Monleón, J.M. Morales, A. Barrasa, J.A. López, C. Vázquez, B.
SC
Celda, Metabolite profiling of fecal water extracts from human colorectal cancer., NMR Biomed. 22 (2009) 342–8.
M AN U
[175] M. Ndagijimana, L. Laghi, B. Vitali, G. Placucci, P. Brigidi, M.E. Guerzoni, Effect of a synbiotic food consumption on human gut metabolic profiles evaluated by (1)H Nuclear Magnetic Resonance
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spectroscopy., Int. J. Food Microbiol. 134 (2009) 147–53. [176] G. Le Gall, S.O. Noor, K. Ridgway, L. Scovell, C. Jamieson, I.T. Johnson, I.J. Colquhoun, E.K. Kemsley, A. Narbad, Metabolomics of
EP
fecal extracts detects altered metabolic activity of gut microbiota in ulcerative colitis and irritable bowel syndrome., J. Proteome Res. 10
AC C
(2011) 4208–18.
[177] D.M. Jacobs, N. Deltimple, E. van Velzen, F.A. van Dorsten, M. Bingham, E.E. Vaughan, J. van Duynhoven, (1)H NMR metabolite profiling of feces as a tool to assess the impact of nutrition on the human microbiome., NMR Biomed. 21 (2008) 615–26. [178] J.C. Lindon, J.K. Nicholson, E. Holmes, J.R. Everett, Metabonomics:
ACCEPTED MANUSCRIPT Metabolic processes studied by NMR spectroscopy of biofluids, Concepts Magn. Reson. 12 (2000) 289–320. [179] A. Smolinska, L. Blanchet, L.M.C. Buydens, S.S. Wijmenga, NMR and
RI PT
pattern recognition methods in metabolomics: from data acquisition to biomarker discovery: a review., Anal. Chim. Acta. 750 (2012) 82– 97.
SC
[180] T.R.H. and, D.O. Koltun, An NMR Strategy for Determination of
Configuration of Remote Stereogenic Centers: 3-Methylcarboxylic
M AN U
Acids, (1998).
[181] H. Whatley, Basic principles and modes of capillary electrophoresis, Clin. Forensic Appl. Capill. Electrophor. (2001) 21–58.
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[182] A. Garcia, B. Olmo, A. Lopez-Gonzalvez, L. Cornejo, F.J. Rupérez, C. Barbas, Capillary electrophoresis for short chain organic acids in faeces Reference values in a Mediterranean elderly population., J.
EP
Pharm. Biomed. Anal. 46 (2008) 356–61.
AC C
[183] T. Toyo’oka, Use of derivatization to improve the chromatographic properties and detection selectivity of physiologically important carboxylic acids, J. Chromatogr. B Biomed. Sci. Appl. 671 (1995) 91– 112.
[184] R.R. Ran-Ressler, S. Devapatla, P. Lawrence, J.T. Brenna, Branched Chain Fatty Acids Are Constituents of the Normal Healthy Newborn Gastrointestinal Tract, Pediatr. Res. 64 (2008) 605–609.
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EP
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SC
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ACCEPTED MANUSCRIPT
ACCEPTED MANUSCRIPT
Table 2: Advantages and disadvantages of the main pre-treatment techniques in scientific reports using GC methods for SCFAs analysis.
centrifugation / filtration / ultrafiltration
Advantages
•
Disadvantages
RI PT
PrePre-treatment
speed
M AN U
•
separation with low temperature
TE D
steam distillation
column overload
•
unspecific results
SC
(direct injection)
•
•
frequent change of column
•
decomposition of acetyl-groups of other sample components
•
unspecific results
•
low recovery rate
•
separation with very low temperature
•
time consuming
acidified sample)
•
sensitive
•
not practical with a lot of samples
•
loss of SCFAs
EP
vacuum distillation (distilled water or
transesterification with different acids
•
direct method
•
column overload
•
fast
•
frequent change of column
•
direct method
•
column overload
•
relatively fast
AC C
simple acidification
ACCEPTED MANUSCRIPT
•
good purification
frequent change of column
•
time consuming (more steps)
•
loss of SCFAs
•
large quantities of reagents (costs)
•
occupational exposure (toxic, flammable,
RI PT
acidification / organic extraction
•
SC
solvent
•
very good purification
AC C
derivatization (chloroformates)
EP
TE D
derivatization (silylation)
M AN U
allergenic, organic solvents)
•
time consuming (more steps)
•
loss of SCFAs
•
large quantities of reagents (cost)
•
occupational exposure (toxic, flammable, allergenic organic solvents)
•
occupational exposure (toxic, flammable, allergenic organic solvents)
ACCEPTED MANUSCRIPT
•
very high purity of sample
•
longer life-span and usage of a
•
and knowledge of technique)
chromatographic system purge and trap technique
•
higher number of volatile compounds
•
extraction capacities of volatiles decrease with a higher molecular mass
AC C
EP
TE D
SCFAs, short-chain fatty acids; SPME, solid phase micro-extraction.
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SC
(compared to SPME)
high cost (fibres, supplementary instrumentation
RI PT
SPME
ACCEPTED MANUSCRIPT
Table 3
Column
filtration
ion-exchange
centrifugation; filtration
ion-exclusion
Detector
References
UV-VIS
Huda-Faujan 2010 [5]
SC
Pretreatment
RI PT
Scientific reports using HPLC methods for SCFAs analysis.
Farnworth 2007 [147]
ion-exchange
UV
Fritz 2005 [148]
ion-exclusion
ECD
Okazaki 2013 [149]
ion-exclusion; RP
conductivity Nagata 2011 [150]
ion-exclusion; RP
conductivity Shimizu 2011 [151], Shimizu 2015 [152]
ion-exclusion; RP
conductivity Tana 2010 [153]
ion-exclusion; RP
conductivity Kato 2004 [154]
ion-exclusion; RP
conductivity Takaishi 2008 [155]
ion-exclusion; RP
conductivity Kanamori 2004 [156]
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VWD
centrifugation; filtration
AC C
EP
TE D
perchloric acid; filtration
ACCEPTED MANUSCRIPT
ECD
ion-exclusion; RP
conductivity Ohigashi 2013 [158]
RI PT
ion-exclusion
Kotani 2009 [157]
cation-exchange
UV;RI
Ferrario 2014 [159]
sulfuric acid; filtration
ion-exclusion
UV
Riezzo 2012 [160], Valerio 2011 [161]
acid; steam distillation
ion-exchange
SC
sulfuric acid; centrifugation; filtration
sulfuric acid; sodium chloride; diethyl ether; acetonitrile; sodium
RP
UV
Underwood 2009 [163]
cation-exclusion
UV
Monira 2010 [164]
UV-VIS
Torii 2010 [165]
TE D
hydroxide; acetonitrile
Bouhnik 2004 [162]
M AN U
sulfuric acid; filtration; 2-NPH-HCl; HCl; hexane; diethyl-ether
UV
perchloric acid
NaOH; centrifugation; chloroform; filtration
AC C
ether; de-ionized water
EP
pyridine; 1-EDC-HCl; 2-NPH-HCl; potassium hydroxide; phosphoric acid; RP
ion-exclusion
absorbance Suzuki 2006 [166]
anion-exclusion
conductivity Wang 2007 [167]
NR, not reported; UV, ultraviolet detector; UV-VIS, ultraviolet visible detector; ECD, electrochemical detector; RP, Reverse Phase column; RI, refractive index detector; VWD, Variable Wavelength Detector.
ACCEPTED MANUSCRIPT
Table 1
RI PT
Scientific reports using GC methods for SCFAs analysis. References
ultrafiltration
Kopečný 2008 [30]a,b
filtration
Chen 2013 [31]a,b, Klein 2008 [32]a(FFAP),b, Fechner 2013 [33]a(FFAP),b,
SC
Pretreatment Pretreatment
M AN U
Roessler 2012 [34]a(FFAP),b, Nistal 2012 [35]a(FFAP),b, Beards 2010 [36]a,b, Turunen 2011 [37]a,b, Kekkonen 2011 [38]a, Finney 2007 [39]a,b Cuervo 2013 [40]c, Salazar 2014 [41]c, Arboleya 2012 [42]c
centrifugation; filtration sodium phosphate
vacuum distillation
phosphoric; vacuum distillation
TE D
copper sulphate, vacuum distillation
Wildt 2007 [45] Fernandes 2013 [46]a(FFAP),b
EP
steam distillation
Muir 2004 [44]
AC C
saline solution
Ben 2008 [43]
Ou 2013 [47]a,b, Wang 2012 [48]a(FFAP), McOrist 2008 [49]a(FFAP), McOrist 2011 [50]a(FFAP), Worthley 2009 [51]a(FFAP),b, Worthley, 2011 [52]a(FFAP),b, Kajander 2007 [53] Abell 2006 [54]
ACCEPTED MANUSCRIPT
Brinkworth 2009 [55]a(FFAP),b, Bird 2008 [56]a(FFAP),b
sulfuric acid; vacuum distillation
Valeur 2010 [57]b, Valeur 2009 [58]b, Tjellström 2012 [59]b, Tjellström
RI PT
orto-phosphoric acid; vacuum distillation
2010 [60]b, Tjellström 2014 [61]b, Tjellström 2013 [62]b, Norin 2004
SC
[63], Midtvedt 2013 [64], Sandin 2009 [65] Weir 2013 [66]c
water extraction; acid
Bouhnik 2007 [67]
ethylene glycol; homogenization; ultrafiltration; hydrochloric acid
M AN U
acidification; centrifugation; filtration
Adams 2011 [68]b Caminero 2012 [69]a(FFAP),b, Costabile 2012 [70]a(FFAP)b, Fava 2013
hydrochloric acid
TE D
[71]a(FFAP),b, Mitsou 2011 [72]a,b, Walton 2010 [73]a,b, Zhao 2006
[26]a(FFAP),b Goossens 2005 [74]a(FFAP),b
hydrochloric acid; diethyl ether
EP
hydrochloric acid; diethyl ether; silylation
AC C
diethyl ether; hydrochloric acid
Karlsson 2010 [75]a Zhang 2013 [76]a(FFAP)
hydrochloric acid; diethyl ether; MTBSTFA with TBDMSCI
Wallace 2015 [77]b
hydrochloric acid; diethyl ether; MTBSTFA
Ramnani 2015 [78]a,b
phosphoric acid; centrifugation phosphoric acid
Achour 2007 [79]b Yen 2011 [80]a,b, Teixeira 2013 [81]a,b
ACCEPTED MANUSCRIPT
Clarke 2011 [82]a(FFAP)
phosphoric acid; oxalic acid
Slavin 2011 [83]
phosphoric acid; diethyl ether
Spiller 2003 [84]
orto-phosphoric acid; diethyl ether
Thompson-Chagoyan 2011 [85]a,b
RI PT
phosphoric acid; sublimation;
SC
lactic acid: enzymatic test
Staudacher 2012 [86]a, Whelan 2009 [87]a, Majid 2011 [88]a, Whelan
phosphoric acid and mercury chloride
M AN U
2005 [89]a, Majid 2014 [90]a, Lefranc-Millot 2012 [91]a(FFAP),b Kumari 2013 [92]a,b
phosphoric acid in mercury chloride; filtration meta-phosphoric acid; methanol
Hovey 2003 [93] Friederich 2011 [94]b
TE D
ultracentrifugation; filtration; formic acid
Holscher 2012 [95]c
formic acid
Bakker-Zierikzee 2005 [96]b
formic acid
EP
lactic acid: heating, enzymatic test
L- and D-lactate: enzymatic test formic acid; methanol methyl esterification (methanol)
AC C
formic acid; methanol
lactic acid: lactate dehydrogenase enzymatic test
Reimer 2012 [97]a,b
Delgado 2006 [98]a(FFAP),b Gostner 2006 [99]a,b
ACCEPTED MANUSCRIPT
Weickert 2011 [100]a,b
perchloric acid in sodium hydroxide; lyophilisation; formic acid in acetone
Mohan 2008 [6]
RI PT
perchloric acid in sodium hydroxide; formic acid in acetone
lactic acid: enzymatic test
Kleessen 2007 [101]a,b
ultrasound; centrifugation; perchloric acid; sodium hydroxide;
sonification; NH2 (amino propyl-) in isopropanol–heptane (SPE); formic acid in diethyl ether lactic acid: perchloric acid in potassium hydroxide; enzymatic test
Tiihonen 2008 [102]a
M AN U
filtration; sonification; sulfuric acid; diethyl ether in sodium sulphate;
SC
liophylization; formic acid and acetone
Maldonado 2010 [103]a,b, Olivares 2006 [104]a,b, Liu 2015 [105]a,b, Sierra
acetate; sodium sulphate
2010 [106]a,b
sulfuric acid; ether lactic acid: methanol, chloroform sodium phosphate buffer; sulfuric acid; ether
EP AC C
sulfuric acid; diethyl ether; calcium chloride
TE D
sodium hydrogen carbonate in argon atmosphere; sulfuric acid, ethyl
Bernal 2013 [107]a,b, Klosterbuer 2013 [108]a, Schneider 2005 [109]b, Schneider 2006 [110]b, Sierra 2015 [111]a,b Woodmansey 2004 [112]a
Macfarlane 2013 [113]a
ACCEPTED MANUSCRIPT
methylation; chloroform (lactate and succinate) Lecerf 2012 [114]a(FFAP),b
oxalic acid; sodium azide; lyophilization
Schwiertz 2010 [115]a,b
oxalic acid
Larsen 2013 [116]a,b
sodium chloride in Tween solution; diethyl ether
Gråsten 2007 [117]a,b
M AN U
SC
RI PT
sulfuric acid
Costabile 2008 [118]a(FFAP),b
acetonitrile
Franc¸ois 2014 [119]a(FFAP),b
diethyl ether
formic acid or phosphoric acid; diethyl ether or dichloromethane or ethyl-
TE D
acetate
Wullt 2007 [121]a , Walton 2012 [122]a
propyl-chloroformate; propanol; pyridine; hexane
EP
silylation (TBDMS derivatives)
SPME (PDMS/CAR or PA, or DVB/CAR/PDMS)
AC C
ethyl chloroformate; ethanol; pyridine; sodium bicarbonate SPME (PDMS/CAR)
Garc´ıa-Villalba 2012 [120]c
Zheng 2013 [123]a,c Gao 2009 [24]c Di Cagno 2011a,c [124], Vitali 2010a,c [125], Ahmed 2013a,c [126], Taneyo Saa 2014 [127]a,c Couch 2015 [128]a,c
ACCEPTED MANUSCRIPT
Kim, 2013 [129]c
Purge and trap sample preparation system
De Preter 2009 [130]c, Windey 2015 [131]c
RI PT
SPME
SC
MTBSTFA, N-Methyl-N-t-butyldimethylsilyl trifluoroacetamide; TBDMSCI, tertbutyldimethylcholorosilane; TBDMS, tert-butyldimethylsilyl; SPE, solid phase extraction; PDMS/CAR, carboxen polydimethylsiloxane; SPME, Solid Phase Micro-Extraction; PA, polyacrylate; DVB/CAR/PDMS,
M AN U
divinylbenzene carboxen polydimethylsiloxane. a
, studies conducted with capillary column: [26,30–39,46–52,55,56,69–76,78,80–82,85–92,97–108,111–128].
a(FFAP)
, studies conducted with flame ionization detector (FID): [26,30–37,39,46,47,51,52,55–62,68–74,78–81,85,91,92,94,96–101,103–107,109–
111,114–119,132]. c
TE D
b
, studies conducted with free fatty acid phase (FFAP) capillary column: [26,32–35,46,48–52,55,56,69–71,74,76,82,91,98,114,118,119].
AC C
EP
, studies conducted with mass spectrometry (MS) detector: [24,40–42,66,95,120,123–131].