Thermal unfolding of calreticulin. Structural and thermodynamic characterization of the transition

Calreticulin (CRT) is a calcium-binding protein that participates in several cellular processes including the control of protein folding and homeostasis of Ca²⁺. Its folding, stability and functions are strongly controlled by the presence of Ca²⁺. The oligomerization state of CRT is also relevant for its functions. We studied the thermal transitions of monomers and oligomers of CRT by differential scanning calorimetry (DSC), circular dichroism (CD) and Fourier transform infrared spectroscopy (FTIR) in the presence and absence of Ca²⁺. We found three and two components for the calorimetric transition in the presence and absence of Ca²⁺ respectively. The presence of several components was also supported by CD and FTIR spectra acquired as a function of the temperature. The difference between the heat capacity of the native and the unfolded state strongly suggests that interactions between protein domains also contribute to the heat uptake in a calorimetry experiment. We found that once unfolded at high temperature the process is reversible and the native state can be recovered upon cooling only in the absence of Ca²⁺. We also propose a new simple method to obtain pure CRT oligomers.     

Histone hyperacetylation can induce unfolding of the nucleosome core particle.

A direct correlation exists between the level of histone H4 hyperacetylation induced by sodium butyrate and the extent to which nucleosomes lose their compact shape and become elongated (62.0% of the particles have a length/width ratio over 1.6; overall mean in the length/width ratio = 1.83 +/- 0.48) when bound to electron microscope specimen grids at low ionic strength (1mM EDTA, 10mM Tris, pH 8.0). A marked proportion of elongated core particles is also observed in the naturally occurring hyperacetylated chicken testis chromatin undergoing spermatogenesis when analyzed at low ionic strength (36.8% of the particles have a length/width ratio over 1.6). Core particles of elongated shape (length/width ratio over 1.6) generated under low ionic strength conditions are absent in the hypoacetylated chicken erythrocyte chromatin and represent only 2.3% of the untreated Hela S3 cell core particles containing a low proportion of hyperacetylated histones. The marked differences between control and hyperacetylated core particles are absent if the particles are bound to the carbon support film in the presence of 0.2 M NaCl, 6mM MgCl2 and 10mM Tris pH 8.0, conditions known to stabilize nucleosomes. A survey of the published work on histone hyperacetylation together with the present results indicate that histone hyperacetylation does not produce any marked disruption of the core particle 'per se', but that it decreases intranucleosomal stabilizing forces as judged by the lowered stability of the hyperacetylated core particle under conditions of shearing stress such as cationic competition by the carbon support film of the EM grid for DNA binding.     

A test of the linear extrapolation of unfolding free energy changes over an extended denaturant concentration range.

Guanidine hydrochloride (GdnHCl) and thermally induced unfolding measurements on the oxidized form of Escherichia coli thioredoxin at pH 7 were combined for the purpose of assessing the functional dependence of unfolding free energy changes on denaturant concentration over an extended GdnHCl concentration range. Conventional analysis of GdnHCl unfolding exhibits a linear plot of unfolding delta G vs [GdnHCl] in the transition zone. In order to extend unfolding delta G measurements outside of that narrow concentration range, thermal unfolding measurements were performed using differential scanning calorimetry (DSC) in the presence of low to moderate concentrations of GdnHCl. The unfolding delta G values from the DSC measurements were corrected to 25 degrees C using the Gibbs-Helmholtz equation and mapped onto the delta G vs [GdnHCl] plot. The dependence of unfolding delta G on [GdnHCl] was found to be linear over the full denaturant concentration range, provided that the chloride ion concentration was kept at a threshold of greater than or equal to 1.5 M. In the DSC experiments performed in the presence of GdnHCl, chloride concentrations were maintained at 1.5 M by addition of appropriate amounts of NaCl. The linear extrapolation method (LEM) gives an unfolding free energy change in the absence of denaturant (delta G degrees N-U) in excellent agreement with the delta G determined by DSC measurement in 1.5 M NaCl. The various methods give a consensus unfolding delta G value of 8.0 kcal/mol at 25 degrees C in the absence of denaturant.(ABSTRACT TRUNCATED AT 250 WORDS)     

A comparative study of the unfolding of the endoglucanase Cel45 from Humicola insolens in denaturant and surfactant.

Cellulases are increasingly being used for industrial purposes, particularly in washing powders, yet little is known of the factors governing the stability of proteins in detergent solutions. We present a comparative analysis of the behavior of the cellulase Cel45 from Humicola insolens in the presence of the denaturant guanidinium chloride and the anionic detergent C12-LAS. Although Cel45 unfolds in GdmCl according to a simple two-state model under equilibrium conditions, it accumulates a transient intermediate during refolding. The four disulfide bonds do not contribute detectably to the stability of the native state. Cel45 is unfolded by very low concentrations of C12-LAS (1-4 mM). An analysis of 16 mutants of Cel45 shows a very weak correlation between unfolding rates in denaturant and detergent; mutants that have the same unfolding rate in GdmCl (within a factor of 1.5) vary 1,000-fold in their unfolding rates in C12-LAS. The data support a simple model for unfolding by detergent, in which the introduction of positive charges or removal of negative charges greatly increases detergent sensitivity, while interactions with the hydrophobic detergent tail contribute to a smaller extent. This implies that different detergent-mediated unfolding pathways exist, whose accessibilities depend on individual residues. Double-mutant cycles reveal that mutations in two proximal residues lead to repulsion and a destabilization greater than the sum of the individual mutations as measured by GdmCl denaturation, but they also reduce the affinity for LAS and therefore actually stabilize the protein relative to wild-type. Ligands that interact strongly with the denatured state may therefore alter the unfolding process.     

Towards a consistent modeling of protein thermodynamic and kinetic cooperativity: how applicable is the transition state picture to folding and unfolding?

To what extent do general features of folding/unfolding kinetics of small globular proteins follow from their thermodynamic properties? To address this question, we investigate a new simplified protein chain model that embodies a cooperative interplay between local conformational preferences and hydrophobic burial. The present four-helix-bundle 55mer model exhibits protein-like calorimetric two-state cooperativity. It rationalizes native-state hydrogen exchange observations. Our analysis indicates that a coherent, self-consistent physical account of both the thermodynamic and kinetic properties of the model leads naturally to the concept of a native state ensemble that encompasses considerable conformational fluctuations. Such a multiple-conformation native state is seen to involve conformational states similar to those revealed by native-state hydrogen exchange. Many of these conformational states are predicted to lie below native baselines commonly used in interpreting calorimetric data. Folding and unfolding kinetics are studied under a range of intrachain interaction strengths as in experimental chevron plots. Kinetically determined transition midpoints match well with their thermodynamic counterparts. Kinetic relaxations are found to be essentially single-exponential over an extended range of model interaction strengths. This includes the entire unfolding regime and a significant part of a folding regime with a chevron rollover, as has been observed for real proteins that fold with non-two-state kinetics. The transition state picture of protein folding and unfolding is evaluated by comparing thermodynamic free energy profiles with actual kinetic rates. These analyses suggest that some chevron rollovers may arise from an internal frictional effect that increasingly impedes chain motions with more native conditions, rather than being caused by discrete deadtime folding intermediates or shifts of the transition state peak as previously posited.     

Direct measurement of the thermodynamic parameters of amyloid formation by isothermal titration calorimetry

Amyloid fibril deposition is associated with over 20 degenerative diseases, including Alzheimer's, Parkinson's, and prion diseases. Although research over the last few years has revealed the morphology and structural features of the amyloid form, knowledge about the thermodynamics of amyloid formation is limited. Here, we report for the first time a direct thermodynamic study of amyloid formation using isothermal titration calorimetry. Beta(2)-microglobulin, a protein responsible for dialysis-related amyloidosis, was used for extending amyloid fibrils in a seed-controlled reaction in the cell of the calorimeter. We investigated the enthalpy and heat capacity changes of the reaction, where the monomeric, acid-denatured molecules adopt an ordered, cross-beta-sheet structure in the rigid amyloid fibrils. Despite the dramatic difference in morphology, beta(2)-microglobulin exhibited a similar heat capacity change upon amyloid formation to that of the folding to the native globular state, whereas the enthalpy change of the reaction proved to be markedly lower. In comparison with the native state, the results outline the important structural features of the amyloid fibrils: a similar extent of surface burial even with the supramolecular architecture of amyloid fibrils, a lower level of internal packing, and the possible presence of unfavorable side chain contributions.     

Studies of the ankyrin repeats of the Drosophila melanogaster Notch receptor. 2. Solution stability and cooperativity of unfolding.

To define the boundaries of the Drosophila Notch ankyrin domain, examine the effects of repeat number on the folding of this domain, and examine the degree to which the modular architecture of ankyrin repeat proteins results in modular stability, we have investigated the thermodynamics of unfolding of polypeptides corresponding to different segments of the ankyrin repeats of Drosophila Notch. We find that a polypeptide containing the six previously identified ankyrin repeats unfolds cooperatively, but is of modest stability. However, inclusion of a putative seventh, C-terminal ankyrin sequence doubles the stability of the Notch ankyrin domain (a 1000-fold increase in the folding equilibrium constant), indicating that the seventh ankyrin repeat is an important part of the Notch ankyrin domain, and demonstrating long-range interactions among ankyrin repeats. This putative seven-repeat polypeptide also shows increases in enthalpy, denaturant dependence (m-value), and heat capacity of unfolding (DeltaC(p)()) of around 50% each, suggesting that deletion of the seventh repeat results in partial unfolding of the sixth ankyrin repeat, consistent with spectroscopic and hydrodynamic data reported in the preceding paper [Zweifel, M. E., and Barrick, D. (2001) Biochemistry 40, 14344-14356]. A polypeptide consisting of only the five N-terminal repeats has stability similar to the six-repeat construct, demonstrating that stability is distributed asymmetrically along the ankyrin domain. These data are consistent with highly cooperative two-state folding of these ankyrin polypeptides, despite their modular architecture.     

The direct measurement of thermodynamic parameters of reactive transient intermediates of the L-glutamate dehydrogenase reaction

In a previous report (Fisher, H. F., Maniscalco, S. J., and Tally, J. (2002) Biochem. Biophys. Res. Commun. 287, 343-347) we demonstrated the capability of the "Le Chatelier forcing method" of producing stable solutions containing substantial amounts of transitory enzyme intermediate complexes that can otherwise be observed only fleetingly in the millisecond time range. The method requires nothing more than running an enzyme reaction using forcing concentrations of reactants against an equally forcing concentration of products until equilibrium is attained. Here we have applied this approach to the measurement of the thermodynamics of several such reactive (and normally transient) intermediate complexes of the bovine liver l-glutamate dehydrogenase-catalyzed reaction. At pH 9.5 and 20 degrees C, we observe both the enzyme-NADPH-alpha-iminoglutarate and enzyme-NADPH-alpha-carbinolamine complexes at concentrations whose sum accounts for 70% of the total enzyme. The pH dependence of these two complexes under equilibrium conditions provides thermodynamic parameters for both the protonated and the unprotonated forms of each of these two entities as well as those of the enzyme-NADP-l-glutamate complex. The equilibrium concentrations of each of these reactive complexes are compared with their corresponding transient steady-state values.     

Thermal features of the bovine serum albumin unfolding by polyethylene glycols.

Albumin showed very poor affinity for polyethylene glycol molecular weight (Mw) 1000 (30 M(-1)) and Mw 8000 (400 M(-1)) (PEG 1000 and PEG 8000). Polyethylene glycol of low Mw favours the ionization of the tyrosine (TYR) residues of albumin. Such variation might be a consequence of the change in dielectric constant at the domain of the protein by PEG binding. PEGs of high Mws stabilize the native compact state of human albumin showing negative preferential interaction with the protein. Interaction between PEGs and albumin is thermodynamically unfavourable, and becomes even more unfavourable for denatured proteins whose surface areas are larger than those of native ones leading to a stabilization of the unfolded state, which is manifested as a lowering of the thermal transition temperature. PEG 8000 perturbs the structure of the protein surface, partially modifying the layer of water and the microenvironment of the superficial aromatic residues (tryptophan, TRP and TYR) which is in agreement with the modifications of the UV spectrum of albumin by PEG 8000 and circular dichroism (CD) spectrum at high temperatures.     

NMR hydrogen exchange of the OB-fold protein LysN as a function of denaturant: the most conserved elements of structure are the most stable to unfolding.

The structure of LysN contains an OB-fold motif composed of a structurally conserved five-stranded beta-barrel capped by a poorly conserved alpha-helix between strands beta3 and beta4. Two additional alpha-helices, unique to the LysN structure, flank the N terminus of the OB-fold. The stability of LysN to unfolding has been investigated with NMR native state hydrogen exchange measurements as a function of guanidinium hydrochloride concentration, and equilibrium unfolding transitions monitored by ellipticity at 222 nm and fluorescence at 350 nm. The spectrophotometric measurements suggest an apparent two-state unfolding transition with DeltaGu(0) approximately 6 kcal/mol and m approximately 3 kcal/(molM). By contrast, NMR hydrogen exchange measurements manifest a distribution of DeltaGu(0) and m values which indicate that the protein can undergo subglobal unfolding. The largest DeltaGu(0) values from hydrogen exchange are for residues in the beta-sheet of the protein. These values, which reflect complete unfolding of the protein, are between 3 and 4 kcal/mol higher than those obtained from circular dichroism or fluorescence. This discrepancy may be due to the comparison of NMR hydrogen exchange parameters measured at residue-level resolution, with spectrophotometric parameters that reflect an unresolved super position of unfolding transitions of the alpha-helices and beta-strands. The largest DeltaGu(0) values obtained from hydrogen exchange for the subset of residues in the alpha-helices of the protein, agree with the DeltaGu(0) values obtained from circular dichroism or fluorescence. Based on the hydrogen exchange data, however, the three alpha-helices of LysN are on average 3 kcal/mol less stable than the beta-sheet. Consistent with the subglobal unfolding of LysN evinced by hydrogen exchange, a deletion mutant that lacks the first alpha-helix of the protein retains a cooperatively folded structure. Taken together with previous results on the OB-fold proteins SN and CspA, the present results for LysN suggest that the most conserved elements of structure in the OB-fold motif are the most resistant to denaturation. In all three proteins, stability to denaturation correlates with sequence hydrophobicity.     

Linear free-energy model description of the conformational stability of uracil-DNA glycosylase inhibitor A thermodynamic characterization of interaction with denaturant and cold denaturation.

The equilibrium unfolding of uracil DNA glycosylase inhibitor (Ugi), a small acidic protein of molecular mass 9474 Da, has been studied by a combination of thermal-induced and guanidine hydrochloride (GdnCl)-induced denaturation. The analysis of the denaturation data provides a measure of the changes in conformational free energy, enthalpy, entropy and heat capacity DeltaCp that accompany the equilibrium unfolding of Ugi over a wide range of temperature and GdnCl concentration. The unfolding of Ugi is a simple two-state, reversible process. The protein undergoes both low-temperature and high-temperature unfolding even in the absence of GdnCl but more so in the presence of denaturant. The data are consistent with the linear free-energy model and with a temperature independent DeltaCp over the large temperature range of unfolding. The small DeltaCp (6.52 kJ.mol-1.K-1) for the unfolding of Ugi, is perhaps a reflection of a relatively small, buried hydrophobic core in the folded form of this small monomeric protein. Despite a relatively low value of DeltaG(H2O), 7.40 kJ.mol-1 at pH 8.3, Ugi displays considerable stability with the temperature of maximum stability being 301.6 K.     

Thermal unfolding of G-actin monitored with the DNase I-inhibition assay stabilities of actin isoforms.

Actin is one of the proteins that rely on chaperonins for proper folding. This paper shows that the thermal unfolding of G-actin, as studied by CD and ultraviolet difference spectrometry, coincides with a loss in DNase I-inhibiting activity of the protein. Thus, the DNase I inhibition assay should be useful for systematic studies of actin unfolding and refolding. Using this assay, we have investigated how the thermal stability of actin is affected by either Ca2 + or Mg2 + at the high affinity divalent cation binding site, by the concentration of excess nucleotide, and by the nucleotide in different states of phosphorylation (ATP, ADP.Pi, ADP. Vi, ADP.AlF4, ADP.BeFx, and ADP). Actin isoforms from different species were also compared, and the effect of profilin on the thermal stability of actin was studied. We conclude that the thermal unfolding of G-actin is a three-state process, in which an equilibrium exists between native actin with bound nucleotide and an intermediate free of nucleotide. Actins in the Mg-form were less stable than the Ca-forms, and the stability of the different isoforms decreased in the following order: rabbit skeletal muscle alpha-actin = bovine cytoplasmic gamma-actin > yeast actin > cytoplasmic beta-actin. The activation energies for the thermal unfolding reactions were in the range 200-290 kJ.mol- 1, depending on the bound ligands. Generally, the stability of the actin depended on the degree with which the nucleotide contributed to the connectivity between the two domains of the protein.     

Thermal stability of peroxidase from the african oil palm tree Elaeis guineensis.

The thermal stability of peroxidase from leaves of the African oil palm tree Elaeis guineensis (AOPTP) at pH 3.0 was studied by differential scanning calorimetry (DSC), intrinsic fluorescence, CD and enzymatic assays. The spectral parameters as monitored by ellipticity changes in the far-UV CD spectrum of the enzyme as well as the increase in tryptophan intensity emission upon heating, together with changes in enzymatic activity with temperature were seen to be good complements to the highly sensitive but integral method of DSC. The data obtained in this investigation show that thermal denaturation of palm peroxidase is an irreversible process, under kinetic control, that can be satisfactorily described by the two-state kinetic scheme, N -->(k) D, where k is a first-order kinetic constant that changes with temperature, as given by the Arrhenius equation; N is the native state, and D is the denatured state. On the basis of this model, the parameters of the Arrhenius equation were calculated.     

Duplicated dockerin subdomains of Clostridium thermocellum endoglucanase CelD bind to a cohesin domain of the scaffolding protein CipA with distinct thermodynamic parameters and a negative cooperativity

Mutagenized dockerin domains of endoglucanase CelD (type I) and of the cellulosome-integrating protein CipA (type II) were constructed by swapping residues 10 and 11 of the first or the second duplicated segment between the two polypeptides. These residues have been proposed to determine the specificity of cohesin-dockerin interactions. The dockerin domain of CelD still bound to the seventh cohesin domain of CipA (CohCip7), provided that mutagenesis occurred in one segment only. Binding was no longer detected by nondenaturing gel electrophoresis when both segments were mutagenized. The dockerin domain of CipA bound to the cohesin domain of SdbA as long as the second segment was intact. None of the mutated dockerins displayed detectable binding to the noncognate cohesin domain. Isothermal titration calorimetry showed that binding of the CelD dockerin to CohCip7 occurred with a high affinity [K(a) = (2.6 +/- 0.5) x 10(9) M(-1)] and a 1:1 stoichiometry. The reaction was weakly exothermic (DeltaHdegrees = -2.22 +/- 0.2 kcal x mol(-1)) and largely entropy driven (TDeltaSdegrees = 10.70 +/- 0.5 kcal x mol(-1)). The heat capacity change on complexation was negative (DeltaC(p) = -305 +/- 15 cal x mol(-1) x K(-1)). These values show that cohesin-dockerin binding is mainly hydrophobic. Mutations in the first or the second dockerin segment reduced or enhanced, respectively, the hydrophobic character of the interaction. Due to partial enthalpy-entropy compensation, these mutations induced only small changes in binding affinity. However, the binding affinity was strongly decreased when both segments were mutated, indicating strong negative cooperativity between the two mutated sites.     

Thermal unfolding of tetrameric melittin: comparison with the molten globule state of cytochrome c.

Whereas melittin at micromolar concentrations is unfolded under conditions of low salt at neutral pH, it transforms to a tetrameric alpha-helical structure under several conditions, such as high peptide concentration, high anion concentration, or alkaline pH. The anion- and pH-dependent stabilization of the tetrameric structure is similar to that of the molten globule state of several acid-denatured proteins, suggesting that tetrameric melittin might be a state similar to the molten globule state. To test this possibility, we studied the thermal unfolding of tetrameric melittin using far-UV CD and differential scanning calorimetry. The latter technique revealed a broad but distinct heat absorption peak. The heat absorption curves were consistent with the unfolding transition observed by CD and were explainable by a 2-state transition mechanism between the tetrameric alpha-helical state and the monomeric unfolded state. From the peptide or salt-concentration dependence of unfolding, the heat capacity change upon unfolding was estimated to be 5 kJ (mol of tetramer)-1 K-1 at 30 degrees C and decreased with increasing temperature. The observed change in heat capacity was much smaller than that predicted from the crystallographic structure (9.2 kJ (mol of tetramer)-1 K-1), suggesting that the hydrophobic residues of tetrameric melittin in solution are exposed in comparison with the crystallographic structure. However, the results also indicate that the structure is more ordered than that of a typical molten globule state. We consider that the conformation is intermediate between the molten globule state and the native state of globular proteins.     

The effect of bilirubin photoisomers on unbound-bilirubin concentrations estimated by the peroxidase method.

Unbound bilirubin is oxidized to nearly colourless substances in the presence of H2O2 or ethyl hydroperoxide and horseradish peroxidase. To predict the risk of kernicterus (degenerated yellow pigmentation of nerve cells), this principle has been widely utilized for estimating the concentration of unbound bilirubin in hyperbilirubinaemic serum. However, the serum contains polar geometric photoisomers of bilirubin. Therefore, to clarify the effect of bilirubin photoisomer concentrations on unbound-bilirubin concentration, the concentration of bilirubin and its photoisomer and of unbound bilirubin in samples obtained from experiments in vivo and in vitro were simultaneously and individually estimated by h.p.l.c. and the peroxidase method. During photoirradiation, both in vivo and in vitro, the serum polar (ZE)-bilirubin IX alpha concentration increased remarkably, but unbound-bilirubin values were not affected at all. However, during experiments in vitro, unbound bilirubin concentrations increased only when concentrations of the rather polar (EZ)- and (EE)-cyclobilirubin IX alpha increased considerably in a human serum albumin-bilirubin solution irradiated with blue light. Thus it is concluded that unbound-bilirubin concentrations, and consequently the initial rate of the peroxidase reaction, is not accelerated by the increase in either (ZE)-bilirubin or (EZ)-cyclobilirubin concentration within the clinically observed range.     

Identification of the testing parameters in high frequency dynamic shear measurement on agarose gels

Dynamic mechanical analysis (DMA) on agarose gels can be used to validate magnetic resonance elastography (MRE) measurements as well as to provide better understanding for the biological responses of cells to the dynamic loadings in cell culture studies. Various parameters potentially affecting the repeatability and accuracy of the DMA shear modulus measurements were investigated systematically in the present study, including sample thickness, shear strain, testing frequency, and compressive clamping strain. The study showed that the thickness of the agarose gel sample must be sufficiently small (1 mm) to prevent the erroneous fluctuation in the measured modulus. The appropriate levels of shear strain (< or = 0.5%) and compressive clamping strain (5-10%) must be applied to overcome the slippage at the gel-clamp interface without causing significant boundary and stress non-uniformity or micro-cracks in the agarose gel sample.     

pH-induced folding/unfolding of staphylococcal nuclease: determination of kinetic parameters by the sequential-jump method.

On the basis of previous stopped-flow pH-jump experiments, we have proposed that the acid- and alkaline-induced folding/unfolding transition of staphylococcal nuclease, in the time range 2 ms to 300 s, follows the pathway N0 in equilibrium with D1 in equilibrium with D2 in equilibrium with D3, in which D1, D2, and D3 are three substates of the unfolded state and N0 is the native state. The stopped-flow "double-jump" technique has been employed to test this mechanism and to determine the rate constants which would not be accessible by the direct pH jump because of the lack of fluorescence signal, i.e., the rates for the conversion of D1 to D2 and of D2 to D3. In the forward jump, a protein solution kept at pH 7.0 was mixed with an acidic or alkaline solution to the final pH of 3.0 or 12.2, respectively. The mixed solution was kept for varying periods of time, called the delay time, tD. A second mixing (the back jump) was launched to bring the protein solution back to pH 7.0. The time course of the Trp-140 fluorescence signals recovered in the back jump was analyzed as a function of tD. Kinetics of the unfolding were found to be triphasic by the double-jump method, contrary to the monophasic kinetics observed by the direct pH jump. Complex kinetics of unfolding are expected with the proposed kinetic scheme.(ABSTRACT TRUNCATED AT 250 WORDS)     

Validation of the measurement of low concentrations of 5-hydroxytryptamine in plasma using high performance liquid chromatography

A sensitive and rapid assay is described for the measurement of low concentrations of 5-hydroxytryptamine (5-HT) present in human platelet-depleted plasma (PDP) using reverse-phase high performance liquid chromatography (HPLC) with fluorimetric detection. With an analysis time of 12 min, this method is particularly useful for large-scale clinical trials investigating small differences in PDP 5-HT concentrations in conditions such as functional gastrointestinal disorders (FGID). The limit of detection and quantification were 1 and 3 nmol/l, respectively, and the calibration curve linear between 1 and 1000 nmol/l. The within-day and between-day precision were 4.3 and <13.6%, respectively.