added
stringlengths 24
24
| created
stringlengths 23
23
| id
stringlengths 3
9
| metadata
dict | source
stringclasses 1
value | text
stringlengths 1.56k
316k
| version
stringclasses 1
value |
|---|---|---|---|---|---|---|
2020-05-18T01:00:17.886Z | 2020-05-15T00:00:00.000 | 218665656 | {
"extfieldsofstudy": [
"Physics"
],
"oa_license": "CCBY",
"oa_status": "HYBRID",
"oa_url": "https://link.springer.com/content/pdf/10.1140/epja/s10050-020-00249-y.pdf",
"pdf_hash": "1fd1d7fd805b1779748e8d907c418ef81f272a18",
"pdf_src": "Arxiv",
"provenance": "20241012_202828_00062_s4wg2_01a5a148-7d10-4d24-836e-fe76b72970d6.zst:6",
"s2fieldsofstudy": [
"Physics"
],
"sha1": "1fd1d7fd805b1779748e8d907c418ef81f272a18",
"year": 2020
} | pes2o/s2orc | Subleading contributions to the nuclear scalar isoscalar currents
We extend our recent analyses of the nuclear vector, axial-vector and pseudoscalar currents and derive the leading one-loop corrections to the two-nucleon scalar current operator in the framework of chiral effective field theory using the method of unitary transformation. We also show that the scalar current operators at zero momentum transfer are directly related to the quark mass dependence of the nuclear forces.
I. INTRODUCTION
The first principles description of nuclei, nuclear matter and reactions is one of the great challenges in contemporary physics with applications ranging from low-energy searches for physics beyond the Standard Model (SM) to properties of neutron stars and neutron star mergers. The currently most efficient and feasible approach along this line relies on the application of suitably taylored effective field theories (EFTs). In particular, an extension of chiral perturbation theory to multi-nucleon systems [1,2], commonly referred to as chiral EFT, has been applied over the last two decades to derive nuclear forces at high orders in the EFT expansion in harmony with the spontaneously broken approximate chiral symmetry of QCD [3,4]. See Refs. [5,6] for the most accurate and precise chiral two-nucleon interactions at fifth order and Refs. [7][8][9][10][11] for a collection of review articles describing the current state-of-the-art in chiral EFT for nuclear forces and selected applications. In parallel with these developments, current operators describing the interactions of nuclear systems with external vector, axial-vector and pseudoscalar sources needed to study electroweak reactions driven by a single photon-or W /Z-boson exchange have been worked out completely through fourth order in the heavy-baryon formulation of chiral EFT with pions and nucleons as the only dynamical degrees of freedom, see Refs. [12,13] for the pioneering studies by Park et al., for our calculations using the method of unitary transformation [18][19][20] and Refs. [21][22][23][24] for an independent derivation by the Jlab-Pisa group in the framework of time-ordered perturbation theory. A direct comparison of the expressions for the current operators derived by different group is hindered by their scheme dependence. However, at least for the two-pion exchange axial-vector currents, our results [16] appear to be not unitarily equivalent to the ones of the Pisa-Jlab group [23], see Ref. [25] for a detailed discussion of the box diagram contribution. We further emphasize that offshell consistency of the electroweak operators derived by our group [14][15][16][17] and the corresponding (unregularized) two- [26,27] and three-nucleon forces [28,29] has been verified explicitly by means of the corresponding continuity equations in Refs. [16,17].
Here and in what follows, we assume exact isospin symmetry with m u = m d ≡ m q . Embedded in the SM, the interactions between quarks and the external vector and axial-vector sources are probed in electroweak reactions involving hadrons or nuclei. Low-energy nuclear systems are nowadays commonly described by solving the many-body Schrödinger equation with the nuclear forces derived in chiral EFT [3,4,7]. An extension to electroweak processes with nuclei requires the knowledge of the corresponding nuclear current operators defined in terms of the functional derivatives of the effective nuclear Hamiltonian in the presence of external fields with respect to v µ (x) and a µ (x) [16]. For the vector, axial-vector and pseudoscalar sources, the corresponding expressions are already available up to fourth chiral order [14][15][16][17]. In this work we focus on the response of nuclear systems to the external scalar source s(x) and thus set v µ = a µ = p = 0. While the scalar currents cannot be probed experimentally within the SM due to the absence of scalar sources, they figure prominently in dark matter (DM) searches in a wide variety of DM models such as e.g. Higgs-portal DM and weakly-interacting massive particles (WIMPs), see [35][36][37] for recent review articles. For example, the dominant interactions of a spin-1/2 Dirac-fermion DM particle χ with the strong sector of the SM is given by the Lagrangian where i denotes the flavor quantum number, G a µν is the gluon field strength, α s is the strong coupling constant and the couplings c i (c G ) determine the strength of the interaction between χ and quarks of flavor i (gluons). Notice that the contributions from coupling to heavy quarks (charm, bottom and top) can be integrated out [38] and the sum in Eq. (1.2) can thus be taken only over the light quark flavors by replacing the coupling constants c i , c G with the corresponding effective ones. Thus, the scalar nuclear currents derived in our paper can be used to describe the interactions of nuclei with DM particles emerging from their isoscalar coupling to the up-and down-quarks ∝ (c u +c d ).
Apart from their relevance for DM searches, the scalar currents are intimately related to quark mass dependence of hadronic and nuclear observables. For example, the pion-nucleon σ-term, σ πN , corresponds to the isoscalar scalar form factor of the nucleon at zero momentum transfer times the quark mass and determines the amount of the nucleon mass generated by the up-and down-quarks. Its value has been accurately determined from the recent Roy-Steiner-equation analysis of pion-nucleon scattering accompanied with pionic hydrogen and deuterium data to be σ πN = (59.1 ± 3.5) MeV [39]. For the status of lattice QCD calculations of σ πN see Ref. [40]. As pointed out, however, in Ref. [41], there is relation between the σ-term and the S-wave πN scattering lengths that so far has not been checked for the lattice calculations. Nuclear σ-terms and scalar form factors of light nuclei have also been studied in lattice QCD, albeit presently at unphysically large quark masses [42,43]. Interestingly, the scalar matrix elements were found in these studies to be strongly affected by nuclear effects (in contrast to the axial-vector and tensor charges), which indicates that scalar exchange currents may play an important role. Last but not least, as will be shown below, the scalar isoscalar currents are directly related to the quark mass dependence of the nuclear forces, a subject that gained a lot of attention in the EFT community in connection with ongoing lattice QCD efforts in the multibaryon sector [44][45][46][47][48][49][50][51][52], a conjectured infrared renormalization group limit cycle in QCD [53,54], searches for possible temporal variation of the light quark masses [55,56] and anthropic considerations related to the famous Hoyle state in 12 C [57][58][59][60].
Clearly, nuclear scalar currents have already been studied before in the framework of chiral EFT, see e.g. [61][62][63][64][65][66][67][68]. For the two-nucleon currents, only the dominant contribution at the chiral order Q −2 stemming from the one-pion exchange has been considered so far. Here and in what follows, Q ∈ {M π /Λ b , p/Λ b } denotes the chiral expansion parameter, M π is the pion mass, p refers to the magnitude of three-momenta of external nucleons, while Λ b denotes the breakdown scale of the chiral expansion. For a detailed discussion of the employed power counting scheme for nuclear currents see Ref. [16]. The two-body scalar current is suppressed by just one power of the expansion parameter Q relative to the dominant one-body contribution. Such an enhancement relative to the generally expected suppression of (A+1)-nucleon operators relative to the dominant A-nucleon terms by Q 2 can be traced back to the vertex structure of the effective Lagrangian and is not uncommon. For example, one-and two-nucleon operators contribute at the same order to the axial charge and electromagnetic current operators, see Table II of Ref. [16] and Table 1 of Ref. [17], respectively. For the scalar operator, the relative enhancement of the two-body terms is caused by the absence of one-body contributions at the expected leading order Q −4 , see e.g. Table III of Ref. [16] for the hierarchy of the pseudoscalar currents. The first corrections to the scalar current appear at order Q −2 from the leading one-loop diagrams involving a single-nucleon line [63]. In this paper we derive the subleading contributions to the two-nucleon scalar isoscalar current operators at order Q 0 . While the one-body current is not yet available at the same accuracy level, using empirical information on the scalar form factor of the nucleon from lattice QCD instead of relying on its strict chiral expansion may, in the future, provide a more reliable and efficient approach. A similar strategy is, in fact, commonly used in studies of electromagnetic processes, see e.g. [69,70] and Ref. [71] for a recent example.
Our paper is organized as follows. In section II, we briefly describe the derivation of the current operator using the method of unitary transformation and provide explicit expressions for the leading (i.e. order-Q −2 ) and subleading (i.e. order-Q 0 ) two-body contributions. Next, in section III, we establish a connection between the scalar currents at zero momentum transfer and the quark mass dependence of the nuclear force. The obtained results are briefly summarized in section IV, while some further technical details and the somewhat lengthy expressions for the two-pion exchange contributions are provided in appendices A and B.
II. TWO-NUCLEON SCALAR OPERATORS
The derivation of the nuclear currents from the effective chiral Lagrangian using the method of unitary transformation is described in detail in Ref. [16]. The explicit form of the effective Lagrangian in the heavy-baryon formulation can be found in Refs. [72] and [73] for the pionic and pion-nucleon terms, respectively. The relevant terms in L N N will be specified in section II D. As already pointed out above, for the purpose of this study we switch off all external sources except the scalar one, s(x). To derive the scalar currents consistent with the nuclear potentials in Refs. [26-29, 31, 32] and electroweak currents in Refs. [14][15][16][17], we first switch from the effective pion-nucleon Lagrangian to the corresponding Hamiltonian H[s] using the canonical formalism and then apply the unitary transformations U Okubo , U η and U [s].
Here and in what follows, we adopt the notation of Ref. [16]. In particular, the Okubo transformations U Okubo [18] is a "minimal" unitary transformation needed to derive nuclear forces by decoupling the purely nucleonic subspace η from the rest of the pion-nucleon Fock space in the absence of external sources. However, as found in Refs. [31], the resulting nuclear potentials ηU † Okubo HU Okubo η, with η denoting the projection operator onto the η-space, are non-renormalizable starting from next-to-next-to-next-to-leading order (N 3 LO) Q 4 . 1 To obtain renormalized nuclear potentials, a more general class of unitary operators was employed in Refs. [31,32] by performing additional transformations U η on the η-space. The explicit form of the "strong" unitary operators U Okubo and U η up to next-to-next-to-leading order (N 2 LO) can be found in Refs. [28,29,31,32]. Nuclear currents can, in principle, be obtained by switching on the external classical sources in the effective Lagrangian, performing the same unitary transformations U Okubo U η as in the strong sector, and taking functional derivatives with respect to the external sources. However, similarly to the above mentioned renormalization problem with the nuclear potentials, the current operators obtained in this way can, in general, not be renormalized. A renormalizable formulation of the current operators requires the introduction of an even more general class of unitary transformation by performing subsequent η-space rotations with the unitary operators, whose generators depend on the external sources. In Refs. [16] and [17], are explicitly given up to N 2 LO. Notice that such unitary transformations are necessarily time-dependent through the dependence of their generators on the external sources. This, in general, induces the dependence of the corresponding current operators on the energy transfer and results in additional terms in the continuity equations [16]. We now follow the same strategy for the scalar currents and introduce additional η-space unitary transformations U [s], U [s] s=mq = η, in order to obtain renormalizable currents. The most general form of the operator U [s] at the chiral order we are working with is given in appendix A and is parametrized in terms of four real phases α s i , i = 0, . . . , 3. The nuclear scalar current is defined via . Solid, dashed and wiggly lines denote nucleons, pions and external scalar sources, in order. Solid dots denote the leading-order vertices from the effective Lagrangians L see [16] for notation. While all the phases remain unfixed, they do not show up in the resulting expressions for the nuclear current given in the following sections. To the order we are working, we therefore do not see any unitary ambiguity.
A. Contributions at orders Q −2
The chiral expansion of the 2N scalar isoscalar current starts at order Q −2 . The dominant contribution is well known to emerge from the one-pion exchange diagram shown in Fig. 1 and has the form where g A and F π are the nucleon axial-vector coupling and pion decay constants, respectively, and q i = p i − p i denotes the momentum transfer of nucleon i. Further, σ i (τ i ) refer to the spin (isospin) Pauli matrices of nucleon i.
Here and in what follows, we follow the notation of our paper [16]. In terms of the Fock-space operatorŜ 2N , the expressions we give correspond to the matrix elements where p i ( p i ) refers to the initial (final) momentum of nucleon i, k is the momentum of the external scalar source and the nucleon states are normalized according to the nonrelativistic relation . Finally, we emphasize that the dependence of the scalar currents on m q , which is renormalization-scale dependent, reflects the fact that in our convention, the external scalar source s(x) couples to the QCD densityqq rather than m qq q. Thus, only the combination m qŜ2N (k) is renormalization-scale independent. This is completely analogous to the pseudoscalar currents derived in Ref. [16], and we refer the reader to that work for more details.
B. One-pion-exchange contributions at order Q 0 Given that the first corrections to the pionic vertices are suppressed by two powers of the expansion parameter and the absence of vertices in L (2) πN involving the scalar source and a single pion, the first corrections to the two-nucleon current appear at order Q 0 . In Fig. 2 we show all one-loop one-pion-exchange diagrams of non-tadpole type that contribute to the scalar current at this order. Similarly, the corresponding tadpole and tree-level diagrams yielding nonvanishing contributions are visualized in Fig. 3. It should be understood that the diagrams we show here and in what follows do, in general, not correspond to Feynman graphs and serve for the purpose of visualizing the corresponding types of contributions to the operators. The meaning of the diagrams is specific to the method of unitary transformation, see [16] for details. Using dimensional regularization, replacing all bare low-energy constants (LECs) l i and d i in terms of their renormalized valuesl i andd i as defined in Eq. (2.118) of [16], and expressing the results in terms of physical parameters F π , M π and g A , see e.g. [15], leads to our final result for the static order-Q 0 contributions to the 2N .
(2.6) e loop function L(q) is defined as apart from the static contributions, we need to take into account for the leading relativistic corrections from tree-level diagrams with a single insertion of 1/m-vertices from the Lagrangian L ⇡N . We stress again to the employed counting for the nucleon mass with m ⇠ ⇤ 2 b /M ⇡ , these contributions are shifted one order lative to the ones emerging from tree-level diagrams with a single insertion of the c i -vertices from L . (2.6) Here, the loop function L(q) is defined as Finally, apart from the static contributions, we need to take into account for the leading relativistic corrections emerging from tree-level diagrams with a single insertion of 1/m-vertices from the Lagrangian L ⇡N . We stress again that due to the employed counting for the nucleon mass with m ⇠ ⇤ 2 b /M ⇡ , these contributions are shifted one order higher relative to the ones emerging from tree-level diagrams with a single insertion of the c i -vertices from L .
Here, the loop function L(q) is defined as Finally, apart from the static contributions, we need to take into acc emerging from tree-level diagrams with a single insertion of 1/m-vertice that due to the employed counting for the nucleon mass with m ⇠ ⇤ 2 b /M higher relative to the ones emerging from tree-level diagrams with a sing in the second line of Fig. 1 one-pion-exchange scalar current operators: where the scalar functions o i (k) are given by (2.6) and the loop function L(k) is defined as Finally, apart from the static contributions, we need to take into account the leading relativistic corrections emerging from tree-level diagrams with a single insertion of the 1/m-vertices from the Lagrangian L πN . Given our standard counting scheme for the nucleon mass m ∼ Λ 2 b /M π , see e.g. [16], these contributions are shifted from the order Q −1 to Q 0 . However, the explicit evaluation of diagrams emerging from a single insertion of the 1/m-vertices into the one-pion-exchange graph in Fig. 1 leads to a vanishing result. Given the relation between the scalar current operator and the nuclear forces discussed in section III, this observation is consistent with the absence of relativistic corrections in the (energy-independent formulation of the) nuclear forces at next-to-leading order.
Last but not least, there are no contributions proportional to the energy transfer k 0 which may appear from the explicit time dependence of the unitary transformations in diagrams shown in Fig. 2.
C. Two-pion-exchange contributions
We now turn to the two-pion exchange contributions. In Fig. 4, we show all diagrams yielding non-vanishing results for the scalar current operator with two exchanged pions. The final results for the two-pion exchange operators read where the scalar functions t i (k, q 1 , q 2 ) are expressed in terms of the three-point function. Their explicit form is given in appendix B. Notice that the (logarithmic) ultraviolet divergences in the two-pion exchange contributions are absorbed into renormalization of the LECs from L (2) N N described in the next section.
D. Short-range contributions
Finally, we turn to the contributions involving short-range interactions. In Fig. 5, we show all one-loop and tree-level diagrams involving a single insertion of the contact interactions that yield non-vanishing contributions to the scalar current. The relevant terms in the effective Lagrangian have the form [32,44] where N is the heavy-baryon notation for the nucleon field with velocity v µ , S µ = −γ 5 [γ µ , γ ν ]v ν /4 is the covariant spin-operator, χ + = 2B u † (s + ip)u † + u(s − ip)u , B, C S,T and D S,T are LECs 2 , . . . denotes the trace in the flavor space, u = √ U , and the 2×2 matrix U collects the pion fields. Further, the ellipses refer to other terms that are not relevant for our discussion of the scalar current operator. The total contribution of the diagrams of Fig. 5 can, after renormalization, be written in the form with the scalar functions s i (k) defined by (2.11) The renormalized, scale-independent LECsD S ,D T are related to the bare ones D S , D T according to with the corresponding β-functions given by 13) and the quantity λ defined as where γ E = −Γ (1) 0.577 is the Euler constant, d the number of space-time dimensions and µ is the scale of dimensional regularization. Clearly, the C T -independent parts of the β-functions emerge from the two-pion exchange contributions discussed in the previous section.
Notice that the LECs C S , C T ,D S andD T also contribute to the 2N potential. However, experimental data on nucleon-nucleon scattering do not allow one to disentangle the M π -dependence of the contact interactions and only constrain the linear combinations of the LECs [44] The LECsD S andD T can, in principle, be determined once reliable lattice QCD results for two-nucleon observables such as e.g. the 3 S 1 and 1 S 0 scattering lengths at unphysical (but not too large) quark masses are available, see Refs. [60] and references therein for a discussion of the current status of research along this line.
Last but not least, we found, similarly to the one-pion exchange contributions, no 1/m-corrections and no energydependent short-range terms at the order we are working. Notice further that the loop contributions to the contact interactions are numerically suppressed due to the smallness of the LEC C T as a consequence of the approximate SU(4) Wigner symmetry [74,75].
III. SCALAR CURRENT AT ZERO MOMENTUM TRANSFER
If the four-momentum transfer k µ of the scalar current is equal zero, one can directly relate the current to the quarkmass derivative of the nuclear Hamiltonian. To see this, we first rewrite the definition of the scalar current in Eq. (2.2) in the form where the nuclear Hamiltonian H eff is defined as and the unitary transformation U [s] satisfies by construction Notice that the last term in the brackets in Eq. (2.2) vanishes for k 0 = 0. On the other hand, we obtain Given the trivial relation the right-most terms in Eqs. (3.1) and (3.4) are equal, and we obtain the relation At the order we are working both commutators in this equation vanish (independently on the choice of unitary phases) leading to In appendix C we demonstrate the validity of Eq. (3.7) for the two-nucleon potential at NLO, see Ref. [44] for the calculation of the quark mass dependence of nuclear forces using the method of unitary transformation.
It is important to emphasize that on the energy shell, i.e. when taking matrix elements in the eigenstates |i and |f of the Hamiltonian H eff corresponding to the same energy, all contributions from the commutator in Eq. (3.6) vanish leading to the exact relation For eigenstates |Ψ corresponding to a discrete energy E, H eff |Ψ = E|Ψ , the Feynman-Hellmann theorem allows one to interpret the scalar form factor at zero momentum transfer in terms of the eigenenergy slope with respect to the quark mass: In particular, for |Ψ being a single-nucleon state at rest, the expectation value on left-hand side of Eq. (3.9) is nothing but the pion-nucleon sigma-term and for an extension to resonances |R , see e.g. Ref. [76].
IV. SUMMARY AND CONCLUSIONS
In this paper we have analyzed in detail the subleading contributions to the nuclear scalar isoscalar current operators in the framework of heavy-baryon chiral effective field theory. These corrections are suppressed by two powers of the expansion parameter Q relative to the well-known leading-order contribution, see Eq. (2.3). They comprise the one-loop corrections to the one-pion-exchange and the lowest-order NN contact interactions as well as the leading two-pion exchange contributions. No three-and more-nucleon operators appear at the considered order. While the two-pion exchange terms do not involve any unknown parameters, the one-pion exchange contribution depends on a poorly known πN LECd 16 related to the quark mass dependence of the nucleon axial coupling g A . It can, in principle, be determined from lattice QCD simulations, see [77,78] for some recent studies. The short-range part of the scalar current depends on two unknown LECs which parametrize the quark-mass dependence of the derivative-less NN contact interactions. In principle, these LECs can be extracted from the quark-mass dependence of, say, the NN scattering length, see Refs. [44-46, 48, 50-52] for a related discussion. Finally, we have explicitly demonstrated that the scalar current operator at vanishing four-momentum transfer is directly related to the quark-mass dependence of the nuclear force. The results obtained in our work are relevant for ongoing DM searches and for matching to lattice QCD calculations in the few-nucleon sector, see e.g. [42,43] for recent studies along this line.
It is important to emphasize that our calculations are carried out using dimensional regularization. For nuclear physics applications, the obtained expressions for the scalar current operator need to be regularized consistently with the nuclear forces, which is a nontrivial task, see Refs. [7,79] for a discussion. Work along these lines using the invariant higher derivative regularization [80] is in progress.
Acknowledgments
We are grateful to Martin Hoferichter and Jordy de Vries for sharing their insights into these topics. At the order we are working, the general structure of the unitary operator U [s] can be written as 2,2 η, Here and in what follows, we use the notation of Ref. [16]. Furthermore, S (κ) n,p denotes an interaction from the Hamiltonian with a single insertion of the scalar current s(x) − m q 3 , n nucleon and p pion fields. The superscripts κ refer to the inverse mass dimension of the corresponding coupling constant given by where d, n and p denote the number of derivatives or pion mass insertions at a given vertex, number of nucleon and pion fields, respectively. Further, c v , c a , c p and c s refer to the number of external vector, axial-vector, pseudoscalar and scalar sources, in order.
(C.5)
It is important to emphasize that in Ref. [44], the short-range LECsD S andD T have been shifted to absorb all momentum-independent contributions generated by the two-pion-exchange. The corresponding shifts forD S andD T are given byD (C.6) Performing the same shifts in the scalar current and using L(0) = 1 and Eq. (B.5) we indeed verify: | v3-fos-license |
2018-04-03T00:19:23.688Z | 2014-02-18T00:00:00.000 | 10805381 | {
"extfieldsofstudy": [
"Medicine"
],
"oa_license": "CCBY",
"oa_status": "GOLD",
"oa_url": "https://journals.plos.org/plosone/article/file?id=10.1371/journal.pone.0084167&type=printable",
"pdf_hash": "4c4d91bc4aed3ca1fe5a489122e013016c5b8e81",
"pdf_src": "PubMedCentral",
"provenance": "20241012_202828_00062_s4wg2_01a5a148-7d10-4d24-836e-fe76b72970d6.zst:7",
"s2fieldsofstudy": [
"Medicine"
],
"sha1": "4c4d91bc4aed3ca1fe5a489122e013016c5b8e81",
"year": 2014
} | pes2o/s2orc | Factors Associated with Impairment of Quadriceps Muscle Function in Chinese Patients with Chronic Obstructive Pulmonary Disease
Background Quadriceps muscle dysfunction is well confirmed in chronic obstructive pulmonary disease (COPD) and reported to be related to a higher risk of mortality. Factors contributing to quadriceps dysfunction have been postulated, while not one alone could fully explain it and there are few reports on it in China. This study was aimed to investigate the severity of quadriceps dysfunction in patients with COPD, and to compare quadriceps muscle function in COPD and the healthy elderly. Methods Quadriceps strength and endurance capabilities were investigated in 71 COPD patients and 60 age-matched controls; predicted values for quadriceps strength and endurance were calculated using regression equations (incorporating age, gender, anthropometric measurements and physical activities), based on the data from controls. Potential parameters related to quadriceps dysfunction in COPD were identified by stepwise regression analysis. Results Mean values of quadriceps strength was 46% and endurance was 38% lower, in patients with COPD relative to controls. Gender, physical activities and anthropometric measurements were predictors to quadriceps function in the controls. While in COPD, forced expiratory volume in 1 second percentage of predicted value (FEV1% pred), nutritional depletion, gender and physical inactivity were identified as independent factors to quadriceps strength (R2 = 0.72); FEV1%pred, thigh muscle mass, serum levels of tumor necrosis factor-alpha (TNF-α) and gender were correlated to quadriceps endurance variance, with each p<0.05. Conclusion Quadriceps strength and endurance capabilities are both substantially impaired in Chinese COPD patients, with strength most affected. For the controls, physical activity is most important for quadriceps function. While for COPD patients, quadriceps dysfunction is related to multiple factors, with airflow limitation, malnutrition and muscle disuse being the main ones.
Introduction
Skeletal muscle dysfunction is well documented as an important systemic manifestation of chronic obstructive pulmonary disease (COPD) and has been recognized as a contributing factor in reduced exercise capacity, impaired quality of life and higher health-care utilization. Furthermore, COPD patients exhibit significant reductions in functional mobility and balance that may affect their ability to perform the activities of daily life. It has been suggested that these reductions in functional performance are related to the muscle dysfunction present in these patients [1]. It is of great interest that the pattern of limb muscle impairment in COPD is different from that seen in the respiratory muscles [2]. There is increasing interest in the role of peripheral skeletal muscles in COPD as it is a potential site of intervention for improving the patient's functional status. Among the reports, quadriceps function assessment was used in the majority of the studies for assessment of peripheral muscles' function as it is readily accessible and is a primary locomotor muscle. Quadriceps dysfunction can be considered in terms of loss of both muscle strength and endurance. Current opinion suggests that the reduction in muscle strength is due to a reduction in cross sectional area while the loss of endurance is due to muscle fiber type changes [3]. Quadriceps muscle dysfunction has been reported to be associated with decreased survival [4], poor functional status, and low quality of life [5]. More importantly, quadriceps muscle strength can better predict mortality than measures of lung function in this population [6]. Improved quadriceps muscle strength and endurance are recognized to underlie much of the increased exercise capacity observed following multidisciplinary pulmonary rehabilitation for COPD [7]. Thus, better understanding of the severity and the factors associated with impairment of quadriceps muscle function in patients with COPD would help develop new preventive interventions in quadriceps dysfunction and therapeutic approaches in the rehabilitation of these patients. However, there are few reports on quadriceps dysfunction in China though there are a large population of COPD patients in China [8]. In recent years, factors related to quadriceps dysfunction have been postulated, such as systemic inflammation, muscle wasting, muscle disuse etc.; while no one can fully explain quadriceps muscle dysfunction in COPD. Therefore, this study was aimed at investigating quadriceps dysfunction in Chinese patients with COPD, and to explore the related underlying factors. We examined in detail the quadriceps' function and endurance, the patients' nutritional status, muscle mass and physical activity and the presence of two cytokines; tumor necrosis factor-alpha (TNF-a) and C-reactive protein (CRP) to examine the potential systemic inflammatory response. In addition, the present study aimed to investigate the predictors for quadriceps functional capabilities in the healthy elderly across an age range comparable to that typically observed in COPD.
Subject selection
The current research was approved by the Research Committee of Human Investigation of Guangzhou Medical University (Approval number: 2011-21). Informed written consent was obtained from each participant. 71 patients with stable COPD were recruited from the outpatients' clinics of Guangzhou Institute of Respiratory Disease (Guangzhou, China) between Mar 2007 to Jun 2009. The diagnosis of COPD was made according to the criteria recommended by the GOLD guidelines [9] with spirometric confirmation of irreversible airflow limitation with post-bronchodilator forced expiratory volume for 1 second (FEV 1 )/forced vital capacity (FVC) ,70%. Significant comorbidities were excluded by medical history, physical check-up and conventional laboratory investigations. All the COPD patients involved in the current study were ex-smokers with abstinence for more than 3 years. Subject exclusion criteria included history of exacerbation in the preceding 3 months, co-morbidities of cardiac, rheumatologic or neuromuscular disorders or unwillingness to participate in the study. Most of the COPD patients were on inhaled corticosteroids (400-800 ug budesonide equivalent dose/ day), none of them were on regular systemic corticosteroids otherwise they would have been excluded; about 15% of patients were naïve to inhaled corticosteroids. 60 subjects for the control group were recruited from the health check-up department of First Affiliated Hospital of Guangzhou Medical University. The criteria for inclusion in the control group were as follows: (1) aged matched to the study group with COPD; (2) normal spirometry; (3) without any respiratory symptoms or other disease affecting quadriceps function; (4) non-smoker or has abstained from smoking for more than 10 years.
Methods
Quadriceps function assessment. Quadriceps functional tests included strength and endurance performance. The quadriceps isometric maximal voluntary contraction force (QMVC) test was performed using the technique described by a previous report [10], with a specially designed chair ( Figure 1). The chair was designed for the following four functions: first, the chair was immovable with being fixed on the floor, while it was comfortable enough and its armrest was strong enough for subjects to exert their maximal force in a sitting position by wrapping their fingers around the armrest; second, there was a strain gauge and load cell (Strainstall, Cowes, UK) installed under the chair so that the quadriceps force could be measured, and the height of the strain load cell was parallel to the ankle of the subjects; third, the strain load cell could be easily dissembled for everyday calibration; fourth, the back of the chair was movable so it could be changed into a bed by laying the back flat for subjects to lie down if necessary. The tests were performed with the subject in a sitting position at 90u hip flexion and knee flexed at 90u over the end of the chair. An inextensible strap was attached around the subject's right leg just superior to the malleoli of the ankle joint. The strap was connected to the strain load cell that was calibrated after each test with weights of known amounts. Subjects were required to try to extend their dominant leg as hard as possible against the inextensible strap. A computer screen was in front of the subjects in order that the force generated was visible to subjects and investigator, so the computer screen served as a positive feedback to help subjects to perform the test. Repeated efforts were made with vigorous encouragement until there was no improvement in the performance, and each effort was sustained for about 3-5 seconds. If maximal values were reproducible (,10% variability) for a consecutive 3 times, i.e., the generated strength reached a plateau, the highest value of the 3 contractions was considered as QMVC [11]. Surface electromyography (sEMG) was recorded for quadriceps muscles of vastus lateralis (VL), rectus femoris (RF), and vastus medialis (VM). The output signals of force and sEMG were recorded via an analogue-digital instrument (Powerlab 8/ 16SP Instruments, Austin, TX, USA) and a personal computer (Apple Computer Inc., Cupertino, CA, USA) running Chart 5.1 software. The quadriceps sEMG amplitude recordings were quantified by using the root-mean-square (RMS). Typical signals of QMVC and sEMG from a normal male subject are shown in Figure 2.
Endurance time. Endurance of the quadriceps was evaluated during an isometric contraction. After 10 minutes of rest following the QMVC maneuvers, subjects were instructed to maintain a tension representing 55%,60% of their own QMVC until exhaustion. The feedback mechanism served by the computer screen helped subjects to maintain the determined submaximal tension. Subjects were strongly encouraged to pursue until tension dropped to 50% QMVC or less for more than 3 seconds. Thus, quadriceps endurance was defined as the time to fatigue (QTf), and the time at which the isometric contraction at 60% of maximal voluntary capacity could no longer be sustained. A sample signal from a normal male control is shown in Figure 3.
Nutritional status assessment. The nutritional status of all subjects was evaluated by using an integrated approach with a modified multiparameter nutritional index (MNI) [12], which consisted of anthropometric measurements and visceral protein levels. Anthropometric measurements included body weight, triceps skinfold thickness (TSF), mid-arm muscle circumference (MAMC). MAMC = mid-arm circumference -p6TSF. Albumin, transferrin and prealbumin plasma concentration were used as visceral protein levels. The MNI score was calculated by: MNI = a+b+c+d+e+f (0 to15). Table 1 shows the variables and point values used for the computation of MNI score.
Quadriceps muscle mass evaluation. The quadriceps muscle mass was evaluated indirectly by anthropometric measurements of the legs [13]. Measurements consisted of quadriceps skin-fold thickness (S) and thigh girth of the subjects. With the patient standing and his weight evenly distributed, the thigh girth was determined on the nondominant side at half the distance between the inguinal crease and a point midway along the patella. Thigh muscle mass was thus evaluated as skinfold corrected thigh girth (CTG), and CTG = mid-thigh girth-pS.
Determination of cytokines. In serum, levels of TNF-a, and CRP were determined. TNF-a level was determined by ELISA kits (Quantikine; R&D Systems, Minneapolis, MN), and CRP by ELISA kits (CHEMICON Int CO. USA).
Level of daily physical activity. The level of daily physical activity (PA) was assessed by using a PA questionnaire [14] adapted for the elderly in China. The questionnaire on habitual PA consisted of 19 items, scored the past 3 year's household activities, sports activities, and other physically active leisure-time activities and gave an overall PA score. An intensity code based on net energetic costs of activities according to Voorrips et al [15] was used to classify each activity. The subjects were asked to describe type of the PA, hours per week spent on it, and period of the year in which the PA was normally performed.
Statistical analyses
Statistical analysis was performed using SPSS 13.0 (SPSS; Chicago, IL) and Minitab 16.0 (Minitab; Techmax Inc.) statistical package for windows. Measurement data were summarized by mean6SD, and categorical data were summarized by number (percentage). P value ,0.05 was considered statistically significant. Two independent-sample t-tests and Chi-square test were used for univariate testing between COPD patients and control subjects. In both control and COPD groups, multiple regression models were developed by stepwise method to determine factors independently contributing to quadriceps strength and endurance, respectively. In the stepwise regression analysis, Alpha-to-Enter 0.15 and Alpha-to-Remove 0.15 were included.
Characteristics of the subjects
The general characteristics of the control subjects and patients with COPD are shown in Table 2. Age and height were
Nutritional status and anthropometric data
All nutritional variables were within the reference values in almost all the control subjects, but below the reference values in most of the COPD patients. MNI sore was significantly higher in COPD patients than in controls (
Level of daily life physical activities
The questionnaire for daily life PA showed that nobody had ever participated in a rehabilitation program among all the participants, while PA scores were significantly lower in COPD patients when compared to controls (table 2), showing a decreased physical activity among the patients.
Quadriceps functional assessment
As expected, there was a gender-related difference in quadriceps strength; QMVC was significantly decreased in females than in males for both COPD patients and controls. When compared with gender-matched controls, the mean values of QMVC and QTf were both significantly reduced in COPD patients, which were also demonstrated for RMS amplitudes of the sEMG signals from VL, RF and VM muscles during the QMVC test (table 3). The mean value of quadriceps strength and endurance was 46% and 38% lower, respectively, in COPD patients than in controls. Among patients with COPD, there was neither a significant difference in QMVC between the steroid-naive patients and those
Serum levels of TNF-a and CRP
Serum levels of TNF-a were significantly increased in patients when compared to controls [(6.9862.50) pg/ml vs.
Correlations
In normal subjects, multivariate stepwise regression analysis suggested that QMVC was predicted by sex (0.386), PA scores (0.279) and weight (0.305), with R 2 of 0.61 (p,0.0001); for endurance time, PA scores (0.519), CTG (0.374), weight (0.617) and sex (20.985) were the contributors to QTf variance, with R 2 of 0.58 (p,0.001). Based on the regression analysis results, we derived 2 predictive equations for quadriceps strength and endurance time from the healthy elderly with an age range from 58-76 years old. The equations describing predicted QMVC force (kg) and QTf (S) were, as follows: In COPD patients, multiple stepwise regression analysis identified that sex, FEV 1 %pred, MNI and PA scores are statistically significant predictors, together explaining 72% of QMVC variance. For endurance time, FEV 1 %pred, CTG, serum TNF-a levels, and sex were predictors to QTf, explaining 44% of the QTf variance. Table 4 shows the factors correlated with QMVC and QTf in COPD patients. Table 5 and table 6 show the standardized coefficients (b) for each predictor variable for QMVC and QTf, respectively, obtained from multiple regression analysis in the COPD group.
Discussion
The main findings of this study are that (1) Both quadriceps strength and endurance capabilities are substantially impaired in Chinese patients with COPD, with strength and endurance being 46% and 38% lower, respectively, in the patients relative to agematched controls; (2) Impairment of quadriceps function correlated with multiple factors, with airflow limitation, malnutrition and muscle disuse taking important roles; (3) Using regression equations generated from a cohort of the healthy elderly across an age range from 58 to 76 years old, we showed that physical activity was an important determinant of quadriceps functional capabilities in healthy individuals. As far as the authors know, this is the first study to characterize quadriceps dysfunction in Chinese patients with COPD and give predictive equations for quadriceps strength and endurance time from the Chinese healthy elderly. Also, this is the first study to investigate the multiple factors related to quadriceps function in both COPD patients as well as in the healthy elderly.
In the present study, the expected gender-related difference in quadriceps strength was observed for both COPD patients and controls, and similar data was also reported by Miller et al [16], indicating that gender difference should be taken into account in assessment of quadriceps function. When compared to gender and age-matched controls, the mean value of QMVC was reduced by 47% and 45%, respectively, in female and male patients; the reduction was more severe than previously reported [17], indicating a more remarkable impairment of quadriceps strength in Chinese patients with COPD. But we should elucidate that most of our study patients had severe and very severe airflow limitation. RMS amplitudes of the sEMG signals from VL, RF and VM muscles were also decreased in COPD patients compared to the controls, supporting a significantly decreased strength in the patients, as muscle strength level could be reflected by the amplitudes of the sEMG signals quantified using RMS. For endurance capability, the values of QTf were lowered 39% and 37% in female and male patients, respectively, when compared to controls. Our data showed that quadriceps muscle strength was impaired to a greater extent than endurance in COPD patients, which was in line with the findings of Zattara-Hartmann et al [18]; on the contrary, Van't Hul et al [19] reported greater impairment in endurance for COPD patients. These conflicting results may be attributed to differences in the severity of airflow limitation between the study patients. The patients studied by Van't Hul et al were all in GOLD stage II to III, while most patients in our study were in GOLD stages III to IV. This explanation is supported by the results of regression correlation analysis in the present study, where b coefficient showed that FEV1%pred more significantly correlated with QMVC than with QTf in COPD patients, demonstrating that quadriceps strength is impaired to a larger extent than endurance in COPD. Remarkably, we found that both QMVC and QTf were significantly correlated with FEV1%pred in patients with COPD. Regarding the relationship of airflow limitation with quadriceps function, similar results have been yielded by some previous studies [10,20,21], though conflicting results were also reported by other studies [22][23][24]. Nevertheless, the highest prevalence of quadriceps weakness was observed in those with the most severe airflow obstruction in our study patients with COPD, which was also demonstrated in a large sample of COPD patients in another study [17], demonstrating that there may be an association between airflow limitation and quadriceps muscle dysfunction. The relationship between quadriceps dysfunction and airflow limitation may have multiple potential explanations. First, the increased cost of breathing as a result of the airflow limitation may well be associated with skeletal muscle weakness in COPD, especially of the lower limb muscles. Second, airflow limitation and the resultant greater respiratory muscle work often leads to respiratory muscle fatigue, which, in turn, increases sympathetic vasoconstrictor activity in the working limb via a supraspinal reflex [25]. The result is a decrease in limb blood flow and a corresponding reduction in oxygen delivery to peripheral muscles, which accelerate the development of quadriceps fatigue. Third, due to airflow limitation and the associated sensation of dyspnea, COPD patients often experience a downward spiral of symptom-induced inactivity and even muscle disuse, which in turn causes muscle structure changes and metabolic derangements, such as a shift from type I to type II skeletal muscle fibers [26], reduced mitochondrial density per fiber bundle [27], and reduced capillary density [28]. Each of these can correlate with a reduced capacity for aerobic metabolism and, ultimately, poorer muscle performance. In addition, due to airflow limitation and the associated impaired gas exchange, patients with COPD have chronic hypoxia to a varying degree, thus a compromised oxygen transport to limb locomotor muscles might be expected. Furthermore, hypoxemia may interfere with muscle differentiation and lead to muscle dysfunction via several pathways. For example, it has been shown that hypoxia inhibits myogenic differentiation through accelerated MyoD degradation and via the ubiquitin proteasome pathway [29]. In addition, hypoxemia might affect the contractile apparatus and enhance muscle oxidative stress.
As for the nutritional status in the COPD patients, although we recruited the patients randomly at the onset of the study, it turned out that 74.65% patients had decreased body weight, with a mean BMI of less than 21 kg/m 2 and MNI score that was significantly elevated, indicating that malnutrition was prevalent in the COPD patients; moreover, the MNI score correlated inversely with QMVC in our patients. A similar study result has been already reported [30], and there is evidence that nutritional supplementation increases muscle strength [31]. In our previous study [32], we have found that skeletal muscle mass is substantially decreased in COPD patients and muscle wasting is the main manifestation of nutritional depletion. In the present study, CTG was identified as a contributor to QTf in both COPD patients and controls. This finding was in line with the study result of the association between muscle loss and increased muscle fatigability in COPD [33], suggesting that muscle wasting is, at least in part, responsible for impairment of quadriceps endurance in COPD.
Muscle wasting is an effect of other pathophysiological changes such as muscle disuse and nutritional depletion. At the same time, the present study derived an equation to predict quadriceps strength and endurance, from the healthy elderly; the data showed a close relationship of PA scores with both QMVC and QTf among control subjects. In the classic description of QMVC measurement in COPD, strength was normalized to body weight [34], and in recent research, QMVC was recognized to be associated with airflow limitation, fat-free mass and age [17]. Partly consistent with that study, our data showed that quadriceps strength was predicted by multiple factors including airflow limitation, nutritional depletion, and muscle wasting and physical inactivity. As far as physical activities were concerned, our study found that the PA score was significantly lower in patients than in controls, which was in keeping with previous studies [35,36]; moreover, our data showed that the PA score had a big effect on both QMVC and QTf in the healthy elderly, based on the standardized b coefficient. Our finding was supported by the classic theory that exercise can improve muscle function while long term inactivity leads to muscle weakness in normal subjects. While in COPD, patients often have a sedentary lifestyle and muscle disuse, which leads to muscle weakness and limited exercise capacity. Our data was also supported by the accumulating study evidence derived from a rehabilitation program, which showed that muscle training can improve muscle strength in COPD patients [37], highlighting a tight link between muscle inactivity and muscle weakness in COPD.
In addition, the present study analyzed the TNF-a levels in serum of the all participants, and found that TNF-a levels were significantly elevated in COPD patients relative to controls; moreover, regression analysis identified TNF-a as one of the contributors to QTf in COPD patients. TNF-a has been recognized as an important cytokine in skeletal muscle wasting [38] as it might compromise muscle function by stimulating muscle protein loss or inducing alterations of muscle proteins catabolism. Our findings, together with previous studies, suggest that systemic inflammation takes an important role in the development of quadriceps dysfunction in COPD. Although one study has found that TNF-a muscle protein levels are decreased in COPD [39] other studies that looked at sputum samples agree with these results [40,41]. With regard to CRP, our study failed to show a significant difference between COPD patients and the controls. In contrast, Broekhuizen et al [42] reported elevated CRP levels in advanced COPD. CRP is an acute-phase reactant, while patients in our study had been stable for 3 months. This may explain why CRP levels were not elevated in our study.
Study Limitations
The current study has several limitations. First, we had the small size of female patients with COPD and relatively small size of male controls, which may offset the accuracy of our study results. Larger scale studies should be conducted in the future to further improve the accuracy of the study results. Second, our patients with COPD were on a variety of inhaled corticosteroids (ICS), which might modify the quadriceps function, thereby interfering with the results. Finally, most of the patients in our study had severe or very severe COPD, such that these data cannot be generalized to patients with mild or moderate disease. Further studies are needed to address these issues.
Conclusions
The present study investigated quadriceps muscle strength and endurance in COPD patients as well as in age-matched healthy elderly; it turned out that the value of quadriceps strength and endurance was 46% and 38% lower, respectively, in COPD patients relative to controls. We draw the conclusion that quadriceps dysfunction is correlated with multiple factors, with airflow limitation, nutritional depletion and muscle disuse taking important roles in its development; while physical activity contributes most to quadriceps function in the healthy elderly. | v3-fos-license |
2019-02-17T14:20:07.089Z | 2018-05-02T00:00:00.000 | 64702668 | {
"extfieldsofstudy": [
"Computer Science"
],
"oa_license": "CCBY",
"oa_status": "GOLD",
"oa_url": "http://downloads.hindawi.com/archive/2018/3140309.pdf",
"pdf_hash": "6ea8305f6433ae49005f03b8cd33d74a42491bde",
"pdf_src": "Anansi",
"provenance": "20241012_202828_00062_s4wg2_01a5a148-7d10-4d24-836e-fe76b72970d6.zst:8",
"s2fieldsofstudy": [
"Engineering",
"Environmental Science"
],
"sha1": "6ea8305f6433ae49005f03b8cd33d74a42491bde",
"year": 2018
} | pes2o/s2orc | Low-Cost SCADA System Using Arduino and Reliance SCADA for a Stand-Alone Photovoltaic System
SCADA (supervisory control and data acquisition) systems are currently employed inmany applications, such as home automation, greenhouse automation, and hybrid power systems. Commercial SCADA systems are costly to set up andmaintain; therefore those are not used for small renewable energy systems. This paper demonstrates applying Reliance SCADA and Arduino Uno on a small photovoltaic (PV) power system to monitor the PV current, voltage, and battery, as well as efficiency. The designed system uses low-cost sensors, an Arduino Uno microcontroller, and free Reliance SCADA software. The Arduino Uno microcontroller collects data from sensors and communicates with a computer through aUSB cable. Uno has been programmed to transmit data to Reliance SCADA on PC. In addition, Modbus library has been uploaded on Arduino to allow communication between the Arduino and our SCADA system by using MODBUS RTU protocol. The results of the experiments demonstrate that SCADA works in real time and can be effectively used in monitoring a solar energy system.
Introduction
For several hundred years, fossil fuels have been consumed as the main source of energy on Earth.As a result, they are now experiencing rapid depletion.Researchers and scientists who understand the importance of renewable energy have dedicated their efforts to the research, expansion, and deployment of new energy sources to replace fossil fuels.
Photovoltaics (PV) are an important renewable energy sources.Also called solar cells, PV are electronic devices that can convert sunlight directly into electricity.The modern forms of PV were developed at Bell Telephone Laboratories in 1954 [1].Despite their promising performance, PV have some limitations, such as depending on factors like longitude, latitude, and weather and being limited to daytime hours to generate power [2].
The SCADA system is software that has been installed in several sites to monitor and control processes, and it is called telemetry importance [3,4].SCADA can monitor real-time electrical data measurements of solar module and batteries and collect data from wind turbines, such as the condition of the gearbox, blades, and electric system [5,6].Moreover, the sun-tracker system has also used the SCADA system to observe the solar insolation and movement of the sun [6].
These days, commercial companies are widespread for monitoring systems such as photovoltaic systems.However, those are quite expensive.For example, SMA Company is a German Company, and it was founded in 1981.It has many products.Some of them are related to monitoring and controlling, for example, Sunny View.It can show all of your system data in good condition, and we can read all data clearly.However, the major problem is that the device is costly; it costs about CA $793 [7,8].
A previous study also shows a data acquisition and visualization system, with storage in the cloud, and it has been applied on a photovoltaic system.In addition, this design was based on embedded computer, and it connected with PV inverters by using RS485 standard, and microcontroller is to read climate sensors but sensors have used web system to show data [9].
Also, a study shows a low-cost monitoring system, it is presented in [10].The system has determined losses in energy production.The paper is based on multiple wireless sensors and low cost, and it used voltage, current, irradiation, and temperature sensors which are installed on PV modules as well.
In this paper, designed SCADA system is of lower price compared with commercial SCADA system, and it delivers the same performance.In order to test this work, the SCADA system is employed for monitoring the parameters of solar energy systems (photovoltaic) in real time, which consist of a solar module, MPPT, and batteries.The parameters are the current and voltage of the photovoltaic (PV) system and the current and voltage of the battery.Data acquisition system is by Arduino controller and sensors.All data are sent to a PC and are shown on a user interface designed by Reliance SCADA.The data are saved on a computer as an Excel file as well.This allows users and operators to monitor the parameters of the PV system in real time.The components of the SCADA system in this paper consist of two parts: hardware and software.
Hardware Design
The proposed Reliance SCADA is designed to monitor the parameters of a small PV system.It is installed at the Department of Electrical Engineering, Memorial University, St. John's, Canada.Figure 1 shows 12 solar panels up to 130 watts and 7.6 amp.Two solar modules are connected in parallel.Therefore, the system shown in Figure 1 consists of 6 sets of 260 watts each.The Reliance SCADA system was designed to be of low cost and can be expanded or modified without the need for major hardware changes in the future.Basic elements of the design are an Arduino Uno controller and sensors, as shown in Figure 2.
Arduino Uno Microcontroller.
Arduino Uno is opensource hardware that is relatively easy to use. Figure 3 shows Arduino Uno, while before MPPT to measure the PV voltage and the other is installed after MPPT to measure the battery voltage.Figure 5 demonstrates how it connects in an electrical circuit with Arduino Uno.
Hardware Setup
Figure 6 shows the hardware setup designed for the SCADA system.
Arduino IDE.
IDE is open-source software that features easy-to-write code that can be uploaded to any board.In this work, we needed to upload a new library on IDE to make a configuration between Arduino Uno and SCADA software by MODBUS RTU protocol.Figure 7 shows how the system works and also shows the code that has been burned on Arduino Uno.
(B) Code.The code has some main functions such as setup() (it is called once when the sketch starts) and loop() (it is called over and over and is heart of sketch).The most important in the code are libraries mentioned initially: regBank.setId()command, regBank.add(),and regBank.set().The purpose of libraries is to connect between Arduino Uno and Reliance SCADA software by MODBUS RTU protocol.regBan.setId() is used to define MODBUS to work as slave.regBank.add()command is used to define addresses of registers which are used to send data to Reliance SCADA on computer.In this work, the addresses were from 30001 to 30005 as mentioned slave.run();}}4.2.Reliance SCADA.Reliance software is employed in numerous technologies for monitoring and controlling systems.It can also be used for connecting to a smartphone or the web.Reliance is used in many colleges and universities around the world for education or scientific research purposes [12].Figure 8 shows a user interface designed by Reliance SCADA software to monitor the parameters of the photovoltaic system.
The user interface has four real-time trends and four display icons to show values as digital numbers.In addition, it
Number
Variable name MODBUS RTU address (1) Voltage of photovoltaic system 0 (2) Current of photovoltaic system 1 (3) Voltage of battery 2 (4) Current of battery 3 (5) Efficiency of MPPT 4 has two buttons and a container.These features are discussed in Results and Discussion.
Communication System
MODBUS library is added to Arduino Uno to allow communication with Reliance SCADA via a USB cable using MODBUS RTU protocol.Table 2 shows the allocation of MODBUS address for MODBUS RTU on Reliance SCADA software, with the MODBUS address for Arduino Uno mentioned in the Arduino code.
Cost of the SCADA System
Most factories that use several systems are looking for a low cost SCADA system to monitor and control their systems remotely.In this paper, the components used are quiet cheap.
Monitring parameters of PV system using Arduino Uno
Voltage Table 3 shows the price (CA dollar) for whole components according to the amazon.cawebsite.
According to Table 3, we found that the whole price of SCADA system was CA$82.This price seems cheap to design SCADA system for monitoring parameters of our system.
Results and Discussion
In this work, the proposed SCADA monitors a solar energy system and several experiments are carried out.The experiments cover the measurement error of the sensor systems which are installed to measure PV current and voltage, battery current and voltage, MMPT efficiency, and SCADA features.
The sensors that are used contain errors, so these errors are calculated with calibrated instruments, as listed in Table 4.
As can be seen in Table 4, the measurement error of current sensors was the highest.The error percentages of the PV current sensor and the battery current sensor are about 3.42% and 3.10%, respectively.Although the error percentages of both voltage sensors were quite low, they were closer to the calibrated instrument.
The monitoring tasks are displayed on the PC.They include the PV parameters as a graph and digital numbers and the MPPT efficiency as digital numbers.Figure 9 shows the user interface of SCADA after the system was operational.
The SCADA system is designed to make an update every minute.As shown in Figure 9, there are four figures: two of them observe the PV voltage and current and the other two monitor the battery voltage and current.The figure also shows that the SCADA system makes updates every minute.
The user interface of SCADA shows five icons displaying values of parameters as digital numbers, and they also make automatic updates every minute.
Our SCADA system has the feature of enabling all data to be easily saved on a computer as an Excel file.To save the data, the user just has to hit the Export-Data icon and then hit the Save-Data icon.These icons are programmed by script to save the data on a PC as an Excel file. Figure 10 shows a screenshot of data saved in Excel.
Also, user interface has a container that shows details.The Arduino connects with SCADA, and it gives warning if there is any error in connection.
The efficiency of MPPT was also monitored.It represents the output power of MPPT over the input power to MPPT. Figure 11 presents MPPT efficiency for various periods of time, with efficiency ranging between 1 and 0.8.
Conclusion
In this paper, a low-cost SCADA system was designed and built with Reliance SCADA software and Arduino Uno.The SCADA system was applied to a stand-alone photovoltaic system to monitor the current and voltage of PV and batteries.The results of the experiments demonstrate that SCADA works in real time and can be effectively used in monitoring a solar energy system.The developed system costs less than $100 and can be modified easily for a different PV system.
Figure 1 :Figure 2 :
Figure 1: Solar panels on the roof of engineering building.
Figure 5 :
Figure 5: Connection drawing of current sensor.
Figure 6 :
Figure 6: Hardware setup of SCADA system.
Figure 9 :
Figure 9: User interface of SCADA while running.
[11]e1shows specifications for the hardware.The license gives permission to anyone to improve, build, or expand Arduino.The original Arduino and its enhancement environment were founded in 2005 in Italy at the Smart Project Company.It has 14 digital input/output pins, 6 of which can be used as analog input/output[11].
2.2.Current Sensor.Current sensors for DC currents must be able to measure a range of currents for PV and batteries between 0 A and 20 A. In this work, an ACS 712 sensor is used for sensing the current.It is designed to be easily used with any microcontroller, such as Arduino.The sensors are based on the Allegro AC712ELC chip.The scale value of ACS 712, which is used in this design, is 20 amp, which is appropriate for sensing current.Two sensors are installed: one a small one.In this work, the voltage sensor is a 25 V-sensor with two resistors of 30 KΩ and 7.3 KΩ.The maximum voltage of either PV or battery is 25 V, so this sensor is appropriate.The output of the voltage sensor is between 0 V and 5 V.This scale is suitable to the Arduino analog inputs.In this experiment, we need two voltage sensors: one is installed
Table 1 :
Specifications of Arduino board.
Table 2 :
Allocation of MODBUS address for MODBUS RTU.
Table 3 :
Price components of SCADA system.
Table 4 :
Measurement errors of sensor system. | v3-fos-license |
2019-01-22T22:23:51.483Z | 2018-12-21T00:00:00.000 | 57013271 | {
"extfieldsofstudy": [
"Biology",
"Medicine"
],
"oa_license": "CCBY",
"oa_status": "GOLD",
"oa_url": "https://doi.org/10.7717/peerj.6132",
"pdf_hash": "4c6ccde31707bd0429229ffa9189cf84161c914a",
"pdf_src": "PubMedCentral",
"provenance": "20241012_202828_00062_s4wg2_01a5a148-7d10-4d24-836e-fe76b72970d6.zst:9",
"s2fieldsofstudy": [
"Biology",
"Medicine"
],
"sha1": "4c6ccde31707bd0429229ffa9189cf84161c914a",
"year": 2018
} | pes2o/s2orc | Non-invasive monitoring of glucocorticoid metabolite concentrations in urine and faeces of the Sungazer (Smaug giganteus)
Developing non-invasive techniques for monitoring physiological stress responses has been conducted in a number of mammal and bird species, revolutionizing field-based endocrinology and conservation practices. However, studies validating and monitoring glucocorticoid concentrations in reptiles are still limited. The aim of the study was to validate a method for monitoring glucocorticoid metabolite concentrations in urine (uGCM) and faeces (fGCM) of the cordylid lizard, the Sungazer (Smaug giganteus). An adrenocorticotropic hormone (ACTH) challenge was conducted on one male and two females with both urine and faecal material being collected during baseline and post-injection periods. Steroid extracts were analysed with four enzyme immunoassays (EIAs)namely: 11-oxoaetiocholanolone, 5α-pregnane-3β-11β-21-triol-20-one, tetrahydrocorticosterone, and corticosterone. A considerable response in fGCM and uGCM concentrations following ACTH administration was observed in all subjects, with the 5α-pregnane-3β-11β-21-triol-20-one and tetrahydrocorticosterone EIAs appearing to be the most suited for monitoring alterations in glucocorticoid metabolite concentrations in S. giganteus using faeces or urine as hormone matrix. Both EIAs showed a significantly higher concentration of glucocorticoid metabolites in faeces compared to urine for both sexes. Collectively, the findings of this study confirmed that both urine and faeces can be used to non-invasively assess adrenocortical function in S. giganteus.
INTRODUCTION
Historically, reptiles have been seen as a vertebrate group with limited importance to the natural environment, with the disappearance of the taxa unlikely to make any noteworthy difference (Zim & Smith, 1953). Thankfully, this sentiment has disappeared as scientists realize the importance of reptiles as an integral part of the ecosystem and thus important indicators of environmental quality (Gibbons & Stangel, 1999). Despite research showing that reptile numbers decline on a similar scale in terms of taxonomic breadth, geographic scope and severity as amphibians (Gibbons et al., 2000), reptiles remain one of the least studied vertebrate groups, being considered of less general interest compared to other fauna (Bonnet, Shine & Lourdais, 2002). The cryptic colorations and nature of reptiles (Zug, Vitt & Caldwell, 2001), as well as the general low population numbers inherent to the taxa (Todd, Willson & Gibbons, 2010), often results in a limited number of individuals available to monitor during a study. A number of factors have been suggested to contribute to the currently recognized decline in reptiles globally (see Todd, Willson & Gibbons, 2010 for a review). However, the direct and indirect effects of factors such as global climate change, disease, and habitat pollution are sometimes difficult to quantify and relate to individual and population health and survivability (Gibbons et al., 2000). In this regard, monitoring physiological stress patterns in reptiles may provide an important insight into the susceptibility of reptiles to population declines when faced with various environmental threats.
Stress is commonly referred to as the stimulus that may threaten, or appear to threaten, the general homeostasis of an individual (Wielebnowski, 2003;Hulsman et al., 2011). The perception of a stressor leads to the activation of the hypothalamic-pituitary-adrenal (HPA) axis and, consequently, to an increase in glucocorticoid (GC) secretion (Sapolsky, Romero & Munck, 2000;Hulsman et al., 2011). An acute increase in GC concentrations can be adaptive in nature, increasing energy availability and altering behavior, while indirectly regulating cardiovascular and metabolic parameters (Romero, 2002;Sapolsky, 2002;Reeder & Kramer, 2005;Walker, 2007). However, prolonged elevation of GC concentrations can lead to a number of deleterious effects, such as the suppression of the immune and reproductive systems, muscle atrophy, growth suppression, and a shortened life span (Möstl & Palme, 2002;Sapolsky, 2002;Charmandari, Tsigos & Chrousos, 2005;Cohen, Janicki-Deverts & Miller, 2007). Thus, monitoring GC concentration in endangered and threatened species can be an important tool for assessing physiological stress in individuals exposed to natural and anthropogenic stressors. Non-invasive hormone monitoring techniques, through the collection of urine or faeces, hold numerous advantages over the traditional use of blood collection. Firstly, there is no need to capture or restrain study animals for sample collection, thus removing any potential stress-related feedback, and thereby also increases safety for both animal subjects and researchers (Romero & Reed, 2005). Further, as a result of the general ease of collection, longitudinal sampling and hormone monitoring of specific individuals are possible (Heistermann, 2010). Finally, hormone metabolite concentrations determined from matrices like faeces, urine, or hair are usually less affected by episodic fluctuations of hormone secretion, as circulating hormone concentrations are accumulating in these matrices over a certain period of time (Vining et al., 1983;Creel, MarushaCreel & Monfort, 1996;Russell et al., 2012). However, prior to the first use of the chosen assays and specific matrices for monitoring physiological stress in a species, it is important that the approach is carefully validated to ensure a reliable quantification of respective GCs (Touma & Palme, 2005). A preferred method of validation is the physiological activation of the HPA axis through the injection of adrenocorticotropic hormone (''ACTH challenge '', (Touma & Palme, 2005), which results in a distinct increase in GC production from the adrenal gland. Collected pre-and post-injection samples are subsequently analyzed to determine which of the tested enzyme immunoassays (EIA) reflects the induced increase in GC concentrations best. Historically, GC patterns in reptiles have been monitored via serum analysis, for example in the red-eared slider turtle (Trachemys scripta elegans, Cash, Holberton & Knight, 1997), the Galapagos marine iguana (Amblyrhynchus cristatus, Romero & Wikelski, 2001), or the tuatara (Sphenodon punctatus, Tyrrell & Cree, 1998). However, some more recent studies attempting to understand the physiological response inherent in reptiles to environmental stressors have already opted for non-invasive hormone monitoring in reptiles, e.g., in Nile crocodiles (Crocodylus niloticus, Ganswindt et al., 2014), the three-toed box turtle (Terrapene carolina triunguis, Rittenhouse et al., 2005), the green anole (Anolis carolinensis, Borgmans et al., 2018) or the green iguana (Iguana iguana, Kalliokoski et al., 2012).
The Sungazer (Smaug giganteus, formerly Cordylus giganteus; Fig. S1) is a cordylid lizard endemic to the grassland of the Free State and Mpumalanga provinces of South Africa (De Waal, 1978;Jacobsen, 1989). It is unique among the cordylid lizards as an obligate burrower rather than rupicolous (Tonini et al., 2016;Parusnath et al., 2017). The species is currently facing large scale habitat degradation and population declines as a result of anthropogenic activities such as agricultural repurposing of its natural habitat, road construction, electricity infrastructure, mining developments, as well as the pet and traditional medicine trade (Van Wyk, 1992;McIntyre & Whiting, 2012;Mouton, 2014). Consequentially, S. giganteus is now listed as vulnerable by the International Union for the Conservation of Nature (IUCN, Alexander et al., 2018).
The aim of the study was to examine the suitability of four enzyme immunoassays (EIA) namely, 11-oxoaetiocholanolone, 5α-pregnane-3β-11β-21-triol-20-one, tetrahydrocorticosterone, and corticosterone, for monitoring adrenocortical function in S. giganteus by determining the stress-related physiological response in faeces and urine following an ACTH challenge test.
Study site and animals
The study was conducted at the SANBI National Zoological Garden (NZG), Pretoria, South Africa (25.73913 • N, 28.18918 • E) from the 24th of November 2017 to the 5th of December 2017. The study animals, consisting of one male (M1:291 g) and two females (F1: 295 g) and F2: 344 g), were housed in individual enclosures within the Reptile and Amphibian Section of the NZG. Individuals were separated by a 1.5 m high wall, which resulted in study animals not being in visual contact with one another. Each enclosure (2 m × 1.5 m) was covered in coarse river sand and included an artificial burrow constructed from fiberglass, UV-light and a water bowl with water available ad libitum. The light regime (13 L: 11 D) and humidity (range: 44-50%) were kept constant throughout the study period. A combination of meal worms and fresh, green vegetables were provided daily to all individuals. Prior to the start of the study, all individuals were given a two-week acclimatization period to the new enclosure and presence of researchers. The limited number of individuals used during the study reflects the availability of study animals in a suitable setting, as well as the difficulty in receiving approval to conduct research on vulnerable and endangered species.
Sample collection and ACTH challenge
In reptiles, urine and faeces can be excreted in unison, though not mixed ( Fig. S2; Singer, 2003); urine is a white, solid substance, compared to the dark, solid faecal component, which allows for the separation of the two matrices with limited levels of cross-contamination (Kummrow et al., 2011). During the entire monitoring period, collected urine and faeces were separated during collection, and the two parts placed into separate 1.5 ml microcentrifuge tubes, sealed, and immediately stored at −20 • C until further processing. Following a two-week acclimatisation period, enclosures were checked for urine and faecal samples during the active period of S. giganteus (6 am-6 pm), for seven days. Cages were checked hourly to limit the effect of bacterial and environmental degradation of urine and faecal samples. In the morning hours of the eighth day all three individuals were injected intramuscularly with 0.45 µg synthetic ACTH g −1 bodyweight (SynACTH R , Novartis, South Africa Pty Ltd) in a 100 µl saline transport. This ACTH dose was chosen as it has been used successfully by a number of studies conducted on amphibian species such as the Fijian ground frog (Platymantis vitiana, Narayan et al., 2010), tree frog (Hypsiboas faber, Barsotti et al., 2017) and the American bullfrog (Rana catesbeiana, Hammond et al., 2018) to evoke a stress response. Subsequently, the individuals were released back into their individual enclosures, with faecal and urine collection continuing until day 15 of the study. The study was performed with the approval of the National Zoological Garden's Animal Use and Care Committee (Reference: P16/22).
Steroid extraction in urine and faecal samples
Urine and faecal samples were lyophilized, pulverized and sifted through a mesh strainer to remove any undigested material, resulting in a fine faecal and urine powder (Heistermann, Tari & Hodges, 1993). Subsequently, 0.050-0.055 g of the respective urine and faecal powder was extracted with 1.5 ml 80% ethanol in water. After vortexing for 15 min, the suspensions were centrifuged for 10 min at 1,600 g and the resulting supernatants transferred into new microcentrifuge tubes and stored at −20 • C until analysis.
Enzyme immunoassay analyses
Depending on the original matrix, steroid extracts were measured for immunoreactive faecal glucocorticoid metabolite (fGCM) or urinary glucocorticoid metabolite (uGCM) concentrations using four different EIAs: (i) an 11-oxoaetiocholanalone (detecting fGCMs with a 5β-3α-ol-11-one structure), (ii) a 5α-pregnane-3β-11β-21-triol-20-one (measuring 3β-11β-diol-CM), (iii) a tetrahydrocorticosterone, and (iv) corticosterone EIA. Details about assay characteristics, including full descriptions of the assay components including cross-reactivities, can be found in Möstl et al. (2002) for the 11oxoaetiocholanalone EIA, Touma et al. (2003) for the 5α-pregnane-3β-11β-21-triol-20one, Palme & Möstl (1997) for the corticosterone EIA and in Quillfeldt & Möstl (2003) for the tetrahydrocorticosterone EIA. Assay sensitivities, which indicates the minimum amount of respective immunoreactive hormone that can be detected at 90% binding, as well as the intra-and inter-assay coefficients of variation of high and low quality controls for each EIA is shown in Table 1. Serial dilutions of extracted faecal and urine samples gave Table 1 The enzyme immunoassay specific parameters used during this study. The sensitivity as well as the intra-and inter-assay coefficient of variation (CV) of the four enzyme immunoassays used during the study.
Enzyme immunoassay
Sensitivity (ng/g dry weight) Intra-assay CV Inter-assay CV displacement curves that were parallel to the respective standard curves in the two assays of choice (5α-pregnane-3β-11β-21-triol-20-one and tetrahydrocorticosterone EIAs), with a relative variation in slope of <4%. All EIAs were performed at the Endocrine Research Laboratory, University of Pretoria, South Africa, as described previously (Ganswindt et al., 2002).
Data analysis
A total of six faecal and urine samples were analyzed for each individual. Individual median fGCM and uGCM concentrations from pre-injection samples were calculated, reflecting individual baseline concentrations. To determine the effect of the ACTH injection on the HPA axis, the fGCM and uGCM concentrations from post-injection samples were converted to percentage response, by calculating the quotient of individual baseline and related fGCM/uGCM samples. In this regard, a 100% (1-fold) response represents the baseline value. Furthermore, the mean absolute deviation (MAD) was calculated for the baseline sample set (pre-injection). The MAD of the particular dataset shows the average distance between each baseline period data point and the calculated mean thereof, which represents the variability of the baseline samples collected. Thus, the lower a MAD value is for a specific EIA, the more stable the assay. Here, the individual baseline uGCM/fGCM concentration was subtracted from all pre-injection fGCM/uGCM values for each EIA-specific data set. The differences were noted as absolute values and the mean of the absolute values calculated, representing the MAD value for each EIA. The MAD values were converted to a percentage deviation value (MAD/Baseline Value*100) to allow for the comparison between EIAs. To determine the effect of the ACTH injection, the absolute change in fGCM and uGCM concentration was determined by calculating the quotient of baseline and post-injection peak fGCM and uGCM samples. MAD values below 15% were regarded as preferable.
The most appropriate EIA for measuring fGCM and uGCM concentrations in the species was chosen by comparing (1) the highest post-injection signal and (2) lowest MAD values observed. Values are given as mean ± standard deviation (SD) where applicable. Analytical statistics and graphical designs were performed using R software (R 3.2.1; R Development Core Team, 2013). Table 2 Urinary and faecal excretion rate, along with the time to peak urinary and faecal glucocorticoid metabolite peaks. The average faecal and urine excretion rate for female and male individuals of the study. Time to peak fGCM and uGCM response, as well as the respective sample numbers, are shown for each study animal. Values are given as mean ± standard deviation.
Defecation rate and MAD results
The average defecation rate (time between defecation events) showed considerable variation between individuals and matrices ( Table 2). The percentage MAD values were considerably lower in all EIAs when analyzing faecal (range: 3.17-15.67%) compared to urine (range: 13.31-56.52%) samples. For faeces, although the corticosterone EIA showed the lowest average percentage MAD value (mean ± SD = 8.37 ± 5.54%), the remaining three EIAs all showed comparable low average MAD levels (range: 9.95-11.02%). In contrast, all four EIAs showed high average percentage MAD levels in urine, with the 11-oxoaetiocholanalone EIA having the lowest average MAD value (mean ± SD = 17.17 ± 5.77%).
Urinary glucocorticoid metabolites analysis
Similar to the fGCM findings, all four EIAs showed a considerable response in uGCM concentrations (149.53%-651.82%) following the ACTH injection (Table 3). For the two females, the 5α-pregnane-3β-11β-21-triol-20-one and tetrahydrocorticosterone EIA showed the highest response, exceeding 340%, in the first collected faecal sample 27 h post ACTH administration (Tables 2 and 3, Fig. 2A & Fig. 2B). Respective uGCM Table 3 The urinary and faecal glucocorticoid metabolite response following ACTH administration in Smaug giganteus. The peak percentage glucocorticoid response in both faeces and urine, across all four enzyme immunoassays tested, in the two female and one male individual following the adrenocorticotropic hormone challenge. Values are given as mean ± SD.
DISCUSSION
In the current study, the defecation rate of study animals were prolonged and varied substantially within and between individuals. Extended defecation rates have been observed in a number of reptile species such as the Italian wall lizard (Podarcis sicula, ∼50 h, (Vervust et al., 2010), veiled chameleon (Chamaeleo calyptratus, ∼96 h, (Kummrow et al., 2011), six striped runner (Cnedmidophurs sexlineatus, 23-26 h, (Hatch & Afik, 1999) and a variety of snake species (45-3,180 h, (Lillywhite, De Delva & Noonan, 2002). Additionally, these studies have also shown high levels of individual variability in terms of gut retention times; for example, Kummrow et al. (2011) observed an individual excretion rate in C. calyptratus ranging from 48-120 h, while Hatch & Afik (1999) found the excretion rate in C. sexlineatus to range from 20-72 h. Understanding species-specific differences and individual variability in faecal and urinary defecation rates are important for a number of reasons. Firstly, the infrequent and extended excretion rate of urinary and faecal material in reptiles may complicate data interpretations (Ganswindt et al., 2014). Furthermore, the movement of urine into the cloaca (urodeum) before moving into the intestines, where urinary and faecal material can be excreted in unison (Singer, 2003), can further complicate the distinction between matrix-specific retention time and steroid hormone metabolite excretion routes.
As it is difficult to collect frequent faecal and urine samples consistently in S. giganteus and other reptile species, it may be advisable to monitor GC metabolite patterns over a longer The MAD values for the four EIAs used in the fGCM analysis indicated low levels of variation from the predetermined baseline values. In contrast to this, the MAD values calculated for the four uGCM EIAs showed high levels of variation from calculated baseline levels. As such, GC metabolite excretion via faeces may be less prone to regular fluctuation than urine, although further research is required to confirm this.
Following ACTH injection, the peak fGCM response was observed in the first faecal sample collected from all study animals. Ganswindt et al. (2014) found peak fGCM concentration, following the ACTH injection, in the first collected faecal sample from C. niloticus. Similarly, Cikanek et al. (2014) stressor. The pooling of faecal material in the reptile gut, over an extended period of time, may explain why peak fGCM responses are observed in the first sample post-injection in reptiles and other infrequent defecators. However, the available literature on reptile fGCM monitoring is limited, with a number of studies failing to highlight when the peak fGCM levels were observed or choosing to pool samples into larger time periods (Rittenhouse et al., 2005). Although all four EIAs displayed considerable peak fGCM responses for both sexes, the tetrahydrocorticosterone and 5α-pregnane-3β-11β-21-triol-20-one EIA performed best in our study, based on (i) EIA stability as seen in the low MAD values and (ii) the magnitude of peak percentage fGCM response following the ACTH injection. As such, both EIAs seem to be suitable for monitoring alterations in fGCM concentration in S. giganteus faecal material.
The peak uGCM concentrations following ACTH administrations were observed in the first and third collected urine sample for the females and male respectively. To our knowledge this is the first study to quantify the uGCM response following the activation of the HPA axis through physiological or biological stressors. In reptiles, the movement of urine into the intestine, and the resulting pooling effect along with faeces over time, may explain why peak uGCM responses were observed within the first collected samples for females and third sample for males. Similar to the fGCM analysis, all four EIAs used during the study were able to monitor alterations in uGCM concentrations following the ACTH administration; the tetrahydrocorticosterone and 5α-pregnane-3β-11β-21-triol-20-one EIAs again showed the highest uGCM response in this regard. With all uGCM MAD values considerably higher than observed for the fGCM analysis, the peak uGCM response values were used to determine EIA suitability; in this regard, both the tetrahydrocorticosterone and 5α-pregnane-3β-11β-21-triol-20-one EIAs were deemed suitable for monitoring alterations in uGCM concentration in S. giganteus urine.
Conclusion
The ability to monitor physiological stress patterns in endangered reptile species, through non-invasive hormone monitoring techniques, offers conservationists an ideal tool which can be implemented within both free-ranging and captive setups with limited effort. With the increase in human-driven factors leading to substantial decreases in reptile populations, the need for such techniques are becoming more important. This study has successfully validated such a technique for monitoring the stress response in S. giganteus in both urine and faeces by using the 5α-pregnane-3β-11β-21-triol-20-one or tetrahydrocorticosterone EIA. Both assays showed low MAD values as well as a considerable response in fGCM and uGCM concentrations following ACTH injection. As such, both sample matrices can be used to monitor physiological stress in S. giganteus. Despite the results of this study, a number of uncertainties need to be addressed by researcher conducting further studies on the topic. Of greatest concern is the observed gut passage time and time to peak fGCM and uGCM concentrations between individuals. Although the time to peak fGCM (24 h) and uGCM (27 h) responses were similar in both females, the monitored male showed a prolonged gut passage time with peak fGCM and uGCM concentrations 81 h and 70 h later, respectively. However, if in fact differences in gut passage time or GC metabolite patterns between individuals or sexes of the species exist is yet to be determined by examining larger study populations. Currently, we recommend collecting only the faecal or urine component for GC metabolite monitoring in S. giganteus. Despite the limitations of this study the findings increased our understanding of stress hormone production, metabolism and excretion pattern in the species. We hope this will encourage and stimulate future research not only on this species, but reptiles in general, especially concerning the non-invasively examining the physiological stress response linked to a host of anthropogenic and natural factors. | v3-fos-license |
2022-01-28T16:58:27.956Z | 2022-01-21T00:00:00.000 | 246336010 | {
"extfieldsofstudy": [
"Medicine"
],
"oa_license": "CCBY",
"oa_status": "GOLD",
"oa_url": "https://www.mdpi.com/1420-3049/27/3/704/pdf",
"pdf_hash": "1bf858fdd82c60b86ae6da884f31eefe6bef1416",
"pdf_src": "PubMedCentral",
"provenance": "20241012_202828_00062_s4wg2_01a5a148-7d10-4d24-836e-fe76b72970d6.zst:13",
"s2fieldsofstudy": [
"Medicine"
],
"sha1": "2f6828dc9719104a798ed71ef65d0c88736d309c",
"year": 2022
} | pes2o/s2orc | The System Profile of Renal Drug Transporters in Tubulointerstitial Fibrosis Model and Consequent Effect on Pharmacokinetics
With the widespread clinical use of drug combinations, the incidence of drug–drug interactions (DDI) has significantly increased, accompanied by a variety of adverse reactions. Drug transporters play an important role in the development of DDI by affecting the elimination process of drugs in vivo, especially in the pathological state. Tubulointerstitial fibrosis (TIF) is an inevitable pathway in the progression of chronic kidney disease (CKD) to end-stage renal disease. Here, the dynamic expression changes of eleven drug transporters in TIF kidney have been systematically investigated. Among them, the mRNA expressions of Oat1, Oat2, Oct1, Oct2, Oatp4C1 and Mate1 were down-regulated, while Oat3, Mrp2, Mrp4, Mdr1-α, Bcrp were up-regulated. Pearson correlation analysis was used to analyze the correlation between transporters and Creatinine (Cr), OCT2 and MATE1 showed a strong negative correlation with Cr. In contrast, Mdr1-α exhibited a strong positive correlation with Cr. In addition, the pharmacokinetics of cimetidine, ganciclovir, and digoxin, which were the classical substrates for OCT2, MATE1 and P-glycoprotein (P-gp), respectively, have been studied. These results reveal that changes in serum creatinine can indicate changes in drug transporters in the kidney, and thus affect the pharmacokinetics of its substrates, providing useful information for clinical use.
Introduction
Drug combination is a joint therapeutic scheme for the treatment of clinical diseases. However, the incidence of drug-drug interactions (DDIs) is remarkably increasing, resulting in a variety of adverse reactions, even threatening human life [1]. Drug transporters are one of the main targets for DDIs. Kidney tissue, the main excretory organ in the body, shows the distribution of drug transporters. Many drugs (including organic anion drugs, organic cationic drugs, and peptide drugs) are mediated by drug transporters concentrated in proximal renal tubules during renal excretion [2]. Once the expression of drug transporters changes, it binds to affect the pharmacokinetics of drugs. Therefore, the Food and Drug Administration and National Medical Products Administration of China have pointed out that eleven drug transporters in the kidneys, including organic anion transporter 1 (OAT1), organic anion transporter 1 (OAT3), organic anion transporter polypeptide 4C1 (OATP4C1), organic cation transporter (OCT2), P-glycoprotein (P-gp), breast cancer resistance protein (BCRP), multi-drug and toxin extrusion protein 1 (MATE1), multi-drug and toxin extrusion protein 2-K (MATE2-K), organic anion transporter 4 (OAT4), multidrug resistance-associated protein 2 (MRP2) and multidrug resistance-associated protein 4 (MRP4), need to be researched for drug applications [3,4].
CKD is widespread in the world, affecting nearly 13% of the population, and CKD has become a global public health problem [5]. According to the online data of the Centers for Disease Control and Prevention, the number of CKD deaths increased by about 12% from 2011 to 2018, ranking ninth in the top ten fatal diseases [6]. Tubulointerstitial fibrosis (TIF) is a common pathological change in CKD progression to end-stage renal disease [7,8]. With kidney damage, CKD is often accompanied by hypertension, cardiovascular disease, diabetes, and other complications. Therefore, combination therapy is a frequent method for patients with CKD [9,10]. In the clinic, the drug administration in patients with CKD is very cautious. Creatinine (Cr) is an endogenous substance that was filtered out through the glomerular [11]. Creatinine clearance (Ccr) is commonly used to evaluate renal function [12,13]. When the drug is eliminated, primarily by glomerular filtration, the clinical administration schedule could be adjusted according to the patient's Cr/Ccr under pathological conditions. However, there have been no clear reports on the changes in drug transporters excreted by drug transporters in vivo, the changes in drug transporters expression in TIF, and the relationship between Cr/Ccr and drug transporters. Glomerular filtration rate (GFR) and proteinuria are still widely used diagnostic indicators, but these two indicators occur late in the disease. Therefore, it is urgently needed to explore the relationship between new indicators and transporters.
Therefore, this study focuses on the relationship between kidney transporters and Cr/Ccr in unilateral urethral obstruction animal model, to provide useful data for the use of clinical drugs and drug combination.
The Renal Parameters in TIF Rats
To observe the dynamics of kidney tissue in TIF rats, the orbital blood and kidneys were harvested on the 4th, 7th, 10th, and 14th days after modeling in the model group. The renal structure was illustrated in Figure 1A. With the increase in modeling time, the right kidney of rats showed an obvious swelling, translucent epidermis, light color, cystic, containing brown turbid liquid. With the increase in modeling time, compared with the control group, the wet weight of the right kidney in the model group increased by approximately 1.51-3.05-fold, and reached the maximum value on the 14th day ( Figure 1B). With the increase in modeling time, the coefficient of right kidney of rats increased by about 1.017-2.507-fold compared with that of the left kidney ( Figure 1C). The measurement of Cr in serum revealed that Cr concentration increased 1.24-1.58 times with the increase in modeling time ( Figure 1D). On the contrary, with the increase in modeling time, Ccr decreased to 39.8-70.7% ( Figure 1E). In the anatomical model group on different days, the ligation kidney of rats was weighed, and the weight obtained was compared with that of the control group. (C) Renal index was calculated as the ratio of the weight of the left kidney and the ligation kidney to body weight. Data were expressed as mean ± SD. L: left kidney, R: ligated kidney. (D) Changes in Cr in serum concentration of rats at different modeling time, compared with the control group. (E) Changes in Ccr in serum concentration of rats at different modeling times, compared with the control group. **** p < 0.0001 *** p < 0.001, ** p < 0.01, * p < 0.05, ns p > 0.05.
Histopathological Findings
The H&E staining and Masson staining were employed to examine the pathological morphology of the tissues as well as fibrocyte collagen precipitation, respectively, as shown in Figure 2A. The H&E results showed that the kidneys in TIF rats exhibited glomerular fibrosis with cystic changes, glomerular enlargement with massive inflammatory cell infiltration, and widening of the renal interstitial space, with the increase in modeling time. They were then scored for pathological damage (Figure 2A), which showed that both kidney injuries increased as modeling time increased. Masson's results indicated that renal tubular dilatation, widening of the renal interstitial space, and an obvious increase in collagen fibers in the renal interstitium were seen in the obstructed side kidneys of the model group compared with the control group ( Figure 2B). As the modeling time increased, the fibrosis area increased by 12%, 19%, 35%, and 38% ( Figure 2B). Histopathological results. Sections of the right kidney of on rats at 4th, 7th, 10th, and 14th days were taken to H&E staining (A). Scale: 600 µm (200×). Histopathological changes in kidney sections were scored as a semi-quantitative percentage of damaged area: 0, normal; 1, cortical area <25%; 2, cortical area 25-50%; 3, the cortical area is 50-75%; 4, cortical area >75%, compared with control group. (B) Sections of the right kidney of on rats at 4th, 7th, 10th and 14th days were taken for Masson staining. Fibrosis area was quantified by Image J Pro Plus 6.0 compared with the control group. **** p < 0.0001 *** p < 0.001, ** p < 0.01.
The Correlation of Renal Transporters and Cr/Ccr in the Pathological State of Renal Fibers
Pearson correlation analysis was utilized to explore the relationship between renal transporter variation and Cr/Ccr under pathological conditions. The analysis result explained that Oct2 and Mate1 were highly negatively correlated with Cr (Pearson coefficient r >0.6, p ≤ 0.05), Among them, Oct2 (r = 0.624, p = 0.000061), Mate1 (r = 0.636, p = 0.0005), Oat2 (r = 0.414, p = 0.013) were moderately related, and the rest were all less than 0.3. Mdr1-α was positively correlated with correlation coefficients lower than 0.5 ( Figure 4A), such as Bcrp (r = 0.49, p = 0.012), which showed a medium relationship, but all others were less than 0.3 without a significant difference. The correlation between Ccr and transporters further confirmed these results. Oct2 (r = 0.601, p = 0.0011) Mate1 (r = 0.434, p = 0.0266), Mdr1-α (r = 0.440, p = 0.0244) ( Figure 4B). In brief, the above results showed that renal transporters were related to Cr and Ccr, and Oct2, Mate1 and Mdr1-α were strongly correlated. Real-time q-PCR analysis showed that the mRNA content of these transporters was 2 −∆∆Ct relative to the mRNA β-actin expression, and Pearson correlation was used to analyze the dynamic changes between the main kidney transporters and Cr. A correlation analysis of the relative size of 2 −∆∆Ct between the changed transporter and β-actin and Ccr was conducted. (B) The mRNA expression of transporter was detected on the 4th, 7th, 10th, and 14th days, and then the correlation between the expression value of transporter and the Ccr rate was analyzed.
The Correlation of Renal Transporters, Cr and Renal Fibers in the Pathological State of Renal Fibers
Further, we conducted a correlation analysis of the dynamic change in Cr and the degree of fibrosis, and the results exhibited that the degree of fibrosis was significantly positively correlated with the dynamic change of Cr (r = 0.736, p ≤ 0.05) ( Figure 5A). We also detected a relationship between transporters and the degree of renal fibrosis. This showed that Oct2 (r = 0.751, p = 0.0001), Mate1 (r = 0.744, p = 0.0002), Mdr1-α (r = 0.597, p = 0.0055) were highly correlated with fibrosis, which were consistent with that of Cr/Ccr ( Figure 5B).
PK of Renal OCT2, MATE1, P-gp Substrates in the TIF Rats
Oct2, Mate1 and Mdr1-α regulate the expression of OCT2, MATE1 and P-gp proteins in vivo. To determine the influence of changes in transporters on pharmacokinetic parameters under pathological conditions, three typical substrates for OCT2, MATE1 and P-gp were selected for pharmacokinetic studies. A methodological verification of the three drugs was conducted (Table 1, Table S1, Figure 6A-C) and the detection method met the methodological requirements. The results exhibited that the AUC of cimetidine (substrate of OCT2) in the model group increased 1.49 times compared with the control group ( Figure 6D). The value of renal clearance (Cl r ) in the model group decreased by 20.5%, which may be linked to the decreased expression of OCT2 protein in the kidney. Digoxin was a typical substrate of P-gp. Its AUC value reduced by 3.138-fold, while Cl r value increased by 2.6-fold, which might be related to the increased expression of P-gp in TIF rats. Ganciclovir is a substrate of MATE1. The AUC of ganciclovir decreased by 11.3%, while Cl r did not significantly change. The pharmacokinetics parameters did not significantly change when ganciclovir was combined with some MATE1 inhibitors or substrates, which may be related to other excretory pathways in vivo.
Discussion
The extensive literature suggests that the expression of kidney transporters in a pathological state will change, for example, under the rat liver ischemia-reperfusion model [14]. This will lead to the up-regulation of MRP and the down-regulation of OCT2, while, for hyperuricemia rats, in acute kidney injury, P-gp, MRP2 and other transporters will be significantly upregulated [15,16]. These changes may be due to the activation or induction of some upstream nuclear receptors under pathological conditions, such as PPAR-α and other nuclear receptors and transcription factors, thereby regulating the expression of downstream transporters [17,18], LXR and FXR are associated with Abcg1 gene and Abc-related protein expression, and its expression can cause changes in downstream transporters. PXR is associated with Slc-related protein expression and Abc-related protein expression, just like Mdr-1α and Slc16a1 [19,20].
Therefore, changes in the body or under certain inflammatory or pathological conditions may cause changes in the expression of some nuclear receptors in the pathway, thus leading to changes in the expression of other transporters [21]. In addition to the nuclear receptors referred to above, some inflammatory factors can also directly affect the expression of transporters. For example, TNF-α can inhibit the transcription of the tubule bile acid transporter Abcb11, bilirubin outlet Abcc2, and sterol transporter Abcg5/8 in intestinal inflammation, cholestasis, or the activation of hepatic macrophages, and thus affect the expression of transporters [22]. Therefore, the present study constructed a classical renal interstitial fibrosis model to explore the changes in renal transporter expression in rats under the TIF model.
Since Cr and Ccr are commonly used indicators to evaluate renal function, this experiment wanted to explore the change rule of Cr and Ccr and the expression of various transporters under the renal interstitial fibrosis model, and whether the expression changes in major transporters in kidney could be inferred through the detection of Cr and Ccr. Therefore, in this paper, the dynamic changes in transport proteins under the TIF model were related to Cr and Ccr by correlation analysis, and transport proteins were found that were highly correlated with Ccr. Transporter inhibitors are compounds that competitively bind or inhibit transporter activity [23,24]. Therefore, in the case of multi-disease combination, there will be interactions between drugs, such as P-gp [25,26], which has a variety of inducers in vivo, including antibacterial drug rifampicin, anti-tumor drug vincristine, doxorubicin, cardiovascular drug verapamil [27], hyperlipidemia drug atorvastatin [28], etc., which can induce the overexpression of P-gp in vivo. As a result, the pharmacokinetics parameters of drugs such as digoxin in vivo are significantly changed, while digoxin has a narrow treatment window, and the blood concentration of digoxin will be greatly reduced in a multi-drug combination, so that digoxin cannot play a therapeutic role. In many studies, the combination of naproxen and other agents with a typical OCT2 substrate (cimetidine) increased the plasma concentration of cimetidine, thereby separately increasing the toxicity of cimetidine [29]. Ganciclovir [30], its pharmacokinetic behavior in some studies [31,32], and part of its MATE1 inhibitors or substrate share, showed no significant change in pharmacokinetics parameters, which may be related to other excretory pathways in the body [33]. In addition, this paper also examines the ligation of the bilateral renal compensatory, where the transporter will affect the elimination of the substrate, and the results showed that the left kidney transporter expression showed no obvious change. We also considered the effect of absorption of drug excretion, in view of the selected sev-eral drugs in the clinic, which are mainly for oral use, and choosing the means of lavage for pharmacokinetics validation. In addition, the specific transporters OCT2, Mate1 and MDR1-α showed a high correlation with renal fibrosis. Therefore, we can deduce the renal fibrosis process from indicators such as blood creatinine/creatinine clearance. Further research will continue to focus on this aspect and deeply explore the mechanism of renal transporter expression changes under pathological conditions.
The study has several advantages and limitations. The advantages include the simplicity pf Cr in serum and Ccr, which can introduce a change in the transporter, as well as the transporter excretion of drug medication guides, without the need for a kidney biopsy. The first limitation is the change in renal fiber and Ccr constant transporter. It is unknown whether his drugs change, as their pharmacokinetics parameters were not studied. Second, the study used TIF rats in the 14th-day group, without considering the changes in pharmacokinetics parameters in other groups. In this study, only rats were used for transport experience, so this was not verified in clinical patients. Therefore, the results of this study may not be applicable to the whole population. As the next step, we will continue to supplement pharmacokinetics experiments to study whether the pharmacokinetics of substrates of several other transporters with a low correlation with Ccr will change, and verify this using in vitro experiments.
Chemicals and Regents
Chloral hydrate (≥99% in purity) was provided by Guangzhou Youbang Biotechnology Co., Ltd. (Guangzhou, China). Cimetidine (≥99% in purity) and irbesartan (internal standard, >98% in purity) ware purchased from Shenzhen upno Biomedical Technology Co., Ltd. (Shenzhen, China). The kit for analysis of blood urea nitrogen (BUN) and Cr was purchased from Nanjing Jiancheng Bioengineering Institute (Nanjing, China). The animal total RNA isolation kit was provided by Foregene Co., Ltd. (Chengdu, China). All other chemical reagents were of chromatographic or analytical grade and were commercially available.
Animals
Healthy male Sprague-Dawley rats (SD rats, aged 7-8 weeks, weight 180-220 g, certification: SCXK-Yue-2016-0041) in specific pathogen-free grade were available from the Experimental Animal Center of Southern Medical University (Guangzhou, China). All experiments followed the National Institutes of Health Guidelines for the Care and Use of Laboratory Animals. All animals were housed in an air-conditioned room with the temperature at 23 ± 2 • C and a relative humidity of 40 ± 5%, under an alternating 12 h dark/light cycle. Animals had free access to food and water throughout the experiment.
Animal Experiment
Thirty SD rats were randomly divided into five groups (n = 6): control and TIF model groups analyzed on the 4th, 7th, 10th and 14th days. The rats in the model group were intraperitoneally anesthetized with 10% chloral hydrate at a dose of 0.3 mL/100 g [34]. Surgery was carried out as previously described. In the model group, the right ureter was exposed and ligated at two points with a 5-0 sterile suture along the lower pole of the right kidney. Then, the ureter was cut to prevent retrograde infection [35]. Two hours later, they were intraperitoneally injected with penicillin (1.6 million units dissolved in 8 mL of normal saline) for two consecutive days. Each rat was subcutaneously injected with 0.30 mL. On the 4th, 7th, 10th and 14th days after operation, blood was taken from the orbit of TIF rats. Urine was taken from the TIF rats placed in the metabolic cage for 24h. Heart, liver, spleen, lung, kidney, intestine and other tissues were dissected. The kidney tissues were weighed and the ratio of kidney weight to body weight was estimated. The right kidney was longitudinally reduced and fixed with paraformaldehyde. The rest was used only for a real-time quantitative polymerase chain reaction (RT-qPCR).
Histology Analysis
The kidney tissue was longitudinally cut, rinsed several times with cold PBS, and fixed overnight with 4% paraformaldehyde. Then, five pieces were cut out after paraffin embedding of the µM section. The renal tissue was stained with H & E at low power (10 × 10) The observation site was determined under a high-power microscope (10 × 20 and 10 × 40) and the target field of vision was selected to take 1-2 pictures. In H & E staining, the degree of renal injury was determined according to the size of glomeruli and the changes in renal tubules [36].
Masson staining: Image Pro Plus 6.0 software was used for quantitative analysis. The degree of renal interstitial fibrosis was evaluated based on the amount of collagen deposition (the percentage of the blue area in the whole cortex). Five different cortical fields were randomly selected from each slice (magnification 200 times). The area of fibrotic lesions was expressed as the percentage of fibrotic area in the whole cortex [37,38].
Detection of mRNA Expression: RT-qPCR
A total of 10-20 mg renal tissue samples were collected into the homogenization tube. Total RNA was extracted according to the protocol of animal tissue total RNA Extraction Kit (Foregene, Chengdu, China). A total of 1000 ng of total RNA was reverse-transcribed into cDNA using Evo m-mlv reverse transcription reagent (Accurate, Shenzhen, China). All subsequent RT-qPCR reactions were performed using 2 × Accurattaq Master Mix (Accurate, China), primer (designed and synthesized by Guangzhou Branch of Beijing Qingke Biotechnology Co., Ltd., Guangzhou, China, Table S2), ddH2O without ribonuclease, reaction volume 20 µL. The PCR was conducted on a rapid real-time PCR system (7500, Thermo Fisher Science, Waltham, MA, USA). At 50 • C The results were analysed under the conditions of C reaction for 3 min, 95 • C reaction for 3 min, 95 • C reaction for 10 s, and 60 • C reaction for 30 sec. The threshold period (CT) was recorded with 7500 fast system software version 2.3, and the multiple changes in mRNA expression were calculated according to the comparative CT method.
Pharmacokinetic Analysis
Thirty rats were split into six groups (n = 5). The rat model of TIF was established by unilateral ureteral obstruction surgical operation under sterile conditions according to previous research. On the 14th day after establishment of the model group, cimetidine (18 mg/kg), ganciclovir (45 mg/kg), and digoxin (5 mg/kg) were given orally in each group, respectively [39]. Blood samples were collected from the retroorbital sinus at the 0 min, 5 min, 15 min, 30 min, 45 min, 90 min, 120 min, 240 min, 360 min, 480 min, 720 min and 1440 min timepoints, and centrifuged immediately after collection (5000 rpm, 8 min), The obtained plasma was stored at −80 • C before any pharmacokinetics analysis. The plasma concentration of cimetidine in SD rats was established by high-performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS).
In a positive ion mode, an API 4000 triple quadrupole tandem mass spectrometer (SCIEX, Framingham, MA, USA) with an ESI (AB SCIEX, Framingham, MA, USA) source was used, and the acquisition and analysis of data were carried out with Analyst 1.6.2 software (Applied Biosystems, Foster City, CA, USA). Multiple reaction monitoring (MRM) parameters for the ganciclovir, cimetidine, digoxin and ebesartan (internal standard, IS) were optimized and are summarized in Table S3. The other ionization parameters were as follows: curtain gas, 20 psi; collision gas, 6 psi; ion source gas 1, 50 psi; ion source gas 2, 50 psi, respectively, with a temperature of 500 • C and an ion spray needle voltage of 5500 V The bioanalytical method validation guidance for industry released by the FDA in 2018 was used to validate the analytical approach used in this study. The selectivity, specificity, accuracy, matrix effects, stability, served as key metrics to affirm the validity of this method [40].
Statistical Analyses
The experimental data were analyzed by Graphpad prism software (San Diego, CA, USA), and the mean value was calculated ± standard deviation (SD). The differences between groups (p < 0.05 and p < 0.01) were analyzed by SPSS 20.0. Dunnett multiple comparison test or LSD test were used for multiple comparison, p < 0.05 was regarded as statistically significant. The results of LC-MS/MS were analyzed by Das 2.0 software. Ccr was calculated with the following formula.
Conclusions
In conclusion, this experiment explored the relationship between major kidney transporters and creatinine, creatinine clearance, and renal fibrosis area. The development of modeling time in the TIF pathological model of rats was studied to infer the relationship between creatinine, creatinine clearance and kidney transporters. The results showed that OCT2 and MATE1 were negatively correlated with creatinine and fibrosis area, and positively correlated with creatinine clearance, while P-gp showed the opposite results. Therefore, we think that Cr/Ccr can be used to infer the transporter expression and renal fibrosis process. Using typical substrates for pharmacokinetic studies, the research results show that, with OCT2 lower expression, substrate cimetidine pharmacokinetic parameters show obvious changes in the body, with a notable rise in AUC and Cmax, while Clr was significantly down-regulated, suggesting that cimetidine excretion was significantly slowed in the TIF model. MATE1 and P-gp substrates showed the opposite results. Therefore, we believe that Cr/Ccr can be used as an indicator of OCT2, MATE1 and P-gp transporter expression, and its changes are significantly correlated with OCT2, MATE1 and P-gp changes, providing data and references for clinical renal disease patients in clinical medication. : Table S1: Methodology of Ganciclovir, Cimetidine and Digoxin, Table S2: The primer sequences of target genes and β-actin, Table S3: Optimized MRM parameters for analytes and IS. | v3-fos-license |
2022-11-20T16:07:28.202Z | 2022-11-01T00:00:00.000 | 253696074 | {
"extfieldsofstudy": [],
"oa_license": "CCBY",
"oa_status": "GOLD",
"oa_url": "https://www.mdpi.com/1422-0067/23/22/14297/pdf?version=1668762465",
"pdf_hash": "473f44f0451180ce890ce04941535dd5c2b10ff0",
"pdf_src": "PubMedCentral",
"provenance": "20241012_202828_00062_s4wg2_01a5a148-7d10-4d24-836e-fe76b72970d6.zst:14",
"s2fieldsofstudy": [
"Biology",
"Chemistry"
],
"sha1": "7ad731214609b3ab5930190cab73ad1c5256a774",
"year": 2022
} | pes2o/s2orc | Potent Activation of Human but Not Mouse TRPA1 by JT010
Transient receptor potential (TRP) ankyrin repeat 1 (TRPA1), which is involved in inflammatory pain sensation, is activated by endogenous factors, such as intracellular Zn2+ and hydrogen peroxide, and by irritant chemical compounds. The synthetic compound JT010 potently and selectively activates human TRPA1 (hTRPA1) among the TRPs. Therefore, JT010 is a useful tool for analyzing TRPA1 functions in biological systems. Here, we show that JT010 is a potent activator of hTRPA1, but not mouse TRPA1 (mTRPA1) in human embryonic kidney (HEK) cells expressing hTRPA1 and mTRPA1. Application of 0.3–100 nM of JT010 to HEK cells with hTRPA1 induced large Ca2+ responses. However, in HEK cells with mTRPA1, the response was small. In contrast, both TRPA1s were effectively activated by allyl isothiocyanate (AITC) at 10–100 μM. Similar selective activation of hTRPA1 by JT010 was observed in electrophysiological experiments. Additionally, JT010 activated TRPA1 in human fibroblast-like synoviocytes with inflammation, but not TRPA1 in mouse dorsal root ganglion cells. As cysteine at 621 (C621) of hTRPA1, a critical cysteine for interaction with JT010, is conserved in mTRPA1, we applied JT010 to HEK cells with mutations in mTRPA1, where the different residue of mTRPA1 with tyrosine at 60 (Y60), with histidine at 1023 (H1023), and with asparagine at 1027 (N1027) were substituted with cysteine in hTRPA1. However, these mutants showed low sensitivity to JT010. In contrast, the mutation of hTRPA1 at position 669 from phenylalanine to methionine (F669M), comprising methionine at 670 in mTRPA1 (M670), significantly reduced the response to JT010. Moreover, the double mutant at S669 and M670 of mTRPA1 to S669E and M670F, respectively, induced slight but substantial sensitivity to 30 and 100 nM JT010. Taken together, our findings demonstrate that JT010 potently and selectively activates hTRPA1 but not mTRPA1.
The chemical compound JT010, 2-chloro-N-(4-(4-methoxyphenyl)thiazol-2-yl)-N-(3methoxypropyl-acetamide, developed by Takaya et al., potently and selectively activates TRPA1 [18]. The half maximum concentration required for TRPA1 activation was reported to be 0.65 nM when applied to human embryonic kidney (HEK) cells expressing human TRPA1 (hTRPA1). In contrast, JT010, even at 1 µM, did not activate TRP vanilloid family type 1, 3, and 4 (TRPV1, TRPV3, TRPV4), TRP melastatin family type 2 and 8 (TRPM2, TRPM8), and TRP canonical family type 5 (TRPC5), suggesting that JT010 is a useful TRPA1 activator as a potential pharmacological tool. Several TRPA1 cysteine residues are targeted by physiological and non-physiological electrophilic compounds that activate the channel [2,19]. Particularly, recent studies have revealed that as a two-step model, cysteine 621 (C621) is critical for channel activation by electrophilic compounds and cysteine 665 (C665) is supportive and important for activation [20,21]. Indeed, hTRPA1 activation by 9,10-PQ is dependent on both C621 and C665 at the N-terminus of the channel [16]. It has also been shown that the C621 mutation in hTRPA1 prevents channel activation by JT010 [18]. Moreover, phenylalanine 669 (F669) is critical for the binding of JT010 to hTRPA1 [22]. Meanwhile, it has been proposed that the attachment of a large electrophile, such as JT010, to C621 is sufficient for the full activation of the channel. Moreover, confirmation of the binding pocket is supported by the interaction between lysine 671 (K671) and the C terminus of the TRP helix of TRPA1 [21]. In contrast, non-electrophilic compounds such as ∆9-tetrahydrocannabinol, nicotine, and menthol activate TRPA1 via different mechanisms [1,23,24]. Therefore, pharmacological tools are important to understand the physiological functions of TRPA1, and extensive efforts have been made to develop highly selective TRPA1 agonists and antagonists.
In this study, we showed that a potent TRPA1 agonist, JT010, at concentrations under 100 nM can activate hTRPA1 but not mTRPA1. A comparison of the JT010-induced response of wild-type and mutant TRPA1s in humans and mice in response to allyl isothiocyanate (AITC) revealed that the mutation of hTRPA1 at position 669 from phenylalanine to methionine (F669M) reduced the sensitivity to JT010, and the double mutation at S669 (serine) and M670 (methionine) of mTRPA1 to glutamine (S669E) and phenylalanine (M670F), respectively, induced weak but substantial sensitivity to JT010. Treatment with JT010 also effectively activated endogenous hTRPA1 in human fibroblast-like synoviocytes (FLSs) with inflammation, but not mTRPA1 in dorsal root ganglion (DRG) cells isolated from mice. Our findings provide novel and important pharmacological evidence that JT010 is a much weaker TRPA1 agonist in mice than in humans.
Results
To confirm the expression of wild-type and mutant TRPA1s in human embryonic kidney (HEK) cells, the TRPA1 channel function was tested by applying AITC at the end of each experiment, except during the application of high JT010 concentrations [25]. Additionally, we confirmed the expression of wild-type and mutant hTRPA1 by Western blotting at the protein level (Supplementary Figure S1A) but failed to find any specific antibody against mTRPA1 (three different antibodies used, not shown). Using HEK cells expressing wild-type hTRPA1 (HEK-hTRPA1), we examined the effects of JT010 on hTRPA1. As shown in Figure 1A, treating HEK-hTRPA1 cells with JT010 at concentrations ranging from 0.3 to 100 nM effectively induced a Ca 2+ response, whereas the treatment failed to evoke any Ca 2+ response in control HEK cells (Supplementary Figure S1B). The doseresponse relationship observed indicated that the half maximum concentration (EC 50 ) required for 50% response was approximately 10 nM ( Figure 1B), suggesting that JT010 is a potent TRPA1 agonist [18]. To confirm whether JT010 also induces the Ca 2+ response of mTRPA1, we applied JT010 to HEK cells expressing wild-type mTRPA1 (HEK-mTRPA1). Surprisingly, applying 10 nM JT010 induced a small Ca 2+ response of mTRPA1 compared with that in control HEK cells ( Figure 1D vs. Supplementary Figure S1B), whereas 100 µM AITC elicited a large response of mTRPA1 and hTRPA1 ( Figure 1C,D). Application of much higher JT010 (1000 nM) concentrations induced a moderate Ca 2+ response of mTRPA1 (Supplementary Figure S1C), suggesting that mTRPA1 is much less sensitive to JT010 than hTRPA1. . At the end of each experiment, 100 μM AITC was applied to confirm hTRPA1 expression. (C,D) JT010 and AITC at 10 nM and 100 μM, respectively, were applied to HEK-hTRPA1 and HEK-mTRPA1 cells, and the measured Ca 2+ response (C) and the peak JT010-and AITC-induced Ca 2+ response (five independent experiments each) (D) are summarized. Two-way analysis of variance (ANOVA): * p = 0.0279, F = 5.85 (species); ** p < 0.0001, F = 84.5 (drugs); * p = 0.0312, F = 5.58 (interaction). Vertical bars = SEM.
To further examine whether JT010 potently activates hTRPA1 but not mTRPA1, we applied 10-100 nM JT010 to HEK-hTRPA1 and HEK-mTRPA1 cells in whole-cell recording mode ( Figure 2). To maintain TRPA1 channel activity during recording, we applied chemical agents in the absence of external Ca 2+ in the standard HEPES-buffered bathing solution (SBS) and the presence of internal Ca 2+ at 0.3 μM in a pipette solution [16]. As shown in Figure 2A-C, exposure of HEK-hTRPA1 cells to JT010 induced inward and outward currents at −90 and +90 mV, respectively, in a concentration-dependent manner. Moreover, a TRPA1 antagonist, A-967079 (A96), at 5 μM abolished JT010-induced currents, demonstrating that JT010 potently activated hTRPA1 channel currents. In contrast, the application of JT010 to HEK-mTRPA1 cells did not induce any clear mTRPA1 channel currents, while 30 μM AITC markedly induced currents at −90 and +90 mV, sensitive to A96 ( Figure 2D-F). Moreover, to exclude the possibility that relatively higher intracellular Ca 2+ (0.3 μM) modifies the effects of JT010 on mTRPA1, HEK cells were internally superfused with only 1 mM EGTA without Ca 2+ (Supplementary Figure S2). Even under these experimental conditions, JT010 did not induce mTRPA1 activation but effectively activated hTRPA1. These results suggest that JT010 is a potent hTRPA1 agonist, but not mTRPA1. . At the end of each experiment, 100 µM AITC was applied to confirm hTRPA1 expression. (C,D) JT010 and AITC at 10 nM and 100 µM, respectively, were applied to HEK-hTRPA1 and HEK-mTRPA1 cells, and the measured Ca 2+ response (C) and the peak JT010-and AITC-induced Ca 2+ response (five independent experiments each) (D) are summarized. Two-way analysis of variance (ANOVA): * p = 0.0279, F = 5.85 (species); ** p < 0.0001, F = 84.5 (drugs); * p = 0.0312, F = 5.58 (interaction). Vertical bars = SEM.
To further examine whether JT010 potently activates hTRPA1 but not mTRPA1, we applied 10-100 nM JT010 to HEK-hTRPA1 and HEK-mTRPA1 cells in whole-cell recording mode ( Figure 2). To maintain TRPA1 channel activity during recording, we applied chemical agents in the absence of external Ca 2+ in the standard HEPES-buffered bathing solution (SBS) and the presence of internal Ca 2+ at 0.3 µM in a pipette solution [16]. As shown in Figure 2A-C, exposure of HEK-hTRPA1 cells to JT010 induced inward and outward currents at −90 and +90 mV, respectively, in a concentration-dependent manner. Moreover, a TRPA1 antagonist, A-967079 (A96), at 5 µM abolished JT010-induced currents, demonstrating that JT010 potently activated hTRPA1 channel currents. In contrast, the application of JT010 to HEK-mTRPA1 cells did not induce any clear mTRPA1 channel currents, while 30 µM AITC markedly induced currents at −90 and +90 mV, sensitive to A96 ( Figure 2D-F). Moreover, to exclude the possibility that relatively higher intracellular Ca 2+ (0.3 µM) modifies the effects of JT010 on mTRPA1, HEK cells were internally superfused with only 1 mM EGTA without Ca 2+ (Supplementary Figure S2). Even under these experimental conditions, JT010 did not induce mTRPA1 activation but effectively activated hTRPA1. These results suggest that JT010 is a potent hTRPA1 agonist, but not mTRPA1. Next, we examined the effects of JT010 on the TRPA1 channels expressed in human and mouse tissues. As inflammatory stimulation with interleukin-1α (IL-1α) transcriptionally induces TRPA1 expression in human FLSs [26,27], we applied JT010 and AITC to human FLSs with or without inflammation. As shown in Figure 3A, both 10 nM JT010 and 100 μM AITC induced substantial Ca 2+ responses in inflammatory FLSs treated with IL-1α for 24 h, but not in control FLSs (vehicle). FLSs sensitive to AITC largely responded to JT010 (74%, 37 cells out of 50 cells) and vice versa (0%, 0 cells out of 40 cells), suggesting that JT010 can activate endogenous hTRPA1 channels in human tissues. In contrast, mouse DRGs sensitive to 100 μM AITC (40 cells out of 45 cells) did not respond to 10 nM Next, we examined the effects of JT010 on the TRPA1 channels expressed in human and mouse tissues. As inflammatory stimulation with interleukin-1α (IL-1α) transcriptionally induces TRPA1 expression in human FLSs [26,27], we applied JT010 and AITC to human FLSs with or without inflammation. As shown in Figure 3A, both 10 nM JT010 and 100 µM AITC induced substantial Ca 2+ responses in inflammatory FLSs treated with IL-1α for 24 h, but not in control FLSs (vehicle). FLSs sensitive to AITC largely responded to JT010 (74%, 37 cells out of 50 cells) and vice versa (0%, 0 cells out of 40 cells), suggesting that JT010 can activate endogenous hTRPA1 channels in human tissues. In contrast, mouse DRGs sensitive to 100 µM AITC (40 cells out of 45 cells) did not respond to 10 nM JT010 (0 cells out of 40 cells), implying that JT010 at less than 10 nM could not activate endogenous mTRPA1 channels in mouse tissue ( Figure 3B). JT010 (0 cells out of 40 cells), implying that JT010 at less than 10 nM could not activate endogenous mTRPA1 channels in mouse tissue ( Figure 3B). As previously reported [18,22], the substitution of cysteine in hTRPA1 at 621 (C621, Figure 4A) with serine (C621S) markedly reduced the response to 10 nM JT010, but not 100 μM AITC ( Figure 4B-D, see also Figure 1B as the control), confirming that C621 is critical for JT010-induced TRPA1 response in humans. This C621 is conserved in mTRPA1 (C622, Figure 4A); therefore, we explored the insensitivity mechanism of mTRPA1 against JT010 using mutant mTRPA1s (Figures 4-6). First, we focused on three cysteines in hTRPA1 that are not conserved in mTRPA1 (Y60, H1023, and N1027, arrow in Figure 4A). To test the possible involvement of cysteines in the sensitivity to JT010, we mutated these residues to cysteine (Y60C, H1023C, and N1027C) and applied JT010 to each mutant. None of these mutants were sensitive to JT010 at 10 nM, whereas all responded to 100 μM AITC ( Figure 4B-D), indicating that these mTRPA1 residues may not determine the responsiveness to JT010. Effects of JT010 on endogenous TRPA1 in human and mouse cells. (A) JT010 at 10 nM and AITC at 100 µM were applied to human FLSs with or without inflammation. FLSs were treated with 10 U IL-1α or vehicle for 24 h and then exposed to JT010 and AITC, and Ca 2+ response was monitored ((A), each representative cell). The peak JT010-and AITC-induced Ca 2+ response (∆ratio) in FLSs with or without IL-1α (six independent experiments each) are summarized in the lower panel. Two-way ANOVA: ** p < 0.0001, F = 167 (pretreatments); ** p < 0.0001, F = 27.0 (drugs); ** p = 0.00015, F = 21.7 (interaction) (B) JT010 at 10 nM and AITC at 100 µM were applied to mouse DRGs, and Ca 2+ response was monitored ((B), a representative cell). The peak JT010-and AITC-induced Ca 2+ response (∆ratio) in DRGs are summarized (lower panel, five independent experiments). Paired Student's t-test: ## p = 0.00042. Vertical bars = SEM.
As previously reported [18,22], the substitution of cysteine in hTRPA1 at 621 (C621, Figure 4A) with serine (C621S) markedly reduced the response to 10 nM JT010, but not 100 µM AITC ( Figure 4B-D, see also Figure 1B as the control), confirming that C621 is critical for JT010-induced TRPA1 response in humans. This C621 is conserved in mTRPA1 (C622, Figure 4A); therefore, we explored the insensitivity mechanism of mTRPA1 against JT010 using mutant mTRPA1s (Figures 4-6). First, we focused on three cysteines in hTRPA1 that are not conserved in mTRPA1 (Y60, H1023, and N1027, arrow in Figure 4A). To test the possible involvement of cysteines in the sensitivity to JT010, we mutated these residues to cysteine (Y60C, H1023C, and N1027C) and applied JT010 to each mutant. None of these mutants were sensitive to JT010 at 10 nM, whereas all responded to 100 µM AITC ( Figure 4B-D), indicating that these mTRPA1 residues may not determine the responsiveness to JT010. Comparison of JT010-induced TRPA1 response among mutants of N-and C-terminal cysteine residues of hTRPA1 and mTRPA1. (A) Alignment of amino acid sequence between hTRPA1 and mTRPA1. C621 and C665 in hTRPA1 (homologous to mTRPA1 C622 and C666) shown by boxes indicate critical cysteines for electrophilic TRPA1 agonist modification. Bold and underlined letters (C59, C1021, C1025 in human, indicated by an arrow) show cysteines substituted in mutant mTRPA1 (Y60C, H1023C, N1027C), whose effect was examined. Yellow color boxes indicate potential critical amino acids for JT010-sensitivity, whose importance is examined in Figures 5 and 6. (B-D) Ca 2+ responses of mutant hTRPA1 with C621S mutation and mutant mTRPA1s with Y60C, H1023C, and N1027C mutations to 10 nM JT010. To confirm the channel expression, 100 μM AITC was applied at the end of the experiment. Each representative Ca 2+ response was superimposed (B) and the peak JT010-and AITC-induced Ca 2+ response (Δratio) is summarized (C, five independent experiments). Paired Student's t-test: ** p < 0.0001, ** p = 0.00092, ** p < 0.0001, and ** p = 0.00064 for hC621S, mY60C, mH1023C, and mN1027C, respectively (D) Ca 2+ response to JT010 was normalized with that to AITC and is summarized. The responses of wild hTRPA1 and mTRPA1 were also included as a comparison (the same data set as Figure 1). Unpaired Student's t-test: ## p < 0.0001. The 'ns' shows no significance by the Tukey-Kramer test. Vertical bars = SEM.
A recent structural analysis of hTRPA1 revealed that the phenylalanine residue at position 669 (F669 shown in yellow in Figure 4A) of hTRPA1 is critical for channel activation by JT010 [22]. Indeed, this phenylalanine is not conserved in mTRPA1 (M670 in mouse; Figure 4A). In addition, the glutamate residue at position 668 (E668 shown in yellow in Fig4A) of hTRPA1 is substituted with serine in mTRPA1 (S669, Figure 4A). Therefore, we examined the involvement of these amino acid residues with different sensitivities to JT010 between hTRPA1 and mTRPA1. As shown in Figure 5A-C, the F669M mutation in hTRPA1 significantly reduced the sensitivity to JT010, but not to AITC, suggesting that M670 in mTRPA1 renders a lower sensitivity to JT010. Particularly, the mutant hTRPA1 with F669M was insensitive to 10 nM JT010 ( Figure 5C). To further confirm the importance of F669, we applied JT010 to mTRPA1 with M670F mutation and double mutants with S669E and M670F mutations ( Figure 6A,B). As 30 μM AITC induced a similar size of TRPA1 currents at +90 and −90 mV, the response of these mutants to JT010 did not differ from that of wild mTRPA1 ( Figure 6C). Furthermore, the relative change in JT010induced TRPA1 currents was analyzed to normalize the current amplitude against the (Y60C, H1023C, N1027C), whose effect was examined. Yellow color boxes indicate potential critical amino acids for JT010-sensitivity, whose importance is examined in Figures 5 and 6. (B-D) Ca 2+ responses of mutant hTRPA1 with C621S mutation and mutant mTRPA1s with Y60C, H1023C, and N1027C mutations to 10 nM JT010. To confirm the channel expression, 100 µM AITC was applied at the end of the experiment. Each representative Ca 2+ response was superimposed (B) and the peak JT010-and AITC-induced Ca 2+ response (∆ratio) is summarized (C, five independent experiments). Paired Student's t-test: ** p < 0.0001, ** p = 0.00092, ** p < 0.0001, and ** p = 0.00064 for hC621S, mY60C, mH1023C, and mN1027C, respectively (D) Ca 2+ response to JT010 was normalized with that to AITC and is summarized. The responses of wild hTRPA1 and mTRPA1 were also included as a comparison (the same data set as Figure 1). Unpaired Student's t-test: ## p < 0.0001. The 'ns' shows no significance by the Tukey-Kramer test. Vertical bars = SEM.
A recent structural analysis of hTRPA1 revealed that the phenylalanine residue at position 669 (F669 shown in yellow in Figure 4A) of hTRPA1 is critical for channel activation by JT010 [22]. Indeed, this phenylalanine is not conserved in mTRPA1 (M670 in mouse; Figure 4A). In addition, the glutamate residue at position 668 (E668 shown in yellow in Fig4A) of hTRPA1 is substituted with serine in mTRPA1 (S669, Figure 4A). Therefore, we examined the involvement of these amino acid residues with different sensitivities to JT010 between hTRPA1 and mTRPA1. As shown in Figure 5A-C, the F669M mutation in hTRPA1 significantly reduced the sensitivity to JT010, but not to AITC, suggesting that M670 in mTRPA1 renders a lower sensitivity to JT010. Particularly, the mutant hTRPA1 with F669M was insensitive to 10 nM JT010 ( Figure 5C). To further confirm the importance of F669, we applied JT010 to mTRPA1 with M670F mutation and double mutants with S669E and M670F mutations ( Figure 6A,B). As 30 µM AITC induced a similar size of TRPA1 currents at +90 and −90 mV, the response of these mutants to JT010 did not differ from that of wild mTRPA1 ( Figure 6C). Furthermore, the relative change in JT010-induced TRPA1 currents was analyzed to normalize the current amplitude against the control before application of JT010 for each TRPA1 ( Figure 6D). The single M670F mutation in mTRPA1 (M670F-mTRPA1) was not sufficient to induce JT010-sensitivity. However, the double mutations of S669E and M670F induced weak but substantial sensitivity to 30 and 100 nM JT010 at +90 mV and 100 nM at −90 mV ( Figure 6D). Nevertheless, the potency of this double mutant against JT010 was much lower than that of F669M-and wild-hTRPA1 ( Figure 6D vs. Supplementary Figure S3). control before application of JT010 for each TRPA1 ( Figure 6D). The single M670F mutation in mTRPA1 (M670F-mTRPA1) was not sufficient to induce JT010-sensitivity. However, the double mutations of S669E and M670F induced weak but substantial sensitivity to 30 and 100 nM JT010 at +90 mV and 100 nM at −90 mV ( Figure 6D). Nevertheless, the potency of this double mutant against JT010 was much lower than that of F669M-and wild-hTRPA1 ( Figure 6D vs. Supplementary Figure S3).
Discussion
In this study, we showed that the potent TRPA1 agonist JT010, which activated hTRPA1 at a concentration range of 0.3 to 100 nM, did not induce clear responses of mTRPA1 in HEK cells with heterologous expression of the channel. In contrast, both hTRPA1 and mTRPA1 showed similar responses to the conventional TRPA1 agonist, AITC. Moreover, JT010 induced the Ca 2+ response of endogenous TRPA1 in human FLSs with inflammation but not in mouse DRG cells. As reported, substitution of F669 in the N-terminus of hTRPA1 to methionine, homologous to mTRPA1 methionine at 670, (F669M-hTRPA1) significantly reduced the response to JT010. In contrast, while a single M670F mutation in mTRPA1 was still insensitive to JT010, the double mutant of mTRPA1 with S669E and M670F mutations induced a weak but substantial response to JT010. Taken together, JT010 is a potent TRPA1 agonist in humans, but not in mice.
We confirmed that JT010, a potent TRPA1 agonist, is an effective hTRPA1 agonist. In experiments measuring Ca 2+ responses and membrane ionic currents, 10-100 nM JT010 Figure 6. S669 and M670 are the potential amino acids that determine the low sensitivity of mTRPA1 to JT010. Cells were superfused with SBS without Ca 2+ and dialyzed with a Cs-aspartate-rich pipette solution including 0.3 µM Ca 2+ . Ramp waveform voltage pulses from −110 to +90 mV for 300 ms were applied every 5 s. (A,B) JT010 was commutatively applied to HEK cells with an M670F substitution in mTRPA1 (A, 670F-mTRPA1) and double substitutions of S669E and M670F (B, 669E, 670F-mTRPA1) to examine the effects on membrane currents at −90 and +90 mV, and the pooled data of the peak currents evoked are summarized (C, four to six independent experiments including the same data set as in Figure 2F). After applying 100 nM JT010, 5 µM A96 was added to block the TRPA1 channel current components. For comparison, 30 µM AITC was used. In the middle and right panels of (A,B), the I-V relationships under each experimental condition are shown. (D) Each current amplitude shown in (C) was normalized to that of the control without JT010 and exhibited the relative amplitude change under each treatment. Dunnett's multiple comparisons test was performed for each TRPA1 gene. * p = 0.0116 and ** p = 0.00829 for 30 and 100 nM JT010, respectively in 669E, 670F-mTRPA1 (+90 mV). * p = 0.0327 for 100 nM JT010 in 669E, 670F-mTRPA1 (−90 mV). Vertical bars = SEM.
Discussion
In this study, we showed that the potent TRPA1 agonist JT010, which activated hTRPA1 at a concentration range of 0.3 to 100 nM, did not induce clear responses of mTRPA1 in HEK cells with heterologous expression of the channel. In contrast, both hTRPA1 and mTRPA1 showed similar responses to the conventional TRPA1 agonist, AITC. Moreover, JT010 induced the Ca 2+ response of endogenous TRPA1 in human FLSs with inflammation but not in mouse DRG cells. As reported, substitution of F669 in the N-terminus of hTRPA1 to methionine, homologous to mTRPA1 methionine at 670, (F669M-hTRPA1) significantly reduced the response to JT010. In contrast, while a single M670F mutation in mTRPA1 was still insensitive to JT010, the double mutant of mTRPA1 with S669E and M670F mutations induced a weak but substantial response to JT010. Taken together, JT010 is a potent TRPA1 agonist in humans, but not in mice.
We confirmed that JT010, a potent TRPA1 agonist, is an effective hTRPA1 agonist. In experiments measuring Ca 2+ responses and membrane ionic currents, 10-100 nM JT010 induced hTRPA1-dependent responses. Indeed, JT010 did not elicit a Ca 2+ response in native HEK (Supplementary Figure S1B). Moreover, it has been reported that JT010, even at 1 µM, does not activate TRPV1, TRPV3, TRPV4, TRPM2, TRPM8, and TRPC5 [18]. While the EC 50 of JT010 against hTRPA1 was 0.65 nM in a cell-based calcium uptake assay, it was~7.6 nM in an electrophysiological study [18,22]. Comparing the pharmacological features of compounds using different assays can be challenging. In this study, the EC 50 of JT010 was estimated to be 3-10 nM against hTRPA1 in Ca 2+ measurements ( Figure 1B) and electrophysiological assays (Figures 2 and 5), strongly supporting that JT010 is a potent hTRPA1 agonist. Moreover, neither 10 nM JT010 nor 100 µM AITC elicited a Ca 2+ response in human FLSs without inflammation. In contrast, both agonists evoked clear responses in the inflammatory FLSs, implying that endogenous TRPA1 in humans can be targeted by JT010. Consistently, injection of JT010 caused pain in humans with a half-maximal effective concentration of 0.31 µM [28], suggesting that JT010 is an effective TRPA1 agonist in vivo in human. In contrast, it has not been determined that JT010 is a weak TRPA1 agonist in vivo in rodents including mouse.
In this study, we confirmed that C621 and F669 are critical for the JT010-induced hTRPA1 activation. When we applied 10 nM JT010 to mutant hTRPA1 with C621S mutation, the Ca 2+ response was abolished ( Figure 4). Consistently, 10 nM JT010 failed to stimulate the Ca 2+ response in C621S mutant cells [18]. Moreover, mutant hTRPA1 with C621S was insensitive to 100 nM JT010 in whole-cell current-recording experiments [22]. Therefore, it is reasonable to assume that the primary binding site of JT010 is the cysteine residue at position 621 of hTRPA1 (Figure 7). Meanwhile, based on the two-step model proposed, whereby C621 is the primary site of electrophile modification and C665 is another modification site for full channel activation, the binding of a small electrophile, AITC, to C665 in hTRPA1 could support the full activation of TRPA1. However, it has been proposed that bulky JT010 can stabilize the open pocket by modifying C621 alone [21]. It is pharmacologically useful to compare JT010 docking sites between hTRPA1 and mTRPA1. In our preliminary docking simulation, the affinity of JT010 against hTRPA1 was weak (∆G = −5.9 kcal/mol). This low affinity cannot explain the high potency of JT010 against hTRPA1 in the previous and present experimental studies [18,22]. Because the docking sites at the highest rank simulated are different from those of the cryo-EM data, it is likely that the docking simulation is limited.
It is clear that mTRPA1 is less sensitive to JT010 than hTRPA1, and JT010 lower than 100 nM hardly activates mTRPA1. Intriguingly, mTRPA1 conserves both C622 (C621 in humans) and C666 (C665 in humans), which are critical cysteines for electrophilic modifications, including those of JT010 ( Figure 4A). Suo et al. found that JT010 covalently binds to C621 of hTRPA1 and interacts with phenylalanine at 612 (F612) and tyrosine at 680 (Y680) of hTRPA1 via CH-π and sulfur-π formation through its thiazol group, respectively [22]. Moreover, the methoxyphenyl group of JT010 potentially interacts with histidine at 614 (H614), proline at 666 (P666), and F669. Mutants with serine at C621 (C621S), alanine at F612 (F612A), alanine at Y680 (Y680A), and alanine at F669 (F669A) mutations exhibited no sensitivity to 100 nM JT010. The importance of isoleucine at 623 (I623), tyrosine at 662 (Y662), and threonine at 684 (T684) of hTRPA1 against JT010 is also clear; the respective mutants dramatically reduce the response to JT010 [22]. Among these important residues, F669 alone is not conserved in mTRPA1, where the homologous residue is substituted with methionine (M670, Fig7), suggesting that this substitution lowers the sensitivity of mTRPA1 to JT010. While the mutant hTRPA1 with F669A mutation was insensitive to 100 nM JT010 [22], the mouse-type mutant with F669M in hTRPA1 retained the response to 30 and 100 nM JT010 ( Figure 5). In contrast, the M670F mutation in mTRPA1 did not induce a clear JT010-dependent response ( Figure 6). This suggests that F669 plays an important role in the response of hTRPA1 to JT010 and that M670 is not a critical determinant of lower sensitivity to JT010 in mTRPA1 (Figure 7). are colored orange and red, respectively. A close-up view of the JT010 binding sites is shown on the right. The methoxyphenyl group of JT010 potentially interacts with the phenyl of F669 in hTRPA1 in a π-π interaction manner. The coordination is shown by the dotted line with a 4.9 Å distance. Out of 100 models, the structure with the lowest zDOPE score (2.69) was adopted (see also Materials and Methods).
It is clear that mTRPA1 is less sensitive to JT010 than hTRPA1, and JT010 lower than 100 nM hardly activates mTRPA1. Intriguingly, mTRPA1 conserves both C622 (C621 in humans) and C666 (C665 in humans), which are critical cysteines for electrophilic modifications, including those of JT010 ( Figure 4A). Suo et al. found that JT010 covalently binds to C621 of hTRPA1 and interacts with phenylalanine at 612 (F612) and tyrosine at 680 (Y680) of hTRPA1 via CH-π and sulfur-π formation through its thiazol group, respectively [22]. Moreover, the methoxyphenyl group of JT010 potentially interacts with histidine at 614 (H614), proline at 666 (P666), and F669. Mutants with serine at C621 (C621S), alanine at F612 (F612A), alanine at Y680 (Y680A), and alanine at F669 (F669A) mutations exhibited no sensitivity to 100 nM JT010. The importance of isoleucine at 623 (I623), tyrosine at 662 (Y662), and threonine at 684 (T684) of hTRPA1 against JT010 is also clear; the respective mutants dramatically reduce the response to JT010 [22]. Among these important residues, F669 alone is not conserved in mTRPA1, where the homologous residue is substituted with methionine (M670, Fig7), suggesting that this substitution lowers the sensitivity of mTRPA1 to JT010. While the mutant hTRPA1 with F669A mutation was insensitive to 100 nM JT010 [22], the mouse-type mutant with F669M in hTRPA1 retained the response to 30 and 100 nM JT010 ( Figure 5). In contrast, the M670F mutation in mTRPA1 did not induce a clear JT010-dependent response ( Figure 6). This suggests that F669 plays an important role in the response of hTRPA1 to JT010 and that M670 is not a critical determinant of lower sensitivity to JT010 in mTRPA1 (Figure 7).
Although the mTRPA1 double mutant with S669E and M670F mutations induced small responses to JT010, its potency was still significantly lower than that of hTRPA1 are colored orange and red, respectively. A close-up view of the JT010 binding sites is shown on the right. The methoxyphenyl group of JT010 potentially interacts with the phenyl of F669 in hTRPA1 in a π-π interaction manner. The coordination is shown by the dotted line with a 4.9 Å distance. Out of 100 models, the structure with the lowest zDOPE score (2.69) was adopted (see also Materials and Methods).
Although the mTRPA1 double mutant with S669E and M670F mutations induced small responses to JT010, its potency was still significantly lower than that of hTRPA1 ( Figure 6D vs. Supplementary Figure S3). As the double mutant included all crucial amino acid residues for the interaction with JT010 proposed, it is notable that the interaction of JT010 with these residues is not sufficient to explain the activation mechanism of TRPA1 by lower JT010 concentration. In contrast, AITC (30 µM) induced large membrane currents of wild-type and all mutant TRPA1s in humans and mice in this study, indicating that large electrophiles like JT010 may have additional interactions with TRPA1. TRPA1 interacts differently with agonists and antagonists from species to species via distinctive molecular mechanisms. A96, a potent TRPA1 antagonist in humans and mice, is a TRPA1 agonist in chicken [29]. Moreover, menthol, a non-electrophile agonist of hTRPA1, inhibits mTRPA1 [23,30]. Particularly, when bulky electrophilic compounds are used, there may be species differences in the response of TRPA1. The basal channel activity was different between hTRPA1 and mTRPA1 under the intracellular dialysis of 0.3 µM Ca 2+ ( Figure 2B,D, 1520.2 ± 301.8 pA vs. 328.35 ± 74.73 pA in mTRPA1 and hTRPA1 at +90 mV, respectively, p < 0.01; −629.55 ± 210.1 pA vs. −87.38 ± 32.58 pA in mTRPA1 and hTRPA1 at −90 mV, respectively, p < 0.05). It is unlikely that this basal activity affected the interaction with JT010 in mTRPA1. Indeed, JT010 at concentrations under 100 nM did not affect mTRPA1, even in the absence of intracellular Ca 2+ , where the basal channel activity was lower (Supplementary Figure S2). When applied to DRG cells isolated from mice, 10 nM of JT010 did not induce any response. Because 100 µM AITC evoked a response in 88% of cells employed, mTRPA1 expression was apparent, suggesting that a lower concentration of JT010 cannot activate endogenous mTRPA1. Notably, the application of 1000 nM JT010 induced a small but substantial Ca 2+ response in HEK-mTRPA1 cells (Supplementary Figure S1C), possibly indicating the interaction of JT010 with C622 of mTRPA1. Nevertheless, it is clear that mTRPA1 is relatively resistant to lower JT010.
In conclusion, we showed that the potent hTRPA1 agonist JT010 could not activate mTRPA1 at concentrations ranging from 0.3 to 100 nM. Moreover, JT010 induced the response of endogenous TRPA1 in human FLSs with inflammation, but not in mouse DRG cells, both of which were sensitive to AITC. As confirmed by the importance of F669 in the N-terminus of hTRPA1 for the JT010-interaction, methionine substitution, which is homologous to mTRPA1 M670, significantly reduced the response to JT010. In contrast, while a single M670F mutation in mTRPA1 was still insensitive to JT010, the double mutant mTRPA1 with S669E and M670F mutations evoked a weak but substantial response to JT010. Taken together, JT010 is a potent TRPA1 agonist in humans, but not in mice.
Cell Culture
HEK cells obtained from the Health Science Research Resources Bank (HSRRB, Osaka, Japan) were maintained in Dulbecco's modified Minimum Essential medium (D-MEM; Sigma-Aldrich, St. Louis, MO, USA) supplemented with 10% heat-inactivated fetal calf serum (FCS; Sigma-Aldrich), penicillin G (100 U/mL, Meiji Seika Pharma Co., Ltd., Tokyo, Japan), and streptomycin (100 µg/mL, Meiji Seika Pharma Co., Ltd., Tokyo, Japan). Human FLSs, which were purchased from Cell Applications (San Diego, CA, USA), were cultured in Synoviocyte Growth medium containing 10% growth supplement, 100 U/mL penicillin G, and 100 µg/mL streptomycin, as described previously [26]. FLSs were maintained at 37 • C in a 5% CO 2 atmosphere. After they reached 70-80% confluence, FLSs were reseeded once every 10 days until nine passages were completed. The cells that grew with a doubling time of 6-8 days after this stage comprised a homogenous population, in which TRPA1 transcriptionally induced by IL-1αwas found to be unaffected. For the experiments, reseeded cells were cultured for 16 days and then exposed to IL-1α or vehicle.
Cell Isolation from DRG in Mice
This study was approved by the Animal Care Committee of Aichi Gakuin University (approval code 21-036 and 22-007) and conducted per the Guiding Principles for the Care and Use of Laboratory Animals approved by the Japanese Pharmacological Society. Male mice weighing 20-30 g were anesthetized with isoflurane and decapitated. Four to five DRGs isolated were washed in phosphate-buffered solution (PBS [in mM]: NaCl 137, KCl 5.4, MgCl 2 1.2, CaCl 2 2.2, Na 2 HPO 4 0.168, KH 2 PO 4 0.44, glucose 5.5, NaHCO 3 4.17, pH7.45) and treated with Ca 2+ -Mg 2+ -free PBS containing 0.05% collagenase (Amano, Nagoya, Japan) and 0.05% dispase (Boehringer Mannheim, Tokyo, Japan). All DRGs were kept in an incubator at 37 • C for 60 min and then the enzyme solution containing isolated DRGs was centrifuged at 1200× g rpm for 10 min. Thereafter, the supernatant was removed and the pellet was resuspended in culture medium (D-MEN with 10% FCS) and gently agitated with a fire-polished wide-pore pipette. Isolated DRG cells were allowed to attach to gelatin-coated glass coverslips in a 35 mm dish and were cultured for 24-48 h at 37 • C in a 5% CO 2 atmosphere, and used within 48 h.
Patch-Clamp Experiments
Whole-cell current recordings were performed as previously described [31]. The resistance of the electrodes was 3-5 MΩ when filled with pipette solution. The Cs + -rich pipette solution contained [in mM] Cs-aspartate 110, CsCl 30, MgCl 2 1, 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) 10, ethylene glycol tetra-acetic acid (EGTA) 10, and Na 2 ATP 2 [adjusted to pH 7.2 with CsOH]. To maintain the activity of TRPA1 currents, intracellular free Ca 2+ concentration was adjusted to a pCa value of 6.5 (0.3 µM Ca 2+ ) by adding CaCl 2 to the pipette solution. In some experiments, the EGTA concentration in the pipette solution was reduced to 1 mM in the absence of CaCl 2 . The membrane currents and voltage signals were digitized using an analog-digital converter (PCI-6229, National Instruments Japan, Tokyo, Japan). WinWCPV5.52 software was used for data acquisition and analysis of whole-cell currents (developed by Dr. John Dempster, University of Strathclyde, UK). The liquid junction potential between the pipette and bath solutions (−10 mV) was calculated. A ramp voltage protocol from −110 mV to +90 mV for 300 ms was applied every 5 s from a holding potential of −10 mV. The leak current component was not subtracted from the recorded current. A standard HEPES-buffered bathing solution (SBS [in mM]: NaCl 137, KCl 5.9, CaCl 2 2.2, MgCl 2 1.2, glucose 14, and HEPES 10 [adjusted to pH 7.4, with NaOH]) was used. All experiments were performed at 25 ± 1 • C.
Measurement of Ca 2+ Fluorescence Ratio
HEK, FLSs, and DRG cells, which were loaded with 10 µM Fura2-AM (Dojindo, Kumamoto, Japan) in SBS for 30 min at room temperature, were superfused with SBS for 10 min, and Fura-2 fluorescence signals were measured at 0.1 Hz using the Argus/HisCa imaging system (Hamamatsu Photonics, Hamamatsu, Japan) driven by Imagework Bench 6.0 (INDEC Medical Systems, Santa Clara, CA, USA). Since the efficacy of gene transfection in HEK cells and the TRPA1 expression level in FLSs and DRG cells were similar but not identical from cell to cell, we collected 50, 5-11, and 8-12 single cells of HEK, FLSs, and DRG cells, respectively, on one coverslip to obtain the average response. We repeated the same protocol with other coverslips to obtain the mean and standard error of the mean (SEM) of independent experiments. In each analysis, the whole cell area was chosen as the region of interest to average the fluorescence ratio.
Modeling
Molecular modeling was performed on UCSF Chimera v1.16 [32] using the modeling software MODELLER v10.3 [33,34]. Using the amino acid sequence of mTRPA1 (ID: Q8BLA8) from UniProt (https://www.uniprot.org/ accessed on 30 August 2022), we modeled the structure of mTRPA1 as a monomer, with JT010-bound hTRPA1 (PBD ID:6PQO) as a template. Of the 100 models, the structure with the lowest normalized Discrete Optimized Protein Energy (zDOPE) score was adopted (Figure 7). The software Autodock-Vina was used to predict the possible binding models of JT010 to hTRPA1 (200 models) and the solutions were ranked according to their binding energy [35]. The grid box for docking model was set to locate C621 at the center and to include all amino acid residues interacted with JT010 (C665, F621, Y680, T684, Y662, I623, and F669 [22]). | v3-fos-license |
2016-05-04T20:20:58.661Z | 2014-08-15T00:00:00.000 | 12696784 | {
"extfieldsofstudy": [
"Medicine"
],
"oa_license": "CCBY",
"oa_status": "GOLD",
"oa_url": "https://ccforum.biomedcentral.com/track/pdf/10.1186/s13054-014-0468-2",
"pdf_hash": "379bba05691ad18ed5a88a161a04eae1ec9a73e2",
"pdf_src": "PubMedCentral",
"provenance": "20241012_202828_00062_s4wg2_01a5a148-7d10-4d24-836e-fe76b72970d6.zst:16",
"s2fieldsofstudy": [
"Medicine"
],
"sha1": "002e8ef4db94e49ac2454831349fa64718d579ba",
"year": 2014
} | pes2o/s2orc | A specialized post-anaesthetic care unit improves fast-track management in cardiac surgery: a prospective randomized trial
Introduction Fast-track treatment in cardiac surgery has become the global standard of care. We compared the efficacy and safety of a specialised post-anaesthetic care unit (PACU) to a conventional intensive care unit (ICU) in achieving defined fast-track end points in adult patients after elective cardiac surgery. Methods In a prospective, single-blinded, randomized study, 200 adult patients undergoing elective cardiac surgery (coronary artery bypass graft (CABG), valve surgery or combined CABG and valve surgery), were selected to receive their postoperative treatment either in the ICU (n = 100), or in the PACU (n = 100). Patients who, at the time of surgery, were in cardiogenic shock, required renal dialysis, or had an additive EuroSCORE of more than 10 were excluded from the study. The primary end points were: time to extubation (ET), and length of stay in the PACU or ICU (PACU/ICU LOS respectively). Secondary end points analysed were the incidences of: surgical re-exploration, development of haemothorax, new-onset cardiac arrhythmia, low cardiac output syndrome, need for cardiopulmonary resuscitation, stroke, acute renal failure, and death. Results Median time to extubation was 90 [50; 140] min in the PACU vs. 478 [305; 643] min in the ICU group (P <0.001). Median length of stay in the PACU was 3.3 [2.7; 4.0] hours vs. 17.9 [10.3; 24.9] hours in the ICU (P <0.001). Of the adverse events examined, only the incidence of new-onset cardiac arrhythmia (25 in PACU vs. 41 in ICU, P = 0.02) was statistically different between groups. Conclusions Treatment in a specialised PACU rather than an ICU, after elective cardiac surgery leads to earlier extubation and quicker discharge to a step-down unit, without compromising patient safety. Trial registration ISRCTN71768341. Registered 11 March 2014.
Introduction
Anaesthesia for cardiac surgery has traditionally been provided with high-dose opioids and long-acting muscle relaxants, in the belief this technique was associated with optimal haemodynamic stability. The resulting prolonged postoperative ventilation and intensive care unit (ICU) length of stay (LOS) were considered acceptable compromises. Rising costs and the need for faster ICU turnover due to increased demand and reduced resources led to reducing the length of ICU stay after cardiac surgery [1,2].
Since the mid-1990s, intensified postoperative rehabilitation has established itself as the optimal approach to patient recovery. Fast-track treatment has become a popular and accepted standard because it allows for early extubation within six hours and consequently reduced LOS in the ICU and hospital [3][4][5]. A significant reduction in time to extubation (ET) without compromising patient's safety has been demonstrated in numerous studies [5][6][7][8][9][10][11]. Zhu et al. described in Cochrane Database Systematic Review a mean reduction of 5.99 hours (2.99 to 8.99 hours) due to implementation of a time-directed extubation protocol without increasing the risk of postoperative complications compared to standard care. Low-dose opioid anaesthesia will reduce mean ET by 7.40 hours (10.51 to 4.29 hours) compared to high-dose opioid anaesthesia [11].
Implementation of a dedicated fast-track protocol that allows not only for earlier extubation but also for earlier transfer from the ICU or post-anaesthetic care unit (PACU) to a step-down unit has been shown to be very effective in reducing ICU-LOS and the total length of hospital stay in retrospective studies [5,6,8]. Zhu et al. showed in a review that low-dose opioid anaesthesia was associated with 3.7 hours (−6.98 to −0.41) lower ICU LOS. Time-directed extubation protocols had 5.15 hours (−8.71 to −1.59) shorter length of stay in the ICU (0.4 to 8.7 hours) compared to conventional groups, as Zhu et al. described, although LOS in hospital was similar in both groups [11].
Utilised in combination, this approach has been associated with both significant cost savings, and also increased ICU bed capacity [12]. Most fast-track treatment protocols for cardiac surgery patients to date, however, have been implemented within the conventional ICU setting.
In general, it is possible to perform an extubation in the operating room (OR) with selected patient groups (OPCAB, MIDCAB and so on). This could make sense if no postoperative care unit is available or the fast-track concept is not continued at the ICU. There is still an ongoing discussion about the advantage of an early extubation in the OR. Straka et al. and Montes et al. were not able to show a reduced ICU LOS in cardiac surgery patients who get extubated in the OR [13,14]. Chamchad et al. found in a non-randomized observational study shorter ICU and hospital LOS. With an average ICU LOS of 27 hours, this study showed no additional benefit compared to early extubation in a PACU/ICU [15].
Nicholson et al. investigated in a randomized trial the effect of immediate extubation after coronary artery bypass graft (CABG) surgery compared to at least three hours ventilation before starting weaning on the pulmonary function. The study was performed in a PACU. They concluded that early extubation will not affect pulmonary function after extubation [16].
Our fast-track concept consists of direct postoperative treatment in a PACU with the primary goal of early extubation, followed by transfer to a step-down unit as soon as specific discharge criteria are met [6].
To the best of our knowledge, no prospective randomized study has been published which compares fast-track treatment in the ICU versus fast-track treatment in the PACU. The hypothesis of the study was that patients treated in the PACU would be extubated earlier, and be discharged to a step-down unit earlier than patients treated in the ICU. Accordingly, the objectives of our study were to compare ET and LOS in the PACU or ICU.
Methods
The study was approved by our local ethics committee (Ethics Committee, Medical Faculty, University of Leipzig, 04107 Leipzig, Reference number 097-2008, trial registration number ISRCTN71768341, http://www.controlled-trials.com/ISRCTN71768341/, registered 11 March 2014), and was conducted as a prospective, randomized, single-blinded, single-centre trial.
For each patient, written informed consent was obtained prior to any protocol-related activities. As part of this procedure, the principal investigator or designee explained orally and in writing the nature, duration, and purpose of the study in such a manner that the patient was aware of the potential risks, inconveniences, or adverse effects that may occur. The patient was informed that he/she was free to withdraw from the study at any time. The patient received all information that was required by local regulations and International Conference on Harmonisation (ICH) guidelines.
During the premedication visit the day before surgery, every patient scheduled to undergo CABG, valve surgery, or combined CABG and valve surgery was screened for inclusion in the study ( Figure 1). Patients who were in cardiogenic shock, were dialysis dependent, or had an additive EuroSCORE of more than 10 were excluded.
The final decision for including or excluding the patient into the fast-track concept was taken by consensus decision between the attending anaesthesiologist and cardiac surgeon at the end of their surgery. Inclusion criteria were: haemodynamically stable (systolic blood pressure >90 mmHg and heart rate <120 bpm; adrenaline or noradrenaline <0.04 mcg/kg/min), normothermic (>36°C core body temperature), and no bleeding. Exclusion criteria followed risk factors identified by Constantinides et al. and Akhtar et al. [17,18]: impaired left ventricular function (ejection fraction below 35%), cardiac assist devices pre-or postoperative and cardiopulmonary instability postoperative (high inotropic support, lactate >5 mmol/l, Horowitz index below 200) After the decision to include the patient into the study, the patient was randomized to either postoperative care in the PACU (n = 100) or ICU (n = 100). For that purpose an envelope was picked out of a box containing 200 sealed envelopes (100 for PACU, 100 for ICU admission) and removed from the box subsequently. A further intra-operative exclusion criterion was lack of an available bed in either the PACU or ICU. In such cases, the patient was not randomized, but was sent to the unit with an available bed, and excluded from the study and further analysis. The medical and nursing staff in the ICU and PACU had been informed about the design and the conduct of the study but were not informed as to which patients were enrolled in the study.
Data collection and analysis was performed by an independent person who was not part of the anaesthetic, surgical or ICU team, and who was not blinded to treatment allocation.
Fast-track anaesthesia protocol
Anaesthetic management consisted of oral premedication with clorazepate dipotassium (20 to 40 mg) the evening before and midazolam (3.75 to 7.5 mg) on the day of surgery. Anaesthesia was induced with fentanyl (0.2 mg) and propofol (1.5 to 2 mg/kg). A single dose of rocuronium (0.6 mg/kg) was used to facilitate intubation. Analgesia was maintained throughout the case with a continuous infusion of remifentanil (0.2 mcg/kg/min), and for hypnosis during the pre-and post-cardiopulmonary bypass (CBP) period sevoflurane (0.8 to 1.1 minimum alveolar concentration (MAC)) was administered whereas during CPB a continuous propofol infusion (3 mg/kg/h) was used. A recruitment manoeuvre was carried out prior to weaning from CPB in order to prevent atelectasis. An external convective warming system with an underbody blanket (Bairhugger™, Arizant Healthcare; Eden Prairie, MN, USA) was used after weaning from CPB to ensure a core temperature of at least 36°C was maintained. For early postoperative analgesia, 1 g paracetamol was administered intravenously to each patient before skin closure. In difference to other studies, we did not include all patients or selected fast-track patients only preoperatively. All patients received the fast-track anaesthesia in the OR. We carefully selected fast-track patients at the end of surgery following the criteria identified as risk factors for fast-track failure [1,17,18]. The final decision to continue the fast-track protocol postoperatively was taken after the end of surgery. As our primary end point was postoperative ventilation time, we defined fast-track failure as postoperative ventilation of more than six hours. That was decided due the literature research where it ranged between three and nine hours [19,20].
Treatment in PACU
All patients were transferred to the PACU intubated, mechanically ventilated with a remifentanil infusion of 0.1 mcg/kg/min. Administration of hypnotic agents was discontinued in the OR.
Postoperative analgesia consisted of an bolus of piritramide (0.1 mg/kg) on discontinuation of the remifentanil infusion, followed by bolus doses as required in 2 to 4 mg aliquots, plus regular paracetamol (1 g every six hours) to achieve a pain score between 2 and 4 on an analogue pain scale from 0 to 10. Patients were extubated when they were conscious and obeyed commands, had stable spontaneous ventilation with pressure support of 10 to 12 cmH 2 O, positive end-expiratory pressure (PEEP) of 5 cmH 2 O, fraction of inspired oxygen (FiO 2 ) of ≤0.4, were haemodynamically stable, not bleeding (≤100 ml/h), and with no significant electrocardiographic abnormalities.
All patients received non-invasive bi-level positive airway pressure ventilation via a face mask for one hour (Elisee 350™, Saime, Savigny-le-Temple, France), immediately after extubation. Initially non-invasive ventilation was commenced at a pressure support of 10 to 15 cm H 2 O and a PEEP of 5 cmH 2 O. The FiO 2 was 0.4. During the period of non-invasive ventilation the pressure support was adapted to patients' needs.
Criteria for discharge to the intermediate care unit (IMC) were that patients must be awake, cooperative, haemodynamically stable (without inotropes) and have both acceptable respiratory pattern and blood gas analysis (pO 2 > 70 mmHg, pCO 2 < 50 mmHg). Chest-X-ray and electrocardiogram were performed in all patients to exclude major pathology.
The physician-to-patient ratio and the nurse-to patientratio were 1:3. The PACU operated daily Monday to Friday from 10:00 to 18:30.
Treatment in ICU
All patients arrived in the ICU intubated, mechanically ventilated with a remifentanil infusion of 0.1 μg/kg/min. Administration of hypnotic agents was discontinued in the OR.
Postoperative analgesia consisted of a bolus of piritramide (0.1 mg/kg) on discontinuation of the remifentanil infusion, followed by bolus doses as required in 2 to 4 mg aliquots, plus regular paracetamol (1 g every six hours). A pain scale was not used on a regular basis for assessing pain. The need for an analgesic medication was estimated by nurses. Extubation criteria were identical to those in the PACU. Non-invasive ventilation after extubation was not implemented routinely. Further treatment in the ICU was determined by the ICU physician according to German guidelines for intensive care treatment in cardiac surgery patients [21]. Criteria for suitability to transfer to IMC were identical to those in the PACU.
The physician-to patient-ratio was 1:12 and the nurseto-patient ratio was 1:2.
Substantial differences in PACU and ICU treatment are listed in Table 1.
Outcomes
Primary end points were ET and PACU/ICU LOS. Secondary outcome measures were hospital LOS, overall length of intensive care treatment (total ICT LOS), in-house mortality, low cardiac output, new-onset cardiac arrhythmia, respiratory failure requiring prolonged ventilation or reintubation and incidences of surgical re-exploration and renal failure.
PACU/ICU LOS is defined as LOS in the PACU or ICU from the end of surgery until discharge to another unit. Additionally, secondary PACU/ICU LOS includes readmissions from step-down units to ICU as well as additional ICU time after transfer from the PACU to ICU based on medical or organisational circumstances.
IMC LOS is defined as LOS in IMC until discharge to a general ward.
Primary ICT LOS is defined as overall length of intensive care treatment (ICT) in PACU/ICU + IMC.
Total ICT LOS is defined as overall length of ICT in the PACU + ICU + IMC including readmission to a unit of higher care grade than a general ward and transfer from the PACU to the ICU.
If patients were transferred from the PACU to the ICU in case of medical or organizational circumstances, they were still analysed as being in the PACU group, although additional ICU LOS was not calculated in PACU/ICU LOS but in secondary PACU/ICU LOS and total ICT LOS. PACU patients who had to stay past 18:30 were admitted to the ICU for further treatment and were evaluated as described above.
Low cardiac output was defined as central venous saturation of <65% with a haematocrit of >30%. Cardiac arrhythmia included atrial fibrillation and atrioventricular block. Acute renal failure was defined as an increase in postoperative serum creatinine of at least three times the preoperative value, or a serum creatinine >150 μmol/l. Stroke was defined as a new transient or permanent motor or sensory deficit of central origin or unexplained coma.
Statistical analysis
Sample sizes were calculated on the basis of data from a previous retrospective study at our institution [6] using SPSS 16.0 (SPSS Inc, Chicago, IL, USA). Using this data, we estimated that ET in the ICU compared to ET time in the PACU would occur four hours later and that the standard deviation would be approximately 500 min. We calculated that 93 patients per group would be required to demonstrate a significant reduction in ET with a power of 90% at significance level of 5%. Accounting for drop-outs and incomplete data, we aimed to recruit 100 patients per group.
Comparisons between the two independent groups (ICU vs. PACU) were performed using the Mann-Whitney U test for continuous data, Mantel-Haenzsel test for categorically ordered data (for example New York Heart Association (NYHA) score) and Fisher's exact test for binary data (for example adverse events). A threshold of 0.05 was considered as significant. All analyses were performed using SPSS 18.0. Continuous parameters were described by median and interquartile range. Categorical data are described by class-wise allocation numbers. Binary data are described as number of events.
The primary end point of this study was time to extubation. We have not adjusted for multiple testing, so other comparisons are considered explorative.
Results
A total of 423 patients consented to participate in the study. All patients were scheduled for CABG, aortic valve replacement (AVR), mitral valve repair/replacement (MVR) or a combination of these procedures ( Table 2). A total of 223 patients were excluded intraoperatively, due to a lack of capacity in either the ICU or PACU (n = 171), or because they were considered unsuitable for fast-track management at the end of their surgery, according to our criteria listed above (n = 52). A total of 200 patients were therefore included in the study from May 2008 until July 2009, 100 in each group. There were significantly more female patients in the PACU group (36 vs. 22, P = 0.04) ( both groups are listed in Table 2. There was no significant difference in type of surgery.
Time to extubation
The median extubation time in PACU group was significantly shorter than in the ICU group (90 min [50; 140] vs. 478 min [305; 643]; P <0.001; Figure 2, Table 4). In the PACU group 97% of the patients were extubated within six hours of admission whereas only 33% of the patients in the ICU group fulfilled the criteria for successful fast-tracking (P <0.001) [5]. In the PACU group five patients required reintubation (three for resurgery, one because of a convulsion, and one for respiratory failure) compared to ten patients in ICU group (five for re-operation, four for respiratory failure, one for cardiopulmonary resuscitation Figure 3, Table 4).
The median LOS in the IMC was 23.0 hours [19.9; 41.8] in the PACU group and 21.0 hours [10.5; 28.8] in the ICU group (P <0.004).
Overall length of ICT in the PACU + ICU + IMC including readmission to a unit of higher care grade than a general ward and transfer from the PACU to the ICU was 30.9 hours [23.9; 59.9] for patients in the PACU group compared to 43.9 hours [24.9; 65.4] for patients in the ICU group (P = 0.08; Figure 4, Table 4). There was no significant difference in median hospital LOS for the PACU group (9 [8; 11]) vs. the ICU group (9 [8; 12] days).
Ninety-one of 100 patients in PACU group were discharged to intermediate care unit whereas nine patients had to be admitted from the PACU to the ICU (Figure 1). Three of these were extubated and haemodynamically stable, and were admitted to the ICU because of lack of available beds in IMC, two patients because of failure to extubate, two patients because of bleeding, and two patients because of cardiac arrhythmia. Four patients in the PACU group had to be admitted from IMC to the ICU (two because of re-thoracotomy, one because of haemodynamic instability, and one because of respiratory failure). A total of 87% of all patients in the PACU group did not require any treatment in the ICU.
Readmission from the general ward to IMC occurred in 13 patients of the PACU group, and was due to: cardiac arrhythmia (n = 4), pleural effusion (n = 5), pneumothorax (n = 2), resurgery (n = 1), and pain control (n = 1), no patient in the PACU group discharged to the ward required readmission to ICU.
In the ICU group five patients required readmission from IMC to ICU, because of respiratory failure (n = 4) and cardiac arrest (n = 1). Two patients were readmitted from the general ward to ICU because they required resurgery. Furthermore, in the ICU group eight patients had to be readmitted from the general ward to IMC because of cardiac arrhythmia (n = 5), neurological deficit (n = 2) and pericardial effusion (n = 1).
Postoperative complications
Postoperative complications for both groups are listed in Table 5. The occurrence of arrhythmias was significantly lower in the PACU group as compared to the ICU group (25 vs. 41, P = 0.02). There was no significant difference in the rate of pleural or pericardial effusions requiring intervention, renal insufficiency or cerebrovascular stroke. The number of patients requiring resurgery (PACU n = 5 vs. ICU n = 11, P = 0.19) was lower in the PACU group (two for implantation of a pacemaker, two for drainage of a haemothorax, one for thrombectomy for deep vein thrombosis) compared to the ICU group (five for drainage of a haemothorax, two for revision of valve after valve replacement, two for implantation of a pacemaker, one thoracotomy for bleeding followed by insertion of extracorporeal membrane oxygenation after resurgery, one for refixation of the sternum).
One patient from the PACU group required ventilation longer than 24 hours vs. seven patients in ICU group (P = 0.07).
Discussion
In our study, we have shown that fast-track treatment of cardiac surgery patients in a dedicated PACU compared to fast-track treatment in the ICU significantly reduces ET (90 vs. 478 min; P <0.001) as well as time to transfer to a step-down unit (LOS PACU 3.3 hours compared to 17.9 hours LOS ICU). We were able to demonstrate a reduction of ventilation time and a significantly reduced utilisation of ICU capacity after cardiac surgery. Although we did not calculate the cost savings, Cheng et al. have clearly shown that early extubation results in reduced costs and better resource utilisation [4]. Hantschel et al. have also demonstrated that postoperative treatment in a PACU after cardiac surgery results in a 52% cost reduction compared to conventional ICU treatment [12]. Opening a PACU for 8.5 hours a day should lead to reduced personnel costs compared to a 24-hour ICU.
An ET of less than six hours after cardiac surgery is considered an important criterion for successful fasttracking after cardiac surgery [4,5]. In the PACU group 97% of the patients fulfilled this criterion but only 33% in the ICU group (P <0.001). In a recent review, Zhu et al. showed that using a low-dose-opioid anaesthesia reduces ventilation times by 7.40 hours. Using a weaning protocol reduced ventilation times by 5.99 hours. In our study, we were able to reduce ventilation times by 6.46 hours, which is comparable to the reduction reported in other studies [11]. Our protocol used low-dose opioid anaesthesia with the short-acting opioid remifentanil. We defined a weaning protocol, which included early stop of anaesthesia, a protocol-driven postoperative pain management and non-invasive ventilation after extubation for at least 60 minutes. Another fast-track criterion is reduced LOS in ICU, usually defined as less than 24 hours [5]. According to this criterion, successful fast-track-treatment was achieved in 95% of the PACU patients compared to 71% patients in the ICU group (P <0.001). Zhu et al. reported in a review a reduction in ICU LOS for low-dose-opioid anaesthesia of 3.7 hours (−6.98 to −0.41) and by using a weaning protocol of 5.15 hours (−8.71 to −1.59) compared to highdose-opioid anaesthesia [11]. In our study, we achieved a reduction in PACU/ICU LOS by 14.6 hours to 3.3 hours. This early discharge to a step-down unit allows using an ICU bed more than once a day. Gooch et al. developed a model of demand elasticity of ICU bed utilization [22]. The authors discussed that ICU beds created their own demand [23]. Under the model of demand elasticity the case mix of patients in the ICU changed depending on bed availability. If enough beds are available or no actual patient needs an ICU bed, it is more likely that patients in the ICU who are not as critically ill do not benefit from ICU stay [23]. By bypassing the ICU for fast-track patients, we possibly reduced this effect of demand elasticity and were able to show a reduction in ICU bed utilization. Still, if we included the readmission and direct transfers from the PACU to the ICU, we found a significant reduction for ICU LOS of 14.4 hours (secondary ICU LOS PACU vs. ICU 3.5 to 17.9 hours).
Published figures for fast-track failure rates range from 11% to 49% depending on the patient population [17,18,24]. In contrast to studies that included all patients undergoing cardiac surgery, our study population was preselected according to our existing fast-track protocol. We primarily excluded patients with a defined risk for fast-track failure during the premedication visit (patients who were scheduled for emergency surgery, were in cardiogenic shock, were dialysis dependent, or had an additive EuroSCORE of more than 10) [1,17,25]. Another explanation for the low fast-track failure rate of 5% for the PACU group is the fact that the final decision for inclusion of the patient to fast-track treatment was made at the end of the surgery. Wong et al. identified need for inotropic support and bleeding as risk factors for delayed extubation as well as delayed LOS in ICU [26]. In our study, 52 out of the 423 patients primarily included were excluded before randomisation because of hemodynamic instability or bleeding at the end of the operation. This underlines the hypothesis that not only careful preselection of potential fast-track patients during the premedication visit is important, but also that re-evaluation of patient suitability at the end of the operation can lead to a reduction of fast-track failure. The relatively high fast-track failure rate for the ICU group (67% time to extubation >6 hours and 29% PACU/ICU LOS >24 hours) may be attributable to several factors: first, the much better physician-to-patient ratio in the PACU (1:3 in the PACU vs. 1:12 in the ICU) allows the physician to effectively implement and manage an early goal-directed therapy. Since the study from Rivers et al. in septic patients we know that early hemodynamic stabilisation is beneficial for the patient and this is certainly also true for cardiac fast-track patients [27]. Several other studies have shown that an early goal-directed fluid management in postoperative cardiac surgery patients results in an improved hemodynamic stability and can reduce ventilation time and ICU LOS [28,29]. Second, due to the fact that one physician in the ICU cares for 12 patients the preselected fast-track patients will not get the same attention as the patient who really needs ICT. One to two severely compromised patients out of the 12 will result in the fact that weaning of the fast-track patient on ICU will be delayed. Kumar et al. have shown that the presence of an intensivist results in reduced ETs [30]. Third, the limited opening times for the PACU may positively motivate the involved staff to treat the patients optimally including early extubation and hemodynamic and respiratory stabilisation so that the patient can be transferred to the IMC for further treatment.
Also, the more focused adherence to the fast-track and enhanced-recovery principles including specifications for medication, postoperative pain control and discharge criteria favours the PACU compared to the ICU. van Mastrigt et al. showed in a meta-analysis that a defined weaning-and-extubation protocol is an important key to reduced intensive care LOS [10]. Although this protocol was the same for the PACU and the ICU, the more disciplined execution of the fast-track protocol and application of non-invasive ventilation in our PACU might be another important factor for success of early extubation. In a prospective randomized study, Zarbock et al. demonstrated a significant reduction in reintubation and readmission to ICU/IMC in cardiac surgery patients using continuous positive airway pressure therapy [31].
We found a lower incidence of reintubation in the PACU with 2.5% (five) vs. 5% in the ICU (ten) patients and a lower readmission rate of the PACU (four) vs. the ICU (seven) patients from step-down unit (IMC) to the ICU without reaching significance. Zhu et al. reported a risk of reintubation in the fast-track group of 1.4% and in the conventional group of 1.7%, [11], which is lower as in our study. However, this study is underpowered to allow any conclusion to the reintubation rate compared to other studies.
The incidences of low cardiac output syndrome, prolonged respiratory insufficiency, cardiac arrest, and death tended to be lower in the PACU group without reaching statistical significance. Because these complications were not primary end points, our study was underpowered for demonstrating significant differences between groups. The incidence of renal failure, stroke, resurgery, and mortality was similar for the PACU and the ICU group. Our study does not allow any conclusion about the safety of our fast-track concept. However, a significantly lower incidence of common postoperative complications for fast-track patients was demonstrated in a prospective study of 1,488 patients by Gooi et al. [3]. Svircevic et al. could not find any evidence for increased risk of adverse outcomes in 7,989 patients undergoing fast-track cardiac surgery [5]. In a recent review, Zhu et al. came to the conclusion that fast-track interventions have similar risks of mortality and major postoperative complications to conventional (not fast-track) care, and therefore appear to be safe in patients considered to be at low to moderate risk [11].
In contrast to other studies on fast-track in cardiac surgery, which included only patients undergoing coronary artery bypass surgery, our patient population was mixed regarding type of operations [4,10,19,32]. More than half of our patient population underwent valve surgery, some of them in combination with CABG. Overall, in our patient population of n = 200 patients only 41.5% were CABG patients (41 vs. 42). A total of 6.5% of all patients (four vs. nine) underwent combined procedures (for example aortic and mitral valve surgery or valve surgery and CABG). We have also shown that fast-track treatment utilising a dedicated PACU can be successfully implemented for different types of cardiac operations.
Limitations of the study
Our demographic data show that there is a significant difference in gender (more female patients in the PACU group). In several studies, female gender was found to be a risk factor for delayed postoperative extubation and prolonged ICU length of stay [1,26]. This might have favoured the ICU group. Anaesthesia and surgery time in the ICU group was significantly longer, but there was no difference in XCL and cardiopulmonary bypass time, which were (amongst others) identified as risk factors for delayed postoperative extubation (>6 hours) and prolonged ICU LOS (>24 hours) [1,26]. Regarding anaesthesia and surgery time, we observed only weak correlations with our outcome variables in both PACU and ICU groups. Hence, it is unlikely that this imbalance in baseline characteristics affects the main conclusion of our study.
Regarding the adverse events, the study was not adequately powered to identify significant differences between the groups.
Conclusions
Our study showed that our fast-track treatment in a dedicated PACU leads to a high rate of success (95%) compared to the ICU (33%). We attribute this difference to better physician-to-patient ratio, allowing for more focused, early postoperative management, and better adherence to an established fast-track protocol. Delaying the decision about patient suitability for fast-track treatment until the end of surgery may also contribute to reducing the incidence of fast-track failures. Running a PACU separated from the ICU in a different part of the hospital, an excellent physician-patient ratio and strong adherence to the fast-track protocol is from our point of view one of the success factors for our study.
Key messages
ET for cardiac surgery patients in a fast-track protocol is significantly shorter in a dedicated PACU than in ICU PACU-LOS is significantly shorter than ICU-LOS | v3-fos-license |
2019-05-27T13:20:36.389Z | 2019-05-27T00:00:00.000 | 165164107 | {"extfieldsofstudy":["Psychology","Medicine"],"oa_license":"CCBY","oa_status":"GOLD","oa_url":"https(...TRUNCATED) | pes2o/s2orc | "A Simple Target Interception Task as Test for Activities of Daily Life Performance in Older Adults\(...TRUNCATED) | v3-fos-license |
2019-08-23T16:21:56.366Z | 2019-07-29T00:00:00.000 | 201393093 | {"extfieldsofstudy":["Psychology"],"oa_license":"CCBYSA","oa_status":"GOLD","oa_url":"http://bircu-j(...TRUNCATED) | pes2o/s2orc | "Development of Contextual-Based Interactive Multimedia of Tema Daerah Tempat Tinggalku of 4 Grade S(...TRUNCATED) | v3-fos-license |
2018-12-03T20:47:41.888Z | 2010-08-05T00:00:00.000 | 54536664 | {"extfieldsofstudy":["Environmental Science"],"oa_license":"CCBY","oa_status":"GOLD","oa_url":"https(...TRUNCATED) | pes2o/s2orc | "Using flushing rate to investigate spring-neap and spatial variations of gravitational circulation (...TRUNCATED) | v3-fos-license |
End of preview. Expand
in Dataset Viewer.
Dolma PeS2o (Creative Commons & Public Domain subset)
This repository contains the Creative Common and public domain subset of open access papers in peS2o . Cutoff date of the collection is October 6, 2024, with train set containing papers up to August 31, 2024.
| Property | Train | Validation |
|---|---|---|
| Whitespace words | 35.4 B | 0.25 B |
| UTF-8 characters | 188 B | 1.3 B |
| Documents | 6.25 M | 39.1 K |
Licenses for documents in this set are distributed as follows:
| License | Train | Validation |
|---|---|---|
| CC-BY | 6,088,325 | 37,754 |
| CC-BY-SA | 120,150 | 1,231 |
| CC0 | 36,373 | 121 |
| public domain | 10,060 | 6 |
The following fields of study are covered (documents belong to one or more field; field of study is determined by Semantic Scholar):
| Field of Study | Train | Validation |
|---|---|---|
| Medicine | 2,435,244 | 23,734 |
| Biology | 1,518,478 | 8,879 |
| Environmental Science | 993,499 | 7,601 |
| Engineering | 656,021 | 5,005 |
| Computer Science | 462,320 | 3,003 |
| Materials Science | 416,045 | 3,166 |
| Physics | 413,461 | 1,285 |
| Chemistry | 406,429 | 2,781 |
| Psychology | 364,441 | 2,126 |
| Education | 220,014 | 1,532 |
| Business | 193,536 | 946 |
| Economics | 185,716 | 921 |
| Agricultural and Food Sciences | 333,776 | 2,013 |
| Sociology | 137,257 | 1,535 |
| Mathematics | 135,676 | 199 |
| Political Science | 106,748 | 378 |
| Geology | 67,258 | 217 |
| Geography | 44,269 | 257 |
| Linguistics | 41,737 | 228 |
| History | 36,848 | 192 |
| Law | 30,888 | 251 |
| Philosophy | 27,518 | 148 |
| Art | 26,658 | 75 |
- Downloads last month
- 92