**1. Introduction**

Escalating urbanization and industrialization has resulted in an increased load demand along with the necessity of higher system stability and reliability, which requires the upgrade and new installation of power transmission schemes (PTSs) [1–5]. Moreover, renewable energy integration [6,7], smart grid development [5,8], and the need of interruption-free operation in the case of faults [8,9] also require the implementation of PTSs within metropolitan areas [10,11]. Researchers have described that conventional PTSs include overhead lines (OHLs) [1,3,5,10,12], underground cables (UGCs) [7,13,14] and gas-insulated lines (GILs) [15–18]. Literature regarding the metropolitan application of PTSs mentioned

that OHLs and UGCs encounter hindrances such as right of way [2,3,19], spatial proximity [9,20,21], aesthetics [19,20], system failure due to prolonged fault clearance time [8,9], corrosion [2,22], trench requirements [14,23], and electromagnetic compatibility (EMC) concerns [2,4,21,24,25]. Further, studies mentioned that conventional GILs also face impediments regarding their implementation in urban vicinities due to their metallic profile, such as their structural rigidity [15,25,26], larger bending radius and lay length [15,16,27], jointing complexities [15,17,27], corrosion protection [16,24,28], requirement of acceleration dampers [17,24,29], and trench development [11,27,30]. Thus, protruding urbanization, despite being a potential load consumer, critically curtails the implementation of conventional PTSs in metropolitan vicinities.

References [11,31–34] reveal that flexible gas-insulated lines (FGILs) comprised of a reinforced thermoplastic enclosure, stranded conductor, and polyurethane (PU) post insulator are a potential candidate for curtailing the intricacies associated with the implementation of conventional PTSs in metropolitan areas. Further, researchers [35–37] have mentioned that flexible cables and enclosures like FGILs are practicable for horizontal directional drilling (HDD)-based underground laying schemes and do not require trench development, which is highly beneficial in urban vicinities. Thus, the simplification of several issues associated with conventional PTSs like right of way, EMC concerns, trench requirement, corrosion protection, and larger land area requirement makes FGILs an appropriate scheme for the subsurface metropolitan application of high-voltage lines. However, researchers have mentioned that the contour irregularity of stranded conductors [38] is a point of concern due to its irregular field distribution [39,40], which results in poor field utilization [17,24,41,42] and augments partial discharge activity [43–45] and streamers [43,45,46]. Moreover, references [17,24,41,42] mentioned that the interdependence of enclosure and conductor sizes apropos of field utilization necessitate critical consideration regarding the dimensional specifications of FGILs in case of any variation in their field utilization. Thus, field irregularity due to stranded conductors in FGILs along with its effect upon dimensional specification needs thoughtful consideration.

Researchers [47–56] mentioned that regarding GILs, irregular field distribution and partial discharge activity due to electrode irregularities could be curtailed by the implementation of a solid dielectric layer on the electrode. However, the implementation of a solid dielectric layer in an FGIL would result in its reduced structural flexibility, which is objectionable regarding their metropolitan applications. A probable solution for conductor irregularity suppression in FGILs could be the implementation of a flexible semiconducting film (SCF) over the stranded conductor. SCFs basically exhibit non-linear conducting characteristics and will facilitate the minimization of the field irregularity and field intensity of FGILs without compromising their structural flexibility. Thus, considering the field irregularity concerns of FGILs, in this research, Autodesk Inventor® was used to model the geometric variants of stranded conductors. These conductor models were then analyzed in COMSOL Multiphysics® regarding electrostatic and dielectric aspects along with the development of the geometrically and electrostatically optimized FGIL model. Considering the performed optimized design, an 11 kV scaled-down model of a 132 kV FGIL was also fabricated in order to practically investigate the electrostatic and dielectric stresses in the FGIL through a high-voltage experimental setup. Simulation and experimental investigations revealed that SCF-coated stranded conductor significantly minimized the field irregularity of the FGIL and improved its dielectric breakdown characteristics.

### **2. Stranded Conductor Geometric Variants**

Stranded conductors are normally discriminated on the basis of strand geometry as well as the compactness technique used in the conductor development [57]. The geometric configuration of stranded conductors used in UGCs and OHLs are specified in Table 1, and Figure 1 represents circular and trapezoidal strand conductors [57].

**Table 1.** Stranded conductors used in conventional power transmission systems.

**Figure 1.** (**a**) Circular strand and (**b**) trapezoidal strand conductors used in conventional power transmission schemes.

### *Electric Field Dispersal Regarding Strand Geometry*

The electric field dispersion in a region normally depends upon electrode geometry, surface irregularity, and gap distribution [58]. The field utilization factor (FUF) gives an idea of the effective utilization of field space and facilitates analysis of the electrostatic stresses imposed upon the dielectric material. In general, the FUF can be evaluated through Equation (1), where *Eavg* denotes the average electric field and *Emax* denotes the maximum electric field. Further, pertaining to its coaxial configuration, the FUF for an FGIL can be calculated through Equation (2), where *R* is the enclosure's radius in millimeters and *r* is the conductor's radius in millimeters [41].

$$
\eta = \frac{E\_{\text{avg}}}{E\_{\text{max}}} \tag{1}
$$

$$F = \frac{r\left(\ln\frac{R}{r}\right)}{R-r} \tag{2}$$

### **3. Design and Analysis**

### *3.1. Dimensional Optimization of FGIL Enclosure Apropos of Stranded Conductor*

The selection of a stranded conductor for an FGIL requires reconsideration regarding enclosure diameter because the FUF for GILs is normally kept in the range of 0.5 to 0.6 and is directly related with their dimensional specifications [24,41,42]. In a standard GIL, enclosure and conductor dimensions are normally selected to have approximately 1 as the solution of the logarithmic expression in Equation (2). That is, the enclosure diameter is approximately three times the conductor diameter [24,41,42]. However, in order to have an optimized enclosure size regarding the required FUF, Equation (2) was rearranged for enclosure dimension and expressed as Equation (3). Considering that Equation (3) appears as an implicit equation, its solution was performed through the Newton–Raphson iterative (NR) method in MATLAB® with the required accuracy up to four decimals and an initial estimate of 50 for the unknown parameter (i.e., enclosure radius). The estimated values and their errors showed a converging trend, and the enclosure radius finally converged in eleven iterations up to the required accuracy. Dimensional appraisal of conductor and enclosure revealed that the enclosure was approximately three times the conductor size and resulted in the achievement of the desired FUF.

$$\mathcal{R} = \operatorname{rep}(\mathbb{P}\left(\left(\frac{R}{r}\right) - 1\right))\tag{3}$$

### *3.2. Electrostatic Field Optimization of the FGIL*

Electric field optimization is obligatory in electrical systems in order to eradicate the prospect of dielectric failure due to partial discharge or gap discharge [58]. Considering the stranded conductor as a potential candidate for pliable GILs and concerning its surface irregularity, detailed electrostatic appraisal is essentially required for the proposed scheme. Protrusions and surface irregularities in stranded conductors may lead to escalated electric fields on the conductor's surface contour, which may result in detrimental partial discharge activity followed by dielectric strength degradation due to streamers [43–45,58]. Thus, considering the importance of field dispersion in pliable GIL, COMSOL Multiphysics®-based electrostatic analysis was performed for the FGIL regarding the stranded conductor specimens given in Table 2 in comparison to existing GILs in order to achieve minimal electrostatic stresses as per the required standards for gas-insulated equipment. The stranded conductors used in the electrostatic examination were developed using Autodesk Inventor®. Dimensional and technical specifications like electrode gap, thickness, and diameter for the conventional and proposed schemes were based upon ASTM B 232, ASTM B 857, and 132 kV GIL standards along with the evaluations of Section 3.1 [59,60]. Table 2 presents the detailed specifications of different conductor specimens used in the electrostatic stress investigation [59,61,62].

**Table 2.** Conductor specimens used in the comparative appraisal.

### 3.2.1. Electrostatic Field Dispersion Apropos of Conventional and Stranded Conductors

Concerning the analysis of the field dispersion along with identification of regions of high electric fields in the proposed GIL scheme, COMSOL Multiphysics®-based models for conventional and pliable GILs were developed and compared regarding the different conductor configurations given in Table 2. Figure 2a,b demonstrates the electric potential and electric field dispersion in a conventional GIL. Figure 3a,b exhibits the electric potential and electrostatic field dispersal in the proposed GIL with a circular strand conductor. Figure 4a,b represents the electric potential and electrostatic field distribution in the proposed GIL with trapezoidal strand conductor. Field dispersion regarding conventional and proposed schemes revealed that stranded conductors resulted in regions of concentrated electric field on the conductor's surface contour. Figure 5a,b represents the enlarged view of such concentrated electric field regions in the FGIL scheme regarding specimen 2 and specimen 3 of Table 2. Critical perusal of Figures 2–5 regarding electric field dispersion reveals that due to protrusions and surface irregularities of the stranded conductors, high electric fields appeared on their surface contour as compared to the conventional scheme with a smooth solid conductor. However, the trapezoidal strand conductor had approximately 10% lower magnitude of maximum electric field stresses due to its relatively smoother profile in comparison to the circular strand conductor. Figure 6 compares the average and maximum electric fields for conventional and proposed GIL schemes regarding the different conductor specimens described in Table 2. Further, Figure 7 compares the FUF for conventional and proposed GIL schemes regarding the different conductor specimens described in Table 2. Detailed analysis of Figures 6 and 7 revealed that the surface irregularity of stranded conductors in the proposed pliable GIL resulted in objectionably high electric fields regarding specimen 2 and specimen 3 in comparison to the conventional scheme regarding specimen 1. Further, the field utilization factor was also reduced by 31% and 23% respectively for specimens 2 and 3 regarding proposed pliable GIL in comparison to specimen 1 regarding the conventional scheme. A probable solution to the above stated problem could be to enlarge the enclosure's diameter or to suppress the conductor's irregularity [63–65]. COMSOL Multiphysics ®-based simulations were performed for this purpose, which revealed that enclosure enlargement resulted in the minimization of the irregular field distribution and reduced the electrostatic stresses on the conductor's surface. However, the FUF was reduced in comparison to the standard allowable limit for GILs because as per GIL standards, the enclosure's diameter should be approximately three times the conductor's diameter in order to acquire an FUF in the permissible range of 0.5 to 0.6 [17,24,41,42]. The violation of the aforementioned constraint regarding enclosure diameter resulted in a poor field utilization factor for the proposed scheme, which is objectionable as per GIL standards. Thus, remedial measures regarding suppression of irregularities in the stranded conductor must be taken in order to achieve the required FUF and eradicate concentrated electric field regions.

**Figure 2.** (**a**) Potential di fference and (**b**) field distribution apropos of a conventional gas-insulated transmission line (GIL) regarding specimen 1 of Table 2.

**Figure 3.** (**a**) Potential difference and (**b**) field distribution apropos of the proposed pliable GIL regarding specimen 2 of Table 2.

**Figure 4.** (**a**) Potential difference and (**b**) field distribution apropos of the proposed pliable GIL regarding specimen 3 of Table 2.

**Figure 5.** Location and magnitude of the maximum electric field for the proposed pliable GIL regarding (**a**) specimen 2 and (**b**) specimen 3 of Table 2.

**Figure 6.** Average and maximum electric field comparison regarding the different conductor specimens described in Table 2.

**Figure 7.** Field utilization factor comparison regarding the different conductor specimens described in Table 2.

### 3.2.2. Contour Irregularity Suppression of Stranded Conductor

Considering the objectionable deviations in the field utilization of the proposed FGIL due to stranded conductors, irregularity suppression essentially needs to be done in order to acquire the required FUF. A probable solution could be the implementation of a silicon carbide (SiC)-impregnated polyester-based SCF of 0.1–0.4 mm thickness on the stranded conductor in order to acquire a relatively smoother conductor profile [39,66,67]. The implementation of such film-coated stranded conductors in gas-insulated equipment necessitates detailed electrostatic and dielectric appraisal, as no published research regarding the implementation of field-graded stranded conductors in gas-insulated equipment exists to date.

### 3.2.3. Electrostatic Field Dispersion Apropos of Film-Coated Stranded Conductors

Concerning the effectivity of irregularity suppression for stranded conductors in terms of field utilization factor and electric field dispersion, SCF-coated stranded conductors were developed using Autodesk Inventor®. Dimensional specifications for the SCF-coated stranded conductors were based upon the ASTM B 232 and ASTM B 857 standards for stranded conductors, and the film thickness was based upon the standard film thickness for power cables [57,61,68]. Detailed specifications of the developed film-coated stranded conductors along with conventional GIL conductor are given in Table 3. Considering the conductor specimens given in Table 3, COMSOL Multiphysics®-based pliable GIL models were developed and analyzed in comparison to existing GIL schemes so as to achieve the desired electrostatic performance per the standards for GILs. Figure 8a,b demonstrates the electric potential and electrostatic field dispersion in the proposed pliable GIL scheme regarding specimen 2 of Table 3 respectively. Figure 9a,b exhibits the electric potential and electrostatic field distribution in the proposed pliable GIL scheme regarding specimen 3 of Table 3 respectively. Figure 10a,b shows the enlarged view of high electric field regions in the proposed FGIL scheme regarding specimens 2 and 3 of Table 3 respectively. Critical perusal of Figures 5 and 10 reveals that surface irregularity suppression resulted in substantial reduction in electrostatic stresses on the surface contour of the stranded conductor, and improved the field distribution for both stranded specimens of Table 3. However, specimen 3 had approximately 6% lower magnitude of maximum electrostatic stresses due to its nearly circular profile in comparison to specimen 2. Figure 11 compares the average and maximum electric fields for the conventional and proposed pliable GIL schemes regarding the respective conductor specimens of Table 3. Further, Figure 12 compares the field utilization factor of conventional and proposed GIL schemes regarding the respective conductor specimens of Table 3. Detailed analysis of Figure 12 reveals that surface irregularity suppression resulted in achieving a relatively better FUF, with a trivial deviation of 7.5% and 1.8% regarding specimens 2 and 3 of Table 3 as compared to specimen 1 of the respective Table. In addition, a smoother conductor profile due to the SCF coating also resulted in substantial electrostatic stress reductions in the conductor contour up to 23% and 21% regarding specimens 2 and 3 of Table 3 in comparison to respective simple stranded conductors of Table 2. Further, in comparison to aluminum conductor steel reinforced (ACSR) and all aluminum alloy conductor (AAAC), due to their compact design, trapezoidal stranded conductors of the aluminum conductor steel supported (ACSS) category exhibit higher ampacity and thermal ratings within the same dimensional specifications [62]. Thus specimen 3 of Table 3 could serve as the optimal candidate regarding thermal, ampacity, and electrostatic requirements along with the desired flexibility for the proposed pliable GIL.

**Table 3.** Conductor specimens used in the comparative appraisal.

**Figure 8.** (**a**) Potential difference and (**b**) field distribution apropos of the proposed pliable GIL regarding specimen 2 of Table 3.

**Figure 9.** (**a**) Potential difference and (**b**) field distribution apropos of the proposed pliable GIL regarding specimen 3 of Table 3.

**Figure 10.** Location and magnitude of the maximum electric field for the proposed pliable GIL regarding (**a**) specimen 2 and (**b**) specimen 3 of Table 3.

**Figure 11.** Average and maximum electric field comparison regarding the different conductor specimens described in Table 3.

**Figure 12.** Field utilization factor comparison regarding the different conductor specimens described in Table 3.

### *3.3. Dielectric Appraisal of FGIL Apropos of the Stranded Conductor*

In order to analyze the breakdown characteristics of the dielectric medium in the stranded-conductor-based FGIL model developed above, an analysis was performed regarding its minimum discharge voltage (i.e., breakdown voltage, BV). Existing methodologies for discharge voltage calculation can be categorized on the basis of the streamer breakdown theory as well as the critical field strength evaluations [58,69]. Both methodologies were incorporated in this research in order to ascertain the practicability of the suggested conductor scheme for a pliable GIL.

### 3.3.1. Breakdown Voltage of the Proposed Configuration Regarding Streamer Breakdown Theory

Per the streamer breakdown theory, a streamer may result in a partial discharge such as a corona or a complete gap discharge, and the associated potential level is considered as the breakdown voltage [58,69]. The minimum breakdown voltage in SF6 insulated equipment can be evaluated by using Equation (4), where *BV* is the breakdown voltage in kV, *P* is the gas pressure in kPa, and *d* is the electrode gap in centimeters [58,69]. However, the effect of electrode surface irregularity should also be considered, as it significantly degrades the minimum breakdown voltage. Considering the conductor's surface irregularity in the model developed above, Equation (5) can be used for the evaluation of the minimum breakdown voltage, where *BV* is the breakdown voltage in kV, *C* is the curvature factor, *d* is the electrode gap in centimeters, *F* is the field utilization factor, *P* is the gas pressure in kPa, and *S* is the electrode roughness factor [58]. Further, considering the case when the dielectric medium comprises sulfur hexafluoride and nitrogen gases at a ratio of 20:80, its breakdown strength will be lesser in comparison to pure SF6 gas, and will depend upon the percentage of SF6 in the mixture. Thus, Equation (6) can be used for the evaluation of possible degradation in the minimum breakdown voltage of the gas mixture [69]. Figure 13 shows the electrode gap comparison between the 132-kV conventional and proposed GIL scheme which is further used in the evaluation of the minimum breakdown voltage for the proposed scheme. Reduction in electrode gap resulted due to the reduction of conductor diameter from 89 mm to 44.5 mm, as the proposed scheme comprises a stranded aluminum conductor whereas the conventional scheme comprises a hollow aluminum conductor. However, the ampacity of both conductors was kept approximately the same. Further, the diameter of the ground electrode was also reduced from 226 to 127.2 mm, as it is based on the dimensional evaluations performed in Section 3.1 regarding the standard field utilization factor as well as the standard dimensional specifications for GILs. Figure 14 represents the breakdown voltage for 100% SF6 content and the respective reduction in this breakdown voltage due to surface irregularity of the stranded conductor using Equations (4) and (5). Moreover, it also highlights the reduction in breakdown voltage due to the reduced SF6 content in the SF6 and N2 gas mixture through Equation (6). Critical analysis of Figures 13 and 14 shows that the BV for the given dimensional and operational specifications of the proposed 132 kV pliable GIL was well above the normal operating voltage and the standard basis insulation level (BIL) value of 132 kV. Thus, per the evaluated dimensional specifications, the proposed FGIL scheme exhibits good dielectric withstand capability.

$$BV = \mathbf{1.321} \cdot (Pd)^{0.915} \tag{4}$$

$$BV = \, 0.8775 \, \text{\${}^{\circ}\$C} \cdot \text{\${}^{\circ}\$C} \cdot \text{\${}^{\circ}\$C} \cdot \text{\${}^{\circ}\$\$} \tag{5}$$

$$m = \text{38.03} \cdot n^{0.21} \tag{6}$$

**Figure 13.** Electrode gap comparison between conventional and proposed flexible GIL (FGIL) schemes.

**Figure 14.** Breakdown voltage (BV) appraisal for the proposed FGIL scheme.

3.3.2. Breakdown Field Strength of the Proposed Configuration Regarding Critical Field Intensity Theory

In the critical field intensity method, the breakdown voltage of a dielectric gas is associated with critical field, electrode gap, FUF, electrode surface irregularity, and gas pressure [69]. According to this theory, the operational and design magnitudes of the electric field should be well below its critical value in order to avoid dielectric breakdown through avalanche [58]. Further, in accordance with GIL standards, the typical allowable design criterion regarding electric field strength is approximately 20 kV/mm, and might be higher such that the influenced region is not substantially enormous [24]. Thus, concerning the practical viability of the proposed 132-kV FGIL scheme, its electrostatic field appraisal regarding operational, design, and critical field values is essentially required. The operational and design values of the electric field in the proposed pliable GIL could be evaluated through Equation (7) by considering the normal operating voltage and standard BIL voltages, respectively [41]. Further, Equation (7) can be rearranged by considering Equations (1), (5), and (6) for the evaluation of the critical electric field as shown in Equation (8) [41,58]. In Equation (8), *BV* is the breakdown voltage and *EC* is the critical electric field as evaluated on the basis of Equations (5) and (6). Figure 15

shows the comparison regarding the operational, design, and critical electric fields for the proposed scheme. Critical perusal of Figure 15 shows that the operational and design values for the electric field were well within limits as specified by the standards for gas-insulated equipment, and both field magnitudes were much less than the critical field value. Thus, per the appraised dimensional specifications, the proposed 132-kV FGIL scheme exhibits good electrostatic stress withstand capability.

$$E\_{\text{max}} = \frac{\mathcal{U}\_0}{r \cdot \ln\left(\frac{R}{r}\right)}\tag{7}$$

$$E\_{\mathfrak{c}} = \begin{array}{c} \frac{BV}{f \cdot (\mathbb{R} - r)} \end{array} \tag{8}$$

**Figure 15.** Electric field appraisal for the proposed FGIL scheme regarding operational, standard BIL, and breakdown voltages.

### *3.4. Field Stress Distribution in Bended Segment of FGIL*

In order to analyze the practicability of the proposed scheme for bent segments, simulation regarding electrostatic stress distribution for FGIL in case of bend was performed in comparison to its equivalent straight model. For the said purpose, 20-m-long straight and bent FGIL models were developed using Autodesk Inventor®, and further field distribution and FUF for the two models were analyzed using COMSOL Multiphysics®. Line bending was performed as per the permissible longitudinal minimum bending radius (LMBR) of reinforced polyvinyl chloride (RPVC), and field distribution as well as FUF analysis for the respective FGIL models was performed regarding axial and radial cross sections. Critical appraisal of bent and straight FGIL models regrading axial cross section revealed a trivial deviation of 0.7% and 0.8% in electric field intensity and FUF, respectively. Moreover, detailed comparison of two FGIL models regrading radial cross section showed a slight deviation of 0.4% and 0.5% in electric field intensity and FUF, respectively. Minimal deviation in the compared models was observed because longitudinal bending as per LMBR limits resulted in negligible circumferential deformation as well as gradual bending. Thus, field magnitude and stress distribution for the bent line segmen<sup>t</sup> were nearly the same as those for the straight line segment.

### **4. Fabrication of the Scaled FGIL Model**

Regarding the practical viability of the proposed scheme, an 11-kV scaled-down model of the 132-kV FGIL was fabricated on the basis of electrostatic modeling by replicating the field distribution of a high-voltage GIL for a scaled down model [17,24,41,42]. Considering the standard FUF and allowable maximum electric field for the 132 kV GIL, dimensional specifications regarding enclosure

and conductor of the 11 kV scaled down model were evaluated per the technique described in Section 3.1. After finalizing the dimensional specifications, the scaled down model was first analyzed and compared with the actual 132 kV GIL model by using the technique described in Sections 3.2 and 3.3 regarding field distribution, field magnitude, FUF, and breakdown characteristics using COMSOL Multiphysics ®. Then, a practical model was developed in order to conduct experimental investigations. RPVC was used as the enclosure material, and braided metallic mesh covered with aluminum foil was placed inside the enclosure as the ground terminal. A stranded aluminum conductor was placed inside the RPVC pipe, and threaded Teflon corks were used to prevent any gas leakage from pipe ends. Further, metallic clamps were placed on Teflon corks in order to avoid cork slippage and gas leakage at high gas pressures. Electrically pretested open cell rebond foam of 105 kg/m<sup>3</sup> density was used to achieve concentric conductor alignment inside the enclosure [34]. A gas charging and discharging system was implemented to pressurize the flexible GIL model at di fferent gas pressures, and to create vacuum. The material and thickness of SCF were selected per the scheme described in Sections 3.2.2 and 3.2.3. The specified FGIL model for high-voltage experimentation was developed for simple as well as SCF-wrapped stranded conductors. Figure 16a,b shows the simple and SCF-coated stranded conductors used in the development of the FGIL models respectively. Figure 17a shows the dimensional specifications of the designed flexible GIL model, and Figure 17b shows the fully developed model.

**Figure 16.** (**a**) Simple stranded conductor and (**b**) semiconducting film (SCF)-coated stranded conductor used in the development of FGIL models.

**Figure 17.** (**a**) Dimensional specifications of the scaled FGIL model and (**b**) the fully developed scaled FGIL model.

### **5. Experimental Setup Development**

Pertaining to the practicability of the proposed scheme, an experimental setup was developed regarding lightning impulse and power frequency disruptive discharge tests for the proposed scheme, as per the IEC 60060-1:2010 standard [70]. FGIL models, fabricated respectively with simple and SCF-coated stranded conductors, were used in this experimental investigation. Concerning the lightning impulse discharge tests, U50 for different GIL specimens was determined by Up–Down method, where for power frequency discharge an average of ten disruptive discharges was considered. A compressor and a pressure control unit were incorporated in order to create a vacuum in the developed GIL model along with the injection of dielectric gas at the desired pressure. Gas-insulated equipment normally utilizes pure SF6 or a mixture of SF6 and N2 at a ratio of 20:80, but the required gas pressure in the latter case was almost doubled as compared to the prior case. Considering the security concerns associated with the high-pressure containment of SF6/N2 mixture at the laboratory level, pure SF6 gas was used, as it would result in a significant reduction of the required gas pressure without compromising the insulation characteristics. A block diagram of the experimental setup is shown in Figure 18. Figure 19a represents the different components used in the high-voltage experimentation, while Figure 19b represents the experimental setup placed in a high-voltage laboratory.

**Figure 18.** Experimental setup for the dielectric strength testing of the developed FGIL models.

**Figure 19.** (**a**) Different components used in the discharge tests and (**b**) the experimental setup for lightning impulse and disruptive discharge tests.

(**a**) (**b**)

### *5.1. Dielectric Breakdown Analysis of the Fabricated FGIL Models*

Experimental analysis apropos of the dielectric breakdown of the developed FGIL models was performed for SF6 and air gases at different gas pressures in order to investigate the power frequency and impulse discharge characteristics of the FGILs.

### 5.1.1. Power Frequency Discharge Test

In order to appraise the dielectric characteristics of the FGILs regarding simple and film-coated stranded conductors, power frequency discharge tests using air and SF6 were performed for the fabricated FGIL models. After creating vacuum in the FGIL models, moisture-free air was filled at different gas pressures from 1 to 2.5 bar, and the discharge voltage was noted for both GIL models. Followed by air, similar power frequency discharge tests were performed regarding SF6 gas for both FGIL specimens at different gas pressures from 1 to 2.5 bar. Figure 20 shows the disruptive discharge test results of air- and SF6-filled simple and film-coated stranded-conductor-based FGIL models under different gas pressures. Critical analysis of Figure 20 reveals that the discharge voltage increased with increasing gas pressure in all cases. However, the FGIL model with a film-coated stranded conductor had relatively higher discharge voltages in air as well as sulfur hexafluoride respectively in comparison to the FGIL model with a simple stranded conductor. Further, pertaining to their higher dielectric strength, SF6-filled FGIL models achieved higher breakdown voltages at the respective gas pressures.

**Figure 20.** Power frequency disruptive discharge investigations regarding air and SF6 for different FGIL models.

### 5.1.2. Lightning Impulse Discharge Test

Concerning the impulse withstand characteristics of the developed models, lightning impulse voltage tests were conducted for SF6- and air-filled FGIL models with simple and SCF-coated stranded conductors. After creating vacuum in the FGIL models, moisture-free air was injected at different gas pressures from 1 to 2.5 bar, and lightning impulse discharge voltage was noted for both GIL models. Followed by air, similar lightning impulse discharge tests were performed regarding SF6 gas for both FGIL models at different gas pressures from 1 to 2.5 bar. Figure 21 represents the test results of SF6- and air-filled simple and film-coated stranded-conductor-based GIL models under different gas pressures. Critical analysis of Figure 21 reveals that the impulse discharge voltage increased with increasing gas pressure in all cases. However, the GIL model with a film-coated stranded conductor had relatively higher discharge voltages regarding air as well as sulfur hexafluoride respectively in comparison to the GIL model with a simple stranded conductor. Further, owing to its higher dielectric strength, SF6-filled GIL models achieved the required BIL value for 11 kV beyond 2 bar pressure. Figure 22 shows the

recorded waveforms of lightning impulse discharge tests regarding air and SF6 gases at a pressure of 2.38 bar.

**Figure 21.** Lightning impulse discharge investigations regarding air and SF6 for different FGIL models.

**Figure 22.** Measured lightning impulse discharge voltage regarding air and SF6 in fabricated FGIL model at 2.38 bar.

### *5.2. Critical Field and Breakdown Field Analysis of Fabricated FGIL Models*

Concerning the GIL, constraints regarding dielectric design require that the (*E*/*P*)*Breakdown* should be relatively lesser than the (*E*/*P*)*Critical* of the respective dielectric gas. The critical reduced field strength (*E*/*P*)*Critical* at (α – η) = 0 regarding air and SF6 per the computations using the BOLSIG+ tool were estimated as 30 kV/cm/bar and 89 kV/cm/bar [41]. Here, η represents the electron attachment rate and α is the coefficient of ionization. Regarding the developed experimental setup, the pressure normalized maximum field strength at *U*50, (*Emax*/*P*)*Breakdown*, could be evaluated by rewriting Equation (7) as Equation (9), where *r* represents the conductor's radius in mm, *R* represents the enclosure's radius in mm, and *P* is the gas pressure in kPa. Figure 23 shows the computations by Equation (9) regarding the experimental findings of lightning impulse discharge characteristics for air- and SF6-insulated FGIL models with simple and SCF-coated stranded conductors. Critical analysis of Figure 23 reveals that the (*E*/*P*)*Breakdown* was lesser than the (*E*/*P*)*Critical* for all scenarios, and furthermore, the SCF coating over the

stranded conductor enhanced the breakdown field level for both air- and SF6-insulated FGIL models. Thus, the developed FGIL models fulfill the above-stated dielectric design requirements for GILs.

$$(E\_{\text{max}} / P)\_{Brackldown} = \frac{lI\_{\\$0}}{r \cdot \ln\left(\frac{R}{r}\right) \cdot P} \tag{9}$$

**Figure 23.** Field strength comparison in fabricated FGIL models regarding air and SF6 at different gas pressures.


**4. Absolute quantification of prospective biomarkers in blood plasma with isotopically labeled internal standards**

Absolute quantification of glycation rates at individual modification sites by means of isotopically labeled standards was for the first time proposed by Zhang and co-workers in 2013 [86]. For the early diagnosis of T2DM glycation at the most abundant human plasma protein HSA was monitored by quantitative analysis of its characteristic tryptic peptides. Thereby, all probands were classified into three groups: T2DM (n = 73), IGT (n = 63), and NGT (n = 253). In this study 18O-labeling was used to screen glucose-sensitive and glucose-insensitive peptides within HSAderived peptides. Glucose-sensitive peptides tested as biomarker candidates for T2DM in a clinical plasma samples, and glucose-insensitive peptide was selected as the internal standards. Three peptides (LDELRDEGK (K190), FKDLGEENFK (K12), and KVPQVSTPTLVEVSR (K414)) showed significant different in their concentrations in the T2DM group compared with the IGT group. Among them two peptides FKDLGEENFK and KVPQVSTPTLVEVSR exhibited significant differences between both NGT/IGTand IGT/T2DM groups indicating that these peptides could be used as potential biomarkers for the early diagnosis of T2DM. It is important to mention that peptides FKDLGEENFK and KVPQVSTPTLVEVSR showed excellent sensitivities (97.23 and 94.47%, respectively) and specificities (93.65 and 98.41%, respectively) between the NGT/IGT groups. For the NGT/IGT and T2DM groups sensitivity of peptides FKDLGEENFK and KVPQVSTPTLVEVSR was 97.06 and 99.27%, respectively, and specificities was 97.23 and 94.47%. It indicates that these peptides could be prospective biomarkers for the early diagnosis of T2DM [86].

The internal standardization with stable isotope-labeled synthetic peptides and related stable isotope dilution techniques are widely used in glycation research [84, 95, 111, 112]. A stable isotope label can be easily introduced in the step of amino acid building block during the synthesis of Amadori-modified peptides [115]. These peptides can be directly used in the stable isotope dilution approach for absolute quantification (AQUA) [116]. Implementation of synthetic Amadori-modified peptides increases robustness, precision and accuracy of biomarkers analysis [117].

In the classical way this strategy was established for glycated peptides by Spiller and co-workers [118], who applied six standard synthetic peptides containing two isotope-labeled residues (introduced by Fmoc-(13C6, 15N) leucine and Fmoc- ( 13C5, 15N) proline) along with Amadori modification, introduced by the global post-synthetic glycation approach [119]. The peptides represented six glycation sites in HSA – TCVADESAENCDKSLHTLFGDK (K64), SLHTLFGDKLCTVATLR (K73), AACLLPKLDELRDEGK (K181), ADLAKYICENQDSISSK (K262), VFDEFKPLVEEPQNLIK (K378) and KLVAASQAALGL (K574). The standard mixtures were spiked (25 μL, 1.2 μmol/L) to the tryptic digest, obtained from five T2DM patients and five non-diabetic individuals and, after BAC enrichment and SPE, analyzed by a hybrid quadrupole-linear ion trap MS in the MRM mode with three specific Q1/Q3 *m/z* ranges (transitions) for each analyte [118].

**77**

*Individual Glycation Sites as Biomarkers of Type 2 Diabetes Mellitus*

All six analytes and corresponding standard peptides could be detected in all ten plasma samples. The quantities varied from 22.6 ± 3.3 to 180.3 ± 8.8 pmol per mg plasma protein. Also it was shown that content of all six glycated peptides were statistically different between the two cohorts. This study demonstrated the applicability of the AQUA technique to quantification of glycation sites in plasma proteins. In agreement with the results of LFQ studies [83], individual HSA glycation sites responded to hyperglycaemia in different ways. Interestingly, significantly higher, in comparison to others, abundance of glycated sites K64 and K378 was observed [118]. For absolute quantification of glycated peptides by the standard isotope dilution technique Prof. Stefanowicz and co-workers used bi-labeled peptides that contained

C6

KKDabK represented the sequence of Ig kappa chain C region protein,

tion of individual glycation sites in blood plasma proteins [120].

Later Spiller *et al.* [97] studied the glycation degrees of 27 glycation sites representing nine plasma proteins in 48 newly diagnosed male T2DM patients and 48 non-diabetic individuals. After protein digestion with trypsin, samples were spiked with concentration-balanced mixture of synthetic 13C,15N-labeled glycated peptides (synthesized according Spiller at al [118]) as internal standards. The quantification was based on MRM using specific transitions for each targeted peptide and isotopelabeled internal peptide standards. The samples from two groups of participants were evaluated by different statistical tests (Kolmogorow-Smirnow, Mann–Whitney, and *t* test), classified by a decision tree algorithm using HbA1c in combination with each glycated peptide. Also to find the best feature set for classification, support vector machine-recursive feature elimination (SVM-RFE) method was performed for all glycated peptides and clinical parameters of participants, including HbA1c, fasting plasma glucose (FPG), body mass index (BMI), etc. The most interesting results were obtained for glycated peptide AVGDKLPECEAVCGKPK (K141) from haptoglobin which half-life time is 2–4 days. The combination of two biomarkers of T2DM glycated K141 of haptoglobin and HbA1cprovided a sensitivity of 94%, a specificity of 98%, and an accuracy of 96% to identify T2DM.But a set of 15 features considering three glycation sites in HSA, K141 in haptoglobin, and 11 routine laboratory measures of T2DM, metabolic syndrome, obesity, inflammation, and insulin

KKDabK corresponded to the HSA sequence were synthesized. The mixture of standard stable isotope-labeled peptides (20 pmol each) was added to aliquots of plasma samples (of 5 T2DM patients and 5 non-diabetic individuals). After tryptic digestion, samples were analyzed by QqTOF-MS. Glycated peptides from plasma samples and standards were annotated in MS scans (*m/z* 400–2000) by their tR, *m/z* and isotopic patterns and confirmed by MS/MS analysis using Orbitrap Elite MS. The corresponding MS profiles clearly represented isotopic patterns of both light and heavy peptides with good sensitivities that gave access to reliable quantification by peak areas of analytes and corresponding internal standards. All three potential biomarker peptides demonstrated a significantly higher (in 1.5–1.9-fold) content in diabetic patients, in comparison to that in non-diabetic controls. The obtained results were cross-validated by label-free quantification performed in an independent RP-HPLC-ESI-QqTOF-MS study [120]. The resulting fold changes were close to those observed with the stable isotope dilution approach, proposed by Spiller et al. [118]. It indicates similar power of the both methods for absolute quantifica-

that cleaves during digestion procedure [115]. Later on we have shown applicability of these bi-labeled glycated peptides for the absolute quantification of individual glycation sites in plasma proteins [120]. Based on the previous label-free quantification [83] the prevalent glycated peptides with biomarker properties were chosen. The Amadori-modified standard peptides DSTYSLSSTLTLSKAmADYE13C6

15N2-lysine, and comprise a dabsyl moiety

15N2-KKDabK and VFDEFKAmPLVEEPQNLI13C6

15N2-

15N2-

*DOI: http://dx.doi.org/10.5772/intechopen.95532*

stable isotope label, introduced as 3

ADLAKAmYICENQDSISS13C6

#### *Individual Glycation Sites as Biomarkers of Type 2 Diabetes Mellitus DOI: http://dx.doi.org/10.5772/intechopen.95532*

*Type 2 Diabetes - From Pathophysiology to Cyber Systems*

**with isotopically labeled internal standards**

assess the predictive potential of these markers. However, on this way, the limitations of label-free approach become critical. Indeed, LFQ is disadvantages for analysis of large cohorts due to sensitivity of electrospray ionization technique (ESI) to multiple factors, which is manifested as matrix effects [113]. Thus, the analysis conditions (e.g. temperature, experimenter, column condition) must be as constant as possible, that is difficult to achieve with big batch sizes. And duration of batch analysis can be rather long, as prolonged gradients are often used to improve peptide separation [114]. To overcome these limitations, absolute quantification strategies can be employed [86].

**4. Absolute quantification of prospective biomarkers in blood plasma**

Absolute quantification of glycation rates at individual modification sites by means of isotopically labeled standards was for the first time proposed by Zhang and co-workers in 2013 [86]. For the early diagnosis of T2DM glycation at the most abundant human plasma protein HSA was monitored by quantitative analysis of its characteristic tryptic peptides. Thereby, all probands were classified into three groups: T2DM (n = 73), IGT (n = 63), and NGT (n = 253). In this study 18O-labeling was used to screen glucose-sensitive and glucose-insensitive peptides within HSAderived peptides. Glucose-sensitive peptides tested as biomarker candidates for T2DM in a clinical plasma samples, and glucose-insensitive peptide was selected as the internal standards. Three peptides (LDELRDEGK (K190), FKDLGEENFK (K12), and KVPQVSTPTLVEVSR (K414)) showed significant different in their concentrations in the T2DM group compared with the IGT group. Among them two peptides FKDLGEENFK and KVPQVSTPTLVEVSR exhibited significant differences

between both NGT/IGTand IGT/T2DM groups indicating that these peptides could be used as potential biomarkers for the early diagnosis of T2DM. It is important to mention that peptides FKDLGEENFK and KVPQVSTPTLVEVSR showed excellent sensitivities (97.23 and 94.47%, respectively) and specificities (93.65 and 98.41%, respectively) between the NGT/IGT groups. For the NGT/IGT and T2DM groups sensitivity of peptides FKDLGEENFK and KVPQVSTPTLVEVSR was 97.06 and 99.27%, respectively, and specificities was 97.23 and 94.47%. It indicates that these peptides could be prospective biomarkers for the early diagnosis of T2DM [86]. The internal standardization with stable isotope-labeled synthetic peptides and

related stable isotope dilution techniques are widely used in glycation research [84, 95, 111, 112]. A stable isotope label can be easily introduced in the step of amino acid building block during the synthesis of Amadori-modified peptides [115]. These peptides can be directly used in the stable isotope dilution approach for absolute quantification (AQUA) [116]. Implementation of synthetic Amadori-modified peptides increases robustness, precision and accuracy of biomarkers analysis [117]. In the classical way this strategy was established for glycated peptides by Spiller

and co-workers [118], who applied six standard synthetic peptides containing

(K73), AACLLPKLDELRDEGK (K181), ADLAKYICENQDSISSK (K262),

three specific Q1/Q3 *m/z* ranges (transitions) for each analyte [118].

15N) proline) along with Amadori modification, introduced by the global post-synthetic glycation approach [119]. The peptides represented six glycation sites in HSA – TCVADESAENCDKSLHTLFGDK (K64), SLHTLFGDKLCTVATLR

VFDEFKPLVEEPQNLIK (K378) and KLVAASQAALGL (K574). The standard mixtures were spiked (25 μL, 1.2 μmol/L) to the tryptic digest, obtained from five T2DM patients and five non-diabetic individuals and, after BAC enrichment and SPE, analyzed by a hybrid quadrupole-linear ion trap MS in the MRM mode with

15N) leucine and Fmoc-

two isotope-labeled residues (introduced by Fmoc-(13C6,

**76**

( 13C5,

All six analytes and corresponding standard peptides could be detected in all ten plasma samples. The quantities varied from 22.6 ± 3.3 to 180.3 ± 8.8 pmol per mg plasma protein. Also it was shown that content of all six glycated peptides were statistically different between the two cohorts. This study demonstrated the applicability of the AQUA technique to quantification of glycation sites in plasma proteins. In agreement with the results of LFQ studies [83], individual HSA glycation sites responded to hyperglycaemia in different ways. Interestingly, significantly higher, in comparison to others, abundance of glycated sites K64 and K378 was observed [118].

For absolute quantification of glycated peptides by the standard isotope dilution technique Prof. Stefanowicz and co-workers used bi-labeled peptides that contained stable isotope label, introduced as 3 C6 15N2-lysine, and comprise a dabsyl moiety that cleaves during digestion procedure [115]. Later on we have shown applicability of these bi-labeled glycated peptides for the absolute quantification of individual glycation sites in plasma proteins [120]. Based on the previous label-free quantification [83] the prevalent glycated peptides with biomarker properties were chosen. The Amadori-modified standard peptides DSTYSLSSTLTLSKAmADYE13C6 15N2- KKDabK represented the sequence of Ig kappa chain C region protein, ADLAKAmYICENQDSISS13C6 15N2-KKDabK and VFDEFKAmPLVEEPQNLI13C6 15N2- KKDabK corresponded to the HSA sequence were synthesized. The mixture of standard stable isotope-labeled peptides (20 pmol each) was added to aliquots of plasma samples (of 5 T2DM patients and 5 non-diabetic individuals). After tryptic digestion, samples were analyzed by QqTOF-MS. Glycated peptides from plasma samples and standards were annotated in MS scans (*m/z* 400–2000) by their tR, *m/z* and isotopic patterns and confirmed by MS/MS analysis using Orbitrap Elite MS. The corresponding MS profiles clearly represented isotopic patterns of both light and heavy peptides with good sensitivities that gave access to reliable quantification by peak areas of analytes and corresponding internal standards. All three potential biomarker peptides demonstrated a significantly higher (in 1.5–1.9-fold) content in diabetic patients, in comparison to that in non-diabetic controls. The obtained results were cross-validated by label-free quantification performed in an independent RP-HPLC-ESI-QqTOF-MS study [120]. The resulting fold changes were close to those observed with the stable isotope dilution approach, proposed by Spiller et al. [118]. It indicates similar power of the both methods for absolute quantification of individual glycation sites in blood plasma proteins [120].

Later Spiller *et al.* [97] studied the glycation degrees of 27 glycation sites representing nine plasma proteins in 48 newly diagnosed male T2DM patients and 48 non-diabetic individuals. After protein digestion with trypsin, samples were spiked with concentration-balanced mixture of synthetic 13C,15N-labeled glycated peptides (synthesized according Spiller at al [118]) as internal standards. The quantification was based on MRM using specific transitions for each targeted peptide and isotopelabeled internal peptide standards. The samples from two groups of participants were evaluated by different statistical tests (Kolmogorow-Smirnow, Mann–Whitney, and *t* test), classified by a decision tree algorithm using HbA1c in combination with each glycated peptide. Also to find the best feature set for classification, support vector machine-recursive feature elimination (SVM-RFE) method was performed for all glycated peptides and clinical parameters of participants, including HbA1c, fasting plasma glucose (FPG), body mass index (BMI), etc. The most interesting results were obtained for glycated peptide AVGDKLPECEAVCGKPK (K141) from haptoglobin which half-life time is 2–4 days. The combination of two biomarkers of T2DM glycated K141 of haptoglobin and HbA1cprovided a sensitivity of 94%, a specificity of 98%, and an accuracy of 96% to identify T2DM.But a set of 15 features considering three glycation sites in HSA, K141 in haptoglobin, and 11 routine laboratory measures of T2DM, metabolic syndrome, obesity, inflammation, and insulin

resistance provided a sensitivity of 98%, a specificity of 100%, and an accuracy of 99% for newly diagnosed T2DM patients. This study shows great potential of glycation sites in plasma proteins providing an additional diagnostic tool for T2DM and elucidating that the combination of these sites with HbA1c and FPG could improve the diagnosis of T2DM. The combination of both biomarkers HbA1c and glycated haptoglobin with half-life times 2 to 4 days is sensitive to long- and short-term fluctuations of blood glucose concentrations [97].

Selected 27 glycated peptides were tested further in the quality of multiple biomarker set [121]. For this plasma samples from 48 patients with duration of T2DM for more than 10 years, 48 non-diabetic individuals and 20 pre-diabetic persons we examined. The strategy of analysis was the same as described above [97]. In longterm controlled T2DM patients, 27 glycated peptides were detected at significantly higher levels and provided moderate diagnostic accuracies (ACCs) from 61 to 79%, resulting in sub-grouping of patients in three distinct clusters. In this study, a feature set of one glycated peptides from haptoglobin (K141) and 6 established clinical parameters provided an ACC of 95%. The same number of clusters was identified in pre-diabetic males (ACC of 95%) using a set of 8 glycation sites (mostly from HSA). Re-examination of all patients present in one cluster showed progression of pre-diabetic state or advanced towards diabetes in the following five years. Overall, the studied glycation sites can play a role of promising biomarkers for sub-grouping pre-diabetic patients to estimate their risk for the development of T2DM [121].

Together with our recent report [84] these work clearly indicate under-explored potential of integrated peptide biomarkers. Moreover, it is well seen, that absolute quantification approaches are preferable, due to their higher precision and lower method-related dispersion. Thus, it is obvious, that after the explorative LFQ-based study, follow-up absolute quantification screening of large cohorts is necessary.

Besides 18O-labeling and AQUA, tag-based labeling approaches were used for quantification of glycation sites. Thus, Qiu*et al*. proposed the use of isobaric tags for relative and absolute quantification (iTRAQ ) to reveal differences in HSA glycation patterns between healthy individuals and diabetic patients [88]. The authors described *in vitro* and *in vivo* experiments have been carried out to evaluate the impact of HSA glycation on the binding to anticoagulant drugs (warfarin and heparin). Plasma samples from 32 diabetic patients and 33 healthy individuals were treated with polyethylene glycol for precipitation of immunoglobulins. After this, the glycated albumin (GA) was separated using BAC. Trypsin digested GA was separated on C18-AQ analytical column and analyzed using a LTQ-Orbitrap Velos Pro MS operated in DDA mode with CID and further with ETD fragmentation. For quantitative analysis, the digested samples were labeled with iTRAQ 8-plex reagents. After labeling, the 8 samples with different mass tags were evenly mixed and glycated peptides with iTRAQ labels were extracted from the pooled samples using BAC. MS analysis was performed on a LTQ-Orbitrap Fusion MS operated in the DDA mode for CID-MS/MS and HCD-MS3 . A total of 49 glycation sites (including 43 glycated lysines and six glycated arginines) on GA were successfully identified using this approach. Among them seven glycation sites, R81, R117, R186, R257, K313, R410 and K541, were discovered for the first time. It is interesting that glycation at sites K4, R81, R117, K439, K519, K538, K541, K557 and K573 were specifically present in diabetic patients, while two sites, R410 and K436, were found only in healthy subjects. Altogether 21 glycation sites were quantified, and 19 of them, including K51, K64, K93, K162, K199, K233, K262, K313, K323, K378, K402, K414, K466, K475, K525, K545, K557, K564 and K574, showed statistically enhanced glycation during diabetes [88].

Examples of methods used for absolute quantification of prospective biomarkers in blood plasma with isotopically labeled internal standards are summarized in **Table 2**.

**79**

**#**

1 2 3 4 5 6 **Table 2.**

*(spec.)*

HSA (K51, K64, K93, K162, K199, K233, K262, K313, K323, K378,

K402,K414, K466, K475, K525, K545, K557, K564 and K574)

Haptoglobin (K141), HSA (K262, K378, K73, K525, K574, K359, K174,

K64), serotransferrin (K683)

Haptoglobin (K141), HSA (K93, K262, and K414)

13C,15N-labelling

13C,15N-labelling

18O-labeling *Overview of methods used for absolute quantification of prospective biomarkers in blood plasma with isotopically labeled internal standards. Accuracy (accur.), sensitivity (sens.), specificity*

ESI-QqLIT ESI-QqLIT

ESI-LTQ-Orbitrap

Statistical difference between control and

[88]

T2DM p < 0.05

Ig kappa chain C region protein (K75), HSA (K286, K402)

*L*-lysine-3

C615N2 and dabsyl moiety

ESI-QqTOF

Significantly higher

[120]

(in 1.5–1.9-fold)

content in T2DM

Sens. 98%, spec.

[97]

100%, accur. 99%

Diagnostic accuracy

[121]

95%

HSA (K64, K73, K181, K262, K378, K574).

HSA (K414)

**Proteins and sites of glycation**

**Peptide labeling**

18O-labeling *L*-leucine-13C6,15N, *L*-proline 13C5,15N

ESI-QqLIT

Statistical difference

[118]

between control and

T2DM: p < 0.0001 or

p < 0.05

**MS** ESI-QqTOF

**Method power**

Sens. 99.27%, spec.

[86]

94.47%

**Ref**

*Individual Glycation Sites as Biomarkers of Type 2 Diabetes Mellitus*

*DOI: http://dx.doi.org/10.5772/intechopen.95532*

#### *Individual Glycation Sites as Biomarkers of Type 2 Diabetes Mellitus DOI: http://dx.doi.org/10.5772/intechopen.95532*

*Type 2 Diabetes - From Pathophysiology to Cyber Systems*

fluctuations of blood glucose concentrations [97].

the DDA mode for CID-MS/MS and HCD-MS3

resistance provided a sensitivity of 98%, a specificity of 100%, and an accuracy of 99% for newly diagnosed T2DM patients. This study shows great potential of glycation sites in plasma proteins providing an additional diagnostic tool for T2DM and elucidating that the combination of these sites with HbA1c and FPG could improve the diagnosis of T2DM. The combination of both biomarkers HbA1c and glycated haptoglobin with half-life times 2 to 4 days is sensitive to long- and short-term

Selected 27 glycated peptides were tested further in the quality of multiple biomarker set [121]. For this plasma samples from 48 patients with duration of T2DM for more than 10 years, 48 non-diabetic individuals and 20 pre-diabetic persons we examined. The strategy of analysis was the same as described above [97]. In longterm controlled T2DM patients, 27 glycated peptides were detected at significantly higher levels and provided moderate diagnostic accuracies (ACCs) from 61 to 79%, resulting in sub-grouping of patients in three distinct clusters. In this study, a feature set of one glycated peptides from haptoglobin (K141) and 6 established clinical parameters provided an ACC of 95%. The same number of clusters was identified in pre-diabetic males (ACC of 95%) using a set of 8 glycation sites (mostly from HSA). Re-examination of all patients present in one cluster showed progression of pre-diabetic state or advanced towards diabetes in the following five years. Overall, the studied glycation sites can play a role of promising biomarkers for sub-grouping pre-diabetic patients to estimate their risk for the development of T2DM [121].

Together with our recent report [84] these work clearly indicate under-explored potential of integrated peptide biomarkers. Moreover, it is well seen, that absolute quantification approaches are preferable, due to their higher precision and lower method-related dispersion. Thus, it is obvious, that after the explorative LFQ-based study, follow-up absolute quantification screening of large cohorts is necessary. Besides 18O-labeling and AQUA, tag-based labeling approaches were used for quantification of glycation sites. Thus, Qiu*et al*. proposed the use of isobaric tags for relative and absolute quantification (iTRAQ ) to reveal differences in HSA glycation patterns between healthy individuals and diabetic patients [88]. The authors described *in vitro* and *in vivo* experiments have been carried out to evaluate the impact of HSA glycation on the binding to anticoagulant drugs (warfarin and heparin). Plasma samples from 32 diabetic patients and 33 healthy individuals were treated with polyethylene glycol for precipitation of immunoglobulins. After this, the glycated albumin (GA) was separated using BAC. Trypsin digested GA was separated on C18-AQ analytical column and analyzed using a LTQ-Orbitrap Velos Pro MS operated in DDA mode with CID and further with ETD fragmentation. For quantitative analysis, the digested samples were labeled with iTRAQ 8-plex reagents. After labeling, the 8 samples with different mass tags were evenly mixed and glycated peptides with iTRAQ labels were extracted from the pooled samples using BAC. MS analysis was performed on a LTQ-Orbitrap Fusion MS operated in

ing 43 glycated lysines and six glycated arginines) on GA were successfully identified using this approach. Among them seven glycation sites, R81, R117, R186, R257, K313, R410 and K541, were discovered for the first time. It is interesting that glycation at sites K4, R81, R117, K439, K519, K538, K541, K557 and K573 were specifically present in diabetic patients, while two sites, R410 and K436, were found only in healthy subjects. Altogether 21 glycation sites were quantified, and 19 of them, including K51, K64, K93, K162, K199, K233, K262, K313, K323, K378, K402, K414, K466, K475, K525, K545, K557, K564 and

Examples of methods used for absolute quantification of prospective biomarkers in blood plasma with isotopically labeled internal standards are summarized in

K574, showed statistically enhanced glycation during diabetes [88].

. A total of 49 glycation sites (includ-

**78**

**Table 2**.

*Overview of methods used for absolute quantification of prospective biomarkers in blood plasma with isotopically labeled internal standards. Accuracy (accur.), sensitivity (sens.), specificity (spec.)*


**1. Introduction**

Monomer and polymer production from renewable feedstocks has become a relevant research target with the aim of providing more environmental friendly solutions to the actual fossil-based market [1–6]. In this framework, 5-(hydroxymethyl)furfural (HMF) is recognized as an ideal platform molecule to develop different green products, since it can be obtained via acid-catalyzed dehydration of biomass-derived sugars [7–9], and, in turn, it can be converted into a wide range of different high added value chemicals [10–13]. Among the HMF products, 2,5-furandicarboxylic acid (FDCA, Scheme 1) has been identified as one of the most interesting [14], since it can be considered as the bioderived counterpart of terephthalic acid for the production of polyesters [15], such as polyethylene 2,5-furandicarboxylate (PEF), being the latter the potential candidate to replace polyethylene terephthalate (PET) in bottle production [16,17]. *Processes* **2020**, *8*, 45 2 of 15

$$\stackrel{\mathsf{\*}}{\mathcal{J}}\_{\mathsf{G}}\bigotimes\_{\mathsf{G}}^{\mathsf{G}\mathsf{T}}\bigotimes\_{\mathsf{G}}^{\mathsf{G}\mathsf{T}\mathsf{S}}\bigotimes\_{\mathsf{G}}^{\mathsf{G}}\bigotimes\_{\mathsf{G}\mathsf{T}\mathsf{S}}^{\mathsf{G}\mathsf{T}}\bigotimes\_{\mathsf{G}}^{\mathsf{G}\mathsf{T}\mathsf{S}}\bigotimes\_{\mathsf{G}}^{\mathsf{G}\mathsf{T}\mathsf{S}}\bigotimes\_{\mathsf{G}}^{\mathsf{G}\mathsf{T}\mathsf{S}}\bigotimes\_{\mathsf{G}}^{\mathsf{G}\mathsf{T}}\bigotimes\_{\mathsf{G}}^{\mathsf{G}}\bigotimes\_{\mathsf{G}}^{\mathsf{G}}$$

**Scheme 1.** 5-(hydroxymethyl)furfural selective oxidation to 2,5-furandicarboxylic acid. **Scheme 1.** 5-(hydroxymethyl)furfural selective oxidation to 2,5-furandicarboxylic acid.

First attempts to industrially convert HMF to FDCA relied on the technologies developed for terephthalic acid production [18]. However, the use of corrosive solvent, homogeneous catalysts and harsh operative conditions forced the investigation of other catalytic systems in order to overcome such constraints. In this regard, precious metal nanoparticles showed their high potential, Au-based systems being the most investigated [19–21]. In the definition of the properties of such catalytic systems, several parameters seem to be fundamental such as: nanoparticle composition and dimension, support/nanoparticle interaction and support surface textural properties. The fine tuning of such parameters can lead to very active systems. For instance, by alloying Au with another metal (such as Cu [22,23] or Pd [24,25]) it is possible to effectively enhance FDCA yield, or by choosing suitable supports catalytic activity can be positively affected [26] and/or base addition can be avoided [27–29]. Besides the textural properties that have been shown to play a prominent role, pore dimension and acid/base sites can be considered of crucial importance [30]. Thus, it is evident that First attempts to industrially convert HMF to FDCA relied on the technologies developed for terephthalic acid production [18]. However, the use of corrosive solvent, homogeneous catalysts and harsh operative conditions forced the investigation of other catalytic systems in order to overcome such constraints. In this regard, precious metal nanoparticles showed their high potential, Au-based systems being the most investigated [19–21]. In the definition of the properties of such catalytic systems, several parameters seem to be fundamental such as: nanoparticle composition and dimension, support/nanoparticle interaction and support surface textural properties. The fine tuning of such parameters can lead to very active systems. For instance, by alloying Au with another metal (such as Cu [22,23] or Pd [24,25]) it is possible to effectively enhance FDCA yield, or by choosing suitable supports catalytic activity can be positively affected [26] and/or base addition can be avoided [27–29]. Besides the textural properties that have been shown to play a prominent role, pore dimension and acid/base sites can be considered of crucial importance [30]. Thus, it is evident that the design and synthesis of materials that possess suitable features can lead to optimal catalytic activity.

the design and synthesis of materials that possess suitable features can lead to optimal catalytic activity. As far as the setups employed to carry out this reaction, batch ones are the most studied. However, the economical sustainability of this kind of approach is still a concern [31]. Thus, to address this issue, efforts must be devoted to process intensification, for instance by evaluating the possibility of developing inexpensive catalytic systems to perform this reaction continuously [32,33], and, in this frame, recent reports have highlighted the interest of both industrial [34,35] and academic research [36,37]. In this context, the use of catalytic membranes is known to provide several advantages, making them efficient tools for applications in several industrial fields [38]. In the case of HMF oxidation, considering that the reaction is carried out under mild operative conditions (70– 120 °C), it is possible that the use of composite polymeric membranes might be advantageous. Along As far as the setups employed to carry out this reaction, batch ones are the most studied. However, the economical sustainability of this kind of approach is still a concern [31]. Thus, to address this issue, efforts must be devoted to process intensification, for instance by evaluating the possibility of developing inexpensive catalytic systems to perform this reaction continuously [32,33], and, in this frame, recent reports have highlighted the interest of both industrial [34,35] and academic research [36,37]. In this context, the use of catalytic membranes is known to provide several advantages, making them efficient tools for applications in several industrial fields [38]. In the case of HMF oxidation, considering that the reaction is carried out under mild operative conditions (70–120 ◦C), it is possible that the use of composite polymeric membranes might be advantageous. Along with all the advantages related to the more traditional inorganic membranes, these materials are characterized by low production costs, ease of handling and tunability of their properties [39].

with all the advantages related to the more traditional inorganic membranes, these materials are characterized by low production costs, ease of handling and tunability of their properties [39]. Electrospinning provides a convenient approach for the preparation and scale-up of membranes made of continuous sub-micrometric fibers characterized by large surface area and porosity [40,41]. This could represent an interesting strategy for the production of catalytic membranes, which can be used in processes for biomass valorization. Briefly, this technology uses electrostatic forces to uniaxially stretch a viscoelastic jet derived from a polymer solution to produce fibers having Electrospinning provides a convenient approach for the preparation and scale-up of membranes made of continuous sub-micrometric fibers characterized by large surface area and porosity [40,41]. This could represent an interesting strategy for the production of catalytic membranes, which can be used in processes for biomass valorization. Briefly, this technology uses electrostatic forces to uniaxially stretch a viscoelastic jet derived from a polymer solution to produce fibers having diameters ranging from a few tenths of nanometers to a few micrometers, collected as nonwovens with mesh porosity typically higher than 80% and pore diameters that can vary from a few to tens of micrometers.

diameters ranging from a few tenths of nanometers to a few micrometers, collected as nonwovens with mesh porosity typically higher than 80% and pore diameters that can vary from a few to tens of micrometers. Electrospun membranes are currently being investigated as materials for the support of heterogeneous catalysts by following different technological approaches, the main one being the production of ceramic fibers that support metal nanoparticles [40]. In this case, a polymer solution containing ceramic precursors and metal salts is electrospun and subsequently heat treated under inert gas to eliminate the organic components and reduce the metal precursor to metal nanoparticles [42–45]. This approach permits the achievement of high catalytic performances by exploiting the high surface area of the fibers but suffers from the high fragility of the completely inorganic nonwoven. Conversely, by keeping unaltered the organic polymeric component, membrane flexibility and Electrospun membranes are currently being investigated as materials for the support of heterogeneous catalysts by following different technological approaches, the main one being the production of ceramic fibers that support metal nanoparticles [40]. In this case, a polymer solution containing ceramic precursors and metal salts is electrospun and subsequently heat treated under inert gas to eliminate the organic components and reduce the metal precursor to metal nanoparticles [42–45]. This approach permits the achievement of high catalytic performances by exploiting the high surface area of the fibers but suffers from the high fragility of the completely inorganic nonwoven. Conversely, by keeping unaltered the organic polymeric component, membrane flexibility and handling can be massively improved. Following this approach, polymeric electrospun nanofibers have been decorated at the surface with metal nanoparticles (NPs) [46,47] in an elegant and effective way that exploits polymer bulk properties and maximizes the catalytic effect. However, NP immobilization at a fiber

handling can be massively improved. Following this approach, polymeric electrospun nanofibers

surface requires a further step in the production process that is time consuming and hardly applicable at the industrial level. Moreover, leaching of metal NPs from a fiber surface cannot be excluded.

In this work, electrospinning is used in a simple and scalable single-step approach for the production of electrospun polymer-inorganic catalytic membranes, potentially suitable for batch and continuous processes. Membranes were manufactured by incorporating preformed Au and Au/Pd NPs (Au/Pd molar ratio 6, which was demonstrated to promote the highest catalytic activity in this reaction [24]) and TiO2 in the starting polymeric solutions. To optimize catalytic activity and stability, two different polymers have been tested—i.e., polyacrylonitrile (PAN) and Nylon 6,6 (NYL)—loaded with Au and alloyed Au/Pd NPs, either directly supported on TiO2 or simply combined with TiO2 during electrospinning. The catalytic activity of catalysts contained in different electrospun membranes towards HMF oxidation to FDCA has been investigated with the goal of highlighting the effect of polymer/inorganic combination on membrane performance. The materials were evaluated in batch experiments to assess the viability for use of electrospun polymer-based catalytic membranes in the conversion of renewable molecules in water.


# **Creating Age-friendly Communities Housing and Technology**

Edited by

Joost van Hoof, Hannah R. Marston, Katie Brittain and Helen Barrie

Printed Edition of the Special Issue Published in *Healthcare*

www.mdpi.com/journal/healthcare


**2. Mathematical Modeling**

The peristaltic (or "sinusoidal") motion of Synovial fluid described by generalized incompressible fluid possesses the Navier-Stokes equations with a viscosity depending on a shear rate and concentration. We must couple this system with one extra convection-diffusion equation for a concentration of hyaluronic acid. The fundamental equations of governing flow with synovial fluid model are described in reference [8] as follows:

$$\text{div}V = 0\tag{1}$$

$$\frac{\partial V}{\partial t} + V \cdot \nabla V + \frac{\nabla p}{\rho} = \frac{2}{\rho} \text{div}(\Theta) \tag{2}$$

$$\frac{\partial \mathcal{C}}{\partial t} = \text{div}(F(\mathcal{C})) - \mathbf{V} \cdot \nabla \mathcal{C} \tag{3}$$

in above equation:

$$F(\mathbb{C}) = D\_{\mathbb{C}} \nabla \mathbb{C}, \; \Theta = \mu(\mathbb{C}, D) \; D \tag{4}$$

in which Δ*V*(*U*,*V*) is velocity, μ is viscosity, *D* is symmetric part of velocity gradient, *P* is pressure, ρ is density, *F* is concentration flux, *C* is concentration of hyaluronan/hyaluronic, and *D*C is constant diffusivity.

Let us focus on two-dimensional peristaltic flows in an asymmetric channel containing width d1 + d2 due to wave traveling in direction of flow with constant velocity *c*. The flow is discussed in Cartesian coordinates. The mass concentrations upon the upper wall are *C*0, whereas on the bottom wall they are *C*1. Peristaltic motion on the upper and lower internal surfaces is recognized as

$$H\_1(X, t) = \,^\vee Y = \,^l d\_1 + b\_1 \cos 2\pi (X - ct) \frac{1}{\lambda} \tag{5}$$

*Coatings* **2018**, *8*, 407

$$H\_2(X,t) = \,^\prime Y = \,^\prime - d\_2 - b\_2 \cos[(X - ct)2\pi + \lambda\phi]\frac{1}{\lambda} \tag{6}$$

To translate the coordinates, we use the same procedure that was used in [13]. The consequent relations of the boundaries of channel are described as

$$h\_1(\mathbf{x}) = y - 1 = \text{ acos}2\pi\mathbf{x} \tag{7}$$

$$h\_2(\mathbf{x}) = y = -b\cos(\phi + 2\pi\mathbf{x}) - d\tag{8}$$

#### *Synovial Fluid Model*

The peristaltic motion of viscous synovial fluid (see [33,34]) with thin film coating at the walls is considered in a two-dimensional channel. The flow patterns corresponding to Models 1 and 2 are markedly different. We shall ignore the detailed discussion here. However, fewer essential points associated with the model are presented. The models under consideration present exciting features. Model 1 is a simple generalized form of a power-law mathematical model for a shear-dependent viscosity that is helpful to define various non-Newtonian fluids in biological and polymer fluid mechanics, food rheology, and geology, to consider the basis of viscosity that affects the concentration of a reactant. Model 2 describes that exponent is a function of concentration.

Model 1: The generalized power-law model and the viscosity are exponentially dependent on concentration, then the Model 1 is written as:

$$\mu(\mathbb{C}, D) = \mu\_0 \mathbf{e}^{\alpha \mathbb{C}} \left( 1 + \gamma^2 \Big| D^2 \Big| \right)^n \tag{9}$$

Model 2: In this model, a shear-thinning index depends upon the concentration (i.e., zero concentration):

$$
\mu(\mathcal{C}, D) = \mu\_0 \left( 1 + \gamma^2 \left| D^2 \right| \right)^{n(\mathcal{C})} \tag{10}
$$

in which,

$$|D| = \sqrt{2\left(\frac{\partial u}{\partial x}\right)^2 + 2\left(\frac{\partial u}{\partial y}\right)^2 + \left(\frac{\partial v}{\partial x} + \frac{\partial u}{\partial y}\right)^2} \tag{11}$$

and

$$m(\mathbb{C}) = -\frac{\mathbf{e}^{\alpha \mathbb{C}} - 1}{2\mathbf{e}^{\alpha \mathbb{C}}} \tag{12}$$

in which *n* is index of shear-thinning comprising values between −0.5 and 0. It is worth mentioning that results of Newtonian fluid are obtained as a particular case of current fluid when *n* = 0.

The governing equations are too arduous to be acquiescent to stability analysis. Therefore, it is necessary to simplify the modeled equations. Make sure that the simplification process is congruous for such problems. Henceforth, we shall assume the long wavelength constraint, i.e., δ 1 and less Reynolds number Re ≈ *O*(1). Now, it is suitable to make the observing equations dimensionless by defining the following ratios:

$$\begin{array}{l} \mathsf{3} = \frac{\mathsf{y}}{\mathsf{d}\_{1}}, a = a^\*(\mathsf{C}\_{1} - \mathsf{C}\_{0}), \ \sigma = \frac{\mathsf{C} - \mathsf{C}\_{0}}{\mathsf{d}\_{1} - \mathsf{C}\_{0}}, \mathsf{7} = \frac{\mathsf{a}}{\|\mathsf{a}\_{0}\|}, \mathsf{7} = \frac{\mathsf{a}}{\|\mathsf{a}\_{1}\|}, \mathsf{7} = \frac{\mathsf{a}\_{1}}{\|\mathsf{a}\|}, \mathsf{8} = \frac{\mathsf{a}\_{1}}{\|\mathsf{a}\|}, \mathsf{a} = \frac{\mathsf{b}\_{1}}{\|\mathsf{a}\|},\\\ \mathsf{b} = \frac{\mathsf{b}\_{2}}{\mathsf{d}\_{1}}, d = \frac{\mathsf{a}\_{2}}{\mathsf{d}\_{1}}, \mathsf{We} = \frac{\mathsf{a}\_{1}}{\mathsf{d}\_{1}}, \ |\mathsf{D}| = \frac{\mathsf{d}\_{1}}{\mathsf{d}\_{1}} |D|, \mathsf{S}\_{\mathsf{c}} = \frac{\mathsf{a}\_{1}}{\|\mathsf{D}\_{\mathsf{c}}}, \ \mathsf{\mathsf{D}} = \frac{\mathsf{a}\_{1}}{\mathsf{d}\_{\mathsf{c}}}, \ \mathsf{Re} = \frac{\mathsf{g} \mathsf{d}\_{\mathsf{c}}}{\mathsf{g}}, \ \mathsf{\mathsf{T}} = \frac{\mathsf{g}}{\mathsf{g}}, \mathsf{T} = \frac{\mathsf{g}}{\mathsf{k}} \end{array} \tag{13}$$

In above expression, *S*c denotes Schmidt number, Re stands for Reynolds number, α represents concentration production, and γ is a material parameter.

The resulting non-dimensional governing equations along with Models 1 and 2 after exempting bar symbols in a wave frame will observe the following form:

$$\frac{\partial v}{\partial y} = -\frac{\partial u}{\partial x} \tag{14}$$

$$\frac{\partial p}{\partial \mathbf{x}} = \begin{cases} \frac{1}{2} \frac{\partial}{\partial y} \left[ (1 + n\sigma) \left\{ 1 + n\mathcal{W} \mathbf{e}^2 \left( \frac{\partial \mathbf{u}}{\partial y} \right)^2 \right\} \frac{\partial \mathbf{u}}{\partial y} \right] & \text{(Model 1)}\\ \frac{\partial^2 \boldsymbol{u}}{\partial y^2} - \frac{n\mathcal{W}\mathbf{e}^2}{2} \frac{\partial}{\partial y} \left[ \sigma \left( \frac{\partial \mathbf{u}}{\partial y} \right)^3 \right] & \text{(Model 2)} \end{cases} \tag{15}$$

Concentration equation for Models 1 and 2 is simplified to the following form:

$$\frac{1}{S\_{\mathbb{C}}} \frac{\partial^2 \sigma}{\partial y^2} = 0 \tag{16}$$

The no slip boundary conditions become:

$$
\mu(h\_1) = -1 \,\upmu(h\_2) = -1, \,\text{\textbullet}(h\_1) = 0, \,\text{\textbullet}(h\_2) = 1 \tag{17}
$$