**1. Introduction**

*They bring it over from Chilac. A lady comes and distributes it among us. All the women know how to embroider; no other way. There is not any work here like in Tehuacan… They pay us little… \$20 to \$25 pesos, depending on what we are stitching.*  *They make anything from dresses to blouses that they resell in Cancun or Matamoros1 . They come with the design already made. We just stitch the pattern. We put up our own string and material. We do two a week, because we each have our own chores to do. My aunts taught me. My mother taught me. We grew up with it because there aren't any jobs. We learned very young. (A. and L. Campos, personal conversation, 14 of February 2020).*

On the various occasions that I visited the Campos2 family in Atempa, Coyomeapan, Puebla, I found a similar scene: three women weaving, sitting in their patio, under the shade of the extending roofs over stone floors. Veronica, Leticia and Antonia (grandmother, daughter and granddaughter) belong to different generations of women from the same family that, among their diverse activities, are also embroiderers. Sitting next to each other, heads down, their gaze focused on the next stitch to color the petals of a flower. The three women barely say anything and only look up to put a new string through the needle. They stitch in a hurry, as they are committed to deliver a shipment with multiple pieces the following day. This performance is replicated in most of the houses in this community of 205 inhabitants [1], since embroidery making is a primary source of income for its residents, in addition to their work in the fields, their trade of products derived from agriculture and livestock, and their paid domestic work.

Atempa is one of forty-two towns in the municipality of Coyomeapan. It is situated in a depression among various hills in the heart of Puebla's Sierra Negra mountain range. Its denizens are indigenous, Nahuatl-speaking first, Spanish-speaking second. The temporal, physical, social, and geographic space that embroidery occupies in these people's lives in Atempa makes me consider how an expression can shed light on their form of life [2]. The field of self-expression within embroidery alludes to an area of content revealing how this practice implicates diverse precepts of precarity, vulnerability, and precarious work dynamics operating from unequal relations in scaled spatial and social capital.

The conditions of Precarity and Marginalization of this town have been studied by academia and institutions of social development, interpreting its findings from public policy or by way of diverse economic variables within a wider geography: regional or municipal, in the best of cases. Of these investigations, we know that the municipality of Coyomeapan, in which the town of Atempa is located, is one of the rural indigenous territories with the most extreme poverty in the state of Puebla [3]. Nevertheless, the majority of these findings had been conducted without considering the local, social and material transformations in concomitance and synchronicity with its actors' practice, who are the protagonists in the phenomena of this investigation. Analyzing the case from this perspective raises questions over the contradictions, the particularities and conditionings that arise among microgeographies when expressing specific lifestyles by these practices [2].

1 Tehuacan is the second most important populated commercial hub in the state of Puebla, after its state capital. It is situated 120 minutes from Coyomeapan, a hillside municipality bordering the state of Oaxaca, and 30 minutes from the peri-urban municipality of San Gabriel Chilac. Cancun is a tourist hub well known for its domestic and foreign vacationers located in the state of Quintana Roo. Matamoros, Tamaulipas, is located on the border with the United States, and is an important commercial cross-border city.

2 The names of the participants have been changed to protect their identity for the sake of this investigation.

*A Hundred Stitches Make a Canvas: How the Practice of Embroidery Relates to Forms of Life… DOI: http://dx.doi.org/10.5772/intechopen.105162*

**Figure 1.** *Embroidery in action.*

In this text, by analyzing the testimonies of embroiderers in Atempa, compiled in a series of interviews and data obtained by way of participatory observation, the ways in which this town connects with other territories through differing levels of domination-subordination in its commercial, social, and spatial relations are revealed. This, among other phenomena, produces ways of life whose constitutive elements are defined by the poverty registry. Not only does this just concern the practice of embroidery, which we can recognize among the dynamics of accumulation, but also the meaning behind its making. A way of its making is defined by time and particular durations; by constitutive spaces through its act of social relations; by specific actors that implement certain techniques, that make available their body, the capacity and their knowledge to complete certain tasks. It is also defined by utilities and objects such as tools and supports, and scenographic objects that bear witness to the acts, whose contribution resides in its capacity to set the stage and contextualize. As a result, the work of textualizing is complicated. Putting into words what appears both simultaneously and on its own rhythm makes impossible the endeavor to deliver the intersubjective experience as it is perceived and expressed by its practitioners ([4], pp. 101–130).

The following lines present, firstly, the context of the investigation in empirical terms, with references of these connections between the concrete practice and other practices, and how their implications are scaled up and down from the local level. In the next segment, spaces for this practice, its materiality and correlation with its capacity to reproduce actions that make embroidering possible are analyzed. Lastly, the connection between these scaled spaces and the meaning of conformity in poverty lifestyles is discussed (**Figure 1**).


*2.6.4 Damage*

Pink stem borer *Sesamia inferens* became a key pest in recent time in cereals and can-do considerable yield loss. Different crops of the family Graminae attacked by this polyphagous pest including rice [44], wheat, pearl millet, finger millet and sorghum [45]. Yield losses caused by *S. inferens* in maize may reach from 25.7 to 78.9% [39].


5. Threats

Canal is distributed from Sorah (Sukkur) to Head Jamrao about 108,960 hectares and Nara Canal was declared in 1972 as a Game Reserve area for the protection of wild animals. The NDWC is also recognized as an essential potential Ramsar Site [1–5]. The different floral habitation distributed in the Nara Desert includes phytoplankton, reed vegetation, herbs, shrubs and trees. The area is ecologically-rich with the faunal biodiversity which includes zooplankton, invertebrates, fishes, amphibians, reptiles, birds, small and large mammals. The NDWC has received high economic, social, floral and faunal habitats, and aquatic biodiversity values because the local communities are directly or indirectly dependent on these natural sustainable resources [1–4, 7,

In the area the water quality and recharged by the Nara Canal is mainly sweet and acceptable ranges of TDS between 500 and 800 ppm except hypersaline lakes. The hypersaline water of desert area is mainly observed brackish which have TDS between 10,000 and 28,000 pp. [5, 36]. The climate of the area is mainly arid having high temperatures and late summer rains observed. The seasonal rainfall is varied and is less than 250–300 mm and rainy season usually starts from June to September. Before the monsoon season, the average temperature is exceeding 45C in the desert region and in the plains of NDWS the average temperature between 30 and 40C. The wetland complex is recognized of great hydrological values as 98% Nara Canal water is used for agriculture and only 2% water is used for domestic and drinking purposes. The wetland complex of Nara Canal is 361.6 km long and 90–135 m wide. The maximum water depth of wetland complex is 7.5 m. The highest water discharges of Nara Canal are reported in

The assessment of physicochemical parameters such as pH and alkalinity revealed that the lake water has mostly remained alkaline during the whole study period except the Nara Canal station due to its input of rain water. The range of hardness was higher in most of the selected lakes of NDWC. The acceptable level of hardness in lake waters is recognized as 200 mg/L by the World Health Organization [34]. The physicochemical parameters of Na and Mg, as well as

the months of May–July and the minimum water flow in the August [1–4, 7].

29, 35] (Figure 7).

76 Community and Global Ecology of Deserts

Figure 7. A view of Nara Canal Khairpur.

Hunting: In the study area, the hunting for recreation is observed common and uncontrolled. However, the region is protected but there is no effective implementation of the wildlife laws for the wild animals. Due to hunting pressure, this is also leading to imbalance between the predator and prey species.

Foraging of Livestock: The large amount of grazing livestock in the area together with the recent climatic changes is degrading the food chain in the ecosystem dynamics.

Cutting trees: In the study area, the cutting of trees in the adjoining desert region for continuous practice of conversion of lands into agricultural fields which is affecting the wild population.

Developmental activities: In this modern era the human population is increasing in higher rate and habitation, the developmental activities in the region and conversion of land for the purpose of agriculture has been damaging the wild habitat and ultimately increasing stress on the existing wildlife.

Acronyms and Abbreviations

EC electrical conductivity

TDS total dissolved solids

Tem temperature

Tur turbidity

Ca calcium

Mg magnesium

Hard hardness

HCO carbonate

Alkal alkalinity

Cl chlorides

Na sodium

SO4 sulfate

Author details

Sindh, Pakistan

Muhammad Saleem Chang

K potassium

BOD biological oxygen demand

NDWC Nara Desert wetland complex

WHO World Health Organization

WWF Worldwide Fund for Nature

Address all correspondence to<EMAIL_ADDRESS>
Department of Science and Technical Education, University of Sindh Jamshoro, Jamshoro,

Ecological and Environmental Assessment of Nara Desert Wetland Complex (NDWC), Khairpur, Sindh-Pakistan

http://dx.doi.org/10.5772/intechopen.78623

79

DO dissolved oxygen

HCO3 bi-carbonate

pH pH

Recommendation:

Controlled hunting: To control the hunting the check posts should be established for keeping vigilance at important points on uncontrolled hunting. Due to shortage of infrastructure in Sindh Wildlife Department like as transport system and staff failed to stop hunting so that the officials must enhance staff and transport. The Wildlife authorities must consider strengthening of Sindh Wildlife Department in the region.

Ecotourism: The area of Nara Wetland Complex is a best site for promoting ecotourism. For sighting of wildlife and bird watching the watch towers at potential points and other facilities should be developed for promoting community based conservation tourism. The people of local community should be participated and benefited from all this tourism activates. From local community the youth should be trained as a volunteers and co-guides. These health activities will provide the incentives to the local community as a source of income generating activity and an alternative livelihood source.

Promote participatory wildlife management and conservation: For promoting participation in wildlife management and conservation the institutional capacity of community based organizations in the region should be developed.


**3. Results**

## *3.1. Grain Refinement*

The results of the grain structure investigation showed very large dendritic grains, the sizes of which were several millimeters (Figure 1a), in the Al0.3Mg1Si alloy without the addition of any transition elements. This is generally characteristic of low-alloyed alloys [35]. Upon the addition of Sc (Figure 1b) and Zr (Figure 1c), the size of the grain structure continues to decrease but not significantly. The scandium concentration is 0.6% which is required to achieve a hypereutectic composition and the formation of primary Al3Sc particle in the liquid [21]. Therefore, in Al0.3Mg1Si0.3Sc alloy grain refinement occurs due to supercooling at the boundaries of the nuclei and the liquid phase [36]. The concentration of zirconium Al0.3Mg1Si0.15Zr reaches a peretectic point of 0.11% [36]. However, significant grain refinement is not observed. It is explained by the fact, that according to [36] at the given concentration, the primary particles of Al3Zr only begin to form in the liquid and a significant grain-refinement effect is observed at a concentration of 0.2%.The concentration of 0.3 Sc and 0.15 Zr is sufficient for the hypereutectic composition and the formation of primary Al3Sc particles in the liquid phase, which leads to a significant appearance of a fine-grained equiaxed structure during casting (Figure 1d).

**Figure 1.** Grain structure after casting (**a**) Al0.3Mg1Si, (**b**) Al0.3Mg1Si0.3Sc, (**c**) Al0.3Mg1Si0.15Zr, (**d**) Al0.3Mg1Si0.3Sc0.15Zr.

A dendritic structure is observed in the Al0.5Mg1.3Si alloy (Figure 2a), as well as in Al0.3Mg1Si. However, the grain size in Al0.5Mg1.3Si is two times smaller. This effect is caused by an increase in the content of Mg and Si, which also leads to supercooling between the nuclei and the liquid phase. Additions of scandium (Figure 2b) or zirconium (Figure 2c) alone only slightly affect the grain size. Their combined use leads to the appearance of fine equiaxed grains which, however, only partially replace the dendritic structure (Figure 2d). The decrease in alloying efficiency will be explained later.

**Figure 2.** Grain structure after casting (**a**) Al0.5Mg1.3Si (**b**) Al0.5Mg1.3Si0.3Sc, (**c**) Al0.5Mg1.3Si0.3Zr, (**d**) Al0.5Mg1.3Si0.3Sc0.15Zr.

Thus, a gradual grain refinement occurs during complex alloying with small additions of zirconium and scandium. The decrease in the grain size is explained, first, by the fact that the growing content of scandium and zirconium causes supercooling between the liquid and the surface of the nuclei formed during solidification thereby facilitating the passing of the latter [36]. This also explains the fact that the grain in Al0.5Mg1.3Si (Figure 3a) is finer than in Al0.3Mg1Si (Figure 3b) after solidification, since the total number of alloying elements increases. With a further increase in the Sc and Zr contents, an almost 10-fold decrease in grain size occurs, mainly because the eutectic composition for Al-Sc-Zr system is reached [37]. As already mentioned, due to the high concentration of Zr and Sc in the Al0.5Mg1.3Si alloys, the grain refinement is more effective than in Al0.3Mg1Si. An explanation of this fact will be given later. Note, however, that no appreciable refinement occurs with an increase in Zr or Sc alone. This is due to the fact that a scandium content of 0.6% is required for the hypereutectic composition and significant grain refinement. At the same time, an increase in Zr content to 0.15% reduces the scandium content which is necessary for the hypereutectic composition to 0.2%. It should be noted that the amount of zirconium required for the overflow reaction of the zirconium content should be about

0.26%. Therefore, it is not efficient for a significant refinement of the grain structure during casting in the concentration range studied here.

**Figure 3.** Grain size at complex alloying with small Sc and Zr additives (**a**) in alloys based on Al0.3Mg1Si, (**b**) on AL0.5Mg1.3Si.

### *3.2. Microhardness Behavior*

The results of microhardness measurements are very interesting. It is generally higher (Figure 4a) in an alloy with a higher silicon content than in an alloy with a low silicon content (Figure 4b). This can be explained by the fact that alloy Al0.3Mg1Si contains more particles of the type (AlSi)3ScZr and/or AlSc2Si2 than Al0.5Mg1.3Si. They can appear due to the fact that the cooling process during casting requires a certain time, which is sufficient for the precipitation of a certain amount of these particles from the supersaturated solid solution [26]. An increased ratio of magnesium and silicon will accelerate the precipitation of these particles from the supersaturated solid solution and thereby increase the microhardness. At the same time, an increase in the zirconium content will reduce the ability to form them upon cooling, since this element reduces the diffusion of scandium [19]. As for the Al0.5Mg1.3Si alloy (Figure 4b), it can be assumed that grain refinement, associated with an increase in the proportion of zirconium and scandium, will have a stronger effect than a decrease in the diffusion of the latter. As mentioned earlier, due to the larger amount of Mg and Si, the refinement of the grain structure will be much stronger. Therefore, the length of grain boundaries is longer. The latter, in turn, serve as an additional source in the formation of secondary metastable particles, contributing to an increase in their number [38]. Note that the effect of the grain structure size can be excluded, since this indicator begins to exert a significant effect on the microhardness when reaching submicron sizes. Even in alloys with the finest grains its size is several microns.

#### *3.3. Phase Diagram Calculation Result*

Figure 5a,b shows polythermal sections of the systems Al-0.3%Mg-1%Si-0.15%Zr- (0–0.4)%Sc (wt. %) and Al-0.5%Mg-1.3%Si-0.15%Zr-(0–0.4)%Sc. Note that alloys with a combined content of scandium and zirconium seem to be the most promising. Firstly, they allow to refine the grain structure which will positively affect the mechanical properties and, secondly, slow down the diffusion of scandium and foster the formation of metastable nanoparticles (AlSi)3ScZr coherent to aluminum matrix, instead of harmful AlSc2Si2. Therefore, polythermal sections with the highest zirconium content are considered. On the whole, phase transformations in both groups of alloys (with different Mg/Si ratios) have common laws. Slightly below 700 ◦C, primary Al3Zr particles begin to precipitate from the liquid. Three phases L + Al3Zr + (Al) are simultaneously present in a narrow range between 653–650 ◦C in Al0.3Mg1Si (Figure 5a) and 650–647 ◦C in Al0.5Mg1.3Si (Figure 6b). After that, Al3Zr dissolves and the ZrSi phase appears instead. With Sc content of up to 0.23%

in Al0.3Mg1Si (Figure 5a) and up to 0.26% in Al0.5Mg1.3Si (Figure 5b), the ZrSi phase is transformed into Zr2Si. At zero scandium content, upon reaching a temperature of 601 ◦C in Al0.3Mg1Si (Figure 5a) and 578 ◦C in Al0.5Mg1.3Si (Figure 5b), the metal finally solidifies. After that an aluminum solid solution and the ZrSi2 phase can be observed in the metal. With an increased concentration of scandium, the solidus temperature gradually decreases up to 0.14% and 0.07% of Sc for Al0.3Mg1Si and Al0.5Mg1.3Si alloys respectively and at higher concentration of Sc the solidus temperatures is slightly increased. When the Sc concentration reaches 0.21% in Al0.3Mg1Si (Figure 5a) and 0.25% Al0.5Mg1.3Si (Figure 5b), L + (Al) + ZrSi2 first transforms into L + (Al) + ZrSi2 + Al3Sc and then finally is solidified as (Al) + ZrSi2 + Al3Sc. When scandium reaches 0.24% in the Al0.3Mg1Si alloys (Figure 6a) and 0.27% in Al0.5Mg1.3Si (Figure 6b), solidification occurs according to the following scheme: L + (Al) + ZrSi2 → L + (Al) + ZrSi2 + Al3Sc → L + (Al) + ZrSi2 + SiSc → (Al) + ZrSi2 + Al3Sc. Upon reaching Sc 0.24% and 0.28% in the alloys Al0.3Mg1Si (Figure 5a) and Al0.5Mg1.3Si (Figure 5b), solidification occurs along an even more complicated path: L + (Al) + ZrSi → L + (Al) + ZrSi + Al3Sc → L + (Al) + ZrSi2 + Al3Sc → L + (Al) + ZrSi2 + SiSc → (Al) + ZrSi2 + SiSc. In addition, in a short interval between 0.23 ÷ 0.24% for Al0.3Mg1Si and 0.27 ÷ 0.28%, solidification occurs according to the following scheme: L + (Al) + ZrSi → L + (Al) + ZrSi2 + Al3Sc → L + (Al) + ZrSi2 + SiSc → (Al) + ZrSi2 + SiSc.

**Figure 4.** Microhardness with complex alloying with small additives Sc and Zr (**a**) in alloys based on Al0.3Mg1Si, (**b**) on Al0.5Mg1.3Si.

It should be noted that the appearance of primary particles in the liquid phase will facilitate grain refinement but not all of them will be equally effective. Primary Al3Zr, and especially Al3Sc, are effective inoculators due to the good correspondence between them and the aluminum matrix. There is no such information about ZrSi2 and SiSc particles. However, it can be assumed that their effectiveness is rather low.

Note that in the Al0.3Mg1Si0.3Sc0.15Zr alloy the combined grain refinement effect of zirconium and scandium does not differ from that observed in pure aluminum and alloys with a high magnesium content [37]. However, in the Al0.5Mg1.3Si0.3Sc0.15Zr alloy at concentrations sufficient to obtain a fine grain structure in pure aluminum and alloys with high magnesium content, a large number of dendritic grains are still observed. A first explanation for this is that Al3Sc and Al3Zr particles are not present at all stages of solidification. They will have a slightly weaker effect than in high-magnesium alloys. It should also be noted that Al3Sc particles, which contribute to efficient grain refinement, act even more efficiently in Al0.3Mg1Si alloys because their formation requires a lower scandium content than in Al0.5Mg1.3Si. This explains the fact that the grain is larger in this group of alloys with a low content of Zr and Sc than in Al0.5Mg1.3Si with a similar content of these elements. However, the grain in Al0.3Mg1Si is refined better with an increase in their concentration. At a low concentration of Sc and Zr, the reason for the finer grain in the

case of Al0.5Mg1.3Si is the aforementioned supercooling at the interface between the nuclei and the liquid phase (arising from the dissolved Mg and Si). However, due to the lower Sc concentration required for the formation of Al3Sc particles during metal solidification, the refinement of the grain structure at high scandium concentrations is more efficient in Al0.3Mg1Si.

**Figure 5.** Polythermal sections (**a**) Al-0.3%Mg-1%Si-0.15%Zr-(0–0.4)% Sc (wt. %) and (**b**) Al-0.5%Mg-1.3%Si-0.15%Zr-(0– 0.4)%Sc (wt. %) of the Al–Mg–Si–Zr–Sc phase diagram calculated using Thermo-Calc software.

**Figure 6.** Al0.3Mg1Si0.3Sc main types of intermetallic compounds (**a**); small dispersoids (**b**); large particles (**c**); result of the energy-dispersive spectroscopy (EDS) investigation of chemical composition of the intermetallic compounds (**d**).

After solidification in both types of alloy, a similar chain of phase transformations is observed. At a low concentration of scandium, the following phase transformations will occur: (Al) + ZrSi2 → (Al) + ZrSi2 + (Si) → (Al) + ZrSi2 + (Si) + Mg2Si. At a higher

concentration of scandium, the following transformations will be observed: (Al) + ZrSi2 + SiSc, (Al) + ZrSi2 + SiSc + (Si); (Al) + ZrSi2 + SiSc + (Si) + Mg2Si. It may seem that the Al0.5Mg1.3Si system is more promising from the point of view of further use, since it has a larger Mg/Si relationship than Al0.3Mg1Si, which decreases the risk of the appearance of harmful particles of the AlSc2Si2-type. However, a lower dissolution temperature of Mg2Si particles in Al0.3Mg1 Si alloys allows one to avoid additional heat treatment. To date, multistage heat treatment has been required to obtain two hardening phases (AlSi)3ZrSc and β" (Mg5Si6) [26]. It consists in homogenization in order to maximize dissolution of Sc, Mg, Si and Zr into a supersaturated solid solution which includes eliminating large non-equilibrium particles. Three-stage annealing is applied after homogenization: The first step is the formation of (AlSi)3Sc particles. Then (AlSi)3ScZr particles are formed on their basis. The third stage consists of the dissolution of phases close to Mg2Si, which at the fourth stage can be isolated in the form of a strengthening β" (Mg5Si6) phase. However, at a low temperature of Mg2Si solubility, the fourth stage of annealing can be avoided, since all the necessary processes will occur at the third stage. Note that, according to the calculations performed in the current Thermo-Calc version, only equilibrium primary particles Al3Zr are present in the alloys studied. The appearance of Al3Zr as solid state equilibrium particle which has D023-structure was not predicted by this calculation. This, however, does not exclude the emerging of metastable particles which have L12 structure and composition close to (AlSi)3Zr.

#### *3.4. Investigation of the Intermetallic Participles by Scanning Electron Microscopy (SEM) Method*

The Al0.3Mg1Si0.3Sc alloy contains two types of particle; the first one can be described as being like Al3Fe2Si2. The second can be interpreted as being like quaternary π phase Fe2Mg7Si10Al18 [39] which in this alloy can form due to there being no equilibrium solidification. Primary intermetallic compounds have both close to round (smaller) and needle-like shapes. Their average size is 12.4 microns. This alloy has an increased amount of fine particles which, among other things, explains its high microhardness (Figure 4a). Considering that no traces of scandium were found in large intermetallic particles with a fraction of its content in the solid solution of only 0.11%, it can be assumed with a high degree of probability that it is in the form of fine particles, the fraction of which is 0.14%. It should be noted that there is a high proportion of magnesium in the supersaturated solid solution. Therefore, it is insufficient for the formation of large intermetallic particles of the Mg2Si type. This chemical composition, as predicted in [25], is extremely unfavorable for the production of strengthening Al3Sc particles, since scandium actively interacts with silicon already during cooling after casting. In this case, Mg is used rather rarely. This means, firstly, the need for a prolonged homogenization (completed by quenching) to dissolve excess Sc and Si at high temperatures. In addition, with a high degree of probability the subsequent heat treatment and aging at 300 ◦C will not be accompanied by the precipitation of Mg2Si-type particles. As a result, silicon will interact only with scandium, which will favor the predominant growth of unwanted AlSc2Si2 particles.

Figure 7 shows the results of electron microscopy for the alloy Al0.3Mg1Si0.3Sc0.15Zr. It contains a number of large primary intermetallic such as Al9Fe2Si2, (Al, Si)3Sc, Al3Sc0.6Zr0.4 with an average size of 11.6 microns. It also contains particles with a composition close to quaternary π phase Fe2Mg7Si10Al18. In this alloy, a fairly large amount of Si and Sc is in solid solution. This suggests that, for a given chemical composition, Si and Sc do not interact so intensely with each other. The presence of particles of both the (Al, Si)3Sc and Al3Sc0.6Zr0.4 types indicates that Sc, upon further heat treatment, will react both with silicon, forming AlSc2Si2 particles, and with zirconium, forming Al3Sc0.6Zr0.4 particles. In this case magnesium also "absorbs" part of the silicon, contributing to the formation of Mg2Si-type particles. Taking into account the results of Thermo-Calc calculations (Figure 5a), it can also be assumed that various Zr and Si compounds will occur during heat treatment. Thus, this alloy seems to be very promising for further study, since excessive silicon will interact both with magnesium and zirconium, as well as various impurities. With proper heat treatment,

this will allow the required amount of Al3ScZr particles which have a L12 structure to be obtained.

**Figure 7.** Al0.3Mg1Si0.3Sc0.15Zr main types of intermetallic compounds (**a**); small dispersoids (**b**); large particles (**c**); result of the EDS investigation of chemical composition of the intermetallic compounds (**d**).

The Al0.5Mg1.3Si0.3Sc alloy contains the main types of large primary non-equilibrium intermetallic compounds Al9Fe2Si2, Fe2Mg7Si10Al18, and (Al, Si)3Sc. Their average size is 10.6 microns. In this case, there are spherical and needle-shaped particles, as well as large elongated eutectic particles arising along the boundaries of dendritic grains. Besides, there are many dispersoids in this alloy occupying 0.2% of the cross-sectional area, but with a rather large size.

In the Al0.5Mg1.3Si0.3Sc0.15Zr alloy, large intermetallic compounds are represented by Fe2Mg7Si10Al18 and (Al, Si)3(Sc) particles with an average diameter of 12.8 microns. There are not so many dispersoids in the alloy, however, they have a size of about 30 nm. Smaller dispersoids with an average size of 20–15 nm were detected using transmission microscopy. Considering also the fact that zirconium can practically not be observed in a supersaturated solid solution and in large intermetallic particles, it can be assumed that the main type of fine particles will be Al3ScZr. Moreover, in this alloy Si reacts sufficiently with magnesium which makes it very promising for further research.

The results of electron microscopy are presented in:

**Figure 8.** Al0.5Mg1.3Si0.3Sc main types of intermetallic compounds (**a**); small dispersoids (**b**); large particles (**c**); result of the EDS investigation of chemical composition of the intermetallic compounds (**d**).

**Figure 9.** Al0.5Mg1.3Si0.3Sc0.15Zr main types of intermetallic compounds (**a**); small dispersoids (**b**); large particles (**c**); result the EDS investigation of chemical composition of the intermetallic compounds (**d**).

**Table 2.** Size and amount of dispersoids in the investigated alloys.

**Table 3.** Size and amount of intermetallic compounds in the investigated alloys.

**Table 4.** Chemical composition of intermetallic particles in the studied alloys.

It must be mentioned that most of the phases listed in Tables 2–4 are non-equilibrium, as result they cannot be predicted by the diagram. The reason for this is the non-equilibrium conditions of solidification and the inevitable presence of iron even in the high frequency batch. However, the diagram on Figure 6 can be very useful for predicting phase transformations after homogenization annealing (for example, combined with quenching) when the composition is close to equilibrium.

#### *3.5. Investigation of the Despersoid by Transmission Electron Microscopy (TEM) Method*

A sufficiently large number of Al3Sc particles is observed in the alloy Al0.3Mg1Si0.3Sc. The size of these particles is 20 nm on average (Figure 10a,b). Particles of a given size are at the coherence loss thresholds. Therefore, some of them are completely coherent (Figure 10d), while others only partially (Figure 10d). The presented data are in good agreement with the data of scanning microscopy, according to which most of the scandium is not in a supersaturated solid solution, and at the same time, it is not observed in large particles of eutectic origin, such as Al0.3Mg1Si0.3Sc0.15Zr. In addition, it is the particles of this type that serve as the main reason for the growth of microhardness (see Figure 4a). It should

also be noted that these particles contain unwanted silicon. However, considering that these particles are fully or partially coherent and considering the increase in microhardness, it is possible with a high degree of probability that they are metastable (AlSi)3Sc, rather than equilibrium harmful AlSc2Si2. The appearance of such particles during solidification in the 6XXX series alloys with small additions of scandium and zirconium as a result of discontinuous precipitation has already been described in many investigations [40,41]. However, it has been described mainly in alloys that do not have such a high excess of silicon. The presence of metastable coherent and semi-coherent particles which precipitate during the cooling process after casting of the Al0.3Mg1Si0.3Sc alloy indicates that they can also be obtained as a result of multistage heat treatment. Thus, the cooling rate achieved in a steel chill mold without zirconium is insufficient so that (AlSi)3Sc-type particles are not precipitated during cooling. It should be noted that in spite the short cooling time of the ingot, a large number of (AlSi)3ScZr dispersoids precipitate in this and other alloys investigated by TEM. This can be explained by the fact that silicon accelerates the kinetics of the precipitation of (AlSi)3ScZr [42].

A different picture of the distribution and chemical composition of fine particles is observed in the Al0.5Mg1.3Si0.3Sc alloy. It also contains the (AlSi)3Sc-type particles but their number is somewhat less than in the Al0.3Mg1Si0.3Sc alloy (Figure 11a). However, in general, they have the same size and the same chemical composition. In addition, needle-shaped particles similar to β" (Mg5Si6) appear (Figure 11b,c).

It should be noted that in spite of the rather short cooling time of the ingot, a large number of Al3ScZr particles precipitate in this (and other alloys investigated by TEM). This can be explained by the fact that silicon accelerates the kinetics of the precipitation of these particles.

Note that emerging of β" actually became possible due to the fact that additional magnesium appeared for the reaction with silicon. Thus, an increase in the Mg/Si ratio will contribute to obtaining an additional synergistic effect between the (AlSi)3Sc and β" (Mg5Si6) particles. In general, the cooling rates after casting in a steel chill mold are also insufficient in this alloy to contain the main elements in a solid solution, and they form hardening phases (AlSi)3Sc and β" (Mg5Si6).

The Al0.3Mg1Si0.3Sc0.15Zr alloy contains a fairly large number of fine coherent or semicoherent (AlSi)3ScZr particles with the L12-structure (Figure 12b–d). At the same time, there is a second type of elongated particles (Figure 12a,c), which can be confused with β" (Mg5Si6), given the rather high content of magnesium as well as silicon in the surrounding solid solution. However, these particles are much longer than β" (Mg5Si6) (from 0.5 to 1 μm). Therefore, a chemical analysis (Figure 12e) shows that they are also discontinuous precipitation of (AlSi)3ScZr particles. This fact is also confirmed by the comparison with the literature data [43], in which discontinuous Al3Sc precipitates are practically identical in shape, size and location of relatively high-angle boundaries compared to the dispersoids observed in this work. It should be noted that, as in Ref. [44], high-angle boundaries serve as a source of discontinuous precipitation of the (AlSi)3ScZr-type dispersoids. When these particles are precipitated, energy is released and the high-angle boundaries begin to move, leaving behind a large number of the (AlSi)3ScZr-type dispersoids. It is noteworthy that dendritic grains, in which intermittent precipitates are found, do not contain L12-nanoparticles (Figure 12c) and vice versa. A possible explanation for this is that in discontinuous precipitation, as in recrystallization [45], there are orientations more favorable for the motion of high-angle boundaries. In grains with an intermittent release of (AlSi)3ScZr, scandium and zirconium become insufficient for the appearance of more coherent particles which have L12-structure. It should be noted that, in the case of the Al0.3Mg1Si0.3Sc alloy, β" (Mg5Si6) particles can be observed together with (AlSi)3Sc particles, in contrast to the L12 and needle shape (AlSi)3ScZr dispersoids. It can also be noted that for the discontinuous and simultaneous precipitation both needle-shaped and L1-structured dispersoids the concentration of scandium in the Al0.3Mg1Si0.3Sc and Al0.5Mg1.3Si0.3Sc alloys is insufficient.

**Figure 10.** (**a**) (AlSi)3Sc coherent and semi coherent particles, (**b**) EDS profile line scan; (**c**,**d**) direct resolution of the crystal lattice with (AlSi)3Sc particles of various sizes.

**Figure 11.** (**a**) Al3Sc coherent and semi coherent particles particles; (**b**) Al3Sc coherent and semi coherent particles + Mg2Si (rod-like) particles (**c**) EDS profile line scan.

**Figure 12.** (**a**,**c**,**d**) Discontinuous precipitation of (AlSi)3ScZr needle-shaped particles; (**b**) (AlSi)3ScZr particles with the L12-structure, (**c**) discontinuous precipitation of (AlSi)3ScZr needle-shaped particles near high-angle boundaries. (**e**) EDS profile line scan.

Mainly coherent or semi-coherent (AlSi)3ScZr-particles, which have the L12-structure (Figure 13a,b), are formed during discontinuous precipitation and are observed in the Al0.5Mg1.3Si0.3Sc0.15Zr alloy. The lack of needle-shape precipitates is most likely due to the limitations of TEM associated with the small area observed. The average grain size in this alloy is 260 μm, while the survey area is 4 μm2, at best. For the alloy Al0.3Mg1.3Si0.3Sc0.15Zr, where the grain is finer, it is easier to find grain boundaries around which intermittent precipitates are concentrated. In the case of Al0.3Mg1Si0.3Sc0.15Zr, the β" (Mg5Si6)-phase is not detected in the alloy. Its absence can be explained by the fact that the bulk of magnesium and silicon form primary Mg2Si-particles at this concentration of zirconium and scandium (see Figure 6).

 **Figure 13.** (**a**,**b**) (AlSi)3ScZr particles with the L12 structure, (**c**) EDS profile line scan.


**2. The BBB and the neurovascular unit**

*Connectivity and Functional Specialization in the Brain*

mechanism known as neurovascular coupling [5].

contributing to neurological effects.

disease (PD) and multiple sclerosis (MS).

the most metabolically active organs in the body, under physiological conditions, the human brain receives 20% of the total basal cardiac output and uses 20% of the body's oxygen and glucose [4]. Energy substrates are consumed by the brain from the blood via transport across the BBB, as the brain lacks a metabolic reservoir to store macromolecules for use when needed. In the mammalian brain, cerebral arteries, arterioles, and capillaries supply CNS with blood in response to neuronal stimuli by increasing the rate of cerebral blood flow (CBF), nutrients and oxygen delivery, a

The neurovascular coupling requires an integrated multicellular response to provide the perfusion needs for neuronal metabolism [5], different cell types are involved in this action, neurons and astrocytes generate mediators that trigger cellular responses in endothelium cells, pericytes, and smooth muscle cells (SMC), which contribute to vascular response in the BBB permeability. Functionally, these interactions are included in the concept of the neurovascular unit (NVU), which comprises various central and peripheral cell types that contribute to BBB structure and function (**Figure 1**) [6, 7]. However, in pathophysiological states, BBB breakdown and dysfunction leads to leakages of harmful blood components into the cerebral parenchyma, cellular infiltration, and aberrant transport and clearance of molecules [8], which is associated with CBF reductions and dysregulation [9],

Here, we first examine the cellular components that underlie the establishment of the BBB in NVU. Then, we focus on the cellular components of BBB and transport physiology. Complementary and in a translational way, examine how BBB breakdown and dysfunction related to acute vascular CNS disorders such as ischemic and hemorrhagic stroke, and BBB breakdown and dysfunction relate to neurological deficits and other pathologies in Alzheimer's disease (AD), Parkinson's

*(A) The multicellular structure of the neurovascular unit (NVU). The BBB is formed by endothelial cells at the level of the cerebral bed (arterial and venous). These endothelial cells interact with perivascular elements, such as the basal lamina (BMs), smooth muscle cells (SMCs) and astrocytic end-feet processes, perivascular neurons and pericytes to form a functional BBB. (B) The core anatomic elements of the NVU. Created with*

**26**

**Figure 1.**

*BioRender.com.*

The NVU is a relatively recent neuroscience concept, representing the structural and functional multicellular relationship between the brain and blood vessels [5]. The cellular components are the endothelial cells (EC), pericytes, perivascular astrocytes, microglia, the basement membrane (BM), and neuron (**Figure 1**) [10]. The NVU components share intimate and complex associations, and these associations have led to their classification as a single functioning unit. The NVU is responsible for the maintenance of a highly selective BBB and cerebral homeostasis, as well as the control of CBF [11]. Each NVU component seems to play a specific and active role, maintaining the dynamic linkages reciprocally under physiological conditions.

**Endothelial cells** are considered the BBB's anatomic basis since they form and tightly seal the wall of all cerebral vessels, thereby building a physical barrier between the blood and the brain parenchyma (**Figure 1**). Two different types of endothelial junctions exist: adherens junctions (AJ) and tight junctions (TJ) [12]. Adherens junctions comprise vascular endothelial (VE) cadherin and neural (N-) cadherin, both acting via homophilic interactions [13]. While VE-cadherin is vital for sealing adjacent endothelial cells, N-cadherin mediates their association with pericytes [13]. TJ contains transmembrane proteins such as claudins, occludins, and junction adhesion molecules, as well as the zona occludens cytoplasmic proteins (ZO). These proteins act collectively to close off interconnecting endothelial cells [14], restricting the paracellular diffusion of hydrophilic substances, even ions; this is a unique feature of the BBB endothelium [11] in the other hand, the neurovascular endothelial cells, in contrast to peripheral endothelial cells, is the low expression of adhesion molecules (e.g. member of the immunoglobulin superfamily VCAM-1), in this sense, immune cells never cross unstimulated BBB in the healthy CNS [15]. Interactions of endothelial cells with other NVU members mediate a decrease in transcytotic activity, downregulation of leukocyte adhesion molecules, and regulation of interendothelial junction stability during development and adulthood [14].

 Structurally, pericytes extend processes from their cell body, covering several endothelial cells (**Figure 1**). In contrast to peripheral tissues, the brain has the highest pericyte to endothelial cell ratio [16]. Pericytes are embedded within the basement membrane (BM) of capillary endothelial cells and are thus centrally positioned between endothelial cells, astrocytes, and neurons [3]. In total, pericytes cover a large cerebral vascular area which can reach up to 40% of the neurovascular surface [17]. One of the main functions of pericytes is the control of the vasoreactivity and cerebral blood flow in response to neuronal activity [18]. As a recently explored example, glutamate induces prostaglandin E2 and nitric oxide release, which leads to actively relaxing pericytes to dilate capillaries [19]. Vascular permeability increases with decreasing pericyte coverage, which is partly due to the regulation of endothelial transcytosis. Moreover, other parts of the NVU are also influenced by pericytes, including neurons, immune cells, and the basement membrane [20].

**Astrocytes** are the most abundant cell type in the brain with a variety of functions. Beyond BBB regulation, they participate in synapse formation, uptake and recycling of neurotransmitters and ions, regulation of extracellular potassium levels, nutrition of neurons, and control of inflammatory responses within the CNS [21]. Astrocytes provide a cellular link between the neuronal circuitry and blood vessels. This neurovascular coupling enables astrocytes to relay signals that regulate blood flow in response to neuronal activity; this includes regulating the contraction/dilation of vascular SMC surrounding arterioles and

capillaries [22, 23]. Astrocytes are also critical cellular support of BBB integrity. Recent molecular studies have shown several molecules released by astrocytes that enhance and maintain barrier tightness, such as cholesterol and phospholipid transporter molecule apolipoprotein E [24, 25]. Release of apolipoprotein E from astrocytes, for example, regulates endothelial TJs by signaling through the low-density lipoprotein receptor related protein 1 (LRP1) on both pericytes and endothelial cells of CNS microvessels [25]. Astrocytes have been identified as essential mediators of BBB formation and function because of purified astrocytes' ability to induce barrier properties in non-CNS blood vessels [26]. Based on these observations, it has been proposed that astrocytes are necessary for the formation of impermeable TJs in the developing vessels of the BBB.

**Microglia** derive from hematopoietic precursors that migrate from the yolk sac into the CNS parenchyma, acting as the brain's main line of defense past the BBB and play a vital role in innate immune responses in the vascular bed and cerebral parenchyma (**Figure 1**) [27], little is known about how microglial-endothelial communications may shape and regulate the homeostatic BBB. However, studies have demonstrated that microglia are associated with endothelial's nascent vessels in the developing brain, and promote the fusion of cells in the stages following vascular endothelial growth factor-mediated induction [28]. Recent studies have shown the activation of microglia in CNS disorders like AD and multiple sclerosis, which are associated with BBB breakdown and neuroinflammation. In these conditions, microglial activation may be both a cause and consequence of BBB dysfunction [20]. Microglia can exist in one of two active states: in the activated pathway, microglia release proinflammatory cytokines like interleukin-1b and tumor necrosis factor-a. Whereas in alternative pathways, microglia are involved in tissue repair, phagocytosing neurons and foreign material, releasing chemokines and vascular endothelial growth factor [29]. On the other hand, brain endothelial cells can also secrete molecules that cause microglial activation [30]. In summary, a complex interplay between systemic and CNS derived immune cells exists at the BBB.

**Basement Membrane:** The vascular tube is surrounded by two basement membranes (BMs), the inner vascular BM, and the outer parenchymal BM (**Figure 1**). The vascular BM is an extracellular matrix secreted by the ECs and pericytes, whereas the parenchymal BM is primarily secreted by astrocytic processes that extend towards the vasculature [31]. These BMs consist of different molecules, including type IV collagen, laminin, heparin sulfate proteoglycans, and other glycoproteins [32]. They provide an anchor for many signaling processes in the vasculature and also constitute an additional barrier for molecules and cells to cross before accessing the neural tissue. Disruption of these BMs by matrix metalloproteinases is an integral part of BBB dysfunction and posterior leukocyte infiltration, which can be observed in many different neurological disorders [32].

**Neurons and interneurons.** Neurons can detect small variations in their supply of nutrients and oxygen and transform these signals into electrical and chemical messages to adjacent interneurons or astrocytes. In response to these signals, necessary adjustment mechanisms are initiated. Due to this phenomenon, neurons are considered NVU's pacemaker [15]. Neurons need to be able to signal to cerebral vessels when their energy demands change. Positive and negative feedback mechanisms exist to regulate cerebral blood flow, accompanied by adjustments of substrate delivery across the BBB, a process known as neurovascular coupling [33]. In this sense, one relevant mechanism for neurovascular coupling is direct innervation of astrocytic processes or the endothelial tube by, amongst others, serotonergic, noradrenergic, cholinergic, and GABAergic neurons [4]. Mechanisms of neurovascular coupling, particularly those that can explain direct molecular effects on BBB integrity, are yet to be established. Future knowledge will be of great interest since

**29**

**Figure 2.**

*brain. Created with BioRender.com.*

*Blood-Brain Barrier Dysfunction in the Detrimental Brain Function*

new therapeutic tools could help modulate intercellular communication in diseases

The BBB is a diffusion barrier essential for the normal function of the CNS. The NVU endothelial cells differ from endothelial cells in the rest of the vascular system by their absence of fenestrations, and for having more extensive junctional molecules, mainly TJ, and sparse pinocytic vesicular transport [34]. These junctional molecules limit the paracellular flux of hydrophilic molecules across the BBB. In contrast, small lipophilic substances (O2 or CO2) can diffuse freely across plasma membranes along their concentration gradient [34]. Nutrients such as glucose and amino acids enter the brain via transporters, whereas receptor-mediated endocytosis mediates larger molecules' uptake, including insulin, leptin, and iron transferrin [35], it is believed that all the components of the BBB are essential for the normal

The Junction complex in the BBB comprises TJ, AJ, and Gap junctions (GJ). The TJ ultrastructurally appear as apparent fusion sites, involving the outer plasma membrane of adjacent endothelial cells [36]. The number of TJ strands, as well as the frequency of their ramifications, varies and consists of three integral membrane proteins: claudin, occludin, and junction adhesion molecules, as well as several other cytoplasmic accessory proteins, including members of the family zonula occludens (ZO-1, ZO-2, ZO-3) and cingulin (**Figure 2**). Cytoplasmic proteins link membrane proteins to actin, for maintaining the structural and functional integrity of the endothelium [36]. The Claudins were identified as the principal component of TJ and are localized exclusively at TJ strands. Claudins bind to other claudins on adjacent endothelial cells to form the primary seal of the TJ [37]. Closest to the

*Basic molecular organization of BBB junctional molecules and transport. The endothelial cells confer unique properties on the BBB. They are the principal line of cerebral vasculature and have numerous junctional molecules such as tight junctions, adherens junction, gap junctions and accessory proteins that limit the passive paracellular diffusion of all but the smallest of solutes and ions. On the other hand, carriers, receptors and active efflux protein mediated transport allow substances such as peptides, amino acids, and glucose to selectively cross the BBB and release toxic substances and drugs into the blood preventing them from entering the*

**3. BBB physiology: building blocks and transport routes in BBB**

*DOI: http://dx.doi.org/10.5772/intechopen.94572*

function, stability, and permeability of the BBB.

linked to vascular dysfunction.

**3.1 BBB junctional molecules**

new therapeutic tools could help modulate intercellular communication in diseases linked to vascular dysfunction.


*5.1. Machine Learning Models Development*

The machine learning models were trained to predict Estatic as a function of the RHOB, DTs, and DTc. The training data were collected from Well-A. Figure 5 compares the actual and estimated Estatic for the training dataset.

**Figure 5.** Actual and estimated static Young's modulus (Estatic) for the training dataset collected from Well-A.

Figure 5 shows that all machine learning models predicted Estatic with very high accuracy. M-FIS predicted Estatic with AAPE of only 0.05% and R of 0.999995. FNN model estimated the Estatic with AAPE and R of 0.78% and 0.999491, respectively, while SVM model estimated Estatic with AAPE of 0.55% and R of 0.999634, and the ANN model predicted Estatic with AAPE of 0.98% and R of 1.000000. The good matching between the actual and estimated Estatic for the training dataset shown in Figure 5 proves the high accuracy of the machine learning models in evaluating Estatic.


**3. Results**

Subject characteristics, including CD4+cell count, CD8+cell count, and CD4/CD8 ratio, weight, body mass index (BMI, in kg/m2 ), are shown in Table 1. At initial screening HIV-infected subjects were significantly lower weight and BMI compared to healthy subjects. There were insignificant changes between initial screening and after 6 months. The range of CD4+cell counts was broad, from 282 to 1830 x 106 cells/L at the initial screening and from 323 to 1687 after 6 months CD4/CD6 ratio was from 0.1 to 1.7 at the initial screening and from 0.1 to 3.4 after 6 months of using food supplements, CD8+cell counts was from 272 to 4525 at the initial screening and from 222 to 4851 after 6 months.

**Table 1.** Effect of antioxidant supplementation on body weight, body mass index and immune function in HIV patients.

Antioxidant status is shown in Table 2. At the baseline HIV-positive patients had significantly lower GSHPX and CAT concentrations compare to healthy subjects (p<0.05), but did not differ after 6 months using food supplements. There were insignificant differences in SOD and MDA measurements and α-Tocopherol concentration at the initial screening, but differed signifi‐ cantly after 6 months compare to HIV-infected and healthy individuals. Changes of biochem‐ ical measurements (alanine aminotranferase, alkaline aminotranferase, bilirubin, albumin and total protein, total holesterol and triglicerides) were not significant between groups of HIVinfected and healthy individuals as well as at the initial screening and after 6 months after use of food supplements. Additionally, two HIV-infected individuals from 26 involved in the study reported that after the use of antioxidant cocktail, gingival bleeding was stopped. One reported this symptom for two years before the study and one reports half a year this problem before study.

in plasma were measured using fluorometric method. Other measurements were also included

Using the SPSS 14.0 for Windows software standard version, statistical analyses were per‐ formed. All group data are expressed as means ± SEMs. The HIV-positive group was compared with the seronegative control subjects by using one way ANOVA. The minimal level of significance was identified at *P* < 0.05. All patients were evaluated with regard to the blood antioxidant system, specifically superoxide dismutase (SOD) and glutathione peroxidase.

Subject characteristics, including CD4+cell count, CD8+cell count, and CD4/CD8 ratio, weight,

subjects were significantly lower weight and BMI compared to healthy subjects. There were insignificant changes between initial screening and after 6 months. The range of CD4+cell counts was broad, from 282 to 1830 x 106 cells/L at the initial screening and from 323 to 1687 after 6 months CD4/CD6 ratio was from 0.1 to 1.7 at the initial screening and from 0.1 to 3.4 after 6 months of using food supplements, CD8+cell counts was from 272 to 4525 at the initial

Weight HIV-infected 26 73.1±2.1 13 74.7±2.8

BMI HIV-infected 26 22.5±0.6 13 23.0±0.8

CD4+ HIV-infected 25 698.4±74.3 21 743.6±87.4 CD8+ HIV-infected 25 1641.9±188.1 21 1341.0±216.1 CD4+/CD8 HIV-infected 25 0.6±0.1 21 0.9±0.2

**Table 1.** Effect of antioxidant supplementation on body weight, body mass index and immune function in HIV

Antioxidant status is shown in Table 2. At the baseline HIV-positive patients had significantly lower GSHPX and CAT concentrations compare to healthy subjects (p<0.05), but did not differ after 6 months using food supplements. There were insignificant differences in SOD and MDA

healthy uninfected 10 82.4±2.6 9 83.3±2.7

Healthy 10 24.7±0.7 9 25.1±0.7

), are shown in Table 1. At initial screening HIV-infected

**Baseline After 6 months**

p<0.05 p<0.05

p<0.05 NS

**n Mean±SE n Mean±SE**

such as catalase and GSHPX.

244 Pharmacology and Nutritional Intervention in the Treatment of Disease

body mass index (BMI, in kg/m2

screening and from 222 to 4851 after 6 months.

**2.3. Statistical analyses**

**3. Results**

patients.

**Table 2.** Effect of antioxidants supplementation on antioxidant enzymes, selenium, vitamin E and lipid peroxidation in HIV patients.

#### **4. Discussion**

There has been increasing interest in the application of antioxidants to many diseases as information is constantly gathered linking the development of HIV to oxidative stress [51]. Antioxidants are believed to function interactively and synergistically to neutralize reactive oxygen species (ROS).

The loss of CD4 T lymphocytes is a central factor in the progression of HIV infection to AIDS. The key role of these cells in regulating and amplifying the immune response means that any decline in their number results in deficits in both humoral and cell-mediated immunity [57]. Both the CD4 percentage and the CD4:CD8 ratio are also affected by changes in the number of CD8 cells, which tends to rise through the course of HIV infection [58].This study demon‐ strates that after supplementation, there is an increase in CD4 T cells in HIV patients. Several research studies have indicated that the apoptosis of CD4 cells contributing to HIV progression does not result solely from HIV infection, but largely from antioxidant imbalances in the host [43, 59, 60]. Activation of latent HIV state can be stimulated in the presence of reactive oxygen species (ROS) through the stimulation of oxygen-responsive transcription factors, specifically

Impact of CoQ10, L-Carnitine and Cocktail Antioxidants on Oxidative Stress Markers in HIV Patients — Mini Review…

http://dx.doi.org/10.5772/58415

247

The mean serum malondialdehyde (MDA) concentrations in this study were significantly elevated in HIV infected patients. Serum concentration of total serum antioxidant status (TAS) was increased after supplementation. Our data indicate that severe oxidative stress occurs in the serum of HIV patients in comparison with controls ones and the inclusion of antioxidants in the therapeutic approach in managing HIV patients may prevent the additional free radicals damage. It has been shown that cells infected with the HIV undergo a significant amplification

− generation. This phenomenon combined with a deficiency in key antioxidant enzymes (SOD and CAT) and a decreased concentration of the antioxidant vitamins, may lead to severe oxidative stress in HIV-infected patients. Consequently these conditions may in turn be responsible for the DNA base modifications observed in this study [43]. Serum MDA adducts also tended to correlate inversely with expression of CD127 on T cells was shown [61]. Study in placenta, umbilical cord blood and infant blood in HIV/ART-exposed infants compared with uninfected controls [62] showed that placental mitochondria malondialdehyde (MDA) concentrations and mtDNA content in placenta and cord blood were similar between groups. Supplementation with antioxidant vitamins prevents oxidative modification of DNA in lymphocytes of HIV-infected patients [44]. Furthermore, as an antioxidant, vitamin E [63]and atherosclerotic lesions contain oxidized lipids [64], therefore, supplementation with vitamin E would decrease heart disease risk. It has been reported that α-tocopherol concentrations in serum are regulated by the α-tocopherol transfer protein and are dependent on serum lipid concentrations because α-tocopherol is non-specifically transported by lipoproteins [63]. Serum micronutrient concentrations along with surrogate markers of atherosclerosis in a cohort of HIV infected adults were studied [65](Falcone et al. 2010), the highest tertile of serum vitamin E concentration was associated with higher common and internal carotid intimamedia thickness (c-IMT) and coronary artery calcium (CAC) scores [65]. The authors concluded that elevated serum vitamin E values may increase the risk of cardiovascular complications in

There appears to be a possible role for antioxidants supplements. However, these supplements cannot effectively eliminate HIV signs and symptoms and the optimal dosage of these nutrients

NF-kB, which induces HIV replication in the infected T-lymphocyte.

of O2

HIV-infected adults [65].

is (whether used alone or in combination) has to defined

All subjects from the current study are at low serum selenium level (which is about 80 – 120 µg /L in Europe), and stayed as that even after use of antioxidants supplements, including selenium. Similar trend was also notice in the selenoenzyme glutathione peroxidase (GSHPx) level among HIV patients. Low serum selenium was defined as a serum level ≤ 85 µg/l [52]. Selenium deficiency, more than any other nutrient, has been documented to correlate with progression and mortality of HIV [53]. Selenium is needed for the proper functioning of the immune system, and appears to be a key nutrient in counteracting the development of virulence and inhibiting HIV progression to AIDS [54]. When taken as a supplement, selenium modulates the cellular response to oxidative stress, inducing a faster restoration of the endogenous antioxidative defence system against the production of reactive oxygen species [55]. Glutathion peroxidase controls the interacellular level of hydrogen peroxide, reducing the formation of reactive oxygen species that can induce lipid peroxidations with consequent damage to the cellular membranes [56].

**Figure 3.** Selenium level of HIV infected individuals and healthy subjects (μ/g Hb)

The loss of CD4 T lymphocytes is a central factor in the progression of HIV infection to AIDS. The key role of these cells in regulating and amplifying the immune response means that any decline in their number results in deficits in both humoral and cell-mediated immunity [57]. Both the CD4 percentage and the CD4:CD8 ratio are also affected by changes in the number of CD8 cells, which tends to rise through the course of HIV infection [58].This study demon‐ strates that after supplementation, there is an increase in CD4 T cells in HIV patients. Several research studies have indicated that the apoptosis of CD4 cells contributing to HIV progression does not result solely from HIV infection, but largely from antioxidant imbalances in the host [43, 59, 60]. Activation of latent HIV state can be stimulated in the presence of reactive oxygen species (ROS) through the stimulation of oxygen-responsive transcription factors, specifically NF-kB, which induces HIV replication in the infected T-lymphocyte.

**4. Discussion**

246 Pharmacology and Nutritional Intervention in the Treatment of Disease

are an ion and a radical.

damage to the cellular membranes [56].

Selenium ug/L

 80 70

60

50

40

30

**Figure 3.** Selenium level of HIV infected individuals and healthy subjects (μ/g Hb)

There has been increasing interest in the application of antioxidants to many diseases as information is constantly gathered linking the development of HIV to oxidative stress [51]. Antioxidants are believed to function interactively and synergistically to neutralize reactive oxygen species (ROS). Reactive oxygen species include molecules like hydrogen peroxide; ions like the hypochlorite ion; radicals like the hydroxyl radical; and the superoxide anion, which

All subjects from the current study are at low serum selenium level (which is about 80 – 120 µg /L in Europe), and stayed as that even after use of antioxidants supplements, including selenium. Similar trend was also notice in the selenoenzyme glutathione peroxidase (GSHPx) level among HIV patients. Low serum selenium was defined as a serum level ≤ 85 µg/l [52]. Selenium deficiency, more than any other nutrient, has been documented to correlate with progression and mortality of HIV [53]. Selenium is needed for the proper functioning of the immune system, and appears to be a key nutrient in counteracting the development of virulence and inhibiting HIV progression to AIDS [54]. When taken as a supplement, selenium modulates the cellular response to oxidative stress, inducing a faster restoration of the endogenous antioxidative defence system against the production of reactive oxygen species [55]. Glutathion peroxidase controls the interacellular level of hydrogen peroxide, reducing the formation of reactive oxygen species that can induce lipid peroxidations with consequent

N = 44 38

HIV infected Healthy

58

59

The mean serum malondialdehyde (MDA) concentrations in this study were significantly elevated in HIV infected patients. Serum concentration of total serum antioxidant status (TAS) was increased after supplementation. Our data indicate that severe oxidative stress occurs in the serum of HIV patients in comparison with controls ones and the inclusion of antioxidants in the therapeutic approach in managing HIV patients may prevent the additional free radicals damage. It has been shown that cells infected with the HIV undergo a significant amplification of O2 − generation. This phenomenon combined with a deficiency in key antioxidant enzymes (SOD and CAT) and a decreased concentration of the antioxidant vitamins, may lead to severe oxidative stress in HIV-infected patients. Consequently these conditions may in turn be responsible for the DNA base modifications observed in this study [43]. Serum MDA adducts also tended to correlate inversely with expression of CD127 on T cells was shown [61]. Study in placenta, umbilical cord blood and infant blood in HIV/ART-exposed infants compared with uninfected controls [62] showed that placental mitochondria malondialdehyde (MDA) concentrations and mtDNA content in placenta and cord blood were similar between groups. Supplementation with antioxidant vitamins prevents oxidative modification of DNA in lymphocytes of HIV-infected patients [44]. Furthermore, as an antioxidant, vitamin E [63]and atherosclerotic lesions contain oxidized lipids [64], therefore, supplementation with vitamin E would decrease heart disease risk. It has been reported that α-tocopherol concentrations in serum are regulated by the α-tocopherol transfer protein and are dependent on serum lipid concentrations because α-tocopherol is non-specifically transported by lipoproteins [63]. Serum micronutrient concentrations along with surrogate markers of atherosclerosis in a cohort of HIV infected adults were studied [65](Falcone et al. 2010), the highest tertile of serum vitamin E concentration was associated with higher common and internal carotid intimamedia thickness (c-IMT) and coronary artery calcium (CAC) scores [65]. The authors concluded that elevated serum vitamin E values may increase the risk of cardiovascular complications in HIV-infected adults [65].

There appears to be a possible role for antioxidants supplements. However, these supplements cannot effectively eliminate HIV signs and symptoms and the optimal dosage of these nutrients is (whether used alone or in combination) has to defined


**3. Conclusions**

*2.4.3. Experimental models of congenital transmission*

36 Leishmaniasis - Trends in Epidemiology, Diagnosis and Treatment

in the liver, spleen, and bone marrow of the offspring [63].

In experimental model in which hamsters were infected with 106

**2.5. Other factors related to substance abuse and work environment**

*2.5.1. Drug use*

*2.5.2. Work environment transmission*

In a murine model of visceral leishmaniasis, twenty 12-week -old female BALB/c mice were infected with *L. infantum*. They were mated 8 weeks later with healthy males, and the females were sacrificed at days 13 and 18 of gestation. The offspring were sectioned in half for PCR analysis. In 15 of the 20 pregnant mice, the parasite was detected by PCR in the spleen. In the offspring, 3 of the 88 placentas and 4 of the 88 pups tested positive for *Leishmania* by PCR [75]. In studies to determine vertical transmission of *Leishmania* in beagles, parasitemia was detected

*sis* during the first week of pregnancy, 24 of 93 (25.8%) of the offspring from infected mothers were PCR positive to *Leishmania*, 2 months after the birth [76]. Furthermore, mice infected with high inoculums of *L. mexicana* strain, known as cause of cutaneous leishmaniasis, showed that all female and their placentas were positive to PCR analysis, and revealed that the infection was present in 39 of 110 offspring of infected mothers, also fetal deaths and resorptions were observed [77]. Then is important to be aware to the fact that leishmaniasis could be transmitted transplacentally and causes fetal resorption, death, and reduction in offspring body weight.

In cases of leishmaniasis infection due to fomites, such as sharp, contaminated objects, the most vulnerable population are illicit drug users. In a Spanish study of syringes used for recreational drug use, it was reported that 32-52% of the syringes were contaminated by *Leishmania*, as determined by PCR. Moreover, 3 different genotypes were identified in multiple samples, confirming that the individuals had shared syringes. Therefore, programs that limit the sharing

As described above, *Leishmania* spp. can be transmitted through fluids such as blood and by contact with animals or even contaminated objects. In all cases, there must be an entry route, which is usually a wound. In staffs dedicated to clinical, diagnostic or medical research, it is not uncommon to find reported cases of *Leishmania* infection, although many such likely go unrecognized [80]. Infections due to accidental exposure can be affected by a variety of factors, including kinematics (e.g., the path and characteristics of exposure and the amount of inoculum), parasite characteristics (e.g., pathogenicity, virulence, viability and infective dose) and host characteristics (e.g., immune status, barrier status and actions following the accident) [80]. However, the possibility of infection due to vector exposure in an endemic region should not be ruled out when performing questioning [81]. The first case of work-related leishmaniasis was reported in 1930, and to date, there have been 12 reported cases of *Leishmania* infection due to accidents at work; these have included 6 different *Leishmania* species, with *L. donovani* being implicated in half of the affected individuals. In these cases, the incubation period ranged

of needles should decrease the infection rate among vulnerable individuals [78-79].

parasites/mL of *L. panamen‐*

The *Leishmania* parasite can survive in a wide range of temperatures and pH conditions, which has allowed it to adapt to the diverse conditions encountered within different vector and host species. In recent years, transmission pathways other than those based on vector species have been described, including invasive procedures for therapeutic purposes, sexual practices, pregnancy, drug practices and work-related accidents among health/research staff members, all of which have led to an increase in the number of reported leishmaniasis cases. However, considering that the latency period of the parasite within the host can last up to one year, there are likely more infected individuals than those that have been reported. Because leishmaniasis is not considered to be a disease that can be transmitted between individuals without the intervention of a vector species, laboratory tests for the presence of these parasites to rule out prospective donors are not carried out prior to in most invasive therapeutic procedures. However, in the case of patients with immunodeficiency, the possibility of contracting the disease is significantly increased. Furthermore, during pregnancy, changes to the immune system can allow for the transmission of the parasite from the mother to the fetus. It should also be noted that maternal-fetal infection can also be of sexual origin. Among drug addicts, it was demonstrated that syringe sharing is a significant source of infection. Finally, in addition to populations exposed in endemic areas, staff working in the health or research sectors should also be considered as populations at risk of acquiring this disease.

Taken together, there is a clear need for the health system to reevaluate the global situation concerning leishmaniasis transmission and to implement strategies to reduce the exposure of individuals to *Leishmania* infections.


**4. Conclusions**

In this work, we presented a structural study of a Cu(II)-H2dtp-based MOF system, in which the rational assembly of Cu(II), dtp2− or Hdtp− as well as auxiliary bdc2− linker give birth to two new MOF materials with the formulae of {[Cu(dtp)](H2O)2}n (**1**) and [Cu(Hdtp)(bdc)0.5]n (**2**). The harvest of 2D "wave-shaped" networks in **1** benefits from the five-coordinative Cu(II), which suggests a low metal ion coordination number in controlling

the formation of the 2D Cu(II)-dtp2− network. The introduction of bdc2− into the Cu(II) dtp2− system results in a 1D chain structure with a "ladder-shape" in **2**. A unique 2D supermolecular layer with a thickness of 7.9 Å is formed by the orderly array of the 1D chain, which may help to broaden the horizon for the assembly of 2D materials through the intermolecular interactions. The air stability of two MOFs is witnessed by PXRD, and the TGA indicates that the thermal stability of the two MOFs is as high as 300 ◦C. The unique structure and relatively good stability of these Cu(II)-MOFs inspired us to further isolate the 2D structures of **1** and **2** as 2D materials for further research.

**Supplementary Materials:** The following supporting information can be downloaded at: https:// www.mdpi.com/article/10.3390/cryst12050585/s1, Table S1: Hydrogen-bonding parameters in **1**; Table S2: Hydrogen-bonding parameters in **2**; Table S3: Selected bond lengths (Å) and angles (o) for **1**; Table S4: Selected bond lengths (Å) and angles (o) for **2**. CCDC 2164393 (**1**) and 2164388 (**2**) contain the supplementary crystallographic data for this paper. These data can be obtained free of charge via www.ccdc.cam.ac.uk/data\_request/cif (accessed on 5 April 2022), by emailing<EMAIL_ADDRESS>or by contacting The Cambridge Crystallographic Data Centre, 12 Union Road, Cambridge CB2 1EZ, U. K.; Fax: +44-1223-336033.

**Author Contributions:** Conceptualization, Z.-W.Z. and L.-Q.W.; methodology, Z.-W.Z., L.-Q.W. and J.-J.L.; validation, J.-J.L., X.-X.C., Q.-W.C. and Y.Q.; formal analysis, J.-J.L., X.-X.C.; Q.-W.C. and Y.Q.; investigation, Z.-W.Z. and L.-Q.W.; resources, Z.-W.Z., L.-Q.W. and J.-J.L.; data curation, J.-J.L.; writing—original draft preparation, Z.-W.Z. and L.-Q.W.; writing—review and editing, Z.-W.Z., L.-Q.W. and Q.C.; visualization, Q.C.; supervision, Z.-W.Z. and L.-Q.W.; project administration, Z.-W.Z. and L.-Q.W.; funding acquisition, Z.-W.Z., L.-Q.W. and J.-J.L. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research was funded by National Natural Science Foundation of China grant number 22161009, Thousands of Young and Middle-aged Backbone Teachers Training Project of Guangxi Colleges and Universities grant number Gui-Jiao 2020-58, University innovation foundation of Guangxi Medicine grant number Gui-Degree 2021 No. 6, Collaborative Innovation Center of Zhuang and Yao Ethnic Medicine grant number 2013 No. 20, Guangxi University of Chinese Medicine Key Project of First-class Discipline Construction grant number 2018XK050, Guangxi Natural Science Foundation grant number 2022GXNSFAA035477.

**Institutional Review Board Statement:** Not applicable.

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** Not applicable.

**Conflicts of Interest:** The authors declare no conflict of interest.

#### **References**


*Article* **Risk of Metabolic Syndrome in Kidney Stone Formers: A Comparative Cohort Study with a Median Follow-Up of 19 Years**

**Robert M. Geraghty 1, Paul Cook 2, Paul Roderick 3 and Bhaskar Somani 4,\***

**Abstract:** Background: Kidney stone formers (SF) are more likely to develop diabetes mellitus (DM), but there is no study examining risk of metabolic syndrome (MetS) in this population. We aimed to describe the risk of MetS in SF compared to non-SF. Methods and Materials: SF referred to a tertiary referral metabolic centre in Southern England from 1990 to 2007, comparator patients were age, sex, and period (first stone) matched with 3:1 ratio from the same primary care database. SF with no documentation or previous MetS were excluded. Ethical approval was obtained and MetS was defined using the modified Association of American Clinical Endocrinologists (AACE) criteria. Analysis with cox proportional hazard regression. Results: In total, 828 SF were included after 1000 records were screened for inclusion, with 2484 age and sex matched non-SF comparators. Median follow-up was 19 years (interquartile range—IQR: 15–22) for both stone formers and stonefree comparators. SF were at significantly increased risk of developing MetS (hazard ratio—HR: 1.77; 95% confidence interval—CI: 1.55–2.03, *p* < 0.001). This effect was robust to adjustment for pre-existing components (HR: 1.91; 95% CI: 1.66–2.19, *p* < 0.001). Conclusions: Kidney stone formers are at increased risk of developing metabolic syndrome. Given the pathophysiological mechanism, the stone is likely a 'symptom' of an underlying metabolic abnormality, whether covert or overt. This has implications the risk of further stone events and cardiovascular disease.

**Keywords:** kidney stones; metabolic syndrome; urolithiasis; nephrolithiasis; kidney calculi; diabetes mellitus

#### **1. Introduction**

Kidney stone disease (KSD) is a costly [1] and increasingly prevalent problem, with the latest USA prevalence (2015–2016) being 10% [2]. Amongst the risk factors for development of KSD, type 2 diabetes mellitus and the metabolic syndrome (MetS) [3] are particularly well described. Both are characterised by high blood glucose and insulin resistance [4] and share common pathophysiologic mechanisms that attributes to the increased risk of KSD, e.g., urinary acidification [5]. This translates to a proportional increase in uric acid stones [6]. Given the MetS pandemic [7], this will lead to worldwide increases in KSD.

The other components of MetS (obesity, hypertension and dyslipidaemia) have all been described, to varying degrees, as carrying increased risk of KSD. There is good epidemiological evidence for the link between obesity and an associated risk of KSD [8]. However, the cause of this increased risk is likely due to the metabolic sequelae of obesity, such as dyslipidaemia and insulin resistance [5].

There is conflicting evidence for the risk of KSD in hypertensives. Unadjusted crude risk demonstrates significantly increased risk of hypertensives becoming stone form-

**Citation:** Geraghty, R.M.; Cook, P.; Roderick, P.; Somani, B. Risk of Metabolic Syndrome in Kidney Stone Formers: A Comparative Cohort Study with a Median Follow-Up of 19 Years. *J. Clin. Med.* **2021**, *10*, 978. https://doi.org/10.3390/jcm10050978

Academic Editor: Wisit Cheungpasitporn

Received: 30 January 2021 Accepted: 18 February 2021 Published: 2 March 2021

**Publisher's Note:** MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

**Copyright:** © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

ers [9,10]. However, on adjustment the increased risk is rendered non-significant [9,10]. This is likely due to the confounding presence of other KSD risk factors, e.g., high blood glucose or dyslipidaemia.

Dyslipidaemia (high serum triglycerides and low high-density lipoprotein) causes demonstrable derangements in 24 h urinary biochemistries [11]. A further sequela of dyslipidaemia is lipotoxicity (abnormal lipid accumulation in tissues) [12]. In the kidney, lipotoxicity reduces ammonium secretion and lowers pH (both risk factors for KSD) [11].

Not only are the components of MetS risk factors for KSD, the reverse is also true. Stone formers are at increased risk of developing both diabetes mellitus [13] and hypertension [14]. As yet however, there is no evidence for increased risk of MetS in stone formers.

The importance of a MetS diagnosis is the increased risk of cardiovascular disease [15]. Although the definition has changed over the years, the consensus across multiple large cohort studies is a 2- to 4-fold increase in risk of cardiovascular disease in those with MetS. This has clinical implications for individuals and populations.

As there has been no study examining the risk of developing MetS in the stone forming population, our primary aim was to describe this risk in stone formers. Our secondary aims were to examine the risk of individual MetS components and risk of MetS per stone type.

#### **2. Methods**

#### *2.1. Definitions*

Metabolic syndrome was defined using the Association of American Clinical Endocrinologists (AACE) criteria [4], which is similar to the more widely used National Cholesterol Education Program Adult Treatment Panel (NCEP ATP) III criteria. It replaces waist circumference with (body mass index) BMI > 25, as waist circumference was not available on electronic records. In addition to AACE criteria, specific treatment for hyperglycaemia or hypertension were included, as well as physician diagnosis (see Table 1). Development of three or more components was defined as incident Metabolic syndrome (MetS). Age of development of MetS defined as age at which 3 or more components present, components assumed to be cumulative, i.e., patients will not lose diabetic or hypertensive, etc., status with increasing age.

Electronic records included all clinical letters, operation notes, test results, diagnoses, treatments, and basic readings including blood pressure, height, and weight.

**Table 1.** Metabolic syndrome definition.

#### *2.2. Study Population*

The cohort consisted of patients with kidney stone disease (KSD) presenting to a tertiary referral hospital referred for metabolic assessment between 1990 and 2007. The study population has been described in a previous cross-sectional study [16] and subsequent cohort study [1]. During this period, stone formers were routinely referred to this clinic by the urology team (both in Southampton and around the region—Dorset, Wiltshire, and Hampshire) and general practitioners. In total, 1000 (from 2801) patients were selected by block randomization after alphabetization of surnames.

Further information on past medical history and subsequent stone recurrence was ascertained retrospectively using hospital and general practice electronic records. The general practice electronic records is downloaded to the Care and Health Information Exchange (CHIE), a large database including data from 172 general practices within Hampshire and the Isle of Wight (95% coverage).

Data collected in retrospect using CHIE: age, sex, past medical history at first presentation, including metabolic syndrome components (see Table 1) and incident metabolic syndrome components. Subsequent stone episodes and stone type were ascertained using a combination of CHIE and hospital records. See Appendix A for stone disease read codes.

Patients who had no documentation (i.e., no evidence of subsequent follow-up or consultation, lived outside or have left Hampshire, or no documentation on CHIE) or had pre-existing metabolic syndrome (MetS) were excluded (see Figure 1).

**Figure 1.** CONSORT flow diagram of patient selection.

#### *2.3. Comparator Population*

Comparator data was supplied by Care and Health Information Analytics (CHIA), the body utilising CHIE data for research, using age (within 5 years), sex, and region matched patients in a ratio of 3:1 once stone formers (SF) had been screened for eligibility. The follow-up period was matched as closely as possible.

Patients with codes associated with KSD (see Appendix A) and previous components of metabolic syndrome were excluded. Data on incident metabolic syndrome components were collected (see Table 1), time defined as initial age to age at which first reached diagnostic criteria for metabolic syndrome component.

Only practices which were present within CHIA on 1st May 2019 were selected to be included. Random patients were selected from this practice cohort. Data on age of development of MetS components and death (if applicable) were extracted.

#### *2.4. Statistical Methods*

SPSS (version 26, IBM, Armonk, NY, USA) and R statistical package version 3.6.3 (R Foundation for Statistical Computing, Vienna, Austria. URL https://www.R-project. org/) (packages: survival and survminer) were used for statistical analysis. Cox propor-

tional hazards model was used to analyse the data, which is presented as hazard ratio (HR) with 95% confidence interval (CI). Time to event was defined as time from presentation to metabolic stone clinic to development of 3 components of metabolic syndrome for both stone formers and comparators. Censoring time was defined as time from presentation to metabolic stone clinic to last CHIE entry or death. We tested the proportional hazards assumptions by calculating Schoenfield residuals and performing a log-rank test.

Subanalyses for 0 and 1 or 2 previous components, as well as stone type. The main outcome measure was adjusted for number of previous components. Chi-squared test was used to compare prior to year 2000 vs. 2000 onwards for components of MetS.

#### *2.5. Sample Size Calculation*

Sample size was calculated estimating a 10% difference (30:40%) in rates of MetS diagnosis between the two groups. Power was set at 80% and significance at 0.05. Sample size was therefore calculated at *n* = 172 per group. Larger numbers have been included to increase power for subanalyses. The 3:1 ratio of controls to cases was used to increase robustness and power.

#### *2.6. Ethical Approval*

Ethical approval for this study was granted by the NHS Bristol Research Ethics Committee (Research ethics committee reference: 18/SW/0185; IRAS ID: 240061).

#### **3. Results**

#### *3.1. Demographics*

There were 828 stone formers and 2484 stone free comparators, with no differences in age or sex between the groups. Stone formers underwent a median 19 years (IQR: 15–22) of follow-up from initial presentation to biochemical clinic. Non-stone formers had data available for the same time period (median 19 years, IQR: 15–22).

There were 361 (43.6%) stone formers who developed metabolic syndrome (MetS), whilst 617 (24.8%) of the stone free comparators developed MetS. Numbers of components and primary stone composition are detailed in Table 2. Deaths were similarly proportioned in the two groups with 113 (13.6%) amongst stone formers, and 366 (14.7%) amongst the comparators.

There were 719 (86.8%) and 2118 (85.3%) stone free comparators without any prior components of MetS. There were 111 (13.4%) and 332 (13.4%) stone free comparators with 1 or 2 components.

**Table 2.** Demographics of stone formers and stone-free comparators.

#### *3.2. Risk of Metabolic Syndrome in Stone Formers*

Stone formers were at significantly increased risk of developing MetS (HR: 1.77; 95% CI: 1.55–2.03, *p* < 0.001) (see Figure 2 and Table 2). This effect was robust to adjustment for presence of previous components (HR: 1.91; 95% CI: 1.66–2.19, *p* < 0.001). This effect was consistent with subanalyses of no previous components (HR: 1.98; 95% CI: 1.69–2.31, *p* < 0.001) and 1 or 2 previous components (HR: 1.54; 95% CI: 1.11–2.14, *p* = 0.011).

**Figure 2.** Kaplan Meier curve with 95% CI (confidence interval) for time to development of metabolic syndrome.

Subanalysis of stone type demonstrated significantly higher risk for stone patients compared to their matched comparators presenting with calcium oxalate (HR: 1.82; 95% CI: 1.53–2.16, *p* < 0.001) and urate stones (HR: 3.87; 95% CI: 2.23–6.72, *p* < 0.001) (see Table 1). Other stone types did not carry significant risk of developing MetS.

Subanalysis of individual components of the metabolic syndrome demonstrated SFs were significantly more likely to develop all bar impaired glucose tolerance on both unadjusted and adjusted analyses (see Table 3). Those with the component pre-existing were excluded.

**Table 3.** Individual components of metabolic syndrome and overall risk. Adjusted for age and sex.

Numbers of patients at follow-up times were as follows: 5-years (control, *n* = 2484; SF, *n* = 828), 10-years (control, *n* = 2481; SF, *n* = 827), 15-years (control, *n* = 1938; SF, *n* = 646), 20-years (control, *n* = 1119; SF, *n* = 373), 25-years (control, *n* = 366; SF, *n* = 122).

There were significantly more patients with previous components of the metabolic syndrome after 2000 than prior (Chi-square, *p* < 0.001), despite this analysis of only those presenting after 2000 still had a significantly increased risk of developing MetS (HR: 2.42, 95% CI: 2.01–2.92, *p* < 0.001). Log rank demonstrated a significant result (*p* < 0.001). Visual

inspection of the Schoenfeld residuals did not demonstrate variation around 0, although it did demonstrate a significant result (global Schoenfeld test, *p* < 0.001) (see Figure 3).

**Figure 3.** Schoenfeld residuals plotted against time. Loess line with 95% CI.

#### **4. Discussion**

This is the first study to examine the risk of metabolic syndrome in stone formers. There was a significant risk (nearly twice as likely) of developing metabolic syndrome in this population, which was more common still in those with uric acid stones.

The main strength of this study is an appropriately powered, significant primary outcome, which is robust to adjustment for previous components. The use of 3:1 matching of study participants to comparators for age and sex, improves power and robustness. Broadly, the sensitivity analyses (log-rank, Schoenfeld residuals and subanalyses) demonstrate results in keeping with the primary outcome.

The major limitation of this study is the risk of under-ascertainment of MetS at baseline (there were only 20 stone formers with MetS), this is reflected in significantly lower MetS components prior to 2000 in both groups. Routine screening of metabolic syndrome components by General Practitioners was not established until after the millennium, which would account for the previously mentioned observation. One would expect a higher number of stone formers to have pre-existing MetS, given that they are more likely to develop KSD [3]. However, the risk of under-ascertainment is likely to be inherent to both groups. We have also adjusted for prior components for both groups, and performed subanalyses on development of MetS with 0, 1, and 2 previous components. All of these analyses demonstrate highly significant results, increasing the likelihood that stone formers are indeed at increased risk of MetS.

There are several other weaknesses to this study. Firstly, the dataset used, Care and Health Information Exchange (CHIE) uses data inputted by general practitioners. Primary care data are known to be more variable and less accurate than secondary care data [17]. It was also not possible to match patient's address' and GP practice's and therefore we were unable to adjust for deprivation. However, the expected results are significant (i.e., urate stones increase risk of MetS and increased risk of recurrence in stone formers with MetS), and therefore there is no risk of type 2 error. Secondly, risk of type 1 error may

be present given the multiple testing in the secondary outcomes, and larger studies are needed to corroborate these findings. Lastly, there may be an argument that stone referrals to a tertiary referral service are not representative of the general stone forming population. However, the recurrence rate is similar to previously documented series (around 40% at 10 years in this cohort) [18], only a small proportion were started on prophylactic medication (16%) and there were similar ratios of stone types to previous series [6,19]. Due to these reasons, we believe this dataset is representative.

The increased risk of MetS in the stone forming population is significant given the rising prevalence of kidney stone disease (KSD), which was 10% in 2015–2016 in USA [2]. This translates into 38.2 million Americans who have had a kidney stone and are therefore at roughly twice the risk of developing MetS, with the associated 2- to 4-fold increased risk in cardiovascular disease [15]. It is should be noted it is unlikely to be all stone formers who develop MetS as there are alternative causes of KSD (genetic, infection, drugs, etc.) that have no association with MetS or cardiovascular disease [20].

It is clear that insulin resistance and renal lipotoxicity are the main drivers of stone formation in the MetS population [11,21]. However, it is not clear why stone formers are at increased risk of developing MetS. Our observation that stone formers are more likely to develop MetS correlates with previous studies on the increased risk of developing diabetes [13] and hypertension [14] in stone formers. Both MetS and DM are characterized by insulin resistance, which leads to urinary acidification and increased uric acid excretion [6,22] with a resulting higher proportion of uric acid stones [23]. Hypertension is also associated with urinary acidification along with hypocitraturia [24], both risk factors for stone formation. However, there is no evidence that kidney stones, or abnormalities in 24 h urinary biochemistry influence the development of MetS or its components.

Intriguingly, the link between KSD and MetS is reflected in the genetics literature. In genome wide association studies, two single nucleotide polymorphisms (rs780093 and rs1260326) within a single gene (*GCKR*) are associated with both KSD [25,26] and MetS [27]. This gene encodes glucokinase regulator protein, which is mainly expressed in the liver [28]. Although not yet demonstrated in functional studies, clinically these variants are associated with higher triglycerides and higher fasting plasma glucose [29], both of which are components of MetS and risk factors for KSD. KSD is therefore likely to be a result of metabolic derangements, given the association with these variants (no renal expression of *GCKR)* and the associated risk of KSD with higher triglycerides, higher fasting plasma glucose and MetS.

If KSD is indeed a symptom of an underlying metabolic derangement, rather than vice versa, then there may be evidence of metabolic dysfunction at presentation. It is unclear in the literature whether there is evidence of insulin resistance or renal lipotoxicity, or its surrogates (dyslipidaemia or high BMI) at this point, and we have discussed the risk of under-ascertainment of MetS components earlier. Interestingly, Sagesaka et al. demonstrated that type 2 diabetes could be predicted up to 10 years before the patient developed the condition using the same factors used to diagnose metabolic syndrome [30]. Unfortunately, they did not examine if the components of MetS rose and fell, respectively, as fasting plasma glucose did.

Futures studies should examine the presence of metabolic syndrome components in stone formers prospectively, examining risk of recurrence with metabolic syndrome and development of metabolic syndrome. The involvement of geno- and phenotype correlations should be considered. Preventative measures for both recurrent stones and components of metabolic syndrome should be trialled. More work also needs to be done on primary prevention and effect on patients quality of life [31,32].

Routine assessment for components of MetS should be standard when assessing a stone formers given the further risk of KSD and, perhaps more importantly, the long-term cardiovascular implications [15].

#### **5. Conclusions**

Kidney stone formers are at increased risk of developing metabolic syndrome, which is commoner with uric acid stones. A stone is likely a 'symptom' of an underlying, perhaps covert, metabolic derangement in idiopathic stone formers given the described pathophysiology.

This increased risk has both individual and health policy implications given the associated cardiovascular outcomes. Assessment for metabolic syndrome should be standard for patients presenting with kidney stones.

**Author Contributions:** Study design/concept: R.M.G., B.S.; Data collection: R.M.G.; Data analysis: R.M.G.; Manuscript draft: R.M.G.; Critical appraisal of manuscript: R.M.G., P.C., P.R., B.S. All authors have read and agreed to the published version of the manuscript.

**Funding:** Robert Geraghty is a National Institute for Health Research funded Academic Clinical Fellow.

**Institutional Review Board Statement:** Ethical approval for this study was granted by the NHS Bristol Research Ethics Committee (Research ethics committee reference: 18/SW/0185; IRAS ID: 240061).

**Informed Consent Statement:** Patient consent was waived as this is a retrospective cohort study. Ethical approval was sought and gained to not gain patients consent to access their records. Ethical approval was granted on the provision that only the authors (who are all involved in these patient's care) had access to the data.

**Data Availability Statement:** As data is identifiable it will not be made available as per ethical approval.

**Acknowledgments:** We would like to thank the Care and Health Information Analytics team for providing the comparator data. We would like to acknowledge Valerie Walker for initiating the Southampton stone database.

**Conflicts of Interest:** The authors declare no conflict of interest.

#### **Appendix A**

Read codes for Kidney stone disease used to exclude patients: a. Readcode version 2 '4G4and below within hierarchy.

b. CTV3

#### **References**


**2.4 Compositional analysis of algal oil**

Algal oil is very high in unsaturated fatty acids [39]. The mentioned fatty acids are present in abundance in algal oil [40].

• Arachidonic acid (AA)

It is an unsaturated, essential long chain (C20) fatty acid and acts as a precursor in the biosynthesis of prostaglandins, thromboxanes, and leukotrienes.

• Eicospentaenoic acid (EPA)

EPA is an omega-3 fatty acid. Omega-3 fatty acid helps lower risk of heart disease, lower triglycerides in the blood, blood pressure, and inflammation.

**Figure 2.**

*Microalgal oil and further trans-esterification to bio-diesel extraction procedure.*

• Docasahexaenoic acid (DHA)

DHA is a long-chain, highly unsaturated omega-3 fatty acid. It affects cell and tissue physiology and function by altering the membrane structure. It also has prominent roles in membrane protein function, cellular signaling and production of lipid mediator.

• Gamma-linolenic acid (GLA)

GLA is an essential fatty acid belonging to omega-6 fatty acid. Omega-6 fatty acids play a crucial role in brain function, growth and development, stimulate skin and hair growth, maintain bone health, regulate metabolism, and maintain the reproductive system.

• Linoleic acid (LA)

It is an essential fatty acid belonging to the omega-3 group. It has been reported to inhibit the synthesis of prostaglandin resulting in reduced inflammation and prevention of certain chronic diseases.