Released this past November, the list is based on data collected from the Web of Science and highlights some of the world’s most influential scientific minds by naming the researchers whose publications over the previous decade have included a high number of Highly Cited Papers placing them among the top 1% most-cited.
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We wish to congratulate all of the researchers named and especially our authors on this amazing accomplishment! We are happy and proud to share in their success!
IntechOpen is proud to announce that 179 of our authors have made the Clarivate™ Highly Cited Researchers List for 2020, ranking them among the top 1% most-cited.
\n\n
Throughout the years, the list has named a total of 252 IntechOpen authors as Highly Cited. Of those researchers, 69 have been featured on the list multiple times.
\n\n\n\n
Released this past November, the list is based on data collected from the Web of Science and highlights some of the world’s most influential scientific minds by naming the researchers whose publications over the previous decade have included a high number of Highly Cited Papers placing them among the top 1% most-cited.
\n\n
We wish to congratulate all of the researchers named and especially our authors on this amazing accomplishment! We are happy and proud to share in their success!
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1. Introduction
\n
The growing resistance of pathogenic bacterial isolates against the traditional chemical antibiotics as well as the resurgent of old disappeared diseases associated with the constant consumers’ demanding of healthier, nutritious and safe food has led the researchers to focus on searching for new, safe and effective molecules. One class of such molecules is the class of polyphenols. Polyphenols are a ubiquitous class of compounds largely present in plants as their secondary metabolites, which are synthesized during their normal development [1] in response to several stressful biotic and abiotic factors [2, 3]. This class of compounds are a much diversified group derived from the amino acids phenylalanine and tyrosine and comprise simple phenols, hydroxybenzoic acids and cinnamic acid derivatives, flavonoids, coumarines, stilbenes and tannins, among others [4–6].
\n
The results from the last decade’s research have shown that polyphenols have important beneficial properties for human health, including antioxidative, antiaging, antibacterial and anti-mutagenic [7–11]. Moreover, the recent evidence of their interaction with proteins, DNA and other biological molecules has enhanced their exploitation for the production of new natural product-derived therapeutic agents. Despite these advantages, several limitations still persist, particularly those related with their extraction efficiency, which affects the large-scale use of some of these substances. The difficulties in screening, extracting, separation and purifying these compounds have increased the development of new and modern methods to address these limitations. In this context, the aim of this chapter is to present an updated review about sources, technologies and methods that have been developed until now to improve the extraction, detection, separation and full characterization of such beneficial compounds, with special emphasis to their possible application in the design of nutraceuticals and functional food products.
\n
2. Foods as natural resources of phenolics
\n
Polyphenols have been exhaustively studied in their different natural matrices such as fruits, vegetables, teas, algae and microalgae and more recently agro-food wastes (peels, seeds, pulps, stems and roots) [12–15]. In the three last decades, there has been a prolific publication of scientific studies showing that plant-derived foods and agro-food wastes from industrial transformation have huge quantities of polyphenols. In Table 1 are summarized some recent studies, and as result from these and other studies, there is a diverse source of polyphenols in plant materials, but both type and amount seem to be highly influenced by their chemical nature, extraction methods, sample particle size, storage time and conditions, as well as by the presence other of interfering substances [25]. Also, their chemical structure and nature vary from simple to highly polymerized substances that include varying proportions of phenolic acids, phenylpropanoids, anthocyanins and tannins, among others [26–28]. Moreover, they might also exist in complex mixtures with carbohydrates, proteins and some quite insoluble high-molecular-weight phenolics [28]. Therefore, the phenolic extraction from plant materials is always a mixture of different steps, and many modifications of a particular method are often needed for the removal of unwanted non-phenolic substances such as waxes, fats, terpenes, pigments (chlorophylls and carotenoids). Solid-phase extraction (SPE) techniques, purification and fractionation based on acidity, are commonly used to remove unwanted non-phenolic substances or even other unwanted phenolics [29].
\n\n
Polyphenols
Source (some examples)
Phenolic acids
Hydroxycinnamic acids
Cereals, coffee, cherries, citrus fruits and juices, peaches, plums, spinach, tomatoes, wheat flour, corn flour, rice flour, potato, olive mill wastewaters, winery sludge from red grapes, artichoke wastewaters, almonds
Hydroxybenzoic acids
Oilseeds, cereals, coffee, cowpeas, wheat flour, black currant, blackberry, raspberry, squash seeds and shell
Flavonoids
Anthocyanins
Grapes, red wine, grape seeds, grape skins, winery by-products, fermented grape pomace, strawberries, back and red currants, raspberries, plums, red cabbage
Chalcones
Apples and apple juices,
Flavanols
Apples, grapes, leeks, tomatoes, curly kale, onions, lettuces, berries, beans, red grapes, black and green tea, red wine and red winery by-products, cider
Flavanones
Citrus fruits, citrus juices, orange peels and seeds wastes
Flavonols
Apples, apple peels, beans, leeks, lettuce, onions, tomatoes, olive leafs, broccoli inflorescences, chestnut, olives and olive fermented pomaces
Most common types of polyphenols found in foods and plant-derived products [14–24].
\n\n\n
Although the recent advances in the technology had providing innovative approaches to obtain enriched polyphenol natural extracts, we must ware that their extraction efficiency will always be dependent of several factors in which the nature of samples and solvent, pH, temperature, light, length of extraction period, particle size, solvent/sample ratio and liquid-liquid or solid-liquid extraction process [25], among others, are the most critical.
\n
3. Methods used in extraction of polyphenols
\n
It is widely accepted that the extraction step is one of the most important stage in isolation of polyphenols, but based in literature, there is no consensus about one single and effective standard extraction method. On contrary, there are several reported methods with very accurate results, and according to the literature in some cases, the solid-liquid extraction with different types of solvents is more adequate [30], and in others, the ultrasound-assisted extraction method (UAE) increases the extraction efficiency [31], while in others, this increment is higher when a microwave-assisted extraction (MAE) is used [32], and advanced methods such as pressurized fluid extraction (PFE), supercritical CO2 extraction (SC-CO2) and enzyme-assisted extraction (EAE) are even better to enhance the content of polyphenols in the extracts [33–39]. Despite this diversity, all have the common fact that the extraction must be conducted carefully but exhaustively with simple, rapid and feasible procedures, and if possible open to automation [40]. In the next paragraphs, we present a summarized information of the most commonly used methods for the extraction of polyphenols in several pant and food matrices.
\n
3.1. Classical solvent extraction
\n
The classical solvent extraction of polyphenols usually includes extraction by maceration and percolation and by successive Soxhlet extraction [41–45].
\n
The maceration, widely used in the past, is nowadays in underuse since other methods are more feasible. It is a simple procedure in which the powdered sample is soaked in an appropriate solvent in a closed container, normally under room temperature with constant or sporadic agitation [41, 46]. In the end of the extraction, the solid parts need to be separated from the solvent, which can be done by filtration, clarification and/or decantation [47]. This method is quite simple to hand but has the main disadvantage of time-consuming, requires a large volume of solvent [41, 42, 48, 49].
\n
Similar to the maceration, the percolation method is characterized by placing the powdered sample in a closed container (normally cylindrical) in which the solvent is discharged from the top towards the bottom in a slow movement (drop wise). [41, 42, 50]. In this case, the filtration is not necessary because the percolator device has itself a filter which placed at the bottom, and we can only collect the final liquid. This method faces the same issues of maceration, which are time-consuming, large volumes of solvent, solubility of polyphenols, particle size of sample and contact time between solvent and sample.
\n
In Soxhlet extraction [41, 42], the powdered samples are sealed in cellulose bags and placed in an extraction chamber located on top of a collecting flask beneath a reflux condenser, and after the addition of the solvent, the system is heated and the solvent condenses after reaching certain level of temperature [51–53]. A reflux occurs continuously. At the end, the liquid extract is collected to the flask positioned beneath the system [51–53]. The Soxhlet extraction is a continuous process with the advantage of being less time and less solvent consuming than the maceration or percolation methods [54]. However, some authors have stated that Soxhlet extraction must be handled carefully because the excess of temperature, always near to the boiling point, can destroy or modify some thermolabile polyphenols [44]. Others reported that Soxhlet extraction is used widely because of its convenience [41, 42, 44, 54]. Although these have variations, all these three methods have the common usage of organic solvents in a solid/liquid ratio. Solvents such as water, methanol, ethanol, acetone, n-hexane, chloroform, propanol and ethyl acetate have been most commonly used for the extraction of polyphenols (Table 2). The difference between solvents resides in their polarity (Figure 1) which affects their capacity in extract phytochemicals. The miscibility of organic solvents (Figure 2) with each other’s or even other types of solvents is another fact to be considered in order to improve the polyphenol extraction yield as shown by several studies [59–63].
4Data collected from Hakansson et al. (2016) [58].
\n\n\n
Figure 1.
Polarity of the organic solvents most commonly used in phytochemicals extraction from natural sources. Adapted with permission from Refs. [55–56].
\n
Figure 2.
Miscibility of organic solvents used in the extraction of phytochemicals from natural sources. Adapted with permission from Refs. [56–58].
\n
In general, organic solvents and their aqueous formulations are mostly used in the extraction of phytochemicals, but it is still no clear which solvent is most adequate for the extraction of polyphenols. For example, acetone showed to be very efficient in the extraction of polyphenols [59] from lychee (Litchi chinenesis Sonn.) flowers in comparison with methanol, ethanol or water. While in walnut (Juglans regia L.) green husks, the highest extraction yield of polyphenols (44.1%) was obtained when water was used as extraction solvent [60]. By other hand, in a recent study [61], it was found that aqueous and organic solvent have a higher extraction efficiency than absolute organic solvents. Similar situation was observed in Phoradendron californicum oak extracts [62], in which aqueous methanol was the solvent most efficient for the extraction of polyphenols.
\n
Based on the literature, there is no consensus about the best solvent to extract polyphenols. However, it has been widely accepted that higher polarity usually means better solubility of polyphenols into extraction solvents; however, differences in the structure of phenolic compounds may be critical for their solubility. Thus, the extraction of polyphenols and other phytochemicals must be prior tested and adapted to the solvent, because the diverse structures of polyphenols, such as multiple hydroxyl groups, conjugated or not with sugars, acids or alkyl groups, interfere in the extraction process. Therefore, it is very difficult to say what type of solvent is better to develop a standard method for all type of polyphenols, but the majority of the authors seem to agree that a good solvent system is the one that allows the maximization of polyphenols extracted without any modifications of their chemical nature. In this context, several factors must be considered when a specific solvent is selected, including (i) solvent power (selectivity); (ii) polarity; (iii) boiling temperature (should be low in order to facilitate removal of the solvent from the product); (iv) reactivity (the solvent should not react chemically with the extract neither should be decomposed quickly); (v) viscosity (low); (vi) stability (should be stable to heat, oxygen and light); (vii) safe in use (should be nonflammable and nontoxic for consumers and environment); (viii) if possible, suitability for reuse; and (ix) compatible with legislation for food applications.
\n
3.2. Advanced methods of extraction
\n
Classical extraction methods are dominated in many laboratory facilities mainly due to its simplicity and low economic cost. Nonetheless, many scientific reports have shown that maceration, percolation and Soxhlet extraction have low efficiency and several environmental issues due to the pollution caused in the environment when large volumes of organic solvents are used. Moreover, the classical extraction often requires a recovery step followed by evaporation to concentrate the extract, which makes it a high time-consuming process. To overcome these constraints, a number of methods have been developed in the last years such as microwave-assisted extraction (MAE); ultrasound-assisted extraction (UAE); supercritical CO2 extraction (SC-CO2); pressurized fluid extraction (PFE); enzyme-assisted extraction (EAE) or even combined approaches. From the hundreds of papers published until now, it seems that these novel extraction techniques can be an interesting choice for classical extraction methods, offering several advantages such as less extraction time length, less volume of solvents, less final toxic residues, higher extraction yields and better reproducibility.
\n
Hundreds of works have been published (some of them listed in Table 3) [63–73] about the use of such methods to improve the extraction yield of polyphenols in different matrices. In the next paragraphs is presented a detailed description of each method.
\n\n
Method
Botanic matrix
Results reported by the authors
References
MAE
Blueberries (Vaccinium corymbosum L.)
The usage of MAE increased the yield of anthocyanins extracted.
Celluclast®, Pectinex® Ultra® and Novoferm® were used to release phenolic compounds from grape wastes. The pretreatment with enzymes increased the yield of polyphenols extracted.
EAE + PLE were applied for the extraction of phytochemicals. The results showed that EAE + PLE enhanced the total phenolic content and the antioxidant capacity.
Identification of phenolic acids, flavonol glycosides in blueberry (Vaccinium corymbosum L.), blackberry (Rubus fructicosus L.), raspberry (Rubus idaeus L.) and cranberry (Vaccinum vitis-idaea L.)
Some recent examples of the usage of HPLC-MS system for separation and quantification of polyphenols in different matrices.
\n\n
3.2.1. The microwave-assisted extraction (MAE)
\n
The MAE is a method that uses energy of microwave radiation to heat solvent in contact with the sample [74–77]. The heat produced increases the diffusivity of the solvent towards the powdered sample, extracting and diffusing the phytochemicals out of the matrix [77]. The disruption of hydrogen bonds, as a result of microwave-induced dipole rotation of molecules, enhances the penetration of the solvent into the matrix, allowing the dissolution of the components into the liquid matrices [78]. This method has the advantage of less consuming time and solvent volumes than the classical approach [77–79]. This method has been largely used for the extraction of monomeric polyphenols (short chains) such as phenolic acids and flavonoids [78–80], but it has been less used to extract polymeric polyphenols such tannins and anthocyanins, because polyphenols with a higher number of hydroxyl-type substituents (long-chains) and those sensitive to higher temperatures (e.g., anthocyanins) may be degraded under MAE extraction conditions [79, 80]. The temperature used for extraction is proportional to the power (watts) and time and inversely proportional to the heat capacity of the solvent and the mass of sample [80]. Higher temperatures and small amounts of sample increase the rate of solvent diffusion and promote faster extraction kinetics [80].
\n
Numerous phytochemical compounds, including polyphenols, have been extracted MAE system as shown in Table 4. It seems that MAE system provides higher polyphenols yield in less consuming time and solvents. However, there are some concerns when the MAE is used to extract polyphenols. Factors such as type of matrix, type and purity of solvent, the microwave application time, power, contact sample surface area and temperature can affect their efficiency. One of the most critical factors is the nature of solvent, which affects not only the solubility of the target components but also the efficiency of all physical process. The choice of solvent must take into account not only the affinity to the target phytochemicals but also the ability to absorb microwave energy [81]. For example, solvents like hexane or dichloromethane, which are transparent to microwaves, do not heat up under microwave [82, 83]; thus, they should not be used in this system. Others, such as ethanol, methanol, or even water, have good microwave absorbing capacity [83], and they get heated up faster; thus, the length of the time and microwave power must be adapted to the solvent to enhance the extraction process without any deleterious effect on thermolabile components.
\n\n\n\n\n
\n
3.2.2. The ultrasound-assisted extraction (UAE) method
\n
The UAE is a very simple method that relies on the mechanical effect caused by the implosion of micro-sized bubbles, which cause a rapid tissue disruption allowing the release of compounds into the solvent [84]. This is a very simple method with relatively low cost, and it can be used on both small laboratory and large industrial scale [84, 85]. The use of UAE has been widely used in the last years in the extraction of polyphenols from different parts of plants such as leaves, stems, stalks, fruits, seeds [85–93]. In general, the experimental procedure involves the use of ultrasounds with frequencies ranging from 20 to 2000 kHz, which increases the permeability of cell walls and produces cavitation.
\n
Several studies have reported that UAE allows a better and faster extraction of polyphenols with less degradation when compared with other extraction methods. For example, UAE shown to be highly efficient in the extraction of carnosic acid and rosmarinic compared to classical methods of extraction [94]. In a recent study [95], the maximum extraction yield of total polyphenols (13.2 mg/g dry weight) from spruce wood bark was obtained when UAE system was used. Also, an increment in anthocyanin content in purple sweet potato was observed when UAE was used [96]. All these studies have in common the same trend: under UAE, the rate speed dissolution of compounds into extraction solvent was always higher, and thus, the solvent volume used and need to extract phytochemicals was lower compared to the classical extraction methods. Based on these studies and others, it seems that UAE has the advantage of being less expensive due to lower solvent volume used, higher amount of samples tested and lower time needed to perform the extraction process. Also, they agree that the lower temperatures and shorter sonication periods (time) are better to enhance the extraction of polyphenols contributing also to the preservation of the thermolabile and unstable compounds. However, some studies [97, 98] reported that sonication for long periods (>40 min) with higher energy levels (above >20 kHz) could have a deleterious effect on phytochemicals due to the decrease of diffusion area and diffusion rate and increased diffusion distance, leading to a global decreased yield of total phenolic and flavonoid content. Moreover, under these conditions might occur the formation of free radicals and consequently undesirable changes in the drug molecules [97].
\n
3.2.3. Pressurized liquid extraction (PLE)
\n
The PLE method, also known as “accelerated solvent extraction (ASE),” is a very recent new technology for phytochemicals extraction including polyphenols, which associates high temperature and pressure [99]. In this method, high level of pressure (normally between 3.3 and 20.3 MPa) is combined with high level of temperatures (between 40 and 200°C) to improve the solubility and desorption of molecules, increasing their movement from matrix into solvents, and thus increasing the yield of polyphenols extracted [54]. According to Nieto et al. [99], the PLE method is an advanced technique that provides a faster extraction processes and requires a small amount of solvents when compared with the classical extraction approach. Moreover, it allows better the usage of water as extraction solvent, which is limited in the other previous methods. The use of water as an extraction solvent in PLE, as so-called subcritical water extraction (SWE), is always possible, particularly when elevated temperatures are used [100]. When temperatures around 200°C are used, a change in the dielectric water properties occurs, and then, the water behaves like a normal organic solvent, increasing their extraction efficiency [101]. The main advantages of PLE often reported by several researchers are cleanness of the extracts that PLE provides in comparison with classical maceration, Soxhlet, MAE and UAE, which results in reduced background noise during the subsequent analytical quantification, is especially important when the LC-MS analysis due to ion-suppression effects [102]. By opposition, the main limitations often reported are the low selectivity towards the analytes during extraction, and many interferents may be extracted during the extraction process, an exaggerated dilution of the analytes, especially when a large number of cycles are used, and the high requirements in instrumentation, which increases their costs [103–105]. However, these limitations in PLE are a well-known extraction technique and have been used for the extraction of polyphenols from several different matrices [106–111].
\n
3.2.4. The supercritical CO2 extraction (SC-CO2)
\n
The SC-CO2 extraction is a process in which the CO2 is used as supercritical fluid and probably is one of the most widely used fluid because it is nontoxic, nonflammable, inert cheap and easily available in high quantity with high grade of purity [112]. SC-CO2 extraction is possible to use different combinations of temperature and pressure [112], making this method one of the most versatile for creating a multitude of end products. Due to the multitude of combinations, low temperatures (31.6°C, the critical point of carbon dioxide) and pressure (7.386 MPa) are needed, and the SC-CO2 has been considered very popular in a lab-scale laboratorial facilities. Moreover, since low temperatures and pressure are used, there is a good preventing of thermal degradation of phytochemicals. The main advantage s of SC-CO2 are [112–116] as follows: (i) more extraction capacity due to their higher diffusion coefficient and lower viscosity than the liquids, which increases a higher mass transfer from solid matrix towards solvents; (ii) it allows higher penetration of solvents into the matrices which increase the effectiveness and polyphenols extraction yield; (iii) it allows different combinations of pressure and temperature and thus allows a better adaptation of the extraction conditions to the different types of food and plant matrices, increasing the solubility of their different components in the supercritical fluids; (iv) it allows the CO2 recycling at the end of the process, without any disgrace of chemical residue to environment at the end of the extraction and separation process.
\n
3.2.5. Enzyme-assisted extraction (EAE)
\n
The EAE is a recent method and is based on the capacity of the enzymes to degrade cell wall components into solvents, in general water, with high stability and high bioactivity [117]. In EAE, the enzymes added to food, plant matrices or agro-food wastes are capable to break and weaken the cell walls, increasing the exposure of their cellular components to extraction [71, 118], and thus increasing the capacity to extract polyphenols from the matrices. In fact, some phytochemicals are dispersed in plant cell cytoplasm, and even, some compounds are bound with the polysaccharide-lignin by hydrogen or hydrophobic chain, which are not accessible with a routine organic solvents [119, 120]. Thus, a previous treatment with enzymes can be the only choice, and an enzymatic pretreatment might be the unique and effective way to release bounded compounds from cells [121].
\n
Cellulases, hemicelullases, pectinases and other enzymes may be used to hydrolyze efficiently the cell wall components, enabling the efficiency of extraction of phenolic compounds. Several papers have been published about the positive effect of EAE on increment of polyphenol extraction yield. In 2012, in a study with grape wastes [71], it was found a strong increment in the release of polyphenols when celluclast®, pectinex® and novoferm® enzymes were used. Similar trends were noted in other works [122, 123] which concluded that EAE should be regarded as an alternative method for improved extraction of insoluble-bound phenolics (linked to carbohydrates and proteins of cell wall matrices) from winemaking by-products. These and many other authors observed that the ability of enzymes to degrade cell walls and membranes enables the extraction efficiency of bioactive compounds, and in several situations, the EAE technology might be the unique way to extract effectively bioactive compounds from foods and agro-industrial by-products. In addition to these advantages, the EAE method has been recognized as one of the most eco-friendly methods, because it uses water as solvent instead of organic chemicals, often toxics [119], and is one of the modern extraction methods that are gaining more attention because of the need for eco-friendly extraction technologies.
\n
3.2.6. Combined approaches
\n
In some circumstances, it is possible to find different studies in which the extraction of phytochemical is done throughout combined methods. This occurs, particularly in situations in which a single extraction method is not as efficient as we would expect, and thus, a combination of extraction processes could be the unique effective method to obtain extracts with different polyphenols.
\n
4. The identification and measurements of phenolics
\n
There is a great diversity of studies about the development of new methods for polyphenol quantification. The high-performance liquid chromatography (HPLC) with or without mass spectrometry (MS) is one of the most commonly applied method to identify and quantify polyphenols. However, the classical spectrophotometric assay is still used, even if their results are limited.
\n
4.1. The classical colorimetric methods
\n
The classical spectrophotometry UV/Vis method [124], even with modifications, is still widely used to measure total phenolic content in plant materials. This method is based on the chemical reduction of polyphenols in an alkaline medium to form a blue chromophore complex (phosphomolybdic/phosphotungstic acid) that can be quantified by visible-light spectrophotometry (at 760 765 nm). Many studies have discussed the advantages and disadvantage of using routinely this method to quantify the level of polyphenols, and most of them seems to agree that although they are easy to perform, low cost, rapid and applicable routinely in the most laboratories, they are not accurate. In addition, the reagents used in the method do not react specifically with only polyphenols, and they react with any reducing substance like ascorbic acid, pigments, aromatic amines and sugars [125], and thus, these methods measure the total reducing capacity and not just the polyphenols compounds. Also, their reagents react with some nitrogen-containing compounds such as hydroxylamine and guanidine [126], thiols, many vitamins and some inorganic ions [127] Therefore, many researchers have chosen to use this method only as an indicative tool of total reduction capacity and not for a specific quantification of polyphenol compounds. However, these methods are still considered useful for a quick and prior screening of numerous samples, and for many applications, a simple measure of total amount of polyphenols is enough.
\n
Similar to total polyphenols, total flavonoids can be measured by spectrophotometry methods, and the AlCl3 method [128, 129] is the most vulgarized method used to determine the total flavonoid content. Vanillin and 4-(dimethylamino)-cinnamaldehyde (DMCA) assays are often used to determine the level of proanthocyanidins, in which the flavonoid catechin is used as standard [130, 131]. Like in total polyphenols, the vanillin or DMCA method can overestimate the amount of total flavonoids present in samples. The proanthocyanidins can also be determined by butanol-HCl [132] and bovine serum albumin (BSA) [133] methods. The butanol-HCl method is based on the cleavage of the flavonoid bonds by hot acid, followed by an auto-oxidation reaction which converts flavan-3-ols into anthocyanidins. The red extract formed has a maximum absorbance at around 550 nm. In the BSA method, the flavonoids complex is dissolved in an alkaline solution (sodium decyl sulphate-triethanolamine) followed by a reaction with ferric chloride solution to form a violet complex with a maximum absorbance at 510 nm.
\n
Another spectrophotometric method widely used in the quantification of polyphenols is the UV/Vis spectrophotometry method to determine the anthocyanin content. The anthocyanins constitute one of the main class of polyphenols largely present in plant samples, particularly in red, blue and black color fruits such as grapes, blueberries, raspberries, redcurrants, blackcurrants, pomegranates and strawberries, among others. The quantification of anthocyanins is in general performed by the differential pH method [134] based on the property of the anthocyanin pigments to change the color with pH, in the wavelength ranging from 490 to 550 nm [134]. The anthocyanins suffer reversible structural modification with a change of pH, and this change allows to estimate spectrophotometrically the total amount of anthocyanins, even in the presence of degraded pigments and other interfering compounds.
\n
All these spectrophotometric methods are considered simple and cheap, but only gives a general estimation about the content of each class of polyphenols but do not allow the quantification of polyphenols individually.
\n
4.2. Chromatography
\n
In the course of the last four decades, several chromatography methods were developed to overcome the main constraints of the classical spectrophotometry methods. The development of new technologies and software led to the appearance of improved methods capable of separation, identification and quantification of phytochemicals individually. These methods are generally based on the principle that a sample is composed of a mixture of components which are separated when the mixture passes through two phases: a mobile (liquid or gaseous) and a stationary (solid, liquid or gel). It is used for the qualitative and quantitative analysis, and the components are separated and analyzed according the properties of a given solution. The great diversity of combinations between the two phases makes possible an existence of several differentiated techniques.
\n
4.2.1. Principles
\n
The basic principles of chromatography are universal [135] and thus widely accepted by all researchers. The main principles are more or less the following ones: (i) chromatography is a physical and chemical method of separating, identification and quantification of different components of samples; (ii) the separation always dependent of the interaction between the components of the mixture with the mobile and stationary phases, and, thus a large combinations of the three are possible; (iii) the interaction of the matrices components with the mix of both phases is influenced by different intermolecular forces including ionic, dipole, nonpolar and effects of specific affinity or solubility; (iv) the mobile phase is generally named as eluent, and the absorbent material, named as stationary phase; (v) the analyte is the compound to be separated; (vi) a chromatogram is the visual output of the chromatograph; (vii) the instrument used for qualitative and quantitative detection of analytes after separation is named as detector; (viii) the separation of components present in the mixture occurs according to the different chemical affinity for the stationary phase, and it happens as the eluent advances on the stationary phase; (ix) the separation of compounds is slower when compounds have strong interaction with the stationary phase and faster when the components have weaker interaction with the stationary phase, and by this, the compounds will be separated from each other as they move over the support material; (x) the component to be analyzed must have solubility with the mobile phase, and different compounds have different retention time values; (xi) the identification and quantification of components in the mixtures are done by comparison with pure commercial standards of known commercial concentration, through analytical curves.
\n
The chromatography can be classified according to several criteria [135, 136], but in general, the chromatography applied in separation, identification and quantification of phytochemicals is classified as:
\n
Gas chromatography (GC), when gas chromatography makes use of a pressurized gas cylinder and a carrier gas (e.g., helium), to carry the solute through the column. The most common detectors used in this type of chromatography are of thermal conductivity and flame ionization detectors. There are three types of GC as follows: (1) gas adsorption, (2) gas-liquid, and (3) capillary gas chromatography.
Liquid chromatography (LC), when a liquid adsorbent is used. This method is used in large-scale applications since adsorbents are relatively inexpensive. There is a liquid-liquid chromatography which is analogous to gas-liquid chromatography. The three types of modern LC are as follows: (1) reverse phase, (2) high performance and (3) size exclusion liquid chromatography, along with supercritical fluid chromatography.
Ion exchange chromatography (IEC), when charged molecules mobile phase passes through the column, until a binding site in the stationary phase appears and retains the molecules. There are two types of ion exchange chromatography: (1) cation exchange in which the stationary phase carries a negative charge, and (2) anion exchange in which the stationary phase carries a positive charge. The method is mainly used in the purification of biological materials.
Affinity chromatography (AC), which is a technique that involves the chemical modification of a given compound by attaching another compound with a specific affinity for the desired molecules. This method requires that the compounds to be analyzed must be inert and easily to modify, and otherwise, it can be very difficult to perform, and a large number of impurities can appear. Therefore, this type of technique is only used in advanced processes of purification.
\n
4.2.2. The use of HPLC-DAD/UV-VIS and HPLC-MS
\n
The high-performance liquid chromatography (HPLC), referred in the past as high-pressure liquid chromatography, like other chromatography methods is a technique used to separate, identify and quantify phytochemicals from plant mixtures and relies on pumps to pass a pressurized solvent containing the plant samples, foods or other matrices through a column filled with a solid adsorbent material (e.g., silica) [137]. The HPLC methods, however, differ from other liquid methods, particularly from “low pressures,” because it uses high pressures (ranging from 50 to 350 bar), while the others normally use the force of gravity to pass the mobile phase through the column [137, 138]. Each component of the samples interacts differently with the adsorbent material of the column, causing a different flow rate for the different components in the mixture, thus leading to the separation of the components as they flow out the column. The columns used in the HPLC methods are made with smaller adsorbent particles size ranging from 2 to 50 µm [137, 138].
\n
Although there is many variations in the HPLC equipment available in the market, the basic HPLC equipment includes a sampler (to carry the sample mixture into the mobile phase), pumps (to deliver mobile phase through the column, with a specific flow) and a detector (such as UV/Vis or photodiode array (PDA), which generates signal proportional to the amount of compound present in the sample mixture [138]. The signal detected allows the identification and quantification of sample components. Each compound detected has a specific retention time; however, due to the interaction strength of interactions between the analytes and stationary phases, the retention time can vary. Nowadays, modern HPLC equipment has a digital processor, which uses a software interface to control the instruments and provides data analysis. Other modern models are equipped with several pumps, which allow different combinations of various solvents at different ratios changing in time, creating a gradient in the mobile phase.
\n
Nowadays, the classic HPLC evolved to HPLC coupled with a mass spectrometry detector (MS), called as LC-MS or HPLC-MS [138]. This new technique allows a more accurate identification which is based on the specific fragmentation of each separated molecule. This enhances the sensitivity and is oriented for the separation of chemicals with specific masses in a complex mixture. The separation of molecules or fragments occurs according to their mass-to-charge ratio in an analyzer by electromagnetic fields. The ions are detected by a qualitative and quantitative analysis, and the signal is processed into mass spectra. The HPLC-MS equipment is in general composed of three modules: (1) an ion source, which converts gas phase sample molecules into ions; (2) a mass analyzer, which sorts the ions by their masses by applying electromagnetic fields; and (3) a detector, which measures the value of the signal detected and provides data for the quantification of each ion present. In the last years, this new method has been strongly implemented in academies in basic research, pharmaceutical and agro-chemical industries to study physical, chemical and biological properties of a great diversity of compounds, as well, and quality control of drugs, foods and natural products. In Table 4 are presented some recent works [139–145] in which HPLC-MS was effectively used for polyphenol characterization of plants and food with very accurate results.
\n
5. Conclusions
\n
This chapter discusses the importance of polyphenols as well as the availability of different methods to extract them from its natural sources. The most widely used methods in the extraction of polyphenols are the classical ones which usually includes maceration, percolation and successive Soxhlet extractions. Although these methods are still in use, they involve long extraction times, huge quantities of solvent, higher accumulation of residues and very limited results. Therefore, new methods such as UAE, MA, PLE, S-CO2 and EAE have been developed in the recent years with very feasible results. Also, the evolution of separation and identification techniques of polyphenols has evolved from a simple colorimetry method to the most advanced chromatography techniques. However, the growing demand for new bioactive molecules from natural sources enhances the continuous search for new and innovative methods to extract and separate new molecules, which never ends.
\n',keywords:"phytochemicals, extraction, determination, colorimetric methods, HPLC, HPLC-MS",chapterPDFUrl:"https://cdn.intechopen.com/pdfs/53539.pdf",chapterXML:"https://mts.intechopen.com/source/xml/53539.xml",downloadPdfUrl:"/chapter/pdf-download/53539",previewPdfUrl:"/chapter/pdf-preview/53539",totalDownloads:3280,totalViews:1465,totalCrossrefCites:11,totalDimensionsCites:15,hasAltmetrics:0,dateSubmitted:"June 1st 2016",dateReviewed:"November 14th 2016",datePrePublished:null,datePublished:"March 15th 2017",dateFinished:null,readingETA:"0",abstract:"The increasing consumers demands to acquire healthier fruits and vegetables as well as the urgency in looking to natural compounds with antioxidant activity and enhanced antimicrobial activity against antibiotic-resistant pathogenic bacterial strains have encouraged a quick expansion of research studies about enhanced phenolic extraction and identification methods. Considering the importance of phenolics as natural compounds with antioxidant and antimicrobial activity, this chapter aims to present the most updated information about extraction methods, ranging from the traditional to the most advanced processes, as well as the access to the modern methods used in the identification and quantification of phenolics. The main goal of this chapter is to provide the reader with a broad view on the different protocols used to extract, identify and quantify phenolic compounds from different kinds of foods, including fruits and vegetables.",reviewType:"peer-reviewed",bibtexUrl:"/chapter/bibtex/53539",risUrl:"/chapter/ris/53539",book:{slug:"phenolic-compounds-natural-sources-importance-and-applications"},signatures:"Alfredo Aires",authors:[{id:"175895",title:"Dr.",name:"Alfredo",middleName:null,surname:"Aires",fullName:"Alfredo Aires",slug:"alfredo-aires",email:"alfredoa@utad.pt",position:null,institution:{name:"University of Trás-os-Montes and Alto Douro",institutionURL:null,country:{name:"Portugal"}}}],sections:[{id:"sec_1",title:"1. Introduction",level:"1"},{id:"sec_2",title:"2. Foods as natural resources of phenolics",level:"1"},{id:"sec_3",title:"3. Methods used in extraction of polyphenols",level:"1"},{id:"sec_3_2",title:"3.1. Classical solvent extraction",level:"2"},{id:"sec_4_2",title:"3.2. Advanced methods of extraction",level:"2"},{id:"sec_4_3",title:"3.2.1. The microwave-assisted extraction (MAE)",level:"3"},{id:"sec_5_3",title:"3.2.2. The ultrasound-assisted extraction (UAE) method",level:"3"},{id:"sec_6_3",title:"3.2.3. Pressurized liquid extraction (PLE)",level:"3"},{id:"sec_7_3",title:"3.2.4. The supercritical CO2 extraction (SC-CO2)",level:"3"},{id:"sec_8_3",title:"3.2.5. Enzyme-assisted extraction (EAE)",level:"3"},{id:"sec_9_3",title:"3.2.6. Combined approaches",level:"3"},{id:"sec_12",title:"4. The identification and measurements of phenolics",level:"1"},{id:"sec_12_2",title:"4.1. The classical colorimetric methods",level:"2"},{id:"sec_13_2",title:"4.2. Chromatography",level:"2"},{id:"sec_13_3",title:"4.2.1. Principles",level:"3"},{id:"sec_14_3",title:"4.2.2. The use of HPLC-DAD/UV-VIS and HPLC-MS",level:"3"},{id:"sec_17",title:"5. 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Analytical Biochemistry. 1978, 91:92–100. doi:10.1016/0003-2697(78)90819-9'},{id:"B132",body:'Porter LJ, Hrstich LN, Chan BG: The conversion of procyanidins and prodelphinidins to cyanidin and delphinidin. Phytochemistry. 1985, 25:223–30. doi:10.1016/S0031-9422(00)94533-3'},{id:"B133",body:'Hagerman AE, Butler LG: Protein precipitation method for the quantitative determination of tannins. Journal of Agricultural and Food Chemistry. 1978, 26:809–12. doi:10.1021/jf60218a027'},{id:"B134",body:'Smith MAL, Marley KA, Seigler D, Singletary KW, Meline B: Bioactive properties of wild blueberry fruits. Journal of Food Science. 2000, 65:352–6. doi:10.1111/j.1365-2621.2000.tb16006.x'},{id:"B135",body:'Weston A, Brown PR: High performance liquid chromatography & capillary electrophoresis: principles and practices. Elsevier Science, Cambridge, Massachusetts, United States 1997.'},{id:"B136",body:'Rouessac F, Rouessac A: Chemical analysis: modern instrumentation methods and techniques. Wiley, Chichester, United Kingdom, 2013.'},{id:"B137",body:'Corradini D: Handbook of HPLC, second edition. CRC Press, Cambridge, Massachusetts, United States. 2016.'},{id:"B138",body:'Niessen WMA, Tinke AP: Liquid chromatography-mass spectrometry general principles and instrumentation. Journal of Chromatography A. 1995, 703:37–57. doi:10.1016/0021-9673(94)01198-N'},{id:"B139",body:'Vagiri M, Ekholm A, Andersson SC, Johansson E, Rumpunen K: An optimized method for analysis of phenolic compounds in buds, leaves, and fruits of black currant (Ribes nigrum L.). Journal of Agricultural and Food Chemistry. 2012, 60:10501–10. doi:10.1021/jf303398z'},{id:"B140",body:'Slatnar A, Mikulic-Petkovsek M, Stampar F, Veberic R, Solar A: HPLC-MSn identification and quantification of phenolic compounds in hazelnut kernels, oil and bagasse pellets. Food Research International. 2014, 64:783–9. doi:10.1016/j.foodres.2014.08.009'},{id:"B141",body:'Badalica-Petrescu M, Dragan S, Ranga F, Fetea F, Socaciu C: Comparative HPLC-DAD-ESI(+)MS fingerprint and quantification of phenolic and flavonoid composition of aqueous leaf extracts of Cornus mas and Crataegus monogyna. Relation to Their Cardiotonic Potential. 2014. 2014, 42. doi:10.10.15835/nbha4219270'},{id:"B142",body:'Diaconeasa ZA, Florica R, Rugin DA, Lucian C, Socaciu C: HPLC/PDA¨CESI/MS identification of phenolic acids, flavonol glycosides and antioxidant potential in blueberry, blackberry, raspberries and cranberries. Journal of Food and Nutrition Research. 2014, 2:781–5. doi:10.12691/jfnr-2-11-4'},{id:"B143",body:'Oliveira BG, Costa HB, Ventura JA, Kondratyuk TP, Barroso MES, Correia RM, Pimentel EF, Pinto FE, Endringer DC, Romão W: Chemical profile of mango (Mangifera indica L.) using electrospray ionisation mass spectrometry (ESI-MS). Food Chemistry. 2016, 204:37–45. doi.10.1016/j.foodchem.2016.02.117'},{id:"B144",body:'Karaaslan NM, Yaman M: Determination of anthocyanins in cherry and cranberry by high-performance liquid chromatography–electrospray ionization–mass spectrometry. European Food Research and Technology. 2016, 242:127–35. doi:10.1007/s00217-015-2524-9'},{id:"B145",body:'Kelebek H: LC-DAD–ESI-MS/MS characterization of phenolic constituents in Turkish black tea: effect of infusion time and temperature. Food Chemistry. 2016, 204:227–38. doi:10.1016/j.foodchem.2016.02.132'}],footnotes:[],contributors:[{corresp:"yes",contributorFullName:"Alfredo Aires",address:"alfredoa@utad.pt",affiliation:'
Centre for the Research and Technology for Agro-Environment and Biological Sciences (CITAB), University of Trás-os-Montes e Alto Douro (UTAD), Vila Real, Portugal
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Ochieng",slug:"joel-w.-ochieng"},{id:"126149",title:"Dr.",name:"Anthony",middleName:null,surname:"Ananga",fullName:"Anthony Ananga",slug:"anthony-ananga"},{id:"137412",title:"Dr.",name:"Violetka",middleName:null,surname:"Tsolova",fullName:"Violetka Tsolova",slug:"violetka-tsolova"},{id:"193798",title:"Dr.",name:"James",middleName:null,surname:"Obuya",fullName:"James Obuya",slug:"james-obuya"}]},{id:"53180",title:"Phenolic Compounds with Anti-virulence Properties",slug:"phenolic-compounds-with-anti-virulence-properties",signatures:"Naybi Muñoz-Cazares, Rodolfo García-Contreras, Macrina Pérez-\nLópez and Israel Castillo-Juárez",authors:[{id:"193519",title:"Dr.",name:"Castillo",middleName:null,surname:"Juárez",fullName:"Castillo Juárez",slug:"castillo-juarez"},{id:"193520",title:"Dr.",name:"García",middleName:null,surname:"Contreras Rodolfo",fullName:"García Contreras Rodolfo",slug:"garcia-contreras-rodolfo"},{id:"193521",title:"MSc.",name:"Pérez",middleName:null,surname:"López Macrina",fullName:"Pérez López Macrina",slug:"perez-lopez-macrina"},{id:"193522",title:"MSc.",name:"Muñoz",middleName:null,surname:"Cazares Naybi",fullName:"Muñoz Cazares Naybi",slug:"munoz-cazares-naybi"},{id:"197805",title:"Dr.",name:"Israel",middleName:null,surname:"Castillo Juárez",fullName:"Israel Castillo Juárez",slug:"israel-castillo-juarez"}]},{id:"52824",title:"Regulatory Mechanism of Skeletal Muscle Glucose Transport by Phenolic Acids",slug:"regulatory-mechanism-of-skeletal-muscle-glucose-transport-by-phenolic-acids",signatures:"Tatsuro Egawa, Satoshi Tsuda, Rieko Oshima, Ayumi Goto, Xiao Ma,\nKatsumasa Goto and Tatsuya Hayashi",authors:[{id:"193107",title:"Dr.",name:"Tatsuro",middleName:null,surname:"Egawa",fullName:"Tatsuro Egawa",slug:"tatsuro-egawa"},{id:"193126",title:"MSc.",name:"Satoshi",middleName:null,surname:"Tsuda",fullName:"Satoshi Tsuda",slug:"satoshi-tsuda"},{id:"193127",title:"Prof.",name:"Katsumasa",middleName:null,surname:"Goto",fullName:"Katsumasa Goto",slug:"katsumasa-goto"},{id:"193128",title:"Prof.",name:"Tatsuya",middleName:null,surname:"Hayashi",fullName:"Tatsuya Hayashi",slug:"tatsuya-hayashi"},{id:"195578",title:"Dr.",name:"Rieko",middleName:null,surname:"Oshima",fullName:"Rieko Oshima",slug:"rieko-oshima"},{id:"195646",title:"Dr.",name:"Ayumi",middleName:null,surname:"Goto",fullName:"Ayumi Goto",slug:"ayumi-goto"},{id:"195708",title:"Prof.",name:"Xiao",middleName:null,surname:"Ma",fullName:"Xiao Ma",slug:"xiao-ma"}]},{id:"54035",title:"Health Benefits of Phenolic Compounds Against Cancers",slug:"health-benefits-of-phenolic-compounds-against-cancers",signatures:"Abdelkader Basli, Nassim Belkacem and Iman Amrani",authors:[{id:"193750",title:"Dr.",name:"Basli",middleName:null,surname:"Abdelkader",fullName:"Basli Abdelkader",slug:"basli-abdelkader"},{id:"195990",title:"Mr.",name:"Belkacem",middleName:null,surname:"Nacim",fullName:"Belkacem Nacim",slug:"belkacem-nacim"},{id:"195991",title:"Dr.",name:"Amrani",middleName:null,surname:"Iman",fullName:"Amrani Iman",slug:"amrani-iman"}]},{id:"53661",title:"Health Status Improved by Aronia Melanocarpa Polyphenolic Extract",slug:"health-status-improved-by-aronia-melanocarpa-polyphenolic-extract",signatures:"Manuela Ciocoiu, Laurentiu Badescu and Magda Badescu",authors:[{id:"193903",title:"Prof.",name:"Manuela",middleName:null,surname:"Ciocoiu",fullName:"Manuela Ciocoiu",slug:"manuela-ciocoiu"},{id:"195685",title:"Dr.",name:"Laurentiu",middleName:null,surname:"Badescu",fullName:"Laurentiu Badescu",slug:"laurentiu-badescu"},{id:"195686",title:"Prof.",name:"Magda",middleName:null,surname:"Badescu",fullName:"Magda Badescu",slug:"magda-badescu"}]}]}]},onlineFirst:{chapter:{type:"chapter",id:"71675",title:"Basic Principles in Microvascular Anastomosis and Free Tissue Transfer",doi:"10.5772/intechopen.91917",slug:"basic-principles-in-microvascular-anastomosis-and-free-tissue-transfer",body:'\n
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1. Introduction to microsugery
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Microvascular transfer is a reconstructive technique based on raising tissues from healthy areas of the body, where an excess or dispensability exists, in advance to transplant them to other regions where they are lacking, mainly after trauma, oncological surgery or chronic infection. A microsurgical transfer from a strict point of view implies a double vascular anastomosis less than 3 mm between vessels in the transferred tissue to the ones in the recipient area [1]. Super-microsurgery would refer to those situations in which anastomoses have a diameter between 0.3 and 0.8 mm [2]. Rigorously speaking, the recipient vessels are those receiving the blood flow and the donors those from which it emanates. From a historical point of view, the compound of the transferred tissues is named free flaps.
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Since its inception, reconstructive techniques have aimed to restore the integrity, form and function of the body [3]. Although plastic surgery is the discipline of medicine that brings together all these techniques, it lacks an anatomical limitation; therefore, its knowledge is widespread according to the diverse body regions through maxillofacial surgery, ophthalmology, hand surgery, etc. For centuries, it was intended to limit the potential damage inflicted to patients by narrowing down the reconstructive options. In this regard, a reconstructive ladder was defined, where the primary closure of the wounds, the cure by secondary intention or the skin grafts were in the lower steps of this ladder and the flaps in the higher [4, 5].
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With the improvement in optical tools, it became easier to perform the vascular anastomoses that allowed free flap transfer and to set up skilled teams. As the tissue transfers became more dynamic and the microsurgery success rates rose, the benefits became more and more evident [5]. It was proven that the transfer of healthy tissues to the hand or head and neck allowed surgeons to achieve faster and better recoveries in areas of high functional demand, also with much more aesthetically acceptable results and lower morbidity. The same happened to breast surgery, where reconstructions with a natural shape and adequate volume could be achieved; the scars were hidden in the distance, and there was no need to use prosthesis. In lower limb osteomyelitis, free muscle flaps became the alternative to amputation. In addition, the advent of perforator flaps, mainly due to the contributions of Song and Koshima [5], thanks to whom it was not necessary to take the underlying muscle to transfer a fasciocutaneous flap, made it possible to further minimise the morbidity of these microsurgical interventions. Finally, a revision of the reconstructive ladder was proposed, the simplicity of the reconstruction would prevail, but pursuing the best aesthetic and functional results. So, a switch to a reconstructive elevator was made. In this way, microsurgical reconstructions became the first-line option for many patients and the technique was extended to a multitude of centres [5, 6].
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2. Basic principles in microsurgery
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2.1 Ergonomy
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Multiple aspects regarding the environment in the operating room and the position are particularly important in microsurgery. It is imperative to have enough field to allow an easy movement. This aspect, which is less substantial in macro-surgery, becomes absolutely fundamental in microsurgery. Mention it at the beginning, does nothing but tries to emphasise its relevance.
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A two-team approach is usually chosen in reconstructive microsurgery, one will raise the flap and the other will set the recipient site where this is going to be transplanted [7]. Therefore, all the time spent planning disposition is properly invested. This is true both for placing the patient in the proper position, and for the surgeon to adopt a comfortable and durable posture. Since the surgery will be prolonged, we must meticulously paddle all bony prominences of the patient and the areas at risk of neurovascular compression. It must be encouraged to take all the necessary anaesthesia monitoring measures at the beginning, just to avoid emergencies or interruptions during delicate stages of surgery. It is also sensible to foresee how the microscope will be arranged in the room.
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The comfort of the surgeon is a must when it comes the time to perform the microvascular anastomosis, primarily regarding the back, scapular and muscular groups. The sutures used usually size about 75–100 μm and the vessel lumen just a few millimetres; therefore, any tremor will greatly hinder the precision and success of the anastomoses. We cannot afford mistakes at any point of the microvascular anastomosis. The surgeon must be perpendicular disposition to the vessels and seated in a self-regulating chair that allows a self-sufficient height adjust. He or she should also be with the feet on a flat surface, the arms supported on a cloth and the hands on some comfortable place of the field to work only with the intrinsic muscles of the hand [1].
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Patience is the cornerstone of microsurgery, calm dissection with no external worries or hurry [8]. For this to be the case, it is essential to be in an easy environment without any tensions among the team members. Fatigue will appear mainly at the most complex moments, well in the middle of long interventions. So, if we do not foresee a comfortable environment with all these details, which may seem insignificant at first, as soon as the least complication appears, the reconstruction will be at high risk. In the case of microsurgical reconstructions, comfort is not a luxury but a must.
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2.2 General conditions
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After having invested enough time planning the operating room configuration, it is time to choose the vessels in the recipient area, since those of the flap are already determined and are assumed to be healthy because of their undamaged origin. It is essential to emphasise that the dissection must be very scrupulous, some groups advocate applying tension to the tissues around the vessel, without any direct pulling or forceps grasping on it, as not to generate any intimal traumas that may cause a thrombotic source [8, 9]. Any injury to the intima of the vessel, unnoticed or not, will expose the subendothelial collagen of the lumen, leading to a thrombotic focus. There are situations where it is impossible not to manipulate the vessel, as it happens in cervical dissections for oncological reasons; in these cases, a high incidence of thrombosis in the recipient vein has been demonstrated [6].
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We must choose healthy vessels, without excessive fibrotic or irradiated tissue around them, this will allow us to perform a clean dissection, achieving a blood-less field. If blood accumulates in the field, we should spare no expense in abundantly rinse the area and review haemostasis. Blood has a red light refraction that deteriorates the sight with usual optical tools and releases procoagulant factors inducing vascular thrombosis [10, 11]. In limbs with previous surgeries or trauma, in case of doubt, we must carry out explorations such as angiography or Doppler, to check the availability of adequate vessels [12, 13, 14, 15]. We should recruit as much vessel length as necessary to prevent any tension in the anastomosis, since the use of vein grafts, although may be needed, should be avoided due to its higher incidence of complications.
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Before sectioning the donor artery to which we are going to transfer our flap, we must ensure that it has a good flow, we should ideally evince pulse [8]. Once sectioned, it will only be valid if we observe the exit of an abundant spurt of pulsatile blood. On the other hand, the vein that receives the blood from the flap in the recipient area must have at least the diameter that the vein of the flap has; otherwise, a bottleneck will form and prevent a good return and a venous congestion may develop in the flap.
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Once the vessels in the receiving area are all set, we proceed to review the haemostasis and the perfusion of our previously dissected flap, then we release and transfer it [8, 10]. We should section the artery first and then the vein, as to avoid any congestion. Then we have to adapt the flap in the recipient area, since after anastomosis the flap will become edematised and its fixation in some deep spaces will be complex. This fixation is a mandatory prior step in all free flaps but in those in which the anastomosis lies in a deeper plane. In the head and neck reconstruction, small and intricate spaces make it advisable to do the fixation at first; but in breast reconstruction, we can only secure it with a gauze before microvascular anastomosis [6].
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When performing the anastomosis, we prefer to adjust each vessel end in a simple microvascular clamp, tension-less approximate both ends and perform the microvascular anastomosis sparing as much proximal dissection as possible between the vessels of the flap pedicle and between the ones of the recipient area. On the other hand, we can place the anastomosis vessels end in a double microvascular clamp and approximate them [16]. The anastomosis should be placed on a rubber contrast and this over a wet gauze to avoid pooling and elevate the anastomosis from the surrounding field, full of thrombogenic debris [10] (Figures 1 and 2).
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Figure 1.
General overview of a microvascular anastomosis. Artery microvascular anastomosis is performed in a blood-less field after vein anastomosis (in a second plane), in a higher position over a rubber medium contrast and wet gauzes. A protected 5F Redon is usually placed under the anastomosis. Dissection of the vessels in each side of the anastomosis is limited, only simple vascular clamps are employed; this eases the one-side-up technique (see below). In this figure, the first two stitches of the triangulation technique are depicted.
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Figure 2.
Examples of the limited pedicle dissection before anastomosis. In the upper picture, we can see a free epigastric flap for an axillary free lymph node transfer. Marked with a star, we see the donor posterior circumflex humeral artery; below it, the inferior epigastric artery of the free flap is shown with both veins at each side. In the lower picture, we see the free flap before the transfer. Marked with a thunder, we see the inferior epigastric pedicle severed before its entrance in the abdominal rectus muscle; the inferior epigastirc vein is marked with a heart, it has been cut near its mouth in the circumflex iliac vein and laid prepared for the lymphovenous anastomosis.
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It is characteristic of the lumen of vessels to show a diameter smaller than that seen before severing them. This phenomenon is known as vasospasm. To mitigate it, we must perform a mechanical dilatation of the vessel lumen with specific dilator forceps and with agents such as lidocaine 1–2% or papaverine 3%, the latter being our preference [8, 9, 10, 11]. Another dilatation technique is to abundantly rinse lumen with heparinised serum (200–300 IU/ml) [11, 12, 13, 14, 15, 16, 17]. It is key to remove the adventitia next to the anastomosis; we usually remove 2–3 mm with cutting technique, by pulling the adventitia over the lumen of the vessel and making a section parallel to the light. Aggressive adventitectomies leave the proximity of the anastamosis lacking vasa vasorum; this can cause ischemia in the vessel wall and, secondary to this, a failure or a pseudoaneurysm. On the other hand, the adventitia is highly thrombogenic, its entry with a knot into the lumen can be disastrous [18]. No technique completely removes the adventitia, but the sharp dissection seems more respectful with the intima [19]. Before carrying out the anastomosis, we must ensure that there are no intimal lesions in the lumen of the vessel, venous valves or branches, that may cause turbulence or resistance to flow in the vicinity of anastomosis [8].
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There is debate about which anastomoses to perform first, whether arterial or venous. If there is no limitation for the position of a vessel deeper than another, as happens in breast reconstruction where the internal mammary vein usually has a more medial position, we can choose any one of them [6, 8]. Many groups choose to start with the arterial anastomosis to minimise isquemia time, taking into account that they do not usually experience added venous congestion. We usually start with the venous anastomosis to avoid any congestion within the flap that can cause a thrombus in its internal circuit. At the time of removing the clamps, once the anastomosis is completed, it is clearly preferable to remove the venous one first.
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2.3 Team
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The use of microsurgical techniques is not limited to reference centres with a high availability of resources, although their routine use is almost exclusive of these. This is due to the disposability of a microsurgical team with several surgeons trained in microvascular anastomosis and free flaps management.
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In this kind of surgery, each mistake has its consequences. So, if we perform these interventions with a very scarce and inexperienced team, these day-long surgeries can be translated into fatigue, nerve-wrecking and inaccuracies. This ultimately will generate failures in the microvascular anastomosis and problems in the perfusion of the flap. Therefore, having a team that allows pauses and relays, without stopping the procedure, is a fundamental element. Likewise, this second fresh team will overcome emergencies (more frequent in the first 48–72 h) or can replace a tired first team. It seems sensible to have at least four microsurgeons, two assistants and two experienced scrub nurses [20].
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A microsurgical team must function as a unit that critically analyses its results, seeking rates of failure lower than 5% in free flaps. Errors and the morbidity of the interventions must be analysed, minimising both. This constant improvement is hard to achieve if several microsurgeons are not available.
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3. Microsurgical tools and instruments
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3.1 Tools
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Microsurgery results from adapting the visual inaccuracy of our naked eye, to the fine movements of our hands. Here is where magnification arises. This can be done by two optical tools: the microscope and the magnifying loupe. In both, good lighting is essential [21, 22].
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Surgical microscopes occupy a large space in the operating room but allow a magnification of up to 40× with greater illumination. In addition, they have pedals to control zoom and focus with the feet. Smaller magnification of 6–12× is usually used for the preparation of the vessels, and then it is increased up to 20× before the microanastomosis. In addition, the microscope gives us a wide range of field and provides the same vision to the surgeon and the assistant, enhancing the collaboration between them [21].
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On the other hand, loupes are very cost-effective and easily transportable visual systems. The most common magnification employed in microsurgery is between 2.5× and 4.5×. In skilled hands, microscope has not proved to be superior versus loupes in achieving high success rates in free tissue transfers [23].
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There are two types of magnifying loupes, on the one hand the compound or Galilean loupes and on the other hand the prismatic. The former consist of two lenses in line, and offer less weight and cost, although their magnification (2.5×) and depth of field are lower. The latter use a prism inside to reach a longer path of light through the lenses, which allows greater magnification and field depth, although they can be darker, heavier, more expensive and fragile [22].
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3.2 Basic microsurgery instruments kit
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The microsurgical instruments have evolved from ophthalmology or jewellery material to extremely specific and precise tools [7]. The basic kit is not made up of too much surgical material. This material should ideally be antireflective and cylindrical to allow its sliding from the index to the middle fingers and facilitate the passage of the needle through the tissues using only the intrinsic musculature. The size of the material should be about 16 cm to facilitate its support in the first hand commissure. In the case of working in very small fields, as in the case of hand surgery, smaller material, about 8 cm, with flat surface may be useful. Nowadays the self-locking material has lost interest, the mere requisite is just to offer little resistance when grasping to preclude any fatigue of the thenar eminence with prolonged use [24].
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The basic kit consists of two scissors, a needle holder and a jeweller forceps. One of the scissors should be curved and round tipped, to be useful to dissect. Other pair should be straight and pointed to perform the adventitectomy and to cut sutures. These pointed scissors should not be used for tissue dissection, because of the possible vessel trauma that they would generate. The jeweller forceps must have a precise closure, with enough contact between surfaces at the tip, just to handle fine sutures of 75 or 100 μm.
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Other instruments that can also be useful are an aspiration system, an irrigation system and a bipolar forceps identical to jeweller forceps but protected. Our preference is to prepare a fixed suction system in the corner of the field, and to avoid introducing traditional aspirators directly over the vessels. Usually we fix a 5F Redon drain in a corner of the field or under the rubber contrast and we keep it connected to soft aspiration, in such way that it rests distant from the area of the anastomosis but does not allow pooling. We also avoid the contact of celluloses or cotton gauzes directly with the lumen of the vessel due to their thrombogenic properties.
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For the lumen irrigation, we use a heparin solution with 200–250 IU/ml [11]. Washing the lumen of the vessel directly can hydrodissect the vessel wall, exposing the subintimal collagen. Therefore, we introduce a blunt-tipped lacrimal cannula into the lumen of the vessel before anastomosis to perform a gentle wash [17]. Likewise, we usually do an irrigation of the flap through the artery with 20 or 30 ml of heparin solution, prior to the transfer; this checks the correct flow in the vascular circuit of the flap.
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3.3 Instruments care
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This delicate material requires little but precise care. First of all, we should avoid falls during surgery or washing, as the tips of the material can be damaged. If this happens, the closure of the material would not be perfect and its functionality would be noticeably reduced. It is also necessary to avoid the tips of the material to be oriented towards the sides in the store box, since movements with the box closed could also damage the tips inadvertently.
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The material should preferably be washed by the scrub nurse or the surgeon himself, who is familiar with it and will be more careful with its handling. A final wash should be done with distilled water and dried with an air gun to prevent rust formation.
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During surgery, the material must always be clean and moist, so that the sutures do not adhere to its surface. Dirty material and damaged tips will cause problems with the suture technique at key moments of the intervention.
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3.4 Sutures
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The most common sutures elected are the 9-0 on a 100-μm needle and the 10-0 on a 75-μm needle. Because of the ease of knotting and the low tissue reaction, the most used material in sutures is nylon. Some authors prefer polypropylene due to a lower tissue reaction, but its knots may be less reliable.
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4. Microsurgery techniques
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There is no stipulated standard on how to perform a microvascular anastomosis, the choice of the specific technique is operator-dependent. However, there are certain issues that we must avoid: a narrowing of the vascular lumen, an irregular distribution of the diameters of the vessels that would generate folds and irregularities, an excessive suture material inside the vascular lumen, and above anything else transmural sutures that bite the posterior wall closing the vascular lumen [24].
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4.1 End-to-end anastomosis
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By far, the most frequently employed technique is the end-to-end anastomosis. Because of its simplicity in less experienced hands, it has one of the lowest failure rates.
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4.1.1 Triangulation
\n
This technique was described by Alexis Carrel in the 1902. His intention was to separate the posterior wall from the anterior, as he realised about the danger of transmural stitches. The technique employed three initial sutures, with 120° separation between each [25]. It was modified with the use of only two initial sutures at 120° or 150° distance, as the posterior side was then longer and also fell away (Figure 3). Finally, it was modified again to propose only two initial sutures at 180°. The rest of the anastomosis will be closed with simple sutures between the initial points [24, 26].
\n
Figure 3.
Triangulation technique, after placing tension between the first two stitches, the longer posterior wall of the anastomosis falls down, precluding transmural stitches.
\n
\n
\n
4.1.2 Continuous
\n
The continuous suture saves time and corrects discrepancies of 2–3 mm in size between vessels, but it has as an inconvenient: the tobacco bag effect. Some authors propose to distribute at first the two vascular lumens with some simple stitches. This technique is not very popular in venous microvascular anastomosis due to its stenosing tendency [24, 26].
\n
\n
\n
4.1.3 Continuous interrupted
\n
The continuous interrupted technique (also known as open-loop technique) is our technique of choice. It combines the safety of simple sutures with the comfort and speed of the continuous ones. It allows to constantly maintain a perfect visualisation of the vascular lumen and at the same time minimises the necessary manoeuvres. In this technique, a continuous suture with a spiral of very wide loops is made, then moved towards a lateral. Finally, each one of the loops is sectioned and knit separately [24] (Figure 4).
\n
Figure 4.
Open-loop technique. Continuous suture of the upper face of the vessel, with very loose loops. Afterwards these loops are divided, and knotted as simple stitches. The posterior wall is depicted sutured first.
\n
\n
\n
4.1.4 One way up
\n
This technique is of first choice when we cannot properly manipulate both the vessels of the microvascular anastomosis, we cannot manage to rotate it in order to carry out the suture of the posterior wall. When performing the one-way-up technique, we begin suturing the posterior side. The needle is introduced from the deep side of the vessel to the intima of the posterior wall and returns through the intima in the lumen of the posterior wall of the opposite vessel. The knots are the same as in simple stitches. After placing three or four stitches in the posterior wall in an inverted fashion, it is easy to perform the remaining stitches in a conventional way. It is important to place the posterior wall stitches close enough to prevent any leakages, as revising the posterior wall is bothering. Lastly, the anterior face is sutured. This technique is one of our preferences as it minimises the incidence of transfixing sutures [24] (Figure 5).
\n
Figure 5.
One-way-up technique. First four to five stitches are placed in the posterior wall in an inverted fashion. It is important to leave only a small gap between the two first knots in this posterior wall, in order to avoid leakages and reviews here. After these first inverted stitches, the rest of them are placed in a conventional simple fashion as depicted in the figure. This technique avoids twisting and injuring the anastomosis.
\n
\n
\n
\n
4.2 End-to-side anastomosis
\n
This type of suture is very useful when there is a great discrepancy between vascular lumens, or when the flow through a vascular axis must be preserved. Therefore, it is very useful in lower limb reconstructions, when one of the vascular axes is damaged or we want to preserve the integrity of all [27]. For example, in head and neck surgery, after a cervical dissection, the high rate of venous thrombosis makes it advisable to choose the internal jugular as recipient vein [6]. In view of the discrepancy between the internal jugular and the vein of any flap, as well as the pertinence of maintaining the flow through the internal jugular, an end-to-side anastomosis is frequently chosen.
\n
To perform this end-to-side anastomosis, we must occlude the flow through the larger vessel that will remain in continuity. Our preference is the use of two rubber loops with a double pass around the vessel. When tensioning these loops, it seems that the damage to the walls of the vessel is inferior than with bulldog or baby Satinsky clamps. Next, by putting traction on the wall of the vessel with a transmural suture, we elongate the wall and make a section with the straight adventitectomy scissors or with a scalpel [27]. The diameter of the hole created must not be greater than the one on the vessel present in the free flap. If possible, the flap is tilted over the anastomosis to suture the posterior face; otherwise, we will use a one-way-up suture technique [28].
\n
\n
\n
4.3 Tips and pearls
\n
\n
It is important to take within each suture a good amount of intima to adequately evert it and expose smooth intima to the vascular lumen, with scarce subendothelial collagen or suture material.
The knots should be flat, placed on one side, with the right pressure just to close the anastomosis, since very tight sutures can cause isquemia and failure.
In case of working with veins of inconsistent walls, to perform an immersion technique, using abundant heparinised serum in the field to open the vascular lumen can be useful.
We should not allow leaks in the anastomosis; these will cease through the formation of an intraluminal thrombus, which can ultimately endanger the entire anastomosis.
At the end of an anastomosis, we must check its permeability, for example by means of a patency test. Other possibility is to make a profuse irrigation through the space left in the microvascular anastomosis before placing the last two stitches, an inflation and slight dilatation of the anastomosis with the heparinised serum evinces the vascular patency (Figure 6). The classic patency test can traumatise the intima.
The learning of these techniques must begin in a laboratory of experimental surgery with animal models [29].
Dilatation of the lumen with specific dilator forceps allows better visualisation of the interior of the vessel, easily recovering the needle at each stitch.
Before passing the entire thread through the anastomosis, the former should be in line with the vessels, and not angulated behind the needle. This precaution will avoid tears and friction on the vessel wall with the thread passage.
Limit the vessel dissection as much as possible (Figures 1 and 2).
\n\n
Figure 6.
Patency test with rinsing. A short Abbocath cannula is introduced in the microvascular anastomosis in between the space left by the two late stitches, we pretend to verify an easy dilatation before finishing the suture. The anastomosis is completed full of heparinised serum, until clamps are released.
\n
\n
\n
\n
5. Couple devices
\n
Since the onset of microsurgery, a great interest was drawn towards the development of suture techniques to perform anastomoses more quickly and automatically, in order to buffer inaccuracies [28]. For this purpose, devices in the form of two metal rings that are coupled, known as coupler devices, were developed.
\n
Currently, its use is widespread, mainly for vein anastomoses, although they have also tested a 100% patency in arterial ones. The vessel is introduced through the ring and the edges are fixed inside-out in the pins arranged in the ring, then the same is done with the other vessel and the hinge of the device, that joins both sides, is closed. The eversion of the edges achieves less exposure of the vascular lumen to foreign material and therefore the rate of thrombogenesis is lower. This eversion of the edges in the case of the arteries is more complex due to the thickness of the vascular wall, which makes its use in arterial anastomosis not so popular [28, 30]. There are coupler devices currently available with built-in systems for flap control, such as Doppler.
\n
Despite their many advantages, they present some drawbacks. Although they have been shown to reduce the time needed to perform the anastomosis, their use involves some complexity and produces some stenosis. On the other hand, they are not recommended in areas with a tendency to infection, with poor vascularisation or to be irradiated.
\n
\n
\n
6. Conclusions
\n
In the search of the best functional and aesthetic results, free tissue transfers have become the gold standard for many of the issues that arise in reconstructive surgery [31, 32]. Clear examples of this are the deep inferior epigastric artery perforator flap in breast reconstruction [7, 8] and the anterolateral thigh flap in head and neck reconstruction [6]. But the success of these techniques does not only depend on an adequate vascular suture, but also on a constellation of details that must be taken into account. These go from the availability of a trained team, to ergonomics, through a scrupulous cleanliness of the surgical field. All this does nothing but stress the importance of patience, good planning, attention to details or even the use of microsurgical checklists in to prevent any error that, however small, can have catastrophic consequences.
\n
\n
Acknowledgments
\n
The authors would like to give special thanks to their chief Dr. Antonio Taboada-Suárez for his support, constant stimuli and generosity and to Dr. Joaquim Megías Barrera for his guidance.
\n
Conflict of interest
The authors declare no conflict of interest.
\n',keywords:"free tissue transplantation, microvascular anastomosis, microsurgery, reconstruction",chapterPDFUrl:"https://cdn.intechopen.com/pdfs/71675.pdf",chapterXML:"https://mts.intechopen.com/source/xml/71675.xml",downloadPdfUrl:"/chapter/pdf-download/71675",previewPdfUrl:"/chapter/pdf-preview/71675",totalDownloads:327,totalViews:0,totalCrossrefCites:0,dateSubmitted:"May 22nd 2019",dateReviewed:"February 27th 2020",datePrePublished:"April 6th 2020",datePublished:null,dateFinished:null,readingETA:"0",abstract:"Free tissue transfer pursues the best functional and aesthetic results in reconstructive surgery. As these techniques completely maximise the donor tissues’ disposability, these treatments have become a first-line option in many situations. When the donor site is taken form the same patient, these surgeries are often referred to as autotransplants. Free tissue transfer sustains in microvascular anastomosis, which are defined by a vessel lumen diameter inferior to 3 mm. Particular attention to some details is important in these techniques, as, for example, to preclude any damage to the vessel walls or any leakage in the microvascular anastomosis. But the success of these techniques does not only depend on an adequate vascular suture, but also on a constellation of details that must be taken into account. These go from the availability of a trained team, to the ergonomics of the surgeon, through the scrupulous cleanliness of the surgical field.",reviewType:"peer-reviewed",bibtexUrl:"/chapter/bibtex/71675",risUrl:"/chapter/ris/71675",signatures:"Ignacio Vila, Iván Couto-González and Beatriz Brea-García",book:{id:"8300",title:"Vascular Biology",subtitle:"Selection of Mechanisms and Clinical Applications",fullTitle:"Vascular Biology - Selection of Mechanisms and Clinical Applications",slug:"vascular-biology-selection-of-mechanisms-and-clinical-applications",publishedDate:"November 11th 2020",bookSignature:"Marcelo",coverURL:"https://cdn.intechopen.com/books/images_new/8300.jpg",licenceType:"CC BY 3.0",editedByType:"Edited by",editors:[{id:"202594",title:"Dr.",name:"Marcelo",middleName:null,surname:"González",slug:"marcelo-gonzalez",fullName:"Marcelo González"}],productType:{id:"1",title:"Edited Volume",chapterContentType:"chapter",authoredCaption:"Edited by"}},authors:null,sections:[{id:"sec_1",title:"1. Introduction to microsugery",level:"1"},{id:"sec_2",title:"2. Basic principles in microsurgery",level:"1"},{id:"sec_2_2",title:"2.1 Ergonomy",level:"2"},{id:"sec_3_2",title:"2.2 General conditions",level:"2"},{id:"sec_4_2",title:"2.3 Team",level:"2"},{id:"sec_6",title:"3. Microsurgical tools and instruments",level:"1"},{id:"sec_6_2",title:"3.1 Tools",level:"2"},{id:"sec_7_2",title:"3.2 Basic microsurgery instruments kit",level:"2"},{id:"sec_8_2",title:"3.3 Instruments care",level:"2"},{id:"sec_9_2",title:"3.4 Sutures",level:"2"},{id:"sec_11",title:"4. Microsurgery techniques",level:"1"},{id:"sec_11_2",title:"4.1 End-to-end anastomosis",level:"2"},{id:"sec_11_3",title:"4.1.1 Triangulation",level:"3"},{id:"sec_12_3",title:"4.1.2 Continuous",level:"3"},{id:"sec_13_3",title:"4.1.3 Continuous interrupted",level:"3"},{id:"sec_14_3",title:"4.1.4 One way up",level:"3"},{id:"sec_16_2",title:"4.2 End-to-side anastomosis",level:"2"},{id:"sec_17_2",title:"4.3 Tips and pearls",level:"2"},{id:"sec_19",title:"5. Couple devices",level:"1"},{id:"sec_20",title:"6. Conclusions",level:"1"},{id:"sec_21",title:"Acknowledgments",level:"1"},{id:"sec_24",title:"Conflict of interest",level:"1"}],chapterReferences:[{id:"B1",body:'\nWei FC, Tay SK. Principles and techniques of microvascular surgery. In: Neligan PC, editor. Plastic Surgery. Vol. 1, Principles. 3rd ed. Elsevier; 2013. pp. 587-621.e10\n'},{id:"B2",body:'\nMasia J, Olivares L, Koshima I, et al. Barcelona consensus on supermicrosurgery. Journal of Reconstructive Microsurgery. 2014;30:53-58\n'},{id:"B3",body:'\nGu YD, Cheng DS, Zhang GM, et al. Long-term results of toe transfer: Retrospective analysis. Journal of Reconstructive Microsurgery. 1997;13(6):405-408\n'},{id:"B4",body:'\nTamai S. History of microsurgery. Plastic and Reconstructive Surgery. 2009;124:e282-e294\n'},{id:"B5",body:'\nEskander A, Kang SY, Teknos TN, et al. Advances in midface reconstruction: Beyond the reconstructive ladder. Current Opinion in Otolaryngology & Head and Neck Surgery. 2017;25(5):422-430\n'},{id:"B6",body:'\nRay E. Head and neck reconstructive surgery. Cancer Treatment and Research. 2018;174:123-143\n'},{id:"B7",body:'\nMurphy BD, Farhadi J, Masia J, et al. Indications and controversies for abdominally-based complete autologus tissue breast reconstruction. Clinics in Plastic Surgery. Jan 2018;45(1):83-91\n'},{id:"B8",body:'\nDancey A, Blondeel PN. Technical tips for safe perforator vessel dissection applicable to all perforator flaps. Clinics in Plastic Surgery. 2010;37(4):593-606\n'},{id:"B9",body:'\nKhouri RK. Avoiding free flap failure. Clinics in Plastic Surgery. 1992;19:773-781\n'},{id:"B10",body:'\nHyza P, Vesely J, Schwarz D, et al. The effect of blood around a flap pedicle on flap perfusion in an experimental rodent model. Acta Chirurgiae Plasticae. 2009;51:21-25\n'},{id:"B11",body:'\nLi X, Cooley BC, Gould JS. Influence of topical heparin on stasis-induced thrombosis of microvascular anastomoses. Microsurgery. 1992;13:72-75\n'},{id:"B12",body:'\nInternational Registry on Hand and Composite Tissue Transplantation–World Exp. 2009. [Accessed: 31 December 2009]\n'},{id:"B13",body:'\nLutz BS, Wei FC, Machens HG, et al. Indications and limitations of angiography before free-flap transplantation to the distal lower leg after trauma: Prospective study in 36 patients. Journal of Reconstructive Microsurgery. 2000;16:187-191\n'},{id:"B14",body:'\nMasia J, Clavero JA, Larranaga JR, et al. Multidetector–Row computed tomography in the planning of abdominal perforator flaps. Journal of Plastic, Reconstructive & Aesthetic Surgery. 2006;59:594-599\n'},{id:"B15",body:'\nIsken T, Alagoz MS, Onyedi M, et al. Preoperative color Doppler assessment in planning of gluteal perforator flaps. Annals of Plastic Surgery. 2009;62:158-163\n'},{id:"B16",body:'\nAcland RD. Microvascular anastomosis: A device for holding stay sutures and a new vascular clamp. Surgery. Feb 1974;75(2):185-187\n'},{id:"B17",body:'\nYan JG, Yousif NJ, Dzwierzynski WW, et al. Irrigation pressure and vessel injury during microsurgery: A qualitative study. Journal of Reconstructive Microsurgery. 2004;20:399-403\n'},{id:"B18",body:'\nLohman R, Siemionow M, Rockwell WB, et al. Acute adverse effects of blunt adventitial stripping. Annals of Plastic Surgery. 1995;35:60-65\n'},{id:"B19",body:'\nKemler MA, Kolkman WF, Slootweg PJ, et al. Adventitial stripping does not strip the adventitia. Plastic and Reconstructive Surgery. 1997;99:1626-1631\n'},{id:"B20",body:'\nHaddock NT, Kayfan S, Pezeshk RA, et al. Co-surgeons in breast reconstructive microsurgery: What do they bring to the table? Microsurgery. 2018;38(1):14-20\n'},{id:"B21",body:'\nNunley JA. Microscopes and microinstruments. Hand Clinics. 1985;1:197-204\n'},{id:"B22",body:'\nBaker JM, Meals RA. A practical guide to surgical loupes. The Journal of Hand Surgery. 1997;22:967-974\n'},{id:"B23",body:'\nShenaq SM, Klebuc MJ, Vargo D. Free–tissue transfer with the aid of loupe magnification: experience with 251 procedures. Plastic and Reconstructive Surgery. 1995;95:261-269\n'},{id:"B24",body:'\nAcland RD, Sabapathy SR. Practice Manual for Microvascular Surgery. 3rd ed. The Indian Society for Surgery of the Hand (ISSH); 2008\n'},{id:"B25",body:'\nCarrel A. La technique operatoire des anastomoses vasculaires et la transplantation des viscères. Lyon Medical. 1902;98:859-863\n'},{id:"B26",body:'\nChen YX, Chen LE, Seaber AV, et al. Comparison of continuous and interrupted suture techniques in microvascular anastomosis. The Journal of Hand Surgery. 2001;26:530-539\n'},{id:"B27",body:'\nGodina M. Preferential use of end–to–side arterial anastomoses in free flap transfers. Plastic and Reconstructive Surgery. 1979;64:673-682\n'},{id:"B28",body:'\nChernichenko N, Ross DA, Shin J, et al. End–to–side venous anastomosis with an anastomotic coupling device for microvascular free–tissue transfer in head and neck reconstruction. The Laryngoscope. 2008;118:2146-2150\n'},{id:"B29",body:'\nKlein I, Steger U, Timmermann W, et al. Microsurgical training course for clinicians and scientists at a German university hospital: A 10–year experience. Microsurgery. 2003;23:461-465\n'},{id:"B30",body:'\nSpector JA, Draper LB, Levine JP, et al. Routine use of microvascular coupling device for arterial anastomosis in breast reconstruction. Annals of Plastic Surgery. 2006;56:365-368\n'},{id:"B31",body:'\nParry SW, Toth BA, Elliott LF. Microvascular free–Tissue transfer in children. Plastic and Reconstructive Surgery. 1988;81:838-840\n'},{id:"B32",body:'\nGermann G, Bickert B, Steinau HU, et al. Versatility and reliability of combined flaps of the subscapular system. Plastic and Reconstructive Surgery. 1999;103:1386-1399\n'}],footnotes:[],contributors:[{corresp:"yes",contributorFullName:"Ignacio Vila",address:"ignacio.vila.garcia@sergas.es",affiliation:'
Department of Plastic Surgery, University Hospital Complex of Santiago, Santiago de Compostela, Spain
Department of Plastic Surgery, University Hospital Complex of Santiago, Santiago de Compostela, Spain
'}],corrections:null},book:{id:"8300",title:"Vascular Biology",subtitle:"Selection of Mechanisms and Clinical Applications",fullTitle:"Vascular Biology - Selection of Mechanisms and Clinical Applications",slug:"vascular-biology-selection-of-mechanisms-and-clinical-applications",publishedDate:"November 11th 2020",bookSignature:"Marcelo",coverURL:"https://cdn.intechopen.com/books/images_new/8300.jpg",licenceType:"CC BY 3.0",editedByType:"Edited by",editors:[{id:"202594",title:"Dr.",name:"Marcelo",middleName:null,surname:"González",slug:"marcelo-gonzalez",fullName:"Marcelo González"}],productType:{id:"1",title:"Edited Volume",chapterContentType:"chapter",authoredCaption:"Edited by"}}},profile:{item:{id:"179899",title:"Prof.",name:"Olga",middleName:null,surname:"Christopoulou",email:"ochris@uth.gr",fullName:"Olga Christopoulou",slug:"olga-christopoulou",position:null,biography:null,institutionString:null,profilePictureURL:"//cdnintech.com/web/frontend/www/assets/author.svg",totalCites:0,totalChapterViews:"0",outsideEditionCount:0,totalAuthoredChapters:"1",totalEditedBooks:"0",personalWebsiteURL:null,twitterURL:null,linkedinURL:null,institution:null},booksEdited:[],chaptersAuthored:[{title:"Comparison of the Land Uses and Sustainable Development in Small Islands: The Case of Skiathos Island, Greece",slug:"comparison-of-the-land-uses-and-sustainable-development-in-small-islands-the-case-of-skiathos-island",abstract:"The Island of Skiathos occupies a total area of 50 km2, accounting for 1.6% of the area of the prefecture of Magnesia and 0.28% of the Region of Thessaly, Greece. The land is hilly and can be divided into farmland, meadows, woodlands, land covered by water and land occupied by settlements and roads. Also, a large part is occupied by burnt areas that resulted from the fire of 2007. The aim of this chapter is to present the evolution of existing land uses at the Island of Skiathos during the past decades. With the contribution of Geographic Information Systems (GIS) and the orthophotomaps, the spatial planning of the land uses can be evaluated for all these years and the total area can also be calculated. Our results are important for understanding the impacts of land uses on ecosystems in the frame of sustainable development. There has been no other research regarding land uses in Skiathos Island in the past, and, also, this is the first digitization of the area. Finally, two sustainable spatial development scenarios for the Island of Skiathos are proposed. The first scenario relates to the results obtained from a prediction (application of the model of automatic cellular) while the second scenario refers to a more realistic model of development with focus on environmental protection and sustainable development.",signatures:"Fani Samara, Stergios Tampekis, Stavros Sakellariou, Olga\nChristopoulou and Athanasios Sfougaris",authors:[{id:"178877",title:"Ph.D. Student",name:"Fani",surname:"Samara",fullName:"Fani Samara",slug:"fani-samara",email:"fanisamara@gmail.com"},{id:"179799",title:"Dr.",name:"Stergios",surname:"Tampekis",fullName:"Stergios Tampekis",slug:"stergios-tampekis",email:"stampeki@gmail.com"},{id:"179898",title:"Ph.D. Student",name:"Stavros",surname:"Sakellariou",fullName:"Stavros Sakellariou",slug:"stavros-sakellariou",email:"stasakel@gmail.com"},{id:"179899",title:"Prof.",name:"Olga",surname:"Christopoulou",fullName:"Olga Christopoulou",slug:"olga-christopoulou",email:"ochris@uth.gr"},{id:"179900",title:"Prof.",name:"Athanasios",surname:"Sfougaris",fullName:"Athanasios Sfougaris",slug:"athanasios-sfougaris",email:"asfoug@agr.uth.gr"}],book:{title:"Urban Agriculture",slug:"urban-agriculture",productType:{id:"1",title:"Edited Volume"}}}],collaborators:[{id:"61187",title:"Prof.",name:"Marina",surname:"Pintar",slug:"marina-pintar",fullName:"Marina Pintar",position:null,profilePictureURL:"//cdnintech.com/web/frontend/www/assets/author.svg",biography:null,institutionString:null,institution:null},{id:"82604",title:"Dr.",name:"Matjaž",surname:"Glavan",slug:"matjaz-glavan",fullName:"Matjaž Glavan",position:null,profilePictureURL:"https://mts.intechopen.com/storage/users/82604/images/3247_n.jpg",biography:"Dr. Matjaž Glavan is an assistant professor of Environmental Planning at Biotechnical Faculty of the University of Ljubljana and the chair for Agrometeorology, Agricultural Land Management, Economics and Rural Development. He completed his BSc degree in Agronomy at the University of Ljubljana, Slovenia, and his MSc degree in Integrated Catchment Management at Cranfield University, UK. He obtained his PhD degree in Biological and Biotechnological Sciences at the University of Ljubljana in 2011. His main field of expertise is integrated water and land management in relation to agriculture. His research focus is also on modelling spatial, economic and environmental impacts of land-use changes and agri-environmental measures on water, soil and land resource quality and quantity. He is also specialized in catchment modelling with the Soil and Water Assessment Tool.",institutionString:null,institution:{name:"University of Ljubljana",institutionURL:null,country:{name:"Slovenia"}}},{id:"178373",title:"Dr.",name:"Ahmed K.",surname:"Ali",slug:"ahmed-k.-ali",fullName:"Ahmed K. Ali",position:null,profilePictureURL:"//cdnintech.com/web/frontend/www/assets/author.svg",biography:null,institutionString:null,institution:{name:"Texas A&M University",institutionURL:null,country:{name:"United States of America"}}},{id:"178797",title:"Dr.",name:"Rozalija",surname:"Cvejić",slug:"rozalija-cvejic",fullName:"Rozalija Cvejić",position:null,profilePictureURL:"//cdnintech.com/web/frontend/www/assets/author.svg",biography:null,institutionString:null,institution:null},{id:"178877",title:"Ph.D. Student",name:"Fani",surname:"Samara",slug:"fani-samara",fullName:"Fani Samara",position:null,profilePictureURL:"//cdnintech.com/web/frontend/www/assets/author.svg",biography:null,institutionString:null,institution:{name:"University Of Thessaly",institutionURL:null,country:{name:"Greece"}}},{id:"179170",title:"Prof.",name:"Majda",surname:"Černič Istenič",slug:"majda-cernic-istenic",fullName:"Majda Černič Istenič",position:null,profilePictureURL:"//cdnintech.com/web/frontend/www/assets/author.svg",biography:null,institutionString:null,institution:null},{id:"179542",title:"Prof.",name:"Bruce",surname:"Dvorak",slug:"bruce-dvorak",fullName:"Bruce Dvorak",position:null,profilePictureURL:"//cdnintech.com/web/frontend/www/assets/author.svg",biography:null,institutionString:null,institution:{name:"Texas A&M University",institutionURL:null,country:{name:"United States of America"}}},{id:"179799",title:"Dr.",name:"Stergios",surname:"Tampekis",slug:"stergios-tampekis",fullName:"Stergios Tampekis",position:null,profilePictureURL:"https://mts.intechopen.com/storage/users/179799/images/system/179799.jpeg",biography:"Dr. Stergios Tampekis (Corresponding author) was born in Ioannina, Greece in August 1978. He is a Doctor of Philosophy degree holder from the Forestry and Natural Environment Department of the Aristotle\nUniversity of Thessaloniki, Greece (2005-2008). He took his two year courses Master of Science degree (2002-2004) from the Forestry and Natural Environment Department of the Aristotle University of\nThessaloniki, Greece. He studied Forestry and Natural Environment at the Aristotle University of Thessaloniki, Greece (1997-2002). He has been a Forest Engineer Researcher at the Hellenic Forest Service, Eastern Attica Research Station, Bureau of Forest Mapping, Greece, since March 2017. He has worked on many research projects (9) since 2004 and has published 13 scientific articles. His research interests are:\na) Multiple-criteria decision analysis techniques (MCDA) for forest operations planning and management, b) Spatially explicit optimization models of forest harvesting operations and transportation systems and c) Environmentally friendly, economically efficient forest operations planning and reduced impact logging.",institutionString:"Hellenic Forest Service",institution:{name:"University Of Thessaly",institutionURL:null,country:{name:"Greece"}}},{id:"179898",title:"Ph.D. Student",name:"Stavros",surname:"Sakellariou",slug:"stavros-sakellariou",fullName:"Stavros Sakellariou",position:null,profilePictureURL:"//cdnintech.com/web/frontend/www/assets/author.svg",biography:null,institutionString:null,institution:null},{id:"179900",title:"Prof.",name:"Athanasios",surname:"Sfougaris",slug:"athanasios-sfougaris",fullName:"Athanasios Sfougaris",position:null,profilePictureURL:"//cdnintech.com/web/frontend/www/assets/author.svg",biography:null,institutionString:null,institution:null}]},generic:{page:{slug:"careers-at-intechopen",title:"Careers at IntechOpen",intro:"
Our business values are based on those any scientist applies to their research. The values of our business are based on the same ones that all good scientists apply to their research. We have created a culture of respect and collaboration within a relaxed, friendly, and progressive atmosphere, while maintaining academic rigour.
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Integrity - We are consistent and dependable, always striving for precision and accuracy in the true spirit of science.
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Integrity - We are consistent and dependable, always striving for precision and accuracy in the true spirit of science.
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Openness - We communicate honestly and transparently. We are open to constructive criticism and committed to learning from it.
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Disruptiveness - We are eager for discovery, for new ideas and for progression. We approach our work with creativity and determination, with a clear vision that drives us forward. We look beyond today and strive for a better tomorrow.
\n\n
What makes IntechOpen a great place to work?
\n\n
IntechOpen is a dynamic, vibrant company, where exceptional people are achieving great things. We offer a creative, dedicated, committed, and passionate environment but never lose sight of the fact that science and discovery is exciting and rewarding. We constantly strive to ensure that members of our community can work, travel, meet world-renowned researchers and grow their own career and develop their own experiences.
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If this sounds like a place that you would like to work, whether you are at the beginning of your career or are an experienced professional, we invite you to drop us a line and tell us why you could be the right person for IntechOpen.
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