Quantitative characterization of the selected tissues wasperformed, and the inter-tissue distribution of metabolites was described.For both varieties, d-mannoheptulose was the major componentin the mesocarp and exocarp, whereas perseitol was predominant inthe seed, followed by sucrose and d-mannoheptulose. Sucrosewas found to be more abundant in seed tissues, with much lower concentrationsin avocado mesocarp and exocarp. Quinic acid showed a predominancein the exocarp, and chlorogenic acid was exclusively determined inexocarp samples.Avocado fruit growthand development, unlike that of other fruits,is characterized by the accumulation of oil and C7 sugars . There are five essentialcarbohydrates which constitute 98% of the total content of solublesugars in this fruit; these are fructose, glucose, sucrose,  The first two subspecies aretypical to tropical highlands, where colder conditions predominate,whereas the West Indian subspecies is originated from tropical lowlands,where the conditions are warmer. There is a wide variety of hybridsamong the different subspecies producing fruits that present differentphysical and sensory properties, including different fruit maturityrates, oil percentages, etc.2Avocado, texture alteration, and changes in the content ofsugars, organics acids, and volatile compounds involved in nutritionalquality, flavor, and aroma.6 In the earlystages of avocado fruit growth and development, more than 40% of themesocarp weight is made up of sugars.7 Acharacteristic of this fruit is the large amount of the less commonheptose (C7) sugars ,which act as respiratory substrates, instead of hexose (C6) sugars, as is typical in most fruits.10 Despite the importance of these carbohydrates, there are still manyaspects to be unraveled about their synthesis, metabolism, transport,and physiological roles. Interesting studies postulate that perseitolacts as a storage sugar (energy source) and d-mannoheptuloseas a transport sugar and sometimes as an energy supplier in the subsequentelicitation of other primary compounds.13 The implication of d-mannoheptulose as a potent inhibitorof hexokinase has also been suggested, preventing the entry of glucoseinto glycolysis, which hinders fruit ripening.14 It has also been reported that these C7 sugars act as antioxidantsin the avocado mesocarp, and, therefore, their initial content atharvest correlates with fruit storability conferring not only carbohydratesto maintain respiration but also protective agents against stress.15Avocadois a fruit with a high metabolic rate that completes itsripening in approximately 7 days at 25 °C after harvest,16 similar is the case of amino acids and organicacids. For decades, they have been determined using gas chromatographycoupled to mass spectrometry (GC–MS) or high-performance liquidchromatography (HPLC) coupled to different detection systems.17 GC–MS allows the simultaneous quantificationof a large number of polar metabolites, with relatively low instrumentalcost and the possibility of using libraries for compound identification.However, in many cases, a derivatization process is required to providethe compounds with sufficient volatility and thermostability.18 As far as HPLC is concerned, the separation of sugars has usuallybeen performed with monosaccharide or NH2 columns (amongothers); sometimes, pre- or post-column derivatization reactions areresorted to, either to improve chromatographic separation or to ensuredetectability.19 Refractive index detector(RID) and evaporative light scattering detector (ELSD) are very suitabledetectors for the determination of sugars, although various LC–MSmethods have been developed over the last 10 years with the aim toquantify one or more classes of polar compounds in different matrices.17 Recently, research using hydrophilic interactionliquid chromatography (HILIC) separations has increased substantially,and a number of stationary phases have been developed for this kindof chromatography. This is mainly due to the growing need to analyzehydrophilic metabolites in a wide variety of scientific fields andto the fact that HILIC provides enhanced retention and separationfor polar compounds and is highly compatible with MS.20 HILIC consists of a polar chromatographic surface  with a mobilephase that includes a water-miscible polar organic solvent (such asacetonitrile) mixed with water, starting with a high percentage oforganic solvent and ending with a high proportion of aqueous phase.21 Interesting applications to determinepolar compounds in diverse matrices can be found in the literature.22The determination of sugars representsan analytical challengedue to their polar nature and the absence of chromophore groups;Table S1—SupportingInformation includes a good number of interesting works that providean overview of the most considered avocado tissues and varieties,metabolites, and methodologies. As can be seen, there are severalmeritorious works dealing with the quantification of sugars and organicacids; however, some of them have considered the determination ofvery few compounds or required the use of two different analyticaltechniques or two chromatographic columns or entailed laborious samplepreparation protocols . No work, so far, has applieda HILIC–MS methodology for the determination of carbohydrates,chlorogenic acid, and quinic acid in avocado tissues. In addition,most of the previous works have been performed mainly in Hass, the preponderant variety in the avocado market worldwide and, consequently,information is lacking for most other avocado varieties. Bacon and Fuerte are green skin varieties used in manycountries as pollinizer for Hass.Focusing on the determinationof carbohydrates and other polarcompounds in avocado, Bacon and Fuerte varieties, determiningthe quantitative levels of C6  andC7 (d-mannoheptulose and perseitol) sugars; and (iii) todescribe the distribution of carbohydrates together with quinic acidand chlorogenic acid in avocado fruit tissues at three different ripeningstages. It is evident that knowing in detail the composition of thedifferent avocado tissues, the distribution of polar metabolites betweentissues of the same fruit and how the ripening process affects themis of undeniable importance and contributes to improve the knowledgeof significant physiological aspects of this tropical fruit. Therefore,this report presents an attractive analytical solution—avoidingthe drawbacks of other methods—for the simultaneous determinationof compounds of interest in avocado and contributes to describe avocadocompositionally and to learn more about the primary post-harvest metabolicprocesses.This workwas approached with multiple objectives: (i) to evaluatethe potential of HILIC–MS to determine polar compounds in avocadotissues; (ii) to characterize samples of mesocarp, exocarp, and seedof 22.1d-mannoheptulose (CAS number 3615-44-9),fructose (57-48-7), glucose (50-99-7), sucrose (57-50-1), quinic acid(77-05-2), and chlorogenic acid (327-97-9) acid were supplied by Sigma-Aldrich. Perseitol (527-06-0) was acquired from Carbosynth. Stock solutions were prepared with specificconcentrations of each metabolite in order to cover the appropriatequantitative ranges for each kind of avocado tissue  was used. Acetonitrile LC–MS grade (ACN) was purchasedfrom Lab-Scan , and ammonium acetate was providedby Sigma-Aldrich . Mobile phases were filtered usinga 0.45 μm Nylaflo nylon membrane acquired from Pall Corporation. Extraction of the analytes of interest was carriedout by using aqueous mixtures of ethanol (EtOH) from Prolabo . Standards of 2.2The sampleswere obtainedfrom a private avocado commercial orchard located in Vélezde Benaudalla, a municipality in the province of Granada, Spain. Thecoordinates of the orchard are latitude: 36° 49′ 55″,North longitude: 3° 30′ 58″ West, with an altitudeabove sea level of 171 meters.Bacon and Fuerte avocados. For both varieties, three different ripening stages weredefined: freshly picked fruits (firmness range >50 N), fruits inanintermediate stage of ripening (50–15 N), and ready-to-eatfruits . Fruit ripening took placeunder identical ambient conditions (20 ± 2 °C). Eighteenfruits were taken (three of each variety at each stage of ripening)which led, considering the three tissues, to a total of 54 samples.The samples were peeled, chopped, frozen, freeze-dried, crushed, homogenized,and stored at −20 °C. Each avocado fruit was consideredindependently, i.e., each avocado was processed and sampled and ledto three samples  to properly studythe distribution of analytes among tissues of a single fruit. Consideringthat each avocado fruit is a unique specimen, it seemed appropriateto treat them as independent, since one of the main goals of thiswork was to explore the distribution of polar metabolites among tissuesof the same fruit.The determination of the aforementionedanalytes was carried out in three tissues—seed, exocarp, andmesocarp—of Bacon fruits were harvestedin late October 2020and Fuerte fruits at the end of November 2020. Percentageof dry matter (DM) was evaluated according to the AOAC 920.151 method23 as soon as the fruits were detached from thetree by taking, at least, 10–15 fruits and calculating themean DM value. DM values for Bacon and Fuerte samples were between 23 and 25% (SD of DM measurements were closeto 1 approx.). The DM values found can be considered normal for thoseavocado varieties in Spain at this time of the harvest season. Accordingto EU regulations, avocados can already be harvested with 21% DM.2.32O, 60:40 (v/v). The mixture was shaken invortex for 3 min; after that, the tubes were introduced into an ultrasoundbath for 30 min, followed by centrifugation at 9000 rpm for 5 min.Once the solid phase and the supernatant were properly separated,the latter was transferred to a flask. The solid residue was extracteda second time, applying the same procedure (a second extraction cycle).Both supernatants were mixed and shaken in vortex for 1 min. Finally,approx. 1 mL of the solution was filtered and transferred to an HPLCvial. Two independent analytical replicates were prepared for eachsample.Metaboliteswere extracted from 0.20 g of lyophilized samples and were mixed with6 mL of EtOH/H2.42O/ACN  for phase A and H2O/ACN  for phase B. Ammonium acetate buffer wasadded to both phases to have the same final concentration in bothbottles (10 mM). The applied elution conditions were: 0 min, 2% Aand 98% B; 5 min, 2% A and 98% B (5 min at isocratic conditions);20 min, 35% A and 65% B; at 21.5 min, the system returned to initialconditions. Each analysis lasted approximately 30 min, taking intoaccount column reequilibration. The MS was operated in a negativemode, and data were acquired in a full scan mode for a mass rangefrom 50 to 1000 m/z. In order toachieve stable ionization, the nebulizer gas (nitrogen) was set at30 psi, dry gas (nitrogen) flow rate at 9 L/min, and temperature at300 °C. The optimum capillary voltage was established at +3200V, and the end-plate offset at −500 V.As the instrumentalplatform, a 1260 Infinity HPLC system  and an Esquire 2000 Ion Trap (IT) MS  coupled by means of an electrospray ionization (ESI) sourcewere used. Chromatographic separation of the different analytes ofinterest was performed using a Fortis HILIC-Diol column , whose dimensions were 2.1 × 150 mm and 1.7 μmparticle size. The column operated at 25 °C, and the injectionvolume was set at 2 μL. The chromatographic flow was fixed at0.3 mL/min. Mobile phases were prepared with water and ACN at differentproportions: HThe software usedto control the LC and MS systems were Agilent ChemStation (AgilentTechnologies) and Esquire Control , respectively.In addition, data processing, management, and representations wereperformed by using DataAnalysis 4.0 software  and Microsoft Excel v 2204.2.5Pure standard solutions as well as avocado tissuesextracts were used for the validation of the method. Linearity, precision,and recovery of the extraction protocol were evaluated.d-mannoheptulose,fructose, glucose, sucrose, and quinic acid, and chlorogenic acid)were prepared in EtOH/H2O, 60:40 (v/v) at ten differentconcentration levels (over the range from the quantification limitto the maximum considered concentration level for each substance)to establish external calibration curves. The concentration of thestock solutions ranged, approximately, from 0.05 to 250 mg/L for fructoseand glucose, from 0.05 to 500 mg/L for d-mannoheptulose,chlorogenic, sucrose, and quinic acid, and 0.1–1350 mg/L forperseitol. Two specific working quantitative ranges or linear dynamicranges were established for each metabolite, except for chlorogenicacid.Solutionsof the seven pure compounds  obtained at the lowest concentration level injected (whichwas different for each compound), estimating the concentration thatgenerated a S/N equal to 3 and 10,respectively.Detection and quantification limits (LOD and LOQ) of eachanalytewere calculated using the signal/noise ratio (Repeatability (intra-day and inter-day) was consideredto assessthe precision of the method; both values were expressed as coefficientof variation (% CV). The intra-day repeatability was obtained fromfive injections of a standard mix (containing the seven selected metabolites)and a quality control (QC) sample  carried out within the same sequence,while inter-day repeatability was obtained from 12 injections performedin different sequences.24The recovery (expressed as percentage)was estimated by subjectingsamples that had already been extracted, as described in In general, it has been establishedthat the matrix effectcanbe considered as negligible if the matrix effect coefficient is foundwithin a range of ±20%.2.6p < 0.05) level between mean values wasdetermined using the Tukey’s test.The data werestatistically analyzed using Statgraphics 19 . A one-way analysis of variance (ANOVA)was performed to compare the results of each analyte for the threeavocados of the same ripening stage and another one to compare fruitsbelonging to different ripening stages. The significance of the differencesat 5%  weretested. The objective was to select the most suitable protocol fromthose considered, which would be valid for all chosen metabolitesand would result in good reproducibility. All evaluated conditionsare summarized in 26 All analytes ionized, giving a prominent signal correspondingto their pseudo-molecular ion, except chlorogenic acid, which alsoshowed a distinctive in-source fragment with m/z 191 ([M – H-162]−). As it willbe described in the following sections, all selected metabolites weredetermined in the three fruit tissues of both varieties, except forchlorogenic acid, which was only quantified in the avocado exocarp.As afirst step, the qualitative examination of the chromatographic profilesobtained was carried out. d-mannoheptulose, chlorogenic acid, perseitol, sucrose,and quinic acid. Owing to the use of a HILIC stationary phase andthe selected elution gradient, the compounds that eluted in the firstminutes were those with a more moderate polarity, and those that elutedin the last part of the analysis were those with the highest polarity.The elution order of the selected analytes was asfollows: fructose,glucose, 27 The term “chlorogenicacids” encompasses a large group of naturally occurring compoundsof which the majority are synthesized in plants by esterificationof a C6–C3 trans-hydroxycinnamic acid with1l-(−)-quinic acid.28 Manyof these compounds, like other polyphenols, are associated with importanthealth benefits and well-known as nutritional antioxidants in plantfoods. It is not easy to describe unambiguously the structures ofacyl-quinic acids that may appear almost identical when drawn in 2Dor projected in 3D.31 In this paper, we focus on the determination of arelevant compound of this category, which is assigned the trivialname of chlorogenic acid (CAS number 327-97-9).28Figure S1—SupportingInformation shows the extracted ion chromatograms (EICs) of the targetanalytes in (A) a standard mix and in (B) an example of an exocarpavocado extract. It seems pertinent to indicate that some other compoundswere detected in our analytical window ; however, aswe were unable to assign a tentative identity, these analytes werenot further considered.Initially, the method was intended to exclusively determine C6and C7 sugars; however, when exocarp, mesocarp, and seed extractswere analyzed, we observed that there were other interesting metabolitesthat could be determined along with sugars. This led to the inclusionof quinic acid and chlorogenic acid as well. The relevance of thesetwo substances in plants is beyond dispute. Quinic acid, among otherfunctions, contributes to the sugar/acid balance and health-givingproperties of the fruits. Moreover, the content of organic acids infruits is closely associated with the activities of the related metabolicenzyme.3.2The applied methodology was evaluated consideringthe analyticalparameters previously described. The numerical results appear in d-mannoheptulose,respectively. The intra-day repeatability, in all cases, presentedvalues lower than 11.4%, whereas inter-day repeatability was consistentlybelow 12.1%. These results can be considered quite adequate, as itshould be noted that HILIC methodologies are generally somewhat lessrobust than those employing reversed-phase LC. The extraction protocolimplemented was also satisfactory, since it led to recovery valuesranging from 97.7 to 100%. The trueness of the method was found between92.3 and 102.2%, and the matrix effect coefficients varied from −10.60to 14.40 for d-mannoheptulose and fructose, respectively,which means that enhancing or suppressing effects were negligible.The LODs obtained ranged between 0.01 and 0.10 mg/Land the LOQsbetween 0.04 and 0.34 mg/L for quinic acid and 3.3Bacon and Fuerte).After verifying that thedeveloped method for the determination of C6 and C7 carbohydrates,as well as quinic acid and chlorogenic acid in avocado tissues, showedacceptable analytical parameters, we proceeded to quantify these analytesin the 108 extracts . The quantitative results  are shown with their corresponding standarddeviation in Table 3 . Nevertheless, some remarks will be made in thisregard (metabolites evolution over ripening) in future paragraphsor sections of this work.After applying one-wayANOVA analyses described above, it was observedthat the concentration values of some analytes showed significantdifferences between avocado fruits at the same ripening stage, whichis understandable, considering that each avocado fruit is a differentspecimen ; in most cases, however,no such significant differences were observed between fruits at thesame ripening stage. When the comparison (one-way ANOVA) was madebetween the different ripening stages , there were not many casesin which significant differences were observed between the quantitativeresults of an analyte for the three ripening stages. Significant differenceswere observed, for example, for chlorogenic acid and glucose in theexocarp of both varieties, for fructose in the exocarp of 37 but what is still largely unknown is how C7 and C6 sugars are allocatedto the different plant tissues in which they are found. In addition,most studies have been performed in Hass and, tothe best of our knowledge, there is no publication describing thecharacterization of the polar compound profiles in Bacon and Fuerte (even less by using HILIC–MS).As discussed in the Bacon and Fuerte varieties. The resultsshow that d-mannoheptulose is the main component in the mesocarp(76.8% for Bacon and 77.2% for Fuerte) and exocarp , and perseitol is predominant in theseed (69.8% for Bacon and 69.3% for Fuerte), followed by sucrose and d-mannoheptulose. This statementcan be made for both varieties and agrees with the published literature.38Fuerte variety), accompanied by an increase of d-mannoheptulosein Bacon and of fructose and glucose in Fuerte.If these percentages were calculated for each ripeningstage, therepresentation would be quite similar for mesocarp and exocarp, but,in unripe avocados, there would be a considerably higher percentageof perseitol. In other words, the percentage of perseitol decreasesin these matrices over the ripening process. For the seed, a decreasein the relative proportion of perseitol is also observed during thesoftening of the fruit . A rather similar situation was observed for thesamples of the same tissue from Fuerte avocados.Possibly, the most marked differences between the two varieties weredetected when comparing sucrose levels in the exocarp, sucrose concentrationsbeing slightly lower in Fuerte.We will begin by commenting on the results for exocarp, thenforseed, and finally for mesocarp tissues. For Bacon and 4.13–8.5 in Fuerte. Seed fructose levels were similar for both varieties, as were sucroselevels, with a slight overall increase in the concentrations of thisanalyte in both varieties throughout ripening (particularly in Fuerte). For the C7 metabolites, the situation was oppositeto that observed in the exocarp, since the concentration of perseitolin the seed was higher than that of d-mannoheptulose, aspreviously described in the literature.42 The values obtained were 51–82 and 1.9–15 mg/g forperseitol and d-mannoheptulose, respectively, in Bacon, and 54–91 mg/g and 3.83–18.9 mg/g forthe same analytes in Fuerte. A similar observationhad been reported in Hass avocado seeds, indicatingthat perseitol was the most abundant carbohydrate, followed by sucrose, d-mannoheptulose, fructose, and glucose.14 Also, Tesfay et al., 2012, made statements in the samedirection in a very interesting piece of work focused on the searchfor the function of carbohydrates in Hass avocados.37In the seed,the ranges found for quinic acid were 1.9–14mg/g in Bacon showed higher C6 concentrationsthan those found in Fuerte, except for sucrose inunripe fruits. These sugars had a quite clear tendency to increaseover ripening in Bacon; however, this tendency couldnot be corroborated for Fuerte. The evolution ofC6 sugars over ripening needs to be studied further, since contradictoryresults are found in the literature depending on the variety and theexperimental design of the study.37d-Mannoheptuloselevels were consistently higher in samples of Bacon variety. Perseitol concentration decreased in both varieties duringfruit softening. Quinic acid concentrations determined for Bacon samples fluctuated from 5 to 11.2 mg/g (increasingslightly during ripening) and from 5.4 to 12.9 mg/g for Fuerte fruits (no clear tendency could be established in this case).The most notable differences between varietieswere observed inmesocarp samples. Figure S2—Supporting Information describesthe average composition of each tissue for Bacon and Fuerte varieties at each ripening level by using radar charts;for better adaptation of the scales and to make visual comparisonfeasible, logarithmic axes have been used. The radar chart or spiderchart offers the opportunity to display multivariate data in the formof a two-dimensional plot (on an axis starting at the center of thegraph) of several quantitative variables.In order to clarify the results discussed in this section, 3.4In order to establish the distribution of each compound among thetissues studied at each ripening stage, representations have beenmade for each metabolite, showing the percentage of the total contentof the fruit found in each tissue. In Bacon, a fact that was more noticeable as thefruit ripened. In Fuerte, however, fructose was foundin a greater proportion in the stone; this fact is more readily discerniblein ripe avocados. Glucose (which is an isomer of fructose) presenteda similar distribution to that of fructose for Bacon fruits, being predominant in the mesocarp. Among the different ripeningstages, the percentage of glucose in mesocarp was higher in the intermediatephase . In Fuerte, glucose prevailedin the seed, particularly in ready-to-eat fruits. In both varieties,sucrose was found to be more abundant in seed tissues, with much lowerconcentrations in mesocarp and exocarp. This is in agreement witha previous study that determined, among other things, the sucroseconcentration in various avocado tissues of the three varieties.38 However, Liu and co-workers reported very similarlevels of sucrose in both mesocarp and seed of Hass avocado fruits.13Among the three matrices, fructosewas found mainly in mesocarpin d-mannoheptulose in Bacon mesocarp washigher than the sum of percentages found in seed andexocarp at the three ripening stages. In Fuerte, d-mannoheptulose was better distributed among the three matrices,increasing in the seed and decreasing in the mesocarp with ripening.In a previous study, it was determined that d-mannoheptuloselevels in the mesocarp of Hass fruits were higherthan those found in exocarp and seed.38 This would be homologous to what was observed in this study for Bacon variety. However, the same authors described thatin the case of Pinkerton and Fuerte avocados, the most notable concentrations of d-mannoheptulosewere found in the exocarp. The latter is what has been evidenced inthe present study for unripe and “medium ripening” Fuerte avocados; in the case of ripe fruits, the concentrationsof d-mannoheptulose in exocarp and seed were very similar.The proportionof d-mannoheptulose as a transport sugar and, insome cases, as an energy supplier in the production of other compounds.The conversion between the two takes place through an aldose enzymepresent in the Calvin cycle. This transformation between aldoses allowsthe supply of transport sugars in the fruit mesocarp.13 It has also been indicated that this transport of sugar in the fruitis part of the mechanism that inhibits fruit ripening on the tree.Furthermore, it is believed that the accumulation of perseitol couldbe closely related to an increase in the synthesis of new C7 sugars.10 The results obtained reinforce the hypothesisthat perseitol is a storage carbohydrate, so this can probably explainwhy the concentration is high in the seed and notably lower in therest of the tissues. Most authors in this field share the hypothesisthat C7 sugars are “multifunctional sugars” and furtherresearch is needed to shed light on this topic and to elucidate themetabolism of heptose carbohydrates in avocado. A study with a largernumber of samples and varieties could confirm the hypothesis thatC7 sugars are widely used during ripening in several functions andshould be observed to decrease as senescence approaches, making thempotential biomarkers of ripening in this fruit.The graphs for perseitol were practically identical for the twovarieties at the three ripening stages. This compound was markedlypreponderant in the seed, especially as the fruits became more mature.As previously stated, perseitol may act as a storage sugar (energysource) and Creole.41For both varietiesand regardless of the ripening stage, quinicacid showed a predominance in the exocarp, where its concentrationrepresented approximately 50% of the total concentration of the threematrices. As pointed out above, chlorogenic acid was found in quantifiablequantities exclusively in the exocarp. Ramos-Aguilar and co-workersalso found no detectable levels of this compound in mesocarp for samplesof ripe fruits from Mexican Bacon, for example, does not necessarilyresemble in its entirety what is established for Fuerte . Thus, the hypothesis wepropose, and which should be confirmed by future research, is thatthe carbohydrates (C6 and C7) in the mesocarp of Fuerte tend to be consumed in metabolic processes, while the sugars inthe flesh of Bacon do not follow the same trend.On the contrary, most of them decrease, except for glucose and fructose,which show a slight increase. It is plausible that, even if all fruitsbelong to the same species, fluctuations and relative levels of primarymetabolites may induce to distinguish one variety from another.The involvement of C6 and C7 sugars in avocado ripeningis irrefutable,so they fluctuate as this process takes place . The data presented indicatethat these fluctuations will depend on the variety studied, and whatis established for Bacon and Fuerte avocados atdifferent stages of ripening. d-Mannoheptulose was the maincomponent in the mesocarp and exocarp, and perseitol was predominantin the seed, followed by sucrose and d-mannoheptulose. Theinter-tissue distribution of sucrose, perseitol, quinic acid, andchlorogenic acid was very similar for Bacon and Fuerte varieties and was not influenced by the ripeningstate, since no significant differences were found during the statisticalanalysis (p ≥ 0.05). However, a differentsituation was observed for fructose, glucose, and d-mannoheptulose,whose partitioning differed greatly between varieties and also duringfruit softening, showing a value of p ≤ 0.05.Considering the important role played by sugars as biomarkers duringthe maturation and ripening processes, it would be interesting tocarry out a more detailed investigation to complete the informationprovided in this contribution, including other varieties and perhapsmore ripening stages that would allow to properly monitor the evolutionover the avocado fruit ripening process.In conclusion, in the present work, the first HILIC–MS methodfor the determination of C6 and C7 sugars in avocado, together withchlorogenic acid and quinic acid, has been developed and validated.The method was applied to the study of 54 samples of different tissuesof Hass is of great interestto diversify the production of avocado varieties in different regionsof the world. Follow-up work on other varieties is necessary to havean ample knowledge of the diversity present in avocado germplasm.This study has provideda reliable and simple to apply analyticalapproach for the simultaneous determination of carbohydrates and othercompounds of interest in avocado. It has also described in detailthe composition of different avocado tissues, the distribution ofpolar metabolites between tissues of the same avocado fruit, and whetherthis distribution is altered during ripening. All these aspects areessential to explore and better comprehend the physiology of thistropical fruit. In addition, the provision of valuable informationon cultivars other than


The rapid global spread of severe acute respiratory syndrome coronavirus (SARS-CoV-2) was met with the rollout of vaccination campaigns at mass vaccination centers. The Palazzo delle Scintille, Milan, was designated by the Lombardy regional administration as a vaccination site with the target of processing about 9,000 users daily.For this observational study, we compared data on vaccinations delivered at the Palazzo delle Scintille with coronavirus disease (COVID-19)-related regional data.Between 25 April 2021 and 28 February 2023, a total of 1,885,822 COVID-19 doses were administered; the mean hourly rate was 289 (247.2), the mean daily rate was 3185.5 (3104.5), the mean user age was 49.5 years (10.7). The Comirnaty vaccine (Pfizer-BioNTech) was most often given . Between 4 December 2021 and 15 January 2022, the daily dose rate was above the maximum daily capacity set by the regional administration.The trend for daily dose rates administered at the Palazzo delle Scintille center was in line with COVID-19-related regional data. The center played a major role in the regional mass vaccination campaign. LoThe Palazzo delle Scintille, located in Milan, the largest of the region’s vaccination sites, was set a daily target of 9,936 vaccinations when operating at maximum capacity . The FonThe aim of the present observational study was: (1) to quantify the longitudinal activity of the MVC expressed as COVID-19 vaccine dose rates by comparing the center’s rates with COVID-19-related regional data; (2) to evaluate the organizational and managerial operations at the MVC; and (3) to describe the administration of influenza vaccination.22.1The Palazzo delle Scintille was selected because of its convenient location and space capacity. The site is served by surface and underground public transport, making it easy to reach from other parts of Milan or the metropolitan area. The center measures 15,500 square meters in floor area, which was subdivided into stations in linear sequence along which users advanced , similar to the pathway layout of other large-scale vaccination centers in Italy and around the world , 14–19. Based on estimated user flow projection from the data recorded on the Poste Italiane platform, the MCV management checked with the pharmacy about the daily availability of doses before scheduling the number of vaccination lines  and modules to be activated each week.2.2The center was staffed by physicians, nurses, and pharmacists for vaccine stockage and administration, as well as support staff, including clerical workers, volunteers, and National Guard members who assisted in directing user flow and data collection. The hospital administrative board oversaw the work of the head manager, the head pharmacist, the chief physician, and the chief nurse employed at our institution, as well as staff from community  and research hospitals  located in the Milan metropolitan area, which collaborated during peak service times.Given the number of people involved in widely diverse tasks, staff turnover, and urgent recruitment, staff training was conducted online, with the aid of operative training in specific stages of operation, roles, and responsibility of each staff member, as well as instruction on electronic data entry. The operative training was updated by integrating information and guidelines issued by national and regional governments and the Italian Medicines Agency to ensure staff received accurate and timely information.2.3Data on daily rates of COVID-19 and influenza vaccination administered at the center were collected via the Poste Italiane website services granted the Lombardy regional administration to support the mass vaccination campaign. The data on the number of COVID-19 vaccinations and SARS-CoV-2-positive swabs recorded for Lombardy were downloaded from the GitHub software platform . Data on33.1Between 25 April 2021 and 28 February 2023, a total of 1,885,822 COVID-19 vaccinations were administered, 957,442 (50.8%) of which were given to males; the hourly dose rate was 289 (247.2) and the daily dose rate was 3185.5 (3104.5) . The mean user age was 49.5 (10.7) years . A total of 139,585 (7.4%) vaccinations were administered to children (age < 18 years) and 431,024 (22.9%) to older adults (age ≥ 60 years). The first dose was given to 490,695 users, the second to 539,049, the third to 701,619, the fourth to 143,477, and the fifth to 10,982 .Starting on 4 December 2021, the center began operating beyond the capacity set by the Lombardy administration , except for two days during the summer months . The meaThe vaccine most often given was Comirnaty , followed by Spikevax , Vaxzevria , Janssen , Comirnaty Pediatric , and Nuvaxovid . Two bivalent vaccines, Comirnaty Plus 10 and Plus B.A. 4–5 and Spikevax B.A. 4–5 , were administered as additional (booster) vaccinations to users aged 12 years and older. Starting in January 2023, Original/Omicron BA.4–5, the bivalent pediatric formulation of Comirnaty, was authorized for use in children aged 5–11 years and given to 77 children  were residents of Lombardy, 95%  were residents of the province of Milan, and 72.9%  were residents of the city of Milan. The proportion of vaccinations given at the center was 8.4%  of the total administered in Lombardy. The daily dose rate was in line with the rates recorded for other vaccination center in Lombardy . The tre3.2During the 2021 winter flu season, a total of 49,045 influenza vaccinations were administered to persons aged 70.2 (7.8) years, on average , making up 2.6%  of the influenza vaccinations administered in Lombardy between October 2021 and February 2022. The vast majority (79.7%) were aged 60 years or older, while children (<18 years) accounted for only 0.1% .During the 2022 winter flu season, 30,688 influenza vaccinations were administered to persons aged 59 (8.8) years, on average ; 69.8% were aged 60 years or older and 1.6% aged under 18 years, with 305 (1%) under age 12 years. 3.3A total of 6,500 physicians and nurses were credentialed via the Poste Italiane web site for administering vaccinations at the center. Physicians and nurses from eight community and research hospitals in the Milan metropolitan area, primary care providers, freelance healthcare providers, and volunteer professionals participated in the vaccine campaign.4The Palazzo delle Scintille MVC worked beyond capacity of the daily dose rate set by the Lombardy administration during December 2021 and early January 2022. User flow gradually decreased starting in February 2022, reflecting the general decline in adherence to the COVID-19 vaccination campaign in Lombardy. The center offered both influenza and COVID-19 vaccination during the 2021 and 2022 winter campaigns; the number of vaccinations was higher for October 2022 than for October 2021.Across the globe, MVCs responded adequately to the COVID-19 public health emergency , 18, 23 During peak user flow periods, people who had booked in advance were given priority and joined a separate queue at the entrance of the center for their appointment. This meant that the number of vaccination lines and sectors had to be adjusted as needed to meet demands within the center’s operating hours and doses available. Finding additional resources among doctors and nurses was the greatest challenge: the majority came to work at the center on their days off. They were paid for their time working at the center.The pandemic struck Lombardy more severely than elsewhere in Europe , 4. The The target daily dose rate at the center was 9,936. Between 4 December 2021 and 15 January 2022 the rate was above capacity probably because users could get a vaccination at the center on a first-come-first-served walk-in basis. The center was busiest when users came for additional vaccinations , 29 and Based on this model, users were categorized according to the dose they came to receive. Users who came to the center for their first or second dose or presented with major medical issues  underwent medical evaluation prior to vaccination by a physician, whereas the remaining users were administered their vaccination by a nurse. This reorganization reduced processing time in response to user experience and dissatisfaction with long waiting times , 30.The daily dose trend appears to partially follow that of SARS-CoV-2 positive cases reported for Lombardy. Starting from the opening of the center through to the deployment of services for booster vaccination during the COVID-19 vaccination campaign , paralleThe total number of influenza vaccinations at the center was lower for 2022 and 2023 than for 2021 and 2022, though more people were vaccinated in October 2022 than in 2021. The difference may be explained by the recommendations issued by the Ministry of Health to move forward to October that year’s influenza vaccination campaign . TogetheOver 7,000 persons were involved in keeping the center running. Clinical staff worked daily shifts of 12, 10 or 6 h depending on daily bookings and user frequency at 144 stations during full operation. Volunteers were vital in relieving clinical staff of nonclinical tasks such as directing use flow and providing help and assistance. The importance of engagement by nonclinical staff in the daily operation of a MVC resides in the increased need for recruiting clinical staff for patient management in hospitals and vaccination campaigns , 19, 41.4.1Set within a regional, national, and international context, the set up and management of the Palazzo delle Scintille COVID-19 MVC provides an example for emergency preparation in the prompt delivery of health and medical services. A limitation to the present study is that we were unable to collect adequate data on the human resources employed in the center’s daily operations; therefore, we cannot draw correlations between daily frequency and constraints on staff availability. Fast vaccination tracks were deployed during the period when the center operated above capacity in an effort to optimize workflow and respond to use dissatisfaction. Unfortunately, we were unable to measure the impact that the organizational model had on these variables due to the lack of data systems for tracking the number of resources involved and the vaccination waiting times.5Between December 2021 and January 2022 the Palazzo delle Scintille MVC operated above capacity set by the regional administration. User frequency reflected the evolution of regulations concerning COVID-19 vaccinations. The experience gained from setting up and managing the Palazzo delle Scintille MVC can inform the establishment of similar MVCs in other regions or countries.The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.JGC: Conceptualization, Data curation, Investigation, Software, Writing – original draft. AB: Conceptualization, Resources, Writing – review & editing. SP: Resources, Writing – review & editing. SG: Formal analysis, Methodology, Visualization, Writing – original draft. FB: Investigation, Writing – original draft. MB: Investigation, Writing – review & editing. LC: Resources, Supervision, Writing – review & editing. AP: Supervision, Writing – review & editing. NT: Supervision, Writing – review & editing. ML: Supervision, Writing – review & editing. SC: Writing – review & editing. MS: Supervision, Writing – review & editing. DL: Conceptualization, Resources, Supervision, Writing – review & editing.


IntroductionMigraine is characterized by persistent headaches and a wide range of symptoms, such as nausea, vomiting, and photophobia. The chance of developing a chronic migraine might be increased by lifestyle variables like obesity, stress, and excessive medication use. According to previous studies in Saudi Arabia, migraines are more common there than they are globally. The study aimed to examine the migraine associations with depression, anxiety, and stress in the population of Makkah City, Saudi Arabia.MethodsThe study employed a descriptive cross-sectional design with a non-probability snowball sampling technique and an online questionnaire that included sociodemographic characteristics, the International Classification of Headache Disorders-3 (ICHD-3) criteria for migraine assessment, and the Depression, Anxiety, and Stress Scale-21 (DASS-21) measure for depression, anxiety, and stress.ResultsOur study included 418 participants, out of whom 73.7% were female and 26.3% were male. Regarding migraine, only 8.9% of participants met the ICHD-3 criteria for migraine headache screening, with a female predominance (78.4%). The study showed a high prevalence of depression, anxiety, and stress among the population , with females having a higher prevalence. Depression, anxiety, and stress had an equal prevalence of 78.4% among migraineurs, which was significantly higher than that of non-migraineurs.ConclusionsThe study found significant associations between migraine and depression, anxiety, and stress. This study provides insights into the association between these conditions. The study's findings suggest the need for screening and management of mental health conditions in patients with migraine. However, extensive efforts are needed to be applied in different cities and demographics for a more precise understanding of the association. Migraine is a primary headache disorder characterized by recurrent attacks of mostly unilateral headaches that are frequently accompanied by nausea, vomiting, and light sensitivity . It is cAccording to the Global Burden of Disease (GBD), headache disorders are the most prevalent and disabling diseases worldwide. The global prevalence of active headache disorders was 52.0%, of which migraine accounted for 14.0% of these cases . The preIn a cross-sectional observational study conducted by Pearl et al. on 567 predominantly female (87.3%) migraine patients, they found a positive correlation between the patient’s Migraine Disability Assessment Scale (MIDAS) and their Patient Health Questionnaire 2 (PHQ-2) . AlQarniAnother study, conducted in Saudi Arabia, assessed 247 migraine patients aged between 16 and 45 years using the Depression Anxiety Stress Scale (DASS-21) questionnaire and found that 73.3% of the patients met the criteria for anxiety, while 70.9% and 72.3% of patients met the criteria for depression and stress, respectively . FurtherAs per the author's knowledge, this was the first study conducted to assess the relationship between migraine headaches and anxiety, depression, and stress in Makkah City, Saudi Arabia, while only a few studies have been conducted worldwide. Therefore, the study aimed to increase understanding of the association of migraine headaches with depression, anxiety, and stress among the population of Makkah City, Saudi Arabia, and to develop more effective strategies for managing these conditions in this specific cultural and environmental context.Study designThe study employed a descriptive cross-sectional study design with a non-probability snowball sampling technique as the sampling method. An online questionnaire (in Arabic) consisting of three parts was used for sociodemographic characteristics (Appendix). In addition, the International Classification of Headache Disorders (ICHD-3) criteria for migraine assessment and the DASS-21 measured the association between depression, anxiety, and stress in migraine patients ,17. The Study populationThe study's target population was general Arabic and English-speaking adults living in Makkah, Saudi Arabia. This study excluded participants who failed to complete the questionnaire, lived outside of Makkah City, and spoke neither Arabic nor English language.Sampling methodologyThe survey was conducted among the general public in Makkah City, Saudi Arabia, from February 26 to April 1, 2023. A written consent form was obtained from all participants before they filled out the questionnaire. According to OpenEpi version 3.1, a sample size of at least 384 participants was considered for a confidence interval level of 95%, an anticipated percentage of frequency of 50%, and a design effect of 1. The questionnaire consisted of three parts and was written in Arabic and English languages. The first part of the questionnaire included sociodemographic characteristics, such as age, gender, nationality, marital status, level of education, occupation, and income. The second part consisted of ICHD-3 migraine criteria. Following that, participants were asked to complete the third part of the questionnaire, which consists of DASS-21 to assess the presence of depression, anxiety, and stress. The anonymity of survey respondents was maintained, and their personal information, such as name, address, phone number, or email address, was not collected in electronic data collection forms; the data were automatically entered into an Excel spreadsheet. After verification and filtering, the data were transferred to BlueSky Statistics version 10.2.1  for analysis.Data analysisData were extracted, reviewed, coded, and entered into BlueSky Statistics version 10.2.1 statistical software. The results were presented as frequencies and percentages. Descriptive statistics were obtained for all sociodemographic variables, including participants' age in years, gender, nationality, marital status, education level, occupation, and family monthly income in Saudi Riyal (SAR), and analysis based on frequency and percent distribution was performed for these sociodemographic variables. Migraine prevalence was estimated using ICHD-3 criteria, and the symptoms' frequencies and percentages were plotted in a graph. As for DASS-21, frequency and percentage were tabulated for depression, anxiety, and stress with the following different levels: normal, mild, moderate, severe, and extremely severe for all participants. The chi-square test was used to assess the association between demographics and migraine, demographics and depression, anxiety and stress, and lastly, depression, anxiety, and stress with migraine. A significant association was determined by a p-value of <0.05.Ethical part and confidentialityConsent was obtained from each participant through the questionnaire. The aims of the research were stated in the questionnaire form for all participants. Additionally, all participants' identities were kept anonymous and confidential. The responses were only accessible to the investigators. Ethical approval was obtained from the Biomedical Ethics Committee of Umm Alqura University (UQU) (#HAPO-02-K-012-2023-02-1476).A total of 567 individuals participated in the study. One participant was excluded considering he was under the age of 18 years, and 148 were excluded because they lived outside of Makkah City, leaving the included sample size at 418. The sociodemographic data in Table Figure Depression prevalence results are presented in Table Table As shown in Table Table The prevalence of stress is shown in Table As indicated in Table The purpose of this cross-sectional study was to determine the relationship between migraine headaches and depression, anxiety, and stress in the Makkah City, Saudi Arabia, population. Our study findings revealed that only 8.9% of the study participants reported experiencing migraines. The study's migraine prevalence is lower than the global prevalence of 14% . This coThe number of participants who reported normal scores regarding depression was 151 (36.1%), 152 (36.4%), and 188 (45%) for anxiety and stress. A larger proportion of participants (63.9%) reported mild, moderate, severe, or extremely severe depression, which is also found to be an alarming increase in prevalence when compared with a previous 2021 study conducted in the city of Jeddah, Saudi Arabia, studying the prevalence of migraine and its effect on QoL among the general population that concluded a depression prevalence of 37.2% . The finOur study found that depression, anxiety, and stress were all significantly associated with migraine, with an equally high prevalence of 78.4% among migraineurs. This is consistent with a previous study conducted among migraine patients in Saudi Arabia, which found abnormal scores for depression (70.9%), anxiety (73.3%), and stress (72.3%) . AnotherThe bidirectional relationship between depression and migraine has been observed in previous studies, with depression being a strong predictor of the progression of migraine . HoweverThe concurrent presence of depression, anxiety, and stress with migraine can significantly affect the QoL of affected individuals. We recommend screening patients with migraines for the presence or development of these mental health conditions, as well as the need to manage them effectively. It is critical to prevent the development of anxiety disorders and depression in migraine patients, which can be accomplished by reducing the number of headache episodes with effective prophylactic pharmacotherapy. We also recommend that further studies be conducted to better understand the relationship between migraine and these mental health disorders in order to develop a more coordinated and direct approach to aid in the diagnosis and management of these conditions.LimitationsDespite making an effort to obtain accurate, precise, and representative outcomes, this study encountered certain limitations. Firstly, collecting data using an online questionnaire has inherent limitations. Second, the majority of the respondents were female (73.7%), Saudi (94.0%), and had a university-level education (76.1%). Another limitation is that the migraine prevalence in the study sample is relatively small (8.9%). These factors may have resulted in unintended biases in the findings. It is important to consider these limitations when interpreting the results of the study and to use caution when generalizing the findings to other populations or contexts. Nonetheless, this study provides insight into the current situation and confirms the outcomes of previous global and local research on the subject. The study used standardized measures to assess migraine headaches, depression, anxiety, and stress, which increases the reliability and validity of the study findings. More research in this area is required, using different methodologies, focusing on different sociodemographic characteristics, and exploring other unexplored regions of Saudi Arabia.Our study provides valuable insights into the association between migraine and depression, anxiety, and stress in the population of Makkah City. The findings highlight the importance of screening and effective management of mental health conditions in patients with migraine to improve their QoL, as the results showed a significant association between migraine and depression, anxiety, and stress. Further studies with a larger sample are warranted in different cities and demographics to better understand the relationship between these conditions and develop more effective interventions.


Pneumonia is a type of lung infection that can be caused by bacteria, viruses, or fungi. It is a serious condition that can affect people of all ages, but it is more dangerous for certain populations, such as the elderly, young children, and people with weakened immune systems. Pneumonia can cause higher risk in patients undergoing surgery, including C-sections. In this case report, we present a pregnant woman who was scheduled for a C-section due to preeclampsia and was initially suspected of concomitant pneumonia. The patient successfully underwent the C-section, but unfortunately experienced a deterioration in her pneumonia after the surgery. She was later admitted to ICU and placed on mechanical ventilation due to the deterioration. Despite the acknowledged risks, including the possibility of death, the patient's family chose to bring the patient home due to their belief that there had been no improvement in the patient's condition and a feeling of resignation. In conclusion, pregnant patients who have pneumonia may require an emergency C-section due to several conditions such as preeclampsia, and the C-section can be conducted successfully. However, it is important for physicians to be aware of the potential for the worsening of pneumonia postoperatively. Post-operative pneumonia is a serious condition that can have a significant impact on the health of patients who have undergone a C-section. Pneumonia is a serious lung infection that can affect all patients, including pregnant women. During pregnancy, the body undergoes many changes such as progesterone and estrogen elevations that can make women more susceptible to infections . The symPneumonia can be a serious concern for pregnant women, as it can lead to significant complications for both the mother and the baby . In seveIn this case report, we present a pregnant woman with suspected pneumonia who was scheduled for a C-section due to preeclampsia. The patient successfully underwent the C-section, but unfortunately experienced pneumonia after the surgery. The purpose of this report is to raise awareness of the potential challenges associated with C-sections in the presence of preeclampsia and suspected pneumonia.A 24-year-old pregnant woman with 38-39 weeks of pregnancy G1P0A0 presented to the emergency department on October 15, 2022 with a chief complaint of contractions 12 hours prior to presentation. Other symptoms from the patient included bilateral lower extremity edema. She denied any symptoms of fever, angina, dyspnea, cough, flu, and changes in bowels habit. The patient denied any contact with a positive COVID-19 patient, history of diabetes, heart failure, kidney disease, and liver disease. No similar symptoms were observed in her family. The patient was previously told that she was suspected of having preeclampsia.The patient was previously diagnosed with pregnancy-related hypertension, which was made around two months prior. She also had previously sought care at a public health center due to swelling in both her extremities. At the public health center, the possibility of preeclampsia was raised and she was referred to a hospital, but unfortunately, she did not follow up on this recommendation.Physical examination on October 15, 2022 showed abnormal vital signs with blood pressure 160/90 mmHg, respiratory rate 22x per minute, heart rate of 90 beats per minute, and temperature of 36.3 degree Celsius. Additionally, peripheral oxygen saturation was 99%. The patient was alert and oriented, with no signs of altered mental status. Upon lung auscultation, decreased breath sounds were heard in the lower right and left lung fields, though there was no evidence of rales or wheezing. The cardiac exam conducted at the emergency ward showed distant heart sounds. Meanwhile, the abdominal exam revealed the presence of contractions, and mild bilateral peripheral edema was observed in the lower extremities.3/µL. The aspartate transaminase (AST) and alanine transaminase (ALT) levels were within normal limits. The rapid COVID-19 antigen test was negative, and all other laboratory tests came back normal. A urine test was also conducted, and it revealed elevated urinary protein with positive 4  on October 15, 2022 showed bilateral pleural effusion, suspected cardiomegaly or pericardial effusion, and suspected mild bilateral lung infiltrate  and suspicion of severe preeclampsia was made. Furthermore, based on the x-ray, the patient was suspected of having pneumonia not related to COVID-19. Initial treatments of the patient were ceftriaxone 1 g every 12 hours, ¼ tablet of misoprostol applied to the posterior fornix, magnesium sulfate injection, nifedipine three times a day at a dose of 10 mg, and methyldopa one time a day at a dose of 250 mg.Due to the presence of severe preeclampsia, the patient was indicated to undergo a C-section procedure. On October 16, 2022, consultations were made with internal medicine and anaesthesiology to assess the patient's tolerance for the surgical procedure. After evaluating the patient, it was determined that the patient was fit to undergo the C-section, which was successfully completed. Post-operation, a complete blood count and other laboratory tests were conducted on October 16, 2022 Table .On October 17, the patient reported dyspnea, which started off mild but soon became severe despite oxygen supplementation. The dyspnea was persistent and not influenced by physical activity or changes in body position. The patient also developed symptoms of a cough and fever.Physical examination on October 17, 2022 showed abnormal vital signs with blood pressure 160/100 mmHg, respiratory rate 40x per minute, heart rate 160 beats per minute, and temperature of 38.3 degree Celsius. Additionally, oxygen saturation deteriorated to 70% room air. There was altered mental status. Lung auscultation revealed decreased vesicular sound on bilateral pulmonary areas. Rales were present during auscultation. Extremity examination showed presence bilateral peripheral edema.On October 17, 2022, another chest x-ray was performed. Comparison of this x-ray with the one taken on October 15, 2022, showed changes in the volume of pleural and pericardial effusions. Overall, the CXR showed bilateral pleural effusion, consolidation on right lung, hilar infiltrates, and presence of air bronchogram on bilateral lower lobes  of leucocyte, elevation of aspartate transaminase (AST) and normal alanine transaminase (ALT). Rapid test COVID-19 antigen was negative and other laboratory tests hyponatremia and hypochloremia. An arterial blood gas analysis was conducted on October 17, 2022, and the results are summarized in Table Complete blood count and other laboratory examinations were performed on October 17, 2022 Table . The patTo further evaluate the patient's condition, the patient was consulted with specialists in internal medicine, pulmonary medicine, and anesthesiology. Based on these consultations, additional treatments were recommended, including oxygen therapy at a flow rate of 15 liters per minute, monitoring of blood gas levels, re-checking of blood laboratory results, and transfer of the patient to the Intensive Care Unit. Furthermore, based on the latest examination data, it was decided to change the patient's treatment plan was adjusted to include meropenem 1 g intravenous, given three times a day, and supportive therapy, including intubation and mechanical ventilation through ventilator. The pulmonologists also recommended acetylcysteine 3x200 mg, and bronchodilator inhalation every eight hours.2 of 100%, and a respiratory rate of 16x/minute.Intubation procedure was conducted using midazolam 5 mg, fentanyl 100 microgram and rocuronium 50 mg post-intubation, physical examination still showed abnormal vital signs, including a blood pressure of 148/95 mmHg, a respiratory rate of 16 breaths per minute, a regular heart rate of 175 beats per minute, and a temperature of 36 degrees Celsius. The patient's peripheral oxygen saturation was 100% on a ventilator mode of synchronized intermittent mandatory ventilation (SIMV) with volume control (VC), tidal volume of 450mL, a ratio of inspiration:expiration of 1:2, positive end-expiratory pressure (PEEP) 5 mmHg, FiOLung auscultation revealed decreased vesicular sound in the right and left lung fields, with rales present. Cardiac auscultation showed distant heart sounds. Abdominal examination showed abdominal breathing. Examination of the extremities showed the presence of bilateral peripheral edema. The patient's diuresis was 1.6 cc/kg body weight per hour.On October 18, 2022, production of a red-black color in the nasogastric tube was observed. Thus, a diagnosis of stress ulcer was made. Thus, the patient was given additional bolus of omeprazole. Furthermore, the patient was given additional treatments, including furosemide, given intravenously two times a day, moxifloxacin, given once a day at a dose of 400 mg, tranexamic acid, given three times a day, bronchodilator inhalation, and a repeat complete blood count. The furosemide was recommended by O&G specialist for postpartum management of severe preeclampsia. However, the internal medicine doctor recommended delaying the administration of loop diuretics until the results of an echocardiogram examination were available.Based on the results of the complete blood count, a working diagnosis of anemia caused by upper gastrointestinal tract bleeding from stress ulcers was established Table . The int2 of 80%, and a frequency of 20. During lung auscultation, a decreased vesicular sound was heard in both the right and left lungs, and rales were present. The cardiac auscultation revealed a distant heart sound. The patient's diuresis was 2.9 cc/kg body weight per hour.On October 19, 2022, the patient's physical examination revealed abnormal vital signs with a blood pressure of 155/108 mmHg on the monitor, a respiratory rate of 27 breaths per minute, a heart rate of 102 beats per minute, and a temperature of 36.2°C. Additionally, the patient's oxygen saturation was 99% on a ventilator mode of SIMV VC, with a tidal volume of 450, an I:E ratio of 1:2, a PEEP of 10 mmHg, a FiOCardiac echocardiography was also performed the same day and the results showed normal ventricle volumes, systolic function, and diastolic function, as well as normal valves. The tricuspid annular plane systolic excursion (TAPSE) was measured at 2 cm, with an ejection fraction (EF) of 54%, normal heart valves, no evidence of thrombus, and no pericardial effusion.Based on the laboratory results, it appeared that the patient still had anemia and had developed acute renal failure Table . The lowOn October 20, 2022, the patient's family made the decision to voluntarily discharge the patient from the hospital and bring her home. Informed consent was obtained from the family. Despite the acknowledged risks, including the possibility of death, the patient's family chose to bring the patient home due to their belief that there had been no improvement in the patient's condition and a feeling of resignation.Pneumonia can have significant effects on a woman's ability to undergo a C-section surgery. Pneumonia is a serious lung infection that causes inflammation and fluid buildup in the lungs, resulting in impaired diffusion. This can be especially concerning in the context of surgery, where a patient needs to be healthy and able to breathe normally in order to undergo the procedure safely.One of the effects of pneumonia on a C-section is that it may delay or even prevent the surgery from taking place . If a woAdditionally, pneumonia can also increase the risks associated with C-section surgery -12. For A difficult issue arises when a patient needs a C-section for an emergency condition, but the patient has pneumonia. In our case, the patient had preeclampsia which was determined to need a C-section, but the presence of pneumonia complicated the case. Very little literature is available describing C-sections in patients with pneumonia. A case report by Lau et al. described an emergency C-section preterm pregnant woman with severe COVID-19 pneumonia and the patient was discharged later on due to improvement . In contPatients who have had a C-section are at an increased risk of developing pneumonia due to several factors ,15. The Our patient also had AKI post-C-section. Patients who have had a C-section are at an increased risk of developing AKI due to several factors, including decreased blood flow to the kidneys, and dehydration ,19. AddiLimitationThe limitations of this case report include the absence of initial hepatic function and kidney function tests. Our hospital lacked some necessary tests such as cholinesterase, and the kidney function test was delayed due to the insurance limitations of the patient. Arterial blood gas was not checked during admission since it was thought at the time there was no need for arterial blood gas due to a lack of dyspnoea symptoms. Additionally, the data on lactate and anion gap were unavailable.Pregnant patients who have pneumonia may require an emergency C-section due to several conditions such as preeclampsia and the C-section can be conducted successfully. However, it is important for physicians to be aware of the potential for the worsening of pneumonia postoperatively. Post-operative pneumonia is a serious condition that can have a significant impact on the health of patients who have undergone a C-section. Early diagnosis and prompt treatment are crucial to minimize the risk of complications and improve the chances of a positive outcome.


Background: The term "pulp stones" refers to conditions of calcification that can appear in any area of the dental pulp. This study aims to assess the prevalence of pulp stones and examine whether there is any connection between their occurrence and factors such as patient age, gender, tooth location, presence of decay, or dental restorations in a western Saudi subpopulation based on cone-beam computed tomography (CBCT).Methodology: 500 patients were randomly selected from the College of Dentistry, Taibah University, Madinah, Saudi Arabia, database. The study involved evaluating 2998 teeth in coronal, axial, and sagittal planes by four dental peer examiners observing and recording data  that included whether pulp stones were present or absent, the teeth group , tooth location (maxillary or mandibular), presence or absence of restoration, presence or absence of caries, and the patient's gender. Chi-square tests were utilized for statistical analysis, and a significance level of p-value ≤0.05 was established.Results: Out of 500 individuals , only 130 individuals (26%) and 278 teeth out of 2998 (9.2%) had pulp stones, with no significant statistical correlation identified between the presence of pulp stones and gender. (P = 0.459). Molars exhibited the greatest incidence of pulp stones , followed by anterior teeth , and premolars had the lowest prevalence . Teeth that showed a higher occurrence of pulp stones were associated with caries  and restorations . The association between pulpal stone and periodontal involvement was seldom significant (0.7%). A significant association was found between the presence of pulp stones and nonintact teeth (P<0.001). There was a statistically significant difference in the distribution of pulp stones between females and males in the maxillary and mandibular  (P = 0.002). Additionally, the frequency of pulp stones was statistically significant when comparing the left and right sides (P<0.001) .Conclusion: Understanding the prevalence and distribution of pulp stones is crucial for dentists and endodontists, as it assists practitioners in devising an appropriate treatment plan for affected teeth that require root canal therapy. One-fourth of the Madinah population was confirmed to have pulp stones, with a higher incidence in molars, caries, and restored teeth. No difference was found between its occurrence and gender. The high prevalence is exhibited in individuals between 45 and 54 years old. However, further studies with equal patient distribution are needed to confirm this observation. In modern dentistry, cone-beam computed tomography (CBCT) has emerged as a valuable diagnostic tool as it surpasses the restrictions of traditional radiographs by providing detailed anatomical information from multiple views, enabling dentists to diagnose and plan treatments more accurately. CBCT offers several significant benefits, including precision, reliability, high-quality resolution, and the ability to produce three-dimensional images without overlapping structures. Due to these advantages, CBCT is increasingly being used in dentistry; it appears that the usage of CBCT for clinical examination of pulp stones is about to become widespread .Pulp stones are areas of calcification that can be present in any region of the dental pulp, whether in primary or permanent teeth; they can also be located in intact, compromised, and potentially even in teeth that have not yet emerged ,3. DurinThe prevalence of pulp stones and their connection to diabetes mellitus and cardiovascular diseases in Saudi Arabia were investigated in a study. A total of 73 patients with cardiovascular disorders were included in the study, of which 24 (32.87%) had pulp stones. Similarly, out of 76 patients with diabetes mellitus, 18 (23.68%) had pulp stones . A studyThis study is a retrospective analysis performed on 500 CBCT images of Saudi patients  who pursued dental treatment for various reasons, such as missed canals, oral surgery, and dental implants. The study was conducted at Taibah University's College of Dentistry Radiology Department, Madinah, in the Western region of Saudi Arabia. Six age groups are included in the study: patients aged 18-24, 25-34, 35-44, 45-54, 55-64, and those older than 65. Images that were unclear or distorted, teeth that had received endodontic therapy, teeth with crowns or posts, root resorption, third molars, and deep carious lesions or restorations that obscured the pulp chamber were excluded. The final sample comprised 500 CBCT images, which were examined to determine the relationship between dental factors and pulp stones. The data observed and recorded includes pulp stone (presence or absence), teeth group , location of tooth (maxillary or mandibular), restoration (presence or absence), caries (presence or absence), and patient's gender. The CBCT images used in the study were obtained and examined at the Radiology Department of Taibah University's College of Dentistry using the CBCT machine (Carestream CS 9300) to obtain all CBCT scans utilized in this study. The parameters of the machine were set to 90 kVp, 4 mA, and 8 seconds of acquisition time for the "Medium Patient" set. For the "Large Patient" set, the settings were 90 kVp, 5 mA, and 8 seconds. When the CBCT was for mandible only, the settings were 90 kVp, 5 mA, and 8 seconds of acquisition time.The data were transferred from the Excel sheet to IBM Corp. Released 2019. IBM SPSS Statistics for Windows, Version 26.0. Armonk, NY: IBM Corp for analysis. Descriptive analyses were carried out to report sample characteristics. Quantitative variables were analyzed as frequencies and percentages. A nonparametric (chi-square) test was used to compare the occurrence of pulp stones with sex, tooth types, associated factors, tooth location, tooth position, and teeth group. The level of significance was set at a p-value ≤0.05.Descriptive analyses were carried out to report sample characteristics. Quantitative variables were analyzed as frequencies and percentages. A nonparametric (chi-square) test was used to compare the occurrence of pulp stones with sex, teeth types, associated factors, tooth location, tooth position, and teeth group. The level of significance was set at a p-value ≤0.05.Among the 500 individuals, 130 were identified as having pulp stones, representing 26%. In addition, one or more pulp stones were identified in 278 (9.2%) teeth through the CBCT examination. Sixty-three (25.2%) males and 67 (26.8) females had pulp stones, with no significant association between gender and the presence of pulp stones (P = 0.459). The pulp stones in the teeth with carious teeth were 74 (26.6%), teeth with restoration were 58 (20.9%), and the least frequent were in teeth affected by periodontal disease (2 (0.7%)). The distribution of pulp stones according to gender is presented in Table The study revealed a significant association between the presence of pulp stones and teeth that were not intact (P<0.001). According to the findings, there is no significant variation between males and females with respect to other risk factors (P=0.470) Table .According to the study, pulp stones were mostly found in molars 238, 85.6%), followed by anterior teeth , and uncommon in premolars   compared to the mandibular teeth , with a significant statistical dissimilarity between females and males (P=0.002). Finally, the cases of pulp stones on the left and right sides showed almost even distribution 136, 48.9%) and , respectively . However, no significant differences were found between males and females (P = 0.289).The prevalence of teeth with pulp stones with age shows that 6.83% of patients aged 18-24 have pulp stones. This also increased in patients aged 25-34 to 10.87%. The highest prevalence of pulp stones was found in the age group range of 45-54; 12.34% of patients had pulp stones. There was a statistically significant difference between the age and the number of teeth with pulp stones (P = 0.003). However, no significant differences were found between males and females (P = 0.471)  in the teeth of a Saudi Arabian population using bitewing radiographs . HamashaIn our findings, pulp stones were found more frequently in females than males. However, this finding is inconsistent with the results of earlier investigations conducted by Hamasha and Darwazeh  and BaghThe pulp stone increases with the patient's age, and some studies indicated that 90% of patients older than 51 have pulp stones. However, in our study, the prevalence of pulp stones in patients older than 51 can be much lower than in most previous studies . This diThe limitation of the current study is that the result did not represent the whole population of Saudi Arabia because it was based on only one center. In order to ensure more reliable results, it is recommended that further studies involve multiple centers. The sample size was more extensive than some of the referred studies; however, a larger sample size is needed to obtain more comprehensive and accurate data. The equal distribution of the sample with age is necessary to ensure an equal distribution of the sample across different age groups.Understanding the prevalence and distribution of pulp stones is crucial for dentists and endodontists, as it assists practitioners in devising an appropriate treatment plan for affected teeth that require root canal therapy. One-fourth of the Madinah population was confirmed to have pulp stones, with a higher incidence in molars, caries, and restored teeth. No difference was found between its occurrence and gender. The high prevalence is exhibited in individuals between 45 and 54 years old. However, further studies with equal patient distribution are needed to confirm this observation.


Dental implant artifacts can compromise the quality of cone-beam computed tomography (CBCT) scans and challenge radiographic detection in surrounding regions. This literature review was conducted to examine the impact of implant angle modification on reducing metal artifacts in CBCT scans.  A scoping review of literature was carried out in PubMed, Embase, Scopus, and Cochrane databases.  Different spatial planes, including alpha, beta, gamma, and phi, along with 0°, 5.2°, 9.8°, 14.5°, 15°, 30°, 45°, 60°, 75°, and 90° angles were studied. Changes in the angular position of implants may reduce metal artifacts and improve the quality of CBCT scans.  Rotating implants within the alpha plane and angling them at 90° in the alpha plane enables reducing dental implant artifacts. Presence of artifacts, namely, spurious visual structures in cone-beam computed tomography (CBCT), can result in impaired image quality and detectability of radiographic details , 2. InteWith the developing use of dental implants as the most common treatment form replacing lost teeth, CBCT has become the standard radiography for thorough pre- and post-operative evaluation of implants and surrounding anatomic structures. However, presence of metal artifacts degrades image quality and hinders assessment of adjacent structures . SeveralGiven the scarce conclusive data regarding the plausible impact of changes in the angular position of implant while scanning on reducing metal artifacts in CBCT scans, this review study was carried out and an effort was made to answer the question whether changing the angular position of implant leads to metal artifact reduction or not.As mentioned, metal artifacts decrease CBCT image quality and prevent evaluation of adjacent structures , 33, 34,The scoping review question was, “How does the modification of the angular position of implants can reduce metal artifact in CBCT scans?”This scoping review is an evaluation of current literature, aiming to assess the evidence and detect existing gaps. A literature search was independently conducted by two individuals on PubMed, Embase, Scopus, and Cochrane databases from October 2022 until June 2023. The search strategy was  with no additional filters. It should be noted that employed keywords were selected based on the MeSH terminology. The competency criteria were collected based on our research question.Inclusion criteria entailed (1) availability of article full text and (2) investigation of the impact of modified angular position of implants on artifact reduction in CBCT scans. We excluded the studies based on the following exclusion criteria: (1) if they were failed to investigate the effect of angle change on reducing the artifacts caused by implants and (2) publication solely in non-English language.The papers were gathered by performing an advanced search on the databases. To fulfil this goal, the keywords were separated with the Boolean operator “AND.” The search was completed using a manual search method. The gathered articles were screened three times. In the first screening, the databases were checked for copies of each paper. In the second screening, the papers were investigated based on their title and abstract. In this case, the papers that were not related to the topic were removed. In the third screening, the full texts of papers were reviewed and the screening process was carried out based on the exclusion criteria. After that, the chosen papers were synthesized according to their qualities. Then, the data of each chosen paper were extracted, including author's name, publication year, implant placement environment, number of implants, width of implants, length of implants, center-to-center distance between implants, anatomical region of the implants, material and brand of the implants, angles, exposure time, voltage, beam currents, study analysis, results, and conclusions. Thematic analysis was employed to analyze the collected data. This analysis is usually used to determine the themes of different topics, analysis, exegesis, description, and conclusion in a systematic review . ArticleThe purpose of this study was to review the impacts of changes in implant angular position on reducing metal artifacts in CBCT images. Reviewed articles were selected from the following databases: PubMed, Embase, Scopus, and Cochrane. The PRISMA-ScR flowchart showing the scoping review process is shown in The effect of various factors on artifacts in CBCT images has been investigated, including metal artifact reduction algorithm (MAR), implant material, voltage, implant position, voxel size, tube current, and field of view (FOV) , 36–39.Many studies have observed MAR to be effective in reducing implant-induced artifacts , 29, 32,Evaluation of the effect of implant material on the number of artifacts has revealed that artifacts resulting from zirconium and titanium implant to hold the highest and least number of artifacts, respectively. There was a moderate chance of the creation of artifacts associated with titanium-zirconium implants , 30, 32.Considering the voltage factor, all studies stated that an increase in kVp limited metal artifacts , 29, 32.Various studies have been conducted on implant position. Machado et al. postulated that artifacts appear more frequently in the mandible than maxilla, and artifacts appear more in the anterior areas than that of the posterior areas . AccordiRegarding the voxel size, previous studies have shown that smaller voxel size caused increased artifact production, while larger voxel size decreased the number of artifacts , 31. TheBased on  the quanIn previous studies, the effect of “changes in the angular position of implants” on the reduction of metal artifacts was examined , 33, 34,In Min and Kim's study in 2021, two dental implants were placed in a homogeneous dental impression material block. The block was scanned with a CBCT scanner at seven different angles  along three different planes. Rotation in the frontal plane was called alpha rotation. The second rotation in the sagittal plane was called beta rotation. The simultaneous rotation in the frontal and sagittal planes was called gamma rotation. Thirteen volumes of interest (VOI) were selected from each axial reconstruction. Gray values (GVs) of each VOIs were measured. Mean differences in GV between the control area and VOIs were calculated. These ΔGVs from different spatial angle were compared and analyzed by Welch's analysis of variance and linear regression. According to the research, increases in alpha angle increase ΔGVs of groups A and B but fail to significantly change group C. Furthermore, any notable changes in ΔGVs were observed for gamma rotation in all VOIs. The study concluded that an increase in alpha angle can reduce interimplant metal artifacts in CBCT images. Therefore, modification of the angle of the patient's head may lead to the reduction of metal artifacts [Z test. Results manifested that for VOI1 and VOI2, as the alpha angle increases, the correlation coefficient increases; however, for VOI3, the correlation coefficient decreases with increasing alpha angle. Therefore, modifying the alpha angle can modify CBCT image quality [The authors in  performe quality .α angle. Since the overlap of highly X-ray absorbing implants may reduce by tilting the jaw, tilting the lower jaw with dental implants can reduce artifacts in CBCT images [In Luckow et al.'s study in 2011, two dental implants were placed in the mandible of a pig, and CBCT was performed based on varying the starting rotation angle of the mandible in the source-detector plane, beam current, accelerating voltage, and angles of the jaw with respect to the source-detector plane. In this research, rotations were performed in three different planes. These rotations were called alpha and beta rotations, respectively, which were performed in the frontal and sagittal planes. Simultaneous rotation in the frontal and sagittal planes was called phi rotation. In alpha and beta rotations, 0°, 5.2°, 9.8°, and 14.5° angles, and phi rotation, 0°, 30°, 45°, and 90° angles were examined. The different datasets were automatically registered with respect to micro-CT data to extract the common volume and the deviance to the predefined standard that characterizes the image quality. The research showed that image quality can be improved by increasing the T images .In the three mentioned articles, the materials of the implants were the same and they were made of titanium. Also, quantification of artifacts was carried out in three articles and the method of measuring artifacts was quantitative , 33, 34.Based on the conclusion of three articles included in this study, among all the introduced plans , alpha plane rotation was the most effective in artifact reduction and an increased alpha angle reduced the number of metal artifacts , 33, 34.Given the small number of papers, as well as the laboratory nature of the studies, it is not possible to highly extend the conclusions to clinical works and more studies in this field are required; besides, more evidence must be collected and clinical research should be carried out.