Membrane filtration is a key technology in dairy processing for the separation of dairy liquids to clarify, concentrate, and fractionate a variety of dairy products. Ultrafiltration (UF) is widely applied for whey separation, protein concentration and standardization, and lactose-free milk production, though its performance can be hindered by membrane fouling. As an automated cleaning process commonly used in the food and beverage industries, cleaning in place (CIP) uses large amounts of water, chemicals, and energy, resulting in significant environmental impacts. This study introduced micron-scale air-filled bubbles  with mean diameters smaller than 5 μm into cleaning liquids to clean a pilot-scale UF system. During the UF of model milk for concentration, cake formation was identified as the dominant membrane fouling mechanism. The MB-assisted CIP process was conducted at two bubble number densities  and two flow rates (130 and 190 L/min). For all the cleaning conditions tested, MB addition largely increased the membrane flux recovery by 31–72%; however, the effects of bubble density and flow rate were insignificant. Alkaline wash was found to be the main step in removing proteinaceous foulant from the UF membrane, though MBs did not show a significant effect on the removal due to the operational uncertainty of the pilot-scale system. The environmental benefits of MB incorporation were quantified by a comparative life cycle assessment and the results indicated that MB-assisted CIP had up to 37% lower environmental impact than control CIP. This is the first study incorporating MBs into a full CIP cycle at the pilot scale and proving their effectiveness in enhancing membrane cleaning. This novel CIP process can help reduce water and energy use in dairy processing and improve the environmental sustainability of the dairy industry. Brucella, Campylobacter, Cryptosporidium, E. coli, Listeria, and Salmonella, and can spread diseases such as tuberculosis and typhus [Milk is a complete food that contains high-quality protein and is an excellent source of calcium and lactose. However, raw milk can carry pathogenic bacteria, including d typhus . Since td typhus .Membrane technology has revolutionized the dairy industry since the 1970s . UltrafiMembrane cleaning is an essential step to maintain membrane performance, increase membrane life span, and reduce the costs of membrane replacement ,5. CleanEscherichia coli and Salmonella Typhimurium on leafy vegetables [Recently, small air bubbles with diameters less than 50 µm, also known as microbubbles (MBs), have distinct properties from millimeter-sized bubbles, including long residence time in liquid, large surface-to-volume ratio, high gas dissolution rate, and generation of high energy and free radicals when they burst ,16. MBs getables , enhancigetables , and intgetables . Due to getables , biofilmgetables , food oigetables , foulinggetables , and biogetables .The objective of this study was to evaluate the effect of MBs on the performance of the CIP operation for a pilot-scale UF system used for milk concentration. Operating parameters studied included the MB number density and flow rate of the cleaning liquid. Moreover, a comparative life cycle assessment (LCA) was performed to quantify the environmental benefits of incorporation of MBs into CIP operation. The results showed that MB-incorporation significantly improved the cleaning performance, in terms of membrane flux recovery, by up to 72%, which led to an up to 37% reduction in the environmental impacts associated with CIP operation.2, two restriction valves , a three-way valve (V.3), two pressure gauges , a flowmeter (F), and a permeate tank. Fifty gallons of model milk were prepared by reconstituting whole milk powder  to a concentration of 0.5% w/v in RO water. The concentration process was conducted by pumping the model milk through the UF system at a flow rate of 130 L/min for 40 min, and the transmembrane pressure (TMP) was controlled at 30 psi. The membrane was conditioned for 30 min with RO water at a flow rate of 130 L/min and TMP of 20 psi before each concentration experiment.Milk concentration was performed using a pilot-scale UF system, as shown in MBs were infused into cleaning liquids using a Nikuni regenerative turbine pump KTM20  where air bubbles are generated via breakup by flow turbulence and vortices. The number of bubbles in the liquid was controlled by air injection, and two air flow rates (5 and 10 L/min) were tested. The number density and mean size of MBs in water were determined using a particle counter . MB-infused alkaline and acid solutions were not characterized in this study due to the limitation of the particle counter which cannot be operated with alkaline and acid solutions, according to the manufacturer.A 7-step CIP process was performed to clean the UF membrane fouled during model milk concentration. After the full CIP process, the cleanliness of the membrane was determined by its water permeate flux, measured at a TMP of 20 psi and a flow rate of 130 L/min over 10 min. The cleaning performance was evaluated based on the recovery of membrane flux, namely the ratio of water permeate flux of the cleaned membrane (tion (1) .(1)Memb3 + 0.2% H3PO4, 55 °C, 20 min), alkaline reconditioning , and final rinse . Between the intermediate rinse and acid wash steps, an additional enzymatic cleaning step using 0.5% Filzym 131  at 43 °C for 45 min) followed by a water rinse step  were applied. Furthermore, a disinfecting step using a 0.05% peroxyacetic-based sanitizer  at 32 °C for 20 min was applied after every three cleaning tests, as recommended by the membrane manufacturer.Repeated concentration-cleaning testing was performed using the same membrane throughout this study. Therefore, to minimize the foulant left on the membrane after every cleaning test, the same CIP process was conducted again but with increased time, temperature, and/or chemical concentration, comprising prerinse , alkaline wash , intermediate rinse , acid wash  − v, which correspond to cake formation, standard blocking, and intermediate blocking mechanisms, respectively [Membrane fouling by the milk model solution was characterized using the modified Hermia’s model (Equations (2) and (3); ).(2)dJdectively .Membrane fouling during the UF of milk is mostly due to the precipitation of microorganisms, proteins, fats, and minerals . To bettAll experiments were performed in triplicate. Data are expressed as mean ± standard deviation and were statistically analyzed by the one-way analysis of variance (ANOVA) followed by Tukey’s test to compare the means of different test groups, with a significance level of 0.05. All statistical analyses were performed using the SAS 9.4 software .In addition to production-based metrics , the effThe goal of this LCA study is to compare the environmental performance of a new CIP method, based on the incorporation of MBs, to that of a conventional CIP process for a pilot-scale UF system employed for whole milk concentration. The results are intended to provide food manufacturers and cleaning system designers and operators with quantitative indicators of the environmental benefits of the new MB-assisted cleaning technology.The simplified concentrated milk production was analyzed from gate to gate, including the concentration and CIP processes. The upstream stages  and milk packaging were not considered. The functional unit (FU) of this comparative LCA was defined as one production cycle that included a 40-min concentration process operated under the UF condition described in The life cycle inventory (LCI) included the resources required for one concentrated milk production cycle, such as milk powder, water, and chemicals, as well as the utilities for pumping and heating. The foreground inventory data were collected from pilot tests, and all the background data including electricity, natural gas, and productions of milk powder and chemicals were adapted from the ecoinvent database v3.0 . ProcessThe environmental impacts of one production cycle were determined using the ReCiPe 2016 Midpoint (E) v1.02 method. This method characterizes eighteen midpoint indicators, including global warming, stratospheric ozone depletion, ionizing radiation, ozone formation, human health, fine particulate matter formation, ozone formation, terrestrial ecosystems, terrestrial acidification, freshwater eutrophication, marine eutrophication, terrestrial ecotoxicity, freshwater ecotoxicity, marine ecotoxicity, human carcinogenic toxicity, human noncarcinogenic toxicity, land use, mineral resource scarcity, fossil resource scarcity, and water consumption. All calculations were conducted using the SimaPro software v8.0.2/h after the first 5 min for initial conditioning then gradually decreased to 28.8 L/m2/h over the rest of the process, as shown in v) and filtration time (t) was measured by the coefficient of determination (R2). Cake formation  was identified as the dominant fouling mechanism of milk concentration in this study, followed by standard blocking (R2 = 0.90) and intermediate blocking (R2 = 0.87). Cake formation is a reversible type of fouling with a layer of foulant loosely attaching to the membrane surface that can be effectively removed by physical cleaning [During the 40-min concentration of model milk, the membrane flux reached 35.1 L/mormation b showingcleaning . Ref. [4cleaning .The MBs generated in water showed a unimodal narrow size distribution. The flow rate of air injection into the Nikuni pump had an insignificant effect on the mean MB diameter, of 4.51 μm for 5 L/min and 4.26 µm for 10 L/min. However, increasing the air flow rate was found to markedly decrease the MB number density, from 10,569 bubbles/mL for 5 L/min to 2021 bubbles/mL for 10 L/min. This could be due to a larger volume of air infused into the water tends to induce the formation of more millimeter-sized bubbles that can rise quickly and burst at the water’s surface.MBs have been reported to aid deposit removal by increasing momentum transfer due to bubble scrubbing  as well Due to the large variation in the protein concentrations of effluents collected from repeated experiments, the amounts of protein removed by the control and MB-assisted CIP processes showed no significant difference for all the cleaning conditions tested. This was primarily caused by the operational uncertainty of the pilot-scale UF system. Specifically, it is difficult to fully drain the cleaning liquid from the membrane housing after each cleaning step, hence the remaining residue is expected to affect the concentration of protein in the cleaning effluent of the following steps. Furthermore, for the MBCA protein assay used for protein concentration measurement, only 1 mL was sampled from the 50-gallon cleaning effluent for each analysis; therefore, a great spatial variation in the protein concentration across such a large volume of cleaning effluent can be expected. MBs have proved effective in removing proteins from microfiltration membrane by pyrolytic decomposition, which was characterized by temperature increases due to adiabatic compression when MBs collapsed . Other sFat has also been found to foul on the membrane during UF of whole milk ; howeverElectricity use was considerably responsible for most of the impacts since coal is the dominant source of electricity generation (88%) in Indiana. Coal-fired thermal power plants release a number of toxins and pollutants into the air, rivers, streams, and lakes, including sulfur dioxide, nitrogen oxide, particulate matter, and mercury . The steThe present study, for the first time, incorporated MBs into a full CIP cycle and investigated their effect on the cleaning of a pilot-scale UF system used for milk concentration under different bubble number densities and flow rates of cleaning liquids. The model milk tested was found to foul on the UF membrane mainly in the form of cake formation. Compared to the control CIP process, MB-assisted CIP showed a higher membrane flux recovery by up to 72%; however, increasing bubble density and flow rate did not show further improvement in this study. Despite the enhanced flux recovery, MBs had an insignificant effect on the amount of protein removed from the UF membrane and no fat removal was detected, which was mainly due to the operational uncertainty of the large-scale CIP process. The LCA on the simplified concentrated milk production revealed that the CIP process predominated most of the environmental impacts, which were mainly contributed by electricity use. Incorporation of MBs into the CIP process can reduce the impact by up to 37%. This study proved that MBs can be easily introduced into conventional CIP operations without major intervention in the existing processing equipment, which is a promising approach to increasing the efficiency of spiral membrane cleaning. Therefore, MB-assisted CIP has great potential for reducing the production costs and environmental footprint of filtered dairy products, including, not only high-protein milk but also whey protein isolate, casein, lactose, etc. Since the interaction between MBs and the milk foulant in the spiral membrane is still unclear, in situ real-time visualization is needed to provide further information about the cleaning mechanism of MB-assisted CIP. Moreover, characterizing the flow field over the spiral membrane with the presence of MBs by computational fluid simulations can help quantify the contact of MBs with foulant and the resulting forces exerted.


Also, the sensor is analytically suitablewith regard to reversibility, resilience, and reproducibility. Thesensing device is validated through a relatively high number of on-bodytests performed with elite athletes cycling and kayaking in controlledenvironments. Correlation outcomes between sweat lactate and otherphysiological indicators typically accessible in sports laboratories are also presented and discussed in relation to the sportperformance monitoring capability of continuous sweat lactate.The chemical digitalizationof sweat using wearable sensinginterfacesis an attractive alternative to traditional blood-based protocolsin sports. Although sweat lactate has been claimed to be a relevantbiomarker in sports, an analytically validated wearable system toprove that has not yet been developed. We present a fully integratedsweat lactate sensing system applicable to in situ perspiration analysis.The device can be conveniently worn in the skin to monitor real-timesweat lactate during sports, such as cycling and kayaking. The noveltyof the system is threefold: advanced microfluidics design for sweatcollection and analysis, an analytically validated lactate biosensorbased on a rational design of an outer diffusion-limiting membrane,and an integrated circuit for signal processing with a custom smartphoneapplication. The sensor covering the range expected for lactate insweat (1–20 mM), with appropriate sensitivity (−12.5± 0.53 nA mM Moreover, thisstrategy provides discrete-type data; hence, it may lose valuablephysiological information and prevent rapid feedback to the athletes.A less invasive approach allowing for continuous and real-time lactatemonitoring is in high demand by sports physiology scientists, athletes,and coaches.Over the past few decades, lactatehas become an important analytical target in several fields, includingthe food industry, clinical diagnostics, and sport science.3 Despite recent studies concluding that sweatlactate is a strong candidate to replace blood analysis for sportsperformance evaluation,5 this remains controversial. Inessence, until the time of writing, all conclusions about sweat lactatehave been drawn from data generated in very distinct sample-basedapproaches, which are subject to significant errors.6 Thus, studies have shown contradictory tendencies for sweatlactate: it increases or decreases with activity evolution, and theexistence and absence of correlation with blood lactate.Sweat offers a promising alternative to blood fordaily analysisperformed on the sports field. Sweat is naturally generated duringany physical activity, collection is non-invasive, and it containsabundant biomarkers related to body conditions, such as lactate.10 Promphet et al. have developed a colorimetric textile-based lactatesensor for on-body measurements.7 The sensoris simple, low cost, and easily fabricated and showed an acceptablelinear response range (LRR) from 1 to 25 mM. However, neither thevalidation of on-body tests nor the effect of sweat accumulation inthe calculated lactate levels was addressed (the device is not a microfluidicone). In addition, the analysis was discrete rather than continuousand required a camera and a portable spectrophotometer. Chen et al.have proposed a flexible electrochemiluminescence platform with arange of response up to 20 mM.11 This sensorwas successfully applied in two on-body tests, and some discrete valueswere obtained. Nevertheless, the degradation and/or leaching of theluminophore material during long-term measurements is a concern, asit may lead to inaccurate results over time.Severallactate sensors have been reported in the literature attemptingto provide trusted on-body data for physiological evidence, includingcolorimetric, electrochemiluminescent, and electrochemical readouts.15 These sensors primarily rely on enzymes, such as lactate oxidase,19 in combination with redox mediators ,14 and employ amperometry for detection. Importantly, most of thesesensors are sensitive to physiological variables and have stability issues, which require correction before the finallactate levels can be provided.14 Potentiometry20 and electrochemical impedance spectroscopy21 have been proposed to avoid using a redox mediator;conductive polymers have been used to develop non-enzymatic sensors;20 and the protective layers or polymeric membranes22 have been addedto improve the overall stability and lifetime. Recently, our grouphas demonstrated that using lipophilic outer membranes significantlyenhances not only the stability but also the LRR of enzymatic lactatebiosensors.23Electrochemicalsensors are one of the most powerful analyticaltools for continuous monitoring of sweat lactate, not only in termsof simplicity, low cost, portability, and easy miniaturization butalso in their analytical performance.24 Thedevice was tested on healthy volunteers engaged in running and demonstratedsuitable agreement with standard analytical techniques. Table S1 in the Supporting Information summarizesthe characteristics of some electrochemical sensors for sweat lactatedetection in terms of analytical performance, level of integration,sampling strategy, validation methodology, physiological evaluation,and the number of on-body assays. As observed, there have been importantadvances in the field, but none of the reported devices reached theanalytical and technological readiness needed for full and reliableapplication in the sports domain. Consequently, sweat lactate sensorshave grown in popularity and importance in the scientific literature,while trustworthy commercial solutions remain in the early stagesof development.In recent studies, thedevelopment of sweat lactate electrochemicalsensors has been combined with advanced sampling approaches  and the integration of electronic hardware andsoftware. For example, Komkova et al. have reported an enzyme-basedlactate biosensor equipped with a capillary casing, as the microfluidicssystem, and wireless electronics.In this paper, we present a fully integratedconcept for a wearablesweat lactate detection device, which is a clear step forward in demonstratingthe significance of wearable sensors and sweat assessment in real-lifescenarios. The concept’s design is based on the combinationof a disposable electrochemical lactate biosensor, a microfluidicsystem for sample collection encompassing perspiration, an electronicboard for wireless data acquisition and processing, and a user-friendlymobile application. On the one hand, the biosensor mechanism permitsaccurate and continuous lactate monitoring in sweat during the entiresports practice period. On the other hand, the reliability and robustnessof the developed system allowed it to be used for sports physiologystudies across different sports disciplines (cycling and kayaking),demonstrating great potential not only within the sports science domainbut also as a possible commercial solution.Figure S1 in the Supporting Information.The system 1a consisThe lactate biosensor comprises a three-electrodesystem  manufactured in a flexible polymericsubstrate. The electronic circuit (designed with KidCAD) applies afixed potential of −0.033 V to the working electrode with respectto the reference (Ag/AgCl electrode) while measuring the current betweenthe working and counter electrodes. An amperometry circuitry readsthe electrochemical sensor, and the microcontroller unit (MCU) isrequired to initiate and communicate with the amperometry circuitry.A built-in Bluetooth low energy transceiver controlled by the MCUwas used to communicate with the custom mobile application.Table S2).All measurements weredone in sports laboratories at an ambient temperature of 20 °Cand 40% relative humidity. Parameters such as height, weight, gender,and the targeted body part were annotated before performing each test.These are provided in the Supporting Information  were monitored through a metabolic chart in mixingchamber mode .The cycling intensity and power output were recorded using a cycleergometer . Before datacollection, each participant performed a 9 min warm-up on the ergometerexercise bike. The warm-up consisted of cycling for 3 min at threedifferent cycling intensities . Then,the training consisted of four 15 min rounds at different intensities,with ca. 2 min of rest between each round. Every 5 min, the subjectwas asked for a Borg scale number to describe their feeling of exhaustion. Blood samples were collected during each restingperiod for lactate and glucose analysis. Sweat samples were also collectedduring the resting periods to validate the lactate measurements providedby the new device.In the cycling tests at Dalarna University(Sweden), the heart rate, VOMax-power tests in cycling and kayaking werecarried out at theSwedish Sport Confederation Performance Laboratory .In this case, blood, sweat, and power output information were recorded.For cycling, a 20 min warm-up and a 20 min self-paced maximal sustainableeffort were performed on a magnetic brake cycle ergometer . Blood samples were collected before and after the warm-upand after finishing the test. The kayaker performed a testing protocolconsisting of paddling on four intensity levels (4 min for each level)and a final self-paced maximal sustainable power step (4 min) usinga kayak ergometer . Blood samples werecollected within 1 min after each step.The fully integratedwearable device for sweat lactate monitoringdeveloped in this paper is presented in The system-level block diagram of the device is given in Supporting Information.The biosensing element follows the “first-generation”concept and consists of three primary parts: the redox mediator, theenzyme, and the external diffusion-limiting layer. The working mechanismis illustrated in Thedevice can be placed  on any partof the body without causing discomfort to the subject while practicingsports. –1, LOD of 0.12 mM, and interceptof −61.7 ± 7.43 nA. Importantly, the biosensor responseincluded the typical concentration interval of sweat lactate.26The electroanalytical performanceof the lactate biosensor was evaluatedin vitro in both batch and flow modes. For the batch mode, the experimentswere accomplished in artificial sweat at a constant stirring rateof 150 rpm. Figure S2 in the Supporting Information). TheLRR was well-maintained (1–20 mM), as well as the slope  and LOD(0.2 mM). The biggest difference was observed in the intercept , which is intrinsic to the variabilityin the fabrication process. Accordingly, each biosensor will requirepre-calibration for further on-body tests.Sensor-to-sensor reproducibility was evaluatedfrom the calibrationgraphs obtained with three identically prepared sensors . The lactate sensor showed a negligible response to thesecompounds, which makes it directly applicable to sweat lactate measurementswithout any potential interference. Moreover, the resilience of thebiosensor was evaluated by inspecting the calibration curves obtainedbefore and after bending the system 10 and 30 times to 45° . Conveniently, the slope and interceptwere only slightly modified after this strong torsion strain, withRSDs of 2.7 and 12.0 after 10 and 30 bends, respectively. Accordingly,the biosensor response is not expected to be affected by subject movementsduring sports.Interference wasassessed by registering the response of the biosensorin 5 mM lactate and then adding 0.1 mM uric acid, 0.1 mM pyruvate,0.1 mM ascorbic acid, or 0.25 mM glucose, which are above typicalconcentrations of these compounds in sweat . Four calibrations were conducted at 22 °C, witha successive decrease in the pH of the solution: 8.1, 6.9, 5.4, and4.3. Then, another three calibrations were performed at a constantpH of 8.1 and varied temperatures: 22, 29, and 36.5 °C. The pHfluctuation caused an overall variation in the slope and sensitivityof ca. 8%, whereas for the temperature, the variations were 10 and23% for the slope and intercept, respectively. On the one hand, thevariation connected to pH changes is much lower than those previouslyreported for other enzyme-based sensors .28 This improvement is likely attributedto the external layer added to the lactate biosensor, which limitsthe number of protons reaching the enzyme from the sample, as suggestedpreviously.9The effect of pH and temperature on the sensorperformance wasalso investigated . However, theRSD for the intercept was >10%, which can be associated with theinherentvariations between electrodes (see above). In any case, the responsebegan to deteriorate 30 days after fabrication.The lifetime of thebiosensor was assessed using a set of fivesimilarly prepared sensors, calibrating each one several days afterbeing prepared: 7, 12, 14, 18, and 29 days. The sensors were storedat 4 °C until they were tested. The five calibration graphs arepresented in –2 min–1).29 Flow ratesadjusted from 0 to 12.5 μL min–1 emulate sweatrates from 0 to 13.7 μL cm–2 min–1 in the microfluidic channel when the area of the microfluidic channel(0.9 cm2) is considered. Thus, a solution containing 5mM lactate was introduced to the cell through a peristaltic pump at10 μL min–1, the flow was maintained, andthe current was developed. Once the steady-state current was reached,the pump provided different flow rates, as shown in Consideringthat the goal of the lactate biosensor is a wearable device for continuouslactate monitoring, the sensor was implemented into the sampling cellto evaluate the performance under flow conditions. This was achievedby connecting a peristaltic pump to the inlet of the cell. Becausethe perspiration rate may vary during physical activity and differencesbetween individuals are expected, the effect of the flow rate on thesensor response was investigated within a range that mimics commonfluctuations in perspiration . Two lactate concentrations within the LRR were investigated,and drifts of 3.3 and 6 nA hectively 3b. The r–1 using artificial sweat as the background. A typicalprofile of the dynamic current at increasing lactate concentrationsand the corresponding calibration graph are depicted in Before each on-body test, the device was calibrated in flow modeusing a peristaltic pump with a constant flow rate of 5 μL minTable S2 in the Supporting Information).Each participant was required to cycle for ca. 76 min divided into10 min of warm-up and four 15 min periods of different intensities(each period followed by 2 min of rest). In parallel to the sweatlactate measurements, sweat and blood samples were collected. Lactatewas determined from all the samples, and glucose was only determinedfrom the blood samples. Also, exhaustion level ,power, heart rate, VO2, and respiratory exchange ratiowere monitored.On-body tests were performedwith the device positioned on two different body parts 4a: four Figure S7 in the Supporting Information. Thesweat lactate was 4.3–5.2 times higher than the blood lactatein zone 2, 2.7–3.7 times higher in zone 3, and 6.2–8.9times higher in the decreasing blood lactate zone.In subject #1 4b, afterIn subject#2 4c, afterFigure S8), when the measurements weretaken on the back (subjects #3–5), sweat lactate tended tobe either almost constant or decreased when the blood lactate increased,coinciding with the trends shown for subject #1. After reaching thelevel of perspiration needed to obtain the measurements from the device,subject #3 presented a lower perspiration level than the rest. Becausethis individual showed almost constant sweat lactate, while the restshowed decreasing lactate, a hypothesis may be drawn. In principle,significant changes in sweat lactate may not be observed by targetinga relatively passive body zone, such as the back, during cycling.Decreasing sweat lactate coinciding with increasing activity intensity(and thus blood lactate) was not expected a priori. Thus, there maybe a dilution of the real (and relatively constant) sweat lactateconcentration caused by high perspiration levels in the individual.This effect has been suggested previously.23Consideringthe lactate profiles observed in the remaining subjects. Thus, all the conclusions drawn in this paper canbe considered valid.More detailed descriptionsand interpretationsof the outcomesobserved for each subject are provided in Table S3 in the Supporting Information.In parallel with the real-time, continuous, and on-bodysweat lactate monitoring using the wearable device, sweat sampleswere collected every 5 min from a body position very close to thedevice. The lactate contents in the samples were analyzed by ion chromatography(IC) and then compared with the discrete values averaged over periodsof 15 min (coinciding with the sampling) from the continuous dataprovided by the wearable device. Although the collected sweat volumewas insufficient to be analyzed by IC in some cases, we assessed atotal of 18 sweat samples. The results are shown in Consideringa cut-off value of 20% for the difference between both techniques,80% of the samples agreed with the calculated sweat lactate, confirmingthe reliability of the measurements. Importantly, significant differencesmay arise from comparing continuous data to discrete profiles, andto address this, we had to use averaged results from the dynamic lactateprofiles, which may induce some errors.t-test was used to estimatewhether the average difference between the results from the wearabledevice and IC is null considering a confidence interval of 95%. Avalue of the t-score (0.47) lower than the criticaltheoretical value (2.11) was obtained, indicating no statistical differencesbetween the results obtained from the two analytical methods. Theboxplot of the difference values , and all thepossible correlations were analyzed. Notably, while we only consideredsweat lactate measurements from the thigh (subjects #2 and #3–6),all the data  were used for the rest of theparameters.The Borg scale,an indicator of the perceived exertion of the body, was selected tounderstand whether sweat lactate can be used as a proxy for sportsperformance. Borg scale grading commonly differs among subjects facinganalogous physical activity owing to different body conditions. Itis a measurement of how hard the subject feels the body working, andit is based on the physical sensations experienced during physicalactivity, which normally coincide with increasing heart rate, respiration(or breathing rate), perspiration, and muscle fatigue; this latteris connected to blood lactate.2, and respiratory quotient) are shownin Figure S9 in the Supporting Information.Importantly, as expected, the perceived exhaustion positively correlatedwith the heart rate (cutoff of 0.6 for a subjective variable). Thisoccurred for the entire pool of samples (n = 70)and when analyzing the data for each subject (Table S5). Indeed, the ratio between the heart rate and theBorg scale was always close to 10 (Table S6), confirming the reliability of the data.32 The respiratory quotient was also found to positively correlatewith the Borg scale in connection with increasing anaerobic respirationand the main metabolism of proteins .33 Once more, this was observed for the entirepool of samples and each subject (Table S7). No clear correlations were found for power, blood glucose, orVO2 .Correlations found for the sweat and blood lactatewith the Borg scale are presented in Tables S8 and S9).Regardinglactate, as shown in In summary, we found promising positivecorrelations between thesweat lactate measured on the thigh with blood lactate, perceivedexertion, heart rate, and the respiratory quotient. The device developedin this study can be used to precisely measure sweat lactate and relatethis to personalized sports performance via the perceived exertionlevel and the heart rate, two parameters that are easy to measure,accessible to professionals and amateurs, and that can be incorporatedinto the software of the device developed in this paper. However,we believe that back measurements could also be targeted upon dilutioncorrection based on additional measurements with a sweat rate sensor.To demonstrate theversatility of the developed system, we explored its use in elitekayakers and cyclists during submaximal and maximal self-paced tests.In kayaking, the upper body musculature plays a critical role, incontrast to cycling, where lower limb musculature consists of thepropulsive muscles. The subject of the kayaking test accomplished fourintensity steps,increasing in power (89–133 watts), followed by 1 min restperiods, before performing the maximal power period. The device startedthe lactate measurements after ca. 5 min. In the warm-up, a clear increase of sweat lactateup to 16.7 mM was observed from 5 to 10 min, followed by a gradualdecrease to 14.3 mM, coinciding with the middle part of the 118 watt-step.This concentration was constant until the end of the step. Then, asimilar trend was found in the 133 watt-step: an initial increaseto 16.4 mM, maintained until the middle of the step, a gradual decreaseto 13.8 mM, and maintenance of this concentration until the end ofthe warm-up. Before the maximal power step, the sweat lactate decreasedto 11.2 mM, suddenly increased to 15.7 mM after some minutes, andremained constant. Interestingly, this latter trend coincided withthe second blood LT. Advantageously, the device was able to monitorchanges in the sweat lactate that may be related to the performanceof the active muscles in the measured body area.The subjectof the cycling test accomplished a free warm-up (powerbetween 120 and 180 watts), 20 min of maximal power exercise (intensityranging from 229 to 400 watts), and a final cool-down period (60 wattsfor 5 min). A free warm-up protocol in which the participants mainlycontrolled the workout power was used. The device started the lactatemeasurements from ca. 28 min, longer than for the kayaker becausekayaking is a whole-body exercise, and thus, more sweating is expected.Hence, the lactate was monitored only during the 20 min self-pacedmaximal sustainable cycling step. The device detected an initial increasein sweat lactate to 4.5 mM, which was constant for 4.5 min beforeincreasing again to 5.4 mM. This concentration was constant for someminutes before rising to 8.4 mM; then, it remained constant untilthe end of the maximal power activity. This last increase seems tocoincide with the second LT. During the calm-down period, sweat lactatewas found to gradually decrease until it reached basal levels.On comparing both activities, cycling revealed three lactate increasesduring the maximal power step in contrast to only one in kayaking.In any case, the final increase coincided with the second blood LT.Both sweat and blood lactate levels were higher in the kayaker thanin the cyclist. For sweat lactate, this could be caused by using amore active muscle area in the kayaker than in the cyclist. Overall,the device provided excellent performance in both cases.Wearable sensors to digitize sweat lactateare positioned as afeasible alternative to traditional blood test protocols. The deviceherein developed demonstrated not only this but also the significanceof sweat lactate in sports performance monitoring. Advantageously,our fully integrated system combines an exhaustive analytical characterizationand rigorous accuracy with outcomes from different on-body cases.The device features an advanced microfluidics design for efficientsweat collection and analysis, lactate biosensors that reversiblycover the expected range for sweat lactate levels with a relativelyfast response time, an integrated electronic circuit for signal processing,and a custom smartphone application. The lactate biosensor responseis negligibly influenced by changes in pH, temperature, flow rate,and certain chemical interferences. Importantly, in cycling, the deviceprovided more engaging results on the thigh than on the back: thisshows the difference between active and passive muscles. We founda positive correlation between the sweat lactate measured from thethigh and blood lactate, perceived exhaustion , heart rate, and the respiratory quotient. Moreover, the lactatemeasurement device is also suitable for monitoring maximal power kayakingand cycling. In terms of commercialization, challenging aspects suchas lifetime, sweat rate correction, and physiological protocols focusedon sweat lactate rather than blood may be tackled.


In the development of bioanalytical LC-MS methods for the determination of drugs in plasma samples in a clinical setting, adequate sample preparation is of utmost importance. The main goals are to achieve the selective extraction of the analytes of interest and attain thorough matrix removal while retaining acceptable ecological properties, cost-effectiveness, and high throughput. Solid-phase extraction (SPE) offers a versatile range of options, from the selection of an appropriate sorbent to the optimisation of the washing and elution conditions. In this work, the first SPE method for the simultaneous extraction of six anticancer drugs used in novel therapeutic combinations for advanced breast cancer treatment—palbociclib, ribociclib, abemaciclib, anastrozole, letrozole, and fulvestrant—was developed. The following sorbent chemistries were tested: octylsilyl (C8), octadecylsilyl (C18), hydrophilic–lipophilic balance (HLB), mixed-mode cation-exchange (MCX and X-C), and mixed-mode weak cation-exchange (WCX), with different corresponding elution solvents. The samples were analysed using LC-MS/MS, with a phenyl column . The best extraction recoveries (≥92.3%) of all analytes were obtained with the C8 phase, using methanol as the elution solvent. The optimised method was validated in the clinically relevant ranges, showing adequate precision (inter-day RSD ≤ 14.3%) and accuracy (inter-day bias −12.7–13.5%). Finally, its applicability was successfully proven by the analysis of samples from breast cancer patients. Palbociclib (PAL), ribociclib (RIB), and abemaciclib (ABE) are newly registered anticancer drugs, inhibitors of the cyclin D-dependent kinases 4 and 6 (CDKi). They are prescribed for the treatment of hormone receptor-positive and human epidermal growth factor 2-negative  breast cancer, in synergistic combinations with endocrine therapy (ET): aromatase inhibitors anastrozole (ANA) or letrozole (LET), or an oestrogen receptor antagonist fulvestrant (FUL). It has been shown that, of all HR+, HER2− patients in recent years 2018–2022), about 70–80% were prescribed with CDKi + ET, while chemotherapy was used in about 15% and ET alone in about 10% of the cases 8–2022, a.The six drugs of interest have a wide array of physical-chemical properties, with the CDKi being weak bases, aromatase inhibitors being neutral and relatively hydrophilic, and FUL being highly lipophilic ,5. Thus,Solid-phase extraction (SPE) is a popular option for the preparation of various types of samples, including human plasma—the most commonly used sample in TDM. Its selectivity, purification efficacy, versatility, and ease of automation are among the most prominent advantages that make it suitable for complex bioanalytical sample preparation .The most common commercially available SPE sorbents are either silica- or polymer-based, with different functional groups and retention mechanisms, such as reversed-phase sorbents similar to chromatographic stationary phases—C18, C8, phenyl, etc.; polymeric hydrophilic–lipophilic balance (HLB) sorbents, comprised of polar pyrrolidone and non-polar divinylbenzene; and ion-exchange sorbents containing ionisable groups, such as the sulfonic and carboxylic groups for cation exchange, or amines for anion exchange ,8,9. To 4OH in MeOH [Thus far, several bioanalytical methods for the determination of ABE, PAL, RIB, ANA, LET, and FUL in human plasma samples have been reported. Most of the methods were developed only for selected combinations of the analytes, with three works focused on all six drugs of interest, applying protein precipitation (PPT) and dispersive liquid–liquid microextraction (DLLME) for sample preparation ,11,12. F in MeOH . The str in MeOH ,27. FuncTo the best of our knowledge, there is no reported protocol for SPE of FUL or a combination of the CDKi with the aromatase inhibitors or FUL from any type of sample thus far. Therefore, the aim of this work was to achieve simultaneous extraction and cleanup with satisfactory extraction yields of all six analytes of interest on a single SPE sorbent. Several different SPE sorbents were tested: reversed-phase: octylsilyl , octadecylsilyl , and hydrophilic–lipophilic balance ; ion-exchange sorbents: mixed-mode cation-exchange , and weak cation-exchange .Reversed-phase sorbents were selected as the most widely applicable sorbents suitable for various types of compounds. Cation-exchange and mixed-mode cation-exchange sorbents were tested due to the basic nature of some of the analytes. Some of these sorbents have already been applied in the literature, but only for the extraction of up to two of the mutually similar drugs of interest. In this phase, all samples were analysed using high-performance liquid chromatography equipped with a diode array and fluorescence detection (HPLC-DAD-FLD). This technique was found suitable for the preliminary experiments due to its simplicity, relatively low cost of operation, and the ability to observe the chromatographic profile of the leftover plasma interferences, thus enabling an easier and broader evaluation of the sample cleanup efficiency.The extraction recoveries were calculated as the ratio of peak areas in spiked plasma samples and standard solutions of the same concentration and are shown in figures as the median and range. Two spiked samples and one blank plasma sample were prepared for each tested condition. Blank sample chromatograms were inspected for matrix interferences at the retention times of the analytes.The structures of the tested reversed-phase and HLB sorbents, with the tentatively highlighted groups responsible for potential intermolecular interactions, are shown in For the experiments, plasma samples were diluted with Milli-Q water in the volume ratio of 4:5. The sorbent-conditioning step included loading with MeOH and Milli-Q water, as per the manufacturer’s instructions . After tThe results are presented in On the C8 sorbent, significantly higher recoveries of all the analytes for all tested conditions except for ACN were observed b. It is Increasing volumes of MeOH were also tested on the C8 sorbent to assess if greater recoveries could be obtained. As discernible from 4OH in MeOH, whereas solvent combinations including HCOOH in MeOH and/or only MeOH, proved favourable, which is also supported by the literature [In contrast to the silica-based C8 and C18 sorbents, Oasis HLB is a polymeric sorbent comprised of vinylpyrrolidone and divinylbenzene groups that enable both hydrophilic, dipole–dipole interactions and hydrophobic, especially π-π interactions, with the compounds from the sample . It is tterature ,25. ANA As shown in 3PO4 and Milli-Q water at a ratio of 5:1:5 . It is meant for the extraction of stronger bases from the sample, assuming they remain protonated at higher pH values of the alkaline washing solvent. Ion exchange occurs when eluting with an acidic eluent and the protons from the solvent switch place with the analytes . HoweverTo determine the best extraction conditions for all six analytes of interest, the most favourable conditions for each sorbent were mutually compared, as summarised in 4OH in MeOH also showed potential for the extraction of the combinations of only CDKi and aromatase inhibitors. However, SPE with a Sep-Pak Vac C8 column, 200 mg/3 mL, eluted with 2 × 750 µL MeOH, proved optimal for all six of the drugs of interest. This method was therefore transferred to LC-MS/MS, as described in As can be seen, the extraction recoveries of FUL were the poorest in most of the tested conditions, except for the C8 eluted with MeOH and the HLB eluted with a combination of MeOH and HCOOH in MeOH. For the other analytes, superior or similar extraction efficacies were achieved with the C8 eluted with MeOH and also with narrower error bars than in some other cases. The C18 sorbent eluted with HCOOH in MeOH and the X-C eluted with a combination of MeOH and NHThe following validation parameters were assessed for the newly developed SPE-LC-MS/MS method: linearity, calibration range, accuracy, precision, selectivity, carry-over, and matrix effects. The stability of the samples at the same storage and working conditions as used in this work has already been confirmed . x2. At least 75% (six) of the calibration standard levels met the criteria that the accuracies of the back-calculated concentrations were within ±15% of the nominal values. The calibration results are summarised in Fresh calibration curves were prepared each time analyte quantitation was planned. All calibration curves were weighted by 1/Accuracy and precision were assessed at three quality control (QC) concentration levels across the linear range: lower limit of quantitation (LLOQ), low, and high, on 10 samples per concentration level within one day and a total of 15 samples per concentration level between days. They were found to be acceptable at all concentration levels . The results are shown in Selectivity was assessed during the method development by reviewing chromatograms of blank plasma samples. The blank chromatogram obtained with the optimised C8-SPE method  showed nThe presence of carry-over was checked by injecting blank samples after the highest calibration concentrations. A slight carry-over of RIB and PAL was observed. An additional blank injection was introduced for the needle and column cleanup, and the needle was washed for 100 s with 50% MeOH after each injection. Thus, the carry-over of RIB and PAL was reduced to less than 3% of the LLOQ .The matrix effects were tested at two QC concentration levels (low and high) in triplicate on plasma samples from six different individuals, including a haemolysed and lipemic plasma, and calculated according to Equation (1):The results are depicted in It should be noted that extensive signal instability was also observed in the case of FUL, which sometimes manifested in a nonlinear response. This phenomenon is attributed to the drug’s high lipophilicity, low ionisation efficiency in the positive ESI mode, and high susceptibility to ionisation effects from any leftover plasma interferences accumulated at the LC-MS interface. Regular, thorough rinsing of the spray shield with 50% i-propanol alleviated the issue to a certain extent; however, the introduction of an isotopically labelled analogue as the internal standard is highly recommended in the case of routine applications in clinical practice. In this work, the main goal was to achieve successful simultaneous extraction of the analytes from plasma samples using SPE and identify potential pitfalls for the method’s application. Further addressing the MS issues and routine application of the method are outside the scope of this paper but will be the subject of our future efforts.To prove the applicability of the method on real patient samples, plasma from four breast cancer patients treated with CDKi + ET combinations was prepared using the developed procedure and quantitated according to a fresh calibration curve. Each of the analytes was present in at least one of the patients. The results were as follows: patient 1: RIB 981.7 ng/mL, ANA 41.7 ng/mL; patient 2: ABE 276.4 ng/mL, LET 69.8 ng/mL; patient 3: PAL 74.2 ng/mL, FUL 21.0 ng/mL; patient 4: PAL 119.3 ng/mL, FUL 14.2 ng/mL. All determined concentrations are within the method’s validated linear ranges as well as within the expected concentration ranges of these drugs in patient plasma ,34,35,36Key parameters of the proposed method and the relevant previously published works are shown in In relation to other methods for these six breast cancer drugs, where samples are prepared using less selective PPT or labour-intensive DLLME ,11,12, t3PO4 (85%) was purchased from T.T.T. ; NH4OH (25%) and Na-Ac were from Alkaloid ; and HCl (37%) was from VWR . Ultrapure water was obtained using a Merck Millipore Milli-Q IQ 7015 system . Analytical grade standards (purity > 97%) of PAL, RIB, and ABE were from Toronto Research Chemicals ; ANA and LET were from Tokyo Chemical Industry ; and FUL was from MilliporeSigma .MeOH for the HPLC and MS, as well as ACN for the HPLC, were purchased from J.T. Baker ; HCOOH for the LC-MS was purchased from Supelco ; H2O:ACN 50:50 v/v. These solutions were mixed and diluted in MeOH to obtain working solutions of the appropriate concentrations. The concentrations of the calibrants used in the method validation are shown in The primary stock solutions of RIB, ABE, ANA, LET, and FUL were prepared to the concentration of 1 mg/mL in MeOH. The primary stock solution of PAL was prepared to the concentration of 225 µg/mL in H2-EDTA anticoagulant. After centrifugation at 1500× g for 10 min, the supernatant was stored short-term at −18 °C and long-term at −80 °C. The plasma was thawed at room temperature for 30 min before any experiments were conducted. Research approvals were obtained from the Ethics Committee of the University of Zagreb Faculty of Pharmacy and Biochemistry  and the Ethics Committee of University Hospital Centre Zagreb .Blood from the patients treated with the drugs of interest, as well as drug-free blood from healthy volunteers, was collected in vials with the KThe tested extraction phases, Oasis MCX 30 mg/1 mL, Oasis HLB 60 mg/3 mL, Oasis WCX 60 mg/3 mL, Sep-Pak Vac C18 200 mg/3 mL, Sep-Pak Vac C8 200 mg/3 mL, and Sep-Pak Vac C8 500 mg/3 mL, were obtained from Waters , while the Strata-X-C 60 mg/3 mL was from Phenomenex . v/v and the samples were diluted with an appropriate solvent. The extractions were carried out using a vacuum manifold  equipped with a vacuum pump. A known volume of the sample eluate  was evaporated to dryness on a vacuum concentrator  and reconstituted in 80 µL of 65% MeOH. Once prepared, the samples were kept on the autosampler at 10 °C for no longer than 10 h.Before commencing with the SPE procedure, blank plasma samples were spiked with a working analyte standard solution at the ratio of plasma:standard 9:1 A Waters XBridge BEH phenyl column, with the dimensions of 150 × 4.6 mm and particle size 2.5 µm, with a corresponding VanGuard phenyl guard column , was used as the stationary phase, thermostated at 35 °C. Ultrapure water (phase A) and MeOH (phase B), both containing 0.1% HCOOH, were used as the mobile phase in the gradient elution. The initial experiments were performed on an Agilent 1100 HPLC equipped with DAD and FLD . The flow rate was set to 0.8 mL/min, and 10 µL of the sample was injected. The applied mobile phase gradient is shown in The method was then transferred to an Agilent 1290 Infinity II UHPLC coupled to a 6470 triple-quadrupole mass spectrometer (QQQ-MS) equipped with an Agilent Jet Stream electrospray (AJS-ESI) source  to achieve the necessary detection sensitivity. With the same stationary and mobile phases, the flow rate was adjusted to 0.6 mL/min using the gradient elution described in In the preliminary studies, the analytes were detected using DAD on 360 nm for PAL, RIB, and ABE, together with FLD at a 212 nm excitation (Ex) and a 310 nm emission (Em) for ANA, LET, and FUL, as previously reported . The AJSData were collected using the Agilent Chemstation 10 software  for the Agilent 1100 HPLC, and the Agilent MassHunter Workstation 10.0 software was used for the data acquisition and qualitative analysis  for the Agilent 1290 UHPLC. Data analyses were performed using Microsoft 365 Excel  and GraphPad Prism 8 .In recent years, the importance of TDM of anticancer drugs has been an emerging topic. An increasing number of bioanalytical methods are being reported in search of fulfilling the first precondition for TDM testing: the development of a reliable procedure for the quantitative determination of drugs-candidates in biological matrices suitable for the busy clinical setting. SPE is currently one of the most common sample preparation techniques due to its versatility. It is simple and fast once optimised; however, its implementation requires extensive method development. In this work, different extraction conditions were tested  for the extraction of six breast cancer drugs, palbociclib, ribociclib, abemaciclib, anastrozole, letrozole, and fulvestrant, from human plasma samples.4OH in MeOH show potential for combinations of only CDKi and aromatase inhibitors. These findings, backed by the detailed analysis of the potential interaction mechanisms, may also be a useful starting point for the development of SPE methods for other structurally similar drugs of interest.Of all the tested conditions, the Sep-Pak Vac C8 (200 mg/3 mL) sorbent eluted with MeOH shows the best overall extraction yields and cleanup for all six drugs of interest, while the Sep-Pak Vac C18 (200 mg/3 mL) eluted with HCOOH in MeOH and the Strata X-C (60 mg/3 mL) eluted with a combination of MeOH and NHThe proposed C8-SPE-LC-MS/MS method is simple and fast, ecologically acceptable, precise, accurate, and sensitive, with adequate, clinically relevant linear ranges. Potential pitfalls in the method development and its application are acknowledged, such as the high levels of interference, regardless of the acceptable extraction yields in the case of the MCX and WCX sorbents, or the observed matrix effects with the C8 sorbent for some of the analytes. This can be avoided in routine clinical settings with regular and thorough ion source rinsing and by using isotopically labelled internal standards. Carry-over was observed during the method development and was resolved with additional column rinsing. Nevertheless, the method’s feasibility is established by its application on four samples from patients treated with the drugs of interest. To the best of our knowledge, this is the first SPE method for the simultaneous extraction of ABE, RIB, PAL, ANA, LET, and FUL from plasma samples, demonstrating satisfactory sensitivity, selectivity, and precision, together with exceedingly high recoveries (≥ 92.3%).


Yucca genus encompasses about 50 species native to North America. Species within the Yucca genus have been used in traditional medicine to treat pathologies related to inflammation. Despite its historical use and the popular notion of its antioxidant and anti-inflammatory properties, there is a limited amount of research on this genus. To better understand these properties, this work aimed to analyze phytochemical composition through documentary research. This will provide a better understanding of the molecules and the mechanisms of action that confer such antioxidant and anti-inflammatory properties. About 92 phytochemicals present within the genus have reported antioxidant or anti-inflammatory effects. It has been suggested that the antioxidant and anti-inflammatory properties are mainly generated through its free radical scavenging activity, the inhibition of arachidonic acid metabolism, the decrease in TNF-α (Tumor necrosis factor-α), IL-6 (Interleukin-6), iNOS (Inducible nitric oxide synthase), and IL-1β (Interleukin 1β) concentration, the increase of GPx (Glutathione peroxidase), CAT , and SOD (Superoxide dismutase) concentration, and the inhibition of the MAPK (Mitogen-Activated Protein Kinase), and NF-κB (Nuclear factor kappa B), and the activation of the Nrf2 (Nuclear factor erythroid 2–related factor) signaling pathway. These studies provide evidence of its use in traditional medicine against pathologies related to inflammation. However, more models and studies are needed to properly understand the activity of most plants within the genus, its potency, and the feasibility of its use to help manage or treat chronic inflammation.The  Yucca genus belongs to the Agavoideae subfamily, a subfamily that is commonly used in traditional medicine thanks to its anti-inflammatory, antimicrobial, and antiparasitic properties [Yucca species in various products, especially in dietary supplements [The operties . It encooperties . These bplements . These bplements .Yucca’s most notable properties is its anti-inflammatory activity. Inflammation is a physiological procedure generated by the immune system in response to tissue injury, stress, pathogens, or toxic compounds [One of ompounds . Howeverompounds , and in ompounds . Oxidatiompounds ; for theompounds .Yucca species, as they are popularly used to treat arthritis since they counteract some effects of this disease. All of the biological properties of plants are due to the high concentration of phytochemicals [Treatment against these diseases is usually anti-inflammatory. Unfortunately, there are some drug-induced side effects that make some treatments inadequate , and thehemicals . HoweverTherefore, the objective of this work is to research the phytochemical composition of the species and the anti-inflammatory and antioxidant properties of these molecules. The literature was explored during the period from 2000 to 2022, although the oldest papers were used for the historical context. In this way, it will be possible to know the possible molecules responsible for these effects and their mechanisms of action.Yucca species are native from North and Central America [Yucca moths, where the moths provide Yucca with pollen while using the flower to oviposit [ America , and the America . The genoviposit . Hybridioviposit .Yucca genus is composed of about 50 species. In particular, “The Plant List” establishes 49 species with an “accepted” status [The literature indicates that the ” status . Althoug” status .Yucca species have been used by natives for many purposes. Some species, such as Yucca schidigera Roezl ex Ortgies, were used for bowstrings, nets, ropes, mats, sandals, and clothing [Yucca elephantipes Regel) at Easter [Yucca glauca Nutt to stimulate hair growth, and in skin conditions [Since ancient times, clothing . It is at Easter . Most imnditions . New Mexnditions . There anditions ,16.These ethnobotanical uses have led to the emergence of research on their biological activities. Starting in 1975, it was tested as a treatment to manage arthritis . Ever siYucca schidigera Roezl ex Ortgies. Platelets are specialized blood cells that play an important role in inflammation. Platelets are capable of upregulating leukocyte functions and releasing proinflammatory cytokines [Yucca schidigera Roezl ex Ortgies significantly decreases the various steps of thrombin-platelet activation [Yucca schidigera Roezl ex Ortgies inhibits COX-1 and COX-2 (Cyclooxygenase-2) in vitro [Despite historical ethnobotanical use, its continuous mention in the literature, and the popular notion of its anti-inflammatory properties, a minimal number of current studies have explored these properties. The most mentioned activity in the literature is the anti-platelet effect of ytokines . It has tivation . The plativation . As can tivation . Then, tin vitro . This isYucca gloriosa L., specifically its inhibitory potential against Ovalbumin-Induced Airway Hyperresponsiveness in mice [Yucca gloriosa L. was administered orally at doses of 50, 100, or 200 mg/kg for 7 days 1 h before each sensitization with ovalbumin. Pretreatment with Yucca gloriosa L. significantly decreased the concentrations of TNF-α, IL-6, interleukin-13 (IL-13), and leucocyte count.A similar effect to that reported for various phytochemicals found within the genus, as can be seen in Yucca gloriosa L. against asthma.The anti-inflammatory effect of  in mice  has alsoSpecifically, the decrease in the TNF-α concentration is the effect with the highest incidence within these compounds. This is expected due to the key role that it plays in inflammation. TNF-α is released by a wide variety of immune cells so that it can bind to its receptors and activate different pathways. Once TNF-α has bound to its receptor, it promotes the formation of a complex capable of activating IκBα kinase (IKK) . Once acAnother of the effects with a high incidence is a decrease in the concentration of IL-6. IL-6 exerts its activity by binding to the IL-6 receptor, thereby activating membrane-bound gp130. This causes JAK enzymes to phosphorylate gp130, thus generating docking sites for proteins, such as STAT3, to bind to and be phosphorylated by JAK enzymes [Yucca gloriosa L. also decreased the concentration of the oxidative markers nitric oxide (NO), myeloperoxidase (MPO), and malonaldehyde (MDA) [In addition to these proinflammatory mediators, treatment with de (MDA) . As can de (MDA) . NO actsde (MDA) . Althougde (MDA) . The maide (MDA) . Howeverde (MDA) . Excesside (MDA) . The abide (MDA) . There aIn addition to NO reduction, many phytochemicals also reported a significant capacity for reducing the concentration of MDA and MPO, known biomarkers of oxidative stress. MPO is a key enzyme in the elimination of pathogens within phagolysosomes of neutrophils, since it generates powerful oxidizing species, such as hypochlorous acid (HOCl), from the catalysis of the reaction of chloride with hydrogen peroxide . HOCl caYucca genus against oxidative stress. A commercially available food additive known as Sarsaponin 30® has been reported to have a protective effect against nitrite-induced oxidative stress in rats [® for 4 weeks prior to the nitrite intoxication in doses of 100 ppm. Said pretreatment reduced the concentrations of MDA and NO in the tissue and in glutathione  and O2, so that subsequently CAT or GPx catalyzes the reduction of H2O2 to water, thus eliminating the oxidative danger of these molecules [Another case with fish was with t status . This isolecules . It shouYucca extracts, the main pathways involved in its anti-inflammatory and antioxidant effect are the inhibition of MAPK, and NF-κB, and the activation of the Nrf2 signaling pathway.As it can be seen through The NF-κB is a family of transcription factors that coordinate one of the most common proinflammatory signaling pathways. Within the phytochemicals in The canonical NF-kB pathway is primarily a response to proinflammatory cytokines, such as TNF-α and IL-1, and it has an important role in chronic inflammatory diseases . The patThe non-canonical NF-kB pathway begins with the activation of the TNFR superfamily members, or the formation of an endosome complex containing NIK, AKT, and MAC, to stabilize and accumulate NIK. The NIK (NF-kB inducing kinase) with IKKα will induce the phosphorylation of the precursor of p52, resulting in the formation of RelB/p52 dimer .Yucca genus that have shown the capacity to suppress this signaling pathway.The MAPK superfamily is one of the major mechanisms used in signaling pathways and is characterized by its activation through the dual phosphorylation on adjacent threonine and tyrosine residues . In inflYucca genus can reduce p38 phosphorylation and inhibit the signaling pathway.The p38 MAPK pathway begins the activation of receptors, such as toll-like receptors, TNFR, or the IL1R superfamily, to inflammatory stimuli. This activation generates the phosphorylation of TRAF 2/3/6 (TNF receptor-associated factor), which in turn activates MAP3Ks, such as TAK1. Then, MAP3K phosphorylates MKK3 or MKK6, and those molecules activate p38 . Many ofThere are 3 types of JNK proteins JNK1 (encoded by MAPK8), JNK2 (encoded by MAPK9), and JNK3 (encoded by MAPK10), where JNK1 and JNK2 are found in almost all cells . This siNrf2 is a transcription factor that regulates the expression of antioxidant and anti-inflammatory proteins, and it is considered a modulator of species longevity . Its antNrf2 is regulated by Kelch-like ECH-associated protein 1 (Keap1) and the Cullin 3 (Cul3) ubiquitin E3 ligase complex. Keap1 sequester Nrf2 and functions as an adaptor, so the Cul3 complex ubiquitinates Nrf2 to facilitate its proteasomal degradation . Nrf2 ca2+), NADP+, and H2O [One gene regulated by Nrf2 is HO-1 (Heme oxygenase 1). The main function of HO-1 is to catalyze Haem (an iron-containing porphyrin) degradation; it uses cytochrome P450 reductase to transform Haem, NADPH, and O2 to biliverdin, carbon monoxide, ferrous iron  assay and an inhibition of 64% in the linoleic acid peroxidation assay. Yucca schidigera Roezl ex Ortgies radical scavenging activity was tested with TEAC  assay and had trolox equivalents (TE) values of 1.78 [Yucca baccata Torr. butanolic extract showed a 29.18 (μg TE/mg) in DPPH assay, 121.8 (μg TE/mg) in TEAC assay, 33.41 (μg TE/mg) in ferric reducing antioxidant power (FRAP) assay, and 156.84 in oxygen radical absorbance capacity (ORAC) assay [Finally, there are relatively abundant reports in the literature on extracts from the activity . Were Yu of 1.78 ,68 and 5 of 1.78 , respectC) assay . These rFor thousands of years, mankind has used plants to treat various ailments. This knowledge has been passed down through hundreds of generations and remains the main form of health care for more than 4 billion people today . PhytochDue to that, in recent years, there has been a trend in countries such as China where plants are being used to generate new drugs. Some phytochemicals are able to modulate inflammation and oxidative stress at the same time, since these two physiological phenomena often share the same pathways and intensify each other. An example of this is that ROS can act as an inflammatory signaling molecule, and in turn, inflammation can induce oxidative stress and reduce cellular antioxidant capacity .Yucca genus can be seen through Section 5.5Out of the documentary research, 365 molecules were found in the literature, of which 92 had antioxidant or anti-inflammatory reported activity. Of these molecules, 51 can be classified as Phenolic Compounds, 13 as Glycosides, 7 as Saponins, 9 as Fatty acids, 5 as Terpenes, 3 as Tocopherol, 2 as Dicarboxylic acid, 1 as Phytosterol, and Xanthones. The antioxidant and anti-inflammatory activities reported in the literature of the phytochemicals found in the Phenolic compounds are phytochemicals that are characterized as containing an aromatic ring bonded to some hydroxyl groups in their structure. Plants can produce a wide variety of phenolic compounds . These cYucca genus, there is great diversity and concentration of phenolic compounds. Specifically, unique phenolic derivatives with potent antioxidant activity have been found in Yucca gloriosa L. (gloriosaols) and Yucca schidigera Roezl ex Ortgies (yuccaols) [Within the uccaols) ,69. Amonuccaols) .Yucca genus and are widely used in the food, pharmaceutical, and cosmetic industries [Yucca schidigera Roezl ex Ortgies, new saponins have been found: Yucca spirostanosides [Saponins are amphiphilic compounds that have a saccharide chain attached to a steroid or triterpenoid . These cdustries . As withanosides .Glycosides are a large structurally diverse group of phytochemicals; they have 2 units a small metabolite (aglycone) and a sugar (glycone) . When plYucca genus, it has been reported that Yucca aloifolia variegate L. contains more saturated fatty acids than unsaturated, constituted mainly by palmitic acid and palmitoleic acid [Fatty acids are lipid structures composed of a long carbon chain with a carboxyl group at one end and a methyl group at the other end . If thiseic acid .Among the phytochemicals that were found at a lower frequency, the terpenes stand out with the anti-inflammatory effect. Terpenes are the most abundant and diverse class of phytochemicals; structurally, they are made up of isoprene molecules . They haYucca phytochemicals in the literature. The same is true regarding the difference between extraction and improvement in phytochemical concentration. Within the reports included here, the great variability caused by the factors previously described is notorious. This can be seen in It is worth noting that the presence by itself of phytochemicals does not guarantee that it will generate the desired biological effect. As with drugs, the quantity of the phytochemical dictates its efficacy. There are many factors that could alter the quantity of phytochemicals. Phytochemicals are mostly generated in response to external stimuli . Thus, aLactobacillus capable of stimulating its activation through small molecules [Yucca, into 4-vinyl-catechol. This is an activator of Nrf2 [Lactobacillus. Thus, the fermentation of phytochemicals through bacteria, such as Lactobacillus, is key for the efficient activation of Nfr2. From the abundance of reports on Yucca phytochemicals activating Nrf2, it could be assumed that other phytochemicals follow the pathway of phytochemicals and are metabolized by bacteria into Nrf2 activators.Finally, another important factor related to the availability of phytochemicals is microbiota. The gut microbiota metabolizes most molecules consumed, including drugs or phytochemicals. In the intestine, phytochemicals are degraded by microbes and absorbed by tissues . Some pholecules . One exa of Nrf2 . It has  of Nrf2 . It alsoYucca genus, there are still many unknowns to be resolved. First, most of the research related to the anti-inflammatory and antioxidant properties has only focused on Yucca schidigera Roezl ex Ortgies and Yucca gloriosa L. [Yucca species. More specifically, to its anti-inflammatory and antioxidant properties and phytochemicals.Although there is research on the anti-inflammatory and antioxidant properties of the riosa L. ,23,27,42riosa L. . Thereforiosa L. . If therriosa L. . Thus, wYucca schidigera Roezl ex Ortgies and Yucca gloriosa L., further study of its use against inflammatory diseases is still needed. In particular, in vitro studies rarely cope with the complexity of human diseases [Yucca, such as ovalbumin-induced airway hyperresponsiveness in mice [Yucca extracts have not been compared. Nor has its effect been compared with that of known treatments. Therefore, the study of Yucca genus against established models of inflammatory diseases is another field without much exploration. Finally, since the discovery of Yuccaol C and its mechanism of action against NO synthesis [Yucca schidigera Roezl ex Ortgies and Yucca gloriosa L., as can be seen in Yucca topic. For the same reason, although there is favorable evidence on the anti-inflammatory and antioxidant capacity of Yucca genus. There is still a lot of research to be done before being able to describe the genus or its phytochemicals as an alternative to treat inflammatory diseases.However, even with the favorable results of the research done with diseases . From wh in mice . Similarynthesis . There hYucca genus encompasses about 40 to 50 species, natives of southern North America. For centuries, it has been used to treat pathologies such as asthma, rheumatism, gonorrhea, sunburns, arthritis, etc. The ethnobotanical use led to the testing of many biological activities, where its antioxidant and anti-inflammatory excels. Unfortunately, there are a limited number of studies, so knowing its composition will provide a better understanding of the molecules responsible for these properties. This is because it is known that the medicinal use of plants is due to its phytochemicals. The documentary research found 92 phytochemicals with reported antioxidant and anti-inflammatory activities. Most of these molecules can be classified as phenolic compounds, glycosides, saponins, or fatty acids. Within these molecules, phytochemicals, such as Yuccaol C, stand out because they are original to the genus and have significant anti-inflammatory and antioxidant activity. The antioxidant and anti-inflammatory properties are mainly generated through free radical scavenging activity, the inhibition of arachidonic acid metabolism, the inhibition of MAPK and NF-κB, and the activation of Nrf2 signaling pathway. The NF-kB pathway is mainly inhibited by phytochemicals through the inhibition of IKK activation and phosphorylation IκBα, and the decrease of NF-kB concentration. The MAPK pathways are mainly inhibited by reducing p38, JNK, and ERK1/2 phosphorylation. Nrf2 is activated by increasing its concentration or inhibiting Keap1. However, there is evidence of the antioxidant and anti-inflammatory activity of some species within the genus, and although it is not abundant, the fact that a great variety of the phytochemicals that compose it present the same activities allows us to assess these properties.


Chronic alcohol use is related to alcoholic cerebellar degeneration that is caused by B1 (thiamine) deficiency and associated with the degeneration of Purkinje cells in the cerebellar cortex.This report aims to present a case with cerebellar ataxia and tremor due to alcoholic cerebellar degeneration that is unexpectedly regressed after starting piracetam infusion treatment in the inpatient Alcohol and Drug Addiction Research, Treatment and Education Center (AMATEM).We investigated the case prospectively. The patient was informed and consent was obtained.A 57-year-old, divorced, retired, male with alcohol use disorder for 48 years  presented to our hospital with upper and lower limb tremors and balance problems for 2 years. Neurological examination revealed dysmetria, cerebellar tremor, hypoesthesia of lower extremities, ataxia so he was unable to tandem-walk. CIWA-Ar score:13. The blood test, including hemogram, biochemical, HbA1C, TFT, serum copper and ceruloplasmin levels, results were all normal. Diazepam 50 mg was started and titrated down by 5 mg per day and discontinued, 600 mg parenteral and 250 mg p.o thiamine initiated for 3 days and continued orally, propranolol 20 mg/day and ecopirin 100 mg/day were continued. Cranial CT, cranial MRI, and EMG were ordered.The patient was consulted to the neurologist for movement disorder.MRI appearance of the cortical sulcus, fissure, cisterna, and cerebellar folia were obvious. The ventricular system was assessed as ecstatic secondary to atrophy. Therefore the alcoholic cerebellar degeneration was diagnosed and increased propranolol to 30 mg/day dose. Furthermore iv infussion piracetam was empirically started at secuencely dose of 60 g/day for 3 days, 45 g/day for 3 days, 30 g/day for 3 days. Upper and lower limb tremor disappeared and ataxa regressed after the treatment was arranged.Image:Image 2:Image 3:Several authors proposed that serotonergic system among other neurotransmitters in the cerebellum might be affected in ACD. Recent studies investigating the use of buspirone in the treatment of ataxia has showed positive results (1-3).The mechanism of action of the piracetam is unclear. Supposed that piracetam binds to the polar head groups of membrane bilayers and induces distinct changes in membrane structure. Its actions in the nervous tissue include indirect modulation of several neurotransmitter systems, neuroprotective and anticonvulsant effects and positive influence on neuronal plasticity.In conclusion, we suggest that high dose piracetam has a potential antiataxic effect in ACD.Cessation of drinking and nutritional supplementation are the only treatments available for ACD. However, gait does not improve in most patients. Pysical therapy, canes, walkers, and wheelchairs are helpful in maintaining mobility (4). Similarly, we observed that the patient’s need for care has been continuing.None Declared


Environmental factors such as diet and lifestyle have been shown to influence the development of some intestinal mucosal lesions that may be precursors of colorectal cancer (CRC). The presence of these alterations seems to be associated with misbalanced immunological parameter levels. However, it is still unclear as to which immunological parameters are altered in each phase of CRC development. In this work, we aimed to study the potential relationships of immunological and metabolic parameters with diet in a CRC-related lesion context. Dietary information was obtained using an annual semi-quantitative food-frequency questionnaire (FFQ) from 93 volunteers classified via colonoscopy examination according to the presence of intestinal polyps or adenocarcinoma. Cytokines, chemokines, and adipokines were determined from serum samples. We observed a reduction in adiponectin according to the damage to the mucosa, accompanied by an increase and decrease in C-X-C motif chemokine ligand 10 (CXCL10) and resistin, respectively, in CRC cases. The presence of aberrant crypt foci (ACF) in the polyp group was associated with higher tumor necrosis factor-alpha (TNF-α) concentrations. Vegetables were directly correlated with adiponectin and resistin levels, while the opposite occurred with red meat. A bioactive compound, soluble pectin, showed a negative association with TNF-α. Future dietary strategies could be developed to modulate specific immunological parameters in the context of CRC. According to GLOBOCAN data, about 1.9 million cases of colorectal cancer (CRC) occurred worldwide in 2020, this type of cancer ranking third in the global incidence of malignant tumors and second in mortality from cancer worldwide . In contA general description of the study sample is presented in p = 0.067). Moreover, the TNF-α/Interleukin(IL)-10 ratio was also increased in patients with polyps and ACF (p = 0.035), thus supporting an inflammatory environment.The presence or absence of ACF in the group of volunteers diagnosed with intestinal polyps was examined to analyze whether, in the presence of ACF, there were differences in immunological parameters . We obseTo explore the association in the sample between immunological parameters and the intake of food groups through diet, Spearman correlations were performed and heatmaps were plotted with the associations obtained . Among t2, while it was inverted with soluble pectin. When evaluating leptin, monounsaturated fatty acids from oils and fats and minerals such as iron, magnesium, selenium, and sodium showed positive correlations with this immunological parameter.We looked deeper into the associations of immunological parameters and diet by analyzing the intake of their constituents, including macro- and micronutrients, bioactive compounds, and xenobiotic compounds . In concNegative correlations were found with the xenobiotic 2-amino-3,4 dimethylimidazo  quinoline (MeIQ). Higher levels of resistin were associated with lower intakes of flavonoids and stilbenes. Soluble pectin displayed a similar negative association with both TNF-α and chemokine CCL2, interestingly similar to what was found in the polyp group with ACF presence. Lipids were the unique compounds inversely associated with IFN-γ. We found that IL-10 was negatively correlated with total protein and animal protein; with lipids; with vitamins such as vitamin A, B12, or B6; with several minerals; and with the xenobiotic benzo(a)pyrene (B(a)P), a polycyclic aromatic hydrocarbon (PAH), the latter also being negatively correlated with cholesterol levels. The correlations of immunological parameters and dietary compounds depending on the diagnosis groups were also evaluated . The intThe present study provides further insight into the existing knowledge on the impact of diet on the immune system at different stages of CRC development. Our findings pointed to CXCL10, adiponectin, and resistin as potential markers involved in the progression of damage on the colorectal mucosa. The association of some dietary components with these immunological parameters may help in the design of dietary strategies aimed at the primary or secondary prevention of this pathology ,23.The decrease in serum adiponectin accompanied by increased levels of CXCL10 in subjects from the polyp and CRC groups as compared to volunteers from the control group is one of the main findings of the present study. Clinically, adiponectin is used as a biomarker in obesity-related diseases, and its level showed an inverse relationship with increasing central adiposity and type 2 diabetes ,25. TherIn our sample population, we did not find any significant correlation of CXCL10 with diet, although when the polyp group was evaluated, a negative correlation with oils and fats was found. Regarding resistin, several authors reported higher levels of this cytokine in different inflammation-related disorders such as atherosclerosis, chronic inflammatory bowel disease, chronic renal disease, systemic lupus erythematosus (SLE), or arthritis ,38,39. TMoreover, when immunological parameters were evaluated in volunteers diagnosed with intestinal polyps and examined as a function of the presence or absence of ACF, TNF-α showed a statistically significant increase in subjects with ACF. Factors such as age, body mass index (BMI), and diet seem to affect the ACF number, which has been proposed to be utilized as a marker for the presence of lesions in the intestine ,43. TNF-It is also important to note that in the population under study, the consumption of alcoholic beverages was higher in both the polyp group and the CRC group, with the intake of ethanol being statistically increased in the polyp group with respect to controls, as previously reported in individuals of this sample population . Some auAmong the limitations of the present study, results from the CRC group should be considered with caution due to the limited sample size. Surprisingly, the subjects in this group were more active than those in the polyp or control group. These findings may be influenced by the small sample size. It cannot be discarded that, as a consequence of the discomfort caused by the development of the disease, they may have attempted to improve their lifestyle. Moreover, the diverse molecular characteristics and pathways of the intestinal polyps should be considered when analyzing higher intragroup variability in those volunteers diagnosed with polyps.This transversal analysis is part of the broader project “Effect of Diet and exposure to XEnobiotics generated during food processing on the genotoxic/cytotoxic capacity of the intestinal Microbiota” (MIXED).g, 15 °C. The resultant aliquots were immediately frozen at −80 °C until analyses. A biopsy of intestinal mucosa from volunteers diagnosed with intestinal polyps was extracted during colonoscopy for examination of ACF at the Pathology Department. In total, 93 participants were included in the study. Subjects were classified into three clinical groups according to their colonoscopy results: control (n = 37), polyp presence (n = 49), and CRC (n = 7).The recruitment of volunteers and collection of blood and tissue samples was carried out from October 2019 to December 2021 by the faculties of the Digestive Section of the Central University Hospital of Asturias (HUCA) and the Carmen and Severo Ochoa Hospital in Asturias, in the north of Spain. Volunteers were selected from among patients who came to the hospital for consultation due to clinical symptoms and among those included in the colon cancer screening program in our region. The exclusion criteria applied to subjects with age under 40 years or over 75 years, as well as those receiving omeprazole, antibiotics, corticoids, or non-steroidal anti-inflammatory drugs. Also, having specific cancer treatment at the time of the study or in the previous two months, previous surgery of the digestive system, autoimmunity, altered thyroid function, or history of diabetes or goiter were considered as exclusion criteria. Those individuals interested in participating were informed of the objectives of the study and signed an informed consent form. Prior to the preparation of the volunteers for colonoscopy, blood samples were taken by venipuncture at proposed intervals between 09.00 and 11.00 after an overnight fast. Blood samples were collected in separate tubes for serum and plasma, immediately kept on ice, and centrifuged at 1000× This project was evaluated and approved by the Regional Ethics Committee of Clinical Research of Asturias (Ref. 163/19) and by the Committee on Bioethics of CSIC (Ref. 174/2020). The procedures were performed in accordance with the fundamental principles set out in the Declaration of Helsinki, the Oviedo Bioethics Convention, and the Council of Europe Convention on Human Rights and Biomedicine, as well as in Spanish legislation on bioethics. Directive 95/46/EC of the European Parliament and the Council of October 1995, on the protection of individuals regarding the processing of personal data, was strictly followed.Dietary information was obtained from patients when they arrived for their colonoscopy results at a medical consultation by means of a personalized interview conducted by trained interviewers. Exceptionally, as a result of the pandemic and COVID-19 restrictions on visitors to hospitals in Spain, some of the surveys were conducted through online tools. For this purpose, a semi-quantitative food-frequency questionnaire (FFQ) was constructed with 155 items. In addition to food and culinary preparations, the specific type of food was recorded, as well as cooking methods and other related information, when necessary. Information relating to dietary assessment has been previously published . The cla2. Subjects were classified into normal weight (18.5–24.9 kg/m2), overweight (25.0–29.9 kg/m2), and obese (30.0 kg/m2), based on the Spanish Society for the Study of Obesity (SEEDO) criteria [Height (m) and weight (kg) were taken via standardized protocols . Body macriteria .Human Essential Immune Response Panel . The detection limits were 0.97 pg/mL, 1.81 pg/mL, 1.28 pg/mL, 0.65 pg/mL, 0.88 pg/mL, 1.45 pg/mL, 2.02 pg/mL, 0.97 pg/mL, 0.77 pg/mL, 0.76 pg/mL, 0.77 pg/mL, and 3.10 pg/mL, respectively. For adiponectin, adipsin, leptin, and resistin determination, the Human Metabolic Panel 1  was used, with detection limits of 41.4 pg/mL, 5.4 pg/mL, 1.6 pg/mL, and 1.4 pg/mL, respectively.To quantify the total antioxidant capacity of serum samples, a spectrophotometric method based on the ferric reducing antioxidant power (FRAP method) was performed using a commercial kit  Assay Kit; ELK Biotechnology, Denver, CO, USA). Total cholesterol was analyzed enzymatically via the cholesterol oxidase e-p-aminophenazone (CHOD-PAP) method using a commercial kit . Circulating levels of cytokines, chemokines, and adipokines were quantified in serum samples using two pre-defined bead-based multiplex assays, following the protocol provided by the manufacturer and using a FACSCanto II flow cytometer . Serum samples were maintained at −80 °C until determinations. IL-4, IL-2, IP-10 (CXCL10), IL-1β, TNF-α, CCL2 (MCP-1), IL-17A, IL-6, IL-10, IFN-γ, IL-12p70, and free active TFGβ1 levels were assessed using the t-tests were performed for categorical and continuous variables regarding a general description of the sample, respectively . The goodness of fit to a normal distribution was checked by means of the Kolmogorov–Smirnov test. As normality of the immunological and dietary variables was not achieved, Mann–Whitney U tests were performed to detect group differences . Correlations were assessed using Spearman rank tests to explore the associations between immunological parameters and food groups and compounds. Heatmaps were generated using the “corrplot” R package version 0.92.The results were analyzed using IBM SPSS software version 25.0  and RStudio software version 1.4.3 . GraphPad Prism 9  and RStudio software were used for graphical representations. Overall, categorical variables were summarized as the number and percentage, and continuous ones were summarized as the mean and standard deviation. Fisher tests and The variation in the levels of adipokines and chemokines with the progression of intestinal mucosal damage revealed the potential use of certain immune parameters as markers of the disease. Moreover, TNF-α concentrations were increased in the presence of ACF, supporting its pro-inflammatory role. The modulation of immunological parameters in patients by shifting to healthy dietary habits could be a non-invasive approach of great interest for future studies.