Groups were age-matched . Serum lipidomics data collected at baseline were clusteredby chemical similarity, and enrichment analyses were used to determine whether serumlevels of any lipid clusters differed between the normal and abnormal CSF groups. In asubset of patients with longitudinal structural MRI , two timepoints of MRI data were used to calculategrey matter annual percent change for each participant. Multivariate partial least squaresanalyses tested for relationships between neuroimaging and lipidomics data which aremoderated by CSF pathology. Our clustering analyses produced 23 serum lipid clusters. Ofthese clusters, six were altered in the abnormal CSF group, including a cluster ofunsaturated phosphatidylcholines. In the subset of participants with longitudinalstructural MRI data, a priori nucleus basalis of Meynert partial leastsquares analyses detected a relationship between unsaturated phosphatidylcholines anddegeneration in the nucleus basalis which is moderated by Alzheimer’s disease CSFpathology (P = 0.0008). Whole-brain grey matter partial least squaresanalyses of all 23 lipid clusters revealed that only unsaturated phosphatidylcholines andunsaturated acylcarnitines exhibited an Alzheimer’s disease-dependent relationship withlongitudinal degeneration . Only the unsaturated phosphatidylcholines predicted basal forebraindegeneration in the whole-brain analyses.Basal forebrain cholinergic neurons are among the first cell types affected byAlzheimer’s disease pathology, but the cause of their early vulnerability is unknown. Thelipid phosphatidylcholine is an essential component of the cell membrane, andphosphatidylcholine levels have been shown to be abnormal in the blood and brain ofAlzheimer’s disease patients. We hypothesized that disease-related changes inphosphatidylcholine metabolism may disproportionately affect basal forebrain cholinergicneurons due to their extremely large size, plasticity in adulthood and unique reliance onphosphatidylcholine for acetylcholine synthesis. To test this hypothesis, we examinedwhether serum phosphatidylcholine levels predicted longitudinal basal forebraindegeneration in Alzheimer’s disease. All data were collected by the Alzheimer’s DiseaseNeuroimaging Initiative. Participants were divided into a normal CSF group (controls;in vivo evidence for a selectiverelationship between phosphatidylcholine and basal forebrain degeneration in humanAlzheimer’s disease, highlighting the importance of phosphatidylcholine to basal forebraingrey matter integrity.Overall, this study provides  Basal forebrain cholinergic neurons are affected in early stages of Alzheimer’s disease,yet the cause of their vulnerability is unknown. Shanks et al. show that disease-relatedchanges in phosphatidylcholine metabolism disproportionately affect the basal forebrain,indicating a unique link to cholinergic function.  Among the most vulnerablecell types are the cholinergic neurons of the basal forebrain.4–8 Post-mortem histology has long implied that basal forebrain degenerationoccurs in individuals with mild cognitive impairment and advanced Alzheimer'sdisease.9–12 More recent longitudinal neuroimaging work13–16and ex vivo histology in suspected early Alzheimer's diseasepatients8 indicatesthat basal forebrain degeneration begins prior to the onset of clinically detectablecognitive impairment, i.e. the presymptomatic stage of Alzheimer's disease. Cognitivelynormal older adults at high risk for Alzheimer's disease according to CSF biomarkers of pTauand Aβ exhibit increased basal forebrain degeneration prior to corticaldegeneration.13–16 Despite the abundant evidence for the selectivevulnerability of the cholinergic basal forebrain to Alzheimer's disease degeneration, thecellular characteristics of cholinergic neurons which contribute to their vulnerabilityremain speculative. One cellular characteristic that is frequently proposed to increasevulnerability to degeneration is the cell’s ‘upkeep cost’, that is, the number of metabolicresources needed for a cell to maintain, repair and build upon its membrane and cytoskeletalstructure.17–20 Under this hypothetical framework, cholinergicneurons would have higher upkeep costs than other neuronal cell types, and age- ordisease-related bottlenecks on the resources needed for upkeep would exacerbate theirvulnerability.Alzheimer’s disease is characterized by the progressive accumulation of amyloid beta (Aβ)and phosphorylated tau (pTau) in the central nervous system,22 One class of lipids in particular, phosphatidylcholines, are animportant structural component of the cell membrane, and act as reservoirs for intracellularsignalling molecules.23–25 An influential theory proposed that cholinergic neurons areselectively vulnerable because they alone additionally use phosphatidylcholines for thebiosynthesis of the neurotransmitter acetylcholine.26 Recent advances in our understanding of themorphofunctional properties of basal forebrain cholinergic neurons have since strengthenedthe plausibility of this theory. Cell type-specific labelling studies of basal forebraincholinergic neurons in mice have revealed the extreme size and extensive branching of theseneurons, which in humans are estimated to exceed 100 m in total length for a singleneuron.28 A cell of this size and complexity would have arelatively high demand for phosphatidylcholines to maintain its membrane. Furthermore,acetylcholine signalling pathways from the basal forebrain are critical for the modulationof the neocortex, acting as a core mechanism for enhancing cortical read-in states duringthe acquisition of novel sensory information and associative learning.29–34 Due to their integral role in modulating attentionand memory encoding, basal forebrain cholinergic neurons therefore likely remain highlyplastic throughout adulthood,35 continuously remodelling their axonalprojections and forming new synapses. Consistent with this idea, the knockdown of axonalphosphatidylcholine synthesis prevents axonal branching in cell culture.36 Moreover, in mouse models of Rhettsyndrome, disease-related deficits in neuroplasticity are rescued by choline supplementationvia increased phosphatidylcholine synthesis.37 Considering their large size and sustained plasticity throughoutadulthood, age- or disease-related disturbances in phosphatidylcholine metabolism maytherefore affect the functioning of basal forebrain cholinergic neurons earlier and moreseverely than other cell types.Dysregulation in lipid metabolism is one possible bottleneck on normal neuronal upkeep.Like all other neurons in the brain, cholinergic neurons rely on lipid pathways to transportcholesterol and phospholipids for cell membrane repair and construction.38 Furthermore,choline-containing lipids are decreased in both the plasma and brain of Alzheimer's diseasepatients,40 indicating that measurements of lipids in the blood mayserve as an appropriate proxy for brain lipids. Here we used highly sensitive and specificCSF biomarkers of Alzheimer’s disease pathology42 to differentiate twoage-matched groups of older adults with normal versus abnormal markers of Alzheimer’spathology.14 We then integrated longitudinal structural MRI datacollected over a 2-year period with a comprehensive panel of serum lipid data collected atMRI baseline. Consistent with our hypotheses, we show that unsaturated phosphatidylcholinespredict longitudinal trajectories of degeneration in the basal forebrain and adjacentbasolateral nuclei in the abnormal CSF group compared to the normal CSF group. Our resultssuggest that a disruption of phosphatidylcholine metabolism is associated with the selectivevulnerability of cholinergic neurons in Alzheimer’s disease.Despite these lines of evidence, the relationship between phosphatidylcholines and basalforebrain cholinergic neuronal integrity remains untested in humans. Non-invasive measuresof phosphatidylcholines can be obtained from the blood and have been shown to reliablydifferentiate older adults at high risk for Alzheimer's disease from age-matchedcontrols.43 The ADNI was launched in 2003 as a public-privatepartnership, led by Principal Investigator Michael W. Weiner, MD. The primary goal of ADNIhas been to test whether serial MRI, PET, other biological markers, and clinical andneuropsychological assessment can be combined to measure the progression of mild cognitiveimpairment and early Alzheimer’s disease. For up-to-date information, see www.adni-info.org. All ADNIparticipants gave informed consent according to the Declaration of Helsinki prior toparticipating in any part of ADNI, and all data collection protocols were approved by theinstitution where the work was performed. To be included in this study, all participantsfrom ADNI needed to have (i) lipidomics data collected at baseline using the UC Davislipidomics platform,44 (ii)information on body mass index (BMI), and (iii) CSF biomarkers of Aβ and pTau. Foranalyses relating lipidomics data to neuroimaging measures, participants needed toadditionally have longitudinal structural MRI data. Following these exclusion criteria, atotal of 313 participants  from ADNI Phase 1 were included in ourlipidomics analyses, and 223 participants  were included in ourneuroimaging analyses. Participant demographics are outlined in Data used in the preparation of this article were obtained from the Alzheimer’s DiseaseNeuroimaging Initiative (ADNI) database (adni.loni.usc.edu).45 Participants were split into groups based on their ratio of CSFpTau/Aβ at baseline. The ratio of pTau/Aβ has been shown to be a sensitive and specificpredictor of Alzheimer's disease pathology and tracks with Aβ PET imaging.42 Participants with ratios of pTau/Aβ below 0.028 are within the normalrange41 and were thereforeconsidered the normal CSF group, while participants with ratios above 0.028 wereconsidered the abnormal CSF group.CSF Aβ and pTau were collected for ADNI and quantified as described previously.46 Participantswith normal CSF but clinically detectable cognitive impairment were excluded from thisstudy because their cognitive dysfunction likely stems from another form of dementia, suchas vascular dementia.47Clinical diagnosis in ADNI is determined using standardized criteria involvingcomprehensive neuropsychological testing and clinical examination by aneurologist.44 Of the 521 measured lipids from the completelipidomics data set available on ADNI, we excluded all unannotated lipids  and one duplicate lipid. The final data set used inthis study consisted of 348 annotated lipids. Participants’ serum levels of each lipidwere standardized via z-score prior to all statistical analyses to allowfor comparisons across lipid species.Serum lipidomics data were collected through untargeted ultra-high-performance liquidchromatography quadrupole time-of-flight mass spectrometry. For information on serumlipidomics data collection and processing in ADNI, see.48 ChemRICH firstmaps each lipid to its most specific Medical Subject Heading term to reflect chemicalontology, then modifies clusters by comparing binary representations of the structure ofcompounds, called substructure fingerprints.49 The Tanimoto coefficient is used to create a pairwise similaritymatrix for all lipids based on their substructure fingerprint, which then undergoeshierarchical clustering. Compounds with over 85% similarity are grouped into the samecluster.48 After clusteringhas been initially performed, Dynamic Tree Cutting50 determines if any sub-clusters exist within thesegenerated clusters. Because ChemRICH only relies on the chemical structure of the lipidsin the data set, it will produce the same lipid clusters in different sets ofparticipants, provided both samples share a common lipidomics platform.ADNI lipids were clustered based on structural similarity and chemical ontology usingchemical similarity enrichment analysis (ChemRICH) with chemical classes as the setdefinition.48First, we compared serum levels of each lipid in the normal and abnormal CSF groups usingindependent samples Wilcoxon tests. The Wilcoxon P values (two-tailed)were aggregated for each lipid cluster to form a distribution. ChemRICH then tests thenull hypothesis that this cluster-level distribution of P values areobtained from a reference uniform P distribution using theKolmogorov–Smirnov (KS) test.48If the KS test rejects the null hypothesis for a lipid cluster, this would indicate that acluster of lipids is significantly altered in the abnormal CSF group compared with thenormal CSF group. For visualization of the direction of change in lipid levels between thenormal and abnormal CSF groups, we additionally determined whether lipids were upregulatedor downregulated in the abnormal CSF group. To do so, we divided the median serum level ofeach lipid in the abnormal CSF group by the median level in the normal CSF group toproduce a fold change estimate.After the formation of lipid clusters, we used the ChemRICH software to perform clusterset enrichment analysis.n = 223) were included in all further analyses. Longitudinalstructural MRI data were collected at baseline and ∼ 6-month follow-ups.51 Preprocessing and annual percentchange calculations were performed as part of a previous study.14 First, all structural MRI imageswere co-registered to tissue probability maps which have been enhanced to improve thecharacterization of the subcortical grey matter.52 Longitudinal structural MRI data were analyzed usingthe SPM12 serial longitudinal pipeline.53 For each participant a within-subject symmetric mid-point averageimage was created using two timepoints (∼ 2 years apart) of their longitudinalT1-weighted imaging data. The interscan interval in years was used toregularize the deformations during the creation of the within-subject mid-point averages.A Jacobian determinant which represents how much each voxel has contracted or expandedcompared with the mid-point average was written out for each time point. Thewithin-subject average images were segmented and bias-corrected using SPM12’s segment withthe enhanced tissue probability maps.52 The grey matter segment of each participant’s mid-point average imagewas multiplied by the Jacobian determinant for each time point to produce longitudinallymodulated grey matter segments which provide an estimate of within-subject change overtime.53 Next, the rigidlytransformed grey matter and white matter segments for each individual were used to createa custom, age-appropriate template for the ADNI population.54 After the creation of the ADNI template, a deformationfield was estimated between the template and the within-subject mid-point average imagefor each participant. Each participant's deformation field was then applied to eachmodulated grey matter segment to warp them into the template space for comparison acrossindividuals. Finally, the voxel-wise annual percent change of grey matter volume wascalculated using the warped longitudinally modulated grey matter segments for eachindividual:A subset of participants with lipidomics, CSF and longitudinal structural MRI data as an a priori region of interest. The NbM consists of Ch4and Ch4p according to Mesulam’s nomenclature.55 The NbM is composed of over 90% cholinergic neurons57 which are known to be especially vulnerable to Alzheimer's diseasepathology.59 A stereotaxic probabilistic mapcreated through post-mortem histology was used to isolate voxels in the NbM.60 The NbM probabilistic map from theleft and right hemispheres was warped from the Montreal Neurological Institute space intothe ADNI population template space. Only voxels that overlapped in 50% of donors were usedin the final NbM mask  correlation analyses.62 PLS is a form of pattern analysis that searches for orthogonallatent variables which represent a significant covariance pattern between two inputdatasets that differs as a function of a grouping variable. Here, we used PLS analysesto assess relationships between lipids and grey matter annual percent change within theNbM region of interest with CSF pathology as the grouping factor. Age and BMI wereincluded as input variables. For a detailed description of the methodologicalimplementation of PLS analysis, see.61To test our 61 and therefore all statistics were performedtwo-tailed. An alpha of P < 0.05 was used in all analyses.Bootstrapping (5000 iterations) was used to derive 95% confidence intervals for eachinput variable to PLS analyses. These confidence intervals demonstrate how reliably eachvariable contributed to latent variable(s) identified in each PLS analysis.61The significance of distinct latent variables in PLS models was assessed using 5000permutation tests. PLS analyses are not directionalNext, we assessed the biochemical and anatomical specificity of any relationshipsdetected between phosphatidylcholines and basal forebrain grey matter integrity. Toassess biochemical specificity, we conducted one PLS analysis per ChemRICH lipidcluster. To assess anatomical specificity, we expanded the search space to thewhole-brain grey matter. With the exception of the search region, all inputspecifications for these PLS analyses were the same as the NbM analyses.https://ida.loni.usc.edu. The ChemRICHanalysis was performed in R. The code to perform ChemRICH can be found online . Longitudinal structural MRIpreprocessing was performed in MATLAB using SPM12 (https://www.fil.ion.ucl.ac.uk/spm/software/download/). PLS analyses wereperformed in MATLAB version 2019b, using PLS software which is freely available (https://www.rotman-baycrest.on.ca).All raw data used in this study are freely available from n = 223) participants additionally had to have two structural MRIscans which were collected ∼2 years apart. Only participants with complete data across allthree modalities were included. For all analyses, participants were broken into a normalCSF group  and an abnormal CSF group . Thenormal CSF group consists of cognitively normal individuals without CSF-based evidence ofAlzheimer’s disease pathology, while the abnormal CSF group consists of participants withvarious clinical cognitive diagnoses  and CSF biomarkers consistent with Alzheimer’s disease. P > 0.05 for all). In the full sample of participants and the MRIsubset, the frequency of carriers of the apolipoprotein E (APOE) ε4allele was significantly higher in the abnormal CSF group (P < 0.001in both the full sample and MRI subset). Consistent with expectations, the abnormal CSFgroup in both participant samples had worse memory and executive functioning performancethan the normal CSF group . Test statistics andP values for between-group comparisons of demographic and clinicalvariables are presented in ADNI participants included in this study had CSF pTau/Aβ and serum lipidomics data whichwere collected at baseline. For our analyses using structural MRI data, shared between unsaturated phosphatidylcholines anddegeneration of the NbM. In prior post-mortem histological59 and in vivo neuroimaging63–66 research, the NbM has been consistently identified as the mostvulnerable nucleus of the basal forebrain to neurodegeneration. Previous research hasdetected relationships between individual lipid species and grey matter degeneration usingunivariate statistical methods.68 However, such asgenes and proteins, lipid species are highly interrelated and form complex regulatorynetworks.70 For this reason, we used PLS toexamine the multivariate relationships of biochemically defined ChemRICH lipid clusterswith longitudinal grey matter degeneration. PLS analyses used permutationtesting61 to test if thepattern of covariance between lipids and brain degeneration differs by a grouping factor.When this is true, significant latent variable(s) will be produced.After determining that serum unsaturated phosphatidylcholines are significantly alteredin individuals with abnormal CSF pTau/Aβ, we used PLS correlation analyses61These salience maps are similar to z-scores, where values farther fromzero represent a stronger relationship between baseline lipids and grey matterdegeneration in a given voxel.PLS analyses can have three possible outcomes: no significant latent variables, onesignificant latent variable, or multiple significant latent variables. If there is nosignificant latent variable, then either no multivariate relationship is detected ineither group or the multivariate relationships are symmetrical in both groups (nullhypothesis is supported). Alternatively, if one or more significant latent variables isdetected, then a significant multivariate relationship exists between baseline lipids andlongitudinal grey matter degeneration which differs as a function of CSF group.Significant latent variables are then expressed as a salience map, which quantifies themultivariate relationship between baseline lipids and longitudinal grey matterdegeneration at each voxel.71 and neurodegeneration,72–75 we additionally included these variables in the PLS analyses toexamine their contribution to the covariance pattern.For our primary analyses, participants were split by CSF group to determine whether thereis a relationship between serum lipid levels and longitudinal grey matter degenerationwhich is moderated by CSF-confirmed Alzheimer's disease pathology. Due to the impact ofage and BMI on lipid metabolisma priori NbM PLS analyses onthe unsaturated phosphatidylcholines, as these lipids were the only phosphatidylcholinecluster which was significantly dysregulated in the abnormal CSF group (P = 0.0008 on 5000 permutationtests). Correlations between all lipids in the unsaturated phosphatidylcholine cluster andtheir bootstrapped confidence intervals are shown in Although the ChemRICH clustering analysis delineated two clusters of phosphatidylcholines(saturated and unsaturated), we focused our SF group . Consistin vivo PET studies with the 18F-Fluoroethoxybenzovesamicol([18F]-FEOBV) radiotracer, which binds to the vesicular acetylcholinetransporter, has revealed that the densest cholinergic innervations in the human braininclude the striatum, lateral and medial temporal cortices, cingulate cortex, insula, andamygdala.77 Our recent work suggests thatlongitudinal degeneration within the NbM covaries with cortico-amygdalar topographies ofboth structural degeneration and cholinergic denervation, and, that this covariationreflects the organization of the basal forebrain cholinergic projections.47 We therefore conducted PLS analysesexamining the multivariate relationship between baseline phosphatidylcholines andlongitudinal grey matter degeneration throughout the entire brain, with the predictionthat this relationship would be most robustly expressed by targets of the basal forebraincholinergic projections.The basal forebrain sends dense cholinergic projections to multiple brain regions. RecentP = 0.0022 on 5000 permutations). 78 This salience map depicts theexpression of the PLS latent variable relating unsaturated phosphatidylcholines to greymatter degeneration throughout the brain. Grey matter voxels that have significantsaliences after FDR correction are voxels that exhibit a significant, reliablerelationship with unsaturated phosphatidylcholines as a function of pTau/Aβ pathology. Thesalience map demonstrates that in addition to the basal forebrain, unsaturatedphosphatidylcholines predict degeneration as a function of pathological pTau/Aβ incortical regions that closely match the projections of the cholinergic system.77 Consistent with the [18F]-FEOBV PET work, these regionsinclude the basal forebrain, caudate nucleus, thalamus, hippocampus, insula, temporalcortex, and anterior and posterior cingulate. Overall, in line with our NbM analysis, ourwhole-brain PLS analysis suggests that unsaturated phosphatidylcholines are important forthe integrity of the basal forebrain and cortical and subcortical areas targeted by itsprojections.The whole-brain grey matter PLS analysis revealed one significant latent variable for theunsaturated phosphatidylcholines  and longitudinal degeneration throughout thewhole-brain grey matter tissue compartment.P = 0.0022 on 5000 permutations) and unsaturated acylcarnitines(P = 0.0018 on 5000 permutations) were the only lipid clusters thatproduced a significant latent variable . In contrast, the unsaturated phosphatidylcholines alone predict differencesin grey matter degeneration between the normal and abnormal CSF groups (P= 0.020 on 5000 permutations). The Sørensen–Dice coefficient was used to compare thesimilarity between the unsaturated phosphatidylcholine salience maps with and without ageand BMI, where a value of 1 reflects two identical salience maps, and a value of 0represents no overlap between the two salience maps. Because the Sørensen–Dice coefficientcompares binary maps, we binarized each salience map at its FDR-correctedz-critical value prior to comparison. The Sørensen–Dice coefficient was0.73, indicating that the expression of the phosphatidylcholine latent variable is highlysimilar with and without the inclusion of age and BMI. Thus, the observed relationship islargely driven by the unsaturated phosphatidylcholines, whereas the relationship betweenunsaturated acylcarnitines and grey matter degeneration is highly driven by interactionswith age and BMI.Prior work has shown that age, BMI and sex strongly influence the lipidome.2 = 4.87, P = 0.034). To do so, we conducted PLSanalyses where participants were grouped by biological sex, regardless of CSF pathology.In these analyses, a significant latent variable demonstrates that the relationshipbetween grey matter degeneration and lipids in a cluster differs as a function of sex.Inputs to the sex PLS analysis were otherwise identical to the CSF analysis.Finally, we investigated whether the relationship observed between unsaturatedphosphatidylcholines and grey matter degeneration was driven by imbalances in the sexeswithin the normal and abnormal CSF groups . The salience map , only the unsaturated phosphatidylcholines weredysregulated in the abnormal CSF group and predicted basal forebrain degeneration. Non-humananimal studies suggest multiple roles of unsaturated phosphatidylcholines in normal andabnormal brain conditions. For instance, emerging evidence indicates thatphosphatidylcholines containing polyunsaturated fatty acids can attenuate Aβ inducedneurotoxicity in Alzheimer’s disease models by reducing inflammation and increasingautophagic clearance mechanisms.90 and phospholipid synthesis.91 Importantly, Novakova et al.91 found that impairment of cholinetransport occurred at low (100 nm) concentrations of Aβ. Because free choline is a precursorfor the synthesis of phosphatidylcholines92 and is additionallyused to synthesize acetylcholine in cholinergic neurons,94 earlyaccumulation of Aβ could create a bottleneck on resources necessary for both structural andfunctional maintenance of cholinergic neurons.Our understanding of the mechanisms by which phosphatidylcholine levels become abnormal inAlzheimer’s disease is incomplete. However, two lines of evidence have pointed to linksbetween Aβ pathology and phosphatidylcholine metabolic pathways. For instance, cell culturework indicates that accumulation of Aβ can decrease choline flux into the cell through itstransporters for both acetylcholine synthesis96 and the transport of phosphatidylcholine,97 and carrying the ε4 allele of the APOEgene is the largest genetic risk factor for late-onset Alzheimer’s disease.99 Studies have also reported increased degeneration of the basalforebrain cholinergic system in APOE4 carriers compared withnon-carriers.100 Unsurprisingly, the abnormal CSFgroup contained the majority of the APOE4 carriers in our sample, with onlyeight normal CSF individuals having one or more ε4 alleles. Because of the important role ofAPOE4 in the pathophysiology of Alzheimer’s disease and cholinergic dysfunction, it ispossible that the combination of Alzheimer’s proteopathies and genetic risk conferred byAPOE4 combine to produce the strongest relationship between unsaturatedphosphatidylcholines and basal forebrain degeneration. In line with this idea, the APOE4protein induces lipid droplet formation in induced pluripotent stem cell-derived astrocytesand yeast, leading to morphological defects.101 These defects are specific to APOE4 (not resulting from Aβ) and arerescued by choline supplementation via increased phosphatidylcholine synthesis through theKennedy pathway. Because of the importance of choline and phosphatidylcholine metabolism onAPOE4-induced lipid dysfunction, we may therefore see that APOE4 moderatesthe relationship between unsaturated phosphatidylcholines and basal forebrain degenerationin our abnormal CSF group. Although we did not have the sample size to examine interactionsbetween APOE4 and CSF, future large-scale studies could further investigatethis relationship.This observed relationship between Aβ and lipid dysregulation may be linked to APOE, thebrain’s major lipid transporter. APOE is involved in both the clearance of Aβ68 The present study extends upon previous human researchby using multivariate PLS techniques and integrating multimodal data including serum, CSF,and longitudinal structural MRI within the same individuals. Our findings extend the work byToledo et al.,85who found differences in blood levels of phosphatidylcholines in individuals within the samecognitive group when split by CSF Aβ. Together, these more recent findings stress the importance ofintegrating both CSF biomarkers and cognitive data to stratify patients when studying thehuman lipidome in Alzheimer’s disease.Examining the associations between single plasma analytes and pathological features ofAlzheimer's disease has been vital in identifying lipid species that are involved inAlzheimer's disease. However, failing to account for the complex interrelationships thatexist among lipids that are structurally or physiologically similar may cause lipiddysregulation which occurs at a network level to be overlooked. Previous studies in humanshave linked various types of blood lipids to Alzheimer's disease clinical diagnosis, CSFproteopathies, cognitive scores and neuroimaging measures using univariate statisticalmethods.n = 84)or Alzheimer’s disease (n = 52). Preclinical individuals(n = 25) represent a minority of this subset. We were therefore unableto stratify out preclinical individuals to investigate whether a relationship betweenunsaturated phosphatidylcholine levels and longitudinal basal forebrain degenerationexists at the earliest stage of the disease. Future studies can address this question withincreased sample sizes and the incorporation of molecular imaging techniques. For example,PET imaging with the [18F]-FEOBV radiotracer can measure damage to cholinergicaxons. Interestingly, axonal damage may precede damage to cholinergic cell bodies asquantified by structural MRI measurements of basal forebrain volume.6 [18F]-FEOBV binds selectively to the vesicularacetylcholine transporter,102 aprotein that is expressed in cholinergic nerve terminals.104[18F]-FEOBV PET in preclinical participants would therefore give an earlier,cell type-specific measurement of cholinergic damage which is lacking in the currentstudy. Our longitudinal structural MRI measurements of basal forebrain volumetry measuredegeneration in a region that is largely but not exclusively cholinergic.105 To mitigate this limitation, we included a prioriregion of interest analyses using the NbM which is composed of over 90% cholinergicneurons.56 Nonetheless,using [18F]-FEOBV PET to determine whether phosphatidylcholines predictcortical cholinergic denervation in cognitively normal older adults with pathological CSFpTau/Aβ warrants further research.A primary limitation of this study is the presence of both early and advanced stages ofAlzheimer’s disease in our abnormal CSF group. Most participants with abnormal CSF in theMRI subset had a clinical diagnosis of mild cognitive impairment (107 measurements oflipids in the serum may not be specifically related to the degeneration of vulnerable celltypes in the brain, and instead more generally index the severity of Alzheimer’s disease.If this were the case, multiple serum lipid clusters would be expected to predict a commonpattern of widespread neurodegeneration typical of Alzheimer’s disease. In our analyses,only one lipid cluster predicted longitudinal degeneration over and above age andBMI—unsaturated phosphatidylcholines—and this pattern was expressed in the basal forebrainnuclei and known targets of its projections. The biochemical and anatomical specificity ofthese findings argues against the possibility of blood–lipidome wide dysfunctionpredicting a general pattern of Alzheimer’s disease degeneration.Finally, the conclusions that can be drawn about a relationship between unsaturatedphosphatidylcholines and basal forebrain integrity would be strengthened by a lipidomicsdata set that was collected in the brain or CSF. Only serum lipidomics data was availablein the ADNI Phase 1 population at the time of this study. Because of the separation ofcentral and peripheral lipid metabolism,Overall, our study was to our knowledge the first to report that unsaturatedphosphatidylcholines are related to longitudinal basal forebrain volumes in living adultswith probable Alzheimer's disease. Our findings are in line with the hypothesis thatcholinergic basal forebrain structural integrity is related to the availability ofphosphatidylcholines, although the exact mechanism of this link in the central nervoussystem remains to be elucidated.fcac318_Supplementary_DataClick here for additional data file.


BackgroundHigh-sensitivity cardiac troponin (hs-cTn) assays have significantly improved the early detection of myocardial injury and the diagnosis of acute coronary syndrome (ACS). Different diagnostic algorithms exist for the interpretation of hs-cTn in the management of patients with suspected ACS. This study analysed the diagnostic efficacy of hs-cTn using serial and single measurements while also shedding light on the challenges associated with the use of this assay.Methods We reviewed 189 results belonging to 120 unique patient episodes and records for troponin tests performed in a two-week period obtained from the West Cumberland Hospital, North Cumbria Integrated Care (NCIC), Whitehaven, England. These troponin tests were carried out based on the NCIC trust guidelines for the use of troponin assays in the management of acute coronary syndrome (ACS). A positive troponin test is defined using the NCIC trust guidelines and the National Academy of Clinical Biochemistry (NACB) guidelines. The case notes of the unique patients were reviewed to determine the outcome, which is defined as the clinical diagnosis on discharge of the patient following a cardiologist review. These outcomes were then used to calculate the sensitivity, specificity, and predictive values. We also determined the alternate diagnosis for false-positive tests.ResultsUsing both guidelines to assess the clinical effectiveness of the troponin assay yields slightly varying results, with the single positive test of NACB demonstrating a higher sensitivity of 92.8% (>71.4%) and a slightly better negative predictive value of 97.8% (>96%). However, using the serial troponin measurements as per the NCIC trust guideline demonstrates a better specificity of 95.2% (>42.4%) and a positive predictive value of 66% (>17.5%). False positive results are identified, which are due to alternate diagnoses such as stable angina, myocarditis, heart failure, sepsis, and malignancy.Conclusion High-sensitivity troponin (hs-cTn) assays play a crucial role in the early detection and management of patients with suspected ACS. This study supports evidence that serial troponin measurements are more diagnostically accurate than single troponin measurements. Although hs-cTn assays offer significant advantages, there remain challenges and limitations that require careful interpretation and clinical correlation. Acute coronary syndrome (ACS) encompasses a spectrum of clinical conditions that result from reduced blood flow to the heart muscle due to compromised coronary blood supply . The obsConventional and high-sensitivity troponin assaysCommercial immunoassays that detect cardiac troponins in blood are now widely available to clinicians to aid in the diagnosis of ACS. Manufacturers use the terms "sensitive" and "high sensitivity" to describe the sensitivity of their assays for marketing purposes . HoweverThe improved sensitivity, specificity, and diagnostic performance of high-sensitivity assays have led to their incorporation into clinical guidelines for the diagnosis and management of ACS ,4. They Troponin measurement is now the cornerstone of the diagnosis of MI . AccordiWhat is a positive troponin result?The 99th Percentile RuleAccording to the National Academy of Clinical Biochemistry (NACB) guideline issued in 2007 , "in theNorth Cumbria Integrated Care Trust GuidelineWe will also be analysing the results based on the NCIC trust guideline, which considers troponin levels to be consistent with an MI if the first troponin is <50ng/L with a subsequent >10ng/L rise or fall on the repeat sample or the first troponin is >50ng/l with a >20% rise or fall on the repeat sample . This alHowever, the question arises as to what the ideal interval should be for repeating the test. There are at least six algorithms. The 0h/3h algorithm from the European guidelines and the two-hour advanced diagnostic pathway use risk score prediction tools, whereas the 0h/2h, 0h/1h European Society of Cardiology (ESC), modified 0/1h ESC, and the current US state-of-the-art algorithms (six- and 12-hour troponins) do not include risk prediction tools. No guideline supports one above the other . The NCIAim and objectivesMost hospitals now have replaced conventional cTn tests with the new fifth-generation hs-cTn T and I assays, which can detect troponin at concentrations 10- to 100-fold lower than conventional assays [Given the variability in the diagnostic accuracy of troponin, it is of interest to continually evaluate the diagnostic and prognostic performances of this assay to ensure standards are maintained regarding the use of troponin assays in the early detection and management of ACS. In this study, we aim to calculate the sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) of the hs-cTnT using the two different aforementioned guidelines as a way of measuring diagnostic performance.Study design and populationThis study followed an initial departmental audit regarding the appropriate use of troponin tests in ACS management. It was a retrospective cohort study that involved analysing all troponin tests requested on admission to the emergency assessment unit and following up with the individual patients to the point of diagnosis and eventual discharge on that particular admission. The study was approved by the trust's clinical effectiveness and audit team.We obtained a patient episode list from the NCIC trust laboratory records for troponin tests performed in the two weeks between April 1-14, 2021, on patients admitted to the emergency admission unit. We reviewed 189 results and input them into our data tool. These troponin tests belonged to 120 patient episodes at West Cumberland Hospital, Whitehaven, England.The inclusion criteria were all patients who were admitted into the emergency admission unit and had a troponin test done within the selected two-week period. Patients who were discharged without admission from the emergency department or who did not require a troponin test were excluded from the study.Clinical assessmentWe reviewed patient information stored on the Clinical Portal, laboratory results, and post-discharge cardiologist clinic letters. We also requested patients’ medical folders from the medical records department when further information was required.All information was collected retrospectively. We compared the initial sets of troponin tests with the eventual outcome on discharge.Outcome measuresThe primary outcome was the diagnostic performance for the rule-out of ACS at the index event for both strategies. A positive test was considered using both guidelines.Any troponin test above the lower threshold limit in the first 24 hours; first troponin <50ng/L and >10ng/L rise or fall on repeat sample; or first troponin >50ng/L and >20% rise or fall on repeat sampleA positive outcome was a diagnosis of ACS following a cardiologist review during the current admission. We also looked into the other causes of false-positive results for these tests.Explanation of statistical measuresWhen interpreting the results of a troponin assay, several statistical measures are used to evaluate its diagnostic performance , as brieIt is important to note that sensitivity and specificity are inherent characteristics  of the tPatient and public involvementThe patients or the public were not involved in the design, conduct, reporting, or dissemination plans of our research.Diagnostic and prognostic performanceThe National Academy of Clinical BiochemistryAcute myocardial infarction (AMI) was diagnosed in 14 (3.6%) of the 120 patients reviewed using the NACB guidelines. Among the 46 patients who had negative test results, only one patient eventually had a diagnosis of ACS on discharge. Further, 13 out of the 14 patients who were diagnosed with ACS on discharge tested positive using the guideline definition. Hence, this approach had a higher sensitivity of 92.8% and an NPV of 97.8%  for both parameters, as illustrated in Figure High-sensitivity cardiac troponin assays have revolutionised the diagnosis and management of ACS. They have significantly improved the early detection of myocardial injury and the diagnosis of myocardial infarction (MI) compared to conventional troponin assays. These hs-cTn assays can detect very low concentrations of cardiac troponin T (cTnT) and troponin I (cTnI), allowing for more rapid diagnosis and risk stratification of patients with suspected acute coronary syndromes (ACS) . TroponiSeveral large-scale clinical trials have demonstrated the benefits of using hs-cTn assays for the diagnosis of MI and the risk stratification of patients with ACS. The High-Sensitivity Troponin in the Evaluation of Patients with Acute Coronary Syndrome (High-STEACS) trial  and the Recent developments in hs-cTn assays have focused on enhancing the analytical performance, including precision, accuracy, and reproducibility, as well as reducing the time to diagnosis. Rapid diagnostic algorithms relying on serial measurements of hs-cTn have been developed and shown to expedite the diagnosis and management of patients with suspected ACS . For insHowever, new-generation high-sensitivity troponin assays (hs-cTn) still present some limitations. This has necessitated the search for novel biomarkers to rule in and rule out myocardial MI in chest pain patients early, the most notable being the heart-type fatty acid binding protein (h-FABP); however, studies on h-FABP as a reliable marker for MI diagnosis have been largely disappointing .This study has raised some issues regarding troponin use in clinical management, which can impact the interpretation of results and clinical decision-making. These are listed below.False-Positive ResultsThe increased sensitivity of hs-cTn assays can detect small elevations of cTn levels in conditions other than ACS. This was more noticeable when we used the NACB single measurement guideline for a positive test, which gave a high percentage of false positive results of 82.4%. This is because the troponin assay cannot differentiate between a true type I MI secondary to a plaque rupture or various other mimics of plaque, including demand ischemia . In our Variability in the URLThere is variability in the 99th percentile URL among different human populations and across assays, which may impact the diagnostic accuracy. This explains the need to make sure cutoff limits are studied and designated for a specific population. This helps to accurately determine what would constitute a positive test to ensure low false positives and negatives ,18.Diagnostic Challenges in Early PresentersPatients presenting very early after symptom onset may have troponin levels below the assay’s detection limit or within the reference range ,18. In sInterpretation of Serial ChangesThe rise and/or fall of hs-cTn levels is an essential component of the diagnostic criteria for MI . HoweverAnalytical and Biological VariabilityDespite the improved precision of hs-cTn assays, there is still some degree of analytical variability between different assays and manufacturers, which may impact the clinical interpretation of results . ConsequStrengths and limitations of this studyThe limitations of this study include the NCIC trust’s hs-cTnT assay cutoff of 12 ng/L. The researcher is aware that there are other troponin assays with lower cutoffs (3-5 ng/L) for which a single baseline hs-cTn T measurement improves sensitivity for AMI markedly and can be used as a rule-out test in patients presenting more than three hours after symptom onset . Also, tHowever, despite these limitations, the study was done randomly, polling all troponin results within a set period without knowledge of the clinical information, reducing bias and increasing the internal validity of the results.The measurement of cTn is now considered the gold standard for the diagnosis of MI and plays a vital role in the risk stratification and management of patients with ACS. The study demonstrated significantly higher specificity (92.8% >71.4%) and positive predictive values (66% >17.5%) for serial troponin measurements over single measurements, with comparable negative predictive values (96% <97.8%) and lesser sensitivity (71.4% >92.8%). This supports evidence that serial troponin measurements are more diagnostically accurate than single troponin measurements. However, although hs-cTn assays offer significant advantages, there remain challenges and limitations, such as high false-positive rates, requiring careful interpretation and clinical correlation.


Predictors of spastic TT were neuromuscular scoliosis (p = 0.03), type of etiology , spasticity , presence of dystonia (p = 0.001), and epilepsy . The predictive model’s average accuracy, sensitivity, and specificity reached 82%. The model’s accuracy aligns with recent studies on applying machine learning models in the clinical field.Insufficient postural control and trunk instability are serious concerns in children with cerebral palsy (CP). We implemented a predictive model to identify factors associated with postural impairments such as spastic or hypotonic truncal tone (TT) in children with CP. We conducted a longitudinal, double-blinded, multicenter, descriptive study of 102 teenagers with CP with cognitive impairment and severe motor disorders with and without truncal tone impairments treated in two specialized hospitals . Clinical and functional data were collected between 2006 and 2021. TT-PredictMed, a multiple logistic regression prediction model, was developed to identify factors associated with hypotonic or spastic TT following the guidelines of “Transparent Reporting of a multivariable prediction model for Individual Prognosis or Diagnosis”. Predictors of hypotonic TT were hip dysplasia ( Cerebral palsy (CP) is a set of nonprogressive postural and motor control disorders caused by injury in the early stages of brain development . PosturaChildren with CP often show deficits in the development of postural control. In the motor dysfunction of children with CP, postural control problems, including those of the trunk, play a significant role. The exact nature of the deficits is only known to a limited extent. There are many studies on postural control in children with CP in the literature, but most focus on standing ,5. The iIn terms of muscle recruitment strategies, postural control can be considered to consist of two levels. The first or basic level is direction specificity, meaning the forward body sway is counteracted by the activation of dorsal muscles. The second level consists of fine-tuning the postural adjustment, for example, in the number of muscles activated, the recruitment order, and the use of anticipatory adjustments. In infants at high risk of CP, no increase in direction specificity was observed between 6 and 18 months . Co. Co® 20.e < 0.2)  were useWe aimed to predict each patient’s probability of having hypotonic or spastic TT, using the glm function of the open source software R 4.2.2 .Dependent variables were the presence of hypotonic, spastic, or normal TT. Independent variables were the type of spasticity (SP), etiology (ET), presence of epilepsy (E), dystonia (D), hip dysplasia (H), neuromuscular scoliosis (NS), gender (SE), MACS, GMFCS, and EDACS.We divided the patient data into a “training set” and a “test set” according to the statistical learning theory described by Vapnik ,15. The As usual, the specificity, sensitivity, and accuracy of the predictions were described in terms of true positive (TP), true negative (TN), false negative (FN), and false positive (FP). Specificity was considered as the percentage of actual negatives that were correctly identified as such. Sensitivity was identified as the percentage of actual positives identified as such. Accuracy was described as the precision of the measurement system in relation to reproducibility and repeatability and was calculated as (TP + TN)/(TP + TN + FP + FN) .Through the R predict function glm, we tested the model’s predictive ability on a new test set. For each patient in the test set, it produced a probability (Prob) of having hypotonic or hypertonic TT in the form of Prob [TT = yes|glm (ET + NS + SP + D + SE + E+ H + MACS + GMFCS + EDACS)], where 0 < Prob < 1. The threshold was established for the decision limit. If Prob [TT = yes|glm (ET + NS + SP + D+ SE + E + H+ MACS + GMFCS + EDACS)] > threshold, the presence of hypotonic or spastic TT was predicted. Thresholds from 0.1 to 0.8 were tested, and the results’ specificity, sensitivity, and accuracy were compared . For exan = 78) had MACS ≥ 4 (mean 4.3 ± SD 1.4) and GMFCS ≥ 4 (mean 4.3 ± 1.2). Concerning trunk movement skills, 59% (n = 60) had only static sitting balance, 37% (n = 38) possessed dynamic sitting balance, 4% (n = 4) had good postural balance, and none achieved the maximum TIS score of 23 points. TCMS showed comparable results . Half of the subjects were unable to walk for functional mobility, 47% (n = 48) were able to walk with aids, and only 2% (n = 2) could walk self-sufficiently. Regarding lower limb function, 49% (n = 48) had considerable difficulty, 35% (n = 36) had severe difficulty, 18% (n = 18) had modest difficulty, and none had mild or no difficulty. Regarding postural ability, 59% (n = 60) could not sit or needed support to maintain an aligned sitting position, and 29% (n = 30) were able to sit and could move. In comparison, 12% (n = 12) could shift their weight laterally and leave the sitting position. results . Trunk mThe neurological status of epilepsy, dystonia, spasticity, neuromuscular scoliosis, and hip dysplasia is shown on n = 27) of patients, and 56% (n = 57) showed reduced hip abduction <20°. Radiologic hip morphology indicated that 61% (n = 62) had normal or near-normal hips, 11% (n = 11) had dysplastic hips, 6% (n = 6) had dysplasia with mild subluxation, 4% (n = 4) had moderate to severe subluxation, and 4% (n = 4) had dislocated hips , severe scoliosis (p = 0.0024), epilepsy (p = 0.0043), dystonia (p = 0.0466), and manual, gross motor, and eating disorders (p = <0.0001) : the presence of hip dysplasia (<0.0001) .p = 0.01, OR = 23.18;Hip dysplasia: p = 0.01, OR = 16.98;Manual disability: p = 0.03, OR = 18.5;Male gender: p = 0.05, OR = 6.17;Gross motor function: p = 0.05, odds ratio (OR) = 0.15.Type of etiology: postnatal > perinatal > prenatal causes, Multivariate regression reported that the factors most associated with hypotonic TT were the following: Factors associated with spastic TT were the following:p = 0.001, OR = Infinite;Presence of dystonia: p < 0.001, OR = Infinite;Type of etiology: prenatal > perinatal > postnatal causes, p = 0.009, OR = Infinite;Type of epilepsy: controlled > refractory, p = 0.03, OR = Infinite;Neuromuscular scoliosis: p = 0.05, OR = Infinite.Type of spasticity: quadri/triplegia > diplegia > hemiplegia, The best multivariate model scored 84% accuracy, 71% sensitivity, and 90% specificity .p-value as a predictor of truncal tone disorder. This means that the significance of SE, H, GMFCS, MACS, and ET in predicting hypotonic truncal tone and H, NS, ET, SP, D, and E in predicting hypertonic truncal tone is very probable, with a p-value < 0.05.The “Pr(>|z|)” column at the far right of Although many publications about impaired postural control exist, we have not found specific comparative studies on the hypotonic and spastic trunk muscles in subjects with CP using a predictive model. Hence, we developed, tested, and applied a machine-learning model to identify factors associated with hypotonic or spastic TT in teenagers with CP. Previous studies have identified high levels of GMFCS ,25,26,30For CP children with trunk hypotonia, the difficulty in achieving upper limb ability and gross motor skills entails significant clinical implications. Thus, it is recommended that, in such children, trunk control ability be assessed when evaluating upper limbs and gross motor functions .In line with the literature ,39,40, wThe interconnection between trunk control, leg muscle strength, and selectivity for gait capacity in children with CP has been shown . It indiIn addition to well-known factors, we identified the following new predicting variables of hypotonic and spastic TT: type of etiology, male gender, presence of dystonia, and epilepsy. However, neuromuscular scoliosis and hip dysplasia could be associated factors rather than etiologic.We underlined the link between trunk muscle tone disorder and type of etiology: antenatal and perinatal rather than postnatal causes seem to be risk factors of spastic TT. The inverse association has been found for hypotonic TT. Because the prediction model has a low odds ratio and there are no studies on this, future research is needed to confirm this finding.On the orthopedic side, previous studies stated that teenagers with CP, neuromuscular scoliosis , and hipAnother novelty concerns male CP subjects, particularly at risk of hypotonic TT compared to female participants. Given the small number of study participants and the lack of the relevant literature, we are very cautious in interpreting this link. However, we intend to explore this aspect with a larger cohort of participants in the future. On the neurologic side, we found that CP subjects with dystonia and epilepsy (refractory > controlled) are more at risk of spastic TT than the control group. Epilepsy and dystonia could result from the comorbidity of a deficient nervous system with musculoskeletal disorders; encephalopathies causing epilepsy commonly affect motor control. This could cause truncal or postural tone disorders. The association between epilepsy and truncal tone disorders can be explained by early cortical injury ,43. The Identifying the type of postural instability (spastic versus hypotonic) in CP as early as possible should allow us to anticipate and personalize rehabilitation treatments. Infants with truncal hypertonicity at 3 months have significantly lower Bayley motor and mental scores at 18 months than infants with normal truncal tone. However, infants with lower extremity hypertonicity at 3 months are no different developmentally at 18 months from infants with normal tone. Infants with truncal or lower extremity hypotonicity fared the worst developmentally. Therefore, there is a high incidence of abnormal muscle tone in premature infants up to 18 months of age and early truncal tone abnormalities are associated with a worse developmental outcome . This deUsing a prediction model to identify risks and factors associated with the type of TT (hypotonic/spastic) in children with moderate–severe CP. This will help clinicians anticipate and focus on rehabilitation strategies and provide guidance toward implementing optimal levels of trunk assistance during rehabilitation .Trunk control should be one of the principal focus areas in testing and intervention in this population. So far, the assessment of truncal muscle tone in CP subjects has only focused on the correlation between trunk muscle deficit and functional abilities. Results from our study indicate that further characteristics related to the trunk’s hypotonia or spasticity should be considered. These factors should be evaluated when planning rehabilitation programs for CP children with trunk tone disorder.Early identification of those at risk for TT instability would help the child achieve independence. Adolescents with the highest independence in motor activity are further interested in conducting activities. The interest in performing activities is associated with maximum independence in motor performance, personal values, and social environment support. Thus, physical rehabilitation for adolescents with CP is recommended, as it can improve their motivation .The main clinical implications of our findings include:Children and families who face the daily consequences of postural control deficits have long demanded more attention from clinicians and researchers. Therefore, it is a surprise that so little consideration has been given to the critical role trunk control can play in the motor deficits these children manifest. Improvements over the past decade have shown that trunk control can be investigated in a clinically meaningful way with greater accuracy .We hope this study will be helpful to in supporting clinical decision making for improving functional skills in CP children with trunk postural control problems. This can be achieved by focusing more closely on the type of TT deficit. This mode of clinical reasoning may be critical in more precisely targeting an intervention to increase functional skills in subjects with CP . The comLimitations of this study include the relatively small number of patients and the retrospective analysis. It is essential to stabilize the predictive performance of the algorithm through a significant increase in the number of patients while keeping the number of independent variables unchanged (<15). Although we limited the number of patients (hundreds) for this study, we intend to implement and refine the model with a more significant number of patients (thousands) in the future. This will help us test and optimize the predictive performance of the model. The limited number of patients also results in potential overfitting of the model, which will be solved by studying a large number of patients ,19.


Interpreting fatalities involving khat is challenging due to a lack of data on cathinone and cathine reference concentrations in postmortem tissues. This study investigated the autopsy findings and toxicological results of fatalities involving khat in Saudi Arabia’s Jazan region from 1 January 2018 to 31 December 2021. All confirmed cathine and cathinone results in postmortem blood, urine, brain, liver, kidney, and stomach samples were recorded and analyzed. Autopsy findings and the manner and cause of death of the deceased were assessed. Saudi Arabia’s Forensic Medicine Center investigated 651 fatality cases over four years. Thirty postmortem samples were positive for khat’s active constituents, cathinone and cathine. The percentage of fatalities involving khat was 3% in 2018 and 2019 and increased from 4% in 2020 to 9% in 2021, when compared with all fatal cases. They were all males ranging in age from 23 to 45. Firearm injuries (10 cases), hanging (7 cases), road traffic accident (2 cases), head injury (2 cases), stab wounds (2 cases), poisoning (2 cases), unknown (2 cases), ischemic heart disease (1 case), brain tumor (1 case), and choking (1 case) were responsible for the deaths. In total, 57% of the postmortem samples tested positive for khat only, while 43% tested positive for khat with other drugs. Amphetamine is the drug most frequently involved. The average cathinone and cathine concentrations were 85 and 486 ng/mL in the blood, 69 and 682 ng/mL in the brain, 64 and 635 ng/mL in the liver, and 43 and 758 ng/mL in the kidneys, respectively. The 10th–90th percentiles of blood concentrations of cathinone and cathine were 18–218 ng/mL and 222–843 ng/mL, respectively. These findings show that 90% of fatalities involving khat had cathinone concentrations greater than 18 ng/mL and cathine concentrations greater than 222 ng/mL. According to the cause of death, homicide was the most common fatality involving khat alone (77%). More research is required, especially toxicological and autopsy findings, to determine the involvement of khat in crimes and fatalities. This study may help forensic scientists and toxicologists investigate fatalities involving khat. Khat is the common name for the plant known by its scientific name as Catha edulis (Vahl) Forssk. ex Endl. Typically, its leaves are chewed to produce stimulant effects such as alertness, euphoria, and increased motor activity . The twoThe most potentially toxic effects of khat consumption are hyperthermia, insomnia, anorexia, constipation, urinary retention, hypertension, myocardial infarction, and arrhythmia ,7,8,9. HBoth cathine and cathinone are basic compounds with pKa values of 9.37 and 7.55, respectively, and are susceptible to postmortem redistribution . As a paThe toxicity of khat exposure has been demonstrated by previous studies, and the possibility of lethality cannot be ruled out. To rule out an overdose as the cause of death, toxicologists must evaluate and interpret the substance concentrations present in postmortem samples. Substance concentrations must be quantified, reported, and published to expand the limited published evidence on toxicological findings for fatalities involving khat. The autopsy findings and other circumstances surrounding death are also important indicators of substance-related deaths. The results of an autopsy finding will also be important for figuring out what concentrations of cathine and cathinone are dangerous and which are fatal. Studying the prevalence of fatalities involving khat is also important for prevention, treatment, and education. The purpose of this study were (a) to analyze fatalities involving khat in Jazan, Saudi Arabia, and (b) to explore the disposition of cathine and cathinone in postmortem tissues.All fatal forensic cases received at the Poison Control and Medical Forensic Chemistry Center in Jazan, Saudi Arabia, from the 1st of January 2018 to the 31st of December 2021 were retrospectively evaluated. The data were collected from the OTARR electronic system using the data collection form. All acquired data regarding the toxicological study results and accident summary, including the manner of death in the forensic cases involving khat, were analyzed. These data included forensic cases that are confirmed as positive for the presence of cathine and cathinone and excluded the forensic cases that are negative for the presence of cathine and cathinone.All information regarding the autopsy finding, manner, and causes of death in fatalities involving khat within the period of study was revised, defined, and recorded by the medical forensic examiner experts at the Jazan Forensic Medicine Center. The autopsy finding data in the autopsied cases were clearly defined and tabulated. The manner of death was classified as follows: suicidal, homicidal, accidental, and undetermined. The identification of hazards for cathine and cathinone, as well as forensic cases involving their exposure, have been evaluated.Drugs of abuse including amphetamines, cocaine, cannabinoids, opiates, barbiturates, and benzodiazepines in all autopsied samples are primarily screened via immunoassay analysis using the RANDOX system . Volatiles including methanol, ethanol, isopropanol, and acetone are routinely analyzed using headspace-gas chromatography. Using liquid chromatography–mass spectrometry, the immunoassay positive results for amphetamine-type stimulants  were confirmed and quantified, while gas chromatography–mass spectrometry was used to exclude other drugs.g using a Heraeus Labofuge 400 centrifuge . The supernatant was then mixed with 1 mL of phosphate buffer pH 6 and vortexed for solid phase extraction (SPE) using DAU extraction columns . Prior to the extraction procedure, one milliliter of each sample’s homogenate was spiked with 20 μL of a prepared stock solution of 3,4-methylenedioxymethamphetamine (MDMA) at a concentration of 50 μg/mL as an internal standard. SPE columns were preconditioned with 3 mL methanol and 3 mL deionized water and equilibrated with 1 mL phosphate buffer at pH 6. Upon sample load completion, SPE columns were washed with 3 mL of deionized water, 1 mL of 0.1 M acetic acid, and 3 mL of methanol and dried for 10 min under a nitrogen stream. Next, cathine, cathinone, and MDMA were eluted into 12 mL glass tubes with 3 mL of a mixture of dichloromethane, isopropanol, and ammonium hydroxide . Then, one drop of 0.1 M hydrochloric acid (HCl) was added into each tube, and all elutions were evaporated to dryness under nitrogen stream. Finally, all samples were reconstituted with 100 μL of the aqueous proportion of the mobile phase (10 mM ammonium formate with 0.11% formic acid) for LCQ Fleet Ion Trap LC/MS analysis.Cathine and cathinone concentrations were identified and quantified via the liquid chromatography–ion trap mass spectrometry (LCQ Fleet Ion Trap LC/MS) method . For sample preparation, one gram of each organ tissue was homogenized in one milliliter of deionized water and then centrifuged for 15 min at 3000× Calibration samples were prepared by spiking 1 mL of blank tissue homogenates with cathine, cathinone, and MDMA . Six calibration points were prepared at concentrations of 50, 100, 250, 500, 1000, 2000, 3000, 4000, and 6000 ng/mL of cathine and cathinone and 1000 ng/mL MDMA as the internal standard. Postmortem samples were diluted prior to extraction and reanalyzed when concentrations exceeded the upper limits of quantification. Three quality control samples were prepared by spiking 1 mL of the blank homogenate tissues with cathine and cathinone at the concentrations of 100, 250, and 500 ng/mL and the concentration of 1000 ng/mL of MDMA as an internal standard.m/z 152 for cathine, m/z 150 for cathinone, and m/z 194 for MDMA. Cathine, cathinone, and MDMA are fragmented in the collision cell with helium gas in the Pulsed q collision-induced dissociation (PQD) mode into m/z 134 and 117 for cathine, m/z 132 and 105 for cathinone, and m/z 163 and 135 for MDMA. The PQD values for cathine and cathinone were 19 and 22 for MDMA. Qualitative and quantitative analyses were performed using X-Caliber Software. The analytical method employed in this study was developed and validated in our laboratory, and it is already routinely used to analyze amphetamine and related substances, with in-house modifications [An LCQ Fleet Ion Trap LC/MS system , employing an LCQ fleet mass analyzer coupled with a Surveyor Auto-Sampler and a Surveyor Quaternary Pump and managed by X-Caliber Software , was used. Briefly, 10 μL of each sample was injected by an autosampler. The chromatographic separation of cathine, cathinone, and MDMA was achieved with a HPLC column , using mobile phase A  and mobile phase B . Gradient elution was performed as follows: 0–1 min, 100% A; and 1–7.5 min, 80% A; 7.5–8.5 min, 50% A, 8.5–9.5 min, 0% A; 9.5–10.5 min, 50% A; and 10.5–11.5 min 100% A. Flow rate was 300 μL/min, and the injection volume was 5 μL. The electrospray ion source (ESI), as an optimized tuning profile of ATS, runs in positive ionization mode with 5 kV spraying voltage, 275 °C capillary temperature, and sheath gas value 30. The mass analyzer runs in the scan mode, scanning at ications .v:v). Thereafter, the columns were washed with 3 mL of methanol, 2 mL of the second elution  was added, and the sample was dried under nitrogen. All samples were reconstituted with methanol (100 μL), then vortexed, and transferred to GC/MS autosampler vials for GC/MS analysis.The general unknown screening analysis was performed via gas chromatography–mass spectrometry (GC/MS) analysis . Each sample (one ml of a homogenate sample) was mixed with 1 mL of the phosphate buffer (pH 6). One milliliter of the homogenate sample was extracted via the solid phase extraction (SPE) technique using DAU extraction columns  according the manufacture’s instructions. For instance, columns were conditioned with 3 mL of methanol and then 3 mL of deionized water and equilibrated with 1 mL of the phosphate buffer (pH 6). Two milliliters of each sample was loaded and allowed to pass slowly. Then, the columns were washed with 3 mL of deionized water followed by 1 mL of 0.1 M acetic acid and allowed to dry for 15 min under a flow of air. The first elution was collected by adding 2 mL of ethyl acetate/hexane  separation column had the following properties: 30 m length, internal diameter (ID) 0.25 mm, and film thickness 0.25 μm. Helium was used as the carrier gas with 1 mL/min flow rate. A total of 2 μL of each sample was injected into the splitless mode at an injection port with a temperature of 260 °C. The GC thermal program started at 80 °C and lasted 1.5 min. The thermal program then increased the temperature at the initial ramp to 210 °C at a rate of 30 °C/min and then slowed to 20 °C/min to reach the final temperature of 320 °C, which was held for 11 min. Electron ionization (EI) was used as the ion source in MS, and the analysis was carried out in scanning mode with an electron energy of 70 eV. The temperature of the ion source and transfer line was set at 230 °C. The mass spectral libraries from Wiley and the National Institute of Standards and Technology (NIST) were used to identify the GC/MS mass spectra of unknown substances.All variables were categorized and tabulated using descriptive statistics. Means, standard error of mean (SEM), and median were presented. SigmaPlot for Windows Version 11.0 was used to analyze all of the data.The Forensic Medicine Center in Jazan, Saudi Arabia, investigated 651 fatal cases over four-year period. Thirty of the cases had postmortem samples positive for cathinone and cathine, the active ingredients in khat. They were all males ranging in age from 23 to 45. Firearm injuries (10 cases), hanging (7 cases), road traffic accidents (2 cases), head injury (2 cases), stab wounds (2 cases), poisoning (2 cases), unknown (2 cases), ischemic heart disease (1 case), brain tumor (1 case), and choking (1 case) were responsible for the deaths. The toxicological analysis of cathinone and cathine in fatalities involving khat was performed and summarized .The average cathinone and cathine concentrations were 85 and 486 ng/mL in the blood, 1009 and 12,616 ng/mL in the urine, 69 and 682 ng/mL in the brain, 64 and 635 ng/mL in the liver, and 43 and 758 ng/mL in the kidney. The blood concentrations of cathinone and cathine ranged from 18 to 218 ng/mL and from 222 to 843 ng/mL, respectively. In 90% of fatalities involving khat, cathinone concentrations greater than 18 ng/mL and cathine concentrations greater than 222 ng/mL were detected. The retention time for cathine, cathinone and MDMA were 4.4, 4.5, and 5.1 min, respectively. The limits of quantification (LOQ) and detection (LOD) were both 36 ng/mL. Precision and accuracy were within 20% standard deviation and 20% bias, respectively. The extraction recovery ranged from 80 to 90%, with less than 20% carryover. At doses of 100 ng/mL and 6000 ng/mL, the matrix effects (suppression/enhancement) were within the acceptable limits (25%).The urine samples had the highest 90th percentile concentrations of cathinone (2134 ng/mL) and cathine . A stacked bar chart of fatalities involving khat alone or khat in combination with other drugs in different manners of death cases is presented in The khat plant (Catha edulis (Vahl) Forssk. ex Endl.) is listed by the Saudi Food and Drugs Authority (SFDA) as a prohibited plant under the Act, whereas cathinone and cathine are listed in Schedules I and III, respectively, of the 1971 United Nations Convention on Psychotropic Substances. Khat, on the other hand, is cultivated in Yemen, where it is widely consumed. However, in the Jazan region, which is close to the Yemeni border, 21.4% of students and 28.7% of all surveyed individuals reported that they were currently chewing khat. In Yemen, khat is commonly cultivated and consumed by 68% of the surveyed Yemenis ,25,26.In 2020, khat was the most frequently seized plant-based substance, according to the United Nations Office on Drugs and Crime . Khat accounts for 55% of all plant-based substances seized between 2016 and 2020. Saudi Arabia accounted for the most total khat seizures in 2019 . In addiFew articles have documented fatalities associated with khat, and there is currently no established reference range for cathinone and cathine that is toxic and lethal ,30,31. CThe median blood concentrations of cathinone and cathine were 40 and 470 ng/mL, respectively, exceeding the median blood levels of cathinone and cathine in previously published forensic non-fatal road traffic accidents  ,35,36. TCathine and cathinone should be included in all routine toxicological investigations, especially in homicidal cases, because the number of fatalities involving khat has already increased. This study reveals the number of deaths associated with khat use, both alone and in combination with other drugs. Which demonstrate that homicidal patterns were observed in a greater proportion of cases involving khat alone (77%) than cases involving khat in combination with other drugs (23%), and that suicidal pattern was observed in a greater proportion of cases involving khat in combination with other drugs (71.5%) than cases involving khat alone (28.5%).Khat use has been associated with a higher risk of myocardial infarction ,39,40,41Hepatotoxicity, on the other hand, has been observed in multiple studies, particularly among chronic khat chewers. Multilobular necrosis, canalicular cholestasis, an enlarged liver with decreased echogenicity, cirrhotic and portal fibrosis, jaundice with elevated liver enzymes, and severe liver injury with an elevated international normalized ratio were all characteristics of khat-related hepatic toxicity ,45,46. HKhat use has also been associated with mental disorders including anxiety, irritability, aggression, psychosis, paranoia, dysphoria, depression, insomnia, hallucinations, and delusions ,48,49. IIn the current study, homicide and suicide were the leading manners of death in fatalities involving khat alone and khat in combination with other drugs, respectively. In fatalities involving khat in combination with other drugs, particularly amphetamine, suicide was the most common manner of death. This study has limitations due to the small sample size and the fact that only non-natural cases were sent for toxicological analysis. In addition, it is essential to determine the relationship between khat consumption, violent crime, and fatalities. Therefore, a study is now underway to examine the role of khat in the toxicology of violent death over a ten-year period (2014 to 2023) and to establish reference ranges for cathinone and cathine in fatalities involving khat, which will represent only the fatal cases intoxicated with both cathinone and cathine.In spite of the limitations described above, these findings suggest that fatalities involving khat are cause for concern, particularly among young people with a higher propensity for homicide and firearm injuries. Therefore, any health-based crime prevention strategy in Jazan has to consider khat use.This study determined the average levels of cathinone and cathine in 30 cases of fatalities involving khat. This information may help forensic toxicologists and pathologists interpret toxicological investigations, but it should not be interpreted independently of other evidence, such as the death investigation and autopsy results. To create the body of literature necessary for more precise determinations of the role of khat in crimes and death investigations, additional research is required, particularly involving toxicological investigative and autopsy findings.


Rejection was associated with lower ApoAII (p = 0.021), ApoB100 (p = 0.032) and ApoM levels (p = 0.025). Lower adipsin levels were detected in those patients receiving desmopressin (p = 0.037); moreover, lower leptin levels were found in those patients receiving glucocorticoid therapy (p = 0.045), and higher T3 levels were found in those patients treated with L-thyroxine (p = 0.047) compared to those patients not receiving these hormone replacement therapies. IL-5 levels were significantly associated with UNOS-D score (p = 0.004), Heart Donor Score (HDS) and Adapted HDS (p < 0.001). The monitoring of immunological and metabolic changes in donors after brain death may help in the prediction of potential complications after heart transplantation, thus potentially optimizing donor heart allocation.The proinflammatory cascade that is activated at the time of brain death plays a crucial role in organ procurement. Our aim of this study was to explore the relationship between the clinical outcome of orthotopic heart transplantation, as well as cytokine and apolipoprotein profiles of the pericardial fluid obtained at donation. Interleukin, adipokine and lipoprotein levels in the pericardial fluid, as well as clinical data of twenty donors after brain death, were investigated. Outcome variables included primary graft dysfunction, the need for posttransplantation mechanical cardiac support and International Society for Heart and Lung Transplantation grade ≥ 2R rejection. Hormone management and donor risk scores were also investigated. Lower levels of IL-6 were observed in primary graft dysfunction (median: 36.72 [IQR: 19.47–62.90] versus 183.67 [41.21–452.56];  Orthotopic heart transplantation (HTX) has been used as an optimal treatment for end-stage heart failure. Strict selection criteria and the application of score systems for the determination of urgency status on the waiting list have further aided in decreasing adverse outcomes . DespiteFurthermore, optimal procurement after brain death also plays an important role in the outcome of HTX. Due to the organ shortage, expanded donor selection criteria have been used . In thesSystemic inflammatory reaction syndrome (SIRS) is triggered after brain death. This process initiates or increases the immune reaction . The relWe hypothesized that the extent of immunological injury can be quantified and that we can obtain an in-depth assessment of heart function if pericardial fluid samples are analyzed. The aim of this study was to explore the relationship between the cytokine and apolipoprotein (Apo) profiles in the pericardial fluid of the donor heart and the clinical outcomes of orthotopic HTX, including PGD, organ rejection and mortality. Pericardial hormone concentrations were also determined in relation to hormone management and donor risk scores.2, respectively. In most of the cases, the cause of brain death was trauma  with or without subdural hematoma. Further descriptive characteristics of the donors are summarized in The median age of the donors was 30 years, and 5 donors (25%) were women (n = 5). There was gender mismatch in 15% (n = 3) of the transplantations, and the medians of the donors’ and recipients’ body mass index (BMI) values were 25.40 and 25.65 kg/mThe median scores of the patients were 0 (IQR: 0–0.75), 16 (IQR: 15–21), and 3.77 (IQR: 3.69–4.38) for the UNOS-D, HDS, and adapted HDS scores, respectively. Each donor risk score correlated with the IL-17A and IL-5 levels. Apo I and Apo A II levels negatively correlated with adapted HDS scores. The UNOS-D score correlated with adiponectin levels. The data are shown in After transplantation, six patients (30%) needed postoperative MCS, and five patients had PGD. Five patients had ISHLT Grade 2R rejection.p = 0.029). IL-6 levels were also significantly lower in the MCS group (p = 0.043). The detailed comparisons are shown in p= 0.045 in the vasoplegic and the nonvasoplegic patients, respectively)  . In patip = 0.044 and p = 0.018 in the PGD and rejection analyses, respectively). Recipient UNOS scores were not different in the investigated outcomes. We have found that ApoM , ApoAII  correlated with the age of the donors. The data are shown in Donor age was higher in the PGD and rejection analyses among those who suffered these adverse events compared to those who did not . Moreover, the administration of hydrocortisone or methylprednisolone was associated with lower leptin levels in comparison to those who were not treated with glucocorticoid . Leptin levels had no significant correlation with body mass index . In L-thyroxine-substituted patients, higher pericardial fluid T3 levels were measured compared to those who were not treated with L-thyroxine . No significant correlation was found between the number of substituted hormones and interleukin levels .  did not receive any replacement, 7 donors (35%) received 1 hormone replacement, 2 patients (10%) received 2 types of hormone therapy and 5 donors (25%) had desmopressin-steroid and L-thyroxin replacement 48 h before explantation. The adipsin level was significantly lower in donors who received desmopressin therapy compared to those who did not have desmopressin replacement therapy . .Significant correlations were found between several immunological and laboratory or echocardiography parameters. The detailed results are shown in We found that the donor pericardial IL-6 level was lower in the transplanted patients who had PGD or MCS support than in those patients who had not received support. Patients who had rejections had significantly higher apoprotein levels. In the case of hormone replacement, desmopressin therapy caused lower adipsin levels, and steroid treatment was associated with lower leptin levels. IL-17A and IL-5 levels correlated with the UNOS, HDS and aHDS donor scores.Brain death has been shown to cause a significant acceleration of immunological reactions, which is characterized by the expression of cytokines and adhesion molecules, followed by the infiltration of leukocytes . The resThe main goal of HTX is to decrease mortality and to secure an improved quality of life after the procedure. Primary graft dysfunction still has a high occurrence rate and is the leading cause of early transplantation-related mortality . The defIn recent decades, the UNOS and the Heart Donor Score have been developed for a better quantitative description of donor hearts ,7,10. WeHormone replacement and/or hormone resuscitation can be an important treatment tool for donor optimization . After bIn addition to PGD, rejection is a feared complication after transplantation. Due to immunotherapy and established monitoring guidelines, the number of acute heart rejections has slightly decreased in recent decades . RejectiThe main limitation of this study was that the pericardial fluid samples of the recipients were not available. Therefore, the bilateral interactions could not be investigated in this study. Serum samples were not obtained in parallel with the pericardial fluids. Moreover, the small population size and the heterogeneity of the donors resulted in lower statistical power. However, a large number of cytokines and lipoproteins were measured, and the samples were obtained from the pericardium, which is in close proximity to the donor organ. The donor population represented the brain death patients procured by the national guidelines. Authors should discuss the results and how they can be interpreted from the perspective of previous studies and of the working hypotheses. The findings and their implications should be discussed in the broadest context possible. Future research directions may also be highlighted.Well-characterized pseudonymized human pericardial fluid and cell-depleted pericardial fluid samples of 20 randomly-chosen donors from the time period between February 2013 and December 2017 were obtained from the Transplantation Biobank of the Heart and Vascular Center at Semmelweis University, Budapest, Hungary . BetweenDonor and recipient variables were retrieved from the National and Eurotransplant-based donor data report form and from electronic medical records from our institutional databank. The following data on donors were collected and analyzed: age, sex, height, weight, body mass index, cause of brain death, overall length of stay (LOS), donor management time at the intensive care unit (ICU), diabetes mellitus, hypertension, smoking, drug abuse, active malignant tumor, serum sodium, serum potassium, serum chloride, serum glucose concentration, blood urea nitrogen (BUN), blood group, urine output (mL/kg/h), administration of inotropic and vasoactive medications , ejection fraction of the heart, interventricular septum thickness in end-diastole and left and right atrial and ventricular diameters. Hormone replacement therapy  administered in the last 48 h before procurement was also added to the variables. Patients who received more than one dose of hydrocortisone or methylprednisolone before procurement were considered as treated patients. Description of the study is shown in Due to the relatively small sample size of our study, the UNOS score was calculated for donors, recipients and overall individuals. The donor-specific UNOS (UNOS D) score includes donor age , total ischemic time , sex mismatch (1 point), and donor diabetes mellitus (1 point). According to these criteria, the UNOS-D score was calculated, and three UNOS-D risk groups were formed: low (score: 0), intermediate (score: 1 or 2), and high (score: ≥3). The recipient-specific UNOS score considered the following parameters: age , body mass index , mean pulmonary artery pressure , total bilirubin , creatinine , previous transplant, previous cancer, and pretransplant mechanical ventilation (each 2 points) or mechanical circulatory support (non-continuous-flow 2 points) . The totThe original Heart Donor Score (HDS) includes the following 12 donor characteristics: age, cause of death , drug overdose, intoxication, carbon monoxide intoxication, meningitis, respiratory disorders, subarachnoid bleeding and sepsis), compromised history , hypertension, cardiac arrest, left ventricular ejection fraction (LVEF), valve function, left ventricular hypertrophy (LVH), coronary angiography, serum sodium, norepinephrine support and dopamine/dobutamine support . The ada® System, BD Biosciences, San Jose, CA, USA) were used for the collection of pericardial fluid samples. Cells were removed via centrifugation . The cell-free samples were recentrifuged , and these supernatants were divided into three 500 µL aliquots and stored in liquid nitrogen until use.After sternal splitting and opening of the pericardium, ACD (Tri-sodium Citrate with Citric Acid and Dextrose) vacutainer tubes  were used for the quantification of pericardial fluid cytokines, adipokines and lipoproteins according to the manufacturer’s instructions.® Kit of Cloude Clone; T3 and T4 Human ELISA Kits Abcam, Cambridge, UK) according to the manufacturer’s instructions. Oxidative damage to pericardial fluid proteins was assessed by determining the free SH content expressed in cysteine equivalents via the DTNB  method (Thermo Scientific Pierce Ellman’s Reagent).Pericardial fluid oxidized low-density lipoprotein (oxLDL), triiodothyronine (T3) and thyroxine (T4) concentrations were determined via ELISA  . Severe p value < 0.05 was considered to be statistically significant.Data are expressed as the median and interquartile range (first–third [IQR]). Differences between the groups were assessed by the nonparametric Mann‐Whitney U test for continuous variables. The association between outcomes and inflammatory variables was tested via Spearman’s correlation. Comparisons between interleukins (IL), apolipoproteins and donor scores were conducted by performing a one-way analysis of variance on the ranks test. Data were analyzed by using IBM-SPSS 22.0 software . All of the statistical tests were two-sided, and a In conclusion, IL-5 levels correlated with the UNOS, HDS and adapted HDS donor scores. PGD and the need for MCS were associated with lower IL-6 in the donors before explantation. Our results indicate that immunological and metabolomics markers should be measured to obtain more information about the apparently well-conditioned donor heart. The measurement is inexpensive and can be easily performed in the laboratory before transplantation. More in-depth knowledge of the immunological and metabolic changes after brain death may help in treatment optimization and donor procurement.