Diagnostic Accuracy of the Telephone Interview for Cognitive Status for the Detection of Dementia in Primary Care.

PURPOSE: Cognitive diagnostic work-up in primary care is not always physically feasible, owing to chronic disabilities and/or travel restrictions. The identification of dementia might be facilitated with diagnostic instruments that are time efficient and easy to perform, as well as useful in the remote setting. We assessed whether the Telephone Interview for Cognitive Status (TICS) might be a simple and accurate alternative for remote diagnostic cognitive screening in primary care. METHODS: We administered the TICS (range, 0-41) for 810 of 1,473 older people aged 84.5 (SD, 2.4) years. We scrutinized electronic health records for participants with TICS scores ≤30 and for a random sample of participants with TICS scores >30 for a dementia diagnosis using all data from the Prevention of Dementia by Intensive Vascular Care (preDIVA) trial for 8-12 years of follow-up. We used multiple imputation to correct for verification bias. RESULTS: Of the 810 participants, 155 (19.1%) had a TICS score ≤30, and 655 (80.9%) had a TICS score >30. Electronic health records yielded 8.4% (13/154) dementia diagnoses for participants with TICS ≤30 vs none with TICS >30. Multiple imputation for TICS >30 yielded a median of 7/655 (1.1%; interquartile range, 5-8) estimated dementia cases. After multiple imputation, the optimal cutoff score was ≤29, with mean sensitivity 65.4%, specificity 87.8%, positive predictive value 11.9%, negative predictive value 99.0%, and area under the curve 77.4% (95% CI, 56.3%-90.0%). CONCLUSIONS: In the present older population, the TICS performed well as a diagnostic screening instrument for excluding dementia and might be particularly useful when face-to-face diagnostic screening is not feasible in family practice or research settings. The potential reach to large numbers of people at low cost could contribute to more efficient medical management in primary care.

Unexpected Arabinosylation after Humanization of Plant Protein N-Glycosylation.

As biopharmaceuticals, recombinant proteins have become indispensable tools in medicine. An increasing demand, not only in quantity but also in diversity, drives the constant development and improvement of production platforms. The N-glycosylation pattern on biopharmaceuticals plays an important role in activity, serum half-life and immunogenicity. Therefore, production platforms with tailored protein N-glycosylation are of great interest. Plant-based systems have already demonstrated their potential to produce pharmaceutically relevant recombinant proteins, although their N-glycan patterns differ from those in humans. Plants have shown great plasticity towards the manipulation of their glycosylation machinery, and some have already been glyco-engineered in order to avoid the attachment of plant-typical, putatively immunogenic sugar residues. This resulted in complex-type N-glycans with a core structure identical to the human one. Compared to humans, plants lack the ability to elongate these N-glycans with ß1,4-linked galactoses and terminal sialic acids. However, these modifications, which require the activity of several mammalian enzymes, have already been achieved for Nicotiana benthamiana and the moss Physcomitrella. Here, we present the first step towards sialylation of recombinant glycoproteins in Physcomitrella, human ß1,4-linked terminal N-glycan galactosylation, which was achieved by the introduction of a chimeric ß1,4-galactosyltransferase (FTGT). This chimeric enzyme consists of the moss α1,4-fucosyltransferase transmembrane domain, fused to the catalytic domain of the human ß1,4-galactosyltransferase. Stable FTGT expression led to the desired ß1,4-galactosylation. However, additional pentoses of unknown identity were also observed. The nature of these pentoses was subsequently determined by Western blot and enzymatic digestion followed by mass spectrometric analysis and resulted in their identification as α-linked arabinoses. Since a pentosylation of ß1,4-galactosylated N-glycans was reported earlier, e.g., on recombinant human erythropoietin produced in glyco-engineered Nicotiana tabacum, this phenomenon is of a more general importance for plant-based production platforms. Arabinoses, which are absent in humans, may prevent the full humanization of plant-derived products. Therefore, the identification of these pentoses as arabinoses is important as it creates the basis for their abolishment to ensure the production of safe biopharmaceuticals in plant-based systems.

A synthetic protein as efficient multitarget regulator against complement over-activation.

The complement system constitutes the innate defense against pathogens. Its dysregulation leads to diseases and is a critical determinant in many viral infections, e.g., COVID-19. Factor H (FH) is the main regulator of the alternative pathway of complement activation and could be a therapy to restore homeostasis. However, recombinant FH is not available. Engineered FH versions may be alternative therapeutics. Here, we designed a synthetic protein, MFHR13, as a multitarget complement regulator. It combines the dimerization and C5-regulatory domains of human FH-related protein 1 (FHR1) with the C3-regulatory and cell surface recognition domains of human FH, including SCR 13. In summary, the fusion protein MFHR13 comprises SCRs FHR11-2:FH1-4:FH13:FH19-20. It protects sheep erythrocytes from complement attack exhibiting 26 and 4-fold the regulatory activity of eculizumab and human FH, respectively. Furthermore, we demonstrate that MFHR13 and FHR1 bind to all proteins forming the membrane attack complex, which contributes to the mechanistic understanding of FHR1. We consider MFHR13 a promising candidate as therapeutic for complement-associated diseases.

Pooling individual participant data from randomized controlled trials: Exploring potential loss of information.

BACKGROUND: Pooling individual participant data to enable pooled analyses is often complicated by diversity in variables across available datasets. Therefore, recoding original variables is often necessary to build a pooled dataset. We aimed to quantify how much information is lost in this process and to what extent this jeopardizes validity of analyses results. METHODS: Data were derived from a platform that was developed to pool data from three randomized controlled trials on the effect of treatment of cardiovascular risk factors on cognitive decline or dementia. We quantified loss of information using the R-squared of linear regression models with pooled variables as a function of their original variable(s). In case the R-squared was below 0.8, we additionally explored the potential impact of loss of information for future analyses. We did this second step by comparing whether the Beta coefficient of the predictor differed more than 10% when adding original or recoded variables as a confounder in a linear regression model. In a simulation we randomly sampled numbers, recoded those < = 1000 to 0 and those >1000 to 1 and varied the range of the continuous variable, the ratio of recoded zeroes to recoded ones, or both, and again extracted the R-squared from linear models to quantify information loss. RESULTS: The R-squared was below 0.8 for 8 out of 91 recoded variables. In 4 cases this had a substantial impact on the regression models, particularly when a continuous variable was recoded into a discrete variable. Our simulation showed that the least information is lost when the ratio of recoded zeroes to ones is 1:1. CONCLUSIONS: Large, pooled datasets provide great opportunities, justifying the efforts for data harmonization. Still, caution is warranted when using recoded variables which variance is explained limitedly by their original variables as this may jeopardize the validity of study results.

Stable Protein Sialylation in Physcomitrella.

Recombinantly produced proteins are indispensable tools for medical applications. Since the majority of them are glycoproteins, their N-glycosylation profiles are major determinants for their activity, structural properties and safety. For therapeutical applications, a glycosylation pattern adapted to product and treatment requirements is advantageous. Physcomitrium patens (Physcomitrella, moss) is able to perform highly homogeneous complex-type N-glycosylation. Additionally, it has been glyco-engineered to eliminate plant-specific sugar residues by knock-out of the ß1,2-xylosyltransferase and α1,3-fucosyltransferase genes (Δxt/ft). Furthermore, Physcomitrella meets wide-ranging biopharmaceutical requirements such as GMP compliance, product safety, scalability and outstanding possibilities for precise genome engineering. However, all plants, in contrast to mammals, lack the capability to perform N-glycan sialylation. Since sialic acids are a common terminal modification on human N-glycans, the property to perform N-glycan sialylation is highly desired within the plant-based biopharmaceutical sector. In this study, we present the successful achievement of protein N-glycan sialylation in stably transformed Physcomitrella. The sialylation ability was achieved in a Δxt/ft moss line by stable expression of seven mammalian coding sequences combined with targeted organelle-specific localization of the encoded enzymes responsible for the generation of ß1,4-galactosylated acceptor N-glycans as well as the synthesis, activation, transport and transfer of sialic acid. Production of free (Neu5Ac) and activated (CMP-Neu5Ac) sialic acid was proven. The glycosidic anchor for the attachment of terminal sialic acid was generated by the introduction of a chimeric human ß1,4-galactosyltransferase gene under the simultaneous knock-out of the gene encoding the endogenous ß1,3-galactosyltransferase. Functional complex-type N-glycan sialylation was confirmed via mass spectrometric analysis of a stably co-expressed recombinant human protein.

Associations of Subjective Memory Complaints and Simple Memory Task Scores With Future Dementia in the Primary Care Setting.

PURPOSE: Family physicians need simple yet comprehensive algorithms to discriminate between community-dwelling older persons who are at increased risk of dementia and those who are not. We aimed to investigate associations between incident dementia and responses to a single question regarding subjective memory complaints (SMC) combined with scores on 2 simple memory tests that are easy to use in the primary care setting. METHODS: Analyses were based on data from 3,454 community-dwelling older persons who participated in the 6- to 8-year Prevention of Dementia by Intensive Vascular Care (preDIVA) trial, yielding 21,341 person-years of observation. Participants were considered a single cohort. We used Cox models to assess separate and combined associations of SMC, an imperfect score on the Mini-Mental State Examination delayed recall item (MMSE-5), and an imperfect score on the Visual Association Test (VAT) with future dementia. RESULTS: Subjective memory complaints alone were associated with future dementia (hazard ratio [HR] = 3.01; 95% CI, 2.31-3.94; P <.001), as were the MMSE-5 (HR = 2.14; 95% CI, 1.59-2.87; P <.001) and VAT (HR = 3.19; 95% CI, 2.46-4.13; P <.001) scores. After a median follow-up of 6.7 years, the occurrence of dementia ranged from 4% to 30% among persons with SMC, depending on the MMSE-5 and VAT scores. These test scores did not substantially alter the association with future dementia for persons without SMC. CONCLUSIONS: In persons with SMC, the strength of the association between future dementia and an imperfect MMSE-5 score depends substantially on the VAT score.

Recombinant Production of MFHR1, A Novel Synthetic Multitarget Complement Inhibitor, in Moss Bioreactors.

The human complement system is an important part of the immune system responsible for lysis and elimination of invading microorganisms and apoptotic body cells. Improper activation of the system due to deficiency, mutations, or autoantibodies of complement regulators, mainly factor H (FH) and FH-related proteins (FHRs), causes severe kidney and eye diseases. However, there is no recombinant FH therapeutic available on the market. The first successful recombinant production of FH was accomplished with the moss bioreactor, Physcomitrella patens. Recently, a synthetic regulator, MFHR1, was designed to generate a multitarget complement inhibitor that combines the activities of FH and the FH-related protein 1 (FHR1). The potential of MFHR1 was demonstrated in a proof-of-concept study with transiently transfected insect cells. Here, we present the stable production of recombinant glyco-engineered MFHR1 in the moss bioreactor. The key features of this system are precise genome engineering via homologous recombination, Good Manufacturing Practice-compliant production in photobioreactors, high batch-to-batch reproducibility, and product stability. Several potential biopharmaceuticals are being produced in this system. In some cases, these are even biobetters, i.e., the recombinant proteins produced in moss have a superior quality compared to their counterparts from mammalian systems as for example moss-made aGal, which successfully passed phase I clinical trials. Via mass spectrometry-based analysis of moss-produced MFHR1, we now prove the correct synthesis and modification of this glycoprotein with predominantly complex-type N-glycan attachment. Moss-produced MFHR1 exhibits cofactor and decay acceleration activities comparable to FH, and its mechanism of action on multiple levels within the alternative pathway of complement activation led to a strong inhibitory activity on the whole alternative pathway, which was higher than with the physiological regulator FH.

A network approach on the relation between apathy and depression symptoms with dementia and functional disability.

BACKGROUND: Studies on the association between depression and dementia risk mostly use sum scores on depression questionnaires to model symptomatology severity. Since individual items may contribute differently to this association, this approach has limited validity. METHODS: We used network analysis to investigate the functioning of individual Geriatric Depression Scale (GDS-15) items, of which, based on studies that used factor analysis, 3 are generally considered to measure apathy (GDS-3A) and 12 depression (GDS-12D). Functional disability and future dementia were also included in our analysis. Data were extracted from 3229 participants of the Prevention of Dementia by Intensive Vascular care trial (preDIVA), analyzed as a single cohort, yielding 20,542 person-years of observation. We estimated a sparse network by only including connections between variables that could not be accounted for by variance in other variables. For this, we used a repeated L1 regularized regression procedure. RESULTS: This procedure resulted in a selection of 59/136 possible connections. GDS-3A items were strongly connected to each other and with varying strength to several GDS-12D items. Functional disability was connected to all three GDS-3A items and the GDS-12D items "helplessness" and "worthlessness". Future dementia was only connected to the GDS-12D item "memory problems", which was in turn connected to the GDS-12D items "unhappiness" and "helplessness" and all three GDS-3A items. CONCLUSION: Network analysis reveals interesting relationships between GDS items, functional disability and dementia risk. We discuss what implications our results may have for (future) research on the associations between depression and/or apathy with dementia.

Healthy ageing through internet counselling in the elderly (HATICE): a multinational, randomised controlled trial.

BACKGROUND: Although web-based interventions have been promoted for cardiovascular risk management over the past decade, there is limited evidence for effectiveness of these interventions in people older than 65 years. The healthy ageing through internet counselling in the elderly (HATICE) trial aimed to determine whether a coach-supported internet intervention for self-management can reduce cardiovascular risk in community-dwelling older people. METHODS: This prospective open-label, blinded endpoint clinical trial among people age 65 years or over at increased risk of cardiovascular disease randomly assigned participants in the Netherlands, Finland, and France to an interactive internet intervention stimulating coach-supported self-management or a control platform. Primary outcome was the difference from baseline to 18 months on a standardised composite score (Z score) of systolic blood pressure, LDL cholesterol, and body-mass index (BMI). Secondary outcomes included individual risk factors and cardiovascular endpoints. This trial is registered with the ISRCTN registry, 48151589, and is closed to accrual. FINDINGS: Among 2724 participants, complete primary outcome data were available for 2398 (88%). After 18 months, the primary outcome improved in the intervention group versus the control group (0·09 vs 0·04, respectively; mean difference -0·05, 95% CI -0·08 to -0·01; p=0·008). For individual components of the primary outcome, mean differences (intervention vs control) were systolic blood pressure -1·79 mm Hg versus -0·67 mm Hg (-1·12, -2·51 to 0·27); BMI -0·23 kg/m2 versus -0·08 kg/m2 (-0·15, -0·28 to -0·01); and LDL -0·12 mmol/L versus -0·07 mmol/L (-0·05, -0·11 to 0·01). Cardiovascular disease occurred in 30 (2·2%) of 1382 patients in the intervention versus 32 (2·4%) of 1333 patients in the control group (hazard ratio 0·86, 95% CI 0·52 to 1·43). INTERPRETATION: Coach-supported self-management of cardiovascular risk factors using an interactive internet intervention is feasible in an older population, and leads to a modest improvement of cardiovascular risk profile. When implemented on a large scale this could potentially reduce the burden of cardiovascular disease. FUNDING: European Commission Seventh Framework Programme.

Detecting Mild Cognitive Deficits in Parkinson's Disease: Comparison of Neuropsychological Tests.

BACKGROUND: Numerous neuropsychological tests and test versions are used in Parkinson's disease research, but their relative capacity to detect mild cognitive deficits and their comparability across studies are unknown. The objective of this study was to identify neuropsychological tests that consistently detect cognitive decline in PD across studies. METHODS: Data from 30 normed neuropsychological tests across 20 international studies in up to 2908 nondemented PD patients were analyzed. A subset of 17 tests was administered to up to 1247 healthy controls. A 2-step meta-analytic approach using standardized scores compared performance in PD with normative data. RESULTS: Pooled estimates of the differences between PD and site-specific healthy controls identified significant cognitive deficits in PD patients on 14 test scores across 5 commonly assessed cognitive domains (attention or working memory, executive, language, memory, and visuospatial abilities), but healthy control performance was statistically above average on 7 of these tests. Analyses based on published norms only, as opposed to direct assessment of healthy controls, showed high between-study variability that could not be accounted for and led to inconclusive results. CONCLUSIONS: Normed neuropsychological tests across multiple cognitive domains consistently detect cognitive deficits in PD when compared with site-specific healthy control performance, but relative PD performance was significantly affected by the inclusion and type of healthy controls versus the use of published norms only. Additional research is needed to identify a cognitive battery that can be administered in multisite international studies and that is sensitive to cognitive decline, responsive to therapeutic interventions, and superior to individual cognitive tests. © 2018 International Parkinson and Movement Disorder Society.

Association of Apathy With Risk of Incident Dementia: A Systematic Review and Meta-analysis.

Importance: Fear of dementia is pervasive in older people with cognitive concerns. Much research is devoted to finding prognostic markers for dementia risk. Studies suggest apathy in older people may be prodromal to dementia and could be a relevant, easily measurable predictor of increased dementia risk. However, evidence is fragmented and methods vary greatly between studies. Objective: To systematically review and quantitatively synthesize the evidence for an association between apathy in dementia-free older individuals and incident dementia. Data Sources: Two reviewers conducted a systematic search of Medline, Embase, and PsychINFO databases. Study Selection: Inclusion criteria were (1) prospective cohort studies, (2) in general populations or memory clinic patients without dementia, (3) with clear definitions of apathy and dementia, and (4) reporting on the association between apathy and incident dementia. Data Extraction and Synthesis: PRISMA and MOOSE guidelines were followed. Data were extracted by 1 reviewer and checked by a second. Main Outcomes and Measures: Main outcomes were pooled crude risk ratios, maximally adjusted reported hazard ratios (HR), and odds ratios (OR) using DerSimonian-Laird random effects models. Results: The mean age of the study populations ranged from 69.2 to 81.9 years (median, 71.6 years) and the percentage of women ranged from 35% to 70% (median, 53%). After screening 2031 titles and abstracts, 16 studies comprising 7365 participants were included. Apathy status was available for 7299 participants. Studies included populations with subjective cognitive concerns (n = 2), mild cognitive impairment (n = 11), cognitive impairment no dementia (n = 1), or mixed cognitive and no cognitive impairment (n = 2). Apathy was present in 1470 of 7299 participants (20.1%). Follow-up ranged from 1.2 to 5.4 years. In studies using validated apathy definitions (n = 12), the combined risk ratio of dementia for patients with apathy was 1.81 (95% CI, 1.32-2.50; I2 = 76%; n = 12), the hazard ratio was 2.39 (95% CI, 1.27-4.51; I2 = 90%; n = 7), and the odds ratio was 17.14 (95% CI, 1.91-154.0; I2 = 60%; n = 2). Subgroup analyses, meta-regression, and individual study results suggested the association between apathy and dementia weakened with increasing follow-up time, age, and cognitive impairment. Meta-regression adjusting for apathy definition and follow-up time explained 95% of heterogeneity in mild cognitive impairment. Conclusions and Relevance: Apathy was associated with an approximately 2-fold increased risk of dementia in memory clinic patients. Moderate publication bias may have inflated some of these estimates. Apathy deserves more attention as a relevant, cheap, noninvasive, and easily measureable marker of increased risk of incident dementia with high clinical relevance, particularly because these vulnerable patients may forgo health care.

Apathy is associated with incident dementia in community-dwelling older people.

OBJECTIVE: To assess whether apathy and depressive symptoms are independently associated with incident dementia during 6-year follow-up in a prospective observational population-based cohort study. METHODS: Participants were community-dwelling older people in the Prevention of Dementia by Intensive Vascular Care trial, aged 70-78 years, without dementia at baseline. Apathy and depressive symptoms were measured using the 15-item Geriatric Depression Scale (GDS-15). Dementia during follow-up was established by clinical diagnosis confirmed by an independent outcome adjudication committee. Hazard ratios (HRs) were calculated using Cox regression analyses. Given its potentially strong relation with incipient dementia, the GDS item referring to memory complaints was assessed separately. RESULTS: Dementia occurred in 232/3,427 (6.8%) participants. Apathy symptoms were associated with dementia (HR 1.28, 95% confidence interval [CI] 1.12-1.45; p < 0.001), also after adjustment for age, sex, Mini-Mental State Examination score, disability, and history of stroke or cardiovascular disease (HR 1.21, 95% CI 1.06-1.40; p = 0.007), and in participants without depressive symptoms (HR 1.26, 95% CI 1.06-1.49; p = 0.01). Depressive symptoms were associated with dementia (HR 1.12, 95% CI 1.05-1.19), also without apathy symptoms (HR 1.16, 95% CI 1.03-1.31; p = 0.015), but not after full adjustment or after removing the GDS item on memory complaints. CONCLUSIONS: Apathy and depressive symptoms are independently associated with incident dementia in community-dwelling older people. Subjective memory complaints may play an important role in the association between depressive symptoms and dementia. Our findings suggest apathy symptoms may be prodromal to dementia and might be used in general practice to identify individuals without cognitive impairment at increased risk of dementia.

Moss-Produced, Glycosylation-Optimized Human Factor H for Therapeutic Application in Complement Disorders.

Genetic defects in complement regulatory proteins can lead to severe renal diseases, including atypical hemolytic uremic syndrome and C3 glomerulopathies, and age-related macular degeneration. The majority of the mutations found in patients with these diseases affect the glycoprotein complement factor H, the main regulator of the alternative pathway of complement activation. Therapeutic options are limited, and novel treatments, specifically those targeting alternative pathway activation, are highly desirable. Substitution with biologically active factor H could potentially treat a variety of diseases that involve increased alternative pathway activation, but no therapeutic factor H is commercially available. We recently reported the expression of full-length recombinant factor H in moss (Physcomitrella patens). Here, we present the production of an improved moss-derived recombinant human factor H devoid of potentially immunogenic plant-specific sugar residues on protein N-glycans, yielding approximately 1 mg purified moss-derived human factor H per liter of initial P. patens culture after a multistep purification process. This glycosylation-optimized factor H showed full in vitro complement regulatory activity similar to that of plasma-derived factor H and efficiently blocked LPS-induced alternative pathway activation and hemolysis induced by sera from patients with atypical hemolytic uremic syndrome. Furthermore, injection of moss-derived factor H reduced C3 deposition and increased serum C3 levels in a murine model of C3 glomerulopathy. Thus, we consider moss-produced recombinant human factor H a promising pharmaceutical product for therapeutic intervention in patients suffering from complement dysregulation.

Reference intervals for red cell variables and platelet counts in infants at 2, 5 and 13 months of age: a cohort study.

AIMS: To derive reference values for red cell variables and platelet counts from a cohort of infants sampled at precise ages during the first 13 months of life. METHODS: Blood counts, reticulocyte counts and zinc protoporphyrin concentrations were obtained from healthy term infants of North European ancestry at 2, 5 and 13 months of age. RESULTS: Mean cell volume (MCV) and mean cell haemoglobin (MCH) values did not differ significantly between 5 and 13 months and MCH concentration was unaffected by age. Values of all other variables at any one age differed significantly from those at the other two. Haemoglobin, mean cell haemoglobin, zinc protoporphyrin and platelet values (95% ranges) at 2 (n=119), 5 (n=97) and 13 months (n=42) were, respectively, 91-125, 101-129 and 105-133 g/L; 28.6-33.1, 24.5-28.7 and 24.3-28.7 pg; 36-116, 25-91 and 27-57 micromol/mol haem; and 216-658, 241-591 and 209-455×10(9)/L. At 2 and 5 months, respectively, 26.9% and 10.8% of subjects had platelet counts >500×10(9)/L. Reticulocyte counts at 2 months and MCV and MCH values at 5 months were significantly higher in girls. In boys, red cell distribution width values were significantly higher at 5 months, and zinc protoporphyrin values at both 2 and 5 months. CONCLUSIONS: These findings indicate the value of obtaining reference data at precise ages during infancy and confirm and extend earlier reports indicating a gender difference in laboratory measures used to assess iron status in early infancy.

Clinical significance of azathioprine active metabolite concentrations in inflammatory bowel disease.

BACKGROUND AND AIMS: There are conflicting reports on the role of azathioprine (AZA) thioguanine nucleotide (TGN) metabolites in optimising therapy for inflammatory bowel disease (IBD). The aim of this study was to investigate TGN intrapatient variation, and the relationship between TGN concentrations and disease activity in IBD patients taking long term constant dose AZA. METHODS: TGN and methylmercaptopurine nucleotide (MeMPN) concentrations were measured at intervals over a two year period. Disease activity was assessed at each clinic visit using the Crohn's disease activity index or Walmsley simple index for ulcerative colitis. RESULTS: Serial TGNs were measured in 159 patients (3-14 TGN assays, median 6). Intrapatient variation in TGN concentrations was 1-5-fold (median 1.6); the incidence of non-compliance was 13%. At the end of two years, 131 patients were evaluable at TGN steady state. Of this group, patients who remained in remission had significantly higher mean TGN concentrations than those patients who developed active disease (median TGNs 236 v 175, respectively; median difference 44 pmol (95% confidence interval 1-92); p = 0.04). MeMPN concentrations were not related to AZA efficacy or toxicity. CONCLUSIONS: This study has shown that lower TGN concentrations were linked to the development of active disease, and that TGNs may act as useful markers of compliance. However, it is clear that repeat TGN measurements are required for an unambiguous index of active metabolite exposure. In view of the high intrapatient variability in TGN production over time, TGN measurements may not be currently advocated for routine clinical use.