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References | ADVERSE EVENTS, ADVERSE EVENT | HBsAg (Summary of subject disposition in Phase I study.Percentage change in HBsAg in Phase I study. (Mean HBsAg and HBsAg clearance rate at the end of the animal study.Summary of demographics and baseline characteristics in the Phase I clinical study.Max: maximal; Min: minimal; SD: standard deviation.Summary of adverse events (AEs) in the Phase I study.AE: adverse event; SAE: serious adverse event; ALT: alanine transaminase; AST: aspartate aminotransferase. Note: (1) % = percentage of patients with n as the denominator. | PMC10778875 | |
Subject terms | OLP | The use of so-called ‘smart drugs’ such as modafinil to improve cognitive performance has recently attracted considerable attention. However, their side effects have limited user enthusiasm. Open-label placebo (OLP) treatment, i.e., inert treatments that are openly disclosed to individuals as having no active pharmacological ingredient, has been shown to improve various medical symptoms and conditions, including those related to cognitive performance. OLP treatment could therefore be an exciting alternative to pharmacological cognitive enhancers. Here, we used a randomized-controlled design to investigate the effect of a 21-day OLP treatment on several sub-domains of cognitive performance in Open Access funding enabled and organized by Projekt DEAL. | PMC10636058 | |
Introduction | Cognitive performance is a vital aspect of daily functioning, and crucial for educational and occupational successFor example, students with a positive treatment expectation showed improvements in a standard IQ testIn search of strategies to overcome the ethical and legal limitations of deceptive placebos and increase trust in healthcare providersSo far, studies in healthy individuals assessing cognitive enhancement through OLPs are still scarce, but results of existing deceptive placebo studies point towards an interesting divide in this effect: While subjective markers (e.g., the subjective perception of one’s own performance) indicate improvements under OLP, this improvement is not reflected in objective performance parameters | PMC10636058 | ||
Materials and methods | PMC10636058 | |||
Clinical trial registration | The monocentric, randomized controlled study was registered in the German Clinical Trials Register on 30/10/2019 (Deutsches Register Klinischer Studien; DRKS; ID DRKS00019203; | PMC10636058 | ||
Ethics approval statement | All study procedures were approved by the University Duisburg-Essen Ethics Committee (19-8874-BO). The study was conducted in accordance with the principles of Good Clinical Practice | PMC10636058 | ||
Participants | allergies | COLOR BLINDNESS, RECRUITMENT, ALLERGIES | Recruitment and data collection took place from October 2019 to March 2020 at the University Hospital Essen in Germany. Participants were recruited via flyers and posters at regional universities and the University Hospital Essen, via an in-house participant database and via social media. Those between 18 and 60 years of age were eligible to take part. Exclusion criteria comprised severe chronic or acute clinical conditions, regular substance use of cannabinoids, cocaine, or amphetamines in the last 4 weeks, color blindness, known allergies or intolerance against the ingredients of the placebo medication, pregnancy or breastfeeding, alcohol consumption on the testing days or the day before, or participation in other (medication-related) studies in the three weeks of testing. All participants underwent an initial phone interview and gave written, informed consent prior to participation. All participants received 50 € after study completion, plus the bonus gained in the Instrumental Learning Task (means and standard deviations are always reported as We collected data from 100 participants. Data of those participants who had only completed the Baseline assessment were discarded ( | PMC10636058 |
Procedure | OLP | The study followed a single-blind, between-subjects design with two sessions (Baseline and Test; see also Fig. Overview of the study design. First, all tasks were completed before participants completed the questionnaires. Tests and questionnaires were then completed in German and in a randomized order, except the d2 which was administered first and the learning task which was always administered last. Trait questionnaires/tasks were only administered at the Baseline (*), all state measures were acquired twice. Each box either contained 45 placebo pills (Zeebo Relief from Zeebo Effect, LCC; white-blue pills filled with silicon dioxide, titanium dioxide and microcrystalline cellulose) in their original packaging (OLP group), or filler material of similar weight but no pills (CTR group). Both groups also received a note that stated the group they had been allocated to, OLP intake instructions (OLP group) or the instructions to do nothing (CTR group) and a question asking about their satisfaction with the group assignment. We chose this approach to control for test–retest training effects over time. The OLP group was fully informed that the pills were placebos and instructed to take one tablet twice daily for 21 days, starting with the day after the Baseline session. This procedure and duration of the OLP intervention had been successfully established in previous work | PMC10636058 | |
Baseline measures | At the Baseline session, we collected demographic data (age, gender, employment, years of education, medication intake and chronic conditions) from each participant as well as an in-house health questionnaire, including data on participants’ use of alcohol, caffeine, nicotine and other substances, to monitor changes in health status and validate the absence of exclusion criteria. To make sure the two groups were comparable in terms of intelligence, we administered the | PMC10636058 | ||
Outcome measures | fatigue | To investigate the effects of OLPs on cognitive performance, we aimed to assess a wide range of complex cognitive abilities while balancing feasibility, time constraints, and participant fatigue. We thus included five standardized, well-established tasks measuring the following aspects of cognitive performance, which have shown robust effects in previous work: mental processing conflicts (Stroop task), divided attention (Dual task), working memory (2-back task), learning of gains and losses (Instrumental learning task), and attention and concentration (d2 task).Both groups received the same standardized instructions for each measure. All tasks and questionnaires were completed in German either on a computer or in a paper–pencil format, as indicated below. For all tasks, we excluded trials with reaction times below 200 ms | PMC10636058 | |
Objective measures | The The In the The The | PMC10636058 | ||
Subjective measures | OLP, ’ | SECONDARY | After each of the tasks described above, participants were asked to rate their cognitive performance using computerized Subsequently, we administered five standardized questionnaires, which were preregistered as secondary outcomes, and measured different psychological parameters at both time points: The Lastly, at the Test session, participants were asked to indicate their satisfaction with their group allocation. The question ‘Are you satisfied with your group allocation (placebo or control group)?’ was answered on a VAS from 0 to 100 with the anchors ‘no, not at all’ and ‘yes, absolutely’. They were also asked in a yes-or-no question, whether they would have preferred to be in the other group. The OLP group was then asked a few questions about their adherence to the placebo intake (how regularly they took the placebo capsules, whether they thought the placebo intake improved aspects such as concentration, “mental freshness”, mood, well-being, sleep, and bodily complaints, whether they would recommend the OLP intake to friends or family and whether they would like to continue the OLP intake; each on a VAS from 0 to 100 with the anchors ‘no, not at all’ and ‘yes, very’). | PMC10636058 |
Data acquisition and analysis | Computerized tasks were administered in a random order using Presentation (version 18.0, Neurobehavioral Systems, Inc. Berkeley, CA, USA), except the learning task which was always administered last. Questionnaires were administered via LimeSurvey (version 4.3.15In order to test whether the two groups differed in any of the measures prior to the treatment phase, we compared their task and questionnaires scores at Baseline. To this end, we computed Welch’s two-sample For all tests, an initial alpha error probability of Following the reviewer’s comments, we exploratorily re-ran our primary analyses with gender as a factor and report those results in the Supplement (Tables | PMC10636058 | ||
Results | PMC10636058 | |||
Baseline group comparison and manipulation checks | All Baseline group differences and respective statistics are reported in Table Participants in the OLP group indicated taking the OLPs regularly (93.37 ± 7.34 ( | PMC10636058 | ||
Effects of OLP intake on objective task measures | First, we analyzed participants’ performance in the objective performance measures (Stroop, dual, 2-back, instrumental learning, and d2). Figures Evidence for training effects, but evidence of absence for any OLP effects on exemplary primary objective outcome measures: (Evidence for time effects, but evidence of absence for group differences in exemplary primary subjective outcome measures regarding (Contrary to our expectations, none of our primary objective outcome measures demonstrated any group effects (main effects or interactions; all | PMC10636058 | ||
Effects of OLP intake on subjective task measures | OLP | Next, we analyzed participants’ subjective evaluation of their objective performance, namely effort invested, satisfaction with their performance, and perceived change from Baseline to Test.Regarding effort, we again found no effect of OLP intake on these outcomes and Bayesian analyses supported the null over the alternative hypothesis with anecdotal to substantial evidence (main effects of or interactions with group; all Regarding participants’ subjective assessment of their satisfaction with their own performance, the OLP group reported higher overall satisfaction, independent of time point in the d2 task as indicated by a main effect of group (Regarding participants’ subjective assessment of whether their cognitive performance changed in any of the objective tasks from Baseline to Test, we found no significant group effects or other differences and anecdotal (dual, 2-back), but mostly substantial (d2, Stroop, learning) evidence for the null compared to the alternative hypothesis regarding (all | PMC10636058 | |
Effects of OLP intake on subjective state questionnaires | depression, anxiety, OLP | Lastly, we analyzed the state questionnaires on anxiety, depression, mood, stress, well-being, sleep, and movement/sport activity, which participants had completed before and after OLP intervention. Mirroring the above analyses, we found no OLP effects on any of these outcomes (see Table In a post-experimental questionnaire at Test, participants had a low belief in OLP induced performance improvement in the computer tasks ( | PMC10636058 | |
Discussion | cognitive functioning, fatigue, OLP | Here, we investigated the performance-enhancing effects of OLPs using an extensive battery of subjective and objective cognitive performance measures and well-being. Our analyses suggest that this treatment had no significant impact on any of the outcomes, compared to a no-treatment control condition. As expected, participants’ performance generally improved from Baseline to Test in most tasks, most likely reflecting training effects in tasks assessing attention and concentration, working memory, and instrumental learningThe lack of objective improvements in cognitive performance is not unusual. Previous studies using deceptive placebos also yielded mixed results for objective parameters in healthy participantsThe absence of any improvement in subjective outcome including perceived performance and satisfaction therewith as well as no improvements of general well-being, however, is surprising given that many studies in clinical, but also non-clinical samples report subjective improvement under OLP treatment. Importantly, this also applies to trials with no changes in objective measures, which indicates that the perception of the patient or healthy participant can deviate from measurable outcome. In contrast to objective outcome parameters which are judged against an external criterion, subjective outcomes require the individual to compare any perceived changes in the relevant metric (e.g., processing speed in a cognitive task) to an internal standard to decide whether the change is meaningful, i.e., significantly different from noise. This threshold is likely to be influenced by the individual’s motivation to categorize changes as meaningful—particularly when they are subtle in nature. A strong motivation will lower the threshold whereas a weak motivation will lead to a higher threshold. Furthermore, the change must be attributed to the treatment (as opposed to coincidence). Several of our subjective outcome measures seem to indicate a rather critical assessment of their treatment in the OLP group which could reflect a high threshold to detect change. Participants in this group were significantly less satisfied with their group allocation than the control group, would have preferred to be in the control group, would not recommend OLPs to others and provided low ratings on their willingness to continue the treatment. Moreover, 5% of participants were excluded because they indicated that they had taken the OLP less than 70% of the time and 12% did not attend the second session.Psychological theories consider motivation or attitude towards an entity as the product of two components—expectancy and valueExpectancy as the second determinant has been a focus of several investigations into factors determining placebo outcome in the past. For deceptive placebo treatments, beneficial effects are thought to depend on an individual’s positive treatment expectationExplicit verbal information about the nature and potential benefits of placebos are a key component of many OLP studiesOur findings have to be interpreted in light of some limitations. First, our sample size was smaller than anticipated. To explore the robustness of our findings, we therefore decided to also apply Bayesian statistics. These analyses provided direct relative evidence for the null vs. the alternative hypothesis. Moreover, to ensure that the results we report are also of clinical significance, we were mainly interested in medium to large effects which can also be detected in a smaller sample such as ours. Second, not all tasks were administered in a randomized order. Especially for the learning task, which was always administered last, we therefore cannot rule out that the results are confounded by an increasing level of fatigue. However, as our findings compare two groups, such an effect (and also training effects) should occur in both groups. Third, we chose established cognitive tasks that had been successfully implemented in previous work and which are sensitive to performance improvement through OLPs while avoiding ceiling effects (e.g., 2-back vs. 3-back). It remains to be investigated whether more cognitively demanding tasks (e.g., 3- or 4-back) would be even more sensitive to the effect of OLPs. Fourth, previous work has shown that a 3-week treatment with OLPs is sufficient to induce effectsTogether, our findings do not support the assumption that OLPs can enhance cognitive functioning in healthy adults, and thereby showcase potential limitations of OLP treatments. Instead, we highlight important considerations for future studies aiming to include OLP treatments. Our proposed framework that follows expectancy-value considerations of motivation suggests several trajectories for future research to gain a more comprehensive understanding of the mechanisms and effects of OLP treatments and predictors for an individual’s response. First, the role of expectancy, value and costs, their respective determinants and interactions need to be investigated in more detail. A better understanding could not only help patient stratification but also aid the systematic investigation of differences in OLP responses between clinical and non-clinical groups and between objective and subjective outcome measures. Second, information about individual expectation, value and cost estimations could inform computational models which promise to reveal (neural) mechanisms underlying observable behaviour. Lastly, a deeper understanding could guide efforts to improve OLP outcome. For instance, expectations might be easier to modulate than costs, which are often outside the control of the practitioner. Information provided before OLP treatment could therefore focus on raising expectations while managing costs or may be tailored to fit the needs or preferences of the individual patient. | PMC10636058 | |
Supplementary Information | The online version contains supplementary material available at 10.1038/s41598-023-45979-3. | PMC10636058 | ||
Acknowledgements | SCHMIDT | The authors thank Sigrid Elsenbruch for valuable input on the results and discussion. They also thank Liane Schmidt for providing the code of the instrumental learning task and for helpful scientific exchange during the project. | PMC10636058 | |
Author contributions | Author contributions are listed according to the CRediT statement | PMC10636058 | ||
Funding | Open Access funding enabled and organized by Projekt DEAL. The work is funded by the Deutsche Forschungsgemeinschaft (DFG, German Research Foundation—Project-ID 422744262—TRR 289). None of the funders had any role in study design, data collection and analysis, interpretation, writing or decision to publish. | PMC10636058 | ||
Data availability | The data used for the analysis has been uploaded to the Open Science Framework project ( | PMC10636058 | ||
Competing interests | The authors declare no competing interests. | PMC10636058 | ||
References | PMC10636058 | |||
1. Introduction | Hyperglycemia, prediabetes, diabetes, inflammation, hyperglycemia, prediabetic, causes cell damage | HYPERGLYCEMIA, VASCULAR COMPLICATIONS, INFLAMMATION, DIABETES MELLITUS, HYPERGLYCEMIA, OXIDATIVE STRESS, DISEASES, COMPLICATIONS, ENDOTHELIAL DYSFUNCTION | The most significant reactive α-dicarbonyl RCS involved in the pathomechanism of glycation and related diseases is methylglyoxal (MGO). Hyperglycemia promotes the generation of MGO and leads to the formation of advanced glycation end products (AGEs). Therefore, MGO trapping and glycation inhibition appear to be important therapeutic targets in prediabetes, diabetes, and in the early prevention of hyperglycemic complications. Peppermint leaf is commonly used as herbal tea, rich in polyphenols. Eriocitrin, its predominant component, in a double-blind, randomized controlled study reversed the prediabetic condition in patients. However, the antiglycation activity of this plant material and its polyphenols has not been characterized to date. Therefore, the aim of this study was to evaluate the ability of a peppermint leaf dry extract and its polyphenols to inhibit non-enzymatic protein glycation in a model with bovine serum albumin (BSA) and MGO as a glycation agent. Peppermint polyphenols were also evaluated for their potential to trap MGO in vitro, and the resulting adducts were analyzed by UHPLC-ESI-MS. To relate chemical composition to glycation inhibitory activity, the obtained peppermint extract was subjected to qualitative and quantitative analysis. The capability of peppermint leaf polyphenols to inhibit glycation (27.3–77.2%) and form adducts with MGO was confirmed. In the case of flavone aglycones, mono- and di-adducts with MGO were observed, while eriodictyol and eriocitrin effectively produced only mono-adducts. Rosmarinic acid and luteolin-7-Non-enzymatic glycation is a reaction between the carbonyl groups of reducing sugars or their derivatives such as reactive carbonyl species (RCS) and the amino, guanidino, or thiol groups of some biomolecules including peptides, proteins, lipoproteins, and nucleic acids. Several physiological products of sugar autooxidation are known, as well as intermediates from glucose and fructose metabolism characterized by the presence of two adjacent carbonyl groups (α-dicarbonyl compounds). These compounds are ketoaldehydes or dialdehydes and are currently called RCS due to their high reactivity. The most important ketoaldehyde involved in the pathomechanism of non-enzymatic glycation and related diseases is methylglyoxal (MGO). Long-term hyperglycemia promotes the production of MGO, and this significantly increases glycation, leading, among other things, to the formation of irreversible advanced glycation end products (AGEs). AGEs can bind to specific receptors (RAGE) in the cell membrane and consequently trigger the nuclear factor κB (NF-κB) signaling pathway, which induces inflammation and oxidative stress and causes cell damage. In patients with diabetes mellitus, blood proteins such as hemoglobin and serum albumin are particularly vulnerable to glycation. MGO-derived AGEs have been shown to play a pivotal role in the onset and progression of vascular complications of diabetics. Methylglyoxal thus appears to be a significant contributor to endothelial dysfunction by increasing oxidation, as well as inducing inflammation and apoptosis [Several substances of natural origin that can inhibit non-enzymatic protein glycation in the BSA-methylglyoxal and BSA-glucose systems are known. Such potential has been described, among others, for the polyphenols of rooibos, green and black tea [Peppermint (The diverse chemical composition of Therefore, the aim of our study was to evaluate the ability to inhibit the process of non-enzymatic protein glycation by a peppermint leaf dry extract and its major polyphenolic components in a model with bovine serum albumin and methylglyoxal as a glycation agent. Peppermint polyphenols were also evaluated for their potential to capture MGO in vitro, and the resulting adducts were analyzed by UHPLC-ESI-MS. The effect of individual extract components on the observed antiglycation properties was also investigated. | PMC10056224 |
2. Results and Discussion | digestive ailments | METABOLIC DISORDERS | Peppermint leaf is a popular non-caffeine alternative to green or black tea. It is also used therapeutically for common digestive ailments. Current research indicates that it may also be effective in certain metabolic disorders. There is evidence from an animal model supporting its potential to lower blood levels of glucose, triacylglycerols, cholesterol and LDL (low-density lipoprotein) [Among the pharmacologically active components of The antioxidant potential of peppermint leaf was established using various methods. These properties included iron (III) reduction, iron (II) chelation, DPPH radical scavenging, and the ability to inhibit phospholipid peroxidation catalyzed by iron (III)-ascorbate [ | PMC10056224 |
2.1. Polyphenolic Profile of Peppermint Leaf Dry Extract | In the present study, a dry extract was obtained from a water infusion of peppermint leaf using the solid-phase extraction (SPE) method. SPE is one of the simplest yet most efficient and versatile techniques for concentrating samples and extracts. With properly selected parameters, it also allows partial purification of the concentrated material from substances of high polarity such as salts and sugars. The peppermint leaf dry extract, containing partially purified polyphenols, was subjected to compositional analysis by UHPLC-ESI-MS against authentic flavonoid and phenolic acid standards. The qualitative composition of polyphenols is shown in The content of the major components of the extract was determined using the HPLC-DAD method previously developed and validated for plant materials in the Lamiaceae family. This method has been used successfully in several studies [ | PMC10056224 | ||
2.2. Anti-Glycation Activity of Peppermint Leaf Dry Extract and Its Polyphenolic Components | obesity, inflammation, hypertension | OBESITY, HYPERCHOLESTEROLEMIA, CARDIOVASCULAR DISEASE, HYPERLIPIDEMIA, HYPERTENSION, INFLAMMATION, IMPAIRED GLUCOSE TOLERANCE, DISORDERS, HYPERGLYCEMIA, INSULIN RESISTANCE, ADIPOSITY, METABOLIC SYNDROME, OXIDATIVE STRESS, TYPE 2 DIABETES | Excessive consumption of simple sugars, glucose and fructose, is one of the reasons for the epidemic of obesity, type 2 diabetes, metabolic syndrome, and cardiovascular disease. Non-enzymatic and enzymatic oxidation of monosaccharides leads to excessive production of RCS, carbonyl stress and related oxidative stress. The consequence is inflammation and malfunction of many tissues and organs, including the liver, kidneys, blood vessels and nervous tissue. These disorders mutually reinforce each other and the accompanying pathologies accumulate with increasing age, which is associated with the progression of cardiometabolic factors such as insulin resistance, impaired glucose tolerance, hyperglycemia, hyperlipidemia, hypercholesterolemia, hypertension and central adiposity [Along with HSA, BSA is the most commonly used in model studies of compounds with antiglycation, anti-AGE and anti-RCS potential. ICThe anti-AGE effect of polyphenols is related to their protective action against chemical modifications of substrates involved in the glycation reaction, such as binding to groups involved in the initiation of glycation or inhibiting the oxidation of simple sugars and ketoamines (Amadori products). A key component of this effect is the ability to capture glycation- or oxidation-initiating factors (RCS, ROS) [ | PMC10056224 |
2.3. MGO-Trapping Potential of Peppermint Leaf Polyphenols | Several synthetic medicines and plant polyphenols are known to have the capacity for MGO scavenging [With the help of UHPLC-ESI-MS, we examined the ability of To sum up, (1) non-methylated flavones demonstrated substantially higher antiglycation and MGO-trapping potential than non-methylated flavanones. The difference in anti-MGO activity of luteolin and eriodictyol, as well as luteolin-7-A trapping test conducted on peppermint leaf extract confirmed the observations gained for its individual compounds. Since the predominant component of The possible heterocyclic structures of the mono- and di-methylglyoxal adducts of luteolin, apigenin, eriodictyol and eriocitrin are shown in Eriocitrin is a 7- | PMC10056224 | ||
3. Materials and Methods | PMC10056224 | |||
3.1. Chemicals | POLAND, ALDRICH | Methanol, acetonitrile (LC gradient grade and LC-MS grade), water (LC-MS grade), DMSO, 98–100% formic acid, methylglyoxal (MGO, 40% in water), and bovine serum albumin (BSA) were purchased from Merck–Sigma–Aldrich (Poland–Sigma-Aldrich, Poznań, Poland). Glacial acetic acid, NaCl, KCl, Na | PMC10056224 | |
3.2. Authentic Standards | Eriodictyol (CAS No. 552-58-9), luteolin (CAS No. 491-70-3), luteolin-7-Stock solutions (1 mg/mL) for quantitative analysis were made by dissolving 2–5 mg of flavonoid in 2–5 mL of methanol. Working standard solutions in the range of 10–250 g/mL (6 measurement points for each pattern) were prepared by mixing with 50% aq. methanol ( | PMC10056224 | ||
3.4. Chemical Composition of Peppermint Leaf Dry Extract | ICH | The presence of flavonoids and phenolic acids in peppermint leaf dry extract was confirmed by the UHPLC-ESI-MS method described previously by Bodalska et al. [The content of the dominant compounds was determined by HPLC-DAD. This method was developed and optimized previously by Fecka and Turek [The HPLC-DAD method was validated according to ICH guidelines for linearity, detection and quantification limits, intra- and inter-day precision. Calibration curves for the quantified compounds were determined from 6 measurement points, and double injections were performed for each concentration. The correlation coefficients of the calibration curves (The content of individual polyphenols (mg/g dry extract) was determined using the external standard method based on the area of the corresponding peaks. Polyphenol content was then converted to micromolar concentrations. The contents of flavonoids, phenolic acids, and polyphenols and their percentages in the polyphenol fraction (non-volatile compounds) were also calculated. | PMC10056224 | |
3.5. Antiglycation Non-Enzymatic Assay (BSA-Methylglyoxal Model) | REGRESSION | For this purpose, 21.2 μM bovine serum albumin was incubated with 0.5 mM methylglyoxal and 1–1.5 mM of the test compound or peppermint leaf dry extract (1–3 mg/mL) in 100 mM PBS solution, pH 7.4, with 0.02% sodium azide. The reaction mixture was shaken (50 rpm) at 37 °C, for 7 days, in closed vials without light. The fluorescent intensity of MGO-mediated AGEs formed during incubation was analyzed using a Synergy HTX Multi-Mode Microplate Reader (BioTek Instruments Inc., Winooski, VT, USA) at a wavelength of 360 nm for excitation (λThe percentage contribution of each component to the activity of the peppermint leaf dry extract was calculated from regression equations for the relationship between the concentration [μM] and % inhibition. These were then related to the overall effect exerted by the extract. | PMC10056224 | |
3.6. MGO Trapping and Adduct Analysis | The direct MGO-trapping capacity of peppermint polyphenols was investigated according to the method of Shao et al. [ | PMC10056224 | ||
3.7. Analysis of the Polyphenol-MGO Adducts | RS | Adducts of peppermint polyphenols with MGO were analyzed by UHPLC-ESI-MS using a Thermo Scientific Dionex UltiMate 3000 UHPLC system (Thermo Fisher Scientific; Waltham, MA, USA) incorporated with a Compact ESI-QTOF-MS (Bruker Daltonics; Bremen, Germany), a quaternary pump (LPG-3400D) and an UltiMate 3000 RS autosampler (WPS-3000). The reaction mixtures were separated on a Kinetex C18 column (150 × 2.1 mm, particle size 2.6 μm) (Phenomenex; Torrance, CA, USA) at 40 °C (a temperature-controlled column compartment, TCC-3000). The mobile phases were ( | PMC10056224 | |
3.8. Statistical Analysis | All data are presented as mean ± standard deviation (SD). Data were analyzed using the Shapiro-Wilk test to assess the normality of distribution, followed by one-way analysis of variance (ANOVA) with Tukey’s multiple comparison test using the GraphPad Prism 9 software, and | PMC10056224 | ||
Supplementary Materials | The following supporting information can be downloaded at: Click here for additional data file. | PMC10056224 | ||
Author Contributions | Conceptualization, I.F.; Methodology, Validation, Formal analysis, Investigation, Data curation, I.F. and K.B.; Writing—original draft preparation, I.F., K.B. and A.K.; Writing—review and editing, I.F. and A.K.; Visualization, K.B. and I.F.; Supervision, I.F.; Project administration, and Funding acquisition, A.K. All authors have read and agreed to the published version of the manuscript. | PMC10056224 | ||
Institutional Review Board Statement | Not applicable. | PMC10056224 | ||
Informed Consent Statement | Not applicable. | PMC10056224 | ||
Data Availability Statement | Data supporting reported results are available from the corresponding author. | PMC10056224 | ||
Conflicts of Interest | The authors declare no conflict of interest. | PMC10056224 | ||
Sample Availability | Samples of the compounds will be available by arrangement with the I.F. | PMC10056224 | ||
Supplementary Information | tumor, fatigue, cancer, toxicities, prostate, pain, mCRPC | TUMOR, ADVERSE EVENTS, CANCER, DISEASE, METASTATIC CASTRATION-RESISTANT PROSTATE CANCER, PROSTATE, METASTASES, COLD | Metastatic castration-resistant prostate cancer (mCRPC) is an immunologically cold disease with dismal outcomes. Cryoablation destroys cancer tissue, releases tumor-associated antigens and creates a pro-inflammatory microenvironment, while dendritic cells (DCs) activate immune responses through processing of antigens. Immunotherapy combinations could enhance the anti-tumor efficacy. This open-label, single-arm, single-center phase I trial determined the safety and tolerability of combining cryoablation and autologous immature DC, without and with checkpoint inhibitors. Immune responses and clinical outcomes were evaluated. Patients with mCRPC, confirmed metastases and intact prostate gland were included. The first participants underwent prostate cryoablation with intratumoral injection of autologous DCs in a 3 + 3 design. In the second part, patients received cryoablation, the highest acceptable DC dose, and checkpoint inhibition with either ipilimumab or pembrolizumab. Sequentially collected information on adverse events, quality of life, blood values and images were analyzed by standard descriptive statistics. Neither dose-limiting toxicities nor adverse events > grade 3 were observed in the 18 participants. Results indicate antitumor activity through altered T cell receptor repertoires, and 33% durable (> 46 weeks) clinical benefit with median 40.7 months overall survival. Post-treatment pain and fatigue were associated with circulating tumor cell (CTC) presence at inclusion, while CTC responses correlated with clinical outcomes. This trial demonstrates that cryoimmunotherapy in mCRPC is safe and well tolerated, also for the highest DC dose (2.0 × 10The online version contains supplementary material available at 10.1007/s00262-023-03421-7. | PMC10264291 |
Keywords | Open access funding provided by University of Bergen (incl Haukeland University Hospital) | PMC10264291 | ||
Introduction | death, cancers, tumor, cancer, malignancies, tumors, mCRPC, Cancer | CANCERS, TUMOR, CANCER, SOLID TUMORS, MALIGNANCIES, IMMUNE TOLERANCE, RENAL CELL CARCINOMA, METASTATIC PROSTATE CANCER, TUMORS, METASTATIC CASTRATION-RESISTANT PROSTATE CANCER, SKIN MALIGNANT MELANOMA, CANCER | Mortality rates remain high for patients who develop metastatic castration-resistant prostate cancer (mCRPC) [The immunological checkpoint inhibitor therapies, the cytotoxic T-lymphocyte protein 4 (CTLA-4) inhibitor, ipilimumab and programmed cell death protein 1 (PD-1) inhibitor, pembrolizumab, have demonstrated marked effects on progression-free survival (PFS) in malignant melanoma, renal cell carcinoma, and cancers with DNA repair deficiencies [An essential part of the immune response is mediated by dendritic cells (DCs), which are antigen-presenting cells that in their immature state recognize and process tumor-associated antigens. Early-phase trials of DC-based cancer immunotherapies have demonstrated the treatment as safe and feasible with anti-tumor immune activation, but with limited clinical responses [The capacity of malignancies to inactivate DCs and effector T cells and evade the circulating antitumor immune responses is challenging the development of immune-modulating therapies. The T cell subset, regulatory T-lymphocyte (Treg), plays a dual role of mediating immune tolerance and restricting the antitumor immunity [The heterogeneous expression of molecular targets on cells within and between tumors induces different treatment sensitivities and accordingly poses a challenge for targeted therapies. Previously, in vitro matured DCs have been trained to destroy mainly tumor cells expressing preselected antigens, but additionally generate bystander effects, where untreated cells are biologically altered by stress signals from directly treated cells to mirror these by exhibiting similar effects like reduced cell survival and genomic instability [Cancer cell heterogeneity is one of the most fundamental problems of cancer therapy. By combining cryoablation with intratumoral DC injection, cryoimmunotherapy (CryoIT) exposes the entire individualized collection of tumor-associated antigens to immature DCs (iDCs), which consequently are positioned to tackle the cellular heterogeneity. Cryoablation is a process where tissue destruction is initiated by freezing solid tumors to -40 °C in one or several freeze–thaw cycles. Cryoablation has been investigated in localized, locally recurrent, and metastatic prostate cancer [The primary aim of this phase I clinical trial of pre-treated mCRPC was to examine the safety and tolerability of the novel combination of cancer tissue cryoablation and intratumoral injection of autologous iDCs with and without immunological checkpoint inhibitor enhancement. Secondary and explorative endpoints included time to progression, survival, and monitoring of immune responses and circulating tumor cells. | PMC10264291 |
Materials and methods | PMC10264291 | |||
Participants | hypersensitivity | ONCOLOGY, HYPERSENSITIVITY, METASTASES, IMMUNODEFICIENCY | Eligible patients had mCRPC with metastases and an Eastern Cooperative Oncology Group (ECOG) performance status of 0–1, adequate organ function, no known hypersensitivity to vaccines or components of the cell therapy, and no contraindications to surgery (Supplementary S1B). The exclusion criteria contained immunodeficiency, other active malignancy, and recent or ongoing anti-tumor treatment. Prior radiotherapy and treatment with androgen deprivation and androgen receptor targeting drugs were accepted (Supplementary S1C). | PMC10264291 |
Study design and data capture | prostate, pain | PROSTATE | This phase I open-label interventional study recruited patients at Haukeland University Hospital, Bergen, Norway. The study consisted of two study parts, investigating the safety and tolerability of combining either cryoablation and iDC treatment (first part), or cryoablation, iDC treatment and immune checkpoint inhibitor therapy (second part), illustrated in Fig. Study overview. Generally, participants attended 14 study-specific visits from inclusion to 52 weeks after the CryoIT, with a follow-up period of 72 months. The CryoIT procedure was performed during visit 4 (day 0). Medications, vital signs, and results from the physical examinations and routine laboratory blood analyses were protocolled. Safety assessments were performed during all post-treatment visits. Imaging was performed concomitantly by MRI of the spinal column, pelvic region and prostate gland, whole-body PET/CT with F18-fluorodeoxyglucose, and radionuclide bone scan with [Moreover, health-related quality of life (HRQoL) forms, visual analog scale (VAS) pain measurements, and blood samples were collected as depicted in Fig. | PMC10264291 |
Procedures | prostate | PROSTATE | Leukapheresis was performed 14 days prior to CryoIT followed by monocyte enrichment using the ELUTRATM System (Supplementary S2). Autologous iDCs were produced according to protocol (Supplementary S2).Cryoablation of the prostate was performed under general anesthesia and ultrasound guidance as a cyclic freeze–thaw process (Supplementary S3). Directly prior to the first freezing cycle, eight core prostate biopsies were sampled transrectally. Succeeding cryoablation, the pre-specified number of viable iDCs was injected intratumorally into the prostate. All patients received cyclophosphamide, 300 mg/m2 intravenously three days prior to CryoIT, and low oral doses (50–100 mg/day) metronomically every other week from week 2 to week 26 post-treatment to avoid Treg increases that might diminish incipient immune responses (Supplementary S5, Fig. | PMC10264291 |
Assessment of safety and toxicity | toxicity, toxicities | ADVERSE EVENT | AEs were recorded, and their severity graded according to the Common Terminology Criteria for Adverse Events v3.0. Clinical investigators evaluated the severity and relatedness of any registered AEs to the trial participation, study drug(s) and ablative procedure. Any AEs that could be related to trial participation were defined as treatment emergent. Any severe AEs, defined as AEs ≥ grade 3, were reported to the sponsor within 48 h. To establish the maximum tolerated dose and recommended phase II trial dose of iDCs, the toxicity was defined as dose limiting if patients experienced any persistent grade 4 toxicity. Maximum tolerated dose was defined as the dose level below which dose limiting toxicities were seen in ≥ 1 of 3 subjects. Given the observed tolerability of DC-based therapy in part 1, subjects in part 2 were treated with the highest iDC dose. Dose-limiting toxicity of cryoablation plus iDC treatment was evaluated separately ( | PMC10264291 |
Assessment of response | tumor, Pain, pain, prostate cancer tumor, Cancer | CANCER, DISEASE, TUMOR | Radiologic responses were assessed by internal expert review. MRI and PET/CT images were evaluated according to the RECISTv1.1. criteria [Prostate-specific antigen (PSA) levels should change at least 25% and display an absolute increase/decrease of 2 ng/mL to define PSA-related progression or improved disease status [Lactate dehydrogenase (LDH) and alkaline phosphatase (ALP), blood-based markers of prostate cancer tumor load and skeletal involvement, were measured sequentially at the routine laboratory.Circulating tumor cells (CTCs) were enumerated pre-treatment and during all follow-up visits using the CellSearch® System (Menarini Silicon Biosystems) according to the producer's standardized procedure (Supplementary S8). CTC responses were grouped as CTCs = 0 or CTCs > 0 independent of pre-treatment values. The response was estimated for two time points: at first available CTCs measurements after the CryoIT, and two weeks after treatment.Patient-reported outcomes were collected using the European Organization for Research and Treatment of Cancer (EORTC) QLQ-C30 questionnaire [Pain was evaluated by VAS pain logs prior to treatment, and from week two after CryoIT (Supplementary S6). | PMC10264291 |
Translational outcomes | tumor | TUMOR | Flow cytometry analyses of immune cells were conducted according to local routine (Supplementary S7) utilizing BD FACSCanto II (3 lasers) and BD FACSDiva software v.8.0.1 (BD Biosciences).Dedicated uropathologists re-examined the formalin-fixed paraffin-embedded (FFPE) primary diagnostic biopsies, assigning all samples a histologic subtype and International Society of Urological Pathology (ISUP) grade group. Furthermore, FFPE hematoxylin- and eosin-stained slides from all eight study biopsies, collected prior to CryoIT from each participant, were examined and graded (Supplementary S8).For each participant, the study biopsy with the most tumor tissue and the highest Gleason pattern was selected for immunohistochemistry analyses for T cells. The corresponding fresh frozen biopsy was selected for the two genetic panels and the T-cell receptor (TCR) sequencing (Supplementary S8 and S10).The biopsied tissue was stained for the four MMR proteins: MSH2, MSH6, PMS2 and MLH1, using the platform Ventana BenchMark Ultra (Roche, Basel, Switzerland) and detection system OptiView (Supplementary S9). MSI was diagnosed when minimum one of four MMR proteins was unstained in tumor nuclei, while positive staining < 10% classified as equivocal.DNA was isolated from each sample using the Qiagen AllPrep DNA/RNA Mini Kit (Qiagen, Hilden, Germany). Targeted parallel sequencing for library preparation (Agilent SureSelect XT-kit, Agilent) was performed on DNA from tumor tissue and matched peripheral blood. For the 360 gene panel targeted enrichment was performed using RNA baits (SureSelect, Agilent) against the coding regions of 360 cancer-related genes [TCR clonotypes were enumerated according to sequence counts. The 200 clonotypes with the highest sequence counts two and six weeks after CryoIT were compared separately with results from pre-treatment samples. Novel clonotypes were those that were undetectable prior to, but detectable after the CryoIT. For pre-treatment samples, the maximum clonal frequencies were used. Expanded clonotypes were pre-treatment clonotypes that expanded > fivefold after treatment. The sum of the frequencies of all identified TCR clonotypes in the entire data set defined the clonal space, while the clonal space percentage of a clonotype was calculated as the percentage of the total number of identified clonotypes. | PMC10264291 |
Data processing and bioinformatics analysis | tumor | TUMOR | For the 360 gene panel analyses, raw sequence data were aligned to the human reference genome (Build-UCSC hg19) using BWA. Somatic single nucleotide variants were detected by application of CaVEMan and insertions/deletions were detected using Pindel [The tumor mutational burden (TMB) analyses were performed by the TSO500 assay (NextSeq platform, Illumina) at the Science for Life Laboratory, Uppsala University, Sweden, (Supplementary S9) according to the producer´s protocol.Bioinformatic processing of the TCR sequencing results was performed by HS Diagnomics (Berlin, Germany). The libraries were based on a 2-step PCR system using gene-specific primers for | PMC10264291 |
Statistical considerations | toxicity | In this phase I clinical trial, sample sizes were not based on statistical methods. The aim of both trial parts was to evaluate the toxicity profile rather than formally to demonstrate any efficacy endpoints. The database cut-off date was April 30, 2021, when all participants had reached 24 months of follow-up, and OS data were updated last October 5, 2022.Descriptive statistics were used to categorize AEs according to their nature and severity. Furthermore, descriptive statistics were applied to demographic data, CTC results, and generation of the global HRQoL spider plot and line graphs, to demonstrate changes in TCR clonotype counts and longevity, and to depict percentage changes by waterfall plots.Unless otherwise stated, all statistical testing between groups was made by Fisher’s exact tests, two-sided Mann–Whitney U tests, or Kruskal–Wallis tests.Statistical significance was defined as PFS and OS were estimated at database cut-off with corresponding 95% confidence intervals estimated by the Kaplan–Meier method and independent groups compared by log-rank tests. Data analyses were performed either in R v.3.6.0 or higher [ | PMC10264291 | |
Results | PMC10264291 | |||
Safety | nausea, urinary tract reactions, pelvic osteomyelitis, death | COLD SYMPTOMS, EVENTS, ADVERSE EVENTS, ADMINISTRATION SITE REACTIONS | In total, 73 AEs were reported, whereof 32 were possibly or probably associated with participation in the trial. No dose-limiting toxic effects were recorded. The most frequently reported events were mild or moderate urinary tract reactions, administration site reactions, nausea, and influenza-like and common cold symptoms. Only two grade 3 AEs associated with the treatment were reported: one urine retention requiring hospitalization and one pelvic osteomyelitis requiring protracted intravenous antibiotics before full recovery (Table Treatment-emergent adverse eventsData are numbers in all treated patients. Treatment-emergent adverse events definitely, probably, or possibly attributed to cryoablation and/or iDC and/or PD-1i/CTLA-4i are shown. No grade 4 or grade 5 adverse events were attributed to treatment as definite, probable, or possible. iDC = immature dendritic cells. CTLA-4i = cytotoxic T-lymphocyte-associated protein 4 inhibitor. PD-1i = programmed cell death protein 1 inhibitor | PMC10264291 |
Response to therapy | prostatic tissue tumors, skeletal lesions, visceral disease, tumor | DISEASE PROGRESSION, TUMOR | At inclusion, 17/18 participants had skeletal lesions visible on scintigraphy, while 16/18 patients had evaluable visceral disease in the form of prostatic tissue tumors. Several patients had PET-positive lymph nodes. Long-term (> 46 weeks) clinical benefit were achieved by 6/18 (33%) participants. Out of 12, 1 patient who progressed was not followed radiologically due to rapid disease progression and malaise (Fig. When changes were measured two and six weeks after CryoIT, differences were observed between participants who had progressed radiologically and those with clinical benefit 22 weeks after treatment (PSA (Clinical, laboratory and radiological outcomes. All patients with pre-treatment CTCs showed either transient CTC decreases which lasted months for several patients (pre-treatment CTC ≥ 5/7.5, Changes in circulating tumor cells (CTC) and T cell receptor (TCR) clonotypes. Two weeks after the intervention, CTC numbers were available for 12/18 (67%). The CTC response analyses identified longer PFS for those with complete disappearance of CTCs (Figure Health-related quality-of-life measurements. Time in weeks since trial inclusion (baseline) is illustrated along the x-axis of all plots. | PMC10264291 |
Translational outcomes | MSS, cancer, tumor, tumors | CANCER, TUMOR, TUMORS | While higher baseline ALP levels correlated with higher tissue expression of Tregs (TCR sequencing of blood samples revealed that the median number of novel and > fivefold expanded clonotypes was 35.5 (IQR 27.5–63.5) after two weeks, compared to 70.5 (IQR 37.0–125.25) six weeks after CryoIT (Fig. While TCR clonotypes found in pre-treatment tumor biopsies could be identified among the clones detected in blood sampled pre- and post-treatment, less than 1% of the newly identified or expanded clonotypes was detected in the biopsy samples (Supplementary Table S8).Both the custom 360 gene and the TSO500 gene panels identified the tumors as MSS with low TMB, and immunohistochemical analyses of MSI status by MMR proteins confirmed MSS results. Most biopsies demonstrated low tumor cell fractions (< 20% cancer cells) (Fig. Results of the genetic and protein expression analyses of the tumor tissues. | PMC10264291 |
Discussion | tumor, prostate cancer, tumors, mCRPC, prostate cancers | TUMOR, TUMORS, SOLID TUMORS, PROSTATE CANCER | In this pioneering mCRPC clinical trial, CryoIT was found to be safe and tolerable. This is consistent with the well-defined safety profile of cryoablation in solid tumors [For the total cohort, PFS was 10.5 months while median OS was 40.7 months. In this early phase trial, no controls were included. In a study including mCRPC patients with diverse risk profiles, median PFS were 11.1 months (IQR 3.7–21.8) versus 8.2 months (3.5–16.6) in the less detrimental and more disadvantageous group, respectively, while corresponding OS were 41.8 (23.4–53.6) and 28.4 (17.9–41.9) months [The associations discovered in this trial between CTC response and PFS in prostate cancer correspond with previous findings in phase III trials, where either conversion from CTC counts ≥ 5 to CTC < 5 or total disappearance indicated favorable responses to therapy [The high proportion of expanded or new TCR clonotypes in blood, both two and six weeks after CryoIT, and the minimal intrapatient clonotype overlap could imply an treatment-related expansion or generation of TCR clonotypes with a time-dependent broadening of the peripheral clonotype repertoire. Several recent publications have found that expanded clonotypes in peripheral blood following immune checkpoint inhibition were different from pre-treatment tumor-infiltrating T-cell clonotypes and seemed to be replenished from outside of the tumor [Patients reported consistently high values in the functional and Global Health Status/HR-QoL domains and low values in symptom domains, but those with pre-treatment CTC reported a worse HRQoL over time. In contrast to other trials [All the patients had MSS tumors with low TMB. This finding is in accord with previous studies identifying 1–2% of prostate cancers as MSI and/or TMB high [This work may be limited by a small sample size, and lack of sequential tumor biopsies and comparator groups. While utilization of low doses of cyclophosphamide could impact the CTC numbers, the doses applied are considerably lower than those required for a general cytotoxic effect. We applied imaging guidelines published prior to trial commencement in 2015. As guidelines were updated as new treatment and technologies developed during the trial period, the application of older versions could limit interpretation of radiologically based outcomes. Second line hormonal treatment and early chemotherapy was fully introduced in Norway during the enrolment period and time-to-event comparisons with historical controls will therefore be imprecise. Despite these limitations, the findings encourage a next phase CryoIT trial, powered to estimate potential treatment efficacy.In conclusion | PMC10264291 |
Supplementary Information | Below is the link to the electronic supplementary material.Supplementary file1 (DOCX 14027 KB) | PMC10264291 | ||
Acknowledgements | CCBIO, HUS, Cancer | CANCER | The authors would like to thank the patients and the following persons who participated in the clinical trial: Bendik Nordanger, Department of Pathology, Haukeland University Hospital (HUS); Ingeborg Winge and Reidun Kristin Kopperud, Centre for Cancer Biomarkers CCBIO, University of Bergen; Andrine Sandøy, Department of Urology, HUS; Hua My Hoang, Rebecca Nguyen, Misbah Sabir, and Jørn Skavland, Department of Clinical Science, University of Bergen; Turid Kirsti Hønnåshagen, Lisbeth Johanne Skoge, and Dag Josefsen, Department of Cellular Therapy, Norwegian Radium Hospital; Ingvill Marie Christensen Curran, Torunn Ervik, Anne Kari Jahn Stephansen, Jannike Lundervik Selen, and Torhild Lahn-Johannessen, Department of Immunology and Transfusion Medicine, HUS; Steffen Hennig and Sigrid Schaper, HS Diagnomics, Berlin; the staff at Clinical Genomics Uppsala at Uppsala University, Sweden. Furthermore, the authors thank Dr. Patrick Juliebø-Jones for help with the linguistic revision. | PMC10264291 |
Author contribution | KHK, BTG, AMØ, HR, and CB designed the trial. AH, LARR, BA, KF, CB, and SIH included patients and performed all trial visits and clinical data collection. DB developed and taught the clinical staff the cryoablative procedures. KHK, AMØ, KF, JRO, and WA performed the sample preparation and laboratory analyses. SHK, GKM, IB, and GK performed the leukapheresis and production of autologous immature dendritic cells. EKK performed the flow cytometry analyses. LARR, AH, TMH, and MB. analyzed the radiologic images. AB, OJH and LAA performed the histological review, the immunohistochemical staining and evaluations, and related statistics including survival analyses. SK performed the genetic assay analyses. KP and SR performed the CTC analyses. KHK, CB, AB, BG, SK, AH, LARR, and LCVT analyzed the data. LCVT, AH, BTG, KHK, AH, and CB wrote the manuscript. All authors read and approved the final manuscript. | PMC10264291 | ||
Funding | Cancer | CANCER | Open access funding provided by University of Bergen (incl Haukeland University Hospital). This work received funding from Helse Vest (grant numbers 912062, 911626, 911747, 911582, 911778, 912226, and 980058), the Helse Vest Strategic grants of Personalized Therapy (grant number 303484) and the Bergen Stem Cell Consortium (grant number 502027). This work was partly supported by the Research Council of Norway through its Centres of Excellence funding scheme (project number 223250), the Research Council of Norway NFR BEHANDLING programme (project number 287829), the Norwegian Cancer Society (project number 197984), and the Bergen Research Foundation/Trond Mohn Stiftelse. | PMC10264291 |
Data availability | tumor, Cancer | TUMOR, CANCER | The data from circulating tumor cell enumeration, TCR sequencing data from sorted T cells including the R code and flow cytometry data used during this study are available through the Alden Cancer Therapy II (kalland@uib.no) for non-commercial research purposes. The joint data access committee of Vestlandets Innovasjonsselskap AS and Alden Cancer Therapy II will grant data access and enter into appropriate data access agreement subject to any applicable ethical approvals upon review of project proposals. | PMC10264291 |
Declarations | PMC10264291 | |||
Competing interests | Cancer | THOMSEN, CANCER | Dr. Gjertsen reports other from Kinn Therapeutics AS, other from Alden Cancer Therapeutics 2 AS, personal fees from BerGenBio AS, personal fees from Novartis AS, personal fees from Seattle Genetics Inc., personal fees from Pfizer, non-financial support from Roche, non-financial support from MSD, outside the submitted work. Dr. Kalland reports grants from Research Council of Norway, grants from The Norwegian Cancer Society, during the conduct of the study; In addition, Dr. Kalland has a patent PCT/EP2017/077698 pending and is the CEO of and owns Stocks in the Company Alden Cancer Therapy II AS that has sponsored the trial registered at ClinicalTrials.gov Identifier: NCT02423928 and which is published in the presently submitted manuscript. He has received no salary or remuneration from Alden Cancer Therapy II AS. Dr. Øyan owns stocks in the Company Alden Cancer Therapy II AS that has sponsored the trial registered at ClinicalTrials.gov Identifier: NCT02423928 and which is published in the presently submitted manuscript. She has received no salary or remuneration from Alden Cancer Therapy II AS. Dr. Gabriel reports grants from Norwegian Cancer Society during the conduct of the study. Dr Thomsen reports personal fees from Bayer and AstraZeneca, sitting on the advisory board for Eisai Co., and financial support from AstraZeneca to a researcher-initiated trial. Dr Beisland reports personal fees from Pfizer, sitting on advisory boards for BMS and MSD, and having travel expenses covered by Olympus. The remaining authors have no conflicts of interest to report. | PMC10264291 |
Ethics approval | The protocol was approved by the National Regional Ethics Committee (REK 2014/1052) with amendments during 2015–2019, as well as the Norwegian Medicines Agency. The trial is registered in ClinicalTrials.gov (NCT02423928). | PMC10264291 | ||
Consent to participate | Written informed consent was obtained from all patients. | PMC10264291 | ||
Consent to publish | Not applicable. | PMC10264291 | ||
References | PMC10264291 | |||
Purpose | Iodine deficiency disorder, IDD | IODINE DEFICIENCY DISORDER | Iodine deficiency disorder (IDD) is an ongoing worldwide recognized problem with over two billion individuals having insufficient iodine intake. School-aged children and pregnant women are often target groups for epidemiological studies, but there is a lack of knowledge on the general adult population. The aim of this study was to assess the iodine status among a Portuguese public university staff as a proxy for the adult working population. | PMC10117252 |
Methods | The population study covered 103 adults within the iMC Salt randomized clinical trial, aged 24–69 years. Urinary iodine concentration was measured spectrophotometrically using the Sandell–Kolthoff reaction. Iodine food intake was assessed using a 24-h dietary recall. The contribution of discretionary salt to the iodine daily intake was assessed through 24-h urinary sodium excretion (UIE) and potentiometric iodine determination of household salt. | PMC10117252 | ||
Results | The mean urine volume in 24 h was 1.5 L. The median daily iodine intake estimated from 24-h UIE was 113 µg/day, being lower among women ( | PMC10117252 | ||
Conclusion | IODINE DEFICIENCY | This study contributes new knowledge about iodine status in Portuguese working adults. The results revealed moderate iodine deficiency, particularly in women. Public health strategies and monitoring programs are needed to ensure iodine adequacy in all population groups. | PMC10117252 | |
Keywords | Open access funding provided by FCT|FCCN (b-on). | PMC10117252 | ||
Introduction | Iodine deficiency disorder, IDD | IODINE DEFICIENCY DISORDER | Iodine is an essential micronutrient, primarily obtained from the diet, indispensable to thyroid hormone production and key for the metabolism regulation of mammals [Iodine deficiency disorder (IDD) is still an ongoing worldwide recognized problem, with over two billion individuals having inadequate iodine intake [In Portugal, studies reported that the median urinary iodine concentration (UIC) is generally adequate in school-aged children [Since about 90% of ingested iodine is excreted in the urine, the median UIC is recognized as a good biomarker of short-term iodine status in groups [The main sources of iodine in the human diet are marine foodstuffs—fish, shellfish, algae and sea salt. Water, milk, vegetables and processed foods, such as bread and margarine, are also potential iodine sources [Universal salt iodization is preconized by the WHO, the United Nations Children's Fund (UNICEF) and the Iodine Global Network (IGN) as a safe, cost-effective and sustainable way to tackle IDD [Therefore, the aim of this study was to evaluate the iodine nutritional status of the public University of Porto staff, as a proxy for the Portuguese adult working population. Additionally, it was intended to demonstrate the potential of occupational health appointments in the education and monitoring of the iodine subject. | PMC10117252 |
Materials and methods | PMC10117252 | |||
Population and study design | hypotensive, urinary incontinence, liver disease | DISORDER, THYROID DISEASE, KIDNEY DISEASE, URINARY INCONTINENCE, LIVER DISEASE, RENAL INFECTION, ACUTE CORONARY SYNDROME, HEART FAILURE, HYPOTENSIVE | The participants of the study were recruited to the iMC Salt randomized controlled trial, designed to decrease salt intake inadequacy. The iMC Salt study was designed to assess the effectiveness of interventions to reduce salt consumption among consumers. The intervention consisted of using the Salt Control H equipment by the participants at home to control the salt quantity used for cooking all meals during the intervention period. Salt Control H is a dispenser that offers doses of salt according to the number of persons and the age (child or adult) of the consumers. The study was an 8-week randomized controlled trial, with follow-up to week 35. Participants were recruited among the staff of the university through the annual mandatory occupational health appointments. The exclusion criteria were pregnancy, hypotensive disorder, renal infection, kidney disease, urinary incontinence, acute coronary syndrome, severe liver disease or heart failure, member of the faculty that promotes the study (i.e., Faculty of Nutrition and Food Sciences), and not using salt for cooking. The primary outcome of this study was the difference in 24-h urinary sodium (as a proxy of salt intake) between the intervention and the control group from the baseline to the end of the intervention (week 8). A detailed description of the methods of the iMC Salt study has been published elsewhere [The iodine nutritional status was assessed for the baseline participants group, after validation of the collected 24-h urine samples, and additional exclusion of participants with previously diagnosed thyroid disease. A sample size of | PMC10117252 |
24-h urine collection | As previously reported [ | PMC10117252 | ||
Estimation of dietary and discretionary salt iodine content | Food intake was assessed using a 24-h dietary recall. Participants were asked to recall all foods and beverages consumed the day before (time of urine collection) and to estimate the portion size with the help of a picture book. The conversion of food intake into iodine was performed using the Food Processor Plus software (ESHA Research, Inc., Salem, OR USA) adapted for the Portuguese population [The food were categorized into 15 major food groups [In the context of the iMC Salt study, the total sodium consumption was assessed through 24-h urinary excretion and a 24-h dietary recall [ | PMC10117252 | ||
Determination of urinary iodine and creatinine concentration | Urinary iodine concentration was assessed spectrophotometrically, using a modification of the Sandell–Kolthoff reaction, with an initial ammonium persulfate digestion [Urinary creatinine, measured by the kinetic Jaffé reaction [The researcher who performed the urinary measurements was not aware of which group within the iMC Salt trial the participant was allocated to. | PMC10117252 | ||
Ethics | Ethical approval for the study was obtained from the Ethics Committee of the Centro Hospitalar Universitário São João, trial registration number NCT03974477. All participants signed an informed consent statement before the start of the study. | PMC10117252 | ||
Statistical analysis | Analysis of the data was performed using STATISTICA software (V.7.0 Stat Soft Inc., Tulsa, OK, USA). Descriptive statistics are presented as mean with standard deviation (SD) and medians, with 25th and 75th percentiles.Spearman’s correlation coefficient was used for the comparison of iodine intake estimated from 24-h urine and 24-h dietary recall. Bland–Altman plots were constructed to evaluate the agreement between the two iodine intake measurement techniques applied.Mann–Whitney One-way analysis of variance with the Kruskal–Wallis test and Dunn’s multiple comparison post-test was used to explore differences between groups. The level of significance was defined as a | PMC10117252 | ||
Results | PMC10117252 | |||
Relationship between the two methods for estimating iodine intake | SD | The median iodine intake estimated from the 24-h dietary recall (58 µg/day) was significantly lower than the levels determined from the UIE (113 µg/day, The agreement between different determination approaches was assessed through a Bland–Altman plot (Fig. Bland–Altman plot, mean iodine intake estimated from 24-h urine iodine excretion (UIE) and the 24-h dietary recall (24-h DR) versus the difference between the methods (24-h DR—UIE). The red line indicates the mean difference in iodine intake estimates, whereas the red dashed line represents the limits of agreement from − 1.96 SD to + 1.96 SD | PMC10117252 | |
Sources of iodine from the 24-h dietary recall | The evaluation of the estimated food intakes and iodine sources showed that dairy, including yoghurt and milk products, was the main source of iodine (Fig. The contribution (%) from food groups to the iMC Salt participant’s total iodine intake by sex | PMC10117252 | ||
Estimated iodine intake from household salt | Integrated into the iMC Salt randomized controlled trial, designed to decrease the salt intake inadequacy, the iodine content in the household salt samples was also analyzed. All the provided 47 salt samples (mostly sea salt) analyzed contained detectable iodine, with an average concentration of 14 mg I/kg (SD 7) (Fig. Iodine levels (mg I/kg) found in household salt samples from the iMC Salt intervention group. Green circles—salt samples complying with the WHO and the Portuguese criteria; orange circle—salt samples complying only with the WHO criteria; gray circles—salt samples that did not comply with WHO or the Portuguese criteria; gray dashed line—iodine range according to the Portuguese legal criteria for salt iodization (19–27 mg I/kg); red solid line—WHO-recommended level of iodine for salt iodization (15 mg I/kg)Forty-five percent of the samples (21/47) presented iodine concentration below the minimum threshold preconized by WHO (15 mg I/kg). Moreover, only 15% (7/47) of the salt samples showed iodine between the established levels in the non-mandatory iodine fortification Portuguese law (19–27 mg I/kg) [ | PMC10117252 | ||
Discussion | UIE, goiter, iodine deficiency | GOITER, IODINE DEFICIENCY | To the best of our knowledge, this is the first study on the iodine status of Portuguese working adults, using gold standard methodology.Based on previous epidemiological studies performed in school-aged children [With a median 24-h UIC of 66 μg/L, 30% of the population showing 24-h UIC below 50 μg/L, and a daily iodine intake of 112 μg/day, this study revealed that the adult working population had a mild iodine deficiency according to the WHO guidelines for iodine status (median UIC < 100 μg/L plus > 20% of the population with a UIC ≤ 50 μg/L), and the EFSA iodine intake threshold of 150 μg/L. In the literature is argued that the extrapolation from the observed correlation between increasing goiter and a UIE < 100 µg/day to a spot sample UIC below 100 µg/L, as indicative of ID, may have been applicable in children but was not correct for adults [From a public health perspective, the mild iodine deficiency found in this study reinforces the need to implement supplementation strategies for the whole, but especially for women of childbearing age to meet the increased needs in a future pregnancy and secure a normal development of the fetus. Indeed, iodine inadequacy was found in Portuguese pregnant women more than a decade ago [Despite the lower value displayed by the 24-h recall approach, both methodologies applied to estimate the daily iodine intake correlated well. The discrepancy found could be explained by incomplete information on iodine content in foods in the national databases and the self-reported 24-h recall. The 24-h dietary recall indicated that 56.7% of the total iodine intake comes from ingested food. Although the intestinal absorption of ingested iodine is considered to be high (more than 90%), the iodine concentration in water and foods is highly variable [Cereals, fruit, vegetables, and legumes are generally poor iodine sources, and the levels present depend on the amount of iodine in the soil where the plants are grown. Worldwide, 30% of the population lives in areas with iodine-deficient soil [The contribution of the discretionary salt to the daily iodine intake was 43% (52 μg), estimated by the difference between the total daily intake (mean 121 μg/day based on UIE), and the iodine amount provided by food (mean 69 μg/L based on the 24-h dietary recall). In the IMC Salt study, the average salt intake at baseline was 8 g/day [Considering the sodium-healthy diet, as preconized by WHO [The iodization of the salt used in households is the primary strategy to prevent iodine deficiency at a population level [If an iodoprophylaxis was followed, with the fortification of the household and food industry salt, and retaining the average contribution of iodine from food estimated in this study, the daily iodine intake would be 168–199 µg in a sodium-healthy diet, already accounting for the inevitable 20% lost during cooking [ | PMC10117252 |
Study strengths and limitations | The iodine intake was estimated simultaneously from both the 24-h urinary iodine excretion and a detailed 24-h dietary recall for each participant. Although cumbersome, the 24-h urinary iodine excretion is considered the best biomarker to assess and monitor the recent iodine intake of a given population [A major limitation of the study was the relatively small sample size. The data collection was performed during the COVID-19 pandemic, and despite the efforts made to solve the inherent implications, it was impossible to achieve the sample size initially estimated [ | PMC10117252 | ||
Conclusions | IODINE DEFICIENCY, DISEASES | To the best of our knowledge, this was the first study reporting on the iodine status in Portuguese working adults, using gold standard methodology. The results revealed a moderate iodine deficiency, with higher expression in women. Dairy was described as the major iodine source in the diet. Although the discretionary salt accounted for almost half of the iodine intake, the unfortified household marine salt failed to provide the required daily intake. Taking into consideration the present iodine status of the country, and the WHO major priority in salt reduction to tackle non-communicable diseases, it is pivotal to design public health strategies and monitoring programs to ensure iodine adequacy in all population groups. In this setting, the mandatory occupational health appointments that this study took advantage of have proven to be an asset for future monitoring programs and in education toward the use of iodized salt. | PMC10117252 | |
Acknowledgements | The authors were grateful to all University of Porto workers that participated in the study. | PMC10117252 | ||
Author contributions | TSS | Conceptualization: AAB; CG; PM; OP. Methodology: AM; CG. Validation: AM, AAB. Formal analysis: AM. Investigation: AM; CG; PM; OP; PP; TSS; PN. Resources: AAB; CG. Data curation: AM; AAB; MR. Writing—original draft preparation: AM. Writing—review and editing: AAB; CG; PM; OP; PP; TSS; PN. Supervision: AAB; CG. Project administration: AAB; CG. Funding acquisition: AAB; CG; OP. All authors have read and agreed to the published version of the manuscript. | PMC10117252 | |
Funding | Open access funding provided by FCT|FCCN (b-on). This work was also partially supported by the Project IMC Salt (POCI-01-0145-FEDER-029269), co-financed by COMPETE 2020, Portugal 2020 and the European Union through the ERDF, and by FCT through national funds. The CITAB is supported by FCT/UIDB/04033/2020. The ITR is supported by LA/P/0064/2020. | PMC10117252 | ||
Data availability | The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request. | PMC10117252 | ||
Declarations | PMC10117252 | |||
Conflict of interest | The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results. | PMC10117252 |
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