Subclinical hypothyroidism in pregnancy - assessment of offspring thyroid status and mitochondrial robustness to stress.

Subclinical hypothyroidism's clinical implications on pregnancy are controversial. Consequently, thyrotropin (TSH) cutoff-values for pregnancy are continuously a subject for debate. In subclinical hypothyroidism, altered levels of thyroid hormones may affect mitochondrial function.Objectives were i) to analyze thyroid hormone levels in offspring of women with and without subclinical hypothyroidism ii) to analyze mitochondrial "robustness" in terms of MTG/TMRM ratio in pregnant women and their offspring in relation to thyroid function and iii) to perform differentiate analyses on different TSH thresholds to determine the importance of cutoff-values to results.Pregnant women were included by blood collections prior to a planned cesarean section, and cord samples were collected after delivery. Thyroid status (analyzed by Siemens Healthcare Diagnostics by an electrochemical luminescent immunoassay based on LOCI-technology) grouped the women and their offspring in euthyroid or subclinical hypothyroid, with groups established from previous recommended third-trimester cutoff-value (TSH > 3.0 mIU/L) and the recently recommended cutoff-value in Denmark (TSH > 3.7 mIU/L). Flow cytometric measurements of mitochondrial function in mononuclear blood cells with the fluorophores TetraMethylRhodamine Methyl Ester (TMRM) and Mitotracker Green (MTG) were used to evaluate mitochondrial robustness as the MTG/TMRM ratio.No significant differences in mitochondrial robustness between euthyroid and subclinical hypothyroid cohorts were observed, irrespective of TSH-cutoff applied. Maternal and cord MTG/TMRM ratios were positively correlated. Cord-TSH was elevated in subclinical hypothyroid offspring, independent of TSH cutoff applied. Cord-TSH was associated with maternal TSH-level, maternal smoking and cord arterial-pH.

Expressions of mitochondria-related genes in pregnant women with subclinical hypothyroidism, and expressions of miRNAs in maternal and cord blood.

BACKGROUND: Subclinical hypothyroidism in pregnancy and definition by upper thyrotropin (TSH) cutoff are controversial. As mitochondria are influenced by thyroid hormones, the purpose in this study was to measure expression of mitochondria-related genes in euthyroid and subclinical hypothyroid pregnant women to obtain more knowledge of potential metabolic consequences of maternal subclinical hypothyroidism. In addition, we wished to test if applied TSH-cutoff significantly changed our results of expressed gene-levels. Moreover, we aimed to identify potential microRNA-biomarkers for subclinical hypothyroidism - markers that could be traced to offspring as well. METHODS: From a cohort of at-term pregnant women undergoing planned cesarean section, 77 women had expression levels of the mitochondria-related genes Peroxisome Proliferator-activated Receptor-γ coactivator-1ß (PGC-1ß), mitochondrial Transcription Factor A (TFAM), Superoxide Dismutase 2 (SOD2) and Nuclear Respiratory Factor 2 (NRF-2) determined by qPCR from blood sampled in prior to delivery. Two TSH-cutoff levels defining subclinical hypothyroidism (> 3.0 and > 3.7 mIU/L) were applied for the procession of results, generating two data analyses of the same cohort. In 22 pairwise maternal-cord samples (subclinical hypothyroid/euthyroid-rate 0.5, TSH-cutoff > 3.0 mIU/L), microRNA-expressions (miRNA) were analyzed. RESULTS: All gene expressions were lower in the subclinical hypothyroid group regardless of applied TSH-cutoff, but insignificant except for PGC-1ß at TSH cutoff > 3.0 mIU/L. Two miRNAs (hsa-let-7d-3p and hsa-miR-345-5p) were upregulated in blood from women and offspring (cord blood) with subclinical hypothyroidism. CONCLUSIONS: A trend towards decreased mitochondrial gene expressions in subclinical hypothyroidism were demonstrated. The miRNAs hsa-let-7d-3p and hsa-miR-345-5p might be potential markers of maternal subclinical hypothyroidism. However, larger studies are needed to verify the findings.

Long-term stability of thyroid peroxidase antibody (anti-TPO) in serum in the Danish General Suburban Population Study.

OBJECTIVES: We evaluated the long-term stability of thyroid peroxidase antibody (anti-TPO). METHODS: In the Danish General Suburban Population Study (GESUS), serum samples were biobanked at -80 °C during 2010-2013. In a paired design with 70 subjects, we compared anti-TPO (30-198 U/mL) measured on fresh serum on Kryptor Classic in 2010-2011 (anti-TPOfresh) with anti-TPO remeasured on frozen serum (anti-TPOfrozen) on Kryptor Compact Plus in 2022. Both instruments used the same reagents and the anti-TPOn automated immunofluorescent assay, which was calibrated against the international standard NIBSC 66/387, based on the Time Resolved Amplified Cryptate Emission (TRACE) technology from BRAHMS. Values greater than 60 U/mL are regarded as positive in Denmark with this assay. Statistical comparisons included Bland-Altman, Passing-Bablok regression, and Kappa statistic. RESULTS: The mean follow-up time was 11.9 years (SD: 0.43). For anti-TPOfrozen vs. anti-TPOfresh, the line of equality was within the confidence interval of the absolute mean difference [5.71 (-0.32; 11.7) U/mL] and the average percentage deviation [+2.22% (-3.89%; +8.34%)]. The average percentage deviation of 2.22% did not exceed analytical variability. Passing-Bablok regression revealed both a statistically significant systematic and proportional difference: Anti-TPOfrozen=-22.6 + 1.22*(anti-TPOfresh). Frozen samples were correctly classified as positive in 64/70 (91.4%; Kappa=71.8%). CONCLUSIONS: Anti-TPO serum samples in the range 30-198 U/mL were stable after 12-years of storage at -80 °C with an estimated nonsignificant average percentage deviation of +2.22%. This comparison is based on Kryptor Classic and Kryptor Compact Plus, which used identical assays, reagents, and calibrator, but for which the agreement in the range 30-198 U/mL is unclarified.

Absorption, metabolism and excretion of once-weekly somapacitan, a long-acting growth hormone derivative, after single subcutaneous dosing in human subjects.

Somapacitan is a reversible albumin-binding growth hormone (GH) derivative in clinical development for once-weekly administration in patients with adult GH deficiency (AGHD) and children with GH deficiency (GHD). To date, the use of somapacitan in AGHD or severe AGHD has been approved in the USA and Japan, respectively. This study (ClinicalTrials.gov, NCT02962440) investigated the absorption, metabolism and excretion, as well as the pharmacokinetics (PK), of tritium-labelled somapacitan ([3H]-somapacitan). Seven healthy males received a single subcutaneous dose of 6 mg somapacitan containing [3H]-somapacitan 20 MBq. Blood, serum, plasma, urine, faeces, and expired air were collected for radioactivity assessment. Metabolites were identified and quantified in plasma and urine collected. The PK of plasma components were determined, and the radioactive peaks of the most abundant plasma metabolites and urine metabolites were selected for analysis. Twenty-eight days after dosing, 94.0% of the administered dose was recovered as [3H]-somapacitan-related material, most of which was excreted in urine (80.9%); 12.9% was excreted in faeces, and an insignificant amount (0.2%) was exhaled in expired air. PK properties of [3H]-somapacitan-related material appeared to be consistent across plasma, serum and blood. Three abundant plasma metabolites (P1, M1 and M1B) and two abundant urine metabolites (M4 and M5) were identified. The total exposure of intact somapacitan accounted for 59% of the total exposure of all somapacitan-related material, P1 accounted for 21% and M1 plus M1B accounted for 12%. M4 and M5 were the most abundant urine metabolites and accounted for 37% and 8% of the dosed [3H]-somapacitan radioactivity, respectively. No intact somapacitan was found in excreta. Two subjects had six adverse events (AEs); all were mild in severity and unlikely to be related to trial product. The majority of dosed [3H]-somapacitan (94%) was recovered as excreted metabolites. Urine was the major route for excretion of somapacitan metabolites, followed by faeces, and exhalation in expired air was negligible. The low molecular weights of identified urine metabolites demonstrate that somapacitan was extensively degraded to small residual fragments that were excreted (fully biodegradable). The extensive metabolic degradation and full elimination of metabolites in excreta were the major clearance pathways of somapacitan and the key elements in its biological fate. A single dose of 6 mg somapacitan (containing [3H]-somapacitan) in healthy male subjects was well tolerated with no unexpected safety issues identified.

Mitochondrial energetics and contents evaluated by flow cytometry in human maternal and umbilical cord blood.

Background: Mitochondrial dysfunction may relate to metabolic disorders. The relation between maternal and fetal mitochondrial function needs attention due to heritage.Objectives: To evaluate the use of the staining methods TetraMethylRhodamine Methyl Ester (TMRM) and Mitotracker Green (MTG) for flow cytometric measurements of umbilical cord blood mitochondrial function. Methods: 53 euthyroid at-term pregnant women and their offspring were included by blood collections. The offspring had blood drawn from the clamped umbilical cord. Flow cytometry with MTG, TMRM and Propidium Iodide were performed the following day. A cell count (antibody coating and flow cytometry) was performed for 9 maternal and cord samples. As a quality control, blood of 32 healthy donors was evaluated by flow cytometric analyzes same day as sampling and the following day to test stability of the measurements.Results: Cord mitochondrial measurements were lower than maternal. Maternal and cord mitochondrial function were positively correlated, especially reflected by MTG fluorescence-intensity (FI). Samples stored presented with very changed fluorescence patterns. However, the fluorescence intensity ratios MTG/TMRM of stained white blood cells were related within same day measurements, depicting an extensive and common bioenergetic cellular change.Conclusion: Cord blood flow cytometry by MTG- and TMRM- staining is possible with fluorescence intensity positively correlated to maternal fluorescence intensity. Storage of blood triggers mitochondrial dynamics. The methods are applicable with certain reservations, and they benefit from their non-invasive character compared to mitochondrial evaluation by muscle-biopsies.

Reduced Gene Expression of Peroxisome Proliferator-Activated Receptor Gamma Coactivator-1α in Whole Blood in Euthyroid Patients One Year After Hemithyroidectomy for Benign Euthyroid Goiter.

We have previously reported decreased thyroid function within the laboratory reference range and changes in mitochondrial function after hemithyroidectomy. Peroxisome proliferator-activated receptor γ coactivator-1α (PGC-1α) and coactivator-1ß (PGC-1ß) are key regulators of mitochondrial biogenesis and function. The aim was to examine the influence of hemithyroidectomy on the longitudinal change in mRNA expression of these genes. In addition, we measured longitudinal changes in mRNA expressions of the mitochoncrial-related genes nuclear factor erythroid-derived 2-like 2 (NFE2L2), mitochondrial transcription factor A (TFAM), and sodium dismutase 2 (SOD2). Twenty-eight patients were examined before and 1, 3, 6, and 12 months after hemithyroidectomy for benign euthyroid goiter. Thyroid stimulating hormone (TSH) and thyroid hormones were measured, and whole blood gene expression of PGC-1α, PGC-1ß, NFE2L2, TFAM, and SOD2 was examined by reverse transcription quantitative Polymerase Chain Reaction. We used mixed effect regression models to investigate changes in gene expression with time. Averaged over all follow-up visits, TSH increased (p=0.001), tT3 declined (p=0.01), and fT4/tT3 ratio increased (p=0.03) over one-year follow-up, but fT4 remained unchanged. Averaged over all follow-up visits, whole blood PGC-1α levels (p<0.001) and SOD2 (p=0.009) levels declined, but PGC-1ß, TFAM, and NFE2L2 did not change over one-year follow-up. The study demonstrates significant downregulation of whole blood PGC-1α and SOD2 gene expressions in hemithyroidectomized patients with a concomitant increase in TSH concentration within the reference range. Thus, hemithyroidectomized patients may likely have impaired mitochondrial function.

Corrigendum to "Impaired Fertility Associated with Subclinical Hypothyroidism and Thyroid Autoimmunity: The Danish General Suburban Population Study".

[This corrects the article DOI: 10.1155/2015/132718.].

Absorption, metabolism and excretion of the GLP-1 analogue semaglutide in humans and nonclinical species.

Semaglutide is a human glucagon-like peptide-1 analogue in clinical development for the treatment of type 2 diabetes. The absorption, metabolism and excretion of a single 0.5mg/450µCi [16.7MBq] subcutaneous dose of [3H]-radiolabelled semaglutide was investigated in healthy human subjects and compared with data from nonclinical studies. Radioactivity in blood, plasma, urine and faeces was determined in humans, rats and monkeys; radioactivity in expired air was determined in humans and rats. Metabolites in plasma, urine and faeces were quantified following profiling and radiodetection. The blood-to-plasma ratio and pharmacokinetics of both radiolabelled semaglutide-related material and of semaglutide (in humans only) were assessed. Intact semaglutide was the primary component circulating in plasma for humans and both nonclinical species, accounting for 69-83% of the total amount of semaglutide-related material, and was metabolised prior to excretion. Recovery of excreted radioactivity was 75.1% in humans, 72.1% in rats and 58.2% in monkeys. Urine and faeces were shown to be important routes of excretion, with urine as the primary route in both humans and animals. Semaglutide was metabolised through proteolytic cleavage of the peptide backbone and sequential beta-oxidation of the fatty acid sidechain, and metabolism was not confined to specific organs. Intact semaglutide in urine accounted for 3.1% of the administered dose in humans and less than 1% in rats; it was not detected in urine in monkeys. The metabolite profiles of semaglutide in humans appear to be similar to the profiles from the nonclinical species investigated.

Cognitive assessment at bedside for iPad: A preliminary validation of a novel cognitive test for stroke patients.

INTRODUCTION: Cognitive impairments are frequent in stroke. Cognitive testing is important for research, prognostication and planning in sub-acute stroke, but poses difficulties due to aphasia, hemineglect, hemiplegia and fatigue. We present the first steps towards a validation of a novel iPad-based test battery: Cognitive Assessment at Bedside for iPad (CABPad). PATIENTS AND METHODS: Stroke patients and age matched healthy controls were tested with CABPad including tests for aphasia, neglect, episodic memory, attention span, executive function, working memory, mental speed, anosognosia, motor speed and depression. A re-test was performed after 1 month. Furthermore, a group of stroke patients was tested with CABPad and traditional neuropsychological tests. RESULTS: Fifty-four patients and 48 healthy controls were included in the first phase. Fifty-three patients (98%) were able to complete at least one test and 50 (92%) all tests at the first test point. Mean test duration in patients was 39 min (range 30-60). We found significant differences in test results at baseline between the two groups. Episodic memory mean difference: 8.5 (95% confidence interval: 4.3, 12.7). Symbol Digit Coding mean difference: 16.3 (95% confidence interval: 10.8, 21.7). The second phase included 16 patients. We found adequate to excellent correlation in the majority of the tests. The CABPad Speech Comprehension test and the Auditory Word Recognition subtest of the Western Aphasia Battery correlated with r = 0.82, p < 0.001. CONCLUSION: CABPad is useful for cognitive testing in stroke patients. It is easy to use for the examiner and patients alike. Immobile patients can be tested at bedside, irrespectively of upper extremity paresis, and the assessment can be performed in a relatively short timespan.

Impaired Fertility Associated with Subclinical Hypothyroidism and Thyroid Autoimmunity: The Danish General Suburban Population Study.

INTRODUCTION: The aim of this study was to estimate the significance of TSH, thyroid peroxidase antibody (TPOAb), and mild (subclinical) hypothyroidism in women from The Danish General Suburban Population Study (GESUS) on the number of children born, the number of pregnancies, and the number of spontaneous abortions. METHODS: Retrospective cross sectional study of 11254 women participating in GESUS. Data included biochemical measurements and a self-administrated questionnaire. RESULTS: 6.7% had mild (subclinical) hypothyroidism and 9.4% prevalent hypothyroidism. In women with mild hypothyroidism TPOAb was significantly elevated and age at first child was older compared to controls. TSH and TPOAb were negatively linearly associated with the number of children born and the number of pregnancies in the full cohort in age-adjusted and multiadjusted models. TSH or TPOAb was not associated with spontaneous abortions. Mild (subclinical) hypothyroidism was associated with a risk of not having children and a risk of not getting pregnant in age-adjusted and multiadjusted models. Prevalent hypothyroidism was not associated with the number of children born, the number of pregnancies, or spontaneous abortions. CONCLUSION: Impaired fertility is associated with TSH, TPOAb, and mild (subclinical) hypothyroidism in a Danish population of women.

Weight Gain and Serum TSH Increase within the Reference Range after Hemithyroidectomy Indicate Lowered Thyroid Function.

Background. Weight gain is frequently reported after hemithyroidectomy but the significance is recently discussed. Therefore, the aim of the study was to examine changes in body weight of hemithyroidectomized patients and to evaluate if TSH increase within the reference range could be related to weight gain. Methods. In a controlled follow-up study, two years after hemithyroidectomy for benign euthyroid goiter, postoperative TSH and body weight of 28 patients were compared to preoperative values and further compared to the results in 47 matched control persons, after a comparable follow-up period. Results. Two years after hemithyroidectomy, median serum TSH was increased over preoperative levels (1.23 versus 2.08 mIU/L, P < 0.01) and patients had gained weight (75.0 versus 77.3 kg, P = 0.02). Matched healthy controls had unchanged median serum TSH (1.70 versus 1.60 mIU/L, P = 0.13) and weight (69.3 versus 69.3 kg, P = 0.71). Patients on thyroxin treatment did not gain weight. TSH increase was significantly correlated with weight gain (r = 0.43, P < 0.01). Conclusion. Two years after hemithyroidectomy for benign euthyroid goiter, thyroid function is lowered within the laboratory reference range. Weight gain of patients who are biochemically euthyroid after hemithyroidectomy may be a clinical manifestation of a permanently decreased metabolic rate.

Persistent cellular metabolic changes after hemithyroidectomy for benign euthyroid goiter.

BACKGROUND: The significance of perturbations of thyroid-stimulating hormone (TSH) and thyroid hormones within the laboratory reference ranges after hemithyroidectomy is unknown. Our aim was to examine changes in TSH and thyroid hormones after hemithyroidectomy for benign euthyroid goiter, focusing on tissue response by examining the mitochondrial membrane potential (MMP) of peripheral blood mononuclear cells (PBMCs) and basal oxygen consumption (V˙O2). MATERIALS AND METHODS: In a prospective study on 28 patients and controls, we examined serum TSH and thyroid hormones before hemithyroidectomy and 1, 3, 6 and 12 months after hemithyroidectomy for benign euthyroid goiter. In the hemithyroidectomy group, flow cytometry was used to measure the MMP of tetramethylrhodamine methyl ester (TMRM)- and MitoTracker Green (MTG)-stained PBMCs, and V˙O2 was measured by an Oxycon Pro apparatus. RESULTS: One year after hemithyroidectomy, TSH had increased from a median of 0.97 mIU/l (interquartile range, IQR: 0.69-1.50 mIU/l) to 2.10 mIU/l (IQR: 1.90-3.00 mIU/l; p < 0.001); free thyroxine (fT4) had decreased from a median of 16.0 pmol/l (IQR: 14.9-17.0 pmol/l) to 14.8 pmol/l (IQR: 14.1-16.4 pmol/l; p = 0.009), whereas total triiodothyronine variations did not differ from those in controls. Concomitantly, the MMP of TMRM- and MTG-stained PBMCs was increased by 58% (p < 0.001) and 22% (p = 0.008), respectively. V˙O2 was increased by 14% (p = 0.01). CONCLUSION: Hemithyroidectomy for benign euthyroid goiter induced persistently increased TSH and decreased fT4, sustained mitochondrial hyperpolarization and increased V˙O2. Our results demonstrate a decrease after hemithyroidectomy of the metabolic state to which the individual is adapted, with persistent cellular metabolic changes in a hemithyroidectomized patient group which is normally considered clinically and biochemically euthyroid.

Ferrous bisglycinate 25 mg iron is as effective as ferrous sulfate 50 mg iron in the prophylaxis of iron deficiency and anemia during pregnancy in a randomized trial.

OBJECTIVE: To compare the effects of oral ferrous bisglycinate 25 mg iron/day vs. ferrous sulfate 50 mg iron/day in the prevention of iron deficiency (ID) and iron deficiency anemia (IDA) in pregnant women. DESIGN: Randomized, double-blind, intention-to-treat study. SETTING: Antenatal care clinic. SAMPLE: 80 healthy ethnic Danish pregnant women. METHODS: Women were allocated to ferrous bisglycinate 25 mg elemental iron (Aminojern®) (n=40) or ferrous sulfate 50 mg elemental iron (n=40) from 15 to 19 weeks of gestation to delivery. Hematological status (hemoglobin, red blood cell indices) and iron status (plasma iron, plasma transferrin, plasma transferrin saturation, plasma ferritin) were measured at 15-19 weeks (baseline), 27-28 weeks and 36-37 weeks of gestation. MAIN OUTCOME MEASURES: Occurrence of ID (ferritin <15 µg/L) and IDA (ferritin <12 µg/L and hemoglobin <110 g/L). RESULTS: At inclusion, there were no significant differences between the bisglycinate and sulfate group concerning hematological status and iron status. The frequencies of ID and IDA were low and not significantly different in the two iron groups. The frequency of gastrointestinal complaints was lower in the bisglycinate than in the sulfate group (P=0.001). Newborns weight was slightly higher in the bisglycinate vs. the sulfate group (3601±517 g vs. 3395±426 g, P=0.09). CONCLUSIONS: In the prevention of ID and IDA, ferrous bisglycinate was not inferior to ferrous sulfate. Ferrous bisglycinate in a low dose of 25 mg iron/day appears to be adequate to prevent IDA in more than 95% of Danish women during pregnancy and postpartum.

Pregnancy-induced alterations in mitochondrial function in euthyroid pregnant women and pregnant women with subclinical hypothyroidism; relation to adverse outcome.

BACKGROUND: It is well documented that overt hypothyroidism is associated with adverse pregnancy outcomes, but studies of subclinical hypothyroidism have demonstrated conflicting results. OBJECTIVE: Thyroid hormones are known to regulate mitochondrial function, and the aim of this study was to examine the possible relationship of subclinical hypothyroidism and mitochondrial dysfunction to adverse pregnancy outcomes in pregnant women. METHODS: Women in their third trimester of pregnancy (n = 113) who did not receive thyroid medication were included in this cross-sectional study. All participants were interviewed, and their thyroid status was determined. All participants had concentrations of thyroid hormones (fT4 and tT3) within the reference range. In addition to thyroid status, mitochondrial membrane potential (MMP) and reactive oxygen species (ROS) were measured by flow cytometry. To establish a reference range of MMP and ROS, a group of euthyroid, nonpregnant women were used as euthyroid controls. Adverse pregnancy outcome was defined as preterm delivery, preeclampsia, placental abruption, Apgar score <7 points 1 minute after birth, or postpartum hemorrhage. RESULTS: The prevalence of subclinical hypothyroidism among pregnant women was 17% (n = 19), and the number of overall adverse pregnancy outcomes was increased (p = 0.02) compared with that in euthyroid pregnant women. Preeclampsia, poor Apgar score, and postpartum hemorrhage were more frequent in the subclinical hypothyroidism group than in the euthyroid group (p = 0.04, p = 0.001 and p = 0.03, respectively), and more women showed prolonged gestation and gave birth later than 41 weeks of gestation than in the euthyroid group (p = 0.04). Compared with euthyroid, nonpregnant controls, a physiological upregulation of mitochondrial function was observed in euthyroid pregnant women. This was impaired in pregnant women with subclinical hypothyroidism. Compared with euthyroid, nonpregnant controls, pregnant women had increased ROS regardless of their thyroid status. CONCLUSION: We speculate that the unfavorable effects on mitochondrial function in women with subclinical hypothyroidism may be associated with higher prevalence of adverse pregnancy outcomes.

Association of lipoprotein levels with mortality in subjects aged 50 + without previous diabetes or cardiovascular disease: a population-based register study.

OBJECTIVE: This study aimed to investigate the association of lipoprotein and triglyceride levels with all-cause mortality in a population free from diabetes and cardiovascular disease (CVD) at baseline. The European Guidelines on cardiovascular disease prevention state that in general total cholesterol (TC) should be < 5 mmol/L (190 mg/dL) and low-density lipoprotein cholesterol (LDL-C) should be < 3 mmol/L (115 mg/dL). DESIGN: A population-based register study in the period 1999-2007 including 118 160 subjects aged 50 + without statin use at baseline. All-cause mortality was related to lipoprotein and triglyceride levels and adjusted for statin use after inclusion. RESULTS: All-cause mortality was lower in the groups with TC or LDL-C above the recommended levels. Compared with subjects with TC < 5 mmol/L, adjusted hazard ratios for the group aged 60-70 years ranged from 0.68 (95% confidence interval (CI) 0.61-0.77) for TC 5-5.99 mmol/L to 0.67 (95% CI 0.59-0.75) for TC 6-7.99 mmol/L and 1.02 (95% CI 0.68-1.53) for TC ≥ 8 mmol/L in males and from 0.57 (95% CI 0.48-0.67) to 0.59 (95% CI 0.50-0.68) and 1.02 (95% CI: 0.77-1.37) in females. For triglycerides, ratios compared with the group < 1 mmol/L in the females aged 60-70 years ranged from 1.04 (95% CI 0.88-1.23) to 1.35 (95% CI 1.10-1.66) and 1.25 (95% CI 1.05-1.48) for triglycerides 1-1.39 mmol/L, 1.4-1.69 mmol/L, and ≥ 1.7 mmol/L, respectively. Statin treatment after inclusion provided a survival benefit. CONCLUSION: These associations indicate that high lipoprotein levels do not seem to be definitely harmful in the general population. However, high triglyceride levels in females are associated with decreased survival.

Synthesis of tocopheryl succinate phospholipid conjugates and monitoring of phospholipase A2 activity.

Tocopheryl succinates (TOSs) are, in contrast to tocopherols, highly cytotoxic against many cancer cells. In this study the enzyme activity of secretory phospholipase A(2) towards various succinate-phospholipid conjugates has been investigated. The synthesis of six novel phospholipids is described, including two TOS phospholipids conjugates. The studies revealed that the TOS conjugates are poor substrates for the enzyme whereas the phospholipids with alkyl and phenyl succinate moieties were hydrolyzed by the enzyme to a high extent.

Prostaglandin phospholipid conjugates with unusual biophysical and cytotoxic properties.

The synthesis of two secretory phospholipase A(2) IIA sensitive 15-deoxy-Delta(12,14)-prostaglandin J(2) phospholipid conjugates is described and their biophysical and biological properties are reported. The conjugates spontaneously form particles in the liposome size region upon dispersion in an aqueous buffer and both phospholipids are hydrolyzed by phospholipase A(2), but with different conversion rates and extent of hydrolysis. The cytotoxicity was evaluated in HT-29 and Colo205 cells and the conjugates induced cell death in the presence of phospholipase A(2) and surprisingly also in the absence of the enzyme.

Synthesis of small molecules with high scaffold diversity: exploitation of metathesis cascades in combination with inter- and intramolecular Diels-Alder reactions.

Our knowledge of the biological relevance of regions of chemical space is shaped, in large part, by the synthetic accessibility of small molecules. Historically, however, chemists have explored chemical space in an exceptionally uneven and unsystematic way. We have previously demonstrated that metathesis cascade chemistry may be harnessed to yield small molecule collections with high scaffold diversity. Here, we describe the extent to which inter- and intramolecular Diels-Alder reactions, when used in conjunction with metathesis cascades, can extend the range of molecular scaffolds that are accessible. A range of metathesis substrates was prepared from combinations of two or three building blocks. Metathesis cascades were exploited to "reprogram" the molecular scaffolds. In many cases, the metathesis products were 1,3-dienes, which were potential substrates for either inter- or intramolecular Diels-Alder reactions. The synthesis and functionalisation of the products was often facilitated by fluorous tagging, for example by using a "safety-catch" linker that we have developed. It was demonstrated that, in certain cases, Diels-Alder reactions could extend the range of molecular scaffolds that may be prepared by using metathesis cascade reactions.

Liposomal formulation of retinoids designed for enzyme triggered release.

The design of retinoid phospholipid prodrugs is described based on molecular dynamics simulations and cytotoxicity studies of synthetic retinoid esters. The prodrugs are degradable by secretory phospholipase A(2) IIA and have potential in liposomal drug delivery targeting tumors. We have synthesized four different retinoid phospholipid prodrugs and shown that they form particles in the liposome size region with average diameters of 94-118 nm. Upon subjection to phospholipase A(2), the lipid prodrugs were hydrolyzed, releasing cytotoxic retinoids and lysolipids. The formulated lipid prodrugs displayed IC(50) values in the range of 3-19 microM toward HT-29 and Colo205 colon cancer cells in the presence of phospholipase A(2), while no significant cell death was observed in the absence of the enzyme.

Thyroid hormone effect on human mitochondria measured by flow cytometry.

BACKGROUND: Mitochondrial function may be impaired in a number of diseases including metabolic syndrome, cardiovascular disease and endocrine disorders. Therefore it is important to be able to measure mitochondrial function in human cells. PURPOSE: The aim of the present study was to evaluate a method to measure mitochondrial function in human derived cells, which also would reflect regulation by thyroid hormones. METHODS: The MDA-MB-231 cell line (a human breast cancer cell line) was incubated with bioactive iodothyronines (T(4), 3'-3, 5-T(3), 3, 5-T(2)) 50 nmol/l for 3 h. Mitochondrial membrane potentials (MMP) were measured by a flow cytometer after staining with Tetramethylrhodamine methyl ester (TMRM). Also, the effect of TRIAC (a stimulator of thyroid hormone nuclear receptors) and L-Carnitine (an inhibitor of thyroid hormone passage into the nucleus) was examined. FINDINGS: It was possible to measure mitochondrial membrane potential (MMP) in human derived cells and to examine thyroid hormone effects using flow cytometry. Bioactive iodothyronines increased mitochondrial membrane potential. TRIAC had no effect and L-Carnitine only inhibited T(4) stimulation of membrane potential. CONCLUSION: Flow cytometry may be a valuable method for examining and testing mitochondrial function in human cells. Our findings demonstrate increase of mitochondrial membrane potential and an extra nuclear short time effect of 3, 5-T(2) on mitochondrial activity.