Hypothyroid women have persistently higher oxidative stress compared to healthy controls.

Objective: Some studies suggest that hypothyroidism is associated with increased oxidative stress. Urinary excretion of 8-oxo-7,8-dihydroguanosine (8-oxoGuo) and 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodG) represents whole-body RNA and DNA oxidation, respectively. These biomarkers have only been explored sparsely in patients with thyroid disorders. Methods: In 45 Danish women with newly diagnosed hypothyroidism, we compared 8-oxoGuo and 8-oxodG before or shortly after initiating levothyroxine with the excretion rates at euthyroidism. We also compared the excretion of 8-oxoGuo and 8-oxodG in the patients after restored euthyroidism with 18 healthy control subjects. Results: Compared with baseline, none of the biomarkers changed significantly in the patients after becoming euthyroid. The geometric mean of 8-oxoGuo was 1.63 (95% CI: 1.49-1.78) nmol/mmol creatinine at baseline and 1.67 nmol/mmol at euthyroidism (95% CI: 1.53-1.83) (P = 0.39), while that of 8-oxodG was 1.28 nmol/mmol creatinine at baseline (95% CI: 1.14-1.44) and 1.32 nmol/mmol at euthyroidism (95% CI: 1.18-1.48), respectively (P = 0.47). The relative mean differences were 0.97 (95% CI: 0.91-1.04) for 8-oxoGuo and 0.97 (95% CI: 0.88-1.06) for 8-oxodG. At baseline, multiple linear regression revealed a positive association between free thyroxine and both biomarkers (8-oxoGuo, P < 0.001; 8-oxodG, P = 0.04). Furthermore, 8-oxoGuo was positively associated with age (P = 0.04) and negatively associated with thyrotropin (P = 0.02). In the control group, the geometric mean of 8-oxoGuo was 1.23 nmol/mmol creatinine (95% CI: 1.07-1.42), while that of 8-oxodG was 1.04 nmol/mmol creatinine (95% CI: 0.88-1.23). Thus, compared with control subjects, euthyroid patients showed a significantly higher level of both 8-oxoGuo (P < 0.001) and 8-oxodG (P = 0.03). Conclusion: In hypothyroid women, no significant effect of levothyroxine treatment on the oxidative stress biomarkers 8-oxoGuo and 8-oxodG could be demonstrated. However, the excretion of these biomarkers was significantly higher than in healthy controls.

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.

On measurements of glucagon secretion in healthy, obese, and Roux-en-Y gastric bypass operated individuals using sandwich ELISA.

Glucagon is a key regulator of metabolism and is used in the diagnostic of neuroendocrine tumors. Accurate measurement of glucagon requires both extreme sensitivity and specificity since several peptides are derived from the same proglucagon precursor encoding part of the glucagon sequence and given that glucagon circulates in picomolar concentrations. A sandwich ELISA was recently developed and extensively evaluated; however, this method may not be accurate when measuring glucagon in patients with an enhanced production of proglucagon-derived peptides as seen after Roux-en-Y gastric bypass (RYGB). To overcome this, a modified version of the ELISA was developed. In this study, we evaluate an unmodified and a modified version of the ELISA in healthy individuals, individuals with obesity, and finally in two cohorts of patients following RYGB surgery using different nutrient stimuli to assess glucagon dynamics. Finally, in vitro spike-in recoveries using native glucagon and proglucagon-derived peptides were performed in buffer and in plasma. Our data support that both versions of the ELISA accurately capture endogenous and exogenous glucagon in healthy individuals and in individuals with obesity. However, the unmodified version of the assay may overestimate glucagon levels in patients following RYGB in line with minimal but consistent cross-reactivity to oxyntomodulin and glicentin that both are 50-fold increased after RYGB. Importantly, we did not find any changes between the two protocols at fasted conditions and therefore diagnostics of glucagonomas is not affected by the choice of assay procedure nor the surgical history of the patient (RYGB).

Follistatin secretion is enhanced by protein, but not glucose or fat ingestion, in obese persons independently of previous gastric bypass surgery.

Follistatin is secreted from the liver and is involved in the regulation of muscle mass and insulin sensitivity via inhibition of activin A in humans. The secretion of follistatin seems to be stimulated by glucagon and inhibited by insulin, but only limited knowledge on the postprandial regulation of follistatin exists. Moreover, results on postoperative changes after Roux-en-Y gastric bypass (RYGB) are conflicting with reports of increased, unaltered, and lowered fasting concentrations of follistatin. In this study, we investigated postprandial follistatin and activin A concentrations after intake of isocaloric amounts of protein, fat, or glucose in subjects with obesity with and without previous RYGB to explore the regulation of follistatin by the individual macronutrients. Protein intake enhanced follistatin concentrations similarly in the two groups, whereas glucose and fat ingestion did not change postprandial follistatin concentrations. Concentrations of activin A were lower after protein intake compared with glucose intake in RYGB. Glucagon concentrations were also particularly enhanced by protein intake and tended to correlate with follistatin in RYGB. In conclusion, we demonstrated that protein intake, but not glucose or fat, is a strong stimulus for follistatin secretion in subjects with obesity and that this regulation is maintained after RYGB surgery.NEW & NOTEWORTHY Circulating follistatin and activin A were studied after intake of isocaloric protein, fat, or glucose drinks in subjects with obesity with and without previous Roux-en-Y gastric bypass (RYGB). Protein intake enhanced follistatin similarly in both groups, whereas glucose and fat ingestion did not change follistatin. Activin A was lower after protein compared with glucose in RYGB. The novel finding is that protein intake, but neither glucose nor fat, stimulates follistatin secretion independently of previous RYGB.

Circulating Glucagon 1-61 Regulates Blood Glucose by Increasing Insulin Secretion and Hepatic Glucose Production.

Glucagon is secreted from pancreatic α cells, and hypersecretion (hyperglucagonemia) contributes to diabetic hyperglycemia. Molecular heterogeneity in hyperglucagonemia is poorly investigated. By screening human plasma using high-resolution-proteomics, we identified several glucagon variants, among which proglucagon 1-61 (PG 1-61) appears to be the most abundant form. PG 1-61 is secreted in subjects with obesity, both before and after gastric bypass surgery, with protein and fat as the main drivers for secretion before surgery, but glucose after. Studies in hepatocytes and in ß cells demonstrated that PG 1-61 dose-dependently increases levels of cAMP, through the glucagon receptor, and increases insulin secretion and protein levels of enzymes regulating glycogenolysis and gluconeogenesis. In rats, PG 1-61 increases blood glucose and plasma insulin and decreases plasma levels of amino acids in vivo. We conclude that glucagon variants, such as PG 1-61, may contribute to glucose regulation by stimulating hepatic glucose production and insulin secretion.