Description and validation of an equilibrium dialysis ID-LC-MS/MS candidate reference measurement procedure for free thyroxine in human serum.

OBJECTIVES: Free thyroxine (FT4) in serum is routinely measured in clinical practice to diagnose and monitor thyroid disease. Due to its concentration in picomolar range and the delicate equilibrium of free and protein-bound T4, accurate measurement is challenging. As a consequence, large inter-method differences in FT4 results exists. Optimal method design and standardization of the FT4 measurement is therefore necessary. The IFCC Working Group for Standardization of Thyroid Function Tests proposed a reference system with a conventional reference measurement procedure (cRMP) for FT4 in serum. In this study, we describe our FT4 candidate cRMP and its validation in clinical samples. METHODS: This candidate cRMP is based on equilibrium dialysis (ED) combined with determination of T4 with an isotope-dilution liquid chromatography tandem mass-spectrometry (ID-LC-MS/MS) procedure and was developed according to the endorsed conventions. Its accuracy, reliability, and comparability was investigated using human sera. RESULTS: It was shown that the candidate cRMP adhered to the conventions and its accuracy, precision, and robustness were adequate in serum of healthy volunteers. CONCLUSIONS: Our candidate cRMP measures FT4 accurately and performs well in serum matrix.

Measuring steroids in hair opens up possibilities to identify congenital adrenal hyperplasia in developing countries.

OBJECTIVE: Patients with congenital adrenal hyperplasia (CAH) in developing countries have limited access to appropriate laboratory facilities for diagnosis and follow-up. The aim of this study is to evaluate steroid measurement in hair as a diagnostic tool to identify and monitor CAH in these patients. DESIGN: A method was developed to measure steroids in hair, the stability of steroids in hair was assessed, and the concentration range in healthy volunteers was determined. Hair samples of patients, before and after starting therapy, were transported at ambient temperature to The Netherlands for analysis. PATIENTS: Twenty-two Indonesian CAH patients and 84 healthy volunteers participated. MEASUREMENTS: Cortisol, 17-hydroxyprogesterone (17OHP), androstenedione, and testosterone in hair were measured by liquid chromatography with tandem mass spectrometry. RESULTS: Steroids in hair could be measured and remained stable (<4.9% deviation) for at least 3 weeks at 4°C and 30°C. In each of the untreated patients, hair concentrations of 17OHP (9.43-1135 pmol/g), androstenedione (36.1-432 pmol/g), and testosterone (2.85-69.2 pmol/g) were all above the upper limit of the corresponding range in healthy volunteers; 5.5 pmol/g, 13 pmol/g, and 1.8 pmol/g, respectively. After starting glucocorticoid treatment, the steroid concentrations in the hair of CAH patients decreased significantly for androstenedione (73%) and testosterone (59%) after 6 months. CONCLUSIONS: CAH could be confirmed in Indonesian patients based on the concentration of 17OHP, androstenedione, and testosterone in hair, and a treatment effect was observed. These findings open up opportunities to diagnose and/or monitor CAH in developing countries with a simple noninvasive technique.

Diurnal salivary androstenedione and 17-hydroxyprogesterone levels in healthy volunteers for monitoring treatment efficacy of patients with congenital adrenal hyperplasia.

OBJECTIVE: Treatment of congenital adrenal hyperplasia (CAH) patients with glucocorticoids is often challenging since there is a delicate balance between over- and undertreatment. Treatment can be monitored noninvasively by measuring salivary androstenedione (A4) and 17-hydroxyprogesterone (17-OHP). Optimal treatment monitoring requires the establishment of reference values in saliva. DESIGN: A descriptive study. PATIENTS: For this study saliva of 255 healthy paediatric and adult volunteers with an age range of 4-75 years old was used. MEASUREMENTS: We developed a sensitive liquid chromatography-tandem mass spectrometry method, assessed salivary A4 and 17-OHP stability, and measured A4 and 17-OHP concentrations in saliva collected in the morning, afternoon, and evening. RESULTS: We quantified A4 and 17-OHP concentrations in the morning, afternoon, and evening and demonstrated that there is a significant rhythm with the highest levels in the morning and decreasing levels over the day. A4 and 17-OHP concentrations display an age-dependent pattern. These steroids remain stable in saliva at ambient temperature for up to 5 days. CONCLUSIONS: Good stability of the steroids in saliva enables saliva collection by the patient at home. Since salivary A4 and 17-OHP display a diurnal rhythm and age-dependent pattern, we established reference values for both children and adults at three time points during the day. These reference values support treatment monitoring of children and adults with CAH.

Psychopathic traits influence threat avoidance in a community sample independent of testosterone.

Psychopathy is a personality construct that encompasses a constellation of traits reflecting emotional dysfunction and antisocial behavior. Individuals with elevated levels of psychopathic traits have shown abnormal affective processing. Studies with psychopathic offenders suggested that this is a result of altered automatic social approach-avoidance tendencies. The goal of the current study was to increase the insight into the underlying mechanism of affective processes in community-dwelling individuals with a high level of psychopathic traits by studying approach and avoidance behavior in an experimental setting. Furthermore, given its link with aggression and threat approach, testosterone was measured to investigate a possible mediatory role. Eighty-seven healthy individuals performed a computerized affective approach-avoidance task in which they pushed or pulled emotional faces using a joystick. The results showed that high levels of psychopathic traits corresponded with diminished threat avoidance to angry faces, as was found previously in psychopathic offenders. Although endogenous testosterone was positively associated with the level of psychopathic traits, it did not mediate the effect of psychopathic traits on threat avoidance. We propose that an increased understanding of the interplay between different neuroendocrine mechanisms could lead to a better insight into the underlying mechanism of abnormal threat avoidance in individuals with high levels of psychopathic traits. (PsycInfo Database Record (c) 2021 APA, all rights reserved).

FSH isoform pattern in classic galactosemia.

Female classic galactosemia patients suffer from primary ovarian insufficiency (POI). The cause for this long-term complication is not fully understood. One of the proposed mechanisms is that hypoglycosylation of complex molecules, a known secondary phenomenon of galactosemia, leads to FSH dysfunction. An earlier study showed less acidic isoforms of FSH in serum samples of two classic galactosemia patients compared to controls, indicating hypoglycosylation. In this study, FSH isoform patterns of five classic galactosemia patients with POI were compared to the pattern obtained in two patients with a primary glycosylation disorder (phosphomannomutase-2-deficient congenital disorders of glycosylation, PMM2-CDG) and POI, and in five postmenopausal women as controls. We used FPLC chromatofocussing with measurement of FSH concentration per fraction, and discovered that there were no significant differences between galactosemia patients, PMM2-CDG patients and postmenopausal controls. Our results do not support that FSH dysfunction due to a less acidic isoform pattern because of hypoglycosylation is a key mechanism of POI in this disease.

Transient human anti-mouse antibodies (HAMA) interference in CA 125 measurements during monitoring of ovarian cancer patients treated with murine monoclonal antibody.

OBJECTIVE: To investigate the influence of human anti-mouse antibodies (HAMA) on serial CA 125 measurements in serum of patients with epithelial ovarian cancer following single intraperitoneal (IP) therapy with Yttrium-90-labeled human milk fat globule 1 murine monoclonal antibody ((90)Y-muHMFG1) as part of a large international randomized phase III trial. METHODS: We monitored CA 125 concentrations in longitudinally collected serum samples from 224 patients after IP (90)Y-muHMFG1 (study group) and from 223 patients who received standard treatment (control group). Serum samples of 22 study patients with increased CA 125 concentrations were selected and subjected to affinity chromatography to study HAMA interference in CA 125 measurements. RESULTS: CA 125 serum concentrations at weeks 1, 4 and 8 were significantly higher in the study group than in the control group. In the first 8 weeks after IP (90)Y-muHMFG1 administration significantly more patients of the study group (144/224) demonstrated CA 125 concentrations above the upper limit of normal of 23 U/mL, as compared to those of the control group (37/223). Affinity chromatography of serum with high CA 125 values in the first 8 weeks confirmed HAMA interference in CA 125 measurements while after 24 weeks this HAMA interference could no longer be detected. CONCLUSIONS: This is the first study to demonstrate that clinical trials applying murine monoclonal antibodies may be flawed by a transient HAMA effect, which should be considered when monitoring ovarian cancer patients with CA 125 measurements.

Adrenergic receptor stimulation attenuates insulin-stimulated glucose uptake in 3T3-L1 adipocytes by inhibiting GLUT4 translocation.

Activation of the sympathetic nervous system inhibits insulin-stimulated glucose uptake. However, the underlying mechanisms are incompletely understood. Therefore, we studied the effects of catecholamines on insulin-stimulated glucose uptake and insulin-stimulated translocation of GLUT4 to the plasma membrane in 3T3-L1 adipocytes. We found that epinephrine (1 microM) nearly halved insulin-stimulated 2-deoxyglucose uptake. The beta-adrenoceptor antagonist propranolol (0.3 microM) completely antagonized the inhibitory effect of epinephrine on insulin-stimulated glucose uptake, whereas the alpha-adrenoceptor antagonist phentolamine (10 microM) had no effect. When norepinephrine was used instead of epinephrine, the results were identical. None of the individual selective beta-adrenoceptor antagonists (1 microM, beta(1): metoprolol, beta(2): ICI-118551, beta(3): SR-59230A) could counteract the inhibitory effect of epinephrine. Combination of ICI-118551 and SR-59230A, as well as combination of all three selective beta-adrenoceptor antagonists, abolished the effect of epinephrine on insulin-stimulated glucose uptake. After differential centrifugation, we measured the amount of GLUT1 and GLUT4 in the plasma membrane and in intracellular vesicles by means of Western blotting. Both epinephrine and norepinephrine reduced insulin-stimulated GLUT4 translocation to the plasma membrane. These results show that beta-adrenergic (but not alpha-adrenergic) stimulation inhibits insulin-induced glucose uptake in 3T3-L1 adipocytes, most likely via the beta(2)- and beta(3)-adrenoceptor by interfering with GLUT4 translocation from intracellular vesicles to the plasma membrane.

Role of hexosamines in insulin resistance and nutrient sensing in human adipose and muscle tissue.

It has been proposed that the hexosamine pathway acts as a nutrient-sensing pathway, protecting the cell against abundant fuel supply, and that accumulation of hexosamines represents a biochemical mechanism by which hyperglycemia and hyperlipidemia induce insulin resistance. We hypothesized that if an increased flux through the hexosamine pathway caused insulin resistance in humans, the hexosamine levels should be increased in adipose and/or muscle tissue in insulin-resistant subjects, such as patients with type 2 diabetes and obese individuals. In addition, we reasoned that if the hexosamine pathway were a nutrient-sensing pathway, hexosamine levels in adipose and skeletal muscle tissue should be correlated with levels of circulating nutrients, such as glucose and free fatty acids (FFAs) and leptin concentrations. In a human cross-sectional study of 55 patients [20 with type 2 diabetes mellitus (DM) and 21 normal-lean (NL) and 14 normal-obese (NO) subjects] who underwent hip replacement surgery, adipose and muscle tissue biopsies were obtained and analyzed for levels of hexosamines [UDP-N-acetylglucosamine (UDP-GlcNAc) and UDP-N-acetylgalactosamine] and hexoses (UDP-glucose and UDP-galactose). Fasting plasma glucose, glycosylated hemoglobin, serum insulin and homeostasis model assessment calculations, serum lipids, and leptin were measured on the same day. Hexosamines were not elevated in adipose and muscle tissue of patients with type 2 DM compared with NL and NO subjects (UDP-GlcNac DM vs. NL vs. NO, 3.3 +/- 2.3 vs. 2.2 +/- 2.1 vs. 3.0 +/- 2.0 nmol/g tissue in adipose tissue and 8.1 +/- 2.9 vs. 7.8 +/- 2.8 vs. 7.6 +/- 2.8 nmol/g tissue in muscle tissue, respectively). Hexosamines in adipose tissue were positively correlated with circulating levels of FFA (UDP-GlcNAc, r = 0.33, P < 0.05; UDP-N-acetylgalactosamine, r = 0.41, P < 0.01). Adipose tissue UDP-GlcNAc was correlated with leptin levels (r = 0.33; P < 0.05). No such relationship was identified in muscle tissue. In conclusion, these findings argue against a pathophysiological role of the hexosamine pathway in insulin resistance in humans but support the hypothesis that the hexosamine pathway in adipose tissue, not in muscle, is a FFA-sensing pathway and could be involved in the regulation of leptin expression.

Regulation of GLUT1-mediated glucose uptake by PKClambda-PKCbeta(II) interactions in 3T3-L1 adipocytes.

Members of the PKC (protein kinase C) superfamily play key regulatory roles in glucose transport. How the different PKC isotypes are involved in the regulation of glucose transport is still poorly defined. PMA is a potent activator of conventional and novel PKCs and PMA increases the rate of glucose uptake in many different cell systems. In the present study, we show that PMA treatment increases glucose uptake in 3T3-L1 adipocytes by two mechanisms: a mitogen-activated protein kinase kinase-dependent increase in GLUT1 (glucose transporter 1) expression levels and a PKClambda-dependent translocation of GLUT1 towards the plasma membrane. Intriguingly, PKClambda co-immunoprecipitated with PKCbeta(II) and did not with PKCbeta(I). Previously, we have described that down-regulation of PKCbeta(II) protein levels or inhibiting PKCbeta(II) by means of the myristoylated PKCbetaC2-4 peptide inhibitor induced GLUT1 translocation towards the plasma membrane in 3T3-L1 adipocytes. Combined with the present findings, these results suggest that the liberation of PKClambda from PKCbeta(II) is an important factor in the regulation of GLUT1 distribution in 3T3-L1 adipocytes.

Hexosamines are unlikely to function as a nutrient-sensor in 3T3-L1 adipocytes: a comparison of UDP-hexosamine levels after increased glucose flux and glucosamine treatment.

Whether the hexosamine biosynthesis pathway acts as a nutrient-sensing pathway is still unclear. Glucose is directed into this pathway by GFAT. Because the activity of GFAT is tightly regulated, we examined whether UDP-hexosamine levels can increase significantly and dose-dependently in response to elevated glucose concentrations. In glucosamine-treated 3T3-L1 adipocytes, inhibition of insulin-stimulated glucose uptake was highly correlated with UDP-hexosamine levels (r = -0.992; p < 0.0001 for UDP-GlcNAc and r = -0.996; p < 0.0001 for UDP-GalNAc). Incubation of 3T3-L1 adipocytes with 0.1 microM insulin for 24 h in medium containing 1 and 5 mM glucose increased the rate of glucose uptake by 365% and 175% compared to untreated cells, respectively. This increase was not observed when the cells were incubated for 24 h with insulin in medium containing 10 or 25 mM glucose. However, treatment of cells with insulin and 1, 5, 10, or 25 mM glucose resulted in similar increases in levels of UDP-GlcNAc and UDP-GalNAc that always amounted to approx 30-40% above baseline values. This led us to conclude that despite exposure of adipocytes to conditions of extreme and prolonged glucose disposal, the increases in cellular UDP-hexosamines were minimal and not dependent on the extracellular glucose concentration. Taken together, our results are in line with the hypothesis that in glucosamine-treated adipocytes UDP-hexosamines influence insulin-stimulated glucose uptake. However, our observations in glucose-treated adipocytes argue against the possibility that UDP-hexosamines function as a nutrient-sensor, and question the role of the hexosamine biosynthesis pathway in the pathogenesis of insulin resistance.

Inhibition of protein kinase CbetaII increases glucose uptake in 3T3-L1 adipocytes through elevated expression of glucose transporter 1 at the plasma membrane.

The mechanism via which diacylglycerol-sensitive protein kinase Cs (PKCs) stimulate glucose transport in insulin-sensitive tissues is poorly defined. Phorbol esters, such as phorbol-12-myristate-13-acetate (PMA), are potent activators of conventional and novel PKCs. Addition of PMA increases the rate of glucose uptake in many different cell systems. We attempted to investigate the mechanism via which PMA stimulates glucose transport in 3T3-L1 adipocytes in more detail. We observed a good correlation between the rate of disappearance of PKCbetaII during prolonged PMA treatment and the increase in glucose uptake. Moreover, inhibition of PKCbetaII with a specific myristoylated PKCbetaC2-4 peptide inhibitor significantly increased the rate of glucose transport. Western blot analysis demonstrated that both PMA treatment and incubation with the myristoylated PKCbetaC2-4 pseudosubstrate resulted in more glucose transporter (GLUT)-1 but not GLUT-4 at the plasma membrane. To our knowledge, we are the first to demonstrate that inactivation of PKC, most likely PKCbetaII, elevates glucose uptake in 3T3-L1 adipocytes. The observation that PKCbetaII influences the rate of glucose uptake through manipulation of GLUT-1 expression levels at the plasma membrane might reveal a yet unidentified regulatory mechanism involved in glucose homeostasis.

Muscle uridine diphosphate-hexosamines do not decrease despite correction of hyperglycemia-induced insulin resistance in type 2 diabetes.

Animal studies suggest that overactivity of the hexosamine pathway, resulting in increased UDP-hexosamines [UDP-N-acetylglucosamine (UDP-GlcNAc) and UDP-N-acetylgalactosamine (UDP-GalNAc)] is an important mechanism by which hyperglycemia causes insulin resistance. This study was performed to test this hypothesis in patients with type 2 diabetes mellitus (DM). Eight obese patients with uncontrolled DM type 2 and severe insulin resistance were treated with iv insulin for 28 +/- 6 d aimed at euglycemia. Before and after iv insulin treatment, insulin sensitivity was measured using a hyperinsulinemic euglycemic clamp, and a muscle biopsy was taken for measurement of UDP-GlcNAc, UDP-GalNAc, UDP-glucose, and UDP-galactose levels. Also, isoelectric focusing patterns of serum transferrin and the urinary excretion of glycosaminoglycans as measures of final products of the hexosamine pathway were examined. After euglycemia, insulin resistance improved, as demonstrated by an increase in the glucose infusion rate during the clamp from 12.7 +/- 5.6 to 22.4 +/- 8.8 micro mol/kg.min (P < 0.0005) and a decrease in insulin requirement from 1.7 +/- 0.9 to 1.1 +/- 0.6 U/kg.d (P < 0.005), whereas metabolic control improved. Surprisingly, both UDP-GlcNAc, from 8.81 +/- 1.21 to 12.31 +/- 2.52 nmol/g tissue (P < 0.005), and UDP-GalNAc concentrations, from 4.49 +/- 0.85 to 5.89 +/- 1.55 nmol/g tissue (P < 0.05) increased. Isoelectric focusing patterns of serum transferrin and excretion of glycosaminoglycans were similar before and after euglycemia. In conclusion, after amelioration of hyperglycemia- induced insulin resistance, UDP-hexosamines increased in skeletal muscle of patients with type 2 DM. These results do not support the hypothesis that accumulation of products of the hexosamine pathway plays a major role in hyperglycemia-induced insulin resistance.