Peter A. Elder

Plasma adiponectin in overweight, nondiabetic individuals with or without insulin resistance

Aim:  Adiponectin is a protein produced exclusively by adipocytes with putative insulin‐sensitizing and anti‐atherogenic properties. This cross‐sectional study investigated the relationship between plasma adiponectin and a range of anthropometric, glycaemic, lipid and inflammatory parameters in overweight and obese subjects expressing characteristics of the metabolic syndrome.

Production of a monoclonal antibody to cortisol: Application to a direct enzyme-linked immunosorbent assay of plasma

Cortisol mouse monoclonal antibodies were produced and characterized. Of the four clones studied, supernatant from one clone (A2), compared with other cortisol monoclonal antibodies, showed minimal cross-reactivity to other C21 steroids and was suitable for the direct determination of cortisol in plasma by enzyme-linked immunosorbent assay using a standard 96-well microtiter plate. The enzyme-linked immunosorbent assay uses the immobilized antigen approach, in which cortisol in plasma samples or standards competes with immobilized steroid for antibody-binding sites. After washing, the cortisol antibody bound to the wells of the microtiter plate is detected with antimouse immunoglobulin conjugated to horseradish peroxidase. Following further washing, o-phenylenediamine substrate is added. The enzyme-linked immunosorbent assay is robust and semiautomated. The mean +/- SD recovery from plasma was 97% +/- 6%. Precision studies on three different plasma pools showed mean coefficients of variation of 7.6% and 8.6% for within- and between-assay variation, respectively. The satisfactory performance criteria allow its use in the routine laboratory.

An enzyme-linked immunosorbent assay (ELISA) for plasma cortisol

A direct ELISA for plasma cortisol is described which is carried out in a standard 96 well microtitre plate. In this ELISA cortisol-thyroglobulin conjugate is immobilised to the microtitre plate and competes with cortisol in the standard or plasma sample for antibody binding sites. Following washing the rabbit cortisol antibody bound to immobilised cortisol is incubated with peroxidase labelled goat antirabbit IgG. Following further washing o-phenylenediamine is added, colour developed, and the plate read at 492 nm on a standard ELISA plate reader. This ELISA shows good agreement with RIA and its sensitivity, specificity and precision allow its use in the routine steroid laboratory.

An enzyme-linked immunosorbent assay (ELISA) for plasma testosterone

A rapid, single extraction ELISA for testosterone in plasma is described, using a standard 96 well microtitre plate. Testosterone is covalently bonded to bovine thyroglobulin and passively adsorbed in guanidine hydrochloride to the ELISA plate, giving an immobilised antigen approach which simplifies subsequent assay standardisation for steroid hormone assays. The addition of standard, sample and first antibody (rabbit anti-testosterone), which is unique for each different assay, is followed by a general procedure which includes washing, addition of peroxidase labelled goat antirabbit IgG, further washing and finally, addition of o-phenylenediamine substrate with colour development and reading of the plate at 492 nm on an automatic ELISA processor. The ELISA assay is compared to a testosterone RIA with 125I-label and has similar specificity and precision to the latter with a quicker processing time, and is more cost effective. The added advantages that ELISA assays confer over RIAs in terms of isotope purchase and disposal make this an ideal procedure for use in a routine steroid laboratory.

Biovariability of plasma adiponectin.

Abstract Background: Adiponectin is a cytokine produced by adipose tissue with insulin sensitising and anti-atherosclerotic effects. Low plasma adiponectin levels are used as a marker of the metabolic syndrome and incipient type 2 diabetes. Methods: We carried out a series of studies to determine the short- and long-term variability of plasma adiponectin levels, including diurnal and post-prandial changes. These investigations also included examining the effect of frozen storage on plasma adiponectin levels. Results: A nested study in 10 overweight subjects with the metabolic syndrome and 10 age- and sex-matched controls showed intra-subject variation in adiponectin levels over a 30-day period of 12.2% and 18.8%, respectively, equivalent to reference change values of 1.7 and 3.6μg/mL. In non-obese subjects, plasma adiponectin levels varied minimally over a 15-month period (baseline, 8.3±2.9μg/mL vs. +15months, 8.2±3.0μg/mL, p=0.95) and showed only minor diurnal and post-prandial changes (pre-meal, 8.2±3.0μg/mL vs. 3h post-prandial, 8.3±3.1μg/mL, p=0.60). The adiponectin assay had an intra-assay variation of 8.8%, with storage at −30°C for 33months or three cycles of freezing and thawing having no discernible effect on adiponectin levels. Conclusions: These results demonstrate that plasma adiponectin levels have relatively low biovariability and that adiponectin can be sampled fasting or non-fasting to provide a reliable marker of insulin resistance and incipient type 2 diabetes. Clin Chem Lab Med 2006;44:1264–8.

Caution on the use of saliva measurements to monitor absorption of progesterone from transdermal creams in postmenopausal women

OBJECTIVES To determine the levels of progesterone in plasma, red cells and saliva as well as pregnanediol-3-glucuronide excretion in postmenopausal women using transdermal progesterone creams. METHODS A double-blind placebo controlled study was carried out using 24 postmenopausal women. Creams (placebo, 20 or 40 mg progesterone/g) were applied twice daily for 3 weeks followed by 1 week without before a further 3-week treatment. Morning samples were collected at 0, 1, 3, 4, 7 and 8 weeks for analysis. RESULTS There were small increases in plasma progesterone levels and pregnanediol-3-glucuronide excretion compared to the placebo group and red cell progesterone levels never exceeded plasma levels during progesterone cream use. Saliva progesterone levels were very high and variable in the progesterone cream groups compared to the placebo group and presented a paradox to the usual relationship observed between plasma and saliva progesterone in premenopausal women. CONCLUSION The absorption of progesterone from transdermal creams is low and we caution against the use of saliva measurements to monitor progesterone absorption. The low systemic absorption of progesterone may not be due to peripheral conversion by 5 alpha-reductase(s). We also conclude that the low level of progesterone associated with red cells suggests they are not important in the delivery of progesterone to target tissues.

Adiponectin attenuates endothelial dysfunction induced by oxidised low-density lipoproteins

We investigated whether the adipocytokine, adiponectin, protected the endothelium against damage induced by oxidised low-density lipoprotein cholesterol (oxLDL). Human umbilical vein endothelial cells were cultured with either 200 or 350 μg/ml oxLDL, with or without adiponectin purified from human serum (12 μg/ml). Cellular oxidative status was assessed by measuring reactive oxygen species (ROS), peroxynitrite and glutathione (GSH) levels, while cell function was evaluated by measuring nitric oxide (NO) levels and immunohistochemical examination of proteins in the adherens cell junction. At a concentration of 200 μg/ml, oxLDL induced a small increase in ROS and peroxynitrite levels, a two-fold increase in GSH levels and no changes in NO levels or localisation of proteins in the adherens junction. However, 350 μg/ml of oxLDL induced a marked increase in ROS and peroxynitrite levels, a four-fold reduction in GSH levels and a significant decrease in NO levels and disruption of the adherens junctions. Addition of adiponectin to the cultures resulted in maintenance of normal ROS, peroxynitrite and GSH levels, with no change in either NO levels or protein localisation in the adherens junction. This study demonstrates that adiponectin protects against endothelial dysfunction and cellular disruption induced by oxLDL, with this effect being due, in part, to maintenance of intracellular GSH levels.

An enzyme-linked immunosorbent assay (ELISA) for plasma progesterone: immobilised antigen approach

A single extraction ELISA for plasma progesterone is described using the fixed antigen approach. Progesterone is covalently coupled to bovine thyroglobulin and adsorbed onto a 96-well microtitre plate in guanidine hydrochloride. The assay, performed on an automatic ELISA processor, follows an established methodology used for other steroid hormones analysed in this laboratory with concomitant advantages in assay standardisation, cost structure and result throughput. A comparison with an established RIA shows the assay to be rapid, of similar specificity and accuracy with a sensitivity of less than 0.5 nmol/l and is suitable for use in a routine endocrine laboratory for determination of luteal function.

Serum 25-OH Vitamin D2 and D3 are Stable under Exaggerated Conditions

Vitamin D analysis is increasingly performed with HPLC–tandem mass spectrometry instead of RIA, and hence samples are frequently sent to more specialized centers for analysis. We therefore investigated the stability of both 25-OH vitamin D3 (25-OH D3) and 25-OH vitamin D2 (25-OH D2) in serum stored under extreme conditions that would likely exceed those normally encountered in sample transit and storage. After our study received ethics committee approval, we collected 200 mL of blood from a single donor who had given written informed consent and had been previously been determined to have adequate concentrations of 25-OH D3. After centrifugation of the blood sample the serum was removed and supplemented with 50 nmol/L of authentic 25-OH D2 to boost endogenous concentrations. We then transferred 1.0-mL portions into 5-mL clear polystyrene tubes. The sample-containing tubes were capped, stored at −20 °C, and subsequently subjected to various treatments in replicates of 5. These treatments included multiple freeze-thaw cycles (1 to 5 cycles), 8 days at ambient temperature under various conditions, and brief exposure to artificial ultraviolet light. The ambient-temperature storage conditions were as follows: 1 set of 5 samples was left on the laboratory bench uncovered and exposed to fluorescent light and diffuse …

Plasma retinol-binding protein is not a marker of insulin resistance in overweight subjects: A three year longitudinal study

OBJECTIVES This longitudinal study investigated whether or not plasma retinol-binding protein (RBP), recently referred to as RBP4, was a marker of insulin resistance in overweight subjects. METHODS We measured anthropometric markers as well as RBP, fasting glucose and insulin in 206 overweight subjects and repeated these measurements 36 months later. Subjects were grouped according to fasting plasma glucose concentration at baseline and 36 months. RESULTS Subjects (n=51) with a normal basal fasting glucose (<5.6 mmol/L) who developed impaired fasting glucose (IFG) 3 years later (>or=5.6 mmol/L) showed a highly significant increase in both fasting insulin and insulin resistance, but importantly no change in plasma RBP. This group had a significant increase in body mass index (BMI). Subjects (n=101) with a normal fasting glucose at both baseline (<5.6 mmol/L) and 36 months showed no significant change in fasting insulin, insulin resistance, RBP or BMI. The remaining subjects had impaired basal fasting glucose and were not analysed on a group-wise basis. Overall, RBP correlated significantly, but inversely, with anthropometric measures, but not with fasting glucose, insulin or insulin resistance. CONCLUSIONS This is the first report of a long-term longitudinal study on RBP and the major finding is that subjects who developed insulin resistance showed no change in plasma RBP. On the basis of our results we consider that RBP cannot be construed as a marker of insulin resistance in overweight humans.