Serum and follicular fluid testosterone concentrations do not correlate, questioning the impact of androgen supplementation on the follicular endocrine milieu.

Basic research into a possible link between serum and follicular fluid androgen concentrations to detemine whether androgen supplementation in low responders affects follicular endocrine milieu is still lacking. Ninety-seven women (aged 28-43 years) undergoing one natural IVF cycle without any hormone stimulation were analysed. Serum and follicular fluid were collected at the time of follicle aspiration, and the concentrations of LH, total testosterone, oestradiol, dehydroepiandrosterone and anti-Mullerian hormone (AMH) were determined. Serum LH (P = 0.003) and AMH (P = 0.026) concentrations, and follicular fluid AMH (P = 0.015) decreased with increasing age. Within follicular fluid, total testosterone was correlated with oestradiol (P < 0.001) and AMH (P = 0.010); LH correlated with AMH (P = 0.005). Correlation analysis of serum and follicular fluid hormone concentrations revealed that LH, oestradiol and AMH correlated (P < 0.001), whereas testosterone did not. Testosterone serum concentrations did not correlate with other follicular fluid hormones, such as dehydroepiandrosterone, oestradiol and AMH, whereas serum LH correlated with follicular flulid AMH (P < 0.008). Follicular fluid hormone concentrations seem to be independent from serum testosterone. Therefore, it is questionable whether an increase in serum testosterone concentration by androgen supplementation could improve the follicular endocrine milieu.

Determination of carbohydrate-deficient transferrin in human serum by two capillary zone electrophoresis methods and a direct immunoassay: comparison of patient data.

Data obtained with two CZE assays for determining carbohydrate-deficient transferrin (CDT) in human serum under routine conditions, the CAPILLARYS CDT and the high-resolution CEofix (HR-CEofix) CDT methods, are in agreement with patient sera that do not exhibit interferences, high trisialo-transferrin (Tf) levels or genetic variants. HR-CEofix CDT levels are somewhat higher compared to those obtained with the CAPILLARYS method and this bias corresponds to the difference of the upper reference values of the two assays. The lower resolution between disialo-Tf and trisialo-Tf observed in the CAPILLARYS system (mean: 1.24) compared to HR-CEofix (mean: 1.74) is believed to be the key for this difference. For critical sera with high trisialo-Tf levels, genetic variants, or certain interferences in the beta-region, the HR-CEofix approach is demonstrated to perform better than CAPILLARYS. However, the determination of CDT with the HR-CEofix method can also be hampered with interferences. Results with disialo-Tf values larger than 3% in the absence of asialo-Tf should be evaluated with immunosubtraction of Tf and possibly also confirmed with another CZE method or by HPLC. Furthermore, data gathered with the N Latex CDT direct immunonephelometric assay suggest that this assay can be used for screening purposes. To reduce the number of false negative results, CDT data above 2.0% should be confirmed using a separation method.

Determination of carbohydrate-deficient transferrin in human serum with capillary zone electrophoresis. Sample preparation strategies for the removal of interferences caused by increased levels of immunoglobulins.

Capillary zone electrophoresis (CZE) in fused-silica capillaries is an effective analytical approach for the separation and determination of the transferrin (Tf) isoforms and thus carbohydrate-deficient transferrin (CDT) in human serum. Sera of patients with progressed liver cirrhosis are prone to interferences in the beta region which prevent the proper determination of CDT by CZE without additional sample preparation. Efforts to identify, reduce or even eliminate these interferences have been undertaken. Data obtained by ultrafiltration, affinity subtraction procedures using protein A, protein L and antibodies against immunoglobulins or Tf, and immunopurification of Tf suggest that the interferences in the patient sera are caused by increased levels of IgA and IgM and are best eliminated by immunopurification. Avian IgY antibody spin column immunocapture of serum Tf followed by CZE analysis of the stripped and concentrated fraction is shown to provide an attractive approach for CDT monitoring in sera with beta region interferences.

Agreement between HbA1c measured by DCA 2000 and by HPLC: effects of fetal hemoglobin concentrations.

BACKGROUND: In subjects with type 1 diabetes, persisting elevations of fetal hemoglobin (HbF) have been demonstrated. This study evaluated whether HbF levels typically seen in type 1 diabetes (up to 3%) interfere with glycohemoglobin determinations using a common immunologic method (DCA 2000). METHODS: HbA(1c) was measured by high-performance liquid chromatography (HPLC) using a Diamat analyzer in 90 type 1 diabetics with parallel determinations of HbF. Results were compared with HbA(1c) concentrations obtained using DCA 2000. RESULTS: Reproducibility was good for both methods with coefficients of variation <5% and correlation between the two methods was good (r(2)=0.939, p<0.0001). Mean difference between the two methods was small (0.007%). Limits of agreement varied between -0.92% and +0.93% (95% confidence interval [95% CI]) and constant bias (intercept: 0.73 95% CI 0.28-1.18) as well as a proportional bias (slope: 0.92 95% CI 0.87-0.97) were detected. At low concentrations of HbF, the DCA 2000 immunologic method tended to underestimate and at higher concentrations tended to overestimate HbA(1c) when compared with Diamat. Stepwise linear regression with HbA(1c) (DCA 2000) as dependent variable included HbA(1c) (Diamat) and HbF in the model (r(2)=0.946, p<0.0001), explaining 94.6% of the variability of HbA(1c) (DCA 2000). Partial correlation coefficient between HbA(1c) (DCA 2000) and HbF corrected for HbA(1c) (Diamat) was 0.337 (p=0.0012). CONCLUSIONS: DCA 2000 allowed measurements of HbA(1c) rapidly and with precision adequate for clinical purposes. However, agreement with Diamat results was comparatively weak with both constant as well as proportional biases. The 95% limits of agreement between Diamat and DCA 2000 fell within a range that significantly limited traceability between these two methods; therefore, the two methods should not be used interchangeably. Small but persistent elevations of HbF concentrations were identified as a significant cofactor, which may be relevant for limited traceability between the two methods.

Capillary zone electrophoresis with a dynamic double coating for analysis of carbohydrate-deficient transferrin in human serum. Precision performance and pattern recognition.

Capillary zone electrophoresis (CZE) with a dynamic double coating permits the simultaneous, individual, quantitative determination of transferrin (Tf) isoforms in human serum and thus carbohydrate-deficient transferrin (CDT), the most specific marker available today for the detection of chronic, excessive alcohol intake. CZE of serum Tf was carefully evaluated using the P/ACE MDQ with fused-silica capillaries of 50 microm I.D. and 60.2 cm total length, the CEofix CDT kit and the instrumental conditions recommended by the kit manufacturer. The precision performance assessed over a 20-day period according to the internationally accepted NCCLS EP5-A guidelines revealed the CZE assay as being highly reproducible with within-run and total precision being dependent on the Tf isoform level and RSD values ranging between 2.2 and 17.6%. Inter-day RSD values for asialo-Tf were noted to be between 9.8 and 11.5% and for disialo-Tf between 3.8 and 8.6%, whereas those for CDT levels of 0.87 and 4.31% of total Tf were determined to be 8.6 and 3.4%, respectively. The RSD values for trisialo-Tf, tetrasialo-Tf, pentasialo-Tf and hexasialo-Tf were found to be between 0.4 and 4.1%. Tf patterns are recognized and identified via detection times of Tf isoforms (intra-day and inter-day RSD values < 1.0% and < 1.7%, respectively), immunosubtraction of Tf and enzymatic sequential cleavage of sialic acid residues. Furthermore, heterozygous Tf BC and Tf CD variants are assigned via spiking with a known mixture of Tf isoforms (e.g. the serum of a healthy Tf C homozygote). Among the non-Tf peaks monitored, the CRP peak detected shortly before disialo-Tf was identified by immunosubtraction and peak magnitudes were found to correlate well with immunochemically determined CRP serum levels. The CZE assay with dynamic double coating could thereby be shown to be sensitive enough to determine elevated CRP levels in human serum. Furthermore, unusual peaks in the gamma-region were identified by customary serum protein CZE, immunosubtraction CZE and immunofixation.

Signalling shortcuts: cell-surface receptors in the nucleus?

Do cell-surface growth-factor receptors and their ligands accumulate in the nucleoplasm under physiological conditions? And, if so, how do they get there and what function do they serve in this location? Recent advances have provided tantalizing hints to the answers to these questions, and hold the key to identifying a new mode of signal transduction.