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Objective: To study possible mechanisms for the inhibition of cytochrome P450 C21 (steroid 21-hydroxylase) enzyme activity by P450 C21 autoantibodies (Abs) in vitro. Design: Two possible mechanisms for the inhibition of P450 C21 enzyme activity by P450 C21 Abs were studied: (a) conformational changes in the P450 C21 molecule induced by Ab binding and (b) the effects of Ab binding to P450 C21 on the electron transfer from the nicotinamide adenine dinucleotide phosphate reduced (NADPH) cytochrome P450 reductase (CPR) to P450 C21. Methods: The effect of P450 C21 Ab binding on the conformation of recombinant P450 C21 in yeast microsomes was studied using an analysis of the dithionite-reduced CO difference spectra. The effect of P450 C21 Abs on electron transfer was assessed by analysis of reduction of P450 C21 in the microsomes in the presence of CO after addition of NADPH. Results: Our studies confirmed the inhibiting effect of P450 C21 Abs on P450 C21 enzyme activity. Binding of the Abs did not induce significant change in the P450 C21 peak at 450 nm (native form) and did not produce a detectable peak at 420 nm (denatured form) in the dithionite-reduced CO difference spectra. This indicated that conformation of P450 C21 around the heme was not altered compared with the native structure. However, incubation of the P450 C21 in yeast microsomes with P450 C21 Ab inhibited the fast phase electron transfer from the CPR to P450 C21. Conclusions: Our observations suggested that the mechanism by which P450 C21 Abs inhibit P450 C21 enzyme activity most likely involves inhibition of the interaction between the CPR and P450 C21.European Journal of Endocrinology 152 95-101

Objective: To study the interaction between human steroid 21-hydroxylase (21-OH) and monoclonal antibodies (MAbs) to 21-OH directed to 3 different epitopes recognised by 21-OH autoantibodies characteristic of autoimmune Addison's disease. Design: Build comparative structural models of 21-OH, 21-OH MAbs and complexes of 21-OH -21-OH MAbs and study the effects of 21-OH MAbs on 21-OH enzyme activity. Then, analyse the relationship between sites important for binding of 21-OH MAbs and 21-OH autoantibodies and sites important for 21-OH enzyme activity. Methods: Variable (V) regions of 21-OH MAbs (M21-OH1, M21-OH3, M21-OH5) were sequenced and models of the MAbs built using structures of antibodies in the database as templates. A comparative model of 21-OH was built using the crystal structure of rabbit cytochrome p450 2c5/3LVdH as template. 21-OH enzyme activity was measured in terms of conversion of [ 3 H]progesterone to deoxycorticosterone and the effect of purified MAb IgGs on 21-OH enzyme activity was assessed. Results: M21-OH1, M21-OH3 and control MAb had no effect on 21-OH enzyme activity with 88.8%^24% (n ¼ 6), 86.7%^7.6% (n ¼ 6) and 86.5%^10.6% (n ¼ 6) of activity remaining in the presence of the respective IgGs. This was consistent with the epitopes for M21-OH1 and M21-OH3 being located distant from 21-OH enzyme active sites in our 21-OH model. The epitope for M21-OH5 which inhibited 21-OH enzyme activity (48.5^8.3% activity remaining; P , 0.001 compared with control MAb IgG) was found close to the redox protein binding site in our 21-OH model. Conclusions: A comparative model of 21-OH has been produced. Analysis of experimental data in the context of the model suggests that M21-OH5 inhibits 21-OH enzyme activity through interference with redox protein binding. European Journal of Endocrinology 153 949-961

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Objective: The influence of age on exophthalmos and thyrotrophin receptor antibodies (TRAb) was studied in untreated patients with Graves’ disease (GD). Exophthalmos measurement is useful for GD diagnosis in elderly Japanese patients. Subjects and methods: Protrusions of the eyes (exophthalmic values: EV) and TRAb were studied in 123 untreated patients with GD and 560 controls. Patients with GD and controls were divided into seven age groups. The receiver operating characteristic (ROC) analysis was done to evaluate the clinical usefulness of EV for GD diagnosis. Findings: The differences of EV between patients with GD and controls were significant in those aged 60 years and over (≥ 60 years). ROC analysis showed that the sensitivity and specificity of EV were high in elderly groups (≥ 60 years), but not in the others (< 60 years). Conclusion: Exophthalmic values have clinical usefulness for GD diagnosis in elderly Japanese. An EV has a diagnostic value for GD in elderly patients (≥ 60 years).