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Fractionation of serum IgA in patients with IgA nephropathy, alcoholic cirrhosis and systemic lupus erythematosus (SLE)
ABSTRACTS OF ANNUAL MEETING 1978
321
APPLICATION OF GC-MS TO ESTIMATION AND CHARACTERIZATION OF METABOLIC PROFILES OF STEROID EXTRACTS
G. E. JOANNOU, W. J. HENSLEY & J. R. TURTLE Prince Alfred Hospital, Sydney
Biochemistry and Endocrinology Department, Royal
The urinary steroid metabolites of 2 male siblings with congenital adrenal hyperplasia of a suspected 11 fJhydroxylase deficiency have been studied in our laboratory by gas chromatography (GC) and gas chromatographymass spectrometry (GC-MS). Preliminary identification of steroids is based on the direct comparison of GC retention indices (RI) with those of reference compounds. The use of at least two oxime-trimethyl-silyl derivatives not only enhances the reliability of the method when RI values are used for the sole criteria of identification but can afford some structural characterization of unknowns. Mass spectrometry is used for absolute confirmation. The profile pattern of this syndrome is distinguished from that of normal subjects by greatly elevated secretion rates of atypical neutral steroids. The tentative identification of a pregnantetrol present in both these extracts has also been achieved. THE ROLE OF L'A-ANDROSTENEDIONE IN THE ASSESSMENT OF PATIENTS WITH DISORDERS OF SEXUAL DEVELOPMENT
J. MONTALTO, H. E. DAVIES, J. F. CONNELLY, G. WARNE & H. N. B. WETTENHALL Clinical Biochemistry, Royal Children's Hospital, Melbourne
Department of
The development of a radioimmunoassay for '" 4-androstenedione ('" 4-A.D.) in plasma has enabled the establishment of reference ranges In prepubertal boys and girls. Application of this assay in a variety of patients has shown the following: (I) An elevated plasma'" 4-A.D. in untreated patients with congenital virilizing adrenal hyperplasia (CV.A.H.) due to 21 hydroxylase deficiency. The "'4-A.D. levels were lower than the corresponding levels of 17 rxhydroxyprogesterone (17 OHP). (2) In 2 cases of lip-hydroxylase deficiency'" 4-A.D. showed higher levels than 17 OHP, but lower than those of II deoxycortisol. (3) Elevated values were also seen in 2 infants with suspected testicular feminization and in 4 children with early pubic hair development. (4) In patients with CV.A.H. on treatment, preliminary studies suggest that "'4-A.D. in conjunction with 17 OHP may clearly define whether patients are receiving optimal steroid therapy. FRACTIONATION OF SERUM IgA IN PATIENTS WITH IgA NEPHROPATHY, ALCOHOLIC CIRRHOSIS AND SYSTEMIC LUPUS ERYTHEMATOSUS (SLE)
A. M. WOOTTON*, P. E. McKENZllot, A. A. GORMLyt, A. R. CLARKSONt, M. E. COLES· & A. J. WOODROFFEj" ·Department of Clinical Chemistry, Institute of Medical and Veterinary Science, Adelaide and t Renal Unit, Royal Adelaide Hospital Deposits of IgA and C3 are found in renal biopsies from patients with IgA nephropathy, alcoholic cirrhosis and SLE. Many of these patients have raised serum IgA levels and it has been suggested that the glomerular deposits may be IgA aggregates rather than soluble immune complexes (lC). The molecular size of serum IgA was therefore determined in control subjects and in patients with these diseases using gel filtration (Sephadex G-200 and Sepharose CL-6B) and sucrose density gradient (10-60%) ultracentrifugation. The fractions were assayed for IgA, IgG, IgM and C3 by laser nephelometry and for IC by'solid phase Clq RIA. Patients with alcoholic cirrhosis and SLE had increased amounts of > 7S IgA and IC There was no increase in polymeric IgA in the majority of patients with IgA nephropathy by 30% had IC The data suggests that the serological and renal findings in these diseases result from IC rather than IgA aggregates. ASSAY OF SERUM FREE T., FREE T, AND FREE rT, BY EQUILIBRATION DIALYSIS AND RADIOIMMUNOASSA Y
R. G. SYMONS & M. L. WELL BY Adelaide
Department of Clinical Chemistry, The Queen Elizabeth Hospital,
The use of free (t) thyroid hormone assays to assess thyroid function is a controversial issue. We have therefore established assays for free T4, free T J and free rTJ with a view to further investigating their role in the evaluation of thyroid disease. Serum was dialyzed against isotonic HEPES buffer. Aliquots of dialysate were assayed for T 4, T J or rT, by radioimmunoassay employing high specific activity tracer, high titre antisera and charcoal as separant. Mean concentrations offT4 and fTJ in euthyroid subjects were found to be similal', being 10.8 ± 4.4 pmoljl and 6.4 ± 0.8 pmoljl respectively. Mean frT J concentration was 1.3 ± 0.8 pmoljl. All subjects in an untreated clinically
321
APPLICATION OF GC-MS TO ESTIMATION AND CHARACTERIZATION OF METABOLIC PROFILES OF STEROID EXTRACTS
G. E. JOANNOU, W. J. HENSLEY & J. R. TURTLE Prince Alfred Hospital, Sydney
Biochemistry and Endocrinology Department, Royal
The urinary steroid metabolites of 2 male siblings with congenital adrenal hyperplasia of a suspected 11 fJhydroxylase deficiency have been studied in our laboratory by gas chromatography (GC) and gas chromatographymass spectrometry (GC-MS). Preliminary identification of steroids is based on the direct comparison of GC retention indices (RI) with those of reference compounds. The use of at least two oxime-trimethyl-silyl derivatives not only enhances the reliability of the method when RI values are used for the sole criteria of identification but can afford some structural characterization of unknowns. Mass spectrometry is used for absolute confirmation. The profile pattern of this syndrome is distinguished from that of normal subjects by greatly elevated secretion rates of atypical neutral steroids. The tentative identification of a pregnantetrol present in both these extracts has also been achieved. THE ROLE OF L'A-ANDROSTENEDIONE IN THE ASSESSMENT OF PATIENTS WITH DISORDERS OF SEXUAL DEVELOPMENT
J. MONTALTO, H. E. DAVIES, J. F. CONNELLY, G. WARNE & H. N. B. WETTENHALL Clinical Biochemistry, Royal Children's Hospital, Melbourne
Department of
The development of a radioimmunoassay for '" 4-androstenedione ('" 4-A.D.) in plasma has enabled the establishment of reference ranges In prepubertal boys and girls. Application of this assay in a variety of patients has shown the following: (I) An elevated plasma'" 4-A.D. in untreated patients with congenital virilizing adrenal hyperplasia (CV.A.H.) due to 21 hydroxylase deficiency. The "'4-A.D. levels were lower than the corresponding levels of 17 rxhydroxyprogesterone (17 OHP). (2) In 2 cases of lip-hydroxylase deficiency'" 4-A.D. showed higher levels than 17 OHP, but lower than those of II deoxycortisol. (3) Elevated values were also seen in 2 infants with suspected testicular feminization and in 4 children with early pubic hair development. (4) In patients with CV.A.H. on treatment, preliminary studies suggest that "'4-A.D. in conjunction with 17 OHP may clearly define whether patients are receiving optimal steroid therapy. FRACTIONATION OF SERUM IgA IN PATIENTS WITH IgA NEPHROPATHY, ALCOHOLIC CIRRHOSIS AND SYSTEMIC LUPUS ERYTHEMATOSUS (SLE)
A. M. WOOTTON*, P. E. McKENZllot, A. A. GORMLyt, A. R. CLARKSONt, M. E. COLES· & A. J. WOODROFFEj" ·Department of Clinical Chemistry, Institute of Medical and Veterinary Science, Adelaide and t Renal Unit, Royal Adelaide Hospital Deposits of IgA and C3 are found in renal biopsies from patients with IgA nephropathy, alcoholic cirrhosis and SLE. Many of these patients have raised serum IgA levels and it has been suggested that the glomerular deposits may be IgA aggregates rather than soluble immune complexes (lC). The molecular size of serum IgA was therefore determined in control subjects and in patients with these diseases using gel filtration (Sephadex G-200 and Sepharose CL-6B) and sucrose density gradient (10-60%) ultracentrifugation. The fractions were assayed for IgA, IgG, IgM and C3 by laser nephelometry and for IC by'solid phase Clq RIA. Patients with alcoholic cirrhosis and SLE had increased amounts of > 7S IgA and IC There was no increase in polymeric IgA in the majority of patients with IgA nephropathy by 30% had IC The data suggests that the serological and renal findings in these diseases result from IC rather than IgA aggregates. ASSAY OF SERUM FREE T., FREE T, AND FREE rT, BY EQUILIBRATION DIALYSIS AND RADIOIMMUNOASSA Y
R. G. SYMONS & M. L. WELL BY Adelaide
Department of Clinical Chemistry, The Queen Elizabeth Hospital,
The use of free (t) thyroid hormone assays to assess thyroid function is a controversial issue. We have therefore established assays for free T4, free T J and free rTJ with a view to further investigating their role in the evaluation of thyroid disease. Serum was dialyzed against isotonic HEPES buffer. Aliquots of dialysate were assayed for T 4, T J or rT, by radioimmunoassay employing high specific activity tracer, high titre antisera and charcoal as separant. Mean concentrations offT4 and fTJ in euthyroid subjects were found to be similal', being 10.8 ± 4.4 pmoljl and 6.4 ± 0.8 pmoljl respectively. Mean frT J concentration was 1.3 ± 0.8 pmoljl. All subjects in an untreated clinically