Variety in growth hormone determinations due to use of different immunoassays and to the interference of growth hormone-binding protein.

More than 30 years after their introduction, growth hormone (GH) immunoassays showed the poorest inter-laboratory agreement of the various hormone assays evaluated in 1998 by the UK National External Quality Assessment Scheme, in which different laboratories using different assays reported that analyses of identical samples differed two- to threefold in value. There is therefore an urgent requirement and desire within the scientific community, particularly within centres diagnosing and treating GH deficiency and acromegaly, to resolve this problem and to develop a GH assay(s) that measures solely all of the relevant components of circulating GH immunoreactivity. The main confounders in the estimation of GH levels (now that the use of GH standards other than that recommended by the World Health Organization has largely been eliminated) are GH heterogeneity, anti-GH antiserum binding site specificity and interference from circulating high-affinity GH-binding protein (GHBP). The effects of these factors are closely related. The present study investigates these factors, focussing on the influence of GHBP and antibody binding site specificity on various assays for GH. The findings lead the authors to suggest that a solution to the problem may be to develop a GH assay that measures specifically and solely all serum 22 kDa GH, as this is the major circulating fraction and carries the dominant GH bioactivity.

Synthesis of stereoisomers and isoforms of a tryptic heptapeptide fragment of human growth hormone and analysis by reverse-phase HPLC and capillary electrophoresis.

The amino acid sequence Asn-Gly has at pH 7 a tendency to induce deamidation of asparagine to aspartic acid via the formation of a cyclic imide. This imide opens up to yield Asp-Gly or the isoaspartic acid (isoAsp) form, isoAsp-Gly. Both isomers may be found in their L-form or D-form. Like Asn-Gly, the sequence Asp-Gly has a tendency for isomerization and racemization via the formation of a cyclic imide intermediate. When human growth hormone is digested with trypsin, one of the fragments is a heptapeptide (amino acid residues 128-134) containing the amino acid sequence Asp-Gly (amino acid residues 130 and 131). This heptapeptide, as well as stereoisomers and isoforms where L-Asp was replaced by D-Asp, L-isoAsp, D-isoAsp or the L-cyclic imide, respectively, has been synthesized and used as a standard to achieve separation of the five forms by capillary electrophoresis and by reverse-phase HPLC. Capillary electrophoresis analysis was performed in uncoated capillaries by the use of aspartic acid/cyclodextrin buffers at low pH. The elution order of the aspartic-acid-containing heptapeptides was D-Asp, L-Asp, L-isoAsp, D-isoAsp and L-cyclic imide. Reverse-phase HPLC analysis was performed on a C18 column by the use of a shallow acetonitrile gradient in trifluoroacetic acid/water. The elution order was D-isoasp, L-isoASp, L-Asp, D-Asp and L-cyclic imide. Human growth hormone samples were degraded by incubation at high temperature and analyzed for their potential content of isomerization and racemization products. Only L-forms of aspartic acid and isoaspartic acid of the heptapeptide fragment were found.

Characterisation of a trisulphide derivative of biosynthetic human growth hormone produced in Escherichia coli.

A novel protein derivative has been found during process development of biosynthetic human growth hormone; it has been characterised as human growth hormone with a Cys182-Cys189 trisulphide bridge. We have not been able to find a previous report in the literature about this kind of derivative. The characterisation was obtained partly on the full-length derivative and partly on a tryptic fragment of the derivative. The full-length derivative was characterised by reduction with 1,4-dithiothreitol followed by electrospray mass spectrometry, treatment with cysteine and measurement of hydrogen sulphide liberation upon cysteine treatment. The tryptic fragment from peptide mapping was characterised by amino acid analysis, amino acid sequencing and mass spectrometry. All data indicated an extra sulphur atom in the Cys182-Cys189 cystine bridge.

Development of an optimized refolding process for recombinant Ala-Glu-IGF-1.

Denatured and reduced N-terminal extended insulin-like growth factor-1 (AE-IGF-1) was purified from Escherichia coli extracts and subjected to in vitro folding. The renaturation process was shown to be a function of the redox potential of the solution. Folding by different methods had no significant effect on the renaturation. A maximal yield of 60% (w/w) was obtained. The folded AE-IGF-1 was enzymatically converted to IGF-1. The major by-product (20% w/w) was identified as scrambled IGF-1. Enzymatic digestion at alkaline and acidic pH suggested two possible disulphide bond arrangements; (i) Cys6-Cys47, Cys18-Cys61, Cys48-Cys52; or (ii) Cys6-Cys52, Cys18-Cys61, Cys47 and Cys48 being in their reduced forms. Energy minimization and molecular modelling suggested that the scrambled IGF-1, having reduced cysteines at positions 47 and 48, was the energetically most stable conformation of the two.

A practical approach to high performance capillary electrophoresis with biosynthetic human growth hormone as a model protein.

Biosynthetic human Growth Hormone (B-hGH) is a protein comprising 191 amino acids. The molecular weight is 22,125 and the isoelectric point is close to pH 5. Due to the ready availability of closely related analogues B-hGH was used as a model protein thus allowing for the demonstration and evaluation of the high resolution capability of high performance capillary electrophoresis (HPCE). The same apparatus was used throughout the experiments and an optimum signal-to-noise ratio was found at 200 nm. Linearity was observed between peak area, retention time and the hGH concentration or sample introduction time. Baseline separation of hGH, desamido hGH and didesamido hGH was obtained. Examples showing analysis with 1 million theoretical plates per meter, high speed separation, simultaneous analysis of multiple samples, sample stacking, hGH tryptic digest, and hGH lysate are reported. The use of electrophoretic velocities instead of apparent velocities for peak identification is illustrated.

Alternative mobile phases for the reversed-phase high-performance liquid chromatography of peptides and proteins.

The use of a high content of acetic acid as mobile phase additive for the reversed-phase high-performance liquid chromatography (RP-HPLC) of several proteins and extracts of biological tissues was evaluated for a divinylbenzene (DVB)-based stationary phase, and the separations obtained with acetic acid gradients in acetonitrile, isopropanol or water were compared with classical polypeptide RP-HPLC on silica C4 with trifluoroacetic acid (TFA)-acetonitrile. The separation patterns for recombinant derived interleukin-1 beta (IL-1 beta) on the C4 column eluted with TFA-acetonitrile and the DVB column eluted with acetic acid-acetonitrile were similar, but only the polymeric column was able to separate the components present in an iodinated IL-1 beta preparation. Neither eluent had any harmful effect on the biological activity of IL-1 beta isolated after RP-HPLC. Several standard proteins could be separated when the polymeric column was eluted with acetic acid gradients in acetonitrile, isopropanol or water and, although the separation efficiency with acetic acid in water was lower than that in combination with classical organic modifiers, insulin, glucagon and human growth hormone (hGH) were eluted as sharp, symmetrical peaks. The recoveries of insulin and hGH were comparable for all three mobile phases (80-90%). The separation patterns obtained from a crude acetic acid extract of a normal and a diabetic, human pancreas analysed using acetic acid gradients with or without organic modifiers were found to be similar and comparable to those obtained on a silica C4 column eluted with an acetonitrile gradient in TFA. The principal differences resulted from the use of different UV wavelengths (215 nm for TFA-acetonitrile, 280 nm for acetic acid). Acetic acid extracts of recombinant derived hGH-producing Escherichia coli were separated on the DVB column eluted with an acetic acid gradient in water. Although the starting material was a highly complex mixture, the hGH isolated after this single-step purification was surprisingly pure (as judged by sodium dodecyl sulphate-polyacrylamide gel electrophoresis). Consequently several (pure) polypeptides and complex biological samples were separated on a polymeric stationary phase eluted with acetic acid gradients in water without the use of organic modifiers.

Strategies for determination of disulphide bridges in proteins using plasma desorption mass spectrometry.

Disulphide bridges have been assigned in three different proteins by locating possible disulphide-linked peptides in enzymic digests of the proteins based on their molecular weight determined by plasma desorption mass spectrometry. Different strategies have been employed including in situ reduction of the nitrocellulose-bound peptides and confirmation of peptide identity by methyl esterification reactions or Edman degradation. The latter was needed for identification of glycosylated disulphide-linked peptides. For insulins cleavage between cysteine residues in close proximity was not possible; but a combination of molecular mass information, enzymic cleavage with two different enzymes and sequence analysis including identification of di-phenylthiohydantoin-cystine could ensure an unambiguous assignment of the disulphide bridges.

Identification of aprotinin degradation products by the use of high-performance capillary electrophoresis, high-pressure liquid chromatography and mass spectrometry.

A preparation of bovine aprotinin, bovine pancreatic trypsin inhibitor, was subjected to high-performance capillary electrophoresis (HPCE) analysis and the purity was calculated to be approximately 80%. The two dominating contaminants were integrated to approximately 7% each as compared to the intact molecule. Characterization by high-pressure liquid chromatographic (HPLC) and mass spectrometric analysis was carried out on digests of the reduced and alkylated molecules. The contaminants were identified as truncated aprotinin, missing one and two amino acids, respectively, at the C-terminus. No such structures were identified in similar amounts in preparations of recombinant aprotinin by HPLC or HPCE.

[Hypoglycemic effects of equimolar quantities of semisynthetic human and porcine insulin in insulin-dependent diabetic patients]. / Den hypoglykaemiske effekt af aekvimolaere maengder semisyntetisk humant og porcint insulin hos insulinafhaengige diabetiske patienter.

In some diabetic patients, transition from porcine insulin (PI) to human insulin (HI) has resulted in alterations or non-appearance of the usual hypoglycaemic symptoms. The authors have, therefore, undertaken a double-blind cross-over trial with induction of hypoglycaemia with semisynthetic HI and PI administered in equimolar quantities to eight insulin-dependent men. Prior to induction of hypoglycaemia, the blood glucose (BG) was stabilized at approximately 6 mmol/l. At the time 0, the BG was 5.9 (5.4-7.1) mmol/l and 6.2 (4.8-7.1) mmol/l (NS), BS-nadir was 2.1 (1.3-2.9) mmol/l and 2.1 (1.3-2.8) mmol/l PI versus HI. At the time -15 minutes and at BG-nadir, assessment of symptoms and neuropsychological testing were performed. The total symptom scorings were identical for the two types of insulin, the maximum intraindividual difference was +1 on a scale from 0 to 3. Cognitive function was reduced significantly in hypoglycaemia with no differences for the types of insulin. The conclusion is that, in insulin-dependent diabetic patients, no differences in hypoglycaemic effects were observed between PI and HI after intravenous administration of equimolar quantities.

High-performance liquid chromatographic separation of membrane proteins isolated from erythrocyte ghosts.

Membrane proteins extracted from erythrocyte ghosts with sodium dodecyl sulphate (SDS), 3-(3-cholamidopropyl)-dimethylamminopropane sulfonate (CHAPS) or octylglucoside have been analyzed in various reversed-phase high-performance liquid chromatographic systems. Only SDS was able to solubilize considerable amounts of membrane proteins with mol.wt. greater than 15,000 daltons, but these membrane proteins were recovered in poor yield from a silica-based C4 column eluted with an acetonitrile gradient in trifluoroacetic acid (TFA). A resin-based phenyl column eluted with a similar TFA-acetonitrile gradient was found to be a better choice with respect to the recovery of membrane proteins with mol.wt. greater than 15,000 daltons, and when this column was eluted with an acetic acid gradient with increasing amounts of acetonitrile, erythrocyte ghost membrane proteins solubilized in SDS (mol.wt. 10,000-200,000 daltons) were separated in six major and several minor components with satisfactory recovery.

NPH insulin administration by means of a pen injector.

In an open randomized cross-over study 50 patients with insulin-dependent diabetes were allocated to 3 months of treatment with NPH insulin either by means of a pen injector (Insuject-X) or by conventional syringes. The needle of the NPH pen injector was removed immediately after use to avoid possible leakage of solvent. Ambulatory control was assessed every 6 weeks, including blood sampling (HbA1c and insulin antibodies) and recording of hypoglycaemia. NPH insulin containers were collected for insulin potency measurement by HPLC. A seven-point blood glucose profile was performed fortnightly by means of home blood glucose monitoring. At the end of the 6 months a questionnaire was completed. No significant changes occurred in HbA1c (difference 0.1 +/- 0.7 (SD)%), blood glucose profile, or the incidence of hypoglycaemic episodes on the two regimens. The concentration of NPH insulin in the containers remained constant. All but two of the patients preferred to continue to use the pen injector. This NPH pen injector is a reliable and efficacious tool which may also prove more convenient for the patients.

Recovery of polypeptides after reversed-phase high-performance liquid chromatography.

Mass recovery of individual polypeptides may be estimated under various practical conditions. With the purpose of obtaining rapid and reliable standard procedures for recovery measurements, we have compared five individual methods utilizing a silica-based stationary phase [Nucleosil C18 (7 microns)/ammonium sulphate-perchlorate-acetonitrile, pH 3.0] and a resin-based stationary phase (TSK Phenyl 5 PW RP/ammonium phosphate-acetonitrile, pH 7.0). The recoveries of insulin (6 kilodaltons), human growth hormone (22 kilodaltons) and human serum albumin (68 kilodaltons) estimated under five different experimental conditions were found to be concordant. Variations in column load, flow-rate, gradient shape and column dwell time and addition of cyclame did not increase the (reduced) recovery of serum albumin and growth hormone.