Hans Erik Sjöberg and Sigbritt Werner
Bengt Karlberg, Sven Almqvist and Sigbritt Werner
Effects of single rapid iv injections of synthetic pyroglutamyl-histidyl-proline amide (TRH) in doses of 100 and 200 μg in nine normal subjects and patients with pituitary and thyroid disorders were evaluated by clinical observation and serial measurements of serum levels of human thyrotrophin (HTSH), cortisol, growth hormone (STH), insulin and PBI. An onset of fatigue was observed in about half of the patients six to eight hours after the injection of TRH and lasted several hours.
A rise in serum HTSH was detectable within 10 min after the administration of 100 μg of TRH in all normal subjects, reaching a maximum in 15 min in seven of these nine individuals and at 30 and 120 min in two subjects; and then gradually decreased over the next two hours, when the mean level was still above the baseline. Three of nine time courses of serum HTSH showed two peaks, a first at 5 to 15 min and a second at 30 to 120 min. Increases in serum PBI were measurable within 120 min after TRH.
Multiple effects of TRH on HTSH, cortisol and STH levels in serum were found in six of nine normal subjects. The percentage increases were frequently larger for STH than for HTSH levels and the effect was heterogeneous on the time courses of these two hormones.
An increase in serum HTSH occurred in four untreated patients with acromegaly, and a slow and prolonged response in two of three patients with primary hypothyroidism after 200 μg of TRH. One woman with Sheehan's syndrome showed no rise in serum HTSH at 100 μg of TRH but a moderate rise at 200 μg of TRH. Another patient with an operated chromophobe adenoma and panhypopituitarism responded normally to 100 μg of TRH.
Clinical studies involving the use of various synthetic derivatives of TRH are needed to evaluate the clinical response of the tripeptide.
Bengt Karlberg, Sven Almqvist and Sigbritt Werner
Peter Brostedt, Marguerite Luthman, Leif Wide, Sigbritt Werner and Paul Roos
Seven highly purified dimeric forms of human pituitary growth hormone, composed of the monomeric forms 20 K hGH, 22 K hGH and 24 K hGH linked together by noncovalent or covalent bonds, have been characterized by an in vitro bioassay (the Nb2 assay), a radioreceptor assay and a radioimmunoassay. Considerable differences in the ability to displace labelled recombinant hGH were observed in the radioreceptor assay. The seven dimeric forms varied over a range between 22 K hGH (most effective) and 20 K hGH. The three covalently-linked dimeric forms had nearly identical affinity constants. The mitogenic action of all but one of the hGH dimers in the Nb2 assay was in the same mutual order as the receptor binding activity in the radioreceptor assay. In the RIA, all dose-response curves were parallel except for those obtained with 20 K hGH and with the dimeric form (20 K-20 K)hGH. In this assay, dimeric variants of the constituents 22 K hGH and 24 K hGH were approximately twice as active as 22 K hGH on a molar basis, suggesting about the same affinity between the antibodies and each of the monomeric forms. Determination of the amino acid compositions of the dimeric forms provided support for their content of monomeric constituents as established earlier by electrophoretic analysis.
Marguerite Luthman, Ella Wallerman, Paul Roos and Sigbritt Werner
The aim was to investigate the bioactivity of a high-molecular weight human growth hormone, identified following molecular sieve chromatography of serum. Nine patients with pituitary disease and GH insufficiency were studied. All patients had non-detectable levels of immunoreactive GH, <0.2 μg/l, in diurnal serum profiles. GH bioactivity was determined before and after size-fractionation of serum. The bioassay is based on the finding that a rat lymphoma cell line, Nb2, proliferates in the presence of lactogens. GH and PRL immunoreactivities were measured by radioimmunoassays. Pronounced GH immunoreactivity was found in fractions of sera from 7 out of the 9 patients and of 2 of 4 control sera, particularly in fractions corresponding to the elution volume of high-molecular weight proteins (>160 kD). PRL immunoreactivity was only detected in fractions corresponding to the elution volume of monomeric PRL. Unfractionated serum had a dose-dependent mitogenic effect on the Nb2 cells. GH-antibodies could not inhibit this effect. Fractions of serum obtained from the patients stimulated Nb2 cell division as well. The mitogenic effect of serum fractions could be inhibited by GH-antibodies. Thus, high-molecular weight GH circulating in patients with GH insufficiency were shown to exert a GH-specific bioactivity in vitro after size-fractionation.
Staffan Gröndal, Barbro Eriksson, Lars Hagenäs, Sigbritt Werner and Tore Curstedt
The urinary steroid profile was determined in 24 patients with adrenocortical carcinoma. Seventeen of the patients had Cushing's syndrome, virilization or feminization, and 7 had no signs of endocrine disease. Seven of the 11 patients still alive are free of disease, after a follow-up period of 5-75 months. The steroid profile varied widely between the patients with adrenocortical carcinoma. Patients with Cushing's syndrome had increased levels of cortisol metabolites and those with virilism had raised excretion of androgen metabolites. Six of the patients with adrenocortical carcinoma showed normal values of these metabolites. In 23 of the 24 patients the excretion of 3β-hydroxy-5-ene steroids and/or metabolites of cortisol precursors, such as tetrahydro-11-deoxycortisol, were significantly increased, compared with healthy controls or patients with adrenal adenomas. These findings suggest a relative deficit or low activity of 3β-hydroxysteroid dehydrogenase/Δ5-4 isomerase and/or 1 1β-hydroxylase in tumour tissue. In the single patient where the steroid profile failed to indicate malignancy, hypercortisolism was seen and the tumour mass was small. The steroid excretion normalized after radical surgery and decreased in patients responding to chemotherapy. During recurred disease the metabolites of 3β-hydroxy-5-ene steroids and/or cortisol precursors increased, but in some patients the excretory pattern then was different from that seen before treatment.
Marguerite Luthman, Ingileif Jónsdóttir, Bo Skoog, Inga-Lena Wivall, Paul Roos and Sigbritt Werner
A radioimmunoassay based on a monoclonal antibody, Mc-ab 1, which was raised against growth hormone but cross-reacted with human placental lactogen yielded higher GH immunoreactivity levels in serum than one based on a polyclonal antiserum. This discrepancy was noted in subjects with normal GH secretion as well as in patients with GH insufficiency. To characterize this GH immunoreactivity detected by Mc-ab 1, affinity purification and molecular sieve chromatography of serum were performed. High molecular weight proteins with GH immunoreactivity were found with both techniques. These proteins were associated with carbohydrates. Affinity cross-linking showed specific binding of radiolabelled GH to high molecular weight proteins in the serum. After fractionation of serum, the GH immunoreactivity became detectable by the polyclonal antiserum assay as well as by an immunoradiometric assay. GH immunoreactive material with an approximate mass of 80 kD was subjected to isoelectric focusing. When GH immunoreactive fractions at pH 5 were re-chromatographed, GH immunoreactivity was recovered in the elution volume corresponding to monomeric GH. Our results show that sera from normal subjects as well as from patients with deficient GH secretion contain notable amounts of high molecular weight GH which is undetectable by antibodies generally used for GH measurements, but which can be revealed after fractionation of serum.