Technological difficulties in ghrelin and obestatin assays

W trakcie kilkuletnich doświadczeń wykonano ponad 1000 radioimmunologicznych oznaczeń greliny i obestatyny, przy których wykazano liczne trudności techniczne. Inną trudnością była niezwykle wielka rozbieżność wyników greliny w publikacjach różnych autorów. Celem pracy jest omówienie tych problemów. Tylko niektóre peptydy podwzgórza i przewodu pokarmowego są możliwe do oznaczania we krwi krążącej, bowiem część neuropeptydów nie przekracza bariery krew–mózg. Niektóre hormony przewodu pokarmowego występują w niezwykle małych stężeniach. Wymaga to wykonania żmudnych i pracochłonnych ekstraktów, w trakcie których dochodzi do strat części peptydów. Ponadto peptydy są nietrwałe i łatwo ulegają rozkładowi. Są one także wrażliwe na wysokie temperatury, stąd niezbędne jest zachowanie niskich temperatur otoczenia w trakcie wykonywania oznaczeń oraz dodawanie do osocza substancji hamujących aktywność enzymów. Peptydy mogą występować w postaci izoform oraz form aktywnych i nieaktywnych, co utrudnia ocenę wyników badań, bowiem obie formy mogą mieć odmienne działanie biologiczne. Jednak stosowane w zestawach RIA przeciwciała są poliklonalne i nie różnicują tych izoform. Brak międzynarodowych standardów uniemożliwia porównywanie wyników badań różnych ośrodków naukowych. Dlatego w każdej serii oznaczeń powinna być dołączona grupa kontrolna osób zdrowych w celu sprawdzenia, czy wyniki stężeń peptydów osób chorych różnią się statystycznie od uzyskanych u osób zdrowych. (Endokrynol Pol 2011; 62 (4): 336–339)In recent years we have performed more than 1,000 radioimmunoassays of ghrelin and obestatin. In these assays, we have encountered several technological obstacles. Another difficulty was the enormous discrepancy of plasma ghrelin results published by different authors. The aim of this article is to comment on these problems. Not all peptides of the hypothalamus and intestines are present in blood circulation. Several neuropeptides do not cross the blood-brain barrier, and several gastrointestinal peptides are present in extremely low concentrations in the blood. That requires time-consuming and laborious extraction. In these procedures, considerable amounts of peptides may be lost. In addition, these peptides are very unstable and prone to enzymatic degradation. This makes it mandatory to add enzymatic inhibitors to plasma samples. The peptides are also unstable in elevated temperatures, hence the assays should be performed in air-conditioned laboratories and the kits should be transported in proper low temperature conditions. Peptides may appear in several isoforms of different biological activity, but antibodies routinely used in these assays are polyclonal and do not differentiate between these forms. This complicates clinical evaluation of the results. To date, there are no international standards of ghrelin, obestatin or other active peptides, probably because of their extreme instability. Because of technological difficulties, the results of peptide assays performed in different scientific research institutions vary greatly and cannot be compared to each other. This disadvantage may be partially diminished by including samples of healthy subjects in each assay run to check whether the peptide concentrations of the patients differ significantly from that of control subjects. (Pol J Endocrinol 2011; 62 (4): 336–339

Steroid 21-hydroxylase is a major autoantigen involved in adult onset autoimmune Addison's disease

AbstractAn adrenal-specific protein reacting with autoantibodies in the sera of patients with adult onset Addison's disease has been purified from human adrenal glands. The protein, mol.wt. 55K, has the biochemical characteristics of steroid 21-hydroxylase and reacts on Western blots with rabbit antibodies to recombinant 21-hydroxylase. Absorption of the native human 55K adrenal protein with human adrenal autoantibodies prevented the subsequent reaction of the 55K protein with rabbit antibodies to 21-hydroxylase in Western blot analysis. In addition, human adrenal autoantibodies reacted with recombinant 21-hydroxylase expressed in yeast. These data indicate that the adrenal specific enzyme steroid 21-hydroxylase is a major autoantigen involved in adult onset autoimmune Addison's disease

Premature ovarian failure and ovarian autoimmunity

Premature ovarian failure (POF) is defined as a syndrome characterized by menopause before the age of 40 yr. The patients suffer from anovulation and hypoestrogenism. Approximately 1% of women will experience menopause before the age of 40 yr. POF is a heterogeneous disorder with a multicausal pathogenesis involving chromosomal, genetic, enzymatic, infectious, and iatrogenic causes. There remains, however, a group of POF patients without a known etiology, the so-called "idiopathic" form. An autoimmune etiology is hypothesized for the POF cases with a concomitant Addison's disease and/or oophoritis. It is concluded in this review that POF in association with adrenal autoimmunity and/or Addison's disease (2-10% of the idiopathic POF patients) is indeed an autoimmune disease. The following evidence warrants this view: 1) The presence of autoantibodies to steroid-producing cells in these patients; 2) The characterization of shared autoantigens between adrenal and ovarian steroid-producing cells; 3) The histological picture of the ovaries of such cases (lymphoplasmacellular infiltrate around steroid-producing cells); 4) The existence of various autoimmune animal models for this syndrome, which underlines the autoimmune nature of the disease. There is some circumstantial evidence for an autoimmune pathogenesis in idiopathic POF patients in the absence of adrenal autoimmunity or Addison's disease. Arguments in support of this are: 1) The presence of cellular immune abnormalities in this POF patient group reminiscent of endocrine autoimmune diseases such as IDDM, Graves' disease, and Addison's disease; 2) The more than normal association with IDDM and myasthenia gravis. Data on the presence of various ovarian autoantibodies and anti-receptor antibodies in these patients are, however, inconclusive and need further evaluation. A strong argument against an autoimmune pathogenesis of POF in these patients is the nearly absent histological confirmation (the presence of an oophoritis) in these cases (< 3%). However, in animal models using ZP immunization, similar follicular depletion and fibrosis (as in the POF women) can be detected. Accepting the concept that POF is a heterogenous disorder in which some of the idiopathic forms are based on an abnormal self-recognition by th