Bestimmung der Bindung von Trijodthyronin an Serumproteine mittels Dextran-Gel-Filtration

Zusammenfassung 1. Es wird eine Methode zur gleichzeitigen Bestimmung des sog. freien und des proteingebundenen Anteils von in vitro zugesetztem L-Trijodthyronin-¹³¹Jod im Serum mittels Dextran-Gel-Filtration angegeben. In der beschriebenen Form ist diese Technik für die routinemäßige Anwendung in der Klinik zur Bestimmung der Bindungs- und Transportverhältnisse von Trijodthyronin geeignet.2. In sog. Verdrängungsversuchen wurde nichtmarkiertes Trijodthyronin dem Inkubationsgemisch von Serum und L-Trijodthyronin-¹³¹Jod zugesetzt. Die zugesetzten Trijodthyroninmengen erschöpfen die Gesamtbindungskapazität der Serumproteine in dem gewählten Konzentrationsbereich keineswegs. Im Gegensatz zum Verhalten der prozentualen Anteile des sog. freien und des proteingebundenen Trijodthyronins steigt die absolute Menge des proteingebundenen Trijodthyronins dabei steil an. Man findet eine Kurve, die nicht einer einfachen Sättigunskurve entspricht, sondern eine Resultante aus Sättigungskurven verschiedener Trijodthyronin-bindender Proteine und Verdrängungskurven kompetitiv gebundener Substanzen (z.B. Thyroxin) darstellt.3. Dextran-Gel wirkt nicht als einfaches Molekülsieb für Trijodthyronin. Es greift vielmehr durch Adsorptionsvorgänge kompetitiv in die Serumproteinbindungsverhältnisse des Trijodthyronins ein. Die physiologische Bedeutung des sog. freien Anteils an Trijodthyronin wird diskutiert.4. Die Methode zur Bestimmung des proteingebundenen Jods (PB¹²⁷I) mittels alkalischer offener Veraschung (Barker) wurde technisch vereinfacht und bezüglich ihrer Reproduzierbarkeit untersucht. Die¹³¹Jodausbeute aus zugesetztem L-Thyroxin-¹³¹Jod lag bei diesem Verfahren bei ca. 80%.

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Summary 1. A method allowing the simultaneous determination in serum of the socalled free and the protein bound part of 1-triiodothyronine-¹³¹I added in vitro, using dextran gel filtration, is presented. Assessment of serum protein binding and transport of triiodothyronine can be conveniently performed for clinical purposes by this procedure.2. In socalled discharge experiments non-labelled triiodothyronine is added to incubation mixtures of serum and l-triiodothyronine-¹³¹I. The amount of added triiodothyronine did not exhaust the total binding capacity of serum proteins for triiodothyronine. In contrast to the behaviour of the percentages of socalled free and protein bound triiodothyronine the absolute amount of protein bound triiodothyronine was rising linearly with rising concentrations of triiodothyronine added. The curve obtained was interpreted as resulting from saturation curves of different triiodothyronine binding proteins and discharge curves of competitively bound substances, e.g. thyroxine.3. Dextran gel is not acting merely as molecular sieve for triiodothyronine, but rather competing actively with serum proteins for triiodothyronine, adsorbing the latter. The physiological rôle of the socalled free triiodothyronine is discussed.4. With addition of l-thyroxine-¹³¹I 80% of¹³¹iodine was recovered, when the method ofBarker for determination of serum protein bound¹²⁷iodine (open alkaline ashing) was used.

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Mit Unterstützung der Deutschen Forschungsgemeinschaft.

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Herrn Prof. Dr. Dr. h. c.Carl Krauspe in Verehrung zum 70. Geburtstag gewidmet.     

Permeation of human ovarian tissue with cryoprotective agents in preparation for cryopreservation

The recent improvements in the treatment of cancer by chemo- and radiotherapy have led to a significant increase in the survival rates of patients with malignant disease, but at the expense of distressing side effects. One major problem, especially for younger patients, is that aggressive therapy destroys a significant proportion of the follicular population, which can result in either temporary or permanent infertility. Freeze-banking pieces of ovarian cortex prior to treatment is one strategy for preserving fecundity. When the patient is in remission, fertility could, theoretically, be restored by autografting the thawed tissue at the orthotopic site or by growing isolated follicles to maturity in vitro. Recent studies have found good follicular survival in frozen-thawed human ovarian tissue but to optimize the process an effective cryopreservation method needs to be developed. An essential part of such a technique is to permeate the tissue with a cryoprotectant to minimize ice formation and the extent of this equilibration is an important determinant of post-thaw cellular survival. In the current study, we have investigated the diffusion of four cryoprotective agents into human tissue at both 4 degrees C and 37 degrees C. We have also studied the effect of adding different concentrations of the non penetrating cryoprotective agent, sucrose, to the freezing media using the release of lactate dehydrogenase as a measure of its protective effect. At 4 degrees C propylene glycol and glycerol penetrated the tissue significantly slower than either ethylene glycol or dimethyl sulphoxide. At the higher temperature of 37 degrees C all four cryoprotectants penetrated at a faster rate, however concern about enhanced toxicity prevents the use of these conditions in practice. Thus, the results suggest that the best method of preparing tissue for freezing is exposure for 30 min to 1.5 M solutions of ethylene glycol or dimethyl sulphoxide at 4 degrees C; this achieved a mean tissue concentration that was almost 80% that of the bathing solution. We also report that the addition of low concentrations of sucrose to the freezing medium does not have a significant protective effect against freezing injury.      

Homozygosity mapping of achromatopsia to chromosome 2 using DNA pooling

Achromatopsia is an autosomal recessive disease of the retina, characterized clinically by an inability to distinguish colors, impaired visual acuity, nystagmus and photophobia. A genome-wide search for linkage was performed using an inbred Jewish kindred from Iran. To facilitate the genome-wide search, we utilized a DNA pooling strategy which takes advantage of the likelihood that the disease in this inbred kindred is inherited by all affected individuals from a common founder. Equal molar amounts of DNA from all affected individuals were pooled and used as the PCR template for short tandem repeat polymorphic markers (STRPs). Pooled DNA from unaffected members of the kindred was used as a control. A reduction in the number of alleles in the affected versus control pool was observed at several loci. Upon genotyping of individual family members, significant linkage was established between the disease phenotype and markers localized on chromosome 2. The highest LOD score observed was 5.4 (theta = 0). When four additional small unrelated families were genotyped, the combined peak LOD score was 8.2. Analysis of recombinant chromosomes revealed that the disease gene lies within a 30 cM interval which spans the centromere. Additional fine-mapping studies identified a region of homozygosity in all affected individuals, narrowing the region to 14 cM. A candidate gene for achromatopsia was excluded from this disease interval by radiation hybrid mapping. Linkage of achromatopsia to chromosome 2 is an essential first step in the identification of the disease-causing gene.      

Genetic and biochemical characterization of Malassezia pachydermatis with particular attention to pigment-producing subgroups.

The aim of the study was the characterization of Malassezia pachydermatis and its pigment-producing subgroup using biochemical tests and RAPD. It was of interest to determine whether particular RAPD patterns could be used to indicate pigment production, as well as a close genetic relatedness to Malassezia furfur. Therefore, 210 strains of M. pachydermatis were examined for morphology, catalase and ss-glucosidase activity, lipid and carbohydrate assimilation and the tryptophan-dependent synthesis of pigments. Of these, 114 strains were subjected to RAPD analyses. A multivariate logistic regression model was applied to classify M. pachydermatis isolates regarding their pigment production by using genetic and biological parameters. Biological and RAPD findings showed a high biological and genetic diversity within the species M. pachydermatis and within pigment producers. RAPD analysis revealed 28 genotypes within 114 strains tested. Pigment producing strains could not be assigned to a common RAPD profile, but a genetic relatedness of pigment-producing M. pachydermatis with M. furfur can be assumed. A particular RAPD pattern allowed statistically significant probability of pigment production (P<0.001) and might be used as a tool to rapidly detect pigment producing M. pachydermatis, e.g. in Malassezia-associated pityriasis versicolor. The reported method is useful for identification of pigment producing M. pachydermatis isolates and has advantages over established tests.     

High vs low anxiety-related behavior rats: an animal model of extremes in trait anxiety

In addition to their robust difference in trait anxiety, as illustrated by a variety of behavioral tests, HAB and LAB rats differ in their stress coping strategies, the former being more susceptible and vulnerable to stressor exposure and preferring more passive strategies. HAB rats of either gender show signs of a hyper-reactive hypothalamic-pituitary-adrenocortical (HPA) axis, thus resembling psychiatric patients. As shown by in situ hybridization and microdialysis in freely behaving animals, both the expression and release of vasopressin in the hypothalamic paraventricular nucleus are higher in HAB than in LAB rats, thus contributing to the HPA axis hyperdrive. Accordingly, in HAB animals, administration of a V1 receptor antagonist normalized the pathological outcome of the dexamethasone/corticotropin-releasing hormone test and triggered behavioral changes toward reduced anxiety and active stress coping. Pharmacological validation has revealed signs of depressive-like behavior, as HAB but not LAB rats have shown more active stress coping behavior and a normalized HPA axis after treatment with paroxetine. Of interest, this antidepressant reduced the hypothalamic overexpression of vasopressin; this novel mechanism of action is likely to contribute to paroxetine effects on both behavioral and neuroendocrine parameters. Cross-mating and cross-fostering paradigms showed that the divergent emotionality in HAB vs. LAB rats is determined genetically, rather than postnatally through maternal behavior. As the behavioral and neuroendocrine phenotyping pointed to the vasopressin gene as a candidate gene critically involved in anxiety, preliminary genetic approaches have been focused on this gene, revealing single nucleotide polymorphisms (SNPs) in the promotor area of the vasopressin gene in HAB, but not LAB rats. HAB/LAB rats are thus proving to be a unique animal model to identify and characterize neurobiological, neuroendocrine, and genetic correlates of trait anxiety, and perhaps depression, in humans.     

Blood glucose measurement by infrared spectroscopy

For the development of an implantable artificial endocrine pancreas, a sensor for blood glucose measurement is needed providing a long-term stability. This goal can be achieved by the application of infrared spectroscopy which, unlike electrochemical sensors, responds directly to the glucose molecule. An investigation under physiological conditions revealed five glucose absorption bands in the near and middle infrared range. These are 1040, 1085, 1109, 1160 and 1365 cm-1. Only the 1040 cm-1 frequency coincides with none of the other infrared-active blood substances like proteins, lipids and urea. Nevertheless, the other absorption bands too, especially the 1109 cm-1 frequency, can be used for blood glucose measurement, if the superimposed absorptions are compensated. Methods for the compensation have been found. Technically feasible embodiments of an infrared glucose sensor are described.     

Hormone-active intradural spinal metastasis of a prolactinoma — A case report

Summary A 44-year-old woman developed acute severe visual field defects and was operated on a macroprolactinoma. Since complete resection of the tumor was not possible, radiotherapy was performed and in addition to hormone replacement therapy, bromocriptine (up to 60 mg daily) was started without however complete normalization of PRL levels. Four years later PRL levels increased to 10⁵ µU/ml despite continuation of dopamin agonist (mesulergin) treatment. As shown by ophthalmological examination and computer tomography there were no signs of regrowth of the pituitary tumor. At that time the patient complained of severe lumbar pain and myelography revealed a tumor mass in the spinal cord (L₁–L₂). Since the spinal tumor was not removable, laminectomy was performed. Histology and immunohistochemistry demonstrated a metastasis of the prolactinoma. Radiotherapy and bromocriptine in extreme doses (140 mg daily) together with an antiestrogen were not able to improve the neurological deficits (paraparesis) and to lower the PRL levels. This case of a metastasis of a prolactinoma after operation, radiotherapy, and dopamin agonist treatment stresses the importance of close surveillance of patients with prolactinomas without PRL normalization during dopamin agonist therapy and shows for the first time the possibility of ectopic PRL production due to an intradural spinal metastasis.Abbreviations ACTH adrenocorticotropin - FSH folliclestimulating hormone - GH growth hormone - Gy Gray - L lumbar - LH luteinizing hormone - µU/ml microunits per milliliter - mU/ml milliunits per milliliter - PRL prolactin - TBG thyroxine-binding globulin - TSH thyrotropin - T₄ thyroxine - T₃ triiodothyronine     

Multiple cerebral metastases: detectability with Gd-DTPA-enhanced MR imaging

Sixteen patients with suspected cerebral metastases were studied with magnetic resonance (MR) imaging before and after the intravenous administration of 0.1 mmol/kg of gadolinium diethylenetriaminepenta-acetic acid. The images were interpreted blindly by two neuroradiologists; all clinical, radiologic (computed tomographic and MR imaging), and pathologic data were reviewed to arrive at a final "best diagnosis," which was then compared with the prior blinded interpretations. Of seven patients found to have multiple metastases, six (86%) had at least one tumor nodule depicted by postinfusion MR imaging that was missed by one or both observers on review of preinfusion images alone. Lesions missed on preinfusion studies were usually small nodules hidden by or not detected next to regions of high-signal edema thought to be related to the adjacent tumor nodule. The authors believe that contrast enhancement improves detection of metastatic foci with MR imaging and that the findings indicate broader implications for the detection of multiple lesions from other causes.