Serum Carrier Protein Research Articles

Bioengineered lipophilic Ru(III) complexes as potential anticancer agents.

Lipid-conjugated Ru(III) complexes - designed to obtain lipophilic analogues of the low molecular weight derivative AziRu, which is a NAMI-A-like anticancer agent - have been synthesized and fully characterized. A detailed biophysical investigation, including multiple, integrated techniques, allowed determining their molecular and self-assembling properties in aqueous solutions mimicking the extracellular environment, showing that our design produced a protective effect from hydrolysis of the Ru(III) complexes. In vitro biological experiments, carried out in comparison with AziRu, demonstrated that, among the novel lipophilic Ru(III) complexes synthesized, the compounds derivatized with palmitic and stearic acid, that we named PalmiPyRu and StePyRu respectively, showed attractive features and a promising antiproliferative activity, selective on specific breast cancer phenotypes. To get a deeper insight into their interactions with potential biomacromolecular targets, their ability to bind both bovine serum albumin (BSA), an abundant serum carrier protein, and some DNA model systems, including duplex and G-quadruplex structures, has been investigated by spectroscopic techniques. Inductively coupled plasma-mass spectrometry (ICP-MS) analysis of the ruthenium amount incorporated in human MCF-7 and MDA-MB-231 breast cancer cells, after incubation in parallel experiments with PalmiPyRu and AziRu, showed a markedly higher cell uptake of the lipophilic Ru(III) complex with respect to AziRu. These data confirmed that the proper lipidic tail decorating the metal complex not only favoured the formation of aggregates in the extracellular media but also improved their cell membrane penetration, thus leading to higher antiproliferative activity selective on breast cancer cells.

Characterization of non-radiolabeled Thyroxine (T4) uptake in cryopreserved rat hepatocyte suspensions: Pharmacokinetic implications for PFOA and PFOS chemical exposure

The alteration of thyroxine (T4) cellular uptake by an environmental chemical can serve as a contributing factor in thyroid hormone (TH) disruption. Herein, we describe a non-radiolabeled (LC-MS/MS) oil-filtration technique designed to characterize the mechanism(s) responsible for T4 cellular uptake in cryopreserved rat hepatocyte suspensions. The environmental chemicals perfluorooctanoate (PFOA) and perfluorooctane sulfonate (PFOS) were evaluated for their effect on T4 hepatic uptake. At 37 °C, hepatic assays demonstrated saturable kinetics with increasing T4 concentrations, while a linear uptake rate consistent with passive diffusion was detected at 4 °C. Carrier-mediated (37–4 °C) transport of T4 was the predominant hepatic uptake process versus passive diffusion. Cyclosporin A (CsA) chemically inhibited T4 hepatic uptake, whereas PFOA/PFOS displayed no inhibition of T4 translocation. Increasing PFOA/PFOS concentration levels with the T4 serum carrier-protein transthyretin (TTR) present resulted in a dose-response increase in T4 hepatic uptake rates, correlating with increased T4 free fraction values. Hepatic assays conducted in the presence of PFOA/PFOS and TTR displayed an enhanced first-order T4 hepatic uptake rate consistent with carrier-mediated transport. These in vitro findings characterizing increased T4 hepatic uptake provides mechanistic insight regarding decreased T4 serum levels (hypothyroxinemia) previously observed within in vivo rodent studies following perfluorinated chemical exposure.

A novel biocompatible NiII tethered moiety as a glucose uptake agent and a hit against methicillin-resistant Staphylococcus aureus

In the efforts to develop a biocompatible transition metal complex as a drug alike for some of the prevailing non-communicable diseases (NCDs) and communicable diseases (CDs), a novel binuclear NiII compound [{NiII(hpdbal-sbdt)}2] (2) has been synthesized by the reaction of Ni(OAc)2.4H2O and H2hpdbal-sbdt (1) [1 is a dibasic tridentate ONS2− donor Schiff base ligand obtained by the condensation of 2-hydroxy-5-(phenyldiazenyl)benzaldehyde (Hhpdbal) and S-benzyldithiocarbazate (Hsbdt)]. Both ligand 1 and compound 2 were structurally characterized in the solid and solution state using various spectroscopic techniques like ATIR, 1H NMR, 13C NMR, TGA, FESEM, EDS and CHNS analysis. The antidiabetic activity of H2hpdbal-sbdt (1) and [{NiII(hpdbal-sbdt)}2] (2) were assessed using 2-NBDG uptake assay. The assay results showed 85% and 95% of fluorescent glucose uptake by insulin resistant HePG2 cells treated with compounds 1 and 2 respectively. The 2-NBDG uptake by the cells treated with the compound 2 was observed to be comparable to the standard antidiabetic drug metformin. Compounds 1 and 2 were also tested against five bacterial and two fungi strains in order to evaluate pathogen killing activity. Compound 2 showed significant inhibitory action towards the methicillin-resistant Staphylococcus aureus (MRSA) strain with an MIC value of 2 μg/mL whereas the ligand 1 was found to be inactive. Furthermore, the interactive nature of compound 2 with a model serum carrier protein bovine serum albumin (BSA) was studied using a multi-spectroscopic approach which provided an insight into the nature and extent of binding, conformational changes and the quenching of amino acid residues of the protein.

Amyloid seeding of transthyretin by ex vivo cardiac fibrils and its inhibition

Each of the 30 human amyloid diseases is associated with the aggregation of a particular precursor protein into amyloid fibrils. In transthyretin amyloidosis (ATTR), mutant or wild-type forms of the serum carrier protein transthyretin (TTR), synthesized and secreted by the liver, convert to amyloid fibrils deposited in the heart and other organs. The current standard of care for hereditary ATTR is liver transplantation, which replaces the mutant TTR gene with the wild-type gene. However, the procedure is often followed by cardiac deposition of wild-type TTR secreted by the new liver. Here we find that amyloid fibrils extracted from autopsied and explanted hearts of ATTR patients robustly seed wild-type TTR into amyloid fibrils in vitro. Cardiac-derived ATTR seeds can accelerate fibril formation of wild-type and monomeric TTR at acidic pH and under physiological conditions, respectively. We show that this seeding is inhibited by peptides designed to complement structures of TTR fibrils. These inhibitors cap fibril growth, suggesting an approach for halting progression of ATTR.

Value of prealbumin in assessment of nutrition for critically ill patients

* Correspondence: mehmetagilli@yahoo.com To the Editor, We read with great interest the article published in a recent issue of the Turkish Journal of Medical Sciences by Dumlu et al. entitled “A general consideration of the importance of nutrition for critically ill patients” (1). We thank Dr Dumlu and colleagues for their valuable investigation evaluating the nutritional status of intensive care unit patients retrospectively. They claimed that prealbumin levels of the patients had a tendency to increase after the provision of nutritional additives. We think that some important issues should be discussed while assessing serum prealbumin. Prealbumin is a serum carrier protein for thyroxine and retinol. Previous studies suggested that several disorders such as type 2 diabetes mellitus, anorexia nervosa, bulimia nervosa, pneumonia, tuberculosis, acute phase response (inflammation, infection, malignancy, trauma), ankylosing spondylitis, rheumatoid arthritis, protein losing enteropathy, Kawasaki disease, Helicobacter pylori infection, major depression, thyroid diseases, and liver diseases could affect serum prealbumin levels (2,3). In their paper, Dumlu et al. did not mention these contributing diseases. It is not obvious whether altered prealbumin levels were due nutrition or the course of the diseases. Secondly, several drugs such as nonsteroidal antiinflammatory drugs, anabolic steroids, corticosteroids, progestational agents, estrogens, and antithyroid drugs could alter serum prealbumin levels (2,4). Dietary food supplements such as omega-3 fatty acids, vitamin A, vitamin C, and zinc could also change these levels (5,6). In this respect, the authors should define whether the participants used these kinds of drugs and dietary supplements in their recent history. In addition, alcohol use is another confounder that should be described (7). Lastly, Bruguerolle et al. showed that a circadian rhythm was detected for serum prealbumin (8). We think that it is essential to express sampling time for measurement of serum prealbumin to provide reliable data. Therefore, interpretation of results in their current form seems problematic. In conclusion, we think that the study by Dumlu et al. contributes valuable data to the medical literature. However, clarifying these concerns would provide a clearer picture to readers. Received: 08.03.2015 Accepted/Published Online: 21.04.2015 Printed: 30.07.2015 Letter to the Editor

Maternal serum protein profile and immune response protein subunits as markers for non‐invasive prenatal diagnosis of trisomy 21, 18, and 13

To use proteomics to identify and characterize proteins in maternal serum from patients at high-risk for fetal trisomy 21, trisomy 18, and trisomy 13 on the basis of ultrasound and maternal serum triple tests. We performed a comprehensive proteomic analysis on 23 trisomy cases and 85 normal cases during the early second trimester of pregnancy. Protein profiling along with conventional sodium dodecyl sulfate polyacrylamide gel electrophoresis/Tandem mass spectrometry analysis was carried out to characterize proteins associated with each trisomy condition and later validated using Western blot. Protein profiling approach using surface enhanced laser desorption/ionization time-of-flight mass (SELDI-TOF/MS) spectrometry resulted in the identification of 37 unique hydrophobic proteomic features for three trisomy conditions. Using sodium dodecyl sulfate polyacrylamide gel electrophoresis followed by Matrix Assisted Laser Desorption Ionization - Time of Flight/Time of Flight (MALDI-TOF/TOF) and western blot, glyco proteins such as alpha-1-antitrypsin, apolipoprotein E, apolipoprotein H, and serum carrier protein transthyretin were identified as potential maternal serum markers for fetal trisomy condition. The identified proteins showed differential expression at the subunit level. Maternal serum protein profiling using proteomics may allow non-invasive diagnostic testing for the most common trisomies and may complement ultrasound-based methods to more accurately determine pregnancies with fetal aneuploidies.

Expressions of Insulin-Like Growth Factor Receptor-1 and Insulin-Like Growth Factor Binding Protein 3 in Advanced Non–Small-Cell Lung Cancer

BackgroundThe insulin-like growth factor (IGF) pathway plays an important role in cell proliferation, differentiation, and apoptosis, and IGF induces those effects mainly through IGF receptor-1 (IGF-1R). The activities of IGF are strictly regulated by a family of IGF binding proteins (IGFBP), especially IGFBP3, a major serum carrier protein for IGF. Patients and MethodsBetween January 2006 and February 2009, in our hospital, 191 patients were histologically diagnosed as having non–small-cell lung cancer (NSCLC), and 74 patients were treated by chemotherapy alone. We examined immunohistochemical expression of both IGF-1R and IGFBP3 in 68 patients who were definitively diagnosed as having adenocarcinoma or squamous cell carcinoma among the 74 patients. ResultsThe clinical characteristics of the included patients were as follows: median age was 68 years (range, 29-86 years); men vs. women, 40 vs. 28; stage III vs. IV, 18 vs. 50; performance status 0-1 vs. 2-4, 58 vs. 10; smoker vs. non-smoker, 44 vs. 24; and squamous cell carcinoma vs. adenocarcinoma, 13 vs. 55. Expression of IGF-1R and IGFBP3 was observed in 37 (54%) and 11 patients (16%), respectively. IGF-1R expression was detected more frequently in patients with squamous cell carcinoma (100%) than in patients with adenocarcinoma (44%) (P < .001), although IGFBP3 expression was not significantly associated with any clinical variables. Among all factors, including IGF-1R and IGFBP3 expression, IGF-1R was significantly associated with response to chemotherapy (P = .028) and performance status was significantly associated with overall survival (P < .001). ConclusionsHigh sensitivity of IGF-1R to squamous cell carcinoma (100%) in this study and another study encourages the use of IGF-1R antibody in the pathologic diagnosis between squamous cell and non-squamous cell carcinoma when using small biopsy specimens.

Clinical significance of type 1 insulin-like growth factor receptor and insulin-like growth factor binding protein-3 expression in squamous cell carcinoma of head and neck

6036 Background: Type 1 insulin-like growth factor receptor (IGF-1R) plays an important role in the growth and apoptosis of cancer cell. The activities of IGF-1R are modulated by a family of high-affinity insulin-like growth factor binding proteins (IGFBPs), of which IGFBP-3 is the major serum carrier protein. The expression and significance of IGF-1R and IGFBP-3 in squamous cell carcinoma of the head and neck (SCCHN) is unknown. Methods: This study explored immunohistochemical expression of IGF-1R and IGFBP-3 in tumor samples from 131 patients with surgically resected SCCHN. Results: The positive expression of IGF-1R and IGFBP-3 was observed in 96 (73.3%) and 117 (89.3%) patients. There were no significant differences in baseline characteristics between the positive and negative expressed groups of IGF-1R and IGFBP-3. With the median follow up of 53.5 months, 3 year progression free (PFS) and overall (OS) survival rate was 64.0% and 72.9%, neither IGF-1R nor IGFBP-3 expression had prognostic values in the whole cohort. Thirty-seven (67.3%) of 55 stage IV patients demonstrated IGF-1R positivity. The subgroup analysis of in stage IV patients showed the poor PFS ( = 0.029) and OS (pp = 0.025) in IGF-1R positive groups. In a multivariate analysis using IGF-1R expression and other clinicopathological parameters, the IGF-1R expression was remained as an independent factor for PFS (p = 0.037, HR = 2.816, 95% C.I. 1.065 - 7.446) and OS (p = 0.034, HR = 3.169, 95% C.I. 1.089 - 9.225). Conclusions: Our work exhibited that IGF-1R expression is frequent in SCCHN patients and related with poor survival in advanced stage, this could support the attempts using the IGF-1R inhibitor for treatment of SCCHN. No significant financial relationships to disclose.

Hypogonadism and diabetes

Clinicians have been aware of the increased prevalence of low testosterone levels in patients with type II diabetes for several years, but how these two conditions are associated is difficult to determine. Older age and obesity may be factors, as both are associated with type II diabetes and both decrease testosterone levels. Sex hormone-binding globulin (SHBG), the major serum carrier protein for testosterone, also may have an impact. SHBG levels fall with obesity and increase with aging. Some studies indicate lower SHBG levels in type II diabetes. Most of the differences in testosterone levels between diabetic and nondiabetic patients may be due to reduced SHBG, rather than reduced testosterone production. However, free testosterone levels fall with increasing age and obesity, rendering many type II diabetic patients testosterone deficient. Testosterone replacement may improve insulin sensitivity in hypogonadal, overweight men with type II diabetes by altering body composition, but studies are conflicting.

Biomarker amplification by serum carrier protein binding.

Mass spectroscopic analysis of the low molecular mass (LMM) range of the serum/plasma proteome is a rapidly emerging frontier for biomarker discovery. This study examined the proportion of LMM biomarkers, which are bound to circulating carrier proteins. Mass spectroscopic analysis of human serum following molecular mass fractionation, demonstrated that the majority of LMM biomarkers exist bound to carrier proteins. Moreover, the pattern of LMM biomarkers bound specifically to albumin is distinct from those bound to non-albumin carriers. Prominent SELDI-TOF ionic species (m/z 6631.7043) identified to correlate with the presence of ovarian cancer were amplified by albumin capture. Several insights emerged: a) Accumulation of LMM biomarkers on circulating carrier proteins greatly amplifies the total serum/plasma concentration of the measurable biomarker, b) The total serum/plasma biomarker concentration is largely determined by the carrier protein clearance rate, not the unbound biomarker clearance rate itself, and c) Examination of the LMM species bound to a specific carrier protein may contain important diagnostic information. These findings shift the focus of biomarker detection to the carrier protein and its biomarker content.

Variant-sequence transthyretin (isoleucine 122) in late-onset cardiac amyloidosis in black Americans.

After the age of 60, isolated cardiac amyloidosis is four times more common among blacks than whites in the United States; 3.9 percent of blacks are heterozygous for an amyloidogenic allele of the normal serum carrier protein transthyretin in which isoleucine is substituted for valine at position 122 (Ile 122). We hypothesized that the high prevalence of transthyretin Ile 122 is at least partially responsible for the increased frequency of senile cardiac amyloidosis among blacks. Paraffin blocks of cardiac tissue were obtained from an earlier study of 52,370 autopsies in Los Angeles and were examined by immunohistochemical and DNA analyses. Samples were available from 32 of 55 blacks and 20 of 78 whites over 60 years of age with isolated cardiac amyloidosis and from two control groups (228 cases). Transthyretin amyloidosis was identified in 31 of the 32 cardiac-tissue samples from the black patients and in 19 of the 20 samples from the white patients. Six of the 26 analyzable DNA samples (23 percent) from the black patients and none of the 19 samples from the white patients were heterozygous for the Ile 122 variant. Four of 125 DNA samples obtained at autopsy (3.2 percent) from a second, more recent, age-matched cohort of blacks without amyloidosis at the same institution were heterozygous for the transthyretin Ile 122 allele. On reexamination the cardiac tissue from these four patients contained small amounts of amyloid not detected at the initial autopsies. All subjects with the Ile 122 variant had ventricular amyloid. The assessment of elderly black patients with unexplained heart disease should include a consideration of transthyretin amyloidosis, particularly that related to the Ile 122 allele.

Impaired growth in Rabson-Mendenhall syndrome: lack of effect of growth hormone and insulin-like growth factor-I.

Mutations in the insulin receptor gene cause the severe insulin-resistant syndromes leprechaunism and Rabson-Mendenhall syndrome. There is no accepted therapy for these inherited conditions. Here we report the results of recombinant human GH (rhGH) and recombinant human insulin-like growth factor-I (rhIGF-I) treatment of a male patient, Atl-2, with Rabson-Mendenhall syndrome. The patient was small for gestational age, had premature dentition, absence of sc fat, acanthosis nigricans, fasting hypoglycemia and postprandial hyperglycemia, and extremely high concentrations of circulating insulin (up to 8500 microU/mL). Fibroblasts and lymphoblasts established from this patient had reduced insulin binding, which was 20-30% of the control value. Binding of epidermal growth factor, IGF-I, and GH to the patient's fibroblasts was normal. The growth of fibroblasts cultured from patient Atl-2 in vitro was intermediate between that of fibroblasts from patients with leprechaunism and control values. The patient's growth curve in vivo was far below the fifth percentile despite adequate nutrition. To stimulate growth, therapy with rhGH was initiated, the rationale being to stimulate hepatic IGF-I production and IGF-I receptor signaling, and bypass the inherited block in insulin receptor signaling. Therapy with rhGH (up to 0.5 mg/kg.week) did not improve growth and failed to increase the levels of circulating IGF-I and IGF-binding protein-3 over a 14-month period. As rhGH could not stimulate growth, rhIGF-I (up to 100 micrograms/kg.day) was given by daily sc injection. No increase in growth velocity was observed over a 14-month period. These results indicate that both GH and IGF-I fail to correct growth in a patient with severe inherited insulin resistance. The lack of efficacy of IGF-I treatment may be related to multiple factors, such as the poor metabolic state of the patient, the deficiency of serum carrier protein for IGF-I, an increased clearance of the growth factor, IGF-I resistance in target cells at a receptor or postreceptor level, or an inhibitory action of the mutant insulin receptors on IGF-I receptor signaling.

Affinity differences for vitamin D metabolites associated with the genetic isoforms of the human serum carrier protein (DBP).

Human vitamin D binding protein (DBP) displays considerable polymorphism with 120 described alleles. Among these, three alleles are frequently observed, Gc 1F (pI 4.94-4.84), Gc 1S (pI 4.95-4.85) and Gc 2 (pI 5.1). Differences between these genetic forms of the protein in affinity for vitamin D metabolites have been detected by electrophoretic methods. The constant affinity (Ka) values determined in this study confirm these differences. The affinities of six rare variants were also examine. Those of the DBP genetic forms to the vitamin D derivatives 25-OH-D3 and 1,25-(OH)2-D3 seem to be related to the isoelectric point of the proteins: a high affinity corresponding to a low isoelectric point. The Gc 1A9 and 1A11 mutants were associated with higher affinity for the vitamin D derivatives and the Gc 1C1 and 1C21 mutants were deficient.

Prostate-specific antigen (PSA) is an insulin-like growth factor binding protein-3 protease found in seminal plasma.

Insulin-like growth factor binding protein-3 (IGFBP-3), the major serum carrier protein for the IGFs, is absent from Western ligand blots of seminal plasma, but is detectable by RIA. IGFBP-3 protease activity has recently been described in pregnancy serum. We investigated the possibility that seminal plasma contains an IGFBP-3 protease, by incubating seminal plasma with 125I-labeled human IGFBP-3. Seminal plasma was found to have potent IGFBP-3 protease activity with a cleavage pattern different from that of pregnancy serum. Prostate-specific antigen (PSA) is a serine protease found in semen. Autoradiographs measuring IGFBP-3 protease activity demonstrated that purified PSA cleaved IGFBP-3, yielding a cleavage pattern identical to that of seminal plasma. IGFBP-2 and -4 in seminal plasma were not degraded by PSA. Cleavage of IGFBP-3 by PSA resulted in a marked reduction in the binding affinity of the fragments to IGF-I, but not IGF-II. We speculate that PSA may serve to modulate IGF function within the reproductive system or in prostate cancer by altering IGF-IGFBP-3 interactions.

Delayed allograft rejection in mice transgenic for a soluble form of the IL-4 receptor

Accumulating evidence suggests that in serum and other biological fluids, cytokine binding is a property associated with soluble proteins, including a high-affinity soluble version of the IL-4 receptor (sIL-4R). While it is tempting to speculate that sIL-4R might act as a serum carrier protein or serve to inhibit or modulate IL-4 action, specific biological roles for sIL-4R remain to be established. To further assess the immunoregulatory and therapeutic potential of sIL-4R and other soluble receptors, we have created transgenic mice which constitutively express elevated levels of biologically active sIL-4R. Phenotypic characterization of lymphoid organs in sIL-4R transgenic mice revealed normal numbers of B and T cells and normal surface marker expression. Splenic lymphocytes displayed normal in vitro activities as measured by the PFC response and generation of cytotoxic T cells. In addition, antigen-specific IgE and IgG 1 in vivo responses were similar in control and transgenic mice. Despite the apparent developmental normality of the sIL-4R transgenic mice, these animals were markedly deficient in the ability to reject cardiac allografts, suggesting that IL-4 is critical for the generation of alloreactivity. The results further suggest that the ability of sIL-4R to regulate IL-4 activities may be under the control of complex interactions that remain to be elucidated.

Vitamin D-metabolites from human plasma and mass spectrometric analysis by fast heavy ion induced desorption

D-vitamin metabolites have been isolated from human serum employing chromatographic techniques. The serum carrier protein for vitamin D (DBP) was first isolated by immunosorbent chromatography. Lipid ligands associated with DBP were then extracted with hexane and separated by high pressure liquid chromatography (HPLC). Detection of vitamin D metabolites by their absorbance of ultraviolet light is not sufficiently sensitive to monitor all vitamin D derivatives from a few millilitres of serum. Therefore, further analyses are necessary to quantitate these compounds. We have begun to develop a mass spectrometric method to achieve a reliable, quantitative procedure. As a first step towards this goal a number of pure samples of vitamin D compounds have been studied in a time-of-flight mass spectrometer based on fast heavy ion induced desorption. All vitamin D compounds examined could be detected and identified by their molecular ion and fragment spectra.

Insulin-like growth factor and its carrier protein in hypopituitary and hypothyroid children and adults.

SUMMARYUtilizing radioligand assays for the somatomedin, insulin‐like growth factor (IGF), and its carrier protein (CP), we have compared their serum levels in normal subjects with those in patients with hypopituitarism and hypothyroidism. The mean (±SEM) serum IGF concentration in children (n= 32, 2–13 yr, 929 ± 46 μU/ml) was virtually identical to that in adults (n= 20, 18–50 yr, 916·29 μU/ml), while CP levels in the former (1·8 ± 0·1 mg/ml) were significantly lower (P &lt; 0·01) than in the latter (2·9 ± 0·1 mg/ml). Both serum IGF and CP were significantly decreased (P &lt; 0·001) in hypopituitary children (n= 5, 6–8 yr, 377 ± 52 μU/ml and 0·9 ± 0·1 mg/ml respectively). Likewise, serum IGF and CP were significantly reduced (P &lt; 0·05) in hypothyroid children (n= 10, 1–15 yr, 497 ± 24 μU/ml and 0·8 ± 0·1 mg/ml respectively) and adults (n= 12, 25–55 yr, 723 ± 40 μU/ml and 1·8 ± 0·2 mg/ml respectively). Growth hormone therapy (4 days) significantly increased (P &lt; 0·05) serum IGF in hypopituitary children (336 ± 80 to 559 ± 59 μU/ml) but did not change the CP level (0·7 ± 0·2 to 0·7 ± 0·1 mg/ml). Thyroxine therapy (4–8 months) significantly increased (P &lt; 0·05) both serum IGF and CP levels in hypothyroid children (497 ± 24 to 666 ± 38 μU/ml, and 0·8 ± 0·1 to 1·0 ± 0·1 mg/ml respectively) but had no effect in adults (723 ± 40 to 937 ± 132 μU/ml, and 1·8 ± 0·2 to 2·1 ± 0·2 mg/ml respectively). These studies indicate that growth retardation in children with hypopituitarism and hypothyroidism is associated with significant reductions in serum IGF and CP levels that are corrected, at least in part, by adequate hormone replacement therapy.

The Effect of Hypothyroidism on Growth, Serum Growth Hormone, the Growth Hormone-Dependent Somatomedin, Insulin-Like Growth Factor, and Its Carrier Protein in Rats*

To study the possible mechanisms involved in growth retardation associated with hypothyroidism, serum T4, GH, the GH-dependent somatomedin, insulin-like growth factor (IGF), and its carrier protein (CP) were measured in hypothyroid rats and their age-matched controls. Three groups of rats were studied: infant, immature, and adult. Marked hypothyroidism (serum T4, less than 1 microgram/dl) was produced in experimental animals by providing them with drinking water containing 0.05% propylthiouracil. Infant and immature hypothyroid rats weighed markedly less than normal controls and had significantly reduced serum levels of GH, IGF, and CP. Normal adult rats, treated with propylthiouracil for 60 days, also weighed considerably less than control animals and exhibited a significant drop in serum GH, IGF, and CP during this period. The administration of bovine GH to hypothyroid adult rats for 7 days did not restore either IGF or CP levels to normal, indicating that their decrease in serum was, in part, a direct result of hypothyroidism per se. These results indicate that serum levels of GH, IGF, and CP are at least partly under thyroid hormone control. Furthermore, these studies suggest that the growth retardation associated with hypothyroidism may be mediated through somatomedin activity.

Insulin-like growth factors I and II: some biological actions and receptor binding characteristics of two purified constituents of nonsuppressible insulin-like activity of human serum.

The insulin‐like growth factors I and II, two purified constituents of nonsuppressible insulinlike activity opf human serum, have nbeen compared with regard to some bniological actions in vitro and some receptor‐binding characteristics. In the presence of an excess of insulin‐antibodies the insulin‐like growth factors I and II (a) stimulate the net gas exchange in rat adipose tissue (fat pad assay) to a similar extent; (b) enhance 3‐O‐methylglucose transport, glucose oxidation and lipogenesis from glucose in rat adipocytes, with slight but significant differences in potency; (c) inhibit lipolysis in adipose tissue from fastedrefed rats, again with slight potency differences. In adipose toissue and adipocytes the biological activity of insulin‐like growth factors I and II is between 1/35 and 1/125 of that of insulin on a molar basis. Insulin‐like growth factors I and II are equally potent mitogens (stimulation of DNA‐synthesis and cell multiplication in cultured chick embryo fibroblasts). Insulin‐like growth factors I and II stimulate 35SO42‐ incorporation into rat costal cartilage with minor differences in potency. In rat adipocytes, insulin‐like growth factors I and II compete with insulin for binding to thje insulin receptor. However, their potencies in ‘displacing’125I‐labeled insulin are between 75 (for factor II) and 290 times (for factor I) lower than that of insulin. In addition, the factors I and II bind to binding sites specific for the factors and for which insulin does not compete. Considerable differences between factors I and II exist regarding their specific binding and their potencies in competing for binding of labeled factors I and II. In chick embryo fibroblasts and isolated chick embryo chondrocytes the insulin‐like growth factors I and II compete more or less equally for binding to a binding site specific for the factors and which appears to mediate their effects on cell growth and sulfation. Indsulin‐like growth factors I and II bind specifically to a partially purified carrier protein of human serum. Pronounced differences in specific binding and their potencies of competition are observed. Thus, the insulin‐like growth factors I and II, two structurally closely related polypeptides, display more or less similar activities in various biological systems in vitro. In all of these, their biological potency can be reasonably correlated with their potency of competition either for the insulin receptor (fat cells) or for a specific binding site (fibroblasts and cyhondrocytes). In some of the radioactive ligand systems, where the biological function of binding sites for insulin‐like growth factor is still unknown (such bindings sites are found in fat cells and on the serum carrier protein), clear cut differences between factors I and II are apparent.

NSILA-carrier protein abolishes the action of nonsuppressible insulin-like activity (NSILA-S) on perfused rat heart.

Human serum in a concentration of 10% in the perfusion medium failed to increase glucose uptake by the isolated perfused rat heart, indicating that nonsuppressible insulin-like activity (NSILA) in whole serum was inactive in this system. When NSILA-carrier protein was added to partially purified NSILA-S, its biological activity on the rat heart disappeared. In contrast, the action of insulin was not affected by the presence of NSILA-carrier protein. Binding of 125I-labelled NSILA-S to rat heart was inhibited by NSILA-carrier protein. 125I-labelled insulin binding was not inhibited. These results support the hypothesis that NSILA-S bound to serum carrier protein is a large molecular compound which does not readily diffuse out of the capillary bed and therefore does not exert insulin-like effects in vivo.