Actovegin®: a biological drug for more than 5 decades.

Actovegin(®) is a biological drug manufactured from a natural source: it is a calf blood hemodialysate. Its therapeutic benefits stem from a variety of pharmacodynamic actions that can be summarized to a common goal, i.e. the enhancement of cellular metabolism; this results from an insulin-like activity mediated by Inositol-phospho-oligosaccharides. Actovegin(®) results in beneficial effects in several pathophysiological clinical settings including malfunction of the blood circulation and trophic disturbances in the brain, impairment of peripheral blood circulation and associated diseases, dermal transplants and acute and chronic wounds. Here, we give an overview of the pharmacodynamic actions of calf-blood hemidialysate and its beneficial effects in a variety of clinical settings.

Postnatally elevated levels of insulin-like growth factor (IGF)-II fail to rescue the dwarfism of IGF-I-deficient mice except kidney weight.

This study tested whether elevated levels of IGF-II in the postnatal period can rescue the dwarfism in IGF-I-deficient mice. Heterozygous Igf1 mutant mice [I(+/-) II(wt)] were crossed with heterozygous Igf1 mutant, phosphoenolpyruvate carboxykinase promoter IGF-II transgenic mice [I(+/-) II(tg)], and [I(+/+) II(wt)], [I(+/+) II(tg)], [I(-/-) II(wt)], and [I(-/-) II(tg)] offspring were investigated. IGF-II levels were 11- and 6-fold higher in male and female [I(-/-) II(tg)] vs. [I(-/-) II(wt)] animals. Western ligand blot analysis revealed markedly reduced activities of 30- and 32-kDa IGF binding proteins (IGFBPs) (most likely IGFBP-1 and IGFBP-2) and the 39- to 43-kDa IGFBP-3 double band in serum from IGF-I-deficient mice. These binding proteins were partially restored by overexpression of IGF-II. Analysis of weight data from the early postnatal period until d 60 showed that, in the absence of IGF-I, elevated levels of IGF-II have no effect on body weight gain. A detailed analysis of body proportions, bone parameters, and organ weights of 60-d-old mice also failed to show effects of IGF-II with one important exception: in Igf1 mutant and also Igf1 intact male mice, IGF-II overexpression significantly increased absolute (+32.4 and +28.6%; P < 0.01) and relative kidney weights (+29.0 and +22.4%; P < 0.001). These changes in kidney weight were associated with reduced phosphorylation of p38 MAPK. In summary, our genetic model shows that substantial amounts of IGF-II in the circulation do not rescue the postnatal growth deficit of IGF-I-deficient mice but increase absolute and relative kidney weights of normal and IGF-I-deficient male mice, suggesting a gender-specific role of IGF-II for kidney growth.

Insulin-like growth factors and binding proteins in early milk from mothers of preterm and term infants.

Breast-fed preterm infants often show a better outcome, partly ascribed to the benefit of insulin-like growth factors (IGFs) and their binding proteins (IGFBP). We compared IGF-I, IGF-II, IGFBP-2 and IGFBP-3 levels, measured by radioimmunoassays in milk samples from 30 mothers of preterm (<31 weeks) and from 19 mothers of term (>37 weeks) infants at days 7 and 21 postpartum. Proteolysis of IGFBP-2 within mother's milk and digestion of (125)I-IGF-II and (125)I-IGFBP-2 by gastric juice from neonates were assessed by electrophoretic techniques. Mean concentrations did not differ between preterm and term milk: IGF-I (2.8 +/- 0.2 vs. 2.3 +/- 0.1 ng/ml), IGF-II (12.0 +/- 0.4 vs. 12.2 +/- 0.5 ng/ml), IGFBP-3 (100.0 +/- 5.1 vs. 80.0 +/- 5.8 ng/ml), but did so for IGFBP-2 (3,144 +/- 172 vs. 2,428 +/- 188 ng/ml, p < 0.02). Immunoblots revealed 42% (p < 0.05) more IGFBP-2 fragments of 14 and 25 kDa in preterm milk. Incubation with gastric juice caused cleavage of (125)I-IGFBP-2 and partial cleavage of (125)I-IGF-II. Mutual complexation protected IGF-II and IGFBP-2 from cleavage, suggesting that both are likely to arrive in the bowel in an intact form to exert promotive effects. The results provide further evidence that IGFBP-2 and IGF-II in breast milk are relevant factors for the early development of preterm infants.

IGF-independent effects of IGFBP-2 on the human breast cancer cell line Hs578T.

There is evidence that insulin-like growth factor-binding protein (IGFBP-2), a modulator of the actions of IGFs, also has IGF-independent effects in human tumor cell lines. These involve specific binding of IGFBP-2 to alpha5beta1-integrin, followed by alterations in the phosphorylation status of downstream signaling molecules. Previously, IGFBP-2 has also been shown to be associated with cell proliferation, adhesion and migration. Here, we investigated direct effects of IGFBP-2 on apoptosis and alterations in the expression of related proteins. The breast cancer cell line Hs578T, which shows no IGFBP-2 production of its own and is independent of the IGF-I receptor, was treated with human recombinant IGFBP-2 in order to study the changes in gene expression induced by IGFBP-2. The methods employed for this purpose were oligonucleotide microarrays, real-time RT-PCR, western blotting, and immunoassays. Out of the 440 genes covered by the Oligo GEArray Human Cancer Microarray OHS-802, the expression of 77 genes was directly influenced by IGFBP-2. By the use of real-time quantitative RT-PCR, the gene expression of Nuclear Factor (NF)kappaB, p53, transforming growth factor beta (TGF beta-1), LAMB1 (Laminin, Beta 1), Bcl-2, and IIp45 was found to be significantly upregulated (by 1.2- to 3.05-fold; all P < 0.001). Accordingly, NFkappaB, p53, and TGF beta-1 proteins, as measured by Western blotting and immunoassay, were upregulated > 1.5-fold. By using an ELISA-based and a flow cytometry-based apoptosis assay, IGFBP-2 was found to have a pro-apoptotic effect on Hs578T cells. Our results suggest that IGFBP-2-induced gene expressions are of functional significance for proliferation, cell adhesion, cell migration and apoptosis, and showed that IGFBP-2 can promote apoptosis in tumor cells independent of IGF.

IGF-II transgenic mice display increased aberrant colon crypt multiplicity and tumor volume after 1,2-dimethylhydrazine treatment.

In colorectal cancer insulin-like growth factor II (IGF-II) is frequently overexpressed. To evaluate, whether IGF-II affects different stages of tumorigenesis, we induced neoplastic alterations in the colon of wild-type and IGF-II transgenic mice using 1,2-dimethylhydrazine (DMH). Aberrant crypt foci (ACF) served as markers of early lesions in the colonic mucosa, whereas adenomas and carcinomas characterized the endpoints of tumor development. DMH-treatment led initially to significantly more ACF in IGF-II transgenic than in wild-type mice. This increase in ACF was especially prominent for those consisting of > or =three aberrant crypts (AC). Nevertheless, adenomas and adenocarcinomas of the colon, present after 34 weeks in both genetic groups, were not found at different frequency. Tumor volumes, however, were significantly higher in IGF-II transgenic mice and correlated with serum IGF-II levels. Immunohistochemical staining for markers of proliferation and apoptosis revealed increased cell proliferation rates in tumors of IGF-II transgenic mice without significant affection of apoptosis. Increased proliferation was accompanied by elevated localization of beta-catenin in the cytosol and cell nuclei and reduced appearance at the inner plasma membrane. In conclusion, we provide evidence that IGF-II, via activation of the beta-catenin signaling cascade, promotes growth of ACF and tumors without affecting tumor numbers.

Evaluation of direct and indirect markers to assess the androgen status in healthy males during aging.

Evaluation of the male androgen status requires a marker that reflects the biologically active fraction of plasma testosterone. The serum sex hormone-binding globulin (SHBG) concentration is not suitable here because of its wide inter-individual scatter. As potential biological markers of the active testosterone fraction we compared indirect methods calculated on the basis of SHBG and total testosterone measured by fully automated IMMULITE 2000 assays (DPC, Los Angeles, CA, USA), and total testosterone alone with direct free testosterone measured by RIA (DPC). Indirect methods were the free androgen index FAI, calculated free testosterone cFT, and calculated bio-available testosterone cBT. Further androgens measured were DHEAS and androstenedione. Blood samples were collected from a cohort of 446 healthy men aged between 20-99 years. All parameters except SHBG decreased significantly during aging. The direct free testosterone assay was significantly correlated with the indirect androgen parameters. This is in accordance with earlier results using LC-MS as the gold standard method. The strongest correlation was seen with cBT/measured albumin (r=0.750), though the direct testosterone RIA does not measure the entire unbound fraction of testosterone, and total testosterone can rapidly be measured with an automated assay system. It was found that a fixed albumin concentration of 43 g/L is a reasonable calculation basis for cBT in subjects of <70 years. In the elderly >70 years or persons with known pathologies of the androgen axis, it is commendable to measure the albumin concentration individually. In conclusion, calculated bio-available testosterone (cBT) is the best marker to reflect the bioactive testosterone fraction, i.e. the androgen status in males.

Effects of insulin-like growth factors and insulin-like growth factor binding protein-2 on the in vitro proliferation of peripheral blood mononuclear cells.

Increasing evidence has implicated that insulin-like growth factors (IGFs), polypeptides structurally related to proinsulin, are involved in the function and development of the immune system. To probe the relevance of IGF binding protein 2 (IGFBP-2) in T-cell activation and proliferation, we studied the role of IGFBP-2 in anti-CD3 monoclonal antibody (mAb)-activated peripheral blood mononuclear cells (PBMCs). Secretion of IGF-I, IGF-II, and IGFBP-2 by PBMCs from healthy adult donors was determined by radioimmunoassays (RIAs). The PBMC proliferative response after stimulation with anti-CD3 mAb and exposure to increasing concentrations of IGF-I, IGF-II, IGFBP-2, and anti-IGFBP-2 were determined by bromodeoxyuridine enzyme-linked immunosorbent assay. Observations were tested for significance by paired t-tests. We demonstrate an increase in IGFBP-2 secretion associated with both activation of PBMC by anti-CD3 mAb and increasing cell density. Incubation with exogenous IGFBP-2 increased the proliferation of PBMCs, whereas anti-IGFBP-2 had an antiproliferative effect on PBMCs that was reversed by simultaneous exposure to IGFBP-2. The stimulatory activity of IGFBP-2 (1-10 ng/ml) on anti-CD3 mAb-activated PBMCs was similar to that of IGF-I and IGF-II (1-100 ng/ml), with the mean increase in PBMC proliferative response ranging between 150% and 160% for IGFBP-2 (p = 0.03), 150% and 170% for IGF-I (p < 0.01), 133%-161% for IGF-II (p < 0.01), and 157% and 175% for IGF-I + IGF-II (p < 0.01). Thus, our data strongly suggest a role for IGFBP-2 as a local growth factor contributing to the proliferation and activation of mononuclear cells.

Insulin-like growth factor binding protein 2 (IGFBP-2) separates hypertrophic and hyperplastic effects of growth hormone (GH)/IGF-I excess on adrenocortical cells in vivo.

GH and IGF-I are capable of inducing cellular hypertrophy and/or hyperplasia. Chronic overexpression of GH in transgenic mice results in systemically and locally increased IGF-I levels and in disproportionate overgrowth, including adrenocortical enlargement and corticosterone hypersecretion. Using PEPCK-bovine GH transgenic (G) mice, we demonstrate that adrenal enlargement involves both hypertrophy (44%) and hyperplasia (50%) of zona fasciculata cells. To clarify whether IGFBP-2 affected cell volume and number, we crossed hemizygous G mice with hemizygous CMV-IGFBP-2 transgenic (B) mice, generating G mice, B mice, GB double transgenic mice, and nontransgenic controls (C). The absolute weight of the adrenal glands was significantly increased in 5-wk- and 4-month-old G mice vs. C and B mice. IGFBP-2 overexpression in GB mice reduced this effect of GH excess by 26% and 37% in 5-wk- and 4-month-old animals, respectively. GH-induced hypertrophy of zona fasciculata cells was completely abolished by IGFBP-2 overexpression in GB mice whereas hyperplasia was not affected. Basal and ACTH-induced plasma corticosterone levels of 4-month-old G mice, but not of GB mice, were two- to threefold increased compared with C mice. Plasma ACTH levels were similar in all groups. Our data show that IGFBP-2 potently separates hypertrophic and hyperplastic effects of GH/IGF-I excess on adrenocortical cells.

Stability of insulin-like growth factor (IGF)-I and IGF binding protein (IGFBP)-3 measured by the IMMULITE automated chemiluminescence assay system in different blood specimens.

Insulin-like growth factor I (IGF-I) and IGF binding protein 3 (IGFBP-3) are measured to diagnose disorders of the somatotropic axis in children and adults. In clinical studies samples for IGF-I and IGFBP-3 measurement must often be stored for months and sent to specialized laboratories. Therefore, we tested the stability of IGF-I and IGFBP-3 in whole blood, serum and plasma from 12 volunteers at 4 degrees, 22 degrees, and 37 degrees C for several hours and at -25 degrees C for several months. The effect of only one protease inhibitor (Aprotinin = Trasylol, Bayer, Germany ) on IGF-I and IGFBP-3 measured in the automated IMMULITE assay system (DPC, Los Angeles) was tested. IGF-I and IGFBP-3 were stable in heparinized whole blood, plasma and serum at 22 degrees C up to 24 hours. IGF-I was stabilized by aprotinin for up to 72 hours at 37 degrees C. Factor concentrations were not altered after storage at -25 degrees C for at least 12 months. Recognition of IGFBP-3 fragments by the antibody used in the automated IGFBP-3 IMMULITE was excluded by measurement in 26 sera from pregnant women which usually contain IGFBP-3 fragments. In conclusion, samples for measurement of IGF-I and IGFBP-3 should be kept on ice and cooled if shipment takes more than 48 hours or alternatively 5000 IU/ml aprotinin should be added. IMMULITE assays are also valid to measure IGF-I and IGFBP-3 after at least 12 months storage at -25 degrees C.

Reference intervals for testosterone, androstenedione and SHBG levels in healthy females and males from birth until old age.

The measurement of androgen levels is important in the follow-up of sexual development and in the diagnosis of disturbances of the gonadal function in children and adults. The aim of this study was to evaluate the age dependence of the serum concentrations of testosterone, androstenedione, and SHBG from birth until old age using the IMMULITE 2000 automated assay system (DPC, Los Angeles). Testosterone and androstenedione median levels were very high during the first weeks of life due to residual maternal hCG and decreased to low basal levels around the detection limit of the assay. With the onset of puberty around the age of 10 years both parameters increased strongly, reaching a maximum at about 17 years (testosterone: > 20-fold in boys, 2-fold in girls; androstenedione: 10-fold in boys, 5-fold in girls). In both girls and boys, we measured a decline in the SHBG medians during sexual maturation. This decline was more pronounced in boys (median 78.3 to 26.2 nmol/l from Tanner stage 1 to 5) since the higher androgen levels are thought to down-regulate SHBG. In male adults a continuous decrease was seen for testosterone from a median of 16.1 nmol/l in age group 21-30 years to 9.7 nmol/l in the age group > 70 years. In women the testosterone levels which were only about 5% of that of men from the same age group decreased only slightly, starting from a median of 0.9 to 0.6 nmol/l. In both sexes androstenedione levels decreased continuously during aging. In contrast to the androgen levels, the median SHBG levels increased steadily in men from 20.8 to 44.5 nmol/l, while the median SHBG levels in women decreased from 78.3 to 44.5 nmol/l in the age group of 61-70 years. Interestingly, the SHBG levels rose again in women of the group > 70 years. The reference intervals elaborated here may help in the assessment of the status of sexual development, and to diagnose pathologies of the gonadal axis or hypogonadism during aging.

The early dehydroepiandrosterone sulfate rise of adrenarche and the delay of pubarche indicate primary ovarian failure in Turner syndrome.

Pubarche without thelarche has been taken as clinical evidence that adrenarche is independent of gonadarche in females. This study examines whether the course of adrenarche [rise of serum dehydroepiandrosterone sulfate (DHEAS)] and pubarche (Tanner stage PH2) is independent from ovarian function. Serum DHEAS levels (n = 867) were longitudinally measured in 111 girls with Turner syndrome between 1990 and 2002. Of these, 22 had spontaneous puberty onset (Tanner stage B2), and 45 had primary ovarian failure (POF). Serum DHEAS levels were assayed by chemiluminescence and compared with those of healthy girls (n = 322; age range, 3-17 yr in both groups). Between the ages of 7 and 17 yr, girls with Turner syndrome had significantly higher age-related DHEAS levels than normal girls (P

Neuroprotective and anti-oxidative effects of the hemodialysate actovegin on primary rat neurons in vitro.

The recently described therapeutic benefits of the hemodialysate actovegin on neuropathic symptoms in diabetic patients with symptomatic polyneuropathy suggest a neuroprotective activity of the drug. To elucidate the possible cellular mechanism of the pharmacological effects of actovegin, we investigated its effects on cultured primary rat neurons in vitro. Primary neurons were cultured for up to 10 days in the presence of increasing doses of actovegin (0.3-1,000 mg/l). Total cell number, dendrite length and the number of excitatory synapses, i.e., the amount of the synaptic V-Glut1 protein, were measured by immunocytochemistry followed by fluorescence microscopy. The apoptotic level in neurons after induction of apoptosis by amyloid peptide Aß(25-35) was assessed by the level of activated caspase-3. In addition, the capability of the neurons to diminish oxidative stress was assessed by measuring the cellular level of reactive oxygen species ROS in the presence of actovegin. Actovegin treatment yielded an increased maintenance of neuronal cells and total number of synapses and could lower the level of activated caspase-3 in a dose-dependent manner. Dendrite lengths were not significantly affected. In addition, actovegin reduced the cellular level of ROS in cultured neurons. The cellular effects observed suggest neuroprotective and anti-oxidative effects of the drug Actovegin(®), which could at least partially explain its therapeutic benefits.

Children with acute Perthes' disease have asymmetrical lower leg growth and abnormal collagen turnover.

BACKGROUND: Abnormalities in distal growth and low levels of insulin-like growth factor (IGF)-I have been reported in children with Perthes' disease. Our aim was to establish whether the acute phase of Perthes' disease is associated with abnormalities of growth, of bone or of collagen turnover. METHODS: We performed a cross-sectional study of 15 children (3-11 years of age, 13 boys) at acute presentation and a longitudinal cohort study of 9 children. We measured (1) the lengths of both lower legs (by knemometry) at weeks 1, 2, 6 and 12, (2) height and weight at presentation and at the second-year follow-up, and (3) levels of IGF-I, IGFBP-3, collagen markers and bone alkaline phosphatase at weeks 1 and 12, and in year 2. RESULTS: Height SD scores were normal at presentation but declined thereafter. Lower leg growth was not impaired at presentation but was asymmetrical, ceased during weeks 2-6, and then resumed symmetrically. Patients had persistently low IGF-I, low soft tissue collagen synthesis and enhanced collagen breakdown compared with age- and sex-related reference data. Markers of bone formation increased during follow-up. INTERPRETATION: Acute changes in lower leg growth reflected differential weight bearing, then immobilization and remobilization. Persistently low IGF-I may have contributed to low soft tissue collagen synthesis and growth. Changes in bone formation markers most likely reflected bone healing.

Reference ranges for serum concentrations of lutropin (LH), follitropin (FSH), estradiol (E2), prolactin, progesterone, sex hormone-binding globulin (SHBG), dehydroepiandrosterone sulfate (DHEAS), cortisol and ferritin in neonates, children and young adults.

The aim of this study was to establish reference ranges for children (neonates to young adults), for serum lutropin (LH), follitropin (FSH), estradiol (E2), progesterone, prolactin, sex hormone-binding globulin (SHBG), dehydroepiandrosterone sulfate (DHEAS), cortisol and ferritin, using the nonisotopic, automated chemiluminescence immunoassay system, Immulite (DPC). Serum samples from 762 children (369 female; age 1 day to 19 years) were examined. Of these, 381 were classified as pubertal. Due to non-normal distribution, the 2.5th, 50th and 97.5th percentiles (central 95% interval) were calculated for each group. Statistical differences between the reference ranges were analyzed with respect to age, sex and the stage of sexual maturation. The median concentrations of E2, prolactin, progesterone, DHEAS, cortisol and ferritin were higher during the first 2 weeks post-partum than thereafter. The largest difference was seen with prolactin, which showed up to 27-fold higher values during this period. In contrast, before the onset of puberty, hardly any sex difference was observed and all analyte concentrations remained relatively constant, apart from SHBG which increased steadily after the neonatal period. The increase of gonadal activity in females with the onset of sexual maturation included an increase in LH and FSH, which was accompanied by a strong increase in E2, progesterone and prolactin. Cortisol increased to a lesser extent during puberty. In males, the increase in the median concentrations of the hormones was smaller, except for DHEAS. The concentration of ferritin was high in the neonatal period but did not change during sexual maturation. Our findings agree with earlier studies. The calculated reference intervals can be used to assess the development of children, particularly for measurements performed by the Immulite and Immulite 2000 chemiluminescence assay systems.

Endocrine alterations in the aging male.

The recent increase in the elderly population, current health trends and awareness of age-related changes in the male endocrine system, have led to discussions about the role of the hormonal changes in the aging process in males. Better prevention and treatment of suboptimal health status and age-related diseases in aging men are based on an improved understanding of aging, particularly of the significance of age-associated hormonal changes. The aims of this study were 1) to evaluate the age dependence of the serum concentrations of the following important hormonal parameters in adult males using the IMMULITE 1 automated assay system (DPC, Los Angeles): testosterone, dehydro-epiandrosterone sulfate (DHEAS), estradiol (E2), sex hormone binding globulin (SHBG), lutropin (LH), follitropin (FSH), cortisol, prolactin, thyroid stimulating hormone (TSH), free triiodothyronine (fT3), free thyroxine (fT4) and the growth hormone-dependent parameters insulin-like growth factor (IGF-I) and IGF-binding protein-3 (IGFBP-3) and 2) to derive the following parameters: calculated free testosterone (cFT), ratio of calculated free testosterone to total testosterone (% cFT) and free androgen index (FAI). We found a significant decrease between the 21-30-year age group and the > 70-year age group for total testosterone (-42.4%), FAI (-65.5%), cFT (-60.0%), % cFT (-30.0%), DHEAS (-71.9%), E2 (-35.4%), TSH (-23.6%), IGF-I (-40.3%) and IGFBP-3 (-26.5%). Since the decreases in the FAI and cFT were greater than that of total testosterone and because these derived parameters reflect the biologically active fraction of testosterone, FAI and cFT are better markers for androgen deficiency in males. In contrast, a significant increase with age was observed for SHBG (+61.2%), LH (+40.0%), FSH (+98.3%) and cortisol (+54.2%). No significant alterations with age were observed for prolactin, fT3 and fT4. The study demonstrates that determining complete profiles of the androgenic, gonadotropic, adrenocortical, thyroid, pituitary and growth hormone/IGF endocrine axes in middle-aged and elderly men may be helpful in obtaining a correct clinical diagnosis for various hormonal disorders.

IGFs, IGFBPs, IGF-binding sites and biochemical markers of bone metabolism during differentiation in human pulp fibroblasts.

OBJECTIVE: To investigate the role of the insulin-like growth factors (IGF) system during the differentiation of human pulp-derived fibroblasts (HPF). METHODS: Primary HPF were cultured for 24 days in DMEM medium with IGF-I or IGF-II (50 ng/ml each). Cell growth and morphology, alkaline phosphatase (ALP) activity, the concentration of free deoxypyridinoline (DPD), IGF-I, -II, IGFBP-2 and -3 were studied. The number of (125)I-IGF-I binding sites was estimated by Scatchard analysis. RESULTS: Light-microscopically visible nodules emerged during differentiation. Simultaneously, the ALP activity increased steadily between days 8 and 24, while the DPD concentration decreased by about 50%. The HPF produced high concentrations of IGF-II (2.00-1.30 microg/10(6) cells) but low IGF-I, IGFBP-2. IGFBP-2 was not changed, IGFBP-3 increased by 65% during differentiation. The number of IGF binding sites increased from 8,500 +/- 55 per cell (day 8) up to 22,000 +/- 570 (day 24). CONCLUSION: The increasing number of IGF-binding sites accompanied by alterations in the biochemical bone markers during the differentiation of HPF suggests an autocrine/paracrine role for the IGFs in the formation of dentinal hard tissue.

Reference ranges for two automated chemiluminescent assays for serum insulin-like growth factor I (IGF-I) and IGF-binding protein 3 (IGFBP-3).

Assays for insulin-like growth factor I (IGF-I) and IGF-binding protein 3 (IGFBP-3) have become essential tools in the diagnostic work-up of disorders of the somatotropic axis in children and adults. The aim of this study was to evaluate the automated IMMULITE IGF-I and IGFBP-3 assays and to establish reference limits--central 95% intervals, median, 0.1 and other centiles as clinically relevant--as a function of age from 797 females and 787 males, from the first week of life through the ninth decade. Pubertal children were classified by sex and by sexual maturation (Tanner stage). IGF-I and IGFBP-3 levels were also assayed in 20 pediatric patients each with growth hormone deficiency (GHD) and Turner syndrome (UTS), before and during 12 months of recombinant growth hormone (rhGH) therapy, as well as in 11 adult patients with GHD and seven with acromegaly before therapy. Both the IGF-I and IGFBP-3 assays were accurate, specific and sufficiently sensitive to measure IGF-I and IGFBP-3 in serum with good linearity and recovery. In the IGF-I assay, potential interference from IGFBPs was eliminated by blocking with excess IGF-II. Circulating IGF-I and IGFBP-3 concentrations, and their ratio IGF-I/IGFBP-3, were age-dependent, showing low levels immediately after birth, a typical pubertal peak for girls and boys, and a pronounced decline after puberty, reaching a plateau in early adulthood. In adults IGF-I and IGFBP-3 levels decreased smoothly but steadily with age. Children with GHD and UTS had low circulating IGF-I and IGFBP-3 levels which increased to normal reference limits under therapy with rhGH. Adult GHD patients showed IGF-I levels below the age-related median; untreated acromegalic patients mostly had IGF-I and IGFBP-3 levels above the age-related 97.5th centile. In conclusion, the automated IMMULITE IGF-I and IGFBP-3 assays are reliable tools in the diagnosis of pathologies of the GH/IGF axis and in the follow-up of their therapies.

Pilot study of elevated levels of insulin-like growth factor-binding protein-2 as indicators of hepatocellular carcinoma.

BACKGROUND/AIMS: Insulin-like growth factor-binding protein-2 (IGFBP-2) is expressed in many malignant tissues, and elevated serum levels can be indicators of tumour activity in addition to conventional tumour markers. Our aim was to evaluate the role of IGFBP-2 levels together with insulin-like growth factor (IGF)-I, IGF-II and IGFBP-3 in the diagnostic work-up of patients with hepatocellular carcinoma (HCC). METHODS: In 50 (39 males, 11 females) histologically confirmed and TNM-graded patients with HCC who had not received adjuvant chemotherapy, the basal serum levels of IGF-I, IGF-II, IGFBP-3, IGFBP-2 and alpha-fetoprotein (AFP) were measured. The median age of the patients was 66 (37-84) years, body mass index was normal (25 (35-16) kg/m2). RESULTS: The levels of IGF-I, IGF-II and IGFBP-3 were diminished, as is the case when nutrition, hepatic function and growth hormone (GH) secretion are decreased. The levels of AFP and IGFBP-2 were markedly high. In 37 cases, IGFBP-2 levels were above the age-related norm, and in 40 cases AFP levels were also elevated. In 3 cases, both AFP and IGFBP-3 were normal, and in 4 cases AFP was high but IGFBP-2 normal, whereas in 10 cases AFP was normal but IGFBP-2 was high. CONCLUSIONS: In addition to AFP, IGFBP-2 appears to be a suitable marker for the evaluation of the serological status of HCC patients. A longitudinal study during disease management is required to assess the full potential of IGFBP-2 measurements as a marker.

Age-dependency of insulin-like growth factors, insulin-like growth factor-binding proteins, and acid labile subunit in plasma and wounds of surgical patients.

Wound problems are common in the elderly. We hypothesized that age-related decrements in blood levels of components of the insulin-like growth factor (IGF) system are reflected in the wound environment. In this prospective, observational study IGF-I, IGF-II, IGF-binding protein-2, IGF-binding protein-3, and acid labile subunit were measured by immunoassays in the wound fluid and plasma of young (23.5 +/- 3.3 years) and elderly (78.9 +/- 6.2 years) patients before and daily for 4 days after elective surgery. IGFs, IGFBP-3, and acid labile subunit in plasma were significantly lower in the elderly group (p < 0.0001). The decrements of these proteins in plasma were reflected in corresponding decrements of 25-70% in the wound fluid of elderly patients (p < 0.0001). Additionally, bioavailability of IGF-I was less in the aged. The IGF parameters in the wound displayed a constant ratio with those of blood, suggesting that blood contributes a major share of the IGF that enters the wound during the initial phase of healing. The current data adds to accumulating evidence that a decline in the IGF system in aged patients contributes to the healing deficits observed in the elderly.

Reference levels of insulin-like growth factor I in the serum of healthy adults: comparison of four immunoassays.

The measurement of insulin-like growth factor-I (IGF-I) has become an essential tool for diagnosing growth hormone deficiency and acromegaly, as well as for monitoring the efficacy of treatment in these disorders. The latter aspect gains significance in the light of epidemiological studies which indicate a relationship between IGF-I levels and the incidence of certain malignancies. We aimed to evaluate the performance of widely implemented IGF-I assays by testing four representative, commercially available immunoassays. Thus, four parallel determinations of the IGF-I levels of 427 healthy blood donors aged between 18 and 79 years were conducted. Apart from divergent performance criteria, the assays also differed systematically. These differences were, however, linear and of lower magnitude among the lower ranges. We conclude that despite the wide variance among commercially available IGF-I assays, which principally involve assay-specific normative data, each of the implemented assays was robust and thus an appropriate tool in the diagnostic work-up of growth hormone deficiency in adult life, when IGF-I levels are low.