Secretion of an insulin-like growth factor-I-related protein by human breast cancer cells.

Somatomedin activity is required for proliferation of normal cells; recently somatomedin activity in the cellular environment was shown to be necessary for expression of the transformed phenotype. We demonstrate here that authentic serum-derived insulin-like growth factor-I (IGF-I) stimulates the proliferation of four human breast cancer cell lines, MCF-7, MDA-MB-231, ZR-75-1, and Hs578T in serum-free monolayer culture and that each of these lines produces and secretes an IGF-I-related growth factor. The two highly tumorigenic estrogen-independent cell lines, MDA-MB-231 and Hs578T, produced 2- to 10-fold more IGF-I than did two estrogen responsive cell lines, MCF-7 and ZR-75-1, which are not tumorigenic in the absence of estrogen. These breast cancer cells also secrete a Mr 50,000 binding activity which partially obscured detection of IGF-I by radioimmunoassay. Acid-ethanol extraction allowed dissociation of the high molecular weight complex; whereupon, fully immunoreactive IGF-I comigrated on acid gel exclusion chromatography with authentic human serum-derived IGF-I. Radioimmunoassay displacement curves for breast cancer cell line-derived IGF-I were parallel to those for authentic IGF-I. Northern blot analysis of mRNA from four breast cancer cell lines demonstrated specific hybridization with a human IGF-I probe corresponding to one of the two major transcripts seen in human liver mRNA. These data suggest that breast cancer cell line-derived IGF-I is similar to liver-synthesized, serum-derived IGF-I.

Dietary phosphorus and 1,25-dihydroxyvitamin D metabolism: influence of insulin-like growth factor I.

Hypophysectomy abolishes the increase in serum 1,25-dihydroxyvitamin D [1,25-(OH)2D] induced by restriction of dietary phosphorus. Administration of GH increases circulating insulin-like growth factor I levels (IGF-I) and restores, in part, the responsiveness of serum 1,25-(OH)2D to restriction of dietary phosphorus. To determine whether the GH-dependent increase in serum 1,25-(OH)2D induced by restriction of dietary phosphorus is mediated by IGF-I, we measured the serum concentration of 1,25-(OH)2D in hypophysectomized rats treated with either GH (100 micrograms/day) or recombinant human IGF-I (150 micrograms/day) and fed either a normal or low phosphorus diet for 6 days. Restriction of dietary phosphorus in sham-hypophysectomized rats increased serum 1,25-(OH)2D from 97 +/- 13 to 251 +/- 36 pg/ml, or 159%, but had no effect on serum 1,25-(OH)2D in hypophysectomized rats. Restriction of dietary phosphorus in rats receiving GH increased, (P less than 0.001) serum 1,25-(OH)2D from 52 +/- 8 to 133 +/- 18 pg/ml, or 156%. Restriction of dietary phosphorus in rats receiving IGF-I increased (P less than 0.001) serum 1,25-(OH)2D from 33 +/- 5 to 94 +/- 11 pg/ml, or 185%, an increase equivalent to that observed in animals receiving GH. For a given diet, no significant differences were seen between the serum concentrations of 1,25-(OH)2D in animals receiving GH or IGF-I. These data indicate that IGF-I can restore the increase in serum 1,25-(OH)2D induced by restriction of dietary phosphorus to the same degree as GH. This strongly suggests that the GH-dependent increase in serum 1,25-(OH)2D induced by restriction of dietary phosphorus is mediated by IGF-I.

Megakaryocytes endocytose insulin-like growth factor (IGF) I and IGF-binding protein-3: a novel mechanism directing them into alpha granules of platelets.

Lysis of platelets releases the contents of the alpha-granules, which contain growth factors, including insulin-like growth factor I (IGF-I) and IGF-binding protein-3 (IGFBP-3). We investigated the mechanism by which IGF-I and IGFBP-3 appeared in the alpha-granules with a goal of modulating their levels in platelets to affect platelet functions. Reverse transcription-PCR was initially used to test whether megakaryocytes contained IGFBP-3 and IGF-I messenger RNA transcripts. We found that megakaryocytes did not express the IGFBP-3 gene, but did have IGF-I messenger RNA. We subsequently investigated whether they incorporated IGFBP-3 and IGF-I by the process of endocytosis and packaged them into the alpha-granules. This hypothesis was tested in two ways. 1) We examined whether during pregnancy in the rat the alpha-granule content for IGFBP-3 paralleled the changes in plasma IGFBP-3 levels caused by the pregnancy-induced IGFBP-3 protease. The alpha-granule contents of both IGFBP-3 and IGF-I declined in parallel to the plasma changes in pregnant rats and returned to normal postpartum. As the binding protein protease acts extracellularly, endocytosis of the IGF-I:IGFBP-3 complex from the extracellular fluid by megakaryocytes was suggested. 2) We tested whether an IGF-I:IGFBP-3 complex comprised of human IGF-I and IGFBP-3 (recombinant 28.7 kDa) injected i.v. appeared in rat platelet alpha-granules. Hypophysectomized rats were injected i.v. with 5.24 mg of a 1:1 complex of IGF-I:IGFBP-3. After 24 h, platelet lysates were prepared and analyzed for IGFBP-3 by Western ligand blotting, and IGF-I was determined by RIA. Platelet lysates of the treated animals showed a prominent new band at approximately 28 kDa, whereas control rats were negative. In addition, the alpha-granule IGF-I concentration increased from 0.38 to 1.9 ng/1 x 10(9) platelets. These results indicate that the IGF-I:IGFBP-3 complex is taken up by megakaryocytes and packaged into the alpha-granules of platelets and demonstrate how the contents of IGF-I and IGFBP-3 in platelets can be modulated by their plasma concentrations. As reverse transcription-PCR has shown that the IGF-I, but not the IGFBP-3, gene is expressed by megakaryocytes, there may be two mechanisms for directing IGF-I into the alpha-granules of platelets.

The mechanism of the action of growth hormone on vitamin D metabolism in the rat.

GH has been shown to stimulate intestinal calcium absorption in rats and humans. We have investigated in rats whether GH might affect intestinal calcium absorption by stimulating the production of 1,25-dihydroxyvitamin D3 [1,25-(OH2D3], the active metabolite of vitamin D3. The tissue distribution of [3H]1,25-(OH2D3 8-40 h after iv injection of [3H]25-hydroxyvitamin D3 ([3H]25OHD3) was measured in sham controls, hypophysectomized, and GH-treated hypophysectomized rats. Since the plasma disappearance rate of iv [3H]1,25-(OH)2D3 was not significantly affected by hypophysectomy, the recovery of [3H]1,25-(OH)2D3 after [3H]25OHD3 administration was taken to be an indirect measure of renal 25-OHD3-1-hydroxylase. Hypophysectomy was found to reduce the recovery of [3H]1,25-(OH)2D3 from serum and intestinal mucosa by 70 +/- 2% (range). A 6-day course of GH treatment of hypophysectomized rats restored the formation of 1,25-(OH)2D3 to normal, and a significant effect was noted within 2 days, before any increase in renal weight was detectable. No other pituitary hormones appeared to be necessary. The markedly atrophic intestinal mucosa of hypophysectomized rats incorporated iv [3H]1,25-(OH)2D3 normally. However, it remains to be determined whether 1,25-(OH)2D3 alone can correct the decreased calcium transport in hypophysectomized rats.

Local injections of human or rat growth hormone or of purified human somatomedin-C stimulate unilateral tibial epiphyseal growth in hypophysectomized rats.

The growth-promoting actions of GH are thought to be mediated by somatomedin(s) (Sms), but unilateral bone growth in hypophysectomized (HX) rats given local injections of human (h) GH has been reported. Using slightly different methods, we have confirmed these results with purified hGH and have extended them to show that four daily injections directly into the tibial epiphyseal plate of 1 or 5 micrograms/day purified rat GH or recombinant DNA-derived hGH, but not of hPRL (6 micrograms/day) caused significant cartilage growth compared to that of the vehicle-injected contralateral tibia in rats that had been HX 14 days before the first injection. Thus, it is unlikely that the effects of GH are due to contaminating growth factors in the GH preparations, because the bacteria-derived preparation of hGH, which is unlikely to have such contaminants, was also active. Furthermore, we have shown that similar injections of 100 or 500 ng/day purified hSm-C caused unilateral tibial growth in rats HX 8 days, but not 14 days, before the first injection. These results demonstrate that both GH and Sm-C have direct growth-promoting effects on cartilage in vivo and are compatible with the theory that GH may act by stimulating local SM synthesis.

Insulin-like growth factor binding protein-3 is present in the alpha-granules of platelets.

The lysis of the alpha-granules of platelets has been shown to release insulin-like growth factors (IGFs). One of the major regulators of the activity of IGFs is the IGF binding proteins (IGFBPs), but IGFBPs were thought to be absent from platelet alpha-granules. Human platelets were isolated, and the contents of the alpha-granules were released by thrombin digestion. IGFBP-3 was identified in the lysate by RIA and a ligand-binding Western blot; no other IGFBPs were observed. Platelet IGFBP-3 was present as the characteristic glycosylated doublet at 45 and 43 kilodaltons on ligand-binding Western blot, but was not associated with the acid-labile subunit in the alpha-granule. Thus, the IGFs in platelet alpha-granules coexist with IGFBP-3, which can play a major role in regulating and targeting the actions of IGFs when released by lysis.

Somatomedin-C substitutes for insulin for the growth of mammary epithelial cells from normal virgin mice in serum-free collagen gel cell culture.

We investigated the effect of somatomedin C (SM-C) on the growth of mouse mammary ductal epithelial cells in collagen gel culture. Epithelial cells, isolated by collagenase digestion of whole glands, were placed into primary serum-free collagen gel cell culture for 10-12 days, during which SM-C was added alone or in combination with other growth-promoting factors. Previous work has shown that these cells require a superphysiological concentration of insulin (10 micrograms/ml) for optimum growth in serum-free medium (a 1:1 mixture of Ham's F-12 and Dulbecco's Modified Eagle's medium) containing epidermal growth factor (EGF). When SM-C (1-250 ng/ml) alone was added to serum-free basal medium containing EGF, it stimulated growth (at concentrations greater than 25 ng/ml) to at least the same extent as insulin at 10 micrograms/ml. There was no additive stimulation of growth when optimal concentrations of insulin and SM-C were added together. The nonadditive stimulation at optimal concentrations of these hormones may indicate that the previous requirement for a superphysiological concentration of insulin for maximum growth was due to low affinity binding of insulin to the SM-C receptor. Rat insulin-like growth factor II (Collaborative Research) at 50-200 ng/ml did not stimulate growth in the presence or absence of insulin. SM-C could not stimulate growth alone. The presence of EGF or mammogenic hormones (progesterone and PRL) was required.

Insulin-like growth factor-I reverses the impairment of wound healing induced by corticosteroids in rats.

Corticosteroids are widely used therapeutic agents that have as a major side-effect the impairment of wound healing. Two hypotheses were tested: 1) that antiinflammatory corticosteroids decrease the local insulin-like growth factor-I (IGF-I) response to injury; and 2) that locally administered IGF-I would overcome methylprednisolone-mediated suppression of healing. The IGF-I concentration was measured in fluid from wire mesh cylinder wounds in rats given saline or methylprednisolone im. Rats receiving 8 and 16 mg had decreased wound IGF-I levels of 32% and 56%, respectively, compared to the saline-injected controls. IGF-I was infused (15 micrograms/day) by osmotic minipumps into wire mesh cylinder wound chambers of methylprednisolone (8 mg)-treated rats for 7 and 14 days. Methylprednisolone decreased wound DNA to 21%, hydroxyproline to 30%, and total protein to 5% of the values found in saline-infused controls. A 14-day treatment with IGF-I completely reversed the effect of methylprednisolone and increased DNA, hydroxyproline, and total protein to 216%, 109%, and 96% of saline control values, respectively. In conclusion, corticosteroids depressed wound IGF-I concentrations in rats, and an infusion of IGF-I into wound chambers reversed the corticosteroid-induced impairment of wound healing, as determined by the DNA, hydroxyproline, and total protein contents in wound chambers.

Somatomedin generating activity of the 20,000-dalton variant of human growth hormone.

The 20,000-dalton variant of human growth hormone stimulated production of somatomedins in the hypophysectomized rat. Somatomedin activity was measured by a radioreceptor assay. The results indicate that the insulin--like activities of human growth hormone that are greatly diminished in the variant are not prerequisites for somatomedin production.

Induction of epidermal growth factor-related polypeptides by 17 beta-estradiol in MCF-7 human breast cancer cells.

MCF-7 human breast cancer cells release polypeptides related to insulin-like growth factor-I (IGF-I) and epidermal growth factor (EGF) into serum-free culture medium (CM). Treatment of MCF-7 cells with 17 beta-estradiol, which is required in vivo for MCF-7 tumor growth in the nude mouse and stimulates the MCF-7 growth rate in vitro, resulted in selectively enhanced growth factor activities in CM. Autostimulatory growth-promoting activity was elevated at least 2-fold, and EGF-like polypeptides were elevated 5-fold but IGF-I immunoreactivity was not elevated. Several species of estrogen-induced receptor-reactive EGF-like polypeptides, suggestive of high molecular weight transforming growth factor alpha, were detected after gel exclusion chromatography of CM extracted with 1 M acetic acid. A 30,000 mol wt peak of EGF receptor competing activity comigrated with a peak of autostimulatory and fibroblast-transforming activity. It is possible that estradiol stimulation of MCF-7 growth and/or tumor formation may depend on induction of EGF-related polypeptide growth factors. EGF-I- and EGF-related polypeptides may act together as autocrine or paracrine growth factors in breast cancer.

Prolactin but not growth hormone stimulates 1,25-dihydroxyvitamin D3 production by chick renal preparations in vitro.

We studied the effect of PRL from two species (bovine and turkey) and GH from two species (bovine and turkey) on 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] production by two whole cell preparations from vitamin D-deficient chick kidneys (slices and tubules). We observed that 8 ng/ml turkey PRL stimulated 1,25(OH)2D3 production by renal tubules and slices. Ovine PRL had a similar effect on 1,25(OH)2D3 production but at higher concentrations. In contrast, neither bovine GH nor turkey GH stimulated 1,25(OH)2D3 production appreciably at doses up to 1000 ng/ml. The effect of PRL on 1,25(OH)2D3 production by renal tubules required a 3-h preincubation, although its effect on 1,25(OH)2D3 production by renal slices was immediate. We conclude that PRL, but not GH, directly stimulates 1,25(OH)2D3 production by the chick kidney.

Lack of response of serum somatomedin to hyperprolactinemia in humans.

Because animal experiments have suggested that PRL might regulate the serum somatomedin (SM) concentration, the effect of sustained hyperprolactinemia on the serum SM level was studied in patients with proven pituitary microadenomas. PRL was determined by RIA. SM was measured on the same sample by a human placental membrane radioreceptor assay in which all SMs cross-react. The mean serum SM (+/-SE) in 16 females with elevated PRL levels from 68--21,000 ng/ml was 0.97 +/- 0.08 U/ml. This was not statistically different from that of 29 normal women, (P greater than 0.2). The mean SM for the 3 male patients with serum PRL levels from 570--5,050 ng/ml was in the lower range for normal males. There was no correlation in either group between the serum SM and PRL levels. These results indicate that PRL is not a major regulator of the serum SM concentration in man.

Basal and regulated secretion of insulin-like growth factor binding proteins in osteoblast-like cells is cell line specific.

Local secretion of insulin-like growth factor binding proteins (IGFBPs) may modulate the effects of IGF-I and IGF-II on bone cell metabolism and proliferation. Several osteoblast-derived cell lines are currently used as interchangeable models to study IGFBP production, although it is unknown whether findings in one cell line can be extrapolated to another cell line or to normal human osteoblasts. In this study, we examined by Western ligand blotting both basal and regulated secretion of IGFBPs in vitro in 1) normal human osteoblast-like (hOB) cells cultured from explants of human trabecular bone; 2) an SV40-transformed hOB (HOBIT) cell line; and 3) several human (U-2, MG-63, TE-85) and rat (ROS 17/2.8 and UMR-106-01) osteosarcoma cell lines. Constitutively, hOB and HOBIT cells produced a similar pattern of IGFBPs, while all other cell lines produced their own unique pattern of IGFBPs. The two rat cell lines differed from the human cell lines as well as from each other. The response to hormonal stimulation also varied among the cell lines. Treatment of hOB and HOBIT cells with IGF-I resulted in a 2-fold increase in medium levels of IGFBP-3; IGF-I decreased levels of 24-kilodalton (kDa) IGFBP in hOB cell-conditioned medium. In addition, IGF-I markedly increased levels of the 29/32/34 kDa IGFBP triplet in U-2 cells, but had little or no effect in the other human and rat osteosarcoma cell lines. PTH increased a 29-kDa IGFBP apparent only in UMR 106-01 cell-conditioned medium, whereas GH had no direct effect on IGFBP secretion in any of the osteoblast-like cells tested. We conclude that basal and regulated secretion of IGFBPs from osteoblast-like cells is cell-line specific. Spontaneously transformed human or rat osteoblast-like cells provide unique model systems to study features of distinct IGFBPs and their regulation; however, hOB cells and their derivatives may be more appropriate models for understanding the regulation of IGFs in human bone.

In vivo actions of insulin-like growth factor-I (IGF-I) on bone formation and resorption in rats.

The in vivo action of insulin-like growth factor-I on bone metabolism has been studied using a new model. Insulin-like growth factor-I (IGF-I) was continuously infused into the arterial supply of the right hindlimb of ambulatory rats for up to 14 days and the effects on cortical and trabecular bone formation and the number of osteoclasts were determined by histomorphometric techniques. The contralateral limb acted as an internal control. IGF-I infusion significantly increased cortical bone formation (p less than 0.01). Trabecular bone was increased 22% (p = 0.07), but the infusion was only for seven days. These effects of IGF-I were age dependent, being absent in young, rapidly growing animals, but present at least until one year of age. IGF-I appears to be a purely anabolic hormone for bone formation, since it significantly stimulates osteoblasts and decreases the number of osteoclasts. Thus, although IGF-I mediates the growth-promoting effect of growth hormone, it does not mediate growth hormone's action on bone resorption.

Characterisation of insulin-like growth factor-binding protein-3 binding to a novel receptor on human platelet membranes.

Insulin-like growth factor-binding protein-3 (IGFBP-3) is an important regulator of insulin-like growth factor (IGF) bioavailability and IGF-independent growth responses. IGFBP-3 is stored within the alpha granules of platelets, permitting its rapid and concentrated delivery at sites of platelet lysis. Previous studies have demonstrated a lack of mRNA for IGFBP-3 in platelets and in the megakaryocytes from which platelets are formed, indicating that IGFBP-3 is endocytosed from the extracellular milieu. In this study, the binding of IGFBP-3 to platelet membranes was characterised to determine whether specific cell-surface IGFBP-3 receptors exist that might account for IGFBP-3 uptake into the alpha granules by megakaryocytes. IGFBP-3 binding to platelets was saturable, requiring at least 4.6 nM (125)I-labelled IGFBP-3 to occupy all binding sites present on 100 microg of platelet membranes. Non-linear regression analysis revealed the presence of a single class of high-affinity binding sites for IGFBP-3 on platelets, with a K(d) between 2.6x10(-10) and 8.0x10(-10) M and 1.51-4.89x10(11) binding sites/mg of platelet membrane. Kinetic analysis of (125)I-IGFBP-3 binding to platelet membranes demonstrated a forward rate (k(on)) of 8.1x 10(8) per M per min. The reverse rate constant (k(off)) was calculated to be 1.6x10(-1) per min (t(1/2)=4.2 min) and confirmed experimentally to be 3.3x10(-1) per min (t(1/2)=2.1 min). Binding of (125)I-IGFBP-3 to platelet membranes was inhibited in a dose-dependent manner by recombinant Escherichia coli IGFBP-3. In contrast, rat IGFBP-4 was not able to compete with (125)I-IGFBP-3 for platelet binding sites. Additionally, concentrations of IGF-I ranging from a 15-fold to a 40 000-fold molar excess caused a consistent 20% reduction in (125)I-IGFBP-3 binding. The mechanism of this slight reduction is unknown, but suggests that IGF-I does not compete directly with IGFBP-3 for receptor binding sites. However, it does not preclude the possibility that IGF-I may be endocytosed into the alpha granules as part of an IGF-I-IGFBP-3 complex. These results demonstrate the presence of high-affinity binding sites for IGFBP-3 on human platelet membranes. The nature and kinetics of the binding reaction are characteristic of a receptor-ligand interaction. This receptor may be involved in the endocytosis of circulating IGFBP-3 by megakaryocytes for packaging within the alpha granules of platelets. It is unknown if it is present in other tissues.

Expression, purification and characterization of the structure and disulfide linkages of insulin-like growth factor binding protein-4.

Insulin-like growth factor binding protein-4 (IGFBP-4), like the other five IGFBPs, is a critical regulator of the activity of insulin-like growth factor (IGF)-I and IGF-II. However IGFBP-4 seems to be the only IGFBP with no potential to enhance the mitogenic actions of the IGFs. IGFBP-1 to -3 and -5 each contain 18 conserved cysteine residues, IGFBP-6 lacks two of the twelve N-terminal cysteines, while IGFBP-4 has two additional cysteines in the central region. A plasmid was constructed to express rat IGFBP-4 as a thioredoxin fusion protein that included a hexahistidine sequence to permit affinity purification. The fusion protein was expressed in E.coli, purified using nickel-chelate affinity chromatography and cleaved by tobacco etch virus (TEV) protease to produce mature rat IGFBP-4 with an additional glycine residue at the N-terminus. Final purification was achieved by further nickel affinity chromatography and reverse phase HPLC. The isoelectric points of the recombinant IGFBP-4 were the same as those of the non-glycosylated isoforms of IGFBP-4 in rat serum. The binding affinities of the recombinant protein and IGFBP-4 secreted by rat cells to IGF-I were compared using a newly developed binding assay. No significant difference could be detected, consistent with proper folding of the recombinant protein. This indicates that glycosylation of IGFBP-4 does not affect its binding to IGF-I. Using mass spectrometry and tandem mass spectrometry no differences between authentic and recombinant IGFBP-4 could be detected. Eight of the ten disulfide linkages have been determined, including linkages of conserved cysteine residues not previously identified in other IGFBPs. Numbering the cysteine residues sequentially from the N-terminus only the disulfide connectivity of C1, C2, C5 and C6 could not be determined. However, C1 is not linked to C1 and C5 is not linked to C6. The established linkages were C3 to C8, C4 to C7, C9 to C 11, and C10 to C12. The two cysteines in the non-conserved mid-region unique to IGFBP-4 (C13 and C14) are linked together. Linkage of the C-terminal cysteine residues is identical to that of IGFBP-2, -5 and -6 (C15 to C16, C17 to C18 and C19 to C20). The central flexible core of IGFBP-4, containing two additional cysteines may contribute to its unique biological action.

Effect of bovine growth hormone and a partially pure preparation of somatomedin on various growth parameters in hypopituitary dwarf mice.

The growth-promoting effects of a partially purified preparation of somatomedin (12.7 units/mg) were compared with those of various doses of bovine GH (5, 20 and 80 micrograms/day) when injected into hypopituitary dwarf mice. Growth parameters studied were body-weight and tail-length velocities (calculated as the slope of a regression line fitted to daily measurements against time), uptake of 35SO4(2-) into costal cartilage in vivo and organ weights (heart, liver and kidney). In the first experiment somatomedin (6.4 units/day), bovine GH and 0.9% NaCl were injected once daily in a volume of 0.1 ml for 10 days. Treatment with bovine GH promoted a significant dose-dependent increase in body-weight and tail-length velocities and 35SO4(2-) uptake into costal cartilage in vivo. Somatomedin also promoted a significant increase in body-weight velocity and 35SO4(2-) uptake, both responses were between that observed with the lowest dose of bovine GH and control values. Somatomedin did not promote increase in tail-length velocity. Organ weights did not differ significantly between any of the treatment groups when expressed as mg/g body weight. In the second experiment somatomedin (a daily total of 21.6 units/day) and 0.9% NaCl were injected three times per day in a volume of 0.033 ml, bovine GH was again injected once daily in a volume of 0.1 ml, and the treatment period was 12 days. As in the first experiment all doses of bovine GH and somatomedin promoted a significant increase in body-weight velocity. These results are consistent with the somatomedin hypothesis.

Insulin-like growth factors I and II in healthy women with and without established osteoporosis.

We measured serum concentrations of insulin-like growth factors I and II (IGF-I and IGF-II) by radioimmunoassay in 107 healthy women aged 28-78 years and in 116 women with established osteoporosis. The women with established osteoporosis were randomized to a 1-year double-blind, placebo-controlled treatment with continuous estrogen/progestogen, anabolic steroids, salmon calcitonin or placebo and the IGFs were measured every 6 months. Women less than 35 years of age had 29% higher levels of IGF-I (p < 0.001) as compared to women above that age. For women more than 35 years of age, we found no correlation between IGF-I and age (r = 0.02). Correspondingly, we found no significant changes in serum IGF-I in 10 women, who were followed with serial measurements of IGFs every 3 months from 2 years before to 1 year after menopause; IGF-II revealed no correlation with age (r = 0.04). In the group of 116 women with established osteoporosis, IGF-I was 30% lower (p < 0.01) as compared to a group of 19 height-, weight- and age-matched nonfractured women (mean age 64 years). The IGF-II levels were equal in the two groups. Over the 1-year therapeutic intervention period, an increase in IGF-I of 13-15% (p < 0.05) was seen in the nandrolone decanoate-treated group. The same tendency was seen for hormone replacement therapy, although it was not significant. In conclusion, the serum level of IGF-I is high in young women, when peak bone mass is attained, and low in postmenopausal women with established osteoporosis.

Isoflurane for prolonged sedation in the intensive care unit; efficacy and safety.

OBJECTIVE: To compare isoflurane with midazolam for prolonged sedation in ventilated patients. DESIGN: Randomised controlled study. SETTING: General intensive care unit in university teaching hospital. PATIENTS: Sixty patients aged 17-80 years who required mechanical ventilation for more than 24 h. INTERVENTIONS: Sedation with either 0.1-0.6% isoflurane in an air-oxygen mixture (30 patients) or a continuous infusion of midazolam 0.02-0.20 mg/kg/h (30 patients). Sedation was assessed initially and hourly thereafter on a six point scale. The trial sedative was stopped when the patient was ready for weaning from ventilatory support. MEASUREMENTS AND RESULTS: Measurements were made of haemodynamic, respiratory and biochemical variables regularly during the period of sedation and for a week after stopping the sedative agent. There was no difference in any of the physiological or biochemical variables recorded between the two groups. Patients sedated with isoflurane recovered more rapidly and were weaned from mechanical ventilation sooner than those sedated with midazolam. CONCLUSIONS: Isoflurane is a useful agent for prolonged sedation of ventilated patients and does not have any adverse effect on the cardiorespiratory system or on hepatic, renal or adrenal function.

The relation between human fetal growth and fetal blood levels of insulin-like growth factors I and II, their binding proteins, and receptors.

OBJECTIVE: To determine the relation between normal human fetal growth and the levels of insulin-like growth factors (IGF-I, IGF-II), their receptors, and IGF binding protein-3 (IGFBP-3) in both the maternal and fetal compartments. METHODS: Serum samples were obtained from normal pregnant women (n = 52) and their fetuses (n = 32) via funipuncture at 21-34 weeks' gestation (mean 29 +/- 4.3) and from term neonates (n = 20) between 38-41 weeks (mean 39 +/- 0.9). Neonates were divided into two groups: the "large" group, whose weights were above the mean for gestational age, and the "small" group, whose weights were below the mean. Aliquots of amniotic fluid (AF) and serum samples were analyzed for levels of IGF-I, IGF-II, and IGFBP-3. Type 1 IGF receptors were assayed from placental extracts of first-trimester elective abortions and from term deliveries. RESULTS: Fetal IGF-I serum levels remained stable throughout most of pregnancy until 34 weeks' gestation (56 +/- 30 ng/mL). Thereafter, IGF-I increased significantly until term (79 +/- 8 ng/mL) (P < .05). Fetal IGF-II levels were relatively unchanged from 23 weeks to term except for a significant increase at 34 weeks. Fetal serum levels of IGFBP-3 averaged 0.8 +/- 0.05 microgram/mL up to 30 weeks' gestation and then increased slightly toward term, at 0.96 +/- 0.05 micrograms/mL. At term, the levels of IGF-I and IGF-II in the AF were not different from the levels in the neonatal serum, but were lower (P < .005) than those in maternal blood. All placental tissue obtained from first-trimester terminations of pregnancy assayed positive for IGF type 1 receptors. There was a direct correlation between neonatal weight and the levels of IGF-I (P < .02), but not with the levels of IGF-II. There were no significant correlations between newborn weights and IGFBP-3, or maternal serum levels of IGF-I and IGF-II. Amniotic fluid IGF-I and IGF-II levels were almost similar to fetal serum levels. CONCLUSION: These data demonstrate the presence of type 1 receptors and the bioavailability of IGF-I, IGF-II, and IGFBP-3 throughout pregnancy. Insulin-like growth factor-I is shown to be adjunctively and directly associated with fetal size in normal pregnancies. The precise role that IGFs play in deviant fetal growth or whether IGFs can be used to treat reduced fetal growth remains unknown and awaits further investigation.