ACVR and ECVDI consensus statement for the standardization of the abdominal ultrasound examination.

This consensus statement is designed to provide a standard of care document and describes the ACVR and ECVDI definition for performing a standard abdominal ultrasound examination in dogs and cats. The ACVR and ECVDI define a standard abdominal ultrasonographic examination as a complete exam of the abdominal organs which is appropriately documented. The consensus statement intends to provide guidance to veterinary sonographers and veterinarians for the performance and documentation of high-quality diagnostic ultrasound examination. The document may also serve as a teaching aid for veterinary students, veterinarians, and residents in diagnostic imaging who seek proficiency in diagnostic ultrasound. Finally, it may serve an additional role in educating the public as to what a high-quality abdominal ultrasound examination should entail.

Individualizing FSH dose for assisted reproduction using a novel algorithm: the CONSORT study.

The CONSORT dosing algorithm individualizes recombinant human FSH (r-hFSH) doses for assisted reproduction technologies, assigning 37.5 IU increments according to patient characteristics: basal FSH, body mass index, age and antral follicle count. A prospective, uncontrolled, international, 18-centre, pilot study of normo-ovulatory women aged 18-34 years inclusive undergoing a long agonist treatment protocol was performed. Follitropin alfa filled-by-mass (GONAL-f®) dose was assigned by the algorithm and was intended to be altered only for risk of ovarian hyperstimulation syndrome (OHSS). Primary end-point was number of oocytes retrieved. Dose groups containing ≥5 patients were analysed: 75 IU (n = 48), 112.5 IU in = 45), 150 IU (n = 34), 187.5 IU (n = 24), 225 IU (n = 10). Cancellations due to inadequate response were higher than expected in the 75 IU group (12/48). Overall, a median of 9.0 oocytes were retrieved (8.5, 8.0, 10.0, 12.0 and 8.0 in the 75, 112.5, 150, 187.5 and 225 IU groups respectively). Clinical pregnancy rates/cycle started were 31.3, 31.1, 35.3, 50.0 and 20.0%, respectively (overall, 34.2%). Two patients had severe OHSS. Use of the CONSORT algorithm achieved an adequate oocyte yield and good pregnancy rates in this preliminary study. Adjustment of the algorithm could reduce cancellation rates.

The period prevalence of oral lichen planus in a cohort of patients with vulvar lichen sclerosus.

BACKGROUND: Lichen sclerosus and lichen planus are chronic inflammatory mucocutaneous disorders that may coexist. OBJECTIVE: The aim of this study was to estimate the period prevalence of oral lichen planus in a cohort of patients with vulvar lichen sclerosus and to document their clinical characteristics. METHODS: We report a series of cases of vulvar lichen sclerosus presenting to two dermatologist-led vulvar clinics in Oxfordshire, England between 1997 and 2007 with coexistent clinical signs of oral lichen planus. RESULTS: Thirteen cases with coexistent vulvar lichen sclerosus and oral lichen planus were identified, of which five had oral biopsies. Four oral biopsies showed histological features consistent with lichen planus. One oral biopsy was not diagnostic but compatible with oral lichen planus. No cases of oral lichen sclerosus were identified. The period prevalence of oral lichen planus was 6 per 1000 cases of vulvar lichen sclerosus. CONCLUSION: The period prevalence of oral lichen planus in women with vulvar lichen sclerosus (0.6%) is similar to that reported for oral lichen planus in the general population (1-2%).

Individualizing FSH dose for assisted reproduction using a novel algorithm: the CONSORT study.

The CONSORT dosing algorithm individualizes recombinant human FSH (r-hFSH) doses for assisted reproduction technologies, assigning 37.5 IU increments according to patient characteristics: basal FSH, body mass index, age and antral follicle count. A prospective, uncontrolled, international, 18-centre, pilot study of normo-ovulatory women aged 18-34 years inclusive undergoing a long agonist treatment protocol was performed. Follitropin alpha filled-by-mass (GONAL-f) dose was assigned by the algorithm and was intended to be altered only for risk of ovarian hyperstimulation syndrome (OHSS). Primary end-point was number of oocytes retrieved. Dose groups containing >or=5 patients were analysed: 75 IU (n = 48), 112.5 IU (n = 45), 150 IU (n = 34), 187.5 IU (n = 24), 225 IU (n = 10). Cancellations due to inadequate response were higher than expected in the 75 IU group (12/48). Overall, a median of 9.0 oocytes were retrieved (8.5, 8.0, 10.0, 12.0 and 8.0 in the 75, 112.5, 150, 187.5 and 225 IU groups respectively). Clinical pregnancy rates/cycle started were 31.3, 31.1, 35.3, 50.0 and 20.0%, respectively (overall, 34.2%). Two patients had severe OHSS. Use of the CONSORT algorithm achieved an adequate oocyte yield and good pregnancy rates in this preliminary study. Adjustment of the algorithm could reduce cancellation rates.

Signalment, history, and outcome of cats with gastrointestinal tract intussusception: 20 cases (1986-2000).

OBJECTIVE: To determine signalment, history, and outcome of cats with gastrointestinal tract intussusception and to identify physical examination, diagnostic imaging, surgical, histologic, and necropsy findings in affected cats. DESIGN: Retrospective case series. ANIMALS: 20 cats with intussusception. PROCEDURES: Medical records were evaluated for information on signalment; history; physical examination, diagnostic imaging, surgical, histologic, and necropsy findings; and outcome. RESULTS: Ten cats were < 1 year old, and 9 were >or= 6 years old. Anorexia (14/17), lethargy (12/17), and vomiting (12/17) were the most common reasons for examination. Dehydration (13/18), poor body condition (12/18), signs of abdominal pain (8/18), and an abdominal mass (8/18) were the most common physical examination findings. Abdominal radiography revealed intestinal obstruction in all 10 cats in which it was performed; abdominal ultrasonography revealed intussusception in all 7 cats in which it was performed. The most common intussusception was jejuno-jejunal (8/20), and no intussusceptions were found proximal to the duodenum. Eleven of 13 cats that underwent laparotomy required intestinal resection and anastomosis. Histologic examination revealed intestinal lymphoma or inflammatory bowel disease in 7 of 8 cats >/= 6 years old and idiopathic intussusception in 7 of 8 cats < 1 year old. CONCLUSIONS AND CLINICAL RELEVANCE: Results suggested that in cats, intussusception has a bimodal age distribution, is most commonly jejuno-jejunal, often requires surgical resection and anastomosis, is often associated with alimentary lymphoma or inflammatory bowel disease in older cats, and is readily diagnosed by means of ultrasonography.

Cost-utility analysis of 21-gene assay for node-positive early breast cancer.

Background: For women with lymph node (ln)-positive, estrogen receptor-positive, and her2 (human epidermal growth factor receptor 2)-negative breast cancer (bca), current guidelines recommend treatment with both hormonal therapy and chemotherapy. The 21-gene Recurrence Score (rs) assay might be helpful in selecting patients with bca who can be spared chemotherapy when they have 1-3 positive lns and a lower risk of recurrence. In the present study, we performed a cost-utility analysis comparing use of the 21-gene rs assay with current practice from the perspective of a Canadian health care payer. Methods: A Markov model was developed to determine costs and quality-adjusted life-years (qalys) over a patient's lifetime. Patient outcomes in both study groups were examined based on published clinical trials. Costs were derived primarily from published Canadian sources. Costs and outcomes were discounted at 1.5% annually, and costs are reported in 2016 Canadian dollars. A probabilistic analysis was used, and the model parameters were varied in a sensitivity analysis. Results: The results indicate that use of the 21-gene rs assay was less costly ($432 less) and more effective (0.22 qalys) than current practice. The probabilistic analysis revealed that 70% of the 10,000 simulated incremental cost-effectiveness ratios were in the southeast quadrant. The results were sensitive to the probability of a low rs and to the probability of receiving chemotherapy in the low-risk rs category and in current practice. Conclusions: Use of the 21-gene rs assay could be a cost-effective strategy for Ontario patients with estrogen receptor-positive, her2-negative early bca and 1-3 positive lns.

Changes in insulin sensitivity and glucose metabolism during therapy with recombinant human growth hormone in short children born small for gestational age show a negative correlation with baseline measurements.

Recombinant human growth hormone (rhGH) is an effective therapy for children with short stature born small for gestational age (SGA); however, insulin resistance can develop during treatment. This retrospective analysis assessed the effect of rhGH treatment (0.067 mg/kg/day) on glucose metabolism and insulin secretion in children with short stature born SGA, and measured whether baseline characteristics correlated with changes in insulin resistance or glucose sensitivity during treatment. Baseline glucose area under the concentration-time curve (AUC) was negatively correlated with the change in glucose AUC (p<0.001). Similar negative correlations were seen between baseline insulin AUC and the change in insulin AUC during treatment (p<0.001); and between baseline HOMA-IR (homeostatic model of insulin resistance) and the change in HOMA-IR during treatment (p<0.001). Small but significant changes, not thought to be clinically significant, were seen in indicators of insulin sensitivity during rhGH treatment. Glucose levels remained within the normal range during oral glucose tolerance testing.

Predictive factors and a corresponding treatment algorithm for controlled ovarian stimulation in patients treated with recombinant human follicle stimulating hormone (follitropin alfa) during assisted reproduction technology (ART) procedures. An analysis of 1378 patients.

BACKGROUND: Identifying parameters that can accurately predict the response to controlled ovarian stimulation (COS) would be of great benefit in assisted reproductive technology (ART) procedures. An analysis was undertaken with the objective of determining whether specific factors could optimally predict a response to stimulation in ART, and to then develop a corresponding treatment algorithm that could be used to calculate the optimal starting dose of recombinant human follicle stimulating hormone (r-hFSH; follitropin alfa) for selected patients. METHODS: The overall population consisted of 2280 normo-ovulatory ART patients from 11 randomised clinical trials. However, for the final analysis population, only patients less than 35 years of age who received r-hFSH monotherapy (N = 1378) were included. RESULTS: Backwards stepwise regression modelling indicated that predictive factors for ovarian response included basal FSH, BMI, age and number of follicles < 11 mm at baseline screening. The concordance probability index was 59.5% for this model. CONCLUSIONS: In the largest data series so far analysed to determine predictive factors of ovarian response, basal FSH, BMI, age and number of follicles < 11 mm at screening were the most important variables in ART patients less than 35 years of age who were treated with r-hFSH monotherapy. Using these four predictive factors, a follitropin alfa starting dose calculator was developed that can be used to select the FSH starting dose required for an optimal response. The relevance of this dose calculator will be evaluated in a prospective clinical trial.

Noninvasive monitoring of murine tumor blood flow during and after photodynamic therapy provides early assessment of therapeutic efficacy.

PURPOSE: To monitor tumor blood flow noninvasively during photodynamic therapy (PDT) and to correlate flow responses with therapeutic efficacy. EXPERIMENTAL DESIGN: Diffuse correlation spectroscopy (DCS) was used to measure blood flow continuously in radiation-induced fibrosarcoma murine tumors during Photofrin (5 mg/kg)/PDT (75 mW/cm2, 135 J/cm2). Relative blood flow (rBF; i.e., normalized to preillumination values) was compared with tumor perfusion as determined by power Doppler ultrasound and was correlated with treatment durability, defined as the time of tumor growth to a volume of 400 mm3. Broadband diffuse reflectance spectroscopy concurrently quantified tumor hemoglobin oxygen saturation (SO2). RESULTS: DCS and power Doppler ultrasound measured similar flow decreases in animals treated with identical protocols. DCS measurement of rBF during PDT revealed a series of PDT-induced peaks and declines dominated by an initial steep increase (average +/- SE: 168.1 +/- 39.5%) and subsequent decrease (59.2 +/- 29.1%). The duration (interval time; range, 2.2-15.6 minutes) and slope (flow reduction rate; range, 4.4 -45.8% minute(-1)) of the decrease correlated significantly (P = 0.0001 and 0.0002, r2= 0.79 and 0.67, respectively) with treatment durability. A positive, significant (P = 0.016, r2= 0.50) association between interval time and time-to-400 mm3 was also detected in animals with depressed pre-PDT blood flow due to hydralazine administration. At 3 hours after PDT, rBF and SO2 were predictive (P < or = 0.015) of treatment durability. CONCLUSION: These data suggest a role for DCS in real-time monitoring of PDT vascular response as an indicator of treatment efficacy.

The cause of neuronal degeneration in Alzheimer's disease.

Alzheimer's disease is associated with a specific pattern of pathological changes in the brain that result in neurodegeneration and the progressive development of dementia. Pathological hallmarks common to the disease include beta-amyloid plaques, dystrophic neurites associated with plaques and neurofibrillary tangles within nerve cell bodies. The exact relationship between these pathological features has been elusive, although it is clear that beta-amyloid plaques precede neurofibrillary tangles in neocortical areas. Examination of the brains of individuals in the preclinical stage of the disease have shown that the earliest form of neuronal pathology associated with beta-amyloid plaques resembles the cellular changes that follow structural injury to axons. Thus, the development of beta-amyloid plaques in the brain may cause physical damage to axons, and the abnormally prolonged stimulation of the neuronal response to this kind of injury ultimately results in the profound cytoskeletal alterations that underlie neurofibrillary pathology and neurodegeneration. Therapeutically, inhibition of the neuronal reaction to physical trauma may be a useful neuroprotective strategy in the earliest stages of Alzheimer's disease.

Interleukin-12 inhibits angiogenesis and growth of transplanted but not in situ mouse mammary tumor virus-induced mammary carcinomas.

We examined the ability of recombinant murine interleukin-12 (rmIL-12) to inhibit the vasculature and growth of mammary carcinomas arising in situ in mouse mammary tumor virus (MMTV)-infected female C3H/HeN mice. Although it is a potent antiangiogenic and antitumor agent in many transplanted murine tumor models, rmIL-12 failed to inhibit the vascularity, reduce the perfusion, or alter the growth of these autochthonous carcinomas. Factors intrinsic to these tumor cells were unlikely to be responsible for therapy failure. This is because primary cells derived from these carcinomas responded to IFN-gamma, and rmIL-12 was effective against transplanted tumors arising from Mm5MT cells, a line established from a MMTV-induced mammary carcinoma in C3H mice. Factors intrinsic to the mice that host the autochthonous mammary carcinomas were also not responsible for failure, because they sponsored rmIL-12 antiangiogenic and antitumor effects against transplanted K1735 murine melanoma tumors. Instead, the autochthonous nature of the mammary carcinomas and their possession of a high percentage of mature, pericyte-covered vessels that are resistant to therapeutic regression may be responsible. This is supported by the observation that transplanted Mm5MT tumors had a lower proportion of pericyte-covered vessels and responded to rmIL-12 therapy. These results point to significant differences between the vasculature of transplanted and autochthonous murine tumors and indicate that their susceptibility to antivascular therapy may differ substantially.

Rescue of hypoxia-inducible factor-1alpha-deficient tumor growth by wild-type cells is independent of vascular endothelial growth factor.

In tumors, rapid cell proliferation associated with deficient vascularization leads to areas of hypoxia. Tumor hypoxia has direct consequences on clinical and prognostic parameters and is a potential therapeutic target. The hypoxic response depends critically on hypoxia-inducible factor-1alpha (HIF-1alpha) in pathological (e.g., tumorigenesis) as well as physiological (e.g., development and wound healing) processes. By s.c. injection of HIF-1alpha(-/-) embryonic stem (ES) cells in nude mice, we were able to demonstrate the role of HIF-1alpha in cell differentiation of teratocarcinomas. HIF-1alpha(+/+) tumors grow fast and preferentially form neuronal tissue, whereas HIF-1alpha(-/-) tumors show delayed growth and favorably form mesenchyme-derived tissue. Mixing wild-type and HIF-1alpha(-/-) ES cells in the same tumor at a ratio as low as 1:100, we showed that HIF-1alpha(+/+) cells can rescue the growth of mixed tumors although these tumors are not significantly different phenotypically or genotypically from the original HIF-1alpha(-/-) tumors. Interestingly, these results are not restricted to teratocarcinomas: they were confirmed with mixtures of Hepa1/Hepa1C4 cells (where HIF-1beta is mutated), demonstrating that growth changes are not related to differences in differentiation observed within teratocarcinomas. We also showed that despite lower mRNA expression, vascular endothelial growth factor protein status in HIF-1alpha(-/-) and mixed tumors does not significantly differ from the HIF-1alpha(+/+) tumors. Moreover, we demonstrated that tumor vascularization remains proportional to vascular endothelial growth factor protein levels, but that hypoxic up-regulation of this growth factor is not the decisive factor influencing tumor growth. Differences in levels of apoptosis are not responsible for alteration in growth because poly(ADP-ribose) polymerase cleavage, a hallmark of the apoptotic process, was similar in HIF-1alpha(+/+), HIF-1alpha(-/-), and mixed tumors. Our data demonstrate that the HIF-1alpha-dependent response of a few cells is capable of sustaining the growth of the whole tumor, probably through the secretion of factors up-regulated under low oxygen conditions.

Doppler ultrasound imaging detects changes in tumor perfusion during antivascular therapy associated with vascular anatomic alterations.

Noninvasive monitoring of antiangiogenic therapy was performed by serial power Doppler ultrasound imaging of murine tumors treated with recombinant interleukin 12, the results of which were correlated with assessments of tumor vascularity by microscopy. Growth of established K1735 tumors, but not of IFN-gamma-unresponsive K1735.N23 variants, was suppressed by treatment. Serial Doppler imaging of K1735 tumor vascularity during treatment revealed a progressive change from a diffuse perfusion pattern to a more punctate distribution. Quantitative analysis of the images revealed that color-weighted fractional average, representing overall tumor perfusion, consistently decreased in these tumors, primarily because of a decrease in fractional tumor cross-sectional area carrying blood flow. In contrast, these parameters increased in nonresponsive tumors during treatment. Confocal microscopy of thick tumor sections revealed a reduction in the density and arborization of vessels labeled in vivo by fluorochrome-conjugated lectin with effective treatment. Immunohistological examination of thin tumor sections confirmed the preferential loss of small vessels with successful therapy. Similar changes in tumor vascular anatomy and perfusion were also observed during recombinant interleukin 12 treatment of two other responsive murine tumor types. These results indicate that power Doppler ultrasound is a sensitive, noninvasive method for reporting functional consequences of therapy-induced vascular anatomical changes that can be used to serially monitor tumor perfusion and efficacy of antivascular therapy in clinical trials.

Regulation of insulin-like growth factor-binding protein-1 in rat serum.

Previous studies support a role for insulin-like growth factor-binding protein-1 (IGFBP-1) in modulating insulin-like growth factor (IGF) availability for glucose homeostasis. We have developed a radioimmunoassay (RIA) for rat IGFBP-1 (rIGFBP-1) and have examined the regulation of circulating levels by nutritional and hormonal status. Rabbit antisera were raised against pure rIGFBP-1, and an assay was established with a sensitivity of 50 pg. In the rat, serum IGFBP-1 concentrations decrease with increasing developmental age. They were highest in fetal rat serum, exceeding 4 mg/L, and decreased to < 0.1 mg/L in adult animals. Serum rIGFBP-1 levels increased during fasting, 6-fold after 24 h and 18-fold after 48 h, and were suppressed to levels identical to ad libitum-fed control rats within 2 h of refeeding. Fasting levels were > 2-fold higher in female than male animals. IGFBP-1 concentrations were suppressed by > 50% in two rat models of insulin resistance. Levels increased in STZ-induced (streptozotocin) diabetes and were suppressed to normal with insulin treatment. Exercise stimulated rIGFBP-1 concentrations in fasting animals. On immunoblotting after SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis), rIGFBP-1 in serum appeared as a doublet with molecular masses at 31 and 33 kD. The components of this doublet did not vary across the range of experimental conditions. These observations indicate that the pattern of regulation of rIGFBP-1 is similar to that seen in previous studies of human IGFBP-1, with age, sex, and nutritional status being important regulators.(ABSTRACT TRUNCATED AT 250 WORDS)

Evaluation of quantified contrast-enhanced color and power Doppler ultrasonography for the assessment of vascularity and perfusion of naturally occurring tumors in dogs.

OBJECTIVE: To investigate subjective and computerized methods of evaluation of color Doppler (CD) and power Doppler (PD) ultrasonographic images (obtained before and after administration of contrast medium) for quantitative assessment of vascularity and perfusion of various naturally occurring tumors in dogs. SAMPLE POPULATION: 34 tumors in 34 dogs. PROCEDURE: Tumors in dogs were examined via CD and PD ultrasonography before and after i.v. injection of a microbubble contrast agent (pre- and postcontrast examinations, respectively). Images were digitized for subjective assessment of vessel density and vascular pattern and computer-aided assessment of parameters of vascularity (fractional area [FA]) and perfusion (color-weighted FA [CWFA] and mean color-weighted FA [CWFA] and mean color level). RESULTS: With both analysis methods, more vessels were identified in precontrast PD ultrasonographic images than in precontrast CD ultrasonographic images. Moreover, compared with values for precontrast PD ultrasonography, FA, CWFA, and mean color level were higher for postcontrast PD ultrasonography. In postcontrast images, there was a significant association between vessel densities determined through subjective and computerized assessments. Although sample size was small, vascularity of squamous cell carcinomas was significantly greater than that of other tumor types. Ten of the 19 softer than issue that sarcomas had low vessel density with minor contrast enhancement. With increasing gross tumor volume, FA and CWFA decreased for all Doppler ultrasonographic methods. CONCLUSIONS AND CLINICAL RELEVANCE: Higher values of the ultrasonographic parameters representing vascularity and perfusion of tumors in dogs were determined via PD ultrasonography after administration of contrast medium than via PD or CD ultrasonography without administration of contrast medium.

Markers of epidermal differentiation expressed by rat keratinocytes cultured by a modified feeder layer technique.

A broad range of analytical methods has been used to investigate the expression of key differentiation markers in keratinocytes cultured by a modified feeder layer technique. Cultures were stratified and showed many of the features characteristic of epidermal differentiation in vivo including tonofilaments, desmosomes, loss of organelles and thickening of the plasma membrane to form the cornified envelope. Profilaggrin synthesis was detected by 32P-incorporation and the presence of filaggrin suggested that it was broken down by the normal route. Staining with the lectin from Ulex europeus revealed the presence of a fucose-containing cell-surface glycoprotein. Keratin synthesis was shown by 3H-leucine incorporation and keratins were analysed by two-dimensional gel electrophoresis in comparison with those from different levels of the epidermis. Quantitative and qualitative differences were found between in vivo and in vitro epidermal differentiation. In particular, cornified envelope numbers were low, in keeping with the observation by electron microscopy of only one layer of cells with this structure. The absence of a true stratum corneum in vitro was also indicated by the virtual absence of histidase activity and stratum corneum keratins. The keratin species present in vitro most closely resembled those of the basal cells of the epidermis, although even in this case differences were observed. The evidence as a whole is consistent with the belief that epidermal cells do synthesise in vitro many of the important proteins involved in differentiation, but that they nevertheless do not develop a true keratinised stratum corneum.

Fibronectin in cultured rat keratinocytes: distribution, synthesis, and relationship to cytoskeletal proteins.

The aim of this study was to investigate whether epidermal cells can synthesise fibronectin and whether the distribution of this glycoprotein is related to the adhesion and cytoskeletal organisation of these cells. The production of fibronectin by newborn rat epidermal cells was shown by indirect immunofluorescence staining of cultures grown in the absence of a feeder layer using an antiserum which had been cross-adsorbed with foetal calf serum proteins to remove antibodies which recognised serum fibronectin. The distribution of fibronectin in areas of cell-cell and cell-substratum contact, characteristically in the form of short radial stitches, was examined in more detail using immunoelectron microscopy with colloidal gold as marker. This showed the close proximity of fibronectin to the cell membrane, with the ventral surface and fine cellular processes showing the heaviest labelling, and also revealed evidence of a relationship between external fibronectin and internal structure in epidermal cells. Immunofluorescence showed that tonofilaments (keratin) and microtubules were present as fibrillar arrays but were not related to fibronectin distribution. Vimentin and desmin were absent. Actin was distributed as a circumferential bundle of filaments, with finer stands running radially to the edge. The latter were reminiscent of the radial fibronectin stitches and a spatial correspondence between fibronectin and actin was confirmed by double-label immunofluorescence which revealed many instances of overlap and colinearity of actin and fibronectin filaments. The ability of keratinocytes to produce fibronectin suggests that these cells can contribute to the formation of the basement membrane in skin. The localisation of fibronectin and its close association with actin also suggests that it is involved in keratinocyte adhesion and is related to the internal organisation of these cells.

Bioavailability of insulin-like growth factors (IGFs) in rats determined by the molecular distribution of human IGF-binding protein-3.

Insulin-like growth factor-binding protein-3 (IGFBP-3) forms a 140-kilodalton (kDa) complex with an acid-labile glycoprotein after complexing with IGF-I or IGF-II. To investigate the bioavailability of circulating IGFs in the rat, we determined the molecular distribution and disappearance of an iv bolus of human IGFBP-3 (130 micrograms/kg). Serum fractions were assayed after size-separation chromatography for human (h) and rat (r) IGFBP-3 with specific RIAs. Within 2 min of injection, 52.9 +/- 1.6% of the hIGFBP-3 appeared in a 140-kDa complex. This form remained in the circulation for hours, whereas hIGFBP-3 in 50- and 30-kDa forms disappeared within 30-60 min. The rapid complexing of hIGFBP-3 to the 140-kDa form did not occur in vitro. Measurement of endogenous rIGFBP-2 indicated that there was minimal exchange of hIGFBP-3 with rIGFBP-3 in the 140-kDa complex within the 60 min of the in vivo study. To determine the importance of IGF-I in regulating the molecular distribution of hIGFBP-3 in serum, the experiment was repeated in streptozotocin-diabetic rats, with total IGF-I levels 50% lower than control values. In these animals, 40.1 +/- 5.2% of the hIGFBP-3 appeared in the 140-kDa complex 2 min after injection, significantly less than that in controls (P < 0.05), and hIGFBP-3 disappeared more quickly from the circulation than in controls (P < 0.05). After coinjection of recombinant human IGF-I, the retention of hIGFBP-3 in the circulation was prolonged in both control and diabetic animals. Because IGFBP-3 must bind IGF before it can associate with the acid-labile subunit, these findings indicate a much greater availability of IGFs to the circulation than previously estimated.

Regulation of rat insulin-like growth factor-binding protein-1: the effect of insulin-induced hypoglycemia.

Rat insulin-like growth factor-binding protein-1 (rIGFBP-1) was purified from H4IIE rat hepatoma cells by IGF-I affinity chromatography and reverse-phase HPLC. A rabbit antiserum (B2) was raised to rIGFBP-1 and a RIA established. Immunoreactive IGFBP-1 was present in rat amniotic fluid and in the medium conditioned by isolated rat hepatocytes and HTC rat hepatoma cells. To study the effect of hypoglycemia, fasting female Wistar rats were anesthetized and cannulated for multiple venous sampling after the administration of insulin or saline. Serum IGFBP-1 rose in adrenal intact rats from < 0.1 micrograms/ml to a maximum of 1.41 +/- 0.23 micrograms/ml approximately 120 min after insulin administration. Compared to adrenal-intact rats, adrenalectomized animals demonstrated a delayed rIGFBP-1 response to hypoglycemia and did not appear to have reached a maximum at 180 min. A slow rise in rIGFBP-1 levels throughout the sampling period was seen after saline injection in both adrenal-intact and adrenalectomized animals. Glucose, corticosterone, rat insulin, and human insulin levels were measured and none, alone, appeared responsible for the observed rIGFBP-1 responses. We conclude that 1) rIGFBP-1 is stimulated in response to hypoglycemia in a similar manner to glucose counterregulatory hormones, 2) an adrenal factor is required for an early rIGFBP-1 response to hypoglycemia, and 3) neither circulating glucose nor insulin levels, alone, are responsible for the observed patterns of response.