Anomalous adenosine cyclic 3':5'-monophosphate responses to glucagon in patients with hepatocellular carcinoma.

Glucagon resistance has been reported in rat hepatoma models. We studied the responses to glucagon challenge in 35 patients with hepato-cellular carcinoma. They have increased cyclic AMP and decreased glucose responses to glucagon (2 mg) challenge when compared with normal controls. Possible explanations for increased cyclic AMP responses include special membrane properties of hepatoma cells and increased adrenergic stimulation of adenylate cyclase during hypoglycemia. Decreased glucose responses are most apparent in patients with overt hypoglycemia. This may be related to a number of postulates, including depleted glycogen store of liver, impaired glycogenolysis, fatty metamorphosis, or insulin-like activities secreted by hepatoma. In this study, the increased cyclic AMP responses do not support the postulation that glucagon receptors are damaged in hepatocellular carcinoma.

The characterization, regulation, and function of insulin receptors on osteoblast-like clonal osteosarcoma cell line.

The properties and regulation of insulin receptors on monolayers of cultured clonal osteoblastic rat osteosarcoma UMR-106 cells and human osteosarcoma U20S cells were studied. Confluent cultures of UMR-106 cells bound lactoperoxidase-labeled, HPLC-purified [125I]A-14-monoiodinated insulin in a reversible, saturable, and specific manner. Binding was related inversely to the incubation temperature. Prolonged period of steady-state binding was achieved at all temperatures studied. Competition curves demonstrated half-maximal inhibition of [125I]insulin binding at an unlabeled insulin concentration of about 1 nM. Scatchard analysis of the binding data was curvilinear, suggesting negative cooperativity, and revealed that UMR-106 osteoblasts contained about 87,000 receptor sites per cell according to a two-site model. Bound [125I]insulin dissociated from osteoblasts with a t1/2 of about 15 minutes at 22 degrees C. The dissociation curve was multiexponential, and the addition of native insulin accelerated the dissociation of intact but not degraded [125I]insulin. Preincubation with 125 nM insulin for 1 h induced 70% loss of binding sites and reduced total insulin bound by 30%. When monolayers were treated with the lysosomotropic agent chloroquine, a 40% increase in cell-associated radioactivity that could not be dissociable in fresh buffer was observed. The use of an energy depleter, sodium fluoride, completely inhibited the effects of chloroquine. Similar results were obtained for human osteosarcoma U20S cells except that the number of receptor sites was far less than that of UMR-106 cells. Insulin increased collagen synthesis at a half-maximal concentration of 1 nM. To conclude, cultured rat and human osteoblasts possess insulin receptors that exhibit kinetic properties and specificity similar to those of other insulin target cells. Receptor-bound insulin is internalized and degraded by a chloroquine-sensitive, energy-requiring reaction. Insulin receptor on bone cells modulates the synthesis of collagen and this role may be important in bone homeostasis.

Parathyroid hormone receptors in human dermal fibroblasts: structural and functional characterization.

Previous studies have established the presence of parathyroid hormone (PTH)-sensitive adenylate cyclase activity in cultured human skin fibroblasts. The present study was undertaken to identify and quantitate PTH receptors directly in such cells. Human dermal fibroblast cell line CRL 1564 was found to possess specific binding sites for [125I]PTH(1-34). These sites bound PTH selectively; bovine and human PTH(1-34) and PTH(1-84) competed for [125I]PTH(1-34) binding sites, whereas the unrelated peptides calcitonin, insulin, AVP, angiotensin II, and ACTH(1-24) were inactive even at micromolar concentrations. Competitive binding experiments demonstrated the presence of binding site heterogeneity. These data fit a "two-site" model (p less than 0.001) in which one binding component has high affinity (Kd = 2.5 ng/ml = 0.6 nM) and low capacity (10(4) sites/cell) while the other has low affinity (Kd = 5.9 micrograms/ml = 1.5 microM) and high capacity (greater than 10(7) sites/cell). Similar high- and low-affinity [125I]bPTH(1-34) binding sites were seen also in CRL 1564 membranes containing a PTH-responsive adenylate cyclase. The Kd of the high-affinity sites was identical to the concentration of unlabeled bPTH(1-34) (4.2 ng/ml = 1.0 nM) required to half-maximally elevated cyclic AMP in CRL 1564 cells. Affinity labeling of specific PTH binding sites revealed the presence of multiple components with Mrs of 85, 70, 40, 33, and 23 kD on SDS-PAGE. Competition experiments did not disclose structurally discrete high- and low-affinity sites. Thus, structurally homologous PTH receptors in human skin fibroblasts apparently can assume two affinity states: (i) a high-affinity state coupled to adenylate cyclase and (ii) a low-affinity state that may represent uncoupled receptors.

Desensitization of parathyroid hormone receptors on cultured bone cells.

Administration of excessive amounts of parathyroid hormone (PTH) in the treatment of osteoporosis can reverse the beneficial effects of a low-dose, intermittent regime. To investigate the direct actions and the possible cellular mechanisms of PTH in inducing desensitization of PTH receptors, we studied the effects of desensitization on rat osteoblastic UMR-106 cells. When the osteoblasts were preincubated with bPTH-(1-34), complete refractoriness to a subsequent challenge with the hormone developed within 1 h and at hormone concentrations as low as 5 nM. When osteoblasts thus desensitized were incubated in hormone-free medium, recovery of the cAMP responses began within 2 h and reached maximum after 16 h. Cycloheximide did not affect the process of desensitization. [Nle8,Nle18,Tyr34]bPTH-(3-34)amide significantly impaired the desensitization process by PTH-(1-34) but did not have stimulatory effect on cAMP responses. No significant heterologous desensitization was obvious after preincubation with isoprenaline (50 microM), prostaglandin E1 (50 microM), or prostaglandin E2 (50 microM) for 2 h. Binding experiments with [125I]PLP-(1-36)amide after desensitization revealed that there was an approximate twofold decrease in receptor affinities as analyzed by Scatchard analysis, showing that the decrease in affinity was prominent in the process of desensitization. When the cells were treated with monensin during desensitization, PTH challenge after desensitization produced significantly lower cyclic AMP responses. Recovery after desensitization occurred over a period of 16 h. Inclusion of monensin, but not cycloheximide, impaired the recovery. The results show that homologous desensitization of rat osteoblasts to PTH is brought about by the occupancy of receptors by PTH-(1-34) but not by cAMP generation itself.(ABSTRACT TRUNCATED AT 250 WORDS)

The use of glucagon challenge tests in the diagnostic evaluation of hypoglycemia due to hepatoma and insulinoma.

We previously found that patients with hypoglycemia due to chronic renal and liver disease had anomalous metabolic responses to glucose and glucagon stimulation. In this study we evaluated the use of glucagon (2 mg, iv) tests in the diagnosis of spontaneous hypoglycemia secondary to hepatocellular carcinoma (HCC) and insulinoma. Twenty-one normal subjects, 45 patients with HCC (11 with hypoglycemia), and 14 patients with insulinoma (all with hypoglycemia) were studied. The fasting blood glucose level was low in all patients with hypoglycemia. The fasting plasma insulin and C-peptide concentrations were high in patients with insulinoma and low in patients with HCC and hypoglycemia. The blood glucose responses to glucagon administration were less than normal in patients with HCC and hypoglycemia and within normal limits in patients with insulinoma. The insulinoma patients had increased plasma insulin and C-peptide responses to glucagon despite having low blood glucose levels. Compared with the HCC patients without hypoglycemia, HCC patients with hypoglycemia had impaired plasma insulin and C-peptide responses. The fasting hypoglycemia, hypoinsulinemia, and impaired insulin/C-peptide responses to glucagon in patients with hepatoma and hypoglycemia presumably reflect the production of insulin-like substances by the hepatoma. We conclude that glucagon administration results in characteristic responses in these groups of patients and can be of use in the diagnosis of spontaneous hypoglycemia secondary to hepatoma or insulinoma.

25-Hydroxycholecalciferol and insulin-like growth factor I are determinants of serum concentration of osteocalcin in elderly subjects with and without spinal fractures.

Osteocalcin is one of the major proteins in the osseous matrix. To evaluate the determinants and thus the physiological control of osteocalcin production in normal and osteoporotic subjects, the serum levels of osteocalcin, 25-hydroxycholecalciferol (25-OH-D3), and insulin-like growth factor I (IGFI) were measured by radioimmunoassay in 44 subjects over 60 years old. Circulating osteocalcin, 25-OH-D3, and IGFI were 0.28 +/- 0.10 nmol/L (1.65 +/- 0.96 ng/mL), 70.5 +/- 25.1 nmol/L (28.3 +/- 10.1 ng/mL), and 23.8 +/- 12.0 nmol/L in 27 healthy controls respectively, as compared with 0.09 +/- 0.09 nmol/L (0.52 +/- 0.52 ng/mL), 48.2 +/- 19.9 nmol/L (19.35 +/- 7.91 ng/mL), and 16.56 +/- 6.96 nmol/L in 17 patients with spinal fractures. Significant correlation was found between osteocalcin and 25-OH-D3 level, as well as between osteocalcin and IGFI. The results show that IGFI and 25-OH-D3 are important determinants for serum concentration of osteocalcin in elderly subjects with and without spinal fractures.

Vitamin D status among patients with fractured neck of femur in Hong Kong.

Vitamin D deficiency leads to secondary hyperparathyroidism initially and then to mild osteomalacia, both of which conditions may be aymptomatic and may predispose to bone fracture. To assess the importance of vitamin D deficiency in predisposing to fractured neck of femur, we studied the vitamin D status, dietary intake and socio-economic characteristics in 69 patients with fractured neck of femur (group A), 28 normal subjects with age above 60 (group B), and 101 normal volunteers (group C). Patients with fractured neck of femor had significantly lower levels of serum 25-hydroxy-cholecalciferol compared with subjects of groups B and C. There is no statistically significant difference in other biochemical parameters, including calcium, phosphate, and alkaline phosphatase. Patients with fractured neck of femur and with 25-hydroxycholecalciferol below 20 ng/mL were characterized by a home-bound and/or institutionalized life-style, smaller living place, and limited access to open space. To conclude, hypovitaminosis D is a common problem among elderly patients with fractured neck of femur in Hong Kong. The fact that such vitamin D deficiency is associated with muscle weakness may contribute to falls, and thus indirectly account for an increased rate of hip fractures over the normal control.

The importance of parathyroid hormone in inhibition of collagen synthesis and mitogenesis of osteoblastic cell.

Loss of bone substance is a common manifestation of hyperparathyroidism. This suggests that parathyroid hormone (PTH) plays an important role as to bone mass. To investigate the mechanism underlying this change in bone mass, I studied the effects of PTH on collagen synthesis and mitogenesis of UMR-106 rat osteoblastic osteosarcoma cells. PTH inhibits the mitogenesis of UMR-106 rat osteosarcoma cells, the half-maximal concentration being 10(-8) to 10(-7) M, which is similar to the EC50 for cyclic AMP accumulation. Cyclic AMP, whose intracellular concentration was increased by PTH, plays a role in the modulation of mitogenesis, as shown by the comparable inhibitory effects of 8-bromoadenosine-3',5'-cyclic AMP (10(-4) M), forskolin (10(-7) M), and the phosphodiesterase inhibitor, IBMX (10(-5) M). PTH, in a similar concentration range, directly inhibited collagen synthesis. Concurrent with the suppression of collagen synthesis, the amounts of a1(I) and a2(I) collagen mRNA decreased proportionately. The results show that PTH modulates collagen synthesis at the transcriptional level. I concluded that parathyroid hormone inhibits the mitogenesis of osteoblasts as well as collagen synthesis by these cells. The decreases in the number of osteoblasts and the amount of collagen synthesis contribute to the loss of bone substance in hyperparathyroidism.

The first documented outbreak of trichinellosis in Hong Kong Chinese.

An outbreak of trichinellosis occurred in a Chinese family after the ingestion of inadequately cooked pork. Diagnosis was established by the demonstration of antibodies against Trichinella spiralis larval antigen by crossed immunoelectrophoresis and enzyme-linked immunosorbent assays, and living larvae in muscle biopsy. The main presenting symptom of these patients was pyrexia. Typical clinical features of Trichinella infection such as periorbital edema and severe myalgia were either minimal or absent. A history of having eaten insufficiently cooked meat is thus extremely important for correct diagnosis. We believe that this is the first documented report of a Trichinella outbreak in Chinese living in Hong Kong.

Analgesic effect of intranasal salmon calcitonin in the treatment of osteoporotic vertebral fractures.

Spinal collapse is a painful complication of osteoporosis. In this study, the analgesic effect of salmon calcitonin, administered intranasally (200 IU per day), was assessed in 18 patients with acute collapse of one to four vertebrae. Pain was evaluated by a descriptive pain scale (ie, Keele's) as well as by the daily consumption of analgesic drugs. When compared with placebo, intranasal salmon calcitonin significantly relieved pain and occasioned a decrease in the consumption of analgesic drugs. No major side effects were reported by the patients under study.

Subclinical hyponatremia, hyperkalemia and hypomagnesemia in patients with poorly controlled diabetes mellitus.

Diabetes mellitus is associated with disturbances in electrolyte metabolism. We studied 68 patients with insulin-requiring diabetes mellitus followed up in the Home Monitoring Clinic in order to assess the relationship between electrolyte disturbances and severity of diabetes. There is a significant correlation of serum sodium (r = -0.323, P less than 0.01), potassium (r = 0.416, P less than 0.001), magnesium (r = -0.292, P less than 0.02) with fasting glucose. Thus, in patients with high fasting blood glucose, sodium and magnesium tend to be lower while potassium is higher. Among the three parameters, only serum magnesium significantly correlates with the level of hemoglobin A1 (r = -0.356, P less than 0.001) and thus may be related to the long-term control of diabetes. On the other hand, the hyponatremia and hyperkalemia are more likely to be related to short-term metabolic control as reflected by fasting blood glucose. To conclude, minor changes in electrolytes have been found in a group of 68 Chinese patients receiving insulin. There is a remote possibility that these electrolyte changes may influence the chemical events responsible for long-term diabetic complications.

Effects of diets with high carbohydrate content on diabetic hyperlipidaemia and microalbuminuria.

Recent dietary recommendations for patients with diabetes mellitus have focussed on the liberalisation of carbohydrate intake to at least 50% of total calories. However, there are reports that this diet may cause adverse metabolic effects as a result of high intake of carbohydrate. Whether this high-carbohydrate diet will exacerbate hypertriglyceridaemia, which increases the risk of atherosclerosis, and diabetic microalbuminuria, which predisposes to progressive renal failure, is unknown. The dietary intake of 28 patients with insulin-dependent diabetes mellitus was assessed by diet histories with both questionnaire surveys and 3-day recall methods. The dietary contents of different constituents were graded according to the type and amount of food typically eaten, and the frequency of their consumption in the past 6 months. Twelve patients were found to have a high dietary intake of carbohydrate and this was confirmed by detailed assessment of food intake records over a 3-day period. The carbohydrate intake of these 12 patients amounted to at least 55% of total calories. Total calorie intake, body weight, mean blood pressure, glycaemic control, and glomerular filtration rate were similar between the 12 patients with high intake of carbohydrate and the other 16 patients with low intake. Urinary protein and albumin appearance as measured by dye binding and immunoassay, fasting cholesterol, triglyceride and high-density lipoprotein cholesterol were also comparable between the two groups. This study provides evidence that a high-carbohydrate diet in the treatment of diabetes mellitus is not associated with significant alterations in the amount of microalbuminuria or in hypertriglyceridaemia.

Rapid diagnosis of trichinosis by percutaneous muscle biopsy.

A new rapid diagnostic procedure for trichinosis is described. Percutaneous needle biopsy of the medial gastrocnemius muscle was used to obtain tissue for examination by a compression method. This procedure may facilitate early diagnosis and treatment of symptomatic trichinosis.

Acute intravascular hemolysis due to accidental formalin intoxication during hemodialysis.

A patient accidentally exposed to formalin during hemodialysis developed acute intravascular hemolysis. In vitro study showed that formalin has direct oxidant action on red blood cells. Formalin intoxication should be recognized as a cause of acute hemolysis during hemodialysis and is not invariably fatal, provided further exposure is stopped in time and the complications prevented.

Effects of selective and non-selective beta-blockers on the alanine and free fatty acid responses to glucagon challenge in hemodialysis patients.

Hypoglycemia is frequently reported in hemodialysis patients on propranolol. The effects of beta-blockers on the glucose, alanine and free fatty acid responses to glucagon challenge (2 mg) in fourteen hemodialysis patients were studied. Patients on propranolol had impaired glycemic response while those on metoprolol had decreased response only before dialysis. There was a significant negative correlation between propranolol level (log-transformed) and glycemic responses, suggesting that propranolol has direct effect on the latter. Hemodialysis patients had fasting alanine levels comparable to those of normal subjects. After glucagon challenge, the decrements in alanine level had no consistent relationship to the impaired glycemic response. Thus suppression of gluconeogenesis is unlikely to be the prime cause of propranolol-induced hypoglycemia. The basal free fatty acid levels were significantly lower among hemodialysis patients on propranolol. This limited availability of free fatty acid as well as the inhibitory effect on hepatic glucagon-stimulated glucose output may contribute to the predisposition to hypoglycemia in dialysis patients given propranolol. The results suggest that metoprolol has less interference on energy substrate supply in hemodialysis patients under fasting conditions.

Effects of propranolol and hemodialysis on the response of glucose, insulin, C-peptide and cyclic AMP to glucagon challenge.

Hypoglycemia is frequently reported in hemodialysis patients on propranolol. We studied the influence of propranolol on carbohydrate metabolism in sixteen hemodialysis patients (eight on propranolol and eight not) with pre-dialysis oral glucose tolerance tests (OGTT) and pre- and post-dialysis glucagon tests. Propranolol was shown to have significant suppressive effect on the insulin response during OGTT. With glucagon challenge pre- and post-dialysis, despite a similar cAMP response, patients on propranolol had significantly lower glucose response than those not receiving propranolol and it is postulated that this is due to post-receptor post-cAMP inhibition of hepatic glycogenolysis. Moreover, while the glucose response in patients not on propranolol significantly increased after dialysis, no significant change was found in patients on propranolol. The significantly higher C-peptide, insulin and cAMP responses to glucagon challenge in the hemodialysis patients as compared to normal controls may be due to delayed clearance of glucagon, and the further increased responses after dialysis may be due to the removal of a dialyzable factor suppressing these responses.

Humoral hypercalcemia of hepatocellular carcinoma associated with elevated levels of parathyroid-hormone-related-peptide.

Parathyroid-hormone-related-peptide (PTHrP) has been shown to be important in the pathogenesis of hypercalcemia of malignancy. The malignancies most commonly associated with hypersecretion of PTHrP include squamous cell carcinoma and breast cancer. The pathogenesis of hypercalcemia associated with hepatocellular carcinoma was evaluated in this study. Two male patients with hepatocellular carcinoma were found to have severe hypercalcemia at initial presentation. PTHrP was measured with radioimmunoassay in these two patients before treatment as well as in nine other patients with hepatocellular carcinoma but without hypercalcemia. The levels were markedly elevated in the two patients with hypercalcemia (29.3 and 32.1 pM), but were less than 5 pM in the nine patients without hypercalcemia. These results suggest that PTHrP was important in the pathogenesis of hypercalcemia associated with hepatocellular carcinoma in these men.

Hypoglycaemia and insulin resistance in uraemia associated with insulin fragments.

The pathogenetic role of insulin fragments in spontaneous hypoglycaemia and insulin resistance in chronic renal failure is discussed. Four different evidences are presented. In our studies, a dialysable 'toxin' with insulin-like actions was found in uraemia. Indeed small polypeptide fragments of hormones with potent activities have been recently identified in uraemic plasma. The varying bioactivities of different insulin fragments can explain the coexistence of insulin resistance and hypoglycaemia in these patients. As shown by our studies, removal of these fragments by haemodialysis results in normalisation of metabolic disturbances. However actual proof is difficult with the present technological problems.

Logistic regression model for the assessment of microalbuminuria and microproteinuria in insulin-dependent diabetic patients at risk.

Diabetic microalbuminuria, which predisposes to the irreversible macroproteinuria and terminal renal failure, has been shown to be reversible by stringent metabolic control. In this study, using logistic regression, 23 patients with insulin-dependent diabetes mellitus were evaluated. Basing on the reported biochemical changes in patients with diabetes mellitus, the relationship between microalbuminuria and serum biochemistries was assessed. Four biochemical parameters were shown to be significantly related to the amount of microalbuminuria and microproteinuria. The microalbuminuria as assayed by rocket immunoelectrophoresis is closely correlated with the microproteinuria as measured by the Coomassie dye binding method and both have a significant relationship with plasma creatinine, bicarbonate, albumin and globulin as assessed by the Minitab computer program and the Biomedical Data Package--Logistic Regression computer program. The relationship with bicarbonate and creatinine can be due to a decrease in glomerular filtration rate associated with more advanced microproteinuria. The relationship with albumin and globulin is the result of impaired albumin synthesis by the liver in diabetes mellitus. From these data, two simple equations which can provide a relative risk factor with rough quantitative assessment for microproteinuria and microalbuminuria are derived. Thus, from these four parameters available from relatively simple blood biochemistries, assessment can be made of the severity of coexisting diabetic proteinuria. The measurement of the latter is not readily available and is more costly and tedious. This assessment together with the finding of a decrease in albumin, slight increase in creatinine and decrease in bicarbonate associated with the more severe microalbuminuria can be used to alert the diabetologist in implementing tighter metabolic control and other interventional methods before irreversible macroproteinuria develops.

Total and regional bone mineral densities in women with Colles' fractures: a comparative study with normal matched controls.

Total body and regional bone mineral densities (BMDs) were measured in 34 women with past Colles' fracture and 34 age- and sex-matched controls using the Norland XR-26 dual energy X-ray bone densitometer. The results showed that in patients with Colles' fracture affecting the left forearms, the BMD at the ultradistal 2.5 cm region was significantly lower in the right forearm when compared with the left. This difference was not statistically significant among patients with fractures affecting the right or both forearms. The patients were also found to have lower BMDs in the femoral regions (0.600 +/- 0.010 g/cm2 in patients versus 0.655 +/- 0.019 g/cm2 in controls), pelvis (0.679 +/- 0.009 g/cm2 in patients versus 0.728 +/- 0.020 g/cm2 in controls) and spine (0.710 +/- 0.018 g/cm2 in patients versus 0.780 +/- 0.030 g/cm2 in controls) when compared with the controls. No such difference could be demonstrated in the head, trunks or arms. These data suggested that women with past Colles' fracture might be more prone to fractures of spine and femoral regions. Bone mineral densities in the weight-bearing regions, including femur and spine correlated strongly with each other (femoral neck versus lumbar spine, r = 0.64, p < 0.0001). Sites from the same anatomic regions, namely the femoral regions had highly correlated BMD values (femoral neck versus Ward's triangle, r = 0.91, SEE = 0.05, p < 0.0001), while poorer correlation was found among unrelated regions, such as between left ultradistal forearm and femoral neck (r = 0.43, SEE = 0.10, p < 0.05).