Exposure to cadmium and persistent organochlorine pollutants and its association with bone mineral density and markers of bone metabolism on postmenopausal women.

Environmental contaminants such as cadmium and persistent organochlorine pollutants have been proposed as risk factors of osteoporosis, and women may be at an increased risk. To assess associations between exposure to cadmium and two different POPs (2,2',4,4',5,5'-hexachlorobiphenyl CB-153, 1,1-dichloro-2,2-bis(p-chlorophenyl)-ethylene p,p'-DDE), on one hand, and bone effects, on the other, in a population-based study among postmenopausal (60-70 years) Swedish women with biobanked blood samples. The study included 908 women and was designed to have a large contrast of bone mineral densities, measured with a single photon absorptiometry technique in the non-dominant forearm. Biochemical markers related to bone metabolism were analyzed in serum. Exposure assessment was based on cadmium concentrations in erythrocytes and serum concentrations of CB-153 and p,p'-DDE. Cadmium was negatively associated with bone mineral density and parathyroid hormone, positively with the marker of bone resorption. However, this association disappeared after adjustment for smoking. The major DDT metabolite (p,p'-DDE) was positively associated with bone mineral density, an association which remained after adjustment for confounders, but the effect was weak. There was no evidence that the estrogenic congener (CB-153) was associated with any of the bone markers. In conclusion, no convincing associations were observed between cadmium and POPs, on one hand, and bone metabolism markers and BMD, on the other.

Blood-borne factors possibly associated with post-operative nausea and vomiting: an explorative study in women after breast cancer surgery.

BACKGROUND: The pathophysiology behind post-operative nausea and vomiting (PONV) is still not fully understood, especially with respect to gender. According to PONV risk scores, female gender is the strongest predictor for PONV. The risk for PONV after general anaesthesia for breast cancer surgery is 50-80%. The aim of the present explorative study was to identify blood-borne factors that might be associated with the development of PONV in women undergoing breast cancer surgery as a basis for further studies. METHODS: Fifty patients were enrolled prospectively in the study. A standardized sevoflurane-based anaesthetic was used. Blood samples for the analysis of vasopressin, gastrin, cholecystokinin, epinephrine, norepinephrine, dopamine, serotonin, platelet count and blood glucose were taken at six pre-determined time points peri-operatively, and PONV was assessed during 24 h. RESULTS: PONV was found in 27 of 47 patients completing the study. Patients with PONV had a larger variability of the platelet count (P = 0.001), a reduced platelet count on the first post-operative day (P = 0.02) and a less pronounced relationship between the platelet count and whole blood serotonin (P = 0.004) compared with non-PONV patients. A lack of a decrease in epinephrine levels in response to the induction of anaesthesia (P = 0.03) and increased levels of vasopressin (P < 0.001), epinephrine (P = 0.005) and blood glucose (P = 0.004) were observed in the early post-operative period in PONV patients. CONCLUSION: Three different platelet-associated factors and an altered epinephrine pattern were found to be associated with the occurrence of PONV after breast cancer surgery.

Production and characterization of antibodies for the specific determination of the opioid peptide Met5-Enkephalin-Arg6-Phe7.

Endogenous opioids serve as modulators of neuroendocrine and immune system processes, the investigation of which calls for high-specificity radioimmunoassays (RIAs). This study focuses on the development and use of a specific radioimmunoassay for the opioid peptide Met5-Enkephalin-Arg6-Phe7 (MEAP), the C-terminus part of proenkephalin A. Antibodies were raised in four rabbits and investigated in terms of titre, avidity and specificity, followed by finding ideal conditions for these antibodies in RIA. MEAP concentrations were determined in crude extracts of rat hypothalamus, dorsal root ganglia, adrenals and ankle using this standardized assay after an oxidizing process. At reverse-phase high-pressure liquid chromatography (HPLC), the position of immunoreactive material from rat hypothalamus eluted as two peaks out of which one was compatible with that of synthetic MEAP. All rabbits exhibited individual differences in relative immune response and time of its onset. The avidity constant was 10 times higher on a molar basis for ab 4108 compared with ab 4182. There was no cross-reactivity for ab 4182 to related peptides, such as enkephalins and dynorphin B, and negligible background values for ab 4108. The relative levels ofimmunoreactive MEAP from the CNS versus peripheral tissues contrasted in accordance with current knowledge. It is suggested that reports with RIA results should include characterization of antibodies, extraction procedures, standard curves and compositions of buffers. Furthermore, the results should preferably be expressed in relation to total protein content.

Bioavailability and dose-dependent anti-tumour effects of 9-cis retinoic acid on human neuroblastoma xenografts in rat.

Neuroblastoma, the most common extracranial solid tumour in children, may undergo spontaneous differentiation or regression, but the majority of metastatic neuroblastomas have poor prognosis despite intensive treatment. Retinoic acid regulates growth and differentiation of neuroblastoma cells in vitro, and has shown activity against human neuroblastomas in vivo. The retinoid 9-cis RA has been reported to induce apoptosis in vitro, and to inhibit the growth of human neuroblastoma xenografts in vivo. However, at given dosage, the treatment with 9-cis RA caused significant toxic side effects. In the present study we investigated the bioavailability of 9-cis RA in rat. In addition, we compared two different dose schedules using 9-cis RA. We found that a lower dose of 9-cis RA (2 mg day(-1)) was non-toxic, but showed no significant effect on tumour growth. The bioavailability of 9-cis RA in rat was 11% and the elimination half-life (t1/2) was 35 min. Considering the short t1/2, we divided the toxic, but tumour growth effective dose 5 mg day(-1) into 2.5 mg p.o. twice daily. This treatment regimen showed no toxicity but only limited effect on tumour growth. Our results suggest that 9-cis RA may only have limited clinical significance for treatment of children with poor prognosis neuroblastoma.

GLP-1 inhibits gastric emptying of water but does not influence plasma.

BACKGROUND: Glucagon-like peptide-1 (GLP-1) has been shown to inhibit gastric emptying of a caloric load but the effect on a non-caloric load is unknown. METHODS: Seven healthy men were studied after an over-night fast. Thirty min before the intake of 330 ml radioactively labeled water either GLP-1 (0.75 pmol/kg/min) or saline was administered intravenously and continued for 75 min. Scintigraphic gastric emptying was performed for 45 min and plasma samples were obtained for analysis of vasopressin, sodium, osmolality, GLP-1, insulin, and glucose. In addition, electric field stimulation of human gastric muscle strips was done. RESULTS: The median (range) percent water retained in the stomach, 45 min after intake of water, was 96% (68%-98%) and 12% (2%-42%) (P = 0.02) during infusion of GLP-1 and saline, respectively. Additionally, GLP-1 did not affect basal tone or contractile response of gastric muscle strips to electric field stimulation or acetylcholine (ACh). There was no change in plasma concentrations of vasopressin, sodium, or plasma osmolality during GLP-1 compared to saline infusion. CONCLUSION: GLP-1 has a profound inhibitory effect on the gastric emptying of water in man, but no short-term effect on water homeostasis. No effect was seen on contractility of gastric muscle strips suggesting an indirect action on gastric emptying.

Limited neuropeptide Y precursor processing in unfavourable metastatic neuroblastoma tumours.

Neuropeptide Y (NPY) is found at high concentrations in neural crest-derived tumours and has been implicated as a regulatory peptide in tumour growth and differentiation. Neuroblastomas, ganglioneuromas and phaeochromocytomas with significant concentrations of NPY-like immunoreactivity were investigated for different molecular forms of NPY and for significance of proNPY processing. Gel-permeation chromatography identified intact NPY (1-36) in all tumours, whereas proNPY (69 amino acids) was detected only in control adrenal tissue and malignant neuroblastomas. Purification of NPY-like immunoreactivity in tumour extracts and structural characterization revealed that both NPY (1-36) and the truncated form NPY (3-36) was present. The degree of processing of proNPY to NPY in tumour tissue was lower in advanced neuroblastomas with regional or metastatic spread (stage 3 and 4) (n = 6), (41%, 12-100%, median, range), compared to the less aggressive stage 1, 2 and 4S tumours (n = 12), (93%; 69-100%), (P= 0.012). ProNPY processing of less than 50% was correlated with poor clinical outcome (P = 0.004). MYCN oncogene amplification was also correlated to a low degree of proNPY processing (P = 0.025). In summary, a low degree of proNPY processing was correlated to clinical advanced stage and poor outcome in neuroblastomas. ProNPY/NPY processing generated molecular forms of NPY with known differences in NPY-receptor selectivity, implicating a potential for in vivo modulation of NPY-like effects in tumour tissue.

Metal-bone interactions.

Recent studies indicate that lead and cadmium may exert both direct and indirect actions on bone turnover, indirectly via kidney dysfunction, and directly on osteoblast and osteoclast function. Increased blood lead concentrations, most likely as a result of an increased bone turnover, have been detected in pregnant, lactating, and menopausal women. Lead exposure has also been negatively associated with children's growth in stature. Both lead and cadmium are nephrotoxic and can disturb vitamin D metabolism. Cadmium has been shown to induce kidney damage and osteoporosis/osteomalacia at long-term high-level exposure. A negative association between cadmium dose and bone mass has recently been detected in both occupationally and environmentally exposed people at relatively low cadmium exposure.

Evaluation of kits for measurement of the soluble transferrin receptor.

Three commercially available kits for determination of the soluble serum transferrin receptor (sTfR), R&D Systems, UK, Ramco Laboratories, USA and Orion, Finland were compared with respect to practicability, comparability and ability to discriminate between iron deficient and non-iron deficient subjects. Serum samples representing different concentrations of sTfR were tested. The three kits involved virtually the same laboratory procedures except for a predilution step for Ramco. Both the absolute amounts and the units (mg/L and nmol/L) differed among the kits, emphasizing the need for internationally accepted reference material and comparable units. The correlation coefficients were 0.974 (Ramco and R&D), 0.769 (R&D and Orion) and 0.759 (Ramco and Orion), indicating a lower comparability for Orion compared to the other two kits. The differences between the kits may be attributed to uncertainties in the reference intervals and to variations in kit format. This may have implications for studies of the usefulness of sTfR as a marker of iron deficiency.

Serum transferrin receptor: a specific marker of iron deficiency in pregnancy.

BACKGROUND: Current markers of iron deficiency tend to be less reliable in pregnancy. OBJECTIVE: Our aim was to study the usefulness of soluble serum transferrin receptor (sTfR) as a marker for iron deficiency during early and late gestation and to define iron status in 254 pregnant Swedish women. DESIGN: We performed a cross-sectional and longitudinal evaluation of sTfR in comparison with concentrations of serum ferritin and hemoglobin in blood collected around gestational weeks 11 and 36. RESULTS: The specificity of sTfR was 100%. The sensitivity in relation to both anemia and depleted iron stores was approximately 70%, but this figure is less reliable because of few samples. sTfR in early pregnancy was low: 11% of women had a value below the reference interval. sTfR increased significantly from early to late pregnancy even in the group of women with persisting iron stores. In late pregnancy, 14% of women developed tissue iron deficiency and 5% had iron deficiency according to a combination of all 3 markers. CONCLUSIONS: sTfR seems to be a specific and sensitive marker of iron deficiency in pregnancy and may have advantages over serum ferritin and hemoglobin. The low sTfR concentration in early gestation seems to be caused by reduced erythropoiesis, whereas the increase from early to late pregnancy reflects increased erythropoiesis, and in case of iron deficiency, also tissue iron deficiency. Further studies are needed to verify whether decreased erythropoiesis reduces the possibility of detecting iron deficiency during early gestation by sTfR.

Levels and molecular forms of chromogranins in human childhood neuroblastomas and ganglioneuromas.

The chromogranins are a class of acidic proteins found in large secretory granules of neuroendocrine tissues and tumors derived from them. We measured the relative amounts and characterized the molecular forms of two members of this family, i.e. chromogranin A and secretogranin II, in 14 neuroblastomas and five ganglioneuromas. In all the tumors investigated significant amounts of chromogranin A and secretogranin II were found. Neuroblastomas contained two times and ganglioneuromas 45 times more secretogranin II compared to chromogranin A. Both proteins were processed in these tumors to a great extent to smaller peptides, only limited amounts of intact chromogranin A or secretogranin II were present. In general, proteolytic processing of secretogranin II to the small neuropeptide secretoneurin was more complete than that of chromogranin A to the peptide GE-25. Proteolytic processing of both chromogranins as well as the total amounts of these proteins were unrelated to tumor staging.

Standardization of HPLC measurements of pyridinium crosslinks.

Measurement of the total amount of the two crosslinks, pyridinoline and deoxypyridinoline, can be made simultaneously by high pressure liquid chromatography preceded by an acidic hydrolysis of the sample and an extraction on cellulose columns. To compensate for the losses and variation in the extraction procedure and increase precision and accuracy a suitable internal standard should be used. A simple procedure to isolate a compound to be used as an internal standard is discussed. With this compound a total, long term coefficient of variation of 7% and 5% for the two crosslinks is achieved. The present inter-laboratory variation reflected by the big differences in reference intervals is probably due to the presence of several different ways to purify and quantify crosslinks to be used as calibrators. Recently the molar absorptivity for both pyridinoline and deoxypyridinoline in the ultra violet range has been defined. This may be the first step towards the creation of a primary international reference material.

Somatostatin in neuroblastoma and ganglioneuroma.

Neuroblastoma, a childhood tumour of the sympathetic nervous system, may in some cases differentiate to a benign ganglioneuroma or regress due to apoptosis. Somatostatin may inhibit neuroblastoma growth and induce apoptosis in vitro and was therefore investigated. Using a radioimmunoassay, we found that all ganglioneuromas contained high somatostatin concentrations (> 16 pmol/g), significantly higher than neuroblastomas (n = 117, median 2.8 pmol/g), healthy adrenals, Wilms' tumours, phaeochromocytomas and other neuroendocrine tumours (P < 0.001). Neuroblastomas contained more somatostatin than control tumours (P < 0.001-0.05). Neuroblastomas amplified for the MYCN oncogene contained less somatostatin than non-amplified tumours (1.2 pmol/g versus 4.0 pmol/g, respectively; P = 0.026). In a clinically unfavourable neuroblastoma subset (age > 12 months, stage 3 or 4) 16 children with high concentrations of somatostatin in primary tumours had a better prognosis than 23 with low somatostatin (46.7% versus 0% survival at 5 years, P < 0.005). Scintigraphy using 111In-pentetreotide identified tumours expressing high-affinity somatostatin receptors in vivo. However, no significant correlation was found between somatostatin receptor expression and peptide content in 15 tumours. Similarly, human SH-SY5Y neuroblastoma xenografts grown in nude rats showed low somatostatin concentrations, but were positive for somatostatin receptor scintigraphy. Treatment of these rats with the somatostatin analogue octreotide seemed to upregulate in vivo receptor expression of somatostatin and vasoactive intestinal peptide more effectively than 13-cis retinoic acid. In conclusion, somatostatin in neuroblastoma is associated with differentiation to benign ganglioneuromas in vivo and favourable outcome in advanced tumours. Furthermore, somatostatin receptor scintigraphy may identify tumours with high-affinity receptors in children that might benefit from targeted therapy using synthetic somatostatin analogues.

Concurrent analysis of neuropeptides and biogenic amines in brain tissue of rats treated with electroconvulsive stimuli.

We developed a method for measuring neuropeptides and monoamines in the same rat brain tissue and applied this method to study the effects of electroconvulsive stimuli (ECS) on these compounds. Rats were treated with repeated ECS or sham ECS. After sacrifice by focused microwave irradiation, brains were dissected and immediately frozen. The tissues were extracted in acetic acid. After lyophilization the samples were reconstituted in phosphate buffer and divided in three fractions: (1) was further purified on a cation-exchange column before catecholamines were measured on a high-performance liquid chromatography (HPLC) system, (2) for measuring serotonin on the HPLC system, (3) for measuring peptide concentrations by specific radioimmunoassays. Confirming our previous findings, ECS significantly increased neuropeptide Y-like immunoreactivity (-LI) in hippocampus and frontal cortex and neurokinin A-LI in the hippocampus, while no changes in substance P- and neurotensin-LI were detected. New findings were a decrease in noradrenaline concentrations in the frontal and occipital cortex and hippocampus, an increase in dopamine concentrations in the frontal and occipital cortex and no serotonin change. In summary, we have developed methods to measure both peptides and monoamines in the same brain tissue specimens, and have shown that ECS leads to changes in both neuropeptides and classical neurotransmitters in distinct brain regions.

Lipoprotein(a): levels in a Swedish population in relation to other lipid parameters and in comparison with a male Sri Lankan population.

OBJECTIVE: To evaluate differences in Lipoprotein (a) [Lp(a)] concentrations between a Swedish and Sri Lankan population. METHODS: The distribution of Lp(a) and its relation to other lipid parameters, measured with an automated turbidimetric method, in 4646 Swedes (1944 females and 2702 males) undergoing health screening and 757 randomly selected Sri Lankan males (667 non-CHD and 80 CHD subjects) was evaluated. RESULTS: The distribution was highly skewed towards low values in both the Swedish population and the Sri Lankan male population. The Swedish population had a median of 0.16 g/L (reported as total mass) whereas the Sri Lankan population median of 0.06 g/L was much lower. For the Swedes, there was a small significant difference of 0.03 g/L between the sexes (F < M; p < 0.001) and Lp(a) was significantly higher in subjects > 50 years of age in both sexes (p < 0.002(F); p < 0.02(M)). 29% had Lp(a) values > 0.30 g/L. In the Sri Lankan males population Lp(a) was also significantly higher in subjects > 50 years of age (p < 0.009) but only 7% had an Lp(a) concentration of > 0.30 g/L. In the CHD subgroup, though not significant, subjects > 50 years of age had a lower Lp(a) concentration, indicating that Lp(a) may be a more significant risk factor in younger subjects. Both the Swedish female and male hypercholesterolemic subgroups had significantly higher Lp(a) concentrations than normolipemic subgroups and the male hypertriglyceridemic subgroups significantly lower Lp(a) concentrations than normolipemic. Great differences in Lp(a) levels are thus found between the two populations. The differences are similar in normolipemic subjects and probably they reflect mainly genetic differences. Lipid/lipoprotein concentrations were also found to differ. It is being investigated if this reflects differences in CHD prevalence. CONCLUSION: Our data support the importance of including Lp(a) measurements when assessing the risk profile for premature development of CHD in the individual patient.

Somatostatin and vasoactive intestinal peptide (VIP) in neuroblastoma and ganglioneuroma: chromatographic characterisation and release during surgery.

Neuroblastomas and ganglioneuromas frequently produce somatostatin (SOM) and vasoactive intestinal peptide (VIP), and elevated concentrations in tumour tissue are associated with favourable outcome. Both somatostatin and VIP have been shown to have an autocrine effect on tumour growth and differentiation in vitro, and VIP may cause clinical symptoms when released systemically. Using gel-permeation chromatography and specific radioimmunoassays, we further characterised somatostatin-like immunoreactivity (SOM-LI) and VIP-like immunoreactivity (VIP-LI) in neuroblastoma and ganglioneuroma tumour tissue. The major part of SOM-LI and VIP-LI in both neuroblastoma and ganglioneuroma represents the biologically active forms SOM-28, SOM-14 and VIP-2, respectively. 21 children with neuroblastoma and ganglioneuroma were monitored with serial plasma samples during surgery. In 8 children with measurable concentrations of SOM-LI, all showed increased concentrations during tumour manipulation (P = 0.004) that subsequently decreased below preoperative levels in all but one case (P = 0.06). The only child presenting with diarrhoea showed the highest preoperative plasma VIP-LI in the study (54 pmol/l). 2 children with increased concentrations of VIP-LI preoperatively showed a rapid decrease after surgical tumour removal. These findings indicate a systemic release from the tumours. It is concluded that plasma and tumour tissue from children with neuroblastoma and ganglioneuroma contain biologically active molecular forms of somatostatin and vasoactive intestinal peptide. These peptides may bear significance both for specific symptoms in certain patients as well as influencing tumour growth and differentiation in vivo.

Pancreastatin immunoreactivity in favourable childhood neuroblastoma and ganglioneuroma.

Neuroblastoma and its benign counterpart, ganglioneuroma, are tumours of the sympathetic nervous system, and known to produce and release various regulatory peptides. In this study, pancreastatin, a 52 amino acid regulatory peptide derived from chromogranin A, was analysed in plasma and tumour tissue from 15 children with neuroblastoma and one with ganglioneuroma. Detectable pancreastatin immunoreactivity (> 1.9 pmol/l) was found in plasma in 13 of 15 children with highest concentrations in samples from children with favourable outcome (P < 0.05). In tumour tissue, non-metastatic tumours showed higher concentrations of pancreastatin immunoreactivity (P < 0.05). However, the highest concentrations were detected in tumours from children with favourable prognosis, regardless of clinical stage at presentation (P < 0.01). Serial plasma samples from one child with neuroblastoma and one with ganglioneuroma were investigated and showed significant systemic release of pancreastatin immunoreactivity during surgical manipulation of tumours with high pancreastatin concentrations. It is concluded that pancreastatin immunoreactivity may be detected in plasma samples and tumour extracts from children with neuroblastoma and ganglioneuroma. Systemic release during surgery implied tumour origin of elevated plasma pancreastatin. Furthermore, higher pancreastatin concentrations correlate with tumour differentiation, localised clinical stage and a favourable outcome for children with these tumours. It is suggested that pancreastatin in plasma and tumour tissue may be utilised as a marker indicating favourable tumour behaviour.