Blood carnitine concentrations are increased after surgical stress.

Elevated plasma carnitine concentrations are demonstrated in surgically stressed and partially hepatectomized rats. The response to surgical stress was observed only when the rats were fed, suggesting that exogenous sources of carnitine may be required for maintaining tissue carnitine concentrations during stress. The results are discussed with respect to the changes in fatty acid metabolism which are associated with these conditions.

Effects of surgical stress on the response of hepatic carnitine metabolism to 48 h starvation in the rat.

Rats subjected to laparotomy and handling of the liver were starved for 48 h, starting either immediately after surgery or 48 h later. Surgery enhanced the rise in plasma non-esterified fatty acid concentrations after starvation without affecting the responses of blood or liver ketone bodies. Thus in surgically stressed rats, blood and liver ketone body concentrations were inappropriately low for the blood fatty acid concentrations. In the control rats, starvation increased hepatic carnitine concentrations, mainly through increases in short-chain acylcarnitine. Surgical stress decreased or abolished these increases. This may possibly contribute to the blunted ketonaemic response observed after surgery.

Liver carnitine metabolism after partial hepatectomy in the rat. Effects of nutritional status and inhibition of carnitine palmitoyltransferase.

This study examined the effects of partial hepatectomy on hepatic carnitine and acylcarnitine concentrations in fed or 24 h-starved partially hepatectomized (PH) or sham-operated (SO) rats at 1 or 4 days after surgery. The ratio of free to esterified carnitine was low in fed PH rats at day 1: the low ratio was increased to the SO value when mitochondrial fat oxidation was inhibited by 2-tetradecylglycidate. Starvation (24 h) increased plasma [non-esterified fatty acid] in PH or SO rats, the increases being greater at day 1 than at day 4. Hepatic [long-chain acylcarnitine] were also increased. These latter increases were a consequence of increased mitochondrial fat oxidation since they were not observed in PH or SO rats treated with 2-tetradecylglycidate. Whereas the starvation-induced increase in long-chain acylcarnitine was associated with increased [ketone body] in livers of SO rats at both day 1 and day 4 after surgery, [ketone body] was inappropriately low for the steady-state long-chain [acylcarnitine] in livers of PH rats at the first post-operative day. This was not a consequence of a decrease in [total carnitine] in the liver. The results are discussed with reference to the role of the liver in determining the relative proportions of the fat fuels available for extrahepatic tissues and the effects of liver cell proliferation on hepatic triacylglycerol metabolism.

Transcription of the prorenin gene in normal and diseased breast.

The angiotensin II type 1 (AT1) receptor is present in a wide variety of human and animal tissues, and is particularly abundant in epithelial cells. Because of this, and because it is known that tissue renin angiotensin systems (RASs) exist that have specific local functions, we investigated the expression and localisation of components of the RAS in normal and diseased breast tissue. Using a monoclonal antibody to the AT1 receptor, immunocytochemistry confirmed that the AT1 receptor was characteristically distributed in ductal epithelial cells in both normal and malignant tissue, and in most, although not all, cells in invasive tumours. Transcription of prorenin mRNA was studied by in situ hybridisation, using a DIG-ddUTP tail-labelled probe specific for the human prorenin gene. In normal tissue, and in cases of ductal carcinoma in situ, prorenin mRNA was distributed in myoepithelial cells and in a band of connective tissue cells completely surrounding the AT1-containing ductal epithelial cells. This prorenin transcribing tissue was disrupted and attenuated in invasive tumours, and in some of these, prorenin mRNA transcription could not be detected at all. Functions ascribed to the tissue RASs include regulation of mitosis and tissue modelling, as well as fluid and electrolyte transport. The results presented here strongly suggest the possibility that a tissue RAS may also be present in the breast, closely coupled to the provision of angiotensin II to the AT1 receptors in ductal epithelial cells. This mechanism is disrupted in cancer.

The nature and significance of multiple isoforms of the oestrogen receptor in breast tumours.

Oestrogen receptors (ERs) in breast tumours are highly heterogeneous. In previous studies we have shown that at least four isoforms may exist. These migrate in isoelectric focusing (IEF) gels to isoelectric points (pI values) 6.1, 6.3, 6.6 and 6.8. Of these the first (pI 6.1) corresponds to the 8S isoform as detected by sucrose gradient fractionation, while the others all sediment at 4S. In a series of 66 breast tumours it was found that those at pI 6.3 and pI 6.8 were significantly correlated with the presence of progesterone receptors. To characterize the isoforms more fully, ER isoforms labelled by [3H]oestradiol binding were fractionated by IEF. The results were compared with those obtained after sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotting using the H222 anti-ER monoclonal antibody. In other experiments, tumour ER isoforms were covalently labelled with [ring-3H] tamoxifen aziridine and separated by IEF. The individual isoforms were electroeluted from the IEF gel and further analysed by SDS-PAGE and non-denaturing PAGE. In summary, the evidence shows that the isoforms of pI values 6.3, 6.8 and 6.6 have molecular masses of 50, 65 and 70 kDa respectively. In addition, all three of these isoforms, i.e. the pI 6.3, 6.8 and 6.6 isoforms, could form dimers. We conclude that the three isoforms sedimenting at 4S have the capacity to form dimers and thus may have the potential for binding to oestrogen response elements in the genome.

Methods for detecting apoptosis in thyroid diseases.

Over the last few years, the importance of apoptosis in determining the fate of thyrocytes in autoimmune thyroid disease has been the topic of intense investigation. It is now clear that thyrocytes from patients with Hashimoto's thyroiditis are destroyed as a result of an apoptotic process. However, there is no general consensus on whether the intrathyroidal lymphocytes or the thyrocytes themselves are responsible for their death. The use of a wide range of techniques has contributed to the assessment of this process both in situ on thyroid sections and in vitro on thyroid cell preparations. The apoptosis field of research is rapidly evolving and as the pathways to cell death become unravelled, novel methods will emerge. As each technique offers some advantage, it is critical to know the most suitable method for a specific study. Equally, each method also has intrinsic limitations. Thus, to achieve reliable results, it is necessary to use more than one technique per study. In addition, techniques related to the measurement of the expression of pro-apoptotic and anti-apoptotic genes have been contributing to the study of the susceptibility of the cells to apoptosis and/or to their ability to kill themselves or neighbouring cells. In this review we will focus on the most relevant techniques.

Significance of the 8S complex in oestrogen receptor recognition.

Previous studies from this laboratory have drawn attention to discrepancies between enzyme-linked immunoassay (EIA) and steroid binding assay (SBA) in the analysis of oestrogen receptors (ER) in breast tumours. In particular, EIA values were at least 3-fold higher than SBA values in tumours which also contained progesterone receptors (PR) when both 4 and 8S isoforms of the ER are present. To test the influence of these isoforms on the two assay systems, the relationships between the oestrogen receptor (ER) values obtained by EIA and SBA were examined in tumour cytosols prepared in the presence of molybdate and protease inhibitors to prevent degradation of the 8S form. Under these conditions, values for ER were the same by EIA and SBA (slope = 1.08, r = 0.886, n = 25) when EIA was performed using low salt phosphate buffer instead of the high salt-containing Abbott-diluent provided with the kit. However, after disruption of the 8S assembly using high K+ concentration, the slope of the regression was 6.37, r = 0.865, n = 25. Using ER from rat uterus, EIA was also performed on intact 8S oligomers, on 8S ER dissociated by high salt, and on glycerol density gradient-fractionated 4S ER. The identity of the ER oligomers and components was confirmed by glycerol density gradient fractionation, and by isoelectric focussing. For the 4S ER, EIA gave similar values whether using low or high salt phosphate buffer. However EIA values for the 8S form were 2-fold higher when the supplied diluent was used than when the assay was performed in low salt buffer. The amount of oestradiol which could be extracted was affected by the different conditions used. Addition of KCl or trypsin to disrupt the 8S ER caused an increase in the amount of extractable oestradiol compared with control values (control = 52 +/- 4.0, high KCl = 91 +/- 4.4, trypsin = 152 +/- 7.5, pg oestradiol/mg protein). We conclude that further antibody binding sites are revealed from the 8S ER form after its disaggregation by high salt. The steroid extraction data also suggests the possibility that tightly bound steroid is retained within the 8S ER structure, and released by 8S disaggregation. Both of these may contribute to the differences between EIA and SBA values.

Progesterone receptor induction by danazol in cultured cancer cells and the rat uterus.

We have previously reported that clinical trials relating to the use of danazol in the management of benign breast disease show a positive correlation between favourable clinical response and an induction of progesterone receptors in the affected tissue which is maintained for a period of at least 6 months subsequent to the cessation of treatment. Further studies designed at elucidating more clearly the actions of danazol at the cellular and molecular levels have confirmed that progesterone receptors are down-regulated by short-term progestin action at the level of the mRNA transcript, but that danazol is subsequently able to produce an enhanced cellular response, inducing progesterone receptors in the presence of oestrogenic agents. Uteri from danazol-treated rats showed a doubling of progesterone receptor concentrations compared with the control uteri. In the mammary cancer cell line T-47D, cells treated with danazol had increased progesterone receptor concentrations of 558.4 +/- 32.0 compared with 152.6 +/- 7.0 fmol/mg protein in the control cells. In both cases, these inductions were observed following a period of progesterone receptor suppression. Short-term molecular studies on T-47D cells indicated that progesterone and danazol initially inhibit mRNA transcription, but that 24 h after treatment an induction is observed. This is especially marked in the danazol-treated cells.

Early time course of the acute phase protein response in man.

The rate at which the acute phase protein response occurred after both major and minor surgery was explored. Increases in the plasma concentration of C-reactive protein (CRP), alpha-1-acid glycoprotein (alpha 1 AG) and fibrinogen were not detected until 6-8 h after the initial incision. The peak concentration of CRP occurred at 48 h and that of fibrinogen at 96 h; alpha 1 AG concentrations rose rapidly until 48 h followed by little change until about 120 h. Although there was widespread variation in the concentrations of individual proteins in patients, severity of injury did not seem to have a significant effect on the time course of the change. Plasma cortisol concentration and the total white blood cell count (WBC) reached their peaks before the acute phase proteins, cortisol at 6 h and WBC at 12 h.

Base deficit after major trauma directly relates to neutrophil CD11b expression: a proposed mechanism of shock-induced organ injury.

OBJECTIVE: To determine whether expression of neutrophil integrin receptors is related to the degree of post-traumatic shock. DESIGN: Data were collected prospectively on patients with major trauma admitted to the surgical intensive care unit. SETTING: Denver General Hospital, Colorado. PATIENTS AND PARTICIPANTS: 17 severely injured adults. MEASUREMENTS AND RESULTS: The mean fluorescence intensity and per cent positive of neutrophil integrin receptors CD11 b, CD18 and CD11 a, and systolic blood pressure, blood transfusion, lactate and base deficit as indices of shock. CD11 b expression on circulating neutrophils was increased 6 and 12 h after trauma. After correcting for the other shock indices, base deficit predicted CD11 b expression at 12 h. CD11 b expression was negatively correlated with the circulating neutrophil count. CONCLUSIONS: The degree of metabolic acidosis after trauma correlates directly with CD11 b receptor expression on circulating neutrophils. This relation may be the mechanism whereby post-traumatic shock results in neutrophil sequestration and neutrophil-mediated organ injury and failure.

The beta-2 microglobulin urine-to-serum ratio: an early marker of renal dysfunction in primary hyperparathyroidism.

Human beta-2 microglobulin (beta-2 M) is released into body fluids as a result of cell turnover, excreted by the kidney, and catabolized in the proximal tubule. Urinary excretion rates constitute a sensitive index of renal tubular function. The beta-2 M urine-to-serum ratio was measured in 25 patients with primary hyperparathyroidism (15 preoperative and 10 treated conservatively) in addition to 20 age- and sex-matched control subjects. The ratio was found to be significantly higher in both the operative and the conservatively managed groups compared to controls (p less than 0.05, Mann-Whitney U test). After surgical excision of a single parathyroid adenoma in the 15 operative cases, the beta-2 M urine-to-serum ratio fell to normal limits. These preliminary findings indicate that the urine-to-serum beta-2 M ratio may be of value in detecting change in renal function in asymptomatic patients with hyperparathyroidism. Further studies are indicated to establish whether these subtle changes are associated with long-term morbidity.

Death of the autoimmune thyrocyte: is it pushed or does it jump?

Programmed cell death or apoptosis is central both in physiology during development and in disease. The mechanism of apoptosis is under the control of antiapoptotic survival genes of the Bcl-2 family and proapoptotic death receptors of the TNF superfamily (Fas, TNFR, TRAILR). Following death signal, the death receptor binds to its own receptor and initiates, through binding of adaptors, a cascade of events mediated by the autoproteolytic activation of specific enzymes called caspases. This enzyme activation is ultimately responsible for the dissembly of basic nuclear and cytoplasmic cell structures leading to cell death. In certain cell systems, antiapoptotic genes of the Bcl-2 family prevent the proapoptotic pathway. One of their roles is to maintain mitochondrial function integrity. In autoimmune destructive thyroiditis high levels of apoptosis have been demonstrated particularly within the destructed follicles near the infiltrated areas in comparison to Graves' disease and non autoimmune glands. In Hashimoto's thyroiditis Fas expression has been found increased on thyrocytes and in vitro can be modulated by proinflammatory cytokines. FasL expression on thyrocytes remains controversial. Thyroid cells from Graves' disease and multinodular glands are known to kill Fas expressing target cells although Hashimoto's thyrocytes are not efficient effector cells. Intrathyroidal lymphocytes from Hashimoto's thyroids maintain functional killer activity. These findings would suggest that intrathyroidal lymphocytes could be responsible for thyrocyte death in vivo. Whether this mechanism is Fas/FasL, TRAIL/TRAILR dependent can not be confirmed as specific blocking reagents were not able to inhibit cell induced death. In Hashimoto's thyroiditis an impairment of Bcl-2 and Bcl-X anitapoptotic genes on thyrocytes has also been detected. Bcl-X expression can be down-regulated in vitro by incubation with cytokines. These findings suggest that thyrocyte death may not exclusively be the result of specific interactions between death receptor and their ligands but it may involve simultaneous impairment of protective genes of the Bcl-2 family. Whether the impairment of the Bcl-2 family is a direct consequence of environmental stimuli or is the result of an intrinsic thyrocyte (mitochondrial?) alteration is as yet not known.

Thyrocyte targets and effectors of autoimmunity: a role for death receptors?

In Hashimoto's thyroiditis, thyrocytes die by apoptosis. Whether this is the result of impaired antiapoptotic gene expression or hyperexpression of proapoptotic signals or other mechanisms is not fully established. Following the suggestion that thyrocytes from Hashimoto's glands die by a fratricidal killing mediated by Fas/Fas ligand, we have investigated whether thyroid cells from different clinical conditions are able to kill Fas-expressing target cells. We have studied whether this effector ability was mediated by Fas/Fas ligand, perforin or other death receptors/ligands, i.e., tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)/tumor necrosis factor-related apoptosis-inducing ligand receptor (TRAIL-R). We have confirmed that thyroid preparations can kill Fas-expressing HUT78 targets through apoptosis. Cell death was only partially dependent on Fas/Fas ligand but it was trypsin-sensitive. Blocking perforin did not affect Fas-expressing target killing while caspase inhibitors had a consistent although limited effect. Thyroid cells were not sensitive to TRAIL/TRAIL-R. We have also found that both thyrocytes and lymphocytes from Graves' disease thyroids were effective at killing autologous and heterologous Fas-expressing targets. Conversely, killing of these targets could be shown only with lymphocytes (but not with thyrocytes) from Hashimoto's glands. In Hashimoto's thyroiditis, thyrocytes were poorly functional while lymphocytes were able to operate as effectors. It is envisaged that thyrocyte death in Hashimoto's would result from autologous thyrocyte killing perpetrated by lymphocytes. Death receptors/ligands would appear to play a role. However, a caspase-independent mechanism may also coexist and contribute to cell death in Hashimoto's thyroiditis.

Integrin beta1 upregulation in MCF-7 breast cancer cells by angiotensin II.

AIMS: Integrins are a major family of cell adhesion molecules whose function is perturbed in tumour invasion and metastasis. Angiotensin II (A II) is well-known in the systemic control of water and electrolyte homeostasis and haemodynamics, but recent evidence points to an additional local renin-angiotensin system (RAS) with possible long-term trophic effects including carcinogenesis. METHODS: The effect of angiotensin II on MCF-7 human breast cancer cell line integrin expression was evaluated with immunocytochemistry (ICC) and immunoprecipitation (IP). RESULTS: The experiments demonstrated a 1.40 +/- 0.14-fold increase in beta, integrin expression on MCF-7 cells following treatment with A II. CONCLUSIONS: These findings report the first evidence of an association between integrins and the RAS in human breast cancer cells and suggest a novel research avenue for future anti-metastatic strategies, through the manipulation of cell adhesion mechanics, in the management of invasive human breast cancer.

The effects of infusion of amino acids alone or in combination with glucose and fat on thyroid hormone and other metabolite interactions in surgery.

The effects of infusion of amino acids alone or in combination with carbohydrate and lipid on triiodothyronine (T3) and reverse triiodothyronine (rT3) status, substrate availability and metabolism in surgically-stressed and clinically euthyroid patients were examined. The characteristic post-operative decline in T3 concentration was observed in all patient groups (p < 0.001) (8 patients in each group). However, infusion with amino acids alone retarded the decline in T3; the decrease was lower than those found in the controls and the mixed nutrition group and restoration of T3 values was incomplete, even by the sixth post-operative day. The pattern of increase in rT3 was similar in all 3 patient groups. However, the T3/rT3 ratio dropped to its lowest level on day 1 in the control group and the mixed infusion group (p < 0.001), with a complete restoration by the sixth post-operative day, whereas in the amino acids infused group the ratio was lowest on day 2 (p < 0.001) and was still significantly low by day 6 compared with the pre-operative value (p < 0.001). The findings, in the group infused with amino acids of a less prominent hyperglycaemia, with the significant slowing of T3 response and the significant correlation of rT3 with the plasma glucose, indicate a link between hyperglycaemia and the thyroid hormone response to surgical trauma. No correlations were found between thyroid hormone or urea concentrations, and the blood concentrations of free fatty acids, 3-hydroxybutyrate, or urea; or between thyroid hormone and the percentage of total urea nitrogen excretion. Results show that the changes in fat metabolism after operation are unlikely to be responsible for the changes in T3 and rT3. In conclusion, whereas the post-operative response of T3 concentration can be partially modified by the nutrition regimen employed, that of rT3 is largely related to surgical stress.

Somatic change in the estrogen receptor gene associated with altered expression of the progesterone receptor.

Using a fragment of the estrogen receptor (ER) cDNA as a probe, Southern blots of HindIII-digested genomic DNA, from breast tumours and non-cancerous tissue, revealed a 1.6 kb hybridizing fragment (1.6KbHF) in 3 out of 30 tumours, and in 31 out of 80 mastectomy samples (16 sectors from each of 5 patients), which was significantly correlated with the absence of progesterone receptor. In 4 of the patients, sectors which showed the 1.6KbHF co-existed in the same breast with sectors which did not. Neither the use of higher concentrations of the restriction enzyme, not high salt extraction of DNA changed the abundance of the 1.6KbHF relative to other bands. Similar studies on EcoRI digests of the same DNA samples failed to show any variants. As a further control, hybridization of the same Southern blots of HindIII-digested DNA, with a cytochrome P450IIA3 cDNA, also failed to show variants in these samples. It is concluded that the presence of the 1.6KbHF represents a somatic change in the nucleus of these samples which may have functional importance.

A potential diagnostic application of magnetization transfer contrast: an in vitro NMR study of excised human thyroid tissues.

A series of freshly excised thyroid tissues was analysed using a nuclear magnetic resonance spectrometer and then subjected to routine histo-pathology examination. Whilst simple T1 values for normal tissue and goitre are not significantly different, the degree of intra-subject T1 and T1s variability is shown to be an indicator of benign thyroid disease. Using data collected from an inversion-recovery sequence performed with and without magnetization transfer, a magnetization transfer rate constant was calculated for each tissue sample. These data suggest that this parameter may provide in vivo discrimination between follicular cancer and follicular adenoma.

The magnetization transfer characteristics of human breast tissues: an in vitro NMR study.

A series of freshly excised human breast tissues was analysed using a nuclear magnetic resonance spectrometer and then subjected to routine histopathology examination. Tissues comprised normal parenchymal, adipose, fibrocystic, fibroadenoma and malignant types. An inversion-recovery sequence performed both with and without magnetization transfer allowed T1, T1s, M0 and Ms values to be obtained. From this information, the magnetization transfer rate constant, K, was calculated for each tissue sample. These data show that T1s provided greater discrimination between neoplasic and normal tissues than did T1. However, neither T1s nor K values provided a means of discriminating between benign and malignant disease.

Measurement of total body nitrogen and oxygen by irradiation with cyclotron neutrons and 'delayed' gamma ray counting.

Measurement of total body nitrogen is assuming increasing importance in the nutritional evaluation of seriously ill patients. Nitrogen has been previously measured either by counting (i) the annihilation radiation from 13N immediately after neutron irradiation with 14 MeV neutrons or (ii) the 'prompt' gamma rays from thermal neutron capture by 14N during irradiation with 14 MeV neutrons or with those produced by isotopic sources or a cyclotron. The present work describes studies into the feasibility of measuring 13N produced by irradiation with a neutron beam from the MRC Cyclotron. A complication of this method is that 13N is also produced in a reaction with 16O. Direct measurement of oxygen by use of the reactions 16O(n, p)16N or 16O(n, 2n)15O enables this interference to be estimated. The former reaction is possible with both 14 meV and cyclotron-produced neutrons but the 7.1 s half-life of 16N requires detectors to be placed in or very close to the irradiation site. In our particular circumstances this is not possible but the more energetic cyclotron neutron spectrum allows the production of 15O which has a half-life of 2.05 min and can be measured in a remote whole-body counter. A disadvantage with the cyclotron beam, in comparison with 14 MeV neutrons, is that a higher dose is required for similar accuracy. A reproducibility of about 4% is obtained with a dose equivalent of 0.01 Sv.

Effects of hyperthyroidism on stimulation of [1-14C]oleate oxidation to 14CO2 in isolated hepatocytes from fed rats by the catecholamines, vasopressin, and angiotensin II.

Possible effects of adrenaline, noradrenaline, vasopressin, and angiotensin II to increase 14CO2 production from [1-14C]oleate were examined in hepatocytes from fed L-triiodothyronine (T3)-treated or control rats. Rates of 14CO2 production were decreased and rates of ketogenesis increased in hepatocytes from T3-treated rats. These changes were accompanied by a marked shift of the 3-hydroxybutyrate:acetoacetate concentration ratio towards acetoacetate. Rates of glucose and lactate release were decreased. Whereas the Ca2+-mobilizing hormones increased 14CO2 production from [1-14C]oleate by 64-84% with hepatocytes from control rats, they increased 14CO2 production from [1-14C]oleate by on 24-32% with hepatocytes from T3-treated rats. The magnitude of the response to the Ca2+-mobilizing hormones in hepatocytes from T3-treated rats was increased by the addition of 3-mercaptopicolinate, an inhibitor of phosphoenolpyruvate carboxykinase, to the incubation medium (increases of 52-88%). In the presence of 3-mercaptopicolinate, the 3-hydroxybutyrate:acetoacetate concentration ratio in hepatocytes from fed, T3-treated rats was similar to that in hepatocytes from control rats in the absence of 3-mercaptopicolinate. The results demonstrate that hyperthyroidism per se does not lead to a loss of sensitivity, in terms of oleate oxidation, either to the catecholamines or to vasopressin and angiotensin II. The impaired ability of hepatocytes from T3-treated rats to respond to these hormones is a consequence of decreased net glycolytic flux or a more oxidized mitochondrial redox state.