Contribution of local regeneration of glucocorticoids to tissue steroid pools.

11ß-Hydroxysteroid dehydrogenase 1 (11ßHSD1) is a drug target to attenuate adverse effects of chronic glucocorticoid excess. It catalyses intracellular regeneration of active glucocorticoids in tissues including brain, liver and adipose tissue (coupled to hexose-6-phosphate dehydrogenase, H6PDH). 11ßHSD1 activity in individual tissues is thought to contribute significantly to glucocorticoid levels at those sites, but its local contribution vs glucocorticoid delivery via the circulation is unknown. Here, we hypothesised that hepatic 11ßHSD1 would contribute significantly to the circulating pool. This was studied in mice with Cre-mediated disruption of Hsd11b1 in liver (Alac-Cre) vs adipose tissue (aP2-Cre) or whole-body disruption of H6pdh. Regeneration of [9,12,12-2H3]-cortisol (d3F) from [9,12,12-2H3]-cortisone (d3E), measuring 11ßHSD1 reductase activity was assessed at steady state following infusion of [9,11,12,12-2H4]-cortisol (d4F) in male mice. Concentrations of steroids in plasma and amounts in liver, adipose tissue and brain were measured using mass spectrometry interfaced with matrix-assisted laser desorption ionisation or liquid chromatography. Amounts of d3F were higher in liver, compared with brain and adipose tissue. Rates of appearance of d3F were ~6-fold slower in H6pdh-/- mice, showing the importance for whole-body 11ßHSD1 reductase activity. Disruption of liver 11ßHSD1 reduced the amounts of d3F in liver (by ~36%), without changes elsewhere. In contrast disruption of 11ßHSD1 in adipose tissue reduced rates of appearance of circulating d3F (by ~67%) and also reduced regenerated of d3F in liver and brain (both by ~30%). Thus, the contribution of hepatic 11ßHSD1 to circulating glucocorticoid levels and amounts in other tissues is less than that of adipose tissue.

11ß-HSD1 plays a critical role in trabecular bone loss associated with systemic glucocorticoid therapy.

BACKGROUND: Despite their efficacy in the treatment of chronic inflammation, the prolonged application of therapeutic glucocorticoids (GCs) is limited by significant systemic side effects including glucocorticoid-induced osteoporosis (GIOP). 11ß-Hydroxysteroid dehydrogenase type 1 (11ß-HSD1) is a bi-directional enzyme that primarily activates GCs in vivo, regulating tissue-specific exposure to active GC. We aimed to determine the contribution of 11ß-HSD1 to GIOP. METHODS: Wild type (WT) and 11ß-HSD1 knockout (KO) mice were treated with corticosterone (100 µg/ml, 0.66% ethanol) or vehicle (0.66% ethanol) in drinking water over 4 weeks (six animals per group). Bone parameters were assessed by micro-CT, sub-micron absorption tomography and serum markers of bone metabolism. Osteoblast and osteoclast gene expression was assessed by quantitative RT-PCR. RESULTS: Wild type mice receiving corticosterone developed marked trabecular bone loss with reduced bone volume to tissue volume (BV/TV), trabecular thickness (Tb.Th) and trabecular number (Tb.N). Histomorphometric analysis revealed a dramatic reduction in osteoblast numbers. This was matched by a significant reduction in the serum marker of osteoblast bone formation P1NP and gene expression of the osteoblast markers Alp and Bglap. In contrast, 11ß-HSD1 KO mice receiving corticosterone demonstrated almost complete protection from trabecular bone loss, with partial protection from the decrease in osteoblast numbers and markers of bone formation relative to WT counterparts receiving corticosterone. CONCLUSIONS: This study demonstrates that 11ß-HSD1 plays a critical role in GIOP, mediating GC suppression of anabolic bone formation and reduced bone volume secondary to a decrease in osteoblast numbers. This raises the intriguing possibility that therapeutic inhibitors of 11ß-HSD1 may be effective in preventing GIOP in patients receiving therapeutic steroids.

Therapeutic glucocorticoids prevent bone loss but drive muscle wasting when administered in chronic polyarthritis.

BACKGROUND: Patients with rheumatoid arthritis (RA) experience extra-articular manifestations including osteoporosis and muscle wasting, which closely associate with severity of disease. Whilst therapeutic glucocorticoids (GCs) reduce inflammation in RA, their actions on muscle and bone metabolism in the context of chronic inflammation remain unclear. We utilised the TNF-tg model of chronic polyarthritis to ascertain the impact of therapeutic GCs on bone and muscle homeostasis in the context of systemic inflammation. METHODS: TNF-tg and wild-type (WT) animals received either vehicle or the GC corticosterone (100 µg/ml) in drinking water at onset of arthritis. Arthritis severity and clinical parameters were measured, serum collected for ELISA and muscle and bone biopsies collected for µCT, histology and mRNA analysis. In vivo findings were examined in primary cultures of osteoblasts, osteoclasts and myotubes. RESULTS: TNF-tg mice receiving GCs showed protection from inflammatory bone loss, characterised by a reduction in serum markers of bone resorption, osteoclast numbers and osteoclast activity. In contrast, muscle wasting was markedly increased in WT and TNF-tg animals receiving GCs, independently of inflammation. This was characterised by a reduction in muscle weight and fibre size, and an induction in anti-anabolic and catabolic signalling. CONCLUSIONS: This study demonstrates that when given in early onset chronic polyarthritis, oral GCs partially protect against inflammatory bone loss, but induce marked muscle wasting. These results suggest that in patients with inflammatory arthritis receiving GCs, the development of interventions to manage deleterious side effects in muscle should be prioritised.

Novel H6PDH mutations in two girls with premature adrenarche: 'apparent' and 'true' CRD can be differentiated by urinary steroid profiling.

CONTEXT: Inactivating mutations in the enzyme hexose-6-phosphate dehydrogenase (H6PDH, encoded by H6PD) cause apparent cortisone reductase deficiency (ACRD). H6PDH generates cofactor NADPH for 11ß-hydroxysteroid dehydrogenase type 1 (11ß-HSD1, encoded by HSD11B1) oxo-reductase activity, converting cortisone to cortisol. Inactivating mutations in HSD11B1 cause true cortisone reductase deficiency (CRD). Both ACRD and CRD present with hypothalamic-pituitary-adrenal (HPA) axis activation and adrenal hyperandrogenism. OBJECTIVE: To describe the clinical, biochemical and molecular characteristics of two additional female children with ACRD and to illustrate the diagnostic value of urinary steroid profiling in identifying and differentiating a total of six ACRD and four CRD cases. DESIGN: Clinical, biochemical and genetic assessment of two female patients presenting during childhood. In addition, results of urinary steroid profiling in a total of ten ACRD/CRD patients were compared to identify distinguishing characteristics. RESULTS: Case 1 was compound heterozygous for R109AfsX3 and a novel P146L missense mutation in H6PD. Case 2 was compound heterozygous for novel nonsense mutations Q325X and Y446X in H6PD. Mutant expression studies confirmed loss of H6PDH activity in both cases. Urinary steroid metabolite profiling by gas chromatography/mass spectrometry suggested ACRD in both cases. In addition, we were able to establish a steroid metabolite signature differentiating ACRD and CRD, providing a basis for genetic diagnosis and future individualised management. CONCLUSIONS: Steroid profile analysis of a 24-h urine collection provides a diagnostic method for discriminating between ACRD and CRD. This will provide a useful tool in stratifying unresolved adrenal hyperandrogenism in children with premature adrenarche and adult females with polycystic ovary syndrome (PCOS).

Should we be giving high concentration oxygen to all patients treated in an ambulance?

Oxygen is one of the most widely used drugs. It is important to recognise that oxygen administration carries risks as well as benefits. While adequate oxygen saturation of arterial blood is an important factor in tissue oxygen delivery, oxygen administration to patients with chronic obstructive pulmonary disease can lead to decompensated type II respiratory failure. In this debate, Dr Lavery makes the case that high concentration oxygen should be given to all patients treated in an ambulance, while Professor Corris argues against this position.

11beta-Hydroxysteroid dehydrogenase type 1 regulates insulin and glucagon secretion in pancreatic islets.

AIMS/HYPOTHESIS: Exposure to excess glucocorticoid is associated with pancreatic beta cell damage and decreased glucose-stimulated insulin secretion (GSIS). Inactive glucocorticoids (cortisone, 11-dehydrocorticosterone) are converted to active cortisol and corticosterone by 11beta-hydroxysteroid dehydrogenase type 1 (11beta-HSD1), which requires NADPH as cofactor, which is generated by hexose-6-phosphate dehydrogenase (H6PDH). We investigated the localisation and activity of 11beta-HSD1 within pancreatic islets, and determined its functional role in the regulation of insulin and glucagon secretion. METHODS: mRNA expression of 11beta-HSD1 (also known as HSD11B1), glucocorticoid receptor and H6PDH (also known as H6PD) in human pancreas and murine islets was examined by real-time PCR. 11beta-HSD1 protein levels were examined by immunohistochemistry and immunofluorescence. 11beta-HSD1 activity was assessed in intact tissue and isolated islets of wild-type (WT) and both 11beta-Hsd1- and H6pdh-null mice. Glucagon secretion and insulin secretion were analysed by RIA and ELISA respectively in isolated murine islets incubated with dexamethasone. RESULTS: 11beta-HSD1 co-localised with glucagon in the periphery of murine and human islets, but not with insulin or somatostatin. Dexamethasone, 11-dehydrocorticosterone and corticosterone induced a dose-dependent decrease in GSIS and glucagon secretion following low glucose stimulation. Reduction of 11beta-HSD1 activity with specific inhibitors or in experiments carried out in H6pdh-null mice reversed the effects of 11-dehydrocorticosterone, but had no effect following treatment with corticosterone. CONCLUSIONS/INTERPRETATION: Local regeneration of glucocorticoid via 11beta-HSD1 within alpha cells regulates glucagon secretion and in addition may act in a paracrine manner to limit insulin secretion from beta cells.

The corticosteroid metabolic profile of the mouse.

Data are presented on the urinary corticosteroid metabolic profile of the mouse strain 129/svJ. Through the use of GC/MS we have characterized, or tentatively identified corticosterone (Kendall's compound B) metabolites of both the 11beta-hydroxy and 11-carbonyl (compound A) series in urine. Full mass spectra of the methyloxime-trimethylether derivatives of 15 metabolites are included in the paper as an aid to other researchers in the field. Metabolites ranged in polarity from tetrahydrocorticosterone (THB) to dihydroxy-corticosterone with dominance of highly polar steroids. We found that prior to excretion corticosterone can undergo oxidation at position 11beta, reduction at position 20 and A-ring reduction. Metabolites retaining the 3-oxo-4-ene structure can be hydroxylated at position 6beta- as well as at an unidentified position, probably 16alpha-. Saturated steroids can be hydroxylated at positions 1beta-, 6alpha-, 15alpha- and 16alpha. A pair of hydroxy-20-dihydro-corticosterone metabolites (OH-DHB) were the most important excretory products accounting for about 40% of the total. One metabolite of this type was identified as 6beta-hydroxy-DHB; the other, of similar quantitative importance was probably 16alpha-hydroxy-DHB. The ratio of metabolites of corticosterone (B) to those of 11-dehydro-corticosterone (A) was greater than 9:1, considerably higher than that for the equivalent "human" ratio of 1:1 for cortisol to cortisone metabolites. Results from this study allowed the evaluation of 11beta-hydroxysteroid dehydrogenase (11beta-HSD) activity in mice with deleted glucose-6-phosphate transporter (G6PT). These mice had attenuated back-conversion of A to B resulting in an increased ratio of A-metabolites to B-metabolites [Walker EA, Ahmed A, Lavery GG, Tomlinson JW, Kim SY, Cooper MS, Stewart PM, 11beta-Hydroxysteroid dehydrogenase type 1 regulation by intracellular glucose-6-phosphate, provides evidence for a novel link between glucose metabolism and HPA axis function. J Biol Chem 2007;282:27030-6]. We believe this study is currently the most comprehensive on the urinary steroid metabolic profile of the mouse. Quantitatively less steroid is excreted in urine than in feces by this species but urine analysis is more straightforward and the hepatic metabolites are less subject to microbial degradation than if feces was analyzed.

An evaluation of protocolised weaning on the duration of mechanical ventilation.

Using a before and after study design, we compared protocolised weaning from mechanical ventilation with usual non-protocolised practice in intensive care. Outcomes (duration of mechanical ventilation, duration of intubation, intensive care stay) and complications (re-intubations, tracheostomy, mortality) were compared between baseline (Phase I) and following implementation of protocolised weaning (Phase II). Over the same period, we collected data in a second (reference) unit to monitor practice changes over time. In the intervention unit, outcomes were longer in Phase II compared with Phase I (all p < 0.005). When adjusted for admission APACHE II score and diagnostic category, only intensive care stay remained significantly longer (p = 0.002). There were significantly more tracheostomies in Phase II (p = 0.004). The reference unit demonstrated no statistically significant differences in study outcomes or complications between Phases. Protocolised weaning did not reduce the duration of mechanical ventilation and was not associated with an increased rate of re-intubation or intensive care unit mortality.

Thrombotic thrombocytopenic purpura secondary to Streptococcus.

We describe a 16 year old female who developed thrombotic thrombocytopenic purpura (TTP) following infection due to Streptococcus. Initially presenting a fever and systemic upset she progressed to develop dialysis dependent acute renal failure, seizures, thrombocytopenia and a haemolytic anaemia--the pentad of features seen in TTP. Prior to the diagnosis she was found to have unexplained and previously undescribed MRI findings of diffuse increased signal intensity in the white matter of the left cerebellar hemisphere posteriorly and also increased signal intensity in the overlying cortex. She was commenced on plasmapheresis, and her anaemia, thrombocytopenia, creatinine and LDH all fully responded. In addition, she had no further seizures following plasmapheresis and has not relapsed to date. We review both the rare association of TTP and streptococcal infection, and the neuroradiological findings described in the literature. This is only the third case report describing TTP following streptococcal infection, and only the second in the era of plasmapheresis.

Molecular basis for the apparent mineralocorticoid excess syndrome in the Oman population.

11beta-Hydroxysteroid dehydrogenase type 2 (11beta-HSD2) plays a crucial role in converting hormonally active cortisol to inactive cortisone, thereby conferring specificity upon the mineralocorticoid receptor (MR). Mutations in the gene encoding 11beta-HSD2 (HSD11B2) account for an inherited form of hypertension, the syndrome of "Apparent Mineralocorticoid Excess" (AME) where cortisol induces hypertension and hypokalaemia. We report five different mutations in the HSD11B2 gene in four families from Oman with a total of 9 affected children suffering from AME. Sequence data demonstrate the previously described L114Delta6nt mutation in exon 2 and new mutations in exon 3 (A221V), exon 5 (V322ins9nt) and for the first time in exon 1 (R74G and P75Delta1nt) of the HSD11B2 gene. These additional mutations provide further insight into AME and the function of the 11beta-HSD2 enzyme. The prevalence of monogenic forms of hypertension such as AME remains uncertain. However, our data suggests AME may be a relevant cause of hypertension in certain ethnic groups, such as the Oman population.

Association studies between microsatellite markers within the gene encoding human 11beta-hydroxysteroid dehydrogenase type 1 and body mass index, waist to hip ratio, and glucocorticoid metabolism.

Two isozymes of 11beta-hydroxysteroid dehydrogenase (11beta-HSD) interconvert active cortisol (F) and inactive cortisone (E). 11beta-HSD1 is an oxo-reductase (E to F) expressed in several glucocorticoid target tissues, including liver and adipose tissue, where it facilitates glucocorticoid-induced gluconeogenesis and adipocyte differentiation, respectively. We have isolated a full-length HSD11B1 genomic clone; the gene is more than 30 kb in length, not 9 kb in length as previously reported, principally due to a large intron 4. Two polymorphic (CA)(n) repeats have been characterized within intron 4: a CA(19) repeat 2.7 kb 3' of exon 4 and a CA(15) repeat 3 kb 5' of exon 5. The microsatellites, CA(19) and CA(15), were PCR amplified using fluorescent primers and were genotyped on an ABI 377 DNA sequencer from DNA of 413 normal individuals enrolled in the MONICA study of cardiovascular risk factors and 557 Danish men (ADIGEN study), of whom 234 were obese [body mass index (BMI), >/=31 kg/m(2) ] at draft board examination and 323 were randomly selected controls from the draftee population with BMI below 31 kg/m(2) (mean +/- SE, 21.7 +/- 0.41). Genotypic data from the normal MONICA cohort was compared with gender, 5beta-tetrahydrocortisol+5alpha-tetrahydrocortisol/tetrahydrocortisone ratio, and waist to hip (W:H) ratio. When analyzed by allele length (0, 1, or 2 short alleles) for the CA(19) marker, there was a trend toward a higher 5beta-tetrahydrocortisol+5alpha-tetrahydrocortisol/tetrahydrocortisone ratio (P = 0.058) and an increased W:H ratio (2 vs. 0.1 short; P(c) = 0.10) with overrepresentation of short alleles. The opposite was true for the CA(15) locus, with longer alleles at this locus predicting increased 11beta-HSD1 activity, particularly in females. Genotypic data from the ADIGEN case-control population was compared with clinical markers of obesity such as BMI and W:H ratio. There was no significant difference in the distribution of either microsatellite marker between lean and obese groups. Allele distributions were binomial, as seen for the MONICA cohort, and the data were split accordingly (zero, one, or two short alleles). No significant association was seen between grouped alleles and the clinical parameters. No association was observed between HSD11B1 genotype and BMI in either population. These data suggest that 11beta-HSD1 is not a major factor in explaining genetic susceptibility to obesity per se. However, weak associations between HSD11B1 genotype, increased 11beta-HSD1 activity, and W:H ratio suggest that polymorphic variability at the HSD11B1 locus may influence susceptibility to central obesity through enhanced 11beta-HSD1 activity (E to F conversion) in visceral adipose tissue.

Deliberate self-poisoning with dapsone. A case report and summary of relevant pharmacology and treatment.

We report the case of a 14-year-old girl who deliberately ingested 8-9 g of dapsone and presented with severe methaemoglobinaemia and altered mental status. Prompt treatment with repeated doses of methylene blue and organ support brought about control of the methaemoglobinaemia and averted organ failure.

The effect of air fluidised bed therapy on hypermetabolism in intensive care unit patients.

Major trauma is associated with a hypermetabolic (increased resting energy expenditure) and hypercatabolic (negative nitrogen balance) response. Studies have suggested that nursing injured patients at higher temperatures reduces the metabolic response (Ryan & Clague 1990). In this study the energy expenditure and urinary nitrogen excretion of major trauma patients was examined when nursing them on an air-fluidised bed at 32 degrees C using a crossover study design. Patients were randomised into two groups. Group A patients (n = 8) remained on a standard intensive care unit bed at an environmental temperature of 22 degrees C while Group B patients (n = 6) were placed on an air-fluidised bed operating at 32 degrees C. On days 4 and 5 after injury, energy expenditure and urinary nitrogen excretion were measured. On day 6, Group A patients transferred to an air-fluidised bed and Group B patients to a standard bed. On days 7 and 8, energy expenditure and urinary nitrogen excretion were again measured. Within group comparisons of energy expenditure and urinary nitrogen excretion were made for days 3 and 4 (period 1) and days 7 and 8 (period 2). In both groups, mean energy expenditure was significantly increased in the second period irrespective of whether air-fluidised bed therapy preceded or followed a period on a standard bed. We concluded that nursing intensive care patients on an air-fluidised bed at 32 degrees C did not influence energy expenditure or urinary nitrogen.

The provision of adult intensive care in Northern Ireland with reference to the role of high dependency care.

In 1991 an audit of Intensive Care Services was carried out by the Northern Ireland Intensive Care Group. In conjunction with this regional overview, all patients in the Regional Intensive Care Unit, (RICU) in the Royal Victoria Hospital were assessed daily, over a 10 month period in 1990-91 and classified as conforming to either intensive care or high dependency status. These data were then used to compare adult intensive care service in Northern Ireland with recent national and international recommendations on intensive care. Ten units in Northern Ireland were surveyed. In regard to national or international guidelines, all ten were deficient to some degree. Four units had significant deficiencies; small patient numbers, lack of 'dedicated' 24 hr medical cover and or deficiencies in the provision of appropriate monitoring and or equipment. There was a large diversity in casemix among the ten units surveyed which suggested differing admission criteria. The bed occupancy of RICU was 100%. Refused admissions constituted a further 13% of unresourced workload. The lack of physically separate, dedicated high dependency unit facilities meant that 26% of bed days were devoted to HDU care (usually for "improved" intensive care unit patients not yet ready for discharge to a general ward. Achieving nationally recommended intensive care standards (on a regional basis) is probably only possible if a number of the smaller intensive care units are redesignated as high dependency units, and patients requiring intensive care are concentrated in a smaller number of larger ICUs. This will increase the frequency of interhospital transfer of critically ill patients.

The gastric tonometer. A valuable monitor of splanchnic perfusion?

Minimally invasive assessment of the adequacy of perfusion of the gastrointestinal tract has become clinically feasible with the availability of the gastric tonometer. This modified nasogastric tube permits calculation of the pH of the gut mucosal cells; a low tissue pH may indicate tissue hypoxia due to regional hypoperfusion. Such regional hypoperfusion is often undetected by other monitors and, if it occurs intra-operatively, may result in a poor outcome following major surgery. In critical illness, the splanchnic area seems to be particularly vulnerable to hypoperfusion and such a regional oxygen deficit is implicated in the causation of organ dysfunction/failure. Recent studies have begun to define the circumstances in which splanchnic tissue acidosis develops and several therapies have been proposed to reverse the regional oxygen deficit. This review seeks to clarify whether or not the tonometer is a valuable addition to our current monitoring aids.

Low gastric intramucosal pH: incidence and significance in intensive care patients.

Monitoring of gastric intramucosal pH (pHi) is advocated in critical illness to detect tissue acidosis due to regional hypoperfusion. However, the number of patients who may benefit from such monitoring remains unclear and the relationship between low pHi and outcome requires further definition. Sixty consecutive patients with Acute Physiology and Chronic Health Evaluation (APACHE II) scores < 30 were studied throughout ICU stay to investigate the incidence of low pHi (< 7.32 for > or = 1 hour), its relationship to outcome, and temporally associated clinical events. pHi was measured 2 to 6 hours post-ICU admission and 8-hourly thereafter. Forty-four patients (73%) exhibited low pHi. Fourteen patients died in ICU with 13 deaths occurring in the low pHi group (P = 0.05). Length of ICU stay was greater in the low pHi group (P = 0.02). The development of low pHi was temporally associated with maximal sepsis score, weaning from assisted ventilation and commencement of enteral feeding.

Correction of splanchnic oxygen deficit in the intensive care unit: dopexamine and colloid versus placebo.

Correction of the splanchnic oxygen deficit indicated by low gastric intramucosal pH (pHi < 7.35) appears to reduce ICU mortality. Dopexamine hydrochloride is in clinical use for this purpose but its efficacy has not been fully investigated. We report the results of a prospective, randomized, placebo-controlled study with a crossover design to assess the efficacy of dopexamine in correcting low pHi. Twelve patients in whom pHi < 7.32 was detected during eight-hourly monitoring were randomized to receive either incremental dopexamine (4-6 micrograms/kg/min) with colloid or 5% dextrose for three hours prior to crossover. There was no difference in pHi between treatments despite cardiovascular effects during dopexamine infusion. There was, however, a time-related increase in pHi suggesting a beneficial effect of conventional therapy. Dopexamine hydrochloride at 4-6 micrograms/kg/min in conjunction with colloid is not a clinically useful therapy to correct the splanchnic oxygen deficit indicated by low pHi.

Intensive care management of multiple-organ dysfunction due to falciparum malaria in a married couple.

A married couple presented simultaneously with malignant tertian malaria and rapidly developed septicaemia and severe multiple-system organ failure. Despite schizonticidal treatment and multisystem support in intensive care the husband died. The selection of chemoprophylactic agents for this couple was not ideal and the duration of therapy before exposure to risk was inadequate. Severe infection with plasmodium falciparum is life-threatening and requires early diagnosis. It is best managed in an intensive care unit where continuous assessment may enable rapid detection of clinical deterioration and allow appropriate treatment to be instituted. The diagnosis should be considered in symptomatic patients who have travelled through areas where malaria is endemic. Recognised guidelines for the prescription of malarial chemoprophylaxis should be followed to ensure adequate protection.