Addison's disease associated with hypokalemia: a case report.

BACKGROUND: Primary adrenal insufficiency (Addison's disease) is a rare medical condition usually associated with hyperkalemia or normokalemia. We report a rare case of Addison's disease, coexisting with hypokalemia, requiring treatment. CASE PRESENTATION: In this case, a 42-year-old man was admitted to the intensive care unit with a history of loss of consciousness and severe hypoglycemia. His blood tests showed metabolic acidosis, low concentrations of cortisol 6 nmol/L (normal 68-327 nmol/L), and high plasma adrenocorticotropic hormone 253 pmol/L (normal 1.6-13.9 pmol/L), and he was diagnosed with primary adrenal insufficiency. Surprisingly, his serum potassium was low, 2.3 mmol/L (normal 3.5-5.1 mmol/L), requiring replacement over the course of his admission. Computed tomography scan of the adrenal glands showed features suggestive of unilateral adrenal tuberculosis. Investigations confirmed renal tubulopathy. The patient responded favorably to cortisol replacement, but never required fludrocortisone. CONCLUSIONS: Coexistence of hypokalemia with Addison's disease is unusual. We recommend investigation of the cause of hypokalemia in its own right, if it occurs with primary adrenal insufficiency.

We are tired of 'adrenal fatigue'.

There are numerous articles in the lay press discussing the existence and under-diagnosis of adrenal fatigue. Proponents of adrenal fatigue state that millions of people have under-active adrenal glands due to repeated stressors, resulting in numerous nonspecific symptoms such as fatigue, insomnia, joint pain and weight gain, among others. In studies to date, proposed methods to assess adrenal fatigue have produced conflicting results and the methodology for assessing the hypothalamic-pituitary-adrenal (HPA) axis has often been inappropriate. Furthermore, only a minority of studies have actually examined the HPA axis. Current evidence does not support the existence of adrenal fatigue or the usefulness of supplements to support adrenal function.

Nodal disease predicts recurrence whereas other traditional factors affect survival in a cohort of South African patients with differentiated thyroid carcinoma.

BACKGROUND AND AIM: Information on patients with differentiated thyroid carcinoma in South Africa is limited. The objective of this study was to review demographics and tumour characteristics in a cohort of patients with differentiated thyroid carcinoma, presenting to Groote Schuur Hospital and evaluate risk factors for recurrence and survival. PATIENTS AND METHODOLOGY: Retrospective demographic and clinical data were collected on all patients referred between January 2003 and December 2013. Prognostic factors for recurrence free survival and cancer specific survival were assessed using univariate and multivariate analyses. RESULTS: The total number of patients was 231.The median age at presentation was 44 years and 82% were female patients. The pathological sub-types were papillary (60.6%), follicular (38.9%) and poorly differentiated (0.5%). Total thyroidectomy was performed in 191 patients and 30 patients required neck dissections. A total of 171 (74%) patients received 131Iodine. The recurrence free and cause specific survival rates at 10 years were 83 and 91%, respectively. Nodal disease at presentation was the only significant risk factor for recurrence (p <  0.001) on multivariate analysis. Significant risk factors for cause specific mortality were age ≥ 45 years (p = 0.006), follicular pathology (p = 0.004), extra-thyroid extension (p = 0.013) and residual tumour (p = 0.004). CONCLUSIONS: Consistent with international trends, patients with differentiated thyroid carcinoma treated at Groote Schuur Hospital had a favourable prognosis. The known risk factors associated with recurrence and survival in this South African cohort were consistent with those reported in developed countries.

Salivary Cortisol and Cortisone do not Appear to be Useful Biomarkers for Monitoring Hydrocortisone Replacement in Addison's Disease.

Salivary cortisol has been used to monitor hydrocortisone replacement in patients with Addison's disease (AD). Since salivary cortisol is metabolised to salivary cortisone, it may be an adjunctive analyte to assess adequacy of hydrocortisone replacement in patients with AD. We aimed to characterise the exposure of salivary cortisol and cortisone in patients and healthy controls. We measured salivary cortisol and cortisone by liquid chromatography-tandem mass spectrometry and constructed a day curve (08:00 until 24:00 h) with 16 time points in 25 AD patients taking their usual hydrocortisone dose and in 26 healthy controls. The median (interquartile range) area under the curve (AUC) for cortisol was not different for patients, compared with controls [55.63 (32.91-151.07) nmol*min*l-1 vs. 37.49 (27.41-52.00) nmol*min*l-1; p=0.098, respectively], whereas the peak cortisol Cmax was higher in patients [32.61 (5.75-146.19) nmol/l vs. 8.96 (6.96-12.23) nmol/l; p=0.013], compared with controls. The AUC for cortisone [23.65 (6.10-54.76) nmol*min*l-1 vs. 227.73 (200.10-280.52) nmol*min*l-1; p≤ 0.001, respectively], and peak cortisone Cmax was lower in patients than in controls [11.11 (2.91-35.85) nmol/l vs. 33.12 (25.97-39.95) nmol/l; p=0.002]. The AUC for salivary cortisol and salivary cortisone were not correlated with any measures of hydrocortisone dose. The time-course and AUC of salivary cortisol were similar between Addison's patients and healthy controls. Patients had substantially lower salivary cortisone AUC, compared to healthy controls. Salivary cortisol AUC and pharmacokinetics were not related to hydrocortisone dose and thus are not likely useful markers for the adequacy of hydrocortisone replacement.

HLA similarities indicate shared genetic risk in 21-hydroxylase autoantibody positive South African and United States Addison's disease.

Genetic similarities between patients from the United States and South African (SA) Addison's Disease (AD) strengthen evidence for genetic association. SA-AD (n = 73), SA healthy controls (N = 78), and US-AD patients (N = 83) were genotyped for DQA1, DQB1, DRB1, and HLA-B alleles. Serum was tested for the quantity of 21OH-AA and IFNα-AA at the Barbara Davis Center. Although not as profound as in US-AD, in SA-AD 21OH-AA + subjects the predominantly associated risk haplotypes were DRB1*0301-DQB1*0201 (DR3), DRB1*04xx-DQB1*0302 (DR4), and the combined DR3/4 genotype. DQB1*0302 associated DRB1*04xx haplotypes conferred higher risk than those DRB1*04xx haplotypes associated with other DQB1 alleles. We found negative association in 21OH-AA + SA-AD for DQA1*0201-DQB1*0202 and DQA1*0101-DQB1*0501 vs SA controls, and positive association for DQA1*0401-DQB1*0402 vs US-AD. Apart from the class II DR3 haplotype, HLA-B8 did not have an independent effect; however together DR3 and HLA-B8 conferred the highest risk vs 21OH-AA negative SA-AD and SA-controls. HLA-B7 (often with DR4) conferred novel risk in 21OH-AA + SA-AD vs controls. This study represents the first comparison between South African and United States AD populations utilizing genotyping and serology performed at the same center. SA-AD and US-AD 21OH-AA + patients share common HLA risk haplotypes including DR4 (with HLA-B7) and DR3 (with HLA-B8), strengthening previously described HLA associations and implicating similar genetic etiology.

Male and female hypogonadism are highly prevalent in South Africans with Addison's disease.

Hypogonadism may complicate Addison's disease (primary hypoadrenalism), but prevalence and metabolic sequelae of hypogonadism in Addison's disease are poorly described. We recruited patients from the South African Addison's disease national registry who received stable replacement doses of hydrocortisone and had no acute illness. Male biochemical testosterone deficiency was defined as an early morning basal testosterone<9.9 nmol/l and premature ovarian failure (POF) when menopause occurred before 40 years of age. Cardiometabolic risk variables were measured in males only. Male hypogonadism prevalence was 33% (14/42), and 10 patients had newly diagnosed hypogonadism. Two untreated patients had elevated FSH or LH (>10 or 12 IU/l). Testosterone deficiency did not correlate with age, disease duration or hydrocortisone dose. Untreated male hypogonadal subjects had a higher (mean ± standard deviation) BMI compared to eugonadal subjects 29.2 ± 4.9 kg/m(2) vs. 24.7 ± 3.4 kg/m(2) (p=0.01) and a higher median (interquartile range) high-sensitive-CRP 6.4 (2.5-14.0) mg/l vs. 1.45 (0.6-2.8) mg/l (p=0.002). There were no differences between the 2 groups in lipids, lipoproteins and fasting glucose. The median (interquartile range) DHEAS was lower in the hypogonadal 0.31 (0.27-0.37) µmol/l, compared with the eugonadal group 0.75 (0.50-1.51) µmol/l (p=0.005). POF was documented in 11% of female patients. Male testosterone deficiency was highly prevalent in this cohort and was primarily due to secondary hypogonadism. Only BMI and hs-CRP were increased in untreated male hypogonadal subjects. Male and female hypogonadism appears to be a common complication of Addison's disease and may contribute to its morbidity.

Increased cardiovascular risk in South African patients with Addison's disease.

Patients with Addison's disease (AD) are believed to be at risk for cardiovascular disease (CVD). South Africa, like the rest of the developing world is experiencing an increase in CVD and patients with AD may be at double the risk of their peers. We wished to explore AD patients' CVD risk factors. A cross-sectional nationwide study in South Africa of patients with AD was conducted. A cohort of 147 patients with AD and 147 healthy control subjects were matched by age, gender, ethnicity, and BMI as far as was possible. Lipoproteins and highly-sensitive C-reactive-protein (hs-CRP) were the main outcome measures. AD patients had significantly higher triglycerides; (p=0.001), lower HDLC (p<0.001), higher hs-CRP (p<0.001), and more small dense LDL; (p=0.002) than controls. Nonesterified fatty acids were lower in patients (p<0.001). Approximately 65% [95% confidence interval (CI 55.6-72.4%)] had hypercholesterolaemia, 75% (CI 64.8-81.2%) had low HDLC, and 75% (CI 68.0-84.1%) had a higher LDLC. Thirteen percent of AD patients had diabetes mellitus, but none of the risk factors differed from the nondiabetics. Only HDLC correlated positively with daily hydrocortisone dose (r=0.32; p=0.005). In conclusion dyslipidaemia is common in South African AD patients; CVD risk assessment and intervention are probably warranted in the management of these patients.

Salivary cortisol day curves in Addison's disease in patients on hydrocortisone replacement.

Using salivary cortisol (SC) measurements, cortisol exposure in Addison's disease patients on hydrocortisone replacement was determined and compared with healthy controls. Cortisol pharmacokinetics was assessed in 31 patients with Addison's disease on replacement hydrocortisone doses (median daily dose 20 mg; range 5-50 mg) and 30 healthy control subjects. Saliva samples (n=16) were collected between 08:00 and 00:00 h in 1 day, using a passive drool technique. Cortisol exposure was evaluated by noncompartmental approach. In the patients, cortisol exposure was significantly higher than in controls: median inter-quartile range (IQR) peak cortisol (C(max)) 174.5 (59.3-837.0) vs. 6.50 (4.7-19.3) nmol/l, p=0.0001; area under the curve (AUC) 390.1 (177.1-928.9) vs. 21.4 (14.6-28.4) minutes*nmol/l, p=0.0001, trough cortisol level (C(min)) 0.49 (0.49-0.96) vs. 0.49 (0.49-0.49) nmol/l, p=0.02, occurring at 480.0 (0.1-660.0) vs. 405.0 (180.0-570.0) min, p=0.56. First peak cortisol was 174.5 (53.0-754.7) vs. 6.27 (3.90-8.47) nmol/l, p=0.0001 and second peak cortisol 18.90 (5.22-76.9) vs. 3.12 (1.76-4.79) nmol/l, p=0.0001. The time to first peak cortisol differed between the 2 groups, 30 (30-75) vs. 0.1 (0.1-30) minutes; p=0.0001. At doses studied, hydrocortisone replacement therapy results in cortisol pharmacokinetics being markedly different from endogenous cortisol profiles in healthy control subjects. Addison's disease patients had significantly higher SC levels compared to healthy control subjects.

Investigation of glucocorticoid receptor polymorphisms in relation to metabolic parameters in Addison's disease.

BACKGROUND: Uncertainty exists whether glucocorticoid receptor (GCR) polymorphisms play a role in steroid-related side effects in Addison's disease (AD) patients on hydrocortisone. The polymorphisms Bcll and N363S appear to increase sensitivity to cortisol, while the ER22/23EK polymorphism has been associated with resistance to cortisol. METHOD: One hundred and forty seven AD patients, and gender, and ethnicity-matched controls were recruited in South Africa. Three polymorphisms in the GCR were studied, using PCR followed by restriction fragment length analysis. Associations with BMI, lipids, glucose and inflammatory markers were investigated. RESULTS: In both patients and controls, the Bcll polymorphism occurred more frequently in whites than in other ethnic groups studied but was not associated with any of the metabolic parameters tested. The ER22/23EK polymorphism was associated with an increased BMI in both patients (29.4 vs 24.7  kg/m²) and control subjects (26.3 vs 24.2  kg/m²). The ER22/23EK polymorphism was also associated with lower LDL cholesterol in control subjects (3.46 vs 3.93  mmol/l) and in patients (3.52 vs 4.10  mmol/l). N363S was associated with increased BMI in controls 29.9  kg/m² vs wild type 24.8  kg/m². Median hydrocortisone doses were greater in patients heterozygous for either ER22/23EK 30.0  mg or N363S 25.0  mg polymorphisms than in wild type patients 20.0  mg (both comparisons). CONCLUSION: Alterations in lipids, BMI and hydrocortisone dose were associated with two polymorphisms. Further larger studies are warranted to corroborate these findings.

Use of AFLP and PFGE to discriminate between Salmonella enterica serovar Typhimurium DT126 isolates from separate food-related outbreaks in Australia.

In 2001 Salmonella enterica serovar Typhimurium definitive phage-type (DT) 126 was isolated at higher frequency in Australia compared to other S. Typhimurium phage types and in comparison to previous years. Associated with this increase was the implication of this phage type in a number of food-related outbreaks. We compared fluorescent amplified fragment length polymorphism (FAFLP) to pulsed-field gel electrophoresis (PFGE), the current 'gold standard' for molecular typing of Salmonella for the discrimination between outbreak-associated isolates and epidemiologically unrelated DT126 strains. FAFLP showed a greater ability to discriminate between isolates than PFGE, with 16 groups of clusters or individual isolates with < 90% similarity to each other compared to three groups as determined by PFGE. Both methods were able to discriminate between isolates from two separate outbreaks in South Australia and isolates associated with an outbreak at a restaurant in New South Wales. The resolving power of both methods was not sufficient to separate all epidemiologically unrelated DT126 isolates from the outbreak isolates. We conclude that amplified fragment length polymorphism is a useful tool to assist in the discrimination of S. Typhimurium DT126 isolates.

Genetic diversity and biological control activity of novel species of closely related pseudomonads isolated from wheat field soils in South Australia.

Rhizobacteria closely related to two recently described species of pseudomonads, Pseudomonas brassicacearum and Pseudomonas thivervalensis, were isolated from two geographically distinct wheat field soils in South Australia. Isolation was undertaken by either selective plating or immunotrapping utilizing a polyclonal antibody raised against P. brassicacearum. A subset of 42 isolates were characterized by amplified 16S ribosomal DNA restriction analysis (ARDRA), BIOLOG analysis, and gas chromatography-fatty acid methyl ester (GC-FAME) analysis and separated into closely related phenetic groups. More than 75% of isolates tested by ARDRA were found to have >95% similarity to either Pseudomonas corrugata or P. brassicacearum-P. thivervalensis type strains, and all isolates had >90% similarity to either type strain. BIOLOG and GC-FAME clustering showed a >70% match to ARDRA profiles. Strains representing different ARDRA groups were tested in two soil types for biological control activity against the soilborne plant pathogen Gaeumannomyces graminis var. tritici, the causative agent of take-all of wheat and barley. Three isolates out of 11 significantly reduced take-all-induced root lesions on wheat plants grown in a red-brown earth soil. Only one strain, K208, was consistent in reducing disease symptoms in both the acidic red-brown earth and a calcareous sandy loam. Results from this study indicate that P. brassicacearum and P. thivervalensis are present in Australian soils and that a level of genetic diversity exists within these two novel species but that this diversity does not appear to be related to geographic distribution. The result of the glasshouse pot trial suggests that some isolates of these species may have potential as biological control agents for plant disease.

Involvement of Ets, rel and Sp1-like proteins in lipopolysaccharide-mediated activation of the HIV-1 LTR in macrophages.

The HIV-1 promoter was used as a model to identify transcription factors involved in LPS-dependent transcription in RAW 264 murine macrophages. Expression plasmids for Ets-2 and PU.1 trans-activated the HIV-1 LTR and recombinant PU.1 and an Ets-2 DNA binding domain/GST fusion protein bound to the 5' kappa B site of the HIV-1 enhancer. Ets-2 mRNA was LPS-inducible in RAW 264 cells and LPS stimulated phosphorylation of threonine 72 residue within the Ets-2 pointed domain. Induction of Ets-2 and other LPS-responsive transcription factors was also observed upon addition of plasmid DNA, which complicates interpretation of transient transfections. The proximal promoter region, containing two Sp1 sites, was also LPS-responsive. We propose that the kappa B elements and the tandem Sp1 sites act as LPS response elements and that kappa B-mediated LPS action involves Ets and rel factors.

Interaction between PU.1 and another Ets family transcription factor promotes macrophage-specific Basal transcription initiation.

Numerous macrophage-restricted promoters lack TATA boxes or other conventional initiation motifs but contain high affinity binding sites (PU boxes) for the macrophage-restricted Ets family transcription factor PU.1. In RAW264 murine macrophages, multimerized PU boxes were not active as enhancers when placed upstream of a minimal promoter. To model their role in basal promoters, we inserted PU boxes into a promoterless luciferase reporter plasmid. Two sites, regardless of orientation, were necessary and sufficient to direct reporter gene expression in transient transfections of the RAW264 macrophage-like cell line. This activity was absent in transfected 3T3 fibroblasts but could be induced by PU.1 coexpression. Both the model promoter and the macrophage-specific mouse and human c-fms promoters were activated in RAW264 cells by other Ets family transcription factors, Ets-2 and Elf-1. In fibroblasts, the effects of PU.1 and Ets-2 were multiplicative, whereas overexpression of PU.1 in RAW264 cells reduced activation of c-fms or model promoters by the other Ets factors. The PU.1 and Ets-2 binding sites of the mouse c-fms promoter have been located by DNase footprinting. A conserved Ets-like motif at the transcription site, CAGGAAC, that bound only weakly to PU.1, was identified as an additional critical basal c-fms promoter element. Comparison of studies on the model promoter, c-fms and other myeloid promoters provides evidence for a conserved mechanism that involves three separate and functionally distinct Ets-like motifs.

Regulation of CSF-1 receptor expression.

Cells of the mononuclear phagocyte lineage possess receptors for macrophage colony-stimulating factor (CSF-1) encoded by the c-fms protooncogene and respond to CSF-1 with increased survival, growth, differentiation, and reversible changes in function. The c-fms gene is itself a macrophage differentiation marker. In whole mount analyses of mRNA expression in embryos, c-fms is expressed at very high levels on placental trophoblasts. It is detectable on individual cells in the yolk sac around 8.5 to 9 days postcoitus, appears on isolated cells in the head of the embryo around 9.5 dpc, and appears on numerous cells throughout the embryo by day 10.5. The extent of c-fms expression is much greater than for other macrophage-specific genes including lysozyme and a macrophage-specific protein tyrosine phosphatase. Our studies of the cis-acting elements of the c-fms promoter have indicated a key role for collaboration between the macrophage-specific transcription factor, Pu.1, which functions in determining the site of transcription initiation, and other members of the Ets transcription factor family. This is emerging as a common pattern in macrophage-specific promoters. We have shown that two PU box elements alone can function as a macrophage-specific promoter. The activity of both the artificial promoter and the c-fms promoter is activated synergistically by coexpression of Pu.1 and another Ets factor, c-Ets-2. A 3.5kb c-fms exon 2 promoter (but not the 300bp proximal promoter) is also active in a wide diversity of tumor cell lines. The interesting exception is the melanoma cell line K1735, in which the promoter is completely shut down and expression of c-fms causes growth arrest and cell death. The activity of the exon 2 promoter in these nonmacrophages is at least as serum responsive as the classic serum-responsive promoter of the c-fos gene. It is further inducible in nonmacrophages by coexpression of the c-fms product. Unlike other CSF-1/c-fms-responsive promoters, the c-fms promoter is not responsive to activated Ras even when c-Ets-2 is coexpressed. In most lines, production of full length c-fms is prevented by a downstream intronic terminator, but in Lewis lung carcinoma, read-through does occur, and expression of both c-fms and other macrophage-specific genes such as lysozyme and urokinase becomes detectable in conditions of serum deprivation.

Opposing actions of c-ets/PU.1 and c-myb protooncogene products in regulating the macrophage-specific promoters of the human and mouse colony-stimulating factor-1 receptor (c-fms) genes.

The receptor for macrophage colony stimulating factor (CSF-1), the c-fms gene product, is a key determinant in the differentiation of monocytic phagocytes. Dissection of the human and mouse c-fms proximal promoters revealed opposing roles for nuclear protooncogenes in the transcriptional regulation of this gene. On the one hand, c-ets-1, c-ets-2, and the macrophage-specific factor PU.1, but not the ets-factor PEA3, trans-activated the c-fms proximal promoter. On the other hand c-myb repressed proximal promoter activity in macrophages and blocked the action of c-ets-1 and c-ets-2. Basal c-fms promoter activity was almost undetectable in the M1 leukaemia line, which expressed high levels of c-myb, but was activated as cells differentiated in response to leukemia inhibitory factor and expressed c-fms mRNA. The repressor function of c-myb depended on the COOH-terminal domain of the protein. We propose that ets-factors are necessary for the tissue-restricted expression of c-fms and that c-myb acts to ensure correct temporal expression of c-fms during myeloid differentiation.

Transcription of individual genes in eukaryotic cells occurs randomly and infrequently.

Experimental evidence is presented indicating that the expression of a lacZ reporter gene driven by the HIV-1 long terminal repeat in a series of stably transfected, cloned macrophage cell lines occurs in a very small proportion of cells. The proportion of cells expressing lacZ, rather than the level of expression in each cell, is regulated by external stimuli such as LPS and phorbol ester. Based upon these and published data we propose that transcription in eukaryotic cells occurs in short pulses interspersed by long periods of inactivity of indeterminate duration. Transcriptional regulation is envisaged as involving changes in the probability rather than the rate of transcription. A probabilistic model of transcription may explain many biological phenomena, such as stem cell division and clonogenic activity, heterogeneous gene expression among clonal cell populations, retroviral latency and cell cycle progression, which appear to involve stochastic decisions.

Comparison of the expression and function of the transcription factor PU.1 (Spi-1 proto-oncogene) between murine macrophages and B lymphocytes.

The expression of mRNA encoding the DNA-binding protein PU.1 (Spi-1) is restricted to B lymphocytes and macrophages. The role of PU.1 in tissue-specific transcriptional regulation in the two cell types was examined by co-transfection of a PU.1 expression plasmid with vectors containing B cell (IgH enhancer) or macrophage-specific (c-fms) transcription control elements. Cotransfection of the PU.1 expression plasmid in MOPC31C B cells trans-repressed the IgH enhancer but trans-activated the c-fms promoter. The latter was insufficient to overcome a block to transcription elongation that determines macrophage-specific c-fms gene expression. In the macrophage line RAW264, PU.1 had no effect on the c-fms promoter, but trans-repressed the activity of a c-fms reporter plasmid containing the transcription attenuator. The effects of PU.1 in both cell types were distinct from those of c-ets-2, a related factor, which trans-activated the c-fms promoter in both B cells and macrophages but also repressed the IgH enhancer. PU.1 was shown to be one of several nuclear proteins that bound a critical cis-acting element of the IgH enhancer, microB, but analysis of nuclear extracts of a wide range of B cell and macrophage lines demonstrated a strong correlation between macrophage phenotype and nuclear PU.1 expression. The data suggest that differences in nuclear PU.1 expression and function between macrophages and B cells may play a role in lineage divergence.