Observational studies on macroprolactin in a routine clinical laboratory.

BACKGROUND: It is now recommended that all samples with raised prolactin should be examined for the presence of macroprolactin. We performed a retrospective review of our experience of macroprolactin to determine the incidence and the natural history of macroprolactin. METHODS: A retrospective study of macroprolactin was made in a large clinical laboratory. Macroprolactin was measured on those samples where it is requested and where the total prolactin is >1000 mIU/L. Prolactin was measured using the Siemens Centaur and macroprolactin was measured following polyethylene glycol (PEG)-precipitation. RESULTS: The incidence of macroprolactin in samples where the total prolactin was >1000 mIU/L was 36/670 (5.4%). During this period, 12,064 samples were received for prolactin analysis. Over the period since 2006, 22 subjects had a sample with an isolated macroprolactin measurement followed by another sample without macroprolactin after a median period of 0.46 years. Twenty-five subjects had multiple consecutive measurements of macroprolactin lasting a median period of 2.1 years. Fourteen subjects had more than six samples which had been subjected to PEG precipitation. In these subjects, the reproducibility of PEG precipitation over a median of 6 years was 1.1% CV (recovery 75% [26-110] (median [range])). CONCLUSIONS: The presence of macroprolactin can change over time and we cannot advise that once a test for macroprolactinemia has been performed that it is not necessary to repeat the investigation if a subsequent sample is hyperprolactinemic; nor can one assume that macroprolactin will not develop even if it has been excluded previously.

Differential remodelling of mitochondrial subpopulations and mitochondrial dysfunction are a feature of early stage diabetes.

Mitochondrial dysfunction is a feature of type I and type II diabetes, but there is a lack of consistency between reports and links to disease development. We aimed to investigate if mitochondrial structure-function remodelling occurs in the early stages of diabetes by employing a mouse model (GENA348) of Maturity Onset Diabetes in the Young, exhibiting hyperglycemia, but not hyperinsulinemia, with mild left ventricular dysfunction. Employing 3-D electron microscopy (SBF-SEM) we determined that compared to wild-type, WT, the GENA348 subsarcolemma mitochondria (SSM) are ~ 2-fold larger, consistent with up-regulation of fusion proteins Mfn1, Mfn2 and Opa1. Further, in comparison, GENA348 mitochondria are more irregular in shape, have more tubular projections with SSM projections being longer and wider. Mitochondrial density is also increased in the GENA348 myocardium consistent with up-regulation of PGC1-α and stalled mitophagy (down-regulation of PINK1, Parkin and Miro1). GENA348 mitochondria have more irregular cristae arrangements but cristae dimensions and density are similar to WT. GENA348 Complex activity (I, II, IV, V) activity is decreased but the OCR is increased, potentially linked to a shift towards fatty acid oxidation due to impaired glycolysis. These novel data reveal that dysregulated mitochondrial morphology, dynamics and function develop in the early stages of diabetes.

Clinical Evaluation of Assays for Plasma Renin Activity and Aldosterone Measurement by Liquid Chromatography-Tandem Mass Spectrometry.

BACKGROUND: Aldosterone and renin are pivotal hormones in the regulation of salt and water homeostasis and blood pressure. Measurement of renin and aldosterone in serum/plasma is essential for the investigation of primary hyperaldosteronism (PA) and monitoring of glucocorticoid replacement therapy. METHODS: We report 2 LC-MS/MS methods developed to measure aldosterone and plasma renin activity (PRA). PRA was determined by endogenous enzymatic generation of angiotensin I using 150 µL of sample. Generated angiotensin I was purified by solid phase extraction prior to chromatographic separation and mass spectrometry. Aldosterone measurement required 300 µL of sample extracted with MTBE prior to LC-MS/MS analysis. RESULTS: The PRA method was linear (1.2-193 nmol/L), sensitive (LLOQ = 1.2 nmol/L), precise (CV = 4.1%), and specific (no cross reactivity for a number of structurally similar steroids). Dilutional linearity and recovery (84%) were acceptable. Accuracy was confirmed by comparison against our current RIA method. The aldosterone method had equally acceptable performance characteristics. Reference ranges in 110 healthy normotensive subjects were: PRA 0.2-3.7 nmol/L/h and aldosterone 50-950 pmol/L. Consecutive patients (n = 62) with adrenal incidentalomas shown to have no functional adrenal disease; their post overnight 1 mg dexamethasone test values were: PRA 0.2-2.6 nmol/L/h and aldosterone 55-480 pmol/L. Serum aldosterone values after 2 liter saline suppression were-normal subjects (n = 17): 78-238 pmol/L and confirmed primary hyperaldosteronism (n = 25): 131-1080 pmol/L. CONCLUSIONS: We have developed robust assays for PRA and aldosterone with appropriate clinical evaluation. These assays are now in routine practice in the UK.

The validation of an in vitro colonic motility assay as a biomarker for gastrointestinal adverse drug reactions.

Motility-related gastrointestinal adverse drug reactions (GADRs), such as constipation and diarrhea, are some of the most frequently reported adverse events associated with the clinical development of new chemical entities, and for marketed drugs. However, biomarkers capable of detecting such GADRs are lacking. Here, we describe an in vitro assay developed to detect and quantify changes in intestinal motility as a surrogate biomarker for constipation/diarrhea-type GADRs. In vitro recordings of intraluminal pressure were used to monitor the presence of colonic peristaltic motor complexes (CPMCs) in mouse colonic segments. CPMC frequency, contractile and total mechanical activity were assessed. To validate the assay, two experimental protocols were conducted. Initially, five drugs with known gastrointestinal effects were tested to determine optimal parameters describing excitation and inhibition as markers for disturbances in colonic motility. This was followed by a "blinded" evaluation of nine drugs associated with or without clinically identified constipation/diarrhea-type GADRs. Concentration-response relationships were determined for these drugs and the effects were compared with their maximal free therapeutic plasma concentration in humans. The assay detected stimulatory and inhibitory responses, likely correlating to the occurrence of diarrhea or constipation. Concentration-related effects were identified and potential mechanisms of action were inferred for several drugs. Based on the results from the fourteen drugs asssessed, the sensitivity of the assay was calculated at 90%, with a specificity of 75% and predictive capacity of 86%. These results support the potential use of this assay in screening for motility-related GADRs during early discovery phase, safety pharmacology assessment.

Optimization of urinary pteridine analysis conditions by CE-LIF for clinical use in early cancer detection.

The second leading cause of death in the US is cancer and early discovery of the disease has translated into reduced fatality rates. We have identified and performed a systematic investigation of a method for urinary pteridine analysis by using CE-LIF, which is believed to possess the potential to diagnose the presence of cancer even earlier than existing methodologies. Through system enhancements, we have been able to improve the resolution of the two least resolved sets of peaks (6,7-dimethylpterin versus 6-biopterin and D-(+)-neopterin versus 6-hydroxymethylpterin) from 0.85 to 2.48 and 0.90 to 3.58, respectively. Additionally, we have discovered that the preparation of the urine samples in previous works was inadequate, and we have corrected the method to fully oxidize the pteridines in the urine, resulting in significantly less variability in quantification and greater ease of defining p-values for healthy versus cancer patients. Finally, we have performed validation steps of spike and recovery and short-term aging studies to demonstrate the method's robustness. As a result, we present an optimized and validated method ready for transfer from discovery phase to clinical trial that can potentially act as a non-invasively pre-screening test for cancer.

Social tenants' health: evaluating the effectiveness of landlord interventions.

BACKGROUND: The National Health Service (NHS) scores well internationally on access to healthcare. But access has been measured on methods likely to undersample the more disadvantaged. Social landlords have access to more disadvantaged groups and may be able to improve health outcomes for their tenants and reduce their NHS usage by simple interventions. METHODS: This is a randomised controlled trial of 547 London social housing 'general needs' tenants over 50 years of age. Participants were given a health assessment, then split into a control group or one of two treated groups. Following early assessment 25 participants had to be withdrawn to receive intensive treatment because of currently untreated major health problems. Participants were followed up over 18 months and changes in health outcomes and NHS usage measured. RESULTS: Compared with the control the most intensively treated group showed non-significant improvements on health outcomes but a significant reduction in NHS resource use, on conventional costings worth some £760 per person. All 25 participants transferred to the most intensively treated group after their early health assessments showed improvement on all health outcomes at final assessment, but these improvements were not statistically significant. CONCLUSIONS: Drawing a sample from disadvantaged but not the most seriously disadvantaged groups in London revealed 4.5% of the population to have very serious untreated health problems. The reason for lack of treatment was mainly non-registration with a general practitioner or psychiatric issues. Simple interventions to a targeted group were found to produce significant reductions in NHS usage and other, although non-significant, health benefits. TRIAL REGISTRATION NUMBER: ID ISRCTN96259142.

Methods to estimate access to care and the effect of interventions on the outcomes of congenital disorders.

In the absence of intervention, early-onset congenital disorders lead to pregnancy loss, early death, or disability. Currently, lack of epidemiological data from many settings limits the understanding of the burden of these conditions, thus impeding health planning, policy-making, and commensurate resource allocation. The Modell Global Database of Congenital Disorders (MGDb) seeks to meet this need by combining general biological principles with observational and demographic data, to generate estimates of the burden of congenital disorders. A range of interventions along the life course can modify adverse outcomes associated with congenital disorders. Hence, access to and quality of services available for the prevention and care of congenital disorders affects both their birth prevalence and the outcomes for affected individuals. Information on this is therefore important to enable burden estimates for settings with limited observational data, but is lacking from many settings. This paper, the third in this special issue on methods used in the MGDb for estimating the global burden of congenital disorders, describes key interventions that impact on outcomes of congenital disorders and methods used to estimate their coverage where empirical data are not available.

Chromosomal disorders: estimating baseline birth prevalence and pregnancy outcomes worldwide.

Chromosomal disorders, of which Down syndrome is the most common, can cause multi-domain disability. In addition, compared to the general population, there is a higher frequency of death before the age of five. In many settings, large gaps in data availability have hampered policy-making, programme priorities and resource allocation for these important conditions. We have developed methods, which overcome this lack of data and allow estimation of the burden of affected pregnancies and their outcomes in different settings worldwide. For example, the methods include a simple equation relating the percentage of mothers 35 and over to Down syndrome birth prevalence. The results obtained provide a starting point for consideration of services that can be implemented for the care and prevention of these disorders.

Rare single gene disorders: estimating baseline prevalence and outcomes worldwide.

As child mortality rates overall are decreasing, non-communicable conditions, such as genetic disorders, constitute an increasing proportion of child mortality, morbidity and disability. To date, policy and public health programmes have focused on common genetic disorders. Rare single gene disorders are an important source of morbidity and premature mortality for affected families. When considered collectively, they account for an important public health burden, which is frequently under-recognised. To document the collective frequency and health burden of rare single gene disorders, it is necessary to aggregate them into large manageable groupings and take account of their family implications, effective interventions and service needs. Here, we present an approach to estimate the burden of these conditions up to 5 years of age in settings without empirical data. This approaches uses population-level demographic data, combined with assumptions based on empirical data from settings with data available, to provide population-level estimates which programmes and policy-makers when planning services can use.

Adaptation to altitude as a vehicle for experiential learning of physiology by university undergraduates.

In this article, an experiential learning activity is described in which 19 university undergraduates made experimental observations on each other to explore physiological adaptations to high altitude. Following 2 wk of didactic sessions and baseline data collection at sea level, the group ascended to a research station at 12,500-ft elevation. Here, teams of three to four students measured the maximal rate of oxygen uptake, cognitive function, hand and foot volume changes, reticulocyte count and hematocrit, urinary pH and 24-h urine volume, athletic performance, and nocturnal blood oxygen saturation. Their data allowed the students to quantify the effect of altitude on the oxygen cascade and to demonstrate the following altitude-related changes: 1) impaired performance on selected cognitive function tests, 2) mild peripheral edema, 3) rapid reticulocytosis, 4) urinary alkalinization and diuresis, 5) impaired aerobic but not anaerobic exercise performance, 6) inverse relationship between blood oxygen saturation and resting heart rate, and 7) regular periodic nocturnal oxygen desaturation events accompanied by heart rate accelerations. The students learned and applied basic statistical techniques to analyze their data, and each team summarized its results in the format of a scientific paper. The students were uniformly enthusiastic about the use of self-directed experimentation to explore the physiology of altitude adaptation and felt that they learned more from this course format than a control group of students felt that they learned from a physiology course taught by the same instructor in the standard classroom/laboratory format.

Comparison of Lipoprotein Electrophoresis and Apolipoprotein E Genotyping in Investigating Dysbetalipoproteinemia.

Dysbetalipoproteinemia is often associated with apolipoprotein E2E2 homozygosity; however, lipoprotein electrophoresis may also be used to assist in the diagnosis. The aim of this study was to compare apolipoprotein E (apo E) genotyping and lipoprotein electrophoresis in investigating dysbetalipoproteinemia. Data were collected over a three-year period from a lipid clinic in a tertiary referral centre and reviewed for apo E genotyping and lipoprotein electrophoresis. Sixty-two patients had both apo E genotyping and lipoprotein electrophoresis. Of these, 16 patients showed broad beta band on electrophoresis. However, only 3 of them had apo E2E2 homozygosity on genotyping. Lipoprotein electrophoresis and apo E genotyping results showed poor concordance. This was primarily due to visual interpretation error of lipoprotein electrophoresis which may over diagnose dysbetalipoproteinemia.

Bone-specific alkaline phosphatase by immunoassay or electrophoresis: their use in clinical practice.

BACKGROUND: Measurement of bone-specific alkaline phosphatase (BALP) may be useful in diagnosing and monitoring metabolic bone disease. This study aimed to evaluate the BALP immunoassay and compare it with electrophoresis (densitometry) for the quantitation of BALP. METHODS: Metra BALP immunoassay kits were used for the method comparison. BALP was also quantitated by electrophoresis (densitometry) in seven patients with active Paget's disease. RESULTS: Immunoassay results did not correlate well with densitometrically quantitated BALP, as there was a statistically significant (p<0.01), negative bias (22%) for results obtained by immunoassay compared with those derived by densitometry. Possible interference in the immunoassay with other isoforms of alkaline phosphatase (ALP) such as liver, placental and intestinal was also observed. The Metra BALP immunoassay is quoted as having an upper dynamic limit of 140 U/L and recommends that samples only require dilution above this level; we observed inconsistent results upon dilution of samples below this level. CONCLUSIONS: Immunoassay and electrophoresis did not correlate well for BALP quantitation. Possible interference with other isoforms of ALP was observed with the BALP immunoassay. The accuracy of the BALP immunoassay is questionable at higher concentrations.

Chylothorax diagnosis: can the clinical chemistry laboratory do more?

BACKGROUND: Chylothorax is a rare anatomical disruption of the thoracic duct associated with a significant degree of morbidity and mortality. Diagnosis usually relies upon lipid analysis and visual inspection of the pleural fluid. However, this may be subject to incorrect interpretation. The aim of this study was to compare pleural fluid lipid analysis and visual inspection against lipoprotein electrophoresis. METHODS: Nine pleural effusion samples suspected of being chylothorax were analysed. A combination of fluid lipid analysis and visual inspection was compared with lipoprotein electrophoresis for the detection of chylothorax. RESULTS: There was 89% concordance between the two methods. Using lipoprotein electrophoresis as gold standard, calculated sensitivity, specificity, negative predictive value and positive predictive value for lipid analysis/visual inspection were 83%, 100%, 100% and 75%, respectively. CONCLUSION: Examination of pleural effusion samples by lipoprotein electrophoresis may provide important additional information in the diagnosis of chylothorax.

CSF xanthochromia: correlation with brain imaging and its usefulness as an out-of-hours test.

BACKGROUND: Subarachnoid haemorrhage (SAH) is a spontaneous bleed into the subarachnoid space which is investigated by CT head and cerebrospinal fluid (CSF) xanthochromia. The aim of this study was to compare CSF xanthochromia with brain imaging and evaluate the need for out of hours (OOH) testing for CSF xanthochromia. METHOD: Discharge summaries and brain imaging of patients with positive xanthochromia screen were reviewed over 12 months retrospectively. Timings of CSF xanthochromia and hospital discharge of 30 consecutive patients with negative xanthochromia screen were also examined. RESULTS: From 289 xanthochromia requests, 23 were positive but only 2 patients had an actual bleed. In 30 consecutive negative xanthochromia results, all brain imaging results suggested no bleed. Eight of these requests were performed OOH, and as a result, 1 patient was discharged early. CONCLUSIONS: CSF xanthochromia has a poor positive predictive value (8.7%) but has a good negative predictive value (100%) for SAH. Analysis of CSF xanthochromia OOH, in patients with negative CT head, did not speed up the discharge process from the hospital.

Role of advanced glycation end products in cardiovascular disease.

Advanced glycation end products (AGEs) are produced through the non enzymatic glycation and oxidation of proteins, lipids and nucleic acids. Enhanced formation of AGEs occurs particularly in conditions associated with hyperglycaemia such as diabetes mellitus (DM). AGEs are believed to have a key role in the development and progression of cardiovascular disease in patients with DM through the modification of the structure, function and mechanical properties of tissues through crosslinking intracellular as well as extracellular matrix proteins and through modulating cellular processes through binding to cell surface receptors [receptor for AGEs (RAGE)]. A number of studies have shown a correlation between serum AGE levels and the development and severity of heart failure (HF). Moreover, some studies have suggested that therapies targeted against AGEs may have therapeutic potential in patients with HF. The purpose of this review is to discuss the role of AGEs in cardiovascular disease and in particular in heart failure, focussing on both cellular mechanisms of action as well as highlighting how targeting AGEs may represent a novel therapeutic strategy in the treatment of HF.

Investigation of urinary pteridine levels as potential biomarkers for noninvasive diagnosis of cancer.

BACKGROUND: Biomarkers are good potential tools for early cancer diagnosis. Here we have analyzed eight different pteridines in the urine samples of cancer patients and compared them with samples from healthy subjects. Pteridines are important cofactors in the process of cell metabolism, and they have recently become a focal point of cancer screening research because certain pteridine levels have been shown to reflect the presence of cancers. METHODS: We analyzed 8 pteridines; 6,7-dimethylpterin, 6-biopterin, d-(+)-neopterin, 6-hydroxymethylpterin, pterin, isoxanthopterin, xanthopterin and pterin-6-carboxylic acid using a house-built high-performance capillary electrophoresis with laser-induced fluorescence detection (HPCE-LIF). The levels of pteridines were reported as a ratio of pteridine to creatinine. Statistical hypothesis testing was conducted and P values were calculated to analyze the data. RESULTS: Among the eight pteridines studied, 6-biopterin, 6-hydroxymethylpterin, pterin, xanthopterin, and isoxanthopterin levels were significantly higher in samples from cancer patients than in those from healthy subjects. Further, xanthopterin and isoxanthopterin levels were compared in breast cancer and lung cancer patients, but no significant difference was observed. CONCLUSION: Some pteridine levels can be used as biomarkers for noninvasive diagnosis of cancer; however, more data is needed to support this hypothesis.