Method design and validation for the determination of uranium levels in human urine using high-resolution alpha spectrometry

Quantification of uranium in human urine is a valuable technique for assessing occupational and public exposure to uranium. A reliable method has been developed and validated in the ARPANSA Radiochemistry Laboratory by means of standard radiochemical separation and purification techniques and measurement using high-resolution alpha spectrometry. This method can be used to evaluate the levels of naturally occurring ²³⁴U, ²³⁵U and ²³⁸U in urine. Method design and validation is the process of defining an analytical requirement, and then confirming that the method under consideration has performance capabilities consistent with what the application requires. The method was designed to measure levels down to 2 mBq/day of total uranium, corresponding to approximately 1/100th of the annual committed effective dose of 20 mSv. Validation tests were developed to assess selectivity, accuracy, recovery and quantification of uncertainty. The radiochemical recovery of this method was measured using ²³²U tracer. The typical minimum detectable concentration for total uranium for 24-h urine samples is approximately 0.6 mBq/day or 0.019 μg/day.

IEF pattern classification-derived criteria for the identification of epoetin-delta in urine

Epoetin-δ (Dynepo™ Shire Pharmaceuticals, Basing stoke, UK) is a synthetic form of erythropoietin (EPO) whose resemblance with endogenous EPO makes it hard to identify using the classical identification criteria. Urine samples collected from six healthy volunteers treated with epoetin-δ injections and from a control population were immuno-purified and analyzed with the usual IEF method. On the basis of the EPO profiles integration, a linear multivariate model was computed for discriminant analysis. For each sample, a pattern classification algorithm returned a bands distribution and intensity score (bands intensity score) saying how representative this sample is of one of the two classes, positive or negative. Effort profiles were also integrated in the model. The method yielded a good sensitivity versus specificity relation and was used to determine the detection window of the molecule following multiple injections. The bands intensity score, which can be generalized to epoetin-α and epoetin-β, is proposed as an alternative criterion and a supplementary evidence for the identification of EPO abuse.

Effect of a low sodium DASH diet, including red meat on blood pressure in post-menopausal women

Background – The DASH type dietary pattern which consists of high fruit, vegetable and dairy products and low saturated fat, is “base-producing” but restricts red meat with no clear justification.
Objective – To compare the BP-lowering effect of Vitality diet (VD), a moderately low sodium, “base” producing modified DASH diet, containing 6 serves/week of lean red meat to a “ high carbohydrate, low fat diet (HCLF diet), with a higher dietary acid load in post-menopausal women.
Design – Ninety-five hypertensive post-menopausal women (46 VD and 49 HCLF) completed a 14-wk randomised parallel study. Home BP was measured daily. Repeat 24-h dietary records and 24-h urine samples were collected fortnightly. Dietary acid load, expressed as potential renal acid load (PRAL), was calculated from nutrient intakes.
Outcomes – During the intervention, the VD group had an average daily consumption of 85 g cooked red meat. They had a mean (± SEM) reduction of 38 ± 7 mmol/d in urinary sodium excretion (P <0.0001), and a 7 ± 4 mmol/d increase in urinary potassium (P = 0.0681), with an estimated 23.1± 2.3 mEq/d lower PRAL than the HCLF group (P <0.0001). The fall in systolic pressure in the VD group tended to be greater by 3 ± 2 mmHg (P = 0.08) than the fall in systolic pressure seen with the HCLF diet. A greater BP-lowering effect of VD was observed among those taking anti-hypertensive medication (n = 17) with a greater 5.5 ± 2.7 mm Hg (P = 0.0518) reduction of systolic BP and greater reduction in diastolic BP by 3.6 ± 1.7 mm Hg (P = 0.0388) compared to the HCLF diet. However, no relationship between BP and PRAL was observed.
Conclusions – A low sodium DASH type dietary pattern with the inclusion of lean red meat was effective in reducing BP in post-menopausal women, particularly in those taking anti-hypertensive medication. This dietary pattern could be recommended for this group who are at increased risk of cardiovascular disease.
This study was funded by Meat & Livestock Australia.

Relationship of heart failure patients' knowledge, perceived barriers, and attitudes regarding low-sodium diet recommendations to adherence.

The purposes of this study were to describe heart failure patient perceptions regarding instructions received for following a low-sodium diet and the benefits, barriers, and ease and frequency of following the diet. A total of 246 patients with heart failure referred from academic medical centers in the United States and Australia participated in the study. A subset of 145 patients provided 24-hour urine samples for sodium excretion assessment. While most (80%) patients reported receiving recommendations to follow a low-sodium diet, their recall of specific instructions was poor. Although the majority (75%) reported following a low-sodium diet most or all of the time, 24-hour urine sodium excretion indicated that only 25% of patients were adherent. Patients who reported being more adherent, however, had lower urine sodium excretion levels. Attitudes regarding difficulty in and perceived benefits of following the diet were not related to sodium excretion. Data on attitudes and barriers provided guidance for strategies to improve adherence.

Effects of exercise on the isoelectric patterns of erythropoietin

Objectives: Recombinant erythropoietin has a strong impact on aerobic power and is therefore one of the most potent doping agents in endurance sports. The anti-doping control of this synthetic hormone relies on the detection, in the urine, of its isoelectric pattern, which differs from that of the corresponding natural hormone, the latter being typically more acidic than the former. However, a small number of natural urinary patterns, referred to as atypical patterns, are less acidic than the dominant form. Based on anecdotal evidence, the occurrence of such patterns seems to be related to particular strenuous exercises. This study aimed to demonstrate this relation using a strenuous exercise protocol.

Design: Seven athletes took part in a training protocol including a series of supramaximal short-duration exercises. Urine and blood samples were collected throughout the protocols.

Settings: World Cycling Center, Aigle, Switzerland, and research laboratories.

Participants: Seven top-level athletes (cyclists) were involved in this study.

Main Outcome Measures: Erythropoietin (EPO) isoelectric patterns were obtained by submitting blood and urine samples to isoelectric focusing. Additional protein dosages were performed.

Results: Supramaximal short-duration exercises induced the transformation of typical urinary natural EPO patterns into atypical ones. None of the obtained atypical patterns fulfilled the 3 criteria mandatory for reporting an adverse analytical finding. Serum EPO patterns were not affected by the exercises that caused the transformation of urinary patterns.

Conclusion: An exercise-induced transient renal dysfunction is proposed as a hypothetic explanation for these observations that rely on parallel investigations of proteinuria in the same samples.

(1)H-NMR analysis of the human urinary metabolome in response to an 18-month multi-component exercise program and calcium-vitamin-D3 supplementation in older men

The musculoskeletal benefits of calcium and vitamin-D3 supplementation and exercise have been extensively studied, but the effect on metabolism remains contentious. Urine samples were analyzed by (1)H-NMR spectroscopy from participants recruited for an 18-month, randomized controlled trial of a multi-component exercise program and calcium and vitamin-D3 fortified milk consumption. It was shown previously that no increase in musculoskeletal composition was observed for participants assigned to the calcium and vitamin-D3 intervention, but exercise resulted in increased bone mineral density, total lean body mass, and muscle strength. Retrospective metabolomics analysis of urine samples from patients involved in this study revealed no distinct changes in the urinary metabolome in response to the calcium and vitamin-D3 intervention, but significant changes followed the exercise intervention, notably a reduction in creatinine and an increase in choline, guanidinoacetate, and hypoxanthine (p < 0.001, fold change > 1.5). These metabolites are intrinsically involved in anaerobic ATP synthesis, intracellular buffering, and methyl-balance regulation. The exercise intervention had a marked effect on the urine metabolome and markers of muscle turnover but none of these metabolites were obvious markers of bone turnover. Measurement of specific urinary exercise biomarkers may provide a basis for monitoring performance and metabolic response to exercise regimes.

24-h urinary sodium excretion is associated with obesity in a cross-sectional sample of Australian schoolchildren

Emerging evidence indicates that dietary Na may be linked to obesity; however it is unclear whether this relationship is independent of energy intake (EI). The aim of this study was to assess the association between Na intake and measures of adiposity, including BMI z score, weight category and waist:height ratio (WHtR), in a sample of Australian schoolchildren. This was a cross-sectional study of schoolchildren aged 4-12 years. Na intake was assessed via one 24-h urine collection. BMI was converted to age- and sex-specific z scores, and WHtR was used to define abdominal obesity. In children aged ≥8 years, EI was determined via one 24-h dietary recall. Of the 666 children with valid urine samples 55 % were male (average age 9·3 (sd 1·8) years). In adjusted models an additional 17 mmol/d of Na was associated with a 0·10 higher BMI z score (95 % CI 0·07, 0·13), a 23 % (OR 1·23; 95 % CI 1·16, 1·31) greater risk of being overweight/obese and a 15 % (OR 1·15; 95 % CI 1·09, 1·23) greater risk of being centrally obese. In the subsample of 8-12-year-old children (n 458), adjustment for EI did not markedly alter the associations between Na and adiposity outcomes. Using a robust measure of daily Na intake we found a positive association between Na intake and obesity risk in Australian schoolchildren, which could not be explained by total energy consumption. To determine whether this is a causal relationship, longitudinal studies, with high-quality measures of Na and EI, are required.

Profiling of polar metabolites in biological extracts using diamond hydride-based aqueous normal phase chromatography

Highly polar metabolites, such as sugars and most amino acids are not retained by conventional RP LC columns. Without sufficient retention low concentration compounds are not detected due ion suppression and structural isomers are not resolved. In contrast, hydrophilic interaction chromatography (HILIC) and aqueous normal phase chromatography (ANP) retain compounds based on their hydrophilicity and therefore provides a means of separating highly polar compounds. Here, an ANP method based on the diamond hydride stationary phase is presented for profiling biological small molecules by LC. A rapid separation system based upon a fast gradient that delivers reproducible chromatography is presented. Approximately 1000 compounds were reproducibly detected in human urine samples and clear differences between these samples were identified. This chromatography was also applied to xylem fluid from soyabean (Glycine max) plants to which 400 compounds were detected. This method greatly increases the metabolite coverage over RP-only metabolite profiling in biological samples. We show that both forms of chromatography are necessary for untargeted comprehensive metabolite profiling and that the diamond hydride stationary phase provides a good option for polar metabolite analysis.

Randomized controlled study of the urinary excretion of biophenols following acute and chronic intake of olive leaf supplements

Olive leaf supplement was characterised by HPLC and administered to healthy young adults over 28 d (three tablets or equivalent liquid dose per day), or in a single bolus dose of three tablets (or liquid equivalent). Oleuropein was the major biophenol in the extracts. There are no data on the excretion of urinary end-products of the metabolism of the olive leaf biophenols. Following both chronic and acute ingestion neither oleuropein, nor its hydrolysis product, hydroxytyrosol, were detected in urine samples. However, glucuronic acid conjugates, derived from oleuropein aglycone were detected in all urine samples up to 6 h following acute ingestion. The data suggest that oleuropein is bioavailable, which is a necessary pre-condition for bioactivity.

Association between parent and child dietary sodium and potassium intakes as assessed by 24-h urinary excretion

The aim of this study was to assess the association between parent and child sodium (Na) and potassium (K) intake as assessed by 24-h urinary excretion (24hUE). Primary school children and their parent(s) provided one 24-h urine sample and information on cooking and children's discretionary salt use. Valid urine samples were provided by 108 mothers (mean age 41.8 (5.1) (SD) years, Na 120 (45) mmol/day) (7.0 g/day salt equivalent) and 40 fathers (44.4 (4.9) years, Na 152 (49) mmol/day (8.9 g/day salt), and 168 offspring (51.8% male, age 9.1 (2.0) years, Na 101 (47) mmol/day (5.9 g/day salt). When adjusted for parental age, child age and gender a 17 mmol/day Na (1 g/day salt) increase in mother's 24hUE was associated with a 3.4 mmol/day Na (0.2 g/day salt) increase in child's salt 24hUE (p = 0.04) with no association observed between father and child. Sixty-seven percent of parents added salt during cooking and 37% of children added salt at the table. Children who reported adding table salt had higher urinary excretion than those who did not (p = 0.01). The association between mother and child Na intake may relate to the consumption of similar foods and highlights the importance of the home environment in influencing total dietary sodium intake.

Effects of a community-based salt reduction program in a regional Australian population

BACKGROUND: Salt reduction is a public health priority but there are few studies testing the efficacy of plausible salt reduction programs.

METHODS: A multi-faceted, community-based salt reduction program using the Communication for Behavioral Impact framework was implemented in Lithgow, Australia. Single 24-h urine samples were obtained from 419 individuals at baseline (2011) and from 572 at follow-up (2014). Information about knowledge and behaviors relating to salt was also collected.

RESULTS: Survey participants were on average 56 years old and 58 % female. Mean salt intake estimated from 24-h urine samples fell from 8.8 g/day (SD = 3.6 g/day) in 2011 to 8.0 (3.6) g/day in 2014 (-0.80, 95 % confidence interval -1.2 to -0.3;p < 0.001). There were significant increases in the proportion of participants that knew the recommended upper limit of salt intake (18 % vs. 29 %; p < 0.001), knew the importance of salt reduction (64 % vs. 78 %; p < 0.001) and reported changing their behaviors to reduce their salt intake by using spices (5 % vs. 28 %; p < 0.001) and avoiding eating out (21 % vs. 34 %; p < 0.001). However, the proportions that checked food labels (30 % vs. 25 %; p = 0.02) fell, as did the numbers avoiding processed foods (44 % vs. 35 %; p = 0.006). Twenty-six percent reported using salt substitute at the end of the intervention period and 90 % had heard about the program. Findings were robust to multivariable adjustment.

CONCLUSIONS: Implementation of this multi-faceted community-based program was associated with a ~10 % reduction in salt consumption in an Australian regional town. These findings highlight the potential of well-designed health promotion programs to compliment other population-based strategies to bring about much-needed reductions in salt consumption.

Salt intakes, knowledge, and behavior in Samoa: monitoring salt-consumption patterns through the World Health Organization's Surveillance of Noncommunicable Disease Risk Factors (STEPS)

This project measured population salt intake in Samoa by integrating urinary sodium analysis into the World Health Organization's (WHO's) STEPwise approach to surveillance of noncommunicable disease risk factors (STEPS). A subsample of the Samoan Ministry of Health's 2013 STEPS Survey collected 24-hour and spot urine samples and completed questions on salt-related behaviors. Complete urine samples were available for 293 participants. Overall, weighted mean population 24-hour urine excretion of salt was 7.09 g (standard error 0.19) to 7.63 g (standard error 0.27) for men and 6.39 g (standard error 0.14) for women (P=.0014). Salt intake increased with body mass index (P=.0004), and people who added salt at the table had 1.5 g higher salt intakes than those who did not add salt (P=.0422). A total of 70% of the population had urinary excretion values above the 5 g/d cutoff recommended by the WHO. A reduction of 30% (2 g) would reduce average population salt intake to 5 g/d, in line with WHO recommendations. While challenging, integration of salt monitoring into STEPS provides clear logistical and cost benefits and the lessons communicated here can help inform future programs.

Maternal creatine in pregnancy: a retrospective cohort study.

OBJECTIVE: To estimate creatine concentrations in maternal plasma and urine, and establish relationships with maternal characteristics, diet and fetal growth. DESIGN: Retrospective cohort study. SETTING: Lyell McEwin Hospital, Adelaide, Australia. POPULATION: A biobank of plasma and urine samples collected at 13, 18, 30 and 36 weeks' gestation from 287 pregnant women from a prospective cohort of asthmatic and non-asthmatic women. METHODS: Creatine was measured by enzymatic analysis. Change in creatine over pregnancy was assessed using the Friedman test. Linear mixed models regression was used to determine associations between maternal factors and diet with creatine across pregnancy and between creatine with indices of fetal growth at birth. MAIN OUTCOME MEASURES: Maternal creatine concentrations, associations between maternal factors and creatine and between creatine and fetal growth parameters. RESULTS: Maternal smoking, body mass index, asthma and socio-economic status were positively and parity negatively associated with maternal plasma and/or urine creatine. Maternal urine creatine concentration was positively associated with birthweight centile and birth length. After adjustment, each μmol/l increase in maternal urinary creatine was associated with a 1.23 (95% CI 0.44-2.02) unit increase in birthweight centile and a 0.11-cm (95% CI 0.03-0.2) increase in birth length. CONCLUSIONS: Maternal factors and fetal growth measures are associated with maternal plasma and urine creatine concentrations. TWEETABLE ABSTRACT: Maternal creatine is altered by pregnancy; fetal growth measures are associated with maternal creatine concentrations.

Direct processing of clinically relevant large volume samples for the detection of sexually transmitted infectious agents from urine on a microfluidic device

Urine is a preferred specimen for nucleic acid-based detection of sexually transmitted infections (STIs) but represents a challenge for microfluidic devices due to low analyte concentrations. We present an extraction methodology enabling rapid on-chip nucleic acid purification directly from clinically relevant sample volumes up to 1 ml and subsequent PCR amplification detection.