Charles Turner

Rapid and specific detection of clinically significant haemoglobinopathies using electrospray mass spectrometry–mass spectrometry

Increasing demand for population screening for the haemoglobinopathies gives rise to a requirement for high throughput systems, which allow for cost effective, rapid, sensitive and specific screening of clinically significant haemoglobins. We have developed a practical and efficient approach using tryptic digestion and electrospray triple quadrupole mass spectrometry–mass spectrometry (MSMS) in multiple reaction monitoring acquisition mode for the identification of the clinically important haemoglobin variants, S, C, DPunjab, OArab, and E. A total of 200 blood samples, comprising 52 haemoglobin AA, 57 AS (sickle cell trait), 44 AC (C trait), 16 SC (SC disease), 14 SS (sickle cell disease), 10 AE (E trait), 2 ADPunjab (DPunjab trait) and 1 each of AOArab (OArab trait), CC (C disease), DPunjabDPunjab (DPunjab disease), OArabOArab (OArab disease), and EE (E disease), have been analysed in parallel with existing phenotype and molecular methods. All haemoglobin variants were correctly identified by MSMS, with no false positives or false negatives. The system detects both heterozygotes and homozygotes and has potential applications in neonatal and antenatal screening.

Blood Pressure Control and Left Ventricular Mass in Children with Chronic Kidney Disease

BACKGROUND AND OBJECTIVES Heart disease is a major cause of death in young adults with chronic kidney disease (CKD). Left ventricular hypertrophy (LVH) is common and is associated with hypertension. The aims of this study were to evaluate whether there is a relationship between LVH and BP in children with CKD and whether current targets for BP control are appropriate. DESIGN, SETTING, PARTICIPANTS, & MEASUREMENTS In this single-center cross-sectional study, 49 nonhypertensive children, (12.6 ± 3.0 years, mean GFR 26.1 ± 12.9 ml/min per 1.73 m²) underwent echocardiographic evaluation and clinic and 24-hour ambulatory BP monitoring. LVH was defined using age-specific reference intervals for left ventricular mass index (LVMI). Biochemical data and clinic BP for 18 months preceding study entry were also analyzed. RESULTS The mean LVMI was 37.8 ± 9.1 g/m²·⁷, with 24 children (49%) exhibiting LVH. Clinic BP values were stable over the 18 months preceding echocardiography. Patients with LVH had consistently higher BP values than those without, although none were overtly hypertensive (> 95th percentile). Multiple linear regression demonstrated a strong relationship between systolic BP and LVMI. Clinic systolic BP showed a stronger relationship than ambulatory measures. Of the confounders evaluated, only elemental calcium intake yielded a consistent, positive relationship with LVMI. CONCLUSIONS LVMI was associated with systolic BP in the absence of overt hypertension, suggesting that current targets for BP control should be re-evaluated. The association of LVMI with elemental calcium intake questions the appropriateness of calcium-based phosphate binders in this population.

Biomarkers of rapid chronic kidney disease progression in type 2 diabetes

Here we evaluated the performance of a large set of serum biomarkers for the prediction of rapid progression of chronic kidney disease (CKD) in patients with type 2 diabetes. We used a case-control design nested within a prospective cohort of patients with baseline eGFR 30-60 ml/min per 1.73 m(2). Within a 3.5-year period of Go-DARTS study patients, 154 had over a 40% eGFR decline and 153 controls maintained over 95% of baseline eGFR. A total of 207 serum biomarkers were measured and logistic regression was used with forward selection to choose a subset that were maximized on top of clinical variables including age, gender, hemoglobin A1c, eGFR, and albuminuria. Nested cross-validation determined the best number of biomarkers to retain and evaluate for predictive performance. Ultimately, 30 biomarkers showed significant associations with rapid progression and adjusted for clinical characteristics. A panel of 14 biomarkers increased the area under the ROC curve from 0.706 (clinical data alone) to 0.868. Biomarkers selected included fibroblast growth factor-21, the symmetric to asymmetric dimethylarginine ratio, β2-microglobulin, C16-acylcarnitine, and kidney injury molecule-1. Use of more extensive clinical data including prebaseline eGFR slope improved prediction but to a lesser extent than biomarkers (area under the ROC curve of 0.793). Thus we identified several novel associations of biomarkers with CKD progression and the utility of a small panel of biomarkers to improve prediction.

Symmetric dimethylarginine, an endogenous marker of glomerular filtration rate, and the risk for microalbuminuria in young people with type 1 diabetes

Objectives To perform a cross-sectional comparison of endogenous markers of glomerular filtration rate (GFR) (plasma symmetric dimethylarginine (SDMA) and estimated GFR (eGFR)) with a direct measure of GFR (using the plasma clearance of Inutest (In-GFR)), and a longitudinal evaluation of these markers in relation to the development of microalbuminuria, in young people with type 1 diabetes (T1D). Methods Longitudinal stored blood samples (n=1105) were available from 417 young people from the Oxford Regional Prospective Study (an inception cohort of 527 children followed for a median of 10.3 (interquartile range 7.1–12.3) years), for measurement of SDMA and creatinine. Additional annually collected data on anthropometric parameters, HbA1c, insulin dose and three early morning albumin:creatinine ratios were available. In-GFR was measured in a representative subgroup of 183 subjects. Main outcome measures SDMA and eGFR. Results SDMA and eGFR were significantly and similarly associated with In-GFR (r=−0.38 and r=0.36, p<0.001). Overall SDMA levels were lower in microalbuminuric (n=116) than normoalbuminuric subjects (n=301) (0.385±0.063 vs 0.412±0.059 µmol/l, p<0.001), probably reflecting hyperfiltration. The pattern of change in SDMA levels with age differed between microalbuminuric and normoalbuminuric subjects. Whereas SDMA levels declined in both groups until the age of 16 years, thereafter they tended to rise only in microalbuminuric subjects, probably reflecting a decline in renal function. Conclusions In this longitudinal study of young people with T1D, measurement of SDMA, in contrast to eGFR, proved to be a reliable marker in identifying changes in filtration rates associated with the development of microalbuminuria (MA).

Investigating FGF-23 concentrations and its relationship with declining renal function in paediatric patients with pre-dialysis CKD Stages 3–5

BACKGROUND The aims of our study were to investigate (i) the prevalence of elevated fibroblast growth factor-23 (FGF-23), (ii) the relationship between FGF-23 concentrations and level of renal dysfunction and (iii) the main determinants of elevation of FGF-23 concentration in children with pre-dialysis chronic kidney disease (CKD) Stages 3-5. METHODS In this single-centre prospective observational study, 71 children with pre-dialysis CKD Stages 3-5, aged 11.9 ± 3.1 years, had FGF-23 levels measured. Anthropometry and routine laboratory investigations were measured. RESULTS Fourteen (19.7%) patients had normal FGF-23 concentrations defined as < 50 ng/L. FGF-23 [median (interquartile range)] concentrations were 78.7 (55.6-137.6) ng/L and following log transformation normalized data with log FGF-23 [mean (SD)] values of 1.96 ± 0.4 ng/L. Log FGF-23 concentrations had a negative reciprocal relationship with estimated glomerular filtration rate (eGFR) (P < 0.0001) and 1,25 vitamin D3 levels (P = 0.01) and a positive relationship with phosphate (P = 0.03) and percent fractional excretion of phosphate (P = 0.01) but not with log-intact parathyroid hormone (PTH) (P = 0.22). Multiple linear regression demonstrated a strong relationship between log FGF-23 and eGFR only. CONCLUSIONS Elevated FGF-23 concentrations were observed in the majority of a carefully managed cohort of children with non-dialysis CKD with a dominant effect on FGF-23 concentrations with glomerular filtration rate (GFR). These data allow the potential confounding effects of PTH and phosphate elevation with declining GFR to be removed, leaving a clearer picture of the FGF-23-GFR relationship.

Gut Permeability in Autism Spectrum Disorders

To test whether gut permeability is increased in autism spectrum disorders (ASD) by evaluating gut permeability in a population‐derived cohort of children with ASD compared with age‐ and intelligence quotient‐matched controls without ASD but with special educational needs (SEN).

Stability of metabolites in dried blood spots stored at different temperatures over a 2-year period

BACKGROUND Quantitative LC-ESI-MS/MS, developed from newborn screening, is increasingly used for targeted metabolite profiling. Dried blood spots (DBS) provide easily obtainable biological samples but long-term stability data are sparse. DBS were stored at ambient temperature (room temperature [RT]; 21°C), -20 and -80°C. Metabolites were analyzed at 12 time points (0-104 weeks) by LC-ESI-MS/MS, using fully quantitative stable isotope dilution. RESULTS Principal component analysis showed alterations in metabolite stability at different temperatures, with major changes only at RT. Univariate analysis for individual analytes demonstrated increases or reductions in concentration. CONCLUSION Significant changes are observed in certain DBS metabolites at RT, which are attenuated or not present when frozen. These data will help to inform the design, analysis and interpretation of future DBS studies.

Asymmetric dimethylarginine in young people with Type 1 diabetes: a paradoxical association with HbA1c

Diabet. Med. 28, 685–691 (2011)

Nitric oxide and l-arginine metabolism in a devascularized porcine model of acute liver failure

In acute liver failure (ALF), the hyperdynamic circulation is believed to be the result of overproduction of nitric oxide (NO) in the splanchnic circulation. However, it has been suggested that arginine concentrations (the substrate for NO) are believed to be decreased, limiting substrate availability for NO production. To characterize the metabolic fate of arginine in early-phase ALF, we systematically assessed its interorgan transport and metabolism and measured the endogenous NO synthase inhibitor asymmetric dimethylarginine (ADMA) in a porcine model of ALF. Female adult pigs (23-30 kg) were randomized to sham (N = 8) or hepatic devascularization ALF (N = 8) procedure for 6 h. We measured plasma arginine, citrulline, ornithine levels; arginase activity, NO, and ADMA. Whole body metabolic rates and interorgan flux measurements were calculated using stable isotope-labeled amino acids. Plasma arginine decreased >85% of the basal level at t = 6 h (P < 0.001), whereas citrulline and ornithine progressively increased in ALF (P < 0.001 and P < 0.001, vs. sham respectively). No difference was found between the groups in the whole body rate of appearance of arginine or NO. However, ALF showed a significant increase in de novo arginine synthesis (P < 0.05). Interorgan data showed citrulline net intestinal production and renal consumption that was related to net renal production of arginine and ornithine. Both plasma arginase activity and plasma ADMA levels significantly increased in ALF (P < 0.001). In this model of early-phase ALF, arginine deficiency or higher ADMA levels do not limit whole body NO production. Arginine deficiency is caused by arginase-related arginine clearance in which arginine production is stimulated de novo.

The measurement of urinary hydroxyurea in sickle cell anaemia

Hydroxyurea is increasingly used in the treatment of sickle cell disease (SCD) although there is little evidence on how best to monitor treatment and compliance. It is also not known why 10–50% patients do not benefit from the drug and whether some of this resistance is because of pharmacokinetic factors. We have developed an assay using mass spectrometry (MS) to measure urinary concentrations of hydroxyurea. We have used this assay to study 12 children and six adults with SCD taking hydroxyurea and found that urinary hydroxyurea was present for at least 12 h following tablet ingestion. Thirty‐five urine samples were analysed that were expected to contain hydroxyurea, based on the reported timing of the last dose and hydroxyurea was detected in 29 (83%) of these. There were also marked differences in urinary hydroxyurea concentrations, suggesting pharmacokinetic variability may explain some of the differences in response to hydroxyurea. Urine samples were also analysed by MS for penicillin metabolites and 43 of the 57 (75%) contained phenoxyacetate, suggesting the ingestion of penicillin within the last 12 h. These assays are potentially useful to study hydroxyurea metabolism further, develop optimal dosing regimes and monitor compliance with treatment.