Liquid chromatography-tandem mass spectrometry of porphyrins and porphyrinogens in biological materials: separation and identification of interfering poly(ethylene) glycol by travelling wave ion mobility spectrometry/tandem mass spectrometry.

Biological and clinical samples for porphyrin and porphyrinogen analyses by liquid chromatography-tandem mass spectrometry (LC-MS/MS) are often contaminated with poly(ethylene)glycol (PEG), which complicates the interpretation of mass spectra and characterisation of new porphyrin metabolites. Two contaminating PEG molecules (m/z 833 and m/z 835) were completely separated from uroporphyrin I (m/z 831) by travelling wave ion mobility spectrometry and characterised by tandem mass spectrometry. One of the PEG species (m/z 835) also co-eluted with uroporphyrinogen I (m/z 837) and was unresolvable by travelling wave ion mobility spectrometry/MS, therefore contaminating the MS/MS mass spectra owing to isotope distribution. These PEG species, with the [M + H](+) ions at m/z at 833 and/or m/z 835, co-eluted with uroporphyrin I and uroporphyrinogen I by LC-MS/MS and could be wrongly identified as uroporphomethenes.

Rapid development of sensitive, high-throughput, quantitative and highly selective mass spectrometric targeted immunoassays for clinically important proteins in human plasma and serum.

OBJECTIVES: The aim of this study was to develop high-throughput, quantitative and highly selective mass spectrometric, targeted immunoassays for clinically important proteins in human plasma or serum. DESIGN AND METHODS: The described method coupled mass spectrometric immunoassay (MSIA), a previously developed technique for immunoenrichment on a monolithic microcolumn activated with an anti-protein antibody and fixed in a pipette tip, to selected reaction monitoring (SRM) detection and accurate quantification of targeted peptides, including clinically relevant sequence or truncated variants. RESULTS: In this report, we demonstrate the rapid development of MSIA-SRM assays for sixteen different target proteins spanning seven different clinically important areas (including neurological, Alzheimer's, cardiovascular, endocrine function, cancer and other diseases) and ranging in concentration from pg/mL to mg/mL. The reported MSIA-SRM assays demonstrated high sensitivity (within published clinical ranges), precision, robustness and high-throughput as well as specific detection of clinically relevant isoforms for many of the target proteins. Most of the assays were tested with bona-fide clinical samples. In addition, positive correlations, (R2 0.67-0.87, depending on the target peptide), were demonstrated for MSIA-SRM assay data with clinical analyzer measurements of parathyroid hormone (PTH) and insulin growth factor 1 (IGF1) in clinical sample cohorts. CONCLUSIONS: We have presented a practical and scalable method for rapid development and deployment of MS-based SRM assays for clinically relevant proteins and measured levels of the target analytes in bona fide clinical samples. The method permits the specific quantification of individual protein isoforms and addresses the difficult problem of protein heterogeneity in clinical proteomics applications.

Total protein, albumin and low-molecular-weight protein excretion in HIV-positive patients.

BACKGROUND: Chronic kidney disease is common in HIV positive patients and renal tubular dysfunction has been reported in those receiving combination antiretroviral therapy (cART). Tenofovir (TFV) in particular has been linked to severe renal tubular disease as well as proximal tubular dysfunction. Markedly elevated urinary concentrations of retinal-binding protein (RBP) have been reported in patients with severe renal tubular disease, and low-molecular-weight proteins (LMWP) such as RBP may be useful in clinical practice to assess renal tubular function in patients receiving TFV. We analysed 3 LMWP as well as protein and albumin in the urine of a sample of HIV positive patients. METHODS: In a cross-sectional fashion, total protein, albumin, RBP, cystatin C, and neutrophil gelatinase-associated lipocalin (NGAL) were quantified in random urine samples of 317 HIV positive outpatients and expressed as the ratio-to-creatinine (RBPCR, CCR and NGALCR). Exposure to cART was categorised as none, cART without TFV, and cART containing TFV and a non-nucleoside reverse-transcriptase-inhibitor (TFV/NNRTI) or TFV and a protease-inhibitor (TFV/PI). RESULTS: Proteinuria was present in 10.4 % and microalbuminuria in 16.7 % of patients. Albumin accounted for approximately 10 % of total urinary protein. RBPCR was within the reference range in 95 % of patients while NGALCR was elevated in 67 % of patients. No overall differences in urine protein, albumin, and LMWP levels were observed among patients stratified by cART exposure, although a greater proportion of patients exposed to TFV/PI had RBPCR >38.8 µg/mmol (343 µg/g) (p = 0.003). In multivariate analyses, black ethnicity (OR 0.43, 95 % CI 0.24, 0.77) and eGFR <75 mL/min/1.73 m2 (OR 3.54, 95 % CI 1.61, 7.80) were independently associated with upper quartile (UQ) RBPCR. RBPCR correlated well to CCR (r2 = 0.71), but not to NGALCR, PCR or ACR. CONCLUSIONS: In HIV positive patients, proteinuria was predominantly of tubular origin and microalbuminuria was common. RBPCR in patients without overt renal tubular disease was generally within the reference range, including those receiving TFV. RBP therefore appears a promising biomarker for monitoring renal tubular function in patients receiving TFV and for distinguishing patients with normal tubular function or mild tubular dysfunction from those with severe renal tubular disease or Fanconi syndrome.

Genomic and metabolomic patterns segregate with responses to calcium and vitamin D supplementation.

Inter-individual response differences to vitamin D and Ca supplementation may be under genetic control through vitamin D and oestrogen receptor genes, which may influence their absorption and/or metabolism. Metabolomic studies on blood and urine from subjects supplemented with Ca and vitamin D reveal different metabolic profiles that segregate with genotype. Genotyping was performed for oestrogen receptor 1 gene (ESR1) and vitamin D receptor gene (VDR) in fifty-six postmenopausal women. Thirty-six women were classified as low bone density as determined by a heel ultrasound scan and twenty women had normal bone density acting as 'controls'. Those with low bone density (LBD) were supplemented with oral Ca and vitamin D and were classified according to whether they were 'responders' or 'non-responders' according to biochemical results before and after therapy compared to controls receiving no supplementation. Metabolomic studies on serum and urine were done for the three groups at 0 and 3 months of therapy using NMR spectroscopy with pattern recognition. The 'non-responder' group showed a higher frequency of polymorphisms in the ESR1 (codons 10 and 325) and VDR (Bsm1 and Taq1), compared with to the 'responders'. The wild-type genotype for Fok1 was more frequent in those with LBD (70 %) compared with the control group (10 %). Distinctive patterns of metabolites were displayed by NMR studies at baseline and 3 months of post-treatment, segregating responders from non-responders and controls. Identification of potential 'non-responders' to vitamin D and Ca, before therapy, based on a genomic and/or metabolomic profile would allow targeted selection of optimal therapy on an individual basis.

Lymph node involvement and surgical approach in parathyroid cancer.

BACKGROUND: The best surgical approach to parathyroid cancer is disputed. Recommendations vary and are built on incoherent evidence. High rates of recurrence and death require an in-depth review of underlying findings. METHODS: This retrospective study includes 11 patients with parathyroid cancer who underwent surgery with central and/or lateral neck dissection by a single surgeon between 2005 and 2010. The diagnosis was based on histopathological criteria in all patients. Patterns of lymph node and soft tissue involvement of these and formerly reported patients were analysed based on full-text review of all published cases of parathyroid cancer. RESULTS: In this series only 1 of 11 patients (9.1%) manifested lymph node metastasis. In the literature, lymph node metastases have been reported in only 6.5% of 972 published patients, or in 32.1% of the 196 in whom lymph node involvement was assessed by the authors. They were, with few exceptions, localised in the central compartment. Recurrence in soft tissue is more frequent than in locoregional lymph nodes. CONCLUSION: Oncological en bloc clearance of the central compartment with meticulous removal of all possibly involved soft tissues, including a systematic central lymph node resection, may improve outcomes and should be included in the routine approach to the suspicious parathyroid lesion. There is no need for a prophylactic lateral neck dissection.

Postprandial bone turnover is independent of calories above 250 kcal.

BACKGROUND: The mechanisms causing bone turnover after food intake have not yet been elucidated. Several gut hormones are secreted in the postprandial phase, proportional to meal calorie content, and possibly one or more of these could influence bone turnover. The aim of this study was to investigate bone turnover in proportion to graded-calorie and fixed calcium containing meals. METHODS: A group of healthy volunteers were given six meals with calories varying from 250 to 3000 kcal on different occasions. All the meals contained 500 mg of calcium. C-telopeptide type I collagen (CTX) was measured before and 180 min after each meal. RESULTS: All meals significantly reduced CTX between 35.8 +/- 5.6% and 44.8 +/- 3.8%. No significant difference in CTX was however apparent for the different calorie containing meals. Observed differences suggest a trend to greater CTX suppression with lower protein and higher fat content of meals. CONCLUSION: Changes in CTX are not proportional to calorie contents when the meals contain 500 mg of calcium. Further studies should now determine whether patients with increased bone resorption would benefit from multiple small meals to slow down the rate of bone loss.

High-dose fish oil and antioxidants in Crohn's disease and the response of bone turnover: a randomised controlled trial.

Crohn's disease is associated with altered bone turnover that may be influenced by nutritional status, the systemic inflammatory response, cytokine production by circulating (peripheral blood) mononuclear cells (PBMC) and antioxidant micronutrient intake. High-dose fish oil is associated with reductions in disease relapse and inflammatory markers, and modulates PBMC function. The effect of fish oil plus antioxidants on bone turnover and PBMC function (the production of interferon-gamma and prostaglandin E2) in Crohn's disease was investigated in a randomised-controlled trial. Patients with currently or recently raised biochemical markers of inflammation (C-reactive protein > or = 6.9 mg/l or erythrocyte sedimentation rate > or =18 mm/h) received fish oil (providing 2.7 g/d EPA and DHA) and antioxidants (vitamins A, C and E, and Se) (n 31) or placebo (n 30) for 24 weeks. Bone turnover was assessed by measuring the concentrations of urinary deoxypyridinoline (bone resorption) and serum osteocalcin (bone formation). Fish oil plus antioxidants were associated with increases in EPA, DHA Se in plasma (all P < 0.01), and with a reduction in interferon-gamma production by mitogen-stimulated PBMC, which demonstrated a negative correlation with deoxypyridinoline/creatinine:osteocalcin ratio (r - 0.33, P = 0.009). There were no differences between the groups at 24 weeks in the response of deoxypyridinoline or osteocalcin or their ratio, or in nutritional status. Dietary supplementation in Crohn's disease with high intakes of EPA and DHA, as fish oil, plus antioxidants was associated with a modulated production of interferon-gamma by PBMC but not altered indices of bone turnover.

Bone mineral density and height gain in children with chronic cholestatic liver disease undergoing transplantation.

BACKGROUND: Osteodystrophy is a well-described complication of chronic liver disease. Previous reports in adults and children undergoing liver transplantation (LT) were discordant, with the former showing no improvement of bone disease in the first year after transplantation and the latter demonstrating remarkable benefit from it. Our aim was to perform a pilot study on osteodystrophy in children undergoing LT and evaluate the contribution of growth on bone mineral density (BMD) changes. METHODS: We studied six patients (two male), with a median age at transplantation of 8.8 (range 3.8-16.6) years. Indications for transplantation were biliary atresia and progressive familial intrahepatic cholestasis (three patients each). BMD was studied with dual-energy x-ray absorptiometry and biochemical markers of liver and bone function in patients before and at 3, 6, and 12 months after LT. RESULTS: Median L2-L4 spinal BMD was 0.54 g/cm2 (range 0.29-0.87) before LT, and 0.58 g/cm2 (0.27-0.86) at 3 months, 0.66 g/cm2 (0.36-1.00) at 6 months, and 0.76 g/cm2 (0.44-1.02) at 12 months after LT (P=0.005). Median height was 133 (range 93-167) cm before LT, and 134 (93-167) at 3 months, 136 (97-167) at 6 months, and 139 (102-167) at 12 months after LT. There was direct correlation between height gain and total body BMD improvement (r=0.929, P=0.007). CONCLUSION: BMD in children with chronic cholestatic liver disease improves remarkably by 12 months after LT. Catch-up growth in children can account for the different effect of LT on bone density between adult and pediatric populations in the first year after surgery.