A longitudinal study of the blood and urine metabolome of Vipera berus envenomated dogs.

Envenomation of dogs by the common European adder (Vipera berus) is associated with high morbidity. The cytotoxic venom of Vipera berus contains enzymes with the potential to cause acute kidney injury, among other insults, however robust biomarkers for such effects are lacking. A prospective observational follow-up study of naturally envenomated dogs and controls was conducted to fill knowledge gaps regarding canine Vipera berus envenomation, attempt to identify novel biomarkers of envenomation and related kidney injury, and elucidate potential long-term effects. Blood and urine samples were analyzed with a global metabolomics approach using liquid chromatography-mass spectrometry, uncovering numerous features significantly different between cases and controls. After data processing and feature annotation, eight features in blood and 24 features in urine were investigated in order to elucidate their biological relevance. Several of these are associated with AKI, while some may also originate from disturbed fatty acid ß-oxidation and soft tissue damage. A metabolite found in both blood and a venom reference sample may represent identification of a venom component in case dogs. Our findings suggest that envenomated dogs treated according to current best practice are unlikely to suffer permanent injury.

Tear and Saliva Metabolomics in Evaporative Dry Eye Disease in Females.

Accurate diagnosis of dry eye disease (DED) is challenging, and even today there is no gold standard biomarker of DED. Hypothesis-free global metabolomic studies of tears from DED patients have great potential to discover metabolites and pathways affected in the pathophysiology of DED, and to identify possible future biomarkers. These metabolites and biomarkers could be important for diagnosing and monitoring disease as well as for new therapeutic targets and strategies. As DED is associated with dry mouth, this study aimed to perform metabolomic analyses of tears and saliva from patients with decreased tear film break-up time but normal Schirmer test, and age-matched controls with both tear production and stability within physiological range. We applied strict inclusion criteria to reduce sampling bias in the metabolomic analyses and selected only age-matched females with Schirmer test values between 10-15 mm/5 min. The tear film analysis arm included 19 patients (with tear film break-up time 0-5 s) and 12 controls (with tear film break-up time 10-30 s), while the salivary analysis arm consisted of a subset which included 18 patients and six controls. Metabolomic analyses were performed using liquid chromatography and high-resolution mass spectrometry. Analyses using a global database search detected a total of 56 metabolites in tear samples that were significantly different between the groups. Of these, several have known associations with DED. These metabolites are present in meibum and have anti-oxidative characteristics or associations with the ocular microbiome, and altered concentrations suggest that they may play a significant role in DED associated with decreased tear film stability. In saliva, hypotaurine levels were lower among patients with tear film instability. In this pilot study, we found different levels of several metabolites in patients with decreased tear film break-up time that may have associations with DED. Future studies are required to replicate our findings and clarify the exact roles of these metabolites.

Continuous age- and sex-adjusted reference intervals of urinary markers for cerebral creatine deficiency syndromes: a novel approach to the definition of reference intervals.

BACKGROUND: Urinary concentrations of creatine and guanidinoacetic acid divided by creatinine are informative markers for cerebral creatine deficiency syndromes (CDSs). The renal excretion of these substances varies substantially with age and sex, challenging the sensitivity and specificity of postanalytical interpretation. METHODS: Results from 155 patients with CDS and 12 507 reference individuals were contributed by 5 diagnostic laboratories. They were binned into 104 adjacent age intervals and renormalized with Box-Cox transforms (Ξ). Estimates for central tendency (µ) and dispersion (σ) of Ξ were obtained for each bin. Polynomial regression analysis was used to establish the age dependence of both µ[log(age)] and σ[log(age)]. The regression residuals were then calculated as z-scores = {Ξ - µ[log(age)]}/σ[log(age)]. The process was iterated until all z-scores outside Tukey fences ±3.372 were identified and removed. Continuous percentile charts were then calculated and plotted by retransformation. RESULTS: Statistically significant and biologically relevant subgroups of z-scores were identified. Significantly higher marker values were seen in females than males, necessitating separate reference intervals in both adolescents and adults. Comparison between our reconstructed reference percentiles and current standard age-matched reference intervals highlights an underlying risk of false-positive and false-negative events at certain ages. CONCLUSIONS: Disease markers depending strongly on covariates such as age and sex require large numbers of reference individuals to establish peripheral percentiles with sufficient precision. This is feasible only through collaborative data sharing and the use of appropriate statistical methods. Broad application of this approach can be implemented through freely available Web-based software.

PGM3 mutations cause a congenital disorder of glycosylation with severe immunodeficiency and skeletal dysplasia.

Human phosphoglucomutase 3 (PGM3) catalyzes the conversion of N-acetyl-glucosamine (GlcNAc)-6-phosphate into GlcNAc-1-phosphate during the synthesis of uridine diphosphate (UDP)-GlcNAc, a sugar nucleotide critical to multiple glycosylation pathways. We identified three unrelated children with recurrent infections, congenital leukopenia including neutropenia, B and T cell lymphopenia, and progression to bone marrow failure. Whole-exome sequencing demonstrated deleterious mutations in PGM3 in all three subjects, delineating their disease to be due to an unsuspected congenital disorder of glycosylation (CDG). Functional studies of the disease-associated PGM3 variants in E. coli cells demonstrated reduced PGM3 activity for all mutants tested. Two of the three children had skeletal anomalies resembling Desbuquois dysplasia: short stature, brachydactyly, dysmorphic facial features, and intellectual disability. However, these additional features were absent in the third child, showing the clinical variability of the disease. Two children received hematopoietic stem cell transplantation of cord blood and bone marrow from matched related donors; both had successful engraftment and correction of neutropenia and lymphopenia. We define PGM3-CDG as a treatable immunodeficiency, document the power of whole-exome sequencing in gene discoveries for rare disorders, and illustrate the utility of genomic analyses in studying combined and variable phenotypes.

Liquid chromatography-tandem mass spectrometry determination of oxalate in spot urine.

BACKGROUND: For assessment of total oxalic acid (OX) status, reliable quantification of OX in both urine and plasma is important. For urine, but not plasma, a commercial kit is available. We have recently described a LC-MSMS method for OX in plasma. The aim of the present study was to evaluate the usefulness of this assay for urine. We also wanted to evaluate if 24 h urine collection could be substituted by OX/creatinine-ratio (U-OX/crea) in spot-urine, and establish precursory reference intervals for U-OX/crea in children and adults. METHODS: Acidified urines were analysed and relevant validation parameters assessed. Diurnal excretion patterns were investigated in nine healthy volunteers on self-chosen diets. For method comparison, 29 urine samples were analysed with both the present method and a commercial urine-oxalate kit. Precursory reference values for U-OX/crea in children and adults (N=103, 1 month-76 years) were calculated. RESULTS: The within-batch coefficient of variation (CV) was 2.5% and a relative recovery of 97% in urine spiked with 5-200 micromol/L OX was found. The LC-MSMS method gave 7.9% higher OX values compared to the kit. No significant diurnal pattern of U-OX/crea was observed. U-OX/crea in children decreases with age, with no gender dependency. In adults no age variation was found, but females had somewhat higher U-OX/Crea compared to males. CONCLUSION: The LC-MSMS method has proven useful for urinary OX quantification. Random spot-urine samples can be used. Age-dependent reference limits for U-OX/crea must be applied in children, in contrast to adults.

Plasma oxalate following kidney transplantation in patients without primary hyperoxaluria.

BACKGROUND: Patients with primary hyperoxaluria may need repeated kidney transplants due to damage from oxalic acid (oxalate) deposits. However, oxalate may also be potentially harmful in all transplant recipients. Determinants of oxalate following transplantation have not been well studied. METHODS: Two hundred and twelve recipients admitted for transplantation were included in the study. Blood samples for measurement of oxalate and other relevant laboratory parameters were collected at baseline and subsequently 10 weeks after transplantation. For oxalate determination, samples were obtained in 99, 167 and 54 patients out of the 212 at baseline, at follow-up and at both time points, respectively. We examined the bivariate association between plasma oxalate at transplantation and preemptive transplantation, time on dialysis, recipient age, creatinine, urea, phosphate, haemoglobin, PTH, albumin and calcium. Oxalate 10 weeks after transplantation was tested likewise including also laboratory parameters at baseline, primary non-function, rejection episodes, live versus deceased donor, donor age and GFR at follow-up. RESULTS: Median plasma oxalate concentration at transplantation was 35.0 micromol/L [95% confidence interval (95% CI) = 10.4-93.9] and 98% of the values were above normal limits (2.6-11.0). Oxalate concentration after 10 weeks was 9.0 micromol/L (4.0-25.5), still 37% being above the upper normal value. Multiple regression analysis revealed established dialysis treatment (P = 0.002) and creatinine (P < 0.000001) as independent positive determinants of oxalate at transplantation. Oxalate at 10 weeks was negatively associated to (51)Cr-EDTA absolute GFR (P = 0.023) and positively associated to donor age (P = 0.027) and plasma creatinine at 10 weeks (P = 0.03). CONCLUSION: At transplantation, plasma oxalate was on average three times increased and above the upper normal limit in 98% of patients and were still above normal in 37% after 10 weeks. The reduction after 10 weeks is determined by GFR and donor age. Whether increased plasma oxalate following kidney transplantation may have long-term consequences needs further study.