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Aim To examine transaminitis in obese children, its association with glucose metabolism and the metabolic syndrome and the response to weight loss through lifestyle modification. Methods 216 children (90 male), aged 2.9–17.6 (median 12.4 years) with median body mass index (BMI) SD score (SDS) of 3.36 (range 1.92–6.22) attending a hospital obesity clinic in Bristol (UK) underwent an oral glucose tolerance test (OGTT) with fasting lipid and liver profile. Auxological measures included weight, height, waist circumference, percentage body fat. Parental history of type 2 diabetes (T2DM) was recorded. The 2007 International Diabetes Federation definition of metabolic syndrome was used. 90 children undergoing a trial of lifestyle modification to improve weight were re-assessed at 12 months. Results 34/216 (16%) children had raised alanine aminotransferase (ALT) (>40 IU/l) with greater prevalence in boys (23% vs 10%, p=0.01) and in those with a parental history of T2DM (30% vs 13.2% p=0.019). Patients with transaminitis were more likely to fulfil the criteria for metabolic syndrome (p<0.001) and have subtle abnormalities in glucose metabolism during an OGTT with elevated glucose levels at 90 (p=0.041) and 120 (p=0.014) min and a greater glucose area-under-the-curve (p=0.014). Improvement in BMI SDS over 1 year correlated with improvement in ALT levels (τ = −0.29, p<0.001). Conclusion Prevalence of transaminitis is significant in obese children and is associated with components of the metabolic syndrome. Lifestyle-based improvement in BMI SDS offers an effective tool for correcting transaminitis and should remain the central component of therapy.

Pompe disease is a lysosomal and neuromuscular disorder caused by deficiency of acid alpha-glucosidase (GAA), and causes classic infantile, childhood onset, or adulthood onset phenotypes. The biochemical diagnosis is based on GAA activity assays in dried blood spots, leukocytes, or fibroblasts. Diagnosis can be complicated by the existence of pseudodeficiencies, i.e., GAA variants that lower GAA activity but do not cause Pompe disease. A large-scale comparison between these assays for patient samples, including exceptions and borderline cases, along with clinical diagnoses has not been reported so far. Here we analyzed GAA activity in a total of 1709 diagnostic cases over the past 28 years using a total of 2591 analyses and we confirmed the clinical diagnosis in 174 patients. We compared the following assays: leukocytes using glycogen or 4MUG as substrate, fibroblasts using 4MUG as substrate, and dried blood spots using 4MUG as substrate. In 794 individuals, two or more assays were performed. We found that phenotypes could only be distinguished using fibroblasts with 4MUG as substrate. Pseudodeficiencies caused by the GAA2 allele could be ruled out using 4MUG rather than glycogen as substrate in leukocytes or fibroblasts. The Asian pseudodeficiency could only be ruled out in fibroblasts using 4MUG as substrate. We conclude that fibroblasts using 4MUG as substrate provides the most reliable assay for biochemical diagnosis and can serve to validate results from leukocytes or dried blood spots.

Neonatal jaundice is a common and severe disease in premature infants with Glucose-6-Phosphate Dehydrogenase (G-6-PD) deficiency. The World Health Organization (WHO) has recommended screening for G-6-PD deficiency in newborns for early recognition as well as to prevent unwanted outcomes in a timely manner. The present study aimed to assess a point-of-care, careSTARTTM G6PD biosensor as a quantitative method for the diagnosis of G-6-PD deficiency. Factors influencing the evaluation of G-6-PD enzyme activity were examined in 40 adults, including ethylenediaminetetraacetic acid (EDTA) anticoagulant, hematocrit concentration, storage temperature and time. Analytic performance of the careSTARTTM G6PD biosensor was evaluated in 216 newborns and compared with fluorescent spot test (FST) and standard quantitative G-6-PD enzyme activity (SGT) assay. The results of factors affecting the G-6-PD enzyme activity showed that the activity determined from finger-prick was not statistically different from venous blood (p = 0.152). The G-6-PD value was highly dependent on the hematocrit and rose with increasing hematocrit concentration. Its activity was stable at 4°C for 3 days. Reliability analysis between the careSTARTTM G6PD biosensor and SGT assay showed a strong correlation with a Pearson's correlation coefficient of 0.82 and perfect agreement by intraclass correlation coefficient (ICC) of 0.90. Analysis of the area under the Receiver Operating Curve (AUC) illustrated that the careSTARTTM G6PD biosensor had 100% sensitivity, 96% specificity, 73% positive predictive value (PPV), 100% negative predictive value (NPV) and 97% accuracy at 30% of residual activity. While the diagnostic ability for identifying G-6-PD deficiency had 78% sensitivity, 89% specificity, 56% positive predictive value (PPV), 96% negative predictive value (NPV) and 88% accuracy when stratified by gender. The careSTARTTM G6PD biosensor is an attractive option as a point-of-care quantitative method for G-6-PD activity detection. Quantification of G-6-PD enzyme activity in newborns is the most effective approach for the management of G-6-PD deficiency to prevent severe jaundice and acute hemolysis.

Background Measurement of adenosine deaminase (ADA) can provide information about cell-mediated immunity. This report’s objective was to study the enzymatic activity of total ADA (tADA) and its isoenzymes ADA1 and ADA2 in canine, equine, porcine, and bovine serum and saliva and their changes in different inflammatory situations in each species. Besides, an automated method for ADA2 measurement was developed and validated. Results tADA was present in serum and saliva of healthy animals of the four species. Erythro-9-(2-hydroxy-3-nonyl) adenine (EHNA) concentration of 0.47 mM was needed for ADA1 inhibition in canine and porcine samples (serum and saliva) and bovine saliva, whereas for equine saliva 0.94 mM was needed. ADA2 activity was not detected in bovine serum and was very low or absent in equine serum and bovine saliva. An automated procedure to measure ADA2 consisting of adding EHNA to a commercial reagent for tADA measurement provided repetitive (coefficients of variation < 8.8% in serum and < 10% in saliva) and accurate (linearity of serial sample dilutions with R 2  > 0.90) results, being equivalent to a manual incubation of the sample with EHNA at a similar concentration. Salivary tADA, as well as ADA1 and ADA2, were higher in dogs with leishmaniosis, horses with acute abdominal disease and pigs with lameness than in healthy animals. tADA and isoenzymes in saliva showed a positive significant correlation with serum ferritin in dogs ( r  = 0.602, P  < 0.01; r  = 0.555, P  < 0.05; and r  = 0.632, P  < 0.01; respectively for tADA, ADA1 and ADA2) and serum C-reactive protein in pigs ( r  = 0.700, P  < 0.01, for both tADA and ADA1; r  = 0.770, P  < 0.001, for ADA2), whereas salivary ADA2 significantly correlated with serum amyloid A in horses ( r  = 0.649, P  < 0.01). In cows, salivary tADA and ADA1 significantly increased after calving, correlating with total white blood cell count ( r  = 0.487, P  < 0.05, for both tADA and ADA1). Conclusions The activity of total ADA and its different isoenzymes, can be measured in serum and saliva of dogs, horses, pigs and cows by a simple and fast procedure described in this report. When measured in saliva, these analytes correlated with other biomarkers of inflammation and it could potentially be used as a biomarkers of inflammation and immune activation in the species of this study.

Introduction:Potential hepatotoxicity associated with disease-modifying antirheumatic drugs (DMARDs) requires laboratory monitoring. In patients with rheumatoid arthritis (RA) or psoriatic arthritis (PsA), the incidence of elevated alanine aminotransferase/aspartate aminotransferase (ALT/AST) enzymes associated with methotrexate (MTX), leflunomide (LEF) and MTX+LEF versus other DMARDs was examined.Methods:Patients with RA and PsA enrolled in the Consortium of Rheumatology Researchers of North America (CORRONA) initiating DMARDs were identified. Abnormalities were identified when either was 1- or 2-fold times above the upper limits of normal (ULN). Odds ratios (OR) between MTX/LEF dose and elevated ALT/AST enzymes were estimated using generalised estimating equations. Interaction terms for use of MTX+LEF quantified the incremental risk of the combination compared with each individually.Results:Elevated ALT/AST levels (>1× ULN) occurred in 22%, 17%, 31% and 14% of patients with RA receiving MTX, LEF, MTX+LEF or neither, respectively; elevations were 2.76-fold (95% CI 1.84 to 4.15) more likely in patients with PsA. Elevations >2× ULN occurred in 1–2% of patients on MTX or LEF monotherapy compared with 5% with the combination. After multivariable adjustment and compared with either monotherapy, the combination of MTX and LEF was associated with a greater risk according to MTX dose used as part of the combination: MTX 10–17.5 mg/week, OR 2.91 (95% CI 1.23 to 6.90); MTX ⩾20 mg/week, OR 3.98 (95% CI 1.72 to 9.24).Conclusions:Abnormal ALT/AST levels developed in 14–35% of patients with RA or PsA initiating DMARD therapy. The risks were incrementally greater in those with PsA and in those receiving MTX (⩾10 mg/day) + LEF. These findings should help inform monitoring for potential hepatotoxicity in these patient populations.

Enzyme assays for high-throughput screening and enzyme engineering, which are often based on derivatives of coumarin, nitrophenol, fluorescein, nitrobenzofurazane or rhodamine dyes, can be divided into two categories: those that depend on labelled substrates, and those that depend on sensing the reactions of unmodified substrates. Labelled substrates include, for example, fluorogenic and chromogenic substrates that generate a reporter molecule by β-elimination, fluorescence resonance energy transfer (FRET) substrates and isotopic labels for enantioselectivity screening. By contrast, endpoint sensing can be done using amine reagents, fluorescent affinity labels for phosphorylated proteins, or synthetic multifunctional pores. Sensing assays can also be done in real time by using, for example, aldehyde trapping to follow vinyl ester acylation in organic solvent or calcein–copper fluorescence for sensing amino acids. The current trend is to assemble many such assays in parallel for enzyme profiling and enzyme fingerprinting.

BackgroundLow alanine aminotransferase (ALT) blood levels are known to be associated with frailty and increased risk of long-term mortality in certain populations. However, the contribution of this marker to long-term outcome has not been assessed in patients with chronic coronary heart disease.ObjectiveThe aim of the current study was to assess the association between low ALT values and long-term, 22.8-year, all-cause mortality in this population.ParticipantsWe examined the association of low ALT (<17 IU/l) with long-term all-cause mortality in the Bezafibrate Infarction Prevention (BIP) Registry population.Key resultsAppropriate laboratory and survival data were available for 6,575 patients, without known liver pathology, included in the BIP registry, with a median follow-up period of 22.8 years. The cumulative probability of all-cause mortality was significantly higher in the low ALT group compared with patients with higher ALT levels (65.6 % vs. 58.4 %; log-rank p < 0.001). Consistently, multivariate analysis, adjusted for multiple established predictors of mortality in this population, demonstrated that low ALT is independently associated with 11 % greater long-term (22.8 years) mortality risk [HR 1.11 (95 % confidence interval: 1.03–1.19; adjusted p < 0.01)].ConclusionsLow ALT levels are associated with increased long-term mortality among middle-aged patients with stable coronary heart disease. This association remained statistically significant after adjustment for other well-established risk factors for mortality in this population.

Background: Newborn screening for deficiency in the lysosomal enzymes that cause Fabry, Gaucher, Krabbe, Niemann–Pick A/B, and Pompe diseases is warranted because treatment for these syndromes is now available or anticipated in the near feature. We describe a multiplex screening method for all five lysosomal enzymes that uses newborn-screening cards containing dried blood spots as the enzyme source. Methods: We used a cassette of substrates and internal standards to directly quantify the enzymatic activities, and tandem mass spectrometry for enzymatic product detection. Rehydrated dried blood spots were incubated with the enzyme substrates. We used liquid-liquid extraction followed by solid-phase extraction with silica gel to remove buffer components. Acarbose served as inhibitor of an interfering acid α-glucosidase present in neutrophils, which allowed the lysosomal enzyme implicated in Pompe disease to be selectively analyzed. Results: We analyzed dried blood spots from 5 patients with Gaucher, 5 with Niemann–Pick A/B, 11 with Pompe, 5 with Fabry, and 12 with Krabbe disease, and in all cases the enzyme activities were below the minimum activities measured in a collection of heterozygous carriers and healthy noncarrier individuals. The enzyme activities measured in 5–9 heterozygous carriers were approximately one-half those measured with 15–32 healthy individuals, but there was partial overlap of each condition between the data sets for carriers and healthy individuals. Conclusion: For all five diseases, the affected individuals were detected. The assay can be readily automated, and the anticipated reagent and supply costs are well within the budget limits of newborn-screening centers.

BackgroundPatients with Fabry disease (FD) show left ventricular hypertrophy (LVH) mimicking hypertrophic cardiomyopathy (HCM) of sarcomeric origin and might benefit, if detected early, from specific enzyme replacement therapy. The prevalence of FD in patients with LVH of 13 mm or greater, screened using the leucocyte alpha-galactosidase A (α-gal A) activity test, a technique that is difficult to apply routinely, ranged from 0% to 6%.ObjectiveTo screen systematically for FD in patients with a diagnosis of HCM (LVH ≥15 mm) in primary cardiology practice, a validated, physician-friendly α-gal A assay was used on dried blood spots using a filter paper test.Design and patientsA cohort of 392 adults (278 men) followed for HCM were screened for FD. A standard blood test was used for confirmation in nine men in whom the α-gal A result was 40% or less.ResultsFour men (1.5%; 1.8% of men ≥40 years vs 0% <40 years; all with α-gal A <30%), but no women, were diagnosed with FD. Index cases presented with diffuse but asymmetric LVH, with severe obstruction in one case and frequent high-grade atrioventricular conduction block necessitating a pacemaker in three cases. Family screening identified eight additional cases. Genotyping was performed successfully on DNA extracted from the filter papers.ConclusionIn male patients diagnosed as having HCM, pure FD cardiac variants are not exceptional and can be specifically identified using a simple filter-paper test. The sensitivity of this test is low in female patients.

Background Aldehyde dehydrogenases belong to a superfamily of detoxifying enzymes that protect cells from carcinogenic aldehydes. Of the superfamily, ALDH1A1 has gained most attention because current studies have shown that its expression is associated with human cancer stem cells. However, ALDH1A1 is only one of the 19 human ALDH subfamilies currently known. The purpose of the present study was to determine if the expression and activities of other major ALDH isozymes are associated with human ovarian cancer and ovarian cancer sphere cultures. Methods Immunohistochemistry was used to delineate ALDH isozyme localization in clinical ovarian tissues. Western Blot analyses were performed on lysates prepared from cancer cell lines and ovarian cancer spheres to confirm the immunohistochemistry findings. Quantitative reverse transcription-polymerase chain reactions were used to measure the mRNA expression levels. The Aldefluor® assay was used to measure ALDH activity in cancer cells from the four tumor subtypes. Results Immunohistochemical staining showed significant overexpression of ALDH1A3, ALDH3A2, and ALDH7A1 isozymes in ovarian tumors relative to normal ovarian tissues. The expression and activity of ALDH1A1 is tumor type-dependent, as seen from immunohistochemisty, Western blot analysis, and the Aldefluor® assay. The expression was elevated in the mucinous and endometrioid ovarian epithelial tumors than in serous and clear cell tumors. In some serous and most clear cell tumors, ALDH1A1 expression was found in the stromal fibroblasts. RNA expression of all studied ALDH isozymes also showed higher expression in endometrioid and mucinous tumors than in the serous and clear cell subtypes. The expression of ALDH enzymes showed tumor type-dependent induction in ovarian cancer cells growing as sphere suspensions in serum-free medium. Conclusions The results of our study indicate that ALDH enzyme expression and activity may be associated with specific cell types in ovarian tumor tissues and vary according to cell states. Elucidating the function of the ALDH isozymes in lineage differentiation and pathogenesis may have significant implications for ovarian cancer pathophysiology.