Clinical impact of ceruloplasmin levels at ANCA-associated vasculitis diagnosis.

OBJECTIVES: Ceruloplasmin is an inhibitor of myeloperoxidase (MPO) activity that plays an important role in the pathophysiology of anti-neutrophil cytoplasmic antibody-associated vasculitis (AAV). This study aimed to evaluate the prognostic impact of serum level of ceruloplasmin at diagnosis in patients with anti-MPO antibody-positive AAV. METHODS: This retrospective monocentric study in Caen University Hospital involved all consecutive adult anti-MPO antibody-positive patients with microscopic polyangiitis or granulomatosis with polyangiitis, diagnosed between January 2010 and January 2022 with available serum sample at inclusion. Patients outcomes were analyzed from two subgroups constituted according to the median serum level of ceruloplasmin. The same analyses were then performed in anti-proteinase 3 (PR3) antibody-positive patients. RESULTS: Within the 92 patients analyzed, 50 patients had anti-MPO antibodies with a median ceruloplasmin level of 0.44 [quartiles 1-3, 0.40-0.49] g/L and a median Birmingham Vasculitis Activity Score of 19 [14-22]. After a median follow-up period of 40 [22-86] months, 13 (26%) patients had died: 10 (40%) in the low ceruloplasmin group and 3 (12%) in the high ceruloplasmin group (p = 0.03), with a significantly worse survival rate in the low ceruloplasmin group (p = 0.021). No significant differences in relapse rate or renal failure was observed between the two groups. The same analyses performed in the group of AAV patients with anti-PR3 antibody did not show any differences. CONCLUSION: In anti-MPO AAV patients, serum level of ceruloplasmin at diagnosis seems to be associated with a significant impact on survival.

Phenotypic and genetic characterization of children with Wilson Disease from Northeast China.

BACKGROUND: Wilson disease (WD) is an autosomal recessive inherited disease caused by ATP7B variants and characterized by copper metabolism defects. However, children with WD are often asymptomatic, making the clinical diagnosis difficult. Therefore, more accurate methods are required for clinical diagnosis. The objective of this study was to highlight the phenotypic and genetic characteristics of children with WD in northeast China. METHODS: We retrospectively analyzed the clinical data and gene sequencing results of 65 children with WD from January 1, 2014, to December 31, 2022, at the Shengjing Hospital of China Medical University. All data refer to the time of diagnosis before treatment. RESULTS: The median age at diagnosis was 5 years (range 1.2-15 years). In 50 cases (50/65, 76.9%) patients, routine physical examinations revealed only abnormal liver function. However, they had a significantly negative (p < 0.05) Kayser-Fleischer ring (KF). Children with acute liver failure had significantly increased 24 h urinary copper excretion (p < 0.05). We detected 46 genetic variants of ATP7B, including seven novel variants. The most frequent variant was p.R778L with an allele frequency of 38.7%. Phenotype-genotype correlation analysis suggested that p.R778L was significantly associated with lower serum ceruloplasmin levels and higher zinc levels (p < 0.05). The loss-of-function (LOF) variant was associated with significantly lower albumin levels (p < 0.05). CONCLUSION: Most children with WD are asymptomatic, which makes early diagnosis of WD difficult. Therefore, clinical and laboratory characteristics as well as genetic testing are essential. p.R778L is the most frequent variant of ATP7B in China and may play an important role in lowering serum ceruloplasmin levels.

Discussion on the relationship between the distribution characteristics of TCM syndrome types and related objective indicators in hepatolenticular degeneration.

Hepatolenticular degeneration is a rare treatable autosomal recessive inherited copper metabolism disorder with a diverse clinical phenotype and an exceptionally complex pathogenesis. Early definitive phenotypic diagnosis and targeted treatment are major challenges worldwide. In this study, we strictly followed the "National Standards of the People's Republic of China - Terminology of Traditional Chinese Medicine Clinical Diagnosis and Treatment (Syndrome Part)," "Chinese Medicine Nomenclature," and the clinical investigation-determined traditional Chinese medicine syndrome differentiation standards at Anhui University of Chinese Medicine to select 6 of the most common traditional Chinese medicine syndrome differentiations. This study retrospectively analyzed 107 patients admitted between 2019 and 2023 with Wilson's disease based on real-world data. After testing for normal distribution and homogeneity of variance, corresponding analysis of variance was selected, followed by post hoc multiple comparisons. Of the selected 25 objective influencing factors, 22 exhibited normal distribution, while red blood cells, hemoglobin, and type IV collagen did not pass the homogeneity of variance test. After analysis of variance, the factors ceruloplasmin (CP) and copper oxidase (SCO) showed significant differences among patients with different traditional Chinese medicine syndromes (P < .05), with partial η2 for CP being 0.13 > 0.06 and for SCO being 0.143 > 0.14. Post hoc multiple comparison results indicated significant differences in CP and SCO among patients with certain traditional Chinese medicine syndromes (P < .05). There were significant differences in the factors CP and SCO among patients with different traditional Chinese medicine syndromes. Significant differences were observed in the copper blue protein factor between damp-heat syndrome and liver and kidney deficiency syndrome, liver and kidney deficiency syndrome and liver and kidney yin deficiency syndrome, liver and kidney deficiency syndrome and phlegm heat and wind syndrome, as well as liver and kidney deficiency syndrome and syndrome of phlegm and blood stasis (P < .05). Significant differences were also found in the SCO factor between damp-heat syndrome and liver and kidney deficiency syndrome, liver and kidney deficiency syndrome and liver and kidney yin deficiency syndrome, liver and kidney deficiency syndrome and phlegm heat and wind syndrome, and liver and kidney deficiency syndrome and syndrome of phlegm and blood stasis (P < .05).

Wilson's disease in Sardinian population: The experience of a pediatric referral center.

BACKGROUND AND OBJECTIVES: Wilson's disease (WD) in children and adolescents is predominantly asymptomatic or oligo-symptomatic. The symptoms are nonspecific and difficult to distinguish from other hepatic or neuropsychiatric disorders. In this study, we present the experience of a pediatric referral center for WD diagnosis and treatment. PATIENTS AND METHODS: We retrospectively analyzed clinical and laboratory data from 99 patients with WD of Sardinian origin, including physical examination, laboratory biochemical testing, liver biopsy, and genetic analysis. RESULTS: Patients were prevalently oligo-symptomatic or asymptomatic. The median age of diagnosis was 8.78 years. Ceruloplasmin values were lower than normal values in all analyzed patients. Twenty-four-hour urinary copper levels were higher than 40 µg/24-h in 92/96 patients. In all analyzed patients with the exception of one, liver copper was higher than 250 µg/g of dry weight but all had >75 µg/g of dry weight. Statistical analysis showed correlation between the age at diagnosis, serum copper, and 24-h urinary copper. Correlation was also found between serum copper and 24-h urinary copper. Molecular analysis of ATP7B gene allowed complete characterization in all the analyzed patients. CONCLUSION: A high index of clinical suspicion and biochemical tests including liver tests, serum ceruloplasmin, and basal 24-h urinary copper excretion and genotype determination are key to WD diagnosis. The long experience that a referral center for WD possesses is an important factor in making WD diagnosis a more accurate process. Studies in animal models on WD could be used as a guide to further investigate the molecular mechanisms that regulate copper metabolism and influence the natural history of WD.

Clinical characteristics and prognosis of early diagnosed Wilson's disease: A large cohort study.

BACKGROUND AND AIMS: Few studies have focused on the outcomes of Wilson's disease (WD) diagnosed before age of 5 years. This study aimed to summarize the clinical features of early diagnosed WD and analyse treatment outcomes and the risk factors associated with treatment failure. METHODS: A total of 139 children confirmed with WD before 5 years were enrolled in this study. Only patients with follow-up over 1 year were analysed with Kaplan-Meier survival analysis. The composite outcomes included death, progression to liver failure or acute hepatitis, development of renal or neurological symptoms and persistent elevation of alanine aminotransferase (ALT). The treatment failure was defined as occurrence of at least one of above outcomes. RESULTS: Among 139 WD patients at diagnosis, two (1.4%) WD patients presented with symptomatic liver disease, whereas 137 (98.6%) were phenotypically asymptomatic, including 135 with elevated ALT and 2 with normal liver function. Median serum ceruloplasmin (Cp) was 3.1 mg/dL, and urinary copper excretion was 87.4 µg/24-h. There were 71 variants identified in the the copper-transporting ATPase beta gene, and 29 were loss of function (LOF). 51 patients with LOF variant were younger at diagnosis and had lower Cp than 88 patients without LOF. Among 93 patients with over 1 year of follow-up, 19 (20.4%) received zinc monotherapy, and 74 (79.6%) received a zinc/D-penicillamine combination therapy. 14 (15.1%) patients underwent treatment failure, and its occurrence was associated with poor compliance (p < .01). CONCLUSIONS: Cp is a reliable biomarker for early diagnosis, and zinc monotherapy is an effective treatment for WD during early childhood. Good treatment compliance is critical to achieve a favourable outcome.

Phenotype and molecular characterization of Wilson's disease in Morocco.

BACKGROUND AND STUDY AIMS: In Morocco the prevalence of Wilson disease (WD) and the spectrum of mutations are not known. The aim of the present study was to estimate the prevalence of WD in Morocco, to evaluate the phenotype among a large cohort of WD patients, and to characterize ATP7B variants in a subgroup of WD patients. PATIENTS AND METHODS: We collected data from 226 patients admitted to five university hospital centers in Morocco between 2008 and 2020. The diagnosis was based on clinical manifestations, function tests and biochemical parameters. The genotype was characterized in 18 families diagnosed at the University Hospital Center of Marrakesh, by next generation sequencing. RESULTS: The mean annual prevalence in Morocco was 3.88 per 100,000 and the allele frequency was 0.15 %. Among the 226 patients included (121 males and 105 females), 196 were referred for a hepatic or neurological involvement and 30 were asymptomatic. The mean age at diagnosis was 13 ± 5.1 years (range: 5 - 42 years). Consanguinity was found in 63.3 % of patients. The mean duration of illness was 2.8 ± 1.9 years. Kayser-Fleischer rings were found in 131 (67.9 %) of 193 patients. Among the 196 symptomatic patients, 141/159 (88.7 %) had low serum ceruloplasmin (<0.2 g/L) and a high 24-hours urinary copper (>100 µg/day) was found in 173/182 (95.1 %) patients. The initial treatment was D-penicillamine in 207 patients, zinc acetate in five, zinc sulfate in five, and nine patients were not treated; 60/207 (29 %) patients have stopped treatment. A total of 72 patients died; the mortality rate was 31.9 %. Eight different ATP7B variants were identified among the 18 patients studied, of which two were novel (p.Cys1104Arg and p.Gln1277Hisfs*52), and six previously published (p.Gln289Ter, p.Cys305Ter, p.Thr1232Pro, p.Lys1020Arg, p.Glu583ArgfsTer25 and c.51+4A>T). All informative patients were homozygous for the disease-causing mutation. CONCLUSION: In Morocco, a high prevalence due to consanguinity and a high mortality rate due to the difficulty of diagnosis and lack of treatment were observed in WD patients. NGS sequencing identified new ATP7B variants in WD patients from Morocco.

Diagnosing aceruloplasminemia: navigating through red herrings.

A 58-year-old female was found to have hyperferritinemia (Serum ferritin:1683 ng/mL) during work-up for mild normocytic anemia. Transferrin saturation(TSAT) was low-normal. Magnetic resonance imaging (MRI) abdomen showed evidence of hepatic iron deposition. Liver biopsy showed 4 + hepatic iron deposition without any evidence of steatosis or fibrosis. Quantitative liver iron was elevated at 348.3 µmol/g dry liver weight [Reference range(RR): 3-33 µmol/g dry liver weight]. She was presumptively diagnosed with tissue iron overload, cause uncertain. A diagnosis of ferroportin disease (FD) was considered, but the pattern of iron distribution in the liver, mainly within the hepatic parenchyma (rather than in the hepatic Kupffer cells seen in FD), and the presence of anemia (uncommon in FD) made this less likely. She was treated with intermittent phlebotomy for over a decade with poor tolerance due to worsening normocytic to microcytic anemia. A trial of deferasirox was done but it was discontinued after a month due to significant side effects. During the course of treatment, her ferritin level decreased. Over the past 1.5 years, she developed progressively worsening neurocognitive decline. MRI brain showed areas of susceptibility involving basal ganglia, midbrain and cerebellum raising suspicion for metabolic deposition disease. Neuroimaging findings led to testing for serum copper and ceruloplasmin levels which were both found to be severely low. Low serum copper, ceruloplasmin levels and neuroimaging findings led us to consider Wilson disease however prior liver biopsy showing elevated hepatic iron rather than hepatic copper excluded the diagnosis of Wilson disease. After shared decision making, ceruloplasmin gene analysis was not pursued due to patient's preference and prohibitive cost of testing. The diagnosis of aceruloplasminemia was ultimately made. The biochemical triad of hyperferritinemia, low-normal TSAT and microcytic anemia should raise the possibility of aceruloplasminemia. Since neurological manifestations are rare in most inherited iron overload syndromes, neurological symptoms in a patient with tissue iron overload should prompt consideration of aceruloplasminemia as a differential diagnosis.

Determination of copper status by five biomarkers in serum of healthy women.

BACKGROUND: The essential trace element copper is relevant for many important physiological processes. Changes in copper homeostasis can result from disease and affect human health. A reliable assessment of copper status by suitable biomarkers may enable fast detection of subtle changes in copper metabolism. To this end, additional biomarkers besides serum copper and ceruloplasmin (CP) concentrations are required. OBJECTIVES: The aim of this study was to investigate the emerging copper biomarkers CP oxidase (CPO) activity, exchangeable copper (CuEXC) and labile copper in serum of healthy women and compare them with the conventional biomarkers total serum copper and CP. METHOD AND MAIN FINDINGS: This observational study determined CPO activity, the non CP-bound copper species CuEXC and labile copper, total serum copper and CP in sera of 110 healthy women. Samples were collected at four time points over a period of 24 weeks. The concentrations of total serum copper and CP were within the reference ranges. The comparison of all five biomarkers provided insight into their relationship, the intra- and inter-individual variability as well as the age dependence. The correlation and Principal Component Analyses (PCA) indicated that CP, CPO activity and total copper correlated well, followed by CuEXC, while the labile copper pool was unrelated to the other parameters. CONCLUSIONS: This study suggests that the non-CP-bound copper species represent copper pools that are differently regulated from total copper or CP-bound copper, making them interesting complementary biomarkers to enable a more complete assessment of body copper status with potential relevance for clinical application.

Determination of copper and other trace elements in serum samples from patients with biliary tract cancers: prospective noninterventional nonrandomized clinical study protocol.

BACKGROUND: Biliary tract cancers (BTCs) are usually diagnosed at an advanced stage, when the disease is incurable. Currently used tumor biomarkers have limited diagnostic value for BTCs, so there is an urgent need for sensitive and specific biomarkers for their earlier diagnosis. Deregulation of the homeostasis of trace elements is involved in the carcinogenesis of different cancers, including BTCs. The objective of the study is to determine/compare the total concentrations of copper (Cu), zinc (Zn) and iron (Fe) and the proportions of free Cu and Cu bound to ceruloplasmin (Cp) and the isotopic ratio of 65Cu/63Cu in serum samples from healthy volunteers and cancer patients using inductively coupled plasma-mass spectrometry-based methods (ICP-MS). PATIENTS AND METHODS: In this prospective, noninterventional, nonrandomized study 20 patients and 20 healthy volunteers will be enrolled to identify serum Cu, Zn and Fe levels, Cu isotopic fractionation as a predictive biomarker of response to systemic therapy of BTCs, which will be evaluated by computed tomography. Newly developed analytical methods based on ICP-MS will be applied to metal-based biomarker research in oncology. CONCLUSIONS: In the study the comparison of the total concentration of selected trace elements, the proportion of free Cu and Cu bound to Cp and the isotopic ratio of 65Cu/63Cu in serum samples from healthy volunteers and cancer patients will be conducted to provide the foundation for the development of a BTC cancer screening methodology and the data on their usability as a potential predictive biomarker for BTCs of response to systemic therapy.

Non-ceruloplasmin-bound copper and copper speciation in serum with extraction using functionalized dendritic silica spheres followed by ICP-MS detection.

The quantification of non-ceruloplasmin-bound copper (NCBC) and total copper in biological fluids is highly required for understanding the correlation of copper with various physiological processes and diseases. In the present work, we developed dendritic spherical silica particles functionalized with EDTA, shortly as DMSPs-EDTA, from the hydrolysis of tetraethyl orthosilicate with the aid of structure-directing agents and subsequent modification of EDTA. DMSPs-EDTA serves as adsorbent with abundant binding sites to facilitate efficient extraction of NCBC. The retained NCBC on DMSPs-EDTA may be readily recovered by stripping with HNO3 (2 mol L-1). By hyphenating with ICP-MS detection, it provides a limit of detection of 1.3 pmol for NCBC. The degradation of ceruloplasmin with 200 mmol L-1 H2O2 releases the bound copper as NCBC to distribute among other ligands, which may be efficiently retained by the adsorbent and facilitate the detection of total copper. The linear ranges of 0.21-10 µmol L-1 and 0.42-30 µmol L-1 were derived for the detection of NCBC and total copper. The recovery rates for spiked NCBC or total copper in serum were derived to be 97-108% and 94-102%, respectively. The analysis of serum for a healthy subject resulted in 1.8 µmol L-1 NCBC and 9.5 µmol L-1 total copper. In addition, the proportions of 8.5-12% for NCBC were derived from the serum of healthy adults, while those for the patients with lung, hepatocellular and esophageal carcinoma were found to be 10-12%, illustrating no obvious difference against the normal group.

Age and Serum Creatinine Can Differentiate Wilson Disease Patients with Pseudonormal Ceruloplasmin.

Methods: We retrospectively screened individuals with serum Cp ≥ 140 mg/L from 1032 WD patients who were hospitalised for the first time. Logistic regression analyses were performed in a case-control study between the WD cohort and another liver disease cohort to explore the independent risk factors for WD diagnosis and establish a regression model to identify them. The follow-up medical records of the WD cohort were subjected to mixed-effects model analysis in a longitudinal study to discover factors associated with Cp normalisation. Results: Eighty-six WD patients and their 353 medical records and another 98 non-WD liver disease patients were included in the present study. Cp normalisation was significantly associated with the copper burden and liver function indexes, such as urinary copper, γ-glutamyltransferase, and albumin (p ≤ 0.001). Logistic regression analysis showed that age and serum creatinine (p ≤ 0.001) were independent risk factors associated with WD. The AUC value of the regression model in the total cohort was 0.926 (p ≤ 0.001). At a cutoff value of ≥0.617 and ≥-1, the positive and negative predictive values were both 90.8% for WD. Conclusion: Increased serum Cp in WD patients is related to excessive copper burden and hepatic injury, and common tests can effectively distinguish WD patients from other liver injury patients.

[Wilson's disease: overview]. / Enfermedad de Wilson.

Wilson's disease (WD) is an uncommon hereditary disorder caused by a deficiency in the ATP7B transporter. The protein codified by this gene facilitates the incorporation of the copper into ceruloplasmin. Therefore, WD accumulates copper primary in the liver and secondary in other organs, such as the central nervous system. It represents a wide spectrum of disease, ranging from being asymptomatic in some patients to promote an acute liver failure in others. The diagnosis requires a combination of clinical signs and symptoms, as well as some diagnostic tests such as the measurement of serum ceruloplasmin, the urinary excretion of copper, the liver biopsy or the genetic testing. The treatment must be maintained lifelong and includes some drugs such as chelating agents (penicillamine and trientine) and inhibitors of the copper absorption (zinc salts). Lastly, the liver transplant should be an option for patients with end-stage liver disease.

Expression of Ceruloplasmin in the Peripheral Blood of Patients With Drug-Resistant Epilepsy.

This study was performed to detect the expression of ceruloplasmin in the peripheral blood of patients with drug-resistant epilepsy and explore the mechanisms of iron metabolism disorder in drug-resistant epilepsy. Peripheral blood was collected from 32 patients with drug-resistant epilepsy, labeled the drug-resistant group; 30 patients who were drug responsive, labeled the drug-responsive group; and 34 healthy people, named the normal group.The expression levels of ceruloplasmin mRNA and ceruloplasmin protein in the peripheral blood of the 3 groups were detected using real-time fluorescence-based quantitative polymerase chain reaction and Western blot. The differences in the expression of ceruloplasmin mRNA of different seizure frequencies and types, electroencephalogram abnormal discharges, and different medication methods were analyzed and compared. The relative expression of ceruloplasmin mRNA and ceruloplasmin protein in the drug-resistant epilepsy group was significantly higher than that in the drug-responsive group (P = .002 and .010, respectively) and higher in the drug-responsive group compared with the normal group (P = .014 and .005, respectively). The relative expression of ceruloplasmin mRNA in patients with epilepsy using different medication methods was statistically significant (P = .001). Patients who received a combination of 2 or 3 drugs exhibited a higher expression than those treated with single-drug treatment, whereas those who received a combination of 3 drugs had a higher expression than those with 2 drugs (P = .013, .001, and .011, respectively). There was no significant difference in the relative expression of Cp mRNA in patients with epilepsy with different seizure frequencies and types and abnormal electroencephalogram discharges (all P > .05). The increased expression of ceruloplasmin in the peripheral blood of patients with drug-resistant epilepsy was closely related to the different medication methods, but no obvious correlation with epileptic seizure frequencies or types and abnormal electroencephalogram discharges was identified. The increased expression of ceruloplasmin enhanced iron oxidative damage and may be the potential mechanism of drug-resistant epilepsy and may be one of the drug resistance indicators for combination drugs when treating drug-resistant epilepsy.

Calculated parameters for the diagnosis of Wilson disease.

Introduction: The diagnosis of Wilson disease (WD) is plagued by biochemical and clinical uncertainties. Thus, calculated parameters have been proposed. This study aimed to: (a) compare the diagnostic values of non-caeruloplasmin copper (NCC), NCC percentage (NCC%), copper-caeruloplasmin ratio (CCR) and adjusted copper in WD; and (b) derive and evaluate a discriminant function in diagnosing WD. Methods: A total of 213 subjects across all ages who were investigated for WD were recruited. WD was confirmed in 55 patients, and the rest were WD free. Based on serum copper and caeruloplasmin values, NCC, NCC%, CCR and adjusted copper were calculated for each subject. A function was derived using discriminant analysis, and the cut-off value was determined through receiver operating characteristic analysis. Classification accuracy was found by cross-tabulation. Results: Caeruloplasmin, total copper, NCC, NCC%, CCR, adjusted copper and discriminant function were significantly lower in WD compared to non-WD. Discriminant function showed the best diagnostic specificity (99.4%), sensitivity (98.2%) and classification accuracy (99.1%). Caeruloplasmin levels <0.14 g/L showed higher accuracy than the recommended 0.20 g/L cut-off value (97.7% vs. 87.8%). Similarly, molar NCC below the European cut-off of 1.6 umol/L showed higher accuracy than the American cut-off of 3.9 umol/L (80.3% vs. 59.6%) (P < 0.001). NCC%, mass NCC, CCR and adjusted copper showed poorer performances. Conclusion: Discriminant function differentiates WD from non-WD with excellent specificity, sensitivity and accuracy. Performance of serum caeruloplasmin <0.14 g/L was better than that of <0.20 g/L. NCC, NCC%, CCR and adjusted copper are not helpful in diagnosing WD.

Pseudocholinesterase as a Biomarker for Untreated Wilson's Disease.

The aim of this study was to demonstrate that pseudocholinesterase (CHE) serum level is a useful diagnostic biomarker for untreated Wilson's disease (WD). Between 2013 and 2019, about 75 patients were referred to the outpatient department of the University of Düsseldorf with suspected Wilson's disease. In 31 patients with suspected Wilson's disease (WD-SUS-group), WD was excluded by means of investigations other than analysis of blood and urine. A total of 27 parameters of blood and urine in these 31 patients were compared to those of 20 de novo patients with manifest WD (WD-DEF-group), which parameter showed the highest significance level of difference between the WD-DEF-group and the WD-SUS-group. Thereafter, receiver operating characteristics (ROC-curves) were analyzed to evaluate which parameter showed the largest area under the curve (AUC) to detect WD. Finally, a logistic regression analysis was performed to analyze which combination of parameters allowed the best classification of the 51 patients either into the WD-DEF-group or into the WD-SUS-group. CHE showed the highest significance level for a difference between the WD-DEF- and WD-SUS-group, had the highest AUC, and, in combination with ceruloplasmin, allowed 100% correct classification. Without CHE, no other combination of parameters reached this level of correct classification. After the initiation of treatment, which regularly results in an improvement in CHE, the high diagnostic accuracy of this biomarker was lost. Cholinesterase turns out to be an excellent biomarker for differentiation between untreated de novo patients with manifest WD and heterozygotic gene carriers.

Influence of Short and Medium Distance Road Transport on the Acute Phase Proteins in Horses.

Horse transport is a common practice and is usually associated as a cause of stress in animals, with consequences for their well-being. There are several of evidence that stress can increase an acute phase response. The aim of this study was to verify whether the road transport of horses over distances of 50 and 300 kilometers induces changes in the values of acute phase proteins. To do this, the serum SDS-PAGE was performed and the bands obtained were identified by mass spectrometry (MALDI-TOF). The blood samples were collected in tubes without anticoagulant to obtain the serum, and the evaluations occurred before the road transportation (T0), immediately after the journey (T1), six hours later (T2), and 24 hours (T3), 48 hours (T4), 72 hours (T5), 96 hours (T6), 120 hours (T7) and 144 hours (T8) after the end of the trip. All analyzes were performed using the Minitab 17 statistical package, and significance was considered when P<0.05. The APPs found through SDS-PAGE and properly identified were α2-macroglobulin, ceruloplasmin, transferrin, albumin, α1-antitrypsin, haptoglobin, apolipoprotein alpha 1, and α1-acid glycoprotein. No differences were observed in the concentration values between 50 and 300 km or between the moments after each route. The distances covered with the horses were not challenging enough to provoke an acute phase response reflected in changes in APPs.

[Phenotypes and ATP7B gene variants in 316 children with Wilson disease].

Objectives: To summarize the clinical phenotypes and the variation spectrum of ATP7B gene in Chinese children with Wilson's disease (WD) and to investigate their significance for early diagnosis. Methods: Retrospective analysis was performed on the clinical data of 316 children diagnosed as WD in Guangzhou Women and Children's Medical Center during the period from January 2010 to June 2021. The general situations, clinical manifestations, lab test results, imaging examinations, and ATP7B gene variant characteristics were collected. The patients were divided into asymptomatic WD group and symptomatic WD group based on the presence or absence of clinical symptoms at the time that WD diagnosis was made. The χ2 test, t test or Mann-Whitney U test were used to compare the differences between groups. Results: Among the 316 children with WD, 199 were males and 117 were females, with the age of 5.4 (4.0, 7.6) years at diagnosis; 261 cases (82.6%) were asymptomatic with the age of 4.9 (3.9, 6.4) years; whereas 55 cases (17.4%) were symptomatic with the age of 9.6 (7.3, 12.0) years. The main symptoms invloved liver, kidney, nervous system, or skin damage. Of all the patients, 95.9% (303/316) had abnormal liver function at diagnosis; 98.1% (310/316) had the serum ceruloplasmin lever lower than 200 mg/L; 97.7% (302/309) had 24-hour urine copper content exceeding 40 µg; only 7.4% (23/310) had positive corneal K-F rings, 8.2% (23/281) had abnormal MRI signals in the lenticular nucleus, and all of them had symptoms of damage in liver, kidney or nervous system. Compared with the group of symptomatic WD, asymptomatic group had higher levels of serum alanine aminotransferase and lower levels ceruloplasmin and 24-hour urine copper [(208±137) vs. (72±78) U/L, (55±47) vs. (69±48) mg/L, 103 (72, 153) vs. 492 (230, 1 432) µg; t=9.98, -1.98, Z=-4.89, all P<0.001]. Among the 314 patients completing genetic sequencing, a total of 107 mutations in ATP7B gene were detected, of which 10 are novel variants, and 3 cases (1.0%) had large heterozygous deletion (exons 10 to exon 11) in ATP7B gene. The percentage of missense mutation in asymptomatic WD children was significantly higher than that in symptomatic WD (81.5% (422/518) vs. 69.1% (76/110), χ²=8.47, P<0.05). WD patients carrying homozygous variant of c.2 333G>T had significantly low levels of ceruloplasmin than those not carrying this variant ((23±5) vs. (61±48) mg/L, t=-2.34, P<0.001). Conclusions: The elevation of serum ALT is an important clue for early diagnosis of WD in children, while serum ceruloplasmin and 24-hour urine copper content are specific markers for early diagnosis of WD. In order to confirm the diagnosis of WD, it is necessary to combine the Sanger sequencing with multiplex ligation-dependent probe amplification or other testing technologies.

Copper-histidine therapy in an infant with novel splice-site variant in the ATP7A gene of Menkes disease: the first experience in South East Asia and literature review.

Menkes disease (MD) is an X linked recessive multi-systemic disorder of copper metabolism, resulting from an ATP7A gene mutation. We report a male infant aged 4 months who presented with kinky hair, hypopigmented skin, epilepsy and delayed development. Magnetic resonance imaging (MRI) of brain demonstrated multiple tortuosities of intracranial vessels and brain atrophy. Investigation had showed markedly decreased serum copper and ceruloplasmin. The novel c.2172+1G>T splice-site mutation in the ATP7A gene confirmed MD. He was treated with subcutaneous administration of locally prepared copper-histidine (Cu-His). Following the therapy, hair manifestation was restored and serum ceruloplasmin was normalised 1 month later. Despite the treatment, epilepsy, neurodevelopment and osteoporosis still progressed. He died from severe respiratory tract infection at the age of 9.5 months. These findings suggest that the benefit of Cu-His in our case is limited which might be related to severe presentations and degree of ATP7A mutation.

Efficacy of Different Diagnostic Test for Identifying Wilson's Disease.

Wilson disease is an autosomal recessive disorder in which copper pathologically accumulates primarily within the liver, brain and other tissues. It can presents clinically as liver disease, as a progressive neurological disorder or as psychiatric illness. The wide array of clinical manifestations of WD can lead to misdiagnosis with subsequent greater risk of irreversible damage to liver and brain. Many tests can be used to investigate patients of Wilson disease, including serum free copper, 24 hours urine copper estimation, hepatic copper estimation and genetic mutation testing. But there is no single ideal diagnostic test that can exclude or confirm the disease with certainty. The aim of the study was to find out the efficacy of different diagnostic test for the diagnosis of Wilson disease. This cross-sectional analytical study was conducted at department of Paediatric Gastroenterology and Nutrition of Bangabandhu Sheikh Mujib Medical University, Dhaka, Bangladesh from January 2016 through January 2018. A total of 56 cases of Wilson disease and 39 patients with a liver disease other than WD were studied. Wilson disease was diagnosed by Leipzig score. Along with other physical findings and laboratory investigations slit lamp eye examination for KF ring, serum ceruloplasmin and 24 hour urinary copper excretion were done. The mean age of WD patients was 9.69±2.37 years, male female ratio was 1:1. Serum ceruloplasmin level was significantly lower in WD patient (p<0.001). Median of 24 hour urinary copper in WD was 702.75µg/ 24 hr (range119-11210µg/24 hour) and in non WD group it was 77.41µg/24 hour (range 20.0-478µg/24 hour) and the difference between them is statistically significant (p=0.001). The sensitivity of KF ring was 82.1% and specificity was 100%. The sensitivity, specificity, positive predictive value, negative predictive value and accuracy of serum ceruloplasmin were 98.2%, 92.3%, 94.8%, 97.2% and 95.7% respectively. The sensitivity, specificity, positive predictive value, negative predictive value and accuracy of 24 hour urinary copper were 100%, 63%, 80% and 85.1% respectively. The sensitivity, specificity, positive predictive value, negative predictive value and accuracy of KF ring, serum ceruloplasmin and basal 24 hour urinary copper excretion when combined together came out to be 70.4%, 100%, 100%, 59.3% and 79.3% respectively. This study result showed that serum ceruloplasmin and 24 hour urinary copper can be used as a screening test for the diagnosis of Wilson disease.

Quantification of human plasma metalloproteins in multiple sclerosis, ischemic stroke and healthy controls reveals an association of haptoglobin-hemoglobin complexes with age.

Advanced analytical methods play an important role in quantifying serum disease biomarkers. The problem of separating thousands of proteins can be reduced by analyzing for a 'sub-proteome', such as the 'metalloproteome', defined as all proteins that contain bound metals. We employed size exclusion chromatography (SEC) coupled to an inductively coupled plasma atomic emission spectrometer (ICP-AES) to analyze plasma from multiple sclerosis (MS) participants (n = 21), acute ischemic stroke (AIS) participants (n = 17) and healthy controls (n = 21) for Fe, Cu and Zn-metalloproteins. Using ANOVA analysis to compare the mean peak areas among the groups revealed no statistically significant differences for ceruloplasmin (p = 0.31), α2macroglobulin (p = 0.51) and transferrin (p = 0.31). However, a statistically significant difference was observed for the haptoglobin-hemoglobin (Hp-Hb) complex (p = 0.04), being driven by the difference between the control group and AIS (p = 0.012), but not with the MS group (p = 0.13), based on Dunnes test. A linear regression model for Hp-Hb complex with the groups now adjusted for age found no statistically significant differences between the groups (p = 0.95), but was suggestive for age (p = 0.057). To measure the strength of association between the Hp-Hb complex and age without possible modifications due to disease, we calculated the Spearman rank correlation in the healthy controls. The latter revealed a positive association (r = 0.39, 95% Confidence Interval = (-0.05, 0.83), which suggests that either the removal of Hp-Hb complexes from the blood circulation slows with age or that the release of Hb from red blood cells increases with age. We also observed that the Fe-peak corresponding to the Hp-Hb complex eluted ~100 s later in ~14% of all study samples, which was not correlated with age or disease diagnosis, but is consistent with the presence of the smaller Hp (1-1) isoform in 15% of the population.