Urinary Coproporphyrin Research Articles

Plasma and urinary CP I and CP III concentrations in chimeric mice with human hepatocytes after rifampicin administration.

The interest in transporter-mediated drug interactions has been increasing in the field of drug development. In this study, we measured the plasma and urinary concentrations of coproporphyrin (CP) I and CP III as endogenous substrates for organic anion-transporting polypeptide (OATP) using chimeric mice with human hepatocytes (PXB mice) and examined the influence of an OATP inhibitor, rifampicin (RIF). CP I and CP III were actively taken up intracellularly, and RIF inhibited the uptake in a concentration-dependent manner for both CP I and CP III in human hepatocytes (PXB-cells). Single doses of RIF at 10 and 30 mg/kg were orally or intravenously administered to PXB mice and wild-type ICR mice. Plasma concentrations (AUC0-8h) of CP I increased in both mice. However, a marked increase in CP III was only observed in ICR mice, after intravenous administration of RIF at 30 mg/kg. The IC50 values of RIF for intracellular CP I/III uptake and the unbound plasma concentrations of RIF suggested that the increase in plasma CP I is associated with the exposure of RIF to OATPs. The 24-h cumulative urinary excretions of CP I and CP III increased in both mice, but more markedly in PXB mice. Thus, RIF increased the plasma and urinary concentrations of CP I and CP III in the mice, as reported in humans, and CP I may be a more sensitive biomarker of OATP-mediated drug interactions in PXB mice.

The role of laboratory diagnostics in the assessment of occupational lead exposure

Introduction: Industrialization and urbanization led to a significant increase in the environment. Lead inhibits the activity of numerous enzymes, triggers oxidative stress, and causes protein biosynthesis dysregulation. Inhalation of lead particles is the most common route of intoxication associated with occupational exposure. This study aims to evaluate laboratory methods and biomarkers in the assessment of lead exposure. Methods: For non-experimental qualitative research, available scientific articles in English published in the relevant databases (MEDLINE and ScienceDirect) were used. The database search was performed using the keywords “Laboratory diagnostics”, “occupational exposure”, and “lead”. Results: Atomic absorption spectrometry (AAS) is the gold standard in laboratory monitoring of occupational lead exposure. Inductively coupled plasma with mass spectrometry is a commonly used method described as more sensitive than AAS due to its low detection limit. Lead concentrations can be determined in various samples, but blood and urine are the most commonly used in laboratory practice. The most important exposure biomarker is the enzyme δ-aminolevulinic acid dehydratase (ALAD) in the blood, which is characterized by progressive inactivation by lead and a negative correlation with its concentration. The concentration of urinary delta-aminolevulinic acid (δ-ALA-U) reflects the state of impaired enzyme function in heme biosynthesis. In addition, determining blood zinc protoporphyrin and urinary coproporphyrin levels significantly aids in assessing occupational lead exposure disorders. Conclusion: The availability of the laboratory methods used and the biomarker specificity and sensitivity play an important role in the adequacy of lead exposure monitoring. Accurate determination of ALAD and δ-ALA-U concentrations, along with other biomarkers, is critical for assessing individuals exposed to lead.

Occupational lead intoxication from terracotta tiles manufacturing: a case study

Abstract We report the case of a 63-year-old terracotta tiles manufacturer who presented with acute abdomen and normocytic anaemia. The patient presented with elevated levels of urinary delta-aminolaevulinic acid without any increase in the levels of urine porphobilinogen or urine coproporphyrin. Detection of elevated lead blood levels (1939 µg/dL one month before hospital admission in the occupational medicine clinic and 44.70 µg/dL at hospital admission, values come from two different laboratories) confirmed the diagnosis of chronic lead poisoning due to occupational exposure. Chelation therapy with D-penicillamine resulted in the improvement of clinical symptoms and lead blood levels. Clinicians should be aware that lead poisoning caused by occupational exposure can still occur sporadically in terracotta tiles manufacturing workers, lead poisoning being a neglected diagnosis in abdominal pain.

The combined action of lead and physical load in a subchorionic experiment on rats

Introduction. Exposure to lead associated with working conditions (especially in mining and metallurgical production) is usually combined with muscular work. However, the influence of this combination on the pathological process development has not been thoroughly studied. Materials and methods. During six weeks, white outbred male rats received intraperitoneal lead acetate at a dose of 11 mg / kg, corresponding to 1/20 LD50, three times a week and/or five times a week physical exercises (forced running for 10 minutes at a speed of 25 m/min). Results. As can be seen from the results, lead caused statistically significant shifts in 33.5% of the organism’s status indices, pointing to the onset of a moderate subchronic intoxication. Content of coproporphyrin in urine and percentage of reticulocytes in the peripheral blood increased, the average content of haemoglobin in the erythrocyte decreased. Under physical activity the harmful effect of lead enhanced in 23.7% of indices and weakened in 28.9% of indices. We did not find changes in 47.4% of indices. Conclusion. Lead intoxication changes ambiguously under the influence of physical exercises. The type of combined effect of lead toxicity and physical activities depends on the outcome by which this type is assessed, on this outcome’s level and the dose ratio.

Clinical, Biochemical, and Molecular Characterization of Neonatal-Onset Dubin-Johnson Syndrome in a Large Case Series From the Arabs.

Background: There are only a few case reports and small case series on neonatal-onset Dubin–Johnson syndrome (DJS), particularly from Far-East Asia, Iranian and Moroccan Jews, and Europe.Objectives: In this first study from the Arabs and the largest series reported to date, we characterized the clinical, laboratory, and molecular features and outcome of gene-confirmed neonatal-onset DJS.Methods: We reviewed our database of 533 cases of neonatal cholestasis that presented to our center during the period from 2008 to 2019. We identified neonates with a disease-causing mutation in ABCC2 gene.Results: Twenty-eight neonates with DJS were diagnosed (5.3%). All of the 28 were full-term, well looking neonates without hepatosplenomegaly, with cholestasis, and normal liver synthetic function since the 1 week of life that resolved within 3–6 months of age, followed by a benign course punctuated by recurrent episodes of jaundice in 43% during a median follow up period of 9.25 (range 2.5–14 years). Alanine aminotransferase levels were within normal range in 26 patients (92%) and mildly elevated in two patients. ALT levels were significantly lower in neonates with DJS than in other cases with neonatal cholestasis from other causes (p < 0.001). The median urinary coproporphyrin I% was 88% (IQ1–IQ3 = 84.2–92.7%). We identified four homozygous variants in the ABCC2 gene (from 22 unrelated families), one splicing variant (c.3258+1G>A; p.?), and three were missense variants; two of which were novel missense variants [c.1594G>A (p.Glu532Lys) and c.2439G>C (p.Lys813Asn)]. The p.Gly758Val mutation has occurred in 23 patients (from 19 unrelated families).Conclusions: Our study suggests that normal ALT-cholestasis in a well-looking neonate should trigger evaluation for DJS. The p.Gly758Val variant in ABCC2 is the most predominant mutation among Arabs with “founder effects.” Identification of the predominant ABCC2 variant in any population is likely to facilitate rapid molecular analysis by future targeting of that specific mutation.

Sustained hyperbilirubinemia following clarithromycin administration

Abstract Rationale: Hyperbilirubinemia is observed in drug-induced liver injury of cholestatic type, but direct inhibition of transporter proteins which facilitates bilirubin transport also causes hyperbilirubinemia. Here we present a case of sustained hyperbilirubinemia after the administration of clarithromycin. Patient concerns: A 52-year-old man was referred to our hospital because of sustained hyperbilirubinemia. Both contrast-enhanced computed tomography and Magnetic resonance cholangiopancreatography did not reveal any abnormality. But his liver was not enhanced in the hepatocellular phase of Gd-EOB-DTPA MRI. Indocyanine Green (ICG) retention rate at 15 minutes (ICG(R15)) was 58%, urinary coproporphyrin was 324 μg/g Cr, and serum bile acid was 283 nmol/mL. Technetium-99m galactosyl human serum albumin (99mTc-GSA) liver scintigram revealed that his liver function was normal (blood clearance ratio (HH15), 0.572; and hepatic uptake ratio (LHL15), 0.948) Diagnoses: These findings suggested that hyperbilirubinemia in the present case was caused by impaired organic anion-transporting polypeptide (OATP) 1B1 and 1B3 activity. Drug-induced lymphocyte stimulation test (DLST) for clarithromycin was positive. Among the drugs prescribed before the presentation of hyperbilirubinemia, clarithromycin is the only agent that has been reported to reduce the OATP1B1/1B3 transporter activity. Interventions: The patient continued taking oral UDCA, but hyperbilirubinemia did not improve. Bilirubin absorption therapy was performed twice. Outcomes: Following bilirubin absorption therapy, hyperbilirubinemia resolved and the bilirubin levels became normal. With the decrease in serum bilirubin, the liver was normally enhanced on the hepatocellular phase of Gd-EOB-DTPA MRI. ICG(R15). Urinary coproporphyrin, and bile acid levels returned to normal range (ICG(R15) of 9.2%, urinary coproporphyrin of 58 μg/g Cr, and bile acid of 13.5 nmol/mL). Lessons: The present case indicates that uptake of Gd-EOB-DTPA in the liver, ICG retention test, serum bile acid levels combined with urinary coproporphyrin can be a potential marker of OATP transporter activity and help in differential diagnosis of sustained hyperbilirubinemia.

Dubin-Johnson Syndrome as Differential Diagnosis for Neonatal Cholestasis.

Dubin-Johnson syndrome (DJS) is an autosomal recessive disorder in which multidrug-resistance-associated protein 2 (MRP2) deficiency causes an excretion disorder of conjugated bilirubin from hepatocytes into bile canaliculi. Its clinical presentation as neonatal cholestasis (NC) is rare but represents an important differential diagnosis. We aimed to define DJS-specific characteristics in NC, in particular in contrast to biliary atresia (BA) patients, and to highlight diagnostic tools that can help to avoid invasive diagnostic tests. We performed a review of case records from 2006 to 2020 and compared 4 DJS patients to 26 patients with proven BA consecutively diagnosed from 2014 to 2017. DJS was diagnosed by urine coproporphyrin analysis (UCA) and by genetic analysis (GA) for disease-associated ABCC2 variants. Four male patients with NC were diagnosed with DJS by UCA and GA. DJS patients presenting as NC showed significantly lower values for aspartate aminotransferase (AST) (P < 0.001), for alanine aminotransferase (ALT) (P = 0.002) and for gamma-glutamyl transferase (GGT) (P < 0.001) compared with BA patients. Other examinations, however, could not clearly discriminate them (e.g.: stool colour, serum bile acids, total serum bilirubin). DJS is not only a rare differential diagnosis in NC with a suspicious phenotype (almost normal AST, ALT) but also shows overlapping features with BA. It should, therefore, be considered in every infant with NC and an atypical liver enzyme pattern to protect patients from unnecessary, invasive examinations. For this, UCA is a fast and reliable diagnostic tool. Confirmation based on GA is recommended. DJS patients have a good long-term prognosis.

Причинно-наслідковий аналіз формування анемій у дітей

The purpose of the study was to determine the main causal factors in the formation of anemia in children of Ukraine, depending on the morphometric changes in erythrocytes of blood, indicators of iron metabolism for the formation of a risk group for oncohematological diseases. Material and methods. 770 children were examined: 724 with anemia, 46 with acute lymphoblastic leukemia. We studied the parameters of the erythrocyte lineage of hematopoiesis, morphometric parameters of erythrocytes, indicators of iron metabolism (serum iron, ferritin, transferrin, the transferrin saturation with iron, hematocrit, the content of δ-aminolevulinic acid and coproporphyrin in urine, pituitary thyroid stimulating hormone depending on the type of somatic pathology. Anemic states were distributed taking into account the average erythrocyte volume and the average hemoglobin content in the erythrocyte, and dividing diagnoses microcytic-hypochromic or normocytic-normochromic anemia, respectively. Results and discussion. The study showed that in children with normocytic-normochromic anemia, the number of erythrocytes and hematocrit were lower than in patients with microcytic-hypochromic anemia, while average erythrocyte volume, average hemoglobin content, serum iron, serum ferritin and transferrin saturation with iron were higher. The number of reticulocytes in the peripheral blood in all the examined subjects was standard. That is, the anemic conditions in children differed in ferrokinetic parameters, in particular, with and without iron deficiency. Taking into account the age of the children and the reasons for the development of anemia, a third of the girls of puberty with microcytic-hypochromic anemia had menorrhagias. In children under 6 years of age with normocytic-normochromic anemia, diseases of the gastrointestinal tract were more often registered; in the older 6 years – gastrointestinal diseases, helminthiasis and allergic reactions compared with patients with microcytic-hypochromic anemia. The development of normocytic-normochromic anemia in children and the functioning of the gastrointestinal tract were influenced by drug treatment for chronic pathology in the body. All children with anemia had an irrational diet. Porphyria was diagnosed in 3.8% of children with microcytic-hypochromic anemia. In 12.7% of children with normocytic-normochromic anemia, the serum thyroid stimulating hormone level was at the upper limit of the reference value (mean 3.3±0.6) mU/L), which correlated with a reduced number of erythrocytes in blood (r = -0.65) and increased values of average erythrocyte volume (r = 0.41) and average hemoglobin content (r = 0.35), and indicates changes in the erythrocyte lineage of hematopoiesis associated with the initial manifestations of thyroid hypofunction. An excess of iron was observed in 7.1% of older boys with normocytic-normochromic anemia, which requires additional examination. In patients with acute lymphoblastic leukemia were diagnosed with normocytic-normochromic anemia of varying severity. The serum ferritin level was (272.1±28.4) ng/ml and was significantly higher than in children with normocytic-normochromic anemia. In 12 of 46 patients, transferrin saturation with iron was increased and amounted to (70.2±2.3)%. Moreover, the higher the level of serum iron and serum ferritin, the higher was the transferrin saturation with iron (rs = 0.5; rs = 0.85). An inverse correlation was established between transferrin saturation with iron, patient survival (rs = -0.45) and a higher probability of death (rs = -0.46). Conclusion. Children with normocytic-normochromic anemia require in-depth examination and constitute a risk group for the development of myelodysplastic syndrome and leukemia

Developmental changes in urinary coproporphyrin ratio in premature infants.

Premature infants have a high concentration of conjugated bilirubin in their blood, although they have a poor glucuronide conjugation of bilirubin. This may be due to developmental changes in the function of adenosine triphosphate binding cassette subfamily C member 2, which is involved in the cellular export of conjugated bilirubin. In the present study, we examined the developmental changes in the urinary coproporphyrin I/(urinary coproporphyrin I+ urinary coproporphyrin III) ratio (UCP (I/ [I + III])), a known biomarker for adenosine triphosphate binding cassette subfamily C member 2 function, in premature infants. Twenty-one premature infants born between 25 and 32weeks of gestation were included in the study. Urine samples were collected within 24h of birth, and at 1week and 3-4weeks after birth. The samples were analyzed by high-performance liquid chromatography to calculate UCP (I/ [I + III]) to examine its association with postnatal age and corrected gestational age. Subjects were excluded if they had liver dysfunction, cholestasis, urinary tract infection, or chromosomal abnormalities. The average UCP (I/ [I + III]) within 24h of birth, at 1week, and at 3-4weeks after birth was 0.84, 0.61, and 0.65, respectively. The UCP (I/ [I + III]) within 24h of birth was significantly higher than that measured at 1week or 3-4weeks after birth. There was no significant correlation between UCP (I/ [I + III]) and the corrected gestational age. The UCP (I/ [I + III]) was higher within 24h of birth. It decreased 1week after birth and remained low without any significant changes for up to 4weeks after birth.

Dubin\u2013Johnson syndrome and intrahepatic cholestasis of pregnancy in a Sri Lankan family: a case report

BackgroundDubin–Johnson syndrome and intrahepatic cholestasis of pregnancy are rare chronic liver disorders. Dubin–Johnson syndrome may manifest as conjugated hyperbilirubinemia, darkly pigmented liver, presence of abnormal pigment in the parenchyma of hepatocytes and abnormal distribution of the coproporphyrin isomers I and III in the urine. Intrahepatic cholestatic jaundice of pregnancy presents as pruritus, abnormal liver biochemistry and increased serum bile acids.Case presentationA Sri Lankan girl presented with recurrent episodes of jaundice. She had conjugated hyperbilirubinaemia with diffuse, coarse brown pigments in the hepatocytes. Urine coproporphyrin examination suggested Dubin–Johnson syndrome. Genetic studies confirmed missense homozygous variant p.Trp709Arg in the ATP-binding cassette sub-family C member 2 gene ABCC2 that encodes the Multidrug resistance-associated protein 2 that causes Dubin–Johnson syndrome. The gene study of the mother revealed the same missense variant in ABCC2/MRP2 but with a heterozygous status, and in addition a homozygous missense variant p.Val444Ala in the ATP-binding cassette, sub-family B member 11 gene ABCB11 that encodes the bile salt export pump.ConclusionDubin–Johnson syndrome should be considered when the common causes for conjugated hyperbilirubinaemia have been excluded, and patient has an increased percentage of direct bilirubin relative to total bilirubin concentration. Its early diagnosis prevents repeated hospital admissions and investigations. Knowledge of a well known homozygous variant in ABCB11 gene could help in the management of pregnancy.

Human Biomonitoring of Lead Exposure.

After a chronic exposure, lead accumulates in the human body, especially in bones and teeth. Critical effects of lead affect the nervous system, reproduction, fertility as well as genotoxicity and carcinogenicity [1]. Analyses of lead concentrations in human biological material are performed using inductively coupled plasma mass spectrometry and atomic absorption spectrometry, but also electrochemical methods and X-ray fluorescence spectroscopy. The predominant sample matrices include blood and bone, as well as urine, hair, nail, and saliva. To characterize first biological effects, diverse parameters are discussed as "biomarkers of effect". These include δ-aminolevulinic acid dehydratase (ALAD) and erythrocyte porphyrins (EPs) in blood as well as δ-aminolevulinic acid (ALA) in urine and plasma and coproporphyrin in urine. However, biomarkers of effect alone are not sufficiently sensitive for an early detection of a health impairment caused by lead. In summary, lead in blood is the most prominent and best validated biomarker for a lead exposure. A recommended diagnostic strategy for revealing lead-induced effects is the determination of lead in whole blood combined with the analysis of different effect parameters like ALA and coproporphyrin in urine and ALAD and zinc protoporphyrin (ZPP) in blood.

Developmental characteristics of urinary coproporphyrin I/(I + III) ratio.

The ratio of urinary coproporphyrin (UCP) I to total urinary coproporphyrin I and III [UCP {I/(I + III)]] serves as a biomarker of the ATP-binding cassette, sub-family C, member 2 (ABCC2) function. The aim of this study was to clarify the characteristics of the developmental pattern of UCP [I/(I + III)] in order to estimate ABCC2 function in children, especially in the neonatal period, by measuring it throughout the entirety of childhood. Measurement of UCP [I/(I + III)] was done high-performance liquid chromatography, using urine samples collected from children from 1 day to 15 years old, involving one sample per child. Urine samples from children with liver and kidney disease and urinary tract infection were excluded. UCP [I/(I + III)] varied widely in infants younger than 6 months old, and was ≥0.3 in 80% of the infants. In contrast, it decreased to <0.30, the lowest, at 1-2 years old. In the 0-6-month-old group, no significant correlation was noted between postnatal age and UCP [I/(I + III)], but a moderate inverse correlation was noted between corrected gestational age and UCP [I/(I + III)]. UCP [I/(I + III)] is inversely correlated with corrected gestational age and is lowest at 1-2 years old. This suggests that ABCC2 activity is correlated with corrected gestational age and is highest at 1-2 years old.

Methotrexate and actinomycin D chemotherapy in a patient with porphyria: a case report

BackgroundDespite their broadly recommended use as chemotherapeutic agents, the porphyrogenicity of methotrexate and actinomycin D have not been confirmed. Accordingly, it is not known whether these agents are safe for use in patients with porphyria.Case presentationIn this report, we present a case of an invasive mole with lung metastasis in a 49-year-old Japanese woman who had previously been diagnosed with acute intermittent porphyria at 27 years of age but had no recent history of acute intermittent porphyria attacks. Her serum human chorionic gonadotropin level was elevated 1 month after hysterectomy, and she was referred to our center for chemotherapy. After she received 100 mg of methotrexate, drug eruptions were observed starting on day 3 and grew progressively worse. Erythema and mucosal erosion spread throughout her body, whereupon she was administered prednisolone. In addition, our patient experienced febrile neutropenia and required granulocyte colony- stimulating factor treatment. No changes in our patient’s urinary coproporphyrin or uroporphyrin levels were detected during this entire episode. Methotrexate was replaced by actinomycin D (0.5 mg/body intravenously on days 1–5 every 2 weeks). After five uneventful cycles of actinomycin D, our patient achieved and maintained a normal serum human chorionic gonadotropin level for 3 years.ConclusionsMethotrexate and actinomycin D did not induce acute porphyric attacks in this patient with acute intermittent porphyria; however, severe adverse effects were noted with methotrexate. Although further investigation is required, our data suggest that these agents are nonporphyrinogenic and can therefore be used to treat patients with comorbid porphyria.

Liver Damage Induced by Succinylacetone: A Shared Redox Imbalance Mechanism between Tyrosinemia and Hepatic Porphyrias

To show liver failure mediated by 5-aminolevulinic acid (ALA), a heme precursor accumulated in inborn and acquired porphyrias, rats were treated with succinylacetone methyl ester (SAME). Treated rats underwent the expected ALA accumulation in plasma, liver and urine as a result from inhibition of ALA dehydratase (ALAD) activity. The enzyme activity decreased concomitantly with diminished urinary coproporphyrin levels. Additionally, liver protein carbonyls, iron and ferritin were higher in groups treated with a lower concentration of SAME whereas malondialdehyde was higher in the group treated with a higher ester dose. Consistent with these biochemical data, chronic treatment SAME was associated with induced oxidative subcellular and tissue damage revealed by cytosolic and mitochondrial changes within the liver cells. Altogether, these data expand the understanding of the direct biochemical mechanism for liver cell death in hepatic inborn disorders by generating excess ALA and may foster future therapeutic-driven strategies to preserve liver function.

Implications of oxidative stress in occupational exposure to lead on a cellular level

The aim of this study was to determine oxidative alterations leading to cellular dysfunctions in Pb-exposed subjects by evaluating damage to all major classes of biomolecules in the cell, lipid peroxidation, protein and DNA damage and determine relationships between parameters of Pb toxicity and specific biomarkers of oxidative damage.Analysis was conducted of smelter workers with high blood Pb and urine aminolevulinic acid levels and slightly elevated values of coproporphyrin and erythrocyte protoporphyrin IX. Significant decreases of thiol groups and increases in carbonyl groups as protein degradation end products, and of nitrite were detected. Elevated rates of lipid peroxidation and rises in the activities of the antioxidant enzymes Cu–Zn superoxide dismutase and catalase were also observed. Both enzymes showed positive correlations with the blood lead levels and urine coproporphyrin, while thiol groups correlated negatively with the same indices. The genotoxic potential of lead was manifested through an increased number of DNA-damaged cells. Increased activities of serum lactate dehydrogenase isoenzymes indicated cellular damage in the lungs, kidneys, and liver. These lead-induced impairments should be taken into consideration in the assessment of Pb-related health hazards.

Direct assay of uroporphyrin and coproporphyrin in human urine by reverse-mode field amplified sample injection-sweeping and micellar electrokinetic chromatography

In this study, an on-line stacking capillary electrophoresis (CE) method, reverse-mode field amplified sample injection equipped with sweeping micellar electrokinetic chromatography (RMFASI-sweeping MEKC), was established for direct determination of uroporphyrin and coproporphyrin in human urine. Porphyrins playing a very important role in the biosynthesis of heme, chlorophyll and other important enzymes are a series of important molecules in organism. Therefore, determination of porphyrin metabolites, uroporphyrin and coproporphyrin, was very important for clinical survey of some diseases. In this study, the urine sample after simple dilution could be directly analyzed by this on-line stacking CE method. The optimal CE separation buffer was 70mM phosphate buffer at pH 3. Before sample injection, a water plug was introduced (2.5psi for 10s), and then the samples were loaded by electrokinetic injection (−10kV, 200s). Finally, the phosphate buffer (70mM, pH 3) containing 100mM SDS was served as the sweeping buffer to stack and separate the analytes at −20kV. The calibration curves were linear over a range of 15–200ng/ml for uroporphyrin, and 300–1000ng/ml for coproporphyrin. The coefficient of correlation (r) in intra-batch (n=5) and inter-batch (n=5) analysis was above 0.983. The LODs (S/N=3) were 5 ng/ml for uroporphyrin, and 100ng/ml for coproporphyrin. The absolute values of relative standard deviation (RSD) and relative error (RE) in intra-batch (n=5) and inter-batch (n=5) assays were less than 8.6% showing the good precision and accuracy. The stacking method was successfully applied in real urine sample and feasible for serving as a tool for detection of uroporphyrin and coproporphyrin in clinical.

Simultaneous Determination of Five Porphyrins in Human Urine and Plasma Using High Performance Liquid Chromatography-Tandem Mass Spectrometry

A specific and sensitive liquid chromatography-electrospray ionization tandem mass spectrometry method (LC-ESIMS/MS) was developed and validated for the simultaneous quantification of porphyrins (coproporphyrin, pentacarboxylporphyrin,hexacarboxylporphyrin, heptacarboxylporphyrin, and uroporphyrin) in human plasma and urine. Acidified plasma samplesand urine samples were prepared by using liquid-liquid extraction using ethyl acetate and protein precipitation with acetonitrile,respectively. The separation was achieved onto a Synergi Fusion RP column (150 mm × 2.0 mm, 4 μm) with a gradient elutionof mobile phase A (0.1% formic acid in 2 mmol/L ammonium acetate, v/v) and mobile phase B (20% methanol in acetonitrile, v/v) at a flow rate of 450 μL/min. Porphyrins and the internal standard (IS), coproporphyrin I-15N4, were detected by a tandemmass spectrometer equipped with an electrospray ion source operating in positive ion mode. Multiple reaction monitoring(MRM) transitions of the protonated precursor ions and the related product ions were optimized to increase selectivity and sensitivity. The proposed method was validated by assessing selectivity, linearity, limit of quantification (LOQ), precision, accuracy,recovery, and stability. The calibration curves were obtained in the range of 0.1-100 nmol/L and the LOQs were estimated as0.1 nmol/L for all porphyrins. Results obtained from the validation study of porphyrins showed good accuracy, precision, recovery,and stability. Finally, the proposed method was successfully applied to clinical studies on the autism spectrum disorder(ASD) diagnosis of 203 Korean children.

HBsAg-positive patient with Dubin-Johnson Syndrome: a case report

Dubin-Johnson Syndrome (DJS) is a rare autosomal recessive liver disorder characterized by chronic or intermittent conjugated, mild hyperbilirubinemia, and is caused by deficiency of multidrug resistance protein 2. DJS, is often an occult entity which until various factors accentuate hyperbilirubinemia, resulting in clinically apparent disease. In this study, we present a HBsAg-positive patient with DJS, who has been diagnosed based on elevated urinary coproporphyrin I isomer. The case was a 21-year-old male who complained about dizziness. His laboratory data revealed a chronic HBsAg sero-positivity and a mild conjugated/unconjugated hyperbilirubinemia; remaining analytical results were normal. Any pathological changes of liver and biliary tree were not observed on ultrasonography. Due to darkbrownish pigmentation demonstrated on liver biopsy reminded the DJS. Total porphyrin concentration in urine sample was within the reference range, diagnosis of DJS was confirmed by increased urine coproporphyrin I/total ratio (97%). Currently, DJS is diagnosed by demonstrating characteristic signs of the DJS on multiple diagnostic tests (e.g. histochemical examination of liver biopsy material and/or cholescintigraphic imaging). Although definitive diagnosis of this disorder mostly relies on ABCC2 gene mutation analysis, determination of urinary isomers of coproporphyrin may relatively be an easily accessible and strong biochemical indicator for diagnosis of DJS.

Novel mutations in the Dubin–Johnson syndrome gene ABCC2/MRP2 and associated biochemical changes

A patient with sepsis and jaundice was admitted for diagnosis and treatment. Associated biochemical changes included increased C-reactive protein, conjugated bilirubin and gamma-glutamyltransferase, the duration of which was protracted. High urine coproporphyrin isomer-1 and immunostaining of liver tissue suggested Dubin-Johnson syndrome. DNA sequencing using polymerase chain reaction amplification of the ABCC2 gene revealed the patient to have a compound heterozygous variant of MRP2, a molecule involved in canalicular transport of bilirubin. There was a history of jaundice since infancy.

Biological Monitoring Among Workers Exposed to Inorganic Lead and Its Compounds

Objective: To explore the association between lead biomarkers and their deviations in the circumstances of occupational exposure, and influence of life style factors. Material and Methods: We performed cross-sectional study using 60 workers occupationally exposed to lead compared with 60 controls. All examinees were assessed by Questionnaire, and laboratory testing concerning blood lead level (BLL), activity of delta-aminolevulinic acid dehydratase (ALAD) in blood, concentration of delta-aminolevulinic acid (ALA) and coproporphyrin in urine, reticulocytes and erythrocytes with basophilic stippling (EBS). Results: The mean values of BLL and ALA were significantly higher, and mean ALAD activity was significantly lower in lead workers than in controls. Lead workers also had a higher rate of abnormal BLL, ALAD, and ALA, significant for BLL and ALAD. The average BLL values among exposed workers and controls in men were significantly higher. There was strong inverse correlation between distribution of ALAD values in exposed workers due to their BLL values. Significant correlation with mean ALAD values was shown for alcohol consumption, form of compounds, and use of protecting equipment, whereas with mean BLL values was shown for age, gender, exposure duration, smoking, and alcohol consumption. Conclusion: The data confirmed the association between occupational exposure and lead biomarkers abnormalities.