Primary carnitine deficiency with severe acute hepatitis following rotavirus gastroenteritis.

Rotavirus infection is a major cause of gastroenteritis, which occurs mainly in children. Liver dysfunction due to rotavirus gastroenteritis has been reported; however, acute hepatitis due to this disease is very rare. We present a rare case in which rotavirus gastroenteritis led to sequential diagnosis of acute hepatitis and systemic primary carnitine deficiency (CDSP) in a 1-year-old girl. The patient's symptoms (hypoglycemia, hepatomegaly, and elevated levels of serum transaminases and creatinine kinase) suggested a steatosis causing liver dysfunction. She was initially considered to have a beta oxygenation defect or secondary carnitine deficiency caused by pivalic acid-containing antibiotics; however, repetitive carnitine analysis and free carnitine clearance measurement confirmed primary carnitine deficiency (carnitine transporter deficiency). Children with severe liver dysfunction due to rotavirus infection and presenting with liver steatosis should undergo blood acyl carnitine analysis to detect potential carnitine or other beta oxidation deficiencies, especially if newborn screening for these diseases is not available.

Hypocarnitinemia Observed in an Infant Treated with Short-Term Administration of Antibiotic Containing Pivalic Acid.

Carnitine is a water-soluble amino acid derivative required for ß-oxidation of long-chain fatty acids. In carnitine cycle abnormalities and low carnitine states, fatty acid ß-oxidation is inhibited during fasting, resulting in hypoglycemia. Pivalic acid is a substance used in prodrugs to increase absorption of parent drugs, and antibiotics containing pivalic acid are frequently used as wide spectrum antibiotics for pediatric patients in Japan. Pivalic acid released after absorption is conjugated with free carnitine to form pivaloylcarnitine, which is then excreted in urine. As a consequence, long-term administration of pivalic acid containing antibiotics has been associated with depletion of free carnitine, inhibition of energy production and subsequent hypoglycemia. Here we report a case of a 23-month-old boy treated with an antibiotic containing pivalic acid for 3 days for upper respiratory tract infection. Laboratory data at referral indicated hypoglycemia, decreased free carnitine and elevated five-carbon acylcarnitine. Isomer separation confirmed the major component of increased five-carbon acylcarnitine to be pivaloylcarnitine, thereby excluding the possibility of a genetic metabolic disorder detected with similar acylcarnitine profile. The level of carnitine was normal when the antibiotic was not administered. Our case shows that the use of antibiotics containing pivalic acid in young children requires consideration of hypocarnitinemia, even with short-term administration.

Newborn screening for carnitine palmitoyltransferase II deficiency using (C16+C18:1)/C2: Evaluation of additional indices for adequate sensitivity and lower false-positivity.

BACKGROUND: Carnitine palmitoyltransferase (CPT) II deficiency is one of the most common forms of mitochondrial fatty acid oxidation disorder (FAOD). However, newborn screening (NBS) for this potentially fatal disease has not been established partly because reliable indices are not available. METHODS: We diagnosed CPT II deficiency in a 7-month-old boy presenting with hypoglycemic encephalopathy, which apparently had been missed in the NBS using C16 and C18:1 concentrations as indices. By referring to his acylcarnitine profile from the NBS, we adopted the (C16+C18:1)/C2 ratio (cutoff 0.62) and C16 concentration (cutoff 3.0nmol/mL) as alternative indices for CPT II deficiency such that an analysis of a dried blood specimen collected at postnatal day five retroactively yielded the correct diagnosis. Thereafter, positive cases were assessed by measuring (1) the fatty acid oxidation ability of intact lymphocytes and/or (2) CPT II activity in the lysates of lymphocytes. The diagnoses were then further confirmed by genetic analysis. RESULTS: The disease was diagnosed in seven of 21 newborns suspected of having CPT II deficiency based on NBS. We also analyzed the false-negative patient and five symptomatic patients for comparison. Values for the NBS indices of the false-negative, symptomatic patient were lower than those of the seven affected newborns. Although it was difficult to differentiate the false-negative patient from heterozygous carriers and false-positive subjects, the fatty acid oxidation ability of the lymphocytes and CPT II activity clearly confirmed the diagnosis. Among several other indices proposed previously, C14/C3 completely differentiated the seven NBS-positive patients and the false-negative patient from the heterozygous carriers and the false-positive subjects. Genetic analysis revealed 16 kinds of variant alleles. The most prevalent, detected in ten alleles in nine patients from eight families, was c.1148T>A (p.F383Y), a finding in line with those of several previous reports on Japanese patients. CONCLUSIONS: These findings suggested that CPT II deficiency can be screened by using (C16+C18:1)/C2 and C16 as indices. An appropriate cutoff level is required to achieve adequate sensitivity albeit at the cost of a considerable increase in the false-positive rate, which might be reduced by using additional indices such as C14/C3.

Prevalence of Fabry disease in dialysis patients: Japan Fabry disease screening study (J-FAST).

BACKGROUND: In Fabry disease, progressive glycolipid accumulation leads to damage in kidney and other organs. This study was designed to determine the prevalence rate of Fabry disease in Japanese dialysis patients. METHODS: All dialysis patients agreeing to Japan Fabry disease screening study (J-FAST) with informed consent were selected except for Fabry disease. The screening was performed by a method of measuring plasma and/or leukocytes lysosomal α-galactosidase A protein level and α-galactosidase A activity. If positive, genetic analysis was carried out upon patient's agreement. RESULTS: J-FAST dealt with 8547 patients (male 5408, female 3139). At the tertiary examination, 26 out of 8547 patients were found to be positive. Six out of 26 patients could not accept genetic analysis because of death. Remaining 20 patients agreed with genetic analysis; then 2 patients (male 2, female 0) had a variation of the α-Gal gene and 11 patients showed E66Q variations. Therefore, the frequency of Fabry disease in J-FAST was 0.04 % (2/5408) in males and 0 % (0/3139) in females, and then 0.02 % (2/8547) in all patients. The presumptive clinical diagnoses of end-stage kidney disease (ESKD) were 10 chronic glomerulonephritis, 7 diabetic nephropathy, 3 unknown etiology, 3 nephrosclerosis, 1 gouty nephropathy, 1 autosomal dominant polycystic kidney disease and 1 renal tuberculosis among 26 tertiary positive patients. Two male Fabry patients were initially diagnosed as nephrosclerosis and chronic glomerulonephritis. CONCLUSIONS: The prevalence rate of Fabry disease in J-FAST was 0.02 %. Moreover, Fabry disease could not be ruled out as the clinical diagnosis of ESKD.

Elevation of pivaloylcarnitine by sivelestat sodium in two children.

BACKGROUND: Sivelestat sodium (sivelestat), a neutrophil elastase inhibitor, is used to treat acute respiratory distress syndrome (ARDS). We report two cases that developed elevated C5-acylcarnitine (C5-AC) levels following treatment with sivelestat. Case 1 was a 14-day-old female infant born at 25 weeks and 1 day of gestation who was treated with sivelestat for the prophylaxis of Wilson-Mikity syndrome soon after birth. Isovaleric acidemia (IVA) was suspected based on a newborn screening using tandem mass spectrometry (MS/MS). Her C5-AC level was elevated to 4.49 µM (cut-off, <1.0) after treatment with sivelestat. Case 2 was a 4-year-old female with pneumocystis pneumonia that developed during chemotherapy for disseminated medulloblastoma. Sivelestat was given for the complication of ARDS. Her C5-AC level increased (1.09 µM) after eight days of treatment with sivelestat. RESULTS: In both cases, IVA was ruled out because isovalerylglycine was not observed in the urinary organic acid analysis. Case 1 was associated with carnitine deficiency (C0 9.16 µM; reference value, 10-60). Liquid chromatography-MS/MS confirmed elevated pivaloylcarnitine (PVC) in both cases. DISCUSSION: Similar to antibiotics containing pivalic acid (PVA), sivelestat contains PVA, which has the potential to cause secondary carnitine deficiency. In addition, elevated PVC can lead to false positive findings of IVA in newborns screened using MS/MS.

The High Relevance of 21-Deoxycortisol, (Androstenedione + 17α-Hydroxyprogesterone)/Cortisol, and 11-Deoxycortisol/17α-Hydroxyprogesterone for Newborn Screening of 21-Hydroxylase Deficiency.

CONTEXT: There are limited reports on the detailed examination of steroid profiles for setting algorithms for 21-hydroxylase deficiency (21OHD) screening by liquid chromatography-tandem mass spectrometry (LC-MS/MS). OBJECTIVE: We aimed to define an algorithm for newborn screening of 21OHD by LC-MS/MS, measuring a total of 2077 dried blood spot samples in Tokyo. METHODS: Five steroids (17α-hydroxyprogesterone [17αOHP], 21-deoxycortisol [21DOF], 11-deoxycortisol [11DOF], androstenedione [4AD], and cortisol [F]) were included in the panel of LC-MS/MS. Samples from 2 cohorts were assayed: Cohort A, 63 "screening positive" neonates who were referred to an endocrinologist (n = 26 with 21OHD; n = 37 false-positive; obtained from 2015 to 2020); and Cohort B, samples (n = 2014) with 17αOHP values in the 97th percentile or above, in the first-tier test with 17αOHP ELISA from 2020 to 2021. RESULTS: Analysis of Cohort A revealed that the 3 indexes 21DOF, 11DOF/17αOHP, and (4AD + 17αOHP)/F had higher area under the curve (AUC) values (0.999, 0.997, 0.989, respectively), while the 17αOHP AUC was lower (0.970). Accordingly, in addition to 17αOHP, the 3 markers were included for defining the screening algorithm. The assay of Cohort B revealed that the new algorithm gave 92% of predicted positive predictive value without false-negative cases. We also determined the reference values for the 5 steroids at 4 to 7 days after birth, according to sex and gestational age (GA), revealing extremely low levels of 21DOF at any GA irrespective of sex differences. CONCLUSION: Our study demonstrated the high relevance of 21DOF, (4AD + 17αOHP)/F, and 11DOF/17αOHP, rather than 17αOHP, for 21OHD screening.

Minimum requirement of donor cells to reduce the glycolipid storage following bone marrow transplantation in a murine model of Fabry disease.

BACKGROUND: Fabry disease (FD) is a lysosomal storage disorders characterized by a deficiency of the lysosomal enzyme, α-galactosidase A. This results in the accumulation of glycolipids, mainly globotriaosylceramide (GL-3), in the lysosomes of various organs. Although bone marrow transplantation and hematopoietic stem cell-based gene therapy can offer the potential of a curative therapeutic outcome for FD, the minimum requirement of donor cells or gene-corrected cells to reduce GL-3 levels is not known. METHODS: Lethally-irradiated FD mice were transplanted intravenously with normal bone marrow cells (Ly5.1 positive) mixed with those of FD mice (Ly5.2 positive) at various ratios to investigate the level of engraftment and enzyme activity necessary to effect a reduction in GL-3 storage. RESULTS: Chimerism of whole white blood cells of recipients' peripheral blood remained stable at 8 weeks after transplantation, and chimerism of granulocytes, monocytes, B cells and T cells was equal to that of white blood cells. GL-3 levels were significantly reduced in the lung and heart of animals with a 30% and 50% chimera, respectively. The extent of reduction in these mice was almost identical to that with 100% chimera. CONCLUSIONS: In FD mice, reconstitution with 100% donor cells is not required to obtain a therapeutic effect following bone marrow transplantation. These results suggest that a 30% gene correction might be sufficient to reverse disease manifestations in FD.

Development of Second-Tier Liquid Chromatography-Tandem Mass Spectrometry Analysis for Expanded Newborn Screening in Japan.

To minimize false-positive cases in newborn screening by tandem mass spectrometry in Japan, practical second-tier liquid chromatography-tandem mass spectrometry analyses have been developed using a multimode ODS column with a single set of mobile phases and different gradient elution programs specific to the analysis of acylcarnitines, acylglycines, amino acids, and organic acids. Most analyses were performed using underivatized samples, except for analysis of methylcitric acid, and careful conditioning of the column was necessary for analyses of organic acids. Our second-tier tests enabled us to measure many metabolites useful for detection of target disorders, including allo-isoleucine, homocysteine, methylmalonic acid, and methylcitric acid. We found that accumulation of 3-hydroxyglutaric acid was specific to glutaric acidemia type I and that the ratio of 3-hydroxyisovaleric acid to 3-hydroxyisovalerylcarnitine was useful to detect newborns of mothers with 3-methylcrotonyl-CoA carboxylase deficiency. Data from the analysis of short-chain acylcarnitine and acylglycine were useful for differential diagnosis in cases positive for C5-OH-acylcarnitine or C5-acylcarnitine.

Plasma globotriaosylsphingosine as a biomarker of Fabry disease.

Fabry disease is an X-linked genetic disorder caused by a deficiency of alpha-galactosidase A (GLA) activity. As enzyme replacement therapy (ERT) involving recombinant GLAs has been introduced for this disease, a useful biomarker for diagnosis and monitoring of therapy has been strongly required. We measured globotriaosylsphingosine (lyso-Gb3) and globotriaosylceramide (Gb3) in plasma samples from ten hemizygous males (six classic and four variant cases) and eight heterozygous females with Fabry disease, and investigated the responses of plasma lyso-Gb3 and Gb3 in a male Fabry patient who had undergone ERT for 4years to determine whether plasma lyso-Gb3 and Gb3 could be biomarkers of Fabry disease. The results revealed that plasma lyso-Gb3 was apparently increased in male patients and was higher in cases of the classic form than those of the variant one. In Fabry females, plasma lyso-Gb3 was moderately increased in both symptomatic and asymptomatic cases, and there was a correlation between the increase in lyso-Gb3 and the decrease in GLA activity. As to plasma Gb3, the levels in the variant Fabry hemizygotes and Fabry heterozygotes could not be distinguished from those in the controls, although those in the classic Fabry hemizygotes were increased. The plasma lyso-Gb3 level in the Fabry patient who had received ERT was elevated at the baseline and fell more dramatically on ERT than that of Gb3. Plasma lyso-Gb3 could thus be a potential biomarker of Fabry disease.

Clinical Application of Liquid Chromatography Tandem Mass Spectrometry Using Dried Blood Spot as a More Rapid Method for Determination of Methylmalonic Acid, Propionylcarnitine, and Total Homocysteine

Abstract Methylmalonic acidemia (MMA) should be diagnosed in early infancy and receive appropriate management promptly after the diagnosis to prevent severe complications leading to death. At present, a newborn screening (NBS) method using tandem mass spectrometry (MS/MS) identifies suspected patients with MMA by elevated propionylcarnitine. In addition, a liquid chromatography tandem mass spectrometry (LC/MS/MS) method using dried blood spot is effective to detect some metabolites as a second-tier test, and reduces the false-positive rate in NBS. However, these tests were only used in screening, and not applied as an examination for evaluating treatment. Herein, we describe a 57-day-old girl with MMA under treatment with cobalamin who had elevated urinary methylmalonic acid levels. We applied the LC/MS/MS method with a separation column to evaluate her cobalamin responsiveness, and discovered an insufficient cobalamin dose earlier than would have been possible using other methods. Based on the current data, this method seems to be applicable for the follow-up of the treatment of MMA patients. However, this should be confirmed with more experience with a larger number of cases and a wider spectrum of disorders.

Non-invasive high-risk screening for Fabry disease hemizygotes and heterozygotes.

The most appropriate time for screening for Fabry disease (FD) is school age. For this reason, we developed non-invasive methods for measuring urinary alpha-galactosidase A (alpha-gal A) protein, using enzyme-linked immunosorbent assay (ELISA), and for globotriaosylceramide (GL-3), using tandem mass spectrometry (MS/MS). We measured these two biomarkers in the urine of previously diagnosed FD hemizygotes and heterozygotes, and in controls. All the classic FD hemizygotes were clearly distinguished from controls by either method alone, and combining the two assays produced 96% sensitivity for detecting heterozygotes. To assess the utility of these methods for screening school children and adults at high risk of FD, a pilot study was conducted. To distinguish FD from 432 controls, cut-off values for alpha-gal A protein and GL-3 were set at the 5th and 95th centile values of the controls, respectively. Among the high-risk patients, the measurements exceeded the cut-off values for both biomarkers in male and female subjects and were strong indicators for Fabry hemizygotes and heterozygotes. However, we recommend that if the results of the first measurements exceed the cut-off values for only one of these biomarkers, another urine sample should be requested for re-assay to confirm the result.

Influence of antibody formation on reduction of globotriaosylceramide (GL-3) in urine from Fabry patients during agalsidase beta therapy.

Two recombinant human agalsidase preparations are available for treatment of Fabry disease. We assayed urinary GL-3 (uGL-3) concentration in seronegative and seropositive patients receiving agalsidase beta (1mg/kg). Antibody formation and residual enzyme activity were strongly correlated. Normalization of uGL-3 was achieved more efficiently in seronegatives. But different from previous reports, reduction of uGL-3 level was observed in some seropositive patients.

Non-invasive screening method for Fabry disease by measuring globotriaosylceramide in whole urine samples using tandem mass spectrometry.

Fabry disease is an X-linked sphingolipidosis due to a deficiency of alpha-galactosidase A, which leads to the accumulation of globotriaosylceramide (GL-3) in several organs. When recombinant human alpha-galactosidase A is intravenously administered repeatedly before the patient develops permanent tissue damage, there is evidence that the accumulation of GL-3 is decreased in some organs and that the clinical symptoms are alleviated in some patients. However, Fabry disease is rare and many patients are not diagnosed until adulthood after irreversible tissue damage has occurred. Our group has developed a simple and non-invasive screening method for Fabry disease that measures total GL-3 in whole urine samples by tandem mass spectrometry. Using this method, we found that the concentration of GL-3 in whole urine sample from hemizygous patients, including pre-symptomatic young children with classic type Fabry disease, was significantly higher than that in controls. The mean concentration of GL-3 in urine from heterozygotes with symptoms was significantly higher than control concentrations, but GL-3 levels in the urine from 2 out of 8 heterozygotes of classic type Fabry disease were within control levels. An asymptomatic 14-year old hemizygote in the family of a cardiac variant did not have elevated urinary GL-3. Therefore, screening for the classic type and probably renal variant of Fabry disease is possible by measuring urinary GL-3, using our method. The early diagnosis of cardiac variant hemizygotes and some heterozygotes with all types of Fabry disease will not be possible using our method. We propose that this procedure can be used as a reliable, non-invasive, simple method for general and high-risk population screening for hemizygotic patients with the classic type and probably renal variant of Fabry disease.