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Glyceroluria and Neonatal Hemochromatosis

Barić, Ivo*; Zibar, Karin*; Ćorić, Marijana; Santer, René; Bonilla, Silvana§; Bilić, Karmen||; Buljević, Andrea Dasović*; Benjak, Vesna*; Fumić, Ksenija||; Whitington, Peter F.§

Journal of Pediatric Gastroenterology and Nutrition: October 2012 - Volume 55 - Issue 4 - p e126–e128
doi: 10.1097/MPG.0b013e3182347a26
Case Reports

*Department of Pediatrics, University Hospital Center Zagreb

Department of Pathology and Cytology, University Hospital Center Zagreb, Zagreb, Croatia

Department of Pediatrics, University Medical Center Hamburg-Eppendorf, Hamburg, Germany

§Department of Pediatrics, Feinberg School of Medicine of Northwestern University, Chicago, IL

||Clinical Institute for Laboratory Diagnostics, University Hospital Center Zagreb, Zagreb, Croatia.

Address correspondence and reprint requests to Karin Zibar, MD, Division for Metabolic Diseases, Department of Pediatrics, University Hospital Center Zagreb, Kišpatićeva 12, 10000 Zagreb, Croatia (e-mail:

Received 1 April, 2011

Accepted 25 August, 2011

The work was supported by Ministry of Science, Education and Sports of the Republic of Croatia Grant 108-1081870-1885.

The authors report no conflicts of interest.

Glycerol is an important intermediate in carbohydrate and lipid metabolism. Glyceroluria has been described in humans as a consequence of urine contamination, in patients with fructose-1,6-diphosphatase deficiency (1) and in glycerol kinase deficiency (2). In knockout mice it occurs with glycerol-3-phosphate dehydrogenase deficiency (3) and in proximal tubule injury due to aquaporin-7 gene (AQP7) deletion (4). Neonatal hemochromatosis (NH) is a severe liver disease with intrauterine onset caused in most cases by fetomaternal alloimmunity (5,6). The typical clinical presentation is that of neonatal liver failure, and diagnosis is achieved by showing siderosis of extrahepatic tissues (7). Proximal tubular dysgenesis has been reported in association with NH (8–10). Because renal tubular development depends upon an intact renin-angiotensin-aldosterone system (11,12), it has been proposed that severe fetal liver injury in NH could impair proximal renal tubular development via impaired hepatic angiotensinogen production (13). We report on 2 siblings who died of neonatal sepsis and multiorgan failure, the second of which had proven alloimmune NH. The unique clinical feature of the second child was significant glyceroluria, which may be explained by proximal renal tubular dysgenesis.

A male patient was born at term to a G1 mother after an unremarkable pregnancy. The parents were healthy and nonconsanguineous. At the age of 5 days he presented in septic shock, became anuric, and required mechanical ventillation. He was somnolent with mild hepatosplenomegaly. Laboratory test results showed thrombocytopenia (42 × 109/L), anemia (Hb 104 g/L), and coagulopathy (prothrombin time 21% of normal plasma, activated partial thromboplastin time 110 seconds, fibrinogen 1.47 g/L). Despite all therapeutic attempts the child died at the age of 6 days due to Escherichia coli sepsis and multiorgan failure. The autopsy reported profound liver fibrosis.

A girl patient was born at term with an unremarkable delivery. The mother, now G4, had 2 early spontaneous miscarriages in the interim. The newborn had thrombocytopenia (67 × 109/L) and mild facial dysmorphy (high forehead, beaked nose, and widely spaced eyes). At the age of 7 hours she became hypotonic and pale and at the age of 30 hours developed septic shock. She was atonic with no respirations, hypotensive (35/14 mmHg), and anuric. She had hemorrhagic vomiting and mild hepatomegaly. Laboratory workup revealed hypoglycemia (1.6 mmol/L), lactic acidemia (10.3 mmol/L), hyperammoniemia (196.9 μmol/L), thrombocytopenia (52 × 109/L), anemia (Hb 97 g/L), severe coagulopathy (prothrombin time <10% of normal plasma, activated partial thromboplastin time > 120 seconds, fibrinogen <0.3 g/L), and hypoalbuminemia (total protein 30 g/L, albumin 19.5 g/L), hyperbilirubinemia (total bilirubin 153 μmol/L, conjugated bilirubin 48 μmol/L), and mildly increased aminotransferases (AST 339 U/L, ALT 75 U/L). Glycerol content of the urine was found to be significantly increased (1.45 and 4.5 mmol/L at the age of 2 and 7 days, respectively, normal <0.2 mmol/L). Plasma triglyceride concentration was 0.85 mmol/L (normal <1.24). Otherwise, organic acids showed only increased excretion of lactate and pyruvate, which were considered secondary phenomena. Succinylacetone was absent. Plasma amino acids showed unspecific changes pointing liver damage. Reducing substances in urine were negative. Plasma samples for iron and iron storage studies were not taken. Chromosome analysis was normal. Abdominal ultrasound showed enlarged liver, ascites, and large hyperechoic kidneys with erased corticomedullar border. The child died at 7 days of multiorgan failure resistant to intensive therapy, including dialysis. Autopsy revealed the NH phenotype with extensive liver siderosis and injury, in combination with siderosis of heart, pancreas, thyroid, and renal tubules. Liver and kidney glycogen contents were normal. Special studies performed on the autopsy materials confirmed alloimmune liver injury (Fig. 1) and proximal renal tubular dysgenesis (Fig. 2).





Glyceroluria was detected by organic acids analysis using gas chromatography-mass spectrometry of trimethylsilyl derivatives (14). Quantitative determination of glycerol in urine was carried out by triglyceride test kit measuring glycerol after triglycerides hydrolysis (Triglyceride R2; in vitro diagnostic reagent; Olympus Life Science Research Europa GmbH, Lismeehan, Clare, Ireland).

Paraffin-embedded liver tissue was used for detection of alloimmune liver injury (5) and renal tubular dysgenesis (13). Liver sections were treated with monoclonal antibody to human SC5b-9 neoantigen (TCC-neoantigen; monoclonal antibody to human SC5b-9 neoantigen; Quidel Corp, San Diego, CA). Kidney sections were treated with antibody to epithelial membrane antigen (EMA; antihuman monoclonal mouse epithelial membrane antigen; Dako Corporation, Carpinteria, CA) and fumaryl acetoacetate hydrolase (FAH; polyclonal antibody provided by RM Tanguay, Ste-Foy, Canada), markers for distal and proximal renal tubules, respectively; followed by treatment with appropriate biotinylated antibody (Vector Laboratories, Burlingame, CA) followed by development with VECTASTAIN ABC reagent (Vector Laboratories) according to the manufacturer's instructions.

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Glyceroluria in the second child was a puzzling clinical finding. She and her brother (patient 1) presented at an early age with what appeared to be septic shock and both died of multiorgan failure. The autopsies of both infants showed severe liver disease, in patient 2 phenotypically NH. The materials from patient 1 could not be recovered to examine for NH. In neither case did the autopsy findings provide causation for glyceroluria in the second child. We could exclude glyceroluria due to contamination because urine was carefully taken by a catheter. Analysis of the FBP1 gene affected in fructose-1,6-diphosphatase deficiency did not show any mutation within the coding sequence and in adjacent intronic segments, which made that condition unlikely in a nonconsanguineous family. Furthermore, it was unlikely that glyceroluria was related to glycerol kinase deficiency because that is an X-linked disorder, and the second child was a girl without chromosomal abnormalities. We assumed that circulating amounts of glycerol should be cleared easily by normal kidneys. Because it is known that there is a selective effect of NH on proximal tubules (13), that AQP7 is a glycerol reabsorbing carrier protein in proximal straight tubules (15), and that AQP7 knockout mice show marked glyceroluria (4), we concluded that glyceroluria in our patient was the result of the shortage in the amount of AQP7 transporters secondary to proximal renal tubular dysgenesis.

The histopathologic hallmark of renal tubular dysgenesis in NH is paucity or absence of proximal tubules without coexistent necroinflammatory disease (15). A close relation has been established between renal tubular dysgenesis and NH-related fetal liver injury (13): the worse the loss of hepatocytes, the greater the degree of tubular dysgenesis. Both of these infants showed severe liver injury with marked fibrosis, consistent with fetal onset of severe liver disease. The second patient was shown to have complement-mediated hepatocyte injury typical of gestational alloimmune liver disease, the cause of most cases of NH (5,6). We assume the first patient's condition was also NH due to alloimmune liver injury, but the autopsy materials could not be retrieved to confirm this.

Aquaporins are membrane proteins that allow water transport across the cell membrane (15). Among numerous aquaporins, AQP7, an aquaglyceroporin, is expressed on the apical membrane of proximal straight tubules (S3 segment). It is responsible mainly for glycerol reabsorption and plays a minor role in water permeability. Sohara et al (4) generated AQP7 knockout mice that showed marked glyceroluria without elevation of plasma glycerol concentrations. This indicates that glyceroluria in AQP7 knockout mice is not due to the increase of plasma glycerol concentration, but is rather a result of impaired glycerol reabsorption in the kidney (4). It is still not clear whether AQP7 is the only glycerol reabsorption pathway present along the nephron, but other glycerol-reabsorbing mechanisms, if they existed, could not completely compensate for the lack of AQP7 in this knockout mouse model.

To our knowledge, this is the first report of glyceroluria in association with NH. Glyceroluria provided evidence for functional proximal tubular impairment in this case in which dysgenesis was confirmed postmortem. Other diseases, which could present both with liver failure and renal tubular impairment in the first days of life, such as inherited disorders of mitochondrial energy production, galactosemia, tyrosinemia type 1, and some rare congenital disorders of glycosylation, seemed unlikely because of negative laboratory findings, lack of typical clinical signs, or the presence of the disease already at birth. In addition, in these disorders proximal renal tubular dysfunction is due to a toxic metabolic defect or the lack of energy and not due to dysgenesis as in NH. The clinical diagnosis of NH can be quite difficult (16). This case suggests that the finding of glyceroluria in association with liver failure in a newborn should alert clinicians to the diagnosis of NH. The report also stresses the need for urinary organic acids analysis using gas chromatography-mass spectrometry in a patient with unexplained neonatal liver disease. This could improve detection and diagnosis of NH, which is important because medical therapy may improve outcome (17). It is also important to make a proper diagnosis to help their mothers, who would benefit from preventive application of intravenous immunoglobulin in future pregnancies (18).

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