Hironori Nagasaka

Craniotabes in Normal Newborns: The Earliest Sign of Subclinical Vitamin D Deficiency

CONTEXT Craniotabes in otherwise normal neonates has been regarded as physiological and left untreated. OBJECTIVE Our objective was to investigate the role of vitamin D deficiency in the development of craniotabes in normal neonates. DESIGN AND SETTING Newborn screening of craniotabes was conducted at the single largest obstetrical facility in Kyoto, Japan. Follow-up study at 1 month was conducted at Kyoto University Hospital. SUBJECTS A total of 1120 consecutive normal Japanese neonates born in May, 2006, through April, 2007, were included in the study. MAIN OUTCOME MEASURES The incidence of craniotabes was scored each month. Neonates with craniotabes were followed up at 1 month with measurements of serum calcium, phosphorus, alkaline phosphatase (ALP), intact PTH, 25-OH vitamin D (25-OHD), urinary calcium, phosphorus, creatinine, and hand x-rays. RESULTS Craniotabes was present in 246 (22.0%) neonates, and the incidence had obvious seasonal variations, highest in April-May and lowest in November. At 1 month, infants with craniotabes had significantly higher serum ALP compared with normal neonates; 6.9% of them had elevated intact PTH over 60 pg/ml, and 37.3% had 25-OHD less than 10 ng/ml. When separately analyzed according to the method of feeding, 56.9% of breast-fed infants showed 25-OHD less than 10 ng/ml, whereas none of formula/mixed-fed infants did, and breast-fed infants had significantly higher serum PTH and ALP compared with formula/mixed-fed infants. SUMMARY These results suggest that craniotabes in normal neonates is associated with vitamin D deficiency in utero, and the deficiency persists at 1 month in many of them, especially when breast-fed.

Unique character and metabolism of high density lipoprotein (HDL) in fetus

Lipid and lipoprotein profiles, and enzymes for the lipid metabolism were compared between cord and adult blood. Consistent with previous reports, the major lipoprotein in cord blood was high-density lipoprotein (HDL), and that in adult blood was low-density lipoprotein (LDL). The level of apolipoprotein E (apo E) in cord blood was almost equivalent to that in adult blood, while other apolipoproteins and lipids were all lower than the adult level. In cord blood, apo E-rich HDL cholesterol represented more than 30% of total HDL cholesterol (around 11% in adult), and the concentration was about twice of that in adult blood. This apo E-rich HDL cholesterol was poorly esterified (E/T 56%) compared with that in adults (93%). The lecithin:cholesterol acyltransferase (LCAT) activity in cord blood was extremely low, while the activity and mass of cholesteryl ester transfer protein (CETP) were higher than those in adult blood. The apo E genotype did not show influences on total cholesterol, LDL cholesterol, total HDL cholesterol, and apo E rich HDL cholesterol levels in cord blood, as opposed to those in adult blood. The association of D442G mutation of the CETP gene with the increased HDL cholesterol in adult blood was not seen in cord blood. Rather, the mutation was associated with low total cholesterol and LDL cholesterol levels in cord blood. These results indicate that, in fetus, the character and metabolism of HDL, especially of apo E-rich HDL cholesterol, are distinct from those in adults.

X-inactivation pattern in the liver of a manifesting female with ornithine transcarbamylase (OTC) deficiency

Ornithine transcarbamylase (OTC) deficiency is the most common urea cycle disorder. It is X‐linked and hemizygous new‐born males usually suffer fatal hyperammonemia. In contrast, carrier females manifest variable phenotypes, ranging from asymptomatic carriers to those with severe hyperammonemia. In order to understand the correlation between X‐inactivation status and the clinical phenotype of carrier females with this disorder, we analyzed the X‐inactivation pattern of peripheral blood leukocytes in a family consisting of a clinically normal mother and two daughters with severe manifestation. In addition, we obtained tissue samples from various parts of the liver of one of these daughters and analyzed X‐inactivation patterns and the residual OTC activities. The X‐inactivation of peripheral blood leukocytes was nearly random in these carrier females and showed no correlation with the disease phenotype. However, the X‐inactivation of the liver was much more skewed and correlated well with the OTC activity of all samples. Interestingly, the degree of X‐inactivation varied considerably, even within the same liver.

Immunohistochemical analysis of Mallory bodies in Wilsonian and non-Wilsonian hepatic copper toxicosis

Patients with Wilsons disease (WD), Indian childhood cirrhosis (ICC), and idiopathic copper toxicosis (ICT) develop severe liver disease morphologically characterized by ballooning of hepatocytes, inflammation, cytoskeletal alterations, and Mallory body (MB) formation, finally leading to mostly micronodular cirrhosis. The pathogenesis of MBs in copper toxicosis is still unresolved. Immunohistochemical analysis of MBs in different types of copper intoxication revealed that keratin, p62, and ubiquitin are integral components. Thus MBs associated with copper intoxication resemble those present in alcoholic steatohepatitis (ASH) and nonalcoholic steatohepatitis (NASH). p62 is a multifunctional immediate early gene product that, on the one hand, is involved in stress‐induced cell signaling (particularly that of oxidative stress) by acting as an adapter protein linking receptor‐interacting protein (RIP) with the atypical protein kinase C. On the other hand, p62 binds with high affinity to polyubiquitin and ubiquitinated proteins. In conclusion, p62 accumulation in WD, ICC, and ICT and deposition in MBs indicates a central role of protein misfolding induced by oxidative stress in copper‐induced liver toxicity. By sequestering potentially harmful misfolded ubiquitinated proteins as inert cytoplasmic inclusion bodies (e.g., as MBs), p62 may be a major player in an important cellular rescue mechanism in oxidative hepatocyte injury. (HEPATOLOGY 2004;39;963–969.)

Experimental evidence that phenylalanine is strongly associated to oxidative stress in adolescents and adults with phenylketonuria

Few studies have looked at optimal or acceptable serum phenylalanine levels in later life in patients with phenylketonuria (PKU). This study examined the oxidative stress status of adolescents and adults with PKU. Forty PKU patients aged over fifteen years were enrolled, and were compared with thirty age-matched controls. Oxidative stress markers, anti-oxidant enzyme activities in erythrocytes, and blood anti-oxidant levels were examined. Nitric oxide (NO) production was also examined as a measure of oxidative stress. Plasma thiobarbituric acid reactive species and serum malondialdehyde-modified LDL levels were significantly higher in PKU patients than control subjects, and correlated significantly with serum phenylalanine level (P<0.01). Plasma total anti-oxidant reactivity levels were significantly lower in the patient group, and correlated negatively with phenylalanine level (P<0.001). Erythrocyte superoxide dismutase and catalase activities were higher and correlated significantly with phenylalanine level (P<0.01). Glutathione peroxidase activity was lower and correlated negatively with phenylalanine level (P<0.001). The oxidative stress score calculated from these six parameters was significantly higher in patients with serum phenylalanine of 700-800 μmol/l. Plasma anti-oxidant substances, beta-carotene, and coenzyme Q(10) were also lower (P<0.001), although the decreases did not correlate significantly with the phenylalanine level. Serum nitrite/nitrate levels, as stable NO products, were higher together with low serum asymmetric dimethylarginine, as an endogenous NO inhibitor. Oxidative stress status is closely linked with serum phenylalanine levels. Phenylalanine level in should be maintained PKU below 700-800 μmol/l even in adult patients.

Comprehensive molecular analysis of Japanese patients with pediatric-onset MODY-type diabetes mellitus

Yorifuji T, Fujimaru R, Hosokawa Y, Tamagawa N, Shiozaki M, Aizu K, Jinno K, Maruo Y, Nagasaka H, Tajima T, Kobayashi K, Urakami T. Comprehensive molecular analysis of Japanese patients with pediatric‐onset MODY‐type diabetes mellitus.

Relationship between oxidative stress and antioxidant systems in the liver of patients with Wilson disease: Hepatic manifestation in wilson disease as a consequence of augmented oxidative stress

The role of oxidative stress in the pathogenesis of liver disease in Wilson disease (WD), a genetic disorder characterized by excess hepatic deposition of copper that generates free radicals, remains unclear. This study investigates oxidative stress on the liver and hepatic antioxidant responses in WD using liver specimens from affected patients showing mild liver damage (group I, n = 3), moderate or greater liver damage (group II, n = 5), and fulminant hepatic failure (group III, n = 5) and from asymptomatic carriers (n = 2). Decreased ratios of reduced glutathione (GSH) to oxidized glutathione (GSSG) and increased thiobarbituric acid reactive substance (TBARS), a lipid peroxidation product, were found in every affected patient, especially in group II and III patients. Activities and protein expressions of Mn-dependent superoxide dismutase (Mn-SOD), CuZn-dependent superoxide dismutase (CuZn-SOD), and catalase were decreased in all patients, especially in group III patients. Glutathione peroxidase (GPx) activity was decreased only in group III patients. Asymptomatic carriers without any clinical manifestations showed normal TBARS level and GSH/GSSG ratio with increases in both GSH and GSSG levels. Their CuZn-SOD, Mn-SOD, and catalase activities were increased. These results suggest that excessive copper-derived oxidants contribute to development and progression of liver disease in WD.

Improved liver function and relieved pruritus after 4-phenylbutyrate therapy in a patient with progressive familial intrahepatic cholestasis type 2.

To examine the effects of 4-phenylbutyrate (4PB) therapy in a patient with progressive familial intrahepatic cholestasis type 2. A homozygous c.3692G>A (p.R1231Q) mutation was identified in ABCB11. In vitro studies showed that this mutation decreased the cell-surface expression of bile salt export pump (BSEP), but not its transport activity, and that 4PB treatment partially restored the decreased expression of BSEP. Therapy with 4PB had no beneficial effect for 1 month at 200 mg/kg/day and the next month at 350 mg/kg/day but partially restored BSEP expression at the canalicular membrane and significantly improved liver tests and pruritus at a dosage of 500 mg/kg/day. We conclude that 4PB therapy would have a therapeutic effect in patients with progressive familial intrahepatic cholestasis type 2 who retain transport activity of BSEP per se.

AP2 adaptor complex mediates bile salt export pump internalization and modulates its hepatocanalicular expression and transport function

The bile salt export pump (BSEP) mediates the biliary excretion of bile salts and its dysfunction induces intrahepatic cholestasis. Reduced canalicular expression of BSEP resulting from the promotion of its internalization is one of the causes of this disease state. However, the molecular mechanism underlying BSEP internalization from the canalicular membrane (CM) remains unknown. We have shown previously that 4‐phenylbutyrate (4PBA), a drug used for ornithine transcarbamylase deficiency (OTCD), inhibited internalization and subsequent degradation of cell‐surface‐resident BSEP. The current study found that 4PBA treatment decreased significantly the expression of α‐ and μ2‐adaptin, both of which are subunits of the AP2 adaptor complex (AP2) that mediates clathrin‐dependent endocytosis, in liver specimens from rats and patients with OTCD, and that BSEP has potential AP2 recognition motifs in its cytosolic region. Based on this, the role of AP2 in BSEP internalization was explored further. In vitro analysis with 3×FLAG‐human BSEP‐expressing HeLa cells and human sandwich‐culture hepatocytes indicates that the impairment of AP2 function by RNA interference targeting of α‐adaptin inhibits BSEP internalization from the plasma membrane and increases its cell‐surface expression and transport function. Studies using immunostaining, coimmunoprecipitation, glutathione S‐transferase pulldown assay, and time‐lapse imaging show that AP2 interacts with BSEP at the CM through a tyrosine motif at the carboxyl terminus of BSEP and mediates BSEP internalization from the CM of hepatocytes. Conclusion: AP2 mediates the internalization and subsequent degradation of CM‐resident BSEP through direct interaction with BSEP and thereby modulates the canalicular expression and transport function of BSEP. This information should be useful for understanding the pathogenesis of severe liver diseases associated with intrahepatic cholestasis. (HEPATOLOGY 2012;55:1889–1900)

Determination of asymmetric dimethylarginine, an endogenous nitric oxide synthase inhibitor, in umbilical blood.

Endothelial cells produce nitric oxide (NO), a potent vasodilator. Asymmetric dimethylarginine (ADMA) is an endogenous inhibitor of NO synthase. Little is known about the potential physiological roles of ADMA in a perinatal setting. This study measures concentrations of ADMA in umbilical blood using enzyme-linked immunosorbent assay and those of NO as nitrite/nitrate (NOx(-)) using the Griess assay. Their relationship to the degree of prematurity and maternal clinical condition is examined. Results show that ADMA concentrations in umbilical blood from control newborns were about twice as high as those of lactating women, healthy children, and healthy adults. Umbilical blood NOx(-) concentrations from control newborns were about half of those of lactating women, healthy children, and healthy adults. Consequently, the levels of ADMA relative to NOx(-) were about 4-fold higher in umbilical blood from control newborns than in blood from lactating women, healthy children, and healthy adults. Furthermore, the umbilical blood ADMA concentrations and the ratios of ADMA to NOx(-) in newborns were higher according to their birth prematurity and lower birth weight. The umbilical ADMA concentrations were independent of the delivery mode and maternal preeclampsia. We infer that the high ADMA levels play physiological roles in maintaining vascular tone and blood redistribution to vital organs during birth, thereby favoring the circulatory transition from fetal to neonatal life.