Masayuki Miwa

Two-Step Biochemical Differential Diagnosis of Classic 21-Hydroxylase Deficiency and Cytochrome P450 Oxidoreductase Deficiency in Japanese Infants by GC-MS Measurement of Urinary Pregnanetriolone/ Tetrahydroxycortisone Ratio and 11β-Hydroxyandrosterone

BACKGROUND The clinical differential diagnosis of classic 21-hydroxylase deficiency (C21OHD) and cytochrome P450 oxidoreductase deficiency (PORD) is sometimes difficult, because both deficiencies can have similar phenotypes and high blood concentrations of 17α-hydroxyprogesterone (17OHP). The objective of this study was to identify biochemical markers for the differential diagnosis of C21OHD, PORD, and transient hyper 17α-hydroxyprogesteronemia (TH17OHP) in Japanese newborns. We established a 2-step biochemical differential diagnosis of C21OHD and PORD. METHODS We recruited 29 infants with C21OHD, 9 with PORD, and 67 with TH17OHP, and 1341 control infants. All were Japanese and between 0 and 180 days old; none received glucocorticoid treatment before urine sampling. We measured urinary pregnanetriolone (Ptl), the cortisol metabolites 5α- and 5β-tetrahydrocortisone (sum of these metabolites termed THEs), and metabolites of 3 steroids, namely dehydroepiandrosterone, androstenedione (AD4), and 11β-hydroxyandrostenedione (11OHAD4) by GC-MS. RESULTS At a cutoff of 0.020, the ratio of Ptl to THEs differentiated C21OHD and PORD from TH17OHP and controls with no overlap. Among metabolites of DHEA, AD4, and 11OHAD4, only 11β-hydroxyandrosterone (11HA), a metabolite of 11OHAD4, showed no overlap between C21OHD and PORD at a cutoff of 0.35 mg/g creatinine. CONCLUSIONS A specific cutoff for the ratio of Ptl to THEs can differentiate C21OHD and PORD from TH17OHP and controls. Additionally, the use of a specific cutoff of 11HA can distinguish between C21OHD and PORD.

Measurement of reference intervals for urinary free adrenal steroid levels in Japanese newborn infants by using stable isotope dilution gas chromatography/mass spectrometry.

BACKGROUND In newborn infants, there are no reference intervals for urinary free steroids, which are thought to reflect the bioavailable fraction of steroids in the blood. We establish a method for simultaneous measurement of urinary free adrenal steroids such as pregnenolone, progesterone, 16α-hydroxyprogesterone, 17α-hydroxyprogesterone, 21-deoxycortisone, 21-deoxycortisol, dehydroepiandrosterone, androstenedione, and 11β-hydroxyandrostenedione by using stable isotope dilution gas chromatography/mass spectrometry (SID-GC/MS) and determined the reference intervals for urinary levels of free adrenal steroids in Japanese newborn infants. METHODS Newborn pooled urine was used for validation. Spot urine samples were collected from 67 full-term Japanese newborn infants (34 male and 33 female infants) at 3-4 days of age to determine reference intervals. The extracted and purified free steroids were delivered with heptafluorobutyric anhydride and analyzed by SID-GC/MS. RESULTS We validated a SID-GC/MS method with good repeatability and recovery rate. The preliminary reference intervals (median [range], μmol/mol creatinine) were as follows: pregnenolone, 4.2 (0.7-31.6); progesterone, 0.5 (not detected (n.d.)-0.6); 16α-hydroxyprogesterone, 1.4 (n.d.-10.3); 17α-hydroxyprogesterone, 1.1 (n.d.-1.9); 21-deoxycortisone, n.d. (n.d.-n.d.); 21-deoxycortisol, n.d. (n.d.-n.d.); dehydroepiandrosterone, 2.2 (0.6-27.3); androstenedione, 0.7 (n.d.-5.2); and 11β-hydroxyandrostenedione, 2.9 (n.d.-26.7). CONCLUSIONS We established a reliable SID-GC/MS method and were able to determine preliminary reference intervals for 9 urinary free adrenal steroids in newborn infants.

Usefulness of measurement of urinary N-terminal pro-brain natriuretic peptide in neonatal period

N-terminal pro-brain natriuretic peptide (NT-proBNP) is an N-terminal peptide dissociated from proBNP when BNP is generated from proBNP. Its molecular size is about 8500 kDa, and it has no physical activity. It is a sensitive marker for the early diagnosis and evaluation of heart failure, as its blood level increases from a mild stage of cardiac dysfunction. Serum NT-proBNP in childhood was reported by Schwachtgen. Some references have investigated the usefulness of urinary NT-proBNP, however, it is still very controversial. We measured the serum and urinary NT-proBNP levels in neonates, and evaluated their correlation and the usefulness of urinary NT-proBNP. The 36 serum and urine samples were collected at the age of 0–25 days from 9 neonates with a birthweight of 659–3330 g (median: 2864 g), who were born at 25–41 (median: 37) weeks’ gestation at Keio University Hospital between October 2007 and June 2008. Each NT-proBNP concentration was determined by ElectroChemi-Luminescence Immunoassay using modular analytics (Roche Diagnostics Co. Ltd, Tokyo, Japan). The serum and urinary sample needed is just 200 mL, and it takes about 20 min for analysis. Neonates who showed severe neonatal asphyxia, congenital heart disease, congenital malformation, or chromosomal anomaly were excluded. Urine samples were collected within 2 h before or after blood collection. Written informed consent was obtained from the neonates’ parents prior to the study. Serum level of NT-proBNP was in agreement with the normal range in the neonatal period reported by Schwachtgen. A significant correlation with a coefficient of determination of 0.548 was observed in the regression equation obtained by the least squares method between the serum and urinary NT-proBNP levels. The serum and urinary level of NT-proBNP was not normally distributed in each group. Furthermore, a low level of NT-proBNP was frequently observed, and we evaluated these data with a natural logarithm. As shown in Figure 1, similar significant correlation with a coefficient of determination of 0.703 was observed by the same method. NT-proBNP is considered to be excreted primarily via the kidney, and its metabolic routes via the muscle, liver, etc., have also been reported in adults. However, in neonates, the metabolic route via the muscle or liver may be negligible because of the small muscle mass and immature metabolic function of the liver. Blood samples cannot be collected in sufficient amounts from neonates, such as premature infants. The results of this study suggest that the cardiac load can be evaluated non-invasively according to the trend of changes in the urinary NT-proBNP level in neonates, especially extremely low-birthweight infants.

Association of Maternal Age to Development and Progression of Retinopathy of Prematurity in Infants of Gestational Age under 33 Weeks

Aim. To find predictive and indicative markers of risk for development of retinopathy of prematurity (ROP) and its progression to the stage requiring laser treatment, in premature infants whose gestational age (GA) was under 33 weeks. Methods. We retrospectively reviewed medical records of 197 premature infants born in 2005–2010 whose GA < 33 weeks and underwent eye screening at Keio University Hospital. The association between candidate risk factors and development or progression of ROP was assessed. Results. Among the 182 eligible infants (median GA, 29.1 weeks; median birth weight (BW), 1028 g), 84 (46%) developed any stage of ROP, of which 45 (25%) required laser treatment. Multivariate analysis using a stepwise method showed that GA (P = 0.002; 95% confidence interval (CI), 0.508–0.858), BW (P < 0.001; 95% CI, 0.994–0.998), and lower maternal age (P = 0.032; 95% CI, 0.819–0.991) were the risk factors for ROP development and GA (P < 0.001; 95% CI, 0.387–0.609) and lower maternal age (P = 0.012; 95% CI, 0.795–0.973) were for laser treatment. The odds ratio of requiring laser treatment was 3.3 when the maternal age was <33 years. Conclusion. ROP was more likely to be developed and progressed in infants born from younger mother and low GA.

Classic and non-classic 21-hydroxylase deficiency can be discriminated from P450 oxidoreductase deficiency in Japanese infants by urinary steroid metabolites.

Abstract. We previously reported a two-step biochemical diagnosis to discriminate classic 21-hydroxylase deficiency (C21OHD) from P450 oxidoreductase deficiency (PORD) by using urinary steroid metabolites: the pregnanetriolone/tetrahydrocortisone ratio (Ptl / the cortisol metabolites 5α- and 5β-tetrahydrocortisone (sum of these metabolites termed THEs), and 11β-hydroxyandrosterone (11OHAn). The objective of this study was to investigate whether both C21OHD and non-classic 21OHD (C+NC21OHD) could be biochemically differentiated from PORD. We recruited 55 infants with C21OHD, 8 with NC21OHD, 16 with PORD, 57 with transient hyper-17α-hydroxyprogesteronemia (TH17OHP), and 2,473 controls. All infants were Japanese with ages between 0–180 d. In addition to Ptl, THEs, and 11OHAn, we measured urinary tetrahydroaldosterone (THAldo) and pregnenediol (PD5). The first step: by Ptl with the age-specific cutoffs 0.06 mg/g creatinine (0–10 d of age) and 0.3 mg/g creatinine (11–180 d of age), we were able to differentiate C+NC21OHD and PORD from TH17OHP and controls (0–10 d of age: 0.065–31 vs. < 0.001–0.052, 11–180 d of age: 0.40–42 vs. < 0.001–0.086) with 100% sensitivity and specificity. The second step: by the 11OHAn/THAldo or 11OHAn/PD5 ratio with a cutoff of 0.80 or 1.0, we were able to discriminate between C+NC21OHD and PORD (1.0–720 vs. 0.021–0.61 or 1.8–160 vs. 0.005–0.32, respectively) with 100% sensitivity and specificity. Ptl, 11OHAn/THAldo, and 11OHAn/PD5 could differentiate between C+NC21OHD and PORD in Japanese infants.

Metabolic acidosis due to continuous drainage of massive chylous pleural effusion in two neonates

There are two types of metabolic acidosis: one resulting from acid accumulation, and the other from HCO3 loss. The latter type is often associated with renal or intestinal HCO3 loss. We report two neonates who presented with metabolic acidosis due to continuous drainage of chylous pleural effusion and required sodium bicarbonate. Case 1: after normal pregnancy and threatened premature labor, baby 1 was born at 31 weeks 2 days of gestation with a birthweight of 1344 g. Because there was no spontaneous breathing, the child was put on a respirator. Bilateral pleural effusion gradually increased, and chest drainage was started at the age of 16 days. Because chylous pleural effusion fluctuated between 100 and 500 mL per day, physiological saline and albumin were given continuously to maintain the circulating plasma volume. The baby was diagnosed with congenital myotonic dystrophy, and the pleural effusion was considered associated with this condition. Octreotide and prednisolone were given to reduce the pleural effusion but no response was observed, and the baby died at the age of 126 days. Metabolic acidosis persisted throughout the clinical course. Arterial blood gas analysis before sodium bicarbonate was as follows: pH, 7.066; pCO2, 56.8 mmHg; base excess, -12.9 mEq/L; Na, 133 mEq/L; K, 5.2 mEq/L; Cl, 111 mEq/L; anion gap (AG), 9.9 mEq/L. Normal anion gap suggested that the metabolic acidosis was due to HCO3 loss. Stools were small in quantity, excluding the loss of HCO3 from the gastrointestinal tract. The urinary pH was 4.91 at a time when the serum pH was 7.03, 5.04 at a time when the serum pH was 7.38, excluding renal tubular acidosis. The dose of sodium bicarbonate required for pH adjustment changed with fluctuations in the amount of pleural effusion, suggesting HCO3 loss into the pleural effusion. It is not possible to measure HCO3 in the pleural effusion directly. Instead, we measured pH of the pleural effusion several times, and this was close to serum pH. For example, pH of pleural effusion was 6.992 at a time when the serum pH was 7.066, and 7.393 at a time when the serum pH was 7.336. We therefore estimated HCO3 loss assuming that HCO3 in the pleural effusion is the same as serum HCO3. The estimated HCO3 loss into the pleural effusion approximated the HCO3 dose given (Fig. 1a). Case 2: baby 2 developed pleural effusion from 30 weeks of gestation, and underwent thoracentesis with basket catheter placement. The infant was born at 33 weeks 4 days of gestation with a birthweight of 2055 g, and was placed on a respirator. Chest drainage for pleural effusion was started at the age of 2 days. Pleural effusion fluctuated between 100 and 500 mL per day, and the circulating plasma volume was adjusted with physiological saline, albumin, fresh frozen plasma (FFP), and gammaglobulin. At the age of 37 days or later, physiological saline was replaced with Lactated Ringer. The pleural effusion subsided after octreotide, thoracic duct ligation, and intrapleural infusions of autologous blood, and the baby was discharged at the age of 111 days. As in baby 1, metabolic acidosis with a normal anion gap was observed, requiring supplementation with sodium bicarbonate. Clinical and laboratory findings excluded HCO3 loss from the kidney or gastrointestinal tract. The estimated HCO3 loss into the pleural effusion approximated the HCO3 dose given (Fig. 1b). Metabolic acidosis with a normal anion gap is generally associated with HCO3 loss in the kidney or gastrointestinal tract. Siegler et al. reported that a 3-month-old infant with massive chylous pleural effusion resulting from postoperative thoracic duct injury presented with metabolic acidosis. The two infants reported here differed regarding the cause of chylous pleural effusion, the underlying disease, and the clinical course, but the clinical and laboratory findings suggest that the loss of HCO3 into the pleural effusion led to metabolic acidosis. We speculate that the continuous drainage of a large amount of chylous pleural effusion resulted in a reduction in plasma HCO3, and the circulating plasma volume was maintained with physiological saline and albumin, leading to dilutional metabolic acidosis. Baby 2 did not require sodium bicarbonate at the age of 23 days or later because of FFP use. At the age of 37 days or later, physiological saline was replaced with Lactated Ringer. This may have eliminated the need for sodium bicarbonate, but it was difficult to evaluate the effect of Lactated Ringer because of the decrease of pleural effusion. In the present study we estimated HCO3 loss to the pleural effusion by using serum HCO3, because serum pH always nearly equaled that of pleural effusion. But this does not mean that serum HCO3 concentration always equals that of the pleural effusion. This is a limitation of the study. It is hoped that a method to measure HCO3 concentration of the pleural effusion Correspondence: Emi Okishio, MD, Division of Neonatology, Department of Pediatrics, Keio University School of Medicine, 35 Shinanomachi, Shinjuku-ku, Tokyo 160-8582, Japan. Email: e_oxy@yahoo.co.jp Received 22 January 2012; revised 6 June 2012; accepted 20 June 2012. bs_bs_banner

Hepatocyte growth factor levels of cord blood in healthy term newborns

Hepatocyte growth factor (HGF) has been reported recently to be a prognostic factor of acute pulmonary disorders. Regarding the clinical relationships of HGF with neonates, Khan et al reported the cord blood serum HGF level in neonates in 1996.1 Since the serum HGF level in normal neonates was 0.66 to 1.33 ng/dl, which was about three times higher than the adult reference value, and increased with the gestational age, they …

C-reactive protein in extremely low birth weight infants using evanescent wave immunoassay to make normative standards after birth

We evaluated CRP in ELBW up to 7 days old using the evanescent wave immunoassay (EVIA). EVIA is a high-sensitive CRP assay method capable of measuring CRP over 4 mg/dL with an interval of 0.1 mg/dL. This system is not influenced by bilirubin, hemoglobin, or lipid. The assay can be performed with 50 mL of whole blood, and the assay time is 10 min. The subjects were 108 ELBW infants born in our NICU between January 2002 and July 2008. Neonates who died within 1 month, those who were transferred to another hospital, those with chromosomal anomalies or congenital malformation, and those who underwent exchange transfusion were excluded. Those infants who exhibited symptoms suggestive of inflammation including fever, oliguria, hypotension, and frequent apnea, those with grade III–IV intraventricular hemorrhage, those with tension pneumothorax, those with necrotizing enterocolitis, and those with required surgical treatment, the data obtained until the day before the diagnosis were used. Those who showed no symptom of infection but were administered antibiotics prophylactically until negative blood cultures were confirmed were not excluded. Written parental informed consent was obtained before this study. Clinical information was collected retrospectively by examining medical records. Blood was collected daily between days 0 and 7 from the ELBW infants. CRP was measured by EVIA using EVANET EV20 (Nissui Pharmaceutical Corporation, Tokyo, Japan). Standard curves employing 50, 90, 95 percentile values were prepared from the accumulated data using Microsoft Excel. Standard values were obtained from the data of 85 of the 108 ELBW infants. The median gestational age of the 85 infants was 27 weeks and 4 days, the median birth weight was 800 g and male-female ratio was 33:52. The median Apgar score at 5 min was 6. Seventy-four infants were born by cesarean section. A transient, increase in CRP was observed in the ELBW infants as in the term infants. (Fig. 1) There have been few reports on continuous evaluation of the CRP value in neonates from immediately after birth. However, these few reports were of term infants. The CRP value fluctuated daily after birth in the ELBW as well as term infants, and temporary increases were also observed. It is well known that maternal serum CRP does not shift across placenta. The results obtained are useful for the early detection of bacterial infections and avoidance of unnecessary antibiotic therapy in ELBW infants.