The effects of bilirubin on brain energy metabolism during normoxia and hypoxia: an in vitro study using 31P nuclear magnetic resonance spectroscopy

Available evidence from in vitro studies suggests that the neurotoxic effects of bilirubin may be exacerbated by, or even require, additional factors such as hypoxia or asphyxia. The aim herein was to use 31P nuclear magnetic resonance spectroscopy to study the effects of bilirubin on brain energy metabolism in vitro under conditions of normoxia and hypoxia. 31P nuclear magnetic resonance spectra were acquired from guinea pig cerebral hemisphere slices during superfusion with solutions containing bilirubin and albumin in 5:1 molar ratio. The effects of bilirubin at concentrations between 400 nmol/liter and 120 mumol/liter were studied under normoxic conditions. Bilirubin caused no apparent disruption in brain energy metabolism during normoxia. The combined effects of bilirubin (40 mumol/liter) and hypoxia were studied. Hypoxia alone led to a steady state reduction in the phosphocreatine to inorganic phosphate peak-height ratio to 0.30 (0.27-0.32) [mean (range) n = 3]. Bilirubin (40 mumol/liter) in the presence of hypoxia caused a further reduction in the phosphocreatine to inorganic phosphate ratio to 0.18 (0.17-0.20) [mean (range) n = 3, p less than 0.01, analysis of variance] which was rapidly reversed on returning to normoxia. These results demonstrate that bilirubin at the concentration studied requires hypoxia, in addition, to cause a measurable disturbance of brain energy metabolism. The nature of this interaction is unknown, but it may reflect the effect of intracellular acidosis on bilirubin solubility or the oxygen dependence of brain mitochondrial bilirubin oxidase.     

Brain energy state and lactate metabolism during status epilepticus in the neonatal dog: in vivo 31P and 1H nuclear magnetic resonance study

The purpose of these experiments was to determine whether flurothyl-induced status epilepticus causes progressive decline of brain high-energy phosphates and progressive increase in brain lactate in neonatal dogs who are paralyzed and oxygenated. In vivo 31P nuclear magnetic resonance spectroscopic measurements showed that the fall in brain pH occurred early in the course of seizure. The decline in phosphocreatine was more gradual, i.e. 50% reduction, during the 1st h of seizure. There was no reduction in ATP during the 3 h of status epilepticus. In vivo 1H nuclear magnetic resonance measurement of brain lactate disclosed a steep rise that stabilized by 60 min. Brain and blood lactate were closely related during the initial phase of seizure, suggesting rapid efflux of lactate from brain or systemic production of lactate. Blood lactate exceeded brain lactate after 1 h of status epilepticus. The new steady state for cerebral phosphocreatine and lactate during status epilepticus was achieved much more slowly during neonatal status epilepticus than has been reported during status epilepticus in the adult experimental animal. The lack of change in ATP during 3 h of seizure indicates that brain energy state is not radically altered during prolonged seizure if oxygenation is maintained.     

Bilirubin-induced changes in brain energy metabolism after osmotic opening of the blood-brain barrier.

Acute and residual effects of blood-brain barrier disruption and bilirubin on brain metabolism were studied in a rat model after osmotic opening of the blood-brain barrier under pentobarbital anesthesia. Arabinose (1.5 M) was infused via the right external carotid artery over 30 s, resulting in opening of the barrier within the right hemisphere. Two min later, bilirubin was infused i.v. over 3 min, raising the serum bilirubin concentration to 37-44 mg/dL (633-752 mumol/L). The animals were euthanized at 15 min or 4 h by freezing the brain in situ. Opening the blood-brain barrier produced small changes in cerebral energy metabolism in some animals at 15 min. Compared with saline-infused control animals, two out of nine rats had decreased brain phosphocreatine and three out of nine developed increased brain lactate levels. Infusion of bilirubin in rats with a disrupted blood-brain barrier produced profound decreases in brain energy metabolites, glucose, and glycogen and a markedly increased lactate/pyruvate ratio at 15 min. The markedly increased lactate in the presence of normal or low pyruvate in bilirubin-treated animals indicates accumulation of NADH and probably reflects severe mitochondrial dysfunction. Four h after the arabinose/bilirubin infusions, the barrier would be expected to be repaired and bilirubin levels were negligible, but two out of five arabinose and three out of six bilirubin rats continued to have severely altered brain metabolism indicating residual brain injury in some animals.     

Clearance of bilirubin from rat brain after reversible osmotic opening of the blood-brain barrier

Utilizing multicomponent spectrophotometry, we assayed the bilirubin content of rat cerebral hemispheres. With this assay, we determined the clearance of bilirubin from the rat brain following reversible, osmotic opening of the blood-brain barrier. Clearance was rapid, with a half-time of 1.7 h. This half-time was the same as that for clearance of bilirubin from the serum, suggesting that brain bilirubin was removed by transport or diffusion back into the general circulation. Osmotic opening does not damage brain tissue. Thus, in the undamaged rat brain, bilirubin is rapidly cleared, in contrast to its persistence in autopsy-proven human kernicterus. The potential for clearance of bilirubin from human neonatal brain should be considered, especially in the absence of underlying tissue damage.     

Cerebral energy metabolism in insulin induced hypoglycemia in newborn piglets: in vivo31P-nuclear magnetic resonance spectroscopy

The effect of insulin induced hypoglycemia on cerebral energy metabolism was examined in four newborn piglets. Cerebral energy metabolism was assessed using in vivo ³¹P-nuclear magnetic resonance spectroscopy. It was demonstrated that the normal level of phosphocreatine/inorganic phosphate (PCr/Pi), an indicator of phosphorylation potential, was maintained at a blood glucose level of 40 mg/dL or above, whereas when blood glucose was reduced to less than 40 mg/dL, PCr/Pi rapidly decreased in parallel with this. Below the critical blood glucose level of 40 mg/dL, a positive correlation (y = 0.02x + 0.632; r = 0.668; P < 0.001) existed between blood glucose and PCr/Pi. In the present investigation, a reduction of blood glucose level to 20 mg/dL or lower resulted in a PCr/Pi of less than 1, indicating a state of cerebral energy failure. The intracellular pH (pHi) was 7.08 ± 0.05 at the onset and 7.15 ± 0.07 in the hypoglycemic state, indicating no significant difference between the two groups. The present study has clarified that cerebral energy failure occurs when the blood glucose level is about 20 mg/dL or lower. The critical point of blood glucose exists to maintain brain energy metabolism.     

Cerebral energy metabolism in phenylketonuria: findings by quantitative In vivo 31P MR spectroscopy

Both severe impairments of brain development in untreated infants and acute reversible neurotoxic effects on brain function are clinical features of phenylketonuria (PKU). For determining whether impairments of cerebral energy metabolism play a role in the pathophysiology of PKU, quantitative in vivo 31P magnetic resonance spectroscopy (MRS) was performed in a supratentorial voxel of 11 adult PKU patients and controls. Peak areas of inorganic phosphate; phosphocreatine; alpha-, beta-, and gamma-ATP; NAD; phosphomonoesters; phosphodiesters; and a broad phospholipid signal were converted to millimolar concentrations. Mg2+, pH, ADP, the phosphorylation potential, and the relative velocity of oxidative metabolism V/Vmax were derived. Clinical evaluation included mutation analysis, neurologic investigation, intelligence testing, magnetic resonance imaging, and concurrent plasma and brain phenylalanine (Phe), the last by 1H-MRS. Phe loading was performed in five patients with an oral dose of 100 mg/kg body wt L-Phe monitored by spectral EEG analysis. Under steady-state conditions, 31P-MRS revealed normal values for ATP, phosphocreatine, NAD, phosphomonoesters, phosphodiesters, Mg2+, and pH in PKU. ADP (+11%) and the phosphorylation potential (+22%) were increased. Peak areas of inorganic phosphate (-22%) and phospholipid (-8%) were decreased. ADP correlated with concurrent plasma (r = 0.65) and brain (r = 0.55) Phe. During the Phe load, blood Phe levels increased steeply. EEG revealed slowing of background activity. The phosphorylation potential decreased, whereas ADP and V/Vmax increased. In vivo 31P-MRS demonstrated subtle abnormalities of cerebral energy metabolism in PKU in steady-state conditions that were accentuated by a Phe load, indicating a link between Phe neurotoxicity and imbalances of cerebral energy metabolism.     

31P nuclear magnetic resonance study of the effect of hypoxemia on neonatal status epilepticus

Prolonged neonatal seizures are often accompanied or exacerbated by hypoxemia. To determine the effects of hypoxemia on neonatal status epilepticus, we determined cerebral blood flow and cerebral metabolic state in groups of neonatal dogs subjected to hypoxia, to seizures during normoxia, or to seizures during hypoxia. The compensatory increase in cerebral blood flow was greatest in animals subjected to seizures during normoxia and somewhat less pronounced in animals made hypoxic. However, blood flow failed to increase in forebrain structures when animals were subjected to the combination of seizures and hypoxia. Accordingly, levels of adenosine triphosphate in forebrain (measured both by in vitro enzymatic analysis and by in vivo phosphorus-31 nuclear magnetic resonance spectroscopy) were depleted to the greatest degree in animals who were seizing while hypoxic. In addition, brain glucose was significantly reduced only in the seizure-hypoxia group. Systemic factors such as hypoxemia may play a critical role in the disruption of cerebral energy balance during neonatal status epilepticus.     

A study of galactose intolerance in human and rat liver in vivo by 31P magnetic resonance spectroscopy.

An oral load of 20 mg/kg galactose produces significant changes in the 31P magnetic resonance spectrum of the liver of a galactosemic patient. The peak at 5.2 ppm (which includes inorganic phosphate and galactose-1-phosphate) increased on two occasions to about twice its original size 60 min after galactose administration. An oral load of 10 mg/kg galactose given to a second patient produced no discernible changes at 30 min. We have also used an animal model of galactose intolerance, in which galactose metabolism in rats was blocked by the acute administration of ethanol. Studies in vivo and in vitro showed that the increase in the peak at 5.2 ppm was largely due to galactose-1-phosphate. We have shown in this preliminary study that small amounts of galactose can produce significant elevation of hepatic galactose-1-phosphate, which can be detected by 31P magnetic resonance spectroscopy.     

In vivo 31P nuclear magnetic resonance measurement of chronic changes in cerebral metabolites following neonatal intraventricular hemorrhage

The purpose of this study was to determine whether cerebral metabolic changes occur after intraventricular hemorrhage in the newborn. Five babies with bilateral grade 3 to 4 intraventricular hemorrhage were compared with 15 preterm infants without intraventricular hemorrhage. Cerebral high-energy phosphorus metabolites and intracellular pH were measured with in vivo 31P nuclear magnetic resonance spectroscopy. Spectra were collected initially within the first 2 weeks of life, and then every other week until discharged from the hospital. The phosphocreatine to inorganic phosphate ratio and the phosphocreatine to adenosine triphosphate ratio were significantly lower in the group with intraventricular hemorrhage, but differences in intracellular pH were not significant. Differences between babies with and without intraventricular hemorrhage varied with postconceptional age: in those with intraventricular hemorrhage, the phosphocreatine to adenosine triphosphate ratio was decreased at all postconceptional ages, and the phosphocreatine to inorganic phosphate ratio was lower in babies with intraventricular hemorrhage and younger than 30 weeks. Results of this study confirm the presence of chronic metabolic changes following intraventricular hemorrhage which may exacerbate neurologic damage after intraventricular hemorrhage in the newborn.     

Hypotonia of rickets: a sequential study by P-31 magnetic resonance spectroscopy

To address the role of high-energy phosphorus compounds in the hypotonia of vitamin D-dependent rickets, nuclear magnetic resonance spectra were obtained sequentially from resting gastrocnemius muscle of a 10-month-old infant with rachitic hypotonia during supplementation with vitamin D, calcium, and phosphorus. During the initial weeks of treatment, the hypotonia resolved before evidence of epiphyseal mineralization. Over the early treatment period, the muscle phosphocreatine/beta-adenosine triphosphate [PCr/beta-ATP] ratio increased from 2.7-2.8 [wk 1-2] to 3.9-4.5 [wk 7-9]. The PCr/beta-ATP ratio for 6-month-old normal infant gastrocnemius and adult forearm were 4.0 and 5.7, respectively. Muscle strength appeared to recover concomitantly with an increase in retained muscle phosphorus and high-energy phosphate compounds, and with relative increase in the muscle phosphocreatine to ATP ratio. The synchrony of clinical recovery may relate to the recovery kinetics of these metabolic changes.     

Glutamate in cerebral tissue of asphyxiated neonates during the first week of life demonstrated in vivo using proton magnetic resonance spectroscopy

We tested the hypothesis that glutamate (Glx) levels as demonstrated by proton magnetic resonance spectroscopy ((1)H-MRS) are elevated in brain tissue of neonates with severe hypoxic-ischemic encephalopathy (HIE). Studies were performed in 26 neonates (median gestational age 40.5 weeks, range 36.7-42.4 weeks; median birth weight 3,360 g, range 2,180-4,200 g). The median postnatal age at the time of testing was 2.5 days (range 1-7 days). HIE was scored according to Sarnat as grade I (n = 4), grade II (n = 15) or grade III (n = 7). Results for neonates with mild to moderate HIE (group 1) were compared to those with severe HIE (group 2). After magnetic resonance imaging, (1)H-MRS was performed in a single volume of interest including the basal ganglia. An echo time of 31 ms was used. After curve-fitting procedures, peak area ratios of different brain metabolites were calculated. The median total Glx/N-acetylaspartate ratio was 1.21 (range 0.64-3.25) in group 1 versus 1.55 (range 1.10-2.75) in group 2 (p = 0.035). The median total Glx/choline ratio was 1.33 (range 0.71-2.52) in group 1 versus 2.14 (range 1.21-3.55) in group 2 (p = 0.019). We concluded that during the first days of life, Glx was elevated in the basal ganglia of neonates with severe HIE.     

紫绀型先天性心脏病患儿脑组织磁共振波谱研究

目的 应用氢质子磁共振波谱(1H-MRS)技术评估紫绀型先天性心脏病患儿脑代谢的变化.方法紫绀型先天性心脏病患儿29例,按不同的血氧饱和度(SaO2)分为紫绀组14例(SaO2为76% ～ 85%)、严重紫绀组15例(SaO2为65% ～ 75%);另选血氧饱和度正常、年龄性别相匹配的对照组儿童30例.应用场强为1.5T超导型磁共振扫描仪分别对这些儿童的右侧基底核区(感兴趣区20 mm × 20 mm × 20 mm 范围)进行氢质子磁共振波谱图像采集.检测指标为N-乙酰天冬氨酸(NAA)、乳酸(Lac)和肌酸(Cr),计算并比较紫绀型先天性心脏病患儿与对照组儿童、严重紫绀组与紫绀组患儿之间的NAA/Cr和Lac/Cr比值的差异.采用SPSS11.0统计软件进行统计分析.结果 紫绀型先天性心脏病患儿的NAA/Cr比值显著低于对照组(P ＜ 0.05),而Lac/Cr比值则明显高于对照组(P ＜ 0.001),差异均有统计学意义.严重紫绀组患儿的NAA/Cr比值也较紫绀组低(P ＜ 0.001),而Lac/Cr比值则较紫绀组高(P ＜ 0.001),差异均有统计学意义.结论 紫绀型先天性心脏病患儿脑组织存在NAA及Lac代谢异常,其改变程度与缺氧严重程度相关,提示慢性缺氧影响紫绀型先天性心脏病患儿的脑代谢.这将有助于阐明紫绀型先天性心脏病患儿脑功能障碍的发生机制,为早期诊断、及时干预,进而提高这些患儿的远期生活质量提供理论依据.     

磁共振波谱测定癫痫患儿脑内代谢变化及其临床意义的研究

目的 了解癫痫患儿脑内代谢变化的特点。方法 应用Ｅｌｓｃｉｎｔ２．０Ｔ超导型太共振仪,对２５例癫痫、５例难治性癫痫患儿脑内Ｎ－乙酰天门冬氨酸（ＮＡＡ）、肌酸和磷酸肌酸（Ｃｒ）、及含胆碱化合物（Ｃｈｏ）的浓度进行测定,并以１０例非颅内疾病患儿作为对照。结果 癫痫组致二内ＮＡＡ及ＮＡＡ／（Ｃｒ＋Ｃｈｏ）比值明显低于病灶对侧（Ｐ〈０．０１）,难治性癫痫病程越长,临床发作次数越频繁,ＮＡＡ／（Ｃｒ＋Ｃｈｏ     

Localised proton magnetic resonance spectroscopy of the brain after perinatal hypoxia: a preliminary report

Objectives. Perinatal hypoxic ischaemic injury is a significant cause of neurodevelopmental impairment. The aim of this study was to evaluate localised proton magnetic resonance spectroscopy (¹H-MRS) after birth asphyxia. Materials and methods. Thirty newborn infants suspected of having perinatal asphyxia (Apgar score < 3) were studied. The mean gestational age was 37 weeks, mean age at the MR examination was 18 days and mean weight was 2.9 kg. A 1.5-T unit was used for imaging and spectroscopy. None of the babies had mechanically assisted ventilation. No sedation was used. Axial T1-weighted and T2-weighted images were obtained. ¹H-MRS was recorded in a single voxel, localised in white matter, using a STEAM sequence. Results. Image quality was good in 25 of 30 babies. ¹H-MRS was performed in 19 of 30 subjects, with adequate quality in 16. Choline, creatine/phosphocreatine and N-acetylaspartate peaks and peak-area ratios were analysed. Lactate was detected in four infants. The N-acetylaspartate/choline ratio was lower in infants with an impaired neurological outcome, but the difference was not statistically significant. Conclusions. This study suggests that ¹H-MRS may be useful for assessing cerebral metabolism in the neonate. A raised lactate level and decreased N-acetylaspartate/choline ratio may be predictive of a poor outcome. However, in our experience this method is limited by the difficulty in performing the examination during the first hours after birth in critically ill babies, the problems related to use of a monovoxel sequence, the dispersion of the ratios and the lack of determination of the absolute concentration of the metabolites. Received: 30 March 1998 Accepted: 24 September 1998     

The effect of diazepam on neonatal seizure: in vivo 31P and 1H NMR study

It is assumed that when anticonvulsants arrest seizure, there is rapid return of brain high energy phosphates and brain lactate to control values. To test this hypothesis, diazepam was administered to neonatal dogs during flurothyl-induced seizure. In vivo 31P nuclear magnetic resonance spectroscopy disclosed that diazepam quickly arrested electrographic seizure and restored brain phosphocreatine and inorganic phosphate to baseline values. In contrast, in vivo 1H nuclear magnetic resonance spectroscopic measurements showed that arrest of seizure with diazepam did not return brain lactate to control values. The sustained increase in cerebral blood flow and prolonged elevation of brain lactate, acetate, valine, and succinate in the postictal period indicate that metabolic recovery of the brain occurs over an extended period of time after the normalization of EEG, phosphocreatine, and brain pH.     

The value of nuclear magnetic resonance in the study of iron overload in thalassemia patients

In patients with thalassemia treated by long-term chronic blood transfusions who survive beyond the first ten or twenty years of life but received no or inadequate chelating therapy during the first years, evaluation of iron overload and its consequences on tissues may prove an arduous task. MRI is a non-invasive means of measuring the amount of iron in the liver and the consequences of the iron overload on the heart and other tissues. For this purpose, MRI is more satisfactory than CT scan studies. In this investigation, 20 patients with thalassemia major underwent MRI. Multiple spin echos were used to allow determination of the transversal relaxation constant T2. This constant, expressed in ms, is related to the concentration of iron in the liver as in the following expression: (C) = 5 410/T2-110. MRI studies disclosed a discrepancy between the severity of hepatic hemosiderosis and development of decompensated iron overload cardiomyopathy. In a unique case, in which a heart transplant and two MRI studies were performed, the severe iron overload that failed to respond to several years of subcutaneous chelating therapy was more than halved by intensive intravenous chelation through a central catheter. MRI studies of the heart provide valuable morphologic and functional data. Although the amount of iron in the myocardium cannot as yet be quantified, modifications of the transversal relaxation time provide information on the severity of the overload and the presence of other myocardial alterations.     

The effects of moderate hypothermia on energy metabolism and serum inflammatory markers during laparotomy

The objective of this study was to investigate energy metabolism of the gut and liver as well as serum inflammatory cytokines following exploratory laparotomy at moderate hypothermia. Two groups of rats were studied, (n=6–8/group); laparotomy at normothermia for 120 min and laparotomy at hypothermia (32–33°C) for 120 min. Study 1: Intestinal glucose, succinate, lactate, phosphocreatine, and ATP as well as hepatic glucose, succinate, lactate, and ATP were measured in terms of micromole per gram using magnetic resonance spectroscopy. Study 2: Serum levels of TNF-α, IL-1β, LPS-inducible chemokine (LIX), and sICAM-1 were measured by ELISA. Histology of the gut and liver were interpreted. Data are expressed as mean and SEM. In Study 1, laparotomy at hypothermia caused an increase in intestinal glucose levels (0.78±0.03 vs. 1.29±0.11, P=0.0012) with a decrease in hepatic lactate levels (0.82±0.04 vs. 0.44±0.06, P<0.001). There were no differences in the other metabolites between the two groups. In Study 2, there were no differences in serum TNF-α, IL-1β, LIX, or sICAM-1 between the two groups. Histological features of the gut and liver among groups were comparable. In conclusion, the intestine and liver react to hypothermia differently. However, levels of high-energy phosphates in both organs are not affected by hypothermia suggesting adequate energy for the organs. It is unlikely that hypothermia induces either systemic inflammatory response or hypoxic damage to the intestine and liver in this model.     

早产儿与足月儿静息态功能磁共振默认网络的研究

目的 探讨早产儿与足月儿静息态功能磁共振成像(RS-fMRI)脑默认网络的差异及意义.方法 选择2012-2013年第三军医大学大坪医院儿科收治的新生儿进行RS-fMRI扫描筛查.通过采用低频振幅(ALFF)算法观察和比较早产儿组与足月儿组不同大脑区域ALFF的增强及减弱.结果 研究期间共招募85例新生儿,排除39例脑损伤显著和图像预处理不符合标准者,共46例新生儿的结果纳入后续研究分析.17例早产儿与29例足月儿的RS-fMRI结果显示,与足月儿相比,早产儿大脑内侧前额叶皮层、额下回、眶额度层、壳核、前颞叶、中间颞叶、前扣带回、后扣带回、中扣带回、苍白球、旁海马皮质脑区ALFF减弱,楔前叶、距状裂皮质ALFF增强.结论 静息状态下,早产儿在额叶和颞叶等脑区功能活动显著减低,但部分集中在视觉区域的功能活动增强,提示早产儿脑内可能存在补偿效应.     

Cohen syndrome. Description of a new case and study of the central nervous system using nuclear magnetic resonance

The Cohen syndrome is characterized by dysmorphic face, obesity, narrow hands and feet and mild mental retardation. So far only 42 cases have been described in literature. The Authors describe a patient who presented some cerebral anomalies at the MRI examination. In particular the MRI showed a large sellar cavity compared to the size of the hypophysis.