# Correlation Analysis of TSB Level and Globus Pallidus-Related Metabolite Indexes of Proton Magnetic Resonance Spectroscopy in the Newborn with Neonatal Jaundice

Authors: Chaoyan Liu; Jieyu Zhang; Zhao Zhang; Yang Li; Zhilei Kang
Journal: Evidence-Based Complementary and Alternative Medicine
 (2022)

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## Abstract

Objective. To investigate the correlation between serum total serum bilirubin (TSB) levels and globus pallidus-related metabolic indexes of proton magnetic resonance spectroscopy (1H-MRS) in the newborn with neonatal jaundice. Methods. 50 children with neonatal jaundice admitted to our hospital from January 2019 to January 2021 were recruited and assigned to a mild condition group (TSB < 221 μmol/L, n = 16), a moderate condition group (221 μmol/L ≤ TSB < 3 42 μmol/L, n = 18), and a severe condition group (342 μmol/L ≤ TSB < 428 μmol/L, n = 16) based on peak TSB. The differences in globus pallidus-related metabolic indexes of 1H-MRS between the groups were compared and their correlation with TSB levels was analyzed. Results. The three groups had comparable N-acetylaspartic acid (NAA)/creatine (Cr), choline (Cho)/Cr, lactic acid (Lac)/Cr, and ml/Cr levels (P>0.05), while there were statistical differences in glutamine (Glx)/Cr levels (P<0.05). The severe condition group showed the highest levels of neuron-specific enolase (NSE), creatine kinase-MB (CK-MB), and troponin (cTnl), followed by the moderate group, and then the mild group (P<0.05). The TSB level is positively correlated with the 1H-MRS metabolic index Glx/Cr. Conclusions. The serum TSB level is correlated with the 1H-MRS metabolic index Glx/Cr in the newborn with neonatal jaundice, and the levels of TSB and Glx/Cr provide a reference for the diagnosis of bilirubin encephalopathy.


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## Body

## 1. Introduction

Neonatal jaundice is common in newborns and is caused by abnormal bilirubin metabolism which increases the level of bilirubin in the blood and results in symptoms such as jaundice of the skin, mucous membrane, and sclera [1–3]. Excessive serum total serum bilirubin (TSB) can affect cell metabolism, interfere with neuronal synaptic transmission, and damage the cells [4]. Bilirubin easily accumulates on the cranial nerve nuclei, such as the globus pallidus, the basal nucleus of the thalamus, and the brainstem nucleus, and causes abnormal neurological changes in the nervous system. In severe cases, it is associated with acute bilirubin encephalopathy (ABE) and sequelae such as dyskinesia, hearing impairment, and enamel dysplasia [5–7]. The relative characteristic MRI manifestation of ABE is a symmetrical short T1 signal in the globus pallidus, but it also occurs in premature infants and neonates with hypoxic-ischemic encephalopathy, which results in a relatively high incidence of false positives results in the diagnosis of ABE using conventional MRI [8, 9]. Proton magnetic resonance spectroscopy (1H-MRS) can quantitatively analyze the metabolism and biochemical changes of living tissues noninvasively, reflecting pathophysiological changes in brain lesions at the neurophysiological and molecular levels, which provides rich biochemical and metabolic information for clinical purposes [10–12]. This study explores the correlation between TSB levels in newborns with neonatal jaundice and 1H-MRS metabolic indicators. The details of the study are as follows.


## 2. Methods

### 2.1. General Materials

A total of 50 children with neonatal jaundice admitted to our hospital from January 2019 to January 2021 were recruited. There were 22 male newborns and 28 female newborns, with a gestational week from 35 to 41 weeks and an average gestational week of (39.2 ± 1.3) weeks; jaundice occurs 2–14 days after birth, with an average of 6.7 ± 3.5 days. In terms of the cause of jaundice, there were 26 cases of infection, 11 cases of maternal and child blood type incompatibility, 5 cases of glucose-6-phosphate dehydrogenase (G-6-dP) deficiency, and 8 cases of other causes.According to the peak TSB, the patients were assigned to a mild condition group (TSB < 221μmol/L) (16 cases), a moderate condition group (221 μmol/L ≤ TSB < 342 μmol/L) (18 cases), and a severe condition group (342 μmol/L ≤ TSB < 428 μmol/L) (16 cases).


### 2.2. Diagnostic Criteria

Patients with jaundice occurring 24 hours after birth; with a serum TBIL level of full-term newborns exceeding the percentile value of 95th day old or a TBIL exceeding the percentile value of the 75th day of age in the presence of high-risk factors (hemolysis, asphyxia, and acidosis); and with a TBIL daily increase of >85 μ·Mol/L or hourly increase of >8.5 μ·mol/L were diagnosed as neonatal jaundice.


### 2.3. Inclusion Criteria and Exclusion Criteria

Patients who were diagnosed with neonatal jaundice after examination; with a gestational age of ≥35 weeks; with complete clinical data; and who knew the purpose and the procedures of the experimental study and signed an informed consent form were included. This study was approved by the Hengshui People’s Hospital ethics committee, No. HSH5719.Patients with congenital neurological dysplasia or neurological diseases; with neonatal pneumonia, biliary atresia, and other system diseases; with unclear 1H-MRS images or with serious artifacts in the IH-MRS images were excluded.


### 2.4. Methods

A 10% chloric acid hydrate solution (0.5–1.0 mL/kg) enema was used for sedation and hypnosis 30 minutes before the examination, a routine MRI examination was performed after the patients fell asleep with their heads fixed by a cotton pad to avoid movement during the examination. The Siemens Verio3.0T magnetic resonance scanner was used, with horizontal axis T1WI as the positioning image and the collected volumetric interest area placed in the pallidus on both sides. The multivoxel level fixed-point resolution 1H-MRS was conducted, and the scanning parameters were TR1300 ms, TE40 ms, the field of view 80 mm × 80 mm, voxel size 10 mm × 10 mm × 10 mm, and matrix 256 × 256. The scanning duration was 5 min. The T1WI sequence of the cranial MRI scan showing a symmetrical high signal in both globus pallidus is a characteristic imaging manifestation of hyperbilirubin encephalopathy. The T1WI sequence of cranial MRI scan shows symmetrical hyperintensity in the globus pallidus on both sides.Metabolites of globus pallidus were measured, including creatine (Cr), glutamine (Glx), N-acetylaspartic acid (NAA), choline (Cho), inositol (mI), and lactic acid (Lac). The collected data was calculated by the postprocessing computer and NAA/Cr, Cho/Cr, Glx/Cr, mI/Cr, and Lac/Cr were calculated using Cr as a reference.When the newborn was admitted to the hospital, 2 ml of heel blood was collected and placed in a vacuum tube. The blood sample was centrifuged to obtain the serum, and the serum was stored in a refrigerator at −20°C for assays. Electrochemiluminescence technology was used to detect the level of neuron-specific enolase (NSE), and the enzymatic rate method was adopted to determine the level of creatine kinase-MB (CK-MB). The instrument was a Roche E170 automatic immunoassay analyzer, and the supporting reagents were provided by Roche. The chemiluminescence method was used to measure the serum troponin (cTnl) level, and the detection instrument was a UniCel dxl 800 automatic chemiluminescence immunoassay analyzer produced by Beckman Company. The above-mentioned tests were carried out in accordance with the operating instructions on the kit and testing equipment.For the treatment of neonatal jaundice, our hospital often uses Chen Zibaihuang lotion for bathing, once a day, for 15–30 minutes each time. The main formula of the lotion is: herba artemisiae scopariae, Gardenia, Rhubarb, Poria,Atractylodes, Astragalus, citrus, radix isatidis, Salvia, Pueraria, Licorice; for children with abdominal distension and vomiting, Pinellia, caulis Bambusae, and taeniis were added to stop vomiting. The dregs are boiled in water for washing. First, the nails were manicured in the incubator, then the hands were washed and warmed, and then the whole body bath was performed. During the treatment, the skin condition of the children should be given attention. For children with allergies, bathing should be stopped in time and appropriate symptomatic treatment should be carried out.


### 2.5. Indicators

The content and changes of NAA/Cr, Cho/Cr, Glx/Cr, mI/Cr, and Lac/Cr were compared, and the correlation between serum TSB level and 1H-MRS metabolic index was analyzed.The levels of NSE, CK-MB, and cTnl in groups with different serum TSB levels were compared.


### 2.6. Statistical Methods

All the data obtained in this study was analyzed using the SPSS20.0 software, and GraphPad Prism 7 (GraphPad Software, San Diego, USA) was used to plot figures. The count data are expressed as (n (%)) and analyzed using the chi-square test. The measurement data are expressed as mean ± SD; one-way ANOVA was used for comparison between multiple groups; and LSD-t test was used for two-by-two comparisons. Differences were considered statistically significant at P<0.05.


## 2.1. General Materials

A total of 50 children with neonatal jaundice admitted to our hospital from January 2019 to January 2021 were recruited. There were 22 male newborns and 28 female newborns, with a gestational week from 35 to 41 weeks and an average gestational week of (39.2 ± 1.3) weeks; jaundice occurs 2–14 days after birth, with an average of 6.7 ± 3.5 days. In terms of the cause of jaundice, there were 26 cases of infection, 11 cases of maternal and child blood type incompatibility, 5 cases of glucose-6-phosphate dehydrogenase (G-6-dP) deficiency, and 8 cases of other causes.According to the peak TSB, the patients were assigned to a mild condition group (TSB < 221μmol/L) (16 cases), a moderate condition group (221 μmol/L ≤ TSB < 342 μmol/L) (18 cases), and a severe condition group (342 μmol/L ≤ TSB < 428 μmol/L) (16 cases).


## 2.2. Diagnostic Criteria

Patients with jaundice occurring 24 hours after birth; with a serum TBIL level of full-term newborns exceeding the percentile value of 95th day old or a TBIL exceeding the percentile value of the 75th day of age in the presence of high-risk factors (hemolysis, asphyxia, and acidosis); and with a TBIL daily increase of >85 μ·Mol/L or hourly increase of >8.5 μ·mol/L were diagnosed as neonatal jaundice.


## 2.3. Inclusion Criteria and Exclusion Criteria

Patients who were diagnosed with neonatal jaundice after examination; with a gestational age of ≥35 weeks; with complete clinical data; and who knew the purpose and the procedures of the experimental study and signed an informed consent form were included. This study was approved by the Hengshui People’s Hospital ethics committee, No. HSH5719.Patients with congenital neurological dysplasia or neurological diseases; with neonatal pneumonia, biliary atresia, and other system diseases; with unclear 1H-MRS images or with serious artifacts in the IH-MRS images were excluded.


## 2.4. Methods

A 10% chloric acid hydrate solution (0.5–1.0 mL/kg) enema was used for sedation and hypnosis 30 minutes before the examination, a routine MRI examination was performed after the patients fell asleep with their heads fixed by a cotton pad to avoid movement during the examination. The Siemens Verio3.0T magnetic resonance scanner was used, with horizontal axis T1WI as the positioning image and the collected volumetric interest area placed in the pallidus on both sides. The multivoxel level fixed-point resolution 1H-MRS was conducted, and the scanning parameters were TR1300 ms, TE40 ms, the field of view 80 mm × 80 mm, voxel size 10 mm × 10 mm × 10 mm, and matrix 256 × 256. The scanning duration was 5 min. The T1WI sequence of the cranial MRI scan showing a symmetrical high signal in both globus pallidus is a characteristic imaging manifestation of hyperbilirubin encephalopathy. The T1WI sequence of cranial MRI scan shows symmetrical hyperintensity in the globus pallidus on both sides.Metabolites of globus pallidus were measured, including creatine (Cr), glutamine (Glx), N-acetylaspartic acid (NAA), choline (Cho), inositol (mI), and lactic acid (Lac). The collected data was calculated by the postprocessing computer and NAA/Cr, Cho/Cr, Glx/Cr, mI/Cr, and Lac/Cr were calculated using Cr as a reference.When the newborn was admitted to the hospital, 2 ml of heel blood was collected and placed in a vacuum tube. The blood sample was centrifuged to obtain the serum, and the serum was stored in a refrigerator at −20°C for assays. Electrochemiluminescence technology was used to detect the level of neuron-specific enolase (NSE), and the enzymatic rate method was adopted to determine the level of creatine kinase-MB (CK-MB). The instrument was a Roche E170 automatic immunoassay analyzer, and the supporting reagents were provided by Roche. The chemiluminescence method was used to measure the serum troponin (cTnl) level, and the detection instrument was a UniCel dxl 800 automatic chemiluminescence immunoassay analyzer produced by Beckman Company. The above-mentioned tests were carried out in accordance with the operating instructions on the kit and testing equipment.For the treatment of neonatal jaundice, our hospital often uses Chen Zibaihuang lotion for bathing, once a day, for 15–30 minutes each time. The main formula of the lotion is: herba artemisiae scopariae, Gardenia, Rhubarb, Poria,Atractylodes, Astragalus, citrus, radix isatidis, Salvia, Pueraria, Licorice; for children with abdominal distension and vomiting, Pinellia, caulis Bambusae, and taeniis were added to stop vomiting. The dregs are boiled in water for washing. First, the nails were manicured in the incubator, then the hands were washed and warmed, and then the whole body bath was performed. During the treatment, the skin condition of the children should be given attention. For children with allergies, bathing should be stopped in time and appropriate symptomatic treatment should be carried out.


## 2.5. Indicators

The content and changes of NAA/Cr, Cho/Cr, Glx/Cr, mI/Cr, and Lac/Cr were compared, and the correlation between serum TSB level and 1H-MRS metabolic index was analyzed.The levels of NSE, CK-MB, and cTnl in groups with different serum TSB levels were compared.


## 2.6. Statistical Methods

All the data obtained in this study was analyzed using the SPSS20.0 software, and GraphPad Prism 7 (GraphPad Software, San Diego, USA) was used to plot figures. The count data are expressed as (n (%)) and analyzed using the chi-square test. The measurement data are expressed as mean ± SD; one-way ANOVA was used for comparison between multiple groups; and LSD-t test was used for two-by-two comparisons. Differences were considered statistically significant at P<0.05.


## 3. Results

### 3.1. Comparison of Metabolic Indexes

The three groups had comparable NAA/Cr, Cho/Cr, Lac/Cr, and ml/Cr levels (P>0.05), while there were statistical differences in Glx/Cr levels (P<0.05). (Table 1).Table 1
The relative content of each metabolite under globus pallidus 1H-MRS (x¯ ± s).
GroupCasesNAA/CrCho/CrLac/CrGlx/Crml/CrMild161.49 ± 0.281.35 ± 0.360.38 ± 0.160.96 ± 0.200.58 ± 0.19Moderate181.53 ± 0.341.39 ± 0.330.40 ± 0.151.26 ± 0.29a0.62 ± 0.21Severe161.58 ± 0.371.43 ± 0.410.44 ± 0.171.40 ± 0.35ab0.66 ± 0.23F0.7130.3650.5879.8600.577P0.4580.6310.560<0.010.566Note: Compared with Mild,P<0.05; compared with Moderate, P<0.05.


### 3.2. Comparison of NSE Levels

The severe condition group showed the highest NSE level, followed by the moderate group, and then the mild group (P<0.05). (Figure 1).Figure 1
Comparison of NSE levels,∗∗∗ indicated P<0.001.


### 3.3. Comparison of CK-MB Levels

The severe condition group showed the highest CK-MB level, followed by the moderate group, and then the mild group (P<0.05). (Figure 2).Figure 2
Comparison of CK-MB levels,∗ indicated P<0.05.


### 3.4. Comparison of cTnl Levels

The severe condition group showed the highest cTnl level, followed by the moderate group, and then the mild group (P<0.05). (Figure 3).Figure 3
Comparison of cTnl levels,∗∗∗ indicated P<0.001.


### 3.5. Correlation between Serum TSB Level with 1H-MRS Metabolic Index

TSB level is positively correlated with the 1H-MRS metabolic index Glx/Cr (Figure4).Figure 4
Correlation between serum TSB level and 1H-MRS metabolic index.


## 3.1. Comparison of Metabolic Indexes

The three groups had comparable NAA/Cr, Cho/Cr, Lac/Cr, and ml/Cr levels (P>0.05), while there were statistical differences in Glx/Cr levels (P<0.05). (Table 1).Table 1
The relative content of each metabolite under globus pallidus 1H-MRS (x¯ ± s).
GroupCasesNAA/CrCho/CrLac/CrGlx/Crml/CrMild161.49 ± 0.281.35 ± 0.360.38 ± 0.160.96 ± 0.200.58 ± 0.19Moderate181.53 ± 0.341.39 ± 0.330.40 ± 0.151.26 ± 0.29a0.62 ± 0.21Severe161.58 ± 0.371.43 ± 0.410.44 ± 0.171.40 ± 0.35ab0.66 ± 0.23F0.7130.3650.5879.8600.577P0.4580.6310.560<0.010.566Note: Compared with Mild,P<0.05; compared with Moderate, P<0.05.


## 3.2. Comparison of NSE Levels

The severe condition group showed the highest NSE level, followed by the moderate group, and then the mild group (P<0.05). (Figure 1).Figure 1
Comparison of NSE levels,∗∗∗ indicated P<0.001.


## 3.3. Comparison of CK-MB Levels

The severe condition group showed the highest CK-MB level, followed by the moderate group, and then the mild group (P<0.05). (Figure 2).Figure 2
Comparison of CK-MB levels,∗ indicated P<0.05.


## 3.4. Comparison of cTnl Levels

The severe condition group showed the highest cTnl level, followed by the moderate group, and then the mild group (P<0.05). (Figure 3).Figure 3
Comparison of cTnl levels,∗∗∗ indicated P<0.001.


## 3.5. Correlation between Serum TSB Level with 1H-MRS Metabolic Index

TSB level is positively correlated with the 1H-MRS metabolic index Glx/Cr (Figure4).Figure 4
Correlation between serum TSB level and 1H-MRS metabolic index.


## 4. Discussion

The metabolic function of the newborn is immature, and when bilirubin production exceeds excretion, the skin and sclera will usually show yellow staining, i.e., neonatal jaundice. Severe hyperbilirubinemia may cause neonatal ABE [13, 14]. At present stage, the diagnosis of hyperbilirubinemia is usually based on the serum TSB level, which is also an important reference for risk assessment, intervention, and prevention of ABE [15–17]. MRS is an imaging technology based on the resonance signal of atomic nuclei within a magnetic field and can measure the signal of compounds according to the chemical shift principle. It can analyze the tissue metabolism and physiological and biochemical changes caused by the disease, which is of significance for the early diagnosis of the pathogenesis of bilirubin encephalopathy [18–20]. At present, MRS can detect atoms such as 1H, 13C, and 31P and has the highest sensitivity for detecting 1H. It measures the levels of metabolites such as NAA, Cho, mI, and Glx and is widely used in the clinical trials of neonatal neurological diseases [21, 22]. Cr is a metabolic marker with stable content in the body and it is used as a reference for measuring the peak changes in other metabolites. The TSB level is positively correlated with the 1H-MRS metabolic index Glx/Cr.In this study, the three groups had comparable NAA/Cr, Cho/Cr, Lac/Cr, and ml/Cr levels (P>0.05), while there were statistical differences in Glx/Cr levels (P<0.05); the serum TSB level is positively correlated with Glx/Cr. The reason may be that high serum TSB levels could lead to an increase in ml, which is associated with an imbalance in the regulation of cell osmotic pressure in brain tissue and further results in neuronal edema. The elevated Glx/Cr may be due to the cytotoxic effect of bilirubin inhibiting the function of astrocytes, the continuous release of excitatory amino acids such as glutamine, and the intense concentration of excitatory amino acids such as glutamine in the interstitial space of nerve cells, resulting in nerve cell damage and death. The study by Lin et al. [23] found that the toxic effect of high bilirubin has the most pronounced impact on glutamine metabolism, and Glx/Cr showed great potential as an auxiliary diagnostic index for the diagnosis of bilirubin encephalopathy. Relevant studies have shown that NSE reflected the damage of bilirubin to neurons, and CK-MB and cTnl were indicators that could precisely reflect myocardial injury [1]. This study analyzed the levels of NSE, CK-MB, and cTnl in groups with different serum TSB levels and found that the severe condition group showed the highest levels of NSE, CK-MB, and cTnl, followed by the moderate group, and then the mild group (P<0.05), which is consistent with the results of prior literature [24], in which elevated serum TSB levels led to increased levels of CK-MB, cTnl, and NSE. The more severe the damage to cardiomyocytes, the higher the level of NSE, which has a certain impact on nerve capacity.In the present study, the serum TSB level is positively correlated with the 1H-MRS metabolic index Glx/Cr. Bilirubin tends to accumulate in brain nuclei, such as the globus pallidus, basal ganglia of the thalamus, and brainstem nucleus, leading to nervous system abnormalities, which are considered an important factor in neonatal death and sequelae. Excessive elevation of serum total bilirubin, especially indirect bilirubin, impairs the synaptic transmission function of neurons, resulting in cell membrane damage, neuronal death, and neonatal bilirubin encephalopathy. The above results all suggested that serum levels of NSE, CK-MB, and cTnl levels were correlated with globus pallidus metabolites. However, no relevant studies have directly shown the relationship between these indicators, and further studies are required for verification.The formula of the lotion used in this study: herba artemisiae scopariae is the main medicine for cholagogue and removing jaundice; Gardenia, Rhubarb clears Sanjiao, clears blood heat; Poria,Atractylodes, and Astragalus invigorate the spleen and remove dampness; radix isatidis clears heat and detoxifies; salvia combined with rhubarb can promote blood circulation and detoxification; puerariae can also be used to induce spleen and all the above are ministerial medicines; licorice can be used as an auxiliary medicine. Studies have shown that the active ingredient of herba artemisiae scopariae is p-hydroxyacetophenone, which is a natural penetration enhancer that allows fatty drugs to penetrate into the skin in an undissociated form and can further effectively promote drug absorption due to the thin and tender skin of newborns [1]. Hydrotherapy has positive effects on neonatal nerve, endocrine, and metabolism. It can stimulate vagus nerve activity, increase the release of hormones such as gastrin, and promote intestinal peristalsis [24].


## 5. Conclusions

In summary, the serum TSB level is correlated with the 1H-MRS metabolic index Glx/Cr in the newborn with neonatal jaundice, and the levels of TSB and Glx/Cr provide a reference for the diagnosis of bilirubin encephalopathy.



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*Source: 9785584-2022-07-04.xml*