Evaluation of Serum Levels of Trace Elements in Children with Biliary Atresia and their Correlation with Liver Histopathology.

BACKGROUND: Children with biliary atresia (BA) have impaired metabolism of trace elements (TEs) (i.e., zinc, copper, selenium, and manganese), leading to an alteration in the serum levels. However, this alteration in serum level has any correlation with liver histopathological changes is not yet clear.
MATERIALS AND METHODS: This prospective study attempts to evaluate the preoperative serum levels of TE in comparison to controls and its correlation with liver histology in children with BA. Kasai portoenterostomy (KPE) and liver biopsy were performed in all cases. On liver histology, various parameters assessed and were graded according to predefined criteria. Serum levels of TE were determined again 12 weeks post-KPE and compared with the preoperative levels.
RESULTS: Mean(±standard deviation [SD]) preoperative serum Zn, Cu, Se, and Mn levels (in μg/dl) in BA patients were 41.6 ± 12.8, 130.6 ± 12.8, 50.0 ± 10.0, and 32.0 ± 20.0, respectively; in controls, these levels were 77.9 ± 13.7, 133.7 ± 13.7, 87.0 ± 13.0, and 8.0 ± 5.5, respectively. Mean postoperative levels in all patients were 68.5 ± 19.0, 91.7 ± 19.0, 79.0 ± 19.0, and 28.0 ± 12.0, respectively. Mean(±SD) postoperative serum Zn, Cu, Se, and Mn levels in BA patients with bile excretion were 73.8 ± 14.9, 83.6 ± 13.8, 85.0 ± 15.0, and 26.0 ± 10.0, respectively, whereas in those with no bile excretion, they were 40.6 ± 12.8, 134.0 ± 23.0, 49.0 ± 11.0, and 44.0 ± 16.0, respectively. In liver histology, specific parameters showed correlation with high Mn and low Zn levels.
CONCLUSION: Serum TE levels are altered in children with BA and the establishment of successful biliary drainage may change the subsequent postoperative serum concentration. Serum Zn and Mn levels can signify specific histopathological liver changes and the extent of liver damage. Copyright:

INTRODUCTION

Biliary atresia (BA) is one of the causes of neonatal cholestasis and characterized by progressive fibrosis of extrahepatic and intrahepatic bile ducts, leading to cirrhosis of the liver. In normal children, the metabolism of trace elements (TEs), especially zinc (Zn), copper (Cu), selenium (Se), and manganese (Mn), require normal liver function and patent enterohepatic circulation for maintaining their normal concentration in serum and at their target site. Each TE has its own unique metabolic pathway, storage site, and target organ. In BA, because of poor liver function, TE metabolism becomes compromised and because of cholestasis with impaired enterohepatic circulation, their concentration (in serum as well as in target organ) also altered.[1] There are contrary reports about the serum concentrations of TE in patients with liver disease. Studies that have correlated liver histological changes with TE levels are available but have not provided any definite correlation between the two.[2] This study has been designed to determine the serum concentration of these TE and correlate them with liver histopathological parameters.

MATERIALS AND METHODS

This was an observational prospective study which was conducted in the department of pediatric surgery at a tertiary care center. Twenty-five children diagnosed with BA were included in the study. Exclusion criteria included children with a history of major bowel resection, any metabolic disease, intake of medication containing TE, top/formula-fed babies, refusal of consent, and those who were lost to follow-up or died before 3 months of surgery. Twenty-five children of the same age group, with no hepatobiliary or gastrointestinal disease, who were admitted in the pediatric surgery ward during the same period, were included in the control group. This study was approved by the institutional ethics committee.

Preoperatively, the patients underwent standard investigations, including biochemical liver function tests, ultrasonography, and hepatobiliary iminodiacetic acid (HIDA) scan. Peroperative cholangiogram was considered diagnostic for BA, and all diagnosed patients were treated by the Kasai portoenterostomy (KPE) procedure. A wedge biopsy of the liver was taken during KPE and examined for various histopathological parameters. Serum levels of TE (Zn, Cu, Se, and Mn) were determined preoperatively and again at 12 weeks postoperatively along with the HIDA scan.

Determination of serum levels of trace elements

With all aseptic precautions, 3 ml of blood was collected in TE free tubes. These blood samples were centrifuged for 5 min at 3000 rpm. Serum was separated, collected in TE free Eppendorf tubes, and stored at −20°C until the analysis. Serum samples (100 μl) were diluted 50-fold in 1% HNO3to make the total volume of 5 ml. Gallium was used as an internal standard. TE concentrations in the serum samples were measured using atomic absorption spectrophotometer; inductively coupled plasma mass spectrometry (X Series 2 – Thermo Scientific) equipment was used.

Histopathological examination and grading of liver biopsy specimens

All the liver biopsy samples were examined by a single senior pathologist (MKS) who was blinded for the tests. The histopathological parameters were assessed, and changes were graded with the criteria presented in Tables 1 and 2.[3]

Table 1

Histological parameters and grading of liver biopsy

Factors	Features	Grading
Cholestasis	Canalicular	1
	Hepatocytes	2
	Centrilobular	3
Hepatocellular damage	Ballooning	1
	Feathery degeneration	2
	Necrosis	3
Bile duct proliferation	Mild	1
	Moderate	2
	Severe	3
Bile duct fibrosis	Mild	1
	Moderate	2
	Severe	3
Bile duct inflammation	Mild	1
	Moderate	2
	Severe	3
Portal edema	Mild	1
	Moderate	2
	Severe	3
Portal inflammation	Mild	1
	Moderate	2
	Severe	3

Table 2

Classification of portal fibrosis

Grades	Features
Grade 0	No fibrosis
Grade 1	Mild fibrosis confined to portal area
Grade 2	Moderate fibrosis in portal-portal area (P-P) bridging fibrosis
Grade 3	Severe expansive fibrosis with widened P-P bridging fibrosis
Grade 4	Liver cirrhosis with reconstruction of hepatic lobules

The TE levels were calculated in μg/dl, and the mean ± standard deviation values were tabulated separately for each TE in the following groups: (i) preoperative, (ii) postoperative (iii) postoperative and HIDA +ve for excretion, (iv) postoperative and HIDA −ve for excretion, and (v) control. The TE levels were also correlated with the severity of histological changes in the liver. The data analysis was performed using Stata software version 9.0 (Stata Corp LP, Texas, USA). Mann–Whitney U-test, Pearson's test, and Fisher's exact test were used, and a value of P < 0.05 was considered statistically significant.

RESULTS

The study group comprised 25 children; 22 males (88%) and 3 females (12%) with a mean age of 3.14 months (range 1–5 months). The control group comprised 25 children; 18 males (72%) and 7 females (28%) with a mean age of 3.52 months (range 2–5 months). Both groups were comparable (P = 0.157). All patients with BA underwent KPE and liver biopsy. Postoperatively, the HIDA scan showed excretion suggestive of a patent bilioenteric anastomosis in 21 children and in 4 children, the HIDA scan did not show excretion suggestive of the nonpatent bilioenteric anastomosis.

Serum level of trace elements in cases and controls

Zinc

The mean level of Zn in the preoperative group was lower than in the postoperative group (P = 0.001), but even in the postoperative group, it was lower than the controls but statistically not significant (P = 0.05). The increase in Zn level was significant in the postoperative group with HIDA +ve (P = 0.0001), while no significant change in the postoperative group with HIDA −ve (P = 0.05).

Copper

The mean level of Cu in the preoperative group was significantly higher than in the postoperative group (P = 0.001) but no significant difference to control values (P = 0.56). The decrease in Cu level was significant in the postoperative group with HIDA +ve (P = 0.0001) while no significant change in the postoperative group with HIDA −ve (P = 0.1) [Table 3].

Table 3

Serum trace elements level in different groups

Group (n=number of children)	Mean±SD
	Zn (µg/dl)	Cu (µg/dl)	Se (µg/dl)	Mn (µg/dl)
Preoperatively (n=25)	41.6±12.8	130.6±12.8	50.0±10.0	32.0±20.0
Postoperatively (n=25)	68.5±19.0	91.7±19.0	79.0±19.0	28.0±12.0
HIDA +ve (n=21)	73.8±14.9	83.6±13.8	85.0±15.0	26.0±10.0
HIDA −ve (n=4)	40.6±12.8	134±23.0	49.0±11.0	44.0±16.0
Control (n=25)	77.9±13.7	133.7±13.7	87.0±13.0	8.0±5.5

Zn: Zinc, Cu: Copper, Se: Selenium, Mn: Manganese, SD: Standard deviation, HIDA +ve: HIDA scan +ve for biliary excretion (patent bilioenteric pathway), HIDA –ve: HIDA scan −ve for biliary excretion (nonpatent bilioenteric pathway)

Selenium

The mean level of Se in the preoperative group was significantly lower than in the postoperative group (P = 0.001) and as compared with control values (P = 0.0001). There was no difference in the postoperative group as compared to the control group (P = 0.1). The increase in Se level was significant in the postoperative group with HIDA positive (P = 0.0001), while no significant change in the postoperative group with HIDA negative (P = 0.35) [Table 3].

Manganese

As depicted in Table 3, the mean level of Mn in the preoperative and postoperative groups were not significantly different (P = 0.19), but the mean value of the preoperative group and the postoperative group were significantly higher than the control group. The decrease in Mn level was significant in the postoperative group with HIDA +ve (P = 0.024) and increase in level was significant in the postoperative group with HIDA −ve (P = 0.005).

Correlation between specific histopathological parameter on liver biopsy with individual trace element level [Table 4]

Cholestasis, hepatocellular damage, bile duct fibrosis, and portal fibrosis

There was a significant association of cholestasis, hepatocellular damage, bile duct fibrosis, and portal fibrosis with Mn levels (P = 0.001, 0.0336, 0.032, and 0.014, respectively) i.e., higher Mn levels were seen with more severe grades of these parameters. This suggests Mn toxicity with higher grades of liver damage.

Portal inflammation

It had a correlation with Zn level, i.e., if the grade of portal inflammation was high, the Zn level was low (P = 0.04). This suggests a Zn deficiency state in the presence of portal inflammation.

Bile duct proliferation, bile duct inflammation, and portal edema

There was no significant correlation of the above-mentioned parameters with any of the TE levels.

DISCUSSION

TEs are essential for the growth and development of infants and children. Apart from specific functions, the majority of them are anti-oxidants and function at the cell level. The liver plays an important part in their metabolism, and some of them depend on the enterohepatic circulation for their optimal effects.

Zinc (Zn) is the most ubiquitous of all TEs involved in human metabolism. It is essential for prenatal and postnatal development and is vital for numerous metabolic activities. Zn deficiency is a leading cause for susceptibility to infections and diarrhea. The daily requirements, 3 mg/day for infants, and up to 8 mg/day for older children are usually met from dietary sources. However, deficiency can occur from decreased dietary intake or it may be associated with chronic liver disease. Thus, in children with BA, the Zn levels are likely to be low preoperatively and become worse with decreased dietary intake. Following absorption from the intestines, Zn is bound with albumin and ferritin. The portal system carries absorbed Zn directly to the liver and then released into systemic circulation for delivery to other tissues. The loss of Zn through the gastrointestinal tract accounts for approximately half of all Zn eliminated from the body, but most of it is reabsorbed, and this process is an important point in the regulation of Zn balance; poor bile excretion reduces the reabsorption of Zn.[45] Zn is also known to be antagonistic with Cu and iron. Thus, low levels of Zn may allow excessive Cu accumulation; this is cytotoxic and results in fibrosis in hepatic tissues leading to cirrhosis.[1] Plasma levels of Zn have not been found to correlate with age, do not appear to be related to repeated surgical procedures or to episodes of cholangitis.[5] In the present study, children with BA had lower serum Zn levels preoperatively, and serum levels improved after surgery. Hence, children require extra Zn supplementation in the preoperative period, while in the postoperative phase, once enterohepatic circulation is established Zn levels improve gradually.

Copper (Cu) is an essential TE which is necessary for respiratory functions. It is absorbed into the body through the intestinal mucosa and transported through the portal blood to the liver. Much of that taken up by the liver is incorporated into ceruloplasmin, released into the blood, and delivered to tissues. Most endogenous Cu is lost through the bile, being excreted into the gastrointestinal tract.[6] As the major route of copper (Cu) excretion is bile, and in BA, there is no bile flow that leads to the accumulation of Cu in the liver;[1] this was evident from higher levels seen with higher grades of liver damage in the study group children [Table 4]. Accumulation of Cu in the liver is known to be a causative factor in Wilson's disease.

Table 4

Correlation of different grades of specific histopathological parameter to individual trace element (median) level

Histopathological parameter	Grade	Number of patients (%)	µg/dl
			Zn	Cu	Se	Mn
Cholestasis	1	11 (44)	42.4	119.0	47.3	16.8
	2	14 (56)	42.2	129.6	52.2	40.4
Hepatocellular damage	1	2 (8)	43.7	108.2	17.8	46.9
	2	12 (48)	41.4	138.9	20.75	48.9
	3	11 (44)	43.5	125.0	40.8	52.6
Bile duct proliferation	1	1 (4)	57.9	119.0	12.8	72.0
	2	16 (64)	42.2	133.5	26.6	47.4
	3	8 (32)	40.4	126.5	34.4	51.65
Bile duct inflammation	1	4 (16)	48.2	122.4	53.15	33.6
	2	4 (16)	42.7	128	47.3	24.8
	3	17 (68)	22.9	123.2	51.2	48.6
Bile duct fibrosis	1	1 (4)	58.7	116	50.3	12.8
	2	8 (32)	41.7	117.4	49.0	17.6
	3	16 (64)	42.6	129.6	51.0	36.0
Portal inflammation	1	4 (16)	46.0	133.2	50.0	23.0
	2	16 (64)	42.5	128.4	46.0	26.7
	3	5 (20)	26.1	125.0	52.8	42.6
Portal edema	1	11 (44)	42.4	125.0	51.8	28.8
	2	14 (56)	42.2	139.0	46.02	26.6
Portal fibrosis	1	1 (4)	58.7	116.1	50.3	12.8
	2	11 (44)	43.7	136	50	19
	3	13 (52)	37.8	128	50	40

Zn: Zinc, Cu: Copper, Se: Selenium, Mn: Manganese

Selenium (Se) is also an essential TE, which acts as an anti-oxidant; it is necessary for cellular functions dependent on the use of thyroxine. The most common dietary source is cereals, and the daily requirement is approximately 55 μg. The serum levels in children with any disease usually decrease because of the consumption of Se against oxidative stress. Furthermore, poor intake and malabsorption contribute to low levels.[7] Serum Se concentration in cirrhotic children has been found to be significantly lower when compared with normal children.[8] Low preoperative levels are suggestive of oxidative stress in the body. Once the surgery is performed, the improved levels of Se are suggestive of decreased oxidative stress as well as improved absorption. In the present study, HIDA −ve children continued to show low levels, indicative of ongoing liver pathology.

Manganese (Mn) is another essential TE, which acts as an antioxidant and is necessary for proper enzymatic functions. In children, the daily requirement of 0.5–2.0 mg is usually provided by cereals, leafy vegetables, and drinking water. Similar to Cu, the route of excretion for Mn is bile and decreased bile flow with intrahepatic cholestasis may result in hepatic accumulation of Mn.[2] The Mn levels in children in this study were higher than normal both preoperatively and postoperatively, although HIDA +ve children showed some decrease in the levels as compared to HIDA −ve children. Thus, serum levels may reflect the accumulation of Mn in the liver. There is a risk for latent toxicity of Mn in these children and serial serum levels along with MRI of the brain have been proposed to detect the condition.[9]

The liver architecture in BA is preserved during the first few weeks of life, but occlusion of the extrahepatic ducts leads to the widening of the portal tracts with edema and increased amounts of fibrous tissue. The proliferation of bile ductules is accompanied by bile stasis both within canaliculi and hepatocytes.[1011]

In the study group children, low Zn levels were shown to correlate with higher grades of portal inflammation. Portal inflammation is not specific for BA and indicates only an ongoing inflammatory process. It is suggested that Zn may have a protective role in inflammation.

Higher Mn levels correlated with cholestasis, hepatocellular damage, bile duct fibrosis, and portal fibrosis. Cholestasis leads to decreased bile secretion that leads to decreased Mn excretion and hence an increased serum level. While Cu is also secreted through bile but in cholestasis, it accumulates in the liver, and hence, serum levels do not represent the actual status of Cu in the liver. When liver storage sites are saturated, then only high copper might reflect in serum levels.[1213] Previous studies have also shown that there is no correlation between serum copper level with hepatic copper concentration.[1213] Bile duct fibrosis and portal fibrosis are an indicator of persistent or progressive hepatic injury and represent a histological hallmark of chronic liver disease regardless of the cause. Serum Mn level correlated well with the chronicity of the disease. Serum Mn can be used to assess the severity of liver damage in the preoperative period and in early follow-up for the effectiveness of surgery. In long-term follow-up, normalization of Mn level can indicate good liver function, stable Mn level indicates slow ongoing deterioration of liver function, and increasing levels either immediately after surgery, or later at follow-up are suggestive of deterioration of liver status and the development of cirrhosis. Hence, Mn can evolve as an important tool to assess liver status in BA children.

CONCLUSION

Serum TE levels are altered in children with BA and the establishment of successful biliary drainage may change the subsequent postoperative serum concentration. Serum Zn and Mn levels can signify specific histopathological liver changes and the extent of liver damage.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.