Journal of Pediatric Gastroenterology & Nutrition:
Original Articles: Hepatology and Nutrition
Continuous 13C-Methacetin Breath Test Differentiates Biliary Atresia From Other Causes of Neonatal Cholestasis
Shteyer, Eyal*; Lalazar, Gadi†; Hemed, Nilla†; Pappo, Orit‡; Granot, Esther||; Yerushalmi, Baruch¶; Gross, Eitan§
*Pediatric Gastroenterology Unit, Department of Pediatrics
†Liver Unit, Department of Medicine
‡Department of Pathology
§Department of Pediatric Surgery, Hadassah-Hebrew University Medical Center, Jerusalem
||Kaplan Medical Center, Rehovot
¶Soroka Medical Center, Ben Gurion University of the Negev Beer Sheva, Israel.
Address correspondence and reprint requests to Eyal Shteyer, MD, Pediatric Gastroenterology Unit, Department of Pediatrics, Hebrew University-Hadassah Medical Center, Jerusalem, Israel (e-mail: email@example.com).
Received 23 March, 2012
Accepted 7 June, 2012
This article is dedicated to the memory of Dr Eitan Gross, who passed away after the article was accepted—a great surgeon and an exceptional human being.
The present study was PI (E.S.) initiated. The MBT machine and the substrates were provided by Exalenz Ltd.
The authors report no conflicts of interest.
Background and Aim: Distinguishing biliary atresia (BA) from other causes of neonatal cholestasis (NC) is challenging. Continuous BreathID 13C-methacetin breath test (MBT) is a novel method that determines liver function. Methacetin is metabolized uniquely by the liver and 13CO2 is measured passively, through a nasal cannula in the exhaled breath. The aim of this study was to assess the ability of MBT to differentiate BA from other causes of NC.
Methods: MBT was performed in infants with NC before any invasive procedure. Percent dose recovered (PDR) peak and time to peak (TTPP) of 13C recovered were correlated with blood test results and degree of fibrosis on liver biopsy.
Results: Fifteen infants were enrolled in the study. Eight were eventually diagnosed as having BA. MBT showed that infants with NC from various causes reached the PDR peak after 44.5 ± 6.7 minutes, whereas infants with BA reached the PDR peak value after 54.7 ± 4.3 minutes (P < 0.005). This suggested low cytochrome P450 1A2 activity in the BA group. The area under the curve (AUC) was 0.95 (95% confidence interval [CI] 0.83–1), sensitivity of 88%, and specificity of 100%.
Conclusions: This pilot study shows that MBT can differentiate between BA and other causes of NC by time to peak of methacetin metabolism. The results suggest that MBT may be used as part of the diagnostic algorithm in infants with liver disease. Larger-scale studies should be conducted to confirm these initial observations.
See “Heavy Breathing: A Step to Noninvasive Assessment of Hepatic Fibrosis and Function in Infants and Children” by Narkewicz on page 2.
Neonatal cholestasis (NC) affects approximately 1 in 2500 births and is defined as conjugated hyperbilirubinemia that occurs in the newborn period (1). The differential diagnosis for NC is wide, but several, potentially treatable disorders should be ruled out early to avoid progressive damage. These infants undergo numerous diagnostic tests including laboratory tests, abdominal imaging, and percutaneous liver biopsy in an attempt to diagnose surgical, such as biliary atresia (BA), genetic, and metabolic conditions. Timely diagnosis and treatment of infants with BA has significant bearing on prognosis, but often diagnosis is delayed because of late referral or lengthy and cumbersome investigations. Unfortunately, clinical features and the routine liver function blood tests fail to distinguish BA from other causes of NC. The criterion standard for diagnosis is intraoperative cholangiogram, but often physicians will thoroughly investigate the infant before sending to the operating room.
Breath tests have been used in the past for assessment of liver status in patients with acute and chronic liver disorders (2–4). Levels of exhaled metabolites of a labeled substrate correlate with the degree of hepatic injury and metabolic hepatic state. Various substrates, such as 13C-phenylalanine (PheBT), 13C-galactose (GBT), 13C-caffeine and indocyanine green, aminopyrine, and methacetin, were shown to reflect hepatic metabolism (5–7). Methacetin, the substrate used in our study, is rapidly metabolized by cytochrome P450 1A2 (CYP1A2) and is considered a good substrate for evaluating CYP1A2 enzyme activity (7,8). Low CYP1A2 was shown to correlate with advanced hepatic fibrosis in patients with hepatitis C (9) and other forms of liver diseases (10). Furthermore, it was shown to play a role in the inflammatory response during sepsis (11). This led us to hypothesize that CYP1A2 function may be able to differentiate between infants with BA and other causes of NC.
Although breath tests using different substrates may adequately assess liver function, their clinical use has been limited. A major drawback in using traditional breath tests is the cumbersome method of isotopic ratio mass spectrometry. This method requires prolonged testing and analysis, imposes patient inconvenience, and delivers limited data points. In contrast, the BreathID continuous online 13C-methacetin breath test (MBT, Exalenz, Modiin, Israel) is based on the measurement of 12CO2 and 13CO2 concentrations by molecular correlation spectroscopy (MCSTM) that can detect variations of <1 of 1000 in the 13CO2/12CO2 ratio (12,13). Furthermore, the BreathID system continuously samples passive breath so that patient compliance is not required. This holds a major advantage in the evaluation of infants and young children. The aim of the present study was to assess the ability of the MBT to differentiate BA from other causes of NC.
Between April 2008 and September 2011, infants from 3 medical centers were recruited to our study. Inclusion criteria consisted of age 2 weeks or younger, with elevated total bilirubin of >34 μmol/dL of which >20% was direct. All legal guardians gave written informed consent for participation in the study. The study was conducted with strict adherence to the principles of the Declaration of Helsinki, and was approved by the institutional review board (IRB) committees and the Israel Ministry of Health Committee for Human Clinical Trials.
All of the infants underwent work-up for infectious, metabolic, endocrine, and genetic diseases. The results of their basic work-up were correlated to the MBT results. These included a complete blood count, coagulation tests (prothrombin time and partial thromboplastin time), and levels of aspartate aminotransferase (AST), alanine aminotransferase (ALT), alkaline phosphatase (ALP), γ-glutamyltranspeptidase (GGTP), lactate dehydrogenase, albumin, and total and direct bilirubin. Routine biochemical tests were performed with commercially available kits at the Hadassah Medical Center core laboratory.
Liver biopsy was obtained either by open biopsy, during a planned Kasai procedure, or by percutaneous liver biopsy. Biopsies were ultrasound guided, under local anesthesia (lignocaine 1%) and sedation. Specimens obtained by means of Menghini needles, diameter 1.6 mm, had an average length of 20 ± 5 mm (range 15–25 mm), and representative according to accepted standards. Biopsy specimens were fixed with formalin, embedded in paraffin, and stained with haematoxylin and eosin. All sections were reviewed by an expert pathologist blinded to the patient's clinical data and breath-test results.
Necroinflammatory score was graded using the modification of Ishak score based on periportal or periseptal interface hepatitis (piecemeal necrosis) (0–4), confluent necrosis (0–6), focal (spotty) lytic necrosis, apoptosis, focal inflammation (0–4), and portal inflammation (0–4) (14). Fibrosis was staged using the Ishak (modified Histological Activity Index) fibrosis score on a scale from 0 to 6 (14).
13C-Methacetin Breath Test
Following a 4-hour fast, patients were connected to the breath-testing unit via a nasal cannula (IDcircuit, Oridion Medical, Jerusalem, Israel) and received, by mouth, 3 mg/kg of N-(4-methoxy-13C-phenyl)acetamide (methacetin; Isotec, Fort Myers, FL) dissolved in 150 mL of water. Breath samples were continuously collected by the BreathID device (Exalenz) before and for 60 minutes after the labeled substrate was administered to the patient. The 13CO2/12CO2 ratio in the breath samples was determined every 2 to 3 minutes. During the test period patients continued fasting and remained at rest to eliminate any variability in CO2 production owing to the ingestion of food or physical activity. MBT was performed after blood tests and abdominal ultrasound to exclude bile duct obstruction or any other malformations (choledocal cysts), and before any other invasive procedure such as liver biopsy or endoscopic retrograde cholangiopancreatography (ERCP).
Analysis of Breath Test Data
Results obtained from the device were expressed as percentage of administered dose of 13C recovery (percent dose recovered [PDR]) and the cumulative percentage of 13C recovery over time (CPDR) at 20, 30, and 60 minutes after ingestion of methacetin, respectively, as well as the PDR peak, and time to peak PDR (time to peak [TTPP]). PDR refers to the rate at which the 13C substrate is metabolized and exhaled, expressed as percentage per hour. PDR is based on the change in 13C/12C ratio for each patient, taking into account their specific parameters affecting overall CO2, normalizing the results for weight, height, and dose (15) CPDR is the numeric integral of PDR, and describes the total percentage of substrate metabolized at any given accumulated time. The BreathID device plots the PDR and CPDR in real time and the PDR peak value is then calculated.
To assess the ability of the breath test to differentiate BA from NC several tests were used: Student t test to compare the various measures between the groups; and graphical box plots to depict the differentiation visually. The average is presented as the middle black line and the rectangles formed around represent the 50% (second and third quintile) of the groups. Finally a receiver operating characteristic (ROC) curve was calculated. The overall performance was analyzed by using each possible cutpoint; the resulting sensitivity and specificity of the test for identification of BA can be indicated as a point on a graph. The area under the ROC curve (area under the curve [AUC]) is a numerical test to assess the overall discriminatory ability of the above-mentioned measures.
Fifteen infants were enrolled into the study. Eight were diagnosed with BA based on liver biopsy and intraoperative cholangiogram. In the other 7 infants NC was the result of neonatal hepatitis in 2, and cytomegalovirus hepatitis, Down syndrome with paucity of intrahepatic bile ducts, HIV hepatitis, progressive familial intrahepatic cholestasis type 2, and Alagille syndrome. The latter group, with no BA, will be referred to as the NC group. The demographic, clinical, and laboratory characteristics are summarized in Table 1.
Assessment of Serum Blood Test Parameters and Fibrosis
Table 2 depicts difference in values of various parameters between BA and NC groups. There was no significant difference in age, bilirubin levels, and serum aminotransferases. Weight was significantly lower in the NC group (4.7 ± 0.6 vs 4.07 ± 0.1, P < 0.04). GGTP was significantly higher in the BA group (907 ± 761 vs 176 ± 169, P < 0.03). Albumin was higher in the BA group, but was not significantly different (39 ± 5 vs 32 ± 5.7, P < 0.07). Fibrosis score was higher in the BA group, with an average grade 4 to 5 in infants with BA and 0 to 1 in the NC group. Fibrosis scores correlated with TTPP, showing r = −0.94 (95% confidence interval [CI] 0.35–0.99, P = 0.013).
MBT Differentiates Between BA and NC
When assessing MBT parameters (Fig. 1), time to peak PDR (TTPP) was significantly higher in the BA group (Fig. 1A) versus NC group (Fig. 1B) (54.7 ± 4.3 vs 44.5 ± 6.7, respectively, P < 0.005). There was no significant difference in other MBT parameters (Fig. 1), but all were low compared with normal established in adults. Figure 2 shows box plots depicting the differentiation of BA from NC by TTPP showing no overlap between groups. The overall performance of MBT was analyzed by ROC curve. For each possible cutpoint, the resulting sensitivity and specificity of the test for identification of BA can be indicated as a point on a graph. The area under the ROC curve (AUC) for TTPP is 0.95 (Fig. 3), with 88% sensitivity and 100% specificity at 52-minute cutoff. GGTP, weight, and albumin were also able to differentiate between the groups, but with lower AUC scores (0.91%, 0.81%, and 0.78%, accordingly).
In the present pilot study we showed that MBT differentiated between BA and other causes of NC. Timely diagnosis of BA is of particular importance because delayed surgical correction is associated with inferior long-term prognosis (16,17). Radioisotope excretion studies, for example, with N-tert-butyliminodiacetic acid (TEBIDA) show absent or reduced excretion into the intestine, but does not distinguish BA from other causes of severe intrahepatic cholestasis such as Alagille syndrome (18). Endoscopic retrograde cholangiopancreatography to visualize the biliary tract is occasionally needed when the diagnosis is unclear, but it is technically difficult in infants and its use is confined to large centers (19). Magnetic resonance retrograde cholangiopancreatography (MRCP) can only identify bile ducts luminal patency of 1 mm in diameter or greater, and is of limited use in the diagnosis of BA (20). To date, the method of choice for diagnosing BA is liver histology. Findings of varying degrees of portal tract fibrosis, edema, ductular proliferation, and bile plugs support BA, whereas evidence of giant cell transformation suggests other causes of neonatal hepatitis. Timing of the liver biopsy, especially before 6 weeks of age, may not show typical features of BA and serial biopsy samples may be necessary for definite diagnosis (21). Equivocal histopathologic features suggest alternative conditions, such as α-1-antitrypsin deficiency, Alagille syndrome, neonatal sclerosing cholangitis, cystic fibrosis, or exposure to total parenteral nutrition (TPN), all of which can mimic BA (22–24). Thus, a simple, noninvasive test is greatly needed to differentiate BA from other nonsurgical diseases.
In the present study, we assessed the methacetin breath testing as a tool to differentiate BA from other causes of NC. To date MBT has been performed in adults only, even though Methacetin itself has been used in the past in children. Methacetin breaks down into acetaminophen and 13CO2 by cytochrome P450 1A2 with a single O-dealkylation step (8). Urinary methacetin had been previously used as a measure for liver detoxification capacity of children from heavily polluted industrial areas (25). Urinary methacetin was also used to assess the maturity of the hepatosomal mono-oxygenation and glucuronidation activities in preterm and term newborns (26). In previous studies, MBT was performed using isotopic ratio mass spectrometry and was correlated with fibrosis and overall liver function (12,27,28). In the present study, we used a novel technology (the BreathID continuous online 13C-MBT, Exalenz) that enabled to perform breath testing in infants. In addition to being less cumbersome, the continuous system has an inherent advantage over mass spectrometry in its ability to identify the PDR peak and time to PDR, which are often missed when noncontinuous measurements are used. Furthermore, being fully automatic and using an internal capnograph, the system mitigates the risk of potential human errors and ensures that the appropriate part of the breath sample is collected. These properties provide clear benefits to its use in infants and children, which otherwise would not be able to undergo the test.
The present study clearly demonstrates that MBT can accurately identify infants with BA with 88% sensitivity and 100% specificity that can be performed easily in small infants with no adverse effects. Other measures such as GGTP, albumin, and weight were also different in the BA group. GGTP was shown by others to be higher in infants with BA (29,30), and should prompt further workup. It should be noted that, unlike other studies, in our study only the TTPP was different between the 2 groups. Animal studies showed that all measures of the MBT were significantly different when comparing hepatitis and fibrosis (31). In human adults all MBT measures differ significantly in patients with hepatitis C (32) and other chronic liver diseases (3). All of the infants in our study had extremely low PDR peak, which was not significantly different between BA and NC. This finding may be because of the extremely low CYP1A2 levels in neonates (33,34) and not necessarily because of fibrosis. It may be that in such low levels of enzyme MBT is not sensitive enough to detect significant changes in MBT measures other the TTPP. A larger number of infants may show a significant difference in other measures. Furthermore, the present issue is likely to be solved by performing MBT in healthy infants as controls, but owing to the ethical issues, this was not done in the present study. Nevertheless, even only the difference in TTPP suggests that the metabolism of Methacetin between BA and NC patient groups is different and most probably due to low CYP1A2 activity and slower metabolism of methacetin in infants with BA. This may be because of several reasons. First, it may be that advanced fibrosis leads to decreased cytochrome enzyme activity. This was shown by Nakai et al (9), who demonstrated that CYP1A2 expression is decreased as fibrosis increases. The decreased CYP1A2 may also play a role in inflammation, and even in augmentation of proinflammatory cytokine production (11). This may be also the case in BA, in which the inflammatory process has a pivotal role in the disease pathogenesis (35,36). On the contrary, it may be that initial levels of CYP1A2 play a role in the pathogenesis and the natural history of BA. CYP1A2 mRNA content shows up to 40-fold variability between individuals, and may not be of importance in healthy subjects (37). Ratanasavanh et al (33) found no staining of CYP1A2 in fetal livers, and variable staining in infants and older children. There was considerable variation in the protein expression at all ages. Thus, further investigations regarding the role of CYP1A2 are important to elucidate its role in the pathogenesis and progression of BA.
Our cohort is not ideal owing to the multiple diagnoses in the control group, in which a wide range of laboratory results may be found. Nevertheless, this variety actually strengthens the fact that MBT is able to detect infants with high suspicion of BA, as a screening tool, regardless of the wide differential diagnosis. Although our study is small, because of the relatively rarity of NC, the results were significant with no overlap in time to peak between infants with BA and other causes of cholestasis. It may be noted that the test was conducted for 60 minutes, during which most infants with BA did not reach the peak metabolism. It may be that a longer period of testing may be of additional value. In addition, future studies should include normal controls, although for the purpose of only differentiating BA this is not essential.
In summary, the results of this pilot study suggest that an on-line 13C-MBT system may provide a simple tool for timely decision making in infants with NC. Furthermore, we show here that the CYP1A2 may have a role in the pathogenesis of BA. At this point, it should not replace the traditional workup, but it does have additional value in the initial investigation. Further large-scale studies with normal controls are required to confirm our preliminary results and assess the validity of this test.
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biliary atresia; diagnosis; methacetin breath test; neonatal cholestasis
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