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INTRODUCTION
Aspartate aminotransferase (AST) and alanine aminotransferase (ALT) are serum markers of hepatocellular damage in chronic hepatitis, and have been used to determine the initiation of antiviral treatment for chronic hepatitis B (CHB) in some regions.1,2 AST is less specific for liver damage because high levels are found in various types of organ damage, including the liver, heart, and skeletal muscle. AST is also found in red blood cells. Therefore, isolated AST elevations are difficult to interpret in the setting of chronic liver disease if extrahepatic causes are excluded. Macro-AST might be considered in such rare cases, in which increased serum AST activity could be due to immunoglobulin complexes with AST but without significant liver inflammation.
Macroenzymes are enzymes in plasma that form high-molecular-mass complexes, either by self-polymerization or by associating with other plasma components. They are usually complexes of normal enzymes with immunoglobulin. The binding of immunoglobulins to circulating enzymes can elevate activity by reducing inactivation, clearance, or excretion.3 Despite little evidence that macroenzymes indicate the presence of disease, increased plasma enzymatic activity could cause diagnostic confusion, particularly in the setting of underlying liver disease. Therefore, it is meaningful to detect their presence using simple methods in clinical settings. This paper presents two cases of prominent AST elevations relative to the ALT levels, in which the origin of AST was determined to be macro-AST using simple methods, such as polyethylene glycol (PEG) precipitation and the refrigeration storage test.
1. Methods
The plasma of both patients underwent PEG precipitation and refrigerated storage to investigate the cause of the disproportionate elevations in AST.3
1) PEG precipitation test
The patient’s plasma was prepared from blood, and the initial enzymatic activity was measured. In the construction of reference ranges of PEG-precipitable activity (%PPA), the patient’s plasma (200 μL) was added to either 200 μL of PEG 6000 (240 g/L in 9 g/L saline) or 200 μL of saline (9 g/L), held at room temperature for 10 min, and centrifuged at 3,000 ×g for 10 min at room temperature. The enzymatic activity was measured in the supernatant (A1) and saline dilution (A2).
Each PEG precipitation test was repeated twice and three times in Cases 1 and 2, respectively. The control sample paired with Case 1 was measured once, whereas the control samples for Case 2 were measured in triplicate to evaluate the assay reproducibility. The mean of replicate measurements was used for the %PPA calculation, with coefficients of variation (CV) of <2% and <20% in Cases 1 and 2, respectively. These variations were considered acceptable for reproducibility in enzyme activity assays (CV <10% excellent; 10–20% acceptable). %PPA was calculated as [(A₂−A₁)/A ₂]×100, and a value >73% was considered positive for macro-AST.4 The same operator performed all replicate assays using the same analyzer to minimize inter-operator variability.
2) Refrigeration storage test
The AST activity was measured in patients (Cases 1 and 2) and control samples stored at 4°C on days 0, 5, and 7. The control samples were obtained from four individuals (Controls 1–4) without macro-AST, representing different clinical backgrounds: a 21-year-old man with transient chest pain, a 66-year-old woman admitted for breast cancer, a 68-year-old woman with cryptogenic cirrhosis, and a 36-year-old woman with a hematologic malignancy. The AST activity was measured in the patient and control samples stored at 4°C on days 0, 5, and 7 without freeze–thaw cycles. The same operator measured each sample once per time point using the same analyzer. The AST activity in all control samples remained stable during storage, confirming the absence of macro-AST
CASE
1. Case 1
A 48-year-old woman was referred because of persistent elevations in her serum AST levels. She was diagnosed with CHB in her 20s. Since then, she had not experienced acute hepatic inflammation and had not taken antiviral treatment for CHB. Her AST levels were higher than 200 U/L seven years ago. One month earlier, her blood test revealed a hemoglobin level of 12.9 g/dL, AST 281 U/L, ALT 16 U/L, ALP 48 U/L, GGT 7 U/L, albumin 4.3 g/dL, total bilirubin 0.9 mg/dL, positive HBsAg, negative HBeAg, and HBV DNA 3.89×102 IU/mL. Her mother had CHB. The patient was not taking specific drugs or herbal medications. She performed regular exercises three times a week. She did not feel muscle pain or muscle weakness. Her physical examination revealed a height and body weight of 159 cm and 53 kg, respectively. The initial laboratory findings were as follows: AST 310 U/L, ALT 17 U/L, ALP 48 U/L, GGT 9 U/L, albumin 4.6 g/dL, total bilirubin 0.9 mg/dL, PT INR 1.02, creatine kinase 79 U/L, thyroid-stimulating hormone 1.16 mIU/L, free T4 1.16 ng/dL. HBV DNA 389 IU/mL, anti-HCV (-), and IgG 1,658 mg/dL. The FibroScan showed a transient elastography value of 4.9 kPa and a controlled attenuated parameter value of 247 dB/m. Abdominal ultrasonography revealed normal hepatic parenchymal echo and multiple hepatic cysts.
She was assessed as having inactive CHB, no evidence of significant liver fibrosis, and isolated AST elevation without evidence of muscle injury, hemolysis, or hypothyroidism. Therefore, the physician performed a PEG precipitation test and refrigeration storage test to evaluate macro-AST.
The PEG precipitation test was repeated twice, yielding consistent results (%PPA=100%). The mean AST %PPA was 100% and 68.1% in the patient and control, respectively (Table 1). During refrigerated storage, the AST activity decreased from 310 U/L to 74 U/L (−76 %) over seven days, confirming macro-AST. In contrast, the AST levels in the controls revealed similar values over time (Fig. 1). Accordingly, the isolated increase in AST activity in this patient was attributed to macro-AST activity. After one year, the follow-up laboratory findings were AST 196 U/L, ALT 13 U/L, HBV DNA 116 IU/mL, and transient elastography 3.7 kPa.
2. Case 2
A 68-year-old man had several operations for vertebral diseases such as a herniated intervertebral disc, vertebral stenosis, and a compression fracture. He was then administered antibiotics, such as cefazolin and ciprofloxacin, for infectious spondylitis. Before undergoing additional vertebral surgery, he showed an abnormal liver function profile. Therefore, he was referred to the Department of Gastroenterology for an evaluation of the preoperative risk. He had been diagnosed with CHB four months earlier and had not taken an antiviral agent. He had no episodes of severe hepatitis and was a social drinker. His morphology revealed an elevated body mass index of 29. The laboratory findings showed hemoglobin 10.9 g/dL, AST 236 U/L, ALT 20 U/L, ALP 147 U/L, GGT 145 U/L, albumin 2.4 g/dL, total bilirubin 1.0 mg/dL, PT INR 1.5, and creatine kinase 17 U/L. The abdominal pelvic CT presented a mildly atrophic liver with a blunted border and a large amount of visceral fat. A further evaluation for viral hepatitis showed positive HBsAg, negative HBeAg, and positive IgG anti-HBc, and an HBV DNA level of 2.25×105 IU/mL. The FibroScan revealed a transient elastography value of 8.9 kPa and a controlled attenuated parameter value of 317 dB/m, indicative of F3 fibrosis and S2 steatosis. No laboratory findings suggestive of hemolytic anemia were noted. The tissue culture from vertebral material showed Candida parasilosis, so fluconazole treatment was initiated.
Conducting a PEG precipitation test and refrigeration storage test was decided because of the prominent AST-dominant abnormal liver profile. Three replicate assays were performed in the PEG precipitation test. The mean AST %PPA was 46.7±19.3%, below the 73% threshold (Table 2). The mean±standard deviation for the control samples (n=3) was 68.1 ± 0.9%. Refrigerated storage showed only a 5–9% decline in AST over seven days, excluding macro-AST. The observed variability (CV≈19 %) was within the acceptable range for reproducibility in enzymatic assays (Fig. 1). The changes in AST activity were not prominent in either test. Therefore, it was inferred that the AST elevation in this patient was not related to macro-AST and might be suggestive of hepatocellular damage in the immune-active phase of CHB. Accordingly, treatment with tenofovir alafenamide, an anti-HBV agent, was also started based on this result.
After two months of antiviral treatment, the patient's values were AST 28 U/L, ALT 8 U/L, and HBV DNA was not detected.
DISCUSSION
Together with ALT, AST is a serum marker of hepatocellular damage in acute or chronic hepatitis because when hepatocyte cell membranes break down or the permeability increases, intracellular AST can leak into the blood. Therefore, some clinical practice guidelines mention ALT and AST as inflammatory markers to determine the initiation of antiviral treatment in CHB.1,2 Nevertheless, AST is not specific to the liver, and can originate from other sources such as the heart, skeletal muscle, and red blood cells. Therefore, an extrahepatic origin of AST must be investigated when AST is elevated more than ALT in liver function tests. In addition, AST could be higher than ALT because of mitochondrial-origin AST released in patients with alcoholic hepatitis. After excluding extrahepatic causes and alcoholic hepatitis, it is difficult to interpret an AST-dominant liver profile. In this case, macro-AST could be considered.
Macroenzymes are enzymes in plasma that form high-molecular-mass complexes, either by self-polymerization or by associating with other plasma components.5 They have been described for most enzymes routinely measured in the clinical laboratory, and are usually complexes of normal enzymes with immunoglobulin. Immunoglobulin binding to circulating enzymes can lead to increased activity, probably by reduced inactivation, clearance, or excretion. Therefore, exaggerated elevations in specific enzymatic activity by these macroenzymes might cause diagnostic confusion. Macro-AST is a rare macroenzyme, and its elevation might also pose interpretive challenges in the setting of chronic liver disease. Therefore, it is necessary to determine the presence of macro-AST in cases of AST-dominant elevations in liver function tests using easy and efficient methods. Several techniques for detecting macroenzymes involve complex procedures requiring highly specialized chromatography, electrophoresis, or ultracentrifugation equipment, which is not usually available in hospital laboratories. Nevertheless, PEG precipitation and the refrigeration storage test are relatively simple techniques that have been successfully applied to detect macroenzymes.3
The two clinical cases mentioned above are good examples of identifying macro-AST in CHB patients with AST-dominant elevations compared with ALT levels. In Case 1, the patient had persistently elevated AST levels with normal ALT levels for decades, but the HBV DNA level was low. She did not drink, and there was no evidence of heart or skeletal muscle injury or hemolysis. After PEG precipitation and refrigerated storage, the AST activity decreased abruptly, and the HBV DNA level remained low. Therefore, the high AST activity was attributed to macro-AST associated with immunoglobulin rather than hepatocellular damage. In contrast, in Case 2, the patient experienced infectious spondylitis and AST-dominant increases compared to ALT, with a high HBV viral load. The AST activity did not decrease sufficiently with PEG precipitation and refrigerator storage, but decreased after the antiviral treatment for CHB. Therefore, the AST elevation was not due to macro-AST, but the exact origin of high AST activity could not be determined.
Several case reports about macro-AST have been reported (Table 3).6-17 These cases of macro-AST were reported in patients aged 6 to 82 years. Most of them were evaluated when their AST elevation was detected incidentally, with levels 2 to 10 times the upper limit of the normal range. Chung et al.7 reported a case of chronic hepatitis C in which the serumㄷ AST levels were markedly elevated, but the liver biopsy revealed only mild chronic hepatitis, showing a discrepancy that was ultimately explained by macro-AST. Foust et al.8 reported similar AST elevations in patients initially suspected of having chronic hepatitis, in which the liver biopsies revealed steatosis and mild necro-inflammation. Thus, a systematic differential diagnosis of various causes must be performed if a patient with chronic hepatitis shows prominent AST elevation. After excluding damage to the liver, muscles, and erythrocytes, the presence of macro-AST should be considered as one of the causes.
Several methods have been described to confirm macro-AST, such as gel electrophoresis,7 immunoprecipitation with specific antibodies,5 gel filtration chromatography and immunoelectrophoresis,16 as well as PEG precipitation and the refrigeration storage test,3 which were applied in the present cases. Even when specific facilities for PEG precipitation are unavailable, which is the case in most hospitals, the refrigeration storage test can be performed easily. In the present cases, the PEG precipitation and refrigeration storage test could differentiate an AST elevation caused by macro-AST from other causes. Therefore, the refrigeration storage test might be a useful tool for this purpose.
Replicate PEG precipitation assays were performed to confirm the reproducibility of the AST measurements. Case 1 revealed complete AST precipitation in duplicate tests (CV <2%), while Case 2 showed moderate variations (CV≈19%), which is acceptable for reproducibility in enzymatic activity assays. These findings support the reliability of the PEG precipitation method in differentiating macro-AST from a true hepatocellular injury.
In conclusion, macro-AST should be considered in the differential diagnosis of cases with a dominant AST elevation on routine testing, particularly in an evaluation for chronic hepatitis. Physicians can avoid unnecessary invasive assessments of liver disease and effectively and safely manage patients by elucidating this puzzling abnormal liver function profile.
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