Isolated elevation of aspartate aminotransferase (AST) in an asymptomatic patient due to macro‐AST

Abstract

Background

A rare and benign cause of isolated aspartate aminotransferase (AST) increase is due to the presence of macro aspartate aminotransferase (macro‐AST). Macro‐AST is not usually considered by clinicians, leading to a large number of unnecessary and even invasive tests before diagnosed. Most commonly used laboratory methods for detection of macro‐enzymes are precipitation with polyethylene glycol (PEG), ultracentrifugation and gel filtration chromatography (GFC).

Methods

We report a case of an asymptomatic patient with persistent isolated AST elevation. PEG precipitation and an alternative method based on the low stability of this macro‐enzyme were performed on the patient serum.

Results

Macro‐AST was early detected by the laboratory observing a significant decrease in AST concentrations when the serum is stored at 4ºC.

Conclusions

Macro‐AST detection based in this method can be very useful as screening, especially in those laboratories where other technologies are not available. Macro‐AST must be considered as cause of unexplained isolated AST elevations before prior investigations to avoid the consequent cost and potential harm to the patient. In our case, early detection of macro‐AST with this method avoided unnecessary treatment and invasive test such as liver biopsy.

1. INTRODUCTION

The elevation of transaminases, alanine aminotransferase (ALT) and aspartate aminotransferase (AST), is usually associated with liver injury but it can also be found in cardiac, renal or muscular diseases, drug or alcohol consumption and haemolytic anaemia.

Isolated elevation of AST without symptoms or any alterations is relatively uncommon in healthy population. A rarely reported and benign cause of AST increase is due to the presence of macro aspartate aminotransferase (macro‐AST).1 Macro‐AST is a high molecular mass macro‐enzyme produced by the formation of complexes with immunoglobulins (mainly Ig G and Ig M) or other plasma components.2 Being a rare condition, it is not usually considered by clinicians, leading to a large number of unnecessary and even invasive tests before diagnosed.3 Laboratory methods for detection of macro‐enzymes most commonly used are precipitation with polyethylene glycol (PEG), ultracentrifugation and gel filtration chromatography (GFC).4

We present a case of an asymptomatic patient with persistent isolated elevation of AST in which early detection of the presence of macro‐AST based on the low stability of this macro‐enzyme avoided the realisation of further studies.

2. CASE DESCRIPTION

A 50‐year‐old female patient referred to our hospital due to persistent AST elevation in range of 368‐532 U/L (reference range: 10‐50) over the past year. Patient was asymptomatic with normal physical examination. There was no relevant medical or family history and she denied any previous drugs or alcohol consumption.

Initial laboratory results showed an elevation of AST serum activity (494 U/L) with the rest of liver function tests and enzymes in normal range: ALT 28 U/L (7‐56), gamma glutamyl transpeptidase 14 U/L (9‐36), creatine kinase 146 U/L (0‐190), lactate dehydrogenase 179 U/L (0‐248), amylase 45 U/L (20‐120), total protein 7.2 g/dL (6.6‐8.3), albumin 4.1 g/dL (3.5‐5.2), bilirubin 1.02 mg/dL (0.3‐1.2) and alkaline phosphatase 92 U/L( 43‐115). Viral serological markers for hepatitis, Cytomegalovirus and Epstein‐Barr virus were negative. Abdominal ultrasonography showed a liver image of normal size and contours. Further investigations for autoimmune hepatitis, Wilson's disease, hemochromatosis, myopathies, celiac and thyroid diseases were normal.

Due to the absence of symptoms and the negativity of the tests, clinicians consulted the case with the laboratory and we suggested to considerate the presence of macro‐AST as a possible cause for this increase.

As we did not have in our hospital at that time methods for detection of macro enzymes, we sent an aliquot of the serum sample to an external laboratory for identification of macro‐AST by chromatographic technique (GFC) with negative results for macro‐AST complexes and quantification of AST in normal range (37 U/L), when our previous result on the same sample was 494 U/L.

Despite the negative result, having no explanation for the decrease in AST levels, we conducted an extensive review of literature and it raised our initial suspicion of the presence of macro‐AST since five days had passed from the moment of extraction until the next analysis. Some authors have reported the low stability of this form of the enzyme, with greater degradation when the sample is refrigerated.2

We decided to confirm the presence of macro‐AST in our hospital laboratory by measuring if AST activity decreases in time with the sample stored at 4ºC and by PEG precipitation. For both studies we requested a new sample from the patient and used five serum from other patients with AST elevated above 100 U/L as controls.

2.1. Method 1

AST activity in serum and five control samples were measured every 24 hours for seven consecutive days to evaluate the degradation of the enzyme while the sample and controls were stored at 4ºC. Quantitative determination of AST was performed by kinetic UV method with pyridoxal phosphate according to International Federation of Clinical Chemistry (IFCC) recommendations with AU400 analyser (Beckman Coulter Inc., Brea, CA, USA).

The results are shown in Figure 1, in the patient sample we observed a continuous decrease in AST activity. One week after extraction, AST serum levels have decreased in 88.1% while in all the controls studied have remained stable and not varied in more than 1%.

Figure 1.

Refrigerated serum. AST in patient sample decreased from 469 U/L on day one to 55.7 on day 7 while in the control samples AST remained on the same levels

2.2. Method 2

Precipitation with PEG was carried out following the procedure described by Patteet et al modified by shortening the centrifugation time to 10 minutes at 3000 rpm as similar results are obtained.4 Quantitative determination of AST was performed in the supernatant (AST PEG). We also diluted the original serum at half with physiological saline solution and performed AST determination (AST DIL) for the calculations.

The results (Table 1) are expressed as percentage of AST recovered after precipitation (AST Recovery %) using the formula 100 × [AST PEG/AST DIL] and percentage of PEG Precipitation activity (PPA), PPA = 100 × [(AST DIL − AST PEG)/AST DIL].

Table 1.

Precipitation with PEG

	AST (U/L) Original serum	AST (U/L) Frozen serum	AST DIL ½ (U/L) Original serum	AST DIL ½ (U/L) Frozen serum	AST PEG (U/L) Original serum	AST PEG (U/L) Frozen serum	AST RECOVERY (%) Original serum	AST RECOVERY (%) Frozen serum	%PPA Original serum	%PPA Frozen serum
Patient	469	371.6	236	169.6	4.4	2.9	1.86	1.71	98.14	98.29
Control 1	152	151.6	76.1	75.8	55.1	54.8	72.40	72.30	27.59	27.70
Control 2	218.1	217.2	108.6	107.9	62.9	65.9	57.92	61.08	42.08	38.93
Control 3	198.2	198.6	99.8	99.2	65.3	62.3	65.43	62.80	34.57	37.20
Control 4	354.4	355.9	178.2	177.4	91.3	90.4	51.23	50.96	48.77	49.04
Control 5	258.9	259.4	128.3	126.2	84.5	81.6	65.86	64.66	34.14	35.34

In the patient serum we obtained an AST activity of 469 U/L, after precipitation with PEG, AST activity decreased to 4.4 U/L, AST RECOVERY was 1.86% (considered positive <40%) and PPA 98.1% ( considered positive >73%) while in all the control samples AST RECOVERY was above 51% (range 51.2‐72.4) and %PPA below 48% (range 27.6%‐48.8%).

To evaluate the stability of the sample for PEG precipitation method we stored a frozen aliquot of the patient serum and five control samples at −20ºC for 7 days repeating the procedure with these results: (Table 1) In the patient sample, AST activity was 371.6 U/L (AST recovery 1.71%, %PPA 98.3), AST after precipitation with PEG decreased to 2.9 U/L. In the control samples % PPA was below 49% (range of 27.7‐49.0) and the AST recovery above 50% (range 50.9%‐72.3%).

Based on these results, we concluded the presence of macro‐AST as the most probable cause for AST increased levels and no further tests or treatment where taken on the patient. She continued asymptomatic for the past year follow up controls with AST levels elevated in same range.

3. DISCUSSION

Macro‐AST is a rare phenomenon of unknown pathogenesis with only a few cases reported in literature, prevalence seems to be lower than other form of macro‐enzymes like creatin kinase, lactate dehydrogenase or amylase.5 The presence of macro‐AST as other macro‐enzymes is generally considered a benign process, however long‐term effects on the patient or association with other pathologies are still unknown.6

There are multiple methods for detection of macro‐enzymes, the most common are by precipitating the complexes of macro‐enzymes with PEG, ultracentrifugation and gel filtration chromatography, which is considered the standard reference method but not available in most laboratories, due to its complexity and elevated price.7

Precipitation with PEG is an economic and simple method used by many laboratories for screening of different form of macro‐enzymes although it has some limitations such as low specificity, the need of confirmation by other methodology and the lack of consensus to establish reference ranges.8 Results can be expressed by AST levels decrease, AST recovery after precipitation and most commonly by the PEG precipitable activity (% PPA).4 Several authors suggest a PPA cut‐off point of 73% while others suggest using a range from 67.1% to 82.2%.9 Samples above these intervals should be considered as high probability presence of macro‐AST. In our patient, PEG precipitation confirmed the presence of macro‐AST with 98.1% PPA.

Davidson et al in 2003 described for the first time the fast degradation of this form of the enzyme when stored at 4ºC, a few years later, Castiella et al suggested that the use of this characteristic on a macro‐AST screening method could be useful to laboratories that do not have PEG or more complex methodologies, even know that the low frequency of macro‐AST makes it very hard to reproduced it in a larger population. Both the authors found that in the presence of macro‐AST, when the sample was stored at 4ºC for one week, there was a decrease in AST levels above 90%. In the absence of macro‐AST, AST in serum is stable for four weeks when stored at 4°C and up to 3 months at ≤20°C10, 11 therefore a significant decrease in AST concentrations will point to a macro‐AST.

Physiopathology mechanism for this lower stability in some forms of macro‐AST remains unclear although some authors suggested it could be related to the gradual precipitation of enzyme complexes.8

This method in our patient showed a decrease of 46% in AST levels after 24 hours, 72% after 48 hours and 88.1% after 1 week. In the control samples used, the decrease after one week was lower than 0.72%.

Due to the low prevalence of macro‐AST and the lack of studies, it is not possible to confirm the validity of this method. An extensive review of the literature showed that all cases previously reported coincide with the instability of the enzyme described by Davidson except for two cases reported in 2010 by Chtioui et al and Maan et al in 2017, with no significant decrease in AST levels after one week stored at 4ºC that these authors suggested could be due to the heterogeneity of the complexes of the macro‐AST.

Although is not a well validated method, being so simple and low cost, it could be very useful as screening, especially in those laboratories where other technologies are not available.12 In case of suspicion of macro‐AST, a 65% or higher decrease of AST levels after 48 hours stored at 4ºC, or even equal or higher than 40% after 24 hours should be considered as highly suggestive for macro‐AST. A negative result, however , would require confirmation by other methods.

For storage conditions of the sample, we recommend freezing at ≤20°C to reduce the degradation of the enzyme. In our patient, we froze the serum for one week and quantificated AST levels after PEG precipitation with %PPA and AST recovery like previous results.

This case reminds us that macro‐AST should be considered by clinicians and laboratory as cause of unexplained isolated AST elevations before carrying out additional tests to avoid the consequent cost and potential harm to the patient. In our case for example, before concluding with the presence of macro‐AST, a liver biopsy was scheduled.

ETHICAL APPROVAL

Written consent was obtained from the patient.

CONFLICT OF INTEREST

The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

AUTHORS’ CONTRIBUTIONS

AGR researched literature, conceived the study and wrote the first draft of the manuscript. All authors reviewed and edited the manuscript and approved the final version of the manuscript

González Raya A, Coca Zúñiga R, Martín Salido E. Isolated elevation of aspartate aminotransferase (AST) in an asymptomatic patient due to macro‐AST. J Clin Lab Anal. 2019;33:e22690 10.1002/jcla.22690