Abstract
Macroenzymes are enzymes in plasma that have a higher molecular mass than the corresponding enzyme normally present under (patho) physiological conditions. Macro species have been described for most routinely measured enzymes, but with only a few reports of macro species with aspartate aminotransferase (AST), and in particular very few reports in children and adolescents. Routine biochemical analysis in a 15-year-old girl presenting with lower back pain revealed an isolated raised AST as part of a liver function test profile. Polyethylene glycol precipitation and gel filtration chromatography showed this to be a macro species.
Case report
A 15-year-old girl was referred by her general practitioner (GP) to the Paediatric Outpatient Department with a seven-month history of lower back pain. The pain was not well localized. The pain involved the lower spine with radiation round to both sides and was aching in nature. There was no associated stiffness or restriction of movement. Examination revealed vague tenderness in the lumbar and thoracic vertebrae. There were no gastrointestinal symptoms. She was on Microgynon 30 for heavy and irregular periods.
As part of her investigations requested by her GP, she had a blood sample taken for measurement of her liver function tests (LFTs), assay carried out on Abbott Architect 8200. This showed an isolated raised aspartate aminotransferase (AST) activity (Abbott assay 7D81-20) (Table 1).
Blood sample results
Further analysis on another sample taken one month later showed normal full blood count and film, normal concentrations of serum sodium, potassium, urea and creatinine, and normal activities of creatine kinase (CK), lactate dehydrogenase (LDH) and amylase but with the isolated raised AST persisting at 342 U/L (calculated globulins = 30 g/L).
At this point, the anonymized case was offered for discussion on the Association for Clinical Biochemistry Mailbase Discussion List (
The sample showed that 99% of AST activity (normal 25–60%) was precipitated with polyethylene glycol (PEG, final concentration 125 g/L) indicating the presence of macro-AST.
The presence of macro-AST was confirmed by gel filtration chromatography which showed a peak of macro-AST contributing > 95% of the total activity, eluting much earlier than AST in a control serum (Figure 1). Gel filtration chromatography was carried out with a 1.6 × 40 cm column of Sephacryl S-300 (Pharmacia). The column was applied with 0.5 mL serum and eluted with Tris/saline buffer pH 7.4 at a flow rate of 0.5 mL/min and 25 fractions of approximately 1.5 mL were collected. AST activity and albumin concentrations in the fractions were measured using the standard Roche assays on the Modular P unit, but in the case of albumin a larger sample volume was used to compensate for the lower concentrations found in the fractions. In this chromatography system, normal serum AST elutes in the same fractions as serum albumin while macro-AST elutes earlier due to its increased molecular mass. The peak of albumin serves as a convenient marker of molecular mass.
Gel filtration results showing aspartate aminotransferase (AST) activity in patient's serum in fractions of eluent compared to AST control. AST activity normally elutes with albumin
Discussion
The phenomenon of macroenzymes was first recognized in 1964. 1 Macroenzymes are enzymes in plasma that have a higher molecular mass than the corresponding enzyme normally present under (patho) physiological conditions. They may arise through self-polymerization or by association with other plasma components, in particular immunoglobulins. 2 Macro species have been described for most routinely measured enzymes, in particular for amylase and creatine kinase with only a few reports of macro species with AST, the first case of macro-AST being reported in 1978. 3,4 Macro species have been less well recognized in children than in adults with fewer than 10 children and adolescents previously reported with macro-AST. 5
AST is a widely distributed enzyme in the body and can be elevated in disease of liver, muscle, kidney and red cells. The normal alanine aminotransferase (ALT), CK, urea and electrolytes (U + E) and renal ultrasound, LDH and full blood count and blood film made the source of the elevated AST in this patient unlikely to be from these areas. The use of PEG as a simple and effective test for the detection of macroenzymes has previously been described in the detection of macro-AST and other macroenzyme species. 6
The presence of a macroenzyme does not appear to have pathological significance in itself, but may be associated with some diseases. IgA-complexed AST has been found to be associated with liver malignancies but as a minor component of the total AST activity. 7 When macro-AST has been described as the major component and the cause of an isolated elevated serum AST activity, as in the case we present here, it has been found to be an AST–IgG complex. 3,7,8 The reason for the formation of such complexes remains unclear, although an autoimmune phenomenon has been suggested. 8
This case illustrates the need to consider a macroenzyme species as a cause of a persistent elevated AST to avoid unnecessary and costly investigation in these patients. In the case of this patient the clinician concerned discussed the AST result with the laboratory prior to any further investigations.
AST is a widely distributed enzyme in the body and hence investigations need to first be directed at excluding these common sources prior to the investigations for a possible macro species. LFTs including ALT measurement to check for a liver source, CK to check for a muscle source and LDH to check for a red cell source should be carried out.
The reason for the fall in AST from the initial value of 407 U/L to 342 U/L was not clear (the only other biochemical change in that period was a fall in her calculated globulins of 3 g/L). The patient has improved symptomatically. Nine months following her initial presentation her isolated raised AST activity persists at 340 U/L (calculated globulins = 29 g/L). The patient declined to have further blood samples taken to monitor the AST activity and to further characterize the nature of the macroenzyme.
Finally, although AST and ALT remain part of the LFT profile offered by many laboratories in the UK, it has been argued recently in a draft proposal by the Scientific Committee of the Association for Clinical Biochemistry that AST should be removed from the profile. 9 The problems caused by macro-AST may be a further argument for this change in practice since macro-ALT seems to be much less common. 2