Case Report

A case of aspartate aminotransferase macroenzyme

J Cabrera-Abreu1, R Jain2, P Robinson1, S Edees2 and T Staughton1
Department of Clinical Biochemistry; 2Department of Paediatrics, Royal Berkshire Hospital, Craven Road, Reading RG1 5AN, UK Corresponding author: Dr J Cabrera-Abreu. Email: jose.cabrera-abreu@royalberkshire.nhs.uk
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Abstract
Aspartate aminotransferase (AST) macroenzyme can result in elevated AST activity in patients with no disease. This case report describes a six-year-old boy who presented to his family doctor with a history of chronic constipation and lower back pain. Routine blood tests showed normal creatine kinase activity (CK), normal alanine aminotransferase activity (ALT) but raised AST. The patient was referred to a hospital paediatrician for further investigation of the abnormal AST and back pain. The raised AST was conrmed as the only biochemical abnormality. Further investigation with polyethylene glycol, followed by measurement of AST in the supernatant, showed undetectable enzyme activity. The sample was sent to a specialist laboratory where it was analysed by Sephacryl S300 gel ltration. This procedure conrmed the presence of a high molecular mass form of AST. AST macroenzyme should be considered as a cause of isolated AST increase, which may avoid further costly investigations. Ann Clin Biochem 2008; 45: 320 322. DOI: 10.1258/acb.2007.007063

Case study
A six-year-old boy of mixed race, 50% Caucasian and 50% Afro-Caribbean, presented with a six-month history of mid back pain to his family doctor. The pain occurred in the morning and in the evening, but did not limit his normal activities. The child was otherwise healthy with a normal appetite. His current medication is lactulose and sodium picosulphate. Past medical history showed that the patient had been investigated for an asymmetrical skull at the age of 10 months ( plagiocephaly). He also suffers from recurrent constipation and had an episode of rectal bleeding due to an anal ssure when he was three years old. On examination he looked thin, with no evidence of kyphosis or scoliosis and no pain in his spine with a full range of movements in back and legs. Thoracolumbar spine X-ray showed no abnormality. Baseline laboratory investigations showed that the only result outside the age-related reference ranges was aspartate aminotransferase (AST, which is offered as part of our initial patient assessment, sample 1, Table 1). Urine culture showed ,10,000 cfu/mL. The biochemist suggested that the raised AST might be exercise-induced and recommended repeat testing with creatine kinase (CK). The patient was seen again by his family doctor 12 days later still complaining of back pain. The patients mother stated that there had not been any recent fall, muscular injury or overexertion. Laboratory tests analysed on that day conrmed the presence of a raised AST (sample 2, Table 1). The patient was referred by the family doctor to the General Paediatrics Outpatient Clinic. His back pain
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persisted. Cardiovascular, respiratory and abdominal examination were normal. Spinal examination revealed no tenderness. Further laboratory tests showed a similar pattern to the previous ones (sample 3, Table 1). At that time, the differential diagnosis included myositis secondary to a Mycoplasma pneumoniae or a viral infection, but the clinical history was not suggestive of it. The possibility of macro-AST was suggested by the biochemist. Treatment of serum sample 3 with polyethylene glycol, followed by AST measurement in the supernatant, showed undetectable activity. As this procedure was not standardized, the sample was sent to another laboratory where it underwent Sephacryl S300 gel ltration (Pharmacia Fine Chemicals Co, Uppsala, Sweden). This technique conrmed the presence of a high molecular mass form of AST. The immunoglobulin class associated with the AST was not identied. The patient was nally referred to the local Orthopaedics Specialist for consultation regarding his back pain. An MRI scan of his lumbar spine was performed which showed no abnormalities.

Discussion
When trying to make the differential diagnosis of a raised liver enzyme in a child, the clinician should be aware that the reference ranges for gamma glutamyl transferase (GGT) and alkaline phosphatase are statistically different from those of adults. In the case of AST, although age-related reference range differences have been reported, it is unclear whether they are statistically signicant: adult

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Table 1 A case of macro aspartate aminotransferase (AST) isoenzyme AST (U/L) Reference range Sample 1 (3 March 2006) Sample 2 (15 March 2006) Sample 3 (11 April 2006) ,46 113 97 100 ALT (U/L) ,56 21 17 19 Alkaline phosphatase (U/L) 129 386 184 175 159 Total bilirubin mmol/L 3 22 4 2 2 CK (U/L) ,374 233 118 215

AST, aspartate aminotransferase; CK, creatine kinase; ALT, alanine aminotransferase

(45 U/L); ages 1 to 3 (60 U/L), 5 to 6 (50 U/L), 7 to 9 (40 U/L) and 10 to 11 (60 U/L).1 In our patient, all AST readings were above the age-related reference range. The effect of race on CK and AST enzyme activities should also be considered. It is well known that the upper limit of the reference range for CK in Afro-Caribbeans is approximately 1.8 times higher than in Caucasians. However, this difference has not been described for AST. Pathological conditions with raised AST are usually classied into hepatic, muscular (cardiac or skeletal) or red blood cell origin. Normality of the other main liver function tests in our patient made the possibility of a liver origin extremely unlikely. The possibility of muscle exercise as the reason for the raised AST was initially suggested as children of this age are usually very active. The normal CK activity was not suggestive of this possibility but it did not rule out a muscular source completely as CK has a wide interindividual variation (CV, coefcient of variation 67%).2 This means that CK can be elevated above the patients usual activity but still be within the population reference range. To rule out this possibility, repetition of liver function tests and CK was recommended. Another reported cause of raised AST and CK in paediatrics is myositis secondary to mycoplasma pneumonia or viral infection (benign acute childhood myositis).3 However, myositis usually follows an acute upper respiratory infection and there was no evidence that this had occurred. Red blood cell origin was considered to be unlikely as the clinical history, full blood count and normal bilirubin concentration were not suggestive of haemolytic anaemia. AST macroenzyme, also called macro-AST, has previously been described.4 6 This is usually a complex formed between AST and immunoglobulins G or A which has a longer half-life than non-complexed AST and, as a consequence of this, gives rise to an elevated AST activity (5 15-fold).4 The condition is rare and similar to other macroenzyme varieties such as macro-CK and macroamylase in the way that they do not appear to be associated with specic diseases.4 Thus, Fortunato et al. 4 described a group of 10 children with asymptomatic and persistently increased concentration of AST, four of which were due to macro-AST. These four patients were thoroughly investigated for liver, muscle and red-cell diseases but no

abnormalities were found. Liver function tests were monitored at 0.5 1-year intervals for a period ranging from 0.5 to 6.5 years and remained normal except for a persistently raised AST. The children remained in a healthy state. More recently, a macro-AST case on a 3.5-year-old child was reported by Wiltshire et al. 5 Reports of macro-AST on adults do not show the same ndings as in children. Thus, Sturk et al. 6 reported the presence of macro-AST reported on a group of seven healthy adults and in seven adult patients with a variety of diseases such as lung cancer with liver metastases, immunoblastic lymphadenopathy, epigastric distress, acute hepatitis, chronic active hepatitis, colitis and uterine bleeding. Moriyama reported on a group of 34 adult patients, most of which had AST Immunoglobulin A complexes and hepatological malignancies or chronic liver disease. Thus, it has been strongly suggested that the immunoglobulin-A complexed-AST is frequently found in association with liver malignancies.7 A relationship between macro-LDH and autoimmune disease has been detected but not been rmly established.5 No such relationship has been found for macro-AST. Sephacryl S300 gel ltration has been described as a useful method for the investigation of macroenzymes.8 In this technique, the high molecular weight form of AST elutes earlier than the usual AST fraction. This technique does not identify the immunoglobulin class associated with the AST. Incubation of serum with antihuman antiserum against the specic immunoglobulin followed by precipitation has been used to identify the immunoglobulin type.8 Other methods that have proved to be useful in the screening of isoenzymes include polyethylene glycol precipitation and isoenzyme electrophoresis.9 Detection of macro-AST has become even less frequent following discontinuation of AST as a test in the panel of routine liver function in the majority of UK biochemistry laboratories. However, AST is still offered on request for the investigation of haemolytic and muscular disorders. Paediatricians and other clinicians should be suspicious of the existence of macroenzymes when they come across patients with an isolated increase in a single enzyme that does not t the clinical condition.

ACKNOWLEDGEMENT

We thank Mr M. Fahie-Wilson, Consultant Biochemist, Biochemistry Department, Southend Hospital, Essex, for the Sephacryl S300 gel ltration analysis and interpretation of Macro-AST.

REFERENCES 1 Soldin S, Brugnara C, Gunter K, Hicks J, eds. Pediatric Reference Ranges. Washington, D C: AACC Press, 1997:1 181 2 Fraser CG. Biological Variation. Interpretation of Clinical Chemistry Laboratory Data. 1st edn. Oxford: Blackwell Scientic Publications, 1986:111 35 3 Belardi C, Roberge R, Kelly M, Serbin S. Myalgia cruris epidemica (benign acute childhood myositis) associated with a Mycoplasma pneumonia infection. Ann Emerg Med 1987;16:579 81

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4 Wiltshire E, Crooke M, Grimwood K. Macro-AST: a benign cause of persistently elevated aspartate aminotransferase. J Pediatr Child Health 2004;40:642 3 5 Fortunato G, Iorio R, Esposito P, Lofrano MM, Vegnente A, Vajro P. Macroenzyme investigation and monitoring in children with persistent increase of aspartate aminotransferase of unexplained origin. J Pediatr 1998;133:286 9 6 Sturk A, Sanders GT. Macro enzymes: prevalence, composition, detection and clinical relevance.J Clin Chem Clin Biochem 1990;28:65 81 7 Moriyama T, Nobuoka M, Makino M. Incidence and properties of aspartate aminotransferase-immunoglobulin complexes in patients with a

high serum aspartate to alanine aminotransferase ratio. Clin Chim Acta 1990;190:47 56 8 OKane M, Telford M, Bell P. Immunoglobulin-AST complex. Ann Clin Biochem 1990;27:82 4 9 Davidson D, Watson J. Macroenzyme detection by polyethylene glycol precipitation. Ann Clin Biochem 2003;40:514 20

(Accepted 19 July 2007)