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Fructose 1,6-bisphosphatase as a marker of
gene, have been described. Affected individuals suffer hepatocellular damage in liver transplantation
from prolonged apnoea when given an otherwise safe J Greenwood, S Reddy, P J Friend, R P Taylor dose of a muscle relaxant. The phenotypic assay is lim- Department of Clinical Biochemistry, John Radcliffe Hospital, Oxford ited in that it cannot reliably distinguish between certain mutations, especially where multiple mutations are pres- Aspartate amino transferase (AST) and alanine amino transferase (ALT) are used as markers of hepatocellular DNA sequencing of two regions of the BCHE gene has damage in liver transplantation but are insensitive to been developed to detect the clinically significant K- and acute changes. When cellular damage does occur levels atypical variants. The method was then used to genotype remain elevated and poorly reflect further damage.
24 patients that had originally had a phenotype assigned Fructose 1,6-bisphosphatase (FBPase) is a key enzyme in based on current methodology. Fourteen of gluconeogenesis and due to its location in the cystol of patients were shown to have more than one mutation the periportal liver cells it has been proposed as an alter- present and five of these patients were reclassified as native marker of hepatocellular damage. We evaluated having potential sensitivity as a result. Two patients with FBPase against conventional liver enzymes in an experi- queried U/AK or A/K phenotypes were shown to be AK/K genotypes. The AK/K genotype is associated with Porcine donor livers were subjected to various periods increased sensitivity to muscle relaxants.
of cold ischaemic injury, group I (n=4) 4 hours, group The newly developed genotyping assay will improve II (n=6) 1 hour and group III (n=4) 0 hours, post donor the current classification, which is based on phenotypic hepatectomy and prior to preservation by normothermic data alone and will add information about multiple extracorporeal sanguineous machine perfusion. FBPase, mutations. Interpreting the genetic background in con- AST and ALT levels were measured in perfusate samples junction with measurements of the actual enzyme activ- taken from the circuit during machine preservation.
ity status will enhance the service providing a more FBPase was analysed enzymatically by monitoring NADPH production at 340 nm as fructose 1,6-bisphos-phate is converted to fructose 6-phosphate. AST and ALTwere analysed using conventional methods.
Prednisolone measurement by liquid
FBPase levels began to increase slowly after reperfu- chromatography: tandem mass spectrometry
sion in all three groups, with the lowest levels being seen in group III which remained below 100 U/L. FBPase in Department of Clinical Biochemistry, Wythenshawe Hospital, groups I and II rose to around 150 U/L at 6 hrs preser- South Manchester University NHS Trust, Southmoor Road, Manchester vation. A similar pattern was seen for both AST and ALT. However, at 8 hrs preservation the FBPase levels Prednisolone is a corticosteroid that is often used for increased in group I to 550 U/L and 750 U/L at 12 and long term suppression of the immune system; for exam- 20 hrs preservation respectively. This was not reflected ple in asthmatics and transplant recipients. With severe in the AST and ALT results which both increased slow- side effects including osteoporosis, diabetes mellitus and ly. The increase in FBPase may be related to graft failure, adrenal suppression it is appreciated that the dose should shown by a decline in bile production, as FBPase rose be kept as low as possible. The monitoring of pred- some hours after bile production fell. Therefore FBPase nisolone would allow the physician to tailor the dose to has the potential to be a sensitive and useful marker of individuals requirements, assess compliance and absorp- tion and improve the outcome of these patients. We havedeveloped a liquid chromatography-tandem mass spec-trometry (LC-MS/MS) assay for the measurement of Development of a genotyping service for the
identification of butyrylcholinesterase variants
Samples (500 µL) and deuterated (d6) prednisolone D Grimberg, S Keeney, A J Pickersgill, M W France, A Cumming (internal standard) were extracted using Waters Oasis¨ Department of Clinical Biochemistry, Manchester Royal Infirmary, HLB columns, the methanol eluant was dried down and the extracted prednisolone was reconstituted in 50:50 The enzyme butyrylcholinesterase (BChE) is essential in mobile phases. The samples were then transferred into a terminating the effect of muscle relaxants, such as 96-deep well microtitre plate, of which 20 µL was inject- suxamethonium. Over 20 mutations, resulting in a ed into the LC-MS/MS system. A Waters Atlantis¨ decrease or loss of enzyme activity within the BCHE column (3.0 mm x 50 mm) was eluted with a step gradient of 50% to 95% methanol containing 2 mmol/L hydrophobic sorting region (residues 70-103) that ammonium acetate and 0.1% (v/v) formic acid, at 0.5 directs the protein to the inter-membrane space. Here we mL/min. The column was operated at ambient tempera- have shown that the residues 1-69 contain all the infor- mation necessary to target YFP to the mitochondria. We The retention times were 2.75 min for prednisolone are currently investigating the role of the hydrophobic and 2.72 min for d6 prednisolone. Cycle time was 5 min.
region in localising CPO to the inter-membrane space.
The transitions used were m/z 361.3>147.1 for pred- This and other findings will help identify some of the nisolone and m/z 367.2>150.3 for d6 prednisolone; molecular defects causing Hereditary Coproporhyria monitored using a Quattro micromass spectrometer. Thebetween-batch precision of the method was 8%, 4% and5% at concentrations of 75 µg/L, 375 µg/L and 750 µg/L Exonic deletions in an 8-year-old boy with
respectively. The within batch precision was <8% for the erythropoietic protoporphyria
same concentrations. The lower limit of detection was 25 µg/L and the assay was liner to 4000 µg/L. There was Department of Medical Biochemistry and Immunology, University negligible suppression of ionisation.
Hospital of Wales, Heath Park, Cardiff CF14 4XW We have developed a robust assay for the measure- Erythropoietic protoporphyria (EPP) normally presents ment of prednisolone. This should allow easy monitoring in childhood with cutaneous photosensitivity caused by of treatment in many patient groups, optimising pred- the build up of protoporphryin in the skin as a result of deficient ferrochelatase activity. Evidence suggests thatclinical expression of classical EPP requires the coinher-itance of a severe ferrochelatase mutation, in trans to Identifying the sequence elements important
the commonly occurring polymorphism, IVS3-48C, for mitochondrial targeting of
which is associated with low enzyme activity.
coproporphyrinogen oxidase
An 8-year-old boy presented with photosensitivity. A diagnosis of EPP was made following demonstration of D Van Der Merwe, A Roberts, M N Badminton Department of Medical Biochemistry and Immunology, University increased concentrations of protoporphyrin in erythro- Hospital of Wales, Heathpark, Cardiff CF14 4XW cytes and plasma. A referral was made for mutation Hereditary Coproporphyria (HCP) is an autosomal dom- detection with a view to genetic counselling.
All eleven exons and flanking intronic regions of the Coproporphyrinogen Oxidase (CPO) activity. CPO is the ferrochelatase gene were analysed by bi-directional sixth enzyme of the haem biosynthetic pathway and is sequencing but no mutation was identified. Samples located within the inter-membrane space of mitochon- from the proband and parents were analysed for the IVS3-48C low expression polymorphism. The haplotypes phyrinogen III to protoporphyrinogen IX. CPO is nuclear of the parents were inconsistent with the probandÕs sam- encoded and synthesised on cytosolic ribosomes as a pre- ple, suggesting the inheritance of a partial gene deletion protein that contains a presequence of 110 amino acids at the amino terminus. The aim of this study was to Deletion studies were undertaken using gene dosage identify the sequence elements necessary to target CPO analysis. Exons 2-11 of the ferrochelatase gene were to mitochondria. We have fused human CPOs containing amplified by PCR in a multiplex reaction, with incorpo- N-terminal and C-terminal deletions, to the amino ter- ration of a fluorescent label and subsequent analysis by minus of yellow fluorescent protein (YFP) and have used gene scanning. Gene dosage was determined by compar- these constructs to investigate the mitochondrial import ison with internal controls. Deletion of exons 3 and 4 in of CPO in human cells. Constructs were transfected by the father and the proband was identified by this lipofection into HeLa cells and their cellular location method. These results explain the clinical expression of imaged by fluorescence and confocal microscopy.
EPP in the proband who, in addition to the deletion, car- Inspection of the CPO presequence predicts a bipartite ries the low expression polymorphism. This is in contrast structure with dual targeting and sorting information: a to the father who, although carrying the deletion, is matrix-targeting signal consisting of a positively charged unaffected due to the absence of the low expression poly- region (residues 1-69) followed by an extended

Source: http://www.focus-acb.org/2004/pdf-downloads/3-bayer-award.pdf

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