Group members
Peter Berman	Chemical Pathologist	Tel 021-406-6392	Room 6.36
Ingrid Baumgarten	Medical Technologist	Tel 021-406-6102	Room 6.36

We have established a number of in vitro assays for the biochemical confirmation of certain inherited metabolic diseases. Each has been prompted by a clinician who sees children presenting with symptoms and signs suggestive of a particular disorder, and who requires biochemical confirmation before instigating specific treatment and/or providing families with prognostic information regarding both the affected child and future pregnancies. For example, the renal unit at Red Cross Children’s Hospital sees children presenting with progressive renal (kidney) failure, associated with accumulation of calcium oxalate crystals. One possible cause for this is an inherited disorder of oxalate metabolism termed ‘Primary Hyperoxaluria type I’. The only effective longterm treatment for this disorder is combined liver and kidney transplant; since the liver is the only organ in which the affected enzyme is expressed, unless the liver is transplanted as well, the new kidney will suffer the same fate as the original. Before embarking on such heroic surgery (such as transplanting an apparently normal liver), it is mandatory that the diagnosis be established with certainty. This is where measuring activity of the affected enzyme in liver biopsy material is so useful. Low activity confirms the diagnosis, and is independent of the specific causative mutation, which would affect a DNA-based diagnostic test.

The source of the material from which the affected enzyme is expressed varies; in the above example it is a small fragment of liver, obtained by biopsy or at post-mortem, and frozen immediately and kept at -80°C to ensure stability. However, if the enzyme is expressed in other cells, one is able to avoid invasive liver biopsies. Examples of other enzyme sources include:

● skin fibroblasts, grown from skin biopsies

● lymphoblasts, derived from whole blood by transformation of circulating lymphocytes with Epstein-Barr virus

● red blood cells, directly obtained from whole blood

Below is a comprehensive list of enzyme assays which we have established in this laboratory, generally at the instigation of a referring clinician who is faced with a diagnostic problem. It includes examples in which different tissues, cells or fluid serve as source of enzyme activity.

● Alanine glyoxalate transferase assay for Primary Hyperoxaluria type I. Performed on liver biopy/autopsy. Urine oxalate measurement also offered as a screening test for the same condition.

● Fatty acid oxidation assay for fatty acid oxidation defects. Performed on skin fibroblasts.

● Glucose-6-phosphatase assay for Glycogen Storage Disease type I (von Gierke’s Disease). Perfomed on liver biopsy/autopsy.

● Cytochrome c oxidase assay for defects in mitochondrial electron transport complex IV (the most common site for defects in electron transport). Performed on skin fibroblasts.

● β-ketothiolase assay for normoglycaemic (i.e. non-diabetic) ketoacidosis in childhood. Performed on skin fibroblasts.

● Succinyl CoA-3-oxoacid-CoA-transferase, performed on the same tissue and for the same indications as β-ketothiolase above.

● Glutamate dehydrogenase for hyperammonaemic hypoglycaemia of childhood. Performed on transformed lymphoblasts.  

● Pyruvate carboxylase for hypoglycaemia of childhood due to defective gluconeogenesis. Performed on skin fibroblasts. 

Example of an enzyme assay ‘made to order’

Maple syrup urine disease (MSUD) is an inherited defect of branched chain-ketoacid dehydrogenase (BCKDH), a multi-enzyme complex responsible for the metabolism of branch chain amino acids, namely valine, isoleucine and leucine. Accumulated branch chain-ketoacids impart a peculiar odour reminiscent of burnt maple syrup to affected infants.

Two and a half years ago a newborn infant presented to Red Cross Children’s Hospital with developmental delay, failure to thrive and profound muscle hypotonia (‘floppiness’). The nursing sister noted a peculiar ‘sweetish, not unpleasant’ smell about the baby, which could not be washed off. Maple  syrup urine disease was suspected, and confirmed biochemically by extremely elevated plasma levels of branch chain amino acids, particularly L-leucine, i.e.

Date                                                     serum concentration (μM)

                                     leucine              isoleucine              valine
           
17/01/2005                     3115                    392                   455
Normal Range                   50-160                  26-90                 90-190         

Protein intake was restricted, resulting in the decline of all 3 amino acids in plasma. Isoleucine fell to extremely low levels, which would impair growth in the baby, while leucine remained unacceptably high. This highlights problems of management of this disorder.

24/01/2005                      2142                    194                   234

27/01/2005                      2073                    154                   163

31/01/2005                      1530                      5                     73

The child deteriorated and died soon afterwards, but not before we had taken a skin biopsy from which we successfully established a fibroblast cell line for subsequent study (line F1187).

The next development was that the mother conceived again with the same father, so her chances of having another affected child was 1 in 4 (the disorder is autosomal recessive). Unwilling to accept this risk, she requested a prenatal diagnosis, with the option of a termination of pregnancy should this fetus be affected. Amnion cells (cell of fetal origin) were removed from her uterus and their ability to decarboxylate radiolabelled leucine was compared to control amniocytes from unaffected fetuses and to the fibroblast line taken previously from the now deceased brother (F1187).

The principle behind the assay is to incubate intact cells in medium containing [1-14C]-labelled leucine and trap the released [14C]CO2 on a sodium hydroxide impregnated filter disc. Label trapped on the disc is counted and enzyme activity calculated in relation to the amount of protein used in the assay.

The figure below shows a representative experiment. The Y-axis depicts enzyme activity, A84, A86, A87, and A89 are unaffected control amniocytes, MEAN is the mean of control amniocyte activities, A88 are amniocytes from the fetus in question, and F1187 are fibroblasts from the affected brother. The numbers above the bars indicate activity expressed as a % of the control mean.

We interpret the data as follows: Residual activity of 43% for the suspect amniocytes relative to unaffected controls suggest that the fetus is a heterozygote for the defect (expected activity 50%). This is significantly higher than the 1% observed in a known homozygote (his brother), which serves here as a positive control for the assay (with a caveat that amniocytes and fibroblasts are different cell types, and may have different intrinsic enzyme activities). So based on his heterozygous status, we anticipate that the unborn child will be a healthy carrier of the disorder, rather than suffer from it, and we recommend that the pregnancy be allowed to continue to term.