Amniotic fluid supernatant

Diagnosis is also based on high levels of some extracellular enzyme activities ( -hexosaminidase, arylsulfatase A, a-mannosidase, etc.) in plasma or amniotic fluid supernatant. 

The prenatal diagnosis of the organic acidurias by enzyme assay on cultured amniotic cells is outside the scope of the present book and will not be discussed further however, attention will be given to an alternative approach to the prenatal diagnosis of the organic acidurias, that of direct chemical analysis of the cell-free amniotic fluid supernatant for accumulating metabolites excreted by the foetus into the fluid. 

Pipette 0.2 ml of CSF or amniotic fluid into an Ultrafree 10000 Filter Unit (Millipore), add 40 pi of water, 30 pi of 30% (w/v) TCA, and 1 mg of Mn02 (see above) shake for 5 min at room temperature. Centrifuge for 30 min at 2000 xg. Immediately transfer the clear supernatant into a vial. For sepiapterin determination, samples should be analyzed without oxidation. 

Figure 55-2 Multi-analyte approach to the prenatal diagnosis of methylmalonic acidemia (cb/C complementation group) by metabolite analysis in ceil-free supernatant of amniotic fluid collected at 16 weeks of gestational age. The symbol marks internal standards. A, Determination of total homocysteine by LC-MS/MS (selected reaction monitoring, SRM, transition m/z 136 to m/z 90 and m/z 140 to m/z 94 for the d -labeled internal standard). The concentration of total homocysteine was l5.7pmol/L (0.7 to 2.0pmol/L). B, Determination of methylmalonic acid by LC-MS/MS (SRM, transition m/z 231 to m/z 119 and m/z 234 to m/z 122 for the d3-labeled internal standard). The concentration of methylmalonic acid was 8.7pmol/L, the reference interval for 16 to 19 weeks of gestational age is 0.2 to 0.7)j,mol/L. C, Determination of propionylcarnitine by LC-MS/MS (parent of m/z 85 scan, the [M+H] ion of C3 is m/z 274, m/z 277 for the interna standard). The concentration was 5.6pmol/L (i.5 to l.8pmoi/L),the C3/C4 ratio was 6.9 (0.9 to 2.6).

---