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Using macro- and microanalytical isoelectric focussing techniques for separation of the soluble leukocytic peroxidase (hydrogen donor: p-phenylenediamine) we found 4 main isoenzyme components with pI at 9.6 (9.0), 7.6 (7.5), 6.2 (6.2) and 4.2 (4.7), which exhibited additional heterogeneities. The isoenzymes of the membrane-bound peroxidase displayed a similar pattern. The isoelectric subfractions of peroxidase in controls and patients with late-infantile (Jansky-Bielschowsky), juvenile (Spielmeyer-Sjögren) and adult (Kufs) types of neuronal ceroid-lipofuscinosis did not reveal any significant differences. Based on these findings, a deficiency of an isoenzyme component cannot be held responsible for producing neuronal ceroid-lipofuscinoses, neither in patients with normal nor in patients with reduced total activity of leukocyte peroxidase.

Human leukocytes contain a peroxidase fraction soluble in 0.1 M phosphate buffer and an insoluble peroxidase component with 10--15 times higher specific activity which can be extracted by 0.1 M phosphate buffer + 0.2% Triton X-100 + 0.2% sodium taurocholate and sonication. Both enzyme components have been estimated spectrophotometrically with the substrate hydrogen peroxide (final concentration 1 mM) and the hydrogen donor p-phenylenediamine (final concentration 28-55 mM) within the first 60 sec. The pH-optimum of the soluble and membrane-bound leukocyte peroxidase is at pH 7.0 with a second smaller peak at pH 5.5. Using 0.2 M boric acid/0.05 M sodium borate buffer (pH 7.6) instead of phosphate buffer a 40%-50% increase of enzyme activity can be achieved. In two patients with the juvenile form of neuronal ceroid-lipofuscinosis (type Spielmeyer-Vogt) the activity of soluble leukocyte peroxidase was considerably reduced, in one patient with the late infantile form (type Jansky-Bielschowsky) the activity was just below the normal range, and in two patients with the adult form (type Kuf) activity was normal. In all patients the activity of membrane-bound leukocyte peroxidase was not significantly altered. Only one of four heterozygotes for the juvenile type had deficient values of the soluble enzyme. The variability of the peroxidase findings in patients and carriers with neuronal ceroid-lipofuscinosis make it uncertain whether this represents the primary enzymic defect.

A biochemical difference is found between the mucolipidoses II and III which may be correlated with their clinical phenotypes. In homogenates of mass-cultured I-cells from patients with MLII (I-cell disease), the residual specific activity of beta-galactosidase is between 3 and 5 times lower than that in the I-cells from patients with MLIII (pseudopolydystrophy). This difference is confirmed in several coverslip culture experiments where conditions of inoculation, propagation, harvest and enzyme assays are rigidly controlled. MLIII cells also hydrolyse the natural substrates asialofetuin-(H)3-galactoside and GM1- (H)3-galactoside more easily. This observation offers support to the hypothesis that beta-galactosidase may play a role in the physiopathology of these mucolipidoses.

A healthy adult female was found to have low levels of hexosaminidase A in serum, leukocytes and fibroblasts when these were assayed with artificial substrates. Fibroblast assay with GM2 ganglioside gave values consistent with a Tay-Sachs heterozygote. Studies of this non-Jewish family revealed evidence for segregation of two mutant alleles for hexosaminidase A.

Two hexosaminidase components, separable by starch-gel electrophoresis and possessing both beta-D-N-acetylglucosaminidase and beta-D-N-acetylgalactosaminidase activity, are present in human tissues. One of these, hexosaminidase component A, is absent in brain, liver, kidney, skin, cultured skin fibroblasts, blood plasma, and leukocytes from nine patients with Tay-Sachs disease. Hexosaminidase assay may facilitate the early diagnosis of individuals homozygous for Tay-Sachs disease.

Fifteen pregnant women with a 25 percent risk of delivering a child with Tay-Sachs disease were monitored by amniocentesis and hexosaminidase A assays of amniotic fluid, uncultured amniotic cells, and cultured amniotic cells. Tay-Sachs disease was diagnosed prenatally in six fetuses; the diagnosis was confirmed in one child after birth and in five fetuses after therapeutic abortion. Prenatal diagnosis indicated the absence of Tay-Sachs disease in nine other fetuses; this diagnosis was confirmed postnatally in six, three are still in utero.

A profound deficiency (10- to 30-fold) of beta-galactosidase activity was found in tissues (liver, spleen, kidney, and brain) from two patients with generalized gangliosidosis; this deficiency is demonstrated as a failure to cleave both p-nitrophenyl-beta-D-galactopyranoside and ganglioside GM(1) labeled with C(14) in the terminal galactose. We believe that this enzymic defect is responsible for the accumulation of ganglioside GM(1) and is the fundamental enzyme defect in generalized gangliosidosis.