Total Hexosaminidase Activity Research Articles

GM2 gangliosidoses: evaluation of clinical, biochemical and genetic findings of patients with three novel mutations

Purpose: The aim of this study is to evaluate the diagnosis characteristics, clinic findings, phenotypical and genotypical features of children with GM2 gangliosidoses.
 Materials and Methods: The file records of 14 patients diagnosed with GM2 gangliosidoses in our clinic were retrospectively reviewed. The GM2 gangliosidoses diagnosis was confirmed by determining the levels of serum total hexosaminidase and β-hexosaminidase activity with genetic analysis. 
 Results: We identified a total of seven different mutations, three of which were novel (one in the HEXA gene and two in the HEXB gene) in 14 patients. We found a high frequency of c.1100_1111del (p.Gly367_Tyr370del) mutation in HEXA affected patients. The mean age at diagnosis was 13.46.3 months and 14.24.2 months for patients with Tay–Sachs disease (TSD) and Sandhoff disease (SD) respectively. Neuroregression was present in 92.9% of our patients. Of the 14 patients, 11 had epilepsy, 10 had developmental delay, 6 had hyperacusis, 6 had cherry-red spots and 6 had macrocephaly, but none of the patients had organomegaly. 
 Conclusion: GM2 gangliosidoses disease should be considered for children with developmental regression and/or delay. For early diagnosis, enzyme analysis and gene detection should be performed in children with suspected GM2 gangliosidoses in the presence of clinical findings.

Incidence and carrier frequency of Sandhoff disease in Saskatchewan determined using a novel substrate with detection by tandem mass spectrometry and molecular genetic analysis

Sandhoff disease is a rare progressive neurodegenerative genetic disorder with a high incidence among certain isolated communities and ethnic groups around the world. Previous reports have shown a high occurrence of Sandhoff disease in northern Saskatchewan. Newborn screening cards from northern Saskatchewan were retrospectively screened in order to investigate the incidence and determine the carrier frequency of Sandhoff disease in these communities. PCR-based screening was conducted for the c.115delG (p.(Val39fs)) variant in the HEXB gene that was previously found in 4 Sandhoff disease patients from this area. The carrier frequency for this allele was estimated to be ~1:27. MS/MS-based screening of hexosaminidase activity along with genetic sequencing allowed for the identification of additional variants based on low total hexosaminidase activity and high % hexosaminidase A activity relative to c.115delG carriers. In total 4 pathogenic variants were discovered in the population (c.115delG, c.619A>G, c.1601G>T, and c.1652G>A) of which two are previously unreported (c.1601G>T and c.1652G>A). The combined carrier frequency of these alleles in the study area was estimated at ~1:15. Based on the number of cases of Sandhoff disease from this area we estimate the incidence to be ~1:390 corresponding to a child being born with the disease every 1–2years on average. The results from our study were then compared with variants in the HEXB gene from the genomes available from the 1000 Genomes project. A total of 19 HEXB variants were found in the 1092 genomes of which 5 are suspected of having a deleterious effect on hexosaminidase activity. The estimated carrier frequency of Sandhoff disease in Saskatchewan at 1:15 is more than 3 times higher than the carrier frequency in the global sample provided by the 1000 Genomes project at 1:57.

Hypermethylation contributes to down-regulation of lysosomal β-hexosaminidase α subunit in prostate cancer cells

β-Hexosaminidase, involved in degradation of glycoproteins and glycosphingolipids, is altered in several tumours leading to enhanced migration capacity. To date, the expression of the β-hexosaminidase isoenzymes in prostate cancer cells has not been elucidated. By using PC3, LNCaP, DUCaP, MDAPCa 2b, and hyperplasic prostate (BPH-1) cell lines, we analysed the β-hexosaminidase activity in each cell line and determined β-hexosaminidase α subunit gene expression in PC3, LNCaP, and BPH-1. We then investigated the methylation status of the gene promoter and determined the cellular responses of PC3 and LNCaP after transfection with β-hexosaminidase α subunit. We found that each prostate cancer cell line had a decrease in total hexosaminidase activity and that the lack of hexosaminidase A activity, observed in PC3 and LNCaP cells, was associated with mRNA disappearance. The HEXA promoter region in LNCaP and PC3 cell lines had methylated CpG islands, as confirmed by 5'-Aza-2'-deoxycitidine treatment, in PC3 cells, used as cell cancer model. We also tested, the involvement of hexosaminidase A in the migration capacity by migration assay using Hex α subunit-transfected PC3. Finally, we found that, after Hex α subunit transfection, both PC3 and LNCaP were less susceptible to exogenous ceramide treatment. Results indicate a likely contribution of the lysosomal enzyme to the acquisition of cancerous features.

Expanding the spectrum of HEXA mutations in Indian patients with Tay–Sachs disease

Tay–Sachs disease is an autosomal recessive neurodegenerative disorder occurring due to impaired activity of β-hexosaminidase-A (EC 3.2.1.52), resulting from the mutation in HEXA gene. Very little is known about the molecular pathology of TSD in Indian children except for a few mutations identified by us. The present study is aimed to determine additional mutations leading to Tay–Sachs disease in nine patients confirmed by the deficiency of β-hexosaminidase-A (<2% of total hexosaminidase activity for infantile patients) in leucocytes. The enzyme activity was assessed by using substrates 4-methylumbelliferyl-N-acetyl-β-d-glucosamine and 4-methylumbelliferyl-N-acetyl-β-d-glucosamine-6-sulfate for total-hexosaminidase and hexosaminidase-A respectively, and heat inactivation method for carrier detection. The exons and exon–intron boundaries of the HEXA gene were bi-directionally sequenced on an automated sequencer. ‘In silico’ analyses for novel mutations were carried out using SIFT, Polyphen2 and MutationT@ster software programs. The structural study was carried out by UCSF Chimera software using the crystallographic structure of β-hexosaminidase-A (PDB-ID: 2GJX) as the template. Our study identified four novel mutations in three cases. These include a compound heterozygous missense mutation c.524A>C (D175A) and c.805G>C (p.G269R) in one case; and one small 1bp deletion c.426delT (p.F142LfsX57) and one splice site mutation c.459+4A>C in the other two cases respectively. None of these mutations were detected in 100 chromosomes from healthy individuals of the same ethnic group. Three previously reported missense mutations, (i) c.532C>T (p.R178C), (ii) c.964G>T (p.D322Y), and (iii) c.1385A>T (p.E462V); two nonsense mutations (i) c.709C>T (p.Q237X) and (ii) c.1528C>T (p.R510X), one 4bp insertion c.1277_1278insTATC (p.Y427IfsX5) and one splice site mutation c.459+5G>A were also identified in six cases. We observe from this study that novel mutations are more frequently observed in Indian patients with Tay–Sachs disease with clustering of ~73% of disease causing mutations in exons 5 to 12. This database can be used for a carrier rate screening in the larger population of the country.

Identification of novel mutations in HEXA gene in children affected with Tay Sachs disease from India.

Tay Sachs disease (TSD) is a neurodegenerative disorder due to β-hexosaminidase A deficiency caused by mutations in the HEXA gene. The mutations leading to Tay Sachs disease in India are yet unknown. We aimed to determine mutations leading to TSD in India by complete sequencing of the HEXA gene. The clinical inclusion criteria included neuroregression, seizures, exaggerated startle reflex, macrocephaly, cherry red spot on fundus examination and spasticity. Neuroimaging criteria included thalamic hyperdensities on CT scan/T1W images of MRI of the brain. Biochemical criteria included deficiency of hexosaminidase A (less than 2% of total hexosaminidase activity for infantile patients). Total leukocyte hexosaminidase activity was assayed by 4-methylumbelliferyl-N-acetyl-β-D-glucosamine lysis and hexosaminidase A activity was assayed by heat inactivation method and 4-methylumbelliferyl-N-acetyl-β-D-glucosamine-6-sulphate lysis method. The exons and exon-intron boundaries of the HEXA gene were bidirectionally sequenced using an automated sequencer. Mutations were confirmed in parents and looked up in public databases. In silico analysis for mutations was carried out using SIFT, Polyphen2, MutationT@ster and Accelrys Discovery Studio softwares. Fifteen families were included in the study. We identified six novel missense mutations, c.340 G>A (p.E114K), c.964 G>A (p.D322N), c.964 G>T (p.D322Y), c.1178C>G (p.R393P) and c.1385A>T (p.E462V), c.1432 G>A (p.G478R) and two previously reported mutations. c.1277_1278insTATC and c.508C>T (p.R170W). The mutation p.E462V was found in six unrelated families from Gujarat indicating a founder effect. A previously known splice site mutation c.805+1 G>C and another intronic mutation c.672+30 T>G of unknown significance were also identified. Mutations could not be identified in one family. We conclude that TSD patients from Gujarat should be screened for the common mutation p.E462V.

Effect of sample collection, temperature and time of storage on β-galactosidase and total hexosaminidase activities in dried blood collected on filter paper

Dried blood spots (DBS) on filter paper is a valuable sampling technique in clinical chemistry, but the stability of enzymes used in the diagnosis of lysosomal storage diseases (LSDs) needs to be evaluated. In a first experiment, blood from 20 subjects was collected using a syringe without additives and distributed into EDTA tubes, heparin tubes, and spotted on filter paper for the comparison of sampling effects. In a second experiment, blood from 30 healthy subjects was spotted on filter paper and analyzed for β-galactosidase and total hexosaminidase activities after storage of the samples at different temperatures for up to 180 days. Initially, we observed that enzyme activities were the same, independent of the collection method. When DBS was stored at 37°C the activity of β-galactosidase dropped to 85% of the initial value after 180 days (p<0.05). At all other temperatures (-20°C, 4°C and 25°C), the results were within the methodological error. Total hexosaminidase activity did not change significantly during the entire study period and at different storage temperatures. The two enzymes investigated in the present study may be stored for up to 17 days (β-galactosidase) or 180 days (total hexosaminidase) until analysis without loss of activity.

Connective tissue alterations in women with pelvic organ prolapse and urinary incontinence

Alterations in collagen synthesis and metabolism have previously been reported in patients with pelvic organ prolapse (POP) and/or urodynamic stress incontinence (USI). Since urinary incontinence does not always associate with POP, the objective of this study was to examine connective tissues from patients with USI plus POP, and patients with prolapse only. Biopsies from the uterosacral ligaments were obtained during the operation from POP patients (n =28), and from continent women (control group, n =12) who underwent surgery for other benign reasons. POP patients were classified following urodynamic tests and symptom questionnaire with respect to the presence (n =14) or absence (n =14) of USI. N-terminal propeptides of collagen (PINP and PIIINP), TGF-beta and leptin were measured in plasma. Hydroxyproline and glycosaminoglycan (GAGs) concentrations and total hexosaminidase activity were measured in tissue samples. Histological sections were prepared using Masson's trichrome technique, and digitised solutions were used for imaging provided by Soft Imaging System GmBh. Statistical evaluations were made by the Kruskal-Wallis test. A significant decrease in hydroxyproline content was found in USI+POP women in comparison to controls (p<0.05). In contrast, histopathological examination revealed an increased density of collagen in USI+POP patients. Hexosaminidase activity was decreased in both groups with POP, but no change in the amount of GAGs was observed. Markers of collagen synthesis (PINP, PIIINP), and factors related to the collagen synthesis (TGF-beta, leptin) remained unaltered. Our biochemical and morphological findings suggest a different organisation of collagen fibres in tissues of patients with USI+POP, when compared with both the controls and the POP patients.

Common HEXB polymorphisms reduce serum HexA and HexB enzymatic activities, potentially masking Tay-Sachs disease carrier identification

A DNA-proven Tay-Sachs disease (TSD) carrier and his brother were found to have serum percent Hexosaminidase A (%HexA) enzymatic activities in the non-carrier range, while the leukocyte %HexA profiles clearly identified them as TSD heterozygotes. Both their serum HexA and HexB enzymatic activities were below reference range, suggesting inheritance of mutations in both the HEXA (α-subunit) and HEXB (β-subunit) genes. DNA sequencing revealed that both individuals, carried the common HEXA 1277_1278insTATC mutation, and two common HEXB polymorphisms: [619A>G (+) delTG]. To determine if these HEXB polymorphisms reduce HexA and HexB enzymatic activities, 69 DNA samples from subjects previously screened enzymatically in both serum and leukocytes for TSD carrier status were selected for either high, mid-range or low serum Total Hex (defined as the sum of HexA and HexB) activities and were tested for the HEXB mutations. Further, three additional TSD carriers ascertained by the atypical pattern of normal serum %HexA but carrier leukocyte %HexA, were found to have the [delTG (+) 619A>G] genotype. In addition, the frequency of the [delTG (+) 619A>G] genotype was significantly higher ( P < 0.01) in subjects with low serum HexB enzymatic activities. Given the high frequency of the [delTG (+) 619A>G] haplotype in the Ashkenazi Jewish population (approximately 10%), up to 10% of TSD carriers may have normal serum %HexA values with low total Hex. Accordingly, serum %HexA should not be the sole criterion used for carrier status determination. Where total Hex activity is reduced, further testing with leukocyte Hex profiles is indicated.

Thermodynamic study of β- N-acetylhexosaminidase enzyme heterogeneity in human seminal plasma

Background It has been suggested that the activity of β- N-acetylhexosaminidase (Hex) in seminal plasma may be used as a biochemical marker of azoospermia. The purpose of our study was to evaluate this hypothesis using a thermodynamic procedure developed to determine total Hex activity and that of its isoenzymes in this biological fluid. Methods Using the substrate 3,3′-dichlorophenolsulphoftaleinil N-acetyl-β- d-glucosaminide, a highly significant difference ( p<0.001) is found between the activation energy of Hex A (41.5 kJ/mol) and of Hex B (72.3 kJ/mol), making it possible to determine the activity of these isoenzymes from the apparent activation energy of the total Hex in seminal plasma. Results A significant difference between the normozoospermic and azoospermic groups was only found for Hex A isoenzyme activity ( p<0.05), although with considerable overlapping between the values of both groups. Significant partial correlations were found for the total Hex, Hex A and Hex B activities with the immobile spermatozoa count ( p<0.01) and for total Hex and Hex B with the dead spermatoza count ( p<0.05). In turn, Hex A had a significant partial correlation with the live spermatozoa count ( p<0.05); however, Hex activity in seminal plasma of acromosomal origin appears to be of little importance in quantitative terms. Conclusions It was not possible to confirm that total Hex activity in seminal plasma, or even of its isoenzymes Hex A and Hex B, is a suitable biochemical marker of azoospermia (efficiency≤67%). The thermodynamic procedure described may be a useful alternative for the study of the Hex enzyme heterogeneity in spermatozoa.

Does Serum Hexosaminidase Activity Play a Role in the Diagnosis of Strangulated Bowel Obstruction? An Experimental Study

Strangulation is associated with an increased risk of mortality and morbidity in patients with mechanical bowel obstruction. The accurate and early recognition of the presence of strangulation is important to allow safe nonoperative treatment. A number of studies have shown that there was no single and reliable test to detect or exclude the presence of strangulation. The aim of this study was to evaluate the role of serum hexosaminidase (Hex) levels in recognition of strangulation in an experimental model of closed loop small bowel obstruction. Forty-two Wistar albino rats were divided into four groups: I, control (n = 5); II, sham laparotomy (n = 5); III, simple obstruction (n = 16); and IV, strangulation groups (n = 16). Activity levels of total Hex and its fractions (Hex A and B) were assayed in serum samples obtained from rats after 3 and 8 hr. Samples of small bowel were also evaluated histologically. Histological evaluation of bowel sections obtained from the strangulation group after 8 hr, revealed transmural hemorrhagic infarction in all animals with a mean +/- SD total Hex activity of 978.25 +/- 150 nmol/hr/ml, which was significantly higher than that in the other groups (P < 0.001). Although sections of bowel from the strangulation group after 3 hr showed severe ischemic injury, the activities of total Hex, Hex A, and Hex B were not different from those of the control, sham, and simple obstruction groups. Histological examination of these groups did not show any sign of ischemia. Total Hex, Hex A, and Hex B activities in the strangulation group were all significantly greater than the activities seen in the simple obstruction group (P < 0.001, for all). In conclusion, increased serum hex levels indicate irreversible transmural infarction only in the late period of strangulation in the closed loop small bowel obstruction model. It seems unuseful for detecting reversible and/or irreversible ischemia in the early period of strangulation.

Plasma and peripheral leukocyte β-N-acetylhexosaminidase isoenzymes and disease activity in rheumatoid arthritis

Objectives: Recently it has been suggested that serum β-N-acetylhexosaminidase (Hex) could be a joint destruction marker in rheumatoid arthritis (RA) patients. However, a large amount of serum Hex activity has its source from platelets, and the blood platelet-count is often increased in RA, which may have masked the significance of the results. The purpose of this study was to investigate the relationship between plasma activity of Hex and disease activity or severity. Design and Methods: In 51 patients with RA, with an evolution period for the illness of 10.9 ± 1.2 yr (range 1-40 yr), we determined the total Hex activity together with its Hex A and B isoenzymes in plasma and in mononuclear (MN) and polymorphonuclear (PMN) leukocytes. Results: The plasma activity of total Hex and Hex B isoenzyme was slightly higher in the group of patients studied ( p < 0.01), together with the specific activity of total Hex, Hex A and B in PMN leukocytes ( p < 0.001) than in the control group. No significant correlation was found between plasma or leukocyte Hex and the radiologic evaluation of the disease (Sharp’s modified method), or the patient’s functional capacity (modified Health Assessment Questionnaire). Likewise, a significant correlation between Hex activity and laboratory inflammation markers (C reactive protein, sialic acid, erythrocyte sedimentation rate) or the evolution time of the disease was not found. Conclusions: The plasma activity of total Hex, or even of its isoenzymes Hex A and Hex B, does not appear to be a reliable marker of erosion and cartilage degradation in RA patients. Liver function appears to be the major determinant for the plasma Hex activity in these patients.

Fenotipo infantil tardío y juvenil de la variante B1 de gangliosidosis GM2

Variant B1 is a rare form of GM2-gangliosidosis characterized by the presence of a mutation in the hexosaminidase A gene (HEXA) leading to a defect in the catalytic region of the alpha-subunit of beta-hexosaminidase A (alpha beta heterodymer). The mutated Hex A has almost normal activity against the natural synthetic substrates (4-methylumbelliferyl-N-acetyl-beta-D-glucosamine, 4MU-NAG) but is unable to hydrolyse GM2-ganglioside and the sulphated synthetic substrates (4MU-NAGS). The first and more frequent mutation described in the alpha-subunit gene associated to B1 variant GM2-gangliosidosis was a G533-->A transition (DN allele) resulting in Arg178His substitution. Here, we report the clinical, enzymatic and molecular characterization in two variant B1 late infantile and juvenile cases. Both cases presented regression of mental skills leading to dementia, epilepsy and severe motor impairment with dystonic involuntary movements and quadriplegia. In the late infantile case (death at 5 years and 8 months), cherry-red spot was also present. Enzymatic assays were performed in fibroblasts, leukocytes and serum and confirmed the abnormally low beta-hexosaminidase A activity against sulphated substrate despite a normal or nearly normal total hexosaminidase activity (unsulphated substrates). The patient with the late infantile phenotype was found to be compound heterozygote for the DN allele whilst the juvenile form was homozygote for that mutation. Variant B1 form of GM2-gangliosidosis is a rare and heterogeneous condition that must be kept in mind when evaluating neurodegenerative disorders associated with speech or gait disturbances, dystonia, seizures and pyramidal features.

Detection of Tay-Sachs disease carriers among individuals with thermolabile hexosaminidase B.

The determination of hexosaminidases A and B in most programmes for Tay-Sachs disease carrier detection is based on their different heat sensitivity (hexosaminidase A is the heat labile isoenzyme). This routine cannot be employed for individuals who also possess a thermolabile hexosaminidase B. In Israel, 0.6% of the screened samples have a labile hexosaminidases B (about 110 each year) and the assessment of their hexosaminidase A activity has hitherto been based on isoenzyme separation by ion exchange chromatography. The latter requires relative large serum samples, and the individuals must usually be reappointed. In order to avoid the thermal treatment we have used the alternative technique, which employs two substrates with different specificities for the two isoenzymes: 1. The fluorogenic substance, 4-methylumbelliferyl-N-acetyl-glucopyranoside, which measures total hexosaminidase activity and 2. the derivative, 4-methylumbelliferyl-N-acetyl glucosamine-6-sulphate, which is considerably more specific toward hexosaminidase A. Hexosaminidase A activity was expressed as a ratio of total activities (the ratio of the assay with 4-methylumbelliferyl-N-acetyl glucosamine-6-sulphate to that with 4-methyllumbelliferyl-N-acetyl-glucopyranoside). Using the results from 65 obligate heterozygotes for Tay-Sachs disease, we established our reference ranges for assigning the genotypes with respect to the Tay-Sachs gene. Comparison of the results from 182 unrelated and randomly chosen sera screened by the ratio method and by heat inactivation, showed a very high correlation (r = 0.996). Sixty eight sera with thermolabile hexosaminidase B were tested by ion exchange chromatography and by the double substrate method, and they yielded identical diagnoses with regard to the Tay-Sachs locus.(ABSTRACT TRUNCATED AT 250 WORDS)

Immunoassay of β-hexosaminidase isoenzymes in serum in patients with raised total activities

Enzyme immunoassay (EIA) methods with monoclonal antibodies specific for β-hexosaminidase (Hex) isoenzymes A and B in human serum are presented. The proportion of Hex A obtained with the EIA-methods was similar to that found with ion-exchange chromatography and isoelectric focusing (about 60% of total Hex activity). The EIA-method for Hex B reacted to a similar extent with Hex B, Hex P and intermediate forms. A highly significant correlation was obtained between total Hex activity in human sera assayed with a conventional enzyme substrate method and the total Hex activity obtained as the sum of Hex A and Hex B analyzed with the EIA-methods. In sera from individuals with increased total activity of Hex there was a positive correlation between total activity of Hex, and percent of Hex B found with the EIA-methods. Investigation of sera from pregnant women and sera from patients with liver cirrhosis and cholestasis showed that the increase in total activity in these patient groups was mainly due to an increase of the isoenzyme forms reacting with the EIA-method for Hex B.

Adult onset motor neuronopathy in the juvenile type of hexosaminidase A and B deficiency

Two sisters presented with progressive muscle cramps, as well as wasting and weakness of the legs with onset after age 20. They also showed intention tremor of the upper extremities and dysarthria starting during the first decade. The older patient also had fasciculations; the younger, hyperreflexia. Total plasma beta-hexosaminidase (Hex) activity with 4-methylumbelliferyl-acetyl-glucosamine as substrate was reduced to 1.4% and 2.7% of the control in the 2 patients, respectively. Hex A activity measured by 4-methylumbelliferyl- N-acetylglucosamine-6- O-sulphate as substrate was 9.9% and 12.8% of the mean control value in the 2 patients, respectively. Hex B activity was undetectable in both patients. Leukocyte total Hex activity was 7–8% of normal; residual Hex A activity in the 2 patients was 17.8% and 16.3% of normal controls, respectively. Fibroblastic residual Hex A activity in the 2 patients was 9.6% and 22% of normal mean value, respectively. Appendiceal ganglion cells contained membranous cytoplasmic bodies in the younger patient. Thin layer chromatography of the appendiceal extract from one patient ( III 2 ) showed a marked increase of GM 2 ganglioside, and some increase of GM 3 ganglioside. Northern blots performed on fibroblast cell lines from both patients for the demonstration of alpha and beta locus messenger RNA showed no difference between patients and control. These patients have a rare form of adult-onset progressive motor neuron disease presumably due to abnormal beta subunits, causing severe deficiency of both Hex A and Hex B. The phenotypic expression of this disease is similar to motor neuron disease due to alpha locus mutations, which suggests that the Hex A deficiency, even though only a partial one, may be the important pathogenic factor.

Characterization of a new model of GM2-gangliosidosis (Sandhoff's disease) in Korat cats.

We have detected a disorder in Korat cats (initially imported from Thailand) that is analogous to human Sandhoff's disease. Pedigree analysis indicates that this disease in an autosomal recessive disorder in the American Korat. Postmortem studies on one affected cat showed hepatomegaly that was not reported in the only other known feline model of GM2-gangliosidosis type II. Histologic and ultra-structural evaluation revealed typical storage vacuoles. There was a marked deficiency in the activity of hexosaminidase (HEX) A and B in affected brain and liver as compared to controls. Electrophoresis of a liver extract revealed a deficiency of normal HEX A and B in the affected animals. The blocking primary enzyme immunoassay verified the presence of antigenically reactive HEX present in affected cat livers in quantities slightly elevated with respect to the normal HEX concentration in control cats. In leukocytes, obligate heterozygotes had intermediate levels of total HEX activity with a slight increase in the percent activity due to HEX A. Indeed, 4 of 11 phenotypically normal animals in addition to four obligate heterozygotes appear to be carriers using this assay. Affected brain and liver compared with control brain and liver contained a great excess of bound N-acetylneuraminic acid in the Folch upper-phase solids; thin-layer chromatography showed a marked increase in GM2-ganglioside. In summary, we have characterized the pedigree, pathology, and biochemistry of a new feline model of GM2-gangliosidosis which is similar to but different from the only other known feline model.

Tay-Sachs disease heterozygote detection in Brazil: comparison between tears and leukocytes as beta-hexosaminidase A source.

Hexosaminidase A activities were determined in tears and peripheral leukocytes of carriers and noncarriers for Tay-Sachs Disease (TSD) as a percentage of total hexosaminidase activity. Correlation between enzyme activities in tears and leukocytes was highly significant (r = 0.75, p less than 0.01). Compared to leukocytes, screening in tears revealed 100% sensitivity, 100% negative predictive value, 86% specificity and 80% positive predictive value. These results indicate that tears are a satisfactory material for mass-screening of TSD-carrier state, but positive results must be confirmed in peripheral leukocytes. Seven heterozygotes were detected among 298 young Ashkenazi Jewish volunteers screened, giving an adjusted frequency of the TSD gene of 0.024.

Hexosaminidase: A marker for intestinal gangrene in necrotizing enterocolitis

Detection of intestinal ischemia, prior to necrosis, is a major clinical problem. The lysosomal acid hydrolase, hexosaminidase (HEX), is known to be elevated in intestinal infarction. To determine if this enzyme could differentiate between partial intestinal ischemia and full-thickness intestinal gangrene, the following rat study was designed. Partial segmental intestinal ischemia was created by ligating alternate vascular bundles over a short (6 vessel) segment of the small-bowel mesentery, and complete segmental intestinal vascular occlusion was achieved by ligating the blood supply to the ileocecal segment. Preoperative serum HEX values were obtained from 15 animals. The rats were separated into one sham-operated and two intestinal ischemia groups. At four hours after surgery HEX values were determined. Total HEX activity was significantly elevated four hours after insult in both partial and complete intestinal ischemia, (P less than 0.005 and P less than 0.001 respectively). Total HEX activity was greater in complete intestinal ischemia than in partial ischemia, (P less than 0.05). Three neonates with intestinal perforation, secondary to necrotizing enterocolitis, were evaluated. The mean preoperative HEX activity was 1421 nmol/hr/mL serum and the mean post-resection HEX activity was 808 nmol/hr/mL serum. These data suggest that serum HEX activity may be a good marker for intestinal gangrene in neonates with necrotizing enterocolitis.

N-acetyl- β-hexosaminidase activity in human breast milk

1. 1. The lysosomal enzyme, N-acetyl- β-hexosaminidase (HEX) is present in human breast milk. It is composed predominantly of “A” (heat-labile) and “B” (heat-stable) isozymes which coelute with the corresponding major serum isozymes on DE-52 ion-exchange chromatography. 2. 2. Total HEX activity in “early” milk obtained at 2.8 ± 1.4 weeks post partum, is approx. 2.5-fold higher (87 ± 29 nmol/60'/mg protein, n = 10) than that of pregnancy serum (35.7 nmol/60'/mg protein) prior to delivery. 3. 3. These levels increase to greater than 3-fold (110 ± 20 nmol/60'/ mg protein, n = 13) as the milk matures (10.3 ± 4.2 weeks). 4. 4. The specific activity of HEX A in the milk changes little with time post partum, because absolute levels are decreasing simultaneous with decreases in milk protein. 5. 5. In contrast, HEX B specific activity is increased, as absolute levels (per volume) remain constant in the face of decreasing milk protein content. 6. 6. These changes result in a high degree of correlation ( r = 0.81) between time of lactation and % HEX A observed.

Sex differences in serum levels of N-acetyl-β-hexosaminidase in infancy: Correlation of enzyme activity with testosterone levels

Serum levels of N-acetyl-β-hexosaminidase (HEX) (EC 3.2.1.30) activity in infants display a sexual dimorphism. Total HEX activity in males between 1 and 3 months of age is significantly elevated over female levels (male (M), 1535 ± 300 nmol/60 min/ml; female (F), 1150 ± 203, P < 0.0005), and the A (labile) isozyme constitutes a significantly lower proportion of the total activity present (M, 56.0 ± 4.2, N = 24; F, 64.3 ± 4.6, N = 21, P < 0.0005). These findings led us to investigate the relationship between testosterone concentration and HEX activity in serum. Samples from male (N = 36) and female (N = 33) infants between 1 and 6 months of age were included. In both sexes, a high degree of correlation (P < 0.0005) was observed between testosterone and total HEX (M, r = 0.71; F, r = 0.73), HEX A (M, r = 0.68; F, r = 0.56) and HEX B (M, r = 0.68; F, r = 0.72). An inverse relationship exists between testosterone levels and % A: M, r = −0.56; F, r = −0.38 (P < 0.0025 and 0.025, respectively). In contrast, no correlation between HEX levels and testosterone was evident in either male or female adults ( r = 0.20 and 0.18, respectively). These data implicate testosterone in the regulation of HEX activity during the early months of human development.