Screening anaphylactoid components of Shuang Huang Lian Injection by analyzing spectrum-effect relationships coupled with UPLC-TOF-MS.

Shuang Huang Lian Injection (SHLI) has been used in China for over 30 years as an effective and widely used Chinese herbal prescription to treat acute respiratory infectious. SHLI has, however, caused many severe anaphylactoid reactions. It is important to identify the potential anaphylactoid components of SHLI. Spectrum-effect relationships were used to explore potentially anaphylactoid components. Based on the original herbal formula, honeysuckle, Fructus Forsythiae and Radix Scutellariae extracts were prepared and combined in appropriate proportions. The preparations were then injected into the caudal vein of rats to obtain in vivo serum samples for pharmacological evaluation and fingerprint analysis. The release rate of ß-hexosaminidase from RBL-2H3 cells and plasma histamine level was used as the pharmacological index. Chromatographic fingerprint analysis identified 22 common peaks. Regression analysis and correlation analysis were used to calculate the relationships between the peaks and the pharmacological effects and identified peaks 5, 6, 11, 12 and 17 as likely anaphylactoid agents. The correlated peaks were identified by comparing the fingerprints with in vitro samples and reference standard samples and the structure was identified by UPLC-TOF-MS. This study established a prospective method to clarify the anaphylactoid components in SHLI, which would provide guidances for screening anaphylactoid components in other traditional Chinese medicine injections.

[Clinical and molecular characteristics of a child with juvenile Sandhoff disease].

OBJECTIVE: To explore the clinical features and molecular mutation of HEXB gene in a case with juvenile Sandhoff disease. METHOD: We retrospectively reviewed the clinical, neuroimaging and biochemical findings in this Chinese child with juvenile Sandhoff disease. Hexosaminidase A and hexosaminidase A & B activities were measured in blood leukocytes by fluorometric assay. HEXB gene molecular analysis was performed by PCR and direct sequencing. RESULT: The 9-year-old boy was admitted for psychomotor regression. He presented slowly progressive gait disorder and dysarthria during the last three years. Cranial MRI revealed a marked cerebellar atrophy with normal intensity in the thalamus and basal ganglia. Brain MRS showed normal in the thalamus and basal ganglia. Hexosaminidase A was 69.5 (mg·h) [normal controls 150-360 nmol/(mg·h)], hexosaminidase A & B activity was 119 nmol/(mg·h)[normal controls 600-3 500 nmol/(mg·h)], confirming the diagnosis of Sandhoff disease. The patient was a compound heterozygote for a novel deletion mutation c.1404delT (p. P468P fsX62) and a reported mutation c.1509-26G>A. CONCLUSION: The clinical features of juvenile Sandhoff disease include ataxia, dysarthria and cerebellar atrophy. The enzyme assay and molecular analysis of HEXB gene can confirm the diagnosis of Sandhoff disease. The novel mutation c.1404delT(p. P468P fsX62) is a disease-related mutation.

[Isoforms A and B of lysosomal N-acetyl-beta-D-hexosaminidase in serum and urine of parenterally fed patients]. / Izoenzymy A i B lizosomalnej N-acetylo-beta-D-heksozoaminidazy w surowicy i moczu chorych zywionych pozajelitowo.

UNLABELLED: Parenteral nutrition entails numerous metabolic complications resulting from food bypass of the gastrointestinal tract. Up to now have not been established all complications of parenteral nutrition, despite intensive research and clinical observations. Knowledge of the biochemical changes resulting from parenteral nutrition is essential to effective prevention, early detection and effective treatment of the metabolic disorders induced by parenteral nutrition. The aim of the study was to evaluate the catabolism of glycoconjugates of parenterally fed patients, reflected by the activity of N-acetyl-beta-D-hexosaminidase (HEX): HEX A and HEX B isoenzymes in serum and urine. MATERIAL AND METHODS: Samples of blood and urine were collected from 23 patients: before intravenous alimentation, at start, as well as of fifth and tenth day of parenteral nutrition. The activity of HEX A and HEX B in serum and urine was determined by the colorimetric method of Zwierz et al. as modified by Marciniak et al. The activity of urinary HEXA and HEX B has been calculated per 1 mg of creatinine. RESULTS: The activity of serum HEXA significantly decreased at fifth day, in comparison to the activity before parenteral alimentation, and significantly increased at tenth day of parenteral nutrition. The activity of HEX B in serum increased significantly at fifth and tenth day of the parenteral nutrition. CONCLUSIONS: Parenteral nutrition alter the catabolism of glycoconjugates, reflected by significant changes in serum HEX A and HEX B activities. Urine was the not appropriate material to evaluate the catabolism of glycoconjugates in view of HEX A and HEX B activities.

GM2 gangliosidoses in Spain: analysis of the HEXA and HEXB genes in 34 Tay-Sachs and 14 Sandhoff patients.

The GM2 gangliosidoses are autosomal recessive lysosomal storage diseases caused by a deficiency of the ß-hexosaminidase A enzyme. This enzyme is composed of two polypeptide chains designated the α- and ß- subunits and it interacts with the GM2 activator protein. The HEXA and HEXB genes encode the α-subunit and the ß-subunit, respectively. Mutations in these genes are causative of Tay-Sachs disease (HEXA) and Sandhoff disease (HEXB). We analyzed the complete HEXA gene in 34 Spanish patients with Tay-Sachs disease and the HEXB gene in 14 Spanish patients with Sandhoff disease. We identified 27 different mutations, 14 of which were novel, in the HEXA gene and 14 different mutations, 8 of which unreported until now, in the HEXB gene, and we attempted to correlate these mutations with the clinical presentation of the patients. We found a high frequency of c.459+5G>A (IVS4+5G>A) mutation in HEXA affected patients, 22 of 68 alleles, which represent the 32.4%. This is the highest percentage found of this mutation in a population. All patients homozygous for mutation c.459+5G>A presented with the infantile form of the disease and, as previously reported, patients carrying mutation p.R178H in at least one of the alleles presented with a milder form. In HEXB affected patients, the novel deletion c.171delG accounts for 21.4% of the mutant alleles (6/28). All patients with this deletion showed the infantile form of the disease. The Spanish GM2 gangliosidoses affected patients show a great mutational heterogeneity as seen in other inherited lisosomal diseases in this country.

An open-label Phase I/II clinical trial of pyrimethamine for the treatment of patients affected with chronic GM2 gangliosidosis (Tay-Sachs or Sandhoff variants).

Late-onset GM2 gangliosidosis is an autosomal recessive, neurodegenerative, lysosomal storage disease, caused by deficiency of ß-hexosaminidase A (Hex A), resulting from mutations in the HEXA (Tay-Sachs variant) or the HEXB (Sandhoff variant) genes. The enzyme deficiency in many patients with juvenile or adult onset forms of the disease results from the production of an unstable protein, which becomes targeted for premature degradation by the quality control system of the smooth endoplasmic reticulum and is not transported to lysosomes. In vitro studies have shown that many mutations in either the α or ß subunit of Hex A can be partially rescued, i.e. enhanced levels of both enzyme protein and activity in lysosomes, following the growth of patient cells in the presence of the drug, pyrimethamine. The objectives of the present clinical trial were to establish the tolerability and efficacy of the treatment of late-onset GM2 gangliosidosis patients with escalating doses of pyrimethamine, to a maximum of 100 mg per day, administered orally in a single daily dose, over a 16-week period . The primary objective, tolerability, was assessed by regular clinical examinations, along with a panel of hematologic and biochemical studies. Although clinical efficacy could not be assessed in this short trial, treatment efficacy was evaluated by repeated measurements of leukocyte Hex A activity, expressed relative to the activity of lysosomal ß-glucuronidase. A total of 11 patients were enrolled, 8 males and 3 females, aged 23 to 50 years. One subject failed the initial screen, another was omitted from analysis because of the large number of protocol violations, and a third was withdrawn very early as a result of adverse events which were not drug-related. For the remaining 8 subjects, up to a 4-fold enhancement of Hex A activity at doses of 50 mg per day or less was observed. Additionally marked individual variations in the pharmacokinetics of the drug among the patients were noted. However, the study also found that significant side effects were experienced by most patients at or above 75 mg pyrimethamine per day. We concluded that pyrimethamine treatment enhances leukocyte Hex A activity in patients with late-onset GM2 gangliosidosis at doses lower than those associated with unacceptable side effects. Further plans are underway to extend these trials and to develop methods to assess clinical efficacy.

The activity of selected glycosidases in salivary gland tumors.

The monitoring of the patients after salivary gland tumors surgery is an important clinical issue. Still imperfect diagnostic procedures also remain a challenge for searching new sensitive and specific biomarkers of neoplastic processes in salivary glands. The aim of the presented study was an the assessment of the activity of HEX, with its isoforms HEX-A and HEX-B, GLU, GAL, MAN and FUC in salivary gland tumor tissues in comparison to a healthy salivary gland tissues taken during autopsy. A group of 42 patients with benign and malignant salivary gland tumors, aged 25-65 were examined. Fragments of salivary gland tumor tissue, fragments of healthy tissue removed during autopsy, blood serum and saliva were collected from patients with salivary gland tumors and healthy volunteers. In salivary gland tumor tissue the activity of HEX, HEX-A, HEX-B, GAL, FUC was considerably higher than in comparison to healthy salivary gland tissue and ascending trend of activity of GLU, MAN was also noticed. The activity of all lysosomal exoglycosidases in blood serum in patients with salivary gland tumors was considerably higher in comparison to healthy volunteers blood serum. The considerably higher activity of HEX, HEX-A, GLU, GAL, MAN, FUC and descending trend of activity of HEX-B were noticed in saliva of patients with salivary gland tumors in comparison to healthy volunteers. The assessment of HEX in blood serum and saliva of patients with salivary gland tumor can be possibly used in diagnostics and monitoring of salivary glands tumors.

N-acetyl-beta-D-hexosaminidase and its isoenzymes A and B in blood serum and urine, as a potential colon cancer markers.

BACKGROUND/AIMS: Evaluation of N-acetyl-beta-D-hexosaminidase (HEX), and its isoenzymes A (HEX A) and B (HEX B) activity in blood serum and urine as potential markers of colorectal cancer. METHODOLOGY: The study was performed in blood serum and urine of 32 patients with adenocarcinoma, 6 with adenocarcinoma mucinosum of the colon, and 20 healthy people. The activity of HEX, HEX A and HEX B was determined in blood serum and urine by spectrophotometric method of Marciniak et al. The concentration of CEA was determined in blood serum by immunoenzymatic method (MEIA). The concentration of protein was assessed by the Lowry method, whereas the concentration of creatinine in urine by the Jaffe method (without deproteinization). RESULTS: A significant increase in the concentration of HEX, HEX A and HEX B activity was proved in serum and urine of patients with colon adenocarcinoma. In patients with colon adenocarcinoma mucinosum, the higher activity of HEX was revealed in blood serum compared to healthy people, and the significantly higher activity of HEX and HEX B expressed as pKat/mg of creatinine, was found in urine. We observe a significant increase in the activity of HEX, HEX A and HEX B expressed in pKat/mg of creatinine was found in urine of patients bearing tumor of diameter 6.0-7.0 cm in comparison to patients with tumor of diameter 4.0-5.0 cm. CONCLUSIONS: The present study results suggest that determination of HEX, HEX A and HEX B activity in blood serum and urine may be used to detect colon cancer in its early stages. However, the use of HEX, HEX A and HEX B activity in oncological diagnostics requires further studies on a larger group of patients.

Isoenzymes A and B of N-acetyl-beta-D-hexosaminidase in serum and urine of patients with pancreatic cancer.

BACKGROUND/AIMS: Adenocarcinoma is the most frequent malignant tumor of the pancreas. Biochemical diagnostics of pancreatic adenocarcinoma is based on determination of carcinoma antigen (CA 19-9) in the blood. Determination of N-acetyl-beta-hexosaminidase (HEX) in the serum and urine was used in diagnosis of renal and gastric cancers. Therefore the aim of our research was to estimate N-acetyl-beta-hexosaminidase (HEX) and its isoenzymes (HEX A and HEX B) in the serum and urine as potential markers of pancreatic cancer. METHODOLOGY: Serum and urine samples were collected from 15 patients with adenocarcinoma of the pancreas and 15 healthy persons. The activity of N-acetyl-beta-hexosaminidase and its isoenzymes (A and B) was determined by a colorimetric method of Zwierz et al. Absorbancy of the yellow product of the colorimetric reaction was determined on the microplate reader EL(x)800 produced by BIO-TEK. The concentration of HEX, HEX A and B was expressed in pKat/mL, and the specific activity in pKat/mg of protein. Protein concentration was determined in the serum by the biuret and in the urine by the Lowry method, respectively, and expressed in mg/mL. RESULTS: The concentration and specific activity of HEX and its isoenzyme A were significantly higher in the serum and urine of pancreatic cancer patients in comparison with the concentration and specific activity in the serum and urine of healthy people. CONCLUSIONS: The results suggest that the activity of HEX and its isoenzyme A determined in the serum and urine can be used as a potential marker of pancreatic adenocarcinoma.