UHPLC-MS/MS method for pharmacokinetic and bioavailability determination of five bioactive components in raw and various processed products of Polygala tenuifolia in rat plasma.

CONTEXT: Sibiricose A5 (A5), sibiricose A6 (A6), 3,6'-disinapoyl sucrose (DSS), tenuifoliside A (TFSA) and 3,4,5-trimethoxycinnamic acid (TMCA) are the main active components of Polygala tenuifolia Willd. (Polygalaceae) (PT) that are active against Alzheimer's disease. OBJECTIVE: To compare the pharmacokinetics and bioavailability of five active components in the roots of raw PT (RPT), liquorice-boiled PT (LPT) and honey-stir-baked PT (HPT). MATERIALS AND METHODS: The median lethal dose (LD50) was evaluated through acute toxicity test. The pharmacokinetics of five components after oral administration of extracts of RPT, LPT, HPT (all equivalent to 1.9 g/kg of RPT extract for one dose) and 0.5% CMC-Na solution (control group) were investigated, respectively, in Sprague-Dawley rats (four groups, n = 6) using UHPLC-MS/MS. In addition, the absolute bioavailability of A5, A6, DSS, TFSA and TMCA after oral administration (7.40, 11.60, 16.00, 50.00 and 3.11 mg/kg, respectively) and intravenous injection (1/10 of the corresponding oral dose) in rats (n = 6) was studied. RESULTS: The LD50 of RPT, LPT and HPT was 7.79, 14.55 and 15.99 g/kg, respectively. AUC 0- t of RPT, LPT and HPT were as follows: A5 (433.18 ± 65.48, 680.40 ± 89.21, 552.02 ± 31.10 ng h/mL), A6 (314.55 ± 62.73, 545.76 ± 123.16, 570.06 ± 178.93 ng h/mL) and DSS (100.30 ± 62.44, 232.00 ± 66.08, 197.58 ± 57.37 ng h/mL). The absolute bioavailability of A5, A6, DSS, TFSA and TMCA was 3.25, 2.95, 2.36, 1.17 and 42.91%, respectively. DISCUSSION AND CONCLUSIONS: The pharmacokinetic and bioavailability parameters of each compound can facilitate future clinical studies.

Disaccharidase Deficiencies in Children With Chronic Abdominal Pain.

OBJECTIVES: Carbohydrate intolerance or malabsorption has been suggested as a cause of chronic abdominal pain (CAP) in a subset of patients. We aimed to evaluate disaccharidase deficiencies in children with functional CAP and to correlate deficiencies with clinical features. METHOD: Patients presenting to the gastroenterology clinic at Children's Hospital of Wisconsin with abdominal pain prospectively completed a detailed demographic, history, and symptom questionnaire. The CAP cohort included those with at least 1 month of symptoms. Data on disaccharidase activity and histology of endoscopic biopsies were collected retrospectively. Only patients with normal histology were included in the study. The association between groups with low disaccharidases and clinical features was examined. RESULTS: A total of 203 pediatric patients with CAP were included. The mean (SD) age was 11.5 (3.1) years, and 32.5% were male. The percentages of abnormally low disaccharidase levels using the standard laboratory cutoffs were lactase, 37%; sucrase, 21%; glucoamylase, 25%; and palatinase, 8%. Thirty-nine percent of the patients with low lactase also had low sucrase, and 67% of the patients with low sucrase had low lactase. There was no significant difference in the activities of any of the disaccharidases or sucrase/lactase ratio in relation to age. Also, no association was found between stool consistency, stool frequency, or location of pain and low disaccharidase activity. CONCLUSIONS: A large proportion of patients with CAP have deficiencies in disaccharidases. Bowel frequency, vomiting, or location of pain was no different between groups, suggesting that these clinical features cannot be used to predict disaccharidase deficiencies.

Presenting Pattern of Pediatric Celiac Disease.

OBJECTIVES: Celiac disease (CD) is an autoimmune disease that can be complicated by impaired nutrition and growth. With the development of sensitive serologic tests, safe endoscopy, and efforts to educate primary care physicians, more children are diagnosed as having CD. The aim of this study is to evaluate the pattern of the presentation of pediatric CD in western New York. METHODS: Chart review of pediatric patients with CD was undertaken. Patients' demographics, presenting features, disaccharidase assay (DA), celiac serology, and Marsh score were reviewed from patients seen at the Digestive Diseases and Nutrition Center, State University of New York at Buffalo from January 2003 through March 2013. RESULTS: A total of 165 patients with CD were evaluated. Mean age was 10.7 ± 4.3 years, 76 male patients. The presenting features were abdominal pain (n = 87, 52.7%), constipation (n = 65, 38.9%), diarrhea (n = 52, 31.1%), family history of first-degree relative (n = 47, 28.1%), diabetes mellitus type 1 (n = 37, 22.2%), failure to thrive (n = 36, 21.8%), reflux (n = 25, 15.1%), vomiting (n = 24, 14.5%), fatigue (n = 15, 9%), short stature (n = 9, 5.4%), thyroid disease (n = 9, 5.4%), Down syndrome (n = 8, 4.8%). We found no correlation between Marsh score and serum tissue transglutaminase (tTG) immunoglobulin (Ig) A level at diagnosis and no correlation between DA and serum tTG IgA level, presenting feature and tTG IgA level, presenting feature and Marsh score, tTG IgA and DA, or between the age and the presenting feature. CONCLUSIONS: Children newly diagnosed as having CD in western New York presented most frequently with abdominal pain and constipation and were older at the time of diagnosis than those described in the classical presentation of CD. We speculate that our patients may have a different long-term natural history and risk factors than originally described for patients with CD.

Low trehalase activity is associated with abdominal symptoms caused by edible mushrooms.

BACKGROUND: The purpose of the study was to evaluate whether maldigestion of trehalose causes abdominal symptoms and which available diagnostic method best distinguishes intolerant from tolerant subjects. METHODS: A 25-g oral trehalose load test was performed in 64 subjects. The 19 experiencing clear symptoms constituted the trehalose-intolerant subjects. Changes from base-line levels of blood glucose, breath hydrogen, and methane and symptoms were recorded after the test. Trehalase activity was determined in serum and on a duodenal biopsy specimen obtained by endoscopy. RESULTS: Intolerant subjects were best differentiated from tolerant subjects by changes in breath gases (hydrogen and methane) and duodenal trehalase to sucrase ratio. The change in breath gases correlated inversely with duodenal trehalase activity, duodenal trehalase to sucrase ratio, and plasma trehalase activity. The correlation between serum and duodenal trehalase activities was on the order of 0.6. Two subjects were found to have trehalase deficiency. CONCLUSIONS: It is obvious that trehalose maldigestion can cause symptoms similar to those of lactose maldigestion and intolerance. Three factors control the genesis of symptoms: 1) the activity of small-bowel trehalase: if it is low, trehalose is maldigested and more trehalose is passed into the colon; 2) the maldigested trehalose, which causes osmotic water flow into the colon, resulting in loose stools and diarrhea; and 3) most importantly, the microflora of the colon, from which symptoms will arise if there are bacteria capable of producing gases from maldigested trehalose. If colonic bacteria cannot produce gases, then distention of the abdomen and intestinal gas expulsion as eructations and flatus will not occur.

[Determination of disaccharidase activity in rat serum, bile, urine and various tissues by high-performance liquid chromatography with differential refractometric detection].

A simple and high sensitive method for the determination of disaccharidase in various biological samples of rats by high-performance liquid chromatography (HPLC) with a differential refractometric detection was developed. This method, as compared with the Dahlqvist method, has the following advantages; 1) There is no necessity for the elimination of biological components which have an influence on the determination of glucose. 2) Only a little enzyme sample is used. 3) The detection limit of a reaction product, glucose, by the HPLC method is about 10 times higher than that by the Dahlqvist method. 4) The maltase activity in the small intestinal mucosa estimated by HPLC method is positively correlated with that estimated by the Dahlqvist method (r = 0.9661, p less than 0.001, n = 8). 5) This method developed in the present study is applicable to not only the small intestinal mucosa, but also serum, bile, urine and various tissues.

One-hour blood-xylose test in diagnosis of cow's milk protein intolerance.

In this prospective study, 18 consecutive children suspected of cow's milk protein intolerance (C.M.P.I.) on clinical grounds were investigated before and after challenge with cow's milk. One-hour blood-xylose, serum-IgE, eosinophil count, serum-complement (C3) and a jejunal biopsy specimen for histology and disaccharidase activity were obtained from all patients before challenge. Serum-complement was measured again 24 h after the beginning of the challenge and the first three tests were repeated on the fifth day. A second jejunal biopsy was obtained in 9 of the first 10 children. 15 of the 18 children were considered to have C.M.P.I. The one-hour blood-xylose test emerged as a simple and valuable test in the diagnosis of C.M.P.I. A significant drop (51--81%) in the one-hour blood-xylose level was observed in all 15 patients. In all patients but one it dropped below 25 mg/dl. Mean value (+/- S.D.) for the group was 47.5 +/- 11.1 mg/dl before and 17.2 +/- 4.2 mg/dl after cow's milk ingestion.

[Insulin and disaccharidases levels of the small intestine of the rat (author's transl)]. / Insulina e níveis das dissacaridases do intestino delgado do rato.

It was taken 32 male Wistar rats, weighting between 130 g and 150 g, free feeding, to study the total and specific activities of lactase, invertase and maltase of small intestine of rats. The animals were divided by chance in 3 experimental and 1 control group. 1. group--Aloxanic diabetes rats: treated with 1 unit of NPH insulin every day: after the 4th day of aloxane administration, all rats were killed. 2. group--Aloxanic diabetes rats--treated for 5 days with 1 unit of NPH insulin every day; after the 5th day until the 7th they were treated with 4 units of NPH insulin and were also killed. 3. group--Hyperinsulinism rats--Normal rats were treated for 4 days with 4 units of NPH insulin every day. After the 5th day they were killed. 4. group--Control group--Normal rats, free feeding. They were observed during 4 days and were also killed. The results showed that none difference was observed in the 4 groups of rats about the total and specific activities of lactase, invertase and maltase of the small intestine. In this study, all the animals with aloxanic diabetes were treated with insulin. Then, it is possible that the insulin inhibited the stimulator effect of the diabetes upon the dissacaridases of the small intestine of the rats.

Human leucocyte alpha-L-fucosidase.

1. Human alpha-L-fucosidase (EC 3.2.1.51) was studied from leucocytes, urine and serum. 2. The leucocyte and urine enzymes are similar in many properties (KM, pH optimum, electrophoretic pattern, heat stability). 3. The serum alpha-L-fucosidase differs from the leucocyte and 4rine enzyme wit,respect to: electrophoretic pattern, pH optimum and heat stapility. 4. The molecular weight of leucocyte alpha-L-fucosidase was determined to be 80 000 +/- 5000. 5. Cu2+, Hg2+ and PCMB are strong inhibitors of leucocyte alpha-L-fucosidase. This inhibition could be completely reversed by beta-mercaptoethanol, indicating that thiol groups are essential for catalytic activity.

Neutral alpha-mannosidase activity in human serum.

Two types of alpha-mannosidase (alpha-D-mannoside mannohydrolase, EC 3.2.1.24) with neutral pH optima exist in serum. The activity with an optimum between pH 6.0 and 6.4 is similar to alpha-mannosidase C, described earlier in tissues. The second activity, with a pH optimum between pH 5.2 and 5.8 is the dominant form in serum. These two forms can be differentiated from each other by gel-filtration, chromatography on DEAE-cellulose or chromatography on Concanavalin-A Sepharose. Using the chromatographic techniques, the serum type neutral activity co-elutes with the acidic forms of the enzyme. However, these two forms can be easily distinguished by effect of pH, heating or inhibition by the substrate methyl-alpha-D-mannopyranoside. The presence of the serum type alpha-mannosidase activity is discussed with respect to mannosidosis, a lysosomal storage disease.

Plasma alpha-L-fucosidase: presence of a low activity variant in some normal individuals.

The expression of a variant alpha-L-fucosidase has been determined in both plasma and leukocytes. This variant enzyme is characterized by unusually tow actuvity in plasma, an altered pH activity profile, and increased heat lability. Individuals possessing this low activity variant in plasma have usual levels of alpha-L-fucosidase activity in leukocytes. The leukocyte enzyme also shows increased heat lability and has an altered pH activity profile. Gel filtration of plasma alpha-L-fucosidase revealed both a high molecular weight form, alpha-L-fucosidase I, and a low molecular weight form, alpha-L-fucosidase II. Only alpha-L-fucosidase II could be detected upon gel filtration of the variant enzyme from plasma. Both control and variant leukocyte alpha-L-fucosidase revealed two peaks by gel filtration but the proportion of alpha-L-fucosidase I was reduced in the variant enzyme sample. Alpha-L-fucosidase II from both control and variant plasma and leukocyte samples was heat labile and possessed little activity at pH 4.0 Alpha-L-fucosidase I from control plasma and leukocytes was more stable to heating and possessed more activity at pH 4.0 than alpha-L-fucosidase II. In contrast, alpha-L-fucosidase I from the variant leukocyte sample was heat labile and had little activity at pH 4.0 thus resembling alpha-L-fucosidase II. These results suggest that the variant is the consequence of an altered allele which affects alpha-L-fucosidase expression in all tissues.

[Lysosomal enzyme activity in the serum of rats with experimental lead poisoning]. / Aktovnost' lizosomal'nykh fermentov v syvorotke krovi drys pri éksperimentalnoi intoksikatsii svintsom

Examination of the effect of experimental lead poisoning on permeability of lysosomal membranes in albino rats demonstrated activation of lysosomal enzymes (alpha-manosidase and beta-acetylglucosaminidase) in the blood serum as soon as the third day after daily administration of lead acetate (20 mg/kg). Apparently damage of the lysosomal membrane played an important role in the pathogenesis of lead poisoning.

[L-alanyl-L-proline-dipeptidase and glycyl-L-valine-depeptidase in malabsorption syndrome]. / L-Alanyl-L-Prolin-Dipeptidase und Glyzyl-L-Valin Dipeptidase beim Malabsorptionssyndrom

On the basis of comparative determinations of the activities of dipeptidases and disaccharidases of the mucous membrane of the small intestine (proximal jejunum) clear correspondences between the morphological findings and the biochemical parameters were the result. L-alanyl-L-prolin-dipeptidase and glycyl-L-valin-dipeptidase as well as lactase, saccharase, maltase and trehalase were determined in altogether 45 children with various malabsorption syndromes of different age in different stages of disease. Diminutions of the activity of the dipeptidases were to be proved analogously to maltase, saccharase and lactase, too, in most cases of subtotal or total villous atrophy. From the results conclusions may be derived to the restricted ability of protein absorption in chronic disease of the small intestine.

Biochemical and genetic studies of plasma and leukocyte alpha-L-fucosidase.

Deficiency of alpha-L-fucosidase in plasma and leukocytes has been demonstrated in three patients affected with fucosidosis. The measurement of plasma fucosidase activity alone is of little diagnostic value. Several normal individuals were found to have extremely low plasma alpha-L-fucosidase activity, but normal activity in leukocyte preparations. The low plasma enzyme activity exhibited by clinically normal individuals appears to be an inherited characteristic. The plasma enzyme was found to be different from that of leukocytes in terms of electrophoretic mobility.

[Alpha-L-fucosidase activity in human blood]. / Al'fa - L-Fukozidaznaia aktivnost' cheloveka.

The alpha-L-fucosidase activity was studied in blood plasma and cells. In leucocytes and blood plasma of healthy persons the fucosidase activity varied considerably. The fucosidase activity in blood was not altered under pathological conditions other than fucosidosis. The fucosidase activity did not depend on sex and age of the persons studied. No correlation was observed between the values of the fucosidase activity. Two forms of fucosidase (alpha-L-fucosidase I and alpha-L-fucosidase II) were found in human leucocytes and blood plasma. Isoelectric focusing experiments indicates multiple forms alpha-L-fucosidase from human serum. The possibility is discussed of the use of human blood plasma and cells not only for estimation of the total fucosidase activity but also for studies of different forms of the enzyme.

[Changes in the serum activity of lysosome enzymes in rats after application of the insecticide propoxur (Baygon)]. / Promeni v serumnata aktivnost na niakoi lizozomni enzimi u plukhove sled prilaganeto na insektitsida propoksur (baigon).

The effect of repeated peroral introduction (4 times) of 1/4 and 1/20 LD50 of monomethylcarbamate insecticide Propoxur (Baygon) on the activity of two lysosomal enzymes (beta-N-acetylglucosaminidase and alpha-mannosidase) is studied in the serum of white male rats with starting weight 300 g. Upon using the higher dose, a statistically significant rise (p less than 0.02) of the activity of either of the enzymes is established. The results obtained are interpreted as a sign of impaired permeability of the lysosomal and cell membrane.