Determination of adenosine phosphates in whole blood by capillary zone electrophoresis.

The pool of chemical energy in an organism represented by high-energy compounds can be assessed by means of adenosine triphosphate (ATP) determination in whole blood and tissues. The elegant manner for the determination of adenosine phosphates (ATP, ADP, AMP) in a single assay is offered by the technique of capillary zone electrophoresis. For this purpose, the BioFocus 3000 Capillary Electrophoresis System (BIO-RAD Laboratories, Inc., Hercules, CA, USA) was used. For the construction of calibration curves, pure preparations of ATP, ADP and AMP were analyzed. The method was used for adenosine phosphates determination in the umbilical blood samples from physiological and immature newborns. Capillary zone electrophoresis enables a specific and simultaneous determination of adenosine phosphates and, thus, monitoring of unusual metabolic situations.

[The human genome with special emphasis on chromosome 1]. / Lidský genom se zvlástním zretelem k chromozomu c. 1.

The development of knowledge of human genomes to the present stage was influenced by three specific genetic methodologies. Chromosomology after the beginning of the fifties, genetics of somatic cells, which began in the mid-sixties and molecular genetics which started at the end of the seventies (1). All methodologies contributed in their time to the mapping of genes and the construction of gene maps. The submitted review is devoted in particular to chromosome 1. Information pertaining to gene mapping helps to elucidate the evolution, organization of controlling mechanisms of the pathogenesis of hereditary disease, neoplasias, malformations for prenatal and premorbid diagnosis and perspectively for gene therapy.

Urinary amylase isoenzymes and amylase polymorphism variants in families with diabetes mellitus type 1.

Insulin-dependent diabetes mellitus type 1 is an autoimmune disease of pancreatic beta-cells with a certain genetic predisposition that is not yet clear. In spite of the confirmed association of diabetes mellitus type 1 with several HLA haplotypes it is considered that other loci must be involved for total genetic susceptibility to the disease. The relationship of insulin deficiency and decreased pancreatic amylase activity suggests that insulin itself is a direct activator of amylase gene expression. Endocrine pancreatic function was monitored by the indirect non-invasive method of urinary pancreatic amylase activity determination (expressed in percentage of total amylase activity) in diabetic children, their parents, healthy sisters and brothers, and in a separate group of women with diabetes type 1 of over 20 years duration. The incidence of hereditary amylase polymorphism variants in these subjects was also ascertained. Decreased pancreatic amylase activity in urine (under 58%) was found to be a characteristic trait in diabetics, and a susceptibility trait in asymptomatic family members. Normal pancreatic amylase activity (66.7 +/- 5.4%) is rare in diabetic patients type 1, but may be seen as a favourable prognostic trait, representing resistance to diabetic complications. The results support the suggestion that hereditary predisposition to the disease is inherited from the father rather than the mother, and that heterozygous amylase polymorphism variants protect their carriers against diabetes mellitus type 1.

Haplotype distribution at the phenylalanine hydroxylase locus in PKU families from the Moravian area of Czechoslovakia.

The analysis of 21 families affected with classical phenylketonuria (PKU) from the Moravian area of Czechoslovakia has revealed 12 different RFLP haplotypes. Nine and eight haplotypes were associated with the normal and with the mutant alleles, respectively. Most normal alleles are associated with haplotype 1 (42.9%). Almost 80% of all mutant alleles are confined within only three haplotypes (1, 2 and 4). There was a strong association between haplotype 2 and the Czech mutant alleles (61.9% of the mutant alleles compared with 4.8% of the normal alleles). There was linkage disequilibrium between this haplotype and the R408W mutation in exon 12. Two mutant haplotypes 7 were found and in both cases they were tightly linked with G272ter mutation. Our finding is in agreement with observations in other Eastern European countries. These data provide further support for the theories of the spread of the R408W mutation from east to west in European populations.

[Liver diseases in children with alpha 1-antitrypsin deficiency in infancy]. / Onemocnení jater u detí s deficitem alfa-1-antitrypsinu v kojeneckém veku.

The authors evaluate the health status of children with alpha-1-antitrypsin deficiency, focused on liver disease in infant age. The children were selected by neonatal screening. Of 21 children one had severe neonatal hepatitis with progression to cirrhosis, 2 children had clinically apparent jaundice to the age of two months, 6 children had elevated total bilirubin and transaminase levels without clinical signs of the disease, 12 of the remaining children had no clinical and laboratory signs of liver disease. In the discussion the authors compare the results with data published abroad.

[Screening for alpha 1-antitrypsin deficiency in neonates]. / Screening deficitu alfa-1-antitrypsinu v novorozeneckém veku.

In a two-year investigation 113,274 children were screened for alpha-1-antitrypsin deficiency. An original and cheap method was used. In children with an alpha-1-antitrypsin values lower than 1.5 g/l the phenotype was assessed. In 120 neonates alpha-1-antitrypsin was assessed by screening and also quantitatively. The physiological range of alpha-1-antitrypsin for neonates is: 1.4-3.32 g/l. A low incidence of alpha-1-antitrypsin in the Czechoslovak population, as compared with investigations abroad, was revealed. The authors discuss the possibility to extend screening from the clinical, ethical and economic aspect to the entire republic.

[Insulin, glucose, proteins and amylase in the saliva of obese children]. / Inzulín, glukóza, bílkoviny a amyláza ve slinách obézních detí.

The authors examined the insulin, glucose, total protein concentrations and amylase activity in the saliva of normal (n = 7) and obese subjects (n = 14) before and after a meal. The variability of the values of the investigated parameters in different subjects is considerable. During repeated examinations of the same normal subjects after a prolonged time interval the responses under similar condition in saliva is 17.7 +/- 13.8 microU/ml, when the mean maximum in the 120th minute is 24.7 +/- +13.9 microU/ml. The glucose concentration is on average 2.1 +/- 1.3 mg/dl, total protein 279.5 +/- 53.2 mg/dl and the amylase activity 226 +/- 133 thousand U/l. In the dynamics of the investigated parameters in obese subjects the concentration of insulin and the other parameters are on average higher than the maximum insulin level in normal children, and in three obese children they were more than four or five times higher. The gradual progressive hypersecretion of insulin may thus imply a disposition for type II diabetes mellitus at a later age.

Polymorphic DNA haplotypes at the phenylalanine hydroxylase (PAH) locus in European families with phenylketonuria (PKU).

DNA haplotype data from the phenylalanine hydroxylase (PAH) locus are available from a number of European populations as a result of RFLP testing for genetic counseling in families with phenylketonuria (PKU). We have analyzed data from Hungary and Czechoslovakia together with published data from five additional countries--Denmark, Switzerland, Scotland, Germany, and France--representing a broad geographic and ethnographic range. The data include 686 complete chromosomal haplotypes for eight RFLP sites assayed in 202 unrelated Caucasian families with PKU. Forty-six distinct RFLP haplotypes have been observed to date, 10 unique to PKU-bearing chromosomes, 12 unique to non-PKU chromosomes, and the remainder found in association with both types. Despite the large number of haplotypes observed (still much less than the theoretical maximum of 384), five haplotypes alone account for more than 76% of normal European chromosomes and four haplotypes alone account for more than 80% of PKU-bearing chromosomes. We evaluated the distribution of haplotypes and alleles within these populations and calculated pairwise disequilibrium values between RFLP sites and between these sites and a hypothetical PKU "locus." These are statistically significant differences between European populations in the frequencies of non-PKU chromosomal haplotypes (P = .025) and PKU chromosomal haplotypes (P much less than .001). Haplotype frequencies of the PKU and non-PKU chromosomes also differ significantly (P much less than .001. Disequilibrium values are consistent with the PAH physical map and support the molecular evidence for multiple, independent PKU mutations in Caucasians. However, the data do not support a single geographic origin for these mutations.(ABSTRACT TRUNCATED AT 250 WORDS)

Determination of the molecular weight of alpha-amylase, as the enzyme-inhibitor complex, using thin layer gel filtration on Sephadex.

Thin layer gel filtration on Sephadex was performed as a simple method for recognition of the small molecular weight differences of human alpha-amylases from different sources. Activity was located with dry chromogenic substrate, using a replica technique. Undesirable interaction between the gel matrix and substrate binding sites on the enzyme, which causes an anomalous decrease in the migration rate of the enzyme protein, was suppressed by preincubation of the enzyme with appropriate inhibitor. Gradual masking of substrate binding sites of the enzyme by increasing concentrations of amylase inhibitors resulted in two distinct migration rates for the enzyme in thin layer gel filtration. This suggests the existence of two substrate binding sites in the enzyme molecule. Together with thin layer gel affinity chromatography on a mixture of Sephadex and ConA-Sepharose, the method yielded useful data on the molecular weight of amylase and its glycosylated forms and served as a valuable tool for the differential diagnosis of macroamylasaemia.

beta-Glucuronidase activity in amniotic fluid.

A bimodal distribution of the beta-glucuronidase activities was observed within a group of amniotic fluid samples obtained from women by transabdominal amniocentesis in the third trimester of complicated gestations: lower enzyme activities were found in the larger subgroup and extremely high enzyme activities in the smaller subgroup. There was no relation between the enzyme activity and pregnancy complications. However, a sex-relationship was observed, in that all the children from all the pregnancies showing very high enzyme activities were boys. This may be due to the known stimulating effect of androgens on the specific activity of beta-glucuronidase.

Urinary isoamylases in juvenile diabetics.

An anomalous ratio of salivary to pancreatic amylase activities has been observed in urine from juvenile diabetics. Decreased pancreatic amylase activity in urine from these subjects appears to be a characteristic trait.