Clearance of phenylalanine ammonia-lyase from normal and tumor-bearing mice.

Yeast phenylalanine ammonia-lyase was administered i.p. to normal and tumor-bearing mice, and its clearance from plasma was studied. Single and multiple weekly injections at dosages of 10,20,50 and 100 units/kg were administered to C57BL female, C57BL X DBA/2F1 male, and A/J female mice. L5178Y murine lymphoblastic leukemia, B16 melanoma, BW10232 adenocarcinoma, and 15091A anaplastic carcinoma were implanted 7 to 11 days prior to enzyme injection in the appropriate host. After a single injection, the average plasma half-lives of phenylalanine ammonia-lyase were 18 to 24 hr in all groups studied. While the other tumors had no effect on the plasma level of phenylalanine ammonia-lyase after a single injection, L5178Y murine lymphoblastic leukemia and 15091A anaplastic carcinoma significantly depressed the maximal level of phenylalanine ammonia-lyase attained in the plasma. After repeated injections of phenylalanine ammonia-lyase, the initial plasma enzyme level was significantly reduced when 20 units/kg were administered, and the clearance of the enzyme from the plasma was greatly accelerated regardless of the amount administered. Furthermore, in tumor-bearing mice, the rate of clearance was significantly more rapid than in the appropriate non-tumor-bearing control.

Induction of porphobilinogen oxygenase and porphobilinogen deaminase in rat blood under conditions of erythropoietic stress.

Porphobilinogen is the substrate of two enzymes: porphobilinogen deaminase and porphobilinogen-oxygenase. The first one transforms it into the metabolic precursors of heme and the second diverts it from this metabolic pathway by oxidizing porphobilinogen to 5-oxopyrrolinones. Rat blood is devoid of porphobilinogen-oxygenase under normal conditions while it carries porphobilinogen-deaminase activity. When the rats were submitted to hypoxia (pO2 = 0.42 atm) for 18 days, the activity of porphobilinogen-oxygenase appeared at the tenth day of hypoxia and reached the maximum at the 14-16th day. It decreased to a half after 2 days (half-life of the enzyme) and disappeared after 4 days of return to normal oxygen pressure. Porphobilinogen-deaminase activity increased after the first day of hypoxia, reached a maximum at the 14-16th day and did not decrease to normal values until the 15th day after return to normal oxygen pressure. The activities of both porphobilinogen-oxygenase and porphobilinogen-deaminase were induced by administration of erythropoietin. When rats were made anaemic with phenylhydrazine, porphobilinogen-oxygenase activity also appeared in the blood cells. Although the reticulocyte concentration was higher when compared to that obtained under hypoxia, the activities of the oxygenase obtained under both conditions were comparable. Porphobilinogen-deaminase activity was always closely related to the reticulocyte content. The appearance of porphobilinogenase-oxygenase under the described erythropoietic conditions was due to a de novo induction of the enzyme, as shown by its inhibition with actinomycin D and cycloheximide. Porphobilinogen-oxygenase as well as porphobilinogen-deaminase were present in the rat bone marrow under normal conditions. Their activities increased in phenylhydrazine treated rats. The properties and kinetics of porphobilinogen-oxygenase from the rat blood and bone marrow were determined and found it differ in several aspects.

Evidence of abnormality of lymphocyte uroporphyrinogen synthase in family members of patients with lymphoproliferative diseases.

Patients with active lymphoproliferative diseases (LPD) were shown to have high activity of lymphocyte uroporphyrinogen synthase (L-UROS), the enzyme which converts porphobilinogen to uroporphyrinogen. The mean L-UROS activity of 64 first-degree relatives of patients with LPD was significantly higher than that of a control group and 45% of these relatives had pathological values of L-UROS. L-UROS activity was also determined in the spouses of 2 patients and was pathologically elevated in both. The pattern of pathological values among family members may indicate the presence of a communicable agent.

Recurrence risk estimation of acute intermittent porphyria based on analysis of porphobilinogen deaminase activity: a Bayesian approach.

Red cell porphobilinogen deaminase is known to be an indicator of the carrier state for acute intermittent porphyria (AIP). This enzyme was assayed in three groups of individuals at least 15 years old: 105 affected individuals or obligate carriers, 234 unaffected first-degree relatives of patients, and 217 unrelated control persons. Analysis of the distribution of the control enzyme activities suggested presence of three commingled distributions. Also, the overlap between carrier-group and control-group values must be taken into account for genetic counseling of relatives whose enzyme activity lies within the overlap. A Bayesian approach is proposed to derive risks for these individuals, using the observed carrier and control distributions. The method is illustrated by deriving risks for a family from our sample.

Protoporphyrinaemia and decreased activities of 5-aminolevulinic acid dehydrase and uroporphyrinogen I synthetase in erythrocytes of a Vitamin B6-deficient epileptic boy given valproic acid and carbamazepine.

Carbamazepine (CBMZP) has been implicated as an inhibitor of the activities of 5-aminolaevulinic acid dehydratase (ALA-D) and uroporphyrinogen I synthetase (URO-S). In an epileptic boy undergoing long-term treatment with valproic acid (VPA), 1.3 g/d, CBMZP, 0.9 g/d and folic acid, 7.5 mg/d, decreased activities of ALA-D and URO-S coincided with increased levels of erythrocyte protoporphyrin (EP) in the absence of Pb poisoning, iron depletion and erythropoietic protoporphyria. A progressive fall in plasma pyridoxal 5'-phosphate (B6-P) to 7.7 nmol/L (lower reference limit, 14.6 nmol/L) prompted implementation of pyridoxine HCl (B6-HCl), 87.5 mg/d followed by administration of both B6-HCl and preformed B6-P (50 mg/d each). This permitted the eventual withdrawal of VPA and a net reduction of CBMZP to 450 mg/d. During these manipulations, ALA-D and URO-S activities, EP and urinary porphyrins and their precursors were measured serially. An assay system utilizing red cell ALA-D for generation of porphobilinogen (PBG) from added ALA at pH 7.4 was used for determination of ALA-D and URO-S activities in separate aliquots of the same assay mixture both in the absence and presence of Zn and dithiothreitol (DTT). One unit (U) for ALA-D = 1 nmol PBG/L RBC/s; for URO-S = 1 nmol porphyrin/L/s; minimum normal level for ALA-D = 135 U; for URO-S = 6 U. B6-HCl alone entailed increases in ALA-D and URO-S prior to any reduction of CBMZP. After administration of both B6-HCl and B6-P and withdrawal of VPA, the overall increase in ALA-D was from 54.59 to 197.2 U (-Zn; -DTT) and from 50.76 to 217.3 U (+Zn; +DTT). The overall increase in URO-S was from 2.67 to 8.90 U (-Zn; -DTT) and from 3.02 to 8.66 U (+Zn; +DTT). During stepwise reduction of VPA, EP remained elevated to values as high as 2.48 mumol/L (upper reference limit, 1.33 mumol/L). Only after permanent withdrawal of VPA did concentrations of EP fall to normal levels. Values for porphyrins and their precursors in urine were normal throughout. Since both VPA and B6-P are strongly protein-bound, it is suggested that VPA displaced B6-P from protective protein binding sites and that the resulting deficit in B6-P (rather than CBMZP) reduced ALA-D and URO-S activities via primary reduction of ALA-synthetase activity. Increases in EP emerge as a hitherto unappreciated effect of VPA warranting further investigation.

Variations in erythrocyte uroporphyrinogen I synthetase activity in non porphyrias.

The activity of erythrocyte uroporphyrinogen I synthetase has been measured in various cases of non-porphyric affections. The results indicate a diminution of this activity in some of the studied cases of chronic renal insufficiency and chronic polyarthritis. This modulation in activity mitigates against its use as a diagnostic criterion of acute intermittent porphyria. On the other hand, an increase in urosynthetase activity has been noted in acute and especially chronic hepatic affections. This increase seems to be connected with the severity of the hepatic affection. The relationship is illustrated particularly in the case of viral hepatitis associated to an AIP, where the increasing activity of the urosynthetase masks for many weeks the congenital deficiency peculiar to this AIP. Our study thus indicates that the diagnosis of AIP based on the activity of the urosynthetase must take into account the pathological context in which the investigation is realised.

The spectrophotometric determination of uroporphyrinogen I synthetase activity.

A simple spectrophotometric method for uroporphyrinogen I synthetase in erythrocytes is described. Results obtained on intermittent acute porphyria patients and carriers are similar to the results obtained with fluorimetric methods. Reproducibility, relationship between enzyme activity and enzyme concentration, and effect of time on enzymatic activity are described.

A filter paper dry blood spot procedure for acute intermittent porphyria population screening by use of whole blood uroporphyrinogen-I-synthase assay.

A filter paper dry blood spot procedure for the determination of whole blood uroporphyrinogen-I-synthase (UIS) activity is presented. The method is based on the concept of enzyme specific activity, the enzyme activity being related to the haemoglobin concentration of the assay sample. The diagnostic capacity with regard to the acute intermittent porphyria (AIP) gene carrier state is shown to be equivalent to that of a washed red cell reference method. On grounds of easy capillary blood sampling, uncomplicated and safe mail specimen transport and simple laboratory reception routines, the method is stated to be well adapted for use in AIP preadolescent population screening.

ELISA for measuring porphobilinogen deaminase in human erythrocytes.

An ELISA method has been developed to quantitate human porphobilinogen deaminase in erythrocyte lysate. The antiserum used in the assay was raised against the erythropoietic form of human porphobilinogen deaminase. The IgG fraction was characterized by use of immunoblotting technique, rocket immunoelectrophoresis and immunotitration and shown to be monospecific. The measuring range of the method was from 4 ng to 50 pg. Intra- and inter-assay coefficients of variation were 6% and 7%, respectively. Erythrocyte lysates from 97 apparently healthy individuals were assayed giving a mean erythrocyte porphobilinogen deaminase protein concentration of 150 +/- 28 SD (micrograms/g Hb) and a specific enzyme activity of 750 +/- 140 SD (nkat/g). Eight patients with acute intermittent porphyria were also investigated. A decreased concentration of enzyme protein, i.e. 84 +/- 13 SD (micrograms/g Hb) with a normal specific activity, was found.

Erythrocyte uroporphyrinogen I synthase activity as an indicator of acute porphyria.

The pre-clinical diagnosis of acute intermittent porphyria (AIP) is important because acute attacks can be brought about by drugs, liver toxins, hormonal changes and diet. There also may be no obvious precipitating agent. The discovery that the activity of uroporphyrinogen I synthase (URO-S) activity in the red blood cells of patients with AIP is half that found in normal persons is of great value in diagnosing this disorder and also appears useful in detecting patients with the latent disease who have normal urinary delta-aminolevulinic acid and porphobilinogen excretion. It also appears to distinguish other types of porphyria from acute intermittent porphyria. It must also be recognized that some red blood cells URO-S determinations will yield indeterminate results; therefore, repeat assays, including examination of kinship, will improve discrimination and confidence in the final diagnosis.

[Erythrocyte levels of ALA-dehydrase and uro-synthetase in chronic renal insufficiency (author's transl)]. / Estudio de los niveles eritrocitarios de ala-dehidrasa y uro-sintetasa en la insuficiencia renal crónica.

Erythrocyte levels of ala-dehydrase and uro-synthetase as well as hematocrit, hemoglobin, iron levels and serum iron binding capacity were studied in a group of 28 patients with chronic renal failure in hemodialysis and compared to those of a control group. As is usual, hematocrit, hemoglobin an iron levels were significantly decreased, as were ala-dehydrase levels, while uro-synthetase levels were within normal levels. These results are interpreted as a blockade of the biosynthetic pathway of the protoporphyrins, which lends support to the hypothesis of an existing defect in hemoglobin synthesis in patients with chronic renal failure.

[Effect of phenazepam and seduxen on the presence of histidase and urokinase in the blood]. / Vliianie fenazepama i seduksena na poiavlenie v krovi gistidazy i urokaninazy.

Liver tissue specific enzymes histidase and urokinase were found in blood of 160 rats after intraperitoneal administration of phenazepam at the doses of 1.0 and 2.5 mg/200 g of body weight as well as of diazepam at the dose of 2.0 mg/200 g. Administration of products of diazepam enzymatic degradation caused also the liberation of these enzymes from liver tissue into blood.

[Specific enzymes in the diagnosis of liver damage in burns]. / Spetsificheskie fermenty v diagnostike porazhenii pecheni pri ozhogovoi bolezni.

The activity of a number of enzymes and the content of the protein and protein fractures in the blood serum was assessed in 128 patients with burns and in 31 normal people. The activity of histidase and arginase were established to be more informative and to more correctly reflect the function of the liver in association with the degree of burn disease, square surface and depth of the injury.