Study of the molecular mechanism of decreased liver synthesis of albumin in inflammation.

Hypoalbuminemia in inflammatory disorders is not an infrequent finding. However, little is known about albumin synthesis in these patients. In the present study we have measured the albumin synthesis in four patients with inflammatory diseases using the [14C]carbonate technique. Because inflammation causes a decreased albumin synthesis and this decreased synthesis could not be related to a reduced amino acid supply, we have also examined the possible molecular mechanisms of reduced albumin synthesis during inflammation using in vivo and in vitro experiments in rats. In rats with turpentine-induced inflammation, serum albumin concentration and liver albumin mRNa level were markedly decreased. These changes could not be reproduced by administration of fibrinogen-, or fibrin-degradation products, or several hormones, such as corticosteroids, growth hormone, and adrenaline. However, monocytic products, especially interleukin 1, postulated to be important mediators of the inflammatory response, reduced albumin synthesis and liver albumin messenger RNA content but not total protein synthesis in rats in vivo and in primary cultures of rat hepatocytes. These findings suggest that monocytic products play an important role in reduced albumin synthesis during inflammation.

Decreased plasma albumin concentration results in increased volume of distribution and decreased elimination of midazolam in intensive care patients.

The pharmacokinetic parameters of 16 patients in the intensive care unit, sedated with midazolam, were evaluated. A large variation was observed in the plasma concentration of midazolam and between the plasma concentration of midazolam and its metabolite 1-hydroxymethylmidazolam glucuronide. The plasma albumin concentration governs the volume of distribution of midazolam. Decreased plasma albumin concentration (25 gm/L) results in an increased volume of distribution and a decreased elimination rate of midazolam. The observed plasma concentration ratio between the parent drug and its metabolite 1-hydroxymethylmidazolam glucuronide is governed by the variables of protein binding, the metabolic rate of midazolam, and the renal clearance of the glucuronide metabolite itself (which can be considered as a measure of the kidney function of the patient).

Important role of tryptophan on albumin synthesis in patients suffering from anorexia nervosa and hypoalbuminemia.

The rate of albumin synthesis and the serum levels of amino acids were measured in three patients suffering from anorexia nervosa. In comparison to the control group the rate of albumin synthesis was lower for the two patients with hypoalbuminemia and the serum levels of valine, isoleucine and tryptophan also were decreased. Intravenous administration of tryptophan alone failed to increase the serum albumin level in one patient. In another patient, a 3-week period of infusion of amino acids without tryptophan failed to correct the rate of albumin synthesis or the serum albumin level. The serum albumin level rose from 37.1 g/liter to 46.8 g/liter after the infusion of amino acids plus tryptophan. The reduced supply of amino acids as the most important factor in the decrease in albumin synthesis and the important role of tryptophan in the regulation of albumin synthesis are discussed.

Estimation of radiation dosage and transmutation effect of 14-C involved in measuring rate of albumin synthesis with 14-C-carbonate.

For direct measurement of the rate of albumin synthesis Na2 14CO3 was used intravenously. The assessment of the radiation hazard involved in the study was based on the knowledge of the minimum dose of Na214CO3 necessary for a sufficient incorporation of 14C in the guanidine-C of arginine in albumin to obtain measurable radioactivity. By measurement of expired 14CO2 and excreted 14C-urea in the urine during a 5-hr period following intravenous administration of Na214CO3 in five subjects, some quantitative data on 14C retention and radiation dosage were obtained. In comparison with animal studies, the rate of expiration of 14CO2 in man is slower. About 50% of the total radioactivity injected was lost through the respiratory route in the first hour. The total amount of expired 14C during the 5 hr of investigation was about 75% of the injected dose for the five subjects. The amount of 14C excreted as urinary 14C-urea during the 5 hr of investigation is very small in comparison with the expired 14C; it was only about 0.5% of the dose injected. The total absorbed radiation dose after complete elimination of 14C from the body was calculated with various assumptions. The extra risk of genetic damage due to disintegration of retained 14C in comparison with that of natural 14C in the body during 30 living years is about 50%.

Effect of immunoneutralisation of cholecystokinin on bombesin-stimulated pancreatic enzyme secretion in the rat.

Infusion of bombesin stimulates plasma cholecystokinin (CCK) and pancreatic enzyme secretion in various species, including the rat. This study was undertaken in two groups of four conscious rats with a cannulated pancreatic duct to determine the role of endogenously released CCK in mediating the effect of bombesin on pancreatic enzyme secretion. Infusion of 2 ml CCK antiserum or normal rabbit serum for 40 min was followed 10 min later by infusion of 18 pmol/kg bombesin for 30 min and after an interval of 90 min by infusion of 24 pmol/kg CCK for 30 min. After administration of control rabbit serum, pancreatic protein secretion increased by 3.2 +/- 1.0 mg/30 min during bombesin and 4.0 +/- 1.5 mg/30 min during CCK, while the plasma CCK increments were 1.7 +/- 0.5 pM and 7.0 +/- 0.9 pM for the bombesin and CCK infusions, respectively. Immunoneutralisation with the CCK antiserum did not significantly affect bombesin-stimulated pancreatic protein secretion (3.6 +/- 1.3 mg/30 min), but almost abolished the pancreatic protein response to CCK (0.5 +/- 0.2 mg/30 min). It is therefore concluded that CCK is not an important mediator of the stimulatory effect of bombesin on the pancreas in the rat.

An enzyme immunoassay for the determination of lipase in human duodenal fluid.

The application of a commercially available enzyme immunoassay method for the determination of lipase concentration in human duodenal fluid is reported. Variations in the test procedure have been investigated. A sample incubation and a conjugate incubation of 1 h at 37 degrees C are necessary. There is substantial agreement between the determination of lipase in duodenal fluid measured according to this enzyme immunoassay and a turbidimetric method. The output of pancreatic lipase, alpha-amylase, and trypsin in four healthy volunteers during infusion of stepwise increasing doses of cholecystokinin was measured. A parallel increase of the output of these three enzymes has been observed, and there is a correlation between the activity concentrations of alpha-amylase and trypsin and the content of lipase in the various samples. This study shows that lipase in duodenal fluid can be easily and reliably determined with this enzyme immunoassay.

Evaluation of tests of exocrine and endocrine pancreatic function in older patients with cystic fibrosis.

In order to determine the value of noninvasive tests in the analysis of pancreatic function in cystic fibrosis, 14 older cystic fibrosis patients were studied by a set of noninvasive tests of exocrine and endocrine pancreatic function. The tests, comprising trypsin, total amylase, pancreatic isoamylase, lipase, pancreatic polypeptide (PP), glucose and insulin in fasting serum, PP, glucose and insulin in postprandial serum, and p-aminobenzoic acid (PABA) excretion in urine, were compared to fecal fat excretion after discontinuation of pancreatic enzyme supplementation. Eleven of the 14 patients were found to have a fecal fat excretion of more than 7 g/day. Serum levels of trypsin, pancreatic isoamylase and lipase, and the urinary excretion of PABA showed significant negative correlations with fecal fat excretion. Endocrine pancreatic function was abnormal in the majority of patients with fibrocystic disease. Although serum trypsin, postprandial PP, and urinary PABA excretion were the most sensitive tests for severe exocrine pancreatic insufficiency, the differences in sensitivity were rather modest. Therefore, the type of test to be selected for clinical use is mainly dependent upon factors as accessibility, simplicity, patient's acceptability, and costs.

Role of cholecystokinin in bombesin-stimulated pancreatic enzyme secretion in conscious rats.

Since bombesin is a potent stimulus of cholecystokinin (CCK) secretion, it is assumed that the stimulatory effect of bombesin on pancreatic protein secretion is mediated by CCK. This study was undertaken to determine in the conscious rat with a cannulated pancreatic duct the role of CCK in the stimulation of pancreatic protein secretion by bombesin. Infusion of 18 pmol/kg/30 min of bombesin into rats stimulated pancreatic protein secretion from 6.7 +/- 1.1 to 9.9 +/- 0.4 mg/30 min (p less than 0.05). This stimulation of pancreatic protein secretion was accompanied by a significant increase in plasma CCK, measured by a specific and sensitive radioimmunoassay, from 3.2 +/- 0.2 to 4.7 +/- 0.2 pM (p less than 0.01). When a similar plasma CCK increment as during infusion of bombesin (1.5 +/- 0.2 pM) was achieved by infusion of 6 pmol/kg/30 min of exogenous CCK (1.6 +/- 0.3 pM), pancreatic protein secretion increased only from 6.9 +/- 0.7 to 7.6 +/- 0.7 mg/30 min (p less than 0.05). To achieve a pancreatic protein secretion similar to that during bombesin, large doses of exogenous CCK (24 pmol/kg/30 min) were needed, resulting in considerably higher plasma CCK concentrations of 10.9 +/- 0.7 pM. It is concluded that CCK is unlikely to play a significant role in the stimulation of pancreatic protein secretion by bombesin in the rat.

An enzymic method for the determination of the glycine/taurine ratio of conjugated bile acids in bile.

A method is described in which the ratio of the glycine- to taurine-conjugated bile acids (G/T ratio) in bile is determined. After pretreatment of the bile for removal of the lipids, the bile acids are deconjugated enzymically with choloylglycine hydrolase (EC 3.5.1.24). The amino acids taurine and glycine are liberated in this way, after appropriate isolation, taurine and/or glycine are then determined with ninhydrin, enabling the establishment of the G/T ratio. A nearly complete hydrolysis was obtained for 6 conjugated bile acids, while the recovery of these acids when added to hog or ox bile was quantitative. The mean G/T ratio for hog bile, ox bile and human B-bile was 6.3, 2.5 and 2.0, respectively. The amount of total, free and conjugated bile acids can be determined by this method, combined with the 3alpha-hydroxysteroid dehydrogenase technique for bile acid determination described by Iwata and Yamasaki [1]. A high G/T ratio was observed in 3 cases of Crohn's disease in the small bowel, but the extent of deconjugation in B-bile was lower than in duodenal fluid. The determination of the G/T ratio can be complementary to our knowledge of the metabolism of bile salts in certain gastro-intestinal disorders.

Standardization of the alkaline phosphatase determination with human placental phosphatase.

The influence of changes in temperature and salt concentrations on the enzymic activity of 3 different alkaline phosphatase preparations has been examined. Beef liver phosphatase is labile; the activity is already lost by lyophilization. Hog intestinal and especially human placental phosphatase are more stable. In contrast to reconstituted sera no preincubation is necessary to restore the enzymic activity. A lyophilized human placental phosphatase, kept at room temperature, showed no change in activity in a period of about one year. A mean value of enzymic activity of 133 U/l was observed with a coefficient of variation of 2.4% for 12 determinations. It is suggested to assign a value to such a preparation and to use it as a primary standard in the determination of the alkaline phosphatase.

Serum versus heparinized plasma for alanine aminotransferase and aspartate aminotransferase of normal individuals.

The influence of heparin on the estimation of aminotransferases was investigated in a reaction medium containing phosphate or Tris buffer. Heparin scarcely affects the ASAT determination, but the ALAT activity in plasma is about 70% of the activity in serum, when the enzyme determination is carried out in Tris buffer. There exists a lag phase due to which the decrease in absorbance with the incubation time is nonlinear. When the Tris buffer is omitted, the same lag phase is observed in the ALAT determination. This lag phase is not caused by the indicator enzyme LDH. Addition of phosphate to the incubation medium without any buffer abolishes this lag phase. The possibility exists that heparin influences the structure of the proteins present in plasma and that these proteins, in their turn, decrease the enzymatic activity of ALAT.

Relationship between plasma pyridoxal-5'-phosphate concentration and the apoenzyme content of serum aminotransferases in patients with a renal allograft.

In 76 patients with a renal allograft the plasma concentration of pyridoxal-5'-phosphate, the activity concentrations of aspartate aminotransferase and alanine aminotransferase and the percentage stimulation of both enzymes have been investigated. Generally, the concentration of the coenzyme was decreased in the patient group compared to normal individuals. The percentage increase of aspartate aminotransferase is the same for the patients as for the normal group. However, the stimulation of alanine aminotransferase is greatly increased for the patient group except for those patients who received cyclosporine A instead of azathioprine. The highly significant inverse correlation between the plasma pyridoxal-5'-phosphate concentration and the percentage stimulation of the enzymatic activity of aspartate aminotransferase and alanine aminotransferase observed before for a normal group, is not present in our patient group. The percentage increase is independent on the activity concentration of the enzymes. It is concluded that the high stimulation of alanine aminotransferase depends on the administration of azathioprine.

Behaviour of L-gamma-glutamyl-4-nitroanilide and L-gamma-glutamyl-3-carboxy-4-nitroanilide with respect to gamma-glutamyltransferases of different origin.

In this paper we compare the measurement of catalytic activity concentrations of gamma-glutamyltransferase with the non-carboxylated and the carboxylated substrate in preparations of different origin. Fresh human sera, commercial test sera and preparations of gamma-glutamyltransferase purified from human liver, porcine kidney and bovine kidney were used as sample materials. When assayed with both substrates preparations of gamma-glutamyltransferase from bovine kidney behaved in a different manner as did the enzyme in preparations from human liver or porcine kidney and the enzyme in fresh human sera. On account of the results obtained with both substrates we classified the commercial test sera for their enrichment using multi-inductive component analysis. The differences observed for the various methods of determination seem to have significance in quality control.

Apoenzyme content of serum aminotransferases in patients with a renal allograft treated with cyclosporine A and azathioprine.

In 16 patients with a renal allograft the activity concentrations of aspartate aminotransferase and alanine aminotransferase and the percentage stimulation of both enzymes were investigated. After the transplantation the patients received prednisone and cyclosporine A as immunosuppressive therapy, while exactly 3 months after the date of transplantation prednisone and azathioprine were given as immunosuppressives. In the first period, the percentage increase of the activity concentration of aspartate aminotransferase and alanine aminotransferase upon supplementation of pyridoxal-5'-phosphate in vitro were similar to that of healthy individuals. In the second period, however, the percentage increase of the activity concentration of alanine aminotransferase was much higher than that of aspartate aminotransferase. Cyclosporine A given during a period of about 400 days did not influence the percentage increase of both enzymes. It is concluded that the high stimulation of alanine aminotransferase in the second period depends on the presence of azathioprine or its metabolites in serum.

Serum trypsin, alpha-amylase and lipase during bombesin stimulation in normal subjects and patients with pancreatic insufficiency.

The effect of infusion of bombesin (60 pmol/kg 20 min) on pancreatic enzymes in serum was studied in 13 normal subjects and 12 patients with pancreatic insufficiency. In normal subjects administration of bombesin induced large increases in serum trypsin (p less than 0.01), while serum total alpha-amylase and pancreatic alpha-amylase did not change and serum lipase showed only a modest rise (0.01 less than p less than 0.05). Patients with pancreatic insufficiency had significantly lower serum concentrations of all enzymes studied (p less than 0.01) and in such patients bombesin did not change the concentrations of pancreatic enzymes in serum. It is concluded that determination of the serum trypsin response to bombesin may be of help in the diagnosis of pancreatic insufficiency.

Physiological plasma concentrations of cholecystokinin stimulate pancreatic enzyme secretion and gallbladder contraction in man.

The present study was undertaken to determine whether infusion of cholecystokinin (CCK) to plasma concentrations comparable to those found after a meal stimulates pancreatic enzyme secretion and gallbladder contraction. Plasma CCK concentrations were measured by radioimmunoassay using antibody T204, which binds to all carboxyl-terminal CCK-peptides containing the sulfated tyrosine region. Ingestion of a standardized test meal in 7 normal subjects induced significant increases in plasma CCK from 2.0 +/- 0.2 pmol/l to levels between 4.6 +/- 0.6 and 7.3 +/- 1.0 pmol/l (p less than 0.05-p less than 0.0005). Infusion of 2.5 pmol/kg X h CCK 33 resulted in significant increases in plasma CCK from 2.0 +/- 0.2 to 3.9 +/- 0.3 pmol/l (p less than 0.0005). This infusion of CCK induced significant increases in trypsin secretion from 0.5 +/- 0.1 to 1.4 +/- 0.2 KU/15 min (p less than 0.005) and in bilirubin output from 1.6 +/- 0.7 to 30.3 +/- 8.0 mumol/15 min (p less than 0.05). It is concluded that physiological plasma concentrations of CCK stimulate pancreatic enzyme secretion and gallbladder contraction in man.

Influence of pyridoxal-5'-phosphate on temperature relationship of aspartate aminotransferase and alanine aminotransferase.

The influence of pyridoxal-5'-phosphate on the temperature relationship of aspartate aminotransferase and alanine aminotransferase has been determined. On varying the reaction temperature from 25 degrees to 45 degrees both enzymes showed small but significant deviations from a linear Arrhenius relationship. Supplementation with pyridoxal-5'-phosphate in general enhanced the catalytic activity to the same degree over the entire temperature range. Our results corroborate the findings of Rej but not those of Jung et al.

Excretion and metabolism of a series of xanthines by the turtle Pseudemys scripta elegans.

The xanthines paraxanthine, xanthine, and uric acid are absorbed completely by the turtle Pseudemys scripta elegans and metabolised presumably into CO2 and NH3. No intermediate metabolites are excreted. Caffeine, hydroxycaffein, theobromine, hydroxytheobromine, and hydroxypraxanthine are absorbed and excreted in different amounts or percentages of the dose administered. Intermediate metabolites of these compounds are not excreted. The turtle Pseudemys scripta elegans may, therefore, be ammonotelic.