Creatine kinase determination: a European evaluation of the creatine kinase determination in serum, plasma and whole blood with the Reflotron system.

We evaluated a new dry-reagent carrier system for the determination of creatine kinase (EC 2.7.3.2) activity, Reflotron CK, with special attention to analytical performance with whole blood. We found a good within series imprecision. The median coefficient of variation was 3.1% for Reflotron CK (blood, serum and plasma) and 0.9% for the automatic analysers (serum and plasma only). The between-days imprecision with Reflotron CK (median CV: less than or equal to 3%) was similar to that for the comparison method on different analysers. Fresh samples of human blood, plasma and serum were examined by Reflotron CK and by a N-acetylcysteine activated creatine kinase method in six different clinical laboratories and in the Evaluation Department of Boehringer Mannheim GmbH. The correlation between these methods was excellent (r greater than or equal to 0.99), the median systematic deviation (bias) for all samples being smaller than -5%. Haematocrits between 0.25 and 0.50, haemolysis up to 6 g/l haemoglobin, and icteric samples with bilirubin concentrations up to 0.2 g/l showed no interference. No drug in therapeutic concentration was found to affect the Reflotron CK results; ascorbic acid, calcium dobesilate and sulphamethoxazole lowered the values only when present in high concentrations. Reflotron CK may be considered as a suitable alternative for decentralized testing sites, especially in situations where creatine kinase results are needed quickly.

Influence of sulphasalazine and mesalazine on cellular and biochemical oxygen metabolite production. Effect of in vivo administration and an in vitro analysis.

Oxidative damage has been implicated in the pathogenesis of inflammatory bowel disease. In the present study sulphasalazine and mesalazine (5-aminosalicylic acid) in vitro were shown to possess scavenging activity and to attenuate the production of oxygen metabolites by neutrophils. In a double-blind randomized crossover study, with five patients with inflammatory bowel disease in remission and four healthy controls, we evaluated the influence of in vivo administration of sulphasalazine and mesalazine on the neutrophil oxygen metabolite production in vitro. Apart from a small but significant increase in the neutrophil H2O2 and O2 production by sulphasalazine, in particular in controls, in vivo administration of both drugs hardly affected the oxygen metabolite-producing capacity of the cells. This observation was confirmed by in vitro preincubation of neutrophils with the drugs and subsequent oxygen metabolite production analysis. It is concluded that sulphasalazine and mesalazine do not influence the oxidative capacity of neutrophils, but scavenge and attenuate the production of oxygen metabolites when present in the immediate surroundings of the cells. Thus, protection against oxidative damage is definitely one of the modes of action of these drugs.

Complexes of creatine kinase and immunoglobulins in serum identified by a nonimmune binding method.

We have developed a method for identifying IgG-complexed creatine kinase (CK; EC 2.7.3.2) (IgG-CK) and IgA-complexed CK (IgA-CK) in serum. We used immobilized Protein G to bind IgG-CK and immobilized jacalin to bind IgA-CK, leaving noncomplexed CK in solution. The noncomplexed CK and total CK were measured kinetically. The results are reported as CK bound to immobilized Protein G and CK bound to immobilized jacalin. We validated the method by using sera determined immunochemically to contain IgG-CK, IgA-CK, mitochondrial CK (CKmt), and free CK-BB. We demonstrated concomitant binding of CK and approximately 99% of IgG, and of CK and approximately 87% of IgA. For CK bound to immobilized Protein G and to immobilized jacalin, intra- and interassay precisions ranged from 2.5% to 9.6%, and detection limits were less than 9 318 U/L in 40 sera containing IgG-CK, and CK bound to immobilized jacalin ranged from 10 to 59 U/L in eight sera containing IgA-CK. These ranges represent the activities of immunoglobulin-bound CK in the sera. In 13 sera containing CKmt and in eight sera containing free CK-BB, the binding of CK was less than 9 U/L. Evidently, this method is useful for identifying IgG-CK and IgA-CK in serum.

Inhibition of cell mediated cytotoxicity by sulphasalazine: effect of in vivo treatment with 5-aminosalicylic acid and sulphasalazine on in vitro natural killer cell activity.

Decreased cell mediated cytotoxicity occurs frequently in inflammatory bowel disease, particularly in patients with active disease. It is not clear, however, whether this decrease is caused by the disease or is a consequence of the medical treatment. In this study we evaluated the effect of in vivo treatment with 5-aminosalicylic acid and sulphasalazine on the in vitro natural killer cell activity in five patients with inflammatory bowel disease in remission and in four healthy control subjects in a double blind randomised crossover trial preceded and separated by four weeks of treatment with placebo. The natural killer cell activity was significantly impaired in 67% (six of nine subjects) after four weeks' sulphasalazine treatment and tended to be related to subjects with a slow acetylator phenotype. In contrast, 5-aminosalicylic acid treatment caused only a marginal reaction in the natural killer cell activity in 22% (two of nine subjects). The inhibitory effects were found to be reversible since the decreased natural killer cell activity was completely restored after placebo treatment in all subjects. In conclusion, in vivo treatment with sulphasalazine inhibits the in vitro natural killer cell activity and this seems to be mediated by the sulphapyridine moiety. This phenomenon may contribute to the low natural killer cell activity found in patients with active inflammatory bowel disease.

Pancreatic carcinoma with polyarthritis, fat necrosis, and high serum lipase and trypsin activity.

A 46 year old white man presented with subcutaneous and intramedullary fat necrosis, destructive polyarthritis, and osteolytic bone lesions, complicating a poorly differentiated adenocarcinoma of the tail of the pancreas with metastases in the liver and omentum. There was a 100-fold increase in serum lipase and trypsin activity. His condition deteriorated rapidly, was characterised by rapid tumour growth, formation of ascites, a 20 kg weight loss, extensive subcutaneous fat necrosis, and fistula formation in the left calf. Treatment with 5-fluorouracil 300 mg/m2 on days 1-5 and doxorubicin 50 mg/m2 and cisplatin 100 mg/m2 on day 1, every three weeks, was well tolerated and resulted in rapid clinical improvement. After three courses of treatment a partial remission was seen and after seven courses further improvement occurred with a return to normal of serum lipase and trypsin activity. One year after starting chemotherapy the tumour relapsed but responded again to chemotherapy (epirubicin 40 mg/m2 and carboplatin 300 mg/m2 on day 1, every three weeks).

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.

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.

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.

Gamma-glutamyltransferase: evaluation of a new method.

The determination of gamma-glutamyltransferase using a new donor substrate, L-gamma-glutamyl-3,5-dibromo-4-hydroxyanilide, was evaluated in two laboratories. Between-run and within-run precision are excellent and comparable with those of other methods for the determination of gamma-glutamyltransferase. The activity concentrations of gamma-glutamyltransferase from human sera obtained with the new donor substrate were slightly higher than those obtained with L-gamma-glutamyl-4-nitroanilide. The reverse was found for sera enriched with gamma-glutamyltransferase of animal origin. Temperature conversion factors (activity 37 degrees C/activity 30 degrees C) are given. The advantages of this method are discussed. The new assay appears to provide a good alternative for the method with L-gamma-glutamyl-4-nitroanilide or one of its derivatives as substrate.

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).

Comparison of two methods for the determination of the isoenzymes of alpha-amylase in serum and urine.

In this paper we describe our results of the determination of the isoenzymes of alpha-amylase in serum and urine with two monoclonal antibodies, using 4,6-ethylidene-protected 4-nitrophenylmaltoheptaoside as substrate. For comparison we have used the wheat-germ inhibitor method with blue starch as substrate. The differences observed between each method were small. The technique using monoclonal antibodies is easy and reliable and can, therefore, replace the wheat-germ inhibitor method.

Some thermodynamic parameters of pancreatic and salivary alpha-amylase in serum.

Using monoclonal antibodies and 4,6-ethylidene-protected 4-nitrophenylmaltoheptaoside as substrate we determined the Arrhenius slope, the apparent energy of activation and the apparent enthalpy changes of total amylase, pancreatic amylase and salivary amylase in serum. The Arrhenius slope and hence the other thermodynamic parameters of pancreatic amylase are significantly different from those of total amylase or salivary amylase, but identical to those obtained for purified human pancreatic amylase. Similarly, the thermodynamic parameters of serum salivary amylase activity are the same as those of the purified enzyme. Temperature conversion factors are given for amylase and its isoenzymes. The results are discussed briefly.

Evaluation of a new alpha-amylase assay using 4.6-ethylidene-(G7)-1-4-nitrophenyl-(G1)-alpha-D-maltoheptaoside as substrate.

The determination of alpha-amylase activity using an ethylidene-blocked 4-nitrophenyl-maltoheptaoside (EPS) has been evaluated in five laboratories on eight different analysers at 25 degrees C, 30 degrees C and 37 degrees C. The protecting ethylidene group inhibits hydrolysis at the non-reducing end of the substrate molecule by the auxiliary enzyme, alpha-glucosidase. The combined reagent is therefore stable for at least 10 days at 2-8 degrees C. HEPES is used, because the molar absorbance of 4-nitrophenol is independent of temperature in the presence of this buffer. Compared with the method using unprotected substrate 4-nitrophenyl-alpha-D-maltoheptaoside (4NP-G7), the present method is equal or better with respect to the imprecision, linearity and interlaboratory transferability of results in human and control sera. Since the protected and unprotected substrates differ in their turnover rate, the new assay yields activities which differ from those of the 4-nitrophenyl-alpha-D-maltoheptaoside method. Based on the homogeneous results obtained in method comparisons between EPS and 4-nitrophenyl-alpha-D-maltoheptaoside, and in order to maintain the 4-nitrophenyl-alpha-D-maltoheptaoside reference values, a conversion factor was derived to eliminate the above differences: activityEPS x 2.50 = activity4NP-G7. The temperature and instrument independence of this relationship was demonstrated in a total of 720 human sera and plasmas.

Reference ranges for alpha-amylase in serum and urine with 4,6-ethylidene-(G7)-1-4-nitrophenyl-(G1)-alpha,D-maltoheptaoside as substrate.

Reference ranges for alpha-amylase in serum, spontaneously voided urine, and 24 h urine were determined, using 4,6-ethylidene-(G7)-1-4-nitrophenyl-(Gl)-alpha,D-maltoheptaoside as the substrate (EPS method), at 25, 30, and 37 degrees C. The measured values were evaluated with and without the use of a factor which converts the results of the alpha-amylase EPS method into values comparable to those obtained with the alpha-amylase PNP method (substrate: 4-nitrophenyl-alpha,D-maltoheptaoside); comparison with the established reference ranges of the PNP method was therefore possible. The values for urine sometimes deviated markedly from the PNP reference ranges, but the values for serum showed close agreement. With the use of the conversion factor, the following reference ranges are proposed for the new alpha-amylase method: Serum (186 males and 131 females): up to 120 U/l (25 degrees C), up to 160 U/l (30 degrees C), and up to 220 U/l (37 degrees C). Spontaneously voided urine: up to 600 U/l (n = 323, 25 degrees C), up to 800 U/l (n = 373, 30 degrees C), and up to 1000 U/l (n = 373, 37 degrees C). 24 h urine: up to 450 U/24 h (n = 90, 25 degrees C), up to 650 U/24 h (n = 129, 30 degrees C), and up to 900 U/24 h (n = 129, 37 degrees C).

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.

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.

Some thermodynamic parameters and temperature-conversion factors for determining alpha-amylase concentration in serum.

We measured the relationship between measured activity concentration and temperature for alpha-amylase (EC 3.2.1.1), using 10 different substrates. At 25-37 degrees C the Arrhenius plot was linear. The activation energy ranged from 33.3 kJ.mol-1 with maltoheptaose as substrate to 54.4 kJ.mol-1 with the Blue-Starch method. Activation energy was lowest for substrates having seven glucose moieties, the ones most suitable for determining alpha-amylase--i.e., the presence of more or fewer glycosyl units increased the activation energy. Substrates with blocked groups at the nonreducing end of the oligosaccharide chain were not considered here, because the relative reaction rates obtained with these substrates were less than those obtained with nonblocked substrates. We also determined temperature-conversion factors for alpha-amylase reaction with the various substrates. The results are discussed in relation to thermodynamic parameters of some other enzymes, e.g., creatine kinase and alkaline phosphatase.

The penetration of ofloxacin into lung tissue.

After a single oral 600 mg dose, ofloxacin concentrations were measured in lung tissue, whole blood and plasma in 11 patients undergoing thoracotomy for a bronchial malignancy. To correct for blood contamination in the tissue samples, the tissue haemoglobin content was measured using a method based on the binding of haemoglobin by haptoglobin. Ofloxacin concentrations in plasma and whole blood did not differ significantly. The calculated blood content in the tissue samples was 0.12 +/- 0.05 ml/g lung tissue. After correction for blood admixture, the mean lung tissue concentration 2 h after administration of ofloxacin was 17.7 +/- 9.2 micrograms/g. At the same time the mean plasma concentration was 8.7 +/- 4.2 mg/l (P less than 0.02). The high concentration of ofloxacin obtained in lung tissue does not result from the preparation technique. After a single 600 mg dose the tissue concentrations proved to exceed MIC values for most pathogens frequently involved in respiratory tract infections.