The role of interferon-gamma in the increased tuberculosis risk in type 2 diabetes mellitus.

As patients with diabetes mellitus are at increased risk of developing tuberculosis, we hypothesized that this susceptibility to mycobacterial infection is due to a defective Th1-cytokine response. To explore this hypothesis, we examined four groups of subjects in Indonesia: 23 patients with tuberculosis, 34 patients with tuberculosis and diabetes, 32 patients with diabetes only and 36 healthy controls. Ex-vivo production of interferon (IFN)gamma, tumour necrosis factor-alpha and interleukin (IL)-1beta, 6, 10, -12 and -4 was measured following stimulation with Mycobacterium tuberculosis, Escherichia coli lipopolysaccharide and phytohaemagglutinin. Patients with active tuberculosis were found to have lower IFNgamma levels and a higher production of other pro-inflammatory cytokines and IL-4, both in the presence and absence of diabetes. Diabetes patients without tuberculosis, however, showed strongly reduced non-specific IFNgamma production, which is essential for inhibition of the initial growth of M. tuberculosis. Our data suggest that a defective non-specific immune response in diabetes may contribute to an increased susceptibility to develop tuberculosis.

Reduced production of immunoregulatory cytokines in vitamin A- and zinc-deficient Indonesian infants.

OBJECTIVE: To determine effects of vitamin A, zinc and iron deficiency in Indonesian infants on the ability to produce immunoregulatory cytokines. DESIGN, SETTING AND SUBJECTS: Immunological assessment was done in 59 infants participating in a cross-sectional nutritional survey in rural West Java, Indonesia. Production of T-helper cell type-1 (Th1, cell-mediated) cytokines interferon-gamma (IFN-gamma), interleukin-12 (IL-12), interleukin-18 (IL-18) and T-helper cell type-2 (Th2, humoral) cytokine interleukin-6 (IL-6) were measured after stimulation with lipopolysaccharide and phytohemagglutinin in an ex vivo whole blood culture system. Circulating neopterin concentrations were determined as an indicator of in vivo macrophage activity. RESULTS: Of the infants, 48% were vitamin A deficient, 44% were anemic (with 17% having iron deficiency anemia), and 17% were zinc deficient. Vitamin-A deficient infants had significantly reduced ex vivo production of IFN-gamma, but also significantly higher circulating neopterin concentrations. Production of IFN-gamma and IL-12 were strongly correlated, IFN-gamma and IL-18 production were not. Zinc deficiency was accompanied by significantly reduced white blood cell counts and reduced ex vivo production of IL-6. Iron status was not related to cytokine production. CONCLUSIONS: This study shows that in vitamin A deficiency there is Th1 dominance in a steady state, combined however with impairment of the Th1 response after stimulation, whereas in zinc deficiency, there is a decreased Th2 response. Overall, vitamin A deficiency and zinc deficiency have marked albeit different effects on the immunocompetence of infants, affecting both cell-mediated and humoral components of the immune system.

Disease-specific ex vivo stimulation of whole blood for cytokine production: applications in the study of tuberculosis.

As a simple method for field studies to assess the cytokine-status of patients with tuberculosis (TB), the use of whole blood instead of isolated cells has advantages, especially since the risk of contamination is minimal. Therefore, cytokine production in whole blood cultures was determined using non-specific and disease-specific stimuli. Heparinized blood from healthy volunteers was either incubated in closed vacutainer tubes or in tissue culture wells after dilution in culture medium. Dose-response and kinetics were investigated for the production of TNFalpha, IL-1beta, IL-1ra, IL-10 and IFNgamma. Patients with TB and healthy individuals were examined for IFN-gamma production in whole blood. In the absence of a stimulus, the production of cytokines is negligible in whole blood cultures. LPS induces the production of TNFalpha, IL-1beta, IL-1ra and IL-10; PHA induces the production of IFNgamma and IL-10. Live BCG induces the production of proinflammatory cytokines, irrespective of tuberculin skin status. In contrast, PPD and MTB-culture filtrate induce production of IFNgamma in skin-test positive and not in skin-test negative healthy subjects. Five out of 13 patients with TB had a low antigen-specific IFNgamma production, suggestive of a minimal or absent specific T-cell response. For most purposes, cultures in closed vacutainer tubes are optimal. If one wishes to focus on T-cell cytokines or if only small volumes of blood are available, dilution of whole blood in culture medium before incubation in tissue culture wells may be preferable.

Plasma patterns of tumor necrosis factor-alpha (TNF) and TNF soluble receptors during acute meningococcal infections and the effect of plasma exchange.

In 39 patients with acute meningococcal infections, the plasma concentrations of tumor necrosis factor-alpha (TNF) and its soluble receptors (sRs) TNFsR-p55 and TNFsR-p75 were measured from admission till recovery. At admission, patients with shock had significantly higher TNF, TNFsR-p55, and TNFsR-p75 values than patients without shock. In addition, during the first 24 hours, patients with shock had higher TNFsR-p75 to TNFsR-p55 ratios, indicating that in shock the increase of TNFsR-p75 exceeds that of TNFsR-p55. TNF measured more than 12 hours after admission failed to differentiate between shock and nonshock because TNF concentrations normalized within 12-24 hours. However, because concentrations of TNFsRs remained elevated for 5-6 days, at that time plasma TNFsRs still differentiated between shock and nonshock. Plasma exchange or whole blood exchange (PEBE), performed in 20 patients with shock, accelerated the decrease of plasma TNFsRs. However, because of a rebound after each PEBE session, the overall half-lives of both TNFsRs were not affected by PEBE.

Elective orthopedic surgery, a model for the study of cytokine activation and regulation.

Induction and downregulation of cytokine production occurs after contact with various inflammatory stimuli. To elucidate the early events after a physical stressor, we studied these processes in 13 patients undergoing elective total hip replacement surgery. In these patients we followed the plasma concentrations of tumour necrosis factor alpha (TNF), interleukin 1beta (IL-1beta), IL-6 and IL-1 receptor antagonist (IL-1Ra), mRNA for these cytokines in peripheral blood cells (PBCs), and the lipopolysaccharide(LPS)-stimulated ex vivo production of these cytokines in whole blood cultures (WBCs). Plasma TNF and IL-1beta were not affected by the surgical procedure, although IL-1beta mRNA levels in PBCs increased significantly. Plasma IL-6 and IL-1Ra increased from 2 to 3 h post-incision onwards. The LPS-induced ex vivo production in WBCs of TNF, IL-1beta and IL-6 decreased from 2 h post-incision; that of IL-1Ra increased. Downregulation of TNF production was not associated with lower TNF-mRNA suggesting post-transcriptional regulatory processes. In contrast, downregulation of IL-1beta production was associated with significantly lower IL-1beta mRNA, suggesting both post-transcriptional and transcriptional mechanisms. Remarkably, the increase of plasma IL-6 occurred when the IL-6 production ex-vivo in WBCs was maximally downregulated. This suggests that other immunocompetent cells and not PBCs are the source for plasma IL-6 and that the IL-6 production in those other cells might be regulated differentially.

The pattern of interleukin-1beta (IL-1beta) and its modulating agents IL-1 receptor antagonist and IL-1 soluble receptor type II in acute meningococcal infections.

Interleukin-1beta (IL-1beta) is considered an important mediator in the pathogenesis of septic shock or bacterial meningitis. Its activity is specifically modulated by IL-1 receptor antagonist (IL-1Ra) and IL-1 soluble receptor type II (IL-1sRII). We now describe the time-course of IL-1beta and these modulating agents in 59 patients with acute meningococcal infections, the prototype human disease of acute endotoxin exposure. Plasma IL-1beta was increased only in severe shock and normalized within 12 to 24 hours, indicating that patients were admitted in an early stage of cytokine activation. Increased IL-1beta values in cerebrospinal fluid (CSF) were confined to patients with meningitis. Plasma IL-1Ra was elevated in both shock and nonshock patients, extremely high values being measured in severe shock. High concentrations of IL-1Ra in CSF were found in meningitis. Plasma IL-1Ra peaked shortly after IL-1beta and decreased steeply in 1 to 2 days, followed by sustained moderately elevated levels in shock patients. Interestingly, IL-1sRII showed a completely different pattern. At admission, both nonshock and shock patients manifested a similar moderate increase of plasma IL-1sRII. However, during recovery plasma IL-1sRII further increased reaching maximal concentrations 3 to 5 days after admission, 1 to 2 days after normalization of IL-1Ra. In shock patients this increase was more prominent than in nonshock patients. It is hypothesized that this increase in plasma IL-1sRII can be explained by a synergistic effect of dexamethasone and endotoxin. A second interesting observation was that, unlike the pattern in plasma, IL-1sRII levels in CSF paralleled those of IL-1beta and IL-1Ra. This suggests different modulation of IL-1beta activity in the subarachnoid space and the plasma compartment. We conclude that: (1) During the early stage of meningococcal infections IL-1Ra modulates IL-1 activity, whereas during recovery IL-1sRII may be more important. (2) Modulation in CSF and in the plasma compartment are differentially regulated.

The effects of dexamethasone and chlorpromazine on tumour necrosis factor-alpha, interleukin-1 beta, interleukin-1 receptor antagonist and interleukin-10 in human volunteers.

Tumour necrosis factor-alpha (TNF-alpha) and interleukin-1 beta (IL-1 beta) are pro-inflammatory cytokines that play an important role in severe infections, whereas IL-1 receptor antagonist (IL-1ra) and IL-10 are anti-inflammatory cytokines that counteract their effects. Chlorpromazine and dexamethasone protect mice against lethal endotoxaemia by decreasing circulating concentrations of TNF-alpha and IL-1 beta. We investigated whether administration of chlorpromazine or dexamethasone to human volunteers is able to modulate the lipopolysaccharide (LPS)-stimulated cytokine production capacity in whole blood. Blood samples were taken before and several time-points after medication. Circulating cytokine concentrations were low in all samples. LPS-induced TNF-alpha and IL-1 beta production in whole blood was inhibited by dexamethasone treatment, while chlorpromazine had no effect. When peripheral blood mononuclear cells were stimulated in vitro with LPS, the addition of chlorpromazine (1-100 ng/ml) had no modulatory action on TNF-alpha, IL-1 beta, IL-1ra or IL-10 synthesis. The chlorpromazine concentrations measured in circulation of volunteers were eight to 40 times lower than the concentrations shown to be effective in mice. In conclusion, chlorpromazine inhibits TNF-alpha and IL-1 beta production in mice at concentrations that cannot be reached in humans, thus precluding its usage in clinical anti-cytokine strategies. In contrast, dexamethasone is an effective inhibitor of pro-inflammatory cytokine production.

Pro- and anti-inflammatory cytokines in healthy volunteers fed various doses of fish oil for 1 year.

Dietary supplementation with n-3 fatty acids from fish oil alleviates inflammation in various chronic inflammatory disease states. Reductions in the production of pro-inflammatory cytokines interleukin 1 beta (IL-1 beta), tumour necrosis factor alpha (TNF-alpha), and interleukin 6 (IL-6) have been seen in humans after short-term n-3 fatty acid supplementation. We investigated long-term effects of dietary n-3 fatty acids on circulating cytokine concentrations and on ex vivo stimulated whole-blood production of IL-1 beta, TNF-alpha and interleukin 1 receptor antagonist (IL-1Ra), the naturally occurring antagonist of IL-1. A total of 58 monks with a mean age of 56 years were randomized into four groups and their diets were supplemented with 0, 3, 6, or 9 g of fish oil, providing 0, 1.06, 2.13 or 3.19 g of n-3 fatty acids per day. Subjects received equal amounts of saturated fatty acids, vitamin E and cholesterol. Compliance was excellent and erythrocyte fatty acid profiles closely reflected the amounts of n-3 fatty acids ingested. In the group receiving 9 g of fish oil per day, no influence of n-3 fatty acids on circulating cytokine concentrations was observed relative to placebo. Endotoxin-stimulated whole-blood cytokine production was measured at 26 and 52 weeks after the start and at 4, 8 and 26 weeks after cessation of supplementation. In all groups, the production of IL-1 beta and IL-1Ra was higher during supplementation than afterwards. However, no differences in cytokine production were noted between the placebo group and the various treatment groups at any point in time. Our results suggest that long-term supplementation of fish oil does not affect ex vivo cytokine production in man.

Lymphocyte subsets, apoptosis, and cytokines in patients with chronic fatigue syndrome.

Whether immunologic abnormalities correlate with fatigue severity and functional impairment in chronic fatigue syndrome (CFS) was investigated. Blood mononuclear cells were immunophenotyped and circulating ex vivo-produced cytokines were measured in 76 CFS patients and 69 healthy matched controls. Expression of CD11b on CD8 cells was significantly decreased in CFS patients. However, the previously reported increased expression of CD38 and HLA-DR was not confirmed. There was no obvious difference in apoptosis in leukocyte cultures, circulating cytokines, and ex vivo production of interleukin (IL)-1 alpha and IL-1 receptor antagonist. Endotoxin-stimulated ex vivo production of tumor necrosis factor-alpha and IL-beta was significantly lower in CFS. The immunologic test results did not correlate with fatigue severity or psychologic well-being was measured by Checklist Individual Strength, Beck Depression Inventory, and Sickness Impact Profile. Thus, these immunologic tests cannot be used as diagnostic tools in individual CFS patients.

Endurance run increases circulating IL-6 and IL-1ra but downregulates ex vivo TNF-alpha and IL-1 beta production.

We investigated whether a 6-h endurance run changes cytokine plasma concentrations and lipopolysaccharides (LPS) stimulated ex vivo production of cytokines in a whole blood culture of 19 well-trained athletes. The average distance covered was 65.1 +/- 8.64 (SD) km. At the end of the exercise, the mean plasma concentration of interleukin-1-receptor agonist (IL-1ra), which was 188 pg/ml 24 h before finish, increased to 886 pg/ml (P < 0.0005). The mean plasma interleukin-6 concentration increased from 18.5 +/- 4.2 to 71.5 +/- 33.3 pg/ml (P < 0.0001). The increase of neutrophils correlated with the increase of IL-1ra concentrations (r = 0.58, P < 0.005). We could not detect an effect of exercise on plasma concentrations of interleukin-1 beta (IL-1 beta) or tumor necrosis factor-alpha (TNF-alpha). The ex vivo LPS-stimulated production of IL-1 beta in athletes 24 h before the run was significantly higher than in sedentary controls. Exercise induced a decrease of LPS-stimulated production of IL-1 beta and TNF-alpha, whereas production of IL-1ra was unchanged. These results show that prolonged exercise elicits a selective downregulation of the proinflammatory cytokine production and upregulation of the cytokines IL-1ra and interleukin-6.

Correlation between proinflammatory cytokines and antiinflammatory mediators and the severity of disease in meningococcal infections.

Pro- and antiinflammatory cytokines and mediators were measured in 39 patients with acute life-threatening meningococcal infections classified into 3 groups: A, meningitis without shock (n = 20); B, meningitis with shock (n = 9); and C, shock without meningitis (n = 10). The plasma concentrations of proinflammatory endotoxin, tumor necrosis factor-alpha (TNF-alpha), interleukin (IL)-6, and IL-8 and antiinflammatory cytokines and mediators IL-1 receptor antagonist, IL-10, and soluble TNF receptors p55 and p75 were strongly associated with this classification; the highest concentrations were in group C. IL-4 was not measurable. IL-1 beta was increased only in rapidly fatal cases. In addition, cerebrospinal fluid (CSF) was analyzed in 21 patients for TNF-alpha and its soluble receptors. In CSF, these compounds were mainly increased in group A, reflecting an intrathecal compartmentalized cytokine production. It is concluded that both pro- and antiinflammatory mediators are simultaneously increased and are strongly associated with a classification based on simple clinical parameters.

Cytokine activation during attacks of the hyperimmunoglobulinemia D and periodic fever syndrome.

The hyperimmunoglobulinemia D and periodic fever (hyper-IgD) syndrome is typified by recurrent febrile attacks with abdominal distress, joint involvement (arthralgias/arthritis), headache, skin lesions, and an elevated serum IgD level (> 100 U/mL). This familial disorder has been diagnosed in 59 patients, mainly from Europe. The pathogenesis of this febrile disorder is unknown, but attacks are joined by an acute-phase response. Because this response is considered to be mediated by cytokines, we measured the acute-phase proteins C-reactive protein (CRP) and soluble type-II phospholipase A2 (PLA2) together with circulating concentrations and ex vivo production of the proinflammatory cytokines interleukin-1 alpha (IL-1 alpha), IL-1 beta, IL-6, and tumor necrosis factor alpha (TNF alpha) and the inhibitory compounds IL-1 receptor antagonist (IL-1ra), IL-10, and the soluble TNF receptors p55 (sTNFr p55) and p75 (sTNFr p75) in 22 patients with the hyper-IgD syndrome during attacks and remission. Serum CRP and PLA2 concentrations were elevated during attacks (mean, 213 mg/L and 1,452 ng/mL, respectively) and decreased between attacks. Plasma concentrations of IL-1 alpha, IL-1 beta, or IL-10 were not increased during attacks. TNF alpha concentrations were slightly, but significantly, higher with attacks (104 v 117 pg/mL). Circulating IL-6 values increased with attacks (19.7 v 147.9 pg/mL) and correlated with CRP and PLA2 values during the febrile attacks. The values of the antiinflammatory compounds IL-1ra, sTNFr p55, and sTNFr p75 were significantly higher with attacks than between attacks, and there was a significant positive correlation between each. The ex-vivo production of TNF alpha, IL-1 beta, and IL-1ra was significantly higher with attacks, suggesting that the monocytes/macrophages were already primed in vivo to produce increased amounts of these cytokines. These findings point to an activation of the cytokine network, and this suggests that these inflammatory mediators may contribute to the symptoms of the hyper-IgD syndrome.

Patterns of proinflammatory cytokines and inhibitors during typhoid fever.

Cytokines and inhibitors in plasma were measured in 44 patients with typhoid fever. Ex vivo production of the cytokines was analyzed in a whole blood culture system with and without lipopolysaccharide (LPS). Acute phase circulating concentrations of cytokines (+/- SD) were as follows: interleukin (IL)-1 beta, < 140 pg/mL; tumor necrosis factor-alpha (TNF alpha), 130 +/- 50 pg/mL; IL-6, 96 +/- 131 pg/mL; and IL-8, 278 +/- 293 pg/mL. Circulating inhibitors were elevated in the acute phase: IL-1 receptor antagonist (IL-1RA) was 2304 +/- 1427 pg/mL and soluble TNF receptors 55 and 75 were 4973 +/- 2644 pg/mL and 22,865 +/- 15,143 pg/mL, respectively. LPS-stimulated production of cytokines was lower during the acute phase than during convalescence (mean values: IL-1 beta, 2547 vs. 6576 pg/mL; TNF alpha, 2609 vs. 6338 pg/mL; IL-6, 2416 vs. 7713 pg/mL). LPS-stimulated production of IL-1RA was higher in the acute than during the convalescent phase (5608 vs. 3977 pg/mL). Inhibited production of cytokines during the acute phase may be due to a switch from a proinflammatory to an antiinflammatory mode.

Differential expression of proinflammatory cytokines and their inhibitors during the course of meningococcal infections.

Circulating concentrations of tumor necrosis factor-alpha (TNF), interleukin (IL)-1 beta, IL-6, IL-1 receptor antagonist (IL-1ra), and soluble TNF receptors p55 (sTNFr-55) and p75 (sTNFr-75) and ex vivo production of TNF, IL-1, IL-6, and IL-1ra using a whole blood culture system were measured during the acute and convalescent stages of meningococcal infection. Circulating TNF and IL-1 were below detection level, whereas IL-6 and IL-1ra, sTNFr-55, and sTNFr-75 were increased at admission. The ex vivo production of proinflammatory cytokines TNF, IL-1, and IL-6 was suppressed at admission and restored gradually during recovery. On the contrary, the production of the antiinflammatory IL-1ra was increased at admission. The elevated concentrations of both IL-1ra and sTNFr early in the course of infection suggest a regulatory role for these antiinflammatory compounds. The observed down-regulation of the ex vivo production of TNF, IL-1, and IL-6 and up-regulation of the production of IL-1ra in the acute stage may indicate a protective regulation mechanism.

Cytokine patterns in patients after major vascular surgery, hemorrhagic shock, and severe blunt trauma. Relation with subsequent adult respiratory distress syndrome and multiple organ failure.

OBJECTIVE: This study investigates the course of serum cytokine levels in patients with multiple trauma, patients with a ruptured abdominal aortic aneurysm (AAA), and patients undergoing elective AAA repair and the relationship of these cytokines to the development of adult respiratory distress syndrome (ARDS) and multiple organ failure (MOF). SUMMARY BACKGROUND DATA: Severe tissue trauma, hemorrhagic shock, and ischemia-reperfusion injury are pathophysiologic mechanisms that may result in an excessive uncontrolled activation of inflammatory cells and mediators. This inflammatory response is thought to play a key role in the development of (remote) cell and organ dysfunction, which is the basis of ARDS and MOF. METHODS: The study concerns 28 patients with multiple trauma, 20 patients admitted in shock because of a ruptured AAA, and 18 patients undergoing elective AAA repair. Arterial blood was serially sampled from admission (or at the start of elective operation) to day 13 in the intensive care unit, and the serum concentrations of tumor necrosis factor-alpha (TNF-alpha), interleukin (IL)-1 beta, and IL-6 were determined. RESULTS: Twenty-two patients died, 15 within 48 hours and 7 after several weeks, as a result of ARDS/MOF. At hospital admission and after 6 hours, these nonsurvivors had significantly higher plasma TNF-alpha and IL-1 beta levels than did the survivors. At the same measuring points, TNF-alpha and IL-1 beta were significantly more elevated in patients with ruptured AAA than in traumatized patients. However, IL-6 was significantly higher in the traumatized patients. In 10 patients, ARDS/MOF developed, and 41 had an uncomplicated course in this respect. Those with ARDS/MOF exhibited significantly different cytokine patterns in the early postinjury phase. TNF-alpha and IL-1 beta levels were higher mainly on the first day of admission; IL-6 concentrations were significantly elevated in patients with ARDS/MOF from the second day onward. The latter cytokine showed a good correlation with the daily MOF score during the whole 2-week observation period. CONCLUSIONS: In the early postinjury phase, higher concentrations of these cytokines are associated, not only with an increased mortality rate, but also with an increased risk for subsequent ARDS and MOF. These data therefore support the concept that these syndromes are caused by an overwhelming autodestructive inflammatory response.

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.

Two dry-reagent systems evaluated for determinations of enzyme activities.

We evaluated the Kodak Ektachem DT60/DTSC and the Boehringer Mannheim Reflotron for measuring activity concentrations of six enzymes. The Ektachem CVs for low concentrations of aspartate aminotransferase and alanine aminotransferase were high. As compared with the Ektachem and a routine "wet-chemistry" system, values for aspartate aminotransferase and alanine aminotransferase were lower as measured by the Reflotron, because no pyridoxal 5'-phosphate is included in the Reflotron slides. Activity concentrations of gamma-glutamyltransferase and creatine kinase measured with the Ektachem and with the routine procedure did not agree well, possibly because the Ektachem gave too-high results for the enzyme activities. Assays of several commercial test sera indicated that test results by the dry-chemistry and routine procedures are not interconvertible. This contrasts with our previous experience, in which between-test agreement for several analytes was acceptable.

Influence of ambient temperature on the Ektachem DT60 results for total bilirubin and total protein.

We evaluated the Kodak Ektachem DT60 analyzer with the DTE module in a two-month clinical trial before its introduction in the Netherlands. At ambient temperatures that differed from that at which the DT60 was calibrated, aberrant behavior for total bilirubin and total protein assays was observed. At subnormal temperatures the former gave higher results than expected, and the latter, lower results. We found a similar effect for total protein determined by the manual biuret reaction at different temperatures, when results were calculated from calibration at a fixed temperature. Total bilirubin assayed by the manual diazobilirubin method showed no such effect. Although the DT60 analyzer is equipped with a temperature-regulated incubator, we conclude that the manufactures's recommended room temperature range for these assays should be narrowed and the three-month calibration period adjusted according to external circumstances.

Stability test of six enzymes for internal quality control.

The daily quality control for the determination of the catalytic activity concentrations of enzymes is an important aspect in clinical chemistry. Instead of the expensive, commercially available control sera, we have looked for a simple, reliable and cheap method for the quality control of enzyme determinations. Commercially available enzymes were suspended in an albumin solution and ampoules were filled with 1.0 ml of these various solutions. The ampoules were stored at 4 degrees C or -20 degrees C. Once a week, during 10 months, catalytic activities of these enzyme-albumin solutions were determined together with the same activities in freshly reconstituted control sera. Aspartate aminotransferase, alanine aminotransferase, alkaline phosphatase, creatine kinase and gamma-glutamyltransferase were determined at 30 degrees C according to well-described methods. alpha-Amylase was determined with the Phadebas method at 37 degrees C. Except for creatine kinase, the stability and reliability of these enzyme solutions are fully comparable with control sera during the experimental period. The catalytic activity concentration of creatine kinase decreased slowly during the 10 months. The enzyme solutions react in the same manner as commercial test sera on changes in the reaction conditions for the enzyme determinations. The conclusion seems justified that these enzyme solutions can be used for the daily quality control of the enzyme determinations instead of control sera.

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.