Female patients with symptoms of urinary tract infection (n = 1136) were studied in primary healt... more Female patients with symptoms of urinary tract infection (n = 1136) were studied in primary health care with respect to (a) clinical symptoms as predictors of bacteriuria; (b) relation between aetiological agent and clinical picture, especially for P-fimbriated Escherichia coli; and (c) clinical findings in cases with 10(2)- less than 10(5) CFU/ml of E. coli. Prevalence of bacteriuria (greater than or equal to 10(5) CFU/ml) was 61%. Concurrence of urgency/frequency and dysuria, short duration of symptoms and hematuria increased the probability of bacteriuria and were also significantly more frequent among cases with low counts of E. coli (10(2) less than 10(5) CFU/ml in pure culture or mixed flora) than among cases with sterile urine, indicating an aetiological role of E. coli in many of those cases. Infections with P-fimbriated E. coli were as benign as the P-fimbriae-negative. The rate of P-fimbriation was 29% in specimens containing greater than or equal to 10(5) CFU/ml of E. coli, 30% among specimens with less than 10(5) CFU/ml in pure culture and 10% in specimens containing less than 10(5) CFU/ml of E. coli in mixed culture. Patients infected with Klebsiella, Enterobacter or Proteus did not show a higher rate of previous urinary tract disease or anomalies.
Journal of Bioluminescence and Chemiluminescence, Jul 1, 1989
Rapid tests for bacteriuria have the highest value, if the test result is available while the pat... more Rapid tests for bacteriuria have the highest value, if the test result is available while the patient is with the doctor. At the bacteriological laboratory rapid testing of samples obtained by mail may be cost‐effective but is of little clinical value. In a previous study performed at a health care centre using conventional urine culture as a reference the ATP test came out as the most reliable one among several rapid bacteriuria tests. The present study was performed to see how the ATP test could be fitted into the routine of the health care centre. Female patients with UTI symptoms were asked to deliver a urine sample to the health care centre laboratory and to wait for the result before seeing the doctor. After having the symptoms confirmed the doctor based the diagnosis on the ATP value. A low ATP value ruled out UTI and a high ATP value confirmed UTI. In patients with an intermediary ATP value (10–50 nmol/I) a positive nitrite test was used to confirm UTI. Only those patients with intermediary ATP values and negative nitrite test had to wait for conventional urine culture. Thus in most patients the decision on antibiotic therapy or not was based on clinical symptoms and ATP results only. Antibiotics (trimethoprim) were given as single dose or as a conventional 7‐day regime in a double‐blind comparison. The correlation between the ATP method and conventional culture was good. Although results of the present study are promising the ATP test as performed is too complicated to become widely accepted at health care centres. However, the dipstick version of the ATP test at present being developed will make the method ideally suited for rapid bacteriuria testing at health care centres and similar doctor's surgery situations.
A semiautomatic luminometric method for determination of small amounts of free fatty acids (FFA) ... more A semiautomatic luminometric method for determination of small amounts of free fatty acids (FFA) released from human adipocytes in vitro is described. Bovine serum albumin (BSA) is used as acceptor of free fatty acids in the incubation medium of isolated fat cells. The assay involves pretreatment with the detergent sodium dodecyl sulfate (SDS) to liberate the free fatty acids from the bovine serum albumin before activation by acyl-CoA synthetase (ACS) (EC 6.2.1.3). This is followed by oxidation of the resulting thioesters by acyl-CoA oxidase (ACO). The H2O2 formed is subsequently measured in a horseradish peroxidase (HRP) (EC 1.11.1.7)-catalyzed luminol reaction. The assay is linear in the interval of 0.01-1 nmol in the cuvette corresponding to 2-200 microM in the sample, and 25 samples are automatically assayed in the luminometer within 75 min. FFA release could easily be studied in a small incubation volume (200 microliters) of very diluted (10(4) cells/ml) human adipocyte suspensions. Samples (25 microliters) containing 0.25% BSA from incubates of adipose tissue cells did not interfere with the standard curve. The analytical interference from different factors that could be used in studies of lipolysis was investigated. No interference was observed up to the following concentrations: 5 microM epinephrine, 5 microM norepinephrine, 80 microM isoproterenol, 1 mM insulin, 2.5 mM propranolol, 5 mM phentolamine, and 5 microM ascorbate. Results obtained with the present assay were highly correlated (r = 0.997) with those obtained by a 260-times less sensitive spectrophotometric kit method.(ABSTRACT TRUNCATED AT 250 WORDS)
In conventional kinetic substrate assays the standard curve is plotted as observed reaction rate,... more In conventional kinetic substrate assays the standard curve is plotted as observed reaction rate, upsilon obs, versus added substrate concentration, Sadd, and has a linearity limited to Sadd much less than Km. From this plot the blank reaction rate, upsilon bl, is easily estimated but not the contaminating substrate concentration, Scon, present in reagents (unless it is the only blank source). Thus the actual substrate concentration, S = Scon + Sadd, cannot be estimated as required for the various linear plots based on the Michaelis-Menten equation. We have derived an expression, (upsilon obs - upsilon bl)/Vapp = Sadd/(Kmapp + Sadd), containing only those parameters measured for a conventional standard curve (Vapp and Kmapp are obtained from a plot of (upsilon obs - upsilon bl) versus (upsilon obs - upsilon bl)/Sadd). A plot of (upsilon obs - upsilon bl)/Vapp versus Sadd/(Kmapp + Sadd) can be used as a standard curve with the following advantages over the conventional standard curve: (a) For all kinetic substrate assays it is identical and connects the points (0, 0) and (1, 1). Thus deviations from true Michaelis-Menten kinetics or erroneous kinetic constants are easily detected. (b) Since it is linear even above Km, the analytically useful range is considerably extended. (c) For assays with a wide dynamic range it can be used in lin-lin or log-log form. The procedure is illustrated for a kinetic assay of glycerol (Kmapp = 40 mumol/liter). The plot was found to be entirely linear in the range 0.07-100 mumol/liter (glycerol concentration in cuvette).
Bacteriuria defined with urine culture as the reference method was compared with bacterial adenos... more Bacteriuria defined with urine culture as the reference method was compared with bacterial adenosine triphosphate (ATP) measurements of cystoscopical urine samples from 216 men. Discrepant samples were studied by reculturing and microscopy of the sediment. Results show a good correlation (sensitivity 1.0 and specificity 0.95) between bacteriuria greater than or equal to 10(4) cfu/ml and bacterial ATP greater than 10 nmol/l measured by bioluminescence. Samples with macroscopic hematuria may influence the ATP value and give false positive results.
Scandinavian Journal of Clinical & Laboratory Investigation, 1998
In microdialysis, samples from the extracellular fluid are analysed in the outgoing dialysate. By... more In microdialysis, samples from the extracellular fluid are analysed in the outgoing dialysate. By adding ethanol to the ingoing perfusate and following its exchange in the dialysate, an outflow/inflow ratio is obtained. This ratio has been shown to correlate with the tissue blood flow. An automatic luminometric ethanol assay that quantifies the light produced from three coupled enzyme reactions is described. Alcohol dehydrogenase catalyses the oxidation of ethanol with formation of NADH, which is monitored using NADH:FMN oxidoreductase and bacterial luciferase. Each assay can be individually calibrated by measuring the increased rate of NADH formation through the addition of a known amount of ethanol. The linear range of the assay was 0.05-10 mmol l-1 in microdialysate samples. The within-run imprecision was 1.4% CV in 10 mmol l-1 samples, and ethanol recovery was almost 100%. The assay was applied to microdialysates that were generated from probes implanted in the vastus lateralis muscle in fasting subjects (n = 12) and perfused with 5 mmol l-1 ethanol at 3 microliter min-1. An outflow/inflow steady-state ratio of 27% (range 19-47%) appeared after about 1 h, followed by a within-run variability of 4.7% CV (range 2.5-7.7% CV), as calculated from samples collected every 15 min up to 4 h after stabilization. In conclusion, a sensitive ethanol assay, suitable for studies of microcirculatory exchange of solute molecules over the dialysis probe is presented.
European Journal of Clinical Microbiology & Infectious Diseases, Feb 1, 1991
Determination by bioluminescence of the bacterial adenosine triphosphate (ATP) level in urine was... more Determination by bioluminescence of the bacterial adenosine triphosphate (ATP) level in urine was evaluated as a method for detection of bacteriuria in 1126 women with symptoms of UTI and 530 attending for follow-up. Conventional urine culture was used as reference method. The criterion for bacteriuria was growth of greater than or equal to 10(5) cfu/ml, giving a prevalence of 0.60. ATP levels of less than 10 nmol/l and greater than 50 mmol/l indicated abacteriuria and bacteruiria, respectively, whereas intermediate concentrations required culture if the nitrite test was negative. With this diagnostic strategy the sensitivity and specificity was 0.96 and 0.90 at the first visit and 0.90 and 0.98 at follow-up. With some methodological improvement the ATP test could be useful in medium-sized and small laboratories.
Journal of Bioluminescence and Chemiluminescence, Jul 1, 1989
A sensitive, specific, and partly automatic method for the analysis of free fatty acids is descri... more A sensitive, specific, and partly automatic method for the analysis of free fatty acids is described. The assay involves activation of free fatty acids by acyl‐CoA synthetase (EC 6.2.1.3) followed by oxidation of the thioesters by acyl‐CoA oxidase. The H2O2 formed is determined in a reaction catalysed by horseradish peroxidase (EC 1.11.1.7) using luminol as electron donor. The assay has a linear range of 0.05 to 5 nmol of different free fatty acids (C10‐C18) in the original sample. The efficiency of the method toward capric, lauric, myristic, palmitic, palmitoleic, stearic, oleic, and linoleic acid measured as recovery of light emission compared to that of H2O2 standards, was over 90%. AffiGel 501 was used to covalently bind the free thiol group in CoASH eliminating interference of this substance in the peroxidase‐luminol reaction.
A kinetic bioluminescence assay with optimized reagent conditions has been developed for applicat... more A kinetic bioluminescence assay with optimized reagent conditions has been developed for application as a screening test for increased creatine kinase (CK) activities in dried blood spots. This test is used for the early detection of Duchenne muscular dystrophy (DMD) in a voluntary CK screening program in West Germany. Of the 176,600 boys tested up to December 31, 1984, 48 who were less than 6 months old had certain or probable DMD (frequency 1: 3679). In 1983 and 1984, the rate of false positive results was 0.016% for a cut-off activity 300 U/liter and 0.061% for a cut-off activity 180 U/liter. Long-term counseling is offered to families of newly detected DMD patients in order to facilitate the aims of the screening program, namely, avoidance of secondary cases in affected families, early professional care for the sick child, and the early opportunity to make the appropriate decisions for a life with an handicapped child. Two types of a benign hereditary blood anomaly were also detected by CK screening (CK-BB inside erythrocytes or thrombocytes).
Publisher Summary This chapter describes the steps involved in estimation of biomass in growing c... more Publisher Summary This chapter describes the steps involved in estimation of biomass in growing cell lines by luminometric ATP assay. Methods for studying the reliability of the results are described. In particular, estimation of energy charge (EC) by a new method for assay of ATP, ADP, and AMP was found to be valuable. The activity of several adenosine nucleotide converting enzymes as a function of the energy charge—that is, (ATP + 0.5 ADP) / (ATP + ADP + AMP)—indicates that the energy charge in intact metabolizing cells should be stabilized in the interval 0.8–0.9. By measuring all three adenine nucleotides, the measure of the physiological status of the cells is obtained that provides information on whether ATP can be used to estimate biomass or if the energy metabolism is disturbed. By the method described in the chapter, all three adenine nucleotides are determined in a single aliquot from the cell extract. Measurement of energy charge gives a reliable estimation of the accuracy of using ATP as a biomass parameter. Growth monitoring by assay of adenine nucleotides includes the following steps: (1) cultivation of cells, (2) sampling and pretreatment of samples, (3) extraction of adenine nucleotides from cells, (4) luminometric assay of adenine nucleotides, and (5) correlation to other biomass estimations.
Publisher Summary This chapter focuses on the determination of creatine kinase (CK) isoenzymes in... more Publisher Summary This chapter focuses on the determination of creatine kinase (CK) isoenzymes in human serum by an immunological method using purified firefly luciferase. CK consists of two subunits, M and B. It appears in different tissues as the isoenzymes MM (muscle type), BB (brain type), and MB (hybrid type). Several techniques for determination of the isoenzyme pattern of creatine kinase in serum have been developed. The isoenzymes may be electrophoretically separated on cellulose acetate, polyacrylamide, agar or agarose and assayed either directly in the gel or after elution of the CK bands. Separation may also be achieved by ion-exchange chromatography on micro-columns of DEAE-Sephadex. Selective inactivation of isoenzymes of CK by precipitating antibodies are described by Jockers-Wretou and Pfleiderer. The chapter discusses the combination of the sensitivity of the luciferase method with the specificity of the immunological method of Neumeier et al.
Single-dose trimethoprim treatment of cystitis was compared with 7-day treatment in a randomized ... more Single-dose trimethoprim treatment of cystitis was compared with 7-day treatment in a randomized double-blind study in primary health care. Consecutive female patients (613) with symptoms of lower urinary tract infection (UTI) and positive bacteriuria screening tests were enrolled. In 502 cases UTI was confirmed by urine culture. Follow-up was performed twice, after 2-3 and 5-6 weeks. Short-term efficacy could be evaluated in 425 cases and accumulated efficacy in 344. Short-term efficacy was 82% for single-dose and 94% for 7-day treatment (P less than .001). Accumulated efficacy was 71% for single-dose and 87% for 7-day therapy (P less than .001). Fewer adverse reactions were noted with single-dose therapy (not significant). The cure rate for UTI caused by P-fimbriated Escherichia coli was not different from that of other E. coli infections. Infections with Staphylococcus saprophyticus showed a lower cure rate than E. coli infections with the single-dose regimen (P less than .05 for short-term efficacy).
Female patients with symptoms of urinary tract infection (n = 1136) were studied in primary healt... more Female patients with symptoms of urinary tract infection (n = 1136) were studied in primary health care with respect to (a) clinical symptoms as predictors of bacteriuria; (b) relation between aetiological agent and clinical picture, especially for P-fimbriated Escherichia coli; and (c) clinical findings in cases with 10(2)- less than 10(5) CFU/ml of E. coli. Prevalence of bacteriuria (greater than or equal to 10(5) CFU/ml) was 61%. Concurrence of urgency/frequency and dysuria, short duration of symptoms and hematuria increased the probability of bacteriuria and were also significantly more frequent among cases with low counts of E. coli (10(2) less than 10(5) CFU/ml in pure culture or mixed flora) than among cases with sterile urine, indicating an aetiological role of E. coli in many of those cases. Infections with P-fimbriated E. coli were as benign as the P-fimbriae-negative. The rate of P-fimbriation was 29% in specimens containing greater than or equal to 10(5) CFU/ml of E. coli, 30% among specimens with less than 10(5) CFU/ml in pure culture and 10% in specimens containing less than 10(5) CFU/ml of E. coli in mixed culture. Patients infected with Klebsiella, Enterobacter or Proteus did not show a higher rate of previous urinary tract disease or anomalies.
Journal of Bioluminescence and Chemiluminescence, Jul 1, 1989
Rapid tests for bacteriuria have the highest value, if the test result is available while the pat... more Rapid tests for bacteriuria have the highest value, if the test result is available while the patient is with the doctor. At the bacteriological laboratory rapid testing of samples obtained by mail may be cost‐effective but is of little clinical value. In a previous study performed at a health care centre using conventional urine culture as a reference the ATP test came out as the most reliable one among several rapid bacteriuria tests. The present study was performed to see how the ATP test could be fitted into the routine of the health care centre. Female patients with UTI symptoms were asked to deliver a urine sample to the health care centre laboratory and to wait for the result before seeing the doctor. After having the symptoms confirmed the doctor based the diagnosis on the ATP value. A low ATP value ruled out UTI and a high ATP value confirmed UTI. In patients with an intermediary ATP value (10–50 nmol/I) a positive nitrite test was used to confirm UTI. Only those patients with intermediary ATP values and negative nitrite test had to wait for conventional urine culture. Thus in most patients the decision on antibiotic therapy or not was based on clinical symptoms and ATP results only. Antibiotics (trimethoprim) were given as single dose or as a conventional 7‐day regime in a double‐blind comparison. The correlation between the ATP method and conventional culture was good. Although results of the present study are promising the ATP test as performed is too complicated to become widely accepted at health care centres. However, the dipstick version of the ATP test at present being developed will make the method ideally suited for rapid bacteriuria testing at health care centres and similar doctor's surgery situations.
A semiautomatic luminometric method for determination of small amounts of free fatty acids (FFA) ... more A semiautomatic luminometric method for determination of small amounts of free fatty acids (FFA) released from human adipocytes in vitro is described. Bovine serum albumin (BSA) is used as acceptor of free fatty acids in the incubation medium of isolated fat cells. The assay involves pretreatment with the detergent sodium dodecyl sulfate (SDS) to liberate the free fatty acids from the bovine serum albumin before activation by acyl-CoA synthetase (ACS) (EC 6.2.1.3). This is followed by oxidation of the resulting thioesters by acyl-CoA oxidase (ACO). The H2O2 formed is subsequently measured in a horseradish peroxidase (HRP) (EC 1.11.1.7)-catalyzed luminol reaction. The assay is linear in the interval of 0.01-1 nmol in the cuvette corresponding to 2-200 microM in the sample, and 25 samples are automatically assayed in the luminometer within 75 min. FFA release could easily be studied in a small incubation volume (200 microliters) of very diluted (10(4) cells/ml) human adipocyte suspensions. Samples (25 microliters) containing 0.25% BSA from incubates of adipose tissue cells did not interfere with the standard curve. The analytical interference from different factors that could be used in studies of lipolysis was investigated. No interference was observed up to the following concentrations: 5 microM epinephrine, 5 microM norepinephrine, 80 microM isoproterenol, 1 mM insulin, 2.5 mM propranolol, 5 mM phentolamine, and 5 microM ascorbate. Results obtained with the present assay were highly correlated (r = 0.997) with those obtained by a 260-times less sensitive spectrophotometric kit method.(ABSTRACT TRUNCATED AT 250 WORDS)
In conventional kinetic substrate assays the standard curve is plotted as observed reaction rate,... more In conventional kinetic substrate assays the standard curve is plotted as observed reaction rate, upsilon obs, versus added substrate concentration, Sadd, and has a linearity limited to Sadd much less than Km. From this plot the blank reaction rate, upsilon bl, is easily estimated but not the contaminating substrate concentration, Scon, present in reagents (unless it is the only blank source). Thus the actual substrate concentration, S = Scon + Sadd, cannot be estimated as required for the various linear plots based on the Michaelis-Menten equation. We have derived an expression, (upsilon obs - upsilon bl)/Vapp = Sadd/(Kmapp + Sadd), containing only those parameters measured for a conventional standard curve (Vapp and Kmapp are obtained from a plot of (upsilon obs - upsilon bl) versus (upsilon obs - upsilon bl)/Sadd). A plot of (upsilon obs - upsilon bl)/Vapp versus Sadd/(Kmapp + Sadd) can be used as a standard curve with the following advantages over the conventional standard curve: (a) For all kinetic substrate assays it is identical and connects the points (0, 0) and (1, 1). Thus deviations from true Michaelis-Menten kinetics or erroneous kinetic constants are easily detected. (b) Since it is linear even above Km, the analytically useful range is considerably extended. (c) For assays with a wide dynamic range it can be used in lin-lin or log-log form. The procedure is illustrated for a kinetic assay of glycerol (Kmapp = 40 mumol/liter). The plot was found to be entirely linear in the range 0.07-100 mumol/liter (glycerol concentration in cuvette).
Bacteriuria defined with urine culture as the reference method was compared with bacterial adenos... more Bacteriuria defined with urine culture as the reference method was compared with bacterial adenosine triphosphate (ATP) measurements of cystoscopical urine samples from 216 men. Discrepant samples were studied by reculturing and microscopy of the sediment. Results show a good correlation (sensitivity 1.0 and specificity 0.95) between bacteriuria greater than or equal to 10(4) cfu/ml and bacterial ATP greater than 10 nmol/l measured by bioluminescence. Samples with macroscopic hematuria may influence the ATP value and give false positive results.
Scandinavian Journal of Clinical & Laboratory Investigation, 1998
In microdialysis, samples from the extracellular fluid are analysed in the outgoing dialysate. By... more In microdialysis, samples from the extracellular fluid are analysed in the outgoing dialysate. By adding ethanol to the ingoing perfusate and following its exchange in the dialysate, an outflow/inflow ratio is obtained. This ratio has been shown to correlate with the tissue blood flow. An automatic luminometric ethanol assay that quantifies the light produced from three coupled enzyme reactions is described. Alcohol dehydrogenase catalyses the oxidation of ethanol with formation of NADH, which is monitored using NADH:FMN oxidoreductase and bacterial luciferase. Each assay can be individually calibrated by measuring the increased rate of NADH formation through the addition of a known amount of ethanol. The linear range of the assay was 0.05-10 mmol l-1 in microdialysate samples. The within-run imprecision was 1.4% CV in 10 mmol l-1 samples, and ethanol recovery was almost 100%. The assay was applied to microdialysates that were generated from probes implanted in the vastus lateralis muscle in fasting subjects (n = 12) and perfused with 5 mmol l-1 ethanol at 3 microliter min-1. An outflow/inflow steady-state ratio of 27% (range 19-47%) appeared after about 1 h, followed by a within-run variability of 4.7% CV (range 2.5-7.7% CV), as calculated from samples collected every 15 min up to 4 h after stabilization. In conclusion, a sensitive ethanol assay, suitable for studies of microcirculatory exchange of solute molecules over the dialysis probe is presented.
European Journal of Clinical Microbiology & Infectious Diseases, Feb 1, 1991
Determination by bioluminescence of the bacterial adenosine triphosphate (ATP) level in urine was... more Determination by bioluminescence of the bacterial adenosine triphosphate (ATP) level in urine was evaluated as a method for detection of bacteriuria in 1126 women with symptoms of UTI and 530 attending for follow-up. Conventional urine culture was used as reference method. The criterion for bacteriuria was growth of greater than or equal to 10(5) cfu/ml, giving a prevalence of 0.60. ATP levels of less than 10 nmol/l and greater than 50 mmol/l indicated abacteriuria and bacteruiria, respectively, whereas intermediate concentrations required culture if the nitrite test was negative. With this diagnostic strategy the sensitivity and specificity was 0.96 and 0.90 at the first visit and 0.90 and 0.98 at follow-up. With some methodological improvement the ATP test could be useful in medium-sized and small laboratories.
Journal of Bioluminescence and Chemiluminescence, Jul 1, 1989
A sensitive, specific, and partly automatic method for the analysis of free fatty acids is descri... more A sensitive, specific, and partly automatic method for the analysis of free fatty acids is described. The assay involves activation of free fatty acids by acyl‐CoA synthetase (EC 6.2.1.3) followed by oxidation of the thioesters by acyl‐CoA oxidase. The H2O2 formed is determined in a reaction catalysed by horseradish peroxidase (EC 1.11.1.7) using luminol as electron donor. The assay has a linear range of 0.05 to 5 nmol of different free fatty acids (C10‐C18) in the original sample. The efficiency of the method toward capric, lauric, myristic, palmitic, palmitoleic, stearic, oleic, and linoleic acid measured as recovery of light emission compared to that of H2O2 standards, was over 90%. AffiGel 501 was used to covalently bind the free thiol group in CoASH eliminating interference of this substance in the peroxidase‐luminol reaction.
A kinetic bioluminescence assay with optimized reagent conditions has been developed for applicat... more A kinetic bioluminescence assay with optimized reagent conditions has been developed for application as a screening test for increased creatine kinase (CK) activities in dried blood spots. This test is used for the early detection of Duchenne muscular dystrophy (DMD) in a voluntary CK screening program in West Germany. Of the 176,600 boys tested up to December 31, 1984, 48 who were less than 6 months old had certain or probable DMD (frequency 1: 3679). In 1983 and 1984, the rate of false positive results was 0.016% for a cut-off activity 300 U/liter and 0.061% for a cut-off activity 180 U/liter. Long-term counseling is offered to families of newly detected DMD patients in order to facilitate the aims of the screening program, namely, avoidance of secondary cases in affected families, early professional care for the sick child, and the early opportunity to make the appropriate decisions for a life with an handicapped child. Two types of a benign hereditary blood anomaly were also detected by CK screening (CK-BB inside erythrocytes or thrombocytes).
Publisher Summary This chapter describes the steps involved in estimation of biomass in growing c... more Publisher Summary This chapter describes the steps involved in estimation of biomass in growing cell lines by luminometric ATP assay. Methods for studying the reliability of the results are described. In particular, estimation of energy charge (EC) by a new method for assay of ATP, ADP, and AMP was found to be valuable. The activity of several adenosine nucleotide converting enzymes as a function of the energy charge—that is, (ATP + 0.5 ADP) / (ATP + ADP + AMP)—indicates that the energy charge in intact metabolizing cells should be stabilized in the interval 0.8–0.9. By measuring all three adenine nucleotides, the measure of the physiological status of the cells is obtained that provides information on whether ATP can be used to estimate biomass or if the energy metabolism is disturbed. By the method described in the chapter, all three adenine nucleotides are determined in a single aliquot from the cell extract. Measurement of energy charge gives a reliable estimation of the accuracy of using ATP as a biomass parameter. Growth monitoring by assay of adenine nucleotides includes the following steps: (1) cultivation of cells, (2) sampling and pretreatment of samples, (3) extraction of adenine nucleotides from cells, (4) luminometric assay of adenine nucleotides, and (5) correlation to other biomass estimations.
Publisher Summary This chapter focuses on the determination of creatine kinase (CK) isoenzymes in... more Publisher Summary This chapter focuses on the determination of creatine kinase (CK) isoenzymes in human serum by an immunological method using purified firefly luciferase. CK consists of two subunits, M and B. It appears in different tissues as the isoenzymes MM (muscle type), BB (brain type), and MB (hybrid type). Several techniques for determination of the isoenzyme pattern of creatine kinase in serum have been developed. The isoenzymes may be electrophoretically separated on cellulose acetate, polyacrylamide, agar or agarose and assayed either directly in the gel or after elution of the CK bands. Separation may also be achieved by ion-exchange chromatography on micro-columns of DEAE-Sephadex. Selective inactivation of isoenzymes of CK by precipitating antibodies are described by Jockers-Wretou and Pfleiderer. The chapter discusses the combination of the sensitivity of the luciferase method with the specificity of the immunological method of Neumeier et al.
Single-dose trimethoprim treatment of cystitis was compared with 7-day treatment in a randomized ... more Single-dose trimethoprim treatment of cystitis was compared with 7-day treatment in a randomized double-blind study in primary health care. Consecutive female patients (613) with symptoms of lower urinary tract infection (UTI) and positive bacteriuria screening tests were enrolled. In 502 cases UTI was confirmed by urine culture. Follow-up was performed twice, after 2-3 and 5-6 weeks. Short-term efficacy could be evaluated in 425 cases and accumulated efficacy in 344. Short-term efficacy was 82% for single-dose and 94% for 7-day treatment (P less than .001). Accumulated efficacy was 71% for single-dose and 87% for 7-day therapy (P less than .001). Fewer adverse reactions were noted with single-dose therapy (not significant). The cure rate for UTI caused by P-fimbriated Escherichia coli was not different from that of other E. coli infections. Infections with Staphylococcus saprophyticus showed a lower cure rate than E. coli infections with the single-dose regimen (P less than .05 for short-term efficacy).
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