Br. J. clin. Pharmac. (1990), 30, 861-869
Leukotrienes in the sputum and urine of cystic fibrosis children ANTHONY P. SAMPSON', DAVID A. SPENCER2, COLIN P. GREEN2, PRISCILLA J. PIPER' & JOHN F. PRICE2 'Department of Pharmacology, Hunterian Institute, Royal College of Surgeons of England, Lincoln's Inn Fields, London WC2A 3PN and 2Departments of Thoracic Medicine and Paediatrics, King's College School of Medicine and Dentistry, Bessemer Road, London SE5 9PJ
1 Leukotrienes (LTs) are potent pro-inflammatory mediators with actions relevant to the pathophysiology of cystic fibrosis (CF), including increased mucus production, bronchoconstriction, leucocyte chemotaxis, and increased vascular permeability. We have therefore investigated the potential role of LTs in children with CF. Leukotriene E4 levels were assessed in the urine of 30 normal (N) children (aged 1.3-12.7 years) and 30 CF children (1.6-14.3 years). Sputum from 13 of the CF children was analysed from LTB4, LTC4, LTD4, and LTE4. LTs were separated by reversed-phase h.p.l.c. and quantitated by radioimmunoassay. 2 Urinary LTE4 levels were log normally distributed, with geometric mean values (95% confidence intervals) of N: 88.4 (71.3-111) pmol mmol-1 creatinine (n = 30), and CF: 112 (70.6-177) pmol mmol-1 creatinine (n = 30; P > 0.05). Of the CF subjects, 33% had urinary LTE4 levels above 200 pmol mmol-1 creatinine, compared with 3.3% of the N children. 3 In sputum, mean (± s.e. mean) LT concentrations were (pmol g-1), LTB4: 44.3 ± 10.8, LTC4: 4.9 ± 1.3, LTD4: 1.8 ± 0.9, and LTE4: 67.7 ± 18.9 (n = 13). 4 Urinary LTE4 levels correlated significantly with sputum LTE4 levels (r = 0.673, P = 0.012), and with sputum levels of total cysteinyl-LTs (r = 0.660, P = 0.014). 5 In conclusion, total cysteinyl-LT content in sputum is 10-fold higher than previously reported, consisting primarily (91%) of LTE4. The high levels of LTE4 and LTB4 in sputum suggest involvement of LTs in the pathophysiology of CF. Urinary LTE4 levels may prove useful as a marker for cysteinyl-LT production in sputum.
Keywords leukotrienes cystic fibrosis sputum urine Introduction
Cystic fibrosis is the most common lethal genetic bolites of arachidonic acid, with pro-inflamdisorder among Caucasians, affecting approxi- matory actions of relevance to the pathophysiomately one in 2500 live births. The autosomal logy of CF. They are generated primarily by recessive gene responsible for the condition leads inflammatory leucocytes in response to a wide to disturbances in the function of secretory variety of immunological and non-immunological epithelia, resulting in excessive bronchial mucus stimuli. Leukotriene B4 is a potent chemotactic secretion, reduced pancreatic enzyme secretion, and chemokinetic agent in vitro for neutrophils and malabsorption in the gastrointestinal tract. (Ford-Hutchinson et al., 1980) and eosinophils Leukotrienes (LTs) are 5-lipoxygenase meta- (Czarnetzki & Rosenbach, 1986), and at higher Correspondence: Dr A. P. Sampson, Department of Thoracic Medicine, King's College School of Medicine,
Bessemer Road, London SE5 9PJ
861
862
A. P. Sampson et al.
concentrations causes neutrophil degranulation and superoxide generation (Feinmark et al., 1981). In vivo, exogenous LTB4 causes accumulation of neutrophils and eosinophils in human skin (Camp et al., 1984). The cysteinyl-LTs (LTC4, LTD4, and LTE4) contract bronchial smooth muscle in vitro (Samhoun & Piper, 1983) and in vivo (Barnes et al., 1984), promote secretion of mucus from human bronchial strips in vitro (Coles etal., 1983), and have a direct action on post-capillary venules to increase vascular permeability (Woodward et al., 1983). An abnormality in the regulation of arachidonic acid release from lymphocyte membrane phospholipids of CF subjects has been demonstrated (Carlstedt-Duke et al., 1986), and an increased proportion of arachidonic acid has been found in CF bronchial phospholipids (Gilljam et al., 1986). These effects might lead to an elevation in arachidonic acid turnover and putatively an increased generation of LTs from inflammatory leucocytes in CF. We have therefore used reverse-phase high
performance liquid chromatography (RP-h.p.l.c.) and selective radioimmunoassays (r.i.a.) to investigate the concentrations of LTs in the biological fluids of children with CF. Urinary excretion of LTE4 was assessed as a marker of endogenous cysteinyl-LT generation in CF children and in a matched group of normal children. The relationship of urinary LTE4 concentrations to the levels of LTB4, LTC4, LTD4, and LTE4 in the sputum of children with CF was also investigated.
LTs that can occur at acid pH (Fitzpatrick et al., 1983), and to optimise recoveries from Sep-Pak purification (Osborne et al., 1983). Urine samples were collected into sterile polypropylene containers on ice. Aliquots (2 ml) were analysed for creatinine content. Tritiumlabelled LTE4 (4 nCi) was added to the remaining aliquots (25 ml) as an internal standard, followed by 4 vol of ice-cold ethanol. Sputum samples were collected into sterile containers on ice, weighed, and [3H]-LTB4, [3H]-LTC4, [3H]-LTD4, and [3H]-LTE4 (2 nCi) added before homogenisation in 4 vol ethanol for 2 min (UltraTurrax homogeniser). Preliminary experiments demonstrated that the use of ice-cold ethanol prevents further generation of LTs following collection and during homogenisation. All samples were left at 40 C for 30 min to allow protein denaturation. Precipitated protein and cell debris were removed by centrifugation at 12000 g for 6 min at 40 C, and the supernatants were evaporated to dryness in vacuo (Buchi rotoevaporator). Leukotrienes in the samples were partially purified on C18 Sep-Pak cartridges; the samples were reconstituted in distilled water (5 ml), and loaded onto a cartridge pre-washed with methanol (5 ml) and distilled water (5 ml). The cartridges were washed successively with distilled water (5 ml), petroleum ether (5 ml), and chloroform (5 ml). Leukotriene-containing material was eluted with methanol (5 ml), filtered (Millex 0.45 FLM), evaporated to dryness, and stored at -20° C prior to reverse-phase h.p.l.c.
Reverse-phase chromatography Methods
Samples were reconstituted in 0.5 ml of the degassed h.p.l.c. solvent system, consisting of Subject selection methanol/distilled water/glacial acetic acid (69/ 31/0.01 v/v/v), adjusted to pH 5.7 with 10% CF children attending clinics at King's College NH4OH, and pumped (1 ml min-) through Hospital, Camberwell, and the Hospital for Sick a C18 guard column and a Spherisorb ODS2 Children, Great Ormond Street, London, analytical column (300 x 4.6 mm). Fractions donated samples of sputum and urine. Parental (1 ml) were collected for up to 25 min. Aliquots permission was obtained in all cases. All CF (0.8 ml) of each fraction were added to Aquasolchildren had raised sweat sodium levels (> 70 2 scintillant (5 ml), and counted for at least 2 min mmol 1-1) and evidence of airways obstruction. each in a Canberra-Packard 2100 scintillation None was receiving oral or inhaled cortico- counter to identify the radiolabelled peaks of the steroids. internal standards, and to allow calculation of Normal children had no history of atopy or of the recoveries of each LT in individual samples. recent upper respiratory tract infection, and The remaining aliquots of the fractions (0.2 ml) voluntarily donated urine samples with parental were evaporated to dryness in vacuo, and stored consent. at -20° C prior to radioimmunoassay. Extraction of samples
Radioimmunoassays
Extractions were performed at neutral pH to prevent the 11-trans isomerisation of cysteinyl-
Radioimmunoassay of LTs B4, C4, and D4 was performed using a double antibody technique.
Leukotrienes in cystic fibrosis Samples were reconstituted in the assay buffer (300 ,ul), and aliquots (100 ,ul) were assayed in duplicate. The samples were incubated for 2 h at 40 C in phosphate-buffered saline (400 Pd) with the radiolabelled LT (4 nCi) and the appropriate anti-LT serum, before addition of the goat antirabbit serum (Bio-Rad). Bound radiolabel was removed by centrifugation for 2500 g for 20 min. LTs were quantitated by interpolation on the standard curve (0.05-50 pmol/tube). The antiserum for LTB4 (gift of Dr J. Rokach) showed negligible cross-reactivity for 6-transLTB4, (SS, 12S)-LTB4, 20-hydroxy-LTB4, 20carboxy-LTB4, 5-HETE, cysteinyl-LTs, and arachidonic acid (Rokach et al., 1984). Fifty percent displacement of radiolabel occurred at an LTB4 concentration of 2.1 pmol/tube. The LTC4/LTD4 antiserum recognised (5S, 6R)-LTC4 and (5R, 6R)-LTC4 (100%), LTC4sulphone (68.9%), and LTD4 (29.4%), but did not cross-react with LTB4, 6-trans-LTB4, (5S, 12S)-LTB4, 20-hydroxy-LTB4, 20-carboxy-LTB4, LTE4, N-acetyl-LTE4, 5-HETE, various prostanoids, arachidonic acid, or glutathione (< 0.05%). Fifty percent displacement of radiolabel occurred with LT concentrations of 0.36 pmol/tube (LTC4) and 1.02 pmolltube (LTD4). The LTE4 radioimmunoassay was performed using an antiserum (gift of Dr B. Peskar) which recognised LTE4 (100%), LTC4 (90%), LTD4 (280%), N-acetyl-LTE4 (320%), and 11-transLTE4 (30%), but not LTB4 (< 0.005%), various prostanoids, arachidonic acid, or glutathione. Samples were incubated overnight at 4° C with [3H]-LTE4 (2.5 nCi) and the antiserum in TrisHCl buffer (600 ,ul; pH 7.4) containing gelatine (0.1% w/v), before addition of dextran-coated charcoal to separate unbound [3H]-LTE4. Standard LTE4 was quantitated in the range 0.02-10 pmol/tube; fifty percent binding occurred at a concentration of 0.57 pmol/tube. The LT immunoreactivity that co-eluted from h.p.l.c. with the respective [3H]-LT internal standards was corrected for recovery, and for the proportion of the total sample assayed. Urinary LTE4 immunoreactivity (in pmol) was further corrected for the individual urinary creatinine (CR) levels. Sputum LT levels were expressed as pmol of LT per gram wet weight of sputum. Comparisons between means (MannWhitney U-test) and correlation coefficients were performed using the MINITAB statistical package (Minitab Inc., State College, PA, USA). Materials The following were used: LTB4 antiserum (gift of Dr J. Rokach, Merck Frosst Laboratories, Canada), LTE4 antiserum (gift of Dr B. Peskar,
863
University of Bochum, FRG), LTB4, LTC4, LTD4, and LTE4 (Chiral Organics), [3H]-LTB4 (32 Ci/mmol), [3H]-LTC4 (39 Ci mmol-1), [3H]LTD4 (38.4 Ci mmol-F), and [3H]-LTE4 (38.4 Ci mmol-1) (New England Nuclear), Aquasol-2 scintillant (DuPont UK Ltd.), absolute ethanol, h.p.l.c.-grade methanol (FSA Laboratories), chloroform, petroleum ether (BDH Chemicals Ltd), Tris-HCl, gelatine, dextran, activated charcoal (Sigma Chemical Co.), goat anti-rabbit IgG (Biorad). Results
Sensitivity and specificity of radioimmunoassays The theoretical lower detection limits were calculated from the lowest LT concentration on the straight-line portion of the r.i.a. standard curves, corrected for the mean recovery of each LT from urine or sputum, and for the proportion of each sample assayed. The theoretical detection limit of LTE4 in urine was 9 pmol mmol-1 creatinine. In sputum, the detection limits for LTB4, LTC4, LTD4, and LTE4 were 0.5 pmol -1, 0.9 pmol g1-, 1.4 pmol g-1, and 0.6 pmol g- respectively. The linearity of measurements of LTE4 in urine was assessed by assaying samples of normal unne containing exogenous LTE4 (0, 0.03, 0.1, 0.3, 1.0 ng ml-'; n = 4). The mean coefficient of variation over this range (equivalent to 0-250 pmol mmol-1.CR) was 33%. The levels detected correlated significantly (r = 0.924, P < 0.001, n = 20) with the amount added (Figure 1). The 1.8
l1.6 y = 0.292 + 0.983 x
1.4 1
Ea,
0)
1.0
0, 0.8 U O It 0.6
?F
)F ,F
*
r= 0.924 P
Leukotrienes in the sputum and urine of cystic fibrosis children ANTHONY P. SAMPSON', DAVID A. SPENCER2, COLIN P. GREEN2, PRISCILLA J. PIPER' & JOHN F. PRICE2 'Department of Pharmacology, Hunterian Institute, Royal College of Surgeons of England, Lincoln's Inn Fields, London WC2A 3PN and 2Departments of Thoracic Medicine and Paediatrics, King's College School of Medicine and Dentistry, Bessemer Road, London SE5 9PJ
1 Leukotrienes (LTs) are potent pro-inflammatory mediators with actions relevant to the pathophysiology of cystic fibrosis (CF), including increased mucus production, bronchoconstriction, leucocyte chemotaxis, and increased vascular permeability. We have therefore investigated the potential role of LTs in children with CF. Leukotriene E4 levels were assessed in the urine of 30 normal (N) children (aged 1.3-12.7 years) and 30 CF children (1.6-14.3 years). Sputum from 13 of the CF children was analysed from LTB4, LTC4, LTD4, and LTE4. LTs were separated by reversed-phase h.p.l.c. and quantitated by radioimmunoassay. 2 Urinary LTE4 levels were log normally distributed, with geometric mean values (95% confidence intervals) of N: 88.4 (71.3-111) pmol mmol-1 creatinine (n = 30), and CF: 112 (70.6-177) pmol mmol-1 creatinine (n = 30; P > 0.05). Of the CF subjects, 33% had urinary LTE4 levels above 200 pmol mmol-1 creatinine, compared with 3.3% of the N children. 3 In sputum, mean (± s.e. mean) LT concentrations were (pmol g-1), LTB4: 44.3 ± 10.8, LTC4: 4.9 ± 1.3, LTD4: 1.8 ± 0.9, and LTE4: 67.7 ± 18.9 (n = 13). 4 Urinary LTE4 levels correlated significantly with sputum LTE4 levels (r = 0.673, P = 0.012), and with sputum levels of total cysteinyl-LTs (r = 0.660, P = 0.014). 5 In conclusion, total cysteinyl-LT content in sputum is 10-fold higher than previously reported, consisting primarily (91%) of LTE4. The high levels of LTE4 and LTB4 in sputum suggest involvement of LTs in the pathophysiology of CF. Urinary LTE4 levels may prove useful as a marker for cysteinyl-LT production in sputum.
Keywords leukotrienes cystic fibrosis sputum urine Introduction
Cystic fibrosis is the most common lethal genetic bolites of arachidonic acid, with pro-inflamdisorder among Caucasians, affecting approxi- matory actions of relevance to the pathophysiomately one in 2500 live births. The autosomal logy of CF. They are generated primarily by recessive gene responsible for the condition leads inflammatory leucocytes in response to a wide to disturbances in the function of secretory variety of immunological and non-immunological epithelia, resulting in excessive bronchial mucus stimuli. Leukotriene B4 is a potent chemotactic secretion, reduced pancreatic enzyme secretion, and chemokinetic agent in vitro for neutrophils and malabsorption in the gastrointestinal tract. (Ford-Hutchinson et al., 1980) and eosinophils Leukotrienes (LTs) are 5-lipoxygenase meta- (Czarnetzki & Rosenbach, 1986), and at higher Correspondence: Dr A. P. Sampson, Department of Thoracic Medicine, King's College School of Medicine,
Bessemer Road, London SE5 9PJ
861
862
A. P. Sampson et al.
concentrations causes neutrophil degranulation and superoxide generation (Feinmark et al., 1981). In vivo, exogenous LTB4 causes accumulation of neutrophils and eosinophils in human skin (Camp et al., 1984). The cysteinyl-LTs (LTC4, LTD4, and LTE4) contract bronchial smooth muscle in vitro (Samhoun & Piper, 1983) and in vivo (Barnes et al., 1984), promote secretion of mucus from human bronchial strips in vitro (Coles etal., 1983), and have a direct action on post-capillary venules to increase vascular permeability (Woodward et al., 1983). An abnormality in the regulation of arachidonic acid release from lymphocyte membrane phospholipids of CF subjects has been demonstrated (Carlstedt-Duke et al., 1986), and an increased proportion of arachidonic acid has been found in CF bronchial phospholipids (Gilljam et al., 1986). These effects might lead to an elevation in arachidonic acid turnover and putatively an increased generation of LTs from inflammatory leucocytes in CF. We have therefore used reverse-phase high
performance liquid chromatography (RP-h.p.l.c.) and selective radioimmunoassays (r.i.a.) to investigate the concentrations of LTs in the biological fluids of children with CF. Urinary excretion of LTE4 was assessed as a marker of endogenous cysteinyl-LT generation in CF children and in a matched group of normal children. The relationship of urinary LTE4 concentrations to the levels of LTB4, LTC4, LTD4, and LTE4 in the sputum of children with CF was also investigated.
LTs that can occur at acid pH (Fitzpatrick et al., 1983), and to optimise recoveries from Sep-Pak purification (Osborne et al., 1983). Urine samples were collected into sterile polypropylene containers on ice. Aliquots (2 ml) were analysed for creatinine content. Tritiumlabelled LTE4 (4 nCi) was added to the remaining aliquots (25 ml) as an internal standard, followed by 4 vol of ice-cold ethanol. Sputum samples were collected into sterile containers on ice, weighed, and [3H]-LTB4, [3H]-LTC4, [3H]-LTD4, and [3H]-LTE4 (2 nCi) added before homogenisation in 4 vol ethanol for 2 min (UltraTurrax homogeniser). Preliminary experiments demonstrated that the use of ice-cold ethanol prevents further generation of LTs following collection and during homogenisation. All samples were left at 40 C for 30 min to allow protein denaturation. Precipitated protein and cell debris were removed by centrifugation at 12000 g for 6 min at 40 C, and the supernatants were evaporated to dryness in vacuo (Buchi rotoevaporator). Leukotrienes in the samples were partially purified on C18 Sep-Pak cartridges; the samples were reconstituted in distilled water (5 ml), and loaded onto a cartridge pre-washed with methanol (5 ml) and distilled water (5 ml). The cartridges were washed successively with distilled water (5 ml), petroleum ether (5 ml), and chloroform (5 ml). Leukotriene-containing material was eluted with methanol (5 ml), filtered (Millex 0.45 FLM), evaporated to dryness, and stored at -20° C prior to reverse-phase h.p.l.c.
Reverse-phase chromatography Methods
Samples were reconstituted in 0.5 ml of the degassed h.p.l.c. solvent system, consisting of Subject selection methanol/distilled water/glacial acetic acid (69/ 31/0.01 v/v/v), adjusted to pH 5.7 with 10% CF children attending clinics at King's College NH4OH, and pumped (1 ml min-) through Hospital, Camberwell, and the Hospital for Sick a C18 guard column and a Spherisorb ODS2 Children, Great Ormond Street, London, analytical column (300 x 4.6 mm). Fractions donated samples of sputum and urine. Parental (1 ml) were collected for up to 25 min. Aliquots permission was obtained in all cases. All CF (0.8 ml) of each fraction were added to Aquasolchildren had raised sweat sodium levels (> 70 2 scintillant (5 ml), and counted for at least 2 min mmol 1-1) and evidence of airways obstruction. each in a Canberra-Packard 2100 scintillation None was receiving oral or inhaled cortico- counter to identify the radiolabelled peaks of the steroids. internal standards, and to allow calculation of Normal children had no history of atopy or of the recoveries of each LT in individual samples. recent upper respiratory tract infection, and The remaining aliquots of the fractions (0.2 ml) voluntarily donated urine samples with parental were evaporated to dryness in vacuo, and stored consent. at -20° C prior to radioimmunoassay. Extraction of samples
Radioimmunoassays
Extractions were performed at neutral pH to prevent the 11-trans isomerisation of cysteinyl-
Radioimmunoassay of LTs B4, C4, and D4 was performed using a double antibody technique.
Leukotrienes in cystic fibrosis Samples were reconstituted in the assay buffer (300 ,ul), and aliquots (100 ,ul) were assayed in duplicate. The samples were incubated for 2 h at 40 C in phosphate-buffered saline (400 Pd) with the radiolabelled LT (4 nCi) and the appropriate anti-LT serum, before addition of the goat antirabbit serum (Bio-Rad). Bound radiolabel was removed by centrifugation for 2500 g for 20 min. LTs were quantitated by interpolation on the standard curve (0.05-50 pmol/tube). The antiserum for LTB4 (gift of Dr J. Rokach) showed negligible cross-reactivity for 6-transLTB4, (SS, 12S)-LTB4, 20-hydroxy-LTB4, 20carboxy-LTB4, 5-HETE, cysteinyl-LTs, and arachidonic acid (Rokach et al., 1984). Fifty percent displacement of radiolabel occurred at an LTB4 concentration of 2.1 pmol/tube. The LTC4/LTD4 antiserum recognised (5S, 6R)-LTC4 and (5R, 6R)-LTC4 (100%), LTC4sulphone (68.9%), and LTD4 (29.4%), but did not cross-react with LTB4, 6-trans-LTB4, (5S, 12S)-LTB4, 20-hydroxy-LTB4, 20-carboxy-LTB4, LTE4, N-acetyl-LTE4, 5-HETE, various prostanoids, arachidonic acid, or glutathione (< 0.05%). Fifty percent displacement of radiolabel occurred with LT concentrations of 0.36 pmol/tube (LTC4) and 1.02 pmolltube (LTD4). The LTE4 radioimmunoassay was performed using an antiserum (gift of Dr B. Peskar) which recognised LTE4 (100%), LTC4 (90%), LTD4 (280%), N-acetyl-LTE4 (320%), and 11-transLTE4 (30%), but not LTB4 (< 0.005%), various prostanoids, arachidonic acid, or glutathione. Samples were incubated overnight at 4° C with [3H]-LTE4 (2.5 nCi) and the antiserum in TrisHCl buffer (600 ,ul; pH 7.4) containing gelatine (0.1% w/v), before addition of dextran-coated charcoal to separate unbound [3H]-LTE4. Standard LTE4 was quantitated in the range 0.02-10 pmol/tube; fifty percent binding occurred at a concentration of 0.57 pmol/tube. The LT immunoreactivity that co-eluted from h.p.l.c. with the respective [3H]-LT internal standards was corrected for recovery, and for the proportion of the total sample assayed. Urinary LTE4 immunoreactivity (in pmol) was further corrected for the individual urinary creatinine (CR) levels. Sputum LT levels were expressed as pmol of LT per gram wet weight of sputum. Comparisons between means (MannWhitney U-test) and correlation coefficients were performed using the MINITAB statistical package (Minitab Inc., State College, PA, USA). Materials The following were used: LTB4 antiserum (gift of Dr J. Rokach, Merck Frosst Laboratories, Canada), LTE4 antiserum (gift of Dr B. Peskar,
863
University of Bochum, FRG), LTB4, LTC4, LTD4, and LTE4 (Chiral Organics), [3H]-LTB4 (32 Ci/mmol), [3H]-LTC4 (39 Ci mmol-1), [3H]LTD4 (38.4 Ci mmol-F), and [3H]-LTE4 (38.4 Ci mmol-1) (New England Nuclear), Aquasol-2 scintillant (DuPont UK Ltd.), absolute ethanol, h.p.l.c.-grade methanol (FSA Laboratories), chloroform, petroleum ether (BDH Chemicals Ltd), Tris-HCl, gelatine, dextran, activated charcoal (Sigma Chemical Co.), goat anti-rabbit IgG (Biorad). Results
Sensitivity and specificity of radioimmunoassays The theoretical lower detection limits were calculated from the lowest LT concentration on the straight-line portion of the r.i.a. standard curves, corrected for the mean recovery of each LT from urine or sputum, and for the proportion of each sample assayed. The theoretical detection limit of LTE4 in urine was 9 pmol mmol-1 creatinine. In sputum, the detection limits for LTB4, LTC4, LTD4, and LTE4 were 0.5 pmol -1, 0.9 pmol g1-, 1.4 pmol g-1, and 0.6 pmol g- respectively. The linearity of measurements of LTE4 in urine was assessed by assaying samples of normal unne containing exogenous LTE4 (0, 0.03, 0.1, 0.3, 1.0 ng ml-'; n = 4). The mean coefficient of variation over this range (equivalent to 0-250 pmol mmol-1.CR) was 33%. The levels detected correlated significantly (r = 0.924, P < 0.001, n = 20) with the amount added (Figure 1). The 1.8
l1.6 y = 0.292 + 0.983 x
1.4 1
Ea,
0)
1.0
0, 0.8 U O It 0.6
?F
)F ,F
*
r= 0.924 P
