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The latency of serum acute phase proteins in meningococcal septicemia, with special emphasis on lactoferrin
Clinica Chimica Ada, Elsevier
136 (1984) 173-178
173
CCA 02754
The latency of serum acute phase proteins in meningococcal septicemia, with special emphasis on lactoferrin Tore J. Gutteberg
*, Bjarn Haneberg
and Trond Jorgensen
Institutes of Clinical Medicine and Medical Biology, University of Tromse, and Medical Department B, University of Bergen (Norway) (Received June 1st; revision September 22nd, 1983) Key words: Mkngococcal
septicemia; Operative trauma; Lacioferrin; C-reactive protein; a, -Aniittypsin; a,-Antichymottypsin; Orosomucoid; Haptoglobin
Summary
Serum lactoferrin concentrations were elevated in almost all children with meningococcal septicemia, in whom the disease had been clinically apparent for less than 18 hours, while the concentrations were normal or only moderately elevated in patients who had had the disease longer before being admitted. Concentrations of C-reactive protein (CRP) were markedly elevated, even with a time lapse of less than six hours, making this the most suitable parameter for the early diagnosis of severe meningococcal infection. Following an operative injury on children the lactoferrin concentrations changed very little. More than six hours after an operation, however, a marked increase in CRP-values was observed, possibly indicating differentiation of this response from that of bacterial infection. The concomitant study of serum cut-antitrypsin, qantichymotrypsin, orosomucoid and haptoglobin did not uncover results of great significance with regard to early changes.
Introduction
Meningococcal septicemia is almost always fatal unless prompt medical action is taken. Since the clinical signs at the early stage may not be specific for the disease, there is a need for rapid additional diagnostic aids. Among the acute phase reactants, serum C-reactive protein (CRP) is most useful in diagnosis of septicemia * Send correspondence to: Dr. Tore J. Gutteberg, Bameavdelingen, N-9012 Regiosykehuset i Tromse, Norway. 0009-8981/84/$03.00
0 1984 Elsevier Science Publishers B.V.
174
of the newborn (Gutteberg et al, unpublished observations). Serum concentrations of CRP are also elevated on admission, or during the first week of bacterial meningitis [1,2]. A measurable CRP-response is similarly evident only a few hours after operative injuries, and is followed in time by q-antichymotrypsin, cu,-antitrypsin, q-acid glycoprotein or orosomucoid, and haptoglobin reactions [3,4]. There are indications that lactoferrin may also represent an acute phase protein (Gutteberg et al, unpublished). We have examined the initial serum samples from children just admitted with meningococcal septicemia, for lactoferrin, CRP, cr,-antitrypsin, cu,-antichymotrypsin, orosomucoid and haptoglobin. The concentrations of these proteins were compared with the time when the disease started or was first noticed. Similar comparisons were made with the time after a standardised operative procedure in children. The aim was to find out whether there are grounds to use any of these parameters in the early diagnosis of meningococcal septicemia. Materials and methods Individuals Twenty-one infants and children, aged 2-14 years (mean and median ages 7 years), with meningococcal septicemia were included in the study. The diagnosis was based on the demonstration of meningococci in blood and/or spinal fluid from 17 of them, while in four the diagnosis was made because of a typical clinical picture with hemorrhagic skin lesions. In each case the time was noted from onset of symptoms, mainly fever. Sera, obtained on admission to hospital, were separated within 2 h after venepuncture and stored at - 20°C until used. Ten children aged 4-14 years (mean and median ages 6 years), who had undergone ureteral reimplantation because of vesicoureteral reflux, were also studied. At the time of operation they were afebrile and well, and none of them had had episodes of urinary tract infections within the last 3 months. They had all received ampicillin therapy from shortly before the operation. Blood samples were obtained before and at intervals after the operation through an indwelling cannula which was kept for 2 days, and serum was immediately separated and stored at -20°C. Samples could then easily be obtained from all these patients up to 33 h following the operation. Informed consent was obtained from the parents. As controls served 19 healthy infants and children, 1 week to 11 years of age (mean and median age 5 and 4 years, respectively). Sera from these controls were obtained and stored as for the meningococcal patients. Both serum and EDTAplasma were obtained from healthy adults to evaluate the lactoferrin determination. Serum proteins Serum lactoferrin concentrations were determined by a modified radioimmunoassay described previously [5]. Briefly, purified human lactoferrin (Behringwerke AG, Marburg-Lahn, FRG) was iodinated by the chloramine-T method [6], and the resulting specific radioactivity was of the order of 3-6 X lo5 cpm per pg. 50 ng 1251-lactoferrin were mixed with 100 ~1 of the serum to be tested, in tubes previously
175
coated with lactoferrin-specific antibodies (Behringwerke AG). These antibodies had been isolated by elution from a lactoferrin-Sepharose (4B) affinity column, using a mixture consisting of 4 mol/l urea, 0.5 mol/l NaCl and 0.1 mol/l sodium acetate at a pH of 4.5. After overnight incubation at 37°C the tubes were washed four times with 0.154 mol/l NaCl, and the remaining radioactivity was counted. Standard curves were obtained in the range of 0.55200 mg/l lactoferrin, which on SDS polyacrylamide gel electrophoresis [7] showed only one major band. Purified lactoferrin provided by Dr. A.-B. Kolsto, National Institute of Public Health, Oslo, corresponded to our standards. The day-to-day variation of the method was 22%, and the coefficient of variation was 12.5%. The serum concentrations of CRP, a,-antitrypsin, a,-antichymotrypsin and orosomucoid were determined by single radial immunodiffusion (Behringwerke AG). Haptoglobin was determined spectrophotometrically [8]. Because of the limited amounts of blood available for testing, the complete series of proteins could not be determined in all patients. The Wilcoxon test for paired samples was used for calculation of statistical significance of proportional differences. Results and discussion
Lactoferrin serum concentrations were elevated in all but one of the 11 meningococcal patients who had been ill for less than 18 h (Fig. 1). It is therefore likely that
. La-i--.. ..-..
Lactoterrm
CRP.
LOO mgll
400 mg/l
1 . l
100
.
l
.
.
_.
IO
:. .
.
.
.
.
.
.
4L.-_-----
1 0
6
12
16 2‘. hours
0
days
6
12
18 2~ hours
2
3
L
5
6
days
Fig. 1. Concentrations of lactoferrin in the first sera obtained from children with meningococcal septicemia (0) in relation to delay from the onset of symptoms or signs, and from children who had undergone reimplantation of ureter (0) in relation to time after operation. The vertical lines indicate SD. The horizontal stippled line represents the upper limit (mean+ 2 SD) in healthy infants and children. Fig. 2. Concentrations of CRP in sera from patients with meningccoccal septicemia; legend as in Fig. 1, except that the stippled line indicates the upper limit of the range seen in healthy individuals.
176
lactoferrin acts like an acute phase protein. The moderately elevated and normal serum levels in patients who had been ill for 24 h or more can be explained by the hypothesis that these patients had a less fulminant form of the disease, making admission less urgent. However, only a moderate increase in lactoferrin concentrations was seen during the first 27 h following an operative injury. Only the increase from 0 to 6 h was statistically significant (p < 0.05). Possibly, the lactoferrin response discriminates between damage caused by severe bacterial infection and that resulting from operative injury. On average, serum lactoferrin concentrations were 26% higher than those in plasma which have previously been used by others for determinations [5]. Although this difference was significant, serum levels were used throughout our study. Storing blood for up to 5 days at 4’C before separation of serum influenced our results only slightly, giving 10% higher values than in serum separated immediately after venepuncture. It is therefore unlikely that technical factors were responsible for the above trends in lactoferrin measurements. The concentrations of CRP were elevated in all our meningococcal patients (Fig. 2), confirming our unpublished observations on infants with neonatal septicemia, and the observations by others on patients with bacterial meningitis [1,2]. Moreover, high levels of CRP were found even in our patients who had been ill for less than 6 h. Even though this finding can be explained by the acutely ill patients having had the disease for a longer period than symptoms or signs had been registered, it indicates that CRP is a valuable tool in the early diagnosis of severe meningococcal infection. The time lag and the marked CRP response within the first 24 h after an operative injury also agree with previous observations [3,4]. The difference in response during the first few hours of meningococcal disease and after the operative
*,antitrypsin
d, antichymotrypsin
6 hours
days
Fig. 3. Concentrations of a,-antitrypsin upper limit, legend as in Fig. 1.
12
A 18 24-i hours
in sera from patients with mening ococcal
Fig. 4. Concentrations of a,-antichymotrypsin the upper limit, legend as in Fig. 1.
days septicemia,
and the
in sera from patients with meningococca 1 septicemia,
and
177 Orosomucoid
Haptoglobin
. .
0.1' -0
J
6
12
16 2A hours
2
3
4
0.1 --
5 6 days
Fig. 5. Concentrations of orosomucoid upper limit, legend as in Fig. 1. Fig. 6. Concentrations of haptoglobin limit, legend as in Fig. 1.
0
6
12
16 2L hours
2
in sera from patients with meningococcal
in sera from patients with meningococcal
3
4
5
6
days
septicemia,
and the
septicemia, and the upper
also suggests that this is due to a difference in strength or quality of stimulation. This has been found useful in uncovering septic complications following gastrectomy [lo], or in patients operated upon to place or revise shunts for hydrocephalus [9]. The early responses of a,-antitrypsin, a,-antichymotrypsin, orosomucoid and haptoglobin as a result of severe meningococcal infections were evidently not very marked, and elevated serum levels were the exception rather than the rule (Figs. 3-6). Following operative injury, serum cY,-antichymotrypsin changed most rapidly, while the increases of the other proteins during the first 27 h were negligible. Moreover, none of these proteins seemed to discriminate between the damage from meningococcal disease and the operative injury. In conclusion, therefore, CRP seems to be the most valuable of the acute phase proteins presently tested in allowing a tentative diagnosis of severe meningococcemia to be ruled out.
injury
Acknowledgement We are grateful
to Dr. Anne-Brit
Kolsto
for supplying
purified
lactoferrin.
References 1 Belfrage S. Plasma protein pattern in course of acute infectious disease. Acta Med Stand 1963; suppl 395: 1-169. 2 Peltola HO. C-reactive protein for rapid monitoring of infections of the central nervous system. Lancet 1982; 1: 980-982. 3 Aronsen K-F, Ekelund G, Kindmark C-O, Laurel1 C-B. Sequential changes of plasma proteins after surgical trauma. Stand J Clin Lab Invest 1972; 29; suppl 124: 127-136.
178 4 Fischer CL, Gill CW. Acute-phase proteins. In: Ritzmann, Dawels, eds. Serum protein abnormalities. Diagnostic and clinical aspects. Boston: Little Brown, 1975: 331-350. 5 Bennett RM, MohIa C. A solid-phase radioimmunoassay for the measurements of lactoferrin in human plasma: variations with age, sex and disease. J Lab Clin Med 1976; 88: 156-166. by chloramine-T. 6 Sonoda S, Schlamowitz M. Studies of ‘251-trace labelling of immunoglobulin-G Immunochemistry 1970; 7: 885-898. 7 Laemmh UK. Cleavage of structural proteins during the assembly of the head of bacterophage T4. Nature 1970; 227: 680-685. 8 Tarukoski PH. Quantitative spectrophotometric determination of haptoglobin. Stand J Chn Lab Invest 1966; 18: 80-86. 9 Bayston R. Serum C-reactive protein test in diagnosis of septic complications of cerebrospinal fluid shunts for hydrocephalus. Arch Dis Child 1979; 54: 545-548. 10 Werner M. Serum protein changes during the acute phase reaction. Clin Chim Acta 1969; 25: 299-305.
136 (1984) 173-178
173
CCA 02754
The latency of serum acute phase proteins in meningococcal septicemia, with special emphasis on lactoferrin Tore J. Gutteberg
*, Bjarn Haneberg
and Trond Jorgensen
Institutes of Clinical Medicine and Medical Biology, University of Tromse, and Medical Department B, University of Bergen (Norway) (Received June 1st; revision September 22nd, 1983) Key words: Mkngococcal
septicemia; Operative trauma; Lacioferrin; C-reactive protein; a, -Aniittypsin; a,-Antichymottypsin; Orosomucoid; Haptoglobin
Summary
Serum lactoferrin concentrations were elevated in almost all children with meningococcal septicemia, in whom the disease had been clinically apparent for less than 18 hours, while the concentrations were normal or only moderately elevated in patients who had had the disease longer before being admitted. Concentrations of C-reactive protein (CRP) were markedly elevated, even with a time lapse of less than six hours, making this the most suitable parameter for the early diagnosis of severe meningococcal infection. Following an operative injury on children the lactoferrin concentrations changed very little. More than six hours after an operation, however, a marked increase in CRP-values was observed, possibly indicating differentiation of this response from that of bacterial infection. The concomitant study of serum cut-antitrypsin, qantichymotrypsin, orosomucoid and haptoglobin did not uncover results of great significance with regard to early changes.
Introduction
Meningococcal septicemia is almost always fatal unless prompt medical action is taken. Since the clinical signs at the early stage may not be specific for the disease, there is a need for rapid additional diagnostic aids. Among the acute phase reactants, serum C-reactive protein (CRP) is most useful in diagnosis of septicemia * Send correspondence to: Dr. Tore J. Gutteberg, Bameavdelingen, N-9012 Regiosykehuset i Tromse, Norway. 0009-8981/84/$03.00
0 1984 Elsevier Science Publishers B.V.
174
of the newborn (Gutteberg et al, unpublished observations). Serum concentrations of CRP are also elevated on admission, or during the first week of bacterial meningitis [1,2]. A measurable CRP-response is similarly evident only a few hours after operative injuries, and is followed in time by q-antichymotrypsin, cu,-antitrypsin, q-acid glycoprotein or orosomucoid, and haptoglobin reactions [3,4]. There are indications that lactoferrin may also represent an acute phase protein (Gutteberg et al, unpublished). We have examined the initial serum samples from children just admitted with meningococcal septicemia, for lactoferrin, CRP, cr,-antitrypsin, cu,-antichymotrypsin, orosomucoid and haptoglobin. The concentrations of these proteins were compared with the time when the disease started or was first noticed. Similar comparisons were made with the time after a standardised operative procedure in children. The aim was to find out whether there are grounds to use any of these parameters in the early diagnosis of meningococcal septicemia. Materials and methods Individuals Twenty-one infants and children, aged 2-14 years (mean and median ages 7 years), with meningococcal septicemia were included in the study. The diagnosis was based on the demonstration of meningococci in blood and/or spinal fluid from 17 of them, while in four the diagnosis was made because of a typical clinical picture with hemorrhagic skin lesions. In each case the time was noted from onset of symptoms, mainly fever. Sera, obtained on admission to hospital, were separated within 2 h after venepuncture and stored at - 20°C until used. Ten children aged 4-14 years (mean and median ages 6 years), who had undergone ureteral reimplantation because of vesicoureteral reflux, were also studied. At the time of operation they were afebrile and well, and none of them had had episodes of urinary tract infections within the last 3 months. They had all received ampicillin therapy from shortly before the operation. Blood samples were obtained before and at intervals after the operation through an indwelling cannula which was kept for 2 days, and serum was immediately separated and stored at -20°C. Samples could then easily be obtained from all these patients up to 33 h following the operation. Informed consent was obtained from the parents. As controls served 19 healthy infants and children, 1 week to 11 years of age (mean and median age 5 and 4 years, respectively). Sera from these controls were obtained and stored as for the meningococcal patients. Both serum and EDTAplasma were obtained from healthy adults to evaluate the lactoferrin determination. Serum proteins Serum lactoferrin concentrations were determined by a modified radioimmunoassay described previously [5]. Briefly, purified human lactoferrin (Behringwerke AG, Marburg-Lahn, FRG) was iodinated by the chloramine-T method [6], and the resulting specific radioactivity was of the order of 3-6 X lo5 cpm per pg. 50 ng 1251-lactoferrin were mixed with 100 ~1 of the serum to be tested, in tubes previously
175
coated with lactoferrin-specific antibodies (Behringwerke AG). These antibodies had been isolated by elution from a lactoferrin-Sepharose (4B) affinity column, using a mixture consisting of 4 mol/l urea, 0.5 mol/l NaCl and 0.1 mol/l sodium acetate at a pH of 4.5. After overnight incubation at 37°C the tubes were washed four times with 0.154 mol/l NaCl, and the remaining radioactivity was counted. Standard curves were obtained in the range of 0.55200 mg/l lactoferrin, which on SDS polyacrylamide gel electrophoresis [7] showed only one major band. Purified lactoferrin provided by Dr. A.-B. Kolsto, National Institute of Public Health, Oslo, corresponded to our standards. The day-to-day variation of the method was 22%, and the coefficient of variation was 12.5%. The serum concentrations of CRP, a,-antitrypsin, a,-antichymotrypsin and orosomucoid were determined by single radial immunodiffusion (Behringwerke AG). Haptoglobin was determined spectrophotometrically [8]. Because of the limited amounts of blood available for testing, the complete series of proteins could not be determined in all patients. The Wilcoxon test for paired samples was used for calculation of statistical significance of proportional differences. Results and discussion
Lactoferrin serum concentrations were elevated in all but one of the 11 meningococcal patients who had been ill for less than 18 h (Fig. 1). It is therefore likely that
. La-i--.. ..-..
Lactoterrm
CRP.
LOO mgll
400 mg/l
1 . l
100
.
l
.
.
_.
IO
:. .
.
.
.
.
.
.
4L.-_-----
1 0
6
12
16 2‘. hours
0
days
6
12
18 2~ hours
2
3
L
5
6
days
Fig. 1. Concentrations of lactoferrin in the first sera obtained from children with meningococcal septicemia (0) in relation to delay from the onset of symptoms or signs, and from children who had undergone reimplantation of ureter (0) in relation to time after operation. The vertical lines indicate SD. The horizontal stippled line represents the upper limit (mean+ 2 SD) in healthy infants and children. Fig. 2. Concentrations of CRP in sera from patients with meningccoccal septicemia; legend as in Fig. 1, except that the stippled line indicates the upper limit of the range seen in healthy individuals.
176
lactoferrin acts like an acute phase protein. The moderately elevated and normal serum levels in patients who had been ill for 24 h or more can be explained by the hypothesis that these patients had a less fulminant form of the disease, making admission less urgent. However, only a moderate increase in lactoferrin concentrations was seen during the first 27 h following an operative injury. Only the increase from 0 to 6 h was statistically significant (p < 0.05). Possibly, the lactoferrin response discriminates between damage caused by severe bacterial infection and that resulting from operative injury. On average, serum lactoferrin concentrations were 26% higher than those in plasma which have previously been used by others for determinations [5]. Although this difference was significant, serum levels were used throughout our study. Storing blood for up to 5 days at 4’C before separation of serum influenced our results only slightly, giving 10% higher values than in serum separated immediately after venepuncture. It is therefore unlikely that technical factors were responsible for the above trends in lactoferrin measurements. The concentrations of CRP were elevated in all our meningococcal patients (Fig. 2), confirming our unpublished observations on infants with neonatal septicemia, and the observations by others on patients with bacterial meningitis [1,2]. Moreover, high levels of CRP were found even in our patients who had been ill for less than 6 h. Even though this finding can be explained by the acutely ill patients having had the disease for a longer period than symptoms or signs had been registered, it indicates that CRP is a valuable tool in the early diagnosis of severe meningococcal infection. The time lag and the marked CRP response within the first 24 h after an operative injury also agree with previous observations [3,4]. The difference in response during the first few hours of meningococcal disease and after the operative
*,antitrypsin
d, antichymotrypsin
6 hours
days
Fig. 3. Concentrations of a,-antitrypsin upper limit, legend as in Fig. 1.
12
A 18 24-i hours
in sera from patients with mening ococcal
Fig. 4. Concentrations of a,-antichymotrypsin the upper limit, legend as in Fig. 1.
days septicemia,
and the
in sera from patients with meningococca 1 septicemia,
and
177 Orosomucoid
Haptoglobin
. .
0.1' -0
J
6
12
16 2A hours
2
3
4
0.1 --
5 6 days
Fig. 5. Concentrations of orosomucoid upper limit, legend as in Fig. 1. Fig. 6. Concentrations of haptoglobin limit, legend as in Fig. 1.
0
6
12
16 2L hours
2
in sera from patients with meningococcal
in sera from patients with meningococcal
3
4
5
6
days
septicemia,
and the
septicemia, and the upper
also suggests that this is due to a difference in strength or quality of stimulation. This has been found useful in uncovering septic complications following gastrectomy [lo], or in patients operated upon to place or revise shunts for hydrocephalus [9]. The early responses of a,-antitrypsin, a,-antichymotrypsin, orosomucoid and haptoglobin as a result of severe meningococcal infections were evidently not very marked, and elevated serum levels were the exception rather than the rule (Figs. 3-6). Following operative injury, serum cY,-antichymotrypsin changed most rapidly, while the increases of the other proteins during the first 27 h were negligible. Moreover, none of these proteins seemed to discriminate between the damage from meningococcal disease and the operative injury. In conclusion, therefore, CRP seems to be the most valuable of the acute phase proteins presently tested in allowing a tentative diagnosis of severe meningococcemia to be ruled out.
injury
Acknowledgement We are grateful
to Dr. Anne-Brit
Kolsto
for supplying
purified
lactoferrin.
References 1 Belfrage S. Plasma protein pattern in course of acute infectious disease. Acta Med Stand 1963; suppl 395: 1-169. 2 Peltola HO. C-reactive protein for rapid monitoring of infections of the central nervous system. Lancet 1982; 1: 980-982. 3 Aronsen K-F, Ekelund G, Kindmark C-O, Laurel1 C-B. Sequential changes of plasma proteins after surgical trauma. Stand J Clin Lab Invest 1972; 29; suppl 124: 127-136.
178 4 Fischer CL, Gill CW. Acute-phase proteins. In: Ritzmann, Dawels, eds. Serum protein abnormalities. Diagnostic and clinical aspects. Boston: Little Brown, 1975: 331-350. 5 Bennett RM, MohIa C. A solid-phase radioimmunoassay for the measurements of lactoferrin in human plasma: variations with age, sex and disease. J Lab Clin Med 1976; 88: 156-166. by chloramine-T. 6 Sonoda S, Schlamowitz M. Studies of ‘251-trace labelling of immunoglobulin-G Immunochemistry 1970; 7: 885-898. 7 Laemmh UK. Cleavage of structural proteins during the assembly of the head of bacterophage T4. Nature 1970; 227: 680-685. 8 Tarukoski PH. Quantitative spectrophotometric determination of haptoglobin. Stand J Chn Lab Invest 1966; 18: 80-86. 9 Bayston R. Serum C-reactive protein test in diagnosis of septic complications of cerebrospinal fluid shunts for hydrocephalus. Arch Dis Child 1979; 54: 545-548. 10 Werner M. Serum protein changes during the acute phase reaction. Clin Chim Acta 1969; 25: 299-305.