Vox Sang. 34: 329-334 (1978)
Serum Pregnancy-Associated a,-Glycoprotein Levels in Autoimmune Diseases C . H . W . Horne, B . von Schoultz, C . M . Towler, M.-G. Damber, T . Stigbrand and Lillian M . Gibb Department of Pathology, University Medical Buildings, Foresterhill, Aberdeen, and Department of Physiological Chemistry and Department of Obstetrics and Gynaecology, University of Umeli, Umei
Abstract. Serum pregnancy-associated a,-glycoprotein (a,-PAG) levels have been measured in patients with a variety of proven non-organ specific and organ-specific autoimmune diseases and in age and sex-matched hospital outpatient and inpatient controls. Significantly lower a,-PAG levels were only observed in patients with thyrotoxicosis and in patients with atrophic gastritis accompanied by megaloblastic anaemia. In the latter case a significant inverse correlation between a,-PAG and haemoglobin levels was noted.
Introduction
Although tissue damage appears to increase the serum a2-PAG concentration, it Pregnancy-associated a,-glycoprotein (a2- is, as yet, uncertain whether inflammation PAG; synonyms: pregnancy zone protein is responsible for a similar increase. In this [PZ protein], pregnancy associated macro- study we have attempted to evaluate the efglobulin [PAM], a,-AP glycoprotein [SP,], fect of chronic inflammatory disease states a,-pregnoglobulin), a high molecular weight on the serum a,-PAG concentration. Autoglycoprotein, is a normal constituent of most immune diseases are generally regarded as normal male and female sera [14]. With in- good examples of chronic inflammatory discreasing age a moderate rise in its serum ease, and since a2-PAG is known to have concentration is observed in both normal immunosuppressive properties [13, 15, 171 males and females [12]. The reason for this it seemed worthwhile investigating the reincrease is unclear. A small increase in the lationship between a,-PAG levels and the serum levels can also be observed in pa- presence or absence of autoimmune disease. tients subjected to surgical trauma [8]. Un- We have accordingly measured the serum like classical acute-phase reactants, how- a,-PAG concentration in patients with variever, this increase is delayed, being maximal ous organ-specific and non-specific autoby post-operative day 7 and for this reason immune diseases and compared these to age ap-PAG cannot be regarded as a classical and sex-matched hospital inpatient and outpatient controls. acute-phase reactant.
330
Hornehon Schoultz/Towler/Damber/Stigbrand/Gibb
Table I ~~
Number of cases
Mean age
Range
1 Hashimoto's disease myxoedema Controls
14 (1 1F/3M)
56.2 55.7
36-74 36-74
2 Thyrotoxicosis Controls
14 (1 2F/2M)
44.7 43.8
19-72 18-71
3 Idiopathic adrenalitis Controls
11 (9F/2M)
31.3 31.6
17-41 15-43
4 (i) Atrophic gastritis with IF antibodies Controls
25 (1 8F/7M)
67.6
35-91
66.0
34-88
68.6
12-86
69.1
10-83
Organ-specificdiseases
(ii) Atrophic gastritis without IF antibodies Controls Non-organ specific diseases 1 Rheumatoid arthritis Controls
29 (I 9F/10M)
53.3 53.8
9-77 9-77
2 Chronic active hepatitis Controls
28 (21F/7M)
48.8 49.1
17-77 18-78
3 Systemic lupus erythematosus Controls
14 (14~)
41.9 42.1
18-57 18-58
4 Primary biliary cirrhosis Controls
28 (26F/2M)
53.8 53.3
32-75 31-75
Patients and Methods Serum samples were selected from the files of the Regional Immunopathology Laboratory. All sera had been stored at -20 OC (for up to 4 years) before assay. The sera from patients with various autoimmune diseases were chosen on the following basis: Organ-Specific Diseases (a) Hashimoto's disease andlor myxoedema high titre antibodies to thyroglobulin based on precipitin and tanned red cell tests and strongly positive test for thyroid microsomal antibodies (fluorescent antibody technique, FAT) [2].
(b) Thyrotoxicosis - clinically hyperthyroid with strongly positive test for thyroid microsomal antibodies (FAT). (c) Idiopathic adrenalitis - strongly positive test for antibodies to adrenal cortex cells (FAT). (d) Atrophic gastritis. (i) Strongly positive test for antibodies to gastric parietal cells (FAT) and positive test for antibodies to intrinsic factor (Ardeman and Chanarin) [l]. (ii) Strongly positive test for antibodies to gastric parietal cells (FAT) and negative test for antibodies to intrinsic factor. Non-Organ Specific Diseases (a) Rheumatoid arthritis - polyarthritis with positive latex fixation test.
Serum Pregnancy-Associated a,-Glycoprotein
33 1
(b) Chronic active hepatitis - clinical and biochemical evidence of chronic liver disease together with strongly positive test for antibodies to smooth muscle (FAT). (c) Systemic lupus erythematosus - clinical evidence of systemic disease together with high titre antibodies to nuclear constituents (FAT) and detectable antibodies to double-stranded DNA (antiDNA kit, Radiochemical Centre, Amersham). (d) Primary biliary cirrhosis - clinical, biochemical and serological evidence of disease and strongly positive test for antimitochondrial antibodies (FAT).
discarded and the DASP particles containing the bound radioactivity, after three washes in normal saline, were counted in a conventional well-type gamma counter. The counting time was 1-2min. In the statistical analysis of results, differences in the mean serum a,-PAG concentrations were evaluated using a paired ‘t’ test. Since the values were distributed asymmetrically about the mean, comparisons were made following logarithmic transformation of the values [lo].
Control samples were also obtained from the files of the Immunopathology Laboratory. They consisted of serum samples from hospital inpatients and outpatients who were known not to have autoimmune disease and who were thought not to be receiving any steroid therapy. Samples were matched for age ( 53 years) and sex and for storage time (k 2 months). The serum a,-PAG concentration was measured using a double antibody solid phase radioimmunoassay technique [ 5 ] . 125I-labelled a2-PAG was prepared by the method of Hunter and Creenwood [9],and usually yielded preparations with a specific activity of 50 pCVfig. The immunoadsorbent was a sheep anti-rabbit y-globulin coupled to cellulose particles (DASP, Organon, Oss, Holland). The monospecific rabbit antiserum to a,PAG was prepared as described previously [3]. The assay volume was 300 yl. The a,-PAG antiserum (100,d) was used at a dilution of 1:150,000. Purified a,-PAG standards (100 P I ) giving a final range of 15.6-2,000 ng/ml or serum samples (100 PI) were added to the assay. Samples found to contain more than 2,000ng/ml were diluted as appropriate. The labelled a,-PAG (1OOpl) was used at a concentration of approximately 50 ng/rnl. All reagents were diluted in the phosphate buffer described by Wide et al. [18]. All standards and test samples were assayed in duplicate. After incubation at room temperature for 2 h, the bound and free a,-PAG were separated using the second antibody (DASP) [6]. 1 ml of a 5.5-ml concentrate of DASP diluted to 100ml with buffer was mixed with each sample and rotated at room temperature for 16h. After centrifugation (1,000 g for 2 min) the supernatant was
Table11 shows the serum a2-PAG concentrations (mean k 1SD) in both test and control groups. Only in two groups is there a significant difference between the test and control sera. The mean a,-PAG concentration in those patients with thyrotoxicosis is significantly lower (p < 0.05) than the age and sex-matched controls. Similarly those patients with atrophic gastritis who also had intrinsic factor antibodies have significantly lower a2-PAG levels (p < 0.05) than the matched controls. However, no such difference was observed in the group with atrophic gastritis with no intrinsic factor antibodies. An inverse correlation (r = 0.51 ;p
Serum Pregnancy-Associated a,-Glycoprotein Levels in Autoimmune Diseases C . H . W . Horne, B . von Schoultz, C . M . Towler, M.-G. Damber, T . Stigbrand and Lillian M . Gibb Department of Pathology, University Medical Buildings, Foresterhill, Aberdeen, and Department of Physiological Chemistry and Department of Obstetrics and Gynaecology, University of Umeli, Umei
Abstract. Serum pregnancy-associated a,-glycoprotein (a,-PAG) levels have been measured in patients with a variety of proven non-organ specific and organ-specific autoimmune diseases and in age and sex-matched hospital outpatient and inpatient controls. Significantly lower a,-PAG levels were only observed in patients with thyrotoxicosis and in patients with atrophic gastritis accompanied by megaloblastic anaemia. In the latter case a significant inverse correlation between a,-PAG and haemoglobin levels was noted.
Introduction
Although tissue damage appears to increase the serum a2-PAG concentration, it Pregnancy-associated a,-glycoprotein (a2- is, as yet, uncertain whether inflammation PAG; synonyms: pregnancy zone protein is responsible for a similar increase. In this [PZ protein], pregnancy associated macro- study we have attempted to evaluate the efglobulin [PAM], a,-AP glycoprotein [SP,], fect of chronic inflammatory disease states a,-pregnoglobulin), a high molecular weight on the serum a,-PAG concentration. Autoglycoprotein, is a normal constituent of most immune diseases are generally regarded as normal male and female sera [14]. With in- good examples of chronic inflammatory discreasing age a moderate rise in its serum ease, and since a2-PAG is known to have concentration is observed in both normal immunosuppressive properties [13, 15, 171 males and females [12]. The reason for this it seemed worthwhile investigating the reincrease is unclear. A small increase in the lationship between a,-PAG levels and the serum levels can also be observed in pa- presence or absence of autoimmune disease. tients subjected to surgical trauma [8]. Un- We have accordingly measured the serum like classical acute-phase reactants, how- a,-PAG concentration in patients with variever, this increase is delayed, being maximal ous organ-specific and non-specific autoby post-operative day 7 and for this reason immune diseases and compared these to age ap-PAG cannot be regarded as a classical and sex-matched hospital inpatient and outpatient controls. acute-phase reactant.
330
Hornehon Schoultz/Towler/Damber/Stigbrand/Gibb
Table I ~~
Number of cases
Mean age
Range
1 Hashimoto's disease myxoedema Controls
14 (1 1F/3M)
56.2 55.7
36-74 36-74
2 Thyrotoxicosis Controls
14 (1 2F/2M)
44.7 43.8
19-72 18-71
3 Idiopathic adrenalitis Controls
11 (9F/2M)
31.3 31.6
17-41 15-43
4 (i) Atrophic gastritis with IF antibodies Controls
25 (1 8F/7M)
67.6
35-91
66.0
34-88
68.6
12-86
69.1
10-83
Organ-specificdiseases
(ii) Atrophic gastritis without IF antibodies Controls Non-organ specific diseases 1 Rheumatoid arthritis Controls
29 (I 9F/10M)
53.3 53.8
9-77 9-77
2 Chronic active hepatitis Controls
28 (21F/7M)
48.8 49.1
17-77 18-78
3 Systemic lupus erythematosus Controls
14 (14~)
41.9 42.1
18-57 18-58
4 Primary biliary cirrhosis Controls
28 (26F/2M)
53.8 53.3
32-75 31-75
Patients and Methods Serum samples were selected from the files of the Regional Immunopathology Laboratory. All sera had been stored at -20 OC (for up to 4 years) before assay. The sera from patients with various autoimmune diseases were chosen on the following basis: Organ-Specific Diseases (a) Hashimoto's disease andlor myxoedema high titre antibodies to thyroglobulin based on precipitin and tanned red cell tests and strongly positive test for thyroid microsomal antibodies (fluorescent antibody technique, FAT) [2].
(b) Thyrotoxicosis - clinically hyperthyroid with strongly positive test for thyroid microsomal antibodies (FAT). (c) Idiopathic adrenalitis - strongly positive test for antibodies to adrenal cortex cells (FAT). (d) Atrophic gastritis. (i) Strongly positive test for antibodies to gastric parietal cells (FAT) and positive test for antibodies to intrinsic factor (Ardeman and Chanarin) [l]. (ii) Strongly positive test for antibodies to gastric parietal cells (FAT) and negative test for antibodies to intrinsic factor. Non-Organ Specific Diseases (a) Rheumatoid arthritis - polyarthritis with positive latex fixation test.
Serum Pregnancy-Associated a,-Glycoprotein
33 1
(b) Chronic active hepatitis - clinical and biochemical evidence of chronic liver disease together with strongly positive test for antibodies to smooth muscle (FAT). (c) Systemic lupus erythematosus - clinical evidence of systemic disease together with high titre antibodies to nuclear constituents (FAT) and detectable antibodies to double-stranded DNA (antiDNA kit, Radiochemical Centre, Amersham). (d) Primary biliary cirrhosis - clinical, biochemical and serological evidence of disease and strongly positive test for antimitochondrial antibodies (FAT).
discarded and the DASP particles containing the bound radioactivity, after three washes in normal saline, were counted in a conventional well-type gamma counter. The counting time was 1-2min. In the statistical analysis of results, differences in the mean serum a,-PAG concentrations were evaluated using a paired ‘t’ test. Since the values were distributed asymmetrically about the mean, comparisons were made following logarithmic transformation of the values [lo].
Control samples were also obtained from the files of the Immunopathology Laboratory. They consisted of serum samples from hospital inpatients and outpatients who were known not to have autoimmune disease and who were thought not to be receiving any steroid therapy. Samples were matched for age ( 53 years) and sex and for storage time (k 2 months). The serum a,-PAG concentration was measured using a double antibody solid phase radioimmunoassay technique [ 5 ] . 125I-labelled a2-PAG was prepared by the method of Hunter and Creenwood [9],and usually yielded preparations with a specific activity of 50 pCVfig. The immunoadsorbent was a sheep anti-rabbit y-globulin coupled to cellulose particles (DASP, Organon, Oss, Holland). The monospecific rabbit antiserum to a,PAG was prepared as described previously [3]. The assay volume was 300 yl. The a,-PAG antiserum (100,d) was used at a dilution of 1:150,000. Purified a,-PAG standards (100 P I ) giving a final range of 15.6-2,000 ng/ml or serum samples (100 PI) were added to the assay. Samples found to contain more than 2,000ng/ml were diluted as appropriate. The labelled a,-PAG (1OOpl) was used at a concentration of approximately 50 ng/rnl. All reagents were diluted in the phosphate buffer described by Wide et al. [18]. All standards and test samples were assayed in duplicate. After incubation at room temperature for 2 h, the bound and free a,-PAG were separated using the second antibody (DASP) [6]. 1 ml of a 5.5-ml concentrate of DASP diluted to 100ml with buffer was mixed with each sample and rotated at room temperature for 16h. After centrifugation (1,000 g for 2 min) the supernatant was
Table11 shows the serum a2-PAG concentrations (mean k 1SD) in both test and control groups. Only in two groups is there a significant difference between the test and control sera. The mean a,-PAG concentration in those patients with thyrotoxicosis is significantly lower (p < 0.05) than the age and sex-matched controls. Similarly those patients with atrophic gastritis who also had intrinsic factor antibodies have significantly lower a2-PAG levels (p < 0.05) than the matched controls. However, no such difference was observed in the group with atrophic gastritis with no intrinsic factor antibodies. An inverse correlation (r = 0.51 ;p
