J Comp Physiol B (1992) 162:168 171
J o u r n a l of Systemic, C o m p a r a t i v e ~,~.e~,o~,. and EnvironPhysiology
B .,e.,., Physiology
9 Springer-Verlag 1992
Fetuin: an acute phase protein in cattle K.M. Dziegielewska l'*, W.M. Brown 1, C.C. Gould 2, N. Matthews 1, J.E.C. Sedgwick 1, and N.R. Saunders ~ 1 Clinical Neurological Sciences (Developmental) Group, Southampton General Hospital, Southampton, $09 4XY, England 2 G.N. Gould and Partners, Veterinary Surgeons, 10 Languard Road, Southampton, England Accepted August 19, 1991
Summary. Fetuin is a plasma protein present in high concentrations during fetal development in animals of the order Artiodactyla. Its role is not known. The human homologue of fetuin - a2HS glycoprotein - has been shown to be a negative acute phase protein in adult plasma. In the present study, the concentration of fetuin was measured in the serum of healthy cattle (Boris boris) and in animals with various injuries and inflammatory disorders. The levels were decreased by 30 % in pregnancy but increased up to 10-fold in some trauma cases. A significant negative correlation between the concentrations of fetuin and albumin has also been found. Thus, fetuin appears to be a positive acute phase protein in cattle. Key words: Fetuin a2HS glycoprotein - Acute phase proteins - Cattle, Boris boris -
Introduction One of the predominant proteins in plasma of developing animals of the order Artiodactyla is the a-globulin fetuin. The concentration of fetuin in plasma of late gestation calves is around 10 mg. m1-1, which is nearly 50% of the total plasma protein concentration (Abe et al. 1976). In the adult, the levels of fetuin fall by an order of magnitude, but it still remains a substantial contributor to the plasma protein concentration (Bergmann et al. 1962). However, information about fetuin expression in adult animals is very limited and no systematic studies have been conducted to look at the range of concentrations in normal animals or possible changes induced by physiological conditions such as pregnancy or pathological conditions such as trauma. The role of fetuin is not clear, and based on in vitro studies a diverse range of properties has been proposed. Abbreviations: c%HS, c~2HSglycoprotein; AP, acute phase
* To whom offprint requests should be sent
These include antiproteolytic activity, lymphocytestimulating properties, thyroid hormone binding [see Dziegielewska et al. (1987) for references] and lipid binding properties (Kumbla et al. 1989). Only recently has the connection been made between human plasma protein a 2 HS glycoprotein (a2HS) and fetuin. Fetuin is the bovine (or other members of the Articodactyla) homologue of human a2HS (Dziegielewska et al. 1990). The function of a2HS is also not known (see Discussion), but in vivo it has been shown that it is a negative acute phase (AP) protein [see Daveau et al. (1988) for references]. Both fetuin and a2HS belong to the cystatin superfamily together with stefins, kininogens, cystatins and histidine-rich glycoprotein (Kellermann et al. 1989; Dziegielewska et al. 1990). Another member of this family, T kininogen, in the rat is a major positive AP protein (Gauthier et al. 1988). How the individual members of this family evolved to acquire their respective functions is not known, although some suggestions for T kininogen have been proposed (Gauthier et al. 1988). The AP reaction is the response to inflammation or trauma, triggered by various events including infection, wounding and chronic disease, that takes place primarily in the liver (e.g. Koj 1985). It results in a characteristic increase or decrease in the concentration of some plasma proteins termed positive or negative AP reactants. The AP protein patterns vary from one species to another. These changes in plasma levels are preceded by corresponding alterations in liver m R N A concentrations as well as changes in the rates of secretion of some AP proteins (Heinrich et al. 1990). a2HS concentrations in humans are known to change in trauma [negative AP protein, Lebreton et al. (1979)], increase in some bone disorders (e.g. osteogenesis imperfecta, Dickson et al. (1983)], and decrease in others [e.g. Paget's disease, Ashton and Smith (1980)]. In this study we have estimated the concentration range of fetuin in adult bovine plasma in normal healthy animals and in animals with various injuries and inflammatory disorders. This study indicates that bovine fetuin in vivo is a positive AP reactant.
K.M. Dziegielewska et al.: Fetuin: an acute phase protein in cattle
169
Materials and methods
Table 1. Concentrations of total protein, albumin and fetuin (rag- ml- 1) in cattle sera
Cattle sera samples were obtained from a local veterinary surgery (C.C. Gould), ADAS (Ministry of Agriculture, Fisheries and Food, Veterinary Investigation Centre, Winchester) and from Hampshire Cattle Breeders Society. Samples were collected from healthy cattle (cows and bulls), healthy cows in various stages of pregnancy, cows that had aborted spontaneously in the previous 24 h, and from animals with various injuries and infections; in this last group samples were collected within a few days of the onset of injury or illness. The reasons for the abortions were not known individually but were generally associated with bacterial or viral infections. No samples were available from animals suffering from specific bonerelated diseases. Protein measurements. Concentrations of albumin and fetuin in individual samples were measured using radial immunodiffusion (Mancini et al. 1965) in 1% w/v agarose in TRIS-barbitone buffer (pH 8.6). Antibodies used were anti-cow albumin (Dakopatts) and anti-calf fetuin (Dakopatts). The amounts of antibody used were 2.0 ~tl 9cm -z ofanti-fetuin and 3.0 ~tl 9cm -z of anti-albumin. Standards used were bovine albumin (ICN, Pentex) and calf fetuin (Hoechst). A set of three dilutions of the standard was always used on each plate. The range of standard concentrations was 0.05-0.5 mg - ml-1 for both proteins. Plasma samples were diluted 20-fold for fetuin estimation and 1000-fold for albumin. All samples were diluted in 0.9 % NaC1 and applied in 5-gl volumes in duplicate. The immunoprecipitation was left to complete for 48 h at room temperature and the diameter of each ring was measured using a dark-field vision box (Shandon Southern Products) and a graticule with subdivisions down to 0.1 mm. The variation between duplicates was less than 5%. The variations in the diameter of the same protein standard from day to day did not exceed 5%. Total protein concentrations were measured using the method of Bradford (1976) and Sigma Protein Standard (code no. $4~10). Protein solution containing 1-10 gg protein in a volume of 0.1 ml was mixed with 1.0 ml of protein reagent and the absorbance at 595 nm was measured. The known concentration of protein (Sigma Protein Standard) was plotted against the absorbance, resulting in a standard curve used to determine the protein content of the unknown plasma. All cattle plasmas were diluted 1:1000 in 0.9% NaC1. Stat&tical analys&. The 109 cattle sera samples were divided into the four groups described above and mean serum total protein, mean serum fetuin and mean serum albumin concentrations were calculated for each group together with standard errors of the means. The fetuin ratio (fetuin concentration divided by total protein concentration) was calculated for each animal. The values for each group were also compared using non-parametric methods (involving medians rather than means) due to the distribution of the data. Kruskal-Wallis one-way analysis of variance by ranks and Wilcoxon rank sum tests were used.
n Normal
18
Pregnant
16
Postabortion Other trauma
60 15
Mean SEM Mean SEM Mean SEM Mean SEM
Total protein
Albumin Fetuin
75.2 :~ 4.2 74.3 =k 2.7 71.1 • 1.9 60.9 i 2.6
38.4 + 2.3 41.6 4- 2.0 33.0 • 2.0 40.0 -4- 4.3
0.57 4-0.04 0.39 -4-0.01 0.98 • 0.89 =k0.21
SEM, Standard error of the means; n, number of animals in each group m o s t s a m p l e s were collected f r o m a n i m a l s in their p r i m e m i l k i n g a n d b r e e d i n g stage r a n g i n g f r o m 2 to 7.5 years. I n the " p r e g n a n t " g r o u p , the c o n c e n t r a t i o n o f fetuin was l o w e r b y n e a r l y 30%. This was n o t a s s o c i a t e d with a general decrease in t o t a l p r o t e i n c o n c e n t r a t i o n ( T a b l e 1). I n the o t h e r two g r o u p s ( p o s t - a b o r t i o n a n d t r a u m a ) t h e m e a n c o n c e n t r a t i o n s o f fetuin were h i g h e r b y a f a c t o r o f 2 ( T a b l e 1, Fig. 1). N o t all a n i m a l s g r o u p e d in the two p a t h o l o g i c a l c a t e g o r i e s h a d i n c r e a s e d fetuin levels. This m a y be a reflection o f the t i m i n g o f the collection o f samples, since the responses o f several o t h e r A P p l a s m a p r o t e i n s h a v e b e e n s h o w n to be t i m e - r e l a t e d [see S c h r e i b e r (1987) for references]. T h e m e d i a n fetuin r a t i o s o f the f o u r g r o u p s o f cattle were f o u n d to be significantly different (P=- 0.0012, analysis o f variance). T h e m e d i a n " p o s t - a b o r t i o n " r a t i o (1.19%) was significantly h i g h e r t h a n b o t h the m e d i a n " n o r m a l " a n d " p r e g n a n t " ratios (0.78% a n d 0.53%, P = 0 . 0 4 8 6 a n d P = 0 . 0 0 0 6 , respectively, W i l c o x o n r a n k s u m test). T h e r e were insufficient n u m bers a n d t o o large a v a r i a b i l i t y in the i n d i v i d u a l values h o w e v e r to s h o w a n y difference b e t w e e n the m e d i a n 2.0
1.5 Q.
Results C o n c e n t r a t i o n s o f t o t a l p r o t e i n , a l b u m i n a n d fetuin were m e a s u r e d in s e r u m f r o m a d u l t cattle a n d d a t a f r o m the f o u r g r o u p s a r e p r e s e n t e d in T a b l e 1. T h e m e a n c o n c e n t r a t i o n o f t o t a l p r o t e i n a n d o f a l b u m i n in all f o u r c a t e g o r i e s was similar a n d w i t h i n the r a n g e o f values r e p o r t e d p r e v i o u s l y ( A b e et al. 1976). I n the " n o r m a l " g r o u p the levels o f fetuin were also similar to t h o s e r e p o r t e d b y B e r g m a n n et al. (1962), a n d there was n o significant difference in fetuin c o n c e n t r a t i o n b e t w e e n the s a m p l e s f r o m cows a n d bulls. A l s o , there were n o detectable a g e - r e l a t e d differences in s e r u m values f o r fetuin in the a d u l t a n i m a l s . T h i s m a y be a reflection o f the fact t h a t
1.0 o~
v
c
/3_
0.5
0
Normal
Pregnant
Postabortion
Other trauma
Fig. 1. Fetuin as proportion of total protein expressed as mean values :k standard errors of the means (error bars). Median values were also compared using non-parametric methods
170
K.M. Dziegielewskaet al. : Fetuin: an acute phase protein in cattle
.o : o . . 9
5
.am
-"-'----.-.23' 9
9
9 ,
0 in
-o-
.,, 9
9
,,,
9
"
,
0
(3
'
2'0
'
4()
'
60
'
Albumin (mg- m1-1) Fig. 2. Scatterplot of serum fetuin versus serum albumin concentrations in four groups of adult cattle,with simplelinearregressionline for all data. There was a highly significant negative correlation between fetuin and albumin (r= -0.3554, P= -0.0004). 9 Normal; 9 Trauma; [] Pregnant; 9 Abortion "trauma" (0.98%) and median "normal" ratios even though the mean fetuin ratio was higher in the "trauma" group than the "post-abortion" group. The median ratio in the "pregnant" group was significantly lower than that in the "normal" group (P
J o u r n a l of Systemic, C o m p a r a t i v e ~,~.e~,o~,. and EnvironPhysiology
B .,e.,., Physiology
9 Springer-Verlag 1992
Fetuin: an acute phase protein in cattle K.M. Dziegielewska l'*, W.M. Brown 1, C.C. Gould 2, N. Matthews 1, J.E.C. Sedgwick 1, and N.R. Saunders ~ 1 Clinical Neurological Sciences (Developmental) Group, Southampton General Hospital, Southampton, $09 4XY, England 2 G.N. Gould and Partners, Veterinary Surgeons, 10 Languard Road, Southampton, England Accepted August 19, 1991
Summary. Fetuin is a plasma protein present in high concentrations during fetal development in animals of the order Artiodactyla. Its role is not known. The human homologue of fetuin - a2HS glycoprotein - has been shown to be a negative acute phase protein in adult plasma. In the present study, the concentration of fetuin was measured in the serum of healthy cattle (Boris boris) and in animals with various injuries and inflammatory disorders. The levels were decreased by 30 % in pregnancy but increased up to 10-fold in some trauma cases. A significant negative correlation between the concentrations of fetuin and albumin has also been found. Thus, fetuin appears to be a positive acute phase protein in cattle. Key words: Fetuin a2HS glycoprotein - Acute phase proteins - Cattle, Boris boris -
Introduction One of the predominant proteins in plasma of developing animals of the order Artiodactyla is the a-globulin fetuin. The concentration of fetuin in plasma of late gestation calves is around 10 mg. m1-1, which is nearly 50% of the total plasma protein concentration (Abe et al. 1976). In the adult, the levels of fetuin fall by an order of magnitude, but it still remains a substantial contributor to the plasma protein concentration (Bergmann et al. 1962). However, information about fetuin expression in adult animals is very limited and no systematic studies have been conducted to look at the range of concentrations in normal animals or possible changes induced by physiological conditions such as pregnancy or pathological conditions such as trauma. The role of fetuin is not clear, and based on in vitro studies a diverse range of properties has been proposed. Abbreviations: c%HS, c~2HSglycoprotein; AP, acute phase
* To whom offprint requests should be sent
These include antiproteolytic activity, lymphocytestimulating properties, thyroid hormone binding [see Dziegielewska et al. (1987) for references] and lipid binding properties (Kumbla et al. 1989). Only recently has the connection been made between human plasma protein a 2 HS glycoprotein (a2HS) and fetuin. Fetuin is the bovine (or other members of the Articodactyla) homologue of human a2HS (Dziegielewska et al. 1990). The function of a2HS is also not known (see Discussion), but in vivo it has been shown that it is a negative acute phase (AP) protein [see Daveau et al. (1988) for references]. Both fetuin and a2HS belong to the cystatin superfamily together with stefins, kininogens, cystatins and histidine-rich glycoprotein (Kellermann et al. 1989; Dziegielewska et al. 1990). Another member of this family, T kininogen, in the rat is a major positive AP protein (Gauthier et al. 1988). How the individual members of this family evolved to acquire their respective functions is not known, although some suggestions for T kininogen have been proposed (Gauthier et al. 1988). The AP reaction is the response to inflammation or trauma, triggered by various events including infection, wounding and chronic disease, that takes place primarily in the liver (e.g. Koj 1985). It results in a characteristic increase or decrease in the concentration of some plasma proteins termed positive or negative AP reactants. The AP protein patterns vary from one species to another. These changes in plasma levels are preceded by corresponding alterations in liver m R N A concentrations as well as changes in the rates of secretion of some AP proteins (Heinrich et al. 1990). a2HS concentrations in humans are known to change in trauma [negative AP protein, Lebreton et al. (1979)], increase in some bone disorders (e.g. osteogenesis imperfecta, Dickson et al. (1983)], and decrease in others [e.g. Paget's disease, Ashton and Smith (1980)]. In this study we have estimated the concentration range of fetuin in adult bovine plasma in normal healthy animals and in animals with various injuries and inflammatory disorders. This study indicates that bovine fetuin in vivo is a positive AP reactant.
K.M. Dziegielewska et al.: Fetuin: an acute phase protein in cattle
169
Materials and methods
Table 1. Concentrations of total protein, albumin and fetuin (rag- ml- 1) in cattle sera
Cattle sera samples were obtained from a local veterinary surgery (C.C. Gould), ADAS (Ministry of Agriculture, Fisheries and Food, Veterinary Investigation Centre, Winchester) and from Hampshire Cattle Breeders Society. Samples were collected from healthy cattle (cows and bulls), healthy cows in various stages of pregnancy, cows that had aborted spontaneously in the previous 24 h, and from animals with various injuries and infections; in this last group samples were collected within a few days of the onset of injury or illness. The reasons for the abortions were not known individually but were generally associated with bacterial or viral infections. No samples were available from animals suffering from specific bonerelated diseases. Protein measurements. Concentrations of albumin and fetuin in individual samples were measured using radial immunodiffusion (Mancini et al. 1965) in 1% w/v agarose in TRIS-barbitone buffer (pH 8.6). Antibodies used were anti-cow albumin (Dakopatts) and anti-calf fetuin (Dakopatts). The amounts of antibody used were 2.0 ~tl 9cm -z ofanti-fetuin and 3.0 ~tl 9cm -z of anti-albumin. Standards used were bovine albumin (ICN, Pentex) and calf fetuin (Hoechst). A set of three dilutions of the standard was always used on each plate. The range of standard concentrations was 0.05-0.5 mg - ml-1 for both proteins. Plasma samples were diluted 20-fold for fetuin estimation and 1000-fold for albumin. All samples were diluted in 0.9 % NaC1 and applied in 5-gl volumes in duplicate. The immunoprecipitation was left to complete for 48 h at room temperature and the diameter of each ring was measured using a dark-field vision box (Shandon Southern Products) and a graticule with subdivisions down to 0.1 mm. The variation between duplicates was less than 5%. The variations in the diameter of the same protein standard from day to day did not exceed 5%. Total protein concentrations were measured using the method of Bradford (1976) and Sigma Protein Standard (code no. $4~10). Protein solution containing 1-10 gg protein in a volume of 0.1 ml was mixed with 1.0 ml of protein reagent and the absorbance at 595 nm was measured. The known concentration of protein (Sigma Protein Standard) was plotted against the absorbance, resulting in a standard curve used to determine the protein content of the unknown plasma. All cattle plasmas were diluted 1:1000 in 0.9% NaC1. Stat&tical analys&. The 109 cattle sera samples were divided into the four groups described above and mean serum total protein, mean serum fetuin and mean serum albumin concentrations were calculated for each group together with standard errors of the means. The fetuin ratio (fetuin concentration divided by total protein concentration) was calculated for each animal. The values for each group were also compared using non-parametric methods (involving medians rather than means) due to the distribution of the data. Kruskal-Wallis one-way analysis of variance by ranks and Wilcoxon rank sum tests were used.
n Normal
18
Pregnant
16
Postabortion Other trauma
60 15
Mean SEM Mean SEM Mean SEM Mean SEM
Total protein
Albumin Fetuin
75.2 :~ 4.2 74.3 =k 2.7 71.1 • 1.9 60.9 i 2.6
38.4 + 2.3 41.6 4- 2.0 33.0 • 2.0 40.0 -4- 4.3
0.57 4-0.04 0.39 -4-0.01 0.98 • 0.89 =k0.21
SEM, Standard error of the means; n, number of animals in each group m o s t s a m p l e s were collected f r o m a n i m a l s in their p r i m e m i l k i n g a n d b r e e d i n g stage r a n g i n g f r o m 2 to 7.5 years. I n the " p r e g n a n t " g r o u p , the c o n c e n t r a t i o n o f fetuin was l o w e r b y n e a r l y 30%. This was n o t a s s o c i a t e d with a general decrease in t o t a l p r o t e i n c o n c e n t r a t i o n ( T a b l e 1). I n the o t h e r two g r o u p s ( p o s t - a b o r t i o n a n d t r a u m a ) t h e m e a n c o n c e n t r a t i o n s o f fetuin were h i g h e r b y a f a c t o r o f 2 ( T a b l e 1, Fig. 1). N o t all a n i m a l s g r o u p e d in the two p a t h o l o g i c a l c a t e g o r i e s h a d i n c r e a s e d fetuin levels. This m a y be a reflection o f the t i m i n g o f the collection o f samples, since the responses o f several o t h e r A P p l a s m a p r o t e i n s h a v e b e e n s h o w n to be t i m e - r e l a t e d [see S c h r e i b e r (1987) for references]. T h e m e d i a n fetuin r a t i o s o f the f o u r g r o u p s o f cattle were f o u n d to be significantly different (P=- 0.0012, analysis o f variance). T h e m e d i a n " p o s t - a b o r t i o n " r a t i o (1.19%) was significantly h i g h e r t h a n b o t h the m e d i a n " n o r m a l " a n d " p r e g n a n t " ratios (0.78% a n d 0.53%, P = 0 . 0 4 8 6 a n d P = 0 . 0 0 0 6 , respectively, W i l c o x o n r a n k s u m test). T h e r e were insufficient n u m bers a n d t o o large a v a r i a b i l i t y in the i n d i v i d u a l values h o w e v e r to s h o w a n y difference b e t w e e n the m e d i a n 2.0
1.5 Q.
Results C o n c e n t r a t i o n s o f t o t a l p r o t e i n , a l b u m i n a n d fetuin were m e a s u r e d in s e r u m f r o m a d u l t cattle a n d d a t a f r o m the f o u r g r o u p s a r e p r e s e n t e d in T a b l e 1. T h e m e a n c o n c e n t r a t i o n o f t o t a l p r o t e i n a n d o f a l b u m i n in all f o u r c a t e g o r i e s was similar a n d w i t h i n the r a n g e o f values r e p o r t e d p r e v i o u s l y ( A b e et al. 1976). I n the " n o r m a l " g r o u p the levels o f fetuin were also similar to t h o s e r e p o r t e d b y B e r g m a n n et al. (1962), a n d there was n o significant difference in fetuin c o n c e n t r a t i o n b e t w e e n the s a m p l e s f r o m cows a n d bulls. A l s o , there were n o detectable a g e - r e l a t e d differences in s e r u m values f o r fetuin in the a d u l t a n i m a l s . T h i s m a y be a reflection o f the fact t h a t
1.0 o~
v
c
/3_
0.5
0
Normal
Pregnant
Postabortion
Other trauma
Fig. 1. Fetuin as proportion of total protein expressed as mean values :k standard errors of the means (error bars). Median values were also compared using non-parametric methods
170
K.M. Dziegielewskaet al. : Fetuin: an acute phase protein in cattle
.o : o . . 9
5
.am
-"-'----.-.23' 9
9
9 ,
0 in
-o-
.,, 9
9
,,,
9
"
,
0
(3
'
2'0
'
4()
'
60
'
Albumin (mg- m1-1) Fig. 2. Scatterplot of serum fetuin versus serum albumin concentrations in four groups of adult cattle,with simplelinearregressionline for all data. There was a highly significant negative correlation between fetuin and albumin (r= -0.3554, P= -0.0004). 9 Normal; 9 Trauma; [] Pregnant; 9 Abortion "trauma" (0.98%) and median "normal" ratios even though the mean fetuin ratio was higher in the "trauma" group than the "post-abortion" group. The median ratio in the "pregnant" group was significantly lower than that in the "normal" group (P
