Veterinary Research Communications

, Volume 33, Issue 5, pp 431–438

Serum protein pattern in ewe with pregnancy toxemia

Authors

    • Department of Biochemistry, Faculty of Veterinary MedicineUniversity of Ondokuz Mayis
  • Gulay Ciftci
    • Department of Biochemistry, Faculty of Veterinary MedicineUniversity of Ondokuz Mayis
Original Article

DOI: 10.1007/s11259-008-9189-9

Cite this article as:
Yarim, G.F. & Ciftci, G. Vet Res Commun (2009) 33: 431. doi:10.1007/s11259-008-9189-9

Abstract

Pregnancy toxemia is a metabolic disease of pregnant ewes which causes significant economic losses in sheep industry. The pathophysiology and metabolic changes of this disorder remain poorly understood. We conducted this study to describe the serum protein pattern associated with the pregnancy toxemia in ewes. In this study, the electrophoretic pattern of serum proteins of 15 ewes with naturally occuring pregnancy toxemia and 12 ewes with uncomplicated pregnant were investigated by Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis (SDS-PAGE). Serum protein patterns were mainly characterized by four bands and located in the 76 kDa, 66 kDa, 55 kDa and 29 kDa both diseased and control groups. The percent of the 66 kDa, 55 kDa and 29 kDa proteins were decreased (P < 0.001 for 66 kDa; P < 0.01 for 55 kDa and P < 0.05 for 29 kDa) while 76 kDa (P < 0.05) protein was significantly increased (P < 0.001) in ewes with pregnancy toxemia relative to controls. Positive correlations were found between activities of liver enzymes and percentage of the distribution in 76 kDa, 55 kDa proteins. In contrast, there was a negative correlation between the 66 kDa protein and liver enzymes. In conclusion, the results of this study demonstrate that the percentages of the 76 kDa, 66 kDa, 55 kDa and 29 kDa proteins are significantly altered in ewes with pregnancy toxemia. However, further studies are needed to explore the potential role of these alterations in the pathophysiology in ewe with pregnancy toxemia.

Keywords

EwePregnancy toxemiaProtein patternSerum

Abbreviations

AST

aspartate aminotransferase

ALT

alanine aminotransferase

GGT

gamma-glutamyl transferase

kDa

kilodalton

SDS-PAGE

sodium dodecyl sulfate polyacrylamide gel electrophoresis

Introduction

Pregnancy toxemia, also known as pregnancy disease, twin lamb disease, ketosis or preeclampsia, is a pregnancy-specific syndrome that frequently affects pregnant ewes characterized by anorexia, hypoglycemia, ketonemia, ketonuria, proteinuria, incoordination, weakness, recumbency and mental dullness (Andrews 1997). It is seen more often in the last 2 to 4 weeks of pregnancy in ewes carrying a large single fetus, twins or triplets and is one of the leading causes of both maternal and fetal/neonatal morbidity and mortality (Andrews et al. 1996). Mortality is high in ewes with pregnancy toxemia, only 20% of preeclamptic ewes recovering without treatment (Thomson 1956). Although intense research for the underlying mediators of this disease remains unclear, a key role for high carbohydrate demand of multiple fetuses and negative energy balance in late pregnancy have been hypothesized (Hay and Baird 1991).

Serum proteins predictable changes in response to inflammation, trauma, malignancy, necrosis, infarction, and chemical injury in animals (Kaneko et al. 1997). Serum protein electrophoresis commonly is used to diagnosis, evaluating and monitoring a variety of disease and conditions (David 2003). Although a specific diagnosis can rarely be made with serum protein electrophoresis, processes of diseases can be recognized by serum protein patterns and alterations to these patterns are associated with a variety of different conditions and diseases (Ritzman and Daniels 1982). It has been established that the serum protein patterns are changed in viral (Giordano et al. 2004), bacterial (Harrus et al. 1996) and parasitological (Camacho et al. 2005) infections of animals. Several studies have focused on various metabolic alterations arising in the ewe pregnancy toxemia (Cantley et al. 1991; Henze et al. 1994; Sargison et al. 1994; Harmeyer and Schlumbohm 2006). However, no reference data on serum protein pattern of in ewe with pregnancy toxemia were available in the literature we reviewed. In the present study, therefore, serum protein patterns were compared between ewes with naturally occuring pregnancy toxemia and uncomplicated pregnant.

Materials and methods

Animal selection

Twenty seven ewes of the Karayaka breed aged between 3 and 5 years and weighing 40–50 kg with 18–20 gestational weeks participated in the study. Blood samples were collected from 15 ewes with naturally occuring pregnancy toxemia and from 12 ewes with uncomplicated pregnant. Pregnancy toxemia was diagnosed with clinical examination and biochemical results of the affected ewes. It was defined if they met the criteria of having body temperature lower than 37.0°C, heart rate higher 110 beats/min, respiratory rate lower than 10 breaths/min, proteinuria of 3 g or more in 24 hours, strong positive urine test for ketones, serum uric acid was >1.5 mg/dL, elevated liver function enzymes, a ketone smell breath, poor body condition, aimless walking, opisthotonos, blindness, lapping, head pressing, tremors and convulsions.

Serum preparation

Blood samples were obtained from each ewe by venipuncture and allowed to clot for at least 30 min at room temperature. Serum was removed by centrifugation at 1150 g for 10 minutes at 4°C. The serum samples were portioned into cryovials and stored at −80°C until assayed.

Serum biochemistry analysis

Serum samples were thawed at 21°C just prior to assay. Assays for serum total protein, albumin, aspartate aminotransferase (AST), alanine aminotransferase (ALT) and gamma-glutamyl transferase (GGT), urea, uric acid and creatinine were performed using autoanalyser (Autolab, AMS Srl, Selective Access) by commercial kits (Sigma-Aldrich Chemie GmbH, Germany) according to the manufacturer’s instruction. Serum globulin level was calculated by subtracting the value of albumin from total protein concentration.

Electrophoretic analysis

SDS-PAGE was performed as described by Laemmli (1970). The serum were mixed with a sample buffer containing 0.0625 M Tris Cl, pH 6.8, 2% SDS, 10% glycerol, 5% 2-mercapto-ethanol and 0.01% bromphenol blue. The samples were boiled for 5 min and stored at −80°C until electrophoretic analysis. Serum samples of 10 µL were applied to gels. Electrophoretic analysis was carried out using vertical slab gel electrophoresis apparatus (Thermo EC 120, New York, USA). Both separating and stacking gels were prepared as 10%. The molecular weight of each protein fraction was determined by using molecular weight range marker (Sigma-Aldrich Chemie GmbH, Germany) as standard. After electrophoresis, the gels were stained with 0.25% coomassie brillant blue in 90 mL of methanol:water (1:1 V/V) and 10 mL glacial acetic acid, and were destained in methanol:water:acetic acid (45:45:10) (Sambrook et al. 1989). Molecular weights are expressed in kilodalton (kDa).

Statistical evaluation of the data

Data are presented as mean±SD of each study group. Groups were compared using the Mann-Whitney U-test (Rao 1973). A value of P < 0.05 was considered as statistically significant.

Results

Serum biochemical findings

The serum protein and enzyme results in the preeclamptic and uncomplicated pregnant ewes were given in Table 1. Serum total protein, albumin and globulin concentrations were 5.2 ± 0.3 g/dL, 2.1 ± 0.2 g/dL and 3.1 ± 0.2 g/dL in preeclamptic ewes; were 6.8 ± 0.5 g/dL, 3.2 ± 0.2 g/dL and 3.6 ± 0.2 g/dL in uncomplicated pregnant ewes, respectively. Significant differences were found between preeclamptic and control ewes for serum total protein, albumin and globulin concentrations (P < 0.001). The albumin-to-globulin (A/G) ratio was also significantly lower in preeclamptic group (0.7 ± 0.1) than that recorded in the control group (0.9 ± 0.1) (P < 0.01).
Table 1

Some biochemical findings in preeclamptic (n = 15) and uncomplicated pregnant (n = 12) ewes

Parameter

Preeclamptic

Uncomplicated pregnant

Total protein (g/dL)

5.2 ± 0.3***

6.8 ± 0.5

Albumin (g/dL)

2.1 ± 0.2***

3.2 ± 0.2

Globulin (g/dL)

3.1 ± 0.2***

3.6 ± 0.2

A/G

0.7 ± 0.1**

0.9 ± 0.1

AST (U/L)

129.9 ± 14.6***

41.1 ± 8.1

ALT (U/L)

49.3 ± 9.8***

25.0 ± 6.4

GGT (U/L)

43.1 ± 11.9***

22.3 ± 4.9

Urea (mg/dL)

37.5 ± 3.6*

34.2 ± 2.2

Uric acid (mg/dL)

1.8 ± 0.4*

1.5 ± 0.2

Creatinine (mg/dL)

1.7 ± 0.3*

1.4 ± 0.2

A/G: Albumin/Globulin. Asterisks indicates the presence of significance, ***P < 0.001, **P < 0.01 and *P < 0.05 (Mann-Whitney U-test)

AST, ALT and GGT activities were 129.9 ± 14.6 U/L, 49.3 ± 9.8 U/L and 43.1 ± 11.9 U/L in the preeclamptic group and 41.1 ± 8.1 U/L, 25.0 ± 6.4 U/L and 22.3 ± 4.9 U/L in the control group, respectively. All enzyme activities of preeclamptic ewes were significantly higher compared to controls (P < 0.001). Mean serum urea, uric acid and creatinine concentrations in the preeclamptic ewes (37.5 ± 3.6 mg/dL; 1.8 ± 0.4 mg/dL and 1.7 ± 0.3 mg/dL, respectively) were mildly higher than that recorded in the uncomplicated pregnants (34.2 ± 2.2 mg/dL; 1.5 ± 0.2 mg/dL and 1.4 ± 0.2 mg/dL, respectively) (P < 0.05).

Electrophoretic findings

Electrophoretic patterns of serum from preeclamptic and uncomplicated pregnant ewes are shown in Fig. 1. These patterns were mainly characterized by four bands located in 76 kDa, 66 kDa, 55 kDa and 29 kDa in both two groups. The electrophoretograms of the preeclamptic ewes revealed a decreased percentage of the distributions in 66 kDa (29.8 ± 2.2% vs. 47.6 ± 2.1%, P < 0.001), 55 kDa (16.6 ± 0.8% vs. 18.2 ± 1.7%, P < 0.01) and 29 kDa (19.4 ± 2.7% vs. 21.4 ± 2.2%, P < 0.05) proteins compare to uncomplicated pregnant ewes. The percent of the 76 kDa (27.1 ± 2.9% vs. 9.6 ± 2.3%, P < 0.001) protein was increased relative to control (Fig. 2, Table 2). The relationship between percentage of the serum proteins and biochemical findings was shown in Table 3. Positive correlation was found between the percentage of the distribution in 76 kDa protein and liver enzymes. In contrast, there were negative correlations between the 66 kDa and 55 kDa protein percentages and liver enzymes (Table 3). No significant correlations were determined between the percentage of 29 kDa protein and serum biochemical findings.
https://static-content.springer.com/image/art%3A10.1007%2Fs11259-008-9189-9/MediaObjects/11259_2008_9189_Fig1_HTML.gif
Fig. 1

Representative serum protein patterns of preeclamptic (n = 15) and uncomplicated pregnant (n = 12) ewes

https://static-content.springer.com/image/art%3A10.1007%2Fs11259-008-9189-9/MediaObjects/11259_2008_9189_Fig2_HTML.gif
Fig. 2

Representative serum protein electrophoretograms of preeclamptic (A) and uncomplicated pregnant (B) ewes

Table 2

Percentage of the serum protein distribution in preeclamptic and uncomplicated pregnant ewes

Molecular weight

Preeclamptic (n = 15)

Uncomplicated pregnant (n = 12)

76 kDa

27.1 ± 2.9***

9.6 ± 2.3

66 kDa

29.8 ± 2.2***

47.6 ± 2.1

55 kDa

16.6 ± 0.8**

18.2 ± 1.7

29 kDa

19.4 ± 2.7*

21.4 ± 2.2

Asterisks indicates the presence of significance between tow groups, ***P < 0.001, **P < 0.01 and *P < 0.05 (Mann-Whitney U-test)

Table 3

Degree of correlations between biochemical findings and percentage of the serum proteins in preeclamptic ewes (n = 15)

 

tp

alb

glob

AST

ALT

GGT

urea

ua

cre

76 kDa

66 kDa

55 kDa

29 kDa

tp

1.00

.687**

.726*

−.746**

−.718**

−.600*

.127

−.015

−.201

−.603*

.753**

−.689**

−.291

alb

.687**

1.00

.000

−.902**

−.965**

−.906**

.034

.118

−.316

−.669**

.910**

−.879**

−.337

glob

.726**

.000

1.00

−.174

−.075

.030

.143

−.131

.023

−.197

.176

−.116

−.082

AST

−.746**

−.902**

−.174

1.00

.847**

.784**

−.035

−.233

.365

.871**

−.921**

−.918**

.405

ALT

−.718**

−.965**

−.075

.847**

1.00

.894**

−.165

−.172

.172

.586*

−.835**

−.812**

.362

GGT

−.600*

−.906**

.030

.784**

.894**

1.00

−.044

−.117

.314

.625*

−.833**

−.756**

.279

urea

.127

.034

.143

−.035

−.165

−.044

1.00

.368

.396

.055

.033

−.097

−.363

ua

−.015

.118

−.131

−.233

−.172

−.117

.368

1.00

.413

−.249

.201

−.252

−.485

cre

−.201

−.316

.023

.365

.172

.314

.396

.413

1.00

.442

−.349

.302

−.196

76 kDa

−.603*

−.669**

−.197

.871**

.586*

.625*

.055

−.249

.442

1.00

−.819**

.794**

.243

66 kDa

.753**

.910**

.176

−.921**

−.835**

−.833**

.033

.201

−.349

−.819**

1.00

−.941**

−.359

55 kDa

−.689**

−.879**

−.116

−.918**

−.812**

−.756**

−.097

−.252

.302

.794**

−.941**

1.00

.285

29 kDa

−.291

−.337−

.082

.405

.362

.279

−.363

−.485

−.196

.243

−.359

.285

1.00

tp: total protein, alb: albumin, glob: globulin, ua: uric acid, cre: creatinin. **P < 0.01 and *P < 0.05 (Pearson’s correlation)

Discussion

Serum proteins are most common biochemical indicators measured routinely for diagnosis or monitoring disease activity; alterations of their concentrations and patterns, although not specific, may be of diagnostic significance in inflammatory, infectious and metabolic diseases (Ritzman and Daniels 1982). Although pregnancy toxemia may affect protein metabolism directly or indirectly, data regarding alterations of patterns and concentrations of serum protein in ewes with naturally occuring pregnancy toxemia have been lacking. Because pregnancy toxemia is one of the leading causes of maternal and fetal death and economical losses in sheep industry, we were interested in investigating the serum protein alterations including concentration and the electrophoretic distribution, that might play a pathophysiological role in this disorder.

It has been proposed that ovine pregnancy toxemia affects the maternal liver, kidneys and brain (Hay and Baird 1991; Jeffrey and Higgins 1992). Hepatic lipidosis with subsequent impairment of liver function usually occurs resulting from excessive liver gluconeogenesis and fat mobilization to maternal and fetal energy demand usually occurs in diseased ewes (Hay and Baird 1991). Affected proximal tubular epithelium and adrenal cortex of the kidney in this disease were described (Tontis and Zwahlen 1987). In this study, the most common serum biochemical findings of preeclamptic ewes were abnormalities in hepatic and renal function tests. These results indicate impaired hepatic and renal functions in diseased animals. The current study showed that serum concentration of albumin was significantly lower in preeclamptic than in uncomplicated pregnant ewes. The lower serum albumin in pregnancy toxemia reconfirms result from earlier study made in women preeclampsia (Gojnic et al. 2004). On the other hand, Honger (1966) demonstrated that albumin catabolism is elevated in preeclampsia. Decreased serum albumin concentration and A/G ratio in ewes with pregnancy toxemia may also be explained by hepatic and renal failure resulting associated with pregnancy toxemia. Furthermore, total protein and albumin concentrations were negatively correlated with serum enzyme activities in preeclamptic ewes. However, no significant correlations were found between serum globulin concentrations and enzyme activities in preeclamptic group.

Several investigators have demonstrated the usefulness of serum protein electrophoresis for the initial evaluation of various clinical or pathological conditions in animals (Harrus et al. 1996; Altintas et al. 2001; Giordano et al. 2004; Camacho et al. 2005). Numerous studies have focused on mediators associated with the metabolic changes arising in ewe pregnacy toxemia (Marteniuk and Herdt 1988; Scott et al. 1995; Harmeyer and Schlumbohm 2006), however, there are no data regarding a serum protein pattern in ewes with pregnancy toxemia. In the work presented here, we have demonstrated that the patterns of ewes with pregnancy toxemia and uncomplicated pregnant were mainly characterized by four bands located in 76 kDa, 66 kDa, 55 kDa and 29 kDa. The percentage of 76 kDa protein in the preeclamptic ewes have been determined to be approximately 2.8 times higher than that in the uncomplicated pregnant. Furthermore, percentages of 66 kDa, 55 kDa and 29 kDa proteins in all sheep were significantly lower than that of healthy. Positive correlation was found between the percentage of the 76 kDa protein and liver enzymes despite there were negative correlations between the 66 kDa and 55 kDa proteins and liver enzymes suggesting a relationship between liver function and serum protein electrophoresis in pregnancy toxemia. This could be the consequence of disturbed synthesis of these proteins by the liver response to pregnancy toxemia. As albumin has a molecular weight around 66 kDa (depending on the species) and it is the major serum protein would be that the predominant band on SDS-PAGE is albumin. The decrease in the 66 kDa band (albumin) negatively correlated with liver enzymes is indication for liver damage/injury often associated with decreased serum albumin levels.

In summary, the present study demonstrated that the percentages of the 76 kDa, 66 kDa, 55 kDa and 29 kDa proteins are significantly altered in ewes with pregnancy toxemia compare to uncomplicated pregnant. Based on these findings, it is likely to speculate that altered protein patterns and concentrations may be associated with hepatic dysfunction and renal failure in ewe with pregnancy toxemia. However, further studies are needed to explore the potential role of these alterations in the pathophysiology in the ewe with pregnancy toxemia.

Acknowledgement

We would like to thank Melda Aytekin, DVM, for her contribution to the diagnosis of the pregnancy toxemia in ewes.

Copyright information

© Springer Science+Business Media B.V. 2008