Measurement of vitamin A, vitamin E, selenium, and L-lactate in dogs with and without osteoarthritis secondary to ruptured cranial cruciate ligament

Abstract

This study compared vitamin A, vitamin E, selenium (Se), and L-lactate in blood and synovial fluid in 2 groups of 6 dogs; a control group (without OA) and an osteoarthritic group with spontaneous cranial cruciate ligament rupture and OA. Concentrations of vitamin E were significantly higher in serum than in synovial fluid in both OA (P = 0.006) and control (P = 0.0008) groups. Vitamin E concentration in synovial fluid was significantly higher in the OA group than in the control group (P = 0.009). Concentrations of Se were significantly higher in serum than in synovial fluid in both OA (P = 0.003) and control (P = 0.0006) groups. There were no significant differences in levels of Se, vitamin A, and L-lactate between the 2 groups. This is the first study to show an increased concentration of vitamin E in the synovial fluid of dogs with OA compared with dogs that did not have OA.

Introduction

Osteoarthritis (OA) is a common clinical condition in veterinary medicine that seriously affects the quality of life of many dogs (1). This syndrome is reported to affect 20% of the canine population older than 1 year of age (2). Multiple etiologies are suspected to contribute to the formation of OA, including defective articular cartilage structure and biosynthesis, joint trauma, joint instability, congenital and developmental abnormalities, and inflammatory conditions (3). Oxidative damage to essential cell components caused by oxygen free radicals is a mechanism in the pathobiology of degenerative joint disease (4).

Observational and epidemiological studies suggest that diets deficient in antioxidants may be associated with an increased incidence of OA or more rapid disease progression (5–7). However, there are no studies in the literature that have conclusively demonstrated this to be the case in OA in dogs. In addition, there are no studies that have compared levels of antioxidants such as vitamin A, vitamin E, and Se and the oxidative stress marker L-lactate between serum and synovial fluid in dogs with and without OA. The goal of this study, therefore, was to compare levels of vitamin A, vitamin E, Se, and L-lactate in the blood and synovial fluid of dogs with OA [as a result of spontaneously induced cranial cruciate ligament (CCL) rupture] with those in the blood and synovial fluid of dogs without OA.

Materials and methods

Two groups of animals were studied: a control group without OA consisting of 6 beagle dogs, 3 males and 3 females (3.2 ± 1.2 y old and weighing 14 ± 1.7 kg) having no signs of OA following orthopedic and radiological examinations; and an OA group consisting of 6 client-owned large breed dogs having OA secondary to unilateral ruptured cranial cruciate ligament, 2 Rottweilers, 1 Labrador retriever, and 3 mongrel dogs (5.3 ± 3.1 y old and weighing 32.5 ± 5.4 kg). Unilateral CCL rupture in dogs with OA was diagnosed on the basis of clinical findings of hind limb lameness, palpable cranial drawer movement, positive results for a tibial compression test, presence of a medial buttress at the stifle joint, and standard mediolateral radiographic views. Radiographs were evaluated in accordance with modified criteria described elsewhere such as joint effusion, osteophytosis, narrowed joint spaces, and subchondral bone sclerosis (8). All dogs with OA had complete CCL rupture confirmed during the surgical treatment (tibial plateau leveling osteotomy). Both groups were fed a diet containing 10 IU of vitamin A, 500 IU/kg body weight (BW) of vitamin E, and 0.37 mg/kg BW of Se for 2 mo. All samples were collected after the 2-month period with the same diet. Animal care procedures followed the guidelines of the Canadian Council on Animal Care.

The dogs from the control group were used for abdominal end-stage surgery laboratory procedures and were euthanized at the end of this teaching laboratory while still under general anesthesia. Dogs in the OA group were received at the small animal hospital of the Faculty of Veterinary Medicine during June–July 2009 for lameness consultation and were diagnosed as having OA with CCL. Only animals which had similar osteophytic lesions at the stifle joint during radiological examination were retained for this study. All stifle joints were grossly inspected to identify macroscopic changes of OA (osteophytes, fibrillation, cartilage vascular invasion).

Blood (5 mL) from the jugular vein was drawn into red-topped tubes after the animals were sedated prior to general anesthesia, and immediately placed into a covered ice bucket to protect the samples from heat and ambient light. Blood was centrifuged for 15 min at 2000 × g, the serum was removed and stored in a cryotube at −80°C.

Synovial fluid samples from the stifle joint were collected under general anesthesia because the animals in the control group were submitted to castration and ovariohysterectomy procedures. Dogs in the OA group had the synovial fluid collected during the corrective procedure for the CCL rupture. The dogs were sedated with an IM injection of acepromazine maleate (Propofol; Glycopyrrolate Sandoz Canada, Toronto, Ontario), 0.05 mg/kg body weight (BW), hydromorphone (Sandoz Canada), 0.2 mg/kg BW, and glycopyrrolate (Glycopyrrolate; Sandoz Canada), 0.005 mg/kg BW. Animals were induced with propofol (Diprivan; AstraZeneca Pharmaceuticals Wilmington Delaware, USA), 6 mg/kg BW and immediately intubated. General anesthesia was maintained with 2% isoflurane. All cranial surfaces of the stifle joints were aseptically prepared before puncture. A 22-gauge needle connected to a 5-mL syringe was inserted into the stifle joint lateral or medial to the patella tendon, at the mid-distance between the patella and the insertion of the patellar tendon at the tibial tuberosity. The synovial fluid samples were centrifuged at 2400 × g for 15 min to remove any cell debris, and stored at −80°C.

Vitamins were analyzed by high-performance liquid chromatography (HPLC) reverse phase based on the method published by Gueguen et al (9), and adapted by our laboratory. Selenium was measured by a HPLC modified method reported by Hawkes and Kutnink (10). Conditions of compliance criteria and quality control included: coefficient of determination of standard curve (r) > 0.98, relative standard deviation (RSD) ≤ 20%, limit of detection (LOD) = 85% to 115% and quantification limit (LQO) = 80% to 120%.

L-lactate in whole blood and synovial fluid was measured by the Lactate Pro meter (Akray Inc., Kyoto, Japan). The kit is supplied with a check strip (to confirm that the analyzer is operating correctly) and a calibration strip that provides a non-quantitative indication of instrument accuracy. In addition, commercially available control solutions containing known lactate concentrations were used prior to any L-lactate analysis to help ensure quality control of the Lactate Pro device.

A general linear model with repeated measurements was performed, with OA (with or without OA) as between subject factor and compartment (serum/whole blood or synovial fluid) as within-subject factor. All mean values are reported with standard error (± SD). P-values for pairwise comparisons between treatment means were adjusted with the sequential Bonferroni correction.

Results

All samples were successfully collected and tested. The owners reported that there had been no additional clinical changes or modification of the recommended diet and exercise management before the surgical procedures and sample collection. Vitamin A concentrations showed no significant differences between the OA group and control in serum (P = 0.03; non-significant after Bonferroni sequential correction) and in synovial fluid (P = 0.60). Also, there were no differences in vitamin A concentration between serum and synovial fluid in the OA group and in the control goup (P = 0.03 non-significant after Bonferroni sequential correction; P = 0.53, respectively) (Table 1).

Table 1.

Mean values [standard deviation ± (SD)] of concentration of vitamin A, vitamin E, selenium, and L-lactate in serum and synovial fluid samples of normal (n = 6) and osteoarthritic dogs (n = 6). P-values* are significant after the sequential Bonferroni correction applied to the whole table.

	Dogs without OA		Dogs with OA		P-value
	1. Serum whole blood	2. Synovial fluid	3. Serum whole blood	4. Synovial fluid	1 versus 2	3 versus 4	1 versus 3	2 versus 4
Vitamin Aa (μmol/L)	5.14 ± 3.97	1.94 ± 1.77	2.07 ± 0.64	1.24 ± 0.47	0.03	0.53	0.03	0.60
Vitamin Ea (μmol/L)	36.4 ± 20.0	2.4 ± 0.45	54.9 ± 21.6	29.3 ± 5.42	0.0008*	0.006*	0.06	0.009*
Seleniuma (μmol/L)	3.44 ± 66	1.57 ± 0.98	2.93 ± 0.63	1.42 ± 0.09	0.0006*	0.003*	0.22	0.71
L-lactateb (mmol/L)	2.72 ± 0.32	1.70 ± 0.44	2.08 ± 0.68	2.30 ± 0.85	0.39	0.74	0.39	0.52

^aVitamin A, vitamin E, and selenium were measured in serum.

^bL-lactate was measured in whole blood.

Concentrations of vitamin E were significantly higher in serum than in synovial fluid in OA dogs (P = 0.006) and in control dogs (P = 0.0008). Concentrations of vitamin E showed no significant differences between control and OA groups (P = 0.06); vitamin E concentration was significantly higher in synovial fluid of dogs in the OA group compared with that in dogs in the control group (P = 0.009) (Table 1).

Concentrations of Se were significantly higher in serum than in synovial fluid in both OA (P = 0.003) and control (P = 0.0006) groups. There were no significant differences between serum Se (P = 0.22) or synovial fluid Se concentrations (P = 0.71) in both groups.

L-lactate concentrations showed no significant differences between OA dogs and control, in serum (P = 0.39) and in synovial fluid (P = 0.52). Also, there were no significant differences between the concentrations in serum and synovial fluid in OA and control groups (P = 0.39; P = 0.74, respectively) (Table 1).

Discussion

To the authors’ knowledge, this is the first study that has compared serum and synovial fluid concentrations of antioxidants such as vitamin A, vitamin E, and Se and the oxidative stress marker L-lactate in dogs suffering OA of the stifle joint. This study showed a significantly higher concentration of vitamin E in the synovial fluid of dogs with OA compared with dogs without OA. This result was unexpected, as vitamin E is normally thought to neutralize, and in the process, be consumed by free radical formation in inflammatory oxidative conditions such as OA. This finding is inconsistent with results previously reported in the human literature that have shown significantly lower concentrations of vitamin E in synovial fluid of OA patients compared with normal patients (11). Despite the lower synovial vitamin E concentration, plasma levels of vitamin E in OA patients did not differ significantly from those in patients free of OA in the same human study. The authors concluded that there was more consumption of vitamin E in the OA joint through its role in terminating lipid peroxidation process than in the normal joints (11,12). However, one can speculate on a possible increased mobilization of vitamin E in OA joints because of the need for this vitamin to neutralize free radicals produced in situ and for the stability of cell membranes and their protection against peroxidation (13).

Our results indicate that vitamin E and Se concentrations were significantly higher in serum than in synovial fluid. These results are in accordance with previous investigations in which higher total Se content was typically found in the kidney, liver, and serum, with lesser amounts in all other tissues or body fluids (14). Transcellular fluids such as synovial and cerebrospinal fluids have less Se concentration than serum. On the other hand, vitamin E is most concentrated in membrane-rich cell fractions such as mitochondria and microsomes (15).

In this study there was no significant difference between the 2 groups in regards to the synovial or whole blood L-lactate levels. This is surprising, considering the strong suspicion that an oxidative process secondary to hypoxia induced by inflammation is believed to be occurring in patients with OA (16). Lactate is an end-product of glycolysis and its production would be expected to be increased in the presence of anaerobic conditions.

Serum and synovial fluid concentrations of vitamin A and Se did not vary between OA and control groups. However, depletion of vitamin A and Se was expected in the OA group, because vitamin A inhibits the inflammatory cascade in OA via the inhibition of both interleukin-1 (IL-1) and tumor necrosis-alpha (TNFα) induced matrix metallopeptidase (MMP)-1 and MMP-13 protein production and enzyme activities in human chondrocytes (17). Selenium depletion is expected in inflammatory sites; however, the lack of difference found in our study is supported by the study of Yazar et al (18), who showed no difference in Se concentration in synovial fluid and plasma in individuals suffering from OA compared with patients with rheumatoid arthritis.

This study showed unusual results: an increase in vitamin E in synovial fluid of dogs with OA and no differences in vitamin A, Se and L-lactate concentrations in serum and synovial fluid of dogs with OA compared with dogs without OA. However, further investigation is required to determine the influence of clinical factors such as magnitude of the individual inflammatory response, chronicity of the disease and animal lifestyle. We could also not exclude influence of signalment on the results, despite the uniformity in the methodology of synovial fluid collection, processing and analysis. To our knowledge, there is no study that has investigated the relationship between these potential factors with the changes on the canine synovial fluid. Thus a controlled study with a larger number of animals is warranted to confirm the higher concentration of vitamin E in the synovial fluid of dogs with OA compared with dogs without OA. CVJ