Changes in plasma lipoprotein during the oestrous cycle of the bitch

Research in Veterinary Science 1994.56, 82-88

Changes in plasma lipoprotein during the oestrous cycle of the bitch L. G. DOWNS, University of Bristol, Department of Medicine, Medical School Unit, Southmead Hospital, Bristol BSIO 5NB, V. ZANI, J. M. WILLS, Waltham Centre for Pet Nutrition, Melton Mowbray, Leicester, LE14 4RT, S. M. CRISPIN, University of Bristol, Department of Veterinary Medicine, Langford House, Bristol BS18 7DU, C. H. BOLTON, University of Bristol, Department of Medicine, Medical School Unit, Southmead Hospital, Bristol BSI O5NB

Five bitches were kept under controlled conditions of diet and exercise for up to four months. They were monitored at regular time intervals for fasting plasma concentrations of cholesterol, triacylglycerols and apolipoproteins AI and B. Lipoprotein cholesterol and triacylglycerol concentrations were also determined in the fasting plasma. Vaginal cytology and plasma progesterone were monitored at weekly intervals to determine the oestrous state of the bitches. Lipoprotein and apolipoprotein concentrations remained steady during anoestrus but large increases and wide fluctuations were shown in the concentrations of both cholesterol and triacylglycerols during metoestrus. Metabolic studies involving the measurement of lipids in canine blood must take into account the stage of oestrus of any bitches involved.

lipoprotein metabolism and cardiovascular risk have been made in humans and are reviewed by Godsland et al (1987). The administration of exogenous sex hormones has been shown to influence lipoprotein metabolism in rats and monkeys (Tikkanen and Nikkilfi 1987) through their action on hepatic lipase. The complex interactions between several different hormones, with changing concentrations over the short time span (21 to 28 days) of the human female cycle, and the plasma lipoproteins are very difficult to interpret. This is largely because the half-life of lipoproteins is measured in days, thus the half-life of high density lipoprotein (HDL) is 3.8 tO 4.0 days and low density lipoprotein (LDL) is 4.7 days (Scanu and Hughes 1962, Solyom et al 1971). The longer follicular and luteal phases in the bitch may provide a good model to study the effects of sex hormones on plasma lipoproteins by permitting more half-lives of LDL and HDL to elapse during each phase. The aim of this study was to investigate the influence of the oestrous state of the bitches on their plasma lipoprotein concentrations by monitoring them under conditions of controlled diet, exercise and lifestyle.

THE hormonal changes occurring during the oestrous cycle of the bitch have been studied extensively and reviewed by Concannon (1986), Edqvist et al (1975) and Jochle and Anderson (1977). The interval between cycles and the duration of the oestrous phases can vary considerably between individual bitches, with the complete cycle varying from five to 12 months. The pattern and concentrations of the sex hormones throughout the cycle have been well established, but no previous studies have described the relationship between endogenous sex hormones and canine plasma lipids. Studies in women have shown changes in plasma lipoprotein composition during the menstrual cycle (Kim and Kalkhoff 1979). Many studies of the interactions between sex hormones and plasma

Materials and methods One labrador and four beagle bitches belonging to the Waltham Centre for Pet Nutrition, Melton Mowbray, aged between two and eight years and of normal weight for their breed and sex, were used for this study. They were maintained on identical diets of a commercial canned dog food and 82

Lipoproteins in bitches mixer with a standardised exercise and feeding regime for two weeks before and for the duration of the monitoring period.

Sampling procedure Vaginal smears. Vaginal smears were collected weekly from the bitches using a 115 mm x 10 mm stick soaked in 0.15 M sodium chloride for several minutes before being inserted into the vestibule to a depth of approximately 4 cm (Christie et al 1972). The stick was gently pressed onto the wall of the vagina to collect the exfoliated cells. The cellular material was smeared across a slide and immediately fixed in methanol (95 per cent). The slides were stained by the Papanicolaou method (1942) to demonstrate the maturation of the squamous epithelial cells by a colour change from blue to red. Using the classification system of Bell et al (1973) a differential microscopic examination of the stained vaginal smears for the presence of parabasal, intermediate and superficial exfoliated cells, as described by Hughes and Dodds (1968), and the presence of red blood cells and neutrophils, permitted an accurate designation of the stages of oestrus. When pro-oestrus was recognised smears were taken every two days until metoestrus, then weekly sampling was resumed. The ten'ninology used in this study for the various stages of the canine oestrous cycle is as described by Concannon (1986). Blood samples. Blood samples (7-5 ml) were taken into ethylenediamine tetra-acetic acid (EDTA) coated tubes from the cephalic vein of either leg, after a 12- to 14-hour overnight fast. Initially, this was done on two consecutive days, then every two days until the state of the oestrous cycle was confirmed by vaginal cytology, then continued once every four days for between 48 and 140 days. After separation by centrifugation and the addition of sodium azide as a preservative (0.5 mg m1-1 plasma), plasma was stored at 4°C before lipoprotein fractionation, which was completed within four days of the blood sample being taken. A portion of plasma every eighth day was stored at -20°C for progesterone assay. Lipoprotein fractionation Plasma lipoproteins were isolated by a combination of precipitation and ultracentrifugation.

83

Plasma (180 gl), density 1.006 g m1-1, was centrifuged at 132,000 g for 3.5 hours in a Beckman Airfuge to isolate the very low density lipoprotein (VLDL) supematant which was removed by tube slicing (adapted from Havel et al 1955). Sodium phosphotungstate/magnesium chloride solution was added to a second sample of plasma (0.5 ml) to precipitate the apolipoprotein B containing lipoproteins, VLDL and LDL. The resulting supernatant is equivalent to HDL (Fehily et al 1988). Verification for the suitability of human fractionation methods to canine plasma was carried out. Canine plasma prestained with Sudan black was ultracentrifuged in a density gradient (1.22 1.006) (Demacker et al 1983). The VLDLfloated at the top of the gradient (d 1.006) in the same position as human VLDL. The precipitation of the apolipoprotein B containing lipoproteins was shown to be complete, by the absence of apolipoprotein B from the supernatant, by immunonepholometry using antigens specific to canine apolipoprotein B. The total cholesterol and triacylglycerols in plasma and lipoproteins were measured enzymatically using Boehringer test kits numbers 236691 and 644200, respectively. Cholesterol and triacylglycerols were assayed in plasma, VLDL and HDL. The values for LDL were obtained by subtraction. The apolipoproteins AI and B were assayed in whole plasma by immunonepholometry using a Hyland PDQ laser nepholometer (Rosseneu et al 1983, Steinberg et al 1983). Antigenically pure canine apolipoprotein AI and lipoprotein B (d 1.04 to 1.053 g m1-1) which contains apolipoprotein B as its sole protein component, were prepared from pooled canine HDL and LDL, respectively, by chromatography (Shore and Shore 1973, Midwinter 1980). The purity of the antigens was confirmed using isoelectric focusing and sodium dodecyl sulphate polyacrylamide gel electrophoresis, and in addition the homogeneity of the apolipoprotein aI was checked by high performance liquid chromatography (Weinberg et al 1988). Antibodies were raised in sheep and did not cross-react with other canine apolipoproteins or albumin. Calibration standards were prepared by the addition of purified apolipoprotein AI and lipoprotein B in known concentrations to lipoprotein free canine plasma, obtained by the removal of all lipoproteins by ultracentrifugation of fresh whole canine plasma at d 1-21 g m1-1. The lipoprotein free plasma was tested for the presence of apolipoproteins by

84

L. G. Downs, V. Zani, J. M. Wills, S. M. Crispin, C. H. Bolton

isoelectric focusing. It was found to be free of apolipoprotein AI as long as the ultracentrifugation at d 1.21 was carried out within 24 hours of the blood being taken. Coefficients of variation for all the methods which included ultracentrifugation, were less than 10 per cent, and those which did not, were less than 6 per cent. ~ ------e ..... -- -o--- --I--

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85

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choosing day zero as the first vaginal smear designated as oestrus by the decrease in the number of red blood cells, the high number of superficial epithelial cells and the absence of parabasal cells and neutrophils.

86

L. G. Downs, V. Zani, J. M. Wills, S. M. Crispin, C. H. Bolton Cholesterol

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The apolipoprotein AI levels in all the animals showed wide variations and no discernible patterns could be demonstrated. The apolipoprotein B levels in the anoestrous phase were more steady, with wider variations recorded during the metoestrous phase, coinciding with the increases in LDL cholesterol levels. Discussion

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cholesterol, with low standard deviations of 0.29 and 0.42 mmol litre -1, respectively, were recorded. Figs 1 and 2 show changes in the two anoestrous bitches over the 40 to 80 days before prooestrus. Three beagle bitches were monitored throughout the pro-oestrus, oestrus and metoestrus. All three showed high plateau values of progesterone during metoestrus (Fig 3). The mean values for total plasma cholesterol of 7.83, 7.34 and 8.01 mmol litre -1, were higher than those of the anoestrus bitches. The standard deviations were also greater (1.36, 1.25 and 0.93 mmol litre -1, respectively). The large variations in lipids recorded through the bitches' oestrus cycle contrasted with the small weekly variation in anoestrous bitches (Figs 4, 5

ApoAI Apolipoproteins

The effect of sex hormones on human lipoprotein metabolism has been studied over many years ....**,'*" but their specific actions are still unclear, with the ,." '... .../i,~./~* ~..,: . "--..~ i • .~ findings of one study often seeming to contradict ~'300 6 another. Statements that androgens reduce plasma HDL concentrations and increase LDL concentraE200" tions, and conversely oestrogen increasing HDL and decreasing LDL, now appear over-simplifica100 tions at best. Most of these studies have monitored the effect of the administration of synthetic hor0 mones to human beings. These synthetic steroids -40 -20 Pro- 13 Days 2'0 4'0 have structural modifications that may change Anoestrus OestrusOestrus Metoestrus their properties compared to the naturally occurFIG 6: Lipoprotein profile of cholesterol, triacylglycerols and ring hormones (Godsland et al 1987). apolipoproteins AI and B in beagle bitch 5 during pro-oestrus, Attempts to elucidate the effects of the natural oestrus and metoestrus cycle of female sex hormones on the lipoprotein Two bitches (one labrador, one beagle) profile have also given conflicting results. Kim remained in anoestrus for most of the monitoring and Kalkhoff (1979) showed significant changes period as confirmed by vaginal cytology and pro- in the lipoprotein profiles of the women they studgesterone levels of under 3.0 nmol m1-1. The total ied, with total cholesterol lower during the luteal plasma cholesterol and triacylglycerol concentra- phase due to a decrease in LDL cholesterol, and tions were within the 95 per cent confidence inter- apolipoprotein B lowest at ovulation. Conversely, vals recorded for the respective breeds (Downs et an earlier study by Adlercreutz and Tallqvist al 1993). Small variations in the total plasma (1959) had reported cholesterol was highest at Apo B

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Lipoproteins in bitches

ovulation, while Demacker et al (1982) found no significant changes throughout the cycle. Relating any observed changes to the plasma concentrations of a specific sex hormone is very difficult. Although the number of bitches studied was small (five) the changes in plasma lipoprotein profile over the oestrous cycle were very marked. Environmental factors were absent from, or controlled, in the present study. Examination of exfoliated cells from the genital tract is a reliable means for determining the stages of the canine oestrous cycle, and the progesterone concentrations, where available, confirm the cytological description of the bitches. The fluctuations recorded during anoestrus were small in comparison to the increases in plasma cholesterol concentrations during metoestrus. Previous work from this laboratory in male dogs has shown that there is no significant variation with time in the lipoprotein fractions or the concentrations of apolipoprotein AI and B. The distribution of cholesterol within the lipoprotein classes started to alter during pro-oestrus, with LDL cholesterol increasing and HDL cholesterol decreasing. This decrease in the main cholesterol carrier, HDL, in dogs, may be analogous to the decrease in LDL cholesterol recorded in women during the follicular phase (Kim and Kalkhoff 1979). Concannon (1986) showed that oestrogen in the dog increased during pro-oestrus to a peak concentration at the end of pro-oestrus. The luteinising hormone peak occured as oestrogen declined. He postulated that this decline in oestrogen and increase in progesterone concentration facilitated the release of luteinising hormone and the start of oestrus.

Between 10 and 15 days after the commencement of metoestrus the total plasma cholesterol and triacylglycerol concentrations increased and large fluctuations were recorded. The cholesterol content of LDL and HDL were both increased and VLDL and LDL triacylglycerol increased. Elevated concentrations continued for 20 days after the progesterone concentrations declined. During metoestrus oestrogen concentrations are declining and progesterone is the dominant sex hormone of the luteal phase. The raised plasma lipid values during metoestrus may contribute to the increase in cases of corneal lipidosis in bitches after oestrus, whelping and during lactation (Crispin and Barnett 1983). It is important that if lipid or lipoprotein ana-

87

lyses are required from bitches, their stage of oestrus must be known; only during anoestrus can the lipid concentration be considered to be basal. Thus one in three random samples from bitches may provide unrepresentative results for plasma lipid assessment.

Acknowledgements Financial support from the Waltham Centre for Pet Nutrition, and the care of the dogs by the staff of the Waltham Centre for Pet Nutrition are gratefully acknowledged. We also wish to thank Mrs Maggie Hopton, who performed some of the lipid analyses at the University of Bristol.

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L. G. Downs, V. Zani, J. M. Wills, S. M. Crispin, C. H. Bolton

JOCHLE, W. & ANDERSON, A. (1977) The oestrous cycle of the dog: a review. Theriogenology 7, 113-140 KIM, H. J. & KALKHOFF, R. (1979) Changes in iipoprotein composition during the menstrual cycle. Metabolism 28, 663-668 MIDWINTER, C. A. (1980)An investigation of very low density lipoprotein apoprotein. PhD thesis, University of Bristoi PAPANICOLAOU, G. N. (1942) A new procedure for staining vaginal smears. Science 95, 438-439 ROSSENEU, M., VERCAEMST, R., STEINBERG, K. K. & COOPER, G. R. (1983) Some considerations of methodoIogy and standardisation of apolipoprotein B immunoassays. Clinical Chemistry 29, 427-433 SCANU, A. M. & HUGHES. W. L. (1962) Further characterisation of the human serum d 1.063-1.210, alpha 1 lipoproteins. Journal of Clinical Investigation 41, 1681- 1689 SHORE, V. G. & SHORE, B. (1973) Heterogeneity of human plasma very low density lipoprotein. Separation of species differing in pro-

tein components. Biochemistry 12, 502-507 SOLYOM. A., BRADFORD, R. H. & FURMAN, R. H. (1971) Methyl testosterone effect on apolipoprotein A and albumin metabolism in canine serum. American Journal of Physiology 221, 1587-1595 STEINBERG, K. K., COOPER, G. R., GRAISER, S. R. & ROSSENEU, M. (1983) Some considerations of methodology and standardisation of apolipoprotein A[ immunoassays. Clinical Chemistry 29, 415-426 TIKKANEN, M. J. & NIKKILA, E. A. (1987) Regulation of hepatic lipase and serum lipoproteins by sex steroids. American Heart Journal 113, 562-567 WEINBERG, R., PALT, N. C. &DAGUE, B. (1988) Analytic and preparative separation of apolipoproteins AI, AII, and AIV by reversed-phase HPLC. Journal of Lipid Research 29, 8 i9-824

Received February 19,1993 Accepted Augztst 18, 1993