PTH-related peptide (PTH-rP) has recently been discovered to exist in high concentrations in milk. The development of a commercial RIA for PTH-rP has allowed ...
0013-7227/91/1296-2815$03.00/0 Endocrinology Copyright © 1991 by The Endocrine Society
Vol. 129, No. 6 Printed in U.S.A.
Parathyroid Hormone-Related Peptide Content of Bovine Milk and Calf Blood Assessed by Radioimmunoassay and Bioassay* JESSE P. GOFF, TIMOTHY A. REINHARDT, SUNG LEE, AND BRUCE W. HOLLIS U.S. Department of Agriculture, Agricultural Research Service, National Animal Disease Center, Metabolic Diseases and Immunology Research Unit (J.P.G., T.A.R.), Ames, Iowa 50010; INCStar Corp. (S.L.), Stillwater, Minnesota 55082; and the Departments of Pediatrics and Biochemistry and Molecular Biology, Medical University of South Carolina School of Medicine (B. W.H.), Charleston, South Carolina 294253
ABSTRACT. PTH-related peptide (PTH-rP) has recently been discovered to exist in high concentrations in milk. The development of a commercial RIA for PTH-rP has allowed us to extend these studies. We measured the PTH-rP content of milk from 42 Jersey cows from a single farm in various stages of lactation. Colostrum (first milk) contained 56 ± 12 ng/ml immunoreactive PTH-rP (iPTH-rP). The iPTH-rP contents of milk 1, 2, 3, 5, 7, and 9 months into lactation were 77 ± 19, 59 ± 14, 57 ± 10, 106 ± 11, 119 ± 16, and 168 ± 17 ng/ml, respectively. Plasma was obtained from 7 Jersey calves at birth and at intervals after the ingestion of colostrum. No iPTH-rP was detected in the plasma at birth. Two hours after the ingestion of colostrum, the iPTH-rP content of plasma was 81 ± 25 pg/ml. The plasma iPTH-rP concentration continued to increase to 384 ± 84 pg/ml at 7 h and peaked at 444 ± 84 pg/ml
P
TH-RELATED peptide (PTH-rP) production by neoplastic tumor cells is one cause of the humoral hypercalcemia of malignancy syndrome observed in humans and animals (1). The amino-terminal region of PTH-rP is structurally similar to the calcitropic PTH (2). Both synthetic, amino-terminal PTH-rP-(l-34) and full-length recombinant PTH-rP-(l-141) are potent agonists of PTH receptors in several bioassay systems (35). PTH-rP or PTH-rP mRNA has recently been demonstrated in nonneoplastic tissues, such as skin keratinocytes (6), fetal and adult parathyroid glands (7-9), placenta (9), and adrenal glands (9). PTH-rP mRNA was also found in the lactating mammary gland (10), suggesting that PTH-rP would be found in milk. Budayr et al (11) found, by RIA and bioassay, that human, rat, and bovine milk contained 40-70 ng eq PTH-rP-(l-34)/ ml milk. Thurston et al. (12) have confirmed these findings and also found PTH-rP present in milk from goats, Received July 22,1991. Address requests for reprints to: Dr. Jesse P. Goff, U.S. Department of Agriculture, Agricultural Research Center, National Animal Disease Center, P.O. Box 70, Ames, Iowa 50010. * No endorsements are herein implied.
12 h after birth. Two calves were sampled through the 60th hour after birth, at which time plasma iPTH-rP was 483 ± 36 pg/ml. The biological activity of the PTH-rP in milk and plasma was assessed by its ability to stimulate cAMP accumulation in ROS 17/2.8 cells. The specificity of this response was determined by the ability of antiserum to PTH-rP to block the activity. The biological activity of the milk samples was between 31-95% of the activity suggested by immunoassay. Biologically active PTHrP could not be detected in any of the calf plasma samples. These results confirm the presence of biologically active PTHrP in milk and suggest that the iPTH-rP is capable of being absorbed. However, our results indicate that the biological activity of the PTH-rP is nearly completely absent once in the systemic circulation. (Endocrinology 129: 2815-2819, 1991)
pigs, mice, opossum, and water buffalo. Ratcliffe et al. (13) and Law et al. (14) have observed multiple fractions of milk with PTH-rP activity by immunoassay and bioassay, suggesting that PTH-rP exists in milk in multiple forms. Law et al (14) determined that bovine milk PTHrP exists as peptides corresponding to PTH-rP-(1-108) and PTH-rP-(l-Hl). The role of PTH-rP in milk is unknown. One suggestion is that PTH-rP plays a role in the transport of calcium across the mammary gland epithelium during milk formation (10, 14). In this study we determined the influence of stage of lactation on bovine milk PTH-rP concentration and the appearance of immunoreactive PTH-rP in plasma of calves after suckling. Materials and Methods Milk and plasma samples Milk samples were obtained from 36 Jersey cows in 6 different stages of lactation. All of these cows were fed an alfalfa, corn silage, and grain mixture to support lactation. Colostrum samples from 7 Jersey and 7 Holstein cows fed a gestation diet of alfalfa and grain were also obtained. Milk samples were 2815
PTH-rP CONTENT OF MILK AND CALF BLOOD
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stored frozen until assay. The milk calcium concentration was determined by atomic absorption spectrophotometry (15). Heparinized blood samples with 400 kallikrein inhibitor units Trasylol (Mobay Chemicals, New York, NY) added per ml were obtained from nine Jersey calves at birth and at various intervals after birth. Calves received 2 liters colostrum within the first hour after birth (n = 7) or 6 h after birth (n = 1). One Jersey calf was fed 2 liters of a soybean-based milk replacer (Isomil, Ross Laboratories, Columbus, OH) within the first hour after birth and colostrum 10 h after birth. Plasma samples were stored frozen until assay. PTH-rPRIA Plasma and milk immunoreactive PTH-rP (iPTH-rP) contents were determined using a commercial kit (PTH-rP 125I Kit, catalog no. 24200, INCStar Corp. Stillwater, MN). This kit uses polyclonal goat serum directed against human PTHrP-(l-40) and recombinant human PTH-rP-(l-84) as standard. The epitope for the PTH-rP antibody is the segment of the PTH-rP molecule encompassed by amino acid residues 2029. PTH-rP in milk was partially isolated by adding 100 ^1 2 M acetic acid to 1 ml milk and centrifuging. The supernatant was diluted with assay buffer before RIA. PTH-rP bioassay ROS 17/2.8 cells were grown to confluency in RPMI-1640 medium (25 mM HEPES with 10% fetal calf serum and insulin) in 16-mm diameter wells. Triamcinolone (108 M) was added to the medium for 48 h before use of the cells to up-regulate receptors for PTH (and presumably PTH-rP) on the cells (16). Aliquots of plasma or milk were added to RPMI-1640 medium with triamcinolone and 1 mM isobutylmethylxanthine to a final volume of 1 ml. Goat polyclonal antibody against PTH-(1-34) was added to the samples to bind and inactivate any PTH that might be in the sample. Graded amounts of synthetic PTH-rP (1-34) (lot 016855, Peninsula Laboratories, Belmont, CA) were added to medium or pooled plasma from colostrum-deprived calves, allowed to incubate for 30 min at 24 C, and used to generate standard curves for the cAMP response of ROS 17/ 2.8 cells to PTH-rP stimulation. To initiate the assay, the old RPMI medium was removed, and the RPMI medium with plasma, milk, or PTH-rP standard added was placed in the well. Wells were incubated at 24 C for 30 min. The medium was removed from the wells, and the wells were rinsed with 0.01 M PBS. Intracellular cAMP was extracted from the cells with acidic ethanol (20 mM HC1) at -20 C. An aliquot of the ethanol extract was dried down in a vacuum centrifuge (Savant Instruments, Farmingdale, NY) and assayed for cAMP by RIA (17) using an antibody generously donated by Dr. Eduardo Slatopolsky, Washington University (St. Louis, MO).
Results Milk PTH-rP Colostrum contained relatively low levels of iPTH-rP, averaging 50.3 ± 4.7 ng/ml in Holstein cows and 56.6 ± 12.2 ng/ml in Jersey cows. Milk iPTH-rP concentrations ranged from 31-234 ng/ml (Fig. 1). Jersey milk iPTH-
3 4 5 6 Month of Lactation
FIG. 1. Milk iPTH-rP and calcium concentrations as a function of stage of lactation.
rP concentrations were lowest during the third month of lactation and highest at the end of lactation. The PTHrP biological activity of colostrum was approximately half the activity found in milk of cows in late lactation. This activity was nearly completely blocked by the addition of 5 ix\ undiluted antiserum against PTH-rP; the same antiserum used to develop the RIA kit (Table 1). The PTH-rP content of the milk and colostrum samples, as determined by bioassay, was in most cases similar to the level of PTH-rP determined by RIA (Table 2). However, in two milk samples taken from cows in late lactation, the biological activity was only 31% and 47% of the level determined by RIA, suggesting that milk of cows in late lactation may contain a high percentage of biologically inactive fragments of PTH-rP. There was no correlation between milk or colostrum calcium and biological or immunological PTH-rP content. Calf plasma PTH-rP iPTH-rP was not detected (
