Changes in Serum Parathyroid Hormone–Related Protein in Breastfed Preterm Infants

Abstract

Background:

Parathyroid hormone–related protein (PTHrP) has the ability to activate parathyroid hormone receptors and cause hypercalcemia. In a previous study we have demonstrated high concentrations of PTHrP in both term and preterm human milk (HM). PTHrP intestinal absorption and its influence upon calcium homeostasis of the preterm infant have not been studied yet. This study assessed the correlation between PTHrP concentrations in preterm HM and PTHrP in maternal and neonatal serum.

Study Design:

We collected samples of expressed HM obtained from 16 mothers of preterm infants (25–34 weeks of gestation) and drew blood samples from both mothers and infants on postpartum days 2 and 10. PTHrP concentrations were measured by two-site immunoradiometric assay. Blood calcium (Ca), phosphorus (P), and alkaline phosphatase (ALP) concentrations were also measured.

Results:

Neither maternal nor neonatal PTHrP serum concentrations varied significantly after 10 days of breastfeeding. There was a correlation between PTHrP concentrations in maternal serum and HM concentrations (R² = 0.24, p = 0.04), but not between HM and neonatal serum concentrations or between PTHrP concentrations in HM and preterm serum concentrations of Ca, P, and ALP.

Conclusions:

Despite high concentrations of PTHrP in preterm HM, serum concentrations of PTHrP of breastfed preterm infants did not increase over time. There was no correlation between PTHrP concentrations in HM and neonatal serum Ca concentration.

Introduction

Parathyroid hormone (PTH)–related protein (PTHrP) was first described in the late 1980s as the cause of humoral hypercalcemia of malignancy.^1,2 This 17,000-dalton adenylate cyclase-stimulating protein has the ability to activate the PTH/PTHrP receptor in a paracrine and autocrine fashion.^1,3,4 As opposed to PTH, which is produced only by the parathyroid gland, PTHrP is produced by a wide range of normal and pathological tissues, including embryonic tissue. PTHrP acts by activating proliferation, differentiation, apoptosis, and smooth muscle relaxation.^5–7 The PTHrP concentration is elevated in breast tissue during pregnancy and lactation and has been shown to be slightly elevated in maternal serum during that period. PTHrP is produced in significant amounts by the breast tissue during lactation.^6,7 Its concentration in human milk (HM) is about 1,000–10,000 times higher than in maternal plasma and about 100 times higher than in the plasma of patients with hypercalcemia of malignancy.^8,9 We had recently demonstrated high PTHrP concentrations in preterm HM.¹⁰ PTHrP concentrations in the placenta and cord blood immediately after birth have also been described earlier, but with mixed results.^11,12

It has been suggested that PTHrP has a role in maintaining serum calcium (Ca) homeostasis in the neonate.⁶ Newborns, particularly preterm ones, experience a transient fall in Ca concentrations soon after birth,^13,14 occurring to a considerable extent in formula-fed infants and less so in breastfed infants.¹⁴ Some investigators have suggested that the subsequent rise in Ca is related to a PTH-like effect of PTHrP because there is no change in PTH concentrations.⁶

It is not known whether there is any systemic absorption of PTHrP by the newborn intestinal tract. Some studies demonstrated that peptides from HM are absorbed because of low gastric proteolytic activity and high permeability of the infant's intestinal mucosa.¹⁵ Thus, we undertook this study to assess changes in PTHrP serum concentrations after birth. We intended to determine whether there is any correlation between PTHrP concentration in preterm HM and PTHrP in maternal and neonatal serum, as well as several other parameters of Ca homeostasis. We specifically hypothesized that human milk PTHrP might influence Ca homeostasis by affecting its serum concentration in the neonate.

Subjects and Methods

Population

The study was approved by local and national ethics committees, and informed consent was obtained from both parents of the preterm infants for their newborn's participation and from the mothers for their own participation. We collected two samples of expressed HM from mothers of preterm infants (25–34 weeks of gestation): one sample on postpartum day 2 (colostrum) and one on postpartum day 10. Milk samples were taken during daylight hours and stored immediately at −70°C until PTHrP analysis was performed. Blood samples were concomitantly drawn from both mothers and infants for measurement of PTHrP, Ca, phosphorus (P), and alkaline phosphatase (ALP) concentrations. Serum samples were stored at −70°C until analysis.

Laboratory methods

Concentrations of PTHrP in HM and serum samples were measured by a two-site immunoradiometric assay (DSL-8100 Active Irmakit, Diagnostic Systems Laboratories Inc., Webster, TX). Samples (0.2 mL) of frozen milk or serum were used for the assay procedure. The milk samples were thawed and diluted to 1:80 with deionized water. We used a commercial kit developed primarily for measurements of PTHrP in plasma. We validated this assay for breastmilk whey. The recovery was 98 ± 8% (n = 5) when 1,600 pg of human PTHrP was added to milk samples and assayed. The sensitivity of the assay was 2 pmol/L for breastmilk and 0.3 pmol/L for serum. There was no cross-reactivity with PTH, while the inter-assay coefficient of variation is 4.4% and the intra-assay coefficient of variation is 4.7%, according to the manufacturer's studies.

Statistical analyses

Because PTHrP was not normally distributed, we applied logarithmic transformation to the data obtained in order to achieve normality. Comparison between measurements and groups was done using the paired t test and Fischer's exact test. Correlations were calculated using Pearson correlations and the Mann–Whitney test. A p value of <0.05 was considered to be significant.

Results

Demographic data of the study participants are presented in Table 1. Seventeen premature infants were originally recruited. One had early-onset sepsis and was not included in the analysis. Another infant, born at 25 weeks of gestation, developed late complications of prematurity and died at the age of 45 days. His data were included. None of the mothers discontinued breastfeeding during the interval between the two measurements. The PTHrP measurements are presented in Table 2. There was no significant change over time in PTHrP serum concentrations in either the maternal or in the infant group. The PTHrP concentrations in HM were higher on postpartum day 10 compared with postpartum day 2, but the difference did not reach a concentration of significance (3,849 pg/L vs. 2,031 pg/L, respectively; p = 0.084). PTHrP serum concentrations of the preterm infants were significantly higher compared with the maternal serum concentrations on postpartum day 10 (1.949 pg/L vs. 1.446 pg/L, respectively; p = 0.004). There was a significant correlation between the PTHrP concentrations in HM and the PTHrP serum concentrations of the mothers (R² = 0.24, p = 0.04), but not between the PTHrP concentrations in HM and the infants' PTHrP serum concentrations (R² = 0.04, p = 0.25). Maternal serum PTHrP concentrations were inversely correlated to their newborn's birthweight (R² = 0.36, p = 0.04) but not to gestational age.

Table 1.

Demographic Characteristics of Mothers and Infants (N = 16 Pairs)

Characteristic	Value
Maternal age (years)
Mean (SD)	35.2 (5.5)
Median	37
Range	27–48
Gender of infant [n (%)]
Male	9 (56)
Female	7 (44)
Gestational age (weeks)
Mean (SD)	31.7 (2.2)
Median	32
Range	25–34
Birth weight (g)
Mean (SD)	1,854 (420)
Median	1,826
Range	713–2,470

Table 2.

Parathyroid Hormone–Related Protein Measurements in Human Milk and Serum

	PTHrP concentration (pmol/L)
	Postpartum day 2 ^a	Postpartum day 10 ^b	p
Maternal blood
Mean	1.39	1.44	0.821
Median	1.29	1.5
Interquartile range	1.05–1.5	1.0–1.78
Maternal milk
Mean	2,031	3,849	0.084
Median	1,120	2,488
Interquartile range	576–2,760	1,124–7,580
Neonatal blood
Mean	1.77	1.94	0.451
Median	1.61	1.95
Interquartile range	1.12–1.95	1.37–2.28

The level of significance for the difference between the PTHrP in the maternal and neonatal blood on postpartum day 2 was p = 0.068.

The level of significance for the difference between the PTHrP in the maternal and neonatal blood on postpartum day 10 was p = 0.004.

PTHrP, parathyroid hormone–related protein.

There was no change over time (postpartum day 2 vs. postpartum day 10) in the P and ALP serum concentrations of the mothers or of their preterm infants, while the Ca concentrations in the neonates did increase significantly over time (8.66 mg/dL vs. 9.81 mg/dL; p = 0.004). No correlation was found between the PTHrP concentration in HM and serum to Ca, P, and ALP concentrations either in the postpartum women or in their offspring.

Discussion

We have shown that despite high concentrations of PTHrP in preterm HM, serum concentrations of PTHrP of breastfed preterm infants do not increase over time. The PTHrP concentrations that were demonstrated in our study were only slightly above the normal adult range.¹⁶ The lack of difference in PTHrP concentrations between the measurements on postpartum days 2 and 10 despite extremely high concentrations in HM may suggest that there is no significant absorption of the protein from the infant's gastrointestinal tract. The differences between maternal and neonatal PTHrP concentrations (in cord blood) have been demonstrated before, and the authors proposed that there is a maternal–fetal gradient.¹² Our results do not support this association. The half-life of PTHrP is on the order of a few hours, so the finding of neonatal concentrations being higher than maternal concentrations probably represents endogenous production of PTHrP in the neonate. This assumption is supported by Papantoniou et al.,¹⁷ who demonstrated that there had been no change in embryonic PTHrP concentrations throughout pregnancy.

The finding of large amounts of PTHrP in HM raises a theoretical question about the biological role of this peptide in HM. Infant formula has small amounts of the equivalent bovine PTHrP, suggesting it is probably not essential for intestinal tract development after birth. The preterm intestinal tract is very vulnerable to the development of necrotizing enterocolitis. A protective effect of HM against this devastating disease is well known, although the mechanism has yet to be determined.^18–21 The existence of PTH/PTHrP receptors in the intestinal mucosa has been known for years.²² The presence of high amounts of PTHrP in preterm HM might have some paracrine effect on the maturation of the preterm intestinal mucosa. PTHrP in breast tissue acts as a promoter of proliferation in mammary glands and as a vasodilator of smooth muscle cells.²³ Hirota et al.²⁴ demonstrated a rise in serum PTHrP in lactating mothers 1 month after birth and speculated that this rise is caused by systemic absorption from breast tissue. This observation was not confirmed in our study. We did find a correlation between HM and maternal serum concentrations, which may imply the possibility that PTHrP is being absorbed systemically.

The main limitation of the current study is the lack of a control group of preterm infants fed mainly by infant formula. We were not able to recruit this group because of the successful promotion of HM feeding to preterm infants in our institution.

In conclusion, we have found in this study that despite high concentrations of PTHrP in preterm HM, serum concentrations of PTHrP of breastfed preterm infants did not increase over time and that there was no correlation between PTHrP concentrations in HM and neonatal Ca serum concentration. We speculate that there is no significant absorption of PTHrP from the preterm infant intestinal tract to the infant systemic circulation and that there is no role for PTHrP in the preterm infants' Ca homeostasis. The possible effect of PTHrP on the preterm intestinal mucosa that might promote differentiation and proliferation warrants further investigation.

Footnotes

Disclosure Statement

No competing financial interests exist.

References

Burtis

, Wu

, Bunch

et al. Identification of a novel 17,000-dalton parathyroid hormone–like adenylate cyclase-stimulating protein from a tumor associated with humoral hypercalcemia of malignancy. J Biol Chem, 1987; 262:7151–7156.

Moseley

, Kubota

, Diefenbach-Jagger

et al. Parathyroid hormone–related protein purified from a human lung cancer cell line. Proc Natl Acad Sci USA, 1987; 84:5048–5052.

Martin

, Moseley

, Gillespie

. Parathyroid hormone–related protein: Biochemistry and molecular biology. Crit Rev Biochem Mol Biol, 1991; 26:377–395.

De Papp

, Stewart

. Parathyroid hormone–related protein: A peptide of diverse physiologic functions. Trends Endocrinol Metab, 1993; 4:181–187.

Danks

, Trivett

, Power

et al. Parathyroid hormone–related protein in lower vertebrates. Clin Exp Pharmacol Physiol, 1998; 25:750–752.

Burton

, Moniz

, Quirke

et al. Parathyroid hormone–related peptide: Expression in fetal and neonatal development. J Pathol, 1992; 167:291–296.

Halapas

, Tenta

, Pantos

et al. Parathyroid hormone–related peptide and cardiovascular system. In Vivo, 2003; 17:425–432.

Namgung

, Tsang

. Bone in the pregnant mother and newborn at birth. Clin Chim Acta, 2003; 333:1–11.

Khosla

, Johansen

, Ory

et al. Parathyroid hormone–related peptide in lactation and in umbilical cord blood. Mayo Clin Proc, 1990; 65:1408–414.

10.

Lubetzky

, Weisman

, Dollberg

et al. Parathyroid hormone–related protein in preterm human milk. Breastfeed Med, 2010; 5:67–69.

11.

Saxe

, Dean

, Gibson

et al. Parathyroid hormone and parathyroid hormone–related peptide in venous umbilical cord blood of healthy neonates. J Perinat Med, 1997; 25:288–291.

12.

Thiebaud

, Janisch

, Koelbl

et al. Direct evidence of a parathyroid related protein gradient between the mother and the newborn in humans. Bone Miner, 1993; 23:213–221.

13.

Hsu

, Levine

. Perinatal calcium metabolism: Physiology and pathophysiology. Semin Neonatol, 2004; 9:23–36.

14.

Tsang

, Light

, Sutherland

et al. Possible pathogenetic factors in neonatal hypocalcemia of prematurity. The role of gestation, hyperphosphatemia, hypomagnesemia, urinary calcium loss, and parathormone responsiveness. J Pediatr, 1973; 82:423–429.

15.

Baldi

, Ioannis

, Chiara

et al. Biological effects of milk proteins and their peptides with emphasis on those related to the gastrointestinal ecosystem. J Dairy Res, 2005; 72,Spec No:66–72.

16.

Jacques

. Interpretation of Diagnostic Tests, 7^th. Lippincott Williams and Wilkins: Philadelphia, 2000; 597–601.

17.

Papantoniou

, Papapetrou

, Antsaklis

et al. Circulating concentrations of immunoreactive parathyroid hormone–related protein and intact parathyroid hormone in human fetuses and newborn. Eur J Endocrinol, 1996; 134:437–442.

18.

Schanler

. The use of human milk for premature infants. Pediatr Clin North Am, 2001; 48:207–219.

19.

Lee

, Polin

. Treatment and prevention of necrotizing enterocolitis. Semin Neonatol, 2003; 8:449–459.

20.

Caplan

, MacKendrick

. Inflammatory mediators and intestinal injury. Clin Perinatol, 1994; 21:235–46.

21.

Lucas

, Cole

. Breast milk and neonatal necrotising enterocolitis. Lancet, 1990; 336:1519–1523.

22.

Ito

, Ohtsuru

. [Parathyroid hormone–related peptide (PTHrP) and PTH/PTHrP receptor in the gastrointestinal tract] Nippon Rinsho, 1996; 54:1104–1108.

23.

Yamamoto

, Duong

, Fisher

et al. Suckling-mediated increases in urinary phosphate and 3′,5′-cyclic adenosine monophosphate excretion in lactating rats: Possible systemic effects of parathyroid hormone–related protein. Endocrinology, 1991; 129:2614–2622.

24.

Hirota

, Anai

, Miyakawa

. Parathyroid hormone–related protein levels in maternal and cord blood. Am J Obstet Gynecol, 1997; 177:702–706.