African-American Bone Metabolism and Lactation Study

Pregnancy and lactation are both states of altered maternal calcium and bone metabolism which may have a significant impact on the development of peak bone mass. While these two states are characterized by different hormonal environments, both have been associated with significant bone loss. The maternal hormonal mechanism for providing calcium to meet the needs of the developing fetus appear different from those that meet the needs of lactation. During pregnancy, the 30 gm of calcium required by the fetus comes predominantly from an increase in maternal intestinal calcium absorption which is mediated by 1,25 dihydroxy vitamin D and other factors. Several studies have measured total and free 1,25 dihydroxy vitamin D through pregnancy and find the values nearly double. Serum Parathyroid Hormone (PTH) levels fall to about 10-30% of the mean non-pregnant value in the first trimester and then increase to the mid-normal range by term, while ionized calcium remains normal throughout pregnancy. Parathyroid Hormone-related Protein (PTHrP) levels gradually increase throughout pregnancy although the source (maternal, fetal, or placental) remains unclear. Most studies of bone metabolism in humans during pregnancy have measured changes in markers of bone turnover rather than bone density to avoid radiation exposure to the fetus. These studies have been confounded by several variables such as the effects of hemodilution in pregnancy, altered glomerular filtration rates (GFR), degradation and clearance of markers by the placenta, which may cloud the results. Some of these studies report an increase in urinary markers of bone resorption from early to mid pregnancy while bone formation markers decrease and then rise before term. Importantly, no one has assessed state-of-the-art markers of bone formation such as type 1 Procollagen N-terminal Propeptide (P1NP) in pregnancy or lactation.

During lactation in humans, it is estimated that 600 to 1000 ml of milk are produced a day with daily calcium loss of 200 to 400 mg. In contrast to pregnancy, a majority of this calcium comes from demineralization of the maternal skeleton, and is probably predominately mediated by PTHrP in the setting of low estrogen. PTHrP levels are significantly higher in lactating women than non-lactating controls while intact PTH is reduced by approximately 50% during the first several months of lactation. The source of the PTHrP is likely the mammary gland, as PTHrP levels are elevated 10,000 fold in milk and circulating maternal PTHrP levels are increased further with suckling. This is also supported by a mouse model in which the tissue-specific ablation of the PTHrP gene in the lactating mammary gland resulted is a decrease in bone loss during lactation. When PTHrP enters the maternal circulation, it stimulates maternal bone resorption from the skeleton and renal tubular resorption of calcium. PTHrP indirectly suppresses PTH as ionized calcium rises to upper levels of normal. 1,25 dihydroxyvitamin D levels fall to within the normal range during lactation, although they have been reported to be higher in lactating than non-lactating postpartum women. Intestinal absorption of calcium also returns to normal during the post-partum period. Serial bone density measurement (BMD)obtained during lactation show a fall of 3-10% in trabecular bone (spine, hip, femur) with a smaller 1-2% loss at cortical bone. Both losses are far greater that than that seen in early postmenopausal women, or in women receiving gonadotropin releasing hormone (GnRH) agonist therapy. This implies that it is not only the fall in estrogen that mediates bone loss during lactation. The bone loss during lactation seems to be transient as there is rapid recovery of bone density in postpartum women with weaning and the resumption of menses.

Markers of bone resorption have been measured in urine in several prospective studies of lactation in humans where they have been reported to be elevated 2-3 fold. However, these results may be confounded by a decrease in GFR and volume contraction that may occur during lactation compared to pregnancy. Surprisingly, more reliable markers of bone resorption measured in serum (CTX and NTX) have not been measured in a controlled lactation study. Markers of bone formation as measured by osteocalcin (Oc) and bone specific alkaline phosphatase (BSAP) have generally been reported to be higher during lactation. However, these results are difficult to interpret as BSAP is not a very sensitive marker of bone formation. Recent data has emerged suggesting that Oc may measure bone resorption as well as formation. The current most accurate measure of bone formation is serum amino-terminal telopeptides of procollagen 1 (P1NP), which until recently had not been measured in a control study of lactating women. We are currently analyzing data from a controlled study of lactation in Caucasian, Hispanic, and Asian women.

Most of the studies on bone metabolism during pregnancy and lactation have been performed in Caucasian females. Most studies have excluded African-Americans due to the unique differences in bone metabolism known to occur in this population which may create wider statistical variation in the study results.

It is well established that bone metabolism in African American subjects differs from the Caucasians in several aspects. The mean 25-hydroxyvitamin D levels are lower in African-Americans primarily due to reduced vitamin D production in the skin associated with increased pigmentation as well as reduced intake of vitamin D. As a consequence, African-Americans have relative secondary hyperparathyroidism and resultant higher levels of PTH, and 1, 25 dihydroxyvitamin D, and lower urinary calcium excretion. One would expect that patients with secondary hyperparathyroidism would result in increased bone turnover and decreased bone mass. This is not what is observed in African Americans though. African Americans have higher bone density and lower fracture risk as compared to Caucasians. Moreover, biochemical markers of bone turnover in African-Americans are in general lower than in Caucasians, particularly for bone formation, as measured by osteocalcin. On the other hand, there is normal or increased renal absorption of calcium. Based on these, Dr. Normal Bell proposed that the black skeleton is resistant to the effects of PTH, while renal sensitivity is maintained or even enhanced. This hypothesis is further supported by the observation of significantly lower bone resorption (as measured by cross-linked N-telopeptide of procollagen I (NTx), cross-linked C-telopeptide of type I collagen (CTx), and free deoxypyridinoline (DPD) and normal to low renal excretion of calcium in response to PTH infusion in African American as compared with Caucasian women.

This is a prospective cohort study of post-partum lactating, non-lactating, and healthy control African-American women who are not currently or have not recently been pregnant. The investigators hope to estimate the measurable differences in bone formation and resorption by comparing blood and urine samples from African-American lactating women to African-American non-lactating postpartum women and normal controls. 100 female volunteers between the ages of 21-45 years will be recruited to achieve 75 evaluable subjects or 25 in each of the three groups. There are two out-patient visits, each 6-8 weeks apart. The visits will take place at the University of Pittsburgh Medical Center (UPMC) - Clinical \& Translational Research Center (CTRC) located on 6 North East (6 NE) in Montefiore Hospital. The post-partum subjects will be recruited during their last trimester of pregnancy or at their 6-week post-partum visit. Healthy controls will be recruited and matched to a breast-feeding postpartum woman who has already completed the study. Visit 1 for all postpartum women will take place at 6-8 weeks after delivery and Visit 2 at 14 weeks after delivery. Healthy control subjects will have two study visits at similar intervals to their matched breast-feeding subject (1.5 to 2 months). For all subjects, an informed consent will be obtained at the first visit, before any study procedures are done: including a medical history and measurement of vital signs, an intake history about menstrual cycles, pregnancy, and if applicable, delivery and breast or bottle feeding events. Subjects will complete a dietary calcium intake questionnaire. Blood collections are obtained at each visit for measurements of the following: serum ionized and total calcium, phosphorus, creatinine, albumin, PTH(1-34), PTH(1-84), PTHrP (1-36), 25-hydroxy Vitamin D by High Performance Liquid Chromatography (HPLC), including D2 and D3, 1,25 (OH)2 vitamin D, estrogen status, Luteinizing Hormone (LH), Follicle-stimulating Hormone (FSH), Estradiol, and markers of bone metabolism (e.g. NTX, CTX, P1NP, Osteocalcin (OC), BSAP, Insulin-like growth factor-1 (IGF-1), and selected cytokines of the interleukin-6 (IL-6). Urine samples will be obtained for calcium, creatinine and phosphorus. A urine pregnancy test will be done at each visit for normal controls. Post-partum women will have a urine pregnancy checked at Visit 2 only, to avoid false positives that are likely at Visit 1 due to the recently completed pregnancy. Thyroid Stimulating Hormone (TSH) will be checked at Visit 1 to exclude those women who have significant alterations in thyroid functioning that could interfere with bone metabolism.