African-American Bone Metabolism and Lactation Study
African-American Bone Metabolism and Lactation Study
The primary aim of this study is to obtain measures of amino-terminal telopeptides of
 procollagen 1 (P1NP), a marker of bone formation, in lactating and non-lactating post-partum
 African-American women both at 6-8 and at 12-14 weeks post-partum, and to compare these
 values to those of normal controls. The secondary aim is to obtain at the same time points,
 measurements of Parathyroid Hormone-related Protein (PTHrP), additional markers of bone
 turnover [e.g. N-telopeptide of collagen cross-links (NTx), C-telopeptide of collagen
 cross-links (Ctx),bone specific alkaline phosphatase (BSAP) and osteocalcin (OC)], calcium
 and vitamin D metabolism in these subjects. These results will be compared with a
 non-African-American cohort of post-partum women and normal controls.

 The investigators hypothesize that African-American lactating women will have increased bone
 turnover when compared to non-lactating postpartum women and normal controls. The
 investigators further hypothesize that bone turnover is increased in lactating women
 independent of race.
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.
Inclusion Criteria:

 -  Group 1: Post-partum (singleton pregnancy) African-American women who are exclusively
 breastfeeding, defined as 1 or fewer bottles of supplemental formula/day.

 -  Group 2: Post-partum (singleton pregnancy) African-American women are non-lactating,
 which is defined as bottle-feeding or having weaned their baby from breastfeeding for
 at least 4 weeks prior to study.

 -  Group 3: Controls- Healthy non-pregnant African-American women will be race and
 age-matched to the breast-feeding women in group one. They may not have been lactating
 or pregnant within the last year.

 Exclusion Criteria:

 -  Subjects with cardiac, hypertensive, vascular, renal (serum creatinine of >1.5),
 pulmonary, endocrine, musculoskeletal, hepatic, hematologic or malignant or
 rheumatologic disease will be excluded from the study.

 -  Smokers and those with a history of significant alcohol or drug abuse are excluded.

 -  Baseline hypertension (systolic BP > 160 mm/Hg) or hypotension (systolic BP < 90
 mm/Hg).

 -  Subjects taking any chronic medications except stable doses of thyroid hormone,
 prenatal, vitamin supplements, or oral contraceptives.

 -  Those who have received any investigational drug in past 90 days will be excluded from
 the study.

 -  Women who are currently pregnant will be excluded from the study. Women who became
 pregnant by In Vitro Fertilization IVF or any hormonal manipulation (i.e. fertility
 drugs such as clomid ®) are also excluded, as they may have an altered pre-pregnant
 hormonal state. All women will have a urine pregnancy test performed at each of the
 two study visits and must not be pregnant in order to continue in the study. Subjects
 are not allowed to donate blood between study visits.